PMID- 16657731 TI - In vivo phytochrome reversion in immature tissue of the alaska pea seedling. AB - Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red absorbing phytochrome loss under continuous red light. PMID- 16657732 TI - On the mechanism and stoichiometry of the oxidation of hydrazine by illuminated chloroplasts. AB - Hydrazine can support a rapid oxygen uptake in illuminated chloroplasts. The oxygen uptake rate is inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea but is only slightly increased by added methyl viologen, and little H(2)O(2) is produced. The pH optimum for hydrazine-dependent oxygen uptake is much higher than that of the Hill reaction. Addition of Mn (II) increases the rate of oxygen uptake in the light and causes the reaction to continue in the dark, the dark rate being dependent on the duration of the preceding light period. Flash yield experiments show that at least six electrons are transferred from hydrazine per flash compared to one electron per flash when water is the electron donor.We conclude that most of the oxygen uptake in the presence of hydrazine is due to a direct oxidation of the hydrazine by oxygen which is catalyzed by some factor, possibly Mn (III), produced by chloroplasts in the light. Other artificial electron donors are shown to support such artefactual oxygen uptake to varying extents. PMID- 16657733 TI - Identification and quantitative analysis of the volatile substances emitted by maturing cotton in the field. AB - When atmosphere from cotton plants (Gossypium hirsutum L., var. Deltapine Smoothleaf) was condensed by passing it over the expansion coil of an air conditioner and three 1-hour collections per day (early morning, noon, and late afternoon) were made, the total essential oils were found to consist of 50 to 60% beta-bisabolol (I(k) 1660) and gamma-bisabolene (I(k) 1550) and 30 to 40% geraniol (I(k) 1250), myrtenal (I(k) 1328), nerolidol (I(k) 1520), and beta caryophyllene oxide (I(k) 1590). As the plant matured, trans-2-hexanol was produced in concentrations of 7 to 27%. Before fruiting, beta-bisabolol made up as much as 60% of the total essential oil transpired by the plants, and as the concentration of beta-bisabolol increased, that of gamma-bisabolene decreased. PMID- 16657734 TI - Effects of air flow rate, storage temperature, and harvest maturity on respiration and ripening of tomato fruits. AB - The interactive effects of aeration rate, storage temperature, harvest maturity, and storage duration on respiration and ripening of tomato fruits (Lycopersicum esculentum var. Roma) were studied. Slow aeration rate strongly reduced the climacteric but did not affect ripening. Low temperature slowed ripening and reduced respiratory rates, but low temperature did not delay attainment of the climacteric maxima. The effect of air flow rate on the content of CO(2) in the fruits' internal atmospheres was investigated. The possibility that CO(2) is not the primary cause of respiratory inhibition under slow air flow rate is discussed. PMID- 16657735 TI - Promotion of indoleacetic Acid oxidase isoenzymes in tobacco callus cultures by indoleacetic Acid. AB - Indoleacetic acid oxidase in tobacco callus cultures (Nicotiana tabacum L., cv. White Gold) was composed of at least two groups of isoenzymes, which were distinctly different in electrophoretic mobilities and in responses to growth substances. Indoleacetic acid had dual effects; at low concentrations it promoted the development of two fast-migrating indoleacetic acid oxidase isoenzymes, but at high concentrations it increased the level of other indoleacetic acid oxidase isoenzymes with low and moderate electrophoretic mobilities. However, indoleacetic acid was not unique in such effects; 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were effective at concentrations lower than that of indoleacetic acid.A correlation was apparent between the relative levels of the two groups of indoleacetic acid oxidase isoenzymes and the rate and type of growth, as affected by auxins. The development of the fast-migrating anionic indoleacetic acid oxidase isoenzymes was accompanied by a type of growth characterized by rapid growth rate, high water content, and friable tissues. On the other hand, a further increase in the isoenzymes of slower migrating rate was associated with growth retardation.The indoleacetic acid-mediated increase of the fast-migrating indoleacetic acid oxidase isoenzymes was dependent on the level of kinetin, suggesting a multiple control by different types of growth substances. The inhibition of the formation of the fast-migrating isoenzymes by actinomycin D and cycloheximide suggests a requirement for both RNA and protein synthesis. PMID- 16657736 TI - The sites of photoconversion of protochlorophyllide to chlorophyllide in barley seedlings. AB - The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably delta amino-levulinic acid synthetase) is not longer than 10 minutes.The rapid phase of chlorophyll formation can be effected by a series of brief (15 second) pulses of light spaced at least 5 minutes apart. When longer dark intervals are used, no increase is observed in the yield of chlorophyll per pulse. We interpret the findings to indicate that the photoconversion takes place at distinct enzymatic sites whose concentration does not increase during a period of 4 hours following the initial illumination. The sites can be used repeatedly with a turnover time determined by the removal of the product chlorophyllide and the synthesis and placement of a new protochlorophyllide molecule. PMID- 16657737 TI - Plant Nucleases: III. Polyacrylamide Gel Electrophoresis of Corn Ribonuclease Isoenzymes. AB - Isoenzymes of RNase were detected in plant extracts after polyacrylamide gel electrophoresis with a new buffer system. The gels were incubated in an RNA solution, then dipped for 30 seconds into 0.2% toluidine blue. The method is rapid and is sensitive to very small amounts of RNase. The effects of buffers and ethylenediaminetetraacetate on the different enzymes are illustrated by photographs and scans of the gels.RNase I, from endosperms and roots, was the fastest-moving corn RNase. Two isoenzymes of corn RNase II, from microsomes, were detected in the hybrid WF9 x M14, while each parental inbred had one of the isoenzymes. Three isoenzymes of corn Nuclease I, from crude mitochondria, had about the same mobility as the RNase II isoenzymes, but were inhibited by ethylenediaminetetraacetate. RNases were also detected in tobacco and wild carrot tissue cultures. PMID- 16657738 TI - Detrimental effect of rust infection on the water relations of bean. AB - Bean plants (Phaseolus vulgaris L.) infected with the rust Uromyces phaseoli became unusually susceptible to drought as sporulation occurred. Under the conditions used (1,300 ft-c, 27 C, and 55% relative humidity) such plants wilted at soil water potentials greater than -1 bar, whereas healthy plants did not wilt until the soil water potential fell below -3.4 bars. Determinations of leaf water and osmotic potentials showed that an alteration in leaf osmotic potential was not responsible for the wilting of diseased plants. When diffusive resistance was measured as a function of decreasing leaf water content, the resistance of healthy leaves increased to 50 sec cm(-1) by the time relative water content decreased to 70%, whereas the resistance of diseased leaves remained less than 8 sec cm(-1) down to 50% relative water content. Apparently, water vapor loss through cuticle damaged by the sporulation process, together with the reduction in root to shoot ratio which occurs in diseased plants, upset the water economy of the diseased plant under mild drought conditions. PMID- 16657739 TI - Nucleic Acid Metabolism in Germinating Onion: I. Changes in Root Tip Nucleic Acid during Germination. AB - Nucleic acid synthesis in the G(1) cell population of the 1-millimeter apex of the Allium cepa embryo was studied during the initial 73 hours of germination. Quantitative data indicate that the total amount of RNA per cell began to increase after 18 hours of germination while the initial DNA per cell increase did not occur until some 20 hours later. Polyacrylamide gel electrophoresis patterns of (3)H-uridine-labeled total nucleic acid samples indicated that synthesis of all detectable RNA fractions present in the pre-emergent 1 millimeter apex (i.e., cytoplasmic and "chloroplast-like" RNA) began at approximately the same time (18 hours). Synthesis of the various cytoplasmic RNA fractions continued throughout the germination period. Data indicating synthesis of the "chloroplast-like" RNA were obtained only for the initial 36 hours of germination. Specific radioactivity of (3)H-uridine-labeled total nucleic acid increased during the first 41.5 hours of germination but then decreased while the accumulation of RNA per cell continued to increase throughout the 73-hour period. In addition, a method is described which reduced the bacterial contamination of Allium seed to a level not detectable by incorporation of radioactive precursors into bacterial ribosomal RNA. PMID- 16657740 TI - Enhancement of protein synthesis in isolated chloroplasts by irradiation of fern gametophytes with blue light. AB - Irradiation of the gametophytes of Pteridium aquilinum with blue light led to a nearly 5-fold increase in the amino acid-incorporating activity of isolated chloroplasts. The blue light effect was not due to increased synthesis of ATP or other energy donors by the chloroplasts but was probably related to an increased production of chlorophyll and photosynthetic enzymes. PMID- 16657741 TI - (+)-abscisic Acid content of spinach in relation to photoperiod and water stress. AB - Levels of (+)-abscisic acid present in the long-day plant spinach (Spinacia oleracea L., cv. Savoy Hybrid 612) grown under different photoperiodic regimes were measured in purified extracts by optical rotary dispersion. When plants were transferred from short to long days, the abscisic acid content increased 2- to 3 fold. This rise in the level of abscisic acid took place during the 1st long day. Abscisic acid levels of plants under short days as well as under long-day conditions were higher at the end of the 8-hour high intensity light period than at its beginning.The growth retardant AMO-1618 [2'-isopropyl-4'-(trimethyl ammonium chloride)-5'-methylphenyl piperidine-1-carboxylate], which strongly reduces the gibberellin content of spinach under long days, did not affect the abscisic acid content.When water was withheld from plants until wilting symptoms appeared, the abscisic acid content increased more than 10-fold over that of turgid plants. There was no evidence that the sudden rise of abscisic acid level during wilting was due to release from a water-soluble bound form.Bioassays of crude acidic extracts in the wheat coleoptile section test did not indicate the presence of other specific growth inhibitors besides abscisic acid. It is concluded that abscisic acid does not function as an endogenous regulator of stem growth and flower formation in the long-day plant spinach. PMID- 16657742 TI - Metabolism of separated leaf cells: I. Preparation of photosynthetically active cells from tobacco. AB - Suspensions of mesophyll cells, prepared from tobacco leaves by treatment with pectinase, fixed CO(2) by photosynthesis. The products of carbon assimilation were similar for both cells and intact tissue. The cells sustained a constant fixation rate for 20 to 25 hours. For optimal CO(2) fixation, enzymatic maceration of the tissue was accomplished in 0.8 m sorbitol, but photosynthesis was optimal in 0.6 m sorbitol at pH 7 to 7.5. A hypertonic environment during maceration, which results in cell plasmolysis, is essential to maintain intact plasmalemmas and hence photosynthetically active cells. For sustained CO(2) fixation, light intensities below 500 foot-candles were required. Higher light intensities (to 1000 foot-candles) gave high initial rates of CO(2) fixation, but the cells bleached and were inactive on prolonged incubation. At pH 7.0 the bicarbonate concentration at maximal velocity of CO(2) fixation was about 1.5 mm and the apparent Km for bicarbonate was 0.2 mm. PMID- 16657743 TI - Diffusible and Extractable Growth Regulators in Normal and Dwarf Shoot Apices of Peach, Prunus persica Batsch. PMID- 16657744 TI - Ethylene production and enzyme induction in excised plant tissues. PMID- 16657745 TI - Effects of cytokinins on growth and auxin in coleoptiles of derooted Avena seedlings. PMID- 16657746 TI - Lateral transport of ions into the xylem of corn roots: I. Kinetics and energetics. AB - A technique is described for study of the kinetics of lateral transport of ions across single roots of corn, Zea mays, in short term experiments under steady state conditions. The kinetics of chloride transfer to the vessels reflected the kinetics of absorption of chloride by the root cells. Efflux from the root vacuoles contributed to only a small extent to transport of chloride into the exudate. Lateral transport of chloride was inhibited by bromide at chloride concentrations in the ranges of both mechanisms 1 and 2 in a manner implicating competition. The uncoupler carbonylcyanide m-chlorophenylhydrazone used at 1 mum caused transfer of chloride to cease almost immediately at both low and high concentrations of chloride. Oligomycin depressed transport of chloride to the vessels within 10 to 15 minutes after application at 2 micrograms per milliliter. Inhibition by oligomycin was 75% at 0.5 mm chloride and 55% at 5 mm.It is concluded that lateral transport of chloride across corn roots is mediated by the dual mechanisms of ion absorption which reside in the plasmalemma. Transfer of chloride is inhibited by bromide and depends upon ATP as energy source. Chloride moves from the plasmalemma, the site of carriermediated absorption, to the xylem vessels by way of the symplasm. There is no evidence in these experiments that lateral transport of chloride in corn roots is governed by diffusion at any concentrations of chloride used in these experiments. PMID- 16657747 TI - Lateral Transport of Ions into the Xylem of Corn Roots: II. Evaluation of a Stelar Pump. AB - When an excised corn (Zea mays) root pretreated with chloride was exposed for 10 minutes to pulse labeling with (30)Cl and then transferred to unlabeled chloride, the activity in the xylem exudate reached a maximum about 4 minutes after pulse labeling was discontinued and then declined sharply. The rate at which labeled chloride was transported across the root into the xylem and basipetally therein was on the order of 75 to 250 centimeters per hour. Consequently, symplasmic movement of chloride in corn roots is fast and may not be rate-limiting in transfer from the root surface to the xylem. Experiments on pulse labeling with (22)Na gave similar results. A large fraction of the absorbed (22)Na was not translocated into the exudate but was tightly sequestered in a cell compartment, probably the vacuole.Electron probe analysis was used to reveal the pattern of potassium distribution in cross sections taken 10 to 11 millimeters from the tip. The cytoplasm and vacuoles of the xylem parenchyma cells accumulated potassium to a much greater extent than cortical and other stelar cells. Ultrastructural studies showed that the cytoplasm of the xylem parenchyma cells contains numerous membrane systems. It was concluded that the xylem parenchyma cells secrete ions from the symplasm into the conducting vessels, and it was suggested that this secretion is driven across the plasmalemma by a carrier-mediated transport. PMID- 16657748 TI - Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue. AB - Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome. PMID- 16657749 TI - l-Phenylalanine Ammonia-Lyase (Maize): Evidence for a Common Catalytic Site for l Phenylalanine and l-Tyrosine. AB - l-Phenylalanine ammonia-lyase (E.C. 4.3.1.5) from maize is active with l-tyrosine and l-phenylalanine and exhibits atypical Michaelis-Menten kinetics with both substrates. With phenylalanine as a substrate, the pH optimum is 8.7 and with tyrosine, 7.7. The estimated Km at high substrate concentrations is 0.27 mm for phenylalanine and 0.029 mm for tyrosine. However, the V(max) with phenylalanine is eight times higher than the V(max) with tyrosine when both are measured at pH 8.7, and 7 times higher when both are measured at their pH optima. The following evidence leads us to the conclusion that there is a common catalytic site for both substrates: (a) It is impossible to appreciably alter the ratio of the two activities during purification and isoelectric focusing. (b) The ratio of the products formed in mixed substrate experiments is in good agreement with the ratio predicted from the estimated Km values. (c) NaBH(4) reduces both activities to the same degree and l-phenylalanine, l-tyrosine, cinnamate, and p-coumarate protect both activities against NaBH(4) reduction to the same degree. In contrast, the enzyme isolated from potato, which does not act on l-tyrosine, is not protected against reduction by either l-tyrosine or p-coumarate. However, both enzymes appear to have a dehydroalanine-containing prosthetic group. PMID- 16657750 TI - Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds. AB - alpha-Amylase levels in intact seeds of barley (Hordeum vulgare L. cv. Himalaya) reach a maximum at 3 to 4 days of germination while gibberellin levels continue to increase beyond 6 days of germination. In contrast to its effect on half seeds, gibberellic acid does not increase the total amount of alpha-amylase produced in germinating seeds. The inability of gibberellic acid to stimulate alpha-amylase production is not related to its availability; rather, evidence suggests that a factor(s) in whole seeds prevents further enhancement of alpha amylase formation and accumulation. Hydrolysis products accumulate in the subaleurone space of the endosperm of germinating seeds up to concentrations of 570 milliosmolar. Chromatography of these hydrolysis products indicate the presence of maltose and glucose. Calculations based on reducing sugar determinations show that glucose accounts for as much as 57% of the solutes present in the endosperm fluid. Both maltose and glucose in the range of 0.2 to 0.4 M effectively inhibit the production of alpha-amylase by isolated barley aleurone layers. This inhibition is quantitatively similar to that brought about by solutions of polyethylene glycol and mannitol. On the basis of these data we propose that hydrolysis products which accumulate in the starchy endosperm of germinating seeds function to regulate the production of hydrolytic enzymes by the aleurone layer. PMID- 16657751 TI - Chlorophyll, Ribulose-1,5-diphosphate Carboxylase, and Hill Reaction Activity in Developing Leaves of Populus deltoides. AB - The synthesis of chlorophyll and ribulose diphosphate carboxylase as well as the development of Hill reaction activity were followed in expanding Populus deltoides leaves and related to photosynthetic patterns. Total chlorophyll, which was not correlated with photosynthetic rate in expanding leaves, decreased slightly with age in very young leaves, due to a decrease in chlorophyll b, but then increased linearly. The ratio of chlorophyll a to b, which rose sharply in young leaves, was highly correlated with the onset of net photosynthesis. Hill reaction activity was very low in young leaves and did not increase significantly until leaves were about half expanded. Ribulose diphosphate carboxylase activity increased in a sigmoid fashion with leaf ontogenesis and closely paralleled development of the photosynthetic system. The study demonstrates the importance of chlorophyll a and Calvin cycle enzyme synthesis to photosynthetic development in expanding leaves. PMID- 16657752 TI - Auxin Transport within Intact Dormant and Active White Ash Shoots. AB - Transport of indoleacetic acid-1(14)C following application to the buds of intact white ash (Fraxinus americana L.) shoots proceeds at a velocity of about 1.3 centimeters per hour in actively growing seedlings, but only 0.3 centimeter per hour in dormant seedlings. The rapid movement is metabolically controlled, and at 1 C or in a nitrogen environment it is reduced to 0.2 centimeter per hour, suggesting that the slower movement is due to diffusion. The transport profile for growing shoots shows a logarithmic decrease in activity in stems treated for 3 hours. However, over longer treatment intervals, especially after 12 hours, a steady state of recoverable activity occurs in the more basal stem segments. Cold treated shoots acquire the capacity for rapid transport 7 days after they are placed into favorable growing conditions, at which time dormancy callose disappears from the phloem, respiratory activity of the stem tissue increases, and mitotic reactivation occurs in the bud. Following shoot reactivation, the velocity and amount of exogenously supplied indoleacetic acid transported remained relatively uniform until the onset of the succeeding dormant period. Five per cent, or less, of the applied tracer moves into the shoot, with substantial portions remaining as indoleacetic acid. PMID- 16657753 TI - A Role for Zinc in the Structural Integrity of the Cytoplasmic Ribosomes of Euglena gacilis. AB - Zinc deficiency in dark-grown Euglena gracilis Klebs, Z strain Pringsheim, results in the disappearance of cytoplasmic ribosomes. In contrast, ribosomes in zinc-sufficient Euglena are conserved, do not undergo turnover, and can be demonstrated at any stage of growth. The zinc content of ribosomes from zinc deficient Euglena just prior to ribosomal disappearance is 300 to 380 micrograms of zinc per gram rRNA as compared to 650 to 1280 micrograms of zinc per gram rRNA in ribosomes from zinc-sufficient cells. Ribosomal disappearance is believed to involve a generalized disintegration process related to the lower content of zinc in the ribosomes. Reappearance of ribosomes requires the addition of zinc. It is proposed that adequate zinc may be essential for normal tertiary and quaternary structure of the cytoplasmic ribosomes of Euglena. PMID- 16657754 TI - The effect of freezing on thylakoid membranes in the presence of organic acids. AB - The effect of salts of organic acids on washed and non-washed chloroplast membranes during freezing was investigated. Thylakoids were isolated from spinach leaves (Spinacia oleracea L.) and, prior to freezing, salts of various organic acids or inorganic salts or both were added. Freezing occurred for 3 to 4 hours at -25 C. After thawing membrane integrity was investigated by measuring the activity of cyclic photophosphorylation.At very low NaCl levels (1 to 3 mm, washed thylakoids) salts of organic acids either could not prevent membrane inactivation in the course of freezing (succinate) or were effective only at relatively high concentrations (0.1 m or more of acetate, pyruvate, malate, tartrate, citrate). If NaCl was present at higher concentrations (e.g., 0.1 m) some organic acids, e.g. succinate, malate, tartrate, and citrate, were able to protect frost-sensitive thylakoids at surprisingly low concentrations (10 to 20 mm). Other inorganic salts such as KCl, MgCl(2), NaNO(3) could also induce protection by organic acids which otherwise were ineffective or poorly effective. For effective protection, a more or less constant ratio between inorganic salt and organic acid or between two or more organic acids had to be maintained. Departure to either side from the optimal ratio led to progressive inactivation.The unspecificity of the protective effect of organic acids suggests that these compounds protect colligatively. There are also indications that, in addition, more specific interaction with the membranes contributes to protection. At temperatures above the freezing point, the presence of salts of organic acids decreased the rate of membrane inactivation by high electrolyte concentrations. PMID- 16657755 TI - Phosphoglycerate as a hill oxidant in a reconstituted chloroplast system. AB - The ability of intact chloroplasts to evolve oxygen when illuminated in the presence of 3-phosphoglycerate is lost following osmotic shock but may be largely restored by the addition of ATP, catalytic amount of nicotinamide adenine dinucleotide phosphate, ferredoxin, and a soluble fraction derived from chloroplasts. In the presence of uncoupling agents and ATP, the rate is faster than that supported by intact chloroplasts. In the absence of uncoupling agents, the ATP requirement may be partially met by photophosphorylation. PMID- 16657756 TI - Chilling injury and nucleotide changes in young cotton plants. AB - The effects of chilling at 3 to 5 C on the nucleotide composition of roots and leaves of cotton (Gossypium hirsutum L.) seedlings were determined. Chilling decreased the concentration of nucleotides, especially di- and triphosphates, in both leaves and roots. Chilling also caused an increase in free nucleosides. The results are interpreted to mean that general phosphorolytic activity is associated with chilling injury rather than damage to the phosphorylating mechanisms alone. Hardening at 10 to 20 C prior to chilling protected the seedlings against subsequent chilling injury and prevented nucleotide losses. PMID- 16657757 TI - Interactions of lipoidal materials and a pyridazinone inhibitor of chloroplast development. AB - Formation of chloroplast pigments was inhibited, and free fatty acids accumulated in mustard (Brassica juncea [L.] Coss.) cotyledons and in barley (Hordeum vulgare L.) first leaves developed after treatment with 4-chloro-5- (dimethylamino)-2- (alpha, alpha, alpha-trifluoro-m-tolyl) -3 (2H) -pyridazinone. The inhibitor reduced the amount of fatty acids found in polar lipids (galactolipids) of barley chloroplasts and increased the amount in nonpolar lipids while having little effect on total content of bound fatty acids. The inhibition of chlorophyll formation was circumvented by D-alpha-tocopherol acetate, phytol, farnesol, and squalene, and by unsaturated fatty acids and their methyl esters. The protective action can be explained partially by an interaction external to the plant whereby 4-chloro-5- (dimethylamino) -2- (alpha, alpha, alpha-trifluoro-m-tolyl) -3 (2H) pyridazinone partitioned out of the aqueous phase and into the lipid phase, thus limiting availability of the inhibitor to plants. However, the amount of inhibitor reaching the cotyledons of tocopherol-protected mustard seedlngs was still in excess of the amount necessary to cause white foliage, but it failed to produce the effect. Tocopherol treatment did not prevent the 4-chloro-5- (dimethylamino) -2- (alpha, alpha, alpha-trifluoro-m-tolyl) -3 (2H) -pyridazinone induced buildup of fatty acids in mustard cotyledons but did partially circumvent the effect in barley leaves. The amount of linolenic acid relative to linoleic acid was reduced in barley leaves and chloroplasts by 4-chloro-5- (dimethylamino) -2- (alpha, alpha, alpha-trifluoro-m-tolyl) -3 (2H) -pyridazinone action and this effect was circumvented by tocopherol. PMID- 16657758 TI - Response of Respiration of Tobacco Leaves in Light and Darkness and the CO(2) Compensation Concentration to Prior Illumination and Oxygen. AB - The course of respiration in control leaves of tobacco (Nicotiana tabacum L.) that were illuminated 4 to 5 hours and then darkened 0.25 to 10 hours and in tobacco leaves starved of carbohydrate by 14 hours or more of darkness was measured as CO(2) efflux in light and darkness into CO(2)-free atmospheres containing 0.04, 2.23, 21, 40, and 100% O(2).Control and starved leaves showed a postillumination burst of CO(2) that differed from the usual rapid postillumination burst of control leaves in its onset and duration and its sensitivity to O(2) and starvation. Relative to control leaves, starved leaves lacked the initial postillumination burst of photorespiration and the O(2) stimulated CO(2) efflux in the light, but these characteristics were regained when starved leaves were permitted to assimilate CO(2) from air or N(2) for as little as 20 minutes. Five respiratory patterns of CO(2) evolution, two in the light and three in darkness, were identified by manipulating light, darkness, starvation, and O(2) concentration.The CO(2) compensation concentrations varied with O(2) but were alike in control and starved leaves regardless of the presence or absence of photorespiratory characteristics and rates of respiration in light and darkness. PMID- 16657759 TI - The Role of Various Regions of the Bean Hypocotyl on Red Light-induced Hook Opening. AB - Measurement of various zones on the concave half of etiolated Phaseolus vulgaris L. (cv. Black Valentine) hypocotyls has shown that growth at the basal portion of the elbow and the contiguous upper portion of the shank was stimulated earliest by red light. Growth of these two zones was unaffected by the tissue of the convex half but was inhibited by tissue distal to them. The inhibition was alleviated by the continuous presence of shank tissue below the growing zones. Based on cuts made halfway through the hypocotyl at positions above, below, or between the two zones of growth, it is suggested that cells at the inner portion of the upper shank control in some way the light-induced growth of the elbow cells directly above. PMID- 16657760 TI - Auxin and red light in the control of hypocotyl hook opening in beans. AB - Evidence is presented to support the suggestion that endogenous auxinlike substances participate in controlling the unbending of the hypocotyl hook of Phaseolus vulgaris L. (cv. Black Valentine). An acidic indole was detected in hook diffusates by fluorometry; triiodobenzoic acid, an inhibitor of auxin transport, prevented red light-induced unbending, and indoleacetic acid can be substituted for tissue just above the elbow region as an inhibitor of opening. Indoleacetic acid also stimulated growth of shank cells, and red light increased the sensitivity of this tissue to the hormone. A small red light-induced stimulation of auxin transport through the inside half of the hypocotyl shank was observed and may be related to light-induced unbending of the hook. PMID- 16657761 TI - Changes of photorespiratory activity with leaf age. AB - We have discovered that younger leaves do not exhibit the same large increase in photosynthesis as do older ones when the oxygen tension is lowered. This phenomenon was observed in various tobacco and citrus species and indicated less photo-respiration in young leaves. Activities of glycolate oxidase and of glyoxylate reductase were found to be significantly lower in younger leaves than in more mature ones from the same plant. Assays of glycolate content revealed the same trend. Levels of phosphoenolpyruvate carboxylase were only slightly higher in the younger leaves. This finding lessens the possibility of differing enzymatic pathways for CO(2) fixation. It is concluded that the small photorespiratory activity of young leaves can be attributed to a lower activity of the photorespiratory enzymes and perhaps also to a slightly higher rate of internal recycling of CO(2). PMID- 16657762 TI - The induction of phenylalanine ammonia lyase and phaseollin by 9-aminoacridine and other deoxyribonucleic Acid intercalating compounds. AB - Bean pod tissue (Phaseolus vulgaris L. var. Top Crop) is induced to produce phaseollin when challenged with various microorganisms. The pods react in the same manner when challenged with 9-aminoacridine. This compound also caused an increase in concentrations of phenylalanine ammonia lyase, an enzyme of the phaseollin synthesizing pathway. Both the synthesis of phenylalanine ammonia lyase and phaseollin are subject to inhibition by actinomycin D, cycloheximide, or 6-methylpurine. The results suggest that both phaseollin production and increased phenylalanine ammonia lyase, when induced by 9-aminoacridine, require newly synthesized RNA and protein.The concentration of 9-aminoacridine optimal for synthesis of phaseollin and PAL (0.5 mg/ml) does not increase the rate of total protein synthesis. However, there is a differential effect of 9 aminoacridine on synthesis of certain protein fractions. Optimal concentrations of 9-aminoacridine induce phaseollin and phenylalanine ammonia lyase synthesis while reducing the net synthesis of RNA during the period of induction.The planar three-ring structure of 9-aminoacridine appears to be a desirable feature for phaseollin and phenylalanine ammonia lyase induction. Similar compounds, all DNA intercalators, having dimethylamino, diethylamino, amino, or 9-alkylamino substitutions of a three-ring acridine skeleton, are also inducers of phenylalanine ammonia lyase and phaseollin synthesis.It is suggested that 9 aminoacridine and other DNA intercalators function as inducers of phaseollin and phenylalanine ammonia lyase synthesis by reacting with the DNA template. PMID- 16657763 TI - Energy budgets and temperatures of nyctinastic leaves on freezing nights. AB - Temperatures of exposed horizontal and vertical soybean leaves (Glycine max [L.] Merr. var. Chippewa) were measured on calm, clear nights with temperatures near freezing. Average leaf-air temperature differences for 5 nights were -1.5 C and 1.0 C for horizontal and vertical leaves respectively. The horizontal leaves were cooler than the vertical leaves. The mean of all observed horizontal-vertical leaf temperature differences was -0.5 C with a maximum average for 1 night of 0.8 C, while maximum differences theoretically attainable in similar leaves were calculated to be -1.7 C. No differences were observed in the extent of frost damage in horizontal and vertical leaves. The apparent reduction in frost damage in vertical leaves observed by Charles Darwin was probably caused by his method of using corks to hold the horizontal leaves and not by leaf orientation. Theoretical considerations and the experimental results indicate that nyctinastic leaf movements probably do not provide significant protection from frost for any plants. PMID- 16657764 TI - Abscission: the role of ethylene modification of auxin transport. AB - The role of ethylene-mediated reduction of auxin transport in natural and ethylene-induced leaf abscission was studied in the cotton (Gossypium hirsutum L., cv. Stoneville 213) cotyledonary leaf system. The threshold level of ethylene required to cause abscission of intact leaves was between 0.08 and 1 mul/l with abscission generally occurring 12 to 24 hours following ethylene fumigation. The threshold level of ethylene required to reduce the auxin transport capacity in the cotyle-donary petiole paralleled that required for stimulation of abscission. In plants where cotyledons are allowed to senesce naturally there is a decline in auxin transport capacity of petioles and increase in ethylene synthesis of cotyledons. The visible senescence process which precedes abscission requires up to 11 days, and increases in ethylene production rates and internal levels were detected well before abscission. Ethylene production rates for entire cotyledons rose to 2.5 mmu1 g(-1) hr(-1) and internal levels of 0.7 mul/l were observed. These levels appear to be high enough to cause the observed decline in auxin transport capacity. These findings, along with those of others, indicate that ethylene has several roles in abscission control (e.g., transport modification, enzyme induction, enzyme secretion). The data indicate that ethylene modification of auxin transport participates in both natural abscission and abscission hastened by exogenous ethylene. PMID- 16657765 TI - Deoxyribonucleic Acid synthesis in root cap cells of cultured roots of convolvulus. AB - Isolated cultured roots of Convolvulus arvensis L. were incubated in 0.2 microcurie per milliliter methyl-(3)H-thymidine for 14 hours, for 64 hours, or for 14 hours followed by transfer to fresh nutrient medium without tritiated thymidine. Autoradiographs of serial, longitudinal sections of roots which were continuously incubated with tritiated thymidine showed that cells of the root cap columella did not undergo DNA synthesis after their formation from the root cap initials. In roots pulse-labeled with tritiated thymidine, the movement of labeled cells through the root cap columella was followed. Labeled cells were displaced at a constant rate of 72 microns per day over a period of 6 to 9 days before they were sloughed off from the root cap. The specialized role of the root cap cells in relation to their distinctive metabolism and longevity is discussed. PMID- 16657766 TI - Regulation of nitrate reductase in excised barley roots. AB - When excised barley roots (Hordeum distichum L.) are appropriately pretreated, the level of nitrate reductase in the roots increases upon exposure to nitrate. Relatively low levels of nitrate (10 mum) gave maximum induction of nitrate reductase. This increase was inhibited by inhibitors of protein and RNA synthesis, indicating that de novo protein synthesis is probably involved. Induction of nitrate reductase by nitrate is partially prevented by the inclusion of ammonium, an eventual product of nitrate reduction, in the incubation medium. Under the experimental conditions used, ammonium did not inhibit the uptake of nitrate by excised barley roots. It is concluded, therefore, that ammonium, or a product of ammonium metabolism, has a direct effect on the synthesis of nitrate reductase in this tissue. PMID- 16657767 TI - Regulation of Nitrate Reductase in Chlorella vulgaris. PMID- 16657768 TI - A simple method for synthesis of ferric ethylenediaminetetraacetate in a pure and stable form. PMID- 16657769 TI - The effects of water stress on some membrane characteristics of corn mitochondria. PMID- 16657770 TI - Effect of scopoletin on two anodic isoperoxidases isolated from tobacco tissue culture w-38. PMID- 16657771 TI - The climacteric in ripening tomato fruit. AB - Phosphofructokinase is identified as the regulator reaction activated at the onset of the climacteric rise in respiration of the ripening tomato fruit (Lycopersicon esculentum Mill). The concentration of ATP in the fruit increases to a maximum value after the climacteric peak of respiration is past. Orthophosphate is proposed as the most probable activator of phosphofructokinase in the ripening fruit.Fifteen hours after infiltrating tomato fruit with orthophosphate, the rate of respiration increased and remained high until the end of the experiment, 45 hours after infiltration. In experiments where tomato plants were grown at various nutrient levels of P, the rate of respiration when fruit harvested at the mature-green stage reached the respiratory climacteric was correlated with the concentration of orthophosphate in the fruit at the end of the experiment. These results are consistent with the hypothesis that stimulation of phosphofructokinase through increasing concentration of orthophosphate in the cytoplasm of the fruit contributes to the climacteric rise in respiration. PMID- 16657772 TI - Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles. AB - Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile. PMID- 16657773 TI - An interpretation of cell growth curves. AB - It is shown that typical cell elongation curves with time, as reported in the literature, are the consequence of a very simple trend of relative elemental elongation rate with time. PMID- 16657774 TI - Growth and Respiratory Response of Fig (Ficus carica L. cv. Mission) Fruits to Ethylene. AB - Growth in diameter of the fig (Ficus carica L. cv. Mission) fruit takes place in three distinct periods; two periods (I and III) of rapid growth are separated by a period (II) of slow growth. With respect to exposure to ethylene, the fruit exhibits a two phase response. Ethylene inhibits fruit growth in phase A (period I), the period of cell division, stimulates growth in early phase B (early period II), and stimulates both growth and ripening during the remainder of phase B (late period II and period III). The adverse effect of exogenous ethylene on fruits during phase A is thought to be due to inhibition of cell division. The gradual transition occurring in the response of fruits during phase B was interpreted in terms of carbohydrate level in the fruits.The onset of period III and a respiratory climacteric rise was preceded by or concomitant with a sudden burst of endogenous ethylene synthesis. This, together with the fact that exogenous ethylene applied at the proper stage of fruit growth triggers both ripening and the climacteric rise, leads to the conclusion that ethylene is the causal agent. In other words, the data support the concept that ethylene is a growth hormone that initiates a chain of metabolic and physiological events leading to fig fruit ripening. PMID- 16657775 TI - The Isolation and Characterization of d-Glucose 6-Phosphate Cycloaldolase (NAD Dependent) from Acer pseudoplatanus L. Cell Cultures: Its Occurrence in Plants. AB - A soluble enzyme system from suspension cultures of Acer pseudoplatanus L. converts d-glucose 6-phosphate to myoinositol. A Mg(2+)-dependent phosphatase, present in the crude extract, hydrolyzes the product of the cyclization, myoinositol monophosphate, to free myoinositol. Further purification of the enzyme system by precipitation with (NH(4))(2)SO(4) followed by diethylaminoethyl cellulose chromatography eliminates the phosphatase and makes it necessary to add alkaline phosphatase to the reaction mixture in order to assay for free myoinositol. Gel filtration on Sephadex G-200 increases the specific activity of the cycloaldolase to 8.8 x 10(-4) units per milligram protein (1 unit = 1 micromole of myoinositol formed per minute). The cycloaldolase has an absolute requirement for nicotinamide adenine dinucleotide and a maximum activity at pH 8 with 0.1 mm nicotinamide adenine dinucleotide. The reaction rate is linear for 2.5 hours when d-glucose 6-phosphate is below 4 mm and has a K(m) of 1.77 mm. The diethylaminoethyl cellulose-purified enzyme is stable for 6 to 8 weeks in the frozen state. PMID- 16657776 TI - Influence of cycloheximide on the synthesis and utilization of amino acids in suspension cultures. AB - Cells from 4-days old suspension cultures of Paul's Scarlet rose were incubated with acetate-U-(14)C for 10 minutes. After washing, cells were incubated for 2 hours in growth medium in the presence and absence of cycloheximide. The (14)C content of individual amino acids in the soluble form and in protein were determined at the end of the 10 minute pulse and at intervals thereafter in control cells and those treated with cycloheximide. During the period following the pulse there was a 3-fold increase in the (14)C content of protein in control cells; no such increase occurred in the presence of cycloheximide.In the control cells there was a net increase in the (14)C content of eight individual amino acids during this period. For six of these, the corresponding increases in the presence of cycloheximide were curtailed by the following amounts: arginine 98%, lysine 94%, isoleucine 93%, threonine 82%, valine 49%, and proline 36%. This is interpreted to mean that the synthesis of each of these amino acids was slowed down when their incorporation into protein was prevented. PMID- 16657777 TI - Photosynthetic enhancement at high light intensities. AB - Enhancement of photosynthetic oxygen evolution by Chlorella pyrenoidosa (211-8b) decreased almost linearly as the rate of photosynthesis was increased. The observed decrease was predicted most accurately by a model assuming exponential rate versus intensity curves as opposed to a model assuming hyperbolic light curves. The decrease in enhancement was not changed significantly upon changing from 0.03% to 5% carbon dioxide. PMID- 16657778 TI - Glycolate: Dichlorophenolindophenol Oxidoreductase in Chlamydomonas reinhardtii. AB - GLYCOLATE: dichlorophenolindophenol oxidoreductase levels in Chlamydomonas reinhardtii are not under the immediate control of the CO(2) tension to which the cells are exposed. The enzyme is synthesized initially in 5% CO(2) in air at a similar rate to that in air. It disappears from the cell under nitrogen limitation. PMID- 16657779 TI - Glucose metabolism of embryos and endosperms from deteriorating barley and wheat seeds. AB - Changes in glucose utilization into CO(2) and ethanol-insoluble material were followed in whole seeds, embryos, and endosperms of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) which had reached different levels of deterioration through accelerated aging treatments. Excised embryos from deteriorated wheat seeds had reduced respiration and glucose utilization into ethanol-insoluble material but not into CO(2). These treatments had no effect on respiration of excised endosperms, although they reduced utilization of glucose into ethanol-insoluble material and CO(2). Changes in metabolic activity of whole seeds in response to deterioration treatments are difficult to interpret because they represent the sum of the changes that take place in the embryos and endosperms. Changes in respiration and glucose utilization in these two tissues neither proceed at the same rate nor go in the same direction during deterioration.Incubation of excised embryos and endosperms in glucose-(14)C under N(2), as compared to air, affects the utilization but not the uptake of glucose. Embryos produce more (14)CO(2) and less labeled ethanol-insoluble material under N(2) as compared to air. The responses of endosperms to N(2) are of a much lower magnitude than those of embryos. PMID- 16657780 TI - Translocation of nickel in xylem exudate of plants. AB - Topped plants of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), corn (Zea mays), carrot (Daucus carota), and peanut (Arachis hypogaea) were treated with 0.5 to 50 micromolar Ni (containing (63)Ni) in nutrient solutions. Xylem exudate was collected for 10 hours or, in the case of corn, for 20 hours at 5-hour intervals. Electrophoresis of nutrient solution distributed all Ni cathodically as inorganic Ni(2+). Low concentrations of Ni in tomato exudate migrated anodically, presumably bound to organic anion (carrier). However, this carrier became saturated at about 2 micromolar Ni in exudate, and excess Ni ran cathodically. Most of the Ni in cucumber, corn, carrot, and peanut exudate ran anodically, and its migration rate was identical for all exudates. Peanut root sap contained 14 to 735 micromolar Ni. The anodic Ni carriers in root sap and exudate appear identical. The carrier in root sap became saturated near 100 micromolar Ni, as shown by cathodic streaking of Ni exceeding that concentration. It appears that all five species translocate low concentrations of Ni in the same anionic form. PMID- 16657781 TI - A Nonproteolytic "Trypsin-like" Enzyme: Purification and Properties of Arachain. AB - By the use of the proteolytic substrates benzoyl-dl-arginine-p-nitroanilide and benzoyl-l-arginine ethyl ester the enzyme arachain has been purified 325-fold from acetone powders of ungerminated peanuts. The pH optimum for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was 8.1 in tris buffer, and for benzoyl-l arginine ethyl ester was 7.5 using N - 2 - hydroxyethylpiperazine - N' - 2 - ethanesulfonic acid buffer. The purest fraction showed one main band with one to three minor bands on disc gel electrophoresis. The major protein component had an S(20,w) of 6.20. The energy of activation for the hydrolysis of benzoyl-dl arginine-p-nitroanilide was calculated to be 16 kilocalories. The Michaelis constant for benzoyl-dl-arginine-p-nitroanilide was 10 micromolar and for benzoyl l-arginine ethyl ester was 110 micromolar. The enzyme showed essentially no activity with casein, dimethyl casein, or bovine serum albumin as substrates. A large number of peptides were hydrolyzed by the enzyme, only l-leucyl-l-tyrosine being resistant of the peptides tested. The results suggest that arachain is not a "trypsin-like" protease but is a peptide hydrolase. PMID- 16657782 TI - The photosynthetic unit in chlorella measured by repetitive short flashes. AB - Apparent size of the photosynthetic unit in Chlorella pyrenoidosa was estimated by the method of Emerson and Arnold: rate of oxygen evolution was measured under repetitive saturating flashes of about 10-microsecond duration separated by dark periods of 0.033 to 0.100 second. Cells used were taken from six steady state cultures maintained at different light intensities. Cell characteristics included a variation in chlorophyll content from 1 to 5%. Apparent size of the photosynthetic unit varied systematically with chlorophyll content in the range of 1560 to 2350 chlorophylls per O(2) per flash. Values for unit size showed no unusual statistical distribution and were not changed significantly by addition of low level background light at 645 or 705 nanometers. Maximal rate of unit turnover, calculated from light-saturated rate and unit size, varied inversely with chlorophyll content in the range of 70 to 180 per second. PMID- 16657783 TI - Correlation between the Suppression of Glucose and Phosphate Uptake and the Release of Protein from Viable Carrot Root Cells Treated with Monovalent Cations. AB - Treating carrot (Daucus carota L.) discs with ice-cold NaCl solutions for 30 minutes caused three effects that appear to be functionally related: the exchange of tissue Ca(2+) and Mg(2+) for Na(+), the release of protein, and the suppression of active uptake of glucose and orthophosphate. Cyclosis continued apparently unabated after treatment with NaCl at concentrations of up to 0.25 m, so the cells remained viable and energetically competent. The correlation between the release of Ca(2+) and Mg(2+) and release of protein, and between these effects and the suppression of glucose and orthophosphate uptake, supports the hypothesis that divalent cations maintain, and monovalent cations disrupt, linkages between the outer cell surface and proteins required for active solute uptake. Calcium preserved uptake activity only when it was added in time to prevent the release of protein. Cells gradually recovered some glucose uptake activity after it had been completely inactivated by treatment with 0.25 m NaCl. This recovery occurred in the absence of added Ca(2+). It was inhibited by puromycin and so appears to require some protein synthesis. Beet (Beta vulgaris L.) discs were more resistant than carrot discs to treatment with NaCl solutions, thus reflecting the difference in tolerance of the two species to sodicity. PMID- 16657784 TI - Role of molybdenum in nitrate reduction by chlorella. AB - Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH(2)-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.Competition studies with tungstate corroborate these conclusions and indicate that the only role played by molybdenum in Chlorella is connected with the reduction of nitrate to nitrite. Tungsten seems to act by replacing molybdenum in the nitrate reductase complex, thus rendering inactive the FNH(2)-nitrate reductase portion of the nicotinamide adenine dinucleotide nitrate reductase complex. PMID- 16657785 TI - The effect of red and far red light on the desaturation of Fatty acids in barley leaves. AB - Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-(14)C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly. PMID- 16657786 TI - Water potential gradient in a tall sequoiadendron. AB - With an elevator installed in a 90-meter tall Sequoiadendron to collect the samples, xylem pressure potential measurements were made approximately every 15 meters along 60 meters of the tree's height. The measured gradient was about -0.8 bar per 10 meters of height, i.e., less than the hydrostatic gradient. Correction of the xylem pressure potential data by calibration against a thermocouple psychrometer confirmed this result. Similar gradients are described in the literature in tall conifers at times of low transpiration, although a different sampling technique was used. If the data in the present study and those supporting it are typical, they imply a re-evaluation of either the use of the pressure chamber to estimate water potential or the present theories describing water transport in tall trees. PMID- 16657787 TI - The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black eyed Peas. AB - Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed. PMID- 16657788 TI - Contributions of photosynthesis and phytochrome to the formation of anthocyanin in turnip seedlings. AB - Turnip seedlings (Brassica rapa L.) irradiated for 24 hours with radiation at 720 nanometers synthesize chlorophyll a and anthocyanin. Antimycin A and 2,4 dinitrophenol, which are known to reduce cyclic photophosphorylation, also reduce anthocyanin synthesis. Noncyclic photophosphorylation is inhibited by 3-(3,4 dichlorophenyl)-1,1-dimethylurea and o-phenanthroline. These compounds promote cyclic photophosphorylation and anthocyanin synthesis. On the basis of these findings it is suggested that the photomorphogenic response of anthocyanin synthesis in turnip seedlings arises in part through photosynthetic activity.Phytochrome involvement in turnip seedling photomorphogenesis is evidenced by the photoreversibility of anthocyanin synthesis in response to 5 minute irradiations with red or far red light. The inhibition of anthocyanin synthesis by 2,4-dinitrophenol does not arise from a destruction of phytochrome photoreversibility.It is suggested that plant photomorphogenic responses to prolonged far red irradiations arise through the photochemical activation of at least two pigment systems; namely, the photosynthetic pigments and phytochrome. PMID- 16657789 TI - Studies on the Biosynthesis and Metabolism of delta-Aminolevulinic Acid in Chlorella. AB - The regulation of chlorophyll synthesis in Chlorella was examined at the level of the formation and metabolism of delta-aminolevulinic acid. delta-Aminolevulinic acid synthetase activity could not be detected in broken cell preparations, and exogenously supplied delta-aminolevulinic acid was taken up only in the presence of dimethylsulfoxide, with a corresponding production of porphobilinogen.The delta-aminolevulinic acid dehydratase of Chlorella has a pH optimum of 7.8 and at this pH the Michaelis constant for delta-aminolevulinic acid is 0.13 millimolar.delta-Aminolevulinic acid excretion in the presence of levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, allowed measurement of the relative rates of delta-aminolevulinic acid synthesis under various growth conditions. A mutant which requires light for chlorophyll synthesis also requires light for delta-aminolevulinic acid accumulation in the presence of levulinic acid. This same mutant has 40% of the delta-aminolevulinic acid dehydratase activity of the wild-type Chlorella during growth in the dark on glucose.The necessity for protein synthesis during chlorophyll synthesis is due primarily to the requirement for protein synthesis during delta-aminolevulinic acid formation.It is concluded that the rate of chlorophyll formation and the cellular chlorophyll content are regulated by the relative rates of synthesis and breakdown of an enzyme responsible for delta-aminolevulinic acid biosynthesis and that this enzyme has an in vivo lifetime of about 30 minutes. PMID- 16657790 TI - Cytokinins extracted from pinto bean fruit. AB - Extracts from various parts of Phaseolus vulgaris L. var. Pinto plants were found to exhibit cytokinin activity with the highest levels present in extracts of the fruit tissue. To separate the cytokinins present in the fruit, extracts were chromatographed in several solvent systems. Bioassays of chromatograms indicated the presence of active factors in those regions of migration associated with several known N-6-substituted aminopurines.Ultraviolet and mass spectral studies confirm the presence of a mixture of N-6-substituted aminopurines in the active material isolated by cation exchange resin chromatography. The evidence strongly supports the conclusions that dihydrozeatin is naturally occurring as the free base and possibly as a ribonucleoside. Further, zeatin appears to occur mainly as a glycosidic compound which is altered by KMnO(4) treatment and is hydrolyzed under acid conditions. PMID- 16657791 TI - Formation of glycolate by a reconstituted spinach chloroplast preparation. AB - A reconstituted preparation requiring fructose 6-phosphate, transketolase, triphosphopyridine nucleotide, ferredoxin, fragmented spinach chloroplasts, and light capable of forming glycolate at rates of about 10 micromoles per milligram of chlorophyll per hour has been characterized. The glycolaldehyde-transketolase addition product could be substituted for fructose 6-phosphate and transketolase. The stoichiometry of the reaction was: 1 mole of fructose 6-phosphate consumed for each mole of glycolate and of reduced triphosphopyridine nucleotide produced. Evidence was presented indicating that glycolate formation was coupled to the photosystems of the photosynthetic electron transport chain. Synthesis of glycolate is envisaged as the result of either (a) a reaction between the upper two carbon atoms derived from fructose 6-phosphate and an uncharacterized oxidant generated by photosystem 2 or (b) hydrogen peroxide produced by the reoxidation of reduced triphos-phopyridine nucleotide or reduced ferredoxin by molecular oxygen. PMID- 16657792 TI - Amylases in developing barley seeds. AB - The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with alpha-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of beta-amylase. PMID- 16657793 TI - Inhibition of glycolipid biosynthesis in chloroplasts by ozone and sulfhydryl reagents. AB - The metabolism of uridine 5'-pyrophosphate-galactose by spinach (Spinacia oleracea) chloroplast preparations was inhibited by ozone. The formation of digalactosyl diglyceride and trigalactosyl diglyceride was inhibited much more than the formation of monogalactosyl diglyceride, steryl glycoside, and acylated steryl glycoside. Essentially identical results were obtained when glycolipid synthesis was inhibited by N-ethyl maleimide, p-hydroxymercuribenzoate, and CdCl(2). Iodoacetate and iodoacetamide affected neither the total incorporation of sugar from uridine 5'-pyrophosphate-galactose nor distribution of the incorporated sugar in the various glycolipids.Ozone reacted with model membrane systems prepared with egg lecithin. In the absence of reduced glutathione, products included malonaldehyde and hydrogen peroxide. In the presence of glutathione, malonaldehyde was still produced, but the glutathione was oxidized and no peroxide was detected. When these studies were extended to chloroplast preparations, it was also found that malonaldehyde was produced and glutathione was oxidized.It was concluded that ozone inhibits glycolipid biosynthesis in chloroplast preparations by way of oxidation of enzyme sulfhydryl groups but that this reaction may be a secondary effect of oxidation of unsaturated fatty acids. PMID- 16657794 TI - Studies of electron transport in dry and imbibed peanut embryos. AB - The respiration of isolated peanut (Arachis hypogea) embryos has been studied with dry and wet embryos and mitochondria prepared after various times of imbibition. Dry seeds respire slowly, apparently via a respiratory chain which is deficient in cytochrome c. Cytochrome c-deficient mitochondria have been prepared from the embryos up to 16 hours following imbibition. These mitochondria can metabolize reduced nicotinamide adenine dinucleotide and succinate, without respiratory control by ADP, but they do phosphorylate. Added cytochrome c increases both respiration and phosphorylation of these embryonic mitochondria. When growth starts, mitochondria appear which are similar to those isolated from other mature plant tissues; they have respiratory control and can actively metabolize succinate, malate, and reduced nicotinamide adenine dinucleotide. These latter mitochondria contain a concentration of cytochrome c comparable to that found in mitochondria isolated from other mature plant tissues. It is suggested that the earliest type of mitochondria may be required to control respiration in the dry and the recently wetted embryo. PMID- 16657795 TI - The rate of photorespiration as measured by means of oxygen uptake and its respiratory quotient. AB - Oxygen recycling inside photosynthesizing leaves was found to amount to less than 0.3% of the oxygen consumed by photorespiration under natural conditions, provided the influence of buildup of oxygen released by photosynthesis into the external air was taken into consideration. When this is related to the amounts of photorespired CO(2), which had been previously found to be reabsorbed by photosynthesis, it appears that previous respiratory quotients reported for photorespiration were underestimated. For the same reason the photosynthetic quotient was overestimated. Actually, quotients of photorespiration and of photosynthesis approach the more normal range of respiratory quotients int the dark. The oxygen recycling was calculated according to an electrical analogue to oxygen flow.The determination of photorespiration, when measured by oxygen uptake, can be more accurate than that by CO(2) measurement. However, recycling of oxygen occurs in larger amounts at lower oxygen and higher CO(2) concentrations, as well as under conditions of high resistance to transpiration. PMID- 16657796 TI - Ethylene control of anthocyanin synthesis in sorghum. AB - Light-induced anthocyanin synthesis in Sorghum vulgare L. seedlings was both promoted and inhibited by ethylene treatment. The rate of anthocyanin formation in sorghum tissue was dependent upon the time of ethylene treatment in relation to light exposure and the stage of the anthocyanin synthesis process. Those plants receiving ethylene treatment during the early lag phase of anthocyanin synthesis had higher anthocyanin content at 24 hours than control plants receiving no ethylene treatment. Plants receiving ethylene treatment after the lag phase had lower anthocyanin content at 24 hours than control plants receiving no ethylene treatment. PMID- 16657797 TI - The role of peroxidase isozymes in resistance to wheat stem rust disease. AB - In common with other disease situations, rust-resistant wheat leaves show a large increase in peroxidase activity during infection. Peroxidase isozymes from healthy or infected lines of wheat (Triticum aestivum L.) near isogenic for resistance and susceptibility to race 56 of Puccinia graminis tritici were separated by gel electrophoresis and the activity of each was estimated by photometric scanning. In order to ensure that the activity of isozymes observed on gels reflected the changes found in peroxidase enzymes assayed spectrophotometrically in extracts, a study was made of extraction procedures, substrates, and reaction conditions for both types of enzyme measurements. Of the 14 isozymes detected in both healthy and infected leaves, increases in only 1 (isozyme 9) were associated consistently with the development of resistant disease reaction at 20 C. Additional evidence was obtained to show that this isozyme can account for the increased peroxidase activity observed in extracts from resistant plants. When plants with high induced peroxidase activity due to resistance at 20 C were treated with ethylene or transferred to 25 C, they reverted to complete susceptibility. However, the disease-induced activity of isozyme 9 did not fall. The data suggest that, in this case, the association of peroxidase with resistance was a consequence of, not a determinant in, resistance. PMID- 16657798 TI - Inhibitory Effect of Carbohydrate on Flowering in Lemna perpusilla: III. Effects of Respiratory Intermediates, Amino Acids, and CO(2). Glucose 6-Phosphate Dehydrogenase Activity. AB - A study was done on the effects of various factors on carbohydrate inhibition of flowering and on in vitro activity of glucose 6-phosphate dehydrogenase in Lemna perpusilla 6746 grown on dilute Hutner's medium in short days. Autoclaving decreased the flower inhibitory effect of sucrose but increased the effects of glucose and fructose. Sucrose inhibition was mimicked by CO(2) and was partially reversed by agitation of the cultures. Inhibition by sucrose was also partially reversed by ATP and intermediates of the tricarboxylic acid cycle, the glycolytic pathway, or the pentose phosphate pathway. Tartaric acid was inactive. Glycine, l alanine, l-aspartate, l-asparagine, l-serine, l-glutamate, and l-glutamine were active, whereas l-cysteine, l-arginine, l-lysine, l-leucine, l-isoleucine, l proline, l-tyrosine, l-tryptophane, and l-phenylalanine were not. Incubation of cultures on distilled water during a single inductive long night prevented flowering. This inhibition was partially reversed by l-alanine and glucose 6 phosphate. There was a correlation between carbohydrate inhibition of flowering and enhancement of glucose 6-phosphate dehydrogenase. Possible mechanisms for the carbohydrate inhibition of flowering are discussed. PMID- 16657799 TI - An examination of methods used to assay potato tuber invertase and its naturally occurring inhibitor. AB - Confirming an earlier report, it was shown that the endogenous inhibitor of potato tuber invertase forms an essentially undissociable complex with the enzyme. Consequently, several previous analyses of potato tuber invertase which were based on equations derived for highly dissociable enzyme-inhibitor complexes are presumed to be in serious error. The complex formation proceeded slowly, requiring approximately 1 day to reach completion at 2 C, and 1 hr at 37 C. Allowing complex formation to reach completion before assaying enzyme activity did not affect the noncompetitive nature of the inhibition.Contrary to previous reports that inhibitor could be selectively inactivated through foaming in a blender, both enzyme and inhibitor appeared to be denatured by such treatment. Foaming accomplished by passing nitrogen gas bubbles through extracts gave more favorable results. PMID- 16657800 TI - Graphical determination of relative concentrations of enzyme and endogenous inhibitor by dilution. PMID- 16657801 TI - The Incorporation of S-Labeled Sulfate into Carrageenan in Chondrus crispus. PMID- 16657802 TI - An inexpensive technique for measuring carbon monoxide formation in plants. PMID- 16657803 TI - Effects of intermediates of the photosynthetic carbon reduction cycle on carbon metabolism in spinach chloroplasts illuminated on filter paper discs. PMID- 16657804 TI - Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides. AB - Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide(650)) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide(632)) which is not. Dark-grown leaves placed in solutions of delta-aminolevulinic acid accumulate protochlorophyllide(632). Protochlorophyllide(650) and protochlorophyllide(632) can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide(650) in the dark, even in the presence of excess delta-aminolevulinic acid, except when a stockpile of protochlorophyllide(632) is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide(650) regeneration is accompanied by a decrease in protochlorophyllide(632). These experiments suggest that protochlorophyllide(632) may be converted to protochlorophyllide(650).Cycloheximide inhibited greening only after an "action dependent" delay, requiring a predictable minimal period of illumination. This inhibition could be relieved for a time by feeding delta-aminolevulinic acid. PMID- 16657805 TI - A partial characterization of an autolytically solubilized cell wall glucan. AB - Incubation of purified cell wall fragments from corn (Zea mays) coleoptiles results in solubilization of some of the wall dry matter. The portion of the weight loss due to enzymatic autolysis is due mainly to solubilization of a glucan and, to a small extent, to liberation of free glucose. No other carbohydrate wall components or sugars other than glucose are solubilized despite the high concentrations of, for example, galactans, arabans, and xylans in the walls. The glucan has been partially characterized and found to be a lichenan like polymer composed of 1-->3 and 1-->4 linked glucosyl units. PMID- 16657806 TI - Ethylene Production by Plant Cell Cultures: Variations in Production during Growing Cycle and in Different Plant Species. AB - Suspension cultures of Rosa sp., soybean (Glycine max L.), wheat (Triticum monococcum L.), sweet clover (Melilotus alba Desc.), Haplopappus gracilis Nutt., and rue (Ruta graveolens) produced ethylene. The amount varied with the species. The rate of formation in rose and Haplopappus cells paralleled growth but accelerated when the stationary phase was reached, after which the rate declined sharply. Light was not required for ethylene production. Exogenous ethylene could not replace 2,4-dichlorophenoxyacetic acid or naphthalineacetic acid in the cell cultures, and there was no stimulation of growth in the normal medium. Ethylene at 20 mm reduced growth of Ruta and rose cells by 30 and 20%, respectively. The amounts of ethylene produced by the cultures do not affect growth. PMID- 16657807 TI - Ethylene production by plant cell cultures: the effect of auxins, abscisic Acid, and kinetin on ethylene production in suspension cultures of rose and ruta cells. AB - Cell suspension cultures of Ruta graveolens (rue) and Rosa sp. produce ethylene. Both cultures grow at a high rate in hormone-free media. The rose cells are undifferentiated while the Ruta cells differentiate and form shoots after extended culture in hormone-free medium. Addition of 2,4-dichlorophenoxyacetic acid stimulated ethylene production in Ruta cells but not in rose cells. Abscisic acid (ABA) inhibited growth and ethylene production in rose, but only ethylene production in Ruta cells. Addition of kinetin reversed the inhibition by abscisic acid in the rose cells but not in the Ruta cells. The results suggested a distinct physiological difference between the two cultures. The Ruta cells responded to the growth regulators in a manner similar to whole plants. PMID- 16657808 TI - Regulation of Auxin Levels in Coleus blumei by Ethylene. AB - An investigation of the effects of ethylene pretreatment on several facets of auxin metabolism in Coleus blumei Benth "Scarlet Rainbow" revealed a number of changes presumably induced by the gas. Transport of indoleacetic acid-1-(14)C in excised segments of the uppermost internode was inhibited by about 50%. Decarboxylation of indoleacetic acid-1-(14)C by enzyme breis was not affected by the pretreatment. Levels of extractable native auxin in upper leaf and apical bud tissue of the pretreated plants were approximately one-half of those present in untreated plants. The rate of formation of auxin from tryptophan by enzyme breis from pretreated plants was approximately one-half that occurring in incubation mixtures containing the enzyme system from untreated plants. The conjugation of indoleacetic acid-1-(14)C in a form characterized chromatographically as indoleacetylaspartic acid was increased 2-fold in the upper stem region of plants pretreated with ethylene. PMID- 16657809 TI - Control of senescence in marchantia by phytochrome. AB - Mature green tissue of Marchantia polymorpha L. bleaches markedly when placed in continuous darkness for 4 days but remains green when given daily 1-hour photoperiods of white light. The tissue, however, is induced to bleach when each daily 1-hour photoperiod is terminated with a brief irradiation with far red light. The bleaching does not occur when each irradiation with far red light is followed by a brief irradiation with red light. The bleaching is taken as an index of senescence since the loss of chlorophyll in the bleached tissue is accompanied by a breakdown of cell organelles and cytoplasm. Phytochrome is clearly implicated in the control of senescence by light. It was also found that 5 minutes of red light given once a day was as effective as the 1-hour photoperiods with white light in preventing the bleaching and that bleaching was induced when each daily 5-minute irradiation with red light was followed by a 10 minute irradiation with far red light. PMID- 16657810 TI - The effects of drought stress on respiration of isolated corn mitochondria. AB - Mitochondria were isolated from etiolated corn shoots (Zea mays L.) that were stressed to a measured water potential. The rates of mitochondrial respiration in state III, state IV, and without phosphate or ADP on a milligram protein basis decreased as water stress increased with succinate, malatepyruvate, or reduced nicotinamide adenine dinucleotide as substrates. Coupling (as determined by respiratory control and ADP/O ratios) did not decrease with increasing water stress. At water potentials greater than -35 bars all respiration had ceased. PMID- 16657811 TI - Gibberellin-induced Fertility in the Fern Ceratopteris thalictroides (L.) Brongn. AB - Treatment of the homosporous fern Ceratopteris thalictroides L. sporophyte with gibberellic acid results in fertile fronds appearing 3 to 5 weeks after treatment. The hetero-blastic leaf sequence is markedly reduced, and the entire life cycle can be completed in 3 months on defined medium. Treatment with gibberellic acid also stimulates the accumulation of starch in mature leaves. PMID- 16657812 TI - Legumin Synthesis in Developing Cotyledons of Vicia faba L. AB - The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation. PMID- 16657813 TI - Effect of metabolic inhibitors and temperature on uptake and translocation of ca and k by intact bean plants. AB - The dependence of Ca uptake and translocation by intact roots of Phaseolus vulgaris on concurrent root metabolism was investigated using (45)Ca-labeled Hoagland solutions at one-half and one-twentieth strength (2.5 and 0.25 mM Ca(2+)). Adsorbed and absorbed (45)Ca fractions in the roots were distinguished on the basis of the time course of exchange with the outer solution. Uptake of (42)K, of which the characteristics are better known, was measured for comparison. The absorbed (45)Ca fraction showed a markedly nonlinear increase with time in contrast to the near linear increase in (42)K. Exposure of roots to cyanide, arsenate, 2,4-dinitrophenol, or low temperatures caused only slight reductions in (45)Ca absorption by roots, but significant reductions of (42)K. In all treatments involving inhibitors and low temperatures, the translocation to shoots of both (45)Ca and (42)K was strongly inhibited. The conclusion that much of the absorbed (45)Ca fraction in the root tissue is taken up by processes which are not rate-limited by metabolism is discussed. PMID- 16657814 TI - Influence of Temperature and Seed Ripening on the in-vivo Incorporation of CO(2) into the Lipids of Oat Grains (Avena sativa L.). AB - To elucidate the influence of growth temperature and of stage of maturity on lipid synthesis in seeds, oat plants (Avena sativa nuda L., variety NOS) were fed with (14)CO(2) at different stages after flowering, and the (14)C-incorporation into the grain lipids was determined at 2, 24, and 48 hours after the end of (14)CO(2)-application. By changing growth temperature from 12 C to 28 C after the application of (14)CO(2) to intact plants, a higher (14)C-labeling of saturated fatty acids was found at the higher temperature. At 28 C, palmitic and stearic acids contained 23% and 9% respectively of total fatty acid-(14)C shortly after the (14)CO(2)-application, whereas at 12 C the corresponding values were 19% and 4%, respectively. Within 2 days (14)C-activity of saturated fatty acids decreased at both temperatures, but to a lesser degree at 28 C. The higher (14)C-labeling of saturated fatty acids and its lower decrease within 2 days at 28 C clearly show a direct influence of temperature on fatty acid biosynthesis in oat grains.At all stages of grain growth, oleic acid had the highest (14)C-activity of all fatty acids shortly after the (14)CO(2)-application. However, (14)C activity of oleic acid rapidly decreases in favor of linoleic acid. With increasing maturity, the intensity of lipid synthesis in the grains decreases; simultaneously, the relative amount of (14)C-saturated fatty acids increases primarily at the expense of (14)C-oleic acid. These tendencies, which were observed in oat plants grown at day temperatures of 12 C during seed development, seem to be paralleled by lipid synthesis in younger grains grown at day temperatures of 28 C. This indicates an indirect influence of growth temperature on lipid synthesis in oat grains during maturation. PMID- 16657815 TI - Boundary layer resistance and temperature distribution on still and flapping leaves: I. Theory and laboratory experiments. AB - If the evaporation is uniform on a flat exposed leaf, forced convection will also be nearly uniform, and the leaf temperature will vary with the square root of the distance from the leading edge. Then the resistance expressed in terms of the proper, i.e., average, temperature has the same value as the resistance of a leaf at uniform temperature. Compared to a steady laminar flow, the turbulence of a realistic wind decreases the resistance by a constant factor of about 2.5. The same constant factor was observed whether the leaf was flapping or not, when the wind velocity was not too low. PMID- 16657816 TI - Effect of antimycin a on photosynthesis of intact spinach chloroplasts. AB - Low concentrations (0.5-10 mum) of antimycin A were shown to increase the rate of CO(2) fixation, O(2) evolution and inorganic phosphate esterification in intact spinach (Spinacia oleracea) chloroplasts. The increase was highest when the light intensity was saturating. Stimulation was independent of the bicarbonate concentration and was accompanied by an enhancement in the synthesis of glycerate 3-phosphate with a decrease in dihydroxyacetone phosphate. The antibiotic decreased the Michaelis constant of the chloroplast but not of ribulose 1,5 diphosphate carboxylase for bicarbonate. It was suggested that antimycin A is affecting that portion (outer envelope) of the intact chloroplast which contains the enzyme mechanism for controlling the pace of CO(2) fixation. PMID- 16657817 TI - Stomatal opening quantitatively related to potassium transport: evidence from electron probe analysis. AB - When stomata of Vicia faba opened (from a stomatal aperture of about 2 micrometers to one of 12 micrometers) the solute content of the guard cells increased by 4.8 x 10(-12) osmoles per stoma. During the same time an average of 4.0 x 10(-12) gram equivalents of K(+) were transported into each pair of guard cells. This amount of K(+), if associated with dibasic anions, is sufficient to produce the changes in guard cell volume and osmotic pressure associated with stomatal opening. Analysis of Cl, P, and S showed that these elements were not transported in significant amounts during stomatal opening. This finding suggests that the anions balancing K(+) were predominantly organic. K(+) was specifically required because no other elements, likely to be present as cations, were found to accumulate in appreciable quantities in guard cells of open stomata. PMID- 16657818 TI - Hydroxyproline arabinosides in the plant kingdom. AB - The hydroxyproline-O-arabinosyl linkage is present in cell walls of selected tissues representing green plants from algae to angiosperms. PMID- 16657819 TI - Growth and nutrient uptake by soybean plants in nutrient solutions of graded concentrations. AB - Soybean plants (Glycine max L. Merr. var. Hawkeye), grown in nutrient solutions maintained at graded concentrations showed a large response in both shoot dry weight and total ion uptake. Growth rate was dependent upon nutrient concentration, even when quantity of nutrient was not limiting. Peak periods for absorption of specific ions at certain growth stages were not exhibited. Rates of ion uptake by soybeans were generally proportional to the growth rate during the period of major growth. It is suggested that a dilute nutrient solution could provide sufficient nutrients for adequate root growth prior to major shoot growth, at which time a more concentrated nutrient solution is needed. PMID- 16657820 TI - Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings. AB - The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.In seedlings grown in the dark for 10 days, the reserve lipid and the glyoxylate cycle enzyme activities persist for a longer time than in the light; correlated with this, there is a continued association of the microbodies with the lipid bodies. When these dark-grown seedlings are then exposed to 51 hours of the light-dark cycle, peroxisomal marker enzymes increase rapidly in activity, and the microbodies become appressed to chloroplasts. We conclude that the characteristic association observed between glyoxysomes and lipid bodies reflects their mutual involvement in net gluconeogenesis through the conversion of fatty acids to carbohydrate, while the close spatial relationship observed between peroxisomes and chloroplasts at later stages of development reflects their mutual involvement in glycolate metabolism.Although glyoxysomal enzyme activities are dropping rapidly while peroxisomal enzyme activities are increasing rapidly during the transition period in the light, the electron microscopic evidence does not indicate that glyoxysomes are being degraded or peroxisomes are being formed. Since in the dark-grown seedlings the activities of peroxisomal enzymes remain low and do not increase as they do in the light, an opportunity is afforded to compare quantitatively any changes in numbers of microbodies per cell with the changes in activities of glyoxysomal enzymes. It is found that the magnitude of the decrease in numbers of microbodies is considerably less than that of the decrease in glyoxysomal enzyme activities between days 4 and 10. When the cotyledons are exposed to light, peroxisomal enzyme activities increase greatly, but again there is no ultrastructural evidence for the synthesis of a new population of microbodies to accommodate this increase. These results allow us to conclude that the developmental transition from glyoxysomal to peroxisomal function almost certainly does not involve the actual replacement of one population of microbodies by another. Rather, the transition probably occurs within existing particles, either by a sequential functioning of two different kinds of microbodies or by a change in enzyme complement within a single population. Our findings with both light- and dark grown cotyledons favor the latter possibility. The cytoplasmic invaginations into microbodies seen during greening of both light-grown cotyledons and etiolated cotyledons exposed to light may be morphological manifestations of the mechanism by which the microbodies lose or gain enzymes. PMID- 16657821 TI - Incorporation of C-Kaurene into the Gibberellin of a Higher Plant (Pharbitis nil Chois). AB - Enzymic formation of (14)C-kaurene from 2-(14)C-mevalonate was carried out with a cell-free system of Cucurbita pepo L. It was shown that either heating of the enzyme system or the addition of the growth retardants (2-chloroethyl) trimethylammonium chloride and 2'-isopropyl-4' (trimethylammonium chloride)-5' methylphenyl piperidine-1-carboxylate prevented the synthesis of (14)C-kaurene. Experiments in which (14)C-kaurene was applied to seedlings of Pharbitis nil revealed that the kaurene is converted to at least two compounds present in the acidic ethyl acetate fraction, containing free gibberellins, as well as in the second acidic ethyl acetate fraction, containing the released bound gibberellins. One of the compounds cochromatographed with gibberellic acid; the other compound is possibly a break-down product of gibberellic acid with no biological activity. PMID- 16657822 TI - 3-Phosphoglycerate Phosphatase in Plants: II. Distribution, Physiological Considerations, and Comparison with P-Glycolate Phosphatase. AB - 3-Phosphoglycerate phosphatase and phosphoglycolate phosphatase were found in leaves of all 52 plants examined. Activities of both phosphatases varied widely between 1 to 20 micromoles per minute per milligram chlorophyll. Plants were grouped into two categories based upon the relative ratio of activity of 3 phosphoglycerate phosphatase to phosphoglycolate phosphatase. This ratio varied between 2:1 to 4:1 in the C(4)-plants except corn leaves which had a low level of 3-phosphoglycerate phosphatase. This ratio was reversed and varied between 1:2 to 1:6 in all C(3)-plants except one bean variety which had large amounts of both phosphatases. By differential grinding procedures for C(4) plants a major part of the 3-phosphoglycerate phosphatase was found in the mesophyll cells and P glycolate phosphatase in the bundle sheath cells. Phosphoglycolate phosphatase, but not 3-phosphoglycerate phosphatase, was located in chloroplasts of C(3)- and C(4)- plants. Formation of 3-phosphoglycerate phosphatase increased 4- to 12-fold during greening of etiolated sugarcane leaves. This cytosol phosphatase displayed a diurnal variation in sugarcane leaves by increasing 50% during late daylight hours and early evening. It is proposed that the soluble form of 3 phosphoglycerate phosphatase is necessary for carbon transport between the bundle sheath and mesophyll cells during photosynthesis by C(4)-plants. In C(3)- and C(4)-plants this phosphatase initiates the conversion of 3-phosphoglycerate to serine which is an alternate metabolic pathway to glycolate metabolism and photorespiration. PMID- 16657823 TI - 3-Phosphoglycerate Phosphatase in Plants: III. Activity Associated with Starch Particles. AB - A particulate form of 3-phosphoglycerate phosphatase represents about 20% of this activity in spinach (Spinacia oleracea var. Longstanding Bloomsdale) leaves. By differential and isopycnic sucrose density gradient centrifugation, all the particulate activity was found in starch grains that pelleted through 2.5 m sucrose. This particulate phosphatase was extremely stable, had a pH optimum of 5.8, and an apparent Michaelis constant (3-phosphoglycerate) of 9 x 10(-4)m. No cation requirement for activity could be demonstrated, and the enzyme was inhibited by 0.5 mm Zn(2+) or Cu(2+). The enzyme was most active in catalyzing the hydrolysis of 3-phospho-d-glycerate, but it was not substrate specific. The phosphatase from the starch grains could not be removed by washing, dialysis, homogenization, or treatment with a French pressure cell, but it was solubilized by prolonged sonication or by addition of 0.25 m MgCl(2), when the particles were suspended in 0.8 m sucrose. The solubilized enzyme was partially purified. The properties of the enzyme on the particles or after solubilization were similar to those previously described for the cytosol form of this phosphatase. It is conjectured that the phosphatase of the starch grain regulates glucan synthesis by controlling a 3-phosphoglycerate pool which is an effector for ADP-glucose pyrophosphorylase. PMID- 16657824 TI - The respiratory chain of plant mitochondria: x. Oxidation-reduction potentials of the flavoproteins of skunk cabbage mitochondria. AB - The oxidation-reduction potentials of the flavoproteins of skunk cabbage (Symplocarpus foetidus) mitochondria have been measured under anaerobic conditions by means of a combined spectrophotometric or fluorimetric potentiometric method. Five components were resolved whose oxidation-reduction reactions corresponded to two-electron changes, as expected for flavoproteins. The midpoint potentials at pH 7.2 are as follows, listed in order of increasingly negative potential: +170 millivolts, +110 millivolts, +20 millivolts, -70 millivolts, and -155 millivolts. The most negative component was highly fluorescent; the other components could only be identified by their characteristic absorbance changes. In addition to these components, which are mitochondrial, variable amounts of a very highly fluorescent flavoprotein with a midpoint potential of -215 millivolts was found. This component appears to be extra-mitochondrial. The same midpoint potential values at pH 7.2 were obtained with mitochondria in the uncoupled state as in mitochondria energized with ATP in the absence of phosphate. PMID- 16657825 TI - Distribution and Variation of Indole Glucosinolates in Woad (Isatis tinctoria L.). AB - The exceptionally high levels in woad (Isatis tinctoria L.) of three indolic goitrogens, namely glucobrassicin, neoglucobrassicin, and glucobrassicin-1 sulfonate, permit the facile study of their distribution in the plant and their changes during its development. Woad seeds contain as much as 0.23% fresh weight of glucobrassicin but no other indole glucosinolate, while 1-week-old seedlings also contain substantial amounts of neoglucobrassicin and glucobrassicin-1 sulfonate in their shoots whether grown in the light or dark. The sulfonate is not found in roots, and light depresses neoglucobrassicin levels in shoots. Sterile root cultures synthesize glucobrassicin and neoglucobrassicin, and significant quantities of these were even found to be excreted by the roots of intact sterile seedlings in culture. This may explain the long known deleterious effect of woad and other cruciferous crops on subsequent plantings and the observation could be of ecological importance. Long term changes in levels of all three substances in the plant are similar and are compatible with earlier suggestions that the compounds could be auxin precursors at the time of flower stem elongation. Since sterile seedlings readily incorporate (35)SO(4) (2-) into indole glucosinolates and relative specific radioactivities suggest that glucobrassicin is the precursor of the other two compounds, pathways of goitrogen biosynthesis should be relatively easily determined in this material. PMID- 16657826 TI - Ethylene air pollution: effects of ambient levels of ethylene on the glucanase content of bean leaves. PMID- 16657827 TI - Changes in the Pattern of Some Isoenzymes of the Corolla after Pollination in Phalaenopsis amabilis Blume. PMID- 16657828 TI - Anion absorption by plants: a unary interpretation of "dual mechanisms". PMID- 16657829 TI - Further Comparisons between Carbowax 6000 and Mannitol as Suppressants of Cucumber Hypocotyl Elongation. AB - Stirring largely eliminated the greater effectiveness of Carbowax 6000 compared to mannitol in reducing cucumber (Cucumis sativus L.) hypocotyl section elongation. Stirring increased elongation in water but reduced elongation in growth regulator solution. Shrinkage of cells without plasmolysis in hypertonic Carbowax solutions indicates that Carbowax 6000 did not penetrate cell walls. Sections prevented from elongating during pretreatment exposure to saturated air grew as much as those not pretreated. Sections pretreated with isotonic Carbowax responded similarly, but sections pretreated with isotonic mannitol grew less. PMID- 16657830 TI - Influence of s-Triazines on Some Enzymes of Carbohydrates and Nitrogen Metabolism in Leaves of Pea (Pisum sativum L.) and Sweet Corn (Zea mays L.). AB - Foliar applications of 2 milligrams per liter of 2-chloro-4,6-bis (ethylamino)-s triazine, 2-methylmercapto-4-ethylamino-6-isobutylamino-s-triazine, and 2-methoxy 4-isopropylamino-6-butylamino-s-triazine caused increases in the activities of starch phosphorylase, pyruvate kinase, cytochrome oxidase, and glutamate dehydrogenase 5, 10, and 15 days after treatment in the leaves of 3-week-old seedlings of pea (Pisum sativum L.) and sweet corn (Zea mays L.). The results indicate that sublethal concentrations of s-triazine compounds affect the physiological and biochemical events in plants which favor more utilization of carbohydrates for nitrate reduction and synthesis of amino acids and proteins. PMID- 16657831 TI - Extracellular hydrolysis of starch in sugarcane cell suspensions. AB - Evidence is presented for the increased excretion of amylolytic enzymes into a sugarcane cell culture medium when starch was substituted for sucrose as an energy source. The excretion was further enhanced by the inclusion of 1 mum gibberellic acid in the nutrient medium. The growth rate of the cells increased after they became adapted to starch relative to cells grown on sucrose, but the rate of amylolytic enzyme excretion remained unaltered. Amylolytic enzymes in the medium included alpha-amylase but the identity of one or more other enzymes related to starch hydrolysis remains in doubt. PMID- 16657832 TI - Lipid biosynthesis by isolated barley chloroplasts in relation to plastid development. AB - The effect of seedling age and of the time of greening on the incorporation of 1 (14)C-acetate into lipids by isolated barley (Hordeum vulgare cultivar Svalof's Bonus) plastids was examined. The fatty acid synthesizing capacity of plastids isolated from 5-day-old seedlings did not increase markedly from zero to 36 hours of greening nor was a light stimulation of fatty acid synthesis observed. However, an increasing capacity for fatty acid synthesis and an increasing light stimulation of this process with greening were attained by the plastids isolated from 7-, 9-, and 11-day-old seedlings.Plastids of 7-day-old dark-grown plants, which were illuminated at 2 foot-candles showed increasing capacity of (14)C acetate incorporation with significant flow into phospholipids and sulfolipid, low flow into digalactosyl diglyceride, and considerable flow into 6-methyl salicylic acid. Exposure of these plants to high light intensity for an hour resulted in chloroplasts that after isolation had a 10-fold increased capacity to incorporate (14)C label into digalactosyl diglyceride, while the flow of (14)C label into phospho- and sulfolipids was unaltered, and that into 6-methyl salicylic acid was drastically curtailed.With plastids from 7-day-old dark-grown plants in early stages of greening, essentially all the (14)C label in the stroma fraction could be accounted for by 6-methyl salicylic acid, while the membrane lipids only contained small amounts of (14)C label. As greening proceeded, the flow of (14)C label into 6-methyl salicylic acid diminished sharply, and the lipids of the lamellar systems became increasingly labeled.Only palmitic and oleic acids were main sites of (14)C label in the membrane lipids.The activity of acetyl CoA carboxylase present in plastids of 5- and 7-day-old dark-grown plants fell sharply as the etioplasts differentiated into chloroplasts. PMID- 16657833 TI - Nonstomatal inhibition of photosynthesis in sunflower at low leaf water potentials and high light intensities. AB - The inhibition of photosynthesis at low leaf water potentials was studied in soil grown sunflower to determine the degree to which photosynthesis under high light was affected by stomatal and nonstomatal factors. Below leaf water potentials of 11 to -12 bars, rates of photosynthesis at high light intensities were insensitive to external concentrations of CO(2) between 200 and 400 microliters per liter. Photosynthesis also was largely insensitive to leaf temperature between 10 and 30 C. Changes in CO(2) concentration and temperature had negligible effect on leaf diffusive resistance. The lack of CO(2) and temperature response for both photosynthesis and leaf diffuse resistance indicates that rates of photosynthesis were not limited by either CO(2) diffusion or a photosynthetic enzyme. It was concluded that photosynthesis under high light was probably limited by reduced photochemical activity of the leaves at water potentials below -11 to -12 bars. PMID- 16657834 TI - Antagonism of some gibberellin actions by a substituted pyrimidine. AB - From a comparison of the effects of seven growth retardants and abscisic acid (ABA) on various growth systems, it was found that the gibberellin-regulated growth of lettuce hypocotyls was uniquely inhibited by the growth retardant, alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidine methanol (EL-531). Auxin regulated growth of coleoptile sections was inhibited by Phosfon and only slightly by EL-531 and Alar. Cytokinin-regulated growth of Xanthium cotyledons showed little or no inhibition by any of the retardants. ABA was inhibitory in all three types of tests. The distinctive effects of EL-531 against gibberellin stimulated growth and the general ability of gibberellic acid to relieve EL-531 inhibition suggest that this retardant acts in part against the gibberellin stimulated growth system, but at a locus which discriminates between growth and nongrowth functions of gibberellic acid. It shows little or no antagonism of gibberellin actions which do not involve growth: the barley endosperm test and the Rumex leaf senescence test. PMID- 16657835 TI - Amylase synthesis and stability in crested wheatgrass seeds at low water potentials. AB - Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of alpha-amylase nor impair the ability of seeds to resume its synthesis when moistened again. beta-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at -40 atmospheres. Seeds synthesized alpha-amylase at 0, -20, and -40 atmospheres, but not at -60 atmospheres. At 0 and -20 atmospheres, the log of alpha-amylase activity was linearly related to hastening of germination. But at 40 atmospheres, seeds synthesized alpha-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of alpha-amylase. It is concluded that inhibition of alpha-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials. PMID- 16657836 TI - Dissociation of N(2) Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea. AB - The dissociation of N(2) gas-induced monomeric ribosomes from the pea root was studied by varying the concentration of KCl (or NH(4)Cl) and MgCl(2) in the presence of dithiothreitol. These monoribosomes were shown to dissociate completely into subunits at 0.5m KCl or NH(4)Cl in the presence of 5 mm MgCl(2). The 40S subunits were more susceptible to structural change in KCl than were the 60S subunits. On the other hand, the 60S subunits appeared to be more labile to NH(4)Cl.The activity of the subunits relative to aminoacyl-tRNA binding and peptide bond formation was investigated using subunits derived from 0.5 m KCl (or NH(4)Cl) in the absence and presence of 5 mm MgCl(2). The 40S subunits were active in aminoacyl-tRNA binding only when dissociated in the presence of MgCl(2). The 40S and 60S subunits combined in the presence of poly U were active in incorporation of (14)C-phenylalanine from (14)C-phenylalanyl-tRNA only when dissociation was achieved in the presence of 5 mm MgCl(2). The KCl-dissociated subunits were much more active in protein synthesis than NH(4)Cl-dissociated subunits. PMID- 16657837 TI - The Necessity for beta-Carotene in the 518 Nanometer Absorbance Change. AB - The identity of the pigment responsible for the light-induced 518 nanometer absorbance change was investigated by extraction and reconstitution of spinach chloroplasts. Heptane extraction of carotene and quinones from lyophilized chloroplasts removes absorbance changes at 518 and 475 nanometers activated by both laser flash and continuous illumination. Some electron transport activity is always present, even in carotene- and quinone-depleted chloroplasts, but the light-induced pH increase disappears following the first extraction step. Readdition of pure beta-carotene partially restores the 518 and 475 nanometer absorbance changes. PMID- 16657838 TI - Probing a membrane matrix regulating hormone action: I. The molecular length of effective lipids. AB - Auxin-induced pea (Pisum sativum) stem section elongation is enhanced at levels of 3 to 40 micromolar by six new classes of alkane derivatives additional to those described earlier, providing that length of their molecules exceeds 20 A. Increasingly longer homologous series of alkyl chlorides, bromides and iodides, alkyl benzenes, dialkyl ethers, and sulfides show a logarithmically linear increase in specific activity above this length, reaching an optimum near 28 to 30 A. Longer dialkyl ethers and sulfides are less effective, while steroids, or alkanes with substituents at both ends, are ineffective.Neither common metabolism nor common physical properties, other than over-all length of active molecules, seem to explain these results. However, the dimensions of the most abundant phospholipid of etiolated peas, 1-palmitoyl-2-linoleoyl-3-sn-phosphatidyl choline are such that a monolayer of this lecithin would contain cavities 20 A long. It is postulated that lipids of this length or longer are active in the pea assay by forcing apart lecithin molecules, changing the charge distribution or chelating properties of a regulatory membrane.Steroid dimensions also match the cavity in lecithin monolayers and steroids would be maintained within such a cavity by hydrogen bonds. The name lipometrin for lipids whose physiological activity varies with length is proposed, among these, substances active in the pea bioassay could be termed oleanimins. PMID- 16657839 TI - Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination. AB - In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.Total activity of the peroxisomal enzymes increased slowly in the dark during the first 4 days of germination and thereafter remained at a constant level of activity in the dark or increased 2-fold in 24 hours of light. The specific activties of glycolate oxidase, hydroxypyruvate reductase, and serine-glyoxylate aminotransferase in the isolated microbody fraction increased about 10-fold between days 2 and 4 in the dark and then remained constant or increased again 10 fold after an additional 48 hours in the light.The total activity of the common microbody marker, catalase, developed similarly to isocitrate lyase, but decreased only 72% by day 9. The specific activities of enzymes (catalase, malate dehydrogenase, and aspartate aminotransferase) common to both microbody systems were 10- to 1000-fold greater than those of other enzymes. It is proposed that malate and aspartate may be involved in hydrogen transport between microbodies and other cellular sites.Glutamate-glyoxylate aminotransferase was very active in microbodies from castor bean endosperm and sunflower cotyledons. The specific activity of this aminotransferase developed similarly to glyoxysomal enzymes in the dark but further increased in the light, as did peroxisomal enzymes.The microbody fraction of castor bean endosperm germinated in the dark for 5 days contained both glyoxysomal and peroxisomal enzymes of similar specific activity.Adjacent to the microbody fraction on sucrose gradients from sunflower cotyledons were etioplasts at slightly lower densities and protein bodies at similar and higher densities. Their presence in the microbody fractions resulted in artificially low specific activities. PMID- 16657840 TI - Dehydration, water fluxes, and permeability of tobacco leaf tissue. AB - Removal of the lower epidermis from a tobacco leaf allows a faster and wider range of water fluxes, without damaging the mesophyll. It also permits a more direct examination of the photosynthetic potential of the tissue at various levels of hydration.The rehydration rate of leaf discs is essentially linear. It decreases with leaf age and is correlated with the rate of dehydration, but it is independent of the tissue's water potential, as estimated by the isopiestic method. The hydraulic permeability coefficient of water influx is directly related to water potential of the tissue, suggesting a mechanism for the regulaton of the hydration level of the leaf tissue.The "energy of activation" of rehydration amounts to about 9 kilocalories per mole at intermediate dehydration, but it greatly declines following water loss in excess of 600 milligrams per gram fresh weight. The excessive dehydration is also characterized by a major increase in permeability (monitored by efflux of ions and materials absorbing ultraviolet light) and by a parallel decrease in photosynthetic activity. The interrelationship of these effects of excessive dehydration is discussed. PMID- 16657841 TI - Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. AB - An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO(3) (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals.By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase. It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction.THIS CONCLUSION WAS SUPPORTED BY EXPERIMENTS THAT INCLUDED: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue.The level of nitrate reductase activity obtained with the in vitro assay is higher (2.5- to 20-fold) than with the in vivo assay for most plant species. The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species. The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted. PMID- 16657842 TI - Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials. AB - Reduced osmotic potentials inhibited the rate of CO(2) fixation by isolated intact spinach (Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO(2) fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO(2), as CO(2) concentrations had no effect on the relative inhibition induced by the lowered potential.A procedure was developed for the determination of several enzymes of the photosynthetic carbon reduction cycle in the intact chloroplast without their being extracted. The activities of the combined three enzymes: ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and ribulose-1,5-diphosphate carboxylase and of ribulose-1,5-diphosphate carboxylase alone were found to be inhibited at low osmotic potentials. Analysis of the photosynthetic products showed that the formation of glycerate-3-phosphate was inhibited to a greater extent than the formation of all other products.CO(2) fixation was partly resumed when chloroplasts were returned from a 0.67 m sorbitol to a 0.33 m sorbitol solution, regardless whether the transfer occurred in the light or in the dark. PMID- 16657843 TI - Lignin formation in wheat coleoptile cell walls: a possible limitation of cell growth. AB - Four growth-influencing compounds-hydroxyproline, 2,2'-dipyridyl, 2 chloroethylphosphonic acid, and indoleacetic acid-were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mm but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth. PMID- 16657844 TI - Indoleacetic Acid biosynthesis in Avena coleoptile tips and excised bean shoots. AB - Avena coleoptiles did not elongate when incubated with tryptophan under sterile conditions. Indole, anthranilic acid, and tryptamine promoted elongation. Under the same conditions, the tissue converted tryptophan-(14)C to IAA-(14)C. More IAA (14)C was produced from indole-(14)C than from tryptophan-(14)C; however, the free tryptophan content of the tissue was also greatly increased by the indole treatment. Tryptophan-(14)C was readily taken up by the tissue but was mainly incorporated into protein and did not increase the free tryptophan level. When bean shoots were labeled with tryptophan-(14)C or indole-(14)C, the label incorporation into IAA-(14)C was very nearly the same. In this tissue the free tryptophan level in the tryptophan-(14)C and indole-(14)C treatments was also about equal. These results suggest that failure of exogenously supplied tryptophan to promote the elongation of Avena coleoptiles is a result of its predominant incorporation into protein and consequent unavailability for conversion to IAA. PMID- 16657845 TI - Carbon dioxide compensation points of leaves and stems and their relation to net photosynthesis. AB - The interactions between CO(2) and H(2)O vapor exchange of the leaf and respirant organs like stems were studied in tobacco plants. The results were analyzed according to a suggested model. Good agreement between open and closed system measurements supported the validity of the model.The measured over-all resistance to CO(2) of a leaf and a stem enclosed in a measuring cuvette was the same as the measured resistance of the leaf when measured alone provided the resistance of the stem to CO(2) is relatively high. The combined CO(2) compensation concentration of a leaf and stem having high resistance to CO(2) was higher than the CO(2) compensation point of the leaf alone, by the magnitude of rate of CO(2) evolution from the stem multiplied by the overall resistance of the leaf.CO(2) evolution into CO(2)-free air was found to be higher in light than in dark in leaves, while the reverse was true for stems. It was concluded that normally the CO(2) compensation point of a leaf is unaffected by stomata and boundary layer resistance while the combined CO(2) compensation point of a leaf and a stem differs in its nature since it represents a steady state of photosynthesis in which stem contribution, I(a), is equal to net photosynthesis, I(s). Interpretation of the experimental data shows tht respiration in the light is unaffected by external CO(2) concentration (at the range of 0-300 mul liter) and by intensity of photosynthesis. PMID- 16657846 TI - Behavior of Corn and Sorghum under Water Stress and during Recovery. AB - Corn (Zea mays L.) and sorghum (Sorghum vulgare, Pers.) plants were grown in a vermiculite-gravel mixture in controlled environment chambers until they were 40 days old. Water was withheld until they were severely wilted, and they were then rewatered. During drying and after rewatering stomatal resistance was measured with a diffusion porometer each morning, and water saturation deficit and water potential were measured on leaf samples. The average resistance of the lower epidermis of well watered plants was lower for corn than for sorghum. When water stress developed, the stomata began to close at a higher water potential in corn than in sorghum. The stomata of both species began to reopen normally soon after the wilted plants were rewatered, and on the 2nd day the leaf resistances were nearly as low as those of the controls. The average leaf water potential of well watered corn was -4.5 bars; that of sorghum, -6.4 bars. The lowest leaf water potential in stressed corn was -12.8 bars at a water saturation deficit of 45%. The lowest leaf water potential in stressed sorghum was -15.7 bars, but the water saturation deficit was only 29%. At these values the leaves of both species were tightly rolled or folded and some injury was apparent. Thus, although the average leaf resistance of corn is little lower than that of sorghum, corn loses much more of its water before the stomata are fully closed than does sorghum. The smaller reduction in water content of sorghum for a given reduction in leaf water potential is characteristic of drought-resistant species. PMID- 16657847 TI - Correlation between Polyribosome Level and the Ability to Induce Nitrate Reductase in Dark-grown Corn Seedlings. AB - Nitrate reductase can be induced in excised shoots of 3-day-old dark-grown Zea mays (var. WF9 x M14) seedlings in the absence of light. In contrast, leaves of 10-day-old dark-grown seedlings require a light treatment in order to induce enzymatic activity. Leaves of 10-day-old dark-grown seedlings contain a very low level of polyribosomes while 3-day-old shoots contain a very high level of polyribosomes. There is a gradual loss of polyribosomes from 3 to 10 days and a gradual loss of in vitro protein synthetic activity of the ribosome preparations. The loss of polyribosomes and decrease in their amino acid-incorporating activity correlate positively with the loss of ability to induce nitrate reducase activity as leaves of dark-grown corn seedlings age. These results corroborate and extend our previous results, in that light is not required for nitrate reductase induction per se in leaves of dark-grown seedlings but is required to reactivate the protein synthetic apparatus of older leaves. PMID- 16657848 TI - The development of photophosphorylation and photosynthesis in greening bean leaves. AB - Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10(-5)m 3-(3,4-dichlorophenyl)-1,1 dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1 dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10(-5)m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened. PMID- 16657849 TI - Development of Pyrimidine-metabolizing Enzymes in Cotyledons of Germinating Peas. AB - Mechanisms controlling conversion of orotic acid-6-(14)C to uridine-5'-phosphate in cotyledons of germinating Alaska peas (Pisum sativum L.) were investigated. The content of 5-phosphoribosyl-1-pyrophosphate was very low in dry seeds, increased to a maximum after about 12 hours of imbibition, and then rapidly declined. Orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase activities more than doubled during the first 24 hours of germination and then also decreased. These results do not account for the continuous increases of orotate anabolism in such cotyledons as we observed previously. The initial increases in activities of these two enzymes were unaffected by cycloheximide, while the subsequent decreases were less rapid in the presence of this inhibitor. Activities of cotyledonary cytidine deaminase and uridine hydrolase also increased during imbibition, but the activity of only the latter showed a decrease after imbibition was completed. Cycloheximide inhibited the initial rapid increase in uridine hydrolase activity but had little effect on its subsequent decline. Cycloheximide had only slight inhibitory effects on the development of cytidine deaminase activity during the first 62 hours. The evidence suggests that uridine hydrolase might be synthesized de novo during the first few days of germination, but that the other three enzymes might not be. PMID- 16657850 TI - Immediate and subsequent growth responses of maize leaves to changes in water status. AB - Elongation of intact young leaves of maize was found to be dynamically dependent on soil water supply. With adequate water, elongation was remarkably constant but slowed when the water potential of the soil in pots dropped from -0.1 to -0.2 bar and stopped when it dropped to -2.5 bars. The corresponding range of leaf water potential was -2.8 to -7 bars. Elongation resumed in less than a few seconds after a mildly water-stressed plant was rewatered.The effects on leaf elongation of step-wise changes in water potential of the root solution were determined. When the water potential of the root medium suddenly decreased below 0 bar, growth stopped initially and then resumed at a lower rate. When the water potential was suddenly increased back to 0 bar, growth accelerated transitorily to a high rate before slowing to the steady state rate. These results suggest an increase in cell extensibility during water stress.Leaves stressed for 1 or more days attained after rewatering almost the length of the control leaves. Growth rate after rewatering did not exceed that of the control at the corresponding developmental stage except during the short transitory rapid phase lasting only a fraction of an hour.As stress developed, growth stopped before carbon dioxide assimilation decreased noticeably. Upon the release of mild and short stress, the transitory rapid growth completely made up for the reduced elongation during stress, suggesting that metabolic processes for cell expansion might have proceeded unchecked during the stress period.The sensitivity and rapidity of response to changes in water status all point to the direct role of water in growth; its uptake provides the physical force for cell enlargement. PMID- 16657851 TI - Isolation of microbodies from plant tissues. AB - Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm(3) in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues. PMID- 16657852 TI - Lateral transport of auxin mediated by gravity in the absence of special georeceptor tissue. PMID- 16657853 TI - Lack of cytokinin activity of quinolinol sulfate. PMID- 16657854 TI - Altered Cell Microfibrillar Orientation in Ethylene-treated Pisum sativum Stems. PMID- 16657855 TI - Effects of free sterols, steryl ester, and steryl glycoside on membrane permeability. PMID- 16657856 TI - Synergistic action of ethylene with gibberellin or red light in germinating lettuce seeds. PMID- 16657857 TI - Some effects of decenylsuccinic Acid on isolated corn mitochondria. AB - The effects of decenylsuccinic acid on the swelling and respiratory capacities of mitochondria isolated from etiolated corn (Zea mays L., Wf9 x M14) shoots were studied. Decenylsuccinic acid (0.1 mM to 1.0 mM) inhibited the oxidation of succinate and malate-pyruvate, stimulated the oxidation of reduced nicotinamide adenine dinucleotide, and uncoupled phosphorylation. The swelling of isolated corn mitochondria, as determined by percentage of transmittance changes, was stimulated by decenylsuccinic acid in potassium chloride reaction media and in sucrose reaction media without bovine serum albumin. In a diaphorase (2, 6 dichlorophenolindophenol as acceptor) reaction with intact mitochondria, only the dehydrogenation rate of malate was reduced by the addition of decenylsuccinic acid. The dehydrogenation of reduced nicotinamide adenine dinucleotide or of succinate was either not affected or was stimulated depending on the diaphorase reaction medium. The oxygen uptake of mitochondria oxidizing N, N, N', N' tetramethyl-p-phenylenediamine diHCl and ascorbate was inhibited at decenylsuccinic acid concentrations greater than 0.5 mM.The results presented lead to the hypothesis that the primary effect of decenylsuccinic acid on isolated corn mitochondria is on the physical properties of the membranes and that decenylsuccinic acid-affected stimulation or inhibition of respiration results from the physical disruption of the membrane. These results appear to be consistent with those previously reported in whole plant studies. PMID- 16657858 TI - Auxin and ribonucleic Acid synthesis in pea stem tissue as studied by deoxyribonucleic Acid-ribonucleic Acid hybridization. AB - The ability of auxin to alter gene transcription in pea (Pisum sativum L.) stem tissues has been investigated by means of DNA/RNA hybridization-competition techniques. In order to obtain reproducible hybridization with total nucleic acid preparations from plants it was found necessary to remove interfering substances, probably polysaccharides; this was accomplished by methoxyethanol extraction and precipitation with cetyltrimethylammonium bromide. When purified in this fashion, plant nucleic acids could be made to form hybrids which showed both species specificity and high thermal stability.No change in hybridizable RNA of stem sections in response to auxin could be detected over a 2- to 24-hour period, regardless of the auxin level employed. In contrast, when large doses of auxin were applied to intact pea seedlings, definite changes in the hybridizable RNA of stem tissue were detected both 8 and 24 hours after treatment. Many of the 2,4-D induced species of RNA which were present at 24 hours were also present at 8 hours. Sections apparently lack a factor needed for the manifestation of the auxin effect on RNA synthesis.Since the hybridization assay employed does not measure all cellular RNA, it is still possible that certain RNA species may be synthesized in sections in response to auxin. However, the auxin promotion of cell elongation in such sections is clearly not associated with changes in hybridizable RNA such as have been reported for several hormonal responses in animal systems. PMID- 16657859 TI - Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination. AB - Rapid increases in activities and components of mitochondrial particles isolated from cotyledons of Pisum sativum var. Alaska during the early stage of germination are described. Respiratory rate of the cotyledons increased rapidly as hydration proceeded. A similar but slightly delayed increase in respiratory activity of the isolated mitochondrial fraction was observed. The respiratory control ratio and adenosine 5'-pyrophosphate/oxygen ratio rose during imbibition. Cytochrome oxidase and malate dehydrogenase activities in the mitochondrial fraction increased during the initial phase of imbibition. The increase seemed to precede that in respiratory activity. A significant activity of cytochrome oxidase and most of the malate dehydrogenase activity in the cotyledons were present in the postmitochondrial fraction in the case of the dry seeds. Mitochondrial protein and phospholipid also increased during imbibition, and the rise in the components seemed to concur with that in respiratory activity. The mechanism of mitochondrial development during imbibition is discussed. PMID- 16657860 TI - Isolation of spherosomes (oleosomes) from onion, cabbage, and cottonseed tissues. AB - Subcellular particles, identical in appearance to spherosomes observed in situ, were isolated from onions (Allium cepa L.) and cabbages (Brassica capitata L.). They were minute spherules about 1 micron in diameter, filled with an evenly stained osmiophilic matrix and delimited by a single, fineline membrane 20 to 30 A thick."Spherosomes" isolated from onions and cabbages were compared with "oil droplets" isolated from cottonseeds. Morphologically, they were similar, even at ultrastructural levels. Their chemical compositions were also similar: both types of particles containing primarily lipid with very little phospholipid or protein. Neither type of particle possessed acid phosphatase activity.These results indicate that oil-droplets of oleaginous tissues correspond to spherosomes of nonoily tissues. Therefore, both types of particles should be referred to by the same name. Since these particles are rich in lipids, it is suggested that the name "spherosome" be abandoned in favor of "oleosome," which is also entitled to priority. PMID- 16657861 TI - Stem diameter in relation to plant water status. AB - An instrument containing a linear variable differential transformer was constructed to obtain continuous, nondestructive measurements of both short term changes in stem diameter and long term growth. In cotton plants, stem diameter, leaf water potential, and leaf relative water content are all closely related to net radiation at the top of the canopy. Leaves from the east and west sides of a plant show slight, but consistent differences in diurnal water potential patterns.Stem diameter and leaf water potential are not related by a single valued function, since there is a diurnal hysteresis between the two, and growth causes an increase in diameter each night. However, the instrument can be used to monitor long term stem diameter growth. PMID- 16657862 TI - "Disaggregation" of phytochrome in vitro-a consequence of proteolysis. AB - The relationship between a large molecular weight (9S) and a small molecular weight (4.5S, 60,000 molecular weight) species of phytochrome was examined to determine if the larger species was an aggregate of the smaller. Alterations of pH, salt concentration, or phytochrome concentration did not cause any observable formation of the large form from the small form. However, in partially purified phytochrome extracts from Secale cereale L. and Avena sativa L., the large form was converted to the small form over time at 4 C in the dark. This breakdown was inhibitable by the protease inhibitor phenylmethanesulfonyl fluoride. When highly purified large molecular weight rye phytochrome was incubated with a neutral protease isolated from etiolated oat shoots, the large phytochrome was converted to the small form without qualitative visible absorbancy changes. The effect of the oat protease could be mimicked by a wide variety of commercial endopeptidases, including trypsin. Examination of the trypsin-induced breakdown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that as the size of the photoreversible unit changes from large to small, the size of its constituent polypeptide chains is reduced from 120,000 to 62,000 molecular weight. These experiments provide evidence that the endogenous breakdown observed in extracts is a result of contaminant protease and, consequently, that the small molecular weight species of phytochrome is an artifact due to proteolysis. PMID- 16657863 TI - The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria. AB - Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P(i)) and ATP are present. No reduction occurs in the absence of P(i), even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.In the absence of P(i) and presence of oligomycin, oxidation of succinate by the alternative cyanide insensitive oxidase pathway, in the presence of sulfide to inhibit cytochrome oxidase, does not reduce endogenous NAD, either in the aerobic steady state or in anaerobiosis. Under these conditions, only the reversed electron transport pathway from succinate to endogenous NAD is active and ATP cannot interact with the respiratory chain. The source of energy for NAD reduction must come from the respiratory chain, and this result shows that oxidation of succinate through the alternate pathway does not provide this energy. PMID- 16657864 TI - Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago. AB - Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO(3) (-) and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO(3) (-), either by amplifying the effect of low endogenous NO(3) (-) levels, or by making NO(3) (-) available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO(3) (-) and benzyladenine, additive responses were obtained. The effects of NO(3) (-) and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO(3) (-), as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO(3) (-) was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO(3) (-) and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO(3) (-). At the time of the second exposure to benzyladenine, no NO(3) (-) was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO(3) (-). PMID- 16657865 TI - Involvement of Photosynthetic Carbon Reduction Cycle Intermediates in CO(2) Fixation and O(2) Evolution by Isolated Chloroplasts. AB - The photosynthetic carbon reduction cycle intermediates can be divided into three classes according to their effects on the rate of photosynthetic CO(2) evolution by whole spinach (Spinacia oleracea) chloroplasts and on their ability to affect reversal of certain inhibitors (nigericin, arsenate, arsenite, iodoacetate, antimycin A) of photosynthesis: class I (maximal): fructose 1, 6-diphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, ribose-5-phosphate; class 2 (slight): glucose 6-phosphate, fructose 6-phosphate, ribulose-1, 5-diphosphate; class 3 (variable): glycerate 3-phosphate. While class 1 compounds influence the photosynthetic rate, they do not lower the Michaelis constant of the chloroplast for bicarbonate or affect strongly other photosynthetic properties such as the isotopic distribution pattern. It was concluded that the class 1 compounds influence the chloroplast by not only supplying components to the carbon cycle but also by activating or stabilizing a structural component of the chloroplast. PMID- 16657866 TI - The Rate of Photorespiration during Photosynthesis and the Relationship of the Substrate of Light Respiration to the Products of Photosynthesis in Sunflower Leaves. AB - Single attached leaves of sunflower (Helianthus annus L. "Mennonite") were supplied (14)CO(2) of constant specific radioactivity in gas mixtures containing various CO(2) and O(2) concentrations. The (14)CO(2) and CO(2) fluxes were measured concurrently in an open system using an ionization chamber and infrared gas analyzer.The rate of photorespiration (5.7 +/- 0.3 mg CO(2).dm(-2).(-1)) during photosynthesis in 21% O(2) at 25 C and 3,500 footcandles was over three times the rate of dark respiration and was independent of CO(2) concentrations from 0 to 300 mul/l. The steady rate of CO(2) evolution into CO(2)-free air was about 30% lower. Low oxygen (1%) inhibited both (14)CO(2) and CO(2) evolution, both during photosynthesis and in CO(2)-free air in the light.At 300 mul/l CO(2) apparent photosynthesis was inhibited 41% by 21% O(2). Two-thirds of the inhibition was due to the inhibition of true photosynthesis by oxygen and one third due to the stimulation of photorespiration. At 50 mul/l CO(2), where the percentage inhibition of apparent photosynthesis by 21% oxygen was 92%, photorespiration accounted for two-thirds of the total inhibition.The rate of (14)CO(2) uptake by the leaf decreased about 30 seconds after the introduction of (14)CO(2), indicating that (14)CO(2) was rapidly evolved from the leaf. The rate of (14)CO(2) evolution increased rapidly with time, the kinetics depending on the CO(2) concentration. The high specific radioactivity of the (14)CO(2) evolved during photosynthesis or in the early period of flushing in CO(2)-free air showed that the substrate for light respiration was an early product of photosynthesis. From the measurement of (14)CO(2) and CO(2) evolution into CO(2)-free air over a longer time period it was apparent that at least three compounds, each of decreased (14)C content, could supply the substrate for light respiration.Based on a consideration of the specific radioactivity of (14)CO(2) evolved under a variety of conditions, it is suggested that total CO(2) evolution in the light or photorespiration is composed of two processes, dark respiration and light respiration. Light respiration is a process that only occurs in the light, persists for some time on darkening, and metabolizes substrates that are quite different from those of dark respiration. PMID- 16657867 TI - Growth-limiting Proteins in Relation to Auxin-induced Elongation in Lupin Hypocotyls. AB - The role of protein synthesis in auxin-induced cell elongation in lupin hypocotyl segments was studied using cycloheximide. Cycloheximide inhibited protein synthesis by 9 minutes. Experiments adding cycloheximide at various times before and after indolyl-3-acetic acid are reported. Estimates of the relative amounts of growth-limiting protein(s), and a first order rate constant for the apparent turnover of the growth-limiting protein(s) were made. It was shown that there is a sizeable growth promotion by auxin after protein synthesis has essentially ceased. It is concluded that the initial phases of auxin action do not require protein synthesis but that its action depends on the existing pool of growth limiting proteins which is rapidly depleted, and protein synthesis is then required for continued elongation. PMID- 16657868 TI - Effect of exposure to subfreezing temperatures on ethylene evolution and leaf abscission in citrus. AB - Citrus leaves exposed to subfreezing temperatures evolved ethylene at rates between 0.1 and 38.3 microliters per kilogram fresh weight per hour whereas untreated leaves evolved between 0.01 and 0.50 microliter per kilogram fresh weight per hour. Leaves not injured by freezing temperatures did not abscise, and ethylene evolution was near normal after 2 days. Freeze-injured leaves continued evolving high ethylene levels 4 or 5 days subsequent to freeze injury, and many of the freeze-killed leaves abscised. Supportive evidence suggested freeze induced ethylene was involved in freeze-induced leaf abscission; whereas freeze inhibited abscission was not due to a lack of ethylene but injury to other metabolic systems necessary for abscission. PMID- 16657869 TI - Phospholipids in the Uredospores of Uromyces phaseoli: I. Identification and Localization. AB - Utilizing paper, thin layer and gas chromatography, the phospholipids of dormant and germinating spores have been isolated and identified. The identifications were based upon agreements of retardation factor values between the unknowns and reference compounds and their derivatives. Quantitative analysis of components, color reactions and specific labeling experiments were also used to support certain identifications. At least three, and usually more, criteria were used for each phospholipid that was definitively identified.The major phospholipids of Uromyces phaseoli are phosphatidylcholine and phosphatidylethanolamine. Diphosphatidylglycerol, or cardiolipin, phosphatidylinositol, and another phosphoinositide were present as minor components. In germinating spores, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylserine, and phosphatidic acid were detected in trace amounts. The presence of these common intermediates in the biosynthesis of phospholipids indicates that phospholipid synthesis is active during the germination process. Except for the absence of phosphatidylglycerol, the types of phospholipids present are similar to the host plant. Germ tube wall preparations were found to contain phosphatidylcholine and phosphatidylethanolamine in about the same proportion as that observed in resting spores, while the proportion of diphosphatidylglycerol was about three times higher. An unidentified phosphorous containing lipid was also a significant component of the total phospholipids extracted from germ tube walls. PMID- 16657870 TI - Phospholipids in the Uredospores of Uromyces phaseoli: II. Metabolism during Germination. AB - The levels and types of phospholipids changed in distinct phases during the germination of (32)P-labeled uredospores of Uromyces phaseoli. During the first 20 minutes of germination, the phospholipid content dropped to 40% of the pregermination level. Between 2 and 3 hours, phospholipid levels increased to approximately 80% of the pregermination levels, and after germination for 5 hours, catabolism had reduced the (32)P-lipids to about the same level observed prior to the first anabolic phase. A second anabolic phase was observed between 5 and 10 hours of germination. Phosphatidylcholine and phosphatidylethanolamine, the major phospholipids, did not undergo anabolism and catabolism at the same rates during germination. Only small quantities of the more polar phospholipids were released to the germination media.Germinating uredospores were capable of utilizing l-methionine-methyl-(14)C, d,l-(14)C serine, (14)C-choline, and (14)C ethanolamine for the synthesis of phospholipids. The active one-carbon units from methionine and serine appear to be involved in the methylation of phosphatidylethanolamine to form phosphatidylcholine. Preformed ethanolamine and choline may be incorporated into these two phospholipids also. Evidence for the synthesis of phosphatidylserine, which is subsequently decarboxylated to yield phosphatidylethanolamine, was obtained. PMID- 16657871 TI - Phytochrome-controlled Nyctinasty in Albizzia julibrissin: III. Interactions between an Endogenous Rhythm and Phytochrome in Control of Potassium Flux and Leaflet Movement. AB - Prolonged irradiation during appropriate parts of the diurnal cycle promotes the opening of Albizzia julibrissin leaflets. Leaflets also open without illumination, but such opening starts later and is slower and less complete. Opening in the dark is accompanied by lower potassium efflux from dorsal pulvinule motor cells but equal or greater potassium movement into ventral motor cells than occurs during opening in the light. Far red-absorbing phytochrome inhibits opening in the dark, indicating that its action is similar during endogenously controlled opening and nyctinastic closure; i.e., a high far redabsorbing phytochrome level is associated with low potassium content in ventral motor cells, high potassium content in dorsal motor cells, and a small angle between leaflets.When open leaflets are darkened, there is an immediate and large potassium flux into dorsal motor cells. This is initially independent of red and far red preirradiation, but prior red light appears to promote continued potassium movement into dorsal cells during the latter part of a 90-minute dark period. The situation in ventral motor cells is different; here the effect of prior red or far red irradiation on potassium efflux is evident after 10 minutes of darkness. Phytochrome controls the direction of potassium movement in ventral motor cells during the early part of the dark period (to 25 minutes); potassium moves out of ventral motor cells if leaflets are preirradiated with red light and into these same cells if leaflets are preirradiated with far red light. Kinetic data are consistent with the suggestion that potassium leaving ventral cells enters dorsal cells. However, there must be an additional source of potassium entering dorsal cells since this potassium movement precedes potassium efflux from ventral cells.Pulvinules excised from the lamina or rachilla open and close in response to light and darkness and also move during extended periods of constant intensity light or uninterrupted darkness. This shows that the photoreceptor controlling opening and the oscillator controlling endogenously rhythmic leaflet movement are localized in the pulvinule. In addition, all the potassium that enters expanding cells during leaflet movement and the energy for potassium transport must be available from within the pulvinule.If leaflets are darkened late in the photoperiod, they close more rapidly and show lower inhibition by far red preirradiation or anaerobic conditions. A rhythmic increase in potassium efflux from ventral motor cells appears to be the basis for rhythmic promotion of nyctinastic closure. We suggest this is due to a rhythmic increase in the leakiness of ventral motor cells. PMID- 16657872 TI - Sodium absorption by intact sugar beet plants. AB - Sodium absorption by intact sugar beet plants (Beta vulgaris) was found to be mediated by at least two distinct mechanisms when uptake was studied over a wide range of Na and K concentrations. The first mechanism operates at low Na concentrations (<1 milliequivalent per liter); presence of K completely blocks this mechanism for Na. The second mechanism operates at high Na concentrations (>1 milliequivalent per liter), transporting Na as well as K; but apparently this mechanism is not active for Na absorption in young sugar beet plants up to the 10 leaf stage. PMID- 16657873 TI - Abscisic Acid and cytokinin contents of leaves in relation to salinity and relative humidity. AB - The question is raised whether the hormonal modifications in a plant exposed to osmotic root stress result directly from the decrease in water potential of the root environment or from disturbances of the plant's water balance.Tobacco plants were held for 24 hours under either high or low relative humidities, with or without salt. The amount of abscisic acid in the leaves of salinized plants rose markedly in low, but not in high, relative humidity. No change in the amount of extractable cytokinins was detected in any treatment. It is tentatively suggested that variations in the water content of leaves constitute a primary signal for modification of plant hormonal balance. PMID- 16657874 TI - Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum. AB - The host-specific toxin of Helminthosporium carbonum (C(32)H(50)N(6)O(10)) was hydrolyzed by 6 n HCl to yield a number of alpha-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1:2. Two other unstable alpha amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. The finding of an alpha,beta unsaturated linkage in H. carbonum toxin explains the instability of the compound and may also account for its specific toxicity. PMID- 16657875 TI - Factors Affecting the Water-sensitive Phase of Flowering in the Short Day Plant Lemna perpusilla. AB - The flowering of Lemna perpusilla strain 6746 is inhibited by daily transfers to water for short periods during a sensitive phase. Supplementing the water with Ca(NO(3))(2) partially reverses the inhibition of flowering while MgSO(4) increases the inhibition. The inhibition by MgSO(4) is overcome by low concentrations of Ca(NO(3))(2). Flower-promoting activity was detected in water and in MgSO(4) solutions that had been incubated with plants under dark but not light conditions. The prevention of this effect by light appears to be photosynthetic rather than to depend on phytochrome. The activity is destroyed by autoclaving but not by brief boiling. This loss of a flower-promoting material may explain the inhibiting effect on flowering by transfers to water. PMID- 16657876 TI - Adenosine diphosphoglucose-starch glucosyltransferases from developing kernels of waxy maize. AB - TWO ADENOSINE DIPHOSPHOGLUCOSE: alpha-1,4-glucan alpha-4-glucosyl-transferases were extracted from kernels of waxy maize harvested 22 days after pollination and separated by gradient elution from a diethylaminoethyl-cellulose column. Both fractions could utilize amylopectin, amylose, glycogen, maltotriose and maltose as primers. The rate of glucose transfer from adenosine diphosphoglucose to rabbit liver glycogen of fraction II was 78% of the rate of glucose transfer to amylopectin, but with fraction I the rate of transfer of glucose to rabbit liver glycogen was 380% of that observed to amylopectin. Glucan synthesis in the absence of added primer was found in fraction I in the presence of 0.5 m sodium citrate and bovine serum albumin. The unprimed product was a methanol precipitable glucan with principally alpha-1,4 linkages and some alpha-1,6 linkages, and its iodine spectrum was similar to that of amylopectin. PMID- 16657877 TI - Entrainment of Lemna CO(2) Output Through Phytochrome. AB - The entrainability of Lemna perpusilla CO(2) output by periodic 15 minute red (R) and far red (F) illuminations was tested in low nitrate medium. R every 8 hour, symbolized R/R/R, gives a flat output (no entrainment) as does F/F/F. However, R/ /- (R every 24 hour) entrains rapidly, and F/-/- does so as well, in a similar manner. The effects of R/R/- and F/F/- also resemble each other closely. Entrainment by R/F/F or R/R/F is rapid and indifferent to order of presentation, e.g., R/F/F and F/R/F lead to the same steady state. Typical phytochrome reversals occur, e.g., R,F/F/F holds output flat, while F,R/F/F entrains in the manner of R/F/F. Blue (B) light acts like R in schedules such as B/F/F but like F in schedules such as B/R/R. In all schedules studied, the zeitgeber (primary synchronizer) appears to be the sharpest transition from a low to a high level of far red-absorbing phytochrome that occurs with a 24-hr periodicity. Thus in entrainment, and by inference in photoperiodic timing, the level of far red absorbing phytochrome at any time may be less significant than the succession of levels of which it is a part, a conclusion that implies the existence of a "scanning" mechanism that compares levels of far red-absorbing phytochrome at various times of day. PMID- 16657878 TI - Conversion of N-(Delta-Isopentenyl)adenosine to Adenosine by Enzyme Activity in Tobacco Tissue. AB - By using [8-(14)C]-N(6)-(Delta(2)-isopentenyl)adenosine as a substrate, we detected the presence of enzyme activity in a crude extract of tobacco tissue that converts this cytokinin into adenosine. The extract also contains strong hydrolase activity that converts adenosine to adenine. PMID- 16657879 TI - Microcalorimetric Measurements of the Heat Evolution and Its Sensitivity to Dinitrophenol during Different Stages of Synchronous Cultures of Scenedesmus. AB - The applicability of the LKB flow microcalorimeter in a microalgal system was tested. Respiration and its sensitivity to 2,4-dinitrophenol during different stages of a synchronous culture of Scenedesmus obtusiusculus were studied. In cells harvested during the first 8 hours of illumination, heat evolution was 10 to 15 microwatts per 10 million cells and then rose to a peak of about 80 microwatts per 10 million when the cells were harvested in the beginning of the dark period, just before the onset of the divisions. Calculated per milligram dry weight, the highest values were about 140 microwatts, also obtained early in the dark period, after which the values dropped. They were roughly 90 microwatts per milligram at the onset of light and a little below 70 microwatts per milligram after 8 hours of illumination. Thus, the synthesis of new cell material does not give rise to actively respiring mitochondria during the first half of the light period, after which the situation is reversed. Low concentrations of dinitrophenol increased heat evolution, and high concentrations were inhibitory. Cells from the early stages of development were more sensitive to dinitrophenol than older cells. Young cells gave a transient peak of heat evolution even when the steady state was inhibited. Coupled oxidative phosphorylation seems necessary for substrate mobilization and transport to the mitochondria of the young cells. Possibly, young cells use mainly fatty acids and old cells carbohydrates as their main substrate for respiration. PMID- 16657880 TI - Influence of soil water stress on evaporation, root absorption, and internal water status of cotton. AB - Diurnal variations in leaf water potential, diffusion resistance, relative water content, stem diameter, leaf temperature, and energy balance components were measured in cotton (Gossypium hirsutum L. var. Lankart 57) during drought stress under field conditions. A plot of leaf water potential against either relative water content or stem diameter during the 24-hour period yielded a closed hysteresis loop. The relation between cell hydration and evaporation is discussed.Despite low soil water potential in the main root zone, significant plant evaporation rates were maintained. Root absorption rates as a function of soil depth were calculated from water content profiles measured with a neutron probe. The maximal root absorption rate of 3.5 x 10(-3) day(-1) occurred at the 75-centimeter depth, well below the main root zone.Stomatal resistance of individual leaves during the daylight hours remained nearly constant at 2.5 seconds centimeter(-1) even though leaf water potentials approached -30 bars. A growth chamber study indicated stomatal closure occurred at potentials near -16 bars. Possible implications of high soil water stress in relation to stomatal function and growth are discussed. Based on an energy balance method, the actual to potential plant evapotranspiration ratio was 0.43 for the 24-hour period, indicating partial stomatal closure. A surface resistance, r(s), of 4.0 seconds centimeter(-1) was calculated for the incomplete canopy with the use of the energy balance data. Alternatively, a canopy resistance of 1.3 seconds centimeter(-1) was attained from a relationship between leaf area and stomatal resistance of individual leaves. If the soil resistance was assumed to be very large and the canopy resistance was weighted for the fractional ground cover of the crop, the calculated surface resistance was 4.3 seconds centimeter(-1). Under these conditions, the two independent estimates of r(s) were in essential agreement. PMID- 16657881 TI - Efficiency of Glasshouse Pot Experiments Rotating versus Stationary Benches. AB - Pot cultures of rice (Oryza sativa L., var. Colusa) in flooded soil were used to measure comparative uniformity of growth where mutual shading was the primary source of variation. Data were analyzed according to various divisions of each layout into blocks, sections, and replicate pots to compare statistical efficiencies.Experiments on stationary benches were subject to large border effects that were not suitably compensated for by any of the systems of replication tested. On the turntable, which carried 24 pots in single file, all plans of analysis tested were acceptable and resulted in coefficients of variability ranging from 2.5 to 4.5%.Consideration of these results, along with the cost of increased replication and the fact that use of a turntable minimizes the effects of environmental variability inherent in greenhouses, leads to the conclusion that the turntable provides more efficient use of greenhouse space for precision experiments than do stationary benches. PMID- 16657882 TI - Effect of Wilting on Carbohydrates during Incubation of Excised Bean Leaves in the Dark. AB - Excised bean (Phaseolus vulgaris) leaves were used to compare the changes in various carbohydrates during dark incubation in a wilted and turgid condition. After a rapid (less than 1 hour) wilt, leaves were incubated at a constant water content (75% of original fresh weight).The starch content of the wilted leaves decreased faster than it did in the turgid leaves. This accelerated starch loss was accompanied by an increase in free (alcohol-soluble) sugars, while no similar increase was observed in the turgid leaves. The increase in free sugars in the wilted leaves during accelerated starch loss was sufficient to account for the decreased starch content in wilted leaves compared to turgid leaves. Thus, total carbohydrate decreased at the same rate in both wilted and turgid leaves during dark incubation.The free sugar which accumulated in the wilted leaves was mainly sucrose. There was no increase in sucrose content of turgid leaves during incubation, although there was a rapid loss of starch. Glucose and fructose also increased during incubation, but there was no difference between the wilted and turgid leaves. These results suggest that wilting of detached leaves caused a conversion of starch to sucrose in the dark. PMID- 16657883 TI - Membrane transformations in aging potato tuber slices. AB - When potato tuber slices (Solanum tuberosum L.) are incubated with radioactive choline, labeled membrane-bound phospholipids are formed. If potato slices are aged for 0 to 24 hours before exposure to radioactive choline, the distribution of the labeled phospholipids undergoes both quantitative and qualitative changes. Quantitatively, there is a marked increase in the total lipoidal radioactivity with aging time. Qualitatively, there is a shift in the kinds of subcellular fractions that are being labeled. Fresh slices incorporate most of the lipoidal radioactivity in the microsomes. Slices aged for 9 hours incorporate most of the label in a fraction consisting of single membrane-bound cisternae, which are presumed to be dictyosomal fragments. Slices aged for 24 hours before incubation with radioactive choline incorporate the greater portion of the label in this same fraction, but a significant portion of the label is found in a heavier, mitochondria-containing fraction. PMID- 16657884 TI - The effects of diethyl pyrocarbonate on the stability and activity of plant polyribosomes. PMID- 16657885 TI - Ethylene-stimulated Synthesis of Ribosomes, Ribonucleic Acid, and Protein in Developing Fig Fruits. PMID- 16657886 TI - Rapid Respiratory Changes Due to Red Light or Acetylcholine during the Early Events of Phytochrome-mediated Photomorphogenesis. AB - Two millimeter long secondary root tips of etiolated mung bean (Phaseolus aureus) plants were given 4 minute consecutive treatments of darkness, red light, far red light, and acetylcholine during darkness. We studied the effects of these treatments on exogenous (H(+)) changes, ATP utilization, O(2) uptake, P(1) levels, and ATPase activity. Red light and acetylcholine increased the level of P(1), O(2) uptake, and exogenous H(+), but decreased ATP concentrations. Darkness and far red light caused the amount of ATP to increase and decreased the O(2) uptake and P(1) level. O(2) uptake of both excised root tips and isolated mitochondria was promoted by acetylcholine levels of the same order of magnitude that promoted the other photomimetic phenomena. ADP-O ratios indicated that acetylcholine did not cause an appreciable decrease in ATP synthesis. The total ATPase activity remained constant throughout all treatments. Ouabain caused no adhesion to negatively charged glass in the dark, while the inhibitors valinomycin, atractyloside, digitoxin, gramicidin, and oligomycin caused immediate adhesion. All of the inhibitors prevented release from the glass. In red light ouabain increased adhesion, whereas the other inhibitors caused caused immediate and complete adhesion.These data seem to imply that one of the functions of the phytochrome-mediated response to red light in roots, regulated by acetylcholine, is to cause the rapid utilization of ATP pools; far red light appears to inhibit this utilization. PMID- 16657887 TI - The Influence of Carbonylcyanide-m-chlorophenylhydrazone and 3-(3,4 Dichlorophenyl)-1,1-dimethylurea on the Fusion of Primary Thylakoids and the Formation of Crystalline Fibrils in Bean Leaves Partially Greened in Far Red Light. PMID- 16657888 TI - Studies on cytokinin production by Rhizobium. AB - Cytokinin was released into the medium by cultures of both Rhizobium japonicum and R. leguminosarum. Calculations show that the amount of cytokinin released during the logarithmic phase of growth by R. japonicum would be sufficient to initiate the cortical cell divisions necessary to form a root nodule. The substance released by R. japonicum was identified as a zeatin-like compound on the basis of paper chromatography in four solvent systems. Two solvents clearly separated the rhizobial product from N(6)-Delta(2)-isopentenyladenine and its ribonucleoside. The predominant intracellular cytokinin found in both enzymatic hydrolysates of sRNA and alkaline hydrolysates of total RNA also was similar to zeatin. PMID- 16657889 TI - The Release of Potassium and Sodium from Young Excised Roots of Zea mays under Various Efflux Conditions. AB - The release of potassium and sodium from excised roots of Zea mays having similar contents of potassium and sodium was studied. At low temperature (2 C) the efflux rates of both cations were very similar, but at higher temperature (20 C) the potassium release was reduced considerably, whereas the sodium release was hardly affected. Also, under anaerobic conditions the potassium efflux rate was nearly as high as the sodium efflux rate, but with normal O(2) supply the potassium release was reduced to about one-fifth. Since a changing efflux medium compared with a constant efflux medium had no great influence upon the sodium release but influenced the potassium release very much, it is assumed that the low potassium release under normal metabolic conditions is due to a reabsorption of effluxed potassium from free space. For sodium this reabsorption is of minor significance, as the uptake potential of maize roots for sodium is very poor. It is concluded that the release of potassium and sodium is a diffusion process and that the cell membranes have rather similar diffusivities for these two cations. PMID- 16657890 TI - Onset of deoxyribonucleic Acid synthesis in germinating wheat embryos. AB - Germinating wheat embryos (Triticum vulgare var. Florence) synthesize proteins before the onset of DNA synthesis. The onset of DNA replication occurs at about 15 hours of germination and was shown to depend on proteins synthesized before 9 hours of germination with the use of blasticidin S, a specific inhibitor of protein synthesis. A 10-fold increase in the activity of DNA-dependent DNA polymerase was found in extracts derived from germinated embryos, as compared to the activity found in extracts from ungerminated embryos. PMID- 16657891 TI - Auxin-induced Conjugation Systems in Peas. AB - Pretreatment of pea (Pisum sativum var. Alaska) sections with any active auxin induces an enzyme which forms aspartate conjugates of exogenously supplied indoleacetic acid, naphthaleneacetic acid, or benzoic acid. Whereas induction of this system is an absolutely auxin-specific process, another enzyme, which forms benzoylmalic acid, is induced both by auxins and by physiologically inactive aromatic carboxylic acids. Induction of both enzymes is abolished by low levels of RNA and protein synthesis inhibitors. The induction specificities and other characteristics of the two systems are compared. PMID- 16657892 TI - Developmental studies on microbodies in wheat leaves : I. Conditions influencing enzyme development. AB - Catalase, glycolate oxidase, and hydroxypyruvate reductase, enzymes which are located in the microbodies of leaves, show different developmental patterns in the shoots of wheat seedlings. Catalase and hydroxypyruvate reductase are already present in the shoots of ungerminated seeds. Glycolate oxidase appears later. All three enzymes develop in the dark, but glycolate oxidase and hydroxypyruvate reductase have only low activities. On exposure of the seedlings to continuous white light (14.8 x 10(3) ergs cm(-2) sec(-1)), the activity of catalase is doubled, and glycolate oxidase and hydroxypyruvate reductase activities increase by 4- to 7-fold. Under a higher light intensity, the activities of all three enzymes are considerably further increased. The activities of other enzymes (cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase) are unchanged or only slightly influenced by light. After transfer of etiolated seedlings to white light, the induced increase of total catalase activity shows a much longer lag-phase than that of glycolate oxidase and hydroxypyruvate reductase. It is concluded that the light-induced increases of the microbody enzymes are due to enzyme synthesis. The light effect on the microbody enzymes is independent of chlorophyll formation or the concomitant development of functional chloroplasts. Short repeated light exposures which do not lead to greening are very effective. High activities of glycolate oxidase and hydroxypyruvate reductase develop in the presence of 3-amino-1,2,4-triazole which blocks chloroplast development. The effect of light is not exerted through induced glycolate formation and appears instead to be photomorphogenetic in character.In senescing leaves excised from the plants decreases in activity of glycolate oxidase, and hydroxypyruvate reductase follow with some delay the decrease in chlorophyll content. The activity of catalase, however, is maintained at high levels, especially when the detached shoots are kept in light. PMID- 16657893 TI - Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations. AB - Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm(3) and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm(3). During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm(3). However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm(3) and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm(3) was found when the dark grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole. PMID- 16657894 TI - Determination of the Rates of Protein Synthesis and Degradation in Lemna minor. AB - Attempts to measure the rates of synthesis and degradation of protein in plant tissues with isotopes are complicated by the presence of at least two pools of amino acids, only one of which contributes to the synthesis of protein. Direct measurement of the protein precursor pool is thus difficult. This paper shows that one solution to this problem is to assume that the amino-acyl transfer RNA is the strict precursor of protein amino acid. By using labeled methionine, the variation with time of the specific radioactivities of methionine bound to RNA and protein have been examined under two different growth conditions in Lemna minor. From these data rates of flux of methionine into and out of protein may be easily determined.A second method of determining the rates of protein synthesis and degradation assumes that the specific radioactivity of the methionyl transfer RNA in label chase conditions is effectively zero. Evidence is presented to support this contention, and the rates determined by this method agree with those calculated by the first method. PMID- 16657895 TI - Control of the Protein Turnover Rates in Lemna minor. AB - The control of protein turnover in Lemna minor has been examined using a method described in the previous paper for determining the rate constants of synthesis and degradation of protein. If Lemna is placed on water, there is a reduction in the rate constants of synthesis of protein and an increase (3- to 6-fold) in the rate constant of degradation. The net effect is a loss of protein from the tissue. Omission of nitrate, phosphate, sulfate, magnesium, or calcium results in increases in the rate constant of degradation of protein.An unusual dual effect of benzyladenine on the turnover constants has been observed. Treatment of Lemna grown on sucrose-mineral salts with benzyladenine results in alterations only in the rate constant of synthesis. Treatment of Lemna grown on water with benzyladenine alters only the rate constant of degradation. Abscisic acid on the other hand alters both rate constants of synthesis and degradation of protein together. Inclusion of growth-inhibiting amino acids in the medium results in a reduction in the rate constants of synthesis and increases in the rate constant of degradation of protein. It is concluded that the rate of turnover of protein in Lemna is very dependent on the composition of the growth medium. Conditions which reduce growth rates also reduce the rates of synthesis of protein and increase those of degradation. PMID- 16657896 TI - Regulation of aromatic amino Acid biosynthesis in higher plants: I. Evidence for a regulatory form of chorismate mutase in etiolated mung bean seedlings. AB - Etiolated mung bean seedlings were examined for chorismate mutase activity. Evidence for the occurrence of two forms of the enzyme (designated CM-1 and CM-2) was obtained by ammonium sulfate fractionation, anion exchange cellulose chromatography, and isoelectric focusing. The two forms showed distinctly different properties, as CM-1 was inhibited by phenylalanine and tyrosine and activated by tryptophan, but inhibition by phenylalanine and tyrosine was reversed by tryptophan. The other form, CM-2, was unaffected by any of the three aromatic amino acids. Isoelectric points of the two forms were CM-1, pH 4.6, and CM-2, pH 5.6. The molecular weights estimated by molecular sieving on Sephadex G 200 were CM-1, 50,000, and CM-2, 36,000. PMID- 16657897 TI - Changes in Molecular Weight of Cellulose in the Pea Epicotyl during Growth. AB - A procedure is described for preparing cellulose nitrate from pea tissues (Pisum sativum L. var. Alaska) in quantitative yield, undegraded and uncontaminated by other polysaccharides. The average degree of polymerization of this product, estimated from viscosity measurements, increased during cell growth and development from a value of about 5000 glucose units in the apical meristem (plumule plus hook) to values near 8000 in fully grown maturing tissues (>20 mm from apex). The cellulose content per cell also increased (approximately 10-fold) during growth in these tissues, as did particulate glucan synthetase activity (3 fold rise). Since the yield of soluble cellulase activity is known to decrease from high values in the meristem to barely detectable amounts in mature tissues, it is suggested that the relative levels and properties of these hydrolytic and synthetic enzyme activities control the amount and degree of polymerization of cellulose formed during cell expansion in the pea epicotyl.Degree of polymerization distribution patterns showed that a low molecular weight component of cellulose (degree of polymerization < 500) was prominent in young tissues whereas high molecular weight components (degree of polymerization > 7000) predominated in mature tissues. Also, cellulose which was formed from radioactive sucrose during 30 minutes of incubation showed a remarkably similar degree of polymerization distribution to cellulose which was present in the tissue at the time of synthesis. It is concluded that new and old parts of the epicotyl cellulose framework are subject to constant modification and equilibration by cellulose-metabolizing enzymes. PMID- 16657898 TI - The role of protein synthesis in the senescence of leaves: I. The formation of protease. AB - The senescence of oat leaves has been studied by following the loss of chlorophyll and protein and the increase of alpha-amino nitrogen, after detachment and darkening. Protein synthesis and the amounts of proteolytic enzymes in the leaves have been determined directly. The process of senescence is shown to be a sequential one in which protein synthesis,most probably the formation of a proteolytic enzyme with l-serine in its active center, is of prime importance. The evidence is as follows. Firstly, l-serine specifically enhances senescence, especially in presence of kinetin. Secondly, cycloheximide, which inhibits protein synthesis in other systems, delays senescence and prevents the serine enhancement. Although requiring higher concentrations, cycloheximide can be as effective as kinetin in inhibiting senescence. It is shown directly that cycloheximide prevents protein synthesis in oat leaves under the same conditions as when it prevents senescence. Thirdly, leaves have been shown to contain two proteinases, with pH optima at 3 and 7.5, whose activity increases during senescence, even though the total leaf protein is decreasing. The amounts of both these enzymes present after 3 days are clearly increased by serine, and are greatly decreased by cycloheximide or by kinetin. The role of kinetin in delaying senescence thus may rest on its ability to suppress protease formation. PMID- 16657899 TI - Migration of Calcium and Its Role in the Regulation of Seismonasty in the Motor Cell of Mimosa pudica L. AB - Volume and conformational changes of the contractile tannin vacuoles of the abaxial motor cells of the primary pulvinus of Mimosa pudica L. parallel the seismonastic leaf movement. Since such changes in cells and organelles of animal systems are often regulated by calcium, we studied Ca(2+) movement in the motor cells and tissue. By fixation with Lillie's neutral buffered formalin, followed by staining with alizarin red sulfate (ARS), calcium was localized in the tannin vacuoles of the motor cells of the primary pulvinus. After treatment with ethylenediaminetetraacetate, 8-hydroxyquinoline, and several other calcium complexing or extracting agents, the color reaction due to alizarin red sulfonate was no longer present. By using an analytical method, it was shown that the effluent from stimulated pulvini has significantly more Ca(2+) than that from unstimulated controls. Ten millimolar LaCl(3) inhibits recovery of the tannin vacuole in vivo in 10 mm CaCl(2) or in distilled water. Quantitative data obtained by microspectrophotometry demonstrated calcium migration during the bending movement of the primary pulvinus. In the adaxial motor cells a small amount of calcium migrates from the tannin vacuole, and calcium on the cell wall moves to the central vacuole. In the abaxial half, a large amount of calcium from the tannin vacuole moves to the central vacuole of the motor cell. It is probable that the calcium binds to the microfibrillar contents of the central vacuole. These observations support the contention that Ca(2+) migrates between the surface of the tannin vacuole and the inside of the central vacuole. The recovery and maintenance of the tannin vacuole in the spherical form may play a role in maintaining turgor in the motor cells of the abaxial half of the primary pulvinus of Mimosa. PMID- 16657900 TI - Phytochrome-mediated Induction of Phenylalanine Ammonia-Lyase in Mustard Seedlings: A Contribution to Eliminate Some Misconceptions. AB - The present report shows that the effect of red and far red light on the synthesis of phenylalanine ammonia-lyase can be ascribed to the action of phytochrome Pfr. This is true not only for short term irradiation but also for the action of continuous far red light. In the latter case, the model worked out by Hartmann implicating some excited species of phytochrome has to be taken into account. PMID- 16657901 TI - Sugar transport in immature internodal tissue of sugarcane: I. Mechanism and kinetics of accumulation. AB - Transmembrane sugar transport into immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L.) is a metabolically regulated process as evidenced by its sensitivity to pH, temperature, anaerobiosis, and metabolic inhibitors. All sugars studied-glucose, fructose, galactose, sorbose, glucose 6 phosphate, 3-O-methylglucose, and 2-deoxy-d-glucose-were apparently transported via the same carrier sites since they competed with each other for uptake. External concentrations of these sugars at one-half V(max) were in the range of 3.9 to 8.4 nm. Preliminary data indicated that phosphorylation may be closely associated with glucose transport. The dominant intracellular sugar after 4-hours incubation was sucrose when glucose, glucose-6-P, or fructose was the exogenously supplied sugar; but when galactose was supplied, only 28% of intracellular radioactivity was in sucrose. Sorbose, 3-O-methylglucose, and 2-deoxy-d-glucose were not metabolized. Thus, by using these analogs, transport could be studied independently of subsequent metabolism, effectively eliminating a complicating factor in previous studies. PMID- 16657902 TI - Glutamate dehydrogenase from apodachlya (oomycetes). AB - A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya. PMID- 16657903 TI - Nucleic Acid and protein changes in relation to cold acclimation and freezing injury of korean boxwood leaves. AB - Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.Exposure of hardy boxwood plants to warm temperatures resulted in a rapid loss in cold resistance and a rapid synthesis of nucleic acids as judged by (32)P incorporation.Following a killing frost to Korean boxwood leaves, there was a rapid decrease in all nucleic acid fractions which was attributed to nuclease activity. Within 5 hours there was no measurable soluble RNA and ribosomal RNA. Tenaciously bound RNA was somewhat more persistent. PMID- 16657904 TI - Induction and regulation of chloroplast replication in mature tobacco leaf tissue. AB - Chloroplast replication was induced in mature tobacco leaf tissue (Nicotiana tabacum L.) by culturing leaf discs on a sterile medium composed of salts and sucrose. Chloroplast replicaton is greatly enhanced by the addition of kinetin to this medium. Kinetin also enhances cell enlargement, but cell division does not occur. Chloroplast replication is nonsynchronous and proceeds most rapidly when the cell enlargement rate decreases. Chloroplast replication is light-dependent, but cell enlargement occurs in both light and dark. Chloroplast replication resumes when discs cultured in the dark are returned to the light. It appears that chloroplast replication is related to cell expansion. The possibility of inducing synchronous replication of chloroplasts in tobacco cells is discussed. PMID- 16657905 TI - Potassium Deficiency-induced Changes in Stomatal Behavior, Leaf Water Potentials, and Root System Permeability in Beta vulgaris L. AB - Studies of the water relations of potassium deficient sugarbeet plants (Beta vulgaris L.) revealed two factors for stomatal closure. One component of stomatal closure was reversible by floating leaf discs on distilled water to relieve the water deficit in the leaves; the other component was reversible in the light by floating the leaf discs on KCl solution for 1 hour or more. Potassium-activated stomatal opening in the light was observed when the guard cells were surrounded by their normal environment of epidermal and mesophyll cells, just as observed by previous workers for epidermal strips. Leaf water potentials, like stomatal apertures, appear to be strongly related to leaf potassium concentration. Potassium-deficient plants have a greatly decreased root permeability to water, and the implications of this effect on stomatal aperture and leaf water potential are discussed. In contrast, petiole permeability to water is unaffected by potassium treatment. PMID- 16657906 TI - Phytochrome action on the timing of cell division in adiantum gametophytes. AB - When filamentous protonemata of Adiantum capillus-veneris L. precultured under continuous red light were transferred to the dark, the apical cell divided about 24 to 36 hours thereafter. The time of the cell division was delayed for several hours by a brief exposure to far red light given before the dark incubation. The effect of far red light was reversed by a small dose of red light given immediately after the preceding far red light. The effects of red and far red light were repeatedly reversible, indicating that the timing of cell division was regulated by a phytochrome system. When a brief irradiation with blue light was given before the dark incubation, the cell division occurred after 17 to 26 hours in darkness. A similar red far red reversible effect was also observed in the timing of the blue light-induced cell division. Thus, the timing of cell division appeared to be controlled by phytochrome and a blue light-absorbing pigment. PMID- 16657907 TI - Regulation of sulfate uptake by excised barley roots in the presence of selenate. AB - Active transport of SO(4) (2-) and SeO(4) (2-) has been evaluated during 60-hour contact of barley roots with nutrient solutions containing either (35)SO(4) (2-) or (75)SeO(4) (2-), or both ions, at 0.1 milli-equivalent per liter. In the SO(4) (2-) solution the time course of active transport follows a straight line; if SeO(4) (2-) is also present transport is strongly inhibited after 20 to 30 hours for both ions. The S-Se uptake ratio remains 1.4 during the 60 hours; S-Se ratio shifts from 3.0 to 3.3 in proteins and falls to 0.6 in free amino acids. S-Se discrimination is mainly operating at the level of amino acid incorporation into proteins. The presence of Se-amino acids blocks this incorporation and brings about an accumulation of free amino acids; at the same time carrier activity is inhibited. The addition of methionine or Se-methionine causes a 60 to 80% inhibition of the active transport. PMID- 16657908 TI - Malate dehydrogenase isoenzymes from cotton leaves: molecular weights. AB - Malate dehydrogenase isolated from leaves of the cotton plant (Gossypium hirsutum L.) appears in the form of several isoenzymes. Four of the isoenzymes found in cotton leaf extracts appear to be charge isomers with a molecular weight of approximately 60,000. A fifth malate dehydrogenase isoenzyme found in leaf extracts has a molecular weight of approximately 500,000. Under appropriate conditions it is possible to form this high molecular weight isoenzyme from at least one of the smaller isoenzymes. In addition, malate dehydrogenase isoenzymes of approximately 700,000 and 130,000 molecular weight have been observed under some conditions, although these isoenzymes do not appear in the crude cotton leaf preparations. The relationship of this heterogeneity with respect to size and to the discrepancies in the number and size of malate dehydrogenase isoenzymes reported from plant tissues may be significant. PMID- 16657909 TI - A rapid effect of kinetin on rehydration of tobacco leaf tissue. AB - Partly dehydrated tobacco leaf tissue (Nicotiana rustica), stripped of the lower epidermis, was used to study the effect of kinetin on the rate of rehydration. Depending on the rate of rehydration in untreated tissue, kinetin either increased or decreased rehydration rates. The response to kinetin was very rapid and could be discerned in less than 2 minutes. On extensive dehydration, the tissue lost the capacity to respond to kinetin. Salinity stress, which decreases the endogenous level of cytokinins in the plant, conditions the leaf to stimulation of rehydration by kinetin.It is suggested that cytokinins may play a role in controlling water permeability of the leaf tissue. PMID- 16657910 TI - Interactions of light and a temperature shift on seed germination. AB - Germination of Rumex obtusifolius L. seeds is potentiated to an observable degree in 2 minutes by a single shift in temperature from 20 to 35 C. Half-maximal potentiation requires less than 32 minutes at the higher temperature. Similar sensitivities to shifts in temperature were observed for seeds of Barbarea vulgaris, R.Br. B. verna (Mill.) Asch., and Lepidium virginicum L. A shift in temperature interacts strongly with change in form of phytochrome induced by light on germination of the four kinds of seeds. The potentiated effects for R. obtusifolius are only moderately affected by 40 mum cycloheximide. Both the temperature shift and light actions are apparently independent of processes of synthesis necessary for growth. PMID- 16657911 TI - Promotion of crown-gall tumor growth by lysopine, octopine, nopaline, and carnosine. AB - The growth of crown-gall tumors on primary bean leaves (Phaseolus vulgaris L. cv. "Pinto") was promoted by the addition of d-lysopine, d-octopine, l-carnosine, or nopaline. Assayed on tumors induced by Agrobacterium tumefaciens strain B6, the relative activity was octopine = carnosine > lysopine >> nopaline; assayed on tumors induced by A. tumefaciens strain T-37, which induces tumors which form nopaline, the relative activity was nopaline = octopine = carnosine > lysopine. From one to three applications of carnosine or octopine gave equal additive increments in tumor growth, showing that a continual supply of these substances is required to maintain an increased rate of growth. At concentrations above 0.1 mm, pairs of these growth-promoting substances were less active than when applied singly. Inhibition of octopine-induced growth was obtained by applying 0.01 mm carnosine with 1 mm octopine and partial inhibition was obtained when carnosine was added 10 hr after octopine. Equimolar mixtures of lysopine, octopine, and carnosine, however, were at least as active in promoting tumor growth as any of the compounds added singly at equivalent concentrations. The activity of 0.1 to 0.5 mm lysopine, octopine, and carnosine was inhibited, respectively, by 1 mml lysine, l-arginine, and l-histidine and this inhibition was limited in each case to the basic amino acid corresponding to that of the growth factor. Arginine fully inhibited octopine-induced tumor growth when applied as much as 6 hr after octopine, indicating that this inhibition was not due to prevention of octopine uptake. Although four separate substances were found which promoted tumor growth, the molecular specificity required for activity of each compound was high. Evidence is presented which suggests that a tumor growth-promoting substance extracted from tumorous leaves is a carnosine-like derivative of l-histidine. PMID- 16657912 TI - Influence of ammonium and nitrate nutrition on enzymatic activity in soybean and sunflower. AB - Under conditions of controlled pH, nitrate and ammonium are equally effective in supporting the growth of young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var., Mammoth Russian) plans. Soybean contains an active nitrate reductase in roots and leaves, but the low specific activity of this enzyme in sunflower leaves indicates a dependency upon the roots for nitrate reduction. Suppression of nitrate reductase activity in sunflower leaves may be due to high concentrations of ammonia received from the roots. Nitrate reductase activity in leaves of nitrate-supplied soybean and sunflower follows closely the distribution of nitrate reductase. For the roots of both species, glutamic acid dehydrogenase activity was greater with ammonium than with nitrate. The glutamic acid dehydrogenase of ammonium roots is wholly NADH-dependent, whereas that of nitrate roots is active with NADH and NADPH. In leaves, an NADPH-dependent glutamic acid dehydrogenase appears to be responsible for the assimilation of translocated ammonia and ammonia formed by nitrate reduction.In soybean roots ammonia is actively incorporated into amides, much of which remains in the roots. Sunflower roots are less active in amide formation but transfer much of it, together with ammonia, into the shoots. Glutamine synthetase activity in leaves is 20- to 40-fold lower than in roots.Glucose-6-phosphate dehydrogenase activity appears to be correlated with the activity of the nitrate reducing system in roots, but not in leaves. PMID- 16657913 TI - Influence of ammonium and nitrate nutrition on the pyridine and adenine nucleotides of soybean and sunflower. AB - Total pyridine nucleotide concentration of root tissue for young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var. Mammoth Russian) plants is the same with either ammonium or nitrate, but nitrate results in an increased proportion of total oxidized plus reduced NADP (NADP[H]) seemingly at the expense of NAD. The activity of NADH- and NADPH-dependent forms of glutamic acid dehydrogenase is correlated with the ratio of total oxidized plus reduced NAD to NADP(H). The low NAD: NADH ratio maintained in nitrate roots despite active NADH utilization via nitrate reductase and glutamic acid dehydrogenase may be the result of nitrate-stimulated glycolysis. Nitrate roots also maintain a high level of NADPH, presumably by the stimulatory effect of nitrate utilization on glucose 6-phosphate dehydrogenase activity. In the presence of nitrate rather than ammonium, the highly active nitrate-reducing leaves of soybean show a greater proportion of total pyridine nucleotide in the form of NADP(H) than do the inactive leaves of sunflower.For all tissues examined, ammonium nutrition yields a higher concentration of total adenine nucleotide than is found with nitrate. The data indicate the production of a higher level of metabolites that enter into purine synthesis with ammonium than with nitrate. Glutamine synthetase activity can be correlated with the concept that enzymes utilizing ATP for biosynthetic purposes increase in activity in accordance with the energy level of the cell. PMID- 16657914 TI - Canopy and Seasonal Profiles of Nitrate Reductase in Soybeans (Glycine max L. Merr.). AB - Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior to full expansion and declined with leaf position lower in the canopy. Total nitrate reductase activity per leaf was also highest in the upper-most fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages.Nitrate reductase activity of soybeans grown in hydroponic systems was significantly higher than activity of adjacent soil grown plants at later growth stages, which suggested that under normal field conditions the potential for nitrate utilization may not be realized. Nitrate reductase activity per gram fresh weight per hour and nitrate content were positively correlated over the growing season with plants grown in either soil or solution culture. Computations based upon the nitrate reductase assay of plants grown in hydroponics indicated that from 1.7 to 1.8 grams N could have been supplied to the plant via the nitrate reductase process. The harvested seed contained 1.1 to 1.2 grams N per plant. Thus, based on previous estimates of approximately 32% of the final N distribution being in the vegetative plant parts, the estimated input of reduced nitrogen via the enzyme assay was in agreement with the actual N accumulation.The amount of calculated N(2)-fixation by nodules per season with plants grown in hydroponics was less than 2% of the computed nitrate reduced via leaf nitrate reductase. Thus, the level of nitrate in the nutrient solution appeared to be quite inhibitory to N(2)-fixation. PMID- 16657916 TI - Artifacts in the Embedment of Water-soluble Compounds for Light Microscopy. AB - The relative effectiveness of freeze-drying and freeze-substitution in accurately preserving the distribution of water-soluble compounds in plants tissues was evaluated. Specimens with a known distribution of water-soluble materials (sucrose-infiltrated pith blocks and red beet tissue) were freeze-dried or freeze substituted and embedded in paraffin, Epon, or methacrylate for microscopic examination. Dried water-soluble compounds were extremely sensitive to only traces of water, which caused severe shrinkage of the vacuolar contents. In spite of care taken to maintain dry conditions, freeze-drying was always accompanied by marked shrinkage of the water-soluble compounds away from the cell walls. Freeze substitution was successful only if the solvents and embedding media were thoroughly dried and if infiltration was done in a dry box. Other artifacts were also noted, although they were less severe. Artifacts comparable to those seen in pith blocks and in red beet tissue were observed in the contents of pumpkin sieve tubes. PMID- 16657915 TI - Cytokinins in seedling roots of pea. AB - The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. The terminal 0- to 1-millimeter root tip contained 43 to 44 times more free cytokinin on a fresh weight or a per cell basis than the next 1- to 5-millimeter root segment. Extracts of more proximal segments behind the tip contained no measurable free cytokinin. Acid hydrolysates of transfer RNA exhibited reproducible cytokinin activity. Bioassays revealed that the predominant amounts of free cytokinin and that present in transfer RNA were restricted to the extreme root tip. There was approximately 27 times more free cytokinin than the amount detected in transfer RNA in root apices. PMID- 16657917 TI - The Retention of Water-soluble Compounds during Freeze-Substitution and Microautoradiography. AB - Freeze-substitution and Epon embedment were quantitatively evaluated for their effectiveness in retaining water-soluble metabolites in plant tissues. Roughly 99% of the 80% (v/v) ethanol-extractable radioactivity in photosynthetically labeled soybean leaf discs and in petiole fragments containing translocated (14)C was retained during freeze-substitution in acetone or propylene oxide and embedment in Epon. Substantially more activity was lost from (14)C-sucrose infiltrated pith blocks, but most or all of this loss came from the block surface. The procedure was effective for a sucrose concentration as low as 0.004%. Sections floated on water retained most of their (14)C-sucrose, and high resolution autoradiographs could easily be prepared without resorting to dry procedures. Embedded (14)C-sucrose was apparently chemically unreactive, since there was no loss of radioactivity when sections were stained with the periodic acid-Schiff reagent, nor did the embedded sucrose show staining. PMID- 16657918 TI - Uptake and Metabolism of 2,2-bis-(p-Chlorophenyl-1,1,1-trichloroethane (DDT) by Marine Phytoplankton and Its Effects on Growth and Chloroplast Electron Transport. AB - The effects of DDT (2,2-bis-(p-chlorophenyl)-1, 1, 1-trichloroethane) on the growth of seven marine phytoplankters, representative of five algal divisions, were studied. At a concentration of 80 parts per billion (0.23 mum) DDT, growth of Dunaliella tertiolecta was unaffected, and there was slight, if any, influence on the development of Cyclotella nana, Thalassiosira fluviatilis, Amphidinium carteri, Coccolithus huxleyi, and Porphyridium sp. Skeletonema costatum exhibited a 9 day lag before cell division commenced, the rate of growth subsequently being the same as in the control (no DDT). A further inoculation of this culture of S. costatum into 80 parts per billion DDT gave another 9-day lag before initiation of normal growth.The ability of marine phytoplankton to metabolize DDT varied. DDE (2,2-bis-(p-chlorophenyl)-1, 1-dichloroethylene) was the only significant hexane-soluble metabolite detected. It occurred in cells of S. costatum, C. nana, T. fluviatilis and D. tertiolecta. Maximum degree of conversion was 7.5% and was based on the total DDT found in the cell-water system of 9-day D. tertiolecta cultures. The total amount of DDT recovered from cultures in 2- to 3-week experiments ranged from 63.5% for T. fluviatilis to 90.7% for S. costatum. The amount of DDT found associated with the cells, collected by centrifugation, in the cell-water system ranged from 70.8 to 99.5%.Chloroplast particles were isolated from a "resistant" species, D. tertiolecta. Noncyclic electron flow, as measured by ferricyanide reduction, was inhibited by DDT and DDE, and could explain growth inhibition in other phytoplankters. Fifty percent inhibition occurred at 20 mum DDT. Sensitivity of phytoplankton to toxic hydrophobic chlorinated hydrocarbons may be dependent upon penetration of the molecules to active sites within membranes. PMID- 16657919 TI - Membrane transport of sugars in cell suspensions of sugarcane: I. Evidence for sites and specificity. AB - Sugar uptake by sugarcane cells in suspension culture was measured over short incubation time spans (5 seconds to 4 minutes), and membrane transport rates were calculated. A relatively high proportion of labeled products in cell extracts after incubation of cells with (14)C-glucose for 5 seconds was sugar phosphates (56%); fructose and sucrose began to appear after 15 and 30 seconds, respectively. Galactose and 3-O-methylglucose competed appreciably with glucose uptake, but ketohexoses and pentoses did not; there was no detectable uptake of sucrose. It is postulated that besides endogenous phosphorylation and further metabolism of glucose the configuration of the hydroxyl on the carbon-2 may be important for efficient membrane transport. The cells had a particularly high affinity for glucose and 3-O-methylglucose (Km = 15 and 16 mum, respectively). PMID- 16657920 TI - Increased Ethylene Production during Clinostat Experiments May Cause Leaf Epinasty. AB - Ethylene production from tomato (Lycopersicum esculentum L. cv. Rutgers) plants based on a clinostat doubled during the first 2 hours of rotation. Carbon dioxide blocked the appearance of leaf epinasty normally associated with plants rotated on a clinostat. These results support the idea that epinasty of clinostated plants was due to increased ethylene production and not to the cancellation of the gravitational pull on auxin transport in the petiole. PMID- 16657921 TI - Characteristics of the process of enzyme release from secretory plant cells. AB - Secretion-the outward movement of molecules across the plasmalemma-of alpha amylase by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers is an energy dependent process that is not directly dependent upon protein synthesis or RNA synthesis and does not appear to be under the direct control of gibberellic acid or abscisic acid. Release-the movement of the secreted alpha-amylase molecules through the walls into the surrounding medium-is apparently diffusion limited and is markedly dependent upon the presence of ions. PMID- 16657922 TI - Flowering and Growth Response of Peanut Plants (Arachis hypogaea L. var. Starr) at Two Levels of Relative Humidity. AB - Peanut plants (Arachis hypogaea L. var. Starr) grown under two different relative humidities were used in all experiments. All plants were germinated and grown to flowering in the greenhouse. At this time, one group was moved to a growth room under 95% relative humidity. After 50 days the humidity of the growth room was lowered to 50%. The second group was moved into a growth room at 50% relative humidity and after 50 days the humidity was raised to 95%.Flowering rates of plants under high humidity were greater than rates of those plants under low humidity. Flowering was stimulated by transfer from low to high humidity, and these plants set the largest percentage of pegs, maintained a high rate of ethylene production by 2-centimeter peg sections, a high growth rate of intact pegs, and they had a higher mean content of gibberellins than plants transferred from a high to a low humidity. The plants in the high to low transfer had the least number of flowers, formed the lowest percentage of pegs, had about 50% less ethylene production by 2-centimeter peg sections, and the peg growth rate declined about 50%.Maximum ethylene production occurred during initial stages of peg growth (1- to 5-millimeter sections), and gibberellin content was generally higher in these peg sections. Thus, high humidity enhanced flowering, peg formation, and peg growth rate. A causal relationship between these effects of high humidity and the growth regulator status of the developing fruit is indicated. PMID- 16657923 TI - Alternative fruiting pathways in phycomyces. AB - Developmental distinctions between giant and dwarf fruiting bodies of Phycomyces blakesleeanus (Burgeff) were studied by means of size measurements and growth analyses. Histograms of fruiting body lengths showed a bimodal distribution, with peaks around 0.3 millimeter (dwarfs) and 30 millimeters (giants). Individual cultures contain both giants and dwarfs. Differences between giants and dwarfs appear in the first phase of development; the apex of the giant is tapered, whereas the dwarf apex is dome-shaped. Probable cytological distinctions at this stage are cited in discussion. The dwarfs terminate enlargement upon expansion of the sporangium, thus lacking the subsequent phase of rapid elongation (stage IV) that contributes 90% of the length in the case of giants. It was concluded that P. blakesleeanus maintains two developmental patterns for asexual fruiting, with dwarfs and giants differing radically in growth regulation. PMID- 16657924 TI - Movement of C-Labeled Assimilates into Kernels of Zea mays L: I. Pattern and Rate of Sugar Movement. AB - Carbon-14, photosynthetically fixed in leaves of Zea mays L. and translocated to developing kernels, passed through specialized basal endosperm cells prior to movement into the starchy endosperm and embryo. Radioactivity migrated in the endosperm at a maximum rate of 2.7 millimeters per hour, and there was no difference in the rate of movement in kernels treated 14 to 30 days after pollination.Sucrose contained over three-fourths of the radioactivity in the kernal base (fruit stalk) 1 to 6 hours after (14)CO(2) treatment of the plant. Conversely, in the basal endosperm three-fourths of the radioactivity was in glucose and fructose. A high proportion of the radioactivity was retained in the monosaccharides of the starchy endosperm the first 3 hours after the (14)CO(2) treatment. With additional time after treatment there was a decline in the percentage of radioactivity in the monosaccharides and an increase in sucrose (14)C. From these data we suggest that translocated sucrose is cleaved to glucose and fructose during entry into the endosperm and that the monosaccharides diffuse throughout the endosperm. Once the sugars arrive in the cells active in starch synthesis, they are rapidly converted to sucrose which in turn is utilized in the synthesis of starch. PMID- 16657925 TI - Movement of C-Labeled Assimilates into Kernels of Zea mays L: II. Invertase Activity of the Pedicel and Placento-Chalazal Tissues. AB - Invertases of the placento-chalazal and pedicel tissues are much more active than invertase from the pericarp of Zea mays L. kernels 12 to 40 days after pollination. Sucrose synthetase was not detected in the pedicel or placento chalazal tissues. Sucrose content and percentage increased in the pedicel with advancing kernel age. Hexoses accounted for over half of the sugars extracted from the placento-chalazal tissues. These data are consistent with the hypothesis that sucrose translocated to the pedicel is hydrolyzed by acid invertase(s) prior to entry of sugar into the endosperm tissue. The placentochalazal tissue appears to be the primary site of sucrose inversion with the pedicel invertase contributing more or less to this process depending on kernel age. PMID- 16657926 TI - Subcellular Localization of Zinc and Calcium in Bean (Phaseolus vulgaris L.) Tissues. AB - Two bean (Phaseolus vulgaris L.) cultivars differing in growth responses to zinc were examined for differences in uptake and subcellular localization of (65)Zn during a 15-day growth period. The zinc-sensitive cultivar Sanilac showed initially a much higher rate of absorption, which declined after 24 hours. The zinc-tolerant cultivar Saginaw showed a slow but steady rate of absorption for 10 days. In roots as well as in stem callus tissues of both cultivars, three-fourths of the absorbed (65)Zn was localized in the "cytoplasmic" supernatant fractions (containing ribosomes and vacuolar sap). Very little (less than 7%) (65)Zn was localized in the cell wall fraction. There was a much greater proportion of the absorbed (65)Zn localized in root mitochondria and nuclei of the zinc-sensitive Sanilac than in the zinc-tolerant Saginaw. Stem callus tissues, however, did not show such cultivar differences in zinc accumulation at the sub-cellular level.Calcium-45 distribution in the cultivar Redkote showed preferential affinity for cell walls both in roots and stem cellus tissue. The percentage of absorbed (45)Ca associated with cell wall fractions of roots gradually increased with a corresponding decline in the percentage of radioactivity associated with the cytoplasmic fraction. At 15 days, 57% of the total (45)Ca in the roots was localized in the cell walls. A similar, albeit less pronounced, preferential (45)Ca sorption on cell walls occurred for the callus tissue. Calcium-45 also accumulated at later stages in the nuclear fraction, but there was no mitochondrial accumulation. PMID- 16657927 TI - Photocontrol of anthocyanin synthesis: I. Action of short, prolonged, and intermittent irradiations on the formation of anthocyanins in cabbage, mustard, and turnip seedlings. AB - Red far red reversibility (phytochrome control) of anthocyanin synthesis can be easily demonstrated for the response induced by short (5 minutes) and relatively short (4 hours) irradiation. Red far red reversibility of the response induced by longer irradiations can be demonstrated by the use of cyclic irradiations alternating short exposures to red and far red light.The level of anthocyanin formed during the dark incubation period following exposure to light depends upon the duration of the irradiation and becomes proportionally smaller as the length of the irradiation increases.Production of anthocyanins under cyclic irradiations depends upon the total energy applied and upon the length of the dark interval between successive irradiations.The relative efficiencies of radiations in various spectral ranges change with changes in the length of the irradiations. PMID- 16657928 TI - Photorespiratory phenomena in maize: oxygen uptake, isotope discrimination, and carbon dioxide efflux. AB - Concurrent O(2) evolution, O(2) uptake, and CO(2) uptake by illuminated maize (Zea mays) leaves were measured using (13)CO(2) and (18)O(2). Considerable O(2) uptake occurred during active photosynthesis. At CO(2) compensation, O(2) uptake increased. Associated with this increase was a decrease in O(2) release such that a stoichiometric exchange of O(2) occurred. The rate of O(2) exchange at CO(2) compensation was directly related to O(2) concentration in the atmosphere at least up to 8% (v/v).When illuminated maize leaves were exposed to saturating CO(2) concentrations containing approximately equal amounts of (12)CO(2) and (13)CO(2), the latter was taken up more rapidly, thus depressing the atom% (13)C in the atmosphere. Moreover, upon exposure to CO(2) containing 96 atom% (13)C, there occurred a directly measurable efflux of (12)CO(2) from the leaves for at least 15 minutes. During this period an equimolar evolution of (16)O(2) and uptake of (13)CO(2) was observed. Thereafter, although the rate of (16)O(2) evolution remained unchanged, the rate of (13)CO(2) uptake declined markedly, suggesting continual (13)C enrichment of the photorespiratory substrate.It is concluded that a finite photorespiratory process occurs in maize and that the CO(2) generated thereby is efficiently recycled. Recycling maintains the internal CO(2) concentration at a level difficult to detect by most photorespiratory assays. PMID- 16657929 TI - An Ascorbate-induced Absorbance Change in Chloroplasts from Violaxanthin De epoxidation. AB - A new ascorbate-induced chloroplast absorbance change which has the characteristics of a carotenoid shift is described. The absorbance change was light-dependent at pH 7 but not at pH 5. The difference spectra for the light and dark changes were similar, showing a large absorbance peak at 505 nanometers, smaller peaks near 468 and 437 nanometers, and a sharp valley around 483 nanometers. The absorbance change is assigned to violaxanthin de-epoxidation because various conditions affected the absorbance change and violaxanthin de epoxidation similarly, and the difference spectrum resembled the spectrum of zeaxanthin minus violaxanthin in organic solvent.Nigericin with KCl inhibited the light-dependent change at 505 nanometers. This effect, as well as the dark change at pH 5, indicated that de-epoxidation requires an acidic condition in chloroplasts. The effects of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, 2,6 dichlorophenolindophenol, and phenazine methosulfate indicated that the chloroplast acidification which mediates the 505 nanometers change is derived from hydrogen-ion transport linked to photosystem 1. Thus the 505 nanometers change could serve as an endogenous probe for chloroplast acidification and an indirect indicator of hydrogenion transport. At pH 5, the role of ascorbate appears to be to provide the reducing potential necessary for reductive de epoxidation of violaxanthin. At pH 7, ascorbate could have an additional effect of stimulating electron transport and hence the hydrogen-ion transport necessary for de-epoxidation.In contrast to leaves and algae, de-epoxidation in chloroplasts was irreversible under the conditions investigated. Under some conditions other absorbance changes which were apparently due to chlorophyll were superimposed on the de-epoxidation change. The relationship of these ascorbate induced changes to other absorbance changes observed in chloroplasts and green algae remains to be determined. PMID- 16657930 TI - Saturation Kinetics of the Velocity of Stomatal Closing in Response to CO(2). AB - Stomatal closing movements in response to changes from CO(2)-free to CO(2) containing air were recorded in leaf sections of Zea mays using air flow porometers. The response to CO(2) was fast; the shortest lag between the application of 300 microliters CO(2) per liter of air and the beginning of a stomatal response was 3 seconds. The velocity of stomatal closing increased with CO(2) concentration and approached its maximal value between 10(3) and 10(4) microliters CO(2) per liter of air. The CO(2) concentration at which the closing velocity reached half its maximal value was approximately 200 microliters CO(2) per liter of air, both in the light and in darkness. This indicates that the mechanism of stomatal responses to CO(2) is the same in both light regimes and that the range of stomatal sensitivity to changes in CO(2) concentration coincides with the range of CO(2) concentrations known to occur in the intercellular spaces of illuminated leaves. PMID- 16657931 TI - Action Spectrum of the Photoinduced Sexual Stage in the Fungus Nectria haematococca Berk. and Br. var cucurbitae (Snyder and Hansen) Dingley. AB - An action spectrum was determined for the photoinduced formation of perithecia in a homothallic strain of Nectria haematococca Berk. and Br. var. cucurbitae (Snyder and Hansen) Dingley. Dose-response curves for perithecial formation were obtained from 340 to 510 nanometers at 10-nanometer intervals. Radiation longer than 510 nanometers was not effective for inducing perithecial formation. The action spectrum indicated peaks of activity near 360, 440, and 480 nanometers with shoulders near 420 and 460 nanometers. Minima occurred near 350 nanometers, 390 to 410 nanometers, and 470 nanometers. The general shape of this action spectrum appears to be similar to those obtained for many different near ultraviolet-blue-sensitive organisms in which a flavoprotein molecule was postulated to be the photoreceptor. PMID- 16657932 TI - Photosynthetic Activities of the Halophilic Alga Dunaliella parva. AB - Dunaliella parva, a unicellular halophilic alga, was found to evolve oxygen photosynthetically only in the presence of a high osmolar concentration. Cell free preparations were obtained by placing the cells in a medium of low osmolarity. The fragments obtained showed a high photoreducing and photophosphorylating activity except for their inability to catalyze all ferredoxin dependent photoreactions. Placing the cells in a medium of intermediate osmolarity produced a "chloroplast" preparation which maintained some capacity for O(2) evolution and CO(2) fixation, while possessing the ability to catalyze the photoinduced reduction of ferricyanide. Enzymic and photosynthetic reactions of cell-free preparations from D. parva were inhibited, rather than stimulated, by the salt concentration optimal for growth. These results were interpreted as indicating the existence of a steep NaCl gradient in vivo between the medium and the cell compartments which are not permeable to salt. PMID- 16657933 TI - Is nicotinamide adenine dinucleotide phosphate an obligatory intermediate in photosynthesis? AB - The site of action of the inhibitors disalicylidenepropanediamine and pyrophosphate was more closely defined as acting on ferredoxin. Three inhibitors which act on the electron transport path between ferredoxin and NADP: disalicylidenepropanediamine, pyrophosphate, and phosphoadenosinediphosphate ribose, had no effect on photosynthesis in cell free preparations of Dunaliela parva at concentrations which completely inhibited the enzymic activity on which each inhibitor acts. The addition of disalicylidenepropanediamine to dark-grown Euglena gracilis cells prevented the light-induced formation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, but not of photosynthesis, chlorophyll synthesis, or NAD-dependent glyceraldehyde-3-phosphate dehydrogenase.The above results are interpreted as indicating that, at least under some conditions, a reduced product of photosystem I preceding ferredoxin in the electron transport path can serve as the reductant of CO(2) in photosynthesis. PMID- 16657934 TI - The problem of reduced nicotinamide adenine dinucleotide oxidation in glyoxysomes. AB - NADH is generated in glyoxysomes both in the glyoxylate cycle and in beta oxidation. No system has yet been described which would oxidize NADH in these organelles. A series of oxidants which might function by coupling NADH oxidation to O(2) through endogenous carriers in the glyoxysomes was examined. Oxidation was brought about by ferricyanide or dichlorophenol-indophenol, but it was shown that this "diaphorase" activity is probably a contaminant. Hydroxypyruvate reductase (NAD-linked) is present in the glyoxysomes, and at very high substrate concentrations (>10 mm) this enzyme can also transfer electrons from NADH to glyoxylate. However, it is most unlikely that this concentration of glyoxylate is ever approached in glyoxysomes, where the malate synthetase would compete on much superior terms. The maximum rates of NADH oxidation observed in the presence of ferricyanide or glyoxylate are only a fraction of those required to reoxidize NADH at the rate occurring in vivo. PMID- 16657935 TI - Effects of ethylene on potato tuber respiration. AB - Treatment of potato tubers (Solanum tuberosum L.) with ethylene gas causes a rapid rise in their respiration rate, reaching 5 to 10 times the rate of untreated tubers over 30 hours of treatment and then falling slowly. The response shows a lag of 8 hours, and more than 24 hours of exposure is required for maximum effect; the temperature optimum is near 25 C. In sensitivity to low concentrations and dependence on temperature, the phenomenon is similar to the effect of ethylene on the respiration of climacteric and nonclimacteric fruits. Treated potato tubers returned to air recover their sensitivity to ethylene more slowly than do nonclimacteric fruits (e.g., mature green oranges). It is proposed that the respiratory rise characteristic of ripening in climacteric fruits and of the wound response in plant tissues is induced by a rise in endogenous tissue ethylene. PMID- 16657936 TI - Salt responses of enzymes from species differing in salt tolerance. AB - Enzymes which are affected by the addition of inorganic salts during in vitro assay were extracted from salt-sensitive Phaseolus vulgaris, salt-tolerant Atriplex spongiosa, and Salicornia australis and tested for sensitivity to NaCl. In each case malate dehydrogenase, aspartate transaminase, glucose 6-phosphate dehydrogenase, and isocitrate dehydrogenase showed NaCl responses similar to those found for commercially available crystalline enzymes from other organisms. Enzymes extracted from plants grown in saline cultures showed no important changes in specific activity or salt sensitivity. Interaction of pH optima and NaCl concentrations suggests that enzymes may differ in the way they respond to salt treatment. PMID- 16657937 TI - Salt responses of carboxylation enzymes from species differing in salt tolerance. AB - This paper reports effects of salts on in vitro activity of phosphoenolpyruvate carboxylase and ribulose-1,5-diphosphate carboxylase, isolated from species differing in salt tolerance.Inhibition of phosphoenolpyruvate carboxylase by the inorganic salts KCl, NaCl, and Na(4)SO(4) depended on the source of the enzyme. Phosphoenolpyruvate carboxylase isolated from leaves of C(4) plants was extremely sensitive to inorganic salts, whereas the enzyme extracted from roots of C(4) plants or from both shoots and roots of C(3) plants was much less sensitive. Ribulose-1,5-diphosphate carboxylase was less salt-sensitive than the phosphoenolpyruvate carboxylases. Differences in salt sensitivity of carboxylases were observed over a wide pH range. The results suggest substantial physical chemical differences between phosphoenolpyruvate carboxylases functioning in photosynthesis and in CO(2) dark fixation.Among C(4) species, phosphoenolpyruvate carboxylase from halophytic species was more salt-sensitive than that from a salt sensitive species. This anomaly, between in vitro response of enzymes and growth response of the plants, is briefly discussed. PMID- 16657938 TI - Cytochrome Oxidase Activity in Cell-free Preparations from Blue-Green Algae. PMID- 16657939 TI - Photophosphorylation by mesophyll and bundle sheath chloroplasts of c(4) plants. PMID- 16657940 TI - The Isolation of Proplastids from Roots of Vicia faba. PMID- 16657941 TI - Microinjection of phycomyces: selection of a strain for possible biological assay of photoreceptor pigment. PMID- 16657942 TI - No stomatal response to ethylene. PMID- 16657943 TI - Solubility of beta-Glucosidase in Homogenates of Sweetclover Leaves and Bean Hypocotyls. PMID- 16657944 TI - Dark germination and seedling growth in monocots and dicots of different photosynthetic efficiencies in 2% and 20.9% o(2). PMID- 16657945 TI - Metabolism of 2-C-(+/-)-abscisic Acid in excised bean axes. AB - Excised embryonic bean axes (Phaseolus vulgaris, var. White Marrowfat) rapidly metabolize 2-(14)C-(+/-)-abscisic acid to two compounds, M-1 and M-2, which have very low growth-inhibitory activity. Chemical tests indicate the M-1 and M-2 are not previously described abscisic acid metabolites. M-2 accumulates in the axes and evidence is presented for the hypothesis that abscisic acid --> M-1 --> M-2. Zeatin, which partially reverses the abscisic acid-mediated growth inhibition of axes, neither decreases abscisic acid uptake nor causes any major changes in its metabolism. It was observed that axes transferred from abscisic acid-containing solutions to buffer resume control rates of fresh weight increase while still containing considerable quantities of abscisic acid. PMID- 16657946 TI - Activity and metabolism of C-(+/-)-abscisic Acid derivatives. AB - Several radioactive analogues of abscisic acid have been tested for their growth inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-(14)C abscisic acid and the cis- and trans-1',4'-diols of 2-(14)C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar. PMID- 16657947 TI - A Cell Wall-degrading Endopolygalacturonase Secreted by Colletotrichum lindemuthianum. AB - Cultures of Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner have been induced to secrete an endopolygalacturonase (polygalacturonide glycanohydrolase EC3.2. 1.15). This enzyme has been brought to a high state of purity by ion exchange, gel filtration, and agarose affinity chromatography. The enzyme has optimal activity at pH 5, has an apparent molecular weight as determined by gel filtration of about 70,000, and prefers polygalacturonic acid to pectin as its substrate. The enzyme, while hydrolyzing only 1% of the glycosidic bonds, reduces the viscosity of a polygalacturonic solution by 50%. Nevertheless, the initial as well as the final products of polygalacturonic acid hydrolysis are predominantly tri- and digalacturonic acid and, to a lesser extent, monogalacturonic acid. The purified enzyme catalyzes the removal of about 80% of the galacturonic acid residues of cell walls isolated from suspension cultured sycamore cells (Acer pseudoplatanus) as well as from the walls isolated from 8-day-old Red Kidney bean (Phaseolus vulgaris) hypocotyls. PMID- 16657948 TI - Light Activation of Mg-dependent Adenosine 5'-Triphosphatase in Isolated Euglena Chloroplasts. AB - Enhancement of Mg(2+)-dependent ATPase activity in Euglena gracilis chloroplasts by light in the presence of a sulfhydryl compound has been demonstrated. A number of uncouplers and energy transfer inhibitors were studied for their effects on the light enhancement of ATPase activity simultaneously with their effects on photophosphorylation. Results suggest that the light-enhanced ATPase activity in Euglena chloroplasts is an energy-initiated process and that the energy is supplied through electron flow upon illumination of the chloroplasts. However, by studying the difference in their response toward the various uncouplers and inhibitors, it seems that the two processes (photohydrolysis of ATP and photophosphorylation) share only the latter part of their energy-transferring pathway. PMID- 16657949 TI - Anion absorption by etiolated wheat leaves after vacuum infiltration. AB - Chloride and phosphate uptake by leaf segments of green and etiolated 7-day-old seedlings of Triticum aestivum L. cultivar Capelle is enhanced by light. In the range from 1.0 to 10 millimolar KCl, maximal rates of uptake were obtained with 1.5-millimeter segments. Above 1.5 millimeters, ion diffusion through the cut edge was the rate-limiting factor, uptake being proportional to the amount of cut edge, but vacuum infiltration of the tissue overcame this limitation, allowing uptake to be independent of segment length. No deleterious effects of vacuum infiltration were observed, and it is suggested that the technique offers a convenient alternative to existing methods in the study of foliar absorption. PMID- 16657950 TI - Cyclopropane Fatty acids in relation to earliness in spring and drought tolerance in plants. AB - Long chain cyclopropane fatty acids were observed in the sulfolipid fraction extracted from leaves of the early spring plants Galanthus nivalis L. and Anthriscus silvestris L. (Hoffm.). The content of cyclopropane fatty acids with 25 carbon atoms appeared to be clearly correlated with earliness in spring, and it ranged from 68% (G. nivalis L., snow drop) to 0.5% (wheat). Several long chain cyclopropane fatty acids were found in the drought-tolerant Corynephorus canescens (L.) P.B., exclusively in the phosphatidyl choline fraction. PMID- 16657951 TI - Modification of the cytokinin promotion of deoxyisoflavone synthesis in soybean tissue. AB - Soybean tissue incubated in liquid media formed daidzin in response to cytokinin when the media contained 0.1 m sucrose but formed another, unidentified compound when the media contained 0.6 m sucrose or mannitol. The cytokinin effect in either setup was detectable only after a lag period of several hours. Addition of possible precursors of the compounds being synthesized or of related compounds did not alter the lag period to any great extent. Trans-cinnamic acid did perhaps shorten it a little as did preincubation of the tissue in the basal medium before the addition of kinetin. Several inhibitors of RNA synthesis substantially reduced the production of daidzin and the other compound. Protein synthesis inhibitors were also effective except that cycloheximide and puromycin actually promoted synthesis of the unknown compound while inhibiting that of daidzin. The results do not give a clear impression as to how the cytokinins are involved in the synthesis of the compounds, but are at least suggestive that cytokinin is involved in RNA or protein synthesis. PMID- 16657952 TI - The Respiratory Chain of Plant Mitochondria: XII. Some Aspects of the Energy linked Reverse Electron Transport from the Cytochromes c to the Cytochromes b in Mung Bean Mitochondria. AB - The cytochromes c of mung bean (Phaseolus aureus) mitochondria become reduced when sulfide, a cytochrome oxidase inhibitor free from uncoupling side effects, is added to the aerobic mitochondrial suspension in the absence of added substrate. The cytochromes b remain largely oxidized. Subsequent addition of ATP results in partial oxidation of the cytochromes c and partial reduction of the cytochromes b due to ATP-driven reverse electron transport through the second site of energy conservation, or coupling site, of the respiratory chain. Cytochrome a is also oxidized under these conditions, but there is no concomitant reduction of the flavoprotein components, of ubiquinone, or of endogenous pyridine nucleotide. The reaction is abolished by oligomycin. The reducing equivalents transported from the cytochromes c and a in ATP-driven reverse electron transport are about 2-fold greater than those which appear in the cytochromes b. It is suggested that the equivalents not accounted for are present in a coupling site enzyme at the second site of energy conservation which interacts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbance changes with redox state over the wavelength range examined. With succinate present, reverse electron transport can be demonstrated at both coupling sites in both the aerobic steady state and in anaerobiosis. ATP-driven reverse electron transport in anaerobiosis maintains cytochrome a 30% oxidized while endogenous pyridine nucleotide is 50% reduced.When mung bean mitochondria, oxidizing succinate in the presence of sulfide through the alternate, cyanide- and sulfide-insensitive terminal oxidase, become anaerobic, cytochrome b(557), which has remained largely oxidized, becomes slowly reduced. The slow reduction is observed in coupled, energized mitochondria and in uncoupled mitochondria; the time course parallels the reduction of cytochrome a(3) under the same conditions. It appears that sulfide-liganded, oxidized cytochrome a(3) may be in close enough proximity to cytochrome b(557) in the membrane to inhibit the reduction of the latter. PMID- 16657953 TI - Tannins as gibberellin antagonists. AB - Fourteen chemically defined hydrolyzable tannins and six impure mixtures of either condensed or hydrolyzable tannins were found to inhibit the gibberellin induced growth of light-grown dwarf pea seedlings. The highest ratio of tannins to gibberellic acid tested (1000: 1 by weight) inhibited from 80 to 95% of the induced growth for all tannins tested except for two monogalloyl glucose tannins which inhibited only 50% of the induced growth. The lowest ratio tested (10: 1) inhibited the induced growth by less than 25% except for the case of terchebin where 50% inhibition was found. The inhibition of gibberellin-induced growth was found to be completely reversed by increasing the amount of gibberellin in three cases tested. Tannins alone did not inhibit endogenous growth of either dwarf or nondwarf pea seedlings. Eight compounds related to tannins, including coumarin, trans-cinnamic acid, and a number of phenolic compounds were also tested as gibberellin antagonists. Most of these compounds showed some inhibition of gibberellin-induced growth, but less than that of the tannins. At the highest ratio (1000: 1) the greatest inhibition was 55%; at the lowest ratio (10: 1) no more than 17% was observed. These compounds did not inhibit endogenous growth, and the inhibition of gibberellin-induced growth could be reversed by increasing the amount of gibberellin in two cases tested.Six chemically defined tannins were found to inhibit hypocotyl growth induced by gibberellic acid in cucumber seedlings. Growth induced by indoleacetic acid in the same test was not inhibited. The highest ratio of tannin to promotor tested gave strong inhibition of gibberellic acid-induced growth, but actually enhanced the growth induced by indoleacetic acid. This difference in action suggests a specificity between the tannins and gibberellic acid. PMID- 16657954 TI - Studies with deoxyribonucleic Acid from blue-green algae. AB - DNA from two blue-green algae was isolated and characterized. The buoyant densities, thermal denaturation and renaturation, thermal melting values, base compositions, sedimentation coefficients, and molecular weights were determined. Blue-green algal DNA renatured extensively and at a comparable rate to that of bacterial DNA. The similarities among the kinds of DNA from bacteria and blue green algae were interpreted to reflect a close relationship. PMID- 16657955 TI - A method for testing the specificity of inhibitors of deoxyribonucleic Acid synthesis in growth studies. PMID- 16657956 TI - An Action Spectrum for Light-induced Primordium Formation in a Basidiomycete, Favolus arcularius (Fr) Ames. AB - The action spectrum for the initiation of fruiting (primordium formation) in Favolus arcularius was determined on the equal response basis. The detectable effect of light was observed in the region between 350 to 560 nanometers, showing six distinct peaks at 374, 398, 424, 446, 480, and 514 nanometers. The half maximum response is reached with 1.8 x 10(8) ergs per cm(2) at the most effective wavelength, 398 nanometers. Since the inhibitors, diphenylamine and quinacrine, had no consistent effect on the primordium formation, it is suggested that the possible photoreceptor pigment(s) may be neither carotenoid nor flavinoid.Comparing the action spectrum with those for some other fungi, the possibility that the photoreceptor system of this fungus may consist of two pigments is discussed. PMID- 16657957 TI - Gibberellin Substitution for the Requirement of the Cotyledons in Stem Elongation in Pisum sativum Seedlings. AB - The removal of the cotyledons from 8-day-old light-grown Pisum sativum cv. Alaska seedlings caused a reduction in the rate of stem elongation to 50% of the intact control value. Gibberellic acid restored the stem elongation rate of decotylized plants to the level of the intact controls. The effect of decotylization was to lower both the rate of node formation and the rate of internode elongation. The steady state rate of internode elongation was reduced to 50% of the control rate by decotylization. Applied gibberellic acid did not restore the normal rate of node formation nor the lag in internode elongation caused by decotylization, but gibberellic acid did restore the normal steady state rate of internode elongation. Analysis of variance demonstrated an interaction between the cotyledons and applied gibberellic acid. 2-Isopropyl-4-dimethylamino-5-methyl phenyl-1-piperidine carboxylate methyl chloride inhibited internode elongation to the same extent in both intact and decotylized plants. The results indicate that the cotyledons are an effective source of gibberellin for the young pea seedling. PMID- 16657958 TI - Stomatal conductance of differentially salinized plants. AB - Stomatal resistance was measured daily with a stomatal diffusion porometer during a 4-week period in leaves of bean (Phaseolus vulgaris L., var. Bush Blue Lake) and barley (Hordeum vulgare L., var. Liberty) plants having roots equally split between two differentially salinized nutrient solutions. The stomatal conductance (reciprocal of stomatal resistance) of plants with half their roots in saline solutions was intermediate between the stomatal conductances of plants grown in nonsaline solutions and those grown in saline solutions. PMID- 16657959 TI - Hormonal control of polyribosome formation in barley aleurone layers. AB - The addition of abscisic acid to barley (Hordeum vulgare L. cv. Himalaya) aleurone layers at the same time as gibberellic acid completely prevents the gibberellin-induced increases in the percentage of polysomes, the formation of polyribosomes, and the synthesis of alpha-amylase, even when the molar concentration of gibberellic acid is four times greater than the concentration of abscisic acid. The addition of abscisic acid to aleurone cells producing alpha amylase (midcourse addition) inhibits the further synthesis of alpha-amylase and decreases the percentage of polysomes but does not change the number of ribosomes per cell.The removal of gibberellic acid from aleurone layers during the midcourse of alpha-amylase production arrests alpha-amylase synthesis and decreases the percentage of polysomes. Readdition of gibberellic acid causes the reinitiation of the synthesis of alpha-amylase and a return of the percentage of polysomes to the original level.The incubation of aleurone layers with 5 fluorouracil inhibits the secretion of alpha-amylase. The changes in polysomes isolated from cells treated with either fluorouracil or actinomycin D correlate with the changes in enzyme synthesis caused by the addition of these inhibitors.Gibberellic acid and abscisic acid affect both the conversion of monosomes to polysomes and the synthesis of new ribosomes. The gibberellin stimulated increases in the number of ribosomes and the percentage of polysomes are probably a prerequisite for the hormone induction of enzyme synthesis. PMID- 16657960 TI - Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria. AB - Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation. PMID- 16657961 TI - Changes in Cell Membrane Permeability in Sunflower Hypocotyls Infected with Sclerotinia sclerotiorum. AB - Influx and efflux of water and urea and electrolyte leakage are less for sunflower (Helianthus annuus) hypocotyl sections above lesions caused by Sclerotinia sclerotiorum than for those from healthy plants. Urea uptake by sections above lesions is reduced (celery, squash, and tomato) or unchanged (bean) in other hosts after Sclerotinia infection. Efflux of urea from sunflower hypocotyls is biphasic, suggesting diffusion in series from two cellular compartments (cytoplasm and vacuole). Efflux during the fast phase was 7 to 20 times greater than that during the slow phase. No difference was noted in urea efflux from healthy and diseased tissues during the slow phase. However, efflux during the fast phase from diseased tissues was slower than from healthy tissues, suggesting that the increased resistance to diffusion of urea in host cells above lesions resides in the plasmalemma. Water movement across cell membranes of healthy and diseased sunflower hypocotyls was reduced when tissues were treated with p-hydroxymercuribenzoate. PMID- 16657962 TI - Adenosine triphosphatase of bean plastids: its properties and site of formation. AB - Extracts of bean (Phaseolus vulgaris L.) etioplasts and chloroplasts contain a dithiothreitol-activated Ca(2+)-dependent adenosine triphosphatase which is inhibited by Dio-9. The chloroplast and etioplast enzymes have identical R(F) values upon disc gel electrophoresis. Optimum extraction of the enzyme from either plastid preparation is accomplished with 1 mm ethylenediamine tetraacetic acid. Photophosphorylation capacity can be partially restored to depleted chloroplast preparations by addition of either the chloroplast or etioplast extract. These results suggest that the adenosine triphosphatase from etioplasts and chloroplasts represents a modified coupling factor for photophosphorylation.The specific activity of the adenosine triphosphatase in the extracts of plastids increases upon greening of etiolated plants due to protein synthesis. This light-induced increase is inhibited by both chloramphenicol and cycloheximide, specific inhibitors of chloroplastic and cytoplasmic protein synthesis. There is no accumulation of adenosine triphosphatase in postribosomal supernatants of cycloheximide or chloramphenicol treated leaves. The results indicate that both the chloroplastic and the cytoplasmic ribosomal systems are required for the formation of the chloroplast adenosine triphosphatase. PMID- 16657963 TI - Generation and suppression of microsomal ribonuclease activity after treatments with auxin and cytokinin. AB - RNase activity was assayed in subcellular fractions of apical regions of Pisum sativum L. var. Alaska epicotyls after seedling decapitation and treatments with various growth regulators. High concentrations of applied indoleacetic acid caused a marked increase to occur in the RNase activity level associated with "heavy" microsomes, e.g., a 20-fold rise per unit RNA or protein in 3 days. This rise could be abolished by treating with the cytokinin benzyladenine along with indoleacetic acid. Nevertheless, indoleacetic acid and benzyladenine acted synergistically in their abilities to evoke swelling and net synthesis of RNA and protein. Polysomal profiles prepared after treatment with indoleacetic acid plus benzyladenine showed less degradation than profiles from any other treatment. It is concluded that auxin generates and cytokinin suppresses the activity of a particular membrane-bound RNase which can control turnover of the auxin-evoked polysomes required for growth in peas. Synergism between the two hormones in this system may be explained by the action of one to increase RNA synthesis and the other to decrease RNA destruction. PMID- 16657964 TI - Inhibition of chloroplast reactions with phenylmercuric acetate. AB - Phenylmercuric acetate is a selective inhibitor of the photosynthetic activities of isolated spinach (Spinacia oleracea) chloroplasts. At 5 mum concentration of phenylmercuric acetate, photophosphorylation is inhibited. At 33 mum phenylmercuric acetate, ferredoxin is inactivated. Ferredoxin-NADP oxidoreductase is 50% inhibited at 100 mum phenylmercuric acetate. Photosystem II reactions are 50% inhibited at 150 mum phenylmercuric acetate and very much higher cooncentrations-500 mum-are needed to approach complete inhibition. Phenylmercuric acetate inhibition of photosystem II appears to be selective, blocking a site between the 3-(3,4-dichlorophenyl)-1,1-dimethyl urea sensitive site and the site inactivated by high concentrations of tris buffer. PMID- 16657965 TI - Studies on Effect of Certain Quinones: I. Electron Transport, Photophosphorylation, and CO(2) Fixation in Isolated Chloroplasts. AB - The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro 1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.CO(2) fixation was inhibited by all the quinones to varying degrees. In chloroplasts treated with 06K-quinone or benzoquinone, CO(2) fixation was inhibited to a greater extent than the photoreduction of ferricyanide or ATP formation, indicating the possibility that the two quinones may also inhibit certain reactions in the carbon reduction cycle. The effect of dichlone and chloranil, but not of 06K-quinone, was overcome by the addition of reduced glutathione. The quinones caused an increase in the proportion of (14)C incorporated into 3-phosphoglyceric acid and a reduction in the amount of glycolic acid. PMID- 16657966 TI - Studies on Effects of Certain Quinones: II. Photosynthetic Incorporation of CO(2) by Chlorella. AB - The effects of various quinone herbicides and fungicides on the photosynthetic (14)CO(2) fixation and the incorporation of (14)C among the products of photosynthesis in Chlorella pyrenoidosa was investigated. Addition of 30 mum 2,3 dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K quinone), or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited CO(2) fixation, whereas 1,4-benzoquinone had no effect. Treatment with 3 mum or higher concentrations of dichlone, 06K-quinone or 1,4-benzoquinone also produced marked changes in the pattern of (14)C distribution. A noticeable effect was an increase in the proportion of (14)C in sucrose and glycine accompanied by a reduction in (14)C lipids and glutamic acid. These changes appear to occur as a result of shifts in the flow of carbon along various biosynthetic pathways of photosynthetic CO(2) fixation. It is suggested that inactivation of coenzyme A and shortage of reduced triphosphopyridine nucleotide in the quinone-treated cells inhibited the synthesis of lipids and glutamic acid, thereby diverting more carbon into sucrose and glycine. PMID- 16657967 TI - Development of chlorophyll and hill activity. AB - A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding delta-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with delta aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.We conclude that the development of photosystem II is limited by the synthesis of proteins in both the cytoplasm and the plastid, not by chlorophyll synthesis. Prolonged illumination is necessary for the development of oxygen evolution. PMID- 16657968 TI - Chloroplast and Cytoplasmic Enzymes: IV. Pea Leaf Fructose 1,6-Diphosphate Aldolases. AB - Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 muM for the chloroplast, 21 muM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 muM for the chloroplast enzyme, 18 muM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf. PMID- 16657969 TI - The levels of soluble nucleotides in wheat aleurone tissue. AB - The content of soluble nucleotides in aleurone layers isolated from mature wheat (Triticum aestivum var. Olympic) grain was investigated. The most abundant nucleotides were adenosine triphosphate, uridine triphosphate, and uridine diphosphoglucose. Smaller amounts of guanosine triphosphate, cytidine triphosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide were also identified. The levels of some of these nucleotides were increased after incubation of the tissue under certain conditions.Nucleotide levels were measured at intervals during incubation of aleurone layers in water. The changes observed are discussed in relation to a response by the tissue to wounding. PMID- 16657970 TI - The metabolism of soluble nucleotides in wheat aleurone layers treated with gibberellic Acid. AB - The metabolism of soluble nucleotides was investigated in wheat (Triticum aestivum var. Olympic) aleurone layers treated with gibberellic acid. Whereas nucleotide levels were relatively unaffected by the treatment, a transient increase was observed in the incorporation of (32)P. The effect was maximal 30 to 60 minutes after gibberellic acid was administered, and by 180 minutes incorporation was lower than in the control. The greatest changes were detected in the nucleoside triphosphates, particularly in cytidine triphosphate. The findings are discussed in relation to the mode of action of gibberellic acid. PMID- 16657971 TI - Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts. AB - Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO(2) in the chloroplasts, nor does it stimulate CO(2) fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO(2) fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen. PMID- 16657972 TI - Inhibition of Sterol Biosynthesis by 2-Isopropyl-4-dimethylamino-5-methylphenyl-1 piperidine Carboxylate Methyl Chloride in Tobacco and Rat Liver Preparations. AB - 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride, 90%, applied to rootless tobacco (Nicotiana tabacum cv. Samson) seedlings inhibits the incorporation of (14)C-mevalonate into sterols. Concomitantly, the retardant causes the accumulation of squalene-2,3-epoxide, an intermediate in sterol biosynthesis. The results with tobacco are identical to those produced by the retardant in cell-free rat liver preparations. PMID- 16657973 TI - Four universal forms of chlorophyll a. AB - We have matched the red absorption band measured at -196 C in a variety of chloroplast preparations with four major component curves representing forms of chlorophyll a having peaks at 661.6, 669.6, 677.1, and 683.7 nanometers. Chloroplast fractions enriched in one or the other of the two photochemical systems both contain these four major components, but system 1 preparations contain relatively more chlorophyll a 684. Chlorophyll a 677 and chlorophyll a 684 have greater bandwidths in system 1. Bands at longer wavelengths near 693 and 704 nanometers also often occur, but with far smaller heights than the above major bands. The longer wavelength bands are more common in system 1 than in system 2. In system 1 the half-widths of the four major bands in typical spectra average 11.3, 10.0, 10.3, and 10.8 nanometers while in system 2 they are 11.6, 9.8, 9.4, and 9.6 nanometers. Some spectra with sharper and some with wider bands were found, but the wavelengths were identical. PMID- 16657974 TI - Induction and development of increased ion absorption in corn root tissue. AB - Washing excised or intact primary roots of corn (Zea mays L., WF9 x M14) in aerated distilled water or dilute salt solutions for 2 hours induced doubling of the rate of accumulation of various nutrient ions and solutes. This response to washing depended upon aerobic metabolism, but involved no increase in aerobic respiration. Excision of root tissue was not required as the effect could be obtained with intact root systems. Increased phosphate absorption followed after a lag period of 30 to 40 minutes and continued for 6 hours before leveling off at about 3.5 times the initial rate. Chloramphenicol was not inhibitory to the development of increased absorption, while inhibitors of RNA and protein synthesis were. Auxins and kinetin were also inhibitory, but so was the antiauxin, 2,4,6-trichlorophenoxyacetic acid.Kinetic analyses of phosphate and potassium ((86)Rb) absorption showed no change in apparent Michaelis constant. Electron microscopic examination and analysis for membrane protein and lipid phosphate content after washing showed no proliferation of membrane or other changes in cell structure and composition. It appears that washing augments or activates existing membrane transport mechanisms by processes involving protein synthesis. PMID- 16657975 TI - Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue. AB - Washing of excised corn (Zea mays L., variety WF9xM14) root tissue is accompanied by an increase in (Mg(2+) + K(+))-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed. PMID- 16657976 TI - Does Gibberellic Acid Stimulate Seed Germination via Amylase Synthesis? PMID- 16657977 TI - Temperature and Sulfur Nutrition Effects on CO(2) Compensation Values in Barley, Peas, and Rape. PMID- 16657978 TI - De Novo Synthesis of Ribonuclease and beta-1,3-Glucanase by Aleurone Cells of Barley. PMID- 16657979 TI - Comparison of in vitro and in vivo assays for nitrate reductase in soybean leaves. PMID- 16657980 TI - Membrane electrical potentials in the cortex and stele of corn roots. PMID- 16657981 TI - Involvement of Acetylcholine in Phytochrome-mediated Processes. PMID- 16657982 TI - 3-(3,4-Dichlorophenyl)-1,1-dimethylurea Effect on Cytochrome b(559) Photooxidation and Q Reduction at Temperatures Near 0 C. PMID- 16657983 TI - Sterols and Chloroplast Structure of Cyanidium caldarium. PMID- 16657984 TI - Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays. AB - The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.Chloroplast fragments isolated from bundle sheath cells showed a light-dependent reduction of potassium ferricyanide, 2, 6 dichlorophenolindophenol, mammalian cytochrome c, plastocyanin, and Euglena cytochrome c(552). These activities were inhibited by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea at 1.25 micromolar. However, the photoreduction of NADP from water was extremely low or absent, except in chloroplasts from very young leaves, and the capacity for NADP reduction appeared to be related to the degree of grana formation.Photosystem I activity was present in bundle sheath chloroplast preparations at all stages of leaf growth and senescence examined. However, the activity was lower than in isolated mesophyll chloroplasts. NADPH diaphorase activity was comparable in both types of chloroplast.Chloroplasts isolated from bundle sheath cells of plants grown under a variety of conditions, including continuous and intermittent light, high and low light intensities, and high temperature, exhibited photosystem II activity. PMID- 16657985 TI - Photoreduction and Oxidation of Cytochrome f in Bundle Sheath Cells of Maize. AB - The photo-oxidation of cytochrome f (cytochrome c(554)) in bundle sheath cells isolated from leaves of maize (Zea mays var. DS 606A) has been compared with that in intact maize leaf and in isolated pea leaf cells (Pisum sativum L.). In all cases, illumination with red light caused a negative absorbance change at 554 nm which was attributed to the oxidation of cytochrome f. The extent of this change was greater using monochromatic red light at wavelengths above 700 nm compared with wavelengths below 700 nm. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea abolished this difference in bundle sheath cells. After illumination for 1 minute or longer in bundle sheath cells, reduction of cytochrome f in the dark was rapid only if the wavelength of the illuminating light was below 700 nm. In the presence of 3 (3,4-dichlorophenyl)-1, 1-dimethlyurea, reduction was slow after illumination at all wavelengths.Cytochrome f photo-oxidation was also followed in cells of a mutant of Chlamydomonas reinhardi, ac-21, which has isolated chloroplasts that exhibit photochemical reactions similar to those shown by isolated bundle sheath chloroplasts. No evidence was obtained for photoreduction of cytochrome f in the mutant.It was concluded that in the chloroplast of the intact bundle sheath cell of maize there is electron flow between photosystem II and cytochrome f resulting in photoreduction of the cytochrome. PMID- 16657986 TI - Nicotinamide adenine dinucleotide phosphate photoreduction from water by agranal chloroplasts isolated from bundle sheath cells of maize. AB - Photoreduction of NADP from water in agranal chloroplasts isolated from the leaf bundle sheath cells of Zea mays (var. DS 606A) or Sorghum bicolor (var. Texas 610) was dependent upon addition of plastocyanin as well as ferredoxin. Activity was further increased by the addition of ferredoxin NADP-reductase. Saturation for plastocyanin was reached at about 6 micromolar. In contrast, grana-containing chloroplasts isolated from leaf mesophyll cells of these plants or from pea (Pisum sativum L.) leaves did not require either plastocyanin or ferredoxin NADP reductase for NADP photoreduction from water, although with some preparations plastocyanin stimulated the activity.Photosystem I activity, which was low in washed preparations of bundle sheath chloroplasts, was also stimulated by plastocyanin. The effect of plastocyanin on photosystem I activity in the grana containing chloroplasts was similar to that on NADP photoreduction from water.In the presence of plastocyanin, the rates of NADP photoreduction from water were about the same in the agranal and granal chloroplasts, but photosystem I activity was considerably higher in bundle sheath chloroplasts. In these chloroplasts photosystem II appeared to limit the rate of NADP photoreduction.The results indicated that the agranal bundle sheath chloroplasts reduced plastocyanin via photosystem II and oxidized it via photosystem I. Both types of maize chloroplast photoreduced oxidized plastocyanin, but in the presence of methyl viologen, reduced plastocyanin was photo-oxidized only by the bundle sheath chloroplasts. PMID- 16657987 TI - Protein Synthesis in Cotyledons of Pisum sativum L: I. Changes in Cell-Free Amino Acid Incorporation Capacity during Seed Development and Maturation. AB - The changes in protein content of pea cotyledons have been followed during the period from 9 to 33 days after flowering. Initially protein content increased gradually with a rapid period of deposition occurring between days 21 and 27 after flowering. After the 28th day the rate of accumulation of protein declined as the seed dehydrated and matured. At maturity the pea cotyledon contained approximately 25% protein which was divided into albumins and globulins in the ratio of 1:1.4.Analytical data and the incorporation of exogenously supplied (14)C-leucine indicated that albumins were synthesized early in cotyledon development whereas globulin synthesis predominated with increasing maturity.Ribosomal preparations extracted from seeds during the period of rapid protein synthesis contained a high percentage of polysomes. Preparations from older cotyledons with a declining capacity for protein synthesis had few polysomes and an abundance of monosomes. The amino acid-incorporating capacity of ribosomal preparations from cotyledons of varying age was related to the polysomic content. The phenylalanine-incorporating capacity of ribosomal preparations from mature pea seed could be stimulated by the addition of polyuridylic acid. The distribution of polysomes and the in vitro incorporation data suggested that protein synthesis could be partially restricted by the availability of messenger RNA at maturity.However, reciprocal mixing experiments of supernatant and ribosomal fractions from cotyledons of different developmental age indicated that the supernatant fractions have varying capacities to stimulate in vitro amino acid incorporation. Thus the possibility of the regulation of protein synthesis at the translational level was not precluded. PMID- 16657988 TI - The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L. AB - The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6 day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination. PMID- 16657989 TI - Postillumination respiration of maize in relation to oxygen concentration and glycolic Acid metabolism. AB - Prior illumination in CO(2)-free air enhances a respiration from maize (Zea mays L.) leaves different in onset and duration from the postillumination burst of photorespiration. The course of respiration after brief illumination of attached leaves was measured as CO(2) efflux in darkness into CO(2)-free atmospheres with four O(2) concentrations. The peak of CO(2) efflux following illumination was suppressed by 2.23% O(2), was completely eliminated by 0.04% O(2), and was not stimulated by 40% O(2) compared with air. Compared with air, steady dark respiration was suppressed by 0.04% O(2) but was not affected by 2.23% nor 40% O(2). Excision and subsequent uptake of distilled water through the vascular system nearly eliminated the enhanced respiration.Several metabolites fed to excised leaves through the vascular system during illumination doubled or tripled the respiration of maize in darkness. The sensitivity to 2.23% O(2) of the respiration of glycolic acid in the dark imitated the sensitivity to O(2) of attached leaves.The respiration of glycolic acid was inhibited by alpha-hydroxy-2 pyridinemethanesulfonate.While attached leaves and leaves fed glycolic acid both released little CO(2) into CO(2)-free air in bright light, declining illuminance caused a larger and prompter CO(2) efflux from leaves fed glycolic acid than from attached leaves. Leaves fed glycolic acid plus 3-(p-chlorophenyl)-1, 1-dimethyl urea released more CO(2) into CO(2)-free air in bright light than did controls fed glycolic acid. PMID- 16657990 TI - Fat Metabolism in Higher Plants: XLVII. The Effect of Nitrite and Other Anions on the Formation of Unsaturated Fatty Acids by Isolated Chloroplasts. AB - Intact spinach and barley chloroplast normally incorporate (14)C-acetate into palmitate and oleate as the major (14)C fatty acids. Addition of nitrite markedly altered the relative patterns of the products with the appearance of stearate, a drop in oleate, but no marked change in palmitate. Arsenite greatly increased appearance of palmitate with a concomitant decrease in the C(18) fatty acids. The effect of other anions was also examined. Spinach and barley plants grown under different nitrogen nutritional conditions also served as sources of chloroplasts, and their activities suggest a correlation between nitrite reductase activity and stearate accumulation. PMID- 16657991 TI - The Influence of Low Substrate Sodium Levels upon the Free Amino Acid Content of Cotton Leaves. AB - The sodium nutrition of cotton (Gossypium hirsutum L.) was investigated. Plants were grown in purified nutrient solutions within a chamber designed to minimize sodium contamination. Three nutrient solutions were employed that contained in microequivalents/liter: (a) 0.17 Na, (b) 43.5 Na and (c) 0.75 Cs, 14.41 Li, and 1.17 Rb. All solutions had adequate potassium. Total free amino acids were increased by sodium. Leaves from plants grown in the high sodium solution contained significantly more free asparagine, arginine, and methionine than leaves from plants of the other treatments. The other alkali metals had little or no effect on concentrations of the free amino acids. An unknown was tentatively identified as argininosuccinic acid. PMID- 16657992 TI - Short term metabolism of urea and purine cytokinins. AB - Approximately 20 to 25% of the cytokinin benzyladenine (BA) taken up by soybean tissues in culture is converted to a stable, long-lived derivative which contains BA as part of its structure. This derivative may be metabolically related to 6 benzylamino-9-beta-d-ribofuranosylpurine 5'-monophosphate (BAMP). In in vivo incubations of 2 hours or less, we recover only BA, benzyladenosine, and BAMP. Benzyladenosine never accounts for more than 10% of the total radioactivity while BAMP builds up to about 20% of the total within 2 to 4 hours. After this period it begins to disappear, and a new, unidentified substance arises at a rate which roughly parallels the loss of BAMP. After about 48 hours this substance, which has good cytokinin activity, accounts for some 20 to 25% of the total radioactivity and persists at this level for at least 60 days. In the meantime the remainder of the BA, as well as benzyladenosine and BAMP, disappear completely. In addition, evidence is presented which suggests that the urea cytokinins are not active as such but first are metabolically transformed into other substances. PMID- 16657993 TI - The dark reactions of rye phytochrome in vivo and in vitro. AB - The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo. The effects of various inhibitors of an in vitro phytochrome-degrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 molecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics. PMID- 16657994 TI - Partial Purification and Characterization of a Phytochrome-degrading Neutral Protease from Etiolated Oat Shoots. AB - A factor catalyzing the in vitro degradation of oat phytochrome in crude extracts has been shown to be a proteolytic enzyme. The enzyme, an endoprotease, has been purified about 600-fold from dark-grown oat shoots by chromatography on ion exchange and molecular seive gels. The pH-activity curve is broad, with a maximum around pH 6.4. The enzyme is apparently dependent on the presence of reduced sulfhydryl groups for activity: low concentrations of reductants stimulate it, while inhibition has been obtained with a variety of sulfhydryl antagonists. High ionic strength conditions are inhibitory. A molecular weight of 61,500 has been estimated, though autolysis may yield smaller active fragments. An enzyme with similar properties has been isolated from imbibed oat seeds, light-grown oat shoots, and dark-grown rye shoots. PMID- 16657995 TI - Two effects of prolonged far red light on the response of lettuce seeds to exogenous gibberellin. AB - Prolonged far red irradiation of imbibed lettuce seeds (Lactuca sativa L. cv. Grand Rapids) makes them unresponsive to subsequent treatment with gibberellin. It has been found that this effect is overcome by supplying gibberellin buffered at a low pH. On the basis of this and other evidence it is suggested that an extended far-red exposure causes a loss of gibberellin sensitivity through an effect on the permeability of the endosperm. In seeds treated simultaneously with gibberellin and far red light, the hormone is taken up at the beginning of the irradiation, but its action is suspended until the seeds are placed in the dark. PMID- 16657996 TI - Light-induced Changes in the Ultrastructure of Pea Chloroplasts in Vivo: Relationship to Development and Photosynthesis. AB - Light-induced structural changes of chloroplasts and their lamellae were studied in leaves of Pisum sativum L., cv. Blue Bantam, using electron microscopy. Upon illumination of 14-day-old plants with 2000 lux, the chloroplasts decreased in thickness by about 23% with an accompanying increase in electron scattering by the stroma. Concomitantly, the average thickness of granal lamellae (thylakoids) decreased from 195 +/- 4 angstroms in the dark to 152 +/- 4 angstroms in the light, and this change was half-saturated at only 50 lux. Lamellar flattening at 50 lux and its reversal in the dark both had half-times of a minute or less. The thickness of a partition (a pair of apposed lamellar membranes) was 140 +/- 9 angstroms in both the light and the dark, indicating that the observed light induced change was in the volume enclosed within the thylakoid. The effect of illumination could be inhibited by various uncouplers of photophosphorylation but not by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, suggesting that it depended on ATP (or its precursor). In the presence of 0.5 micromolar nigericin, the thickness of the granal lamellae increased in the light to 213 +/- 3 angstroms; this may reflect an uptake of K(+) into an osmotically responding space within the thylakoids.During development, the capacity of the chloroplasts to flatten upon illumination increased in parallel with the amount of chlorophyll per gram of leaf and the number of lamellae per chloroplast. In contrast, the capacity of the leaves to fix CO(2) lagged nearly 2 days behind the development of chlorophyll. CO(2) fixation developed in parallel with the stacking of the lamellae into grana, supporting the contention that such organization is related to the linkage of photosystem II to photosystem I. PMID- 16657997 TI - Phosphorylated and nucleotide sugar metabolism in relation to cell wall production in Avena coleoptiles treated with fluroride and peroxyacetyl nitrate. AB - Coleoptile sections of Avena sativa L. were pretreated with sodium fluoride or peroxyacetyl nitrate at levels which inhibit auxin-induced growth but did not affect glucose uptake or CO(2) production when postincubated for 30 minutes in a (14)C-glucose medium without auxin. Labeling of metabolites involved in cell wall synthesis was measured. Peroxyacetyl nitrate decreased labeling, and it was concluded that the pool size of uridine di-phosphoglucose, sucrose, and cell wall polysaccharides decreased compared to control. The changes suggest that peroxyacetyl nitrate inactivated sucrose and cell wall synthesizing enzymes including cellulose synthetase and decreased cell growth by inhibiting production of cell wall constituents. Fluoride treatment had no effect on production of cell wall polysaccharides, with or without indoleacetic acid stimulation of growth. The only change after fluoride treatment was a decrease in uridine diphosphoglucose during incubation without indoleacetic acid, a decrease that disappeared when indoleacetic acid was present. It was concluded that some other aspect of cell wall metabolism, not determined here, was involved in fluoride induced inhibition of growth. PMID- 16657998 TI - Nitrate reductase of rice seedlings and its induction by organic nitro-compounds. AB - Nitrate simultaneously induced NADH- and NADPH-nitrate reductase activities in rice seedlings. Chloramphenicol, other organic nitro-compounds such as o nitroaniline and 2,4-dinitrophenol and nitrite also induced nitrate reductase in rice seedlings. The nitrate- or nitrite-induced nitrate reductase could accept electrons more efficiently from NADH than NADPH. However, when this enzyme was induced by organic nitro-compounds, it could accept electrons more efficiently from NADPH than NADH. PMID- 16657999 TI - Glutamate dehydrogenase from pumpkin cotyledons: characterization and isoenzymes. AB - Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH(4) (+) or alpha-ketoglutarate. The soluble enzyme was more sensitive to NH(4) (+) inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found. PMID- 16658000 TI - The relationship of the peroxidative indoleacetic Acid oxidase system to in vivo ethylene synthesis in cotton. AB - Since peroxidase and manganese have been implicated in both auxin destruction and ethylene production, the effect of auxins and high tissue levels of manganese on the peroxidative indoleacetic acid oxidase system and the internal level of ethylene was determined in cotton (Gossypium hirsutum L. cv. Watson GL-7). The highest level of manganese tested produced manganese toxicity symptoms, including necrotic lesions, accompanied by an increase in internal ethylene levels at about 15 days after treatment initiation. Statistically significant increases in indoleacetic acid oxidase and peroxidase activity were first observed 2 days later and were paralleled by tissue manganese levels above 7.4 milligrams per gram dry weight and internal ethylene levels of 0.77 microliters per liter air. Eight hours after application of 2,4-dichlorophenoxyacetic acid or indoleacetic acid, the internal levels of ethylene were increased to above 6.6 microliters per liter air in cotton plants, and levels of this magnitude were maintained for a 72 hour period of observation. Modification of peroxidase and indoleacetic acid oxidase activity in auxintreated plants definitely occurred well after the elevation of internal ethylene levels. While ethylene levels and indoleacetic acid oxidase activity were increased by both experimental approaches, the earlier appearance of increased ethylene indicates that the peroxidative indoleacetic acid oxidase system in cotton is not involved in ethylene synthesis or that this enzyme is not the rate-limiting factor when ethylene synthesis is increased. Ethylene, as well as auxin destruction, may be involved in some of the long term plant responses to toxic levels of manganese. The findings also suggest that auxin-induced ethylene may play a role in the elevation of peroxidase and indoleacetic acid oxidase activity eventually seen in extracts of plants treated with auxins. The data support the assumption that the enzymatic portion of the indoleacetic acid oxidase system in cotton is a peroxidase. PMID- 16658001 TI - Phytochrome Control of Another Phytochrome-mediated Process. AB - The phytochrome-mediated attachment of root tips of mung bean (Phaseolus aureus) and barley (Hordeum vulgare) to glass is affected by the prior exposure of hydrated seeds or seedlings to red or far red radiation. Prior irradiation of seeds or seedlings of mung bean with red light promotes attachment, while far red light promotes detachment of root tips. Similar exposure of barley seeds and seedlings to red light accentuates detachment, while far red light accentuates attachment of root tips. Red-far red light reversibility of the pretreatments indicates phytochrome control.Indoleacetic acid concentrations of 10 nM or higher appear to mimic the effects of far red light pretreatments in both mung bean and barley roots tips. PMID- 16658002 TI - Sugar Accumulation in Sugarcane: Carrier-mediated Active Transport of Glucose. AB - The rate-limiting reaction for glucose uptake in storage tissue of sugarcane, Saccharum officinarum L., appears to be the movement of glucose across the boundary between the free space and the metabolic compartments. The mechanism for uptake of glucose across this boundary has been studied using 3-O-methyl glucose, an analogue of glucose which is not metabolized by sugar-cane tissue.This analogue is taken up by sugarcane storage tissue at a similar rate to glucose. Its rate of uptake follows Michaelis-Menten kinetics, Km = 1.9 mm, and it is competitively inhibited by glucose, Ki = 2 to 3 mm. Glucose uptake is similarly inhibited by 3-O-methyl glucose. Uptake of 3-O-methyl glucose is energy-dependent and does not appear to be the result of counterflow of glucose.It is concluded that glucose and 3-O-methyl glucose uptake across the boundary between the free space and the metabolic compartment in this tissue is mediated by an energy dependent carrier system capable of accumulating the sugars against a concentration gradient. PMID- 16658003 TI - Translocation of carbon-14 in sugarcane plants supplied with or deprived of phosphorus. AB - Translocation of radioactive carbon from the fed part of a blade to the rest of the plant was impaired by a deficiency in phosphorus only when the level of phosphorus was low enough to decrease growth.Phosphorus deficiency decreased the percentage of inorganic phosphorus more than that of organic phosphorus, indicating little effect of phosphorus deficiency upon phosphorylation.Thus phosphorus deficiency did not reduce phosphorylation enough to affect translocation. Evidence for the involvement of phosphorylation in translocation obtained from studies with potassium deficiency, light intensity and quality, 3 (p-chlorophenyl)-1, 1-dimethylurea and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, and carbon dioxide suggests that noncyclic photosynthetic phosphorylation provides the energy for phototranslocation. PMID- 16658004 TI - Anaerobic Accumulation of gamma-Aminobutyric Acid and Alanine in Radish Leaves (Raphanus sativus, L.). AB - In leaves, the anaerobic accumulation of alanine was accompanied by a loss of aspartate, and these changes preceded gamma-aminobutyrate accumulation and glutamate loss. Changes in keto acid content did not appear to be the cause of amino acid changes. Accumulation of gamma-aminobutyrate was due to acceleration of glutamate decarboxylation and arrest of gamma-aminobutyrate transamination. Changes in enzyme content did not explain the changes in reaction rates in vivo. Most of the aspartate may be converted anaerobically to alanine via oxalacetate and pyruvate. PMID- 16658005 TI - In Vivo and In Vitro Studies on gamma-Aminobutyric Acid Metabolism with the Radish Plant (Raphanus sativus, L.). AB - Labeled glutamate was rapidly converted to gamma-aminobutyrate in intact, excised radish (Raphanus sativus L., var. Champion) leaves. Labeled gamma-aminobutyrate was metabolized via succinate and the Krebs cycle and was not carboxylated to form glutamate. Administration of carbon-14 and tritium-labeled succinate indicated that less than 10% of the gamma-aminobutyrate formation occurs by amination of succinic semialdehyde. Therefore, most gamma-aminobutyrate formation must be via glutamate decarboxylation.Radish leaf extracts were more active in catalyzing transamination between gamma-aminobutyrate and pyruvate than that between gamma-aminobutyrate and alpha-ketoglutarate. Glutamate decarboxylase was approximately 20 times more active than gamma-aminobutyrate: pyruvate transaminase. Succinic semialdehyde dehydrogenase was found in the extracts, and NAD was much more active as a hydrogen acceptor than NADP. No reduction of succinate to succinic semialdehyde by the NAD-linked dehydrogenase could be demonstrated. The following pH optima were determined: glutamate decarboxylase, 5.9; gamma-aminobutyrate: pyruvate transaminase, 8.9; succinic semialdehyde: NAD dehydrogenase, about 9.0. PMID- 16658006 TI - Variations in sodium uptake along primary roots of corn seedlings. AB - Entry of Na(+) into segments of the apical 8-centimeter portion of corn (Zea mays) roots was investigated and analyzed for each centimeter segment separately. Influence of temperature in the 0 C to 30 C range was well described by the Arrhenius equation [U = A exp (-Ea/RT)]. Values of A and Ea differed for each segment, tending to lessen with increasing distance from root apex. Time course of Na(+) entry was followed up to 70 minutes. Time relations of the process fit well the expression U = m [1 - exp (-nt)]. Calculated maximal uptake capacity (m) diminished with increasing distance from the apex. The data presented indicate that sodium uptake mechanisms vary qualitatively and quantitatively along corn roots. Thus, the use of entire roots for characterization of uptake mechanisms should be reassessed. PMID- 16658007 TI - 4-Hydroxycinnamic Acid Hydroxylase and Polyphenolase Activities in Sorghum vulgare. AB - Green shoots and first internodes of Sorghum vulgare var. Wheatland milo contain three phenoloxidase activities separable by means of Sephadex G-100 gel filtration. Two of these are found only in green leaves. I, eluted at the void volume and presumably a high molecular weight form, has both monophenol and diphenol oxidase functions; II, an intermediate molecular weight form displays only a diphenol function; III, a low molecular form found only in first internodes, catalyzes the hydroxylation of 4-hydroxycinnamic acid to caffeic acid and may have a weak diphenol activity. The hydroxylase activities of peaks I and III were completely inhibited by boiling or by 1 millimolar diethyldithiocarbamate and were partially inhibited by 1 millimolar KCN. The time courses of the two monophenol activities differ in that the activity of internode tissue was linear for at least 3 hours while that of the leaf began to decrease after 15 minutes. Both O(2) and a suitable electron donor were obligatory. At pH 6, ascorbic acid and 2-amino-4-hydroxy-6, 7-dimethylpteridine were the best electron donors, while NADPH was less effective. The diphenol oxidase functions of forms I and II in leaf preparations were not identical. The activity of I was less stable than that of II. While both were more active with chlorogenic acid, the ratio of activity with chlorogenic acid to that with 3,4 dihydroxyphenylalanine was less than 50 for I and greater than 50 for II. PMID- 16658008 TI - Protein Metabolism in Cultured Plant Tissues: III. Changes in the Rate of Protein Synthesis, Accumulation, and Degradation in Cultured Pith Tissue. AB - During the transition of tobacco (Nicotiana tabacum) pith tissue to callus tissue, there were changes in the composition of the soluble amino acid pools, in the distribution of amino acids between pool and protein, and in the synthesis, accumulation, and degradation of proteins. The size of the leucine pool decreased from 90 nanomoles per gram fresh weight in fresh pith to 20 nanomoles in 24-hour cultured pith, followed by a return to 90 nmoles in pith cultured longer than 5 days. The latter value is the same as that reported for exponentially growing callus cells. Many other pool amino acids changed as dramatically. However, they always approached callus levels after 5 days of culturing. The total amino acid content of pith tissue (the sum of both pool and protein) remained unchanged during culturing. The value for total amino acid content (34 to 42 nanomoles per gram fresh weight) was also similar to that found in callus. The distribution of amino acids between pool and protein did change during culturing. The transition of pith tissue with 88% of its total amino acids free in the soluble pool to callus with 92% of its amino acids in protein was further characterized by changes in protein metabolism. Both protein synthesis and accumulation increased over the first 50 hours in culture to a maximum rate of 45 milligrams protein synthesized gram protein(-1) hour(-1). After 50 hours in culture, the rate of protein accumulation decreased to equal the rate of fresh weight accumulation (10 mg g(-1) hour(-1)). However, protein synthesis continued at a high rate for several days, suggesting protein degradation was turned on by this time. By 5 days protein synthesis had decreased to a rate similar to that of callus. PMID- 16658009 TI - Induced changes in permeability of plant cell membranes to water. AB - The half-time for THO equilibration was three times longer for a living carrot (Daucus carota L.) cylinder than for a dead one. Furthermore, the energy of activation of THO flux was more than twice as high for the living cylinder. Passage through living membranes thus constitutes a rate-limiting step for THO flux in carrot tissue.CO(2) increased the half-time (t((1/2))) for THO equilibration. Treatment with abscisic acid or with tertiary butanol decreased t((1/2)). In neither case was the selective permeability of the membrane destroyed.p-Chloromercuribenzoate and carbonyl cyanide m-chlorophenylhydrazone, if supplied together with abscisic acid or tertiary butanol, abolished their action. If abscisic acid or butanol was first allowed to act alone, its effect was stable to subsequent treatment with the inhibitors. p-Chloromercuribenzoate and carbonyl cyanide m-chlorophenylhydrazone at concentrations at which they affected abscisic acid and butanol action, did not influence THO flux in control tissue. At considerably higher concentrations, however, 2, 4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone raised t((1/2)). The CO(2) effect was very rapidly reversible. Full reversal of the butanol effect required 3 hours, and that of abscisic acid required 4 days. PMID- 16658010 TI - Photosynthetic carbon metabolism of isolated corn chloroplasts. AB - Chloroplasts have been isolated from 4- to 6-day-old corn (Zea mays) leaves capable of assimilating 45 micromoles CO(2) per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of (14)C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix CO(2) at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate. PMID- 16658011 TI - Effect of pH on Penetration of Naphthaleneacetic Acid and Naphthaleneacetamide Through Isolated Pear Leaf Cuticle. AB - Penetration of naphthaleneacetic acid through enzymatically isolated upper pear (Pyrus communis L. cv. Bartlett) leaf cuticle increased as the donor pH was decreased. Naphthaleneacetamide penetration was not influenced by donor pH. The effect of pH on naphthaleneacetic acid penetration was reversible. Higher receiver (simulated leaf interior) pH favored penetration of naphthaleneacetic acid. Changes in the degree of dissociation, and hence polarity, as controlled by hydrogen ion concentration was the prime factor in the response of naphthaleneacetic acid to pH. At pH values lower than the pK (4.2 for naphthaleneacetic acid), the molecule was primarily undissociated, lipophilic, and penetrated into the cuticle; whereas, at pH values above the pK naphthaleneacetic acid was ionized, hydrophilic, and penetrated the cuticle with difficulty or not at all. Data presented are consistent with the hypothesis that naphthaleneacetic acid and naphthaleneacetamide penetration through the cuticle takes place by diffusion. PMID- 16658012 TI - The Correlated Appearance of Prolamellar Bodies, Protochlorophyll(ide) Species, and the Shibata Shift during Development of Bean Etioplasts in the Dark. AB - The structure and physiology of the etioplast was investigated in developing primary leaves of 3- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Increase in total protochlorophyll(ide) content followed that of leaf fresh weight. In 3- to 4-day-old bean leaves more than 50% of the protochlorophyll(ide) is in the form of protochlorophyll(ide) 628, which is nontransformable by light. Most of the transformable pigment is protochlorophyll(ide) 635, with smaller amounts of protochlorophyll(ide) 650. During leaf development from the 3rd to the 7th day phototransformable protochlorophyll(ide) with an absorbance maximum at 650 nm accumulates faster than nontransformable protochlorophyll(ide) or protochlorophyll(ide) 635. This increase in protochlorophyll(ide) 650 is correlated with the formation and enlargement of prolamellar bodies.The transformable protochlorophyll(ide) is converted by light directly to chlorophyll(ide) 672 in young leaves which do not yet have prolamellar bodies, and chlorophyll(ide) 672 may arise largely from the protochlorophyll(ide) 635. In older leaves the protochlorophyll(ide), largely protochlorophyll(ide) 650, is converted to chlorophyll(ide) 683, and a Shibata shift results in a change in the wavelength of absorption to 672 nm. The increase in protochlorophyll(ide) 650, the formation of prolamellar bodies, and the presence of the Shibata shift appear to be closely correlated. A model is briefly presented to provide a unified interpretation of these findings, including certain similarities between dark-grown Euglena cells and 3- to 4-day-old etiolated bean leaves. PMID- 16658013 TI - A study of ethylene in apple, red raspberry, and cherry. AB - High ethylene levels were associated with flower abscission in apple (Malus sylvestris) and cherry (Prunus avium and Prunus cerasus), "June drop" of immature cherries, and harvest drop of apple and red raspberry (Rubus idaeus). However, an increase in ethylene content was not associated with June drop of apples and harvest drop of cherries. During the period of fruit ripening on the plant, the largest increases in ethylene occurred in apple flesh and red raspberry receptacular tissue. Ethylene remained low throughout the period of sweet and tart cherry ripening. The data obtained indicated marked ethylene gradients between adjacent tissues. Increases of ethylene in some tissues may have resulted from ethylene diffusion from adjacent tissues containing high levels of ethylene. PMID- 16658014 TI - Involvement of Ethylene in Phytochrome-mediated Carotenoid Synthesis. AB - Accumulation of carotenoid pigments in the shoot apex of etiolated pea (Pisum sativum cv. Alaska) seedlings is completely prevented by ethylene. Under certain conditions carotenoid synthesis is normally controlled by endogenously produced ethylene. The gas completely inhibits carotenoid synthesis induced either by continuous white light or brief illumination with red light, but only partially inhibits light-induced chlorophyll formation. Far red illumination followed by red illumination reverses the action of red light on carotenoid synthesis. Red light-induced carotenogenesis is partly or wholly caused by phytochrome-mediated inhibition of ethylene biosynthesis. PMID- 16658015 TI - Distribution and activation of the Golgi apparatus in geotropism. AB - We find a differential distribution of dictyosomes in the avascular tip cells of the oat (Avena sativa) coleoptile upon geostimulation. A differential activation (increased vesicle production) of the dictyosomes with respect to gravity also occurs, but only in the tip cells of the lower tissues. Similar differences in distribution and activation of dictyosomes occur also in cells subjacent to the avascular tip (1st and 5th millimeter from the apex) of both the upper and lower half-tissues. When only the outer epidermal cells below the apex are considered, the differential distribution and activation of dictyosomes occur only in the lower outer epidermis. The changes in distribution of dictyosomes begin at 6 minutes, or sooner, from the start of geostimulation, before the onset of geotropism. The kinetics of amyloplast sedimentation and Golgi movement do not appear to differ in the cells of the avascular tip. We suggest that the Golgi participates in, and possibly initiates, the differential elongation of cells of geotropically stimulated coleoptiles. PMID- 16658016 TI - Characteristics of hook formation by bean seedlings. AB - Explants were isolated from 6-day-old etiolated bean seedlings (Phaseolus vulgaris L. cv. Black Valentine) containing the cotyledons with 4 mm of hypocotyl just below the node and/or the epicotyl. During incubation on distilled water, uneven growth of the hypocotyl or epicotyl occurred resulting in the formation of a hook. The more rapid growth of the side which became convex was not dependent upon the presence of the slower growing concave side. It was concluded that the main axis has an intrinsic capacity for asymmetric growth. The growth leading to hook formation was inhibited by alpha-naphthaleneacetic acid at concentrations above 0.2 milligram per liter. PMID- 16658017 TI - Abscisic Acid: correlations with abscission and with development in the cotton fruit. AB - Abscisic acid was measured in developing cotton fruit (Gossypium hirsutum) by means of gas-liquid chromatography. High levels of abscisic acid occurred in correlation with abortion and abscission of young fruit, with low germination of immature seed, and with senescence and dehiscence of mature fruit. Declining or low levels of abscisic acid occurred in correlation with the period of most rapid fruit growth and with high germination of immature and mature seed. Young fruit of cultivar Acala 4-42 contained about twice as much abscisic acid as young fruit of cultivar Acala SJ-1, and this difference is correlated with a higher rate of young fruit abscission in Acala 4-42. Young fruit abscising late in the fruiting season contained about twice as much abscisic acid as young fruit abscising early in the fruiting season. PMID- 16658018 TI - Photophosphorylation Can Provide Sufficient Adenosine 5'-Triphosphate to Drive K Movements during Stomatal Opening. PMID- 16658019 TI - Effect of Germanium and Utilization of Boron in Tomato (Lycopersicon esculentum Mill.). PMID- 16658020 TI - Photochrome-induced Adhesion of Mung Bean Root Tips to Platinum Electrodes in a Direct Current Field. PMID- 16658021 TI - Absence of phytochrome participation in chlorophyll synthesis in euglena. PMID- 16658022 TI - An effect of water stress on ethylene production by intact cotton petioles. PMID- 16658023 TI - The Site of Synthesis of Two Chloroplast Cytochromes in Chlamydomonas reinhardi. PMID- 16658024 TI - Rehydration of Phytochrome in Imbibing Seeds of Amaranthus retroflexus L. PMID- 16658025 TI - Relationship Between alpha-Tocopherol and alpha-Tocopherylquinone in Mineral deficient Maize Plants. PMID- 16658026 TI - Mechanisms of hormone action: use of deuterated ethylene to measure isotopic exchange with plant material and the biological effects of deuterated ethylene. AB - We observed no exchange between deuterated ethylene (C(2)D(4)) and the hydrogen of pea seedlings (Pisum sativum L. cv. Alaska). This suggests that bonding forces in which exchange could readily occur are not important in the physiological action of ethylene. Deuterated ethylene was just as effective as normal ethylene in inhibiting the growth of pea root sections. These results indicate that splitting carbon to hydrogen bonds did not occur during ethylene action. PMID- 16658027 TI - Mechanism of Ethylene Action: Biological Activity of Deuterated Ethylene and Evidence against Isotopic Exchange and cis-trans-Isomerization. AB - Deuterated ethylene was used to study the mechanism of ethylene action in etiolated pea seedlings (Pisum sativum L. cv. Alaska). No apparent differences were observed in the biological activity of tetradeuteroethylene (C(2)D(4)) and ordinary ethylene (C(2)H(4)) using the pea stem straight growth assay. The absence of an isotopic effect is discussed in relation to the possibility that ethylene binds to a metal or that carbon to hydrogen bonds of ethylene are broken during its mechanism of action.Analyses by gas chromatography of gas samples obtained from chambers containing intact etiolated pea plants exposed to 2 microliters of C(2)D(4) per liter of air for up to 5 days resulted in no detectable exchange between the deuterium atoms of C(2)D(4) and the hydrogen atoms of the tissue. Similarly, infrared spectra of gas samples obtained from chambers containing plants exposed to either cis or trans-C(2)D(2)H(2) indicated that no conversion had occurred to the corresponding trans or cis isomer. These results suggest that the mechanism of ethylene action does not involve an intermediate ethylene complex resulting in hydrogen exchange or cis-trans isomerization during a possible catalytic activation of the receptor site(s). PMID- 16658028 TI - A Phytotoxic Glycopeptide from Cultures of Corynebacterium insidiosum. AB - Cultures of Corynebacterium insidiosum produce an extra-cellular phytotoxic glycopeptide that possesses the ability to wilt plant cuttings. Wilt induced by this glycopeptide is directly dependent upon time and upon concentration with measureable wilt occurring in 40 nm solutions in 1 hour. The organism produces 1.3 grams toxin/liter of culture medium. The toxin was purified, and the physical, chemical, and biological properties were measured. The glycopeptide has an empirical formula of C(108)H(226)O(132)N based on 1 atom of nitrogen. The molecular weight as estimated by light scattering and column gel chromatography indicated values approximating 5 x 10(6). The toxin does not dissociate into small molecular weight subunits when treated with 8 m urea or 30% pyridine.The toxin has a specific optical rotation of [alpha](5460 A) (34.5 C) = -166 degrees , an intrinsic viscosity of 0.2307 dl/g, and decomposes at 260 C. It has a blue chromophore due to copper chelation at a concentration of 75 moles copper/mole toxin. Mannose, glucose, galactose and l-fucose, with trace amounts of rhamnose and an unidentified reducing sugar, comprise 83.1% of the toxin. An unknown organic acid appearing chemically similar to a keto-deoxy organic acid comprises 8.8% of the toxin. Lysine(2), arginine(1), aspartic acid(1), threonine(1), serine(1), glutamic acid(1), glycine(2), alanine(2), valine(2), leucine(2), and isoleucine(1), form a single peptide with glycine as the sole NH(2)-terminal amino acid. The peptide-carbohydrate linkage appears to be of a glycosidic nature involving the -OH of threonine. This single peptide composes 2.6% of the toxin, and there are 77 moles peptide/mole of purified glycopeptide. PMID- 16658029 TI - Solution-flow in the Phloem: I. Theoretical considerations. AB - A mathematical model for the reversible exchange of THO between the sieve tube lumen and its surrounding phloem tissue is used to explain the difference between the apparent velocities of THO and (14)C-sucrose transport observed when both are supplied simultaneously. Theoretically predicted results show a close correlation with those obtained experimentally. This model may be used in evaluating previous work in which THO was used as a tracer. The calculations support the existence of a mass flow of sugars in aqueous solution along the path. PMID- 16658030 TI - Solution-Flow in the Phloem: II. Phloem Transport of THO in Beta vulgaris. AB - Translocation profiles along the path were studied using a modified flap-feeding technique for the simultaneous application of THO and (14)C-sucrose. A re evaluation of a mathematical model for phloem transport with reversible lateral exchange of tracer along the path indicates that lower apparent velocities for THO as compared to labeled carbohydrate are primarily due to extensive lateral exchange of THO along the conduction path. Path-chilling experiments support the concept that THO and (14)C-sucrose exhibit different lateral exchange characteristics. The data presented are consistent with a solutionflow mechanism. PMID- 16658031 TI - The conversion of 2-hydroxyethylhydrazine to ethylene. AB - It was shown that 2-hydroxyethylhydrazine is a source of ethylene in a chemical model system. Treatment of pea seedlings produces physiological responses similar to that of ethylene. The hypothesis is advanced that 2-hydroxyethylhydrazine effects this response in pea seedlings via the release of ethylene, as was suggested earlier for its effect on abscission and flowering. PMID- 16658032 TI - Nutrient status and mycorrhizal enhancement of water transport in soybean. AB - Mycorrhizal soybean (Glycine max L. Merr. var. Harosoy-63) plants have lower resistances to water transport than nonmy-corrhizal plants after 4.5 weeks of growth. Although resistances of whole plants differ by 40%, there were no differences in the resistances of stems plus leaves, indicating that the major effect of the mycorrhizae was to reduce the resistance of the roots. Since the fungitoxicant, p-chloronitrobenzene, had no effect on resistances to water transport, reduced resistances were probably not caused by a direct modification of the transport pathway by the fungus. Differences in resistance between mycorrhizal and nonmycorrhizal soybean were essentialy eliminated by the application of nutrients to the soil. Thus, lowered resistances of mycorrhizal roots growing in soil with low levels of nutrients probably resulted from the enhanced nutrient status of the plant brought about by the fungus. Mycorrhizal infection increased growth at both low and high nutrient levels. PMID- 16658033 TI - Significance of enhancement for calculations based on the action spectrum for photosynthesis. AB - Calculations of the errors involved in measuring "photo-synthetically active radiation" in different ways are based on the assumption that the photosynthetic rate of a leaf in "white" light is equal to the sum of the products of (a) the photo-synthetic rate per unit of incident energy flux (action spectrum) by (b) the spectral energy flux distribution of the white light, the products being summed over all wavelengths at which the action spectrum is greater than zero. The calculations are valid only if the effects of different wavelengths are independent and additive. Although interactions are well documented in photo synthesis ("enhancement"), tests showed that the photosynthetic rates of leaves of six species, in four different types of white light, were within +/-7% of the rates calculated in this way. PMID- 16658034 TI - Ion transport induced by polycations and its relationship to loose coupling of corn mitochondria. AB - Treatment of corn mitochondria (Zea mays L., WF9 (Tms) x M14) with polycations (protamine, pancreatic ribonuclease, or polylysine) releases acceptorless respiration if phosphate is present. Concurrently, there is extensive active swelling which is reversed when respiration is uncoupled or stopped. Mersalyl, the phosphate transport inhibitor, blocks both the release of respiration and the active swelling. Diversion of energy into phosphate transport lowers respiratory control and ADP: O ratios. This response is termed "loose coupling" in distinction to "uncoupling" in which energy is made unavailable for either transport or ATP formation. Corn mitochondria as used here are endogenously loose coupled to some extent, and show state 4 respiration linked to active transport.The action of polycations can be partially mimicked by lowering pH of the suspending medium; both give swelling in sucrose medium and increased light absorbancy. Triton X-100, a nonionic detergent, will uncouple and accentuate active swelling, but unlike polycations it will not release state 4 respiration. Calcium ion acts something like polycation in activating phosphate transport and releasing respiration, and polycation appears to block entry of Ca(2+).It is speculated that neutralization of certain negatively charged acid groups on the membranes by polycations increases permeability to solutes and decreases coulombic repulsion of phosphate in approaching transport sites. In consequence, respiration rates and active transport rates increase. PMID- 16658035 TI - Isolation of Indole-3-ethanol Oxidase from Cucumber Seedlings. AB - Previous work in this laboratory has shown that cucumber (Cucumis sativus L.) seedlings contain large amounts, relative to other indolic compounds, of extractable indole-3-ethanol (IEt); tracer studies have established that IEt is metabolized to IAA. We have now succeeded in isolating an enzyme from these seedlings which catalyzes the oxidation of IEt to indole-3-acetaldehyde (IAAld). The identification of the product as IAAld was based on solvent partitioning of the free aldehyde and its bisulfite adduct and radiochromatography following incubation of enzyme with (14)C-IEt. A novel, quantitative colorimetric test for IAAld was also developed utilizing the Salkowski reagent. Partial purification of the enzyme was achieved by salt gradient chromatography on Bio-Rex 70, heating the preparation to 70 C, and chromatography on Sephadex G-150. This purification procedure yielded an enzyme activity purified in excess of 3000-fold, and studies on a standardized Sephadex column suggest a molecular weight of the enzyme of approximately 105,000. The reaction was found to proceed only aerobically; and, in the absence of other electron acceptors, O(2) appears to be reduced to H(2)O(2). The enzyme has nearly maximum activity from pH 8 to 11. PMID- 16658036 TI - Some counteractive effects of antitranspirants. AB - Some previously unexpected effects of antitranspirants on stomata and plant growth are described, and their implications are discussed. Antitranspirant films increase the resistance to diffusion of water vapor from stomata. This increases leaf water potential and results in wider stomatal apertures immediately under the film, as well as on those parts of a partially treated leaf which are not covered by the film. Antitranspirants are expected to curtail growth via reduced photosynthesis. However, by increasing plant water potential they can enhance the growth of fruits and shoots. PMID- 16658037 TI - The occurrence of nitrate reductase in leaves of prunus species. AB - Nitrate reductase was found in leaves of apricot Prunus armeniaca, sour cherry P. cerasus, sweet cherry P. avium, and plum P. domestica, but not in peach P. persica, from trees grown in sand culture receiving a nitrate containing nutrient solution. Nitrate was found in the leaves of all species. Nitrate and nitrate reductase were found in leaves of field-grown apricot, sour cherry, and plum trees. The enzyme-extracting medium contained insoluble polyvinylpyrrolidone, and including dithiothreitol or mercaptobenzothiazole did not improve enzyme recovery. Inclusion of cherry leaf extract diminished, and peach leaf extract abolished, recovery of nitrate reductase from oat tissue. Low molecular weight phenols liberated during extraction were probably responsible for inactivation of the enzyme. The enzyme from apricot was two to three times as active as from the other species. Both nicotine adenine diphosphopyridine nucleotide and flavin mononucleotide were effective electron donors. The enzyme was readily induced in apricot leaves by 10 mm nitrate supplied through the leaf petiole. PMID- 16658038 TI - Photoperiodic Effects on the Emanation of Volatiles from Alfalfa (Medicago sativa L.) Florets. AB - Alfalfa (Medicago sativa L.) plants acclimated to photoperiods of 18 hours light, 6 hour dark in plant growth chambers exhibited a daily cyclic pattern of floret volatile emanation with a maximum emanation of about 6.5 nanograms of hydrocarbons/floret.30 minutes. This maximum was reached about 6 to 8 hours into the light period. After 8 hours of light, emanation of volatiles decreased rapidly to less than 0.1 ng/floret.30 min even though light and temperature remained constant. Under continuous illumination, only a small increase of volatile emanation occurred during the following 24 hours. It appeared that a dark period was necessary to promote floret volatile emanation. Floret volatile emanation was drastically affected for at least 7 days following a photoperiod change. A photoperiod change caused 6-fold concentration oscillations every 2 hours. The results are interpreted on the basis of a very active floral metabolism controlled by photoperiodically induced rhythms. PMID- 16658039 TI - Requirement for extraction of polyribosomes from barley tissue. AB - The isolation of barley (Hordeum vulgare L.) polyribosomes, showing minimal degradation effects of endogenous RNase, required a buffer adjusted to pH 8.0 and containing 0.40 m KCl in addition to common extraction components. The extracted polyribosomes were characterized in sucrose gradients by their conversion to monosomes when incubated with pancreatic RNase and by their dependence on adequate amounts of Mg(2+) during extraction and analysis. Factors which contributed to polyribosome stability were evaluated by the relative sedimentation rates of aggregates in sucrose gradients. Tissue extraction at KCl concentrations less than 0.40 m and below pH 8.0 resulted in an appearance of larger amounts of ribosomes in the less dense region of the sucrose gradient after centrifugation. The addition of 10 mm dithiothreitol was partially effective in preventing the loss of higher polymerized states of polyribosomes at KCl concentrations below 0.40 m. Extractions conducted at KCl concentrations greater than 0.40 m and at pH 8.0 reduced the amount of ribosomes obtained from the tissue. The monosome portion of the polyribosomal profile was partially dissociated into subunits when the tissue was extracted in 0.60 m KCl. A similar effect on monosomes was obtained when polyribosomes were incubated with cycloheximide and 0.40 m KCl, a result not observed by use of a combination of 0.10 m KCl and the drug or 0.40 m KCl alone. PMID- 16658040 TI - Turnover of Phospholipids in Normal and Phosphorus-deficient Spirodela. AB - When (32)P(1) was supplied as a 15-minute pulse to normal Spirodela oligorrhiza plants, the first phospholipid to become fully labeled was phosphatidic acid. Phosphatidyl glycerol reached maximum labeling before the other major phospholipids. In phosphorus-deficient plants, however, phosphatidyl glycerol became labeled much more slowly than either phosphatidyl choline or phosphatidyl ethanolamine, and also the proportion of phosphatidyl glycerol present was smaller. Thus, phosphatidyl glycerol synthesis is sensitive to phosphorus deficiency. Since most of the phosphatidyl glycerol present in Spirodela was localized in the chloroplast, this effect appeared to be specifically one on chloroplast composition. The phosphorus-deficient chloroplast had a 60% lower phospholipid content and a normal phospholipid pattern, but the phospholipid which was present was apparently cycling much less rapidly. Zeatin, which ameliorates the visual symptoms of phosphorus deficiency, also reduces the effect of phosphorus deficiency on phospholipid synthesis. PMID- 16658041 TI - Biochemical and Enzymatic Changes in Apple Leaf Tissue during Autumnal Senescence. AB - The biochemical changes occurring during the natural senescence of apple leaf tissue (Pyrus malus L., Golden Delicious) coincided with specific changes in the environment. Protein, sugars, and total nitrogen began declining in leaf tissue when the daylength first became less than 14 hours in the second week of August. The activity of triose phosphate dehydrogenase declined shortly afterwards, while the activities of malate dehydrogenase, glutamic dehydrogenase, and aspartate aminotransaminase increased. Chlorophyll, DNA, RNA, and fresh weight began declining when the daylength first became less than 12 hours at the end of September. At the same time sugars and the activities of RNase, polyphenol oxidase, and proteolytic enzymes began increasing. Protein synthesis, total nitrogen, and the activities of malate dehydrogenase, glutamic dehydrogenase, and aspartate aminotransaminase began declining rapidly and amino acids began to accumulate after the first frost of the year. RNase, polyphenol oxidase, and proteolytic activity reached their highest specific activities after the first frost. PMID- 16658042 TI - Biochemical Changes in the Rice Grain during Germination. AB - Changes in the content of starch, protein, and RNA and in the activity of their hydrolases in the rice endosperm (Oryza sativa L., variety IR8) were determined during the first week of germination without added nutrient both in the dark and in the light. Changes were generally more rapid in the dark than in the light. Oxygen uptake and RNase activity started to increase and the root protruded on the second day, followed by the coleoptile on the third day, and the primary leaf on the fourth day. ATP level was at a maximum on the fourth day. The activity of amylases and R enzyme increased progressively, but that of phosphorylase tended to decrease during starch degradation. A new alpha amylase isozyme band appeared during germination. Glucose was the major product of starch degradation. Sucrose, maltose, maltotriose, raffinose, and fructose were also detected. Protease activity reached a maximum on the fifth or sixth day and closely paralleled the increase in soluble amino N and soluble protein.In embryoless seed halves with 0.12 muM gibberellin As, peak protease activity occurred in 2.5 days and peak alpha amylase activity on the fifth day of incubation. The production of alpha amylase, protease, and R enzyme was inhibited by 40 muM cycloheximide, but only alpha amylase and R enzyme were inhibited by 20 mug/ml actinomycin D. PMID- 16658043 TI - Involvement of Peroxidase and Indole-3-acetic Acid Oxidase Isozymes from Pear, Tomato, and Blueberry Fruit in Ripening. AB - Protein extracts were obtained from climacteric fruits (pear, tomato) and nonclimacteric fruits (blueberry) during various stages of ripening. The use of a gel electrophoresis technique revealed a consistent reinforcement in indoleacetic acid oxidase but not in peroxidase isozymes during ripening. The significance of the results is discussed in relation to the resistance of fruits to ripening and ethylene action. PMID- 16658044 TI - Intraspecific measurements of photorespiration. AB - The relative magnitudes of (a) CO(2) compensation concentration, (b) zero CO(2) intercept of the CO(2) response curve, (c) O(2) suppression of net photosynthesis, (d) differential (12)CO(2) and (14)CO(2) uptake, and (e) (14)CO(2) efflux into CO(2)-free air were determined in the dry bean (Phaseolus vulgaris L.) varieties Michelite-62 (M-62) and Red Kidney (RK). In comparing the two varieties for each of the above processes, there were three categories of response, M-62 > RK, M-62 = RK, and M-62 < RK. Since these processes did not give the same relative difference for the two varieties being studied, it was concluded that these phenomena cannot validly be used to estimate the magnitude of photorespiration, although they do identify its presence. The results suggest that photorespiration is but one component of O(2) inhibition of net photosynthesis and that photorespiration itself has two or more component metabolic pathways. PMID- 16658045 TI - Extraction and Reconstitution of Photosystem II. AB - Hill activity (oxygen evolution with ferricyanide as the electron acceptor), light-induced absorbance changes at liquid nitrogen temperature associated with the primary activity of photosystem II, and fluorescence yield changes at both low temperature and room temperature were measured with lyophilized spinach chloroplasts before and after extraction with hexane and reconstitution with beta carotene and plastoquinone A. Extraction eliminated the Hill activity, and both beta-carotene and plastoquinone A were required for maximal restoration of activity to the reconstituted chloroplasts.Extraction also eliminated the light induced absorbance changes at -196 C due to the photoreduction of C-550 and photooxidation of cytochrome b(559), and reconstitution with beta-carotene and plastoquinone A restored the low temperature photoreactions. However, only beta carotene was essential for the restoration of the photoreactions. Cytochrome b(559) was modified, as a result of the extraction, to a lower redox potential, autooxidizable form and remained as such after reconstitution with beta-carotene. The beta-carotene-restored chloroplasts showed the photoreduction of C-550 but not the photooxidation of cytochrome b(559) because the cytochrome was already oxidized. When beta-carotene-reconstituted chloroplasts were suspended in buffer containing ascorbate prior to freezing, the cytochrome b(559) was reduced and could be photooxidized by irradiation at low temperature. After reconstitution with beta-carotene plus plastoquinone A the cytochrome b(559) was partially restored to its original high potential form and was in the reduced state so that both the photoreduction of C-550 and the photooxidation of cytochrome b(559) occurred on irradiation of the beta-carotene plus plastoquinone A-reconstituted chloroplasts. Reconstitution with plastoquinone A alone had essentially no effect on restoring the photoreactions.The fluorescence yield of dark-adapted lyophilized chloroplasts at -196 C showed an irreversible increase of about 2.5 fold during irradiation. After extraction the fluorescence yield of the chloroplasts was high (at the maximal light-induced level of the lyophilized control chloroplasts) and showed very little change in the light. Reconstitution with beta-carotene alone restored some fluorescence quenching which was relieved by irradiation at low temperature. Reconstitution with plastoquinone A alone restored a high degree of quenching, but this quenching was not relieved by light at low temperature. Fluorescence emission spectra at -196 C showed that the fluorescence of variable yield in the lyophilized and beta-carotene-reconstituted chloroplasts involved only the 680 and 695 nm emission bands but not the larger 730 nm emission band, whereas the irreversible quenching in plastoquinone A reconstituted chloroplasts involved all wavelengths of emission. Extraction of the chloroplasts also eliminated the sharp 695 nm emission band at low temperature, and reconstitution with beta-carotene partially restored it.The fluorescence yield changes at room temperature differed from the low temperature measurements in that the strong fluorescence quenching restored to the plastoquinone A-reconstituted chloroplasts was relieved by light and reappeared in the dark. Thus plastoquinone A appeared to be much more effective than beta carotene in restoring the fluorescence of variable yield in room temperature measurements. However, it is argued from the results at low temperature that the quenching in plastoquinone A-reconstituted chloroplasts, which is probably due to the oxidized form of the quinone, is nonspecific and a different quenching mechanism from that which obtains in normal chloroplasts.The results suggest that extraction with hexane removes plastoquinone A, which interrupts electron transport, and beta-carotene, which disrupts the primary photochemical activity of photosystem II. Reconstitution of the extracted chloroplasts with beta carotene alone restores C-550 and the primary photochemical activity of photosystem II, and when the photosystem II reaction centers are restored the additional requirement of plastoquinone A for the Hill reaction can be demonstrated. PMID- 16658046 TI - Terminal Oxidases of Chlorella pyrenoidosa. AB - In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O(2)) of 2.1 +/- 0.3 mum, while the second oxidase has a Km (O(2)) of 6.7 +/- 0.5 mum, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8 hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake. PMID- 16658047 TI - Induction of phenylalanine ammonia lyase and pisatin by photosensitive psoralen compounds. AB - The psoralen compounds, xanthotoxin and 4,5', 8-trimethylpsoralen, when activated, increased phenylalanine ammonia lyase (PAL) activity and the synthesis of pisatin in excised pea pods. Pods presoaked 1 hr with 4,5',8-trimethylpsoralen and then irradiated 4 minutes with 366 nanometer ultraviolet light had twice as much PAL activity 3 hours after irradiation and 12 times as much PAL activity 20 hours after irradiation as the pods of the water-treated control. Increases in PAL activity and pisatin synthesis were not obtained with 4,5',8 trimethylpsoralen, xanthotoxin, or 366 nanometer light treatment alone. 4,5',8 Trimethylpsoralen in combination with the irradiation treatment (366 nanometers) enhanced the rate at which l-leucine is incorporated into various fractions of soluble proteins in excised pods 8 hours after treatment. This treatment decreased the rate at which orotic acid is incorporated into RNA. The increase in PAL activity induced by irradiated psoralens was prevented when 6-methylpurine (0.5 milligram per milliliter) or cycloheximide (10 micrograms per milliliter) was applied immediately following the irradiation period. Possible functions of psoralen compounds in plants are discussed. PMID- 16658048 TI - Characterization of Short Time Labeled Adenosine Monophosphate-rich Ribonucleic Acids of Soybean. AB - The total population of newly synthesized (32)P-AMP-rich RNA has been separated into two major types based on repeated fractionation on methylated albumin kieselguhr columns. The purified D-RNA which elutes, under our experimental conditions, primarily in the salt gradient has a GMP/AMP ratio of about 0.8 and an AMP + UMP content of about 56 mole per cent. The purified TB-RNA which preferentially remains bound to the column in the salt gradient has a GMP/AMP ratio of about 0.4 to 0.45 and an AMP + UMP content of about 65 mole per cent. In addition to being distinguished by their fractionation on the methylated albumin kieselguhr column and base composition analysis, purified D-RNA and TB-RNA have different size distributions on sucrose gradient and acrylamide gel fractionation, are differentially associated with polyribosomes and have different stabilities in the tissue. PMID- 16658049 TI - Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport. AB - The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V(max) of 1.3 mumoles/gram fresh weight.2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When (14)C-glucose was supplied exogenously, (14)C-glucose 6-phosphate and (14)C-glucose were the first labeled compounds to appear in the tissue; no (14)C-sucrose was detected until after 60 second incubation. After 15-second incubation in (14)C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6 phosphate; trace amounts of (14)C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments. PMID- 16658050 TI - The requirement for sodium as a micronutrient by species having the c(4) dicarboxylic photosynthetic pathway. AB - Six species having characteristics of plants with the C(4) dicarboxylic photosynthetic pathway, Echinochloa utilis L. Ohwi et Yabuno (Japanese millet), Cynodon dactylon L. (Bermuda grass), Kyllinga brevifolia Rottb., Amaranthus tricolor L. cv. Early splendour, Kochia childsii Hort., and Portulaca grandiflora Hook (rose moss), responded decisively to 0.1 milliequivalent per liter NaCl supplied to their culture solutions initially containing less than 0.08 microequivalent per liter Na. Chlorosis and necrosis occurred in leaves of plants not receiving sodium. Portulaca failed to set flower in the sodium-deficient cultures. Under similar conditions Poa pratensis L. (Kentucky blue grass) having characteristics of the C(3) photosynthetic pathway made normal growth and did not respond to the addition of sodium. It is concluded from these results and previously reported work that sodium is generally essential for species having the C(4) pathway but not for species with the C(3) pathway. PMID- 16658051 TI - Plants under Climatic Stress: III. Low Temperature, High Light Effects on Photosynthetic Products. AB - An investigation has been made of the combined effects of low temperature and high light on the level of several photosynthetic products in the leaves of a group of plants differing widely in their tolerance to this stress. Starch levels in these plants after chilling are dependent on the time of day that temperatures are lowered and seem related to rates of CO(2) assimilation under this stress. Prolonged low-temperature, high-light treatment (10 C at 160 wm(-2)) of Sorghum bicolor induced a rapid starch hydrolysis after a lag of some 24 hours. Differing rates of starch loss at the cellular level and a rapid migration of chloroplasts toward the base of upper mesophyll cells were also seen in leaves of this stress sensitive species.Chilling increased the level of almost all free amino acids in tolerant and in semi-tolerant species, while amino acids related to intermediates of the C(4)-pathway show a sharp or transitory decrease in Sorghum. These and other changes observed in Sorghum suggest that some time- and temperature dependent blockages develop in the interconversion of C(4)-pathway intermediates and possibly in the flow of other intermediates to and from the sites of C(4) photosynthesis.Levels of ATP in the leaves of Sorghum, Paspalum, and Amaranthus increased at night and following chilling and did not fall until pronounced necrosis of the leaves commenced. PMID- 16658052 TI - Mode of Action of the Toxin from Pseudomonas phaseolicola: I. Toxin Specificity, Chlorosis, and Ornithine Accumulation. AB - The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%.Protection from and reversal of toxin-induced chlorosis in bean leaves was attempted with several amino acids. Aside from protection with l-citrulline which was previously reported, only l-arginine-HCl and to a minor extent l-leucine and l-glutamine showed protection from chlorosis. l-Citrulline and l-arginine-HCl (but not l glutamine and l-leucine) also reversed toxin-induced chlorosis.Ultrastructurally, cells from toxin-treated chlorotic tissues showed no observable changes as compared to nontreated tissues. This, together with the ability of the two amino acids to reverse chlorosis, indicated that the toxin causes a reversible biochemical lesion in treated tissue.While tissues from bean plants inoculated with P. phaseolicola showed a large accumulation of ornithine, toxin-treated tissues showed no accumulation of ornithine. The latter finding indicated that in addition to the ornithine carbamoyltransferase inhibitor, the pathogen may produce inhibitors of other ornithine metabolizing enzymes in inoculated tissues. PMID- 16658053 TI - Mode of Action of the Toxin from Pseudomonas phaseolicola: II. Mechanism of Inhibition of Bean Ornithine Carbamoyltransferase. AB - A chlorosis-inducing toxin of Pseudomonas phaseolicola was examined for inhibition of ornithine carbamoyltransferease prepared from acetone powder of bean (Phaseolus vulgaris L.) plants. The enzyme has a pH optimum at 8.5, involves a ternary complex reaction mechanism, and shows Michaelis constants of 5.0 mm and 1.7 mm for ornithine and carbamoylphosphate, respectively. Assuming reversible catalysis, Michaelas constants of 11 mm and 3.3 mm are calculated for citrulline and arsenate. Toxin induces allosteric competitive inhibition in relation to carbamoylphosphate and a noncompetitive mode of inhibition in relation to ornithine, except at high toxin concentrations where uncompetitive inhibition is observed. In the backward assay, competitive inhibition is observed for both arsenate and citrulline. Inhibition is increased with preincubation time and shows saturation kinetics with regard to toxin concentration. PMID- 16658054 TI - Penetration of stomata by liquids: dependence on surface tension, wettability, and stomatal morphology. AB - Wettability of the leaf surface, surface tension of the liquid, and stomatal morphology control penetration of stomata by liquids. The critical surface tension of the lower leaf surface of Zebrina purpusii Bruckn. was estimated to be 25 to 30 dyne cm(-1). Liquids having a surface tension less than 30 dyne cm(-1) gave zero contact angle on the leaf surface and infiltrated stomata spontaneously while liquids having a surface tension greater than 30 dyne cm(-1) did not wet the leaf surface and failed to infiltrate stomata. Considering stomata as conical capillaries, we were able to show that with liquids giving a finite contact angle, infiltration depended solely on the relationship between the magnitude of the contact angle and the wall angle of the aperture. Generally, spontaneous infiltration of stomata will take place when the contact angle is smaller than the wall angle of the aperture wall. The degree of stomatal opening (4, 6, 8, or 10 mum) was of little importance. Cuticular ledges present at the entrance to the outer vestibule and between the inner vestibule and substomatal chamber resulted in very small if not zero wall angles, and thus played a major role in excluding water from the intercellular space of leaves. We show why the degree of stomatal opening cannot be assessed by observing spontaneous infiltration of stomata by organic liquids of low surface tension. PMID- 16658055 TI - Inhibition of Photosystem II in Isolated Chloroplasts by Lead. AB - Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition. PMID- 16658056 TI - Changes in Mitochondrial Properties Associated with Chloroplast Development in Jack Bean (Canavalia ensiformis [L] DC.). AB - Procedures were developed to isolate actively respiring mitochondria from jack bean (Canavalia ensiformis [L.] DC.) leaves and cotyledons. Consistent respiratory control values of about 2 for succinate oxidation were obtained in preparations from etiolated cotyledons. Jack bean mitochondria oxidized malate with high respiratory control (2 to 5) and ADP/O (up to 2.8).Mitochondria purified from greening leaves and cotyledons exhibited rapid increases in respiratory control for up to 4 hours of light exposure followed by a decrease in activity. ADP/O values remained constant at about 1.2 while oxygen consumption per unit mitochondrial protein increased constantly during the first 14 hours of greening in preparations from cotyledons. The changes in mitochondrial respiratory activity during greening were correlated with the reversible appearance of mitochondria in the condensed conformation observed by electron microscopy. Both isolated mitochondrial pellets and cotyledon mitochondria in situ were shown to exhibit these changes in conformation during greening. PMID- 16658057 TI - Comparison of endogenous gibberellins and of the fate of applied radioactive gibberellin a(1) in a normal and a dwarf strain of Japanese morning glory. AB - The effect of application of GA(3) on hypocotyl growth, the endogenous GAs, and the metabolism of applied (3)H-GA(1) were investigated in relation to dwarfism and light-mediated growth inhibition in the normal (tall) strain Violet and the dwarf strain Kidachi of Japanese morning glory (Pharbitis nil). GA(3) applied in a wide concentration range (10(-9) to 10(-3)m) to 4-day-old seedlings caused great extension of the hypocotyls in light-grown plants of both the normal and the dwarf strain. However, the dwarf strain did not attain the same length as the normal one at any given GA(3) concentration, even when saturation was reached. Dark-grown plants of the dwarf strain responded to GA(3), although relatively much less than light-grown ones; dark-grown plants of the normal strain showed no GA(3) response at all.The levels of free GAs in both strains remained more or less constant in both dark- and light-grown plants up to 18 days after germination. The levels of bound GA in dark- as well as light-grown plants of both strains increased after germination, reached a maximum at the 9th day after germination, and then rapidly declined again. The period of increase coincided with rapid elongation of the hypocotyl and the expansion of the cotyledons.The dwarf strain, Kidachi, contained less endogenous gibberellins, particularly bound gibberellins, than the normal strain, Violet. Dark-grown plants of both strains contained less bound GAs than light-grown plants.Applied (3)H-GA(1) was metabolized to the same extent in both dwarfs and normals, on the one hand, and in both dark- and light-grown plants, on the other. This metabolism involved binding as well as breakdown of the (3)H-GA(1). PMID- 16658058 TI - Abscisic Acid and stomatal regulation. AB - The closure of stomata by abscisic acid was examined in several species of plants through measurements of CO(2) and H(2)O exchange by the leaf. The onset of closure was very rapid, beginning at 3 minutes from the time of abscisic acid application to the cut base of the leaf of corn, or at 8 or 9 minutes for bean, Rumex and sugarbeet; rose leaves were relatively slow at 32 minutes. The timing and the concentration of abscisic acid needed to cause closure were related to the amounts of endogenous abscisic acid in the leaf. Closure was obtained in bean leaves with 8.9 picomoles/cm(2). (+)-Abscisic acid had approximately twice the activity of the racemic material. The methyl ester of abscisic acid was inactive, and trans-abscisic acid was likewise inactive. The effects of stress on levels of endogenous abscisic acid, and the ability of very small amounts of abscisic acid to cause rapid closure suggests that stomatal control is a regulatory function of this hormone. PMID- 16658059 TI - Cytokinins in pisum transfer ribonucleic Acid. AB - Five cytokinin-active ribonucleosides have been isolated from the transfer RNA of 7-day-old green pea shoots (Pisum sativum L. var. Alaska). Ultraviolet spectroscopy and mass spectrometry have been used to identify 6-(3-methyl-2 butenylamino)-9-beta-d-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino) 2-methylthio-9-beta- d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2 butenylamino)-9-beta-d-ribofuranosylpurine. The latter was separated into the cis and trans-isomers by thin layer chromatography. The fifth cytokinin is indicated to be 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-d -ribofuranosylpurine on the basis of its chromatographic properties. PMID- 16658060 TI - Effect of filipin on liposomes prepared with different types of steroids. AB - The action of the polyene antibiotic filipin on the permeability of liposomes prepared with lecithin and several plant and other steroids was studied. The effect of filipin was found to be dependent upon the type of steroid incorporated into the membrane. The interaction of filipin with steroids was found to be related both to the functional group at the 3-position and the aliphatic chain of the steroid. Complex formation of the steroid with filipin in aqueous solutions, as detected by ultraviolet spectrophotometry, correlated with the ability of filipin to increase the permeability of the liposomes.From these results it was suggested that the action of filipin could be used as a diagnostic tool to determine the presence of different steroids in plant membranes. Differences in the sensitivity of subcellular organelles to filipin may be due to differences in the steroid composition of their membranes. PMID- 16658061 TI - The Effect of Light upon Development in Potato Tissue Slices. PMID- 16658062 TI - On the involvement of acetylcholine in phytochrome action. PMID- 16658063 TI - Diphenylcarbazone: a reagent for photosystem 1 activity in chloroplast fragments. PMID- 16658064 TI - Pectin methylesterase and polygalacturonase in avocado fruit at various stages of development. PMID- 16658065 TI - Effect of light on basipetal movement of indoleacetic Acid in green stem sections of coleus. PMID- 16658066 TI - A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers. AB - The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and alpha-amylase synthesis: (a) synthesis of alpha amylase took place in parallel with incorporation of label into gibberellic acid RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for alpha amylase synthesis to occur but that alpha-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for alpha-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control. PMID- 16658067 TI - Lateral movement of water and sugar across xylem in sugarcane stalks. AB - Laterally connected vascular bundles in the nodes of sugarcane (Saccharum species cv. Pindar) stalks allow a rapid redistribution of water across the stalk should the vascular continuity be partly disrupted. Tritiated water supplied to the roots exchanged rapidly between the xylem and storage tissue so that net movement up the stalk was slow. The half-time for exchange in a labeled stalk was about 4 hours so that the entire water content of a sugarcane stalk can turn over at least once in a single day. No rapid flux of sugar between xylem and phloem or xylem and storage tissue was detected. Functional xylem contained only low sugar concentrations: less than 0.3% w/v in the stalk and less than 0.02% w/v in the leaf. Previous reports of high sugar levels (9% w/v) in sugarcane stalk xylem reflect some degree of xylem blockage followed by a slow equilibration with free space sugars in the storage tissue. PMID- 16658068 TI - The Mechanism of the Scopoletin-induced Inhibition of the Peroxidase Catalyzed Degradation of Indole-3-acetate. AB - The naturally occurring coumarin, scopoletin, has been found to modify horseradish peroxidase rapidly to give a stable, spectroscopically distinguishable form of the enzyme. Peroxidase treated with scopoletin is less active in reactions with molecular oxygen and indole-3-acetic acid. Kinetic data for the degradation of this growth regulator were obtained with a continuously monitored fluorometric procedure. Lineweaver-Burk plots of the reciprocal rate of degradation against the reciprocal substrate concentration were markedly curved in the presence of the inhibitor, scopoletin. Excess indole-3-acetate restored the scopoletin-treated enzyme to a reactive state. In the presence of molecular oxygen, concentrations of indole-3-acetic acid which were at least 10-fold greater than the inhibitor concentration led to the rapid oxidation of the coumarin and converted peroxidase to compound III as expected from previous studies. This form of the enzyme is the catalytically active species in the oxidative degradation of the growth regulator. The kinetically preferential reaction of scopoletin or related coumarins with peroxidase and the suppression of indole-3-acetic acid degradation may provide a possible control mechanism over the oxidative degradation of indole-3-acetate by this plant enzyme. PMID- 16658069 TI - Gravity and intracellular differences in membrane potentials of plant cells. AB - The electrical potentials across the cell membranes of the lower parts of Zea mays coleoptile cells are about 2 millivolts more negative than across the upper parts. This electrical polarization with respect to gravity occurs when coleoptiles are oriented with their apical ends either up or down and seems independent of the magnitude of the potential when the potential is modified by other treatments. PMID- 16658070 TI - Chloroplast structure and function in tissue cultures of a c(4) plant. PMID- 16658071 TI - Plant Growth Inhibitor from Abrus precatorius Seeds. PMID- 16658072 TI - Evaluation of edge injection errors in the floating leaf disk method of measuring leaf tissue water deficit. PMID- 16658073 TI - The nitrate reductase of chlorella: species or strain differences. PMID- 16658074 TI - Interaction of carbon dioxide and ethylene in overcoming thermodormancy of lettuce seeds. AB - The combination of ethylene with CO(2) will completely overcome the thermodormancy of lettuce (Lactuca sativa L.) seeds at 35 C. This combination is effective if it is added to seeds either at the start or after several days of imbibition. The action of ethylene is dependent upon the CO(2) level present in the atmosphere surrounding the seeds. When CO(2) is trapped by KOH the ethylene effect is essentially nil. PMID- 16658075 TI - The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill. AB - Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen production, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing acid-dependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria.A comparison of enzyme activities in extracts from Aloe leaves and from other plants and studies of leaf anatomy and chloroplast morphology revealed typical characteristics of C(3)-, as well as C(4)-, plants in Aloe. PMID- 16658076 TI - Large Scale Isolation and Purification of Eyespot Granules from Euglena gracilis var. bacillaris. AB - Large volumes of eyespot granules were isolated from homogenates of Euglena gracilis Klebs var. bacillaris Pringsheim by flotation centrifugation in a Beckman Ti-15 zonal rotor, and were further purified by centrifugation in a swinging bucket rotor. Examination with the electron microscope showed the eyespot granules to be free from other cellular material. Freezing had no apparent effect on the structure or on the absorption properties of the eyespots. Absorption spectra of pure fractions of eyespot granules free of chloroplast contamination showed the previously reported curves in the range of 360 to 520 nanometers, as well as a peak at 660 to 675 nanometers. The procedure for the large scale isolation of eyespot granules from Euglena gracilis is compared with other methods which have employed conventional centrifugation, and the significance of the use of zonal rotors for isolating large quantities of pure eyespot granules is discussed. PMID- 16658077 TI - Meristematic Activity during Adventitious Root Primordium Development: Influences of Endogenous Auxin and Applied Gibberellic Acid. AB - Intact brittle willows (Salix fragilis L.) were treated so that developing adventitious root primordia in the stems would be subjected to elevated gibberellic acid or reduced endogenous auxin levels. Observations were made of primordia that were initiated during the experiments and of primordia that were established before the experiments began. The results indicated that as primordia became older and contained more cells, auxin basipetally transported in the stem seemed to be of less importance in determining cell number per primordium. Thus, established primordia depended upon this auxin to a lesser extent than primordia which were being initiated. These observations were explained on the basis of differential contributions during primordium development of cell division in the cambium of the stem and in the primordia themselves. As opposed to the effects of reduced auxin levels, applied gibberellic acid reduced the cell number per primordium most in established primordia. Initiating primordia were least affected by gibberellic acid treatment. Gibberellic acid treatment seemed mainly to reduce intraprimordium cell division, on which continued development of established primordia most depends. Seemingly, at least in brittle willow, applied gibberellic acid blocks the action of auxin in primordium development subsequent to the initiation phase. PMID- 16658078 TI - Purification and properties of adenosine diphosphoglucose pyrophosphorylase from sweet corn. AB - A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg(2+) and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1 phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 x 10(-4), 3.2 x 10(-5), 3.3 x 10(-5), and 6.2 x 10(-4)m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 x 10(-7)m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate. PMID- 16658079 TI - Phycomyces: growth responses of the sporangium. AB - During the development of the sporangiophore of the fungus Phycomyces blakesleeanus there occurs a period of several hours when the sporangiophore does not elongate; instead, its "growth" is diverted into the formation of a sporangium at its top. This period of head formation is called stage II. Clearly, growth has not ceased but rather the geometry of the growing area has changed from that of a cylinder to a sphere. The growing sphere is found to have properties similar to the stage IV growing zone in that it functions as a sensory receptor and effector. The growing sporangium responds to both light (light head response) and humidity (wet head response). A model is presented giving a possible mechanism by which the ultimate size of the sporangium is regulated. PMID- 16658080 TI - Regulation of ribulose diphosphate formation in vivo by light. AB - Light-dependent formation of ribulose-1,5 diphosphate is completely inhibited by low concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which do not severely affect cyclic photophosphorylation. Also in Scenedesmus mutant number 11, capable of cyclic photophosphorylation, cellular ribulose-1,5 diphosphate levels do not increase upon illumination. When mutant cells are H(2) adapted, however, a light-dependent formation of ribulose-1,5 diphosphate is observed in the presence of H(2). From these results it has been concluded that at least part of the Calvin cycle does not operate in the dark, since a reductant is lacking which is generated in the light. PMID- 16658081 TI - Photoperiodic Entrainment Patterns in the CO(2) Output of Lemna perpusilla 6746 and of Several Other Lemnaceae. AB - The CO(2) output of Lemna perpusilla 6746 in "skeleton photoperiods" consisting of alternating 10(1/2)-hour and 13-hour dark periods separated by (1/4)-hour illuminations was recorded under stable high and low nitrate conditions. The phase relationship finally attained between light schedule and output is the same regardless of which dark period is given first, but entrainment is more rapid (as is flowering) with an initial 13-hour dark period. In all respects other than bistability-the assumption of two different stable phase relationships depending on the initial dark period-both flowering and the course of CO(2) output conform to Pittendrigh's model derived from Drosophila eclosion rhythms, confirming the view that an endogenous circadian rhythm, or biological clock, underlies the photoperiodic control of flowering in this plant. Experiments with rigorous temperature control show that earlier results with long light exposures were in part due to temperature changes; in consequence, it is clear that entrainment patterns with high nitrate differ even more from those in low nitrate than was previously evident, and not simply by the addition of a "nitrate peak." Other Lemnaceae tested with a few simple light-dark schedules in both types of media show a variety of responses, with no obvious correlation to photoperiodic response type. PMID- 16658082 TI - Sugar accumulation in sugarcane: role of cell walls in sucrose transport. AB - A new method was devised to measure the concentration of sucrose in the free space of sugarcane stalks. The free space includes the aqueous phase of cell walls. The method differs substantially, in principle, from one used previously, but it gave the same result, namely, that sucrose concentrations in the free space can approach the level in the bulk of the tissue. PMID- 16658083 TI - Ribonucleic Acid Synthesis Associated with a Developmental Change in the Gametophyte of Pteridium aquilinum. AB - When bacteria are subjected to step-down conditions, there is an enhanced production of a messenger-type of RNA for a short time after the shift. A cultural shift, which appears to be similar to step-down, is described for gametophytes of Pteridium aquilinum. When the cultures are grown in white light, a population of rapidly dividing cells is produced, whereas in red light cell elongation predominates. If the cultures growing in white light are shifted to red light, a transition occurs which involves a rapid decrease in growth and nucleic acid synthesis. In particular, there is a marked decline in RNA synthesis for a short time following the shift and prior to the initiation of the new mode of growth. It appears that this observed change in RNA synthesis is related to the initiation of the new mode of growth. PMID- 16658084 TI - Carbohydrate translocation in sugar beet petioles in relation to petiolar respiration and adenosine 5'-triphosphate. AB - Earlier studies have shown that the retarding effect of low petiolar temperatures on sucrose transport through sugar beet (Beta vulgaris L.) petioles is markedly time-dependent. Although the initial effect of chilling the petiole to near 0 C is severely inhibitory, translocation rates soon recover (usually within about 2 hours) to values at or near the control rate. In the present studies, selected metabolic parameters were measured simultaneously with translocation. No stoichiometric relationships among petiolar sucrose transport, petiolar respiration (CO(2) production), and calculated petiolar ATP turnover rates were evident. It appears that the major sources of energy input energizing carbohydrate transport in sieve tubes function mainly at either loading or unloading sites and not at the level of individual sieve-tube elements. PMID- 16658085 TI - In vitro measurement of pollen tube growth inhibition. AB - A method for estimating inhibition of pollen tube growth was developed. Pollen is placed in straight lines on an agar surface where it responds uniformly and predictably to aqueous solutions of germination-inhibiting substances located in wells at the ends of the lines. A scale of ratings, roughly corresponding to serial, doubled concentrations of inhibiting substances, was devised. Water soluble organic solvents are relatively noninhibitory, salts are variable, and metabolic inhibitors have strong inhibitory effects. Pollens differ in their susceptibility to inhibition and in their response to particular substances. PMID- 16658086 TI - A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides. AB - A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH(4) to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex 1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH(4) to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined. PMID- 16658087 TI - Spherosome membranes: half unit-membranes. AB - Spherosomes are bounded by unusual single-line "membranes" which measure 2 to 3.5 nanometers in width, contrasted to the well known tripartite unit-membranes which measure 6 to 8.5 nanometers in over-all thickness. Juxtaposed externally (from the side addressing the hyaloplasm), two spherosomal membranes adjoin to form a thicker single line, but apposed internally (the sides that contact stored lipid) two single-line membranes touch to form a tripartite structure resembling a unit membrane. Morphologically, we interpret the single-line membranes of spherosomes as half unit-membranes whose polar surfaces face the hyaloplasm and whose lipoidal nonpolar surfaces contact internal storage lipid.Corroboration of this interpretation was shown biochemically by demonstrating the presence of membrane structural protein in peanut spherosomes. In addition, an immunological identity between membrane protein isolated from spherosomes of quiescent seeds and membrane protein extracted from the mitochondrial fraction of 10-day germinated seedlings was observed. We conclude that the atypical, single-line membranes bounding spherosomes are in fact biological membranes that correspond to half unit-membranes. PMID- 16658088 TI - A condensation type porometer for field use. AB - A double cup transpiration porometer is described which simultaneously measures the resistance to water vapor diffusion from the upper and lower epidermises of a leaf. In use the cups are dried, the porometer is clamped on the leaf, and the transit time required for water vapor to condense on a cold (0 C) reflective surface in each cup is recorded. Since the sensing element remains at constant temperature, only transit time and leaf temperature are required to calculate leaf resistance. PMID- 16658089 TI - Peroxidase isozymes of first internodes of sorghum: tissue and intracellular localization and multiple peaks of activity isolated by gel filtration chromatography. AB - Electrophoretic analyses using Sepraphore III strips indicate the presence of a minimum of five bands of peroxidase activity detectable with o-dianisidine and H(2)O(2) in extracts from first internodes of Sorghum vulgare var. Wheatland milo. Three of these isozymes were anodic and two were cathodic forms at pH 8.3. The relative amounts of these forms are compared in zero time and incubated excised internodes, stelar and cortical tissues of internodes, and in other parts of the plant. Localization of these isozymes with respect to walls and cytoplasm was characterized by differential centrifugation after grinding of the internodes and by an in situ extraction of walls by centrifugation after vacuum infiltration. Using the latter in situ method, 32% of the total activity of the fast moving cathodic form was exchanged from the wall after infiltration with 50 mm CaCl(2). Only trace amounts of the other isozymes were localized in the walls of the cortex. The isozymes were eluted as two peaks from columns of Sephadex G 100 and three peaks from Agarose A-15m. Although such groupings may be due to asymmetric molecules and ionic interactions as well as to molecular weight differences, they may indicate associations with complexes or membranes of different cytoplasmic constituents. PMID- 16658090 TI - Changes in indoleacetic Acid oxidase isoenzymes in tobacco tissues after treatment with 2,4-dichlorophenoxyacetic Acid. AB - 2,4-Dichlorophenoxyacetic acid had a multiple effect on the development of indoleacetic acid oxidase isoenzymes in tobacco callus tissues (Nicotiana tabacum, cv. White Gold) cultured in vitro, and the development of these isoenzymes was differentially associated with growth promotion or inhibition depending on the concentration of 2,4-dichlorophenoxyacetic acid. At low concentrations (0.1 to 1 mum) it promoted the development of a fast migrating isoenzyme A(5) accompanied by stimulation of a tumor-type growth. At high concentrations (10 to 100 mum), it inhibited the development of the fast migrating isoenzymes but promoted a sharp rise in others with slower electrophoretic mobilities, which was accompanied by growth inhibition. The implications are that 2,4-dichlorophenoxyacetic acid might alter the level of endogenous auxins through its dual effects on the oxidase isoenzyme system. PMID- 16658091 TI - Regulation of cell division and cell enlargement by turgor pressure. AB - Isolated radish (Raphanus sativus L., var. Red Prince) cotyledons were incubated in growth medium plus graded concentrations of mannitol (-1 to -16 bars) for 28 hours. At the end of the incubation period, turgor pressures were measured using thermocouple psychrometers. Cell division, as measured by DNA increase, was greatly stimulated by increasing turgor from 5 to 6 bars. Cell enlargement was stimulated as turgor increased above 3 bars. The critical turgor pressure for increased cell division thus appeared significantly greater than that for increased cell enlargement. PMID- 16658092 TI - Pollen germination and tube growth: dependent on carbon dioxide and independent of ethylene. AB - The influence of ethylene and CO(2) on pollen germination and tube growth was investigated employing ventilated culture systems. Ethylene had no effect on pollen germinability or tube growth. Germinating pollen did not produce a detectable amount of ethylene (less than 0.1 nl/g.hr). Supplementing the cultures with CO(2) caused a marked increase in germination and tube growth. The half maximal response for germination was less than 0.5%. CO(2) levels ranging from 1.08 to 2.22% were found in the internal cavity of lily styles. CO(2) derived from stylar metabolism may, therefore, modulate pollen tube growth thus integrating the events leading to fertilization. PMID- 16658093 TI - High photosynthetic rate of a chlorophyll mutant of cotton. AB - In a chlorophyll mutant (virescent) and wild-type cotton (Gossypium hirsutum L.), a number of photosynthetic parameters have been measured and compared with those published for other chlorophyll mutants. (a) The photosynthetic rates at 230 w/m(2) (400-700 nm) from a tungsten lamp were 36.8 mg CO(2) fixed/dm(2).hr (virescent) and 39.5 mg CO(2) fixed/dm(2).hr (wild-type). On a chlorphyll basis, the photosynthetic rates were 36.8 and 12.1 mg CO(2) fixed/mg chl.hr, respectively. (b) The photosynthetic rates at 13 w/m(2) (400-700 nm) from a tungsten source were 7.1 mg CO(2) fixed/dm(2).hr (virescent) and 7.4 mg CO(2) fixed/dm(2).hr (wild-type). On a chlorophyll basis, the photosynthetic rates were 6.0 and 1.4 mg CO(2) fixed/mg chl.hr, respectively. (c) The chlorophyll a/b ratios of the virescent and wild-type leaves were 3.3 and 4.1 (d) The chlorophyll/carotenoid ratios for the virescent and wild-type leaves were 3.2 and 7.3, respectively. (e) The photosynthetic carbon metabolism of the chlorophyll mutant was through the reductive pentose phosphate cycle. (f) The CO(2) compensation points for the virescent and wild-type plants were similar. (g) The mutant and wild-type leaves have the same quantum yield in the red part of the visible spectrum, but the virescent leaves have a lower quantum yield in the blue part of the spectrum. (h) Virescent and wild-type leaves contain similar levels on a protein basis of several reductive pentose phosphate cycle enzymes. PMID- 16658094 TI - Nuclear gene affecting greening in virescent peanut leaves. AB - Chlorophyll synthesis induced by continuous illumination of dark-grown seedlings has been followed in wild-type and virescent peanut leaves. Compared to the wild type leaves, chlorophyll synthesis in the virescent leaves shows a 72-hour lag period before the onset of a phase of rapid chlorophyll accumulation. The development of chloroplast grana and the activity of many enzymes of the reductive pentose phosphate cycle, phosphoenolpyruvate carboxylase, and malate dehydrogenase are reduced in the virescent leaves during the lag phase of chlorophyll accumulation. Although nucleic acid synthesis in the virescent leaves in normal, there is a distinctly lower rate of protein synthesis. The low level of protein synthesis during the lag period might limit the synthesis of a factor(s) essential for the development of both cell and chloroplast constituents. PMID- 16658095 TI - Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus. AB - l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 mum. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d glucosamine acts as a feedback inhibitor of the enzyme. PMID- 16658096 TI - Nonrandom bioelectrical signals in plant tissue. AB - The results of investigations on nonevoked bioelectrical activity in the India rubber tree (Ficus elastica) are presented. Metal electrodes inserted into the plant issue were used as the ionic-to-electronic conduction converting elements. Nonevoked pulse bursts were observed with amplitudes in the 10 to 200 microvolts range. An upper limit value of the cell refractory period has been estimated from the maximum pulse frequency observed. PMID- 16658097 TI - A C-assay for photorespiration in aquatic plants. AB - The (14)C-assay developed by Zelitch to evaluate photorespiration was modified for use in submersed aquatic plants in the laboratory and in situ. Results in laboratory cultures of Najas flexilis suggest that photorespiration occurs but is limited in comparison to terrestrial C(3) plants and is related to the rate of diffusion of CO(2) from the plant and to the carrying capacity of water for dissolved oxygen. PMID- 16658098 TI - Photoconversion of riboflavin to lumichrome in plant tissues. AB - Free flavins have been extracted from shoots of etiolated corn (Zea mays L., var. Burpee Snowcross) and from yeast cells and separated from other substances by absorption on resorcinol-formaldehyde resin and talc columns and by thin layer chromatography. Riboflavin was the only free flavin present. Extracts of etiolated shoots of oats (Avena sativa L., var. Multiline E-69 and Clinford) yielded riboflavin plus a second free flavin previously demonstrated in oats. The areas of the chromatograms expected to contain lumichrome were completely clear. After illumination of any of the three organisms with artificial light (1100 ft c) or sunlight for 6 hours, lumichrome (7,8-dimethylalloxazine) was found. In corn shoots after irradiation by sunlight, the amount of lumichrome present was equivalent to 2.5% of the total free flavin. Lumichrome was identified by thin layer chromatography in six solvent systems (including two two-dimensional systems), by its characteristic fluorescence in acetic acid, by its absorption spectrum, and by formation of a characteristic hydrate in ammonia-containing solutions. A comparison was made with in vitro photolysis of riboflavin and the possible role of photolysis of riboflavin (either free or bound) and of lumichrome formation in photo-responses of plants is discussed. Placing the shoots in the dark for 4 hours after irradiation in sunlight for 6 hours led to no detectable loss of the lumichrome which had been formed. PMID- 16658099 TI - Sensitivity to stimulation, a component of the circadian rhythm in luminescence in gonyaulax. AB - A new method for the stimulation of bioluminescence in the dinoflagellate Gonyaulax polyedra is described. With this technique, in which cells flow through a capillary coil, it is possible to graduate the intensity of the stimulus by varying the flow rate. In continuous darkness, the threshold stimulus for cells in the middle of the day phase is greater than that for cells in the middle of the night phase. Some evidence suggests heterogeneity of sensitivity to stimulation among either cells or individual luminescent sources within a cell. At stimulus intensities much above threshold, the luminescence of both day- and night-phase cells is proportional to the number of cells within the capillary coil. Night-phase cells emit about 14 times as much light as do day-phase cells in continuous darkness.Single bioluminescent flashes from cells were recorded with a high speed camera. No significant difference in flash kinetics was found between cells in the day and the night phase in continuous darkness. Cells in the night phase emit a flash three to five times brighter than that from day-phase cells. About twice as many flashes are recorded in a given time from a population of night-phase cells.The activity of both luciferin and luciferase have been shown to vary rhythmically. The differences in threshold and number of flahses are evidence for a second component of the circadian rhythm in luminescence, a rhythm in sensitivity to stimulation. PMID- 16658100 TI - Ethylene evolution from cucumber plants as related to sex expression. AB - Ethylene evolved from monoecious and gynoecious cucumber (Cucumis sativus) plants grown under short and long day conditions was determined. More ethylene was evolved from floral buds and apices bearing buds than from whole seedlings of comparable weight. More ethylene also was evolved from apices of the gynoecious than from those of the monoecious type. Furthermore, quantities evolved from female buds were greater than from male ones and plants grown under short day conditions which promote femaleness evolved more ethylene than those grown under long day conditions. The data suggest that ethylene participates in the endogenous regulation of sex expression by promoting femaleness. PMID- 16658101 TI - The metabolism of organic acids by a marine pennate diatom. AB - Cocconeis diminuta, a marine benthic diatom, metabolizes acetate and lactate (14)C. In the light, the major product was lipid, whereas in the dark, CO(2) was the major product. Analysis of proteins synthesized in the presence of acetate or lactate showed that radioactivity was incorporated predominantly into the glutamate family of amino acids and those amino acids related directly to the substrate. Light and dark assimilation of substrate was inhibited slightly by 3 (3',4'-dichlorophenyl)-1,1-dimethylurea and 2,4-dinitrophenol. 3-(3',4' Dichlorophenyl)-1,1-dimethylurea caused a pattern of metabolism of acetate in the light characteristic of that which occurs in the dark. Monofluoroacetic acid inhibited assimilation considerably in the dark, but less in the light. The level of enzymes of the tricarboxylic acid cycle and NADH-oxidase were found to be about the same as those in other autotrophs. The metabolism of acetate and lactate is discussed in relation to the autotrophic mode of nutrition of Cocconeis diminuta. PMID- 16658102 TI - The influence of gibberellic Acid on the permeability of model membrane systems. AB - Gibberellic acid increases the permeability of model membranes composed of various plant-source lipids, a sterol, and dicetyl phosphate. As a result of hormone treatment, the flux of uncharged molecules such as glucose or sucrose, or charged ions such as chromate, through the model membranes (liposomes or micelles) is increased. The revelance of this finding to the in vivo effects of the hormone is briefly discussed. PMID- 16658103 TI - Comparison of the effectiveness of glycolic Acid and glycine as substrates for photorespiration. AB - Considerable evidence exists that the carboxyl-carbon atom of glycolic acid is the primary source of the CO(2) produced during photorespiration by leaves of many species of plants, including tobacco. Experiments were conducted to determine whether glyoxylate or glycine, both products of glycolic acid metabolism, is the more immediate precursor of photorespiratory CO(2).Illuminated tobacco leaf disks were floated on 18 mm solutions of glycolate-1-(14)C or glycine-1-(14)C in CO(2)-free air. The (14)CO(2) released and the (14)C content of several postulated intermediates were determined when the substrate solutions were provided alone or with one of the following: 9 mm alpha-hydroxy-2-pyridine methanesulfonic acid, an inhibitor of the oxidation of glycolate to glyoxylate; 9 mm isonicotinyl hydrazide, an inhibitor of the conversion of glycine to serine; or 18 mm nonradioactive glycine or glycolate with the other radioactive substrate.Both inhibitors decreased the rate of photorespiration in tobacco leaf disks by the (14)C-assay. The alpha-hydroxy-2-pyridine-methanesulfonic acid severely blocked (14)CO(2) production and labeling of the glycolate pathway from glycolate-1-(14)C. Isonicotinyl hydrazide had little effect on the (14)CO(2) released from glycine-1-(14)C although the glycine to serine conversion was severely inhibited.These results and other data in the literature indicate that the glycolate pathway of carbohydrate metabolism does not supply sufficient CO(2) during the synthesis of serine from glycine to account for the rates of photorespiration observed in many species. A direct decarboxylation of glyoxylate is more likely the main source of photorespiratory CO(2). PMID- 16658104 TI - Comparison of zeatin indoleacetate with zeatin and indoleacetic Acid in the tobacco bioassay. AB - Zeatin indole-3-acetate, 6-[4-(indole-3-acetoxy)-3-methyl-trans-2 butenylamino]purine, is at least as effective as zeatin on a molar basis in satisfying the cytokinin requirement for growth and bud formation in tobacco bioassays. It is less effective than indole-3-acetic acid and is needed as a variable function of the cytokinin concentration for satisfying the optimal requirement of an auxin. Comparisons of the types of growth and yield of tissue obtained with serial concentration of the ester and with equimolar mixtures of its free base and acid indicate that the relative requirement for auxin changes with the concentration of cytokinin and is related to the types of callus growth and differentiation which occur. The results also suggest that the ester serves as a source of auxin only after modification, presumably by hydrolysis to indoleacetic acid. PMID- 16658105 TI - Effect of Ethylene on Cell Division and Deoxyribonucleic Acid Synthesis in Pisum sativum. AB - Ethylene and supraoptimal levels of 2,4-dichlorophenoxyacetic acid inhibit the growth of the apical hook region of etiolated Pisum sativum (var. Alaska) seedlings by stopping almost all cell divisions. Cells are prevented from entering prophase. The hormones also retard cell division in intact root tips and completely stop the process in lateral buds. The latter inhibition is reversed partially by benzyl adenine. In root tips and the stem plumular and subhook regions, ethylene inhibits DNA synthesis. The magnitude of this inhibition is correlated with the degree of repression of cell division in meristematic tissue, suggesting that the effect on cell division results from a lack of DNA synthesis. Ethylene inhibits cell division within a few hours with a dose-response curve similar to that for most other actions of the gas. Experiments with seedlings grown under hypobaric conditions suggest that the gas naturally controls plumular expansion and cell division in the apical region. PMID- 16658106 TI - Effects of Ethylene and 2,4-Dichlorophenoxyacetic Acid on Cellular Expansion in Pisum sativum. AB - Ethylene inhibits growth in the subhook region of intact etiolated pea seedlings (Pisum sativum, var. Alaska) by reducing the capacity of the polar auxin transport system supplying auxin to the tissue. Application of 0.1 mm 2,4 dichlorophenoxyacetic acid reverses the growth inhibition caused by ethylene, and stimulates formation of sufficient gas to induce a swelling response in the absence of applied ethylene. Added ethylene causes a further swelling response but no change in growth rate when 0.1 mm 2,4-dichlorophenoxyacetic acid is present. If ethylene produced in response to 0.1 mm 2,4-dichlorophenoxyacetic acid is removed by hypobaric conditions, tissue swelling is prevented but the growth rate is not altered. Reducing the pressure also does not affect the growth rate of control plants. A higher concentration of 2,4-dichlorophenoxyacetic acid (1 mm) acts in a similar manner except that it also depresses growth through direct herbicidal action, whereas 0.1 mm 2,4-dichlorophenoxyacetic acid stimulates growth. Applied and auxin-induced ethylene prolong the phase of cellular expansion in both etiolated and light-grown seedlings. As long as ethylene is present, growth continues, glucose is incorporated into the cell wall, and the wall weight increases in proportion to tissue fresh weight. When ethylene is removed, glucose incorporation into the cell wall decreases and growth ceases. PMID- 16658107 TI - Relation of Phytochrome-enhanced Geotropic Sensitivity to Ethylene Production. AB - Brief exposure of etiolated pea (Pisum sativum cv. Alaska) seedlings to red light enhances subsequent development of geotropic curvature of the stem. Both this response and inhibition of ethylene production by red light become maximal 8 hours after illumination. Very low concentrations of applied ethylene inhibit development of geotropic curvature, whereas hypobaric treatment enhances geotropic sensitivity by removing endogenous ethylene. Increased geotropic sensitivity after illumination is accompanied by increased lateral migration of (3)H-indoleacetic acid in response to gravity, and ethylene inhibits this lateral migration. It is suggested, therefore, that red light-enhanced geotropic sensitivity is caused by increased lateral auxin transport resulting from a reduction in ethylene production after illumination. PMID- 16658108 TI - The Gas Exchange of Hydrogen-adapted Algae as Followed by Mass Spectrometry. AB - A mass spectrometer inlet and an oxygen electrode in the same vessel allowed the continuous recording of the gases exchanged (H(2), CO(2), O(2)) by hydrogenase containing anaerobically adapted Scenedesmus obliquus strain D(3) (Gaffron) and Chlorella fusca Shihira et Krauss (= pyrenoidosa) 211-15. A light intensity which produces more photosynthetic oxygen than the cells can re-reduce to water leads to de-adaptation and the substitution of normal photosynthesis for photoreduction. The sequence of these metabolic events was recorded in a matter of a few minutes. Upon exposure of these adapted algae to light, an evolution of hydrogen lasting up to 60 seconds preceded any other light-dependent gas exchange. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, this initial hydrogen production was inhibited approximately 50%, pointing to a contribution of electrons by photosystem II. At very low hydrogen tensions (0.1 microliter per milliliter), a balance between light-induced production and absorption of hydrogen was observed in normal, unpoisoned algae. Addition of either glucose or inhibitors of phosphorylation increased the release of hydrogen in the light very considerably. When the light was turned off the algae consumed the remaining amount of hydrogen, only to release it again upon illumination. This reversible hydrogen exchange persisted even when any concomitant carbon dioxide exchange had been abolished. PMID- 16658109 TI - The development of photosynthesis in a greening mutant of chlorella and an analysis of the light saturation curve. AB - Photosynthetic oxygen evolution considerably precedes the rise in chlorophyll during the greening of a yellow mutant of Chlorella vulgaris. Dark-grown cells required 20 times more light to saturate photosynthesis than light-grown or normal cells. The chlorophyll appears to add first to active reaction centers, then to fill in a more general antenna. The carotenoid pigments seem to add more randomly to the reaction centers. The shape of the light saturation curves can be explained with the assumption that an excitation in the antenna can reach several reaction centers. The efficiency of the total unit is constant during the greening process. PMID- 16658110 TI - Photosynthesis in Eurasian Watermilfoil (Myriophyllum spicatum L.). AB - Gas exchange of Eurasian watermilfoil (Myriophyllum spicatum L.) indicated a near zero CO(2) compensation point and a high temperature optimum for photosynthesis. These properties are characteristic of plants fixing CO(2) by a beta carboxylation mechanism. Operation of the Calvin cycle was shown and no evidence for beta-carboxylation was obtained. These results indicate that near-zero CO(2) compensation points are not dependent on a beta-carboxylation mechanism. PMID- 16658111 TI - Indoleacetic Acid oxidase: a dual catalytic enzyme? AB - The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme. PMID- 16658112 TI - Polyamines in soybeans. AB - Putrescine, spermidine, and spermine were three main polyamines isolated from soybeans and partially characterized. Occurrence of polyamines in soybeans was established by separating trichloroacetic acid extracts of soybeans by cationic exchange column chromatography, identification with thin layer chromatography, paper electrophoresis, mass spectral analysis, reactions with ninhydrin and Dragendorff reagents, and spectrophotometric characteristics. Soybeans contained a minimum of 29.0 micrograms of polyamines per gram of full-fat flour. The alcohol-soluble fraction of soybeans contained polyamines also. Resting seeds contained spermidine in higher concentration than either putrescine or spermine. Spermine appeared to be present in lowest concentration. Preliminary experiments suggested that some polyamines were possibly in bound forms. PMID- 16658113 TI - Promotion of peroxidase activity in the cell wall of Nicotiana. AB - Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5 triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5 triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin. PMID- 16658114 TI - Biochemical and Biophysical Characteristics of a Photosynthetic Mutant of Euglena gracilis Blocked in Photosystem II. AB - A photosynthetic mutant of Euglena gracilis, Z strain, thought to be blocked in the electron transport chain between the two photosystems and to have a missing or nonfunctional primary acceptor for photosystem II, was further studied and characterized. The data from low temperature fluorescence spectra, delayed light emission, and electron paramagnetic resonance support the previous work.The mutant was shown to lack plastoquinone A and possibly cytochrome b(559) but to possess plastoquinone B and a higher complement of carotenoids, especially xanthophylls, than the wild type.The results are consistent with the postulated role of plastoquinone as the primary electron acceptor for photosystem II and as responsible for the electron paramagnetic resonance signal II. The abundance of xanthophylls in the mutant can be explained by the protective role of carotenoids against photosensitized reactions. PMID- 16658115 TI - Structural Characteristics of a Photosynthetic Mutant of Euglena gracilis Blocked in Photosystem II. AB - The techniques of thin sectioning and freeze etching were employed in comparing the chloroplast structure of the wild type and photosynthetic mutant P(4) of Euglena gracilis, Z strain. The mutant chloroplasts were characterized by a lack of thylakoid pairing even under high salt conditions. In addition the mutant thylakoids were more varied in size and fewer in number than those of the wild type. No differences between the mutant and wild type were observed in the size and distribution of the particles within the chloroplast membranes seen by the freeze-etching technique.These structural abnormalities do not appear to be correlated per se with the absence of plastoquinone A in the mutant but may be related to a different lipid composition observed in the mutant. PMID- 16658116 TI - Effects of medium composition and carbon dioxide on circadian conidiation in neurospora. AB - Efforts to significantly perturb the timing mechanism, and thus the period, of the rhythm responsible for circadian conidiation in bd, a strain of Neurospora crassa, by altering the medium composition have been unsuccessful. Various salt solutions, sugars, and amino acids do, however, have pronounced effects on growth and conidiation, and thus on the expression and persistence of rhythmicity.Aeration stimulates conidiation in growth-tube cultures, thereby allowing experiments which demonstrate that nearly all strains and species of Neurospora are capable of circadian conidiation. These results extend to Neurospora the generalization that physiological and developmental regulation in all eukaryotic organisms has a circadian component. Aeration also increases the persistence of circadian conidiation suggesting that the damping of rhythmicity previously observed on certain media represents the cessation of conidiation, rather than the stopping of a timing mechanism.Aeration is apparently effective in that it maintains CO(2) levels in growth-tube cultures below a critical concentration. Carbon dioxide was shown to inhibit conidiation in both wildtype and bd strains, with the latter being about 200 times more resistant than the former. PMID- 16658117 TI - Purification and Partial Characterization of a Glucan Containing Indole-3-acetic Acid. AB - The "bound auxin" of Zea mays, first described by Berger and Avery (Amer. J. Bot. 1944; 31: 199-203) has been purified and partially characterized. It is an indole 3-acetic acid-containing, high molecular weight, lipophilic cellulosicglucan. The indole-3-acetic acid is in ester linkage as evidenced by indoleacetamide formation upon ammonolysis. The glucan is of variable chain length and comprises, in general, 35 to 50 per cent of the dry weight of the compound. The glucosidic residues are beta 1 --> 4 linked and are hydrolyzed by cellulase. Mild acid hydrolysis produces cellobiose and cellotriose. Other components, as yet unidentified, of the compound are described. PMID- 16658118 TI - Action Spectrum of Cytochrome f Photooxidation in Greening Bean Leaves. PMID- 16658119 TI - The biosynthesis of metal porphyrins by subchloroplastic fractions. PMID- 16658120 TI - Equal Quantal Spectra for the Effect of Light on the Growth of Conidiophores and for the Induction of a Circadian Rhythm of Zonation in Sclerotinia fructicola (Wint.) Rehm. PMID- 16658121 TI - Protein synthesis by isolated etioplasts and chloroplasts from pea and wheat and the effects of chloramphenicol and cycloheximide. AB - Etioplasts capable of incorporating (14)C-leucine into protein have been isolated from dark-grown pea and wheat plants. The requirements for leucine incorporation for etioplasts were similar to those for chloroplasts. An ATP-generating system, Mg(2+), and GTP were required. The amino-acid-incorporation activity of etioplasts from wheat was comparable to that of chloroplasts on an RNA basis, whereas the activity of pea etioplasts was about 50% of the activity of pea chloroplasts. The incorporation of leucine into protein by etioplasts and chloroplasts from pea and wheat was inhibited by chloramphenicol, and to a slight extent by cycloheximide. PMID- 16658122 TI - In situ measurement of root-water potential. PMID- 16658123 TI - Effect of abscisic Acid and its interactions with other plant hormones on ethylene production in two plant systems. PMID- 16658124 TI - Solute potentials of sucrose solutions. PMID- 16658125 TI - The Activity of beta-Ecdysone in Four Gibberellin Bioassays. PMID- 16658126 TI - Light-stimulated Production of a Chloroplast-localized System for Protein Synthesis in Euglena gracilis. AB - Chloroplasts and proplastids isolated respectively from autotrophic and dark adapted cells of Euglena gracilis strain Z incorporated (14)C-l-leucine into protein. In each case the incorporation was inhibited by chloramphenicol (50% inhibition at about 5 mug/ml for chloroplasts and 30 mug/ml for proplastids), but not appreciably by cycloheximide at concentrations up to 200 mug/ml. Chloroplasts from autotrophic cells incorporated leucine into protein at rates of about 10 pg leucine per mg RNA in one minute, but isolated proplastids were only 5 to 10% as active. When dark-adapted cells were illuminated there was little increase in the activity of the chloroplast fraction during the first 12 hr. Between 12 and 24 hr, when there was a rapid increase in the rate of synthesis of chlorophyll, the capacity of the chloroplast fraction for protein synthesis increased markedly. Suppression of the formation of a chloroplast-localized system for protein synthesis by treating the cells with chloramphenicol and the lack of such an effect with cycloheximide suggests that certain of the proteins which form part of a functional chloroplast system for protein synthesis are themselves synthesized within the chloroplasts. PMID- 16658127 TI - A Re-evaluation of Soybean Leaf Photorespiration. AB - Recalculations of soybean photorespiration indicate that mean rates are closer to 16.1 than 5.6 milligrams of CO(2) per square decimeter per hour as previously reported. Photorespiration of soybean thus amounts to at least a 30% carbon turnover of light-saturated photosynthesis. Photorespiration showed no significant relationship to net photosynthesis. Negative correlations were found between CO(2) efflux and stomatal resistance as well as between corrected photorespiration and residual intracellular resistance of the leaf to CO(2) uptake. PMID- 16658128 TI - Lipid Composition of Pea and Bean Leaves during Chloroplast Development. AB - The changes in composition of the complex lipids were followed during the greening of dark-grown pea (Pisum sativum) and bean (Phaseolus vulgaris) seedlings. No significant changes in glycerolipid concentrations in the leaves were observed during the early stages of greening (0-8 hour for peas and 0-12 hour for beans). On further greening, there was an increase in the proportion of galactolipids and a decrease in the phospholipids. The fatty acid composition of the galactolipids remained constant during 24 hours of greening, but there was a slight increase in alpha-linolenic acid at 72 hours in the bean. The percentage of alpha-linolenic acid in the phospholipids and in sulfolipid showed a marked increase between 24 and 72 hours in the bean. Trans-delta(3)-hexadecenoic acid was the major fatty acid of phosphatidyl glycerol in bean leaves at 72 hours, but it was barely detectable at 24 hours. The lipid composition of greening leaves is discussed in relation to the fine structure and photochemical activity of the developing plastids. PMID- 16658129 TI - Control and Kinetics of Branch Root Formation in Cultured Root Segments of Haplopappus ravenii. AB - Branch root formation required only the presence of minerals, sucrose as a carbon source, and an auxin. The number of primordia formed was a function of auxin concentration. With naphthaleneacetic acid at 0.1 mg/l, up to 60 or more branches were formed per centimeter of Haplopappus ravenii root segment. Under our conditions, pea root segments formed only five or six branches per centimeter, but tomato and radish, like H. ravenii, formed large numbers of branches. Cytokinin inhibited branch formation, while gibberellic acid was without effect. Vitamins were not required for branch formation, although they enhanced elongation. Up to 5 days were required for the maximum number of stable branch primordia to form under the influence of naphthaleneacetic acid. If naphthaleneacetic acid was withdrawn earlier, fewer branch primordia developed. The requirement for a lengthy exposure to naphthaleneacetic acid, the kinetics of the response, and the ease with which naphthaleneacetic acid could be rinsed out of the tissue with consequent cessation of branch root formation, were similar to other hormone-regulated developmental systems. Anatomical and cytological studies were made of segments exposed for various times to auxin. The segments were mostly diarch, and branches formed obliquely to protoxylem poles. While primarily only pericycle-endodermis cells divided, both these and cortex cells responded in the first 24 hours exposure to naphthaleneacetic acid with enlarged nuclei and nucleoli, and a few cortical cells divided. Maximum nucleus and nucleolus size was reached approximately 9 hours after exposure to naphthaleneacetic acid. Branches rarely elongated more than 5 cm before their meristems died. The H. ravenii culture is maintained only by the frequent formation of new naphthaleneacetic acid-induced branches. PMID- 16658130 TI - An intermediate in the synthesis of glucobrassicins from 3-indoleacetaldoxime by woad leaves. AB - Leaves of woad (Isatis tinctoria L.) were found to incorporate efficiently tritiated indoleacetaldoxime and (35)S from (35)S-l-cystine into glucobrassicin and sulfoglucobrassicin. Time course of incorporation of (35)S from (35)S-cystine into the glucosinolates indicated that glucobrassicin was formed first and then sulfoglucobrassicin. Simultaneous administration of tritiated indoleacetaldoxime and (35)S-cystine gave doubly labeled glucobrassicin and sulfoglucobrassicin. About twice as much (35)S was present in sulfoglucobrassicin as compared to glucobrassicin per unit of (3)H incorporated, indicating that a second, probably oxidized, atom of (35)S was later introduced into sulfoglucobrassicin. However, the (35)S incorporated from cystine into both glucosinolates during the first 8 hours of metabolism was almost exclusively in the divalent sulfur moiety. The incorporation patterns of (35)S and titritated indoleacetaldoxime into the glucosinolates suggested a fast turnover of glucobrassicin in the metabolizing leaves.A new indolic, sulfur-containing neutral compound X was found to accumulate in woad leaves when administered (3)H-3-indoleacetaldoxime and cold cystine or (35)S-cystine and cold 3-indoleacetaldoxime. This accumulation was enhanced about 2- to 2.5-fold by the simultaneous administration of postassium selenate, an inhibitor of biological sulfation processes. Selenate also appeared to inhibit the conversion of glucobrassicin to 1-sulfoglucobrassicin. Partially purified compound X was efficiently converted (56-60%) to glucobrassicin and 1 sulfoglucobrassicin on readministration to woad leaves, indicating it to be a precursor of the glucosinolates. Compound X, on treatment with myrosinase, slowly yielded a less polar, indolic, sulfur containing compound Y and glucose. Compound Y decomposed with time into indoleacetonitrile suggesting that it may be indoleacetothiohydroximate. Compound X has been tentatively assigned the structure of desthioglucobrassicin, the nonsulfated form of glucobrassicin. PMID- 16658131 TI - Intracellular distribution of mitochondria after geotropic stimulation of the oat coleoptile. AB - The number of mitochondria is greater in the bottom than in the top of cells in geotropically stimulated oat (Avena sativa) coleoptiles. In the avascular tip and outer epidermis of subapical regions this difference occurs only in the lower tissues. These inequalities are found both in the KMnO(4) (-) and in the glutaraldehyde-fixed tissues; however, they are significant only in the former. Also, the number of mitochondria scored is consistently lower when the tissues were fixed in KMnO(4). These results suggest that mitochondria undergo a small degree of sedimentation after geostimulation, a redistribution reduced by the slower fixation with glutaraldehyde. Differences in mitochondrial number begin later than those in the amyloplast and the Golgi apparatus after geotropic stimulation. The cells in the avascular-tip region (a region having an important role in geotropism) have two to three times more mitochondria than the subapical cells. PMID- 16658132 TI - Isolation of Plastids from Sunflower Cotyledons during Germination. AB - Plastids from cotyledons of sunflower (Helianthus annus L.) seedlings, germinated in the dark or in the light, were isolated by isopycnic sucrose density gradient centrifugation. At all stages of development the whole plastids contained triose phosphate isomerase, NADPH-glyoxylate reductase, and l-dihydroxyphenylalanine oxidase, which were used as marker enzymes. At the beginning of germination the isopycnic density of whole plastids (proplastids) was about 1.22 g cm(-3). During development of proplastids into etioplasts in the dark, their isopycnic density increased to 1.26 g cm(-3). During exposure of germinating seedlings to white light for 2 days, the isopycnic density of whole plastids decreased from 1.26 to 1.22 g cm(-3). These changes in isopycnic density of plastids on sucrose density gradients are consistent with changes in the plastid ultrastructure caused by the protein-rich prolamellar body or by the lipid-rich thylakoids. Broken plastids (thylakoids), determined by the main peak of chlorophyll, increased in isopycnic density from less than 1.14 to about 1.17 g cm(-3) during illumination. During germination no major changes occurred in the isopycnic density of mitochondria. Microbodies had an isopycnic density of 1.24 g cm(-3) in very early stages of germination, and their density increased to 1.265 g cm(-3), when glyoxysomal enzymes reached maximum development. PMID- 16658133 TI - Boundary Layer Resistance and Temperature Distribution on Still and Flapping Leaves: II. Field Experiments. AB - The forced convection of heat from reed (Phragmites communis) leaves was observed in their natural environment. The leaves were painted with liquid crystals, which displayed or indicated their temperature without any interference with natural air flow. Temperature differences as large as 15 C were observed between the leading and trailing edges of the nontranspiring, painted leaves. The turbulence of the natural wind decreased the boundary layer resistance around the leaf to about 40% of the resistance in a laminar steady wind. PMID- 16658134 TI - Radial transport of sodium and chloride into tomato root xylem. AB - Transport of Na and Cl across exuding tomato (Lycopersicon esculentum Mill.) roots was determined as a function of ambient NaCl concentrations in the ranges of both systems 1 and 2. Kinetics of radial transport under steady-state conditions and the effect of dinitrophenol indicate that Na and Cl were transported by two different mechanisms. Sodium was neither accumulated against a concentration gradient nor directly inhibited by dinitrophenol from diffusing into the xylem. Chloride was accumulated in the xylem and its transport was nearly completely blocked by dinitrophenol. A comparison of the radial transport isotherms for Na and Cl for intact and decapitated plants indicates that the separate mechanisms were not unique to excised roots. It is concluded that radial Na transport in tomatoes was facilitated by a passive convective type process with the rate-limiting barrier located at the outer cortical plasmalemma. Chloride transport in both concentration ranges involved, either directly or indirectly, a metabolic mechanism. Absorption and retention of Na in the root tissue was negligible. Chloride was accumulated by the tissue but was unaffected by dinitrophenol. PMID- 16658135 TI - Sterol Changes during Germination of Nicotiana tabacum Seeds. AB - The identity, composition, and concentration of the total, free, esterified, and glycosidic sterol fractions were determined during germination of tobacco seeds. The total, free, and esterified sterols increased, with stigmasterol and campesterol accounting for most of the increase. Steryl glycosides decreased during germination, and stigmasteryl and sitosteryl glycosides showed the largest decrease. During germination, sitosterol was the major sterol in all fractions but stigmasterol and campesterol showed the greatest changes. The fatty acid composition of the steryl esters and acylated steryl glycosides most closely resembled the di- and triglycerides. PMID- 16658136 TI - The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum: I. Utilization of Injected Stigmatic Exudate. AB - A quantity of labeled stigmatic exudate, collected from detached Lilium longiflorum (cv. Ace) pistils labeled with d-glucose-1-(14)C, was fractionated on Sephadex G-100 and the polysaccharide component, G-100-I, was injected into the hollow styles of unlabeled detached pistils (cv. Ace) which had been removed on the day after anthesis from the plant. Injected pistils were immediately cross pollinated with L. longiflorum (cv. No. 44) pollen. Eighty-four hours later, pistils were dissected to recover the pollen tubes, expended exudate, and labeled tissues of the stigma and style. Distribution of label revealed that at least 25% of the carbohydrate substance in excised pollen tubes was derived from G-100-I. The composition of expended exudate adhering to pollen tubes, of pollen tube cytoplasm, and of pollen tube walls suggests that utilization of exudate by growing pollen tubes involves uptake and incorporation into pollen tube cytoplasm of exudate polysaccharide fragments followed by extensive metabolism of at least a portion of the incorporated carbohydrate prior to its utilization for pollen tube wall biosynthesis. Results suggest the presence of at least two polysaccharide components in G-100-I, one which resists major degradation following injection into the style and another which undergoes measurable degradation both before and after entry into the pollen tube. PMID- 16658137 TI - Changes in the Ribonuclease Activity of Flax Cotyledons following Inoculation with Flax Rust. AB - There was a significant increase in the ribonuclease activity of both resistant (Bombay) and susceptible (Bison) varieties of flax (Linum usitatissimum L.) 3 to 4 days after inoculation with flax rust (Melampsora lini [Pers.] Lev., race No. 3). A second and much greater increase in the activity of this enzyme occurred only in the susceptible host at later stages of disease development. While a similar increase in ribonuclease level was also caused by mechanical injury, evidence is presented showing qualitative differences between the enzyme from parasitized tissue and that from the mechanically injured cotyledons. Comparison of the enzyme from healthy and inoculated cotyledons and from flax rust revealed the presence of a relatively unstable component and some unique catalytic properties in the enzyme from inoculated cotyledons. PMID- 16658138 TI - The relative amounts and identification of some 2,4-dichlorophenoxyacetic Acid metabolites isolated from soybean cotyledon callus cultures. AB - Soybean (Glycine max L.) cotyledon callus grown on radioactive 2,4 dichlorophenoxyacetic acid (2,4-D-1-(14)C) as an auxin produced 2,4-D metabolites, which qualitatively and quantitatively changed with time. Water soluble fractions from the tissue exhibited a steady increase in radioactivity during the course of 24 days. Following beta-glucosidase treatment, at least eight aglycones were obtained from the water soluble fraction of the tissue after 8 days. The metabolite, 4-hydroxy-2,5-dichlorophenoxyacetic acid was the most abundant aglycone during the entire 32 day growth period while 4-hydroxy-2,3 dichlorophenoxyacetic acid was detected as a minor metabolite. Radioactivity in the ether soluble acidic fractions reached a maximum of 82% of the total in the tissue after 2 days. The level then decreased to 44% by the end of 24 days. A total of seven ether soluble components were detected. In addition to 2,4-D glutamic acid, which was detected in high amounts after 24 hours, 2,4-D aspartic acid was found to be the most abundant ether soluble metabolite after longer time periods. Mass spectral data and a fragmentation pattern are presented for 2,4-D aspartic acid. PMID- 16658139 TI - Effects of Hydroxylamine on Photosystem II: II. Photoreversal of the NH(2)OH Destruction of O(2) Evolution. AB - The inactivation of O(2)-evolving centers by NH(2)OH extraction was shown to be reversible. This reversal required light and manganese. This light-induced restoration of active O(2)-evolving centers was analyzed using three green algae and the blue-green alga, Anacystis nidulans. The following results were obtained: [List: see text]. PMID- 16658141 TI - Antagonistic effects of high and low temperature pretreatments on the germination and pregermination ethylene synthesis of lettuce seeds. AB - Red light-induced germination of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C was inhibited if the seeds were first imbibed at 30 C for 36 hours. This effect was counteracted by exogenous ethylene and associated with a reduction in the rate at which the seeds produced ethylene throughout the pregermination period. A chilling treatment reversed the effect of a prior imbibition at 30 C on both germination and ethylene production. The possibility that the pretreatments influence germination through their effects on ethylene production is discussed.Other evidence presented indicates that the inability of seeds to germinate at supraoptimal temperature is not due either to a rapid loss of far red-absorbing phytochrome or to an inadequate capacity for ethylene synthesis. It was also shown that a chilling treatment potentiated germination at high temperature without affecting the ethylene synthetic capacity of the seeds. PMID- 16658140 TI - The Respiratory Chain of Plant Mitochondria: XIV. Ordering of Ubiquinone, Flavoproteins, and Cytochromes in the Respiratory Chain. AB - The effect of initial oxygen concentration on the rate and extent of oxidation of the respiratory chain carriers of anaerobic mitochondria from mung bean (Phaseolus aureus) seedlings was examined. The substrate was succinate, with malonate added to give malonate to succinate ratios of 6 to 12, thereby minimizing the flow of reducing equivalents from substrate and insuring maximal extent of oxidation of the carriers. The ratio of oxidizing equivalents available from oxygen to reducing equivalents available from reduced ubiquinone, designated the equivalents ratio, varied from 30 to 1. Cytochromes aa(3) and c(547) have unaltered oxidation half-times, designated t((1/2) on), as the equivalents ratio is reduced from 30 to 3, and the extent of oxidation is decreased by about 25%. The time of the oxidation-reduction cycle induced by the oxygen pulse, calculated from the point of half oxidation to that of half reduction and designated t((1/2) off), decreases 200 fold with this reduction in equivalents ratio. The oxidation half-time, t((1/2) on), for ubiquinone is unaltered by decreasing the equivalents ratio from 6 to 1; the value of t((1/2) off) decreases only 30% while the extent of oxidation decreases 50%. The values of t((1/2) on) and t((1/2) off) and the extent of oxidation of cytochrome b(553) and flavoprotein Fp(ha) were all much reduced at low equivalents ratios. The results, plus results from previous studies, indicate that there is the following linear sequence of components in the plant respiratory chain:Dehydrogenase.UQ.Fp(ha).b(553). c(549).a.a(3)Cytochrome b(557) and the low potential flavoproteins do not fit into this linear sequence. PMID- 16658142 TI - Effect of cetyltrimethylammonium bromide on the activity of particulate starch synthetase from potato tuber. AB - The action of some detergents on the incorporation of glucose from uridine diphosphate glucose or adenosine diphosphate glucose into the potato tuber starch grain was studied. It was found that the cationic detergent, cetyltrimethylammonium bromide, produces a rapid binding of both sugar nucleotides to the grain and a great increase in the incorporation of glucose into the polysaccharide. Kinetic constants of starch synthetase are also modified, there being an affinity increase for both sugar nucleotides. Neutral detergents are without effect and anionic detergents are inhibitors. PMID- 16658143 TI - Obligatory reduction of ferric chelates in iron uptake by soybeans. AB - The contrasting Fe(2+) and Fe(3+) chelating properties of the synthetic chelators ethylenediaminedi (o-hydroxyphenylacetate) (EDDHA) and 4,7-di(4-phenylsulfonate) 1, 10-phenanthroline (bathophenanthrolinedisulfonate) (BPDS) were used to determine the valence form of Fe absorbed by soybean roots supplied with Fe(3+) chelates. EDDHA binds Fe(3+) strongly, but Fe(2+) weakly; BPDS binds Fe(2+) strongly but Fe(3+) weakly. Addition of an excess of BPDS to nutrient solutions containing Fe(3+)-chelates inhibited soybean Fe uptake-translocation by 99+%; [Fe(II) (BPDS)(3)](4-) accumulated in the nutrient solution. The addition of EDDHA caused little or no inhibition. These results were observed with topped and intact soybeans. Thus, separation and absorption of Fe from Fe(3+)-chelates appear to require reduction of Fe(3+)-chelate to Fe(2+)-chelate at the root, with Fe(2+) being the principal form of Fe absorbed by soybean. PMID- 16658144 TI - Plant Carbonic Anhydrases: I. Distribution of Types among Species. AB - On the basis of polyacrylamide gradient gel electrophoresis of leaf extracts from 24 species of higher plants, two main forms of carbonic anhydrase (EC 4.2.1.1) were recognized; the "dicotyledon" type and the "monocotyledon" type. More than one band of enzyme was found on gels from most species, suggesting the possibility of carbonic anhydrase isoenzymes in higher plants. PMID- 16658145 TI - Plant Carbonic Anhydrases: II. Preparation and Some Properties of Monocotyledon and Dicotyledon Enzyme Types. AB - Carbonic anhydrase (EC.4.2.1.1) was purified from leaves of the dicotyledon Pisum sativum L. (56-fold) and from leaves of the monocotyledon Tradescantia albiflora Kunth. (24-fold). The molecular weight of the Pisum enzyme was estimated to be 188,000 +/- 8,000 with subunit sizes of 28,000 +/- 3,000 and 56,600 +/- 3,500. It contained 1 mole zinc per 32,500 +/- 2,000 g protein. The molecular weight of the Tradescantia enzyme was estimated to be 42,000 +/- 2,000 with a subunit size of 27,500 +/- 2,200. It contained 1 mole zinc per 34,000 +/- 2,000 g protein. The two enzyme preparations were different in specific activity, stability in solution, and sensitivity to sulfonamides and inorganic anions. Gel electrophoresis separated each purified preparation into two active enzyme bands. PMID- 16658146 TI - Inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate. AB - 6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5 diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis. PMID- 16658147 TI - Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis. AB - The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.Only aspartate, alanine, glycine, serine, threonine, and lysine became labeled during photosynthetic (14)CO(2) fixation by isolated chloroplasts: the label in aspartate represented over 60% of the total (14)C found in the amino acids. Glutamate dehydrogenase, glutamate-oxaloacetate, and glutamate-pyruvate aminotransferases were present in the chloroplasts, but no other transferase activities from glutamate could be detected. The chloroplasts were able to synthesize a total of 17 other protein amino acids from either alanine or aspartate, but no synthesis of leucine by aminotransferase reactions could be detected. The synthesis of aspartate was studied in more detail. The enzyme systems required for the generation of oxaloacetate from triose phosphate were virtually absent from the chloroplasts but present in the leaf cytoplasmic fraction. Addition of either a leaf "cytoplasmic" fraction or an oxaloacetate generating system resulted in an increased proportion of the total (14)C fixed being found in the amino acid fraction during photosynthetic (14)CO(2) fixation.It is suggested that the supply of oxaloacetate from the cytoplasm is one of the important factors controlling the synthesis of amino acids by the chloroplast. PMID- 16658148 TI - Phytochrome-controlled Nyctinasty in Albizzia julibrissin: IV. Auxin Effects on Leaflet Movement and K Flux. AB - Indole-3-acetic acid, alpha-naphthylacetic acid, and 2,4-dichlorophenoxyacetic acid (0.001 to 1.0 mm) inhibit the nyctinastic closure of excised Albizzia leaflet pairs; antiauxins and auxin analogs are ineffective, and the auxin effects seem not to be mediated by ethylene. Indoleacetic acid (0.001 to 0.1 mm) also promotes rhythmic opening in the dark, but is ineffective during that phase of rhythmic closure ("leaky phase") which is insensitive to azide. At these concentrations, all of the indoleacetic acid effects are reversible upon transfer of the tissue to water and are linked to alteration of potassium flux in pulvinule motor cells.A supraoptimal concentration of indoleacetic acid (1 mm) inhibits rhythmic opening as well as nyctinastic closure, although it has little or no effect on potassium flux in motor cells. These inhibitions cannot be completely reversed by transferring the leaflets to water.Although indoleacetic acid (0.01 to 1.0 mm) inhibits leaflet opening and potassium flux in dorsal and ventral motor cells when leaflets are transferred from darkness to light, it has no effect during other portions of the light period, implying that changes in endogenous auxin do not control leaflet angle in the light. Neither does auxin seem to be involved in the phytochrome-regulated process, since it does not alter phytochrome control of leaflet movement or potassium flux. However, endogenous auxin probably plays an important role in controlling potassium flux into ventral motor cells during the opening phase of rhythmic leaflet movement in the dark. PMID- 16658149 TI - Microbody enzymes and carboxylases in sequential extracts from c(4) and c(3) leaves. AB - A seven-step sequential grinding procedure was applied to leaves of Atriplex rosea, Sorghum sudanense, and Spinacia oleracea to study the distribution of carboxylases and microbody enzymes. In the extracts from C(4) species there were 7- to 10-fold reciprocal changes in specific activities of ribulose-1, 5 diphosphate carboxylase and phosphoenolpyruvate carboxylase. No such changes occurred in sequential extracts from spinach. No inhibitors of ribulose-1, 5 diphosphate carboxylase were detected when the mesophyll extracts of Sorghum were assayed together with spinach extracts. These results reaffirm the conclusion of others that phosphoenolpyruvate carboxylase is largely confined to the mesophyll in these species and ribulose-1, 5-diphosphate carboxylase to the bundle sheath. The specific activities of glycolate oxidase and hydroxypyruvate reductase in bundle sheath extracts were two to three times those in mesophyll fractions. Catalase behaved similarly in Atriplex rosea but in Sorghum the specific activity was virtually the same in all fractions. From the relative amounts of these enzymes present, and comparison with the data obtained from spinach, it is concluded that typical leaf peroxisomes are present in the bundle sheaths of both C(4) species and in the mesophyll of Atriplex rosea. The relative enzyme activities in the mesophyll of Sorghum suggest that the microbodies there are of the non-specialized type found in many nongreen tissues. The activities of the microbody enzymes in the bundle sheath of Sorghum seem quite inadequate to support photorespiration. PMID- 16658150 TI - Phytochrome transformation in lettuce seed irradiated at various temperatures. AB - Phototransformation of phytochrome in lettuce seeds (Lactuca sativa L. var. Grand Rapids) was examined by testing germination responses of seeds irradiated at various temperatures. Temperature variations from 0 to 50 C had no influence on the germination of partially hydrated seeds (about 15% water content) irradiated with either red or far red light prior to imbibition. At -15 C far red light more effectively retarded germination than red light promoted it. No effective phototransformation was detected at -79 C or -196 C. PMID- 16658151 TI - Influence of Light Intensity on Reductive Pentose Phosphate Cycle Activity during Photoheterotrophic Growth of Rhodospirillum rubrum. AB - Light intensity during growth affects the proportion of carbon dioxide fixed by the reductive pentose phosphate cycle relative to that incorporated via C(4) acids in acetate phototrophs of Rhodospirillum rubrum. With cells grown at high light intensity (9000 lux) the specific activities of ribulose-1, 5-diphosphate and propionyl CoA carboxylases were increased compared with cells grown at low light intensity (1500 lux), although pyruvate carboxylase activity was unaltered.Kinetic experiments with cells assimilating acetate at high light intensity showed that when the cells had been grown at high light intensity there was a rapid incorporation of (14)CO(2) into phosphate esters compared with cells grown at low light intensity and fixing (14)CO(2) while assimilating acetate at low light intensity. The percentage of the total radioactivity present in phosphate esters plotted against time gave a negative slope for high light conditions compared with a positive slope for low light conditions. High light grown cells assimilating acetate at high light intensity showed the greatest combined rate of (14)CO(2) fixation via the reductive pentose phosphate cycle and C(4) acids, and this corresponded to the shortest mean generation time. When cells were grown at high light intensity and allowed to assimilate (14)CO(2) at high light intensity but in the stationary phase, the pattern of (14)CO(2) fixation resembled that for low light-grown cells assimilating acetate and fixing (14)CO(2) at low light intensity, showing that both acetate assimilation and high light intensity were necessary for the rapid incorporation of (14)CO(2) via the reductive pentose phosphate cycle. PMID- 16658152 TI - Multiple forms of glucosephosphate isomerase in maize. AB - Three apparently different glucosephosphate isomerases are found in the developing seeds of maize (Zea mays L.). Glucosephosphate isomerase I is found in both the endosperm and embryo. It is separable by column chromatography from glucosephosphate isomerase II of the developing endosperm and glucosephosphate isomerase III of the developing embryo and is further distinguished from them by heat stability, temperature activation, and relative insensitivity to the presence of zinc ions in the reaction mixture. Glucosephosphate isomerases II and III elute in the same fractions from diethylaminoethyl cellulose columns but are distinguished by electrophoretic mobility and reaction to the presence of adenosine 5'-triphosphate in the reaction mixture. All three isomerases give multiple banding patterns on electrophoresis. An extensive investigation of the conditions generating additional electrophoretic species and chromatographically separable minor activity peak (Ia) from glucosephosphate isomerase I has shown that these transformations are enhanced by dialysis, column chromatography, ammonium sulfate fractionation, and treatment with urea. The transformations are retarded by the presence of mercaptoethanol during these operations. We concluded that the multiple banding pattern seen on electrophoresis of glucosephosphate isomerase I prepared by certain procedures is artifactual. In germinating seeds of maize, glucosephosphate isomerases I and III are detectable, but II is not. It is possible that glucosephosphate isomerase II specifically catalyzes a step in starch biosynthesis. PMID- 16658153 TI - Phytochrome control of growth cessation and initiation of cold acclimation in selected woody plants. AB - Short day enhancement of cold acclimation in twigs of Cornus (red-osier dogwood), Weigela, and Pyracantha (firethorn) was studied using dark interruptions with red or with red-far red radiation. Hardiness was estimated by freezing stem tissues to preselected temperatures and evaluating injury electrolytically. Dark-period interruptions with red radiation suppressed cold acclimation in Cornus and Weigela. When red light was followed by far red light, suppression was relieved. No radiation control of acclimation was found with Pyracantha. The short day enhancement of cold acclimation in Cornus and Weigela appears to be phytochrome mediated. PMID- 16658154 TI - Translocation of Photosynthate in Curly Top Virus-infected Tomatoes. AB - Photosynthate translocation in single leaflets of healthy and curly top virus infected tomatoes was investigated using (14)C as a marker. The amount of radioactivity found in plant parts not exposed to (14)CO(2) was substantially lower in diseased than in healthy plants. The time lag for the appearance of (14)C in the petiole was considerably longer in the infected plants than in the healthy. The kinetics of disappearance of (14)C from the lamina during the 24 hour period following labeling showed a strong retention of recent assimilates within the diseased leaf, not accompanied by increased immobilization into insoluble forms. Sucrose was the predominant compound participating in photosynthate transport in both healthy and diseased leaves. The amount of (14)CO(2) fixed was approximately 40% lower in curly top virus-infected leaves than in healthy leaves. PMID- 16658155 TI - Effect of Transpiration-reducing Chemicals on Growth, Flowering, and Stomatal Opening of Tomato Plants. AB - Phenyl mercuric acetate, 8-hydroxyquinoline, N-dimethylamino succinamic acid, or 2-chloroethyl trimethyl ammonium chloride were sprayed on 37-day-old tomato (Lycopersicon esculentum Mill. cv. Sioux) plants seven times at weekly intervals. Plants of nearly normal appearance resulted with all treatments except 2 chloroethyl trimethyl ammonium chloride. There was no change in leaf number, but 2-chloroethyl trimethyl ammonium chloride increased the number of flowers. 2 Chloroethyl trimethyl ammonium chloride and phenyl mercuric acetate caused earlier flowering. Yield was not affected significantly. Stomatal opening was reduced 80% immediately after spraying with phenyl mercuric acetate or 2 chloroethyl trimethyl ammonium chloride, but 6 days after spraying, the reduction in stomatal opening was only 30 to 40%. Wilting was delayed 8 days by phenyl mercuric acetate and 4 days by 2-chloroethyl trimethyl ammonium chloride and N dimethyl amino succinamic acid treatments, when water was withheld 59 days after the final spray application. PMID- 16658156 TI - Isolation of an enzyme system which will catalyze the glycosylation of extensin. AB - Enzymes which catalyze the glycosylation of the cell wall protein extensin using uridine diphosphate l-arabinose-(14)C as a substrate are present in a crude extract prepared from suspension cultured sycamore cells (Acer pseudoplatanus L.). This enzyme system sediments when the crude extract is subjected to centrifugation at 37000g. A base hydrolysate of the product contains a mixture of hydroxyproline-arabinosides which are electrophoretically and chromatographically identical to those obtained by hydrolysis of extensin isolated from the cell wall. The hydroxyproline-rich protein used as an acceptor in the glycosylation reactions is present in the particulate fraction. In addition, evidence is presented which indicates that hydroxyproline-rich tryptic peptides prepared from the cell wall can also be used as an acceptor by this enzyme system. The presence of Mg(2+) or Mn(2+) in the reaction mixture increases the enzyme-catalyzed incorporation of arabinose into extensin by about 1.4 times. About two-thirds of the product mixture is composed of arabinose-containing compounds which have not been identified. Some of these products appear to be hydroxyproline-glycosides which have not been previously reported. PMID- 16658157 TI - Estimation of the transport and carboxylation components of the intracellular limitation to leaf photosynthesis. AB - A model is presented which enables gas exchange data to be used to partition the intracellular resistance to leaf photosynthesis into carboxylation and transport components. A basic assumption is that the over-all kinetics of the carboxylation reaction fit the Michaelis-Menten equation.The model was tested for cotton (Gossypium hirsutum L., var. Deltapine Smoothleaf), where photorespiration was suppressed by using gas mixtures containing less than 1.5% oxygen. It was concluded that the transport resistance formed the major component of the intracellular resistance for the plants studied. However, in some cases the major intracellular factor limiting photosynthesis, at an ambient CO(2) concentration of 600 ng cm(-3), was the carboxylation system, which was close to saturation. PMID- 16658159 TI - Volatile metabolites controlling germination in buried weed seeds. AB - Velvetleaf (Abutilon theophrasti Medic), morning glory (Ipomoea purpurea [L.] Roth), and wild mustard (Brassica kaber [D.C.] L. C. Wheeler) seeds exhibited decreased germination with increased planting depth in soil. Flushing the soil for 2 minutes each day with air overcame the inhibition. A sealed in vitro system was used to sample the volatile components produced by weed seeds. Inhibition of seed germination was accompanied by decreased O(2) levels and production of volatile metabolites identified as acetaldehyde, ethanol, and acetone. The effectiveness of these compounds in reducing germination was dependent on O(2) levels. PMID- 16658158 TI - Effect of light and gibberellin on ribonucleic Acid species of pea stem tissues as studied by deoxyribonucleic Acid-ribonucleic Acid hybridization. AB - The ability of gibberellin and light to alter gene transcription in dwarf pea (Pisum sativum L., var. Progress No. 9) stem issues has been investigated by means of DNA/RNA hybridization-competition techniques. Distinct changes in hybridizable RNA are caused by a 24-hour pretreatment of the seedlings with light, but no changes in RNA were detected up to 50 hours after treatment of the seedlings with gibberellin. Gibberellin is similar to auxin in its ability to induce stem growth without causing detectable changes in hybridizable RNA. PMID- 16658160 TI - Mitochondrial tyrosyl transfer ribonucleic Acid in soybean seedlings. AB - Soybean seedlings were examined for the presence of mitochondrial tRNA. Tyrosyl transfer tRNAs from whole cells, from a well characterized mitochondrial preparation, and from a snake venom phosphodiesterase-treated mitochondrial preparation, were compared by reverse phase chromatography. It was concluded that none of the three previously reported tRNA(Tyr) species were mitochondrial. Rather, a fourth tRNA(Tyr) species, eluting somewhat later, was of mitochondrial origin. Mitochondrial tRNA(Tyr) was chromatographically similar to Escherichia coli tRNA(Tyr). PMID- 16658161 TI - False broomrape: a physiological disorder caused by growth-regulator imbalance. AB - False broomrape on tobacco (Nicotiana tabacum) can be induced by applying cytokinins to the roots, by causing an increase in the cytokinin-auxin ratio in the roots, by removing the apical and auxillary buds, or by applying extracts from tobacco with false broomrape to the roots of healthy plants. It can be prevented by treating debudded plants with auxin. PMID- 16658162 TI - Mechanism of the Photochemical Activity of Isolated Chloroplasts: VII. Inactivation of a Photoact by Dilute Ferricyanide Ion. PMID- 16658163 TI - Mitotic Index and Cell Cycle of Lemna perpusilla under Different Photoperiods. PMID- 16658164 TI - Internal Ethylene Levels during Ripening and Climacteric in Anjou Pears. PMID- 16658165 TI - Abscission: potentiating action of auxin transport inhibitors. AB - Reduction in petiolar auxin transport has been proposed as one of the functional actions of endogenous or exogenous ethylene as it regulates intact leaf abscission. If this hypothesis is correct, auxin-transport inhibitors should hasten the rate or amount of abscission achieved with a given level of ethylene. Evidence presented here indicates that the hypothesis is correct. Three auxin transport inhibitors promoted ethylene-induced intact leaf abscission when applied to specific petioles or the entire cotton plant (Gossypium hirsutum L., cv. Stoneville 213). In addition, the transport inhibitors caused rapid abscission of leaves which usually do not abscise under the conditions employed. No stimulation of abscission occurred during the initial 3 to 5 days after plants were treated with transport inhibitors unless such treatments were coupled with exogenous ethylene or that derived from 2-chloroethylphosphonic acid. However, vegetative cotton plants did abscise some of their youngest true leaves during the 2nd and 3rd weeks of exposure to transport inhibitor alone. Taken as a whole, the results indicate that reducing the auxin supply to the abscission zone materially increases sensitivity to ethylene, a condition which favors a role of endogenous ethylene in abscission regulation. Such a role of ethylene indicates the importance of auxin-ethylene interactions in the over-all hormone balance of plants and specific tissues. PMID- 16658166 TI - Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota). AB - A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation.The melting temperature of ammi and carrot DNA in 0.15 m NaCl and 15 mm trisodium citrate buffer, pH 7.0, was 84.0 C and 84.5 C, respectively. The molecular weight for ammi DNA was 1.43 x 10(8), and for carrot DNA it was 1.56 x 10(8). Ammi DNA exhibited a single band at 1.690 grams per cubic centimeter in CsCl, whereas carrot DNA showed two bands, one at 1.693 grams per cubic centimeter and another at 1.706 grams per cubic centimeter. Ammi DNA consisted of a doublestranded form, since denaturation of the DNA caused a complete upward shift of 0.020 grams per cubic centimeter. PMID- 16658167 TI - Auxin transport: a new synthetic inhibitor. AB - The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid 1-(14)C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-(14)C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-(14)C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid.DPX1840 also inhibited the lateral displacement of auxin. In horizontally placed stem sections of Helianthus annuus L. pretreated with DPX1840, the ratio of radioactivity from indoleacetic acid-1-(14)C in the upper versus the lower halves of the sections following basipetal indoleacetic acid-1-(14)C transport was approximately 50:50, whereas in the corresponding controls it was approximately 40:60.The data indicate that many of the characteristic effects of DPX1840 on plants, especially those which are known to involve auxin (e.g., epinasty, abscission, apical dominance, tropism), are due, at least in part, to its effects on auxin transport. PMID- 16658168 TI - Investigations of Canavanine Biochemistry in the Jack Bean Plant, Canavalia ensiformis (L.) DC: II. Canavanine Biosynthesis in the Developing Plant. AB - The canavanine content of developing leaves of jack bean, Canavalia ensiformis (L.) DC., increases during leaf development. The leaf possesses the enzymes required for synthesizing canavanine by a cyclic series of reactions analogous to the ornithine-urea cycle. This reaction series involves the sequential formation of canaline, O-ureidohomoserine, and canavaninosuccinic acid. PMID- 16658169 TI - Effect of Time, Water Flow, and pH on Centripetal Passage of Radiophosphorus across Roots of Intact Plants. AB - The effects of time, rate of the water flow, and ambient pH on centripetal passage of radiophosphorus across intact bean roots to the xylem were studied. Isotope which completed passage and entered the xylem stream, as well as amounts delivered to the plant top, served to measure centripetal passage.Centripetal passage of radiophosphorus increased parabolically reaching a maximum after 1 hr and maintained this level during the 2nd hr. This pattern was consistent for all conditions studied. The curve suggested that passage did not progress as an abrupt front, but rather that it occurred through a phosphorus pool before reaching the xylem.Differences in rate of water flow through test plants, accomplished by adjusting the humidity of the foliage environment, did not significantly affect centripetal passage of radiophosphorus. Water flow did, however, profoundly influence composition of the xylem stream by altering the solvent to isotope ratio.Centripetal passage of radiophosphorus was not affected by solution pH in the acid range (pH 4.8, 5.2, 6.4), but was inhibited in the more alkaline range (pH 7.0, 7.5, 8.0). The similarity of these findings to those in the literature for phosphorus uptake by individual cells suggests that cell uptake may constitute the primary rate-limiting step in the over-all process of ion passage to the xylem. PMID- 16658170 TI - The distribution of carbonic anhydrase and ribulose diphosphate carboxylase in maize leaves. AB - Extraction of maize (Zea mays) leaves by progressive grinding under suitably protective conditions yields total carbonic anhydrase activities (4800 units per milligram chlorophyll) comparable to the activity in spinach (Spinacia oleracea) leaves. The total ribulose diphosphate carboxylase activity was also equal to or greater than the best literature values for maize. Of the total leaf carbonic anhydrase, 72.5% on a chlorophyll basis was present in the mesophyll cells and 14.2% in the bundle-sheath cells. The distribution of the total leaf ribulose diphosphate carboxylase between the mesophyll and bundle-sheath cells was 42.0 and 48.7% respectively. There was three times as much total chlorophyll in extracts of the mesophyll cells compared with the bundle-sheath cells of maize. Similar results for the above distribution of the two enzymes were found using a differential grinding technique. The possible function of carbonic anhydrase in photosynthesis is discussed. The equal distribution of ribulose diphosphate carboxylase activity between the mesophyll and bundle-sheath cells casts doubt upon the hypothesis that a rigid biochemical compartmentation exists between these cell types in maize. PMID- 16658171 TI - Interrelationships of ethylene and abscisic Acid in the control of rose petal senescence. AB - The role of abscisic acid and ethylene in the senescence of rose petals cv. Golden-Wave was examined. A rise in ethylene evolution, followed by an increase in the level of abscisic acid was observed. The presence of abscisic acid in rose petals was established, using different chromatography systems, several bioassays, and immunoassay. External application of ethylene accelerated senescence and induced a rise in endogenous abscisic acid-like activity. Application of abscisic acid promoted senescence, but suppressed ethylene production. The data suggest that the participation of these two hormones in the control of senescence is via the same pathway. The possibility of interrelationship between abscisic acid and ethylene was tested and experimental evidence in favor of this hypothesis is presented. It was suggested that ethylene affects senescence in rose petals by inducing an increase in abscisic acid activity, which in turn may control ethylene evolution, via a feedback mechanism. PMID- 16658172 TI - Phosphate-induced Stimulation of Acceptorless Respiration in Corn Mitochondria. AB - By use of the organic mercurial mersalyl to block phosphate transport, it has been shown that only a small fraction of the respiratory increase of corn mitochondria in response to additions of inorganic phosphate is due to energy expended in phosphate accumulation. Most of the respiratory release occurs from accelerated turnover of the coupling mechanism with internal phosphate in an oligomycin-sensitive reaction. Addition of ADP to mersalyl-blocked mitochondria depletes internal phosphate in ATP formation and respiration declines. Arsenate produces the same responses as phosphate but is more effective in respiratory release.Inhibition of the ADP-ATP antiporter with atractyloside shows that the increased respiration with internal phosphate is not due to turnover of ADP acceptor through exogenous ATPase.Use of valinomycin to facilitate movement of K(+) greatly accelerates the rate of phosphate swelling, but there is no consistent correlation between respiration and swelling. In the absence of phosphate, valinomycin dramatically releases respiration with only trivial swelling.The data indicate that loose coupling in due only fractionally to energy expenditure in ion transport. An explanation consistent with the observations can be derived by assuming that both a high energy intermediate (I approximately X) and a proton motive force-or its electrochemical equivalent-arise at coupling sites. PMID- 16658173 TI - Adaptation to quantum flux by the emerson photosynthetic unit. AB - The size of the Emerson photosynthetic unit was measured in Chlorella pyrenoidosa strain no. 252 grown at light intensities between 50 and 1000 foot candles. The Emerson photosynthetic unit changed from a minimum size of 1970 molecules chlorophyll a + b/O(2) per flash in cells grown at 1000 foot candles to a maximum size of 3150 molecules chlorophyll a + b/O(2) per flash for cells grown at 50 foot candles. The size changes were interpreted as a partial adaptation where the trapping center antenna responded to changes in incident light intensity. Light induced changes in chlorophyll content and size of the Emerson photosynthetic unit were directly related.Two strains of Chlorella pyrenoidosa adapted by growth to 500 foot candles were then illuminated at the reduced light intensity of 50 foot candles. Emerson photosynthetic unit size (Emerson strain) increased from 2110 molecules Chlorophyll a + b/O(2) per flash at time zero to a maximum size of 3160 after 65 hours at 50 foot candles. The Emerson photosynthetic unit size for strain 252 transferred from 500 to 50 foot candles was 2260 at zero hours and 3650 after 50 hours at 50 foot candles. Emerson photosynthetic unit sizes for similar cultures which remained at 500 foot candles were almost constant in size. Oxygen yield per flash per cell was nearly constant whereas Emerson photosynthetic unit size increased in cells moved to the reduced incident light intensity. PMID- 16658174 TI - Dark Fixation of CO(2) by Crassulacean Plants: Evidence for a Single Carboxylation Step. AB - Malic acid isolated from Bryophyllum pinnatum (Lamk.) Oken (B. calycinum Salisb.), Bryophyllum tubiflorum Harv., Kalanchoe diagremontiana Hamet et Perrier and Sedum guatamalense Hemsl. after dark (14)CO(2) fixation was degraded by an in vitro NADP-malic enzyme technique. In the short term (5 to 30 seconds) the malic acid was almost exclusively labeled in the C-4 carboxyl carbon (greater than 90%). The percentage of (14)C in the C-4 carboxyl of malic acid declined slowly with time, reaching 70% in B. tubiflorum and 54% in B. pinnatum after 14 hours of exposure to (14)CO(2). It was found that malic acid-adapted Lactobacillus arabinosus may seriously underestimate the C-4 carboxyl component of label in malic acid-(14)C. The amount of substrate which the bacteria can completely metabolize was easily exceeded; there was a significant level of randomization of label even when beta-decarboxylation proceeded to completion, and in extended incubation periods, more than 25% of label was removed from malic acid-U-(14)C. The significance of these findings in relation to pathways of carbohydrate metabolism and malic acid synthesis in Crassulacean acid metabolism is discussed. PMID- 16658175 TI - Phloem Translocation and Heat-induced Callose Formation in Field-grown Gossypium hirsutum L. AB - Phloem translocation rates in field-grown cotton (Gossypium hirsutum L.) dropped from morning to afternoon and continued to decline toward evening, except that recovery occurred following the hottest afternoon when the maximum temperature was 44 C. Water deficits increased from morning to evening, and severity of deficits generally were proportional to daytime heating. Water stress contributed toward reducing translocation but was not always the governing factor. Callose breakdown appeared to be slower than heat-induced synthesis, and in the evening callose still reflected the influence of high afternoon temperatures. Translocation was considerably reduced when about 50% or more of the hypocotyl sieve plates had large amounts of callose. While heat-induced callose may have reduced translocation because of sieve plate pore constriction, temperatures of 39 to 44 C appeared to inhibit an additional component of translocation as well, possibly in the leaf blade. PMID- 16658176 TI - The Formation of beta, 1 --> 4 Glucan from UDP-alpha-d-Glucose Catalyzed by a Phaseolus aureus Enzyme. AB - Particulate enzyme preparations from Phaseolus aureus hypocotyls catalyze the formation of an alkali insoluble beta, 1 --> 4 linked [(14)C]-glucan using UDP alpha-d [(14)C]-glucose as substrate. Particulate enzymes prepared from root tissue also catalyzed the production of beta, 1 --> 4 glucan. UDP-beta-d-[(14)C] glucose would not serve as a substrate for these enzymes. The presence or absence of beta, 1 --> 4 glucan synthetase activity was independent of tissue source, substrate concentration, or homogenization method.The particulate enzyme also catalyzes the formation of a beta, 1 --> 3 linked glucan from UDP-d glucose which is usually soluble in hot alkali. The solubility of the beta, 1 --> 3[(14)C] glucan decreased when the enzyme was obtained from hypocotyls germinated at a higher temperature. The water-soluble material resulting from the catalyzed reaction includes a large proportion of what appears to be [(14)C]-laminaribiose, and smaller proportions of [(14)C]-laminaritriose and [(14)C]-glucose. There is no detectable quantity of [(14)C]-cellobiose or [(14)C]-cellotriose in the water soluble material. PMID- 16658177 TI - Photophosphorylation and Carbon Dioxide Fixation by Chloroplasts Isolated from Populus deltoides. AB - A system has been developed for the isolation of photosynthetically active chloroplasts from leaves of Populus deltoides. A high proportion of the chloroplasts appeared intact. The maximum rates of different photosynthetic processes were as follows: CO(2) fixation 3.5 micromoles per milligram chlorophyll per hour, noncyclic ATP synthesis 10 micromoles per milligram chlorophyll per hour, and cyclic ATP synthesis 300 micromoles per milligram chlorophyll per hour. PMID- 16658178 TI - Relationship between Ethylene and the Growth of Ficus sycomorus. AB - The relationship between ethylene and growth was investigated in Ficus sycomorus L. A marked increase in ethylene emanation preceeded all the phases of rapid growth and ripening of the syconium.Gashing of fig during the 16th to 22nd day of syconium development induced a 50-fold increase in the rate of ethylene emanation within the 1st hour, and a 2- to 3-fold increase in diameter and weight, followed by ripening within 3 days. Application of ethylene, Ethephon, and auxins caused the same effects as wounding. Since the auxin and Ethephon induced ethylene emanation, it is concluded that ethylene is mainly responsible for the marked morphological changes caused by gashing. The stage of slow growth of this fruit is characterized by slow emanation of ethylene, low sensitivity to exogenous ethylene, and no morphological responses to gashing. PMID- 16658179 TI - Physiology of Oil Seeds: IV. Role of Endogenous Ethylene and Inhibitory Regulators during Natural and Induced Afterripening of Dormant Virginia-type Peanut Seeds. AB - To further elucidate the regulation of dormancy release, we followed the natural afterripening of Virginia-type peanut (Arachis hypogaea L.) seeds from about the 5th to 40th week after harvest. Seeds were kept at low temperature (3 +/- 2 C) until just prior to testing for germination, ethylene production, and internal ethylene concentration. Germination tended to fluctuate but did not increase significantly during the first 30 weeks; internal ethylene concentrations and ethylene production remained comparatively low during this time. When the seeds were placed at room temperature during the 30th to 40th weeks after harvest, there was a large increase in germination, 49% and 47% for apical and basal seeds, respectively. The data confirm our previous suggestion that production rates of 2.0 to 3.0 nanoliters per gram fresh weight per hour are necessary to provide internal ethylene concentrations at activation levels which cause a substantial increase of germination. Activation levels internally must be more than 0.4 microliter per liter and 0.9 microliter per liter for some apical and basal seeds, respectively, since dormant-imbibed seeds containing these concentrations did not germinate. Abscisic acid inhibited germination and ethylene production of afterripened seeds. Kinetin reversed the effects of ABA and this was correlated with its ability to stimulate ethylene production by the seeds. Ethylene also reversed the effects of abscisic acid. Carbon dioxide did not compete with ethylene action in this system. The data indicate that ethylene and an inhibitor, possibly abscisic acid, interact to control dormant peanut seed germination. The inability of CO(2) to inhibit competitively the action of ethylene on dormancy release, as it does other ethylene effects, suggests that the primary site of action of ethylene in peanut seeds is different from the site for other plant responses to ethylene. PMID- 16658180 TI - Transport of sodium into the xylem exudate of tobacco. AB - When tobacco (Nicotiana tabacum L. var. Virginia Gold) plants were pretreated with Na ((22)Na) several days before detopping, from 2.3 to 4.9% of Na previously accumulated in roots appeared in the xylem exudate in 7 days after detopping. Na from the external medium, however, was readily transported to the exudate. Moreover, the amount of the pretreatment Na that was transported to the exudate was not influenced by the presence of Na in the external medium. When Na was present in the external medium after detopping, about 4% (with an NaNO(3) post treatment) to 10% (with an NaCl post treatment) of the Na transported to the xylem in the 7 days following detopping originated in the vacuoles. Nitrate salts of K or Na in the external medium after detopping resulted in transport of large quantities of the respective cation to the exudate, but not in increased transport of the pretreatment Na. A much larger percentage of the K that was accumulated after detopping than of the Na similarly accumulated was transferred to the xylem exudate. PMID- 16658181 TI - Studies on Chloroplast Development and Replication in Euglena: III. A Study of the Site of Synthesis of Alkaline Deoxyribonuclease Induced during Chloroplast Development in Euglena gracilis. AB - During chloroplast development in Euglena, the activity of a specific DNase, Euglena alkaline DNase, increases in a manner similar to that of chlorophyll synthesis, but without the lag customarily associated with the early hours of chlorophyll synthesis. The increase in Euglena alkaline DNase activity is not inhibited by chloramphenicol or by streptomycin, but is inhibited by cycloheximide. Euglena alkaline DNase activity is present in a group of aplastidic substrains which contain carotenoids. These results are interpreted to mean that this chloroplast-related DNase is synthesized in the cytoplasm, and that the genetic information for this enzyme is probably nuclear.It is also shown that different bleached substrains exhibit substantial variation, both in total carotenoids and in Euglena alkaline DNase activity. These results are discussed in terms of the possibility that a cytoplasmic photoreceptor system is influencing the light-induced increase in Euglena alkaline DNase activity. PMID- 16658182 TI - Equilibrium constants and photosynthetic enhancement. AB - The linear (Myers) and nonlinear (Joliot-Kok) models of photosynthesis were used to predict enhancement. With an apparent equilibrium constant of 1000 between the two light reactions, both models predict a minimal enhancement of about 1.0. However, with an equilibrium constant of 5, both models predict a minimal enhancement significantly greater than 1.0. Consequently, with an equilibrium constant of 5, neither model can account for the observed enhancement values of 1.0 near 685 nanometers in Chlorella. Also, with an equilibrium constant of 5, enhancement significantly greater than 1.0 is predicted between two short wavelengths or between two long wavelengths; neither is observed. PMID- 16658183 TI - Stimulation by Ethylene of Chlorophyll Biosynthesis in Dark-grown Cucumber Cotyledons. AB - Five-day-old etiolated cucumber (Cucumis sativus L. var. Alpha Green) cotyledons produced more chlorophyll over a 4-hour illumination period after a prolonged exposure (12 to 72 hours) in the dark to ethylene concentrations ranging from 0.1 to 10 mul/l. Intact seedlings and excised cotyledons responded in the same way to this treatment. This effect does not involve a shortening of the lag phase of chlorophyll accumulation. Exposure of cotyledons to ethylene during the illumination period did not produce the same stimulatory effect on chlorophyll synthesis and, under certain conditions, chlorophyll synthesis was slightly inhibited by exposure to ethylene in the light. PMID- 16658184 TI - The Ultrastructure of Chloroplasts in Mineral-deficient Maize Leaves. AB - The ultrastructure of mesophyll chloroplasts in full-nutrient and mineral deficient maize (Zea mays) leaves was examined by electron microscopy after glutaraldehyde-osmium tetroxide fixation. Nitrogen, calcium, magnesium, phosphorus, potassium, and sulfur deficiencies were induced by growing the plants in nutrient culture. Distinctive chloroplast types were observed with each deficiency. Chloroplasts from nitrogen-deficient plants were reduced in size and had prominent osmiophilic globules and large grana stacks. Magnesium deficiency was characterized by the accumulation of osmiophilic globules and the progressive disruption of the chloroplast membranes. In calcium deficiency, the chloroplast envelope was often ruptured. Chloroplasts from potassium- or phosphorus-deficient plants possessed an extensive system of stroma lamellae. Sulfur deficiency resulted in a pronounced decrease of stroma lamellae, an increase in grana stacking, and the frequent occurrence of long projections extending from the body of the chloroplast. These morphological changes were correlated with functional alterations in the chloroplasts as measured by photosystem I and II activities. In chloroplasts of the nitrogen- and sulfur-deficient plants an increase in grana stacking was associated with an increase in photosystem II activity. PMID- 16658185 TI - Some Biochemical Characteristics of Chloroplasts from Mineral-deficient Maize. PMID- 16658186 TI - Coumarin-induced Tuber Formation on Excised Shoots of Solanum tuberosum L. Cultured in Vitro. PMID- 16658187 TI - Promotion of cell elongation in Avena coleoptiles by acetylcholine. PMID- 16658188 TI - Auxin control for orientation of pea roots grown on a clinostat or exposed to ethylene. PMID- 16658189 TI - Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck. AB - A study of the growth-inhibiting effect of chlorate on the Berlin strain of Chlorella vulgaris Beijerinck provided complete confirmation of the theory of chlorate toxicity first proposed by Aberg in 1947. Chlorate was toxic to the cells growing on nitrate, and relatively nontoxic to the cells growing on ammonium. The latter cells contained only 0.01 as much NADH-nitrate reductase as the nitrate-grown cells. Chlorate could substitute for nitrate as a substrate of the purified nitrate reductase with Km = 1.2 mm, and V(max) = 0.9V(max) for nitrate. Bromate, and to a much smaller extent, iodate, also served as alternate substrates. Nitrate is a reversible competitive inhibitor of chlorate reduction, which accounts for the partial reversal, by high nitrate concentrations, of the observed inhibition of cell growth by chlorate. During the reduction of chlorate by NADH in the presence of purified nitrate reductase, there was a progressive, irreversible inhibition of the enzyme activity, presumably brought about by the reduction product, chlorite. Both the NADH-nitrate reductase activity and the associated NADH-cytochrome c reductase activity were inactivated to the same extent by added chlorite. The spectral properties of the cytochrome b(557) associated with the purified enzyme were not affected by chlorite. The inactivation of the nitrate reductase by chlorite could account for the toxicity of chlorate to cells grown on nitrate, though the destruction of other cell components by chlorite or its decomposition products cannot be excluded. PMID- 16658190 TI - Endogenous metabolism of fungus spores: stimulation by physical and chemical means. AB - Endogenous respiration of spores of the fungus Myrothecium verrucaria can be stimulated up to over-10 fold by diverse chemicals or by physical treatments. Greatest effects were caused by azide (12-fold at 250 mum) and by 2,4 dinitrophenol (7-fold at 300 mum). Marked stimulation was also caused by 10 mum silver (5-fold), 30 mum pentachlorophenol (6-fold), 10 mum carbonyl cyanide m chlorophenyl hydrazone (4.5-fold) and 10 mum merthiolate (4-fold). Physical treatments such as heat (50 C), freezing, and sonication at sublethal levels were also stimulatory. Stimulation by azide or dinitrophenol was much greater in young than in old spores, whereas response to other chemicals and to freezing was relatively unaffected by spore age. In older spores the effect of azide was no greater than some other inhibitors. During incubation with azide, the endogenous trehalose reserves decreased and changes in free amino acids occurred, both increases and decreases. Thus anabolic as well as catabolic changes occur as evidenced also by the germination of a few (up to 5%) spores. The mechanisms of stimulation must be varied and complex. Permeability changes in the membrane confining endogenous reserves are proposed as a common initial cause. Additional changes in characteristics of membranes of other subcellular particles, as well as enzymic phenomena such as uncoupling of oxidative phosphorylation, are presumably involved in instances where greater stimulation occurs. The data are consistent with the hypothesis that dormancy in these spores results from separation of substrates from metabolic enzymes and more specifically that metabolites are sequestered rather than enzymes. PMID- 16658191 TI - Role of Protein Synthesis in the Senescence of Leaves: II. The Influence of Amino Acids on Senescence. AB - When the first leaf of the oat (Avena sativa) seedling is detached and placed in the dark, yellowing and proteolysis take place rapidly. The earlier finding that d-serine promotes this process has led to a further study of the controlling roles of several amino acids. Since the action of serine was found to be more powerful in presence of kinetin than alone, the effects of other amino acids have been restudied in presence of kinetin. Cysteine emerges as a moderately strong promotor of senescence, with glycine and alanine having definite but weaker effects. The serine effect is antagonized by arginine, especially in presence of kinetin, and so is the cysteine effect. This is considered to indicate that these two amino acids act in the same way. The antagonism exerted by arginine is in turn antagonized by canavanine. The protease activities at two pH regions which increase in the oat leaf during senescence react to both p-chlorimercuri phenylsulfonate and to phenylmethyl-sulfonyl fluoride, and thus may contain both SH and OH groups. The amounts of both these enzyme activities formed in the leaf during 3 days in the dark are increased over 50% by pretreatment with serine, and this increase is very largely prevented by arginine. The amounts of soluble proteins left in the leaf vary as expected in the opposite sense. It is deduced that control of the new formation of proteases plays an important part in senescence. A suggestion is made as to the mechanism of control of senescence in leaves. PMID- 16658192 TI - Cell Wall Regeneration around Protoplasts Isolated from Convolvulus Tissue Culture. AB - Protoplasts of Convolvulus arvensis L. tissue culture regenerated a wall-like structure within 3 days in culture. Although unusually electron dense and atypically amorphous in the electron microscope, this structure could be digested with Myrothecium cellulase but was resistant to protease, a Rohm and Haas pectinase, and a beta-1, 3-exoglucanase just like the original wall. A cytochemical test for callose was negative. Wall regeneration required a readily metabolized external carbon source and was not inhibited by a high concentration of cycloheximide, puromycin, or actinomycin D. Protoplast budding was correlated with the wall regeneration, and the latter was related quantitatively to the sucrose concentration in the medium. Although a concentration of 1 mum 2,4 dichlorophenoxy acetic acid is used normally for both general culture of the tissue and for wall regeneration, concentrations of 0 and 0.1 mm, which are highly deleterious to growth, have no appreciable effect on the incidence of the wall-like structure regenerated around protoplasts. The ability of protoplasts to undergo cell wall regeneration was decreased when they were cultured in the presence of proteolytic enzymes. PMID- 16658193 TI - The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis. AB - The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg(2+) concentration is 3 mm. Heavy metal ions (Cu(2+), Hg(2+), Ni(2+), Zn(2+)), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO(3) (-) was also the same (16 to 19 mm). PMID- 16658194 TI - Effects of Germination on NA-K-stimulated Adenosine 5'-Triphosphatase and ATP dependent Ion Transport of Isolated Membranes from Cotyledons. AB - A purified membrane fraction featuring ATPase activity was isolated from cotyledon tissue of Phaseolus vulgaris at different stages of germination. The fraction is enriched in both basal and Na(+)-K(+)-stimulated ATPase and is relatively free of contamination by fragments of mitochondrial membrane and microsomes. The isolated membranes have been tentatively identified as partially purified plasma membrane.The specific activities of the basal- and cation sensitive ATPases were high in membrane preparations from young cotyledon tissue but decreased with advancing senescence. Electron microscopy of the preparation showed that the isolated membranes were of primarily vesicular conformation. These vesicles proved to be capable of extruding Na(+) and K(+) in the presence of ATP. Moreover, the degree of ATP-dependent extrusion varied during germination in a pattern that resembled variations during the same period in the Na(+)-K(+) stimulated ATPase of the isolated fraction. This indicates that as the level of cation-sensitive ATPase on the membrane rises or falls, there is a corresponding change in the ability of the membrane to actively translocate Na(+) and K(+). PMID- 16658195 TI - Photoregulation of Nicotinamide Adenine Dinucleotide Kinase Activity in Cell-free Extracts. AB - This article gives evidence that NAD kinase activity is controlled by the action of phytochrome. The NADP level rapidly increased in the cotyledons of seedlings of Pharbitis nil strain Violet (a short day plant), when the inductive dark for flowering was interrupted with a 5-minute illumination of red light. Illumination with far red light immediately after illumination with red light counteracted partly the effect of the latter.A partially purified phytochrome preparation from the uppermost internodes including the hook of Pisum sativum var. Alaska contained NAD kinase activity, and this increased greatly on illumination with red light. The effects of red and far red light on NAD kinase activity were reversible. The Km value of NAD kinase in the phytochrome preparation for NAD was 1.84 mm in the dark and 0.90 mm under red light. A broad optimum of the NAD kinase reaction and its photopotentiation appeared in the region of pH 6.6 to 8.6.The NAD kinase activity in a phytochrome preparation from the coleoptiles of Avena sativa was also activated by red light.It was suggested that the Pfr type of phytochrome may contribute to the decrease of the Km value of NAD kinase reaction by causing a conformational alteration of NAD kinase. PMID- 16658196 TI - Cytochemical localization of catalase in glyoxysomes isolated from maize scutella. AB - The localization of catalase in isolated maize scutellum glyoxysomes was investigated by means of the diaminobenzidine histochemical reaction. Only the membranes of the glyoxysomes become heavily stained after incubation with diaminobenzidine and H(2)O(2). If the glyoxysomes are lysed with Tricine buffer at pH 9, 70% of the catalase is solubilized, while the remaining 30% is tightly bound to an insoluble fraction formed mostly by glyoxysomal membranes. This suggests that catalase may be present also in the matrix of the glyoxysomes. The lack of staining of the matrix with diaminobenzidine is probably due to the high concentration of catalase in the membranes of the organelles. PMID- 16658197 TI - The Regulatory Properties of Purified Phaseolus aureus Sucrose Synthetase. AB - Phaseolus aureus sucrose synthetase, purified to homogeneity, was assayed in the presence of a variety of biological compounds to test for possible regulatory effectors. The oxidized form of nicotinamide adenine dinucleotide phosphate, as well as indoleacetic acid, gibberellic acid, and pyrophosphate were found to activate the forward reaction (sucrose degradation) and inhibit the reverse reaction (sucrose synthesis). The reduced form of nicotinamide adenine dinucleotide phosphate antagonizes the effect of the oxidized form. Fructose 1 phosphate and divalent cations inhibit the forward and activate the reverse reaction. Pyrophosphate and fructose 1-phosphate are effective only in the presence of magnesium chloride. Uridine triphosphate inhibits both the forward and reverse reactions. All effectors except gibberellic acid are active only in the millimolar range of concentrations; maximal stimulation for any effector is approximately 2-fold. The effects of combinations of effectors are roughly additive. Using pyrophosphate in the presence of magnesium chloride as an effector, results of kinetic studies offer a model by which an effector can activate an enzymatic reaction in one direction and inhibit in the reverse direction. PMID- 16658198 TI - The Role of a d-Mannosyl-Lipid as an Intermediate in the Synthesis of Polysaccharide in Phaseolus aureus Seedlings. AB - Particulate preparations from Phaseolus aureus produce a d-mannosyl-lipid when treated with GDP-d-mannose. This lipid complex appears to be an active d-mannose donor, and some investigators have proposed that its role might be an obligatory intermediate in mannan synthesis of higher plants. When the partially purified d mannosyl-lipids, isotopically labeled in the d-mannose moiety, were treated with particulate enzymes under a variety of conditions, a negligible amount of material was produced that behaved as a polysaccharide. Endogenous, particle bound d-mannosyl-(14)C-lipid prepared from P. aureus particles readily transferred d-mannose to GDP to yield GDP-d-mannose and was hydrolyzed to free d mannose when treated briefly with 0.01 n HCl at 100 C. The d-mannosyl-lipid, therefore, exhibits active d-mannose transfer potential in its endogenous state. When endogenous glycosyl-lipid was incubated in the absence of GDP-d-mannose (14)C, little or no polysaccharide was produced. It was, instead, slowly degraded to d-mannose. Addition of several different unlabeled sugar nucleotides had no effect on the results. Our studies to date, therefore, offer no evidence that the mannosyl-lipid is an obligatory precursor of polysaccharide. PMID- 16658199 TI - Studies of l-Cysteine Biosynthetic Enzymes in Phaseolus vulgaris L. AB - In higher plants the biosynthesis of l-cysteine from l-serine, acetylCoA, and sulfide requires serine transacetylase and O-acetylserine sulfhydrylase. The distribution of these enzymes in kidney bean (Phaseolus vulgaris L. cv. Red Kidney) seedlings was determined. Between one-third and two-thirds of the serine transacetylase activity was associated with mitochondria, whereas all of the O acetyl-serine sulfhydrylase activity was present in the soluble fraction of cell homogenates. In a 14-day plant approximately two-thirds of the O-acetylserine sulfhydrylase activity and approximately one-half of the serine transacetylase activity was found in the leaves.Sulfur-deficient plants were grown to determine the effect of sulfur status on the levels of cysteine biosynthetic enzymes. Total extractable serine transacetylase activity was not affected by sulfur deficiency; in contrast, there was an increase in O-acetylserine sulfhydrylase activity under these conditions. PMID- 16658200 TI - l-Phenylalanine Ammonia-lyase (Maize): Partial Purification and Response to Gibberellic Acid and Cycloheximide of l-Phenylalanine and l-Tyrosine Ammonia lyase Activities. AB - Extracts of maize leaf sheath tissue deaminate both l-phenylalanine and l tyrosine. The activities with both substrates are enhanced by treating the plant with gibberellic acid. Both activities decrease rapidly at the same rate when tissue is incubated in a moist atmosphere, and this decrease can be slowed by treatment with cycloheximide. The ratio of the activities was constant throughout a series of purification steps which included acetone and ammonium sulfate precipitation, and passage through an agarose column. The two activities could not be separated by isoelectric focusing. These results support our earlier conclusion that both activities occur at the same catalytic site. PMID- 16658201 TI - Nitrate absorption and assimilation in ryegrass as influenced by calcium and magnesium. AB - The absorption and assimilation patterns of (15)NO(3) (-) supplied as the Ca(2+) and Mg(2+) salts to intact ryegrass (Lolium perenne) seedlings were compared. No statistically significant effect of ambient cation on the amounts of (15)NO(3) ( ) absorbed was observed in the initial six hours, but during the subsequent six hours, absorption from Ca((15)NO(3))(2) exceeded that from Mg ((15)NO(3))(2).Lower rates of (15)NO(3) (-) assimilation were found in roots exposed to Mg((15)NO(3))(2) than in those exposed to Ca((15)NO(3))(2). It was proposed that Mg(2+) initiated a restriction in (15)NO(3) (-) reduction in roots, probably as a consequence of a Mg(2+)-induced physiological Ca(2+) deficiency. Lower (15)N translocation rates were also observed from Mg((15)NO(3))(2). These effects of Mg(2+) in depressing (15)NO(3) (-) assimilation and translocation occurred prior to an effect on (15)NO(3) (-) uptake.In shoots, larger amounts of reduced (15)N products occurred with Ca((15)NO(3))(2) than with Mg((15)NO(3))(2). It was concluded that this was due to enhanced translocation of (15)NO(3) (-) (and possibly its reduced products) in presence of Ca(2+) rather than to specific cation effects on (15)NO(3) (-) assimilation in the shoots. PMID- 16658202 TI - Two components of auxin transport. AB - The transport of indoleacetic acid-1-(14)C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t((1/2)) of about 30 minutes) and a slow transport system (t((1/2)) about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid. PMID- 16658203 TI - Biochemical Pathway of Stress-induced Ethylene. AB - Ethylene production from beam and tobacco leaves increased rapidly following the application of toxic compounds such as CuSO(4), Endothal, and ozone. Treatments which increased ethylene evolution also increased the conversion of U-(14)C methionine into ethylene. Cycloheximide inhibited the production of chemical stress-induced ethylene. These results suggest that ethylene is produced by the same biochemical pathway forming basal ethylene, auxin-induced ethylene, or that produced during the ripening of climacteric fruit. PMID- 16658204 TI - Auxin activity of 3-methyleneoxindole in wheat. AB - A product of the enzymatic oxidation of indole-3-acetic acid, 3 methyleneoxindole, is at least 50-fold more effective than indole-3-acetic acid in stimulating the growth of wheat (Triticum vulgare, red variety) coleoptiles. Ethylenediaminetetra-acetic acid can antagonize the growth-stimulating properties of the parent compound, indole-3-acetic acid, presumably by chelating Mn(2+), which is required for the enzymatic oxidation of indole-3-acetic acid. The growth stimulating effect of 3-methyleneoxindole, a product of the blocked reaction, on the other hand, is still evident in the presence of ethylenedia-minetetraacetic acid. In the presence of 2-mercaptoethanol, indole-3-acetic acid fails to stimulate the elongation of wheat coleoptiles. The property of binding to sulfhydryl compounds including 2-mercaptoethanol is unique to 3-methyleneoxindole among indole-3-acetic acid and its oxidation products. These findings suggest that 3-methyleneoxindole is an obligatory intermediate in indole-3-acetic acid induced elongation of wheat coleoptiles. PMID- 16658205 TI - Enzymatic dehydration of 3-hydroxymethyloxindole. AB - Crude and partially purified extracts of wheat (Triticum vulgare, red variety) germ catalyze the dehydration of 3-hydroxymethyloxindole to 3-methyleneoxindole. Examination of the ultraviolet absorption spectrum of a reaction mixture consisting of either the extract or partially purified enzyme and 3 hydroxymethyloxindole, shows that this oxindole has undergone complete dehydration to 3-methyleneoxindole. TPNH-linked 3-methyleneoxindole reductase, also a constituent of the wheat germ extract, can be separated from the dehydrase by passage through an Agarose 15 column. Utilizing these partially purified enzymes, it can be demonstrated that the dehydrase activity found in wheat germ is a discrete enzymatic function. PMID- 16658206 TI - The Binding of Indole-3-acetic Acid and 3-Methyleneoxindole to Plant Macromolecules. AB - Homogenates of pea (Pisum sativum L., var. Alaska) seedlings exposed to (14)C indole-3-acetic acid or (14)C-3-methyleneoxindole, an oxidation product of indole 3-acetic acid, were extracted with phenol. In both cases 90% of the bound radioactivity was found associated with the protein fraction and 10% with the water-soluble, ethanol-insoluble fraction. The binding of radioactivity from (14)C-indole-3-acetic acid is greatly reduced by the addition of unlabeled 3 methyleneoxindole as well as by chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid to 3-methyleneoxindole. Chlorogenic acid does not inhibit the binding of (14)C-3-methyleneoxindole. The labeled protein and water-soluble, ethanol-insoluble fractions of the phenol extract were treated with an excess of 2-mercaptoethanol. Independently of whether the seedlings had been exposed to (14)C-indole-3-acetic acid or (14)C-3-methyleneoxindole, the radioactivity was recovered from both fractions in the form of a 2-mercaptoethanol-3 methyleneoxindole adduct. These findings indicate that 3-methyleneoxindole is an intermediate in the binding of indole-3-acetic acid to macromolecules. PMID- 16658207 TI - Ethylene-induced Pea Internode Swelling: Its Relation to Ribonucleic Acid Metabolism, Wall Protein Synthesis, and Cell Wall Structure. AB - Exposure of etiolated pea (Pisum sativum L. var. Alaska) subapical sections to ethylene disrupts normal polar cell expansion, but fresh weight increase is little affected and the tissue expands radially, swelling. Ethylene has no effect on gross ribonucleic acid metabolism before or during the period when swelling occurs, but incorporation of (14)C-proline and leucine into wall-associated protein is markedly inhibited after an initial 3-hour lag period which precedes swelling. Ethylene affects the composition of this protein, altering the proline hydroxyproline ratio. The gas also alters the optical birefringence pattern of the cell wall, indicating that the cellulose microfibrillar orientation has been changed. PMID- 16658208 TI - Temperature dependence of photosynthesis in cotton. AB - Cotton plants (Gossypium hirsutum L., var. Deltapine Smooth Leaf) were grown under controlled environmental conditions over a range of day/night temperatures from 20/15 to 40/35 C. Their photosynthetic characteristics were then measured over a comparable temperature range. Net photosynthesis tended stongly to be greatest, and intracellular resistance to CO(2) transport to be lowest, when the measurement temperature corresponded to the daytime growth temperature, suggesting pronounced acclimation of the plants to the growth temperature. The preferred growth temperature was close to the 25/20 C regime, since net photosynthesis of these plants, regardless of measurement temperature, was higher and intracellular resistance lower, than in plants from any other regime.Ribulose diphosphate carboxylase activity per unit leaf area was highest in plants grown at 25/20 C, but did not show pronounced changes with growth temperature. Glycolate oxidase activity decreased and NADH-malate dehydrogenase activity tended to increase with increasing growth temperature. In contrast, changes in carbonic anhydrase activity with growth temperature showed a general similarity to changes in photosynthetic rate. This may suggest that the "chemical resistance" component of the intracellular resistance bears a relationship to the amount of carbonic anhydrase in the leaf. PMID- 16658209 TI - Phytochrome-controlled Nyctinasty in Albizzia julibrissin: V. Evidence against Acetylcholine Participation. PMID- 16658210 TI - Comparative freezing patterns in stems of cherry and azalea. AB - Ice formation in stems, as determined by means of an electrophoretic mobility technique, occurs much more rapidly in azalea than in sour cherry. The difference is more marked in the bark than in the wood. Disrupting the structure of the tissues completely eliminates differences in freezing patterns, although gross anatomical differences do not appear to account for differences in species response. Microscopic examination of frozen stems indicated that little redistribution of water occurred during freezing in azalea, and the tissues were disrupted as these crystals developed. In cherry, on the other hand, water diffused to nucleating centers where crystal growth was not opposed, giving rise to "glaciers." PMID- 16658211 TI - Location of glycoproteins that contain glucosamine in plant tissues. AB - When radioactive d-glucosamine is provided to Acer pseudoplatanus cells in liquid culture in order to label those glycoproteins that contain amino sugars, it is incorporated predominantly into a crude cell wall fraction. This observation was confirmed histologically by preparing autoradiographs of thin tissue sections from plasmolyzed cells. Highly purified cell wall material from unlabeled cells has also been shown to contain small amounts of glucosamine. Similarly, about one half of the amino sugar recovered from cultured cells of Nicotiana tabacum is present in their cell walls. In corn roots, however, the labeled glycoproteins that are formed after glucosamine incorporation are predominantly cytoplasmic and not deposited outside the protoplast. PMID- 16658212 TI - Content of adenosine phosphates and adenylate energy charge in germinating ponderosa pine seeds. AB - An average of 540 picomoles of total adenosine phosphates was found in the embryo of mature seeds of ponderosa pine (Pinus ponderosa Laws.) and 1140 picomoles in the gametophyte. Adenylate energy charges were 0.44 and 0.26, respectively. After stratification, total adenosine phosphates increased 7-fold and 6-fold in embryo and gametophyte, respectively, and energy charges rose to 0.85 and 0.75. During germination, total adenosine phosphates increased to a 20-fold peak on the 9th day in gametophytic tissue, parallel with the peak of reserve regradation and organellar synthesis, and then decreased. In embryo and seedling, total adenosine phosphates elevated 80-fold with two distinct oscillating increases of AMP and ADP. The oscillating increases occurred before the emergence of radicle and cotyledons during which the highest mitotic index prevailed in all tissues. Energy charges fluctuated between 0.65 at the rapid cell dividing stage to 0.85 at the fully differentiated stage of the seedling, while energy charges remained around 0.75 in the gametophyte. These data indicated that the content of adenosine phosphates of germinating seeds reflects growth, organogenesis, and morphogenesis, and that a compartmentalized energy metabolism must exist in dividing and growing plant cells. PMID- 16658213 TI - Isolation and characterization of membranes from the cultivated mushroom. AB - The membranes of sporophore cap tissue from the cultivated mushroom, Agaricus bisporus (Lange) Sing., were isolated using discontinuous sucrose gradient ultracentrifugation of a tissue homogenate. A membrane-rich fraction was concentrated at the 1.16/1.18 g/cc interface and a mitochondria-rich fraction at the 1.18/1.20 g/cc interface. The membrane fraction was judged to be greater than 90% membrane vesicles by electron microscopy. The protein to lipid ratio of the membrane fraction was 1.1; the molar ratio of sterol to phospholipid was 0.77. The specific radioactivity of a Mg-activated ATPase was 2.5 times greater in the membrane fraction than in the homogenate. No 5'-nucleotidase or Na-K-Mg-activated ATPase activity was observed. PMID- 16658214 TI - Photosynthetic properties of permaplasts of anacystis. AB - A treatment procedure using lysozyme and ethylenediaminetetracetic acid gave intact but permeable cells (permeaplasts) of Anacystis nidulans. Rates of electron transport from water to carbon dioxide, ferricyanide, 2,6-dichlorophenol indophenol, benzoquinone, and methyl viologen, and from reduced indophenol to methyl viologen were measured as a function of treatment time. Rates of oxygen evolution in complete photosynthesis and electron flow from water to methyl viologen showed rapid and parallel decline with treatment time. Electron flow from water to ferricyanide and from reduced indophenol to methyl viologen increased during the first half hour of treatment (phase 1) to 60 to 80% of the original photosynthetic rate. Longer treatment (phase 2) resulted in decreased rate of ferricyanide reduction but not in rate of methyl viologen reduction from indophenol. Electron flow from water to quinone was two to three times higher than for complete photosynthesis in intact cells. It remained high during phase 1 and declined during phase 2. Phase 1 permeaplasts apparently retain high activity for photosystems 1 and 2 photoreactions. PMID- 16658215 TI - Effects of proline and carbohydrates on the metabolism of exogenous proline by excised bean leaves in the dark. AB - Proline was metabolized when vacuum infiltrated into starved bean (Phaseolus vulgaris L.) leaves from plants previously in the dark for 48 hours, but an equivalent increase in protein proline was not observed. When (14)C-proline was infiltrated into starved leaves, a large percentage of the (14)C was recovered in other amino acids, organic acids, and CO(2), in addition to that recovered as protein proline. However, extensive oxidation of proline was observed only if enough proline was added to increase substantially the endogenous concentration of proline. Increasing the endogenous concentration did not affect the amount of proline that was incorporated into protein.When added to leaves from plants previously in the light of near saturating intensity for more than 16 hr, very little (14)C-proline was oxidized, even when a sufficient amount of proline was added to increase the endogenous concentration. Adding sucrose to starved leaves along with the proline slowed down the oxidation of proline. Thus, it appears that some carbohydrate or intermediate of carbohydrate metabolism may inhibit the oxidation of proline in leaves.Based on kinetics of labeling, the pathway of proline oxidation was by conversion to glutamic acid and subsequent metabolism to intermediates in the Krebs cycle and to CO(2). PMID- 16658216 TI - Auxin-induced Changes in Avena Coleoptile Cell Wall Composition. AB - Sugar and uronic acid residues were derived from wall polysaccharides of oat (Avena sativa, var. Victory) coleoptiles by means of 2 N trifluoroacetic acid, 72% sulfuric acid, or enzymic hydrolysis. The products of hydrolysis were reduced and acetylated to form alditol acetates which were analyzed using gas chromatography. Time-course studies of auxin-promoted changes in various wall fractions indicate that when exogenous glucose was available, increases in certain wall constituents paralleled increases in length. However, under conditions where exogenous glucose was not available, and where wall synthesis was limited, such correlations with growth were not apparent. Under these latter conditions total wall weight initially increased slightly, then decreased. These changes in weight were the net of increases in cellulose and some noncellulosic constituents and a decrease of over 75% in noncellulosic glucose. When coleoptile sections were preincubated without exogenous glucose for 8 hours to deplete endogenous wall precursors and subsequently treated with auxin, there were no detectable increases in wall weight. There was instead an auxin-promoted decrease in wall weight, and this decrease paralleled a decrease in noncellulosic glucose. There were no significant changes in other wall components. The auxin-promoted decreases in noncellulosic glucose are interpreted as a possible step in the mechanism of growth. PMID- 16658217 TI - Freezing injury in potato leaves. AB - Time-temperature profiles of freezing leaves from frost-resistant (Solanum acaule Bitt.) and frost-susceptible (Solanum tuberosum L. subsp. tuberosum Hawkes) types of potatoes did not reveal any major differences. The pattern of change in resistance of leaves to low voltage, low frequency current during freezing was different in the frost-resistant and susceptible leaves. In tissue sections from both types of leaves, cells freeze extracellularly at cooling velocities lower than 5 C per minute. Cells from leaves of resistant plants showed a higher osmotic pressure but not a higher water permeability than those from susceptible plants. The extent of injury caused by even very slow freezing was greater than that caused by equivalent isopiestic desiccation, particularly in susceptible leaves. The higher osmotic pressure in cells of leaves from resistant plants can account for the greater desiccation resistance but not for the frost resistance observed. PMID- 16658218 TI - Biocarbonate Effect on the Photophosphorylation Catalyzed by Chromatophores Isolated from Chromatium Strain D: XII. Structure and Function of Chloroplast Proteins. AB - Photophosphorylation catalyzed by chromatophores prepared from Chromatium strain D was stimulated by bicarbonate. The stimulative effect was pH dependent and the lower the pH the more marked the activation. At pH 8.0, bicarbonate (8 mm) exhibited a negligible effect, whereas at pH 7.0 approximately a 5-fold activation was observed. The apparent activation constant of bicarbonate was determined to be approximately 10.2 mm, at which concentration approximately a 7 fold activation of photophosphorylation was observed. PMID- 16658219 TI - Aftereffects of low and high temperature pretreatment on leaf resistance, transpiration, and leaf temperature in xanthium. AB - Leaf resistance for water vapor (total diffusion resistance minus boundary layer resistance), transpiration, and leaf temperature were measured in attached leaves of greenhouse-grown Xanthium strumarium L. plants that had been pretreated for 72 hours with high (40 C day, 35 C night), or low (10 C day, 5 C night) air temperatures. Measurements were made in a wind tunnel at light intensity of 1.15 cal cm(-2) min(-1), air temperatures between 5 and 45 C, and wind speed of 65 cm sec(-1). Leaf resistances in low temperature pretreated plants were higher (8 to 27 sec cm(-1)) than in controls or high temperature pretreated plants (0.5 to 3 sec cm(-1)) at leaf temperatures between 5 and 25 C. Thus, the pretreatment influenced stomatal aperture. PMID- 16658220 TI - An Explanation for the Difference in Photosynthetic Capabilities of Healthy and Beet Yellows Virus-infected Sugar Beets (Beta vulgaris L.). AB - Sugar beets (Beta vulgaris L.) infected with the Beet Yellows Virus exhibit lower rates of net photosynthesis at light saturation than do healthy plants. These Pn reductions were correlated with increases in leaf resistance to water vapor loss. Theoretical analyses demonstrated that, although the leaf resistance to water vapor loss increases could account for a major part of the net photosynthesis decreases, some other aspect of leaf functioning also was debilitated by infection. Both the levels and the activities of ribulose-1, 5-diP carboxylase were less on a leaf area basis in extracts from infected leaves than from healthy ones. Soluble carbohydrates accumulate in Beet Yellows Virus-infected leaves, but inhibiting translocation in several ways provided no evidence in support of the hypothesis that the accumulation of photosynthates in leaves has a direct, short term, feed-back effect upon the photosynthetic rate. PMID- 16658221 TI - Polyribosomes from peas: an improved method for their isolation in the absence of ribonuclease inhibitors. AB - Profiles of polyribosomes were obtained from etiolated stem segments of Pisum sativum L. var. Alaska isolated in various buffers. Tissue homogenized in a medium containing 0.2 m tris-HCl, pH 8.5, 0.2 m sucrose, 30 mm MgCl(2), and 60 mm KCl yielded polyribosomes exhibiting far less degradation than tissue homogenized in conventional media containing tris-HCl at lower ionic strength and pH. A further decrease in degradation was found when polyribosomes were sedimented through a sucrose pad buffered at pH 8.5 prior to centrifugation. Increased separation was obtained using heavy (125-500 mg/ml), linear sucrose gradients. Using these techniques, messenger RNA species bearing up to 12 ribosomes (dodecamers) were resolved, with messenger RNA chains bearing 9 ribosomes (nonamers) being the most abundant (having the highest absorption peak). The data presented suggest that buffer of high ionic strength and high pH was more effective in preventing degradation of polyribosomes than was diethyl pyrocarbonate and, furthermore, that ratios involving large polyribosomes (hexamers and larger) were more accurate indices of degradation than were ratios involving total polyribosomes. PMID- 16658222 TI - The level of phytohormones in monoecious and gynoecious cucumbers as affected by photoperiod and ethephon. AB - The endogenous levels of auxin, gibberellin, and inhibitors were followed in monoecious and gynoecious cucumber (Cucumis sativus L.) plants, and in plants treated with the ethylene-releasing compound Ethephon (2-chloroethyl phosphonic acid). Higher auxin inhibitor and lower gibberellin levels were associated with female tendency. The endogenous level of gibberellin and auxin decreased in Ethephon-treated plants. Application of Ethephon induced a rise in abscisic acid. Root application of abscisic acid promoted female tendency of gynoecious cucumbers grown under conditions which increase maleness. High CO(2) levels, which are known to antagonize ethylene, increased maleness of gynoecious cucumbers. The possibility of interrelationship between gibberellin, auxin, ethylene, and abscisic acid on sex expression are discussed. PMID- 16658223 TI - Acid-bath Phosphorylation in Heptane-extracted Chloroplasts. AB - When spinach (Spinacia oleracea) chloroplast fragments were lyophilized and extracted with n-heptane, acid-bath phosphorylation was significantly reduced. Phosphorylation could be restored to these chloroplasts, if the extracted material (containing quinones, carotenoids, and other lipids) was added back to dry preparation before rehydration. The results reported suggest it was not quinones which were required for restoration of acid-bath phosphorylation but lipids. It is suggested that the lipids which were extracted by n-heptane serves as structural role in the thylakoid membrane. PMID- 16658224 TI - Effects of cytokinins on the respiration of soybean callus tissue. AB - A technique which incorporates a brief blending step to disperse callus tissue into small clumps of cells was developed, and the effects of cytokinins on respiration of soybean (Glycine max [L.] Merrill var. Acme) callus tissue prepared in this way were studied. Adenine alone did not affect respiration, but kinetin and zeatin showed effects correlating with their reported effects on growth of this tissue; after about 3 hours both hormones promoted respiration at concentrations which promote growth, while kinetin, but not zeatin, also exhibited inhibition at higher concentrations. Studies with 2,4-dinitrophenol led to the suggestion that although the respiration of this tissue is largely under the control of ATP levels, kinetin does not exert its control on respiration through effects on ATP levels or oxidative phosphorylation during the monitoring period. Further inhibitor and substrate studies provided evidence that the promotion of respiration by kinetin results from an increase in substrate entering the tricarboxylic acid cycle, perhaps by an effect on pyruvate metabolism.The inhibition of respiration by high concentrations of kinetin is partially due to effects on oxidative phosphorylation or ATP utilization, but 70% of the inhibition cannot be attributed to this. PMID- 16658225 TI - A possible role of divalent manganese ions in the photoinduction of phenylalanine ammonia-lyase. AB - Divalent Mn ions cause an increase in the level of phenylalanine ammonia-lyase in gherkin hypocotyls. With the exception of Mg ions, which had a small effect, no other metal ion has so far been found which could replace the Mn ion in this respect. Invertase and peroxidase were not significantly affected by the Mn treatment. The increase in phenylalanine ammonialyase activity is explained by the removal, under the influence of Mn ions, of hydroxycinnamic acids, which cause repression of phenylalanine ammonia-lyase synthesis and/or inactivation of phenylalanine ammonia-lyase. Arguments are advanced for the hypothesis that photochemical transformations of Mn complexes are involved in the photoinduction of phenylalanine ammonia-lyase in dark-grown gherkin seedlings. PMID- 16658226 TI - Chloride accumulation by mung bean root tips: a low affinity active transport system at the plasmalemma. AB - Net uptake of Cl(-) into root tips of mung bean (Phaseolus aureus) increases steadily with increasing external concentrations from 1 to 60 mm. Membrane potentials were measured to determine the equilibrium concentration of Cl(-) in the tissue which could be due to diffusion. This concentration was readily exceeded in both the relatively nonvacuolate tips (0 to 1 mm) and the vacuolate, mature upper sectons (1 to 11 mm) of the roots. The activity coefficient of both cytoplasmic and vacuolar Cl(-), measured with Cl(-) sensitive microelectrodes, was approximately the same as that of a pure KCl solution of the same concentration. It is concluded that the "second mechanism" of ion uptake involves a large increase in the rate of active transport at the plasmalemma as the external concentration is increased above 1 mm. PMID- 16658227 TI - Influence of decenylsuccinic Acid on water permeability of plant cells. AB - Decenylsuccinic acid altered permeability to water of epidermal cells of bulb scales of Allium cepa and of the leaf midrib of Rhoeo discolor. Water permeability, as determined by deplasmolysis time measurements, was related to the dose of undissociated decenylsuccinic acid (mm undissociated decenylsuccinic acid x minute). No relationship was found between permeability and total dose of decenylsuccinic acid, or dose of dissociated decenylsuccinic acid, suggesting that the undissociated molecule was the active factor in permeability changes and injury.At doses which did not damage cells (0.0008 to 0.6 [mm of the undissociated molecule x minute]) decenylsuccinic acid decreased water permeability. At higher doses (e.g., 4 to 8 [mm x minute]) injury to cells was common and decenylsuccinic acid increased permeability. Doses above the 10 to 20 (mm x minute) range were generally lethal. The plasmolysis form of uninjured cells was altered and protoplasmic swelling occasionally was observed. The dose dependent reversal of water permeability changes (decreased to increased permeability) may reflect decenylsuccinic acid-induced changes in membrane structure. Reported effects of decenylsuccinic acid on temperature dependence of permeability and frost resistance were not verified. PMID- 16658228 TI - N-(Delta-Isopentenyl)adenosine: Its Occurrence as a Free Nucleoside in an Autonomous Strain of Tobacco Tissue. AB - Cytokinins from both the free nucleoside pool and the transfer RNA have been isolated and identified in a habituated strain of tobacco pith callus (Nicotiana tabacum [L] var. Wisconsin 38). The transfer RNA of this strain contains both N(6)-(Delta(2)-isopentenyl) adenosine and N(6)-(4-hydroxy-3-methylbut-2-cis-enyl) adenosine. The trans-hydroxylated derivative is absent from the transfer RNA of this dark-grown tissue. N(6)-(Delta(2)-Isopentenyl)-adenosine was identified as a component of the free nucleoside pool in concentrations of about 10 micrograms per kilogram of tissue.The cytokinin-requiring tissue grown in the presence of N(6)-furfurylaminopurine contains less than 85 nanograms per kilogram of N(6) (Delta(2)-isopentenyl) adenosine. This is the detectable limit under the conditions of the experiment. PMID- 16658229 TI - Translocation and Metabolism of Ricinine in the Castor Bean Plant, Ricinus communis L. AB - Ricinine-3,5-(14)C (N-methyl-3-cyano-4-methoxy-2-pyridone) administered to senescent leaves of Ricinus communis L. was translocated to all other tissues of the plant. Developing fruit and especially seeds were found to be labeled the most rapidly. Young growing leaves and other developing tissues of the plant imported ricinine from the senescent leaves much more quickly than mature leaves. Relative intensities of the radioactive ricinine imported and deposited in various tissues indicate a possible functional role of ricinine in the castor bean plant. Data on N-demethyl ricinine presented here may stimulate interest in the possible physiological role of the ricinine to N-demethyl ricinine interconversion. PMID- 16658230 TI - Immunological Comparisons of Chymotrypsin Inhibitor I among Several Genera of the Solanaceae. AB - Microcomplement fixation was employed to compare the immunological differences that occur between purified inhibitor I from potato tubers, the four purified protomers that comprise it, and inhibitor I from tuber and leaf extracts. Total inhibitors of chymotrypsin and trypsin in leaves of seven genera of the Solanaceae were identified by enzymatic assay. In leaves of three genera, Solanum, Lycopersicum, and Datura, chymotrypsin inhibitor I was identified immunologically. In petals of all seven genera inhibitor I was also identified immunologically. With the microcomplement fixation technique inhibitor I from leaf or petal extracts of eight Solanaceae genera were compared. An immunological relationship of inhibitor I among seven of these genera was established. PMID- 16658231 TI - Use of fluid fluorocarbons to study freezing in plant tissues. PMID- 16658232 TI - The Contrast between Active Transport and Diffusion of Indole-3-acetic Acid in Coleus Petioles. PMID- 16658233 TI - Enhanced Activity of the Soluble Ribonucleic Acid Polymerase from 2,4 Dichlorophenoxyacetic Acid-treated Maize Seedlings. PMID- 16658234 TI - Effect of Urea on Ammonium-dependent Synthesis of Carbamyl Phosphate during Spore Germination of Geotrichum candidum. AB - The specific activities of enzymes catalyzing the ammonium-dependent carbamyl phosphate synthesis (NH(3)-CPS) and the glutamine-dependent carbamyl phosphate synthesis (GLN-CPS) were increased during germination by approximately 5-and 1.7 fold respectively in the presence of 35 mm urea. The increase of NH(3)-CPS and GLN-CPS levels occurred immediately after the onset of germination and prior to the appearance of germ tube. Ammonium also stimulated the NH(3)-CPS activity, but the induction caused by urea was about three times higher than that by ammonium.Both NH(3)-CPS and GLN-CPS were highly labile. NH(3)-CPS was obtained free of GLN-CPS after (NH(4))(2)SO(4) fractionation and diethylaminoethyl cellulose chromatography. The optimum pH for NH(3)-CPS was 8.5 as opposed to broad pH optimum of pH 5.6-8 for the reverse reaction. The Km values obtained for NH(3), glutamine, and carbamyl phosphate were 12 mm, 0.5 mm, and 0.083 mm, respectively. PMID- 16658235 TI - Phytochrome Control of Germination of Rumex crispus L. Seeds Induced by Temperature Shifts. AB - High germination of curly dock (Rumex crispus L.) seeds is evident after suitable imbibition and temperature shift treatment, but germination at constant temperatures fails without an input of far red-absorbing form of phytochrome. Preliminary imbibitions at high temperatures (30 C) sharply reduce germination induced by temperature shifts. High germination may be restored by low energies of red radiation, or by brief far red adequate for the photosteady state. Prolonged far red during imbibition also nullifies temperature shift-induced germination. After prolonged far red, high germination may be restored by red radiation of an energy dependent upon the duration of the far red treatment. The evidence supports the conclusion that dark germination induced by temperature shifts arises from the interaction of pre-existent far red-absorbing form of phytochrome in the mature seeds with the temperature shift. PMID- 16658237 TI - Further comparative studies of pectin esterase in relation to leaf and flower abscission. AB - The possible role of pectin esterase in abscission of bean and coleus leaves was re-examined using improved methods for its extraction. Its possible role in abscission of flowers of tobacco and leaves of soybean. cotton, mulberry, magnolia, and euonymus was also examined. In seven of the eight species, no detectable changes in abscission zone pectin esterase activities immediately preceded abscission. Therefore, pectin esterase probably plays no causal role in abscission in these species.In coleus, activity in the abscission zone's distal portion decreased by one-half at most and remained unchanged in its proximal portion with abscission's onset. Also accompanying abscission in coleus was a decrease in the proportion of abscission zone pectin esterase extractable with 3 m sodium chloride at pH 7. These changes were not a general concomitant of senescence as they were not detected in the middle of abscising coleus petioles. Pectin esterase may play a role in coleus leaf abscission. PMID- 16658236 TI - Synthesis and turnover of nitrate reductase in corn roots. AB - THE INDUCTION AND REINDUCTION OF NITRATE REDUCTASE IN ROOT TIP OR MATURE ROOT SECTIONS SHOW ESSENTIALLY A SIMILAR PATTERN: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6 methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t(1/2) = 3 hours) than in mature root sections (t(1/2) = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme. PMID- 16658238 TI - Studies on Phytoalexins: The Relationship between Actinomycin D and Ribonucleic Acid Synthesis during the Induction of Phaseollin in the French Bean (Phaseolus vulgaris L.). AB - Actinomycin D stimulated phaseollin production in endocarp tissues of the French bean (Phaseolus vulgaris L.), maximum production being obtained with 25 to 30 micrograms per milliliter of antibiotic. Under these conditions, net incorporation of (3)H-uridine into total cell ribonucleic acid was inhibited by more than 80% over a 6-hour induction period. If allowance was made for a 2-hour lag in the action of actinomycin D, inhibition of incorporation was greater than 95%. Contrary to other reports, no evidence was obtained of an increased formation of any specific ribonucleic acid fraction. Actinomycin D applied in the cold (4 C) was not found to be effective in stimulating phaseollin production. When applied in this way, actinomycin D did not affect induction of phaseollin by a fungal peptide, Monilicolin A, although ribonucleic acid synthesis was inhibited by more than 95%. It is suggested that the induced formation of phytoalexins may not be dependent on increased ribonucleic acid synthesis as has previously been claimed.Other experiments indicated that the apparent effects of actinomycin D on ribonucleic acid synthesis could be influenced by the choice of precursor used to label ribonucleic acid and by the order of addition of precursor and antibiotic to the plant tissue. These effects were only observed in the period immediately following application of actinomycin D. It is suggested that such effects could critically influence the results obtained in short term experiments and may explain some differences in reported action of actinomycin D from different laboratories. PMID- 16658239 TI - Abscisic Acid in relation to mineral deprivation. AB - Tobacco (Nicotiana rustica) plants growing in half-strength Hoagland solution were deprived of nutrients by being transferred to distilled water. The abscisic acid content of leaves in the mineral-deprived plants rose continuously throughout the 7 days of the experimental period. However, although the content of ABA rose within 24 hours, a decline in growth and leaf-chlorophyll were discernible only after the 4th day of mineral deprivation. As anticipated, mineral-deprived (stressed) plants exhibit "resistance" to lack of aeration in the root medium, similar to that shown in salt-stressed plants or plants that were pretreated with absiscic acid. When the mineral-deprived plants were returned to half-strength Hoagland, the content of leaf abscisic acid declined to the prestressed level and the "resistance" to lack of root aeration disappeared.These results indicate that an increase in abscisic acid may be induced by conditions unfavorable to growth and not exclusively by conditions affecting the plant's water balance. In addition, the work also indicates that mineral deficiency is associated with significant modification in the hormonal balance of the plant. PMID- 16658240 TI - Polysome Formation in Light-controlled Dormancy. AB - Lettuce (Lactuca sativa) seeds var. Grand Rapids could be maintained many weeks in the dark without germination. Following illumination with white light, a gradual increase in polyribosome population up to the time of germination was demonstrated by sucrose gradient centrifugation. Polysomes could not be detected in imbibed seeds maintained continuously in the dark. Thus, polysome formation and therefore the capacity for a high rate of protein synthesis required for germination and growth, is not associated with the process of imbibition, but is dependent upon the seeds having received the dormancy-breaking stimulus of illumination. PMID- 16658241 TI - Iron-stress Response in Mixed and Monocultures of Soybean Cultivars. AB - Hawkeye (Fe-efficient) and PI-54619-5-1 (Fe-inefficient) soybeans (Glycine max [L.] Merr.) were grown in mixed and monoculture nutrient solutions to evaluate an inhibitory effect of PI-54619-5-1 on the uptake of Fe by Hawkeye. The ability of Hawkeye to take up Fe (Fe-stress response) was dependent on the degree of Fe stress (Fe deficiency) and was not the result of an inhibitory substance released by PI-54619-5-1 in mixed culture (Hawkeye + PI-54619-5-1).The degree of Fe stress in Hawkeye was dependent on the amount of Fe taken up by the plant. Hawkeye took up more Fe and developed less Fe stress in mixed than in monoculture because in mixed culture PI-54619-5-1 did not utilize Fe as efficiently as Hawkeye which allowed more Fe to be available per HA plant. Thus, Fe-stress response, rather than any inhibitory substances produced by PI-54619-5-1 plants, controls the uptake of Fe in Hawkeye soybean. PMID- 16658242 TI - Proline Content and Metabolism during Rehydration of Wilted Excised Leaves in the Dark. AB - Excised bean (Phaseolus vulgaris) leaves were used to measure changes in proline content and proline metabolism during rehydration in the dark after the leaves had been incubated in the dark 24 hours in a wilted condition.The increase in nonprotein proline which occurs in wilted leaves stopped immediately upon rehydration, and thereafter levels of proline declined. The rate of decline and the fate of the metabolized proline depended on the amount of carbohydrate present. When the level of carbohydrate was relatively high, the rate of decline in proline content was slow, and the proline was converted mainly to protein proline. When the level of carbohydrate was relatively low, the rate of proline decline was rapid and a large percentage of the proline was oxidized to CO(2) and other amino acids in addition to conversion to protein proline.The results indicate that nonprotein proline can be oxidized when the level is increased by endogenous synthesis. Proline oxidation has been observed when the levels are increased by adding exogenous proline. Oxidation of endogenous proline is inhibited by carbohydrates in the leaf, as is true of oxidation of exogenous proline. PMID- 16658243 TI - Regulation of salt respiration in carrot root slices. AB - In slices of carrot-phloem parenchyma washed for 7 days in water at 20 C, 50 mm KCl stimulates respiration by up to 100% of the ground respiration within 4 minutes of application. The data presented imply that ADP liberated in the cytoplasm as a consequence of KCl accumulation first stimulates a regulator reaction requiring ADP (phosphoglycerate kinase). Thereafter, the point of control alternates between this reaction and the phosphofructokinase reaction, forming a sequence of enzyme stimulations which continue after the new steady state of increased respiration is established. KCl induces a similar sequence in slices washed for 3 days, but it is completed within 3 minutes, and metabolite oscillations are not so marked. In slices washed for 2 days, KCl stimulates respiration by less than 10%, and the sequence of regulator reactions does not occur. Phosphoglycerate kinase is the only enzyme stimulated within 3 minutes of applying KCl to these slices. Contrary to previous reports, KCl frequently stimulates the respiration of freshly prepared slices by 10 to 30%. PMID- 16658244 TI - Phytochrome-mediated Electric Potential Changes in Oat Seedlings. AB - Brief exposures to red light induce far red-reversible changes of 5 to 10 millivolts magnitude in the upper 1 centimeter of etiolated Avena coleoptiles. The changes begin within 15 seconds of the start of illumination and they continue for at least 12 minutes. The changes were measured using a flowing solution of 10 mm KCl to contact the surface of the coleoptile. A dark-grown coleoptile shows no change in response to far red light unless it first receives red light treatment. The second of two red light exposures is ineffective without an intervening far red treatment. Some characterization of these electric responses to light is presented. PMID- 16658245 TI - Effect of gamma-Irradiation on the Biosynthesis of Carotenoids in the Tomato Fruit. AB - Fruits of the lutescent tomato genetic line were exposed to gamma-radiation at different stages of maturity to determine the effect of ionizing radiation on carotenoid synthesis in the ripening fruit. Irradiation generally resulted in the inhibition of carotenogenesis. The effect was more pronounced at the higher dosage and in less mature fruit. Lycopene synthesis was inhibited more extensively than beta-carotene synthesis. The total carotenoid content was also generally lower in irradiated fruits. It was proposed that the beta-carotene in the tomato fruit is formed by a pathway not involving lycopene. PMID- 16658246 TI - The cupric ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts. AB - Strong inhibition of uncoupled photosynthetic electron transport by Cu(2+) in isolated spinach chloroplasts was observed by measuring changes in O(2) concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu(2+) to the chloroplast membranes resulted in removal of Cu(2+) from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu(2+) binding to inhibitory sites. Preincubation in the dark resulted in Cu(2+) binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.When the photosystems were assayed separately, photosystem I was more resistant to inhibition than photosystem II. The most sensitive site to the inhibitor was the oxidizing side of photosystem II. PMID- 16658247 TI - Cytokinins: synthesis and biological activity of geometric and position isomers of zeatin. AB - Geometric and position isomers of zeatin and of ribosylzeatin and other compounds closely related to zeatin have been tested in the tobacco (Nicotiana tabacum var. Wisconsin No. 38) bioassay. None was more active than zeatin itself. There was a much greater difference in activity (> 50-fold) between trans- and cis-zeatin than between trans-isozeatin [6-(4-hydroxy-2-methyl-trans-2-butenylamino) purine] and cis-isozeatin [6-(4-hydroxy-2-methyl-cis-2-butenylamino) purine], the latter being less active than cis-zeatin and trans-isozeatin. Higher concentrations were required for equivalent callus growth stimulated by the 9-ribosyl derivatives, which followed an order of decreasing activity: ribosyl-trans-zeatin > ribosyl cis-zeatin > ribosyl-trans-isozeatin > ribosyl-cis-isozeatin, corresponding roughly to that of the bases. The effect of side chain, double bond saturation was to diminish the activity, and in the dihydro series the shift of the methyl group from the 3- to the 2-position in going from dihydrozeatin to dihydroisozeatin [6-(4-hydroxy-2-methylbutylamino) purine] resulted in a 70-fold decrease in activity. cis-Norzeatin [6-(4-hydroxy-cis-2-butenylamino) purine], which was less than one-fifth as active as cis-zeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3,6 dihydro-1,2-oxazin-2-yl) purine] was about 1/100 as active as cis-norzeatin. These findings delineate completely the effect on the cytokinin activity of zeatin of variation in side chain geometry, presence and position of the methyl substituent, presence and geometry of hydroxyl substitution, presence of the double bond, and of side chain cyclization. PMID- 16658248 TI - Localization of crystals in diseased oats treated with uranyl acetate. AB - Uranyl acetate, a suppressor of victorin-induced electrolyte leakage in oat leaves when applied together with, or before, victorin, also suppressed victorin induced changes in ultra-structure. Uranyl crystallized in cell walls and near the plasmalemma of vascular cells, but was excluded from the protoplasm. Fewer crystals occurred near the plasmalemma when leaves were allowed to take up uranyl and victorin simultaneously than when uranyl alone was absorbed, but deposition in cell walls was similar in the two treatments. No differences in crystal distribution were found in uranyl-treated leaves which subsequently took up either water or victorin. The most striking effect of prolonged exposure to uranyl was increased vesicular activity in the protoplasm, formation of complex concentric membranes, and tonoplast damage. Following victorin treatment, uranyl post-treatment was ineffective in suppressing electrolyte leakage or preserving normal cellular ultrastructure. More severe ultrastructural damage was found in victorintreated leaves after uranyl post-treatments than after post-treatment with water, a result of victorin-induced damage which facilitates uranyl entry into the protoplasm. PMID- 16658249 TI - Use of uncoupling acridine dyes as stoichiometric energy probes in chloroplasts. AB - In a suspension of spinach chloroplasts the fluorescence of atebrin and other uncoupling acridine dyes is quenched upon energization which is associated with a proportional binding of the dyes to the organelles. There is a stoichiometric relation between the amount of dye bound and the actual steady state level of energy. When the concentration of atebrin is increased in energized chloroplasts the fluorescence is completely quenched until a certain concentration is attained above which the response sharply declines. Such titrations with atebrin were carried out under conditions of partial electron transport governed by photosystems I and II, in the presence of 3-(3,4-dichlorophenyl)-1, 1 dimethylurea and cyanide, respectively, and of complete electron transport governed by the two photo-systems. The sum of the saturating amounts of atebrin obtained in these partial electron flow systems equals that obtained in the complete system. This lends strong support to the view that two sites of energy conservation are coupled to the linear photosynthetic electron transport.When ATP was the energy donor the saturating amounts of atebrin were the same in both control and cyanide-treated chloroplasts, indicating that the energy-conserving mechanism was unimpaired in the latter.Removal of the chloroplast-coupling factor by ethylenedia-minetetraacetate treatment leads to inhibition of the probe responses, which can be restored again in recoupled chloroplasts. PMID- 16658250 TI - Ethylene synthesis in lettuce seeds: its physiological significance. AB - The germination and pregermination ethylene production of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C after a red light treatment are inhibited if the seeds are first imbibed at 30 C for 36 hours. In this study, low concentrations of ethylene were found to enhance the germination of seeds pretreated at 30 C more than that of untreated controls. In the presence of high concentrations of ethylene, pretreated seeds and controls germinated at a similar rate. These results are consistent with the view that a prolonged imbibition at 30 C inhibits germination at a lower temperature through its effect on the ethylene production of the seeds. As a further test of the hypothesis, estimates were made of the pregermination ethylene content of untreated seeds and pretreated seeds incubated in the presence of sufficient ethylene to make them germinate as rapidly as untreated seeds. The values obtained were 0.65 and 0.74 nanoliter of ethylene per gram (dry weight) of seeds, respectively. PMID- 16658251 TI - Induction of indoleacetic Acid synthetases in tobacco pith explants. AB - Formation of indoleacetic acid synthetases in tobacco pith explants was determined by following the growth of tissue cultures under conditions of indole 3-acetic acid (IAA) deprivation and by measuring the enzymatic conversion of tryptophan to IAA in the cultures. The pith explants obtained from the parent plant (Nicotiana glauca) and from basal regions of the tumor-prone hybrid (N. glauca x N. langsdorffii) both show a requirement for exogenous IAA for growth initiation in culture. The parent pith requires the constant presence of added IAA for continued growth, but hybrid pith, after initial treatment with IAA, will grow without further additions. IAA synthetases are detected in the cell homogenates of hybrid pith explants cultured with either continuous or initial IAA addition. These observations indicate that IAA may induce its own production. In contrast, IAA synthetases are not found in the parent pith under comparable culture conditions. Besides IAA, nonhormonal compounds such as indole and tryptophan are also capable of stimulating growth of hybrid pith, possibly through the induction of IAA synthetases needed for IAA formation. Indole and tryptophan are, however, inactive in growth promotion of the parent pith. These results suggest that the genomic expression of IAA synthetase formation is more stringently controlled in N. glauca than in the tumorprone hybrid. PMID- 16658252 TI - Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C(4) Dicarboxylic Acid Pathway of Photosynthesis. AB - The activities of certain enzymes related to the carbon assimilation pathway in whole leaves, mesophyll cell extracts, and bundle sheath extracts of the C(4) plant Panicum miliaceum have been measured and compared on a chlorophyll basis. Enzymes of the C(4) dicarboxylic acid pathway-phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase-were localized in mesophyll cells. Carbonic anhydrase was also localized in mesophyll cell extracts. Ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, and ribulose diphosphate carboxylase-enzymes of the reductive pentose phosphate pathway-were predominantly localized in bundle sheath extracts. High activities of aspartate and alanine transaminases and glyceraldehyde-3-P dehydrogenase were found about equally distributed between the photosynthetic cell types. P. miliaceum had low malic enzyme activity in both mesophyll and bundle sheath extracts.Isolated bundle sheath cells were capable of converting aspartate to oxalacetate at rates approaching the aspartate transaminase activity of bundle sheath extracts. The bundle sheath cells had a light induced CO(2) fixation of 23 mumoles of CO(2)/mg chl.hr in the absence of exogenous substrates.The photorespiratory enzymes, hydroxypyruvate reductase and glycolic oxidase, were about 3 fold higher in bundle sheath extracts than in mesophyll extracts when compared on a chlorophyll basis. PMID- 16658253 TI - Detergent extraction of enzymes from tobacco leaves varying in maturity. AB - Enzyme activities of tobacco leaves were compared in detergent extracts. Highest levels of chlorogenic acid oxidase, malate-NAD oxidoreductase, and acid phosphatase were obtained from bud tissues. Peroxidase activity was least in young leaves and highest in senescent leaves yellowed with Ethrel. Peroxidase zymograms obtained by means of disc electrophoresis showed differences in isozyme composition among all five samples examined. Although protease was found in material extracted from buds, upper, middle, and lower leaf positions, none could be demonstrated in Ethrel-treated mature leaves. PMID- 16658254 TI - Elongation responses of the oat shoot to blue light, as measured by capacitance auxanometry. AB - An auxanometer based on capacitance micrometry is described. It does not require light or contact with the plant, and will detect changes in length of the Avena coleoptile of 1 micrometer in 3 to 5 seconds. The field employed, 1 kilohertz at a gradient of 5 volts per centimeter, does not affect the growth rate of the coleoptile. Vertical illumination with blue light at fluences of 1 to 50 ergs.cm( 2) cause an accelerated rate of coleoptile extension, beginning within 10 min after exposure. At higher exposures, the previously described growth inhibition becomes dominant. PMID- 16658255 TI - Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroides. AB - The ultrastructure of sectioned cells of mutant and wild type Rhodopseudomonas spheroides has been examined by electron microscopy. The characteristic vesicles associated with the presence of bacteriochlorophyll were found in wild type cells grown with low aeration. These were also found in mutant TA-R which forms bacteriochlorophyll under high aeration. None of the mutants with blocks in bacteriochlorophyll synthesis contained intracytoplasmic membrane. These included mutant 8-17 which accumulates bacteriochlorophyllide but fails at the phytolation step. We conclude that the intact bacteriochlorophyll molecule, or some particular membrane protein associated with it, is needed for the development of the characteristic intracytoplasmic membrane system in R. spheroides. PMID- 16658256 TI - Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomonas spheroides. AB - Phytol has been separated and identified in the unsaponifiable lipid fraction from wild type Rhodopseudomonas spheroides, but it was not detected in mutant strains blocked at various stages of bacteriochlorophyll synthesis. Incorporation of (14)C-acetate into phytol paralleled bacteriochlorophyll synthesis in suspensions of the wild type incubated anaerobically in the light. The addition of chloramphenicol inhibited both processes. It is concluded that phytol formation is tightly coupled to the synthesis of the pyrrole component of bacteriochlorophyll. PMID- 16658257 TI - Decrease of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenase Activities in the Xylem of Populus gelrica on Budding. AB - The activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases, transketolase, phosphoglucose isomerase, and fructose 6-phosphate kinase were studied in extracts of wintering poplar (Populus gelrica) xylem. The xylem of wintering poplar showed high levels of transketolase, glucose 6-phosphate, and 6 phosphogluconate dehydrogenases. On recommencement of growth, the two dehydrogenase activities decreased. The three remaining enzymes appeared to be unchanged. In spring and early summer, glucose 6-phosphate dehydrogenase of the xylem was extremely low. On the other hand, 6-phosphogluconate dehydrogenase, which also became lower during the metabolic shift from winter to spring, was readily detected, and was several times higher than glucose 6-phosphate dehydrogenase throughout the year. The low dehydrogenase activities lasted into late October and then appeared to resume their original activity. A shift of metabolism at the beginning of growth was also observed by measuring the amount of sugar phosphates, soluble amino acids and amides, and proteins in the xylem. In contrast to the decrease of the two dehydrogenases and soluble proteins at the time of budding, incorporation of lysine-U-(14)C into the xylem protein ramained constant. A method to transfuse radioactive compounds into a section of stem was described. PMID- 16658259 TI - Ethylene: role in fruit abscission and dehiscence processes. AB - Two peaks of ethylene production occur during the development of cotton fruitz (Gossypium hirsutum L.). These periods precede the occurrence of young fruit shedding and mature fruit dehiscence, both of which are abscission phenomena and the latter is generally assumed to be part of the total ripening process. Detailed study of the dehiscence process revealed that ethylene production of individual, attached cotton fruits goes through a rising, cyclic pattern which reaches a maximum prior to dehiscence. With detached pecan fruits (Carya illinoensis [Wang.] K. Koch), ethylene production measured on alternate days rose above 1 microliter per kilogram fresh weight per hour before dehiscence began and reached a peak several days prior to complete dehiscence. Ethylene production by cotton and pecan fruits was measured just prior to dehiscence and then the internal concentration of the gas near the center of the fruit was determined. From these data a ratio of production rate to internal concentration was determined which allowed calculation of the approximate ethylene concentration in the intact fruit prior to dehiscence and selection of appropriate levels to apply to fruits. Ethylene at 10 microliters per liter of air appears to saturate dehiscence of cotton, pecan, and okra (Hibiscus esculentus L.) fruits and the process is completed in 3 to 4 days. In all cases some hastening of dehiscence was observed with as little as 0.1 microliter of exogenous ethylene per liter of air. The time required for response to different levels of ethylene was determined and compared to the time course of ethylene production and dehiscence. We concluded that internal levels of ethylene rose to dehiscence-stimulating levels a sufficience time before dehiscence for the gas to have initiated the process. Since our data and calculations indicate that enough ethylene is made a sufficient time before dehiscence, to account for the process, we propose that ethylene is one of the regulators of natural fruit dehiscence, an important component of ripening in some fruits. Our data also suggest a possible involvement of ethylene in young fruit abscission. PMID- 16658258 TI - Water Stress Enhances Ethylene-mediated Leaf Abscission in Cotton. AB - Abscission of cotyledonary leaves from cotton (Gossypium hirsutum L. cv. Stoneville 213) seedlings occurred following relief from water stress. The amount of abscission was related to the magnitude of the plant water deficit. Leaf abscission promoted by exogenous ethylene was enhanced in seedlings subjected to water stress. Treatment with ethylene (2.0 to 3.2 microliters of ethylene per liter of air for 24 hours) raised the threshold plant water potential required to induce abscission from -17 to -7 bar, indicating that the stress caused the tissue to become predisposed to ethylene action. Based on the abscission response curve for seedlings treated with ethylene while under water stress, this apparent predisposition was developed as the plant water potentials reached the -7 to -10 bar range. The abscission-promoting effects of ethylene in combination with water stress were reversed with 15% CO(2) at plant water potentials above -12 bar, but the CO(2) reversal was lost at lower water potentials. These results are compatible with the concept that ethylene plays a regulatory role in leaf abscission induced by water stress. PMID- 16658260 TI - Ethylene: Response of Fruit Dehiscence to CO(2) and Reduced Pressure. AB - These studies were conducted to determine whether ethylene serves as a natural regulator of fruit wall dehiscence, a major visible feature of ripening in some fruits. We employed treatments to inhibit ethylene action or remove ethylene and observed their effect on fruit dehiscence. CO(2) (13%), a competitive inhibitor of ethylene action in many systems, readily delayed dehiscence of detached fruits of cotton (Gossypium hirsutum L.), pecan (Carya illinoensis [Wang.] K. Koch), and okra (Hibiscus esculentus L.). The CO(2) effect was duplicated by placing fruits under reduced pressure (200 millimeters mercury), to promote the escape of ethylene from the tissue. Dehiscence of detached fruits of these species as well as attached cotton fruits was delayed. The delay of dehiscence of cotton and okra by both treatments was achieved with fruit harvested at intervals from shortly after anthesis until shortly before natural dehiscence. Pecan fruits would not dehisce until approximately 1 month before natural dehiscence, and during that time, CO(2) and reduced pressure delayed dehiscence. CO(2) and ethylene were competitive in their effects on cotton fruit dehiscence. All of the results are compatible with a hypothetical role of ethylene as a natural regulator of dehiscence, a dominant aspect of ripening of cotton, pecan, and some other fruits. PMID- 16658261 TI - Serological Characterization of the Glycolate-oxidizing Enzymes from Tobacco, Euglena gracilis, and a Yellow Mutant of Chlorella vulgaris. AB - An antiserum to tobacco glycolate oxidase has been prepared by injection of the purified enzyme into rabbits. Double gel diffusion tests between the antiserum and purified antigen and also with a crude tobacco preparation gave a single immunoprecipitation band. Crude extracts of Euglena gracilis Z Klebs, containing glycolate dehydrogenase, and of Chlorella vulgaris 211-11h/20, containing glycolate oxidase, also formed single bands with the tobacco antiserum. The algal bands were identical and showed partial identity with the tobacco band. The antiserum inhibited the glycolate oxidase activities of the tobacco and Chlorella extracts but did not affect Euglena glycolate dehydrogenase activity. PMID- 16658262 TI - pH Dependence and Cofactor Requirements of Photochemical Reactions in Maize Chloroplasts. AB - The pH dependence of the photoreduction of ferricyanide and the photoreduction of NADP from water and photosystem I activity have been compared in isolated chloroplasts from mesophyll and bundle sheath cells of Zea mays. The maximum activity of photoreduction of ferricyanide occurs at pH 8.5 in isolated mesophyll chloroplasts. The addition of methylamine does not cause a marked shift in the pH maximum, but brief sonication lowers the pH maximum to 7.0. In contrast, isolated bundle sheath chloroplasts have a pH maximum at 7.0 and the shape of the pH versus activity curve is similar to that of sonicated mesophyll chloroplasts. When photoreduction of ferricyanide by the isolated chloroplasts is measured at their pH maxima, the values for bundle sheath chloroplasts are about half those of methylamine-treated mesophyll chloroplasts on a chlorophyll basis.The pH maxima for the photoreduction of NADP from water and photosystem I activity are similar in both mesophyll and bundle sheath chloroplasts with maximum activity occurring at pH 7.0 in both cases. In the presence of added plastocyanin and ferredoxin NADP-reductase, the photosystem I activities of both sonicated mesophyll and sonicated bundle sheath chloroplasts are significantly higher than those of the unsonicated preparations. On a chlorophyll basis, photosystem I activity of bundle sheath chloroplasts is at least twice that of mesophyll chloroplasts. PMID- 16658264 TI - Polar Indole-3-acetic Acid Diffusion in Nonliving and Model Systems. AB - Polar indole-3-acetic acid movement was observed in killed plant segments and in artificial model systems. The polar diffusion of indole-3-acetic acid was observed in tissue killed by chemical or physical means in an agar-plant system and in a multicelled Plexiglas dialysis chamber containing hypocotyl tissue gradients or gradients of anion exchange material.It was suggested that polar indole-3-acetic acid movement in excised segments may be a reflection of the diffusion of indole-3-acetic acid through asymmetric gradients of materials with binding characteristics somewhat similar to ion exchange. PMID- 16658263 TI - Reversible abolition of enhancement in isolated spinach chloroplasts. AB - The photosynthetic evolution of oxygen by isolated chloroplasts of Spinacia oleracea L. was studied using a modulated oxygen electrode. The enhancement effect, measured as the increase in the relative quantum yield of 650-nanometer light due to the presence of 710-nanometer light, was profoundly influenced by the concentration of inorganic cations in the bathing medium. Chloroplast fragments immersed in a solution containing a very low concentration of MgCl(2) or KCl, did not display enhancement but could be made to do so by raising the concentration of MgCl(2) to 3 mm, or that of KCl to 35 mm. This change in the enhancement properties was completely reversible. The maximum value of enhancement in a MgCl(2) solution appeared to occur at a concentration between 15 and 30 mm, while in KCl, the enhancement effect increased almost linearly up to a concentration of 100 mm.The appearance of the initial oxygen transient, the burst, was also controlled by the concentration of inorganic cations. Increasing concentrations of MgCl(2) or KCl caused the burst to grow in size in a way which was linearly related to the corresponding increase in enhancement. PMID- 16658265 TI - Effects of cycloheximide on amino Acid biosynthesis in corn roots. PMID- 16658266 TI - Complementation of isolated mitochondria from several wheat varieties. PMID- 16658267 TI - Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels. AB - Soluble ADPglucose-alpha-glucan 4-alpha-glucosyltransferase (starch synthetase), ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase were assayed in extracts from developing kernels of maize (Zea mays). Normal, waxy and amylose-extender maize at stages of development ranging from 8 days to 28 days after pollination were studied. Shrunken-4 maize at the 22-day stage was also studied. There is adequate activity of both ADPglucose pyrophosphorylase and starch synthetase at all stages of development to account for the synthesis of starch. Thus all starch could be synthesized via the ADPglucose pathway. High levels of UDPglucose pyrophosphorylase and of phosphorylase activities were also found at all stages of development. The possible role of phosphorylase in starch synthesis could not be discounted. The levels of phosphorylase, ADPglucose pyrophosphorylase, starch synthetase, and UDPglucose pyrophosphorylase activities in shrunken-4 kernels were about 20 to 40% of that found in normal maize kernels. It appears that the mutation in shrunken-4 affects the activities of more than one enzyme. The defective starch synthesis seen in this mutant could be due to the low activities of ADPglucose pyrophosphorylase and starch synthetase rather than the low activity of phosphorylase. PMID- 16658268 TI - Effect of light on ribonucleic Acid metabolism in greening maize leaves. AB - The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either (32)P-phosphate or double labeling with (14)C- and (3)H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.Sucrose density gradient analyses of ribosomes and of extracted RNA did not reveal light-induced incorporation of label into messenger-like RNA associated with polyribosomes during brief illumination. However, newly produced RNA which seems to be neither ribosomal RNA nor transfer RNA is detectable after illumination for 2.5 hours or longer. PMID- 16658269 TI - Photosynthetic Production of Hydrogen Peroxide by Anacystis nidulans. AB - A sensitive assay based upon fluorescence of scopoletin allowed continuous recording of H(2)O(2) production in illuminated intact cells of Anacytis nidulans. Onset of illumination was followed by a 5 to 10 second lag, a burst of very rapid production continuing for up to 5 minutes, and finally a slow and continuing steady rate of H(2)O(2) production. Size of the H(2)O(2) burst was decreased by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, by low O(2), and by certain Calvin cycle intermediates; it was increased by high light intensity, CO(2) depletion, Calvin cycle inhibitors (as iodoacetamide), cold shock, carbonyl cyanide m-chlorophenylhydrazone, and certain organic acids as glycolate). The H(2)O(2) burst was explained by the following hypothesis: a low potential reductant is produced more rapidly than it can be used in the normal pathway to CO(2) reduction and, instead, reacts with oxygen. H(2)O(2) production is regarded as a metabolic defect observable in Anacystis most dramatically during the transition from a very low rate of oxidative dark metabolism to a high rate of photosynthetic metabolism. PMID- 16658270 TI - Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings. AB - Mevalonic acid-2-(14)C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-(14)C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-(14)C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover. PMID- 16658271 TI - Chlorophyll Formation in Greening Bean Leaves during the Early Stages. AB - In the evolution of the absorption spectrum of etiolated bean leaves (Phaseolus vulgaris L.) following illumination, a rapid photoconversion of 50% or more of the active protochlorophyllide at room temperature is followed by a shift of the chlorophyll(ide) absorption maximum: C(678)--> -->C(684)-->C(672 nm). Kinetic studies at 2 C and the absence of an isosbestic point provide evidence for an intermediate between C(678) and C(684). A dramatically different evolution is observed following the photoconversion of only 5 to 30% of the active protochlorophyllide at room temperature. C(672) appears within 30 seconds, and no subsequent dark shift occurs during the following 90 minutes. At 0 C, conversion of 5% of the active protochlorophyllide produces a new species, C(676), which converts progressively to C(672) within 10 minutes. We interpret the results in terms of two photochemical steps operating in series for the complete conversion of active protochlorophyllide. Furthermore, there appears to be competition between an irreversible, terminal dark shift and the second light reaction. We propose a scheme based on dimers of protochlorophyllide reduced stepwise to dimers of chlorophyllide in two successive light reactions. The intermediate mixed protochlorophyllide-chlorophyllide dimer absorbs at 676 nm and displays a much faster dissociation to monomers than does the chlorophyllide-chlorophyllide dimer. PMID- 16658272 TI - Nitrate Uptake by Dark-grown Corn Seedlings: Some Characteristics of Apparent Induction. AB - Five-or six-day old seedlings of corn (Zea mays L.) were exposed to 0.25 mm Ca(NO(3))(2), 1.0 mm sodium 2-[N-morpholino]-ethanesulfonate, 5 mug Mo per liter and 50 mug of chloramphenicol per ml at pH 6. Nitrate uptake was determined from depletion of the ambient solution. The pattern of nitrate uptake was characterized, after the first 20 minutes, by a low rate which increased steadily to a maximal rate by 3 to 4 hours. Transfer of nitrate to the xylem did not totally account for the increase. Development of the maximal accelerated rate did not occur at 3 C with excised roots nor with seedlings whose endosperm had been removed. Use of CaCl(2) rather than Ca(NO(3))(2) resulted in a linear rate of chloride uptake during the first 4 hours, and chloride uptake was not as restricted by endosperm removal as was nitrate uptake.Nitrite pretreatments or the addition of cycloheximide (2 mug ml(-1)), puromycin (400 mug ml(-1)) and 6 methylpurine (0.5 mm) restricted maximal development of the accelerated nitrate uptake rate. Actinomycin D (20 mug ml(-1)) inhibited the rate only after about three hours exposure. The RNA and protein synthesis inhibitors also restricted nitrate reductase induction in the apical segments of the root tissue. The data suggest that development of the maximal accelerated rate of nitrate uptake depended upon continuous protein synthesis, and the hypothesis that synthesis of a specific nitrate transport protein must occur is advanced. But the alternative hypothesis, i.e., that induction of nitrate reductase (and/or a consequence of the act of nitrate reduction) provided the required stimulus, remains tenable. PMID- 16658273 TI - Some factors regulating auxin translocation in intact bean seedlings. AB - Several factors which influence the translocation patterns of stem-injected indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and 2,4,5 trichlorophenoxyacetic acid in bean seedlings (Phaseolus vulgaris L. cv. Stringless Greenpod) were characterized. The acropetal translocation of auxin from the site of injection is markedly sensitive to concentration in the range of 1.0 to 5.0 micrograms per plant. The antiauxin p-chlorophenoxyisobutyric acid enhanced translocation of 2,4,5-trichlorophenoxyacetic acid to the growing shoots and primary leaves. Translocation to the roots was unaffected by p chlorophenoxyisobutyric acid while leaching of 2,4,5-trichlorophenoxyacetic acid into the nutrient solution was enhanced slightly. Steam girdling experiments revealed that translocation to the primary leaves was in the xylem. The protein synthesis inhibitor, cycloheximide, inhibited accumulation of 2,4,5 trichlorophenoxyacetic acid in young shoots, epicotyls, and roots and enhanced accumulation in the primary leaves. The relative exchangeability of auxin between xylem and phloem is discussed in terms of regulation of auxin movements in intact bean seedlings. PMID- 16658274 TI - Isolation from Rhizobium for factors which transfer electrons to soybean nitrogenase. AB - Rhizobium japonicum grown anaerobically with nitrate produced several factors capable of transferring electrons from spinach chloroplasts lacking photosystem II to soybean root nodule nitrogenase. The same organism grown aerobically produced no factors with detectable activity. It is concluded that the bacterium is potentially capable of producing these proteins in the low oxygen conditions of the leguminous root nodule. PMID- 16658275 TI - A Reversible Conversion of Phototransformable Protochlorophyll(ide)(656) to Photoinactive Protochlorophyll(ide)(656) by Hydrogen Sulfide in Etiolated Bean Leaves. AB - The relationship of phototransformable protochlorophyll-(ide) to photoinactive protochlorophyll(ide) has been studied in the primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Subjecting the leaves to an atmosphere of H(2)S causes an immediate loss of phototransformable protochlorophyll(ide)(650) and a simultaneous increase in photoinactive protochlorophyll(ide)(633). When such leaves are returned to air or N(2), the absorbance at 650 nm increases, whereas the absorbance at 633 nm decreases and photoactivity is restored. The reversion of protochlorophyll-(ide)(633) to protochlorophyll(ide)(650) is one-half complete in 3 minutes at 22 C in 8-day-old leaves. Ninety-five per cent recovery of protochlorophyll(ide)(650) is obtained when exposure to H(2)S is less than 3 minutes in duration; longer periods reduce the reversion capacity proportionately. The leaves are relatively undamaged by brief exposures to H(2)S, as judged by electron microscopy and by their ability to synthesize chlorophyll under continuous illumination. Hydrogen sulfide has no immediate effect upon the absorption properties of a partially purified preparation of the protochlorophyll(ide) holochrome, an etioplast suspension, or leaves subjected to freezing and thawing. Compounds such as HCN and HN(3) cause an irreversible conversion of protochlorophyll(ide)(650) to protochlorophyll(ide)(633) with total loss of photoactivity. Sulfhydryl agents, such as beta-mercaptoethanol and cysteine, cause a slow, irreversible transformation of the photoactive pigment to the photoinactive form and inhibit the ability of the leaves to synthesize chlorophyll under continuous illumination. The results obtained suggest that H(2)S may alter the interaction between the source of hydrogens on the protein moiety of the holochrome and the chromophore in vivo by reducing a disulfide bond in the protein, thereby causing a reversible conformational change in the complex. PMID- 16658276 TI - Hormonal control of isoperoxidases in lentil embryonic axis. AB - Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes. PMID- 16658277 TI - Indoleacetic Acid and abscisic Acid antagonism: I. On the phytochrome-mediated attachment of mung bean root tips on glass. AB - The phytochrome-mediated attachment of mung bean (Phaseolus vulgaris L., var. Oklahoma 612) root tips on glass is quickly affected by indoleactic acid and abscisic acid at concentrations of 10 nm or less. Indoleacetic acid induces detachment, whereas abscisic acid induces attachment. Both plant regulators rapidly antagonize the action of the other. None of several cytokinins, gibberellins, and ethylene tested over a wide range in concentration had any effect on either attachment or detachment of root tips. It is postulated that phytochrome could control the endogenous levels of indoleacetic acid and abscisic acid and perhaps other hormones under certain circumstances, that this action is the first process initiated by phytochrome, and that indoleacetic acid and abscisic acid act on the plasmalemma to bring about opposing changes in the surface electric charges of plant cells. PMID- 16658278 TI - Indoleacetic Acid and Abscisic Acid Antagonism: II. On the Phytochrome-Mediated Attachment of Barley Root Tips on Glass. AB - The effects of low concentrations (10 nm or less) of indoleacetic acid and abscisic acid on the phytochrome-mediated attachment of barley root (Hordeum vulgare L., var. Compana) tips on glass have been found to be opposite to those reported for mung bean root tips. Unlike their effects on mung bean root tips, indoleacetic acid causes attachment while abscisic acid causes detachment of barley root tips. Both hormones specifically and rapidly counteract the action of the other. No effect on the photoreversibility of attachment was observed when several gibberellins, cytokinins, and ethylene were added to the assay medium over a wide concentration range. PMID- 16658280 TI - The Role of ATP in Mechanically Stimulated Rapid Closure of the Venus's Flytrap. AB - When the midribs of untreated traps of Dionaea muscipula are frozen in liquid nitrogen after rapid closure, they contain significantly less ATP than those frozen before closure. Exogenous ATP causes a significant increase in the rate of mechanically stimulated trap closure. Illuminated traps close faster than those kept in the dark. The traps of plants placed in 100% O(2) close much faster than do air controls, while 100% CO(2) inhibits closure. It is concluded that ATP is probably the native source of potential energy for contraction of the trap's midrib, and that if the endogenous ATP titer is increased by oxidative phosphorylation or an exogenous source, the trap will close faster. PMID- 16658279 TI - The Structure of Plant Cell Walls: I. The Macromolecular Components of the Walls of Suspension-cultured Sycamore Cells with a Detailed Analysis of the Pectic Polysaccharides. AB - This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus). These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9% oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16% rhamnogalacturonan, and 21% xyloglucan.The structures of the pectic polymers (the neutral arabinan, the neutral galactan, and the acidic rhamnogalacturonan) were obtained, in part, by methylation analysis of fragments of these polymers which were released from the sycamore walls by the action of a highly purified endopolygalacturonase. The data suggest a branched arabinan and a linear 4-linked galactan occurring as side chains on the rhamnogalacturonan. Small amounts or pieces of a xyloglucan, the wall hemicellulose, appear to be covalently linked to some of the galactan chains. Thus, the galactan appears to serve as a bridge between the xyloglucan and rhamnogalacturonan components of the wall.The rhamnogalacturonan consists of an alpha-(1 --> 4)-linked galacturonan chain which is interspersed with 2-linked rhamnosyl residues. The rhamnosyl residues are not randomly distributed in the chain but probably occur in units of rhamnosyl- (1 - > 4)-galacturonosyl- (1 --> 2)-rhamnosyl. This sequence appears to alternate with a homogalacturonan sequence containing approximately 8 residues of 4-linked galacturonic acid. About half of the rhamnosyl residues are branched, having a substituent attached to carbon 4. This is likely to be the site of attachment of the 4-linked galactan.The hydroxyprolyl oligo-arabinosides of the hydroxyproline rich glycoprotein contain 3-linked, 2-linked, and terminal arabinosyl residues. The structure of the hydroxyprolyl oligo-arabinosides deduced from our methylation studies agrees with the structure reported for similar oligosaccharides. PMID- 16658281 TI - The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells. AB - The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues. PMID- 16658282 TI - The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension cultured Sycamore Cells Based on the Interconnections of the Macromolecular Components. AB - Degradative enzymes have been used to obtain defined fragments of the isolated cell walls of suspension-cultured sycamore cells. These fragments have been purified and structurally characterized. Fragments released from endopolygalacturonase-pretreated cell walls by a purified endoglucanase and the fragments extracted from these walls by urea and alkali provide evidence for a covalent connection between the xyloglucan and pectic polysaccharides. Fragments released by a protease from endopolygalacturonase-endoglucanase-pretreated cell walls provide evidence for a covalent connection between the pectic polysaccharides and the structural protein of the cell wall. Based on these interconnections and the strong binding which occurs between the xyloglucan and cellulose, a tentative structure of the cell wall is proposed. PMID- 16658283 TI - Wound-induced Proteinase Inhibitor in Tomato Leaves: Some Effects of Light and Temperature on the Wound Response. AB - Wounding of single leaflets of young tomato (Lycopersicum esculentum var. Bonnie Best) plants causes the release of a proteinase inhibitor inducing factor. This factor is rapidly transported throughout the plant where it causes accumulation of inhibitor I, a potent inhibitor of several serine proteinases from both animals and microorganisms. The wound-induced accumulation of inhibitor I is both light- and temperature-dependent. In total darkness no accumulation results from wounding. The accumulation exhibits a linear dependence upon light up to 300 foot candles. At 600 foot candles and above, the response is maximal. In light the wound response possesses an unusual temperature dependence with an optimum rate of accumulation near 36 C. Below 20 C no accumulation occurs. The over-all process contains two light- and temperature-dependent steps, one involving wounding and transport, the other involving accumulation. PMID- 16658284 TI - Interactions between Gibberellic Acid, Ethylene, and Abscisic Acid in Control of Amylase Synthesis in Barley Aleurone Layers. AB - Gibberellic acid-induced alpha-amylase synthesis in barley (Hordeum vulgare L.) aleurone layers was inhibited by abscisic acid, and the inhibition was partly removed by additional gibberellic acid alone and by ethylene alone. Together additional gibberellic acid and ethylene almost eliminated abscisic acid inhibition of amylase synthesis. Time course studies of these phenomena showed that the effect of abscisic acid, ethylene, and varying concentrations of gibberellic acid on the course of amylase synthesis were either to speed up or slow down the whole process and not to affect the lag phase or the linear phase separately. The data are discussed in relation to previous studies of abscisic acid-gibberellic acid interaction. PMID- 16658285 TI - Comparative immunochemistry of phytochrome. AB - Partially purified high molecular weight preparations of phytochrome, estimated to be close to 440,000 molecular weight based upon chromatography through a calibrated Bio-Gel P-300 column, were obtained from Garry and Newton oats (Avena Sativa L., cv. Garry and cv. Newton), rye (Secale cereale L., cv. Balbo), barley (Horedum vulgare L., cv. Harrison), and pea (Pisum sativum L., cv. Alaska) by a sequence of three chromatographic steps: brushite, diethylaminoethyl cellulose, and Bio-Gel P-300. No significant differences were observed between these preparations during purification or subsequent handling. In addition, a low molecular weight form of phytochrome was purified from Garry oats. Two specific antisera against a low molecular weight form of phytochrome (60,000 molecular weight) obtained from etiolated Garry oat seedlings are characterized and used to compare the phytochrome preparations. Double diffusion assays indicated antigenic identity between all preparations except that pea phytochrome yielded a spur when compared to oat phytochrome. Micro complement fixation assays yielded complete identity between Garry and Newton oat phytochrome, reduced activity with rye and barley phytochrome, and a complete lack of activity with pea phytochrome at the serum dilutions assayed. Immunoelectrophoretic assays indicated that all high molecular weight phytochrome preparations were homogeneous by this criterion and that there were only slight differences between the preparations in electrophoretic mobility. Large and small forms of phytochrome isolated from Garry oats were found to be very similar antigens when tested with the anti-small phytochrome sera, although the small form was observed to electrophorese at a much slower rate than the large. PMID- 16658286 TI - Immunological Determination of the Relationship between Large and Small Sizes of Phytochrome. PMID- 16658287 TI - Effect of hormone antagonist on the rooting of shoot cuttings. PMID- 16658288 TI - Abscisic Acid effect on root exudation related to increased permeability to water. PMID- 16658289 TI - Changes in certain aminoacyl transfer ribonucleic Acid synthetase activities in developing pea roots. AB - Tyrosyl-, arginyl-, leucyl-, and phenylalanyl-tRNA synthetase activities were measured in extracts from three root sections of 3-day-old pea seedlings. The sections 0 to 2, 3 to 7, and 8 to 22 millimeters from the root tip were chosen to represent the regions of cell division, elongation, and maturation, respectively. The specific activity for each aminoacyl-tRNA synthetase was highest in the 0- to 2-millimeter section and lowest in the 8 to 22 millimeter section. The changes in specific activity between the sections, however, varied with the particular synthetase. Tyrosyl-tRNA synthetase from each section was fractionated into two activity regions on a diethylaminoethyl cellulose column. Approximately 10, 22, and 44% of the total tyrosyl-tRNA synthetase activity in the 0 to 2, 3 to 7, and 8- to 22-millimeter sections, respectively, were associated with the first tyrosyl-tRNA synthetase region; the remaining activity was located in the second tyrosyl-tRNA synthetase region. Only one activity region for arginyl-tRNA synthetase was detected by diethylaminoethyl cellulose column fractionation. PMID- 16658290 TI - Photoperiodism and Enzyme Activity: Balance between Inhibition and Induction of the Crassulacean Acid Metabolism. PMID- 16658291 TI - Ultrastructural transformations in bean inner mitochondrial membranes. AB - The ultrastructure of inner membrane-matrix mitochondria isolated from bean (Phaseolus vulgaris) shoots was examined in different metabolic states. Gross ultrastructural transformations analogous to the condensed-to-orthodox configurational changes reported in mammalian mitochondria are observed on transistion from nonrespiring to respiring metabolism. With the induction of oxidative phosphorylation, the particles remain in the orthodox configurational state. The reverse orthodox-to-condensed configurational changes observed in mammalian preparations does not occur. Optically monitored absorbancy studies with bean particles show a substrate-supported P(i)-induced swelling under the same conditions that induce the condensed-to-orthodox ultrastructural transformation. The swelling is associated with the net uptake of K(+) and P(i) as well as a small P(i)-induced respiratory stimulation. When phosphorylation is initiated with these swollen particles, the optically monitored volume remains unchanged. Thus a positive correlation exists between the ultrastructural configuration and the osmotic volume changes, which supports the conclusion that configurational changes reflect internal osmotic adjustments. PMID- 16658292 TI - Stomatal Behavior and Water Status of Maize, Sorghum, and Tobacco under Field Conditions: I. At High Soil Water Potential. AB - Diurnal changes in the vertical profiles of irradiance incident upon the adaxial leaf surface (I), stomatal resistance (r(s)), leaf water potential (psi), osmotic potential (pi), and turgor potential (P) were followed concurrently in crops of maize (Zea mays L. var. Pa 602A), sorghum (Sorghum bicolor [L.] Moench var. RS610), and tobacco (Nicotiana tabacum L. var. Havanna Seed 211) on several days in 1968 to 1970 when soil water potentials were high. In all three crops the r(s), measured with a ventilated diffusion porometer, the psi, measured with the pressure chamber, the pi, measured with a vapor pressure osmometer, and the calculated P, decreased from sunrise to reach minimum values near midday and then increased again in the afternoon. The diurnal range of all the variables was greater for leaves in the upper canopy than for those in the lower canopy. P was observed to decrease with decreasing psi, but never became zero. Sorghum had a higher P at a psi of, say -10 bars, than did maize, and maize had a higher P than tobacco at the same psi. Moreover, at the same psi the upper leaves in all canopies had a higher P than the lower leaves. When compared at high irradiances, r(s) did not increase as psi declined to -13, -15, and -10 bars or as P declined to 0.3, 3.5, and 1.2 bars in maize, sorghum, and tobacco, respectively. The relation between r(s) and I in the upper, nonsenescent leaves of all three crops fits a hyperbolic curve, but the response varied with species and leaf senescence. The adaxial and abaxial epidermises had the same response of r(s) to I in maize and sorghum, whereas in tobacco the adaxial epidermis had a higher r(s) than the abaxial epidermis at all values of I. At equal values of I, tobacco had the lowest leaf resistance (r(l)) and maize had the highest r(l). Senescent maize leaves had nonfunctional stomata, whereas the lowermost sorghum leaves had higher stomatal resistances on average than the other leaves. PMID- 16658293 TI - Far Red and White Light-promoted Utilization of Calcium by Seedlings of Phaseolus vulgaris L. AB - The cotyledons and embryo axes of seeds of Phaseolus vulgaris L. cv. Pinto contained 16% of the total calcium in the seed. The remaining 84% was in the testas. There was no evidence that calcium in testas was used in seedling growth or that calcium was leached from seedlings during growth.An external supply of calcium decreased the incidence of hypocotyl collapse (a severe symptom of calcium deficiency), increased the calcium content of all organs, and increased the dry weight of all organs except cotyledons. Light treatments decreased the incidence of hypocotyl collapse and increased the calcium content and dry weight of all organs except cotyledons and hypocotyls.White light was more effective than far red light for decreasing incidence of hypocotyl collapse. Usually the effects of white light and far red light on the calcium content and dry weight of organs were similar, and usually those of white light were quantitatively greater than those of far red light. It is suggested that the light-promoted effects were associated with photomorphogenesis and that differences in data obtained with white light and far red light could be associated with photosynthesis. PMID- 16658294 TI - Effects of phosphorus deficiency on the photosynthesis and respiration of leaves of sugar beet. AB - Phosphorus deficiency was induced in sugar beet plants (Beta vulgaris L. var. F5855441), cultured hydroponically under standardized environmental conditions, by removal of phosphorus from the nutrient supply at the ten leaf stage 28 days after germination. CO(2) and water vapor exchange rates of individual attached leaves were determined at intervals after P cutoff. Leaves grown with an adequate nutrient supply attained net rates of photosynthetic CO(2) fixation of 125 ng CO(2) cm(-2) sec(-1) at saturating irradiance, 25 C, and an ambient CO(2) concentration of about 250 mul l(-1). After P cutoff, leaf phosphorus concentrations decreased as did net rates of photosynthetic CO(2) uptake, photorespiratory evolution of CO(2) into CO(2)-free air, and dark respiration, so that 30 days after cutoff these rates were about one-third of the control rates. The decrease in photosynthetic rates during the first 15 days after cutoff was associated with increased mesophyll resistance (r(m)) which increased from 2.4 to 4.9 sec cm(-1), while from 15 to 30 days there was an increase in leaf (mainly stomatal) diffusion resistance (r(l)') from 0.3 to 0.9 sec cm(-1), as well as further increases in r(m) to 8.5 sec cm(-1). Leaf diffusion resistance (r(l)') was increased greatly by low P at low but not at high irradiance, r(l)' for plants at low P reaching values as high as 9 sec cm(-1). PMID- 16658295 TI - Light and calcium interactions in chlorella inhibited by sodium chloride. AB - Analysis of NaCl toxicity in Chlorella sorokiniana showed decreased growth rates, increased dry weight per cell, increased intracellular Na(+) and Cl(-), more total chlorophyll per cell, a decreased chlorophyll a to chlorophyll b ratio, increased rates of O(2) evolution, and decreased rates of CO(2) fixation when the extracellular concentration of NaCl was increased from zero to 0.3 m. Cultures did not grow at concentrations greater than 0.3 m NaCl unless 10 mm calcium salts were present. Inclusion of that concentration of Ca(2+) extended the tolerance to 0.5 m NaCl before growth stopped. Increasing the light intensity from 1.2 to 9.4 mw/cm(2) increased growth rates for cultures in 0.10 to 0.45 m NaCl. At 14 mw/cm(2) added Ca(2+) reduced growth rates of cultures in 0.3 m NaCl compared to controls without added Ca(2+). Maximal growth rates for cultures in NaCl media were achieved by addition of 10 mm CaSO(4) and maintenance of the light intensity at 9.4 mw/cm(2). The maximal growth rate of the organism was 9.6 doublings/day achieved at 2.7 mw/cm(2) for control cultures. In 0.3 m NaCl the growth rate was 4.3 doublings/day at 2.7 mw/cm(2) and 8.2 doublings/day at 9.4 mw/cm(2) with 10 mm CaSO(4) added.Increasing light intensities from 2.7 to 9.4 to 14 mw/cm(2) decreased intracellular Na(+) in cells cultured in 0.3 m NaCl medium without added Ca(2+) and increased Cl(-) uptake in cells cultured in 0.3 m NaCl medium with and without added Ca(2+). For cells cultured in 0.3 m NaCl medium at 14 mw/cm(2) intracellular Na(+) was 0.68 meq/g dry weight with Ca(2+) added and 0.81 meq/g dry weight without Ca(2+) added. Addition of Ca(2+) at 2.7 mw/cm(2) reduced intracellular Na(+) to similar values. It is postulated that energy requirements for active Na(+) exclusion were reduced by addition of Ca(2+) allowing more energy to be used for cell growth resulting in increased growth rates.O(2) evolution and CO(2) fixation studies indicated that increased photosynthetic energy, probably actuated by a high proton gradient accompanying Cl(-) influx and uncoupled from CO(2) fixation, was available for maintenance of cellular integrity and active control of intracellular ionic ratios. The O(2) evolving capacity was destroyed at 12 and 29 mw/cm(2) for cells cultured in 0.3 m NaCl medium respectively with and without the addition of Ca(2+). Control cultures continued producing O(2) at light intensities up to 115 mw/cm(2). PMID- 16658296 TI - Endosperm protein of wheat seed as a determinant of seedling growth. AB - Seed of a Mexican semidwarf wheat (Triticum aestivum L. cv. Inia 66), was obtained from a nitrogen fertilizer field trial grown in Mexico. A high positive correlation was obtained between seed protein content and seedling dry weight after 3 weeks growth (r = +0.92(**)). The seedling dry weight was positively related to the protein content of the aleurone layer and endosperm, but not to the embryo. Small, 35 milligrams, high protein seeds (4.7 milligrams protein per seed) produced larger seedlings than large, 45 milligrams, low protein seeds (4.3 milligram protein per seed). There was no difference in the weight or protein content of embryos from low and high protein seeds and their growth was similar. Composite seeds of the two protein levels were produced by transferring embryos from one endosperm type to the other. After 4 weeks, there was no difference between the different embryo types grown on the same endosperm type. High protein endosperm produced more vigorous seedlings regardless of the embryo type grown on it, indicating that the factor(s) responsible for the greater growth of high protein seed is in the endosperm. PMID- 16658297 TI - Auxin inhibition of ripening in bartlett pears. AB - The effect of indoleacetic acid and 2,4-dichlorophenoxyacetic acid on the ripening of intact mature-green pears (Pyrus communis var. Bartlett) was investigated using a vacuum infiltration technique.The effects of indoleacetic acid and 2,4-dichlorophenoxyacetic acid at concentrations of 0.01, 0.1, and 1.0 mm each were studied on softening, degreening, and on ethylene and CO(2) evolution. Softening and degreening were inhibited increasingly in response to increased concentrations of indoleacetic acid. This inhibitory property was amplified by 2,4-dichlorophenoxyacetic acid at concentrations comparable to those of indoleacetic acid. Application of the auxins also prevented the climacteric rise in respiration, but stimulated ethylene synthesis. Despite the presence of elevated ethylene levels, the inhibitory auxin effect was predominant.It is proposed that endogenous auxins in fruit represent a resistance factor in ripening and must be inactivated before ripening can occur. PMID- 16658298 TI - The origin and turnover of organelle membranes in castor bean endosperm. AB - The origin and turnover of organelle membranes in castor bean (Ricinus communis L. var. Hale) endosperm was examined using choline-(14)C as a phospholipid precursor. On sucrose gradients three major particulate fractions were separated; a light membranous fraction (density 1.11-1.13 gram per cm(3)), the mitochondria (1.18 gram per cm(3)), and the glyoxysomes (1.24 gram per cm(3)). Choline-(14)C was readily incorporated into lecithin in all three particulate fractions, but the light membranous fraction became labeled first. Incorporation continued into all three fractions for 6 hours, at which time the available choline-(14)C had been completely used. Subsequently, (14)C was lost from the three components at distinctly different rates. When an excess of unlabeled choline was added after 1 hour (pulse-chase experiment), incorporation of choline-(14)C into glyoxysomes and mitochondria continued for three hours, but at a diminishing rate. This was followed by a period in which the (14)C content of the mitochondria declined at a rate expected, if the half life of lecithin in the membrane were about 50 hours and that of the glyoxysomes 10 hours. These values are close to those calculated from the experiments in which no chase was used. The labeling in the light membrane fraction behaved differently from that of the mitochondria and glyoxysomes following the chase of unlabeled choline. Incorporation continued for only 1 additional hour, and then the (14)C content declined sharply in the subsequent 4 hours. The early kinetics and subsequent interrelationships are those expected if the lecithin in the membranes of mitochondria and glyoxysomes originates in components of the light membrane fraction. PMID- 16658299 TI - Development of enzymes in the cotyledons of watermelon seedlings. AB - Changes in hypocotyl length, cotyledon weight, lipid content, chlorophyll content, and capacity for photosynthesis have been described in seedlings of Citrullus vulgaris, Schrad. (watermelon) growing at 30 C under various light treatments. Corresponding changes in the levels of 19 enzymes in the cotyledons are described, with particular emphasis on enzymes of microbodies, since during normal greening, enzymes of the glyoxysomes are lost and those of leaf peroxisomes appear. In complete darkness enzymes of the glyoxysomes reach a peak at 4 days and decline as the fat is depleted. Enzymes of mitochondria and of glycolytic pathways also peak at 4 to 5 days and either remain unchanged or decline to a lesser extent. Exposure to light at 4 days, when the cotyledons emerge, results in a selectively greater destruction of enzymes of the glyoxylate cycle; chlorophyll synthesis and capacity for photosynthesis increase in parallel, and there is a striking increase in the activities of chloroplast enzymes and in those of the leaf peroxisomes, hydroxypyruvate reductase and glycolate oxidase. The reciprocal changes in enzymes of the glyoxysomes and of leaf peroxisomes can be temporally dissociated, since even after 10 days in darkness, when malate synthetase and isocitrate lyase have reached very low levels, hydroxypyruvate reductase and glycolate oxidase increase strikingly on exposure to light and the cotyledons become photosynthetic. Furthermore, the parallel development of enzymes of leaf peroxisomes and functional chloroplasts is not immutable, since hydroxypyruvate reductase and glycolate oxidase activity can be elicited in darkness following a 5-minute exposure to light at day 4 while chlorophyll does not develop under these conditions. PMID- 16658300 TI - Formation of carbon monoxide and bile pigment in red and blue-green algae. AB - Five blue-green and one red algal species produced carbon monoxide during photosynthetic growth. The blue-green algae synthesized CO and phycocyanobilin in equimolar quantities at identical rates. The red alga, Porphyridium cruentum, incorporated Delta-aminolevulinic acid-5-(14)C into phycoerythrobilin and CO. The ratio of the specific radioactivity of phycoerythrobilin to that of CO, and the kinetics and stoichiometry of phycocyanobilin and CO formation suggest that linear tetrapyrroles in plants are derived by the porphyrin pathway via the intermediate formation of heme. The similarity between bile pigment production in algae and in mammalian systems is discussed. PMID- 16658301 TI - Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase: I. Their Regulation Under Continuous Red and Far Red Light in the Cotyledons of Sinapis alba L. AB - The development of NADP- and NAD-dependent glyceraldehyde 3-phosphate dehydrogenase and NADH-specific glyoxylate reductase was followed in Sinapis alba cotyledons grown in the dark or under continuous red and far red light. All three enzyme activities are promoted by light, continuous far red light being more than twice as effective as continuous red light. The activities of the NADP glyceraldehyde 3-phosphate dehydrogenase and glyoxylate reductase increase in the far red light from 36 to 96 hours. They remain constant until at least 120 hours after sowing and are respectively 11 and 6 times higher than the maximum dark activities. Contrary to this, the activity of the NAD-glyceraldehyde 3-phosphate dehydrogenase is scarcely more than doubled under continuous far red irradiation relative to its maximal dark level, and its time course curve is displaced along the time axis, with the activity increasing between 24 and 72 hours after sowing.The increase in activity of NADP-glyceraldehyde 3-phosphate dehydrogenase and glyoxylate reductase is inhibited by d-threo-chloramphenicol but not by the l threo isomer at concentrations of 200 micrograms per milliliter or less, whereas the slight inhibitory effect of chloramphenicol on the NAD-glyceraldehyde 3 phosphate dehydrogenase is not stereospecific. The three enzyme activities are inhibited by cycloheximide.When Knop's solution is used as growth medium it strongly promotes NADP-glyceraldehyde 3-phosphate dehydrogenase activity in vivo and is twice as effective in the red light as in the far red light and dark. The activity of NAD-glyceraldehyde 3-phosphate dehydrogenase is only slightly and almost equally enhanced by Knop's solution in the dark, red, and far red light.These results are consistent with the following conclusions. [List: see text]. PMID- 16658302 TI - Action Spectra for Guard Cell Rb Uptake and Stomatal Opening in Vivia faba. AB - Abaxial epidermal strips, containing guard cells as the only viable cells, were prepared from leaves of Vicia faba following a period in darkness, and floated, under CO(2)-free air, on 2 mm RbCl + 0.1 mm CaCl(2) labeled with (86)Rb(+). Under white light (high pressure mercury vapor lamp), stomatal opening in these strips approached its maximum at less than 0.02 calorie per square centimeter per minute. Under light of different wavelengths, 20 nanometers apart, and at a low quantum flux density of 7 x 10(14) quanta per square centimeter per second, Rb(+) uptake and stomatal opening were activated only in the blue and long ultraviolet regions, with a peak at 420 to 460 nanometers. The action spectrum suggests that the underlying process is not photosynthesis. At higher quantum flux density (38 x 10(14) quanta per square centimeter per second), uptake and opening also responded to red (600-680 nanometers) and somewhat to green light, with a minimum at 540 to 560 nanometers, indicating a possible involvement of the photosynthetic process. This light-induced opening appeared not to be mediated by a lowering of CO(2) concentration, since CO(2)-free air was used in all treatments and controls. Stomatal opening paralleled Rb(+) uptake in all cases. This constitutes further evidence for the potassium transport hypothesis of stomatal movement.In the abaxial surface of leaf discs under air of normal CO(2) concentration, stomatal opening in white light approached its maximum at an intensity similar to that for epidermal strips. At both quantum flux densities, the action spectra for opening in leaf discs were very similar to those for epidermal strips. Thus, these light-linked processes for stomatal opening are likely to be the same in leaves as in epidermal strips. PMID- 16658303 TI - Apple leaf senescence: leaf disc compared to attached leaf. AB - Attached apple leaves (Pyrus malus L., Golden Delicious) began to lose protein in early August as the first sign of senescence. Apple leaf discs prepared from samples before early August gained protein for up to 7 days after detachment. After early August, the loss of protein from leaf discs was no greater than the loss from attached leaves in 7 days. The loss of chlorophyll from leaf discs began over 2 months before attached leaves began to lose chlorophyll naturally and before leaf discs lost protein. Leaf discs from presenescent leaves did not senesce significantly faster when maintained in darkness instead of 12 hours of light. In general, the loss of protein and chlorophyll from apple leaf discs after 7 days was much less than for most other leaf types studied. PMID- 16658304 TI - Rapid growth inhibition of Avena coleoptile segments by abscisic Acid. AB - An angular position sensing transducer was used to make continuous measurements of elongation of a column of Avena sativa coleoptile segments. Elongation stimulated by 2 mum indoleacetic acid was inhibited by 0.1 mm abscisic acid with a latent period of about 4 or 5 minutes at pH 6.0, 30 C. Full growth inhibition was not established until about 1 hour after the addition of the abscisic acid. The same degree of final growth inhibition could be obtained under the above conditions using 10 muM and 1 muM abscisic acid, but the latent period was longer. Pretreatments with abscisic acid affected the growth rate but did not extend the latent period of a subsequent response to auxin. The short term kinetics of inhibition by abscisic acid were not similar to those of any of the inhibitors of RNA and protein synthesis tested in this system. PMID- 16658305 TI - Isolation of Mesophyll Cells from Sedum telephium Leaves. AB - A technique is described for mechanically isolating metabolically active individual spongy mesophyll cells from the Crassulacean acid metabolism plant, Sedum telephium. Mature leaves are selected at about 2 PM when acidity is low, and three different media are used to reduce the problem of leaf acidity and to maintain isotonic conditions. The upper and lower epidermis is peeled from chilled leaves and the leaves are suspended in a buffered "soaking medium," then gently ground with a mortar and pestle. Cells and debris are separated using a "washing medium," with cells being filtered through a 136 micron net and collected on an 80 micron net. Cells then are suspended in a "cell suspension medium" and concentrated by centrifugation. Approximately 2 hours are required for the isolation procedure, and activity in CO(2) fixation is constant for up to 4 hours after isolation. Microscopic examination shows about 65% of the isolated cells appear intact and unplasmolyzed and are similar to leaf msophyll cells. The yield of cells on a leaf chlorophyll basis is about 1%. A light micrograph of the isolated cells is given.The isolated cells upon addition of phosphoenolpyruvate, 2-phosphoglycerate, and ribulose-1, 5-diphosphate fix CO(2) as rapidly as intact leaves; however, without exogenous substrate the cells only fix CO(2) between 10 and 20% of intact leaves. The temperature and pH optima for cellular CO(2) fixation in the presence of phosphoenolpyruvate is 35 to 45 C and 7.5 to 9.0, respectively. The light and dark portions of CO(2) fixation with the isolated cells are considered in relation to a scheme for net CO(2) fixation by Crassulacean acid metabolism plants. PMID- 16658306 TI - Effects of 2-Butylmalonate, 2-Phenylsuccinate, Benzylmalonate, and p Iodobenzylmalonate on the Oxidation of Substrates by Mung Bean Mitochondria. AB - The effect of inhibitors of carboxylic acid anion transport on the oxidation of substrates by mung bean (Phaseolus aureus) mitochondria was investigated. The oxidation of malate in the presence of either glutamate or cysteine sulfinate was inhibited by 2-butylmalonate, 2-phenylsuccinate, benzylmalonate, and p iodobenzylmalonate in both intact and broken mitochondria. The oxidation of succinate, on the other hand, was inhibited in intact but not in broken mitochondria. The oxidation of reduced nicotinamide adenine dinucleotide was inhibited only by p-iodobenzylmalonate. This inhibition occurred only in coupled mitochondria and could be reversed by the addition of adenosine diphosphate.These results suggest that the compounds studied inhibit the entry of succinate and probably also of malate into mung bean mitochondria. All four compounds inhibit the enzyme (s) which oxidize malate while p-iodobenzylmalonate also interferes with oxidative phosphorylation. PMID- 16658307 TI - Lipid transformations in greening and senescing leaf tissue. AB - Analyses were made of chlorophyll a and b and fatty acids (18:3, 18:2, 18:1, 18:0, 16:2, 16:1, and 16:0) of greening and senescing leaf tissue. Those dark grown tissues given a prior treatment of red, far red, or red followed by far red light showed similar increases in chlorophylls and linolenate (18:3) when exposed to continuous white light. In contrast, green barley (Hordeum vulgare L.) leaves placed in the dark lost chlorophylls and fatty acids, especially 18:3. Senescing cocklebur (Xanthium strumarium L.) leaf tissue showed a decline in chlorophyll and fatty acids, especially again 18:3. Abscisic acid, but not sucrose, accelerated these senescent changes. Radioactive acetate incorporation into the galacto-lipids and phospholipids of senescing cocklebur leaf tissue increased and then the radioactivity of the lipids decreased in senescent tissues. PMID- 16658308 TI - Activation of polyphenol oxidase of chloroplasts. AB - Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or 18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30 minutes. No single peptide from the digested trypsin was found to be the sole activating factor. About 0.25 mug of trypsin activated 50% the polyphenol oxidase activity in a standard chloroplast assay containing 2.1 mug of chlorophyll. Treatment of spinach chloroplasts with tris buffer or ethylenediamine tetraacetate extracted the ATPase activity, but the polyphenol oxidase activity remained with the broken plastids. However these treatments increased the latent polyphenol oxidase activity 50- to 100-fold.Chloroplasts from a second group of plants, including alfalfa, wheat, oats, peas, and sugarcane leaves, oxidized dihydroxyphenylalanine at a rate of 11 to 120 mumoles x mg(-1) chlorophyll x hr(-1). Polyphenol oxidase in these chloroplasts required a low intensity of red light for activity. Fifty or 75% activation of the oxidase in wheat chloroplasts required 4 to 6 foot candles of light and more light was required for alfalfa chloroplasts. Blue or far red light were ineffective. Trypsin was inhibitory. Upon aging chloroplasts from wheat leaves, but not alfalfa or peas, for 5 to 7 days at 4 C the total polyphenol oxidase activity did not increase, but the activation characteristics changed to those of chloroplasts from the spinach group. Chloroplasts from a third group of plants, including bean, tomato, and corn leaves, slowly oxidized dihydroxyphenylalanine in the dark and exhibited no latency. PMID- 16658309 TI - Functional and Structural Changes in Senescing Populus deltoides (Bartr.) Chloroplasts. AB - Chloroplasts isolated from Populus deltoides leaves were used to study age dependent changes in the rate of cyclic photophosphorylation. Single leaves were used to measure CO(2) fixation by leaf discs, chlorophyll concentration, and ATP synthesis. The ability of chloroplasts to synthesize ATP diminished steadily from the time of full leaf expansion, regardless whether the results are expressed on a leaf area or chlorophyll basis. This decline in the rates of ATP synthesis was paralleled by the decline in the rate of CO(2) fixation. The results suggest that the efficiency of the membrane-bound ATP synthesizing system declines with age. PMID- 16658311 TI - Appearance of Three Chloroplast Isoenzymes in Dark-grown Pea Plants and Pea Seeds. AB - Activity peaks characteristic of the chloroplastic Calvin cycle enzymes triose phosphate isomerase, ribose 5-phosphate isomerase, and fructose 1,6-diphosphate aldolase are found in isoelectric focusing patterns of dark-grown pea (Pisum sativum) seedlings and seeds. Apparently, in this higher plant these three chloroplastic isoenzymes can be formed in the absence of light and of chloroplast formation. PMID- 16658310 TI - Studies on the Control of the Rhythm of Photosynthetic Capacity in Synchronized Cultures of Euglena gracilis (Z). AB - Synchronous cell division in Euglena gracilis (strain Z) was obtained in 24-hour light cycles consisting of 10 hours of light and 14 hours of darkness; cell division was restricted to the dark period. Photosynthetic capacity was found to vary in a cyclic manner during the cell cycle, reaching a peak 2 hours before the onset of darkness. Light reactions were investigated during the cell cycle to determine what role they played in the control of the observed rhythmic changes in capacity. Light-saturation curves showed no major change in the light-limited region. No fluctuations were found in Hill reaction activity or photoreduction of methyl red during the cell cycle. These results imply that the reactions comprising photosystems I and II do not generate the capacity rhythm.Some of the photosynthetic dark reactions were also followed during the cell cycle in an attempt to determine their possible role in the control of the rhythm of photosynthetic capacity. The activity of ribulose-1, 5-diphosphate carboxylase showed no correlation with the rhythm. On the other hand, the activity of glyceraldehyde-3-phosphate dehydrogenase was found to parallel the change in photosynthetic rate under various growth conditions. The rhythm in photosynthetic capacity could be effectively divorced from the cell cycle itself by placing cultures in high frequency light cycles (LD: 2,4) or in stationary growth-phase conditions. If synchronously dividing cultures previously grown in LD: 10, 14 were released into continuous dim illumination and constant temperature, the rhythm of capacity persisted for only one full cycle. PMID- 16658312 TI - d-Glucose 6-Phosphate Cycloaldolase: Inhibition Studies and Aldolase Function. AB - d-Glucose 6-phosphate cycloaldolase is inhibited 83% by 0.66 mm EDTA and stimulated 1.7-fold by 0.6 mm KCl. Dihydroxyacetone phosphate, an analog of the last three carbons in the proposed intermediate, d-xylo-5-hexulose 6-phosphate, acts as a partially competitive inhibitor. Treatment with NaBH(4) in the presence of dihydroxyacetone phosphate does not cause permanent inactivation as would be expected if a Schiff base were being formed. In these properties it resembles a type II, metal-containing aldolase. Photooxidation in the presence of Rose Bengal inactivates this enzyme. NAD(+) partially protects against this photooxidation. Cells grown on medium lacking myoinositol had four times as much enzyme activity as cells grown on medium containing 100 mg of myoinositol per liter. PMID- 16658313 TI - Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass. AB - Experiments with intact plants of Lolium perenne previously grown with (14)NO(3) (-) revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched (15)NO(3) (-). The exuded (14)NO(3) ( ) was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the (14)NO(3) (-) was observed. Influx of (15)NO(3) (-) was significantly greater than influx of (14)NO(3) (-) from solutions of identical NO(3) (-) concentration. Transferring plants to (14)NO(3) (-) solutions after a six-hour period in (15)NO(3) (-) resulted in efflux of the latter. Presence of Mg(2+), rather than Ca(2+), in the ambient (15)NO(3) (-) solution resulted in a decidedly increased rate of (14)NO(3) (-) efflux and a slight but significant increase in (15)NO(3) (-) influx. Accordingly, net NO(3) (-) influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO(3) (-) within unstirred layers from the passive pathway to the active uptake site. PMID- 16658314 TI - The mechanism of freezing injury in xylem of winter apple twigs. AB - In acclimated winter twigs of Haralson apple (Pyrus Malus L.), a lag in temperature during cooling at a constant rate was observed at about -41 C by differential thermal analysis. The temperature at which this low temperature exotherm occurred was essentially unaffected by the cooling rate. During thawing there was no lag in temperature (endotherm) near the temperature at which the low temperature exotherm occurred, but upon subsequent refreezing the exotherm reappeared at a somewhat higher temperature when twigs were rewarmed to at least 5 C before refreezing. These observations indicate that a small fraction of water may remain unfrozen to as low as -42 C after freezing of the bulk water in stems. The low temperature exotherm was not present in twigs freeze-dried to a water content below 8.5% (per unit fresh weight), but it reappeared when twigs were rehydrated to 20% water. When freeze-dried twigs were ground to a fine powder prior to rehydration, no exotherm was observed. Previous work has shown that the low temperature exotherm arises from xylem and pith tissues, and that injury to living cells in these tissues invariably occurs only when twigs are cooled below, but not above the temperature of the low temperature exotherm. This study revealed that the low temperature exotherm resulted from the freezing of a water fraction, that the freezing of this water was independent of the freezing of the bulk water, that the exotherm was associated with some gross structural feature but not the viability of the tissue, and that injury to living cells in the xylem and pith was closely and perhaps causally related to the initial freezing of this water. PMID- 16658315 TI - Compartmental utilization of carboxyl-C-tripalmitin by tissue homogenate of pine seeds. AB - A tissue homogenate of megagemetophyte of germinating seeds of Jeffrey pine (Pinus Jefferii Grev. and Balf.) was incubated with sonication-dispersed and albumin-carried (14)C-tripalmitin in order to elucidate the sequential and quantitative role of cellular organelles in utilizing lipid reserve in seeds. After 5 minutes at 30 C, 25% of the tracer was localized in the fat body fraction, 9% in the pellet containing mitochondria and glyoxysomes, 14% in the supernatant, and 2% was found as CO(2). Radioactivity increased with time of incubation in the latter three fractions indicating the forward direction of utilization. Fat bodies contained mainly lipases and hydrolyzed the tracer to palmitate with diglyceride and monoglyceride as intermediates. About two-thirds of the palmitate had left the fat bodies in 5 minutes and entered the pellet fraction within which the tracer was distributed 1:2 in mitochondria and glyoxysomes, respectively. Longer incubation reduced the ratio to 1:3 while both organelles acquired more radioactive intermediates. Labeled acetyl-CoA and intermediate of beta-oxidation were found in both organelle-containing fractions. The supernatant fraction contained radioactive diglycerides, monoglycerides, palmitate, sterol esters, and phospholipids, indicating lipase activity and direct utilization of fatty acid for the synthesis of sterol esters and polar lipids. PMID- 16658316 TI - Polyribosomes Conserved during Desiccation of the Moss Tortula ruralis Are Active. AB - During desiccation of the moss Tortula ruralis (Hedw.) (Gaertn, Meyer and Scherb) polyribosomes are conserved. On rehydration, protein synthesis is rapidly resumed. In the presence of protein synthesis initiation inhibitors ribosome run off from the conserved polyribosomes takes place, confirming that these retain their activity as intact structures during desiccation. PMID- 16658317 TI - Multiphasic absorption of glucose and 3-o-methyl glucose by aged potato slices. AB - The isotherm for glucose absorption by aged potato (Solanum tuberosum var. Russet Burbank) discs shows four distinct phases in the concentration ranges 1.0 to 75 mum, 75 mum to 1.5 mm, 1.5 to 15 mm, and 15 to 100 mm, respectively. Each segment of the multiphasic isotherm, when plotted reciprocally by the method of Lineweaver and Burk or of Hofstee, without regard for uptake in earlier phases, indicates absorption rate to be a hyperbolic function of concentration. The observations suggest that glucose uptake is carrier-mediated, and that the transport barrier undergoes a series of all-or-none transformations at critical external concentrations, yielding successive new and higher values for the parameters Km and V(max) 3-O-Methyl glucose, a nonmetabolizable analogue of glucose, shows the same multiphasic absorption isotherm, with Km values essentially similar to those for glucose uptake, and V(max) values somewhat lower than those for glucose absorption. Whereas the first three phases of the absorption isotherm are taken to reflect passage across the plasma membrane, the fourth phase may reflect kinetics of glucose or 3-O-methyl glucose transport to the vacuole. PMID- 16658318 TI - A red-far red reversible effect on uptake of exogenous indoleacetic Acid in etiolated rice coleoptiles. AB - The uptake and accumulation of exogenous indoleacetic acid-(14)C by intact rice coleoptiles were examined. The absorption of exogenous indoleacetic acid was controlled by phytochrome, while the subsequent accumulation of this indoleacetic acid in various portions of the coleoptile was complex, and the effect of red light in this system was small compared to the alteration of the uptake of indoleacetic acid by red light. The absorption of indoleacetic acid exhibited two phases: the first occurring during the first 3-hour portion of the incubation was an inhibition, while the second was a promotive effect at about the 5th hour of incubation. Both of these effects were red, far redreversible, implicating phytochrome in this effect. Neither the destruction nor the immobilization of this exogenous indoleacetic acid apeared to be greatly affected by red light irradiation. The principal interaction between phytochrome and indoleacetic acid appears to occur during the absorption of exogenous indoleacetic acid. This effect may be related to the control by phytochrome of the amount of auxin which diffuses from coleoptile tips. PMID- 16658319 TI - Alternate pathways of glycolate synthesis in tobacco and maize leaves in relation to rates of photorespiration. AB - After a preliminary period in light, leaf disks floated on 10 mm alpha-hydroxy-2 pyridinemethanesulfonic acid to inhibit glycolate oxidase accumulate glycolate at average initial rates of 67 micromoles in tobacco and 8 micromoles per gram fresh weight per hour in maize under optimal conditions in air. In the presence of (14)CO(2), the glycolate synthesized has a high specific radioactivity in illuminated tobacco and a low one in maize. Isonicotinic acid hydrazide also inhibits glycolate oxidation and causes a slow accumulation of glycolate in maize but not in tobacco, while it inhibits glycolate synthesis in tobacco but not in maize. Radioactive carbon in acetate-2-(14)C and especially pyruvate-3-(14)C is incorporated predominantly into the C-2 of glycolate in both species, but the specific radioactivity is much greater in maize. Glyoxylate-2-(14)C is readily converted to glycolate-2-(14)C in both species. The addition of phosphoenolpyruvate stimulated glycolate formation in maize and inhibited its synthesis in tobacco, and in the presence of (14)CO(2) the specific radioactivity in glycolate-(14)C was decreased greatly by the added phosphoenolpyruvate only in maize.Thus, unsymmetrically labeled glycolate is mainly synthesized from pyruvate 3-(14)C by a slow pathway in maize. Tobacco possesses an additional rapid pathway that produces equally labeled glycolate more directly from fixed CO(2) during photosynthesis. Glycolate is believed to be the primary substrate of photorespiration, and sufficiently rapid rates of glycolate synthesis have been observed in tobacco to account for this function. Hence the high rates of photorespiration observed in tobacco leaves compared with maize result partly from differences between these species in the pathway of glycolate synthesis. PMID- 16658320 TI - Blue Light Interference in the Phytochrome-controlled Germination of the Spores of Cheilanthes farinosa. AB - Short exposure of the spores of Cheilanthes farinosa to low intensity red light promotes their germination, which is not reversed by a subsequent exposure to far red light. Germination is, however, inhibited by blue light administered before or after red light. Inhibition of germination by blue light is annulled by exposure to a higher intensity of red light, and germination of the repromoted spores is inhibited by far red light. Mutual photoreversibility of germination is also observed in repromoted spores irradiated successively with far red and red light. Although germination appears to be basically under phytochrome control, it is postulated that the presence of a blue light-absorbing pigment interferes with phytochrome transformations in the spores. PMID- 16658321 TI - Effects of calcium and kinetin on growth and cell wall composition of pea epicotyls. AB - Kinetin and CaCl(2), in the presence of indoleacetic acid, promoted lateral expansion of epicotyls of decapitated and derooted Alaska pea seedlings (Pisum sativum L.) and inhibited their elongation. This growth response was correlated with the development of cell walls unusually rich in pectic uronic acids. Epicotyls in calcium-auxin solutions continued to enlarge and to add new wall material long after tissues in auxin only had stopped. Longitudinal enlargement, associated with the development of walls poor in pectic uronic acids, was favored by KCl, MgCl(2), and ethylenediaminetetraacetate. The last of these agents promoted the loss of (45)Ca from the epicotyls. Seedings grown in vermiculite moistened with CaCl(2), KCl, or MgCl(2) solutions did not differ in appearance or in the composition of their walls. They responded similarly to experimental treatment except that the decapitated epicotyls of the MgCl(2)-grown plants suffered an absolute loss of pectic uronate when incubated in that salt. PMID- 16658322 TI - Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid. AB - Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4 dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used (14)C-aspartate, which was then separated from the (14)C ureidosuccinate product by Dowex 50 (H(+) form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5'-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings. PMID- 16658323 TI - Aspartokinase from wheat germ: isolation, characterization, and regulation. AB - Aspartokinase has been isolated from wheat germ and a preliminary survey made of its properties in a partially purified extract. The enzyme has an absolute requirement for ATP and a divalent metal ion. The phosphate donor can be either ATP or GTP, but other nucleotides are ineffective. Both magnesium and manganese will activate the enzyme, whereas calcium shows a trace amount of activity. The enzyme has a Km of 16.7 mm for aspartate, 1.2 mm for ATP, and 3.3 mm for MgCl(2). Lysine inhibits the reaction at fairly low concentrations, and threonine inhibits at high concentrations. Other amino acids which are derived from aspartate (methionine, homoserine, threonine, and isoleucine) have little effect. When lysine and threonine are added together, they show a concerted inhibition of the reaction. The enzyme is also stabilized against heat inactivation by lysine and threonine together but not by either when added separately. It is suggested that aspartokinase from plants is a regulatory enzyme and exhibits a concerted feedback mechanism. PMID- 16658324 TI - Aspartokinase in Lemna minor L: Studies on the in Vivo and in Vitro Regulation of the Enzyme. AB - The growth of Lemna minor was followed by means of frond number, fresh weight, and dry weight measurements in the presence of various amino acids at a concentration 0.25 mm. Lysine inhibited growth but not to the same extent as threonine and homoserine. Isoleucine was also an inhibitor of growth. In the presence of methionine there was some growth for 2 to 3 days, but by 5 days most of the plants appeared to be dead. When lysine and threonine were added together, there was no growth at all, and the plants were dead after 5 days. This effect of lysine + threonine could be reversed by adding methionine or homoserine to the growth medium.The isolated aspartokinase from Lemna showed inhibition by lysine and higher concentrations of threonine. When these amino acids were added together at low concentrations, there was a concerted inhibition of the aspartokinase. It is suggested that some effects of amino acids on the growth of L. minor can be explained on the basis of a concerted feedback control of aspartokinase. PMID- 16658325 TI - In vivo assay of nitrate reductase in cotton leaf discs: effect of oxygen and ammonium. AB - Factors affecting nitrate reduction by leaf discs of cotton (Gossypium hirsutum L.) were investigated. When incubated in 30 mm nitrate, discs reduced nitrate much more slowly under air or O(2) than under N(2). Inhibition by O(2) did not occur at nitrate levels of 100 mm or greater. Treatment with arsenate had little effect under N(2) but stimulated nitrate reduction under air. Similarly, ammonium inhibited nitrate reduction, with the inhibition being partially relieved by arsenate. Uptake of nitrate was unaffected by ammonium. The NAD/NADH ratio increased in response to both oxygen and ammonium. The effects of these treatments on nitrate reduction can be explained by competition with nitrate for NADH generated by glycolysis. PMID- 16658326 TI - Physiology and Ultrastructure of an Oxygen-resistant Chlorella Mutant under Heterotrophic Conditions. AB - The oxygen-resistant strain of Chlorella sorokiniana (Shihira and Krauss), distinguished by its ability to grow autotrophically under high partial pressures of oxygen, was studied and partially characterized in heterotrophic culture. Ultrastructural analysis of glucose-grown oxygen-resistant strain and wild type cells reveals that osmiophilic deposits (possibly polyphosphate) are present only in wild type, while oxygen-resistant strain apparently contains increased amounts of starch and endoplasmic reticulum. Of major physiological significance are the observations that: (a) oxygen-resistant strain requires 6 to 8 days to completely adapt to dark, heterotrophic conditions, whereas wild type acclimates in 1 day; (b) oxygen-resistant strain is resistant to high oxygen tension only when grown on glucose, but not on acetate; and (c) the respiratory rate, but not the photosynthetic rate, of heterotrophic oxygen-resistant strain is abnormal compared to wild type. PMID- 16658327 TI - Benzyladenine-induced Movement of C-Labeled Photosynthate into Roots of Vitis vinifera. AB - Roots of Vitis vinifera L., were treated with benzyladenine when the plant shoots were 38 cm long. Seventy-two hours after benzyladenine treatment, apical or basal leaves on separate shoots were exposed to (14)CO(2). Control shoots received (14)CO(2) but no benzyladenine. Application of benzyladenine directed (14)C photosynthate to roots, but a small amount of radioactivity was detected in the shoot tip when (14)CO(2) was administered to an apical leaf. Distribution of radioactivity among the sugar, organic acid, and amino acid fractions was altered by benzyladenine treatment. In all parts of plants with roots treated with benzyladenine and apical leaf fed (14)CO(2), the percentage of the total label in the sugar fraction comprised of fructose was generally more than twice that in control plants. PMID- 16658328 TI - Characteristics of Nitrate Reduction in a Mutant of the Blue-Green Alga Agmenellum quadruplicatum. AB - Characteristics of nitrate reduction in terms of nitrite production in an N methyl-N'-nitro-N-nitrosoguanidine-induced mutant of the blue-green alga Agmenellum quadruplicatum are described. Following induction of nitrate reduction a linear rate of nitrite production proportional to cell concentration was observed. Rate of nitrite production and growth rate showed similar responses to pH, temperature, and light intensity. If required, only trace amounts of carbon dioxide were necessary for nitrite production. Atmospheres of oxygen or nitrogen inhibited production of nitrite. In addition, a low but constant rate of nitrite production was observed in the dark. Nitrite production by mutant AQ-6 was studied in terms of photosynthesis. As nitrite production proceeded, rate of photosynthesis declined. Ultraviolet irradiation and 3-(3,4-dichlorophenyl)-1, 1 dimethylurea poisoning did not prevent nitrite production. The action spectrum of nitrite production was chlorophyll a-like. PMID- 16658329 TI - The Stoichiometry of Respiration-driven Potassium Transport in Corn Mitochondria. AB - Determinations were made for corn (Zea mays L., WF9-Tms x M14) mitochondria of the stoichiometric relationship between K(+) transport and bond energy produced in respiration (K(+)/ approximately ratio). With inward pumping of potassium acetate activated by NADH oxidation, the initial rate of K(+) transported into the sucrose inaccessible space varied between 0.58 and 0.97 K(+)/ approximately , assuming 2 high energy bond equivalents per NADH oxidized. Only small amounts of H(+) were ejected. Valinomycin did not alter the ratio.Efflux pumping of K(+) in KCl media gave a K(+)/ approximately of 0.92. Substituting sucrose for KCl caused a drop in respiration and H(+) ejection, indicating a requirement for cation efflux pumping in acceptorless respiration. Valinomycin added to the KCl medium accelerated passive swelling but did not impair subsequent efflux pumping, although respiration was partially uncoupled. PMID- 16658330 TI - Alteration in cell permeability as a mechanism of action of certain quinone pesticides. AB - The permeability of the Chlorella pyrenoidosa membrane was studied by following the efflux of (14)C-intracellular material from cells which had been allowed to incorporate (14)CO(2) photosynthetically. It was observed that the efflux increased upon treatment with low concentrations (3-30 muM) of 2, 3-dichloro-1, 4 naphthoquinone (dichlone), 2-amino-3-chloro-1, 4-naphthoquinone (06K-quinone), and 2, 3, 5, 6-tetrachloro-1, 4-benzoquinone (chloranil). Dichlone caused a greater loss of intracellular material than chloranil or 06K-quinone. The rate of loss as well as the total loss of (14)C increased with an increase in the concentration of the quinones. In the dichlone-treated cells, the leakage was observed within 1 minute of the addition of the chemical and the effect on cell permeability was irreversible. Cells exposed to dichlone in the light or under anaerobic conditions released significantly greater amounts of (14)C-material than cells treated in the dark or under aerobic conditions. The aqueous ethanol soluble fraction of the cell was found to be the source of the released material. The proportion of the ethanol-soluble (14)C that leaked out of the cell varied with the time of (14)C-assimilation prior to treatment with dichlone. In the dichlone-treated cells, practically all the (14)C-sucrose, alanine, glutamine, serine, and glycine leaked out, whereas glutamic, aspartic, succinic, and fumaric acids were lost only partially. Essentially no (14)C-lipids were lost from the cells during dichlone treatment.The extreme rapidity of the effect of dichlone on permeability and the low concentrations at which dichlone acted suggest that the cell membrane may be a primary site of action of dichlone, and that the metabolic changes observed in dichlone-treated Chlorella may be due to the changes in the cell membrane structure. PMID- 16658331 TI - Effect of petiole anoxia on Phloem transport in squash. AB - Translocation of (14)C-labeled assimilates in Early Prolific Straightneck squash (Cucurbita melopepo torticollis Bailey) through a 15-centimeter oxygen-deficient zone of the petiole was studied as a function of varying periods of anaerobiosis (N(2) atmosphere). Initiation of anaerobic conditions caused an immediate and rapid decline in translocation to about 35 to 45% of the pretreatment rate within 30 to 40 minutes. This inhibition response (first inhibition response) was transient, however, and full recovery to the pretreatment rate occurred during the ensuing 60 to 90 minutes. Following this adaptation response to anaerobic conditions, translocation continued unimpaired for extended periods of time, approaching, and in some cases exceeding, 24 hours. The second inhibition response was permanent and could not be reversed by supplying air during a subsequent 20-hour period. PMID- 16658332 TI - Mechanism of inhibition of translocation by localized chilling. AB - Arrhenius plots of translocation velocity as a function of petiole temperature show a marked increase in temperature dependence below 10 C in bean (a chilling sensitive species) but not in sugar beet (chilling-resistant). The increased temperature dependence below 10 C was not observed for cytoplasmic streaming or oxygen uptake in bean. Bean petioles were served to release pressure in order to determine whether sieve tubes are obstructed in cold-treated petioles. The resulting pressure release caused serious displacement of the crystalline protein bodies in the sieve tubes of petioles at 25 C, but in those locally cooled to 0 C for 30 minutes little displacement occurred, indicating obstruction in the latter. An ultrastructural study of sieve tubes in tissue frozen rapidly in situ and dehydrated by freeze substitution revealed that treatment at 0 C for 30 minutes caused structural alteration and displacement of the cytoplasmic material lining the sieve tube wall resulting in occlusion of sieve plates. The sieve plates of the control petioles at 25 C were generally clear of obstructions. The results indicate that inhibition of translocation by chilling in chilling sensitive plants results from physical blockage of sieve plates rather than from direct inhibition of a metabolic process which drives translocation. PMID- 16658333 TI - Changes in respiration, photosynthesis, adenosine 5'-triphosphate, and total adenylate content of ozonated pinto bean foliage as they relate to symptom expression. AB - The effect of 0.25 to 0.30 microliter per liter ozone on photosynthesis and respiration and on the ATP and total adenylate content of the primary leaves of pinto beans (Phaseolus vulgaris L.) was examined. Changes in these parameters over a 72-hour time period were correlated with the development of symptoms of ozone toxicity. Toxicity symptoms normally appeared within 24 hours. The content of ATP and total adenylates increased immediately following a 3-hour exposure to ozone. Photosynthesis was depressed initially, but returned to normal within 24 hours. Respiration was not always altered initially, but it was significantly stimulated within 24 hours. We interpret the results to mean that the changes in adenylate content and photosynthesis are early events in the initiation of ozone damage and that the change in respiration is a consequence rather than a cause of cellular injury. PMID- 16658334 TI - Action Spectra for Chromatic Adaptation in Tolypothrix tenuis. AB - The dark synthesis of biliproteins in the blue-green alga Tolypothrix tenuis is controlled by brief light treatments. Green light potentiates synthesis of phycoerythrin and red light potentiates synthesis of phycocyanin. Red reverses the effect of green and vice versa. Action spectra for the red and green effects were obtained for the wavelength region 320 nanometers to 710 nanometers, at 10 nanometer intervals. The principal action band in the red peaks at 660 nanometers, with a half-band width of 58 nanometers and an accompanying shortwave band at 360 nanometers. The green action band peaks at 550 nanometers, with a half-band width of 76 nanometers, and a shortwave band at 350 nanometers. Chromatic adaptation and another photomorphogenic response in the blue-green algae are discussed in terms of possible regulation by a photoreversible pigment recently isolated from Tolypothrix. PMID- 16658335 TI - Photophosphorylation during Chloroplast Development in Red Kidney Bean: II. Photophosphorylation and Photoreduction Appear Concomitantly but Initially are Uncoupled. AB - Cyclic phosphorylation with phenazine methosulfate and noncyclic phosphorylation and reduction with ferricyanide were detected in isolated chloroplasts from greening bean leaves after 3 to 4 hours of illumination. Activity commenced when rapid synthesis of chlorophyll was initiated. Rates of photophosphorylation were comparable to mature levels by 15 to 18 hours of development. Photoreduction of ferricyanide attained a peak value by 12 hours of illumination and subsequently fell to normal levels by 15 to 18 hours. With ferricyanide, the P/e(2) ratios were initially less than 0.1 but were close to 1.0 after 18 hours of illumination. The data suggested that photosystems I and II appeared concomitantly in the chloroplast but were not fully operative until later in development. Proplastids and immature chloroplasts exhibited high capacities to reduce ferricyanide in the dark. The rates of dark reduction rapidly diminished to low levels by 15 hours of illumination when normal rates of photochemical activity were observed. After a 2-to 3-day lag, a rapid increase in leaf fresh weight was noted at the time total chlorophyll content reached steady state values on a fresh weight basis. With fresh weight as an index of growth, primary leaves completed their development after 6 to 7 days of illumination. PMID- 16658336 TI - Studies on Auxin Protectors: XI. Inhibition of Peroxidase-Catalyzed Oxidation of Glutathione by Auxin Protectors and o-Dihydroxyphenols. AB - Commercial horseradish peroxidase, when supplemented with dichlorophenol and either manganese or hydrogen peroxide, will rapidly oxidize glutathione. This peroxidase-catalyzed oxidation of glutathione is completely inhibited by the presence of auxin protectors. Three auxin protectors and three o-dihydroxyphenols were tested; all inhibited the oxidation. Glutathione oxidation by horseradish peroxidase in the presence of dichlorophenol and Mn is also completely inhibited by catalase, implying that the presence of Mn allows the horseradish peroxidase to reduce oxygen to H(2)O(2), then to use the H(2)O(2) as an electron acceptor in the oxidation of glutathione. Catalase, added 2 minutes after the glutathione oxidation had begun, completely inhibited further oxidation but did not restore any gluthathione oxidation intermediates. In contrast, the addition of auxin protectors, or o-dihydroxyphenols, not only inhibited further oxidation of gluthathione by horseradish peroxidase (+ dichlorophenol + Mn), but also caused a reappearance of glutathione as if these antioxidants reduced a glutathione oxidation intermediate. However, when gluthathione was oxidized by horseradish peroxidase in the presence of dichlorophenol and H(2)O(2) (rather than Mn), then the inhibition of further oxidation by auxin protectors or o-dihydroxyphenols was preceded by a brief period of greatly accelerated oxidation. The data provide further evidence that auxin protectors are cellular redox regulators. It is proposed that the monophenol-diphenol-peroxidase system is intimately associated with the metabolic switches that determine whether a cell divides or differentiates. PMID- 16658337 TI - Influence of hydrogen fluoride fumigation on the water economy of soybean plants. AB - The influence of hydrogen fluoride fumigation on water economy was studied using soybean (Glycine max [L] Merr.). Fumigation caused partial stomatal closure in 1 hour and practically complete closure within 4 hours. The transpiration rate was greatly reduced by fumigation, while the leaf temperature was increased. Water potential increased after 1 day but fell drastically when necrosis occurred. Effects of interrupted fumigation during the day were somewhat less severe in all respects than those of continuous fumigation; nighttime fumigation caused only minor effects. Fluoride uptake was also much less from nighttime than from daytime fumigations. PMID- 16658338 TI - Adenosine triphosphate content and seed vigor. AB - ATP content in imbibed seeds is significantly correlated with seedling size in fatty, starchy and proteinaceous seeds, and it indicates viability in seed lots. ATP content thus appears to be a useful biochemical index of seed vigor. PMID- 16658339 TI - Abolishment of Specific Toxicity of Host-Specific Toxin of Helminthosporium carbonum by Electrolytic Reduction. AB - The host-specific toxin produced by the plant pathogen, Helminthosporium carbonum Ullstrup is normally about 300 times as toxic for corn plants susceptible to the disease as it is for disease-resistant plants. Electrolytic reduction of the toxin destroys this specificity, rendering the material no more toxic for disease susceptible plants than for disease resistant ones. PMID- 16658340 TI - Time of response of coleoptiles to kinetin. PMID- 16658341 TI - Acetylcholine Inhibition of Phytochrome-mediated Increases in a Flavonoid and in Phenylalanine Ammonia-Lyase Activity of Etiolated Barley Plumules. PMID- 16658342 TI - Possible errors in quantitative determination of phenylalanine ammonia-lyase activity by spectrophotometric methods. AB - A possible error in spectrophotometric determination of cinnamate, the product of phenylalanine ammonia-lyase activity, using nonpurified protein extracts has been shown.Under optimal conditions for phenylalanine ammonia-lyase activity, with borate buffer and in the presence of alpha-keto acids, phenylpyruvate is produced and its enol tautomer-borate complex formed, strongly absorbs at 290 nanometers. PMID- 16658343 TI - The interpretation of studies on rapidly labeled ribonucleic Acid in higher plants. PMID- 16658344 TI - Scanning electron microscope observations of herbicide dispersal using cathodoluminescence as the detection mode. PMID- 16658345 TI - Anaerobic nitrite production by plant cells and tissues: evidence for two nitrate pools. AB - Tobacco (Nicotiana tabacum L. cv. Xanthi) XD cells containing nitrate and nitrate reductase stopped producing nitrite after approximately 1 hour when incubated under anaerobic conditions. The cessation of nitrite production was not due to an inactivation of the nitrate reducing system. This was shown by the ability of the cells to resume anaerobic nitrite production at a rate similar to the initial rate of nitrite production upon exposure to nitrate, monohydroxy alcohols or pyrazole. Cessation of nitrite production also could not be attributed to leakage of nitrate from the cells. Although some nitrate did leak from the cells, most of the nitrate was still in the cells by the time anaerobic nitrite production ceased. We infer the existence of a small metabolic pool and a large storage pool of nitrate, such that nitrite production ceases when the metabolic pool is depleted of nitrate. The metabolic pool of nitrate in tobacco cells decreased 170 fold as the culture aged from 3 to 5 days. However, total cellular nitrate during this period remained relatively constant.Anaerobic nitrite production by barley (Hordeum vulgare) aleurone layers and corn (Zea mays) leaf sections also ceased after only a small fraction of endogenous nitrate was reduced and resumed again upon addition of exogenous nitrate. In contrast to that found with tobacco cells, the metabolic pool of nitrate in corn leaf sections remained constant with age, while total endogenous nitrate increased. These results were interpreted to mean that higher plants in general contain metabolic and storage pools of nitrate, the properties of which vary with species and physiological variables. PMID- 16658346 TI - Soybean polyamines: separation and characterization of cadaverine. AB - Cadaverine in soybeans was separated by ion exchange chromatography from other polyamines previously identified. Identification of cadaverine was based on ion exchange separation, thin layer chromatography, paper electrophoresis, mass and nuclear magnetic resonance spectral analyses. Since the molecules of putrescine and cadaverine are so similar, separation and identification of the two components are difficult. Their R(F) values on thin layer chromatography are close, although cadaverine produces a bluish purple color when sprayed with ninhydrin reagent, while putrescine forms a purple color. Separation likewise is poor by paper electrophoresis, gas chromatography, and gel filtration. The mass spectra of cadaverine and putrescine have m/e peaks at 30, 43, 45, 56, 73, 85, 102 and 30, 43, 59, 71, 88, respectively. The m/e peaks differentiate one compound from the other. Nuclear magnetic resonance spectra and their integration curves show that cadaverine contains two types of methylene protons (10 total) in 3:2 ratio while putrescine produces two types (8 total) in 1:1 ratio. Polyamines occur at levels of micrograms per gram of soybeans with spermidine present in the largest quantity. PMID- 16658347 TI - Ethylene, light, and anthocyanin synthesis. AB - Ethylene control of anthocyanin formation functions only through light-initiated synthesis pathways of the rapid synthesis phase. Treatment with ethylene in the dark had no effect on dark anthocyanin synthesis in red cabbage (Brassica oleracea L.). Pretreatment of both red cabbage and sorghum (Sorghum vulgare L.) with ethylene for 24 hours in the dark did increase the rate of synthesis when the tissue was placed in the light. Light-initiated anthocyanin synthesis is inhibited by ethylene when the tissue is returned to the dark. PMID- 16658348 TI - Photosynthetic phosphoenolpyruvate carboxylases: characteristics of alloenzymes from leaves of c(3) and c(1) plants. AB - A detailed comparison of green leaf phosphoenolpyruvate carboxylases from the C(4)-species Atriplex spongiosa and the C(3)-species Atriplex hastata revealed significant physical and kinetic differences. The two alloenzymes can be separated by anion exchange chromatography but have comparable molecular weights (350,000). Maximal velocity estimates were 38.0 and 1.48 micromoles per minute per milligram of chlorophyll for the carboxylases of A. spongiosa and A. hastata, respectively. Km phosphoenolpyruvate estimates were 0.49 and 0.08 mm for the C(4)A. spongiosa and C(3)A. hastata and the Km Mg estimates were 0.33 mm for the C(4) species and 0.017 mm for the C(3) species. The activity of the phosphoenolpyruvate carboxylase of A. spongiosa is more sensitive to chloride and phosphate than the phosphoenolpyruvate carboxylase of A. hastata, but both are equally sensitive to Mg chelating substances such as ATP, ADP, and citrate if assayed at their respective Km Mg values. A survey of the phosphoenolpyruvate carboxylases from 18 C(4) and C(3) species resulted in mean maximal velocity estimates of 29.0 +/- 13.2 and 1.50 +/- 0.57 micromoles per minute per milligram of chlorophyll for the C(4) species and C(3) species, respectively. Km phosphoenolpyruvate estimates were 0.59 +/- 0.35 mm and 0.14 +/- 0.07 mm for the C(4) and C(3), and Km Mg estimates were 0.50 +/- 0.30 and 0.097 +/- 0.057 mm for C(4) and C(3). All differences between means were significant at the 0.01 confidence level, supporting our hypothesis that the phosphoenolpyruvate carboxylase alloenzymes of C(4) and C(3) plants are functionally different and are associated with different photosynthetic roles. Both function in the photosynthetic production of C(4) acids, the phosphoenolpyruvate carboxylase of C(4) species largely producing malate or aspartate (or both) as a photosynthetic intermediate and the phosphoenolpyruvate carboxylase of C(3) species producing malate or aspartate (or both) as a photosynthetic product. PMID- 16658349 TI - Multiple forms of plant phosphoenolpyruvate carboxylase associated with different metabolic pathways. AB - The physical and kinetic properties of multiple forms of phosphoenolpyruvate carboxylase were studied in leaves of C(4) and C(3) species, their F(1) and F(3) hybrids, in greening maize leaves, in Crassulacean acid metabolism plants, and in nongreen root tissues. Four different forms are suggested: a C(4) photosynthetic phosphoenolpyruvate carboxylase with high Km for phosphoenolpyruvate ( approximately 0.59 mm), Km Mg ( approximately 0.5 mm), and V(max) ( approximately 29 micromoles per minute per milligram of chlorophyll); a C(3) photosynthetic phosphoenolpyruvate carboxylase with low Km for phosphoenolpyruvate ( approximately 0.14 mm), Km for Mg ( approximately 0.097 mm), and V(max) (1.5); a Crassulacean acid metabolism type with low Km for phosphoenolpyruvate (0.14 mm), and high V(max) (14 micromoles per minute per milligram of chlorophyll); and a nongreen or nonautotrophic type with low Km for phosphoenolpyruvate, Km for Mg, and low V(max). In closely related species or within species, the types can be differentiated by anion exchange column chromatography. Each of the four forms is associated with a different metabolic pathway: the phosphoenolpyruvate carboxylase of C(4) species for malate generation as a photosynthetic intermediate, the phosphoenolpyruvate carboxylase of C(3) species in malate generation as a photosynthetic product, the phosphoenolpyruvate carboxylase of Crassulacean acid metabolism species in malate generation as a CO(2) donor for photosynthesis during the subsequent light period, and a nongreen or root type producing malate for ionic balance and reduced nicotinamide adenine dinucleotide phosphate generation. The data in this paper in conjunction with published information support the notion of different molecular forms of a protein functioning in different metabolic pathways which have common enzymic steps. PMID- 16658350 TI - Embryo Development in Phaseolus vulgaris: II. Analysis of Selected Inorganic Ions, Ammonia, Organic Acids, Amino Acids, and Sugars in the Endosperm Liquid. AB - Endosperm liquid bathes the embryo of Phaseolus vulgaris from the heart stage through the late cotyledon state. This liquid was aspirated from many ovules of the same stage, pooled, and analyzed for the following constituents and parameters: [List: see text]. PMID- 16658351 TI - Dark Reversion of Phytochrome in Lettuce Seeds Stored in a Water-saturated Atmosphere. AB - Dark reversion of the far red-absorbing form of phytochrome, which does not occur in dry lettuce (Lactuca sativa var. Grand Rapids) seeds, appears to take place in seeds stored in a water-saturated atmosphere. The water content (approximately 70% after 10 days) of such seeds is insufficient to support germination; however the treatment enhances germination in seeds stored for 1 to 5 days, but this enhancement subsequently disappears, and the effect of extended storage (up to 28 days) is inhibiting. The half-time for dark far red-absorbing phytochrome reversion is 7 to 8 days, and at this time it can be completely reversed by exposing the seeds to a flash of red light. Storage of more than 7 to 8 days decreases red light enhancement of germination. PMID- 16658352 TI - Kinetics of stress relaxation properties of oat coleoptile cell wall after geotropic stimulation. AB - This study describes the stress relaxation of the cell wall of oat (Avena sativa) coleoptiles after different periods of geotropic stimulation. The upper and lower tissues (with respect to gravity) of geotrophically stimulated coleoptiles exhibit different wall properties. The lower tissues are less resistant to deformation than the upper. The ratio of stress to strain is significatly less in the lower than in the upper tissue. Similarly, the relaxation time and the minimum relaxation time, derived from the Maxwell model which describes the physical characteristics of polymers, are also shorter in the lower tissue. However, the maximum relaxation time shows no difference between the upper and lower tissues of a geotropically stimulated coleoptile. The differences between the tissues begin at about 8 minutes after the commencement of stimulation, similar to the time for the initiation of dictyosome redistribution, and precede the onset of geotropism. The above responses of the cell wall of the lower tissue are similar to those induced by indoleacetic acid. The parameters of wall properties of the coleoptiles of both the control and the geostimulated fluctuate rhythmically with time. The periodic changes in wall properties of the coleoptile are compared to other cyclic physiological phenomena. PMID- 16658353 TI - Changes in Phospholipid Composition of a Winter Wheat Cultivar during Germination at 2 C and 24 C. AB - Evaluation of various solvent systems for lipid extraction of wheat Triticum aestivum L. cv. Rideau seeds showed that boiling 2-propanol followed by the Bligh Dyer procedure was the most efficient method, with respect to lipid yield and ability to inactivate lipolytic enzymes. Ten phospholipids were identified in dry seeds; the major components being phosphatidylcholine, lysophosphatidylcholine, N acyl lysophosphatidyl-ethanolamine, N-acylphosphatidylethanolamine, and phosphatidylethanolamine. After growth for 1 week (2 C) or 31 hours (24 C), the proportions of phosphatidylethanolamine + lysophosphatidic acid and phosphatidic acid increased, lysophosphatidylcholine decreased, and the remaining phospholipids showed little change. At 5 weeks (2 C) or 72 hours (24 C), the seedlings showed 5-fold increases in the proportion of phosphatidic acid largely at the expense of phosphatidylcholine, small decreases in N-acyl lysophosphatidylethanolamine and N-acylphosphatidylethanolamine, and significant increases in lysophosphatidylcholine. The changes in phosphatidic acid and phosphatidylcholine are interpreted as being partially due to increasing phospholipase D activity during germination. In general, the phospholipid composition was similar in morphologically equivalent seedlings grown at 2 C or 24 C. The increased membrane content in seedlings grown at 2 C does not reflect any preferential synthesis of individual phospholipids. PMID- 16658354 TI - Growth, greening, and phytochrome in etiolated spirodela (lemnaceae). AB - Two species of Spirodela were grown aseptically in a simple mineral medium containing sucrose. Weak red light (15 erg cm(-2) sec(-1)) enhanced dark growth of S. oligorrhiza, whereas weak far red light (15 erg cm(-2) sec(-1)) when given after the red light reduced this effect.The lag period in chlorophyll accumulation (20-24 hours) could be shortened by a brief red light illumination, at both 25 C and 0 C, 12 to 24 hr before the exposure to continuous white light. The effect of red light in shortening the lag phase was diminished when high intensity of far red light (10(3)-10(4) erg cm(-2) sec(-1)) was given simultaneously with the red light.Spectrophotometric studies showed detectable amounts of phytochrome in the plants, part of which decayed immediately during a short illumination by red light. The other relatively stable, phytochrome decayed only after prolonged illumination. PMID- 16658355 TI - Factors affecting electrical impedance of internodal stem sections. AB - A sample holder was designed and built to facilitate measuring the magnitude and phase angle of the electrical impedance of internodal stem sections from Cornus stolonifera Michx. A nonpolarizing, electrically conducting manganese dioxidecarbon paste used between the stem sample and the electrodes of the sample holder allowed measurement of impedance at frequencies from 50 hertz to 500 kilohertz without electrode polarization or electrical interference. The impedance magnitude was linearly dependent on the sample length, but this dependence was minimized by computing a normalized impedance magnitude. The normalized impedance magnitude (Z(nf)) was calculated using the impedance magnitude (Z) at any specified frequency (f) and the impedance magnitude at 500 kilohertz (Z(500 khz)) in the following formula: Z(nf) = (Z - Z(500 khz))/Z(500 khz). The normalized impedance magnitude was sensitive to injury produced by boiling and peeling the sample. Electrical impedance measurements on the bark and wood separately demonstrated that they have different electrical properties. PMID- 16658356 TI - A Comparison of Nitrite Reductase Enzymes from Green Leaves, Scutella, and Roots of Corn (Zea mays L.). AB - Nitrite reductase from green leaves of corn (Zea mays L.) is eluted from a diethylaminoethyl-cellulose column in one peak of activity by a chloride gradient, while nitrite reductase from scutellum tissue is resolved into two peaks of activity, apparently representing two forms of the enzyme NiR1 and NiR2. One of these (NiR2) elutes at the same concentration of chloride as the leaf nitrite reductase. Roots and etiolated shoots also exhibited both forms of the enzyme, however, lesser amounts of NiR1 is extractable from these tissues than from scutellum. Comparison of green leaf nitrite reductase with NiR2 from scutellum tissue shows similar or identical properties with respect to molecular weight, isoelectric point, electron donor requirements, inhibition properties, pH optima, thermal stability, and pH tolerance. The significance of these similarities in relation to probable differences in the biochemical mechanism of nitrite reduction between leaf and scutellum tissues is discussed. Although ferredoxin is considered, with some reservations, to be the electron donor for nitrite reductase in green tissue, the reductant for nongreen tissue is not known. The possibility that nitrite reductases from green and non-green tissues uses the same electron donor, in vivo, is considered. PMID- 16658357 TI - Reduction of water permeability in potato tuber slices by cyanide, ammonia, 2,4 dinitrophenol, and oligomycin and its reverse by adenosine 5'-triphosphate and cytidine 5'-triphosphate. AB - Five millimolar KCN reduced water permeability in 1-millimeter thick slices of potato tuber (Solanum tuberosum L.). One-tenth millimolar ATP and CTP prevented or reversed the reduced permeability. UTP and GTP were not effective. Five millimolar ammonium carbonate or 0.1 millimolar 2,4-dinitrophenol also reduced water permeability, but ATP and CTP were only partially effective in reversing the reduced permeability. Oligomycin, 5 micrograms per milliliter, reduced water permeability, and the reduction was reversed by ATP and CTP. ATP and CTP appear to be involved in maintaining the structure of water pathways into the cell. PMID- 16658358 TI - Host-Pathogen Interactions: VI. A Single Plant Protein Efficiently Inhibits Endopolygalacturonases Secreted by Colletotrichum Lindemuthianum and Aspergillus Niger. AB - Endopolygalacturonases have been purified from the extracellular enzymes of Colletotrichum lindemuthianum and Aspergillus niger. A protein, purified from Red Kidney (Phaseolus vulgaris) beans for its ability to inhibit the endopolygalacturonase secreted by C. lindemuthianum, inhibits the A. niger endopolygalacturonase almost as efficiently as it inhibits the C. lindemuthianum enzyme. PMID- 16658359 TI - beta-Cystathionase In Vivo Inactivation by Rhizobitoxine and Role of the Enzyme in Methionine Biosynthesis in Corn Seedlings. AB - Rhizobitoxine has previously been shown to inactivate irreversibly beta cystathionase isolated from spinach. In the present studies, rhizobitoxine was shown to inhibit partially beta-cystathionase of spinach and corn seedlings in vivo. An activity of 30 to 40% of normal remained in toxin-treated seedlings of both spinach and corn. Possible reasons for the partial inhibition are discussed.Rhizobitoxine-treated and control corn seedlings were allowed to assimilate (35)SO(4) (2-) for 3 or 6 hours, and the radioactivity incorporated into sulfur amino acids at these times was studied. The most striking effect of rhizobitoxine was an increase (up to 22-fold) in radioactive cystathionine. Accumulation of radioactivity in methionine was only slightly inhibited by rhizobitoxine treatment. The results strongly suggest that the transsulfuration pathway contributes to methionine biosynthesis, and that metabolism via this pathway is impaired, but not entirely eliminated, by rhizobitoxine treatment.The present data do not permit decisions about the relative contributions of the transsulfuration and the direct sulfhydration pathways to methionine biosynthesis, or whether the pathological effects of rhizobitoxine are due chiefly to inhibition of beta-cystathionase. PMID- 16658360 TI - Effects of Oxygen and Respiratory Inhibitors on Induction and Release of Dormancy in Aerial Tubers of Begonia evansiana. AB - Effects of O(2) and some respiratory inhibitors on the induction and release of bud dormancy were examined with the aerial tubers of different ages of Begonia evansiana Andr. Oxygen was needed not only for tuber sprouting but also during the chilling process at 2 to 5 C to break tuber dormancy. If the mature tubers were exposed to blue light during the chilling period, their dormancy was strikingly released even by the chilling given under an O(2) concentration as low as 3%. Blue light pretreatment promoted photo-sprouting of immature tubers only when given under lower O(2) concentrations. On the other hand, red light became effective in inducing dormancy in the immature tubers and in prolonging dormancy in the mature tubers as O(2) tension was increased. This was also the case with the induction of dormancy in the immature tubers by exposing them to a lower temperature (17 C) in the dark. The development of dormancy was suppressed by 2,4 dinitrophenol, p-nitrophenol, and sodium azide. PMID- 16658361 TI - The effect of wilting on proline metabolism in excised bean leaves in the dark. AB - The effects of wilting on the fate of proline and on the rates of nonprotein proline formation and utilization have been determined in excised bean leaves. Wilting did not alter the fate of exogenously added (14)C-l-proline (2 mm) in either non-starved leaves (from plants previously in the light) or starved leaves (from plants previously in the dark). The fate of proline in nonstarved leaves was protein synthesis and in starved leaves was protein synthesis and oxidation to other compounds.Wilting caused an increase in non-protein proline formation, possibly including release by proteolysis and synthesis from precursors in both starved and nonstarved leaves. Wilting caused a decrease in proline utilization in nonstarved leaves by decreasing protein synthesis. In starved leaves, wilting caused an increase in the rate of proline utilization but this is due to the higher content of proline in wilted leaves compared to the turgid leaves which causes more proline utilization by oxidation. Thus, the primary effects of wilting which lead to the accumulation of proline were to decrease protein synthesis and to increase proline formation. The source of the proline is not known but the increased formation due to wilting is not affected by the carbohydrate content of the leaf. The role of carbohydrates is to prevent the loss of accumulating proline by oxidation. PMID- 16658362 TI - Studies on the mechanism of glycerate 3-phosphate synthesis in tomato and maize leaves. AB - The net carbon incorporation in maize (Zea mays) and tomato (Lycopersicum esculentum) leaves was mainly the result of the carboxylation of ribulose 1,5 diphosphate. In both of these organisms synthesis of glycerate 3-phosphate was studied during short chase experiments (2 or 3 seconds in (14)CO(2) then 8 to 27 seconds in unlabeled CO(2)). Changes in the radioactivity in the individual carbon atoms of glycerate 3-phosphate, malate, and aspartate are consistent with the formation, in both leaves, of 2 molecules of glycerate 3-phosphate for each CO(2) molecule incorporated. The CO(2), before reacting with ribulose 1,5 diphosphate, is first incorporated in an intracellular CO(2) pool which has a different composition according to the species. This pool is constituted in tomato by volatile compounds (50 nanomoles per gram of fresh weight) more or less in equilibrium with atmospheric CO(2). In maize the pool consists of carbon atoms 4 of malate and aspartate (for at least 80% of the pool) and volatile compounds which correspond, in all, to 540 nanomoles per gram of fresh weight where atmospheric CO(2) enters through an irreversible reaction. PMID- 16658363 TI - Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots. AB - A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis. PMID- 16658364 TI - An extracellular macromolecular complex from the surface of soybean suspension cultures. AB - A cell-free, insoluble cell wall fraction is described which floats on the surface of suspension cultures of Glycine max L. Merrill var. Acme cells. Its accumulation is governed by both the shaking speed and the medium volume, a shaking speed of 110 to 120 revolutions per minute with a medium volume of about 100 to 120 milliliters in a 250-milliliter flask being optimal. Various factors which could control the accumulation of the complex were tested and are discussed, and scanning electron micrographs of the complex being released from the cell surface are presented.The composition of the complex by weight is 46% galacturonic acid, 36% protein, 11% lignin (apparent), 4.4% arabinose, 2% ash, and 0.5% methyl ester. Evidence for an intimate relationship between the uronic acid and protein fractions is presented. The protein contains hydroxyproline, and the bulk of it is tightly bound to the complex, although a portion can be removed with high salt treatments. PMID- 16658365 TI - Protein metabolism in leaves and developing grains of rices differing in grain protein content. AB - Four semi-dwarf rices (Oryza sativa L.) differing in percentage of grain protein, grown in a flooded field receiving basal N fertilization, had a peak activity of root glutamate dehydrogenase 4 weeks after transplanting. A lower peak occurred during panicle formation 10 weeks after transplanting. The percentage of N of the active leaf blades was also highest 4 weeks after transplanting. The activity of nitrate reductase in the leaf blades was low and decreased after transplanting.Among the three rices with similar grain yield, the rice with high percentage of protein tended to translocate more leaf N to the developing grains than the rices with average grain protein content. The leaf blades of the former also had lower rates of leucine incorporation during grain development but higher protease activity than leaves of the rices with average protein content. Developing grains of the rices with high percentage of protein tended to have higher levels of soluble protein, free amino N, and protease, and a faster rate of leucine incorporation than grains of the IR3 rice with average percentage of protein, regardless of grain yield. PMID- 16658366 TI - Evidence for Two Photoreactions and Possible Involvement of Phytochrome in Light dependent Stomatal Opening. AB - Leaves of the xantha mutant of Helianthus annuus have a higher rate of transpiration and a lower diffusive resistance in the light than in the dark. Stomates of this nonphotosynthetic mutant open in the light and close in the dark.Comparative studies of tobacco, xantha mutant, and wild-type sunflower stomatal opening over a range of light intensities in isolated portions of the spectrum reveal two patterns of response: (a) a low intensity opening in the green and far red characterized by partial opening, absence of a threshold, and saturation of the response at low light intensities; (b) a high intensity response in the blue characterized by a threshold (intensities greater than 100 microwatts per square centimeter needed for opening) and a linear opening response at higher incident light intensities. In xantha mutant stomates only the low intensity system appears to be operational, while both low and high intensity systems are present in the wild-type sunflower and tobacco.Red light has an inhibiting effect on stomatal opening in both mutant and wild-type sunflowers. They require prior exposure to far red for opening to occur in red light. This redfar red antagonism suggests the involvement of phytochrome. PMID- 16658367 TI - Effect of gibberellic Acid and actinomycin d on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells. AB - Analysis of structural changes in barley aleurone cells during germination or following incubation of isolated layers in gibberellic acid with or without actinomycin D revealed extensive development of rough endoplasmic reticulum. Following the assembly of stacked rough endoplasmic reticulum, vesiculation occurred mainly in basal regions of the cell, resulting in a polar distribution of rough endoplasmic reticulum vesicles. It is postulated that these vesicles are involved in protein secretion, because smooth vesicles, derived from the rough endoplasmic reticulum, apparently become appressed to the plasma membrane. The increased alpha-amylase in the ambient medium and in cell homogenates correlated directly with formation and subsequent vesiculation of the rough endoplasmic reticulum. Furthermore, when cells were treated with actinomycin D and gibberellic acid, alpha-amylase synthesis was inhibited by 45% and secretion by 63%. These cells were characterized cytologically by large areas of disarrayed segments of fragmented rough endoplasmic reticulum, corresponding to a high intracellular level of alpha-amylase. In addition, small lipid bodies common to the segmented regions of rough endoplasmic reticulum were surrounded by fine fibrous material, short segments of rough endoplasmic reticulum, and free ribosomes, suggesting that actinomycin D had interfered with development and organization of rough endoplasmic reticulum. PMID- 16658368 TI - Cytokinins in Rhizopogon roseolus Secretion of N-[9-(beta-d-Ribofuranosyl-9H) Purin-6-Ylcarbamoyl]Threonine into the Culture Medium. AB - Rhizopogon roseolus excretes the transfer RNA component, N-[9-(beta-d ribofuranosyl-9H) purin-6-ylcarbamoyl]threonine (Ado-CO-thr) into the culture medium. This compound was proposed to be the archetype of ureidopurines exhibiting cytokinin activity. The amount of Ado-CO-thr isolated from the medium is about one-twentieth the amount of ribosylzeatin that can be isolated. PMID- 16658369 TI - trans-Ribosylzeatin: Its Biosynthesis in Zea mays Endosperm and the Mycorrhizal Fungus, Rhizopogon roseolus. AB - When [8-(14)C]-N(6)-(Delta(2)-isopentenyl) adenosine is incubated with the endosperm of corn (2 weeks after pollination), it is converted to [(14)C]-N(6)-(4 hydroxy-3-methylbut-2-trans-enyl) adenosine, trans-ribosylzeatin. This biosynthetic step, N(6)-(Delta(2)-isopentenyl) adenosine to ribosylzeatin, also occurs in the mycorrhizal fungus, Rhizopogon roseolus. PMID- 16658370 TI - New photoresponses of phycomyces. AB - The influence of light on asexual fruiting and mycelial growth of Phycomyces blakesleeanus Burgeff was studied by means of fruiting body counts and size measurements in cultures on solid media under varied incubation conditions. Five types of photoresponses were shown by ATCC Strain 8743a: (a) photoinduction of giant sporangiophores; (b) interference by light with an endogenous system that otherwise induces fruiting when the mycelium approaches the rim of the Petri dish; (c) inhibition of mycelial growth rates by light; (d) inhibition of dwarf sporangiophore induction by light; and (e) postponement by light of death in clones maintained by serial transfer at low temperature. A second strain, designated G5, showed responses comparable to (a), (b), and (d). The magnitudes of the responses were greatly affected by temperature of incubation and available nitrogen (asparagine) supply. The photoinduction of giant sporangiophores could be demonstrated with light of wavelengths between 380 and 480 nanometers but not with 520 nanometers or above. At 480 nanometers, light doses as small as 40 ergs per square centimeter were effective in inducing giant sporangiophores in strain 8743a. PMID- 16658371 TI - Partial Characterization of C(x) Cellulase and Cellobiase from Ripening Tomato Fruits. AB - Cellulolytic enzymes were studied in extracts from the locular contents of ripening fruits of Lycopersicon esculentum var. KC-146. When acting on carboxymethyl cellulose, the enzyme preparations were capable of decreasing the viscosity of the reaction mixture and generating reducing groups, oligosaccharides, and glucose. Cellobiose cellotriose, cellotetrose, and cellopentose also served as substrates for glucose production.These properties were due to the presence in the extracts of two cellulolytic enzymes-C(x) cellulase (E.C. 3.2.1.4) and cellobiase- (E.C. 3.2.1.21). No evidence was found for the presence of exocellulase or C(1), an enzyme which initiates enzymatic attack on insoluble cellulose.Cellobiose was found to be weakly inhibitory to C(x) cellulase. d-Glucono-1,5-lactone competitively inhibited cellobiase. The K(m) for cellobiose was 0.34 mm, and the K(i) for gluconolactone was 23 muM. PMID- 16658372 TI - Carotenoid composition of spinach chloroplast grana and stroma lamellae. AB - Stroma lamellae and grana stacks prepared by French press rupture of spinach (Spinacia oleracea) chloroplasts contain similar amounts of beta-carotene on a protein basis. The grana fraction has considerably more xanthophylls than does the stroma fraction. Total carotenoid to chlorophyll ratios are similar for both fractions. PMID- 16658373 TI - Phycomyces: Mechanical Behavior of Stage II and Stage IV. PMID- 16658374 TI - Organic Acid Changes in the Epidermis of Vicia faba and Their Implication in Stomatal Movement. AB - Considerable evidence indicates that the increase in guard cell turgor resulting in stomatal opening is brought about by active K(+) uptake into guard cells. Only a small increase in inorganic anions appears to accompany the increase in K(+). A plausible explanation is that organic acids are produced within guard cells and act as counterions, whereas the H(+) produced are exchanged for K(+).This hypothesis was tested by using different levels of ambient CO(2) in light to control stomatal aperture and at the same time measure changes in organic acid production in the epidermis of Vicia faba. Epidermal strips were used, quickly frozen in liquid nitrogen, and later extracted.A positive correlation was found between stomatal resistance (r(s), indirect measure of stomatal aperture) and CO(2) level. With decreases in r(s), total titratable acidity increased. The organic acids, glyceric, malic, and citric, in the epidermis, as measured by gas chromatography of trimethylsilyl derivatives, increased. Changes in glucose or sucrose were not found. These analyses provided evidence that organic acid production in the epidermis is associated with stomatal opening. PMID- 16658375 TI - Partial Purification of the NADH-Nitrate Reductase Complex from Chlorella pyrenoidosa. AB - The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO(3) (-) in the NADH-nitrate reductase assay are 10 mum, 2.6 mum, and 0.23 mm, respectively. Heat treatment exerts varying effects on the enzymatic activities associated with the nitrate reductase complex. PMID- 16658376 TI - A Simple Method to Differentiate between alpha- and beta-Amylase. PMID- 16658377 TI - Stomatal penetration by liquids. PMID- 16658378 TI - Circadian Rhythms and the Induction of Flowering in Sinapis alba. PMID- 16658379 TI - Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: I. Kinetic Aspects. AB - Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose dependent beta-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 mum IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate xylose as substrates, and to a lesser extent with guanosine diphosphoglucose dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control. PMID- 16658380 TI - Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: II. Metabolic Requirements. AB - The 2- to 4-fold rise in particle-bound beta-glucan synthetase (uridine diphosphate-glucose: beta-1, 4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca(2+) at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by alpha, alpha' dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor. PMID- 16658381 TI - Rhythmicity in the Basipetal Transport of Indoleacetic Acid through Coleoptiles. AB - (14)C-Indoleacetic acid was applied to coleoptiles of corn (Zea mays) and oat (Avena sativa). The coleoptiles were detached from the endosperms at 6-minute intervals after indoleacetic acid application, and the radioactivity was determined in successive 2-millimeter regions. The rate (per cent per minute) of basipetal transport of indoleacetic acid is periodic in various regions of the coleoptile, with a period of about 20 minutes. The possible relation of this cyclic phenomenon to other rhythmic processes of similar periodicities is discussed. A distinct acropetal transport (against the concentration gradient) from the subapical region to the apical 2-millimeter region of the coleoptile was detected.The velocity of indoleacetic acid transport differs in different regions of the coleoptile. Within an entire coleoptile the velocity can be divided into three classes for corn, 41 millimeters per hour (apical), 13 millimeters per hour (mid), and 34 millimeters per hour (base), and 2 classes for oats, 28 millimeters per hour (apical) and 14 millimeters per hour (remainder). An inverse relationship between the velocity of indoleacetic acid transport, and the growth rate of the coleoptile is discussed. Corn coleoptiles exceed oat coleoptiles both in rate and in velocity of IAA transport. PMID- 16658382 TI - Loss of membrane transport ability in leaf cells and release of protein as a result of osmotic shock. AB - Osmotic shock severely reduces the ability of aged strips of Phaseolus vulgaris leaves to take up alpha-aminoisobutyric acid, an amino acid analogue which is known to be transported by a specific mechanism. Cold osmotic shock, i.e., transfer from 0.5 m sucrose at 25 C to H(2)O at 2 C, decreases alpha aminoisobutyric acid uptake almost to zero. Substitution of 10(-3)m ethylenediaminetetraacetate for the sucrose, i.e., treatment which does not involve plasmolysis, produces a similar, but less severe, effect.About 3.5% of the total cell protein is released as a result of cold osmotic shock, by far the greater proportion being liberated into the water during the second stage of the shock treatment. Ethylenediaminetetraacetate and other shock treatments also bring about protein release, and the amount released is correlated with degree of depression of subsequent alpha-aminoisobutyric acid uptake.Shock tissue is capable of recovering a large proportion of its uptake capacity during subsequent immersion in 10(-4)m CaSO(4).Separate estimation of alpha-aminoisobutyric acid influx and efflux showed that the marked effect of shock on net flux is largely attributable to a reduction in influx, and not to an increase in efflux. This and other results indicate that the shock effect on net flux is not due to nonspecific damage to membranes bringing about "leakiness."The fact that alpha aminoisobutyric acid uptake is reduced to near zero by treatment which allows the cells to retain over 95% of their protein suggests that the shock phenomenon is analogous to that in bacteria, and that the small fraction of protein lost may be closely involved in the transport mechanism. PMID- 16658383 TI - Changes in cellulase and pectinase activities in fruit tissues and separation zones of citrus treated with cycloheximide. AB - Cellulase activity increased in separation-zone tissues 1 day after "Valencia" orange (Citrus sinensis [L.] Osbeck) was treated with 20 micrograms per milliliter cycloheximide. Exocellulase was detected only in the separation zones of treated fruit, whereas endocellulase was present in zones from both treated and control fruit. Endocellulase activity in separation-zone tissue of treated fruit was nearly three times as great as that in control tissues. Cellulase activity was restricted to separation-zone tissue. Pectinase and an albedo macerating factor activity were very low and were not influenced by the treatment. The cycloheximide effect in these experiments was apparently caused by ethylene produced by wound tissue. PMID- 16658384 TI - The hormone content of ripening grape berries and the effects of growth substance treatments. AB - Berries on field-grown Vitis vinifera cv. Doradillo were treated at different times during stage II with benzothiazole-2-oxyacetic acid or 2 chloroethylphosphonic acid, and measurements were made of their growth and hormone content. The concentration of ethylene was low during stage II and declined as berries ripened. Both 2-chloroethylphosphonic acid and benzothiazole 2-oxyacetic acid caused increases in ethylene concentration, yet they had varying effects on ripening: the former applied at the start of stage II and the latter applied 1 week before the end of stage II delayed ripening, while 2 chloroethylphosphonic acid applied at the end of stage II hastened ripening.The abscisic acid content of berries increased as they ripened, and the effects of 2 chloroethylphosphonic acid and benzothiazole-2-oxyacetic acid on abscisic acid levels were correlated with the effect of these compounds on ripening. The roles of abscisic acid and ethylene in the regulation of the ripening of grapes are discussed. PMID- 16658385 TI - Mathematical Analysis of Plant Growth zea mays Primary Roots. AB - A new mathematical treatment is given to the concept of relative elemental growth rate, the limiting value at a point of the relative rate of tissue growth. The new point of view requires consideration of a coordinate system in which each point on the root surface is defined by its initial position on the root axis. The resultant technique of plotting growth parameters provides the ability to distinguish between waves of growth propagating toward the root tip away from cells and waves which tend to propagate along with the cells. Thus it is possible to model the plant growth-regulatory process in terms of auxin and other regulators, considering both cell-associated maxima of regulator concentration and maxima which propagate through the tissue. PMID- 16658386 TI - Embryoless Wheat Grain: A Natural System for the Study of Gibberellin-induced Enzyme Formation. AB - Yorkstar wheat, grown in New York State, has a high percentage (10-11) of grains without embryos. The embryoless grains have viable aleurone layers and show no sign of injury. These grains are able to support alpha-amylase synthesis only in the presence of gibberellin A(3) (GA(3)). In the absence of GA(3) some protein synthesis occurs in embryoless grains during the early hours of soaking, indicating that such activity occurs prior to and independent of GA(3) induction of alpha-amylase. The level of beta-amylase on a dry weight basis is the same in embryoless and normal grains and decreases with time of soaking. In the presence of GA(3), beta-amylase decreases at a slower rate. Isoenzymes of alpha-amylase from GA(3)-treated embryoless and normal grains show quantitative as well as qualitative differences. Cycloheximide (60 mug/ml) completely inhibits the synthesis of alpha-amylase by embryoless grains. Of the RNA synthesis inhibitors, actinomycin D (60 mug/ml) was ineffective while 6-methylpurine (60 mug/ml) gave 65% inhibition without decreasing the number of isoenzymes. PMID- 16658387 TI - Morphogenesis in selaginella: auxin transport in the stem. AB - Selaginella willdenovii Baker is a prostrate vascular cryptogam with a dorsiventral stem. At each major branching of the stem apex a dorsal and a ventral angle meristem is formed. The ventral meristem becomes determined as a root, and the dorsal meristem as a shoot. The present investigation examined the distribution and transport of (14)C-indoleacetic acid through stem tissues as a basis for the pattern of meristem determination. Externally applied indoleacetic acid is transported into receiver blocks with a velocity of 12 millimeters per hour. Much of the auxin becomes immobilized in the tissue and is not transported. The polar ratio of auxin transport is approximately 2. Auxin is transported equally on the dorsal and the ventral sides of the stem axis, and the auxin flux in vascular tissue is twice that in the cortex. In the branch junctions twice as much auxin is transported on the dorsal side as on the ventral side, and this is held to be the consequence of the lateral branch vascular tissue connecting with the dorsal and median, but not with the ventral vascular strand of the stem axis. PMID- 16658388 TI - Chloroplast maintenance and partial differentiation in vitro. AB - Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These cofactors were previously shown to be essential for protochlorophyll and chlorophyll biosynthesis. Ultrastructural changes were monitored by electron microscopy. The following observations were made. (a) Crude homogenates contained agents which degraded etioplasts and developing chloroplasts. (b) Added cofactors were essential for the maintenance of the membrane structure; they were also implicated in the transformation of the prolamellar body in the absence and presence of light. (c) Light pretreatment of the cotyledons improved the maintenance of the developing chloroplast membranes during subsequent in vitro incubation. (d) In the presence of the cofactors, grana formation appeared to take place in the absence of nuclear-cytoplasmic control. PMID- 16658389 TI - Biosynthesis and accumulation of microgram quantities of chlorophyll by developing chloroplasts in vitro. AB - Developing chloroplasts were incubated under conditions previously shown to induce protochlorophyll and chlorophyll biosynthesis, as well as chloroplast maintenance and partial differentiation in vitro. In the presence of air, delta aminolevulinic acid, coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate, microgram quantities of chlorophyll accumulated after 1 hour of incubation. Part of the chlorophyll was not extractable in organic solvents; it is referred to as bound chlorophyll. The amount of bound chlorophyll depended on the degree of cotyledon greening at the time of plastid isolation. Etioplasts with or without a lag phase of chlorophyll biosynthesis synthesized nonphototransformable protochlorophyll and smaller amounts of extractable chlorophyll. As the greening of excised cotyledons progressed, more of the chlorophyll became bound before and after in vitro incubation. It is suggested that this increase in the fraction of bound chlorophyll reflects the biosynthesis of membrane-bound chlorophyll receptor sites. In the absence of cofactors, chlorophyll biosynthesis was blocked and porphyrins accumulated, indicating damage of the chlorophyll biosynthetic chain. It is concluded that chlorophyll accumulation constitutes a potentially convenient tool for the study of thylakoid membrane biogenesis in vitro. PMID- 16658390 TI - Anion transporters in plant mitochondria. AB - The swelling of potato (Solanum tuberosum L.) mitochondria in isosmotic ammonium salts of phosphate, chloride, malate, succinate, and citrate was investigated by measuring light scattering. Potato mitochondria swell spontaneously in ammonium phosphate, and this swelling can be inhibited in N-ethylmaleimide. They swell in ammonium malate or succinate only after the addition of inorganic phosphate and in ammonium citrate only after the addition of both phosphate and a dicarboxylic acid. Pentylmalonate inhibits swelling in ammonium citrate solutions by competing for dicarboxylate entry. The results indicate that potato mitochondria possess a phosphate-hydroxyl carrier, a dicarboxylate carrier, and a tricarboxylate carrier. PMID- 16658391 TI - Light-induced Changes in the Pattern of Protein Synthesis during the Early Stages of Greening of Etiolated Maize Leaves. AB - The effect of light on protein synthesis during the early stages of greening of etiolated maize (Zea mays) leaves was studied using double labeling with leucine and fractionation of proteins by gel filtration and acrylamide gel electrophoresis. The incorporation of labeled leucine into a relatively small number of plastid proteins is effected within the first 30 to 60 minutes of illumination. These proteins do not accumulate with time. When illumination is prolonged, additional proteins are effected.Experiments using inhibitors of protein synthesis suggest that at least some of the proteins effected by 1 hour of illumination might be synthesized in the cytoplasm and not in plastids. Actinomycin D inhibits the incorporation of labeled leucine into some of the protein fractions, but enhances the incorporation into other fractions far above the effect exerted by light. PMID- 16658392 TI - The genes for cytoplasmic ribosomal ribonucleic Acid in higher plants. AB - The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 x 10(6) molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction. PMID- 16658393 TI - Further studies on the photosynthesis of carrot tissue cultures. AB - The influence of kinetin and sucrose on the photosynthetic activity of carrot (Daucus carota) tissue cultures in relation to growth was investigated. The results showed that light contributes heavily to the growth of tissue cultures measured in terms of fresh and dry weight and cell division activity. In light, the fresh weight, dry weight, and number of cells per explant were about or more than doubled. This indicated that after the development of chloroplasts, carrot tissue cultures can grow autotrophically at least as far as energy and carbon are concerned. Kinetin was shown to have an important role in developing the photosynthetic apparatus and photosynthetic activity of tissue cultures as manifested by the increase of chlorophyll content (60%), Hill activity (about 3 fold), and (14)C-fixation from NaH(14)CO(3) (about 20%). On the other hand, the presence of sucrose in the medium reduced the chlorophyll content by about 30% and (14)C-fixation from NaH(14)CO(3) in the soluble fraction by about 60%. A possible correlation between the influence of kinetin on sugar uptake and the effect of kinetin on (14)C-fixation from NaH(14)CO(3) was discussed. PMID- 16658394 TI - Probing a Membrane Matrix Regulating Hormone Action: II. The Kinetics of Lipid Induced Growth and Ethylene Production. AB - Lipids which are active oleanimins, i.e., those which stimulate respiration and auxin-induced cell elongation of pea stem sections, also initiate a period of ethylene formation in them after a lag period of at least 1 hour. Production of ethylene requires auxin, is inhibited by cycloheximide and dinitrophenol applied during or before the lag period, and is greatly stimulated by lipids longer than 20 Angstroms in length such as heptadecyl-benzene, chloro- or iodohexadecane, triolein, and vitamins E and K(1), but not by the shorter chloro- and iododecane. beta-Stigmasterol at 10 to 40 mum concentrations depresses both oleanimin-induced growth and ethylene formation.The effect of oleanimins on the growth rate steadily declines and disappears after 6 hours, whereas oleanimin induction of ethylene stays at a high level until it rapidly disappears after 6 hours. Nongrowing second internode sections also produce ethylene on oleanimin treatment, so ethylene formation is not dependent on cell elongation even though it requires auxin. Preincubation with heptadecylbenzene or auxin does not change the delay of an hour or more before ethylene is produced, whereas increases in growth are noted at the earliest measurements. Oleanimins stimulate growth at less than optimal auxin concentration, even as low as 20 nm, where a proportional ethylene formation is not noted. It is concluded that ethylene formation is not causally related to growth in these tissues.The decline in oleanimin-induced ethylene formation is not changed by renewal of the incubation medium, and sucrose which is required to maintain growth for 20 hours does not influence growth or ethylene formation up to 6 hours. l-Methionine increases ethylene formed after heptadecylbenzene treatment, but unexpectedly, malonate is much more effective.Auxin concentrations supraoptimal for growth cause no growth rate reductions for the first 10 hours, but they greatly enhance oleanimin-induced ethylene formation even when heptadecylbenzene is added after 6 hours. Applied ethylene even at concentrations much above those produced by the tissue itself fails to stimulate or inhibit short term pea stem section growth. It is concluded that the effect of oleanmins on growth is not mediated by ethylene. The similarities in concentration and molecular dimensions of these structurally diverse lipids which simultaneously stimulate respiration, growth, and ethylene formation, suggest a single site of action located in a regulatory membrane. PMID- 16658395 TI - "Survival" of mitochondria in vitro: physical and energy parameters. AB - Isolated mitochondria have been maintained active and coupled for 72 hours at 25 C. Survival (retention of respiratory control) is a function of incubation temperature and dependent upon aeration and substrate. ATP does not entirely substitute for substrate, indicating a need for products of active metabolism other than energy. An improvement in respiratory control is often observed during the first several hours of incubation. Sedimentation and resuspension at 24-hour intervals prolonged survival. As revealed by electron microscopy, mitochondria maintained their basic structure during a 72-hour period at 25 C.Survival is a dynamic, energy-requiring process and must be distinguished from so-called "aging" of organelles at ice temperatures. As a manifestation of partial autonomy, survival may prove useful in assessing aspects of mitochondrial function and the mitochondrial-cellular interrelationship. PMID- 16658396 TI - Control of the formation of amylases and proteases in the cotyledons of germinating peas. AB - Protease activity increased in attached cotyledons of germinated peas (Pisum sativum L. cv. Alaska) as the stored proteins declined but did not increase in excised cotyledons incubated for the same length of time. Cotyledons of seeds germinated in the presence of a casein hydrolysate solution developed less protease activity than did those germinated on water. These results suggest that accumulation of amino acids regulates the protease level in the cotyledons of germinating peas.In contrast to protease, alpha- and beta-amylase increased during incubation of excised pea cotyledons. Their increase was inhibited by abscisic acid. Abscisic acid did not inhibit (14)C-leucine incorporation into protein or reduce the respiratory rate in the cotyledons; hence, its effect on amylase formation was not the result of a general inhibition of metabolism. An ether-soluble acid fraction, which would contain any abscisic acid present in the material, inhibited amylase formation more when it was obtained from imbibed seeds than when it was obtained from cotyledons of seeds germinated for 10 days. These and other results suggest that amylase formation in germinating peas is regulated by abscisic acid. PMID- 16658397 TI - The role of light and growth regulators in the opening of the dentaria petiolar hook. AB - The phenomenon of the etiolated hook is not restricted to the hypocotyl of the dicotyledenous plant (e.g., Phaseolus) but appears to serve a similar, adaptive function in the petioles of certain rhizomatous plants. The commonly employed regulants of hypocotyl hook opening were tested for their effect on the petiolar hook of Dentaria diphylla. The hook was found to require both light (red light promoted, far red inhibited) and the intact leaf for opening. The leaf requirement was fully replaced by gibberellic acid (0.04% in lanolin) but only in light; cobalt chloride (0.1-1.0 mm) promoted a partial opening in dark with or without leaf; and coumarin (1 mm), indoleacetic acid (1-4% in lanolin), and ethylene 10 microliter per liter all inhibited opening of hooks with or without lamina. The absolute requirement for light and leaf tissue and the replacement of proximal tissue by GA(3) alone represent marked differences in the physiology of hypocotyl and petiolar hooks. These differences are believed to indicate the necessity for concomitant leaf maturation in petiolar hook opening. PMID- 16658398 TI - Reproductive Biology of Selaginella: I. Determination of Megasporangia by 2 Chloroethylphosphonic Acid, an Ethylene-releasing Compound. AB - Control clumps of Selaginella wallacei Heiron., sprayed with distilled water with Tween 20, produced a high proportion of microsporangia. Similar clumps sprayed with 2-chlorethyl-phosphonic acid, and ethylene-releasing compound (Ethephon), at 7.65 and 76.5 mg/liter produced almost exclusively megasporangia. Treatment of Selaginella pallescens (Presl) Spring with Ethephon at 34 mg/liter caused the production of megasporangia in the microsporangiate files of the strobili. The possibility that ethylene may be involved in the regulation of heterospory in Selaginella is discussed. PMID- 16658399 TI - Chemical composition of tobacco leaves altered by near-ultraviolet and intensity of visible light. AB - Low energies of near-ultraviolet radiation (300-400 nanometers), applied simultaneously with visible radiation to Nicotiana tabacum L. during daily illumination periods, increased levels of chlorogenic acid isomers, total soluble phenolics, alkaloids, and soluble sugars in expanding leaf lamina compared with controls that had near-ultraviolet filtered out. However, total nitrogen concentrations decreased. The responses to near-ultraviolet were interrelated with intensity of visible light. The presence of near-ultraviolet (which accounted for less than 4% of the total light energy) along with visible light resulted in component concentration differences similar to those caused by much greater increases of visible light without near-ultraviolet. PMID- 16658400 TI - Floral stimulus movement in perilla and flower inhibition caused by noninduced leaves. AB - Photoperiodic control of flowering in the short day plant Perilla involves the transmission of a floral stimulus from induced leaves to the shoot apex. We have studied the basipetal movement of this stimulus and of (14)C-labeled assimilates in plants with an induced leaf (donor) grafted into the uppermost internode of a vegetative plant in which the axillary shoots at various nodes along the stem function as receptors.In agreement with earlier autoradiographic studies by Chailakhyan and Butenko (Transl. Fiziol. Rast. 1957, 4:426-438), a quantitative relationship was found between flower formation and the transport of labeled assimilates from the donor leaf into receptor shoots. The presence of noninduced leaves (i.e., leaves maintained on long day) on the receptor plant prevents flowering and assimilate import by shoots in the axils of the long day leaves and by shoots at nodes in the same orthostichy lower down on the stem. All these vegetative shoots import assimilates from the long day leaves.Both the floral stimulus and the inhibitory effects of long day leaves are translocated basipetally over long distances and in specific parts of the vascular system which correspond to specific orthostichies of the stem. Movement in the xylem of the stain safranin indicates that assimilates from the long day leaves and from the donor can be transported in distinct regions of the vascular bundles in the stem. The pattern of assimilate distribution evidently depends on the pattern of vascular anatomy and on the relative contribution by the various sources of assimilates.A light intensity close to that saturating for photosynthesis saturates long day inhibition of flowering. Moreover, under the appropriate experimental conditions, inhibition is not only caused by leaves in long day, but also by those in short day or darkness.Apparently the floral stimulus moves in parallel with the assimilates in the phloem of Perilla, so that the pattern of assimilate distribution from different leaves determines the pattern of movement of the floral stimulus. It is, therefore, concluded that long day inhibition of flowering in Perilla is the result of competition between assimilate streams originating in the donor and long day leaves. A specific transmissible inhibitor is most likely not involved in the control of flowering in Perilla. PMID- 16658401 TI - Indole-3-ethanol Oxidase: Kinetics, Inhibition, and Regulation by Auxins. AB - We report the further characterization of indole-3-ethanol oxidase from cucumber seedlings. The effects of various inhibitors suggest that the enzyme may be a flavoprotein with a metal ion and sulfhydryl groups required for full activity. Indole-3-acetaldehyde, a product of the reaction, inhibits the enzyme. This inhibition is overcome by O(2) but not by indole-3-ethanol, indicating that the kinetic mechanism of the enzyme is a ping-pong Bi-Bi. The enzyme undergoes cooperative interactions with indoleethanol, yielding Hill coefficients as high as 2.96. Gibberellins are without effect on the enzyme, but it is inhibited by several acidic indoles possessing growth-promoting activity and by two synthetic auxins, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid. Increasing concentrations of indoleacetic acid (IAA) brought about a slight reduction in the indoleethanol concentration producing halfmaximal velocity. Increasing levels of indoleethanol decreased the concentration of IAA required for half-maximal inhibition. At low concentrations of indoleethanol, low levels of IAA activated rather than inhibited. The effect of IAA was not overcome at higher levels of indoleethanol. These results may be interpreted as showing that IAA is a noncompetitive inhibitor which binds to that conformation of the enzyme which also binds indoleethanol. The significance of these interactions for the regulation of IAA biosynthesis is discussed. PMID- 16658402 TI - Diurnal variations in photosynthetic products and nitrogen metabolism in expanding leaves. AB - Expanding leaves of Capsicum frutescens L. cv. California Wonder, Cucumis melo L. cv. Hales Best, and Citrus sinensis L. Osbeck cv. Washington Navel showed a marked diurnal periodicity in the incorporation of (14)C from photosynthetically fixed (14)CO(2) into amino acids. Incorporation was virtually nil at the beginning of the photoperiod, reached a maximum in the 6th to 7th hour and decreased during the latter part of the photoperiod. In Capsicum frutescens this was apparently a reflection of the availability of reduced nitrogen controlled by the activity of nitrate reductase in the leaves. This also controlled the periodicity of the incorporation of (14)C into fraction I protein. Possible control mechanisms and the relation of nitrogen metabolism to the periodicity of leaf expansion growth are discussed. PMID- 16658403 TI - Membrane-bound Adenosine Triphosphatase Activities of Oat Roots. AB - Homogenates of oat (Avena sativa cv. Goodfield) roots contained at least five membrane-associated adenosine triphosphatase (ATPase) activities. The membrane bound ATPases were separated on sucrose gradients and distinguished by membrane density, pH optima, sensitivity to monovalent salts, and substrate specificity.A membrane fraction sedimenting at low centrifugal force (13,000g) contained two ATPase activities at pH 9.0. One membrane ATPase was coincident with cytochrome c oxidase activity and had a density of 1.18 grams per cubic centimeter. This membrane system was identified as mitochondria. The other pH 9.0 ATPase in this fraction occurred at a density of 1.16 grams per cubic centimeter. The identity of this membrane is unknown.Three additional ATPases were in a membrane fraction sedimenting at high centrifugal forces (13,000-80,000g). One membrane ATPase coincided with NADH-cytochrome c reductase activity, had a density of about 1.09 grams per cubic centimeter, and was equally active at pH 6.0 and 9.0. A second membrane ATPase of the 13,000 to 80,000g fraction had a density of 1.13 grams per cubic centimeter and was more active at pH 9.0 than at pH 6.0. A third membrane ATPase had greater activity at pH 6.0 than at pH 9.0, and the membrane had an apparent density of 1.17 grams per cubic centimeter on the sucrose gradient. This ATPase was especially sensitive to KCI. The identity of the membranes which contain ATPases is discussed in relation to the distribution of other enzymes on the gradient. PMID- 16658404 TI - The presence of ribulose 1,5-diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans. AB - Ribulose 1,5-diphosphate carboxylase was detected in extracts of germinating castor bean (Ricinus communis var. Hale) endosperms. This is the first report of this enzyme in a nonphotosynthetic (no chlorophyll) plant tissue. Radioactive 3 phosphoglyceric acid has been identified as the principle product resulting from the enzymatic condensation of (14)C-bicarbonate and ribulose-1,5-diP in endosperm extracts. The Km values of bicarbonate and ribulose-1,5-diP for the endosperm carboxylase are 1.14 x 10(-2)m and 7.5 x 10(-5)m, respectively. The carboxylase activity peaks at 4 days in endosperms of castor beans germinated in the dark. The specific activity of the carboxylase at this stage of germination is 4.3 mumoles of 3-phosphoglycerate formed/mg protein.hr. The presence of ribulose-1,5 diP carboxylase and other enzymes of the reductive pentose phosphate pathway show the potential of this pathway in castor bean endosperms. PMID- 16658405 TI - The Phosphorylation of Ribosomal Protein in Lemna minor. AB - Sterile cultures of Lemna minor have been labeled with (32)P(1), and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with (32)P(1), calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome.The phosphorylated protein is found in both polysomes and "derived" monomers and appears to be located in the ribosomal small subunit. Its apparent molecular weight is 42,000. Addition of growth-inhibiting concentrations of abscisic acid does not alter the apparent degree of labeling of this protein in 5 hours, but after 24 hours of treatment the total protein phosphorus was reduced from 0.75 atom of phosphorus per ribosome to 0.36 atom of phosphorus per ribosome. PMID- 16658406 TI - Studies on chlorogenic Acid biosynthesis in sweet potato root tissue in special reference to the isolation of a chlorogenic Acid intermediate. AB - Marked polyphenol production takes place in root tissue of sweet potato, Ipomoea batatas Lam. cv. Norin 1, in response to slicing. A possible intermediate, tentatively termed compound V, of chlorogenic acid biosynthesis was isolated from the root tissue administrated with t-cinnamic acid-2-(14)C. Compound V was proved to be an ester whose acid moiety was t-cinnamic acid, and the hydroxyl group bearing moiety appeared to be a carbohydrate. Compound V was suggested to be the first intermediate after t-cinnamic acid involved in the chlorogenic acid biosynthetic pathway by the following three results. (a) label of t-cinnamic acid 2-(14)C was distributed in compound V first, then transferred to chlorogenic acid and isochlorogenic acid, isomers of dicaffeoylquinic acid; (b) specific radioactivity of compound V increased prior to that of the fraction containing chlorogenic acid and isochlorogenic acids and decreased prior to that of the latter; and (c) label of compound V was efficiently incorporated into chlorogenic acid and isochlorogenic acid. PMID- 16658407 TI - Induction of deoxyribonucleic Acid synthesis in potato tuber tissue by cutting. AB - Incorporation of (32)P-orthophosphate was found in the DNA fraction of aerobically incubated potato discs when examined by methylated albumin kieselguhr column chromatography. The estimation of DNA content of the discs was by a method developed for starchy tissues and showed that the incorporation of (32)P was due to net synthesis of DNA. The DNA content of a disc rapidly increased after a lag period of about 12 hours. The increase continued during the entire test period although at a lower rate during the later period of aging. DNA synthesis was further examined by measuring the rate of incorporation of (3)H-thymidine. The striking similarity which was found between changes in the rate of DNA accumulation and in the rate of (3)H-thymidine incorporation indicates that the incorporation of (3)H-thymidine actually represents the net synthesis of DNA. Although the experiments with microautoradiography revealed that DNA synthesis occurred exclusively in nuclei, no signs of cell division were detected by microscopic observation. DNA synthesis in potato discs was further examined by using inhibitors of protein and RNA synthesis and was sensitive to those inhibitors. The significance of the present results is discussed in relation to the role of wounding in the induction of DNA synthesis. PMID- 16658408 TI - Mechanics of the Compression Wood Response: II. On the Location, Action, and Distribution of Compression Wood Formation. AB - A new method for simulation of cross-sectional growth provided detailed information on the location of normal wood and compression wood increments in two tilted white pine (Pinus strobus L.) leaders. These data were combined with data on stiffness, slope, and curvature changes over a 16-week period to make the mechanical analysis. The location of compression wood changed from the under side to a flank side and then to the upper side of the leader as the geotropic stimulus decreased, owing to compression wood action. Its location shifted back to a flank side when the direction of movement of the leader reversed. A model for this action, based on elongation strains, was developed and predicted the observed curvature changes with elongation strains of 0.3 to 0.5%, or a maximal compressive stress of 60 to 300 kilograms per square centimeter. After tilting, new wood formation was distributed so as to maintain consistent strain levels along the leaders in bending under gravitational loads. The computed effective elastic moduli were about the same for the two leaders throughout the season. PMID- 16658409 TI - Effects of potassium deficiency on the photosynthesis and respiration of leaves of sugar beet. AB - Sugar beet plants (Beta vulgaris L. var. F5855441) were germinated and cultured under standardized environmental conditions for 28 days. Potassium deficiency was then induced by withholding K from the culture solution. Changes in CO(2) and water vapor exchange rates and surface temperatures of individual attached leaves were measured with time after K cut-off, along with changes in the concentrations of the leaf minerals K, Na, Ca, Mg, Fe, Mn, Cu, and Zn. During the 1st week after K cut-off the concentration of Na in the leaf blade increased from 200 to 1000 milliequivalents per kilogram dry matter while K decreased from 1500 to 300 milliequivalents per kilogram. During the subsequent 2 weeks, both Na and K concentrations decreased. The concentrations of other leaf minerals, except Mn, were little affected by K cut-off. Photosynthetic CO(2) uptake per unit area decreased linearly with time after cut-off and attained one-third of the control rate after 21 days. Low K apparently decreased photosynthesis through an increase in mesophyll resistance to CO(2) (r(m)) from 2.8 to 5.3 seconds per centimeter in 21 days. Leaf (mainly stomatal) diffusion resistance (r'(1)) increased only slowly during the first 15 days from 0.3 to 0.5 second per centimeter, eventually reaching 1.6 seconds per centimeter at 21 days. Low K progressively decreased the photorespiratory evolution of CO(2) into CO(2)-free air, but steadily increased the rate of CO(2) evolution in dark. PMID- 16658410 TI - Photosynthetic carbon metabolism in isolated maize bundle sheath strands. AB - Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO(2) per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic (14)CO(2) fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath (14)CO(2) fixation. After 5 minutes of photosynthesis in (14)C-bicarbonate, 95% of the incorporated (14)C was found in compounds other than C(4)-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of (14)C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of "malic" enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP(+)-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize. PMID- 16658411 TI - Metabolic regulation in the senescing tobacco leaf: I. Changes in pattern of p incorporation into leaf disc metabolites. AB - Changes in phosphate metabolism were explored in discs from tobacco (Nicotiana tabacum) leaves of three contrasting types: green leaves which were fully expanded and attached to the plant, leaves which had yellowed following excision and dark starvation, and leaves which had yellowed while attached to the plant. 2,4-Dinitrophenol at 10(-5)m stimulated the respiration rate of discs from green and yellow-detached leaves only slightly, but markedly stimulated that of discs from yellow-attached leaves. Following a 10-minute uptake period the incorporation of (32)P-orthophosphate into phosphate esters and lipids of discs from yellow-detached leaves was resistant to 2,4-dinitrophenol, whereas in discs from green and yellow-attached leaves it was inhibited by 2,4-dinitrophenol. Incorporation into a salt-soluble fraction containing unidentified nucleotide material showed converse behavior in that it was stimulated by 2,4-dinitrophenol in discs from green and yellow-attached leaves; in discs from yellow-detached leaves it was resistant to 2,4-dinitrophenol. In discs from yellow-detached and yellow-attached leaves there was a shift in the labeling pattern of phosphate esters toward increased label in hexose phosphates at the expense of adenine nucleotides, 3-phosphoglycerate, and phosphoenolpyruvate. It is concluded that incorporation into phosphate esters in discs from yellow-detached leaves is by substrate level phosphorylation coupled to enhanced aerobic glycolysis. In discs from yellow-attached leaves, on the other hand, incorporation depends on oxidation phosphorylation, and it is suggested that the shift in labeling pattern is caused by senescence-induced changes in activity of glycolytic enzymes. PMID- 16658412 TI - Metabolic Regulation in the Senescing Tobacco Leaf: II. Changes in Glycolytic Metabolite Levels in the Detached Leaf. AB - Yellowing of detached mature tobacco leaves standing in water in the dark was accompanied by a strong "climacteric rise" in respiration rate. During this period the ATP level and energy charge of the adenylate system also rose. The levels of glycolytic intermediates between glucose 1-phosphate and triose phosphates rose, those between 3-phosphoglycerate and phosphoenolpyruvate fell, and pyruvate rose. On the assumption of a drop in NAD/NADH ratio, as found by other workers in wheat leaves, the reverse crossover between triose phosphates and 3-phospholglycerate was attributed to inhibition of glyceraldehyde 3 phosphate dehydrogenase. The forward crossover between phosphoenolpyruvate and pyruvate was taken to indicate activation of pyruvate kinase, possibly by fructose diphosphate. Secondary large rises in pyruvate and fructose diphosphate occurred well after the climacteric peak had been passed. No evidence was found for participation of phosphofructokinase in metabolic control in the yellowing leaf. Possible limitations to the use of the crossover theorem in the present situation, such as changes in compartmentation and in flux through branch points, are emphasized. PMID- 16658413 TI - Differences in Lipid Composition between Undifferentiated and Mature Maize Chloroplasts. AB - Lipid compositions of undifferentiated maize (Zea mays) chloroplasts, capable of fixing CO(2), were compared with the lipid compositions of mature chloroplasts, which do not fix CO(2), located in both the mesophyll and bundle sheath cells. The major lipids found in all three chloroplast types were the glycolipids, monogalactosyl diglyceride and digalactosyl diglyceride, followed by decreasing amounts of sulfolipid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol, and diphosphatidyl glycerol. Quantitative differences in lipid components were observed among the chloroplast types. The mesophyll and bundle sheath maize chloroplasts differed in their chlorophyll a/chlorophyll b ratios (2.27 and 4.13 respectively) and their content of glycolipid relative to chlorophyll (51.8% glycolipid to 20.9% chlorophyll and 84.5% glycolipid to 10.1% chlorophyll respectively). A comparison between the lipid compositions of maize mesophyll chloroplasts and mesophyll chloroplasts obtained from spinach, sugar beet, and tobacco showed many similarities. PMID- 16658414 TI - Effects of some rare elements on nicotine content of the tobacco plant. AB - Fifty-four rare elements were tested for their effects on the nicotine level of tobacco (Nicotiana tabacum L.) plants grown in solution culture. Be, Cu, Pd, Pt, and Sm definitely increased nicotine yield (over 25%), whereas Bi, Co, Ho, Pb, Ni, Rb, Ag, Tl, Sn, U. V. and Zr definitely decreased nicotine yield. Cs, Er, Li, Rh, Ru, Se, Sr, Ti, and Yb possibly increased (less than 25%) nicotine yield, whereas As, Ce, Cr, Dy, Gd, I, Mo, Nd, Re, Ta, and Th possibly decreased nicotine yield. Other elements including Al, Ge, Au, Hf, In, Ir, La, Lu, Hg, Os, Pr, Sc, Te, Tb, Tm, W, and Zn showed no significant effects. PMID- 16658415 TI - Use of dextran-40 gradients for separation of pea cotyledon mitochondria into different fractions. AB - A crude pea (Pisum sativum L. var. Homesteader) mitochrondrial preparation was divided into two equal parts. One part was layered on a Dextran-40 step gradient, and the other on a sucrose step gradient, and they were centrifuged to obtain different bands of particles. The densities at which the particles banded and the mitochondrial respiratory activities of the particles were determined. Dextran-40 density gradient centrifugation resulted in a better separation of mitochondrial populations than did sucrose density gradient centrifugation. Separation by sucrose density gradient centrifugation may not be according to the true densities of the particles. On the other hand, the use of gradients of Dextran 40, a solute of low osmotic potential, facilitated separation of particles acording to their true densities. Such mitochondria showed better respiratory control ratio and ADP:0 values, than those isolated by sucrose density gradient centrifugation. PMID- 16658416 TI - Failure of maleic hydrazide to act as a sulfhydryl or carbonyl reagent. PMID- 16658417 TI - Modified photoelectric device for recording leaf movements. PMID- 16658418 TI - Change of leaf dimensions and air volume with change in water content. PMID- 16658419 TI - Nitrate Reductase Activity and Polyribosomal Content of Corn (Zea mays L.) Having Low Leaf Water Potentials. AB - Desiccation of 8- to 13-day-old seedlings, achieved by withholding nutrient solution from the vermiculite root medium, caused a reduction in nitrate reductase activity of the leaf tissue. Activity declined when leaf water potentials decreased below -2 bars and was 25% of the control at a leaf water potential of -13 bars. Experiments were conducted to determine whether the decrease in nitrate reductase activity was due to reduced levels of nitrate in the tissue, direct inactivation of the enzyme by low leaf water potentials, or to changes in rates of synthesis or decay of the enzyme.Although tissue nitrate content decreased with the onset of desiccation, it did not continue to decline with tissue desiccation and loss of enzyme activity. Nitrate reductase activity recovered when the plants were rewatered with nitrate-free medium, suggesting that the nitrate in the plant was adequate for high nitrate reductase activity. The rate of decay of nitrate reductase activity from desiccated tissue was essentially identical to that of the control, in vivo or in vitro, regardless of the rapidity of desiccation of the tissue. Direct inactivation of the enzyme by the low water potentials was not detected. Polyribosomal content of the tissue declined with the decrease in water potential, prior to the decline in nitrate reductase activity. Changes in ribosomal profiles occurred during desiccation, regardless of whether the tissue had been excised or not and whether desiccation was rapid or slow. Reduction in polyribosomal content did not appear to be associated with changes in ribonuclease activity. Nitrate reductase activity and the polyribosomal content of the tissue recovered upon rewatering, following the recovery in water potential. The increase in polyribosomal content preceded the increase in nitrate reductase activity. Recovery of enzyme activity was prevented by cycloheximide.Based on these results, it appears that nitrate reductase activity was affected primarily by a decrease in the rate of enzyme synthesis at low leaf water potentials. PMID- 16658420 TI - The role of cyclic photophosphorylation in vivo. AB - When cyclic photophosphorylation is inhibited in Chlorella vulgaris cells by carbonylcyanide-trifluoromethoxy phenylhy-drazone, photosynthetic CO(2)-fixation under anaerobic conditions exhibits a distinct lag. Under the same conditions, the light-dependent formation of ribulose diphosphate shows also this lag. It is concluded that cyclic photophosphorylation is required to fill up the pools of phosphorylated intermediates of the Calvin cycle at a time when noncyclic photophosphorylation cannot yet efficiently operate. Under aerobic conditions, the initial energy demand can be accommodated by respiratory ATP or cyclic photophosphorylation or both. Evidence for stoichiometric participation of cyclic photophosphorylation in photosynthesis is still lacking. PMID- 16658421 TI - Vanadium Uptake by Plants: Absorption Kinetics and the Effects of pH, Metabolic Inhibitors, and Other Anions and Cations. AB - The kinetics of vanadium absorption by excised barley (Hordeum vulgare L., cv. Eire) roots were investigated with respect to ionic species of V in solution, time and concentration dependence, Ca sensitivity, and interaction with various anions, cations, and pH levels. The role of metabolism in V absorption was also studied using anaerobic treatment (N(2) gas) and chemical inhibitors (NaN(3), KCN, or 2,4-dinitrophenol). Approximately one-third of the labeled V initially taken up by excised roots was desorbed to a constant level after 45 min in unlabeled V solutions. The rate of absorption of labeled V from 5 mum NH(4)VO(3) solutions containing 0.5 mm CaSO(4) was constant for at least 3 hours. Omission of Ca resulted in a 72% reduction in V uptake when compared to controls with 0.5 mm CaSO(4). The rate of uptake of V was highest at pH 4 but dropped to a very low level at pH 10. It was relatively constant between the pH levels of 5 and 8 at which the VO(3) (-) ion is the predominant ionic species in solution. The rate of absorption of V was followed as a function of concentrations from 0.5 to 100 mum NH(4)VO(3). It was found to be a linear function of concentration and did not follow saturation kinetics. Absorption experiments carried out with labeled V from either N(a)VO(3) or NH(4)VO(3) sources gave similar results. No anion studied (i.e. HPO(4) (2-), HAsO(4) (2-), MoO(4) (2-), SeO(4) (2-), SeO(3) (2-), CrO(4) (2-), BO(3) (3-), No(3) (-), and Cl(-)) interfered appreciably (i.e. less than 30% inhibition) with the absorption of labeled V. Anaerobic treatment of absorption solution with N(2) gas did not inhibit V absorption by excised roots. The results obtained using chemical inhibitors were not consistent. It was concluded that V is not actively absorbed by excised barley roots. PMID- 16658422 TI - Biochemical Studies on Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination: Effects of Antibiotics on the Development. AB - l-Leucine-U-(14)C was incorporated into mitochondrial protein in pea (Pisum sativum var. Alaska) cotyledons during the imbibing stages. Incorporation was almost completely inhibited by cycloheximide but not by chloramphenicol. Both antibiotics did not affect increases in mitochondrial activities and components of the cotyledons during imbibition. Therefore, mitochondrial development seems to be achieved by a transfer of protein pre-existing in the cytoplasm into the mitochondria rather than by de novo synthesis of mitochondrial protein. Cycloheximide stimulated an increase in bile saltsoluble protein of mitochondria in imbibing pea cotyledons. The recovery of cytochrome oxidase activity after sucrose density gradient centrifugation was enhanced, and the morphological properties of mitochondria were altered by cycloheximide. PMID- 16658423 TI - Changes in Amount of Polyphenols and Activity of Related Enzymes during Growth of Tobacco Flower and Capsule. AB - Developmental stages of tobacco (Nicotiana tabacum L. cv. Burley 21) flower and capsule were correlated with tissue contents of polyphenols and activities of phenylalanine ammonialyase, polyphenoloxidase, and peroxidase. Chlorogenic acid, scopolin, and scopoletin were present in most tissues, whereas rutin and two dihydroxyphenolic glycosides concentrated primarily in the corolla and placenta, respectively. Ovules contained only chlorogenic acid. As development progressed, polyphenols accounted for nearly 15% of the dry weight in the green capsule of field-grown plants. Fertilization triggered a rapid increase of chlorogenic acid in the ovary. When l-phenylalanine-U-(14)C was fed to the detached green capsules and capsule parts, an incorporation of radioactivity into chlorogenic acid and dihydroxyphenolic glycosides occurred which suggested in situ synthesis of these compounds. This was subtantiated by a positive correlation between phenylalanine ammonia-lyase activity and polyphenol accumulation. High polyphenoloxidase activity was associated mainly with the ovary, whereas peroxidase activity was maximal during senescence of all tissues. Polyacrylamide gel slab electrophoresis revealed five cathodic bands and one diffuse zone with poly-phenoloxidase activity in flower extracts. Two anodic poly-phenoloxidase isozymes appeared only in the fertilized ovary. Among 17 peroxidase isozymes, six cathodic forms were present throughout floral development, and the anodic ones increased in number and activity at the later stages of capsule growth. PMID- 16658424 TI - A role for calcium in auxin transport. AB - The basipetal transport of the auxin, indoleacetic acid, in sunflower stem sections is markedly suppressed by washing the tissue in ethylenediaminetetraacetate, and transport is restored by subsequent application of calcium solutions. The ethylenediaminetetraacetate treatment is shown to result in the removal of substantial amounts of calcium from the tissue, and the restoration of transport is distinctive for calcium solutions, lesser effects being observed for magnesium and lanthanum, and little effect for monovalent cations. The calcium effects are interpreted as indicating that the auxin transport system depends upon structural or functional features of cellular membranes which involve calcium in a manner analogous to the transport of inorganic ions. PMID- 16658425 TI - Inhibition of abscission by calcium. AB - An inhibition of abscission in bean petiole explants is reported for additions of calcium salts, especially at concentrations between 10(-3) and 10(-1)m. Magnesium is less effective, and other commmon macronutrients are ineffective in inhibiting abscission. Evidence from timing experiments indicates that the calcium inhibition may act on the stage I or the "juvenile" stage of the explant, and that the inhibition of abscission may result from a retardation of senescence development in the pulvinar tissues of treated explants. PMID- 16658426 TI - Regulation of Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase in Germinated Spores of Geotrichum candidum. AB - Germinating spores of Geotrichum candidum produce only a nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase. Synthesis of glutamate dehydrogenase was repressed by the presence of ammonia, whereas urea, glutamate, or glutamine were ineffective. The enzyme was not subject to catabolite repression and was localized in the cell sap fraction. The glutamate dehydrogenase has been purified 93-fold and showed maximal activity at pH 8.2 in the forward and reverse directions. When measuring the initial reaction rate at pH 7.2, a variety of tricarboxylic acid cycle intermediates displayed additive and unidirectional activation of the reductive amination reaction and inhibition of the oxidative deamination reaction. The modulating effects were pH-dependent and diminished at alkaline pH values. Substrate inhibition exerted by alpha ketoglutarate was strongest at neutral pH.Rate-concentration plot with respect to alpha-ketoglutarate at pH 7.2 was sigmoidal, and the enzyme was entirely inactive at substrate concentrations lower than 1 mm. In the presence of fumarate, the enzyme did not exhibit the cooperative interaction described above and was markedly activated at alpha-ketoglutarate concentrations lower than 1 mm. The tricarboxylic acid cycle intermediates released the enzyme from substrate inhibition by alpha-ketoglutarate. Double reciprocal rate-concentration plots displayed noncompetitive inhibition between succinate and NADP and a mixed noncompetitive and competitive inhibition between succinate and glutamate. The binding order of substrates in the reductive amination reaction was NADPH, alpha ketoglutarate, and subsequently ammonia. PMID- 16658427 TI - Rehydration versus Growth-induced Water Uptake in Plant Tissues. AB - Experiments show that the rate of water uptake by living tissues external to mature xylem of cotton stems (Gossypium hirsutum L. Auburn 7-683) is very similar to the corresponding curves for leaf tissue. In both cases one obtains a two phase curve with phase I corresponding to passive rehydration and phase II pertaining to active growth.A theory of water movement in plant tissue first proposed by Philip allows one to make a more rigorous distinction than made previously between phase I and phase II. This theory is applied explicitly to water uptake by leaf disks and results in a simple expression for the time required for phase I completion. Because the time required varies as the square of the disk radius, it is essential to use a standad disk size in water uptake studies of a particular tissue.Additional analysis indicates that clear temporal distinction cannot be made between phase I and phase II. Different portions of the leaf disk rehydrate at significantly different rates, resulting in a grey zone with phase I and phase II occurring simultaneously in different parts of the disk. PMID- 16658428 TI - Isocitrate lyase in green leaves. AB - Isocitrate lyase (EC 4.1.3.1) has been demonstrated in crude dialyzed extracts of healthy spinach (Spinacia oleracea) leaves from commercial sources and wheat (Triticum aestivum) and maize (Zea mays) leaves stored in darkness in the cold room for 1 week. The products of the reaction were identified as glyoxylate and succinate, the former by its phenylhydrazone, and the latter traced by isotopic labeling and cochromatography. Fresh spinach extracts contain a mixture of at least two endogenous inhibitors of isocitrate lyase activity and one of them is proteinaceous. The endogenous inhibitor(s) is thermostable and retains 50% of its inhibitory effect even after boiling for 10 minutes. Dark starvation of the leaves removes the inhibition, due possibly to autolysis of the inhibitor(s). The inhibitor(s) can also be removed by filtration through Sephadex gels. The crude extract from spinach shows double pH optima in phosphate buffer at pH 7.4 and pH 8.0. The apparent Km at pH 7.4 was 0.1 mm. Oxaloacetate, dl-malate, succinate, 3 phosphoglycerate, and glycolate at 10 mm concentration inhibited, but ribulose 1,5-diphosphate activated enzymic activity. PMID- 16658429 TI - Effects of Kinetin on the Permeability of Allium cepa Cells. AB - Permeability changes of Allium cepa cells were studied by a plasmometric method. Onion epidermis was floated in phosphate buffer solution with kinetin (2.5 milligrams/liter), or buffer without kinetin as a control, for 10 hours. The treated and control tissues were then transferred to one of the following four permeants: thiourea, urea, maloamide, dimethylurea.Kinetin treatment increased the permeability of onion epidermal cells to thiourea and urea. The kinetin effect on permeability to malonamide and dimethylurea was not significant. It is suggested that kinetin might affect the protein component of the cell membrane. PMID- 16658430 TI - Diurnal trends in net photosynthetic rate and carbohydrate levels of soybean leaves. AB - A study was made of diurnal trends in net photosynthetic rate and carbohydrate levels of unifoliolate leaves of soybean (Glycine max L. Merrill) under constant environmental conditions (50,000-lux light intensity, 24.5 C air temperature, 60% relative humidity, and 300 microliters of CO(2) per liter of air).Net photosynthetic rate remained relatively constant between 4 and 10 hours after the lights were turned on but then gradually declined to 85% of this rate by the end of the 16-hour photoperiod. The decline in net photosynthetic rate was due to increases in both stomatal diffusion resistance and residual resistance to CO(2).The decline in net photosynthetic rate began when the rate of starch accumulation began to decline rapidly. At this time, there also appeared to be an increase in soluble carbohydrate level. The results suggest that when a high starch level was reached, further starch synthesis was impaired, leading to an increase in soluble carbohydrate level and, consequently, a reduction in net photosynthetic rate. PMID- 16658431 TI - The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva. AB - Dunaliella parva, a green halophilic alga, was found to accumulate very large amounts of intracellular glycerol. Through measurements of the intracellular volume the internal concentration of glycerol was calculated and found to be around 2.1 m in cells cultured in 1.5 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular glycerol varied accordingly, reaching its new osmotic equilibrium after about 90 minutes. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. The above results indicate the existence of a new type of algal osmoregulation, in which the osmotic balance depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. PMID- 16658432 TI - Increase in Free and Bound Abscisic Acid during Natural and Ethylene-induced Senescence of Citrus Fruit Peel. AB - Free and bound abscisic acid and neutral growth inhibitors were detected in citrus fruit peel using two bioassays and gasliquid chromatography. None of the inhibitor components seems to be directly associated with chloroplasts or chromoplasts in citrus fruit peel.Green, nonsenescent fruits contain mostly neutral inhibitors and relatively low amounts of free and bound abscisic acid. Upon harvest and storage in ethylene, 50 microliters per liter, both free and bound abscisic acid accumulate rapidly, attaining within 24 hours a level of 1 microgram per gram. After 48 hours bound abscisic acid reaches a much higher level than free abscisic acid. Fruits allowed to senesce on the tree follow a similar course of abscisic acid accumulation, attaining finally a 10: 1 ratio of bound to free abscisic acid. PMID- 16658433 TI - Mechanism of Chilling Injury in Sweet Potato: X. Change in Lipid-Protein Interaction in Mitochondria from Cold-stored Tissue. AB - Seventy per cent of the phospholipid in mitochondria from sweet potato roots was removed by aqueous acetone treatment. The amount of phospholipid that could be rebound to these lipid-depleted mitochondria roughly corresponded to the amount of phospholipid in untreated mitochondria. The activities of NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, cytochrome oxidase, and succinoxidase in lipid-depleted mitochondria were restored by addition of mitochondrial phospholipid to about 60, 50, 15, and 35%, respectively, in comparison to untreated mitochondria. The capacity of lipid-depleted mitochondria from 14-day cold-stored tissue to bind mitochondrial phospholipid from healthy tissue was lower than that from healthy tissue. However, there was no large difference in activities of NADH-cytochrome c oxidoreductase and succinate cytochrome c oxidoreductase between both phospholipid rebound lipid-depleted mitochondria from healthy and 14-day cold-stored tissues. On the other hand, activity of succinoxidase in phospholipid rebound lipid-depleted mitochondria from 14-day cold-stored tissue was decreased by about 50% of that from healthy tissue. Furthermore, the capacity of lipid-depleted mitochondria from 2-day cold stored tissue to bind mitochondrial phospholipid from healthy tissue was higher than that from healthy tissue. PMID- 16658434 TI - The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris). AB - The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.The present paper describes the structure of a xyloglucan present in the walls and in the extracellular medium of suspension-cultured Red Kidney bean (Phaseolus vulgaris) cells and compares the structure of the bean xyloglucan with the structure of the sycamore xyloglucan. Although some minor differences were found, the basic structure of the xyloglucans in the cell walls of these distantly related species is the same. The structure is based on a repeating heptasaccharide unit which consists of four residues of beta-1, 4-linked glucose and three residues of terminal xylose linked to the 6 position of three of the glucosyl residues. PMID- 16658435 TI - The Metabolism of Hormones during Seed Germination and Release from Dormancy: III. The Effects and Metabolism of Zeatin in Dormant and Nondormant Ash Embryos. AB - Zeatin and zeatin-9, beta-ribonucleoside enhance the germination of dormant ash embryos. While the first macroscopic signs of germination appear only after about 72 hours, 12 hours of exposure to 50 mum zeatin is as effective as continuous incubation. There must be barriers against transport out of the embryos since 8 (14)C-zeatin and its metabolites, zeatin-9, beta-ribonucleoside, the 5'-mono and the suspected di- and triphosphates, accumulate against a concentration gradient. Zeatin ribonucleoside is about as effective as zeatin in enhancing embryo germination, yet the internal 8-(14)C-zeatin level is lower by a factor of about 50 when the ribonucleoside is fed. The physiological effects of zeatin and abscisic acid on the germination of ash embryos are antagonistic. There is, however, no evidence that abscisic acid has a significant effect on 8-(14)C zeatin uptake or conversions. PMID- 16658436 TI - Response of Nicotiana mesophyll protoplasts of normal and tumorous origin to indoleacetic Acid in vitro. AB - Enzymatically isolated mesophyll protoplasts of the two normal, nontumor-forming parent species Nicotiana glauca and N. langsdorffii and two of their tumor-prone interspecific hybrids were maintained in a 0.5 m mannitol solution supplemented with various concentrations of auxin (indoleacetic acid) and the growth inhibitor abscisic acid. The bursting response of protoplasts in medium containing indoleacetic acid in physiological concentrations showed that protoplasts from the tumorous hybrids tolerate auxin in up to 30 times higher concentrations than protoplasts from parent plants. The "survival" of all protoplast preparations in comparable abscisic acid containing media was significantly greater than that in the indoleacetic acid supplemented solutions. Protoplasts in vitro respond with bursting only after the external indoleacetic acid concentrations reach levels comparable to those of endogenous auxins present in these cells. The data are discussed in conjunction with previous observations on uptake and maintenance of indoleacetic acid levels in tumorous Nicotiana tissues. PMID- 16658437 TI - Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes. AB - Three DNA buoyant density species (nuclear, 1.692 g cm(-3); mitochondria 1.705 g cm(-3); and proplastid, 1.713 g cm(-3)) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm(-3) species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis. PMID- 16658438 TI - Mechanism of naphthaleneacetic Acid conjugation: no effect of ethylene. AB - Formation of naphthaleneacetic acid-glucose (NAGLu) in detached leaves, floating on alpha-naphthaleneacetic acid-1-(14)C (NAA, 0.05 microcurie per milliliter, 3.1 mum)-buffer solution (phosphate-citrate, pH 4.2) began immediately while there was a 2- to 4-hour lag before NAA-asparatate (NAAsp) could be detected. Subsequent increase in the NAAsp conjugate reflected a decrease in free NAA to 1 to 2% of the total radioactivity taken up. Pretreatment with 31 mum(12)C-NAA for 18 hours doubled NAAsp formation after transfer for 4 hours to (14)C-NAA. Pretreatment with ethylene, as ethephon (up to 400 milligrams per liter) or ethylene gas (10 microliters per liter), did not induce NAAsp formation. In the presence of NAA, ethylene had no effect on NAA conjugation. Similarly, CO(2) (5%) did not modify the formation of the conjugates. Rhizobitoxine (1.87 mum) inhibited NAA-induced ethylene production but did not prevent NAA-induced formation of NAAsp. We concluded that the conjugation of NAA with aspartic acid is not mediated by ethylene. PMID- 16658439 TI - The osmotic potential of polyethylene glycol 6000. AB - Osmotic potential (psi(s)) of aqueous solutions of polyethylene glycol 6000 (PEG 6000) was curvilinearly related to concentration. At given concentrations, psi(s) increased linearly with temperature. The effects of concentration and temperature on psi(s) of PEG-6000 solutions differ from those for most salts and sugars and apparently are related to structural changes in the PEG polymer. Measurements of psi(s) with thermocouple psychrometers are more negative than those with a vapor pressure osmometer, with the psychrometer probably giving the more nearly correct psi(s) for bulk solutions. An empirical equation permits calculation of psi(s) from known concentrations of PEG-6000 over a temperature range of 15 to 35 C. Viscometery and gravimetric analysis are convenient methods by which the concentrations of PEG-6000 solutions may be measured. PMID- 16658440 TI - Purification of oat and rye phytochrome. AB - A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A(280) nm/A(665) nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants. PMID- 16658441 TI - Partial characterization of oat and rye phytochrome. AB - Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (sigma(200)) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a sigma(200) value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein. PMID- 16658442 TI - Immunochemistry of phytochrome. AB - Rabbit antibody was elicited against purified oat phytochrome polypeptides. Immunodiffusion and immunoelectrophoresis indicated the antibody elicited was predominantly a single precipitin system. No antigenic difference was detected between red-absorbing phytochrome and far red-absorbing phytochrome. Crude preparations of rye and corn phytochrome showed a line of identity when cross reacted with oat polypeptide phytochrome; pea phytochrome showed a line of partial identity. Precipitin reactions with purified rye phytochrome analyzed with sucrose density gradient centrifugation and immunodiffusion confirmed that the same class of determinants was available to the antibody when the protein was known to be in a state which had not undergone extensive proteolytic attack. PMID- 16658443 TI - Inhibition of Low pH-induced Elongation in Avena Coleoptiles by Abscisic Acid. AB - An angular position-sensing transducer was used to make continuous measurements of acid-induced elongation of Avena sativa coleoptile segments. Elongation rates at pH 4.5 (5 mm succinate buffer) were about 5-fold greater than those at pH 6.0. Buffered 0.1 mm abscisic acid produced a partial decrease of the growth rate. Pretreatments with abscisic acid buffered at pH 6.0 usually caused a further reduction of the elongation response when the coleoptile segments were subsequently placed in buffer at pH 4.5 containing abscisic acid. Abscisic acid did not completely prevent the pH effect in any of these experiments, and the brief latent period of the pH response was not affected by abscisic acid treatments. At pH 4.5, where the inhibitory effect of ABA was maximum, low pH induced elongation was also inhibited by KCN and HgCl(2). These results suggest that pH-(4.5) induced elongation in this system may be dependent on some metabolic processes and that abscisic acid-induced inhibition of this elongation may involve an interaction with these processes. PMID- 16658444 TI - Ethylene, a regulator of young fruit abscission. AB - In an earlier study we reported that detached cotton flowers produced sufficient ethylene before the period of natural abscission to suggest that ethylene might be a natural regulator of young fruit abscission. The present report explores this probability further. Intact cotton (Gossypium hirsutum L.) fruits produced ethylene at rates as high as 36 mul ethylene/kg fresh wt.hr during the 2 days before they abscised. Direct measurements of ethylene in gas samples withdrawn from fruits indicated that production of 1 mul ethylene/kg fresh wt.hr is equivalent to an internal concentration of approximately 0.1 mul/l. Fumigation of fruiting cotton plants with only 0.5 mul/l caused 100% abscission of young fruits and floral buds within 2 days. This correlated with the estimated endogenous levels of ethylene. Reduced pressure, which reduced the internal levels of ethylene, delayed abscission of young fruits and leaves, a result which supports our conclusion from this study- that ethylene is one of the regulators of young fruit abscission in cotton. PMID- 16658445 TI - Effects of Chronic Internal beta-Radiation from Photoassimilated CO(2) on the Retention and Distribution of C in Young White Pine Plants. AB - Eastern white pines (Pinus strobus L.) in their 3rd year of growth photoassimilated 35, 110, 220, or 400 microcuries of (14)CO(2) on a single occasion in the spring when needle expansion was occurring. Once assimilated into organic products and translocated to various sinks, the retained (14)C represented an internal chronic source of ionizing radiation to the plant. About 2.5 months later, the pines were harvested, and the distribution of (14)C activity was determined. In addition, new needle lengths, fresh weights, and rates of processes involving CO(2) exchange were measured.All parameters measured were affected in those pines which initially assimilated 220 or 400 microcuries of (14)CO(2), whereas no significant differences were observed between control plants and those initially assimilating 35 microcuries. Plants incorporating 110 microcuries were intermediate in their responses. The percentage of assimilated (14)C retained by the plants decreased as the initial activity of assimilated (14)C increased, with the losses of (14)C occurring through both respiration and the shedding of needles. The pines which assimilated 35 microcuries of (14)CO(2) retained about 70% of the (14)C; those which assimilated 400 microcuries retained only about 28%. The distribution of the (14)C activity recovered from all the plants was essentially the same; about 65% of the total (14)C recovered was in the new needles and 13% in the roots. PMID- 16658446 TI - Hexokinase from maize endosperm and scutellum. AB - Hexokinase (EC 2.7.1.1) was isolated from endosperm and scutellum of developing and germinating maize (Zea mays) seeds. With fructose as the variable substate, Michaelis constant values for the scutellum enzyme were about onethird those of the endosperm enzyme (0.05 versus 0.15 mm), and no developmental differences were observed. With glucose as the variable substrate, Michaelis constant values were all in the range 0.1 to 0.2 mm. The enzyme preparation from germinating scutellum was studied further; when glucose was varied over a wide range, a Michaelis constant of 3.4 mm was observed in addition to the much lower Michaelis constant noted above. This low affinity binding of glucose may have regulatory significance and may indicate the presence of a glucokinase in addition to hexokinase. PMID- 16658447 TI - Proposed Model for the Peroxidase-Catalyzed Oxidation of Indole-3-acetic Acid in the Presence of the Inhibitor Ferulic Acid. AB - Linear increments in ferulic acid concentration produce logarithmic increases in the ferulic acid-induced lag periods prior to the peroxidase-catalyzed oxidation of indole-3-acetic acid in a system containing 2,4-dichlorophenol and MnCl(2) in acetate buffer at pH 5.6. Maintaining the ratio of indole-3-acetic acid to ferulic acid constant at 100 while linearly raising the ferulic acid concentration results in linear increases in the lag period. Both indole-3-acetic acid and ferulic acid are substrates of horseradish peroxidase in the presence of H(2)O(2), and indole-3-acetic acid competitively inhibits the oxidation of ferulic acid. A model for the enzymatic oxidation of indole-3-acetic acid catalyzed by peroxidase is proposed. PMID- 16658448 TI - Photocontrol of the germination of onoclea spores: I. Action spectrum. AB - Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm(-2); sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylls and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1 dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process. PMID- 16658449 TI - Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: IV. De Novo Synthesis of Sucrose 6-Phosphate Synthetase in Scutellum. PMID- 16658450 TI - A Rapid Phytochrome-dependent Reduction of Nicotinamide Adenine Dinucleotide Phosphate in Particle Fraction from Etiolated Bean Hypocotyl. PMID- 16658451 TI - Thymidine-phosphorylating Activity in gamma-Plantlets: Separation of Onset of Activity from Deoxyribonucleic Acid Synthesis. PMID- 16658452 TI - Effect of salinity on the time course of wheat seedling growth. PMID- 16658453 TI - Induction of abscission at hypobaric pressures. AB - The use of hypobaric pressures has increased the precision of abscission research by enabling us to differentiate between abscission action of ethylene and abscisic acid. When cycloheximide is sprayed on fruit attached to trees, enhanced levels of ethylene occur in the fruit and, subsequently, the fruit abscises. When ethylene in the fruit is eliminated by hypobaric pressures, the fruit does not abscise. Thus, ethylene is the effector of fruit abscission that results from cycloheximide treatment. When abscisic acid is applied to the fruit through stem uptake and ethylene is removed by hypobaric pressures, rapid fruit abscission occurs, which is presumably caused by abscisic acid itself. Thus, either ethylene or abscisic acid will induce abscission of citrus. Likewise, the abscission of debladed petioles of Coleus plants appears to be effected either by ethylene or abscisic acid. PMID- 16658454 TI - Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during the Cell Expansion Phase of Cotyledon Development in Vicia faba L. AB - In Vicia faba L., the tissue specific proteins, legumin and vicilin, are synthesized during the cell expansion phase of cotyledon development. During this growth period, RNA and nuclear DNA increase 8- to 10-fold. (3)H-Uridine and (3)H adenosine are incorporated into ribosomal RNA, both 25S and 18S, and into transfer RNA. DNA isolated from cotyledons in the cell division phase of growth has been compared with DNA isolated from cotyledons undergoing expansion growth. Results indicate that the DNA increase involves replication of the whole genome (endoreduplication). PMID- 16658455 TI - Mechanism of a Synergistic Effect of Kinetin on Auxin-induced Ethylene Production: Suppression of Auxin Conjugation. AB - In hypocotyl segments of mung bean (Phaseolus mungo L.) seedlings, exogenously supplied indoleacetic acid was rapidly conjugated mainly into indoleacetylaspartic acid, which was inactive in inducing ethylene production. Kinetin is known to stimulate indoleacetic acid-induced ethylene production. The mechanism of kinetin action on indoleacetic acid-induced ethylene production by hypocotyl segments of mung bean seedlings was studied in relation to indoleacetic acid uptake and indoleacetic acid metabolism. Kinetin enhanced indoleacetic acid uptake during the initial 2-hour incubation and markedly suppressed the conversion of indoleacetic acid to indoleacetic acid conjugates throughout the whole 7-hour incubation. As a result, there was more free indoleacetic acid and less conjugated indoleacetic acid in the segments treated with kinetin than in those receiving no kinetin. A close relationship was demonstrated between the rate of ethylene production and the level of free indoleacetic acid, which was regulated by kinetin. PMID- 16658456 TI - Multiple forms of acidic endopeptidase from germinated barley. AB - An endopeptidase preparation from germinated barley Hordeum vulgare L., cv. Trophy, purified by affinity chromatography and density-gradient electrofocusing, consisted of three or four components. The preparation was only partly resolved by electrofocusing, with evidence of three possible components (pI 4.15, 4.28, and 4.37). Gel filtration on Sephadex G-75 yielded an asymmetrical peak, the major part of which corresponded to a molecular weight of 14,100, with evidence of one larger and two smaller components. The activity of the preparation was sulfhydryl-dependent; cysteine was the most effective of several sulfhydryl compounds tested. The preparation was sensitive to O(2) in the absence of metal chelating agents and was inhibited by sulfhydryl reagents. It showed very narrow concentration tolerances for both cysteine and a substrate, N,N dimethylhemoglobin. The Km value on N,N-dimethylhemoglobin at pH 3.8 was 0.064 to 0.067% (w/v) substrate; V(max) was 0.80 to 0.83 A(340) per hour. Normal enzyme activity and molecular-size distribution were observed when the endopeptidases were extracted in the inhibited state and subsequently reactivated, thus ruling out the possibility that the enzymes might be autolytic artifacts that arose during extraction and purification. PMID- 16658457 TI - Increased Activity of Chromatin-bound Ribonucleic Acid Polymerase from Soybean Hypocotyl with Spermidine and High Ionic Strength. AB - Optimal activity of chromatin-bound RNA polymerase from soybeans is obtained with 1 mm Mn(2-), but only when high ionic strength or polyamines are included in the medium. Such inclusion does not increase the Mg(2+) activation of the polymerase, but it does lower the concentration needed for optimum activity from 10 mm to 1 mm. Mg(2-) activation is inhibited by added Mn(2+), and the inhibition is relieved by high ionic strength or spermidine. The RNA polymerase with either cation is almost entirely polymerase I at low and high ionic strength as evidenced by insensitivity to alpha-amanitin. Treatment of soybean seedlings with 2,4-dichlorophenoxyacetic acid does not change these characteristics; although the activity rises 3- to 4-fold.It is suggested that chromatin as prepared here may be a selected fraction enriched in polymerase I, which is activated by either Mg(2+) or Mn(2+), and that the Mn(2-) inhibition of activity is due to a known reaction of Mn(2-) with DNA which can be relieved by high ionic strength. PMID- 16658458 TI - On the nature of the physiological responses of Avena stem segments to gibberellic Acid treatment. AB - Gibberellic acid was found to cause elongation in Avena sativa (oat) stem segments whether it was applied continuously or as a short pulse. The shorter the pulse time became, the higher was the gibberellic acid concentration needed to cause elongation; the segmental growth apparently depends upon the amount of gibberellic acid taken up by the segments. Avena segments showed a decreased growth response to gibberellic acid if the treatments were initiated at increasingly later times after excision from the plant. This decreased responsiveness to gibberellic acid was inhibited by low temperature (0-4 C), but accelerated by anaerobiosis. On the other hand, growth stimulation by a gibberellic acid pulse at the start of incubation was not altered by cold treatment but was nullified by a nitrogen atmosphere. Both the readiness of the segments for growth stimulation by gibberellic acid and its action in promoting growth clearly involve temperature-dependent, aerobic metabolism.Segments were able to use glucose, fructose, sucrose, and glycerol but not malate, citrate, pyruvate, glycine, or glutamine as substrate for growth. Since final elongation without exogenous substrate was highly correlated with initial content of total carbohydrate and reducing sugar, carbohydrates seem to be the major endogenous growth substrates.The Avena segments are composed of three distinct morphological units: node, internode, and encircling leaf sheath. Although the node and leaf sheath do not grow, they must be present for maximal growth of the internode. Quantitative assessment of the roles of each part suggests that a substance other than gibberellin or sugar is necessary for maximal internodal growth and that this substance may be channeled from the leaf sheath to the internode through the anastomosing vascular tissue of the node. PMID- 16658459 TI - Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots. AB - Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. "Nauty") root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO(2) partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme. PMID- 16658460 TI - Influence of phenolic acids on ion uptake: I. Inhibition of phosphate uptake. AB - The influence of naturally occurring phenolic acids on phosphate uptake by barley (Hordeum vulgare L. cv. Karlsberg) roots was examined using (32)P-labeled phosphate. Without exception, all compounds tested, namely, benzoic, 2 hydroxybenzoic, 4-hydroxybenzoic, 3,4-dihydroxybenzoic, 3,4,5-trihydroxybenzoic, 4-hydroxy-3-methoxybenzoic, 4-hydroxy-3,5-dimethoxybenzoic, cinnamic, 2 hydroxycinnamic, 4-hydroxycinnamic, 3,4-dihydroxycinnamic, 4-hydroxy-3 methoxycinnamic, and 4-hydroxy-3,5-dimethoxycinnamic acids, inhibited uptake.The degree of inhibition correlated well with the lipid solubility of these phenolic compounds. This as well as kinetic data support the hypothesis of inhibition through altered membrane properties. PMID- 16658461 TI - Evidence for lack of turnover of ribulose 1,5-diphosphate carboxylase in barley leaves. AB - Turnover of ribulose 1,5-diphosphate carboxylase in barley leaves (Hordeum vulgare L.) was followed over time in light and dark. The enzyme was degraded in prolonged darkness and was resynthesized after the plants were returned to light. Labeling with (14)C showed that simultaneous synthesis and degradation (turnover) did not occur in light. In contrast, the remaining soluble protein was turned over rapidly in light. Although ribulose 1,5-diP carboxylase can be both degraded and synthesized, these processes seem not to occur simultaneously, but can be induced independently by changing environmental conditions. PMID- 16658462 TI - Gametogenesis in Chlamydomonas eugametos: I. Light Requirements. AB - Male and female mating types of Chlamydomonas eugametos Moewus show an absolute light requirement for gametogenesis. Increasing light intensity from 0.3 to 1.2 mw cm(-2) during nitrogen starvation (a precondition for gametogenesis) caused an increase in gametogenesis throughout a 28-hour period. Gametogenesis was measured by determining the percentage of paired cells after a 1-hour mixing period. Light requirements for the male and female differed. There was a 9-hour lag period in gametogenesis in the male, but no lag in the female. Gametogenesis was reduced 50% in the female and 90% in the male when 6.0 mum 3-(3,4-dichlorophenyl)-1, 1 dimethyl-urea was in the N-starvation medium. Sodium acetate, 1.8 mm, in the N starvation medium increased gametogenesis in both mating types and eliminated the 9-hour lag in the male for cells irradiated for 3, 6, 9, 12, 15, 18, or 23 hours during the last part of a 23-hour N-starvation period. Sodium acetate concentrations higher than 1.8 mm inhibited the mating process. 3-(3,4 Dichlorophenyl)-1, 1-dimethylurea inhibition of gametogenesis was decreased in the male but increased in the female, when sodium acetate was added to the N starvation medium. These results indicate a nonphotosynthetic as well as a photosynthetic role for light in the gametogenesis of both mating types. Also, the male will not undergo gametogenesis unless a required amount of energy is provided either in the medium or through photosynthesis. PMID- 16658463 TI - Enzymatic fractionation of carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants. AB - The carbon atoms of glucose and malate in C(4) plants are 2 to 3 per thousand enriched in (12)C with respect to atmospheric CO(2); whereas these intermediates in C(3) plants are 15 to 18 per thousand enriched with (12)C with respect to atmospheric CO(2). The enzymatic synthesis of malate from phosphoenolpyruvate and bicarbonate in preparations of leaves of Sorghum bicolor, Haygrazer result in a carbon isotope fractionation of about 3 per thousand. The enzymatic synthesis of phosphoglyceric acid from ribulose 1,5-diP and CO(2) in these preparations (contaminated with carbonic anhydrase) at 24 C and 37 C result in a carbon isotope fractionation of 33.7 per thousand and 18.3 per thousand, respectively. These data are consistent with the conclusion that the small enrichment of (12)C in the carbon atoms of malate and glucose (with respect to atmospheric CO(2)) in leaves of Sorghum bicolor, Haygrazer occurs at the phosphoenolpyruvate carboxylase step. PMID- 16658464 TI - Partial Purification and Properties of d-Glucosamine 6-Phosphate N Acetyltransferase from Phaseolus aureus. AB - d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 x 10(-2)m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg(2+) was required for maximum activity of the enzyme. Mn(2+) and Zn(2+) were inhibitory, while Co(2+) had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg(2+) on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 x 10(-4)m and 7.1 x 10(-4)m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 x 10(-4)mp hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme. PMID- 16658465 TI - Effect of Low Levels of Calcium on Exudation of Sugars and Sugar Derivatives from Intact Peanut Roots under Axenic Conditions. AB - The effects of 10, 20, 35 and 50 mg of Ca(2+) per liter on the qualitative and quantitative exudation of sugars from roots of 5-week-old peanut plants, Arachis hypogaea L., grown axenically in nutrient solutions, were measured. Nutrient solutions in which plants had been growing were collected at weekly intervals for 4 weeks, sugars in them were measured by gasliquid chromatography of the trimethylsilyl derivatives. Arabinose, ribose, xylose, fructose, mannose, glucose, galactose, mannitol, galacturonic acid, inositol, sucrose, and five unknowns were found. Qualitative and quantitative differences in exudates were correlated with age of the plants and calcium level. Four times more sugar was exuded at 10 mg than at 50 mg of Ca(2+) per liter but no significant differences in growth were observed. Ion efflux measurements suggested that low levels of Ca(2+) increased root cell membrane permeability. PMID- 16658466 TI - Salt-stimulated Adenosine Triphosphatase from Smooth Microsomes of Turnip. AB - The turnip (Brassica rapa L.) microsome fraction contains both a Mg(2+)-inhibited acid phosphatase and a salt-stimulated Mg(2+)-activated ATPase. However, as the pH optimum of the ATPase was 8.0 to 8.5, the acid phosphatase activity could be eliminated by assaying at or above pH 7.8. The ATPase was concentrated in a fraction equivalent to the smooth microsomal membranes and was not due to fragments of mitochondria. The salt-stimulated activity showed specificity for anions rather than cations. The activity was further stimulated by carbonyl cyanide m-chloro-phenylhydrazone (CCCP), 2,4-dinitrophenol, valinomycin, nigericin, and NH(4)Cl. There was a synergistic effect between CCCP and valinomycin. Activity was insensitive to oligomycin phlorizin, ouabain, and atractylate. Based on similarity to the chloroplast ATPase, it was proposed that this ATPase was situated on the outside of the vesicle.It is suggested that the ATPase is involved in the movement of ions, particularly anions, and may be related to the anion accumulation mechanism, which is known to occur across the tonoplast of such tissues. PMID- 16658467 TI - The Relation between Photophosphorylation and Delayed Light Emission in Chloroplasts. AB - One millisecond delayed light emission has been studied in isolated coupled lettuce (Lactuca sativa var. romaine) chloroplasts. Delayed light emission was increased upon addition of ferricyanide or 2,3,5,6-tetramethyl-p-phenylene diamine. In the presence of ferricyanide, the magnitude of the signal was increased by the addition of ADP (in the absence of orthophosphate), ATP, DI0-9, or phlorizin. The signal was also increased by the addition of NaCl and by the addition of NH(4)Cl in the presence of a high NaCl concentration. The signal of delayed light emission was decreased by the addition of gramicidin, valinomycin, and by the addition of NH(4)Cl in the presence of a low NaCl concentration.Phosphorylation, whether started by addition of ADP or by addition of glucose plus hexokinase plus ATP, caused a significant decrease in delayed light emission. It was concluded that the magnitude of delayed light emission reflects the size of the proton motive force across the thylakoid membrane. Calibration of delayed light emission by creating KCl gradients indicated that the value for the electrochemical potential gradient for H(+) in the presence of ferricyanide was at least 155 millivolts decreasing to 134 millivolts after the onset of phosphorylation. PMID- 16658468 TI - Estimation of nitrogenase using a colorimetric determination for ethylene. AB - Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene. PMID- 16658469 TI - Malate Dehydrogenases of Pisum sativum: Tissue Distribution and Properties of the Particulate Forms. AB - Mitochondria and leaf microbodies isolated from leaves of pea (Pisum sativum) by sucrose density gradient centrifugation were each shown to have a unique form (isoenzyme) of malate dehydrogenase (EC 1.1.1.37) based on chromatographic and kinetic properties. Root organelle preparations were shown to contain only a mitochondrial malate dehydrogenase with physical and kinetic properties similar to the leaf form. The absence of a detectable root microbody malate dehydrogenase similar to the leaf enzyme, which is intermediate in electrophoretic and chromatographic properties between the mitochondrial and soluble isoenzymes, was confirmed by diethylaminoethyl cellulose column chromatography and starch-gel electrophoresis of total homogenates from leaf and root tissue. These findings tend to support the role of the leaf microbody isoenzyme in a pathway unique to photosynthetic tissue. PMID- 16658470 TI - Light-induced Ethylene Production in Sorghum. AB - Ethylene production was induced in sections of dark-grown Sorghum vulgare L. seedlings by treatment with light in the blue and far red regions of the light spectrum. The action spectrum closely resembled the previously reported spectra for high irradiance response; thus, light-induced ethylene production is probably a high irradiance response with phytochrome as the initial photoreceptor. PMID- 16658471 TI - Quantifying Rhythmic Movements of Albizzia julibrissin Pinnules. AB - The cosinor technique, previously applied to studies of animal rhythms, is used to assess the circadian rhythm in pinnule movement of Albizzia julibrissin. The method provides a quantitative approach for studying rhythm properties of either intact or excised pinnules. Phase shifting in A. julibrissin, as compared to the usually slower phase shifts of various circadian phenomena in the insect, bird, and mammal, occurs within 1 day or two. Rhythm adjustment in the pinnules takes place more rapidly when the lighting regiment is advanced than when it is delayed. PMID- 16658472 TI - The role of phytochrome in an interaction with ethylene and carbon dioxide in overcoming lettuce seed thermodormancy. AB - Ethylene and CO(2) were used to control induction of germination in thermodormant lettuce seed (Lactuca sativa L.). These experiments ultimately showed that germination depends on the presence of an active form of the phytochrome. The phytochrome system is functional and stable at 35 C, a temperature which completely inhibits germination. Phytochrome responses to red or far red light and darkness showed that this inhibition of germination under light must be due to some other block(s) rather than to a direct inactivation of the phytochrome system itself. A postred radiation increase in lettuce seed germination that is not reversed by far red light was observed. The CO(2) requirement for C(2)H(4) action is not due to a change in the medium's pH; addition of C(2)H(4) plus CO(2) at the start of imbibition did not result in as much germination as when they were added several hours after imbibition. This reduction in germination, when the gases are added at the start of imbibiton, is due to CO(2). PMID- 16658473 TI - Effects of carbon dioxide on activity of apple mitochondria. AB - Effects of CO(2) on mitochondrial activity of apple (Malus pumila Mill. var. Richared Delicious) were studied in two ways. Immediate effects were determined by imposing 3 to 18% CO(2)-bicarbonate mixtures on isolated apple mitochondria, and long term effects were determined by extracting mitochondria from apples that had been stored for intervals in atmospheres containing 6 or 12% CO(2) plus 3% O(2). The CO(2)-bicarbonate systems had immediate and broad effects on mitochondrial oxidations: 18% CO(2) stimulated malate oxidation about 10%; suppressed alpha-ketoglutarate, citrate, and NADH oxidations about 10%; and suppressed fumarate, pyruvate, and succinate oxidations about 32%. The effects of lower CO(2) concentrations varied with substrates. Mitochondria isolated from fruit stored in 6 or 12% CO(2) possessed a reduced capacity to oxidize added succinate or NADH, but retained a marked sensitivity to CO(2)-bicarbonate mixtures. Respiratory control in these mitochondria was somewhat reduced, but CO(2) had not acted as a strong uncoupling agent. PMID- 16658474 TI - Effects of Potassium Deficiency on the Photosynthesis and Respiration of Leaves of Sugar Beet under Conditions of Low Sodium Supply. AB - Sugar beet plants (Beta vulgaris L. cv. F58-554H1) were germinated and cultured under standardized environmental conditions. The effects of K deficiency on photosynthetic and respiratory CO(2) exchange rates of attached leaves were studied under conditions of low Na supply by withholding both Na and K from the culture medium at cut-off (28 days after planting). Potassium and Na concentrations in the leaf blade and petiole decreased rapidly during the 8 days after cut-off, then more slowly.Photosynthetic CO(2) uptake per unit leaf area decreased rapidly with time after cut-off to 23% of the control rate in 17 days. Mesophyll resistance to CO(2) (r(m)) increased sharply after cut-off, r(m) eventually attaining 8.3 sec cm(-1). Leaf (mainly stomatal) diffusion resistance, r(1)', also increased rapidly from 4 days after cut-off, reaching 1.9 sec cm(-1) 13 days later. The photorespiratory evolution of CO(2) into CO(2)-free air decreased progressively after cut-off, but the rate of dark respiratory CO(2) evolution increased. It was concluded that withholding Na as well as K at cut-off increased the deleterious effects of K deficiency on photosynthesis and stomatal opening. PMID- 16658475 TI - Effects of gibberellic Acid and sucrose on the growth of oat (Avena) stem segments. AB - Gibberellic acid induced growth in Avena (oat) stem segments within 35 minutes after hormone application. The total elongation elicited by gibberellic acid was greater than 15 times the control growth. The sensitivity of the segments to low concentrations of gibberellic acid (1 pmole) and the specificity of the segments to the gibberellin class of hormones suggest that oat stem segments would be a valuable tool for gibberellin bioassays. Both gibberellic acid-induced growth and control growth are temperature-dependent and showed a Q(10) of two or greater. Although the most apparent effect of gibberellic acid was to promote the uptake of water into the internode, the hormone also promoted transport of endogenous substrate and the uptake of exogenous substrate into the growing region. The growth promotion was accomplished without an apparent increase in osmotic pressure. PMID- 16658476 TI - Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells. AB - A procedure for the separation of proplastids free of mitochondria from dark grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100A in diameter. No larger particles, typical of light-grown chloroplasts, were observed. PMID- 16658477 TI - Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings. AB - The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalofs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b 559(HP)). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80 fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.Plastids showed substantial photochemical oxygen evolution after a seedling greening time of 1 hour. However, a comparison of the relative activity of leaves and plastids at 2 hours and 24 hours of greening suggests that there was some inactivation of greening plastids during isolation. Appreciable photosystem I activity was observed as early as 15 minutes of greening.The synthesis of cytochrome b-559(HP) during greening does not correlate with the onset of oxygen evolution. Cytochrome b-559(HP) was absent from etioplasts and in most preparations of 2-hour plastids. The average amount of cytochrome b-559(HP) at 2 hours of greening was well below the level needed to provide 1 molecule of the carrier for each functional photosynthetic chain. The results suggest that cytochrome b-559(HP) is not essential for oxygen evolution. Cytochrome f, cytochrome b(6), and the low potential form of cytochrome b-559 were present in the etioplast. There was little increase in the levels of these cytochromes during 24 hours of greening. PMID- 16658478 TI - Malate dehydrogenase isoenzymes in division synchronized cultures of euglena. AB - Sucrose density gradient centrifugation of broken cell suspensions of autotrophically grown Euglena gracilis Klebs. has allowed the separation of chloroplasts, mitochondria, and peroxisomes. Chlorophyll was taken as a marker for chloroplasts, fumarase and succinate dehydrogenase for mitochondria, and glycolate oxidoreductase for peroxisomes. Peaks of malate dehydrogenase (l-malate NAD oxidoreductase, EC 1.1.1.37) activity were found in the mitochondrial and peroxisomal fractions. Acrylamide gel electrophoresis showed specific isoenzymes in the mitochondrial and peroxisomal fractions and a third isoenzyme in the supernatant. The mitochondrial isoenzyme which had a Km (oxaloacetate) of 30mum was inhibited by oxaloacetate concentrations above 0.17 mm, an inhibition of 50% being given by 0.9 mm oxaloacetate. The peroxisomal isoenzyme had a Km (oxaloacetate) of 24 mum, was inhibited by oxaloacetate concentrations above 0.13 mm, 50% inhibition being given by 0.25 mm oxaloacetate. Malate dehydrogenase activity in the supernatant did not show inhibition by increasing oxaloacetate concentration, the Km (oxaloacetate) being 91 mum.In division synchronized cultures of Euglena, all three isoenzymes of malate dehydrogenase were synthesized over the light phase of the cycle. Darkening light phase cultures did not affect malate dehydrogenase activity. The addition to cultures of cycloheximide at a concentration previously shown to inhibit protein synthesis on Euglena cytoplasmic ribosomes completely inhibited increase in malate dehydrogenase activity over the cell cycle. Malate dehydrogenase activity was unaffected by the addition of chloramphenicol in amounts known to inhibit preferentially protein synthesis on 70S ribosomes. PMID- 16658479 TI - Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies. AB - Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and "malic enzyme" (EC 1.1.1.40) in bundle sheath cells. NADP glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations. PMID- 16658480 TI - Studies on the 70S Ribosomal Content of a Plastid Mutant in Gossypium hirsutum. AB - Analysis of a mutation in cotton (Gossypium hirsutum), which is maternally inherited, revealed that the completely white sectors of leaves were deficient in the 70S class ribosomes, whereas the yellow sectors exhibited the same level of the latter as in the green leaves. PMID- 16658481 TI - Growth of Rice Root-derived Callus Tissue in Suspension Culture. PMID- 16658482 TI - Control of CO(2) Fixation by Cell-free Extracts of Chlamydomonas reinhardtii. AB - Contrary to earlier reports, CO(2) fixation by extracts of Chlamydomonas is inhibited by glutamate and aspartate. These amino acids and some organic acids are shown to be inhibitors of phosphoenolpyruvate carboxylase. Inorganic phosphate is shown to activate CO(2) fixation, but there is a time lag before inorganic phosphate exerts its full activating effect. PMID- 16658483 TI - Effects of water stress on the indoleacetic Acid oxidase activity in wheat leaves. PMID- 16658484 TI - Germination of Phaseolus vulgaris: IV. Patterns of Protein Synthesis in Excised Axes. AB - Soluble proteins from excised Phaseolus vulgaris axes incubated for 1 hour in (3)H or (14)C- amino acid mixtures at different times during the period leading up to initiation of cell elongation were compared by acrylamide gel electrophoresis. Differences in electrophoretic patterns were found when proteins from axes incubated during the 1st hour of imbibition were compared with proteins from axes incubated during the hour when cell elongation was initiated. These differences greatly diminished by the 2nd hour of imbibition which suggests that they were due primarily to incomplete axis imbibition. A 5-hour actinomycin D treatment which reduced amino acid incorporation by 40% in the 5th hour had no apparent effect on the electrophoretic pattern during that hour. PMID- 16658485 TI - Elimination of bacterial contamination from onion seed. PMID- 16658486 TI - Chloroplast response to low leaf water potentials: I. Role of turgor. AB - The effect of decreases in turgor on chloroplast activity was studied by measuring the photochemical activity of intact sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves having low water potentials. Leaf turgor, calculated from leaf water potential and osmotic potential, was found to be affected by the dilution of cell contents by water in the cell walls, when osmotic potentials were measured with a thermocouple psychrometer. After the correction of measurements of leaf osmotic potential, both the thermocouple psychrometer and a pressure chamber indicated that turgor became zero in sunflower leaves at leaf water potentials of -10 bars. Since most of the loss in photochemical activity occurred at water potentials below -10 bars, it was concluded that turgor had little effect on the photochemical activity of the leaves. PMID- 16658487 TI - Chloroplast Response to Low Leaf Water Potentials: II. Role of Osmotic Potential. AB - Electron transport in chloroplasts isolated from desiccated sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves was compared with electron transport in sunflower chloroplasts in sorbitol-containing media having various osmotic potentials. In media having low osmotic potentials and dichloroindophenol as electron acceptor, the activity for electron transport was inhibited, but the inhibition was much less than that due to comparable desiccation in vivo. The inhibition at low osmotic potentials was rapidly reversed by returning the chloroplasts to media having high osmotic potentials, but the activity of chloroplasts from desiccated tissue showed no reversal when the chloroplasts were placed in media having high osmotic potentials. Nevertheless, the inhibition of chloroplast activity due to desiccation in vivo was basically reversible, because chloroplasts recovered quickly when they were rehydrated in vivo. The large differences between desiccation in vivo and exposure to low osmotic potential in vivo indicate that osmotic solutions did not reproduce the effects of tissue desiccation. It is concluded that decreases in the Gibbs free energy of water due to decreased osmotic potentials probably have only a small effect on electron transport in chloroplasts from desiccated tissue and do not account for the major effects of leaf desiccation on electron transport. PMID- 16658488 TI - Substrate Activation of beta-(1 --> 3) Glucan Synthetase and Its Effect on the Structure of beta-Glucan Obtained from UDP-d-glucose and Particulate Enzyme of Oat Coleoptiles. AB - UDP-d-glucose, at a micromolar level in the presence of MgCl(2) and oat (Avena sativa) coleoptile particulate enzyme which contains both beta-(1 --> 3) and beta (1 --> 4) glucan synthetases, produces glucan with mainly beta-(1 --> 4) glucosyl linkages. An activation of beta-(1 --> 3) glucan synthetase by UDP-d-glucose and a decrease in the formation of beta-(1 --> 3) glucan in the presence of MgCl(2) have been observed. However, at high substrate concentration (>/= 10(-4)m), the activation of beta-(1 --> 3) glucan synthetase is so pronounced that the formation of beta-(1 --> 3) glucosyl linkage predominates in synthesized glucan regardless of the presence of MgCl(2). These observations may explain the striking shift in the composition of glucan of particulate enzyme from a beta-(1 -> 4) to beta-(1 --> 3) glucosyl linkage when UDP-d-glucose concentration is raised from a low concentration (/= 10( 4)m).Besides UDP-d-glucose, CDP-d-glucose can also serve as substrate for the formation of beta-(1 --> 3) glucan in the presence of beta-(1 --> 3) synthetase. PMID- 16658489 TI - Abscission: support for a role of ethylene modification of auxin transport. AB - Three types of whole plant experiments are presented to substantiate the concept that an important function of ethylene in abscission is to reduce the transport of auxin from the leaf to the abscission zone. (a) The inhibitory effect of ethylene on auxin transport, like ethylene-stimulated abscission, persists only as long as the gas is continuously present. Cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants placed in 14 mul/l of ethylene for 24 or 48 hours showed an increase in leaf abscission and a reduced capacity to transport auxin; but when returned to air, auxin transport gradually increased and abscission ceased. (b) Ethylene induced abscission and auxin transport inhibition show similar sensitivities to temperature. A 24-hour exposure of cotton plants to 14 mul/l of ethylene at 8 C resulted in no abscission and no significant inhibition of auxin transport. Increasing the temperature during ethylene treatment resulted in a progressively greater reduction in auxin transport with abscission occurring at [unk]27 C where auxin transport was inhibited over 70%. (c) Auxin pretreatment reduced both ethylene-induced abscission and auxin transport inhibition. No abscission occurred, and auxin transport was inhibited only 18% in cotton plants which were pretreated with 250 mg/l of naphthalene acetic acid and then placed in 14 mul/l of ethylene for 24 hours. In contrast, over 30% abscission occurred, and auxin transport was inhibited 58% in the corresponding control plants.Collectively, the results presented here and elsewhere indicate that ethylene regulates the sensitivity of the cells in the abscission zone to the more direct actions of the gas (e.g., enzyme induction, secretion) by reducing auxin transport. PMID- 16658490 TI - An arginyl-transfer ribonucleic Acid protein transferase from cereal embryos. AB - Embryos from rice (Oryza sativa L. var. Bluebonnet) and wheat (Triticum aestivum L.) contain an aminoacyl-tRNA protein transferase which transfers arginine from arginyl-tRNA to the N terminus of a protein acceptor. The activity was measured in vitro in a reaction mixture containing embryo supernatant fraction, buffer, sulfhydryl reagent, and arginyl-tRNA. It was not dependent on the usual cofactors for ribosomal protein synthesis, nor was it sensitive to cycloheximide or puromycin. However, the activity was inhibited by ribonuclease. The enzyme was purified 33-fold from a crude homogenate of rice embryos. An apparent endogenous substrate from rice embryos was prepared free of transferase activity; however, the transferase was not purified sufficiently to show absolute dependence on the presence of this endogenous substrate. PMID- 16658491 TI - Metabolism of Separated Leaf Cells: III. Effects of Calcium and Ammonium on Product Distribution During Photosynthesis with Cotton Cells. AB - Separated mesophyll cells from cotton (Gossypium hirsutum var. Stoneville 1613 Glandless) were isolated with pectinase and mechanical agitation. The separated cells had rates of light-dependent CO(2) fixation between 50 to 100 mumoles CO(2) per mg chlorophyll per hour. The presence of Ca(2+) in the incubation medium did not significantly affect the type of photosynthetic products formed, but 2 mm Ca(2+) did cause a 50% decrease in the appearance of photosynthetic products in the incubation medium. The movement of all types of products (sugars, organic, and amino acids) out of the cells was reduced similarly by the Ca(2+). Light had no affect on the movement of products out of the cells, whereas 1 mm ethylenediaminetetra-acetate greatly increased the movement. The addition of 1.6 mm NH(4)Cl to the cell suspensions caused a large increase in the amount of fixed (14)C appearing in the amino acid fraction and a decrease in the sugar fraction. These metabolic changes in the cells were reflected in the movement of products out of the cells so that the incubation medium also contained a larger amount of label in amino acids and a smaller amount in sucrose. Although the cell plasma membrane restricted the movement of soluble products, it did not discriminate significantly between the types of products moved. PMID- 16658492 TI - Promotion of seed germination by cyanide. AB - Potassium cyanide at 3 mum to 10 mm promotes germination of Amaranthus albus, Lactuca sativa, and Lepidium virginicum seeds. l-Cysteine hydrogen sulfide lyase, which catalyzes the reaction of HCN with l-cysteine to form beta-l cyanoalanine, is active in the seeds. beta-l-Cyanoalanine is the most effective of the 23 alpha amino acids tested for promoting germination of A. albus seeds. Aspartate, which is produced by enzymatic hydrolysis of asparagine formed by hydrolysis from beta cyanoalanine, is the second most effective of the 23 amino acids. Uptake of aspartate-4-(14)C is much lower than of cyanide.Radioactive tracer in K(14)CN shows uptake of about 1.5 mumoles of HCN per gram of A. albus and L. sativa seeds after 20 hours of imbibition. Extracts of the seeds gave high (14)C activity in beta-cyanoalanine, asparagine, and aspartate. The acid-hydrolyzed protein extract gave high activity only in aspartate. Tests were negative for free cyanide in the seed. Respiration of the seed is inhibited more than 75% by KCN and by KN(3) at 10 mm. Azide at greater than 1.0 mm inhibits the promotion of germination by cyanides. Neither 0.1 mm KCN nor KN(3) inhibit O(2) consumption, whereas lower concentrations promote germination. It is concluded that the high rate of utilization of cyanide in the reaction to form beta-l-cyanoalanine and the subsequent incorporation into protein limit any inhibition of oxygen consumption. The promotion of seed germination is substrate-limited by asparagine-aspartate, which is required for protein synthesis. PMID- 16658493 TI - Response of carbon dioxide fixation to water stress: parallel measurements on isolated chloroplasts and intact spinach leaves. AB - Application of water stress to isolated spinach (Spinacia oleracea) chloroplasts by redutcion of the osmotic potentials of CO(2) fixation media below -6 to -8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO(2) fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO(2) fixation to osmotic potential of the fixation medium was found with chloroplasts isolated from dehydrated leaves-namely, fixation rate was inhibited at -8 bars, compared with -16 or -24 bars.Low leaf water potentials were found to inhibit CO(2) fixation of intact leaf discs to almost the same degree as they did CO(2) fixation by chloroplasts isolated from those leaves. CO(2) fixation by intact leaves was decreased by 50 and 80% when water potentials were reduced from -7.1 to -9.6 and from -7.1 to -17.6 bars, respectively. Transpiration was decreased by only 40 and 60%, under the same conditions. However, correction for the increase in leaf temperature indicated transpiration decreases of 57 and 80%, similar to the relative decreases in CO(2) fixation.Despite the 4-fold increase in leaf resistance to CO(2) diffusion in the gas phase when the water potential of leaves was reduced from -6.5 to -14.0 bars, an additional increase of about 50% in mesophyll resistance was obtained. CO(2) concentration at compensation also increased when leaf water potential was reduced. PMID- 16658494 TI - The effect on net photosynthesis of pedigree selection for low and high rates of photorespiration in tobacco. AB - A normal appearing plant with a low rate of photorespiration (ratio of (14)CO(2) released light/dark = 1.6) was found in an unselected tobacco (Nicotiana tabacum) cultivar. The plant was self-pollinated, and further selections were made on several successive generations. Excised leaves from the progeny of the selections were examined for photorespiration and net CO(2) assimilation in normal air during photosynthesis. Similar measurements were made of plants derived from selfed parents with high rates of photorespiration (ratio of (14)CO(2) released light/dark = 3.0 or greater). Efficient photosynthetic plants (greater than 22.0 mg of CO(2) dm(-2) hr(-1)) with low rates of photorespiration produced a larger proportion of efficient progeny (about 25%) than did selfing inefficient plants (about 6%), but this proportion did not increase in successive generations.Wide variations in photorespiration and photosynthesis were observed within populations of normal appearing plants grown in the same greenhouse environment. Several plants had photosynthetic rates as great as 25 mg of CO(2) dm(-2) hr(-1) coupled with light/dark ratios below 2.0.The characteristics of two representative contrasting plants were studied more extensively and were fairly constant, irrespective of leaf position on the stalk, leaf size, or time of sampling within approximately a 2-week period. One plant with a mean light/dark ratio of 1.7 showed a mean net photosynthesis of 23.4 mg of CO(2) dm(-2) hr(-1), while an inefficient plant with a light/dark ratio of 3.7 had an average photosynthetic rate of only 17.0 mg CO(2) dm(-2) hr(-1). The dark respiration was similar in these plants. Thus decreasing the photorespiratory rate by about one half increased net photosynthesis by 38%. The results suggest that diminishing photorespiration, in an otherwise suitable genetic background, will result in large increases in net photosynthesis and plant productivity. PMID- 16658495 TI - Ethylene Production and Respiratory Behavior of the rin Tomato Mutant. AB - Little or no change in ethylene or CO(2) production occurred in rin tomato mutant fruits monitored for up to 120 days after harvest. Of the abnormally ripening tomatoes investigated, including "Never ripe" (Nr Y a h, Nr c l(2) r), "Evergreen" (gf r) and "Green Flesh" (gf), only rin did not show a typical climacteric and ethylene rise.Fruits from F(1) plants resulting from reciprocal crosses between rin and normal plants apeared to ripen normally, but when compared to normal fruit, their ripening was delayed as measured by ethylene and CO(2) production and color change. These fruits produced only one-third to one half as much ethylene at the peak of production compared to normal fruits.Exogenous ethylene or propylene treatment did not stimulate ethylene production by rin fruits but did stimulate CO(2) production. The CO(2) stimulation persisted only in the presence of the exogenous olefins. Stimulation of CO(2) production could be repeated several times in the same fruit. Wounding stress stimulated both ethylene and CO(2) production in rin fruits. It was concluded that rin tomato fruits behave like nonclimacteric fruits. PMID- 16658496 TI - Comparative studies on tobacco pith and sweet potato root isoperoxidases in relation to injury, indoleacetic Acid, and ethylene effects. AB - Sweet potato (Ipomoea batatas) root parenchyma and tobacco (Nicotiana tabacum) stem pith, both known to increase peroxidase activity after excision, differed from each other in their isoperoxidase patterns and in the isoperoxidase responses to injury and exogenous ethylene.In potato root sections, the injury dependent peroxidase increase was due to an induction of two isoenzymes, as well as to a promotion of some constitutive ones. In tobacco pith, this increase was entirely due to seven isoperoxidases not detectable, or detectable only in traces, immediately after excision. Actinomycin D did not inhibit the development of any isoperoxidases in the potato root sections and strongly repressed the development of all injury-induced isoenzymes in tobacco pith. Cycloheximide totally inhibited the development of all isoperoxidases in both species, with the exception of two injury-enhanced isoenzymes in root parenchyma.In root sections, indoleacetic acid had a weak inhibitory effect on one injury-induced isoperoxidase only, whereas in tobacco pith it inhibited the development of the injury-induced, as well as the constitutive, isoperoxidases.Exogenous ethylene did not induce, enhance, or significantly suppress any of the tobacco pith isoenzymes, whereas in potato root sections, it suppressed slightly the development of the injury-induced, had no effect on some of the injury-enhanced, and greatly promoted some of the injury-unaffected or-enhanced isoperoxidases. Removal of ethylene stopped the ethylene-dependent peroxidase increase without affecting the injury-induced increase. When applied to intact potato roots, ethylene did not induce any new isoperoxidases and promoted the same constitutive isoenzymes as it did in root sections.Thus, the tissue peroxidase response to ethylene seems independent of its response to injury. Differences between tissue species in their response to ethylene may depend on the presence or absence of isoperoxidases sensitive to ethylene. The inhibition of injury-dependent peroxidase development by indoleacetic acid cannot be explained by an ethylene induced inhibition. PMID- 16658497 TI - Enzymes of phospholipid metabolism in the endoplasmic reticulum of castor bean endosperm. AB - The intracellular location of several enzymes concerned with phospholipid metabolism was investigated by examining their distribution in organelles separated on sucrose gradients from total homogenates of castor bean (Ricinus communis var. Hale) endosperm. The enzymes phosphatidic acid phosphatase, CDP diglyceride-inositol transferase, and phosphatidyletha-nolamine-l-serine phosphatidyl transferase were all primarily or exclusively confined to membranes of the endoplasmic reticulum. These results and those reported previously on lecithin synthesis establish a major role of the endoplasmic reticulum in phospholipid and membrane synthesis in plant tissues. PMID- 16658498 TI - Circular dichroism spectra of granal and agranal chloroplasts of maize. AB - Granum-containing chloroplasts from mesophyll cells of maize (Zea mays L. var. MV 861) leaves exhibited circular dichroism spectra with a large double signal; peaks at 696 nm (+) and 680 nm (-). In the circular dichroism spectra obtained with agranal chloroplasts of bundle sheath cells, this large double signal is absent. Separation of grana lamellae, in a medium of low salt concentration and in KSCN solution, resulted only in a slight decrease of the amplitude, while upon treatment with digitonin the large double signal disappeared. A negative signal of the chlorophyll b peak at 654 nm was observed in the case of both granal and agranal chloroplasts, and it was not affected either by low salt or by digitonin treatment. A positive peak at about 515 nm was higher in granal than in agranal chloroplasts. PMID- 16658499 TI - Optical parameters of leaves of 30 plant species. AB - Optical parameters (absorption coefficient k, infinite reflectance Rinfinity, scattering coefficient 8) are tabulated for seven wavelengths and analyzed for statistical differences for 30 plant species. The wavelengths are: 550 nm (green reflectance peak), 650 nm (chlorophyll absorption band), 850 nm (infrared reflectance plateau), 1450 nm (water absorption band), 1650 nm (reflectance peak following water absorption band at 1450 nm), 1950 nm (water absorption band), and 2200 nm (reflectance peak following water absorption band at 1950 nm).Thick, complex dorsiventral (bifacial mesophyll) leaves such as rubber plant, begonia, sedum, and privet had lower Rinfinity values than thinner, less complex dorsiventral leaves (i.e., soybean, peach, bean, rose) or essentially centric (undifferentiated mesophyll) sorghum and corn leaves. Infinite reflectance was negatively correlated with leaf thickness (-0.734(**)).Thick, complex dorsiventral leaves (crinum, oleander, privet, rubber plant, sedum) had higher (p 0.01) k values than thinner, less complex dorsiventral leaves (i.e., soybean, rose, peach) or essentially centric sorghum, sugarcane, and corn leaves. A coefficient of 0.718(**) was obtained for the correlation of k values with leaf thickness values.Complex dorsiventral oleander, orange, and crinum leaves had higher (p 0.01) 8 values than less complex dorsiventral (i.e., onion, begonia, banana) or centric leaves (i.e., corn and sugarcane). The scattering coefficient was not correlated with leaf thickness. PMID- 16658500 TI - Characterization of Plasma Membrane-associated Adenosine Triphosphase Activity of Oat Roots. AB - ATPase activity of plasma membranes isolated from oat (Avena sativa L. cv. Goodfield) roots was activated by divalent cations (Mg(2+) = Mn(2+) > Zn(2+) > Fe(2+) > Ca(2+)) and further stimulated by KCl and a variety of monovalent salts, both inorganic and organic. The enzyme exhibited greater specificity for cations than anions. The presence of Mg(2+) was necessary for KCl stimulation. Ca(2+) was ineffective in replacing Mg(2+) for activation of plasma membrane ATPase, but it did activate other membrane-bound ATPases. The pH optima for Mg(2+) activation and KCl stimulation of the plasma membrane ATPase were 7.5 and 6.5, respectively.The plasma membrane ATPase showed little synergistic effects of K(+) and Na(+), and it was only slightly sensitive to ouabain. Oligomycin did not inhibit the ATPase, while N,N'-dicyclohexylcarbodiimide was a potent inhibitor of the enzyme.The apparent Km for Mg(2+) activation (0.84 mm) of the ATPase was about twice that of the apparent Km for ATP (0.38 mm). The effect of KCl in stimulating the enzyme was not on the apparent Km values for ATP and Mg(2+) but rather on maximum velocity. The kinetics of KCl stimulation of the plasma membrane ATPase were similar to the kinetics of (42)K(+) influx into oat roots and neither followed the Michaelis-Menten equation but rather were best described by a single activity curve with continually changing kinetic parameters. These results support the concept that cation transport at the plasma membrane of root cells is coupled to a cation-activated ATPase which is functional from low (0.01 mm) to high (50.0 mm) concentrations of KCl. PMID- 16658501 TI - Properties of an Aminotransferase of Pea (Pisum sativum L.). AB - A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With alpha ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other alpha-keto acids tested, pyruvate and oxalacetate were more active than alpha-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and alpha-ketoglutarate as co substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan. PMID- 16658502 TI - Some aspects of the regulation of arginine biosynthesis in soybean cell cultures. AB - The levels of the activities of argininosuccinate synthetase and argininosuccinate lyase were measured in soybean (glycine max L. var. Mandarin) cell suspension cultures grown in the presence or absence of exogenous arginine. In some experiments, actinomycin D or cycloheximide were also added to the cultures, at critical stages of their growth. The results obtained led to the conclusion that activity of argininosuccinate synthetase is subject to significant inhibition by levels of arginine similar to those found to occur within the cells. Argininosuccinate lyase activity appeared to be enhanced, when arginine levels were increased above those occurring physiologically. Both enzymes appeared to be subject to inactivation, possibly via proteolysis. PMID- 16658503 TI - Growth of plants in solution culture containing low levels of chromium. AB - Chromium was not required for normal growth of romaine lettuce (Lactuca sativa L. subsp. longifolia), tomato (Lycopersicon esculentum Mill.), wheat (Triticum aestivum L.), or bean (Phaseolus vulgaris L.) in solution culture containing 3.8 x 10(-4) muM Cr. Plants grown on this purified nutrient solution contained an average of 22 ng Cr/g dry weight. Duckweed (Lemna sp.) grew and reproduced normally on a dilute nutrient solution containing 3.8 x 10(-5) muM Cr. PMID- 16658504 TI - Comparative effects of hydrogen ions, carbon dioxide, and auxin on pea stem segment elongation. PMID- 16658505 TI - Application of chemicals in organic solvents to dry seeds. AB - Various chemicals were applied to dry seeds by means of organic solvents. The gibberellic acid-treated (1 mm) lettuce seeds (Lactuca sativa L.) germinated nearly 100% in the dark even after prolonged storage, and those treated with abscisic acid (1 mm or 0.5 mm) failed to germinate in the light. The seedlings emerging from morphactin-treated (1 mm) cucumber seeds (Cucumis sativus L.) exhibited profound changes in morphology. Different combinations of hormones applied to lettuce seeds caused a promotion or an inhibition of germination. Germination promotion or inhibition studies showed that the applied chemicals could be removed by washing with an organic solvent or water. Progressively larger amounts of chemicals were removed with increasing periods of washing. Thus the chemical appeared to penetrate the seed to some degree. The potential of the organic solvent method is discussed. PMID- 16658506 TI - An excellent source of vegetative buds for use in plant hormone studies on apical dominance. AB - When studying the role of plant hormones in the control of growth at apical meristems, it is often difficult to obtain needed amounts of physiologically uniform buds. A source and method are described for obtaining sufficient quantities of large, uniform buds and for the treatment of the buds with indoleacetic acid and kinetin. Buds from the root system of Euphorbia esula L. were grown in Petri plates, with agar suspending the short root sections from which they emanate. Plant hormones are applied by their incorporation in the agar. The effect of various concentrations of indoleacetic acid and kinetin on bud growth was examined. PMID- 16658507 TI - Flow of Chromium into Apple Fruit during Development. AB - A continuous flow of chromium into apples occurs throughout their growth. This ready movement of chromium into the apple is similar to that reported for essential trace elements such as boron, zinc, iron, copper, and manganese but differs from the restricted movement reported for mercury and cadmium. PMID- 16658508 TI - Polarity and rate of transport of cyclic adenosine 3,5'-monophosphate in the coleoptile. AB - Transport of tritiated cyclic AMP in the coleoptile of oats (Avena sativa) and corn (Zea mays) is polar, with basipetal to acropetal ratios of 4.0 and 3.2, respectively. The rate of transport is approximately that of indoleacetic acid. The linear velocity of transport, however, is at least five times that of auxin. A loss in transport polarity of the nucleotide occurs in subapical tissues within several hours after decapitation of the coleoptile, accompanied by a decrease in transport rate. The loss in polarity is not reversed by exogenous auxin, but the reduction in transport is. Auxin also inhibits the uptake of cyclic AMP. Exogenous cyclic AMP is metabolized rapidly by coleoptile tissues. If cyclic AMP does have a cellular function in the coleoptile, its transport behavior is compatible with that of a hormone. PMID- 16658509 TI - Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues. AB - Preparations of TPN-linked nonreversible d-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible d-glyceraldehyde 3 phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The properties of the enzyme were similar from each of these sources: the Km values for d-glyceraldehyde 3-phosphate and TPN were about 20 mum and 3 mum, respectively. The enzyme activity was inhibited by l glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and phosphohydroxypyruvate. Activity was found predominantly in photosynthetic and gluconeogenic tissues of higher plants. A light-induced, phytochrome-mediated increase of enzyme activity in a photosynthetic tissue (pea shoots) was demonstrated. Appearance of enzyme activity in a gluconeogenic tissue (endosperm of castor bean, Ricinus communis) coincided with the conversion of fat to carbohydrate during germination. In photosynthetic tissue, the enzyme is located outside the chloroplast, and at in vivo levels of triose-phosphates and pyridine nucleotides, the activity is probably greater than that of DPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase. Several possible roles for the enzyme in plant carbohydrate metabolism are considered. PMID- 16658510 TI - Bicarbonate ion as a critical factor in photosynthetic oxygen evolution. AB - Bicarbonate ion, not dissolved CO(2) gas, is shown to increase 4- to 5-fold the rate of dichlorophenol indophenol reduction by isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed chloroplasts continue to exhibit bicarbonate dependent 2,6-dichlorophenol indophenol reduction. Bicarbonate is shown to act close to the oxygen-evolving site, i.e. prior to the electron donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis. PMID- 16658511 TI - De novo synthesis of phytochrome in pumpkin hooks. AB - Phytochrome becomes density labeled in the hook of pumpkin (Cucurbita pepo L.) seedlings grown in the dark on D(2)O, indicating that the protein moiety of the pigment is synthesized de novo during development. Red light causes a rapid decline of the total phytochrome level in the hook of etiolated seedlings but upon return to the dark, phytochrome again accumulates. These newly appearing molecules are also synthesized de novo. Newly synthesized phytochrome in both dark-grown and red-irradiated seedlings is in the red-absorbing form. Turnover of the red-absorbing form is indicated by the density labeling of phytochrome during a period when the total phytochrome level in the hook of dark-grown seedlings remains constant. However, it was not possible to determine whether this results from intracellular turnover or turnover of the whole cell population during hook growth. PMID- 16658512 TI - Turnover of phytochrome in pumpkin cotyledons. AB - By using density labeling, it was found that the protein moiety of phytochrome is synthesized de novo in the red-absorbing form in cotyledons of dark-grown pumpkin (Cucurbita pepo L.) seedlings, as well as those irradiated with red light and returned to the dark. The rate of synthesis appears to be unaffected by the light treatment. Turnover of the red-absorbing form was also detected in dark grown seedlings using density labeling, while turnover of the far red-absorbing form is already implied from the well known "destruction" observed in irradiated seedlings. In both cases, true degradation of the protein is involved, but the rate constant of degradation of the far red-absorbing form may be up to two orders of magnitude greater than that of the red-absorbing form. The data indicate that, in pumpkin cotyledons, phytochrome levels are regulated against a background of continuous synthesis through divergent rate constants of degradation of the red and far red-absorbing forms and the relative proportions of the two forms present. PMID- 16658513 TI - Effect of water on the ripening of pericarp disks from tomato fruits. AB - Color change, a measure of the ripening of pericarp disks of tomato fruits (Lycopersicon esculentum Mill. cv. Moneymaker), was delayed by osmotic water uptake. An even greater delay occurred when substances from the disks were allowed to leach out or to diffuse into agar, indicating the existence of a water soluble substance(s) necessary for the ripening process. Osmotic solutions, allowing for more leaching, were more inhibitory to color development than the same amount of distilled water. The ripening process of tomato fruit disks can thus be disturbed by such processes as washing, infiltration, or incubation with solutions. PMID- 16658514 TI - The interaction of respiration and photosynthesis in induction of nitrate reductase activity. AB - The respiration and photosynthesis requirement for induction and maintenance of nitrate reductase activity was determined on leaves of Hordeum vulgare L. In this induction, glucose substituted for light in both dark-grown and carbohydrate depleted green leaves. Oxygen appeared to be required for induction in all cases studied. In light and under N(2), 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely inhibited induction, presumably by inhibiting the production of O(2), Hence, under N(2) the leaves appeared to utilize both the O(2) produced by photosynthesis and the CO(2) produced by respiration. CO(2) fixation can then produce both photosynthate to drive the induction and terminal electron acceptors to allow photosynthetic electron flow. This possibility was further suggested by the observation that CO(2) was an absolute requirement for induction in carbohydrate-depleted barley leaves. Results obtained with respiratory inhibitors also indicated that respiration drove the induction of nitrate reductase.Exogenously supplied glucose also substantially slowed the loss of nitrate reductase that occurred when barley leaves were placed in darkness. It is presumed that glucose allowed the synthetic or activation phase of the induction to proceed more rapidly. Our results support the hypothesis that one of the main effects of light may be to supply photosynthate to support respiration, which then drives the induction process. PMID- 16658515 TI - The Effect of Arsenate and Other Inhibitors on Early Events during the Germination of Lettuce Seeds (Lactuca sativa L.). AB - The effect of arsenate, arsenite, 2,4-dinitrophenol, and anaerobiosis on early events in seed germination was investigated using both intact and punched seeds of lettuce (Lactuca sativa L.). It was found that punching the seed removes penetration barriers to the entrance of inhibitors without an undue loss of germination or light responses. The kinetics of the action of germination inhibitors were established by 2-hour pulse experiments. Arsenate and 2, 4 dinitrophenol have very different kinetics. The inhibition of germination in punched seeds by arsenate given in conjunction with phosphate compared with the lack of inhibition of arsenate plus phosphate on the growing seedling, suggest a distinct metabolic change in the germinating embryo at some time between the onset of germination and subsequent seedling growth. PMID- 16658516 TI - Organic acids and iron translocation in maize genotypes. AB - Translocation of Fe was studied in WF9 (Fe-efficient) and ys(1)/ys(1) (Fe inefficient) maize (Zea mays L.) genotypes. Iron-deficient WF9 translocated more Fe to the tops than Fe-deficient ys(1)/ys(1). Malate and citrate contents of root saps increased nearly 2-fold and aconitate increased over 4-fold in both genotypes as Fe of nutrient solutions increased from 0.1 to 3 milligrams per liter. Relative acid contents in root saps were as follows: malate > aconitate > citrate. Citric acid concentrations in stem exudates were nearly the same as in root sap. Malic acid concentrations were considerably lower in exudates than in root saps, and only a trace of aconitic acid was detected in the exudates. The concentration of Fe was 7-fold higher in exudate of WF9 than in exudate of ys(1)/ys(1) and the concentration of exudate P was about the same for both genotypes.Electropherograms of WF9 stem exudates showed that (59)Fe moved toward the anode as (59)Fe-citrate. Exudates of ys(1)/ys(1) contained insufficient (59)Fe to produce radiographs. When (59)Fe was added in vitro to ys(1)/ys(1) stem exudate, the (59)Fe moved as (59)Fe-citrate, indicating that sufficient citric acid was present in the exudate to chelate the Fe. Effectiveness of citric, isocitric, trans-aconitic, and malic acids in moving (59)Fe electrophoretically in acetate, citrate, isocitrate, trans-aconitate, and malate buffers was studied. Malic, acetic, and trans-aconitic acids were ineffective in moving Fe from the origin. Citric acid moved Fe anodically whenever present on the electropherogram and successfully competed with the other acids for Fe.Results with ys(1)/ys(1) roots indicate an absence of an efficient mechanism for transporting Fe from cortical cells to the xylem. If Fe can reach the xylem stream, the ys(1)/ys(1) genotype should be as efficient as WF9 in moving Fe to the leaves. PMID- 16658517 TI - Incorporation of C-Leucine into Apple Leaf Protein and Its Inhibition by Protein Synthesis Inhibitors during Growth and Senescence. AB - Of the total (14)C-leucine taken up by intact apple (Pyrus malus L., Golden Delicious) leaf discs, 44 to 62% is incorporated into protein from June to early October. Of this amount, an average of 35% is released by mild, room temperature acid hydrolysis. Prior to mid-August when leaf protein begins to decline, 15 to 20% of the (14)C-leucine incorporated into protein occurs in water-(buffer) soluble protein, of which only 3% is released by mild acid hydrolysis. After mid August, 40% of the label in protein occurs in soluble protein. The specific radio activity of the soluble protein increases by 4- to 5-fold after mid-August, while that of total protein increases by less than 2-fold. In presenescent leaves (before the decline of protein in August) 20 micrograms per milliliter cycloheximide inhibits the incorporation of (14)C-leucine into protein by 71%, and 20 micrograms per milliliter chloramphenicol inhibits it by 30%. In senescing leaves, cycloheximide inhibits (14)C-leucine by 85% or more, while chloramphenicol inhibits it by less than 15%. Coincident to the initial decline of leaf protein, chloramphenicol greatly loses its ability to inhibit the incorporation of (14)C-leucine into apple leaf protein. At all leaf ages, chloramphenicol increases the loss of chlorophyll from apple leaf discs. The effect of cycloheximide on leaf disc senescence changes with leaf age: in early season samples, it increases the loss of chlorophyll; in mid-season samples, it has no effect; and in late season samples, it retards the loss of chlorophyll. PMID- 16658518 TI - Fat Metabolism in Higher Plants: LVII. A Comparison of Fatty Acid-Synthesizing Enzymes in Chloroplasts Isolated from Mature and Immature Leaves of Spinach. AB - Chloroplasts isolated from immature leaves of spinach (Spinacia oleracea) differ in enzyme levels from those isolated from mature leaves. On a chlorophyll basis, immature chloroplast preparations had 5- to 6-fold higher capacity to synthesize fatty acids from 2-(14)C-acetate compared to plastids isolated from mature leaves. This difference was correlated with higher activities for the enzymes, acetyl coenzyme A synthetase, malonyl coenzyme A synthetase, acetyl coenzyme A carboxylase, and oleyl coenzyme A transferase in plastid pressates obtained from immature leaves. Disrupted chloroplast preparations from both mature and immature leaves retained the ability to incorporate 2-(14)C-acetate into fatty acids in a pattern similar to that by isolated chloroplasts. 2-(14)C-Acetate, 2-(14)C-acetyl coenzyme A, 2-(14)C-malonate, and 1,3-(14)C malonyl coenzyme A were readily incorporated into a number of fatty acids. Moreover, the synthesis of oleate by chloroplast pressates from these substrates was strongly inhibited by KCN, flavin adenine mononucleotides and dinucleotides, and anaerobic conditions, while linolenic acid synthesis was unaffected by these compounds. PMID- 16658519 TI - Mechanism of plant growth stimulation by naphthenic Acid: effects on nitrogen metabolism of phaseolus vulgaris L. AB - Fourteen-day-old Phaseolus vulgaris L. cv. Top Crop (bush bean) plants were sprayed with the plant growth stimulant, potassium naphthenate (20 mm). Seven days after treatment the contents of glutamic acid dehydrogenase, glutamic oxaloacetic transaminase, nitrate reductase, glutamine synthetase, and cytochrome oxidase in the trifoliate leaf blades of treated plants were significantly larger, and the specific activity of the last four was significantly greater. Potassium nephthenate (1 mum) in the assay solutions did not significantly alter the activity of these enzymes in the cell-free extracts of untreated plants. Leaf discs from treated plants did not incorporate (14)C-leucine into protein more actively. The protein content of leaves of treated plants was 15.3% greater, and the percentages of 16 individual amino acids in the hydrolysates of the proteins of control and treated plants showed numerous differences. The major changes were greater percentages of glutamic acid, glycine, and proline, and smaller values of arginine, lysine, tyrosine, and leucine in protein of treated plants. The content of ethanol-soluble (free) amino acids was greater by 7.5%. The principal changes in content of these acids were larger percentages of arginine and lysine, and smaller values for glutamic acid, serine, and proline in the leaves of potassium naphthenate-treated plants. The content of DNA, measured 1, 2, and 3 weeks after a foliar application of potassium naphthenate, was not significantly different from that of untreated plants, but the amount of RNA was significantly greater at all three times of measurement. The number and weight of green pods per plant 30 days after potassium naphthenate application were significantly larger, suggesting that the stimulative action of potassium naphthenate was in progress at the times of the assays. A mechanism, involving a genetic and a metabolic phase, is suggested for the stimulation of plant growth by naphthenate. PMID- 16658520 TI - Rhythmic Differences in the Basipetal Movement of Indoleacetic Acid between Separated Upper and Lower Halves of Geotropically Stimulated Corn Coleoptiles. AB - Rhythmic fluctuation in the basipetal movement of auxin occurs in corn (Zea mays) coleoptiles oriented either in the vertical or in the horizontal position. This periodicity of transport rate varies from region to region in a horizontal coleoptile. Between an upper and lower half coleoptile (with respect to gravity), the comparable regions in the coleoptile do not exhibit similar periods. The velocity of transport also varies from region to region along a geostimulated coleoptile. In the upper half coleoptile, the velocities are 29 millimeters per hour (tip), 8 millimeters per hour (mid), and 30 millimeters per hour (base); in the lower, 41 millimeters per hour (tip), 12 millimeters per hour (mid) and 12 millimeters per hour (base).During the first 24 minutes of transport, there is a reduction of basipetal movement of (14)C-indoleacetic acid in the lower half coleoptile. This may be causally related to the initial downward geotropic curvature in oat and corn coleoptiles reported by others. However, about 30 minutes after donor removal a significant reduction of basipetal transport of indoleacetic acid occurs in the upper half coleoptiles. PMID- 16658521 TI - Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips. AB - Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 mum and 10 mum). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 mum) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 mum) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production. PMID- 16658522 TI - Early Actions of Gibberellic Acid on the Embryo and on the Endosperm of Avena fatua Seeds. AB - Gibberellic acid at 0.1 mum stimulates amylase synthesis in dormant Avena fatua seeds without inducing germination; at 0.5 mm it enhances biosynthesis of proteins and RNA in both the embryo and the endosperm and utilization of the endosperm sugars by the embryo. These events occur in early hours (0-14th hour) and prior to germination, which begins 24 hours after gibberellic acid application. These observations are in agreemeent with the concept that in cereal grains gibberellic acid has two morphological sites of actions: the embryo and the endosperm, and that germination (radicle protrusion) is not caused by gibberellic acid-induced amylase synthesis in the endosperm. PMID- 16658523 TI - Ethylene, carbon dioxide, and anthocyanin synthesis. PMID- 16658524 TI - Plants under Climatic Stress: V. Chilling and Light Effects on Radiocarbon Exchange between Photosynthetic Intermediates of Sorghum. AB - Patterns of radiocarbon exchange between photosynthetic intermediates of the chilling sensitive Sorghum bicolor were modified by exposure to a combined environmental stress of low temperature (10 C) and moderate light levels (170 w.m(-2), visible). Pulse chase experiments with(14)CO(2) showed that this stress initially slowed the release of photosynthetically absorbed radiocarbon from malate. Further exposure caused an increased proportion of the radiocarbon to accumulate in aspartate. This trend continued, so that after 30 hours, some 80% of absorbed radiocarbon remained in aspartate after 1 minute of chasing and subsequent release of carbon into the C(3) cycle was very slow. In Sorghum, chilling combined with light seemed to cause a restriction in an early step of the C(4) pathway before ultrastructural changes could be detected in the mesophyll chloroplasts. PMID- 16658525 TI - Vanadium and plant nutrition: the growth of lettuce (lactuca sativa L.) and tomato (lycopersicon esculentum mill.) plants in nutrient solutions low in vanadium. AB - Lettuce (Lactuca sativa L.) and tomato (Lycopersicon esculentum Mill.) plants were grown in purified nutrient solutions with and without the addition of 50 nanograms per milliliter V. These experiments showed that lettuce and tomato plants can be grown to maturity on nutrient solutions containing less than 0.04 nanogram per milliliter V with tissue concentrations of less than 2 to 18 nanograms per gram V. Growth and dry matter yield were comparable to those of plants grown on nutrient solutions containing 50 nanograms per milliliter with tissue levels of V from 117 to 418 nanograms per gram. Thus if V is an essential element for lettuce and tomato plants, the adequate tissue level would be less than 2 nanograms per gram V derivable from a growth medium containing less than 0.04 nanogram per milliliter V. PMID- 16658526 TI - Inactivity of 3-methyleneoxindole as mediator of auxin action on cell elongation. AB - The recently reported growth-promoting ability of 3-methyl-eneoxindole was examined in order to test the hypothesis that indole-3-acetic acid acts as a growth promoter only after oxidative conversion to 3-methyleneoxindole. Methyleneoxindole was synthesized from indole-3-acetic acid and N bromosuccinimide, and its identity was confirmed by ultraviolet absorption, infrared absorption, mass spectrometry, and melting point. Methyleneoxindole was found to lack growth-promoting activity in coleoptile and pea (Pisum sativum) stem segments. Chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid, was found to have no inhibitory effect on growth promotion by indole-3 acetic acid. It is concluded that 3-methyleneoxindole is inactive as a growth promoter and therefore does not mediate the action of auxin on cell elongation. PMID- 16658527 TI - The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum: II. Production and Utilization of Exudate from Stigma and Stylar Canal. AB - Detached pistils of Lilium longiflorum were labeled with d-glucose-U-(14)C 24 hours after anthesis and then sampled for the next 6 days to determine the appearance of label into exudate from the stylar canal and the stigmatic surface of the pistil. Results were obtained with unpollinated cv. Ace pistils and pollinated cv. Ace pistils, selfed or crossed (cv. Croft pollen). Limited data were also obtained on cv. Croft pistils, selfed or crossed (cv. Ace pollen).Exudate appeared in the canal and on the stigmatic surface soon after anthesis. In unpollinated pistils it continued to accumulate for about 5 days in the canal and for the full term of the experiment, 7 days, on the stigmatic surface. Canal exudate eventually mixed with stigmatic exudate in unpollinated pistils. Pollination interrupted the flow of exudate, and a portion of the pistil secretion product was diverted toward the synthetic requirements of the developing pollen tubes. Two days after pollination, the specific radioactivity of pollen tube cytoplasm had reached a level comparable to canal exudate. Twenty four hours later, the specific radioactivity of pollen tube wall substance was about 80% of the value found in canal exudate. Similar patterns of (14)C incorporation and similar carbohydrate contents were obtained from self- and cross-pollinated pistils, although the former contained pollen tubes of less than one-half the length of the latter. PMID- 16658528 TI - Effects of Filipin and Cholesterol on K Movement in Etiolated Stem Cells of Pisum sativum L. AB - Filipin, a polyene antibiotic known to induce leakage of materials from various cells, depresses K(+) and NO(3) (-) uptake in etiolated pea epicotyl segments. Filipin concentrations which strongly reduce K(+) influx have little effect on efflux; however, high concentrations enhance K(+) efflux. Filipin has no effect on respiration rates or cell electropotentials; its action is presumed to be on the cell membranes. Cholesterol, but not a thiol-protecting agent (dithiothreitol), enhances K(+) influx and counteracts the inhibition by filipin. Although this effect of cholesterol may be due to an interaction with filipin in the outer solution, there is reason to believe that its major effect is to impart stability to the membrane; filipin is believed to act by interfering with sterol stabilization of phospholipid layers. The predominant native sterols of etiolated pea stem (Pisum sativum L. var. Alaska), which cholesterol probably mimics, are beta-sitosterol, campesterol, and stigmasterol. PMID- 16658529 TI - Isolation and Characterization of Glucosamine-containing Storage Glycoproteins from the Cotyledons of Phaseolus aureus. AB - Cotyledons of Phaseolus aureus contain protein-bound glucosamine which is metabolized during germination. The glucosamine is present in storage glycoproteins, and these are concurrently metabolized along with the glucosamine. These glycoproteins are associated with protein bodies. Characterization of the glucosamine-containing storage proteins showed them to be identical with vicilin and legumin, the major storage proteins of the Leguminosae. Phaseolus aureus vicilin has a sedimentation constant of 8.0S and is made up of four nonidentical subunits. It contains 0.2% glucosamine and 1% mannose. Legumin has a sedimentation constant of 11.3S and is made up of three nonidentical subunits. It contains about 0.1% glucosamine. PMID- 16658530 TI - The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling. AB - In a study on 3-day maize (Zea mays) seedlings, grown on nitrate, requirements were established for the maximum extraction and optimum stabilization of nitrate reductase in vitro. With the primary root, 5 mm cysteine were required in the extraction medium, but for the scutellum, which has a high level of endogenous thiol, the use of additional thiol resulted in a reduced yield of a more labile enzyme. Activity of the root and scutella nitrate reductase was obtained with either NADH or NADPH, but that of the root enzyme with NADPH was only demonstrated in the absence of phosphate.Before leaf expansion, the nitrate reductase in the maize seedling was mainly in the scutellum. The enzyme present in the primary root was predominantly in the apical region (0-2 mm). In contrast, glutamate dehydrogenase was concentrated in the mature basal region of the root (30-60 mm). A high level of nitrate (approximately 100 mm) was required to saturate the induction of nitrate reductase in the root tip, mature root, and scutellum. The concentration of nitrate required to give half the maximum level of enzyme induced was the same for each region (29 mm).After leaf expansion, more than 90% of the nitrate reductase was in the shoot, mainly in the leaf blade, and a marked decrease occurred in the level of the enzyme in the scutellum. A large proportion of the glutamate dehydrogenase was still found in the root. PMID- 16658531 TI - A nitrate reductase inactivating enzyme from the maize root. AB - The nitrate reductase in the mature root extract of 3-day maize (Zea mays) seedlings was relatively labile in vitro. Insoluble polyvinylpyrrolidone used in the extraction medium produced only a slight increase in the stability of the enzyme. Mixing the mature root extract with that of the root tip promoted the inactivation of nitrate reductase in the latter. The inactivating factor in the mature root was separated from nitrate reductase by (NH(4))(2)SO(4) precipitation. Nitrate reductase was found in the 40% (NH(4))(2)SO(4) precipitate, while the inactivating factor was largely precipitated by 40 to 55% (NH(4))(2)SO(4). The latter fraction of the mature root inactivated the nitrate reductase isolated from the root tip, mature root, and scutellum. The inactivating factor, which has a Q(10) 15 to 25 C of 2.2, was heat labile, and hence has been designated as a nitrate reductase inactivating enzyme. The reduced flavin mononucleotide nitrate reductase was also inactivated, while an NADH cytochrome c reductase in nitrate-grown seedlings was inactivated but at a slower rate. The inactivating enzyme had no influence on the activity of nitrite reductase, glutamate dehydrogenase, xanthine oxidase, and isocitrate lyase. The activity of the nitrate reductase inactivating enzyme was not influenced by nitrate and was also found in the mature root of minus nitrate-grown seedlings. PMID- 16658532 TI - Rhythmic Leaflet Movement in Albizzia julibrissin: Effect of Electrolytes and Temperature Alteration. AB - The rhythmic movement of darkened Albizzia leaflets is accompanied by K(+) flux in pulvinule motor cells whose turgor changes control opening and closing. The azide-sensitive open phase is promoted by an increase in temperature from 16 to 33C (Q(10) = 3), implying active transport of K(+) ions during this period. The azide-insensitive closed phase is less temperature-sensitive and has a Q(10) less than 1, implying diffusion or some other physical process as the predominant pathway of K(+) flux at this time. Thus rhythmic leaflet movement is probably due to oscillation in active K(+) transport or membrane permeability or both. External electrolytes (0. 1 n) alter leaflet angle during the open, but not the closed, phase of the rhythm. All chlorides except NH(4) (+) promote opening, with divalent more effective than monovalent ions. Some anions promote and others inhibit opening; activity is not correlated with charge. It is likely that electrolytes alter leaflet movement by altering K(+) flux, accomplishing this by interacting with key macromolecules in motor cell membranes.Pfr phytochrome dampens the amplitude of rhythmic leaflet movement; this process is temperature sensitive (Q(10) = 2) and unaltered by 0.1 n salt solutions. Although K(+) flux is a common basis for phytochrome and rhythmic control of leaflet movement, different mechanisms are clearly involved. PMID- 16658533 TI - Hormonal control of orthophosphate incorporation into phospholipids of barley aleurone layers. AB - Gibberellic acid added to isolated barley aleurone layers enhances orthophosphate incorporation into chloroform-methanol-soluble compounds. The effect is measurable at 4 to 6 hours after the addition of gibberellic acid and reaches a maximum after 8 to 12 hours. The increase in the rate of orthophosphate incorporation is 3- to 5-fold over the rate in control layers incubated without gibberellic acid.The gibberellic acid enhancement of the rate of phospholipid labeling is inhibited within 1 to 2 hours by cycloheximide, 6-methylpurine, and abscisic acid.The increase in labeling of phospholipids occurs throughout the subcellular fractions rather than being restricted to a specific fraction or organelle. The increase in radioactivity in phospholipids as shown by thin layer chromatography is due to a proportional increase in all phospholipids.The enhancement by gibberellic acid of the rate of phospholipid labeling may be required for the subsequent production of gibberellic acid-induced hydrolases. PMID- 16658534 TI - Flower Formation in Excised Tobacco Stem Segments: III. Deoxyribonucleic Acid Content in Stem Tissue of Vegetative and Flowering Tobacco Plants. AB - A method has been developed that extracts DNA from stem tissue of flowering tobacco plants, Nicotiana tabacum cv. Wis. 38. The DNA content of stem tissue from a flowering tobacco plant is correlated with its capacity to flower in vitro. Stem segments known to form 100% floral buds contain 10 times more DNA per gram fresh weight than segments that form 5% floral buds and 95% vegetative buds, and in the uppermost 28 centimeters of flowering tobacco plant stems the DNA content decreases roughly in parallel with the floral gradient.((3)H)-Thymidine incorporation into DNA in the internodes is inhibited by attached leaves in flowering tobacco plants but promoted in vegetative plants. Low indoleacetic acid concentrations (2.8 and 5.7 mum) inhibit DNA synthesis in internodes of defoliated stems of flowering tobacco plants to the same extent as attached leaves, whereas the same concentrations of indoleacetic acid promote DNA synthesis in internodes of defoliated stems of vegetative plants. The optimal concentration (11.4 mum) of indoleacetic acid supplied to defoliated stems of vegetative plants increased the rate of DNA synthesis to 4 times the rate in defoliated stems without indoleacetic acid treatment. This increase more than compensates for the promotion of DNA synthesis by the young leaves. Thus, the opposite effects of young leaves on DNA synthesis in internodes of flowering and vegetative tobacco plants may be related to their auxin content. PMID- 16658535 TI - Regulation of invertase levels in Avena stem segments by gibberellic Acid, sucrose, glucose, and fructose. AB - Gibberellic acid and sucrose play significant roles in the increases in invertase and growth in Avena stem segments. About 80% of invertase is readily solubilized, whereas the rest is in the cell wall fraction. The levels of both types of invertase change in a similar manner in the response to gibberellic acid and sucrose treatment. The work described here was carried out with only the soluble enzyme. In response to a treatment, the level of invertase activity typically follows a pattern of increase followed by decrease; the increase in activity is approximately correlated with the active growth phase, whereas the decrease in activity is initiated when growth of the segments slows. A continuous supply of gibberellic acid retards the decline of enzyme activity. When gibberellic acid was pulsed to the segments treated with or without sucrose, the level of invertase activity increased at least twice as high in the presence of sucrose as in its absence, but the lag period is longer with sucrose present. Cycloheximide treatments effectively abolish the gibberellic acid-promoted growth, and the level of enzyme activity drops rapidly. Decay of invertase activity in response to cycloheximide treatment occurs regardless of gibberellic acid or sucrose treatment or both, and it is generally faster when the inhibitor is administered at the peak of enzyme induction than when given at its rising phase. Pulses with sucrose, glucose, fructose, or glucose + fructose elevate the level of invertase significantly with a lag of about 5 to 10 hours. The increase in invertase activity elicited by a sucrose pulse is about one-third that caused by a gibberellic acid pulse given at a comparable time during mid-phase of enzyme induction, and the lag before the enzyme activity increases is nearly twice as long for sucrose as for gibberellic acid. Moreover, the gibberellic acid pulse results in about three times more growth than the sucrose pulse. Our studies support the view that gibberellic acid, as well as substrate (sucrose) and end products (glucose and fructose), play a significant role in regulating invertase levels in Avena stem tissue, and that such regulation provides a mechanism for increasing the level of soluble saccharides needed for gibberellic acid-promoted growth. PMID- 16658536 TI - Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach. AB - A shortage in the zinc supply to spinach (Spinacia oleracea L.) drastically reduced carbonic anhydrase levels with little effect on net CO(2) uptake per unit leaf area, except with the most severe zinc stresses. Under these conditions, carbonic anhydrase was below 10% and photosynthesis 60 to 70% of the control levels. When photosynthesis was measured at a range of CO(2) supply levels, zinc deficient leaves were less efficient at 300 to 350 microliters per liter CO(2) and above, but the same as controls at lower CO(2) levels. This suggests that carbonic anhydrase does not affect the diffusion of CO(2), and that the effect of zinc deficiency was on the photosynthetic process itself. Our evidence does not support the hypothesis that carbonic anhydrase has some role in facilitating the supply of CO(2) to the sites of carboxylation within the chloroplast. PMID- 16658537 TI - Cholinesterases from Plant Tissues: II. Inhibition of Bean Cholinesterase by 2 Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine Carboxylate Methyl Chloride (AMO-1618). AB - 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO-1618) inhibits the activity of a cholinesterase isolated from mung bean (Phaseolus aureus) roots at concentrations comparable to those which retard growth and inhibit development of secondary roots of bean seedlings. Fifty per cent inhibition of the cholinesterase activity occurred at 0.21 mm. Inhibition of the cholinesterase is non-competitive and irreversible. The growth retardant seems to be a specific cholinesterase inhibitor since it has no effect on non choline esterases. PMID- 16658538 TI - Deferral of leaf senescence with calcium. AB - In view of the possibility that senescence may be a consequence of the deterioration of membrane compartments in the cells of leaves, calcium was studied as a possible agent which might defer senescence. The senescence of corn leaf discs was deferred by added calcium, and the effect was additive to the cytokinin deferral of senescence. Likewise, the senescence of Rumex leaf discs was deferred by added calcium, and the effect was additive to the gibberellin deferral of senescence. Detailed experiments with corn leaf discs established that the increase in apparent free space associated with senescence was completely prevented by calcium. An increase in hydraulic permeability during senescence was likewise demonstrated, and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Each of the measured functions of leaf senescence (chlorophyll content, protein decrease, apparent free space increase, and hydraulic permeability increase) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cellular membranes. PMID- 16658539 TI - Plastid differentiation, acyl lipid, and Fatty Acid changes in developing green maize leaves. AB - Plastid differentiation, acyl lipid, and fatty acid composition have been followed in successive 2-cm sections from the base (youngest tissue) to the tip (oldest tissue) of green Zea mays (maize) leaves grown under a normal diurnal light regime. Although the youngest cells (0-4 cm from the leaf base) had only proplastids with one or two grana, they contained chlorophylls a and b, monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. In the more mature sections, the plastids increased in size 5-fold, and differentiation into mesophyll and bundle-shealth chloroplasts had occurred. Concomitantly, the levels of all the lipids increased with the exception of phosphatidylcholine and phosphatidylethanolamine which decreased. With increasing cell maturity, the percentage of linolenic acid increased in all the individual acyl lipids, but palmitic acid remained constant in phosphatidylcholine, phosphatidylethanolamine, and sulfolipid. The Delta(3t)-hexadecenoic acid was only detectable in the phosphatidylglycerol of the most mature maize tissue. PMID- 16658540 TI - Triparanol Inhibition of Sterol Biosynthesis in Chlorella ellipsoidea. AB - The sterol composition of C. ellipsoidea was markedly changed when this alga was grown in the presence of 1 mug/g triparanol. Triparanol appears to inhibit the removal of 14alpha-methyl group, the second alkylation at C-24, Delta(7) reductase, and Delta(8) --> Delta(7)-isomerase. The effect of triparanol in Chlorella is much more diversified than the specific effect originally assigned to it in animals. PMID- 16658541 TI - Turgor-dependent Changes in Avena Coleoptile Cell Wall Composition. AB - The effects of reduced turgor pressure on growth, as measured by cell elongation, and on auxin-mediated changes in cell walls, as measured by analyses of wall composition, were examined using Avena coleoptile segments. Although moderate (1 4 bar) decreases in turgor resulted in a progressive decline in growth proportional to the decrease in turgor, the major auxin-induced change in wall composition, a decrease in noncellulosic wall glucose, was unaffected. Severe (5 8 bar) decreases, however, did inhibit this auxin effect on the wall, and with turgor decreases of 9 bars or more this auxin effect was no longer apparent. The results show that turgor pressure is required for this auxin-mediated wall modification and also that this modification of wall glucose occurs at turgor pressures less than those required for wall extension. Changes in other wall components were generally unaffected by altering turgor pressure. PMID- 16658542 TI - Separation and characterization of endopolygalacturonase and exopolygalacturonase from peaches. AB - Two polygalacturonases (PG I and PG II) have been separated from extracts of ripe peaches (Prunus persica) by chromatography on Sephadex G-100. PG I hydrolyzes polygalacturonic acid from the nonreducing ends of the molecules, releasing galacturonic acid as the product. It functions optimally at pH 5.5, requires Ca(2+) for activity, and hydrolyzes low molecular weight substrates most rapidly. In contrast, PG II cleaves the molecular chain of the substrate randomly with a pH optimum at about 4. This enzyme is most reactive with substrates of intermediate molecular weight. It catalyzes the release of water-soluble, but 70% ethanol-insoluble, pectin from washed peach cell walls. PMID- 16658543 TI - Photochemical properties of mesophyll and bundle sheath chloroplasts of maize. AB - Several photochemical and spectral properties of maize (Zea mays) bundle sheath and mesophyll chloroplasts are reported that provide a better understanding of the photosynthetic apparatus of C(4) plants. The difference absorption spectrum at 298 K and the fluorescence excitation and emission spectra of chlorophyll at 298 K and 77 K provide new information on the different forms of chlorophyll a in bundle sheath and mesophyll chloroplasts: the former contain, relative to short wavelength chlorophyll a forms, more long wavelength chlorophyll a form (e.g. chlorophyll a 693 and chlorophyll a 705) and less chlorophyll b than the latter. The degree of polarization of chlorophyll a fluorescence is 6% in bundle sheath and 4% in mesophyll chloroplasts. This result is consistent with the presence of relatively high amounts of oriented long wavelength forms of chlorophyll a in bundle sheath compared to mesophyll chloroplasts. The relative yield of variable, with respect to constant, chorophyll a fluorescence in mesophyll chloroplasts is more than twice that in bundle sheath chloroplast. Furthermore, the relative yield of total chlorophyll a fluorescence is 40% lower in bundle sheath compared to that in mesophyll chloroplasts. This is in agreement with the presence of the higher ratio of the weakly fluorescent pigment system I to pigment system II in bundle sheath than in mesophyll chloroplast. The efficiency of energy transfer from chlorophyll b and carotenoids to chlorophyll a are calculated to be 100 and 50%, respectively, in both types of chloroplasts. Fluorescence quenching of atebrin, reflecting high energy state of chloroplasts, is 10 times higher in mesophyll chloroplasts than in bundle sheath chloroplasts during noncyclic electron flow but is equal during cyclic flow. The entire electron transport chain is shown to be present in both types of chloroplasts, as inferred from the antagonistic effect of red (650 nm) and far red (710 nm) lights on the absorbance changes at 559 nm and 553 nm, and the photoreduction of methyl viologen from H(2)O. (The rate of methyl viologen photoreduction in bundle sheath chloroplasts was 40% of that of mesophyll chloroplasts.). PMID- 16658544 TI - Peroxidase Activity in the Abscission Zone of Bean Leaves during Abscission. AB - Peroxidase activity and localization in the abscission zone of bean leaves were studied histochemically and by gel electrophoresis. Deblading of bean leaves resulted in an increase in peroxidase activity in the abscission zone 2 to 4 days after deblading with highest activity just prior to separation. In debladed plants, the cell division in six to eight layers of cells preceded separation. An ethylene treatment (8 microliters per liter) induced separation of debladed petioles in approximately 24 hours and of intact plants in 36 to 48 hours. Ethylene treatment produced similar results in both debladed and intact plants. In ethylene-treated plants, whether debladed or not, enzyme localization was restricted to only two to three layers of cells with no cell division apparent prior to separation. Infrequent cell divisions were observed after treatment with 2-chloroethylphosphonic acid (1000 micrograms per liter) (Ethephon); however, other changes were similar to those observed with ethylene. Deblading and ethylene treatment resulted in changes in the six peroxidase isozymes observed in the abscission zone. Only four were observed in samples collected 2 centimeters below the abscission zone. Peroxidase bands IV and V increased significantly in debladed and ethylene-treated plants and peroxidase VI decreased only in debladed plants. The changes in peroxidase activity were invariably observed prior to separation in all treatments. PMID- 16658545 TI - Lipids in rye seedlings in relation to vernalization. AB - Increasing the chilling time from 1 to 8 weeks decreased the time to heading of winter rye (Secale cereale var. Sangaste) to approximate that of the spring variety (Prolific). On a dry weight basis, the total phospholipid content of the embryos was higher in Sangaste but declined in both varieties during chilling. The proportions of the individual phospholipid components were similar for both varieties and showed similar responses during the 8-week chilling period. Phosphatidylcholine declined and phosphatidic acid increased in both varieties during the treatment.During the initial 3 weeks, an increased accumulation of linolenic acid and a corresponding decline in linoleic acid occurred for all the lipid components and then remained relatively stable. The glycolipids were more unsaturated than the phospholipids; however, the amount of linolenate was approximately doubled in both during the treatment. In general, the fatty acid content of the respective lipid classes were similar for both varieties. PMID- 16658546 TI - Changes in the Activity of Some Hydrolases, Peroxidase, and Catalase in the Rice Seed during Germination. AB - A study was made of the changes in activity of enzymes involved in the breakdown of stored phytin, lipid, and hemicellulose in the aleurone layer of rice seed (Oryza sativa L., variety IR8) during the 1st week of germination in the light. Enzyme assays were made on crude extracts from degermed seed, and activities were expressed on a per seed basis. Phytase activity increased within the 1st day of germination. The increase in activity of most other enzymes-phosphomonoesterase, phosphodiesterase, esterase, lipase, peroxidase, catalase, beta-glucosidase, and alpha- and beta-galactosidase-closely followed the increase in protein content. Their peak activities occurred by the 5th to the 7th day. Some enzymes, such as beta-1, 3-glucanase and alpha-amylase, continued to increase in activity after the 7th day. Phytase, beta-1, 3-glucanase, and alpha-amylase followed a similar sequence of production in embryoless seed halves incubated in 0.12 muM gibberellin A(3), but the production of lipase was delayed. PMID- 16658547 TI - Cyclic nucleotide phosphodiesterase activity in barley seeds. AB - Barley seeds (Hordeum vulgare L. cv. Himalaya) contain an enzymatic activity which catalyzes the hydrolysis of adenosine cyclic 3': 5'-monophosphate and adenosine cyclic 2': 3'-monophosphate. A large portion of the enzymatic activity is present in the dry seed, existing in both soluble and particulate form. Secretion of the soluble phosphodiesterase from embryoless seeds is enhanced by gibberellic acid and inhibited by abscisic acid, dinitrophenol, and cycloheximide. Attempts to isolate or detect a phosphodiesterase which specifically hydrolyzes adenosine cyclic 3': 5'-monophosphate were unsuccessful. Inhibition experiments indicate that probably one enzyme is involved in the hydrolysis of both of these substrates. PMID- 16658548 TI - Photosynthetic Reactions in the Marine Alga Codium vermilara: I. CO(2) Fixation and Hill Reaction in Isolated Chloroplasts. AB - Chloroplasts were isolated from the marine alga Codium vermilara (Siphonales). The isolated chloroplasts were active in CO(2) fixation in the light at a rate comparable to the rates obtained by fragments of thalli. Maximal rates of CO(2) fixation by isolated chloroplasts from Codium were obtained in the presence of salt or sorbitol isoosmotic with sea water. The conditions of isolation of Codium chloroplasts are much less stringent than those required for active chloroplasts from higher plants. The isolated chloroplasts comprise a homogeneous population of the intact "class I" type, as based on microscopic observations and on their inability to reduce ferricyanide unless osmotically shocked. The intact chloroplasts are able to reduce p-benzoquinone at a high rate. PMID- 16658549 TI - Dark Release of CO(2) from Higher Plant Leaves. AB - A study was conducted with 48 species of the amount of (14)CO(2) released during the first minute of dark following fixation of (14)CO(2) in the light. Light fixation periods varied from 5 to 60 seconds. The species examined included both monocots and dicots and represented C(4), C(3), and Crassulacean acid metabolism (CAM) photosynthetic types.Total CO(2) uptake was determined as the sum of soluble (14)C (ethanol and water), insoluble (14)C, and (14)CO(2) released during the first minute of dark. Insoluble (14)C made up a very small percent of the total. The percent of soluble (14)C increased with fixation time in C(4) species, while the percent of (14)CO(2) released in the dark decreased. C(3) species released a very small percent of (14)CO(2) in the dark, and this percent remained relatively constant with time of (14)CO(2) fixation whereas C(4) species released a higher percent initially and this percent declined with time of fixation. CAM species were similar to C(4) species.The species are grouped according to the (14)CO(2) released during the first minute of dark with light labeling times of 5 and 60 seconds. These comparisons are shown to separate C(3) from C(4) and CAM species. Further, the data suggest that the origin of the CO(2) in the dark release of C(4) plants is quite different from that of C(3) plants. PMID- 16658550 TI - Specific degradation of a plant leucyl transfer ribonucleic Acid by a factor in the homologous synthetase preparation. AB - Partially purified aminoacyl synthetase preparations from pea roots (Pisum sativum L. var. Alaska) contain a heat-labile factor which can degrade leucyl tRNA(6) (leu) to a new species. The singular electrophoretic and chromatographic mobilities, the isoprenoid nucleoside content, and the charging characteristics of the new species (designated leucyl-tRNA(L) (leu)), suggest that it is a fragment of tRNA(6) (leu) containing at least that portion of the original molecule extending from the 3' terminus to the anticodon. Conversion appears to be highly specific since neither bulk tRNA, the other leucine tRNA subspecies, nor tyrosine, phenylalanine, or tryptophan tRNAs are susceptible to degradation during incubation with the synthetase preparation. PMID- 16658551 TI - Increased electron density of tonoplast membranes in washed corn root tissue. AB - Washing corn (Zea mays L.) root segments for 4 hours results in increased tonoplast electron density in fixed and stained tissue. It suggested that previously reported increases in solute absorption rate with root tissue washing may lie with changes in the properties of the tonoplast. PMID- 16658552 TI - Gibberellic Acid and Ion Release from Barley Aleurone Tissue: Evidence for Hormone-dependent Ion Transport Capacity. AB - The release of potassium, magnesium, and phosphate ions from aleurone cells of barley (Hordeum vulgare L. cv. Himalaya) is a gibberellic acid-dependent process. The release of these ions is preceded by a lag period of 6 to 8 hours after gibberellic acid addition. The effect of gibberellic acid on the release of ions is not mediated through an effect on ion solubilization. Thus, gibberellic acid does not apreciably affect the sum of extracted and released ions relative to controls. Rather, the effect of the hormone is on the release process itself. Inhibitors of oxidative phosphorylation when added with gibberellic acid or at times up to 6 hours after gibberellic acid inhibition release. When these inhibitors are added after ion release has begun, however, rapid efflux of ions occurs. These results suggest a strong correlation between energy levels and ion transport capacity. Inhibitors of RNA and protein synthesis also inhibit gibberellic acid-stimulated ion release. Evidence suggests that RNA and protein synthesis are required to establish and maintain ion release capacity of aleurone cells. PMID- 16658553 TI - Phytochrome destruction: an apparent requirement for protein synthesis in the induction of the destruction mechanism. AB - Examination of the phytochrome destruction reaction as a function of age in etiolated oat (Avena sativa L. cv. Garry) seedlings demonstrates that following illumination of 3-day-old shoots there is a lag, not observed in 4- or 5-day-old oats, prior to the onset of destruction. This light-mediated induction of the phytochrome destruction mechanism in 3-day-old shoots is inhibited by chloramphenicol, actinomycin D, and puromycin suggesting that protein synthesis is required. In 4-day-old shoots, actinomycin D and puromycin do not alter the kinetics of destruction while chloramphenicol partially inhibits the process. Thus, the inhibitors have a specific effect on the induction of the destruction mechanism but not its subsequent operation. PMID- 16658554 TI - Pyruvate kinase, a possible regulatory enzyme in higher plants. AB - A number of plant species were examined for the presence of pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40), and of a phosphatase activity which hydrolyzes phosphoenolpyruvate. Of those examined, only cotton (Gossypium sp. L.) seeds were found to be sufficiently free of the phosphatase to permit a kinetic study of pyruvate kinase.During germination of cotton seeds, pyruvate kinase activity rises for the first 3 days, after which it falls back to its original level. This developmental pattern is characteristic of enzymes involved in the conversion of fat into carbohydrate in fatstoring seeds. The phosphatase also rose rapidly during germination, which precluded the use of extracts from seedlings in the study of pyruvate kinase. No evidence was found for the presence of more than one pyruvate kinase in cotton seedlings.In crude extracts from ungerminated seeds, the enzyme shows slight deviations from normal kinetics with respect to phosphoenolpyruvate, magnesium, and to a lesser extent, ADP. After partial purification of the enzyme by ion exchange chromatography, the enzyme shows normal kinetics. The enzyme is activated by AMP, and inhibited by both ATP and citrate, in both crude and partially purified preparations. It is suggested that cotton seed pyruvate kinase is a regulatory enzyme. PMID- 16658555 TI - The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey. AB - Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O(2) uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate. PMID- 16658556 TI - Influence of Temperature on Sterol Biosynthesis in Triticum aestivum. AB - Sitosterol, campesterol, stigmasterol, and cholesterol were isolated from green wheat (Triticium aestivum var. Monon) seedlings. Sitosterol was the predominant sterol extracted from the shoot, root, and crown tissue. Cholesterol accounted for less that 1% of sterol in shoot tissue with only trace amounts in the root. A temperature change from 10 to 1 C resulted in a general decrease in sitosterol, stigmasterol, and campesterol in the shoot tissue. The cholesterol level was not altered significantly by the temperature change. The sterols in the root responded in a manner very different from those in the shoots. With the reduction in temperature, sterols first decreased and then recovered over a period of 7 to 14 days to levels that were equal to or exceeded the original levels. From these experiments, it would appear that root tissue can acclimate to the lower temperatures and continue sterol synthesis at the normal rate. The level and response of sterols in the crown tissue were intermediate between the root and shoot tissue. At 10 C the crown response was similar to that of root tissue, whereas, at 1 C the response more closely resembled that of the shoot. PMID- 16658557 TI - Phytochrome properties and the molecular environment. AB - In vitro data support a scheme of phytochrome phototransformation involving intermediates in a sequential pathway. The fraction of total phytochrome maintained as intermediate under conditions of pigment cycling as well as the rate of the dark reversion of the far red-absorbing (Pfr) to the red-absorbing form of phytochrome (Pr) has been shown to depend on the molecular environment of the phytochrome molecules. Inverse dark reversion of Pr to Pfr has been observed in vitro. These results contribute toward an understanding of the observed paradoxes between physiological experiments and measurements of the amount and state of phytochrome in vivo. The in vivo spectrophotometric assay measures an average of the properties of phytochrome in different cellular environments, whereas a particular physiological response may be controlled by phytochrome molecules in one particular environment. It is therefore possible that all phytochrome is potentially active and triggers specific responses by virtue of its localization. PMID- 16658558 TI - Mineral nutrient requirements of cucumber seedlings. AB - Mineral nutrient requirements for maximum growth rate of cucumber (Cucumis sativus L.) seedlings are estimated on the basis of three criteria. 1. The optimal weight proportions among the nutrients present in the seedlings. In relation to N = 100, close to 75 K, 13 P, 9 Ca, and 9 Mg are required. 2. The optimal ratio between the nitrogen sources NH(4) and NO(3) in the solution. The best growth is recorded with about equivalent amounts. Nitrate alone is also highly productive. Cucumber prefers nitrate and is sensitive to high ammonium concentrations. 3. The optimal total concentration in the solution corresponds to 200 to 300 milligrams of nitrogen per liter, with the proportions of the nutrients according to criterion 1. Simple growth methods are suggested in which the three criteria are fulfilled.Calcium, especially, is found to accumulate in older leaves. It is proposed that this is not an expression for a physiological requirement for calcium but a mechanism for deposition of excess uptake important in adaptation to calcareous soils. This mechanism also operates at low calcium uptake rates, causing deficiency in the young parts, despite relatively high contents in the old leaves. Cucumber is therefore interpreted to be an obligate calcicole. The results are discussed in relation to similar experiments with birch (Betula verrucosa Ehrh.) seedlings, a species able to grow on very acid soils. PMID- 16658559 TI - Polyribosomes from Peas: II. Polyribosome Metabolism during Normal and Hormone induced Growth. AB - Polyribosomes as large as 10-mers (strands of messenger RNA bearing 10 ribosomes) were isolated from etiolated pea (Pisum sativum L. var. Alaska) stem tissue during all stages of development when methods were used which essentially eliminated ribonuclease activity during extraction. Actively growing tissue, harvested from the apical 10 mm, yielded many large polyribosomes and a low (<20%) proportion of monosomes. Similar tissue, allowed to age by applying lanolin to decapitated apices, showed a progressive decrease in number of larger polyribosomes and an increase in the proportion of monosomes. Hormone treatments, which prolonged growth and delayed aging, delayed the loss in large polyribosomes and the increase in proportion of monosomes. Growth-stimulating hormones, added to previously aged tissue, stimulated the production of many large polyribosomes in pre-existing cells.It is suggested that (a) large polyribosomes occur in all regions of the pea stem, (b) changes in polyribosome distribution appear to precede changes in growth rate, (c) loss of larger polyribosomes is closely related to a decrease in mRNA templates followed more gradually by loss of ribosomes, (d) hormone-stimulated continuation of growth is accomplished through maintenance of available mRNA.Methods are described, involving detailed analysis of polysome distribution, which, although they cannot be used to measure changes in initiation of ribosomes on to mRNA, do permit measurement of the amount of polysomal-associated mRNA present in tissues at different stages of growth. These analyses lead to the further suggestion that hormone stimulation of growth of previously nongrowing tissue is accomplished primarily through an increase in available mRNA prior to synthesis of ribosomes. PMID- 16658560 TI - The velocities of ion transport into and through the xylem of roots: findings with a two-point application pulse-chase technique. AB - Excised corn roots, Zea mays, had radioactively labeled solution applied at two points along their length, for 1 minute, and were then kept in dilute, unlabeled nutrient solution. During this "chase" period, exudate was collected at 1-minute intervals, and its content of radioions was determined. Two pulses of label appeared in succession, originating at the points of application near the cut end of the root and farther from the cut end, respectively. Calculation yields the velocity at which the ions moved radially across the root into the xylem (v(r)) and the velocity at which they moved longitudinally within the xylem (v(l)). For Rb(+) labeled with (86)Rb, v(r) was 1.8 and v(l), 35 cm/hr. For Br(-) labeled with (82)Br, v(r) was 1.4 and v(l), 103 cm/hr. PMID- 16658561 TI - Some Physiological Effects of Viviparous Genes vp(1) and vp(5) on Developing Maize Kernels. AB - The effects of two viviparous genes, vp(1) and vp(5), on development of the maize (Zea mays L.) embryo and endosperm were investigated. Differences between viviparous and normal embryos first appeared at 25 to 30 days after pollination. Increases in fresh weights indicated that viviparous began to grow more rapidly than normal embryos at that time. Amino acids and ethanol-soluble carbohydrates also accumulated more rapidly in viviparous, but a reserve material (lipid) was lower in viviparous than in normal embryos.The fresh and dry weights and total nitrogen content of endosperms from viviparous resembled those of normal seeds until about 30 days after pollination, but were all lower in viviparous after that time. Pronounced differences in alpha-amylase activity were not observed until late in development (40 days after pollination) when the enzyme increased in viviparous seeds only. Developmental changes in viviparous seeds generally resemble those of normally germinating seeds. PMID- 16658562 TI - Phosphoenolpyruvate carboxykinase in plants exhibiting crassulacean Acid metabolism. AB - Phosphoenolpyruvate carboxykinase has been found in significant activities in a number of plants exhibiting Crassulacean acid metabolism. Thirty-five species were surveyed for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, ribulose diphosphate carboxylase, malic enzyme, and malate dehydrogenase (NAD). Plants which showed high activities of malic enzyme contained no detectable phosphoenolpyruvate carboxykinase, while plants with high activities of the latter enzyme contained little malic enzyme. It is proposed that phosphoenolpyruvate carboxykinase acts as a decarboxylase during the light period, furnishing CO(2) for the pentose cycle and phosphoenolpyruvate for gluconeogenesis.Some properties of phosphoenolpyruvate carboxykinase in crude extracts of pineapple leaves were investigated. The enzyme required Mn(2+), Mg(2+), and ATP for maximum activity. About 60% of the activity could be pelleted, along with chloroplasts and mitochondria, in extracts from leaves kept in the dark overnight. PMID- 16658563 TI - Regulation of Nitrate Uptake in Penicillium chrysogenum by Ammonium Ion. AB - A nitrate uptake system is induced (along with nitrate reductase) when NH(4) (+) grown Penicillium chrysogenum is incubated with inorganic nitrate in synthetic medium in the absence of NH(4) (+). Nitrate uptake and nitrate reduction are probably in steady state in fully induced mycelium, but the ratios of the two activities are not constant during the induction period. Substrate concentrations of ammonium cause a rapid decay of nitrate uptake and nitrate reductase activity. The two activities are differentially inactivated (the uptake activity being more sensitive). Glutamine and asparagine are as effective as NH(4) (+) in suppressing nitrate uptake activity. Glutamate and alanine were about half as effective as NH(4) (+). Cycloheximide interferes with the NH(4) (+)-induced decay of nitrate uptake activity. The ammonium transport system is almost maximally deinhibited (or derepressed) in nitrate-grown mycelium. PMID- 16658564 TI - Chromatin-directed Ribonucleic Acid Synthesis: A Comparison of Chromatins Isolated from Healthy, Avirulent Agrobacterium tumefaciens Inoculated, and Crown Gall Tumor Tissues of Vicia faba. AB - Chromatin was extracted from healthy, avirulent Agrobacterium tumefaciens inoculated, and crown-gall tumor Vicia faba internodes of the same age. Chromatin from crown-gall tissue produced 5 times more RNA per 100 micrograms of DNA than chromatin from the healthy tissue. When template availability was compared using chromatin with saturating amounts of Escherichia coli RNA polymerase, chromatin from crown-gall tissue had 36% more available template than the controls. In addition, when gamma-(32)P-ATP was incorporated into the RNA synthesizing reaction mixture, with saturating amounts of E. coli RNA polymerase, there were twice as many RNA chain starts in tumor as in control tissue. PMID- 16658565 TI - Activation and inhibition of ribulose 1,5-diphosphate carboxylase by 6 phosphogluconate. AB - Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl(2) in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 mum 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed. PMID- 16658566 TI - Initiation of Ripening in Bartlett Pear with an Antiauxin alpha(p Chlorophenoxy)isobutyric Acid. AB - A vacuum infiltration technique was used to apply an anti-auxin, alpha-(p chlorophenoxy) isobutyric acid to mature green pears (Pyrus communis var. Bartlett). Application of alpha-(p-chlorophenoxy) isobutyric acid, at 0.02, 0.2, and 2.0 mm progressively accelerated the onset of chlorophyll degradation, softening, and CO(2) evolution. The action of alpha(p-chlorophenoxy) isobutyric acid is apparently independent of ethylene, since the auxin analogue depressed ethylene evolution and could overcome ethylene deficiency in fruit ripening under hypobaric conditions.The auxin analogue decreased the Michaelis constant of indoleacetic acid oxidase in vitro, suggesting that the antiauxin action of alpha (p-chlorophenoxy) isobutyric acid is the acceleration in the breakdown of endogenous auxins in fruit and subsequently the initiation of ripening. PMID- 16658567 TI - Some Physiological Characteristics of the Ethylene-requiring Tomato Mutant Diageotropica. AB - The diageotropica mutant of tomato (Lycopersicon esculentum Mill.) is shown to require exogenous ethylene for normal growth and development. This single gene mutant is characterized by unsupported horizontal growth of shoots and roots, dark green hyponastic leaf segments, thin rigid stems, and primary and adventitious roots which lack lateral roots. Experiments with growth regulators indicate that the mutant does not produce normal amounts of ethylene in response to auxin treatment. Tests with ethylene-producing compounds or ethylene precursors demonstrate that the mutant requires ethylene for normality. Ethylene concentrations as low as 0.005 microliters per liter are capable of completely normalizing mutant characteristics. This mutant with its isogenic parent variety, cv. VFN8, should be a suitable tool for investigating auxin-stimulated ethylene production and their interrelationship in the control of plant morphology and physiology. PMID- 16658568 TI - Occurrence of a Temperature-induced Phase Transition in Mitochondria Isolated from Apple Fruit. AB - Mitochondria were isolated from fruit of six cultivars of apples differing in susceptibility to the physiological disorder, low temperature breakdown. The state 3 rate of succinate-dependent oxygen uptake and the motion of a spin label were measured at from 0 to 25 C. Arrhenius plots of the data showed that the apparent energy of activation of both respiration and motion of the spin label increased abruptly at low temperatures indicative of a temperature-induced phase change in the membrane lipids. The changes were detected with mitochondria from all of the cultivars, but the temperature at which the changes occurred did not correlate with the susceptibility of the cultivars to low temperature breakdown. PMID- 16658569 TI - Translocation of iron from soybean cotyledons. AB - Soybean seeds, Glycine max L. Merrill, were produced by plants treated from anthesis to seed maturity with (59)Fe supplied as ferric ethylenediaminedi (o hydroxyphenylacetate). Seed coats accounted for 7.4% of dry seed weight and had Fe concentrations 5 times greater than the embryos. After germinating 2 days, cotyledons contained 69.6% and radicles 5.0% of original seed Fe. Fractions of seed Fe unavailable to seedlings were: 19.8% in seed coats, 1.7% in germination paper, 0.1% in the water under germinating seeds, and 3.8% unaccounted for. Every 3 days seedlings received nutrient solution without Fe or with 10 mum ferric ethylenediaminedi (o-hydroxyphenylacetate) and developed as deficient Fe or normal Fe plants. The deficient Fe cotyledons on day 18 retained 13% of the labeled Fe originally present. Cotyledons of normal Fe plants retained 50 to 70% of their original Fe. Moreover, cotyledons of the normal Fe plants accumulated externally supplied Fe and finally contained twice the quantity of Fe originally present. Stem exudate collected above cotyledons of deficient Fe plants contained 5.3 mum(59)Fe. Electrophoresis of exudate showed that most of the (59)Fe migrated anodically as a single band and was in the position of ferric citrate. PMID- 16658570 TI - A Variation of C(4) Leaf Anatomy in Arundinella hirta (Gramineae). AB - The species Arundinella hirta L. posseses a striking variation of the leaf anatomy that is characteristic of C(4) grasses. In addition to a sheath of large, bright green cells around the vascular bundles, there are strands of large parenchyma cells which appear identical to the bundle sheath cells and which run parallel to the vascular bundles, but which are not associated with any vascular tissue. This species may be useful for studying the cellular compartmentalization associated with the C(4) pathway and should provide interesting material for determining the role of translocation in the functioning of the C(4) system. PMID- 16658571 TI - Inhibition of postoxidative calcium release in corn mitochondria by inorganic phosphate. AB - Respiration drives the accumulation of a small amount of calcium in corn (Zea mays L.) mitochondria, and this calcium is released when respiration ceases. A postenergized addition of phosphate leads to phosphate uptake and enhaced calcium retention. Oligomycin, KCN, 2,4-dinitrophenol, or mersalyl are without effect on the phosphate-induced calcium retention. Addition of phosphate also inhibits the release of endogenous phosphate which normally accompanies the calcium. It is suggested that passive phosphate uptake retards the release of endogenous phosphate which is complexed with the calcium. PMID- 16658572 TI - Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa. AB - In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% (14)CO(2) or 0.05% (14)CO(2) in air, gassing with 100% O(2) results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% (14)CO(2) than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O(2). PMID- 16658573 TI - Influence of Translocation of Photosynthetic Efficiency of Phaseolus vulgaris L. AB - Measurements of net photosynthesis show that in Phaseolus vulgaris L. the cultivar Michelite-62 exceeds the cultivar Red Kidney in net CO(2) uptake by 23 to 31%. Data on translocation of pulse label indicate that export of a pulse of photosynthetically assimilated (14)C from the source leaf of either M-62 or Red Kidney follows an exponential pattern and shows an initial rapid phase followed by a second slower phase. The steeper slope for both phases in M-62 suggests its rate of translocation of pulse label is higher than that of Red Kidney. Furthermore, only 38% of the (14)C remains in the leaf of M-62 after 8 hours, while Red Kidney retains up to 60% of the label. Leaf autoradiographs obtained after pulse labeling demonstrate a much faster rate of vein loading in M-62 and are considered evidence for the higher translocation efficiency of M-62. These results provide evidence for a positive correlation between photosynthetic efficiency and translocation efficiency in M-62 and Red Kidney and give support to our hypothesis that translocation is one of the important physiological factors controlling the varietal differences in photosynthetic efficiency in Phaseolus vulgaris. PMID- 16658574 TI - Terpene Biosynthesis in Cell-free Extracts and Excised Shoots from Wedgwood Iris. AB - Excised shoots and cell-free extracts prepared from Wedgwood iris (Iris hollandica Hoog. "Wedgwood") shoots metabolized (14)C-labeled mevalonic acid (MVA). By using cell-free extracts, the (14)C from MVA-1-(14)C was recovered as (14)CO(2), while that from MVA-2-(14)C was recovered as neutral terpenes, acid hydrolyzable terpenes, or (14)CO(2). Also, under optimal incubation conditions, 12.8 nanomoles R-MVA-2-(14)C was incorporated into neutral terpenes per milligram fresh weight per hour. In contrast, excised shoots incorporated only 0.58 nanomoles R-MVA-2-(14)C per mg fresh weight per hour. Labeled products identified from the cell-free system were squalene, farnesol, geranylgeraniol, and compounds that are converted to farnesol and geranylgeraniol after alkaline hydrolysis. Squalene and a 4,4-dimethylsterol were identified as products from excised shoots but not the terpene alcohols or the alkaline-hydrolyzable compounds. PMID- 16658575 TI - Correlation between Chlorophyll and Chlorogenic Acid Content in Tobacco Leaves. AB - A positive correlation (r = 0.75, P < 0.01) was obtained between chlorophyll and chlorogenic acid content in the seedling leaves of burley and dark tobaccos. The dark tobaccos contained significantly higher concentrations of both constituents than the burleys. Such a correlation also occurred in a cytoplasmic mutant of chlorophyll-variegated tobacco when the green and yellow laminae were compared. In addition, the activity of phenylalanine ammonia-lyase and polyphenol-oxidase was higher in the green lamina than in the yellow tissue, which coincided with quantitative distribution of chlorogenic acid. Chlorophyll deficiency induced by streptomycin in tobacco seedlings resulted in a progressive decrease in chlorogenic acid content. However, an interruption of streptomycin treatment provoked accumulation of the two compounds. Dark-grown seedlings showed an increase in the content of chlorophyll and chlorogenic acid upon illumination. Incorporation of l-phenylalanine-U-(14)C into chlorogenic acid during leaf greening was drastically reduced owing to the presence of phenylpyruvate; the latter compound is a possible by-product of chlorophyll biosynthesis. This phenomenon was also evident with light-grown leaves. Results suggest that in addition to phenylalanine ammonia-lyase as a key enzyme regulating chlorogenic acid biosynthesis, an alternative pathway involving the conversion of phenylpyruvate to cinnamate may be functional in tobacco leaves. This pathway may bear importance as to higher chlorogenic acid content in dark tobaccos than in burleys. PMID- 16658576 TI - C/C ratio changes in crassulacean Acid metabolism plants. AB - (13)C/(12)C ratios have been found in totally combusted leaves of Crassulacean acid metabolism plants to range from -14 to -33 delta (13)C per thousand compared with a limestone standard. Crassulacean acid metabolism plants apparently utilize both ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase to assimilate atmospheric CO(2) and, depending on environmental conditions, have (13)C/(12)C ratios indicative of either carboxylase or to any intermediate value. The degree of discrimination against (13)C and the resultant (13)C/(12)C ratio from the photosynthetically fixed CO(2) is influenced by environmental conditions and is not a specific and fixed characteristic of a Crassulacean acid metabolism plant. Certain Crassulacean acid metabolism plants may shift their ratios as much as 17 delta (13)C per thousand in specific environments. PMID- 16658577 TI - Functioning of the adenine nucleotide transporter in the arsenate uncoupling of corn mitochondria. AB - Arsenate uncouples mitochondrial respiration in a process stimulated by ADP, inhibited by oligomycin, and competitively inhibited by inorganic phosphate. If mersalyl is added to corn mitochondria to block further transport of accumulated arsenate, the uncoupled respiration continues unabated due to recycling of matrix arsenate. Addition of ADP now inhibits rather than promotes respiration and the mitochondria shrink. It is established by arsenate analyses that arsenate is removed from the matrix. Oligomycin or atractyloside block the removal by inhibiting ADP-arsenate formation or transport, respectively. It is deduced that ADP-arsenate is stable in the membrane and is transported outward for hydrolysis in the external aqueous phase. Hence, ADP-arsenate formed in oxidative phosphorylation is not directly released to the matrix, and a mechanism must exist for its direct transfer to the transporter. PMID- 16658578 TI - Effects of Moisture Deficits on C Translocation in Corn (Zea mays L.). AB - Corn plants (Zea mays L.) were grown in the field on two soils. On a droughty soil, water was withheld from some plants during the grain-filling period while other plants were irrigated. Carbon-14 was fed to the leaves, and translocation to different plant parts was determined. Translocation appeared to be more sensitive to moisture stress than was photosynthesis. More radioactive carbon was retained in both the fed portion and the nonfed portion of the leaf of stressed plants than in nonstressed plants. The stalk segment between the treated leaf and ear-node also contained less radioactivity in stressed plants than in nonstressed plants. On a soil with higher water-holding capacity, moisture stress was imposed on plants by root pruning. Plants under severe stress continued to translocate photosynthetically assimilated (14)C nearly as well as nonstressed plants for 90 minutes. Between 90 and 120 minutes after labeling, there was a major reduction in amount translocated in stressed plants compared to the nonstressed plants. At longer translocation times the rates of translocation appeared again to be more nearly equal. PMID- 16658579 TI - Morphology and Photosynthetic Efficiency of Tobacco Leaves That Received End-of Day Red and Far Red Light during Development. AB - Shaded leaves in plant canopies receive a higher proportion of far red relative to red light than is received by unshaded leaves. Brief end-of-day irradiations with red or far red light, acting through the phytochrome system, reversibly control morphological development of tobacco plants. Leaves that received far red light for 5 minutes at the end of each day during development were longer and narrower than those that received end-of-day red light. The far red treated leaves weighed less, had fewer stomata, and had less chlorophyll per unit area of leaf. Net CO(2) assimilation rates did not differ significantly between red- and far red-treated leaves on an area basis; however, the far red-treated leaves assimilated significantly more CO(2) on a leaf weight basis. PMID- 16658580 TI - Absorption and distribution of high specific radioactivity 2-C-abscisic Acid in cotton seedlings. AB - High specific radioactivity (26.3 mc/mmole) racemic 2-(14)C-abscisic acid was synthesized. An aliquot of abscisic acid, 1.2 x 10(-4)m in aqueous methanolic solution, was applied to the surface of either a cotyledon or the first true leaf of 8- to 32-day-old cotton seedlings (Gossypium hirsutum L.). After various intervals (6-192 hours), the seedlings were processed for autoradiography, counting, and identification of the radioactivity. After 6 hours, radioactivity was observed moving basipetally out of the treated leaf toward the roots. Four days later, radioactivity could be detected throughout the whole seedling. After 8 days, 10% of the recovered radioactivity was found in the roots, and 80% remained in the treated leaf blade. Neither leaf type nor age had any effect on the abscisic acid movement or pattern of distribution. Isolated radioactivity from the roots was identified as abscisic acid, based on comparison with an authentic standard by thin layer chromatography, gas-liquid chromatography, or gas-liquid chromatography-mass spectrometry. PMID- 16658581 TI - Carbon dioxide metabolism in leaf epidermal tissue. AB - A number of plant species were surveyed to obtain pure leaf epidermal tissue in quantity. Commelina communis L. and Tulipa gesnariana L. (tulip) were chosen for further work. Chlorophyll a/b ratios of epidermal tissues were 2.41 and 2.45 for C. communis and tulip, respectively. Phosphoenolpyruvate carboxylase, ribulose 1,5-diphosphate carboxylase, malic enzyme, and NAD(+) and NADP(+) malate dehydrogenases were assayed with epidermal tissue and leaf tissue minus epidermal tissue. In both species, there was less ribulose 1,5-diphosphate than phosphoenolpyruvate carboxylase activity in epidermal tissue whether expressed on a protein or chlorophyll basis whereas the reverse was true for leaf tissue minus epidermal tissue. In both species, malic enzyme activities were higher in epidermal tissue than in the remaining leaf tissue when expressed on a protein or chlorophyll basis. In both species, NAD(+) and NADP(+) malate dehydrogenase activities were higher in the epidermal tissue when expressed on a chlorophyll basis; however, on a protein basis, the converse was true. Microautoradiography of C. communis epidermis and histochemical tests for keto acids suggested that CO(2) fixation occurred predominantly in the guard cells. The significance and possible location of the enzymes are discussed in relation to guard cell metabolism. PMID- 16658582 TI - The effect of greening of sorghum leaves on the molecular weight of a complex containing 4-hydroxycinnamic Acid hydroxylase activity. AB - During the greening of leaves of Sorghum bicolor var. Wheatland milo, the activity of 4-hydroxycinnamic (p-coumaric) acid hydroxylase in pH 6 buffered extracts was shifted from a relatively low to a high molecular weight fraction. Differences between these forms found in etiolated and green leaves were based on differential centrifugation, ammonium sulfate precipitation, and on elution patterns from Agarose A-15m. Both molecular weight forms were precipitated by protamine sulfate at pH 6, and approximately 40 to 80% of the activity of each form was associated with a 500 to 37,000g pellet when tissues were ground at pH 8 in media of either high or low osmotic concentration. Although no fraction with hydroxylase activity was ever found without any chlorogenic acid oxidase activity, the two activities frequently varied independently, and could be partially separated from each other, using the above techniques. Comparisons were made with the very small molecular weight form of 4-hydroxycinnamic acid hydroxylase characteristic of tissues of first internodes. The significance of these results in terms of possible multienzyme complexes capable of converting phenylalanine and tyrosine to cinnamic acid derivatives is discussed. PMID- 16658583 TI - Respiration-independent Binding of SR to Bean Mitochondria. AB - Binding of Sr(2+) to bean mitochondria (Phaseolus vulgaris) shows a dissociation constant of 25 x 10(-6) and results in 40 to 50 nmoles of Sr(2+) bound per mg protein. The binding is partially inhibited by valinomycin plus K(+), 2, 4 dinitrophenol, as well as ruthenium red at a level of the 120 nmoles per mg protein. These compounds also partially inhibit active uptake of Sr(2+). Calcium and Mg(2+) also partially inhibit binding in the same magnitude as previously reported for inhibition of transport. Phosphate which is required for divalent cation transport is without effect on the binding of Sr(2+). The possible role of the observed binding sites in divalent cation transport is discussed. PMID- 16658584 TI - Incorporation of carbohydrate residues into peroxidase isoenzymes in horseradish roots. AB - Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-(14)C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-(3)H and mannose-U-(14)C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of (14)C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems. PMID- 16658585 TI - Participation of ethylene in common purslane response to dicamba. AB - The responses of common purslane (Portulaca oleracea L.) plants to 2-methoxy-3,6 dichlorobenzoic acid (dicamba) were found to be similar in many respects to ethylene fumigation effects. Dicamba and ethylene increased the permeability of cell membranes in purslane tissues. An increased efflux of electrolytes was observed in the bending region of the stems of dicamba-treated plants. Epinastic leaves after dicamba (10 micrograms) and ethylene (microliter per liter) treatments showed an increased efflux of rubidium. The permeability effects were observable within 1 day after dicamba or ethylene application. Protein metabolism in purslane leaves was not influenced by dicamba until 2 days after treatment, as indicated by reduced nitrate reductase activity. Inhibition of phenylalanine-U (14)C incorporation into protein was observed 3 days after treatment. Ethylene reduced both phenylalanine-U-(14)C incorporation into protein and nitrate reductase activity within 1 day. Dicamba caused a rapid increase in ethylene production in purslane plants to levels many times greater than those observed in untreated plants. It was concluded that the dicamba-enhanced production of ethylene is responsible for many of the observed effects of the herbicide. PMID- 16658586 TI - A chamber for applying pressure to roots of intact plants. AB - A chamber was designed to apply up to 20 bars pressure to roots of intact plants. The unique features of this chamber are a split top arrangement to permit enclosing roots of intact plants within the chamber, a circulation coil to control temperature of rooting media, and a valve arrangement to permit changing solution without disturbing the plant. Changes in transpiration in response to changes in the pressure applied to roots of intact pepper plants illustrate one use of the equipment. Well watered plants at low light (0.05 langley/min) were observed to exude water from the leaf margins when 5 bars pressure was applied to the roots. When roots were cut off, a 1 bar pressure caused exudation. Plants with cooled roots or plants in dry soil did not exude water when as much as 6 bars pressure was applied. Transient response of transpiration rates to sudden application and release of pressure was observed in pepper and bean plants but not in rhododendron. The magnitude of this transient response was highly dependent upon light intensity and CO(2) concentration in the aerial environment. PMID- 16658587 TI - Phytochrome Transformation and Action in Seeds of Rumex crispus L. during Secondary Dormancy. AB - Promotion of germination by red light fails after prolonged dark imbibition of Rumex crispus L. seeds, indicative of a secondary dormancy. The degree and rate of inception of the dormancy increases with increasing temperature. Following establishment of the dormancy, germination response to red light can be restored by either prolonged cold treatment or brief high temperature shifts. Loss of phytochrome was not a factor in the initial establishment of the dormancy. When the seeds are in secondary dormancy, the chromophore of phytochrome can be transformed to the far red-absorbing form, but the far red-absorbing form cannot induce germination. The responses to changes in temperature suggested dependence of germination on order left arrow over right arrow disorder transitions in components of the seeds. PMID- 16658588 TI - Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga. AB - Preparations of heterocysts of Anabaena cylindrica Lemm. had 7- to 8-fold higher activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, 2-fold more hexokinase activity, and 0.02 to 0.06 times as much ribulose diphosphate carboxylase and glyceraldehyde 3-phosphate dehydrogenase activities as did whole filaments per milligram soluble protein in cell-free extracts. Time courses of solubilization of glucose 6-phosphate dehydrogenase activity indicated that heterocysts contain 74 to 80% of the total activity of this enzyme in filaments. PMID- 16658589 TI - Purification of enzymatically isolated mesophyll protoplasts from c(3), c(4), and crassulacean Acid metabolism plants using an aqueous dextran-polyethylene glycol two-phase system. AB - Enzymatic digestion of leaf segments with 2% cellulase, in combination with a pectinase in some species, yields intact protoplasts mixed with epidermal tissue, vascular tissue, broken protoplasts, and chloroplasts. Epidermal and vascular tissue are removed with sieves of various porosity. Intact protoplasts in the filtrate are separated from other components by an aqueous two-phase system which consists of dextran-polyethylene glycol, with sorbitol and sodium phosphate. Intact protoplasts partition at the interphase, while chloroplasts and broken protoplasts partition in the lower phase when the separation is facilitated by low speed centrifugation. The optimum conditions for purification of maize mesophyll protoplasts with high yields are centrifugation of the two-phase system at 300g for 6 minutes at 2 C with a mixture including 0.46 m sorbitol, 10 mm sodium phosphate, 5.5% polyethylene glycol 6000, and 10% dextran of average molecular weight of 20,000 to 40,000. The collection of protoplasts at the inter phase was proportional to the amount of chlorophyll added over a wide range of concentrations regardless of the initial contamination of the preparation by other cellular debris. The two-phase system is applicable for protoplast purification from a wide variety of species, including C(3), C(4), and Crassulacean acid metabolism plants, regardless of protoplast size. PMID- 16658590 TI - Steps in the acquisition of photosynthetic competence by plastids of maize. AB - Coupling factor particles are associated with membranes of maize etioplasts (Lockshin et al., 1971. Biochim. Biophys. Acta 226: 366-382). In addition, several, but not all, of the polypeptides found in the photosynthetic lamellae of maize chloroplasts are present in etioplasts.During the photoregulated maturation of chloroplasts from etioplasts, the membranes of the organelle acquire the capacity to carry on acid to base phosphorylation. The acquisition of this ability is closely correlated with the development of osmotic responsiveness and the appearance of a 46,000 dalton polypeptide in the membranes. PMID- 16658591 TI - Development of the Generative Cell Wall in Monotropa uniflora L. Pollen. AB - During an ultrastructural survey of an achlorophyllus dicotyledonous plant, Monotropa uniflora L., a stage of pollen development was encountered which suggests a relationship between the activity of the rough endoplasmic reticulum and the development of the generative cell wall. PMID- 16658592 TI - Purification and some properties of two polyphenol oxidases from bartlett pears. AB - Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K(m) values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V(max) and K(m) values for oxygen and chlorogenic acid were 103 mumoles O(2) uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K(m) values for chlorogenic acid were independent of pH from 3 to 7; the V(max) values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K(i) values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K(i) values of 0.04 and 0.11 mm for enzymes A and B, respectively. PMID- 16658593 TI - Acceleration of the Growth of Camellia sasanqua Pollen by Soaking in Organic Solvent. AB - Camellia sasanqua pollen that had been soaked in acetone or diethyl ether for only 5 minutes grew three to four times longer pollen tubes than unsoaked pollen. Although the acceleration of pollen tube growth was observed when the pollen had been soaked in cold solvents (5 C, -15 C) for 100 days, soaking in warm solvents (30 C, 24 C) caused it to decrease gradually, and the pollen eventually lost the ability to germinate. The acceleration may be caused by removing inhibitor from the pollen grain by the organic solvents. PMID- 16658594 TI - Leaf water content and hormone effects on ribonuclease activity. AB - In barley (Hordeum vulgare) leaves in which the water balance was not hampered, kinetin and abscisic acid effected the well documented decrease and increase, respectively, in RNase activity. When the plants were exposed to water shortage, leaf-water saturation deficit increased steadily, with kinetin enhancing and abscisic acid retarding the rise. Under drought, the pattern of hormonal effects was inverted, with kinetin enhancing RNase activity over and above the activity assayed in abscisic acid-treated leaves. A very close relationship between RNase activity and water saturation deficit was found and significantly, it was maintained irrespective of the hormonal treatment, which in itself markedly modified leaf-water saturation deficit. The inverted effects of kinetin and of abscisic acid on RNase activity under conditions of water shortage were interpreted as resulting primarily from the effects of these hormones on leaf water. It is suggested that under conditions of increased water deficiency in the plant, cell-water supersedes hormonal regulation in effecting RNase activity. PMID- 16658595 TI - Effects of Helminthosporium carbonum Toxin on Nitrate Uptake and Reduction by Corn Tissues. AB - Susceptible corn tissues exposed to the host-specific toxin of Helminthosporium carbonum race 1 reduced more nitrate to nitrite than did control tissues, as measured by an in vivo method. There were no differences in nitrate reductase activities extracted from treated and control tissues and assayed by an in vitro method. Toxin-treated susceptible roots removed nitrate from solution and accumulated it in the tissues twice as fast as did control roots. Uptake by resistant roots was stimulated also, provided approximately 100 times higher concentrations of toxin were used. Toxin-stimulated nitrate uptake occurred in the presence of tungstate, which eliminates nitrate reductase activity. Toxin did not cause leakage of nitrate from roots under these conditions. Thus, toxin enhanced nitrate accumulation was caused by increased nitrate uptake rather than by decreased nitrate metabolism or decreased nitrate leakage. The data indicate that toxin increases the rate of nitrate reduction in vivo by increasing the availability of substrate, not by stimulation of enzyme synthesis. PMID- 16658596 TI - Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots. AB - Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO(3) (-), Na(+), Cl(-), 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na(+) and Cl(-). Toxin did not affect uptake of Na(2) (-), K(+), Ca(2+), phosphate ion (H(2)PO(4) (-) and HPO(4) (-)), SO(4) (-), and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes. PMID- 16658597 TI - Factors affecting the stability and accuracy of the bioassay for the sperm attractant sirenin. AB - Optimal response of the sperm of Allomyces from the highly male strain M16 to the chemotactic agent, sirenin, was shown to occur when the sperm suspension contained 2 mm piparazine-N', N-bis[2-ethane sulfonic acid] buffer, 3 mm CaCl(2), and chelated trace elements. For the male strain M3, the CaCl(2) needed was 3.5 mm with the other two components the same as for M16. The inclusion in the sperm suspension of MgCl(2), KH(2)PO(4), or NH(4)Cl was without effect, except that under certain conditions phosphate was detrimental. The variability of 10 replicate assays was substantially reduced by using sperm in the bioassay at a concentration of 500,000 per ml rather than the former concentration of 100,000 per ml with a concomitant reduction in the concentration of sirenin above the membrane to which the sperm were attracted. PMID- 16658598 TI - The chemotactic activity of various sirenins and analogues and the uptake of sirenin by the sperm of allomyces. AB - The enantiomorphs of sirenin and several analogues of sirenin were tested for their activity in attracting the sperm of Allomyces. Only l-sirenin was active. The uptake of sirenin by the sperm was measured from solutions containing l sirenin in concentrations from 5 to 10,000 nm. Uptake from 5 and 100 nm followed first order kinetics. At higher concentrations the rate of uptake slowed down, and at the very highest ones it stopped after a few minutes. When sperm had taken up all the sirenin from a 5 nm solution, it took approximately 45 minutes for them to regain full responsiveness. After uptake of sirenin by sperm, none could be recovered from them. PMID- 16658599 TI - A Mathematical Treatment of Munch's Pressure-Flow Hypothesis of Phloem Translocation. AB - The steady state solutions of two mathematical models are used to evaluate Munch's pressure-flow hypothesis of phloem translocation. The models assume a continuous active loading and unloading of translocate but differ in the site of loading and unloading and the route of water to the sieve tube. The dimensions of the translocation system taken are the average observed values for sugar beet and are intended to simulate translocation from a mature source leaf to an expanding sink leaf. The volume flow rate of solution along the sieve tube, water flow rate into the sieve tube, hydrostatic pressure, and concentration of sucrose in the sieve tube are obtained from a numerical computer solution of the models. The mass transfer rate, velocity of translocation, and osmotic and hydrostatic pressures are consistent with empirical findings. Owing to the resistance to water flow offered by the lateral membranes, the hydrostatic pressure generated by the osmotic pressure can be considerably less than would be predicted by the solute concentration. These models suggest that translocation at observed rates and velocities can be driven by a water potential difference between the sieve tube and surrounding tissue and are consistent with the pressure-flow hypothesis of translocation. PMID- 16658600 TI - Drought Adaptation in Opuntia basilaris: Significance of Recycling Carbon through Crassulacean Acid Metabolism. AB - Contrasting metabolic regimes operate in Opuntia basilaris Engelm. and Bigelov, before and after precipitation. During periods of drought, atmospheric CO(2) exchange and transpiration are greatly reduced throughout the day/night cycle by stomatal closure and a highly impervious cuticle. The hypothesis is that endogenously produced CO(2) is retained and recycled through dark CO(2) fixation, organic acid transformations, photosynthesis, and respiration. Immediately following precipitation, nighttime stomatal opening is initiated, permitting increased atmospheric CO(2) assimilation and organic acid synthesis. PMID- 16658601 TI - Influence of moisture, heat, and light stress on hydrogen fluoride fumigation injury to soybeans. AB - Soybean (Glycine max (L) Merr.) plants were exposed to a single fumigation with hydrogen fluoride at concentrations sufficient to cause visible injury within 2 days. They were subjected to soil moisture or osmotic stress prior to, during, or after fumigation. Moisture stress before or during fumigation reduced injury because of stomatal closure and reduced fluoride uptake. Moisture stress after fumigation markedly accentuated the injury resulting from a single fumigation compared to plants kept continually under optimum soil moisture conditions. Full sunlight following the fumigation accentuated injury, while shade reduced it. Higher temperatures following fumigation also increased severity of symptoms. PMID- 16658602 TI - Effect of Abscisic Acid on Uptake and Metabolism of [H]Gibberellin A(1) and [H]Pseudogibberellin A(1) by Barley Half-seeds. AB - Uptake and metabolism of 1,2-[(3)H]gibberellin A(1) ([(3)H]GA(1), I) and its 3 hydroxy epimer ([(3)H]pseudoGA(1), II) by barley (Hordeum vulgare L.) half-seeds were measured after 24 hours of incubation, in the presence or absence of abscisic acid in the media. Uptake of both compounds was enhanced by abscisic acid, and abscisic acid enhanced the extent of metabolism of [(3)H]GA(1). However, [(3)H]pseudoGA(1) was not metabolized, even in the presence of abscisic acid. The significance of the stereo-chemistry of the 3-hydroxyl position is discussed. PMID- 16658603 TI - The Effects of Octanoate and Oleate on Isocitrate Lyase Activity during the Germination of Pinus pinea Seeds. AB - The changes of isocitrate lyase levels with respect to the catabolism of triglycerides have been studied during the germination of Pinus pinea seeds. We studied the effects of octanoate, oleate, and inhibitors of protein synthesis on isocitrate lyase during germination. Pyruvate kinase, glucose-6-P-dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase were also assayed. Octanoate and oleate inhibited the isocitrate lyase activity, similarly to cycloheximide, chloramphenicol, and actinomycin, inhibitors of protein biosynthesis. This inhibitory effect is not specific but is strikingly evident with isocitrate lyase. This inhibition was not proportional to the concentration but was proportional to the chain length of oleate and octanoate. PMID- 16658604 TI - Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L. AB - Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene. PMID- 16658605 TI - Proline Uptake and Utilization by Chlorella pyrenoidosa. AB - Conditions for proline uptake and utilization by Chlorella pyrenoidosa Chick are described. Proline is taken up by growing cultures during late log phase growth after depletion of glucose from the medium. However, proline uptake by stationary phase cultures requires the presence of glucose in the medium. The results are consistent with the interpretation that some carbohydrate is required for proline uptake but proline uptake is inhibited by the accumulation of intracellular carbohydrates.Metabolism of proline was by incorporation into protein and oxidation to CO(2) by conversion to glutamic acid and subsequent metabolism of the carbon by the Krebs cycle. PMID- 16658606 TI - Temperature effects on radial propagation of water potential in cotton stem bark. AB - Low temperature affects the lateral movement of water across the xylem-phloem boundary in intact cotton stems. There is a reduction in the effective diffusion coefficient relating free energy flux to water potential gradients with an associated increase in resistance to water flow. Detached phloem and excised leaves do not show this effect of low temperature. Experiments on stem section halves indicate that the effect is probably associated with the cambial region. PMID- 16658607 TI - Induction of Stomatal Closure by Helminthosporium maydis Pathotoxin. AB - Helminthosporium maydis pathotoxin caused a rapid inhibition of photosynthesis in whole leaves of maize having Texas male-sterile cytoplasm but not in leaves having normal cytoplasm. Electron transport, phosphorylation, and proton uptake activities of isolated chloroplast lamellae, from either normal or Texas male sterile cytoplasm leaves, were unaffected by addition of toxin to the reaction mixture. The toxin was found to have a direct effect on stomatal functioning. Rates of transpiration were inhibited in treated leaves at times slightly preceding the observed effects on photosynthesis. In studies with isolated epidermal peels from Texas male-sterile cytoplasm maize leaves, the toxin inhibited light-induced K(+) uptake by guard cells. All effects of the toxin on Texas male-sterile cytoplasm maize tissues were found to be similar to the mode of action of abscisic acid. PMID- 16658608 TI - Structural Development during Germination of Different Populations of Mitochondria from Pea Cotyledons. AB - The crude mitochondrial fraction from pea cotyledons can, from days 1 to 7 of germination, be separated into three fractions by sucrose density gradient centrifugation. When seeds were grown in water (control) or cycloheximide (120 micrograms per milliliter of medium) for 4 days, the originally different populations of mitochondria acquired a uniform density and separated together in band 1 (density, 1.205 grams per milliliter). The oxidative and phosphorylative activities of mitochondria obtained from 4-day-old control and 4-day-old cycloheximide-treated pea seeds were the same. However, mitochondria from pea seeds that were grown in d-threo-chloramphenicol (1.5 milligrams per milliliter of medium) or erythromycin (0.5 milligram per milliliter of medium) for 4 days separate into three bands (fully developed mitochondria in the top band [band 1] and partially developed mitochondria in the lower two bands [bands 2 and 3]). Separation patterns and oxidative and phosphorylative activities were the same for mitochondria separated from 4-day-old cotyledons treated with d-threo chloramphenicol or erythromycin and from 1-day-old cotyledons grown in water. This indicated that these inhibitors prevented the partially developed mitochondria originally in bands 2 and 3 from developing further. In contrast, cycloheximide did not seem to interfere with the mitochondrial structural development. These results along with those obtained from the experiments on the effects of d-threo-chloramphenicol, erthromycin, and cycloheximide on (14)C leucine incorporation into mitochondrial membrane proteins suggest that the increase in mitochondrial activity during germination may be a result of structural development (membrane synthesis) in pre-existing mitochondria. PMID- 16658609 TI - Adenosine 5'-triphosphate-sulfurylase in corn roots and its partial purification. AB - ATP-sulfurylase (ATP-sulfate adenyltransferase, EC 2.7.7.4) was found in nonparticulate fractions of both roots and leaves of Zea mays L. seedlings using two detection methods. Addition of exogenous pyrophosphatase was essential for maximum rates of conversion of (35)SO(4) (2-) to labeled adenosine phosphosulfate in unpurified root extracts, but not in unpurified leaf extracts. In the presence of exogenous pyrophosphatase, the enzyme from roots exhibited specific activities as high as those obtained with the leaf enzyme. The root enzyme was purified 33 fold by centrifugation and column chromatography procedures. Its molecular weight obtained by Sephadex gel filtration was about 42,000. Its Km for pyrophosphate was 7 mum, while for adenosine phosphosulfate, the Km was 1.35 mum. None of the enzyme fractions studied converted adenosine phosphosulfate into detectable amounts of 3'-phosphoadenosine-5'-phosphosulfate. ATP-sulfurylase was also found in roots of corn seedlings grown aseptically. The data suggest that at least the first reaction in sulfate reduction might proceed as effectively in roots as in shoots. PMID- 16658610 TI - Solute distribution in sugar beet leaves in relation to Phloem loading and translocation. AB - The distribution of solutes in the various cells of sugar beet (Beta vulgaris L.) source leaves, petioles, and sink leaves was studied in tissue prepared by freeze substitution. The differences in degree of cryoprotection indicated that sieve elements and companion cells of the source leaf, petiole, and sink leaf contain a high concentration of solute. The osmotic pressure of various types of cells was measured by observing incipient plasmolysis in freeze-substituted tissues equilibrated with a series of mannitol solutions prior to rapid freezing. Analysis of source leaf tissue revealed osmotic pressure values of 13 bars for the mesophyll and 30 bars for the sieve elements and companion cells. The osmotic pressure of the mesophyll of sink leaves was somewhat higher.The sharp concentration increase at the membrane of the sieve element-companion cell complex of the source leaf indicates active phloem loading from the free space at this site. Active loading of the phloem is presumably needed to move the sugar from the chloroplasts of the mesophyll to the sieve tubes against the concentration gradient. The osmotic pressure of the mature sieve element companion cell complex appears to be approximately the same in source leaf, path, and sink leaf tissue. There is a distinct difference in concentration between the mature sieve element-companion cell complex in the sink and the surrounding mesophyll. The solute distribution suggests that sugar is actively accumulated from the free space by the developing sink leaf tissue.The osmotic values observed in the various cells are consistent with the operation of a mass flow mechanism of translocation driven by active phloem loading and by active accumulation of sugar by sink tissues. PMID- 16658611 TI - The Conversion of Photoinactive Protochlorophyllide(633) to Phototransformable Protochlorophyllide(650) in Etiolated Bean Leaves Treated with delta Aminolevulinic Acid. AB - The relationship of phototransformable protochlorophyllide to photoinactive protochlorophyllide has been studied in primary leaves of 7- to 9-day-old dark grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Various levels of photoinactive protochlorophyllide, absorbing at 633 nm in vivo, were induced by administering delta-aminolevulinic acid to the leaves in darkness. Phototransformable protochlorophyllide, absorbing at 650 nm in vivo, was subsequently transformed to chlorophyllide by a light flash, and the regeneration of the photoactive pigment was followed by monitoring the absorbance increase at 650 nm in vivo. A small increase in the level of protochlorophyllide(633) causes a marked increase in the extent of regeneration of protochlorphyllide(650) following a flash. High levels of the inactive pigment species, however, retard the capacity to reform photoactive protochlorophyllide. A nonstoichiometric and kinetically complex decrease in absorbance at 633 nm in vivo accompanied the absorbance increase at 650 nm. The half-time for protochlorophyllide(650) regeneration in control leaves was found to be three times longer than the half time for conversion of chlorophyllide(678) to chlorophyllide(683) at 22 C. The results are consistent with the hypothesis that protochlorophyllide(633) is a direct precursor of protochlorophyllide(650) and that the protein moiety of the protochlorophyllide holochrome acts as a "photoenzyme" in the conversion of protochlorophylide to chlorophyllide. PMID- 16658612 TI - Studies on the Energy-coupling Sites of Photophosphorylation: II. Treatment of Chloroplasts with NH(2)OH Plus Ethylenediaminetetraacetate to Inhibit Water Oxidation while Maintaining Energy-coupling Efficiencies. AB - Artificial electron donors to photosystem II provide an important means for characterizing the newly discovered site of energy coupling near photosystem II. However, water oxidation must be completely abolished, without harming the phosphorylation mechanism, for these donor reactions and the associated phosphorylation to withstand rigorous quantitative analysis. In this paper we have demonstrated that treatment of chloroplasts with hydroxylamine plus EDTA at pH 7.5 in the presence of Mg(2+) followed by washing to remove the amine is a highly reliable technique for this purpose. The decline of the Hill reaction and the coupled phosphorylation during the treatment were carefully followed. No change in the efficiency of phosphorylation (P/e(2) 1.0-1.1) was observed until the reactions became immeasurable. Photosystem I-dependent reactions, such as the transfer of electrons from diaminodurene or reduced 2,6-dichlorophenolindophenol to methylviologen, and the associated phosphorylation were totally unaffected. It is clear that the hydroxylamine treatment is highly specific, with no adverse effect on the mechanism of phosphorylation itself. Benzidine photooxidation via both photosystems II and I in hydroxylamine-treated chloroplasts (electron acceptor, methylviologen; assayed as O(2) uptake) supports phosphorylation with the same efficiency as that observed for the normal Hill reaction (P/e(2) = 1.1). An apparent P/e(2) ratio of 0.6 was computed for the photooxidation of ascorbate. PMID- 16658613 TI - The Effect of an Oxygen-free Atmosphere on Net Photosynthesis and Transpiration of Barley (Hordeum vulgare L.) and Wheat (Triticum aestivum L.) Leaves. AB - Stomata of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) leaves failed to open in the light and close in the dark or respond to changes in the CO(2) concentration of the atmosphere in either light or dark when the leaves were in an O(2)-free atmosphere. In contrast, the expected responses to environmental changes were found in atmospheres containing 1.5% O(2). It appears that O(2) is necessary for both opening and closing of wheat and barley stomata. PMID- 16658614 TI - Polyribosome formation and protein synthesis in imbibed but dormant lettuce seeds. AB - Dormancy is maintained in Grand Rapids lettuce (Lactuca sativa) seeds imbibed on water in darkness at 25 C. Polyribosome formation and protein synthesis occur early in the imbibition phase and considerable polysomal material is also present after 24 and 48 hours, even though the seeds have failed to germinate. Incorporation of labeled leucine into protein following a 24-hour preincubation period shows that these polysomes are active in protein synthesis. PMID- 16658615 TI - Influence of auxin and incubation on the relative level of polyribosomes in excised soybean hypocotyl. AB - The influence of incubation and auxin (2,4-D) on polyribosome level in soybean hypocotyl was studied.A marked drop in the relative level of polyribosomes in excised apical or meristematic tissue (0 to 5 millimeters below the cotyledons) occurred during incubation. The addition of auxin to the incubation medium did not affect polyribosome level. A similar decrease in polyribosome level occurred in excised elongating tissue (5 to 15 millimeters below cotyledons) during incubation. Auxin, however, caused a small but highly reproducible stabilization of polyribosomes in this tissue. There was a rapid, but small, auxin-independent increase in polyribosomes of basal or nongrowing hypocotyl (from 20 to 40 millimeters below cotyledons) during incubation, followed by a larger auxin dependent increase in polyribosomes. While auxin is known to cause an increase in total ribosomes during incubation of the excised basal hypocotyl, the observed transformation from monoribosomes to polyribosomes was not dependent on new ribosome synthesis.Protein synthetic activity (poly U-directed phenylalanine incorporation) of the 80S monoribosomes at low Mg(2+) levels increased during incubation of the excised basal hypocotyl. The increase in ribosome activity was biphasic (an initial auxin-independent phase followed by an auxin-dependent increase in activity) correlating with the biphasic increase in polyribosomes. The enhanced activity of 80S monoribosomes was related, at least in part, to an increase in the level of peptidyl-tRNA associated with the ribosome population. Removal of peptidyl-tRNA from the ribosomes reversed the auxin effect.The hypothesis is advanced that the increase in polyribosomes in response to incubation and to auxin is preceded by and dependent upon the activation of 80S monoribosomes. This activation is in addition to a requirement for continued RNA synthesis, at least in part mRNA, for the transition from monoribosomes to polyribosomes. PMID- 16658616 TI - Turnover of galactosylglycerol and osmotic balance in ochromonas. AB - Osmotic balance in Ochromonas malhamensis is mediated directly by fluctuations in the pool size of alpha-galactosylglycerol (isofloridoside). Chase experiments with glucose-(14)C indicate that the pool is in rapid turnover even at constant size. The turnover rate is related to the pool size. Regulation of the pool size seems to occur at enzymic steps involved in the formation, as well as those involved in the degradation of isofloridoside. PMID- 16658617 TI - Regulation of Succinate Dehyrogenase in Higher Plants: I. Some General Characteristics of the Membrane-bound Enzyme. AB - The spectrophotometric phenazine methosulfate assay of succinate dehydrogenase was adapted to use with cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria with suitable modifications to overcome the permeability barrier to the dye. Procedures in the literature for the isolation and sonic disruption of mitochondria from these sources were modified to assure maximal yield and stability of the enzyme. In tightly coupled mung bean mitochondria, as isolated, about half of the succinate dehydrogenase is in the deactivated state, and the enzyme is further extensively deactivated on sonication or freeze thawing. In cauliflower mitochondria most of the enzyme is in the deactivated form, and little or no further deactivation occurs on sonication or freeze thawing. Incubation of mitochondria from either source with succinate leads to full activation of the enzyme. The energy of activation for the conversion of the deactivated to the activated form in membranal preparations under the influence of substrate is about 30,000 cal/mole, essentially the same value as in animal tissues. Activation of the enzyme also occurs under the influence of a variety of other agents, among which the action of anions as activators is documented in the present paper. Activation is accompanied by the release of very tightly bound oxaloacetate. As in animal tissues, the enzyme appears to contain covalently bound flavin (histidyl 8alpha-FAD), and the turnover number is 19,400 moles of succinate oxidized/mole of histidyl flavin at pH 7.5, 38 C. PMID- 16658618 TI - Regulation of Succinate Dehydrogenase in Higher Plants: II. Activation by Substrates, Reduced Coenzyme Q, Nucleotides, and Anions. AB - The effect of various agents on the activation of succinate dehydrogenase in cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria and in sonicated particles has been investigated. Reduced coenzyme Q(10), inosine diphosphate, inosine triphosphate, acid pH, and anions activate the enzyme in mitochondria from higher plants in the same manner as in mammalian preparations. Significant differences have been detected in the behavior of plant and animal preparations in the effects of ATP, ADP, NADH, NAD-linked substrates, and of 2, 4 dinitrophenol on the state of activation of the dehydrogenase. In mammalian mitochondria ATP activates, whereas ADP does not, and the ATP effect is shown only in intact mitochondria. In mung bean and cauliflower mitochondria, both ATP and ADP activate and the effect is also shown in sonicated and frozen-thawed preparations. In sonicated mung bean mitochondria NADH causes complete activation, as in mammalian submitochondrial particles, but in sonicated cauliflower mitochondria activation by NADH is incomplete, as is also true of intact, anaerobic cauliflower mitochondria. Moreover, neither NAD-linked substrates nor a combination of these with NADH can fully activate the enzyme in cauliflower mitochondria. In contrast to mammalian mitochondria, succinate dehydrogenase is not deactivated in cauliflower or mung beam mitochondria under the oxidized conditions brought about by uncoupling of oxidative phosphorylation by 2,4-dinitrophenol. PMID- 16658619 TI - The formation, isolation, and biological activity of a cytokinin 7-glucoside. AB - The cytokinin, 6-benzylaminopurine, is converted to its 7-glucoside in intact seedlings, organ slices, and tissue cultures from several plants. The ribonucleoside and 5'-ribonucleotide appear transiently, and the general metabolic sequence seems to be nearly identical in the four plant species thus far studied. The glucoside persists for long periods in plant tissues, while all other forms of the cytokinin are rapidly metabolized and disappear within a few hours. A procedure for the isolation in pure form of the glucoside is described. PMID- 16658620 TI - Amino Acid uptake by pea leaf fragments: specificity, energy sources, and mechanism. AB - Amino acid uptake into leaf fragments of Pisum sativum depended on metabolism. Glycine uptake was optimal at 30 C and could be supported by respiration and by photosynthesis. Based on studies with an electron flow cofactor, inhibitors, and uncouplers, the energy source for glycine uptake was apparently ATP.The energy dependent transport of glycine was mediated by a carrier that had a broad specificity for neutral and positively charged l-amino acids. It readily translocated 15 such l-amino acids into the cells, but had a very low affinity for l-aspartate, l-glutamate, d-amino acids, and alpha-aminoisobutyrate. The Ki for competitive inhibition of glycine uptake by another amino acid was equal to the Km for the uptake of that competing species. PMID- 16658621 TI - Absorption of 2,4-Dichlorophenoxyacetic Acid and 3-(p-Chlorophenyl)-1, 1 dimethylurea (Monuron) by Barley Roots. AB - Absorption from culture solution of the herbicides 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 3-(p-chlorophenyl)-1, 1-dimethylurea (Monuron) by excised barley (Hordeum vulgare L.) roots was studied to determine whether absorption was due to an active or a passive mechanism. Herbicide absorption was followed at low temperature, under anaerobic conditions, and in the presence of metabolic inhibitors and compounds of structure similar to that of the herbicide. Total absorption was divided into two phases, exchangeable and nonexchangeable herbicide, by washing the roots for 1 hour following absorption. Absorption of both exchangeable and non-exchangeable 2, 4-D appeared to depend on a supply of metabolic energy which suggests that an active mechanism may be involved. A possible conclusion is that 2, 4-D is absorbed by roots by an adsorption mechanism and that energy is required to maintain the integrity of the absorbing surfaces of the cell. In contrast, absorption of Monuron was independent of an energy supply. It is concluded that the bulk of the Monuron absorbed was taken up passively by diffusion. PMID- 16658622 TI - The Conversion of d-Glucose-6-C to Cell Wall Polysaccharide Material in Zea mays in Presence of High Endogenous Levels of Myoinositol. AB - When corn (Zea mays) roots are supplied with high concentrations of unlabeled myoinositol, the conversion of d-glucose-6-(14)C to cell wall galacturonic acid is significantly reduced compared to controls, although its incorporation into cell wall glucosyl units remains unchanged. This suggests that, in order to be converted to uronic acid, radiolabel from glucose must first pass through the internal myoinositol pool of the roots. PMID- 16658623 TI - The Biosynthesis of (+)-Tartaric Acid in Pelargonium crispum. AB - Metabolic conversion of l-galactono-1, 4-lactone and l-ascorbic acid to (+) tartaric acid and oxalic acid has been studied in Pelargonium crispum, cv. Prince Rupert. Experiments with specifically labeled substrates suggest a path of conversion involving cleavage of l-ascorbic acid, or a metabolic product of l ascorbic acid, between C(2) and C(3), such that oxalic acid arises from the two carbon fragment and (+)-tartaric acid from the four carbon fragment. PMID- 16658624 TI - Polyribosomes from Peas: III. Stimulation of Polysome Degradation by Exogenous and Endogenous Calcium. AB - Attempts were made to isolate microsomes from Pisum sativum L. var. Alaska by low speed centrifugation of a postmitochondrial supernatant made 8 mm in Ca(2+). However, the addition of Ca(2+) in concentrations as low as 1 mm to the postmitochondrial supernatant resulted in extensive polysome degradation. Degradation was dependent on both Ca(2+) concentration and the duration of incubation. Resuspension of isolated polysomes in Ca(2+)-containing buffer did not result in degradation, whereas resuspension in Ca(2+)-containing postpolysomal supernatant did. Both Ca(2+) and a heat-labile factor in the supernatant were required for polysome degradation. The degradation in the homogenate with or without added Ca(2+) could be reduced by (a) dilution with larger volumes of grinding buffer, (b) increasing the concentration of tris-HCl in the grinding buffer, (c) adding diethylpyrocarbonate or ethyleneglycol-bis (2 aminoethylether) tetraacetic acid (a specific calcium chelator) prior to homogenization or immediately after the addition of Ca(2+). Endogenous Ca(2+) can increase the destruction of polysomes during their isolation in this tissue, presumably by activating a ribonuclease. Addition of Ca(2+) is not a useful technique for separating undegraded free and membrane-bound polyribosomes. PMID- 16658625 TI - T-2 toxin decreases logarithmic growth rates of tobacco callus tissues. AB - T-2 toxin, a mycotoxin produced by Fusarium tricinctum, decreases logarithmic growth rates of tobacco (Nicotiana tabacum L.) pith callus tissues. Toxin concentrations as low as 0.003 mum will decrease growth rates; a concentration of 0.081 mum will halt growth completely. Additional exogenous cytokinin will reduce the inhibition by toxin only when the initial cytokinin and toxin concentrations are quite low (about 0.01 mum). When inhibited tissues are transferred to media lacking toxin, they assume the faster, control rates almost immediately. Maximal yields of tissue (yields at the point at which no sugar was detected in the medium) are not affected by toxin concentrations of 0.01 to 0.036 mum. PMID- 16658626 TI - Fusarium tricinctum T-2 Toxin Inhibits Auxin-promoted Elongation in Soybean Hypocotyl. AB - T-2 toxin, a mycotoxin produced by Fusarium tricinctum, inhibited elongation of excised hypocotyl sections of Glycine max var. Hawkeye 63. Auxin-promoted elongation was inhibited more severely than was control elongation, and a 1 hour preincubation of 5 mum toxin prevented the induction of a faster rate of elongation by auxin. While the inhibition of elongation by cytokinin was similar to that of the toxin, the mode of action of the two compounds appeared to be different, i.e. their effects on elongation were additive, and only kinetin promoted radial enlargement. Toxin treatment did not diminish cytokinin-induced radial enlargement. The properties of the plasma membrane, as measured by electrolyte leakage, were not affected by the toxin. PMID- 16658627 TI - Leaf Age and Ethylene-induced Abscission. AB - Ethylene has been generally credited with promoting the abscission of the oldest leaves on a plant first. Vegetative cotton (Gossypium hirsutum L.) seedlings are an exception to this generalization. Under some conditions the younger, apical, unexpanded, or partially expanded leaves abscise before the less young, basal leaves or cotyledons. The degree or extent of apical leaf abscission increases with ethylene concentration and with plant age from 2 to 5 weeks. The response is promoted by auxin transport inhibitors. Usually the leaves which abscise first are those which have just unfolded and ones apical to the opened but unexpanded leaves. With plants with eight or nine leaves and macroscopic leaf buds, after the initial loss of unexpanded leaves, abscission tends to progress downward from the youngest remaining leaves and upward from the oldest leaves. The findings indicate that some characteristic(s) of apical leaves increases their sensitivity to ethylene. The characteristic may be differences in the abscission process between expanded and unexpanded leaves or differences in the hormone complement of the different leaves. Work is under way to modify this young leaf abscission response in an effort to determine its cause. PMID- 16658628 TI - Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L. AB - In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The free amino acid level in detached cotyledons reached a maximum at the 11th day; protease formation leveled off after 50% of the original seed protein was digested. These data suggest that high free amino acid levels may inhibit protease formation.The activity of partially purified protease in aqueous extracts was enhanced by 10 mm 2-mercaptoethanol or cysteine, indicating a sulfhydryl requirement for activation. Protease formation in detached cotyledons was inhibited 30% by 10 mug/ml cycloheximide and 50% by 100 mum abscisic acid. In contrast, alpha-amylase formation was inhibited 90% by 10 mug/ml cycloheximide and 95% by 20 mum abscisic acid. The cycloheximide data suggest that only a part of the protease, but all of the alpha-amylase, is synthesized de novo; the similar pattern of inhibition by abscisic acid emphasizes the concept that protease may exist in two forms. PMID- 16658629 TI - A mechanism for the indirect transfer of photosynthetically reduced nicotinamide adenine dinucleotide phosphate from chloroplasts to the cytoplasm. AB - A triose phosphate/3-phosphoglycerate shuttle for the indirect transfer of photosynthetically reduced NADP from chloroplasts to the cytoplasm has been demonstrated in vitro. Triose phosphate, formed from 3-phosphoglycerate in the chloroplast, was oxidized back to 3-phosphoglycerate outside the chloroplast by the nonreversible d-glyceraldehyde 3-phosphate dehydrogenase reaction which is specific for NADP. The 3-phosphoglycerate could presumably return to the chloroplast to complete the shuttle. The properties of nonreversible d glyceraldehyde 3-phosphate dehydrogenase are considered particularly suitable for effective operation of this shuttle system. PMID- 16658630 TI - Effects of Ammonium Nutrition on Water Stress, Water Uptake, and Root Pressure in Lycopersicon esculentum Mill. AB - Ammonium (NH(4) (+)) nutrition inhibits water uptake and root exudation and decreases leaf water potential of tomato plants grown in solution culture. This inhibition is readily reversible by NO(3) (-) for short term exposures to NH(4) (+); however, recovery is delayed following long term exposures. PMID- 16658631 TI - Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric Chloride. AB - Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl(2) (less than 1 mumole Hg(2+)/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl(2) concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl(2) to these sites.Concentrations of HgCl(2), which inhibit steady state phosphorylation, do not inhibit dark phosphorylation after illumination (X(E)), suggesting that HgCl(2) affects a step in the coupling mechanism prior to the terminal step of ATP formation. PMID- 16658632 TI - Calcium distribution in the abscission zone of bean leaves: electron microprobe x ray analysis. AB - The calcium content and distribution across the abscission zones of (2 chloroethyl) phosphonic acid-treated bean (Phaseolus vulgaris L. var. Contender) leaves were lower and not uniformly distributed as compared to the control. Calcium chloride-treated bean leaves had a higher calcium content, with more calcium localized in the potential abscission layer. Ethephon treatment promoted abscission in both debladed and nondebladed plants; there was a corresponding decrease in calcium in the abscission zone just prior to separation. Deblading of bean leaves under a calcium solution increased the calcium level in the abscission zone and delayed abscission. PMID- 16658633 TI - Short term increases in the cold tolerance of red osier dogwood stems induced by application of cysteine. AB - Bark tissues of Cornus stolonifera stems, treated with cysteine at 24 hours after treatment, survived exposure to -11 C (the tissue temperature) with little or no injury. An initiation of increase in the cold tolerance was usually observed when plants were treated with cysteine at 12 hours after treatment. Neither plants at 36 or 48 hours after treatment nor plants 12 hours before treatment had shown increases in the cold tolerance. They were killed below -5 C, which was the survival temperature of untreated control plants. Two weeks or more of short day induction before cysteine application were required for a significant effect of short term 5 C increase in the cold tolerance. PMID- 16658634 TI - Translocation of sucrose by squash plants. AB - Sucrose-(14)C was fed to the leaf blades of squash plants (Cucurbita pepo L. var. melopepo torticallis Bailey) for 30 or 60 minutes. Petioles of treated plants were cut into sections and extracted. The majority of the (14)C within the petiole was in sucrose rather than stachyose, the sugar that is transported by the squash plants when (14)CO(2) is supplied. This indicates that the phloem loading system of squash plants is not the system that specifies which sugar is transported. PMID- 16658635 TI - Intracellular distribution of hydroperoxide isomerase. AB - Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies. PMID- 16658636 TI - The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide by isolated plant mitochondria. AB - Exogenous NADH oxidation by cauliflower (Brassica oleracea L.) bud mitochondria was sensitive to antimycin A and gave ADP/O ratios of 1.4 to 1.9. In intact mitochondria, NADH-cytochrome c reductase activity was only slightly inhibited by antimycin A. The antimycin-insensitive activity was associated with the outer membrane. Malate oxidation was sensitive to both rotenone and antimycin A and gave ADP/O values of 2.4 to 2.9. However in the presence of added NAD(+), malate oxidation displayed similar properties to exogenous NADH oxidation. In both the presence and absence of added NAD(+), malate oxidation was dependent on inorganic phosphate and inhibited by 2-n-butyl malonate. PMID- 16658637 TI - An Apparatus to Produce Gas Mixtures with Controlled CO(2), O(2), and Water Vapor Concentrations. AB - An apparatus to produce continuous gas mixtures for use in measurements of plant gas exchange is described. A wide range of CO(2) and water vapor concentrations can be provided and O(2) concentration can be varied from 0 to 21%. Changes in the concentrations of the components are accomplished conveniently, rapidly, and independently. With occasional adjustments, CO(2) and O(2) concentrations can be maintained to within +/- 1 mul/l and +/- 0.1%, respectively. Dew point of the gas mixture can be maintained to within +/- 0.05 C. PMID- 16658638 TI - Greater Length of Ribonucleic Acid Synthesized by Chromatin-bound Polymerase from Auxin-treated Soybean Hypocotyls. AB - An investigation has been made of the RNA synthesized by chromatin-bound RNA polymerase from soybean hypocotyls (Glycine max var. Wayne). Polymerase activity is 4- to 5-fold higher with chromatin from tissue treated with 2,4 dichlorophenoxyacetic acid, a synthetic auxin, compared to untreated tissue. Thin layer chromatography of the RNA hydrolysis products and acrylamide gel electrophoresis of the RNA synthesized by the chromatin show that increased activity induced by 2,4-dichlorophenoxyacetic acid is due primarily to the production of longer RNA chains, with only 20 to 50% increase in the number of RNA chains. The observation that 2,4-dichlorophenoxyacetic acid treatment leads to greater rates of RNA synthesis, producing longer chains in unit time, suggests that one manifestation of auxin activity is in activation of RNA polymerase I (ribosomal RNA polymerase). PMID- 16658639 TI - Phytochrome and Seed Germination: VI. Phytochrome and Temperature Interaction in the Control of Cucumber Seed Germination. AB - Phytochrome control of cucumber seed germination is temperature-dependent. A prolonged exposure to radiation from broad spectrum far red sources (Pfr/P = 0.05 to 0.07) prevents germination at temperatures below 20 C. Above 20 C there is no inhibition and it appears as if there is an escape from phytochrome control. However, radiation from a monochromatic, narrow band 730 nanometer source (Pfr/P < 0.02) inhibits germination at temperatures above 20 C. This result supports the idea that, even at high temperatures, Pfr is responsible for the activation of germination. After 4 days of exposure to far red, a short red irradiation is quite effective in promoting germination if temperatures during the dark incubation periods are maintained below 20 C; red becomes effective at temperatures above 20 C. Promotion of germination will take place at a temperature of 25 C or higher without red irradiation. Again, we have an apparent escape from phytochrome control at high temperatures. However, if higher temperatures are used for only short periods, 2 to 6 hours, in combination with short red irradiation, one can demonstrate that activation of germination at high temperatures is still dependent on phytochrome. Phytochrome is probably destroyed during prolonged exposure to far red. Thus, the subsequent short red irradiation establishes levels of Pfr which may not be sufficient to promote germination at low temperatures but are probably adequate at high temperatures. PMID- 16658640 TI - Effect of Heat Shock on Plant Growth and on Lipid and beta-Glucan Syntheses. AB - Primary leaves of 8-day-old Phaseolus vulgaris plants dipped in water at 46.5 to 47.5 C for 2 minutes were found to have decreased synthesis of lipid and beta glucan compared to controls; UDP-glucose was used as substrate by cell-free preparations. As time elapsed, leaf growth, which was inhibited within the first 12 hours recovered in a subsequent 24-hour period. Synthesis of alkali-insoluble glucan by cell-free preparations was stimulated both in absolute and relative terms in the treated leaves at the later time period. PMID- 16658641 TI - Higher plant cell membrane resistance by a single intracellular electrode method. AB - A single intracellular microelectrode technique has been adapted to measure membrane resistance in a higher plant cell. As a direct result of the convenience of this method, which allows relatively long term recordings on a single cell, it has been found that membrane resistance increases for about 30 minutes after cell impalement in Pisum sativum L. cv. Alaska root cortical cells, although cell potential is established at a constant value in less than 2 minutes. It is proposed that these observations imply a regulating feedback loop between electrogenic pump rates and membrane potential. PMID- 16658642 TI - A water potential threshold for the increase of abscisic Acid in leaves. AB - A relationship between abscisic acid concentration and leaf water status is reported. Water potentials were measured in leaves of Ambrosia artemisiifolia L. and Ambrosia trifida L. throughout a period of dehydration of intact plants. Tissues from the same leaves were analyzed for abscisic acid. For both species, abscisic acid began to increase in a critical water potential range (-10 to -12 atmospheres). These data suggest a threshold water potential that stimulates abscisic acid synthesis. The data support the hypothesis that a small change in water potential could affect stomatal resistance to water loss by means of a very sensitive chemical feedback control mechanism. PMID- 16658643 TI - The effect of growth in 99.8 deuterium oxide on the ultrastructure of winter rye. AB - A preliminary report dealing with the ultrastructural effects of culture in a 99.8% D(2)O (deuterium oxide) environment on winter rye (Secale cereale L. cv. Winter) is presented. In general, the cells of D(2)O-cultured seedlings appeared similar to the cells of H(2)O-cultured seedlings. However, differences were found in chloroplast and dictyosome morphology, and ribosome number. PMID- 16658644 TI - Regulation of Hexose Transport in Chlorella vulgaris: Characteristics of Induction and Turnover. AB - Of nine species of unicellular algae tested, Chlorella vulgaris showed the highest inducibility for an active hexose transport system. Whereas the rate of uptake in all other species was increased by induction less than 5-fold, it was increased more than 400-fold in one strain of C. vulgaris. With glucose as inducer, the minimum time necessary to synthesize inducible proteins of the transport system was 15 minutes. The Km for induction with glucose is 5 mum and with 6-deoxyglucose 1 mm. The inducing sugars have to penetrate the cells to be effective.Evidence indicating that regulation of induction occurs at the transcriptional level was obtained. The induction was inhibited by 6 methylpurine. When cells were exposed to induce in the presence of actidione no increase in transport activity could be measured. After removal of actidione as well as the inducer, an increased uptake activity was observed after 30 to 60 minutes. The induced uptake system showed a turnover with a half-life of 4 to 6 hours at 26 C under nongrowing conditions; at 0 C turnover was negligible. Turnover was partly inhibited by anaerobic condition and by actidione; it was accelerated under growing conditions. PMID- 16658645 TI - Translocation of Radioactive Carbon after the Application of C-Alanine and CO(2) to Sunflower Leaves. AB - (14)C-(UL)-l-Alanine was applied to the surface of mature leaves at the second node of sunflower (Helianthus annuus L. cv Commander) plants, under illumination. The alanine was absorbed during a 4-hour period, and some of it was metabolized by the absorbing tissue. After a lag period of about 15 minutes from first application, distribution of (14)C through the plant proceeded in much the same pattern as when (14)CO(2) is assimilated by similar leaves. Most, if not all, of the (14)C exported from the absorbing regions was in sucrose. Only minute amounts appeared in alanine or other amino acids in surrounding parts of the leaf blade or in the petiole, although these were strongly labeled in the tissue absorbing (14)C-alanine.When (14)CO(2) was supplied for 15 minutes to leaves of different ages, amino acids were lightly labeled in the leaf blade. Mature green leaves exported only sucrose. Yellowing leaves on 60-day-old plants exported a variety of substances including amino acids. PMID- 16658646 TI - Activation of 80S Maize Ribosomes by Red Light Treatment of Dark-grown Seedlings. AB - The in vitro protein synthetic activity of 80S ribosomes from leaves of dark grown corn seedlings was enhanced (at low Mg(2+) levels) by a 5-minute red light treatment applied 2 hours prior to tissue harvest. The effect was completely reversed by an immediate brief far red treatment, suggesting that ribosome activation is controlled by the phytochrome system. Experiments in which the interval between light treatment and tissue harvest was shortened indicate that the response was quite rapid. The initial increase in activity was detected within 30 minutes, followed by a rapid increase during the next 1.5 hours. No further increase occurred after 2 to 3 hours.The change in ribosome activity relates, at least in part, to an increase in the level of peptidyl-tRNA associated with ribosomes. Removal of peptidyl-tRNA from light-treated ribosomes also completely reversed the red light effect. Activation of ribosomes by 2 to 3 hours continuous white light (as previously reported) differs from red light activation in that reversal of this response requires salt washing of the ribosomes in addition to removal of peptidyl-tRNA. PMID- 16658647 TI - Inhibition of the photosynthetic capacity of isolated chloroplasts by ozone. AB - Isolated spinach chloroplasts have been used as a model system for studying the interaction of ozone, a component of photochemical smog, with plant membranes. Ozone bubbled into a suspension of isolated chloroplasts inhibits electron transport in both photosystems without uncoupling ATP production. Photosystem I (reduced 2,6-dichlorophenolindolphenol --> NADP(+)) is a little more sensitive than photosystem II (H(2)O --> 2,6-dichlophenolindolphenol). Ozone does not act as an energy transfer inhibitor, since the drop in ATP production and high energy intermediate (measured by amine-induced swelling) is nearly parallel to the decline in electron transport. A reasonable hypothesis is that ozone disrupts the normal pathway of energy flow from light-excited chlorophyll into the photoacts by a disruption of the components of the membrane but not a general disintegration of the membrane. In addition, ozone does not seem to penetrate into the grana region through the outer membrane of intact plastids, since ozone lowers the bicarbonate-supported O(2) evolution but does not affect the rate of ferricyanide reduction in the same plastids after osmotic disruption. This would indicate that the effect of ozone on green plants, at low concentrations, may be due to the interaction of ozone with the first membrane it contacts and not directly with internal metabolic processes. PMID- 16658648 TI - Ribulose Diphosphate Carboxylase from Freshly Ruptured Spinach Chloroplasts Having an in Vivo Km[CO(2)]. AB - The properties of a form of ribulose diphosphate carboxylase having a high affinity for CO(2) have been studied. Its apparent Km(HCO(3) (-)) of 0.5 to 0.8 mm (pH 7.8) and calculated Km(CO(2)) of 11 to 18 mum are comparable to the values exhibited by intact chloroplasts during photosynthesis. This form of the enzyme was released from chloroplasts in hypotonic media and was unstable, rapidly converting to a form having a high Km(HCO(3) (-)) of 20 to 25 mm similar to that for the purified enzyme. Incubation of the enzyme with MgCl(2) and HCO(3) (-) yielded a third form with an intermediate Km(HCO(3) (-)) of 2.5 to 3.0 mm.The low Km form had sufficient activity both at air levels of CO(2) and at saturating CO(2) to account for the rates of photosynthesis by intact chloroplasts. The low Km form could be stabilized in the presence of ribose 5-phosphate, adenosine triphosphate, and MgCl(2), at low temperatures for up to 2 hours. PMID- 16658649 TI - Water-deficit Stress, Ethylene Production, and Ripening in Avocado Fruits. AB - Differential rates of water loss were achieved in picked avocado (Persea americana Mill.) fruits, either by controlling the evaporation rate or by supplying water through the fruit stalk. A negative linear correlation was found between the daily rate of water loss from fruits and their ripening, as determined by the time from harvest to peak of ethylene production. Ripening rate was hastened by 40% in fruits which had lost water at rate of 2.9% fresh weight per day, compared with those which lost only 0.5% per day. PMID- 16658650 TI - A digital diffusion porometer circuit. AB - An automatic digital diffusion porometer circuit is described. The circuit is highly stable with respect to temperature and supply voltage. The device is capable of high timing accuracy over very short measurement intervals, so that measurements may be made rapidly without the danger of stomatal changes occurring during the measurement period. PMID- 16658651 TI - Correlations of Growth Rate and De-etiolation with Rate of Ent-Kaurene Biosynthesis in Pea (Pisum sativum L.). AB - Biosynthesis of the gibberellin precursor ent-kaurene-(14)C from mevalonic acid-2 (14)C was assayed in cell-free extracts of shoot tips of etiolated and light grown Alaska (normal) and Progress No. 9 (dwarf) peas (Pisum sativum L.). During ontogeny of light-grown Alaska peas, kaurene-synthesizing activity increased from an undectectable level in 3-day-old epicotyls to a maximum in shoot tips of 9-day old plants and remained relatively constant thereafter until postanthesis. The capacity for kaurene synthesis in extracts from shoot tips of 10-day-old etiolated Alaska seedlings increased approximately exponentially during the first 12 hr of de-etiolation in continuous high intensity white light and remained relatively constant during the succeeding 24 hr of irradiation. Extracts from light-grown Alaska (normal) shoot tips possessed greater capacity for kaurene synthesis than did extracts from light-grown Progress No. 9 (dwarf) shoot tips. Extracts from shoot tips of either light-grown cultivar displayed greater kaurene synthesizing capacity than was observed in extracts from their dark-grown counterparts. It is concluded that gibberellin biosynthesis in pea shoot tips is subject to partial regulation by factors controlling the rate of biosynthesis of kaurene. PMID- 16658652 TI - Mode of Pisatin Induction: Increased Template Activity and Dye-binding Capacity of Chromatin Isolated from Polypeptide-treated Pea Pods. AB - Increases in phenylalanine ammonia lyase activity and pisatin synthesis were induced in excised pea pods (a) by basic polypeptides such as protamine, histone, lysozyme, cytochrome c, and ribonuclease; (b) by the polyamines spermine, spermidine, cadaverine, and putrescine, and (c) by the synthetic oligopeptides poly-l-lysine, poly-dl-ornithine, and poly-l-arginine.Poly-l-lysine (1 milligram per milliliter, molecular weight 7,200) was utilized as a model inducer of pisatin and phenylalanine ammonia lyase. The poly-l-lysine-induced responses could be inhibited by adding the RNA synthesis inhibitors cordycepin or alpha amanitin to the pods prior to or at the time of inducer application. Cordycepin added 1.5 hours after inducer no longer completely inhibited induction. The application of poly-l-lysine was shown to characteristically change the rate of RNA synthesis within 30 minutes. Ultrastructural changes in pea nuclei were detected within 3 hours, and gross changes in nuclear morphology were apparent at 14 hours after inducer application. The physical appearance of uranyl acetate stained chromatin isolated from poly-l-lysine 2 hours after inducer application differed from that of water-treated tissues. The template properties of chromatin extracted from pods 3 hours after inducer application were consistently superior to control chromatin when assayed with Escherichia coli RNA polymerase (without sigma factor). Chromatin from poly-l-lysine-induced tissue also bound 49% more actinomycin D-(3)H.The DNA-complexing properties of inducer compounds and the induced changes in the template and dye-binding properties of pea chromatin formed the basis for a proposed mode of action for phytoalexin induction. PMID- 16658653 TI - Patterns of food utilization by the germinating lettuce seeds. AB - The embryo excised from seed of Grand Rapids lettuce (Lactuca sativa L.) can be cultured in distilled water. Complete digestion of the endosperm and transfer of nutrients from the endosperm to the embryo occur in the germinating seed with fat as the source of food. The fat is utilized for respiration, synthesis of amino acids, and to a degree, converted to sucrose. (14)C-Glucose administered to the seed is quickly converted to sucrose in the endosperm and translocated to the embryo. Radioactivity associated with the glucose remains predominantly in the carbohydrate fraction, and much of it is incorporated into what is believed to be cell wall polysaccharides. Relatively little isotope is distributed in the amino or organic acids. PMID- 16658654 TI - Factors affecting the reduction of acetylene by Rhizobium-soybean cell associations in vitro. AB - The acetylene reduction assay was used to measure presumed N(2)-reducing activity in Rhizobium-soybean cell associations in vitro. No acetylene reduction was observed in liquid suspensions of these organisms, but cells plated onto an agar medium from a liquid suspension of Rhizobium and soybean cells exhibited acetylene-dependent production of ethylene after 7 to 14 days. Aggregates of soybean cells 0.5 to 2.0 mm in diameter were required for this activity. Decreasing oxygen from 0.20 atm to 0.10, 0.04, or 0.00 atm completely inhibited acetylene reduction. The presence of 2,4-dichlorophenoxyacetic acid or kinetin increased endogenous ethylene production and inhibited acetylene-dependent ethylene production. Acetylene reduction was observed with three out of four strains of R. japonicum tested, and three rhizobial strains, which produce root nodules on cowpeas but not soybeans, formed an association capable of acetylene dependent ethylene production. PMID- 16658655 TI - Alkaloid Changes in Tobacco Seeds during Germination. AB - Nicotine, nornicotine, anabasine, and anatabine, normally found in growing and mature tobacco (Nicotiana tabacum L.) plants, were extracted and quantified from mature tobacco seeds and young tobacco seedlings. The rate of net alkaloid disappearance and accumulation in tobacco seedlings was related to phases of germination.In general, the increased rate of germination associated with higher temperatures also increased the rate of initial loss of alkaloids and the subsequent rate of accumulation of alkaloids. Maximum alkaloid accumulation in 144-hour-old seedlings cultured with 10-hour day occurred at 27 C. Following an 8 hour photoinduction period, seeds germinated in darkness accumulated greater amounts of alkaloids than seeds exposed to light each day. Seeds germinated in darkness for 96 hours, following the 8-hour photoinduction period, and then exposed to light each day accumulated the greatest amounts of alkaloids. PMID- 16658656 TI - Rapid Phytochrome-mediated Changes in Adenosine 5'-Triphosphate Content of Etiolated Bean Buds. AB - This study was designed to determine the effects of red and far red irradiation on ATP metabolism in etiolated bean buds (Phaseolus vulgaris L. var. Red Kidney). Compared to dark controls, red irradiated buds show an initial decline in ATP content at 15 seconds following a 5-minute irradiation. ATP content then rapidly rises to a peak at 1 minute, and then slowly returns to the baseline. The 1 minute promotion of ATP content is red/far red reversible. Acetylcholine does not appear to mimic red light in this system; it causes a marked decrease in ATP content. PMID- 16658657 TI - Ethylene-induced Tropism of Trifolium fragiferum L. Stolons. AB - The hypothesis that ethylene regulates prostrate stem growth was investigated using stolons of strawberry clover (Trifolium fragiferum L. var. Salina). Stolons became erect when treated with ethylene or 2-chloroethylphosphonic acid. Curvature was visibly detectable 2 hours after ethylene treatment, and subsequent stem elongation was rapid. Indoleacetic acid application to prostrate stolons caused only a small transitory curvature persisting less than 48 hours. Indoleacetic acid-stimulated curvature was accompanied by an increase in ethylene evolution. When stolon curvature was induced by placing strawberry clover plants in darkness or by applying gibberellic acid, ethylene production did not parallel stolon curvature. PMID- 16658658 TI - Activation of Protein Synthesis upon Dilution of an Arachis Cell Culture from the Stationary Phase. AB - When a stationary phase cell culture of Arachis hypogaea L. is diluted into fresh media, there occurs a 10-fold increase in the rate of protein synthesis. The kinetics of the activation of amino acid-incorporating capacity show a lag of 10 to 15 minutes with maximal activity reached at 2 hours after dilution. The activation of protein synthesis is oxygen-dependent and is accompanied by a 2- to 4-fold increase in polyribosome content, as well as by a 3- to 4-fold increase in the rate of mRNA synthesis. Ribosomal function, as ascertained by determination of ribosomal transit time, is about 2.5 times more efficient in 2-hour diluted cultures as in cells immediately after dilution. These observations indicate that a very early response in the transition of plant cell cultures from the stationary state is an increased capacity for protein synthesis. At a molecular level, this increase in protein synthetic capacity is due in part to an increased mobilization of mRNA into polyribosomes and in part to a more efficient ribosomal translational capacity. PMID- 16658659 TI - A simple technique for the establishment of nitrogenase in soybean callus culture. AB - A nitrogen-fixing association can be readily established in vitro between Rhizobium and cultured soybean root cells. Plant cells are grown as a thin callus on the surface of solid defined medium containing low levels of inorganic nitrogen and inoculated with bacteria during the active phase of growth. Acetylene reduction activities up to 275 nmoles ethylene/hr.g dry weight of cells have been recorded.The system is compact, uses inexpensive glassware, and facilitates multiple replication of samples. The use of a single medium throughout the experiments avoids unnecessary disturbance to the cells. The development of nitrogenase activity is relatively rapid and its activity can be recorded within a 3-week period. PMID- 16658660 TI - High Energy Charge as a Requirement for Axis Elongation in Response to Gibberellic Acid and Kinetin during Stratification of Acer saccharum Seeds. AB - The growth potential of embryonic axes of Acer saccharum Marsh. increased during moist storage at 5 C but not at 20 C. During the period of increasing growth potential, the oxygen consumption of the axes remained constant. It was possible to distinguish three phases of the stratification-germination process at 5 C with respect to response of the axis to gibberellic acid and kinetin. From 0 to 10 days the growth regulators had no effect on elongation; from 10 to 60 days axis elongation was stimulated; and between day 60 and day 75, when germination had begun, the growth substances were inhibitory. The adenylate energy charge remained low (0.15) in axes of dry dormant seeds but increased to 0.78 following imbibition of water and 10 days of moist storage at 5 C. This phenomenon was not specifically related to low temperature stratification, since a rapid increase in the energy charge of the axes also occurred following imbibition and moist storage at 20 C. The excised axes would elongate in response to the growth substances only when a high energy charge (approximately 0.8) was maintained. PMID- 16658661 TI - Enhancement of Phloem exudation from cut petioles by chelating agents. AB - The photosynthetic assimilates in leaves of Perilla crispa attached to the plant were labeled by treating the leaves with (14)CO(2). When subsequently detached, these leaves exuded a negligible amount of radioactivity from the cut petiole into water. Ethylenediaminetetraacetate (EDTA), citric acid, and ethyleneglycol bis (beta-aminoethyl ether) N,N'-tetraacetate greatly increased exudation of labeled assimilates into a solution bathing the petioles. The optimal concentration of EDTA was 20 mm, and maximal exudation took place between 2 and 4 hours after excision. Up to 22% of the radioactivity fixed in the leaf was exuded into an EDTA solution as compared to an export of 38% from attached leaves. The amount of radioactivity in the exudate was much reduced at low temperature. Presence of EDTA was required in the collecting solution for only 1 to 2 hours; upon transfer to water, exudation continued as in continuous presence of EDTA. Ca(2+) completely inhibited the effect of EDTA.Anatomical studies indicated that callose formation on the sieve plates near the cut surface of the petioles was less in leaves on EDTA than on water.More than 95% of the radioactivity exuded by detached leaves was present in the sugars verbascose, stachyose, raffinose, and sucrose, which are translocated in the phloem of Perilla. Labeled glucose, fructose, and galactinol were detected in the leaf blade and petiole, but not in exudates.The addition of EDTA to a solution bathing the petiole of detached leaves of Chenopodium rubrum and Pharbitis nil also increased the exudation of labeled assimilates. In these two species, label appeared only in a compound that cochromatographed with sucrose.It is concluded that the radioactive products in the solution are actually exuded by the phloem. Possibly EDTA chelates Ca(2+) that otherwise participates in the reactions that seal cut phloem. PMID- 16658662 TI - Steryl Glycoside Formation in Seedlings of Nicotiana tabacum L. AB - Particulate enzyme preparations from tobacco seedlings (Nicotiana tabacum L.) were used in the synthesis of steryl glycoside. The data obtained by measuring cholesterol-4-(14)C incorporation generally agree with results obtained with UDP glucose-(14)C. The in vitro reaction was linear for the first 10 minutes and had a pH optimum of 7.0 to 7.4. Addition of ATP activated while UDP-glucose inhibited slightly the reaction. In short term experiments, the percentage disappearance of endogenous and added sterol was about the same.Intact tobacco seedlings incorporated cholesterol-4-(14)C and sitosterol-4-(14)C into their steryl glycosides. The acylated steryl glycosides were more rapidly labeled than the nonacylated form. After 12 hours of incubation with cholesterol-4-(14)C, about 5% of the radioactivity was recovered as steryl glycoside and 12% as acylated steryl glycoside. Incubation for 12 hours with authentic cholesteryl-(14)C glucoside gave only a 4% acylation, and under these conditions 21% of the radioactivity was recovered as free cholesterol. It is suggested that acylated steryl glycosides may be formed through the acylation of steryl glycosides or the transfer of an acyl-glycosyl group to sterol. PMID- 16658663 TI - Properties of phosphoribulokinase of whole chloroplasts. AB - The ability of intact spinach (Spinacia oleracea) chloroplast preparations to catalyze CO(2) fixation and photophosphorylation was examined. Under conditions optimal for CO(2) fixation, only poor photophosphorylation was observed. Conditions optimal for photophosphorylation were found to be highly inhibitory to the CO(2)-fixing capacity of the intact chloroplast preparation.A method for following the activity of phosphoribulokinase in the intact chloroplast preparation was developed, and conditions for optimal activity were defined. The enzyme was found to be activated 2- to 4-fold by preillumination with a half-time of less than 15 seconds. Activation was inhibited by magnesium ions and selectively by inhibitors of photosynthetic electron transport. We concluded that activation was due to the effect of a photoproduced reductant in a site preceding ferredoxin in the electron transport chain. The photoactivated state of the enzyme decayed in the dark with a half-time of about 8 minutes. PMID- 16658664 TI - Carbon dioxide fixation in the light and in the dark by isolated spinach chloroplasts. AB - Factors affecting CO(2) fixation in the spinach (Spinacia oleracea) chloroplast were investigated. Free magnesium ions are shown to be highly inhibitory for photosynthetic CO(2) fixation in isolated intact spinach chloroplasts. The pH optimum for CO(2) fixation is about 8.5 but is dependent upon the reaction medium. Conditions are defined under which chloroplasts illuminated in the absence of CO(2) accumulate ribulose 1,5-diphosphate, and fix CO(2) in a subsequent dark period when high magnesium ion concentrations are provided. The regulation of photosynthetic CO(2) assimilation by these factors is discussed. PMID- 16658665 TI - Formation of adenosine 5'-phosphorofluoridate and the assay of adenyl cyclase in barley seeds. AB - Barley seed (Hordeum vulgare L.) homogenates contain an apparent enzymatic activity which catalyzes the synthesis of adenosine 5'-phosphorofluoridate from magnesium-adenosine 5'-triphosphate and sodium fluoride. Formation of this compound may interfere with some adenyl cyclase assays which use fluoride as a component of the incubation medium. Neither adenyl cyclase activity nor endogenous adenosine 3': 5'-monophosphate was detected in barley seed homogenates or extracts. PMID- 16658666 TI - Utilization of nucleoside diphosphate glucoses in developing cotton fibers. AB - The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP (14)C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-(14)C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers. PMID- 16658667 TI - A Failure to Detect an Influence of Magnetic Fields on the Growth Rate and Circadian Rhythm of Neurospora crassa. AB - Low strength magnetic fields, 6.36 and 32.25 gauss, were found to have no effect, with one questionable exception, on the circadian rhythm and growth rate of Neurospora crassa. This was true whether the fields were continuous, pulsed 20 minutes daily, or on a 12: 12, on-off cycle. PMID- 16658668 TI - Accumulation and radial transport of ions from potassium salts by cucumber roots. AB - Accumulation of K(+) is insensitive to the anion supplied with it at a solution concentration below 1 mm. Rates of K(+) transport to the xylem from the same solutions are, however, dependent upon the anion present and decrease in the order NO(3) (-) >> Cl(-) > SO(4) (2-). Parallel effects on rates of exudation and anion transport result from kind and concentration of anion supplied and time of exposure to the solution. When high K salt concentrations are used, only linear relationships are found between solution concentrations and transport rates. However, ion concentration in the exudate increases more than external solution concentration, while exudation rate is unaffected. It is suggested that some of the ions transported are from compartments within the cells. At high solution concentrations KNO(3) results in more exudation and in higher ion concentration in the exudate than is found with KCl. PMID- 16658669 TI - Mechanism of Indole-3-acetic Acid Conjugation: No Induction by Ethylene. AB - Formation of indole-3-acetic acid-aspartate in detached primary leaves of cowpea (Vigna sinensis Endl.) floating on (14)C-indole-3-acetic acid (3 muc; 3.15 mum, phosphate-citrate buffer, pH 4.75), almost doubled when leaves were pretreated with 31.5 mum(12)C-indole-3-acetic acid for 17 hr and then transferred to (14)C indole-3-acetic acid for 4 hours as compared with leaves preincubated in buffer only. When leaves were preincubated with ethylene (11.0 and 104 mul/l) instead of (12)C-indole-3-acetic acid, no induction of indole-3-acetylaspartic acid formation was observed, and the rate of indole-3-acetylaspartic acid formation decreased as compared with control leaves. Rhizobitoxine (1.87 mum) inhibited indole-3-acetic acid-induced ethylene production but did not prevent the formation of indole-3-acetylaspartic acid. In view of the similarity of these results and those previously obtained with alpha-naphthaleneacetic acid, it is concluded that ethylene has no role in the auxin-induced indole-3-acetylaspartic acid formation in cowpea leaves. PMID- 16658670 TI - Enzymes related to lactate metabolism in green algae and lower land plants. AB - Cell-free extracts of Chlorella pyrenoidosa contained two enzymes capable of oxidizing d-lactate; these were glycolate dehydrogenase and NAD(+)-dependent d lactate dehydrogenase. The two enzymes could be distinguished by differential centrifugation, glycolate dehydrogenase being largely particulate and NAD(+)-d lactate dehydrogenase being soluble. The reduction of pyruvate by NADH proceeded more rapidly than the reverse reaction, and the apparent Michaelis constants for pyruvate and NADH were lower than for d-lactate and NAD(+). These data indicated that under physiological conditions, the NAD(+)-linked d-lactate dehydrogenase probably functions to produce d-lactate from pyruvate.Lactate dehydrogenase activity dependent on NAD(+) was found in a number of other green algae and in the green tissues of a few lower land plants. When present in species which contain glycolate oxidase rather than glycolate dehydrogenase, the enzyme was specific for l-lactate rather than d-lactate. A cyclic system revolving around the production and utilization of d-lactate in some species and l-lactate in certain others is proposed. PMID- 16658671 TI - Biosynthesis of phosphatidylethanolamine by enzyme preparations from plant tissues. AB - The enzymic utilization of cytidine diphosphoethanolamine in the synthesis of phosphatidylethanolamine is localized in the microsomal fraction of spinach (Spinacia oleracea) leaves. The metal ion requirement can be satisfied by Mn(2+) (saturation approximately 0.6 mm) or Mg(2+) (saturation approximately 25 mm). The enzyme has a pH optimum of 8.0 in the presence of Mn(2+) and 7.5 in the presence of Mg(2+). A Michaelis constant of 20 mum was determined for cytidinediphos phoethanolamine. Enzyme activity was stimulated by thiol compounds and inhibited by thiol reagents. No inhibition was obtained with cytidine monophosphate and Tween 80.The in vitro biosynthesis of phosphatidylethanolamine was inhibited by cytidine diphosphocholine and the biosynthesis of phosphatidylcholine was inhibited by cytidine diphosphoethanolamine. Activities of the two synthetic systems were indistinguishable on the basis of susceptibility to lyophilization and inhibition by thiol reagents. PMID- 16658672 TI - The participation of two rhythms in the leaf movements of xanthium plants given various light-dark cycles. AB - We have shown Xanthium strumarium exhibit two distinct leaf movement rhythms with one occurring in continuous light and presumably related to an endogenous rhythm initiated by the "light-on" signal and the other occurring in continuous dark and presumably related to an endogenous rhythm initiated by the "light-off" signal. Characteristic of the light-on rhythm is a sudden and rapid downward movement of the leaf occurring about 16 hours after the light-on signal. Characteristic of the light-off rhythm is an immediate and sudden upward movement following the light-off signal. Under certain photoperiodic treatments, the two movements seem to be in conflict.The characteristic light-on downward movement was predominant in the light-dark 14: 10 and 16: 8 treatments while the light-off upward movement was predominant in light-dark 8: 16 and 10: 14 treatments. In the light-dark 12: 12 treatment, one movement was predominant in about half of the cases and vice versa. Thus leaf movements of X. strumarium plants given these light-dark treatments were found to fit a hypothesis based on the participation of both a light-on and a light-off rhythm. The over-all leaf movement rhythm may be controlled by what might be called two clocks. Both of these clocks may have essentially the same basic mechanism with one important distinction. One is rephased by the light-on signal and the other by the light-off signal. Furthermore, the leaf movements related to each clock are distinctive.The hypothesis of the participation of a light-on and a light-off rhythm was also found to be applicable to published data (M. Holdsworth, 1959. Effects of day length on the movements of pulvinate leaves. New Phytol. 58: 29-45). Interpretation of the Bauhinia leaf movements along this line is also presented. PMID- 16658673 TI - Comparative Enzymology of the Adenosine Triphosphate Sulfurylases from Leaf and Swollen Hypocotyl Tissue of Beta vulgaris: Multiple Enzyme Forms in Hypocotyl Tissue. AB - ATP sulfurylase activity was partially purified from the swollen hypocotyl of beetroot (Beta vulgaris); activity was measured by sulfate-dependent PPi-ATP exchange. The ATP sulfurylase activity was separated from pyrophosphatase and ATPase activities which interfere with the assay of ATP sulfurylase activity. The ATP sulfurylase activity from hypocotyl tissue was invariably resolved into two approximately equal activities (hypocotyls I and II) by ion exchange chromatography and polyacrylamide gradient gel electrophoresis. Both enzymes catalyzed selenate- and sulfate-dependent PPi-ATP exchange; the affinity of hypocotyl II for these substrates was greater than for hypocotyl I. It is unlikely that the two activities arise by allelic variation or as an artifact of purification; they are most probably isoenzymes. Studies of the subcellular localization of the two hypocotyl enzymes were inconclusive.ATP sulfurylase was also purified from leaf tissue. Ion exchange chromatography resolved the ATP sulfurylase from leaf tissue into a major activity (which accounted for 98% of the total leaf activity) and a minor activity. The major leaf and hypocotyl II ATP sulfurylases were indistinguishable as judged by the properties investigated. PMID- 16658674 TI - Wound healing in potato tuber tissue: phosphon inhibition of developmental processes requiring protein synthesis. AB - Several aspects of wound healing in tuber tissue of potato (Solanum tuberosum var. Kennebec), known to require protein synthesis, are inhibited by 2,4 dichlorobenzyltributylphosphonium chloride (Phosphon D). Cell division was completely blocked by 60 mum Phosphon and markedly reduced by concentrations as low as 3 mum. When applied at the time of wounding, 0.25mm Phosphon completely prevented the wound-induced respiratory increase. Application at 15 hours after wounding arrested respiration at the rate present at that time. The same concentrations of Phosphon inhibited auxin-induced cell expansion of the tissue, protein synthesis as measured by the incorporation of leucine-(14)C into the trichloroacetic acid-insoluble fraction of tissue disks, and the appearance of wound-induced peroxidase isozymes. None of these inhibitory effects of Phosphon could be prevented or reversed by the application of gibberellic acid. All wound induced processes inhibited by Phosphon are also inhibited by cycloheximide. It is suggested that inhibitory effects of Phosphon on wound healing in potato and on other developmental processes in excised plant tissues which cannot be reversed by gibberellin are due to interference with protein synthesis. PMID- 16658675 TI - Physiology of the Yellow-Green 6 Gene in Tomato: A Possible Interrelationship between the Phenotypic Expressions of the Yellow-Green 6 Gene Mutation and the Gibberellins. AB - The yellow-green 6 (yg(6)) mutation in tomato (Lycopersicon esculentum Mill.) is controlled by a single recessive gene with pleiotropic effects. The syndrome of characters associated with the mutation are enhanced stem elongation, reduced chlorophyll content and absence of detectable anthocyanins. We now have shown that the mutant also has fewer lateral roots than the wild type and higher l phenylalanine ammonia-lyase (E.C. 4.3.1.5) activity than the normal tomato. These traits of the mutant closely resemble those induced in many plants by the application of gibberellic acid which suggests that the phenotypic expressions of the mutation might in some manner be related to the endogenous level or activity of the gibberellins. In support of this premise, data are presented which show that the characters of the mutant can be induced in the wild type tomato by application of gibberellic acid. Conversely, several traits of the wild type can be induced in the mutant by an inhibitor of gibberellin hiosynthesis, Phosfon. In addition, an embryoless barley half-seed bioassay for the gibberellins and gas liquid chromatography indicated that the mutant contained at least three times as much total gibberellin as the wild type plant. PMID- 16658676 TI - The Flavin Content of Clovers Relative to Symbiosis with a Riboflavin-requiring Mutant of Rhizobium trifoli. AB - A riboflavin-requiring auxotroph of Rhizobium trifolii (T1/D-his(r)-15) formed ineffective root nodules on red clover and on two cultivars of subterranean clover, but produced almost fully effective nodules on several other cultivars of subterranean clover. Fluorescence and bioassay measurements of the flavin content of the roots and shoots of these cultivars revealed no differences between cultivars which could be correlated with the differences in symbiotic response. The concentration of flavin in nodules formed by the auxotroph (in the absence of riboflavin), by the effective parent strain (T1), or by a partly effective mutant (penicillin-resistant) of T1 was roughly proportional to the effectiveness of the nodules. Effective nodules contained 20 times as much flavin, and ineffective nodules 3 to 4 times as much flavin as non-nodulated root tissue. Approximately 20 to 30% of the flavins in both root and nodule tissue was flavin adenine dinucleotide and 70 to 80% was riboflavin + flavin mononucleotide. Most of the flavin adenine dinucleotide in macerated nodules was associated with host cell fragments, and none was detected in a cell-free fraction. Bacteroids accounted for approximately 20% of flavins in effective nodules and also contained more riboflavin + flavin mononucleotide than cultured rhizobial cells. The total flavin content of noninoculated roots increased from about 1.2 nmoles to 1.7 nmoles flavin/g of tissue after 3 days' exposure to 80 mum riboflavin. Exposure of only the upper or lower portion of preinoculated roots indicated negligible translocation, as effective nodulation occurred only on parts of the root in direct contact with riboflavin. Plants grown in a medium containing combined nitrogen (100 or 300 mum nitrogen added as (NH(4))(2)SO(4)), but no added riboflavin showed an increased root flavin content (about 2.1 nmoles flavin/g tissue) and a partly effective response when inoculated with the mutant. Nitrogen also promoted some upward translocation of exogenous riboflavin in the roots. PMID- 16658677 TI - Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron. AB - Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of alpha, alpha' dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8 hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm alpha, alpha' dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of alpha, alpha'-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with alpha, alpha'-dipyridyl, porphyrin production is inhibited and delta-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of delta-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of delta-aminolevulinic acid and porphyrins. PMID- 16658678 TI - S-acyl glutathione thioesterase of plant tissue. AB - The enzymic hydrolysis of S-acyl glutathione has been observed in extracts of various plant tissues. The richest source found in this study was avocado mesocarp. No enzymic activity was observed with acetyl coenzyme A or acetylthiocholine as substrates. Hydrolytic activity was essentially constant in the pH range 7 to 9. There was a break in the activation energy plot at 25 C with activation energy above that point being 6800 calories and 2600 calories below it. The Michaelis constants for S-acetyl, S-propionyl, and S-butyryl glutathione were 200, 80, and 15 mum, respectively. The data are not consistent with the hypothesis that variation in the phytotoxicity of peroxyacyl nitrates is a function of the ability of the plant to hydrolyze glutathione thioesters. PMID- 16658679 TI - Further studies on the bicarbonate stimulation of photophosphorylation in isolated chloroplasts. AB - The bicarbonate effect in stimulating the rate of photophosphorylation by isolated spinach (Spinacia oleracea var. Virginia blight-resistant savoy) chloroplasts at a pH below the optimum has been re-examined. Its seasonal nature may be related to the hormonal status of the plants. Bicarbonate anions stimulate adenosine 5'-triphosphate synthesis if added in the final, adenosine 5' triphosphate-forming stage of either a postillumination or an acid-base experiment. They also stimulate the membrane-bound, Mg(2+)-dependent adenosine 5' triphosphatase of chloroplasts, and the Ca(2+)-dependent adenosine 5' triphosphatase of detached coupling factor. These and other data point to the interaction between energized thylakoid membranes and the coupling factor as the probable site of action of bicarbonate anions when they stimulate photophosphorylation. PMID- 16658680 TI - Activation of Avena coleoptile cell wall glycosidases by hydrogen ions and auxin. AB - Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of beta-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo beta-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process. PMID- 16658681 TI - Auxin Does Not Alter the Permeability of Pea Segments to Tritium-labeled Water. AB - The possibility of an auxin effect on the permeability of pea (Pisum sativum L. ev. Alaska) segments to tritium-labeled water has been investigated by three separate laboratories, and the combined results are presented. We were unable to obtain any indication of a rapid effect of indoleacetic acid on the efflux of (3)HHO when pea segments previously "loaded" for 90 minutes with (3)HHO were transferred to unlabeled aqueous medium with indoleacetic acid. We were able to confirm that segments pretreated with (3)HHO plus indoleacetic acid for 60 to 90 minutes can show an enhanced (3)HHO release as compared with minus indoleacetic acid controls. However, this phenomenon appears to be due to an increased uptake of (3)HHO during the prolonged indoleacetic acid pretreatment, and therefore we conclude that auxin does not alter the permeability of pea segments to (3)HHO in either short term or long term tests. We confirm previous reports that the uptake of (3)HHO in pea segments proceeds largely through the cut surfaces, and that the cuticle is a potent barrier to (3)HHO flux. PMID- 16658682 TI - Elimination of the lag period in chloroplast development in a chlorophyll mutant of peanuts. AB - The mutation of a nuclear gene in peanut (Arachis hypogaea L.) plants results in a reduced light-dependent development of chloroplast fine structure, soluble protein, ribulose-1, 5-diP carboxylase, NADP-glyceraldehyde-3-P dehydrogenase, fructose-1, 6-diP aldolase, glycerate-3-P kinase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and dark respiration during the 72-hour lag period of chlorophyll synthesis in dark-grown leaves exposed to continuous light. The mutation has pleiotropic affects. Kinetic analysis shows there is also a 72 hour lag period in the light-dependent development of NADP-glyceraldehyde-3-P dehydrogenase and fructose-1, 6-diP aldolase in the mutant leaves, whereas there is no lag in the development of NAD-malate dehydrogenase and dark respiration. There is minimal development of the chloroplast during the 72-hour mutationally induced lag period, but there is pronounced cytoplasmic and mitochondrial activity during this phase. There is a 24-hour lag period in the light-dependent enlargement of the mutant leaves. At the completion of leaf enlargement, chloroplast differentiation is initiated. The mutation does not result in any chloroplast deletions, it only affects the timing of the synthesis of these components.Elimination of the lag period in leaf enlargement and chloroplast development (potentiation) requires a preliminary 72- to 96-hour dark period before exposing the dark-grown leaves to continuous light. There is extensive development of the etioplasts during this dark period. These results establish that the nuclear gene mutation affects the early stages of plastid development and not the light-dependent synthesis of plastid components. The nuclear gene may code for the regulation of the synthesis of a component (nutrient) in the dark (or during the lag phase in the light) which is essential for the development of mesophyll cells and plastids. Although, the chloroplast is a semi-autonomous organelle, nuclear gene control of chloroplast differentiation may not be independent of cellular growth. PMID- 16658683 TI - Internal Water Status of Kinetin-treated, Salt-stressed Plants. AB - Stomatal resistances and turgor pressures were measured during a 12-day period on leaves of bean (Phaseolus vulgaris L. cv. Contender) which were treated with kinetin, were salinized or were treated with kinetin and salinized. Stomatal resistances were highest in salt-stressed plants, and progressively lower in salt stressed and kinetin-treated plants, control plants, and kinetin-treated plants. Turgor pressures were highest in salt-stressed plants, and progressively lower in control plants, kinetin-treated plants, and salt-stressed and kinetin-treated plants. Stomata appeared to be kept open more widely under kinetin treatment than under control conditions, even when turgor pressures were lower in kinetin treated plants than in control plants. PMID- 16658684 TI - Inhibition of Sterol Biosynthesis in Chlorella sorokiniana by Triparanol. AB - When Chlorella sorokiniana was cultured in the presence of 1 mg/1 triparanol succinate, there was a 42% reduction in total sterol concentration. Algal biomass was reduced by approximately the same amount. In addition to the cycloartenol, cyclolaudenol, 24-methyl-pollinastanol, ergosta-5, 7-dien-3beta-ol, and ergosterol that occur in control culture, pollinastanol, 14alpha-methyl-5alpha ergost-8-en-3beta-ol, 5alpha-ergosta-8, 14, 22-trien-3beta-ol, 5alpha-ergosta 8(14), 22-dien-3beta-ol, 5alpha-ergosta-8(9), 22-dien-3beta-ol, 5alpha-ergosta-8, 14-dien-3beta-ol, 5alpha-ergost-8(9)-3n-3beta-ol, 5alpha-ergost-8(14)-en-3beta ol, 5alpha-ergosta-7, 22-dien-3beta-ol, and 5alpha-ergost-7-en-3beta-ol were isolated and identified from triparanol succinate-treated cells. A biosynthetic pathway for sterol biosynthesis in this organism is postulated based on all the sterols that were isolated and identified in triparanol-treated cultures of C. sorokiniana. Cyclolaudenol appears to be the product of the first alkylation at C 24 in this organism rather than the more common 24-methylene cycloartanol. Since 24-methylene sterols are needed for the second alkylation reaction, this would explain the absence of C-29 sterols in C. sorokiniana. Four of the sterols identified in C. sorokiniana are reported for the first time in a living organism. They are: 24-methyl pollinastanol, 5alpha-ergosta-8, 14, 22-trien-3beta ol, 5alpha-ergosta-8(14), 22-dien-3beta-ol and 5alpha-ergost-8(14)-en-3beta-ol. PMID- 16658685 TI - Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T). AB - Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from (14)C-mevalonic acid or (14)C-acetate, permitting preparation of (14)C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility. PMID- 16658686 TI - Rapid estimates of relative water content. AB - Relative water content may be accurately estimated using the ratio of tissue fresh weight to tissue turgid weight, termed here relative tissue weight. That relative water content and relative tissue weight are linearly related is demonstrated algebraically. The mean value of r(2) for grapevine (Vitis vinifera L. cv. Shiraz) leaf tissue over eight separate sampling occasions was 0.993. Similarly high values were obtained for maize (Zea mays cv. Cornell M-3) (0.998) and apple (Malus sylvestris cv. Northern Spy) (0.997) using a range of leaf ages. The proposal by Downey and Miller (1971. Rapid measurements of relative turgidity in maize (Zea mays L.). New Phytol. 70: 555-560) that relative water content in maize may be estimated from water uptake was also investigated for grapevine leaves; this was found to be a less reliable estimate than that obtained with relative tissue weight. With either method, there is a need for calibration, although this could be achieved for relative tissue weight at least with only a few subsamples. PMID- 16658687 TI - Isolation and characterization of organelles from soybean suspension cultures. AB - Whole homogenates from cells of Glycine max grown in suspension culture were centrifuged on linear sucrose gradients. Assays for marker enzymes showed that distinct peaks enriched in particular organelles were separated as follows: endoplasmic reticulum (density 1.10 g/cm(3), NADH-cytochrome-c reductase), Golgi membranes (density 1.12 g/cm(3), inosine diphosphatase), mitochondria (density 1.18-1.19 g/cm(3), fumarase, cytochrome oxidase) and microbodies (density 1.21 1.23 g/cm(3), catalase). In cells which had ceased to grow (stationary phase) only a single symmetrical catalase peak at density 1.23 g/cm(3) was observed on the sucrose gradient. During the phase of cell division and expansion a minor particulate catalase component of lighter density was present; its possible significance is discussed. PMID- 16658688 TI - Actions of gibberellic Acid and phytochrome on the germination of grand rapids lettuce seeds. AB - Red light and gibberellic acid were about equally effective in promoting germination of Grand Rapids lettuce (Lactuca sativa L.) seeds. With initial far red light treatment more than 80% remained dormant in subsequent dark storage. After 2 days of dark storage, red light effectively promoted germination, while gibberellic acid action was weak. With between 2 and 10 days of dark storage, gibberellic acid had little effect, while promotion by red light decreased slowly and finally disappeared. After 10 days of dark storage, both gibberellic acid and red light were required for germination. The dark storage treatment interferes with phytochrome-independent germination processes and cannot be overcome by added gibberellic acid. However, storage may also decrease the effectiveness of endogenous gibberellins. Phytochrome-dependent germination seems to require only low levels of endogenous gibberellin activity or the addition of gibberellic acid. Gibberellins and red light appear to act on germination by regulation of sequential sites of a branched-looped pathway. PMID- 16658689 TI - Sicklepod Surface Wax Response to Photoperiod and S-(2,3 Dichloroallyl)diisopropylthiocarbamate (Diallate). AB - Sicklepod (Cassia obtusifolia L.) leaflet epicuticular fatty acid, fatty alcohol, and hydrocarbon contents were measured by gas-liquid chromatography from plants grown under 10-, 12-, 14-, or 16-hour photoperiods and treated with S-(2,3 dichloroallyl)diisopropylthiocarbamate (diallate) (0, 0.14, 0.28, 0.56, 1.12 kilograms per hectare). As diallate concentration increased, epicuticular fatty acid content decreased. Fatty alcohol content was maximal in plants treated with 0.28 kilograms per hectare diallate and was reduced from that level at herbicide concentrations above or below this rate. Hydrocarbon content patterns were similar to those of the fatty alcohols except that the hydrocarbons at 0.28 kilograms per hectare were 61% of that present in the control, whereas the concentration of fatty alcohols increased to 200% of the control in treatments of 0.28 kilograms per hectare diallate.Photoperiodic influence on the various epicuticular lipid classes showed increased fatty acid and hydrocarbon contents as the photoperiod was lengthened. Fatty alcohols were maximal under 16-hour photoperiods and minimal under 12-hour photoperiods.Photoperiod-diallate concentration interactions were demonstrated in the epicuticular fatty acid, fatty alcohol, and hydrocarbon contents of sicklepod leaflets. The amount of each lipid class present per gram leaf fresh weight was influenced by the rate of diallate applied and the photoperiod under which the plants were grown. Each lipid class responded differently to the combination of treatments. PMID- 16658690 TI - Cytochemical localization of catalase activity in glyoxysomes from castor bean endosperm. AB - Glyoxysomes isolated from castor bean (Ricinus communis L. var. zanzibariensis) endosperm have been stained by the cytochemical diaminobenzidine reaction. The reaction product obtained by preincubation with 3,3'-diaminobenzidine and incubation with the reagent and H(2)O(2) is distributed uniformly throughout the matrix of the organelles. Ricinosomes or dilated cisternae may be completely absent from the organelle preparation or are, at the most, a minor contaminant. PMID- 16658691 TI - Light-controlled Stem Elongation in Pea Seedlings Grown under Varied Light Conditions. AB - There appears to be an orderly transition from one photosensitive state to another in light-controlled stem elongation in the garden pea, Pisum sativum L. cv. Alaska. Stem elongation in dark-grown plants is known to be phytochrome controlled. However, seedlings are insensitive to phytochrome after a red light pretreatment. An action spectrum for inhibition in these seedlings has peaks at 440 and 620 nm. Protochlorophyll is suggested as the photoreceptor. If these red pretreated seedlings are further exposed to white light, sensitivity to 440 to 620 nm light is lost. Promotion by blue-green light is the only photoresponse shown by these seedlings. Inhibition of completely white light-grown seedlings required simultaneous exposure to high intensity blue light and 600 nm light. PMID- 16658692 TI - Irreversible Plastid Loss in Euglena gracilis under Physiological Conditions. AB - Irreversible loss of the ability to develop chloroplasts in Euglena gracilis may develop following transfer from organic medium to defined medium. Requirements for the loss include the absence of light and a temperature of 30 C (the optimal temperature for multiplication) although neither darkness alone nor this temperature alone serves as the bleaching agent. The extent of bleaching of a population can approach 100% but depends heavily on the following conditions: the pH of the defined medium and its phosphate content, the age of the parent culture at transfer, and the length of time spent in the defined medium before cell divisions are permitted. Bleaching is not due to loss of nonreplicating proplastids through "dilution out" as cells divide but appears to be a more direct inactivation of chloroplast differentiation from proplastids. PMID- 16658693 TI - The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues. AB - delta-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. delta-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of delta-aminolevulinic acid dehydrase. The rate of delta aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of delta-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, delta-aminolevulinic acid accumulation ceased.delta Aminolevulinic acid accumulation showed an absolute requirement for oxygen and was inhibited drastically by cyanide and azide, and to a lesser extent by arsenite and malonate. 2,4-Dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, sodium fluoroacetate, and hydroxylamine hydrochloride showed no effect under the conditions tested. Freezing and thawing of the tissue completely prevented the accumulation of delta-aminolevulinic acid.The findings of this investigation are consistent with the hypothesis that delta-aminolevulinic acid is a chlorophyll precursor in higher plants, and that chlorophyll biosynthesis is regulated at the level of the formation of delta-aminolevulinic acid. PMID- 16658694 TI - The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues. AB - delta-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various (14)C-labeled precursors and levulinic acid, a competitive inhibitor of delta aminolevulinic acid dehydrase. The radioactivity in the accumulated delta aminolevulinic acid was measured.The most effective labeled precursors were the 5 carbon dicarboxylic compounds glutamate, glutamine, and alpha-ketoglutarate. (14)C-Labeled glycine and succinate were relatively poor. The carboxyl and the methylene carbons of glycine were incorporated into delta-aminolevulinic acid to about equal extent. The carboxyl carbon of glutamate was incorporated almost as well as the internal carbons of the same compound. These results are inconsistent with the succinyl CoA-glycine succinyl transferase (delta-aminolevulinic acid synthetase) mode of delta-aminolevulinic acid production.When the same experiments were performed on turkey blood (which, as avian blood in general, possesses delta-aminolevulinic acid synthetase), delta-aminolevulinic acid was labeled most effectively from glycine-2-(14)C, moderately well from glycine-1 (14)C and glutamate-3,4-(14)C and not at all from glutamate-1-(14)C.It appears probable that greening higher plant tissues possess an alternate route to delta aminolevulinic acid in which the carbon skeleton of glutamate (and alpha ketoglutarate) is incorporated intact into the first committed metabolite of the chlorophyll pathway. PMID- 16658695 TI - Inhibition of photophosphorylation by kaempferol. AB - Kaempferol, a naturally occurring flavonol, inhibited coupled electron transport and both cyclic and noncyclic photophosphorylation in isolated pea (Pisum sativum) chloroplasts. Over a concentration range which gave marked inhibition of ATP synthesis, there was no effect on basal or uncoupled electron flow or light induced proton accumulation by isolated thylakoids. It is suggested that kaempferol acts as an energy transfer inhibitor. PMID- 16658696 TI - Fluxes of a nonelectrolyte and compartmentation in cells of carrot root tissue. AB - Permeation of thiourea into cells of carrot (Daucus carota L.) storage root discs was found to be governed by its concentration gradient only. The analysis of the course of thiourea efflux from preloaded discs shows two distinct exponential curves, apart from an initial very fast component. Reasons are given for relating the first exponential part of the curve to efflux from the cytoplasm and the second exponential part to efflux from the vacuole.The rate constant for thiourea efflux through the tonoplast is markedly temperature-dependent (activation energy 13.2 kcal mole(-)) and is lowered by 2,4-dinitrophenol and by phenyl mercuric acetate treatments. The rate constant for its efflux through the plasmalemma, on the other hand, has a low activation energy (4.2 kcal mole(-)), which is equal to that of free diffusion of thiourea in aqueous solution, and is not affected by the metabolic inhibitors. Possible reasons for the difference in the properties of the two membranes are discussed. PMID- 16658697 TI - A polygalacturonase from citrus leaf explants: role in abscission. AB - The relationship between polygalacturonase activity and abscission of citrus leaf explants was studied. Determination of polygalacturonase activity in citrus tissues requires concentration of the enzyme, use of a proper assay method, and inhibition of an oxidase present in the extracts which oxidizes the reaction products of the polygalacturonase. The polygalacturonase from citrus leaf explants is an exopolygalacturonase and appears to be a soluble enzyme.Polygalacturonase activity increased during abscission of citrus leaf explants and was localized in the separation layer. Ethylene accelerated the increase in polygalacturonase activity, but its effect was evident only after at least an 8-hour lag period. 2,4-Dichlorophenoxyacetic acid and cycloheximide inhibited abscission and polygalacturonase activity. It is concluded that polygalacturonase, in addition to cellulase, plays a role in abscission. PMID- 16658698 TI - Soluble peroxidase in fluid from the intercellular spaces of tobacco leaves. AB - A high proportion of the soluble peroxidase of tobacco (Nicotiana tabacum L. var. Bottom Special) leaves is found in the fluid obtained by centrifugation of a buffer solution previously infiltrated into the intercellular spaces. Only a very small amount of the cytoplasmic enzyme, glucose 6-phosphate dehydrogenase, is present in this fluid. Specific activity data suggest that an active process is responsible for the transfer of soluble peroxidase to the intercellular space and that the intercellular fluid fraction is not simply composed of material moving out of leaf cells by diffusion. The centrifugation method is a satisfactory means of isolating diluted intercellular fluid for biochemical and physiological investigations. PMID- 16658699 TI - Effect of low temperature on amino Acid metabolism in wintering poplar: arginine glutamine relationships. AB - Analyses of free amino acids in poplar (Populus gelrica) were carried out throughout a year to see the effect of low temperature on a system regulating amino acid metabolism in the tree. The results indicated that during the wintering phase arginine was the major amino acid both in bark and xylem, particularly in xylem, and that at the time of budding and growing glutamine and glutamate became dominant. Changes in the relative levels of glutamine (plus glutamate) and arginine to the total amino acids of the alpha-ketoglutarate family indicated the presence of a regulatory system annually controlling the synthesis between glutamine (plus glutamate) and arginine. The system appeared to be governed and sensitized by low temperatures. Neither a transition of the synthesis from arginine to glutamine (plus glutamate) nor budding occurred in the poplars which spent the winter months in a greenhouse. PMID- 16658700 TI - Response of seedlings of a dwarf and a normal strain of watermelon to gibberellins. AB - Hypocotyl and root elongation in a dwarf and a normal strain of watermelon (Citrullus lanatus [Thunb.] Matsu.) in the absence or presence of different gibberellins was investigated in seedlings grown under gold fluorescent light or in darkness. The normal strain, "Sugar Baby," responded only slightly to the gibberellic acids employed. At appropriate concentrations all of the gibberellic acids were capable of normalizing growth in the monorecessive dwarf strain, WB-2, in darkness or in light. Gibberellins A(4+7) and A(7) were effective in stimulating hypocotyl elongation at concentrations 10 to 15 times lower than that needed for a response to GA(1) or GA(3). Dark-grown dwarfs responded to about a 3 fold lower concentration of GA(4+7) than those grown in light.In contrast to hypocotyl elongation, root elongation was greater in the dwarf than in the normal strain. Concentrations of gibberellic acids which enhanced hypocotyl elongation of WB-2 plants, inhibited root growth proportionately.Anatomically, the response of the dwarf strain to GA(4+7) was primarily in terms of increased cell division. Dark-enhanced elongation of both SB and WB-2 was due almost solely to increased cell elongation. The results suggest that, at least in some species, a major component of dark-enhanced growth (or light-inhibited growth) is physiologically distinct from gibberellic acid-stimulated elongation. PMID- 16658701 TI - Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6 Methoxy Mellein in Carrot Root. AB - The rate of incorporation of (14)C from acetate-1-(14)C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9 fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of (14)C from glucose-U (13)C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of (14)C incorporation from malonate-2-(14)C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of (14)C from acetate-1 (14)C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.There was little or no incorporation of (14)C from acetate or malonate into 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin (6 methoxy mellein) in fresh carrot discs or in discs treated with water, but label was incorporated rapidly in discs inoculated with C. fimbriata or treated with Ethrel. The rate of incorporation of label from acetate into fatty acids was severalfold higher than into 6-methoxy mellein, but the rate of incorporation of label from malonate was approximately equal into fatty acids and 6-methoxy mellein 24 hours after inoculation. The rate of fatty acid synthesis does not appear directly related to the accumulation of 6-methoxy mellein. PMID- 16658702 TI - The Potassium Content of Gonyaulax polyedra and Phase Changes in the Circadian Rhythm of Stimulated Bioluminescence by Short Exposures to Ethanol and Valinomycin. AB - A circadian rhythm in the intracellular level of K(+) in Gonyaulax polyedra is reported. When axenic cultures of Gonyaulax in continuous light (60-75 fot candles) are exposed for 4 hours to 0.1 or 0.2% ethanol, the subsequent free running rhythm in stimulated bioluminescence is phase-shifted, the amount and direction of the shift being dependent on the time in the circadian cycle when cells are treated. The phase-response curve for ethanol closely resembles that for light in similarly maintained cells. When valinomycin (0.1 or 0.2 mug ml(-1)) is present in addition to ethanol, the phase of the bioluminescence rhythm is returned to that of an untreated cell suspension. Valinomycin thus negates the effect of ethanol on phase. The intracellular K(+) level immediately after treatment of a cell suspension for 4 hours with ethanol (0.1%) is about half that of untreated cells. If valinomycin (0.1 mug ml(-1)) is also present during the 4 hour treatment, the intracellular K(+) is only slightly lower than in untreated cells. Increasing the external concentration of K(+) or Na(+) for 4 hours has no effect on the rhythm of stimulated bioluminescence. These results are interpreted as support for the hypothesis that the mechanism by which circadian oscillations are generated involves changes in membrane properties. PMID- 16658703 TI - Phytochrome-dependent Reduction of Nicotinamide Nucleotides in the Mitochondrial Fraction Isolated from Etiolated Pea Epicotyls. AB - When mitochondria isolated from etiolated pea (Pisum sativum cv. Alaska) epicotyls were exposed briefly to red light, their ability to reduce exogenous NADP was enhanced. The red light effect was reversed by far red light. Photoreversible absorbance changes between 730 nm and 800 nm were spectrophotometrically detected in the purified mitochondria and its membrane fraction. The dehydrogenase activity in the mitochondria was heat-labile and was dependent on the presence of magnesium ion and appropriate substrates such as glucose 6-phosphate, isocitrate, pyruvate, 6-phosphogluconate, and succinate. The photoreversible effect was seen only for a few minutes after the irradiation, and was cancelled by hypotonic treatment or addition of Triton X-100. A similar but lesser effect was observed in the pea microsome fraction, whereas no photoreversible response was seen with a supernatant fraction resulting from centrifugation at 10(5)g for 30 minutes. PMID- 16658704 TI - Polar transport of kinetin in tissues of radish. AB - Polar transport of kinetin-8-(14)C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots. PMID- 16658705 TI - Phytochrome stability in vitro: I. Effect of metal ions. AB - Photoreversible phytochrome disappears from etiolated tissue upon actinic irradiation. Such disappearance, of possible physiological importance, involves several processes, at least one of which is accelerated by metals in vivo. Purified phytochrome from oat (Avena sativa L. cv. Garry) coleoptiles is greatly stabilized in vitro by scrupulous removal of metal impurities via chelating agents. Such stabilized phytochrome decays rapidly upon the addition of about 10 mum Hg(2+), Cd(2+), Cu(2+), and Zn(2+), all of which probably act on sulfhydryl groups. Other tested metals and growth factors were much less active or inactive. The metals effective in promoting decay do not affect the Pfr --> Pr reversion process. This supports other evidence indicating the possible physiological importance of phytochrome "decay." PMID- 16658706 TI - Stomatal Behavior and Water Status of Maize, Sorghum, and Tobacco under Field Conditions: II. At Low Soil Water Potential. AB - Diurnal changes in the vertical profiles of irradiance incident upon the adaxial leaf surface (I), leaf resistance (r(1)), leaf water potential (psi), osmotic potential (pi), and turgor potential (P) were followed concurrently in crops of maize (Zea mays L. cv. Pa602A), sorghum (Sorghum bicolor [L.] Moench cv. RS 610), and tobacco (Nicotiana tabacum L. cv. Havanna Seed 211) on several days in 1968 to 1970 when soil water potentials were low. The r(1), measured with a ventilated diffusion porometer, of the leaves in the upper canopy decreased temporarily after sunrise [ approximately 0530 hours Eastern Standard Time] as I increased, but then r(1) increased again between 0700 and 0830 hr Eastern Standard Time as the psi, measured with a pressure chamber, decreased rapidly from the values of 7, -4 and -6 bars at sunrise to minimal values of -18, -22 and -15 bars near midday in the maize, sorghum, and tobacco, respectively. The pi, measured with a vapor pressure osmometer, also decreased after sunrise, but not to the same degree as the decrease in psi, so that a P of zero was reached in some leaves between 0730 and 0800 hours. The lower (more negative) pi of leaves in the upper canopy than those in the lower canopy gave the upper leaves a higher P at a given psi than the lower leaves in all three species; leaves at intermediate heights had an intermediate P. This difference between leaves at the three heights in the canopy was maintained at all values of psi. The r(1) remained unchanged over a wide range of P and then increased markedly at a P of 2 bars in maize, -1 bar in sorghum, and near zero P in tobacco: r(1) also remained constant until psi decreased to -17, -20, and -13 bars in leaves at intermediate heights in maize, sorghum, and tobacco, respectively. In all three species r(1) of leaves in the upper canopy increased at more negative values of psi than those at the base of the canopy, and in tobacco, leaves in the upper canopy wilted at more negative values of psi than those in the lower canopy. PMID- 16658707 TI - Distribution of ribosomal deoxyribonucleic Acid in subcellular fractions of higher plants. AB - Methods are described by which ribosomal DNA can be enriched in subcellular fractions of carrot and soybean. With both carrot and cucumber it was possible to obtain a distinct satellite DNA which contained the rDNA. Hybridization values greater than 0.49% were necessary before a satellite component was observed. Saturation hybridization values for soybean, carrot, and cucumber DNA were 0.2, 0.49, and 1.14%, respectively. These values were increased 1.6- and 2-fold in soybean and carrot, respectively, but enrichment was not obtained for cucumber. PMID- 16658708 TI - Studies on the Energy-coupling Sites of Photophosphorylation: V. Phosphorylation Efficiencies (P/e(2)) Associated with Aerobic Photooxidation of Artificial Electron Donors. AB - The rate of Hill reaction can be measured accurately as O(2) uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O(2) consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O(2) uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O(2) uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O(2)) through both photosystems II and I is practically independent of the donor used-catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e(2) ratio is 1.0 +/- 0.1. Only ascorbate gives a slightly lower value (P/e(2) = 0.9). (NH(2)OH treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O(2)) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e(2) ratio is 0.6 +/- 0.08. PMID- 16658709 TI - Deoxyribonucleic Acid polymerase from wheat embryos. AB - A soluble DNA-dependent DNA polymerase was extracted from wheat embryos. In vitro, the incorporation of radioactive thymidine triphosphate into acid insoluble material is dependent upon the presence of the enzyme, all four deoxyribonucleotide triphosphates, Mg(2+), and a DNA template. Incorporation occurs on native, alkali-denatured, and strictly double-stranded DNA. The in vitro synthesized product is a polydeoxynucleotide with a chain length shorter than the template; it has the same buoyant density as wheat embryo DNA when this DNA is used as template; and it forms a double-stranded complex with the DNA template. These data suggest that the in vitro DNA synthesis catalyzed by proteins extracted from wheat embryos occurs in a semiconservative way. PMID- 16658710 TI - Selective effects of victorin on growth and the auxin response in Avena. AB - Victorin, the pathotoxin from the host-specific pathogen, Helminthosporium victoriae, promotes the growth of coleoptile segments when given at concentrations that are high but which still show selective effects on susceptible and resistant tissue. The latent period in the growth response of both susceptible and resistant tissue is about 3.6 minutes compared to 11.0 minutes in the response of these tissues to auxin. The victorinpromoted rate of elongation of 8-millimeter segments is about 0.2 millimeter per hour in susceptible tissue and about 0.1 millimeter per hour in resistant tissue compared to about 0.4 millimeter per hour in response to auxin. At low concentrations, the toxin has no growth-promoting effect in either susceptible or resistant coleoptile segments. Over a wide range of concentrations, victorin inhibits the growth response of susceptible tissue to auxin completely while having no effect on the response of resistant tissue to auxin.Victorin induces solute leakage in coleoptile tissue of susceptible but not resistant varieties of Avena. In susceptible tissue, solute leakage begins about 2 minutes after the addition of toxin and continues for at least 4 hours.The rapidity and close correlation of toxin effects on membrane permeability (2 minutes) and on growth (3.6 minutes) suggest the possibility that the initial action of victorin in both cases may be at the cell surface, perhaps the plasmalemma. PMID- 16658711 TI - Localization of lead accumulated by corn plants. AB - Light and electron microscopic studies of corn plants (Zea mays L.) exposed to Pb in hydroponic solution showed that the roots generally accumulated a surface Pb precipitate and slowly accumulated Pb crystals in the cell walls. The root surface precipitate formed without the apparent influence of any cell organelles. In contrast, Pb taken up by roots was concentrated in dictyosome vesicles. Dictyosome vesicles containing cell wall material fused with one another to encase the Pb deposit. This encased deposit which was surrounded by a membrane migrated toward the outside of the cell where the membrane surrounding the deposit fused with the plasmalemma. The material surrounding the deposit then fused with the cell wall. The result of this process was a concentration of Pb deposits in the cell wall outside the plasmalemma. Similar deposits were observed in stems and leaves suggesting that Pb was transported and deposited in a similar manner. PMID- 16658712 TI - A study of the control of glycolate excretion in chlorella. AB - Chlorella pyrenoidosa cells grown on 5% CO(2) excreted glycolate when incubated in light with 10 mm bicarbonate, but excreted no glycolate under the same conditions when they were maintained on air for 7 hours prior to the assay. Incubation of 5% CO(2)-grown and air-grown cells with 10 mm isonicotinyl hydrazide or 10 mm alpha-hydroxypyridinemethane sulfonate during the assay stimulated the excretion of glycolate by CO(2)-grown cells severalfold that of air-grown cells.Adaptation of CO(2)-grown Chlorella to growth on air did not affect the levels of glycolate dehydrogenase in the cells and did not affect the rate of dark oxidation and metabolism of exogeneous (14)C-glycolate by the cells. These results indicate that the lack of glycolate excretion by air-grown or air adapted cells of Chlorella cannot be explained by a concomitant change in the level of glycolate dehydrogenase. PMID- 16658713 TI - Amyloplast Size and Number in Gravity-compensated Oat Seedlings. AB - Gravity compensation by the horizontal clinostat increases the diameter of amyloplast starch grains of oat (Avena sativa cv. Victory) coleoptile parenchyma cells, as compared to vertically rotated and stationary controls. In dark-grown coleoptile tip parenchyma cells, measured starch grain sizes exhibit a wide distribution of diameters, from approximately 1.5 to approximately 8.0 mum, but fall into three prominent diameter classes. The compensated tissues from both the tip and the subapical region have more starch grains in the larger, and fewer in the smaller size classes, compared to controls. The total number of starch grains per cell, the total plastid number per cell, and cell volume are unaffected by gravity compensation. Amyloplasts with large starch grains are denser, as well as larger in diameter, than those with smaller starch grains. The amyloplast is considered as a geosensor with an active metabolic role in the geotropic transduction mechanism. PMID- 16658714 TI - An apparent oligomer of malate dehydrogenase from bean leaves. AB - Two forms of malate dehydrogenase of widely differing molecular weight have been examined from primary leaves of Phaseolus vulgaris. In addition to the normal 69,000 molecular weight enzyme, an unusual form of 280,000 molecular weight may be detected by sucrose density gradient centrifugation or gel filtration with Sephadex G-200. Isopycnic density gradient centrifugation showed that both forms of malate dehydrogenase differed markedly from the bulk of the leaf protein by their low bouyant density of 1.261 g/cm(3).High molecular weight (280,000) malate dehydrogenase could be converted to active low molecular weight (69,000) malate dehydrogenase by treatment with 2.5 m CsCl, 1.0 m NaCl, 6 m urea, pH 6.5 or below, or one freeze-thaw cycle. Simple removal of salt or raising the pH were not effective in reforming the high molecular weight malate dehydrogenase after dissociation. The high molecular weight enzyme was not dissociated during prolonged dialysis against 0.1 m NaCl or 0.05 m phosphate, pH 7.0. Calcium at concentrations up to 0.1 m produced no activation or differential response in the two MDH forms.High and low molecular weight malate dehydrogenase were nearly identical in susceptibility to inhibition by various unreactive substrate analogs. However, there was a marked difference in the ability of the two forms of malate dehydrogenase to reduce 3-acetylpyridine-deamino-NAD. This difference in activity was the basis of a convenient assay for determining the ratio of high to low molecular weight malate dehydrogenase in crude extracts. The pH activity profiles and Michaelis constant for malate were nearly identical for the two molecular weight forms.Analysis by polyacrylamide gel electrophoresis revealed one high molecular weight and two low molecular weight malate dehydrogenase zones. Dissociation of high molecular weight malate dehydrogenase resulted in formation of low molecular weight enzyme whose electrophoretic properties differed from the normal low molecular weight forms. PMID- 16658715 TI - Nitrite assimilation and amino nitrogen synthesis in isolated spinach chloroplasts. AB - The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed CO(2) at rates of approximately 100 mumoles hr(-1) mg chlorophyll(-1).The chloroplast-enriched fraction contained the enzymes, nitrite reductase and NADPH-glutamate dehydrogenase, needed for the reduction of nitrite and incorporation of ammonia into glutamate. Kinetic studies showed that the reduction of nitrite by the chloroplast-enriched fraction is light-dependent, and the process proceeds at rates of 6 to 12 mumoles hr(-1) mg chlorophyll(-1). The addition of nitrite to the chloroplast preparation caused a 3-fold increase in the production of alpha-amino nitrogen when compared with the control without nitrite. There was a stoichiometric relation between amino-nitrogen synthesis and nitrite disappearance from the medium. The ratio of amino-nitrogen: NO(2) (-) ranged from 0.6 to 0.9. The initial rate of amino-nitrogen production was faster when (alpha)-ketoglutarate was added to the nitrite reducing chloroplast medium than when it was omitted. However, these high rates were not sustained and the total amino-nitrogen production at the end of a 30-minute period was only slightly higher. These data show that chloroplasts are functionally able and contain the enzyme complement necessary to utilize light energy for the reduction of nitrite to amino nitrogen. Thus, chloroplasts should be considered as a major site for in vivo amino-nitrogen synthesis in green plants. PMID- 16658716 TI - Linear velocity of cyclic adenosine 3',5'-monophosphate transport in corn coleoptiles. AB - The transport of cyclic adenosine 3', 5'-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed. PMID- 16658717 TI - Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves. AB - Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalofs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.Saponin Chl-H extracted from barley leaves illuminated for 15 minutes has an absorption maximum at 670 nm and an apparent molecular weight greater than 100,000. This chlorophyll holochrome has photosystem I activity and it is eluted together with the cytochromes. Saponin holochrome extracted from barley leaves returned to darkness after a light period, contains chlorophyll(ide) and protochlorophyllide complexes. Gel chromatography yields a complete separation of Chl-H (apparent molecular weight > 100,000) and photoactive Pchl-H (63,000).It is proposed that Chl-H dissociates into a chlorophyll(ide) a carrier protein complex and a photoenzyme, before the incorporation of chlorophyll into the lamellar membrane.Spectrofluorimetry on partially photoconverted preparations of saponin holochrome from barley, bean, and pea gave no indication for resonance energy transfer from protochlorophyllide to chlorophyllide. The saponin holochromes gave high polarization values, in contrast with bean holochrome extracted without the aid of detergents and bean leaves. PMID- 16658719 TI - Precursor-Product Relationships during Sulfate Incorporation into Porphyridium Capsular Polysaccharide. AB - This study describes the kinetics of (35)S-incorporation during in vivo sulfate esterification of Porphyridium aerugineum capsular polysaccharide. Techniques were developed to isolate the precursor pool (free sulfate), cell-associated product, and extracellular product. Specific radioactivities of these three fractions were monitored during pulse-chase sequences. Label rapidly appeared in the pool during the pulse, then declined asymptotically during the chase as equilibrium was approached. Efflux of small quantities of isotope from the cell during chase periods was not the result of backleakage, but the result of washing untransported isotope from the free-space. During the pulse, intracellular product was labeled at 25% of the rate at which the pool was labeled. Fully 50% of the label which left the pool was incorporated into the polysaccharide as ester sulfate, indicating that polysaccharide esterification is a major metabolic pathway for sulfate. The specific radioactivity of the extracellular product increased slowly throughout pulse and chase periods.Porphyridium was shown to be highly dependent on exogenous supplies of sulfate, the cells lysing when denied adequate quantities of this nutrient. The free sulfate pool size was measured as 33 x 10(6) sulfate ions per cell in log phase. 3'-Phosphoadenosine-5' phosphosulfate was tentatively identified in the water-soluble cell extract and is thought to be the "activitated" donor for sulfate transfer reactions. PMID- 16658718 TI - Structural and functional responses of wheat mitochondrial membranes to growth at low temperatures. AB - The responses in membrane lipid composition, structure, and function of four cultivars of wheat (Triticum aestivum L.) to growth at low temperature have been investigated. Marked growth temperature-dependent alterations in the fatty acid composition and unsaturation of the mitochondrial phospholipids correlate with changes in respiratory activity in all the varieties. Parameters such as the respiratory control ratio and the phosphorylative efficiency decrease in cold adapted seedlings. Three temperature-dependent structural transitions were identified in the mitochondria by the spin-labeling method. The structural transitions occur at lower temperatures in the cold-grown material. The shift in one transition appears to be quantitatively greater in the winter hardy varieties. Cold-induced changes in all of the other measured parameters were indistinguishable in hardy and nonhardy varieties. The results indicate major involvement of the phospholipid matrix in cold acclimation. A link between cold acclimation and winter survival may exist involving the structural and functional modifications in membrane structure which occur during acclimation. PMID- 16658720 TI - Nicotinamide Adenine Dinucleotide-dependent Proline Dehydrogenase in Chlorella. AB - An NAD-linked dehydrogenase from Chlorella pyrenoidosa Chick catalyzing the conversion of l-proline to Delta(1)-pyrroline-5-carboxylic acid was partially purified. Delta(1)-Pyrroline-5-carboxylic acid was identified as the product by co-chromatography of it and its o-aminobenzaldehyde derivative with authentic compounds. The enzyme is NAD and l-proline specific and is not an oxidase; NADP is not inhibitory. The Michaelis constant for NAD is 0.08 mm and for proline is 0.73 mm. PMID- 16658721 TI - Red light enhancement of the phototropic response of etiolated pea stems. AB - In the subapical third internode of 7-day-old etiolated pea seedlings, the magnitude of phototropic curvature in response to continuous unilateral blue illumination is increased when seedlings are pre-exposed to brief red light. The effect of red light on blue light-induced phototropism becomes manifest maximally 4 or more hours after red illumination, and closely parallels the promotive action of red light on the elongation of the subapical cells. Ethylene inhibits phototropic curvature by an inhibitory action on cell elongation without affecting the lateral transport of auxin. Pretreatment of seedlings with gibberellic acid causes increased phototropic curvature, but experiments using (14)C-gibberellic acid indicate that gibberellic acid itself is not laterally transported under phototropic stimuli. Neither red light nor gibberellic acid treatment has any promotive effect on blue light-induced lateral transport of (3)H-indoleacetic acid. Under conditions where phototropic curvature is increased by red light treatment, low concentrations of indoleacetic acid applied in lanolin paste to the apical cut end of the seedling cause an increased elongation response in subapical tissue. This could explain increased phototropic curvature caused by red light treatment. PMID- 16658722 TI - Characteristics of an Acid protease from maize endosperm. AB - An assay has been developed to measure protease activity in endosperm extracts of maize seeds. With hemoglobin as substrate, the enzyme(s) has a pH optimum of 3.8 and a temperature optimum of 46 C. It also degrades gliadin, edestin, bovine serum albumin, and partially hydrolyzed zein and glutelin under standard assay conditions. The enzyme(s) has endopeptidase activity with all substrates tested. When undenatured zein and glutelin are suspended in an agar gel, both are efficiently degraded. Using this assay, the protease activity increases from day 3 to day 8 after inhibition and then declines. PMID- 16658723 TI - The Hydrolysis of Endosperm Protein in Zea mays. AB - Degradation of the major storage proteins in maize endosperm, zein and glutelin, begins during the 2nd day of germination. The protein most abundant in the mature endosperm is degraded most rapidly. The patterns of protein loss are essentially similar in germinating seeds and excised endosperms. Cycloheximide, added at the beginning of the incubation period, prevents the development of alpha-amylase and protease activities and the disappearance of starch and protein reserves. Late additions (70 hours) of cycloheximide still inhibit the increase in hydrolase activity but have no effect on the hydrolysis of storage reserves. The results indicate that the hydrolytic enzymes are synthesized de novo in the maize endosperm. PMID- 16658724 TI - Distribution and development of nitrate reductase activity in germinating cotton seedlings. AB - Activity of nitrate reductase in roots and cotyledons of cotton seedings (Gossypium hirsutum L. cv. Deltapine 16) increased rapidly on germination, reaching a maximum after 1 day of imbibition. Thereafter, activity declined until emergence and greening of the cotyledons, when it again began to increase steadily. Germinating soybean (Glycine max (L.) Merrill cv. Merit) and sunflower (Helianthus annuus L. cv. Peredovic) seedlings did not show the early peak of activity. The early peak depended on nitrate and was sensitive to cycloheximide, but not to actinomycin D or other inhibitors of RNA synthesis. The second, light dependent increase was sensitive to actinomycin D. In roots, the early peak of activity occurred before any growth. After emergence of the root tip from the seed coat, activity was localized in the terminal 2 millimeters, whether expressed on a fresh weight, protein, or root basis. The difference in activity between the apical (0-2 millimeter) and subapical (2-4 millimeter) segments did not result from differences in nitrate availability, energy supply, or turnover rates of nitrate reductase. Root activity was similar to that of the cotyledons after emergence, in that both were sensitive to actinomycin D. PMID- 16658725 TI - Characterization of a H Efflux from Suspension-cultured Plant Cells. AB - A readily assayed H(+) efflux from sycamore (Acer pseudoplatanus), rye (Lolium perenne), and bean (Phaseolus vulgaris cultivars Red Kidney and Small White) suspension-cultured cells has been detected and partially characterized. The H(+) efflux has been shown to require a source of energy, to be significantly stimulated by Na(+) and Mg(2+) but not by K(+) and Ca(2+), and to have a pH optimum at 7. The study of this H(+) efflux was undertaken because the characteristics of auxin-induced growth and of H(+)-induced growth are sufficiently similar to suggest that a H(+) efflux may be an intermediate in the mechanism of auxin-induced growth. However, the H(+) efflux from these suspension cultured cells was found to be insensitive to exogenously added hormones. PMID- 16658726 TI - Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of Chlorella sorokiniana. AB - Mutants of Chlorella sorokiniana selected for resistance to the methionine analogue ethionine took up ethionine at the same rate as did the wild type strain. Cells of two ethionine-resistant mutants produced severalfold higher levels of free methionine and cysteine than did wild type cells.Exogenous methionine had no apparent effect on free methionine production in a mutant that produces excessive levels of free methionine. Under the same conditions, production of free methionine was relatively inhibited in wild type cells and in a mutant that produces wild type levels of free methionine.The results suggest that free methionine production in the wild type strain is subject to endproduct control, and that this control is lacking in one class of ethionine-resistant mutants. PMID- 16658727 TI - Chloroplast Response to Low Leaf Water Potentials: III. Differing Inhibition of Electron Transport and Photophosphorylation. AB - Cyclic and noncyclic photophosphorylation and electron transport by photosystem 1, photosystem 2, and from water to methyl viologen ("whole chain") were studied in chloroplasts isolated from sunflower (Helianthus annus L. var Russian Mammoth) leaves that had been desiccated to varying degrees. Electron transport showed considerable inhibition at leaf water potentials of -9 bars when the chloroplasts were exposed to an uncoupler in vitro, and it continued to decline in activity as leaf water potentials decreased. Electron transport by photosystem 2 and coupled electron transport by photosystem 1 and the whole chain were unaffected at leaf water potentials of -10 to -11 bars but became progressively inhibited between leaf water potentials of -11 and -17 bars. A low, stable activity remained at leaf water potentials below -17 bars. In contrast, both types of photophosphorylation were unaffected by leaf water potentials of -10 to -11 bars, but then ultimately became zero at leaf water potentials of -17 bars. Although the chloroplasts isolated from the desiccated leaves were coupled at leaf water potentials of -11 to -12 bars, they became progressively uncoupled as leaf water potentials decreased to -17 bars. Abscisic acid and ribonuclease had no effect on chloroplast photophosphorylation. The results are generally consistent with the idea that chloroplast activity begins to decrease at the same leaf water potentials that cause stomatal closure in sunflower leaves and that chloroplast electron transport begins to limit photosynthesis at leaf water potentials below about -11 bars. However, it suggests that, during severe desiccation, the limitation may shift from electron transport to photophosphorylation. PMID- 16658728 TI - Amylases from aleurone layers and starchy endosperm of barley seeds. AB - Amylases from incubated aleurone layers or from starchy endosperm of barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated using acrylamide gel electrophoresis and analytical gel filtration with Sephadex G-200. Electrophoresis of amylase from aleurone layers yields seven visually distinct isozymes with an estimated molecular weight of 43,000. Because each isozyme hydrolyzes beta-limit dextrin azure and incorporates calcium-45, they are alpha amylases. On Sephadex G-200, amylase from the aleurone layers is separated into seven fractions ranging in estimated molecular weights from 45,000 to 3,000. Little or no activity is observed when six fractions are subjected to electrophoresis. Electrophoresis of only the fraction with the estimated molecular weight of 45,000 gave the seven isozymes. The amylases are heat labile and cannot be stabilized by the presence of substrate or by the protease inhibitor, phenylmethylsulfonylfluoride. Electrophoresis of amylase from the starchy endosperm yields nine beta-amylases. Four of these beta-amylases are isozymes with an estimated molecular weight of 43,000. The other five forms of beta-amylase represent molecular aggregates of the four basic beta-amylase monomers. A dimer, a tetramer, and an octamer of beta-amylase can be identified with estimated molecular weights of about 86,000, 180,000 and 400,000, respectively. These estimated molecular weights were confirmed on Sephadex G-200. There are five additional fractions of beta-amylase with estimated molecular weights ranging from 30,000 to 4,000. These fractions are not observed electrophoretically. PMID- 16658729 TI - The analysis of spiral growth in phycomyces using a novel optical method. AB - A conical mirror was designed and used to measure simultaneously the elongational and rotational displacement of a number of markers on the growing zone of the sporangiophore of Phycomyces. The results obtained by this new optical method demonstrate that the rotational rate is roughly proportional to the elongational rate, except in the lower region of the growing zone where a significant amount of rotation occurs without measurable elongation. From the data presented in this report, we have constructed a model that appears to explain the mechanism responsible for the left-handed spiral growth of the developing sporangiophore. PMID- 16658730 TI - Photosynthesis in the Higher Plant, Vicia faba: III. Serine, a Precursor of the Tricarboxylic Acid Cycle. AB - Evidence is presented to support the hypothesis that serine, rather than 3 phosphoglycerate of the Calvin cycle, is a precursor of the tricarboxylic acid cycle during photosynthesis by the higher plant, Vicia faba. Identification of the serine intermediate is based upon a unique C(1) > C(2) > C(3) isotope distribution for that metabolite following the fixation of (14)CO(2). This labeling pattern, while incompatible with an origin either in the Calvin cycle or the glycolate pathway, satisfies a critical criterion for the 3-carbon precursor of the anomalously labeled organic acids. The predominant carboxyl carbon atom labeling of serine reflects either a mixing of two pools of that metabolite, ie., C(1) = C(2) > C(3) and C(1) > C(2) = C(3), or a higher order of complexity in its synthesis. An anomalous C(1) = C(2) > C(3) < C(4) distribution for aspartate, however, suggests an origin by the carboxylation of a 3-carbon intermediate related to serine which has a C(1) = C(2) > C(3) distribution. The latter distribution has been proposed for the serine intermediate of the postulated formate pathway. This pathway is described by the generalized metabolic sequence: CO(2) --> formate --> serine --> organic acids. Corresponding carbon atom distributions for citrate (C(1) > C(2)), aspartate (C(2) > C(3)), and serine (C(2) > C(3)) belie a precursor-product relationship with alanine (C(2) = C(3)), which is a molecular parameter of the Calvin cycle product, 3-phosphoglycerate. PMID- 16658731 TI - The Galactolipid, Phospholipid, and Fatty Acid Composition of the Chloroplast Envelope Membranes of Vicia faba. L. AB - The galactolipid, phospholipid, and fatty acid composition of chloroplast envelope membrane fractions isolated from leaves of Vicia faba L. has been determined. The major lipids in this fraction are: monogalactosyldiglyceride, 29%; digalactosyldiglyceride, 32%; phosphatidylcholine, 30%; and phosphatidylglycerol 9%. The lipid composition of the chloroplast envelope membranes is qualitatively similar to that of the lamellar membranes isolated from the same plastids, but the proportion of each lipid present is very different. The total galactolipid to total phospholipid ratio was 1.6: 1 in the envelope and 11.1: 1 in the lamellae. The monogalactosyldiglyceride-digalactosyl diglyceride ratio was 0.9: 1 in the envelope and 2.4: 1 in the lamellae. Both membranes lack phosphatidylethanolamine.Linolenic acid is the major fatty acid in the envelope lipids representing 63% of the total fatty acid, whereas in the lamellae it represents 83%. The same fatty acids are present in both the envelope and lamellar lipids except the trans-Delta(3)-hexadecenoic acid, which is confined to the lamellar lipids, particularly the phospholipid fraction.A quantitative comparison of the lipid and fatty acid compositions of the envelope with those of mitochondrial and microsomal fractions indicates that the chloroplast envelope has a composition intermediate between that of the chloroplast lamellae and these extrachloroplastic membranes. PMID- 16658732 TI - Persistence of red light induction in lettuce seeds of varying hydration. AB - Incubation of lettuce seeds (Lactuca sativa L. cv. Grand Rapids) in 0.3 m mannitol allows sufficient water uptake to make seeds fully sensitive to red light. But germination is possible only after lowering the osmotic potential of the incubation medium. The red light induction of these incompletely hydrated seeds can be reversed by far red light. Their reversibility declines with time at a slower rate than seeds incubated in water. About half the seeds in 0.3 m mannitol respond to far red light when all seeds in water have escaped control by far red light. Close to 100% of the seeds remain sensitive to far red exposure if 0.6 m mannitol is used as osmoticum. The retention of the original red light stimulus is inversely related to the concentration of the incubation medium.The fresh weight of viable seeds incubated with water or with an osmoticum increases rapidly during the first 5 hours, then remains stationary for about 12 hours. After that only germinating seeds experience a second increase in fresh weight. Heat-killed seeds do not show such a discontinuity in water uptake. PMID- 16658733 TI - Enzymes Involved in the Postharvest Degradation of Sucrose in Beta vulgaris L. Root Tissue. AB - The reducing sugar content of sugar beet (Beta vulgaris L.) roots increased during 30 days of storage at 21 C and 160 days at 5 C as a result of an increase in acid invertase activity. Sucrose synthetase and neutral invertase activities were high at harvest but declined during storage, thus showing no relationship with postharvest reducing sugar accumulation in sugar beet roots. Acid alpha glucosidase activity was detected in fresh roots but showed no activity with sucrose as a substrate. PMID- 16658734 TI - Phospholipid degradation in frozen plant cells associated with freezing injury. AB - A striking degradation of phosphatidylcholine into phosphatidic acid was observed in the cortical tissues of less hardy poplar (Poplus euramericani cv. gelrica), when the tissues were frozen below a lethal temperature. No change in phospholipids was detected during freezing or even after thawing in the cortical tissues of hardy poplar which survived slow freezing to -30 C or even immersion in liquid N(2) after prefreezing to -50 C. The degradation of phosphatidylcholine during freezing appears to be intimately associated with freezing injury. PMID- 16658735 TI - Phycomyces: electrical response to light stimuli. AB - Electrical signals have been detected in response to light excitation of the fungus Phycomyces blakesleeanus. These signals are related to the wavelength and intensity of the stimulus and the growth stage of the fungus. A relationship between the signals and the possible photoreceptor-pigment system is explored. PMID- 16658736 TI - Effect of CO(2) Concentration on Glycine and Serine Formation during Photorespiration. AB - Amount and products of photosynthesis during 10 minutes were measured at different (14)CO(2) concentrations in air. With tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) leaves the percentage of (14)C in glycine plus serine was highest (42%) at 0.005% CO(2), and decreased with increasing CO(2) concentration to 7% of the total at 1% CO(2) in air. However, above 0.03% CO(2) the total amount of (14)C incorporated into the glycine and serine pool was about constant. At 0.005% or 0.03% CO(2) the percentage and amount of (14)C in sucrose was small but increased greatly at higher CO(2) levels as sucrose accumulated as an end product. Relatively similar data were obtained with sugar beet (Beta vulgaris L. cv. US H20) leaves. The results suggest that photorespiration at high CO(2) concentration is not inhibited but that CO(2) loss from it becomes less significant. PMID- 16658737 TI - Comparison of plant cytoplasmic ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis. AB - The electrophoretic mobilities of the cytoplasmic ribosomal proteins of several species of plants were compared using two-dimensional electrophoresis. The total number of proteins as well as the number of acidic and basic proteins in individual species varied markedly. Of the species examined, Triticum aestivum had the highest number of basic cytoplasmic ribosomal proteins and Hordeum vulgare had less than half as many. However, marked similarities were noted in the electrophoretic mobilities of many of the proteins, especially for wheat, rye, and barley and for peas and beans. There was a statistically significant positive correlation between the numbers of basic proteins in the species and their chromosome number. PMID- 16658738 TI - Cell walls of germinating uredospores: I. Amino Acid and carbohydrate constituents. AB - Synthesis of germ tube wall is a major quantitative event during germination penetration of fungi on host plants, but little is known of germ tube composition or metabolic regulation. Sonic oscillation was used to separate germ tubes from germinating uredospores of Uromyces phaseoli var. typica. Uniformly (14)C-labeled wall fractions from both structures were prepared by repeated low speed centrifugation and extraction with polar and nonpolar solvents. Based on amino acid analysis, approximately 6 and 16% of the carbon from uredospore and germ tube walls, respectively, was present in amino acids readily accessible to protease. Covalent linkages between amino acid and carbohydrate of walls was indicated by analysis of fragments prepared by mild hydrolytic procedures and separated by column chromatography and paper electrophoresis. The existence of protein in wall structures may resolve some previous uncertainty about the occurrence of protein biosynthesis during germination of rust fungi. Glucose, mannose, and glucosamine were the only carbohydrate components identified in both germ tubes and uredospore walls but different percentages were observed (germ tubes 28: 16: 16; uredospore 6: 36: 6). In germ tubes, most of the glucosamine was present in linkages hydrolyzed only by strong acid treatment, suggesting chitin-like polymers. In uredospore walls, glucosamine appears to be associated with red uredospore pigment which has properties similar to those of a melanin. Approximately 20% of the carbon in walls could not be identified with known compounds, partially because of degradation during the analytical procedures. PMID- 16658739 TI - Cell Walls of Germinating Uredospores: II. Carbohydrate Polymers. AB - Polymeric carbohydrates in (14)C-labeled germ tube and uredospore walls of Uromyces phaseoli var. typica were studied by permethylation and by enzymatic hydrolysis. The native structure of the uredospore wall limited the effectiveness of both techniques with this wall, but evidence for two distinct polysaccharides was obtained. A linear (1-->3) glucan, containing minor quantities of (1-->6) linkages, may account for most of the glucose in the uredospore wall. A second uredospore polymer was a glucomannan similar to one reported for other rust fungi in that it consisted of approximately equal numbers of beta(1-->3) and beta(1- >4) mannosidic linkages with glucose as a minor component at the nonreducing end. Branching, most likely by (1-->6) mannose links, was low. In contrast to uredospore wall, considerably more germ tube polysaccharide was accessible to enzymes and to methylation. Methylation studies indicate that (1-->3) glucose and mannose bonds occur predominantly. Evidence from hydrolysis with exo- (beta)-(1- >3) glucanase suggests distinct wall regions of beta(1-->3) glycan, highly branched by (1-->6) bonds, as well as wall regions of a glucomannan with alternating (1-->3) glucose and (1-->3) mannose residues. Polymer heterogeneity was indicated by differences in the proportions of mannose, glucose, and galactose as reducing end groups in different solubility fractions. In germ tube walls, but not in uredospore walls, glucosamine apparently existed as part of chitin polymer as evidenced by the isolation of N,N-diacetylchitobiose from chitinase digestion. PMID- 16658740 TI - The chloroplast and cytoplasmic ribosomes of euglena: I. Stability of chloroplast ribosomes prepared by an improved procedure. AB - A new isolation procedure has resulted in an improved yield of stable 68S chloroplast ribosomes from Euglena gracilis var. bacillaris. Chloroplasts are isolated by suspending the cells in buffer I (sorbitol, 250 mm; sucrose, 250 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm) and passing through a French press at less than 1500 pounds per square inch. The crude chloroplasts are purified by three washings with buffer II (sorbitol, 150 mm; sucrose, 150 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm). Stable 68S chloroplast ribosomes are obtained when the isolated chloroplasts are resuspended in ribosome buffer (tris-HCI, pH 7.6, 10 mm; magnesium acetate, 12 mm; KCI, 60 mm) containing spermidine, 0.5 mm; mercaptoethanol, 14 mm; sucrose, 8% (w/w), passed through a French press at 4000 pounds per square inch and extracted with either 0.1% (w/v) sodium deoxycholate or 1.0% (v/v) Triton X-100. At 0 to 4 C in ribosome buffer, the purified 68S chloroplast monosome forms a 53S particle while the 35S particle, an expected product of monosome dissociation, cannot be detected. Spermidine and mercaptoethanol prevent the formation of 53S particles from 68S monosomes. The purified 53S particles derived from 68S monosomes contain 23S RNA as well as a significant amount of 16S RNA, suggesting that this particle may not be a true ribosomal subunit. PMID- 16658741 TI - The Chloroplast and Cytoplasmic Ribosomes of Euglena: II. Characterization of Ribosomal Proteins. AB - Cytoplasmic and chloroplast ribosomal proteins were isolated from Euglena gracilis and analyzed on polyacrylamide gels. Cytoplasmic ribosomes appear to contain 75 to 100 proteins ranging in molecular weight from 10,200 to 104,000, while chloroplast ribosomes appear to contain 35 to 42 proteins with molecular weights ranging from 9,700 to 57,900. This indicates that the cytoplasmic ribosomes are similar in composition to other eucaryotic ribosomes, while chloroplast ribosomes have a protein composition similar to the 70S procaryotic ribosome. The kinetics of light-induced labeling of cytoplasmic ribosomal proteins during chloroplast development has been determined, and the results are compared with the kinetics of ribosomal RNA synthesis. PMID- 16658742 TI - Lipid biosynthesis in green leaves of developing maize. AB - Successive leaf sections from the base to the tip of rapidly developing leaves of 7-day-old maize (Zea mays var. Kelvedon Glory), grown in the light, utilized acetate for fatty acid biosynthesis in a very divergent manner. Basal regions of the leaf containing proplastids synthesized insignificant proportions of unsaturated fatty acids and appreciable proportions of fatty acids with 20 or more carbon atoms. An increase in the light intensity during incubations with acetate-1-(14)C resulted in very little enhancement of either total or polyunsaturated fatty acid biosynthesis in this tissue.When the distal leaf sections, containing mesophyll chloroplasts with well developed grana and bundle sheath chloroplasts without grana, were incubated with acetate at 150 ft-c and 30 C, approximately 30% of the newly synthesized fatty acids were unsaturated (mainly 18: 1 and 18: 2). At 2800 ft-c and 20 C, 60% of the fatty acids were unsaturated and the total synthesis of fatty acids increased 4-fold. No detectable amount of fatty acids with 20 or more carbon atoms were synthesized in this morphologically mature tissue, and the proportions of newly synthesized fatty acids more closely resembled the endogenous fatty acids in the immature tissue.Only 4% of the newly synthesized fatty acids were 18: 3 but most of this was incorporated into monogalactolipid. In the distal sections, 20 to 25% of the newly synthesized fatty acids in monogalactolipid were 18: 3 compared with the endogenous proportions of 85%. The differences in the composition of the newly synthesized fatty acids and the endogenous fatty acids appear to be related very largely to the low rate of 18: 3 biosynthesis from 18: 1 and 18: 2. Phosphatidyl choline and phosphatidyl ethanolamine, with lower proportions of 18: 3, contained radioactive fatty acids which resembled the endogenous composition more closely.Phosphatidyl choline was quantitatively the most important acyl lipid synthesized under both light conditions. In addition, there was considerable stimulation of acetate incorporation into phosphatidyl glycerol and monogalactolipid, especially in the morphologically most mature regions of the tissue at the higher light intensity. PMID- 16658743 TI - Relationship of ribonucleic Acid metabolism in embryo and aleurone to alpha amylase synthesis in barley. AB - RNA metabolism of embryo and aleurone of barley grains (Hordeum vulgare L. cv. Himalaya) was studied to elucidate the role of these tissues in the control of alpha-amylase synthesis and germination. The extent of (3)H-uridine incorporated into various RNA classes of the embryo during the first 12 hours of germination was low but constant. Subsequently, there was a rapid increase in RNA synthesis of all fractions. In the aleurones, after 16 hours, a gradual decrease in (3)H uridine incorporation was observed, and by the time the synthesis of RNA in the aleurones had stopped, alpha-amylase level was at its highest in the grain.On transfer to accelerated aging conditions (43 C; 85% relative humidity), the grains lost their viability within 4 weeks. That this was due to a rapid deterioration of the embryo and not of the aleurone was apparent in studies on alpha-amylase formation, RNA metabolism, and ATP content in grains in various physiological states reported here. Results presented here also reveal a marked influence of the embryo and GA(3) on the quality of the newly synthesized RNAs. Aleurones which lacked the impulse of embryo or GA(3) were capable of synthesizing RNA but these RNAs were less heterodisperse than RNAs from aleurones which were under the influence of an embryo or GA(3). PMID- 16658744 TI - Cell wall and protoplast isoperoxidases in relation to injury, indoleacetic Acid, and ethylene effects. AB - In tobacco (Nicotiana Tabacum) pith, sweet potato (Ipomoea batatas), and carrot (Daucus carota) storage roots, differences were found between cell wall and protoplast peroxidases in their isoenzyme patterns, activity, and reaction to tissue injury.In the pith of elongating tobacco internodes, 90% of total activity was associated with the walls, 80% of which was due to the ionically and covalently bound fractions. With senescence, the increase in activity occurred mainly in the protoplast and the free fraction in the walls. The major protoplast isoperoxidases formed the free wall fraction, and the minor ones formed the wall bound fractions. The minor isoperoxidases were the only ones whose activities were affected by cut injury. Neither senescence nor injury caused qualitative changes in the cell isoperoxidase pattern. In the potato root cells, the isoperoxidases were also distinctive in their distribution and in their reactions to injury as well as to ethylene.In potato and carrot roots, the wall fractions contributed 30 and 95%, respectively, to total peroxidase activity. The injury induced increase in peroxidase activity occurred mainly in the protoplast of tobacco pith and potato roots and almost entirely in the ionically bound fraction of the walls in carrot roots. Ethylene stimulated peroxidase development in potato roots, especially in the protoplast, but had no effect in either tobacco pith or carrot roots. Ethylene did not affect the inhibiting action of indoleacetic acid on peroxidase activity in tobacco pith, nor did indoleacetic acid significantly affect the stimulating action of ethylene in potato roots.Actinomycin D inhibited the injury-dependent development of peroxidase in tobacco pith, stimulated the injury- and ethylene-dependent peroxidase development in potato root, and had no effect on peroxidase development in carrot root. Cycloheximide prevented the increase in peroxidase activity in tobacco pith and potato root, but not in the carrot root. Thus, the enhancement of peroxidase activity resulted from either enzyme activation exclusively or from enzyme synthesis. RNA synthesis seems to be only indirectly related to isoperoxidase synthesis caused by injury or ethylene. PMID- 16658745 TI - The Effect of Light on the Synthesis of Mitochondrial Enzymes in Division synchronized Euglena Cultures. AB - The development of the mitochondrial enzymes fumarase and succinate dehydrogenase has been followed in Euglena cultures division-synchronized by 14-hour light periods alternating with 12-hour dark periods. The activity of both enzymes was unaltered over the light phase, doubled in early dark phase, and thereafter remained constant over the rest of the cycle. The increase in enzyme activity in early dark phase probably represented de novo enzyme synthesis because it was prevented by the addition of cycloheximide at a concentration known to inhibit protein synthesis on Euglena cytoplasmic ribosomes.When division-synchronized cultures were darkened in early light phase, a doubling of both fumarase and succinate dehydrogenase activity resulted, showing that light was repressing enzyme synthesis. The addition of acetate did not have a similar effect to darkening cultures: enzyme activity being unaltered over the light phase of the cycle. Enzyme expression was also unaffected by the addition of 3-(3,4 dichlorophenyl)-1,1-dimethylurea, a potent inhibitor of photosynthetic carbon dioxide fixation. The addition of 6-methylpurine (an inhibitor of transcription) at the beginning of the light phase inhibited enzyme increase in early dark phase, but when added at a later stage of the light phase (hour 8), increase in enzyme activity in early dark phase was unaffected. We concluded that transcription for these enzymes occurs in early light phase but light exerts a post-transcriptional control so that enzyme synthesis does not result until cells enter the dark phase of the cell cycle. PMID- 16658746 TI - Variation in the carbon isotope composition of a plant with crassulacean Acid metabolism. AB - The content of (13)C varies in plants with Crassulacean acid metabolism. Differences up to 3.5 per thousand in the (13)C/(12)C ratios were observed between leaves of different age in the same plant of Bryophyllum daigremontianum. Soluble and insoluble carbon in the same leaf differed up to 8 per thousand, the largest difference occurring in the leaves with the highest Crassulacean acid metabolism activity. Models to account for the isotope discrimination by C(3), C(4), and Crassulacean acid metabolism plants are proposed. PMID- 16658747 TI - Inhibition of ethylene evolution in papaya pulp tissue by benzyl isothiocyanate. AB - Papaya (Carica papaya L.) pulp tissue disks in an incubation medium composed of 0.4 m sucrose evolve ethylene at an optimum pH of 5.25 at 30 C. Disks of young preclimacteric fruit evolve the gas linearly with fruit age until fruit age reaches 4 months. Disks from 5-month-old postclimacteric fruit produce approximately 5-fold more ethylene than disks from 4-month-old fruit. Ethylene evolution by unaged papaya disks is inhibited potently by benzyl isothiocyanate. The compound inhibits production of ethylene by approximately 60% at a concentration of 0.046 mm. However, in aged papaya disks benzyl isothiocyanate causes no inhibition of ethylene production indicating that the compound inhibits the induction of the ethylene-producing system rather than the evolution of the gas per se. Even at a 2-fold higher concentration benzyl isothiocyanate has no effect on respiration of unaged papaya disks. It is proposed that benzyl isothiocyanate may act as an endogenous regulator of ethylene evolution in papaya fruit. PMID- 16658748 TI - Chloroplast Reactions of Photosynthetic Mutants in Zea mays. AB - Three seedling lethal mutants of Zea mays with impaired photosynthesis are described. These recessive mutants were selected on the basis of high chlorophyll fluorescence. They have normal chlorophyll pigmentation but are unable to fix CO(2) fully. Evidence is presented from fluorescence characteristics of isolated chloroplasts that both photosystem I and II mutants were isolated. Using conventional measures of photosynthetic electron transport, we suggest that the photosystem I mutant has limited ability to reduce NADP. The other two mutants are clearly blocked in photosystem II, one possibly lacking the primary electron acceptor. PMID- 16658749 TI - Photoperiodism and enzyme activity: towards a model for the control of circadian metabolic rhythms in the crassulacean Acid metabolism. AB - Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID- 16658750 TI - Activation of the de novo pathway for pyridine nucleotide biosynthesis prior to ricinine biosynthesis in castor beans. AB - The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de novo pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively. PMID- 16658751 TI - Photocontrol of Hook Opening in Cuscuta gronovii Willd. AB - Hook opening in seedlings of Cuscuta gronovii Willd. occurred only after prolonged exposures to blue, red, or far red light. Prolonged far red exposure was less effective than prolonged exposure to red or blue light. Brief far red irradiation inhibited the inductive effect of red light. The far red inhibition was in turn reversed by brief red irradiation. These effects suggest the involvement of two photosystems in the control of hook opening in Cuscuta gronovii Willd.: a phytochrome-mediated system and a separate high energy requirement. PMID- 16658752 TI - The Abolition of the Lag Phase in Greening Cucumber Cotyledons by Exogenous delta Aminolevulinic Acid. AB - Etiolated cucumber cotyledons treated with delta-aminolevulinic acid accumulated protochlorophyllide which was phototransformable to chlorophyll (ide). The phototransformation process in the delta-aminolevulinic acid-treated tissue was markedly temperature-dependent, consistent with the view that this protochlorophyllide must combine with the holochrome apoenzyme before phototransformation can occur.The treatment which effects lag phase removal in control tissue did not affect the course of chlorophyll (ide) accumulation in delta-aminolevulinic acid-treated tissue under either continuous or intermittent illumination. It was concluded that the lag phase in etiolated tissues must reflect the gradual development of the ability to synthesize delta-aminolevulinic acid at an appreciable rate. PMID- 16658753 TI - Chloroplast grana membrane carboxyl groups: their involvement in membrane association. AB - Chloroplast membrane carboxyl groups were modified by carbodiimide activation followed by glycine methyl ester substitution, leaving the derivatized group uncharged. This charge alteration induced a number of effects similar to addition of salts to control chloroplasts suspended in a low salt medium. These include: (a) restacking or multiple membrane association in low salt-treated chloroplasts that lack grana stacks, (b) protection against polycation inhibition of photosystem I electron transfer, (c) reduction of the amount of polycations bound to the membranes, and (d) increased 90 degrees light scattering due to membrane conformational changes. Carboxyl modification also altered acid-induced conformational changes.These effects are interpretated as the results of the reduction in the surface negative charge contributed by carboxyl groups. Membrane structure at both a local, polyelectrolyte level and at the level of membrane membrane interaction (stacking) is controlled in part by these negative, charged groups. PMID- 16658754 TI - Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva. AB - An NADP(+)-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella. PMID- 16658755 TI - Nitrate reductase activity and protein concentration of two populas clones. AB - Nitrate reductase activity and protein percentage of various tree parts of two Populus clones were determined in relation to nitrate ion activity. Nitrogen was supplied as NH(4)NO(3) in a nutriculture system. Wisconsin-5 had significantly greater nitrate reductase activity than Tristis No. 1. Protein percentages of leaf plastochron index 10 leaves (tenth leaf below first leaf lamina exceeding 20 mm in length), bottom leaves, and roots in relation to nitrate ion activity were not appreciably different between clones. The nitrate reductase activity and protein percentage of Tristis No. 1 apex started to level off at the same nitrate ion activity, about 0.09 mm. In Wisconsin-5 apex protein percentage continued to increase at nitrate ion activities where nitrate reductase activity decreases sharply, suggesting that protein nitrogen was being supplied by ammonium ion. The difference in nitrate reductase activity between clones was probably due to genetically determined ability to synthesize nitrate reductase in response to nitrate ion. The expression of nitrate reductase activity was not an index of nitrogen assimilation ability but may be a useful index of growth potential when nitrate ion does not limit nitrate reductase synthesis. PMID- 16658756 TI - Effects of Cycloheximide upon Formation of Ribonucleic Acid Cytidylic and Uridylic Acids. AB - Concentrations of cycloheximide as low as 3 mug/ml inhibited incorporation of labeled orotic acid or uridine into RNA cytidylic acid of soybean (Glycine max) hypocotyl sections. Even lower concentrations of this well known protein synthesis inhibitor interfered with conversion of labeled cytidine into RNA uridylic acid. Both cycloheximide and puromycin inhibited absorption of (3)H phenylalanine and its incorporation into protein, but puromycin did not significantly affect the labeling patterns of RNA cytidylic and uridylic acids when orotic acid-6-(14)C was fed. Results give further support to the hypothesis that cycloheximide inhibits the interconversion of uridine and cytidine nucleotides, presumably by acting as a glutamine antagonist in the glutamine dependent reaction catalyzed by cytidine triphosphate synthetase. PMID- 16658757 TI - Senescence of attached bean leaves accelerated by sprays of silicone oil antitranspirants. AB - During an investigation into the use of oil emulsions in foliar sprays, it was found that silicone oil emulsions accelerated the senescence of the primary leaves of bean (Phaseolus vulgaris) plants. It was shown that accelerated senescence was not a result of the reduced transpiration rates found in silicone sprayed leaves. Furthermore, the silicone oil emulsions did not induce leakiness in plant cell membranes. The senescence-enhancing effect seems to be connected with the ability of the silicone oil emulsions to penetrate into the leaf interior. PMID- 16658758 TI - Effect of Noise on the Mathematical Parameters that Describe Isothermal Seed Germination. AB - A mathematical model is proposed to describe the isothermal germination rate of seeds as a function of time. All environmental parameters were held constant with the exception of the sound-pressure level and the frequency of the impinging sound waves. Each single frequency sound has its own set of mathematical parameters that describe the differential germination rate as a function of time. Frequencies of 100, 1000, 2000, 4000, and 9000 hertz as well as broad band noise, all at 100 decibels, were used in the experiments. PMID- 16658759 TI - Levels of (+/-) Abscisic Acid and Xanthoxin in Spinach under Different Environmental Conditions. AB - The levels of the growth inhibitors(+)-abscisic acid and xanthoxin were determined in the long day plant spinach (Spinacia oleracea L. cv. Savoy Hybrid 612) grown under different environmental conditions. When plants were transferred from light to darkness, the (+)-abscisic acid level always decreased, whereas the xanthoxin content did not change. The (+)-abscisic acid content was higher in plants grown under low than under high relative humidity.Xanthoxin levels were not affected by photoperiod, whereas the (+)-abscisic acid content increased 2 to 3 times upon transferring plants from short day to long day. Shoot tips with young leaves and mature leaves of the same plants analyzed separately did not differ in their inhibitor content when expressed per unit dry weight. No increase in xanthoxin level was observed in wilting plants. In general, the xanthoxin levels of spinach were much less affected by changes in the environment than were those of (+)-abscisic acid. In conclusion, there is no correlation between xanthoxin and (+)-abscisic acid levels in spinach on the one hand, and growth and flowering responses on the other. PMID- 16658760 TI - Dehydroipomeamarone as an Intermediate in the Biosynthesis of Ipomeamarone, a Phytoalexin from Sweet Potato Root Infected with Ceratocystis fimbriata. AB - Recently, we isolated dehydroipomeamarone, a new sesquiterpenoid from sweet potato (Ipomoea batatas Lam.) root tissue infected with Ceratocystis fimbriata (Ell. et Halst.). The purpose of this investigation was to determine whether dehydroipomeamarone was a precursor in the biosynthetic pathway of ipomeamarone. The incorporation of acetate-2-(14)C into ipomeamarone was markedly inhibited by the presence of dehydroipomeamarone. Radioactive dehydroipomeamarone was efficiently converted into ipomeamarone, and the compound was biosynthesized earlier than ipomeamarone according to a time course analysis of the production of the terpenoid. These results support the notion that dehydroipomeamarone is an immediate precursor of ipomeamarone. On the other hand, the production of ipomeamarone was slightly lessened in the presence of dehydroipomeamarone. Thus, the marked reduction of acetate-2-(14)C incorporation into ipomeamarone by dehydroipomeamarone may result from both isotopic dilution and an inhibitory effect by exogenous dehydroipomeamarone. PMID- 16658761 TI - Studies on the respiratory properties of mitochondria isolated from developing winter wheat seedlings. AB - Mitochondria isolated from shoots of 2 days, light- and dark-grown winter wheat (Triticum aestivum L. cv. Rideau) seedlings oxidize alpha-ketoglutarate and l malate with good respiratory control and ADP: O ratios. The efficiency of oxidative phosphorylation, and respiratory control are both reduced significantly when succinate or NADH is employed as substrate. Respiratory control values and ADP: O ratios show a general decline in mitochondria from seedlings of increasing age, whether grown in light or dark. In light-grown seedlings, the decrease in respiratory control with aging is due principally to a decrease in the rate of state 3 respiration, while in dark-grown material, the decrease appears to be due mainly to an increased rate of state 4 respiration. In both light- and dark-grown seedlings, oxygen consumption during state 3 respiration is severely inhibited by oligomycin. During state 4 respiration, 2,4-dinitrophenol stimulates oxygen uptake to a level approximately two-thirds the normal ADP-stimulated rate. PMID- 16658762 TI - Dual effects of ethylene on potato dormancy and sprout growth. AB - Dormant potato tubers (Solanum tuberosum L.) of two cultivars were treated with various concentrations of ethylene gas for various exposure periods. As has been shown by others, ethylene caused a rapid but transient increase in respiration rate, which appeared to be independent of any effects on dormancy. All concentrations tested caused accelerated sprouting, 2 microliters per liter being the most effective. Ethylene exerts a dual effect on potato tubers: it markedly shortens the duration of rest, but it inhibits elongation of the sprouts during extended treatment. Comparing these results with published work on seeds, bulbs, and corms suggests that ethylene must have a significant but as yet unexplained role in rest and dormancy. However, since the most effective ethylene treatment did not equal the response elicited by treatment with ethylene chlorhydrin, other factors must also contribute to termination of rest. PMID- 16658763 TI - Inhibition of phosphorus and water passage across intact roots by polyethylene glycol and phenylmercuric acetate. AB - Application of polyethylene glycol or phenylmercuric acetate to intact bean (Phaseolus vulgaris L., cv. Red Wade) roots inhibited passage of phosphorus across the roots to the xylem. The same results occurred for foliar application of phenylmercuric acetate when time was allowed for absorption and distribution of the chemical in the plant. For both chemicals the inhibition of phosphorus was proportional to or greater than any accompanying restriction on water flow across the root. PMID- 16658764 TI - The Influence of Benzimidazole on the Gametophyte of Thelypteris felix-mas. AB - Benzimidazole has been studied previously by certain investigators as a growth regulator. This compound was found to promote various phases of growth in the gametophyte of the fern Thelypteris felix-mas. Concentrations of benzimidazole in the order of 0.01 mm, 0.02 mm, and 0.1 mm stimulated cell division, an increase in body size, and also rhizoid lengths of the gametophyte. A concentration of 1.0 mm was always inhibitory. PMID- 16658765 TI - Turnover of cell wall polysaccharides in elongating pea stem segments. AB - Turnover of cell wall polysaccharides and effects of auxin thereon were examined after prelabeling polysaccharides by feeding pea (Pisum sativum var. Alaska) stem segments (14)C-glucose, then keeping the tissue 7 hours in unlabeled glucose with or without indoleacetic acid. There followed an extraction, hydrolysis, and chromatography procedure by which labeled monosaccharides and uronic acids were released and separated with consistently high recovery. Most wall polymers, including galacturonan and cellulose, did not undergo appreciable turnover. About 20% turnover of starch, which normally contaminates cell wall preparations but which was removed by a preliminary step in this procedure, occurred in 7 hours. Quantitatively, the principal wall polymer turnover process observed was a 50% decrease in galactose in the pectinase-extractable fraction, including galactose attached to a pectinase-resistant rhamnogalacturonan. Other pectinase-resistant galactan(s) did not undergo turnover. No turnover was observed in arabinans, but a doubling of radioactivity in arabinose of the pectinase-resistant, hot-acid degradable fraction occurred in 7 hours, possibly indicating conversion of galactan into arabinan. None of the above changes was affected by indoleacetic acid, but a quantitatively minor turnover of a pectinase-degradable xyloglucan was found to be consistently promoted by indole-acetic acid. This was accompanied by a reciprocal increase in water-soluble xyloglucan, suggesting that indoleacetic acid induces conversion of wall xyloglucan from insoluble to water soluble form. The results indicate a highly selective pattern of wall turnover processes with an even more specific influence of auxin. PMID- 16658766 TI - Stachyose: an early product of photosynthesis in squash leaves. AB - It was hypothesized that stachyose is translocated by squash because stachyose is supplied to the phloem loading system by the photosynthetic system. To test this hypothesis, (14)CO(2) was supplied to squash leaves. The nonphosphorylated sugars containing (14)C were studied. A large proportion of (14)C appeared in stachyose very early in the time sequence, tending to confirm the hypothesis. PMID- 16658767 TI - Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid. AB - Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 x 10(6) daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 x 16(6) before denaturation, and 0.68 and 1.5 x 10(6) after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde. PMID- 16658768 TI - Host-Pathogen Interactions: VII. Plant Pathogens Secrete Proteins which Inhibit Enzymes of the Host Capable of Attacking the Pathogen. AB - The results presented demonstrate that microbial pathogens of plants have the ability to secrete proteins which effectively inhibit an enzyme synthesized by the host; an enzyme whose substrate is a constituent of the cell wall of the pathogen. The system in which this was discovered is the anthracnose-causing fungal pathogen (Colletotrichum lindemuthianum) and its host, the French bean (Phaseolus vulgaris). An endo-beta-1, 3-glucanase present in the bean leaves is specifically inhibited by a protein secreted by C. lindemuthianum. The cell walls of C. lindemuthianum are shown to be composed largely of a 1, 3-glucan. PMID- 16658769 TI - Use of protein in extraction and stabilization of nitrate reductase. AB - The in vitro instability of nitrate reductase (EC 1.6.6.1) activity from leaves of several species of higher plants was investigated. Decay of activity was exponential with time, suggesting that an enzyme-catalyzed reaction was involved. The rate of decay of nitrate reductase activity increased as leaf age increased in all species studied. Activity was relatively stable in certain genotypes of Zea mays L., but extremely unstable in others. In all genotypes of Avena sativa L. and Nicotiana tabacum L. studied, nitrate reductase was unstable. Addition of 3% (w/v) bovine serum albumin or casein to extraction media prevented or retarded the decay of nitrate reductase activity for several hours. In addition, the presence of bovine serum albumin or casein in the enzyme homogenate markedly increased nitrate reductase activity (up to 15-fold), especially in older leaf tissue. PMID- 16658770 TI - Chloramphenicol as an energy transfer inhibitor in spinach chloroplasts. AB - At concentrations of up to 300 mug/ml both d-threo- and l-threo-chloramphenicol act as energy transfer inhibitors in spinach chloroplasts, in that they inhibit both phosphorylation and phosphorylating electron transport, without affecting the nonphosphorylating electron transport which occurs either in the absence of a phosphate acceptor or in the presence of the uncoupler ammonium chloride. At higher concentrations, there appears to be an additional site of chloramphenicol inhibition of electron transport. If d-threo-chloramphenicol is to be used as a protein synthesis inhibitor in intact chloroplasts or tissues, control experiments with another chloramphenicol isomer seem to be necessary. PMID- 16658771 TI - Isoaccepting Transfer Ribonucleic Acids during Chilling Stress in Soybean Seedling Hypocotyls. AB - Total aminoacylation of glycine and leucine transfer RNAs was compared between chilled and nonchilled hypocotyls of 7-day-old soybean seedlings. Total charging was greater for both specific transfer RNAs from nonchilled sources. Isoaccepting transfer RNA species for glycine and leucine were fractionated using reverse phase column chromatography. Leucyltransfer RNAs were fractionated into six distinct fractions with relatively small shifts appearing in specific fractions between chilled and nonchilled sources. Glycyl-transfer RNAs were fractionated into two distinct fractions with major shifts appearing for both fractions between chilled and nonchilled sources. PMID- 16658772 TI - The role of galactolipids in spinach chloroplast lamellar membranes: I. Partial purification of a bean leaf galactolipid lipase and its action on subchloroplast particles. AB - A galactolipid lipase has been isolated and partially purified from the chloroplast fraction of the primary leaves of Phaseolus vulgaris var. Kentucky Wonder. The lipase hydrolyzed monogalactosyl diglyceride rapidly and phosphatidyl choline relatively slowly. Triolein and p-nitrophenyl stearate were not hydrolyzed.Spinach subchloroplast particles were excellent substrates for the lipase. Initial rates of fatty acid release from subchloroplast particles at 30 C by the lipase as high as 60 microequivalents per minute per milligram protein were observed. At completion of the reaction, about 2.7 microequivalents of fatty acid were liberated per milligram of chlorophyll in the subchloroplast particles, indicating that major amounts of lipid in the particles were rapidly attacked by the lipase.The treatment of subchloroplast particles with the lipase resulted in a rapid inhibition of light-dependent electron flow. This inhibition was largely prevented when the incubation was carried out in the presence of high concentrations of defatted bovine serum albumin. These results suggest that when precautions are taken to prevent the binding of fatty acids to the subchloroplast particles, large amounts of lipid may be removed without a marked effect on electron flow. PMID- 16658773 TI - Freezing of nonwoody plant tissues: I. Effect of rate of cooling on damage to frozen beet root sections. AB - Small cylinders of red beet (Beta vulgaris) root were frozen at various rates. Ultraslow cooling at 0.2 C per hour to -4 C produced little damage, as determined by leakage of pigment and electrolytes, and softening. All of these increased at faster rates of cooling or at lower temperatures. Cooling at the ultraslow rate appears to induce extracellular freezing, resulting in a protective dehydration of the cell contents. PMID- 16658774 TI - Freezing of Nonwoody Plant Tissue: II. Cell Damage and the Fine Structure of Freezing Curves. AB - Temperature recordings of the freezing of plant tissues include two plateaus or regions of reduced slope. During the second of these, small positive spikes were observed. When a completely frozen tissue was thawed and refrozen, neither the second plateau nor the spikes were recorded. Both were present, however, if the initial freezing had been terminated before the second plateau had been reached. The spikes appear to represent the release of heat of crystallization during the freezing of individual cells. Such a freezing and thawing cycle destroys the ability of the cells to remain supercooled in the presence of the ice that is formed as the first plateau is recorded. PMID- 16658775 TI - The Use of an Enzyme Electrode in the Analysis of Indole-3-acetic Acid Oxidase Activity in Avena. AB - A flexible analytical system which allows for the continuous potentiometric monitoring of the disappearance of an electrochemical species, ferrocyanide, by the peroxidase enzyme is described. The ability of peroxidase to mediate the oxidation of indole-3-acetic acid is followed by observing the competition of indole-3-acetic acid with ferrocyanide for the peroxidase enzyme. This is accomplished by examining potentiometrically the decrease in the rate of ferrocyanide oxidation with increasing indole-3-acetic acid concentration. Homogenates of Avena sativa coleoptiles are investigates for both peroxidase and indole-3-acetic acid oxidase activity. Observations are made with respect to H(2)O(2) and ferrocyanide in the presence and absence of indole-3-acetic acid and naphthalene acetic acid and several interpretations of the reaction kinetics are postulated. Solutions previously assayed for indole-3-acetic acid oxidase activity, when dialyzed and reassayed for peroxidase activity, demonstrated an unimpaired ability to oxidize ferrocyanide peroxidatively, suggesting interpretations of the bisubstrate situation which differ slightly from interpretations given in the literature. PMID- 16658776 TI - Kinetics of Activation of Nicotinamide Adenine Dinucleotide Kinase by Phytochrome Far Red-absorbing Form. AB - The effects of exogenous redox cofactors and purine analogues on the activation of the NAD kinase by Pfr were examined. Addition of phenazine methosulfate, flavin mononucleotide, or methylene blue increased the activation of NAD kinase by red light in a partially purified preparation of phytochrome. Phenazine methosulfate and flavin mononucleotide do not absorb light in the red or far red region, so they do not act as light receptors in this activation. Thus they probably intervene in electron transfer between phytochrome and NAD kinase. Addition of kinetin with these compounds increased photopotentiation further. In the presence of phenazine methosulfate and kinetin, the activation of NAD kinase by red light was counteracted by illumination with far red light immediately after red light.The relation between the dose of illumination with red light and the degree of activation of NAD kinase was measured. Illumination for 5 minutes with 800 ergs cm(-2) sec(-1) of red light was sufficient to obtain maximal activation. In the dark, the activated state of NAD kinase was maintained for about 10 min and then rapidly lost. The time of preservation of the activated state was greatly shortened by illumination with far red light, indicating the participation of phytochrome. PMID- 16658777 TI - Cation Penetration through Isolated Leaf Cuticles. AB - The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS(+) >/= Rb(+) > K(+) > Na(+) > Li(+). The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K(+) and H(2)O at pH above 9 were of similar magnitude. At pH 5.5 H(2)O penetration was not affected but that of K(+) was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites. PMID- 16658778 TI - Phenolic acids in wheat coleoptile cell walls. AB - The phenolic constituent of nonvascular cell walls of wheat (Triticum aestivum L.) coleoptiles, which yields vanillin upon nitrobenzene oxidation, is not lignin as I previously claimed. It seems to be mainly ferulic acid bonded to carbohydrate, probably by an ester linkage. The acid is associated with a fraction of the wall rich in arabinose and xylose, although it is not known whether it is esterified directly with these pentose residues. The phenolic carbohydrate complex is released by cellulase, but not by pronase or a mixture of hemicellulases. PMID- 16658779 TI - Cellulase and Abscission in the Red Kidney Bean (Phaseolus vulgaris). AB - Cellulase (beta-1, 4-glucan-glucanohydrolase EC 3.2.1.4) activity in the abscission zone of red kidney bean (Phaseolus vulgaris) was previously shown to exist in at least two different molecular forms. The form of the enzyme which has an isoelectric point of 4.5 is present in both abscising and nonabscising tissue and requires grinding for extraction. Another form of the enzyme which has an isoelectric point of 9.5 is present only in tissue in which the abscission process has been induced. Further, much of this form of cellulase can be removed from the tissue by vacuum infiltration with buffer. Time course studies indicate that while the increase in measurable cellulase activity in tissue which is actively undergoing abscission was due primarily to the appearance of cellulase 9.5, this form of the enzyme cannot be removed by vacuum infiltration until after the breakstrength of the abscission zone has decreased nearly to zero. The intracellular localization of these two forms of cellulase is discussed. PMID- 16658780 TI - Morphogenesis in Selaginella: II. Auxin Transport in the Root (Rhizophore). AB - The rhizophore of Selaginella willdenovii Baker develops from the ventral angle meristem. The morphological nature of this organ has been in dispute. The purpose of this investigation was to obtain physiological evidence to support the contention that the rhizophore is a root and not a shoot. This was accomplished by studying the movement of (3)H-indoleacetic acid and (14)C-indoleacetic acid in Selaginella rhizophores. In 6-millimeter tissue segments, twice as much radioactivity accumulated in acropetal receivers as in basipetal. During 1 hour of transport in intact roots auxin traveled twice as far in the acropetal direction as basipetal. A significant amount of radioactivity transported in the tissue was found to co-chromatograph with cold indoleacetic acid. Decarboxylation accounted for 10% loss of activity from donors. The data provide sufficient physiological evidence that this organ is morphogenetically a root. PMID- 16658781 TI - Proteins of soybean seeds: I. Isolation and characterization of the major components. AB - Soybean (Glycine max) storage proteins were characterized by sedimentation and by polyacrylamide gel electrophoresis under dissociating (8 m urea) and nondissociating conditions. Three sedimenting classes of proteins were found, with sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The coefficients were related to the bands obtained by electrophoretic separation. The results support the idea that relatively few proteins make up the bulk of the seed protein. PMID- 16658782 TI - Proteins of Soybean Seeds: II. Accumulation of the Major Protein Components during Seed Development and Maturation. AB - Fresh weight and dry weight as well as quantitative and qualitative protein changes in the developing soybean (Glycine max) seed were described from 12 days after flowering until maturity. The seed proteins were separated on sucrose density gradients into three major fractions, having average sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The 2.2S sedimenting proteins predominated at very early stages of development (12 days after flowering) and decreased proportionately throughout maturation. The 7.5S and 11.8S components appeared to be synthesized later in maturity and in larger amounts than the 2.2S proteins. Electrophoretic studies on extracts from whole seeds and on isolated protein fractions confirmed the early abundance of proteins in the 2.2S fraction and revealed temporal differences in the accumulation of three components of the 7.5S fraction. The 11.8S sedimenting fraction appeared throughout seed development as a homogeneous protein which accumulated in the seed with a time course similar to that of the total 7.5S protein fraction. PMID- 16658783 TI - Cholinesterases from Plant Tissues: III. Distribution and Subcellular Localization in Phaseolus aureus Roxb. AB - The distribution and localization of cholinesterase in Phaseolus aureus, Glycine max, and Pisum sativum is described. The enzyme is present in roots, leaves, stems, root callus tissue, root cells suspension cultures, and root nodules. Cholinesterase in roots is found primarily in the cell wall. In cell fractionation experiments, at least 95% of the cholinesterase activity is associated with cell wall material. The enzyme can be solubilized by salt solutions, whereas Triton X-100 and sodium deoxycholate solubilize relatively small amounts of the enzyme. Cytochemical techniques have been employed to show the presence of cholinesterase activity at the cell surface and in the cell wall of certain cells of the root. PMID- 16658784 TI - An Apparent Cellulase Complex in Tomato (Lycopersicon esculentum L.) Fruit. AB - Four enzyme-containing fractions were separated by ammonium sulfate fractionation of 2-day, postbreaker tomato (Lycopersicon esculentum L. cv. Manhattan). The pH optima and substrate specificities are reported. The enzymes were identified as a nonspecific beta-glucosidase, an exo-beta-1,4-glucanase, and two endocellulases. Both endocellulase fractions were able to catalyze the hydrolysis of various insoluble cellulose materials.Together, the enzymes studies are capable of completely degrading insoluble cellulose to short chain cellodextrins and glucose. Ripening tomato fruit apears to contain a complex of cellulases similar to that of cellulolytic bacteria and fungi. PMID- 16658785 TI - Energies of freezing and frost desiccation. AB - A stable cellulose paper system was studied to relate water distribution data, as obtained previously from plant tissues, to the analysis of freezing energy. Water distribution data for the cellulose system were obtained by several techniques and were coordinated with calorimetric data. The effect of the cellulose system on the latent heat of freezing was evaluated to estimate activation energies as functions of the amount of associated liquid water. Similar activation energies of water phase transitions in critical plant tissue systems may be heritable characteristics that affect freezing stress. Adhesion energy, that develops between ice and hydrophilic polymer systems as they compete for liquid water in a complex interface, was suggested as one possible source of freezing stress. This does not occur in frost desiccation. PMID- 16658786 TI - Pitfalls in using sodium hypochlorite as a seed disinfectant in C incorporation studies. AB - Seeds sterilized with sodium hypochlorite (NaOCl) retained sufficient amounts to interfere with studies of amino acid metabolism of the sterilized seeds during germination. Repeated washing in water did not remove NaOCl completely. However, soaking the seeds for 10 min in 0.01 n HCl removed NaOCl completely, without reducing germinability.Residual NaOCl reacted with the amino acids and reduced their concentrations in the incubation media. This reaction resulted in high production of CO(2) and low uptake of amino acids by the seeds. Decarboxylation of the amino acids occurred in the incubation medium outside the seed, was independent of the presence of seeds in the reaction, and therefore was not related to amino acid metabolism by the seeds. Effects of NaOCl on uptake, incorporation, and CO(2) production from indoleacetic acid were similar to those of the amino acids studied. PMID- 16658787 TI - Microelectrodes suitable for use in cells with high hydrostatic pressure. AB - Microelectrodes with a 1- to 2-mum tip diameter have been made which are capable of withstanding plant cell hydrostatic pressure on impalement. Filling the electrodes with 1% agar or 5% gelatin in 2 m KCl prevents cytoplasmic contents from moving into the electrode tip on impalement and therefore prevents the irreversible increase in resistance which often occurs. The agar and gelatin electrodes were tested in two fresh water algae, Nitella translucens and Mougeotia sp., and the potentials recorded were found comparable to those recorded with standard 2 m KCl electrodes. PMID- 16658788 TI - Boron-induced Bioelectric Field Change in Mung Bean Hypocotyl. AB - Low concentrations of boron were found to affect the bioelectric field potentials of hypocotyls excised from 4-day-old mung bean (Phaseolus aureus cv. Oklahoma 612) seedlings grown in darkness and irradiated with red light. The significance of this effect is discussed in relation to the possible role of boron in some membrane function. PMID- 16658789 TI - Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis. AB - Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane. PMID- 16658790 TI - Rapid Increase in Adenosine 5'-Triphosphate during Early Wheat Embryo Germination. AB - The ATP content of isolated wheat (Triticum aestivum L. var. Polk) embryos increases 5-fold during the first 30 minutes and 10-fold during the first hour of germination to 80% of maximum. The ATP level remains at approximately 800 nanomoles per gram of tissue during the next 15 hours. ADP, AMP, and total adenosine phosphates decrease between 1 and 6.5 hours, while adenylate energy charge increases from 0.6 to 0.8 and remains constant. The rapid increase in ATP during imbibition is consistent with the energy requirement for polyribosome formation and protein synthesis during the first hours of germination. A method for determining nanomole quantities of ATP in tissue extracts by isotopic dilution of gamma-(32)P-ATP in the hexokinase reaction is outlined. PMID- 16658791 TI - Effects of Red and Far Red Light on the Initiation of Cold Acclimation in Cornus stolonifera Michx. AB - Red and far red light distinctly influence the initial phytochrome-mediated phase of cold acclimation in red-osier dogwood (Cornus stolonifera). Under controlled conditions, short days and end-of-day far red light exposure after long days promote growth cessation, cold acclimation, and subsequent cold hardening of dogwood stems in response to low temperature. Nuclear magnetic resonance absorption spectra of the water in internode stem sections imply that the short day-induced phase of cold acclimation involves a change in tissue hydration, at least in part, due to a substantial reduction in bulk phase water as a result of senescence and loss of water from the pith. Seasonal responses to light and an attempt to induce early acclimation under natural conditions with end-of-day far red light are discussed. PMID- 16658792 TI - Photosynthetic intermediates, the warburg effect, and glycolate synthesis in isolated spinach chloroplasts. AB - Increasing levels of CO(2) have been shown to stimulate the rate of photosynthesis, eliminate the oxygen inhibition of photosynthesis (Warburg effect), and decrease glycolate formation in isolated spinach chloroplasts. Ribose 5-phosphate and fructose 1,6-diphosphate at concentrations of 5 to 10 mum also stimulate the rate of plastid photosynthesis and eliminate the Warburg effect. In contrast to the effect of high CO(2) levels, these sugar phosphates have little effect on glycolate formation. Evidence is presented to show that the level of intermediates of the photosynthetic carbon reduction cycle may influence the Warburg effect in vivo. It is postulated that the formation of glycolate is not the causal factor of the Warburg effect. PMID- 16658793 TI - Effect of carbon dioxide and ethylene on tuberization of isolated potato stolons cultured in vitro. AB - Carbon dioxide stimulates tuberization of isolated potato (Solanum tuberosum L.) stolons cultured in vitro. The stimulatory effect is inhibited by C(2)H(4) which is by itself also inhibitory of tuberization. Furthermore, C(2)H(4) inhibits kinetin-induced tuber initiation. Both the formation and elongation of roots are inhibited by C(2)H(4). The antagonistic actions of CO(2) and C(2)H(4) on tuberization are discussed. PMID- 16658794 TI - Citrate synthetase in mitochondria and glyoxysomes of maize scutellum. AB - Mitochondria and glyoxysomes were isolated from scutella of maize (Zea mays L.) by density gradient centrifugation. Citrate synthetase was partly solubilized from the organelles by sonication. The sonicated organelle suspensions were centrifuged at high speed, and the supernatants were used as enzyme preparations without further purification. The enzymes of the two organelles differ in all properties examined (pH activity curve, Km for substrates, elution volume on Sephadex G-100, mobility on starch gel at pH 7). Both enzymes are inhibited by ATP, but the inhibition is stronger for the mitochondrial enzyme. The inhibition is competitive for the mitochondrial enzyme and noncompetitive for the glyoxysomal enzyme. The glyoxysomal, but not the mitochondrial enzyme, is inhibited 40% with 1 mm ADP and cytidine triphosphate. PMID- 16658795 TI - Prechilling of Xanthium strumarium L. Reduces Net Photosynthesis and, Independently, Stomatal Conductance, While Sensitizing the Stomata to CO(2). AB - Greenhouse-grown plants of Xanthium strumarium L. were exposed in a growth cabinet to 10 C during days and 5 C during nights for periods of up to 120 hours. Subsequently, CO(2) exchange, transpiration, and leaf temperature were measured on attached leaves and in leaf sections at 25 or 30 C, 19 C dew point of the air, 61 milliwatts per square centimeter irradiance, and CO(2) concentrations between 0 and 1000 microliters per liter ambient air. Net photosynthesis and stomatal conductance decreased and dark respiration increased with increasing duration of prechilling. The reduction in net photosynthesis was not a consequence of decreased stomatal conductance because the intercellular CO(2) concentration in prechilled leaves was equal to or greater than that in greenhouse-grown controls. The intercellular CO(2) concentration at which one-half maximum net photosynthesis occurred remained the same in prechilled leaves and controls (175 to 190 microliters per liter). Stomata of the control plants responded to changes in the CO(2) concentration of the air only slightly. Prechilling for 24 hours or more sensitized stomata to CO(2); they responded to changes in CO(2) concentration in the range from 100 to 1000 microliters per liter. PMID- 16658796 TI - The Azolla, Anabaena azollae Relationship: I. Initial Characterization of the Association. AB - Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source.Morphological aspects of the symbiotic association show the confinement of the Anabaena azollae within the leaf cavity of the Azolla. Procedures were established for the isolation of pure preparations of Anabaena azollae and Azolla chloroplasts. It has not yet been possible to grow the isolated alga in independent culture.Photochemical activities of the isolated alga and fern chloroplasts were measured by spectrophotometric assays for photosystems I and II as well as by P700-content (photosystem I) and delayed light emission (photosystem II). In the algal fraction, both photosystems were repressed when compared to freeliving Anabaena cylindrica, but the relative ratio of photosystem I to photosystem II may be appreciably greater in Anabaena azollae. Azolla chloroplasts were generally comparable to spinach chloroplasts.A comparison of the chlorophyll a and b content of Azolla fronds with and without the symbiotic alga resulted in an estimate that in the symbiotic association, the Anabaena azollae accounts for from 7.5 to 15% of the total chlorophyll. PMID- 16658797 TI - The Azolla, Anabaena azollae Relationship: II. Localization of Nitrogenase Activity as Assayed by Acetylene Reduction. AB - Anaerobic (microaerophilic) acetylene reduction by Azolla caroliniana Willd. was dependent on light and saturated at approximately 450 foot candles. Maximum rates of acetylene reduction were 60 nmoles/mg chlorophyll minute. However, rates of 25 to 30 nmoles/mg chlorophyll minute were more common.The growth of Azolla for 35 days with nitrate or urea as a nitrogen source decreased the rate of acetylene reduction approximately 30% compared to controls grown on nitrogen. Prolonged growth on nitrate or urea (6-7 months) resulted in a 90% decrease in the rate of acetylene reduction.The inhibition of acetylene reduction by 3 (3,4 dichlorophenol) 1,1-dimethylurea (12 muM) was not pronounced until the Azolla became depleted of the reserves formed during photosynthesis. The interval required for this depletion was dependent upon pretreatment and varied from 2 to more than 12 hours. Oxygen evolution was inhibited 75% in 10 minutes by the same concentration of 3 (3,4-dichlorophenol) 1,1-dimethylurea.The addition of oxygen, 20% volume per volume, resulted in a 30 to 40% decrease in the rate of acetylene reduction and the onsetof 3(3,4-dichlorophenol) 1,1-dimethylurea inhibition was more rapid then under microaerophilic conditions. The aerobic dark reduction of acetylene was from 10 to 30% of the rate of aerobic reduction in the light.Acetylene reduction activity was absent in fronds freed ofthe symbiotic algae and present in isolated Anabaena azollae. This study shows that the alga is the agent of acetylene reduction and suggests that there is considerable transport of metabolites between the fern and the blue-green alga. PMID- 16658798 TI - Improvements of the nitrite color development in assays of nitrate reductase by phenazine methosulfate and zinc acetate. AB - Nitrate reductase activity is most commonly assayed by measurement of product formation. Excess NADH and factor(s) present in the enzyme extract that interfere with the diazotization and azo color complex of nitrite cause a depression of apparent nitrate reductase activity. Two postassay treatments were found that markedly enhanced the extent of nitrite color formation and apparent nitrate reductase activity. The procedure involves stopping the reaction with zinc acetate (50 mumoles per ml of reaction mix), followed by removal of the precipitate by centrifugation. Presumably the zinc acetate removes extract factor(s) that interfere with color development, because it does not remove the NADH. Phenazine methosulfate (15 nmoles per ml of reaction mix) is added to aliquots of the supernatant and allowed to stand for 20 min at 30 C to oxidize the residual NADH before color development. PMID- 16658799 TI - Phosphoenolpyruvate carboxylase from spinach leaf tissue: inhibition by sulfite ion. AB - Phosphoenolpyruvate carboxylase (EC 4.1.1.31), partially purified from spinach (Spinacia oleracea) leaves, is inhibited by SO(3) (2-) ion. The inhibition is competitive or mixed type with respect to HCO(3) (-) (Ki = 17 mm), and noncompetitive with respect to phosphoenolpyruvic acid (Ki = 11 mm), Mg(2+) (Ki = 10 mm), and Mn(2+) (Ki = 2.4 mm). The inhibitory effect of SO(3) (2-) is more significant in the presence of Mn(2+) than in the presence of Mg(2+). l-Malate, an inhibitor of phosphoenolpyruvate carboxylase activity, and SO(3) (2-) may bind at the same site on the enzyme. Glyoxal bisulfite and glyoxylate bisulfite are equally effective inhibitors of the enzyme activity as SO(3) (2-), but alpha hydroxypyridinemethanesulfonate is a weak inhibitor. The data are discussed in relation to the physiological effect of the air pollutant (SO(2)) on plant leaf metabolism. PMID- 16658801 TI - The Respiratory Chain of Plant Mitochondria: XVI. Interaction of Cytochrome b(562) with the Respiratory Chain of Coupled and Uncoupled Mung Bean Mitochondria: Evidence for Its Exclusion from the Main Sequence of the Chain. AB - Cytochromes b(553), b(557), and b(562) of mung bean (Phaseolus aureus) mitochondria become partially reduced with endogenous substrate on addition of antimycin A to the aerobic mitochondrial suspension. Addition of ATP causes partial reoxidation of the three cytochromes. This partial oxidation by ATP is inhibited by oligomycin and reversed by uncoupler. Ubiquinone does not appear to act as electron acceptor for the oxidation reaction, but a nonfluorescent flavoprotein, or possibly ironsulfur protein, component does appear to act as acceptor. This is consistent with reverse electron transport driven by ATP across the first site of energy conservation of the respiratory chain. Endogenous pyridine nucleotide and the fluorescent flavoprotein with E(m7.2) = -155mv (midpoint potential at pH 7.2, referred to normal hydrogen electrode) in uncoupled mitochondria become reduced in anaerobiosis attained by oxidation of succinate in the absence of respiratory inhibitors of the cytochrome chain, provided that Pi and ATP are present. Under these same conditions, cytochrome b(557) is completely reduced but cytochrome b(562) remains nearly completely oxidized. There is no equilibration across the first site of energy conservation between the carriers on the low potential side and cytochrome b(562) with E(m7.2) = -77mv on the high potential side. It is concluded that cytochrome b(562) is not a part of the main sequence of electron transport carriers of the mitochondrial respiratory chain of plants; it can participate in redox reactions with the respiratory chain in coupled mitochondria but not in uncoupled mitochondria unless antimycin A is present. PMID- 16658800 TI - Inactivation of pea leaf chloroplastic and cytoplasmic glucose 6-phosphate dehydrogenases by light and dithiothreitol. AB - Chloroplastic and cytoplasmic forms of pea (Pisum sativum L.) leaf glucose-6-P dehydrogenase can be separated by gel electrophoresis. Both forms are found in etiolated seedlings. When light-grown plants are illuminated or when crude extracts are treated with dithiothreitol, the chloroplastic and cytoplasmic forms of this enzyme are inactivated. Michaelis-Menten kinetics are obtained for inactivation of the chloroplastic and cytoplasmic glucose-6-P dehydrogenases and for activation of two dehydrogenases involved in photosynthetic carbon metabolism with dithiothreitol. The mechanism for the activation of the two light-activated enzymes appears to be similar to the mechanism for the inactivation of the chloroplastic and cytoplasmic forms of the light-inactivated oxidative pentose phosphate enzyme glucose-6-P dehydrogenase. PMID- 16658802 TI - The Respiratory Chain of Plant Mitochondria: XVII. Flavoprotein-Cytochrome b(562) Interaction in Antimycin-treated Skunk Cabbage Mitochondria. AB - During the transition from the aerobic steady state with succinate as substrate to anaerobiosis, in suspensions of skunk cabbage (Symplocarpus foetidus) mitochondria treated with antimycin A, cytochrome b(562) becomes reoxidized to the extent of about 20%, synchronously with the reduction of cytochrome c(549). This reoxidation occurs in both the absence and presence of m chlorobenzhydroxamic acid, a specific inhibitor for the alternate terminal oxidase of plant mitochondria. A flavoprotein component, amounting to 13% to 15% of the total nonfluorescent mitochondrial flavoprotein, undergoes reduction synchronously with the oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid. This flavoprotein component remains reduced in the presence of cyanide. The half-time for reduction of the flavoprotein component and cytochrome c(549) and for oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid is 2 seconds. The half-times for oxidation of cytochrome c(549) and the flavoprotein component are 2.1 and 170 milliseconds, respectively, during the anaerobic to aerobic transition induced by addition of 14 mum O(2) to the mitochondrial suspensions. The half-time for reduction of cytochrome b(562) under these conditions is 150 milliseconds, synchronous with the flavoprotein component. The synchrony of the flavoprotein oxidation and of the cytochrome b(562) reduction at a rate much slower than that of cytochrome c(549) oxidation implies that, in antimycin treated plant mitochondria, the state of the cytochrome b(562)/antimycin complex is regulated by the redox state of this flavoprotein component, rather than by cytochrome c(549). It is tentatively suggested that these two components are not part of the main sequence of the respiratory chain, but may be part of a multienzyme complex active in the hydroxylation reactions required for ubiquinone biosynthesis in the inner mitochondrial membrane. PMID- 16658803 TI - Influence of opaque-2 and floury-2 genes on formation of proteins in particulates of corn endosperm. AB - Protein-rich subcellular particulates were isolated by zonal centrifugation from homogenates of endosperms of normal, opaque-2, and floury-2 mutant corn (Zea maize) kernels at different stages of development. In early stages the high lysine mutants vary from normal corn by greater production of a glutelin protein not associated with the matrix. This protein is high in lysine and may become a component of matrix glutelin at later stages of maturity. Differences in size and structure of zein-rich protein bodies were observed in the mutant strains when compared with normal corn. Enhanced production of nonmatrix glutelin as well as the reduction in synthesis of lysine-deficient zein is responsible for the improved lysine content of the mutant endosperms at early stages of development. PMID- 16658804 TI - Relationship between the Level of Adenine Nucleotides and the Carboxylation Activity of Illuminated Isolated Spinach Chloroplasts: A Study with Antimycin A. AB - The changes in the levels of intact spinach (Spinacia oleracea L.) chloroplast adenine nucleotides during the time course of light-dependent CO(2) fixation were determined with respect to the effect of antimycin A. This study demonstrated that antimycin A lowered the rate of ATP formation during the induction period of carboxylation. While the steady state levels of ATP and the energy-charge value also decreased in the presence of antimycin, the concomitant increase of the CO(2) fixation activities insured higher ATP turnover rates. Changes in the labeling of CO(2) fixation products during the lag phase suggested a stepwise activation of the Calvin cycle, with fructose 1,6-diphosphate, and ribulose 5 phosphate kinase being activated before ribulose 1,5-diphosphate carboxylase. The possible mechanisms of the enhancement of CO(2) fixation activity by antimycin A in relation to its action on photophosphorylation during the lag phase are discussed. PMID- 16658805 TI - Effect of Water Stress on Ethylene Production by Detached Leaves of Valencia Orange (Citrus sinensis Osbeck). AB - Detached leaves of Valencia oranges, Citrus sinensis Osbeck, emanated ethylene at markedly higher rates and contained more endogenous ethylene when placed under water stress at 55% relative humidity than when placed in water-saturated air. Water stress induced defoliation from detached branches. Relieving the water stress of such leaves by transfer to a mist chamber resulted in lowering of the rates of ethylene emanation to the level occurring in leaves maintained continuously in a mist chamber. This ability to recover from the water stress was evident for only up to 10 to 20 hours of stress, when the relative turgidity of the leaves was 50 to 60%. Beyond that time the level of ethylene emanation of stressed leaves was not lowered by rehydration in a mist chamber; these overstressed leaves could not reabsorb their original water content. Ethylene emanation was in high correlation with the relative turgidity of detached leaves of oranges. PMID- 16658806 TI - Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum. AB - The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm(3). In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm(3), as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm(3) from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism. PMID- 16658807 TI - Isolation of intact plastids from a range of plant tissues. AB - A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sativum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome oxidase and catalase, respectively. For three of the four tissues the level of contamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed. PMID- 16658808 TI - Seed aging: chromosome stability and extended viability of seeds stored fully imbided. AB - Increase in moisture content of seeds of Lactuca sativa L. and Fraxinus americana L. in air-dry storage caused a rapid decline in longevity and an increase in the rate of accumulation of chromosome aberrations. Storage of seeds fully imbibed but unable to germinate allowed a high germination capacity to be maintained for long periods, together with a very low incidence of chromosome aberrations. Seedlings grown from dry-stored seeds showed an increase in morphological abnormalities with length of storage, whereas seedlings from imbibed-stored seeds appeared normal. It is suggested that in dry tissues, enzyme-controlled turnover and repair may be temporarily suspended, and that this may be an important factor in the loss of seed viability in storage. The effect of increasing seed longevity by lowering the moisture content of dry-stored seeds is discussed in relation to this hypothesis. The relevance of the proposal is also discussed in relation to ecological studies. PMID- 16658809 TI - The effect of light on the tricarboxylic Acid cycle in green leaves: I. Relative rates of the cycle in the dark and the light. AB - Excised green leaves of mung bean (Phaseolus aureus L. var. Mungo) were used to determine the effect of light on the rate of endogenous respiration via the tricarboxylic acid cycle. Illumination with white light at an intensity of 0.043 gram calories cm(-2)min(-1) (approximately 8600 lux) of visible radiation (400 700 nm) gave a rate of apparent photosynthesis, measured as net CO(2) uptake, of 21 mg CO(2) dm(-2)hr(-1) which was about 11-fold greater than the rate of dark respiration. The feeding of (14)CO(2) or (14)C-labeled acids of the tricarboxylic acid cycle in the dark for 2 hours was established as a suitable method for labeling mitochondrial pools of cycle intermediates.At a concentration of 0.1 mm 3-(3,4-dichlorophenyl)-1,1-dimethylurea, apparent photosynthesis was inhibited 82%, and the refixation of (14)CO(2) derived internally from endogenous respiration was largely prevented. In the presence of this inhibitor endogenous respiration, measured as (14)CO(2) evolution, continued in the light at a rate comparable to that in the dark. Consequently, under these conditions light induced nonphotosynthetic processes have no significant effect on endogenous dark respiration. Inhibitors of the tricarboxylic acid cycle, malonate and fluoroacetate, were used to determine the relative rates of carbon flux through the cycle in the dark and in the light by measuring the rate of accumulation of (14)C in either succinate or citrate. Results were interpreted to indicate that the tricarboxylic acid cycle functions in the light at a rate similar to that in the dark except for a brief initial inhibition on transition from dark to light. Evidence was obtained that succinate dehydrogenase as well as aconitase, was inhibited in the presence of fluoroacetate. PMID- 16658811 TI - The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: III. A Comparison between Some C(3) and C(4) Plants. AB - The chlorophyll-based specific activity of cytochrome oxidase and three exclusively mitochondrial enzymes of the tricarboxylic acid cycle showed little variation between leaves of C(3) and C(4) plants or between mesophyll and bundle sheath cells of Atriplex spongiosa and Sorghum bicolor. However, a large, light dependent transfer of label from intermediates of the tricarboxylic acid cycle to photosynthetic products was a feature of leaves of C(4) plants. This light dependent transfer of label was barely detectable in leaves of C(3) plants and in leaves of F(1) and F(3) hybrids of Atriplex rosea (C(4)) and Atriplex patula spp hastata (C(3)). The light-dependent transfer of label to photosynthetic products in leaves of C(4) plants was inhibited by the tricarboxylic acid cycle inhibitors malonate and fluoroacetate. The requirement for continued tricarboxylic acid cycle activity was also indicated in experiments with specifically labeled succinate-(14)C. These experiments, together with the distribution of (14)C in glucose prepared from sucrose-(14)C formed during the metabolism of succinate-2,3 (14)C, confirmed that the photosynthetic metabolism of malate and aspartate derived from the tricarboxylic acid cycle, and not the refixation of respiratory CO(2), was the main path of carbon from the cycle to photosynthesis. PMID- 16658810 TI - The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: II. Intermediary Metabolism and the Location of Control Points. AB - Long term feeding of acetate-2-(14)C, (14)CO(2), citrate-1,5-(14)C, fumarate-2,3 (14)C, and succinate-2,3-(14)C to mung bean (Phaseolus aureus L. var. Mungo) leaves in the dark gave labeling predominantly in tricarboxylic acid cycle intermediates. Kinetics of the intermediates during dark/light/dark transitions showed a light-induced interchange of (14)C between malate and aspartate, usually resulting in an accumulation of (14)C in malate and a decrease of it in aspartate. (14)C-Phosphoenolpyruvate also showed a marked decrease during illumination. Changes in other intermediates of the tricarboxylic acid cycle were relatively minor. The kinetic data have been analyzed using the Chance crossover theorem to locate control points during the dark/light/dark transitions. The major apparent control points are located at malate and isocitrate dehydrogenases, and less frequently at citrate synthase and fumarase. These findings are explained in terms of the light-induced changes in adenine nucleotides and nicotinamide adenine dinucleotides. PMID- 16658812 TI - Postharvest response of avocado fruits of different maturity to delayed ethylene treatments. AB - The effect of 10, 100, 1000, 10,000 ppm of ethylene, applied for 3, 6, 12, and 24 hours, on the ripening rate of "Hass" avocado (Persea americana Mill.) fruits at three stages of maturity was investigated. Ethylene treatments were started either immediately after picking or 2 days later. A sharp peak of ethylene production was found to precede full softening by about 2 days and the occurrence of this peak was used to determine ripening rate. Hastening of fruit ripening was much more marked following ethylene treatment which began 2 days after harvesting than immediately after. The difference in response diminished gradually as the fruit became more mature.These results can be explained by the assumption that the factors which prevent avocado ripening on the tree, continue to exert their influence for a limited period after harvest and inhibit the ripening response in the freshly picked fruit. This inhibition decreases with advancing fruit maturity. PMID- 16658813 TI - Inhibition of Cuticular Lipid Biosynthesis in Pisum sativum by Thiocarbamates. AB - Treatment of slices of young pea leaves (Pisum sativum) with muM solutions of alpha-chlorallyl diethyldithiocarbamate, dichloroallyl diisopropylthiocarbamate, or S-ethyldipropylthiocarbamate resulted in inhibition of incorporation of [1 (14)C]acetate into C(31) alkane and C(31) secondary alcohol, very little effect on the synthesis of C(26) and C(28) fatty alcohols, and an accumulation of (14)C in shorter chain cuticular lipids, particularly C(22) acid. Higher concentrations of the thiocarbamates caused inhibition of synthesis of C(26) and C(28) fatty alcohols and an accumulation of label in C(22) acid. Further increase in thiocarbamate concentration resulted in inhibition of C(22) acid synthesis also. The three thiocarbamates at muM concentration also inhibited incorporation of [1 (14)C]stearic acid specifically into C(31) alkane and C(31) secondary alcohol. These results suggest that thiocarbamates reduce cuticular lipid formation by a concentration-dependent inhibition of the various chain-elongating enzyme systems. PMID- 16658814 TI - Endogenous rhythmic activity of photosynthesis, transpiration, dark respiration, and carbon dioxide compensation point of peanut leaves. AB - At 14-hour day length, 25 C leaf temperature, 9 mm Hg vapor-pressure deficit, and 1.17 joules cm(-2) min(-1) irradiance, the diurnal change in daily photosynthesis of the cultivated peanut (Arachis hypogaea L.) is a result of an endogenously controlled circadian rhythm in net photosynthesis which peaks near noon and troughs near midnight. By resetting the day-night light regime, the rhythm rephased in continuous light. The free-running rhythm approximates 26 hours. Both transpiration and dark respiration show similar rhythmicity, with transpiration closely in phase with the rhythm in photosynthesis. The rhythm in carbon dioxide compensation point is approximately 12 hours out of phase, peaking at midnight and troughing at midday. Endogenous changes in stomatal aperture seemed to be the major control of the rhythm in photosynthesis. The activity of ribulose-1,5 diphosphate carboxylase increased during the normal photoperiod, leveling off after 12 hours; however, the activity was not correlated with the rhythmic change in photosynthesis. PMID- 16658815 TI - Vein Loading: The Role of the Symplast in Intercellular Transport of Carbohydrate between the Mesophyll and Minor Veins of Tobacco Leaves. AB - Enzymatically separated leaf tissues of Nicotiana tabacum L., exhibiting good metabolic integrity, were used to evaluate the kinetics of sugar accumulation over the concentration range of 10 to 100 mm. Mesophyll cells exhibited Km values of 16 and 30 mm for glucose and sucrose, respectively; minor veins showed a reverse relationship, with Km values of 58 and 16 mm for glucose and sucrose, respectively. This would suggest that sucrose is preferentially absorbed by the minor vein net. Analysis of V(max) data indicates a reduction in the ability of isolated minor veins to accumulate substrate, implicating a symplastic rather than apoplastic route for intercellular transport. Competition studies demonstrate a common carrier for sucrose and glucose in both tissue types and suggest the presence of a "transport compartment," entry to which is regulated by a critical intracellular sucrose concentration. PMID- 16658816 TI - Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation. AB - Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria. PMID- 16658817 TI - A Direct Comparison between Circadian and Noncircadian Rhythms in Neurospora crassa. AB - A method has been devised for observing both circadian and noncircadian rhythms in a single wild type strain of Neurospora crassa. This method allows a direct comparison of the properties of the two types of rhythm. The circadian rhythm of conidiation always entrains to a light-dark cycle, damps out in constant light, and has a temperature-compensated period length. The noncircadian rhythm of hyphal branching, expressed by the same strain under different environmental conditions, does not entrain to a light-dark cycle, persists in constant light, and its period length is temperature-dependent. These results suggest that the two rhythms have different underlying mechanisms and demonstrate that the differences in the rhythms previously observed in different strains (patch, band, and clock) are not due to genetic differences between these strains but rather are inherent properties of the rhythms themselves. PMID- 16658818 TI - Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue. AB - Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation. PMID- 16658819 TI - Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings. AB - Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO(2) levels ranging from 100 to 800 mul/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO(2) in the dark and maintained in an atmosphere containing 100 mul/l CO(2) accumulated 7-fold more nitrate and had 2 fold more nitrate reductase activity than plants exposed to 600 mul/l CO(2), after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO(2) was high. Plants exposed to increasing CO(2) levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 mul/l CO(2)) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate. PMID- 16658820 TI - Localization of Stem Elongation Control in Cucumis sativus L. AB - Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA(3), GA(4+7), and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA(4+7) was more effective than GA(3). Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA(4+7) or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants. PMID- 16658821 TI - Effects of ethylene and gibberellic Acid on cellular growth and development in apical and subapical regions of etiolated pea seedling. AB - Subhook swelling of 4-day-old etiolated pea seedlings (var. Alaska), caused by 0.5 microliter per liter ethylene, was prevented by preincubation and continued growth in 0.1 mm gibberellic acid (GA). The subhook region exhibited normal elongation and cell size and volume. However, inhibition of elongation and cessation of cell division caused by 0.5 microliter per liter ethylene in the apical hook region of the etiolated pea stem were not overcome by GA. Most of the arrested cells were in G(2). These data suggest a possible interaction of GA and ethylene in cell enlargement in the subhook region of the etiolated pea seedlings. They also suggest a different mode of action by ethylene in the apical hook region where the ethylene effect was not counteracted by GA. PMID- 16658822 TI - Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase: II. Far Red Light-Dependent Development of Glyceraldehyde 3-Phosphate Dehydrogenase Isozyme Activities in Sinapis Alba Cotyledons. AB - Ammonium sulfate chromatography has been employed to separate glyceraldehyde 3 phosphate dehydrogenases (GPD) of Sinapis alba cotyledons of various developmental stages. Cotyledons of dark-grown seedlings possess one major NAD specific enzyme designated NAD-GPD I. Irradiation with continuous far red light leads to a strong increase in NADP-GPD activity and to the formation of a second NAD activity designated NAD-GPD II. These two activities occur in a constant ratio during cotyledon development, and they are eluted together in ammonium sulfate chromatography. In a later stage of cotyledon development the light dependent increase in NAD-GPD II is matched by an equivalent decrease in NAD-GPD I. These data suggest that the chloroplast marker enzyme NADP-GPD (EC 1.2.1.13) also has NAD activity and that the light-dependent formation of this bifunctional enzyme is correlated with activity changes of the NAD-GPD of cytoplasmic glycolysis (EC 1.2.1.12). PMID- 16658823 TI - The structure of plant cell walls: v. On the binding of xyloglucan to cellulose fibers. AB - Cell wall strength is decreased by both auxin treatment and low pH. In a recently proposed model of the plant cell wall, xyloglucan polymers are hydrogen-bonded to cellulose fibrils, forming the only noncovalent link in the network of polymers which cross-link the cellulose fibers. The decreased strength of the cell wall seen upon lowering the pH might be due to an effect of hydrogen ions on the rate of xyloglucan creep along cellulose fibers. This paper investigates binding of xyloglucan fragments to cellulose. At equilibrium, the per cent of nine- and seven-sugar xyloglucan fragments which are bound to cellulose is sensitive to both temperature and the concentration of nonaqueous solvents. However, neither the per cent of xyloglucan fragments bound to cellulose at equilibrium, nor the rate at which the xyloglucan fragments bind to cellulose, is sensitive to changes in hydrogen ion concentration. These results support the hypothesis that, within the cell wall, xyloglucan chains are connected to cellulose fibers by hydrogen bonds, but these results suggest that this interconnection between xyloglucan and cellulose is unlikely to be the point within the wall which regulates the rate of cell elongation. PMID- 16658824 TI - The Structure of Plant Cell Walls: VI. A Survey of the Walls of Suspension cultured Monocots. AB - The primary cell walls of six suspension-cultured monocots and of a single suspension-cultured gymnosperm have been investigated with the following results: (a) the compositions of all six monocot cell walls are remarkably similar, despite the fact that the cell cultures were derived from diverse tissues; (b) the cell walls of suspension-cultured monocots differ substantially from those of suspension-cultured dicots and from the suspension-cultured gymnosperm; (c) an arabinoxylan is a major component (40% or more by weight) of monocot primary cell walls; (d) mixed beta-1,3; beta-1,4-glucans were found only in the cell wall preparations of rye grass endosperm cells, and not in the cell walls of any of the other five monocot cell cultures nor in the walls of suspension-cultured Douglas fir cells; (e) the monocot primary cell walls studied contain from 9 to 14% cellulose, 7 to 18% uronic acids, and 7 to 17% protein; (f) hydroxyproline accounts for less than 0.2% of the cell walls of monocots. Similar data on the soluble extracellular polysaccharides secreted by these cells are included. PMID- 16658825 TI - Structure, gas chromatographic measurement, and function of suberin synthesized by potato tuber tissue slices. AB - The polymeric material (suberin) of the wound periderm of potato tuber slices was analyzed after depolymerization with LiAIH(4) in tetrahydrofuran or BF(3) in methanol with the use of thin layer chromatography, chemical modification, and combined gas-liquid chromatography and mass spectrometry. Fatty acids (C(16) to C(26)), fatty alcohols (C(16) to C(26)), octadec-9-ene-1, 18-dioic acid, and 18 hydroxy-octadec-9-enoic acid were identified to be the major components. Based on the structural information that the two bifunctional C(18) molecules constituted a major portion of suberin, a gas chromatographic method of measuring suberization was developed. This method consisted of hydrogenolysis of powdered tissue followed by thin layer chromatography and gas chromatographic measurement of octadecene-1, 18-diol as the trimethylsilyl ether. With this assay it was shown that the development of resistance to water loss by the tissue slices was directly proportional to the quantity of the bifunctional C(18) molecules, thus providing evidence that a function of suberin is prevention of water loss. PMID- 16658826 TI - The inhibition of soybean metabolism by cadmium and lead. AB - Lead (300 mum) and cadmium (18 mum) inhibit pod fresh weight in soybeans (Glycine max L.) by 35%. Eighteen micromolar cadmium caused a 30% decline in nitrogenase activity by day 52 (the day on which maximum activity was measured) and a 71% inhibition by day 59. The heavy metals depressed photosynthetic rates; when photosynthesis was depressed by 60%, as measured on the day of peak photosynthesis activity, carbohydrate did not accumulate in the nodules. The reduction of pod fresh weight correlated with the effect of lead and cadmium on several other aspects of plant metabolism (shoot, root, leaf, and nodule dry weight; nodule ammonia, protein and carbohydrate content). PMID- 16658827 TI - Reduction in Extractable Deoxyribonucleic Acid Polymerase Activity in Pisum sativum Seedlings by Ethylene. AB - An extract from the apical portion of etiolated seedlings of Pisum sativum L. was used as a test system to examine the action of ethylene on DNA polymerase activity. The extract catalyzed the polymerization of labeled deoxyribonucleoside triphosphates into a trichloroacetic acid-insoluble product. The system required Mg(2+), nicked DNA, and all four deoxyribonucleoside triphosphates for maximum activity. Extracts from plants previously treated with ethylene showed less activity to synthesize DNA than extracts from nontreated plants. Loss of extractable DNA polymerase activity may be due to accumulation of a non competitive inhibitor in the ethylene-treated plants. Treating the extract with ethylene did not affect the polymerase activity. Inhibition of cell division by ethylene observed in this and other tissues may be the result of accumulation of an inhibitor of DNA polymerase. PMID- 16658828 TI - Biosynthesis of Umbelliferose in Aegopodium podagraria. AB - The following reaction leading to the synthesis of the trisaccharide umbelliferose was demonstrated in an enzyme preparation from leaves of Aegopodium podagraria L.: sucrose + UDP-gal-(14)C --> umbelliferose-(14)C + UDP. Neither galactinol nor galactose 1-phosphate could replace UDP-gal. Among 10 different sugars tested only sucrose was a suitable galatosyl acceptor. PMID- 16658829 TI - A Synergistic Stimulation of Avena sativa Coleoptile Elongation by Indoleacetic Acid and Carbon Dioxide. AB - The ability of 0.03% CO(2) to stimulate growth has been investigated using etiolated Avena coleoptile sections maintained in buffered solution. This concentration of CO(2)-stimulated growth after a lag period of 12 to 15 minutes, and a synergistic relationship between indoleacetic acid and CO(2) in stimulating growth has been demonstrated. The response to CO(2) is inhibited by cycloheximide and is lost approximately 10 minutes after exposure to CO(2)-free air. Malate can replace CO(2) in stimulating growth. In the light of these data and recent literature on a growth response of coleoptile sections to CO(2)-saturated solutions, the existence of two mechanisms of CO(2)-simulated growth is proposed. In addition, it is suggested that growth promotion by 0.03% CO(2) is mediated by a process involving dark CO(2) fixation. PMID- 16658830 TI - The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles. AB - The influence of indoleacetic acid, 0.03% CO(2), and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO(2) stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO(2). Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO(2), and that indoleacetic acid increased the incorporation of (14)C-bicarbonate into protein. These data are discussed in the context of CO(2)-stimulated growth of etiolated tissue, and proposals that CO(2)-stimulated growth involves dark CO(2) fixation. PMID- 16658831 TI - Lipase Activities in Castor Bean Endosperm during Germination. AB - Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal "ghosts" at equilibrium density 1.21 g/cm(3) on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed. PMID- 16658832 TI - Supercooling in overwintering azalea flower buds. AB - Differential thermal analysis and nuclear magnetic resonance spectroscopy experiments on whole flower buds and excised floral primordia of azalea (Rhododendron kosterianum, Schneid.) proved that supercooling is the mode of freezing resistance (avoidance) of azalea flower primordia. Increase in the linewidth of nuclear magnetic resonance spectra for water upon thawing supports the view that injury to the primordia occurs at the moment of freezing. Nonliving primordia freeze at the same temperatures as living primordia, indicating that morphological features of primordial tissues are a key factor in freezing avoidance of dormant azalea flower primordia. Differential thermal analyses was used to study the relationship of cooling rate to the freezing points of floral primordia in whole flower buds. At a cooling rate of 8.5 C per hour, primordia in whole buds froze at about the same subfreezing temperatures as did excised primordia cooled at 37 C per hour. At more rapid cooling rates primordia in intact buds froze at higher temperatures. PMID- 16658833 TI - Nucleic Acid and protein metabolism of senescing and regenerating soybean cotyledons. AB - An alternative to the leaf disk system for studies of the metabolism of senescence is described. The progress of senescence of soybean (Glycine max L.) cotyledons is arrested when the epicotyl is removed. Epicotyl removal at 16 or 17 days reversed the decline in nucleic acid, protein, and chlorophyll content in the cotyledon. Epicotyl removal at 18 days did not reverse the decline in the above components, and therefore the progress of cotyledon has passed the point of no return. Cotyledons lost 90% of their nucleic acid and 80% of their protein before senescence became irreversible. The rate of recovery in various macromolecular components after epicotyl removal did not occur in an equal manner. Nucleic acid was regenerated at a faster rate than chlorophyll, which was regenerated at a faster rate than soluble protein. The heavy nucleic acid components (ribosomal and heavy ribosomal messenger fractions) regenerated at greater rates than did the soluble RNA or DNA. No label from (14)CO(2) was incorporated into DNA of the cotyledons when the epicotyl was present but label was incorporated into DNA after epicotyl removal.The parallels between the mechanisms of cotyledon senescence and apical dominance are discussed. PMID- 16658834 TI - l-Phenylalanine Ammonia-lyase Activity in Robinia pseudoacacia Seedlings: I. Cyclic Phenomenon Activity during Continuous Light. AB - Influences of light and darkness on l-phenylalanine ammonia-lyase activity of black locust (Robinia pseudoacacia) seedlings were studied. Light initiates both an increase in enzyme activity (or enzyme synthesis) and a subsequent decrease in activity. These dual influences of light create cyclic changes in l-phenylalanine ammonia lyase activity in continuous light. The cyclic changes in activity (during continued light) cannot be attributed to an endogenous rhythm and rather are related to the changes in balance between the enzyme activity and its inactivating system. PMID- 16658835 TI - Assimilation, Distribution, and Root Exudation of C by Ponderosa Pine Seedlings under Induced Water Stress. AB - The effect of specific levels of induced water stress on the root exudation of (14)C from 9-month-old and 12-month-old ponderosa pine (Pinus ponderosa Laws.) seedlings was examined. Polyethylene glycol (PEG-4000) was used to decrease root solution water potentials by 0, -1.9, -2.6, -5.5, -9.6 and -11.9 bars in either aerated 0.25X Hoagland's nutrient solution or aerated distilled water.Assimilation of (14)CO(2) by plants under stress and subsequent translocation of (14)C label to the roots were both inhibited by a decrease in substrate water potential. Six days after (14)CO(2) introduction essentially no (14)C was detected in the roots of plants maintained at solution potentials of 5.5 bars or below. In subsequent studies (14)CO(2) was introduced 4 days prior to induction of stress. This allowed sufficient time for distribution of (14)C label throughout the root system.Root exudation of (14)C-labeled sugars, amino acids, and organic acids from plants in nutrient solution showed an increase from 0 to 1.9 bars, a decline from -1.9 to about -5.5 bars, and then an increase again from -5.5 to -11.9 bars. As substrate potential decreased, sugars as a percentage of total exudate increased, organic acids decreased and amino acids showed a slight decrease. Marked changes in percentages occurred between 0 and -2.6 bars. The exudation of sugars, amino acids, and organic acids from plants in distilled water showed similar trends in response to water stress as those in nutrient solution, but the quantity of total (14)C exuded was greater. PMID- 16658836 TI - The Respiratory Chain of Chlorella protothecoides: I. Inhibitor Responses and Cytochrome Components of Whole Cells. AB - The respiration and cytochrome properties of "glucose-bleached" Chlorella protothecoides Kruger, Indiana strain 25, were studied. This organism, when grown heterotrophically with high glucose and a low organic nitrogen source, has no chlorophyll, little carotenoid, and diminished chloroplast structure-factors which make it suitable for respiration studies.Whole cell endogenous oxygen uptake rates are either stimulated or only slightly inhibited by cyanide, azide, CO, and antimycin. When these inhibitors are used with m-chlorobenz-hydroxamic acid (mCLAM), an inhibitor of higher plant mitochondrial alternate oxidase, O(2) uptake is inhibited. There is little effect of mCLAM by itself on the rate of oxygen uptake. The inhibition by CO of O(2) uptake in the presence of mCLAM is reversed by light.The cytochrome chain of C. protothecoides consists of cytochromes aa(3), b, and c, as revealed by room temperature difference spectra. In common with mitochondria of higher plants, there is a further reduction of cytochrome b with dithionite. In the presence of antimycin, the cytochromes aa(3) and c are oxidized and cytochrome b is reduced. Cyanide causes a partial reduction of cytochromes aa(3) and c while cytochrome b remains oxidized. This general response is characteristic of higher plant mitochrondria having large amounts of cyanide-resistant respiration. Carbon monoxide spectra reveal one CO combining pigment. The cytochrome b region differs from that of higher plants in that the typical complex spectrum does not appear at low temperature (-190 C).The concentration of cytochrome aa(3) per cell volume was observed during the greening of "glucose-bleached" cells. The concentration of these cytochromes nearly tripled during the 24 hours of the initial stages of greening. PMID- 16658837 TI - The Respiratory Chain of Chlorella protothecoides: II. Isolation and Characterization of Mitochondria. AB - Mitochondria were isolated from glucose-bleached Chlorella protothecoides Kruger, Indiana strain 25. These mitochondria oxidized succinate, NADH, and l-malate at high rates. Oxygen uptake with these substrates was partially inhibited by 1 mmm chlorobenzhydroxamic acid (mCLAM). Respiratory control was seen with succinate as substrate in the presence of mCLAM. The apparent Km for succinate was determined to be 0.83 mm. Chlorella mitochondria catalyzed the oxidation of reduced horse heart cytochrome c. PMID- 16658838 TI - Metabolism of Tritiated Gibberellin A(9) by Shoots of Dark-grown Dwarf Pea, cv. Meteor. AB - Tritium-labeled gibberellin A(9) ((3)H-GA(9)) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA(20), GA(10), 2,3-dihydro-GA(31), and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GA(30) and 2,3-dihydro-GA(31) were produced within 5 hours following (3)H-GA(9) application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after (3)H-GA(9) application. Levels of GA(10) increased with time, and since no GA(10) was produced during the purification procedures, GA(10) was, in all probability, produced from (3)H-GA(9) within the plant tissue. The radioactive interconversion products produced by pea from (3)H-GA(9) have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of (3)H-GA(9) with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu). PMID- 16658839 TI - Change in invertase activity of sweet potato in response to wounding and purification and properties of its invertases. AB - When root tissue of sweet potato (Ipomoea batatas Lam.) was sliced, acid invertase activity, initially absent in freshly sliced tissue, appeared after a 3 to 6-hour lag phase, rapidly reached a maximum in 18 hours, and thereafter decreased. The increase in invertase activity was accompanied by a decrease in sucrose content of the root tissue. Alkaline invertase activity was present in fresh root tissue, but changed little after wounding. Acid invertase in wounded tissue and alkaline invertase in fresh tissue were purified and their properties were investigated. The acid invertase was a ss-fructofuranosidase and was unaffected by substrate or by any of the cations and several metabolites. The alkaline invertase was more specific for sucrose, was inhibited by glucose and glucose 6-phosphate, and displayed non-Michaelis-Menten kinetics. PMID- 16658840 TI - Characterization of cadmium uptake by plant tissue. AB - The uptake of cadmium by excised root tissue of barley (Hordeum vulgare L. cv. Arivat) was investigated with respect to kinetics, concentration, and interactions with various cations. The role of metabolism in Cd absorption was examined using a range of temperatures, anaerobic treatments, and chemical inhibitors. The uptake and distribution of Cd in intact barley plants was also determined. A large fraction of the Cd taken up by excised barley roots was apparently the result of exchange adsorption and was displaced by subsequent desorption with unlabeled Cd, Zn, Cu, or Hg. Another fraction of Cd which could not be displaced by desorption in unlabeled Cd was thought to result from strong irreversible binding of Cd, perhaps on sites of the cell wall. The fraction of the Cd taken up beyond that by exchange adsorption by fresh roots was a linear function of temperature, and inhibited by conditions of low oxygen and by the presence of 2,4-dinitrophenol. It was concluded that this fraction of Cd entered excised barley roots by diffusion. Diffusion, when followed by sequestering, probably accounts for the accumulation of Cd observed in intact barley plants. PMID- 16658841 TI - Phytochrome-mediated Germination Responses in gamma-Irradiated Lettuce Seeds. AB - Sublethal doses of gamma-radiation and far red light have some-what analogous, red light reversible, effects on the germination of lettuce seeds (Lactuca sativa L. var. Grand Rapids). However, the mechanism by which gamma-radiation retards germination appears to differ from that of far red light. Compared to controls, gamma-radiation retarded germination for the first 24 hours; but after 36 or 48 hours of imbibition gemination of treated seeds was higher than that of the controls, whether or not the gamma-irradiated seeds received red or far red light. The effects of gamma-radiation are more pronounced in seeds containing 15% water at the time of treatment than in those containing only 7% water. The promotive action of red light is operative in the presumed absence of cell division in gamma-treated seeds. PMID- 16658842 TI - Seasonal Patterns of Acid Metabolism and Gas Exchange in Opuntia basilaris. AB - Acid metabolism and gas exchange studies were conducted in situ on the cactus Opuntia basilaris Engelm. and Bigel. A pattern of significant seasonal variation was evident. The pattern was controlled by rainfall, which significantly influenced plant water potentials, total gas transfer resistances, and nocturnal organic acid synthesis. In winter and early spring, when plant water stress was mild, stomatal and mesophyll resistances remained low, permitting enhanced nocturnal assimilation of (14)CO(2). The day/night accumulation of acidity was large during these seasons. In summer and fall, plant water stress was moderate, although soil water stress was severe. The nocturnal assimilation of (14)CO(2) was very low during these seasons, even in stems with open stomata, indicating large mesophyll resistances restricting exogenous gas incorporation. The day/night accumulation of acidity was reduced, and a low level of acid metabolism persisted throughout this period. The rapid response to a midsummer rainfall emphasizes the importance of plant water potential as a parameter controlling over-all metabolic activity. The seasonal variations of acid metabolism and gas exchange significantly influenced the efficiency of water use and carbon dioxide assimilation. Periods of maximal efficiency followed rainfall throughout the course of the year. PMID- 16658843 TI - The Influence of Nitrate and Chloride Uptake on Expressed Sap pH, Organic Acid Synthesis, and Potassium Accumulation in Higher Plants. AB - The influence of NO(3) (-) uptake and reduction on ionic balance in barley seedlings (Hordeum vulgare, cv. Compana) was studied. KNO(3) and KCl treatment solutions were used for comparison of cation and anion uptake. The rate of Cl(-) uptake was more rapid than the rate of NO(3) (-) uptake during the first 2 to 4 hours of treatment. There was an acceleration in rate of NO(3) (-) uptake after 4 hours resulting in a sustained rate of NO(3) (-) uptake which exceeded the rate of Cl(-) uptake. The initial (2 to 4 hours) rate of K(+) uptake appeared to be independent of the rate of anion uptake. After 4 hours the rate of K(+) uptake was greater with the KNO(3) treatment than with the KCl treatment, and the solution pH, cell sap pH, and organic acid levels with KNO(3) increased, relative to those with the KCl treatment. When absorption experiments were conducted in darkness, K(+) uptake from KNO(3) did not exceed K(+) uptake from KCl. We suggest that the greater uptake and accumulation of K(+) in NO(3) (-)-treated plants resulted from (a) a more rapid, sustained uptake and transport of NO(3) (-) providing a mobile counteranion for K(+) transport, and (b) the synthesis of organic acids in response to NO(3) (-) reduction increasing the capacity for K(+) accumulation by providing a source of nondiffusible organic anions. PMID- 16658844 TI - Gibberellic Acid-induced Phase Change in Hedera helix as Studied by Deoxyribonucleic Acid-Ribonucleic Acid Hybridization. AB - Applications of gibberellic acid to the mature form of Hedera helix induce morphological reversions to the juvenile form of growth. The juvenile forms produced are stable with time and differ dramatically from the mature in phenotype. DNA-RNA hybridization techniques have been used to study the RNA populations of juvenile, mature and gibberellic acid-treated mature apices. Hybridization competition experiments using RNA extracted by a hot phenol technique and uniformly labeled in vitro with (3)H dimethylsulfate show no qualitative differences between the species of RNA present in juvenile and mature apices. However, differences are observed in the frequency distribution of RNA species using both uniformly labeled or pulse-labeled RNA as a reference. RNA extracted from gibberellic acid-treated mature buds was a less effective competitor than control mature RNA and the difference observed was comparable to that observed between mature and juvenile RNA. These results indicate that at least part of the molecular basis of phase change and gibberellic acid action may involve an alteration in the rate of transcription of certain genes in the apices of the mature form.RNA extracted using the hot phenol procedure contained a fraction of rapidly labeled RNA which was not extractable with cold phenol. When RNA extracted only with cold phenol was used in competition experiments sequences unique to the juvenile were detected and sequences unique to the mature were not detected. Implications of these results in relation to possible post transcriptional control mechanisms are discussed. PMID- 16658845 TI - Protein kinase activity associated with isolated ribosomes from peas and lemna. AB - Protein kinase (ATP:protein phosphokinase) has been found associated with ribosomes prepared from 5-day-old etiolated pea shoots and sterile cultures of Lemna minor. The enzyme catalyzes the phosphorylation of ribosomal proteins in vitro and may be involved in the formation of phosphoproteins in the ribosomes in vivo. Protein kinase sediments with ribosomes during sucrose density gradient centrifugation through a buffer containing 0.02 m KCl. Seventy per cent of the enzymic activity dissociates from the ribosomes in 0.3 m KCl, but the remaining protein kinase remains firmly bound in 0.7 m KCl. Cyclic AMP does not modify the activity of these protein kinases in vitro. Benzyladenine inhibits the protein kinase, but only at high concentrations of this cytokinin. PMID- 16658846 TI - Effects of cation levels of the nutrient medium on the biochemistry of chlorella: I. Concentration series. AB - The effects of variations in nutrient cation levels on the growth and biochemistry of Chlorella were investigated. This study involved concentration series experiments in which the levels of Mg(2+), K(+), and Ca(2+) varied from deficiency to toxicity levels for growth. The nutrient sufficiency concentrations of Mg(2+) and K(+) were 0.08 and 0.10 meq/1, respectively. Deficiencies of Mg(2+) or K(+) reduced the growth rate, as well as cellular total nitrogen and unsaturated fatty acid levels. K(+) deficiency increased total lipid levels, while total fatty acids were unaffected. Increasing Mg(2+) or K(+) concentrations in the nutrient media were accompanied by corresponding increases in growth rate and certain biochemical fractions. Calcium was without effect except at a toxicity level. Cellular sufficiency concentrations for Mg(2+) and K(+) were 0.3 and 1.2% of the dry weight, respectively. PMID- 16658847 TI - Effects of Cation Levels of the Nutrient Medium on the Biochemistry of Chlorella: II. Factorial Experiment. AB - A factorial experiment was designed to study the effects of Mg(2+), K(+), and Na(+) on the growth and biochemistry of Chlorella sorokiniana. Raising Mg(2+) or K(+) concentration in the nutrient medium increased growth rates as well as total N levels and Mg(2+) and K(+) accumulation by the cells. The total N effect was Mg(2+)-dependent-if Mg(2+) was below a certain level in the medium-increasing the K(+) concentration did not raise the total N level of cells. Low nutrient levels of K(+) decreased the levels of unsaturated fatty acids (especially 18:1 and 18:3), while increasing the levels of palmitic acid (16:0), total fatty acids, and total lipid. Increasing nutrient K(+) concentrations were accompanied by increases in levels of some unsaturated fatty acids, with a concomitant reduction in 16:0, total fatty acids and total lipid. Low Mg(2+) levels in the nutrient medium reduced the cellular levels of palmitic acid, total fatty acids, total lipid, and certain unsaturated fatty acids (though this last effect also depended on the nutrient level of K(+)). These relationships indicate that Mg(2+) may be important in the initial steps of fatty acid synthesis, whereas K(+) may be necessary for the formation of certain unsaturated fatty acids. Variations in Na(+) concentration did not have any significant effect on the growth and biochemistry of C. sorokiniana. PMID- 16658848 TI - Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves. AB - Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH(2)PO(4) and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1). PMID- 16658849 TI - The Effect of Temperature on Photo-induced Carotenoid Biosynthesis in Neurospora crassa. AB - The temperature dependence of carotenoid synthesis in Neurospora crassa was investigated. The primary light reaction is independent of temperature, but the amount of carotenoid pigment which subsequently accumulates in the dark is strongly dependent on the temperature during the dark incubation. Carotenoid synthesis shows a sensitivity to both high and low temperatures, and of the temperatures tested, 6 C is optimal. Exposure to temperatures above 6 C for various times immediately following irradiation brings about a temperature dependent reduction in the amount of carotenoid pigment that is synthesized in a total dark incubation time of 24 hours. This sensitivity to incubations at temperatures above 6 C is reduced by either continuous irradiation during the entire time at the higher temperature or by a short irradiation at the end of this period, and the relative effectiveness of these two types of light treatments is presented. Carotenoid production is also sensitive to amino acid analogues and inhibitors of protein synthesis during a critical period after irradiation.It is proposed that the light reaction leads to the production of a compound which can be degraded in a temperature-dependent competitive reaction. This compound (or a product derived from it) can also induce the de novo synthesis of an enzyme (or enzymes) required for carotenoid production. An alternative hypothesis, that a repressor is directly inactivated in the light reaction, can be ruled out by the results presented. PMID- 16658850 TI - Structure and function of developing barley plastids. AB - Five different regions of the first foliage leaf of etiolated barley seedlings were studied with respect to leaf growth, plastid growth and replication, differentiation of etioplasts, and conversion of etioplasts into chloroplasts upon illumination. Ultrastructural changes of the plastids were correlated with chlorophyll synthesis and development of photosynthetic activity as measured by (14)CO(2) incorporation and O(2) evolution. The first foliage leaf has greater linear growth over a longer period of time in the dark than in the light. Only the bottom two regions (4 and 5) are still growing in the 5-day etiolated leaf. Region 4 grows by cell elongation, and region 5 grows by both cell division and elongation. Plastids in all five regions of the leaf are capable of enlarging when exposed to light. This is true both for the intact plant and for excised sections. Plastid replication occurs predominantly in the younger regions of the leaf (regions 3, 4, and 5). The amount of chlorophyll synthesized by different regions in the intact plant is significantly higher (3-40 times) than that made by excised sections. Ultrastructural changes occurring in each region when excised sections are illuminated were classified into five stages involving increased membrane synthesis and appression into grana, and these changes were correlated with the first appearance of photosynthetic activity. The earliest detectable photosynthetic activity occurs in region 1 after 2 hours of illumination when chloroplasts show only a few overlaps in the thylakoids. Plastids in younger regions of the leaf require up to 24 hours of light to form grana and develop photosynthetic activity. Plastids in each region of the leaf are in different stages of development when photosynthesis is initiated, indicating that development of photosynthetic activity is not strictly correlated with a certain stage of plastid development. Membrane appression is not indicative of photosynthetic activity since overlaps are formed in the dark, but it was always present when photosynthetic activity was detectable. Likewise, there does not appear to be any strict correlation between the presence of chlorophyll and membrane appression. These results show that the particular structural and functional correlations that can be made depend to a large degree on age of the tissue. PMID- 16658851 TI - Rapid Inhibition of Auxin-induced Elongation of Avena Coleoptile Segments by Cordycepin. AB - The effects of cordycepin (3'-deoxyadenosine), an RNA synthesis inhibitor, on auxin-induced elongation in Avena coleoptile segments were studied with a position-sensing transducer. Cordycepin rapidly inhibited auxin-stimulated growth in the coleoptile segments whether added before, at the same time as, or after, the 2 mum auxin treatment. Midcourse additions of 100, 50, and 25 mug/ml cordycepin inhibited auxin-promoted elongation in an average of 18, 22, and 35 minutes, respectively. Additions of cordycepin before or at the same time as the auxin treatment partially inhibited the magnitude of the subsequent auxin promoted growth but did not appreciably alter the latent period of the auxin response. It was concluded that if cordycepin is inhibiting the synthesis of RNA required for growth, the decay time for this RNA may be considerably shorter than that suggested in the literature from actinomycin D experiments. Preliminary kinetic evidence indicated that cordycepin does not inhibit auxin-induced elongation by acting as a respiratory inhibitor. Studies in mung bean shoot mitochondria demonstrated that cordycepin has no effect on respiration, respiratory control, or ADP/oxygen ratios. PMID- 16658852 TI - Phosphatidylglycerol synthesis in castor bean endosperm: kinetics, requirements, and intracellular localization. AB - The synthesis of phosphatidylglycerol in castor bean (Ricinus communis var. Hale) endosperm tissue was found to be located in both the endoplasmic reticulum and mitochondrial fractions separated on sucrose density gradients. The enzyme of both fractions attained maximum activity at 5 mm Mn(2+), 0.075% Triton X-100, and pH 7.3. The addition of dithiothreitol produced little effect, but sulfhydryl inhibitors reduced activity in both systems. Cytidine diphosphate-diglyceride exhibited an apparent Michaelis constant for the endoplasmic reticulum enzyme of 2.8 mum and for the mitochondrial enzyme of 2.0 mum; the maximum reaction rate was achieved at about 20 mum. For the second substrate, glycerol-phosphate, the apparent Michaelis constant for both fractions was about 50 mum and maximum velocity was reached at 400 mum. The specific activity of the mitochondrial enzyme was generally twice that of the endoplasmic reticulum. PMID- 16658853 TI - Efficiency and regulation of water transport in some woody and herbaceous species. AB - The efficiency with which plants transport water is related to the water potential differences required to drive water fluxes from the soil to the leaf. A comparative study of two woody and three herbaceous species (Citrus sinensis L. cv. Koethen, Pyrus kawakami L., Helianthus annuus L. cv. Mammoth Russian, Capsicum frutescens L. cv. Yolo Wonder, and Sesamum indicum L. cv. Glauca) indicated contrasts in water transport efficiency. Depression of leaf water potential in response to transpiration increases was found in the woody species; the herbaceous species, however, had more efficient water transport systems and presented no measurable response of leaf water potential to transpiration changes. Different maximum transpiration rates under the same climatic conditions were observed with different species and may be accounted for by stomatal response to humidity gradients between leaf and air. Leaf diffusion resistance in sesame increased markedly as the humidity gradient was increased, while leaf resistance of sunflower responded less to humidity. Stomata appeared to respond directly to the humidity gradient because changes in leaf water potential were not detected when leaf resistance increased or decreased. PMID- 16658854 TI - Water Permeability and Cold Hardiness of Cortex Cells in Cornus stolonifera Michx.-A Preliminary Report. AB - The relationship of freezing resistance to water permeability of cortex cells was studied in stems of red osier dogwood (Cornus stolonifera Michx.). Permeability was estimated by determining the diffusion flux of tritiated water from cortex slices previously equilibrated in tritiated water. Energy of activation and diffusion time comparisons of tritiated water flux from living cortex slices and slices killed by immersion in liquid N(2) verified that intact membranes of uninjured cortex cells limited water flux.Water permeability of living phloem and cortical parenchyma cells increased during the initial (photoperiodically induced) phase of cold acclimation. This accompanied an increase in hardiness from -3 to -12 C. Little if any further increase in permeability was noted during subsequent acclimation to below -65 C.Permeability measurements on nonhardy cortex samples yielded consistent results, but measurements on samples from hardy twigs were often difficult to reproduce. This unexplained variability precludes specific conclusions, but the tritiated water diffusion flux technique may provide an alternative to traditional plasmolytic techniques in studying water permeability in woody plant tissues. PMID- 16658855 TI - Boundary layers of air adjacent to cylinders: estimation of effective thickness and measurements on plant material. AB - Using existing heat transfer data, a relatively simple expression was developed for estimating the effective thickness of the boundary layer of air surrounding cylinders. For wind velocities from 10 to 1000 cm/second, the calculated boundary layer thickness agreed with that determined for water vapor diffusion from a moistened cylindrical surface 2 cm in diameter. It correctly predicted the resistance for water vapor movement across the boundary layers adjacent to the (cylindrical) inflorescence stems of Xanthorrhoea australis R. Br. and Scirpus validus Vahl and the leaves of Allium cepa L. The boundary-layer thickness decreased as the turbulence intensity increased. For a turbulence intensity representative of field conditions (0.5) and for nu(wind)d between 200 and 30,000 cm(2)/second (where nu(wind) is the mean wind velocity and d is the cylinder diameter), the effective boundary-layer thickness in centimeters was equal to [Formula: see text]. PMID- 16658856 TI - Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue. AB - Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-(14)C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3 acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.In the absence of 2,4-dinitrophenol, indoleacetic acid induced ethylene parallels the free indoleacetic acid level within the tissue. However, in the presence of 2,4-dinitrophenol the rate of ethylene production did not correlate with the free indoleacetic acid level. These results indicate that both indoleacetic acid-induced ethylene production and indoleacetic acid conjugation require a continuous supply of ATP, the formation of which was inhibited by 2,4-dinitrophenol. PMID- 16658857 TI - Multiple Forms and Intracellular Localization of Uridine Diphosphate Glucose Pyrophosphorylase in Avena sativa. AB - Uridine diphosphate glucose pyrophosphorylase was isolated separately from Avena sativa leaves, roots, and etiolated coleoptiles and purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography and polyacrylamide gel electrophoresis. There was no difference in the enzyme from the different tissue types with respect to properties exhibited during the purification procedure. A small portion of the enzyme from all three sources was found to be particulate when homogenized in aqueous sucrose media. Characterization of the particulate form by discontinuous sucrose density gradient showed the enzyme to be located at two different densities, one of which corresponded to chloroplasts in the leaves and plastids in the coleoptiles and roots. Homogenization and fractionation of the oat leaves using the nonaqueous media, hexane, and carbon tetrachloride, resulted in 50 to 60% of the enzyme being associated with chloroplasts and the remainder being associated with other membranous material. These data indicate that the enzyme from oat leaves, roots, and etiolated coleoptiles has multiple intracellular locations, and it is suggested that compartmentation of this enzyme may be a mechanism for regulation of uridine diphosphate glucose metabolism in oats. PMID- 16658858 TI - Peroxidases in Tobacco Abscission Zone Tissue: II. Time Course Studies of Peroxidase Activity during Ethylene-induced Abscission. AB - Ethylene-induced abscission in flower pedicels of Nicotiana tabacum L. cv. Little Turkish causes a progressive increase in peroxidase activity during the first 4 hours of a 5-hour time course ethylene treatment period, with decrease in peroxidase activity occurring between 4 hours and 5 hours, when the supernatant extracts of abscission zone segments are tested spectrophotometrically for peroxidase activity, using guaiacol and hydrogen peroxide. Nonethylene-treated tissue has a much lower level of peroxidase activity over the same time course period. In ethylene-treated tissue the decline in break-strength correlates with the beginning of increase in peroxidase activity (3 hours). When the abscission zone area of the pedicel is further divided into proximal, abscission zone, and distal portions, respectively, the ethylene-treated tissue has the highest peroxidase activity in the abscission zone portion, with the maximum peak occurring at 4 hours and decreasing between 4 hours and 5 hours. Acrylamide gel electrophoresis of enzyme breis from ethylene-treated aand nonethylene-treated plants reveals that no new peroxidase isozymes are formed in response to ethylene, indicating an increase in the amount of one or in both of the two already existing isozyme banding patterns. The measurement of protein in the proximal, abscission zone, and distal segments, over a 5-hour ethylene treatment period, indicates that it is being translocated in a distal to proximal direction in the abscission zone pedicel. The possible participatory role for peroxidase in ethylene-induced tobacco flower pedicel abscission are discussed. PMID- 16658859 TI - The development of isocitric lyase activity in germinating cotton seed. AB - In cotyledons of germinating cotton (Gossypium hirsutum L. var. Stoneville 213) seedlings, in the dark, isocitric lyase (EC 4.1.3.1) activity peaks after 2 days and thereafter slowly declines to a negligible value after 8 days. The maximum activity of this enzyme in cotyledons of 2-day-old seedlings was 16.2 mumoles of glyoxylate formed/15 min.10 cotyledon pairs. Actinomycin D at a concentration of 10 mug/ml, if added to the imbibing solution, completely prevents the development of isocitric lyase activity in these germinating seed. In cotyledons of germinating cotton seedlings, in the light, isocitric lyase activity peaks after 2 to 3 days and sharply declines to a negligible value after 4 days. The maximum activity of this enzyme in cotyledons of 2- to 3-day-old seedlings was 13.2 mumoles of glyoxylate formed/15 min.10 cotyledon pairs. Actinomycin D at a concentration of 10 mug/ml, if added to the imbibing solution, severely inhibits the development of enzyme activity.In germinating seed, in the light, the synthesis of chlorophyll and glyceraldehyde-3-P dehydrogenase is also limited by the addition of low concentrations of actinomycin D. The new synthesis of fructose-1, 6-diP aldolase, which is detectable after 1 to 2 days of germination, is inhibited by 10 mug/ml of actinomycin D. We, therefore, conclude that the synthetic events leading to the development of chlorophyll, some glyoxysomal and chloroplast enzymes in germinating cotton seedlings depend on newly transcribed mRNA. PMID- 16658860 TI - Influence of assimilate demand on photosynthesis, diffusive resistances, translocation, and carbohydrate levels of soybean leaves. AB - Rates of net photosynthesis and translocation, CO(2) diffusive resistances, levels of carbohydrates, total protein, chlorophyll, and inorganic phosphate, and ribulose 1,5-diphosphate carboxylase activity were measured in soybean (Glycine max L. Merrill) leaves to ascertain the effect of altered assimilate demand. To increase assimilate demand, the pods, stems, and all but one leaf (the "source leaf") of potted plants were completely shaded for 6 or 8 days and the responses of the illuminated source leaf were monitored. Rate of net photosynthesis in the source leaf of the shaded plants was found to increase curvilinearly to a maximum on the 8th day. The source leaf of the control plants (no sink shading) maintained a constant photosynthetic rate during this period. Vapor-phase resistance to CO(2) diffusion did not vary with treatment, but mesophyll (liquid phase) resistance was significantly lower in the source leaf of the shaded plants.Starch concentration in the source leaf of shaded plants decreased more than 10-fold during the 8-day shading period. In this same period, sucrose concentration rose nearly 3-fold. Conversely, in the source leaf of the unshaded plants, starch concentration remained high (23% of leaf dry weight) and sucrose concentration remained very low (1.2%). When measured on the 8th day of treatment, translocation rate, ribulose 1,5-diphosphate carboxylase activity, and inorganic phosphate concentration were found to be significantly higher in the source leaf of the shaded plants than in the control source leaf.When shaded plants were again illuminated, all measured response trends in the source leaf were reversed. These data indicate that assimilate demand has a marked influence on source-leaf photosynthesis and carbohydrate formation and export. PMID- 16658861 TI - Cellular and ultrastructural changes in mesophyll and bundle sheath cells of maize in response to water stress. AB - Ultrastructural changes were correlated with leaf water potential, relative water content, and abscisic acid levels in the leaf. Mesophyll cells were more prone to damage than bundle sheath cells at a leaf water potential of -18.5 bars. Tonoplast breakdown and cell disruption occurred in 25% of the mesophyll cells. On rewatering, these disrupted cells did not recover. In bundle sheath cells, starch, lost at about -13.5 bars leaf water potential, reappeared within 2.5 hours of rewatering. PMID- 16658862 TI - Evidence Against the Involvement of Galactosidase or Glucosidase in Auxin- or Acid-stimulated Growth. AB - Research on the mode of action of auxin in the promotion of growth has shown that auxin treatment leads to hydrogen ion secretion and wall acidification. It has recently been reported that auxin stimulates cell wall beta-galactosidase activity in Avena coleoptiles, presumably by causing cell wall acidification, since the pH optimum for the enzyme is about 5.0. It has been suggested that enhancement of beta-galactosidase and/or other glycosidase activity mediates growth promotion by auxin or low pH. This hypothesis was tested by examining the effect of inhibitors of beta-galactosidase and beta-glucosidase. Severe inhibition of measureable beta-galactosidase or beta-glucosidase activity was found to have no effect on auxin- or acid-promoted growth. It is concluded that neither beta-galactosidase nor beta-glucosidase plays an important role in short term growth promotion by auxin or acid. The data do not rule out the possibility that some other cell wall glycosidase is involved in auxin or acid action. PMID- 16658863 TI - Effects of indoleacetic Acid on dictyosomes of apical and expanding cells of oat coleoptiles. AB - We found that the auxin-induced growth is mediated through the activation of the dictyosomes (collectively, the Golgi apparatus). Incubation of oat (Avena sativa) coleoptile segments in indoleacetic acid-sucrose-phosphate buffer changes significantly the number of dictyosomes in the expanding cells. A further indication of auxin enhancement of dictyosome activity is a decrease in dictyosomal cisternae (flattened membranous sacs) number. This decrease occurred after 6 minutes of incubation in auxin, and then was followed by a reduction in the organelle number per se. These times are in keeping with the rapid action of auxin-induced cell elongaton, and the latent period of geotropism. In the apical cells, the effect of indoleacetic acid is more subtle and complex. The periods of increased dictyosome utilization and of increased dictyosome synthesis in auxin treated segments alter with those of the control. These observations indicate that dictyosomes not only have a function in cell elongation, but also may participate in processes such as auxin transport and stimuli perception. The expanding cells have five times as many dictyosomes as the cells in the apex. Dictyosome number within a cell appears to be directly proportional to the length of the cell. The fluctuation of dictyosome number and the effect of auxin on the rate of elongation of individual outer epidermis are discussed. PMID- 16658864 TI - Dose-response curves for radish seedling phototropism. AB - Radish seedlings (Raphanus sativus L.) were grown for 4 days in complete darkness, or in white light, or for 3 days in darkness followed by 1 day of red light. Phototropic dose-response curves for the seedlings grown in these three ways were determined with 460-nm light. The dark-grown and red light-treated seedlings responded with positive curvatures of no more than 10 degrees to energy doses in the first positive range and with larger positive curvatures in the second positive dose range. No indifferent or negative curvature was seen with the light intensity used. White light-grown seedlings did not respond to first positive energy doses, but responded as strongly to second positive doses as the other types of seedlings. PMID- 16658865 TI - The effect of molecular size, concentration in nutrient solution, and exposure time on the amount and distribution of polyethylene glycol in pepper plants. AB - Pepper plants Capsicum annuum L. var. California Wonder were grown in nutrient solutions of either -3.0 or -5.0 bars osmotic potential, using polyethylene glycol with molecular weights of 400, 600, 1000, 1540, or 4000 as osmotica. Polyethylene glycol with molecular weights of 1000 or 1540 proved most satisfactory as osmotica to decrease the water potential of nutrient solutions.There was no relationship between the small amount of polyethylene glycol accumulated in the plants and the amount of water transpired. The concentration of polyethylene glycol in the expressed sap of the leaves and the total accumulated was inversely related to molecular weight of polyethylene glycol, was greater at lower osmotic potential of nutrient solution, and increased with time in solution. Except for plants grown in polyethylene glycol 4000, there was more polyethylene glycol in leaves than roots. The indications were that, when the concentration of polyethylene glycol reached a value of 1 to 2 mg per ml, any additional quantity absorbed was transferred to the leaves. The major proportion of polyethylene glycol 4000 absorbed was retained in the roots.The results of Sephadex gel chromatographs showed that the passage of polyethylene glycol through the plants did not alter the average molecular weight. This indicated that there was no selective absorption of small molecules that might be present as contaminates in the commercial product. PMID- 16658866 TI - Role of mitochondria in the origin of chloroplast starch grains: description of the phenomenon. AB - By phase microscopic observation of living palisade parenchyma cells in sections of Nicotiana excelsior leaves from plants previously placed in the dark for 72 hours, 30 to 45 minutes of light is found to induce mitochondria to remain stationary within the concavity of the chloroplasts and become round. Extending the illumination period to 60 to 90 minutes causes the stationary mitochondria in the concavity to change from a translucent to an opaque appearance, the change coinciding with the first appearance of starch as detected by blue staining of the grains with I(2)-KI. It is speculated that an interaction bearing some resemblance to the previously described interaction between mitochondria and the mobile phase of the chloroplasts may also operate in the starch grain phenomenon. PMID- 16658867 TI - Plant growth retardants as inhibitors of sterol biosynthesis in tobacco seedlings. AB - Three plant-growth retardants 2'-isopropy1-4'-(trimethylammonium chloride)-5 methylphenylpiperidine carboxylate (Amo 1618), beta-chloroethyltrimethylammonium chloride, and tributyl-2, 4-dichlorobenzylphosphonium chloride were tested for their effects on sterol production in, and growth of tobacco (Nicotiana tabacum) seedlings. As the concentration of each retardant increased, there was an increased inhibition of the incorporation of dl-2-(14)C-mevalonic acid into sterol (particularly desmethylsterol) fractions and an increased retardation of stem growth. Growth retardation was observed with both single and repeated retardant treatments, and with Amo 1618, in particular, a close quantitative relationship between inhibition of sterol biosynthesis and stem growth was obtained. Gibberellic acid completely overcame retardant effects and application of sterols also restored normal growth. It is concluded that the concept of causality in the relationship between growth retardation and gibberellin biosynthesis is probably premature, since growth retardants have a more general inhibitory action on isoprenoid biosynthesis in plants. PMID- 16658868 TI - Effects of host-specific toxins on electropotentials of plant cells. AB - Host-specific toxins from Helminthosporium victoriae (HV) and Periconia circinata (PC) caused gradual decreases in the negative electropotentials of single cells of susceptible but not of resistant plants. When tissues were held in a standard nutrient solution, the decrease (depolarization) induced by HV toxin was approximately 50 mv/hr; the decrease induced by PC toxin was even more gradual. Changes in ion efflux were detected before changes in electropotential. In contrast, toxin from H. carbonum caused a rapid but transient increase in negative electropotential of cells. Carbonyl cyanide m-chlorophenylhydrazone, which (like other metabolic inhibitors) blocks electrogenic pumps, caused cell electropotentials to decrease by approximately 50 mv within a few minutes. This suggests that HV and PC toxins do not have direct effects on electrogenic pumps, but do affect passive efflux of ions, or electrically neutral ion exchange systems, across the plasma membrane. PMID- 16658869 TI - Phosphate absorption rates and adenosine 5'-triphosphate concentrations in corn root tissue. AB - The correlations between ATP concentration in corn (Zea mays) root tissue and the rate of phosphate absorption by the tissue have been examined. Experimental variation was secured with 2,4-dinitrophenol, oligomycin, mersalyl, l-ethionine, 2-deoxyglucose, N(2) gassing and inhibition of protein synthesis. It is concluded that ATP could be the energy source for potassium phosphate absorption, but only if the transport mechanism possesses certain properties: oligomycin-sensitivity; creation of a proton gradient susceptible to collapse by uncouplers; phosphate transport via a mersalyl-sensitive Pi(-)-OH(-) transporter; good activity at energy charge as low as 0.4; short enzymatic half-life for the ATPase or phosphate transporter; a linked mechanism for K(+)-H(+) exchange transport, possibly electrogenic. PMID- 16658870 TI - Concentrations of Indole-3-acetic Acid and Its Esters in Avena and Zea. AB - An isotope-dilution method has been developed for the assay of free indole-3 acetic acid and ester indole-3-acetic acid as measured by indole-3-acetic acid liberated by mild alkaline hydrolysis. Application of this method to seedlings of Avena sativa and Zea mays indicates the upper limit of free indole-3-acetic acid in Avena to be about 16 mug per kg and in Zea, about 24 mug. The amount of 1 n alkali-labile indole-3-acetic acid in Zea is about 330 mug per kg and there is very little 1 n alkali-labile IAA in Avena. A chemical characterization of the indole-3-acetic acid of Avena and a confirmation of the chemical characterization of the indole-3-acetic acid of Zea is presented. PMID- 16658871 TI - The isolation and partial characterization of a membrane fraction containing phytochrome. AB - If 4-day-old dark-grown zucchini squash seedlings (Cucurbita pepo L. cv. Black Beauty) are exposed briefly to red light, subsequent cell fractionation yields about 40% of the total extractable phytochrome in the far red-absorbing form bound to a particulate fraction. The amount of far red-absorbing phytochrome in the pellet is strongly dependent on the Mg concentration in the extraction medium. The apparent density of the Pfr-containing particles following sedimentation on sucrose gradients corresponds to 15% (w/w) sucrose with 0.1 mm Mg and 40% sucrose with 10 mm Mg. This particulate fraction could be readily separated from mitochondria and other particulate material by taking advantage of these apparent density changes with changes in Mg concentration. Electron microscopy of negatively stained preparations shows that with 1 mm Mg only minute particles are present. These were too small to reveal structural detail with this technique. With 3 mm Mg, separate membranous vesicles between 400 and 600 Angstroms in diameter appear. At higher Mg concentrations, the vesicles aggregate, causing obvious turbity. The effect of Mg on vesicle formation and aggregation is completely reversible. Above 10 mm Mg, vesicle aggregation persists, but the percentage of bound Pfr decreases. PMID- 16658872 TI - In Vivo Properties of Membrane-bound Phytochrome. AB - After a 3-minute irradiation with red light, which saturates the phototransformation from the red light-absorbing form of phytochrome to the far red light absorbing form of phytochrome, about 40% of the phytochrome extractable from hooks of etiolated squash seedlings (Cucurbita pepo L. cv. Black Beauty) can be pelleted as Pfr at 17,000g after 30 minutes. Dark controls yield only 2 to 4% pelletable phytochrome in the form Pr. If a dark period intervenes between red irradiation and extraction, the bound Pfr gradually loses its photoreversibility. The time course for this destruction parallels the time course for phytochrome destruction in vivo following saturating red irradiation. The soluble fraction of phytochrome remains constant. These results suggest that in squash seedlings phytochrome destruction is related exclusively to the fraction which becomes membrane-bound. The induction of phytochrome binding by red light is not completely reversible by far red. In plants given saturating red followed immediately by saturating far red light, 12% of the phytochrome is found in the bound fraction as Pr if the phytochrome extraction is immediate. If a dark period intervenes between red-far red treatment and extraction, the bound phytochrome is released within 2 hours. A model of the binding properties of phytochrome, based on molecular interaction at the membrane is proposed, and possible consequences for the mechanism of action of phytochrome are discussed. PMID- 16658873 TI - Developmental changes in endosperm of germinating castor bean independent of embryonic axis. AB - Dry castor bean (Ricinus communis) seeds were cut transversely into halves and the half without the embryonic axis was placed in moist vermiculite at 30 C for 5 days. The development of the endosperm in the half-bean was found to be qualitatively similar to that in the whole seedling in the appearance of various enzymes of gluconeogenesis, the accumulation of glucose and sucrose as the end products of fat utilization, and the development of subcellular structure. It is concluded that during germination of castor bean, the embryonic axis does not directly control the developmental changes in the endosperm. PMID- 16658874 TI - Rhythmic potassium flux in albizzia: effect of aminophylline, cations, and inhibitors of respiration and protein synthesis. AB - Rhythmic leaflet movement in Albizzia is controlled by rhythmic K(+) flux in pulvinal motor cells. The angle assumed by darkened leaflets during the open phase of the rhythm can be altered by various compounds and changes in temperature; such treatments are ineffective during the closed phase. In all cases, effects on leaflet angle are correlated with and probably a consequence of K(+) flux in pulvinal motor cells. Incubation at low temperature (6C) or on sodium azide (1.0 mm) reduces K(+) in the ventral motor region and increases K(+) in the dorsal motor region, thereby decreasing leaflet angle. Incubation on cycloheximide (0.1 mm) or sodium acetate (0.05 m) inhibits protein synthesis; if the incubation period immediately precedes the opening phase, these compounds prevent both K(+) flux into the ventral motor cells and leaflet opening. Magnesium nitrate (0.05 m), supplied after leaflets have started to open, promotes K(+) secretion from the dorsal motor cells and increases the angle of opening.The data support the following hypothesis. Active K(+) transport into the ventral motor cells and out of the dorsal motor cells leads to opening; K(+) leakage in the opposite directions causes closure; and the interaction of these processes results in a rhythmic oscillation. Proteins in the ventral cell membranes that are required for active transport or membrane integrity turn over rhythmically and are resynthesized before opening. The availability of divalent cations determines the phase relationships between the K(+) rhythms in the dorsal and ventral motor cells.White light phases the rhythm. The "light on" signal turns on a K(+) secreting pump in the dorsal motor cells, while the "light off" signal initiates a period of protein synthesis which in turn leads to active K(+) transport into the ventral motor cells. Aminophylline (0.1-6.0 mm) inhibits white light-promoted opening and nyctinastic closure. PMID- 16658875 TI - Effect of red light on coleoptile growth. AB - The effects of red light in reducing the growth of the oat (Avena sativa L.) coleoptile and the synthesis of auxin in the coleoptile tip are detectable 2 hours after treatment and become more pronounced with time. When the coleoptile tip is supplied with additional tryptophan the synthesis of auxin is doubled both in darkness and when exposed to red light. Treatment of the tip with gibberellic acid or pyridoxal phosphate overcomes the reduction of auxin synthesis caused by red light. The uptake of exogenous indoleacetic acid, at pH 6.5, by coleoptile tissue is doubled by exposure to red light. The effect of red light on coleoptile growth appears to be mediated by phytochrome in the cell membrane which delocalizes the tryptophan utilized for auxin synthesis. PMID- 16658876 TI - Hormone-solute interactions in the lettuce hypocotyl hook. AB - The hypocotyl hook of the lettuce (Lactuca sativa cv. Grand Rapids) seedling is stimulated to a high degree of curvature through a synergistic interaction of ethylene and gibberellic acid in the light. Presentation of various inorganic salts to the seedlings caused extensive alteration of the hormone-induced curvatures, with ammonium and sulfate being the most stimulatory of curvature, and potassium and carbonate being the most inhibitory of curvature. Experiments using organic buffers indicated that the effect was not a pH response. The abilities of various cations and anions to alter the hormonally regulated curvature is suggested as further evidence of solute alteration of hormonal effectiveness. The interpretation is offered that the solutes may be influencing hormonal effectiveness through salting-in and salting-out effects on macro molecules such as proteins. PMID- 16658877 TI - The Metabolism of Oat Leaves during Senescence: I. Respiration, Carbohydrate Metabolism, and the Action of Cytokinins. AB - When the detached first leaves of green or etiolated oat (Avena sativa cv. Victory) seedlings senesce in the dark, their oxygen consumption shows a large increase, beginning after 24 hours and reaching a peak of up to 2.5 times the initial rate by the 3rd day. This effect takes place while the chlorophyll of green leaves, or the carotenoid of etiolated leaves, is steadily decreasing. Kinetin, at a concentration which inhibits the decrease in pigment, completely prevents the respiratory rise; instead, the oxygen consumption drifts downwards. Lower kinetin concentrations have a proportional effect, 50% reduction of respiration being given by about 0.1 mg/l. About one-fifth of the respiratory rise may be attributed to the free amino acids which are liberated during senescence; several amino acids are shown to cause increases of almost 50% in the oxygen consumption when supplied at the concentrations of total amino acid present during senescence. A smaller part of the rise may also be due to soluble sugars liberated during senescence, largely coming from the hydrolysis of a presumptive fructosan. The remainder, and the largest part, of the increase is ascribed to a natural uncoupling of respiration from phosphorylation. This is deduced from the fact that dinitrophenol causes a similar large rise in the oxygen consumption of the fresh leaves or of leaf segments kept green with kinetin, but causes only a very small rise when the oxygen consumption is near its peak in senescent controls. The respiration of these leaves is resistant to cyanide, and 10 mm KCN even increases it by some 30%; in contrast, etiolated leaves of the same age, which undergo a similar rise in oxygen consumption over the same time period, show normal sensitivity to cyanide. The respiratory quotient during senescence goes down as low as 0.7, both with and without kinetin, though it is somewhat increased by supplying sugars or amino acids; glucose or alanine at 0.3 m bring it up to 1.0 and 0.87, respectively.N(6) Benzylaminopurine and Delta-2-isopentenylaminopurine act similarly to kinetin in repressing the respiratory rise, the former being five times as active as kinetin, while the latter has only 1% of the activity of kinetin. Zeatin also powerfully prevents senescence. Because the repression of the respiratory rise is shown by each cytokinin at the concentration at which it inhibits senescence, the action is ascribed in both cases to the maintenance of a tight coupling between respiration and phosphorylation. It is pointed out that such an effect would explain many features of cytokinin action.A change in the methodology of the senescence experiments is described and compared with the method previously used, and the influence of temperature and age of the plants on the course of leaf senescence are presented in detail. PMID- 16658878 TI - Promotion of seed germination by nitrate, nitrite, hydroxylamine, and ammonium salts. AB - Action and uptake of azides, nitrates, nitrites, hydroxylamines, and ammonium salts were measured on germination of Amaranthus albus, Lactuca sativa, Phleum pratense, Barbarea vulgaris, B. verna, and Setaria glauca seeds. Nitrate and nitrite reductase activities were measured in vivo for each of these kinds of seeds. Activities were measured in vitro for catalase, peroxidase, glycolate oxidase, and pyridine nucleotide quinone reductase on extracts of A. albus and L. sativa seeds before and after germination. The enzymic activities measured and the responsiveness of the haemproteins to inhibition by the several compounds indicate that nitrites, azides, and hydroxylamines promote seed germination by inhibition of H(2)O(2) decomposition by catalase. Ammonium salts showed pronounced promotive activity only for B. verna and B. vulgaris seeds, for which they served as metabolic substrates.The promotion of germination is thought to depend on coupling of peroxidase action to NADPH oxidation, which can regulate the pentose pathway of d-glucose 6-phosphate use. Pyridine nucleotide quinone reductase is the possible coupling enzyme. This enzyme and others required for the action are present in the seeds before imbibition of water. PMID- 16658879 TI - Polarity of Thiamine Movement through Tomato Petioles. AB - Thiamine-(14)C moved through petiolar sections of Lycopersicon esculentum Mill. var. Michigan State Forcing with striking similarity in kinetics to auxins and gibberellic acid moving through similar sections of other green plants. Thiamine moved with strong basipetal polarity, at a velocity of 3 to 5 mm per hour, and emerged unchanged into the basal receiver agar block, judging by chromatography. This lends support to the hypothesis that polar movement is a property of several classes of plant hormones, rather than being restricted to the auxins (as previously believed). PMID- 16658880 TI - Comparative Enzymology of the Glyceraldehyde 3-Phosphate Dehydrogenases from Pisum sativum. AB - Glyceraldehyde 3-phosphate dehydrogenases (EC 1.2.1.12 and 1.2.1.13) have been purified from the seed, root, etiolated, and green shoot of peas (Pisum sativum). These enzymes are tetramers of 140,000 daltons, with subunits of 35,000 daltons. The enzymes differ in isoelectric point. The seed enzyme has a pI of 5.1, and the root enzyme has a pI of 4.5. The cytoplasmic enzyme from etiolated shoots is slightly acidic with a pI of 5.7 to 6.1 and is found in two separable forms. The chloroplast enzyme (from green shoots) is most basic with a pI of 8.0.In immunodiffusion experiments, the seed, root, and cytoplasmic enzymes of the etiolated shoot share antigenic homology, while the chloroplast enzyme does not cross react antigenically with the extra-chloroplast enzymes. The antiserum to the pea chloroplast enzyme did, however, cross react with glyceraldehyde 3 phosphate dehydrogenase purified from the spinach chloroplast. Therefore, the chloroplast enzyme is significantly different from the extra-chloroplast enzymes with respect to primary sequence.The NADP analog phosphoadenosine diphosphoribose showed competitive inhibition to the chloroplast enzyme with either pyridine nucleotide. The NAD analog pyridine 3-aldehyde NAD was competitive with respect to the NAD activity but was hyperbolic competitive in the presence of NADP, indicating a complexity in the binding of pyridine nucleotide to the chloroplast enzyme. PMID- 16658881 TI - Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm. AB - Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur. PMID- 16658882 TI - Effect of chilling temperatures on the activities of glyoxysomal and mitochondrial enzymes from castor bean seedlings. AB - Castor bean (Ricinus communis L.) is included among the group of plants sensitive to chilling temperatures. Seedlings of this species were shown to exhibit visible symptoms of this injury, as well as impaired radicle growth and storage product mobilization. Respiration of intact seedlings and oxidation of succinate by isolated mitochondria displayed discontinuities in Arrhenius plots of their reaction velocities, characteristic of chilling species. However, gluconeogenic glyoxysomal enzymes do not display such discontinuities, indicating that there is probably no functional relationship between these enzymes and the glyoxysomal membrane. PMID- 16658883 TI - Assay and Biochemical Properties of the Proteinase Inhibitor-inducing Factor, a Wound Hormone. AB - An assay has been developed for the proteinase inhibitor-inducing factor (PIIF), a wound hormone. PIIF is present in tomato (Lycopersicum esculentum var. Bonnie Best) leaf extracts and induces accumulation of proteinase Inhibitor I when the extracts are supplied briefly to excised leaves that are subsequently incubated in water under constant light. An active water-soluble crude PIIF solution was conveniently prepared from autoclaved and lyophilized tomato leaves. Accumulation of Inhibitor I, induced by crude PIIF, is linear, commencing at about 8 to 10 hours after feeding and continues for several hours. Evidence is presented that the PIIF-induced accumulation of Inhibitor I, determined immunologically, is accompanied by the accumulation of other trypsin and chymotrypsin inhibitors, determined enzymatically. The accumulation of Inhibitor I is inhibited by actinomycin D and cycloheximide but not by chloramphenicol or rifampin. PIIF cannot be replaced by traumatin, indoleacetic acid, gibberellic acid, kinetin, ethylene, or abscisic acid. PIIF activity was not destroyed by incubation with a number of proteolytic, carbohydrase, phosphatase, or pyrophosphatase enzymes. The active substance is insoluble in lipid solvents. PMID- 16658884 TI - beta-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem. AB - Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. beta-1,3- and beta-1, 4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 mum) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mm) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of beta-1, 4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, beta 1, 4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of beta-1, 3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations. PMID- 16658885 TI - Inter-Tissue Correlations in Organ Fragments: Organogenetic Capacity of Tissues Excised from Stem Segments of Torenia fournieri Lind Cultured Separately in Vitro. AB - In order to study the effect of inter-tissue correlations on the organogenetic capacities of various tissues of stem segments of Torenia fournieri Lind, different types of explants were excised and grown separately: epidermis, subepidermal parenchyma, epidermis plus subepidermal parenchyma but devoid of vascular tissue and stem segments devoid of epidermis.The epidermis, normally capable of bud formation in the context of a stem segment, dies if grown in culture alone; however, it can form buds after callus formation if, after excision, it is replaced on the original stem segment. Subepidermal parenchyma, normally mitotically inactive in the stem segment, forms roots when grown alone and buds and roots when grown with the epidermis. A stem segment devoid of epidermis forms only roots. The histological study showed that, in explants composed of epidermal and a few subepidermal parenchyma layers, buds can arise exclusively from the epidermis or from both epidermal and subepidermal cells, whereas roots are exclusively formed by parenchyma tissue.The break in correlations among tissues by the culture of each tissue separately seems to "liberate" masked capacities in certain tissues and to repress the expression of capacities in others. PMID- 16658886 TI - Separation and partial characterization of multiple ribonucleic Acid polymerases from soybean hypocotyl. AB - Two major peaks of RNA polymerase activity have been routinely separated by diethylaminoethyl cellulose chromatography following solubilization from soybean (Glycine max L. var. Wayne) chromatin. The relative amounts of these two peaks depend upon the manner in which the chromatin is purified. Pelleting the chromatin through dense sucrose solutions results in not only a loss of total solubilized RNA polymerase activity but also a selective loss of the alpha amanitin-sensitive form of the enzyme. Peak I elutes from a diethylaminoethyl cellulose column at a KCl concentration of approximately 0.27 m, is insensitive to alpha-amanitin and rifamycin, and has Mg(2+) + Mn(2+) optima of 5 mm and 1.25 mm, respectively. The enzyme is inhibited by KCl concentrations of about 0.03 m or greater. Peak II elutes from the column at a KCl concentration of approximately 0.35 m, is sensitive to alpha-amanitin, insensitive to rifamycin, and has Mg(2+) + Mn(2+) optima of 2 mm and 1.0 mm, respectively. Activity is inhibited by KCl concentrations of about 0.06 m or greater. Both enzymes prefer denatured calf thymus DNA, but peak II exhibits a stronger preference. PMID- 16658887 TI - Separation and partial characterization of two ribonucleic Acid polymerases from pea seedlings. AB - Two DNA-dependent RNA polymerases (ribonucleoside triphosphate:RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from pea (Pisum sativum) seedlings. The enzymes were solubilized by sonication in high salt buffer and were separated by chromatography on diethylaminoethyl cellulose using a linear salt gradient. Polymerase I eluted at 0.10 m (NH(4))(2)SO(4), accounted for about 10% of the recovered activity and was completely insensitive to alpha-amanitin. Polymerase II eluted at 0.14 m (NH(4))(2)SO(4), accounted for the remaining 90% of recovered activity and was strongly inhibited by alpha-amanitin. Both enzymes preferred denatured to native DNA as template, both showed an absolute requirement of divalent cation, and both were sensitive to the ionic strength of the assay medium. The developing pea seedling seems a promising system for studies of possible changes in relative activities and roles of multiple RNA polymerases during eukaryotic development. PMID- 16658888 TI - The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondria. AB - The oxidation of malate, citrate, and alpha-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD(+) to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD(+) were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD(+). PMID- 16658889 TI - Immunological and biochemical studies on isozymes of malate dehydrogenase and citrate synthetase in castor bean glyoxysomes. AB - Rabbit gamma-globulin obtained after the injection of solubilized proteins of castor bean (Ricinus communis L var. Hale) glyoxysomes contains antibodies against some of the glyoxysomal enzymes. The gamma-globulin was shown to inhibit by 50% and 80%, respectively, the in vitro activities of the castor bean glyoxysomal citrate synthetase and malate dehydrogenase. The conditions required for the inactivation are described.The glyoxysomal and mitochondrial citrate synthetase from castor bean endosperm show no significant difference in the above immunological test, in their apparent Michaelis constant values for acetyl coenzyme A and oxaloacetate, or in their sensitivity toward ATP inhibition. In the immunological test, glyoxysomal malate dehydrogenase from castor bean endosperm appeared to be related more closely to the leaf peroxisomal malate dehydrogenase from spinach (Spinacia oleracea L.) and to the glyoxysomal malate dehydrogenase from a variety of other fatty seedlings than to the castor bean mitochondrial or soluble malate dehydrogenase. Unlike the mitochondrial or soluble malate dehydrogenase, the castor bean glyoxysomal malate dehydrogenase is easily inactivated by gentle heat treatment, as was reported previously by other workers for the leaf peroxisomal malate dehydrogenase. Thus, the malate dehydrogenases from various plant microbodies share some common characteristics which distinguish them from other isozymes of malate dehydrogenase. PMID- 16658890 TI - myo-Inositol 1-Phosphate Synthase Inhibition and Control of Uridine Diphosphate-d glucuronic Acid Biosynthesis in Plants. AB - Of the eight intermediates associated with the two pathways of UDP-d-glucuronic acid biosynthesis found in plants, only d-glucuronic acid inhibited myo-inositol 1-phosphate synthase (EC 5.5.1.4), formerly referred to as d-glucose 6-phosphate cycloaldolase. Inhibition was competitive. An attempt to demonstrate over-all reversibility of the synthase indicated that it was less than 5% reversible, if at all. PMID- 16658891 TI - Failure to Detect Cyclic 3', 5'-Adenosine Monophosphate in Healthy and Crown Gall Tumorous Tissues of Vicia faba. AB - Attempts were made to provide proof for the occurrence of cyclic 3',5'-adenosine monophosphate in healthy and crown gall tissues of Vicia faba. Although our purified extracts gave positive readings in the Gilman binding assay for cyclic AMP, they were not digested by a specific cyclic 3',5'-adenosine monophosphate phosphodiesterase from beef heart. The extracts were digested, however, by a partially purified cyclic nucleotide phosphodiesterase from carrot tissue, which attacks both cyclic 2',3'- and 3',5'-nucleotides. The data indicate that the substances detected in the V. faba extracts are perhaps cyclic 2',3'-nucleotides, a possible RNA degradation product. PMID- 16658892 TI - Kaempferol inhibitions of corn mitochondrial phosphorylation. AB - Kaempferol (3, 5, 7, 4'tetrahydroxyflavone) inhibited the rate of state 3 substrate oxidation, but not the state 4 rate. This, along with the kaempferol inhibition of substrate-driven calcium-phosphate deposition, provided evidence that kaempferol was acting specifically on the phosphorylation mechanism and not on electron transfer. Kaempferol, however, did not inhibit ATP-driven contraction while oligomycin did. Comparisons of kaempferol with mersalyl indicated that kaempferol did not inhibit phosphorylation by blocking phosphate transport. Both kaempferol and 2,4-dinitrophenol inhibited calcium-phosphate transport, but kaempferol did not stimulate respiration to the extent that 2,4-dinitrophenol did under acceptorless conditions. Kaempferol had no effect on NADH-driven contraction in a potassium chloride reaction medium. The site of kaempferol effect is thus seen to be unique from oligomycin and more like aurovertin, likely acting before the formation of the phosphorylated high energy intermediate, but not as an uncoupler in the traditional 2,4-dinitrophenol mode. PMID- 16658893 TI - Effects of magnesium deficiency on the photosynthesis and respiration of leaves of sugar beet. AB - The effects of Mg deficiency on the photosynthesis and respiration of sugar beets (Beta vulgaris L. cv. F58-554H1) were studied by withholding Mg from the culture solution and by following changes in CO(2) and water vapor exchange of attached leaves. Leaf blade Mg concentration decreased from about 1200 to less than 200 meq kg(-1) dry matter without change in the rate of photosynthetic CO(2) uptake per unit leaf area, while from 200 to 50 meq kg(-1) the rate decreased to one third. Rates of photorespiratory evolution of CO(2) into CO(2)-free air responded to Mg like those of photosynthetic CO(2) uptake, the rates decreasing to one half, below 200 meq kg(-1). Respiratory CO(2) evolution in the dark increased almost 2-fold in low Mg leaves. Magnesium deficiency had less effect on leaf (mainly stomatal) diffusion resistance (r(1)) than on mesophyll resistance (r(m)); in Mg-deficient plants r(m) increased from 2.9 to 7.1 sec cm(-1), whereas r(1) became significantly greater than the control value only in the most severe instances of Mg deficiency. PMID- 16658894 TI - The occurrence and nature of ornithine carbamoyltransferase in senescing apple leaf tissue. AB - Ornithine carbamoyltransferase (EC 2.1.3.3) activity was detected in apple (Pyrus malus L.) leaf tissue from early June to November. Total activity remained relatively constant at 4.1 mumoles citrulline produced per hour per 10 cm(2) until mid-October when it sharply doubled. Following the first frost of the autumn, the enzyme lost about 80% of its former activity. The enzyme from apple leaf exhibited two pH optima, one at pH 8.6 and the other at pH 7.8, indicating the presence of isozymes or two forms of the enzyme. At pH 8.6, a partially purified enzyme preparation had binding contrasts for its substrates of 6 mm for carbamyl-phosphate and 4.8 mm for ornithine. At pH 7.8, the Km for carbamyl phosphate was 1.9 mm and the Km for ornithine was 1.22 mm. PMID- 16658896 TI - Nuclear magnetic resonance of water in cold acclimating red osier dogwood stem. AB - The pulsed and continuous-wave nuclear magnetic resonance of water in cold acclimating red osier dogwood (Cornus stolonifera Michx) stem showed reduced relaxation times and increased line width. The reduction of relaxation times suggests an over-all restriction in the motional characteristics of the water. The increased line width is not related to a molecular property of the water, but is useful in estimating the initiation of cold acclimation. Biphasic relaxation characteristics may be related to partitioning of the water at the cellular level. The liquid water content of the stem was a weak function of temperature between -25 and -55 C, corresponding to approximately 0.15 gram of water per gram of dry stem. The quantity of unfrozen water at subfreezing temperatures was not strongly dependent on the degree of cold acclimation. It is concluded that the ability of dogwood to survive low temperatures depends on its ability to tolerate diminished quantities of liquid water. PMID- 16658895 TI - Biosynthesis of grana and stroma lamellae in spinach. AB - Park and co-workers (Annu. Rev. Plant Physiol. 22: 395-430) have suggested that stroma lamellae, which perform only photosystem I contain a developing photosystem II which becomes functional upon the folding of these membranes to form grana stacks.The present study was undertaken to test this hypothesis. The lipid and protein constituents of chloroplast membranes were pulse-labeled radioactively with (14)C, and the specific activity of stroma and grana components were studied in a pulse-chase experiment. The components of both grana and stroma lamellae become labeled and decay at similar rates which suggests that the Park and Sane hypothesis is not correct. The results also show large differences in the turnover rates of some chloroplast membrane components supporting the multistep model for membrane synthesis and maintenance in this organelle. PMID- 16658897 TI - Energy Metabolism of Rumex Leaf Tissue in the Presence of Senescence-regulating Hormones and Sucrose. AB - Hormones which inhibit senescence of Rumex leaf tissue in the dark include gibberellin A(3), and the cytokinins 6-benzylamino purine and zeatin. These hormones inhibit respiratory metabolism in this tissue, but do not change the pattern or total amount of oxygen consumption during senescence. Abscisic acid, a senescence accelerator, correspondingly stimulates oxygen consumption. This correlation of senescence rate and respiration rate holds with regard to the hormone concentrations effective and the continued activity of the hormones when added after the lag phase of chlorophyll breakdown. Transfer experiments show that the respiratory inhibition due to gibberellin A(3) and the promotion due to abscisic acid become established within 3 hours of hormone addition. When gibberellin A(3) and zeatin were rapidly added to narrow strips of tissue, no inhibitions of oxygen uptake were observed in the first 12 minutes. Senescence inhibiting concentrations of sucrose strongly stimulate respiratory meabolism, raise the respiratory quotient, and cause inhibition of chlorophyll and protein breakdown which is distinct from the effect of gibberellins or cytokinins.It is proposed that the gibberellins and cytokinins may act by inhibiting a rate limiting process in the metabolism of starving leaf tissue. This rate-limitation hypothesis envisions a possibly subtle action of the hormone which retards but does not alter the pattern of macromolecular changes during cell autolysis. PMID- 16658898 TI - Enhanced inflorescence development in bougainvillea "san diego red" by removal of young leaves and cytokinin treatments. AB - Removal of young leaves and application of the cytokinin, N-benzyla-alpha (tetrahydro-2H-pyran-2yl)-adenine promote inflorescence development in Bougainvillea "San Diego Red". Defoliation greatly increased the amount of assimilate accumulated at the shoot tip 1 to 2 days after treatment. Cytokinin applications further increased the amount accumulated and this increase was apparent 4 days before morphological changes could be detected at the inflorescence axes. Short days promoted inflorescence development and also increased assimilate accumulation at the reproductive axes; thus, it is suggested that the role of short day induction in bougainvillea may be that of redirecting the flow of assimilates, perhaps by its influence on cytokinin synthesis and distribution. PMID- 16658899 TI - Ultrastructural evidence for calcium phosphate deposition by isolated corn shoot mitochondria. AB - This study demonstrates that isolated corn (Zea mays L. Wf9 xM14) shoot mitochondria will accumulate calcium phosphate crystals in vitro that are comparable to those observed in both in vivo and in vitro animal mitochondria. The intensity of deposition, as observed by electron microscopy, increased with incubation time, substrate concentration, and concentration of calcium and inorganic phosphate. Calcium phosphate deposits were initially observed as amorphous, nearly spherical areas that lacked sharp or clear edges, but which were crystalline in nature under heavier loading conditions and longer incubation times. PMID- 16658900 TI - The Suitability of a Quantitative Spectrophotometric Assay for Phenylalanine Ammonia-lyase Activity in Barley, Buckwheat, and Pea Seedlings. AB - It has been suggested by others that the spectrophotometric assay of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm may be complicated by a side reaction involving transamination from phenylalanine onto alpha-keto acids. This would lead to the production of phenylpyruvate which would spontaneously tautomerize and form an enol borate complex absorbing at this wavelength. We find that the inclusion of 1 ml of either 60 mum alpha ketoglutarate or 500 mum phenylpyruvate in our 3-ml reaction mixtures has no significant effect on the spectrophotometric assay of phenylalanine ammonia-lyase in shoots from young seedlings of barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), or pea (Pisum sativum). Although these side reactions may be involved in preparations with very low enzyme activity, the spectrophotometric determination of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm appears to be a reliable and sensitive technique in these seedlings. PMID- 16658901 TI - Starch in Fungi: III. Isolation and Properties of an Amylose-precipitating Factor from Lentinellus Ursinus Fruit Bodies. AB - A factor which precipitates amylose has been isolated from Lentinellus ursinus (Fr.) Kuhner fruit bodies. This factor could be a protein or a polypeptide. Glucose, maltose, and amylopectin do not affect the binding of amylose. Amylose binding is unaffected by temperature (4 to 40 C) or pH (6 to 8.5). PMID- 16658902 TI - Growth Responses to Sodium by Bryophyllum tubiflorum under Conditions Inducing Crassulacean Acid Metabolism. AB - The dry weight yield of plants of Bryophyllum tubiflorum Harvey, a species with Crassulacean acid metabolism characteristics, increased significantly (P < 1%) in response to added sodium (0.1 milliequivalents per liter NaCl was supplied to the culture solution initially containing less than 0.08 microequivalents per liter of Na) when grown under short day (8 hours) conditions but not when grown under long day conditions (16 hours).From results of other work with Crassulacean acid metabolism species, it appears likely that under long day conditions, the plants assimilate CO(2) by the C(3) pathway but under short day conditions by the Crassulacean acid metabolism pathway in which metabolic processes common to those operating in the C(1) dicarboxylic pathway are active. It is suggested that sodium is involved in plants assimilating CO(2) with the C(4) and Crassulacean acid metabolism pathways. PMID- 16658903 TI - On the 50th anniversary of our society. PMID- 16658904 TI - Conceptual developments in photosynthesis, 1924-1974. PMID- 16658905 TI - Plant photobiology in the last half-century. PMID- 16658906 TI - Conceptual developments in metabolic control, 1924-1974. AB - A brief sketch is given of the development of the understanding of the respiratory mechanism in plants over the past 50 years. Against this background the following aspects of control are discussed: (a) nonreversibility of catabolic sequences; (b) compartmentation of reactions and reactants; (c) control by amount of enzyme; (d) control by NAD and NADP; (e) control by ADP supply; (f) pacemaker reactions in glycolysis; and (g) control at branch points: further examples of allostery. PMID- 16658908 TI - Fifty years of plant hormone research. PMID- 16658907 TI - Biological nitrogen fixation, 1924-1974. PMID- 16658909 TI - Conceptual developments in membrane transport, 1924-1974. PMID- 16658910 TI - Fifty years of progress in water relations research. AB - Many of the basic concepts dealing with soil and plant water relationships were in existence 50 years ago, but were inadequately presented in the textbooks of that time. There has been a marked increase in the amount of work done in this field during recent decades, but much of it involves advances in understanding the concepts already in existence. Three of the most important advances in the field of water relations are: (a) acceptance of the term, water potential, to describe the free energy status of water in soil and plants; (b) marked improvement in methods of measuring water potential and stomatal resistance; and (c) use of the concept of water flow in the soil-plant system as analogous to flow of electricity in a conducting system.A number of interesting and important problems remain to be studied. Of these, probably the most important is to learn why mild water stress of less than - 10 bars can affect various enzyme-mediated metabolic processes. Plant scientists in applied fields also need to learn more about the causes of differences in ability to tolerate drought among plants of various kinds. There is uncertainty concerning the relative magnitude of the resistances to water flow in various parts of the soil-plant system and concerning the causes of the apparent changes in resistance to water flow with increase in rate and with time of day. More information also is needed concerning the role of growth regulators synthesized in roots and the importance of the older, suberized roots in the absorption of water and mineral nutrients. PMID- 16658911 TI - Long distance transport. PMID- 16658912 TI - Dependence of nitrite reduction on electron transport chloroplasts. AB - Methyl viologen and phenazine methosulfate (photosystem I electron acceptors), 3 (3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU, electron-transport inhibitor), and methylamine (photophosphorylation uncoupler) were used to study the dependence of nitrite reduction on electron transport in chloroplasts.DCMU, methyl viologen, and phenazine methosulfate markedly inhibited, whereas methylamine stimulated NO(2) (-) reduction in isolated, intact spinach (Spinacia oleracea L.) chloroplasts. The addition of DCMU to leaf sections of spinach and corn, (Zea mays L. var. XL81), incubated with No(3) (-), caused no inhibition of nitrate reduction but inhibited nitrite reduction leading to the accumulation of NO(2) ( ) in the light. The addition of methylamine to comparable leaf sections did not affect either nitrate or nitrite reduction.WE CONCLUDED THAT: (a) nitrite reduction is functionally associated with the electron transport arising from the light reactions of the chloroplast and this provides additional support for the localization of nitrite reductase in the chloroplast; (b) nitrite reduction is associated with photosystem I and ferredoxin is the most likely donor in leaf tissue; and (c) ATP is not involved directly in nitrite reduction. However, ATP synthesis, by regulating electron flow to photosystem I, can affect nitrite reduction in the light. PMID- 16658913 TI - Fat Metabolism in Higher Plants: LXII. Stearl-acyl Carrier Protein Desaturase from Spinach Chloroplasts. AB - Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds. PMID- 16658914 TI - Extraction and partial characterization of cellulases from expanding pea epicotyls. AB - Etiolated pea (Pisum sativum) epicotyls synthesize a buffer-soluble cellulase (cellulase A) and a salt-soluble cellulase (cellulase B) (EC 3.2.1.4) after treatment with high (0.5%) auxin levels. Only cellulase A increased in activity after treatment with low (0.005%) auxin. Cellulase A was released into the supernatant after homogenization of tissue in dilute buffer (buffer-soluble), had a pH optimum at 5.5, was relatively thermostable, and its activity was inhibited by NaCl. Cellulase B was released by 1 m NaCl (salt-soluble) from excised tissue segments or from the insoluble residue remaining after removal of the buffer soluble form. It had a pH optimum at 7.0, was thermolabile, and required salt for maximum activity. When subjected to polyacrylamide gel electrophoresis, the cellulase fraction released by NaCl from excised segments showed two bands of cellulase activity compared to several for the buffer-soluble fraction. Electrophoretic analysis of the buffer and salt-soluble fractions for marker enzymes indicated the presence of malate dehydrogenase activity in all fractions and glutamate dehydrogenase activity in the buffer-soluble fraction only.Exposure of intact pea epicotyls to 70 mul/l of ethylene gas for 3 days did not affect cellulase A activity, but caused a 5-fold increase in cellulase B activity (enzyme released by salt from the buffer-insoluble residue). We concluded that ethylene and auxin generate different forms of cellulase. PMID- 16658915 TI - Cherry fruit abscission: evidence for time of initiation and the involvement of ethylene. AB - Initiation of abscission at the pedicel-fruit zone in the sour cherry (Prunus cerasus L. cv. Montmorency) occurs near the transition of Stage II to Stage III of fruit growth. The preinitiation phase is characterized by a high fruit removal force (FRF) and explants prepared from fruits during this period do not undergo abscission as indexed by a reduction in FRF. Ethylene does not cause a significant reduction in FRF either in attached fruit or in explants prepared during this period. By contrast, after initiation (Stage III of fruit growth), there is a marked decrease in FRF with fruit development, explants prepared from fruits during this period undergo abscission, and ethylene markedly promotes the loss in break-strength. Neither the rate of evolution nor the internal concentration of ethylene in the fruit were correlated with fruit abscission. Similar abscission responses, as indexed by FRF and sensitivity to ethylene, were observed in attached fruit and in detached fruit explants. PMID- 16658916 TI - Relationship between Promotion of Xyloglucan Metabolism and Induction of Elongation by Indoleacetic Acid. AB - Auxin promotes the liberation of a xlyoglucan polymer from the cell walls of elongating pea (Pisum sativum) stem segments. The released polymer can be isolated from the polysaccharide fraction of the water-soluble portion of tissue homogenates, thus providing as assay for this kind of metabolism. Promotion of xyloglucan metabolism by auxin begins within 15 minutes of hormone presentation. The effect increases with auxin concentration in a manner similar to the hormone effect on elongation. However, the xyloglucan effect of auxin occurs perfectly normally when elongation is completely blocked by mannitol. Metabolic inhibitors and Ca(2+), on the other hand, inhibit auxin promotion of elongation and of xyloglucan metabolism in parallel. The results suggest that the changes in xyloglucan reflect the means by which auxin modifies the cell wall to cause elongation. PMID- 16658917 TI - Metabolism of Glutamate in Suspension Cultures of Paul's Scarlet Rose Cells. AB - When specifically labeled glutamate-1-(14)C was provided to 4-day-old rose cells, 87.6% of the (14)C in glutamate recovered from protein was in the number 1 carbon atom of the glutamate molecule. It was concluded that newly absorbed glutamate was incorporated directly into protein without any prior metabolism.A double labeling study with acetate-U-(14)C and glutamate-U-(3)H showed the availability of absorbed glutamate for protein synthesis was equal to that of endogenously synthesized glutamate. PMID- 16658918 TI - Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados. AB - A continuous exposure of intact avocados (Persea americana) to 400 mul/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3 phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase. PMID- 16658919 TI - Apical correlative effects in leaf epinasty of tomato. AB - The influence of the stem apex on leaf curvature was investigated using debudded tomato (Lycopersicon esculentum Mill. cv Anahu) plants and petiole explants, consisting of a section of petiole attached to a section of stem.Decapitation of the main shoot of tomato plants induced hyponasty of petioles in young leaves. Application of auxin in place of the removed apex or fumigation of intact tomato plants with ethylene produced epinastic curvature at the base of the petiole. Simultaneous carbon dioxide treatments prevented the development of petiolar epinasty due to auxin and ethylene treatments. Application of ethylene gas to the decapitated shoot or injection into the stem, induced petiolar epinasty. In a saturating level of ethylene gas, tomato petioles did not respond to indole-3 acetic acid applied to the cut apex. Auxin-induced ethylene production in petiole explants preceded the development of epinasty. Application of indoleacetic acid in lanolin to the entire lower side of the petioles of leaves in situ produced petiole epinasty. Petiolar epinasty due to apically applied indoleacetic acid resulted from differential cell elongation.The auxins indole-3-acetic acid, 2,4 dichlorophenoxyacetic acid, and naphthalene-1-acetic acid induced epinasty when applied apically to decapitated tomato plants, while gibberellic acid, kinetin, abscisic acid, and auxin or gibberellin antagonists had no effect. When such compounds were applied to petiole explants, only indole-3-acetic acid and kinetin caused an increase in ethylene production and the effect of kinetin was relatively weak.Application of 2,3,5-triiodobenzoic acid around the stem did not change the effect on petiolar epinasty of auxin applied to the decapitated shoot or around the stem. Radioautography showed that the label from (14)C-indoleacetic acid applied apically entered the petiole and midrib tissue; however, extraction showed that only a fraction of the label in these tissues was in the form of indoleacetic acid.Removal of leaflets from leaves induced hyponasty in the midrib region, and application of auxin to the leaflet stubs produced midrib epinasty; carbon dioxide did not block the action of auxin in this type of epinasty. Removal of leaflets from leaves did not alter the effect of apically applied auxin on petiolar epinasty.The data are consistent with the hypothesis that the oblique orientation of leaves in tomato plants is influenced by two epinastic responses. Petiolar epinasty is controlled by the apical region on the stem and is due to the action of auxin-induced ethylene; and midrib epinasty is due to an action of auxin other than through ethylene. PMID- 16658920 TI - Uptake of bicarbonate ion in darkness by isolated chloroplast envelope membranes and intact chloroplasts of spinach. AB - Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach (Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO(3) (-) uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO(2) fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO(3) (-) and not CO(2). The significance of HCO(3) (-) uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO(3) (-) uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO(2) fixation, suggesting that HCO(3) (-) uptake by the envelope membrane may regulate photosynthetic CO(2) fixation. PMID- 16658921 TI - Membrane-surfactant Interactions in Lipid Micelles Labeled with l-Anilino-8 naphthalenesulfonate. AB - Sonicated lipid micelles, formed from phospholipids isolated from yolks of fresh hen eggs, were used as a model membrane system for studying the effects of several surfactants on membrane properties. The interactions of the surfactants with the model system were followed through the fluorescence of the hydrophobic probe l-anilino-8-naphthalenesulfonate. The surfactants investigated were polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene thioether (Sterox SK), mono-calcium salt of polymerized aryl alkyl sulfonic acids (Daxad 21), and alkylbenzyl quaternary ammonium halide (AHCO DD 50). All surfactants enhanced fluorescence of the membrane-bound probe, and the degree of this enhancement correlated with the previously established phytotoxicity of these substances. The results indicate that surfactants can produce distinct changes in artificial phospholipid membranes and suggest that this lipid interaction may account for altered membrane permeability characteristics in surfactant-treated plants. The effects are observable for surfactant concentrations as low as 0.0001% (w/v), representing an approximate 10-fold increase in sensitivity for detecting surfactant effects compared with previous results on permeability changes in isolated plant cells. PMID- 16658922 TI - Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation in gradients of silica. AB - Chloroplast suspensions from spinach (Spinacia oleracea L.) were clearly resolved into intact and stripped chloroplasts by isopycnic centrifugation in density gradients of silica sol ("Ludox") and polyethlene glycol. The intact chloroplasts fixed CO(2) and evolved O(2) more rapidly than the crude suspensions; the stripped chloroplasts were inactive. During the photosynthetic fixation of (14)CO(2) in the intact chloroplasts recovered from the gradient, the (14)C label was observed to spread through the photosynthetic intermediate pools, as well as into starch, which indicates that the purified chloroplasts are metabolically competent. This appears to be the first report of the retention of photosynthetic activity following the purification of chloroplasts in density gradients. PMID- 16658923 TI - The Effect of Potassium on the Fixation of Molecular Nitrogen by Root Nodules of Vicia faba. AB - The effect of potassium supply of Vicia faba on the fixation of molecular nitrogen by root nodules was studied by using (15)N-labeled molecular nitrogen. Plants well supplied with potassium showed higher contents of (15)N in the soluble amino fraction and in the protein fraction of various plant organs as compared with plants of a lower potassium status. This effect was evident particularly in the root nodules. Assimilation experiments, carried out with (14)CO(2), revealed that the content of radioactivity in the sugars and amino acids of the root nodules was increased by the potassium supply of the host plants. In particular, the content of (14)C amino acids in the root nodules was influenced beneficially by potassium, which means that potassium favored the provision of reduced nitrogen (NH(3)). It is postulated that the better carbohydrate supply of nodules, by plants well supplied with potassium, results in a higher carbohydrate turnover in the nodules and thus the provision of ATP and reducing electrons required by the nitrogenase is enhanced. PMID- 16658924 TI - The manganese toxicity of cotton. AB - Cotton plants (Gossypium hirsutum. Linn. var. Sankar 4) were grown at normal and toxic levels of substrate manganese, and the altered metabolism of manganese toxic plants was studied. The tissues of plants exposed to toxic levels of manganese had higher activities of peroxidase and polyphenol oxidase, and the activities of catalase, ascorbic acid oxidase, glutathione oxidase and cytochrome c oxidase were lowered. In addition, the high manganese tissue had lower contents of ATP and glutathione but higher amounts of ascorbic acid. The respiration of the partially expanded leaves and the growing tips of toxic plants were depressed when compared to that of the normal tissues. The metabolic changes of manganese toxicity of cotton are placed in the following order: accumulation of manganese in the leaf tissue; a rise in respiration; stimulation of polyphenol oxidase; the appearance of initial toxicity symptoms; the evolution of ethylene and stimulation of peroxidase; the presence of severe toxicity symptoms; the depression of terminal oxidases and respiration; abscission of the growing tip and proliferation of the stem tissue. The early stimulation of polyphenol oxidase may be used to detect potential manganese toxicity. PMID- 16658925 TI - Transition of metabolisms in living popular bark from growing to wintering stages and vice versa: changes in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities and in the levels of sugar phosphates. AB - Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar (Populus gelrica). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins.Normal concentrations of total hexose phosphates in the glycolytic pathway plus 6-phosphogluconate were found to be 288 to 895 mumoles/kilogram dry weight. During the metabolism transition (September and April), a transient and striking increase of 6-phosphogluconate was observed. In September, 6 phosphogluconate reached a level on the order of 10(-4)m and was 4 times that of fructose 6-phosphate. The increase in 6-phosphogluconate coincided with the increase in the glucose 6-phosphate dehydrogenase activity. Coincidentally, with the change of 6-phosphogluconate level, a large deviation of the in vivo ratio of fructose 6-phosphate to glucose 6-phosphate from the known equilibrium constant was observed, showing the relation of pentose phosphate cycle enzyme activity to the control of glycolysis. The ratio of glucose 6-phosphate to glucose 1 phosphate deviated from that predicted. These ratios fluctuated throughout the year and were affected by the growth phases. The levels of pentose phosphate cycle metabolites, except for 6-phosphogluconate, in the bark were extremely low.The level of inorganic phosphate in the living bark throughout the year was dependent upon the growth phases, suggesting the presence of a regulatory mechanism to maintain inorganic phosphate at a given level as the growth phase changed.Sugar phosphate levels of popular twigs or potato tubers (Solanum tuberosum) remained constant after they were stored for 2 weeks at low temperatures, whereas in sweet potato roots (Ipomoea batatas), the level rose to about 9-fold of the control, indicating the presence of a strict regulatory system for the synthesis and catabolism of sugar phosphate in the former two. PMID- 16658926 TI - The location of nitrite reductase and other enzymes related to amino Acid biosynthesis in the plastids of root and leaves. AB - Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate aminotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activity. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts. PMID- 16658927 TI - Activation of ribulose 1,5-diphosphate carboxylase by nicotinamide adenine dinucleotide phosphate and other chloroplast metabolites. AB - Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 10 mm MgCl(2) and 1 mm bicarbonate in the absence of ribulose 1,5-diphosphate, can be further activated about 170% with 0.5 mm NADPH present in the preincubation mixture. NADP(+), NADH, and NAD(+) are ineffective. The activation by NADPH is comparable to that previously seen with 0.05 to 0.10 mm 6-phosphogluconate in that these specific preincubation conditions are required, but the effects of NADPH and 6-phosphogluconate are not additive. Moreover, where higher concentrations of 6-phosphogluconate inhibited the enzyme, higher concentrations of NADPH give a greater activation, saturating at about 1 mm and 200%. Under the specified conditions of preincubation, fructose 1,6-diphosphate has an activation curve similar to that of 6-phosphogluconate, peaking at 0.1 mm and 70%. Above this level, activation decreases, and inhibition is seen at still higher concentrations. Other metabolites tested produced smaller or no effects on the enzyme activity assayed under these conditions. When either reduced NADP or 6 phosphogluconate are present in the preincubation mixture, it becomes possible to determine the Km for bicarbonate using a Lineweaver-Burk plot, and the Km for bicarbonate under these conditions is 2.8 mm, corresponding to 0.3% CO(2) at pH 7.8 and 25 C. PMID- 16658928 TI - Adenosine Phosphates in Germinating Radish (Raphanus sativus L.) Seeds. AB - Changes in concentrations of adenosine phosphates (AMP, ADP, and ATP), oxygen utilization, and fresh weights were measured during the first 48 hours after imbibition of water by quiescent radish seeds (Raphanus sativus L.) at 22.5 C. The changes in ATP concentrations, oxygen utilization, and fresh weights followed a triphasic time course, characterized by a rapid initial increase, which extended from 0 to approximately 1.5 hours, a lag phase from 1.5 to 16 hours, and a sharp linear increase from 16 to 48 hours. In unimbibed seeds, the concentrations of ATP, ADP, and AMP were <0.1, 0.9, and 2.2 nmoles/seed, respectively. After imbibition of water by the quiescent seeds, for 1 hour, the ATP concentration had increased to 2.5, and ADP and AMP concentrations had decreased to 0.3 and 0.1 nmole/seed, respectively. These early changes occurred also in seeds maintained under anaerobic conditions (argon), or when treated with either 5 mm fluoroacetate, or 5 mm iodoacetate. The concentrations of ADP and AMP did not change significantly from 1 to 48 hours. The termination of the lag phase at 16 hours correlated with radicle emergence. Cell division in the radicles was initiated at approximately 28 hours. ATP concentrations in seeds maintained under argon or treated with fluoroacetate remained relatively constant from approximately 2 to 48 hours. In contrast, the ATP concentration of iodoacetate treated seeds decreased curvilinearly from 4 to 48 hours. Oxidative phosphorylation was estimated to have contributed 15, 20, and 65% of the pool ATP at 1.5, 16, and 48 hours, respectively. PMID- 16658929 TI - A Method for Characterizing the Relation between Nutrient Concentration and Flux into Roots of Intact Plants. AB - A method based on the rate of depletion of a nutrient from solution was developed to characterize nutrient flux of plant roots. Nutrient concentration of the solution was measured at a series of time intervals to describe the complete depletion curve. An integrated rate equation, based on a Michaelis-Menten model, was developed and fit to the data of the depletion curve using a least-square procedure. The equation contained values for V(max), the maximum rate of influx; Km, the Michaelis constant; and E, efflux, which were used to describe the relation between solution concentration and net influx rate. Models other than Michaelis-Menten could also be used. The method uses only one plant or group of plants to obtain data over a range of nutrient concentrations, is adapted particularly to the low concentration range, and measures the concentration below which net influx ceases. With this method the plant is in steady state absorption prior to the experiment and continues at this steady state until near the end of the experiment.A procedure was also developed to measure uptake rate at constant concentration by adding nutrients to the pot at a constant rate that matched net influx into the root. This method also provides a means of measuring diurnal fluctuations in net influx rates. PMID- 16658930 TI - Physiological studies with isolated leaf cells: early products of photosynthesis and their metabolic interconversions. AB - A number of plants have been surveyed with respect to isolation by mild grinding in large quantities of leaf cells. The extent of recovery of mesophyll cells per unit leaf area was found to vary with plant species and the method of grinding. Greater than 70% recovery was obtained from the leaves of Canna indica L., Crotalaria Laburnifolia L., and Thunbergia grandiflora Roxb.By pulse-chase time course experiments, the photosynthetically fixed primary carbon compounds of bean leaf cells were not converted into the ethanol-insoluble fraction. About 25% of total (14)C-photoassimilates were found to leak out into the incubation medium. In contrast, Euglena and Chlorella cells incorporated their primary photosynthetic products into cellular macromolecules and the amount of "leak" was very little. (14)C-Leucine supplied to the bean cells was absorbed readily and incorporated into the trichloroacetic acid insoluble fraction. PMID- 16658931 TI - Effects of light intensity and oxygen on photosynthesis and translocation in sugar beet. AB - The mass transfer rate of (14)C-sucrose translocation from sugar beet (Beta vulgaris, L.) leaves was measured over a range of net photosynthesis rates from 0 to 60 milligrams of CO(2) decimeters(-2) hour(-1) under varying conditions of light intensity, CO(2) concentration, and O(2) concentration. The resulting rate of translocation of labeled photosynthate into total sink tissue was a linear function (slope = 0.18) of the net photosynthesis rate of the source leaf regardless of light intensity (2000, 3700, or 7200 foot-candles), O(2) concentration (21% or 1% O(2)), or CO(2) concentration (900 microliters/liter of CO(2) to compensation concentration). These data support the theory that the mass transfer rate of translocation under conditions of sufficient sink demand is limited by the net photosynthesis rate or more specifically by sucrose synthesis and this limitation is independent of light intensity per se. The rate of translocation was not saturated even at net photosynthesis rates four times greater than the rate occurring at 300 microliters/liter of CO(2), 21% O(2), and saturating light intensity. PMID- 16658932 TI - A method to compare the effect of ionic iron and iron chelates in nutrient solution cultures. AB - A method is described by which the effect of chelated Fe can be compared with the effect of ionic Fe in nutrient solution cultures over a prolonged period of time. Plants are grown in two solutions in succession: the one containing all nutrient elements except Fe, the other one containing the Fe compound together with Ca(NO(3))(2). In experiments with soybeans (Glycine maxima (L.) Merr.) and with corn (Zea mays L.) it was shown that a 7-day cycle with the ratio of 4 days nutrient solution to 3 days Fe solution resulted in growth and Fe nutrition similar to plants grown with a normal nutrient solution. PMID- 16658933 TI - Iron requirement and iron uptake from various iron compounds by different plant species. AB - The Fe requirements of four monocotyledonous plant species (Avena sativa L., Triticum aestivum L., Oryza sativa L., Zea mays L.) and of three dicotyledonous species (Lycopersicum esculentum Mill., Cucumis sativus L., Glycine maxima (L.) Merr.) in hydroponic cultures were ascertained. Fe was given as NaFe-EDDHA chelate (Fe ethylenediamine di (O-hydroxyphenylacetate). I found that the monocotyledonous species required a substantially higher Fe concentration in the nutrient solution in order to attain optimum growth than did the dicotyledonous species. Analyses showed that the process of iron uptake was less efficient with the monocotyledonous species. When the results obtained by using chelated Fe were compared with those using ionic Fe, it was shown that the inefficient species were equally inefficient in utilizing Fe(3+) ions. However, the differences between the efficient and the inefficient species disappeared when Fe(2+) was used. This confirms the work of others who postulated that Fe(3+) is reduced before uptake of chelated iron by the root. In addition, it was shown that reduction also takes place when Fe is used in ionic form. The efficiency of Fe uptake seems to depend on the efficiency of the root system of the particular plant species in reducing Fe(3+). The removal of Fe from the chelate complex after reduction to Fe(2+) seems to present no difficulties to the various plant species. PMID- 16658934 TI - Detection and identification of cytokinins produced by mycorrhizal fungi. AB - Several fungi including six species of the genus Rhizopogon, 22 species of Hebeloma and one of Agaricus have been screened for production of cytokinins. The screening was done by culturing cytokinin-requiring soybean callus tissue alongside the fungus on a medium lacking a cytokinin supply. Growth of the soybean callus indicated production of cytokinins by the fungus. Of the fungi tested, only R. ochraceorubens A. H. Smith gave off sufficient cytokinin to be detected. Although a number of mycorrhizal species are now known to make and give off cytokinins, an even larger number apparently do not do so under the conditions of screening employed. An unidentified ectendotrophic species definitely gave off trans-zeatin, which has been crystallized, and probably trans ribosylzeatin. Suillus punctipes (Pk.) Sing. apparently produced the same two cytokinins. PMID- 16658935 TI - A simpler iterative steady state solution of munch pressure-flow systems applied to long and short translocation paths. AB - A simple steady state iterative solution of Munch pressure-flow in unbranched sieve tubes containing only water and sucrose is derived. The iterative equations can be solved on a programmable desk calculator. Solutions are presented for steady state transport with specific mass transfer rates up to 1.5 x 10(-5) mole second(-1) centimeters(-2) (= 18.5 grams hour(-1) centimeters(-2)) over distances in excess of 50 meters. The calculations clearly indicate that a Munch pressure flow system can operate over long distances provided (a) the sieve tube is surrounded by a semipermeable membrane; (b) sugars are actively loaded in one region and unloaded at another; (c) the sieve pores are unblocked so that the sieve tube hydraulic conductivity is high (around 4 centimeters(2) second(-1) bar(-1)); (d) the sugar concentration is kept high (around one molar in the source region); and (e) the average sap velocity is kept low (around 20-50 centimeters hour(-1)). The dimensions of sieve cells in several species of plants are reviewed and sieve tube hydraulic conductivities are calculated; the values range from 0.2 to 20 centimeters(2) second(-1) bar(-1). For long distance pressure-flow to occur, the hydraulic conductivity of the sieve cell membranes must be about 5 x 10(-7) centimeters second(-1) bar(-1) or greater. PMID- 16658936 TI - Multiple amine oxidases in cucumber seedlings. AB - Cell-free extracts of cucumber (Cucumis sativus L. cv. National Pickling) seedlings were found to have amine oxidase activity when assayed with tryptamine as a substrate. Studies of the effect of lowered pH on the extract indicated that this activity was heterogeneous, and three amine oxidases could be separated by ion exchange chromatography. The partially purified enzymes were tested for their activities with several substrates and for their sensitivities to various amine oxidase inhibitors. One of the enzymes may be a monoamine oxidase, although it is inhibited by some diamine oxidase inhibitors. The other two enzymes have properties more characteristic of the diamine oxidases. The possible relationship of the amine oxidases to indoleacetic acid biosynthesis in cucumber seedlings is discussed. PMID- 16658937 TI - Formation of C-Labeled Alanine from Pyruvate during Short Term Photosynthesis in a C(4) Plant. AB - Large amounts of alanine are produced in the first few seconds of photosynthesis in Portulaca oleracea L. The normal precursor-product relationship (phosphoglyceric acid --> pyruvate --> alanine) does not appear to operate in this species since labeling in pyruvate precedes that in phosphoglyceric acid. Pulse-chase experiments show that the alanine is rapidly metabolized. After a 6 second pulse of (14)CO(2), the percentage of (11)C in alanine drops more than 30% in the first 10 seconds of a (12)CO(2) chase period. The percentage of (14)C in the other early-labeled photosynthetic products, aspartate and malate, also decreases during the (12)CO(2) chase. The decrease of label in these compounds is concomitant with an increase in the labeling of sucrose and alanine, which in this case is formed via phosphoglyceric acid. Randomization of label within alanine increases gradually throughout the 2-minute chase.Alpha-keto acids accounts for up to 20% of the total (14)CO(2) incorporated by young leaf tissue. Pyruvate alone accounts for at least 65% of the radioactivity in the keto acid fraction in both young and mature leaves. Other early-labeled keto acids are oxaloacetate and, in young tissue only, hydroxyphenylpyruvate. We propose that, in Portulaca, pyruvate is one of the earliest-labeled photosynthetic products and serves as the immediate precursor of alanine. PMID- 16658938 TI - Studies on soybean nodule senescence. AB - Soybean Glycine max. L. Merr. nodule senescence was studied using the loss of acetylene reduction by intact tap root nodules as its indication. Tap root nodules from two varieties (Calland and Beeson) of field-grown soybeans were used. The specific activities of nitrogenase (micromoles/minute gram fresh weight of nodules) as measured by the acetylene reduction assay decreased abruptly between 58 to 65 and 68 to 75 days after planting the Beeson and Calland soybeans, respectively. Major changes were not detected in dry weight, total nitrogen, and leghemoglobin levels during the period when in vivo nitrogenase activity declined. Ammonium levels in the cytosol of nodules and poly-beta hydroxybutyrate increased moderately just prior to or coincidental with the loss of nitrogenase activity. Neither enzymes that have been postulated to be involved in ammonium assimilation nor NADP(+)-specific isocitrate dehydrogenase exhibited any large changes in specific activities during the initial period when nitrogenase activity declined. PMID- 16658939 TI - Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris. AB - A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K(+) and Mg(2+). Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca(2+) are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6 diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca(2+) dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg(2+).The significance of the properties of pyruvate kinase in the regulation of photosynthetic carbohydrate metabolism, especially in connection with the inability of fructose 1,6-diphosphate to reverse Ca(2+) and ATP inhibitions, is emphasized. PMID- 16658940 TI - Sterol molecular modifications influencing membrane permeability. AB - Various sterols and related steroids were tested for their ability to influence ethanol-induced electrolyte leakage from Hordeum vulgare roots. Cholesterol had the greatest influence and, depending on concentration, it stimulated or inhibited the loss of electrolyte. Cholesterol, however, was ineffective if the roots were pretreated with ethanol. These data suggest that sterols protect rather than restore membrane structure. First, modifications in the cholesterol perhydrocyclopentanophenanthrene ring system suggest that at least one double bond is required for membrane activity. Second, increasing the bulkiness of the C(17) side chain of cholesterol, as shown with campesterol, stigmasterol, and sitosterol, decreased its activity. Apparently for maximum effectiveness the sterol molecule should have a relatively flat configuration. Third, the C(3) hydroxyl group is required for membrane activity since cholesteryl methyl ether, cholest-5-ene-3beta-thiol and cholesteryl halogens were without activity. Exception to the foregoing rule was cholestane which was slightly active but which has neither a C(3)-hydroxyl group nor a double bond in the ring system. PMID- 16658941 TI - Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana. AB - THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000. PMID- 16658942 TI - Lysine Biosynthesis in Barley (Hordeum vulgare L.). AB - Lysine biosynthesis in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following precursors into the endosperm of the seedlings: acetate-1-(14)C; acetate-2-(14)C; pyruvate-1-(14)C; pyruvate-2-(14)C; pyruvate-3-(14)C; alanine-1-(14)C; aspartic acid-1-(14)C; aspartic acid-2-(14)C; aspartic acid-3-(14)C; aspartic acid-4-(14)C; alpha-aminoadipic acid-1-(14)C; and alpha, epsilon-diaminopimelic acid-1-(7)-(14)C. The distribution of activity in the individual carbon atoms of lysine in the different biosynthetic experiments was determined by chemical degradation. The incorporation percentages and labeling patterns obtained are in agreement with the occurrence of the diaminopimelic acid pathway. The results do not fit the incorporation percentages and labeling patterns expected if the alpha-aminoadipic acid pathway was operating. However, the results show that barley seedlings are able to convert a small part of the alpha-aminoadipic acid administered directly to lysine.The labeling pattern of lysine was found to be symmetrical around carbon 4. This indicates that the biosynthetic pathway proceeds via a symmetrical intermediate like ll-alpha, epsilon-diaminopimelic acid, or includes compounds as 2, 3 dihydrodipicolinic acid or Delta(1)-piperideine-2, 6-dicarboxylic acid which probably isomerise with concomitant lack of asymmetry in the labeling. The percentages of incorporation show that both the mesoand ll-forms of alpha, epsilon-diaminopimelic acid are metabolically convertible to lysine in seedlings of barley. PMID- 16658943 TI - Kinetin-induced Changes in delta-Aminolevulinic Acid Dehydratase of Tobacco Callus. AB - Kinetin-induced changes in dry weight, soluble protein content, delta aminolevulinic acid dehydratase activity, and chlorophyll content of two clones of Nicotiana tabacum L. callus were studied. Kinetin brought about a marked increase in the delta-aminolevulinic acid dehydratase activity of nongreen tissue just before induction of greening. The experimental data suggested a possible induction of specific chloroplast protein(s) during the kinetin-induced greening of nongreen tobacco tissue. Kinetin caused a decline in the delta-aminolevulinic acid dehydratase activity and chlorophyll content of the green callus used in the present study. PMID- 16658944 TI - A simple carbon dioxide injection system for photosynthetic studies. AB - A simple carbon dioxide injection system has been developed for the maintenance of CO(2) concentrations in semiclosed cuvette systems suitable for photosynthesis and gaseous pollutant studies. The device injects small volumes of pure carbon dioxide into the cuvette in response to a signal from an infrared gas analyzer. PMID- 16658945 TI - Location of the low temperature water flow barrier in stems. AB - Experiments are described indicating the magnitude and location of the low temperature barrier to lateral water flow in stems of cotton (Gossypium hirsutum L. ;Auburn 7-683'). Rehydration of wilted stem tissues was performed at 6 C and 32 C. Compared with the 32 C control, a 13-fold increase in the rehydration halftime was recorded at 6 C when water entered the secondary phloem tissues across the vascular cambium from the secondary xylem. However, only a 3-fold increase in the rehydration halftime occurred when water entered phloem tissues through the cortex, and most of this increase was due to the higher viscosity of water at the lower temperature. These results show that the cambial region of an intact cotton stem markedly resists the radial flow of water at lower temperatures. This resistance was not demonstated by other stem tissues. PMID- 16658946 TI - Promotion of acetylene reduction by Rhizobium-soybean cell associations in vitro. AB - Nitrogenase activity, determined by the acetylene reduction assay, in soybean cell suspensions infected with Rhizobium, was stimulated significantly by the addition of succinic acid or glutamine. Acetylene-dependent ethylene values as high as 4.6 mumoles of ethylene/gram dry weight.hour were observed. PMID- 16658947 TI - Structure of some cyclohexyl compounds as related to their ability to stimulate plant growth. AB - Aqueous solution, 10 mm, of cyclohexanecarboxylic, cyclohexylacetic, cyclohexylpropionic, cyclohexylbutyric acids (all components of naphthenic acid); cis-1,2-, and trans-1, 4-cyclohexyldicarboxylic acids; 3-cyclohexene-1-carboxylic and cyclohexylsulfamic acids; and cyclohexyl mercaptan were applied to 14-day-old bush bean plants, Phaseolus vulgaris L. cv. Top Crop. Only cyclohexanecarboxylic and cyclohexylacetic acid resulted in a statistically significant (P = 0.05) increase in pod production per plant in all experiments. The stimulation by the first four monocarboxylic acids decreased as the number of methylene groups in the side chain increased from 0 to 3. The effective compounds possessed an H saturated 6-carbon ring with a single carboxyl group attached directly to the ring or separated from it by no more than one methylene group. PMID- 16658948 TI - Induction of Phenylalanine Ammonia-lyase in Strawberry Leaf Disks: Action Spectra and Effects of Wounding, Sucrose, and Light. AB - The increase in phenylalanine ammonia-lyase (PAL) activity in strawberry (Fragaria vesca var. WSU-1232) leaf disks required wounding, sucrose, and light and was cycloheximide-sensitive. In injured leaves and in leaf disks, the highest PAL activity was detected nearest the wounded tissues. Without wounding, no increase in activity was observed when leaves were cultured in sucrose and light.The optimal concentration of sucrose for enzyme activity increase ranged from 0.15 m to 0.4 m. At the suboptimal sucrose concentration, the level of PAL activity was dependent upon the concentration of sucrose. A low but constant level of activity was detected in leaf disks maintained in 0.15 m sucrose and in darkness. Light accelerated the rate of PAL increase but did not change the total level of enzyme activity which was determined by the sucrose concentration.Enzyme activity disappeared rapidly when leaf disks cultured in sucrose and light were transferred to darkness or to water in light. Unlike in Xanthium leaf disks, cycloheximide could not completely inhibit the decay of enzyme activity, suggesting that an inactivating system was synthesized during the induction period, and the activity of the inactivating system increased as the induction period lengthened.The effect of light on accumulation of PAL activity appeared to be linked to photosynthesis. In the presence of 25 mum 3-(3,4-dichlorophenyl)-1,1 dimethylurea, the effect of light on enzyme increase was completely nullified. Addition of 25 mum 3-(3,4-dichlorophenyl)-1,1-dimethylurea to culture medium caused rapid decay of PAL activity from leaf disks which had been previously cultured in sucrose and light. The relation between effect of light and photosynthesis was further demonstrated by the action spectrum. Leaf disks incubated in sucrose and light of different wavelengths exhibited maximum accumulation of PAL activity at two wavelengths (475 nm and 625 nm). Action spectrum for protection against PAL decay exhibited a plateau at 475 to 525 nm and a peak at 625 nm. Action spectra for accumulation and protection against inactivation of PAL activity, therefore, appeared to be very similar to the action spectrum of photosynthesis. PMID- 16658949 TI - Effect of Glucose and Adenosine Phosphates on Production of Extracellular Carbohydrases of Alternaria solani. AB - Production of carbohydrases by Alternaria solani is inhibited by glucose under low growth conditions. In an enriched medium, glucose has little effect on the production of polygalacturonase and cellulase while it still suppresses production of beta-glucosidase. Low levels of all three enzymes were produced in the absence of their respective substrates. Such regulation has been found with many organisms. However, far greater production of these carbohydrases occurred with additions of adenosine phosphates to the growth media. Highest stimulation of enzyme production was by adenosine 5'-phosphate. Adenosine 5'-triphosphate and cyclic 3', 5'-adenosine monophosphate gave lesser amounts. Starvation appears to induce production of extracellular carbohydrases and adenosine 5'-phosphate may have a role in the starvation process. PMID- 16658950 TI - Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers. AB - The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH(4)) or deuterolysis (LiAlD(4)) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16 dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16 hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-(14)C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16 dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-(14)C]palmitic acid was incorporated into C(27), C(29), and C(31)n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C(27), 15%; C(29), 48%; C(31), 38%) was similar to the per cent composition of the alkanes (C(27), 12%; C(29), 43%; C(31), 44%). [1-(14)C]Stearic acid was also incorporated into C(27), C(29), and C(31)n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1 (14)C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C(28), C(30), and C(32) acids into alkanes in 0.5% to 5% yields. [G-(3)H]n-octacosanoic acid (C(28)) was incorporated into C(27), C(29), and C(31)n-alkanes. [G-(3)H]n triacontanoic acid (C(30)) was incorporated mainly into C(29) and C(31) alkanes, whereas [9, 10, 11-(3)H]n-dotriacontanoic acid (C(32)) was converted mainly to C(31) alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes. PMID- 16658951 TI - Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase. AB - Kinetic properties of soybean net photosynthetic CO(2) fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO(2) concentration, and O(2) concentration. With leaves, O(2) inhibition of net photosynthetic CO(2) fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO(2) fixation at higher temperatures was caused by a reduced affinity of the leaf for CO(2) and an increased affinity of the leaf for O(2). With purified ribulose 1,5-diphosphate carboxylase, O(2) inhibition of CO(2) incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O(2) sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO(2) and a slightly increased affinity of the enzyme for O(2). The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO(2) and O(2) provides further evidence that the carboxylase regulates the O(2) response of photosynthetic CO(2) fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO(2) and O(2) fixation in vivo is proposed.Oxygen competitively inhibited carboxylase activity with respect to CO(2), and CO(2) competitively inhibited oxygenase activity with respect to O(2). Within the limits of experimental error, the Michaelis constant (CO(2)) in the carboxylase reaction was identical with the inhibition constant (CO(2)) in the oxygenase reaction, and the Michaelis constant (O(2)) in the oxygenase reaction was identical with the inhibition constant (O(2)) in the carboxylase reaction. The Michaelis constant, (ribulose 1,5 diphosphate) was the same in both the carboxylase and oxygenase reactions. This equality of kinetic constants is consistent with the notion that the same enzyme catalyzes both reactions. PMID- 16658952 TI - Activation of 4-hydroxycinnamate hydroxylase in extracts from sorghum. AB - Three types of activation of 4-hydroxycinnamate hydroxylase have been observed in extracts of Sorghum bicolor. One involves the elimination of a lag period either by increasing the enzyme concentration or by the addition of a catalytic amount of caffeic acid. Two involve increases after freezing of incubation mixtures containing both hydroxycinnamate and ascorbate, one being a rapid but short-lived increase that may be limited to the freeze-thaw period, the other a slower and more sustained effect on the maximum linear rate obtained during the incubation period after thawing. PMID- 16658953 TI - Physiological and Cytological Similarities between Disease Resistance and Cellular Incompatibility Responses. AB - Excised pea pods responded similarly to both the invasion of plant pathogenic fungi and the presence of bean tissue, bean pollen, and mouse tumor cells by synthesizing pisatin and by developing a characteristic yellow-green fluorescence. Both responses were dependent on RNA and protein synthesis. Conversely, the foreign pollen and incompatible fungi were sensitive to the pea pod tissue and were subject to abnormal development.The induction of pisatin and the yellow-green fluorescence development were mediated by multiple compounds of varying sizes released by fungi or mouse tumor cells. The incompatibility between a bean pathogen, Fusarium solani f. sp. phaseoli, and pea pod tissue was hypothesized to occur as a result of the cross contamination of such inducing compounds. PMID- 16658954 TI - Plants under Climatic Stress: VI. Chilling and Light Effects on Photosynthetic Enzymes of Sorghum and Maize. AB - The activity of several photosynthetic enzymes was unaltered by exposure of sorghum or maize to low temperatures (10 C) and light (170 w m(-2)). Two light activated C(4)-pathway enzymes, NADP-malate dehydrogenase and pyruvate Pi dikinase, were reduced in activity, and this was largely attributable to a loss of enzyme rather than to incomplete enzyme activation. Loss of NADP-malate dehydrogenase was more marked in sorghum than in maize, and in both species no loss occurred at 10 C when light levels were reduced from 170 to 50 w m(-2). A light-dependent, low temperature-induced loss of catalase activity was also observed in maize leaves.The rate of in vivo activation of pyruvate Pi dikinase following illumination was reduced at 10 C compared with that at 25 C, but no immediate effect of low temperature on the in vivo activation of NADP-malate dehydrogenease could be measured. A similar differential effect of temperature on the rates of activation of these two enzymes was found in vitro. Arrhenius type plots of pyruvate Pi dikinase from sorghum and maize demonstrated a further sensitivity to low temperature. A sharp increase in the activation energy of this enzyme was observed below 12 C, both in the presence and absence of Triton X-100. No change in the activation energy of maize leaf malic enzyme, NADP-malate dehydrogenase, fructose-1, 6-diphosphate aldolase, or NADP-glyceraldehyde 3-P dehydrogenase occurred over a temperature range of 6 to 30 C.The postillumination time course of pyruvate Pi dikinase activation, net photosynthesis and stomatal opening was followed. Reduction in the rate of response that occurred with decreasing temperature was similar in all cases, and at any one temperature, pyruvate Pi dikinase activation slightly preceded increasing photosynthesis rates. Causal relationships could not, however, be proved. PMID- 16658955 TI - On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls. AB - Irradiation with ultraviolet light causes in the hypocotyl of dark-grown gherkin seedlings the partial conversion of trans-hydroxycinnamic acids to the cis isomers. The trans-hydroxycinnamic acids inhibit the development of phenylalanine ammonia-lyase activity, and the transformation of these compounds to the much less inhibitory cis-isomers forms a ready explanation for the increase in phenylalanine ammonia-lyase activity in the hypocotyl of gherkin seedlings irradiated with ultraviolet light. Arguments are advanced that the increase in phenylalanine ammonia-lyase activity caused by irradiation with blue light is also (at least in part) initiated by trans-cis isomerisation of the hydroxycinnamic acids. PMID- 16658956 TI - Proteinase Inhibitor-inducing Factor in Plant Leaves: A Phylogenetic Survey. AB - Thirty-nine plant species representing 20 families from the four major divisions of plants were surveyed for the presence of proteinase inhibitor-inducing factor activity in leaves or other tissues. Tissue juices were assayed for their capacity to induce accumulation of proteinase inhibitor I in excised tomato (Lycopersico esculentum) leaves. In tissues of only 2 of the 39 species was proteinase inhibitor-inducing factor-like activity not found. The activity was absent in cabbage leaves and celery stalks. Fruiting bodies from one of three fungi genera assayed contained exceptionally large quantities of proteinase inhibitor-inducing factor-like activity. Extracts from Agraricus campestris fruiting bodies contained over 20 times more activity than tomato leaf juice. The survey confirms that substances with proteinase inhibitor-inducing factor-like activity are widespread in the plant kingdom. PMID- 16658957 TI - Photosynthetic adaptation to temperature in c(3) and c(4) grasses: a possible ecological role in the shortgrass prairie. AB - Increasing pretreatment day temperatures of 20, 30, and 40 C resulted in decreased net photosynthesis in Agropyron smithii (C(3)) while in Bouteloua gracilis (C(4)) net photosynthesis was increased. The effect on photosynthesis of increasing analysis temperatures was the same as observed by increasing pretreatment temperatures. Resistance of the stomata and boundary layer were less affected by pretreatment temperatures than were the remaining resistances of a physical and chemical nature. Resistances for A. smithii were increased and those for B. gracilis were decreased by increasing pretreatment temperatures. Phenology of the species in the shortgrass prairie is such that A. smithii has its greatest growth activity during the cool portion of the growth season, whereas B. gracilis is most active in the warm portion. Thus, photosynthetic adaptation to temperature is strongly suggested as a strategy for ecosystem utilization by reduction of interspecific competition. PMID- 16658958 TI - The effect of cyanide and carbon monoxide on the electrical potential and resistance of cell membranes. AB - The rapid reduction in cell electropotentials induced by metabolic inhibitors is strong evidence for an electrogenic ion pump. According to Ohm's law, such a depolarization might be explained by a reduction in electric current, I, with unidirectional transport of a given ion, or an increase in permeability (decrease in resistance). With cells of etiolated seedlings of Pisum sativum L. cv. Alaska and Zea mays cv. Golden Bantam, carbon monoxide inhibition, which occurs only in the dark and is readily reversed by light, allows repeated cycling of depolarization and repolarization; there is no effect on cell membrane resistance. In contrast, cyanide inhibition results in a marked increase in membrane electrical resistance; with cyanide following repeated pulses of current used in measuring cell membrane resistance, the resistance eventually (about 10 minutes) shows an abrupt drop as in the "punch-through" effect reported by H. G. L. Coster (1965. Biophys. J. 5: 669-686). PMID- 16658959 TI - The function of the carbohydrate units of three fungal enzymes in their resistance to dehydration. AB - Glucose oxidase (from Aspergillus niger), glucoamylase (from Rhizopus spp.), and cellulase (from Aspergillus niger) of fungal origin are all glycosylated proteins. Dehydration of the three enzymes to a range of water potentials did not affect their activity. However, when more than 10% of the carbohydrate associated with the molecules was removed by periodate oxidation, the enzymes were highly susceptible to dehydration when compared with oxidized controls. Polyvinyl pyrrolidone and Dextran T500 protected the three enzymes in their oxidized state against the effects of dehydration.The carbohydrate units of the enzymes have a function in protecting the enzymes from dehydration and this may be a contributing factor to the survival of microorganisms in environments of low water potentials. The involvement of water associated with the enzyme molecules due to the presence of carbohydrate units is considered to be the protecting mechanism. PMID- 16658960 TI - Survival of Suspension-cultured Sycamore Cells Cooled to the Temperature of Liquid Nitrogen. AB - Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) which were immersed in liquid nitrogen after prefreezing to the temperatures from -30 to -50 C in the presence of dimethylsulfoxide and glucose as cryoprotective additive could proliferate vigorously when rewarmed rapidly in water at 40 C. For maintaining high viability of the cells after immersion in liquid nitrogen, it seems to be essential to use the cells at the later lag phase or the early cell division phase. This study provides a possibility for long term preservation in liquid nitrogen of plant-cultured lines. PMID- 16658961 TI - Homoserine esterification in green plants. AB - EXTRACTS OF PHYLOGENETICALLY DIVERSE PLANS WERE SURVEYED FOR THEIR ABILITY TO SYNTHESIZE THE FOLLOWING HOMOSERINE ESTERS WHICH ARE POTENTIAL PRECURSORS FOR METHIONINE AND THREONINE SYNTHESIS IN GREEN PLANTS: O-acetyl-, O-oxalyl-, O succinyl-, O-malonyl-, and O-phosphohomoserine. Synthesis of O-acylhomoserine esters was detected only in Pisum sativum L. and Lathyrus sativus L. Extracts of P. sativum, a plant known to accumulate O-acetylhomoserine, catalyzed the specific synthesis of this ester from homoserine and acetyl-CoA. Extracts of L. sativus, a plant known to accumulate O-oxalylhomoserine, catalyzed the specific synthesis of this ester from homoserine and oxalyl-CoA. None of the other plants surveyed, including representatives of the green algae, horsetails, gymnosperms, and angiosperms, catalyzed the synthesis of any of the O-acylhomoserine esters studied. In contrast, synthesis of O-phosphohomoserine by the reaction catalyzed by homoserine kinase was demonstrated in extracts of all plants examined, including the two exceptional legumes.These results suggest that, among the five homoserine esters studied, O-phosphohomoserine is the major activated homoserine derivative in plants. Direct confirmation of the dominant physiological role of O phosphohomoserine in the synthesis of cystathionine in the transsulfuration pathway of methionine biosynthesis in plants has recently been provided (Datko, A. H., Giovanelli, J., and Mudd, S. H. 1974. J. Biol. Chem. 249: 1139-1155). PMID- 16658962 TI - The mode of incorporation of 6-benzylaminopurine into tobacco callus transfer ribonucleic Acid: a double labeling determination. AB - Cytokinin-dependent tobacco (Nicotiana tabacum) tissue was grown in the presence of N(6)-benzyladenine (6-benzylaminopurine) labeled in the phenyl ring with (3)H and in the 8-position of the purine ring with (14)C. The (3)H/(14)C ratio of N(6) benzyladenosine recovered from labeled tobacco callus transfer RNA preparations was compared with the corresponding ratio for N(6)-benzyladenine supplied in the medium. By this means, with suitable controls, the incorporation of the synthetic cytokinin, N(6)-benzyladenine, into the transfer RNA of tobacco callus tissue was shown to involve the intact moiety. The observed level of incorporation was up to one N(6)-benzyladenosine molecule per 10(4) transfer RNA molecules. PMID- 16658963 TI - Phospholipids in the developing soybean seed. AB - The distribution of phospholipids in developing soybean seeds [Glycine max (L.) Merr., var. "Chippewa 64," "Harosoy 63," "Wayne," and "Clark 63"] was followed. From 30 to 60 days after flowering expressed as mole per cent of phospholipid phosphorus phosphatidic acid decreased from 14.8 to 9.1; phosphatidylinositol increased from 0 to 9.1; phosphatidylcholine increased from 8.2 to 9.8; phosphatidylethanolamine increased from 5.3 to 8.6; phosphatidylglycerol increased from 3.2 to 4.8; diphosphatidylglycerol increased from 2.7 to 4.1; and N-acylphosphatidylethanolamine decreased from 65.8 to 54.6. However, from 60 days after flowering to maturity, phosphatidic acid decreased to 0; phosphatidylinositol increased roughly 2-fold; phosphatidylcholine increased roughly 4.7-fold; phosphatidylethanolamine increased 3-fold; N acylphosphatidylethanolamine decreased 11-fold; whereas phosphatidylglycerol and diphosphatidylglycerol remained essentially constant. Percentages of individual phospholipid species were not statistically different between any two varieties at a given time period.Immature soybean cotyledons incubated with (14)C-acetate or -pyruvate demonstrated rapid incorporation into the phospholipid fraction. N acylphosphatidylethanolamine was found to account for nearly 70% of the total radioactivity incorporated by the total polar lipid fraction and greater than 30% of the total radioactivity added. PMID- 16658964 TI - Adenylate energy pool and energy charge in maturing rape seeds. AB - A study of energy state and chemical composition of pod walls and seeds of maturing rape (Brassica napus L.) was conducted on two varieties, Victor and Gorczanski. Total adenosine phosphates, ATP, and adenylate energy charge increased with increasing cell number and cellular synthesis during the early stages, remained high at maximum dry weight accumulation and maximum substrate influx time, and decreased with ripening. A temporal control of energy supply and ATP concentration is evident in developing tissues with determined functions; whereas the association of a high energy charge and active cellular biosynthesis occurs only in tissues with a stabilized cell number. PMID- 16658965 TI - Properties of glucosyltransferase and glucan transferase from spinach. AB - A glucosyl and a glucosyl-glucan transferase activity from spinach (Spinacia oleracea L. var. Matador) leaves have been partially purified and characterized. The latter activity (fraction 1 after diethylaminoethylcellulose chromatography) is responsible for the transfer of glucosyl as well as of maltosyl, maltotriosyl, and higher homologous residues to glucose giving rise to maltose and the correspondingly larger molecules. This fraction also shows beta-amylase activity. The transfer takes place only to glucose; maltose, as well as other alpha-1,4 glucans, serve as donors. The enzyme fraction 2 is amylase-free and catalyzes only the transfer of glucosyl moieties, again with high acceptor specificity to glucose. Maltose and larger alpha-1, 4-glucans, with the exception of maltotriose and maltotetraose, act as donors. The physiological function of these enzymes may be the formation of oligosaccharide primers for starch synthetase or phosphorylase. PMID- 16658966 TI - Control of diurnal variations in photosynthetic products: I. Carbon metabolism. AB - It has been demonstrated previously that the synthesis of amino acids from photosynthetically fixed carbon in leaves of Capsicum annuum L. cv. California Wonder occurs in the middle of the photoperiod. This paper reports experiments which identify control points regulating the carbon flow in these leaves.Estimations have been made of the levels of intermediates between 3 phosphoglycerate and pyruvate and between 3-phosphoglycerate and fructose 6 phosphate in leaves at different times in the photoperiod. Application of the Chance crossover analysis indicates that during periods of amino acid synthesis, pyruvate kinase is activated, possibly by ammonium ions. Fructose diphosphate aldolase could possibly be an additional control point, showing activation when amino acid synthesis has ceased. There was no indication of diurnal periodicity in the activity of fructose diphosphate aldolase. PMID- 16658967 TI - Control of Diurnal Variations in Photosynthetic Products: II. Nitrate Reductase Activity. AB - Nitrate accumulates in the leaves of Capsicum annuum L. cv. California Wonder and the leaf content is dependent on the nitrate level supplied to the roots. There is no consistent diurnal periodicity in the leaf nitrate levels.Nitrate reductase activity exhibits three distinct peaks in the leaves. One in the dark period, a second at the time of the prevailing dawn, and a third 6 hours after the beginning of the photoperiod. The third peak of activity can be induced by a short period of illumination (20 minutes) which is also sufficient to induce the labeling of the amino acid fraction by (14)CO(2). PMID- 16658968 TI - Autoradiographic Examination of Meristems of Intact Boron-deficient Squash Roots Treated with Tritiated Thymidine. AB - Intact roots of boron-sufficient squash (Cucurbita pepo L.) plants, plants entering boron deficiency, and plants recovering from boron deficiency were exposed to tritiated thymidine at the end of the treatment period to label the replicating DNA of root tip cells. Using histological sections, autoradiographs of intact root meristems were prepared. The labeling pattern in +B root tips revealed the presence of a well defined quiescent center. The ability of root tip cells to incorporate label is correlated with the total root elongation during the -B treatment period. A greater amount of total root elongation during boron deficiency and recovery reflects the fact that root tip cells have retained their ability to synthesize DNA and enter mitosis for a longer time. In roots recovering from boron deficiency, cells of the quiescent center were seen to play no part in the recovery process in roots treated for as long as 20 hours in a -B nutrient solution. They were inactive before, during, and after the -B treatment. Cessation of mitosis occurs as early as 6.5 hours after boron is withheld from the nutrient solution while DNA synthesis can occur for as long as 20 hours after withholding boron. It was concluded that boron is essential for continued DNA synthesis and mitotic activity. The absence of boron results in the cessation of mitosis and DNA synthesis within 20 hours from the time boron is withheld. PMID- 16658969 TI - Oscillations in stomatal conductance: the influence of environmental gain. AB - It is supposed that oscillations in stomatal conductance are associated with the dynamic properties of the loop in which rate of evaporation affects, through physiological processes, the aperture of stomata and stomatal aperture in turn affects rate of evaporation. It is therefore predicted that their occurrence must be influenced by the magnitude of what is termed environmental gain: the sensitivity of rate of evaporation to change in leaf conductance to vapor transfer. Two methods of manipulating gain, and their effects on stomatal behavior in cotton (Gossypium hirsutum L. cv. Deltapine Smooth Leaf), are described. In the first, gain was increased by decreasing ambient humidity; in the second, it was made zero by regulating ambient humidity to keep rate of evaporation constant despite changes in conductance. The results are in accord with the supposition. PMID- 16658970 TI - Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties. AB - Purified glutamine synthetase from pea seedlings was most active with Mg(2+) as the metal activator, but Mn(2+) and Co(2+) were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg(2+) saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co(2+) also gave a sigmoidal saturation curve, but when Mn(2+) was varied only slightly sigmoidal kinetics were seen. Addition of Mn(2+), Ca(2+), or Zn(2+) at low concentrations sharply inhibited the Mg(2+) -dependent activity, partially by shifting the pH optimum. Addition of Co(2+) did not inhibit Mg(2+)-dependent activity. The nucleotide triphosphate specificity changed markedly when Co(2+) or Mn(2+) replaced Mg(2+). Using the Mg(2+) dependent assay, the Michaelis constant (Km) for NH(4) (+) was about 1.9 x 10(-3) M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%. PMID- 16658971 TI - An improved method for the isolation of spinach chloroplast envelope membranes. AB - A three-phase, discontinuous sucrose gradient yielded two distinct fractions of envelope membranes from spinach (Spinacia oleracea L.) chloroplasts. Their buoyant densities were 1.08 g cm(-3) and 1.11 g cm(-3). Electron micrographs showed the lighter and heavier fractions to consist primarily of single and double membranes, respectively. The milligrams of lipid-milligrams of protein ratio for the complete envelope membrane (double membrane fraction) was 1.74. Thin layer chromatograms showed that the lipids of the complete envelope membranes were similar to those found in earlier preparations which consisted of single and double membranes. This isolation procedure is superior to earlier methods in that the percentage of complete envelope membranes is greater and the yield is almost three times as great. Enzymatic and chemical analyses and microscopic examination showed the complete envelope membranes were free of bacterial, fungal, microsomal, mitochondrial, and lamellar membrane contamination as well as stromal contamination. The specific activities of nonlatent Mg(2+) dependent ATPase (80 mumoles of phosphate released hr(-1) mg protein(-1)) were about 10-fold higher than those values found with earlier preparations consisting of single and double membranes, indicating that the ATPase is largely lost in preparations containing single membranes. These higher values show that the ATPase is located in the double membrane and probably functions in the transport processes of the envelope membrane. PMID- 16658972 TI - l-Ascorbic Acid Metabolism in Vitaceae: Conversion to (+)-Tartaric Acid and Hexoses. AB - The metabolic fate of l-ascorbic acid-1-(14)C and -6-(14)C has been investigated in two species in two genera of Vitaceae. Results suggest that ascorbic acid metabolism in the Vitaceae involves splitting the 6-carbon chain into 4- and 2 carbon fragments. The former, corresponding to C1 through C4 of ascorbic acid, is further oxidized to tartaric acid while the latter, corresponding to C5 and C6, is recycled into hexose phosphate metabolism. Comparison of these findings with previous observations on the conversion of ascorbic acid to (+)-tartaric acid in Pelargonium crispum clearly reveals two distinct processes of tartaric acid biosynthesis in those plants identified as tartaric acid accumulators. PMID- 16658973 TI - The Effect of Calcium Nutrition of Ethylene-induced Abscission. AB - The influence of calcium nutrition on ethylene-induced abscission was studied by growing cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants for several weeks in nutrient solutions containing 2, 10 (normal level), 15, or 20 meq/l of calcium, and then treating the plants with ethylene. Increasing the calcium level of cotton from 2 to 20 meq/l resulted in a 9-fold increase in the calcium content of the abscission zone and a maximum reduction of 25% in the amount of leaf abscission induced by ethylene (9 mul/l). Bean plants grown on 10, 15, or 20 meq/l calcium solutions showed corresponding increases in the calcium content of the abscission zone but showed no significant differences in the rate of ethyleneinduced abscission. Only at the lowest calcium level of 2 meq/l, where deficiency symptoms became apparent, was a significant effect observed. These results suggest that under normal cultural practices calcium nutrition has little influence on the rate of ethylene-induced abscission. PMID- 16658974 TI - Microenvironmental manipulation of the observed michaelis constant of ribulose diphosphate carboxylase. AB - Urease and ribulose 1, 5-diphosphate carboxylase can be bound to Sepharose to give an immobilized two-enzyme system which catalyzes the reaction urea --> H(2)CO(3) --> phosphoglyceric acid. The observed Km of the system for urea approaches the lower value for urease when carboxylase levels on the gel exceed urease levels. If a similar system operates in the chloroplast, the high Km (H(2)CO(3)) of ribulose 1,5-diphosphate carboxylase may not be metabolically significant. PMID- 16658975 TI - Sterol Accumulation and Composition in Developing Zea mays L. Kernels. AB - Kernels were collected from three maize (Zea mays L.) inbreds from 10 days after pollination until kernel maturity. Sitosterol, campesterol, and stigmasterol were the major sterols at all stages of kernel development. Cholesterol was less than 1% of the dry weight. The three major sterols accumulated during kernel development, but at a rate slower than dry weight. The ratio of the sterols did not vary greatly among the inbreds. At maturity, the three inbreds, Wf9, Oh43, and Ky226, had sterol levels of 325, 228, and 173 micrograms per kernel, respectively. Sitosterol accounted for 75 to 85% of the sterol. The relative amount of stigmasterol decreased during the linear phase of development, while sitosterol increased in the free fraction and campesterol increased in the steryl ester fraction.Free sterols and steryl esters were the major sterol fractions and steryl glycosides and acylated steryl glycosides were only minor components during kernel development. Free sterol content decreased rapidly in two maize inbreds between 10 and 26 days after pollination, but partially recovered in one of the inbreds during final stages of development. In the same two inbreds the steryl ester content reached a maximum during the late stages of linear kernel growth. PMID- 16658976 TI - Cholinesterases from plant tissue: v. Cholinesterase is not pectin esterase. AB - Several properties of the cholinesterase from Phaseolus aureus Roxb. and of pectin (methyl) esterases from both Phaseolus aureus and Lycopersicon esculentum (L.) Mill. are contrasted. Cholinesterase activity is inhibited by all of the concentrations of NaCl tested, from 0.05 m to 0.9 m, a property which differs sharply from published data pertaining to pectin esterase. Although crude preparations of cholinesterase contain pectin esterase activity, further purification by gel filtration of the cholinesterase results in a nearly complete elimination of the pectin esterase activity. The activity of neither the pectin esterase from Lycopersicon esculentum nor that from Phaseolus aureus is affected by 25 mum neostigmine, a potent inhibitor of the cholinesterase activity extracted from Phaseolus aureus. PMID- 16658977 TI - Increase in electrogenic membrane potential with washing of corn root tissue. AB - Washing of corn root tissue increases the electrical potential difference, negative inside, across the membranes of epidermal cells. There is no lag period in the development of the extra potential, and the entire increase is electrogenic as evidenced by collapse with the uncoupler, (p-trifluoromethoxy) carbonyl cyanide-phenylhydrazone.Proton extrusion by the tissue declines with washing but can be reinstituted by the addition of mersalyl, an inhibitor of the Pi-OH(-) antiporter of mitochondrial membranes.It appears that washing may enhance or augment the activity of an electrogenic ion pump, possibly a proton or cation efflux pump. In addition, there may be augmentation of an anion-OH(-) antiporter driven by the proton motive force created. PMID- 16658978 TI - The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed. AB - Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-(14)C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.Dormant intact seeds also metabolize (S)-2-(14)C-abscisic acid during stratification at 5 C or incubation at 25 C. The metabolites appear to be similar to those observed in excised embryos although by 12 days of stratification a fourth metabolite is observed. More than 90% of the (14)C-abscisic acid was metabolized after 26 days of stratification at 5 C or after 12 days of incubation at 25 C. Stratification at 5 C leads to the breaking of dormancy while incubation at 25 C does not. PMID- 16658979 TI - An amphoteric conjugate of [h]gibberellin a(1) from barley aleurone layers. AB - The major metabolite produced during incubation of [(3)H]gibberellin A(1) ([(3)H]GA(1)) with barley aleurone layers is an amphoteric, water-soluble compound tentatively called [(3)H]ampho GA(1). Formation of [(3)H]ampho GA(1) in barley aleurones begins after a period of 2.5 hours. As judged by degradation studies as well as Sephadex column chromatography, GA(1) appears to be linked to a peptide; positions C-3 and C-7 were ruled out as conjugation sites. PMID- 16658980 TI - An early response to gibberellic Acid not requiring protein synthesis. AB - Cell-free extracts from gibberellic acid-treated barley (Hordeum vulgare L. cv. Himalaya) aleurone layers show phosphorylcholine glyceride transferase activity greater than that from control layers. The increase in activity is not prevented by a mixture of amino acid analogs nor by cordycepin under conditions in which it is demonstrated that the analogs and the cordycepin are entering the cells in effective concentrations. We conclude therefore that the GA(3)-dependent increase in phosphorylcholine glyceride transferase activity (which occurs within the first 4 hours of GA(3) treatment) does not require RNA synthesis or protein synthesis. PMID- 16658981 TI - Permeability of seed coats to water as related to drying conditions and metabolism of phenolics. AB - The seed coat of Pisum elatius is normally impermeable to water. When seeds are dried in the absence of oxygen their coats are totally permeable to water. Structural differences are observed between permeable and impermeable seed coats. In the genus Pisum, species with normally impermeable seed coats have a high content of phenolics and of catechol oxidase, while seed coats of P. sativum contain very little catechol oxidase and have a very low content of phenolics. Such differences are not noted in the cotyledons. We hypothesized that during dehydration of seeds, oxidation of phenolic compounds in seed coats through catalysis of catechol oxidase in presence of O(2) might render the seed coats impermeable to water. PMID- 16658982 TI - Comparative Studies on Nitrate Reductase in Agrostemma githago Induced by Nitrate and Benzyladenine. AB - NADH-nitrate reductase activity in excised embryos of Agrostemma githago develops in response to nitrate as well as benzyladenine. Induction of nitrate reductase by benzyladenine was much more susceptible to inhibition by a mixture of amino acid analogues and by cordycepin than induction by nitrate. In contrast, only induction of nitrate-nitrate reductase was decreased by chloramphenicol.NADH cytochrome c reductase and reduced flavin mono-nucleotide-nitrate reductase activities were found to be associated with NADH-nitrate reductase and were induced by both nitrate and benzyladenine. When a partially purified enzyme sample was centrifuged in a linear 5 to 20% sucrose density gradient, a minor and a major band of NADH-cytochrome c reductase activity were observed. NADH-nitrate reductase cosedimented with the major band.The characteristics of nitrate-nitrate reductase and benzyl-adenine-nitrate reductase were compared by four methods but no differences could be detected: (a) Both enzymes sedimented with the same velocity during sucrose density gradient centrifugation. (b) Their distribution among fractions obtained by differential precipitation with (NH(4))(2)SO(4) was identical. (c) The elution profile of nitrate-nitrate reductase and benzyl adenine-nitrate reductase after chromatography on diethyl-aminoethyl Sephadex A 25 columns showed no significant difference. (d) On polyacrylamide gel, the electrophoretic migration of the two enzymes was also identical. PMID- 16658983 TI - Control of Nitrite Reductase Activity in Excised Embryos of Agrostemma githago. AB - When excised embryos of Agrostemma githago were incubated with nitrate, the activities of both nitrate reductase and nitrite reductase were enhanced. By contrast, benzyladenine induced nitrate reductase only. Our data suggest that nitrate affected nitrite reductase activity directly, without first being reduced to nitrite. When the endogenous nitrite production was increased by raising the level of nitrate reductase through simultaneous treatment with nitrate and benzyladenine, the activity of nitrite reductase was not higher than in embryos treated with nitrate alone. On the other hand, tungstate given together with nitrate drastically inhibited the development of nitrate reductase activity without reducing the enhancement of nitrite reductase activity. Nitrite enhanced nitrite reductase activity, though less efficiently than nitrate. PMID- 16658984 TI - Respiration and Gas Exchange in Stem Tissue of Opuntia basilaris. AB - Respiration and gas exchange in the light were studied manometrically with tissue slices from stem material of Opuntia basilaris Engelm. and Bigel. Dark respiration rates were greater in young stems than in mature stems. The timing of the experiment in the day/night cycle influences the magnitude and pattern of respiration and gas exchange in the light. Net dark respiration has a temperature optimum between 35 and 40 C, and is maintained at 60% of the control rate in tissue equilibrated with experimental osmotic potentials of -25 bars. Net gas exchange in the light is regulated by the titratable acidity of the tissue and by the tissue temperature. Increased rates of net CO(2) evolution and net O(2) consumption occur in the light with high levels of titratable acidity and high temperatures. An efflux of CO(2) and influx of O(2) occur following light/dark transitions. These patterns are reversed following dark/light transitions. Similar results were demonstrated at 15, 25, and 35 C, and are interpreted as a mechanism of adaptation to desert environments. PMID- 16658985 TI - Effect of pH and Auxin on Chloride Uptake into Avena Coleoptile Cells. AB - The effect of pH on (36)Cl(-) movement into coleoptile cells (Avena sativa L. cv. Garry) was investigated and compared with effects of indoleacetic acid. (36)Cl(-) uptake, but not efflux, is stimulated when coleoptile sections are placed in media adjusted to pH levels from 5 to 3 after a preincubation period at pH 6.5. The enhancement is seen within 2 minutes, is not correlated with growth, and is completely erased by respiratory inhibitors. In comparison to the acid-induced stimulation, the stimulatory effect of indoleacetic acid on (36)Cl(-) uptake is also not accompanied by accelerated efflux, and indoleacetic acid does not further stimulate (36)Cl(-) uptake into 1-millimeter sections beyond that seen at pH 3.5 without auxin. PMID- 16658986 TI - Partial Purification and Properties of a beta-d-Glucosyltransferase Occurring in Germinating Phaseolus aureus Seeds. AB - A soluble enzyme that transfers d-glucose from uridine diphosphate d-glucose to low molecular weight hydroxyl compounds has been discovered in germinating mung bean (Phaseolus aureus) seeds and purified 220-fold. Phenol and n-butyl alcohol are the most reactive aceptors examined. The reactivities of various acceptors are largely independant of hydroxyl group acidities or of the electronic properties of the acceptors. The space-filling properties or length of the acceptors, on the other hand, appear to be the predominant controlling factor which determines their relative abilities to accept d-glucose, the ideal size being that of phenol and n-butyl alcohol. The enzyme is deactivated by p chloromercuribenzene sulfonate; dithiothreitol imparts considerable stabilization to the enzyme. It does not require exogenously added metal ions but is slightly stimulated by magnesium ion. On the other hand, the enzyme is partially inhibited by 10 mm manganous ion, cobalt ion, ferrous ion, and ethylenediaminetetraacetate. Zinc ion at 10 mm concentrations strongly inhibits the enzyme. The molecular weight of the enzyme, determined by gel filtration, is approximately 62,000. PMID- 16658987 TI - Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose. AB - Incubation of mature sweet corn kernels of Zea mays in dilute solutions of (14)C labeled indole-3-acetic acid leads to the formation of (14)C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O (indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O (indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O (indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of (14)C-labeled indole-3 acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide. PMID- 16658988 TI - Effects of Germination-promoting Substances Given in Conjunction with Red Light on the Phytochrome-mediated Germination of Dormant Lettuce Seeds (Lactuca sativa L.). AB - Ethylene or thiourea can substitute for gibberellic acid but not for red light in breaking the secondary dormancy induced by extended dark storage of fully hydrated lettuce seeds (Lactuca sativa cv. Grand Rapids). After 10 days of storage, ethylene, thiourea, or gibberellic acid applied either separately or in any combination in conjunction with red light induced near maximal germination. When applied separately without red light, none of the substances promoted germination of seeds stored 10 days. Combinations of any two or all three of the substances in the absence of red light induced some germination but no combination was as effective as any single substance given with red light. PMID- 16658989 TI - Influence of Phenolic Acids on Ion Uptake: IV. Depolarization of Membrane Potentials. AB - The membrane potentials of aged, excised barley (Hordeum vulgare L.) root cells were rapidly depolarized by the addition of salicylic acid (o-hydroxybenzoic acid) to the buffered medium bathing root segments. Initial values for membrane potentials were restored very slowly (within 100 minutes) by replacing the phenolic solution by phenolic-free buffer. Several other naturally occurring benzoic and cinnamic acids depolarized cell membrane potentials. The cinnamic acids consistently caused a greater depolarization than the correspondingly substituted benzoic acids. A strong positive correlation was found between the depolarization values (DeltaE) for the benzoic acids and their lipid solubilities. This study supports the hypothesis that the inhibition of ion uptake brought about by naturally occurring phenolic acids is caused by a generalized increase in membrane permeability to inorganic ions. PMID- 16658990 TI - The Metabolism of Oat Leaves during Senescence: II. Senescence in Leaves Attached to the Plant. AB - The course of senescence in the first leaves of light-grown Avena seedlings when attached to the plant has been compared with that previously studied in detached leaves and leaf segments. Proteolysis in the leaf, whether attached or detached, is accompanied by markedly polar basipetal transport of amino acids. This polar transport can be superimposed on the known transport of amino acids towards a locally applied cytokinin. In the intact plant, it results in a strong movement into the roots. The reducing sugars, which are set free in senescence, do not participate appreciably in this polar transport phenomenon.If cytokinin is applied directly to the roots, then, if the plants are 7 days old, it becomes transported upward into the basal parts of the leaf and there delays the senescence process. However, if the plants are 10 days old, a "reverse Mothes effect" occurs, in which senescence of the leaf is actually promoted and part of the amino acids transported into the roots is synthesized into root protein. PMID- 16658991 TI - Growth rate and turgor pressure: auxin effect studies with an automated apparatus for single coleoptiles. AB - Because turgor pressure is regarded as the driving force for cell extension, any general theory of plant growth requires quantitative information on the relationship between steady irreversible growth rate and turgor pressure. To investigate contrasting views of this relation an automated apparatus was constructed which perfused both the outer and inner epidermis of a single coleoptile while its growth rate was continuously recorded. Turgor was altered abruptly by perfusing with solutions of varying tonicity. With specially grown rye coleoptiles the half-time of the osmo-elastic response was reduced to 2 minutes or less. After decay of this response, however, rate continued to change (so as to partially compensate the effects of the turgor shift in question) for 30 to 60 minutes. Only then could a steady rate be taken. A characterization of steady rate versus turgor covering five turgor values for a single coleoptile thus required many hours. The conclusions are as follows. (a) The change in steady rate, per unit change in turgor, was much greater +IAA than -IAA. (b) Both auxin and turgor act to reset an apparent stabilizing system whose presence is shown in the partial compensation of the initial response to turgor shifts. The above "extensibility" changes are operational only. They need not reflect changes in the immediate physical extensibility of the wall; they could reflect changes in a process acting on the wall. (c) The growth rate versus turgor relation shows some hysteresis. PMID- 16658992 TI - An Accounting of Horseradish Peroxidase Isozymes Associated with the Cell Wall and Evidence that Peroxidase Does Not Contain Hydroxyproline. AB - Isopycnic equilibrium centrifugation techniques were used to determine whether any horseradish (Amoracia lapathifolia) peroxidase isozymes were associated with hydroxyproline containing moieties. Purified peroxidase, horseradish root extracts, and peroxidase isozymes released from horseradish root cell walls were tested. In no case could any peak of peroxidase activity be found to band with hydroxyproline.A fluorimetric method for measurement of peroxidase activity was used to determine quantitatively the amount of total peroxidase located on horseradish root cell walls. Twenty per cent of the total peroxidase is found in the cell wall fraction after extraction; 93% of this cell wall associated peroxidase can be removed by washing with 2 m NaCl. Some peroxidase isozymes released by salt washing are not found in the cytoplasmic extract. This indicates that not all of the ionically bound peroxidase represents cytoplasmic contamination. The 1.4% of the total peroxidase activity can thus be considered tightly bound to the cell wall. Of this portion, 75% can be solubilized by treatment with a cellulase preparation. One isozyme is released which was not present in the original cytoplasmic extract. PMID- 16658993 TI - Structural and Physiological Changes in Sugar Beet Leaves during Sink to Source Conversion. AB - The onset of export during leaf development was correlated with changes in metabolism and ultrastructure and with patterns of solute distribution in the developing seventh leaf of sugar beet (Beta vulgaris L.) in order to study the cause of initiation of translocation. Infrared gas analysis of carbon dioxide uptake showed a broad peak for net photosynthesis dm(-2) at 35 to 40% final laminar length. Pulse labeling with (14)CO(2) demonstrated that maximum import of translocate occurred at 25% final laminar length; export was first observed at 35% final laminar length. Between 40 and 50% final laminar length a rapid increase in amount of export occurred, primarily as a result of the increase in the area of leaf which was exporting. Whole leaf autoradiography revealed that onset of phloem loading spread basipetally from the leaf tip; loading was initiated at about 22% final laminar length and was essentially complete by 50% final laminar length. Those areas which clearly exhibited loading no longer imported from other parts of the plant while the area in transition still appeared to import label from source regions.There was little difference between source and sink leaf tissue in the kinetic parameters K(j) and J(max) (30) for uptake of exogenous sucrose supplied via free space. The concentration of solutes in sieve elements and companion cells of the sink leaf was highest in the mature tip area and gradually decreased in the direction of the immature base. There appeared to be no dramatic structural transformation within the phloem of the minor veins that was closely correlated with the time when phloem loading or export began. Rather, there appeared to be a gradual differentiation of phloem which resulted in a sizable proportion of the population of minor vein sieve elements and companion cells attaining maturity in the older sink regions prior to initiation of phloem loading. The area of the leaf undergoing development appeared to exhibit the beginnings of phloem loading 30 to 45 hours prior to onset of export. Import continued into the area in transition until the full level of vein loading was attained. Structural maturation of the phloem and onset of phloem loading are felt to be more preparatory in nature rather than immediately causal events which triggered export.The initiation of export out of a developing leaf, we believe, is the result of the increasing solute content within the sieve element and companion cells of the minor veins, in particular. The higher osmotic pressure in the sieve tubes causes a reversal of the previously inward directed gradient and produces a mass flow, through unobstructed sieve elements, out of the new source region of the leaf. PMID- 16658994 TI - Evidence for active Phloem loading in the minor veins of sugar beet. AB - Phloem loading in source leaves of sugar beet (Beta vulgaris, L.) was studied to determine the extent of dependence on energy metabolism and the involvement of a carrier system. Dinitrophenol at a concentration of 4 mm uncoupled respiration, lowered source leaf ATP to approximately 40% of the level in the control leaf and inhibited translocation of exogenously supplied (14)C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mm inhibited rather than promoted CO(2) production, indicating a mechanism of inhibition other than uncoupling of respiration. The 8 mm dinitrophenol also reduced ATP to approximately 40% of the level in the control source leaf and reduced translocation of exogenous sucrose to approximately 10% of the control. Application of 4 mm ATP to an untreated source leaf promoted the translocation rate by approximately 80% over the control, while in leaves treated with 4 mm dinitrophenol, 4 mm ATP restored translocation to the control level. No recovery of translocation was observed when ATP was applied to leaves treated with 8 mm dinitrophenol. The results indicate an energy-requiring process for both phloem loading and translocation in the source leaf.Application of (14)C-sucrose solutions in a series of concentrations through the upper surface of a source leaf produced a biphasic isotherm for translocation out of the fed region. A similar dual isotherm was obtained for phloem loading with leaf discs floated on (14)C-sucrose solutions. The first and possibly the second phases were attributed to active, carrier-mediated accumulation in the minor vein phloem. Autoradiography of the tissue confirmed that most of the sucrose was localized in the minor veins. Data from uptake through the abraded surface of intact leaves, the most reliable method, were analyzed by the Hofstee method. Kinetic parameters, analogous to Km and V(max) of enzyme studies, were calculated to be: K(j) = 16 mm and J(max) = 70 mug C/min dm(2) or 490 nmoles sucrose/min.dm(2). Rates for phloem loading and translocation of exogenous sucrose are equal to or greater than those observed for compounds derived from photosynthetically fixed CO(2). The data indicate that a free space sucrose concentration in the region of the minor vein phloem of approximately 20 mm can support translocation at the rates commonly observed for photosynthetically produced sugars. PMID- 16658995 TI - Role of free space in translocation in sugar beet. AB - The involvement of the free space in phloem loading of sucrose was studied in sugar beet source leaves (Beta vulgaris, L.). Sucrose, supplied exogenously to the abraded upper surface of leaves at a concentration of 20 mm, was available for translocation at rates similar to those obtained with photosynthesis. The exogenous sucrose substituted as a source of translocate for assimilate derived from photosynthesis when the latter process was disrupted by plasmolysis of the leaf with 0.8 M mannitol. The mesophyll symplast was not completely disrupted by this treatment, however. Data from the sugar uptake experiments indicate that phloem loading can occur from the free space.Isotope trapping of labeled sugars derived from (14)CO(2) was used to intercept and identify sugars passing through the free space prior to phloem loading. Increased translocation rates induced by 4 mm ATP or increased light intensity were accompanied by increased trapping of sucrose but not of glucose. The data support the view that sucrose passes into the free space prior to phloem loading. PMID- 16658996 TI - Isolation of Flower-inducing and Flower-inhibitory Factors from Aphid Honeydew. AB - The aphid Dactynotus ambrosiae Thomas has been allowed to feed on vegetative or flowering plants of the short-day plant Xanthium strumarium L., and the honeydew which they produce is extracted and tested for an effect on flowering using the long-day plant Lemna gibba L., strain G3 for the bioassay. One zone of flower inducing activity and at least two zones of flower-inhibitory activity are consistently obtained from the honeydew extracts. The levels of flower-inducing and flower-inhibitory activity are not demonstrably different in vegetative and flowering honeydew. The honeydew extracts are inactive on Xanthium but do give some flower induction with the short-day plant Lemna perpusilla Torr., strain 6746. The flower-inducing activity is clearly of plant origin and is present in the phloem since the same active material can be obtained from vegetative or flowering Xanthium by methanol extraction, and honeydew produced by aphids feeding on a chemically defined synthetic diet is completely without flower inducing activity. This is the first report of successful flower induction in the long-day plant L. gibba G3 by some means other than long-day treatment. PMID- 16658997 TI - Identification of the Flower-inducing Factor Isolated from Aphid Honeydew as being Salicylic Acid. AB - Honeydew produced by the aphid Dactynotus ambrosiae when feeding on flowering or vegetative plants of the short day plant Xanthium strumarium contains an active substance capable of inducing flowering in the long day plant Lemna gibba G3. In the present study, this active material has been identified as salicylic acid through the use of gas-liquid chromatography and mass, infrared, and ultraviolet spectrometry. Authentic salicylic acid induces flowering in L. gibba G3 under strict short day conditions with an optimal response at about 5.6 mum. The possible significance of salicylic acid for the control of flowering in Xanthium or L. gibba G3 is discussed. PMID- 16658998 TI - The effect of tonicity and metabolic inhibitors on respiration and ripening of avocado fruit slices. AB - The ripening of avocado (Persea americana Mill.) fruit slices was inhibited whether they were floated in water or in buffered aqueous 0.3 m mannitol, 0.25 m KCl, and sucrose. There was no evidence to support the contention that ripening occurred when the tonicity of the bathing medium was increased. Decreased gaseous exchange is considered to be a major cause of this inhibition because by utilizing a technique that afforded better aeration, slices could be water infiltrated and still ripen normally. Metabolic studies on the ripening of slices using this method indicated that several metabolic inhibitors, malonate, cyanide, acetaldehyde, dinitrophenol, and fluoride did not prevent ripening, but that their effect on the respiration pattern was marked. This technique provides a suitable way to study control of ripening at the tissue level. PMID- 16658999 TI - Some effects of applied gibberellic Acid on the synthesis and degradation of lipids in isolated barley aleurone layers. AB - An analysis of the lipids in isolated barley (Hordeum vulgare L.) aleurone layers after 12 hours incubation in the presence or absence of gibberellic acid showed no quantitative or qualitative changes. Longer incubation periods resulted in some lipid degradation which was greater in the presence of 1 mum gibberellic acid.Glycerolipid synthesis was measured in isolated barley aleurone layers during the first 12 hours of incubation in the presence or absence of gibberellic acid by following the incorporation of (3)H-glycerol. No significant effect of the hormone was found on either the incorporation of glycerol into lipids or on the types of lipid being synthesized. PMID- 16659000 TI - Interference in Carotenogenesis as a Mechanism of Action of the Pyridazinone Herbicide Sandoz 6706: Accumulation of C-40 Carotenoid Precursors Inhibition of beta-Carotene Synthesis and Enhancement of Phytoene Epoxidation. AB - The herbicide Sandoz 6706 (4-chloro-5-(dimethylamino)-2-alpha,alpha,alpha, (trifluoro-m-tolyl)-3(2H)-pyridazinone), when applied as a preplant soil treatment at a concentration of 0.05 mug/g reduced the content of beta-carotene and chlorophylls in 21-day-old wheat seedlings (Triticum aestivum L.) by 55% and 29%, respectively, without affecting the fresh or dry matter of the seedlings. At 0.8 mug/g, the herbicide reduced the content of beta-carotene and chlorophyll by as much as 98%, while the fresh weight of the albino seedlings was reduced by only 24%. The effect of the herbicide on chlorophyll b was much stronger than on chlorophyll a. Time course studies of pigment synthesis in Sandoz 6706-treated seedlings showed that chlorophyll, beta-carotene, cyclic xanthophylls, phytoene, phytofluene, and zeta-carotene were accumulating during the first 7 days after sowing. Later on, there was a sharp decline in the content of chlorophyll and beta-carotene and a gradual reduction in the content of phytofluene, zeta carotene, and cyclic xanthophylls; the content of phytoene remained essentially unchanged. Coinciding with the drop in content of beta-carotene and chlorophyll, there was a remarkable increase in the content of epoxy phytoene. It is suggested that Sandoz 6706 might act as an inhibitor of the cyclization reaction in the biosynthetic pathway of carotenoids and that other effects, such as the bleaching of chlorophyll, are a consequence of this inhibition. PMID- 16659001 TI - Localization of Enzymes of Glycolate Metabolism in the Alga Chlorogonium elongatum. AB - Organelles in homogenates from autotrophic cells of Chlorogonium elongatum were separated on linear sucrose gradients. The distribution of enzymes typical of leaf peroxisomes was determined.Whereas more than 60% of the catalase activity was particulate and recovered in microbodies at a mean density of 1.225 g/cm(3) within the gradient, in most experiments only 5 to 10% (as a maximum 30%) of the NAD-dependent hydroxypyruvate reductase was particulate, and this was recovered principally at density 1.19 g/cm(3). This distribution coincides with that of cytochrome oxidase, malate dehydrogenase, and isocitrate dehydrogenase, the mitochondrial markers. Glyoxylate-glutamate aminotransferase and glycolate dehydrogenase showed a similar distribution pattern to that of NAD-dependent hydroxypyruvate reductase. Thus in Chlorogonium the enzymes of the glycolate pathway are not associated with the microbodies that are recovered at density 1.225 g/cm(3).The single large chloroplasts of the Chlorogonium cells are broken during grinding, and this probably accounts for the finding that NADP-glyoxylate reductase was recovered only in the soluble fractions of the gradient. PMID- 16659002 TI - Globulin-specific Proteolytic Activity in Germinating Pumpkin Seeds as Detected by a Fluorescence Assay Method. AB - The proteolytic activities of alpha-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds (Cucurbita moschata) were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates. Casein, glyceraldehyde-3 P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates. Chymotrypsin, trypsin, pepsin, and bromelain exhibited activity against all or almost all of the protein substrates. The activity of 1 mug of alpha-chymotrypsin or trypsin and 100 ng of pepsin could be easily detected by this method of assay within 4 to 5 minutes depending upon the substrate. The enzyme extracted from 3-day germinated pumpkin seeds exhibited strong activity only against pumpkin seed globulin, weak activity against the globulins of squash and cucumber and casein, and no activity against the other protein substrates. Activity against pumpkin globulin was maximal at pH 7.4. When assayed by an increase in ninhydrin-positive products, the enzyme extract from pumpkin seeds also showed strong activity against pumpkin globulin and weak activity against casein. The 1-anilino-8-naphthalenesulfonate fluorescence method was at least 20 times more sensitive than the ninhydrin method and was 10 to 20 times more rapid. PMID- 16659003 TI - Boron Deficiency in Unfertilized Cotton (Gossypium hirsutum) Ovules Grown in Vitro. AB - Boron deficiency and phytohormone interactions have been studied in unfertilized cotton (Gossypium hirsutum) ovules grown in vitro. Such ovules required exogenous indoleacetic acid and/or gibberellic acid for fiber elongation. Boron also was required for maintenance of fiber elongation and normal morphogenesis throughout 14 days of culture. The amount of exogenous boron necessary for maximum fiber elongation varied among experiments, presumably in relation to endogenous boron levels at anthesis. Some ovular epidermal cells distant from the liquid medium could be induced to elongate into fiber even after 6 days in boron-deficient medium in response to the later addition of boron.Boron deficiency, in the presence of exogenous indoleacetic acid, was characterized by lack of fiber development on the inundated ovular surface and reduced fiber growth on the ovular surface exposed to air. In the presence of gibberellic acid, boron deficiency was characterized by complete absence of fiber and callusing of the entire ovular surface. When both indoleacetic acid and gibberellic acid were added, the lack of boron resulted in proliferation of callus laterally and upward from the inundated epidermis, accumulation of brown pigments (presumably phenolic compounds) in the callus, and restriction of fiber to a small area of the upper ovular surface. PMID- 16659004 TI - Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells. AB - A systematic evaluation disclosed the following conditions to be optimum for the isolation of viable protoplasts from cultured cells of Nicotiana tabacum L. ;Bright Yellow' grown in liquid suspensions: (a) the cell culture in the early phase of cell number increase, (b) an enzyme mixture of 1% cellulase "Onozuka" and 0.2% Macerozyme, (c) an enzyme solution pH of 4.7 or 5.7, (d) a 2- to 3-hr incubation period, (e) 5 ml of enzyme solution per 500 mg cells and contained in a 50-ml Delong flask, (f) agitation on a gyrotory shaker at 50 rpm, and (g) 0.3 to 0.8 m mannitol as osmoticum in the cell enzyme mixture. The incubation temperature may be varied from 22 to 37 C. The procedure enabled 30% of the tobacco cells to form protoplasts, 80% of which regenerated cell walls in 4 days and 40% resumed cell division activity when returned to cell culture medium. PMID- 16659005 TI - In vivo molybdate inhibition of sulfate transfer to porphyridium capsular polysaccharide. AB - Active transport of exogenous sulfate into log phase cells of Porphyridium aerueineum followed Michaelis-Menten kinetics, and the apparent Km for sulfate transport is approximately 2.5 x 10(-6)m. Molybdate, also a group VI anion, is a competitive inhibitor of sulfate transport, and the inhibition is freely reversible. Once in the cell, molybdate depresses the rate of sulfate pool utilization by blocking sulfate transfer to polysaccharides destined for secretion to the cell surface. Specifically, molybdate inhibits the formation of adenosine 5'-phosphosulfate and in turn the formation of adenosine 3'-phosphate 5'-phosphosulfate, the activated donor for sulfate transfer reactions. Combined with the previous identification of adenosine 3'-phosphate 5'-phosphosulfate, this is taken as evidence that the adenosine 5'-phosphosulfate/adenosine 3' phosphate 5'-phosphosulfate enzymatic sequence for sulfate activation and sulfate donor reactions is operating in Porphyridium. Thiosulfate is utilized as effectively as sulfate as both a sulfur source for growth and polysaccharide synthesis. PMID- 16659006 TI - Autocatalysis in a reconstituted chloroplast system. AB - In whole plants and intact chloroplasts, photosynthesis does not reach its full rate immediately upon illumination but only after a lag which is believed to reflect an autocatalytic increase in the concentration of carbon cycle intermediates. Autocatalysis has now been observed in a reconstituted system containing envelope-free chloroplasts augmented with ferredoxin and other stromal proteins but only catalytic amounts of ATP and NADP. With ribose 5-phosphate as substrate, the CO(2) dependent O(2) evolution recorded for such mixtures implies rates of "endogenous" or ferredoxin-dependent photophosphorylation as high as 360 mumoles of orthophosphate esterified mg(-1) chlorophyll hr(-1). PMID- 16659007 TI - Endogenous levels of auxin and tumorigenesis in a Nicotiana amphiploid. AB - Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown aseptically on nutrient medium in a controlled environment chamber. At regular intervals after the seeds had been sown, the incidence of tumor formation was scored and plants were harvested. Indole-3 acetic acid was extracted, purified, and assayed spectrofluorometrically. A close correlation in time between onset of tumor formation and decline in endogenous level of indole-3-acetic acid was demonstrated. PMID- 16659008 TI - Penetration of dry seeds with chemicals applied in acetone. AB - Acetone is able to carry chemicals through the seed coats to reach the embryo. The amount of chemical penetrating into the seeds depends on the seed type, permeation time, and the concentration of solution. The chemicals reaching the embryo are biologically effective. PMID- 16659009 TI - Composition and Structure of Protein Bodies and Spherosomes Isolated from Ungerminated Seeds of Sorghum bicolor (Linn.) Moench. AB - Protein bodies and spherosomes have been isolated from mature seeds of Sorghum bicolor (Linn.) Moench by a procedure which successfully disrupts the protein starch complex in the grain. Protein bodies from whole grain are 68% protein and have a distinct border and a monolithic appearance. Those from embryo are 95% protein and have diffuse borders, vacuoles, and appear very granular. Aleurone tissue protein bodies are 46% protein with a structure similar to those from embryo, but possibly are composed of a protein carbohydrate mixture. Spherosomes from all sources are quite similar in composition and structure. They have an average composition of 27% protein, 12% phosphorus, and 8.6% metals. Microscopically, they appear as small vesicles bounded by a wall which is probably composed of protein and the potassiummagnesium salt of phytic acid. PMID- 16659010 TI - Reproductive Growth and Dry Matter Production of Glycine max (L.) Merr. in Response to Oxygen Concentration. AB - Reproductive as well as vegetative parameters of mature soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to altered pO(2) during all or part of the growth cycle were measured. Oxygen concentration was found to be a key factor controlling all phases of reproductive development. Exposure to 5% O(2) from early seedling stage to senescence increased leaf, stem, and root dry weights and reduced seed yields when compared to 21% O(2); exposure to low O(2) during the vegetative growth stage from early seedling to mid-flowering arrested pod but not seed development; exposure from mid-flowering to mid-pod filling almost completely arrested seed but not pod development; exposure from mid-pod filling to senescence arrested seed development at the mid-filling stage.Exposures to 5% O(2) initiated at mid flowering for 1, 2, 3, 5, 10, and 15 days had no effect on seed development when the exposure was up to 3 days and produced almost total arrest when the exposure was 10 or more days, suggesting reversibility. The requirement for O(2) in seed development is independent of CO(2) concentration with similar results produced by subambient O(2) combined with ambient CO(2), elevated CO(2) up to 2000 mul/l or depressed levels of CO(2) with the CO(2)/O(2) ratio as in air. An elevated O(2) atmosphere containing 40% O(2) and ambient or elevated CO(2) inhibited total growth but did not affect the balance of vegetative to reproductive growth.We conclude that an unknown reaction or process requiring at least atmospheric concentrations of O(2) but independent of CO(2) in contrast to photorespiration is necessary for optimization of all phases of reproductive growth and the effect is reversible for exposures of up to 3 days but not for exposures of 10 days or more. We propose that this O(2) phenomenon may be the result of a unique physical process or chemical reaction associated with translocation and accumulation of assimilates in reproductive structures. PMID- 16659011 TI - Influence of Temperature on the Flowering of Lemna perpusilla 6746 Grown under Skeleton Photoperiods. AB - The flowering of Lemna perpusilla Torr. strain 6746 grown under 24-hour skeleton photoperiods consisting of 13- and 10.5-hour dark periods separated by 0.25-hr light pulses is strongly dependent on temperature. When plants are cultured in 50 ml Erlenmeyer flasks containing 20 ml of half-strength Hutner's medium supplemented with 1% (w/v) sucrose maximum, per cent, flowering occurs at 23 C. At temperatures above and below 23 C a marked decline in per cent flowering is seen. PMID- 16659012 TI - Influence of timing and number of consecutive inductive photoperiodic cycles on the flowering of lemna. AB - Requirements for flowering of the short day plant Lemna perpusilla Torr. strain 6746 can be studied by interposition of varying numbers of consecutive short days during 7 days of continuous light. A single inductive cycle can cause the formation of few flowers if it comes during the middle of a 7-day period of continuous light. Three inductive cycles cause 30% or more of the fronds to flower if the cycles are properly spaced in the 7-day period. The fact that timing of the inductive photoperiodic cycles is critical indicates the importance of development time and abortion of evoked floral primordia in the flowering response. These results are particularly useful in studies of processes occurring during induction. PMID- 16659013 TI - Reversal of the effects of a night interruption in lemna by inhibitors of ribonucleic Acid synthesis. AB - The inhibition of flowering of Lemna perpusilla Torr. strain 6746 caused by a light break can be partially reversed by treatment with actinomycin D or 2 thiouracil. Actinomycin D is most active in reversing the response to a light break if the inhibitor is present in the fronds at the time the light break is administered. PMID- 16659014 TI - Multiple forms of invertase in developing oat internodes. AB - Three different invertases are found in the developing internodes of oat (Avena sativa cv. Victory). Two soluble invertases (I and II) are separable on diethylaminoethylcellulose and Sephadex columns. They are further distinguished by their kinetic constants, heat stability, and differences in stability and apparent activity optima in response to pH treatments. Relative activities of the two soluble isozymes change considerably during the developmental stages examined. Invertase I activity rises early and begins to fall after maximal activity is reached at 6 hours of incubation. This early increase in activity accompanies the period of most rapid growth rate of the internode. Invertase II activity does not increase significantly during the first 6 hours of internode extension, but rapidly rises to a maximum activity at 16 hours, then declines. The third form of invertase, bound invertase (III), is present in both immature and mature stem tissue. Its activity increases (by 6 hours) during immature growth stages, decreases considerably with maturation, and remains relatively constant in mature tissue. PMID- 16659015 TI - Effect of Disalicylidenepropanediamine on the Light-dependent Reduction of Carbon Dioxide and Glycerate 3-Phosphate in Intact Spinach Chloroplasts. AB - Disalicylidenepropanediamine (DSPD) at 0.1 to 1 mm levels inhibited light dependent (14)CO(2) assimilation in intact spinach chloroplasts about 50 to 80%, and this inhibition was accompanied by an increased ratio of (14)C-glycerate 3 phosphate to (14)C-glyceraldehyde 3-phosphate. Enzymatic analysis established that DSPD also inhibited the light-dependent reduction of glycerate 3-phosphate in intact spinach chloroplasts. DSPD at 0.5 mm did not inhibit ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, glycerate 3-phosphate kinase, NADP(+) linked glyceraldehyde 3-phosphate dehydrogenase or ribulose 1,5-diphosphate carboxylase. The inhibition of chloroplast (14)CO(2) assimilation by DSPD appeared to be related to the inhibition of the photosynthetic electron transport chain. These observations are consistent with experimental results which demonstrated that DSPD inhibited directly the chloroplast lamellar membrane mediated, light-dependent reduction of ferredoxin (Trebst, A. and M. Burba, 1967, Z. Pflanzenphysiol. 57: 419-433 and Ben-Amotz, A. and M. Avron, 1972, Plant Physiol. 49: 244-248). PMID- 16659016 TI - Characterization of Inositol-containing Phosphosphingolipids from Tobacco Leaves: Isolation and Identification of Two Novel, Major Lipids: N Acetylglucosamidoglucuronidoinositol Phosphorylceramide and Glucosamidoglucuronidoinositol Phosphorylceramide. AB - A method for a large scale extraction of phosphoglycosphingolipids from the leaves of Nicotiana tabacum L. has been developed. The phosphosphingolipid concentrate consists of a dozen or more polar lipids as judged by thin layer chromatography. Two of these lipids were purified by chromatography on porous silica beads and partially characterized. These lipids are formulated as: N acetylglucosamidoglucuronidoinositol phosphorylceramide and glucosamidoglucuronidoinositol phosphorylceramide. Although not fully characterized, the other lipids in the concentrate are inositol-containing phosphosphingolipids with a higher carbohydrate content. PMID- 16659017 TI - Studies on Phospholipid-synthesizing Enzyme Activities during the Growth of Etiolated Cucumber Cotyledons. AB - The enzymatic incorporation of sn-glycerol 3-phosphate into lipid by extracts of cucumber (Cucumis sativus) cotyledons showed an absolute requirement for ATP (saturation 2 mM). The incorporation was stimulated 4-fold by 0.2 mM oleate. Ethyldiaminetetraacetate stimulated the incorporation at concentrations below 1 mM and inhibited at higher concentrations. Mg(2+) did not affect the reaction. Triton X-100 and Cutscum inhibited the reaction, while a third detergent, Span 80, was stimulatory. p-Mercuribenzoate was inhibitory. The enzymatic reaction has a pH optimum in the range of 8.8 to 9.6. The Michaelis constant was 112 muM for sn-glycerol 3-phosphate. The major amount of product was phosphatidic acid, the remainder was diacylglycerol, monoacylglycerol, and an unknown phospholipid.The activity profiles for two glyoxysmal enzymes, malate synthetase and catalase, were compared to the activities of four enzymes involved in phospholipid synthesis. Phosphatidylcholine and phosphatidylethanolamine synthesis paralleled the activity profiles of catalase and malate synthetase, as well as the levels of endogenous diglycerides. sn-Glycerol 3-phosphate incorporation peaked at a later stage of cucumber cotyledon growth than the glyoxysomal enzymes and seemed to be the major pathway of phosphatidic acid synthesis. Diglyceride phosphokinase activity did not reach appreciable levels during the first 11 days of cucumber cotyledon growth. PMID- 16659018 TI - Properties of Photosynthetic Mutants Isolated from Euglena gracilis. AB - Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation. PMID- 16659019 TI - A Method for Producing, Selecting, and Isolating Photosynthetic Mutants of Euglena gracilis. AB - A method was developed for the isolation of photosynthetic mutants of Euglena gracilis. It consists of the following steps. (a) Incubation of the cells under phototrophic conditions in the presence of 3 (3,4-dichlorophenyl)-1, 1 dimethylurea for 1 week. This step caused a drastic reduction in the number of chloroplasts per cell; (b) mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine; (c) phototrophic growth for a few days to allow for phenotype expression; (d) selection by incubation in the presence of arsenate under phototrophic conditions for 2 days; (e) plating and growth under photoorganotrophic conditions; (f) assay of green colonies for ability to evolve oxygen. About 10% of the green colonies were found to be deficient in their ability to evolve oxygen. In principle the method may prove suitable for the isolation of other types of mutants of Euglena. PMID- 16659020 TI - Ozone-induced Fatty Acid and Viability Changes in Chlorella. AB - Ozone-treated cells of the photosynthetic green alga Chlorella sorokiniana var. pacificensis exhibit an exponential decline in viability, as measured by their ability to form colonies on agar plates. Postexposure conditions appear to have little, if any, effect on this rate of decline. Except in young (early exponential phase) cells, culture age did not affect this rate. The decline in cell viability was correlated with the production of malondialdehyde, arising from the oxidative breakdown of an ozonide of unsaturated fatty acid material. The loss of fatty acids is substantiated by gas-liquid chromatography. A loss of 5 x 10(-15) moles of fatty acid per cell corresponds to 75% nonviable cells after 50 minutes of ozone exposure. PMID- 16659021 TI - Oxygen availability in polyethylene glycol solutions and its implications in plant-water relations. AB - The solubility of O(2) in polyethylene glycol 4000 and 6000 solutions of varying concentrations was determined iodimetrically (titrimetrically) and electrochemically using a rotating glassy carbon electrode and a PAR Model 174 Polarograph. The titrimetric determination resulted in the formation of an unexpected precipitate at 2% (w/v) polyethylene glycol corresponding to the approximate critical micelle concentration of the two polyethylene glycol homologs. Beyond 5% polyethylene glycol, O(2) concentration was inversely proportional to polyethylene glycol concentration, and was higher in polyethylene glycol 4000 solutions than in polyethylene glycol 6000. The electrochemical data are a direct measure of O(2) transport to the electrode surface, rather than O(2) activity or concentration. Results indicate that even at relatively high H(2)O potentials, the transport of O(2) to the root surface might be insufficient to meet the plant's respiratory requirements. PMID- 16659022 TI - Isolation of Polysaccharides Sulfated during Early Embryogenesis in Fucus. AB - Beginning 10 hours after fertilization, zygotes of Fucus distichus L. Powell incorporate (35)S into polysaccharides as a sulfate ester of fucose. These sulfated polysaccharides are sequestered in only the rhizoid cell of the two celled embryo and can serve as a marker of cellular differentiation. Zygotes were pulsed at different times after fertilization with Na(2) (35)SO(4) to identify and isolate the fucans localized within the region of cytoplasm destined to become the rhizoid cell. Low molecular weight pools of (35)S were saturated within 60 minutes, with the greatest incorporation into ethanol-soluble and insoluble fractions occurring with 0.1 mm Na(2)SO(4) in the artificial sea water medium. At the time of rhizoid formation, four fucose-containing polysaccharide fractions incorporated (35)S. When each fraction was subjected to diethylaminoethyl chromatography, two components were eluted with KCl that contained over 84% of the fucose and 93% of the (35)S of the particular fraction. Highvoltage paper electrophoresis of each fraction also resulted in the separation of these two major components. Both components from each of the four fractions behaved identically when separated by diethylaminoethyl chromatography and paper electrophoresis. By comparing the incorporation of (35)S into the polysaccharide fractions at 4 and 16 hours after fertilization, the fucan-sulfate components that are localized in the cytoplasm at the time of rhizoid formation were isolated. Although sulfated polysaccharides in brown algae are reported to be very heterogeneous in terms of their sugar composition and complexes with other heteropolymers, we propose that there are two major components that are sulfated during early embryogenesis in Fucus. The location of these two sulfated polysaccharides in different chemical fractions may reflect their subcellular localization (e.g., cytoplasmic vesicles or cell walls), or their association with other heteropolymers. PMID- 16659023 TI - The glycolate pathway and photosynthetic competence in euglena. AB - The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated. PMID- 16659024 TI - The reaction of coumarins with horseradish peroxidase. AB - The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.Peroxidase catalyzed coumarin oxidation requires either an electron donor and molecular oxygen or hydrogen peroxide. If peroxide is present, the reaction is mediated by peroxidase compound II which reacts rapidly and stoichiometrically with scopoletin. Different oxidation products are formed, depending on whether IAA or hydrogen peroxide promotes the reaction. A scopoletin free radical intermediate has been isolated from the peroxide reaction mixture but was not detected in the peroxide-free system.When indole-3-acetate is the electron donor, reduced peroxidase combines with molecular oxygen to give peroxidase compound III. Added scopoletin is cooxidized with indole-3-acetate. Compared to the scopoletin peroxidation, this reaction is slower and yields fewer coumarin oxidation products. The differences observed between the two scopoletin oxidation pathways reflect: (a) the competition between indole-3-acetate and scopoletin for peroxidase compounds; (b) the lower reactivity of scopoletin with peroxidase compound III compared with peroxidase compound II. The peroxide promoted reaction is eliminated by catalase, while the indole-3-acetate initiated oxidation is not affected by excess quantities of either catalase or superoxidase dismutase. PMID- 16659025 TI - Metabolism of H-Gibberellin A(1) and H-Gibberellin A(4) by Phaseolus coccineus Seedlings. AB - [(3)H]-Gibberellin A(1) (GA(1)) and (3)H-GA(4) were applied separately to Phaseolus coccineus seedlings grown under red light. (3)H-GA(1) was converted to a compound with gas-liquid radiochromatography retention times identical to those of GA(8). (3)H-GA(4) underwent conversion to at least three metabolites, none of which corresponded to GA(1-38). The rate of metabolism of (3)H-GA(4) was significantly higher than that of (3)H-GA(1). PMID- 16659026 TI - The control properties of phosphofructokinase in relation to the respiratory climacteric in banana fruit. AB - Glucose 6-phosphate, fructose 6-phosphate, fructose 1, 6-diphosphate, and triose phosphates, and the enzymes phosphofructokinase, aldolase, and glucose 6 phosphate dehydrogenase were extracted from banana fruit (Musa cavendishii, Lambert var. Valery) at the (a) preclimacteric, (b) climacteric rise, (c) climacteric peak, and (d) postclimacteric stages of ripening. The level of fructose 1, 6-diphosphate increased 20-fold whereas the concentration of other intermediates changed no more than 2.5-fold between stages a and c. For these same extracts, phosphofructokinase activity increased 2.5-fold whereas the activity of glucose 6-phosphate dehydrogenase and aldolase changed only fractionally. Substrate saturation studies (fructose 6-phosphate) of phosphofructokinase activity showed a decrease in the [S](0.5) from 5.6 to 1.7 mM betwen stages a and c. The enzyme from both sources seems to be regulated by a negative cooperative effect with the control being more stringent in the enzyme from stage a. The difference in enzyme activity is consistent with the increase in respiratory activity between the two stages. PMID- 16659027 TI - The effect of nucleotides and inhibitors on respiration in isolated wheat mitochondria. AB - The effect of mono-, di-, and trinucleoside phosphates and respiratory inhibitors on respiration in winter wheat (Triticum aestivum L. cv. Rideau) mitochondria has been examined. When added during state 4 respiration, subsequent to addition of ADP, all of the dinucleotides stimulated oxidation and induced respiratory control with all substrates examined. Similar results were obtained with AMP, but other mononucleotides and all trinucleotides did not affect the rate of oxidation. Nucleoside diphosphates did not stimulate respiration when added prior to the addition of ADP, but subsequent addition of AMP, ADP, or ATP re established coupled respiration in the presence of the dinucleotides.The duration of 2, 4-dinitrophenol stimulated respiration during oxidation of alpha ketoglutarate was found to be dependent on the amount of AMP, ADP, or ATP added, either prior, or subsequent to, addition of the uncoupler. The addition of oligomycin during 2, 4-dinitrophenol stimulated respiration reestablished coupled respiration with low ADP/O ratios, when added after addition of ATP or conditions which allow formation of ATP from added ADP. The nucleoside diphosphates, other than ADP, did not stimulate oxidation of alpha-ketoglutarate in the presence of 2, 4-dinitrophenol until a small amount of adenine nucleotide was added to the system. The results suggest that dinucleotides other than ADP, are able to participate in the energy conversion processs of the mitochondria, probably via transphosphorylation reactions. PMID- 16659028 TI - Kinetics and Energetics of Light-driven Chloroplast Glutamine Synthesis. AB - Chloroplasts contain the enzyme glutamine synthetase. Formation of glutamine by isolated chloroplasts is light-dependent and requires an intact outer envelope. Addition of exogenous glutamic acid, as well as nitrogen donors such as nitrite or ammonium, stimulate the synthesis of this amide. Photosynthetic generation of ATP satisfies the light requirement of glutamine synthesis. The process is supported by cyclic as well as noncyclic photophosphorylation. PMID- 16659029 TI - The Structure of Plant Cell Walls: VII. Barley Aleurone Cells. AB - The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls. PMID- 16659030 TI - Citrate cleavage enzymes from developing soybean cotyledons: incorporation of citrate carbon into Fatty acids. AB - Data are presented which demonstrate a citrate cleavage enzyme in the supernatant of a developing soybean (Glycine max L. Merr., var. Harosoy 63) cotyledon homogenate following a 126,000g spin for 2 hours. Activity of the enzyme was observed directly in the supernatant enzyme preparation and in a desalted supernatant preparation by measuring the formation of acetylhydroxamate. Acetylhydroxamate production was dependent on citrate and coenzyme A. The reaction increased with time, citrate, and coenzyme A concentrations.Involvement of the enzyme in lipid synthesis was investigated by the incorporation of carbon from citrate-1,5-(14)C into fatty acids. Incorporation shows a pH optimum at 8.5, a temperature optimum at 30 C, and a dependence on ATP and coenzyme A. The reaction is linear throughout the range of extract concentrations tested and is linear as a function of time for 1 hour. Isotope was distributed primarily in unsaturated fatty acids. PMID- 16659031 TI - Acid Hydrolases and Autolytic Properties of Protein Bodies and Spherosomes Isolated from Ungerminated Seeds of Sorghum bicolor (Linn.) Moench. AB - Protein bodies and spherosomes isolated from mature seeds of Sorghum bicolor (Linn.) Moench have measurable activity of acid protease, alpha-glucosidase, beta glucosidase, beta-galactosidase, phytase, acid pyrophosphatase, p-nitrophenyl phosphatase, and RNase. Protein bodies have largely insoluble activities, and produce soluble protein and soluble amino nitrogen during autolysis. They have the dual function of protein storage and protein catabolism. Spherosomes have considerable amounts of soluble enzymes and autolytically produce soluble amino nitrogen and inorganic phosphate but release little soluble protein. Spherosomes are similar to animal lysosomes but have an additional storage function for protein, phosphorus, and metals. Mature sorghum seed contains the necessary enzymes and substrates to generate two basic metabolites, amino acids and inorganic phosphate. PMID- 16659032 TI - The mechanism of ethylene and cyanide action in triggering the rise in respiration in potato tubers. AB - Ethylene and cyanide induce a sharp increase in respiration in potato tubers (Solanum tuberosum, var. Russet) attended by changes in the glycolytic intermediates which indicate that both gases enhance glycolysis. The level of sucrose also increases in response to both treatments. The data are taken to indicate that both cyanide and ethylene either activate or affect the link between the conventional electron transport chain and the cyanide-insensitive path. It is further proposed that this activation may well be the primary event leading to the rise in respiration. Ethylene increases the level of adenosine 5' triphosphate and it is suggested that because of the 4- to 6-fold increase in the rate of electron flux through site I, which continues to operate in the over-all cyanide-insensitive path, the absolute levels of adenosine 5'-triphosphate will also be expected to increase in the presence of cyanide. The increase in sucrose content is considered to be the consequence of the rise in adenosine 5' triphosphate concentration. PMID- 16659033 TI - Inhibition of in Vivo Conversion of Methionine to Ethylene by l-Canaline and 2,4 Dinitrophenol. AB - l-Canaline, a potent inhibitor of pyridoxal phosphate-mediated reactions, markedly inhibited the conversion of methionine to ethylene and carbon dioxide by apple tissue. A 50% inhibition of methionine conversion into ethylene was obtained with 50 mum canaline and almost complete inhibition with 300 mum canaline. When 2,4-dinitrophenol, an oxidative phosphorylation uncoupler, was fed to apple tissue, it inhibited the conversion of radioactive methionine to ethylene by 50% at a concentration of 60 mum and by 90% at a concentration of 100 mum. Production of labeled carbon dioxide from acetate-1-(14)C was increased by 2,4-dinitrophenol, indicating that the inhibition of ethylene production was due to uncoupling of phosphorylation. Auxin-induced ethylene production by mungbean (Phaseolus mungo L.) hypocotyl sections was similarly inhibited by these inhibitors.These results support the proposal that pyridoxal phosphate is involved in the formation of ethylene from methionine, substantiate the requirement for ATP in ethylene production, and suggest that this ATP requirement occurs in the step (s) between methionine and ethylene. The biosynthetic mechanism probably involves activation of methionine by ATP followed by a pyridoxal phosphate-mediated gamma-elimination. PMID- 16659034 TI - Plasma membrane adenosine triphosphatase of oat roots: activation and inhibition by mg and ATP. AB - ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L. cv. Goodfield) roots was examined in the presence of various concentrations of MgCl(2) and ATP. A Mg(2+): ATP ratio of about 1 was required for maximal activity regardless of the concentrations used; the optimum concentration for both Mg(2+) and ATP was 9 mm. Based on the ATPase activity at different concentrations of complexed Mg.ATP and free ATP, it is concluded that Mg.ATP is the true substrate of this enzyme.Under certain experimental conditions, high concentrations of MgCl(2) and ATP inhibited the plasma membrane ATPase. On the basis of the relative amounts of free and complexed ATP and Mg(2+), it was found that the different moieties caused different amounts of inhibition. Free ATP inhibited the ATPase at concentrations in excess of 2 mm. Mg.ATP concentrations above 11 mm inhibited the enzyme. Free Mg(2+) caused only a slight inhibition of the ATPase.The Km for Mg.ATP was found to vary from 0.64 to 1.24 mm depending on the experimental conditions. This variation is thought to be due to variable amounts of Mg.ATP, which serves as an inhibitor as well as the substrate, and free ATP, which also inhibits the enzyme. PMID- 16659035 TI - The pathway of carbon dioxide fixation in crassulacean plants. AB - Combined gas chromatography-mass spectrometry of malic acid derivatives has been used to show unequivocally that malic acid, synthesized during active acid accumulation in the dark by Kalanchoe daigremontiana Hammet et Perrier in the presence of (13)CO(2) is produced by a pathway involving a single carboxylation. The significance of the finding that crassulacean malate synthesized in the dark and in the presence of (14)CO(2) often contains 66% of the total carboxyl label in carbon atom 4, which has previously been taken to indicate the operation of a double carboxylation pathway or has been dismissed as an artefact, is discussed. PMID- 16659036 TI - Cytokinin effects on growth of quiescent tobacco pith cells. AB - Excised pith tissue from Nicotiana glauca or the tumor-prone hybrid N. glauca x N. langsdorffii has no growth requirement for exogenous cytokinins. Addition of kinetin to cultures of these lines results in growth inhibition at a kinetin concentration 1000-fold lower than the optimal level for kinetin-requiring lines. Cytological comparison of the kinetin-inhibited 2N hybrid and glauca tissues with pith from the kinetin-requiring N. tabacum var. Wisconsin 38 suggests that the nature of the cytokinin action is similar in both situations and that the primary function of cytokinin, when it stimulates growth, may be to curtail cell expansion, thereby facilitating a balance of cell expansion and division requisite for maximal growth. PMID- 16659037 TI - Glycosidases in Cell Wall-degrading Extracts of Ripening Tomato Fruits. AB - Enzyme preparations were obtained from cell wall debris of tomato (Lycopersicon esculentum L. cv. Tropic) fruits at various stages of ripeness and were assayed for glycosidase and polysaccharidase activities. In addition to polygalacturonase (mol wt 40,000), ripening fruits contain beta-galactosidase (mol wt 63,000) and beta-1, 3-glucanase (mol wt 12,000). The beta-glycosidases, unlike polygalacturonase, are active in extracts of green fruits. Placental tissue shows very low polygalacturonase but increasing beta-galactosidase and beta-1, 3 glucanase activities as ripening proceeds. A large change in the susceptibility of the walls to hydrolase action occurs before the stage in which the greatest polygalacturonase activity occurs. The possibility that the beta-glycosidases contribute to the wall modifications that lead to fruit softening is discussed. PMID- 16659038 TI - Force Required to Detach Conidia of Helminthosporium maydis. AB - The force required to break the conidium-conidiophore attachment in Helminthosporium maydis was measured by centrifugation and by a small jet of air. The force which removed half the spores from their stalks was found by both methods to be about 1 x 10(-2) dynes. The corresponding speed of the air jet was about 10 m/sec. Although spores are removed over a range of applied force, most of this spread is accounted for by the variation in spore size. Therefore, the apparent strength of the attachment of conidia to conidiophores, though relatively large, is surprisingly uniform. PMID- 16659039 TI - Transformations of a large aggregate of hydroxycinnamate hydroxylase to lower molecular weight forms by sulfhydryl agents in green leaves of sorghum. AB - The large, partly microsomal aggregate containing 4-hydroxycinnamate hydroxylase activity isolated from green leaves of Sorghum bicolor at pH 6 was obtained instead as intermediate molecular weight forms when green leaves were ground in the presence of 10 mm mercaptoethanol or dithiothreitol. Elution profiles from agarose (Bio-Gel A-15m and A-1.5m) columns indicated that the 4-hydroxycinnamate hydroxylase activity was due either to multiple forms or to a mixture of forms in various stages of dissociation, the largest being eluted just after the void volume from an agarose 1.5m column. The larger form was similar to the major one found previously in etiolated leaves and was precipitated in the same ammonium sulfate fraction. The activity was unstable, but could be reactivated by incubation of the undiluted enzyme extract alone at 30 C prior to the assay. The data indicate that disulfide bonds are involved in the in vivo formation of the large aggregate in green leaves as well as being necessary for the maintenance of optimal activity of smaller polymeric forms in vitro. PMID- 16659040 TI - Photocontrol of the Germination of Onoclea Spores: II. Analysis of Germination Processes by Means of Anaerobiosis. AB - The oxygen requirements during the three phases of photoinduced germination of Onoclea sensibilis L. spores were analyzed by temporarily applying nitrogen atmosphere. The dark preinduction phase, during which the spores imbibe water and establish sensitivity to irradiation, involves an oxidative process which can be reversibly inhibited and stimulated by nitrogen and air, respectively. The induction phase of germination is characterized by a pure photochemical reaction, independent of temperature and oxygen. The postinduction phase, when the photoproduct triggers dark processes eventually leading to the protrusion of the rhizoidal or protonematal cells, involves an oxidative process which occurs within the first 10 hours of this phase. This oxidative process differs in kinetic characteristics from that in the preinduction phase. The oxidative process is inhibited by nitrogen treatment, but following nitrogen inhibition the ability of the spores to germinate can be reinstated by a long period of air intervening between the nitrogen treatment and a second irradiation. This suggests that enzymes or reactants which are needed in the postinduction process decay under anaerobic conditions and are resynthesized when the spores are transferred to air. Spores take up acetocarmine stain towards the latter part of the postinduction phase. Stain uptake is apparently succeeded very closely by cell division, and some time later by protrusion of the germling cells. PMID- 16659041 TI - Malic dehydrogenase from tamarix roots: effects of sodium chloride in vivo and in vitro. AB - Soluble and mitochondrial malic dehydrogenases (MDH) were isolated from root tips of the halophyte Tamarix tetragyna L. grown in the presence and absence of NaCl. The activity of the enzymes isolated from root tips grown in the presence of NaCl was lower than that of the enzymes isolated from roots grown in absence of NaCl. The mitochondrial MDH was much more sensitive to salinity than the soluble MDH. The soluble enzyme from roots grown in NaCl had a higher Km for malate and lower Km for NAD than enzyme from the control roots. Addition of NaCl in vitro at 72 mM significantly stimulated the reductive activity of soluble MDH, while higher NaCl concentrations (240 mM and above) depressed enzyme activity. The inhibition of enzyme activity by various salts was found to be in the order MgCl(2) > NaCl = KCl > Na(2)SO(4). Mannitol at equiosmotic concentrations had no effect. Substrate inhibition, typical for oxaloacetate oxidation, was not observed at high NaCl concentrations in vitro and high substrate concentrations neutralized the inhibitory effect of NaCl. Increased coenzyme concentrations had no effect. In vitro NaCl increased the Km for malate and oxaloacetate already at relatively low concentrations. At the same time NaCl decreased the Km for NAD and NADH. The inhibitory effect of NaCl on enzyme activity seems not to be due to the effect on the Km alone. Soluble and mitochondrial MDH had different responses to pH changes, mitochondrial MDH being more sensitive. Mitochondrial MDH released from the particles had a similar response to that of the entire particles. Changes of pH modified the effect of NaCl on enzyme activity. It was postulated that NaCl apparently induces conformational changes in the enzyme. PMID- 16659042 TI - The effects of digitonin on photochemical activities of isolated chloroplasts. AB - The addition of digitonin to chloroplasts stimulated the rate of oxygen evolution followed by a gradual inhibition. The effect of digitonin was dependent on the digitonin to chlorophyll ratio and on temperature and time. The initial stimulation of oxygen evolution appeared to be a result of uncoupling as digitonin did not stimulate oxygen evolution by uncoupled chloroplasts. The stimulatory effect occurred more rapidly at high digitonin to chlorophyll ratios but the extent of stimulation was low and inhibition occurred soon after addition of the detergent. The inhibition of electron flow by digitonin was due to a site of action near photosystem II which resembled the inhibition reported for tris buffer and resulted in photobleaching. However, digitonin inhibition could not be recovered by washing with reducing agents and was only partially recovered by the addition of artificial electron donors to photosystem II. Electron flow mediated by photosystem I was unaffected by the addition of digitonin but was decreased when the chloroplasts were separated by subsequent centrifuging. This suggested that digitonin solubilizes photosystem I components which remain active in the soluble form. PMID- 16659043 TI - Chloroplast and Cytoplasmic Enzymes: VI. Pea Leaf 3-Phosphoglycerate Kinases. AB - Pea (Pisum sativum) leaf chloroplastic and cytoplasmic 3-phosphoglycerate kinases (ATP: d-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) have similar Michaelis constants for ATP, 0.7 and 0.55 mm, for ADP, 0.18 and 0.22, and for 3-P glycerate, 0.59 and 0.54 mm at low substrate concentrations, and 1.6 and 1.25 mm at high substrate concentrations. Both enzymes are inhibited by ADP and AMP in the ATP-utilizing direction and by ATP and AMP in the ATP-generating direction and are controlled by energy charge. Apparently, whether the cytoplasmic and chloroplastic kinases in the plant cell will participate in the reductive pentose phosphate cycle and gluconeogenesis or in glycolysis will be determined by the environment in the cell compartment and not by the differential properties of the enzymes themselves. PMID- 16659044 TI - Refractive index of soybean leaf cell walls. AB - The refractive index of soybean (Glycine max [L.] Merr.) leaf cell walls was measured by two methods. The refractive index of fully hydrated walls in the living leaf was about 1.415, while that of dried cell walls was about 1.53. The refractive index of the external surface of the living leaf hair was 1.48. PMID- 16659045 TI - The Carotenoid Hydrocarbons of Euglena gracilis and Derived Mutants. AB - An examination of the carotene fractions extracted from Euglena gracilis Z and pressure-bleached Euglena mutants PR-1, PR-2, PR-3, and PR-4 revealed phytoene in mutants PR-1, PR-2, and PR-3. Photosynthetic E. gracilis Z cultured at different light intensities showed no detectable phytoene, nor was phytoene found in mutant PR-4. However, dark-cultured E. gracilis Z yielded readily assayable amounts of phytoene. With the exception of PR-4, in which no C(40) carotenoids were detected, the following carotenes were identified in all from their mass spectra: phytoene, phytofluene, zeta-carotene, beta-zeacarotene, and beta-carotene. Of these, phytoene and beta-zeacarotene had not previously been unequivocally identified in Euglena. PMID- 16659046 TI - Differential regulation of nitrate reductase induction in roots and shoots of cotton plants. AB - The induction of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.) was regulated by several amino acids and by ammonium. Glycine, glutamine, and asparagine strongly inhibited induction of activity by nitrate and also decreased growth of sterile-cultured roots on a nitrate medium. Methionine, serine, and alanine weakly inhibited induction, and 11 other amino acids had little or no effect. Ammonium also decreased induction in root tips, but was most effective only at pH 7 or higher. The optimum conditions for ammonium regulation of induction were identical to those for growth of sterile-cultured roots on ammonium as the sole nitrogen source. Aspartate and glutamate strongly stimulated induction, but several lines of evidence indicated that the mechanism of this response was different from that elicited by the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake.In green shoot tissues, all attempts to demonstrate regulation of induction by amino acids failed. The great difference in observed responses of root and shoot to amino acids suggests that their nitrate reductase activities are regulated differently. Differential regulation of this enzyme is consistent with the responses of root and shoot nitrate reductase activity to nitrate. PMID- 16659047 TI - Partial reactions of photosynthesis in briefly sonicated chlamydomonas: I. Cell breakage and electron transport activities. AB - The cell structure of Chlamydomonas reinhardi is disrupted by brief exposure to sonication. The extent of cell breakage can be determined quickly by cell count with the light microscope. Rates of photochemical activities of briefly sonicated cells approach those reported for higher plant chloroplasts. These activities are a sensitive function of time of sonication and sonic power used. The method of brief sonication is rapid and convenient and gives a stable preparation useful for determining photochemical activities in Chlamydomonas. PMID- 16659048 TI - Partial Reactions of Photosynthesis in Briefly Sonicated Chlamydomonas: II. Photophosphorylation Activities. AB - Briefly sonicated Chlamydomonas reinhardi cells are capable of both cyclic and noncyclic photophosphorylation and, in each case, the maximum rates approach those reported for higher plant chloroplasts. Photophosphorylation coupled to ferricyanide reduction occurs with a P/2e ratio approaching unity.The conditions for optimum activity are similar to those reported for spinach or swiss chard chloroplasts; the major difference is the extreme sensitivity to salt and relative insensitivity to methylamine. Cell preparation, sonication, and assays were all performed at room temperature under conditions suitable for screening a large number of potential mutants deficient in photophosphorylation activity. This method was easily adapted to Euglena gracilis strain Z but not adaptable for Chlorella vulgaris or Scenedesmus obliquus strain D(3). PMID- 16659049 TI - A kinetic analysis of the effects of gibberellic Acid, zeatin, and abscisic Acid on leaf tissue senescence in rumex. AB - Hormones which inhibit senescence in Rumex leaf tissue in the dark include gibberellic acid and the cytokinin zeatin. Abscisic acid accelerates senescence in this tissue. Other workers have proposed that cytokinins, but not gibberellins, interact with abscisic acid in senescing Rumex leaf tissue. The present study reinvestigates the question of interaction using measurements of chlorophyll degradation kinetics as parameters of senescence rate and draws the conclusion that neither zeatin nor gibberellic acid interact with abscisic acid in this system. In support of this conclusion are these results. Zeatin clearly cannot overcome the effects of abscisic acid when hormone solutions are replaced every other day. The kinetics of chlorophyll breakdown for tissue treated with unreplaced saturating zeatin solutions is different from that of tissue exposed to saturating zeatin plus abscisic acid. The observed rates of chlorophyll breakdown for tissue treated with abscisic acid and zeatin agree closely with predicted rates using a multiplicative model for independent action of the two hormones.Zeatin solutions, when replaced every other day, show up to a 550-fold increase in effective concentration in the retardation of senescence. Less than a 10-fold increase could be accounted for by the addition of more zeatin molecules to the tissue. A nonbiological inactivation of zeatin or the production of an inhibitor of zeatin action by the tissue could not be demonstrated. It seems that zeatin is metabolically inactivated or sequestered in this tissue. The possible physiological significance of the inactivation of cytokinins in leaf tissue is discussed. PMID- 16659050 TI - Inactivation of pea leaf phosphofructokinase by light and dithiothreitol. AB - When intact pea (Pisum sativum L.) plants are illuminated, the glycolytic enzyme phosphofructokinase is inactivated. In crude extracts the enzyme is inhibited by dithiothreitol. It would seem that this cytoplasmic enzyme, like glucose-6-P dehydrogenase, is light-inactivated when the enzymes of photosynthetic carbon metabolism are light-activated. PMID- 16659051 TI - Maize Mitochondria: Cytochromes of Fertile and Cytoplasmic Male-sterile Lines. AB - Cytochromes a + a(3), b, and c of mitochondria prepared from fertile and cytoplasmic male-sterile single- and double-cross maize (Zea mays L.) lines were examined by difference spectra at 25 C. The sterile lines contained the full complement of cytochromes, and absorption maxima were identical for each fertile sterile combination. A small, but significant, increase of b cytochromes was detected in each sterile line, whereas the quantities of cytochromes a + a(3) and c were essentially unchanged. The yield of mitochondrial protein per gram of tissue was higher for the single-than for the double-cross, whereas the sterile cytoplasm did not affect ultimate mitochondrial yield. PMID- 16659052 TI - Phytochrome Characterization by Rabbit Antiserum against High Molecular Weight Phytochrome. AB - Both small and large sizes of phytochrome purified from Garry oat (Avena sativa L. ev. Garry) as well as large phytochrome purified from Newton oat (A. sativa L. cv. Newton), rye (Secale cereale L. cv. Balbo), barley (Hordeum vulgare L. cv. Harrison), and pea (Pisum sativum L. cv. Alaska) seedlings are characterized by a specific antiserum against large Garry oat phytochrome. A spur is observed by double diffusion assay against large and small Garry oat phytochrome indicating only partial identity. In micro-complement fixation assays, large Garry oat phytochrome yields greater activity than small Garry oat phytochrome. In addition, the peak of activity is shifted to a higher antigen concentration with small phytochrome. Phytochrome, red-absorbing form, and phytochrome, far redabsorbing form, are indistinguishable by both double diffusion and micro complement fixation assay. The different grass phytochromes are antigenically identical by double diffusion assay. Immunoelectrophoretic analyses of oat and rye large phytochrome, after proteolysis, suggest that there are one or a few regions of the molecule especially susceptible to hydrolysis by a wide variety of endopeptidases. PMID- 16659053 TI - Immunological and physical characterization of the products of phytochrome proteolysis. AB - The relationship between high molecular weight (large) and low molecular weight (small) forms of phytochrome has been shown earlier to be one of proteolysis. The products of such proteolysis are characterized here by chromatography through Bio Gel P-200 using specific antiphytochrome sera as an assay system. Degradation of large oat (Avena sativa L. cv. Garry) phytochrome as phytochrome, red-absorbing form, phytochrome, far red-absorbing form, or under cycling conditions in crude preparations containing one or more proteases, always yields one fragment with the immunochemical, electrophoretic, spectroscopic, and size characteristics of small phytochrome. In addition, other fragments are detected which may account, in part, for the different molecular weight estimates reported by others for purified, photoreversible phytochrome. The small phytochrome produced by proteolysis with trypsin of a purified large phytochrome preparation is similar to that produced by the endogenously derived protease(s). A large (estimated molecular weight = 90,000), apparently nonphotoreversible peptide is also identified which is electrophoretically and immunochemically distinct from small phytochrome. Thus, it seems that small phytochrome may not represent more than approximately one-half of the native molecule. PMID- 16659054 TI - Comparison of Propylene-induced Responses of Immature Fruit of Normal and rin Mutant Tomatoes. AB - Continuous application of propylene to 40 to 80% mature fruits of normal tomato strains (Lycopersicon esculentum Mill.) advanced ripening in fruits of all ages by at least 50%. Although preclimacteric respiration was stimulated by propylene treatment, there was no concomitant increase in ethylene production. Once ripening commenced, the rates of endogenous ethylene production were similar in both propylene-treated and untreated fruits. Continuous exposure to propylene also stimulated respiration in immature fruits of rin, a nonripening mutant. Although respiration reached rates similar to those during the climacteric of comparable normal fruits there was no change in endogenous ethylene production which remained at a low level. Internal ethylene concentrations in attached 45 to 75% mature fruits of rin and a normal strain were similar. It is suggested that the onset of ripening in normal tomato fruit is not controlled by endogenous ethylene, although increased ethylene production is probably an integral part of the ripening processes. PMID- 16659055 TI - Growth of Chlorella in a Nitrate-limited Chemostat. AB - Chlorella pyrenoidosa was grown in a continuous-flow chemostat under nitrogen limited conditions. The population density tended to oscillate very significantly. Net specific growth rate was only approximately a hyperbolic function of nitrate concentration in the chemostat. The best estimate of the half saturation constant for nitrate is 6 mug of nitrogen per liter and it is unlikely that the value is greater than 14 mug per liter or 1 mum nitrate.The dry weight production of cells per unit of nitrogen taken up is a linearly decreasing function of the net specific growth rate with a maximum of 27.1 mg per mg N and a minimum of about 9 mg per mg N. Thus there is considerable storage of nitrogen at high growth rates. Both the dark respiration rate and the rate of photosynthesis at light saturation increase with increasing net specific growth rate. PMID- 16659056 TI - Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm. AB - A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5. PMID- 16659057 TI - Nucleic Acid Metabolism during Germination of Pima Cotton (Gossypium barbadense). AB - The nucleic acid metabolism in long staple Pima cotton (Gossypium barbadense L.) was examined during the first 48 hours of germination at 34 C. Specific activity analysis of pulselabeled nucleic acids indicated that the first significant incorporation of precursors into DNA occurs at approximately 18 hours when the radicles are emerging with a peak of incorporation occurring at 36 hours. Specific activity of total RNA also indicate peak values at 18 hours and 36 hours of germination. Sucrose gradient analysis of pulse-labeled RNA indicated that the synthesis of ribosomal RNA reached a maximum value during the 18-hour period and that there was a shift in transcriptional products during the 30- to 36-hour interval. The extraction of the RNA from pulse-chase studies and from labeled polyribosomes suggest that a significant portion of the RNA synthesized at 36 hours appears to be messenger RNA and at 18 hours is ribosomal RNA. These distinct patterns of metabolism during germination provide a background for studying nucleic acid metabolism during chilling damage. PMID- 16659058 TI - Chromatography of Cytokinins on a Neutral Polystyrene Resin: A Simple Procedure for the Separation of the cis and trans Isomers of Zeatin or Ribosylzeatin. AB - A simple procedure for the separation of the cis and trans isomers of zeatin and ribosylzeatin by column chromatography on a neutral polystyrene resin, Porapak Q, in aqueous ethanol solutions is reported. The method has been used to examine the stereoisomer composition of ribosylzeatin isolated from wheat germ transfer RNA. Chromatographic data for several other cytokinins are also presented. PMID- 16659059 TI - Biochemical effects of technetium-99-pertechnetate on microorganisms. AB - The biochemical effects of technetium-99 as pertechnetate (TcO(4) (-)) were investigated in a variety of microorganisms (a nonsulfur purple bacterium, five blue-green algae, a protozoan, a diatom, two heterotrophic bacteria, a red alga and two green algae). Sensitivity to pertechnetate as measured by growth ranged from marked inhibition at 1 mug Tc/ml (nonsulfur purple bacterium) to no effect at 600 mug Tc ml (both green algae). No correlation between organism type and growth susceptibility to pertechnetate was apparent. The blue-green alga, Agmenellum quadruplicatum strain PR-6, bound technetium-99 to a level of 3 mug/mg dry weight cells (from medium containing 1.5 mm pertechnetate) in the light, but little or none in the dark; cell death occurred only with uptake. Addition of TcO(4) (-) to the medium caused a rapid but temporary increase in ATP levels of PR-6 (in the light only) and Tetrahymena pyriformis strain WH14. Respiration of organisms WH14 and Bacillus subtilis and photosynthesis of organism PR-6 were immediately slowed by the introduction of pertechnetate. Technetium as pertechnetate has a possible biochemical effect on cells, unrelated to its radioactivity or to a general oxidation effect. PMID- 16659060 TI - Water uptake, diameter change, and nonlinear diffusion in tree stems. AB - A diffusion model for phloem swelling and contraction is proposed in which the rate of water movement changes markedly with moisture content. Good agreement between the actual swelling of the phloem of cotton stems and that predicted by the model was obtained. This result implies that water moves more readily into the phloem when it becomes wetter. This model also explains the lag of shrinkage of pine stems behind the water potential of the foliage and predicts that the lag is related to the thickness of the phloem. PMID- 16659061 TI - Photocontrol of Anthocyanin Synthesis: III. The Action of Streptomycin on the Synthesis of Chlorophyll and Anthocyanin. AB - Streptomycin enhances the synthesis of anthocyanins and inhibits the synthesis of chlorophylls and the development of chloroplasts in dark-grown seedlings of cabbage (Brassica oleracea), mustard (Sinapis alba), tomato (Lycopersicon esculentum), and turnip (Brassica rapa) exposed to prolonged periods of irradiation in various spectral regions. These results suggest that the contribution of photosynthesis to light-dependent high irradiance reaction anthocyanin synthesis in seedlings of cabbage, mustard, tomato, and turnip is minimal, if any at all. So far, phytochrome is the only photoreceptor whose action in the control of light-dependent anthocyanin synthesis in seedlings of cabbage, mustard, tomato, and turnip has been satisfactorily demonstrated. PMID- 16659062 TI - The development of microbodies (glyoxysomes and leaf peroxisomes) in cotyledons of germinating watermelon seedlings. AB - The ontogeny of glyoxysomes and leaf peroxisomes has been examined in the cotyledons of germinating watermelon (Citrullus vulgaris) seedlings. Organelles from the cotyledons were extracted by razor blade homogenization and microbodies were separated by sucrose density gradient fractionation. Both kinds of microbodies have the same mean equilibrium density on sucrose gradients.The development of leaf peroxisomes was examined in seedlings transferred to light at 4 days and 10 to 12 days. In seedlings maintained in darkness to the age of 10 to 12 days, glyoxysomal enzymes virtually disappeared, and the losses were paralleled by a corresponding loss in microbody protein. During this period peroxisomal activity was low and changed only slightly. On transfer to light at this stage, the activity of peroxisomal enzymes rose strikingly. The residual glyoxysomal activity disappeared completely, and the developmental pattern of microbody catalase and microbody protein paralleled the light-induced glyoxysomal disappearance.Similar patterns of microbody development were observed when 4-day old dark-grown seedlings with maximum glyoxysomal activities were exposed to light. The activity of the peroxisomal enzymes increased and the glyoxysomal enzymes disappeared at a faster rate than in darkness. These changes were again paralleled by the accelerated demise of microbody catalase and microbody protein. Thus under both conditions glyoxysomes were selectively destroyed during peroxisomal development, and the amount of peroxisomes produced was insufficient to offset the loss of glyoxysomal protein. The results do not support the contention that glyoxysomes are transformed to leaf peroxisomes in developing cucurbit cotyledons and favor the view that the two kinds of microbody arise independently of each other. PMID- 16659063 TI - Ethylene and the annona flower. AB - The annona (Annona hybrida) flower is protogynous, and reaches its male stage about 26 hours after the beginning of the female stage. A rise in ethylene production was found to precede the male stage. Much more ethylene was produced by the reproductive organs than by the petals, with the anthers producing most of the ethylene. Ethylene treatments advanced the male stage, but not the female stage. Exposing flowers to hypobaric pressure postponed the onset of their male stage. Application of various growth substances causing ethylene production by the flower advanced the male stage. PMID- 16659064 TI - Glycolate oxidase content of microbodies as affected by nitrate. AB - Glycolate oxidase is loosely held by microbodies obtained from etiolated barley (Hordeum vulgare L.) leaves depleted of nitrate. Defined centrifugation conditions cause the complete detachment of the enzyme from the microbodies. Addition of nitrate to these plants brings about a greater retention of glycolate oxidase by the microbodies. Synthesis of a nitrate-induced protein seems to be responsible for the enhanced retention of glycolate oxidase. Catalase, on the contrary, is strongly attached to the microbodies under all nutritional and experimental conditions considered. PMID- 16659065 TI - Diurnal variation in algal acetylene reduction (nitrogen fixation) in situ. AB - Diurnal variation in algal nitrogen fixation was studied in Lake Mendota, Wisconsin, during the summers of 1971 to 1973. Approximately two-thirds of the daily acetylene reduction in the surface decimeter occurred before noon. The decline in acetylene reduction (nmoles/liter.hr) near midday was partially because the algae relocated themselves at greater depths. However, acetylene reducing activity (nmoles per A(663) unit chlorophyll a per hour) also decreased as midday approached. Occasionally algae would resurface near the end of the day. On average, acetylene reduction (nmoles per liter per hour) was maximum at about 0900 Central standard time in the top decimeter, and acetylene reduction between 0830 and 0930 Central standard time represented 13% of the total daily acetylene reduction. Furthermore, acetylene reduction in the top decimeter, on average, represented 3.6% of the total acetylene reduction in the column. Calculation of the contribution by nitrogen fixation to a lake's fixed nitrogen budget is discussed. PMID- 16659066 TI - The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Soybean Hypocotyl Cells: Their Relation to Messenger Ribonucleic Acid. AB - Plant ribonucleic acids which have high adenosine monophosphate concentrations were studied. Purified deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid fractions both contained poly-adenosine monophosphate sequences (those from the latter being longer than those from the former); without these poly-adenosine monophosphate sequences their base compositions were the same. The average poly-adenosine monophosphate sequence from purified tenaciously bound ribonucleic acid was 160 residues long, as measured by gel electrophoresis. However, base hydrolysis and chromatography indicated one 3'-nucleoside (adenosine) per 71 nucleotides, giving a chain length of 72 residues. The dominant species in the cytoplasm, as measured by radioactive precursor incorporation, was tenaciously bound ribonucleic acid, whereas deoxyribonucleic acid-like ribonucleic acid was present in greater amounts in the nucleus. This work provides evidence that deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid represent forms of messenger ribonucleic acid in soybean, with deoxyribonucleic acid-like ribonucleic acid residing in the nucleus, perhaps as the messenger ribonucleic acid precursor, and tenaciously bound ribonucleic acid residing, as the active messenger ribonucleic acid, in the cytoplasm. PMID- 16659067 TI - Preparation and properties of chloroplasts depleted of chloroplast coupling factor 1 by sodium bromide treatment. AB - Chloroplasts were treated with 2 m sodium bromide. The resulting particles lost their ATPase activity and chloroplast coupling factor 1 subunits were detected in the supernatant by means of gel electrophoresis and specific antibodies. The chloroplast coupling factor 1 depleted particles show high rates of Hill reaction with pH optimum shifted toward lower pH. The sodium bromide treatment also abolished the light-induced proton uptake. In the presence of N-methylphenazonium methosulfate light-induced proton release, insensitive to uncouplers, was observed. Addition of dicyclohexylcarbodiimide reversed the light-induced pH changes to the normal proton uptake and increased the pH optimum of the Hill reaction. PMID- 16659068 TI - Phosphorylation of Chromatin-associated Proteins in Lemna and Hordeum. AB - Sterile embryos of barley (Hordeum vulgare) and cultures of Lemna perpusilla have been labeled with (32)Pi and the chromatin proteins prepared and separated by acid-urea and sodium dodecyl sulfate gel electrophoresis. Under these conditions chromatin proteins became labeled and the gel radioactivity profiles which were complex indicated a probable minimum of 15 to 20 proteins phosphorylated with molecular weights ranging from 10(4) to 10(5). The majority of the radioactivity, 80 to 90% of the total, is found in the acidic protein fraction and this can be recovered as serine phosphate after partial acid hydrolysis.Nuclei have been isolated from Lemna and barley and found to possess endogenous kinase activity. In vitro labeling of these nuclei with (32)P-adenosine triphosphate indicated that similar proteins appear to become labeled as in vivo labeling with (32)Pi but the proportions of label in each protein were different. PMID- 16659069 TI - Regulation of the Phosphorylation of Chromatin-associated Proteins in Lemna and Hordeum. AB - This paper represents attempts to observe alterations in the pattern of chromatin protein phosphorylation in Lemna and barley (Hordeum vulgare).As judged by in vitro labeling the phosphorylation profile is substantially altered during germination. This may not be the result of specific tissue differentiation, however, because phosphorylation does not differ markedly between the embryonic root and shoot. Treatment of nuclei from germinating embryos with low concentrations of sodium or potassium chloride produced phosphorylation patterns similar but not identical to those found in nuclei from ungerminated embryos.Treatment of Lemna with abscisic acid in vivo causes substantial alterations in the labeling of three protein bands and part of this may be duplicated by labeling isolated nuclei from treated tissue with gamma(32)P-ATP. Some effects of light/dark transition on Lemna chromatin protein phosphorylation are also described. PMID- 16659070 TI - Characterization of adenosine diphosphate glucose pyrophosphorylases from developing maize seeds. AB - Electrophoretic examination of 22-day-old, normal maize (Zea mays L.) endosperm extracts revealed two zones of adenosine diphosphate glucose pyrophosphorylase activity. The enzymes are identical in terms of Km for glucose 1-phosphate and the effect of 3-phosphoglyceric acid on apparent Km for glucose 1-phosphate. Both enzymatic activities increase with increasing doses of the functional alleles at the shrunken-2 and brittle-2 loci. Molecular weight differences between the two electrophoretic species were inferred from sucrose gradient centrifugation. It is suggested that the two bands of activity represent different aggregation states of the same enzyme because under different extraction conditions, only one enzyme is found. Molecular weight estimates of 237,000 and 253,000 were obtained for the smaller enzyme. It is suggested that this enzyme is an aggregate of several subunits. Comparison of the embryo and endosperm pyrophosphorylases showed the embryo activity to be more heat stable and probably independent of direct shrunken-2 or brittle-2 control. PMID- 16659071 TI - Sulfate transport in cultured tobacco cells. AB - Sulfate transport by tobacco (Nicotiana tabacum L. var. Xanthi) cells cultured on either l-cysteine or sulfate as a sole sulfur source was measured. The transport rate on either sulfur source was low during pre-exponential growth, increased during exponential growth, and was maximal in late exponential cells. The initial increase in transport rate was correlated with a decline in the intracellular sulfate, but was not correlated with the amino acid content of the cells which remained relatively constant before the depletion of the endogenous sulfate pool. The previously reported inhibition of sulfate transport by l-cysteine was shown to be caused by an elevation in intracellular sulfate resulting from the degradation of cysteine to sulfate. It is proposed that the intracellular sulfate pool is the major factor regulating the entry of sulfate into tobacco cells. PMID- 16659072 TI - Ethylene-induced Leaf Abscission Is Promoted by Gibberellic Acid. AB - Gibberellic acid (GA(3)) promoted leaf abscission from cotton (Gossypium hirsutum L.) plants exposed to ethylene. With mature plants, only the rate of abscission was increased, but when vegetative plants were exposed to ethylene for 4 days or less, the amount of abscission was increased markedly. Promotion of abscission occurred at near saturating ethylene levels (10 mul/liter), over a wide range of GA(3) concentrations, and with both GA(3) and GA(7).GA(3) promoted abscission when Ethephon was substituted for ethylene and at locations not receiving direct application of GA(3). The magnitude of the abscission promotion by GA(3) was greater than that resulting from auxin transport inhibitors or abscisic acid. The characteristic inhibition of abscission by auxin occurred. The responses suggest that endogenous gibberellins may be involved in rapid abscission of apical leaves from vegetative cotton plants exposed to ethylene. Application of GA(3) may offer an additional option in agricultural manipulation of abscission and dehiscence. PMID- 16659073 TI - Influence of a Ceratocystis ulmi Toxin on Water Relations of Elm (Ulmus americana). AB - Water-soluble glycopeptides isolated from cultures of Ceratocystis ulmi have been reported to be toxins involved in Dutch elm disease. The influence of the glycopeptides on the water relations of Ulmus americana seedlings was tested by placing cut stems in glycopeptide preparations. After 4 hours in 200 micrograms per milliliter toxin the stem conductance of the seedlings was reduced by 79% and the leaf water potential was reduced by 3 bars to that at which the seedlings wilted, the stomata closed, and transpiration decreased. Decrease in stem conductance as the mode of action of the toxin was further confirmed by forcing toxin through the stem and petiole of elm and measuring the effects on stem conductance. High molecular weight dextrans were found to mimic the action of toxin on stem and petiole conductance, and their ability to do so was found to be correlated with their molecular weight. As low as 4 micrograms of toxin or dextrans were found to measurably decrease the stem and petiole conductance of elms. Disruption of the water-conducting system of elms and other plants by small quantities of high molecular weight compounds may be a factor in diseases with wilting symptoms. PMID- 16659074 TI - The effects of water stress on the development of the photosynthetic apparatus in greening leaves. AB - The effects of low and high relative humidity and of polyethylene glycol-induced root water stress on chlorophyll accumulation, on formation of the lamellar chlorophyll-protein complexes, and on the development of photosynthetic activity during chloroplast differentiation were examined. Low relative humidity or polyethylene glycol-induced root water stress (stress conditions) resulted in a 3 to 4 hour lag in chlorophyll accumulation, retarded the rate of chlorophyll b accumulation, and reduced the rate of formation of the light-harvesting chlorophyll a/b protein. All of these effects could be overcome by high relative humidity (nonstress) conditions. Concomitant measurement of leaf water potential showed that under stress conditions greening leaves were subjected to initial water deficits of -8 bars which decreased to -5 bars after 3 to 4 hours of illumination corresponding to the end of the lag phase. Leaves greening under nonstress conditions did not experience leaf water deficits greater than about -5 bars. It seems that the attainment of a minimum leaf water potential of -5 bars may be critical in the control of early chloroplast development. These results demonstrate that the lag phase is not indicative of a programmed event in chloroplast development, but rather is attributable to environmental conditions prevailing during leaf development and greening. PMID- 16659075 TI - Abscission: the initial effect of ethylene is in the leaf blade. AB - The leaf blade of cotton (Gossypium hirsutum L. cv. Stoneville 213) was investigated as the initial site of ethylene action in abscission. Ethylene applied at 14 mul/l to intact 3-week-old plants caused abscission of the third true leaf within 3 days. However, keeping only the leaf blade of this leaf in air during ethylene treatment of the rest of the plant completely prevented its abscission for up to 7 days. This inhibition of abscission was apparently the result of continued auxin production in the blade since (a) the application of an auxin transport inhibitor to the petiole of the air-treated leaf blade restored ethylene sensitivity to the leaf in terms of abscission; (b) repeated applications of naphthaleneacetic acid to the leaf blade of the third true leaf, when the entire plant was exposed to ethylene, had the same preventive effect on abscission of this leaf as keeping its leaf blade in air; and (c) the inhibitory effect of ethylene on auxin transport in the petiole, which is reduced by auxin treatment, was also reduced by placing the leaf blade in air.The reverse treatment of exposing only the leaf blade of the third true leaf to 14 mul/l of ethylene, while the rest of the plant was kept in air, also did not cause abscission for up to 5 days. Auxin transport in the petioles of these leaves, however, was inhibited over 80% within 2 days and this effect presumably accounted for their increased sensitivity to ethylene during the subsequent exposures of the whole leaf to the gas.These results suggest that an initial and essential function of applied ethylene in abscission is to reduce the amount of auxin transported out of the leaf blade. This reduction together with the inhibitory effect of ethylene on auxin transport in the petiole reduces the auxin level at the abscission zone to a point where the cells in this region become responsive to the more direct action of the gas (e.g., enzyme induction and secretion). This sequence of events accounts for the lack of abscission unless ethylene is applied to both the leaf blade and the abscission zone. PMID- 16659076 TI - Effects of Heat Shock on Growth and on Lipid and beta-Glucan Synthetases in Leaves of Phaseolus vulgaris and Vigna sinensis. AB - Primary leaves of intact plants of Phaseolus vulgaris and Vigna sinensis 7 to 9 days old were dipped in water for 2 minutes at 47.4 +/- 0.2 C and 48.7 +/- 0.5 C, respectively. Leaf growth was inhibited within the first 20 hours, and growth partially or completely recovered in a subsequent 20-hour period. Analysis of the glucolipid and beta-1, 3- and beta-1, 4-glucan synthetases showed that the most evident effects of heat shock were the severe inhibition of the synthesis of beta 1, 4-glucosyl glycosidic linkages and stimulation of beta-1, 3-linkage synthesis. The stimulation appeared either immediately after treatment or within the first 20 hours and lasted at least 60 hours after treatment. Phaseolus plants reacted more intensely to the heat shock, reflecting a possible genetic difference in the response mechanisms. PMID- 16659077 TI - Phycomyces: a change in mechanical properties after a light stimulus. AB - Tensile tests were conducted on the photoresponsive stage IVb sporangiophore of the fungus Phycomyces before and after a saturating light stimulus. The results demonstrate that an increase in the mechanical extensibility of the cell wall occurs after the light stimulus. This increase in mechanical extensibility occurs in the growing zone of the sporangiophore. The majority of this increase occurs in the region about 300 umeters beneath the sporangium. PMID- 16659078 TI - High resolution gel electrophoresis of chloroplast membrane polypeptides. AB - In the present study we extend previous work from this laboratory on the polypeptide composition of photosynthetic lamellae. Using a high resolution sodium dodecyl sulfate gel electrophoresis technique, we show that both grana and stroma lamellae have qualitatively very similar polypeptide compositions although some clear quantitative differences are demonstrated. PMID- 16659079 TI - Plant desiccation and protein synthesis: an in vitro system from dry and hydrated mosses using endogenous and synthetic messenger ribonucleic Acid. AB - The conditions and requirements for an in vitro protein synthesizing system from the moss Tortula ruralis are outlined. Using this system the effects of desiccation, achieved quickly or slowly, were studied. Slowly dried moss retained fewer polyribosomes on desiccation but more active ribosomes than rapidly dried moss. Even in the completely desiccated moss the polyribosomes and/or free ribosomes present have retained their synthetic capacities. On rehydration, the slowly dried moss resumed protein synthesis more quickly than moss previously desiccated rapidly. Moss ribosomes are cycloheximide sensitive and chloramphenicol insensitive and thus the major protein synthesis occurs within the cytoplasm on rehydration. Extracted polyribosomes per se can withstand desiccation to a significant extent, suggesting that protection by the cytoplasm might not be necessary. The aquatic moss Hygrohypnum luridum can retain polyribosomal and ribosomal activity during desiccation, but this decreases greatly on rehydration. PMID- 16659080 TI - Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles. AB - The incorporation of adenosine-8-(14)C into adenosine cyclic 3':5'-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-(14)C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the (14)C present in the original homogenate, migrated as adenosine cyclic 3':5'-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5'-AMP. Deamination with NaNO(2) as well as further chromatographical purification also suggested that only a small fraction of the (14)C in the partially purified samples could be in adenosine cyclic 3':5'-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the (14)C radioactivity in marker adenosine cyclic 3':5' monophosphate-containing fractions at any stage of purification during several experiments.In a single experiment, no labeled guanosine cyclic 3':5' monophosphate could be detected in oat coleoptile-first leaf segments incubated in guanosine-8-(14)C either with or without indoleacetic acid. These results do not support the hypothesis that a cyclic nucleotide mediates the action of indoleacetic acid on oat coleoptile extension. PMID- 16659081 TI - Some Morphogenic Differences between Monoecious and Gynoecious Cucumber Seedlings as Related to Ethylene Production. AB - Exposure of gibberellic acid-treated seedlings of a monoecious cucumber cultivar ;Chipper' (Cucumis sativus L.) to ethylene caused thickening of the hypocotyl, inhibited longitudinal growth, and had no effect on fresh weight. Downward curvature of cotyledons was increased by the presence of ethylene. A gynoecious breeding line, ;Gy 3,' had thicker hypocotyls and displayed its cotyledons in a more downward position than ;Chipper'. Excised hypocotyls of the gynoecious seedlings produced three times as much ethylene as did the monoecious Chipper hypocotyls. Thus, ethylene may play a role in the regulation of cucumber seedling morphology. PMID- 16659082 TI - Stimulation of Phospholipid Biosynthesis during Frost Hardening of Winter Wheat. AB - Lipids were labeled with (33)P during frost hardening of two varieties of winter wheat (Triticum aestivum), hardy Kharkov and much less hardy Champlein. The main labeled compounds were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol. With time of incorporation the proportion of the radioactivity incorporated into the lipids increased in phosphatidylcholine, especially in Kharkov and at 1 C. During hardening, phospholipid synthesis was greatly stimulated in Kharkov, but much less in Champlein. The proportion of the phospholipids synthesized changed only little with hardening, with a trend towards an increase in phosphatidylcholine. Increased phospholipid synthesis does not seem to be a prerequisite to hardening in winter wheat. However, a high rate of phospholipid synthesis may be required to maintain frost resistance. PMID- 16659083 TI - Purification and Some Properties of Chlorella fusca Ribulose 1,5-Diphosphate Carboxylase. AB - Ribulose 1,5-diphosphate carboxylase has been purified from extracts of autotrophically grown Chlorella fusca by ammonium sulfate precipitation and centrifugation on a linear sucrose density gradient. The enzyme was homogeneous by the criterion of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 530,000, and it was composed of two types of subunit of molecular weight 53,000 and 14,000. Ribulose 1,5-diphosphate, CO(2), and Mg(2+) had Michaelis constant values of 15 mum, 0.3 mm, and 0.37 mm, respectively. At high bicarbonate concentration (17 mm and 50 mm), 6-phosphogluconate inhibited the enzyme, the inhibition being noncompetitive with respect to ribulose 1,5 diphosphate (Ki 0.065 mm), whereas at low bicarbonate concentration (1 mm), 6 phosphogluconate activated the enzyme. Oxygen was a competitive inhibitor with respect to CO(2), suggesting the enzyme also functions as an oxygenase. This was confirmed by direct assay, a 1: 1 stoichiometry between ribulose 1,5-diphosphate consumed and O(2) uptake being observed. PMID- 16659084 TI - Cyanide-resistant Respiration of Sweet Potato Mitochondria. AB - The oxidation of malate and succinate by sweet potato mitochondria (Ipomoea batatas [L.] Lam.) was blocked only partly by inhibitors of complexes III (2 heptyl-4-hydroxyquinoline-N-oxide) and IV (cyanide and azide). The respiration insensitive to inhibitors of complexes III and IV was inhibited by salicylhydroxamic acid. Essentially complete inhibition was obtained with inhibitors of complex I (rotenone, amytal, and thenoyltrifluoroacetone) and complex II (thenoyltrifluoroacetone). The observations indicated that electrons were transferred to the cyanide-resistant pathway from ubiquinone or from nonheme iron (iron-sulfur) proteins of complexes I and II before reaching the b cytochromes. In contrast, the oxidation of exogenous NADH did not involve the alternate pathway, as indicated by complete inhibition by inhibitors of complexes III and IV and the absence of an effect of inhibitors of complexes I and II. Hence, electrons from exogenous NADH appear to pass directly to complex III in sweet potato mitochondria. PMID- 16659085 TI - Endogenous ethylene and abscisic Acid relative to phytogerontology. AB - Endogenous production of ethylene and endogenous levels of abscisic acid were measured from Hibiscus rosa-sinensis L. abscission zone explants at six stages of development: tight bud, open flower, closed flower, petal abscission, calyx abscission, and peduncle abscission.Explants acropetal and basipetal to the abscission zone produced less ethylene than the abscission zone explants. Ethylene production increased with time both prior to and during abscission, reaching a peak in the later stages of senescence after abscission was complete.Bound abscisic acid was greatest in segments acropetal to the abscission zone at the closed flower stage. Free abscisic acid was double that of bound abscisic acid in the tight bud stage with the basipetal level exceeding that of the acropetal level until flower closure. Acropetal-free abscisic acid began to rise at petal abscission increasing sharply to a peak at calyx abscission. Both free and bound abscisic acid were greatly reduced at peduncle abscission. A relationship of ethylene and abscisic acid to abscission and senescence appears to exist. PMID- 16659086 TI - Substrate specificity of chlorophyllase. AB - Apparent Km and V(max) values were obtained for hydrolysis of methyl and ethyl chlorophyllides a, methyl and ethyl pheophorbide a, and 9-hydroxymethyl pheophorbide a by chlorophyllase from Ailanthus altissima. Analysis of substrate specificity data for chlorophyllase indicates that the presence of a 9-keto group and a methyl alcohol group esterified at the 7-position in chlorophyll derivatives results in maximum binding affinity for substrates. Data on maximum reaction rates indicate that the rate-controlling step of hydrolysis occurs after release of the alcohol from the ester. Probable high affinity chlorophyllase inhibitors can be predicted on the basis of these specificity studies.An improved method for purification of chlorophyllase has been developed. PMID- 16659087 TI - Acid Phosphatase Development during Ripening of Avocado. AB - The activity and subcellular distribution of acid phosphatase were assayed during ethylene-induced ripening of whole fruit or thick slices of avocado (Persea americana Mill. var. Fuerte and Hass). The activity increased up to 30-fold during ripening in both the supernatant fraction and the Triton X-100 extract of the precipitate of a 30,000g centrifugation of tissue homogenates from whole fruit or slices ripening in moist air. Enzyme activity in the residual precipitate after Triton extraction remained constant. The development of acid phosphatase in thick slices ripened in moist air was similar to that in intact fruit, except that enzyme development and ripening were accelerated about 24 hours in the slices. The increase in enzyme activity that occurs in slices ripening in moist air was inhibited when tissue sections were infiltrated with solutions, by aspiration for 2 minutes or by soaking for 2 hours, anytime 22 hours or more after addition of ethylene. This inhibition was independent of the presence or absence of cycloheximide or sucrose (0.3-0.5m). However, the large decline in enzyme activity in the presence of cycloheximide, as compared with the controls, indicated that synthesis of acid phosphatase was occurring at all stages of ripening. PMID- 16659088 TI - Loss of phytochrome photoreversibility in vitro: I. Extraction and partial purification of killer. AB - Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, "Killer," which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described. PMID- 16659089 TI - Action of Nalidixic Acid on Chloroplast Replication in Euglena gracilis. AB - The role of light in nalidixic acid bleaching of Euglena gracilis var. bacillaris was investigated. The kinetics of loss of the chloroplast-associated DNA and the sensitivity of chloroplast replication to ultraviolet light was followed during treatment with nalidixic acid. By using the mutant P(4)ZUL, and 3-(3,4 dichlorophenyl)-, 1-dimethylurea, it was demonstrated that the requirement for light was a functioning photosynthetic electron transport system. Ultracentifugal analysis showed a substantial decrease in chloroplast-associated DNA after 6 hours of treatment with nalidixic acid. Ultraviolet target analysis revealed that the number of chloroplast genomes per cell had been reduced. The possible role of light and implications of the reduction in chloroplast genomes for chloroplast replication are discussed. PMID- 16659090 TI - The effect of adenosine 5'-triphosphate on the shibata shift and on associated structural changes in the conformation of the prolamellar body in isolated maize etioplasts. AB - Isolated maize (Zea mays var. kelvedon glory) etioplasts have been used to investigate the relationships between the spectral shifts and ultrastructural changes which occur during light-induced chloroplast development. After primary photoconversion, the Shibata shift was observed as a change from 680 to 670 nm in the chlorophyllide absorption maximum. When 1.5 nm ATP was added to the incubation medium the maximum was 675 nm even after 3.5 hours of illumination. Difference spectra for this effect indicate ATP inhibition of the Shibata shift. Two bands with maxima at 682 and 669 nm can be used to fit spectra of both ATP treated and control etioplasts, the estimated proportions of chlorophyllide 682 being 36% and 6%, respectively. Quantitative analysis of electron micrographs of the etioplasts showed that the frequency of untransformed prolamellar bodies was also higher in the presence of ATP (73% untransformed compared to 22% in the absence of ATP). A similar correlation was observed when transformation was measured for two etioplast fractions which show the shift to different extents. These results imply that the Shibata shift and prolamellar body transformation are related events, both being inhibited by the presence of ATP. ATP may therefore have an important role in regulating the early stages of plastid development. PMID- 16659091 TI - Photosynthetic action spectra of trees: I. Comparative photosynthetic action spectra of one deciduous and four coniferous tree species as related to photorespiration and pigment complements. AB - Comparative isoenergetic action spectra of net photosynthesis for intact, current year foliage of five tree species were determined from 400 to 710 nm by CO(2) exchange analysis. The blue (400 to 500 nm) peak of net photosynthetic activity for the green broadleaves of red alder (Alnus rubra Bong.) was reduced to a plateau for the green needle-leaves of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) and Sitka spruce (Picea sitchensis [Bong.] Carr.), a shoulder for the blue-green needles of Colorado spruce (Picea pungens Engelm.), and a reduced shoulder for the blue-white needles of Blue spruce (Picea pungens var. hoospii). These differences were attributable neither to a differential blue light stimulation of photorespiration nor to a differential presence of a nonplastid screening pigment. The conifers all had similar carotenoid-chlorophyll ratios, with approximately 50% more carotenoid relative to chlorophyll as compared to red alder. Blue light absorption and low efficiency of energy transfer by the carotenoids probably accounts for the low net photosynthetic activity of the green conifers in blue light as compared to red alder. Leaf form per se (broad versus needle) had no distinguishable influence on these results. PMID- 16659092 TI - Photosynthetic Action Spectra of Trees: II. The Relationship of Cuticle Structure to the Visible and Ultraviolet Spectral Properties of Needles from Four Coniferous Species. AB - The relative reflectance spectra for control and treated (surface wiped) current year foliage of Douglas fir, and Sitka, Colorado, and Blue spruce (Pseudotsuga menziesii [Mirb.] Franco, Picea sitchensis [Bong.] Carr., Picea pungens Engelm., and Picea pungens Engelm. var. hoopsii, respectively) were obtained from 220 to 700 nm. The green color of the control foliage of both Douglas fir and Sitka spruce was unaffected by the treatment whereas the blue-green and blue-white foliage of control Colorado and Blue spruce, respectively, became "green" as a result of the wiping. The relative reflectance curves for all green foliage, including the treated Colorado and Blue spruce, were all very similar with a peak in the green (540-560 nm), minima in the red (660-680 nm) and blue (450-500 nm), and very low reflectivities in the ultraviolet (lambda < 400 nm). In contrast, the control foliage for Colorado and Blue spruce both showed a generally higher relative reflectance over most of the visible spectrum (400-700 nm) with a marked increase in the blue region (400-500 nm). At wavelengths below 420 nm, their relative reflectances increased sharply with decreasing wavelength, the reflectance at 220 nm for Blue spruce being over four times that at 540 nm.Scanning electron microscope examination of the needles' surfaces revealed a system of wax filaments whose complexity correlated with the degree of ultraviolet and blue reflectance.It is concluded that both the bluish appearance (glaucous bloom) and the low relative efficiencies of blue light in photosynthesis of Colorado and Blue spruce result from the selectively enhanced reflection of blue light caused by the presence of the epicuticular wax deposits. The enhanced blue light reflection was shown to be the shoulder of a scattering effect which appeared to peak in the short ultraviolet region below 200 nm. The ecological implications of the results are discussed. PMID- 16659093 TI - Zinc Nutrition and Starch Metabolism in Phaseolus vulgaris L. AB - The effect of Zn nutrition on leaf starch metabolism was studied in two navy bean (Phaseolus vulgaris L.) varieties, Sanilac and Saginaw. Sanilac is much more susceptible to Zn deficiency than is Saginaw. The variables examined in these two strains were starch content, the activity of soluble starch synthetase (ADP glucose: starch alpha-4-glucosyltransferase, EC 2.4.1.b), and the size and number of starch grains. All of these variables decreased during Zn deficiency. The reductions were much greater in Sanilac than in Saginaw. Thus, positive correlations exist between the relative changes in these variables in Sanilac and Saginaw under low Zn and their genotypic difference in growth response to low Zn. These results are taken to purport that the above observations most likely represent characteristic responses to Zn deficiency. We therefore suggest that, as a possible role in plant metabolism, Zn is involved in starch formation. PMID- 16659094 TI - Photoreceptor Pigment for Blue Light in Neurospora crassa. AB - Irradiating the mycelium of Neurospora crassa with moderate intensities of blue light causes a reversible photoreduction of a b-type cytochrome. The action spectrum for the photoreduction of cytochrome b is very similar to the absorption spectrum of flavin pigments. Prolonged irradiation of the mycelium with strong blue light irreversibly bleaches flavin-like pigments and as these pigments are bleached the photoresponse of cytochrome b is lost. We conclude from these and other data that a flavin is the photoreceptor pigment for the photoreduction of cytochrome b. The close similarity between the action spectrum for the photoreduction of cytochrome b and action spectra for a number of physiological photoresponses suggests that this photoreceptor pigment controls a wide variety of photobiological processes in a wide diversity of organisms. PMID- 16659095 TI - Spectral Characterization of the Photoreducible b-Type Cytochrome of Dictyostelium discoideum. AB - Irradiation of a soluble extract from broken cells of Dictyostelium discoideum causes the photoreduction of a b-type cytochrome. The cytochrome b can be separated from cytochrome c, which is also present in the extract, by column chromatography on Brushite, but the cytochrome b is no longer sensitive to light after separation on the column. Low temperature spectroscopy shows that reduced form of the photoreducible cytochrome b has a Soret band at 423 nm and a split alpha band with maxima at 558 and 551 nm similar to the b-type cytochrome in complex II of beef heart mitochondria. PMID- 16659096 TI - The Incorporation of d-Glucosamine into Glycolipids and Glycoproteins of Membrane Preparations from Phaseolus aureus Hypocotyls. AB - Radioactivity from d-glucosamine-(14)C is incorporated into particulate fractions of hypocotyls of Phaseolus aureus (mung bean) seedlings. Polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-solubilized materials revealed that several polypeptide components varying considerably in molecular weight had become radioactive during the incubation. A considerable amount of (14)C was also recovered in lipid. Equilibrium centrifugation of the particulate material, isolated by initial centrifugation at 100,000 times gravity on sucrose density gradients revealed that radioactivity was recoverable in all of the membrane fractions along the gradient. It is suggested that glycoproteins and glycolipids containing amino sugar are normal constituents of such membranes. The ability of the particulate preparations to catalyze the transfer of N-acetyl-d-glucosamine from uridine diphospho-N-acetyl-d-glucosamine to endogenous acceptor material was also tested. Transfer was optimal at around pH 9 and in the presence of 10 mm Mg(2+), and it occurred largely into an unidentified lipid fraction. After equilibrium centrifugation of crude membrane material on sucrose gradients, a number of distinct fractions could be detected which would catalyze the transfer reaction. Uridine diphospho-d-glucose transferase activity showed a similar but not identical distribution along the gradient. PMID- 16659097 TI - De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination. AB - An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a Dnase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination. PMID- 16659098 TI - Rapid effects of indoleacetic Acid and ethylene on the growth of intact pea roots. AB - Root auxanometers were used to determine the growth rates of individual intact primary roots accurately and quickly. The growth of pea (Pisum sativum L.) roots was inhibited by both indoleacetic acid and ethylene within 20 minutes. A supramaximal concentration of ethylene inhibited root growth less than did 5 to 20 mum indoleacetic acid, indicating that inhibition of root growth by auxin was not due only to indoleacetic acid-induced ethylene production. Inhibition of root growth was largely relieved within 60 minutes of removal of both growth regulators. PMID- 16659099 TI - Revised Methods for Purification of Ribosyl-trans-zeatin from Vinca rosea L. Crown Gall Tumor Tissue. AB - Ribosyl-trans-zeatin has been purified from Vinca rosea L. crown gall tumor tissue by using two new sequences of isolation procedures. Identification of the compound has been established by mass spectrometry, ultraviolet absorbancy spectra, chromatographic values, and growth activity. The isolation sequences eliminate the exposures to pH extremes and the strong cation-exchange resin used in the purification reported earlier. The initial extraction procedures have been designed so as to avoid enzymatic alteration or production of active material and to prevent the inclusion in the extracts of nucleic acids which might serve as soures of the small active compounds. The production of ribosylzeatin by the tumor tissue is confirmed and the validity of isolation steps such as the use of cation exchangers is supported. PMID- 16659100 TI - Effects of ethylene, kinetin, and calcium on growth and wall composition of pea epicotyls. AB - Ethylene supplied with indoleacetic acid at 0.1 and 1 mum inhibited elongation and enhanced swelling in epicotyls of decapitated and derooted pea seedlings (Pisum sativum L., var. Alaska). These growth responses were correlated with the development of cell walls rich in weak acid-extractable materials and pectic uronic acids. Ethylene had no effect on the formation of hemicellulose, or hemicellulosic uronic acid. Ethylene stimulated the formation of residual materials at 0.1 mum indoleacetic acid but had little effect at 1 mum. With indoleacetic acid at 10 mum, ethylene modified neither the growth or wall composition appreciably. Growth and wall composition in intact seedlings were modified in similar fashion by ethylene. In intact seedlings ethylene promoted the development of walls high in weak acid-extractable materials and pectic uronic acid. These effects were less impressive in the first 24 hours than in the second 24 hours when the control plants suffered a net loss of these constituents. Ethylene considerably inhibited the formation of hemicellulose and residual wall materials in the apical sections but promoted it in the basal sections of the intact seedlings.Measurements of ethylene production by decapitated and derooted pea seedlings suggest that Ca(2+) and kinetin do not promote swelling through an effect on the formation of ethylene.We propose that cells of ethylene-treated pea epicotyls lack polarity because their walls are abnormally rich in pectic substances. PMID- 16659101 TI - Stomatal Response to Environment with Sesamum indicum. L. AB - Leaf resistance of Sesamum indicum L. increased when the humidity gradient between leaf and air was increased, at moderate temperatures, even though calculated carbon dioxide concentrations within the leaf decreased slightly. Mesophyll resistance remained relatively constant when humidity gradients were changed, indicating that the increases in leaf resistance were mainly caused by reductions in stomatal aperture and that nonstomatal aspects of photosynthesis and respiration were not affected. Low carbon dioxide concentrations inside the leaf decreased but did not eliminate resistance response to the humidity gradient. Internal carbon dioxide concentrations had little effect on resistance in humid air but had moderate effects on resistance with large humidity gradients between leaf and air. Stomatal response to humidity was not present at high leaf temperatures. Effects of humidity gradients on photosynthetic and stomatal responses to temperature suggested that large humidity gradients may contribute to mid-day closure of stomata and depressions in photosynthesis. PMID- 16659102 TI - Dichlorophenylurea-insensitive Reduction of Silicomolybdic Acid by Chloroplast Photosystem II. AB - The photoreduction of silicomolybdate and other heteropoly ions by chloroplasts is insensitive to 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU). Both water and diphenylcarbazide can be used as electron source for the reduction. Three different assays for silicomolybdate reduction are described including oxygen evolution, formation of a reduced heteropoly blue silicomolybdate, or an indirect assay for reduced silicomolybdate by redox indicators, such as ferricyanide or cytochrome c. The effects of detergents and tris washing are consistent with silicomolybdate reduction through photosystem II before the DCMU site. The effects of orthophenanthroline and bathophenanthroline indicate chelator sensitive sites in photosystem II before the site of DCMU action. PMID- 16659103 TI - Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae. AB - delta-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-(14)C from C. caldarium cells which had been incubated with delta-aminolevulinate-4-(14)C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that delta aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-(14)C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled delta-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with delta aminolevulinate-4-(14)C. Succinate-2-3-(14)C appeared to be as good a carbon source of amino acids as did C(4) and C(5) of delta-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of delta aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which delta-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of delta-aminolevulinate catabolism in the organisms employed are discussed. PMID- 16659104 TI - Carbonic anhydrase of spinach: studies on its location, inhibition, and physiological function. AB - Carbonic anhydrase activity was determined in spinach (Spinacia oleracea) leaf organelles isolated on sucrose density gradients and was found to be predominantly in the intact chloroplast fraction. The small amount of activity associated with the mitochondrial fractions was probably due to intact chloroplast contamination. No activity could be associated with the broken chloroplast or microbody fractions. Based upon inhibitor studies, carbonic anhydrase was found to be around 2 mm in the chloroplast. Ethoxzolamide, an inhibitor of carbonic anhydrase, reduced CO(2) fixation in intact chloroplasts. The concentration required to inhibit CO(2) fixation 20 to 40% was in excess of that required to inhibit the purified enzyme. The inhibition was partially reversed by CO(2). Ethoxzolamide had no effect on photosynthetic NADP reduction or photophosphorylation measured by methyl viologen reduction. The physiological role of carbonic anhydrase was shown not to be associated with CO(2) diffusion or CO(2) concentration. It is proposed that other functions of carbonic anhydrase could be the protection against denaturation by transient localized changes in pH or the hydration of compounds other than CO(2). PMID- 16659105 TI - Isotope Fractionation in Photosynthetic Bacteria during Carbon Dioxide Assimilation. AB - The delta (PDB) (13)C values have been determined for the cellular constituents and metabolic intermediates of autotrophically grown Chromatium vinosum. The isotopic composition of the HCO(3) (-) in the medium and the carbon isotopic composition of the bacterial cells change with the growth of the culture. The delta (PDB) (13)C value of the HCO(3) (-) in the media changes from an initial value of -6.6 per thousand to +8.1 per thousand after 10 days of bacterial growth and the delta (PDB) (13)C value of the bacterial cells change from -37.5 per thousand to -29.2 per thousand in the same period. The amount of carbon isotope fractionation during the synthesis of hexoses by the photoassimilation of CO(2) has a range of -15.5 per thousand at time zero to -22.0 per thousand after 10 days. This range of fractionation compares to the range of carbon isotope fractionation for the synthesis of sugars from CO(2) by ribulose 1,5-diphosphate carboxylase and the Calvin cycle.The amount of carbon isotope fractionation during the synthesis of aspartic acid from CO(2) is -24.9 per thousand at time zero and -15.0 per thousand after 10 days of bacterial growth. This amount of fractionation is in the range of carbon isotope fractionation for the synthesis of C(4) amino acids by a double carboxylation through ribulose 1,5-diphosphate and phosphoenolpyruvate carboxylase. PMID- 16659106 TI - Oxidative turnover of auxins in relation to the onset of ripening in bartlett pear. AB - Pears (Pyrus communis var. Bartlett) kept in 100% O(2) showed an increase in the rate of softening, chlorophyll degradation, and ethylene evolution. The O(2) application could overcome, in part, the inhibition of ripening by 1 mm indoleacetic acid. Ripening of pears was also accelerated by the application of solutions containing indoleacetic acid-oxidation products, obtained by an overnight incubation of 0.1 and 1 mm indoleacetic acid with traces of H(2)O(2) and horseradish peroxidase. Although both treatments stimulated ethylene evolution, the promotion of ripening could not be attributed to an indirect ethylene effect. Indoleacetic acid oxidation products obtained in vivo by high O(2) tensions or in vitro by enzymatic degradation may function in the promotion of fruit ripening and the synthesis of ethylene. PMID- 16659107 TI - Protoheme turnover and chlorophyll synthesis in greening barley tissue. AB - Studies in which (14)C-labeled precursors were fed to etiolated barley leaves (Hordeum vulgare L. var. Proctor) yielded chlorophyll and protoheme having similar specific radioactivities. These findings indicate: (a) there appears to be a rapid turnover of protoheme in the absence of net synthesis; (b) both pigments probably originate from a single 5-aminolevulinic acid pool; (c) the efficient utilization of glutamate-1-(14)C and the relatively poor utilization of glycine-2-(14)C suggest that 5-aminolevulinic acid is probably synthesized by a pathway other than 5-aminolevulinic acid synthetase (succinyl CoA-glycine succinyltransferase) in agreement with previously published work; (d) protoheme turnover appears to be faster under conditions which allow for rapid chlorophyll accumulation; (e) difference spectra indicate that mitochondrial cytochromes make a relatively minor contribution to the total heme in barley leaves. These findings are discussed in the light of current knowledge about tetrapyrrole regulation in photosynthetic organisms. PMID- 16659108 TI - Role of Membrane-bound, Fixed-charge Changes in Phytochrome-mediated Mung Bean Root Tip Adherence Phenomenon. AB - The movement of cells and cell fragments in an electric field provided a means for determining the nature of cellular surface charges. We found that changes in ionic strength and particularly changes in Ca(2+) and H(+) in the bathing medium cause changes in the surface charges on the root cap cells in the absence of red light. Red light-induced charge changes are demonstrable only on root cap cells and are reversible with far red light. By osmotically separating the membrane from the wall, we demonstrated that both light-induced and ionically mediated charge changes are associated with the cell membrane and not the cell wall. PMID- 16659109 TI - The Relationship between Satellite Deoxyribonucleic Acid, Ribosomal Ribonucleic Acid Gene Redundancy, and Genome Size in Plants. AB - The buoyant density of ribosomal DNA is similar in species with or without satellite DNA, and in all species examined was distinguishable from that of the satellite DNA. In melon tissues (Cucumus melo) the percentage satellite DNA is not correlated with the percentage hybridization to ribosomal RNA. Satellite DNA sequences do not appear to be dispersed between those coding for ribosomal RNA. There is no correlation between the presence of satellite DNA and high ribosomal RNA gene redundancy, but there is a correlation between satellite DNA and small genome size, which results in a correlation between satellite DNA and a high percentage hybridization to ribosomal RNA. Satellite DNAs are defined as minor components after CsCI centrifugation. PMID- 16659110 TI - l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons: Purification and Properties. AB - ORNITHINE: 2-oxoacid aminotransferase (EC 2.6.1.13) has been purified over 400 fold with a total recovery of 14% from acetone powders of cotyledons of germinating squash (Cucurbita pepo, L.) seedlings. The pH optimum of the transamination between l-ornithine and alpha-ketoglutarate is 8 and the Michaelis constants are 4.7 mm and 6.3 mm, respectively. The enzyme has a molecular weight of 48,000 as determined by gel filtration. The reaction is essentially specific for alpha-ketoglutarate as the amino group acceptor. The enzyme is inhibited very strongly by hydroxylamine, and less severely by NaCN and isonicotinylhydrazide. No inhibition is observed in the presence of 10 mml-cysteine. The energy of activation is 7.6 kcal/mole. The stability of the enzyme preparation is enhanced by the presence of dithioerythritol and glycerol. The enzyme activity of the most purified fraction is stimulated 30% by the addition of pyridoxal phosphate; however, the evidence for the unequivocal involvement of pyridoxal phosphate was inconclusive. PMID- 16659111 TI - Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L. AB - Differential and sucrose density gradient centrifugation established that about 80% of the total arginase activity (EC 3.5.3.1) in cotyledons of germinating broad bean seeds (Vicia faba L.) was present in the mitochondrial fraction. The mitochondrial arginase activity was enhanced considerably by exposure to osmotic shock, by freezing and thawing, or by Triton X-100 treatment. About 10% of the total arginase activity was recovered from the 40,000g supernatant fraction. During seed maturation, arginase activity in the cotyledons decreased to about one-third of its maximal activity, while increasing over 10-fold during subsequent germination. The time courses of mitochondrial arginase, succinate oxidase, and succinate dehydrogenase activities differed considerably during germination. PMID- 16659112 TI - Time-dependent Behavior of a Mathematical Model for Munch Translocation: Application to Recovery from Cold Inhibition. AB - The time evolution of a Munch pressure-flow translocation system is calculated using a numerical computer method. Results are obtained for the time course of the system variables following application of a large resistance in the translocation path, intended to simulate a cold block. The resistance factor required to produce translocation inhibition indicates that even moderate inhibition is primarily due to sieve plate pore block-age, rather than to the viscosity increase. The calculated time for recovery from cold inhibition and the shape of the translocation recovery curve agree with experimental results. The time for translocation recovery and the level of velocity recovery depend on the rate of sucrose unloading in the sink; on the sucrose concentration in the sieve tube; on the position, length, and resistance factor of the cold block; and on the hydraulic conductivities. PMID- 16659113 TI - Carbon dioxide and senescence in cotton plants. AB - Glandless cotton plants (Gossypium hirsutum L. cv. Coker 100) were subjected to the influence of high CO(2)-bicarbonate. The content of protein decreased with no accompanying increase in its degraded products. The decrease in protein was correlated with the low content of chlorophyll and also with the reduced activity of carbonic anhydrase. The initiation of these correlations coincided with the time when the control leaves contained the highest enzyme activity during leaf growth. The high concentration of bicarbonate directly restricted the rate of photophosphorylation and that of the Hill reaction in isolated chloroplasts. The amount of ATP in leaves treated in vivo also diminished. High CO(2) as bicarbonate, however, did not directly inhibit the activity of carbonic anhydrase in vitro. PMID- 16659114 TI - Photosynthetic activity and membrane polypeptide composition of supergranal chloroplasts from plant tissue cultures containing a viruslike particle. AB - Tissue culture cells of Streptanthus tortuosus (Kell.) var. orbiculatus (Greene) Hall (Cruciferae), having a viruslike particle in their nucleoli, the STV cell line, contain "supergranal" chloroplasts. Freeze-fracture studies of chloroplasts of a control cell line, which lacks the viruslike particles, reveal two complementary faces similar to those observed in spinach chloroplasts. Replicas of freeze-fractured STV supergranal chloroplasts, however, show that one membrane face (B) contains widely spaced 80 A particles and the other face (C) is essentially smooth. Isolated STV supergranal chloroplasts lack photosystem II activity as indicated by their inability to reduce dichlorophenolindophenol and are unable to reduce NADP with electrons from photosystem II or from ascorbate reduced dichlorophenolindophenol. However, partial photosystem I activity is indicated by the reduction of methyl viologen with electrons from dichlorophenolindophenol-ascorbate. This supports the concept that there is not a direct correspondence between grana formation and photosystem II activity. Electrophoresis shows that all of the major polypeptide bands present in the STV supergranal chloroplasts are also present in the control chloroplast membranes. One band, molecular weight 33,000, is present in a greatly increased amount in the STV supergranal chloroplast membranes and may be associated with grana stacking. PMID- 16659115 TI - Studies on the mode of action of aminotriazole in the induction of chlorosis. AB - The herbicide 3-amino-1, 2, 4-triazole induces complete chlorosis without any morphogenetic effect on Canna edulis leaves. Comparative analyses of the mineral and biochemical composition and also of the physiological properties of normal and bleached leaf cells establish a high specificity of this herbicide for inhibition of formation of chloroplast membranes. The synthesis of membrane lipids and chloroplastic terpenoid derivatives were specifically inhibited. This compound was found to inhibit photosynthetic O(2) evolution and (14)CO(2) fixation at the whole cell level and ferricyanide reduction in isolated chloroplasts immediately after its application. This inhibition was reversible with the addition of a number of unrelated compounds shown to nullify its herbicidal effect.The mechanism of action of this compound in specifically blocking the development of chloroplast membranes in discussed in terms of its primary action on photosynthetic carbon fixation. PMID- 16659116 TI - Water Conservation in Kalanchoe blossfeldiana in Relation to Carbon Dioxide Dark Fixation. AB - The succulent Kalanchoe blossfeldiana v. Poel. var Tom Thumb was treated on long and short photoperiods for 6 weeks during which short day plants developed thicker leaves, flowered prolifically, and exhibited extensive net dark fixation of carbon dioxide. In contrast, long day plants remained vegetative and did not develop thicker leaves or exhibit net carbon dioxide dark fixation. When examined after the photoperiodic state described, long day plants showed approximately three times more water loss over a 10-day period than short day plants. Water loss is similar during light and dark periods for short day plants but long day plants exhibited two times more water loss during the day than at night. The latter plants also lost three and one-half times more water during the light period than short day plants. The water conservation by short day plants is correlated with conditions of high carbon dioxide dark fixation and effects of its related Crassulacean acid metabolism on stomatal behavior. PMID- 16659117 TI - Involvement of the Golgi Apparatus in the Synthesis and Secretion of Hydroxyproline-rich Cell Wall Glycoproteins. AB - Pulse labeling of carrot root phloem parenchyma (Daucus carota L. cv. Nantes) tissue with (14)C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identity of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as (14)C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major site of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma membrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants. PMID- 16659118 TI - Effect of lanthanum on ion absorption in corn roots. AB - Short term (10 min) influx of (86)Rb-labeled potassium into corn (Zea mays L. WF9 x M14) root segments was inhibited by La (NO(3))(3) or LaCl(3). Half maximal inhibition of K(+) influx from 0.25 mm KCl was obtained with 0.025 mm La(3+). Kinetic analysis indicated the inhibition to be of a competitive nature. With absorption periods exceeding one hour, La(3+) no longer inhibited, but rather stimulated K(+) influx rates. La(3+) was not an inhibitor of (36)Cl or (32)P absorption. Separated cortex and stele absorbed labeled potassium (and phosphate) at comparable rates, and La(3+) inhibited potassium influx in both tissues. The effects of La(3+) on ion absorption were similar to those of Ca(2+), suggesting that the two polyvalent cations act at the same site. Based on this and the observation that La(3+) does not seem to penetrate the plasma membrane, it was concluded that La(3+) and Ca(2+) affect changes in ion transport without entering cells. PMID- 16659119 TI - Hydrogen Ion Entry as a Controlling Factor in the Acid-growth Response of Green Pea Stem Sections. AB - The ability of green pea (Pisum sativum var. Alaska) stem sections to elongate in response to H(+) has been reinvestigated. Contrary to the conclusions of Barkley and Leopold, (Plant Physiol. 1973. 52: 76-78) these sections elongate in response to H(+) whenever H(+) entry through the cuticle is facilitated by slits, holes, or removal. Both live and frozen-thawed sections can undergo acid-induced elongation. Green pea stems behave as predicted by the acid-growth theory. PMID- 16659120 TI - Identification of plant hormones from cotton ovules. AB - An extract from 8-day-old cotton ovules (Gossypium hirsutum L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A(1), A(3), A(4), A(7), A(9), and A(13) in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2 trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine(2iP), 6-(3 methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine(2iPA), and 6-(4-hydroxy-3 methyl-2-trans-butenylamino)-9-beta-d- ribofuranosylpurine (zeatin riboside) in the third fraction. PMID- 16659121 TI - Enzymes of glycerol metabolism in the storage tissues of Fatty seedlings. AB - Various enzymes of glycerol metabolism in the extracts of 5-day-old eastor bean (Ricinus communis L. var. Hale) endosperm and 4-day-old peanut (Archis hypogaea L.) cotyledon were studied. NAD-glycerol dehydrogenase and NAD-alpha glycerolphosphate dehydrogenase were not detected. Glycerol kinase was detected in the soluble fractions and an alpha-glycerolphosphate oxidoreductase was found in the particulate fractions. The particulate fractions were separated into various organelle fractions by sucrose gradient centrifugation and the alpha glycerolphosphate oxidoreductase was shown to be present in the mitochondria. The properties of the castor bean mitochondrial alpha-glycerolphosphate oxidoreductase resembled those of a similar enzyme present in the mitochondria of many animal tissues. A survey showed that the alpha-glycerolphosphate oxidoreductase was present in great amount only in the storage tissues of fatty seedlings but not in other nonfatty plant tissues. It is concluded that in the storage tissues of fatty seedlings, the soluble glycerol kinase and the mitochondrial cytochrome-linked alpha-glycerolphosphate oxidoreductase are the two enzymes responsible for the initial conversion of glycerol to hexose. PMID- 16659122 TI - Changes in Alfalfa Stem Conductance Induced by Corynebacterium insidiosum Toxin. AB - A toxin involved in bacterial wilt of alfalfa (Medicago sativa L.) has been isolated from cultures of the pathogen, Corynebacterium insidiosum, as well as from diseased plants (S. M. Ries and G. A. Strobel. 1972. Physiological Plant Pathology 2: 133-142). The influence of this toxin, a glycopeptide with a molecular weight of 5 x 10(6), on the water relations of alfalfa was examined. It was found that very small amounts of the toxin (2 mug) significantly reduced stem conductance through 15-cm long stems. This decrease in stem conductance caused by the toxin best explains the rapid decrease in transpiration and stomatal conductance and the resultant wilting after alfalfa cuttings have been in 200 mug ml(-1) toxin for 2 hours. Membrane damage resulting in water leakage was ruled out as a factor in the wilting during the 2-hour period. It is postulated that the toxin acts by interfering with water movement through pit membranes. PMID- 16659123 TI - Short interval leaf movements of cotton. AB - Gossypium hirsutum L. cv. Lankart plants exhibited three different types of independent short interval leaf movements which were superimposed on the circadian movements. The different types were termed SIRV (short interval rhythmical vertical), SIHM (short interval horizontal movements), and SHAKE (short stroked SIRV). The 36-minute period SIRV movements occurred at higher moisture levels. The 176-minute period SIHM occurred at lower moisture levels and ceased as the stress increased. The SHAKE movements were initiated with further stresses. The SLEEP (circadian, diurnal) movements ceased with further stress. The last to cease just prior to permanent wilting were the SHAKE movements. PMID- 16659124 TI - Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry. AB - Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF(3) in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of omega-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C(20)) were the dominant components in all six plants. In the alcohol fraction C(18), C(20), C(22), and C(24) saturated primary alcohols were the major components. C(16) and C(18) dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9 ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane 1, 16-dioic acid was the major dicarboxylic acid. The composition of the omega hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18 hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18 hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences. PMID- 16659125 TI - Developmental Changes in Multiple Forms of Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase in Soybean Hypocotyl. AB - The relative levels of multiple RNA polymerases were determined in soybean (Glycine max L. var. Wayne) hypocotyl during various stages of development. The meristematic region of the hypocotyl contains more total polymerase activity per gram fresh weight and a greater proportion of polymerase I relative to II than the differentiated regions. The fully elongated tissue comprising the lower half of the hypocotyl contains mainly RNA polymerase II. The hook region contains a polymerase activity peak which is completely sensitive to alpha-amanitin and partially sensitive to rifamycin SV. This peak is not detectable in other regions of the hypocotyl. Polymerase I is reproducibly separated into a major and a minor component, both being resistant to alpha-amanitin. The two components elute at salt concentrations of 0.2 m and 0.23 m KCl, respectively, while the alpha amanitin-sensitive polymerase (II) elutes at 0.3 m KCl. The polymerase activity peak which is detectable only in the hook region elutes at approximately 0.5 m KCl. Polymerase levels were also determined in water-stressed tissue and in tissue which was harvested after three days of growth instead of the usual four days. PMID- 16659126 TI - A device for comparing callus growth rates in vitro. AB - A device to compare the kinetics of callus growth in vitro is described. Changes in volumes of callus grown in scintillation vials were monitored photometrically without removing the sample from the solid support and medium. It is shown that a fiberglass-paper solid support is superior to a plastic foam solid support for the growth of American chestnut callus. PMID- 16659127 TI - Temperature Sensitivity of the Latent Phase in Ethylene-induced Elongation. AB - The temperature sensitivity is reported for the latent period preceding ethylene induced elongation in the adaxial half of the leaf petiole of Helianthus annuus. When intact plants were exposed to 10 mul of ethylene/l of air over the temperature range 18 to 35 C, the minimum latent time was 62 minutes at 28 C and the maximum was 132 minutes at 18 C. The temperature coefficient, Q(10), changed from 2.1 below 28 C, to 0.7 above. In 100 mul of ethylene/l of air, the latent time was reduced by 14% at 18 C, but was significantly increased at 28 and 38 C. These results show that the latent period in the elongation response of the petiole to ethylene cannot be reduced below about 60 minutes by raising either the leaf temperature or the atmospheric ethylene concentration. PMID- 16659128 TI - Comparative Activity of Isomers of Zeatin and Ribosyl-Zeatin on Funaria hygrometrica. AB - The activities of isomers of zeatin, ribosyl-zeatin, and 6-(gamma,gamma dimethylallylamino) purine (i(6)Ade) on the moss Funaria hygrometrica are compared by measuring the ability of the cytokinins to induce callus or gametophores. The cis- and trans-ribosyl-zeatins were inactive, and therefore this kind of bioassay cannot be used as evidence for the presence or absence of a cytokinin in tests on natural products. PMID- 16659129 TI - The influence of aging conditions on the short term growth of green pea stem segments. AB - Green pea (Pisum sativum L. var. Alaska) stem segments that were aged in buffer responded differently after aging depending on whether they were floating or submerged, or bubbled with air or N(2). Segments aged anaerobically for only 1 to 2 hours at 23 C responded to subsequent aerobic conditions by elongating more rapidly than aerobically aged sections. Longer periods of anaerobic treatment (up to 5 hours at 23 C) caused sections to exhibit an auxin-insensitive growth lag and reversible shrinkage. The shrinkage accelerated upon return to aerobic conditions but reversed after 1 to 2 hours. Green pea stem segments therefore require vigorous aeration during aging and growth measurements. PMID- 16659130 TI - Effect of leaf water deficit on stomatal and nonstomatal regulation of net carbon dioxide assimilation. AB - The effect of leaf water deficit on net CO(2) assimilation was studied under two conditions: in one, the stomata were allowed to contribute to the regulation of CO(2) assimilation; in the other, air was forced through the leaf at a constant rate to overcome the effects of change in stomatal resistance accompanying changes in leaf water deficit. When the stomata were allowed to regulate the gaseous diffusive resistance of the leaf, CO(2) assimilation decreased with increasing leaf water deficit. However, when air was forced through the leaf, the rate of assimilation was not inhibited by increasing leaf water deficit. The results indicate that the inhibition of net CO(2) assimilation with increasing leaf water deficit is a consequence of an increase in the diffusive resistance to gas exchange and not of a change in apparent mesophyll resistance. PMID- 16659131 TI - Effects of irradiance and leaf water deficit on net carbon dioxide assimilation and mesophyll and transport resistances. AB - Rate of net CO(2) assimilation by soil-grown soybean plants were studied over a range of relative leaf water contents at each of four levels of irradiance. There was a large interaction between light level and leaf water deficit on the rate of CO(2) assimilation. The effect of leaf water deficit on assimilation became larger as irradiance increased. Both stomatal resistance to CO(2) transport and mesophyll resistance to CO(2) assimilation increased as leaf-water deficit increased. The increase in both resistance with changing leaf-water content was largest at high irradiance and became smaller as irradiance decreased. Relief of soil-moisture stress by watering induced large oscillations of CO(2) assimilation, stomatal resistance, and mesophyll resistance. The oscillation of the mesophyll resistance occurred in the absence of changes in relative water content and appeared to be related to oscillations in leaf temperature. The observed increase in mesophyll resistance with decreasing leaf-water content under nonoscillative conditions may be caused by changes in leaf temperature rather than leaf water content. PMID- 16659132 TI - Enzyme content of plant microbodies as affected by experimental procedures. AB - The content of specific enzymes in microbodies isolated from tobacco Nicotiana rustica, L. leaves may vary according to the procedure followed during the isolation of the organelles. The type of homogenizing medium, its ionic components and the ratio of medium to tissue during homogenization, affect the over-all yield and relative distribution of each microbody enzyme in different ways. The type of density gradient and the initial acceleration of the centrifuge rotor affect also the enzyme content of sedimenting microbodies. These observations explain some of the conflicting results obtained on the determination of the intracellular location of several enzymes. PMID- 16659133 TI - Metabolism of uronic acids in plant tissues: partial purification and properties of uronic Acid oxidase from citrus leaves. AB - A new enzyme, named uronic acid oxidase, was extracted and purified 67-fold by (NH(4))(2)SO(4) fractionation and CM-Sephadex column chromatography from ethylene treated Shamouti orange (Citrus sinensis L. Osbeck) leaves. The enzyme catalyzes the oxidation of d-galacturonic acid and d-glucuronic acid to the corresponding hexaric acids in the presence of molecular oxygen with the production of H(2)O(2). The pH optimum for the oxidation of d-galacturonic acid and d glucuronic acid is between 7 and 8. The enzyme is highly specific for d galacturonic acid and d-glucuronic acid. It also oxidizes polygalacturonic acid. The apparent Michaelis constant values of the enzyme for d-galacturonic acid and d-glucuronic acid are 0.13 and 0.5 mm, respectively. The molecular weight of the enzyme, as determined by gel filtration, is about 98,000. The enzyme is inhibited by sodium hydrosulfite and other sulfites, indicating that it contains a flavin prosthetic group. PMID- 16659134 TI - Cell Wall and Protoplast Isoperoxidases of Corn Leaves in Relation to Cut Injury and Infection with Helminthosporium maydis. AB - Young leaves of two corn (Zea mays) inbreds with normal and Texas male-sterile cytoplasm, which differed in their susceptibility to Helminthosporium maydis Nisikado and Miyake race T, showed no significant qualitative or quantitative differences in their isoperoxidase patterns. Of six cathodic and four anodic isoenzymes present in the soluble fraction, four and two, respectively, comprised the fraction ionically bound to the cell wall. Peroxidase fractions ionically and covalently bound to the wall constituted about 20% of the total peroxidase activity. No new isoperoxidases were detected in either inbred line in response to cutting, infection, or detachment only and exposure to darkness for 40 hours. Three isoperoxidases, all cathodic, mainly reacted to cutting injury as well as fungal infection. One of the isoperoxidases appeared responsible for the increase in the peroxidase activity of the soluble fraction while the other two were responsible for the increase in that of the fraction ionically bound to the walls. The relative increase in the latter fraction was greater for infected leaves than for mechanically injured ones. No significant differences were found between the two inbreds in their peroxidase reactions to cutting injury or infection. Thus, the corn leaf isoperoxidases were distinctive in their distribution in the cell and in their reaction to injury. Changes in their activity induced by infection may result from a nonspecific response to injury. PMID- 16659135 TI - Cell wall and protoplast isoperoxidases in tobacco plants in relation to mechanical injury and infection with tobacco mosaic virus. AB - Leaves and pith of Turkish, Wisconsin 38, and Samsun NN tobacco (Nicotiana tabacum) varieties, which differ in their sensitivity to tobacco mosaic virus, showed the same qualitative isoperoxidase patterns and a similar distribution of distinctive isoperoxidases between the cell protoplast and wall-free, ionically, and covalently bound fractions. No changes in the qualitative isoenzyme spectrum were found in relation to age, mechanical injury, or leaf infection with tobacco mosaic virus. The distinctive isoperoxidases which reacted to infection were the same as those responsive to mechanical injury, confirming that the enzyme reaction to infection results from a nonspecific response to injury. The increase in peroxidase activity in response to infection or mechanical injury, or both, was greater in young tissue than in the older ones. The great increase in Samsun NN leaves and no increase in those of the two other varieties in response to infection may be due to differences in the degree to which the pathogen affected processes controlling the nonspecific peroxidase reaction to injury. Peroxidase development in the infected Samsun NN leaves was due to isoenzymes which form the wall-bound fraction in very young tissues, and to those which increase in activity with aging in the protoplast and wall-free fractions. In mechanically injured tissue, only the first group of isoenzymes increased in activity. In Samsun NN plants, the increased peroxidase activity in upper intact leaves above the infected ones was only due to isoenzymes whose activity increases with both normal and virus-accelerated senescence. Peroxidase reaction to challenge inoculation in these leaves was the same whether the lower ones were intact, infected and/or mechanically injured. Thus, the induced systemic resistance to tobacco mosaic virus may be due to other than peroxidase factors.In infected tissues, peroxidase was detected in the endoplasmic reticulum, Golgi apparatus, vacuole, cell wall, and intercellular spaces. The Golgi vesicles were often localized near the tonoplast and plasmalemma, fusing with membranes and secreting their contents. The possible "rejuvenating" effects of injury on synthesis and transport of distinctive isoperoxidases are discussed. PMID- 16659136 TI - Metabolism of Tritiated Gibberellins in d-5 Dwarf Maize: I. In Excised Tissues and Intact Dwarf and Normal Plants. AB - Metabolism of [(3)H]gibberellin A(1) ([(3)H]GA(1)) was followed in intact seedlings and excised apices and leaf tissue of both dwarf and normal (tall) plants of d-5 maize (Zea mays L.). The three metabolites produced were tentatively identified as [(3)H]GA(s), [(3)H]GA(s)-glucoside ([(3)H]GA(s)-glu), and [(3)H]GA(1)-X, an unknown.In 3-hour, pulse-labeling experiments with tissues of incubated, expanding leaves, more than 70% of the [(3)H]GA(1) taken up was metabolized to the three products within 12 to 15 hours. [(3)H]GA(1) fed to the roots of 7-day-old seedlings was readily translocated to the leaves, and all three metabolites were found in both roots and leaves. [(3)H]GA(1)-X was the major metabolite in roots, whereas in leaves the major metabolite was [(3)H]GA(s) glu. There were no consistent differences in [(3)H]GA(1) metabolism between dwarf and normal plants, indicating that dwarfism in d-5 maize is not associated with modified GA(1) metabolism.In excised, mature leaf tissue, [(3)H]GA(1) metabolism was slower than in excised, young leaf tissue. Mature leaf tissues produced [(3)H]GA(s)-glu as by far the major metabolite, with [(3)H]GA(s) and [(3)H]GA(1) X as minor metabolites. In contrast, in young leaves the three metabolites appeared sequentially in significant proportions: [(3)H]GA(8) first, followed by [(3)H]GA(s)-glu and, finally, [(3)H]GA(1)-X. PMID- 16659137 TI - Iron deficiency and the structure and physiology of maize chloroplasts. AB - The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency. PMID- 16659138 TI - Degradation of argininosuccinate lyase by a protease synthesized in soybean cell suspension cultures. AB - Suspension cultures of soybean (Glycine max L.) were shown to contain protease activity which could be inhibited by the addition of protease inhibitors such as p-hydroxymercuribenzoate and ethylenediaminetetraacetic acid. The use of these inhibitors, coupled with studies of the rate of degradation of argininosuccinate lyase (argininosuccinate-lyase = l-arginino-succinate arginine-lyase, EC 4.3.2.1) in extracts of cell cultures grown for 24 hours led to the hypothesis that a metal-dependent protease is synthesized by the cells after 24 hours of growth, to remove the lyase enzyme. PMID- 16659139 TI - Studies of storage proteins of higher plants: I. Concanavalin a from three species of the genus canavalia. AB - Concanavalin A, the lectin of the Jack bean, Canavalia ensiformis, was extracted and compared with homologous proteins from Canavalia gladiata and Canavalia maritima. All proteins were bound to Sephadex G-100 and eluted from the gel with buffered glucose solution. Quantitative recoveries indicated that large quantities (23 to 28% of dry seed protein) of these lectins are synthesized by all three species. Antibody preparations made against C. ensiformis lectin failed to discriminate among the three proteins; the pattern of the precipitin bands indicated identical antigenic determinants in the Ouchterlony double-diffusion assay. Native and sodium dodecyl sulfate polyacryl-amide gel electrophoresis also failed to distinguish differences in the proteins. The storage protein active in carbohydrate binding is composed, in each case, of identical subunits. However, the amino acid composition of the subunit chains from the three sources is not identical. In particular, the lectins from C. ensiformis and C. gladiata contain two methionine residues per protein subunit, while only one methionine residue is found in the C. martima lectin. Cyanogen bromide cleavage of the purified subunit from C. maritima yieded two fragments with molecular weights estimated at 20,400 and 4,600, respectively. Amino acid analysis of the separated fragments indicated that the methionine residue at position 130 in C. ensiformis is absent in the lectin from C. maritima. PMID- 16659140 TI - Contribution of Nonautotrophic Carbon Dioxide Fixation to Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose. AB - Bicarbonate-(14)C was provided to 5- and 11-day-old suspension cultures of Paul's Scarlet rose, and the incorporation of (14)C into lipid, protein, amino acids, and organic acids was determined. The rate of bicarbonate uptake was approximately the same by 5- and 11-day-old cells, but the distribution of (14)C among cell constituents was markedly different. In 5-day-old cells a larger proportion of the (14)C entered protein, whereas in 11-day-old cells there was a greater tendency for (14)C to accumulate in malate.The (14)C in protein was distributed among 10 amino acids each having greater than 1% of the total (14)C recovered in protein. The distribution of (14)C among tricarboxylic acid cycle intermediates indicated that the aspartate family of amino acids was synthesized directly from oxaloacetate produced as a result of nonautotrophic CO(2) fixation. However, this was not the sole source of oxaloacetate used for the synthesis of aspartate, for in a double labeling study with bicarbonate-(14)C and acetate-(3)H it was shown that oxaloacetate was drained simultaneously from the tricarboxylic acid cycle for this purpose. PMID- 16659141 TI - Isolation and Characterization of Stemphylin, a Chromone Glucoside from Stemphylium botryosum. AB - A phytotoxic compound was isolated from a liquid culture medium of Stemphylium botryosum, a pathogen of lettuce. The toxin is an amorphous yellow solid with absorbance maxima at 218, 268, and 427.5 nm and exhibits a bathochromic shift in alkaline pH. It has a molecular weight of 370 and an empirical formula of C(17)H(22)O(9). Glucose and aromatic pigments are detected after acid hydrolysis. Based on its spectral and chemical properties, the proposed structure of the toxin is 3-hydroxy- 2,2-dimethyl-5-alpha-d-glucopyranoside-2,3-dihydrochromone, and it has been given the trivial name stemphylin. A linear relationship exists between lesion area and amount of toxin applied to a young lettuce leaf. The relationship between toxin production and the development of disease symptoms is discussed. PMID- 16659142 TI - Postillumination burst of carbon dioxide in crassalacean Acid metabolism plants. AB - Immediately following exposure to light, a postillumination burst of CO(2) has been detected in Crassulacean acid metabolism plants. A detailed study with pineapple (Ananas comosus) leaves indicates that the postillumination burst changes its amplitude and kinetics during the course of a day. In air, the postillumination burst in pineapple leaves generally is exhibited as two peaks. The postillumination burst is sensitive to atmospheric CO(2) and O(2) concentrations as well as to the light intensity under which plants are grown. We propose that the CO(2) released in the first postillumination burst peak is indicative of photorespiration since it is sensitive to either O(2) or CO(2) concentration while the second CO(2) evolution peak is likely due to decarboxylation of organic acids involved in Crassulacean acid metabolism.In marked contrast to other higher plants, the postillumination burst in Crassulacean acid metabolism plants can be equal to or greater than the rate of photosynthesis. Photosynthesis in pineapple leaves also varies throughout a day. Both photosynthesis and the postillumination burst have a daily variation which apparently is a complex function of degree of leaf acidity, growth light intensity, ambient gas phase, and the time a plant has been exposed to a given gas. PMID- 16659143 TI - Regulation of glucose 6-phosphate dehydrogenase in blue-green algae. AB - Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) has been partially purified from Anacystis nidulans and Anabaena flos-aquae by means of ammonium sulfate fractionation and exclusion gel chromatography and the kinetic properties determined.Glucose 6-phosphate dehydrogenase from these blue-green algae exhibits Michaelis-Menten kinetics at pH 6.7. At this pH, Km values of 0.37 mm for glucose 6-phosphate and 10 mum for NADP were determined. At a pH above 7.4, the enzyme exhibits sigmoidal kinetics with respect to glucose 6-phosphate saturation but the saturation curve for NADP remains hyperbolic.ATP is an inhibitor of the enzyme competitively with NADP with a Ki of 2 to 5 mm. NADPH inhibits the enzyme competitively with glucose 6-phosphate. The inhibition curves for NADPH are hyperbolic at pH 6.7 and sigmoidal at pH 8.6.The significance of these in vitro kinetics are discussed relative to in vivo data on the control of glucose 6 phosphate turnover in blue-green algae. PMID- 16659144 TI - Ethylene-enhanced Ion and Sucrose Efflux in Morning Glory Flower Tissue. AB - Rib tissue segments excised from open flowers or buds of Ipomoea tricolor Cav. and floated on aqueous media responded to ethylene treatment by rolling up after 2 to 3 hours; a simple method for quantitating the rolling up is presented. The rolling up response was temperature- and oxygen-dependent and was critically affected by the pH of the medium. The ethylene concentration giving a half maximal response was 0.1 mul/l; continuous ethylene treatment was not required for the response as a 1-hour ethylene exposure enhanced rolling up.Rib segments rolling up during ethylene treatment unrolled when transferred to 0.5 m sucrose, indicating that rolling up was due to asymmetric turgor changes in the segments. Compartmental analysis of (36)Cl(-) efflux from rib segments showed a fast and a slow phase; the slow phase, with a half-time of about 6 hours, is tentatively identified as efflux from the vacuolar compartment. During ethylene treatment, the rate of (36)Cl(-) efflux in the slow phase rose markedly as the rolling up response developed. A similar result was obtained with the efflux of (86)Rb(+). The release of (14)C-metabolites, labeled either by a period of (14)CO(2) fixation in darkness or by exposure to (14)C-(U)-glucose, also increased during ethylene-induced rolling up.These results suggest that ethylene causes an increase in membrane permeability in certain cells of the rib tissue. PMID- 16659145 TI - Concentration dependencies of some effects of ethylene on etiolated pea, peanut, bean, and cotton seedlings. AB - The effects of a series of concentrations of ethylene (10, 20, 40, to 10,240 nl/l) on elongation, diameter, and geotropism of the stems and roots of etiolated seedlings of Pisum sativum L., Arachis hypogea L., Phaseolus vulgaris L., and Gossypium hirsutum L. were measured or observed. Of the 24 possible responses, 4 were unaffected at the concentrations used, 5 were affected slightly, and the remaining responses exhibited a 14-fold range of apparent half-maximum concentration dependencies (i.e. 95 nl/l for the effect on pea epicotyl geotropism to 1350 nl/l for the promotion of cotton hypocotyl diameter). Six or possibly eight of these responses appear to have the same concentration dependencies while the others fell in pairs or as individual responses. The data, if interpreted in a manner analogous to enzyme kinetics, are indicative of more than one primary mechanism for ethylene action in plants. PMID- 16659146 TI - Presence of Two Different Membrane-bound, KCl-stimulated Adenosine Triphosphatase Activities in Maize Roots. AB - Recent publications have indicated that a KCl-stimulated ATPase from cereal roots is specifically associated with plasmalemma-enriched membrane fractions. However, in previous work we found that relatively high specific activities of this enzyme were also associated with a membrane fraction which did not contain plasmalemma. In an attempt to clarify this discrepancy, we have investigated the effect of density gradient composition upon the association of the enzyme with different membrane fractions isolated from the roots of Zea mays L. (WF9 x M14).When roots were fractionated on a single, discontinuous sucrose density gradient the KCl stimulated ATPase activity was concentrated in membrane fractions of relatively high density (sigma = 1.17-1.22) which were enriched in plasmalemma. When fractions were isolated on both Ficoll and sucrose density gradients, KCl stimulated ATPase activity was again found associated with plasmalemma and also in a second membrane fraction which did not contain plasmalemma. This fraction was found at relatively low densities (sigma = 1.08) on the Ficoll gradient. The precise identity of the membrane in this fraction could not be determined.A preliminary investigation into the properties of the two KCl-stimulated ATPase activities indicated that both had acid pH maxima and both displayed similar responses to changes in KCl concentration. PMID- 16659147 TI - Photosynthetic Unit Size during the Synchronous Life Cycle of Scenedesmus. AB - Apparent size of the photosynthetic unit (chlorophyll/O(2) per flash) was estimated by O(2) yield of repetitive short flashes on cell samples taken at various times from a synchronized culture (14-hour light, 10-hour dark) of Scenedesmus obliquus. Unit size was essentially invariant (< 10% variation) with a mean value of 1750 chlorophyll/O(2) per flash. In contrast, the light-saturated photosynthetic rate per unit chlorophyll, or turnover rate of the photosynthetic unit, varied with the life cycle, rising 40% in the first three hours of the light period and decaying slowly thereafter. The results are taken as evidence that the metabolic machinery is subject to far greater control and adjustment than is the photochemical machinery. PMID- 16659148 TI - Isolation and Regeneration of Tobacco Mesophyll Cell Protoplasts under Low Osmotic Conditions. AB - A method is described for the isolation of large numbers of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyll cell protoplasts under relatively low external osmotic conditions. The procedure utilized 0.2 m sucrose as the primary osmoticum and a mixture of 0.5% macerozyme, 4% cellulase, and 2% polyvinylpyrrolidone, pH 5.4. The viability of resultant protoplasts was confirmed through regeneration of fertile plants. Plating and regeneration studies revealed, however, that qualitative and quantitative modifications in plating and differentiation media were necessary for protoplasts prepared in this manner. Over-all, the procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration. In addition, the system should permit studies related to the influence of differing osmoticum levels on a variety of cell functions. PMID- 16659149 TI - The lack of effect of cyclic adenosine 3':5'-monophosphate on Avena coleoptile growth. AB - The effects of cyclic adenosine 3':5'-monophosphate (cAMP) on the growth of Avena coleoptile segments over 4 to 10 hours were monitored with a position sensing transducer. At pH 6, cAMP (0.1 mm with and without 2.5 mm glucose; or 2 mm alone) or dibutyryl cAMP (0.1 mm) was added at the beginning of the experiment, or after about 1 hour or after about 6 or 7 hours. Under all conditions tested, cAMP compounds had little or no effect on coleoptile segment elongation. Inasmuch as cAMP does not duplicate the rapid and vigorous elongation obtained with 2 mum auxin, the hypothesis that cAMP is a mediator of auxin activity is not supported by experimental evidence in this system. This conclusion is dependent upon the assumption that the cAMP compounds penetrated the tissue. PMID- 16659150 TI - Compartmentation in Vicia faba Leaves: I. Kinetics of C in the Tissues following Pulse Labeling. AB - Leaflets of Vicia faba were pulse-labeled with (14)CO(2) to follow the subsequent movement of photosynthate between leaf tissues. Samples were taken during a (12)CO(2) chase, quick frozen, freeze-substituted, and embedded in methacrylate. Paradermal sections provided tissue samples consisting only of upper epidermis, palisade parenchyma, spongy parenchyma and veins, spongy parenchyma, or lower epidermis. Most CO(2) fixation occurred in the palisade parenchyma, but its (14)C content declined rapidly after labeling. Concomitant with the decline of activity in the palisade parenchyma, there was an increase in activity in the spongy parenchyma and upper epidermis and a slow increase in the lower epidermis. Activity in the palisade parenchyma and spongy parenchyma eventually reached similar levels and remained constant. Tissue samples containing veins were consistently the most radioactive, and activity in those samples showed a decline. Very little change occurred in the insoluble fraction from any tissue. The results support previous assumptions regarding the pathway of assimilate transport to the veins, and demonstrate the rapidity of such transport. Sucrose is apparently the principal mobile compound. PMID- 16659151 TI - Compartmentation in Vicia faba Leaves: II. Kinetics of C-Sucrose Redistribution among Individual Tissues following Pulse Labeling. AB - Leaflets of Vicia faba L. were pulse labeled with (14)CO(2) and the kinetics of (14)C-sucrose redistribution among individual tissues was followed. Sucrose specific activity in the whole leaf peaked about 15 minutes after labeling and declined with a half-time of about 80 minutes. In one experiment, leaflet discs taken at various times during the (12)CO(2) chase were quick frozen, freeze substituted, and embedded in plastic. The tissue was sectioned paradermally and sections of palisade parenchyma, of spongy parenchyma, and of spongy parenchyma that contained veins were collected. Water extracts from these sections were assayed for sucrose specific activity. Sucrose specific activity in the palisade parenchyma was higher than that of the spongy parenchyma and reached a maximum in both tissues 9 to 15 minutes after labeling. Sucrose specific activity initially declined rapidly in the palisade parenchyma followed by a period during which little or no loss occurred. Sucrose specific activity in sections containing veins peaked at 15 minutes with a maximum value substantially higher than either mesophyll tissue, indicating that recently synthesized sucrose was preferentially exported from the mesophyll. Decline of activity in these sections containing veins continued for the remainder of the experiment. Sucrose specific activity in lower epidermal peels peaked several minutes after that of the whole leaflet and remained lower. Sucrose specific activity in upper epidermal peels was variable (probably due to contamination), but the limited data suggest that the sucrose specific activity there reached somewhat higher values than those of the lower epidermis. The experiments indicate that each leaf tissue contains a kinetically identifiable sucrose pool (which we refer to as "histological compartmentation"), and that further compartmentation may occur at the intracellular level. A simulation of leaf sucrose compartmentation is presented. PMID- 16659152 TI - Photosynthetic activities of spinach leaf protoplasts. AB - Photosynthetic activities of protoplasts isolated from spinach leaf (Spinacia oleracea L.) were investigated. The protoplasts were stable up to 9 hr, without loss of the original activity of CO(2) fixation (33-75 mumoles CO(2)/mg Chl.hr) and light-dependent O(2) evolution (33-40 mumoles O(2)/mg Chl.hr), when stored in 0.8 m mannitol-0.05 m N-tris (hydroxymethyl)-methylglycine-NaOH buffer, pH 7, at 4 C in dark. The optimum pH of 8.5 for CO(2) fixation reaction carried out in the present experimental condition employed is about the same as that reported for intact spinach chloroplasts. The CO(2) concentration for half-maximal rate of CO(2) fixation by protoplasts. "Km (CO(2))," were determined to be 19.8 mum (pH 7) and 42 mum (pH 8.5) and are similar to those observed for intact spinach chloroplasts. Protoplasts showed postillumination CO(2) fixation. Over-all results indicate that spinach protoplasts are as active as the intact plant leaf tissues in their photosynthetic activities. PMID- 16659153 TI - Light-controlled Leaf Expansion in Peas Grown under Different Light Conditions. AB - Several photosystems control leaf expansion in Alaska peas (Pisum sativum). Phytochrome is known to control expansion in dark-grown peas. But plants exposed briefly to red light are insensitive to phytochrome, an insensitivity that is itself phytochrome-produced. Leaf expansion in these plants is promoted by 440 or 630 nm of light (probably mediated by protochlorophyll). Plants grown in white fluorescent light required simultaneous exposure to high intensity blue and yellow light for promotion of leaf expansion. Since these results parallel studies on light-controlled inhibition of stem elongation, shoot growth as a whole is coordinated by these photosystems. Such coordination might be a mechanism of plant competition for light. PMID- 16659154 TI - Regulation of ribulose 1,5-diphosphate carboxylase by substrates and other metabolites: further evidence for several types of binding sites. AB - Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mm Mg(2+) and low CO(2) concentrations by its substrates (RuDP and CO(2)) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO(3) (-) and Mg(2-), and this inactivity persists for 20 minutes or longer after 1 mm HCO(3) ( ) and 10 mm Mg(2+) are added. Maximum activity requires that the preincubation mixture also include either 0.01 mm 6-PGluA or 0.5 mm NADPH.When the enzyme, following preincubation with HCO(3) (-) and Mg(2+), is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The Ki value for 6-PGluA is 0.02 mm and the Ki value for FDP is 190 mum. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition.In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A(1), A(2), and A(3). RuDP binds very tightly to site I (in the absence of Mg(2+) or HCO(3) (-)), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg(2+) and 1 mm HCO(3) ( ). Mg(2+) and HCO(3) (-) (or CO(2)) bind to site A(3) (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A(1) and NADPH at site A(2) during preincubation with Mg(2+) and HCO(3) (-). With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A(1).The physiological role of this regulation is discussed, particularly with respect to protection of "C-3" plants against oxidation of RuDP to phosphoglycolate. PMID- 16659155 TI - The Toxins of Helminthosporium maydis (Race T) : A Colorimetric Determination of the Toxins, Their Appearance in Culture and in Infected Plants. AB - Host-specific toxins produced by Helminthosporium maydis, race T, are measured quantitatively by a chemical assay procedure involving reaction of the toxins with a sulfuric acidacetic anhydride reagent and measurement of the absorbance of the product at 330 nm. The assay was shown to measure total toxin concentrations after only limited fractionation of the culture medium. Using the assay it was possible to show that the highest amount of toxin per gram of fungus mycelium occurs early in the growth cycle of H. maydis. Toxins I, II, and V are the predominant toxins at these early times both in culture and in infected corn and wheat varieties. Some chromatographic and spectral properties of toxin V, a previously unreported toxin, are described. Since toxin V appears in culture prior to toxins I, II, III and IV, a precursor-product relationship can be suggested. PMID- 16659156 TI - Ozone injury in soybeans: isoflavonoid accumulation is related to necrosis. AB - Fumigation of soybean leaves (Glycine max [L.] Merr. with ozone caused stippling and silvering at the same time that large accumulations of the isoflavonoid compounds daidzein, coumestrol, and sojagol occurred. Nitrogen dioxide and sulfur dioxide caused lesser accumulation of the isoflavonoids, and peroxyacetyl nitrate did not result in significant accumulation. Visible toxicity and chemical changes in ozone-fumigated leaves were similar to the hypersensitive disease defense reaction of soybean leaves to the pathogen Pseudomonas glycinea, except that the phytoalexin hydroxyphaseollin was not produced in the ozone-treated leaves. PMID- 16659157 TI - Ribonuclease in senescing morning glory: purification and demonstration of de novo synthesis. AB - Isolated flower buds and flowers of Ipomoea tricolor went through the same stages of development as those attached to the plant. Ribonuclease activity increased sharply in both cases during the time of flower fading and aging. Affinity chromatography using guanosine diphosphate-Sepharose was employed for fast and efficient purification of ribonuclease. Flowers which were kept on D(2)O during the senescence phase incorporated deuterium into ribonuclease as shown by isopycnic density gradient centrifugation in CsCl, suggesting that ribonuclease was de novo synthesized during aging of the flower. PMID- 16659158 TI - Interaction of kinetin and calcium in relation to their effect on stimulation of ethylene production. AB - Application of kinetin and Ca(2+) caused a striking synergistic increase in ethylene production by mung bean (Phaseolus aureus Roxb) hypocotyl segments. The effect of kinetin on Ca(2+) uptake and of Ca(2+) on the uptake and metabolism of kinetin in relation to their effect on ethylene production was studied. Tracer experiments showed that kinetin greatly increased the uptake of (45)Ca(2+) after 6 hours of incubation. Reciprocally, Ca(2+) stimulated the uptake of kinetin-8 (14)C and remarkably enhanced the metabolism of kinetin-8-(14)C into several polar metabolites. Consequently, the quantity of free kinetin-8-(14)C remaining in Ca(2+)-treated segments was much less than in control segments. A possible mechanism accounting for the synergism between kinetin and calcium on ethylene production is discussed. PMID- 16659159 TI - The effect of carbohydrates and arginine on arginine metabolism by excised bean leaves in the dark. AB - The effect of carbohydrate on arginine utilization by excised bean (Phaseolus vulgaris L. var. Tendergreen) leaves in the dark was studied by adding arginine to leaves differing in carbohydrate levels, and measuring the arginine content of the leaves at intervals. In nonstarved leaves, the arginine content decreased steadily after vacuum infiltration of 10 mm arginine and was essentially completely utilized by 36 hours after infiltration. In starved leaves, the arginine content did not decrease except for a brief period of about 4 hours after infiltration. The distribution of (14)C after adding (14)C-arginine to starved and nonstarved leaves indicated that the presence of carbohydrates in the leaves stimulates the utilization of arginine for protein synthesis and conversion to other amino acids, organic acids, and CO(2) (catabolism). Adding sucrose along with arginine to starved leaves stimulated this utilization of arginine for both protein synthesis and catabolism. This effect of sugar on catabolism is different than results of similar studies done previously with proline.Increasing the concentration of added arginine greatly increased arginine catabolism but had a relatively small effect on utilization of arginine for protein synthesis. This result is the same as similar results from adding different concentrations of proline to excised leaves. PMID- 16659160 TI - Growth and Development of Soybean (Glycine max [L.] Merr.) Pods: CO(2) Exchange and Enzyme Studies. AB - The rates of CO(2) exchange and (14)CO(2) incorporation in the light and dark and the activities of several photosynthetic, photorespiratory, and respiratory enzymes of soybean (Glycine max [L.] Merr. cv. Wye) reproductive structures were determined at weekly intervals from anthesis to pod maturity. At all stages of pod development soybean reproductive structures were found to be incapable of net photosynthesis under the experimental conditions employed, but capable of gross photosynthesis and light-induced (14)CO(2) uptake. Consistent with the lack of net photosynthesis throughout the development of the reproductive structure, the maximum in vitro activity of ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) in pod tissue was only 3% of that in leaf extracts when expressed on a fresh weight basis. We concluded that the major role of the reproductive structure of the soybean with respect to photosynthetic carbon metabolism is the reassimilation of its respiratory CO(2). PMID- 16659161 TI - Polyribosomes from Peas: V. An Attempt to Characterize the Total Free and Membrane-bound Polysomal Population. AB - Attempts were made to isolate and characterize the total population of free and membrane-bound polysomes from the elongating region of dark-grown pea stems (Pisum sativum L.). Partial separation of free from membrane-bound polysomes was achieved by relatively low speed centrifugation of the homogenate. Complete separation was not achieved. Based on analysis of the rRNA content of various subcellular fractions, fractionated tissue yielded greater than 95% of the rRNA found in whole tissue. Approximately 45% of the ribosomal material was membrane bound (released by detergent) and was found in the "wall" (13%), the "nuclear" pellet (2%), and the "mitochondrial" pellet (29%). The remaining 55%, consisting primarily of free polysomes, could be recovered free from membranous material by sedimentation through a dense (700 mg/ml) sucrose pad for 90 hours. The advantages and disadvantages of using sucrose pads for the separation of free and membrane-bound polysomes are discussed. PMID- 16659162 TI - The nature of spontaneous changes in growth rate in isolated coleoptile segments. AB - About 4 hours after they are cut from the seedling, corn (Zea mays L.) coleoptile segments mounted vertically show a strong increase in growth rate. This increase occurs in water or various buffers near pH 7 and is not accompanied by the accumulation of a growth promoter in the medium. The increase in growth rate is prevented by 1 mmp-fluorophenylalanine and is strongly inhibited by 0.1 mmp chlorophenoxyisobutyric acid.The increased growth rate is accompanied by a 95% increase in the ability of tissue extracts to catalyze the conversion of (14)C tryptophan to (14)C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in segments from coleoptiles grown aseptically.The spontaneous increase in growth rate is completely but reversibly inhibited by 1 mum indole-3-acetic acid. Cytokinins have little effect on the spontaneous growth response, whereas gibberellic acid is observed to extend the latent period and reduce the magnitude of the response. It is tentatively concluded that the increase in endogenous growth rate may result from increased auxin production upon derepression of the auxin biosynthesis pathway after isolating the tissue from the normal supply of auxin from the tip. PMID- 16659163 TI - Further Chemical and Morphological Characterization of Chloroplast Membranes from a Chlorophyll b-less Mutant of Hordeum vulgare. AB - A comparative study of peptide composition and freeze-fracture morphology of chloroplast membranes from a chlorophyll b-less mutant and a normal barley plant (Hordeum vulgare L.) is reported in this work. Using a high resolution, discontinuous sodium dodecyl sulfate-acrylamide gel electrophoretic system, we show that the mutant chloroplast membranes not only completely lack the 25 kilodalton peak, which accounts for about 18% of the chloroplast membrane protein in the normal plant, but also exhibit gross reduction in other components at 27.5 and 20-kilodalton regions. Despite such extensive deletions in the peptide composition of the mutant chloroplast lamellae, no alteration could be detected in either density or size of the intramembranous particles, visualized by freeze fracturing. PMID- 16659164 TI - Partial Restoration of the High Rate of Plastid Pigment Development and the Ultrastructure of Plastids in Detached Water-stressed Wheat Leaves. AB - Detached etiolated wheat (Triticum aestivum L. cv. Chris) leaves accumulated plastid pigments at a high rate, developed chloroplasts with stacked thylakoids, and stored plastid starch when wetted on filter paper in light. A moderate water deficit of - 10 bars markedly reduced the accumulation of chlorophyll and carotenoids in the 8-day-old detached leaves during greening. delta Aminolevulinic acid treatment of stressed leaf segments resulted in slightly increased pigment accumulations but benzyladenine application restored plastid pigment formation in stressed tissue to within 15% of the pigment content of the nonstressed detached leaves. The addition of delta-aminolevulinic acid to benzyladenine-treated stressed leaf segments improved both chlorophyll and carotenoid formation to nearly the amounts found in nonstressed leaf tissue. Stressed leaf sections developed plastids that were small, lacked starch, contained few thylakoids per granum, and possessed dilated thylakoids. Benzyladenine application to the stressed leaf segments did not restore normal plastid stacking but benzyladenine induced the formation of extended intergranal lamellae and stimulated pigment accumulations in both stressed and nonstressed detached leaves. Starch was absent in plastids of benzyladeninetreated leaf sections. PMID- 16659165 TI - A Re-evaluation of the Nitrate Reductase Content of the Maize Root. AB - The standard procedure for the in ritro extraction of nitrate reductase from the tip region (0-2 cm) of the primary root of the maize (Zea mays L.) seedling indicated an activity of the enzyme approximately 5-fold higher than that obtained with an in vivo assay. In more mature regions of the primary root the ratio of in vitro to in vivo activity was much lower and in older seedlings was less than unity. The mature root extracts had a more labile nitrate reductase and a higher level of an inactivating enzyme. The use of phenylmethylsulphonyl fluoride in the extraction medium gave only a partial protection of the nitrate reductase from the old root samples. Casein (3%) resulted in a greatly increased yield of nitrate reductase (36-fold with one sample) and a more constant in vitro in vivo activity ratio for all root samples. With casein in the extraction medium, much higher levels of nitrate reductase were recovered from the mature root zone, and the root content of this enzyme was now shown to be quite a significant proportion of the total in the maize seedling. Casein was shown to inhibit the action of the inactivating enzyme on nitrate reductase. Evidence is also presented for a nitrate reductase inactivating enzyme in the maize scutella and leaf tissues and in the roots and shoots of pea seedlings. PMID- 16659166 TI - Water stress and protein synthesis: I. Differential inhibition of protein synthesis. AB - Water stress causes both a qualitative change in the types of proteins produced by Avena coleoptile cells as demonstrated by a double-labeling ratio technique, and a quantitative reduction in the rate of incorporation of leucine into proteins. The osmotica mannitol and Carbowax-4000 cause similar changes in the pattern of protein synthesis showing that these effects are due to water stress rather than to a particular osmoticum. PMID- 16659167 TI - Water Stress and Protein Synthesis: II. Interaction between Water Stress, Hydrostatic Pressure, and Abscisic Acid on the Pattern of Protein Synthesis in Avena Coleoptiles. AB - Water stress causes a reduction in hydrostatic pressure and can cause an increase in abscisic acid in plant tissues. To assess the possible role of abscisic acid and hydrostatic pressure in water stress effects, we have compared the effects of water stress, abscisic acid, and an imposed hydrostatic pressure on the rate and pattern of protein synthesis in Avena coleoptiles. Water stress reduces the rate and changes the pattern of protein synthesis as judged by a double labeling ratio technique, Abscisic acid reduces the rate but does not alter the pattern of protein synthesis. Gibberellic acid reverses the abscisic acid-induced but not the stress-induced inhibition of protein synthesis. The effect of hydrostatic pressure depends on the gas used. With a 19: 1 N(2)-air mixture, the rate of protein synthesis is increased in stressed but not in turgid tissues. An imposed hydrostatic pressure alters the pattern of synthesis in stressed tissues, but does not restore the pattern to that found in turgid tissues. Because of the differences in response, we conclude that water stress does not affect protein synthesis via abscisic acid or reduced hydrostatic pressure. PMID- 16659168 TI - Initial stages in the onset of senescence in tobacco leaves. AB - A marked loss of leucine (14)C incorporation occurred in chloroplasts isolated from Nicotiana rustica L. leaves exposed to 24 hours of darkness. This loss is not due to an initial decline in RNA-synthesis potential of the chloroplasts, as was inferred from the extent of UTP incorporation by the isolated chloroplasts. Upon reillumination of the leaves, leucine incorporation by the isolated chloroplasts reverted to its original level within 3 to 4 hours, hence it is doubtful whether the period of 24 hours after detachment should be regarded as the initial phase of leaf senescence.After 48 and 72 hours of darkness, however, complete recovery of the incorporation activity was not achieved by re illumination of the leaves, representing the apparent onset of an irreversible process. Treatment with kinetin, which markedly delayed the symptoms of senescence in these tobacco leaves, did not prevent the dark-induced decline in chloroplast protein synthesis activity. Nor, up to 24 hours of darkness, did it have any effect on the light-induced complete recovery of this synthesis. Nevertheless, after reilluminating kinetin-treated leaves that had been exposed to darkness for 48 and 72 hours, leucine incorporation in the isolated chloroplasts was resumed at a faster rate and reached a higher level than did the untreated controls. PMID- 16659169 TI - Regulation of chlorophyll synthesis in the green alga golenkinia. AB - Chlorophyll synthesis in Golenkinia is inhibited 10-fold by growth in darkness on acetate or by growth on elevated concentrations of acetate in the light, particularly if the growth medium contains low levels of nitrogen. Glucose has no such inhibitory effect. delta-Aminolevulinic acid, with a maximal effect at 0.01 m, but not its precursors, overrides the inhibitory effect of acetate and darkness, restoring chlorophyll synthesis. Glycine, succinate, and alpha ketoglutarate, the precursors tested, all enter the cell. Cells forming chlorophyll produce significantly more aminolevulinic acid than do cells becoming bleached, further indicating the important regulatory role of this compound. Cyclic AMP has no effect on chlorophyll synthesis. These results are compared with those obtained studying other algae, and a mechanism relating light and acetate to chlorophyll formation is proposed. PMID- 16659170 TI - Energy State and Dinitrogen Fixation in Soybean Nodules of Dark-grown Plants. AB - Dark treatment of 25-day-old greenhouse-grown plants of inoculated soybean (Glycine max var. Chippewa) for 1 day reduced ATP by 70%, sucrose by 60%, total adenosine phosphates by 60%, ATP/ADP ratio by 55%, nitrogenase activity by 50%, and energy charge by 15% in nodules. The close correlation between nitrogenase activity and these energy parameters indicates that they may play a major role in regulating dinitrogen fixation in the symbiotic system. PMID- 16659171 TI - Photosynthetic enhancement studied in intact spinach chloroplasts. AB - The Emerson enhancement effect was evaluated in the intact spinach (Spinacia oleracea var. Long Standing Bloomsdale) chloroplast by monitoring the uptake of (14)CO(2) during illumination by 640 nm and 720 nm lights. Low levels (about 10 mum) of fructose 1,6-diphosphate, ribose 5-phosphate, and glycerate 3-phosphate stimulated the rate of photosynthesis and abolished enhancement values observed in their absence. Concentrations of the two sugar phosphates at levels of 1 mm responded similarly. In contrast, 1 mm glycerate 3-phosphate inhibited the rate of photosynthesis and increased enhancement. The exchange of glycerate 3 phosphate for glyceraldehyde 3-phosphate was speculated to be a factor underlying the decrease in photosynthesis and the increase in enhancement. Glucose 6 phosphate, NADPH, and l-malate did not influence photosynthesis or enhancement.The uncoupler, p-trifluoromethoxyphenylhydrazone, decreased the rate of photosynthesis but did not change the enhancement values. ATP (0.2 to 1 mm) had an occasional stimulating effect on CO(2) fixation but no effect on enhancement. Magnesium ions inhibited photosynthesis and decreased the enhancement values. It was concluded that the enhancement phenomenon reflects events of the photosynthetic carbon reduction cycle as well as the photochemical act. PMID- 16659172 TI - Photocontrol of the Germination of Onoclea Spores: III. Analysis of Germination Processes by Means of Cycloheximide. AB - Possible involvement of protein synthesis in the germination of Onoclea sensibilis spores was investigated by temporarily applying 0.1 mm cycloheximide before and after photoinduction. Cycloheximide was shown to inhibit protein synthesis, but not to act as an uncoupler of respiration. When cycloheximide was added before or shortly after photoinduction, spore germination was inhibited with the half-maximal inhibition attained in 30 to 45 minutes and the maximal inhibition in 2 hours of incubation. When the time of the inhibitor treatment was delayed after photoinduction, the spores escape from the inhibitory effect of cycloheximide slowly during the first 8 hours and abruptly thereafter with a half maximal time of 10 hours. If spores are washed free of exogenous cycloheximide and subsequently irradiated, their ability to germinate can be reinstated in distilled water with a half-maximal time of 12 hours. The kinetics of recovery were identical and of apparent first order, regardless of whether cycloheximide treatments were given before or after photoinduction. These results are interpreted to indicate that the normal course of germination of Onoclea spores requires the continuous synthesis of a short lived enzyme that functions in the germination processes at about 10 hours after photoinduction. The cycloheximide sensitive step follows in the germination processes an anaerobiosis-sensitive step, but precedes the time of acetocarmine uptake or visible signs of protrusion. PMID- 16659173 TI - Purification and partial characterization of barley leucine aminopeptidase. AB - A peptidase acting on Leu-Gly-Gly and Leu-Tyr at pH 8 to 10 was purified about 670-fold from germinated grains of barley (Hordeum vulgare L.). Gel electrophoretic analyses indicated a purity of about 90%. The purified enzyme is remarkably similar to mammalian leucine aminopeptidases (EC 3.4.1.1) both in chemical and in enzymatic properties. It has a sedimentation constant of 12.7S and a molecular weight of about 260,000. The enzyme has a high activity on leucine amide and di- and tripeptides with N-terminal leucine or methionine; leucyl-beta-naphthylamide, in contrast, is hydrolyzed very slowly. The enzyme also liberates N-terminal amino acids from the insulin B chain. The pH optima for the hydrolysis of different substrates depend on the buffers used; highest reaction rates are generally obtained at pH 8.5 to 10.5. Mg(2+) and Mn(2+) ions stabilize (and probably activate) the enzyme. In contrast to mammalian leucine aminopeptidases, the barley enzyme is inactivated in the absence of reducing sulfydryl compounds. PMID- 16659174 TI - Cycloheximide is not a specific inhibitor of protein synthesis in vivo. AB - Cycloheximide is frequently presumed to inhibit specifically the cytoplasmic protein synthesis of eukaryotes. Although previous investigators have shown that it had other effects on the cells of a variety of organisms, these results were frequently presumed to be secondary effects of the inhibition of protein synthesis. This paper shows that a wide range of deleterious effects are produced by cycloheximide on a single organism, Chlamydomonas reinhardi Dangeard. If, protein synthesis is inhibited by nonpermissive conditions in temperature sensitive mutants or with other treatments these "secondary" effects are not produced. Instead, cycloheximide appears to have two or three independent inhibitory effects on the cell. Moreover, in contrast to a number of previous investigations, these results show that protein synthesis is not required for RNA synthesis. Instead the rate of RNA synthesis is actually increased by interference with protein synthesis. PMID- 16659175 TI - Phytochrome-mediated de Novo Synthesis of Phenylalanine Ammonia-Lyase in Cell Suspension Cultures of Parsley. AB - After a preirradiation with ultraviolet light, phenylalanine ammonia-lyase activity in cell suspension cultures of parsley (Petroselinum hortense Hoff.) is controlled by phytochrome (red/far red photoreversibility). Isopycnic CsCl density gradient centrifugation, after labeling with (15)N (90 atom%) under inductive and noninductive conditions, was used to investigate the mode of action of phytochrome in this response. After a 5hour labeling period, a buoyant density shift of 0.009 kg.l(-1) (0.7%) without band-broadening (indicating close to maximal labeling of the enzyme), was observed in irradiated cells. In dark-grown controls, the density shift was 0.004 kg.l(-1) (0.3%), accompanied by significant band-broadening, indicating turnover of about half of the enzyme pool during 5 hours. These results are taken as evidence that phytochrome controls de novo synthesis of this enzyme over a background of basal turnover. PMID- 16659176 TI - The Metabolism of Oat Leaves during Senescence: III. The Senescence of Isolated Chloroplasts. AB - The changes in chlorophyll and protein in senescing chloroplasts isolated from the first leaves of 7-day-old oat (Avena sativa) seedlings have been investigated. In darkness the chlorophyll in these plastids is highly stable, losing only 5 to 10% of its content after 7 days at 26 C. This result contrasts with the behavior of chlorophyll in intact leaves, in which about 80% of the pigment would have disappeared in that time. The protein is less stable than the chlorophyll, though more stable than in the leaf; probably a small amount of protease is present in the plastids. Some protein is also being synthesized in the chloroplasts along with its breakdown; gains of up to 38% in protein and 13% in chlorophyll were observed under different conditions. l-Serine, which actively promotes senescence in the leaf, has only a very slight effect on the chloroplasts, and kinetin antagonizes it. Kinetin also has a small but significant effect in preserving the protein from breakdown. Acid pH somewhat promotes the breakdown, both of chlorophyll and protein. A loss of chlorophyll and protein comparable to that occurring in the senescence of the leaf could not be induced in the chloroplasts by suspending them in malate, in cytoplasmic extract, or in any of a number of enzymes tested alone. Incubation with a mixture of four enzymes was the only treatment which approximated the senescent process in the leaf, causing 34% loss of chlorophyll at pH 5 and 40% loss of protein at pH 7.4, both in 72 hours.In white light, the chlorophyll and the carotenoids, but not the protein, disappear rapidly. This disappearance was shown to be prevented in an atmosphere of nitrogen or in air by a number of reducing agents, of which ascorbic acid was the most effective. It is, therefore, ascribed to photooxidation rather than to normal senescence. PMID- 16659177 TI - C(4) Photosynthesis: Light-dependent CO(2) Fixation by Mesophyll Cells, Protoplasts, and Protoplast Extracts of Digitaria sanguinalis. AB - Mesophyll cells, protoplasts, and protoplast extracts of Digitaria sanguinalis were used for comparative studies of light-dependent CO(2) fixation. CO(2) fixation was low without the addition of organic substrates. Pyruvate, oxaloacetate, and 3-phosphoglycerate induced relatively low rates (10 to 90 mumoles/mg chlorophyll.hr) of CO(2) fixation when added separately. However, a highly synergistic relationship was found between pyruvate + oxaloacetate and pyruvate + 3-phosphoglycerate for inducing light-dependent CO(2) fixation in the mesophyll preparations. Highest rates of CO(2) fixation were obtained with protoplast extracts. Pyruvate, in combination with oxaloacetate or 3 phosphoglycerate induced light-dependent rates from 150 to 380 mumoles of CO(2) fixed/mg chlorophyll.hr which are equivalent to or exceed reported rates of whole leaf photosynthesis in C(4) species. Concentrations of various substrates required to give half-maximum velocities of CO(2) fixation were determined, with the protoplast extracts generally saturating at the lowest substrate concentrations. Chloroplasts separated from protoplast extracts showed little capacity for CO(2) fixation. The results suggest that CO(2) fixation in C(4) mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase.The stimulation of pyruvate-induced CO(2) fixation by oxaloacetate and 3-phosphoglycerate is thought to be due to induction of noncyclic electron transport which generates ATP for the conversion of pyruvate to phosphoenolpyruvate by pyruvate Pi dikinase. The primary products of the substrate-induced CO(2) fixation were oxaloacetate and malate, which provides further evidence for carbon fixation through the beta-carboxylation pathway. High rates of light-dependent CO(2) fixation with a significant percentage of (14)C fixed into malate suggest an efficient operation of both photosystems I and II.The substrate inductions are discussed with respect to the proposed role of the mesophyll cell in C(4) photosynthesis, and schemes suggesting the stoichiometry of energy requirements for photosynthetic carbon metabolism in C(4) mesophyll cells are presented. PMID- 16659178 TI - C(2)H(4): its purification for biological studies. AB - A gas chromatographic technique is described for obtaining ultra high purity (14)C(2)H(4) for use in biological studies. (14)C(2)H(4) purchased from commercial sources contained readily detectable impurities including radioactive acetylene. Following purification on two different columns, no impurities were detected by high sensitivity gas chromatographic analysis. However, shortly thereafter impurities were detected as a result of radiation decomposition. Trapping and immediately regenerating ultra high purity (14)C(2)H(4) from dilute, filtered Hg (CIO(4))(2) solutions did not cause the formation of impurities, whereas additional impurities were formed when unpurified (14)C(2)H(4) was used. Impurities were also formed when ultra high purity (14)C(2)H(4) was stored in such solutions prior to its regeneration or when it was trapped and immediately regenerated from more concentrated Hg(CIO(4))(2) solutions. PMID- 16659179 TI - Transport of Metabolites across Isolated Envelope Membranes of Spinach Chloroplasts. AB - Isolated envelope membranes of spinach chloroplasts (Spinacia oleracea L. var. Viroflay) exhibited selective permeability. Metabolites such as 3 phosphoglycerate, bicarbonate, glyoxylate, and acetate were transported rapidly; 6-phosphogluconate, glycolate, glycine, l-malate, and succinate were intermediate; whereas glucose 6-phosphate, fructose 1,6-diphosphate, and sucrose were hardly transported. Transport rates, metabolite accumulations within the membrane vesicles, and the internal water volume of isolated and in situ envelope membranes were compared and found to show similar trends. PMID- 16659180 TI - Incorporation of mevalonic Acid into ribosylzeatin in tobacco callus ribonucleic Acid preparations. AB - The incorporation of (14)C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of (14)C-2 mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the (14)C-2 mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue. PMID- 16659181 TI - Differential cold tolerance, starch, sugar, protein, and lipid of yellow and purple nutsedge tubers. AB - From measurements of viability after exposure of tubers to natural overwintering in the soil and 6 weeks exposure at 2 C, species cold tolerance of the tubers was ranked in decreasing order: yellow nutsedge ;I' (Cyperus esculentus L.), an ecotype originating in Illinois; yellow nutsedge ;G', an ecotype originating in Georgia; and purple nutsedge (Cyperus rotundus L.). The ratios of unsaturated saturated fatty acids in tuber triglycerides, tuber polar lipids, and leaf polar lipids followed the same order as the cold tolerance rankings, with the most cold hardy species having the highest ratios. Lipid content was less than 1% of dry weight in purple nutsedge tubers, but was from 5 to 7% in both yellow nutsedge tubers. Starch, sugar, and lipid contents increased significantly in the hardy yellow nutsedge ;I' tubers during a 6-week exposure to 2 C, but did not change in the susceptible purple nutsedge tubers; only sugar increased in yellow nutsedge ;G' tubers after this treatment. Protein content was not altered by the 2 C treatment in any of the tubers. Apparently, several factors involving starch, sugar, lipids, and fatty acids are related to the differences in tolerance to cold in these species. PMID- 16659182 TI - Morphological and Photosynthetic Properties of Digitonin-treated Chloroplast Membranes from the Wild-type and ac-5 Strains of Chlamydomonas reinhardi. AB - Chloroplast membranes of wild-type Chlamydomonas reinhardi, treated with digitonin, yield photosystem II-rich and photosystem I-rich fractions; this fractionation is accompanied by a separation of stacked (grana) lamella from unstacked (stroma) lamellae. Poor fractionation of the photosystems occurs when the treated chloroplast membranes derive from the ac-5 strain grown mixotrophically, whereas good fractionation occurs with ac-5 cells grown phototrophically; the mixotrophic cells possess only unstacked membranes, whereas the phototrophic cells possess stacked membranes. We concluded that digitonin fractionation is dependent on the stacked membrane configuration. PMID- 16659183 TI - Comparative studies of glyoxysomes from various Fatty seedlings. AB - The separation of various organelles from cotton cotyledon (Gossypium hirsutum L.), cucumber cotyledon (Cucumis sativus L.), peanut cotyledon (Archis hypogaea L.), pine megagametophyte (Pinus ponderosa Laws), and watermelon cotyledon (Citrullus vulgaris Schrad.) by sucrose density gradient centrifugation was found to be similar to that described for castor bean endosperm (Ricinus communis L.). Equilibrium densities were 1.12 to 1.13 g cm(3) for endoplasmic reticulum, 1.17 to 1.19 g/cm(3) for mitochondria, and 1.25 g/cm(3) for glyoxysomes. Isolated glyoxysomes from different fatty seedlings have striking similar specific activities of individual enzymes. The only exception is alkaline lipase activity which, when assayed with an artificial substrate, varies some 10-fold in glyoxysomes from different fatty seedlings. The properties of individual enzymes in glyoxysomes from different fatty seedlings are qualitatively similar as regard to sub-organelle localization and behavior in the presence of KCl and Triton X 100. In pine megagametophyte, the glyoxysomes and not the mitochondria are the intracellular site for the breakdown of stored lipid. PMID- 16659184 TI - Biosynthesis of Cutin: Enzymatic Conversion of omega-Hydroxy Fatty Acids to Dicarboxylic Acids by Cell-free Extracts of Vicia Faba Epidermis. AB - Long chain dicarboxylic acids are constituents of the protective biopolymers cutin and suberin of plants. Cell-free extracts from the excised epidermis of Vicia faba leaves catalyzed conversion of 16-hydroxy[G-(3)H]hexadecanoic acid to the corresponding dicarboxylic acid with nicotinamide-adenine dinucleotide phosphate as the preferred cofactor. This enzymatic activity, located largely in the 100,000g supernatant fraction, had a pH optimum near 8. This dehydrogenase showed an apparent Km of 1.25 x 10(-5)m and 3.6 x 10(-4)m for 16 hydroxyhexadecanoic acid and NADP, respectively. Modification of the substrate, either by esterification of the carboxyl group or by introduction of another hydroxyl group at C-10, resulted in a substantial (two-thirds) decrease in the rate of reaction, and hexadecanol was not a good substrate. The enzyme was inhibited by thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate. The aldehyde intermediate was trapped by the inclusion of dinitrophenyl hydrazine in the reaction mixture, and the 16-oxo compound was regenerated and identified. Furthermore, synthetic 16-oxo-[G-(3)H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an omega-hydroxyacid dehydrogenase and an omega-oxoacid dehydrogenase. PMID- 16659185 TI - Movement of C-labeled Sugars into Kernels of Wheat (Triticum aestivum L.). AB - An anatomical study of wheat (Triticum aestivum L.) kernels 14 days after anthesis revealed that the tracheary elements of the pericarp vascular bundle are not in direct continuity with those of the rachilla. The phloem was continuous from the rachilla into the crease of the pericarp.Shortly after exposure of the flag leaf to (14)CO(2), relatively high proportion of the (14)C extracted from the pericarp and endosperm was found in glucose and fructose. With additional time, the percentage of (14)C in the monosaccharides declined and that in sucrose increased to a maximum 3 hours after (14)CO(2) exposure. The monosaccharides comprised about one-half of the soluble sugars extracted from the pericarp. Based on these observations, it appeared that sucrose hydrolysis might be prerequisite to sugar movement from the terminal phloem elements in the pericarp and into the endosperm. However, when (14)C-fructosyl-sucrose was injected into the peduncle, there was little additional randomization of the (14)C between the glucose and fructose moieties of sucrose extracted from the pericarp and endosperm compared to the rachis sucrose. If we assume that injected sucose was transported to the kernels via the phloem, then either sucrose moves out of the terminal phloem elements in the pericarp and into the endosperm unaltered, or if hydrolysis and resynthesis are a prerequisite to transport into the endosperm, the products of hydrolysis are not freely available for isomerization. PMID- 16659186 TI - Ribulose diphosphate carboxylase synthesis in euglena: increased enzyme activity after transferring regreening cells to darkness. AB - The transfer of dark-grown cultures of Euglena gracilis Klebs strain Z regreening in the light back into darkness resulted in a dramatic increase in ribulose diphosphate carboxylase activity. On a culture volume basis activity increased 4 fold over a 24-hour dark period, although on a protein basis activity declined because of rapid cell division. Mixed assays with light- and dark-growing cell extracts provided no evidence for the removal of an inhibitor of ribulose diphosphate carboxylase upon transferring regreening cells back to darkness. Although ribulose diphosphate carboxylase activity increased over a 24-hour dark period, there was no concomitant increase in the potential of the cells for photosynthetic carbon dioxide fixation.Higher light intensities than the optimum for ribulose diphosphate carboxylase synthesis during regreening resulted in a greater relative rate of synthesis on transfer to darkness so that the maximum activity of ribulose diphosphate carboxylase reached in the dark was constant, regardless of light intensity during regreening. A tentative hypothesis to explain these results is that the synthesis of the large and small subunits of ribulose diphosphate carboxylase occur at different stages of cell development, light being necessary for the synthesis of the large subunit and also for regulating the synthesis of the small subunit. PMID- 16659187 TI - The Occurrence and Development of Amylase Enzymes in Incubated, De-embryonated Maize Kernels. AB - The development of amylase activity in extracts from de-embryonated and GA(3) treated de-embryonated maize kernels (Zea mays L.) was determined during a 10-day incubation period. The increase in activity was compared with activity extracted from endosperms dissected from germinating whole kernels. Chromatographic analysis of reaction products as well as physicochemical characterization demonstrated that the activities from GA(3)-treated and nontreated tissue were comparable and that part of the activity was attributable to alpha amylase.Concomitant with the increase in activity was the appearance of a number of starch-degrading bands, as evidenced by polyacrylamide gel electrophoresis. Actinomycin-D (20 mug/ml) and cycloheximide (5 mug/ml), when present in the incubation medium at early periods of incubation, were capable of inhibiting the development of amylase activity and of preventing the appearance of the starch degrading bands.The results indicate that the development of alpha-amylase activity in de-embryonated maize kernels is independent of an embryo or an exogenous source of gibberellic acid and suggest that this process involved protein synthesis. PMID- 16659188 TI - Photosynthetic Reactions in the Marine Alga Codium vermilara: II. Structural Studies. AB - Chloroplasts from Codium vermilara, isolated by relatively crude methods, are able to fix CO(2) at rates comparable to the rates of intact plants. Sections in thalli of Codium vermilara show that the chloroplasts are surrounded by a thin layer of cytoplasm. This surrounding layer of cytoplasm, is retained also in isolated chloroplasts, and presumably preserves the intactness of the chloroplast envelope. PMID- 16659189 TI - Sodium-1,2-C Acetate Incorporation in Roots of Frost-hardy and Less Hardy Alfalfa Varieties under Hardening Conditions. AB - When the temperature of incorporation of sodium acetate-1, 2-(14)C into lipids of alfalfa (Medicago media Pers. var. Rambler and Medicago sativa L. var. Caliverde) roots was lowered from 22 C to 1 C, elongation and desaturation of fatty acids and the labeling of phosphatidylcholine were strongly stimulated.Controlled hardening of alfalfa stimulated strongly the incorporation of sodium acetate-1, 2 (14)C into root lipids of the hardy variety Rambler, but only slightly in the case of the frost-sensitive variety Caliverde. When incorporation was done at 1 C at various times of hardening, labeling decreased significantly in linoleic acid with a corresponding increase in oleic acid. Hardening, therefore, repressed specifically the initial low temperature stimulation of oleic acid desaturation, without affecting the stimulation of elongation of palmitic acid and the desaturation of stearic acid at low temperature. The radioactivity in linoleic acid was slightly greater in hardy Rambler than in Caliverde throughout hardening.When feedings were done at 1 C at various times of hardening, labeling of phosphatidylcholine increased in Rambler while it decreased in Caliverde. Throughout the hardening period, when incorporation was done at 1 C, linoleic acid represented a higher percentage of the label in phosphatidylcholine than in phosphatidylethanolamine or triglycerides and its specific radioactivity was much greater in phosphatidylcholine than in phosphatidylethanolamine and triglycerides and in Rambler than in Caliverde. Phosphatidylcholine seems, therefore, to play a special part in linoleic acid synthesis and in its control during the acquisition of frost hardiness. PMID- 16659190 TI - Protein Body Composition in Cuburbita maxima Cotyledons as Determined by Energy Dispersive X-Ray Analysis. AB - Energy dispersive x-ray analysis was used to study the composition of certain protein body components in Cucurbita maxima cotyledons. The globoid crystal was rich in phosphorus, potassium, and magnesium. This elemental composition provides further evidence that the globoid crystal in squash cotyledon protein bodies is composed of phytin, a myoinositol hexaphosphoric acid salt of potassium and magnesium. Calcium, a common component of phytin in many species, was absent or present in only trace amounts in the globoid crystals of squash. Results of analyses of globoid crystals from seeds produced in different parts of North America suggest that there is definite specificity for the cations used in phytin deposition. Variations in soil types and other environmental factors seem not to have influenced the type of cation stored. Energy dispersive x-ray analysis of the proteinaceous regions revealed the presence of phosphorus, sulfur, and a trace of chlorine. Sulfur was expected, due to the presence of some sulfur containing amino acids in the protein. PMID- 16659191 TI - The Interaction between Osmotic- and Pressure-induced Water Flow in Plant Roots. AB - This paper presents a general model for coupled solute and water flow through plant roots based on the thermodynamics of irreversible processes. The model explains in a straight-forward manner such experimentally observed phenomena as changes in root resistance, increased solute flux, and apparent negative resistance, which have been reported for root systems under the influence of a hydrostatic pressure gradient. These apparent anomalies are explained on the basis of the interaction between the osmotic and hydrostatic driving forces and the well known "sweeping away" or dilution effect. We show that with a constant hydraulic conductivity the only features necessary to explain these phenomena are some type of membrane or membranelike structure and a mechanism for actively accumulating solutes. PMID- 16659192 TI - Effects of calcium on the photosynthesis of intact leaves and isolated chloroplasts of sugar beets. AB - Effects of calcium on photosynthesis in sugar beets (Beta vulgaris L. cv. F58 554H1) were studied by inducing calcium deficiency and determining changes in CO(2) uptake by attached leaves, electron transport, and photophosphorylation by isolated chloroplasts, and CO(2) assimilation by ribulose diphosphate carboxylase extracts. Calcium deficiency had no significant effect on leaf CO(2) uptake, photoreduction of ferricyanide, cyclic or noncyclic ATP formation of isolated chloroplasts, or on ribulose diphosphate carboxylase CO(2) assimilation, when the rates were expressed per unit chlorophyll. When expressed per unit leaf area CO(2) uptake increased by about 15% in low calcium leaves. The most noticeable effect of calcium deficiency was reduction in leaf area: low calcium had no effect on dark respiratory CO(2) evolution, on leaf diffusion resistance, or on mesophyll resistance to CO(2). We concluded that only small amounts of calcium are required for normal photosynthetic activity of sugar beet leaves. PMID- 16659193 TI - Polysome Formation in Light-sensitive Common Purslane Seeds. AB - Common purslane (Portulaca oleracea L.) seeds show light-controlled dormancy. Ribosome profiles from dark-incubated seeds consist of 22 to 26% polysomes. Light induces germination and stimulates polysome formation during the 12-hour lag period preceding radicle protrusion. Polysome levels increase to 29, 35, and 41% with exposure to 3, 6, and 9 hours of light, respectively. Although polysomes form on imbibition in the dark, 6 hours of light stimulates a significant increase in polysome formation which is probably related to early stages of radicle elongation. PMID- 16659194 TI - Concurrent comparisons of stomatal behavior, water status, and evaporation of maize in soil at high or low water potential. AB - Concurrent measurements of evaporation, leaf conductance, irradiance, leaf water potential, and osmotic potential of maize (Zea mays L. cv. Pa602A) in soil at either high or low soil water potential were compared at several hours on two consecutive days in July. Hourly evaporation, measured on two weighing lysimeters, was similar until 1000 hours Eastern Standard Time, but thereafter evaporation from the maize in the dry soil was always less than that in the wet soil; before noon it was 62% and by midafternoon, only 35% of that in the wet soil. The leaf water potential, measured with a pressure chamber, was between 1.2 and -2.5 bars and between -6.8 and -8 bars at sunrise (about 0530 hours Eastern Standard Time) in the plants in the wet and dry soil, respectively, but decreased quickly to between -8 and -13 bars in the plants in the wet soil and to less than -15 bars in the plants in the dry soil by 1100 to 1230 hours Eastern Standard Time. At this time, the leaf conductance of all leaves was less than 0.1 cm sec(-1) in the maize in the dry soil, whereas the conductance was 0.3 to 0.4 cm sec(-1) in the leaves near the top of the canopy in the wet soil. The osmotic potential, measured with a vapor pressure osmometer, also decreased during the morning but to a smaller degree than leaf water potential, so that by 1100 to 1230 hours Eastern Standard Time the leaf turgor potential was 1 to 2 bars in all plants. Thereafter, leaf turgor potential increased, particularly in the plants in soil at a high water potential, whereas leaf water potential continued to decrease even in the maize leaves with partly closed stomata. Evidently maize can have values of leaf conductance differing 3- to 4- fold at the same leaf turgor potential, which suggests that stomata do not respond primarily to bulk leaf turgor potential. Evidence for some osmotic adjustment in the plants at low soil water potential is presented. Although the degree of stomatal closure in the maize in dry soil did not prevent further development of stress, it did decrease evaporation in proportion to the decrease in canopy conductance. PMID- 16659195 TI - The Effect of Indole-3-acetic Acid and Other Growth Regulators on the Ripening of Avocado Fruits. AB - Observations were made of the effects of several plant regulators, indole-3 acetic acid, kinetin, abscisic acid, and gibberellic acid, as well as of extracts prepared from leaves and fruit stalks on the respiration pattern, ethylene production, and the number of days to ripen of avocado fruits (Persea americana Mill.). These substances were vacuum infiltrated to insure good penetration and distribution. Kinetin, abscisic acid, gibberellic acid, and the extracts had no effect on either ripening time or on the respiration pattern and ethylene production of the fruits. Indoleacetic acid, however, had a marked effect on ripening. At high concentrations (100 and 1000 mum), indoleacetic acid stimulated respiration and induced preclimacteric ethylene production, resulting in accelerated ripening of the fruits. At the low concentrations (1 and 10 mum), it delayed ripening of fruits and suppressed the climacteric respiration and ethylene production. The results reinforce several previous observations with other fruits that auxins may largely constitute ;resistance to ripening' and may be responsible for the lack of ripening shown by unpicked fruits. PMID- 16659197 TI - Intracellular recordings from phycomyces. AB - Intracellular recordings from the giant sporangiophore of Phycomyces stage II were obtained. The mean transmembrane potential for 30 observations was -119.9 millivolts (negative inside), and it did not change either as a result of a light stimulus or during dark adaptation. Injected depolarizing and hyperpolarizing step currents and steady currents did not produce any avidence of spike activity. We conclude that light transduction and dark adaptation in Phycomyces are not based on alterations of the transmembrane potential. PMID- 16659196 TI - The Effects of pH, Ionic Strength, and Ethylene on the Extraction of Cellulase from Abscission Zones of Citrus Leaf Explants. AB - The solubility of cellulase extracted from the abscission zones of citrus leaf explants (Citrus sinensis L. Osbeck) in sodium phosphate buffer depends on the pH of the extracting solution and, to a lesser extent, on the ionic strength. By increasing molarity from 0.01 to 0.16, the solubility of cellulase increased from 51% to 89% at pH 6.1 and from 70% to 98% at pH 7. In all cases, residual cellulase was further extracted from the pellet by buffer containing 1 m NaCl. Most of the enzymic activity was found in tissues proximal to the separation line, and activity of the cellulase which was soluble in phosphate buffer was closely correlated with abscission at both pH values. When extraction of cellulase at pH 6.1 with phosphate buffer was followed by a reextraction of the pellet with buffer containing 1 m NaCl, the activity of the cellulase soluble in the fortified buffer was also correlated with abscission. Pretreatment of explants with ethylene increased the solubility of cellulase in the phosphate buffer regardless of the pH used at the first extraction. PMID- 16659198 TI - Abscisic Acid stimulates elongation of excised pea root tips. AB - Excised Pisum sativum L. root tips were incubated in a pH 5.2 sucrose medium containing abscisic acid. Elongation growth was inhibited by 100 mum abscisic acid. However, decreasing the abscisic acid concentration caused stimulation of elongation, the maximum response (25% to 30%) occurring at 1 mum abscisic acid. Prior to two hours, stimulation of elongation by 1 mum abscisic acid was not detectable. Increased elongation did not occur in abscisic acid-treated root tips of Lens culinaris L., Phaseolus vulgaris L., or Zea mays L. PMID- 16659199 TI - Absorption and transfer of fe and mn in germinating sorghum. AB - The absorption of Fe from FeSO(4), FeEDTA, and FeEDDHA (ferric ethylenediaminedi (o-hydroxyphenylacetate)), and Mn from MnSO(4), MnEDTA, and MnEDDHA, by germinating sorghum (Sorghum vulgarie Pers. var. M 35-1) was studied. The seeds were found to absorb Fe and Mn from all the sources, and these ions moved to the scutellum, shoot, and root. EDDHA facilitated greater translocation of Fe and Mn from the seed to the shoot and root. The translocation of Fe was more towards the root than to the shoot, whereas it was the reverse in the case of Mn.We observed that the leaves of seedlings treated with Mn were slightly chlorotic. Further studies revealed that Mn did not affect the translocation of Fe, and possibly interfered with Fe utilization in chlorophyll synthesis. PMID- 16659200 TI - Loss of Ribulose 1,5-Diphosphate Carboxylase and Increase in Proteolytic Activity during Senescence of Detached Primary Barley Leaves. AB - Symptoms typical of senescence occurred in green detached primary barley (Hordeum vulgare L.) leaves placed in darkness and in light. Chlorophyll, total soluble protein, ribulose 1,5-diphosphate carboxylase protein and activity each progressively decreased in darkness and to a lesser extent in light. In all treatments most of the total soluble protein lost was accounted for by a decrease in ribulose 1,5-diphosphate carboxylase protein, suggesting that the chloroplast was a major site of degradation early in senescence.Loss of ribulose 1,5 diphosphate carboxylase protein was negatively correlated with an increase in proteolytic activity measured against azocasein. Both rates were exponential, with about a 30% difference in apparent rate constants. Cycloheximide essentially prevented the loss of chlorophyll, ribulose 1,5-diphosphate carboxylase protein, and activity and completely inhibited the increase in proteolytic activity against azocasein. Since chloramphenicol had little effect on the loss of ribulose 1,5-diphosphate carboxylase protein or chlorophyll, or on proteolytic activity against azocasein, it is suggested that the proteolytic activity was developed on cytoplasmic 80 S ribosomes.Kinetin greatly retarded the onset of such symptoms of senescence by inhibiting the losses of chlorophyll and ribulose 1,5-diphosphate carboxylase protein and protected against inactivation of enzymic activity. It also prevented the increase in proteolytic activity measured against azocasein. Incorporation of labeled amino acids into ribulose 1,5-diphosphate carboxylase during its rapid degradation showed that the enzyme was under turnover. The changes in ribulose 1,5-diphosphate carboxylase protein and activity, chlorophyll, soluble protein other than ribulose 1,5-diphosphate carboxylase, proteolytic and esterolytic activity during senescence indicate that senescence is a selective, sequential process. PMID- 16659201 TI - Effect of gibberellic Acid on crown gall tumor induction in aging primary pinto bean leaves. AB - Gibberellic acid was tested for its effect on tumor induction by Agrobacterium tumefaciens in primary pinto bean (Phaseolus vulgaris) leaves in various stages of development. The hormone was found to promote tumor induction in partially aged leaves but did not effect tumor induction in very young leaves or in fully matured leaves. It is suggested that the natural loss of susceptibility to tumor induction in maturing pinto bean leaves is associated with a concomitant loss of endogenous gibberellins and/or a sensitivity to gibberellins. PMID- 16659202 TI - Microbody-marker Enzymes during Transition from Phototrophic to Organotrophic Growth in Euglena. AB - Transfer of Euglena gracilis Klebs Z cells from phototrophic to organotrophic growth on acetate results in derepression of the key enzymes of the glyoxylate cycle, malate synthase and isocitrate lyase, which appear coordinately regulated. The derepression of malate synthase and isocitrate lyase was accompanied by increased specific activities of succinate dehydrogenase, fumarase, and malate dehydrogenase, but hydroxypyruvate reductase activity was unaltered.Isolation of organelles from broken cell suspensions of cells grown heterotrophically on acetate was achieved by isopycnic centrifugation on sucrose gradients. Peaks of mitochondrial enzymes were obtained at equilibrium densities of 1.22 g cm(3) and 1.16 g cm(3), and although significant differences in the distribution of tricarboxylic acid cycle enzymes between these two peaks were not recorded adenosine triphosphatase activity was detected only in the less dense fraction (1.16 g cm(3)) showing this contained damaged mitochondria. The peak of particulate glyoxylate cycle enzymes was at an equilibrium density of 1.25 g cm(3), this being the same as that for glycolate pathway enzymes from phototrophic cells. Citrate synthase, isocitrate lyase, malate synthase, and malate dehydrogenase were all present in this fraction so it was concluded that Euglena glyoxysomes contain a complete glyoxylate cycle. PMID- 16659203 TI - The glyceraldehyde 3-phosphate and glycerate 3-phosphate shuttle and carbon dioxide assimilation in intact spinach chloroplasts. AB - The regulation of CO(2) assimilation by intact spinach (Spinacia oleracea) chloroplasts by exogenous NADP-linked nonreversible d-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) was investigated. This dehydrogenase mediated a glyceraldehyde 3-phosphate/glycerate 3-phosphate shuttle for the indirect transfer of NADPH from chloroplast to the external medium. The rate of NADPH formation in the medium reflected glyceraldehyde 3-phosphate efflux from the chloroplast. Increasing enzyme concentrations stimulated NADP reduction and, in turn, CO(2) fixation. Pyrophosphate increased CO(2) fixation by apparently inhibiting glyceraldehyde 3-phosphate efflux. Increasing the glycerate 3 phosphate concentration above 0.1 mm stimulated glyceraldehyde 3-phosphate efflux but inhibited CO(2) fixation. Addition of up to 0.5 mm orthophosphate enhanced both glyceraldehyde 3-phosphate efflux and CO(2) fixation while each was inhibited by higher orthophosphate concentrations. The mechanism by which the extent of glyceraldehyde 3-phosphate efflux regulated the rate of CO(2) fixation in chloroplasts was discussed. PMID- 16659204 TI - Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase. AB - The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either alpha-mannosidase or N-acetyl-beta-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination. PMID- 16659205 TI - Galactolipid Synthesis in Vicia faba Leaves: I. Galactose, Glycerol, and Fatty Acid Labeling after CO(2) Feeding. AB - The galactose, glycerol, and fatty acids of mono- and digalactosyl diglycerides (MGDG and DGDG) have been separated and analyzed for (14)C activity after (14)CO(2) feeding of Vicia faba leaf discs. Fully expanded and developing leaves were analyzed at time intervals following feeding during continuous illumination. In addition, fully expanded leaves were analyzed after similar times in complete darkness. In all cases, (14)C was incorporated very rapidly into galactose, whereas glycerol and fatty acids were labeled much more slowly and over a longer period of time. The data are consistent with the galactosylation of a diglyceride to MGDG which is in turn galactosylated to DGDG. The data suggest that the formation of diglycerides suitable for galactosylation to MGDG is slow in comparison to the galactosylation process. It is also suggested that DGDG may be formed from more than one pool of MGDG. The complete analysis of the (14)C incorporation into galactose appears to represent the only satisfactory method of comparing galactolipid synthesis by (14)C incorporation. Estimates of comparative rates of synthesis of MGDG and DGDG have been made on this basis. PMID- 16659206 TI - Regulation of cell wall synthesis in Avena stem segments by gibberellic Acid. AB - Gibberellic acid induces (a) increased elongation of Avena sativa stem segments, (b) increased formation of cell wall material, measured on the basis of dry weight, and (c) increased incorporation of (14)C-glucose into all fractions of the cell wall material. This increased incorporation of radioactivity correlates well with increased formation of cell wall material and shows a time-course pattern similar to the time course of the elongation response. Approximately one hour after the application of gibberellic acid, the rates both of growth and of incorporation of radioactivity accelerate to about 2-fold over the control rate. Gibberellic acid does not stimulate the incorporation of labeled glucose into the cell wall material simply by increasing the rate of uptake of glucose by internodal cells. The stimulation of the incorporation of (14)C-glucose into cell wall material, which reflects the stimulation of cell wall synthesis, seems to be an important and relatively early effect of gibberellic acid in this system and probably contributes significantly to the elongation response elicited by the hormone. PMID- 16659207 TI - Carbon-13 Nuclear Magnetic Resonance Analysis of Metabolism in Soybean Labeled by CO(2). AB - Fourier transform (13)C nuclear magnetic resonance spectra have been obtained of intact, fresh soybean ovules (Glycine max L. cv. Dare) harvested from pods subtended by a trifoliolate exposed to (13)CO(2) 1 to 3 days earlier. The high resolution spectra are interpreted in terms of the labeled sugars and lipids in the ovule. Comparison of the spectra taken over the 3-day period permits qualitative estimates of sugar metabolism and rates of lipid synthesis. The spectra also contain information about the distribution of labels within the lipid chains. This information leads to a method of estimating the extent to which glucose degradation in the synthesizing soybean ovule is involved in the reactions of the phosphogluconate pathway. PMID- 16659208 TI - C(4) photosynthesis in tree form euphorbia species from hawaiian rainforest sites. AB - The (13)C (12)C isotope ratios and the leaf anatomy of 18 species and varieties of Euphorbia native to the Hawaian Islands indicated that all possess C(4) photosynthesis. These species range from small prostrate coastal strand shrubs to shrubs and trees in rainforest and bog habitats. The results show that C(4) photosynthesis occurs in plants from a much wider range of habitats and life forms than has been previously reported. PMID- 16659209 TI - Photoaffinity-labeled Auxins: Synthesis and Biological Activity. AB - Two light-sensitive analogs of 2,4-dichlorophenoxyacetic acid, namely 4-azido-2 chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid, have been synthesized for use as auxin photoaffinity labels. The preparation and biological activity of the compounds are described. Both contain the photolabile azido group; the 2,4-substituted compound shows auxin activity and the 3,5-substituted compound does not. These photoaffinity analogs of 2,4-dichlorophenoxyacetic acid may be useful in the identification of the auxin receptor molecules in plant cells and eventually of the receptor sites within these molecules. PMID- 16659210 TI - Interactions of microtubule disorganizers, plant hormones, and red light in wheat coleoptile segment growth. AB - Growth response of coleoptile segments excised from 3-day-old seedlings of wheat (Triticum vulgare cv. Baart) to gibberellic acid, indoleacetic acid, and 2,4 dichlorophenoxyacetic acid, to red light, and to several microtubule disorganizers depends on the initial position of the excised segment in the intact coleoptile. Red light, 660 nm, stimulates the growth of the apical cells, but inhibits markedly the growth of the cells in the basal region of the coleoptile. The effects of red light are independent of sucrose, gibberellic acid, indoleacetic acid, and 2,4-dichlorophenoxyacetic acid, even though these substances themselves markedly affect the growth of the coleoptile segments. Concentractions of the microtubule disorganizers, vinblastine sulfate, cupric chloride, urea, and colchicine, which do not alter significantly the growth of the dark control apical segments, substantially repress the promotive effects of red light or auxin on the increase in length of the apical cells of the coleoptile. This suggests that stimulation by red light and by auxin involves microtubule production. Microtubule disorganizers repress the growth of elongating cells of the coleoptile, yet on the other hand, auxin and irradiation do not alter significantly the response of basal cells to the microtubule disorganizing agents. We hypothesized that light and growth regulators induce polymerization of nonaggregated microtubule subunits, resulting in faster growth. PMID- 16659211 TI - Relation between Mesophyll Surface Area, Photosynthetic Rate, and Illumination Level during Development for Leaves of Plectranthus parviflorus Henckel. AB - The influence of illumination level during leaf development on the mesophyll cell surface area per unit leaf area (A(mes)/A), CO(2) resistances, and the photosynthetic rate was determined for leaves of Plectranthus parviflorus Henckel. The relative importance of A(mes)/A versus CO(2) resistances in accounting for observed changes in photosynthesis was quantitatively evaluated using equations based on analogies to electrical circuits.When the illumination during development was raised from 900 to 42,000 lux, the leaves more than tripled in thickness as the mesophyll cells increased in size and frequency, which caused A(mes)/A to go from 11 to 50. The net rate of photosynthesis at light saturation concomitantly increased 4-fold, reflecting a corresponding decrease in the total resistance for CO(2) movement per unit leaf area. However, the CO(2) resistance per unit area of mesophyll cells remained about 580 seconds per centimeter for leaves grown under 900 to 42,000 lux. Thus, for P. parviflorus, the increased photosynthetic rate for leaves developing under higher illuminations resulted from a higher A(mes)/A, not from changes in the CO(2) resistances within individual mesophyll cells, expressed per unit area of cell surface. Results are discussed in terms of previously observed increases in thickness, internal leaf area, and photosynthetic rates for sun versus shade leaves on various plant species. PMID- 16659212 TI - Stereoisomeric Characterization of Tartaric Acid Produced during l-Ascorbic Acid Metabolism in Plants. AB - Labeled tartaric acids from Pelargonium crispum apices which had been fed l ascorbic acid-6-(14)C and Vitis labrusca and Parthenocissus inserta tissues which had been fed l-ascorbic acid-1-(14)C were examined by chemical means to determine chiral configuration. In each instance, label was associated with (+)-tartaric acid.Similar experiments with labeled tartaric acid from P. crispum which had been labeled with d-glucose-1-(14)C or -6-(14)C led to the same result. No evidence was obtained for formation of labeled meso-tartaric acid in experiments described above. The recent suggestion of H. Ruffner and D. Rast (Z. Pflanzenphysiol. 73: 45-55, 1974) that conversion of l-ascorbic acid to tartaric acid in plants is a nonenzymatic process is re-examined in the light of present findings. PMID- 16659213 TI - Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue. AB - Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, alpha-amanitin, and cycloheximide. alpha-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by alpha-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system. PMID- 16659214 TI - The role of cytokinins in chloroplast lamellar development. AB - The accumulation of chlorophyll, production of two specific lamellar chlorophyll protein complexes, onset of O(2) evolution, and detection of P700 were examined in intact Jack bean (Canavalia ensiformis [L.] D.C.) leaves treated with 10(-5)m kinetin or benzyladenine and allowed to green under low (30-35%) and high (80 85%) relative humidity. In contrast to reports of the promotion of chlorophyll accumulation by cytokinin treatment in excised tissue or cotyledons, intact greening leaves showed neither promotion of chlorophyll accumulation nor alteration in formation of the lamellar chlorophyll-protein complexes or development of photosynthetic function. Furthermore, cytokinin was ineffective in relieving the consequences of low relative humidity water stress on chlorophyll accumulation and on the formation of at least one lamellar chlorophyll-protein. PMID- 16659215 TI - Auxin and the response of pea roots to auxin transport inhibitors: morphactin. AB - The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4 dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding. PMID- 16659216 TI - Carbon dioxide assimilation by leaves, isolated chloroplasts, and ribulose bisphosphate carboxylase from spinach. AB - The relationship between rate of photosynthesis and CO(2) concentration has been reinvestigated using isolated spinach (Spinacia oleracea) chloroplasts. The apparently low CO(2) concentration required for half-maximal photosynthesis is shown to result partly from a ceiling imposed by electron transport. In double reciprocal plots of rate against CO(2) concentration, this ceiling results in departures from linearity at high CO(2) concentrations. If these rate limitations are disregarded in extrapolation the "true" CO(2) concentration required for half maximal carboxylation by intact chloroplasts is approximately 46 mum (CO(2)).When assayed under comparable conditions, ribulose bisphosphate carboxylase from these chloroplasts also shows an apparent Km (CO(2)) of approximately 46 mum, suggesting that its characteristics are not modified by extraction. An improved assay for ribulose bisphosphate carboxylase yielded rates of carboxylation considerably higher than those previously reported, the highest maximal velocities recorded approaching 1000 mumoles CO(2) fixed mg(-1) chlorophyll hr( 1) at 20 C. With such Km and V(max), values the carboxylase would be able to achieve, at concentrations of CO(2) less than atmospheric, rates of CO(2) fixation equal to those displayed by the parent tissue or by the average plant under favorable conditions in its natural environment. PMID- 16659217 TI - Inhibition of Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations. AB - A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift. PMID- 16659218 TI - Perithecial Formation in Gelasinospora reticulispora: IV. Action Spectra for the Photoinduction. AB - Action spectra for photoinduction of perithecia after different lengths of dark period were determined with apically growing mycelium of a sordariaceous fungus Gelasinospora reticulispora. When hyphae were exposed to monochromatic light in near ultraviolet and visible regions, reciprocity between intensity and exposure time was observed within the range of incident energy used. The resulting action spectrum determined after a dark period of 48 hours showed a peak at 460 nm with shoulders at 420 and 480 nm and another peak at 370 nm, indicating minima at 410, 430, and 470 nm. After 72 hours darkness the spectrum was very similar to the above, except that the major peak shifted to 450 nm and the near ultraviolet region was somewhat less effective. In both cases, wavelengths longer than 520 nm showed no effect.In addition, when irradiated with colored lamps, near ultraviolet light was relatively less effective after the 72-hour dark period than after the 48-hour dark period. The reason for this phenomenon is discussed. PMID- 16659219 TI - Nitrate Uptake and Assimilation by Wheat Seedlings during Initial Exposure to Nitrate. AB - Nitrate uptake, reduction, and translocation were examined in intact, 14-day-old, nitrogen-depleted wheat (Triticum vulgare var. Knox) seedlings during a 9-hour exposure to 0.2 mm Ca (NO(3))(2). The nitrate uptake rate was low during the initial 3-hour period, increased during the 3- to 6-hour period, and then declined. By the 3rd hour, 14% of the absorbed nitrate had been reduced, and this increased to 36% by the 9th hour. Shoots accumulated reduced (15)N more rapidly than roots and the ratio of reduced (15)N to (15)N-nitrate was higher in the shoots. A significant proportion of the total reduction occurred in the root system under these experimental conditions. Accumulation of (15)N in ethanol insoluble forms was evident in both roots and shoots by the 3rd hour and, after 4.5 hours, increased more rapidly in shoots than in roots.An experiment in which a 3-hour exposure to 0.2 mm Ca ((15)NO(3))(2) was followed by a 12-hour exposure to 0.2 mm Ca ((14)NO(3))(2) revealed a half-time of depletion of root nitrate of about 2.5 hours. A large proportion of this depletion, however, was due to loss of (15)N-nitrate to the ambient (14)N-nitrate solution. The remaining pool of (15)N-nitrate was only slowly available for reduction. Total (15)N translocation to the shoot was relatively efficient during the first 3 hours after transfer to Ca ((14)NO(3))(2) but it essentially ceased after that time in spite of significant pools of (15)N-nitrate and alpha-amino-(15)N remaining in the root tissue. PMID- 16659220 TI - Parthenocarpic Fruit Development from Grafted Ovaries of Cucuminis sativus L. AB - Reciprocal cleft and pistillate floral bud grafts were made between parthenocarpic ;Fertilla' and nonparthenocarpic ;MSU 713-5' cucumber (Cucumis sativus L.) lines to localize the site for stimulation of parthenocarpic fruit set. No fruit set on ;MSU 713-5' controls, scion grafted to ;Fertilla,' or rootstock with ;Fertilla' as the scion. ;Fertilla' controls, rootstock, and scions all produced parthenocarpic fruit when grafted to ;MSU 713-5.' When pistillate floral buds of ;MSU 713-5' were grafted to ;Fertilla,' no fruit were produced. However, individual immature pistillate buds of ;Fertilla' developed into mature fruits when grafted onto ;MSU 713-5.' Hence, the immature ovary is the site of stimulation for parthenocarpic fruit set in cucumber. PMID- 16659221 TI - Plant Desiccation and Protein Synthesis: II. On the Relationship between Endogenous Adenosine Triphosphate Levels and Protein-synthesizing Capacity. AB - Rehydration of Tortula ruralis in 2,4-dinitrophenol inhibits protein synthesis, polysome formation, and ATP production. Polysomes are conserved intact and are active in vitro in hydrated Tortula placed in this chemical, although in vivo protein synthesis is inhibited. Hydrated moss placed under nitrogen in the dark shows a reduced capacity for ATP and protein synthesis, but polysomes are conserved. During anaerobiosis in light, ATP and protein synthesis are unaffected. Rehydration of slow-dried Tortula in nitrogen in the dark results in reduced in vivo protein synthesis, but not polysome formation; this reduction is much less in the light. Slow-dried moss, but not fast-dried, has a greatly reduced ATP content in the dry state, but this rapidly returns to normal levels on rehydration. The prolonged burst in respiration observed previously on rehydration of Tortula is not paralleled by ATP accumulation. Changes in energy charge in all treatments tested follow the changes in ATP. The aquatic moss, Hygrohypnum luridum, which is intolerant to drought, loses ATP during fast drying and this is not replenished on subsequent rehydration.We consider that the relationship between levels of ATP and protein synthesis is more meaningful during rehydration of mosses (the time when repair to desiccation-induced cellular damage can occur) than during desiccation, and that drought-induced cessation of protein synthesis may not be mediated directly through ATP availability. PMID- 16659222 TI - Solubilization of enzymes from glyoxysomes of maize scutellum. AB - Glyoxysomes isolated from maize scutella (Zea mays L.) were subjected to several disruptive treatments (osmotic shock, resuspension in an alkaline medium, addition of detergent). The damaged glyoxysomes were centrifuged at 89,500g for 40 minutes and several enzymic activities (isocitratase, malate synthetase, catalase, citrate synthetase, malate dehydrogenase) were measured in the supernatant fraction and in the pellet. Isocitratase is the most easily released of all glyoxysomal enzymes closely followed by malate synthetase. Citrate synthetase is in all instances the most insoluble enzyme. All of the enzymes had higher specific activities in the supernatant than in the pellet. These findings suggest that in corn scutellum glyoxysomes none of these enzymes is truly membrane-bound. PMID- 16659223 TI - Transplantation Studies with Immature Fruit of Normal, and rin and nor Mutant Tomatoes. AB - The aim of the work reported herein was to determine whether the lack of normal ripening in fruits of rin and nor tomato mutants is due to the presence of ripening inhibitors or to the lack of ripening factors in the fruit. A fruit tissue transplantation technique was developed for this purpose.Disks of pericarp tissue were transplanted reciprocally between tomato fruits (Lycopersicon esculentum Mill.) of the rin and nor mutants and fruits of ;Rutgers,' a normally ripening cultivar. CO(2) and ethylene evolution rates were measured daily. To test whether materials are translocated between receptor fruits and transplanted disks, fruits were vacuum-infiltrated with (14)C-labeled amino acids and implanted with disks from unlabeled fruits.Normal ripening was not induced in disks of rin and nor tissues implanted in Rutgers fruit although development of yellow or yellow-orange colors associated with senescence of the mutant fruits was accelerated. Disks of Rutgers fruit tissue implanted in fruits of rin and nor ripened normally and concomitant with the intact Rutgers control fruits. The transplanted disks contained 28.8% as much label as found in disks of receptor fruits; thus, significant translocation into the implanted disks occurred. It is concluded that fruits of the mutants do not contain translocatable ripening inhibitors or lack translocatable ripening factors. PMID- 16659224 TI - Peroxidative Activity in Euglena gracilis. AB - Cell-free homogenates of Euglena gracilis contain very low levels of catalase activity as compared to higher plants and some other algae. Purified Euglena cytochrome c acts catalytically as a peroxidase. The observed catalytic activity of cytochrome c in extracts from heterotrophically grown cells was more than enough to account for the observed rates of hydrogen peroxide destruction. The peroxidative activity of Euglena cytochrome c was completely inhibited by 20 mm 3 amino-1,2,4-triazole. PMID- 16659225 TI - Leaf tissue senescence: constant responsiveness to hormones despite a seasonal cycle in senescence rate. AB - During winter, excised leaf tissue from Rumex obtusifolius degrades chlorophyll at twice the summer rate but the plant hormones, gibberellic acid and zeatin, inhibit the senescence rate by a constant percentage, regardless of season. PMID- 16659226 TI - Phytochrome, nitrate movement, and induction of nitrate reductase in etiolated pea terminal buds. AB - The role of phytochrome in the induction of nitrate reductase of etiolated field peas (Pisum arvense L.) was examined. Terminal bud nitrate concentration increased in darkness, and the increase correlated with induction of nitrate reductase following brief exposure of intact plants to red, blue, far red, and white lights. Brief light exposure of intact plants stimulated nitrate uptake and induction of nitrate reductase by terminal buds subsequently excised and incubated on nitrate solution in darkness; exposure of excised buds in contact with nitrate led to less uptake but more induction. Nitrate and nitrate reductase activity both declined during incubation with water, irrespective of light treatment. Nitrate enrichment of intact terminal buds and uptake into excised buds and increases in nitrate reductase activity were all red/far red reversible. Dimethyl sulfoxide (1%, v/v) and sugars (sucrose 0.5%, glucose 1, w/v), although stimulating nitrate uptake into excised tissue in darkness, failed to enhance nitrate reductase activity over dark controls. Phytochrome may regulate nitrate reductase via both nitrate movement and a general mechanism such as enhancement of protein synthesis. PMID- 16659227 TI - Acetyl coenzyme a-glutamate acetyltransferase and N-acetylornithine-glutamate acetyltransferase of chlorella. AB - The enzymic formation of acetylglutamate has been studied in Chlorella vulgaris extracts. Acetyl CoA and N(2)-acetyl-l-ornithine served as substrates for glutamate acetylation whereas acetylphosphate, N(5)-acetyl-l-ornithine, and N(2) acetyl-2,4-diamino butyrate were ineffective. Acetyl CoA-glutamate transacetylase and acetylornithine-glutamate transacetylase activities have been purified over 180-fold with no indication of any separation of activities. The acetyl CoA activity was more labile than acetylornithine activity so that preparations having acetylornithine-glutamate transacetylase activity but no acetyl CoA glutamate transacetylase activity were obtained. The two acetylating activities appear to be properties of one enzyme with one portion more easily denatured.Both acetylating activities had pH optima between 8 and 8.5. The Km value for glutamate was 3 mm for both activities. The Km values were 0.2 mm for acetylornithine and 3.2 mm for acetyl CoA. Arginine inhibited acetyl CoA glutamate transacetylase (Ki = 0.94 mm) and acetylglutamate phosphokinase (Ki = 0.5 mm) but had no effect on acetylornithine-glutamate transacetylase. The lack of an inhibitory effect of proline on any of the three enzymic activities indicates that acetylglutamate is not a normal intermediate in proline biosynthesis. Growth of Chlorella with arginine as a nitrogen source had no effect on enzyme levels, showing that end-product repression is not a control factor in arginine biosynthesis in Chlorella. In Chlorella, arginine controls its own biosynthesis by inhibiting acetylglutamate phosphokinase and controls the level of acetylated intermediates by inhibiting acetyl CoA-glutamate transacetylase. PMID- 16659228 TI - Pea leaf glutamine synthetase: regulatory properties. AB - Of a variety of purine and pyrimidine nucleotides tested, only ADP and 5'AMP significantly inhibited the Mg(2+)-dependent activity of pea leaf glutamine synthetase. They were less effective inhibitors where Mn(2+) replaced Mg(2+). They were competitive inhibitors with respect to ATP, with inhibition constant (Ki) values of 1.2 and 1.8 mm, respectively. The energy charge significantly affects the activity of glutamine synthetase, especially with Mg(2+). Of a variety of amino acids tested, l-histidine and l-ornithine were the most inhibitory, but significant inhibition was seen only where Mn(2+) was present. Both amino acids appeared to compete with l-glutamate, and the Ki values were 1.9 mm for l-histidine (pH 6.2) and 7.8 mm for l-ornithine (pH 6.2). l-Alanine, glycine, and l-serine caused slight inhibition (Mn(2+)-dependent activity) and were not competitive with ATP or l-glutamate.Carbamyl phosphate was an effective inhibitor only when Mn(2+) was present, and did not compete with substrates. Inorganic phosphate and pyrophosphate caused significant inhibition of the Mg(2+) dependent activity. PMID- 16659229 TI - In Vitro Synthesis of Ureidohomoserine by an Enzyme from Jack Bean (Canavalia ensiformis) Leaves. AB - An enzyme was extensively purified from jack bean leaves (Canavalia ensiformis L.) which produced o-ureidohomoserine from l-canaline and carbamyl phosphate. The most highly purified preparations catalyzed both this reaction and citrulline synthesis from ornithine and carbamyl phosphate, and the ratio of the two activities remained nearly constant during purification. When hydrated jack bean seeds were the enzyme source, ornithine carbamyltransferase (EC 2.1.3.3) activity was high but synthesis of ureidohomoserine was barely detectable. Both ornithine carbamyltransferase and the ureidohomoserine synthesizing enzyme had similar Km values for carbamyl phosphate. The purification data suggest that one enzyme may catalyze both reactions in jack bean leaves. PMID- 16659230 TI - Light sensitivity of na, rb, and k absorption by different tissues of bean leaves. AB - Autoradiographs of (22)Na-loaded bean (Phaseolus vulgaris L. cv. ;Brittle Wax') leaf slices showed that most of the tracer was concentrated in vascular tissue. Rubidium-86 was uniformly distributed in slices that had been incubated in darkness; after incubation in the light many small dark spots appeared on the autoradiographs, apparently corresponding with the stomata. Autoradiographs of (42)K-loaded slices showed a rather uniform distribution of the tracer, whether the slices had been incubated in light or in darkness. PMID- 16659231 TI - Production of isoprene by leaf tissue. AB - Isoprene production by Hamamelis virginiana L. and Quercus borealis Michx. leaves was studied. When ambient CO(2) concentrations were maintained with bicarbonate buffers, the rate of isoprene production at 125 microliters per liter of CO(2) was approximately four times that at 250 microliters per liter of CO(2). Isoprene production was drastically inhibited by 97% O(2). Dichlorodimethylphenylurea (0.1 mm), NaHSO(3) (10 mm), and alpha-hydroxy-2-pyridinemethanesulfonic acid (10 mm) inhibited isoprene production but increased the compensation point of the tissue. Isonicotinic acid hydrazide neither inhibited isoprene emission nor increased the compensation point of the tissue significantly. Inhibition of isoprene production does not seem to correlate with stomatal resistance. Isoprene was labeled by intermediates of the glycolate pathway, and similarities are noted between the biosynthesis of isoprene and that of beta-carotene. PMID- 16659232 TI - Foliar Iron Spray Potentiates Growth of Seedlings on Iron-free Media. AB - Growth of bean (Phaseolus vulgaris cv. ;Brittle Wax') seedlings in iron-free media caused large reductions in root size and dye reduction capacity. Shoot growth was also severely retarded and the classical symptoms of chlorosis were observed. A single prophylactic spray of FeSO(4), applied to the primary leaves of 7-day seedlings, enabled subsequent growth to equal that obtained when iron was continuously supplied to the roots over a 12-day period, although chlorophyll levels were lower. By adding a silicone-based surfactant to FeSO(4) spray solutions, the burn damage normally caused to seedlings by such sprays was largely eliminated and the irreversible adsorption of iron increased. Foliar spray of an iron chelate (ferric ethylenediaminedi (o-hydroxyphenylacetic acid)) were less effective than those of FeSO(4). PMID- 16659233 TI - Changes in Enzyme Regulation during Growth of Maize: I. Progressive Desensitization of Homoserine Dehydrogenase during Seedling Growth. AB - The sensitivity of homoserine dehydrogenase (EC 1.1.1.3) to inhibition by the feed-back modifier, l-threonine, was examined in preparations derived from etiolated shoots, roots, and lightgrown tissues of Zea mays L. var. earliking. A progressive decrease in enzyme sensitivity was observed during seedling growth. Enzyme derived from internode tissue retained a greater sensitivity to the effector than enzyme derived from apical portions of etiolated shoots, whereas enzyme from root tips was characteristically more sensitive than that prepared from mature cells of the root. Enzyme desensitization occurred rapidly during culture of excised shoots and the activities of both homoserine dehydrogenase and aspartokinase (EC 2.7.2.4) declined during shoot culture under a variety of conditions. The initial enzyme levels and the characteristic sensitivity of homoserine dehydrogenase were preserved during culture at 5 to 7 C, but desensitization was not prevented by inclusion of cycloheximide in the culture medium.Results of control experiments provide evidence that desensitization occurs in vivo. No alteration of the enzyme properties was detected during extraction or concentration of sensitive or insensitive enzyme or during coextraction of enzyme from mixed populations of different age shoots; nor was a differential distribution of inhibitors or activators indicated during assay of mixed preparations. The change in enzyme sensitivity was apparent under a variety of assay conditions and was not accompanied by changes in the apparent affinity of the enzyme for the substrate, homoserine. It is suggested that systematic changes in the regulatory characteristics of certain enzymes could be an important level of metabolic regulation during cellular differentiation.Three forms of maize homoserine dehydrogenaase were detected after acrylamide gel electrophoresis of samples derived from 72-hr shoots. Similar analysis of samples from older shoots revealed a broad asymmetric band of enzyme activity, suggesting that changes in the relative distribution of specific forms of the enzyme could be related to the growth-dependent changes in the sensitivity of maize homoserine dehydrogenase. PMID- 16659234 TI - Changes in Enzyme Regulation during Growth of Maize: II. Relationships among Multiple Molecular Forms of Homoserine Dehydrogenase. AB - The relative contribution of each of several forms of homoserine dehydrogenase (EC 1.1.1.3) to the total enzyme population in etiolated shoots and in roots of Zea mays L. var. earliking was examined by the use of gel filtration chromatography and disc gel electrophoresis. In enzyme preparations derived from shoots of seedlings grown for 72, 120, or 168 hours, two molecular forms, II and III, which have the same apparent molecular weight but differ in net charge, contributed 75 to 80% of the total enzyme activity. A lower molecular weight species, form I, contributed 20 to 25% of the activity from 72-hour shoots, but was found to decrease concomitantly with a proportional increase in activity contributed by aggregated enzyme form(s) during shoot development. Form I contributed a comparatively larger fraction of the total enzyme activity in preparations of roots of 72-hour seedlings.The characteristic enzyme activity of different tissues was found to be the result of variations in both the amount and the properties of individual forms. Form I was consistently insensitive to inhibition by the feed-back modifier, l-threonine, but evidence is presented which indicates that the regulatory properties of form II and/or form III are systematically altered during shoot growth. The activity of the enzyme forms was also differentially stimulated by monovalent cations, K(+) being the most effective activator; in all cases the potential for activation was correlated with the potential for inhibition. In contrast to these differences among the forms of the maize enzyme, all forms were shown to share a number of common characteristics. Potential factors which could influence the growth-associated changes in homoserine dehydrogenase are discussed briefly. PMID- 16659235 TI - Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T. AB - The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of (14)CO(2) by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of (14)C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO(2) fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO(2) provides a bioassaay for race T toxin which is both reliable and rapid. PMID- 16659236 TI - Studies on the Development and Localization of Catalase and H(2)O(2)-generating Oxidases in the Endosperm of Germinating Castor Beans. AB - During germination of castor bean seeds (Ricinus communis var. Hale), the changes of activity of catalase, uricase, and alpha-hydroxyacid oxidase of the endosperm follow a rise and fall pattern with a peak between day 4 and 5 similar to that observed for the glyoxylate cycle enzymes. After 3 days of germination, most of the activities of these enzymes are recovered from the glyoxysomal fraction separated by isopycnic sucrose density gradient centrifugation.The activities of the oxidases of the glyoxysomal fractions increase somewhat more slowly than those of catalase or isocitrate lyase during the first 3 days of germination. But separation of the oxidative enzyme activities from glyoxysomal isocitrate lyase activity was not achieved by density gradient centrifugation or by density gradient electrophoresis. The sedimentation velocities of these two kinds of enzyme activities were found to be identical. The role and regulation of the glyoxysomal uricase, glycolate oxidase, lactate oxidase, and catalase activity in endosperm cells are discussed. PMID- 16659237 TI - Light-enhanced Chloride Uptake by Wheat Laminae: A Comparison of Chopped and Vacuum-infiltrated Tissue. AB - The time course of (36)Cl(-) uptake from 5 mm KCl by 1.5-mm leaf segments of Triticum aestivum L. seedlings has been determined over 24 hours both in the light and in the dark. A light-enhanced uptake of Cl(-) develops after a few hours. Using whole laminae which have been water-injected by vacuum infiltration, a light-enhanced uptake is apparent from zero time. Uptake values achieved in the light by the two types of tissue are similar but in the dark there is a restricted uptake by the whole infiltrated laminae. It is considered that the slower uptake by whole laminae relative to chopped tissue in the dark is due to cuticular resistance to solute penetration, whereas in the light the impediment is overcome by stomatal opening. A light-enhanced uptake unrelated to stomatal opening is discernible in both tissues. Its energetic basis has not been defined but may be related to substrate exhaustion. The absorption mechanism is not impaired by vacuum infiltration. PMID- 16659238 TI - Effect of vacuum infiltration on photosynthetic gas exchange in leaf tissue. AB - Using a manometric method, photosynthetic oxygen evolution and (14)CO(2) fixation have been determined for leaf tissue of Triticum aestivum L., Hordeum vulgare L., Phaseolus vulgaris L., and Lemna minor L. Approximately similar values in the range 0.2 to 0.4 millimoles grams fresh weight(-1) hour(-1) were obtained for both gases. In tissue subjected to vacuum infiltration, O(2) evolution and (14)CO(2) fixation were barely measurable. It is considered that the elimination of photosynthetic gas exchange results from a decreased supply of CO(2) to the chloroplasts. Chopping wheat laminae also leads to a reduction in photosynthetic gas exchange, slices 1 millimeter or less giving only 10 to 20% of the value for whole tissue. Respiration is unaffected by either treatment. Carbonic anhydrase did not improve photosynthetic gas exchange in infiltrated tissue. The use of sliced or vacuum-infiltrated leaf tissue in photosynthetic studies is discussed. PMID- 16659239 TI - The Effect of ATP on the Photoconversion of Protochlorophyllide in Isolated Etioplasts of Zea mays. AB - The transformation of protochlorophyllide (PChle) into chlorophyllide (Chle) has been studied in isolated etioplasts from Zea mays. ATP (1.5mm) prevented the transformation of photoconvertible PChle 650 to PChle 630 in aged etioplasts. Curve analysis indicated that the ATP effect on photoconvertibility could be entirely accounted for by changes in the proportions of PChle 630 and PChle 650 and examination of the isolated pigments revealed that only unphytylated PChle could be activated for photoconversion by ATP. In etioplasts aged for 5 hours, ATP also stimulated photoconversion of PChle 630 into Chle 670. The process was temperature-sensitive and involved PChle 650 and Chle 680 as intermediates. AMP alone had no effect, but inhibited ATP retardation of PChle loss. ADP had a similar but lesser effect than ATP. The ADP response, but not the ATP response, was considerably enhanced in the presence of an ATP-generating system (phosphoenolpyruvate/pyruvate kinase). UTP, GTP, and CTP gave 40 to 50% of the ATP response with intact etioplasts. In envelope-free etioplasts, ATP gave the greatest response but the other nucleotides were now 80% as effective as ATP. After primary photoconversion, ATP stimulated resynthesis of PChle 650. It is proposed that ATP both gives the holochrome the ability to bind to the PChle molecule and enables additional association of the pigment-protein complex to form PChle 650. PMID- 16659240 TI - Phosphate-dependent Substrate Transport into Mitochondria: Oxidative Studies. AB - The oxidation of malate and citrate by isolated plant mitochondria can be stimulated by the addition of inorganic phosphate. This stimulation (a) is not inhibited by oligomycin or uncouplers; (b) can not be duplicated by addition of adenine nucleotide; (c) is inhibited by 2-n-butylmalonate; and (d) is not evident in detergent-treated mitochondria. Phosphate was required to elicit uncoupler stimulated respiration. It is concluded that these effects of phosphate are attributable to a stimulated rate of substrate penetration into the mitochondria, and do not involve the oxidative phosphorylation process.Disrupting the mitochondria with detergent removed the requirement for phosphate and showed that butylmalonate did not inhibit the respiratory enzymes. PMID- 16659241 TI - Oxygen Isotope Fractionation during Photosynthesis in a Blue-Green and a Green Alga. AB - Oxygen isotope fractionation ((18)O/(16)O) at the natural abundance level has been measured during photosynthesis of a blue-green and a green alga. When sufficient attention is paid to removal of contaminating air O(2) before and during the experiments, then the photosynthetic O(2) evolved, as compared to the water O(2), had an average difference of -0.36% for a blue-green alga and -0.80% for a green alga. These experiments suggest that there is no reason to invoke an inverse isotope effect in photosynthesis as part of the explanation for the (18)O enrichment in atmospheric O(2) relative to O(2) in oceanic waters. In addition, in an indirect way, the experiments also support the argument that the bulk of O(2) evolved during photosynthesis comes from water. A 10% contribution of O(2) arising from CO(2) would have been detectable in the present work. PMID- 16659242 TI - The action of valinomycin in uncoupling corn mitochondria. AB - Valinomycin in the presence of potassium is a potent uncoupler of corn (Zea mays L.) mitochondria, eliminating respiratory control. Valinomycin produces higher steady state potassium phosphate swelling which can be reversed to give active shrinkage if mersalyl is added to block the Pi(-)/OH(-) antiporter. Respiration declines concurrently. Uncouplers accelerate the shrinkage and restore the respiration. The same results can be obtained with sodium phosphate if gramicidin D is substituted as ionophore.It is concluded that valinomycin uncoupling is the result of cyclic salt transport, with influx pumping of potassium phosphate via the Pi(-)/OH(-) antiporter and efflux pumping via a K(+)/H(+) antiporter. The result is a higher level of steady state swelling, rapid turnover of the proton gradient, and uncoupled respiration rates. The level of steady state swelling can be manipulated by varying the valinomycin or K(+) concentrations, with high concentrations favoring activation of the efflux pump.A mosaic membrane model with high resistance for proton and monovalent cation penetration to the cation(+)/H(+) antiporter is used to explain the results. PMID- 16659243 TI - The effects of light intensity and spectral quality on growth and shoot initiation in tobacco callus. AB - The effects of eight different narrow band-emitting fluorescent lamps (371-750 nm) and four commercial broad band-emitting fluorescent sources upon growth and shoot initiation in tobacco callus (Nicotiana tabacum var. Wisconsin 38) have been characterized. Wavelength and intensity are equally important parameters in determining morphogenic changes. Near ultraviolet light (371 nm) was found to stimulate (0.024 mw/cm(2)) or inhibit (above 0.15 mw/cm(2)) callus growth and shoot initiation, depending on the light intensity. Stimulation of growth and shoot production occurs also in blue light region, but at higher intensity than in the near ultraviolet. Red and far red light (up to 1.7 mw/cm(2)) do not appear to affect callus growth or stimulate shoot initiation. The enhancement of callus growth and the stimulation of shoot initiation are controlled by the same near ultraviolet-absorbing photoreceptor system present in a small enough concentration that it cannot be recognized in the absorption spectrum of the intact tissue. Carotenoids, porphyrins, and phytochrome associated with the high irradiance response do not appear to qualify as the photoreceptor. Flavonoids are possible candidates. Radiation emitted by fluorescent lamps outside the near visible region was determined, and we concluded that energy levels were not sufficient to affect the reported results. The spectral output of several commercial lamps in the visible and near visible regions is such that there could be different effects on growth and development of tissue cultures. PMID- 16659244 TI - The Metabolism of Oat Leaves during Senescence: IV. The Effects of alphaalpha' Dipyridyl and other Metal Chelators on Senescence. AB - The senescence of the first leaves of light-grown Avena seedlings when detached and placed in the dark is inhibited by alpha, alpha'-dipyridyl and alpha, alpha', alpha''-tripyridyl at concentrations between 10(-5) and 10(-4) M. Five other chelating agents exert similar inhibiting effects at concentrations 3 to 30 times higher. The senescence of etiolated leaves, as shown by loss of carotenoid and protein, is similarly inhibited. Ethylene-diaminetetraacetate has a similar effect in the dark, though only at 10 mM and above, but in the light it causes bleaching of chlorophyll. It is deduced that an iron-containing system plays an essential part in the initiation of the senescence process. PMID- 16659245 TI - Correlative studies of cell wall enzymes and growth. AB - If cell wall hydrolytic enzymes are involved in extension growth, a correlation may be expected between hydrolytic activity of the cell walls and growth rate of the tissue from which the walls are prepared. Epicotyl sections from 0 to 5 mm, 6 to 10 mm, and 11 to 15 mm below the apical hook of pea seedlings (Pisum sativum var. Alaska) have relative growth rates of 100:15:2, respectively. The relative beta-glucosidase activities (units/mg wall) of cell walls from these sections are respectively, 100:24:23, for walls prepared in glycerol and 100:42:23 for walls prepared in aqueous solution. Thus, there is a correlation between growth rate of the tissue and specific activity of the wall-associated beta-glucosidase. Similar correlations were found for other cell wall-associated hydrolases.Relative cell numbers for the above sections, as determined by counting, were 100:25:16, and with these data it could be calculated that the amount of cell wall beta glucosidase activity per cell is essentially a constant. Thus, for epicotyl sections the amount of enzyme per cell does not change during the process of cell elongation but the specific activity declines as the result of deposition of new wall material. PMID- 16659246 TI - Nitrate translocation by detopped corn seedlings. AB - Five-day-old seedlings of corn (Zealpha mays L.) grown without nitrate were decapitated and exposed to 0.5 mm KNO(3) or 0.5 mm KCl in aerated solutions at 30 C. Uptake of nitrate, chloride, and potassium was determined by replacing solutions hourly and measuring their depletion. Translocation of these ions and of organic nitrogen was determined by hourly analysis of the vascular exudate. Nitrate reduction was estimated by the difference between nitrate uptake and nitrate recovered in the tissue and exudate. Nitrate uptake exhibited its usual pattern of apparent induction resulting in the development of an accelerated uptake phase. Chloride uptake remained fairly constant throughout the experimental period. Translocation of nitrate increased progressively for at least 7 hours whereas chloride translocation reached a maximum about the 3d hour and then declined to a lower rate than nitrate translocation. Nitrate uptake and translocation were restricted by anaerobiosis, by 20 and 40 C relative to 30 C, and by 0.05 mm 6-methylpurine, an RNA-synthesis inhibitor. Accumulation, reduction and translocation of nitrate had different sensitivities to all these factors. The effect of 0.05 mm 6-methylpurine was more detrimental to nitrate translocation and nitrate reduction than to nitrate uptake.Ambient nitrate, relative to chloride, enhanced the exudation volume and the translocation of organic nitrogen within 4 hours from initiation of the experiments. Translocation of nitrate and organic nitrogen decreased shortly after removal of external nitrate. The higher rates of organic nitrogen translocation which occurred during nitrate uptake indicates either (a) rapid translocation of amino acids synthesized from the entering nitrate, or (b) an accelerated rate of protein turnover and a resulting enhancement in translocation of endogenous amino acids. PMID- 16659247 TI - Acceleration of dark reversion of phytochrome in vitro by calcium and magnesium. AB - Rates of dark reversion of the far red-absorbing form of phytochrome, Pfr, to the red-absorbing form, Pr, have been determined in the presence of several salts. Low concentrations of calcium chloride and magnesium chloride (up to 3 mm) accelerated the rate of dark reversion at all stages of purification of phytochrome from etiolated rye (Secale cereale L. cv. Balbo) seedlings. The complex kinetics of the dark reversion could be resolved into two first-order components. The effect of the added divalent cations was on the relative proportion of the fast and slow reacting components, rather than on the rate constants of the two populations. It was possible to reverse the effects of the cations by adding the chelating agents ethylene-bis-(oxyethylene-nitrilo) tetraacetic acid or ethylenediaminetetraacetate. The effect of the divalent cations is not a nonspecific ionic strength effect. The relative proportion of the two populations was also affected by the degree of purity of the phytochrome samples. PMID- 16659248 TI - Ultrastructural Effects of Water Stress on Chloroplast Development in Jack Bean (Canavalia ensiformis [L.] DC). AB - An ultrastructural study has been made to determine to what degree chloroplast differentiation is retarded in leaves of young jack bean (Canavalia ensiformis [L.] DC.) seedlings when they are subjected to mild water stress. Rapid chloroplast differentiation occurred when etiolated seedlings were allowed to green at relative humidities above 85% but not at a relative humidity of 25%. Response to a drop in humidity was rapid. Germination and early development in the dark occurred at 100% relative humidity. At the time of exposure to light, the etioplasts contained well formed prolamellar bodies. Under high relative humidity conditions, transformation of the prolamellar body was well advanced within 2 hours. Under low relative humidity conditions, however, prolamellar body differentiation was extremely retarded for more than 24 hours following the beginning of illumination. PMID- 16659249 TI - Carbon dioxide fixation in isolated kalanchoe chloroplasts. AB - Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 mumoles of CO(2) per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO(2) fixation were primarily those of the photosynthetic carbon reduction cycle. PMID- 16659250 TI - Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins. AB - Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis. PMID- 16659251 TI - Incorporation of C-Photosynthate into Protein during Leaf Development in Young Populus Plants. AB - Gas exchange and protein metabolism were studied in expanding, mature, and near senescent leaves of young clonal Populus x euramericana cv. Wisconsin-5 plants. Dark respiration, CO(2) evolution in the light, and CO(2) compensation concentrations were highest in unexpanded leaves but declined markedly as leaves matured and aged. Net photosynthesis was highest in nearly mature leaves. Fresh weight continued to increase after leaf expansion was complete, whereas soluble protein levels declined. Changes in the distribution of photosynthetically incorporated (14)C indicated that a high level of protein synthesis and rapid formation of structural components occurred only in expanding leaves. Protein turnover was slight in expanding leaves but was substantial after leaves were mature. Expanding leaves synthesized predominantly fraction I protein (ribulose diphosphate carboxylase). However, formation of this protein from photosynthate was slight once leaves matured. PMID- 16659252 TI - Distribution Pattern of Cell Division Centers on the Epidermis of Stem Segments of Torenia fournieri during de Novo Bud Formation. AB - The stem epidermis in Torenia fournieri, which has budding potentialialities, is composed of one cell layer which can be easily separated from the rest of the stem segment at different stages of bud formation. As the buds are formed directly from the epidermis, without intermediate callus formation, it is possible to observe simultaneously the cell division centers over the entire excised epidermal surface. The quantitative analysis at the 6-day stage of bud formation showed that the cell division centers do not have a random distribution on the epidermal surface. With respect to the length of the stem segment, the frequency of cell division centers increases toward the base which is also the direction of auxin transport. With respect to the width, the maximum number of division centers is observed on either side of the median zone. The median zone and the lateral zones have few division centers. An anatomical study showed that the zones with few division centers are the closest to underlying vascular tissue. A more uniform distribution of division centers can be obtained by addition of auxin to the medium. PMID- 16659253 TI - The Site of Cellulose Synthesis: Cell Surface and Intracellular beta-1, 4-Glucan (Cellulose) Synthetase Activities in Relation to the Stage and Direction of Cell Growth. AB - beta-1, 4-Glucan (cellulose) synthetase activity (UDP-glucose: beta-1, 4-glucan glucosyl transferase) present at cell surfaces of growing regions of Pisum sativum epicotyl was assayed by supplying UDP-(14)C-glucose directly to thin slices of tissue. Initial rates of glucosyl transfer under these conditions approached the rates of cellulose deposition observed in vivo in intact tissue at various stages of growth. Normal tissue homogenization procedures destroyed the high surface activity, although a small amount of residual activity (3-10% of total) could be detected in particulate fractions. In homogenates from elongating tissue, the residual activity was almost entirely associated with Golgi membrane. In homogenates of tissue which had ceased elongating, whether because of normal maturation or treatment with ethylene (or high levels of auxin), the activity was present in Golgi plus a membrane fraction rich in smooth endoplasmic reticulum vesicles. It is suggested that cellulose synthetase activity associated with these two organelles represents intracellular enzyme in transit to specific sites of cellulose synthesis and microfibrillar orientation at the cell surface. PMID- 16659254 TI - Studies on Pea Ribosomal Proteins: Conformational and Biological Activity Changes of Ribosomal Subunits Derived by NH(4)Cl Dissociation. AB - Ribosomal subunits prepared by NH(4)Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L(2) and L(9) as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH(4)Cl retained L(2) and L(9), but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl(2)) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH(4)Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L(2) and L(9), showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH(4)Cl (this apparently leads to a preferential detachment of L(2) and L(9) at 0.5 m NH(4)Cl) and that the activity of the purified subunits depends not only on the presence of L(2) and L(9) but also on the organization of these proteins within the 60S subunits. PMID- 16659255 TI - Effects of red light on the growth of intact wheat and barley coleoptiles. AB - The final lengths of intact dark-grown coleoptiles vary with species and cultivar. The growth distribution pattern in the apical 25-mm growing zone and the absolute amount of growth in each zone depend on the age and species of the coleoptile. A comparative study of several cultivars of wheat, Triticum vulgare, and barley, Hordeum vulgare, indicates that the growth distribution pattern in 30 to 38-mm coleoptiles varies with the species and cultivar. In barley, there are two patterns of growth distribution among the several cultivars, whereas in wheat, all cultivars exhibit a common zonal growth pattern. The total growth of coleoptiles, initially 30 to 38 mm in length, during a 24-hour dark incubation period is the same in dark-grown coleoptiles as in those irradiated with 3 minutes of red (660 nm) light prior to the incubation period. The growth distribution pattern in the growing zone of this 30- to 38-mm coleoptile is, however, altered by red light. Growth of the apical 5-mm zone is stimulated by red light and the zonal growth 5 to 10 mm below the apex is only slightly affected, whereas growth in the zones 10 to 15 to 20, and 20 to 25 mm below the apex is inhibited. This growth distribution pattern in irradiated coleoptiles changes as the coleoptile increases in length. The response of a zone following exposure to red light is dependent upon the age of the seedlings irradiated. The over-all effect of red light on growth of the intact coleoptile varies with the length of the coleoptile. In young seedling 20 to 29 mm in length, the cells of the coleoptile can compensate for the effects of red light, with the over-all growth of the dark-grown and irradiated coleoptile about the same. As the seedling grows older, the cells of the coleoptile can no longer make up for the effects of red light, and the over-all effect changes from compensation to pronounced inhibition. PMID- 16659256 TI - Dark CO(2) Fixation in Gladiolus Cormels and Its Regulation during the Break of Dormancy. AB - The increase in dark CO(2) fixation during cold storage of Gladiolus x gandavensis van Houtte-type grandiflorus cormels is used to monitor changes in their state of dormancy. Dark fixation is also promoted by benzyladenine, which breaks cormel dormancy, and is inhibited by abscisic acid and gibberellin A(3), which inhibit cormel germination. The rate of dark fixation by nondormant cormels is five times higher than that in dormant ones. Dark fixation is not due to microorganisms. It is temperature-dependent and can be measured stoichiometrically in vivo. The apex and base of the cormels accumulate more label than the central part. Dark fixation of both dormant and nondormant cormels is also promoted by imbibition in water. The fate of the labeled assimilates was followed by ion exchange chromatography. PMID- 16659257 TI - Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination. AB - The pattern of change in the activity of alcohol dehydrogenase in maize (Zea mays L.) scutellum during seed germination is not altered by 10 mug/ml cycloheximide or 50 mug/ml actinomycin D. The enzyme does not become density labeled when maize seeds are germinated in the presence of D(2)O and (15)NH(4)Cl, indicating that no new alcohol dehydrogenase molecules are synthesized after the onset of germination. However, the activity of an endogenous inhibitor for alcohol dehydrogenase is increased after germination. The increase of this inhibitor is concomitant with the decline of alcohol dehydrogenase activity, indicating that the activity of alcohol dehydrogenase during seed germination is controlled by the level of the inhibitor. PMID- 16659258 TI - Metabolism of Tritiated Gibberellins in d-5 Dward Maize: II. [H]Gibberellin A(1), [H]Gibberellin A(3), and Related Compounds. AB - After 30 minutes of incubation of young leaf sections of d-5 maize (Zea mays L.) in [(3)H]gibberellin A(1) ([(3)H]GA(1)), the metabolite [(3)H]GA(8) was present in significant amounts, with a second metabolite, [(3)H]GA(8)-glucose ([(3)H]GA(8)-glu), appearing soon after. A third [(3)H]GA(1) metabolite, the polar uncharacterized conjugate [(3)H]GA(1)-X, took more than 1 hour to appear. The protein synthesis inhibitor cycloheximide inhibited the production of all [(3)H]GA(1) metabolites, indicating a possible protein synthesis requirement for [(3)H]GA(1) metabolism.By preincubating leaf sections in unlabeled GA(1) before exposure to [(3)H]GA(1) or by reducing the specific radioactivity of the [(3)H]GA(1) supplied, it was possible to reduce greatly the conversion of radioactive GA(1) to [(3)H]GA(8)-glu, without affecting conversion to [(3)H]GA(1) X. Increasing the molar concentration of the [(3)H]GA(1) fed greatly increased the molar yield of [(3)H]GA(1)-X, whereas the molar yields of [(3)H]GA(8) and [(3)H]GA(8)-glu were much less affected.The principal metabolite of [(3)H]GA(3) was a very polar compound having chromatographic properties similar to those of the conjugate [(3)H]GA(1)-X produced from [(3)H]GA(1). The naturally occurring GAs [(3)H]GA(1), [(3)H]GA(3), and [(3)H]tetrahydroGA(3) were metabolized to a much greater extent than were the artifical derivatives [(3)H]ketoGA(1), [(3)H]GA(1)-methyl ester, and [(3)H]pseudoGA(1). Only [(3)H]GA(1) and [(3)H]GA(3), with their identical D ring structures, were converted to [(3)H]GA(1)-X type compounds; [(3)H]-ketoGA(1) and [(3)H]tetrahydroGA(3), with modified D rings, were not converted to this type of conjugate. PMID- 16659259 TI - Phosphoenolpyruvate carboxykinase activity in grape berries. AB - Phosphoenolpyruvate (PEP) carboxykinase activity was found in crude extracts of ;Pinot noir' grape berries. The enzyme required ATP, Mn(2+) plus Mg(2+), a pH of 6.6, and a temperature of 40 C for maximum activity. The range in concentration of oxaloacetic acid needed for maximum phosphoenolpyruvate carboxykinase activity was 5 to 10 mm, and the Km for HCO(3) (-) in the exchange of (14)CO(2) into oxaloacetic acid was 26.8 mm.Changes in the activity of PEP carboxykinase and PEP carboxylase in berries were studied at weekly intervals throughout fruit development. PEP carboxykinase had maximum activity 4 weeks after flowering, and during the following 11 weeks remained relatively constant. The activity of PEP carboxylase was 2- to 4-fold higher than PEP carboxykinase throughout fruit development, and changed little except for a sharp reduction at the onset of ripening. PMID- 16659260 TI - H(2) metabolism in photosynthetic organisms: I. Dark h(2) evolution and uptake by algae and mosses. AB - Dark H(2) metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to H(2) evolution. H(2) evolution was sensitive to disalicylidenepropanediamine. An immediate H(2) uptake was observed in these algae. Immediate dark H(2) uptake but no evolution was observed in the mosses.A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p benzoquinone, NAD, NADP, but not spinach ferredoxin. H(2) evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H(2) evolution and for H(2) uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve H(2). Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H(2) by a different pathway than Chlamydomonas. PMID- 16659261 TI - Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl. AB - A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin treated tissue. The DNA content per nucleus was 4 +/- 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of (3)H-TTP into DNA. PMID- 16659262 TI - Effect of growth in highly salinized media on the enzymes of the photosynthetic apparatus in pea seedlings. AB - The rate of chlorophyll formation in initially etiolated pea seedlings (Pisum sativum) that are growing in the light in salinized media is slower than in similar plants not subjected to salinity. However, the final steady state level of chlorophyll is the same under both conditions. Growth under saline conditions did not change the ratio of dry weight to wet weight in the plant leaves nor the specific concentration of soluble protein in leaf extracts. Changes in the specific activity of 11 enzymes in leaf extracts during growth in the light were measured. At least six of these enzymes are known to be part of the photosynthetic apparatus and that their synthesis is subject to photocontrol. The changes in specific activity that were observed were slower in the salt-treated plants, but the final steady state concentration of each was the same as in the control plants. It is concluded that salinity impairs growth of pea plants but that formation of enzymes and other proteins are always in balance with growth. PMID- 16659263 TI - beta-1, 3-Glucan Synthase from Lilium longiflorum Pollen. AB - A particulate fraction from pollen tubes and ungerminated pollen of Lilium longiflorum incorporated (14)C-glucose from UDP-glucose-(14)C into a lipid fraction and into beta-1, 3-glucan. Partial hydrolysis of the glucan yielded laminaribiose as the only radioactive disaccharide. The preferred substrate was UDP-glucose, and enzyme activity was stimulated by glucose and by beta-linked di- and trisaccharides. Enzyme from growing pollen tubes synthesized beta-1, 3-glucan more rapidly and produced a higher proportion of alkali-insoluble glucan than did enzyme from ungerminated pollen. The onset of pollen tube growth may be dependent on altered activity of beta-1, 3-glucan synthase. PMID- 16659264 TI - Isolation and translation of plant messenger RNA. AB - A fraction of the RNA species isolated from Lemna gibba G-3 consists of molecules with attached sequences of polyadenylic acid. This polyadenylic acid-containing fraction, separated from total RNA by adsorption onto oligothymidylic acid cellulose, was shown to be mRNA by its ability to serve as template in a cell free translation system derived from wheat germ. The products of translation were characterized by electrophoresis. This method permitted the comparison of mRNA from plants grown under different light conditions. Such plants were shown to possess qualitative and quantitative differences in their mRNA complements. PMID- 16659265 TI - Changes in Catechol Oxidase and Permeability to Water in Seed Coats of Pisum elatius during Seed Development and Maturation. AB - In the developing seed coat of Pisum elatius, o-dihydroxyphenols are present in appreciable amounts at all stages of development. However, catechol oxidase activity rises sharply during the later stages of development, shows a further abrupt rise during dehydration of the seed coat, and then decreases. It is suggested that a tanning reaction is induced by the contact of enzyme with its substrate while cell membranes are ruptured, and that this reaction renders the seed coats impermeable. The entire chain of events does not occur in Pisum sativum which has permeable seed coats. PMID- 16659266 TI - Ventilation required to entrain small particles from leaves. AB - Particles are blown from leaves when the wind at the height of the particles exceeds a minimum which is about 5 m/sec for some fungal spores. In the moderate winds typical within a canopy of leaves, the minimum is attained at spore height during brief changes in wind or puffs before the boundary layer grows to particle height. The requisite change in speed to remove spores occurs over a sizeable area only when the speed changes abruptly in a short distance in the direction of the wind. PMID- 16659267 TI - Inhibition of Gibberellic Acid-induced Elongation in Avena Stem Segments by a Substituted Pyrimidine. AB - Avena stem segments, which respond with high amplitude, specificity, and sensitivity to gibberellic acid, were used to study the inhibition of gibberellin induced elongation by the growth retardant alpha-cyclopropyl-alpha-(4 methoxyphenyl)-5-pyrimidine methanol (EL-531). It was found that EL-531 strongly inhibits gibberellic acid-induced elongation in this system at a concentration of 1 mm. From a double-reciprocal plot of elongation and gibberellic acid concentration, it seems that EL-531 and gibberellic acid do not compete reversibly for the same site of action. Also, because EL-531 effectively inhibits elongation in internodal tissue dissected away from the node and leaf sheath, it cannot be acting primarily by inhibiting the synthesis or transport of the leaf sheath factor(s). Because EL-531 causes lateral expansion of the stem segments as well as increased diameters of epidermal cells, in a manner very similar to the effects of colchicine, it is suggested that EL-531 inhibits gibberellic acid induced elongation by somehow interfering with the orientation of the products of cell wall synthesis. PMID- 16659268 TI - Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid. AB - The short term effect of 11.4 mum indoleacetic acid on the incorporation of (methyl-(3)H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 mum) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 mum indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments. PMID- 16659269 TI - Phosphatidylserine synthesis in castor bean endosperm. AB - Phosphatidylserine synthesis by the endoplasmic reticulum fraction isolated from castor bean (Ricinus communis var. Hale) endosperm was assayed by measuring the incorporation of (14)C-l-serine into chloroform-soluble material. Both phosphatidylserine and phosphatidylethanolamine were identified as products. The incorporation required calcium ions and showed an optimum pH of 7.8 in 2 mm CaCl(2). Phosphatidylethanolamine and CDP-diglyceride stimulated the reaction only about 40 to 50% and primary alcohols had relatively little effect on the incorporation. These and other results suggest the synthesis of phosphatidylserine in this tissue occurs by an exchange reaction but the relative roles of phospholipase D and phosphatidylethanolamine: l-serine phosphatidyltransferase remain to be elucidated. PMID- 16659270 TI - Conversion of Signals from Ion-specific Electrodes to Linear Concentrations. AB - This paper describes the assembly (from commercially available components) of an antilog converter, which transforms the output signals of ion-specific electrodes to ionic concentrations suitable for a linear recorder. It responds linearly to cation concentrations from 10 mum to at least 10 mm and can be used for electrodes kept at any temperatures (0 to 50 C). The leakage of K(+) from a unicellular algae (Chlorella sorokiniana) can be induced by Triton X-100, heating, or suspension in a tris buffer and is used to demonstrate the operation of this device. PMID- 16659271 TI - Incorporation of C-photosynthate into major chemical fractions of source and sink leaves of cottonwood. AB - The incorporation and distribution of photosynthetically fixed (14)CO(2) was followed for 48 hours in a recently matured source leaf (LPI 7) and in young expanding source and sink leaves (LPI 4) of cottonwood (Populus deltoides Bartr.). The major chemical constituents of leaf laminae and petioles were separated by sequential solvent extractions and enzyme hydrolyses. Two hours after labeling, about 80% of the (14)C was found in water-alcohol-soluble constituents in the mature source lamina as compared to about 45% in those of the young expanding leaf. In both mature and expanding source leaves the water alcohol-soluble constituents decreased while the CHCl(3)-soluble and -insoluble compounds increased with time. After 48 hours, 7 and 37% of the total (14)C was recovered from structural carbohydrates and from protein + CHCl(3)-soluble fractions, respectively, in the mature source leaf; and 4 and 65%, respectively, in the young source leaf. When the distribution of (14)C among major chemical fractions was calculated on per cent dpm/mg basis, the data showed that a young sink leaf incorporated over twice as much (14)C into structural carbohydrates as a young source leaf (11% versus 4%). However, when calculated on an absolute dpm/mg basis, activity in this fraction of the young source leaf exceeded that in the sink leaf by a ratio of about 11:1 (9528 versus 845 dpm/mg). Thus, most of the material for synthesis of structural carbohydrates was derived from in situ photosynthate.The distribution of (14)C in chemical fractions recovered from petioles was similar to that recovered from their respective laminae, except that petioles incorporated greater amounts (up to 24% of total (14)C) into structural carbohydrates. In contrast to lamina tissue, most of the photosynthate for synthesis of structural carbohydrates in the petioles of young developing leaves was imported from mature leaves farther down the stem. PMID- 16659272 TI - Carbon dioxide compensation points of flowering plants. AB - Carbon dioxide compensation points of several hundred species of monocotyledons and dicotyledons have been measured during the course of various experiments in our laboratory over a period of several years. These have been classified into two groups: high, compensation points of 40 mul/l or greater; and low, compensation points of 10 mul/l or less. They are listed alphabetically both by families and species for monocotyledons and dicotyledons. Only two species did not unequivocally fit into the above established groups. These were Moricandia arvensis (L.) DC., which had an average compensation point of 26 mul/l and Panicum milioides Nees ex Trin., which was variable, but most often equilibrated between 12 to 20 mul/l CO(2). PMID- 16659273 TI - Relationships between Leaf Water Status, Abscisic Acid Levels, and Stomatal Resistance in Maize and Sorghum. AB - A new method for abscisic acid extraction and purification was developed to handle large numbers of small (about 125 milligrams fresh weight) samples of leaf discs. This method enabled short term changes in abscisic acid to be followed in single leaves.Water potentials, stomatal diffusion resistance, and abscisic acid levels were measured in the leaves of maize (Zea mays L. var. Wisconsin 575) and sorghum (Sorghum bicolor, hybrid NK 145) plants subjected to a drought-recovery cycle under controlled environmental conditions. The levels of abscisic acid began to rise, and the stomata closed, over a narrow range of water potential (-8 to -10 bars) in both species. Abscisic acid levels continued to rise after the stomata closed. The maximum amount of abscisic acid extracted from maize leaves was about twice that from sorghum, but this represented a similar proportional increase over the control level.In excised leaves little or no change in abscisic acid levels was found within 60 to 120 minutes after stress was applied (phase I). Subsequently abscisic acid levels rose rapidly to about 20 times the prestress level (phase II). Stomatal closure always preceded the phase II increase in abscisic acid.The aftereffects of stress differed in the two species. In sorghum control levels of both abscisic acid and stomatal resistance were regained within 24 hours of rewatering. In maize abscisic acid levels also fell rapidly, but regained control levels only after 48 hours. The resumption of normal stomatal functioning occurred after a further 24 hours.Impaired stomatal functioning after stress does not appear to be associated with high residual levels of abscisic acid in the leaf. PMID- 16659274 TI - Induction of responsiveness to octopine and lysopine in crown-gall tumors. AB - Bean leaf tumors induced by Agrobacterium tumefaciens strain 181 show enhanced growth in response to octopine or lysopine only if one of these compounds is present during the period of tumor induction. Either compound applied during this period results in tumors which subsequently respond to both. The combined action of the bacterium plus octopine or lysopine at induction is proposed to induce transcription of plant genes coding for enzymes involved in the degradation and/or biosynthesis of octopine and lysopine. PMID- 16659275 TI - Induction of a Sensitive Response to Helminthosporium maydis Race T Toxin in Resistant Mitochondria of Corn (Zea mays L.) by Removal of the Outer Mitochondrial Membrane. AB - Mitochondria isolated from Texas cytoplasmically male sterile (Tms) and normal (N) versions of corn (Zea mays L.) exhibit differential sensitivity to toxin(s) produced by Helminthosporium maydis race T, the causal organism of southern corn leaf blight. Malate dehydrogenase was inhibited by toxin(s) in intact Tms mitochondria but was unaffected in N mitochondria. Removal or rupture of the outer mitochondrial membrane resulted in retention of sensitivity of malate dehy drogenase in Tms mitochondria to toxin(s), and induction of a sensitive response in normally toxin-insensitive N mitochondria. This suggests that a permeability difference in the respective outer membranes of N and Tms mitochondria may affect the passage of toxin(s) to a mitochondrial site of action. Mitochondrial bioassays indicate that more toxin was bound by Tms mitochondria than by N mitochondria; the greatest toxin binding was associated with the inner membrane of Tms mitochondria. PMID- 16659276 TI - Acetylene reduction (nitrogen fixation) and metabolic activities of soybean having various leaf and nodule water potentials. AB - An apparatus was designed that permitted acetylene reduction (N(2) fixation) by root nodules to be measured in situ simultaneously with net photosynthesis, dark respiration, and transpiration of the shoot in soybean plants (Glycine max [L.] Merr. var. Beeson). Tests showed that acetylene reduction was linear with time for at least 5 hours, except for the first 30 to 60 minutes. Endogenous ethylene production did not affect the measurements. Successive determinations of acetylene reduction could be made without apparent aftereffects on the plant.This apparatus was used to investigate the effects of soil flooding and desiccation on acetylene reduction under conditions where soil, nodule, and leaf water potentials could be measured. No acetylene reduction was detectable in flooded soil or in soil desiccated to a water potential of -19.5 bars. Between these extremes, acetylene reduction displayed a sharp optimum. Removing the soil eliminated the inhibitory effects of flooding, suggesting that rates of gas exchange were restricted between the nodules and the atnosphere at soil water potentials above -2 bars.As the soil desiccated further, acetylene reduction decreased, and the decrease was correlated with decreases in photosynthesis and transpiration. Although dark respiration was inhibited, it was not affected to the extent that acetylene reduction, photosynthesis, or transpiration were. Consequently, it was concluded that photosynthesis, transpiration, or some direct effect on the nodules other than that caused by respiration were most likely to account for the inhibition of acetylene reduction at soil water potentials below 2 bars. PMID- 16659277 TI - Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials. AB - The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean (Glycine max [L.] Merr. var. Beeson) using an apparatus that permitted simultaneous measurements of acetylene reduction, net photosynthesis, and transpiration. The inhibition of acetylene reduction caused by low water potentials and their aftereffects could be reproduced by depriving shoots of atmospheric CO(2) even though the soil remained at water potentials that should have favored rapid acetylene reduction. The inhibition of acetylene reduction at low water potentials could be partially reversed by exposing the shoots to high CO(2) concentrations. When transpiration was varied independently of photosynthesis and dark respiration in plants having high water potentials, no effects on acetylene reduction could be observed. There was no correlation between transpiration and acetylene reduction in the CO(2) experiments. Therefore, the correlation that was observed between transpiration and acetylene reduction during desiccation was fortuitous. We conclude that the inhibition of shoot photosynthesis accounted for the inhibition of nodule acetylene reduction at low water potentials. PMID- 16659278 TI - Characterization of Carrot and Tobacco Cell Cultures Resistant to p Fluorophenylalanine. AB - This study describes the isolation and characterization of p-fluorophenylalanine resistant diploid tobacco (Nicotiana tabacum L.) and diploid carrot (Daucus carota L.) cultured cell lines. The p-fluorophenylalanine-resistant tobacco and carrot lines can grow in medium containing p-fluorophenylalanine concentrations 10 to more than 100 times those which inhibit the growth of susceptible cells, respectively. The resistance trait was retained when the cells were grown in a medium lacking the phenylalanine analog for 50 generations. All 14 single cell clones started from the resistant carrot line remained resistant. The resistant lines incorporated much less p-fluorophenylalanine into protein, partially due to a decrease in uptake. In carrots, an increase in the levels of free phenylalanine and tyrosine also apparently contributed to the decreased incorporation of p fluorophenylalanine into protein by increasing the metabolic pool size which diluted the incoming analog and caused a lowered percentage of incorporation, which was observed. Apparently, phenylalanine and tyrosine synthesis was also increased in resistant tobacco lines, since chorismate mutase was found to have greater activity and to be less sensitive to inhibition by phenylalanine, tyrosine, and p-fluorophenylalanine. It appears, however, that phenylalanine and tyrosine do not accumulate above the normal levels in the resistant tobacco cells, as these amino acids were apparently converted into phenolic compounds which were found in higher levels (6 times). The low frequency of appearance, the stability of the trait, and the biochemical nature of the resistance, indicate that the p-fluorophenylalanine resistance found in the carrot and tobacco lines described here is due to a mutation. PMID- 16659279 TI - Ripening and in vitro retention of respiratory control by avocado and pear mitochondria. AB - The retention of respiratory control ("survival") by mitochondria held at 25 C was studied in relation to the ripening of two varieties of avocado (Persea americana Mill. var. ;Fuerte' and ;Hass') and one variety of pear (Pyrus communis. L. var. ;Bartlett') fruit. The survival of avocado mitochondria increased from 8 to 10 hours when isolated from unripe, preclimacteric fruit, to 48 hours when isolated from fully ripe, postclimacteric fruits. Although rates of alpha-ketoglutarate oxidation, respiratory control, and ADP/O decreased somewhat in the postclimacteric phase, survival per se was not affected. Pear mitochondria survived for more than 30 hours regardless of the physiological age of the source.Exposure of postclimacteric avocado mitochondria to a preclimacteric supernatant fraction curtailed their survival. The harmful effect of some unknown substance(s) in the preclimacteric avocado supernatant fraction was confirmed by utilizing pear mitochondria as an independent test system. PMID- 16659280 TI - Water Use Efficiency of Field-grown Maize during Moisture Stress. AB - Theoretical analysis of the CO(2) assimilation and water loss by single leaves suggests that the water use efficiency of C(4) species decreases as stomatal resistance increases. To confirm this hypothesis for a complete maize crop, results from computer simulations and a field experiment were compiled for varying stomatal resistances. A soil-plant-atmosphere model allowed simulations of the many simultaneous interactions between a crop canopy and its environment. The simulations for varying stomatal resistances clearly indicated that as stomatal resistance increased, water use efficiency of the maize crop decreased. The field experiment data also confirmed that water use efficiency was significantly decreased under water stress conditions when stomatal resistance increased. We concluded that management practices for maize, which induce moisture stress conditions resulting in increased stomatal resistance, reduce both crop photosynthetic productivity and water use efficiency. PMID- 16659281 TI - Abscisic Acid Metabolism in Water-stressed Bean Leaves. AB - Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-(14)C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of (14)C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04 mug to approximately 0.5 mug/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA and DPA levels rose throughout the wilt period. PA rose from 0.20 mug to 1.0 mug and DPA from 0.8 mug to over 3 mug/g fresh weight. From these data, we calculated the rate of ABA synthesis to be at least 0.15 mug/hr.g fresh weight during this period. We have interpreted these results to mean that in wilted leaves an elevated level of ABA is maintained because the rate of synthesis and metabolism are both elevated and approximately equal. PMID- 16659282 TI - Quantitative regulation of gene activity in plants. AB - The regulation of activity of ribosomal RNA genes has been studied in euploid and aneuploid varieties of hyacinth. The gene/gene product relationship was determined by measuring the specific gene product (stable rRNA) and the total gene product (protein) in varieties with different dosages of rRNA genes. The results show that the cellular rRNA content is positively regulated, that maximum gene utilization is not normally employed, and that the degree of utilization responds to the total genome rather than to the specific rRNA gene dosage. The mechanism of control of this regulation appears to be at the transcriptional level. PMID- 16659283 TI - Delta-Pyrroline-5-carboxylic Acid Dehydrogenase in Barley, a Proline-accumulating Species. AB - The characteristics of the enzyme Delta(1)-pyrroline-5-carboxylic acid dehydrogenase from etiolated barley (Hordeum distichum) shoots have been examined. The bulk of the enzyme activity was found in the 10,000g pellet fraction, this activity being displayed only after detergent treatment of the suspended pellet. The enzyme was most active at pH 8, and activity was NAD dependent. Enzyme activity was unaffected by either mannitol or sucrose in the reaction mixture up to a concentration of 0.45 m but was strongly inhibited by Cl(-) and, to a lesser extent, SO(4) (2-). The inhibition attributable to KCl was reversed by increasing the concentration of Delta(1)-pyrroline-5-carboxylic acid in the reaction mixture. PMID- 16659284 TI - Additive and synergistic effects of kinetin and ethrel on germination, thermodormany, and polyribosome formation in lettuce seeds. AB - The inhibition of germination of Grand Rapids lettuce (Lactuca sativa L.) seeds at 35 C was removed to a marked extent by kinetin and 2-chloroethylphosphonic acid (ethrel). When both compounds were used together, an additive effect was observed. A synergistic effect was, however, noted when ethrel promoted the kinetin reversal of abscisic acid inhibition of seed germination (light- as well as gibberellic acid-, induced). Both kinetin and ethrel increased the total ribosomal material and the percentage of polyribosomes in lettuce seeds imbibed in the light for 24 hours. A combination of the two compounds showed a synergism in polyribosome formation only at high ethrel concentration. The inability of ethrel to reverse abscisic acid inhibition indicates that kinetin action cannot always be substituted by ethrel. The possible mechanisms involved in the enhanced response by a combination of kinetin and ethrel are discussed. PMID- 16659285 TI - Gibberellin response in lettuce hypocotyl sections. AB - Excised lettuce (Lactuca sativa L.) hypocotyl sections retain the ability to elongate in response to gibberellic acid (GA(3)) addition. In 48 hr at 30 C a GA(3)-treated segment more than doubles while a control segment elongates less than 50%. Auxin has no detectable effect on this system. Sensitivity to GA(3) is not decreased by apex or root removal. Of the experimental variables tested, temperature, sucrose, and preincubation in water affect growth both with and without GA(3). Blue and far red light inhibit growth without GA(3); this inhibition is reversed by GA(3). Potassium chloride stimulates growth of illuminated sections treated with GA(3) but has no effect on control growth. When sections are incubated in the dark, KCl has a promotive effect on elongation. PMID- 16659286 TI - C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions. AB - The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of (14)C(2)H(4) into the tissue and (b) the conversion of (14)C(2)H(4) to (14)CO(2), was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to (14)C(2)H(4) markedly reduced both a and b. Increasing the (14)C(2)H(4) concentration from 0.14 to over 100 mul/l progressively increased the rate of a and b with tissue incorporation being greater than (14)C(2)H(4) to (14)CO(2) conversion only below 0.3 mul/l (14)C(2)H(4). Reduction of the O(2) concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO(2) (5%) severely inhibited (14)C(2)H(4) to (14)CO(2) conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during (14)C(2)H(4) treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b.The possibilities that the incorporation of (14)C(2)H(4) into pea tissues and its conversion to (14)CO(2) is linked to ethylene action, or that it represents a means of reducing the endogenous ethylene level, are discussed.Several problems encountered with the use of polyethylene vials, rubber serum stoppers, Clorox, and microbial contamination are also described. PMID- 16659287 TI - Partial purification of a growth factor from orange juice which affects citrus tissue culture and its replacement by citric Acid. AB - Orange (Citrus sinensis L. Osbeck) tissue cultures required a supply of orange juice to the medium for their vigorous growth. The growth-promoting activity of juice seemed to involve both cell division and cell enlargement. Juice had no promotive activity in bioassays for auxins, gibberellins, and cytokinins. The growth promoting activity of juice was mostly transferred into 1-butanol upon partition at pH 2. Gas chromatographic analysis of this acid 1-butanol fraction revealed large amounts of citric acid and negligible amounts of other organic acids. Supply of pure citric acid to the medium, alone or in combination with different concentrations of juice, indicated that citric acid replaces most of the requirement for juice.It seems that citric acid, which is a natural component of citrus juice, is responsible for the major part of the growth-promoting activity of the juice. The significance of citric acid as a growth factor in tissue cultures and the reasons for the dependence of citrus tissue cultures on external supply of citric acid are discussed. PMID- 16659288 TI - Metabolic Conversion of l-Ascorbic Acid to Oxalic Acid in Oxalate-accumulating Plants. AB - l-Ascorbic acid-1-(14)C and its oxidation product, dehydro-l-ascorbic acid, produced labeled oxalic acid in oxalate-accumulating plants such as spinach seedlings (Spinacia oleracea) and the detached leaves of woodsorrel (Oxalis stricta and O. oregana), shamrock (Oxalis adenopylla), and begonia (Begonia evansiana). In O. oregana, conversion occurred equally well in the presence or absence of light. This relationship between l-ascorbic acid metabolism and oxalic acid formation must be given careful consideration in attempts to explain oxalic accumulation in plants. PMID- 16659289 TI - Host-Pathogen Interactions: VIII. Isolation of a Pathogen-synthesized Fraction Rich in Glucan That Elicits a Defense Response in the Pathogen's Host. AB - A polysaccharide from the fungal pathogen Colletotrichum lindemuthianum causes browning and phytoalexin production when applied to the cut surfaces of bean (Phaseolus vulgaris) cotyledons and hypocotyls. The application of an amount of polysaccharide equivalent to less than 100 ng of glucose will elicit this response in the bean tissues. The polysaccharide has been isolated both from culture filtrates and from the mycelial walls of the fungus. Purification of the polysaccharide involved anion and cation exchange chromatography and gel filtration. The polysaccharide has an apparent molecular weight between 1,000,000 and 5,000,000 daltons, and consists predominantly of 3- and 4-linked glucosyl residues. PMID- 16659290 TI - Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. AB - Storage protein hydrolysis in the cotyledons of germinating mung beans (Phaseolus aureus Roxb.) was examined by histochemical techniques, and the autolytic capacity of isolated protein bodies was studied with biochemical methods. The localization of endopeptidase activity within the cotyledons was studied using an India ink-gelatin film technique. After 24 hours of imbibition, a low level of endopeptidase activity was found throughout the storage tissues of the cotyledons. A marked increase in activity was noted in cells farthest from the vascular bundles 48 to 60 hours after the start of imbibition. The decrease in storage protein followed the same spatial distribution starting in the cells farthest from the bundles. The cotyledons contain a population of cells in various stages of endopeptidase activity enhancement and storage protein degradation. A wave of endopeptidase activity moves progressively through the cotyledons towards the vascular bundles leaving behind areas devoid of stored reserves and low in endopeptidase activity. Observations on the morphology of protein bodies during germination indicate that the membrane surrounding them remains intact, while the reserves disappear. This result suggests that the protein bodies may be undergoing autolysis. To determine whether this may indeed be the case, protein bodies were isolated from the meal of mung bean seeds using an aqueous medium containing 80% glycerol. The protein body preparations and the cytoplasm were assayed for the presence of a number of enzymes which may be involved in the breakdown of the storage proteins. The protein bodies contained all, or nearly all, of the carboxypeptidase, alpha-mannosidase, N-acetyl-beta glucosaminidase, and caseolytic activity. The cytoplasm contained all, or most, of the leucine aminopeptidase and the trypsin-like activity (benzoyl arginine-p nitroanalide as substrate). Incubation of the isolated protein bodies resulted in the release of amino acids. An analysis of the products of hydrolysis indicated that very little, if any, storage protein was being hydrolyzed during the incubation. Hydrolysis of the storage proteins present in the protein bodies was greatly accelerated by the addition of extracts from the cotyledons of 4-day-old seedlings. The results suggest that new enzymic activities not present in the protein bodies isolated from dry seeds must either be activated or synthesized and possibly added to the protein bodies before storage protein breakdown can begin. PMID- 16659291 TI - Studies on the secretion of maize root cap slime: I. Some properties of the secreted polymer. AB - The secreted slime from root cap cells of corn (Zea mays, cv. SX-17) was studied. Production of slime by excised root tips is stimulated by the addition of 40 mM sucrose or fucose and half-strength Hoagland's solution to the incubation medium. Secreted slime was recovered from aqueous solution by precipitation with ethanol. The polymer has a molecular weight greater than 2 x 10(-6) daltons and a density of 1.63 g cm(-3). Protein is not present in material purified by density gradient centrifugation with cesium chloride. Fucose (39%) and galactose (30%) are the principle neutral sugars found in the purified polymer. Galacturonic and glucuronic acids, arabinose, xylose, mannose, and glucose are also present. PMID- 16659292 TI - Studies on the Secretion of Maize Root Cap Slime: II. Localization of Slime Production. AB - The distribution of fucose-containing polysaccharides in apical 1-cm sections of corn (Zea mays cv. SX-17) root tips was analyzed. Fucose-containing polysaccharides were localized predominantly in the apical 1 mm of the root, i.e., in the apical initials and root cap. An analysis of the distribution of incorporated radioactive label from l-fucose[(3)H] gave similar results. After a 2-hr incubation with fucose[(3)H], label was found principally in two components, namely a water-soluble slime fraction and hemicellulose. The incorporation of fucose into the water-soluble, ethanol-insoluble fraction was primarily in the apical 1 mm of the root, whereas incorporation into a water-insoluble, potassium hydroxide-soluble fraction was in the region 2 to 5 mm behind the root cap. Addition of sucrose to the incubation medium during fucose[(3)H] incorporation reduces label uptake but increases the amount of label in the fucose-rich secreted polysaccharide. The utility of fucose as a marker for the secreted polysaccharide was confirmed by demonstrating that no appreciable metabolism of this sugar occurs. PMID- 16659293 TI - Events surrounding the early development of euglena chloroplasts: v. Control of paramylum degradation. AB - The degradation of the storage carbohydrate, paramylum, is induced by light in wild-type Euglena gracilis Klebs var. bacillaris Pringsheim and in a mutant, W(3)BUL, which lacks detectable plastid DNA. Treatment of wild type with cycloheximide in the dark produces 60% as much paramylum breakdown as light, whereas treatment with levulinic acid in the dark yields a slightly greater response than light. Both cycloheximide and levulinic acid produce a greater paramylum breakdown in the light than they do in the dark. Treatment of W(3)BUL with levulinic acid in darkness produces a larger paramylum degradation than light, with values similar to wild type in the light. Treatment of W(3)BUL with cycloheximide induces paramylum degradation in darkness, and as with wild type, light is slightly stimulatory in the presence of both cycloheximide or levulinic acid. Streptomycin brings about only a very small amount of paramylum breakdown in the dark and only slightly inhibits breakdown in the light. Thus paramylum breakdown induced by light does not require the synthesis of proteins on cytoplasmic or plastid ribosomes. A model which explains these results postulates the existence of a protein which inhibits paramylum breakdown. When the synthesis of this protein is prevented either by light, cycloheximide, or by levulinic acid acting as a regulatory analog of delta amino levulinic acid, paramylum breakdown takes place. Because levulinic acid is a better inducer than light in W(3)BUL, W(3)BUL may not be able to form as much delta amino levulinic acid in light as wild type. The small amount of induction by streptomycin is viewed as a secondary regulatory effect attributable to interference with plastid protein synthesis which affects regulatory signals from the plastid to the rest of the cell. PMID- 16659294 TI - Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities. AB - Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome. PMID- 16659295 TI - Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants. AB - The use of mesophyll protoplast extracts from various C(4) species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C(4) photosynthesis. Conditions regulating the formation of the C(4) acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C(4) species, Eleusineindica, an NAD-malic enzyme C(4) species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C(4) species. Light dependent induction of CO(2) fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and alpha-ketoglutarate, or 3-phosphoglycerate induced high rates of CO(2) fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C(4) mesophyll cells. Induction by pyruvate or alanine and alpha-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO(2) fixation was not.Several differences between these species representing the three C(4) groups were observed. Substrate induction of CO(2) fixation in mesophyll protoplast extracts of D. sanguinalis gave malate as a major product; only by an apparent exchange reaction with cold aspartate did substantial label appear in aspartate (up to 53% of labeled products). In contrast, aspartate was a major product when alanine and alpha ketoglutarate served as inducing substrates with E. indica (up to 57%) and U. panicoides (up to 86%). With induction by pyruvate or 3-phosphoglycerate, mesophyll preparations of U. panicoides and E. indica were less effective in forming malate (up to 31% of products) than D. sanguinalis (up to 87% of products). After 2 seconds of whole leaf (14)CO(2) fixation, malate was the major labeled product (57%) with D. sanguinalis, whereas with E. indica and U. panicoides aspartate was the predominant product (73% and 76%, respectively).With mesophyll protoplast extracts of D. sanguinalis, aspartate inhibited CO(2) fixation (about 50% at 0.6 mm), while malate was relatively uninhibitory at comparable concentrations. CO(2) fixation by mesophyll protoplast extracts of E. indica was inhibited by malate (about 50% at 0.6 mm), while aspartate was relatively uninhibitory. With mesophyll preparations of U. panicoides, malate or aspartate (2 mm) caused only slight inhibition of CO(2) fixation. The regulation of aspartate and malate synthesis in C(4) mesophyll cells is discussed relative to initial products of photosynthesis in C(4) species in vivo and species differences in the mechanisms of C(4) photosynthesis. PMID- 16659296 TI - Photomanipulation of phytochrome in lettuce seeds. AB - Seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were imbibed and given either short irradiation with red or far red light prior to drying or dried under continuous red or far red light. Seeds treated with either short or continuous red germinate in darkness, whereas seeds treated with either short or continuous far red require a short exposure to red light, after a period of imbibition, to stimulate germination. Irradiation of dry red seeds with far red light immediately before sowing results in a marked inhibition of germination. This result was predicted since far red-absorbing form phytochrome can be photoconverted to the intermediate P650 (absorbance maximum 650 nm) in freeze dried tissue. A similar far red treatment to continuous red seeds is less effective and it is concluded that in these seeds a proportion of total phytochrome is blocked as intermediates between red-absorbing and far red absorbing form phytochrome, which only form the far red-absorbing form of phytochrome on imbibition. The inhibition of dry short red seeds by far red light can be reversed by an irradiation with short red light given immediately before sowing, confirming that P650 can be photoconverted back to the far red-absorbing form of phytochrome. The results are discussed in relation to seed maturation (dehydration) on the parent plant. PMID- 16659297 TI - Carbon dioxide requirements for phytochrome action in photoperiodism and seed germination. AB - The effect of interrupting darkness with red light in the presence or absence of 0.03% CO(2) was studied in relation to flowering of Xanthium pennsylvanicum and germination of light-sensitive lettuce seeds. The results indicate that CO(2) is essential for red light to be effective in either process. PMID- 16659298 TI - The role of abscisic Acid in cross-adaptation of tobacco plants. AB - Tobacco plants (Nicotiana rustica L.) pre-exposed to leaf dehydration, mineral deprivation, salination, or BO(3) (3-) toxicity exhibited increased resistance to subzero temperature and to reduced oxygen in the root medium. The stressed plants all showed an elevated content of leaf abscisic acid. Upon transfer of mineral deprived and salinated plants to prestress conditions, a decline in leaf abscisic acid content to prestress levels took place together with a loss of the increased resistance to subzero temperature and to deprivation of root oxygen. Treatment with abscisic acid by direct application to the leaves or by addition to the root medium improved leaf resistance to subzero temperature and to deprivation of root oxygen. A common hormone-regulation mechanism involving abscisic acid is suggested for this phenomenon of "cross-adaptation" by which a given stress confers increased resistance to other, apparently unrelated stresses. PMID- 16659299 TI - Phytochrome: A Re-examination of the Quaternary Structure. AB - Highly purified phytochrome samples from rye (Secale Cereale cv. Cougar) were fractionated by ultracentrifugation in isokinetic sucrose density gradients. Three protein species were separated with estimated sedimentation coefficients of 6.5S, 8.0S, and 11.5S. The 6.5S and 8.0S forms contained photoreversible phytochrome and produced a single subunit of 120,000 molecular weight upon reduction and electrophoresis in the presence of sodium dodecyl sulfate. The 11.5S species contained no detectable phytochrome. Reduction and electrophoresis of the 11.5S species in the presence of sodium dodecyl sulfate produced a major polypeptide of 32,000 molecular weight and a minor polypeptide of 48,000 molecular weight. The square tetrameric structures, observed by electron microscopy and previously thought to be phytochrome molecules, were found to be due to the presence of this 11.5S species in phytochrome preparations. PMID- 16659300 TI - Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis. AB - Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s(20, w) at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.The phylogenetic relationship of carboxylase from Euglena and from three higher plants was investigated. In general, the size, subunit formation, and quaternary structure of RUDPcase from the various sources seemed to be similar. A partial immunochemical reaction between anti-RUDPcase serum from Euglena and the enzymes from lettuce, cucumber, and New Zealand spinach suggested that the algal and higher plant carboxylases were related but not identical. This was borne out by amino acid analyses which showed a close correspondence between the large, but not the small, subunits of Euglena and lettuce. PMID- 16659301 TI - Photocontrol of Anthocyanin Synthesis: IV. Dose Dependence and Reciprocity Relationships in Anthocyanin Synthesis. AB - Under continuous far red light, anthocyanin synthesis in young, dark-grown cabbage seedlings (Brassica oleracea cv. Red Acre) is irradiance-dependent and fails to follow the reciprocity (irradiance x time = constant) relationships. Under intermittent far red treatments extended over a prolonged period of time, anthocyanin synthesis becomes dose dependent, and reciprocity relationships are valid. Intermittent far red treatments with short dark intervals between successive irradiations are as effective as continuous treatments, if the total radiation doses applied with the two types of treatments are equal and are applied over equally long periods of time. The high effectiveness of inter mittent treatments, the dose dependence, and the validity of the reciprocity relationships suggest that cycling between red-absorbing form of phytochrome and far red-absorbing form of phytochrome and the formation of electronically excited far red-absorbing form of phytochrome, or the involvement of a second photoreactive system, besides phytochrome, may play only a minor role in high irradiance reaction anthocyanin synthesis brought about by prolonged exposures to far red irradiation. PMID- 16659302 TI - Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension cultured Acer pseudoplatanus Cells. AB - Using suspension-cultured Acer pseudoplatanus cells requiring 2,4 dichlorophenoxyacetic acid for growth, the dependence of the population doubling time and the maximum increase in cell population density on the auxin concentration was studied. It appears that in the range of 2,4 dichlorophenoxyacetic acid concentration from 4 x 10(-8) to 4 x 10(-6) M, the rate of cell division during the logarithmic growth phase is independent of the auxin concentration, while the maximum number of cell generations obtained is limited by the initial auxin concentration. The significance of these two aspects of auxin action are discussed. PMID- 16659303 TI - Influence of Short Term Inhibitor Treatment on the Flowering of Lemna perpusilla 6746. AB - Short term inhibitor treatment can be used to examine the processes that occur during an inductive dark period in the short day plant Lemna perpusilla Torr., strain 6746. Several inhibitors of protein biosynthesis are most effective in reducing per cent flowering when treatment occurs over the 2-hour intervals beginning at the 12th hour or the 14th hour of a 8 (16) photoperiodic cycle. The antimetabolite, 5-fluorouracil, is most effective when treatment occurs early in the dark period. Evidence is cited suggesting that distilled water incubation inhibits flowering by interfering with protein biosynthesis. PMID- 16659304 TI - The occurrence and distribution of poly(a) ribonucleic Acid in soybean. AB - The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T(1) digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T(1) digestion. PMID- 16659305 TI - The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses. AB - Phosphoenolpyruvate (PEP) carboxylase activity in immature ;Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity. PMID- 16659306 TI - Promotion of Xyloglucan Metabolism by Acid pH. AB - Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H(+) secretion theory of auxin action. PMID- 16659307 TI - The regulation of potassium absorption in barley roots. AB - The dynamics of changes in K(+) influx across the plasmalemma and of internal K(+) concentrations [K(+)](1) of intact barley (Hordeum vulgare) roots were examined as the roots were converted from ;high-salt' to ;low-salt' roots. Following the transfer of plants grown in 0.5 mm CaSO(4) solutions plus various concentrations of KCl to 0.5 mm CaSO(4) solutions, influx rates increased and internal K(+) concentrations declined as a function of time and the initial K(+) status of the tissue. The relationship between plasmalemma influx and [K(+)](1) was examined over a wide range of [K(+)](1) values by growing intact plants in various concentrations of KCl. Plasmalemma influx was inversely correlated with the square of [K(+)](1). A model for the regulation of plasmalemma influx by [K(+)](1) is considered. PMID- 16659308 TI - Synthesis of Endosperm Proteins in Wheat Seed during Maturation. AB - The time of synthesis, the molecular weight, and the relative glutamine-glutamate and proline to leucine ratios of the endosperm proteins of wheat (Triticum aestivum L. cv. Logan) were determined using a sodium dodecyl sulfate polyacrylamide gel technique. In general, synthesis of most proteins occurred through much of the maturation of the seed, but past 20 days the rate of synthesis of the high molecular weight proteins declined more rapidly than those of lower molecular weight. The synthesis of at least one protein occurred only late in seed maturation. Several of the high molecular weight proteins had glutamate-glutamine to leucine ratios higher than the remainder of the proteins. No evidence for proteins of a polyglutamine-glutamate and/or proline nature was found. PMID- 16659309 TI - Chemical Basis for Greenbug Resistance in Small Grains: II. Identification of the Major Neutral Metabolite of Benzyl Alcohol in Barley. AB - ((14)C)Benzyl alcohol was administered either by uptake through the roots or by injection directly into the stems of wheat (Triticum aestivum L. em Thell), sorghum (Sorghum bicolor. L Moench) and two strains of barley (Hordeum vulgare L.). One strain of barley was susceptible to the greenbug (Schizaphis granium Rondani), and the other was greenbug-resistant. In all four plants, several radioactive metabolites were formed. The major neutral metabolite has been identified as benzyl-beta-d-glucopyranoside. This glucoside was found to have no biological activity against the greenbug under conditions in which the parent compound, benzyl alcohol, inhibits the reproduction of this insect pest. PMID- 16659310 TI - The effect of sucrose on the differentiation of excised fern leaf tissue into either gametophytes of sporophytes. AB - Excised juvenile leaves of Microgramma vacciniifolia (Polypodiaceae) develop sporophytic regenerants when grown on mineral agar with sucrose. The ratio of sporophytes to gametophytes produced from the leaf tissue increases with higher percentages of sucrose such that at 4% sucrose, the induction of aposporous gametophytes is a rare occurrence. Experiments varying the osmotic potential with sorbitol and those holding the osmotic potential of the culture medium constant while varying the sucrose level indicate that the effect of sucrose on the differentiation of fern leaf tissue into either gametophyte or sporophyte is nutritional rather than osmotic. A significant effect of sucrose in altering the differentiation of fern leaf tissue is the increased rate of senescence promoted by high sucrose concentrations. PMID- 16659311 TI - Osmoregulation in Cotton Fiber: Accumulation of Potassium and Malate during Growth. AB - Kinetics and osmoregulation of cotton (Gossypium hirsutum L.) fiber growth (primarily extension) have been studied. Growth is dependent on turgor pressure in the fiber. It is inhibited when a decrease in the water potential of the culture medium due to an addition of Carbowax 6000, equals the turgor pressure of the fiber. Potassium and malate accumulate in the fiber and reach peak levels when the growth rate is highest. Maximum concentrations of potassium and malate reached in the fiber can account for over 50% of the osmotic potential of the fiber. As growth slows down, levels of potassium and malate decrease and turgor pressure declines. Cotton ovules are capable of fixing H(14)CO(3) (-) in the dark, predominantly into malate. Fiber growth is inhibited by the absence of potassium and/or atmospheric CO(2). We suggest that potassium and malate act as osmoregulatory solutes and that malate, at least in part, arises from dark CO(2) fixation reactions. PMID- 16659312 TI - Isolation of Intact Chloroplasts of Euglena gracilis by Isopycnic Sedimentation in Gradients of Silica. AB - A technique is described for the isolation of structurally intact and partially active chloroplasts from photoheterotrophically grown Euglena gracilis. The separation of intact chloroplasts from stripped chloroplast membranes and other subcellular particles was achieved by sedimentation in continuous, isosmotic density gradients of Ludox AM, a silica sol.The final preparations contained an average of 93% intact chloroplasts and corresponded to approximately 10% of the the chlorophyll of the original cell suspension and 20 to 30% of the chlorophyll layered on the gradients.The chloroplasts obtained were intact by the criteria of ultrastructure, their content of ribulose diphosphate carboxylase, and their activity in a modified Hill reaction assay (U. Heber and K. A. Santarius. 1970. Z. Naturforsch. 25b: 718-727). In addition, the isolated chloroplasts were capable of incorporating amino acids into protein in the light. PMID- 16659313 TI - Sites of auxin action: regulation of geotropism, growth, and ethylene production by inhibitors of auxin transport. AB - The inhibitors of auxin transport-NPA (N-1-naphthylphthalamic acid), DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-a] isoindol-8-one), and TIBA (2,3,5-triiodobenzoic acid)-inhibited geotropism in roots of intact Pisum sativum L. seedlings. NPA and DPX1840 also caused cellular swelling in the roots. The swelling was due to a greater inhibition of elongation than increase in weight and looked identical to the one caused by ethylene. However, ethylene did not act as an intermediate in the action of auxin transport inhibitors because all three failed to stimulate ethylene production and some of their growth-inhibiting effect was retained in the presence of saturating levels of ethylene. In the presence of 10 mum indoleacetic acid the growth-inhibiting effect of auxin transport inhibitors was lost after 18 hours. On the other hand, auxin transport inhibitors did not interfere with the ability of auxin to promote ethylene production. Growth inhibition caused by auxin transport inhibitors was reversible. Pea root sections resumed normal growth following flushing of treated sections with inhibitor-free solutions. Experiments with (14)C-2, 4 dichlorophenoxyacetic acid revealed that the herbicide and auxin transport inhibitors may have the same binding site. It was concluded that a class of structurally dissimilar compounds may share a similar physiological role since they all appear to compete with endogenous auxin for certain binding sites and they all have similar growth-regulating activities. PMID- 16659314 TI - A simple, inexpensive, high pressure liquid chromatographic method for separating cytokinins in plant extracts. AB - Separation of a mixture of the main cytokinins occurring naturally in plant tissues was achieved by high pressure liquid chromatography using insoluble polyvinylpyrrolidone as the solid support. The separation of each cytokinin was first assessed over a range of salt and l-butanol concentrations and pH using a mixture of borate buffer and l-butanol as the mobile phase to determine the conditions necessary for optimum resolution. A discrete separation of zeatin, N-6 (Delta-2-isopentenyl)adenine, their related ribonucleosides, and kinetin was achieved using a simple isocratic elution with 0.025 m borate buffer at pH 6.8 and 4% (v/v) l-butanol. A number of cytokinin-active compounds were detected in cabbage extracts by the Amaranthus betacyanin bioassay using this separation technique. PMID- 16659315 TI - Heat-induced Resistance of Sugarcane to Helminthosporium sacchari and Helminthosporoside. AB - Sugarcane clones susceptible to eye spot disease became resistant to the pathogen or helminthosporoside activity during warm summer months. Exposure of plants to 30 to 35 C temperatures, hot water treatment of excised leaves from 30 to 50 C, preincubation of leaves in an N(2) atmosphere, or preincubation in the presence of protein synthesis inhibitors also induced resistance. Hot water-treated leaves reverted to a susceptible condition. Five to 15 C temperatures, an N(2) atmosphere, or protein synthesis inhibitors prevented this recovery. Results indicate that temperature may influence host tissue sensitivity to helminthosporoside, thus governing disease development throughout the year. PMID- 16659316 TI - Studies on the Growth Effects of the Canaline-Urea Cycle Amino Acids with Lemna minor L. AB - The aquatic microphyte, Lemna minor L., was utilized to assess the relative toxicity and general growth effects of canavanine, canaline, ureidohomoserine (UHS), and canavaninosuccinate (CSA). These amino acids are constituents of the canaline-urea cycle and structural analogues of the ornithine-urea cycle amino acids.Comparative growth studies with L. minor revealed that the canaline-urea cycle amino acids are potent antimetabolites. With the exception of CSA, they are extremely toxic at a concentration of 5 mum. Over a concentration range of 1 to 4 mum, canavanine is the most growth-inhibiting of the canaline-urea cycle amino acids. At or above 5 mum, canavanine and canaline possess comparable toxicity. UHS is less growth-inhibiting than canavanine or canaline, and CSA is the least toxic of the canaline-urea cycle intermediates. PMID- 16659317 TI - Evidence for Cytokinin in Bacterial Leaf Nodules of Psychotria punctata (Rubiaceae). AB - Cytokinin activity based on two bioassays was at least 100-fold higher in Psychotria punctata leaf discs with bacterial nodules than in discs without them. Nodulated discs from young leaves yielded 0.4 to 6 mug of cytokinin (zeatin equivalents) per g fresh weight of leaf tissue, whereas non-nodulated discs from the same leaves yielded 0 to 0.003 mug per g fresh weight. These estimates probably include free-base cytokinins and, if present, any nucleoside cytokinins precipitable by acidic silver nitrate. Cytokinin concentrations in Psychotria leaf nodules appear to be higher than normally found in green leaves of other plants. In l-butanol-acetic acid-water (12:3:5, v/v), the one peak of activity chromatographed with an R(F) similar to zeatin's, but both number and identity of the active substance(s) remain unknown. These findings suggest that a cytokinin is produced by bacteria in leaf nodules of P. punctata and that it is involved in the symbiosis. PMID- 16659318 TI - Promotion of radish cotyledon enlargement and reducing sugar content by zeatin and red light. AB - Effects of zeatin on amino acid and sugar contents of detached radish (Raphanus sativus L.) cotyledons were investigated to determine if accumulation of these solutes contributes to cytokinin-enhanced growth. Protein and amino acid levels were not significantly affected, but in cotyledons incubated in light the hormone caused greater accumulations of reducing sugars than occurred in light controls. Continuous fluorescent light or a few minutes of red light increased both the growth rate and the reducing sugar levels compared to dark controls. A far red treatment following red light overcame the promoting effect of the latter. Amounts of reducing sugars were closely associated with growth under the above conditions. Activity of an unidentified amylase was elevated by continuous light or a red light treatment (nullifiable by far red), suggesting that reducing sugars were derived from starch. Zeatin-treated cotyledons exhibited less amylase activity than did light controls, perhaps implicating cytokinin-stimulated conversion of fats to sugars in light. In darkness zeatin promoted cotyledon growth but did not increase sugar levels nor amylase activity, suggesting that enhanced ion accumulation also contributes to the more rapid water uptake leading to growth. PMID- 16659319 TI - Sulfur dioxide inhibition of photosynthesis in isolated spinach chloroplasts. AB - Photosynthetic oxygen evolution by isolated spinach (Spinacia oleracea L.) chloroplasts approached complete inhibition in the presence of a 5 mm concentration of sulfur dioxide. A similar inhibition was observed in the presence of equimolar concentrations of bisulfite ions, suggesting a parallel mode of action. In contrast, an equimolar concentration of sulfite ions was markedly less inhibitory and sulfate ions caused negligible inhibition of apparent photosynthesis. The mode of action of sulfur dioxide and related sulfur anions in inhibiting photosynthesis was found to be essentially independent of direct hydrogen-ion effects. Supplements of inorganic pyrophosphate lessened the inhibition of oxygen evolution caused by sulfur dioxide and the sulfur anions.Sulfur dioxide and the sulfur anions were almost equally effective in inhibiting cyclic and noncyclic photophosphorylation in chloroplast suspensions. However, the extent of the inhibition of these photosynthetic reactions does not appear sufficient to account for the inhibition of photosynthetic oxygen evolution by sulfur dioxide. PMID- 16659320 TI - Separation of Chlorophyll Degradation from Other Senescence Processes in Leaves of a Mutant Genotype of Meadow Fescue (Festuca pratensis L.). AB - Chlorophyll levels in l-cm sections of the youngest fully expanded leaves of normal (Y) Festuca pratensis L. declined almost to zero over a period of 6 days after excision. Chlorophyll in a mutant genotype (NY) remained near the initial level for the whole of this period. Abscisic acid promoted pigment loss in Y but had no significant effect on chlorophyll in NY. Kinetin retarded pigment loss in Y but was ineffective in NY. Other biochemical changes associated with leaf senescence-reduction in protein content and the appearance of novel isoenzymes of alpha-naphthyl acetate esterases-occurred in both genotypes. Abscisic acid accelerated protein breakdown, whereas kinetin inhibited the loss of protein in both genotypes. The mutation thus appears to be expressed as a highly specific lesion in pigment metabolism. We concluded that pigment breakdown, which is widely used as an index of leaf senescence, may not be an inevitable part of the aging process. PMID- 16659321 TI - Paradoxical Effect of Actinomycin D: Regulation of Synthesis of Wound RNase at Translation in Turnip Tissue. AB - Cutting of tissue sections induces RNase (EC 2.7.7.16) activity (phase I) in white turnip (Brassica rapa L. var. rapa) which peaks in 4 or 7 hours and then declines rapidly (phase II). The increase is inhibited by cycloheximide; also RNase from tissue bathed in 99.8% D(2)O during phase I underwent a large increase in buoyant density, indicating that the increased activity is due to de novo synthesis. Actinomycin D inhibited induction of RNase only if given within the initial 45 minutes after cutting. When it was applied after 45 minutes, it caused enhancement (super-induction) of RNase activity for over 24 hours. The half-time for degradation of RNase during phase I in the presence of cycloheximide and phase II in the presence and absence of cycloheximide is the same, indicating that the decline in RNase activity is due to cessation of synthesis. Also the rate of degradation of RNase remains the same during superinduction, thus indicating that actinomycin D superinduction is due to maintenance of synthesis of RNase rather than inhibition of its rate of degradation. Consistent with this is the fact that actinomycin D superinduction of RNase is inhibited by cycloheximide. The evidence is consistent with the hypothesis that messenger RNA for RNase is long-lived and the decline in RNase is due to transscription of a regulator gene coding for a specific repressor protein during phase I which inhibits RNase synthesis at the level of translation. Superinduction of RNase activity by actinomycin D is explicable in terms of (a) inhibition of synthesis of the mRNA coding for a repressor protein that inhibits translation of RNase specific mRNA, or, (b) differential stability of mRNAs in presence of actinomycin D, and competition among mRNAs for factors rate-limiting to translation, thus favoring synthesis of proteins coded by long-lived messengers. PMID- 16659322 TI - Control of amino Acid synthesis in tissue culture cells. AB - Low concentrations of cycloheximide added to rose suspension culture cells stopped protein synthesis and drastically reduced the incorporation of (14)C into arginine, lysine, isoleucine, threonine, and valine. PMID- 16659323 TI - Phytochrome-mediated Carotenoids Biosynthesis in Ripening Tomatoes. AB - Red light induced and far red light inhibited carotenoid biosynthesis in ripening tomatoes (Lycopersicon esculentum Mill.) when compared to controls kept in the dark. Red illumination following far red illumination reversed the inhibitory action of far red light on carotenoid biosynthesis, suggesting a phytochrome mediated process. Quantitation of individual carotenoids favored the hypothesis of two separate carotenoid biosynthetic pathways in tomatoes. PMID- 16659324 TI - Dynamics of photoassimilated carbon in douglas fir seedlings. AB - The relations between CO(2) uptake, translocation, and carbon accumulation in several vegetative components of Douglas fir seedlings (Pseudotsuga menziesii [Mirb.] Franco) have been quantified using (14)CO(2). Seedlings were exposed to a constant specific radioactivity of (14)CO(2) and a repeating daily pattern of temperature and light for 4 consecutive days. Results of (14)C analysis, which indicated a transitory pattern of photoassimilated carbon movement, were extrapolated to a "steady rate" using a compartment analysis. Accumulation rates of photoassimilated carbon, relative to tissue carbon, were new needles, 0.94%/day, old needles, 1.14%/day, new shoots 0.38%/day, stem, 0.16%/day, and roots, 0.50%/day. Therefore, the source of carbon, the needles, is also the strongest sink. PMID- 16659325 TI - Alkylguanidines as inhibitors of k transport in isolated barley roots. AB - It has been shown that plants can accumulate K(+) through an energy-dependent process. The effect of alkylguanidines, in particular octylguanidine on the uptake of (86)Rb(+) by excised barley roots (Hordeum vulgare var. Apizaco LV-72), has been studied. (86)Rb(+) was used as tracer of K(+). The uptake of (86)Rb(+) which is linear with time and shows saturation kinetics is inhibited by octylguanidine. Half-maximal inhibition of (86)Rb(+) uptake is attained at 50 muM octylguanidine. Octylguanidine induces a decrease in the V(max) of the process and increases the Km of the system for Rb(+). When the effects of various alkylguanidines were studied, the following order of effectiveness was encountered; octylguanidine = hexilguanidine > butylguanidine > ethylguanidine > guanidine. This suggests that guanidines inhibit Rb(+) uptake by interacting through its positively charged guanidinium group with a Rb(+) carrier while the alkyl chain interacts with the hydrophobic milieu of the membrane. PMID- 16659326 TI - Contractile Characteristics of Mimosa pudica L. AB - The force-velocity characteristics of the primary pulvinus of Mimosa pudica have been determined using a new polytonic measurement technique. The contractile characteristics were determined from a modified form of Hill's equation (Hill, A. V. 1938. Proc. Roy. Soc. London B126: 136-195) describing the physiological contractile behavior of animal muscle. The values of the resulting Hill's constants were found to be remarkably similar to those of intact animal muscle and reconstituted contractile collagen. PMID- 16659327 TI - Photocontrol of the Germination of Onoclea Spores: IV. Metabolic Changes during Germination. AB - The changes in levels of metabolites during photoinduced germination of Onoclea sensibilis L. spores are described. Proteins and lipids, which constitute 25 and 20%, respectively, of the unimbibed spores on a dry weight basis, are hydrolyzed at the time of differentiation and elongation of the germling cells and may be utilized for these processes. Sucrose degradation, starch synthesis, and active respiration occur during dark imbibition, but these processes are accelerated by red or far red irradiation. Endogenous sucrose is the probable source of the carbon skeleton for starch synthesis. PMID- 16659328 TI - DPX 1840-induced Organogenesis in Xanthi-nc Tobacco Plants. AB - The synthetic growth regulant DPX 1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H pyrazolo[5, 1-a]isoindol-8-one) induced callus growth and subsequent tissue differentiation on cut surfaces of decapitated Xanthi-nc tobacco plants (Nicotiana tabacum). Callus formation and organogenesis induced by DPX 1840 depended on the presence of leaves. The adventitious meristems developed into either vegetative or flowering shoots. Pedicels that bore single flower buds developed two abscission zones that caused the buds to abscise before anthesis. The various morphological and physiological processes affected by DPX 1840 suggests that this growth regulator affects the endogenous hormonal distribution and/or activity. PMID- 16659329 TI - Mechanism of sulfate transport inhibition by cycloheximide in plant tissues. AB - Inhibition by cycloheximide of sulfate transport in both barley roots (Hordeum vulgare L.) and potato tuber (Solanum tuberosum L.) increases with increasing inhibitor concentration only to a limited extent, depending on the length of the tissue incubation with the inhibitor. In contrast to this, increasing concentrations of dinitrophenol have a rapid and total inhibitory effect on the active transport. Leucine transport in the same tissues is strongly inhibited by dinitrophenol but is not affected by cycloheximide, whereas incorporation into protein is mainly inhibited by cycloheximide. It appears that the mechanism of transport inhibition by cycloheximide in plant tissues consists in stopping new carrier synthesis and not in the disruption of energy flow. Sulfate carriers show comparable decay rates in barley roots and potato tuber, the mean life being shorter than that of the leucine carriers. These appear more stable in roots than in storage tissues. PMID- 16659330 TI - Site of action of inhibitors of carbon dioxide assimilation by whole lettuce chloroplasts. AB - The sites of action of several compounds, reported to inhibit CO(2) fixation by chloroplast preparations were located by developing assays in lettuce chloroplasts to test their effect on partial reactions of the carbon cycle and on carbonic anhydrase. The results indicated that: d, l-glyceral-dehyde and 5'-AMP inhibit phosphoribulose kinase or isomerase. 3-Phosphoglyceric acid and 6 phosphogluconate inhibit ribulose diphosphate carboxylase. Azide, Mg(2+), and nitrite inhibit the activity of carbonic anhydrase of lettuce chloroplasts and light-dependent CO(2) fixation by intact chloroplasts with similar sensitivities. None of these inhibited CO(2) fixation in ruptured chloroplasts. It is suggested that the inhibition by azide, nitrite, and magnesium ions of CO(2) fixation by intact chloroplasts is due to their inhibition of the activity of carbonic anhydrase. PMID- 16659331 TI - Accumulation of delta-Aminolevulinic Acid and Its Relation to Chlorophyll Synthesis and Development of Plastid Structure in Greening Leaves. AB - Levulinic acid inhibited the greening of etiolated maize (Zea mays) and bean (Phaseolus vulgaris) leaves and caused accumulation of delta-aminolevulinic acid (ALA). ALA accumulation in maize was equivalent to the decrease in chlorophyll, over a wide range of experimental conditions. It was saturated at low light intensities and was not limited by the supply of substrates during the early hours of greening. During 20 hours in light, levulinic acid had little effect on the structural development of thylakoids in bundle sheath chloroplasts but significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts. Recrystallization of prolamellar bodies and their reformation was inhibited. Mitochondria appeared not to be affected.The accumulation of ALA in bean leaves differed from that in maize in regard to its time course and the effect of levulinic acid concentration and light intensity. The amount of ALA accumulated exceeded that expected from the degree of inhibition of chlorophyll synthesis. Levulinic acid caused abnormalities in the structural development of bean chloroplasts and marked swelling of mitochondria.Chloramphenicol and cycloheximide inhibited ALA accumulation, while inhibitors of RNA synthesis had no effect. The extent of inhibition depended on the time the inhibitor was applied during the greening process. The use of ALA accumulation as a tool for studying the control of chlorophyll synthesis is discussed. PMID- 16659332 TI - Control of delta-Aminolevulinic Acid and Chlorophyll Accumulation in Greening Maize Leaves upon Light-Dark Transitions. AB - The accumulation of delta-aminolevulinic acid (ALA) was studied in greening maize (Zea mays) leaves which were transferred to darkness and reilluminated after various periods of time. The system synthesizing ALA decays in the dark with a half-life of about 80 minutes. The onset of enzyme decay after transfer to darkness shows a 40-minute lag. The accumulation of ALA in the presence of levulinic acid in leaves transferred to darkness corresponds to that expected from the estimated half-life of the enzyme synthesizing ALA. On the other hand, the accumulation of protochlorophyll upon transfer to darkness in the absence of levulinic acid stops much earlier. It is suggested that a control point exists in the pathway between ALA and protochlorophyll, preventing utilization of the accumulated ALA upon transfer of greening leaves to darkness. This is supported by the observed effects of low intensities of monochromatic light (648 nm) on ALA and chlorophyll accumulation. PMID- 16659333 TI - Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination. AB - Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5- and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%. PMID- 16659334 TI - Relationships between the Transition of the Physical Phase of Membrane Lipids and Photosynthetic Parameters in Anacystis nidulans and Lettuce and Spinach Chloroplasts. AB - The transition of the physical phase of lipids in membrane fragments of a blue green alga Anacystis nidulans was studied by a spin labeling technique. The maximum hyperfine splitting of the electron spin resonance spectrum of the N-oxyl 4', 4'-dimethyloxazolidine derivative of 5-ketostearic acid plotted against the reciprocal of the absolute temperature gave a discontinuity point that was characteristic of a transition of the physical phase of the hydrocarbon region of membrane lipids. The phase transition appeared at approximately 13 or 24 C in the organisms grown at 28 or 38 C, respectively.The temperature dependence curve of chlorophyll a fluorescence in intact cells, membrane fragments, and extracted lipids of Anacystis cells suspended in a buffer solution showed that the fluorescence yield became maximum near the phase transition temperatures. These findings suggest that chlorophyll a in the thylakoid membrane works as a native fluorescence probe for the detection of phase transition.The temperature dependence of photosynthetic electron transport reactions was studied by measuring the oxidoreductive reactions of P700 and by measuring O(2) evolution. Each of the Arrhenius plots of the reaction rates was composed of two straight lines with a break near the phase transition temperatures. The activation energy was always lower above than below the transition temperatures. It is proposed to explain these phenomena that a reaction involving plastoquinone is influenced by the physical state of membrane lipids.The shift between the pigment state 1 and state 2 measured by fluorescence transients also showed a characteristic break in the Arrhenius plots near the phase transition temperatures; below the transition temperatures the shift almost disappeared. This suggests that the configurational change of the thylakoid membrane related to the state 1 and state 2 shift is dependent on the physical state of membrane lipids. In the chloroplasts of lettuce and spinach, on the other hand, there was no break in the Arrhenius plot of the electron transport reactions or of Mg(2+)-induced changes of chlorophyll a fluorescence.It is suggested that the transitions of the hyperfine splitting of the ESR signal, electron transport, and the configurational change, as well as the appearance of the maximum of chlorophyll a fluorescence, in the thylakoid membranes of Anacystis nidulans are all related to the transition of the physical phase of membrane lipids between the liquid crystalline state and the mixed liquid crystal-solid state. PMID- 16659335 TI - In vitro simulation of cytoplasmic membrane senescence in cotyledons. AB - The loss of microsomal NADH-cytochrome c reductase activity (EC 1.6.99.3) in cotyledons, known to accompany germination of Phaseolus vulgaris and thought to reflect the progress of cytoplasmic membrane senescence, can be simulated in an in vitro system in which isolated microsomes from 2-day-old tissue are treated with cytosol fractions (microsomal supernatants). Inactivation of the enzyme is comparatively low when the microsomes are treated for 4 hours with cytosol fractions from 1- and 2-day-old tissue, but increases to about 68% upon treatment with a corresponding fraction from 3-day-old cotyledons. This temporal pattern is consistent with the pronounced in situ decline in NADH-cytochrome c reductase detectable between the 2nd and 4th days of germination. Extensive in vitro inactivation was also effected by cytosol fractions prepared from older tissue, including that harvested after 9 days of germination by which time the cotyledons were beginning to abscise.The degree of inactivation in the in vitro system proved to be proportional to the concentration of cytosol present in the incubation mixture, and also increased with time at fixed cytosol concentrations. Moreover, the rate of in vitro inactivation was some 18-fold greater than that occurring in situ. The data have been interpreted as indicating that cytoplasmic membrane senescence in cotyledons is, in part, mediated by one or more factors in the cytosol, extrinsic agents which are sequestered in situ and released gradually during germination as the pressures of senescence become more intense. PMID- 16659336 TI - Size and specific radioactivity of the [h]thymidine triphosphate pool and DNA synthesis in the radicles of germinating cotton. AB - By relating the specific radioactivity, size, and turnover of the labeled thymidine triphosphate pool to DNA synthesis, it was shown that the 32- to 38 hour interval of cotton seedling (Gossypium barbadense L.) germination was the peak period of DNA synthesis within the radicle tip during a total germination time of 48 hours at 35 C. PMID- 16659337 TI - Comparison of DNA Polymerase of Rhizobium meliloti and Alfalfa Bacteroids. AB - DNA dependent-DNA polymerase activity was established and partially purified from extracts of cultured Rhizobium meliloti, F-28, and nodule bacteroids (R. meliloti, F-28) of alfalfa plants (Medicago sativa). Polymerase activity in the partially purified fractions showed characteristic dependence on Mg(2+), DNA, and a full complement of deoxyribonucleoside triphosphates. DNase activity, preference of "activated" double strand DNA, and inhibition by p chloromercuribenzoate and MnCl(2) were responses common to both systems. The two systems however did exhibit some differences in pH, Mg(2+), and primer optima. Polymerase activity in crude extracts of the cultured bacteria was more stable and had 10- to 18-fold greater specific activity than the bacteroid extracts. Preliminary measurements of specific DNA polymerase activity in crude extracts of cultured Rhizobium japonicum were not significantly higher than that in the crude extracts of soybean nodule bacteroids. A possible correlation between DNA synthesis and the successful establishment of rhizobia-legume symbiosis is discussed. PMID- 16659338 TI - Abscission of Phaseolus and Impatiens Explants: Effects of Ionizing Radiation upon Endogenous Growth Regulators and de Novo Enzyme Synthesis. AB - Stem-petiole explants from the lower pulvinus of the primary leaves of Phaseolus vulgaris L. cv. Red Kidney and from Impatiens sultani Hook cv. Scarlet Baby were exposed to varying dosages of gamma-radiation. With bean, irradiation of 175 to 525 kiloroentgens (kR) significantly accelerated the onset of abscission with a maximum response at 175 to 280 kR. Higher dosages (beginning at 600-700 kR) usually prevented abscission. With Impatiens, 18 to 35 kR significantly accelerated both the onset of abscission and possibly the initial abscission rate; 350 kR cut the time to 100% abscission in half and substantially accelerated the initial abscission rate. Inhibition of abscission in Impatiens was not possible with the available dose rate (35 kR/hour).The accelerating effect of ionizing radiation was apparently due to primarily a reduction in auxin activity, thus accelerating the shift from the relatively ethylene-insensitive stage I to the ethylene-sensitive stage II. In vitro irradiation of exogenous auxin in agar substantially reduced the abscission-delaying effects of the auxin. The low dosage accelerating effects of the ionizing radiation were negated by exogenous auxin application, either prior to or following irradiation. With prior auxin application, a higher concentration of auxin was required to achieve the same delaying effects as with application following irradiation. Apparently due to this reduction in auxin activity, irradiation hastened sensitivity to exogenous ethylene.The accelerating effects of ionizing radiation may be secondarily due to increased levels of endogenous ethylene. With bean tissues, exposures of 262 through 1500 kR of gamma radiation increased the measurable levels of endogenous ethylene released into the ambient air within 2 or 3 days after irradiation.The results of actinomycin D treatment suggested that required de novo RNA synthesis occurred about 4 hours earlier in those Impatiens explants that had received 105 kR gamma radiation than in the nonirradiated controls.An increased mobilization of Ca from the abscission zone may be a contributing factor in the radiation-induced acceleration of abscission. For both bean and Impatiens, exogenous Ca inhibited abscission of irradiated and nonirradiated explants. In addition, a more concentrated Ca solution was required to inhibit abscission of irradiated bean explants, as compared to the nonirradiated controls. PMID- 16659339 TI - Effect of digitonin concentration on electron transport, phosphorylation, and proton uptake by subchloroplast particles. AB - The relative activity of photosystem I subchloroplast particles was dependent on the digitonin concentration used during incubation. At low digitonin concentrations (2 mg digitonin per mg chlorophyll), the particles were greatly enriched in photosystem I activities and showed high rates of cyclic phosphorylation and appreciable light-induced proton uptake. Increasing the concentration of digitonin increased the yield of photosystem I particles but decreased their specific activity of electron transport and of cyclic phosphorylation. The decrease in activity was not related to the degree of enrichment of the pigments associated with photosystem I nor to the degree of separation of the photosystems. Release of plastocyanin was dependent on the digitonin concentration. As the digitonin concentration was increased, the plastocyanin content of the photosystem I particles decreased. The decrease in photochemical activity with increasing digitonin concentration was attributed to the loss of plastocyanin during isolation. PMID- 16659340 TI - Cold hardening in citrus stems. AB - Stem cold hardening developed to different levels in citrus types tested in controlled environments. Exotherms indicated ice spread was more uniform and rapid in unhardened than in cold-hardened stems. All attempts to inhibit the functioning of citrus leaves resulted in less cold hardening in the stems. Citrus leaves contribute a major portion of cold hardening in the wood. PMID- 16659341 TI - Effects of Abscisic Acid and Benzyladenine on Fruits of Normal and rin Mutant Tomatoes. AB - Since ethylene application did not induce ripening in detached fruits of the nonripening mutant rin we initiated studies to determine possible involvement of other hormones. We proposed that the lack of ripening in mutant rin tomato fruit may result from a lack of abscisic acid or from excessive endogenous levels of cytokiuin. Application of abscisic acid (3 x 10(-5)m and 10(-3)m) to detached fruits of a normal strain (Lycopersicon esculentum Mill. cv. ;Rutgers') reduced the time to initiate ripening by about 50%. This acceleration of the onset of ripening appeared not to be due to an increased rate of ethylene production. Abscisic acid did not alter respiration or ethylene production or induce ripening in rin fruit. Ripening in Rutgers fruit was not influenced by treatment with 6 benzyladenine (4.44 x 10(-6)m, 4.44 x 10(-5)m or 1.8 x 10(-4)m). Fruits of the mutant rin showed no response to exogenous BA. However, senescence rates of leaf disks of both Rutgers and rin were significantly inhibited by as little as 10( 7)m exogenous benzyladenine. The results are discussed in relation to previous studies of the physiology of rin fruits and it is concluded that endogenous levels of ABA and cytokinins do not account for the lack of ripening in rin fruit. PMID- 16659342 TI - Ethylene production and respiration in aging leaf segments and in disks of fruit tissue of normal and mutant tomatoes. AB - Leaf segments of tomato plants (Lycopersicon esculentum Mill.) of a normal strain and of two nonripening mutants rin and nor were aged in darkness. Respiration in leaf segments of all strains followed a climacteric-like pattern which was accompanied by a similar pattern of ethylene production. l-Methionine-U-(14)C vacuum-infiltrated into leaf segments at the beginning of the climacteric-like rise in respiration was metabolized to ethylene and CO(2) during the subsequent 48 hours to about the same extent in all strains. Pericarp disks of immature fruits of all strains also metabolized l-methionine-U-(14)C to ethylene and CO(2) to about the same extent during the first 48 hours following cutting and vacuum infiltration. Conversion of methionine to ethylene in disks was much more efficient than in aging leaf segments. The apparent capacity for increased production of ethylene in aging leaf segments and in response to wounding in pericap disks of rin and nor is contrasted with the absence of a respiratory climacteric and an associated large increase in ethylene production during natural aging of intact fruits of these two strains. PMID- 16659343 TI - Possible Involvement of alpha-Farnesene in the Development of Chilling Injury in Bananas. AB - The levels of the sesquiterpene hydrocarbon, alpha-farnesene were found to be higher in green bananas (Musa accuminata AAA) that were more susceptible to the development of chilling injury. The levels of alpha-farnesene were further increased by storage of the fruit at low temperature. PMID- 16659344 TI - Propylene-a competitor of ethylene action. AB - Propylene competed with the ethylene-induced reduction in length growth of the epicotyl of the etiolated garden pea (Pisum sativum L. cv. Alaska). These results constitute further evidence that ethylene acts by attaching itself loosely to a site. PMID- 16659345 TI - Structure of functional soybean sieve elements. AB - Soybean (Glycine max cv. Bragg) petiolar tissue containing translocated (14)C sucrose was quick frozen, freeze-substituted in acetone or propylene oxide and embedded in Epon. This procedure allowed cytological observations on sieve elements whose functional condition could be verified by microautoradiography. Sieve elements and companion cells were essentially free of ice damage. Aside from a P-protein crystal, the central portion of the sieve tube lumen was devoid of stainable content except in the vicinity of sieve plates. Various sized clumps of stacked endoplasmic reticulum (ER) lined the wall. Superficially, the ER "membranes" seemed to consist of parallel arrays of 100 A protein fibrils. Although that possibility could not be excluded, it seemed more likely that the fibrils were actually between ER cisternae and that the lipoprotein ER membrane could not be detected readily due to the loss of lipids during tissue preparation. The amount and distribution of proteinaceous material in the vicinity of sieve plates was variable but, when present, still consisted almost entirely of 100 A fibrils organized into membrane-like arrays. Stacks of ER in various degrees of disorganization and a few 100 A fibrils were found near sieve plates, with some fibrils extending through the pores. However, most (70%) of the sieve plate pores were essentially free from obstruction. The observations favor an osmotically generated pressure flow mechanism of translocation in soybean. PMID- 16659346 TI - Effects of abscisic Acid on growth, RNA metabolism, and respiration in germinating bean axes. AB - The effect of abscisic acid on growth, respiration, the ATP pool, and rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination has been measured in experiments where the duration of abscisic acid application and its concentration have been varied. At concentrations from 10(-7) to 10(-4)m, abscisic acid inhibits synthesis of RNA with maximal inhibition (80%) at 10(-5)m. RNA synthesis is inhibited by abscisic acid at all times examined (12, 18, and 24 hours), but the extent of inhibition is maximal at 18 hours. In 18-hour axes RNA synthesis is inhibited 42%, ATP pool size is reduced 3%, and O(2) consumption is decreased by 6% after 75 minutes of abscisic acid treatment. Inhibition of RNA synthesis is complete by 2 hours of treatment with abscisic acid, and recovery to near control levels occurs by the 3rd hour after removal from abscisic acid. PMID- 16659347 TI - Benzyladenine reversal of abscisic Acid inhibition of growth and RNA synthesis in germinating bean axes. AB - The effect of benzyladenine on growth, ATP pool size and specific radioactivity, and the rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination were measured in experiments where the duration of benzyladenine application and its concentration were varied. Maximum promotion of growth (25%) occurs at 10(-5)m benzyladenine. Maximum promotion of RNA synthesis (44%) occurs at 10(-5)m benzyladenine. Benzyladenine has little effect on the size or specific radioactivity of the ATP pool. Benzyladenine can completely counteract abscisic acid inhibition of growth and RNA synthesis, and these reversals are measurable in 2 hours. GA(1) and GA(3) do not promote growth or counteract abscisic acid inhibition of growth in germinating bean axes in these experiments. PMID- 16659349 TI - Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth. AB - Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea. PMID- 16659348 TI - Enzymes of carbohydrate metabolism in the developing rice grain. AB - The levels of reducing and nonreducing sugars, starch, soluble protein, and selected enzymes involved in the metabolism of sucrose, glucose-1-P, and glucose nucleotides were assayed in dehulled developing rice grains (Oryza sativa L. line IR1541-76-3) during the first 3 weeks after flowering. The level of reducing sugars in the grain was highest 5 to 6 days after flowering. The level of nonreducing sugars and the rate of starch accumulation were maximum 11 to 12 days after flowering, when the level of soluble protein was also the highest. The activities of bound and free invertase, sucrose-UDP and sucrose-ADP glucosyltransferases, hexokinase, phosphoglucomutase, nucleoside diphosphokinase, and UDP-glucose and ADP-glucose pyrophosphorylases were high throughout starch deposition, and were maximum, except for nucleoside diphosphokinase which did not increase in activity, between 8 and 18 days after flowering. Soluble primed phosphorylase and ADP glucose-alpha-glucosyltransferase (starch synthetase) were both present during starch accumulation. Phosphorylase activity was at least 2 fold that of soluble starch synthetase but the synthetase followed more closely the rate of starch accumulation in the grain. The activity of starch synthetase bound to the starch granule also increased progressively with increased starch content of the grain. PMID- 16659350 TI - Preadaptation of protein synthesis in wheat seedlings to high temperature. AB - The optimum temperature of protein synthesis in wheat seedlings (Triticum aestivum L.), measured as (14)C-leucine incorporation, depends on the growing temperature. Plants grown at reduced temperature (4 C) reach their optimum at 27.5 C, whereas plants kept at 36 C have the highest rate of protein synthesis at 35 C. The transition is gradual. The activation energy of protein synthesis for seedlings grown at medium or reduced temperature is lower (about 11 kcal/mole), than for plants grown at higher temperatures (15 keal/mole). The decline of the rate of protein synthesis beyond the temperature optimum is also affected by the growth temperature; only plants kept at 30 or 36 C show a sharp decrease with increasing slope; plants kept at 4, 10, and 20 C exhibit a linear and comparatively moderate decline. PMID- 16659351 TI - Leaf Age as a Determinant in Stomatal Control of Water Loss from Cotton during Water Stress. AB - The stomatal resistance of individual leaves of young cotton plants (Gossypium hirsutum L. var. Stoneville 213) was measured during a period of soil moisture stress under conditions of constant evaporative demand. When plants were subjected to increasing soil water stress, increases in stomatal resistance occurred first on the lower leaves and the stomata on the upper surfaces were the most sensitive to decreasing leaf-water potential. Stomatal closure proceeded from the oldest leaves to the youngest as the stress became more severe. This apparent effect of leaf age was not due to radiation differences during the stress period. Radiation adjustments on individual leaves during their development altered the stomatal closure potential for all leaves, but did not change the within-plant pattern. Our data indicate that no single value of leaf water potential will adequately represent a threshold for stomatal closure in cotton. Rather, the stomatal resistance of each leaf is uniquely related to its own water potential as modified by age and radiation regime during development. The effect of age on stress-induced stomatal closure was not associated with a loss of potassium from older leaves. Increases in both the free and bound forms of abscisic acid were observed in water-stressed plants, but the largest accumulations occurred in the youngest leaves. Thus, the pattern of abscisic acid accumulation in response to water stress did not parallel the pattern of stomatal closure induced by water stress. PMID- 16659352 TI - Ribulose 1,5-Diphosphate Carboxylase Synthesis in Euglena: II. Effect of Inhibitors on Enzyme Synthesis during Regreening and Subsequent Transfer to Darkness. AB - Dark-grown Euglena gracilis Klebs strain Z Pringsheim cells, which have been partially regreened in the light, show a striking, continued synthesis of the chloroplast enzyme ribulose 1,5-diphosphate carboxylase on transfer back into darkness. This dark synthesis of the enzyme was completely prevented by the addition of 15 mug/ml of cycloheximide to the culture medium but was unaffected, for at least 8 hours, by the addition of 1 mg/ml of d-threo-chloramphenicol. The addition of either cycloheximide or d-threo-chloramphenicol to dark-grown cultures at the onset of illumination completely inhibited the light-induced synthesis of ribulose 1,5-diphosphate carboxylase. When cells which had been illuminated in the presence of d-threo-chloramphenicol, and hence were unable to synthesize ribulose 1,5-diphosphate carboxylase, were transferred to darkness in the absence of this inhibitor, synthesis of the carboxylase then occurred. Dark grown cells which had been illuminated in the presence of cycloheximide failed to synthesize the enzyme when placed in the dark in the absence of cycloheximide. The addition of 5-fluorouracil to regreening cultures to prevent light-induced transcriptional steps completely blocked the synthesis of ribulose 1,5 diphosphate carboxylase. PMID- 16659353 TI - Occurrence of Pyruvate Orthophosphate Dikinase in the Succulent Plant, Kalanchoe daigremontiana Hamet. et. Perr. AB - Pyruvate orthophosphate dikinase was detected from Kalanchoe daigremontiana Hamet. et. Perr., a succulent plant with crassulacean acid metabolism. Enzyme activity was similar to that of maize extracts. Two enzymes demonstrating pyruvate orthophosphate dikinase activity from K. daigremontiana and Zea mays were found to be partially identical from enzyme-inhibition and immunoprecipitin tests with maize enzyme antiserum. A time course study demonstrated that pyruvate orthophosphate dikinase activity in leaf extracts was dependent upon exposure of leaves to light. PMID- 16659354 TI - Glucomannan Biosynthesis Catalyzed by Pisum sativum Enzymes. AB - The results of molecular weight studies, structural analysis of the [(14)C]polysaccharides, and enzymic properties indicate that the Pisum sativum guanosine diphosphosphate glucose: glucosyltransferase is an enzymic component involved in the biosynthesis of glucomannan chains. The properties of the Pisum sativum particulate enzyme are essentially identical to the glucomannan synthetase obtained from Phaseolus aureus. Also present in the particulate preparation is an enzyme which catalyzes the formation of a [(14)C]mannolipid, using guanosine diphosphate-[(14)C]mannose as a substrate. The [(14)C]mannolipid is hydrolyzed by treatment with 0.012 m HCl, but is stable to treatment with 0.09 m NaOH. The formation of the [(14)C]mannolipid is apparently reversed by guanosine diphosphate, but not by guanosine monophosphate. The chromatographic mobility of the [(14)C]mannolipid is identical to that of a similar mannolipid synthesized by a Phaseolus aureus enzyme. PMID- 16659355 TI - Effects of osmotic gradients on vacuolar malic Acid storage: a basic principle in oscillatory behavior of crassulacean Acid metabolism. AB - Malate synthesis by CO(2) dark fixation and malate accumulation in the vacuoles of leaf slices of Kalanchoe daigremontiana Hamet et Perrier, a plant performing crassulacean acid metabolism, occurs only in external solutions where the osmotic pressure difference between the cells and the medium is low. Conversely, malate loss from the vacuoles depends on a high osmotic pressure difference between the cells and the medium and is observed in media of low osmotic pressure. This suggests that the diurnal oscillations of malate levels in crassulacean acid metabolism leaf cells are regulated by osmotic gradients. These findings support a model which is introduced to explain how the rhythm of crassulacean acid metabolism may function in the intact plant. PMID- 16659356 TI - Ribosomal Changes during Induction of Cold Hardiness in Black Locust Seedlings. AB - Protein synthesis has been implicated in the cold-hardening process. Ribosomes from cold hardy and nonhardy black locust (Robinia pseudoacacia L.) seedlings were compared to determine if cold acclimation is related to alteration of ribosomal structure. Ribosomal structure, as indicated by thermal melting profiles, appears to be altered during induction of hardiness. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicates at least 17 proteins from hardy seedlings that are different from those of nonhardy seedlings. These different proteins may be partially responsible for the different thermal melting profiles observed. PMID- 16659357 TI - Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls. AB - The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca(2+), kinetin, or ethylene. Both Ca(2+) and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca(2+), kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls. PMID- 16659358 TI - Comparative Study of the Carotenoid Composition of the Seeds of Ripening Momordica charantia and Tomatoes. AB - The total carotenoid concentration of the seeds of Momordica charantia rose about 100-fold from the immature to the ripe stage. The massive increase was almost exclusively attributable to lycopene, which accounted for 96% of the carotenoids of the ripe seeds. The carotenoid pattern of the seed was found to be drastically different from that of the pericarp. The seed, which contained fewer carotenoids, had a total concentration 12 times greater than that in the pericarp at the ripe stage. The acyclic lycopene selectively accumulated in the seed, whereas the cyclic carotenoids, cryptoxanthin, zeaxanthin and beta-carotene, were the principal pigments of the ripe pericarp. The seed of ripe tomatoes reflected the qualitative pattern of the whole fruit. The total carotenoid concentration was, however, much lower and the lycopene content was particularly low. beta-Carotene, having a comparatively high concentration, emerged as the major pigment of the seed. PMID- 16659359 TI - pH Dependence of the Km(CO(2)) of Ribulose 1,5-Diphosphate Carboxylase. AB - The Km(CO(2)) values of ribulose 1,5-diphosphate carboxylase in freshly ruptured spinach (Spinacia oleracea L.) chloroplasts and in the purified form isolated from spinach leaves were found to be pH dependent. Raising the pH of the assay solution produced a substantial decrease in the Km(CO(2)) of both enzyme systems. In freshly ruptured chloroplasts at pH 7.2 the Km(CO(2)) was 25 mum, at pH 8 it decreased to 19 mum, and at pH 8.8 a further decrease to 7 mum was found. With the purified enzyme at pH 7.2 the Km(CO(2)) was 147 mum, while the corresponding Km values for pH 8 and 8.8 were 34 and 15 mum CO(2), respectively. The latter figure approximates the physiological Km(CO(2)) of 10 mum estimated for photosynthesizing leaves and intact chloroplasts. The maximum velocity for both enzyme systems at optimum substrate levels was at pH 8, but the highest calculated rate of CO(2) uptake at atmospheric CO(2) levels occurred at pH 8.8. These results support the proposal that the light-induced efflux of protons out of the chloroplast stroma may be a major factor involved with the reported in vivo light activation of ribulose 1,5-diphosphate carboxylase. PMID- 16659360 TI - Effect of glucose on the induction of nitrate reductase in corn roots. AB - In Zea mays L., addition of glucose to the induction medium has no effect on the induction of nitrate reductase during the initial 3 hours either in root tips (0 10 mm) or mature root sections (25-35 mm). With longer times, higher levels of enzyme activity are recovered from both root segments when glucose is present in the incubation medium. The induction in root tips is saturated by 10 mm NO(3) ( ). Higher concentrations of NO(3) (-) are required for saturation in mature root sections. The response to glucose is seen over a wide range of external NO(3) (-) concentrations.Nitrate reductase activity is lost rapidly when nitrate is withdrawn from the induction medium. Additions of glucose do not prevent this loss. Additions of glucose have no effect on total uptake of NO(3) (-) by the root segments but they increase the anaerobic NO(2) (-) production in both root tips and mature root segments. This latter measurement is considered to be an estimate of an active NO(3) (-) pool in the cytoplasm. Thus the results show that glucose alters the distribution of NO(3) (-) within the root sections. This may be an important factor in controlling the in vivo stability of the enzyme or its rate of synthesis. PMID- 16659361 TI - Partial Purification and Properties of Ornithine Transcarbamoylase from Nostoc muscorum Kutzing. AB - Ornithine transcarbamoylase (carbamoyl phosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) has been partially purified from the blue-green alga Nostoc muscorum Kutzing, an organism in which the enzyme seems to be involved in a bicarbonate-fixing pathway leading to citrulline. Pertinent to possible regulation of this pathway, the enzyme shows hyperbolic substrate kinetics, has a molecular weight estimated at 75,000 daltons, and its catalytic capability is little influenced by a selection of metabolites that might conceivably act as regulators in vivo. Thus it seems unlikely that this enzyme is the control point for bicarbonate fixation. In terms of energy of activation (12.3 kcal/mole), size and Km for carbamoylphosphate, the Nostoc enzyme resembled preparations from liver and higher plants more than preparations from Streptococcus and Mycoplasma. The enzymes from Streptococcus and Mycoplasma are probably specialized for citrulline breakdown rather than citrulline synthesis. The Km for ornithine was 2.5 mm at a saturating concentration of carbamoylphosphate and the Km for carbamoylphosphate was 0.7 mm at an ornithine concentration of 2 mm. Ornithine was inhibitory at concentrations greater than 2 mm. Phosphate was a competitive inhibitor with respect to carbamoylphosphate. The pH optimum for citrulline synthesis was 9.5. PMID- 16659362 TI - Auxin transport inhibitors: fluorescein and related compounds. AB - Fluoresceins are shown to be effective inhibitors of indoleacetic acid transport as measured by the receiver agar block technique, eosin having the same order of activity as 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid, with fluorescein less effective. It is suggested that many of their characteristic effects on plants, especially those which involve auxin, are at least partially due to their effects on auxin transport. PMID- 16659363 TI - Promotion of softening and ethylene synthesis in bartlett pears by 3-methylene oxindole. AB - A purified preparation of 3-methylene oxindole (3-MeOx) was applied to Bartlett pears (Lyrus communis) by vacuum infiltration. The infiltrated fruit were kept at room temperature at atmospheric or at one-twentieth of an atmospheric tension. The rate of softening was markedly enhanced by the application of 0.1 and 1 mum 3 MeOx. At 10 mum 3-MeOx the promotive effect of the compound was diminished. All the employed concentrations of 3-MeOx exceeded the effect of applied ethylene. The enhancement of softening in fruit kept under hypobaric conditions suggests that the action of 3-MeOx is a direct one and not an indirect ethylene effect. 3 MeOx also showed stimulation in the onset of ethylene synthesis, shortening the time required to obtain the peak in ethylene synthesis from 5 days by the control to 3 days by 0.1 mum and 2 days by 1 mum of the applied compound. As with softening, 3-MeOx at 10 mum diminished the rate of ethylene synthesis.The results suggest that 3-MeOx could function as a senescence promoter in fruit. Also, since auxins retard ripening while 3-MeOx promotes ripening, the action of 3-MeOx may be considered as that of an auxin antagonist. The occurrence and the mode of action of 3-MeOx as a possible senescence factor in fruit are discussed. PMID- 16659364 TI - Evidence for Covalently Attached p-Coumaric Acid and Ferulic Acid in Cutins and Suberins. AB - p-Coumaric acid (4-hydroxycinnamic acid) and ferulic acid (4-hydroxy-3 methoxycinnamic acid) have been identified as constituents of cutin. Their reduction products were isolated from a phenolic fraction released from the cutin of the fruits of apple, peach, pear, and two varieties of tomato and apple leaf by treatment with LiAlH(4) or LiAlD(4). They were identified by combined gas chromatography and mass spectrometry. p-Coumaric acid was present in all samples of cutin (0.07-0.53% by weight), whereas only peach and pear cutin contained measurable amounts of ferulic acid (0.007% and 0.035%, respectively). Both p coumaric acid and ferulic acid were identified to be constituents of the insoluble material recovered after partial hydrolysis (12-42% loss) of cutin in 1 m NaOH at 80 C. A significant part (48%) of the p-coumaric acid contained in tomato cutin was contained in the insoluble material recovered after partial degradation (7.4%) of this cutin with 0.01 m NaOH. These data indicate that these phenolic components are tightly (possibly covalently) bound to cutin. Similar analysis of the phenolic fractions from the suberins of potato, sweet potato, turnip, rutabaga, carrot, and red beet revealed that they contained only ferulic acid (0.05-0.22%). Ferulic acid was identified as a constituent of the insoluble material recovered after partial hydrolysis of potato and beet suberins (34% and 32% loss, respectively) in 1 m NaOH at 80 C. A major part (65%) of the ferulic acid contained in potato suberin was contained in the insoluble material recovered after partial (26.8% loss) degradation of this suberin with 0.01 m NaOH. Ferulic acid appears to be tightly (probably covalently) bound to suberin. PMID- 16659365 TI - Endogenous Gibberellins of Pine Pollen: II. Changes during Germination of Pinus attenuata, P. coulteri, and P. ponderosa Pollen. AB - The endogenous gibberellins (GAs) of pollen of Pinus attenuata, P. coulteri, and P. ponderosa were bioassayed at hour 0, 3, 15, 24, 48 and 72 of germination. Dormant pollen showed relatively high GA activity throughout the elution spectrum (i.e. ranging from relatively nonpolar to highly polar). The maximum GA activity was obtained at hour 15 in more polar regions and especially in the zone corresponding to GA(3) (for P. attenuata estimated as 250 micrograms of GA(3)/kilogram pollen). It is probable that the "nonpolar" GAs present in high quantities in dormant pollen and in early stages of germination were converted to "more polar" GAs as germination progressed. The amount of all GAs decreased after hour 15 of germination and by hour 72 no GAs could be detected. Among the species tested P. attenuata showed the highest over-all GA activity. PMID- 16659366 TI - Control by Phytochrome of Cytoplasmic and Plastid rRNA Accumulation in Cotyledons of Mustard Seedlings in the Absence of Photosynthesis. AB - Light stimulates the accumulation of cytoplasmic and plastid rRNA in the cotyledons of mustard (Sinapis alba L.) seedlings. This effect is mediated by phytochrome. Continuous far red irradiation and brief red light pulses (the effect of which is reversible by brief far red light pulses) increase the amount of 1.3 x 10(6) + 0.7 x 10(6) and 1.1 x 10(6) + 0.56 x 10(6) molecular weight rRNA species. Large and small ribosomal subunit RNAs are maintained in a 1:1 molar ratio in both cytoplasm and plastids, irrespective of seedling age and light treatment. Continuous white fluorescent light (which saturates chlorophyll synthesis) is less effective than continuous far red light (essentially no chlorophyll synthesis) in producing the response, indicating that the accumulation of plastid rRNA is independent of the build-up of a functional photosynthetic apparatus. We conclude that the transcription of cytoplasmic and plastid rRNA cistrons is under phytochrome control. PMID- 16659367 TI - Studies with detached lupin root nodules in culture: I. Maintenance and induction of acetylene reduction activity. AB - A method has been developed for culturing detached nitrogen-fixing root nodules of lupin (Lupinus angustifolius L.) on a simple nutrient medium. Under the best conditions devised, the acetylene reduction activity of mature detached nodules was maintained at 10 to 25 nmoles of ethylene hr(-1) mg(-1) fresh weight for 3 days. Under the same culture conditions, immature nodules increased their acetylene reduction activity from 0.01 nmole or less to about 1 nmole hr(-1) mg( 1) fresh weight. PMID- 16659368 TI - Comparison between Biosynthesis of ent-Kaurene in Germinating Tomato Seeds and Cell Suspension Cultures of Tomato and Tobacco. AB - Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures derived from tobacco callus (Nicotiana tabacum L.), tomato callus (Solanum lycopersicum L.), and in germinating tomato seeds. Incubation of extracts derived from the two cell cultures with either isopentenyl pyrophosphate (14)C or with (14)C-labeled mevalonate, followed by alkaline phosphatase hydrolysis, resulted in the formation of trans-geranylgeraniol-(14)C and trans farnesol-(14)C. The corresponding pyrophosphates of trans-geranyl-geraniol-(14)C and trans-farnesol-(14)C were also detected. No detectable amount of ent-kaurene (14)C was produced by these enzymatic preparations when trans-geranylgeranyl (14)C pyrophosphate served as substrate. However, copalyl-(14)C pyrophosphate served as a substrate for the production of ent-kaurene. Cell-free extracts derived from germinating tomato seeds catalyzed the formation of ent-kaurene (14)C from mevalonate-(14)C, isopentenyl-(14)C pyrophosphate, trans geranylgeranyl-(14)C pyrophosphate, and copalyl-(14)C pyrophosphate. PMID- 16659369 TI - Suaeda monoica, a C(4) Plant without Typical Bundle Sheaths. AB - Suaeda monoica Forssk. ex J. F. Gmel was found to possess the C(4) pathway of photosynthesis. The succulent leaves of Suaeda lack a green bundle sheath formation but have a layer of chlorenchyma, containing large and centripetally arranged chloroplasts, which surrounds the water tissue. We suggest that the proximity of a chlorenchymatous cell layer to the vascular bundles is not necessary for the operation of the C(4) pathway. PMID- 16659370 TI - A Mathematical Treatment of Rate Data Obtained in Biological Flow Systems under Nonsteady State Conditions. AB - The problem of determining gas exchange rates from flow system data under nonsteady state conditions is analyzed. A correction factor is presented for obtaining constant rates under nonsteady state conditions. A general formula for obtaining any rate under nonsteady state conditions is also given. Turnover time is defined and discussed in terms of the mathematics presented. The origins of nonsteady states and steady states in flow systems are discussed, as are some of the experimental advantages of working under nonsteady state conditions. PMID- 16659371 TI - The isozymic similarity of indoleacetic Acid oxidase to peroxidase in birch and horseradish. AB - The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found. PMID- 16659372 TI - Technique for the Determination of the Rate of Ethylene Production by Pseudomonas solanacearum. AB - A tube culture system was designed for measurement of ethylene evolved by the phytopathogenic bacterium, Pseudomonas solanacearum. The system consisted of 10 glass tubes joined together in series and coated on the inside surface with a dextrose-peptone-casamino acids agar medium. The system provided a large surface for bacterial growth in relation to the volume of air. The system was seeded with a bacterial suspension (7 x 10(8) cells/ml) drawn through all the tubes by vacuum applied at one end and was then placed in a water bath at 30 C. Air was pumped through the system at 3 ml/min; the outlet was connected directly to the inlet port of a gas sampling loop and ethylene in the sample was determined by gas chromatography.Maximum rate of ethylene production for a fluidal, virulent isolate of P. solanacearum (K60) was 5.5 x 10(-9) moles/min and occurred at the end of lag phase and beginning of stationary phase. Three other fluidal isolates produced ethylene at relatively low rates (2.4-6.4% that of K60). Avirulent, butyrous variants of these isolates grew as well as the virulent forms in most cases, but ethylene production rates per cell were much lower for the avirulent than for the virulent forms. Loss of virulence appears to be accompanied by lower ethylene production.Peak CO(2) production (14.5 mumoles/min) and O(2) consumption (11.7 mumoles/min) for isolate K60 also occurred at the time when the bacterial culture was entering stationary phase. The concentrations of O(2) (11%) and CO(2) (11%) in air present at this time were thought to be neither limiting nor inhibitory to bacterial growth. PMID- 16659373 TI - Nitrate uptake and induction of nitrate reductase in excised corn roots. AB - The characteristics of nitrate uptake and induction of nitrate reductase were studied in excised roots of corn (Zea mays L.). Upon initial exposure to nitrate, the low initial rate of nitrate uptake gradually increased until a steady uptake rate was achieved in 1 to 2 hours depending on the NO(3) (-) concentration. The pattern was observed over a wide range (0.2-5 mm) of nitrate concentrations and was independent of the accompanying cation.The nitrate uptake pattern as a function of increasing external nitrate concentrations (0.2-50 mm) followed saturation type kinetics. The reciprocal plot of the data was not linear but hyperbolic, indicating that more than one Km for nitrate uptake can be resolved from the data. This suggests the existence of either one carrier system with changing kinetic constants or the existence of dual uptake systems. The pattern of induction of nitrate reductase was coincident with the pattern of nitrate uptake as a function of time and increasing nitrate concentrations. The rate of induction of nitrate reductase was regulated by the rate of nitrate flux.Washing the roots for 2 hours enhances nitrate uptake by 2.5-fold over the nonwashed tissue. The presence of nitrate in the washing solution leads to further (3.5 fold over control) increases in the rate of nitrate uptake supporting the contention that nitrate plays a specific role in the induction of the inducible nitrate carrier independent of the washing effect. PMID- 16659374 TI - Stoichiometric Correlation of Malate Accumulation with Auxin-dependent K-H Exchange and Growth in Avena Coleoptile Segments. AB - The action of auxin in the promotion of growth has been suggested in the literature to depend on cell wall acidification. In a former investigation by the present authors the electrochemical balance in auxin-induced proton extrusion was shown to be maintained by potassium net uptake. The present paper reports data demonstrating that the elongation of Avena coleoptile segments is accompanied by an accumulation of malate, which is stoichiometrically correlated with potassium uptake. We concluded that this malate accumulation is required in a mechanism regulating intracellular pH. PMID- 16659375 TI - Energy Supply and Light-enhanced Chloride Uptake in Wheat Laminae. AB - The light-supported component of (36)Cl uptake from 5 mM K(36)Cl by green laminae, either chopped or vacuum-infiltrated, of Triticum aestivum L. seedlings has been determined by subtraction of dark uptake values from light uptake values and the energy sources for the uptake elucidated on the basis of the effects of 3 (3,4-dichlorophenyl)-1,1 dimethylurea (DCMU), carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), antimycin A(1) (AA), and N(2) on light and dark uptake. The light-supported Cl(-1) uptake is shown to be partially inhibited by DCMU or AA but unaffected or stimulated by FCCP or N(2). There is no additive effect on inhibition caused by DCMU + N(2) or FCCP + AA but there is an added inhibition caused by DCMU + AA, DCMU + FCCP, and by FCCP or AA in anaerobic conditions. The effect of these inhibitors on photosynthetic gas exchange of chopped tissue has also been determined. On the basis of the results it is concluded that the uptake of Cl(-), supported in the dark by oxidative phosphorylation, is enhanced by light and may be supported by cyclic and non-cyclic electron-flow energy. Uptake is not obligatorily linked to any one energetic pathway and there may be switching from one source to another. PMID- 16659376 TI - Effect of Temperature on Respiration of Mitochondria and Shoot Segments from Cold hardened and Nonhardened Wheat and Rye Seedlings. AB - The effect of temperature on respiration of mitochondria and tissue segments from three wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar grown at 2 and 24 C has been examined. Discontinuities in Arrhenius plots of respiratory activity against temperature were observed for mitochondria and tissue segments from seedlings grown at both temperatures. The rates of respiration decreased abruptly below the transition temperatures, resulting in increased energy of activation values for respiration. Transition temperatures were observed from 6 to 10 C during tissue segment respiration, and from 10 to 14 C during respiration by isolated mitochondria. Respiratory control and efficiency of phosphorylation were not affected markedly by either reaction temperature or growth temperature of the seedlings. No correlation was observed between the cold hardiness of the cultivars and the temperature at which structural transitions occurred in the mitochondria. Dry matter content of the seedlings increased markedly during growth at 2 C, but no appreciable changes in the levels of mitochondrial protein were observed. The results support the view that changes other than fatty acid unsaturation are involved in the abrupt change in mitochondrial membrane properties at low temperature. PMID- 16659377 TI - Determination of unfrozen water in winter cereals at subfreezing temperatures. AB - The freezing of water in acclimated and nonacclimated cereals was studied using pulsed nuclear magnetic resonance spectroscopy. The quantity of unfreezable water per unit dry matter was not strongly dependent on the degree of cold acclimation. In contrast, the fraction of water frozen which was tolerated by nonacclimated winter cereals and by an acclimated spring wheat (Triticum aestivum L.) was less than in acclimated hardy cereals. The freezing curves had the following form:L(T) = L(0)DeltaTm/T + KL(T) and L(0) are liquid water per unit dry matter at T and 0 C, respectively. DeltaTm is the melting point depression and K is the liquid water which does not freeze. PMID- 16659378 TI - Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons. AB - The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.The results from these studies provide pertinent information toward understanding the succession from glyoxysomal to peroxisomal metabolism in cotyledons of fatty seedlings. The coexistence of two separate microbody types functioning at different stages of development apparently is not the case. The localizations of both marker enzymes within one microbody type strongly suggest that the metabolic transition involves a change in enzyme complement within an ongoing population of microbodies. PMID- 16659379 TI - Effect of oxygen on photosynthesis by spinach leaf protoplasts. AB - The photosynthetic CO(2) fixation by spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts was inhibited by substituting an atmosphere of N(2) with one of either air (21% O(2)) or 100% O(2). The inhibitory effect of 100% O(2) was greater than that of air. The mode of inhibition by 100% O(2) and air was competitive with respect to CO(2); Ki(O(2)) value was 0.32 mM at pH 7 and 0.28 mM at pH 8.5 The labeling patterns of compounds in protoplasts exposed to (14)CO(2) in light after transferring them from N(2) to O(2) atmospheres were examined. There was no detectable (14)CO(2) incorporation into glycolate under anaerobic and O(2) atmospheres; a more marked labeling of glycine occurred under an oxidative environment compared to that under the anaerobic condition, presumably because of a rapid transformation of glycolate to glycine in the protoplasts. PMID- 16659380 TI - Ozone-induced Loss of Intracellular Potassium Ion from Chlorella sorokiniana. AB - The unicellular algae Chlorella sorokiniana was used as a model system to investigate the interaction of ozone with plant cell membranes. Ozone induces K(+) leakage from Chlorella sorokiniana similar to the electrolytic loss observed from many higher plants under stressful conditions. The kinetics of this leakage indicate that ozone initially interacts reversibly (within sec) with sites on membranes allowing a passive efflux of K(+). This efflux ceases within minutes after the ozone stress is removed. This return to normal efflux is very temperature dependent. High intracellular osmolarity seems to be an important criterion of susceptibility to ozone injury in this model system, since rates of ozone-induced K(+) leakage are less when the external osmotic potential is decreased by suspension of the cells in mannitol. Cell interaction with ozone is further complicated by a saturating-type dependence of the K(+) efflux upon ozone concentration within the medium. PMID- 16659381 TI - Photoactivation of NAD Kinase through Phytochrome: Phosphate Donors and Cofactors. AB - The specificities of phosphate donors and the effects of metal chelating agents and divalent metal ions on NAD kinase activation by phytochrome-far red-absorbing form (Pfr) were examined. ATP was the most efficient phosphorylating agent. Uridine 5'-triphosphate, cytidine 5'-triphosphate (CTP), inosine 5'-triphosphate, and guanosine 5'-triphosphate in this order caused significant phosphorylation in the dark. Under red light, striking photoactivation of NAD kinase was obtained with ATP and subsequently CTP.In the presence of exogenous Mg(2+), which is required for NAD kinase activity, alpha-nitroso-beta-naphthol, cyanide, and dimethylglyoxime, strongly inhibited the activation by red light without affecting the level of NAD kinase in the dark.Of the divalent cations tested with the KCN-treated phytochrome preparation, only Co(2+) was effective for photoactivation of NAD kinase. Even when Mg(2+), an essential component of NAD kinase, was added to the assay system, the further addition of Co(2+) was required for the activation of NAD kinase by Pfr. PMID- 16659382 TI - Endogenous abscisic Acid levels in germinating and nongerminating lettuce seed. AB - The concentrations of abscisic acid in Grand Rapids lettuce (Lactuca sativa L.) seeds imbibed under conditions which promote or inhibit germination were determined by electron capture-gas chromatography. The concentration of abscisic acid in dry seeds was 12 to 14 nanograms per 100 milligrams. During 24-hour imbibition, the abscisic acid content diminished more rapidly during conditions which allow germination (25 C in light) than in conditions which inhibited germination (35 C in light or darkness at 25 C). A decrease in endogenous levels of abscisic acid was not always correlated with germination. PMID- 16659383 TI - Red-Far Red Reversible Effect on Polysome Formation in the Embryos of Pinus thunbergii Seeds. AB - Polysome formation in the embryos of Pinus thunbergii seeds was studied. Free ribosomes were dissociated to smaller subunits in a high salt buffer, but the complex ribosomes were not. The free ribosomes could be distinguished from monomer ribosomes derived from polysomes after RNase treatment. The monomer ribosomes present in the embryos of the dark-imbibed seeds were predominantly free ribosomes; very small quantities of polysomes could be detected in the embryos from dark-imbibed seeds. Such polysomes remained at a very low level during dark imbibition at least for a month. The level of polysomes increased 4 hours after a brief exposure to red light. The effect of red light on polysome formation was partially reversed when followed by far red light irradiation. PMID- 16659384 TI - Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls. AB - Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg(2+), the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg(2+), the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg(2+), phytochrome was associated with a "heavy" plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction. PMID- 16659385 TI - Auxin: its role in genetic tumor induction. AB - Seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown on nutrient medium supplemented with indoleacetic acid (IAA) and scored at regular intervals for the incidence of tumor formation. IAA at 2 x 10( 5)m caused a significant reduction in the rate of tumor formation. Plants were also grown on nutrient medium under two different intensities of illumination, and the endogenous level of IAA was determined in 23-day-old seedlings. Those grown under 2000 ft-c of illumination had a higher incidence of tumors and a significantly lower level of endogenous IAA than those grown under 500 ft-c of illumination. A correlation in time between decline in the endogenous level of IAA and onset of tumor formation was demonstrated in greenhouse-grown plants. PMID- 16659386 TI - Promotion of Sorghum Callus Growth by the s-Triazine Herbicides. AB - Growth-promoting action of simazine and other s-triazine herbicides was detected by the use of sorghum (Sorghum bicolor [L]. Moench) callus tissue and the chlorophyll retention test. Soil application of simazine [2-chloro-4, 6 bis(ethylamino)-s-triazine] at sublethal levels nearly doubled the growth promoting action of sorghum root exudates. Treated plants yielded up to 26% more total protein than untreated plants. This indicated that the level of callus growth-promoting action in the root exudate of the plant has a positive effect on its final total protein yield and confirms a positive effect of simazine on total protein content in certain instances. The results may provide a new understanding of the mode of action of s-triazines applied at sublethal levels in increasing protein content and certain enzymic activities of treated plants. It is speculated that the growth-promoting action of these herbicides is hormonal in nature and most likely kinetin-like. PMID- 16659387 TI - Photoautotrophic growth and photosynthesis in tobacco callus cells. AB - Haploid tobacco (Nicotiana tabacum L.) cell cultures derived from quite different cultivars have been grown photoautotrophically in medium lacking sucrose and with 1.6 mum naphthaleneacetic acid and 1.5 mum isopentenylaminopurine. Cells were grown for 5 months on agar medium in Petri plates in air with dry weight increases of 1.5- to 3-fold per month. Callus cells were also grown photoautotrophically for at least three consecutive transfers 3 weeks apart in shallow liquid medium in horizontally placed gas-washing bottles where they were gassed continuously with air or air enriched with CO(2). Raising the CO(2) level in the air surrounding the cells increased the growth rate, and after about 3 weeks in 1% CO(2) the dry weight was approximately 3-fold greater than the inoculum. Growth rates remained about the same after each consecutive transfer. Autotrophic growth with this regime is not restricted to specific clones or cultivars.Photosynthetic measurements in an atmosphere containing (14)CO(2) established that rates of CO(2) assimilation in the callus cells at high CO(2) levels were similar to those of leaves on a chlorophyll basis, but were much slower on a fresh weight basis. Photosynthetic light saturation was achieved at an irradiation of about 125 mueinsteins m(-2) sec(-1) (400-700nm). The availability of photosynthetically dependent haploid cells provides an opportunity to select photosynthetic mutations which can be expressed in plants regenerated from these cells. PMID- 16659388 TI - Effect of gibberellic Acid on the plasticity and elasticity of Avena stem segments. AB - Extensibility characteristics of Avena stem segments treated with gibberellic acid (GA) were investigated in living internodes using a microgrowth method and in partially extracted cell walls subjected to Instron extensometer analysis. Both techniques showed that treatment with GA greatly increases internodal plasticity, but has virtually no effect on internodal elasticity. The increase in plasticity occurred 1 to 2 hours after the initiation of hormone treatment, which is similar to the time of onset of GA-enhanced growth and cell wall synthesis. Cycloheximide was shown to inhibit the effect of GA on plasticity. PMID- 16659389 TI - Effect of Altered pO(2) in the Aerial Part of Soybean on Symbiotic N(2) Fixation. AB - Dry matter accumulation, nitrogen content and N(2) fixation rates of soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to a pO(2) of 5, 10, 21, or 30% and a pCO(2) of 300 mul CO(2)/l or a pO(2) of 21% and a pCO(2) of 1200 mul CO(2)/l during the complete growth cycle were measured. Total N(2)[C(2)H(2)] fixed was increased by CO(2)/O(2) ratios greater than those in air and was decreased by ratios smaller than those in air; the effects on N(2) fixation of decreased pO(2) or elevated pCO(2) were quantitatively similar during the period of vegetative growth. Decreased pO(2) produced a smaller increase then elevated pCO(2) during the reproductive period, presumably because of the decreased sink activity of the arrested reproductive growth under subambient pO(2). At a pO(2) of 5% and a pCO(2) of 300 mul CO(2)/l total N(2) fixed was increased 125% and per cent nitrogen content in the vegetative parts was increased relative to air while that in the seed was decreased. Dry matter production was increased and reproductive growth was arrested as previously reported for plants receiving only fertilizer nitrogen. At a pO(2) of 30% and a pCO(2) of 300 mul CO(2)/l total N(2) fixed was decreased 50% and per cent nitrogen content in the vegetative part was increased relative to air while that in the reproductive structures was unaffected. Dry matter production was similarly decreased in both vegetative and reproductive structures. These effects of altered pO(2) in the aerial part on N(2) fixation are consistent with the hypothesis that the amount of photosynthate available to the nodule may be the most significant primary factor limiting N(2) fixation while sink activity of the reproductive structures may be a secondary factor. PMID- 16659390 TI - Cellulase Activity, Endogenous Abscisic Acid, and Ethylene in Four Citrus Cultivars during Maturation. AB - At maturity, the fruit of two early maturing orange cultivars, Hamlin and Pineapple (Citrus sinensis [L.] Osbeck), contained more ethylene and abscisic acid than the late maturing Valencia and Lamb Summer (C. sinensis [L.] Osbeck) cultivars. Ethylene (up to 95 nl/l in internal atmosphere) and abscisic (50 mug/kg dry weight flavedo) increased most rapidly in Pineapple, leading to increased cellulase activity and loosening of the fruit. Fruit of the two late maturing cultivars contained less than 25 nl/1 ethylene and 40 mug abscisic acid/kg dry weight of flavedo at peak maturity. Cellulase activity and loosening of the fruit of these late maturing cultivars was slight. PMID- 16659391 TI - Temperature regulation of germination in crimson clover seeds. AB - Seeds of Dixie crimson clover (Trifolium incarnatum cv. Dixie) completed germination in 36 hours at 20 C. At 10 C germination was delayed by 24 hours. At 30 C only 20% germinated and the rest remained viable for a long time but not germinable. Different patterns of adenylate energy state and zymograms of acid phosphatase and esterase were observed from seeds grown under the three temperatures for 24 hours. Varied specific activities of protease, alpha-amylase, ATPase, RNase, acid phosphatase, glutamine synthetase, and fumarase were also found. Protein-synthesizing ability was proportional to temperature. These data indicate that temperature regulates seed germination at multiple sites. PMID- 16659392 TI - Distribution and nonphotochemical transformation of phytochrome in subcellular fractions from pisum epicotyls. AB - In etiolated pea (Pisum sativum L. cv. Alaska) shoots about 3% of the total extractable phytochrome was found in the mitochondrial fraction and about 4.5% in the microsomal fraction, while over 70% was soluble in the 105,000g supernatant. The value of Delta(DeltaA) per milligram of protein was significantly higher in the 105,000g supernatant than in these particulate fractions. The percentage conversion of Pr to Pfr was approximately proportional to the total dose of red light in every subcellular fraction tested, unless the dose approached a saturation level. After a brief irradiation of intact shoots with red light at 26 C, each subcellular fraction showed different patterns of dark transformation in vivo at 26 C; that is, the amount of the particulate-bound phytochrome increased immediately after the irradiation, and a reversion of Pfr to Pr was indicated for the first 2 hr in the 12,000g supernatant, but not at all in the mitochondrial and microsomal fractions. The amounts of Pr in the mitochondrial and microsomal fractions did not change during the dark incubation, while those in the 12,000g supernatant increased with time. Similar results were obtained with apical shoot segments after exposure to red light at 0 C and a subsequent dark incubation on moist filter paper at 26 C. PMID- 16659393 TI - Heritable Variation in a Polypeptide Subunit of the Major Storage Protein of the Bean, Phaseolus vulgaris L. AB - Electrophoretic analysis of the major seed protein, G1 globulin, from four strains of Phaseolus vulgaris L. revealed a three-banded pattern for two strains having a high methionine content (BBL 240 and PI 302,542). The other two strains (PI 207,227 and PI 229,815) known to have a lower seed methionine content, had a two-banded subunit pattern for the G1 globulin. Analytical ultracentrifugation confirmed that globulin from the two-banded strains underwent pH-dependent reversible dissociation similar to that previously found for a three-banded cultivar; additionally, the protomer molecular weight showed that three subunits of about 50,000 molecular weight each were present in the G1 globulin of the two banded strain. Gel patterns of G1 globulin from the two strains used as parents, BBL 240 and PI 229,815, showed differences in the largest subunit, which appeared as either a 53,000 molecular weight polypeptide known to be present in the three banded strain, or as a shorter polypeptide having a molecular weight close to 47,000. Analysis of G1 protein from portions of single hybrid seeds showed a banding pattern intermediate between the two- and three-banded types. The subunit pattern from all seeds with intermediate-banded parents segregated in a manner consistent with that expected for control of the polypeptide by a single Mendelian gene. The remaining portions of the seeds were grown to confirm that they represented true crosses. The procedures used are essentially nondestructive, and can be used as a basis for selecting seeds having different protein characters. PMID- 16659394 TI - Cell-free Synthesis of the Major Storage Protein of the Bean, Phaseolus vulgaris L. AB - As seeds of the French bean (Phaseolus vulgaris, L. cv. Tendergreen) mature, a single protein, G1 globulin (analogous to legumin), represents the majority of protein synthesized. Washed polysomes extracted from developing cotyledons had little endogenous activity in amino acid incorporation, but on addition of cell free extracts from wheat germ, active incorporation was obtained, the level being similar to that with viral RNA as messenger. The Mg(2+) optimum for protein synthesis in the presence of bean polysomes was 6 mm compared with 4 mm for synthesis of viral polypeptides in the wheat germ system. Using T-2 toxin as an inhibitor, it was shown that 29% of the incorporation depended on initiation events. Electrophoretic analysis of the total polypeptide products of cell-free synthesis gave a disperse profile. Centrifugation to remove polysome-bound peptides after 60 minutes incubation gave a supernatant having a product with the same electrophoretic mobility as G1 globulin and containing 26% of the radioactivity present in the gel. Protein eluted from this peak was subjected to re-electrophoresis and shown to consist of the three polypeptide subunits characteristic of G1 globulin. PMID- 16659395 TI - Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography. AB - myo-Inositol 1-phosphate synthase (EC 5.5.1.4) is the enzyme which catalyzes the synthesis of the precursor for the myo-inositol oxidation pathway. Rice callus grown in suspension culture provides a good source of plant enzyme. Use has been made of a noncompetitive inhibitor to prepare an affinity column for this enzyme. With this column, the enzyme from rice callus has been purified 1500-fold in a single step, about 9000-fold over-all, to a specific activity of 0.078 units per milligram of protein. This is an order of magnitude greater than previous purifications of the plant enzyme. PMID- 16659396 TI - Temperature dependence of chlorophyll a fluorescence in relation to the physical phase of membrane lipids algae and higher plants. AB - The temperature dependence of the yield of chlorophyll a fluorescence was measured at room temperatures in living algal cells and higher plant chloroplasts. 3-(3',4'-Dichlorophenyl)-1, 1-dimethylurea was added to the samples during the measurements in order to eliminate the influence of photosynthetic photochemical reactions on the fluorescence yield.In Anacystis nidulans the maximum in the curve for the fluorescence yield versus temperature occurred near the temperatures at which the transition of physical phase of the membrane lipids changes from the liquid crystalline to the mixed solid-liquid crystalline states. These findings suggest that the occurrence of a fluorescence maximum in the temperature-dependence curve is an indication of the thermal transition of physical phase of membrane lipids. Similar but less distinct maxima were found in Cyanidium caldarium and Euglena gracilis. No maxima were seen in the curves for chlorophyll a fluorescence versus temperature in Chlorella pyrenoidosa, Plectonema boryanum, Stichococcus bacillaris Phaeodactylum tricornutum, or in chloroplasts of the higher plants, spinach, lettuce, tomato, and Tidestromia oblongifolia.The fluorescence yield of allophycocyanin in Anacystis did not show such a maximum, but did show a steep increase with decreasing temperature below 5 C. the fluorescence yields of chlorophyll a and phycobilins extracted from Anacystis increased monotonously with decreasing temperature. PMID- 16659397 TI - Occurrence of some enzymes in starchy endosperm and hormonal regulation of isoperoxidase in aleurone of wheat. AB - Peroxidase, indoleacetic acid-oxidase, alkaline, and acid phosphatases were detected in dry starchy endosperm (minus aleurone) of wheat grain. The isoperoxidase pattern differed in different parts of the dry grain. Several new isoperoxidases were found in embryos after soaking. The intensity of isoperoxidases in aleurones was enhanced in the presence of embryo or 2 muM GA(3) after 24 hours of soaking, but decreased after 72 hours. Indoleacetic acid and kinetin had no effect on isoperoxidase of aleurone. Actinomycin D and cycloheximide had no effect on isoperoxidases of aleurones from embryonectomized or naturally occurring embryoless grains. However, these two inhibitors increased the intensity of isoperoxidases in aleurones of intact embryonated grains after soaking. PMID- 16659398 TI - Inhibitory action of five tannins on growth induced by several gibberellins. AB - The following tannins, Chinese gallotannin, 1,2,3,4,6-pentagalloyl glucose, chebulinic acid, procyanidin dimers, and procyanidin trimers were tested and found to be antagonists of seven gibberellins (GAs). Each tannin inhibited the growth induced by any of the gibberellins GA(1), GA(3), GA(4), GA(7), GA(9), GA(13), and GA(14) in the dwarf pea assay. Endogenous growth was not affected. The highest ratio of tannin to gibberellin tested (1000:1 by weight) inhibited from 60 to 95% of the induced growth for all tannins and all gibberellins tested. The tannins were particularly inhibitory against GA(4) and GA(14) where a ratio of 10:1 (tannins: GA by weight) resulted in up to 85% growth reduction. Inhibition could be completely reversed by increasing the amount of gibberellin in all combinations studied. The procyanidin dimers and trimers were the first purified components of condensed tannins to be tested in this system and were potent inhibitors particularly against growth induced by GA(4) and GA(14). Inhibition by these compounds along with similar inhibition by previously tested hydrolyzable tannins demonstrates that the effect is general to tannins of all classes. PMID- 16659399 TI - Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds. AB - Asparagine accounted for 50 to 70% of the nitrogen carried in translocatory channels serving fruit and seed of white lupin (Lupinus albus L.). Rates of supply of the amide always greatly exceeded its incorporation as such into protein. An asparaginase (l-asparagine amido hydrolase EC 3.5.1.1) was demonstrated in crude extracts of seeds. In vitro activity was up to 5 mumoles of aspartate formed per hour per gram fresh weight at the apparent Km(Asn) value of 10 mM, and this more than accounted for the estimated rates of asparagine utilization in vivo. Asparaginase activity per seed increased 10-fold in the period 5 to 7 weeks after anthesis, coinciding with early stages of storage protein synthesis in the cotyledons.Double labeled ((14)C (U), (15)N (amide)) asparagine was fed to fruiting shoots through the transpiration steram. Fruit phloem sap analysis indicated that virtually all of the label was translocated to seeds in the form of asparagine. In young seeds (15)N from asparagine breakdown was traced to the ammonia, glutamine, and alanine of endospermic fluid, the (14)C appearing mainly in nonamino compounds. In the cotyledon-filling stage the C and N of asparagine was contributed to a variety of amino acid residues of protein. PMID- 16659400 TI - Water Permeability during Tomato Fruit Development in Normal and rin Nonripening Mutant. AB - This work tested one aspect of the relations between membrane permeability and fruit ripening. Membrane permeability was measured as [(3)H]water efflux rate from preloaded fruit pericarp disks. Different stages of fruit development were compared between two tomato (Lycopersicon esculentum Mill) strains: the normal Rutgers and the isogenic nonripening rin strain. The first significant increase in permeability was measured in Rutgers tissue at 110% of development, after fruit ripening had already begun as indicated by ethylene and CO(2) evolution and lycopene synthesis. The rin did not show any increase in tissue permeability during fruit development or maturation.Our results do not support the idea that the first event of the ripening process is an increase in membrane permeability. Nevertheless, the nonripening mutant fails to show the normal increase in permeability. PMID- 16659401 TI - Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition. AB - An attempt to isolate intact mitochondria from dry pea seeds (Pisum sativum var. Alaska) ended in failure. Cytochrome oxidase in crude mitochondrial fraction from dry seeds was separated into three fractions by sucrose density gradient centrifugation. Two of the fractions contained malate dehydrogenase, whereas the other did not. Equilibrium centrifugation of mitochondrial membrane on sucrose gradients revealed that the membrane from the fraction without malate dehydrogenase was lighter than that from the others. Differences were observed in relative content of phospholipid to protein and in polypeptide composition analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis among the membranes from three fractions and imbibed cotyledons. Membrane from the fraction without malate dehydrogenase was rich in phospholipid and lacking in polypeptides with relatively high molecular weights as compared with that from others. During imbibition, the fraction without malate dehydrogenase and one of the other two disappeared rapidly after a lag phase lasting for at least 1 hour. Concomitantly, active and stable mitochondria increased in the cotyledons. The results were interpreted to indicate that there were at least three types of mitochondria in dry seeds, the membranes of which differed in their biochemical properties, and that the mitochondria became active and stable through assembly of protein into the membranes during imbibition. PMID- 16659402 TI - A rapid technique for the estimation of polynucleotide adenylyltransferase and ribonucleic Acid polymerase in plant tissues. AB - Nucleic acid-dependent polynucleotide adenylytransferase (EC 2.7.7.19) and ribonucleic acid polymerase (EC 2.7.7.6) have been partially purified from maize tissues (Zea mays L.) utilizing ammonium sulfate precipitation and batch diethylaminoethylcellulose chromatography. The technique is applicable to the simultaneous processing of up to eight samples of plant tissue and affords a rapid and reproducible means of assaying these two enzymes from small quantities of kernels or seedlings. The kinetic characteristics of the partially purified enzymes resemble those from more extensively purified preparations. PMID- 16659403 TI - Effect of Gibberellic Acid, Kinetin, and Ethylene plus Carbon Dioxide on the Thermodormancy of Lettuce Seed (Lactuca sativa L. cv. Mesa 659). AB - The effects of gibberellic acid and kinetin with ethylene plus carbon dioxide on the thermodormancy of lettuce seeds (Lactuca sativa L. cv. Mesa 659) at 35 C in the dark were studied. The combination of gibberellic acid plus kinetin with ethylene plus carbon dioxide was most effective in overcoming thermodormancy in these Great Lakes type seeds, alleviating any induced light requirement. Gibberellic acid action required at least a minimal level of ethylene plus carbon dioxide. Kinetin action was independent of ethylene plus carbon dioxide but interacted with the gases when the gases were added. A schematic representation of the interaction is presented. PMID- 16659404 TI - Restoration of Organic Acid Accumulation in Sectioned Leaves of Bryophyllum tubiflorum Harv. AB - When leaves of Bryophyllum tubiflorum were cut into transverse sections, and held at 20 C in the dark, the capacity to accumulate organic acid decreased with decreasing section thickness. In addition, the rate of respiration increased with decreasing section thickness and was unaffected by changes in O(2) concentration above 5% or by the presence (1%) of CO(2). It was concluded that O(2) ventilation is not a controlling factor in respiration. Malonate (0.1 m) and fluoroacetate (0.01 m) restored the capacity of sectioned leaves to accumulate acid to normal levels and depressed respiration in 1-millimeter sections. Acid accumulation in 8 millimeter sections remained essentially constant at 20, 15, and 10 C, and was equal to that in unsectioned leaves, but accumulation in 2-millimeter sections rose to normal levels as the temperature fell to 10 C. Twenty-three additional metabolic inhibitors (none specific to the tricarboxylic acid cycle) were screened, and none promoted acid accumulation in sectioned leaves at 20 C. The results suggest that sectioning stimulates a respiratory sequence which includes the tricarboxylic acid cycle. This sequence in turn competes with the synthesis or accumulation of malic acid. PMID- 16659405 TI - Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons. AB - Developmental and compartmentation studies were used to evaluate the relative roles of the oxidative pentose phosphate cycle, the Calvin cycle, and the glycolysis in cotyledons of radish (Raphanus sativus L.).Glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, glucose-6-P isomerase, and the NAD dependent glyceraldehyde-3-P dehydrogenase were present in high activity in ungerminated seeds, increased about 2-fold during germination in the dark, and were slightly enhanced by light. In contrast, NADP-dependent glyceraldehyde-3-P dehydrogenase was developed to only a small degree in the dark, but increased severalfold in continuous white or far red light. The activity of phosphofructokinase was low throughout germination.The separation of cell compartment-specific isoenzymes showed that, except in ungerminated seeds, the plastid enzyme accounted for 40 to 45% of the total activity of 6-P-gluconate dehydrogenase and for 15 to 20% of glucose-6-P isomerase. The remaining activity was due to the cytosolic isoenzymes. The presence of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase in plastids was also established by their presence in the isolated organelle. The NAD-dependent glyceraldehyde-3-P dehydrogenase was mostly due to the cytosolic isoenzyme, whereas the NAD dependent activity associated with the NADP-dependent glyceraldehyde-3-P dehydrogenase was very small.The data indicate that the enzymes of the oxidative pentose phosphate cycle are present in the cytosol throughout germination. In the plastids these enzymes already became fully developed during early germination in the dark, whereas enzymes of the Calvin cycle increased only in the light. Glycolysis seemed to be of minor importance. PMID- 16659406 TI - Leaf water stress in engelmann spruce: influence of the root and shoot environments. AB - The response of xylem pressure potential of Engelmann spruce (Picea engelmannii Engelm.) to environmental factors was studied in the natural subalpine environment. Data were analyzed in the context of a leaf water potential model based upon the van den Honert model for water transport through the soil-plant atmosphere continuum. At soil temperatures of 10 to 15 C, xylem pressure potential decreased to about -10 bars as the ratio of leaf to air absolute humidity difference to leaf diffusion resistance (an estimate of transpiration) increased. The potentials were slightly lower at all flux rates above zero when the soil temperature was 5 to 10 C, and at temperatures of 0 to 5 C the potentials decreased sharply to as low as -20.4 bars, even though the soil water supply was adequate. The relative viscosity of water and soil to leaf resistances for flow were compared for Engelmann spruce and citrus at low soil temperatures. These comparisons indicated that decreased root permeability was probably not an important factor causing higher stresses in spruce at 5 to 10 C, but for citrus, root permeability became limiting at soil temperatures as high as 13.5 C. Xylem pressure potential was correlated with net radiation during the daytime when soil temperature was above 7 C. Under other conditions, however, xylem potential and net radiation apparently had a different relationship. The relationship between flux density and potential was the same on unshaded and shaded portions of the crown, with differences in potential related to differences in flux density. PMID- 16659407 TI - Mechanical inhibition of hypocotyl elongation induces radial enlargement: implications for cytokinin action. AB - Excised elongating segments from 3-day-old soybean (Glycine max var. Wayne) seedlings radially enlarged when auxin-promoted elongation was mechanically inhibited. Growth was similar to segments treated with auxin plus cytokinin. This observation suggests that cytokinin does not necessarily directly "reorient" cell enlargement. Cytokinin-induced radial cell growth may be a secondary effect of cytokinin's inhibition of auxin-promoted elongation. PMID- 16659408 TI - Photocontrol of fungal spore germination. AB - Germination of Puccinia graminis f. sp. tritici uredospores is inhibited by continuous irradiation. Prehydration of spores enhances both dark germination and photoinhibition. Simultaneous irradiation with ineffective red (653 nanometers) and inhibitory far red light (720 nanometers) results in partial nullification of the inhibition brought about by far red light alone. This result would be consistent with the involveent of a photoreversible pigment system similar to phytochrome, operating via the high irradiance reaction. PMID- 16659409 TI - Isolation of nucleoli and localization of ribonucleic Acid polymerase I from soybean hypocotyl. AB - An effective method for the isolation of nucleoli from auxin-treated soybean (Glycine max, var. Wayne) hypocotyl was developed by polytron homogenization and sucrose gradient centrifugation. The nucleoli expressed only the alpha-amanitin insensitive RNA synthetic activity. This activity chromatographed as RNA polymerase I on DEAE-cellulose. It appears that the plant nucleolus, like the animal nucleolus, is the site of localization for RNA polymerase I. PMID- 16659410 TI - Purification of NADH-Nitrate Reductase by Affinity Chromatography. AB - ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, EC 1.6.6.1) from Chlorella vulgaris has been purified to electrophoretic homogeneity with an overall yield of 60% by a procedure that utilizes blue dextran-agarose as an affinity column. Nitrate reductase binds to blue dextran and is not eluted in the presence of high ionic strength buffer but is rapidly eluted in the presence of mumolar concentrations of NADH. PMID- 16659411 TI - Evidence for Ammonium-dependent de Novo Synthesis of Glutamate Dehydrogenase in Detached Oat Leaves. AB - Glutamate dehydrogenase becomes density labeled through the incorporation of deuterium and (15)N when detached oat leaves (Avena sativa var. Fulghum) are incubated in the presence of ammonia. The enzyme has been isolated by means of DEAE-cellulose chromatography, ammonium sulfate precipitation, isopycnic equilibrium centrifugation, and disc electrophoresis from leaves fed l-methionine (35)S. Radioactivity is incorporated into isozyme 1 of glutamate dehydrogenase, whereas isozyme 2, detected only in the absence of ammonia, has not been labeled. Cycloheximide, chloramphenicol, puromycin, and 6-methyl purine inhibit the elevation of glutamate dehydrogenase by ammonia. It is suggested that the increase in glutamate dehydrogenase activity is due to de novo synthesis of isozyme 1. PMID- 16659412 TI - Changes in Isoperoxidases during Cold Treatment of Dormant Pear Embryo. AB - The number of isoperoxidases and the intensity of certain isozymes increased with increasing periods of stratification of pear (Pyrus communis cv. Bartlett) embryos. The presence of GA(3) or kinetin during stratification enhanced the activity of certain isoperoxidases, and these enhancements were blocked in the presence of ABA which by itself had an inhibitory effect. Enhancement in isoperoxidases of pear embryos during stratification was inhibited by 6 methylpurine and cycloheximide; and in the presence of either of these two inhibitors, stratification failed to release the dormancy. Pear embryos germinated for 3 days showed changes in the pattern of isoperoxidases depending on the length of stratification. PMID- 16659413 TI - Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants. AB - Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed. PMID- 16659414 TI - Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll. AB - An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 x M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K(+)-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K(+)-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.A major peak of ATPase activity at pH 6.5 was observed at low density in Ficoll after nonequilibrium centrifugation in a combination Ficoll-sucrose gradient. Twenty to forty percent of the vesicles in this ATPase fraction stained positively for plasma membranes, and with equilibrium centrifugation the major portion of the ATPase activity shifted to densities in sucrose which were characteristic of plasma membranes. All major vesicular ATPase activities observed in Ficoll or sucrose contained substantial amounts of plasma membranes. For unknown reasons, mitochondria and plasma membranes equilibrated over a broader density range and at lower peak densities in sucrose as a result of equilibrium centrifugation through Ficoll. PMID- 16659415 TI - Effects of Water Stress on the Ultrastructure of Leaf Cells of Sorghum bicolor. AB - The subcellular changes which occurred in sorghum leaves during increasing water stress and subsequent rewatering are described. Stomata were closed, abscisic acid levels were elevated, and the amounts of starch in the bundle sheath chloroplasts were much reduced by - 14 bars leaf water potential. Swelling of the outer chloroplast membrane, and reorganization of the tonoplast to form small vesicles from the large central vacuole, occurred by a leaf water potential of - 37 bars. Complete structural disruption of the tonoplast, as previously described for maize was not found. On rewatering, large amounts of starch reappeared within three hours. These findings strengthen the hypothesis that maintenance of tonoplast integrity is an important factor in the ability of plants to withstand drought. PMID- 16659416 TI - Abscisic Acid Content and Stomatal Sensitivity to CO(2) in Leaves of Xanthium strumarium L. after Pretreatments in Warm and Cold Growth Chambers. AB - The degree of stomatal sensitivity to CO(2) was positively correlated with the content of abscisic acid of leaves of Xanthium strumarium grown in a greenhouse and then transferred for 24 hours or more to a cold (5/10 C, night/day) or a warm growth chamber (20/23 C). This correlation did not exist in plants kept in the greehouse continuously (high abscisic acid, no CO(2) sensitivity), nor in plants transferred from the cold to the warm chamber (low abscisic acid, high CO(2) sensitivity). The abscisic acid content of leaves was correlated with water content only within narrow limits, if at all. At equal water contents, prechilled leaves contained more abscisic acid than leaves of plants pretreated in the warm chamber. There appear to be at least two compartments for abscisic acid in the leaf. PMID- 16659417 TI - Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations. AB - The incorporation of the cytokinin N(6)-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N(6)-benzyladenine-8-(14)C or N(6)-benzyladenine-8-(14)C: benzene-(3)H(G) has been examined. N(6)-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N(6)-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G 200 gel filtration had no effect on the N(6)-benzyladenosine content of the RNA preparation. The distribution of N(6)-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N(6)-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T(2) and phosphatase, or ribonuclease T(2) and a 3'-nucleotidase. N(6)-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T(2) or phosphatase alone or by successive treatment with ribonuclease T(2) and a 5'-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T(1) and pancreatic ribonuclease converted the N(6) benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N(6)-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant. PMID- 16659418 TI - Some regulatory properties of pea leaf carbamoyl phosphate synthetase. AB - Carbamoyl phosphate synthetase of pea shoots (Pisum sativum L.) was purified 101 fold. Its stability was greatly increased by the addition of substrates and activators. The enzyme was strongly inhibited by micromolar amounts of UMP (Ki less than 2 mum). UDP, UTP, TMP, and ADP were also inhibitory. AMP caused either slight activation (under certain conditions) or was inhibitory. Uridine nucleotides were competitive inhibitors, as was AMP, while ADP was a noncompetitive inhibitor. Enzyme activity was increased manyfold by the activator ornithine. Ornithine acted by increasing the affinity for Mg.ATP by a factor of 8 or more. Other activators were IMP, GMP, ITP, and GTP, IMP, like ornithine, increased the Michaelis constant for Mg.ATP. The activators ornithine, GMP, and IMP (but not GTP and ITP) completely reversed inhibition caused by pyrimidine nucleotides while increasing the inhibition caused by ADP and AMP. PMID- 16659419 TI - Attempts to detect cyclic adenosine 3':5'-monophosphate in higher plants by three assay methods. AB - Endogenous levels of cyclic adenosine-3':5'-monophosphate in coleoptile first leaf segments of oat (Avena sativa L.), potato (Solanum tuberosum L.) tubers, tobacco (Nicotiana tabacum L.) callus, and germinating seeds of lettuce (Lactuca sativa L.) were measured with a modified Gilman binding assay and a protein kinase activation assay. The incorporation of adenosine-8-(14)C into compounds with properties similar to those of cyclic AMP was also measured in studies with germinating lettuce seeds. The binding assay proved reliable for mouse and rat liver analyses, but was nonspecific for plant tissues. It responded to various components from lettuce and potato tissues chromatographically similar to but not identical with cyclic AMP. The protein kinase activation assay was much more specific, but it also exhibited positive responses in the presence of compounds not chromatographically identical to cyclic AMP. The concentrations of cyclic AMP in the plant tissues tested were at the lower limits of detection and characterization obtainable with these assays. The estimates of maximal levels were much lower than reported in many previous studies. PMID- 16659420 TI - Manipulation of galactolipid Fatty Acid composition with substituted pyridazinones. AB - The fatty acid composition of the major lipids of the chloroplast membranes, the mono- and digalactosyl diglycerides, can be definably altered with various substituted pyridazinones. Galactolipid fatty acid composition of wheat (Triticum aestivum L.) can be altered so that there is a decrease in linolenic acid accompanied by an increase in linoleic acid without a shift in the relative proportion of saturated to unsaturated fatty acids; the fatty acid composition can be shifted toward a higher proportion of saturated fatty acids; or the fatty acid composition of the monogalactosyl diglycerides can be altered in preference to the digalactosyl diglycerides. Also, the light-mediated parallel accumulation of chlorophyll and linolenic acid can be separated with a substituted pyridazinone. The substituted pyridazinones may be useful tools in clarifying the role the galactolipids and their component fatty acids play in the structure and function of chloroplast membranes in higher plants. PMID- 16659421 TI - Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive gamma-Irradiation of the Seed. AB - Spinach seeds (Spinacia oleracea L.) given massive doses of gamma-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis. PMID- 16659422 TI - The Light-harvesting Chlorophyll a/b-Protein Complex of Chlamydomonas reinhardii. AB - The molecular organization of chlorophyll in Chlamydomonas reinhardii has been shown to be essentially similar to that in higher plants. Some 50% of the chlorophyll in Chlamydomonas reinhardii chloroplast membranes has been shown to be located in a chlorophyll a/b-protein complex. The complex was isolated in a homogeneous form by hydroxylapatite chromatography of sodium dodecyl sulfate extracts of the chloroplast membranes. Its absorption spectrum exhibits two maxima in the red region at 670 and 652 nm due to the presence of equimolar quantities of chlorophylls a and b in the complex. Preparations of the chlorophyll-protein also contain some of each of the carotenoids observed in the intact chloroplast membrane, but not in the same proportions. The native complex (S value = 2.3S) exhibits a molecular weight of 28,000 +/- 2,000 on calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on the basis of its amino acid composition and other data a more probable molecular weight of about 35,000 was calculated. Each 35,000 dalton unit contains three chlorophyll a and three chlorophyll b molecules, and on the average one carotenoid molecule conjugated with probably a single polypeptide of 29,000 daltons. Comparison of spectral and biochemical characteristics demonstrates that this algal chlorophyll protein is homologous to the previously described major light-harvesting chlorophyll a/b-protein of higher plants. It is anticipated that the Chlamydomonas complex functions solely in a light-harvesting capacity in analogy to the function determined for the higher plant component. PMID- 16659423 TI - Influence of Ionic Strength, pH, and Chelation of Divalent Metals on Isolation of Polyribosomes from Tobacco Leaves. AB - A procedure was developed for extracting polysomes from tobacco (Nicotiana sp) leaves. Unexpanded leaves ground in a medium consisting of 200 mm tris-HCl, pH 9, 400 mm KCl, 200 mm sucrose, and 35 mm MgCl(2) yielded larger amounts of polysomes with less degradation than polysomes from leaves extracted with buffers of lower ionic strength or pH. Extraction of polysomes from expanded leaves required the inclusion of ethyleneglycol-bis(2-aminoethyl ether)tetraacetic acid (EGTA, a divalent cation chelator with a high affinity for Ca(2+), Cu(2+), and Zn(2+)). EGTA also improved isolation of polysomes from unexpanded leaves. Addition of 25 mm Ca(2+), Cu(2+), or Zn(2+) to extracts from young leaves precipitated polysomes, and density gradient profiles of polysome preparations from the cation treatments mimicked profiles from expanded leaves which were extracted without EGTA. Polysome precipitation by Ca(2+) was prevented by EGTA. Endogenous Ca(2+) was present in unexpanded leaves in sufficient concentrations (25 mm) to cause some precipitation of polysomes during extraction, and this cation increased by 60% in expanded leaves. Cu(2+) and Zn(2+) were not present in amounts sufficient to cause polysome precipitation. The results show that recovery of polyribosomes may be reduced by divalent cations in leaf tissue, and this can be overcome by chelation of these ions with EGTA. PMID- 16659424 TI - Distribution of Protein-bound Hexosamine in Chloroplasts. AB - Intact chloroplasts of spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) mesophyll cells contained 0.33, 0.50, and 0.14% of bound hexosamine on a protein basis, respectively. Undifferentiated maize chloroplasts contained 0.19%. Values for chloroplast lamellae were, respectively, 0.16, 0.18, 0.12, and 0.06% and for envelope membranes they were 1.6, 2.5, 3.8, and 2.7%. Thus most of the hexosamine of chloroplasts is located in the envelope membrane. PMID- 16659425 TI - Nitrate absorption by barley: I. Kinetics and energetics. AB - The absorption of NO(3) (-) by barley (Hordeum vulgare L.) was investigated by following the disappearance of NO(3) (-). The absorption was related to several parameters: NO(3) (-) and Ca(2+) concentrations, pH, and the presence of various anions. Absorption rate increased with increasing Ca(2+) concentration, reaching a maximum at approximately 5 mm Ca(2+), and was considerably inhibited by NH(4) (+). Absorption was influenced markedly by pH, and little or not at all by anions (Cl(-), Br(-), SO(4) (2-)), and was decreased by respiratory and oxidative phosphorylation inhibitors. PMID- 16659426 TI - Nitrate Absorption by Barley: II. Influence of Nitrate Reductase Activity. AB - The influence of protein synthesis and nitrate reductase activity on nitrate absorption by barley (Hordeum vulgare L.) was investigated. Cycloheximide decreased nitrate absorption. Pretreatment studies showed that cycloheximide affects either energy transfer or nitrate reductase activity or both.Illumination increased plant capacity for nitrate absorption, possibly through increased energy supply and/or increased nitrate reductase activity. There was a positive correlation between nitrate reductase activity and light. Inhibiting the development of nitrate reductase activity by tungstate decreased nitrate absorption.AT LEAST TWO NITRATE TRANSPORT SYSTEMS ARE THUS PROPOSED IN BARLEY: one operating in the dark, with little nitrate reductase activity detectable; and one closely correlated with nitrate reductase activity. Total absorption is the sum of dark absorption and absorption facilitated by nitrate reductase. PMID- 16659427 TI - Pyrophosphorylases in Solanum tuberosum: I. Changes in ADP-Glucose and UDP Glucose Pyrophosphorylase Activities Associated with Starch Biosynthesis during Tuberization, Maturation, and Storage of Potatoes. AB - Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16- to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2- to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.Evidence that UDP glucose and ADP-glucose pyrophosphorylases are separate protein entities was indicated by their (a) activity ratio variations during tuber development and storage, (b) extraction stabilities, (c) morphological localization, (d) separation with ammonium sulfate, (e) pH optima, and (f) differential activation with 3-P-glycerate. PMID- 16659428 TI - Analysis of steady state photosynthesis in alfalfa leaves. AB - A method for carrying out kinetic tracer studies of steady state photosynthesis in whole leaves has been developed. An apparatus that exposes whole leaves to (14)CO(2) under steady state conditions, while allowing individual leaf samples to be removed as a function of time, has been constructed. Labeling data on the incorporation of (14)C into Medicago sativa L. metabolite pools are reported. A carbon dioxide uptake rate of 79 micromoles (14)CO(2) per milligram chlorophyll per hour was observed at a CO(2) level slightly below that of air. Several actively turning over pools of early and intermediate metabolites, including 3 phosphoglyceric acid, glycerate, citrate, and uridine diphosphoglucose, showed label saturation after approximately 10 to 20 minutes of photosynthesis with (14)CO(2) under steady state conditions. Alanine labeling increased more rapidly at first, and then at a lower rate as saturation was approached. Sucrose was a major product of photosynthesis and label saturation of the sucrose pool was not observed. Labeled carbon appeared rapidly in secondary metabolites. The steady state apparatus used has numerous advantages, including leaf temperature control, protection against leaf dehydration, high illumination, known (14)CO(2) specific radioactivity, and provision for control and adjustment of (14)CO(2) concentration. The apparatus allows for experiments of long duration and for sufficient sample points to define clearly the metabolic steady state. PMID- 16659429 TI - Cell isoperoxidases in sweet potato plants in relation to mechanical injury and ethylene. AB - Leaves and storage roots of sweet potato plants (Ipomea batatas) showed the same qualitative isoperoxidase patterns and a similar distribution of distinctive isoperoxidases between the cell protoplast and cell wall free, ionically bound, and covalently bound fractions. No changes in the qualitative isoenzyme spectrum were found in relation to age, mechanical injury, or ethylene action. Thus, as in tobacco plants, the cell isoperoxidases in sweet potato did not reflect the possible differential mRNA synthesis in relation to organ, age, or injury. Transcription does not seem to be a limiting factor in injury- and ethylene dependent peroxidase enhancement during the first 24 hr.The contribution of the wall ionically bound and protoplast fractions was highest in young and old leaves, respectively. In the protoplast and wall ionically and covalently bound fractions, 14, 6, and 5 isoenzyme bands were found; in addition, 4 bands, not detected in the protoplast, were also revealed in the covalently bound fraction. The distinctive "juvenile" and, developing with age, "mature" isoforms were mainly found in the ionically bound and protoplast fractions, respectively.The injury-enhanced and/or ethylene-enhanced peroxidase development was most pronounced in young leaves. Ethylene suppressed some injury-enhanced, had no effect on some other injury-enhanced, and greatly promoted some of the injury unaffected or enhanced isoperoxidases. After ethylene removal, an increase in the "mature" isoforms was found in the protoplast of intact leaves.Electron microscopy of leaves revealed peroxidase in membrane-bound vesicles located mainly in the vacuole; a thin layer of reaction products was also found on the wall's outer surface. No Golgi apparatus were seen in the cells of control or ethylene-treated intact leaves. In ethylene-treated intact or injured leaves accumulations of reaction products between the plasmalemma and wall were also found. Numerous Golgi apparatus with dark stained vesicles were seen in injured, and especially in injured and ethylene-treated leaves; the vacuolar bodies seemed to occur in very great number. PMID- 16659430 TI - Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision. AB - The dark respiration rate of discs from fully expanded tobacco leaves (Nicotiana tabacum) increased linearly with decreasing diameter, the relative increase being independent of leaf age. The wound respiration responsible for this situation reached a plateau within 15 minutes of excision. Metabolite analysis gave evidence for two independent effects, also unrelated to age. The first was a forward crossover between phosphoenolpyruvate and pyruvate which was found as early as 1 minute after excision and persisted for up to 40 minutes. It was attributed to activation of pyruvate kinase by a changed ionic balance resulting from membrane damage, was accompanied by a reverse crossover between triose phosphates and 3-phosphoglycerate, and was localized in the outer region of the discs. The second effect was a rapid rise in hexose monophosphate and ATP levels throughout the discs. After 1 to 10 minutes the ATP/ADP ratio rose strongly for at least 3 hours; after 20 to 40 minutes there was net synthesis of adenine nucleotide as ATP. These results indicate that extrapolation from leaf discs to intact leaves is highly inadvisable. PMID- 16659431 TI - In vivo conversion of 5-oxoproline to glutamate by higher plants. AB - l-(U-(14)C)-5-oxoproline (pyrollidone carboxylic acid or pyroglutamic acid) was infiltrated into detached leaves of a number of species and incubated for 1 to 6 hours. In every case, conversion to labeled glutamate and glutamine was observed. The amount converted varied from 1 to 64% of the total label fed depending on the species. The ratio of glutamate-(14)C to glutamine-(14)C ranged from 5 in Vicia faba to 1 in sugar beet. This ratio could be affected by preinfiltrating various compounds before allowing the uptake of the 5-oxoproline. When l-methionine-dl sulfoximine was prefed to sugar beet leaves, the glutamate-glutamine ratio increased from 1 to 10. Prior treatment of V. faba leaves with azaserine resulted in essentially only labeled glutamine being recovered. Preinfiltration with NaF or ATP gave similar results in that the glutamate-glutamine ratio was greatly decreased. The results are consistent with glutamate being produced from the 5 oxoproline and then being converted to glutamine. PMID- 16659432 TI - Stimulation of ethylene production in the mung bean hypocotyls by cupric ion, calcium ion, and kinetin. AB - The synergistic stimulation of ethylene production by kinetin and Ca(2+) in hypocotyl segments of mung bean (Phaseolus aureus Roxb.) seedling was further studied. The requirement for Ca(2+) in this system was specific. Except for Sr(2+), which mimicked the effect of Ca(2+), none of the following divalent cations, including Ba(2+), Mg(6+), Cu(2+), Hg(2+), Co(2+), Ni(2+), Sn(2+), and Zn(2+), showed synergism with kinetin on ethylene production. Fe(2+), however, showed a slight synergism with kinetin. Some of them (Hg(2+), Co(2+), and Ni(2+)) had a strong inhibitory effect, while others (Zn(2+), Mg(2+), Sn(2+), and Ba(2+)) had a slight or no inhibitory effect on ethylene production in the absence or presence of kinetin.Cu(2+) alone, depending on the concentration applied, stimulated ethylene production with a lag period of about 2 hours and had no synergism with kinetin on ethylene production. When Cu(2+) was applied with Ca(2+), a remarkable synergistic stimulation of ethylene production was observed. Tracer experiments indicated that Cu(2+) enhanced the uptake of (45)Ca(2+) into the tissues during the first few hours of incubation, and this increase of (45)Ca(2+) uptake paralleled the enhancement of ethylene production. When Ca(2+) was applied together with kinetin plus Cu(2+), both the ethylene production and the (45)Ca(2+) uptake were greatly increased over those from the segments treated with Cu(2+) or kinetin alone. The increase in ethylene production as a result of kinetin plus Ca(2+) plus Cu(2+) treatment is equal to the combined increases caused by kinetin plus Ca(2+) and Cu(2+) plus Ca(2+). A possible mechanism accounting for such cooperative effects of Cu(2+), Ca(2+), and kinetin on ethylene production is discussed. PMID- 16659433 TI - Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination. AB - The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of (14)C-labeled glucosamine, it appeared that not all of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins. PMID- 16659434 TI - Nonmetabolic catalysis of C-furfuryl amino purine (kinetin) on plant surfaces, glass, and porcelain ware. AB - Kinetin-8-(14)C degraded rapidly upon drying on living or inert surfaces. However, when care was exercised to avoid taking solutions to dryness during fractionation of plant extracts containing (14)C-kinetin and before partitioning by thin layer chromatography, little degradation occurred. A procedure for 24 hour ethyl acetate partitioning, using a continuous liquid-liquid extractor, which permits nearly complete removal of kinetin from aqueous solutions, is herein described. High natural light intensities in the greenhouse or from fluorescent/incandescent sources greatly enhanced nonmetabolic degradation of kinetin on leaf (Bougainvillea) or glass surfaces, which indicated that this may be a confounding factor in analyzing metabolism of kinetin in plant tissues. One of the degradation products is probably adenine. PMID- 16659435 TI - Transmembrane electropotential in barley roots as related to cell type, cell location, and cutting and aging effects. AB - Transmembrane electropotential difference (PD) was measured in whole roots of barley (Hordeum vulgare L. cvs. Compana and Himalaya). Seedlings were grown 4 to 5 days in aerated 0.5 mm CaSO(4) or a nutrient solution. Measurements of PD were made with roots bathed in CaSO(4), KCl + CaSO(4), or the nutrient solution. The following results were found. (a) There was a radial PD gradient with epidermal cells being 10 to 58 millivolts less negative than cells in the third layer of the cortex (outside to inside). There was no longitudinal PD gradient in the region 0.5 to 4 cm from the root tip, nor was there any difference between the PD of young root hairs and other epidermal cells. (b) Cell PD in excised whole roots was not detectably different from that found in roots attached to the shoot, and was unchanged for 2 hours from excision. (c) In 1-centimeter sections of root, cell PD at the freshly cut surface was depolarized by 90 millivolts from that in the intact root; cells farther than 1 millimeter from the cut surface were not depolarized. The PD of cells at the cut surface became more negative upon aging the segment in 0.5 mm CaSO(4), eventually becoming greater by -25 millivolts than that in cells of intact roots. Cells in segments to which the root tips were attached had less negative PDs after aging than those in subapical segments, indicating a possible hormonal effect. PDs in aged, excised segments are not equivalent to those in intact roots. (d) Creeping of cytoplasm over electrode tips inserted into the vacuole gave measurements of vacuole-to-cytoplasm PD of + 9 millivolts in 0.5 mm CaSO(4) and + 35 millivolts in 1 mm KCl + 0.5 mm CaSO(4). Most of the cell PD was across the plasmalemma. (e) The reducing sugar content of roots in CaSO(4) solution was greater than that of roots in the nutrient solution in which ion uptake, particularly K(+) occurred. PMID- 16659436 TI - Electrochemical gradients and k and cl fluxes in excised corn roots. AB - The compartmental analysis method was used to estimate the K(+) and Cl(-) fluxes for cells of excised roots of Zea mays L. cv. Golden Bantam. When the measured fluxes are compared to those calculated with the Ussing-Teorell flux-ratio equation, an active inward transport of Cl(-) across the plasmalemma is indicated; the plasmalemma K(+) fluxes are not far different from those predicted for passive diffusion, although an active inward transport cannot be precluded. Whether fluxes across the tonoplast are active or passive depends upon the vacuolar potential which is unknown. Assuming no electropotential gradient, the tracer flux ratios are fairly close to those predicted for passive movement. However, if the vacuole is positive by about 10 millivolts relative to the cytoplasm, the data suggest active inward transport for K(+) and outward transport for Cl(-).Fluxes to the xylem exudate were found to be more accurately estimated from the specific radioactivity of the cytoplasm (symplasm) than from the external solution specific radioactivity. The electrochemical gradients for K(+) and Cl(-) between the xylem vessels and the surrounding stelar parenchyma indicate active K(+) and passive Cl(-) movement into the vessels. The data are interpreted as being in accord with radial transport through the symplast into living vessels. PMID- 16659437 TI - Electrical resistances of corn root segments. AB - Longitudinal electrical resistances have been measured on 2-centimeter segments of corn (Zea mays L.) roots, cut at varying distances from the root apex. The segment resistances vary from 400 to 100 kilohms per centimeter along the root length (apex to 18 cm), with the maximum occurring in the 2- to 4-centimeter segment, and decreasing thereafter toward the root base. Measurements of isolated root cortical sleeves and steles show that the pathway of least resistance is in the cortex, which has a greater cross-sectional area; the specific resistance of the older stele is less than that of the cortex. The anatomical state of the xylem cannot be inferred from electrical resistance determinations. PMID- 16659438 TI - Separation and characterization of potato lipid acylhydrolases. AB - Three distinct potato (Solanum tuberosum) lipid acyl-hydrolases have been isolated and characterized. Nonfluorescent esters of the fluorescent alcohols, N methylindoxyl and N-methylumbelliferone, have been used as convenient substrates for lipid acyl-hydrolase estimation. Enzyme I has been shown to be a neutral lipase which favors glyceryl triolein over the di- and monoolein, which shows no activity with phospho- and galactolipids and which favors long chain fatty acid esters of N-methylindoxyl over the butyrate ester. Enzyme II, while attacking glyceryl mono- and diolein, as well as favoring the butyrate ester of N methylindoxyl over the myristate ester, is basically a phospholipid and galactolipid acyl-hydrolase. Enzyme III may reasonably be considered an esterase, since it hydrolyzes glyceryl monoolein exclusively among the neutral lipids, shows minimal activity on phospho- and galactolipids, and hydrolyzes N methylindoxylbutyrate exclusively compared with N-methylindoxyl-myristate. PMID- 16659439 TI - Purification and characterization of an a type phospholipase from potato and its effect on potato mitochondria. AB - A potato (Solanum tuberosum) phospholipid acyl-hydrolase, which - in the pH range 7.5 to 8.5-is at least 10,000 times more effective with phospholipids than with galactolipids, has been purified and characterized. It is a soluble enzyme readily distinguished from a neutral lipid lipase and a third lipid acyl hydrolase which, while acting on phospholipid, shows a decided preference for glyceryl monoolein. The phospholipase in question has a pH optimum of 8.5, is stimulated by Ca(2+) at pH above 7.5 and inhibited by Ca(2+) at lower pH, is not dependent on detergents although stimulated by Triton X-100 to a moderate extent, and remains very active at temperatures close to zero. The phospholipids of intact potato mitochondria are highly susceptible to degradation by potato phospholipase, and it is suggested that this enzyme is involved in the extensive lipid breakdown which occurs in fresh potato slices following cutting, and in the deterioration of mitochondria during their preparation and aging. PMID- 16659440 TI - Water uptake and water diffusivity of seeds. AB - When pea (Pisum sativum) seeds were wetted, a sharp front separated the wet and dry portions, the seeds swelled, and the water content in the wetted portion continued to increase for a long time. A model was proposed and tested that takes into account these three characteristics and in particular does not postulate a constant diffusivity. The parameters of the model are simply the rate of penetration of the wetting front and a swelling factor. PMID- 16659441 TI - Interactive effects of salinity and phosphorus nutrition of the concentrations of phosphate and phosphate esters in mature photosynthesizing corn leaves. AB - The effects of salinity on corn plants (Zea mays L.) are influenced by the concentration of nutrient orthophosphate. Salinity (-2 bars each of NaCl and CaCl(2)) was more injurious in combination with a high concentration of orthophosphate (2 mm) (that gave optimum yields in the absence of salinity) than it was with a lower concentration (0.1 mm). With 2 mm orthophosphate, salinity seemed to damage the plant mechanisms that normally regulate the internal concentration of orthophosphate resulting in excessive accumulation and P toxicity. On the other hand, with 0.1 mm orthophosphate, salinity decreased orthophosphate concentration in mature leaves. This effect was paralleled by decreases in the concentration of adenosine 5'-triphosphate and in the energy charge of the adenylate system, indicating an orthophosphate deficit. Even so, plants survived salinity better under these conditions than in the presence of 2 mm orthophosphate. The data indicated that salinity affected the phosphorylated state of the adenine nucleotides only indirectly through its effect on the concentration of orthophosphate in the cells.Salinity, especially in the presence of 2 mm orthophosphate, resulted in an increase in the concentrations of sugar phosphates in mature photosynthesizing leaves, suggesting that translocation rather than photosynthesis was a limiting process. Decreased translocation could be a secondary effect of decreased growth. However, a decreased translocation rate could cause decreased growth by limiting the supply of essential metabolites reaching growing tissues. PMID- 16659442 TI - Genotypic Responses to Salinity: Differences between Salt-sensitive and Salt tolerant Genotypes of the Tomato. AB - Four ecotypes of the species Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull. from the Galapagos Islands were compared with L. esculentum Mill cv. VF 36 with respect to salt tolerance. The L. cheesmanii ecotype that proved most salt tolerant was selected for detailed comparison with the L. esculentum cultivar. Plants were grown in modified Hoagland solution salinized with synthetic seawater salt mix. Growth rates under saline conditions were examined and amino acid, sugar, total amino nitrogen, free acidity, and Na and K levels in the tissues of the most and least tolerant plants were measured under salt stress and nonstress conditions. Results indicate that all Galapagos ecotypes were far more salt tolerant than was the esculentum cultivar. They could survive in full strength seawater nutrient solution while the esculentum cultivar could not in most cases withstand levels higher than 50% seawater. Growth rates were reduced in both species under saline conditions but the esculentum cultivar was more severely affected. High levels of total amino nitrogen, specific amino acids, and free acidity along with low sodium content were found in the salt stressed VF 36 cultivar. The opposite responses were noted in the salt stressed treatments of the Galapagos ecotype. Tissue sugar levels did not appear to be similarly correlated with salt stress in either species. Potassium content fell sharply during salinization in the Galapagos ecotype while in the esculentum cultivar it declined relatively little even at high levels of salinity. PMID- 16659443 TI - Effect of Salinity on Respiratory Pathways in Root Tips of Tamarix tetragyna. AB - Oxygen uptake in the presence of exogenous glucose was lower in Tamarix root tips grown in saline media than in those grown in Hoagland solution. This effect was not overcome by raising the external glucose concentration.Glucose uptake and CO(2) evolution were depressed in the presence of NaCl. This effect was observed also when roots were exposed to salinity only during growth but not during uptake. Increasing the external concentration of glucose from 0.01 to 1 mm induced only a 10-fold increase in glucose uptake and CO(2) evolution. However, (14)C evolved in CO(2) as percent of (14)C absorbed, remained constant at all salinity treatments, and was similar at both glucose concentrations.Salinity above 120 mm NaCl increased the percentage of absorbed glucose oxidized via the pentose phosphate pathway, but did not affect the glycolytic pathway. At the same time, salinity depressed the glucose-6-P dehydrogenase, pyruvate kinase, and oxidative phosphorylation. These effects become most evident at a salinity level of about - 10 atm (240 mm), a concentration which is rarely exceeded in the root zone of the natural habitat of the plants.We concluded that Tamarix is reasonably well adapted to the conditions of its habitat, and that salinity affects its root metabolism differently than it does that of pea roots. PMID- 16659444 TI - Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots. AB - The effect of a toxin extract of Helminthosporium maydis, race T on K(+) ((86)Rb) uptake by excised root segments of normal (N) and Texas cytoplasmic male-sterile (T) versions of corn inbred W64A was investigated. The uptake of K(+) was inhibited in both N and T roots by the toxin. This was true for both basal (freshly excised) and augmented (pretreated with aeration) K(+) uptake. Augmented uptake was more toxin-sensitive than basal uptake (irrespective of cytoplasm type), and the augmented uptake in T roots was seven to eight times more toxin sensitive than in N roots.Specific zones of roots differed in their basal and augmented K(+) uptake rates as well as their toxin sensitivities. The root apex of T was more sensitive to toxin than the apex of N roots when basal K(+) uptake was measured. In mature zones of the root, T was more sensitive than N when augmented rates were measured. During the development of the augmented K(+) uptake capacity in either N or T roots, the sensitivity to the toxin did not change; uptake in N roots was inhibited by 10 to 25% and uptake in T roots was inhibited by 70 to 80%.The difference in toxin sensitivity of K(+) uptake between N and T roots may be due to N possessing a protective mechanism which is deficient in T. PMID- 16659445 TI - Response of barley aleurone layers to abscisic Acid. AB - Cordycepin, an inhibitor of RNA synthesis in barley (Hordeum vulgare L.) aleurone cells, does not inhibit the gibberellic acid-enhanced alpha-amylase (EC 3.2.1.1.) synthesis in barley aleurone layers if it is added 12 hours or more after the addition of the hormone. However, the accumulation of alpha-amylase activity after 12 hours of gibberellic acid can be decreased by abscisic acid. The accumulation of alpha-amylase activity is sustained or quickly restored when cordycepin is added simultaneously or some time after abscisic acid, indicating that the response of aleurone layers to abscisic acid depends on the continuous synthesis of a short lived RNA. By analysis of the newly synthesized proteins by gel electrophoresis with sodium dodecylsulfate, we observed that the synthesis of alpha-amylase is decreased in the presence of abscisic acid while the synthesis of most of the other proteins remains unchanged. From the rate of resumption of alpha-amylase production in the presence of cordycepin and abscisic acid, it appears that abscisic acid does not have a measurable effect on the stability of alpha-amylase mRNA. PMID- 16659446 TI - Galactolipid Synthesis in Vicia faba Leaves: II. Formation and Desaturation of Long Chain Fatty Acids in Phosphatidylcholine, Phosphatidylglycerol, and the Galactolipids. AB - The labeling kinetics of the fatty acids of phosphatidylcholine (PC), phosphatidylglycerol (PG), monogalactosyldiglyceride (MGDG), and digalactosyldiglyceride (DGDG) were examined after (14)CO(2) feeding and incubation of leaf discs of Vicia faba over 72 hours in continuous light. The results indicate a rapid accumulation and turnover of radioactivity into PC and PG fatty acids (oleic acid in PC and oleic and palmitic acids in PG). Radioactivity accumulates in MGDG and DGDG fatty acids much more slowly and continuously over 72 hours. Most of this activity is found in linoleic and linolenic acids; very little activity is found in the more saturated fatty acids. Little or no desaturation occurs in situ in conjunction with the galactolipids. The results suggest that PC and PG may act as "carriers" for MGDG and DGDG fatty acid synthesis. Analyses of the labeling patterns of the molecular species of MGDG after (14)CO(2) and (14)C-acetate feeding confirm that MGDG is formed by galactosylation of a preformed diglyceride containing predominantly unsaturated fatty acids. PMID- 16659447 TI - Lack of influence of phytochrome on membrane permeability to tritiated water. AB - The water permeability of tissues was investigated by measuring the efflux of (3)HHO from previously loaded (in darkness) etiolated bean buds (Phaseolus vulgaris L. var. Red Kidney), pea epicotyl segments (Pisum sativum L. var. Alaska), and oat coleoptile segments (Avena sativa L. var. Garry). Red light, far red light, or darkness was applied at the time of transfer of tissue from labeled to unlabeled medium. There were no effects of light on half-time for efflux or on the maximum level of radioactivity in the medium. Based on these criteria, phytochrome exerts no apparent control over water permeability. PMID- 16659448 TI - Phytochrome Stability in Vitro: II. A Low Molecular Weight Protective Factor. AB - The decay in vitro of crude and purified phytochrome preparations, measured as decline in spectrophotometrically detectable photoreversibility, can be retarded by the addition of an as yet unidentified "protective factor" from the green tissue of peas. The factor has an apparent molecular weight of 465, is stable at elevated temperatures and to oxygen and high light intensity. It confers optimal protection at pH about 7.8 and is not greatly affected by treatment with hydrolytic enzymes. Protective factor also protects phytochrome against accelerated decay resulting from the addition of Cu(2+); it may therefore function as a metal chelator. PMID- 16659449 TI - Photosynthetic Independence of Light-induced Anthocyanin Formation in Zea Seedlings. AB - Results are reported which support the view that the photosynthetic photosystems are not involved in the high irradiance response (HIR) phenomenon of light dependent anthocyanin biosynthesis in dark-grown Zea mays L. seedlings. A negative correlation between change in greening rates and change in light dependent anthocyanin accumulation rates with age was demonstrated. Lack of chlorophyll synthesis in a strain of maize possessing a temperature-sensitive lesion for chlorophyll synthesis could not be correlated with light-induced anthocyanin accumulation. Furthermore, seedlings totally lacking photosynthetic capabilities, either due to a genetic lesion or to excision of all photosynthetic tissue, had an enhanced rate of photoinduced anthocyanin formation. This evidence indicates that the HIR results in the initiation of processes that are in competition with chloroplast development for substrate in normal, intact seedlings. PMID- 16659450 TI - Bound auxin formation in growing stems. AB - The term "bound auxin" is herein used to describe auxin conjugates insoluble in organic solvents which dissolve indoleacetic acid (IAA) and its derivatives, but hydrolyzable by NaOH to release IAA. Bound auxin from pea stems was fractionated into water-soluble, water-insoluble/NaOH-hydrolyzable, and insoluble components. Formation of bound auxin commenced with 15 minutes of applying exogenous labeled IAA, and progressively increased in amount, relative to IAA uptake, over 6 hours. Formation was not restricted to any particular zone of the stem and occurred in both light- and dark-grown stems. A greater quantity of bound auxin was formed in light-grown stems, reaching 4.2 and 7.7%, of the IAA taken up, in the water soluble and water-insoluble/NaOH-hydrolyzable fractions after 6 hours. The presence of sucrose, during either the IAA treatment or an aging pretreatment had no effect, though 6 hours aging did cause a subsequent increase in the water insoluble fraction of the bound auxin. Bound auxin formation in light-grown stems was dependent on respiratory metabolism, being reduced by KCN. It was also reduced, compared to total uptake, by inhibitors of RNA, and protein synthesis (6 methylpurine and cycloheximide) but only when the inhibitors preceded auxin addition and were present for a 4-hour period. Addition of inhibitors following auxin had no effect, suggesting an early inductive effect of auxin on bound auxin formation. Inhibitors of cell elongation had no effect. Deoxyglucose, an inhibitor of glucan synthesis, had only a small effect on the water-soluble fraction. Bound auxin is an important auxin product in growing plants. Its function is unknown, but some possibilities are discussed. PMID- 16659451 TI - Stepwise generation of the natural oxidant in a reconstituted chloroplast system. AB - Isolated chloroplasts which have lost their envelopes and, in consequence, the soluble components which constitute the stroma, will nevertheless evolve O(2) when supplied with an artificial oxidant (the Hill reaction). They will also evolve O(2) with NADP as the Hill oxidant if supplemented with ferredoxin. With catalytic NADP, continuing O(2) evolution can be maintained by the inclusion of a suitable reaction or reaction sequence which reoxidizes NADPH.In the Benson Calvin cycle the terminal oxidant is glycerate 1,3-bisphosphate which is generated by phosphorylation of 3-phosphoglycerate, its immediate precursor. Experiments with a reconstituted chloroplast system are described in which this reaction sequence is catalyzed by stromal protein and supported by photophosphorylation of catalytic ADP. In the presence of CO(2), 3 phosphoglycerate can be progressively replaced by ribulose 1,5-bisphosphate, ribose 5-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, and finally by glyceraldehyde 3-phosphate. In this last instance the natural oxidant is regenerated from its own reduction product (via the carboxylation step) and the reaction sequence therefore involves the entire photosynthetic carbon cycle. PMID- 16659452 TI - Light Modulation of Enzyme Activity in Chloroplasts: Generation of Membrane-bound Vicinal-Dithiol Groups by Photosynthetic Electron Transport. AB - Inhibitor experiments indicate that photosynthetic electron transport is required for light activation of the pea (Pisum sativum) leaf chloroplast enzymes NADP linked glyceraldehyde-3-phosphate dehydrogenase, NADP-linked malic dehydrogenase, ribulose-5-phosphate kinase and sedoheptulose-1,7-diphosphate phosphatase, and for inactivation of glucose-6-phosphate dehydrogenase. Modulation of the activity of the dehydrogenases and kinase apparently involves a component preceding ferredoxin in the photosynthetic electron transport chain; activation of the phosphatase involves an electron transport component at the level of ferredoxin. Modulation of enzyme activity can be obtained in a broken chloroplast system consisting of membrane fragments and stromal extract. The capacity for light regulation in this system is reduced or eliminated when the membrane fraction is exposed to arsenite in the light or to sulfite in light or dark. Light-generated vicinal-dithiols seem therefore to be involved in modulation of the activity of the enzymes included in this study. PMID- 16659453 TI - Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato. AB - A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 mug of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.Immunological and electrophoretic properties of inhibitor I from tobacco crown gall tumor, callus, etiolated, and variegated tissues were compared. Agar immunodiffusion assays showed no apparent differences among precipitin reaction lines between inhibitor I of tumor, callus, variegated, and etiolated tissues. The immunoelectrophoretic mobilities of inhibitor I of tumor, variegated, and etiolated tissues were the same, but differed from that of either normal or crown gall callus tissues. These results suggest that different isoinhibitors of inhibitor I could account for the observed differences in electrophoretic mobilities, or that modification of the inhibitor has occurred sometime during, or after, its synthesis. PMID- 16659454 TI - Phospholipid synthesis and exchange in castor bean endosperm homogenates. AB - Crude organelle preparations from castor bean (Ricinus communis L.) endosperm rapidly incorporate CDP-((14)C)choline and CDP-((14)C)-ethanolamine into phosphatidylcholine and phosphatidylethanolamine, respectively. Separation of organelles by sucrose density gradient centrifugation following incubation with these substrates demonstrated that most of the (14)C phospholipids thus formed were present in the endoplasmic reticulum membranes, although label was also found in mitochondria, proplastids, and glyoxysomes. The phospholipid synthesizing enzymes, cholinephosphotransferase and ethanolaminephosphotransferase, are exclusively confined to the endoplasmic reticulum membrane fraction, suggesting that the appearance of (14)C-phospholipid in other organelles was due to phospholipid exchange. Phospholipid synthesis was inhibited by the cytoplasmic supernatant fraction. The active inhibitor in this fraction was not identified, but the inhibition was not significantly relieved by either dialyzing or boiling the supernatant. Phosphatidylcholine synthesis showed an absolute requirement for Mg(2+); the Michaelis constant was 1 mm. Ca(2+) was a potent inhibitor of Mg(2+)-stimulated phospholipid synthesis and enhanced the decay of (14)C-phospholipids from pre-labeled membranes, particularly when the membranes were resuspended in the cytoplasmic supernatant.The data are consistent with the concept that the endoplasmic reticulum is a major site of membrane proliferation where structural lipids, and possibly proteins, are inserted into, and thus expand, a pre-existing membrane fraction. Other organelle and cellular membranes could therefore originate from the proliferating endoplasmic reticulum by a process of membrane flow and differentiation. PMID- 16659455 TI - Glycosylated seryl residues in wall protein of elongating pea stems. AB - The protein content of salt-washed cell walls isolated from etiolated stems of Pisum sativum L. approximately doubled during elongation. In the same period the concentration in the wall of hydroxyproline, hydrazine-labile (= presumably glycosylated) serine, valine, tyrosine, lysine, and histidine increased markedly in comparison with other amino acids. After elongation was completed both the amino acid composition and the protein content of the cell wall changed only slightly. The ratio for the wall of hydrazine-labile seryl residues to hydroxyprolyl residues remained constant during and after elongation and was found to be 0.20. A linear relationship was established between the rate of elongation and the concentration in the wall of the hydroxyproline-rich glycoprotein both in vivo and in cut sections incubated in buffer. PMID- 16659456 TI - Changes in Ascorbic Acid Content during Growth and Development of Panicum miliaceum. AB - The present paper deals with changes in the ascorbic acid content of the shoot apex during its transformation from the vegetative to the reproductive state and its further development in Panicum miliaceum var. Samai Co. 1. Seedlings were exposed to 24-hour illumination, natural day, and 8 hours of illumination per day. Ascorbic acid was determined for the growing apex, stem, and leaf of the main shoot and for the individual branches produced on it at successive developmental stages.A several-fold increase in ascorbic acid content of the growing apex of the main shoot of plants grown under 8-hour illumination synchronizes with its transformation from the vegetative to the reproductive state. In plants grown under natural day and continuous illumination, which continue to grow vegetatively, the ascorbic acid content remains at a low level. Moving these plants to 8-hour illumination results in floral initiation and an increase in the ascorbic acid content. The high level of ascorbic acid is maintained through the gametogenic and embryogenic phases, but decreases during seed formation. A rise in ascorbic acid content of branch apices always occurs later than that in the main shoot but the increase again coincides with floral initiation in the branch apex. The ascorbic acid contents of stem and leaf are not significantly affected by the photoperiod. Hence, a massive upsurge in ascorbic acid content synchronizes with the transformation of the vegetative branch and shoot apices to the reproductive state. PMID- 16659457 TI - Effects of kinetin and root tip removal on exudation and potassium (rubidium) transport in roots of honey locust. AB - Exudation, (86)Rb transport, and water permeability were examined in excised roots of honey locust (Gleditsia triacanthos L.) treated by removing the tip 2 mm (tip-cut 2 mm) or tip 8 mm of the root, or by adding kinetin, or by both treatments. Tip removal increased the rate of exudation. Kinetin, 5 x 10(-6)m, inhibited exudation and Rb transport in tip-cut 2-mm roots; the inhibition was reversible. Kinetin inhibition of exudation was initially associated with lower K(Rb) transport and later with decreases in both ion transport and water permeability. Exudation was also inhibited at 10(-10) to 10(-7)m kinetin. Exudation from roots with intact tips was not altered by kinetin until after about 24 hours. Light during the exudation period had no significant (95%) influence on rate of exudation during the first 24 hours whether root tips were cut or kinetin applied.The results suggest the involvement of the root tip in regulating exudation in other parts of the root. This regulation might occur through cytokinin control of water permeability and the rate of ion transport. PMID- 16659458 TI - Effect of glycidate on glycolate formation and photosynthesis in isolated spinach chloroplasts. AB - Glycidate (2,3-epoxypropionate) increased CO(2) photoassimilation in intact spinach (Spinacia oleracea L.) chloroplasts in the presence of various inhibitors of photosynthesis, including O(2), arsenite, azide, iodo-acetamide, and carbonylcyanide 3-chlorophenylhydrazone. Although the mechanism by which glycidate enhances photosynthesis is obscure, the stimulatory effect cannot be ascribed to either an inhibition of glycolate formation, a specific interaction with the O(2) inhibition of photosynthesis, or a direct effect on the ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) reaction. The lack of a differential effect of glycidate on photosynthesis and glycolate formation in the isolated chloroplast was confirmed in whole leaf studies by the CO(2) compensation concentration assay. These results are at variance with the report that glycidate stimulates net photosynthesis in tobacco leaf disks by irreversibly inhibiting glycolate formation and thus photorespiration (Zelitch, I., 1974, Arch. Biochem. Biophys. 163: 367-377). PMID- 16659459 TI - Effect of Benzyladenine, 2,4-Dichlorophenoxyacetic Acid, and d-Glucose on myo Inositol Metabolism in Acer pseudoplatanus L. Cells Grown in Suspension Culture. AB - Suspension cultures of Acer pseudoplatanus L. cells grown for 15 days in medium (T. Murashige and F. Skoog. 1962. Physiol. Plant. 15: 473-497) contained 3% sucrose, 1 mg/l 6-benzylaminopurine (BA), and 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), referred to here as normal media, removed newly added myo-inositol 2-(3)H up to 100 mg/l in 24 hours and utilized up to 20% of this cyclitol for pectin biosynthesis. When the BA content of the growth medium was raised 10-fold, uptake of myo-inositol was drastically reduced and very little was available for pectin biosynthesis. Neither cell growth as measured by packed cell volume or by dry weight, nor monomer composition of pectic polysaccharides was affected by the increased level of cytokinin. Increasing, 2,4-d 10-fold instead of BA had little or no effect on myo-inositol uptake, although it did reduce the amount of myo inositol utilized for pectin biosynthesis. Cells grown 15 days in normal media failed to remove added myo-inositol if 3% d-glucose was included. The net result was similar to that found in cells grown in the high BA condition. If a trace amount of d-galactose-1-(14)C was supplied to cells after 15 days of growth in normal, high BA, or high 2,4-d media, there was no significant variation in uptake and utilization of label among the three growth conditions. PMID- 16659460 TI - Inhibition of ent-Kaurene Oxidation and Growth by alpha-Cyclopropyl-alpha-(p methoxyphenyl)-5-pyrimidine Methyl Alcohol. AB - Growth of Alaska peas (Pisum sativum) is inhibited more than 60% by alpha cyclopropyl-alpha-(p-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) treatment. This growth inhibition can be reversed completely by gibberellic acid application. Cell-free enzyme preparations from pea shoot tips and wild cucumber (Marah oreganus) endosperm were used to test the effects of this substituted pyrimidine on the incorporation of mevalonic acid-(14)C into ent-kaurene and ent kaurenol, respectively. Ancyidol (10(-6)m) completely blocks the conversion of ent-kaurene to ent-kaurenol. This result was confirmed with the wild cucumber endosperm system by testing the direct conversion of labeled ent-kaurene to ent kaurenol. Ancymidol at higher concentrations (10(-3)m) inhibits the incorporation of mevalonic acid-(14)C into ent-kaurene to a lesser extent. It is concluded that one mode of action of this growth regulator is the inhibition of gibberellin biosynthesis. PMID- 16659461 TI - Studies on the Secretion of Maize Root Cap Slime: IV. Evidence for the Involvement of Dictyosomes. AB - The involvement of dictyosomes and their vesicles in secretion of slime by maize root cap cells is demonstrated by kinetic and organelle fractionation experiments using l-fucose as a specific marker for the secreted slime. Pulse-chase experiments show that l-[1-(3)H]fucose is incorporated into two distinct fractions of root cap cells. Incorporation into a water-soluble, ethyl alcohol insoluble fraction of the homogenate has a peak at 20 minutes of chasing followed by rapid loss of label. Seventy per cent of the radioactivity in this fraction is secreted from the tissue during a 2-hour chase period. Incorporation of label from [(3)H]fucose into a water-insoluble fraction is kinetically different suggesting that in situ incorporation of label is occurring into the cell wall. Labeling of the water-soluble, ethyl alcohol-insoluble fraction with an (14)C amino acid mixture differs from that of [(3)H]fucose. Thus, while release of the [(3)H]fucose-containing polymer begins after 10 to 15 minutes of chasing, the release of the (14)C-amino acid polymer is delayed an additional 5 to 10 minutes and occurs at a lower rate. Cesium chloride density gradient centrifugation of secreted material labeled with radioactivity from [(3)H]fucose indicates the presence of only one major component having a buoyant density similar to that of purified root cap slime (1.63 g cm(-3)). Sucrose density gradient centrifugation of homogenates of [(3)H]fucose-labeled root cap tissue shows that radioactivity in nondialyzable material occurs as a broad band between densities 1.12 and 1.18 g cm(-3) with a peak at density 1.15 g cm(-3), the same density at which dictyosomes were localized by electron microscopy. Autoradiography of organelle fractions shows that radioactivity was associated almost exclusively with dictyosomes. PMID- 16659462 TI - Inhibition of linolenic Acid synthesis and modification of chilling resistance in cotton seedlings. AB - The temperature at which cotton seeds (Gossypium hirsutum L.) germinated influenced the fatty acid composition of the polar lipids of developing root tips. Seeds were germinated at 15, 20, 25, and 30 C. As the temperature decreased the linolemic acid content of the polar lipid fraction increased. Sandoz 9785[4 chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone] reduced the low temperature induced increase in linolenic acid content of the polar lipids and reduced seedling ability to withstand 8 C chilling. The results are consistent with the conclusion that chilling resistance in cotton seedlings is related to the level of linolenic acid in the polar lipids in the developing root tips. PMID- 16659463 TI - Events Surrounding the Early Development of Euglena Chloroplasts: 7. Inhibition of Carotenoid Biosynthesis by the Herbicide SAN 9789 (4-Chloro-5-(methylamino)-2 (alpha,alpha,alpha,-trifluoro-m-tolyl)-3-(2H)pyridazinone) and Its Developmental Consequences. AB - The herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(alpha,alpha,alpha-trifluoro-m tolyl-3- (2H)pyridazinone) blocks carotenoid synthesis in growing and resting cells of Euglena at concentrations of 20 to 100 mug/ml without affecting cell viability. Although the inhibition is immediate and complete, in resting cells no decrease in already synthesized carotenoids is found indicating a lack of turnover. In cells growing in the dark, carotenoids are diluted out as the cells divide. Cells dividing in the light in the presence of SAN 9789, eventually lose viability, presumably because of photooxidations usually prevented by carotenoids. During 72 hours of light-induced plastid development in dark-grown resting cells, none of the usual carotenoids increase while phytoene accumulates, indicating that SAN 9789 blocks carotenoid synthesis at this point. Chlorophyll synthesis and membrane formation are also blocked by the herbicide, but these inhibitions appear to be secondary to the inhibition of carotenoid synthesis. That carotenoid levels are strongly correlated with and may control the synthesis of chlorophyll and the formation of plastid membranes is suggested by the following data. (a) If dark-grown dividing cells are placed in the presence of the herbicide for various periods, rested and exposed to light in the presence of the drug, different amounts of carotenoids remain in the cells and the amount of chlorophyll finally synthesized is proportional to the amount of carotenoids present. (b) Photodestruction of chlorophyll is excluded, since the same amounts of chlorophyll are formed at intensities of 10 to 100 foot-candles of light. (c) Photoconversion of protochlorophyll(ide) to chlorophyll(ide) in dark-grown cells is not blocked by the herbicide. (d) Initial rates of chlorophyll synthesis are the same in treated and nontreated cells. (e) The extent of membrane formation appears to parallel the amount of carotenoids present as judged by electron microscopy. PMID- 16659464 TI - Effect of freezing and cold storage on phospholipids in developing soybean cotyledons. AB - Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. "Harosoy 63" were frozen with liquid N(2), dry ice, or stored in a freezer (-20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at -20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with (3)H, and fatty acids with (14)C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing. PMID- 16659465 TI - Respiratory Transition during Seed Germination. AB - Experiments with germinating seeds of Wayne soybean (Glycine max Merr.) show that between the 4th and the 8th hour of germination, respiration experiences a transition from predominantly "alternate" respiration, which is sensitive to salicylhydroxamic acid, to a cyanide-sensitive respiration. The dependence of early germination stages on alternate respiration is reflected in several types of seed functions, including subsequent root growth rate, chlorophyll synthesis, and germination itself. The early period of germination is shown to require a normal O(2) tension, which is no longer a requirement at later stages. The changing sensitivity to cyanide and to salicylhydroxamic acid is found to be common to seven different types of germinating seeds. It is proposed that the alternate pathway of respiration provides something essential for the completion of the earliest stages of seed germination. PMID- 16659466 TI - Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts. AB - Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO(2) reduction and were insensitive to additions of photosynthetic intermediates when CO(2) was available at saturating concentrations. Photosynthetic rates were measured polarographically as O(2) evolution, and the extent of the reduction of substrate was estimated from the amount of O(2) evolved. With CO(2) as substrate, inhibition of photosynthesis by O(2) was dependent on pH. At pH values above 8, rates of O(2) evolution were strongly inhibited by O(2) and only a fraction of the added bicarbonate was reduced before O(2) evolution ceased. The extent of O(2) evolution declined with increasing O(2) concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O(2) levels, but the extent of O(2) evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O(2) evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O(2) concentration. In contrast, there was little effect of O(2) and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O(2). The results are discussed in terms of a balance between photosynthetic O(2) evolution and O(2) consumption by the ribulose diphosphate oxygenase reaction. PMID- 16659467 TI - Selection of diquat resistance photosynthesis mutants from maize. AB - Resistance of a seedling to the herbicide 1,1'-ethylene-2,2'-dipyridylium bromide (diquat) can be used as a selective technique for photosynthesis mutants in Zea mays L. Diquat requires reduction by the light reaction in order to kill leaf cells and, therefore, nonphotosynthetic mutants survive. This technique was tested using known mutants and is applicable to larger samples of plants than previous techniques. Resistance to diquat should allow selection of mutants on the oxidizing side of photosystem II which are not previously available in higher plants. PMID- 16659468 TI - Stem sensitivity and ethylene involvement in phototropism of mung bean. AB - A system is described for the examination of phototropism in the epicotyl of a dicot seedling, mung bean (Phaseolus aureus Roxb.), under conditions approximating nature, including the use of intact, nonetiolated plants exposed to elevated, continuous, white, unilateral light. It is found that in this system perception of the phototropic stimulus by the leaves alone cannot account for the curvature, and that exposure of the stem is also necessary. The phototropic response was found to be strongly altered in nonintact plants. Hypobaric treatment indicates that ethylene may participate in phototropism, possibly by acting as an inhibitor of auxin transport. PMID- 16659469 TI - Osmotic shrinkage as a factor in freezing injury in plant tissue cultures. AB - Haplopappus gracilis and Acer saccharum tissue culture cells are extremely sensitive to freezing injury, and exhibit a decrease in survival from 98% at -1 C to 4% at -3 C (Haplopappus) and 92% at -3 C to 13% at -5 C (Acer) when suspended in distilled H(2)O, seeded at -1 C, and then cooled by 0.1 C/minute. Similar results are obtained when cells are suspended in growth medium. The extent of shrinkage of cells during freezing can be duplicated by exposure of the cells to plasmolyzing solutions of nonpenetrating substances (Delta T(f) = 1.86 phivm). Solutions of sucrose and glycerol that produce extensive plasmolysis cause a decrease in survival within 3 to 5 minutes at room temperature, and the higher the molality to which the cell is exposed the greater the injury. Also, the rate of rehydration of the plasmolyzed cell and of the frozen cell affects its survival, with the slower rate being more beneficial. The close correlation between the decrease in survival at subzero temperatures and the decrease in survival when cells are placed in solutions having osmolalities, which could produce the same extent of shrinkage as these killing temperatures, suggests that this shrinkage is related to freezing injury in tissue culture cells. PMID- 16659470 TI - Peridinin-Chlorophyll a Proteins of the Dinoflagellate Amphidinium carterae (Plymouth 450). AB - The marine dinoflagellate Amphidinium carterae (Plymouth 450) releases several water-soluble peridinin-chlorophyll a proteins after freezethawing. These chromoproteins have a molecular weight of 39.2 x 10(3) and are comprised of noncovalently bound peridinin and chlorophyll a and a nonoligomeric protein. They have distinct isoelectric points and may be resolved into six components by either isoelectric focusing on polyacrylamide gel or ion exchange chromatography. The predominant chromoprotein, which has a pI of 7.5, constitutes about 90% of the extractable peridinin-chlorophyll a protein. It consists of an alanine-rich apoprotein of molecular weight 31.8 x 10(3) stoichiometrically associated with 9 peridinin and 2 chlorophyll a molecules. Additionally, the peridinin-chlorophyll a proteins with pI values of 7.6 and 6.4 were purified and found to have amino acid and chromophore composition essentially identical with the pI 7.5 protein. Peridinin-chlorophyll a protein, pI 7.5, after treatment at alkaline pH was transformed into several more acid pI forms of the protein, strongly suggesting that the naturally occurring proteins are deamidation products of a single protein. Fluorescence excitation and emission spectra demonstrate that light energy absorbed by peridinin induces chlorophyll a fluorescence presumably by intramolecular energy transfer. The peridinin-chlorophyll a proteins presumably function in vivo as photosynthetic light-harvesting pigments. PMID- 16659471 TI - Estimated Drainage of Carbon from the Tricarboxylic Acid Cycle for Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose Cells. AB - The amount of carbon (mumoles of carbon atoms) drained from the tricarboxylic acid cycle for protein synthesis was compared with mumoles of CO(2) released from the cycle at 2-day intervals during the growth of suspension cultures of Paul's Scarlet rose. We concluded that during the period of most rapid protein synthesis (day 0-4) one-sixth as much carbon was drained from the tricarboxylic acid cycle for protein synthesis as was released as CO(2). By day 8, one-thirtieth of the amount of carbon released as CO(2) was incorporated into protein. Net protein synthesis stopped on day 8, but the evolution of CO(2)/culture continued at its maximum rate until day 10.Similar ratios were calculated based on the recovery of (14)C in protein versus CO(2) following a 3-hr provision of labeled substrates to 3-day-old cells (age of maximum protein synthesis). Provision of acetate-1-(14)C and acetate-2-(14)C indicated from one-eighth to an equal amount of carbon was incorporated into protein as was released as CO(2). When (14)C-labeled intermediates of the tricarboxylic acid cycle were provided, the ratio of (14)C incorporated into protein versus that evolved in CO(2) ranged from 1/0.9 to 1/4.9.Following a critical analysis of the methods used, it was concluded that during periods of rapid protein synthesis, a conservative estimate of the amount of carbon drained from the tricarboxylic acid cycle for protein synthesis was one fourth of the amount evolved as CO(2) from the cycle. PMID- 16659472 TI - The Occurrence of Peroxide in a Perennial Plant, Populus gelrica. AB - A large amount of peroxide was found in twigs of poplar, Populus gelrica, which was grown in the field under natural conditions. The peroxide found in xylem and living bark was about 1.2 and 0.5 mumoles per gram dry weight sample, respectively, and served as a substrate both for catalase and cytochrome c peroxidase. PMID- 16659473 TI - Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoe daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism. AB - NAD-specific "malic" enzyme (EC 1.1.1.39) has been isolated and purified 1200 fold from leaves of Kalanchoe daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO(4) (2-), suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn(2+), which cannot be replaced by NADP or Mg(2+). For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn(2+), and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 mumole/hr.mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO(2) for recycling during cessation of gas exchange in periods of severe drought. PMID- 16659474 TI - Catalase, Peroxidase, and Polyphenoloxidase Activities during Rice Leaf Senescence. AB - The activities of catalase, peroxidase, and polyphenoloxidase were studied in attached and detached rice (Oryza sativa L. cv. Ratna) leaves. Catalase activity decreased while peroxidase and polyphenoloxidase activities increased during senescence of both attached and detached rice leaves. Kinetic (5 mum) and benzimidazole (1 mm), which are known to delay the senescence of detached rice leaves, retarded the decrease of catalase activity during detached leaf senescence. On the other hand, these chemicals accelerated the increase of peroxidase and polyphenoloxidase activities over the water control. Total phenolics accumulated in detached and darkened rice leaves, but in attached leaf senescence in light no accumulation of phenolics was observed. PMID- 16659475 TI - Characteristics of a 4-hydroxycinnamate hydroxylase purified from sorghum leaves. AB - A membrane-associated 4-hydroxycinnamate hydroxylase (p-coumarate hydroxylase) from green leaves of Sorghum bicolor has been purified by mercaptoethanol treatment, ammonium sulfate fractionation, and chromatography on hydroxyapatite and agarose 1.5m. Ascorbate (or reduced pyridine nucleotide) is an obligatory electron donor for the hydroxylation of 4-hydroxycinnamate, but not for p-cresol. The most highly purified fraction has a 260/280 ratio of approximately 1 and contains carbohydrate or other orcinol-reacting materials. The hydroxylase enzyme exists in series of aggregated forms at pH 6 ranging from about 60,000 to 1.5 million depending on the ionic strength, but even at high ionic strengths the bulk of the enzyme exists in relatively high molecular weight aggregates. PMID- 16659476 TI - Genotypic variation in carboxylation of tomatoes. AB - The gas exchange characteristics of 24 genotypes of Lycopersicon esculentum Mill. and one of L. minutum were measured with an infrared gas analyzer and dew point hygrometer in an open system. Net carbon exchange (NCE) and transpiration rate were measured at 50, 100, 150, and 300 mu1 1(-1) CO(2), and a regression of NCE versus internal lead [CO(2)] estimates was calculated. The slope of the regression curve at the CO(2) compensation point was used as the measure of carboxylation efficiency (CE). Significant genotypic differences for CE were obtained. Differences in CE did not appear to be due to differences in diffusive resistance defined as the sum of the boundary layer resistance (r(a)) and the stomatal plus cuticular resistance (r(1)). There was no correlation (r = -0.07) between (r(a) + r(1)) and CE. Within groups with nonsignificantly different means for (r(a) + r(1)) there were genotypes with extremes for CE.The zero CO(2) intercept has been used as an indication of photorespiration. Application of this method revealed a strong inverse relationship between CE and the intercept value, indicating either that photorespiration is related directly to CE or that this method is unreliable for estimating photorespiration. The fact that the variation in CE occurs at light saturation suggests that the observed differences in CE and rates of NCE are determined either by: (a) the concentration and/or kinetic properties of the photochemical reaction centers and associated electron transfer components as they affect the supply of NADPH and ATP and consequently the levels of Calvin cycle intermediates; or (b) the concentration and/or kinetic properties of ribulose 1,5-diphosphate carboxylase. PMID- 16659477 TI - Isolation and bicarbonate transport of chloroplast envelope membranes from species of differing net photosynthetic efficiency. AB - A three-phase discontinuous sucrose gradient yielded two fractions of chloroplast envelope membranes from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L., mesophyll and undifferentiated chloroplasts). These species were selected to represent plants with fast photorespiration and slow net photosynthesis, fast photorespiration yet fast net photosynthesis, and slow photorespiration and fast net photosynthesis, respectively. Buoyant densities were 1.08 and 1.11 g cm(-3). The light fraction contained primarily single (incomplete) membrane vesicles and the heavy fraction double (complete) ones. Enzymic, chemical, and electron microscopic examination of the complete envelope membranes showed a lack of microbial, microsomal, mitochondrial, and lamellar membrane contamination as well as stromal contamination. Envelope membranes for all species examined were found to contain 2 to 4% of the total chloroplast protein and yields of about 0.2 to 0.4 mg of protein were obtained from 40 g leaves. An Mg(2+)-dependent nonlatent ATPase, a marker enzyme for chloroplast envelope membranes, had the following activities (mumoles of phosphate released/hr(-1) mg protein(-1)): spinach, 77; sunflower, 163; old maize, 126; and young maize, 87. Bicarbonate transport was directly correlated with levels of ATPase activity in spinach and sunflower envelope membranes. Transport of HCO(3) (-) with sunflower envelope membranes approached that of young maize. PMID- 16659478 TI - Glutamine synthetase regulation by energy charge in sunflower roots. AB - Energy charge [(ATP) + (1/2) (ADP)]/[(ATP) + (ADP) + (AMP)] and glutamine synthetase activity (transferase reaction) of roots increase in a near congruent manner when decotyledonized sunflower plants (Helianthus annuus L. var. Mammoth Russian) are grown in nitrate for 9 days. Replacement of nitrate with ammonium for the final 2 days leads to a higher energy charge and increased enzyme activity. Similar correlations occur when nitrate plants are placed on a zero nitrogen regimen and when they are subjected to continuous darkness. A rank order correlation of 0.72 is obtained for all data. Control concepts such as adenylylation-deadenylylation and ammonium inhibition of enzyme synthesis are not supported by the data. Energy charge-enzyme activity plots support the view that glutamine synthetase of sunflower roots is subject to control by end products of glutamine metabolism. Alanine appears to exert a modulating effect on the regulation of glutamine synthetase by energy charge. PMID- 16659479 TI - Method for growing plants aeroponically. AB - A simple, inexpensive system for growing plants with their roots bathed in nutrient mist is described. The aeroponics system uses a spinner from a home humidifier to propel nutrient solution into a polyethylene-lined plywood box atop which plants are supported on plastic light-fixture "egg crating." Success in growing a number of herbaceous and woody species, including nodulated legumes and nonlegumes, is reported. PMID- 16659480 TI - Calcium Requirement for Indoleacetic Acid-induced Acidification by Avena Coleoptiles. AB - The ionic specificity of IAA-induced acidification by Avena coleoptiles was studied, using zwitterionic, presumably impermeant buffers. The acidification was almost totally dependent on divalent cations with an order of effectiveness of Ca(2+) >/= Sr(2+) > Mn(2+), Mg(2+); whereas other polyvalent cations tested were ineffective. The Ca(2+) response was IAA-dependent. The CaCl(2) concentration was optimal at 0.3 to 1 mm and inhibitory at higher concentrations. Sr(2+) inhibited Ca(2+)-dependent acidification and monovalent cations such as K(+) did not induce additional acidification in the presence of optimal CaCl(2). These data are consistent with a mechanism for IAA-induced acidification involving a Ca(2+) H(+) exchange. PMID- 16659481 TI - Developmental expression of a catalase inhibitor in maize. AB - The expression of an endogenous catalase inhibitor has been studied during development of Zea mays. In the 3-day seedling, the inhibitor is expressed primarily in the scutellum and in the aleurone layer of the endosperm. These tissues also show the highest catalase activity at this stage. Inhibitor expression has also been studied temporally in the scutellum, roots, and shoot over the first 12 days of germination. Inhibitor expression shows an inverse relationship with catalase activity in the scutellum and in the shoot. The relationship is less rigid in the root, due probably to the low levels of inhibitor found in that tissue. The role of the inhibitor in catalase regulation is discussed. PMID- 16659482 TI - Boron uptake by excised barley roots: I. Uptake into the free space. AB - At 2 C, all boron accumulated by excised barley roots (Hordeum vulgare L. cv. Herta) remains in the free space; i.e. active uptake is nil at this temperature. Three component fractions of free space B were apparent: (a) a surface contaminant film of B on blotted roots, (b) water free space B, and (c) B reversibly bound in the cell walls. A stoichiometric release of H(+) from the roots in the presence of B indicated that B was bound by borate complexes with polysaccharides in the cell walls. Polysaccharide-borate complexes are much less stable than those of monosaccharides, and the bound B fraction could be readily removed by rinsing the roots in the presence of a monomeric polyol possessing the necessary cis-diol configuration. Cell wall material separated from excised barley roots had a B binding capacity 66% greater than that of intact roots.A 30 minute rinse in distilled H(2)O or 0.5 mm CaSO(4) was required to remove all cell wall-bound B from the roots after a 30-minute uptake period. Thus, although B in the contaminant surface film and the water free space is rinsed from the roots within 10 minutes, a 30-minute rinse is essential if all reversibly accumulated B is to be removed from the free space. PMID- 16659483 TI - Morphology of Arabidopsis Grown under Chronic Centrifugation and on the Clinostat. AB - Morphological measurements were made on populations of Arabidopsis thaliana grown from seed for 21 days under essentially constant environmental conditions except for the influence of gravitational or centrifugal accelerations. Growth conditions were what had been proposed for experiments in an artificial satellite. Observations are reported for plants grown at normal 1-g upright or on horizontal clinostats and for plants grown on a centrifuge. Increased g-force, up to 15 times normal, was found to have significant but small effects on some morphological end points. The plants' sensitivity to the magnitude of the g-force was much less than to its vector direction.Data from centrifuge experiments (which determined the g-functions for particular characters) were extrapolated to zero-g to predict a set of morphological characteristics of a plant developing in the satellite environment. As an alternative means of predicting properties of a zero-g plant, characteristics of plants grown on horizontal clinostats were measured. The results of these two predictive methods were not in agreement.Clinostat grown plants were morphologically distinct from upright stationary controls. When plants were grown while rotating in the upright position on vertical clinostats they were similar to stationary plants also grown upright, but there were small differences some of which were statistically significant. PMID- 16659484 TI - A Mung Bean Assay for Malformin-induced Growth Stimulation. AB - A bioassay employing green or etiolated cuttings of Phaseolus aureus Roxb. was developed for determining malformin-induced growth stimulation in light. Growth enhancement of green cuttings was more rapid and relatively greater than that of etiolated cuttings. Cuttings from green seedlings responded less as seedlings aged; those from etiolated seedlings responded more. Malformin also stimulated the growth of green or etiolated seedlings in light. Most growth enhancement induced by malformin occurred in the upper 1 cm of the stem. Using green cuttings, malformin stimulated stem elongation relatively more when cotyledons, leaves, or especially apical buds were removed. Although malformin failed to stimulate elongation of 2-cm stem sections "floated" on solutions in Petri dishes, it stimulated elongation of sections when they were upright. High concentrations of indoleacetic acid inhibited growth enhancement by malformin. When gibberellin and malformin were combined, growth enhancement was nearly additive. PMID- 16659485 TI - Changing ratios of phototransformable protochlorophyll and protochlorophyllide of bean seedlings developing in the dark. AB - Protochlorophyll (Pchl) and protochlorophyllide (Pchlide) are at comparable levels in 2-day-old (young) etiolated bean leaves (Phaseolus vulgaris L. var. Red Kidney). During subsequent development in the dark, both pigments increase, but the rate of Pchlide increase is greater than that of Pchl, leading to the commonly observed predominance of Pchlide beyond 7 days (old leaves). Both protopigments are phototransformable to their respective chlorophyll(ide) photoproducts throughout dark development. The rate of protopigment regeneration in young leaves after illumination is rapid and displays no lag, whereas this process in old leaves begins slowly and achieves only about one-fifth the rate of younger leaves. The rate of chlorophyllide esterification is also faster in the younger tissue. Since the proplastid-related properties of young bean leaves are quite similar to those of Euglena, young leaves and Euglena may represent an evolutionarily primitive case compared with older bean leaves which contain etioplasts. Since Euglena and young beans green perfectly well when exposed to light, the extensive modifications associated with prolonged dark growth do not seem to be obligatory for plastid development. The properties of older beans are viewed as being the consequence of prolonged etiolation which may provide a faster rate of plastid development and appearance of photosynthesis as the plant nears the limits of its stored reserves. PMID- 16659486 TI - Studies on lipid synthesis and degradation in developing soybean cotyledons. AB - The metabolic activity of individual lipid classes found in developing soybean cotyledons (Glycine max.) is estimated by determining the degradation rate of the compound under given conditions. Pulse-labeling and dual substrate labeling are used to evaluate this parameter. These studies indicate first order decay kinetics for phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acyl-phosphatidylethanolamine, diglyceride, and zero order kinetics for triglyceride in cotyledons var. "Harosoy 63" at 30 days after flowering. Decay coefficients for acyl groups and lipid-glycerol moieties within specific lipid classes from either method are comparable. Half-life (t((1/2))) calculations from the decay coefficients indicate extremely rapid turn-over rates (0.08 to 3.4 hours at 25 C) and suggest similar turnover rates of acyl groups and lipid-glycerol in diglyceride and all phospholipids except N acylphosphatidylethanolamine where acyl groups are replaced independent of the glycerol moiety. These experiments reveal not only different metabolic activity between lipid components of soybean cotyledons, but also describe a new method for measuring lipid turnover in plants. PMID- 16659487 TI - Phosphatidylcholine synthesis in castor bean endosperm. AB - Three pathways for phosphatidylcholine synthesis were assayed in castor bean (Ricinus communis var. Hale) endosperm. Phosphatidylethanolamine: S adenosylmethionine methyl transferase occurred predominantly in the endoplasmic reticulum fraction, but some activity appeared in the mitochondria. Phosphorylcholine glyceride transferase occurred exclusively in the endoplasmic reticulum. The phosphorylcholine glyceride transferase activity was approximately 20-fold greater than the methylation pathway in the endoplasmic reticulum. No exchange activity was found. The Michaelis constant for the methylation was 31 mum for S-adenosylmethionine; phosphatidylethanolamine promoted the reaction slightly while other intermediates stimulated it by about 50%. The pH optimum was 9. Phosphorylcholine glyceride transferase had a Michaelis constant of 9.7 mum for cytidine diphosphate choline but variable results were obtained from diglycerides. The pH optimum was 7.5 and a divalent cation was required, Mg(2+) giving the greatest stimulation. PMID- 16659488 TI - Interaction of N-Methylphenazinium Methyl Sulfate with the Thylakoids of Illuminated Chloroplasts in the Presence of 3-(3,4-Dichlorophenyl)-1,1 dimethylurea. AB - The light-dependent quenching of the chlorophyll a fluorescence at room temperature by N-methylphenazinium methyl sulfate (PMS) was investigated with isolated chloroplasts inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Other investigators have considered this quenching to be a consequence of the formation of a high energy membrane state related to photophosphorylation.It was found that the fluorescence quenching was accompanied by a reversible bleaching of PMS which could not be attributed to its photo-reduction. Both fluorescence quenching and PMS bleaching, and their dark reversal, were similarly affected by the experimental conditions. In particular, they were only slightly sensitive to the presence of uncouplers of photophosphorylation. However, bleaching and fluorescence quenching were strongly inhibited by uncouplers when the creation of a proton gradient across the thylakoid membrane was made possible by a presence of some PMS in its reduced form.It is suggested that the bleaching of PMS resulted from its binding to the thylakoid membranes when charges became available during conformational changes as a consequence of the light reaction in photo-system I. The same conformational changes were apparently responsible for the fluorescence quenching, but a large pH gradient across the membranes was not essential. PMID- 16659489 TI - Cytokinins in Vinca rosea L. Crown Gall Tumor Tissue as Influenced by Compounds Containing Reduced Nitrogen. AB - Several compounds containing reduced nitrogen markedly increased the yields of cell-division compounds extractable from an A6 Vinca rosea L. crown gall tumor tissue. Casein hydrolysate, several amino acids, and ammonium salts were effective. Both trans-zeatin and ribosyl-trans-zeatin were substantially increased in total amount per culture and in concentration. These two compounds have been identified by several criteria including mass spectra. The reduced nitrogen treatments also caused the appearance of a cytokinin not previously detected in this tissue; it has not yet been identified. The tumor tissue rapidly absorbed [8-(14)C]adenine from a liquid medium. Within 1 hour, the tissue converted some of the adenine to zeatin and ribosylzeatin, and greater degrees of conversion occurred in 2-, 4-, and 8-hour periods. The tissue grown on a medium containing ammonium chloride accumulated considerably greater quantities of the two cytokinins made from the labeled adenine during each incubation period. PMID- 16659490 TI - Interactions between Mitochondria and Chloroplasts in Cells: I. Action of Cyanide and of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea on the Spore of Funaria hygrometrica. AB - The effects of cyanide and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on photosynthesis and respiration of intact chlorophyllic moss (Funaria hygrometrica) spore was investigated. Thirty micromolar cyanide strongly inhibited dark respiration, was without effect on photosynthesis at high light intensities (above the saturation plateau values), and stimulated photosynthesis at low light intensities (below the saturation plateau values). Three hundred nanomolar DCMU inhibited the photosynthesis and was without effect, even under light conditions, on the dark respiration. It seems likely, therefore, that in the chlorophyllic moss spore the cytochrome oxidase pathway is not functioning under high light intensities unless the photosynthesis is inhibited by DCMU. PMID- 16659491 TI - Water deficit and ethylene evolution by young cotton bolls. AB - Ethylene evolution and abscission of young cotton (Gossypium hirsutum L.) bolls were shown, in earlier papers, to increase when plants were subjected to conditions that decreased photosynthesis and sugar content of bolls (dim light, long warm nights). Moisture stress also increased ethylene evolution by young bolls, but it did not decrease their concentrations of fructose, glucose, or sucrose. When detached bolls were incubated for 16 or 24 hours at high or low humidity, their rate of ethylene evolution increased markedly at low humidity and slightly at high humidity. These results suggest that water deficit stimulates ethylene evolution by young bolls directly through partial desiccation, but do not exclude the possibility of a stimulus from moisture-stressed plants. Although attached and detached bolls both lost only a small percentage of their water content, detached bolls lost more for a given rate of ethylene evolution than bolls on moisture-stressed plants. The increased rate of ethylene evolution by young cotton bolls on plants subjected to a water deficit is probably adequate, in many cases, to cause their abscission. PMID- 16659492 TI - Role of the endoplasmic reticulum in glyoxysome formation in castor bean endosperm. AB - Homogenates of the endosperm of castor bean (Ricinus communis var. Hale) were prepared at intervals during germination and fractionated on sucrose gradients. Early in germination when glyoxysomes were being produced, a substantial proportion (50%) of the activities of malate synthetase and citrate synthetase was recovered in the membranes of the endoplasmic reticulum (mean density 1.12 grams per cubic centimeter). This proportion declined to less than 10% at 4 days when the glyoxysomes were fully developed.Gradient fractions challenged by antiglyoxysome-protein antiserum in double immunodiffusion assay revealed strong antigenic response in the endoplasmic reticulum membranes. The results support the view advanced earlier that glyoxysomes are derived directly from the endoplasmic reticulum. PMID- 16659493 TI - Molecular integrity of plant ribosomal ribonucleic Acid. AB - Thermal denaturation of plant ribosomal RNA followed by gel fractionation shows that although a large percentage of molecules contain breaks in the polynucleotide chain, 25S and 18S RNAs do exist as unique molecular species. Values for the rate constant of hydrolysis under routine denaturing conditions are of the order of 10(-7) to 10(-8) sec(- 1) and these are shown not to be a result of ribonuclease activity. This high rate of hydrolysis and the use of insensitive fractionation procedures may account for the reported absence of a 25S rRNA molecule and its apparent conversion to a molecule similar in size to 18S RNA. PMID- 16659494 TI - A Method for Eliminating the Contribution of Agrobacterium tumefaciens to Studies of Nucleic Acid Biosynthesis in Crown Gall Tumors. AB - A procedure is described that permits the use of radiotracer methods for examining crown gall tumors in the presence of the initiating agent Agrobacterium tumefaciens. On the basis of sensitivity testing using the disc diffusion technique and the minimum inhibitory concentration procedure and incorporation of radioactive phosphorus into nucleic acids, it was determined that A. tumefaciens was most sensitive to tetracycline, oxytetracycline, and methacycline. It was further demonstrated that nucleic acid synthesis in tumor tissue from four representative plants including tomato (Lycopersicum esculentum L.), Bryophyllum daigremontiana (Hamet and Perrier), pinto beans (Phaseolus vulgaris L.), and carrot (Daucus carota L.) was unaffected by a concentration of tetracycline (50 mug/ml) which completely inhibited nucleic acid synthesis in the bacterium. On this basis a procedure is described which first eliminates surface contamination and then uses tetracycline to nullify the contribution of A. tumefaciens in radioactive studies using tumors initiated by this bacterium. PMID- 16659495 TI - Effects of red light and ethylene on growth of etiolated lettuce seedlings. AB - Low concentrations of ethylene inhibit hypocotyl elongation of etiolated lettuce seedlings (Lactuca sativa cv. Grand Rapids), whereas red light does not inhibit it. The plumular hook tightens in response to either ethylene or red light. A combination of these two factors gives an additive response. Red light has no effect on ethylene production and red light will cause hook closure even under hypobaric pressure which removes endogenous ethylene. This suggests that ethylene and red light act independently in causing hook closure.These findings differ from those about etiolated beans where red light causes a decrease in ethylene production and a straightening of the plumular hook. PMID- 16659496 TI - Influence of the nutrient medium on the recovery of dividing cells from tobacco protoplasts. AB - Systematic tests resulted in a nutrient solution containing the following, in milligrams per liter, for the culture of protoplasts isolated from Nicotiana tabacum L. callus cells: Murashige and Skoog salts (T. Murashige and F. Skoog, 1962. Physiol. Plant. 15: 473-497); sucrose, 15,000; mannitol, 110,000; alpha naphthaleneacetic acid, 0.6; kinetin, 0-0.1; thiamine.HCl, 10; pyridoxine.HCl, 10; nicotinic acid, 5; myo-inositol, 100; and glycine, 2. In this medium, regeneration of cell wall has been observed in 85% and resumption of cell division among 35% of the protoplast isolates. PMID- 16659497 TI - Studies of sulfate utilization of algae: 15. Enzymes of assimilatory sulfate reduction in euglena and their cellular localization. AB - Crude extracts of wild-type Euglena grown in the light (WTL) or in the dark (WTD) and a mutant lacking detectable plastid DNA (W(3)BUL) contain adenosine 5' phosphosulfate (APS) sulfotransferase. Isotope dilution experiments indicate that adenosine 3'-phosphate 5'-phosphosulfate (PAPS) sulfotransferase is absent.Thiosulfonate reductase, requiring addition of NADH or NADPH but not ferredoxin, and O-acetyl-l-serine sulfhydrylase, the two other enzymes of the bound intermediate pathway of assimilatory sulfate reduction, are also present. Increasing levels of all three enzymes were found in WTL, WTD, and W(3)BUL during logarithmic growth but the various activities were similar at comparable stages of growth in all three types of cell.These results show that the three enzymes are not coded in the chloroplast DNA and are not restricted to Euglena cells having fully developed chloroplasts. Consistent with this, they do not increase during light-induced chloroplast development in resting cells and are found to be enriched in the mitochondrial fraction. Further resolution of this fraction on sucrose gradients shows that the APS sulfotransferase is associated with both the microbody (glyoxysomal) and mitochondrial fractions while the thiosulfonate reductase and O-acetyl-l-serine sulfhydrylase are associated only with the mitochondria. Thus the three known enzymes of the bound pathway of assimilatory sulfate reduction are present in Euglena mitochondria.Although the activity of the entire bound pathway (APS to cysteine) is low in extracts, addition of dithiothreitol which releases free sulfite from the product of the APS sulfotransferase reaction, causes an increase in reduction activity indicating that a sulfite reductase is also present. It remains to be shown which reducing system is the significant one in vivo in Euglena. PMID- 16659498 TI - Indoleacetic Acid synthesis in soybean cotyledon callus tissue. AB - Growth of an auxin-requiring soybean cotyledon callus tissue (Glycine max L., Merr. var. Acme) was promoted by tryptophan, tryptamine, indole, indoleacetamide and, to a very slight degree, anthranilic acid. When tryptophan-3-(14)C was supplied in the growth medium, labeled indoleacetic acid (IAA) was found in both the tissue and the medium. Medium, from which the cells had been removed, was also found to convert labeled tryptophan to IAA. Soybean callus contained 0.044 mumole/g free tryptophan, but this is apparently not available for conversion to IAA. These results suggest that while exogenously supplied trytophan could elevate a specific internal pool where IAA synthesis occurs some of the growth on a tryptophan medium can be accounted for by external conversion. PMID- 16659500 TI - Promotive effects of organic solvents and kinetin on dark germination of lettuce seeds. AB - Significant promotion in dark germination was observed when Grand Rapids lettuce (Lactuca sativa L.) seeds were soaked in acetone or dichloromethane, vacuum dried, and imbibed at 25 C. Permeation of kinetin via these organic solvents further enhanced the dark germination. Those seeds that were affected by acetone and acetone-kinetin treatments and germinated in the dark escaped red-far red photocontrol of germination. Although abscisic acid was not detected in the organic solvent leachates, they did contain other inhibitory substances affecting lettuce seedling growth. In the light, acetone and acetone-kinetin treatments also enhanced the rate of germination and the increased germination by acetone kinetin treatment was correlated with increased polyribosome formation. The possible mechanisms involved in promotion of lettuce seed germination by organic solvents and kinetin are discussed. PMID- 16659499 TI - Action Spectrum between 260 and 800 Nanometers for the Photoinduction of Carotenoid Biosynthesis in Neurospora crassa. AB - An action spectrum for light-induced carotenoid biosynthesis in Neurospora crassa was determined in 4 to 20 nm steps from 260 to 800 nm. Four-day, dark-grown mycelial pads of N. crassa were exposed to varying amounts of monochromatic radiant energy and time. After a 48-hour incubation period at 6 C, carotenoid content was assayed spectrophotometrically in vivo. The action spectrum has maxima at 450 and 481 nm in the visible range and at 280 and 370 nm in the ultraviolet. A pigment synthesized by Neurospora whose absorption spectrum resembles the action spectrum is beta-carotene.A model for the regulation of carotenoid biosynthesis in N. crassa is proposed which describes a mechanism by which beta-carotene could act as a photoregulator. This carotenoid is suggested to be both photoreceptor for and regulator of carotenoid biosynthesis. PMID- 16659501 TI - Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites. AB - The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H(2)O-->methyl viologen, H(2)O- >silicomolybdic acid H(2)O-->ferricyanide, and H(2)O-->dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1 butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H(2)O-->silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl) 1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition. PMID- 16659502 TI - Inhibition of cottonseed germination with abscisic Acid and its reversal. AB - Germination of cottonseed (Gossypium hirsutum L.) was inhibited by abscisic acid. Inhibition was greater when seeds were soaked in abscisic acid for 5 hours and dried prior to germination than when abscisic acid was applied in the germination medium. (2-Chloroethyl)phosphonic acid, gibberellic acid, and kinetin partially overcame the inhibitory action of abscisic acid. Combinations of (2 chloroethyl)phosphonic acid with gibberellic acid or kinetin were more effective than the individual substances. Germination also was partially restored by removal of seed coats. Fusicoccin completely restored germination of abscisic acidtreated seeds. PMID- 16659503 TI - An Increase in Mechanical Extensibility during the Period of Light-stimulated Growth. AB - The sporangiophore of Phycomyces responds to a temporary increase in light intensity with a transient increase in growth rate that begins 2 to 3 minutes after the initiation of the stimulus and continues until approximately the 12th minute. Tensile tests conducted on the stage IVb sporangiophore demonstrate that an increase in mechanical extensibility of the cell wall occurs 2 minutes after the initiation of a light stimulus and continues until approximately the 15th minute. This finding supports the theory that light-stimulated plant cell expansion and rate of expansion is a function of the mechanical extensibility of the cell wall. PMID- 16659504 TI - Nitrate Reductase in Barley Roots under Sterile, Low Oxygen Conditions. AB - Levels of nitrate reductase activity (EC 1.9.6.1.) as high as 11 mumoles nitrite produced/hour gram fresh weight were found in barley (Hordeum vulgare cv. Compana) roots grown under low oxygen conditions. Roots of plants given identical treatment under sterile conditions did not develop the high levels of nitrate reductase activity. The results suggest that the buildup of particulate, reduced viologen-utilizing nitrate reductase reported in barley roots may be caused by bacterial contamination. The nitrate reductase activity in roots grown under low oxygen conditions was not specific for reduced nicotinamide adenine dinucleotide like the assimilatory nitrate reductase (EC 1.6.6.1.) normally found in aerated plant roots. PMID- 16659505 TI - Turbidimetric measurement of plant cell culture growth. AB - Turbidimetric measurement of cultures grown in sidearm flasks was used to measure the growth of plant cells. The turbidity was shown to vary proportionally with cell number and dry weight over time. The effect of different freezing conditions on the growth of the culture was presented to demonstrate the application of the sidearm-turbidity method. PMID- 16659506 TI - Mesophyll Resistance and Carboxylase Activity: A Comparison under Water Stress Conditions. AB - The response of several leaf gas exchange parameters were monitored with decreasing leaf water potential in Phaseolus vulgaris L. leaflets. These included photosynthesis, transpiration, CO(2) compensation point, ribulose 1,5-diphosphate carboxylase activity, boundary layer plus stomatal, and mesophyll resistance to diffusion of CO(2). Mesophyll resistance was calculated under two assumptions: (a) the CO(2) concentration at the chloroplast was zero, and (b) it was equal to the CO(2) compensation point.Contrary to some reports on bean, the estimates of mesophyll resistance (both models) increased with decreasing leaf water potential. Concurrently, the CO(2) compensation point increased and ribulose 1,5 diphosphate carboxylase activity decreased as internal water stress increased.It is suggested that the mesophyll resistance term has been too liberally interpreted in the past and that future use be verified by an indicator of biochemical response to environmental stress such as the assay for ribulose 1,5 diphosphate carboxylase activity. PMID- 16659507 TI - Swelling and Contraction of Mitochondria from Cold-hardened and Nonhardened Wheat and Rye Seedlings. AB - A comparison of mitochondria isolated from 2 and 24 C grown winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) seedlings revealed no correlation between changes in swelling and contraction characteristics and extent of cold hardiness. The swelling response changed markedly due to growth at low temperature, but the change was similar for the four cultivars examined. The swelling response was also observed to change rapidly during aging of isolated mitochondria, either at 2 or 24 C. Spontaneously swollen mitochondria, isolated from 24 C grown seedlings, contracted abruptly upon addition of certain oxidizable substrates, but this response was lost when seedlings were transferred from 24 to 2 C. Studies on the effect of various substrates and respiratory inhibitors on the swelling and contraction responses indicate that inhibitors which reduce or stop electron flow through the electron transport chain also inhibit substrate induced mitochondrial contraction. PMID- 16659508 TI - Seasonal variation in the hormone content of willow: I. Changes in abscisic Acid content and cytokinin activity in the xylem sap. AB - Changes in levels of abscisic acid (ABA) and cytokinin activity in the xylem sap of willow (Salix viminalis, L.) were followed throughout two growth cycles.Growth in spring was preceded by decreasing levels of ABA and an increase in cytokinin activity. The onset of dormancy was associated with low levels of cytokinins and high contents of ABA. A second peak of ABA was found in July which was not related to the dry weight of the sap. The main cytokinin activity in the sap was due to a zeatin riboside-like compound. PMID- 16659509 TI - Effects of sulfur on the photosynthesis of intact leaves and isolated chloroplasts of sugar beets. AB - Effects of sulfur on photosynthesis in sugar beets (Beta vulgaris L. cv. F58 554H1) were studied by inducing sulfur deficiency and determining changes in the photosynthesis of whole attached leaves and of isolated chloroplasts. The rates of photosynthetic CO(2) uptake by intact leaves, photoreduction of ferricyanide, cyclic and noncyclic photophosphorylation of isolated chloroplasts, and the rate of CO(2) assimilation by ribulose diphosphate carboxylase, decreased with decrease in total leaf sulfur from 2500 to about 500 mug g(-1) dry weight. Sulfur deficiency reduced photosynthesis through an effect on chlorophyll content, which decreased linearly with leaf sulfur, and by decreasing the rate of photosynthesis per unit chlorophyll. There was only a small effect of sulfur deficiency on stomatal diffusion resistance to CO(2) until leaf sulfur decreased below 1000 mug g(-1) when stomatal resistance became a more significant proportion of the total diffusion resistance to CO(2). Light respiration rates were positively correlated with photosynthesis rates and dark respiration was unchanged as leaf sulfur concentrations declined. PMID- 16659510 TI - Studies on the carbon dioxide promotion and ethylene inhibition of tuberization in potato explants cultured in vitro. AB - Ethylene inhibited the tuberization of etiolated potato (Solanum tuberosum L. var. Red La Soda) sprout sections cultured in vitro. Carbon dioxide did not overcome the C(2)H(4) inhibition but it was required for normal tuberization. Ethylene totally prevented root formation and development. It inhibited stolon elongation, and caused thickening and diageotropical growth of the stolon. In addition, C(2)H(4) prevented the accumulation of both starch and red anthocyanin which are always present in a tuber. Ethylene also inhibited the kinetin increased tuberization of sprout sections.Three to five days of exposure to CO(2) were required to obtain promotion of tuberization of stolons cultured in vitro. Bicarbonate ion did not affect starch synthetase activity isolated from potato tubers in vitro. The evidence presented suggests that CO(2) gas rather than HCO( ) (3) or CO(2-) (3) ions in equilibrium with dissolved CO(2) was probably responsible for the stimulation. Morphological changes elicited by CO(2) and C(2)H(4) are described and the mechanism of action of both on tuberization is discussed. PMID- 16659511 TI - Effect of powdery mildew infection on photosynthesis by leaves and chloroplasts of sugar beets. AB - Chloroplasts isolated from powdery mildew-infected (Erysiphe polygoni DC) sugar beet leaves (Beta vulgaris L) showed a reduction in the rate of electron transport and in the accompanying ATP formation in noncyclic photophosphorylation (water as electron donor, NADP as electron acceptor) and little or no change in the rate of ATP formation in cyclic photophosphorylation catalyzed by phenazine methosulfate. The inhibition of noncyclic photophosphorylation appeared to lead in the parent leaves to a decreased rate of photosynthetic CO(2) assimilation and a shift in products resulting in a relative increase of amino acids. These changes were accompanied by alterations in chloroplast ultrastructure and by a reduction in the activity of enzymes necessary for the formation of organic acids (phosphoenolpyruvate carboxylase and malate dehydrogenase). These results are similar to the findings of Montalbini and Buchanan (1974 Physiol. Plant Pathol. 4: 191-196) with chloroplasts from rust-infected Vicia faba leaves. PMID- 16659512 TI - Endogenous cytokinins in maturing manzanillo olive fruits. AB - Cytokinin activity was found in mature black olive fruits while in green fruits the activity was markedly lower. The substances with cytokinin activity showed chromatographic properties similar to those of nucleotides. Acid hydrolysis caused a shift of the active zone in the organic phase of 1-butanol-acetic acid water (4:1:5) from R(F) 0.1 to R(F) 0.8. The possible relation of these endogenously appearing cytokinins to anthocyanin accumulation and aging of the fruit is discussed. PMID- 16659513 TI - Additive and Synergistic Growth-inhibiting Properties of the Canaline-Urea Cycle Amino Acids. AB - Growth studies with Lemna minor revealed the additive and synergistic growth inhibiting properties of the canaline-urea cycle amino acids. Simultaneous canavanine and canaline treatment caused an additive reduction in frond production. Ureidohomoserine interacted with canaline or canavanine to affect synergistically L. minor growth by enhancing individual canavanine or canaline toxicity and increasing the additive growth reduction caused by canavanine plus canaline. The ornithineurea cycle amino acids effectively counteracted both the additive and synergistic growth-inhibiting properties of the canaline-urea cycle compounds. PMID- 16659514 TI - Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus. AB - Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B(12) to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B(12), plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly. PMID- 16659515 TI - Pigment Changes Associated with Application of Ethephon ((2 Chloroethyl)phosphonic Acid) to Fig (Ficus carica L.) Fruits. AB - The application of (2-chloroethyl)phosphonic acid (Ethephon) to ;Mission' fig fruits (Ficus carica L.) during late period II of their development stimulated ripening and change in color from green to bluish black within 8 days. Chlorophylls a and b decreased rapidly within 4 days after Ethephon treatment, and degradation continued at a decreasing rate for an additional 4 days, at which time the fruits had attained their maximum diameter and were considered fully ripe. Levels of beta-carotene, lutein, violaxanthin, and neoxanthin decreased in a pattern similar to that of chlorophylls a and b. The rates of beta-carotene and lutein degradation were initially greater than those of the xanthophyll pigments. Degradation rates of the various carotenoids were comparable 4 to 8 days after treatment.There was no measurable anthocyanin synthesis during a 2- to 4-day period following Ethephon treatment. Beyond this lag phase, anthocyanin accumulation was linear, and the amount of pigment synthesized was a function of both light intensity and duration. Although Ethephon promoted the rate of anthocyanin accumulation, it did not increase the total amount of pigment synthesized in treated fruits. Etiolation of fruits from the time of Ethephon treatment until maturity stimulated an increase in growth and completely inhibited anthocyanin production in the skin. Ethephon-treated fruits which ripened while etiolated were larger in diameter and higher in both fresh and dry weights than nonetiolated controls. PMID- 16659516 TI - Membrane lipid metabolism in germinating castor bean endosperm. AB - Castor bean (Ricinus communis L. var. Hale) endosperms, excised after 2 days germination at 30 C, were incubated 5 min to 8 hr with (14)C-acetate and (3)H glycerol. Homogenates were fractionated by sucrose gradient centrifugation. Organelles found to be active in lipid synthesis were the lipid bodies and the endoplasmic reticulum. The products of incorporation in the lipid bodies were (3)H-diglycerides containing (14)C-fatty acids of more than 20 carbons. In contrast, the endoplasmic reticulum produced (3)H-phospholipids as well as (3)H diglycerides rich in (14)C-linoleate. The phospholipids synthesized and their acyl contents were of the types known to be the major components of organelle membranes in this tissue. Phospholipids and diglycerides containing (14)C and (3)H were found in the glyoxysomes and mitochondria subsequent to their appearance in the endoplasmic reticulum. The results show that germinating castor bean endosperm synthesizes membrane lipids de novo from acetate rather than reutilizing stored lipid components directly. It is also apparent that the endoplasmic reticulum is responsible for several steps in membrane lipid production. PMID- 16659517 TI - Polyribosomes from Peas: VI. Auxin-stimulated Recruitment of Free Monosomes into Membrane-bound Polysomes. AB - Auxin treatment of aged pea stems (Pisum sativum L. var. Alaska) caused a decrease in monosomes (especially free monosomes) and an increase in polysomes (especially membrane-bound polysomes). These effects were not duplicated by gibberellic acid or benzyladenine. These auxin-stimulated shifts in polysome distribution commenced at least 9 hours before significant growth took place. It is suggested that this auxin-stimulated incorporation of free monosomes into membrane-bound polysomes might involve increased utilization (through activation or synthesis) of messenger RNA(s) acting as template(s) for synthesis of secretable enzyme(s) involved in growth. PMID- 16659518 TI - In vivo nitrate reduction in relation to nitrate uptake, nitrate content, and in vitro nitrate reductase activity in intact barley seedlings. AB - A study was done to relate the in vivo reduction of nitrate to nitrate uptake, nitrate accumulation, and induction of nitrate reductase activity in intact barley seedlings (Hordeum vulgare L. var. ;Numar'). The characteristics of nitrate uptake in response to both time and ambient concentration of nitrate regulated reduction and accumulation. Uptake, accumulation, and in vivo reduction achieved steady state rates in 3 to 4 hours, whereas extractable (in vitro) nitrate reductase activity was still increasing at 12 hours. In vivo reduction of nitrate was better correlated exponentially than linearly over time with in vitro activity of nitrate reductase. A similar relationship occurred over increasing concentration of nitrate in the ambient solution. The results suggest that the rate of in vivo reduction of nitrate in barley seedlings may be regulated by the rate of uptake at the ambient concentrations of nitrate employed in the study. PMID- 16659519 TI - Relationship between Ethylene Evolution and Senescence in Morning-Glory Flower Tissue. AB - An excised tissue system consisting of corolla rib segments was developed to study the relationship between senescence and ethylene production in morning glory flowers (Ipomoea tricolor). Such segments, isolated 1 or 2 days (day -1 or day -2) before flower opening (day 0) passed through the same developmental phases as did the corresponding tissues of the intact organ. When excised on day 1 and incubated overnight, the rib segments turned from purple to blue and changed from a slightly curled to a flat configuration. On day 0, these segments rolled up during the afternoon and turned purple again, as did the ribs of an intact corolla; the rolling up coincided with an increased rate of ethylene production. Premature rolling up and associated ethylene evolution were induced by ethylene or propylene treatment. When segments were excised on day -2 and incubated overnight, there were no changes in color or shape; during day -1, no spontaneous rolling up and little ethylene evolution occurred. Application of ethylene or propylene to these immature segments elicited rolling up but did not stimulate endogenous ethylene production.Overnight treatment of segments cut on day -1 with 10(-6)m benzyladenine markedly retarded spontaneous rolling up and ethylene evolution, although the response to applied ethylene was only slightly slowed. Overnight treatment of segments cut on day -1 with the ethoxy analog of rhizobitoxine (10(-5) to 10(-4)m) resulted in almost complete (>99%) inhibition of both spontaneous and propylene-induced ethylene evolution. Although spontaneous rolling up was delayed, it was not abolished, and ethylene-induced rolling up was almost unaffected.These data indicate that an ethylene-generating system develops as an integral part of the aging process in flower tissue. Ethylene hastens aging of the flower, but may not play an obligatory role in flower senescence. PMID- 16659520 TI - Methionine metabolism and ethylene biosynthesis in senescent flower tissue of morning-glory. AB - In immature rib segments prepared from morning-glory (Ipomoea tricolor) flower buds, the major soluble metabolite formed from tracer amounts of l-methionine-U (14)C was S-methylmethionine (SMM). In segments of senescing ribs, (14)C was progressively lost from SMM and appeared in free methionine. Immature segments contained about 4 nmoles of free methionine and about 16 nmoles of SMM per 30 segments. As the segments senesced, the methionine content increased about 10 fold while the SMM content remained unchanged; during this time about 0.8 nmole of ethylene was produced per 30 segments. Tracer experiments with l-methionine-U (14)C, l-methionine-methyl-(3)H, and l-homocysteine thiolactone-(35)S indicated that SMM was capable of acting as a methyl donor, and that in senescent segments the methyl group was utilized for methionine production with homocysteine serving as methyl acceptor. Of the 2 molecules of methionine produced in this reaction, 1 was re-methylated to SMM, and the other contributed to the observed rise in the content of free methionine.Internal pools of methionine and SMM were prelabeled (but not significantly expanded) by overnight incubation on 10 mum l-methionine-U (14)C. The specific radioactivity of the ethylene subsequently evolved during the senescence of the segments closely paralleled the specific radioactivity of carbon atoms 3 plus 4 of free methionine extracted from the tissue, demonstrating that methionine was the major precursor of ethylene in this system. The specific radioactivity of carbon atoms 3 plus 4 of extracted SMM was about twice that of the free methionine.Based on these results, a scheme for methionine biosynthesis in senescent rib tissue is presented. The operation of this pathway in the control of ethylene production is discussed. PMID- 16659521 TI - Biosynthesis of wound ethylene in morning-glory flower tissue. AB - Production of wound ethylene was investigated in rib segments excised from flower buds of morning-glory (Ipomoea tricolor). Segments of the ribs were cut from buds 2 days before flower opening, floated overnight on 5 mm KCl solution, and transferred to agar the following morning. These immature segments evolved only a small quantity of ethylene during incubation on agar, with most of the production occurring in the morning. When such segments were wounded mechanically early in the afternoon, the rate of ethylene production rose more than 10-fold within 1 hour and returned to a low rate after about 3 hours.Production of ethylene by both untreated and wounded rib segments was inhibited more than 95% by overnight pretreatment with the ethoxy analog of rhizobitoxine (3 x 10(-5) and 10(-4)m). After overnight exposure of segments to 9 muml-methionine-U-(14)C, the specific radioactivity of the ethylene evolved by untreated and wounded tissue was determined and compared to the specific radioactivities of carbon atoms 3 plus 4 of methionine and S-methylmethionine (SMM) extracted from the segments. The specific radioactivity of methionine was about one-half that of SMM; neither value was significantly affected by wounding. The specific radioactivity of ethylene evolved by untreated tissue was close to that of SMM. In wounded tissue the specific radioactivity of the ethylene evolved was lower, but still above that of methionine. These results are consistent with the interpretations that wound ethylene is synthesized from carbon atoms 3 plus 4 of either SMM or methionine. On the basis of earlier experiments with senescing rib segments, it is suggested that methionine serves as the precursor of the wound ethylene. PMID- 16659522 TI - Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max. AB - The activity and extent of adenylylation of glutamine synthetase was examined in both free-living and bacteroid forms of Rhizobium japonicum in the presence of excess ammonia. Ammonia caused an apparent repression of glutamine synthetase in free-living R. japonicum and adenylylation of the enzyme was also increased. In contrast, neither the activity nor the extent of adenylylation of the bacteroid enzyme was consistently affected by ammonium treatment of bacteroid suspensions. Similar results were obtained after ammonium treatment of soybean plants even though nitrogenase activity was reduced markedly. We have been unable to demonstrate ammonium repression of nitrogenase activity in R. japonicum-Glycine max symbiotic association that is mediated through bacteroid glutamine synthetase. This result is in contrast to the situation in nitrogen-fixing strains of Klebsiella where a role of glutamine synthetase in the regulation of nitrogenase has been reported. PMID- 16659523 TI - Aging progression involving dwarfism and its acceleration by red light in bean hypocotyls. AB - The effect of red light on the aging progression of the bean (Phaseolus vulgaris L.) hypocotyl segment unit was examined in relation to dwarfism using Kentucky Wonder (tall) and Masterpiece (dwarf) varieties. In both plants, red light promoted the elongation of younger zones and inhibited that of mature zones. The zone exhibiting maximum elongation was shifted to the younger zones by red light irradiation regardless of the plant type, but its extent was greater in the dwarf than in the tall. Thus, red light hastens both the beginning of elongation in the younger portion and its termination in the mature portion of the hypocotyl, particularly of the dwarf plant. These red light responses in each zone of both the tall and dwarf hypocotyl units were reversed by subsequent exposure to far red light regardless of the duration and intensity of red light, thus indicating that the hastened aging progression of the hypocotyl by red light is mediated by phytochrome. However, there is no difference in the rate of decay of Pfr between the tall and dwarf hypocotyls.The increased expression of bean dwarfism seems to result from a concerted action of red light upon the maturation of younger portions of the hypocotyls, which is more rapid in the dwarf than in the tall, and upon the elongation of the relatively matured portion, which is more severely inhibited in the dwarf. PMID- 16659524 TI - Interrelations between Environmental Factors and Freezing Resistance of Cabbage Leaves. AB - Rapid wilting of cabbage leaves (Brassica capitata L.), induced by excision of the shoot, induced as rapid and high a degree of freezing resistance as a similar period of hardening at low temperature. Maximum hardening in the leaf was generally associated with the maximum growth rate. On the other hand, exposure of the excised shoot to low temperature while immersed in aerated water failed to harden the plants. In the absence of light, abrupt wilting at room or low temperature induced little or no hardening. With the available equipment, which required the absence of light, freezing temperatures induced little or no hardening above that obtained by nonfreezing low temperature. In fact, the plant frozen at moderate temperatures showed a gradual but steady decrease in freezing resistance. Since these experiments were performed with plants grown in pots, and since they eventually became pot-bound, the results may not apply equally to field-grown plants. PMID- 16659525 TI - Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons. AB - The incorporation of phospholipids specifically labeled with glycerol-2(3)H and acyl-(14)C by whole cell tissues of developing soybean cotyledons (Glycine max L.) reveals that phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, and phosphatidic acid can be metabolized to diglyceride. The diglyceride formed may be recylced into phospholipid or acylated to triglyceride. Diglyceride from phosphatidic acid and phosphatidylethanolamine is used readily in triglyceride biosynthesis compared to the other phospholipids. Incorporation of N-acylphosphatidylethanolamine having [9-10-(3)H(N)]oleic acid esterified at sn-3 in cotyledons shows rapid acyltransfer of (3)H into triglyceride and therefore N acylphosphatidylethanolamine appears to participate in triglyceride biosynthesis as an acyl donor. These studies emphasize phospholipid metabolism in developing soybean cotyledons is a dynamic process which plays a key role in triglyceride formation. PMID- 16659526 TI - Influence of Leaf Starch Concentration on CO(2) Assimilation in Soybean. AB - Net photosynthetic rate, CO(2) compensation concentration, and starch and soluble sugar concentrations were measured in soybean (Glycine max [L.] Merrill) leaves in an attempt to evaluate the effect of carbohydrate concentration on rate of CO(2) assimilation.Plants were grown in a controlled environment room at 23.5 C, 50% relative humidity, 16-hour photoperiod, and quantum flux (400-700 nm) of 510 mueinsteins/m(2).sec (30,090 lux) at plant level. On the 21st day after seeding, plants were subjected for 12.5 hours to one of three CO(2) concentrations (50, 300, or 2000 mul/l) in an attempt to alter leaf carbohydrate levels. Following the CO(2) treatment, gas exchange measurements were made at a CO(2) concentration of 300 mul/l on the lowermost trifoliolate leaf. Immediately after measurement, the leaf was removed and stored at -20 C until carbohydrate analyses were performed.Increasing the CO(2) concentration for 12.5 hours significantly increased leaf starch concentration but not soluble sugar concentration. There was a strong negative correlation between net photosynthetic rate and starch concentration. Net photosynthetic rate declined from approximately 38 to 22 mg CO(2)/dm(2) leaf area.hr as starch concentration increased from 0.5 to 3 mg/cm(2) leaf area. Carbohydrate concentrations had no effect on compensation concentration.The decrease in net photosynthetic rate as starch concentration increased resulted from an increase in mesophyll (liquid phase) CO(2) diffusion resistance. This suggests that starch accumulation may reduce net photosynthetic rate by impeding intracellular CO(2) transport. PMID- 16659527 TI - Plant Desiccation and Protein Synthesis: III. Stability of Cytoplasmic RNA during Dehydration, and Its Synthesis on Rehydration of the Moss Tortula ruralis. AB - RNA species from the haploid gametophyte generation of the moss Tortula ruralis exhibit typical eukaryotic characteristics. The major ribosomal and soluble RNA species are stable during drying and rehydration. RNA synthesis occurs rapidly on reintroduction of the moss to water and incorporation into high molecular weight RNA fractions was detected after 20 to 30 minutes of rehydration and into low molecular weight fractions after 30-60 minutes. Newly synthesized ribosomal RNA was detected in ribosomes within 2 hours of rehydration, but not in polysomes. It is apparent that the ribosomal and transfer RNA conserved during desiccation is involved in the re-establishment of early protein synthesis during subsequent rehydration and that, initially, there is no requirement for newly synthesized material. PMID- 16659528 TI - Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis. AB - Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of (3)H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of (14)C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of (3)H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence. PMID- 16659529 TI - Comparative studies on the induction and inactivation of nitrate reductase in corn roots and leaves. AB - A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH(2)-nitrate reductase and NO(3) (-)-induced NADH cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A x 182E). The three activities were induced in parallel in both tissues when NO(3) (-) was supplied. WO(4) (=) suppressed the induction of NADH- and FMNH(2) nitrate reductase activities in root tips and leaves. The NO(3) (-)-induced NADH cytochrome c reductase activity showed a normal increase in roots treated with WO(4) (=). In leaves, on the other hand, there was a marked superinduction of the NO(3) (-)-induced NADH-cytochrome c reductase in the presence of WO(4) (=).The half-life values of NADH-nitrate reductase and FMNH(2)-nitrate reductase measured by removing NO(3) (-) and adding WO(4) (=) to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO(3) (-) in the induction medium together with WO(4) (=) gave partial protection of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities in both root tips and leaves with a t(0.5) of 6 and 8 hours, respectively. NO(3) (-) also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO(3) (-)-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO(3) (-) protected the loss of NO(3) (-)-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase. PMID- 16659530 TI - Evidence for a Block between Plastoquinone and Cytochrome f in a Photosynthetic Mutant of Lemna with Abnormal Flowering Behavior. AB - Mutant strain 1073 of Lemna perpusilla is concluded to be blocked between plastoquinone and cytochrome f in the photosynthetic electron transport system. The location of the block is based on the following observations of activities in chloroplasts isolated from the mutant and wild-type plants. (a) Relative to wild type, electron flow rates from water to ferricyanide, 2,6-dichlorophenol indophenol or NADP were very low in the mutant, but rates of photosystem I dependent electron flow and cyclic phosphorylation were high. (b) Chlorophyll a fluorescence induction curves for mutant and wild type were similar. (c) Silicomolybdate and lipophilic acceptors in the mutant were photoreduced at rates comparable to wild type. (d) Cytochrome f of the mutant chloroplasts was not reduced by red light, but was oxidized by red or far red light. (e) Reduction of the primary electron acceptor of photosystem II (Q) by ATP-driven reverse electron flow was not observed in the mutant. PMID- 16659531 TI - Isotope Discrimination by Ribulose 1,5-Diphosphate Carboxylase: No Effect of Temperature or HCO(3) Concentration. AB - Carbon 13 isotope discrimination by ribulose 1,5-diphosphate carboxylase from soybean (Glycine max [Merr.] cv. Amsoy) was studied as a function of temperature, bicarbonate concentration, and pH. None of these factors affected the degree of discrimination against (13)C. The average delta(13)C was -28.3%, a value close to that found for whole C(3) plants. The zero temperature response observed here with ribulose 1,5-diphosphate carboxylase corroborates data from whole plants. The lack of effect of bicarbonate concentration on discrimination is consistent with both current theories of alternate forms of carboxylase. PMID- 16659532 TI - Trypsin-induced ATPase Activity in Potato Mitochondria. AB - Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg(2+), 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg(2+)-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60 C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear. PMID- 16659533 TI - Development of ribulose 1,5-diphosphate carboxylase in nonphotosynthetic endosperms of germinating castor beans. AB - Ribulose 1,5-diphosphate (RuDP) carboxylase has been partially purified from dark grown nonphotosynthetic endosperms of germinating castor beans (Ricinus communis var. Hale). The Km values for RuDP, HCO(3) (-), and Mg(2+) are 0.51, 33, and 1.78 mm, respectively. The pH optimum for the carboxylation reaction is pH 7.5. Germination is required for the development of the carboxylase in the endosperms. The enzyme reaches a maximal activity in 4- to 5-day-old dark-grown seedlings (which have an endosperm weight of approximately 0.75 g fresh weight/bean) and then declines. Total endosperm carboxylase activity is 1230 nmoles/min.g fresh weight which is 25 and 50% of the total activity developed in soybean and maize leaves, respectively. Specific activity of the carboxylase in crude soluble endosperm preparations (which contain enzymic and storage protein) is 0.05 mumole/min.mg protein. This is 5 times greater than the specific activity of RuDP carboxylase in soluble preparations from etiolated leaves. During germination the V(max) of the endosperm carboxylase for RuDP increases 10-fold. Development of the enzyme is inhibited 90% by the exposure of the endosperm to 2 mug/ml cycloheximide or 50 mug/ml chloramphenicol. Light (or phytochrome Pfr) is not required for the synthesis of the enzyme. Electron photomicrographs of dark-grown endosperm cells (with peak RuDP carboxylase activity) show proplastids with several invaginations of the inner membrane but no prolamellar-like structures. PMID- 16659534 TI - Events Surrounding the Early Development of Euglena Chloroplasts: VII. Photocontrol of the Source Of Reducing Power for Chloramphenicol Reduction by the Ferredoxin-NADP Reductase System. AB - d(-)threo-Chloramphenicol blocks chlorophyll and plastid protein synthesis in Euglena. During chloroplast development in white light, but not in red, the cells escape from chloramphenicol inhibition and chlorophyll formation is restored. Concomitantly, chloramphenicol is reduced. Reduction of chloramphenicol in an enzyme extract from Euglena requires NADPH and ferredoxin for maximal activity. Methyl viologen replaces ferredoxin, and when chemically reduced, ferredoxin or methyl viologen reduces chloramphenicol directly. This suggests that the enzyme involved is ferredoxin-NADP reductase. In agreement, crude extracts from wild type and W(3)BUL, a mutant lacking detectable plastids and plastid DNA, when separated on acrylamide gels, show a single band which reduces methyl viologen with NADPH, and its mobility is similar in wild type and in mutant W(3)BUL. The reductase is inducible by light and increases 3-fold in wild type in white or red light and 1.5-fold in W(3)BUL in white light. DCMU does not block chloramphenicol reduction in vivo indicating that electrons originate from sources other than photosynthetic electron transport. We infer that chloramphenicol is reduced by ferredoxin which receives electrons via ferredoxin-NADP reductase. The limiting step is not the enzyme but the source of reducing power which can be supplied from the cytoplasm, probably under control of the blue light receptor. Ferredoxin and ferredoxin NADP reductase appear to be coded in the nuclear genome, synthesized on cytoplasmic ribosomes, and join a group of enzymes which cannot be precisely localized, since they may be active anywhere from their site of synthesis in the cytoplasm to their place of deposition in the chloroplast. PMID- 16659535 TI - Identification of Cytokinins of Root Nodules of the Garden Pea, Pisum sativum L. AB - Five cytokinin activities which induced soybean callus proliferation were detected in ethanol extracts of root nodules of the garden pea (Pisum sativum L., cv. Little Marvel). The most active factors among them were identified as zeatin and its riboside on the basis of their mobility on thin layer chromatography in three solvent systems. Smaller activities of zeatin ribotide, isopentenyladenine and its riboside were also detected. Cytokinin activity gradually decreased with the cultivation period, but no qualitative change in the active compounds was found. PMID- 16659536 TI - Effect of removal of the root tip on the development of enhanced rb absorption by corn roots. AB - Samples of primary root tissue of corn (Zea mays L.) were aged either in CaSO(4) solution or in humid air, after which they were immersed for 10 minutes in a solution containing 0.1 mm(86)RbCl. Aging in solution, but not in humid air, enhanced the subsequent rate of Rb(+) absorption. Excision of roots before aging was followed by greater enhancement than when exicision followed aging. The time course of aging of 1-cm segments from different portions of the root showed decreasing response with increasing distance from the root cap. The aging response of apical segments (5-15 mm from the root cap) could be detected within 10 minutes and usually reached a maximum within 2 hours. Rb(+) absorption by apical segments (5-15 mm) aged without the tip (0-5 mm) was more than double that by apical segments whose tips were left attached until the end of the aging period. When apical segments without the tip were aged for 2 hours in the CaSO(4) solution in which seedlings had previously been grown for 24 hours, the rate of absorption was only 63% of samples aged in fresh solution. When apical segments were aged for 2 hours in fresh solution containing excised tips floating free in the solution, the rate of Rb(+) absorption was 20% less than in samples aged in solution containing no excised tips. The data presented in this study are interpreted to indicate that a water-soluble metabolite, originating in the root tip and translocated basipetally, inhibits Rb accumulation. PMID- 16659537 TI - Effects of 35 C heat treatments on photosensitive grand rapids lettuce seed germination. AB - Grand Rapids lettuce (Lactuca sativa L.) seeds were given 35 C heat treatments to increase photodormancy in a subsequent 20 C dark period. Short heat treatments (1 5 hours) induced a significant germination percentage increase of from 16% to over 50% depending on seed lot. With longer heat treatments dark germination percentage was gradually reduced to zero. If given at the end of 35 C, far red or red followed by far red further increased the amount of dark germination.Thermodormancy also delayed red-stimulated germination by 10 hours or more when red was given following a long 35 C treatment. The presence of Pfr was required during this time since far red light remained effective in reversing at least 50% of the red stimulation for up to 16 hours compared to only 4 hours in nonheat-treated seeds. PMID- 16659538 TI - Aggregate formation from short fragments of plant DNA. AB - Large aggregates have been observed after partial reassociation of pea (Pisum sativum L.) DNA preparations sheared to mean single strand fragment lengths as short as 350 nucleotides. At high DNA concentrations and conditions of salt and temperature which require only moderate precision of base pairing, aggregates pelletable by brief centrifugation account for 30 to 40% of the total DNA from peas, while calf thymus DNA reassociated under similar conditions forms less than 10% pelletable structures. In contrast to networks formed during the reassociation of long DNA fragments containing interspersed repetitive sequences, these aggregates contain a high percentage of double-stranded DNA and are enriched in repetitive sequences.Aggregates detectable by centrifugation do not begin to appear until after extensive repetitive sequence reassociation has already occurred. The results are consistent with a model involving secondary reassociation between single-stranded regions ("hanging tails") remaining after initial duplex formation. This process would lead to formation of large multimers of the original fragments, analogous to the large hyperpolymers which have been observed in extensively reassociated prokaryotic DNA. Randomly sheared fragments containing short (about 300 base pairs) repetitive sequences interspersed with single copy DNA would not be expected to hyperpolymerize significantly under these conditions. I suggest, as a working hypothesis, that much of the repetitive sequence DNA in peas is contained in regions considerably longer than 300 base pairs. PMID- 16659539 TI - Glutamine Synthesis and Its Relation to Photophosphorylation in Pisum Chloroplasts: Effects of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and Antimycin A. AB - Illuminated pea (Pisum sativum) chloroplasts can convert glutamate to glutamine using ATP generated by photophosphorylation to drive the glutamine-synthetase reaction. Light-dependent glutamine synthesis is sensitive to 3-(3,4 dichlorophenyl)-1,1-dimethyl urea (DCMU), but only at concentrations higher than are necessary to suppress photoreduction of ferricyanide or phosphoglycerate. Conversely, glutamine synthesis is far more sensitive to antimycin A than is photoconversion of phosphoglycerate to triosephosphate. When 3.8 mm phosphoglycerate is supplied, glutamine synthesis is stimulated in both the presence and absence of antimycin A.These data seem to be consistent with the operation of an endogenous, DCMU-sensitive, phosphorylation process-possibly cyclic-which can support glutamine synthesis in white light under aerobic conditions. The stimulatory effect of phosphoglycerate suggests that noncyclic phosphorylation is initiated or accelerated when this substrate is supplied. This noncyclic process evidently provides ATP over and above the amount required for phosphoglycerate photoreduction, i.e. the ATP/e(2) ratio exceeds 1.0. The additional ATP produced under these conditions is available for glutamine synthesis and lessens its dependence on cyclically (or pseudocyclically) generated ATP. PMID- 16659540 TI - Mercury-induced Ethylene Formation and Abscission in Citrus and Coleus Explants. AB - Mercury vapor induces ethylene formation and abscission in Citrus and Coleus explants. Both responses are markedly greater in the absence of CO(2). The stimulation of these metabolically complex processes indicates that the action of mercury vapor is not consistent with the more popular conception of mercury toxicity. This was manifested in its complete failure to disturb respiratory gas exchange, and in the total absence of any necrosis. Accordingly, the effect of mercury appears to be highly specific. The overall significance of these findings is discussed with respect to physiological, environmental, and methodological aspects. PMID- 16659541 TI - Sequence of Reactivation of Ribonucleic Acid Synthesis during Early Germination of the Maize Embryo. AB - The onset of RNA synthesis in primary roots of germinating Zea mays embryos was studied. Incubation of excised embryos in the radioactive precursor solution was performed after different germination periods.It was observed that there are three successive stages in the reactivation of RNA synthesis. Stage one is characterized by the synthesis in the chromatin of heterodisperse nuclear RNA rich in adenine. Stage two is characterized by the synthesis of heterodisperse nuclear RNA in the chromatin and 2.3 x 10(6) rRNA precursor in the nucleolus. Stage three is characterized by the synthesis of heterodisperse nuclear RNA and rRNA precursor and by the processing of the latter in mature rRNA. PMID- 16659542 TI - Effects of Ancymidol (a Growth Retardant) and Triarimol (a Fungicide) on the Growth, Sterols, and Gibberellins of Phaseolus vulgaris (L.). AB - The effect of the two substituted pyrimidines, ancymidol (a growth retardant) and triarimol (a fungicide) on Phaseolus vulgaris was studied. Both compounds retarded shoot and root elongation as well as increases in fresh weight. Both compounds caused production of ethylene-like responses when given in high dosages or when applied shortly after germination. As growth retardation was shown to occur in the absence of net increase in sterol levels, neither ancymidol nor triarimol apparently retards growth by inhibiting sterol synthesis.Both ancymidol and triarimol treatment drastically reduced the amount of extractable gibberellin like activity in beans. Ancymidol also either induced or enhanced the production of a compound which gave a negative response in the bioassay plant Oryza sativa var. Tan-ginbozu. The addition of gibberellin completely relieved the dwarfing effects of both ancymidol and triarimol in dark-grown beans. It is concluded that ancymidol and triarimol affect a gibberellin-induced growth response, probably by inhibiting gibberellin biosynthesis. PMID- 16659543 TI - Aliphatic Chains of Esterified Lipids in Isolated Eyespots of Euglena gracilis var. bacillaris. AB - Isolated eyespot granules of Euglena gracilis Klebs var. bacillaris Pringsheim contained approximately 6% lipids (based on protein). Separation of the lipid extracts by thin layer chromatography revealed four major fractions: wax esters, triacylglycerols, free fatty acids, and phospholipids. Methanolysis of each fraction yielded between 27 and 29 different fatty acids ranging from 12:0 to 22:6. Acetates of the fatty alcohols of the wax fraction consisted of 11:0 to 18:0 carbon chains, with 14:0 being the major component; unsaturated alcohols were not detected. PMID- 16659544 TI - Autoinhibition of Ethylene Formation in Nonripening Stages of the Fruit of Sycomore Fig (Ficus sycomorus L.). AB - Differences in the mechanism of ethylene emanation of Ficus sycomorus L. during various stages of the fruit development were investigated by enclosing the figs in jars. Two distinct patterns of ethylene emanation were found. Pattern a. in stages not capable of ripening, neither spontaneously nor as a result of physiological treatment (nonripening stages A and C), ethylene concentration in the jar increased linearly for a short time and then remained constant. Pattern b. in stages capable of ripening (ripening stages B, D, and E), the linear increase in ethylene concentration continued for the entire period of measurement. In nonripening stages, ethylene emanation stopped when ethylene concentration in the jar reached a constant value (0.6 mul/l at stage C). Aeration of the figs and the jar renewed ethylene emanation. CO(2) concentration in the jar never exceeded 0.5%. Treatment of stage C figs with 0.6 to 10 mul/l exogenous ethylene caused immediate and complete cessation of ethylene emanation whereas the same treatment did not cause any change in rate of ethylene emanation from figs at the ripening stages B and D. Gashing (wounding) of stage C figs temporarily changed the pattern of ethylene emanation from pattern a to pattern b.We concluded that in the nonripening stages ethylene acts as an autoinhibitor of its own production but this does not occur in the ripening stages. PMID- 16659545 TI - Synergetic Cultures of Glycine max Root Cells and Rhizobia Separated by Membrane Filters. AB - When suspension cultures of actively growing soybean (Glycine max L.) root cells were separated by two or three membrane filters from suspension cultures of the bacteria, a synergetic (cooperative) activation of nitrogenase was observed in the Rhizobium japonicum used in the bacterial side. Either plant cells or plant cell-conditioned medium was needed for this activation to take place. Both acetylene reduction and hydrogen evolution by the activated R. japonicum persisted for several days after removal from the apparatus when (a) a suitable carbon source was provided, (b) oxygen supply was limited, and (c) growth of bacteria was suppressed by lowering of ammonia and nitrate concentrations. Activation could also take place when the bacteria were placed in media to which plant cell-conditioned medium was added. The advantages of this method for studies on symbiosis are discussed. PMID- 16659546 TI - Leaf cell water and enzyme activity. AB - This work supports further the thesis that under conditions of water stress, cell water content may supersede hormonal regulation in effecting enzyme activity, thus becoming a regulatory factor in cellular metabolism. Addition of NaCl to the root medium of barley plants (Hordeum vulgare L.) markedly increased leaf RNase activity parallel to an increase of leaf water saturation deficit (WSD). Kinetin and abscisic acid, applied to the salinated plants, also modified RNase activity, as well as leaf-WSD. The familiar pattern of effects of these hormones on leaf RNase as well as leaf chlorophyll content was inverted, kinetin effected a relative increase in RNase activity and a decrease in leaf chlorophyll, whereas abscisic acid effected a relative decrease in RNase activity and maintained chlorophyll content. A close relationship between enzyme activity and leaf WSD became evident when leaf RNase and protease activities in the salinated plants were plotted against leaf WSD. This close relationship was maintained irrespective of the hormonal treatments, which in themselves markedly modified leaf WSD. As predicted, high relative humidity which relived the leaves from salt induced water stress prevented the salt-induced rise in RNase activity. PMID- 16659547 TI - Simultaneous measurement of oxygen and hydrogen exchange from the blue-green alga anabaena. AB - Two Clark-type polarographic electrodes were used to measure simultaneous H(2) and O(2) exchange from three species of the blue-green alga Anabaena. Maximum H(2) photoevolution from N(2)-fixing cultures of Anabaena required only the removal of dissolved O(2) and N(2); no adaptation period was necessary. No correlation of H(2) photoproduction with photosynthetic O(2) evolution, beyond their mutual light requirement, was found. Hydrogen photoevolution has the following characteristics in common with N(2) fixation in these organisms: DCMU insensitivity; similar white light dependency with very low dark production rates; maximum efficiency in photosystem I light; inhibition by N(2), O(2) and acetylene; and an apparent requirement for the presence of heterocysts. Growth on nitrate medium reduces, and on ammonium medium obliterates, both reactions. Cultures grown under limiting CO(2) conditions have H(2) photoproduction rates proportional to their growth rates. Hydrogenase activity is inferred from H(2) uptake in the dark, but this activity apparently is independent of the photoevolution of H(2) which is ascribed strictly to the nitrogenase system. PMID- 16659548 TI - Specificity of the Antheridogen from Ceratopteris thalictroides (L.) Brongn. AB - Characteristics of the fern antheridogen from Ceratopteris thalictroides (L.) Brongn. are investigated. These are: (a) determination of molecular size (it is readily dialyzable), (b) pK(a) (about 5), (c) movement in thin layer chromatography, and (d) ability to induce dark germination of fern spores. These four characteristics are compared to the same characteristics of three other antheridogens (antheridogens A and B or GA). Molecular size and pK(a) are similar, but, the antheridogens are separable from each other using thin layer chromatography. It was also shown that spore germination is not induced by the Ceratopteris antheridogen, even in its own spores, a characteristic not reported as shared by the other antheridogens. However, the inconsistency of spore germination as an assay for antheridogen is demonstrated. The presence of gametophyte-produced allelopathic substances is also shown. PMID- 16659549 TI - Correlative Aspects of Imposition of Dormancy in Caryopses of Aegilops kotschyi. AB - Caryopses of Aegilops kotschyi Boiss. from different positions within the same spikelet differ greatly in their degree of dormancy. Imposition of this dormancy pattern is a correlative phenomenon within the spikelet, inasmuch as the uppermost developing caryopsis is least dormant and its development is associated with the dormancy status imposed on the lower (one or two) caryopses. Differences in pigmentation of the caryopses follow a corresponding pattern. PMID- 16659550 TI - Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle. AB - Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi permeability of thylakoids. PMID- 16659551 TI - Alterations in Chloroplast Thylakoids during Cold Acclimation. AB - Freeze-fracture electron microscopy reveals a decreased particle concentration on the inner fracture face of acclimated thylakoids, suggestive of some alteration(s) in the hydrophobic region. Sonic oscillation causes a reversal of the altered particle concentration in acclimated thylakoids and suggests that increases in unsaturation of fatty acids can, at most, account for only part of the altered particle concentration. The particles on the inner fracture face of acclimated thylakoids are of one size group (+/- 140 A) as compared to two size groups (+/- 100 A and +/- 165 A) for nonacclimated thylakoids. The paracrystalline array might be associated with the acclimated state of thylakoids. Nonacclimated thylakoids require 50 mm sucrose for maximum protection of light-dependent proton uptake, while acclimated thylakoids require 25 mm sucrose, and the protection afforded acclimated thylakoids during a freeze-thaw cycle is greater. Sucrose is required for alterations in acclimated thylakoids to be manifested. Apparently increased hardiness is not only associated with changes in cellular environment but also alterations in membranes. PMID- 16659552 TI - Lysine Catabolism in Barley (Hordeum vulgare L.). AB - Lysine catabolism in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following tracers into the endosperm of the seedlings: aspartic acid-3-(14)C, 2-aminoadipic acid-1-(14)C, saccharopine-(14)C, 2,6-diaminopimelic acid-1-(7)-(14)C, and lysine-1-(14)C. Labeled saccharopine was formed only after the administration of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings. The metabolic fate of the other tracers administered also supported a catabolic lysine pathway via saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2 aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of l-lysine catabolism in barley seedlings. PMID- 16659553 TI - Hydroxylamine reductase enzymes from maize scutellum and their relationship to nitrite reductase. AB - Three enzymes contribute to the total hydroxylamine reductase activity of corn (Zea mays L.) scutellum extracts. Two of these resemble enzymes previously prepared from leaves, while the third, which accounts for a major part of the activity, appears to have no counterpart in leaf tissue. One of the hydroxylamine reductases found only in small amounts is associated with nitrite reductase and is induced, together with nitrite reductase, by nitrite. The other two enzymes are noninducible by nitrite and can be totally separated from nitrite reductase, which subsequently remains capable of catalyzing the reduction of nitrite to ammonia. Possible causes of the decline of hydroxylamine reductase activity during the induction of nitrite reductase are discussed. PMID- 16659554 TI - Effects of Light, Abscisic Acid, and N-Benzyladenine on the Metabolism of [H]Gibberellin A(4) in Seeds and Seedlings of Lettuce, cv. Grand Rapids. AB - Gibberellin A(4) (GA(4)) can substitute for light in the germination of Grand Rapids lettuce seeds. Seeds imbibed in [(3)H]GA(4) do not convert this to other GAs prior to, or immediately following, visible germination: thus GA(4) alone can promote radicle expansion. Abscisic acid inhibited [(3)H]GA(4)-induced germination, but did not significantly affect [(3)H]GA(4) uptake or metabolism during germination. (6)N-benzyladenine overcame the inhibitory effect of abscisic acid and increased [(3)H]GA(4) uptake, although radicle emergence was delayed somewhat.During hypocotyl extension there was a large conversion of [(3)H]GA(4) to [(3)H]GA(1) in light or darkness, the major conversion site being the growing root. Hypocotyls of dark-grown seedlings contained more [(3)H]GA(1) than those of light-grown seedlings. The apparent inability of exogenous GA(1) to promote greater hypocotyl extension than GA(4) is related to its poorer uptake. Abacisic acid markedly inhibited hypocotyl expansion, root growth, and the conversion of [(3)H]GA(4) to [(3)H]GA(1). PMID- 16659555 TI - Chloroplast Response to Low Leaf Water Potentials: IV. Quantum Yield Is Reduced. AB - Quantum yields were measured for CO(2) fixation by sunflower (Helianthus annuus L.) leaves having various water potentials and for dichlorophenolindophenol photoreduction by chloroplasts isolated from similar leaves having various water potentials. In red radiation, the quantum yield for CO(2) was 0.076 for an attached sunflower leaf at a water potential of -3 to -4 bars but was 0.020 for the same leaf at -15.3 bars. After recovery to a water potential of -5 bars, the quantum yield rose to 0.060. Soybean (Glycine max L. [Merr.]) leaves behaved similarly. Chloroplasts from a sunflower leaf with a water potential of -3.6 bars had a quantum yield for 4 equivalents of 0.079, but when tissue from the same leaf had a water potential of -14.8 bars, the quantum yield of the chloroplasts decreased to 0.028. The decrease could not be attributed to differences in rates of respiration by the leaves or the chlorophyll content or absorption spectrum of the leaves and chloroplasts.The data are the first to demonstrate an effect of low leaf water potential on the quantum yield and they indicate that changes occurred close to the primary photochemical events of photosynthesis. The similarity in response of the leaves and chloroplasts indicates that certain changes in photosynthesis at low water potentials are attributable to the chloroplasts rather than the stomata. PMID- 16659557 TI - Cyclic Photophosphorylation in the Mykotrophic Orhid Neottia nidus-avis. AB - The mykotrophic orchid Neottia nidus-avis (L.) Rich. is not able to evolve oxygen in the light. Plastid preparations from the lip (labellum) of the orchid perform a photosystem I-dependent photoreduction of methylviologen with the artificial electron donor couple 2,6-dichlorophenol indophenol ascorbate. Photosystem II reactions such as the ferricyanide Hill reaction or the photoreduction of 2,6 dichlorophenol indophenol with diphenylcarbazide as the electron donor are not functioning. The plastids exhibit phenazine methosulfate-mediated cyclic photophosphorylation. After infiltration with (32)P-labeled phosphate the labellum forms (32)P-ATP in the light. This rate of ATP formation is enhanced by additional infiltration of phenazine methosulfate prior to illumination. The brown color of the plant is caused by an absorption shift of carotenoids to longer wavelength. By comparison of absorption spectra with the fluorescence excitation spectra of plastid preparations and of the extracted pigments we show that no appreciable energy transfer from carotenoids to chlorophyll occurs. PMID- 16659556 TI - Changes in Microsomal Enzymes and Phospholipid during Dehardening in Stem Bark of Black Locust. AB - Upon dehardening of stem bark of black locust (Robinia pseudoacacia), a significant decrease in phospholipid content on a milligram protein basis was observed in various crude particulate cell fractions. To ascertain this with a defined membrane, microsomal preparations were separated into several membrane fractions on a discontinuous sucrose gradient. Based on the distribution of various enzymes on the gradient, Golgi apparatus membranes, tonoplast, and unidentified membranes containing acid protease were separated with less contamination by other membranes. The subfraction, with an apparent density of 1.10 g/cc, which was enriched in fragmented tonoplast, contained the most phospholipid per milligram protein. Dehardening resulted in a significant quantitative reduction in protein and phospholipid in the submicrosomal fractions. Significant decreases in phospholipid content per milligram protein were observed during dehardening in tonoplast, Golgi apparatus, and unidentified membranes containing acid protease as well as other membrane fractions. During dehardening, marked decreases in inosine diphosphatase and NADH cytochrome c reductase activities were observed, suggesting a marked degradation of the membranes containing those enzymes. The transition of cell membranes from a phospholipid-enriched state to a phospholipid depleted state is apparently involved in the dehardening process concomitant with a decrease in tissue hardiness. PMID- 16659558 TI - Extractant Influence on the Relationship between Extractable Proteins and Cold Tolerance of Alfalfa. AB - The influence of ionic composition and pH of extractant on the relationship between the extracted proteins and the cold tolerance of Vernal and Arizona Common alfalfa (Medicago sativa L.) was examined. Five environments were used to induce different tolerance levels. The quantity of protein extracted from plants was influenced by the hardening environment, cultivar, and ionic composition and pH of 29 extractants. Extractants with a pH below 6 generally extracted less protein.The measured cold tolerance of the plants was correlated with the quantity of protein detected in many of the 14 regions of the electrophoresis gel columns regardless of extractant but was most closely associated with the protein in either region 7 or 8 with nine of ten extractants.The magnitudes of cultivar and hardening effects on quantities of protein detected on various column regions were influenced by choice of extractant. This suggests that proper extractant selection may be vital for study of isozymes by electrophoresis. PMID- 16659559 TI - Role of Galactolipids in Spinach Chloroplast Lamellar Membranes: II. Effects of Galactolipid Depletion on Phosphorylation and Electron Flow. AB - A galactolipid lipase from primary bean (Phaseolus vulgaris) leaves has been used to partially deplete spinach chloroplast inner membranes of their galactolipids. Chloroplasts treated with the lipase in the absence of bovine serum albumin lost 91% of their monogalactosyl diglyceride, 83% of their digalactosyl diglyceride, all of their phosphatidyl choline, but none of their sulfolipid. Electron microscopy of this sections revealed that the treated chloroplasts were greatly enlarged and lacked membrane stacking. Linolenic acid had similar effects on the structure of the chloroplasts. Chlorophyll, carotenoids, and coupling factor 1 remained bound to the treated membranes.To minimize the inhibition of phosphorylation and electron flow by fatty acids released by the lipase, bovine serum albumin (15-24 mg/ml) was added to the lipase incubation mixtures. Bovine serum albumin inhibited the extent, but not the initial rate, of fatty acid release by the lipase. Electron microscopy of chloroplasts treated with the lipase in the presence of bovine serum albumin showed that membrane stacking was partially maintained. Chloroplasts treated with lipase under these conditions retained about 30% of their monogalactosyl diglyceride, 50% of their digalactosyl diglyceride and phosphatidyl choline. The sulfolipid and phosphatidyl glycerol contents were unchanged. Electron flow through photosystems I and II with artificial electron donors and acceptors was not affected by lipase treatment in the presence of bovine serum albumin. In contrast, oxygen evolution and phosphorylation were partially inhibited. These reactions are also very sensitive to fatty acids and it is possible that the inhibition is the result of interaction of fatty acids with the membrane prior to their binding to bovine serum albumin.In view of the irreversible inactivation of electron flow and phosphorylation by fatty acids, it is difficult to assess the role of galactolipids in these processes when a specfic lipase is used to deplete the membrane. PMID- 16659560 TI - Differential Localization of Fraction I Protein between Chloroplast Types. AB - The soluble proteins of C(3) and C(4) mesophyll chloroplasts and C(4) bundle sheath extracts have been analyzed by gel electrophoresis for fraction I protein. Gel scans of soluble protein from C(4) bundle sheath extracts and C(3) mesophyll chloroplasts showed typical fraction I protein peaks that could be identified by ribulose diphosphate carboxylase activity. No such peak was observed for C(4) mesophyll chloroplasts, which also lacked both large and small subunits of ribulose diphosphate carboxylase on sodium dodecyl sulfate gels. The absence of fraction I protein in these chloroplasts was reflected in the soluble protein to chlorophyll ratios, which were roughly 3-fold lower than the ratio obtained for C(3) chloroplasts. The carboxylating enzyme in C(4) mesophyll cells, phosphoenolpyruvate carboxylase, was found to be a major protein in the cytoplasm of C(4) mesophyll protoplasts, and had higher mobility than fraction I protein. PMID- 16659561 TI - Transport and metabolism of h-gibberellin a(1) in dioecious cucumber seedlings. AB - The transport of (3)H-GA(1) through hypocotyl segments of cucumber (Cucumis sativus L.) was found to be nonpolar. The transport of (3)H-GA(1) was increased by pretreatment with relatively high concentrations of either IAA or Ethephon (2 chloroethylphosphonic acid). Hypocotyl segments from plants of a gynoecious genotype transported more (3)H-GA(1) than those of an androecious. The metabolism of (3)H-GA(1) in hypocotyl segments was neither related to the sex genotype of the cucumber plant nor influenced by pretreatment with Ethephon. The primary metabolite of GA(1) was suggested to be GA(8). Two other suspected metabolites were not identified. Differences in the endogenous GA of gynoecious and androecious plants could not be accounted for by transport differences. PMID- 16659562 TI - Interaction between Osmotic- and Pressure-induced Water Flow in Plant Roots. AB - When the pressure gradient across a root alters, there is often an apparent change in the permeability of the root to water. Fiscus (Plant Physiol. 1975. 55: 917-922) has suggested that this can be explained by a simple two-compartment model which takes into account rates of solute uptake into the xylem. A method of testing actual data against the Fiscus model is proposed; this shows that in some cases the apparent changes in permeability cannot be explained by the model. The model is not adequate to predict the amounts of solute reaching the xylem by passive drag: a three-compartment model would be more realistic. PMID- 16659563 TI - Storage Protein Synthesis in Maize: Isolation of Zein-synthesizing Polyribosomes. AB - Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg(2+) contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels. PMID- 16659564 TI - Microencapsulation of chloroplast particles. AB - Chloroplast and photosystem I particles were encapsulated in small spheres (about 20 mum diameter) with an artificial membrane built up by cross-linking amino groups of protamine with toluenediisocyanate. The artificial membrane was permeable to small substrate and product molecules but not to soluble proteins. Photosystem I activity was retained by the encapsulated chloroplast particles. Washed photosystem I particles were encapsulated with the soluble proteins, ferredoxin, and ferredoxin-NADP oxidoreductase, and the microcapsules photoreduced NADP using ascorbate plus dichlorophenolindophenol as the electron donor. The photosystem I particles were also encapsulated with hydrogenase from Chromatium and a very low rate of photoevolution of hydrogen was obtained. The results show that chloroplast membrane fragments can be encapsulated with soluble proteins that couple transfer reactions to the primary photochemical apparatus. PMID- 16659565 TI - Host-Pathogen Interactions: IX. Quantitative Assays of Elicitor Activity and Characterization of the Elicitor Present in the Extracellular Medium of Cultures of Phytophthora megasperma var. sojae. AB - Resistance of soybean (Glycine max L.) seedlings to Phytophthora megasperma var. sojae (Pms) is in part due to the accumulation in infected tissue of a compound which is toxic to Pms. The accumulation of this compound, a phytoalexin called glyceollin, is triggered by infection, but it can also be triggered by molecules, "elicitors," present in cultures of Pms. The ability of the Pms elicitor to stimulate phytoalexin accumulation in soybean tissues has been used as the basis for biological assays of elicitor activity. Two bioassays were developed and characterized in this study of the Pms elicitor. These bioassays use the cotyledons and the hypocotyls of soybean seedlings. The cotyledon assay was used to characterize the extracellular Pms elicitor. This elicitor was isolated from Pms cultures and purified by ion exchange and molecular sieving chromatography. The extracellular Pms elicitor was determined to be a predominantly 3-linked glucan, which is similar in composition and structure to a polysaccharide component of Pms mycelial walls. PMID- 16659566 TI - Host-Pathogen Interactions: X. Fractionation and Biological Activity of an Elicitor Isolated from the Mycelial Walls of Phytophthora megasperma var. sojae. AB - An elicitor of phytoalexin production in soybean (Glycine max L.) tissues was isolated from purified Phytophthora megasperma var. sojae mycelial walls by a heat treatment similar to that used to solubilize the surface antigens from the cell walls of Saccharomyces cerevisiae. The wall-released elicitor is a discrete, minor portion of the P. megasperma var. sojae mycelial walls. The elicitor released from the mycelial walls was divided by diethylaminoethylcellulose and concanavalin A-Sepharose chromatography into four fractions, each having different chemical characteristics. The four fractions were obtained from each of the three races of P. megasperma var. sojae. The corresponding fractions from each of the three races are very similar in composition and elicitor activity. The results suggest that the elicitor activity of each fraction resides in the glucan component of the fraction. Evidence is presented to demonstrate that the elicitors are not race-specific and that the accumulation of glyceollin is not sufficient to account for race-specific resistance. PMID- 16659567 TI - Host-Pathogen Interactions: XI. Composition and Structure of Wall-released Elicitor Fractions. AB - The structures of the four wall-released elicitor fractions isolated from the Phytophthora megasperma var. sojae mycelial walls have been examined. The results demonstrate that fraction I is primarily composed of a branched beta-1,3-glucan, similar in structure to the extracellular elicitors described previously (Ayers, A., J. Ebel, F. Finelli, N. Burger, and P. Albersheim. 1976. Plant Physiol. 57: 751-759). Fractions II and IV are primarily composed of a highly branched mannan containing glycoprotein, with fraction IV richer in protein than fraction II. Fraction III contains, attached to protein, a mixture of the two polysaccharide types found in fraction I and in fractions II and IV. The structural data presented here, in concert with the biological data presented in the previous two papers (Ayers et al. 1976. Plant Physiol. 57: 751-759; 760-765), demonstrate that the only compound produced by P. megasperma var. sojae which contains elicitor activity is the glucan. Evidence is presented that the terminal glycosyl residues of the glucan are required for elicitor activity. In addition, it is demonstrated that 90% of the glucan can be removed enzymically without any loss of biological activity. The active residue of the enzymic digestion is a highly branched 3- and 3,6-linked glucan containing about 4% mannosyl residues. The results presented suggest that the mannosyl residues of the glucan, which represent only about 1% of the undegraded glucan, are likely to participate in the active site of this molecule. The role of elicitors and phytoalexins in host-pathogen interactions is discussed. Evidence for the existence of and possible identity of another factor, which determines race specificity of host-pathogen interactions, is summarized. PMID- 16659568 TI - Host-Pathogen Interactions: XII. Response of Suspension-cultured Soybean Cells to the Elicitor Isolated from Phytophthora megasperma var. sojae, a Fungal Pathogen of Soybeans. AB - The glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, the fungus which causes stem and root rot in soybeans, stimulates the activity of phenylalanine ammonia-lyase and the accumulation of glyceollin in suspension-cultured soybean cells. Nigeran, a commercially available fungal wall glucan, was the only other compound tested which has any activity in this system. Glyceollin is a phenylpropanoid-derived phytoalexin which is toxic to P. megasperma var. sojae. Evidence is presented to support the hypothesis that the action of elicitors in stimulating phytoalexin synthesis is not species or variety specific but, rather, is part of a general defensive response of plants. PMID- 16659569 TI - Lipoxygenase and hydroperoxide lyase in germinating watermelon seedlings. AB - Lipoxygenase (EC 1.13.1.13) was found in seedlings of Citrullus lanatus (Thunb.) Matsum. and Nakai (watermelon). The enzyme has pH optima of 4.4 and 5.5 and is inhibited by 0.2 mM nordihydroguaiaretic acid. It is present in two functional units with estimated molecular weights of 120,000 and 240,000, respectively.A new enzyme, tentatively termed hydroperoxide lyase, has been partially purified from watermelon seedlings. The enzyme, located principally in the region of the hypocotyl-root junction, catalyzes the conversion of 13-l-hydroperoxy-cis-9-trans 11-octadecadienoic acid to 12-oxo-trans-10-dodecenoic acid and hexanal. The hydroperoxide lyase enzyme from watermelon has a molecular weight in excess of 250,000, a pH optimum in the range of 6 to 6.5, and is inhibited by p chloromercuribenzoic acid. Its presence has also been demonstrated in other cucurbits.The maximum activity of both enzymes occurs on the 6th day of germination. The identification of the products of the hydroperoxide lyase reaction suggests that lipoxygenase and hydroperoxide lyase may be involved in the conversion of certain polyunsaturated fatty acids to traumatic acid (trans-2 dodecenedioic acid). PMID- 16659570 TI - Sequential changes in the lipids of developing proplastids isolated from green maize leaves. AB - Changes in lipid composition were followed as a proplastid develops into a chloroplast. Methods were devised for the isolation of developing proplastids from sections of five different ages from the same 7-day-old maize (Zea mays var. Kelvedon Glory) leaf. Electron micrographs illustrate the homogeneity of the five types of plastid suspension, minimal contamination with other cytoplasmic membranes, and the presence of morphologically intact plastids in the proportions 85% (youngest), 85%, 80%, 70% and 60% (oldest), respectively. Both bundle sheath and mesophyll plastids are well preserved in isolation. Plastid numbers were determined from calibration curves of the chlorophyll content of each type of suspension, and lipid values then expressed as nmoles/10(6) plastids. Monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG), sulfoquinovosyl diglyceride, and phosphatidyl glycerol (PG) all increase during plastid development but the rate of increase is different for each lipid. The largest changes are in MGDG (6-fold) and DGDG (4-fold). Phosphatidyl choline shows a continuous decline during plastid development. Phosphatidyl inositol and phosphatidyl ethanolamine were found in all the suspensions in low concentrations (0.4-4.0% of total lipid): calculations showed their presence could not be accounted for by bacterial or mitochondrial contamination. The increase in PG parallels the chlorophyll changes during development and at maturity 1 molecule of PG is present per 3 molecules of chlorophyll. The results are discussed in the context of the molecular structure of the photosynthetic thylakoid membranes. PMID- 16659571 TI - Partial purification and characterization of two Peptide hydrolases from pea seeds. AB - Two peptide hydrolases have been found in pea seeds (Pisum sativum var. Greenfeast) and extensively purified by ion exchange chromatography using benzoyl dl-arginine-p-nitroanilide as substrate. The enzymes which both have molecular weights of 65,000 can be separated by anion exchange chromatography but are otherwise virtually identical in the properties tested. They did not hydrolyze several common protease substrates but readily hydrolyzed small peptides containing basic amino acids on the carboxyl side of these residues. They are completely inhibited by diisopropylfluorophosphate and are inhibited to varying extents by thiol reagents. PMID- 16659572 TI - Photoreduction of sulfur dioxide by spinach leaves and isolated spinach chloroplasts. AB - Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed. PMID- 16659573 TI - Carbohydrate-limited Growth Kinetics of Tobacco (Nicotiana rustica L.) Callus. AB - Logistic curves were fitted to sigmoidal growth data obtained from tobacco (Nicotiana rustica L.) callus grown on media prepared with 0.1, 0.03, 0.01, and 0.003 m sucrose. Analysis of the growth curves indicated that final yields and specific growth rates were influenced by the initial sucrose concentration. Growth yields from the four treatments were similar (0.61 +/- 0.04 gram dry tissue per gram sucrose supplied). Initial specific growth rates exhibited a Michaelis-Menten dependency on initial sucrose concentration such that the V(max) = 0.18 g g(-1) day(-1) and Km = 0.0037 m sucrose. PMID- 16659574 TI - Enhancement of CO(2) Uptake in Avena Coleoptiles by Fusicoccin. AB - When Avena coleoptile segments are immersed in a solution containing H(14)CO(3) ( ), the appearance of label in the tissue is stimulated approximately 3-fold by fusicoccin application. This effect is rapid (1-2 minutes lag time), dependent upon respiratory energy, inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not appreciably altered by cycloheximide treatment. A large percentage of the cellular radioactivity is found in the form of malate. Preliminary experiments indicate that CO(2), as opposed to HCO(3) (-), is the favored species of "CO(2)" taken up by the segments. These results are consistent with the notion that CO(2), presumably by virtue of its fixation and conversion to malic acid, participates in the early events associated with fusicoccin-enhanced acidification of the cell wall region leading to the stimulation of cell extension growth. PMID- 16659575 TI - Effect of Thiamine on Ethanol and Pyruvate Production in Helminthosporium maydis. AB - Growth of the fungus Helminthosporium maydis race T in a basal glucose-l asparagine liquid medium, pH 5, is inhibited by thiamine-HCl. Analysis of the media for organic acids reveals that the extracellular pyruvate concentration decreases as the thiamine-HCl concentration of the medium increases. Extracellular ethanol, in contrast to pyruvate, increases in concentration as the thiamine-HCl concentration of the medium increases under both aerobic and anaerobic conditions.The changes in ethanol and pyruvate levels in the presence of thiamine-HCl occur via a thiamine-mediated increase in the activity of pyruvate decarboxylase but not alcohol dehydrogenase. This increase in pyruvate decarboxylase activity appears to be due to an increase in the quantity of enzyme present rather than an activation of pre-existing enzyme. Whereas thiamine pyrophosphate stimulates pyruvate decarboxylase activity in vitro, thiamine-HCl has no effect. Neither thiamine derivative affects alcohol dehydrogenase activity. The increase in pyruvate decarboxylase activity which accompanies an increase in the thiamine-HCl concentration of the medium is correlated with a decrease in the level of intracellular pyruvate. PMID- 16659576 TI - Additional evidence for separable responses to auxin in soybean hypocotyl. AB - Additional evidence for two separable responses to auxin is presented. The average of 24 control experiments indicated lag times of 12.4 and 35.4 min, and maximum rates of 0.57 and 0.54 mm hr(-1), for the first and second response, respectively. The auxin analog 4-azido-2-chlorophenoxyacetic acid increased the lag time of the second response (but not the first), resulting in the temporal separation of the two responses. Plots of elongation rates against time, taken from the literature, allowed the characterization of the two responses in monocotyls and dicotyls. Study of published rate-time elongation curves showed that the maximum rate of the first response is frequently greater than the maximum rate of the second response; however, the maximum rate of the second response has not yet been shown to exceed the maximum rate of the first response. PMID- 16659577 TI - Chemotaxis of Rhizobium spp. to Plant Root Exudates. AB - Rhizobium spp. show chemotaxis to plant root exudates. Both legumes and non legume root exudates attract the different rhizobia studied. However, the bacteria show a differential response in that they are attracted to the root exudates of some plants and show no attraction toward others. An example of negative chemotaxis was also observed. The trefoil strain of Rhizobium shows chemotaxis which is qualitatively different from that observed in other bacteria in that simple sugars, di-and trisaccharides, dextrans, and amino acids do not attract this bacterium. PMID- 16659578 TI - Relation between Paramylum Content and the Length of the Lag Period of Chlorophyll Synthesis during Greening of Dark-grown Euglena gracilis. AB - Euglena cells, strains Z and bacillaris, were grown in the dark under various nutritional deficiencies. After 3 days of nondivision, cells were transferred to the light, and the following parameters were measured: the paramylum content at the time of illumination (zero time), the rate of paramylum consumption during the first 10 hours of greening, and the length of the lag phase of chlorophyll synthesis. Similar results were obtained with both strains and can be summarized as follows. (a) The use of various nutritional deficiencies allows the control, to a certain extent, of the amount of paramylum present at zero time. (b) The rate of paramylum consumption is proportional to the cellular paramylum content for values in excess of 50 picograms/cell. (c) The length of the lag phase increases rapidly when the cellular content of paramylum decreases below 50 picograms. This period can be greatly diminished by the addition of an exogenous organic carbon source. (d) The amount of paramylum (rate of paramylum consumption x length of lag phase) consumed during the lag phase is around 5 to 10 picograms/cell for cells which contain less than 50 picograms of paramylum/cell. It increases when the cellular paramylum content increases, this increment being more rapid for bacillaris than for Z cells. PMID- 16659579 TI - Influence of Culture Conditions on the Length of the Lag Period of Chlorophyll Synthesis in Preilluminated Dark-grown Euglena. AB - Two parameters of the potentiation process, the lengths of preillumination and of the subsequent dark phase have been determined for Euglena cells which had been grown in the dark under two different culture conditions. In both cases a maximum potentiation response is obtained after a preillumination period of about 2 hours. The minimum length of the dark period necessary for maximum potentiation changes from 1 hour for cells which contain, at the moment of illumination, a high paramylum content, to 6 hours for cells which have a low paramylum content. PMID- 16659580 TI - Involvement of endogenous ethylene in the induction of color change in shamouti oranges. AB - Reducing the level of endogenous ethylene in detached Shamouti oranges (Citrus sinensis L. Osbeck) by means of subatmospheric pressure did not alter the rate of chlorophyll destruction and color changes during the first 8 days after harvest in the presence or absence of exogenous ethylene. Reducing the activity of ethylene by means of CO(2)-known to be a competitive inhibitor for ethylene inhibited chlorophyll destruction and color change in Shamouti oranges ventilated with ethylene, but had no effect on these processes in the absence of applied ethylene. The evidence presented indicates that endogenous ethylene may not be the primary inducer for the natural color change in detached Shamouti oranges. PMID- 16659581 TI - A new class of synthetic auxin transport inhibitors. AB - Auxin transport inhibition by a new class of synthetic plant growth regulants, the 2-(3-aryl-5-pyrazolyl)benzoic acids, was examined in bean (Phaseolus vulgaris L.) using the donor-receiver agar cylinder technique. These compounds can be prepared by the dehydrogenation and ring cleavage of compounds like DPX-1840 (2 (4-methoxyphenyl)-3,3adihydro-8H-pyrazolo[5,1-a] isoindol-8-one) which was previously reported (Plant Physiol. 1972. 50: 322-327) to be a potent inhibitor of auxin transport. These new growth regulators inhibit auxin transport more than DPX-1840 does as evidenced by their consistently greater reduction of basipetal auxin transport capacity in bean when incorporated into the receiver agar cylinder or applied foliarly to intact plants. Direct comparisons of the effect of DPX-1840, its dehydrogenation product (2-(4-methoxyphenyl)-8H-pyrazolo [5,1 a]isoindol-8-one), and its open-ring form (2-(3-(4-methoxyphenyl)-5-pyrazolyl) benzoic acid) on auxin transport indicated the following order of activity: ring open > dehydrogenated form > DPX-1840. DPX-1840-(14)C, applied at 0.5 mg/l to etiolated bean hypocotyl hooks followed by extraction and thin layer chromatography, indicated the biological conversion of DPX-1840 to its open-ring form. Collectively, these results suggest that the biologically active forms of DPX-1840-type compounds are the open-ring (2-(3-aryl-5-pyrazolyl) benzoic acids. PMID- 16659582 TI - Calorimetric studies of the elongation of Avena coleoptile segments. AB - Elongation rate and heat produced by Avena coleoptile segments suspended in sucrose buffer solutions were measured at pH values from 3.5 to 8.5. The caloric efficiency of elongation (CEE) was defined as the ratio of the rate of elongation to the rate of heat production. Elongation and CEE were greatest at intermediate pH values, but heat production (about 1 cal/g.hr) was insensitive to pH within the limits of experimental error (+/-20%). Quantitative agreement was found between the results of previous respiration studies and the rate of heat production in an aerobic atmosphere, which indicates that oxidative metabolism accounts for essentially all energy changes in the cell, so matter flow is a significant component of the bioenergetics of cell function. Indole-3-acetic acid up to 1 mm, produced about a 10-fold increase in elongation rate, a 5-fold increase of the CEE, and a 25% increase in heat production. Above this concentration, sharp drops in both elongation and heat production occurred, without altering the CEE at pH 6.5, but greatly decreasing the CEE at pH 4.5. Elongation and CEE showed marked decreases after 4 hours in an anaerobic atmosphere, but heat production did not exhibit a proportional decrease. These studies indicate that rate of cell elongation in the presence and absence of auxin is not directly proportional to the overall metabolism of the cell. PMID- 16659583 TI - Glucolipids of Zea mays and Pisum sativum. AB - The glucolipids formed upon feeding (U-(14)C)glucose to embryos of Zea mays were partially characterized with respect to: (a) metabolic turnover, (b) acid lability, (c) phosphorus content, (d) chromatographic properties, and (e) hydrolysis products. The chloroform-methanol-soluble assimilated radioactivity was examined specifically for occurrence of a glycosylated prenol phosphate. With the extraction conditions used, no evidence was found for formation of a glucosylated prenol phosphate. Several, as yet unidentified, acid-labile glucolipids undergoing metabolic turnover were observed. Four diglycerides were characterized as hydrolysis products of a fraction that contained (14)C-glucose and phosphorus, and was subject to metabolic turnover. Examination of the 1 butanol-soluble glucolipids from pea (Pisum sativum) seedlings also demonstrated anionic glucolipids, evidencing metabolic turnover but none with the properties of glucosylated prenol phosphate. PMID- 16659584 TI - Auxin biogenesis: subcellular compartmentation of indoleacetaldehyde reductases in cucumber seedlings. AB - Subcellular fractionation of cucumber (Cucumis sativus L.) seedlings was achieved, and two of the enzymes in the auxin biosynthetic pathway were localized. NADH-specific indoleacetaldehyde reductase activity was observed only in the cytosol fractions obtained from separated hypocotyl and cotyledon tissue. In contrast, a portion of the NADPH-specific indoleacetaldehyde reductase activity was associated with a microsomal fraction derived from these tissues. The NADPH-specific indoleacetaldehyde reductase was consistently found to be more firmly associated with the microsomal fraction derived from hypocotyls than with that from the cotyledons. These results indicate a division of the terminal steps of auxin biogenesis into at least two subcellular compartments. PMID- 16659585 TI - Floral activity in solutions of deoxyribonucleic Acid extracted from tobacco stems. AB - For Nicotiana tabacum cv. Wis. 38 plants, the capabilities of solutions containing DNA, extracted from either homogenates of stems in a floral state or nuclei of stems in a vegetative state, to effect flowering of vegetative plants have been studied. Previous work indicates that the DNA from homogenates of stems in a floral state is mainly nuclear. If DNA solutions are supplied to axillary buds of vegetative plants and if the axillary buds are defoliated every 4th day for 12 days, the buds supplied a solution of DNA from stems in a floral state initiate flowers under noninductive conditions, and the buds supplied a solution of DNA from stems in a vegetative state remain vegetative. Heating and rapidly cooling a solution of DNA from stems in a floral state enhances its floral activity. Heating and cooling a DNA solution also results in novel flowers showing up in many treated plants. Novel flowers are more striking in the offspring than in the parents. The capabilities of heated-cooled DNA solution to initiate flowers in noninductive conditions and to cause novel flowers are eliminated completely by treating (before heating and cooling) the DNA solution with deoxyribonuclease. Heated-cooled solutions of DNA extracted from nuclei of either vegetative stems or vegetative leaves contain no floral activity. PMID- 16659586 TI - Glutamate Synthetase in Developing Cotyledons of Pisum sativum. AB - Glutamate synthetase (glutamine[amide]:alpha ketoglutarate amino transferase oxidoreductase) activity has been demonstrated in the developing cotyledons of Pisum sativum L. cv. Burpeeana. The enzyme appears to be soluble and is specific for glutamine as amide donor. The enzyme activity is greater with NADH than with NADPH as electron donor.It is suggested that glutamate synthetase in the developing cotyledon provides a mechanism by which the amide nitrogen of glutamine, from the translocatory stream, is converted into the amino nitrogen of glutamate; transamination reactions involving this synthesized glutamate could provide the amino groups for the biosynthesis of the seed protein amino acids. PMID- 16659587 TI - Purification and partial characterization of a dipeptidase from barley. AB - A peptidase hydrolyzing the dipeptide Ala-Gly optimally at pH 8 to 9 was purified about 3500-fold from germinated grains of barley (Hordeum vulgare L.). According to disc electrophoresis in the presence of sodium dodecyl sulfate, the preparation was about 90% pure.The enzyme preparation hydrolyzed all of the 15 neutral dipeptides tested, but showed no activity on Lys-Gly or Asp-Ala. Three tripeptides and four synthetic aminopeptidase substrates were hydrolyzed at less than 0.1% of the rate for Ala-Gly; inhibition and inactivation tests indicated that these reactions were due to some contaminating aminopeptidase(s). The results suggest that the purified enzyme is a specific dipeptidase. Km values were determined for six dipeptides at pH 8.8; they varied from 0.3 to 15.8 mm. The enzyme was strongly inhibited both by metal chelators and by sulfhydryl reagents. The elution volumes of the enzyme in gel chromatography on Sephadex G 150 and Ultrogel AcA 22 corresponded to molecular weight 130,000 and 175,000, respectively. The migration rate in sodium dodecyl sulfate disc electrophoresis indicated a subunit molecular weight of 50,000.The barley enzyme is remarkably similar to several mammalian and microbial dipeptidases. PMID- 16659588 TI - Evidence for Phloem loading from the apoplast: chemical modification of membrane sulfhydryl groups. AB - The water-soluble, sulfhydryl-specific, chemical modifier p chloromercuribenzenesulfonic acid reversibly inhibited the accumulation of exogenously supplied (14)C-sucrose into leaf discs of Beta vulgaris. P Chloromercuribenzenesulfonic acid treatment did not inhibit photosynthesis or respiration or induce membrane leakage to sucrose, indicating that the site of inhibition was the plasmalemma. The active loading of sucrose and (14)CO(2) derived assimilates into the phloem and their translocation from the source leaf were inhibited by the nonpermeant modifier. Several nonpermeant sulfhydryl group modifiers also inhibited sucrose accumulation into leaf discs while two amino reactive reagents had no effect. The results indicate that sugars are actively accumulated into the phloem from the apoplast and that membrane sulfhydryl groups may be involved. PMID- 16659589 TI - Photosynthetic carbon metabolism of a marine grass. AB - The delta(13)C value of a tropical marine grass Thalassia testudinum is -9.04 per thousand. This value is similar to the delta(13)C value of terrestrial tropical grasses. The delta(13)C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: -11.2 per thousand, 13.1 per thousand, -10.1 per thousand, -11.1 per thousand, and -11.5 per thousand, respectively. The delta(13)C values of malic acid and glucose of Thalassia are similar to the delta(13)C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C(4)-dicarboxylic acid pathway in this marine grass. The inorganic HCO(3) (-) for the growth of the grass fluctuates between -6.7 to -2.7 per thousand during the day. If CO(2) fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a -3 per thousand fractionation between HCO(3) (-) and malic acid), the predicted delta(13)C value for Thalassia would be -9.7 to -5.7 per thousand. This range is close to the observed range of -12.6 to -7.8 per thousand for Thalassia and agree with the operation of the C(4)-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO(3) (-) in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO(2) fixation in C(4) terrestrial plants.Electron microscopy of the leaves of Thalassia reveal thick walled epidermal cells exceedingly rich in mitochondria and C(3)-type chloroplasts. The mesophyll cells have many different shapes and surround air lacunae which contain O(2) and CO(2). The mesophyll cells are highly vacuolated and the parietal cytoplasm contains an occasional chloroplast. This chloroplast contains grana but the lamellar system is not as developed as the system in epidermal chloroplasts. Extensive phloem tissue is present but the xylem elements are reduced in this aquatic grass. The vascular tissue is not surrounded by bundle sheath cells.This work does not establish the exact relation between structure and function in Thalassia, but it does show the C(4)-type photosynthetic carbon metabolism in this grass involves epidermal and mesophyll cells and internally produced O(2) and CO(2) in the air lacunae. PMID- 16659590 TI - Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C(4) Plants. AB - Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C(4) groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD malic enzyme type); and Panicum maximum (phosphoenolpyruvate carboxykinase type). Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase were predominantly localized in mesophyll cells of all the species, except in P. maximum where nitrite reductase had similar activity on a chlorophyll basis, in both mesophyll and bundle sheath cells. NADH-glutamate dehydrogenase was concentrated in the bundle sheath cells, while NADPH-glutamate dehydrogenase was localized in both mesophyll and bundle sheath cells. The activities of nitrate-assimilating enzymes, except for nitrate reductase, were high enough to account for the proposed in vivo rates of nitrate assimilation.Based on the differential centrifugation of cell homogenates of P. miliaceum, mesophyll chloroplasts appear to be the major site of nitrate assimilation since nitrite reductase, glutamine synthetase, glutamate synthase, and NADPH-glutamate dehydrogenase were primarily localized in the chloroplast fraction. Both the glutamine synthetase-glutamate synthase and glutamate dehydrogenase pathways were considered as alternative routes of amino nitrogen synthesis. PMID- 16659591 TI - Cobalt and plant development: interactions with ethylene in hypocotyl growth. AB - Co(2+) promoted elongation of hypocotyl segments of light-grown cucumber (Cucumis sativus) seedlings. Time course and dose response data are presented and interactions with IAA, gibberellin, cyclohexanol, and cotyledons described. Segments without cotyledons responded to Co(2+) only if grown in gas-tight vessels with IAA added. When bases of cotyledons were ringed with an inhibitor of auxin transport, Co(2+) caused no growth promotion in the hypocotyl. Co(2+) prevented lateral swelling of hypocotyls treated with supraoptimal IAA. Removal of ethylene from the atmosphere reduced the Co(2+) response, but Co(2+) did not counteract the inhibitory effect of increased ethylene levels. These results are consistent with the hypothesis that Co(2+) promotes hypocotyl elongation by inhibiting ethylene production. The hypothesis was confirmed by a direct demonstration that Co(2+), at growth-promoting concentrations, powerfully inhibited ethylene production in the cucumber hypocotyl. PMID- 16659592 TI - Effect of protein additives on acetylene reduction (nitrogen fixation) by Rhizobium in the presence and absence of soybean cells. AB - The effect of protein additives on acetylene reduction (N(2) fixation) by Rhizobium associated with soybean cells (Glycine max [L.] Merr.) in vitro was studied. Acetylene reduction was promoted on the basal medium supplemented with 1.4 mg of N/ml supplied as aqueous extracts of hexane-extracted soybean, red kidney beans (Phaseolus vulgaris L.), or peas (Pisum sativum L.). Commercial samples of alpha-casein, or bovine serum albumin also promoted acetylene reduction at a concentration of 1.4 mg of N/ml of basal medium, but egg albumin supplying an equal amount of nitrogen to the basal medium completely suppressed acetylene reduction. Autoclaving the aqueous extract of hexane-extracted soybean meal had no effect on its ability to promote acetylene reduction. The presence of 40 mm succinate decreased acetylene reduction with leguminous proteins supplying 1.4 mg of N/ml but promoted acetylene reduction by Rhizobium 32H1-soybean cell associations on media containing alpha-casein, bovine serum albumin, or egg albumin suppling 1.4 mg of N/ml. Similar results were obtained with both cowpea Rhizobium 32H1 and Rhizobium japonicum 61A96. Pure cultures of Rhizobium 32H1 developed acetylene-reducing activity in the presence of soybean extract on basal agar medium and in vermiculite supplied with N-free mineral salts plus crude soybean meal. The results suggest that in certain situations, free living Rhizobium may reduce N(2) under field conditions. PMID- 16659593 TI - An improved cytokinin bioassay using cultured soybean hypocotyl sections. AB - This paper describes a modified soybean (Glycine max) tissue culture bioassay for cytokinins. Soybean hypocotyls were grown under sterile conditions and sliced into 1-mm sections. Sections were cultured for 5,9,13, or 22 days on a callus medium with zeatin or other cytokinins. The fresh weight of sections increased with the cytokinin concentration from 0.0005 to 1 mum zeatin; 2-fold concentration differences were readily distinguishable at 9 days. The assay should prove to have several advantages over the conventional soybean callus bioassay including convenience, lower variability between tissue samples, and improved resolution. Its specificity is comparable to that of the soybean callus bioassay. PMID- 16659594 TI - Stomatal response of engelmann spruce to humidity, light, and water stress. AB - Stomatal response of Engelmann spruce (Picea engelmannii Engelm.) to environmental conditions was studied in the natural subalpine environment and under controlled laboratory conditions. Stomata of naturally occurring trees responded to the difference in absolute humidity from leaf to air. When foliage was exposed to full sunlight, stomatal conductance decreased as the absolute humidity difference increased. In the shade, where photosynthetically active radiation was 10% of that in full sunlight, stomatal closure at large absolute humidity differences was much more complete. No effect of soil or air temperatures on stomatal aperture was observed in the field, nor were differences among three contrasting sites detected. Under growth chamber conditions, stomata responded to photosynthetically active radiation, but conductances were influenced by leaf-to-air differences in absolute humidity. Leaf water potentials below - 15 bars resulted in lower conductances over a range of humidity and light conditions. Because net photosynthesis under shaded conditions in the natural environment must be very low, stomatal closure could result in considerable savings in water while having a minimum effect on net photosynthesis. PMID- 16659595 TI - Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination: I. Nitrate Reductase and Isocitrate Lyase. AB - Actinomycin D at 10 mug/ml strongly inhibited the increase in isocitrate lyase activity during germination of seeds and 40-day-old embryos of cotton (Gossypium hirsutum L.) when the germination period was preceded by 3 hours of soaking in the inhibitor solution. No inhibition was observed without the presoaking. Induction of nitrate reductase activity by nitrate was never inhibited by actinomycin D under the same conditions, and was frequently stimulated about 50%. Thus, the method of applying actinomycin D to the seeds and ovules could affect interpretation of its action. Abscisic acid at 5 mug/ml blocked production of isocitrate lyase activity in both pregermination treatments, but did not inhibit induction of nitrate reductase activity. Induction of nitrate reductase activity became insensitive to the two inhibitors during ovule maturation, at about 32 days after anthesis. The results indicate that isocitrate lyase, a germination enzyme, is not synthesized on performed mRNA. In this respect, the appearance of activity in cotton resembles that in other species of fatty seeds. In contrast, induction of nitrate reductase activity, which is unnecessary for germination, apparently is not regulated at the level of transcription except in young ovules. PMID- 16659596 TI - Purification and characterization of phosphoenolpyruvate carboxylase from maize leaves. AB - Phosphoenolpyruvate carboxylase has been purified to homogeneity from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. The ratio of specific activities in crude extracts and the purified enzyme suggests that the enzyme is a major soluble protein in the tissue. The enzyme has a sedimentation coefficient (s(20,w)) of 12.3S and a molecular weight, determined by sedimentation equilibrium, of 400,000 daltons. Dissociation of the enzyme and electrophoresis on dodecyl sulfate polyacrylamide gels yields a single stained band which corresponds to a subunit weight of 99,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains.The enzyme yields cooperative rate-concentration plots (Hill number of 2) with phosphoenolpyruvate as the variable substrate at pH 7. This cooperativity disappears in the presence of an activator, glucose-6-P, or by raising the pH of the assay mixture to 8. Glycerol (20%, v/v) exerts a similar effect. The enzyme is also activated in the presence of glycine which causes an increase in V(max) without significant effect on the apparent Km for phosphoenolpyruvate and Hill number. The apparent Km for HCO(3) (-) is 0.02 mm, and the activation constant for Mg(2+) is 1.54 mm at pH 7. There is an abrupt discontinuity in Arrhenius plots and an associated increase in activation energy below 10.8 C. PMID- 16659597 TI - Different mass transfer rates of labeled sugars and tritiated water in xylem vessels and their dependency on metabolism. AB - Solutions of (14)C-sugars in tritiated water or solutions of (14)C- and (3)H sugars were perfused by gravity through the xylem vessels of excised tomato internodes (Lycopersicon esculentum) mostly during 2 hours.Mass flow of a solution in plant vessels is found not to be in conflict with different mass transfer rates of sugar and water molecules. It is explained by individual lateral escape rates from the vessel for each single compound. The escape of tritiated water can be ascribed to diffusion, while the escape of sugars is apparently linked to the metabolism of the surrounding parenchyma cells (Q(10) sucrose uptake = 3.5). Lateral escape rates of sugars from the xylem vessels are in the proportion of sucrose-glucose-fructose (2.4:1.0:0.6). The accessibility of xylem parenchyma cells and their differential permeability to sugars control the longitudinal mass transfer of sugars in the xylem vessels.As sucrose and glucose do not compete for uptake from the vessels by the contiguous cells, separate uptake systems for both may be postulated. PMID- 16659598 TI - Regulation of lignin formation in reed canarygrass in relation to disease resistance. AB - Lignin content and enzymes involved in lignification were measured in leaf discs of reed canarygrass (Phalaris arundinacea L.) inoculated with Helminthosporium avenae and floated on water or solutions of cycloheximide (25 mug/ml). Fungal germ tubes did not penetrate localized lignified swellings, which formed beneath penetration sites, in the outer epidermal wall of discs floated on water. Within 18 hours, inoculated discs on water had higher lignin content and higher activity of the enzymes phenylalanine ammonia lyase, tyrosine ammonia lyase, hydroxycinnamate-CoA ligase and peroxidase than noninoculated discs on water. When inoculated tissues were floated on cycloheximide solutions, increases in lignin content and enzyme activities associated with lignin biosynthesis were inhibited, and the tissue was susceptible to fungal penetration. Lignin biosynthesis at the site of attempted fungal penetration may play an important role in the resistant response of reed canarygrass to leaf-infecting fungi. PMID- 16659599 TI - Soluble and cell wall peroxidases in reed canarygrass in relation to disease resistance and localized lignin formation. AB - The relationship of peroxidases to an inducible disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canarygrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wall-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wall near the site of attempted penetration. When inoculated discs were floated on solutions of cycloheximide (25 mug/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cycloheximide treatment.It was suggested that the resistance mechanism in P. arundinacea involves an induction of cathodic isoperoxidases in challenged tissue. These peroxidases may function in the biosynthesis of lignin at the site of attempted penetration. PMID- 16659600 TI - Aspects of nitrogen metabolism in the rice seedling. AB - The effects of nitrogen source NO(3) (-) or NH(4) (+) on nitrogen metabolism during the first 2 weeks of germination of the rice seedling (Oryza sativa L., var. IR22) grown in nutrient solution containing 40 mug/ml N were studied. Total, soluble protein, and free amino N levels were higher in the NH(4) (+)-grown seedling, particularly during the 1st week of germination. Asparagine accounted for most of the difference in free amino acid level, in both the root and the shoot. Nitrate and nitrite reductase activities were present mainly in the shoot and were higher in the NO(3) (-)-grown seedling, whereas the activity of glutamate dehydrogenase and glutamine synthetase in the root tended to be lower than that of the NH(4) (+)-grown seedling during the 1st week of germination. Glycolate oxidase and catalase activities were present mainly in the shoot. Maximum activity of the above five enzymes occurred 7 to 10 days after germination. Differences in the zymograms of nitrate reductase, glutamate dehydrogenase, and catalase were mainly between shoot and root and not from N source. Nitrite reductase bands were observed only in plants grown in plants grown in NO(3) (-).Ten-day-old seedlings of three rices differing in level of grain protein did not differ in the level of N fractions and of enzyme activities, which were consistent with their differences in grain protein content. PMID- 16659601 TI - Rhythmic Nitrate Reductase Activity in Leaves of Capsicum annuum L. and the Influence of Kinetin. AB - In the expanding leaves of Capsicum annuum L. cv. California Wonder, two of the three peaks of nitrate reductase activity associated with the light period exhibit a circadian rhythm that persists in continuous light.The spray application of kinetin to the whole shoot or to leaves other than the ones used for nitrate reductase assay causes a phase shift in the activity peaks and this has been used in preliminary investigations of the character of the mechanisms controlling the timing of the peaks.There was some indication that the rate of translocation of nitrate from the roots might be involved. The levels of nitrate moving up the stem after kinetin treatment were more dependent upon the rate of sap flow than on the concentration of nitrate in the sap. For this reason, transpiration rates in whole plants were measured after kinetin treatment but no change in pattern was seen that would correlate with the phase shift in nitrate reductase activity.The occurrence of nitrate reductase peaks in excised leaves suggested a leaf-based in addition to a root-or stem-based mechanism in the timing of nitrate reductase activity in the leaves. PMID- 16659602 TI - Starch degradation in isolated spinach chloroplasts. AB - A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with (14)C-starch is described. These intact chloroplasts were incubated aerobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3 phosphate and to maltose. PMID- 16659603 TI - Auxin-induced Changes in the Incorporation of H-Amino Acids into Soybean Ribosomal Proteins. AB - Auxin-induced activation of 80S ribosomes and polyribosome formation in mature soybean (Glycine max var. Hawkeye) hypocotyl (R. L. Travis, J. M. Anderson, and J. L. Key. 1973. Plant Physiol. 52: 608-612) in the presence of a mixture of radioactive amino acids correlates with an increased specific radioactivity of at least three ribosomal proteins; the labeling of one of these increased severalfold above the control level. Results of experiments with 5-fluorouracil and cycloheximide indicated that the proteins in question were synthesized in response to auxin and became associated with pre-existing ribosomes. Ribosome dissociation experiments indicated that these proteins were associated with the 60S ribosome subunit. PMID- 16659604 TI - Light-induced De-epoxidation in Lettuce Chloroplasts: VI. De-epoxidation in Grana and in Stoma Lamellae. AB - Grana and stroma lamellae fractions prepared from illuminated chloroplasts (Lactuca sativa L. var. Manoa) by French press treatment contained less violaxanthin and more zeaxanthin than the corresponding fractions from dark controls. In both fractions, only part of the total violaxanthin was de epoxidized under illumination, and the ratio of de-epoxidized and unchanged violaxanthin was similar. This not only shows that the de-epoxidation system is present in both grana and stroma thylakoids but also that violaxanthin is heterogeneous in both membranes. The presence and similarity of the de epoxidation system in grana and stroma lamellae suggest that the function of the violaxanthin cycle is linked to photosynthetic activities which are common to both types of membranes. PMID- 16659605 TI - Influence of Antitranspirants on Rapeseed (Brassica campestris) Plants under Water-stressed and Nonstressed Conditions. AB - Daily transpiration rate was decreased by low soil moisture, phenylmercuric acetate (PMA) in combination with kaolinite, Mobileaf (a filmforming wax emulsion manufactured by the Mobil Oil Co.), PMA alone, and kaolinite alone in this order. At high soil moisture, dry matter was decreased by PMA but was increased by Mobileaf and by PMA + kaolinite combination. At low soil moisture, dry matter was increased by Mobileaf.Water use efficiency was increased at low soil moisture and by antitranspirants. The relative water content of leaves was reduced by low soil moisture but was increased by the antitranspirants which relieved plant water stress. PMID- 16659606 TI - Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans. AB - Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.The inhibitory activity against the mung bean endopeptidase is due to the presence of two inhibitors which can be separated by chromatography on Sephadex G-100. The two inhibitors have approximate molecular weights of 12,000 and smaller than 2,000 daltons. The large inhibitor coelutes with trypsin inhibitor on Sephadex G-100, but these two inhibitory activities can be separated by means of a trypsin affinity column.The inhibitory activity disappears slowly from crude extracts incubated at 6 C and more rapidly when the extracts are incubated at 25 C or 37 C. The disappearance of inhibitory activity is accompanied by a rise of the endopeptidase activity, but an examination of the kinetics of these two phenomena suggests that they are not causally related. Fractionation of the cellular organelles on sucrose gradients shows that the inhibitory activity is not associated with the protein bodies, but rather with the cytosol. Our results suggest that the endopeptidase inhibitor(s) does not regulate the increase in endopeptidase activity which accompanies germination or the metabolism of storage protein. We, therefore, postulate that the inhibitor(s) may function in protecting the cytoplasm from accidental rupturing of the protease-containing protein bodies. PMID- 16659607 TI - Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue. AB - Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B(6). Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mm sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm(-3)), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs.Heterologous DNA renaturation kinetic analyses were performed in 0.14 m sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B(6) DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot((1/2)) of 2.8, identical to that observed when B(6) DNA was reannealed in the absence of foreign DNA.Reannealing rates of B(6) DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids.The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random (1/5) of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded. PMID- 16659608 TI - Developmental Effects of Zeatin, Ribosyl-Zeatin, and Agrobacterium tumefaciens B(6) on Certain Mosses. AB - Eight species of mosses studied were divided into two groups on the basis of their developmental responses to ribosyl-trans-zeatin and Agro-bacterium tumefaciens B(6). All eight produced either gametophores or callus on the protonema in response to 6-(gamma,gamma-dimethylallylamino) purine and trans zeatin. Three which produced normal gametophores with A. tumefaciens yielded callus or abnormal gametophores with ribosyl-trans-zeatin. Ribosyl-trans-zeatin and A. tumefaciens were relatively ineffective on five other mosses. Characteristics of protonemal growth common to each of these two groups are described. PMID- 16659609 TI - Organelle-specific Isozymes of Aspartate-alpha-Ketoglutarate Transaminase in Spinach Leaves. AB - Four distinct isozymes of aspartate-alpha-ketoglutarate transaminase in a spinach (Spinacia oleracea L.) leaf extract were separated by starch gel electrophoresis. Of the total aspartate-alpha-ketoglutarate transaminase activity, approximately 45% was represented by the chloroplast isozyme, 26% by the cytosol isozyme, 19% by the mitochondrial isozyme, and 3 to 10% by the peroxisomal isozyme. The aspartate-alpha-ketoglutarate transamination activity in the four subcellular compartments behaved similarly. It was freely reversible and alpha-ketoglutarate was preferred to pyruvate or glyoxylate as the amino group acceptor. With glutamate as the amino group donor, oxaloacetate was superior to pyruvate or glyoxylate as the acceptor in chloroplasts, mitochondria, and cytosol, while pyruvate or glyoxylate was preferred to oxaloacetate as the acceptor in peroxisomes. PMID- 16659610 TI - Inhibition of ethylene production by cobaltous ion. AB - The effect of Co(2+) on ethylene production by mung bean (Phaseolus aureus Roxb.) and by apple tissues was studied. Co(2+), depending on concentrations applied, effectively inhibited ethylene production by both tissues. It also strongly inhibited the ethylene production induced by IAA, kinetin, IAA plus kinetin, Ca(2+), kinetin plus Ca(2+), or Cu(2+) treatments in mung bean hypocotyl segments. While Co(2+) greatly inhibited ethylene production, it had little effect on the respiration of apple tissue, indicating that Co(2+) does not exert its inhibitory effect as a general metabolic inhibitor. Ni(2+), which belongs to the same group as Co(2+) in the periodic table, also markedly curtailed both the basal and the induced ethylene production by apple and mung bean hypocotyl tissues.In a system in which kinetin and Ca(2+) were applied together, kinetin greatly enhanced Ca(2+) uptake, thus enhancing ethylene production. Co(2+), however, slightly inhibited the uptake of Ca(2+) but appreciably inhibited ethylene production, either in the presence or in the absence of kinetin. Tracer experiments using apple tissue indicated that Co(2+) strongly inhibited the in vivo conversion of l-[U-(14)C]methionine to (14)C-ethylene. These data suggest that Co(2+) inhibited ethylene production by inhibiting the conversion of methionine to ethylene, a common step which is required for ethylene formation by higher plants.Co(2+) is known to promote elongation, leaf expansion, and hook opening in excised plant parts in response to applied auxins or cytokinins. Since ethylene is known to inhibit these growth phenomena, it is suggested that Co(2+) exerts its promotive effect, at least in part, by inhibiting ethylene formation. PMID- 16659611 TI - Influence of Temperature on Nitrate Metabolism and Leaf Expansion in Soybean (Glycine max L. Merr.) Seedlings. AB - The effect of various day temperatures on NADH-nitrate reductase, NADH- and NADPH glutamate dehydrogenases, nitrate, protein and leaf area, measured at intervals during the ontogeny of the first trifoliolate soybean leaf, was determined. At 32.5 C and 25 C, nitrate concentration, nitrate reductase, and NADPH-glutamate dehydrogenase activities increased concurrently with leaf development and then decreased as leaf maturation progressed. At 40 C, these three components showed no initial increase and the concentration or activities decreased throughout the development of the leaf. The effects of temperature on NADH-glutamate dehydrogenase were the reverse. Rates of protein accumulation were higher at 40 C during the first 2 days of leaf development while higher rates were measured the first 5 days of leaf growth at 32.5 C. At 25 C, protein accumulation was low during the first 3 days of leaf growth, increased in the period of 3 to 5 days, and then declined up to 8 days of leaf development. Leaf expansion progressed at faster rates at 32.5 C and 25 C and at a much slower rate at 40 C. Leaf growth was essentially complete after the fifth day regardless of temperature.In crude leaf homogenates, apparent irreversible inactivation temperatures were 36 C for nitrate reductase and 65 C for NADPH-glutamate dehydrogenase. In vivo studies indicated a lower inactivation temperature for NADPH-glutamate dehydrogenase; however, it was still more heat-tolerant than nitrate reductase.We envisaged that reduced nitrogen supplied by NO(3) (-) assimilation is a factor in leaf expansion. PMID- 16659612 TI - Automatic monitoring of a circadian rhythm of change in light transmittance in ulva. AB - Ulva lactuca L. var. latissima (L.) DeCandolle has a circadian rhythm of visible light transmittance change which is caused by chloroplast orientation. With a continuously recording microphotometer system, clear rhythms could be monitored for up to 10 days. Measuring beam intensity effects on the free running period were seen down to 10(-7) w cm(-2). While these effects complicate the measuring process, they demonstrate that Ulva is very sensitive to light. The free running period in constant darkness at 20 C is 24 to 25 hours. The position in the cell occupied by the chloroplasts when the rhythm damps out can be influenced by light. A method is described by which the times of rhythm maxima can be calculated accurately and objectively from a relatively small number of points. PMID- 16659613 TI - Circadian rhythms of chloroplast orientation and photosynthetic capacity in ulva. AB - Ulva lactuca L. var. latissima (L.) Decandolle and var. rigida (C. Agardh) Le Jolis and U. mutabilis Foyn have a circadian rhythm of chloroplast orientation which results in large changes in the light-absorption properties of the thallus. During the day, the chloroplasts cover the outer face of the cells and absorbance is high. At night, the chloroplasts are along the side walls and absorbance is low. Enteromorpha linza (L.) J. Agardh, E. intestinalis (L.) Link, E. sp., and Monostroma grevillei (Thuret) Wittrock, members of the Ulvales, were not observed to have this rhythmic movement. Chloroplasts, when in the face position, could not be induced to move to the sides by high intensity light up to 80,000 lux. Unrelated to chloroplast position per se and light-absorption efficiency, there is a rhythm of photosynthetic capacity which peaks just before midday and which continues in constant darkness. PMID- 16659614 TI - Carbohydrate and enzymic characterization of a high sucrose sugary inbred line of sweet corn. AB - Reserve carbohydrates were determined on developing endosperm of a new line of sugary maize (Zea mays L.). Other entries, included for comparative purposes, were Midway (sugary), Funks G4646 (starchy), and Illini X-tra Sweet (shrunken-2). Sucrose in the new line, Illinois 677a, was more than twice that of Midway at most stages of development, and reached a maximum of 40% of dry weight at 18 days after pollination. Appreciable phytoglycogen accumulated in Illinois 677a, reaching 30% or more of dry weight as endosperm tissue matured. Thus, Illinois 677a is a typical sugary maize concerning phytoglycogen content, but it resembles shrunken-2 concerning the extent of sucrose accumulation.Enhanced sucrose accumulation by Illinois 677a was not accounted for by altered in vitro activities of invertase, sucrose synthase, UDP-glucose pyrophosphorylase, or ADP glucose pyrophosphorylase. Its normal level of ADP-glucose pyrophosphorylase set Illinois 677a apart from shrunken-2 in which the enzyme was drastically reduced. PMID- 16659615 TI - Regulation of potassium absorption in barley roots: an allosteric model. AB - Plasmalemma influx isotherms for K(+) were measured in the system I concentration range (0.01-0.32 mm), for barley (Hordeum vulgare L.) roots of varying internal K(+) concentration, and Km values for influx calculated. In plants grown for several days in CaSO(4) or in CaSO(4) plus KCl solutions, as well as in plants grown in CaSO(4) for several days and then rapidly loaded with KCl during a pretreatment period, Michaelis constant values were positively correlated with internal K(+) concentrations. Influx of K(+) is shown to be sigmoidally related to internal K(+) concentration and Hill plots of influx data give linear transformations with n = 4. This information is taken as support for an allosteric model for the regulation of K(+) influx in which the "carrier" is envisaged as possessing a single external binding site for K(+) as well as four internal sites for allosteric control of influx. PMID- 16659616 TI - Characteristics of External NADH Oxidation by Beetroot Mitochondria. AB - Mitochondria isolated from fresh red beetroot (Beta vulgaris L.) tissue do not oxidize external NADH with O(2) as the electron acceptor. These mitochondria have a rotenone- and antimycin-insensitive pathway of NADH oxidation associated with the outer membrane and are capable of reducing cytochrome c or potassium ferricyanide. They are also capable of oxidizing internal NADH via the inner membrane electron transport chain with normal rotenone and antimycin sensitivity and ADP/O ratios. They differ from other plant mitochondria in the apparent lack of the NADH dehydrogenase located on the outer surface of the inner membrane. It is shown that this activity develops during the aging of red beetroot slices in aerated dilute CaSO(4) solutions, and is present in the mitochondria isolated from aged tissue.Mitochondria isolated from fresh red beetroot tissue are capable of oxidizing external NADH via a malate-oxaloacetate shuttle system. It is suggested that these mitochondria possess a rapid oxaloacetate-transporting system. PMID- 16659617 TI - Enzymic nature of the protein moiety of protochlorophyllide holochrome. AB - The enzymic nature of the protein moiety of protochlorophyll(ide) holochrome was studied by following the fate of the [(14)C]protochlorophyll(ide) formed when dark-grown barley (Hordeum vulgare) or bean (Phaseolus vulgaris) leaves are incubated in the dark with 3 mm 4-delta-[(14)C]aminolevulinic acid. It was found that: [List: see text]Since turnover of protochlorophyll(ide) was not observed, these results show that there is a free exchange between the old "endogenous" and the new delta-aminolevulinic-acid-induced protochlorophyll(ide) molecules on the active site of the holochrome protein. These results are consistent with the hypothesis that the holochrome protein acts as an enzyme. PMID- 16659618 TI - Effects of Cyanide and Ethylene on the Respiration of Cyanide-sensitive and Cyanide-resistant Plant Tissues. AB - The effects of cyanide and ethylene, respectively, were studied on the respiration of a fully cyanide-sensitive tissue-the fresh pea, a slightly cyanide sensitive tissue-the germinating pea seedling, and a cyanide-insensitive tissue the cherimoya fruit. Cyanide inhibition of both fresh pea and pea seedling respiration was attended by a conventional Pasteur effect where fermentation was enhanced with an accumulation of lactate and ethanol and a change in the level of glycolytic intermediates indicative of the activation of phosphofructokinase and pyruvate kinase accompanied by a sharp decline in ATP level. In these tissues, ethylene had little or no effect on the respiration rate, or on the level of glycolytic intermediates or ATP. By contrast, ethylene as well as cyanide enhanced both respiration and aerobic glycolysis in cherimoya fruits with no buildup of lactate and ethanol and with an increase in the level of ATP. The data support the proposition that for ethylene to stimulate respiration the capacity for cyanide-resistant respiration must be present. PMID- 16659619 TI - Induction of Furano-terpene Production and Formation of the Enzyme System from Mevalonate to Isopentenyl Pyrophosphate in Sweet Potato Root Tissue Injured by Ceratocystis fimbriata and by Toxic Chemicals. AB - When sweet potato (Ipomoea batatas) root tissue was infected by Ceratocystis fimbriata, activity of the enzyme system from mevalonate to isopentenyl pyrophosphate, especially of pyrophosphomevalonate decarboxylase (EC 4.1.1.33), was increased in the noninfected tissue adjacent to the infected region, preceding the furano-terpene production in the infected region. Cutting and incubation of sweet potato slices did not produce furano-terpenes, and only slightly increased the activity of the enzyme system from mevalonate to isopentenyl pyrophosphate. The enzymic activity in diseased tissue was localized in the soluble fraction, and was higher in the tissue from the surface to a depth of about 5 mm with gradual decrease toward the inner part.Mercuric chloride (0.1%, w/v) and sodium dodecyl sulfate (1.0%, w/v) were utilized as model inducers of furano-terpenes and pyrophosphomevalonate decarboxylase. The mercuric chloride- or sodium dodecyl sulfate-induced response was inhibited by administration of cycloheximide to the discs together with the inducer immediately after disc preparation. When cycloheximide or blasticidin S was applied together with the inducer, to the discs 9 hours or more after disc preparation, the induction was not inhibited but rather stimulated. PMID- 16659620 TI - Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva. AB - Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae. PMID- 16659621 TI - Esters of indole-3-acetic Acid from Avena seeds. AB - The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK(a) values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.Dehusked Avena kernels contained 8 mg/kg total IAA of which 5.5% was free and 94.5% esterified. IAA bound through a peptidic linkage was present, but in only trace amounts. PMID- 16659622 TI - Endogenous Gibberellins of Pine Pollen: III. Conversion of 1,2-[H]GA(4) to Gibberellins A(1) and A(34) in Germinating Pollen of Pinus attenuata Lemm. AB - Gibberellins A(1) and A(34) (possibly A(2)) were found as products of metabolism of 1,2-[(3)H]GA(4) during germination of Pinus attenuata pollen. The conversion from GA(4) to GA(1) and GA(34) occurred as hydroxylations at atoms C-13 and C-2 of the ent-gibberellane skeleton, respectively. Percentage interconversion of the GA(4) absorbed was in the range of 0.15 to 0.43% for GA(1) and 1.54 to 3.22% for GA(34). Identifications were made on a gas-liquid chromatograph with radioactive monitoring by comparison with standards. PMID- 16659623 TI - Variation in germination and amino Acid leakage of seeds with temperature related to membrane phase change. AB - Leakages of amino acids and/or fluorescent material as functions of temperature between 15 and 40 C are reported for imbibed seeds of Avena fatua L., Lactuca sativa L., Barbarea vulgaris R. Br., Amaranthus albus L., Abutilon theophrasti Medic., Lychnis alba Mill., Daucus carota L., Setaria faberi Herrm., Setaria viridis (L.) Beauv., and Datura stramonium L. The leakage indicates prominent increase in permeability of the plasmalemma in the 30 to 35 C range for 8 of the 10 kinds of seeds studied. Germination of the seeds at constant temperatures or with daily shifts in temperature is related to the membrane transition temperature for permeation by amino acids. Seeds of A. albus and A. theophrasti, which did not show membrane changes in the 25 to 40 C range, germinated best at 35 to 40 C; the other seeds germinated best below 30 C. Seeds of B. vulgaris showed rapid permeation of limiting membranes upon initial wetting with water, which was indicative of membrane disorder when dry. Leakage under anaerobiosis was observed for S. faberi seeds. PMID- 16659624 TI - In vitro iodination of plant ribonucleic acids. AB - The optimum conditions for in vitro iodination of RNAs have been established which yield specific radioactivities ranging from 10 x 10(4) to 10 x 10(6) cpm/mug. A nomogram has been constructed by correlating specific radioactivities of RNA with concentration of KI, RNA, and (125)I. This nomogram can be used to determine the conditions for the desired specific radioactivities for any unknown RNA. The in vitro iodinated RNA has been compared with in vivo labeled RNA for hybridization characteristics. Competition hybridization between (125)I-labeled chloroplast-rRNA and unlabeled pea (Pisum sativum) chloroplast-rRNA was identical to that found using [(32)P]chloroplast-rRNA. Thermal stability of DNA-(125)I-rRNA hybrids was similar to the thermal stability of DNA-[(32)P]rRNA hybrids. The iodinated RNA was not found to have undergone any changes in its hydrogen-bonding properties. PMID- 16659625 TI - Metabolism of Indole-3-Acetic Acid: II. Oxindole Pathway in Parthenocissus tricuspidata Crown-Gall Tissue Cultures. AB - An indoleacetic acid oxidase preparation from an acetone powder of Parthenocissus tricuspidata crown-gall tissue has been examined. An intermediate in the reaction is 3-hydroxymethyloxindole and nonenzymic conversion of it to 3-methyleneoxindole was observed. Neither reaction mixtures nor 3-methyleneoxindole have any auxin like activity in Avena or wheat coleoptile bioassays. In vivo studies show that although 53% decarboxylation of indoleacetic acid was observed in 48 hours, only a small amount of 3-methyloxindole could be recovered from the medium. The other decarboxylated products remain to be identified but are not 3 hydroxymethyloxindole or 3-methyleneoxindole. PMID- 16659626 TI - Mechanism of Plant Growth Stimulation by Naphthenic Acid: II. Enzymes of CO(2) Fixation, CO(2) Compensation Point, Bean Embryo Respiration. AB - Potassium naphthenate, 20 mm, was applied to the foliage of 14-day-old plants of bush bean, Phaseolus vulgaris L, cv Top Crop, maize, Zea mays L, cv Golden Bantam, spring wheat, Triticum vulgare Vill., cv Neepawa, and a 2 mm solution to 21-day-old plants of sugar beet, Beta vulgaris L, cv CS-43. Seven days after application, the activities of ribulose diphosphate carboxylase and phosphoenolpyruvic carboxylase in leaves of naphthenate-treated bean and maize were greater than in the leaves of untreated plants. The increase in activity of the carboxylases in treated spring wheat lacked statistical significance. At the same time after treatment, the CO(2) compensation point of bean was smaller than that of control plants, as was the average CO(2) compensation point of sugar beet measured at intervals up to 21 days after spraying. Respiratory rates of embryos of bean seeds soaked for 12, 24, and 48 hours in 43.5 mum K naphthenate were greater than those of seeds soaked in water. Ascorbate oxidase activity in bean leaves, determined 7, 14, and 21 days after K naphthenate application, was also stimulated. Foliar application of 10 mm cyclohexanecarboxylic acid to bean was followed in 7 and 14 days by a greater activity of catalase than in control plants. Higher activity of the enzyme, measured 6, 7, 12, and 14 days after spraying, also resulted from K naphthenate application. The results indicate that the higher rates of photosynthesis in naphthenate-treated plants may be due in part to increased rates of CO(2) fixation, and that greater photosynthetic efficiency, together with a more plentiful supply of ATP arising from increased electron flow in respiration, is involved in the greater growth of plants to which naphthenate has been applied. PMID- 16659627 TI - Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts. AB - Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.The envelope membranes were separated into two fractions in the gradients according to their banding densities. Electron micrographs showed that the light envelope fraction consisted mostly of single-membrane vesicles, whereas the heavy envelope fraction consisted of multiple layers of folded membranes. Both envelope fractions were ultrastructurally distinct from the thylakoid membranes. PMID- 16659628 TI - Physiological Characteristics of Photosynthesis and Respiration in Stems of Populus tremuloides Michx. AB - The physiological responses of 6- to 8-year-old aspen (Populus tremuloides Michx.) stems to temperature, light, and CO(2) concentration were investigated in the field throughout the year using infrared CO(2) analysis. Light response studies showed that the rate of gross photosynthesis was linear from 0 to 400 ft c (0 to 1.6 mw/cm(2) of 400-700 nm) with light saturation being reached between 800 to 1400 ft-c (3.2 to 5.6 mw/cm(2) of 400-700 nm). At this light intensity, the respiratory CO(2) loss was reduced to 10 to 15% of dark rates. Net photosynthetic CO(2) uptake was not observed even at intensities as high as 3400 ft-c (13.6 mw/cm(2) of 400-700 nm). The light response curve was similar for both winter and summer stems.During summer months, the respiratory and photosynthetic rates of the aspen stem increased with temperature at a near constant rate between 5 and 35 C. For winter stems, the gross photosynthetic rate increased in a pattern similar to the dark respiratory rate as the temperature rose from 3 to 17 C. Below 0 C and above 17 C, however, the gross photosynthetic rate fell off in relation to the respiratory rate so that the per cent of CO(2) reassimilated decreased from 75% to less than 50%. Measurable bark photosynthetic activity was not observed below -3 C.The gross photosynthetic rate of stems was not affected when the gas passing through the cuvette contained concentrations of CO(2) ranging from 0 to 580 mul CO(2)/l air. PMID- 16659629 TI - In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA. AB - Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.Attempts to correlate protease activity with the increasingly labile nitrate reductase activity in expanding cotyledons were unsuccessful. Instead, when extracts containing stable nitrate reductase were mixed with extracts containing labile nitrate reductase, the latter was stabilized. The nature of the "protector" in the stable extracts is not known. It is heat-stable, but apparently does not function in vivo since nitrate reductase in germinating cotton seedlings rapidly declines following a peak of activity at 24 hours. We suggest that the protector may function by preventing nitrate reductase from dissociating into inactive subunits. PMID- 16659630 TI - Azolla-Anabaena azollae Relationship: IV. Photosynthetically Driven, Nitrogenase catalyzed H(2) Production. AB - The water fern, Azolla caroliniana Willd., containing the symbiotic, heterocystous blue-green alga, Anabaena azollae, has been studied under various growth conditions to characterize its light-dependent production of H(2). The response of H(2) production to N(2) and C(2)H(2) and the absence of a differential effect of m-chlorocarbonyl cyanide phenylhydrazone on H(2) production and C(2)H(2) reduction, coupled with the parallel inhibition of both processes by DCMU imply that the production of H(2) is nitrogenase-catalyzed and ATP-dependent.H(2) was produced by fronds grown under air-CO(2) in the presence or absence of combined nitrogen. When cultured under argon-O(2)-CO(2), only those fronds provided with combined nitrogen remained viable and produced H(2). Fronds grown on nitrate under air plus 2% CO also produced H(2). In comparison to fronds grown on N(2) alone, fronds grown on nitrate had an increased rate of H(2) production relative to C(2)H(2) reduction, and the inhibition of H(2) production by air was less.CO in argon +/- CO(2) resulted in a partial inhibition of H(2) production, whereas CO in argon-CO(2)-C(2)H(2) enhanced H(2) production in fronds grown without combined nitrogen. Our studies strongly indicate that H(2) production is nitrogenase-catalyzed but the possibility that the symbiont contains a hydrogenase cannot be totally excluded. PMID- 16659631 TI - Limitation on the use of the horizontal clinostat as a gravity compensator. AB - If the horizontal clinostat effectively compensates for the influence of the gravity vector on the rotating plant, it should make the plant unresponsive to whatever chronic acceleration may be applied transverse to the axis of clinostat rotation. This was tested by centrifuging plants while they were growing on clinostats. For a number of morphological end-points of development the results depended on the magnitude of the applied g-force. Therefore, gravity compensation by the clinostat was incomplete. This conclusion is in agreement with results of satellite experiments which are reviewed. PMID- 16659632 TI - Analysis of native gibberellins in the internode, nodes, leaves, and inflorescence of developing Avena plants. AB - The native gibberellins (GAs) of various organs of the Avena plant were analyzed by bioassay and gas chromatography-mass spectrometry (GC-MS) after silicic acid partition column chromatography. The major GA of the inflorescence was identified as GA(3) by GC-MS, and this GA also forms the major component of the nodes, p-1 internode, and roots as determined by GLC or chromatography/bioassay. The inflorescence and nodes are the major sources of native GAs, the last two leaves, internode, and roots having significantly lower amounts of GA-like substances. In the internode, less polar GAs predominated at the lag stage of development, whereas by the log and plateau stages, the more polar GAs increased significantly.Since less polar GAs are early in oxidative interconversion sequences, this finding indicates sequential conversion to more polar and probably more active GAs, during log phase growth of the p-1 internode. PMID- 16659633 TI - Light modulation of glucose-6-phosphate dehydrogenase: partial characterization of the light inactivation system and its effects on the properties of the chloroplastic and cytoplasmic forms of the enzyme. AB - Light inactivation of glucose-6-phosphate dehydrogenase within the pea (Pisum sativum L.) leaf chloroplast has a narrow pH optimum between 7.2 and 7.4 and is NADP-sensitive. The pH optimum for dark activation is slightly lower. Inactivation apparently results in a simple decrease in maximal velocity of the chloroplastic and cytoplasmic forms of the enzyme with no concomitant change in pH optimum or K(m) (glucose 6-phosphate). PMID- 16659634 TI - Ribosyl-cis-zeatin in a Leucyl Transfer RNA Species from Peas. AB - tRNA(6) (Leu) in Pisum sativum seed has been purified. This tRNA species contains a cytokinin-active nucleoside and accounts for approximately 7% of the total cytokinin activity in acid hydrolysates of pea tRNA. The cytokinin has been identified as ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino) -9 beta-d-ribofuranosylpurine. PMID- 16659636 TI - Control of Changes in Mitochondrial Activities during Aging of Potato Slices. AB - Aging of slices of potato tuber (Solanum tuberosum L.) in an aerated liquid medium induces a number of changes in mitochondrial activities. A nonphosphorylative, cyanide-insensitive electron transport pathway (alternate pathway) is brought into operation. The rate of oxidation of exogenous NADH increases markedly and the efficiency of phosphorylation with this substrate remains the same as it is in mitochondria isolated from fresh tissue slices. On the contrary, the rates of oxidation of succinate and malate do not increase while lower phosphorylative efficiencies indicate that a fraction of their electrons reaches oxygen through the alternate pathway. Chloramphenicol, a specific inhibitor of the mitochondrial protein-synthesizing system, has no effect whatsoever on these events. However, cycloheximide, which acts on the corresponding cytoplasmic system, prevents both the development of the alternate pathway and the rise in the rate of oxidation of exogenous NADH. These effects are interpreted as showing a specific control of the cytoplasmic protein synthesizing system on the changes in mitochondrial oxidations during aging. PMID- 16659635 TI - Variables Affecting the CO(2) Compensation Point. AB - Some factors influencing dark respiration, photorespiration, and photosynthesis were examined for their effect on the CO(2) compensation point (70 mul/l) of detached soybean (Glycine max) leaf discs. A higher compensation point in young leaves decreased to the constant value after leaf expansion and maturation, but increased again during senescence. The compensation point was 40 to 50% higher in plants grown in the summer than in the winter. The compensation point and dark respiration increased with temperatures above 17 C. Below 17 C dark respiration continued to decrease, but the compensation point did not decrease further. Increasing light intensities did not affect the compensation point.The effect of selected chemicals on the compensation point were surveyed. Some buffer components did not greatly alter the compensation point but organic solvents lowered it. Potassium phosphate and pyrophosphate greatly increased it. Inhibitors of photosynthesis increased the compensation point. Hydroxypyridinemethanesulfonate and sodium bisulfite severely inhibited photosynthesis in soybean leaves, stimulated dark respiration, and increased the compensation point. PMID- 16659637 TI - Ammonium Influence on the Growth and Nitrate Reductase Activity of Paul's Scarlet Rose Suspension Cultures. AB - Suspension cultures of Paul's Scarlet rose were grown in two defined media which differed only in their inorganic nitrogen content. Both possessed equal amounts of NO(3) (+) (24 mm), but differed in that NH(4) (+) (0.91 mm) was present in control medium; whereas, no NH(4) (+) was present in the test medium. A comparison of fresh weight increases over a 14-day growth period showed that NH(4) (+) caused a 2-fold stimulation in growth and governed the pattern of development.Ammonium also caused a 2-fold increase in nitrate reductase activity but had little influence on the activity of representative enzymes from the Embden-Meyerhof pathway or citric acid cycle. Thus NH(4) (+) enhanced the nitrate reductase activity which was correlated with increased growth.Ammonium had no influence on the in vitro activity of nitrate reductase which suggested that the stimulatory influence was due to an increased synthesis of the enzyme. The enhanced synthesis did not appear to be due to an increased availability of NO(3) (+) since the uptake of NO(3) (+) by intact cells was not influenced by the presence of NH(4) (+) during the period of most rapid increase in nitrate reductase activity. PMID- 16659638 TI - Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides. AB - Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. O(2) evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of O(2) evolution was 16 to 25 mum. The degree to which ATP stimulated O(2) evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of O(2) evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of O(2) evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 mumol ATP hydrolyzed mg chlorophyll(-1) hour(-1). The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of O(2) evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO(2) fixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect. PMID- 16659639 TI - Ethylene formation in sugar beet leaves: evidence for the involvement of 3 hydroxytyramine and phenoloxidase after wounding. AB - Ethylene production by sugar beet (Beta vulgaris L.) leaf discs is inhibited by white (or red, >610 nm) light or by wounding. In contrast, in wounded leaf discs, ethylene production is stimulated by light. The effect of light on wounded leaf discs has been studied by using an in vitro system which mimics the loss of compartmentation in the wounded leaf. Chlorophyll-free extracts from sugar beet leaves stimulate the production of the superoxide free radical ion (as a prerequisite for ethylene formation) by illuminated chloroplast lamellae. The substance from the crude leaf extracts which is active in stimulating the production of the superoxide free radical ion has been identified as 3 hydroxytyramine (dopamine). Exogenous dopamine between 5 mum and 100 mum stimulates ethylene formation by illuminated chloroplast lamellae from methional. It also stimulates the production of the superoxide free radical ion, the formation of which apparently involves both a lamellar phenoloxidase and photosynthetic electron transport as a 1-electron donor, and is cyanide sensitive. PMID- 16659640 TI - Abscisic Acid Content, Transpiration, and Stomatal Conductance As Related to Leaf Age in Plants of Xanthium strumarium L. AB - Among the four uppermost leaves of greenhouse-grown plants of Xanthium strumarium L. the content of abscisic acid per unit fresh or dry weight was highest in the youngest leaf and decreased gradually with increasing age of the leaves. Expressed per leaf, the second youngest leaf was richest in ABA; the amount of ABA per leaf declined only slightly as the leaves expanded. Transpiration and stomatal conductance were negatively correlated with the ABA concentration in the leaves; the youngest leaf lost the least amount of water. This correlation was always very good if the youngest leaf was compared with the older leaves but not always good among the older leaves. Since stomatal sensitivity to exogenous (+/-) ABA was the same in leaves of all four age groups ABA may be in at least two compartments in the leaf, one of which is isolated from the guard cells.The ability to synthesize ABA in response to wilting or chilling was strongly expressed in young leaves and declined with leaf age. There was no difference between leaves in their content of the metabolites of ABA, phaseic, and dihydrophaseic acid, expressed per unit weight. PMID- 16659641 TI - Abscisic Acid content of senescing petals on cut rose flowers as affected by sucrose and water stress. AB - Leafless cut Superstar roses (Rosa hyb.) were kept in a 1% sucrose solution. During the first few days of treatment, the abscisic acid content and the water deficit in the petals was higher in treated flowers than in controls kept in water. Later and up to the termination of the flower's life, ABA content and water deficit values were lower in petals of sucrose-treated flowers than in controls. Water stress treatments resulted in higher water deficit values and higher ABA content of petals. An 8-day sucrose treatment following temporary water stress improved the quality of flowers and reduced the level of ABA in the petals. We conclude that the effect which sucrose has on the ABA content of rose petals is at least partly due to its effect on changes in water deficit in the petals. This happens in spite of the fact that rose petals have no stomata, and therefore, ABA is not involved in regulating water balance via the stomata. PMID- 16659642 TI - Enzyme levels in relation to obligate phototrophy in chlamydobotrys. AB - During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.Enzyme assays demonstrated an incomplete tricarboxylic acid cycle in cells growing photoheterotrophically on acetate or phototrophically on carbon dioxide. Although these cells lack alpha-ketoglutarate dehydrogenase and succinate thiokinase, a cyclic flow of acetate carbon is possible in the presence of the glyoxylate cycle enzymes but the yield of adenosine triphosphate from acetate oxidation may be insufficient to support heterotrophic growth, so rendering Chlamydobotrys an obligate phototroph. PMID- 16659643 TI - Effects of inorganic salts on tissue permeability. AB - Inorganic solutes are shown to alter the permeability of root and leaf tissues. Experiments with beet root tissues reveal that CaCl(2) decreases leakage of betacyanin from the tissue, that (NH(4))(2)SO(4) increases leakage, and that each salt can relieve the effects of the other. A comparison of cations and anions shows a range of effects with the various solutes. Experiments with Rumex obtusifolius L. leaf discs reveal that whereas CaCl(2) defers the development of senescence, (NH(4))(2)SO(4) hastens senescence and increases the leakage of materials out of the leaf discs. The solute effect on Rumex obtusifolius L. is prevented by gibberellin. CaCl(2) can relieve the (NH(4))(2)SO(4) effect. The results are interpreted as indicating that the inorganic solutes may serve to alter the permeability of membranes through alterations of interactions between water and macromolecules in the tissues; the interpretation is consistent with the evidence for opposite effects of Ca and NH(4), the effective concentrations being about 10(-3)m, and the reversibility of the effects of one solute by another of opposite stabilization-destabilization effect. PMID- 16659644 TI - Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L. AB - Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO(2) fixation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH(14)CO(3), and the kinetics of (14)C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO(2) fixation is similar in the different mesophyll tissues from which these cells were derived. PMID- 16659645 TI - Potassium-linked Chloride Fluxes during Rhythmic Leaf Movement of Albizzia julibrissin. AB - Transverse sections of Albizzia pulvinules were examined with an electron microprobe to determine ion fluxes associated with turgorcontrolled leaflet movements. K(+) and Cl(-) concentrations are high in the flexor and low in the extensor region of closed pulvini. Both ions migrate out of the flexor and into the extensor during opening as previously described for K(+). The distribution of these elements is significantly correlated in each phase of the rhythmic cycle examined, but only 50 to 60% of the ionic charge of potassium is balanced by chloride. This value increases to 65 to 85% if one considers only the mobile fraction of the potassium.The increase in concentration of both ions in the extensor region precedes the decrease in the flexor, thus indicating that there must be a storage reservoir for K(+) and Cl(-). The inner cortex is suggested as such a reservoir, and plasmodesmata are discussed as a probable pathway for ion movement. PMID- 16659646 TI - Uptake and Metabolism of d-Glucose by Neocosmospora vasinfecta E. F. Smith. AB - Freshly harvested, nongrowing mycelium of Neocosmospora vasinfecta E. F. Smith rapidly absorbed exogenous glucose but converted a greater proportion to trehalose and glucan than to respiratory CO(2). This effect was accentuated in mycelium preincubated for 3.5 hours in water before exposure to glucose. Glucose was absorbed via two uptake systems, both apparently constitutive, with apparent Km values for glucose of 0.02 mm (high affinity) and 2 mm (low affinity). The glucose derivative 3-O-methylglucose (3-O-MG) was also absorbed by two apparently constitutive systems with apparent Km values for 3-O-MG of 0.065 mm and 1.9 mm. Absorption of 3-O-MG by both freshly harvested and preincubated mycelium led to its accumulation. Freshly harvested mycelium lost accumulated 3-O-MG rapidly to water, whereas preincubated mycelium showed reduced or no leakage. The reduction in leakage due to preincubation was prevented by 5 mug/ml cycloheximide in the preincubation medium. Glucose competitively inhibited 3-O-MG uptake via the high affinity system and induced loss of previously accumulated 3-O-MG from preincubated mycelium. The uptake of both glucose and 3-O-MG was associated with a transient alkalinization of the uptake medium. It is concluded that uptake of both glucose and 3-O-MG by at least the high affinity system is energy-linked and probably mediated by proton cotransport. PMID- 16659647 TI - Intracellular localization of glycolate dehydrogenase in a blue-green alga. AB - Glycolate dehydrogenase activity was detected in cell-free extracts of Oscillatoria sp. prepared by osmotic lysis of spheroplasts in 0.05 m potassium phosphate buffer, pH 7.5, containing 0.3 m mannitol. Most of the enzyme activity was found in a particulate fraction and localized in the photosynthetic lamellae after centrifugation in a discontinuous sucrose density gradient. Enzyme activity was detected in this fraction both in the presence and absence of the artificial electron acceptor 2,6-dichlorophenolindophenol (DPIP) and a low rate of O(2) uptake was detected in this lamellar fraction. Activity was lost from the lamellar fraction by repeated washing or by treatment with 0.005% Triton X-100 and the solubilized enzyme activity was DPIP-dependent. The data indicate that both glycolate dehydrogenase and its natural electron acceptor are bound to the photosynthetic lamellae in vivo. In contrast, catalase activity was found in the soluble cytoplasmic fraction. PMID- 16659648 TI - Rapid Auxin-induced Decrease in Free Space pH and Its Relationship to Auxin induced Growth in Maize and Pea. AB - A pH microelectrode has been used to investigate the auxin effect on free space pH and its correlation with auxin-stimulated elongation in segments of pea (Pisum sativum) stem and maize (Zea mays var. Bear Hybrid) coleoptile tissue. Auxin induces a decrease in free space pH in both tissues. In maize coleoptiles, free space pH begins to fall within about 12 minutes of exposure to auxin and decreases by about 1 pH unit by approximately 30 minutes. In pea, pH begins to decrease within an average of 15 to 18 minutes of exposure to auxin and falls by about 0.9 pH unit by approximately 40 minutes. Auxin-stimulated elongation, measured in the same two tissues similarly prepared, appears in maize at the earliest 18 minutes after auxin application, while in pea it appears at the earliest 21 to 24 minutes after auxin application. The auxin analogs p chlorophenoxyisobutyric acid and phenylacetic acid do not stimulate elongation above control levels in maize or pea tissue segments and do not cause a decrease in free space pH in either tissue. These findings are consistent with the acid secretion theory of auxin action. PMID- 16659649 TI - Kinetics of Hormone-induced H Excretion. AB - A study has been made of the kinetics of hormone-induced H(+) excretion from peeled Avena coleoptile sections using a new, simple technique involving direct application of the pH electrode to the surface of the section. Hormone-induced H(+) excretion begins after lags and occurs at rates which are consistent with a role of H(+) in regulating cell elongation. With fusicoccin, H(+) excretion begins within the 1st minute, and an external pH of 5 (optimal for wall loosening) is reached in 5 to 8 minutes, while with auxin the lag averages 14 minutes and pH 5 is reached in 20 to 30 minutes. KCN, which inhibits cell elongation in 3 to 5 minutes, stops H(+) excretion in less than 1 minutes, whereupon the external pH rises sharply. Cycloheximide stops auxin-induced H(+) excretion in 3 to 8 minutes, and the pH then rises slowly. In the absence of hormones, the pH of the extracellular solution comes to equilibrium at 5.6, but the actual pH of the wall solution is probably about 0.3 unit below this due to Donnan effects. PMID- 16659650 TI - Photocontrol of Anthocyanin Synthesis: V. Further Evidence against the Involvement of Photosynthesis in High Irradiance Reaction Anthocyanin Synthesis of Young Seedlings. AB - Streptomycin and chloramphenicol inhibit the development of the photosynthetic apparatus and enhance anthocyanin synthesis in tomato (Lycopersican esculentum, cv. Beefsteak) and red cabbage (Brassica oleracea, cv. Red Acre) seedlings. The two antibiotics do not affect the basic features of light-dependent anthocyanin formation: the relative effectiveness of different irradiance levels and of different spectral regions and the red-far red reversibility of the response are essentially the same in seedlings grown in water or in solution of the two antibiotics. The action of the two antibiotics on anthocyanin synthesis is probably independent of the action of light. The results provide further evidence that the role played by photosynthesis in high irradiance reaction anthocyanin synthesis of young seedlings is only a minor one, if at all. PMID- 16659651 TI - Photosynthetic Rates of Sun versus Shade Leaves of Hyptis emoryi Torr. AB - Leaves on a bush of Hyptis emoryi Torr. varied in length from less than 1 cm when development occurred in full sunlight (e.g. 40 Mjoules m(-2)) to over 7 cm when the total daily solar irradiance was less than 3 Mjoules m(-2). The 1-cm sun leaves were 3-fold higher than the 7-cm shade leaves in chlorophyll per unit area, mesophyll thickness, and the internal to external leaf area ratio (A(mes)/A). The higher A(mes)/A caused a 1.2-cm leaf to have a 3-fold lower CO(2) liquid phase resistance than did a 7.1-cm leaf. Large thin shade leaves captured photosynthetically active radiation effectively (less than 7% passed through), but were not adapted to full sunlight. Specifically, when a 6.9-cm leaf was placed at 910 w m(-2) for 30 min, its temperature exceeded that of the air by nearly 8 C. For the common daytime air temperatures above 30 C for this desert shrub, large shade leaves would have temperatures far in excess of that optimum for photosynthesis for H. emoryi, 29 to 32 C. PMID- 16659652 TI - Cell wall assembly in fucus zygotes: I. Characterization of the polysaccharide components. AB - Fertilization triggers the assembly of a cell wall around the egg cell of three brown algae, Fucus vesiculosus, F. distichus, and F. inflatus. New polysaccharide polymers are continually being added to the cell wall during the first 24 hours of synchronous embryo development. This wall assembly involves the extracellular deposition of fibrillar material by cytoplasmic vesicles fusing with the plasma membrane. One hour after fertilization a fragmented wall can be isolated free of cytoplasm and contains equal amounts of cellulose and alginic acid with no fucose containing polymers (fucans) present. Birefringence of the wall caused by oriented cellulose microfibrils is not detected in all zygotes until 4 hours, at which time intact cell walls can be isolated that retain the shape of the zygote. These walls have a relatively low ratio of fucose to xylose and little sulfate when compared to walls from older embryos. When extracts of walls from 4-hour zygotes are subjected to cellulose acetate electrophoresis at pH 7, a single fucan (F(1)) can be detected. By 12 hours, purified cell walls are composed of fucans containing a relatively high ratio of fucose to xylose and high levels of sulfate, and contain a second fucan (F(2)) which is electrophoretically distinct from F(1). F(2) appears to be deposited in only a localized region of the wall, that which elongates to form the rhizoid cell. Throughout wall assembly, the polyuronide block co-polymer alginic acid did not significantly vary its mannuronic (M) to guluronic (G) acid ratio (0.33-0.55) or its block distribution (MG, 54%; GG, 30%; MM, 16%). From 6 to 24 hours of embryo development, the proportion of the major polysaccharide components found in purified walls is stable. Alginic acid is the major polymer and comprises about 60% of the total wall, while cellulose and the fucans each make-up about 20% of the remainder. During the extracellular assembly of this wall, the intracellular levels of the storage glucan laminaran decreases. A membrane-bound beta-1, 3-exoglucanase is found in young zygotes which degrades laminaran to glucose. It is postulated that hydrolysis of laminaran by this glucanase accounts, at least in part, for glucose availability for wall biosynthesis and the increase in respiration triggered by fertilization. The properties and function of alginic acid, the fucans, and cellulose are discussed in relation to changes in wall structure and function during development. PMID- 16659653 TI - Effects of inhibitors of RNA and protein synthesis on aspartate transcarbamylase activity in etiolated plant tissue. AB - Aspartate transcarbamylase (ATCase) activity declines in etiolated cowpea (Vigna unguiculata L. Walp.) and soybean (Glycine max L. Merr.) hypocotyls between 3 and 11 days after planting. Treating cow-pea hypocotyls with cycloheximide (CH), actinomycin D (AMD), 6-methyl purine (6-MP), or cordycepin increases ATCase activity up to 740, 350, 465, and 305%, respectively, over water-treated controls 48 to 72 hours after treatment. In contrast erythromycin had no effect, and d threo-chloramphenicol (CHL) reduced ATCase activity nearly 40%. CH, AMD, and CHL, whose effects were further characterized, each markedly reduced total RNA synthesis and protein synthesis. Respiration was stimulated by CH and AMD and reduced by CHL. In soybean, CHL-treated tissues and water-treated controls had comparable ATCase activities 48 hours after treatment, while AMD, 6-MP, and CH treatments reduced activities 29, 37, and 78%, respectively. The results suggest that the level of ATCase activity in etiolated cowpea hypocotyls is regulated by a mechanism or mechanisms that are interfered with by inhibition of RNA and protein synthesis. Possibly the mechanism is absent from etiolated soybean hypocotyls. PMID- 16659654 TI - Metabolism As a Function of Water Potential in Air-Dry Seeds of Charlock (Sinapis arvensis L.). AB - A new method is described for studying the metabolism of air-dry seeds. An initial pulse of (14)CO(2) was supplied to seeds maintained in air at controlled low water potentials for 6 months. Seeds were also infiltrated with 2-(14)C acetate and with (14)C-l-leucine at 0 C, redried rapidly at 0 C, and maintained at controlled low water potentials for 4 to 6 weeks. The metabolism of the air dry seeds was a function of the water content of the tissues, which was in equilibrium with the water potential at the seed surface. The fixation of (14)CO(2) and the utilization of 2-(14)C-acetate increased exponentially with water content. The incorporation of (14)C-l-leucine into protein increased linearly with water content. Metabolism was not reduced to a low rate except in air-dry seeds at the lowest water potentials (-1716 to -762 bars) with 4 to 6% water. PMID- 16659655 TI - Effect of cyanide on the plasmalemma potential of mnium. AB - By centrifuging Mnium cuspidatum leaf cells, the cytoplasm can be distinguished from the vacuole and a microelectrode tip can be located unambiguously in the cytoplasm. The site of the electrogenic pump is clearly demonstrated to reside in the plasmalemma as shown by depolarization of the cell electropotential induced by CN(-). PMID- 16659656 TI - Osmotic factors of dehardening in cornus Florida L. AB - The killing temperature for cortical cells from the flowering dogwood changes abruptly from -25 C to -15 C during dehardening. Cell sap concentration, minimum critical cell volume, and osmotically inactive cell volume show a progressive change during dehardening, but only cell sap concentration is correlated directly with the killing temperature, showing the same step change. There is a limit to the extent to which hardy dogwood cells can be osmotically reduced in volume. Beyond this limiting volume, the extracellular osmotically can be increased without further volume reduction. Ultimately the cell succumbs, presumably to an osmotic pressure gradient. Nonhardy cells do not exhibit this resistance to shrinkage. The ability to resist volume reduction is probably a crucial factor in the freezing resistance of dogwood cortical cells. PMID- 16659657 TI - Utilization of Selenocysteine by a Cysteinyl-tRNA Synthetase from Phaseolus aureus. AB - An l-cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus has been purified approximately 200-fold. The enzyme uses selenocysteine as substrate in the ATP-PPi exchange assay; other cysteine analogs were inactive. The molecular weight as determined by Sephadex G-200 column chromatography is about 61,000; sodium dodecyl sulfate and 8 m urea acrylamide gel electrophoresis indicate that the enzyme is a dimer consisting of two identical monomers of molecular weight 30,000. A method for the preparation of selenocysteine from selenocystine is described. PMID- 16659658 TI - Simple photometric auxanometers of high sensitivity. AB - The elongation phase of growth of plant parts, as 1-cm segments excised from wheat coleoptiles here, is very simply recorded photometrically. One or more aligned segments submerged in aerated buffer pushed an Al foil shutter over a slit of light incident on a photodetector such as a solar or photronic cell, connected directly to a recorder, or a photomultiplier tube in a commercial photometer. Convenient kinetic records were obtained at 5 mm per min chart speed when one segment was combined with a 1-mm long slit, or more segments and longer slits, for which a 1-mm chart unit usually exceeded noise and was equivalent to 4 mum growth per cm coleoptile. In the presence of 10 cm of coleoptile segments no replenishment of solution was necessary during kinetic measurements of response in a 50-ml reservoir of IAA more concentrated than 30 nm. PMID- 16659659 TI - Development of Photosystem II Complex during Greening of Chlamydomonas reinhardi y-1. AB - The relative content of organized pigment, active centers, and acceptor pools of photosystem II and their interconnection during the development of the photosynthetic membranes of Chlamydomonas reinhardi y-1 have been measured using the fluorescence induction technique. The degree of connectivity and efficiency of the developing system has been assessed also from measurements of maximal rates, quantum yield, and flash yield of 2,6-dichlorophenolindophenol photoreduction using H(2)O as the electron donor. The results obtained indicate that the process of membrane development in this organism consists of two phases: an initial phase of reorganization and connection between pre-existing components, and a second phase of actual accumulation of newly formed, complete, and active units. The ratio of active centers to Chl remains practically constant throughout the process while the degree of connectivity between the active center and the plastoquinone pool was doubled during the early phase of the greening. In addition the degree of connectivity between the plastoquinone pool and the rest of the electron transport chain increases as demonstrated by a 10- to 20-fold rise in the quantum yield and a 10-fold rise in the maximal rate and the flash yield. The ratio of light harvesting Chl to active centers remains apparently constant during the second phase of the greening as indicated by light saturation experiments and by the constancy of the apparent photosynthetic unit size. Electron donation from H(2)O seems to develop slower than the activity of the rest of the complex as demonstrated by measurements of 2,6 dichlorophenolindophenol photoreduction using 1,5-diphenylcarbazide as the electron donor. The value of all the above parameters which remain constant during the second phase of the greening are comparable to those obtained with membranes of light-grown cells. PMID- 16659660 TI - A potent inhibitor of ethylene action in plants. AB - Ag(I), applied foliarly as AgNO(3), effectively blocked the ability of exogenously applied ethylene to elicit the classical "triple" response in intact etiolated peas (Pisum sativumcv. Alaska); stimulate leaf, flower, and fruit abscission in cotton (Gossypium hirsutumcv. Stoneville 213); and induce senescence of orchids (Hybrid white Cattleya, Louise Georgeianna). This property of Ag(I) surpasses that of the well known ethylene antagonist, CO(2), and its persistence, specificity, and lack of phytotoxicity at effective concentrations should prove useful in defining further the role of ethylene in plant growth. PMID- 16659661 TI - Affinity Chromatography of the Major Seed Protein of the Bean (Phaseolus vulgaris L.). AB - The major globulin of the French bean (Phaseolus vulgaris L.) undergoes a reversible pH-dependent polymerization. At pH values above 6.5, the monomeric form of the protein predominates; and at pH values below 6.5, the protein occurs as a polymer, probably a tetramer. At extremes of pH, the protein dissociates further into peptides. The reversible pH-dependent interaction between globulin subunits is used in this report as the basis for an affinity chromatography procedure for isolation of the globulin. The major globulin from several genetic variants can be obtained in gram quantities and does not indicate the presence of any impurities on discontinuous sodium dodecyl sulfate gel electrophoresis. PMID- 16659662 TI - Cell potentials, cell resistance, and proton fluxes in corn root tissue: effects of dithioerythritol. AB - Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H(+) and K(+) fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.We deduce that dithioerythritol activates a passive H(+)/K(+) antiport, driven in these experiments by the outwardly directed electrochemical gradient of K(+). The net effect on H(+) and K(+) fluxes is believed to reside with the combined activity of a polarized H(+)/K(+) exchanging ATPase and the passive H(+)/K(+) antiport. A model is presented to show how the combined system might produce stable potential differences and K(+) content. PMID- 16659663 TI - An NADP-dependent Glutamate Dehydrogenase in Chloroplasts from the Marine Green Alga Caulerpa simpliciuscula. AB - NADP-dependent glutamate dehydrogenase was partially purified from extracts of the marine siphonous green alga Caulerpa simpliciuscula. The enzyme had an apparent Km NH(4) (+) of 0.4 to 0.7 mm and was highly specific for NADPH, alpha ketoglutarate, and ammonium ions.The bulk of the NADP-glutamate dehydrogenase was isolated with the chloroplast fraction in cell-free preparations of this alga and was released from these "chloroplast fractions" as a soluble enzyme on gentle lysis of chloroplast membranes. PMID- 16659664 TI - Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoe fedtschenkoi. AB - In vitro studies of dark (14)CO(2) fixation with isolated cell aggregates of Kalanchoe fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO(2) fixation, since 4-(14)C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO(2) fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase. PMID- 16659665 TI - NADH- and NAD(P)H-Nitrate Reductases in Rice Seedlings. AB - By use of affinity chromatography on blue dextran-Sepharose, two nitrate reductases from rice (Oryza sativa L.) seedlings, specifically, NADH:nitrate oxidoreductase (EC 1.6.6.1) and NAD(P)-H:nitrate oxidoreductase (EC 1.6.6.2), have been partially separated. Nitrate-induced seedlings contained more NADH nitrate reductase than NAD(P)H-nitrate reductase, whereas chloramphenicol-induced seedlings contained primarily NAD(P)H-nitrate reductase. NAD(P)H-nitrate reductase was shown to utilize NADPH directly as reductant. This enzyme has a preference for NADPH, but reacts about half as well with NADH. PMID- 16659666 TI - In vitro activation of a galactosyl transferase involved in the osmotic regulation of ochromonas. AB - Osmotic regulation in the flagellate Ochromonas malhamensis Pringsheim is mainly mediated by fluctuations in the pool size of alpha-galactosyl-(1-->1)-glycerol (isofloridoside). A regulated key enzyme of isofloridoside metabolism is the galactosyl transferase producing isofloridoside phosphate. The activity of this enzyme in crude extracts can be increased 5- to 20-fold by incubation at pH 6. The activation occurs in a reaction with a Q(10) of 1.5 to 3 and is dependent on time and pH value. Inactivation of the activated form of the enzyme is also time dependent, and is minimal at the pH value at which activation is optimal. The data suggest a regulation of the enzyme by chemical modification due to the action of auxiliary enzymes. PMID- 16659667 TI - Indole-3-acetic Acid in Douglas Fir: Analysis by Gas-Liquid Chromatography and Mass Spectrometry. AB - We sought evidence for the occurrence and seasonal variation of indole-3-acetic acid (IAA) in shoots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco).Collections obtained in December and June were extracted with methanol and diethyl ether. Extracts were purified by solvent partitioning and with Sephadex LH-20. Qualitative and quantitative information was acquired by gas liquid chromatography of methyl, trimethylsilyl, or both derivatives of plant extract components. Analysis was performed with polar (XE-60) and moderately polar (Hi-Eff-8-BP) stationary phases. Results from three collections demonstrated that IAA does occur in Douglas fir and that amounts vary seasonally. Mass analysis of the proposed endogenous IAA peak from two representative extracts supported gas-liquid chromatography data and established the presence of IAA in Douglas fir. PMID- 16659668 TI - Characterization of passive ion transport in plasma membrane vesicles of oat roots. AB - The passive influx and efflux of inorganic ions across plasma membrane vesicles purified from extracts of Avena sativa roots were investigated. Uptake was measured by incubating the vesicles in a radioisotope for various times. The "loaded" vesicles were separated from the external solution by gel filtration. Efflux was measured by dialyzing the preloaded vesicles.Ion transport was differentiated from superficial ion binding by (a) the time course of association of radioisotope with the vesicles; (b) the rate of loss of radioisotope from the vesicles; (c) the linear increase in isotope associated with the vesicles as the external concentration was increased; (d) the enhanced loss of radioisotope from the vesicles induced by Triton X-100; and (e) the low amount of isotope associated with the vesicles at low temperatures.The plasma membrane vesicles were differentially permeable to the alkali cations with the order of decreasing permeation being K(+) > Rb(+) > Cs(+) > Na(+) > Li(+). The relative transport of Rb(+), Na(+), and Cl(-) across the plasma membrane vesicles was about 1.0:0.50:0.18. The permeability coefficient (P) for Rb(+) was estimated to be 0.29 +/- 0.15 x 10(-8) cm/sec.ATP (and ADP) decreased the passive uptake of Rb(+) into the vesicles, however, this effect did not appear to be related to the ATPase of the plasma membrane. PMID- 16659669 TI - Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts. AB - Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues. PMID- 16659670 TI - Cytochemical localization of glycolate dehydrogenase in mitochondria of chlamydomonas. AB - Mildly disrupted cells of Chlamydomonas reinhardi Dangeard were incubated in a reaction medium containing glycolate, ferricyanide, and cupric ions, and then processed for electron microscopy. As a result of the cytochemical treatment, an electron opaque product was deposited specifically in the outer compartment of mitochondria; other cellular components, including microbodies, did not accumulate stain. Incubation with d-lactate yielded similar results, while treatment with l-lactate produced only a weak reaction. Oxamate, which inhibits glycolate dehydrogenase activity in cell-free extracts, also inhibited the cytochemical reaction. These findings demonstrate in situ that glycolate dehydrogenase is localized in mitochondria, and thus corroborate similar conclusions reached on the basis of enzymic studies of isolated algal organelles. PMID- 16659671 TI - Possible Interference by an Acid-stable Enzyme during the Extraction of Nucleoside Di- and Triphosphates from Higher Plant Tissues. AB - Acid extracts from tissues of two solanaceous plants were found to contain a heat labile, nondialyzable factor which hydrolyzes nucleoside di- and triphosphates to nucleoside monophosphates. This acid-resistant factor shows optimal ATP hydrolyzing activity at pH 5, whereas practically no activity was detected below pH 3 and above pH 9. It does not hydrolyze sugar phosphates, nucleoside monophosphates, uridine diphosphoglucose, and phosphoenolpyruvate. In order to estimate quantitatively the amount of nucleoside di- and triphosphates in a plant extract, care must be taken to circumvent possible interference by this factor. This is achieved by carefully maintaining the extract below pH 3. PMID- 16659672 TI - Association of latent cellulase activity with plasma membranes from kidney bean abscission zones. AB - Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane. PMID- 16659673 TI - Cation-stimulated Adenosine Triphosphatase Activity and Cation Transport in Corn Roots. AB - ATPase activity of the plasma membrane fraction from primary roots of corn (Zea mays L. WF9 x M14) was activated by Mg(2+) and further stimulated by monovalent cations (K(+) > Rb(+) > Cs(+) > Na(+) > Li(+)). K(+)-stimulated activity required Mg(2+) and was substrate-specific. Maximum ATPase activity in the presence of Mg(2+) and K(+) was at pH 6.5 and 40 C. Calcium and lanthanum (<0.5 mm) were inhibitors of ATPase, but only in the presence of Mg(2+). Oligomycin was not an inhibitor of the plasma membrane ATPase, whereas N,N'-dicyclohexylcarbodiimide was. Activity showed a simple Michaelis-Menten saturation with increasing ATP.Mg. The major effect of K(+) in stimulating ATPase activity was on maximum velocity. The kinetic data of K(+) stimulation were complex, but similar to the kinetics of short term K(+) influx in corn roots. Both K(+)-ATPase and K(+) influx kinetics met all criteria for negative cooperativity. The results provided further support for the concept that cation transport in plants is energized by ATP, and mediated by a cation-ATPase on the plasma membrane. PMID- 16659674 TI - Photoreduction of O(2) Primes and Replaces CO(2) Assimilation. AB - A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O(2) evolution or CO(2) uptake was observed, O(2) evolution was normal but completely compensated by O(2) uptake. Similarly, when CO(2) uptake was totally or partially inhibited, O(2) evolution proceeded at a high (near maximal) rate. Under all conditions, O(2) uptake balanced that fraction of the O(2) evolution which could not be accounted for by CO(2) uptake.From these observations we concluded that O(2) and CO(2) are in direct competition for photosynthetically generated reducing power, with O(2) being the main electron acceptor during the induction process and under other conditions in which CO(2) reduction cannot keep pace with O(2) evolution. The high rate of the O(2) uptake reaction observed in the presence of iodoacetamide, KCN, or carbonyl cyanide p trifluoromethyoxyphenylhydrazone, suggests that a special high capacity oxidase distinct from ribulose diphosphate oxygenase exists in whole cells. The rapid reduction of molecular O(2) after a period of darkness probably serves as a priming reaction for the photosynthetic apparatus. The high steady state rate of the O(2) cycle in the absence of CO(2) fixation suggests that the regulation of photosynthesis does not involve significant changes in the rate of photochemical electron transport. PMID- 16659675 TI - Photosynthesis in trees: organization of chlorophyll and photosynthetic unit size in isolated gymnosperm chloroplasts. AB - Chloroplasts have been isolated in high yield from several gymnosperms and from two deciduous trees. The organization of chlorophyll in the chloroplasts of these woody species is basically similar to that in angiosperm crop plants and green algae. The tree chloroplasts contain two chlorophyll proteins, the P700 chlorophyll a-protein and the major light-harvesting chlorophyll a/b-protein, the size, spectral characteristics, and function of which are the same as the equivalent complexes previously isolated from other classes of green plants. All the gymnosperms have chlorophyll/P700 ratios (photosynthetic unit sizes) 1.6 to 3.8 times larger than that typically found in crop plants; the deciduous trees have units of intermediary size. The presence of fewer but larger photosynthetic units in the woody species can partially account for their lower photosynthetic rate and explains why their photosynthetic processes saturate at lower light intensities. Chloroplasts of shade needles have large units containing a greater proportion of the light-harvesting chlorophyll a/b-protein than those of sun needles. PMID- 16659676 TI - Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays. AB - The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.The unappressed membranes (stroma lamellae and bundle sheath plastid membranes) were enriched in heavy (60-40 kilodaltons) peptides and depleted in light (31-20 kilodaltons) peptides as compared to stacked grana membranes. The heavier peptides were tentatively identified as subunits of chloroplast coupling factor. These peptides in unappressed membranes were much more resistant to removal by washing with ethylenediaminetetraacetate (under conditions of low ionic strength) than they were in granal membranes.Ribulose-1,5-diphosphate carboxylase was identified on the gels and was localized exclusively in the bundle sheath cells. It is suggested that sodium dodecyl sulfate electrophoresis is a simple method to test for the localization of carboxylase in various C(4) plastid fractions. PMID- 16659677 TI - Nitrogen metabolism in soybean tissue culture: I. Assimilation of urea. AB - Cultured soybean (Glycine max, Kanrich variety) cells grow with 25 mm urea as the sole nitrogen source but at a slower rate than with the Murashige and Skoog (MS) (Physiol. Plant. 15: 473-497, 1962) nitrogen source of 18.8 mm KNO(3) and 20.6 mm NH(4)NO(3). Growth with urea is restricted by 18.8 mm NO(3) (-), 50 mm methylammonia, 10 mm citrate or 100 mum hydroxyurea, substances which are much less restrictive or nonrestrictive in the presence of ammonia nitrogen source. The restrictive conditions of urea assimilation were examined as possible bases for selection schemes to recover urease-overproducing mutants. Since urease has higher methionine levels than the soybean seed proteins among which it is found, such selections may be a model for improving seed protein quality by plant cell culture techniques.Callus will not grow with 1 mm urea plus 18.8 mm KNO(3). Urease levels decrease 80% within two divisions after transfer from MS nitrogen source to 1 mm urea plus 18.8 mm KNO(3). Hydroxyurea is a potent inhibitor of soybean urease and this appears to be the basis for its inhibition of urea utilization by callus cells.Stationary phase suspension cultures grown with MS nitrogen source exhibit trace or zero urease levels. Soon after transfer to fresh medium (24 hours after escape from lag), urease levels increase in the presence of both MS or urea nitrogen source. However, the increase is 10 to 20 times greater in the presence of urea. NH(4)Cl (50 mm) lowers urease induction by 50% whereas 50 mm methylammonium chloride results in more drastic reductions in urea stimulated urease levels. Citrate (10 mm) completely blocks urease synthesis in the presence of urea.Ammonia and methylammonia do not inhibit soybean urease nor do they appreciably inhibit urea uptake by suspension cultures. It appears likely that methylammonia inhibits urea utilization in cultured soybean cells primarily due to its "repressive" effect on urease synthesis.Citrate does not inhibit urease activity in vitro and exhibits only a partial inhibition (0-50% in several experiments) of urea uptake. It appears likely that the citrate elimination of urease production by cultured soybean cells is due to its chelation of trace Ni(2+) in the growth medium. Dixon et al. (J. Am. Chem. Soc. 97: 4131-4133, 1975) have reported that jack bean (Canavalia ensiformis) urease contains nickel at the active site. PMID- 16659678 TI - Characterization of sulfate transport in cultured tobacco cells. AB - Sulfate transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured in liquid medium was investigated.Transport was linear with time, had a sharp pH optimum between 6.5 and 7.5, and obeyed Michaelis-Menten kinetics. The Km varied within the range 2 x 10(-5)m and 4 x 10(-5)m and the maximum velocity was in the range 100 to 400 nanomoles per gram fresh weight.hour.Transport was inhibited more than 90% by 10(-4)m sulfite, thiosulfate, metabisulfite, sulfide, selenate, and chromate, but was inhibited less than 40% by 10(-3)m chloride, nitrate, or phosphate. Selenate was a competitive and sulfide a noncompetitive inhibitor of sulfate transport.The oxidative respiration inhibitors, azide and cyanide, uncoupling reagents, carbonylcyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol, and the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) were all potent inhibitors of transport. Inhibition by CCCP was not prevented by preincubation of cells with dithiothreitol. Removal of CCCP from the transporting medium resulted in a partial resumption of transport, in contrast removal of DCCD had no effect.Sulfate transport was inhibited more than 90% by 10(-4)m mercaptoethanol, dithiothreitol, or d-cysteine and was abolished by either 10( 5)m N-ethylmaleimide or 10(-4)m iodoacetamide. Removal of mercaptoethanol from the transporting medium resulted in a return to maximal rates of transport whereas when either N-ethylmaleimide or iodoacetamide were removed transport remained inhibited.N-ethylmaleimide (10(-5)m) and iodoacetamide (10(-4)m), which inhibited transport completely, induced the efflux of between 70 and 90% of the transported sulfate in 5 hours. Metabolite efflux was induced by the following compounds, which are listed according to their effectiveness, DCCD, CCCP, mercaptoethanol, and selenate. Increasing the concentration of an inhibitor, in excess of that required to inhibit transport 100%, increased the rate of nonspecific metabolite efflux from the cells. PMID- 16659679 TI - Abscisic Acid Levels in Soybean Reproductive Structures during Development. AB - Abscisic acid (ABA) concentrations and growth rates of developing soybean (Glycine max [L.] Merr. cv. Wye) seeds and pod walls were determined from anthesis to maturation using high pressure liquid chromatographic techniques. Developing soybean seeds contain up to 12,200 ng/g fresh weight of ABA compared to 330 ng/g fresh weight for pod walls. In the developing seeds ABA levels correlated with growth rates, being the highest during the most active growth period of seed enlargement, and then decreasing to less than 10 ng/g fresh weight at maturity. Higher levels of ABA were found to occur in the cotyledons and seed coats than the root-shoot axes at 21 days postanthesis. The time required for excised root-shoot axes to initiate growth in liquid culture decreased as seed development progressed and ABA levels of the seeds declined. PMID- 16659680 TI - Carbon isotope ratios in crassulacean Acid metabolism plants: seasonal patterns from plants in natural stands. AB - A year round study of photosynthesis and carbon isotope fractionation was conducted with plants of Opuntia phaeacantha Engelm. and Yucca baccata Torr. occurring in natural stands at elevations of 525, 970, 1450 and 1900 m. Plant water potentials and the daytime pattern of (14)CO(2) photosynthesis were similar for all cacti along the elevational gradient, despite significant differences in temperature regime and soil water status. Carbon isotope ratios of total tissue and soluble extract fractions were relatively constant throughtout the entire year. Additionally, the sigma(13)C values were similar in all plants of the same species along the elevational gradient, i.e. -12.5 +/- 0.86 per thousand for O. phaeacantha and -15.7 +/- 0.95 per thousand for Y. baccata. The results of this study indicate Crassulacean acid metabolism predominates as the major carbon pathway of these plants, which do not facultatively utilize the reductive pentose phosphate cycle of photosynthesis as the primary carboxylation reaction. PMID- 16659681 TI - Correlation of absorbance changes and thylakoid fusion with the induction of oxygen evolution in bean leaves greened by brief flashes. AB - Dark-grown bean leaves (Phaseolus vulgaris) which had been greened for several days in a repetitive series of brief xenon flashes were studied during the initial induction period when O(2) evolution first appears. The induction of O(2) evolution requires actinic irradiation (e.g. 2 mw/cm(2) of red light) and goes to completion in about 8 minutes with a half-time just under 3 minutes. Absorbance measurements on the intact leaves showed that a change of a carotenoid pigment, monitored at 505 nm, was closely correlated with the rate of O(2) evolution during the induction period. Inhibitor studies, however, showed that the absorbance change persisted in the presence of a number of inhibitors which blocked O(2) evolution. Electron microscopy revealed that the primary thylakoids which were unfused in the flashed leaves before induction became fused in pairs or groups of three during the 8-minute induction period. It is postulated that the 505-nm absorbance change of the carotenoid pigment is correlated more directly with the fusion process than with O(2) evolution. Heat treatment (45 C for 5 min) or infiltration with 0.8 m tris, which prevented the fusion process, also prevented the absorbance change.If the leaves were preilluminated for 8 minutes with very weak red light (20 muw/cm(2)) which induced no O(2) evolution, absorbance change, or thylakoid fusion, there was an immediate burst of O(2) evolution at the onset of actinic irradiation and the induction period, as noted by O(2) evolution or by the 505-nm absorbance change, was reduced to 2 minutes (half-time of 40 seconds). It is concluded that the electron transport system in the flashed leaves is blocked at the Mn site between water and photosystem II and that the photoactivation of Mn into the thylakoid membranes occurs during the low light, photoactivation process. After the electron transport chain is thus repaired, ion-pumping mechanisms driven by actinic light may lead to steady-state photosynthesis as well as to thylakoid fusion. PMID- 16659682 TI - Ethylene-induced Formation of ABA in Citrus Peel as Related to Chloroplast Transformations. AB - When mature green harvested Shamouti oranges (Citrus sinensis L. Osbeck) were exposed to 35 mul/liter of ethylene, a 3-fold increase in free abscisic acid (ABA) of the flavedo could be detected after 12 hours and a 10-fold increase after 24 hours, while chlorophyll destruction did not exceed 20%. The increase in free ABA continued up to 24 hours and leveled off. Bound ABA accumulated strongly after 24 hours suggesting that excess of free ABA was being converted into the bound form. Similar increases in ABA upon exposure to ethylene occurred also in fully mature orange fruits which had already lost all of their chlorophyll, in white and green portions of the flavedo of variegated lemons, and in the colorless albedo of Eureka lemons.The synthetic cytokinin benzyladenine which retards chlorophyll breakdown delayed the ethylene-induced accumulation of free and bound ABA. GA(3) had no effect on endogenous ABA levels.Application of (14)C 2-mevalonolactone to fruits resulted in low but distinct labeling of R(F) zones corresponding to ABA markers, in both free and bound acid fractions. However, there were no significant differences in patterns of incorporation between ethylene-treated and control fruits.The results indicate that ABA accumulates in citrus peel upon exposure to ethylene irrespective of the type of plastids present in the tissue. The possible role of chloroplasts in ABA formation is discussed. PMID- 16659683 TI - Production of cell wall hydrolyzing enzymes by barley aleurone layers in response to gibberellic Acid. AB - The cell walls of barley (Hordeum vulgare var. Himalaya) aleurone layers undergo extensive degradation during the tissue's response to gibberellic acid. Previous work had shown that these cell walls consist almost entirely of arabinoxylan. In this study we show that gibberellic acid stimulates endo-beta-1,4-xylanase activity in isolated aleurone layers. In addition, gibberellic acid enhances the activity of two glycosidases: beta-xylopyranosidase and alpha arabinofuranosidase. No gibberellic acid-stimulated cellulase activity was detected. Germination studies showed a similar pattern of enzyme development in intact seeds. PMID- 16659684 TI - Metabolism of [H]Gibberellin A(20) in Light- and Dark-grown Tobacco Callus Cultures. AB - The growth of tobacco callus in culture (previously shown to contain gibberellin [GA]-like substances), and its ability to metabolize [(3)H]-GA(20) were examined. Growth rates, in the presence and absence of exogenously applied GA, were examined in light and dark conditions. Dark-grown callus grew at a much faster rate than light-grown and [(3)H]GA(20) was metabolized much more rapidly in darkness than in light.[(3)H]GA(1) was identified by combined gas-liquid chromatography/mass spectrometry as a major product of [(3)H]GA(20), and was found to be a more potent promoter of tobacco callus growth than GA(20). PMID- 16659685 TI - Purification and characterization of glucose dehydrogenase from a heterotrophically grown blue-green alga. AB - NADP- and NAD-dependent glucose dehydrogenase was partially purified from a dark grown blue-green alga (endophytic Nostoc strain MAC). Polyacrylamide gel electrophoresis established that a single protein possessed dual activity for either NADP or NAD. No other electron acceptor substituted for pyridine nucleotides and no evidence for a flavin prosthetic group was found. Although the Km for NADP was 8.8 mum and for NAD 328 mum, the enzyme was equally active with NAD or NADP at saturating levels of substrates. The enzyme was similar to previously described glucose dehydrogenase in that it had a high Km for glucose (18-20 mm at 35 C) and an alkaline pH optimum of 7.6 to 9.4. PMID- 16659686 TI - Effect of water stress on proline synthesis from radioactive precursors. AB - Barley (Hordeum vulgare L. var. Prior) leaves converted more (14)C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (Delta(1)-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of (14)C glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility. PMID- 16659687 TI - Two elongation responses to auxin respond differently to protein synthesis inhibition. AB - The first and second responses to auxin react differently to the inhibition of protein synthesis by cycloheximide. It was determined that the protein with the shortest half-life, among the several necessary for the first response, is different from its counterpart among the several necessary for the second response. Specifically, the protein half-lives are 28 minutes and 11 minutes for the first and second responses, respectively. PMID- 16659688 TI - Metabolism of Poly(A) in Plant Cells: Discrete Classes Associated with Free and Membrane-bound Polysomes. AB - In the subapical region of dark-grown pea epicotyls about 40% of the total polysomes are associated with membranes. The presence of poly(A) in polysomal mRNA was detected by hybridization of unlabeled RNA with (3)H-poly(U). Both free mRNA and messenger ribonucleoprotein particles in polysomes hybridize with (3)H poly(U) quantitatively. The binding of (3)H-poly(U) to polysomes is increased by treatment with the detergent sodium dodecyl sulfate. Since detergent influenced the (3)H-poly(U) binding more in membrane-bound polysomes than in free, there may be more protein(s) associated with the poly(A) portion of the mRNA in membrane bound polysomes. Analysis of the poly(A) segments isolated from the mRNA of these two classes of polysomes indicates that there are discrete classes of poly(A) and they appear to be differentially associated with free and membrane-bound polysomes. Mean size distribution of poly(A) in free polysomes is larger than in membrane-bound polysomes.Following treatment (2 days) with the plant growth hormone indoleacetic acid, there is a gradual decrease in the mean length of total poly(A), which appears to correspond to a decrease in the size of the polysomes and their associated mRNA. PMID- 16659689 TI - Synthesis of Suberin during Wound-healing in Jade Leaves, Tomato Fruit, and Bean Pods. AB - The structure and composition of the aliphatic monomers of the polymeric material deposited during wound-healing of tomato fruit, bean pods, and Jade leaves were examined. After removing the cuticle-containing layer of tissue, the wounds were healed for 14 days and the resulting surface layer was excised, lyophilized, solvent-extracted, and depolymerized by hydrogenolysis with LiAlH(4) or transesterified with BF(3) in methanol. The products obtained by the chemical depolymerization were subjected to thin layer chromatography and combined gas chromatography and mass spectrometry. The major aliphatic components isolated from the hydrogenolysate of the wound polymer produced by tomato fruit were hexadecane-1,16-diol and octadec-9-ene-1,18-diol, which were shown to be derived from a 1:1 mixture of omega-hydroxy and dicarboxylic acids of the appropriate chain length by LiAlH(4) reduction. Also identified in the wound polymer were long chain (>C(20)) fatty acids and alcohols. This monomer composition is typical of suberin polymers and is in sharp contrast with that of the cutin of tomato fruit which contains dihydroxy C(16) acid as the major aliphatic component. The hydrogenolysis of the wound material from bean pods gave octadecene-1,18-diol as the major aliphatic component, and smaller amounts of hexadecane-1,16-diol and long chain alcohols. Similar treatment of the normal cuticular tissue of these pods gave hexadecane triol, as well as C(16) and C(18) alcohols. Hydrogenolysis of wound material from the Jade leaves gave octadecene-1,18-diol, C(16) and C(22) diols, as well as alcohols from C(16) to C(26), whereas similar treatment of the cutin-containing tissue from these leaves gave C(16) triol as the major aliphatic component. Thus, the major aliphatic monomers of the polymeric material deposited during the wound-healing of bean pods and Jade leaves are very similar to those of suberin, although the natural protective polymer of these tissues is cutin. From these results, it is concluded that suberization is a fundamental process involved in wound-healing in plants, irrespective of the chemical nature of the natural protective polymer of the tissue. PMID- 16659690 TI - Circadian rhythmicity in excised samanea pulvini: I. Sucrose-white light interactions. AB - The rhythmic movement of excised Samanea saman pulvini incubated in H(2)O or 50 mm sucrose was monitored during extended periods of white light (cool white fluorescent, 2,000 ft-c), darkness, or alternating white light (16 hr) and darkness (8 hr). In continuous white light, the rhythm damps at an intermediate angle after only one cycle, whether pulvini are incubated in sucrose or H(2)O. The rhythm also damps after the first cycle when darkened pulvini are incubated in H(2)O, but it persists for several cycles if sucrose is available. Sucrose depresses the mesor (average angle) during extended dark periods in Samanea, as in Albizzia julibrissin, but it increases the mesor if supplied during white light-dark cycles. With the latter irradiation schedule, oscillations persist for several cycles whether pulvini are supplied with H(2)O or sucrose, but closure is incomplete when pulvini are incubated in sucrose. PMID- 16659691 TI - Circadian Rhythmicity in Excised Samanea Pulvini: II. Resetting the Clock by Phytochrome Conversion. AB - Excised Samanea saman pulvini were incubated in H(2)O or 50 mm sucrose in darkness for 100 to 152 hours except for brief exposures to red or far red light, and angles of opening measured periodically. When pulvini are incubated in H(2)O, the rhythm damps in the open position after two to three cycles irrespective of the light treatments, but when sucrose is available, the now persistent oscillations show large red, far red-regulated effects on phase, amplitude, mesor slope, and entrainment. Single red light pulses rephase the rhythm, with a phase response curve that resembles that reported for other plants and animals; such rephasing is prevented by immediately subsequent far red light, indicating that phytochrome is the photoreceptor. Red light pulses repeated every 24 hours entrain the rhythm, and also prevent damping if presented at an appropriate part of the cycle. PMID- 16659692 TI - Citric Acid cycle activity in mitochondria isolated from mung bean hypocotyls. AB - Citric acid cycle activity in mitochondria from mung bean (Phaseolus aureus var. Jumbo) hypocotyls were examined by surveying (a) characteristics of oxidation of cycle intermediates; (b) activities of cycle enzymes in mitochondrial extracts; (c) contents of cycle intermediates and electron transport components in isolated mitochondria; and (d) time-course changes of products formed during oxidation of succinate, malate, and citrate. Isolated mitochondria are deficient in thiamine pyro-phosphate and somewhat so in adenylates, but apparently sufficient in CoA, NAD, and electron transport carriers. Cycle activity in the mitochondria is not directly correlated with the activities of the enzymes measured in extracts. These studies led to the conclusion that the region between malate and citrate is an important regulatory area in citric acid cycle functioning in isolated mung bean mitochondria. PMID- 16659693 TI - Regulation of malate oxidation in isolated mung bean mitochondria: I. Effects of oxaloacetate, pyruvate, and thiamine pyrophosphate. AB - In order to investigate the relationship between malate oxidation and subsequent cycle reactions, the effects of oxaloacetate, pyruvate, and thiamine pyrophosphate on malate oxidation in mung bean (Phaseolus aureus var. Jumbo) hypocotyl mitochondria were quantitatively examined. Malate oxidation was optimally stimulated by addition of pyruvate and thiamine pyrophosphate, whose addition lowered the apparent Km for malate from 5 mm to 0.1 mm. Intermediate analysis showed that the stimulatory effect was correlated with removal of oxaloacetate to citrate. Oxaloacetate added alone was shown not to be metabolized until addition of pyruvate and thiamine pyrophosphate; then oxaloacetate was converted in part to pyruvate and also to citrate. These results establish that malate oxidation in mung bean mitochondria is subject to control by oxaloacetate levels, which are primarily determined by the resultant of the activities of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase. PMID- 16659694 TI - Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria: II. Role of Adenylates. AB - Effects of ADP and ATP on products of malate oxidation in the presence or absence of respiratory inhibitors and an uncoupler were investigated in mitochondria isolated from mung bean (Phaseolus aureus var. Jumbo) hypocotyls. Changes in levels of products from malate oxidation generally correlated directly with changes in oxygen uptake. Effects of ADP and ATP were indistinguishable from each other when respiratory chain activity was limited. We concluded that adenylates indirectly act on malate oxidation via the oxidation-reduction status of the pyridine nucleotides which are linked to the respiratory chain. The possibility of allosteric action of ADP and ATP on malate dehydrogenase activity was examined in both intact mitochondria and a partially purified enzyme preparation. Although small inhibition, 16% with 500 muM ATP and 8% with 500 muM ADP, was observed at pH 9.5, this effect was abolished by the addition of magnesium ions or by lowering the pH to 7.2. We concluded that these adenylate effects are probably not a significant factor in regulation under physiological conditions. Furthermore, the equilibrium constant of malate dehydrogenase (to 1.5 x 10(-5)) in both mitochondria and the partially purified enzyme calculated from the steady state level of NADH formed suggested that the enzyme functions in an equilibrium manner in intact mitochondria. PMID- 16659695 TI - Microbody Malate Dehydrogenase Isozyme in Cotyledons of Cucumis sativus L. during Development. AB - The properties of the microbody malate dehydrogenase (EC 1.1.1.37) (MDH) isozyme from cotyledons of Cucumus sativus L. were compared during development. It is concluded that the isozyme remains unaltered, despite the transition from glyoxysomal to peroxisomal function that occurs during greening of the cotyledons. This conclusion is based on electrophoretic behavior, chromatographic elution from DEAE-cellulose, molecular weight, kinetic behavior, and immunological identity. In most cases, the distinct properties of the other MDH isozymes in the tissue during development provide additional support for an unchanging microbody isozyme. A method for assaying specifically the microbody isozyme was developed; a diluted preparation was assayed spectrophotometrically before and after complete immunological precipitation. The turnover of the microbody MDH isozyme was investigated by a radioactive labeling study. There is incorporation into both glyoxysomal and peroxisomal MDH. Degradation rates do not correspond with either decline of glyoxysomal activity or the continuation of peroxisomal activity. Apparently, the microbody MDH isozyme is continually turned over throughout cotyledon development. PMID- 16659696 TI - Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei. AB - DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to alpha-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 mug/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of alpha-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates. PMID- 16659697 TI - Structure and Some Properties of Soluble 1,3-beta-Glucan Isolated from the Green Alga Caulerpa simpliciuscula. AB - The properties of the soluble beta-glucans formed during photosynthesis of the green siphonous alga Caulerpa simpliciuscula are described. There are two components in the soluble beta-glucan fraction. One has an apparent degree of polymerization of 37 glucose units and the other of 270 glucose units. The beta glucan with the lower apparent molecular weight accounts for most of the mass in the beta-glucan fraction and is similar in properties to soluble laminarins reported in other algal and fungal species. The beta-glucan with the high apparent molecular weight contains most of the radioactivity accumulated in the beta-glucan fraction during short periods of photosynthesis. PMID- 16659698 TI - Potato tuber callus: validation as a biochemical tool. AB - Callus was initiated from explants of tubers of the Norchip cultivar of Solanum tuberosum L. and grown on medium with a single carbon source and without addition of coconut milk, protein hydrolysate, or amino acid. Callus samples were harvested at intervals and compared to mature tubers for which there was good biochemical knowledge.The amino acid spectrum, the glycoalkaloid content, and the properties of the isolated invertase and sucrose synthetase were similar in callus and in tuber. Significantly the level of sucrose synthetase varied with the age of the developing callus just as it did with the age of the developing tuber. Of greater significance, levels of reducing sugars and invertase varied with the age of developing callus and also with time and temperature of storage after the callus has ceased growth. Similar changes occur in intact tubers.Callus and tuber biochemistry differed in the amount of deposited starch and in the absence of potato invertase inhibitor. PMID- 16659699 TI - Changes in anthocyanin and phenolics content of grapevine leaf and fruit tissues treated with sucrose, nitrate, and abscisic Acid. AB - Sucrose (0.04 to 0.12 m) induces accumulation of both total phenolics and anthocyanin in leaf discs of grapevine (Vitis vinifera L.) incubated in intermittent light. Abscisic acid (20 muM) and 2-chloroethyl phosphonic acid (60 mum) act synergistically with the sucrose to enhance its induction of both total phenolics and anthocyanin. The magnitude of this interaction depends on leaf age. Nitrate (30 mm) inhibits sucrose induction of phenolics and anthocyanin. Levels of total phenolics and anthocyanin changed independently. PMID- 16659700 TI - Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Cofactor Requirements. AB - The rates of oxidation of ent-kaur-16-ene to ent-kaur-16-en-19-ol, ent-kaur-16-en 19-al, ent-kaur-16-en-19-oic acid, and ent-kaur-16-en-7alpha-ol-19-oic acid are maximal in microsomes prepared from the endosperm of immature Marah macrocarpus seeds in which the cotyledons are approximately one-half the overall length of the seed. The supernatant fraction remaining from the preparation of the microsomes contains factors which stimulate the rates of oxidation catalyzed by the microsomes. Added TPNH is more effective than added DPNH in meeting the requirement for reduced pyridine nucleotide. A mixture of DPNH, ATP, and TPN(+) is much more effective than DPNH alone. Experiments with 2,4-dinitrophenol as a selective inhibitor indicate that the ATP-stimulated synthesis of TPNH which occurs in these microsomes in the presence of this mixture of coenzymes provide TPNH for use in the mixed function oxidations. Relatively low concentrations of DPNH and TPNH together are much more effective than either alone at equivalent concentration. This is consistent with the involvement of two pathways of electron transfer associated with the mixed function oxidations, one of which preferentially utilizes TPNH and the other favoring DPNH. FAD added to microsomes at an optimal concentration of about 10 mum in the presence of TPNH stimulates the rate of the oxidations; higher concentrations are inhibitory. FMN by itself does not produce this stimulation. However, FMN and FAD added together at low concentrations (0.5 mum each) have approximately the same effectiveness as FAD alone at 10 mum. This suggests a role for both flavin nucleotides in the normal electron transfer pathways associated with these oxidations. Some of the stimulatory properties of the supernatant fraction may be accounted for by its content of reduced pyridine nucleotides, FAD, and FMN; the concentrations of FAD and FMN were determined to be 1.1 mum and 0.4 mum, respectively. However, the effects of the supernatant fraction are not completely explained by its content of these coenzymes since other experiments indicate the presence of a heat labile, nondialyzable stimulatory factor(s) in the supernatant fraction in addition to heat-stable, dialyzable fractors. PMID- 16659701 TI - Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Electron Transfer Components. AB - Cytochrome P-450 and cytochrome b(5) at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b(5) reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b(5) and DPNH-cytochrome b(5) reductase activity were largely separated from one another and from an overlapping mixture of TPNH cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination.The possible participation of these components in the mixed function oxidation of ent-kaur-16-ene and a number of its oxidized derivatives catalyzed by these microsomes is discussed in relation to the model which has been developed to explain the function of analogous components in mixed function oxidase reactions in mammalian liver microsomes. PMID- 16659702 TI - Distribution of Cellular Carbohydrates during Development of the Mycelium and Fruitbodies of Flammulina velutipes. AB - Flammulina velutipes (Curt. ex Fr.) Sing. was grown on potato-glucose solution freed of most starch. Glucose uptake and dry weight changes in the colony indicated that the large fruitbodies derived their substrates partly from glucose remaining in the medium and partly from cellular constituents stored in the mycelium and small fruitbodies. Changes in the amounts of low molecular weight carbohydrates, glycogen, and four cell wall polysaccharide fractions were followed in the mycelium and fruitbodies. Trehalose, arabitol, and smaller amounts of mannitol were the main stored low molecular weight carbohydrates. A large net loss of these compounds occurred in the mycelium and small fruitbodies after their growth ceased. The carbohydrates accumulated in the large fruitbodies, but were also partly metabolized in the colony. Reducing sugars were minor components, and included about 30 to 50% glucose and a small undetermined quantity of fructose. Glycogen was the main storage carbohydrate in the mycelium, and was also stored in the small fruitbodies. It was broken down in both structures during growth of the large fruitbodies which accumulated only small amounts. During the same period, almost 45% of the maximum amount of cell wall polysaccharides were degraded in the small fruitbodies, but not in the mycelium.By feeding (14)C-glucose in replacement medium, incorporation of radioactivity into carbohydrates was followed in the colony during fruit-body development. Total incorporation was highest in trehalose, next highest in glycogen, and the rest was found in polyols and cell wall polysaccharides except for a few per cent which remained in endogenous glucose. In the large fruitbodies, specific radioactivity in glucose was much lower than in trehalose and mannitol. The labeling patterns in the mycelium and large fruitbodies suggested that trehalose, mannitol, and possibly arabitol were translocated into the stipes and pilei. PMID- 16659703 TI - Production of guard cell protoplasts from onion and tobacco. AB - Guard cell protoplasts (GCP) from young cotyledons of onion and tobacco were isolated in culture microchambers where optimal isolating and culture conditions could be determined in situ. The digestion course was quantified by following under polarized light the loss of.retardation of the birefringent cellulose of the guard cells. The assay showed that driselase has a 5-fold higher cellulytic activity than cellulysin. Driselase is, however, harmful to the GCP. Calcofluor staining was less adequate for establishing digestion courses because it increases sharply after exposing guard cells to cellulysin.Osmotic conditions were crucial for GCP survival. Onion guard cells fragment in the presence of strong plasmolyticum (>0.45 m) indicating cytoplasmic connections between neighboring guard cells and/or cytoplasmic attachments to the wall. Tobacco guard cells plasmolyzed with 0.7 m mannitol revealed several areas of strong attachment to the wall which resulted in severe damage to the cells. Healthy tobacco GCP are obtained by an initial digestion with 4% (w/v) cellulysin in 0.23 m mannitol for 2 to 3 hours followed by an increase in the osmoticum to 0.7 m to stabilize the forming protoplasts.Onion GCP were obtained by digesting paradermal slices with 4% (w/v) cellulysin in 0.23 m mannitol. Protoplasts can be osmotically released by replacing the enzyme solution with 0.23 m mannitol at early stages of digestion. They are also available after prolonged digestion (6-12 hours). Paradermal slices also yield mesophyll and epidermal cell protoplasts but they can be selectively washed away if a pure preparation of GCP is desired. Onion GCP have been kept alive in a simple culture solution for up to 10 days. PMID- 16659704 TI - Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: I. Regulation by Nitrate Flux. AB - The roles that leaf nitrate content and nitrate flux play in regulating the levels of nitrate reductase activity (NRA) were investigated in 8- to 14-day old maize (Zea mays L.) plants containing high nitrate levels while other environmental and endogenous factors were constant. The nitrate flux of intact plants was measured from the product of the transpiration rate and the concentration of nitrate in the xylem. NRA decreased when the seedlings were deprived of nitrate. The nitrate flux and the leaf nitrate content also decreased. When nitrate was resupplied to the roots, all three parameters increased.Attempts to alter the nitrate flux by varying transpiration rates were unsuccessful due to a relatively constant rate of delivery of nitrate to the xylem as transpiration rates fell. However, cooling the roots resulted in a decrease in the nitrate flux. Plants with a lower nitrate flux rapidly lost NRA, although the leaf nitrate content was initially unaffected. If the roots remained cool for a long enough time, the leaf nitrate content eventually decreased. Rewarming the roots increased the nitrate flux, leaf nitrate content, and NRA to control levels. When the nitrate flux in excised shoots was varied in three separate ways, decreasing the nitrate flux to the leaves resulted in a rapid decrease in NRA, although leaf nitrate contents were unchanged.These experiments show that the nitrate flux to the leaves from the roots plays a much larger regulatory role than the leaf nitrate content in controlling the level of NRA in intact plants. PMID- 16659705 TI - Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: II. Regulation by Nitrate Flux at Low Leaf Water Potential. AB - Experiments were conducted to determine whether the nitrate flux to the leaves or the nitrate content of the leaves regulated the nitrate reductase activity (NRA) in leaves of intact maize (Zea mays L.) seedlings having low water potentials (psi(w)) when other environmental and endogenous factors were constant. In seedlings that were desiccated slowly, the nitrate flux, leaf nitrate content, and NRA decreased as psi(w) decreased. The decrease in nitrate flux was caused by a decrease in both the rate of transpiration and the rate of nitrate delivery to the transpiration stream. Upon rewatering, the recovery in NRA was correlated with the nitrate flux but not the leaf nitrate content.Recovery depended on protein synthesis, since recovery could be prevented in excised leaves if an inhibitor of protein synthesis was present. However, it also depended on a high nitrate flux, since recovery could be prevented if there was no nitrate flux, despite a relatively high, constant leaf nitrate content, a high psi(w), and the absence of an inhibitor of protein synthesis.The synthesis of NRA could be increased at low psi(w) if seedlings were desiccated in the presence of additional nitrate, which increased the nitrate flux to the leaves. Since the decrease in NRA at low psi(w) could be relieved by increasing the nitrate flux and recovery also depended on nitrate flux, the inhibition of NRA at low psi(3) was not controlled by a direct effect of psi(w) on protein synthesis nor by alterations in the leaf nitrate content, but rather by a decrease in the nitrate flux that in turn regulated the synthesis of the enzyme. PMID- 16659706 TI - Nitrogen Metabolism in Plant Cell Suspension Cultures: II. Role of Organic Acids during Growth on Ammonia. AB - Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, alpha ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from (14)C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes.It appears that the role of organic acids is neither connected to ammonium transport nor to relief of ammonia toxicity, but may be related to the need for additional carbon skeletons for synthesis of amino acids. PMID- 16659707 TI - Failure of Ethylene to Change the Distribution of Indoleacetic Acid in the Petiole of Coleus blumei X frederici during Epinasty. AB - The effect of ethylene on the distribution of applied indoleacetic acid in the petiole of Coleus blumei Benth. X C. frederici G. Taylor has been investigated during the development of epinastic curvature. Using intact plants, (14)C-IAA was applied to the distal region of the leaf lamina and the accumulation of label in the abaxial and adaxial halves of 5 mm petiole sections was determined after 1.5, 3, and 6 hours. Over this period the label was transported out of the lamina into the petiole at a rate of at least 66 mm hr(-1). Of the total amount of label in the petiole sections, 24 to 30% was located in the adaxial half and this distribution was not altered significantly by exposing plants to an atmosphere containing 50 mul/l ethylene. Thus when epinastic curvature is induced by ethylene there is no associated increase in the IAA content of the expanding adaxial half. The role of endogenous IAA in petiole epinasty was studied by restricting its movement with DPX 1840 (3,3a-dihydro-2-[p-methoxyphenyl]-8H pyrozolo{5,1-a}isoindol-8-one). The leaf petioles still showed an initial epinastic response to ethylene. It is concluded that ethylene-induced epinasty is not dependent upon either any change in the transport of IAA or its redistribution within the petiole. PMID- 16659708 TI - Purification and properties of two proteolytic enzymes with carboxypeptidase activity in germinated wheat. AB - Two proteolytic enzymes with carboxypeptidase activity have been isolated from a germinated wheat extract and partially characterized. Both enzymes rapidly released amino acids from hemoglobin and gluten and hydrolyzed carbobenzoxy phenylalanylalanine. The enzymes were inhibited by diisopropylphosphofluoridate, but unaffected by salts, ethylenediaminetetraacetate, and sulfhydryl reagents at lower concentrations, and had molecular weights of approximately 55,000 and 61,000. Analysis of the hydrolysis products of hemoglobin and gluten indicated that both enzymes had broad specificities, including the ability to release proline. PMID- 16659709 TI - Respiratory Chain of Plant Mitochondria: XVIII. Point of Interaction of the Alternate Oxidase with the Respiratory Chain. AB - Oxidation of the respiratory chain carriers of anaerobic, CO-saturated skunk cabbage (Symplocarpus foetidus) mitochondria, by means of an O(2) pulse, proceeds primarily through the cyanide-insensitive alternate oxidase, since the oxidation of cytochromes a and a(3) takes place with a half-time of 3 seconds, corresponding to the rate of dissociation of CO from reduced cytochrome a(3). Ubiquinone and part of the flavoprotein are oxidized within 1 second under these conditions, and this rapid rate of oxidation is strongly inhibited by m chlorobenzhydroxamic acid (mCLAM), a specific inhibitor of the alternate oxidase of plant mitochondria. The rate of ubiquinone oxidation under these conditions in white potato (Solanum tuberosum) mitochondria, which have no alternate oxidase, is the same as that in skunk cabbage mitochondria treated with mCLAM. Ubiquinone, thus identified as the carrier common to both the cytochrome and alternate oxidase pathways, is linked to the alternate oxidase by a flavoprotein of midpoint potential 50 millivolts more negative with which it is in equilibrium. This arrangement provides a switch for diverting electron transport primarily through the cytochrome pathway under state 3 conditions and primarily through the alternate oxidase pathway under state 4 conditions. PMID- 16659710 TI - Development of the Primary Photochemical Apparatus of Photosynthesis during Greening of Etiolated Bean Leaves. AB - Seven-day-old dark-grown bean leaves were greened under continuous light. The amount of chlorophyll, the ratio of chlorophyll a to chlorophyll b, the O(2) evolving capacity and the primary photochemical activities of Photosystem I and Photosystem II were measured on the leaves after various times of greening. The primary photochemical activities were measured as the photo-oxidation of P(700), the photoreduction of C-550, and the photo-oxidation of cytochrome b(559) in intact leaves frozen to -196 C. The results indicate that the reaction centers of Photosystem I and Photosystem II begin to appear within the first few minutes and that Photosystem II reaction centers accumulate more rapidly than Photosystem I reaction centers during the first few hours of greening. The very early appearances of the primary photochemical activity of Photosystem II was also confirmed by light-induced fluorescence yield measurements at -196 C. PMID- 16659711 TI - Lettuce seed germination: modulation of pregermination protein synthesis by gibberellic Acid, abscisic Acid, and cytokinin. AB - Protein synthesis in gibberellin-treated lettuce (Lactuca sativa) seeds has been studied during the lag phase between the beginning of imbibition and the first signs of radicle protrusion. When compared to the water-imbibed controls, both polyribosome populations and radioactive leucine incorporation into protein increase in the embryos of GA(3)- induced seeds early in the imbibition period. Since these results are contradictory to previously published studies, the reasons for the differences are outlined and various alternative possibilities eliminated. The protocol for protein extraction, particularly the speed at which the supernatant from the seed homogenate is cleared, is important for demonstrating the GA(3)-mediated changes. Embryos maintained in the dormant state by abscisic acid still conduct considerable amounts of protein synthesis, and this is enhanced by concentrations of 6-benzylaminopurine which also promote germination. Therefore, the actions of GA(3), abscisic acid, and cytokinin on lettuce seed germination are mediated, directly or indirectly, via protein synthesis. PMID- 16659712 TI - Water Potential and Stomatal Resistance of Sunflower and Soybean Subjected to Water Stress during Various Growth Stages. AB - Plants of two varieties of soybean (Glycine max (L.) Merr.) and two varieties of sunflower (Helianthus annuus L.) were grown in controlled environments and subjected to water stress at various stages of growth. Leaf resistances and leaf water potentials were measured as stress developed. In soybeans the upper leaf surface had a higher resistance than the lower surface at all leaf water potentials and growth stages. Resistance of the upper surface began to increase at a higher water potential and increased more than the resistance of the lower surface. Resistances returned to prestress values 4 days after rewatering. In sunflowers upper and lower leaf surfaces had similar resistances at all water potentials and growth stages. Leaf resistances were higher in sunflower plants stressed before flowering than in those stressed later. Sunflower plants stressed to -16 bars recovered their prestress leaf resistance and water potential a few days after rewatering, but leaves of sunflower plants stressed to -23 bars died. Leaves of soybean and sunflower plants stressed before flowering suffered less injury than those of older plants and sunflowers stressed after flowering suffered more injury than soybeans. PMID- 16659713 TI - Annual Variation in the Sterol Content of Digitalis purpurea L. Seedlings. AB - Seedings from a single lot of Digitalis purpurea L. seeds were germinated in batches over a period of 13 months. A total lipid extract was made which was resolved into esterified and unconjugated plus glycosylated sterol fractions. The amounts of sterol in each fraction and in the total were compared for seedlings germinated at different times of the year. The amount of esterified sterols reached a maximum value from March until June, and a low value from July until January. In January, a sharp increase began which lasted until March. Amounts of unconjugated and glycosylated sterols were elevated from March until June, low from July until October, and on the rise from November until March. These data correlate with an annual cycle in seed germination. The phase of maximum sterol content of seedlings is followed by a period of null germination. PMID- 16659714 TI - Ultrastructural alteration of plant plasma membranes induced by auxin and calcium ions. AB - Ultrastructural changes in isolated and in situ plasma membranes of etiolated soybean hypocotyls (Glycine max L. cv. Wayne) were induced by indole-3-acetic acid (IAA), other auxins, and calcium chloride. Fixed and embedded preparations were stained by a phosphotungstate-chromate procedure to identify and accentuate plasma membrane. Measurements were on micrographs obtained with an electron optical system calibrated and corrected for reproducible and accurate size measurements. Plasma membranes treated for 20 minutes with 1 mum IAA were 10 to 15% thinner than controls. The response to IAA was rapid, reproducible, auxin specific, temperature-dependent, and reversible. Comparable responses were obtained with isolated and in situ membranes. Membranes treated with 0.5 m calcium chloride for 20 minutes were 15 to 20% thicker than controls. Multiple cycles of alternating calcium and IAA treatments yielded membranes with dimensions that reflected the last treatment of the series. The findings show a direct response of plasma membranes to growth regulating agents and provide evidence for a cell-free response of isolated plasma membranes to a hormone. PMID- 16659715 TI - Evidence for an increase in microviscosity of plasma membranes from soybean hypocotyls induced by the plant hormone, indole-3-acetic Acid. AB - The plant hormone indole-3-acetic acid (IAA or auxin) added at a concentration for half-maximal promotion of cell elongation (1 mum) caused an increase of 25% in the fluorescence polarization of the membrane-bound probe N-phenyl-1 naphthylamine, when added to fractions enriched in plasma membranes from soybean hypocotyls (Glycine max L. var. Wayne), with no measurable change in fluorescence lifetime. The amplitude of the polarization increase was maximal in the temperature range 12 to 22 C. The findings provide evidence for a cell-free response of isolated plasma membranes to the hormone and imply that the response involves an increase in the microviscosity of hydrocarbon regions of the membrane. PMID- 16659716 TI - Differential Oxygen Response of Photosynthesis in Soybean and Panicum milioides. AB - The carbon dioxide compensation concentration of Panicum milioides was less than that of soybean over the range of 15 to 35 C. In soybean (Glycine max [L.] Merr. cv. Wayne), the compensation concentration was directly proportional to O(2) concentration. In P. milioides, the compensation concentration was near zero up to 10% O(2) and then increased linearly with higher O(2), although the slope of the response was less than that in soybean. Leaf extracts of P. milioides contained 3-fold higher phosphoenolpyruvate carboxylase activity than soybean leaf extracts. Oxygen inhibition of photosynthesis and carboxy-lation efficiency was less in P. milioides than that observed in soybean. The affinity of P. millioides ribulose-1,5-di-P carboxylase for CO(2) appeared to be slightly greater than that of soybean. The affinity of both enzymes for O(2) was similar. The reduced response of the compensation concentration and photosynthesis to O(2) in P. milioides may be explained by photosynthetic phosphoenolpyruvate carboxylase fixation and by an apparent increased affinity of ribulose-1,5-di-P carboxylase for CO(2). PMID- 16659717 TI - Concentration-dependent Unloading as a Necessary Assumption for a Closed Form Mathematical Model of Osmotically Driven Pressure Flow in Phloem. AB - Previous attempts to model steady state Munch pressure flow in phloem (Christy and Ferrier. [1973]. Plant Physiol. 52: 531-538; and Ferrier et al. [1974]. Plant Physiol. 54: 589-600) lack sufficient equations, and results were produced which do not represent correct mathematical solutions. Additional equations for the present closed form model were derived by assuming that unloading of a given solute is dependent upon the concentration of that solute in the sieve tube elements. Examples of linear and enzymic type unloading mechanisms are given, although other concentration-dependent mechanisms could be substituted. A method for a numerical solution is outlined, and proof of convergence is presented along with some representative data and the speed of computer calculations. The model provides the minimal set of equations for describing the Munch pressure flow hypothesis as it might operate in plants. PMID- 16659718 TI - Photosynthesis, Dark Respiration, and Growth of Rumex patientia L. Exposed to Ultraviolet Irradiance (288 to 315 Nanometers) Simulating a Reduced Atmospheric Ozone Column. AB - Net photosynthesis, dark respiration, and growth of Rumex patientia L. exposed to a ultraviolet irradiance (288-315 nanometers) simulating a 0.18 atm.cm stratospheric ozone column were determined. The ultraviolet irradiance corresponding to this 38% ozone decrease from normal was shown to be an effective inhibitor of photosynthesis and leaf growth. The repressive action on photosynthesis accumulated through time whereas leaf growth was retarded only during the initial few days of exposure. Small increases in dark respiration rates occurred but did not continue to increase with longer exposure periods. A reduction in total plant dry weight and leaf area of approximately 50% occurred after 22 days of treatment, whereas chlorophyll concentrations remained unaltered. PMID- 16659719 TI - Effect of benzyladenine on some enzymes of mitochondria and microbodies in excised sunflower cotyledons. AB - Benzyladenine (BA) increases the rate of expansion of dark-grown sunflower (Helianthus annuus L.) cotyledons. The hormone slightly enhances the development of the two glyoxysomal enzymes, isocitrate lyase and malate synthetase, during the first 3 days of germination and greatly accelerates their decay in the 2 following days. The levels of the peroxisomal enzymes, glycolate oxidase and glyoxylate reductase, are enhanced by BA more than those of the two glyoxysomal enzymes. These effects of BA on microbody enzymes are very similar to those of white light. Mitochondrial enzyme activities are increased to a varying extent by BA: the increase is minimal for fumarase, and maximal for cytochrome oxidase. The level of cytochrome oxidase is enhanced 346% at the 5th day of germination. Also, the rate of O(2) consumption is increased by BA, but the time course of this increased O(2) consumption does not match with that of cytochrome oxidase. Fusicoccin, a fungal toxin, mimics the effect of BA on cotyledon expansion, but fails to duplicate its action on microbody enzymes. This suggests that the effect of BA on microbody enzymes is not closely linked with the mechanism of growth promotion. PMID- 16659720 TI - Photorespiration in c(3) and c(4) plant tissue cultures: significance of kranz anatomy to low photorespiration in c(4) plants. AB - Photorespiration rates in tissue cultures of a C(4) plant, Portulaca oleracea, were compared to those in tissue cultures of a C(3) plant, Streptanthus tortuosus. The C(4) plant tissue cultures have one-half to one-third the photorespiration rate of the C(3) plant tissue cultures and respond to varying O(2) concentrations in a manner typical of C(4) plants. The results suggest that the lack of detectable photorespiration in C(4) plants is not related to leaf anatomy. PMID- 16659721 TI - Water Relations and Photosynthesis of a Desert CAM Plant, Agave deserti. AB - The water relations and photosynthesis of Agave deserti Engelm., a plant exhibiting Crassulacean acid metabolism, were measured in the Colorado desert. Although no natural stomatal opening of A. deserti occurred in the summer of 1975, it could be induced by watering. The resistance for water vapor diffusion from a leaf (R(WV)) became less than 20 sec cm(-1) when the soil water potential at 10 cm became greater than -3 bars, as would occur after a 7-mm rainfall. As a consequence of its shallow root system (mean depth of 8 cm), A. deserti responded rapidly to the infrequent rains, and the succulent nature of its leaves allowed stomatal opening to continue for up to 8 days after the soil became drier than the plant. When the leaf temperature at night was increased from 5 to 20 C, R(WV) increased 5-fold, emphasizing the importance of cool nighttime temperatures for gas exchange by this plant. Although most CO(2) uptake occurred at night, a secondary light-dependent rise in CO(2) influx generally occurred after dawn. The transpiration ratio (mass of water transpired/mass of CO(2) fixed) had extremely low values of 18 for a winter day, and approximately 25 for an entire year. PMID- 16659722 TI - Comparison of in Vivo and in Vitro Assays of Nitrate Reductase in Wheat (Triticum aestivum L.) Seedlings. AB - The effectiveness of the in vivo and in vitro assays for nitrate reductase (NR) in estimating the amounts of reduced N made available to plants was tested against the daily increases in reduced N (Nesslerization) actually accumulated by the plant. With growth-chamber-grown wheat seedlings, the average ratio values (input of reduced N as estimated by the in vitro assay to actual accumulation of N by the plant) were 3.9 for shoots, 3.7 for the roots, and 4.1 for the entire plant, over a 10-day period. With the in vivo assay, the average ratio values were 0.7 for the shoot, 1.8 for the root, and 0.9 for the entire plant. Although the linear regressions between the accumulated N in the plant and the estimated N input (by both in vitro and in vivo assays) were significant and positive, the in vivo assay provided the closest approximation of the actual amount of N accumulated.The in vivo NR assay effectively distinguished between two wheat varieties. The variety known to have the higher percentage of seed protein also had the higher amounts of NR activity.With seedling wheat leaves, the addition of NADH plus a surfactant increased in vivo NR activity approximately 2-fold over comparable controls. Because the tissue contained high levels of nitrate and enzyme, we concluded that reducing potential was the rate-limiting factor in nitrate reduction in situ in these growth-chamber-grown plants. PMID- 16659723 TI - Effect of light and glucose on the induction of nitrate reductase and on the distribution of nitrate in etiolated barley leaves. AB - Barley seedlings grown in the dark with 10 mm KNO(3) have low levels of nitrate reductase activity even though large amounts of No(3) (-) accumulate in the leaves. When the leaves are excised and transferred to the light, there is an increase in nitrate reductase activity both in the presence and absence of exogenous NO(3) (-). When the leaves are transferred to a glucose solution (0.05 m) but kept in the dark, induction of nitrate reductase activity occurs only when fresh NO(3) (-) is added to the system.In dark-grown leaves, there are small traces of NO(3) (-) in a "metabolic pool." Addition of glucose does not alter this distribution. Light, on the other hand, results in an appreciable accumulation of NO(3) (-) in the metabolic pool. There is a linear correlation between nitrate reductase activity and the size of the metabolic NO(3) (-) pool. Our results thus suggest that NO(3) (-) accumulates in a storage pool when seedlings are grown in continuous darkness. The transfer of this NO(3) (-) to an active metabolic pool is mediated by light but not by glucose. We believe that this transfer of NO(3) (-) leads to the induction of nitrate reductase. When NO(3) (-) is included in the medium, both light and glucose increase its incorporation into the metabolic pool. The results suggest two mechanisms for regulating the metabolic NO(3) (-) pool: (a) a transfer from the storage pool which requires light; and (b) a transfer from the external medium which requires either glucose or light. PMID- 16659724 TI - Effects of Chloramphenicol on the Circadian Rhythm of Neurospora crassa. AB - Chloramphenicol, an inhibitor of mitochondrial protein synthesis, shortened the period length of the circadian rhythm in the Timex strain of Neurospora crassa by 2 hours. Both the l(+) threo and d(-) threo optical isomers had the same effect on the period of the rhythm, whereas only the d(-) threo isomer significantly inhibited mitochondrial protein synthesis. Tetracycline, another inhibitor of mitochondrial protein synthesis, did not change the period of the circadian rhythm. The effect of chloramphenicol on the circadian rhythm is, therefore, presumably not directly related to inhibition of mitochondrial protein synthesis, suggesting that chloramphenicol has other effects. PMID- 16659725 TI - Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis. AB - Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C(16:0), 41 and 36%) and linolenic (C(18:3), 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C(18:0), 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative. PMID- 16659726 TI - A New Sensitive Root Auxanometer: Preliminary Studies of the Interaction of Auxin and Acid pH in the Regulation of Intact Root Elongation. AB - A new sensitive root auxanometer is described. The auxanometer represents an adaption of the position-sensor transducer method to measurement of intact root elongation and has the advantages of simplicity and high sensitivity. Experiments with the auxanometer show that auxin begins to inhibit intact pea root elongation within 10 minutes and continues to inhibit elongation for at least 1 hour following a 1-hour treatment with the hormone. Exposure of pea roots to pH 4 results in a 2- to 3-fold increase in elongation rate beginning about 1 minute after acid treatment. Acid-induced elongation continues at a steady rate for at least 160 minutes and can be reinitiated repeatedly by shifting between pH 4 and 6.5. Auxin inhibits acid-induced elongation whether given before or after acidification, and a transient exposure to auxin renders intact roots relatively insensitive to acid for at least 1 hour after withdrawal of the hormone. PMID- 16659727 TI - Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum. AB - Phosphoenolpyruvate carboxykinase, EC 4.1.1.32 (PEPCK), was purified 43-fold from the grass Panicum maximum. Michaelis constants (Km) were determined for the exchange reaction, the carboxylation reaction, and the decarboxylation reaction. The Km values for oxaloacetate and ATP in the decarboxylation reaction were found to be lower than the Km values for the substrates used in the exchange reaction and in the carboxylation reaction. Phosphoenolpyruvate carboxylase was not detectable in the purified PEPCK preparation.Studies on the nucleotide specificity of the oxaloacetate decarboxylation reaction indicate that ATP serves as the best nucleotide for this reaction and that ADP is about 60% as effective as ATP. The pH optimum for decarboxylase activity is near 6.8. The decarboxylation reaction has a divalent cation requirement with both Mn(2+) and Mg(2+) needed for full activity.Temperature curves of the three PEPCK reactions indicate optimum activities between 38 and 45 C. There is a pronounced drop in the decarboxylation and carboxylation activities as the temperature is decreased from these optima. Below 30 C the energy of activation was 8.2 kcal/mol for the decarboxylation reaction.These studies are consistent with the proposal that under physiological conditions PEPCK catalyzes the decarboxylation of oxaloacetate in the bundle sheath cells of Panicum maximum leaves during C(4) dicarboxylic acid photosynthesis. PMID- 16659728 TI - Turgor Pressure Regulation in Valonia utricularis: Effect of Cell Wall Elasticity and Auxin. AB - The electrical membrane resistance rho(0) of the marine alga Valonia utricularis shows a marked maximum in dependence on the turgor pressure. The critical pressure, P(c), at which the maximum occurs, as well as its absolute value, rho(0) (max), are strongly volume-dependent. Both P(c) and rho(0) (max), increase with decreasing cell volume. It seems likely, that these relationships reflect the elastic properties of the cell wall, because the volumetric elastic modulus, epsilon, is also volume-dependent, increasing hyperbolically with cell volume. Both P(c) and rho(0) (max) can be affected by external application of indole-3 acetic acid at concentrations of 2.10(-7)m to 2 .10(-5)m. The critical pressure is shifted by 1.2 to 6 bars toward higher pressures and the maximum membrane resistance increased up to 5.6-fold. During the course of the experiments (up to 4 hours), however, IAA had no effect on the volumetric elastic modulus, epsilon.The maximum in membrane resistance is discussed in terms of a pressure dependent change of potassium fluxes. The volume dependence of P(c) and rho(0) (max) suggests that not only turgor pressure but also epsilon must be considered as a regulating parameter during turgor pressure regulation. On this basis a hypothesis is presented for the transformation of both, a pressure signal and of changes in the elastic properties of the cell wall into alterations of ion fluxes. It is assumed that the combined effects of tension and compression of the membranes as well as the interaction between membrane and cell wall opposingly change the number of transport sites for K(+) providing a turgor-sensing mechanism that regulates ion fluxes. The IAA effects demonstrated are consistent with this view, suggesting that the basic mechanisms for turgor pressure regulation and growth regulation are similar.Any relation connecting growth rate with turgor pressure should be governed by two parameters, i.e. by a yielding pressure, at which cell growth starts, and by the critical pressure, at which it ceases again. PMID- 16659729 TI - Selective Modulation of RNA Polymerase I Activity during Growth Transitions in the Soybean Seedling. AB - RNA polymerase I and II activities were measured in tissues of the soybean (Glycina max, var. Wayne) hypocotyl where dramatic changes in the relative level of RNA synthesis are associated with normal and auxin-induced growth transitions. When assayed in isolated nuclei, the activity of RNA polymerase I changed much more than the activity of RNA polymerase II during these growth transitions. The activity of RNA polymerase I expressed in the nuclei generally showed a positive correlation with the relative level of RNA synthesis (i.e. accumulation) of that tissue. Following solubilization of the RNA polymerases from these isolated nuclei and fractionation of them on DEAE-cellulose, the activity of RNA polymerase I relative to that of RNA polymerase II showed smaller changes during these growth transitions than when assayed in the nuclei. Thus, these data indicate that the activity of RNA polymerase I is significantly modulated in the nucleus, up or down depending upon the growth state, during growth transitions in the soybean in addition to lesser changes which occur in the apparent level of the enzyme. PMID- 16659730 TI - Starch degradation in the cotyledons of germinating lentils. AB - Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the alpha type. Starch is degraded slowly in the first days; during this time, alpha- and beta amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in alpha-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three alpha-amylases, and one beta-amylase. Based on their heat lability or heat stability, two sets of alpha-amylase seem to exist in lentil cotyledons. PMID- 16659731 TI - Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO(2). AB - The N(2)-fixing legume nodule requires O(2) for ATP production; however, the O(2) sensitivity of nitrogenase dictates a requirement for a low pO(2) inside the nodule. The effects of long term exposures to various pO(2)s on N(2)[C(2)H(2)] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO(2) of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O(2) initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO(2) of 0.32 atmospheres had little effect on N(2)[C(2)H(2)] fixation; a pO(2) of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O(2) controls by 95 hours. Re-exposure to ambient pO(2) of plants adapted to nonambient pO(2)s reduced N(2)[C(2)H(2)] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO(2) conditions, and a time period was required to readapt to ambient O(2). It is concluded that the N(2)[C(2)H(2)]-fixing system of intact soybean plants is able to adapt to a wide range of external pO(2)s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO(2) optimal for nitrogenase activity. PMID- 16659732 TI - Dormancy in Seeds of Charlock (Sinapis arvensis L.): Early Effects of Gibberellic Acid on the Synthesis of Amino Acids and Proteins. AB - Charlock (Sinapis arvensis L.) seeds were imbibed with 10 mm GA(3) for 24 hours at 0 C. After equilibration at 25 C, a 5-fold increase in radioactivity in the amino acids labeled from 2-(14)C-acetate was observed within 2 hours. The total amount of amino acids was reduced to half, and the specific radioactivity increased approximately 10-fold, indicating a diversion of metabolites for amino acid and protein synthesis in GA(3)-treated seeds. The rate of incorporation of l (14) C-leucine into protein was doubled. Autoradiographs showed that enhancement of protein synthesis was localized in the shoot and root meristems, the developing vascular tissues, and in the endosperm cells inside the testa. PMID- 16659733 TI - Water stress, rapid polyribosome reductions and growth. AB - Measurements of the water status of various plant tissues exposed to differing levels of salts for 1 hour were made using the recently developed Campbell J-14 press (Logan, Utah). Values obtained with the press were found to correlate well with estimates of relative water content, and experiments with 3-day-old pumpkin seedlings showed that detectable changes in press values of cotyledon tissues could be obtained within 5 minutes following salt- or desiccation-induced stress.Polyribosome levels were measured in tissues from various plant species following short duration water stress. A small reduction in polyribosome percentage was obtained in cotyledons of 3-day-old pumpkin seedlings which were exposed to an osmotic potential (NaCl) of -4 bar for 10 minutes, but more pronounced changes were found after 30 minutes of stress. Shoot tissues of peas, barley, wheat, and safflower following 20- or 30-minute salt- or desiccation induced stress yielded extracts with reduced polyribosome levels; however, 30 minutes of exposure of cotton and pumpkin seedlings to -6 bars did not result in altered polyribosome percentage of extracts from roots. Studies using shoot tissues from pumpkins and peas showed that polyribosome percentages and growth rates of both plants were reduced in proportion to loss of tissue water. These plants differed in their sensitivity to stress in that polyribosome content and growth rate reductions were both nearly twice as severe per unit of water loss in peas as in pumpkins. These data along with those obtained by others suggest that growth rate reductions may be directly proportional to reductions in polyribosome levels during water stress. PMID- 16659734 TI - Turgor pressure sensing in plant cell membranes. AB - Experimental evidence is reviewed which shows that the cell membrane is compressible by both mechanical and electrical forces. Calculations are given which show that significant changes in the thickness of cell membranes can occur as a result of (a) direct compression due to the turgor pressure; (b) indirect effects due to the stretching of the cell wall; and (c) the stresses induced by the electric field in the membrane.Such changes in the membrane thickness may provide the pressure-transducing mechanism required for osmoregulation and growth. An important feature of the model is that this pressure transduction can occur not only in the plasmalemma (where there is a pressure gradient), but also in the tonoplast. PMID- 16659735 TI - Superoxide dismutase in ripening fruits. AB - The levels of superoxide dismutase (SOD) activity in extracts of preclimacteric apple, banana, avocado, and tomato fruits were not greatly different than in extracts of postclimacteric fruits. The results indicate that no major quantitative change in SOD occurs in fruits with or preceding the onset of senescence. Tomato fruit SOD was studied in more detail, and was found largely in the soluble fraction, and to a lesser extent in the mitochondrial and plastid fractions. The soluble fraction was purified by ammonium sulfate fractionation, column chromatography, and isoelectric focusing. Isoelectric focusing separated SOD from contaminating peroxidases. The purified tomato SOD showed an apparent molecular weight of 31,500 determined by gel filtration. Polyacrylamide gel electrophoresis of this preparation indicated two SOD components corresponding to two protein bands, one of which stained more intensely than the other. The purified tomato enzyme was inhibited 90% by 1 mm KCN. PMID- 16659736 TI - Free Methionine Levels in rin and Normal Isogenic Tomato Fruits Ripened in the Field or in Storage. AB - Free methionine levels in rin and normal tomato fruits were determined microbiologically. Similar levels (1750 mug/100 g fresh weight) for mature green fruits of both rin and a normal isogenic line suggest that the lack of ripening of rin fruits is not due to low methionine levels. Methionine levels of mature green rin and normal fruits were 1750 mug/ 100 g fresh weight. Normal fruits ripened either on or off the vine were 2860 and 2500 mug/100 g fresh weight, respectively. The rin fruits which were left on the plant or held in air at 20 C until soft and yellow were significantly lower in methionine than C(2)H(4) treated rin fruits or any normal fruits. Harvested rin and normal fruits held at 20 C in continuously applied ethylene (10 mul/l) had higher methionine levels than comparable air controls; levels in treated rin fruits were significantly higher than those in normal fruits. PMID- 16659737 TI - Diurnal and Seasonal Patterns of Photosynthesis and Respiration by Stems of Populus tremuloides Michx. AB - The photosynthetic and respiratory rates of 5- to 7-year-old aspen stems (Populus tremuloides Michx.) were monitored in the field for 1 year to determine the seasonal patterns. The stem was not capable of net photosynthesis, but the respiratory CO(2) loss from the stem was reduced by 0 to 100% depending on the time of year and the level of illumination as a result of bark photosynthesis. The monthly dark respiratory rate ranged from 0.24 mg CO(2)/dm(2). hr in January to a maximum 7.4 mg CO(2)/dm(2). hr in June. Individual measurements ranged from 0.02 mg CO(2)/dm(2). hr in February to 12.3 mg CO(2)/dm(2). hr in June. Gross photosynthesis followed a pattern similar to the dark respiratory rate. The mean monthly rate was highest in June (1.65 mg CO(2)/dm(2). hr) and lowest in December (0.02 mg CO(2)/dm(2). hr). Individual measurements ranged from 0.0 mg CO(2)/dm(2). hr in winter to 5.5 mg CO(2)/dm(2). hr in July.Winter studies showed that stem respiration continued down to -11 C, the coldest temperature during this study. Upon warning to -3 C, the dark respiratory rate showed a sudden sharp increase (7- to 12-fold) which required many hours to return to normal levels. No measurable photosynthesis occurred below -3 C. Between -3 and 0 C, the maximal photosynthetic rate was reduced to less than 50% of the respiratory rate, but increased to 89% between 5 to 10 C.On a yearly basis, bark photosynthesis in P. tremuloides reduced the stem respiratory CO(2) loss by 28.7% on a daytime basis and an estimated 16 to 18% on a 24-hour basis. PMID- 16659738 TI - Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L. AB - Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration. PMID- 16659739 TI - Regulation of Senescence in Carnation (Dianthus caryophyllus): Effect of Abscisic Acid and Carbon Dioxide on Ethylene Production. AB - Abscisic acid hastened senescence of carnation flowers and this was preceded by stimulation of accelerated ethylene production. Carbon dioxide delayed the onset of autocatalytic ethylene production in flowers regardless of treatment with abscisic acid. Flowers exhibited a low and transient climacteric of ethylene production without wilting while in 4% carbon dioxide and underwent accelerated ethylene production culminating in wilting when removed from carbon dioxide. Hypobaric ventilation, which lowers ethylene to hyponormal levels within tissues, extended flower longevity and largely negated enhancement of senescence by abscisic acid. Supplementing hypobarically ventilated flowers with ethylene hastened senescence irrespective of abscisic acid treatment. Collectively, the data indicate that abscisic acid hastens senescence of carnations largely as a result of advancing the onset of autocatalytic ethylene production. PMID- 16659740 TI - Metabolism of Indole-3-Acetic Acid: III. Identification of Metabolites Isolated from Crown Gall Callus Tissue. AB - The metabolism of labeled indole-3-acetic acid (IAA-2-(14)C) was investigated in Parthenocissus tricuspidata crown gall callus tissue. After 48 hours incubation, 85 to 90% of the supplied IAA was taken up by the tissue, and of that taken up, about 45% was conjugated with five amino acids. The conjugates found were aspartic and glutamic acid (minor ones) as well as glycine, alanine, and valine (major ones). The last four are being reported for the first time as metabolites of IAA. These conjugates were identified through their chromatographic properties, hydrolysis products, and their mass spectra. The possible significance of these amino acid conjugates is discussed. PMID- 16659741 TI - Studies on acidification of media by Avena stem segments in the presence and absence of gibberellic Acid. AB - The rate of acidification of media by Avena stem segments was studied with a titrimeter. GA(3) increased this rate by an average of 17% if supplied to the segments 90 min prior to measurement. GA(3) inhibited the rate by 15% if supplied 10 min prior to measurement. After 90 min incubation, stimulation of elongation had started; at 10 min, GA(3) had not yet started to stimulate elongation in the segments.The acidification rates of the nodes (including the sheath-pulvinus), leaf sheath bases, and the internode bases of the stem segments were determined for plus and minus GA(3)-treated segments. The internode fraction contributes most to modification of the acidification rate, the node-pulvinus fraction less so, and the nongrowing sheath not at all.Acidification rates were measured for segments in different stages of elongation (lag, log, and plateau phases of growth). Segments in these growth stages were obtained from intact plants and from segments preincubated in sucrose and sucrose + GA(3). Segments from all sources which are in the log phase of growth have the highest rates, those in the plateau phase the lowest. For lag and log growth phases, segments preincubated in sucrose + GA(3) show the highest rates, those preincubated in sucrose the lowest rates. The opposite occurs for segments in the plateau phase of growth.Segments stimulated to grow by GA(3) cause the pH of their incubation media to drop to pH 5.15 from an initial pH of 6.5. Nonstimulated segments cause a drop to pH 5.6. Long term growth of the segments is maximal in media buffered to pH 5 in the presence and absence of GA(3).Our results support the idea that GA(3) stimulates an active acidification process in Avena stem segments just after GA(3) starts to stimulate growth in the segments, and that such an acidification process could play an important role in wall-loosening during active growth of the internode. PMID- 16659742 TI - Membrane-bound UDP-Glucose: Lipid Glucosyltransferases from Peas. AB - An enzymic preparation from peas (Pisum satisum), able to form neutral and polar glycosides, is described. The best sugar donor is UDP-glucose and the acceptors are present in the enzymic system.The neutral glycolipids have been characterized as steryl and acylated steryl glucosides. The polar glucolipid had been identified as polyprenyl monophosphate glucose. The glucose is linked to the phosphate in beta configuration. Polar glucolipids are also formed from UDP glucose and exogenous prenylic acceptors, either alpha-saturated, as dolichyl monophosphate, or allylic, as ficaprenyl monophosphate.THE TWO GLUCOSYLATING ACTIVITIES HAVE BEEN PARTIALLY SEPARATED BY DIFFERENTIAL CENTRIFUGATION: the fraction that precipitates at 25,000g has most of the neutral glucolipid synthesizing activity, and the fraction that sediments at 100,000g is rich in polar glucolipid glucosyl transferase activity. This latter activity was strongly dependent on Mg(2+) concentration, the optimum being around 5 to 10 mm. UDP inhibits the reaction and 0.2 to 0.5% (v/v) Triton X-100 has a stimulatory effect. The optimum pH is 7.5. PMID- 16659743 TI - Endogenous abscisic Acid in relation to bud growth in alternate bearing ;valencia' orange. AB - An investigation was conducted into the relation of ABA (cis-trans-abscisic acid) in the dormant buds of alternate bearing ;Valencia' orange (Citrus sinensis [L.] Osbeck) trees. ABA did not appear to be related to alternate bearing but t-ABA (2 transabscisic acid) did. There was 5- to 10-fold more t-ABA than ABA in the buds. There was more t-ABA in the buds of the "on" trees than in the buds of the "off" trees, and a drastic drop in t-ABA in both types of buds as spring growth approached. Bud dormancy and readiness for growth as related to t-ABA are discussed. PMID- 16659744 TI - Immunological similarities of proteinase inhibitors from potatoes. AB - Proteinase inhibitors, purified independently from Japanese, United States, and German potato varieties, and having different physicochemical and inhibitory properties, are shown to be immunochemically similar. These results indicate that the heterogeneity found among proteinase inhibitors from potato tubers is apparently due both to intervarietal, as well as intravarietal, variations in isoinhibitor components. PMID- 16659745 TI - Red Light-enhanced Phytochrome Pelletability: Re-examination and Further Characterization. AB - Red light-enhanced pelletability of phytochrome was observed in extracts of all 11 plants tested: Avena sativa L., Secale cereale L., Zea mays L., Cucurbita pepo L., Sinapis alba L., Pisum sativum L., Helianthus anuus L., Raphanus sativus L., Glycine max (L.) Merr., Phaseolus vulgaris L., and Lupinus albus L. This enhanced pelletability was observed in all 11 plants following in situ irradiation (in vivo binding) but only in Sinapis and Cucurbita after irradiation of crude extracts (in vitro binding). In vivo binding was not strongly dependent upon pH and, with few exceptions, was not markedly sensitive to high salt concentration, whereas in vitro binding was completely reversed by both high pH and high salt concentration. However, both binding phenomena were observed only with a divalent cation in the extract buffer. In vivo binding was further characterized using Avena which showed an increase in pelletability from less than 10% in dark control extracts to more than 60% in extracts of red light-irradiated shoots. The half-life for binding was 40 seconds at 0.5 C and was strongly temperature dependent, binding being complete within 5 to 10 sec at 22 C. If pelletable phytochrome in the far red-absorbing form was photoconverted back to the red absorbing form in situ, phytochrome was released from the pelletable condition with a half-life of 25 minutes at 25 C and 100 minutes at both 13 C and 3 C. No cooperativity in red light-enhanced pelletability with respect to phytochrome-far red-absorbing form was observed. PMID- 16659746 TI - Microbodies from spirogyra: organelles of a filamentous alga similar to leaf peroxisomes. AB - Organelles from Spirogyra cells were separated on a linear sucrose gradient. After centrifugation, most of the protein was found in the top fraction. Two minor protein peaks at density (g/cm(3)) 1.17 and 1.21 were due to chloroplast particles and mitochondria, respectively. Although there was an extremely low concentration of protein at density 1.25 g/cm(3), a major part of the activity of glycolate oxidase was found in this region. The enzyme was able to transfer electrons to O(2) and only lost 12% of its activity in the presence of 1 mm cyanide. As documented by electron micrographs, microbodies moved to density 1.25 g/cm(3) during centrifugation. This observation, as well as the fact that high activities of hydroxypyruvate reductase and catalase were also found at the same density, suggest that the microbodies from Spirogyra are similar to those in green leaves of higher plants. PMID- 16659747 TI - Seasonal changes in reflectance and standing crop biomass in three salt marsh communities. AB - Reflectance of red (656-705 nm) and infrared (776-826 nm) solar radiation and standing crop biomass were measured in three salt marsh communities at intervals of approximately 2 weeks between February and August 1974. Red reflectance declined at the onset of greening in each community and was correlated with standing crop of green biomass. Infrared reflectance increased substantially in the shrub community but less in the grass and sedge communities. The inverse of red reflectance was found to be a reliable predictor of green biomass in sedge and grass communities, but not in a shrub community. PMID- 16659748 TI - Kinetic studies of the thermal decomposition of 2-chloroethylphosphonic Acid in aqueous solution. AB - The decomposition of 2-chloroethylphosphonic acid in aqueous solution has been studied at pH values from 6 to 9 and at temperatures in the 30 to 55 C range. The rate of decomposition is estimated from the rate of formation of ethylene. The rate is proportional to the concentration of the phosphonate dianion and is independent of the hydroxyl ion concentration. The rate constant at 40 C is 1.9 x 10(-4) sec(-1) and the activation energy is 29.8 kcal mol(-1). The rate of reaction is not affected significantly by the presence of potassium iodide or urea (substances which increase the rate of leaf abscission in trees sprayed by 2 chloroethylphosphonic acid). The rate decreases slightly in the presence of low concentrations of magnesium and calcium ions. PMID- 16659749 TI - Protein Bodies from the Endosperm of Castor Bean: Subfractionation, Protein Components, Lectins, and Changes during Germination. AB - Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm(3), 1.30 g/cm(3), and > 1.46 g/cm(3), respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane.The proteins of the protein bodies were resolved into protein components of diverse molecular weights in sodium dodecyl sulfate-acrylamide gel electrophoresis. The protein components of the organelle matrix were distinct from those of the crystalloids. Whereas the matrix proteins had very diverse molecular weights, the crystalloid proteins were mainly composed of several proteins with molecular weights between 50,000 and 60,000 daltons. Also, the matrix proteins were soluble in water while the crystalloid proteins were insoluble in water but soluble in salt solution, thus representing albumins and globulins, respectively. Two of the matrix proteins with molecular weights approximately 120,000 and 65,000 daltons were identified as the phytohemagglutimin and the toxic protein ricin, respectively.During germination, the crystalloid proteins served as the storage protein and went through rapid degradation with smaller polypeptides formed as intermediates. In contrast, the proteins of the matrix under-went a much slower degradation during the same period and did not appear to be storage protein. PMID- 16659751 TI - pH Changes in the Cytoplasm of the Blue-Green Alga Anacystis nidulans Caused by Light-dependent Proton Flux into the Thylakoid Space. AB - The pH in the cytoplasmic and thylakoid spaces of the blue-green alga, Anacystis nidulans, has been determined in the light and in the dark by uptake of 5,5 dimethyloxazolidine-2,4-dione and methylamine into the sucrose-impermeable (3)H H(2)O space, as measured by silicon layer filtering centrifugation.Illumination causes an alkalinization in the cytoplasm which is accompanied by an acidification in the thylakoid space, reflecting light-dependent proton transport across the thylakoid membrane. Under light conditions, a pH gradient of approximately 2.8 between the cytoplasmic and thylakoid spaces has been measured that can be abolished almost completely by addition of the uncoupler, 3 chlorocarbonyl cyanide phenylhydrazone. The pH in the cytoplasm is independent of the pH in the medium. PMID- 16659750 TI - Protein bodies of castor bean endosperm: isolation, fractionation, and the characterization of protein components. AB - Protein bodies in the endosperm of castor bean seeds (Ricinus communis L.) contain phytin globoids and protein crystalloids embedded in an amorphous proteinaceous matrix. The protein bodies are apparently surrounded by a single membrane. The protein bodies were isolated by grinding and centrifuging in glycerol. Such isolated protein bodies were almost identical (after cytological fixation) to those observed in situ, except that the globoids were lost. However, membrane-like structures appear to have surrounded the globoids. Histochemical analysis of the isolated protein bodies showed that carbohydrates (glycoproteins) are localized only in the matrix region.Addition of water to protein bodies in glycerol caused dissolution of the matrix, and release of the globoids and crystalloids. When the crystalloids were centrifuged on sucrose density gradients, they were recovered at an equilibrium density of 1.29 to 1.30 g/ml. The crystalloids were only slightly soluble in most aqueous buffers but were very soluble in sodium dodecyl sulfate, urea, or NaOH solutions.Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and chromatography on ion exchange celluloses show that the protein bodies are composed of one major and several minor anodic proteins. The major protein, along with a few of the minor proteins, is localized in the crystalloids.The major protein (molecular weight 65,000) was converted by mercaptoethanol into subunits with molecular weights of 32,000 and 15,800. It is proposed that the protein is made up of two of the smaller subunits and one of the larger, linked by disulfide bridges. None of the crystalloid proteins appear to be glycosylated.The water-soluble matrix fraction is composed mainly of two proteins, with molecular weights of 12,500 and 10,300 on the gels. Neither is a glycoprotein, and neither can be reduced with mercaptoethanol to give subunits. The soluble fraction also contains other lesser components among which are several glycoprotein lectins. One of these is ricin D, which is the main glycoprotein in the protein bodies. PMID- 16659752 TI - Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts. AB - Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [(35)S]methionine or [(3)H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea. PMID- 16659753 TI - Polygalacturonases Release Cell-Wall-bound Proteins. AB - Purified polygalacturonases from two fungi released proteins from wall fractions prepared from three plant species. Peroxidase activity was associated with the proteins released from the cell walls, and several of the protein fractions released contained hydroxyproline. Cellulase, purified free of pectic enzyme activity, was ineffective in releasing cell wall proteins. Specific inhibition of endopolygalacturonase activity prevented release of the proteins. PMID- 16659754 TI - Relationships between Carbon Dioxide, Malate, and Nitrate Accumulation and Reduction in Corn (Zea mays L.) Seedlings. AB - The observation that exposure of the leaf canopy to increasing concentrations of CO(2) (100-400 mul/l) decreases the influx of nitrate to the leaf blades, but not to the roots or stalks (largely leaf sheaths), was reconfirmed using (15)NO(3) ( ). Decreases in leaf nitrate supply were associated with decreases in induction of nitrate reductase, thus supporting the view that the influx of nitrate to a tissue is a major factor in regulation of the level of nitrate reductase. The whole plant (15)N distribution data show that the CO(2) effects were due to decreased influx of nitrate into the leaf blade rather than CO(2)-enhanced nitrate reduction. The decreases in nitrate accumulation by the leaf blade with increases in CO(2) concentration were only partially accounted for by differences in transpiration. Because the initial malate concentration of root tissue (detopped plants) had no subsequent effect on nitrate uptake, it seems unlikely that high levels of malate induced by CO(2) were responsible for the exclusion of nitrate from the leaf blades.Time course changes in nitrate and malate concentrations in root tissue (detopped plants) during nitrate uptake showed that oxidation of extra malate does not stimulate nitrate uptake and that malate is not specifically required as an energy source at the ion carrier level.The observation that nitrate and malate concentrations in corn leaf blades were negatively correlated was reconfirmed with 25 additional corn genotypes. However, using the same tissue, a higher correlation was obtained between malate plus aconitate and nitrate, suggesting that organic acids other than malate could be involved. The proposal that reduction of nitrate in the leaf is stoichiometrically related to malate production is a valid explanation of the relationship only if malate oxidation does not provide NADH for nitrate reduction. However, addition of malate and NAD to crude extracts (in vitro assay) or malate to leaf blade sections (in vivo assay) caused nitrate reduction. Because of these observations and the known intracellular location of NAD-malate dehydrogenase and nitrate reductase, we believe that malate oxidation is one of the major sources of NADH for nitrate reduction in corn leaf blades in situ. PMID- 16659755 TI - Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): I. Effects of Light and Temperature. AB - The optimum in vivo nitrate reductase (NR) assay medium for soybean (Glycine max [L.] Merr.) leaves was 50 mm KNO(3), 1% (v/v) 1- propanol, and 100 mm potassium phosphate buffer (pH 7.5).Loss of in vivo NR activity from leaves of soybeans exposed to dark was fastest at 40 C and slowest at 20 C. However, by the end of a 16-hr dark period, even those plants exposed to the lowest (20 C) temperature had lost 95% of the initial activity. Upon re-exposure to light, following a 16 hr-30 C dark period, in vivo NR activity increased rapidly to maximum levels after 4 hr light. The rate of increase was proportional to light intensity (6, 16, and 45 klux) and independent of temperature (20, 30, and 40 C).Studies with field-grown soybeans indicated that mighttime temperature (16-27 C) had no effect on the subsequent in vivo NR activity in sunlight at ambient temperature. There was a marked decrease in in vivo NR activity in late afternoon with the field-grown plants. This decrease continued throughout the night with elevated temperature (27 C) while NR activity increased when a cooler (16 C) night temperature was imposed.The changes in in vivo NR activity in response to light and dark treatments were quite rapid and thought to be related to energy limitations as well as enzyme level. PMID- 16659756 TI - Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): II. Energy Limitations. AB - Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO(3) (-), NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO(3) (-)in vivo, -NO(3) (-)in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO(3) (-) to the in vivo NR assay medium increased activity (over that of the NO(3) (-)in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO(3) (-) was rate-limiting. The stimulation of in vivo NR activity by NO(3) (-) was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO(3) (-) was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 mumol NO(2) (-) [g fresh weight, hr](-1)) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.The addition of NADH to the in vivo NR assay medium did not stimulate NR activity, although it was not established that NADH entered the tissue. The addition of glucose, fructose 1,6 diphosphate, pyruvate, citrate, succinate, or malate to the in vivo assay medium significantly increased measurable NR activity of leaf tissue from plants pretreated to extended dark periods at elevated temperature. Glucose additions were most effective, usually stimulating increases 2- to 3-fold greater than the other metabolites. Increased NR activities from the various additives were attributed to production of NADH. The loss of in vivo NR activity in soybeans during darkness appeared to be due to the combination of a net loss of enzyme per se and energy depletion. The subsequent light stimulation of NR activity was likely due to increased availability of reductant energy as well as a net synthesis of the NR enzyme. PMID- 16659757 TI - Malate Dehydrogenase and NAD Malic Enzyme in the Oxidation of Malate by Sweet Potato Mitochondria. AB - Over a range of concentrations from less than 0.1 mm to more than 70 mm, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S(0.5) of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S(0.5) of 41.6 mm.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of S(0.5) values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme. PMID- 16659758 TI - Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L. AB - In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-(14)C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [(14)C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [(14)C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [(14)C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.IT IS POSTULATED THAT TWO PATHWAYS EXIST FOR THE BIOSYNTHESIS OF ACYLATED STERYL GLYCOSIDE: one through steryl glycosides, and the other through an unidentified steroid component. It is the latter pathway which appears to be dominant in the in vivo tobacco system. PMID- 16659759 TI - Cytokinins in Corynebacterium fascians Cultures: Isolation and Identification of 6-(4-Hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine. AB - In addition to the four cytokinins, 6-(3-methyl-2-butenylamino)purine, 6 methylaminopurine and the cis and trans isomers of 6-(4-hydroxy-3-methyl-2 butenylamino)purine, reported earlier from our laboratories, three cytokinin active fractions have been obtained from the aqueous medium of 6-day-old Corynebacterium fascians cultures. One of these has been identified as 6-(4 hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine (2-methylthio-cis-zeatin, c-ms(2)io(6) Ade).The elution volumes of the other two fractions correspond to those of authentic 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-d-ribofuranosyl purine and 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine, indicating the presence of trace amounts of these two ribonucleosides. PMID- 16659760 TI - Regulatory Properties of the ADP-Glucose Pyrophosphorylase of the Blue-Green Bacterium Synechococcus 6301. AB - ADP-glucose was found to be the primary sugar nucleotide used for glycogen synthesis by Synechococcus 6301. ADP-glucose pyrophosphorylase was partially purified 12-fold from this blue-green bacterium. The enzyme was activated 8- to 25-fold by glycerate 3-phosphate. Fructose 6-phosphate, fructose 1,6 bisphosphate, 5'-adenylate, and adenosine diphosphate activated the enzyme, but less than glycerate 3-phosphate. The enzyme was inhibited by inorganic phosphate. The I(0.5) of phosphate was 0.072 mm, and in the presence of 2 mm glycerate 3 phosphate, increased to 1.8 mm. The substrate saturation curves for glucose 1 phosphate and ATP were hyperbolic in both the presence and absence of glycerate 3 phosphate or phosphate. The saturation curve for MgCl(2) was sigmoidal; 2 mm glycerate 3-phosphate decreased the sigmoidicity from a Hill slope n value of 5.6 to 2.8, and increased the MgCl(2) optimum from 3 mm to 6 to 7 mm. PMID- 16659761 TI - Light-dependent Elongation of Anaerobically Maintained Green Pea Stem Segments and Its Implications. AB - Anaerobic conditions reversibly inhibit the elongation of isolated green pea (Pisum sativum L. var Alaska) stem segments. Illumination of segments maintained under anoxia causes a resumption of growth. Polarographic studies show pea stem segments are photosynthetically competent as determined by O(2) evolution. Although O(2) production is totally inhibited by dichlorophenyldimethylurea (DCMU) and dinitrophenol (DNP) inhibits O(2)-dependent growth, neither DCMU nor DNP completely abolishes light-dependent growth, although both reduce the effect markedly. Phenazine methosulfate promotes the growth of anaerobically maintained, illuminated, DCMU-treated segments. The data indicate that the principal effect of light in inducing growth under anaerobic conditions is the photosynthetic provision of O(2) for respiration. There is also some evidence that, at least in the absence of O(2), a small amount of elongation is due to some other light driven process, perhaps cyclic photophosphorylation. PMID- 16659762 TI - Comparison of the photosynthetic characteristics of three submersed aquatic plants. AB - Light- and CO(2)-saturated photosynthetic rates of the submersed aquatic plants Hydrilla verticillata, Ceratophyllum demersum, and Myriophyllum spicatum were 50 to 60 mumol O(2)/mg Chl.hr at 30 C. At air levels of CO(2), the rates were less than 5% of those achieved by terrestrial C(3) plants. The low photosynthetic rates correlated with low activities of the carboxylation enzymes. In each species, ribulose 1,5-diphosphate carboxylase was the predominant carboxylation enzyme. The apparent K(m)(CO(2)) values for photosynthesis were 150 to 170 mum at pH 4, and 75 to 95 mum at pH 8. The K(m)(CO(2)) of Hydrilla ribulose 1,5 diphosphate carboxylase was 45 mum at pH 8. Optimum temperatures for the photosynthesis of Hydrilla, Myriophyllum, and Ceratophyllum were 36.5, 35.0, and 28.5 C, respectively. The apparent ability of each species to use HCO(3) (-) ions for photosynthesis was similar, but at saturating free CO(2) levels, there was no indication of HCO(3) (-) use. Increasing the pH from 3.1 to 9.2 affected the photosynthetic rate indirectly, by decreasing the free CO(2). With saturating free CO(2) (0.5 mm), the maximum photosynthetic rates were similar at pH 4 and 8. Carbonic anhydrase activity, although much lower than in terrestrial C(3) plants, was still in excess of that required to support HCO(3) (-) utilization.Hydrilla and Ceratophyllum had CO(2) compensation points of 44 and 41 mul/l, respectively, whereas the value for Myriophyllum was 19. Relatively high CO(2) compensation points under 1% O(2) indicated that some "dark" respiration occurred in the light. The inhibition of photosynthesis by O(2) was less than with terrestrial C(3) plants. Glycolate oxidase activity was 12.3 to 27.5 mumol O(2)/mg Chl.hr, as compared to 78.4 for spinach. Light saturation of photosynthesis occurred at 600 to 700 mueinsteins/m(2).sec in each species grown under full sunlight. Hydrilla had the lowest light compensation point, and required the least irradiance to achieve the half-maximal photosynthetic rate.Field measurements in a Hydrilla mat indicated that in the afternoon, free CO(2) dropped to zero, and O(2) rose to over 200% air saturation. Most photosynthetic activity occurred in the morning when the free CO(2) was highest and O(2) and solar radiation lowest. The low light requirement of Hydrilla probably provides a competitive advantage under these field conditions. PMID- 16659763 TI - Differential effects of actinomycin d and cordycepin in lettuce seed germination and RNA synthesis. AB - Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using (3)H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. (3)H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited (3)H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of (3)H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination. PMID- 16659764 TI - Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits. AB - Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5 diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH(2)-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits. PMID- 16659765 TI - Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard. AB - Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ. PMID- 16659766 TI - Effects of inorganic solutes on the binding of auxin. AB - The binding of alpha-naphthaleneacetic acid ((14)C-NAA) to pelletable particulates from corn (Zea mays) coleoptiles was found to be influenced by inorganic solutes. La(3+), Ca(2+), and Mg(2+) increased the binding whereas monovalent cations did not. The concentrations of CaCl(2) which increased auxin binding were similar to those which inhibited coleoptile elongation in the presence of auxin. These results are interpreted as suggesting that the alteration of hormonal effectiveness by some inorganic solutes involves alterations in the attachment of the hormone to binding sites in the cell. PMID- 16659767 TI - Anomalous Temperature Dependence of Seedling Development in Some Soybean (Glycine max [L.] Merr.) Cultivars: Role of Ethylene. AB - In Clark and Shelby soybean (Glycine max [L.] Merr.) seedlings, hypocotyl elongation was inhibited and hypocotyl swelling and root dry weight were increased by a temperature of 25 C. At 20 and 30 C, development was normal, as was development of Hawkeye and Mandarin soybean seedlings at all three temperatures. Dry matter distribution at 9 days indicates that inhibition of hypocotyl elongation is not due to a lack of translocation from cotyledons, but to a diversion of dry matter from hypocotyl to root. Ethylene evolution by Clark seedlings at 25 C exceeds that at 20 and 30 C. At all three temperatures, Mandarin seedlings' ethylene evolution is at the same low rates as those of Clark at 20 and 30 C. Clark's enhanced rate at 25 C precedes the deceleration of hypocotyl elongation occurring at 5 days. The abnormal effects of a temperature of 25 C on Clark seedlings' development is partially reversed by CO(2) and GA(3) and can be duplicated in Mandarin by applying ethylene. In Clark, effects of the temperature can be further accentuated by indoleacetic acid, which stimulates ethylene evolution, and by applying ethylene to the seedlings. It is concluded that the temperature-induced symptoms, similar in most respects to the well known "triple response" of legume seedlings, are caused by abnormally high levels of ethylene in tissues of the anomalous cultivars. PMID- 16659768 TI - Abscisic Acid Metabolism by a Cell-free Preparation from Echinocystis lobata Liquid Endoserum. AB - A cell-free enzyme system capable of metabolizing abscisic acid has been obtained from Eastern Wild Cucumber (Echinocystis lobata Michx.) liquid endosperm. The reaction products were determined to be phaseic acid (PA) and dihydrophaseic acid (DPA) by co-chromatography on thin layer chromatograms as the free acids, methyl esters, and their respective oxidation or reduction products. The crude enzyme preparation was separated by centrifugation into a particulate abscisic acid (ABA)-hydroxylating activity and a soluble PA-reducing activity. The particulate ABA-hydroxylating enzyme showed a requirement for O(2) and NADPH, inhibition by CO, and high substrate specificity for (+)-ABA. Acetylation of short term incubation mixtures gave evidence for the presence of 6'-hydroxymethyl-ABA as an intermediate in PA formation. Determinations of endogenous ABA and DPA concentrations suggest that the ABA-hydroxylating and PA-reducing enzymes are extensively metabolizing ABA in the intact E. lobata seed. PMID- 16659769 TI - Contribution of Arginine to Proline Accumulation in Water-stressed Barley Leaves. AB - Barley (Hordeum vulgare cv Prior) leaves converted l-U-(14)C-arginine to labeled proline. Accumulation of radioactivity in proline was greater in wilted leaves, but only after 9 hours of incubation. As the increase in free proline was detectable after only 3 to 6 hours, it is likely that the observed stimulation of proline labeling represents a result rather than a cause of proline accumulation. Furthermore, the loss of total arginine during water stress was only 10 to 15% of the increase in proline. We conclude that arginine probably contributes less than 1% of the carbon in the expanding proline pool of wilted barley leaves. PMID- 16659770 TI - Altered nitrogenous pools induced by the azolla-anabaena azolla symbiosis. AB - The free amino acid and ammonia pools of Azolla caroliniana were analyzed by quantitative column chromatography on columns capable of separating all of the nitrogenous constituents normally found in physiological fluids. Comparisons were made of plants containing symbiotic algae and grown on nitrogen-free media, plants grown on media containing nitrate, and algae-free plants also grown on nitrate media. The major feature of the data was a very high level of intracellular ammonia found in plants which contain N(2)-fixing algal symbionts. In addition to the more usual amino acids, serine and cystathionine were found in the free amino acid pool. PMID- 16659771 TI - Photoperiodic control of apical senescence in a genetic line of peas. AB - An early flowering genetic line of peas (Pisum sativum L.), designated G2, has dominant genes at two different loci, both of which function in short days to greatly extend the reproductive phase and thus to delay apical senescence. Long days (18 hours) promote senescence in this line, but the effect is reversible by reinstatement of short days (9 hours) until 3 to 4 days before the apex senesces. The response to photoperiod was quantitative. Increasing the photoperiod from 14 to 18 hours led to a progressive decrease in the number of nodes formed prior to death of the apex. Induction of senescence was determined by the total number of hours of light and darkness rather than by the length of the dark period. Senescence required flower and fruit development as well as long days. PMID- 16659772 TI - Photoaffinity-labeled Cytokinins: Synthesis and Biological Activity. AB - Two new azidopurine derivatives, 2-azido-N(6)-(Delta(2)-isopentenyl)adenine and 2 azido-N(6)-benzyladenine, have been synthesized as potential photoaffinity labels for probing cytokinin-binding sites. The preparation and the biological activity of these compounds are described. PMID- 16659773 TI - Plant Pyruvate Dehydrogenase Complex: II. ATP-Dependent Inactivation and Phosphorylation. AB - ATP inactivated plant pyruvate dehydrogenase complex (PDC) from broccoli (Brassica oleracea) mitochondria. ATP inactivation of the complex was time dependent and proportional to the ATP concentration. Time-dependent incorporation of (32)P from [gamma(32)P]ATP into trichloroacetic acid-precipitable protein corresponded to the inactivation of the PDC. It is concluded that plant PDC is phosphorylated and inactivated by a PDC kinase. PMID- 16659774 TI - Leaf area partitioning as an important factor in growth. AB - Despite continuing efforts to correlate unit area rates of photosynthesis of crop varieties with growth rates, there has been little or no success. It is reasonable to assume that partitioning of photosynthate into new leaf area is an important component of growth. Accordingly, an expression was developed to measure leaf area partitioning. Using growth analysis techniques, relative growth rates were compared to net assimilation rates, partitioning of daily weight gain into new leaf area, and partitioning of daily weight gain into new leaf weight of nine species grown in growth chambers under three temperature regimes. Day/night temperatures of 21/10, 32/21, and 38/27 C caused large differences in relative growth rates. Relative growth rates were closely correlated with leaf area partitioning in seven of the nine species, but were inversely correlated with leaf weight partitioning for six of the nine species. Relative growth rates were poorly correlated with net assimilation rates for five of the nine species. The product of net assimilation rate times leaf area partitioning is shown to be equal to the relative leaf area expansion rate.These results indicate that growth responses due to temperature shifts were more sensitive to changes in leaf area partitioning or relative leaf area expansion rates than to net assimilation rates. Because changes in leaf area partitioning or relative leaf area expansion rates can have an effect on relative growth rates that overshadow changes in net assimilation rates, and because net assimilation rates are largely a function of unit area rates of photosynthesis, the correlation of unit area rates of photosynthesis with growth should include consideration of leaf area partitioning or relative leaf area expansion rates. PMID- 16659775 TI - Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots: II. Sensitivity of Development of the Augmented Uptake Potential to Toxin and Inhibitors of Protein Synthesis. AB - Basal K(+) uptake in the root midzone region (cm 2 + 3 + 4) of N and T cytoplasmic versions of each of four maize inbreds was equally sensitive to the toxin(s) of Helminthosporium maydis, race T. Basal K(+) uptake in the root apex (0-1 cm) and augmented K(+) uptake in the root midzone were more toxin-sensitive in inbreds W64A(T) and Mo17(T) than in inbreds W64A(N) and Mo17(N). This differential response of N and T cytoplasms to toxins was not found for corresponding cytoplasms of inbreds WF9 and B37.Development of the augmented K(+) uptake rate in midzone segments of W64A(T) was blocked by a toxin concentration which did not affect augmentation development in W64A(N). Augmentation development was more toxin-sensitive in T than in N cytoplasmic versions of all inbreds tested. Fertility-restoring nuclear loci decreased but did not eliminate the toxin sensitivity of augmentation developments as observed in root midzones of inbred A619(T). Chloramphenicol-and/or cycloheximide-sensitive protein synthesis was required for augmentation development, but not for expression of either basal or augmented K(+) uptake. PMID- 16659776 TI - Heavy metal impurities impair the spectrophotometric assay of ribulose bisphosphate carboxylase activity. AB - An inverse relationship between the concentration of ribose 5-phosphate and apparent ribulose bisphosphate carboxylase activity was observed. The Lilley Walker assay spectrophotometric assay, in which the 3-phosphoglyceric acid dependent oxidation of reduced pyridine nucleotide is measured, is shown to be highly sensitive to inhibition by heavy metals. Analysis of the purity of reagents showed that ribose 5-phosphate is often contaminated with lead in sufficient quantity to impair the assay. This noncompetitive inhibition by ribose 5-phosphate is independent of the competitive inhibition of this substrate as an ATP sink as described by Slabas and Walker. A method for checking reagent purity and removing heavy metal contaminants is described. PMID- 16659777 TI - Ethylene Production by Albedo Tissue of Satsuma Mandarin (Citrus unshiu Marc.) Fruit. AB - Isolated albedo tissue of Satsuma mandarin (Citrus unshiu Marcovitch, cv. Owari) fruit produced a large quantity of ethylene during incubation at 26 C in the dark. When sliced, albedo tissue began producing ethylene at an increasing rate until a maximum was reached after incubation for about 30 hours. Aged albedo discs which were capable of producing ethylene, actively converted l-[U (14)C]methionine into both ethylene and carbon dioxide. In fresh tissue, almost no measurable conversion of radioactive methionine into ethylene took place. Conversion of labeled l-methionine into ethylene was totally inhibited by the addition of nonradioactive l-methionine or l-ethionine. It appears possible, from these findings, that methionine is a precursor of ethylene in the aged albedo discs. Ethylene synthesis in the aged albedo tissue was markedly reduced in the presence of cycloheximide, suggesting that there may be a rapid turnover of the ethylene-producing system, and that its formation involves protein synthesis. Actinomycin D exerted no effect. PMID- 16659778 TI - Variations in photosynthetic rates of fourteen coleus cultivars. AB - Fourteen cultivars of coleus (Coleus blumei Benth) were found to vary in chlorophyll content, photosynthetic rate, anthocyanin content and number of stomates.Cultivars ;Gaslight' and ;Velvet Rainbow' had 0.17 and 0.92 mg chlorophyll per g of leaves, fresh weight, respectively.Maximum photosynthetic rate (18.7 mg CO(2)/mg Chl.hr) was found in the leaves of ;Pastle Rainbow' and minimum (2.7) in the ;Velvet Rainbow.' PMID- 16659779 TI - Dihydroxyacetone Kinase Activity in Dunaliella parva. AB - An enzyme catalyzing the phosphorylation of dihydroxyacetone has been identified in the halophilic alga, Dunaliella parva. Since glycerol and glyceraldehyde are not substrates, the enzyme is referred to as dihydroxyacetone kinase. Dihydroxyacetone kinase was purified 9-fold by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. PMID- 16659780 TI - Effect of Glucose and CO(2) on Nitrate Uptake and Coupled OH Flux in Ankistrodesmus braunii. AB - In Ankistrodesmus braunii, in the absence of CO(2), i.e. in CO(2)-free air or N(2), photosynthetic nitrate uptake and nitrate reduction were inhibited, especially at low pH. Under such conditions, glucose stimulated nitrate uptake and reduction to almost the same level in the pH range between 6 and 8.5. CO(2) at 0.03% effected an intermediate pH dependence of nitrate uptake; saturating CO(2) concentration (more than 1%) eliminated the pH dependence, as did glucose, but the rates were enhanced compared with glucose. Glucose and, even more, CO(2), drastically reduced the release of nitrite and ammonia to the medium, the stoichiometry between alkalinization of the medium and nitrate uptake (OH( )/NO(3) (-)) approached 1.Due to the lack of storage vacuoles in Ankistrodesmus, nitrate uptake and nitrate reduction were closely coupled processes whose experimental separation is difficult. The relieving effect of glucose and CO(2) suggests a carrier-mediated nitrate uptake which is more limiting than nitrate reduction and is sensitive to low pH, but which is stabilized by some intermediate originating from an active carbon metabolism. PMID- 16659781 TI - Properties of Kaurene Synthetase from Marah macrocarpus. AB - The kaurene synthetase from immature seeds of Marah macrocarpus (Greene) Greene was partially purified from cell-free homogenates of endosperm by a combination of QAE-Sephadex A-25 chromatography and hydroxyapatite chromatography and freed of contaminating phosphatase activity. The two catalytic activities associated with kaurene synthetase, the cyclization of geranylgeranyl-pyrophosphate to copalyl-pyrophosphate (activity A) and the cyclization of copalyl-pyrophosphate to ent-kaurene (activity B), were not even partially resolved from one another during these procedures. Both activities had identical elution profiles from a calibrated Sepharose 4B column corresponding to a molecular weight less than that of ovalbumin (45,000).The A and B activities had pH optima of 7.3 and 6.9, respectively. Both activities required millimolar concentrations of the following divalent cations in the order: Mg(2+) > Mn(2+) > Co(2+). Activities A and B were both sensitive to inhibition by Hg(2+), Cu(2+), p-hydroxymercuribenzoate, and N ethylmaleimide, but activity B was much more sensitive than activity A. The average value of Km' (apparent Km in the absence of substrate inhibition) for geranylgeranyl-pyrophosphate was 1.6 mum. Values of 0.5 and 0.6 mum were obtained for Km' and Km, respectively, for copalyl-pyrophosphate. The Vm' values for the two activities were similar: 12 and 9 pmol/minute.mug protein for activities A and B, respectively.N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and N,N-dimethylaminoethyl-2,2-diphenylphentyl ether (SKF-3301A), tributyl-2,4 dichlorobenzylphosphonium chloride (Phosfon D), tributyl-2,4 dichlorobenzylammonium chloride (Phosfon S), 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618), 2-(N,N-dimethyl-N heptylammonium bromide)-p-methan-1-ol (Q-58), and 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol (Q-64), at concentrations from 1 to 5 mum, were effective inhibitors of kaurene synthetase activity A. Acetylcholine chloride and 2 chloroethyl-trimethylammonium chloride were effective inhibitors of activity A only at concentrations of 5 mm or greater. Abscisic acid, indole-3-acetate, gibberellin A(1), gibberellin A(3), a mixture of gibberellins A(4) and A(7), gibberellin A(13), and N,N-dimethylaminosuccinamic acid (B995) were not inhibitory at any of the levels tested. None of these compounds was an effective inhibitor of activity B at concentrations less than 0.5 mm. PMID- 16659782 TI - Studies on Sequential Parasitism by Orobanche and Cuscuta on Petunia hybrida: Choline Kinase and Phospholipid. AB - Parasitism by Cuscuta and Orobanche on Petunia hybrida resulted in decreased choline kinase activity and phospholipids in the host shoots. The Cuscuta infected host roots suffered a decline in phospholipid concentration with no appreciable change in enzyme activity, whereas the roots of the Orobanche infected plants exhibited a substantial increase in phospholipid concentration despite a marked lowering in enzymic activity. Superimposition of infection by Cuscuta on Orobanche-infected plants resulted in an increase in both enzyme activity and phospholipid in host shoots; the host roots recorded a decline in phospholipid, although enzyme activity was increased. As compared to the filaments infecting singly, Cuscuta, in sequential infection, registered an increase in phospholipid concomitant with a fall in enzyme activity, whereas the root parasite revealed a lowered enzyme activity and a slight decrease in phospholipid. It is hypothesized that a physiological response to infection by root parasite was an accumulation of phospholipids at the region under infection, and to that by shoot parasite was an uptake of phospholipids by the parasite from the host; this was effected not by de novo synthesis but rather by mobilization from distal regions. PMID- 16659783 TI - Photophosphorylation Associated with Photosystem II: I. Photosystem II Cyclic Photophosphorylation Catalyzed by p-Phenylenediamine. AB - Incubation of spinach chloroplast membranes for 90 minutes in the presence of 50 mm KCN and 100 mum HgCl(2) produces an inhibition of photosystem I activity which is stable to washing and to storage of the chloroplasts at -70 C. Subsequent exposure of these preparations to NH(2)OH and ethylenediaminetetraacetic acid destroys O(2) evolution and flow of electrons from water to oxidized p phenylenediamine, but two types of phosphorylating cyclic electron flow can still be observed. In the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazinemethosulfate catalyzes ATP synthesis at a rate 60% that observed in uninhibited chloroplasts. C-Substituted p-phenylenediamines will also support low rates of photosystem I-catalyzed cyclic photophosphorylation, but p phenylenediamine is completely inactive. When photosystem II is not inhibited, p phenylenediamine will catalyze ATP synthesis at rates up to 90 mumol/hr.mg chlorophyll. This reaction is unaffected by anaerobiosis, and an action spectrum for ATP synthesis shows a peak at 640 nm. These results are interpreted as evidence for the existence of photosystem II-dependent cyclic photophosphorylation in these chloroplast preparations. PMID- 16659784 TI - Ribulose bisphosphate carboxylase: altered genetic expression in tall fescue. AB - A decaploid tall fescue (Festuca arundinacea Schreb) genotype has been found which exhibits net photosynthetic rates of 32 to 41 mg CO(2)/dm(2).hour as opposed to a mean of 22 mg CO(2)/dm(2).hour for 10 hexaploid genotypes. The decaploid genotype exhibited a ribulose 1,5-bisphosphate (RuBP) carboxylase specific activity 1.3- to 2-fold higher than typical tall fescue genotypes. Specific activities of photorespiratory enzymes and nitrate reduction enzymes were lower in the decaploid than the hexaploid genotypes. Results suggest that genetic expression of RuBP carboxylase activity may have been altered to increase the net photosynthesis rate in the decaploid genotype. PMID- 16659785 TI - Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants. AB - The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of (14)C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of (14)C-leucine into trichloroacetic acid precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented. PMID- 16659786 TI - Comparative growth analyses of panicum species with differing rates of photorespiration. AB - Panicum milioides, a naturally occurring species with reduced photorespiration, P. bisulcatum, a C(3) species, and P. miliaceum, a C(4) species, were grown for 4 weeks at altered pO(2) and pCO(2) and several vegetative growth parameters were determined at weekly intervals. Compared to a pO(2) of 10%, a greater O(2) inhibition of the relative growth rate and dry matter production was observed for P. bisulcatum than for P. milioides at both 21% and 40% O(2), whereas little effect of O(2) was noted for P. miliaceum. Similarly, exposures to elevated pCO(2) of 500 and 1000 mu1 CO(2)/liter resulted in a greater stimulation of vegetative growth for P. bisulcatum than for P. milioides, with little effect on P. miliaceum. The CO(2) compensation concentration of P. milioides was less than that of P. bisulcatum over a pO(2) range of 5 to 40%. At 5% O(2), the compensation concentration was relatively O(2)-insensitive, whereas above 5% it increased with increasing pO(2). It is concluded that P. milioides represents the first well documented example of a C(3) plant with reduced photorespiration, based on both leaf CO(2) exchange parameters and growth analyses of dry matter production. PMID- 16659787 TI - Two effects of cytokinin on the auxin requirement of tobacco callus cultures. AB - Cytokinin affects the requirement for auxin of a strain of tobacco callus (Nicotiana tabacum) which is cytokinin-autotrophic when grown on Murashige and Skoog medium with 11.4 mum of indole-3-acetic acid but requires cytokinin 6-(3 methyl-2-butenylamino)purine (i(6) Ade) when grown on the same medium with <3 mum indole-3-acetic acid. As the exogenous concentration of cytokinin (i(6) Ade) is increased, the concentration of indole-3-acetic acid required for growth is decreased. A second effect of cytokinin, observed sporadically in cultures with 2.5 mum or 5 mum i(6) Ade, is the transformation of some of the callus pieces to auxin-autotrophic growth. Strains, both callus-forming and bud-forming tissues, that arise in this manner are not permanently altered in their auxin requirement because subcultures on medium without cytokinin still require exogenous auxin. PMID- 16659788 TI - Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum). AB - Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-(14)C]oleic acid and [1-(14)C]acetate as precursors. In 4-day aged tissue, [1-(14)C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H(4)), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that (14)C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO(4) treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1 (14)C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that (14)C was contained in alkanols and alkane-alpha,omega-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C(20), which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C(16), C(18), C(20), C(22), and C(24)-alpha,omega-diols. Soluble lipids derived from [1-(14)C]acetate in the aged tissue also contained labeled very long acids from C(20) to C(28), as well as C(22) and C(24) alcohols, but no labeled omega-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C(21) and C(23) were the major components with lesser amounts of C(19) and C(25). The amount of (14)C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-(14)C]oleic acid, respectively. Incorporation of [1-(14)C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-(14)C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the alpha-omega-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization. PMID- 16659789 TI - Properties of a Protein Activator of NAD Kinase from Plants. AB - Purification of pea (Pisum sativum) seedling NAD kinase by DEAE-cellulose column chromatography resulted in loss of activity, due to dissociation of an activator from the enzyme. The purified enzyme preparation, which was almost completely inactive, regained the activity when the activator was added back.The activator was purified 320-fold by ion exchange chromatographies. The activator was susceptible to proteolytic enzymes, but not to ribonuclease, glucoamylase or pectinase, indicating that it is of a protein nature. This protein was relatively stable in boiling water, but susceptible to acid or alkali, especially under high temperatures. Restoration of catalytic activity of inactive enzyme was proportional to amounts of the activator added. Gel filtration indicated that molecular weight of the activator was 28,000.The activator was found in extracts from various plants. PMID- 16659790 TI - Roles of Extensibility and Turgor in Gibberellin- and Dark-stimulated Growth. AB - The elongation response elicited by incubating excised hypocotyl sections of lettuce (Lactuca sativa L.) in light in gibberellin (GA) can be enhanced by the addition of Cl(-), Br(-), and NO(3) (-) salts of K(+) and Na(+). Sections incubated in light in the absence of GA do not elongate in response to the addition of salts. In contrast, excised hypocotyls incubated in darkness elongate equally in both GA and water, and their elongation can also be enhanced by KCl treatment. Growth stimulation by the salts of K(+) and Na(+) occurs optimally at 10 mm and the magnitude of the response is proportional to the duration of salt treatment. Although the growth of sections incubated in light in the absence of GA is not enhanced by various salts of K(+) and Na(+), the concentration of these cations exceeds that in GA-treated sections. In dark-grown tissue, uptake of K(+) also occurs in both GA- and H(2)O-treated sections incubated in 10 mm KCl. Since increased osmotic potential resulting from cation uptake does not correlate with growth stimulation resulting from salt treatments, we conclude that increased cell turgor is not the principal driving force for growth in hypocotyl sections. Changes in the extensibility of GA-treated, light-grown tissue and dark-grown tissue incubated with and without GA correlate with the increased growth rate of these sections. Incubation of sections in KCl results only in changes in water potential of sections without having a significant effect on extensibility. When changes in water potential are accompanied by increased extensibility, however, a marked increase in growth rate is observed. PMID- 16659791 TI - Properties and Regulation of Aspartate Kinase from Barley Seedlings (Hordeum vulgare L.). AB - Aspartate kinase (EC 2.7.2.4) has been purified 8-fold and characterized from germinating barley (Hordeum vulgare) seedlings. The enzyme is inhibited 50% by 0.7 mm l-lysine and almost completely at 5 mm. l-Methionine does not affect the enzyme on its own, but at low concentrations (0.1-1 mm) increases the inhibition in the presence of lysine, indicating that the two amino acids act as cooperative feedback regulators. PMID- 16659792 TI - Diurnal variation in n(2) fixation and photosynthesis by aquatic blue-green algae. AB - Rates of (14)CO(2) fixation, O(2) evolution, and N(2) fixation (acetylene reduction) by natural populations of blue-green algae recovered from Lake Mendota were measured at frequent intervals between sunrise and sunset. Photosynthesis and N(2) fixation were depressed during midday when light intensity was greatest. As the light intensity rose, most of the algal population migrated to deeper, light-limited waters where radiation damage would be diminished. As the relative rate of N(2) fixation compared to CO(2) fixation increases with depth, it is suggested that the algae maintain balanced growth by migrating vertically via buoyancy regulation. High concentrations of dissolved O(2) in lake water may inhibit N(2) fixation by enhancing photorespiration. Several factors such as photosynthetic rate, light intensity, dissolved O(2), species composition, and vertical and horizontal migration all affect observed rates of in situ N(2) fixation. PMID- 16659793 TI - Influence of carbohydrates on quantitative aspects of growth and embryo formation in wild carrot suspension cultures. AB - Wild carrot (Daucus carota L.) cell suspensions were grown on a mineral salt medium supplemented with 10 mmmyoinositol in the presence and absence of 2.25 mum 2,4-dichlorophenoxyacetic acid (2,4-D), and a variety of carbon sources.The data obtained on growth and embryo number in the absence of 2,4-D show that wild carrot suspensions were able to utilize sucrose, glucose, fructose, galactose, mannose, maltose, raffinose, or stachyose as a carbon source. A highly significant correlation between dry weight and embryo number was obtained regardless of the carbohydrate source suggesting the involvement of a common intermediate in the metabolism of the various sugars.In the presence of 2.25 mum 2,4-D, embryo formation was suppressed. Time course of dry weights obtained in the presence and absence of 2,4-D show that 2,4-D increased the growth rate of the tissue when glucose, fructose, mannose, or stachyose was used as the carbon source. The growth rates on other sugars remained unchanged under these conditions. PMID- 16659794 TI - Quantum Yields for CO(2) Uptake in C(3) and C(4) Plants: Dependence on Temperature, CO(2), and O(2) Concentration. AB - The quantum yields of C(3) and C(4) plants from a number of genera and families as well as from ecologically diverse habitats were measured in normal air of 21% O(2) and in 2% O(2). At 30 C, the quantum yields of C(3) plants averaged 0.0524 +/- 0.0014 mol CO(2)/absorbed einstein and 0.0733 +/- 0.0008 mol CO(2)/absorbed einstein under 21 and 2% O(2). At 30 C, the quantum yields of C(4) plants averaged 0.0534 +/- 0.0009 mol CO(2)/absorbed einstein and 0.0538 +/- 0.0011 mol CO(2)/absorbed einstein under 21 and 2% O(2). At 21% O(2), the quantum yield of a C(3) plant is shown to be strongly dependent on both the intercellular CO(2) concentration and leaf temperature. The quantum yield of a C(4) plant, which is independent of the intercellular CO(2) concentration, is shown to be independent of leaf temperature over the ranges measured. The changes in the quantum yields of C(3) plants are due to changes in the O(2) inhibition. The evolutionary significance of the CO(2) dependence of the quantum yield in C(3) plants and the ecological significance of the temperature effects on the quantum yields of C(3) and C(4) plants are discussed. PMID- 16659795 TI - Metabolism of Indole-3-acetic Acid: IV. Biological Properties of Amino Acid Conjugates. AB - The biological activity of 20 l-alpha-amino acid conjugates of indole-3-acetic acid (IAA) to stimulate cell elongation of Avena sativa coleoptile sections and to stimulate growth of soybean cotyledon tissue cultures has been examined at concentrations of 10(-4) to 10(-7)m. In the Avena coleoptile test, most of the amino acid conjugates stimulated elongation. Several of the conjugates stimulated as much elongation as IAA but their half-maximum concentrations tended to be higher. Some of the more active conjugates were alanine, glycine, lysine, serine, aspartic acid, cystine, cysteine, methionine, and glutamic acid.In the soybean cotyledon tissue culture test, all of the l-alpha-amino acid conjugates of IAA stimulated growth except for the phenylalanine, histidine, and arginine conjugates. Most of the conjugates produced responses at least as great as that caused by IAA. Conjugates with half-maximum concentrations lower than IAA included cysteine, cystine, methionine, and alanine. These conjugates exceed the IAA-induced callus growth at all tested concentrations. Other conjugates significantly better than IAA at 10(-6)m were serine, glycine, leucine, proline, and threonine. PMID- 16659796 TI - Photochemical dimerization of ferulic Acid by chloroplasts from sorghum. AB - Sorghum (Sorghum bicolor) chloroplasts, lamellar fragments, and Triton X-100 solubilized preparations catalyze a blue and red light-sensitized oxidation of ferulic acid to its beta-beta-linked dimer and its hydrolysis product, the acid dimer. Exogenous superoxide dismutase had no effect, and catalase and 1 to 10 mm KCN inhibited this photooxidative dimerization only in detergent-treated chloroplasts. It is postulated that the final oxidant is H(2)O(2), formed by light-induced photosystem I electron transport, followed by an unidentified peroxidatic activity. The reaction differs, however, from that catalyzed by horseradish peroxidase in the presence of H(2)O(2). PMID- 16659797 TI - Root hair cell enhancement in tissue cultures from soybean roots: a useful model system: in vitro Rhizobium symbiosis. AB - A technique for obtaining large numbers of root hair cells in cell cultures from soybeans is described. The cells were grown on agar containing the Prairie Regional Laboratory B5 (PRL-B5) medium for periods longer than 60 days. Mixed populations of cultured root hair cells and cortical cells were used to study the in vitro association between soybean cells and Rhizobium japonicum. The advantages of these types of root cell cultures in studies of symbiosis are discussed. PMID- 16659798 TI - Dormancy and Impotency of Cocklebur Seeds: IV. Effects of Gibberellic Acid, Benzyladenine, Thiourea, and Potassium Nitrate on the Growth of Embryonic Axis and Cotyledon Segments. AB - Germination of nondormant but impotent small cocklebur seeds (Xanthium pennsylvanicum Wallr.) was promoted profoundly with thiourea or benzyladenine, and slightly with gibberellic acid. Gibberellic acid was ineffective in causing the germination of dormant cocklebur seeds, although thiourea and benzyladenine were effective. Experiments with excised seed pieces showed that the promotive effects of thiourea, benzyladenine, and gibberellic acid on cocklebur seed germination were associated with the enhancement of growth of seed parts; thiourea stimulated predominantly the axial growth, whereas benzyladenine stimulated predominantly the cotyledonary growth.Potassium nitrate or indoleacetic acid had little effect on the initial growth of either axes or cotyledons. Except for gibberellic acid, all of the compounds employed enhanced ethylene production, but in general, the ethylene production seemed more likely to be a consequence of growth rather than a cause of it. We concluded that the chemical regulation of seed germination may be a consequence of the alteration of growth capabilities in either the axes or cotyledons, or both. PMID- 16659799 TI - Dormancy and Impotency of Cocklebur Seeds: VII. Inability of Dormant Cotyledons to Form Chlorophyll. AB - Dormant seeds of cocklebur (Xanthium pennsylvanicum Wallr.) were characterized by the lack of ability to form chlorophyll. Such an inability of cotyledons of the dormant seeds was improved by the application of various factors and reagents which were capable of breaking the dormancy and of increasing cotyledon enlargement. Of these, ethylene, benzyladenine, and high temperature treatments were particularly effective, and, in turn, oxygen enrichment, gibberellic acid, thiourea, carbon doxide, and potassium nitrate were also promotive to the greening of the dormant cotyledons. The effects of benzyladenine, oxygen enrichment, and high temperature were reduced in the presence of absorbents for endogenously evolve carbon dioxide and ethylene. delta-Aminolevulinic acid could not restore their greening ability. PMID- 16659800 TI - Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria. AB - Phosphoenolpyruvate partially inhibits the accumulation of Ca(2+) in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca(2+) uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca(2+) uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca(2+) uptake completely while partially inhibiting NADH-supported Ca(2+) uptake. The oxaloacetate inhibition of NADH-supported Ca(2+) uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca(2+) uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca(2+) uptake. PMID- 16659801 TI - Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds. AB - The tannins chebulinic acid or tara tannin were added to an incubation system in which GA(3) induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA(3) alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA(3), the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA(3) was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA(3) in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA(3). PMID- 16659802 TI - Some properties of 3-phosphoglycerate phosphatase from developing rice grain. AB - Some properties of 3-P-glycerate phosphatase from developing caryopsis of rice (Oryza sativa L., variety IR26) were studied. The enzyme was found to be soluble and not bound to starch, and concentrated mainly in the pericarp-aleurone layer; its maximum activity was at 12 to 14 days after flowering. Contents of 3-P glycerate and chlorophyll were highest in the grain at 7 to 8 days after flowering when starch synthesis was at a maximum. The enzyme was purified about 100-fold by precipitation with 50 to 80% ammonium sulfate, followed by chromatography through Sephadex G-200 and CM-Sephadex C-50. The pH optimum was from 5.7 to 6 and no cation was required for activity. The purified preparation had an apparent Km of 2.85 mm and was inhibited by Cu(2+), Hg(2+), Zn(2+), Fe(3+), molybdate, and F(-). The enzyme also exhibited high activity toward UTP, ATP, and p-nitrophenyl phosphate; moderate activity toward other phosphates; but no activity toward phytate. A molecular weight of about 23,000 was obtained for the 3-P-glycerate peak during gel filtration on Sephadex G-200, which corresponded to a value of 26,000 for the major protein fraction by thin layer gel filtration on Sephadex G-150. Zymograms of the whole extract and semipurified preparations showed two phosphatase bands with 3-P-glycerate as substrate. PMID- 16659803 TI - Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria. AB - A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O(2) uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K(+) permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V(max), probably reflecting a diffusion-limited process.The results indicate that rapid malate and succinate oxidation require the operation of both the phosphate and dicarboxylate transporters, which in turn depend on maintenance of a proton motive force across the inner membrane. In addition, phosphate can stimulate acceptorless malate oxidation by reaction with the coupling mechanism, and in uncoupled mitochondria which are depleted of substrate there is a slow rate of oxidation which appears to be limited by diffusive entry. PMID- 16659804 TI - Plant Cuticles Are Polyelectrolytes with Isoelectric Points around Three. AB - The isoelectric points of isolated cuticles from Citrus aurantium L. (3.15), Prunus armeniaca L. (3.45), and Pyrus communis L. (2.90) leaves were determined from membrane potentials. At pH values below the isoelectric point, cuticular membranes carry a net positive charge and are permselective to anions (determined using (82)Br(-)). Above the isoelectric point, they carry a net negative charge and are permselective to cations (determined using (24)Na(+)). There are no gradients of fixed charges across the cuticular membranes as indicated by the absence of asymmetry potentials. Positive charges in the membranes originate from residues of basic amino acids of proteins or polypeptides contained in a nonextractable form within the cuticle. The exchange capacity of basic fixed groups in the cuticles of six species (Lycopersicon esculentum Mill., Capsicum annuum L. fruit cuticles, and Brassaia spec. leaf cuticles in addition to the above species) varied between 0.010 and 0.025 meq g(-1) cuticle. Fixed acidic groups were donated by residues of acidic amino acids, polygalacturonic acid, and nonesterified -COOH groups of the cutin polymer. At pH 8, total cation exchange capacity as determined using (45)Ca(2+) varied between 0.26 (Citrus) and 0.30 (apricot) meq g(-1). PMID- 16659805 TI - Effect of light quality on the organization of photosynthetic electron transport chain of pea seedlings. AB - The activity of NADP and O(2) photoreduction by water is essentially higher in chloroplasts isolated from pea seedlings (Pisum sativum L.) grown under blue light as compared with that from plants grown under red light. In contrast, the photoreduction of NADP and O(2) with photosystem I only is practically the same or even lower in chloroplasts isolated from plants grown under blue light. The addition of plastocyanin does not affect the rate or the extent of NADP photoreduction by water in the chloroplasts isolated from plants grown under blue light, whereas it sharply activates NADP reduction in the chloroplasts isolated from plants grown under red light. The extent of the light-induced oxidation of cytochrome f is appreciably higher in chloroplasts isolated from plants grown under blue light. Cytochrome b(559) plays the predominant role in the oxidoreductive reactions of these chloroplasts. Furthermore, the fluorescence measurements indicate more effective transfer of excitation energy from chlorophyll to the photosystem II reaction center in chloroplasts isolated from plants grown under blue light.The data reported are interpreted in terms of differential rates of electron transport from water in the two chloroplast preparations. PMID- 16659806 TI - Differences between Adenosine Triphosphatases from Monocotylous and Dicotylous Plants. AB - The mitochondrial membrane-bound ATPases of several plants were investigated. Two distinct types were encountered. The mitochondrial ATPases of castor bean (Ricinus communis var. Zanzibarensis, L.), cauliflower (Brassica oleracea, L.), and scarlet runner (Phaseolus coccineus, L.) were found to be inhibited by oligomycin, to have elevated molecular weights when separated from the organelles by ultrasonication and ammonium sulfate treatment and, subsequent to purification, to be cold-labile. On the other hand, mitochondria isolated from wheat (Triticum aestivum, L.), maize (Zea mays, L.), calla (Zantedeschia aethiopica, Spreng.), and onions (Allium cepa, L.) contain ATPases which, after ultrasonication of the organelles, were virtually insensitive to oligomycin and those molecular weights were as low as about 45,000; in the purified form they were resistant to storage in the cold. The plants whose mitochondria were of the first type, characterized by having ATPases similar to those of the mitochondria of animal tissues and bakers' yeast, belonged to the dicotyledons, whereas the mitochondria of the other type were found in monocotyledonous plants. PMID- 16659807 TI - Inhibitory effect of a rhizobitoxine analog on bud growth after release from dormancy. AB - Application of the ethoxy analog of rhizobitoxine (l-2-amino-4-[2'-aminoethoxy] trans-3-butenoic acid), an inhibitor of ethylene biosynthesis, inhibited growth of apple, crabapple, and apricot buds released from dormancy by chilling or by treatment with benzyladenine. When tea crabapple (Malus hupehensis [Pamp.] Rehd.) buds were sprayed once with 8.8 x 10(-3)m benzyladenine, ethylene production by the buds increased significantly 24 to 48 hours after benzyladenine treatment. Application of the rhizobitoxine analog to the buds at the time of benzyladenine treatment reduced ethylene evolution to the level of the controls for up to 2 weeks after treatment. Increase in bud weight was inhibited also but to a lesser extent. These data suggest that growth of buds is accompanied by ethylene production and that the inhibition of ethylene biosynthesis also inhibits bud growth. Since additional metabolic effects result from the action of the rhizobitoxine analog, no firm conclusions on its role can be drawn at this time. PMID- 16659808 TI - Presence of Polypeptides of Cytoplasmic and Chloroplastic Origin in Isolated Photoactive Preparations of Photosystems I and II in Chlamydomonas reinhardi y-1. AB - Photoactive preparations were obtained by treatment of photosynthetic membranes with lipid micelles and deoxycholate.A photosystem I preparation can be extracted from the membranes by both lipid micelles and deoxycholate, whereas the photosystem II preparation remains in a particulate state.The photosystem II preparation exhibits electron transfer activity measured by photoreduction of 2,6 dichlorophenolindophenol with 1,5-diphenylcarbazide as an electron donor and has a ratio of chlorophyll a/b of 1.4. This preparation contains three major polypeptides of cytoplasmic origin and two of chloroplastic origin.The photosystem I preparation exhibits high rates of methylviologen photoreduction with ascorbate and 2,6-dichlorophenolindophenol as electron donors and does not contain chlorophyll b. This preparation contains two major polypeptides of chloroplastic origin and one of cytoplasmic origin. These results indicate that both cytoplasm and chloroplast contribute polypeptides for the formation of photosystems I and II in Chlamydomonas reinhardi. PMID- 16659809 TI - Preparation of Antibodies against Specific Chloroplast Membrane Polypeptides Associated with the Formation of Photosystems I and II in Chlamydomonas reinhardi y-1. AB - The major Chlamydomonas reinhardi y-1 chloroplast membrane polypeptides-I + II, IV, V(a + b)-have been isolated by use of preparative sodium dodecyl sulfate-gel electrophoresis.Rabbit antisera prepared against these polypeptides interact with intact membranes as well as membrane fractions containing these specific antigens. Antisera against polypeptides I + II partially inactivate photosystem I. Antisera against polypeptide IV prevent inactivation of the Hill reaction and inhibit photooxidation of diphenylcarbazide with dichlorophenolindophenol as an electron acceptor. The protection of the Hill reaction by IV antiserum, measured with whole membranes, is partially hindered by the presence of V antiserum, which, by itself, causes inhibition of diphenylcarbazide photooxidation.We concluded that parts of the primary structure of these polypeptides are exposed in situ and can be recognized in whole membranes by antisera prepared against sodium dodecyl sulfate-denatured antigens. Polypeptides I and II, and polypeptides IV, Va, and Vb are associated with photosystem I and photosystem II, respectively. PMID- 16659810 TI - Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: IV. Effect of Abscisic Acid and Water Content on RNase Activity and RNA. AB - Plants of the wilty tomato (Lycopersicum esculentum) mutant, flacca, and of the normal cultivar Rheinlands Ruhm growing under either "normal" or high humidity were used in this research. Under normal humidity, RNase activity was much higher in mutant plants in which abscisic acid (ABA) and water content were lower than in the normal plant. The mutant also contained less RNA and protein per cell and less soluble RNA relative to ribosomal RNA as compared with the normal genotype. In ABA-treated mutant plants, RNase activity decreased while RNA, protein, the ratio of soluble to ribosomal RNA and water content increased.Under high humidity, RNase activity in mutant plants was decreased, but was still somewhat higher than that in the normal plant, although water saturation deficit was equal in both plant types. Abscisic acid increased RNase activity in the mutant plants. The content of RNA and protein per cell was similar in both types, but the ratio of soluble to ribosomal RNA remained lower in the mutant. In ABA-treated mutant plants, although the content of DNA and RNA per fresh weight was similar to that of control mutant plants, the ratio of RNA to DNA decreased significantly. In addition, ABA caused an increase of the soluble to ribosomal RNA ratio toward the normal value in mutant plants.Contrary to ABA, kinetin increased RNase activity in the mutant under normal humidity and decreased it under high humidity.A similar incorporation of labeled uridine into RNA in normal, mutant, and ABA treated mutant plants under normal humidity suggests that the difference between mutant and normal plants in respect to total, soluble, and ribosomal RNA results not from a different rate of RNA synthesis but from a different rate of RNA degradation, i.e. RNase activity. PMID- 16659811 TI - Mechanism of cyanide inhibition of Phloem translocation. AB - Petiolar application of potassium cyanide inhibited (14)C-assimilate translocation without affecting source leaf photosynthesis or phloem loading of sucrose in Phaseolus vulgaris. The inhibition of transport was correlated with disruption of the structural integrity of the sieve tubes (sieve pore blockage) rather than impairment of a metabolic process in the translocation path driving translocation. PMID- 16659812 TI - Effect of Colored Light on Stomatal Opening Rates of Vicia faba L. AB - The average opening rate of Vicia faba L. stomata was determined over an initial 20-minute light period following darkness. Nonsaturating intensities of broad band red and blue light had similar quantum effectiveness for the promotion of opening, whereas broad band green was about 40% and far red about 5% as effective. The opening rates under saturating red, green, and blue light were the same. Net photosynthesis was measured under various intensities of the same red, green, and blue light spectra. Red and blue light were equally efficient in causing photosynthesis, whereas green was 60% as effective. The light compensation points for the three colors were at higher intensities than those which saturated the opening rate response. These data suggest that only a single pigment system, probably the photosynthetic pigments, is responsible for initiating the light-induced opening response in V. faba stomata. PMID- 16659813 TI - Nitrite Reduction in Reconstituted and Whole Spinach Chloroplasts during Carbon Dioxide Reduction. AB - Nitrite reduction in either whole, isolated spinach chloroplasts (Spinacia oleracea L.) or in reconstituted spinach chloroplasts is stimulated by a short period of photosynthetic CO(2) fixation in the light prior to nitrite addition. With reconstituted chloroplasts, a similar stimulation can be obtained in nitrite reduction without CO(2) fixation by the addition of dihydroxyacetone phosphate or fructose 6-phosphate. Specific intermediate metabolites of the photosynthetic carbon reduction cycle may have a regulatory role in nitrite reduction in chloroplasts in the light. PMID- 16659814 TI - Separation and Some Properties of Large and Small Amyloplasts throughout Development in Barley Endosperm. AB - A method is described whereby amyloplasts from immature barley (Hordeum distichum L.) endosperm could be separated into two populations of large and small amyloplasts at all stages of development. The small amyloplasts had more amylopectin than the large at early stages, but by 60 days after anthesis, the large had the greater proportion of amylopectin. Starch synthetase activity was associated with both types of amyloplast. The nucleotide specificity of the starch synthetase associated with each population varied independently throughout development. At 25 days after anthesis, the large amyloplasts were more susceptible than the small to alpha-amylolysis; however, at 38 and 60 days, the small amyloplasts became more susceptible. PMID- 16659815 TI - Differential counteraction of ethylene effects by gibberellin a(3) and n(6) benzyladenine in senescing citrus peel. AB - Treatment of mature citrus fruit (Citrus sinensis) with ethylene induced rapid chlorophyll destruction, a rise in respiration, a release of free amino acids, an accumulation of reducing sugars, and an appearance of phenylalanine ammonia lyase activity. Gibberellin A(3) (GA(3)) and N(6)-benzyladenine (BA) opposed the effects of ethylene on chlorophyll, amino acids, and to a lesser extent, reducing sugar levels. The ethylene-induced respiratory rise was only slightly modified by GA(3) and BA. Phenylalanine ammonia lyase activity was not affected by GA(3).The antagonism between ethylene and the senescence-delaying regulators GA(3) and BA seems to operate mainly within the chloroplast, but might not be confined to this compartment. The accumulation of reducing sugars exhibits the antagonism although it is not apparently related to the chloroplast. PMID- 16659816 TI - Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids. AB - A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems. PMID- 16659817 TI - Temperature-dependent Response to Indoleacetic Acid Is Altered by NH(4) in Cultured Cotton Ovules. AB - Unfertilized cotton ovules, cultured in vitro, produced fiber in response to indoleacetic acid (IAA) and/or gibberellic acid (GA(3)). Of the two hormones, IAA produced the greater amount of fiber per ovule, and the effect of both hormones applied simultaneously was approximately additive. Depending upon in vitro culture temperature, a variable number of unfertilized cotton ovules produced fiber in response to 5 mum IAA. The response to IAA was "all or none" for each ovule. The percentage of ovules responding to 5 mum IAA ranged from zero at 28 C to 100 at 34 C. The ability to respond at nonpermissive culture temperatures (i.e. 28 to 30 C) was markedly increased by including 2.5 mm NH(4)NO(3) in the KNO(3)-based medium or by increasing concentrations of IAA. The inclusion of NH(4) (+) in the basal culture medium provided only an increased percentage of ovules producing fiber in the presence of IAA, but did not increase fiber production per ovule. Temperature, like GA(3), increased both the number of ovules producing fiber in response to IAA and the amount of fiber produced per ovule. PMID- 16659818 TI - Effect of different killing techniques on early labeled photosynthetic products in c(4) plants. AB - The choice of leaf-killing technique was found to affect significantly the distribution of label among early labeled photosynthetic products in two C(4) plants, Portulaca oleracea and Zea mays. The major effect of these procedures was on the amount of amino acids present, particularly alanine, and the ratio of malate to aspartate. Killing Portulaca leaves in alcohol generally results in more alanine and the predominance of malate over aspartate. When the leaves are killed by immediate freezing, however, aspartate contained more radioactivity than malate, and alanine was present in much reduced amounts. The various methods also differ in the relative amounts of C(3) cycle compounds and other, secondary intermediates which were obtained. PMID- 16659819 TI - Growth and gibberellin a(1) metabolism in excised lettuce hypocotyls. AB - Excised lettuce (Lactuca sativa L. cv. Arctic) hypocotyls retain the ability to elongate in response to exogenously supplied gibberellic acid and gibberellin A(1) (GA(1)). We have studied the relationship between metabolism of GA(1) and elongation in this tissue. In 24 hours at 28 C, hypocotyls treated with 3 mum GA(1) double in length while controls elongate less than 45%. After an exogenous hormone supply is removed, hypocotyls continue to grow faster than untreated controls, although as the hormone application time is decreased, the GA(1) concentration required to effect a given length change increases. [(3)H]GA(1) was used to determine rates of hormone uptake, efflux, and metabolism. In the presence of [(3)H]GA(1), hypocotyls accumulate and metabolize lable for at least 24 hours. When the exogenous label is removed, the amount of acidic GA in the hypocotyl declines rapidly to a constant level while ethyl acetate-insoluble metabolites increase rapidly to a constant level. Lable accumulation and metabolism at any time are proportional to the external GA(1) concentration below 50 mum GA(1). Chromatographic analysis of radioactive compounds present in tissue extracts suggests that unaltered GA(1) is the major component of the acidic ethyl acetate-soluble fraction, and gibberellin A(8) is a minor component. The ethyl acetate-insoluble fraction appears to contain an unidentified GA(1) metabolite with chromatographic properties similar to those of GA(1). The strong retention of accumulated GA(1) confirms the possibility of a continuing requirement for GA(1) during the sustained response to a GA(1) "pulse" but raises the question of accessibility of the stored hormone for growth promotion. PMID- 16659820 TI - Long distance translocation of sucrose, serine, leucine, lysine, and carbon dioxide assimilates: I. Soybean. AB - To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. ;Wells'), (14)C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with (14)C sucrose and (14)CO(2) assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists. PMID- 16659821 TI - Long Distance Translocation of Sucrose, Serine, Leucine, Lysine, and Carbon Dioxide Assimilates: II. Oats. AB - To establish whether several amino acids were equally able to enter the phloem of oat (Avena sativa L.) plants and be transported, several (14)C-labeled amino acids were applied individually to an abraded spot on a fully expanded source leaf. The base of an immature sink leaf was monitored with a GM tube for time and rate of arrival of radioactivity. Transport of (14)C-sucrose and (14)CO(2) assimilates was measured for a comparison. The applied l-serine, l-lysine, and l leucine, as well as sucrose, entered the phloem and were transported to the sink leaf at rates between 1.16 and 1.83 cm/min. Transport velocity for CO(2) assimilates was 1.57 cm/min. A heat girdle near the top of the source leaf sheath blocked most transport, which indicated that transport was primarily through the phloem. Mass transfer rates for amino acids were only 3% as great as that for sucrose, suggesting different mechanisms of entry for sucrose than for amino acids into the phloem. The higher percentage of CO(2) assimilates mobilized to the sink leaf was attributed to the greater surface area of minor veins accessible to loading, as compared to those compounds supplied via an abraded spot. Serine was extensively metabolized in the source leaf, and radioactive products in the sink leaf mirrored those in the source leaf. Most radioactivity of lysine and leucine remained within these compounds in the source, path, and sink tissues. We concluded that there was no barrier to entry of amino acids into the phloem and transport therein. Data do not suggest a specific mechanism for entry of amino acids into the phloem. PMID- 16659822 TI - Phospholipid-synthesizing Enzymes Associated with Golgi Dictyosomes from Pea Tissue. AB - Golgi dictyosomal membranes isolated from pea (Pisum sativum) stem tissue, using a combination of rate zonal and isopycnic sucrose density centrifugation, were shown to bear cytidine diphosphate-choline:diglyceride phosphorylcholinetransferase, CDP-ethanolamine:diglyceride phosphorylethanolaminetransferase, and CTP:phosphorylcholine cytidyltransferase activities. Although the majority of the activity of the phospholipid synthesizing enzymes was associated with the endoplasmic reticulum, the activity found in the Golgi system was about 25% of the total activity. These results suggest that Golgi dictyosomes probably synthesize at least part of the membrane phospholipids that they may need for their secretory function and for dictyosomal proliferation during cell growth, rather than importing this material entirely from the endoplasmic reticulum. PMID- 16659823 TI - Phytochrome and Circadian Clocks in Samanea: Rhythmic Redistribution of Potassium and Chloride within the Pulvinus during Long Dark Periods. AB - Previous investigations with the electron microprobe reveal that the movements of Samanea leaflets are correlated with massive redistribution of K within the pulvinus. Evidence is now presented that Cl moves with K, whether plants are in white light or darkness, whether or not the amplitude of free running oscillations has damped, and whether or not the rhythm has been rephased by phytochrome photoconversion. The mid-extensor to mid-flexor ratio of K + Cl is correlated with leaflet angle under all conditions. Total Cl in both inner cortex and motor region is approximately 0.6 as high as K. The stoichiometry between Cl and the migratory fraction of K is close to, but not precisely 1:1 in all regions of the pulvinus, suggesting that other ions or systems may also be involved in the balancing of electrical charges. PMID- 16659824 TI - Induction of Frost Hardiness in Stem Cortical Tissues of Cornus stolonifera Michx. by Water Stress: I. Unfrozen Water in Cortical Tissues and Water Status in Plants and Soil. AB - Water supply and day length were varied in cold hardiness studies of red osier dogwood plants (Cornus stolonifera Michx.). The frost killing temperature, the content and freezing of stem cortical tissue water along with soil moisture content and tension were evaluated. Seven days of water stress in long and short day photoperiod regimes caused a rapid decrease in soil moisture content and plant water potential. During the same period, the frost hardiness increased from -3 to -11 C. Further water stress treatment had little effect. Control plants in short days showed only a gradual decrease in plant water potential and only gradually increased in frost hardiness while control plants in long days were unchanged. Freezing studies using nuclear magnetic resonance showed that increased hardiness in water-stressed plants resulted from both an increased tolerance of freezing and an increased avoidance of freezing, the latter resulting from higher solute concentration in the tissue solutions. The short day controls also showed similar changes; however, the changes were smaller over the 21 days of the study. PMID- 16659825 TI - Induction of Frost Hardiness in Stem Cortical Tissues of Cornus stolonifera Michx. by Water Stress: II. Biochemical Changes. AB - A decrease of protein, RNAs, and starch, and an increase of sugar were observed in 3-day water-stressed red osier dogwood plants (Cornus stolonifera Michx.) when the frost hardiness increased from -3 to -6 C. As the frost hardiness increased to -11 C after 7 days of treatment, the starch continuously decreased, however, the proteins and RNAs increased with a continuous increase of sugar. Further water stress treatment had little effect on the changes of these chemicals. Control plants in short days showed similar gradual biochemical changes in patterns. From the results of frost hardiness increases, the pattern of biochemical changes, and the mechanism of the increased freezing resistance, it appears that the water stress and short days accomplished essentially the same physiological end(s) in inducing frost hardiness in red-osier dogwood. PMID- 16659826 TI - Model for variable light sensitivity in imbibed dark-dormant seeds. AB - The level of light-induced germination of the seed of common purslane (Portulaca oleracea L.) and curly dock (Rumex crispus L.) changes with dark incubation time prior to brief, low energy, red light treatment. The rate at which phytochrome far red-absorbing form (Pfr) acts in the light-induced population of seeds was measured by quantitating per cent reversals of the red light effect with saturating far red light exposures at successive times after the red light exposure. A linear positive correlation was found between this rate and the final germination level. These results are compatible with a model involving changing levels, during dark incubation, of a component with which Pfr interacts. In this model, germination is initiated after attainment of a certain level of interaction between Pfr and this component. These findings also support the view that the Pfr to Pr decay rate constant and total phytochrome level are stable during dark incubation. PMID- 16659827 TI - Ultrastructural Changes during Swelling and Contraction of Mitochondria from Cold hardened and Non-hardened Winter Wheat. AB - Mitochondria isolated from both 2 and 24 C grown winter wheat (Triticum aestivum L.) undergo spontaneous swelling in isomolar KCI solutions, but only 24 C mitochondria exhibit a substrate-induced contraction response. Electron microscopic examination revealed that 24 C mitochondria have more clearly defined cristae, less matrix material, and are generally more electron-dense than 2 C mitochondria. During swelling, the matrix material of both 2 and 24 C mitochondria expands and the mitochondria become less electron-dense. After partial swelling, 24 C mitochondria contract upon addition of succinate, and regain structural characteristics similar to those of untreated mitochondria. In contrast, mitochondria from 2 C seedlings continue to swell after addition of substrate, and many of the mitochondria become irregular in shape and lose much of their matrix material. A comparison of results obtained from absorbancy measurements, electron microscopy, and a Coulter Counter indicate that swelling and contraction involve changes both in over-all volume, and internal structural characteristics of mitochondria from 2 and 24 C grown seedlings. Electron microscopic examination of shoot cells showed that mitochondria in 24 C grown seedlings possessed more recognizable cristae and greater internal organization than mitochondria in 2 C seedlings. PMID- 16659828 TI - Influence of temperature gradients on leaf water potential. AB - Water potential was monitored at nine locations along single maize (Zea mays L.) leaf blades with aluminum block in situ thermocouple hygrometers. Water potential showed a continuous decrease toward the tip, with a 2- to 4-bar difference between leaf base and tip under both moist and dry soil conditions. The water potential difference between the soil and the leaf base was about 4 bars. Water potentials decreased during the day and during a drying cycle, and increased at night and after irrigation. Heating a band of a leaf to 40 C or cooling it to 7 C had no influence on the water potential of the affected portion when this was corrected for hygrometer output over standard calibrating solutions at the respective temperatures. Heating or cooling a portion of a leaf had neither short nor long term effects on water potential of more distal leaf portions continuously monitored by hygrometers in dew point readout. Water potential fluctuated with an amplitude of about 1.5 bars and an irregular period of 10 to 30 minutes. Measurements with silver foil in situ psychrometers gave similar results. PMID- 16659829 TI - Lipid composition of organelles from germinating castor bean endosperm. AB - Glyoxysome, endoplasmic reticulum, mitochondria, and proplastid fractions were isolated from endosperm of castor beans (Ricinus communis) germinated for 5 days at 30 C. Samples from sucrose density gradients were diluted with 0.15 m KCI and the membranes pelleted. Lipid extracts of these membranes were analyzed for phosphoglyceride, acyl lipid, and sterol content. The endoplasmic reticulum contains 1.24 mumol of phosphoglyceride per mg of protein; the mitochondria, 0.65 mumol/mg; and the glyoxysome membranes, 0.55 mumol/mg. Phosphatidyl choline and phosphatidyl ethanolamine are the most abundant lipids in all membranes studied, accounting for 70% or more of the lipid phosphorus and 50% or more of the fatty acid. Glyoxysome membranes and endoplasmic reticulum also contain phosphatidyl inositol (respectively, 9 and 17% of the lipid phosphorus) and free fatty acids (13% of the total fatty acid in each). Compared with other organelles, mitochondrial membranes have more phosphatidyl ethanolamine relative to phosphatidyl choline and are characterized by the presence of cardiolipin, in which 80% of the fatty acid is linoleate. The relative amounts of linoleate, palmitate, oleate, stearate, and linolenate in each of the phosphotoglycerides are constant regardless of the membrane source. Stimasgasterol and beta sitosterol are present in the membranes (1-9 nmol each/mg protein).The data provide further evidence that glyoxysome membranes are derived from the endoplasmic reticulum but at the same time indicate some differentiation. PMID- 16659830 TI - Adenosine triphosphatase from soybean callus and root cells. AB - The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+) > Sr(2+)) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K(+) > Rb(+) > Choline(+) > Na(+) > Li(+) > NH(4) (+) > Cs(+) > tris(+). Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na(+) over K(+). No synergism between K(+) and Na(+) was found with preparations from either cell source.The pH optimum for ATP hydrolysis in the presence of 50 mm KCl and 3 mm MgSO(4) was 6.5 for both preparations and slightly higher in the presence of 3 mm MgSO(4) alone. The order of nucleotide preference was found to be: ATP >> ADP > GTP > CTP > UTP. Maximal glucan synthetase activity at high (1 mm), but not at low (1 mum), substrate was found to be coincident with the position of this fraction on the sucrose gradient. PMID- 16659831 TI - Developmental Biochemistry of Cottonseed Embryogenesis and Germination: VIII. Free Amino Acid Pool Composition during Cotyledon Development. AB - The composition of the free amino acid pool in embryonic cotton (Gossypium hirsutum) cotyledons is quite distinct from that of endosperm, and that of germinated, greened cotyledons is quite distinct from that of leaves. During germination (including the precocious germination of immature seeds), the pool expands considerably showing a pronounced accumulation of asparagine. The high level of asparagine found in seedling roots and in the cotyledon vascular exudate indicates that this is the major transported amino acid in germination. There is no pool expansion in the presence of abscisic acid. In the presence of actinomycin D, the pool expands, but an enormous accumulation of glutamine takes place. The composition of the pool at any stage is not related to the composition of the isoacceptor transfer RNA pool, nor to the composition of the storage protein. Anaerobiosis leads to an accumulation of aspartate, alanine, and glycine at the expense of asparagine; however, desiccation does not result in an accumulation of proline. Conspicuously high levels of arginine are maintained through embryogenesis and germination. The levels of individual amino acids are presented as nanomol per cotyledon pair and as per cent of total pool. PMID- 16659832 TI - Carbon Dioxide Exchange and Acidity Levels in Detached Pineapple, Ananas comosus (L.), Merr., Leaves during the Day at Various Temperatures, Oxygen and Carbon Dioxide Concentrations. AB - The effects of temperature, O(2), and CO(2) on titratable acid content and on CO(2) exchange were measured in detached pineapple (Ananas comosus) leaves during the daily 15-hour light period. Comparative measurements were made in air and in CO(2)-free air. Increasing the leaf temperature from 20 to 35 C decreased the total CO(2) uptake in air and slightly increased the total CO(2) released into CO(2)-free air. Between 25 and 35 C, the activation energy for daily acid loss was near 12 kcal mol(-1), but at lower temperatures the activation energy was much greater.Increasing O(2) or decreasing the CO(2) concentration decreased the total CO(2) fixation in air, whereas the total CO(2) released in CO(2)-free air was increased. The total acid content remained constant at 20 C, but it decreased progressively with increasing temperature both in air and in CO(2)-free air. The total acid content at 30 C remained constant in 2% O(2) irrespective of CO(2) concentration. The total acid content decreased in 21 and 50% O(2) as the CO(2) increased from 0 to 300, and 540 mul/l of CO(2). The data indicate that photorespiration is present in pineapple. The lack of acid loss in 2% O(2) suggests that light deacidification is dependent upon respiration and that higher O(2) concentrations are required to saturate deacidification. PMID- 16659833 TI - Isolation and Characterization of a Novel p-Coumaric Acid Ester of myo-Inositol from Needles of Taxus baccata. AB - A novel derivative of myo-inositol was isolated from the needles of Taxus baccata and identified as p-coumaric acid ester. By means of its levorotatory optical activity and formation of a borate complex, the compound was designated tentatively as a 1l-4-p-coumaroyl-myo-inositol. Chemotaxonomic investigations have shown this ester to be present in needles of 14 out of 34 tested species of gymnosperms. When (14)CO(2) or (14)C-myo-inositol were fed to young needles of T. baccata, there was a rapid incorporation of label in the p-coumaroyl ester, and a pulse-chase experiment indicated a substantial turnover of this compound over a 4 month period. PMID- 16659834 TI - Effect of Potassium Supply on the Rate of Phloem Sap Exudation and the Composition of Phloem Sap of Ricinus communis. AB - The composition of phloem sap has been investigated in Ricinus communis var. gibsonii, grown for 2 weeks on nutrient solution of low and high potassium content (K(1) and K(2)). Diagonal cuts were made in the bark of the stem resulting in the exudation of clear droplets which mainly consisted of phloem sap. Although the plants at low K (0.4 mm) and high K (1 mm) did not differ in growth, leaf area, height, or stem circumference, the rate of exudation of the high K plants was about twice as high as that of the plants with the lower K supply. This promoting effect of K on exudation did not result in a dilution of organic (sucrose, UDP-glucose, ATP, UTP) and inorganic constituents of the phloem sap. For the following compounds, even significantly higher concentrations in the exudate were observed in the K(2) plants: potassium, raffinose, glucose 6 phosphate, and fructose 6-phosphate. Also, the osmotic pressure of the phloem sap was substantially increased in the higher K treatment. Experiments in which labeled (14)CO(2) was applied to one leaf showed that K had a favorable effect on the assimilation of CO(2), and in particular promoted the export of photosynthates from the leaf. It is suggested that the higher rate of phloem loading in the plants with the better K supply is due to the higher CO(2) assimilation rate and especially to a better provision of ATP required for phloem loading. Higher phloem-loading rates result in higher osmotic pressure in the sieve tubes which probably gave rise to the higher flow rates observed in the plants with improved K supply. PMID- 16659835 TI - Effect of turgor pressure and cell size on the wall elasticity of plant cells. AB - Direct measurements of the volumetric elastic modulus, in, of cells of a higher plant were performed on the epidermal bladder cells of Mesembryanthemum crystallinum using a pressure probe technique. Measurements on giant algal cells (Valonia, Nitellopsis) are given for comparison. Giant celled algae and M. crystallinum bladders have elastic moduli, in, which depend strongly on turgor pressure, P, and on cell volume, V. The in values of Mesembryanthemum bladders range between 5 bar at zero pressure and 100 bar at full turgor pressure (3-4 bar). in increased with cell size (volume) at a given turgor pressure, and this volume dependence was pronounced more in the high pressure range. From the in (P) characteristics, complete volume-pressure curves were obtained for Mesembryanthemum bladders and giant algal cells. The results suggest that the in (P) and in (V) characteristics of all plant cells are similar. The significance of the pressure and volume effects for the water relations and growth processes of plant cells is discussed briefly. PMID- 16659836 TI - Leaf Proteolytic Activities and Senescence during Grain Development of Field grown Corn (Zea mays L.). AB - Some proteolytic enzymes occurring in the leaves of field-grown corn (Zea mays) (B73) were identified and partially characterized. Changes in activities of several proteolytic enzymes and in concentrations of protein and chlorophyll as a function of intraleaf segments (tip to base), leaf position, and leaf senescence during grain development and maturation were followed in crude leaf extracts.The aminopeptidase (not affected by sulfhydryl or fluoride reagents) was most active at pH 7, while the carboxypeptidase(s) (sensitive to fluoride, but insensitive to sulfhydryl reagents) was most active in the acid range, pH 3 to 6. The presence of two or more endopeptidases is indicated. Endopeptidase (caseolytic) activity at pH 5.4 appeared to be stimulated by sulfhydryl groups or EDTA, while caseolytic activity at pH 7.5 was not.Visually, individual leaf senescence starts at the leaf tip and the necrotic (brown) V-shaped area enlarges progressively toward the leaf base. Canopy senescence occurs in two phases. Foliar symptoms are first observed on the bottom leaf and then in sequential order up the plant. Subsequently, senescence occurs on the top leaf and moves downward. These foliar senescence symptoms are paralleled by decreases in exopeptidase activities, protein, and chlorophyll concentrations and by increases in endopeptidase activities.During development and maturation of the grain, both aminopeptidase and carboxypeptidase activity of the middle half of the ear leaf increased (2- to 3-fold) during the onset of the visual reproductive phase (tassel and car emergence). However, during grain development and plant senescence, both activities decreased rapidly and concurrently with the loss of protein and chlorophyll from this leaf section. In contrast, caseolytic activity at both pH 5.4 and 7.5 increased gradually during the early reproductive phase and rapidly with leaf senescence. The fastest rate of increase in caseolytic activities was concurrent with the most rapid loss of protein from the leaves. The coincidence of these events suggests a major role for the caseolytic enzymes in initiating the rapid hydrolysis of leaf protein. PMID- 16659837 TI - Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss. AB - The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded. PMID- 16659838 TI - Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts. AB - After a 5-hour incubation of protoplasts of Nicotiana tabacum L. ;Xanthi' with (3)H-DNA (7.26 mug/ml) from N. tabacum L. ;Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4 dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus resistant gene.A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli(3)H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the (3)H-DNA entered the protoplasts. Competition experiments between the (3)H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded (3)H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the "soluble" fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable (3)H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 x 10(-15)g donor DNA, or approximately half of the E. coli genome. PMID- 16659839 TI - Superoxide dismutases: I. Occurrence in higher plants. AB - Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium. The enzyme is present in the shoots, roots, and seeds of the three species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated a total of 10 distinct forms of the enzyme. Corn contained seven of these forms and oats three. Peas contained one of the corn and two of the oat enzymes. Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme. Some of the leaf superoxide dismutases were found primarily in mitochondria or chloroplasts. Peroxidases at high concentrations interfere with the assay. In test tube assays of crude extracts from seedlings, the interference was negligible. On gels, however, peroxidases may account for two of the 10 superoxide dismutase forms. PMID- 16659840 TI - Superoxide Dismutases: II. Purification and Quantitative Relationship with Water soluble Protein in Seedlings. AB - Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.Superoxide dismutase accounted for 1.6 to 2.4% of the water-soluble protein in seedlings of corn, peas, and oats (Avena sativa L., cv. Au Sable). The superoxide dismutase activity per plant and per milligram water-soluble protein considerably increased during germination of oats and during greening and hook opening of peas. PMID- 16659841 TI - Cold hardiness and deep supercooling in xylem of shagbark hickory. AB - Differential thermal analysis, differential scanning calorimetry, pulsed nuclear magnetic resonance spectroscopy, and low temperature microscopy are utilized to investigate low temperature freezing points or exotherms which occur near -40 C in the xylem of cold-acclimated shagbark hickory (Carya ovata L.). Experiments using these methods demonstrate that the low temperature exotherm results from the freezing of cellular water in a manner predicted for supercooled dilute aqueous solutions. Heat release on freezing, nuclear magnetic resonance relaxation times, and freezing and thawing curves for hickory twigs all point to a supercooled fraction in the xylem at subfreezing temperatures. Calorimetric and low temperature microscopic analyses indicate that freezing occurs intracellularly in the xylem ray parenchyma. The supercooled fraction is found to be extremely stable, even at temperatures only slightly above the homogeneous nucleation temperature for water (-38 C). Xylem water is also observed to be resistant to dehydration when exposed to 80% relative humidity at 20 C. D(2)O exchange experiments find that only a weak kinetic barrier to water transport exists in the xylem rays of shagbark hickory. PMID- 16659842 TI - Supercooling in overwintering azalea flower buds: additional freezing parameters. AB - Results of calorimetric, nuclear magnetic resonance, and low temperature light microscopic studies on supercooled azalea (Rhododendron kosterianum, Schneid.) floral primordia are reported. Heat release during freezing of the supercooled floral primordia is in the range predicted for supercooled pure water. Spin lattice and spin-spin relaxation times measured by pulsed nuclear magnetic resonance spectroscopy decreased after freezing, suggesting that a redistribution of tissue water is associated with injury to the floral primordium. The calorimetric and low temperature microscopy studies showed no detectable ice formation in floral primordia until the major freezing event at low temperature. No resistance to ice growth is found to exist in the primordium tissues, indicating that a freezing barrier or thermodynamic equilibrium exists between the unfrozen primordium and other flower bud parts which contain ice at subfreezing temperatures. PMID- 16659843 TI - Polymorphism of Microbody Malate Dehydrogenase in Opuntia basilaris. AB - Electrophoretic survey of malate dehydrogenase (EC 1.1.1.37) in Opuntia basilaris showed intraspecific polymorphism. Further experiments with microbody malate dehydrogenase-specific antiserum suggest that the polymorphism occurs in microbody malate dehydrogenase independent of the soluble and mitochondrial forms. The pattern of polymorphism is one expected from a two-allele Mendelian system. PMID- 16659844 TI - Changes in Leaf Proteins of Peas, Pisum sativum L., during Development on Deflorated Plants. AB - The soluble (sap) proteins of leaves of pea, Pisum sativum L. cvs. Alaska and Greenfeast, allowed to develop normally or deflowered, to prevent senescence, were separated by isoelectric focusing.There was a decline in certain proteins, with increases in others as the leaves aged but preventing senescence of the whole plant did not alter the pattern of change in leaf proteins. We concluded that whole plant senescence proceeds independently of leaf senescence. PMID- 16659845 TI - Influence of the root tip and the duration of washing on k retention by excised apical root segments of corn. AB - Excised apical segments of corn root (Zea mays) (5-15 mm from the root cap), some with and some without the root tips (0-5 mm) attached, were washed for varying time periods up to 4 hours in 0.5 mm CaSO(4). After washing, tips were removed from those segments washed with tips attached, and then all segments and tips were analyzed for K(+) content. The root tips (0-5 mm) initially contained about twice the K(+) of the apical segments (5-15 mm). The loss of K(+) did not exceed 15% in the tips or 20% in the apical segments. Loss of K(+) was most pronounced during the 1st hour of washing. There was little difference in K(+) content of apical segments washed with tips attached compared with those washed tipless. Thus, the presence of the intact root tip had no consistent influence on the ability of the older root tissue to retain K(+). PMID- 16659846 TI - Wall analyses of lophocolea seta cells (bryophyta) before and after elongation. AB - Lophocolea heterophylla (Schrad.) Dum. (a leafy liverwort) produces sporophytes with seta cells that elongate 50-fold in 3 to 4 days. Wall components of these cells have been characterized by microscopic histochemistry, colorimetry, and gas chromatography of neutral sugars. Seta cell walls are qualitatively similar to primary cell walls of higher plants. The pectic fraction, however, responds differently to standard histochemical staining and extraction. Quantitatively, mannose, fucose, and rhamnose are in higher percentage, and arabinose and xylose are lower than typically found in vascular plants. Hexuronic acids increase on a percentage basis during elongation; pentoses decrease slightly, while hexose levels remain about the same. Increase in total wall carbohydrate after 2,400% elongation of setae was 1.8-fold. PMID- 16659847 TI - Glycoprotein synthesis in plants: I. Role of lipid intermediates. AB - The enzymic processes involved in glycoprotein synthesis have been studied using crude extracts obtained from developing cotyledons of Phaseolus vulgaris harvested at the time of active deposition of vicilin. Radioactivity from GDP [(14)C]mannose can be incorporated by crude extracts into a single chloroform methanol-soluble product as well as into insoluble product(s). Mannose is the sole (14)C-labeled constituent of the lipid. The kinetics of incorporation of (14)C, as determined by pulse and pulse-chase experiments using GDP [(14)C]mannose, as well as direct incorporation from added [(14)C]mannolipid, shows that the mannolipid is an intermediate in the synthesis of the insoluble product(s). The characteristics of the mannolipid are consistent with it being a mannosyl phosphoryl polyprenol. The mannose is apparently attached to the lipid via a monophosphate linkage. Of the radioactivity in the insoluble product(s), about 20% is pronase-digestible during a "pulse experiment." After a chase with unlabeled GDP-mannose, about 40% is pronase-digestible; the other 60% is as yet uncharacterized. A radioactive product soluble in a mixture of chloroform methanol-H(2)O can be extracted from the insoluble residue obtained during a pulse, but is no longer present after a chase. This product may be a lipid oligosaccharide, the final intermediate in glycoprotein synthesis. Data are presented on incorporation from UDP-N-[(14)C]acetylglucosamine into both chloroform-methanol-soluble and -insoluble product(s). The results are consistent with an involvement of lipid intermediates in the glycosylation of protein in this system, and support the concept that the mechanisms of glycoprotein synthesis in higher plants are similar to those which have been reported for mammalian systems. PMID- 16659848 TI - Nonstomatal inhibition of photosynthesis at low water potentials in intact leaves of species from a variety of habitats. AB - Mesophyll resistance to CO(2) uptake was calculated from gas exchange data on intact leaves of 12 species of woody plants. Plants studied were native to habitats ranging from streamsides to deserts. Gas exchange measurements were made at light saturation and constant temperature to eliminate possible effects of light and temperature on estimates of mesophyll resistance. Cuticular transpiration was measured and used in calculation of stomatal resistances from whole leaf transpiration rates. In all species examined, an increase in mesophyll resistance was observed as leaves dried. The increase in mesophyll resistance in all cases occurred at the same water potential as the initial decline in net photosynthesis, and was accompanied by an increase in stomatal resistance. PMID- 16659849 TI - Water stress effects on the content and organization of chlorophyll in mesophyll and bundle sheath chloroplasts of maize. AB - The consequences of drought stress on the organization of chlorophyll into photosynthetic units and on the chlorophyll-protein composition of mesophyll and bundle sheath chloroplasts of Zea mays L. were studied. It was found that the majority of chlorophyll lost in response to water stress occurs in the mesophyll cells with a lesser amount being lost from the bundle sheath cells. All of the chlorophyll loss could be accounted for by reduction in the lamellar content of the light-harvesting chlorophyll a/b-protein, a rather specific target for water stress. The decreased content of this chlorophyll-protein accounts for the elevated chlorophyll a/b ratios and the reduced photosynthetic unit sizes of the two cell types in stressed plants. The implications of the selective catabolism of this major membrane component are discussed. PMID- 16659850 TI - Environmental and genetic components of stomatal behavior in two genotypes of upland cotton. AB - Two parental lines of Upland cotton (Gossypium hirsutum L.) plus their reciprocal F(1), F(2), and backcross populations were field-tested for environmental and genetic components of leaf diffusive resistance. Leaf resistance was measured with a diffusion porometer three times each day during 6 days in August. A large, consistent environmental component was present during the morning, afternoon, and evening, but the genetic component appeared mainly during the afternoon. Leaf water potential indicated that afternoon was the period of highest water stress. Genetic analyses revealed that leaf resistance was associated with both additive and dominance genetic variances, with an estimated narrow sense heritability of 25%. High leaf resistance was completely dominant to low resistance. PMID- 16659851 TI - Characterization of naphthaleneacetic Acid binding to receptor sites on cellular membranes of maize coleoptile tissue. AB - Characteristics of and optimum conditions for saturable ("specific") binding of [(14)C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a K(D) of 5 to 7 x 10(-7)m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg(2+) or Ca(2+) above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor ("supernatant factor") found in maize tissue. PMID- 16659852 TI - Effects of Osmotic Shock on Some Membrane-regulated Events of Oat Coleoptile Cells. AB - Oat coleoptile sections (Avena sativa L. cv. "Garry") were osmotically shocked with 0.5 m mannitol followed by 1 mm Na-phosphate (pH 6.4) at 4 C. This treatment reduced uptake of alpha-aminoisobutyric acid, 3-o-methyl glucose, and leucine by 75 to 90% but inhibited (36)Cl(-) uptake only 30%. Some recovery was observed 1 to 3 hours later. Respiration rates were unaffected by osmotic shock and protein synthesis was reduced 11%.Osmotic shock also stimulated efflux of alpha aminoisobutyric acid and K(+) and led to an increase in conductivity of the solution bathing shocked sections. The transmembrane electropotential of 75% of the shocked cells fell to -20 mv to -45 mv compared with the majority of unshocked cells at -80 mv to -120 mv.We concluded that osmotic shock selectively modifies the plasma membrane. The inhibitions of uptake could be due to removal of specific components of the plasma membrane and/or to the lowered electropotential. PMID- 16659853 TI - Osmotic Shock Inhibits Auxin-stimulated Acidification and Growth. AB - Cells of oat coleoptiles (Avena sativa L. cv. "Garry") have been osmotically shocked in order to observe the effect of alterations of the plasma membrane on some auxin responses. When coleoptile sections were treated sequentially with 0.5 m mannitol and 1 mm Na-phosphate (pH 6.4) at 4 C, polar auxin transport and acidification by 1 mM CaCl(2) were unaffected, but auxin-stimulated acidification and growth were eliminated. Shock treatment also had no effect on acid-stimulated growth or on freezing point depression by the cytoplasm. It is suggested that osmotic shock modifies a portion of the plasma membrane which interacts with auxin and eventually leads to growth. PMID- 16659854 TI - Role of Phenolic Inhibitors in Peroxidase-mediated Degradation of Indole-3-acetic Acid. AB - 7-Hydroxy-2,3-dihydrobenzofuran derivatives, metabolites of a carbamate insecticide carbofuran, and five other phenolic inhibitors of indoleacetic acid (IAA) oxidase interfered with IAA-induced spectral change in the Soret band of horseradish peroxidase (HRP). The onset of IAA degradation required transformed HRP intermediates. The inhibitors, when added before IAA, protected HRP from reacting with IAA, thus preventing formation of highly reactive enzyme intermediates, and consequently, IAA degradation. When added after IAA, the inhibitors quickly reversed the IAA-induced spectral change of HRP and inhibited further IAA degradation.The phenolic inhibitors differed in stability and reactivity. 7-Hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, 3,7-dihydroxy-2,2 dimethyl-2,3-dihydrobenzofuran, catechol, protocatechuic acid, caffeic acid, ferulic acid, and scopoletin belonged to one group which produced only a temporary inhibition to IAA-induced spectral change of HRP and IAA degradation since the inhibitors were metabolized in the reaction. The length of the lag was dependent on the IAA, inhibitor, and enzyme concentrations. 3-Keto-7-hydroxy-2,2 dimethyl-2,3-dihydrobenzofuran and 3-keto-carbofuran belonged to the other group which produced a persistent inhibition.Degradation of IAA required both the heme group and apoprotein of HRP. Reconstituted enzyme from bovine hemin and apoprotein or HRP after unfolding by urea or guanidine treatment were inhibited by the inhibitors in a way similar to the native HRP. The inhibition was reversible by higher concentrations of IAA, but the plot of 1/v versus 1/s and 1/v versus i were curvilinear, reflecting the complex nature of a competitive inhibition. PMID- 16659855 TI - Phloem Pressure Differences and C-Assimilate Translocation in Ecballium elaterium. AB - The role of phloem turgor pressure in (14)C-assimilate translocation in Ecballium elaterium A. Rich was studied. The direction of translocation was manipulated by two methods: darkening, or defoliation, of the upper or lower halves of the shoots. After 24 hours of labeled assimilate movement, sieve tube turgor levels were measured with the phloem needle technique. Distribution of label, determined by autoradiography and counting, revealed a direct correlation between the direction of assimilate transport and the pressure difference. Phloem turgor levels always decreased in the stem of darkened shoots; this resulted in greater pressure differences in the stem between the source leaf receiving (14)CO(2) and treated regions. PMID- 16659856 TI - Effect of Water Stress on Turgor Differences and C-Assimilate Movement in Phloem of Ecballium elaterium. AB - The effects of water stress on pressure differences and (14)C-assimilate translocation in sieve tubes of squirting cucumber Ecballium elaterium A. Rich were studied. Water stress was induced by transfer of plants from culture solution to a polyethylene glycol 6,000 solution having an osmotic potential of 18.2 atm. Sieve tube turgor, turgor differences between source and sink, and translocation rate were decreased. After 260 minutes of translocation, only 19% of the total fixed (14)CO(2) had moved out of the leaf, compared to the control value of 62% after the same period of time. The results suggest that water stress slows translocation by lowering sieve tube turgor differences, which are essential for the pressure flow mechanism of conduction. PMID- 16659857 TI - Photosynthesis in relation to leaf characteristics of cotton from controlled and field environments. AB - In situ and light-saturated net photosynthetic rates per unit leaf area were greater in cotton (Gossypium hirsutum L.) plants grown in pots in the field than in similar plants from a phytotron growth chamber. Light-saturated stomatal resistances did not differ in leaves of similar age and exposure on field and chamber plants; lower photosynthetic rates in chamber leaves were associated with greater mesophyll resistance. Differences in net photosynthetic rates were related to differences in leaf thickness. When the photosynthetic rates were expressed per unit of mesophyll volume or per unit chlorophyll differences between field and chamber plants were much less than when rates were expressed per unit leaf area. Characterization of the chloroplast lamellar proteins showed that the field leaves had smaller photosynthetic units than the chamber leaves. Since the field leaves also contained more chlorophyll per unit area, this resulted in a much larger number of photosynthetic units per unit area in the field leaves. PMID- 16659858 TI - Tumor induction by agrobacterium involves attachment of the bacterium to a site on the host plant cell wall. AB - Cell wall preparations from primary bean leaves were found to inhibit tumor initiation by Agrobacterium tumefaciens strain B6 when inoculated with the bacteria on bean leaves. Membrane fractions from these same leaves were noninhibitory. The cell walls were effective when applied prior to or with bacteria, but application of cell walls about 15 minutes after bacteria did not affect the number of tumors initiated. Much of the inhibitory activity of the plant cell walls was eliminated by pretreatment with dead site-attaching bacteria or with lipopolysaccharide from these bacteria. Cells and lipopolysaccharide from non-site-attaching agrobacteria had no effect on the activity of the plant cell walls. About 30% inhibition of tumor initiation was obtained with plant cell walls at 50 mug/ml dry weight, and at 10 mg/ml dry weight about 70% inhibition was typical. Both early and late appearing tumors were affected by the cell walls, indicating that they do not exclusively affect tumors arising from either small or large wounds. These data show that plant cell walls but not membranes contain surfaces to which A. tumefaciens adheres and these exhibit the specificity typical of the host site to which virulent agrobacteria must attach to induce tumors. It is concluded that some portion of wound-exposed plant cell wall constitutes the host adherence site in Agrobacterium infections. PMID- 16659859 TI - Ammonium and amino acids as regulators of nitrate reductase in corn roots. AB - When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4 azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme. PMID- 16659860 TI - Rapid Hormone-induced Hyperpolarization of the Oat Coleoptile Transmembrane Potential. AB - The effects of the plant growth substances indoleacetic acid (IAA) and fusicoccin on the transmembrane potential of Avena coleoptile cells (at 27-29 C) were studied. Fusicoccin caused hyperpolarization of the membrane potential which started after a lag of less than 20 seconds, and which on average reached -49 mv at an external K(+) concentration of 1 mm and -75 mv at 0.1 mm K(+). IAA caused a hyperpolarization of -25 mv starting after a lag of 7 to 8 minutes. These results suggest that fusicoccin and IAA both activate electrogenic H(+) extrusion. PMID- 16659861 TI - Localization and Characterization of Photosystem II in Grana and Stroma Lamellae. AB - Attempts have been made to identify intramembranous particles observed in freeze fracture electron microscopy as specific functional components of the membrane. The intramembranous particles of the exoplasmic fracture (EF) face of freeze fractured pea (Pisum sativum) chloroplast lamellae are nonuniformly distributed along the membrane. Approximately 20% of the particles are in unpaired membrane regions whereas 80% are localized in regions of stacked lamellae (grana partitions). The EF particles within the grana regions of the chloroplast membrane are of a larger average size than those in stroma lamellae.Photosystem II activity of isolated stroma lamellae is about 20 to 25% of that of grana enriched membrane fragments when measured at high light intensities. The photosystem II activity of stroma lamellae requires higher light intensities for attainment of maximal rates than does that of grana membranes. Lactoperoxidase catalyzed iodination of stacked chloroplast lamellae was used to demonstrate that 75 to 80% of all photosystem II centers are localized in grana partition regions.The data presented support the concept that the intramembranous particles of the EF face visualized on freeze-fractured chloroplast lamellae represent a central photosystem II reaction center complex plus associated light-harvesting chlorophyll protein. The fact that the EF particles of stroma lamellae are smaller than those of grana regions can be directly correlated to the presence of photosystem II units with small antennae chlorophyll assemblies in stroma lamellae. PMID- 16659862 TI - Importance of Time after Excision and of pH on the Kinetics of Response of Wheat Coleoptile Segments to Added Indoleacetic Acid. AB - Segments of coleoptiles of 3-day-old wheat (Triticum x Aestivum L. cv. Kharkov M.C. 22) grown at 24 C were strung on a glass rod and the kinetics of their elongation in 0.01 m K-phosphate buffer was examined photometrically. Measured rates of elongation in response to treatments were corrected by subtraction of endogenous rates. The customary practice of testing the effects of growth regulators added between the two endogenous surges of growth, that is, up to 3 hours after segments were excised from coleoptiles, gave erroneous kinetic data. Rates of response were then limited by the passive penetration of added auxin and the second endogenous surge interfered with late responses. It was necessary to wait for a phase of more rapid but more steady elongation after the second endogenous surge was over, about 4 hours for wheat at 25 C, to attain the active uptake required for nearly synchronous response through the segment. The more active uptake in this steady phase was confirmed with beta-[2-(14)C]indoleacetic acid and it was greater at pH 5 than at pH 7. The degree of dissociation of indoleacetic acid added at pH 7 was an impediment to penetration that could be compensated for by removal of intercellular air. The pH did not influence the endogenous rate of elongation. The dependence of the rate of elongation on the concentration of indoleacetic acid added at pH 5 was bell-shaped with maximum rate at 10 mum indoleacetic acid, in confirmation of previous measurements made over long intervals of time. The relation between the response and suboptimal concentrations was not sigmoid but was indicative of greater binding affinity than previously reported. PMID- 16659863 TI - Involvement of hydrogen peroxide in the regulation of senescence in pear. AB - Endogenous peroxide levels in pear fruit (Pyrus communis) were measured using a titanium assay method, and were found to increase during senescence in both Bartlett and Bosc varieties. Application of glycolic acid or xanthine, serving as substrates for the formation of H(2)O(2), increased the peroxide content of the tissue and accelerated the onset of ripening, as measured by increased softening and ethylene evolution. Application of ethylene also induced increased peroxide levels. Ripening processes were similarly promoted when peroxides were conserved by inhibiting the activity of catalase with hydroxylamine or potassium cyanide. By comparison, the inhibition of glycolate oxidase with alphahydroxy-2 pyridinemethanesulfonic acid decreased the peroxide content of the tissue and delayed the onset of ripening. These results indicate that the onset of ripening correlates with the peroxide content of fruit tissues as occurring under normal conditions or as influenced by the treatments. Hydrogen peroxide may be involved in oxidative processes required in the initiation and the promotion of ripening. PMID- 16659864 TI - Free sugars in relation to starch accumulation in developing rice grain. AB - The changes in sugars (water-soluble carbohydrates) were studied in the developing grain of rice (Oryza sativa L., variety IR28 and IR29) in relation to the role of these sugars as precursors of ADP glucose in starch accumulation. The levels of total sugars, total reducing sugars and free glucose, sucrose and other nonreducing sugars, maltooligosaccharides, and total and nonsucrosyl fructose followed closely the changes in the rate of starch accumulation, in both IR28 and 29; the peak value occurred 9 days after flowering. The level of soluble carbohydrates remained high in the caryopsis and also in milled rice after starch accumulation, suggesting that the supply of sugar precursors does not limit starch accumulation in the rice grain. Because of a higher level of reducing sugars, the level of free sugars in the grain of waxy rice IR29 was higher than that of nonwaxy IR28. PMID- 16659865 TI - Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides. AB - Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum) chloroplasts was inhibited by inorganic pyrophosphate (PPi). Oxygen evolution was also inhibited by high concentrations of orthophosphate (Pi) and the inhibition was relieved by 3-phosphoglycerate. In contrast, the inhibition by PPi was not relieved by 3-phosphoglycerate, indicating that hydrolysis of PPi and accumulation of inhibitory concentrations of Pi were not occurring. In agreement with this suggestion, the percentage of (14)C-labeled products diffusing out of the chloroplasts was increased by Pi but not by PPi. The inhibition of O(2) evolution by PPi was reversed by ATP. The concentration of PPi required for 50% inhibition was 1.2 to 1.4 mm and the subsequent stimulation by ATP was half maximal at 16 to 25 mum. Carbon dioxide-dependent O(2) evolution by spinach chloroplasts, or chloroplasts isolated from older pea plants, was not significantly inhibited by PPi.Chloroplasts were preloaded with (14)C-ATP and release of the labeled nucleotides was measured to assess the activity of adenine nucleotide transport across the inner chloroplast envelope membrane. A rapid exchange was promoted by the addition of exogenous ATP. Addition of PPi also resulted in a release of endogenous nucleotides. We suggest that PPi inhibits CO(2) fixation by entering the chloroplast in exchange for endogenous adenine nucleotides via the transporter on the inner envelope membrane. The subsequent depletion of the internal adenine nucleotide pool would result in decreased CO(2) fixation due to insufficient ATP. Addition of ATP to PPi-inhibited chloroplasts apparently results in uptake of catalytic amounts of ATP and restoration of the internal adenine nucleotide pool thus relieving the inhibition of CO(2) fixation. PMID- 16659866 TI - Photosynthesis by isolated pea chloroplasts: some effects of adenylates and inorganic pyrophosphate. AB - When added singly to chloroplasts isolated from young pea (Pisum sativum) leaves, both inorganic pyrophosphate (PPi) and small quantities (0.2 mm) of ADP inhibit photosynthesis, but when added together they cause a marked stimulation. ATP (at 0.2 mm) is less inhibitory (or not inhibitory) when added alone, but like ADP, stimulates when added in the presence of PPi. This behavior is in marked contrast to that of spinach (Spinacia oleracea) chloroplasts which are normally stimulated rather than inhibited by PPi and which are largely unresponsive to exogenous adenylates. The inhibitory behavior of PPi with pea chloroplasts was observed under conditions where external hydrolysis to Pi is negligible. It is proposed that the exchange of organic and PPi across the chloroplast envelope may be more rapid in chloroplasts from young pea leaves than in chloroplasts from spinach and that interaction between these two processes could account for the principal observations. PMID- 16659867 TI - Evidence for alpha-Tocopherol Function in the Electron Transport Chain of Chloroplasts. AB - The effect of three different stable radicals-2,2-diphenyl-1-picrylhydrazyl, 1,3,5-triphenyl-verdazyl, and galvinoxyl-was studied in photosystem II of spinach (Spinacia oleracea) chloroplasts. Inhibition by the three was noted on dimethylbenzoquinone reduction in presence of 2,5-dibromo-3-methyl-6-isopropyl-p benzoquinone (DBMIB) and on silicomolybdate reduction in presence of 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) in photosystem II and on the H(2)O --> methylviologen reaction encompassing both photosystems. Inhibition of all photosystem II reactions except silicomolybdate reduction could be partially restored by alpha-tocopherol or by 9-ethoxy-alpha-tocopherone but not by other quinones or radical chasers. On this basis, a functional role for alpha tocopherol in the electron transport chain of spinach chloroplasts between the DCMU and DBMIB inhibition sites is postulated. PMID- 16659868 TI - Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera. AB - Proteinase inhibitor-inducing factor (PIIF)-induced accumulation of trypsin inhibitory activity was assayed in leaves of 23 species of plants representing 10 agriculturally important genera. Inhibitory activity was assayed in extracts from attached leaves or from excised leaves supplied through the cut petioles for 30 minutes with extracts containing the wound hormone PIIF, obtained from either tomato leaves or from the leaves of each plant under study. During subsequent incubation in light for 72 hours, PIIF-induced trypsin inhibitory activity accumulated in significant quantities in 10 of the 23 species. Alfalfa accumulated the highest levels of inhibitory activity (340 mug trypsin inhibited/ml leaf juice), followed by tobacco, tomato, potato, strawberry, cucumber, squash, clover, broadbean, and grape. It is suggested that the inhibitors might be classed as allelochemics that are present in certain plants and not others in response to environmental pressures during their evolution. PMID- 16659869 TI - Induction of Root Nodule Senescence by Combined Nitrogen in Pisum sativum L. AB - Root nodule senescence induced by nitrate and ammonium in Pisum sativum L. was defined by determining nitrogenase activity and leghemoglobin content with the acetylene reduction and pyridine hemochrome assays. Root systems supplied with 100 mm KNO(3) or 100 mm NH(4)Cl exhibited a decrease in nitrogenase activity followed by a decline in leghemoglobin content. Increasing the CO(2) concentration from 0.000320 atm to 0.00120 atm had no effect on the time course of root nodule senescence when 20 mm KNO(3) was supplied to the roots; in vitro nitrate reductase activity was detected in leaves and roots, but not bacteroids. Nitrate appeared in leaves, roots, and the nodule cytosol fraction but not bacteroids when 20 mm KNO(3) was supplied to roots. When nitrate entered through the shoots, however, no root nodule senescence was observed, and no nitrate was detected in root or nodule cytosol fractions although nitrate and nitrate reductase were found in leaves. The results suggest that nitrate does not induce root nodule senescence through competition between nitrate reductase and nitrogenase for products of photosynthesis. PMID- 16659870 TI - Phosphorylation of cytokinin by adenosine kinase from wheat germ. AB - Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N(6)-(delta(2)-isopentenyl)adenosine. Electrophoretic analysis indicated that only N(6)-(delta(2)-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5'-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine.The phosphorylation of the cytokinin and adenosine required ATP and Mg(2+). The pH optimum was from 6.8 to 7.2 for both the cytokinin and adenosine. At pH 7 and 37 C the Km and V(max) for the cytokinin were 31 mum and 8.3 nmoles per mg protein per minute, and the values for adenosine were 8.7 mum and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ phosphorylated N(6) (delta(2)-isopentenyl)adenosine. These preparations also phosphorylated N(6) (delta(2)-isopentenyl)adenine when 5-phosphorylribose-1-pyrophosphate was present. PMID- 16659871 TI - Metabolism of Oat Leaves during Senescence: V. Senescence in Light. AB - A comparison has been made of the progress of senescence in the first leaf of 7 day-old oat plants (Avena sativa cv. Victory) in darkness and in white light. Light delays the senescence, and intensities not over 100 to 200 ft-c (1000-2000 lux) suffice for the maximum effect. In such intensities, chlorophyll loss and amino acid liberation still go on in detached leaves at one-third to one-half the rate observed in darkness; however, when the leaves are attached to the plant, the loss of chlorophyll in 5 days is barely detectable. Transfer of the leaves from 1 or 2 days in the low intensity light to darkness, or vice versa, shows no carryover of the effects of the preceding exposure, so that such treatment affords no evidence for the photoproduction of a stable substance, such as cytokinin, inhibiting senescence. Light causes a large increase in invertaselabile sugar and a smaller increase in glucose, and application of 100 to 300 mm glucose or sucrose in the dark maintains the chlorophyll, at least partially. Correspondingly, short exposure to high light intensity, which increased the sugar content, had a moderate effect in maintaining the chlorophyll. However, 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) completely prevents the increases in sugars and yet does not prevent the effect of light on senescence, whether determined by chlorophyll loss or by protein hydrolysis. Light causes a 300% increase in the respiration of detached oat leaves, and kinetin lowers that only partly, but unlike the increased respiration associated with senescence in the dark, the increase in the light is fully sensitive to dinitrophenol, and therefore cannot be ascribed to respiratory uncoupling. The increased respiration in light is prevented by DCMU, parallel with the prevention of sugar formation. It is therefore ascribed to the accumulation of soluble sugars, acting as respirable substrate. Also, l-serine does not antagonize the light effect. For all of these reasons, it is concluded that the action of light is not mediated by photosynthetic sugar formation, nor by photoproduction of a cytokinin. Instead, we propose that light exerts its effect by photoproduction of ATP. The action of sugars is ascribed to the same mechanism but by way of respiratory ATP. This hypothesis unifies most of the observed phenomena of the senescence process in oat leaves, and helps to explain some of the divergent findings of earlier workers. PMID- 16659872 TI - Changes in Metabolite Levels in Kalanchoe daigremontiana and the Regulation of Malic Acid Accumulation in Crassulacean Acid Metabolism. AB - Changes in glucose-6-P, fructose-6-P, fructose-1,6-diP, 6-phospho-gluconate, phosphoenolpyruvate, 3-phosphoglycerate, and pyruvate levels in the leaves of the Crassulacean plant Kalanchoe daigremontiana Hammet et Perrier were measured enzymically during transitions from CO(2)-free air to air, air to CO(2)-free air, and throughout the course of acid accumulation in darkness. The data are discussed in terms of the involvement of phosphoenolpyruvate carboxylase in malic acid synthesis and in terms of the regulation of the commencement of malic acid synthesis and accumulation through the effects of CO(2) on storage carbohydrate mobilization and its termination through the effects of malic acid on phosphoenolpyruvate carboxylase activity. PMID- 16659873 TI - Phosphatidic Acid synthesis in castor bean endosperm. AB - Enzyme assays on organelles isolated from the endosperm of castor bean (Ricinus communis var. Hale) by sucrose density gradient centrifugation showed that palmitoyl-CoA:sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) was localized in the membranes of the endoplasmic reticulum. Mn(2+) was required for activity, but Ca(2+) and Mg(2+) could substitute for Mn(2+) at higher concentrations. The apparent Km was 170 mum for sn-glycerol 3-phosphate and approximately 8 mum for palmitoyl-CoA. The optimum pH range was 7 to 7.5 and the principal reaction product was diacyl-sn-glycerol 3-phosphate (phosphatidic acid). Monoacyl-sn glycerol 3-phosphate (lysophosphatidic acid) was not released as a free intermediate in the reaction. The maximum activity of the enzyme occurred immediately after imbibition, preceding the development of mitochondria and glyoxysomes. PMID- 16659874 TI - Identification and Properties of the Messenger RNA Activity in Chlamydomonas reinhardi Coding for the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase. AB - Properties of the mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi were determined. Large subunit synthesis, directed by RNA from partially purified whole cell extracts, was detected by specific immunoprecipitation of polypeptide products synthesized in a heterologous translation system derived from Escherichia coli. Large subunit synthesis showed sharp RNA concentration dependence in an E. coli translation system, and at optimal RNA concentrations, immunoprecipitable large subunit synthesis accounted for 2% of the total incorporation. Large subunit messenger activity sedimented at 12 to 14S on nondenaturing sucrose gradients and did not bind to oligo(dT)-cellulose suggesting the mRNA is not polyadenylated. The immunoprecipitable products translated in vitro are not complete polypeptide chains, but are smaller peptides identifiable as large subunit fragments by tryptic fingerprint analysis. No immunoprecipitable product was obtained when similar RNA fractions were tested in a wheat germ translation system. PMID- 16659875 TI - Identification and Precipitation of the Polyribosomes in Chlamydomonas reinhardi Involved in the Synthesis of the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase. AB - The size classes of polyribosomes involved in the synthesis of ribulose-1,5 bisphosphate carboxylase large subunit were determined by binding radioiodinated specific antibodies to polyribosomal preparations from Chlamydomonas reinhardi. Antibodies specific to the denatured large subunit and to the native enzyme bound primarily to small polyribosomes (N = two to five ribosomes). The binding of antibodies to small polyribosomes was unexpected since the large subunit is a large polypeptide (molecular weight 55,000) coded for by a corresponding large mRNA (12-14S). Control experiments showed that this unexpected pattern of antibody binding was not a result of messenger RNA degradation, "run-off" of ribosomes from polyribosomes, or adventitious binding of the completed enzyme to a selected class of polyribosomes. In addition, polyribosomes bearing nascent large subunit chains have been immunoprecipitated from small polyribosome fractions. A large RNA species that can direct the synthesis of large subunit in vitro was extracted from small polyribosomes. PMID- 16659876 TI - Cytokinins: Metabolism and Biological Activity of N-(Delta-Isopentenyl)adenosine and N-(Delta-Isopentenyl)adenine in Tobacco Cells and Callus. AB - The cytokinin, N(6)-(Delta(2)-isopentenyl)adenine, is found to be at least 3.3 times as active as N(6)-(Delta(2)-isopentenyl)adenosine in promoting the growth of cytokinin-requiring tobacco (Nicotiana tabacum) callus. Absorption rates of N(6)-(Delta(2)-isopentenyl)adenine and N(6)-(Delta(2)-isopentenyl)adenosine by tobacco cells in liquid suspension do not differ significantly. In these cells, N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate, di-, and triphosphate are synthesized in both cases, but 7-glucosylation occurs significantly only with N(6)-(Delta(2)-isopentenyl)adenine, protecting thereby its N(6)-isopentenyl side chain from cleavage. Degradation by N(6)-side chain removal appears to be intense, leading to the formation of adenine, adenosine, and adenylic nucleotides. Thus, it is suggested that N(6)-(Delta(2)-isopentenyl)adenine-7 glucoside is a protected or storage form of the cytokinin which could account for the higher biological activity of N(6)-(Delta(2)-isopentenyl)adenine than of N(6) (Delta(2)-isopentenyl)adenosine. PMID- 16659877 TI - Patterns of Ethylene and Carbon Dioxide Evolution during Cotton Explant Abscission. AB - The relationship between abscission and the evolution of ethylene and CO(2) was examined in explants and explant segments of cotton seedlings (Gossypium hirsutum L. cv. Acala SJ-1) under both static and flow system conditions, and in the presence and absence of mercuric perchlorate. Explant excision was immediately followed by increased ethylene evolution (wound ethylene); senescence was also accompanied by increased ethylene evolution (senescence ethylene). One or two ethylene peaks were found to interrupt the low background rate of ethylene evolution during the period between excision and senescence. The first intermediate ethylene peak coincided with a rise in CO(2) evolution; however, precedence could not be established. No statistical correlations were discovered between either intermediate ethylene peak and abscission. The best statistical correlation was found between wound ethylene and abscission at 12 hr after excision. No positive correlations were found between senescence ethylene and abscission. Implications of these results for the understanding of the role of ethylene in explant abscission are discussed.Relationships between a number of different explant treatments and ethylene evolution were also examined. Ethylene production in response to indoleacetic acid applications, abscisic acid applications, and different types of wounding is summarized. It was concluded that the results of the standard abscission bioassay (conducted in Petri dishes) have not been influenced by unnatural ethylene accumulations. PMID- 16659878 TI - Cyclic adenosine 3':5'-monophosphate in moss protonema: a comparison of its levels by protein kinase and gilman assays. AB - From the protonema of the moss Funaria hygrometrica (L.) Sibth, a factor indistinguishable from cyclic adenosine 3':5'-monophosphate (cAMP) has been isolated. The factor stimulated the activity of protein kinase from rabbit skeletal muscle and co-chromatographed with authentic cAMP in two solvent systems. Its ability to stimulate protein kinase activity was completely abolished by 3':5'-cyclic nucleotide phosphodiesterase, the rate of inactivation being similar to that of authentic cAMP. Based on these properties, this factor is identified as 3',5'-cAMP. Cyclic AMP could be readily removed from the cells and washing the cells with water reduced the endogenous level of cAMP by 2- to 3 fold. A comparison of cAMP levels by protein kinase and Gilman assays was made. The intracellular levels determined by protein kinase assay were about 7-fold lower than the values obtained by Gilman assay. This discrepancy was due to the presence of unidentified compounds which were completely degraded by 3':5'-cyclic nucleotide phosphodiesterase. Although these displaced labeled cAMP in the Gilman assay, they did not stimulate the protein kinase activity. The protonema may contain cyclic nucleotides other than cAMP; these will not be detected in the protein kinase assay due to the specificity of this reaction. The crude extracts were found to be unsuitable for assaying cAMP by either method. PMID- 16659879 TI - Changes of Template Activity and Proteins of Chromatin during Wheat Germination. AB - Relationships between changes in template activity and composition of chromatin during germination of wheat embyros (Triticum aestivum L.) were investigated. The template activity of chromatin was determined with exogenous DNA-dependent RNA polymerase II (EC 2.7.7.6) prepared from wheat embryos. It was essentially constant for 18 hours of germination, corresponding to 2.5% of that of a native calf thymus DNA. Thereafter, the activity increased 2-fold and 5-fold at 24 and 60 hours of germination, respectively.Chromatin-associated proteins were separated into at least 22 distinct bands by sodium dodecyl sulfate gel electrophoresis throughout 60 hours of germination. Significant changes were observed in two nonhistone proteins, approximate molecular weights 59,000 and 39,000: the amount of the former was constant up to 18 hours, reduced for the period from 18 to 60 hours, and that of the latter was decreased for the period from 18 to 60 hours of germination. No change was observed in the number of histone components by acid-urea gel electrophoresis. PMID- 16659880 TI - Growth of Suspension-cultured Acer pseudoplatanus L. Cells in Automatic Culture Units of Large Volume. AB - A phytostat to mass culture higher plant cells in liquid medium is described. This apparatus allowed the culture in batch, turbidostat and chemostat of 20 liters of cells. Automatic control of cell suspension growth was based on culture turbidity. Changes with time of certain cell characteristics, particularly cell respiration and phospholipid content, indicated that the test time to harvest large amounts of sycamore cells (Acer pseudoplatanus L.) in good physiological state was about 2 days before the end of the exponential phase of growth, when the cell density reached one million cells per milliliter of culture. PMID- 16659881 TI - Nutrition of a developing legume fruit: functional economy in terms of carbon, nitrogen, water. AB - The economy of functioning of the developing fruit of white lupin (Lupinus albus L.) is assessed quantitatively in relation to intake and usage of carbon, nitrogen, and water. Of every 100 units of carbon imported from the parent plant, 52 are incorporated into seeds, 37 into nonmobilizable material of the pod, and the remaining 11 lost as CO(2) to the atmosphere. An illuminated fruit can make net gains of CO(2) from the atmosphere during the photoperiods of all but the last 2 weeks of its life, suggesting that it is active in assimilation of CO(2) respired from pods and seeds. This conservation activity is important to carbon economy.Phloem supplies 98% of the fruit's carbon and 89% of its nitrogen. Most of the xylem's contribution enters early in development. Xylem and phloem supply similar sets of amino compounds, amides predominating. Ninety-six per cent of the fruit's nitrogen becomes incorporated into seeds. Sixteen per cent of the seed's nitrogen is mobilized from the senescing pod.The transpiration ratio of the fruit is 22.5 ml per gram dry matter accumulated. Xylem supplies 60% of a fruit's total water requirement and the equivalent of two-thirds of its transpiration loss. Phloem becomes prominent as a water donor once the seeds start to fill.The fruit exhibits a 31% conversion by weight of organic imports into food reserves of seeds. This entails an intake through vascular channels of 1756 mg sucrose and 384 mg amino compounds and an accumulation in seeds of 412 mg protein, 132 mg oil, and 110 mg perchloric acid-soluble carbohydrate. PMID- 16659882 TI - Induction of Acid Metabolism in Portulacaria afra. AB - Portulacaria afra, a succulent plant, shifts from a predominantly C(3) mode of gas exchange to a typical Crassulacean acid metabolism type CO(2) uptake in response to water or NaCl stress. Control plants in the absence of water stress assimilated CO(2) during the light (about 7-8 mg CO(2) dm(-2) hr(-1)), transpiration (about 1.5 g dm(-2) hr(-1)) was predominantly during the day, stomates were open during the day, and there was little diurnal organic acid fluctuation. Stressed plants showed only dark CO(2) uptake and dark water loss, nocturnal stomatal opening, and an increased diurnal fluctuation of titratable acidity. Within 2 weeks after rewatering, stressed plants returned to the control acid fluctuation levels indicating that the response to stress was reversible. PMID- 16659883 TI - Upsurge in respiration and peroxide formation in potato tubers as influenced by ethylene, propylene, and cyanide. AB - A continuous application of ethylene (10 mul/l) and propylene (500 mul/l) to potato tubers (Solanum tuberosum L.) resulted in an upsurge of respiration and a concomitant rise in peroxides. When applied in 100% O(2), the effect of ethylene and propylene on respiration and peroxide formation was augmented. Hydrogen cyanide (500 mul/l) mimicked the action of ethylene and propylene inducing a respiratory rise and a corresponding increase in peroxides. As with ethylene, the effect of HCN was augmented in high O(2) tensions. The results support the suggestion that ethylene activates the cyanide-insensitive respiratory pathway. PMID- 16659884 TI - Site of Monoterpene Biosynthesis in Majorana hortensis Leaves. AB - Excised epidermis of Majorana hortensis Moench (sweet marjoram) leaves incorporates label from [U-(14)C]sucrose into monoterpenes as efficiently as do leaf discs, while mesophyll tissue has only a very limited capacity to synthesize monoterpenes from exogenous sucrose. These results strongly suggest that epidermal cells, presumably the epidermal oil glands, are the primary site of monoterpene biosynthesis in marjoram. Using a leaf disc assay, it was demonstrated that label from [U-(14)C]sucrose is incorporated into monoterpenes most efficiently in very young leaves. PMID- 16659885 TI - Isolation and Identification of the Precursor of Ethane in Phaseolus vulgaris L. AB - Ethane production by homogenates of Phaseolus vulgaris L. cv. Harvester was studied. The precursor of ethane was identified as linolenic acid. The liberation of ethane was optimum at pH 4.2 and was highest from homogenates of leaves and apical buds. When roots were homogenized in linolenic acid solution, ethane and ethylene production were stimulated. In corn root homogenates, ethylene biosynthesis was stimulated nearly 8-fold by linolenic acid. The enzyme responsible for ethane production from oat root homogenates was soluble and had a high molecular weight. PMID- 16659886 TI - Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize. AB - Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes. PMID- 16659887 TI - Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: I. Carbon Dioxide Photoassimilation and Glycolate Synthesis. AB - The influence of pH upon the O(2) inhibition of (14)CO(2) photoassimilation (Warburg effect) was examined in intact spinach (Spinacia oleracea) chloroplasts. With conditions which favored the Warburg effect, i.e. rate-limiting CO(2) and 100% O(2), O(2) inhibition was greater at pH 8.4 to 8.5 than at pH 7.5 to 7.8. At pH 8.5, as compared with 7.8, there was an enhanced (14)C-labeling of glycolate, and a decrease of isotope in some phosphorylated Calvin cycle intermediates, particularly triose-phosphate. The (14)C-labeling of starch was also more inhibited by O(2) at higher pH. The enhanced synthesis of glycolate during (14)CO(2) assimilation at higher pH resulted in a diminution in the level of phosphorylated intermediates of the Calvin cycle, and this was apparently a causal factor of the increased severity of the Warburg effect.The (14)C-labeling profiles have been interpreted in terms of a "CO(2)"-sensitive as well as a "CO(2)"-insensitive mechanism for glycolate synthesis. Both mechanisms functioned optimally at the higher pH and both responded to O(2). PMID- 16659888 TI - Differential effect of irradiance and nutrient nitrate on the relationship of in vivo and in vitro nitrate reductase assay in chlorophyllous tissues. AB - Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity. PMID- 16659889 TI - Reversible Effects of Toxin from Helminthosporium maydis Race T on Oxidative Phosphorylation by Mitochondria from Maize. AB - Host-selective toxin from Helminthosporium maydis race T inhibited oxidative phosphorylation (AT(32)P formation) and stimulated ATPase activity by mitochondria from male-sterile (T) but not from normal (N) cytoplasm maize (Zea mays L.). Toxin increased the rate of NADH oxidation, but succinate oxidation was slightly, and malate-pyruvate oxidation was strongly inhibited as the associated ATP formation was abolished. There was a 1-minute lag before toxin gave maximal stimulation of NADH oxidation; the responses to 2,4-dinitrophenol and valinomycin were immediate. There was also a delay in the effect of toxin on ATP formation. T mitochondria were more sensitive than were N mitochondria to uncoupling by nigericin plus K(+); there was no evidence, however, that the action of toxin is related to that of nigericin or other ionophores. With NADH as the substrate, the degree of uncoupling increased with increases in toxin concentration up to a saturating level; kinetics of the response suggested reversibility. T mitochondria exposed to toxin for 5 minutes regained normal rates of respiration and of ATP formation when they were washed with toxin-free medium, showing that the uncoupling effect is reversible. Evidently HM-T toxin does not bind firmly to its site(s) of action, in contrast to reports for another hostselective toxin. PMID- 16659890 TI - Effect of aminoethoxy analog of rhizobitoxine on ripening of pears. AB - Ripening reactions in pears (Pyrus communis L.) were differentially affected by an aminoethoxy analog of rhizobitoxine (l-2-amino-4-[2-aminoethoxy]-trans-3 butenoic acid) (AAR). Ethylene production of both ;Anjou' and ;Bartlett' pears was inhibited by AAR. Decrease in firmness, increase in protein N and soluble pectin were delayed by AAR in ;Anjou' but not in ;Bartlett' pears. While loss in malic acid was retarded in ;Anjou' pears, rates of citric acid accumulation and malic acid reduction were not affected by AAR in ;Bartlett' pears. PMID- 16659891 TI - Correlation between Phenylalanine Ammonia Lyase Activity and Phenolic Biosynthesis in p-Fluorophenylalanine-sensitive and -resistant Tobacco and Carrot Tissue Cultures. AB - Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.A comparison of the responses of PAL activity to light treatment and changes in the 2,4 dichlorophenoxyacetic acid concentration in the medium revealed no differences for both tobacco lines. After inoculation of the cells into fresh medium, the rates of nitrate uptake from the medium were similar for both lines as were the rates of protein synthesis. The reason for the increased PAL levels in the resistant tobacco cells remains unknown. PMID- 16659892 TI - Auxin Transport as Related to Leaf Abscission during Water Stress in Cotton. AB - Plant water deficits reduced the basipetal transport of auxin in cotyledonary petiole sections taken from cotton (Gossypium hirsutum L.) seedings. A pulse labeling technique was employed to eliminate complications of uptake or exit of (14)C-indoleacetic acid from the tissue. The transport capacity or the relative amount of radioactivity in a 30-minute pulse which was basipetally translocated was approximately 30% per hour in petioles excised from well watered seedlings (plant water potentials of approximately -4 to -8 bars). No cotyledonary leaf abscission took place in well watered seedlings. Plant water potentials from -8 to -12 bars reduced the transport capacity from 30 to 15% per hour, and although the leaves were wilted, cotyledonary abscission did not increase appreciably at these levels of stress. The threshold water potential sufficient to induce leaf abscission was approximately -13 bars and abscission increased with increasing stress while the auxin transport capacity of the petioles remained relatively constant (15% per hour). The basipetal transport capacity of well watered petioles tested under anaerobic conditions and acropetal transport tested under all conditions were typically less than basipetal transport under the most severe stress conditions. Cotyledonary abscission took place during and 24 hours after relief of stress with little or no abscission taking place 48 hours after relief of stress. Although the water potential returned to -4 bars within hours after rewatering the stressed plants, partial recovery of the basipetal transport capacity of the petioles was not apparent until 48 hours after rewatering, and at least 72 hours was required to return the transport capacity to near normal values. These data support the view that decreased levels of auxin reaching the abscission zone from the leaf blade influence the abscission process and further suggest that the length of time that the auxin supply is maximally reduced is more critical than the degree of reduction. PMID- 16659893 TI - Subcellular localization of cytokinins in transfer ribonucleic Acid. AB - Evidence on the localization of cytokinins in chloroplast tRNA was obtained by comparison of Euglena gracilis var. bacillaris light-grown and dark-grown wild type cultures and chloroplast-bleached mutant strains. The several cytokinins characteristic of tRNA were separated by Sephadex LH-20 column chromatography of the hydrolysates and were quantitatively determined by tobacco bioassays of the eluates. The results indicate that 6-(3-methyl-2-butenylamino)-9-beta-d ribofuranosylpurine (i(6) A) is formed in both the cytoplasmic and chloroplast tRNA, whereas 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-d- ribofurano sylpurine (c-io(6)A) is produced mainly in the cytoplasmic tRNA and 6-(4-hydroxy 3-methyl-2-butenylamino)-2-methylthio-9-beta-d- ribofurano-sylpurine (ms(2)io(6)A) is localized exclusively in chloroplast tRNA. The restriction of the methiolation reaction to the chloroplast is supported by results of radioisotope experiments showing that (35)S-labeled MgSO(4) is incorporated into ms(2)io(6)A in the wild type cultures, but not in the chloroplast-bleached mutant strains. PMID- 16659894 TI - Fractionation of stable carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants. AB - The active species of "CO(2)" and the amount of fractionation of stable carbon isotopes have been determined for a partially purified preparation of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) from corn (Zea mays) leaves. The rates of the enzyme reactions, using substrate amounts of HCO(3) (-), CO(2) or CO(2) plus carbonic anhydrase, show that HCO(3) (-) is the active species of "CO(2)" utilized by PEP carboxylase. The K(m) values for CO(2) and HCO(3) (-) are 1.25 mm and 0.11 mm, respectively, which further suggest the preferential utilization of HCO(3) (-) by PEP carboxylase. The amount of fractionation of stable carbon isotopes by PEP carboxylase from an infinite pool of H(12)CO(3) (-) and H(13)CO(3) (-) was -2.03 per thousand. This enzyme fractionation (delta), together with the fractionation associated with absorption of CO(2) into plant cells and the equilibrium fractionation associated with atmospheric CO(2) and dissolved HCO(3) (-) are discussed in relation to the fractionation of stable carbon isotopes of atmospheric CO(2) during photosynthesis in C(4) plants. PMID- 16659895 TI - Photocontrol of Anthocyanin Synthesis: VI. Spectral Sensitivity, Irradiance Dependence, and Reciprocity Relationships. AB - The spectral sensitivity and the irradiance dependence of anthocyanin synthesis, a "high irradiance response," in cabbage (Brassica oleracea, cv. Red Acre) and tomato (Lycopersicon esculentum, cv. Beefsteak) seedlings exposed to continuous irradiation depend upon the length of the exposure. In cabbage, blue and red are more effective than far red when the irradiations are shorter than 12 hours and less effective than far red when the irradiations are longer than 12 hours. The irradiance dependence is negligible under red and becomes evident under blue and far red red only for exposures longer than 12 hours. Anthocyanin synthesis under intermittent light treatments, of efficiency comparable to that of continuous treatments, obeys the Bunsen-Roscoe reciprocity law and is a function of the dose (irradiance x time), rather than of the irradiance alone. The validity of the reciprocity relationships suggests that only one photoreceptor is responsible for the photocontrol of the response in the blue, red, and far red spectral regions. The characteristics of the response suggest that the photoreceptor is phytochrome, at least in cabbage. PMID- 16659896 TI - On the Relationship between Extracellular pH and the Growth of Excised Pea Stem Segments. AB - Studies with stem segments of peas (Pisum sativum L. var. Alaska) suggest that the pH of the medium bathing elongating tissue does not always reflect intramural (cell wall) conditions or that pH is not a controlling factor in elongation. Peeled, green segments, and peeled or nonpeeled etiolated segments appear to regulate the pH of their bathing medium causing it to become acidified with or without the addition of auxin. The growth rates of segments are greatest during a period before acidification is evident and slow during the time in which the medium becomes acidified. We cannot reproduce the dramatic auxin-induced pH shifts reported in the literature because the control segments are becoming more acid also; but there is some evidence that acidification may occur in response to auxin treatments. K(+) additions mimic the acidifying tendency of auxin but are without growth-promoting effect. Emergent growth (an extremely rapid burst of growth following anaerobic treatments) is not accompanied by a drop in pH of the bathing medium. Proper aeration of the bathing medium in extracellular pH studies is crucial and may explain differences between our results and other published accounts. The data suggest that the techniques used for most extracellular pH studies may not very closely approximate in vivo conditions or properly reflect intramural H(+) concentration fluxes. PMID- 16659897 TI - Hormonal control of somatic embryo development from cultured cells of caraway: interactions of abscisic Acid, zeatin, and gibberellic Acid. AB - The effects of abscisic acid, zeatin, and gibberellic acid on the development of somatic embryos from cultured cells of caraway (Carum carvi L.) were observed.Somatic embryos complete development on a basal medium without exogenous hormones, but some are subject to developmental abnormalities including malformed cotyledons and accessory embryos. Both zeatin and gibberellic acid, especially in combination, stimulate growth and increase the frequency of aberrant forms. Zeatin causes the formation of multiple shoots, leafy and abnormal cotyledons, and in the dark, enlarged hypocotyls; gibberellic acid effects root elongation, polycotyledony, and some callus formation. In contrast, abscisic acid, at concentrations which do not inhibit embryo maturation, selectively suppresses abnormal proliferations. With abscisic acid, and especially in the dark, a high percentage of embryos complete development with two fleshy cotyledons on unelongated axes free of accessory embryos.In the light, zeatin eliminates abscisic acid inhibition while gibberellic acid only partially counters its effect, promoting elongated radicles and green rather than white cotyledons. In the dark, zeatin in combination with abscisic acid stimulates extensive callusing. Gibberellic acid does not reverse the effects of abscisic acid but rather enhances them and can counter the disruptive effects of zeatin.The results demonstrate that the balance between abscisic acid on the one hand and zeatin and gibberellic acid on the other can effectively control somatic embryo development and either disrupt or ensure normal maturation. PMID- 16659898 TI - Rapid isolation of mesophyll cells from leaves of soybean for photosynthetic studies. AB - Mesophyll cells were rapidly isolated from soybean (Glycine max [L.]) leaves using a combined Macerase enzyme-stirring technique. About 50% to 70% of the leaf cells on a chlorophyll basis from 3 grams of leaves could be isolated in 15 minutes. The cells obtained by this method were capable of high rates of photosynthesis even after storage in the dark for periods of up to 9 hours. The CO(2)-saturated rate of photosynthesis increased from 5 mum CO(2)/mg Chl.hour at 5 C to 170 mum CO(2)/mg Chl.hour at 40 C. At atmospheric CO(2) concentration, the rate varied from 5 to 55 mum CO(2)/mg Chl.hour over this temperature range. The reduced temperature response of photosynthesis at low CO(2) concentration was due to an increased Km(CO(2)) of the cells with increasing temperature. The products of photosynthesis in the isolated cells were similar to the products of leaf photosynthesis. PMID- 16659899 TI - Regulation of Senescence in Carnation (Dianthus caryophyllus) by Ethylene: Mode of Action. AB - Carnation (Dianthus caryophyllus) flowers were exposed to 2 mul/l ethylene and examined at intervals to determine the time course of wilting, decrease in water uptake, and increase in ionic leakage in response to ethylene. A rapid decrease in water uptake was observed about 4 hours after initiating treatment with ethylene. This was followed by wilting (in-rolling of petals) about 2 hours later. Carbon dioxide inhibited the decline in water uptake and wilting and this is typical of most ethylene-induced responses. Ethylene did not affect closure of stomates. Ethylene enhanced ionic leakage, as measured by efflux of (36)Cl from the vacuole. This was judged to coincide with the decrease in water uptake. Gassing flowers with propylene initiated autocatalytic ethylene production within 2.4 hours. Since the increase in ethylene production by carnations preceded the increase in ionic leakage and the decline in water uptake by several hours, it is apparent that the change in ionic leakage does not lead to the initial increase in ethylene production as reported (Hanson and Kende 1975 Plant Physiol 55:663 669) in morning glory but may explain the autocatalytic phase of ethylene production. PMID- 16659900 TI - Auxin-binding Sites of Maize Coleoptiles Are Localized on Membranes of the Endoplasmic Reticulum. AB - Sites in maize (Zea mays L.) coleoptile homogenates that reversibly bind naphthalene-1-acetic acid with high affinity and may represent receptor sites for auxins are located primarily on cellular membranes that show the enzymic and buoyant density characteristics of membranes of the rough endoplasmic reticulum. The sites remain attached to the endoplasmic reticulum (ER) membranes after the ribosomes have been stripped off them. Binding sites for naphthylphthalamic acid, an inhibitor of auxin transport, are located on membranes different from those that carry the naphthalene-1-acetic-acid (NAA)-binding sites, and which are probably plasma membrane. The two kinds of binding sites can be largely separated by appropriate density gradient centrifugation. The results raise the possibility that primary auxin action occurs at ER membranes and could represent facilitation of the transfer of hydrogen ions and nascent secretory protein into the ER lumen followed by secretory transport of these products to the cell exterior via the Golgi system. PMID- 16659901 TI - Photosynthesis in c(4) plant tissue cultures: significance of kranz anatomy to c(4) Acid metabolism in c(4) plants. AB - The pattern of photosynthetic carbon metabolism was determined in tissue cultures of Portulaca oleracea. Four-carbon acids are the most heavily labeled photosynthetic products during short term exposure to (14)CO(2), containing greater than 40% of the total radioactivity incorporated. Phosphoglyceric acid and sugars account for only 10% of the label after equal exposure times. Other features of the CO(2) assimilation pattern in Portulaca callus tissue include a relatively large percentage of label located in various minor products throughout the time course studied, and a greater incorporation of (14)C into sugars in tissue cultures than occurs in leaves. Ultrastructurally, the chloroplasts and cells of the callus are like those in the mesophyll cells of Portulaca leaves. The requirement for Kranz anatomy for operation of functional C(4) physiology is discussed. PMID- 16659902 TI - Sequence arrangement in satellite DNA from the muskmelon. AB - Two fractions of a satellite DNA from the muskmelon (Cucumis melo L.) isolated as a unimodal peak from CsCl gradients, differ in melting properties and complexity as estimated by reassociation kinetics. At 49.8 C, all of the low melting fraction was denatured and all of the high melting fraction was native. There were almost no partially denatured molecules detected in the electron microscope at this temperature. This observation provides direct evidence that the two fractions are not closely linked. We conclude that satellite I, the high t(m), low complexity fraction, exists as a 600-nucleotide sequence in blocks of at least 67 tandem repeats. Since the complexity of the low melting fraction, satellite II, is greater than the size of the molecules in our assay, we can only say that the minimum size of each unit of satellite II is 2.5 x 10(7) daltons. It is unlikely that any spacer sequences are interspersed with either satellite.Sequences homologous to those of satellite I were also shown to be present as a minor fraction on 4900 nucleotide pair fragments with main band DNA density. These long main band fragments probably contain in addition at least two repeated sequence elements unrelated to satellite I since they aggregate (form large network structures) when reassociated. Coaggregation of sheared (3)H satellite I with long main band DNA could not be attributed to contamination of main band with long satellite DNA. We interpret the results as an observation of a recently created family of tandemly repeating sequences whose members are beginning to be scattered throughout the genome.We discuss how the aggregation technique may be generally useful for assessing linkage between a minor and a major DNA fraction when both fractions may be present in the initial DNA preparation. Applications for the technique include the search for DNA sequences in the nucleus which are homologous with chloroplast DNA and for Agrobacterium tumefaciens DNA in the nuclei of plant cells transformed to the tumor phenotype by the bacterium. PMID- 16659903 TI - Adenylate metabolism of embryonic axes from deteriorated soybean seeds. AB - RNA and protein syntheses in axes excised from dry soybean (Glycine max L.) seeds at different levels of deterioration were assayed. Low rates of protein synthesis in slightly deteriorated seeds were not due to losses in ribosomal or soluble fraction activities. However, the lowered rates of RNA and protein syntheses of deteriorated seeds were associated with reduced ATP content of the tissues. Adenine and adenosine conversions to ATP were reduced in deteriorated axes, and these reductions were reflected in reduced incorporation of these compounds into RNA. PMID- 16659904 TI - Phytochrome-controlled Hydrogen Ion Excretion by Avena Coleoptiles. AB - A red light-induced, far red reversible stimulation of proton efflux from apical segments of etiolated Avena sativa L. cv. Victory coleoptiles was observed. The acidification responses to red light and also to auxin were not the consequence of respired CO(2). The response to red light was strongly inhibited by cycloheximide and carbonyl cyanide, m-chlorophenyl hydrazone, but mannitol had a stimulatory effect. Red light and auxin applied together yielded a greater than additive response, in comparison to the effects of the two stimuli applied separately. PMID- 16659905 TI - Characterization of Phosphoenolpyruvate Carboxykinase from Pineapple Leaves Ananas comosus (L.) Merr. AB - Phosphoenolpyruvate carboxykinase has been partially purified from pineapple (Ananas comosus [L.]) leaves. Specific activities obtained show it to be a major activity in this tissue. Above 15 C, the respective activation energies for decarboxylation and carboxylation are 13 and 12 kcal/mol. Below 15 C, there are discontinuities in Arrhenius plots with an associated large increase in activation energy. The adenine nucleotides are preferred to other nucleotides as substrates. The apparent Km values in the carboxylation direction are: ADP 0.13 mm, HCO(3) (-) 3.4 mm, and phosphoenolpyruvate 5 mm. In the decarboxylation direction, the apparent Km values are: ATP 0.02 mm, ADP 0.05 mm, and oxaloacetate 0.4 mm. The decarboxylation activity had an almost equal velocity with either ADP or ATP. The pH optima are between 6.8 and 7. Inhibition of the carboxylation reaction by ATP, pyruvate, and carbonic anhydrase was demonstrated. Decarboxylase specific activities are over twice carboxylation activities. The data support a model in which phosphoenolpyruvate carboxykinase is of physiological significance only during the light period and then only as a decarboxylase. PMID- 16659906 TI - Wheat mitochondria: oxidative activity and membrane lipid structure as a function of temperature. AB - Mitochondrial oxidative activity and membrane lipid structure of two wheat (Triticum aestivum L.) cultivars were measured as a function of temperature. The Arrhenius activation energy for the oxidation of both succinate and alpha ketoglutarate was constant over the temperature range of 3 to 27 C. The activation energy for succinate-cytochrome c oxidoreductase activity was also constant over the same temperature range. The concentration of mitochondria in the reaction, the degree of initial inhibition of state 3 respiration, and the time after isolation of mitochondria were each shown to be capable of causing a disproportionate decrease in the rate of oxidation at low temperatures which resulted in an apparent increase in the activation energy of oxidative activity. Using three spin-labeling techniques, wheat membrane lipids were shown to undergo phase changes at about 0 C and 30 C. It is concluded that the membrane lipids of wheat, a chillingresistant plant, undergo a phase transition similar to the transition observed in the membrane lipids of chilling-sensitive plants. For wheat, however, the transition is initiated at a lower temperature and extends over a wider temperature range. PMID- 16659907 TI - In vivo measurement of phytochrome in tomato fruit. AB - Presence of phytochrome in two kinds of tomatoes (Lycopersicon esculentum Mill.), the yellow lutescent strain and cherry tomatoes (L. esculentum Mill. var. cerasiformecv. Red Cherry), was established by measuring the absorption difference spectra of the whole fruit after irradiation with red and with far red light. Phytochrome content was determined in yellow lutescent tomatoes and decreased gradually during the ripening period. PMID- 16659908 TI - Pyruvate and malate transport and oxidation in corn mitochondria. AB - Pyruvate oxidation and swelling in pyruvate solutions by corn (Zea mays) mitochondria were inhibited by alpha-cyano-4-hydroxy-cinnamic acid, an inhibitor of pyruvate transport in animal mitochondria; however, there was no inhibition of pyruvate dehydrogenase activity, and malate and NADH oxidation were not affected. These results suggest the presence of a pyruvate(-)-OH(-) exchange transporter which supplies the mitochondrion with oxidizable substrate. Lactate appears to be transported also, but not dicarboxylate anions or inorganic phosphate. The rate of pyruvate transport was much slower than that of malate, however, and valinomycin was required to elicit appreciable swelling in potassium pyruvate.Malate oxidation contributed significantly to respiration supported by pyruvate plus malate, and malate did not act solely as a "sparker" for pyruvate oxidation. NAD(+)-malic enzyme activity was found in sonicated preparations, and comparison of O(2) consumption with CO(2) released from 1-(14)C-pyruvate indicated that transported malate was being converted to pyruvate, particularly as the malate to pyruvate ratio increased. The results suggest that pyruvate transport becomes limiting under conditions of high energy demand, but that rapid malate transport makes up the difference, supplying pyruvate via malic enzyme and replenishing losses of tricarboxylic acid cycle intermediates. PMID- 16659909 TI - Effects of increased gravity force on nutations of sunflower hypocotyls. AB - A centrifuge was used to provide sustained acceleration in order to study the hypocotyl nutation of 6-day-old Helianthus annuus L. over a range of g-forces, up to 20 times normal g. At the upper end of this g-range, nutation was impeded and at times was erratic evidently because the weight of the cotyledons exceeded the supportive abilities of the hypocotyls.Over the range 1 to 9 g, the period of nutation was independent of the resultant force vector. Over the same g-range, the amplitude of nutation was nearly independent of the chronic g-force.If nutation in sunflower seedlings is an oscillation caused by a succession of geotropic responses which continue to overshoot the equilibrium position (plumb line), we might expect its amplitude to be more sensitive to changes in magnitude of the sustained g-force. In order to preserve the geotropic model of nutation viz. that it is a sustained oscillation driven by geotropic reactions, it is necessary to assume that geotropic response must increase with increasing g most rapidly in the region of the g-parameter below the terrestrial value of 1 g. PMID- 16659910 TI - Selectivity of alkali cation influx across the plasma membrane of oat roots: cation specificity of the plasma membrane ATPase. AB - Influx of alkali cations (Li(+), Na(+), K(+), Rb(+), Cs(+)) across plasma membranes of cells of excised roots of Avena sativa cv. Goodfield was selective, but different, in the absence and in the presence of 1 mm CaSO(4). Ca(2+) reduced the influx rates of all of the alkali cations-especially Na(+) and Li(+). Transport selectivity changed as the external concentrations of the alkali cations increased.Plasma membrane ATPase, purified from Avena sativa roots, was differentially stimulated by alkali cations. This specificity, however, was not altered by Ca(2+) or the external cation concentrations. A close correspondence existed between the relative influx rates of K(+), Rb(+), and Cs(+) and the relative stimulation of the ATPase by these cations. A similar correspondence did not occur for Na(+) and Li(+).Selective cation transport in oat roots could result, in part, from the specificity of the plasma membrane ATPase, but other factors such as specific carriers or porters or differential diffusion rates must also be involved. PMID- 16659911 TI - Subcellular localization of the cyanogenic glucoside of sorghum by autoradiography. AB - The use of microautoradiography at the electron microscopic level indicates that the vacuole is the site of accumulation of the cyanogenic glucoside of Sorghum bicolor. When a specific biosynthetic precursor of dhurrin, p-hydroxy[3,5 (3)H]phenylacetaldoxime, was used, 90% of the tritium label was recovered in the vacuoles of tissue prepared for microautoradiography. l-[3,5-(3)H]Tyrosine and d [1-(3)H(N)]glucose, nonspecific precursors of dhurrin, of differing solubilities and biosynthetic capacity, were also fed to the shoots. The data obtained from these controls indicated that the high recovery of label in the vacuole of aldoxime-fed shoots was not indicative simply of the size of the vacuole, nor was it a result of movement of labeled compounds during preparation of the tissue for electron microscopy. The problem of movement of these labeled compounds during dehydration of tissue was dramatically reduced by chemical dehydration in 2,2 dimethoxypropane in less than 30 minutes rather than with routine dehydration in acetone or alcohol series for 24 hours. PMID- 16659912 TI - myo-Inositol Metabolism in Lilium longiflorum Pollen: Uptake and Incorporation of myo-Inositol-2-H. AB - Germinating Lilium longiflorum pollen absorbs and metabolizes myo-inositol-2-(3)H (MI-2-(3)H) with a pronounced lag when label is supplied from the beginning of germination. If MI-2-(3)H is given after 3 hours of germination, incorporation of labeled metabolic products into pollen tube polysaccharides is constant over a range of 0.56 mm to 2.78 mm MI. When MI-2-(3)H is supplied as a 0.5-hour pulse 3 hours after germination, the proper precursor-product relationship to tube wall polysaccharides is observed. Replacing 10% of the germination media with sigmatic exudate from a compatible cultivar hastens germination and tube elongation. Enhanced MI metabolism accompanies tube growth in this exudate-enriched media. PMID- 16659913 TI - Effect of a myo-Inositol Antagonist, 2-O, C-Methylene-myo-Inositol, on the Metabolism of myo-Inositol-2-H and d-Glucose-1-C in Lilium longiflorum Pollen. AB - 2-O,C-Methylene-myo-inositol (MMO), a myo-inositol (MI) antagonist, inhibits germination and tube elongation of pollen from Lilium longiflorum cv. Ace or 44. The presence of 5 mm MMO in Dickinson's pentaerythritol medium (Plant Physiol. 43:1-8) partially blocks germination. The tubes produced are short and fail to elongate. In the presence of MI, MMO's toxic effect is blocked. As little as 0.56 mm MI will maintain normal germination in the presence of 43 mm MMO, and pollen tubes continue to elongate for 2 to 3 hr. Eventually, the toxic action of MMO prevents further growth. MMO does not inhibit UDP-d-glucose dehydrogenase from lily pollen.Uptake of MI-2-(3)H and incorporation of tritium into galacturonic acid and pentose units of tube wall pectin are blocked by MMO. The site of this inhibition is undertermined. Uptake of d-glucose-1-(14)C and incorporation of (14)C into 70% ethyl alcohol-insoluble polysaccharides of germinated pollen are not blocked by MMO, but distribution of label into polysaccharide product is altered. In MMO-treated pollen, very little (14)C is found in uronic acid or pentose units. At 30 mm MMO, about two-thirds of the carbon flow from d-glucose to these pectic components is interrupted. MMO also alters d-glucose metabolism in the 70% ethyl alcohol-soluble fraction, but the compound involved must still be identified.These results offer fresh evidence of an intermediary role for MI during UDP-d-glucuronate biosynthesis in germinated pollen. PMID- 16659914 TI - Measurement of the Cytoplasmic pH in Nitella translucens: Comparison of Values Obtained by Microelectrode and Weak Acid Methods. AB - A comparison has been made between the use of two types of pH microelectrode and the weak acid method for determining the cytoplasmic pH of Nitella translucens at an external pH of 6. There was good agreement between the value obtained with glass pH microelectrodes (7.54 +/- 0.15 se) and that obtained using the weak acid 5,5-dimethyloxazolidine-2,4-dione (7.42 +/- 0.07 se). Plastic-insulated antimony microelectrodes gave a significantly lower value (6.74 +/- 0.15 se) possibly due to disruption of the insulation by the cell wall. The addition of 1 mM NaN(3) rapidly reduced the pH recorded by the glass pH microelectrodes to about 5.3. A smaller change was observed using the weak acid method. The relevance of this observation to recent work on indoleacetic acid transport is discussed. PMID- 16659915 TI - Biochemistry of Suberization: omega-Hydroxyacid Oxidation in Enzyme Preparations from Suberizing Potato Tuber Disks. AB - A cell-free extract obtained from suberizing potato (Solanum tuberosum L.) tuber disks catalyzed the conversion of 16-hydroxy[G-(3)H]hexadecanoic acid to the corresponding dicarboxylic acid with NADP or NAD as the cofactor, with a slight preference for the former. This omega-hydroxyacid dehydrogenase activity, located largely in the 100,000g supernatant fraction, has a pH optimum of 9.5. It showed an apparent Km of 50 muM for 16-hydroxyhexadecanoic acid. The dehydrogenase activity was inhibited by thiol reagents, such as p-chloromercuribenzoate, N ethylmaleimide, and iodoacetamide, and this dehydrogenase is shown to be different from alcohol dehydrogenase. That 16-oxohexadecanoic acid was an intermediate in the conversion of 16-hydroxyhexadecanoic acid to the corresponding dicarboxylic acid was suggested by the observation that the cell free extract also catalyzed the conversion of 16-oxohexadecanoic acid to the dicarboxylic acid, with NADP as the preferred cofactor. The time course of development of the omega-hydroxyacid dehydrogenase activity in the suberizing potato disks correlated with the rate of deposition of suberin. Experiments with actinomycin D and cycloheximide suggested that the transcriptional processes, which are directly related to suberin biosynthesis and omega-hydroxyacid dehydrogenase biosynthesis, occurred between 72 and 96 hours after wounding. These results strongly suggest that a wound-induced omega-hydroxyacid dehydrogenase is involved in suberin biosynthesis in potato disks. PMID- 16659916 TI - Changes in Enzyme Regulation during Growth of Maize: III. Intracellular Localization of Homoserine Dehydrogenase in Chloroplasts. AB - Extracts of leaf tissue of Zea mays L. seedlings were fractionated on nonlinear sucrose gradients to separate subcellular organelles. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact chloroplasts, as judged by the presence of chlorophyll and triosephosphate isomerase activity. Neither enzyme activity was detected in fractions containing ruptured chloroplasts, mitochondria, or microbodies. Quantitative measurements of enzyme activity and chlorophyll, and electron microscopic analysis of plastid preparations support the conclusion that maize mesophyll chloroplasts contain a significant fraction of the total cellular content of homoserine dehydrogenase.A survey of representative kinetic, regulatory, and physical properties did not reveal any significant differences between enzyme released from isolated, undamaged chloroplasts and that obtained from soluble cellular fractions.Examination of enzyme prepared from chloroplasts of different age seedlings indicated that the sensitivity of homoserine dehydrogenase to inhibition by the feedback modifier l-threonine was progressively diminished during growth of the plants. This systematic change in regulatory properties of the enzyme occurred to the same extent for the enzymes obtained from chloroplasts and soluble fractions. PMID- 16659917 TI - Light-induced Adhesion of Spirogyra Cells to Glass. AB - Adhesion of Spirogyra (tentatively, Spirogyra fluviatilis) cells to glass is described. The cells of an algal filament can adhere to a substrate only when they are located at the end of the filament. Rapid adhesion is induced by blue violet light (blue adhesion) as well as by temperature shift (about 6 C --> about 22 C) or shaking (dark adhesion). Adherent cells detach in 1 hour in the absence of one of these stimuli. Slow adhesion is induced by red light (red adhesion) 1 hour after irradiation, and may be controlled by phytochrome. A cell once caused to adhere by red light does not release from the glass.Adhesion seems to be maintained by a cementing substance, probably qa mucoprotein. A transparent material which appears around the tip of the cell may be the cementing substance. PMID- 16659918 TI - Formation of N-acetylglutamate by extracts of higher plants. AB - The enzymic synthesis of N-acetylglutamate was studied in extracts of higher plant tissues, especially in sugar beet leaves (Beta vulgaris L.). Sugar beet leaves had an enzyme that transferred the acetyl group either from acetyl-CoA or from N(2)-acetylornithine to glutamate. The enzyme was so unstable that special precautions were necessary for its detection and appreciable purification was impossible. The Km values were 2.5 and 0.025 mM for acetyl-CoA and N(2) acetylornithine, respectively. The Km for glutamate was 23 mM with acetylornithine-glutamate transacetylase and 2.7 mM with acetyl-CoA-glutamate transacetylase. The pH optimum for acetyl-CoA-glutamate transacetylase was about 7.2 whereas that for acetylornithine-glutamate transacetylase was about 8.3. Acetylphosphate, N(2)-acetyl-2,4-diaminobutyrate, propionyl-CoA, and succinyl-CoA were not substrates.Arginine inhibited the acetyl-CoA-glutamate transacetylase and acetylglutamate phosphokinase but had no effect on the acetylornithineglutamate transacetylase. Related compounds had either no effect or much less than arginine. Arginine had no effect on enzyme levels.Acetyl-CoA glutamate transacetylase was also found in Raphanus sativus L., Glycine max L. Merr., Arachis hypogaea L., Brassica rapa L., and Pisum sativum L. Acetylornithine-glutamate transacetylase was found in all of the above species plus Zea mays L., Avena sativa L., and Triticum aestivum L. PMID- 16659919 TI - Metabolic regulation of glycolate synthesis, photorespiration, and net photosynthesis in tobacco by L-glutamate. AB - Experiments were undertaken to identify and characterize control mechanisms in tobacco leaf tissue which decrease the relative contribution of photorespiratory CO(2) release and thereby increase net photosynthetic CO(2) fixation. A number of metabolites were supplied to illuminated leaf discs and their effect on the inhibition of glycolate synthesis was measured. Glycolate accumulation, in the presence of alpha-hydroxy-2-pyridinemethanesulfonic acid, was inhibited in leaf discs previously floated on 30 mM solutions of either L-glutamate, L-aspartate, phospho-enolpyruvate, or glyoxylate. The effect of glutamate on glycolate synthesis, which was investigated in detail, was concentration- and time dependent. Glycolate synthesis was inhibited about 40% by treating leaf discs with 30 mM glutamate, and the inhibition continued for more than 4 hours after the glutamate solution was removed.The glutamate inhibition of glycolate synthesis was accompanied by a marked decrease in the rate of photorespiratory CO(2) release and by maximal increases of about 25% in net photosynthetic CO(2) fixation. The products of (14)CO(2) fixation in leaf discs previously treated with glutamate showed a decrease in glycine (26%), serine (12%), and the stronger acids (18%), and an increase in the neutral compounds (26%) in comparison with discs floated only on water.Data are presented which question whether a catabolite of glutamate or the amino acid itself is responsible for the results observed. These experiments support the view that a genetic selection strategy based on the metabolic control of photorespiration would result in large increases in net photosynthetic CO(2) assimilation in species with high rates of photorespiration. PMID- 16659920 TI - Characterization of two isoenzymes of lipoxygenase from bush beans. AB - Two isoenzymes of lipoxygenase, a and b, have been obtained from bush beans (Phaseolus vulgaris) as electrophoretically homogeneous proteins. Both proteins have a molecular weight of 100,000, contain 1 atom of iron, and appear to be composed of a single peptide chain. However, these enzymes appear to differ in some other respects. Thus, lipoxygenase a has an isoelectric point of 6.03 while lipoxygenase b has a value of 5.57. Their pH optima are 5 to 7 and 6.5 to 7, respectively. Both lipoxygenase a and b, when acting on linoleic acid plus the product hydroperoxide, generate what are presumably keto-dienes with an absorption maximum at 280 nm. Whereas lipoxygenase a can catalyze this secondary reaction in the presence of O(2), lipoxygenase b does so only under anaerobic conditions. Lipoxygenase a is stimulated by Ca(2+) while lipoxygenase is not. An unexpected finding is the strong inhibition of lipoxygenase a by Mn(2+) (50% inhibition at 12.5 muM under standard reaction conditions). Lipoxygenase b is inhibited by Mn(2+) but only at concentrations about 250 times greater. PMID- 16659921 TI - Estimation of Osmotic Gradients in Soybean Sieve Tubes by Quantitative Autoradiography: Qualified Support for the MUnch Hypothesis. AB - An attempt was made to evaluate Munch's hypothesis of osmotically generated pressure flow in soybean (Glycine max L.) sieve tubes from velocity measurements and calculations of pressure potentials and sieve tube resistances. Pressure potential was estimated from values for water potentials and osmotic potential. Leaf water potential measurements were made by isopiestic thermocouple psychrometry, while the water potential of the nutrient solution was made with a vapor pressure osmometer. Osmotic potential was measured by first bringing the sucrose pools in the entire plant to the same specific radioactivity by steady state-labeling of the shoot with constant specific radioactivity (14)CO(2) for 5 to 8 hours. Sucrose concentrations in sieve tubes were calculated from the disintegration rate per unit volume in sieve elements as measured by absolute quantitative microautoradiography of freeze-substituted, Eponembedded source (leaf) and sink (root) tissues.Conductivity of the sieve tubes was calculated from measurements of their dimensions in the petiole, stem, and root. The total pressure drop required for pressure flow at the observed velocities was calculated from the conductivity, velocity, and path length.In all experiments, the calculated sucrose concentration in source sieve tubes was greater than that in sink sieve tubes, with an average ratio (source to sink) of 1.79:1. However, the absolute sucrose concentrations (average values of 46.4 mg cm(-3) in the source and 23.9 mg cm(-3) in the sink) would have been insufficient to maintain positive turgor in the sieve elements, and the expected pressure differences would not have accounted for movement at the observed velocities. However, the low values for sucrose concentrations almost certainly were due to loss of sucrose during tissue preparation but, for technical reasons, such loss could not be accurately quantified.Assuming a sucrose concentration sufficient to maintain zero turgor in the root sieve tubes, a xylem water potential gradient (psi(w) [sink] - psi(w) [source]) of 2 bars between source and sink, and the measured ratio of sucrose concentrations in source and sink (1.79:1), the average turgor gradient between source and sink (psi(p) [sink] - psi(p) [source]) would have been about -1.6 to -3.5 bars, which compares favorably with the -1.07 to -2.41 bars average gradient that would have been required to drive translocation at the observed velocities. PMID- 16659922 TI - Role of cytokinins in carnation flower senescence. AB - Stem and leaf tissues of carnation (Dianthus caryophyllus) plants appear to contain a natural antisenescence factor since removal of most of these tissues from cut carnation flowers hastened their senescence. However, kinetin (5-10 mug/ml) significantly delayed senescence of flowers with stem and leaf tissues removed. In addition, the life span of cut flowers with intact (30-cm) stems was increased with kinetin treatment. Peak ethylene production by presenescent flowers was reduced 55% or more with kinetin treatment and was delayed by 1 day. Kinetin-treated flowers were less responsive to applied ethylene (100 mul/l for 3 hours) than untreated flowers. Possible natural roles of cytokinins in carnation flower senescence are discussed. PMID- 16659923 TI - Abscission responses to moisture stress, auxin transport inhibitors, and ethephon. AB - The three abscission-inducing agents - water stress, Ethephon, and auxin transport inhibitors-acted synergistically to promote leaf fall in cotton (Gossypium hirsutum L.). However, the synergism was primarily between stress and Ethephon. Auxin transport inhibitors did not promote the effect of stress alone, only promoted the effect of Ethephon in well watered plants and gave a very small promotion with stress and Ethephon together. Abscission was rapid in stressed plants treated with Ethephon and an auxin transport inhibitor, while leaves fell more slowly from well watered plants treated with Ethephon alone. This suggests that water stress or auxin transport inhibitors influence initial events in abscission; since an auxin transport inhibitor will replace the effect of stress but not Ethephon, an initial event in stress-induced abscission appears to be inhibition of auxin transport. Ethephon promoted lateral bud release, and auxin transport inhibitors did not duplicate that effect alone or promote it in combination with Ethephon. PMID- 16659924 TI - UDP-glucose: Glucan Synthetase in Developing Cotton Fibers: I. Kinetic and Physiological Properties. AB - A uridine diphosphate(UDP)-glucose:glucan synthetase can be demonstrated in detached cotton fibers (Gossypium hirsutum L.) and in an isolated particulate fraction from such fibers. When assayed with detached fibers, the kinetics of the glucan synthetase activity with respect to variation in substrate concentration is complex and indicates activation of the enzyme by the substrate. Activity is stimulated by Ca(2+) or Mg(2+) and beta-linked glucosides; the effect of the beta linked glucosides is to shift the range in which substrate activation occurs to lower concentrations of UDP-glucose. At concentrations of UDP-glucose below 50 mum, addition of uridine triphosphate, in addition to beta-linked glucoside, results in significant stimulation of activity. This effect can be explained by the conversion of uridine triphosphate to UDP-glucose by UDP-glucose pyrophosphorylase, thereby raising substrate concentration to the activating range. In detached fibers, glucan synthetase activity is high at all stages of fiber development. The properties of the glucan synthetase of the isolated particulate fraction closely resemble those of the enzyme assayed in detached fibers; however, in contrast to detached fibers, the ability to detect enzyme activity is more dependent on fiber age, showing maximal activity between 16 and 18 days postanthesis, coincident with the time of rapid onset of secondary wall cellulose deposition. PMID- 16659925 TI - UDP-glucose: Glucan Synthetase in Developing Cotton Fibers: II. Structure of the Reaction Product. AB - The solubility properties, composition, and structure of the radioactive product synthesized from UDP-[(14)C]glucose by a highly active cotton fiber glucan synthetase have been determined. Product obtained under the following three different conditions was analyzed: at high and low substrate concentrations by detached fibers, and at high substrate concentrations with an isolated particulate preparation. The results of acetic and nitric acid digestion, enzyme digestion, total acid hydrolyses, periodate oxidation, partial acid hydrolyses, and methylation analyses all support the conclusion that the product of the glucan synthetase produced under all three assay conditions is a linear beta-(1- >3)-glucan. PMID- 16659926 TI - Interaction of chloroplasts with inhibitors: induction of chlorosis by diuron during prolonged illumination in vitro. AB - A primary symptom of diuron (DCMU) phytotoxicity in plants is the destruction of chlorophyll. To study this process in vitro, chloroplasts from pea leaves (Pisum sativum L.) have been incubated in the light with DCMU for periods of up to 34 hours. The sequence of photodestruction of chlorophylls and carotenoids has been followed to try and establish the nature of the chloroplast protection mechanisms that are destroyed by DCMU. beta-Carotene decays most rapidly, followed by chlorophyll a and xanthophylls which are destroyed in a constant ratio, followed finally by chlorophyll b. Bypassing the DCMU block in the electron transport system with an artificial electron donor provides complete protection against chlorophyll and carotenoid photodestruction. The same protection by this electron donor system is afforded to stroma-free lamellae from which soluble reductants have been removed so that NADPH formation, which has been proposed as an essential part of a protective xanthophyll cycle, is not possible. Both this and the simultaneous loss of chlorophyll a and xanthophylls tend to preclude the breakdown of a xanthophyll cycle from the possible protective mechanisms inhibited or destroyed by DCMU.Cofactors of cyclic electron transport also protect against DCMU-induced photodestruction of pigments. Their concentration dependence for this protection appears to reflect their various abilities to catalyze cyclic photophosphorylation. The extent to which the chlorophylls are destroyed in the major pigment-protein complexes from chloroplasts illuminated with and without DCMU has been measured. In the absence of DCMU, the light harvesting chlorophyll a/b protein complex is destroyed most rapidly. In the presence of DCMU, the losses of chlorophyll a from the photosystem I P700 chlorophyll a protein and the chlorophyll a/b complex are about the same. Chlorophyll losses are matched by simultaneous losses of the protein moieties; spectral analyses show that the remaining chlorophyll a is held in a loose association with the protein. Phenazine methosulfate protects the chlorophyll of the light-harvesting complex in DCMU-treated chloroplasts more than it protects that in photosystem I. Data published on DCMU-induced fluorescence and its quenching are used to interpret the longer term DCMU-induced chlorosis and its protection. By blocking electron transport, conformational changes in the membrane that allow spillover of excitation energy from photosystem II to photosystem I (and quenching of fluorescence by this means) are prevented. The mechanism that normally protects the chloroplast against excessive illumination is then overloaded which impairs the harmless dissipation of absorbed light energy; consequently, the pigments are destroyed. When photosystem I is allowed to function again through cyclic electron flow, a necessary conformational change is believed to be reintroduced that once again allows the harmless dissipation of excitation energy through spillover. A functional electron transport system associated with photosystem I will protect against DCMU-induced chlorosis when the thylakoid membranes are intact, but when the P700-chlorophyll a protein complex is in isolation, there is only a limited degree of protection. PMID- 16659927 TI - Storage Protein Synthesis in Maize: III. Developmental Changes in Membrane-bound Polyribosome Composition and in Vitro Protein Synthesis of Normal and Opaque-2 Maize. AB - Zein synthesis accompanied an increase in large polyribosomes of maize (Zea mays) endosperm cells. The two classes of polyribosomes (free and membrane-bound) had dissimilar size class distributions. Membrane-bound polyribosomes were predominantly large size classes, which were not found in free polyribosomes. The ratio of large membrane-bound polysomes to total membrane-bound polysomes was highest when zein was being synthesized. Appearance of the large polysomes correlated with the onset of zein accumulation in vivo. These large size classes were nearly absent in the opaque-2 mutant at all stages of endosperm development. Similarly, rRNA content was reduced in the mutant from that in normal endosperm development. These differences were associated with reduced in vitro synthesis and in vivo accumulation of zein. PMID- 16659928 TI - Alpha-ketoglutarate supply for amino Acid synthesis in higher plant chloroplasts: intrachloroplastic localization of NADP-specific isocitrate dehydrogenase. AB - Isocitrate dehydrogenase was found in Pisum sativum chloroplasts purified on sucrose density gradients. A chloroplast-enriched pellet obtained by differential centrifugation formed two chlorophyll-containing bands. The lower one containing intact chloroplasts had NADP-specific isocitrate dehydrogenase and triose phosphate isomerase activities. Mitochondria and peroxisomes were observed to band well away from the intact chloroplast region, as indicated by peak activities of fumarase and catalase, respectively. The presence of isocitrate dehydrogenase in chloroplasts suggests that chloroplasts may generate at least some of the alpha-ketoglutarate required for glutamate synthesis. PMID- 16659929 TI - Changes in the Number, Viability, and Amino-acid-incorporating Activity of Rhizobium Bacteroids during Lupin Nodule Development. AB - Between days 10 and 21 after inoculation of Lupinus angustifolius seedlings with Rhizobium NZP 2257, the average nodule fresh weight increased 3-fold and the number of bacteroids per nodule increased more than 10-fold.The viability of Rhizobium bacteroids, as judged by their ability to form colonies on yeast extract agar, declined from about 10% on days 10 and 11 after inoculation to about 0.3% on days 14 to 25. Bacteroid viability was highly sensitive to osmolarity.At optimal pH and K and Mg ion concentrations, the incorporation of (14)C-glycine into isolated bacteroids was also very sensitive to osmolarity, and fell in parallel with bacteroid viability during nodule development.WE SUGGEST THAT AT LEAST TWO PROCESSES CONTRIBUTE TO BACTEROID NONVIABILITY: a reversible change in the cell wall structure occurring between days 10 and 14 after inoculation, and a subsequent irreversible change. PMID- 16659930 TI - Emergent growth: an auxin-mediated response. AB - Restoration of oxygenated conditions following 15 minutes to 2 hours of anoxia causes light-grown pea (Pisum sativum L. var. Alaska) stem segments to elongate 100 to 200% more than continuously aerated segments. This "emergent growth" response takes place in the presence of 5 mm F(-), an inhibitor of anaerobic respiration; therefore, a build-up of glycolytic products does not appear to be the mechanism underlying emergent growth. "Acid growth" does not appear to account directly for the hyperelongation, as extracellular pH does not drop following a return to aerobic conditions. Studies with (14)C-indoleacetic acid indicate that auxin is freed from some previously unavailable pool during O(2) limited treatments. We suggest, therefore, that emergent growth is a response to auxin which is released during anaerobiosis: the newly mobile or diffusible auxin promoting growth when O(2) is no longer limiting. PMID- 16659931 TI - Phloem Loading of Sucrose: pH Dependence and Selectivity. AB - Autoradiographic, plasmolysis, and (14)C-metabolite distribution studies indicate that the majority of exogenously supplied (14)C-sucrose enters the phloem directly from the apoplast in source leaf discs of Beta vulgaris. Phloem loading of sucrose is pH-dependent, being markedly inhibited at an apoplast pH of 8 compared to pH 5. Kinetic analyses indicate that the apparent K(m) of the loading process increases at the alkaline pH while the maximum velocity, V(max), is pH independent. The pH dependence of sucrose loading into source leaf discs translates to phloem loading in and translocation of sucrose from intact source leaves. Studies using asymmetrically labeled sucrose (14)C-fructosyl-sucrose, show that sucrose is accumulated intact from the apoplast and not hydrolyzed to its hexose moieties by invertase prior to uptake. The results are discussed in terms of sucrose loading being coupled to the co-transport of protons (and membrane potential) in a manner consistent with the chemiosmotic hypothesis of nonelectrolyte transport. PMID- 16659932 TI - Carbon Dioxide and Ethylene Control of Spore Germination in Onoclea sensibilis L. AB - Regulation of spore germination in the fern Onoclea sensibilis L. was investigated by applying CO(2) alone and in combination with ethylene. Sterile spores were sown aseptically on Knops solution in loosely capped culture tubes, enclosed individually in 2-liter chambers, and grown under continuous white light. When maintained in enclosed containers with the ethylene-absorbent mercuric perchlorate and with atmospheres enriched up to 2% CO(2) (v/v), spores germinated without any inhibition. Higher levels of applied CO(2) were progressively inhibitory. Inhibition by CO(2) was reversible. When CO(2) was permitted to escape and spores were exposed subsequently to ambient laboratory air, recovery from inhibition occurred within 48 hours. Also, inhibition by CO(2) was specific, since the same degree of inhibition resulted regardless of whether spores were treated with exogenous CO(2) for 48, 72, or 96 hours. The effect on germination of 1 mul/l added ethylene depended upon the amount of applied CO(2). When containers of KOH were enclosed and ambient CO(2) was absorbed, inhibition of germination by 1 mul/l exogenous ethylene was 90%. When CO(2) was applied in concentrations from 0.25 to 1.0% (v/v), CO(2) increasingly antagonized the inhibitory action of 1 mul/l added ethylene. Thus, photoinduced germination of spores was regulated by competitively interacting levels of CO(2) and ethylene. PMID- 16659933 TI - Failure of lactoperoxidase to iodinate specifically the plasma membrane of cucurbita tissue segments. AB - An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable (125)I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g . cm(-3). The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and (125)I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane.Autoradiographic analysis indicates, however, that the (125)I is almost exclusively associated with an amorphous film (possibly phloem-exudate protein) overlying the cut cells at the point of prelabeling excision and along the outer physical surface of the hypocotyl cuticle. No evidence of plasma membrane labeling is apparent. The observed membrane-like behavior of the iodinated material upon cell fractionation is attributed to the preferential posthomogenization association of this material with a particular membrane fraction(s). These data indicate that in addition to the well recognized potential for spurious labeling of the internal cytoplasmic proteins of leaky cells, a further source of ambiguity in surface-labeling experiments should be considered. That is, the potential for labeling extracellular proteins of nonplasma membrane origin but with a capacity to become associated with membranes upon homogenization. PMID- 16659934 TI - Light-induced Polysome Formation in Etiolated Leaves: Kinetics of Inhibition by Antibiotics. AB - Red light-induced, far red light-reversible increase in etiolated bean (Phaseolus vulgaris, var. Asgrow Valentine) leaf polyribosomes was shown to be sensitive to actinomycin D, cycloheximide, and rifampicin inhibition. Actinomycin prevented response to red light if administered simultaneously with a 10-minute illumination, had no immediate effect if given 2 hours after illumination, but was again rapidly inhibitory at 4 and 6 hours. The effects of actinomycin and far red light were more than additive.Cycloheximide consistently inhibited polysome formation within 1 hour at all times tested.Formation of both cytoplasmic and organelle polysomes was depressed by rifampicin, an antibiotic whose action is primarily on RNA synthesis in organelles.The results are interpreted to show that light-triggered polysome formation exhibits a changing need for RNA synthesis and an obligate dependence on protein synthesis during the 1st hours following brief illumination. PMID- 16659935 TI - Carbon dioxide compensation values in citronella and lemongrass. AB - Carbon dioxide compensation values of mature leaves from 10 selections of citronella (Cymbopogon nardus [L.] Rendle) grown at 32/27 or 27/21 C day/night temperatures and three strains of lemongrass (Cymbopogon citratus [D.C.] Stapf. and Cymbopogon flexuosus [D.C.] Stapf.) grown at 8- or 15-hour photoperiods were measured in a controlled environment at 25 C. All leaves had low compensation values but citronella varied from 1.3 to 9.7 mul/liter and lemongrass from 0.7 to 3.5 mul/liter. Lower growing temperature generally resulted in lower compensation values for citronella but there was no consistent photoperiod effect on lemongrass. PMID- 16659936 TI - A possible new nucleoside diphosphatase from the cytosol of soybean and alfalfa nodules. AB - Nucleoside diphosphatase activity has been found in the cytosol of soybean (Glycine max) and alfalfa (Medicago satavia) nodules. The enzyme differs from other diphosphatases in that it does not exhibit a strong preference for one nucleoside diphosphate over another. Very little, if any, diphosphatase activity was detected in root extracts of alfalfa and soybean seedlings. PMID- 16659937 TI - Auxin Has No Effect on Modification of External pH by Soybean Hypocotyl Cells. AB - The cellular adjustment of the pH of the external environment of soybean (Glycine max) hypocotyl elongating cells, frequently assumed to be hydrogen ion secretion when the pH is lowered, is unaffected by auxin. These elongating cells actively adjust the external hydrogen ion concentration (from any pH in the range of 4-8) to pH 5.4 + 0.2. This pH adjustment occurs in a medium which does not contain potassium. Growth-optimum auxin concentrations have no effect on cellular pH adjustment of the external medium, whether added at the beginning of the experiment or after the equilibrium pH is attained. The pH adjustment by the cells occurs rapidly and in spite of the presence of a cuticle. PMID- 16659938 TI - Comparison of Auxin-induced and Acid-induced Elongation in Soybean Hypocotyl. AB - Acid-induced growth was compared to auxin-induced growth. After a transient pH 4 induced increase in the elongation rate was completed, auxin could still induce an enhanced rate of elongation in soybean (Glycine max) hypocotyl segments. This auxin response occurred both when the medium was changed to pH 6 before auxin addition, and when the auxin was added directly to the pH 4 medium. This postacid response to auxin was persistent, and quite unlike a postacid response to acid, which was again shortlived. One mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7) inhibited the first response to auxin (the first response to auxin being similar to the acid response), but not the second response. This did not appear to be simply a hydrogen ion neutralizing effect, however, since a 50-fold increase in buffer concentration at pH 6 did not inhibit the first response. Decrease in the pH of the external medium, previously shown to occur with excised soybean hypocotyl segments, was not affected by auxin. Furthermore, this pH drop, during which the cells appear to be adjusting their external pH to about 5.4, did not result in an increased rate of elongation. Addition of auxin after the equilibrium pH had been attained did not alter the pH, but it did increase the rate of elongation, eliciting a normal auxin response. It was concluded that hydrogen ions do not mediate in long term auxin-induced elongation in soybean hypocotyl. PMID- 16659939 TI - Chemical Composition and Ultrastructure of Suberin from Hollow Heart Tissue of Potato Tubers (Solanum tuberosum). AB - The disorder of potato tubers (Solanum tuberosum var. Russet Burbank) called "hollow heart" is manifested by the occurrence of hollow regions in internal parts of the tuber. The structure and composition of the suberin from the tissue lining of these internal cavities were determined by gas chromatography and mass spectrometry of the LiAlH(4)-hydrogenolysis products. Identification of octadecene-1,18-diol as the major component and the presence of hexadecane-1,16 diol and very long chain (>C(18)) alcohols in the hydrogenolysate showed that the suberin lining the internal cavities is quite similar to that found in the periderm of external wounds and the natural skin. Electron microscopic examination showed similar lamellar structure for the suberin of hollow heart, external wound periderm, and the natural skin of potato tubers. The results show that suberin can develop in a tissue which is not exposed to the external environment. PMID- 16659940 TI - Chloroplast glutathione reductase. AB - Glutathione reductase (EC 1.6.4.2) activity is present in spinach (Spinacia oleracea L.) chloroplasts. The pH dependence and substrate concentration for half maximal rate are reported and a possible role in chloroplasts is proposed. PMID- 16659941 TI - Subcellular Localization and Developmental Changes of Aspartate-alpha Ketoglutarate Transaminase Isozymes in the Cotyledons of Cucumber Seedlings. AB - The total activity of aspartate-alpha-ketoglutarate transaminase in the cotyledons of cucumber (Cucumis sativus L.) seeds increased 17-fold during the first 2 days of germination in darkness and then declined gradually to 20% of the peak activity after 10 days. Exposure of the seedlings to light at day 3 accelerated the decline. The enzyme in the cotyledon extracts from seedlings at various ages was resolved into six distinct isozymes by starch gel electrophoresis. Isozymes 1 and 2 were glyoxysomal isozymes with different developmental patterns. Isozyme 1 followed the developmental pattern of the total enzyme activity in darkness, and was rapidly eliminated upon illumination. Isozyme 2 increased in activity to a peak at day 2 and declined rapidly thereafter, and disappeared completely at day 6; this developmental pattern was independent of light. No major difference in the optimal pH for activity, substrate specificity, and reversibility was observed between isozymes 1 and 2. The combined developmental pattern of isozymes 1 and 2 during germination correlated with that of the glyoxysomes. Isozyme 3 was located in the cytosol and its developmental pattern followed that of the total activity. Isozymes 4,5, and 6 were plastid isozymes and appeared only after 2 days of germination. Unlike many other chloroplast enzymes, the appearance of the chloroplast transaminase isozymes was under temporal control and was independent of illumination. No enzyme activity was detected in isolated mitochondria. The findings illustrate a complicated cellular control system for the appearance of various organelle specific transaminase isozymes and thus the amino acid metabolism during germination. PMID- 16659942 TI - Purification and Partial Characterization of a Lectin from Phaseolus vulgaris. AB - A method is presented for the isolation of a lectin from a Brazilian cultivar of the common bean (Phaseolus vulgaris L.), through extraction in acidic (pH 4.2) medium, fractionation with ammonium sulfate, and chromatography on DEAE cellulose. The lectin was shown to be homogeneous by gel electrophoresis and isoelectric focusing.The molecular weight, determined by osmometry, is 100,250 daltons; the isoelectric point, determined by isoelectric focusing, is pH 5.1; and the extinction coefficient at 280 nm and pH 7 is E(1%) (1 cm) = 7.85.The lectin was shown to be a glycoprotein with 9.11% of neutral sugars and 1.44% of amino sugars. Its amino acid composition was characterized by the absence of methionine and a very low content of half-cystine, with a predominance of acidic and hydroxylated amino acids.The lectin presented no specificity when tested with red blood cells of all the groups of the ABO system. PMID- 16659943 TI - Sites of Abscisic Acid Synthesis and Metabolism in Ricinus communis L. AB - The sites of abscisic acid (ABA) synthesis and metabolism in Ricinus communis L. were investigated by analyzing the levels of ABA and its two metabolites phaseic acid (PA) and dihydrophaseic acid (DPA) in the shoot tips, mature leaves, and phloem sap of stressed and nonstressed plants.Water stress increased the concentration of ABA, PA, and DPA in phloem exudate and also increased the levels of all three compounds in mature leaves and in shoot tips. The latter had a very high DPA content (18.7 mug/g fresh weight) even in plants not subjected to water stress. When young and mature leaves were excised and allowed to wilt, the level of ABA increased in both, demonstrating that leaves at an early stage of development have the capacity to produce ABA.These results have been interpreted to mean that in mature leaves of nonstressed Ricinus plants, ABA is synthesized and metabolized, and that ABA itself, as well as its metabolites, are translocated in the phloem to the shoot tips (sinks). Since DPA, but not ABA, accumulates in the shoot tips, it follows that ABA is metabolized rapidly in the apical region. To what extent ABA present in young leaves of nonstressed plants is the consequence of synthesis in situ and of import from older leaves remains to be determined. PMID- 16659944 TI - Evaluation of a Chemical Method for Assay of Helminthosporium maydis Race T Toxin. AB - A chemical test reported by Karr et al. (Plant Physiol. 55:727) to assay for host specific toxin produced by Helminthosporium maydis race T was evaluated. Preparations from culture filtrates of both race T isolates, containing host specific toxin, and similar preparations from race O isolates, containing no detectable host-specific toxin, gave positive reactions in the chemical assay. Also, preparations containing active or inactive toxin gave equal responses in this test. The procedure does not provide a reliable method for assaying H. maydis race T toxin. PMID- 16659945 TI - Acclimation of Photosynthetic and Respiratory Carbon Dioxide Exchange to Growth Temperature in Atriplex lentiformis (Torr.) Wats. AB - Atriplex lentiformis plants collected from coastal and desert habitats exhibit marked differences in capacity to adjust photosynthetic response to changes in growth temperature. Plants from desert habitats grown at 43 C day/30 C night temperatures had higher CO(2) uptake rates at high temperatures but reduced rates at low temperatures as compared to plants grown at 23 C day/18 C night temperatures. In contrast, growth of the coastal plants at high temperatures resulted in markedly reduced photosynthetic rates at all measurement temperatures.Leaf conductances to CO(2) were not important in controlling either the differences in the temperature dependence of net CO(2) uptake or the differences in photosynthetic capacities at any measurement temperature. At low measurement temperatures, differences in photosynthetic capacities among plants acclimated to the contrasting growth regimes were correlated with differences in leaf ribulose diphophate carboxylase activities. At high measurement temperatures, the improved net photosynthetic performance of the high temperature acclimated desert plants appeared to be due to a combination of decreased respiration rates, decreased temperature dependence of respiration, and an apparent increased thermal stability of photosynthetic CO(2) exchange. PMID- 16659946 TI - Requirements for extraction of polyribosomes from plant callus cultures. AB - A procedure was developed to isolate polyribosomes from plant cell cultures. Tobacco callus extracted in 10 mm MgCl(2), 80 mm KCl, 250 mm sucrose, and 140 mm tris-HCl (pH 8.2) yielded larger amounts of polysomes than cells extracted in higher or lower ionic strength or pH buffers. Optimal conditions for extraction of polysomes from soybean callus were identical except the most suitable pH for recovery was 8.5. Addition of the divalent cation chelator, ethylene glycol-bis(2 aminoethyl ether)-tetraacetic acid (EGTA) to the extraction medium improved polysomal yield from tobacco and soybean cultures. Polysomes were successfully extracted from potato, tomato, corn, and barley cell cultures in extraction medium supplemented with EGTA. PMID- 16659947 TI - Lipid crystallization in senescent membranes from cotyledons. AB - Lipid transition temperatures for rough and smooth microsomal membranes isolated from bean (Phaseolus vulgaris) cotyledon tissue at various stages of germination were determined by wide angle x-ray diffraction. The transition temperatures were established by recording diffraction patterns through a temperature series until a sharp x-ray reflection centered at a Bragg spacing of 4.15 A and denoting the presence of crystalline lipid was discernible. For rough and smooth microsomes from 2-day-old tissue, the transitions occurred at 0 C and 3 C, respectively, indicating that at this early stage in the germination sequence the membrane lipid is entirely liquid-crystalline at physiological temperature. By the 4th day of germination, the transition temperatures had increased to 32 C for smooth microsomes and 35 C for rough microsomes, indicating that at 29 C, which was the growth temperature, portions of the membrane lipid were crystalline. During the later stages of germination, the transition temperature for smooth microsomes continued to rise through 44 C at day 7 to 56 C at day 9, by which time the cotyledons were extensively senescent and beginning to abscise. There was also a dramatic increase in the proportion of membrane lipid in the crystalline phase at 29 C. By contrast, the rough microsomes showed little change in transition temperature and only a slight increase in the proportion of crystalline lipid during this late period in germination. The data indicate that substantial amounts of the lipid is senescing membranes are crystalline even at physiological temperature. Moreover, there is a temporal correlation between the appearance of this crystallinity and loss of membrane function, suggesting that the two may be causally related. PMID- 16659948 TI - Relations between Light Level, Sucrose Concentration, and Translocation of Carbon 11 in Zea mays Leaves. AB - The mechanism of carbon transport in Zea mays leaves was investigated using carbon 11 which is a short lived (half-life 20.4 min) positronemitting isotope. The gamma radiation produced on annihilation allows in vivo or nondestructive measurement of the isotope and the short half-life allows many measurements of translocation to be made on the same leaf within the same day.Carbon 11 produced by the (10)B (d,n)(11)C nuclear reaction was converted to (11)CO(2), fed to a leaf as a short pulse, and assimilated during photosynthesis. The progress of the radioactive pulse along the leaf in the phloem was monitored in several positions simultaneously with counters. The counters were NaI crystals with photomultipliers and the output was amplified, passed to single channel analyzers, and the counts accumulated for 20 seconds every 30 seconds. Corrections were made for the half-life and background radiation by computer, and the results were displayed on a high speed plotter. Information derived from the corrected data included the speed of translocation, the shape of the radioactive carbon pulse, and the influence of light and distance along the leaf on these parameters. The plants were kept under controlled environment conditions during all measurements.A speed was derived from the time displacement of the midpoint of the front of the pulse, measured at two positions along the leaf. This was an apparent mean speed of translocation because it averaged a variation in speed with distance, variation in speed between or within sieve tubes, and it averaged the mean speed of all of the particles in the pulse.A wide range of speeds of translocation from 0.25 to 11 cm min(-1) was observed but most of the variability was due to the variation in light available to the leaf. For example, the speed of translocation was proportional to the light level on either the whole plant or individual leaf. Shading of the leaf established that the light effect was not localized in either the feeding area or in the portion of the leaf on which the measurements were made. It was proposed that the speed was dependent on the proportion of the leaf in the light upstream from the last counter. The speed of translocation was relatively independent of the stage of growth of the plant, age of the leaf, and the time during the diurnal light cycle.Data obtained on the level of the reducing sugars, starch, and sucrose in the leaf were related to the speed of translocation. A biphasic relationship between speed and sucrose concentration in the leaf was established and the high speeds measured during experiments only occurred when sucrose concentrations in the leaf exceeded 8% of the dry weight.The shape of the pulse loaded into and translocated in the phloem was estimated from the half-width of the pulse. The half-width was primarily determined by loading phenomena which resulted in an increase in the half-width from 2 minutes when fed to the leaf to more than 40 minutes in the phloem. In many examples, the pulse continued to broaden with distance along the leaf from the fed region. The half-width was independent of the speed but highly dependent on the light level. PMID- 16659949 TI - Rapid separation and quantification of abscisic Acid from plant tissues using high performance liquid chromatography. AB - Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-(14)C]ABA, of which 98.9% was recovered.Following purification by the preparative HPLC procedure, the ABA in a soybean seed extract was quantified using either GLC with an electron capture detector (GLC-EC) or by analytical HPLC with a UV detector. For soybean seed extracts, two analytical HPLC column packing materials were found adequate: muPorasil and muBondapak-NH(2) (Waters Associates). However, with complex tissue extracts, such as soybean leaf and nodule tissues, only GLC-EC had the necessary selectivity and sensitivity. PMID- 16659950 TI - Nitrogen Metabolism in Soybean Tissue Culture: II. Urea Utilization and Urease Synthesis Require Ni. AB - Potassium citrate (10 mM, pH 6) inhibits the growth of cultured (Glycine max L.) cells when urea is the sole nitrogen source. Ureadependent citrate toxicity is overcome by three separate additions to the growth medium: (a) NH(4)Cl (20 mM); (b) high levels of MgCl(2) (10 mM) or CaCl(2) (5-10 mM); (c) low levels of NiSO(4) (10(-2) mM). Additions of 10(-2) mM NiSO(4) not only overcome citrate growth inhibition but the resultant growth is usually better than urea-supported growth in basal medium (neither added citrate nor added nickel). In the absence of added citrate, exceedingly low levels of NiSO(4) (10(-4) mM) strongly stimulate urea-supported growth in suspension cultures.Citrate does not inhibit growth when arginine is sole nitrogen source. However, cells using arginine have no net urease synthesis in the presence of 10 mM potassium citrate. When 10(-2) mM NiSO(4) is added to this medium, urease specific activity is 10 times that observed in basal medium lacking both citrate and added nickel.Citrate is a chelator of divalent cations. That additional Mg(2+) or Ca(2+) alleviates urea dependent citrate toxicity indicates that citrate is acting by chelation, probably of another trace divalent cation; this is probably Ni(2+) since at 10( 2) mM it overcomes citrate toxicity and at 10(-4) mM it stimulates urea-supported growth in the absence of citrate. That ammonia overcomes citrate toxicity indicates that the trace Ni(2+) is essential specifically for the conversion of urea to ammonia. Ni(2+) stimulation of urease levels in arginine-grown cells supports this contention.In basal medium, soybean cells grow slowly with urea nitrogen source presumably because the trace amounts of Ni(2+) present ( 39 C was slow and comparable to rate of growth; development of the insensitive condition after transfer 39 --> 25 C was rapid, implying rapid in situ alteration. An immediate metabolic effect, observed as a decrease in rate of photosynthetic O(2) evolution measured at growth temperature, was less severe than loss of viability. Continued light incubation under growth conditions led to slow decay in rate of O(2) evolution accompanied by loss of membrane chlorophyll. The multiple effects which comprise the cold shock syndrome appear to be membrane-related phenomena and thereby provide an experimental probe of normal membrane function. PMID- 16659978 TI - Spectral Changes in Anacystis nidulans Induced by Chilling. AB - When Anacystis nidulans, strain TX 20 was grown at 39 C, then rapidly chilled to 0 C, a pigment with a carotenoid-like spectrum was bleached. This effect was not seen when cells which had been grown at 25 C were chilled. The effect seen in 39 C-grown cells was not reversible except under extreme conditions such as heating to near boiling for several minutes. Bleaching could be prevented by prior exposure of cells to glutaraldehyde, but could not be reversed by glutaraldehyde treatment following chilling. The effect occurred upon chilling 39 C-grown cells even after extensive heating at 85 C, a treatment which destroys phycocyanin and metabolic activities. 25 C-grown cells were induced to bleach by chilling when suspended in 50% glycerol. The results are interpreted as indicating a chill induced change in aggregation state of a carotenoid, which changes its specific absorbance. PMID- 16659979 TI - Isolation and Function of Allophycocyanin B of Porphyridium cruentum. AB - Allophycocyanin B was purified to homogeneity from the eukaryotic red alga Porphyridium cruentum. This biliprotein is distinct from the allophycocyanin of P. cruentum with respect to subunit molecular weights, and spectroscopic and immunological properties. The purified allophycocyanin B has a long wavelength absorption maximum at 669 nm at room temperature and at 675 nm at -196 C while the fluorescence emission maximum is at 673 nm at room temperature and 679 nm at 196 C. The emission spectrum of allophycocyanin shifted only 1 nm, from 659 to 660 nm, on cooling to -196 C, and was the same with allophycocyanin crystals as it was with pure solutions of the pigment. Phycobilisomes from P. cruentum have a major fluorescence emission band at 680 nm at -196 C which emanates from the small amount of allophycocyanin B present in the phycobilisomes. Light energy absorbed by the bulk of the biliprotein pigments is transferred to allophycocyanin B with high efficiency. PMID- 16659980 TI - Effect of Light and Chilling Temperatures on Chilling-sensitive and Chilling resistant Plants. Pretreatment of Cucumber and Spinach Thylakoids in Vivo and in Vitro. AB - The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. "Marketer") and spinach (Spinacia oleracea L. "Bloomsdale") were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca(2+)-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca(2+)-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca(2+)-dependent ATPase activity and chlorophyll is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light. PMID- 16659981 TI - Oxygen Inhibition of Photosynthesis: I. Temperature Dependence and Relation to O(2)/CO(2) Solubility Ratio. AB - The magnitude of the percentage inhibition of photosynthesis by atmospheric levels of O(2) in the C(3) species Solanum tuberosum L., Medicago sativa L., Phaseolus vulgaris L., Glycine max L., and Triticum aestivum L. increases in a similar manner with an increase in the apparent solubility ratio of O(2)/CO(2) in the leaf over a range of solubility ratios from 25 to 45. The solubility ratio is based on calculated levels of O(2) and CO(2) in the intercellular spaces of leaves as derived from whole leaf measurements of photosynthesis and transpiration. The solubility ratio of O(2)/CO(2) can be increased by increased leaf temperature under constant atmospheric levels of O(2) and CO(2) (since O(2) is relatively more soluble than CO(2) with increasing temperature); by increasing the relative levels of O(2)/CO(2) in the atmosphere at a given leaf temperature, or by increased stomatal resistance. If the solubility ratio of O(2)/CO(2) is kept constant, as leaf temperature is increased, by varying the levels of O(2) or CO(2) in the atmosphere, then the percentage inhibition of photosynthesis by O(2) is similar. The decreased solubility of CO(2) relative to O(2) (decreased CO(2)/O(2) ratio) may be partly responsible for the increased percentage inhibition of photosynthesis by O(2) under atmospheric conditions with increasing temperature. PMID- 16659982 TI - Oxygen Inhibition of Photosynthesis: II. Kinetic Characteristics as Affected by Temperature. AB - The response of whole leaf photosynthesis of wheat (Triticum aestivum L.) in relation to soluble CO(2) available to the mesophyll cells, under low (1.5%) O(2) at 25, 30, and 35 C, followed Michaelis-Menten kinetics up to saturating CO(2) but deviated at high CO(2) levels where the experimental V(max) is considerably less than the calculated V(max). The affinity of the leaves for CO(2) during photosynthesis was similar from 25 to 35 C with Km (CO(2)) values of approximately 3.5 to 5 muM.In considering the effect of O(2) on photosynthesis at 25, 30, and 35 C where O(2) and CO(2) are expressed on a solubility basis: (a) the effect of O(2) on carboxylation efficiency was similar at the three temperature; (b) increasing temperature caused only a slight increase in kinetic constants Ki(O(2)) and Km(CO(2)), while the ratio of Ki(O(2))/Km(CO(2)) was similar at the three temperatures; and (c) the reciprocal plots of apparent rate of photosynthesis versus (CO(2) - Gamma) at various O(2) levels showed O(2) to be a competitive inhibitor of photosynthesis.A model for separating O(2) inhibition of photosynthesis into two components, direct competitive inhibition and inhibition due to photorespiration, was presented from both simulated and experimental data of photosynthetic response curves to varying CO(2) concentrations at low O(2)versus 21% O(2). The photorespiratory part of O(2) inhibition is considered as a major component at Gamma and increases with increasing temperature and with increase in O(2)/CO(2) solubility ratio. The competitive component of O(2) inhibition is considered as a major component of O(2) inhibition under atmospheric CO(2) levels and is relatively independent of temperature at a given O(2)/CO(2) ratio. PMID- 16659983 TI - Determination of hydraulic and osmotic properties of soybean root systems. AB - An analytical technique which allows the experimental determination of soybean (Glycine max L.) root properties is presented. Two major problems hamper the interpretation of experimental data. These are: (a) the influence of a possible boundary layer which raises the effective value of pi degrees above that of the bulk solution; and (b) the difficulty of obtaining an adequate measure of the internal osmotic pressure except at high values of volume flow rate due primarily to possible exchanges of solutes between the xylem and adjacent tissues. Consideration of these two problems leads to an interpretation of previous models which is reconcilable with the criticisms of Newman (Plant Physiology 1975 57: 738-739).In these experiments, estimates of hydraulic conductivity and reflection coefficient are based on high flow rate data where the osmotic effects are minimized. Because of the difficulties attached to the evaluation of pi(i), at low and moderate flow rates, any technique for evaluating root parameters which depends on knowledge of when the osmotic pressure difference (in bars) is zero will be subject to large errors, at least until both problems metioned above have been adequately resolved.An additional problem which must be dealt with in terminal root segments is the effect of a standing osmotic gradient. It is thought that this is not a serious problem in a complex root system.Transpiration rates are calculated on the basis of leaf and root surface areas and experimentally determined root volume flow. It is shown that root flow rates necessary to sustain high transpiration rates in the shoots are easily accommodated by the model at moderate levels of applied pressure difference. PMID- 16659984 TI - Azolla-Anabaena azollae Relationship: V. N(2) Fixation, Acetylene Reduction, and H(2) Production. AB - In order to characterize the reactions catalyzed by nitrogenase in the Azolla Anabaena association, (15)N(2) fixation, C(2)H(2) reduction, and ATP-dependent H(2) production were measured in both the Azolla-Anabaena complex and in the alga isolated from the complex.The rate of reduction of substrates and of ATP dependent H(2) evolution was determined at various partial pressures of C(2)H(2) and N(2). A pC(2)H(2) of 0.1 atm was nearly optimal for C(2)H(4) production and inhibited H(2) production by 95%. The ratio of C(2)H(2) reduced to N(2) fixed was determined as a function of constant pC(2)H(2) (0.1 atm) and variable pN(2). This ratio decreased with increasing pN(2) and the decrease was correlated with less H(2) production. Ratios obtained at N(2) partial pressures of approximately 0.3, 0.6, and 0.8 atm, respectively, were 3.2, 2.0, and 1.7 for the association and 4.4, 3.0, and 2.5 for the isolated symbiont. Rates obtained for C(2)H(2) reduction, N(2) fixation, and H(2) production were used to obtain an expression of the electron balance in vivo. PMID- 16659985 TI - Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea L. Crown Gall Tumor Tissue. AB - Recently detected but unidentified cytokinin activity in crown gall tumor tissue from Vinca rosea L. grown on media containing sources of reduced nitrogen has now been attributed to two adenine-type cytokinins. These compounds are glucopyranosyl derivatives of zeatin and ribosylzeatin. The substitution in each case is on the isopentenyl chain of the parent compound. Neither of these compounds had activity in the soybean callus bioassay at concentrations lower than 1 nm whereas zeatin had activity at 0.1 nm. PMID- 16659986 TI - Mass Spectroscopic Identification of Cytokinins: Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea Crown Gall. AB - Mass spectrographic and chemical studies of the permethyl and trimethylsilyl ethers of two new cytokinins isolated from Vinca rosea crown gall callus cultures by Peterson and Miller (Plant Physiol 59: 1026-1028) indicate that they are 6-(4 0-beta-d-glucopyranosyl-3-methyl-trans-but-2-enylamino) purine (glucosyl zeatin) and 9-beta-d-ribofuranosyl-6 (4-0-beta-d-glucopyranosyl-3-methyl-trans-but-2 enylamino) purine (glucosyl ribosylzeatin). The nature of the mass spectra of the permethylated cytokinins suggests that these derivatives may have considerable utility in the detection of low levels of cytokinins in plant material. PMID- 16659987 TI - Interaction of boron with components of nucleic Acid metabolism in cotton ovules cultured in vitro. AB - Cotton (Gossypium hirsutum L.) ovules grown in a defined nutrient medium undergo normal morphogenesis, including fiber production. In identical medium lacking boron, ovules callus and accumulate brown substances. Boron deficiency-like symptoms were induced by 6-azauracil and 6-azauridine in ovules growing in boron sufficient media. Other nucleoside base analogs either reduced or had no effect on over-all growth, but did not cause typical boron-deficient callus growth of cotton ovules. Orotic acid and uracil countered the effects of 6-azauracil. Actinomycin D, fluorodeoxyuridine, and ethidium bromide reduced not only fiber production on ovules growing in boron-sufficient media but also callusing of ovules in boron-deficient media.Similarities between symptoms of boron deficiency and 6-azauracil injury, and the ability of uracil to suppress both, suggest that boron deficiency symptoms are related to reduced activity in the pyrimidine biosynthetic pathway. Growth inhibition by most nucleoside base analogs tested, actinomycin D, fluorodeoxyuridine, and ethidium bromide, as compared to callusing brought on by boron deficiency and 6-azauracil, indicates that boron deficiency symptoms are not related to a reduction in nucleic acid biosynthesis. Based on this information, a discussion of the possibility that boron deficiency causes reduced synthesis of UDP-glucose is presented. PMID- 16659988 TI - Induced senescence of intact wheat seedlings and its reversibility. AB - Intact wheat seedlings (Triticum aestivum L.) were induced to senesce by placing them in the dark and at various stages of senescence were placed back in the light and their recoverability observed. Seedlings demonstrated complete recovery of chlorophyll, protein, and rate of photosynthesis after 2 days in the dark, but were unable to recover fully after 4 days. This suggests the onset of an irreversible stage in senescence by day 4. Foliar applied cytokinins delayed senescence, and zeatin at 0.1 mm delayed the onset of the irreversible stage for 6 days. In addition to delaying the loss of total soluble protein, zeatin maintained the net protein recovery capacity of the tissue. Control seedlings, however, lost their potential for net protein recovery at a rate similar to their loss of total soluble protein. Treatment with zeatin had no apparent effect on dark respiration during senescence, and although treatment did delay the loss of membrane permeability to substrate, the change in permeability occurred too late to have a causal role in senescence. PMID- 16659989 TI - Increased Survival and Differentiation of Frozen Herbaceous Plant Organ Cultures through Cold Treatment. AB - Cold treatment of donor carnation plants (Dianthus caryophyllus L.) at 4 C for 3 days or more resulted in a doubling in the percentage of excised, frozen shoot apices which survived freezing and a 6- to 7-fold increase in the percentage which formed leaf primordia or shoots. The optimal freezing parameters for both survival and differentiation were as follows: size of the shoot apex-two to three sets of leaf primordia; dimethylsulfoxide concentration in the freezing solution 5%; time in dimethylsulfoxide prior to freezing->30 minutes; average cooling rate >/=50 C/minute; initial warming rate-about 1450 C/minute. In general, the cells in the meristematic region of the shoot apex remained viable after freezing while those in the leaf primordia did not. Viability of the meristematic cells appears to be maintained by preventing the growth of intracellular ice crystals formed during rapid cooling by rapidly passing the tissue through the temperature zone in which lethal crystal growth occurs (mechanism of Luyet). Applications of this technique are discussed. PMID- 16659990 TI - Early effects of boron deficiency on indoleacetic Acid oxidase levels of squash root tips. AB - The indoleacetic acid (IAA) oxidase activity of root tips of boron-sufficient, deficient, recovering, and IAA-treated boron-sufficient squash plants (Cucurbita pepo L.) was determined. Apical and subapical root sections displayed an increase in IAA oxidase activity between 6 and 9 hours after boron was withheld, and after 24 hours the activity of the apical sections showed a 20-fold increase over +B controls. Root elongation of -B plants was inhibited before an increase in oxidase activity could be detected. Roots of plants subjected to 12 hours of -B treatment and then transferred to +B treatment for recovery regained normal elongation rates and oxidase activity within 18 to 20 hours. IAA treatment of +B plants increased IAA oxidase activity of apical and subapical root sections and also inhibited root elongation and caused symptoms similar to -B treatments.These results have demonstrated the earliest enzymic change for intact boron-deficient plants. The results are in agreement with the theory that boron deficiency symptoms may be the result of supraoptimal endogenous levels of IAA. These high levels of IAA may inhibit cell division and lead to an induction of the IAA oxidase enzyme. PMID- 16659991 TI - White light requirements for stimulation of plant growth by malformin. AB - Over a 3-day period, the minimum white fluorescent light intensity required for malformin-induced growth stimulation of etiolated and green cuttings of Phaseolus aureus was approximately 2.6 x 10(3) and 0.4 x 10(3) ergs/cm(2) . sec, respectively. High light intensities were unable to inhibit the ability of malformin to stimulate growth. Over 3 days, the minimum photoperiod for malformin induced growth stimulation using etiolated and green cuttings and a light intensity of 13.5 x 10(3) ergs/cm(2) . sec was 4 hours and 1 hour, respectively. Malformin must be present in the area of growth stimulation during the time of light treatment. Those changes induced by light and required for malformin induced growth stimulation were estimated to undergo almost complete decay within 1 hour in the dark. By manipulating the experimental technique, it was possible to stimulate the growth of green cuttings with malformin with a 10-min light treatment (13.5 x 10(3) ergs/cm(2) . sec). Although low light intensities and short photoperiods did not allow growth stimulation by malformin using etiolated cuttings, they prevented or alleviated growth inhibition induced by malformin in the dark. PMID- 16659992 TI - Release of Esterase Following Germination of Lettuce Seed (Lactuca sativa L.). AB - Light-insensitive lettuce seeds, Lactuca sativa L. cv. Great Lakes, release esterases for a period following radicle protrusion. Very little or no enzymes are released prior to 24 hours or after 48 hours of germination. As compared to intact seeds, half-seeds readily release esterases and the release is not affected by far red irradiation. Bulk of the released esterases are derived from the endosperm tissue and presumably exists in the intact seed as a component of the extraembryonic fluid. PMID- 16659993 TI - Proteases of senescing oat leaves: I. Purification and general properties. AB - Two proteases active in the senescing first leaves of oat seedlings (Avena sativa cv. Victory) have been purified approximately 500-fold by a combination of ammonium sulfate precipitation, affinity chromatography on hemoglobin-Sepharose, and ion exchange chromatography on DEAE-Sephadex. The enzymes show pH optima of 4.2 and 6.6 with denatured hemoglobin as substrate, and the molecular weights of both are about 76,000. Their optimum temperatures are close to 50 C. Small amounts of a third enzyme, active at pH 3.5, may also be present. The enzyme active at pH 6.6 shows evidence of a sulfhydryl residue in the active site. PMID- 16659994 TI - Accumulation of free ricinoleic Acid in germinating castor bean endosperm. AB - Lipids from the endosperm of germinating castor bean (Ricinus communis var. Hale) were separated by thin layer chromatography and quantitated by gas chromatography. During the later stages of lipid breakdown (4-6 days germination at 30 C), several lipid classes were found in addition to the storage triglycerides, which are triricinoleins for the most part. One was identified as free ricinoleic acid, the proportion of which increased as germination progressed. After 6 days germination, ricinoleic acid comprised more than 30% of the total lipid. The appearance of this fatty acid implies that lipase activity (lipolysis) is not strictly coordinated with beta oxidation in this tissue. PMID- 16659995 TI - Effects of cycloheximide and chloramphenicol on the synthesis of polypeptides found in three subcellular fractions of maize scutellum. AB - The effects of two protein synthesis inhibitors, cycloheximide and chloramphenicol, on the synthesis of mitochondrial proteins in maize (Zea mays) have been studied. The results of these investigations suggest that while most of the mitochondrial proteins are synthesized in the cytoplasm and are subsequently associated with the mitochondrion, several proteins of the mitochondrial inner membrane are synthesized within the mitochondrion. These results are consistent with those observed in several other systems, but not previously reported for higher plants. PMID- 16659996 TI - Metabolic Activity of Isolated Leaf Cells of Phaseolus vulgaris in Relation to Leaf Development. AB - Mesophyll cells were isolated from primary leaves of 5- to 21-day Phaseolus vulgaris plants. The rate of photosynthesis and respiration, and RNA, protein, and lipid synthesis was determined for these cells. Appropriate (14)C substrates and product purification procedures were used for each process prior to liquid scintillation counting. The size of the leaves increased about 5-fold between days 5 and 11, and then remained relatively constant. The greatest increase in size occurred between days 5 and 6. The age of the leaf from which the cells were isolated had a pronounced effect on the rate of all of these processes. The largest changes occurred during the period of leaf expansion (days 5-11). Initially the rate of RNA, protein, and lipid synthesis increased rapidly, maintained a maximum rate for only 1 day (day 6 or day 7), and then declined. The rate of photosynthesis increased more slowly reaching a maximum at day 9, remained relatively constant until day 15, and then declined. The rate of respiration decreased during the first 4 days to low level which was maintained throughout the experiment. The time course patterns of these biochemical processes in isolated cells were similar to those which have been reported for intact leaves. It seems that isolation of leaf cells does not modify their metabolic activity. PMID- 16659997 TI - Continuous, automated acetylene reduction assays using intact plants. AB - An automated method was developed for continuous, in situ determination of acetylene reduction (N(2) fixation) by intact soybean plants (Glycine max [L.]). The culture vessel containing the roots of intact plants grown in sand culture is sealed at the surface and an air-acetylene mixture continuously injected into the root chamber. The effluent gas is automatically sampled and injected into a gas chromatograph. Continuous acetylene assay at intervals as short as 3.5 min may be made over a period of several days, without attention, except for plant watering. Adverse effects of prolonged exposure of the root system to acetylene were mitigated by pulse injection of acetylene for 20 min followed by 40 min of acetylene-free air. Bare root systems can be suspended in a reaction chamber and sprayed with water or nutrient solution; this permits periodic removal of the root system for sampling nodules.In studies lasting several diurnal cycles, acetylene reduction did not decline more than 50% of the maximum rate in light, thus nitrogenase activity depends on concomitant photosynthesis and on carbohydrate from storage pools. PMID- 16659998 TI - Isolation of uridine 5'-pyrophosphate glucuronic Acid pyrophosphorylase and its assay using p-pyrophosphate. AB - A procedure was devised to detect and assay uridine 5'-pyrophosphate (UDP) glucuronic acid pyrophosphorylase in plant extracts. Substrates are UDP glucuronic acid and (32)P-pyrophosphate, and the (32)P-uridine 5'-triphosphate produced is selectively adsorbed to charcoal. The charcoal adsorption procedure is a modification of that used to determine (32)P-adenosine 5'-triphosphate produced by adenosine 5'-pyrophosphate glucose pyrophosphorylase, and the modification greatly improves the retention of uridine 5'-triphosphate. PMID- 16659999 TI - Zinc, Iron, and Chlorophyll Metabolism in Zinc-toxic Corn. AB - Zinc toxicity and Zn-Fe interactions were studied in corn (Zea mays L. var. Barbecue hybrid) grown in hydroponic culture. High Zn greatly reduced the root and shoot fresh weights; increasing Fe largely, but not completely, restored normal growth. Correlation analyses of root and leaf Zn and Fe contents suggested that Zn may interfere with the translocation of Fe; however, Zn toxicity was not associated with a diminished leaf Fe content. Fe did appear to retard both the absorption and the translocation of Zn. The chlorosis of Zn-toxic plants is not attributable to diminshed total leaf Fe; however, this chlorosis is relieved by increasing nutrient Fe. Zn and Fe probably do interact at some site. PMID- 16660000 TI - Changes in biochemical composition of the cell wall of the cotton fiber during development. AB - The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postanthesis) through the period of secondary wall formation, using cell walls derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure. Expressed as a weight per cent of the total cell wall, cellulose, neutral sugars (rhamnose, fucose, arabinose, mannose, galactose, and noncellulosic glucose), uronic acids, and total protein undergo marked changes in content during the elongation period. As a way of analyzing absolute changes in the walls with time, data have also been expressed as grams component per millimeter of fiber length. Expressed in this way for plant-grown fibers, the data show that the thickness of the cell wall is relatively constant until about 12 days postanthesis; after this time it markedly increases until secondary wall cellulose deposition is completed. Between 12 and 16 days postanthesis increases in all components contribute to total wall increase per millimeter fiber length. The deposition of secondary wall cellulose begins at about 16 days postanthesis (at least 5 days prior to the cessation of elongation) and continues until about 32 days postanthesis. At the time of the onset of secondary wall cellulose deposition, a sharp decline in protein and uronic acid content occurs. The content of some of the individual neutral sugars changes during development, the most prominent change being a large increase in noncellulosic glucose which occurs just prior to the onset of secondary wall cellulose deposition. Methylation analyses indicate that this glucose, at least in part, is 3-linked. In contrast to the neutral sugars, no significant changes in cell wall amino acid composition are observed during fiber development.Compositional analyses of cell walls derived from culture-grown fibers indicate that these walls are remarkably similar to those derived from fibers grown on the plant, both in terms of composition and in terms of relative changes in composition during development.A comparison of our results on total cell wall composition and linkages of sugars as determined by a preliminary methylation analysis of unfractionated fiber walls indicates that the primary cell wall of cotton fibers is similar to that of primary cell walls of other dicotyledons and of gymnosperms as reported in the literature. PMID- 16660001 TI - Localization of Acid hydrolases in protoplasts: examination of the proposed lysosomal function of the mature vacuole. AB - The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, beta galactosidase, alpha-glycosidase and beta-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome. PMID- 16660002 TI - Host-Pathogen Interactions: XIII. Extracellular Invertases Secreted by Three Races of a Plant Pathogen Are Glycoproteins Which Possess Different Carbohydrate Structures. AB - The invertase present in the culture fluid of races 1, 2, and 3 of Phytophthora megasperma Drechs. var. sojae A. A. Hildebrand (Pms) were purified until they gave but a single band, whether stained for protein or carbohydrate, after isoelectric focusing in flat bed gels. The sugar compositions of multiple preparations of the purified invertases from each race of this fungal pathogen were determined by quantitative gas chromatography of their alditol acetates. The invertases are composed of about 25% carbohydrate. Mannose and glucosamine make up more than 97% of the carbohydrate portions of the invertases of all three Pms races analyzed, but the ratio of mannose to glucosamine is clearly not the same in each race. The glycosyl linkage compositions of the glucosamine-containing mannans of multiple preparations of the Pms invertases were determined by GC-MS analysis of the partially methylated alditol acetate derivatives. The results of these analyses demonstrate clear quantitative differences between the glycosyl components of the different Pms races. The existence of race-specific carbohydrate structures in the differentially virulent Pms races suggests that these carbohydrates may be involved in determining the specificity of hostpathogen interactions. PMID- 16660003 TI - Transient changes during soybean imbibition. AB - Air dry cotyledons of soybean (Glycine max Merr. var. Wayne) imbibe water rapidly for about 10 minutes followed by a slower, linear rate of uptake. Leakage of solutes out of the coytledon likewise shows an initial rapid period, followed by a slower, nearly linear rate after 5 to 10 minutes; both the rapid and the steady rate leakage are greater for initially drier seeds. Respiratory activity of cotyledons as measured by manometric techniques becomes apparent after about 10 minutes of imbibition while polarographic studies of ground particles suggest that O(2) comsumption begins almost immediately upon wetting. Initial wetting of the seed causes the release of adsorbed gases, and a series of changes in volume of the seed-water mixture are charted. The data are interpreted as indicating that extensive physical changes occur in the first few minutes of water entry, including a rearrangement of membranes changing them from a relatively porous to a less permeable condition, and a release of adsorbed gases which cause an inflation or swelling of the seed. PMID- 16660004 TI - Biosynthesis of Cutin omega-Hydroxylation of Fatty Acids by a Microsomal Preparation from Germinating Vicia faba. AB - omega-Hydroxylation of fatty acids, which is a key reaction in the biosynthesis of cutin and suberin, has been demonstrated for the first time in a cell-free preparation from a higher plant. A crude microsomal fraction (105,000g pellet) from germinating embryonic shoots of Vicia faba catalyzed the conversion of palmitic acid to omega-hydroxypalmitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as alpha- and beta hydroxy acids, the omega-hydroxylation product was identified by gas chromatography on a polyester column and reverse phase, high performance liquid chromatography, two techniques which were shown to resolve the positional isomers. Gas chromatographic analysis of the dicarboxylic acid obtained by CrO(3) oxidation of the enzymic product also confirmed the identity of the enzymic omega hydroxylation product. This enzymic hydroxylation required O(2) and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of omega-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This omega-hydroxylation was inhibited by the classical mixed function oxidase inhibitors such as metal chelators (o-phenanthroline, 8-hydroxyquinoline, and alpha,alpha-dipyridyl), NaN(3) and thiol reagents (N-ethylmaleimide and p chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P(450), the present omega-hydroxylase was inhibited by CO and this enzyme system showed unusually high sensitivity to this inhibition; 10% CO caused inhibition and 30% CO completely inhibited the reaction. Another unusual feature was that the inhibition caused by any level of CO could not be reversed by light (420-460 nm). PMID- 16660005 TI - Equal Expression of the Maternal and Paternal Alleles for the Polypeptide Subunits of the Major Storage Protein of the Bean Phaseolus vulgaris L. AB - Discontinuous sodium dodecyl sulfate slab gel electrophoresis of G1 globulin from several strains of Phaseolus vulgaris L. seed permitted clear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F(1) seed from the cross BBL 240 x PI 229,815 showed four polypeptides on dissociation of the G1 protein; however, the amount of each of the 53,000 and 50,500 subunits was half that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues. PMID- 16660006 TI - Development of ribulose-1,5-diphosphate carboxylase in castor bean cotyledons. AB - Light was not essential for the development of ribulose-1,5-diphosphate carboxylase protein or catalytic activity in the photosynthetic cotyledons of germinating castor beans (Ricinus communis). Cotyledons developing in the dark showed higher activity than those in the light. Returning cotyledons developing in the light to darkness resulted in a significant increase in ribulose-1,5 diphosphate carboxylase activity compared to cotyledons in continuous light. PMID- 16660007 TI - Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L. AB - The metabolism of sucrose to long chain fatty acids in the endosperm of developing castor bean (Ricinus communis L.) seeds requires a combination of cytosolic and proplastid enzymes. The total activity and the subcellular distribution of the intermediate enzymic steps responsible for the conversion of sucrose to pyruvate have been determined. Hexose phosphate synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase. The proplastids contain the necessary complement of glycolytic enzymes to account for the in vivo rates of acetate synthesis from glucose 6-phosphate. These organelles also contain the majority of the cellular 6-phosphogluconate dehydrogenase, transketolase, and transaldolase activities.The consequence of these enzyme distributions is that glucose 6-phosphate or 6-phosphogluconate produced in the cytosol must be transported into the proplastids where conversion to pyruvate occurs. The unique segregation of the two oxidative steps in the pentose phosphate pathway may be required to meet the metabolic needs of these fat-storing seeds. Compartmentation of glucose-6-phosphate dehydrogenase in the cytosol and 6-phosphogluconate dehydrogenase in the proplastids is discussed in light of the NADPH requirements for fatty acid synthesis in these subcellular locations. PMID- 16660008 TI - Photosynthesis In Elodea canadensis Michx: Four-Carbon Acid Synthesis. AB - Experiments to determine the early labeled photosynthetic products in Elodea canadensis show that after 2 seconds of exposure to NaH(14)CO(3), 45% of the (14)C incorporated is located in malate and aspartate. Phosphoglyceric acid and sugars account for 27% of the label during similar exposures. Equivalent amounts of organic acids and C(3) cycle products are present after 8 seconds. Four-carbon acids remain relatively unchanged throughout the first 45 seconds of exposure, while sugars increase in a linear fashion. Enzyme assays indicate that ribulose diphosphate and phosphoenolpyruvate carboxylase enzymes are present in a ratio of approximately 2:1. It appears that E. canadensis is able to synthesize significant amounts of four-carbon acids via beta-carboxylation and this may play a role in maintaining a pH favorable for carboxylation in aquatic plants. PMID- 16660009 TI - Studies on the behavior of the senescence signal in anoka soybeans. AB - Soybean, a monocarpic plant, has been found to undergo rapid senescence as its fruits mature. In soybeans (Glycine max[L.] Merrill) cv. Anoka, foliar senescence begins during the period of most rapid pod-fill (seed growth), and it can be eliminated by surgical removal of the seeds at an early stage of their growth. Experiments in which fruits are removed from some regions of the plant but allowed to remain on other regions have established that the transmission of the senescence signal is limited; it affects mainly those leaves nearest to the nodes bearing the fruits. The implications of this localized signal movement are discussed. PMID- 16660010 TI - Temperature-induced Changes in Hill Activity of Chloroplasts Isolated from Chilling-sensitive and Chilling-resistant Plants. AB - The effect of temperature on Hill activity has been compared in chilling sensitive and chilling-resistant plants. The Arrhenius activation energy (Ea) for the photoreduction of 2,6-dichlorophenolindophenol by chloroplasts isolated from two chilling-sensitive plants, mung bean (Vigna radiata L. var. Mungo) and maize (Zea mays L. cv. PX 616), increased at low temperatures, below 17 C for mung bean and below 11 C for maize. However, the Ea for this reaction in pea (Pisum sativum L. cv. Massay Gem), a chilling-resistant plant, likewise increased at temperatures below 14 C. A second change in Ea occurred at higher temperatures. The Ea decreased above about 28 C for mung bean, 30 C for maize, and 25 C for pea. At temperatures approaching 40 C, thermal inactivation of Hill activity occurred. These results, when taken together with previous results obtained with the chilling-resistant plant barley, indicate that chloroplasts from both chilling-sensitive and chilling-resistant plants can undergo a change in chloroplast membrane activity at low temperatures above freezing and that the presence of such a change in chloroplast membranes is not necessarily correlated with chilling sensitivity. PMID- 16660011 TI - Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts. AB - Starch synthesis in leaves was increased by phosphate starvation or by treatments which decreased cytoplasmic orthophosphate levels (such as mannose feeding). Usually less than 30% of the total carbon fixed during CO(2) assimilation was incorporated into starch in spinach (Spinacia oleracea L.), spinach beet (Beta vulgaris), and tobacco (Nicotiana tabacum) leaves.In isolated spinach chloroplasts, formation of starch from CO(2) was usually less than in leaves. In the absence of significant levels of 3-phosphoglycerate, concentrations of phosphate as low as 1 mm (in the medium) or 10 mm (in the stroma) almost completely inhibited starch synthesis. The inhibitory action of phosphate could be overcome by 3-phosphoglycerate. The controlling factor of starch synthesis appeared to be the ratio of phosphoglycerate to orthophosphate rather than the stromal hexose monophosphate concentration, and it is suggested that this control is exerted via the phosphate translocator and the known allosteric regulation of ADP-glucose pyrophosphorylase. Starch synthesis was also favored by the presence of dihydroxyacetone phosphate and by high light and high temperature. Oxygen was inhibitory, probably owing to carbon drain into glycolate. Starch formation by intact chloroplasts could not be promoted by added glucose or glucose 6 phosphate.Starch mobilization in the dark was promoted by orthophosphate and phosphate-dependent mobilization was inhibited by phosphoglycerate. The principal products of starch breakdown in the presence of phosphate were the transport metabolites dihydroxyacetone phosphate and 3-phosphoglycerate. Formation of these compounds from starch was stimulated by ATP or oxaloacetate. In a phosphate independent reaction, starch was also converted to neutral products such as maltose and glucose. The rates of phosphate-dependent starch degradation phosphorolysis were very much higher than those of starch hydrolysis for which there was no phosphate requirement. PMID- 16660012 TI - Development of the Two Heterogeneous Photosystem II Units in Etiolated Bean Leaves. AB - The development of the photosystem II units in relation to the heterogeneity of their photochemical centers was studied in etiolated bean leaves (Phaseolus vulgaris var. red kidney) greened under continuous or intermittent light. The study was done in order to see whether grana are the loci of the units with the efficient photosystem II activity (alpha units), while the stroma thylakoids are the loci of the units with the less efficient photosystem II activity (beta units), as it has been proposed. In addition, the interrelations between alpha and beta centers have been investigated. It was found that the alpha and the beta centers of photosystem II were present in the first photosynthetic membranes irrespective of the mode of greening of the leaves. The magnitude of their respective photochemical rate constants, K'(alpha) and K(beta), increased with time in continuous light and it reached the steady-state values of the mature chloroplasts within 16 hours, while in intermittent light it remained smaller. The differentiation of the system II units in alpha and beta centers containing units is more evident under conditions of intermittent illumination, i.e. when the rate of chlorophyll biosynthesis is the limiting step for chloroplast development.It is concluded that the heterogeneity of the photochemical centers in system II is an endogenous property of the chloroplast lamellae. The alpha centers and the beta centers develop independently of each other from the beginning of the light-induced greening. They do not share the same pigment beds. The presence of grana, chlorophyll b, and the chlorophyll-protein complex II is not a prerequisite for the formation or development of the alpha centers. The formation of these centers precedes grana formation in greening plastids. PMID- 16660013 TI - Differential effects of sucrose, abscisic Acid, and benzyladenine on shoot growth and callus formation in the abscission zone of excised citrus buds. AB - The omission of sucrose from the basal medium stimulated callus formation in bud explants of Citrus sinensis (L.) Osbeck. Moreover, it increased the abscisic acid induced callus proliferation reported earlier in the presence of 5% sucrose (Altman and Goren, Physiol. Plant. 32: 55, 1974). The inhibition of callus formation by the addition of sucrose was not due to the high osmotic potential of the medium. Benzyladenine induced callus formation slightly, in all sucrose concentrations up to 5%. The high level of sucrose was required, however, for the growth of shoots from buds cultured on both basal and benzyladenine-containing media.The uptake of (14)C-sucrose by bud explants was linear for at least 98 hr, and was enhanced significantly by both abscisic acid and benzyladenine during the initial 24-hour period. Abscisic acid enhanced the absorption of (14)C-sucrose and the accumulation of sugars in buds cultured at high levels of sucrose. More than 50% of the total label accumulated in the callus of abscisic acid-treated explants whereas only 16 and 23% were observed in the growing shoots of control and cytokinin-treated explants, respectively.Results suggested that while sucrose "starvation" induced initial callus formation, high levels retarded further proliferation of the callus. PMID- 16660014 TI - Movement and Endogenous Levels of Abscisic Acid during Water-Stress-induced Abscission in Cotton Seedlings. AB - In an effort to investigate possible involvement of abscisic acid (ABA) in foliar abscission processes, its movement and endogenous levels were examined in cotyledons taken from cotton seedlings (Gossypium hirsutum L.) subjected to varying degrees of water deficit, a condition which initiates leaf abscission. Using a pulse-labeling technique to avoid complications of uptake and exit from the tissue, ABA-1-(14)C movement was observed in both basipetal and acropetal directions in cotyledonary petioles taken from well watered, stressed, and rewatered plants. The label distribution patterns obtained after 1 and 3 hours of transport under all situations of water supply were diffusive in nature and did not change when tested under anaerobic conditions. The transport capacity of the petioles ranged from 3.6 to 14.4% ABA-1-(14)C transported per hour at estimated velocities of 0 to 2 millimeters per hour. Comparison of basipetal and acropetal movement indicated a lack of polarity under all conditions tested. These low transport capacities and slow velocities of movement, when compared to the active transport systems associated with auxin movement, as well as the lack of anaerobic effects and polarity, suggest that ABA movement in cotton cotyledonary petiole sections is facilitated by passive diffusion. Increases in free and bound ABA in the lamina with increased water stress did not correlate with patterns of cotyledonary abscission. Thus, no evidence was found to suggest that ABA is directly involved in stress-induced abscission processes. PMID- 16660015 TI - Hormonal activity in detached lettuce leaves as affected by leaf water content. AB - The interrelationship between water deficiency and hormonal makeup in plants was investigated in detached leaves of romaine lettuce (Lactuca sativa L. cv. ;Hazera Yellow'). Water stress was imposed by desiccating the leaves for several hours in light or darkness at different air temperatures and relative humidity. In the course of desiccation, a rise in abscisic acid content and a decline in gibberellin and cytokinin activity were observed by gas-liquid chromatography, by both the barley endosperm bioassay and radioimmunoassay and by the soybean callus bioassay. Gibberellin activity began to decline in the stressed leaves before the rise in abscisic acid, the rate of this decline being positively correlated with the rate of increase in leaf water saturation deficit. Recovery from water stress was effected by immersing the leaf petioles in water while exposing the blades to high relative humidity. This resulted in a decrease in leaf water saturation deficit, a reduction in abscisic acid content, and an increase in gibberellin and cytokinin activity.Application of abscisic acid to the leaves caused partial stomatal closure in turgid lettuce leaves, whereas treatment with gibberellic acid and kinetin of such leaves had no effect on the stomatal aperture. In desiccating leaves, however, gibberellic acid and kinetin treatment considerably retarded stomatal closure, thus enhancing the increase in leaf water saturation deficit. These results suggest that the effect of desiccation in changing leaf hormonal make-up, i.e. a rapid increase in abscisic acid and a decrease in both cytokinin and gibberellin activity, is related to a mechanism designed to curtail water loss under conditions inducing water deficiency. PMID- 16660016 TI - Mitochondrial Activity and Ethanol Accumulation in Ice-encased Winter Cereal Seedlings. AB - Cold-hardened dark-grown seedlings of winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) are killed during total encasement in ice at -1 C at a rate related to the initial cold hardiness of the cultivars. Few plants remain alive after 7 days of encasement. Nonhardened seedlings are rapidly killed in ice. The respiratory properties of mitochondria isolated from plants after increasing periods of ice encasement decline slowly, and activity is little impaired when intact plants are about 50% killed. Electron microscopy indicates that mitochondrial structure is not disrupted until 3 weeks of ice encasement. Ethanol accumulates in hardened and nonhardened plants in ice, but at levels which are not toxic to the plants. PMID- 16660017 TI - Strong binding of cytochrome C on the envelope of spinach chloroplasts. AB - Yeast cationic ferricytochrome c was able to bind to the spinach (Spinacia oleracea) chloroplast envelope with a low affinity (Kd = 1.1 mum). The total amount of low affinity binding sites was of the order of 50 nmol cytochrome c mg( 1) protein. We gave the evidence that binding of ferricytochrome c to the envelope was electrostatic and that the envelope membranes were strongly negatively charged. Addition of yeast ferricytochrome c to a preparation of intact washed chloroplasts (class I) induced a strong agglutination of chloroplasts. PMID- 16660018 TI - Simultaneous Inhibition of Linolenic Acid Synthesis in Winter Wheat Roots and Frost Hardening by BASF 13-338, a Derivative of Pyridazinone. AB - Treatment of 12-day-old winter wheat (Triticum aestivum) plants with BASF 13-338 {4-chloro-5 (dimethylamino)-2-phenyl-3(2H)-pyridazinone} 36 hours before frost hardening simultaneously and completely inhibits accumulation of linolenic acid in the roots during the hardening period and the acquisition of frost resistance. Increased unsaturation of fatty acids is therefore probably an important part of the mechanism of cold adaptation in winter wheat.BASF 13-338 also prevents the increase in per cent dry weight in roots and shoots during hardening and causes a decrease in root lipid phosphorus and total fatty acids.The concurrent increase in linoleic acid and decrease in linolenic acid in the treated plants, while the level of the other fatty acids is but little affected, suggest that BASF 13-338 specifically inhibits linoleic acid desaturase. PMID- 16660019 TI - Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum. AB - The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C(4) plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3 phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg(2+) chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used. PMID- 16660020 TI - Phosphoserine and phosphohydroxypyruvic Acid: evidence for their role as early intermediates in photosynthesis. AB - Photosynthetic fixation of (14)CO(2) in the bean Phaseolus vulgaris, cv. Pencil Pod Black Wax, resulted in the appearance of labeled compounds that were characterized as phosphoserine and phosphohydroxypyruvate by chromatographic separation and by the synthesis of chemical derivatives. In (14)CO(2)/(12)CO(2) pulse-chase experiments these metabolites demonstrated the rapid pool saturation and depletion of (14)C characteristic of early intermediates in photosynthetic carbon fixation. They were present in sufficient amounts to account for about 35% of total carbon fixed in 1 minute. PMID- 16660021 TI - Inhibition of the Development of Induced Respiration and Cyanide-insensitive Respiration in Potato Tuber Slices by Cerulenin and Dimethylaminoethanol. AB - The interdependence of the development of wound-induced respiration and membrane related phospholipid biosynthesis in potato tuber (Solanum tuberosum var. Russet) slices was established by the use of agents which selectively affect lipid and phospholipid synthesis. Cerulenin, a specific inhibitor of de novo fatty acid synthesis, inhibited the ultimate development of wound-induced respiration and of cyanide resistance only when given in the critical first 10 to 12 hours of slice aging. Similarly, when slices were exposed to the choline analogue dimethylaminoethanol within the first 10 hours, the phospholipid composition of the membrane lipids was drastically altered, the wound-induced respiration in a 24-hr period was substantially curtailed, and the development of cyanide insensitivity was sharply inhibited. These observations indicate that time restricted membrane-related phospholipid synthesis is prerequisite to the development of wound-induced respiration and concurrent cyanide insensitivity. PMID- 16660022 TI - Purification and initial kinetic characterization of different forms of o acetylserine sulfhydrylase from seedlings of two species of phaseolus. AB - Purification of O-acetylserine sulfhydrylase (OASS) from seedlings of two species of Phaseolus reveals the presence in both species of two forms of this enzyme. The isolation and purification procedure gives purification of 7- to 160-fold for individual isoenzymes with specific activities ranging from 33 IU mg(-1) to 775 IU mg(-1) protein.Detailed study of the basic kinetic parameters of the OASS isoenzymes indicates that both forms from Phaseolus vulgaris (which are of about equal specific activity) display substrate inhibition by S(2-) above 1 mm and positive cooperativity at lower concentrations of S(2-). With respect to O acetylserine (OAS), the second substrate of the reaction, one P. vulgaris isoenzyme shows substrate inhibition by OAS concentrations above 10 mm, while the second is unaffected by OAS concentrations up to 50 mm. The isoenzymes from Phaseolus polyanthus (one of which has a specific activity 24 times higher than the other) are slightly and approximately equally inhibited by both S(2-) and OAS. PMID- 16660024 TI - Diffusion mechanism for phyllotaxis: theoretical physico-chemical and computer study. AB - The mechanism for leaf position determination by the diffusion of an inhibitor has been studied in relation to the geometry of leaf positions. A computer model has been constructed for the inhibitor-diffusion process on a cellular cylindrical surface. The behavior of the model has been analyzed mathematically. The main results are: (a) that our model generates most of the phyllotactic patterns observed in nature; and (b) that restraints have been found for permissible values of diffusion rates and decay rates of the hypothetical inhibitor. PMID- 16660025 TI - Ethylene-induced Fine Structure Alterations in Cotton and Sugarbeet Radicle Cells. AB - Electron microscopic examination of cotton (Gossypium hirsutum L.) and sugarbeet (Beta vulgaris L.) radicles subjected to ethylene at 1, 10, 100 microliters per liter and a saturated ethylene atmosphere showed distinct ultrastructural differences compared to aerobic, control tissue and to anaerobic, nitrogen treated radicles. Short term ethylene treatments of 1 hour induced the formation of cisternal stacks of rough endoplasmic reticulum in undifferentiated radicle cells. Similar profiles of rough endoplasmic reticulum were not observed in control radicles, but were found in nitrogen-treated tissues. Additionally, ribosomes in all ethylene-treated radicles were larger than ribosomes in control and nitrogen-treated cells. By ribosome density determinations it was found that the ethylene-treated ribosomes were swollen and less dense. The cell walls of the ethylene-treated tissue were also expanded and less dense than those of control tissue. PMID- 16660023 TI - Properties and subcellular distribution of two partially purified ornithine transcarbamoylases in cell suspensions of sugarcane. AB - The spatially separated forms of ornithine transcarbamoylase (EC 2.1.3.3) of different molecular weights coexist in sugarcane (Saccharum sp.). The smaller form of the enzyme (mol wt 79,000) appears to be cytoplasmic, while a larger form (mol wt 224,000) sedimented with mitochondria. The Km of the cytoplasmic enzyme for ornithine was 3.11 mm, while the enzyme in the mitochondrial fraction had a Km of 0.50 mm for this substrate; both enzymes had similar affinity for carbamoyl phosphate (0.12 mm). Characteristics of the smaller ornithine transcarbamoylase are in keeping with a predominantly catabolic function, those of the enzyme which sediments with mitochondria, with an anabolic function. Only the mitochondrial enzyme was regulated in vivo by exogenous arginine. PMID- 16660026 TI - Adenylate cyclase activity not found in soybean hypocotyl and onion meristem. AB - Tissue, homogenates, and purified cell fractions prepared from hypocotyls of a dicot, soybean (Glycine max), and meristematic tissue of a monocot, onion (Allium cepa), were examined critically for evidence of adenylate cyclase activity. Three assay methods were used: chemical analysis, isotope dilution analysis, and enzyme cytochemistry. In both crude extracts or whole tissue, as well as purified membranes, with or without auxin, no adenylate cyclase was detected by any of the three methods.For plasma membranes, the specific activity was less than 1/40 or 1/25,000 that of rat liver plasma membranes, depending on the assay procedure, i.e. below the limits of detection. Using comparable methods, we could detect neither cyclic adenosine 3':5'-monophosphate nor the phosphodiesterase responsible for its degradation in either purified membranes or homogenates. The results suggest that hormone responses in plants are not generally mediated by a mechanism involving the obligate production of cyclic adenosine 3':5' monophosphate by a plasma membrane associated adenylate cyclase. PMID- 16660027 TI - Genetic characterization and high efficiency photosynthesis of an aurea mutant of tobacco. AB - A new tobacco (Nicotiana tabacum) aurea mutant was isolated from the progeny of a selfed variegated tobacco plant. The new mutant is termed Su/su var. Aurea. If the mutant is selfed, the seeds obtained give rise to four types of plants: green seedlings which correspond to the wild type; yellow-green seedlings which correspond to the earlier described Su/su; yellow seedlings which correspond to the new tobacco aurea mutant Su/su var. Aurea; and white lethal seedlings. The frequency ratio of the four phenotypes is 1:1:1:1. It appears that the mutation is due to two independent nuclear factors, su and aur, both of which have to be present in a heterozygous conditions, Su/su Aur/aur, to give rise to the new aurea phenotype. The aurea mutant Su/su var. Aurea has a reduced photosynthetic unit size which is approximately one-eighth of the wild type. Despite its chlorophyll deficiency, the plant grows well and exhibits maximal photosynthetic rates on a chlorophyll basis which are at least seven times higher than those of the green wild type provided the temperature and the light intensities are high enough. In contrast to the earlier described Su/su, the new mutant does not exhibit more photorespiration than the wild type. It appears that the factor aur causes either repression of photorespiration or an increase in the number of functioning photosynthetic units. PMID- 16660028 TI - C(4) Pathway Photosynthesis at Low Temperature in Cold-tolerant Atriplex Species. AB - Two species of Atriplex were grown under low temperature (8 C day/6 C night) and high temperature (28 C day/20 C night) regimes. The photosynthetic capacity of these plants was studied as a function of temperature in a leaf gas exchange cuvette. Both species showed substantial photosynthetic capacity between 4 and 10 C and this was not enhanced by growth at low temperatures but rather, was somewhat greater in plants grown at higher temperature. Photosynthetic capacity of low temperature-grown plants at high temperature was greater in Atriplex confertifolia (Torr. and Frem.) S. Watts., a native of cool deserts, than in Atriplex vesicaria (Hew. ex. Benth.) from warmer desert areas. Leaves of both species were also subjected to (14)CO(2) pulse-chase and steady-state feeding experiments under controlled temperature conditions. These experiments revealed that the kinetics of carbon assimilation through the intermediates of the C(4) pathway is not substantially disrupted at low temperature in either species. There was, however, a substantial interchange of label between aspartate and malate at low temperature which was not evident at high temperature. There was also an increase in the pool sizes of the C(4) acids involved in photosynthesis of A. confertifolia. Speculation as to the explanation of these changes and their possible significance in promoting low temperature C(4) photosynthesis in these plants is presented. PMID- 16660029 TI - Net Photosynthesis, Electron Transport Capacity, and Ultrastructure of Pisum sativum L. Exposed to Ultraviolet-B Radiation. AB - Pisum sativum L. was exposed to ultraviolet-B (UV-B) radiation (280-315 nm) in greenhouse and controlled environment chambers to examine the effect of this radiation on photosynthetic processes. Net photosynthetic rates of intact leaves were reduced by UV-B irradiation. Stable leaf diffusion resistances indicated that the impairment of photosynthesis did not involve the simple limitation of CO(2) diffusion into the leaf. Dark respiration rates were increased by previous exposure to this radiation. Electron transport capacity as indicated by methylviologen reduction was also sensitive to UV-B irradiation. The ability of ascorbate-reduced 2,6-dichlorophenolindophenol to restore much of the electron transport capacity of the UV-B-irradiated plant material suggested that inhibition by this radiation was more closely associated with photosystem II than with photosystem I. Electron micrographs indicated structural damage to chloroplasts as well as other organelles. Plant tissue irradiated for only 15 minutes exhibited dilation of thylakoid membranes of the chloroplast in some cells. Some reduction in Hill reaction activity was also evidenced in these plant materials which had been irradiated for periods as short as 15 minutes. PMID- 16660030 TI - Purification and Characterization of Sucrose Synthetase from the Shoot of Bamboo Leleba oldhami. AB - A 108-fold purification of the sucrose synthetase from the extract of the shoot of bamboo Lelaba oldhami was achieved by ammonium sulfate fractionation, calcium phosphate gel adsorption, and chromatographic separations on Sephadex G-100 and diethylaminoethyl-cellulose columns. Some properties of this enzyme, namely thermal and pH stabilities, stabilization by aqueous glycerol, pH optimum, substrate specificities, effects of metallic ions, effects of sulfhydryl reagents, molecular weight, sedimentation constants, isoelectric point, and substrate saturation kinetics had been investigated.The substrate saturation kinetics indicated that the enzyme could be an allosteric enzyme with the saccharide substrates (sucrose and fructose) serving as the homotropic allosteric effectors in regulating the biosynthesis and degradation of sucrose. PMID- 16660031 TI - Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum. AB - Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection. PMID- 16660032 TI - Role of Concentration-dependent Unloading in Mathematical Models of Munch Transport. AB - It has been erroneously claimed (Goeschl et al. [1976] Plant Physiol. 58: 556 562) that concentration-dependent unloading is required for a correct mathematical solution for Munch phloem transport. Although it may prove to be physiologically correct, concentration-dependent unloading is not a mathematical necessity. Furthermore, its use in a mathematical model may not be desirable, because there is an infinite family of solutions for any given unloading distribution, irrespective of whether concentration-dependent unloading is assumed. Some illustrative numerical results are presented. PMID- 16660033 TI - Effect of Temperature on Phytochrome-mediated Responses in Seedlings of Mustard. AB - Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.). An investigation was conducted into the effects of temperature on the light promotion of these three photoresponses. When given prior to a light exposure there was either no difference between a 20 and 30 C temperature treatment, or the 30 C treatment resulted in a greater photoresponse. When given subsequent to a light exposure 20 C was far more effective in promoting the responses than was a 30 C treatment. These results are in accordance with the known effects of low temperature on delaying phytochrome-far red absorbing form (Pfr) destruction, thus resulting in more Pfr being available to promote the responses. The difference between temperature treatments was greatest following a short light exposure, and much smaller following a long light exposure. When light was given as intermittent illumination the difference between subsequent temperature treatments was much less than when the same amount of light was given as continuous illumination. PMID- 16660034 TI - Metabolism and distribution of cyclohexanecarboxylic Acid, a plant growth stimulant, in bush bean. AB - A foliar spray of 10 mm cyclohexanecarboxylic acid (CHC), a component of the growth stimulant naphthenic acid, to primary leaves of 14-day-old plants of bush bean, Phaseolus vulgaris L., cv Top Crop, resulted in increased vegetative growth and pod production. One minute after the application of 0.5 mm CHC-7-(14)C (CHC(*)) to a primary leaf, CHC(*) was present within it. The chief conversion of the CHC(*) in the leaf during the interval 15 minutes to 4 hours after the acid had been applied appeared to be CHC(*) to its glucose conjugate (CHC(*)-G), and during 4 to 48 hours, CHC(*)-G to CHC(*)-aspartate and an unknown metabolite. Radioactivity was confined to the leaf for at least 1 hour, but by the 12th hour was detected throughout the plant. In the interval 1 to 4 weeks after CHC(*) application, the mean percentage distribution of radioactivity was: treated leaf, 65.3; roots, 18.8; stem, 7.7; trifoliate leaves, 5.9; flower buds-flowers-pods, 2.3. During this period CHC(*)-G was the most prominent metabolite in all organs; no free CHC(*) was detected. Light favored the movement of CHC(*) conjugates out of the leaf; glucose fed to dark-grown plants substituted for light to some extent but aspartate was relatively ineffective, suggesting the dependence of outward movement on ATP. The presence of the glucose and aspartate conjugates of the acid in all organs of CHC-treated plants and the absence of free CHC from them suggest that one or both conjugates, rather than the acid itself, are involved in growth stimulation. PMID- 16660035 TI - Assay for Helminthosporium maydis Toxin-binding Activity in Plants. AB - A relatively rapid and sensitive assay is described for assessing the binding of Helminthosporium maydis Race T(14) C-toxins I and II to plant components. The technique is a modification of the one of Haddad and Birge (J. Biol. Chem. 250: 299-303, 1975), and utilizes dextran-coated charcoal as an adsorbent for the unreacted toxin and employs a Millipore filter to isolate the protein-toxin complex.Extracts from susceptible corn line W64A Tms possess a protein primarily localized in the cytosol which is relatively heat-insensitive, ficin- and papain sensitive, and binds toxins I and II at half-saturation in the order of 0.1 mm. The toxin-binding activities of the extracts of various corn lines and other species are not correlated to resistance or susceptibility to H. maydis Race T, nor to sensitivity to the toxins. These findings are discussed relative to the function of the binding protein and cellular sensitivity to the toxins. PMID- 16660036 TI - Holographic interferometric measurement of motions in mature plants. AB - Holographic interferometry has been used to plot graphs of the phototropic and geotropic bending undergone by a mature Stapelia variegata Linn over a 5-minute period. The holographic interferometric technique is shown to have the advantage of measuring displacements at least as small as 0.16 micrometers which permits observation of extremely slow plant motions over time periods of a few minutes. In addition, the holographic technique provides a permanent record of displacement information over the entire plant in a single hologram. The short 5 minute period required to produce a holographic interferogram has permitted the monitoring of slow plant motions by recording a series of consecutive holograms at 5-minute intervals over a 75-minute period. The results have been plotted on a graph thereby capturing for the first time such small displacement, velocity, and acceleration of a mature plant as a function of time. PMID- 16660037 TI - Regulation of plant pyruvate dehydrogenase complex by phosphorylation. AB - The ATP-dependent inactivation of the pyruvate dehydrogenase complex (PDC) was examined using ruptured mitochondria and partially purified pyruvate dehydrogenase complex isolated from broccoli and cauliflower (Brassica oleracea) bud mitochondria. The ATP-dependent inactivation was temperature- and pH dependent. [(32)P]ATP experiments show a specific transphosphorylation of the gamma-PO(4) of ATP to the complex. The phosphate attached to the PDC was labile under mild alkaline but not under mild acidic conditions. The inactivated phosphorylated PDC was not reactivated by 20 mm MgCl(2), dialysis, Sephadex G-25 treatment, apyrase action, or potato acid phosphatase action. However, partially purified bovine heart PDC phosphatase catalyzed the reactivation and dephosphorylation of the isolated plant PDC. The ATP-dependent inactivation phosphorylation of the PDC was inhibited by pyruvate. It is concluded that the ATP-dependent inactivation-phosphorylation of broccoli and cauliflower mitochondrial PDC is catalyzed by a PDC kinase. It is further concluded that the PDC from broccoli and cauliflower mitochondria is capable of interconversion between an active (dephosphorylated) and an inactive (phosphorylated) form. PMID- 16660038 TI - Effect of glyphosate on carrot and tobacco cells. AB - The growth of suspension-cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L. cv. Xanthi) cells was inhibited by glyphosate (N [phosphonomethyl]glycine). This inhibition was reversed by adding combinations of phenylalanine, tyrosine, and tryptophan or casein hydrolysate. Casein hydrolysate and phenylalanine + tyrosine + tryptophan were the most effective treatments. Reversal of glyphosate-induced inhibition occurred only if the aromatic amino acids were added during the first 8 days of glyphosate incubation. Glyphosate uptake was not reduced when the aromatic amino acids or casein hydrolysate were added.Even though phenylalanine biosynthesis is a suggested site for glyphosate action, inhibitory levels of glyphosate did not lower free phenylalanine concentrations in carrot cells within 10 days. (14)C-Phenylalanine studies indicated that the metabolic pool size was, likewise, not decreased.In carrot cells total free amino acids increased within 6 hours after glyphosate addition. Cell protein levels declined within 48 hours following glyphosate treatment.Studies on (14)C-thymidine and (14)C-uridine incorporation were complicated by rapid metabolism of these compounds to (14)CO(2). PMID- 16660039 TI - Effects of alpha-Hydroxy-2-Pyridinemethanesulfonic Acid on Photosynthetic Carbon Dioxide Uptake and Stomatal Movements in Excised Tomato Leaves. AB - The effects of 10(-2)m alpha-hydroxy-2-pyridinemethanesulfonic acid (alphaHPMS) on the CO(2) compensation point, photosynthetic CO(2) uptake, CO(2) evolution into CO(2)-free air in light, and stomatal movement, in excised tomato leaves (Lycopersicon esculentum Mill. Eurocross BB-F(1) Hybrid) were studied. It was found that alpha-HPMS had a transient lowering effect on the CO(2) compensation point of treated leaves within the first 5 minutes of application. The net photosynthetic CO(2) uptake was inhibited by alpha-HPMS treatment. The inhibition increased with time and was enhanced in an O(2)-free atmosphere. The CO(2) evolution into CO(2)-free air in light was inhibited by alpha-HPMS. The inhibition was O(2)-dependent because the effect was observed only in 21% O(2) but not in O(2)-free N(2). Stomatal apertures were affected by alpha-HPMS, but the effect was transient and was observed 15 to 30 minutes after the application. The time course of this closure did not account for the observed inhibition of net CO(2) uptake. PMID- 16660040 TI - Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development. AB - In vivo CO(2) fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N(2) fixation. PEP carboxylase activity was predominantly located in the bacteroid containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO(2) fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO(2) fixation in mature nodules was 7.9 nmol hour(-1) mg fresh weight(-1), similar to rates of N(2) fixation and reported values for amino acid translocation.The results suggest that the oxaloacetete used as the primary "carbon skeleton" acceptor for ammonia assimilation and amino acid synthesis in lupin nodules is provided via the PEP carboxylase reaction rather than through the tricarboxylic acid cycle. The source of PEP is presumably glycolysis, while the major source of CO(2) is inferred to be respiration. PMID- 16660041 TI - Dependence of Wound-induced Respiration in Potato Slices on the Time-restricted Actinomycin-sensitive Biosynthesis of Phospholipid. AB - Actinomycin D prevents the full development in a 24-hour period of both wound respiration and cyanide resistance only when given in the first 10 to 12 hours following the cutting of potato tuber (Solanum tuberosum var. Russet) slices. The capacity for choline incorporation into phosphatidylcholine increases with slice aging and is inhibited by actinomycin D in the same time-restricted way. The time restricted effectiveness of actinomycin D applies to the cutting-elicited enhanced synthesis of three critical enzymes of phosphatidylcholine synthesis, namely phosphorylcholine-glyceride transferase, phosphorylcholine-cytidyl transferase, and phosphatidylphosphatase. By contrast, actinomycim D given at any time is without effect on the measurable levels after 24 hours of a selection of glycolytic and mitochondrial respiratory enzymes. Neither succinic dehydrogenase nor cytochrome oxidase activity increases with time in aging potato slices in the presence or absence of chloramphenicol. The foregoing observations emphasize the central role of phospholipid, and ultimately membrane biosynthesis, in the development of wound-induced respiration. PMID- 16660042 TI - Reversal of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition of carbon dioxide fixation in spinach chloroplasts and protoplasts by dicarboxylic acids. AB - 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition of (14)CO(2) fixation in isolated intact spinach (Spinacia oleracea L.) chloroplasts was reversed (by about 34%) by l-malate but not by oxaloacetate (OAA). However, OAA reversed the DCMU inhibition in spinach protoplasts indicating an extrachloroplastic enzyme requirement. Extrachloroplastic OAA reduction was coupled with external dihydroxyacetone phosphate (DHAP) oxidation, and the malate formed from such coupling might then enter the chloroplasts. Evidence was presented using ruptured protoplasts that the export of recently formed 3-phosphoglyceric acid (PGA) out of chloroplasts in exchange for external DHAP was reversed by excess OAA. The PGA/DHAP shuttle across the chloroplast envelope was found to be regulated by the external concentrations of DHAP and OAA. PMID- 16660043 TI - Studies on the mechanism of action of dinitramine: effect on soybean root plasma membrane. AB - The effect of dinitramine, a selective herbicide, on the plasma membrane of the soybean (Glycine max L.) root was studied. Used as marker systems to observe the herbicide effect were two plasma-membrane-specific enzymes, pH 6.5 ATPase and glucan synthetase.The activity of pH 6.5 ATPase decreased significantly in membrane vesicles prepared from roots harvested 15 minutes after treatment with dinitramine. Maximum inhibition occurred in roots harvested 2 hours after treatment. Glucan synthetase activity decreased similarly within 2 hours of treatment.Membrane permeability to (86)Rb was rapidly increased by dinitramine.The activity of pH 6.5 ATPase returned to the control level within 8 hours of treatment with dinitramine.These results show dinitramine's initial site of action to be the plasma membrane, producing an over-all reduction in membrane function through inactivation of membrane-associated proteins. PMID- 16660044 TI - Semipermeable membrane system for subjecting plants to water stress. AB - A system was evaluated for growing plants at reproducible levels of water stress. Beans (Phaseolus vulgaris L.) were grown in vermiculite, transferred to a semipermeable membrane system that encased the root-vermiculite mass, and then placed into nutrient solutions to which various amounts of polyethylene glycol (PEG) 20M were added to control solution water potential. The membrane (Spectrapor 1) had a minimum molecular weight cutoff that excluded the PEG 20M. The plants equilibrated with the nutrient solution within 1 to 4 days, and exhibited normal diurnal water relations. Use of the semipermeable membrane system to induce water stress reduces many of the problems associated with hydroponic media. PMID- 16660045 TI - Immunological Identification of Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles. AB - Proteinase inhibitor I has been identified and quantified in isolated vacuoles from tomato (Lycopersicon esculentum) leaves induced to accumulate inhibitors either by wounding or by supplying excised leaves with the wound hormone, proteinase inhibitor-inducing factor. Proteinase inhibitor II was also identified in the vacuoles but not quantified. Control vacuoles were prepared from unwounded plants that did not contain inhibitors. Vacuole to leaf cell ratios of inhibitors, chlorophyll, and several vacuolar and cytoplasmic enzymes were determined. The inhibitors were found almost entirely in the vacuoles. Acid phosphatase was located in control leaf vacuoles, but was found in both vacuoles and cytoplasm in induced leaves. Carboxypeptidase, induced by wounding, was found distributed between the vacuoles and cytoplasm of induced leaves. Low vacuole to leaf cell ratios of three cytoplasmic markers, triosephosphate isomerase, catalase, and chlorophyll, indicated that the isolated vacuoles were relatively free of intact protoplasts and cell debris. PMID- 16660046 TI - Polypeptide composition of chlorophyll-protein complexes from romaine lettuce. AB - The protein moiety of the two major chlorophyll-protein complexes associated with chloroplast membranes of outer, dark green leaves of a romaine lettuce shoot (Lactuca sativa L. var. Romana) has been analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complex II, also termed light harvesting chlorophyll-protein complex, is shown to consist of a major polypeptide of 25 kilodaltons (kD) and two minor ones of 27.5 and 23 kD. The 25 kD subunit is the single largest polypeptide component of the chloroplast membranes, accounting for about 25% of their total protein. Complex I contains only high molecular weight subunits, the major one being at 67 kD, these subunits representing only a small percentage of the chloroplast membrane total protein.These data, suggesting an oligomeric nature for the apoprotein of these two chlorophyll-protein complexes, are difficult to reconcile with the estimated molecular weights of the native complexes and raise some intriguing questions as to the types of interactions among the components of these major lipoproteins of the photosynthetic membranes. PMID- 16660047 TI - Photosynthesis, growth, and the role of chloride. AB - Previous studies with isolated chloroplasts have indicated that Cl(-) is an essential cofactor for photosynthesis. Considerable support for the postulated Cl(-) requirement in photosynthesis came from the observation that Cl(-) is essential for growth. Data are presented which show that a 60% reduction in growth which occurred in Cl(-) -deficient sugar beet (Beta vulgaris L.) was not due to an effect of Cl(-) on the rate of photosynthesis in vivo (net CO(2) uptake per unit area of attached leaves). The principal effect of Cl(-) deficiency was to lower cell multiplication rates in leaves, thus slowing down their growth and ultimately decreasing their area. The absence of an effect of Cl(-) on photosynthesis in vivo was unlikely to have been due to Cl(-) retention by the chloroplasts because their Cl(-) concentration (measured after nonaqueous isolation) decreased progressively with decrease in leaf Cl(-).An effect of Cl(-) with isolated chloroplasts in vitro, however, was confirmed. Addition of Cl(-) to the reaction medium after washing chloroplasts in EDTA increased the rate of ferricyanide photoreduction 10-fold. This effect of Cl(-) did not appear to be related to the Cl(-) concentration of the chloroplasts since chloroplast Cl(-) was not decreased further by washing in EDTA. It is concluded that Cl(-) has not yet unequivocally been shown to be an essential cofactor for photosynthesis and that the response to Cl(-)in vitro probably does not have a physiological basis. PMID- 16660048 TI - Appearance of Membrane-bound Iron-Sulfur Centers and the Photosystem I Reaction Center during Greening of Barley Leaves. AB - Dark-grown barley (Hordeum vulgare) etioplasts were examined for their content of membrane-bound iron-sulfur centers by electron paramagnetic resonance spectroscopy at 15K. They were found to contain the high potential iron-sulfur center characterized (in the reduced state) by an electron paramagnetic resonance g value of 1.89 (the "Rieske" center) but did not contain any low potential iron sulfur centers. Per mole of cytochrome f, dark-grown etioplasts and fully developed chloroplasts had the same content of the Rieske center. During greening of etioplasts under continuous light, low potential bound iron-sulfur centers appear. In addition, the photosystem I reaction center, as measured by the photooxidation of P700 at 15K, also became functional; during greening the appearance of a photoreducible low potential iron-sulfur center paralleled the appearance of P700 photoactivity.These findings indicate the close association of the low potential iron-sulfur centers with the photosystem I reaction center; they also support the concept that the development of stable charge separation in the photosystem I reaction center requires, in addition to P700, a low potential iron-sulfur center. PMID- 16660049 TI - Regulation of the Biosynthesis of Ent-Kaurene from Mevalonate in the Endosperm of Immature Marah macrocarpus Seeds by Adenylate Energy Charge. AB - The rate of kaurene biosynthesis from mevalonate in a cell-free enzyme preparation from the endosperm of immature seeds of Marah macrocarpus is regulated by adenylate energy charge. The response curve is typical of a biosynthetic energy-utilizing sequence in which the rate of biosynthesis increases sharply as the energy charge is increased above 0.80. ADP proved to be an effective inhibitor of this process. AMP gave no inhibition at concentrations up to 2 mm and orthophosphate gave no inhibition up to 15 mm. Measurement of the pool sizes of intermediates in the sequence showed that the presence of ADP caused an increase in the levels of 5-phosphomevalonate and 5 pyrophosphomevalonate and a decrease in the levels of isopentenyl pyrophosphate and kaurene. These results indicate that pyrophosphomevalonate decarboxylase is the enzyme most subject to regulation by adenylate energy charge. The rate of conversion of isopentenyl pyrophosphate to kaurene and the rate of utilization of mevalonate by mevalonate kinase were not influenced by variations in the adenylate energy charge. PMID- 16660050 TI - Alteration of soybean complex lipid biosynthesis by s-ethyl dipropylthiocarbamate. AB - Soybean (Glycine max [L.] Merr. var. Glabrous D62-7812) plants were grown in aerated Hoagland and Arnon mineral nutrient solution containing 0 or 2.6 mum S ethyl dipropylthiocarbamate (EPTC) in a growth chamber. After 19 days exposure to EPTC, total leaf fresh weight was reduced 18% by 2.6 mum EPTC while total leaf fatty acid content was reduced 63%. Galactolipid content decreased while phospholipid content increased. Linolenic acid content decreased from 67.5% of the leaf total fatty acid content to 31.5% with 2.6 mum EPTC treatment. Equivalent increases were observed in palmitic (+6.3%), stearic (+1.1%), oleic (14.4%), and linoleic (+13.9%) acids. PMID- 16660051 TI - Evolutionary conservation of chloroplast genes coding for the large subunits of fraction 1 protein. AB - Crystalline fraction 1 protein, obtained from four species of Nicotiana, have identical polypeptide compositions and isoelectric points. However, the tryptic peptide map of the large subunit of this protein from N. knightiana and N. paniculata differs from that of N. tomentosa and N. tomentosiformis. Since the large subunits of fraction 1 protein are coded by chloroplast DNA, the difference in their primary structure reflects the structural changes of the chloroplast genes containing the coding information. This indicates that the rate of mutation of chloroplast DNA seems to be higher than predicated from the analysis of isoelectric points of this protein. PMID- 16660052 TI - Polarity of Indoleacetic Acid in young Coleus Stems. AB - Young internodes of Coleus blumei Benth. have long been known for their sizable amount of acropetal indoleacetic acid movement. However, plants of the same clone, under improved growing conditions, now show almost absolute basipetal polarity of (14)C-indoleacetic acid, as measured by liquid scintillation counting of (14)C in the receiver cylinders of agar. The ratio of basipetal to acropetal movement is now as much as 85:1, instead of the 3:1 ratio found years ago under conditions providing slower growth. PMID- 16660053 TI - DNA binding and uptake by nuclei isolated from plant protoplasts: factors affecting DNA binding and uptake. AB - DNA binding and uptake by nuclei isolated from soybean (Glycine max L. Merr.) protoplasts were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drastically with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occurred at pH 6 and the process was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dT]-[dA-dT]) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-l-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake. PMID- 16660054 TI - A rapid, simple method for nuclei isolation from plant protoplasts. AB - A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA. PMID- 16660055 TI - Gibberellic Acid enhancement of DNA turnover in barley aleurone cells. AB - When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [(3)H]thymidine labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse chase studies demonstrated that the newly synthesized DNA is metabolically labile.The buoyant density on CsCl density gradients of hormone-treated aleurone DNA is identical with that of DNA extracted from whole seedlings. After density labeling halfseed DNA with 5-bromodeoxyuridine, a bimodal absorption profile is obtained in neutral CsCl. The light band (1.70 g/ml) corresponds to unsubstituted DNA, while the heavy band (1.725-1.74 g/ml) corresponds to a hybrid density labeled species. GA increases the relative amount of the heavy (hybrid) peak in halfseed aleurone layer DNA, further suggesting that the hormone enhances semiconservative replication in halfseeds.DNA methylation was also demonstrated. Over 60% of the radioactivity from [(3)H-Me]methionine is incorporated into 5 methylcytosine. GA has no effect on the percentage distribution of label among the bases.It was concluded that GA enhances the rate of DNA degradation and DNA synthesis (turnover) in halfseeds, but primarily DNA degradation in isolated aleurone layers. Incorporation by isolated aleurone layers is due to DNA repair. Semiconservative replication apparently plays no physiological role in the hormone response, since both isolated aleurone layers and gamma-irradiated halfseeds respond normally. The hypothesis was advanced that endoreduplication and DNA degradation are means by which the seed stores and mobilizes deoxyribonucleotides for the embryo during germination. PMID- 16660056 TI - Response to chilling of tomato seedlings and cells in suspension cultures. AB - Tomato cell suspensions and seedlings (Lycopersicon esculentum) responded comparably when exposed to chilling temperatures (10 C or below). Seedling growth and cellular activities related to cell viability and culture growth (triphenyltetrazolium chloride reduction, fluoroscein diacetate uptake, and hydrolysis) were sharply diminished below 10 C. Arrhenius plots of the respiratory O(2) consumption by both seedlings and cell suspensions had a break at 10 C, as is characteristic for chilling-sensitive species. The acyl chains that were found in the phospholipids of both cell cultures and seedlings were similar. These results indicate the potential usefulness of plant suspension cultures for studies of chilling injury. PMID- 16660057 TI - Oxaloacetate as the Source of Carbon Dioxide for Photosynthesis in Bundle Sheath Cells of the C(4) Species Panicum maximum. AB - 3-Mercaptopicolinic acid (3-MPA), an inhibitor of phosphoenolpyruvate carboxykinase, was employed to study the role of organic acid decarboxylation during C(4) photosynthesis. Treatment of detached Panicum maximum leaves with 5 mm 3-MPA inhibited photosynthesis 70 to 75%. Oxygen was found to have no effect on the degree of inhibition. The postillumination (14)CO(2) burst associated with P. maximum photosynthesis was almost abolished by 5 mm 3-MPA. The turnover rates of malate and aspartate during C(4) photosynthesis were severely reduced as well as the rates of formation of C(3) cycle intermediates in P. maximum leaves treated with 3-MPA. These results are interpreted as direct evidence for the fixation of CO(2), arising from the decarboxylation of oxaloacetate, by the C(3) cycle in bundle sheath cells of P. maximum leaves. PMID- 16660058 TI - Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations. AB - Kinetin, N(6)-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N(6)-furfuryladenine-8-(14)C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N(6)-furfuryladenosine (fr(6)A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr(6) A moieties. The extent of kinetin incorporation was four times greater than that observed for N(6)-benzyladenine. The radiochemical purity of the recovered fr(6) A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr(6) A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N(6) furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative. PMID- 16660059 TI - C(2)H(4): Its Incorporation and Oxidation to CO(2) by Cut Carnations. AB - Cut carnations (Dianthus caryophyllus L. cv. ;Improved White Sim') were exposed to ultra high purity (14)C(2)H(4) (20 mul/1) during flower opening and senescence to study its incorporation and metabolism. During treatment precautions were taken to exclude inhibitory volatiles from rubber serum stoppers which were identified as CS(2) and COS. As with the pea seedling (Nature 1975, 255:144-147), cut carnations incorporated (14)C(2)H(4) into ethanol-soluble tissue metabolites and oxidized the hormone to (14)CO(2). Oxidation increased from 0.5 to 3 dpm . mg dry wt(-1).6 hr(-1) during the period of flower opening and early petal wilt. As severe petal wilt set in, and the ovary increased in size and dry weight, oxidation increased to a peak of nearly 29 dpm . mg dry wt(-1).6 hr(-1). Concomitant with this peak was a similar rise in the rate of (14)C(2)H(4) incorporation into the petals, peduncle, bracts, and sepals. Much higher rates of incorporation were found for the reproductive and receptacle tissues. Incorporation into these tissues steadily increased during flower opening reaching a peak of over 160 dpm . mg dry wt(-1) . 6 hr(-1) just before full bloom. This peak preceded a peak of endogenous ethylene production while the (14)C(2)H(4) oxidation peak followed it. PMID- 16660060 TI - Characteristics of tomato cell wall degradation in vitro: implications for the study of fruit-softening enzymes. AB - The in vitro degradation of green tomato (Lycopersicon esculentum L.) cell walls by an extract of ripe fruits was characterized. The susceptibility of isolated walls to enzymolysis varied considerably among the different cultivars tested. Wall solubilization in vitro appeared to be nearly as extensive as that which accompanies fruit ripening. The solubilized material was primarily polyuronide; smaller amounts of neutral sugar were released. Gel filtration chromatography indicated that an endopolygalacturonase was the only enzyme in the citrate extract able to hydrolyze isolated cell walls. However, this polygalacturonase in vitro did not lead to the substantial (40-60%) decrease in wall galactose which was observed in situ. This difference between in vitro and in situ wall modification is discussed in terms of the possible involvement of other wall hydrolases in fruit softening. PMID- 16660061 TI - Concentration of Indole-3-acetic Acid and Its Derivatives in Plants. AB - Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed. PMID- 16660062 TI - Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence. AB - Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue. PMID- 16660063 TI - Effect of ethylene on the endogenous cytokinin and gibberellin levels in tuberizing potatoes. AB - High concentrations of 2-chloroethylphosphonic acid inhibited tuberization on aged potato tubers (Solanum tuberosum) that had been predisposed to the little tuber disorder. As a result of this treatment sprouts developed which contained relatively high levels of endogenous gibberellins and which elongated normally. The endogenous cytokinin levels in the different treatments did not change appreciably. It is suggested that tuberization is prevented by ethylene either as a direct inhibition of cell division or that it prevents the endogenous cytokinins from functioning. Irrespective of the mode of action of ethylene, cell division apparently is the primary process affected, the result being that storage tissue required for the accumulation of starch is not formed. PMID- 16660064 TI - Reversal of induced dormancy in lettuce by ethylene, kinetin, and gibberellic Acid. AB - The germination of lettuce seeds (Lactuca sativa L., cv. Premier Great Lakes) was significantly inhibited by high temperature (32 C), 0.1 mM abscisic acid or 0.4 M mannitol. Ethylene (16 mul/1 of air) partially reversed the dormancy induced by all three inhibitors but only in the presence of 1 mM gibberellic acid (GA) or light. Neither ethylene plus GA nor ethylene plus light were able to promote germination when thermal inhibition was imposed at 36 C. Addition of 0.01 mM kinetin to the ethylene plus GA or light reversed thermodormancy at 36 C. The dormancy imposed by abscisic acid was also reversed by kinetin. Kinetin was unable to reverse the osmotic dormancy imposed by mannitol. The reversal of osmotic dormancy by ethylene or ethylene plus GA was actually inhibited by kinetin but only in the light. Kinetin apparently stimulates cotyledonary growth in the presence of light, and this growth may compete for certain metabolites critical to radicle growth and subsequent germination. Kinetin and ethylene, as demonstrated primarily in the thermodormancy at 36 C and in osmotic dormancy, appear to regulate a common event(s) leading to germination but through mechanisms unique to each respective growth regulator. The regulation of germination by ethylene is absolutely dependent upon an interaction with GA and/or light. PMID- 16660065 TI - Plastocyanin as the possible site of photosynthetic electron transport inhibition by glutaraldehyde. AB - Treatment of spinach chloroplasts with glutaraldehyde causes an inhibition in the electron transport chain between the two photosystems. Measurements of O(2) flash yields, pH exchange, and fluorescence induction show that the O(2) evolving apparatus, photosystem II and its electron acceptor pool are not affected. The behavior of P700 indicates that its reduction but not its oxidation, is severely inhibited. Cytochrome f is still reducible by photosystem II but also slowly oxidizable by photosystem I. The sensitivity of isolated plastocyanin to glutaraldehyde further supports the conclusion that glutaraldehyde inhibits at the plastocyanin level and thereby induces a break between P700 and cytochrome f. PMID- 16660066 TI - Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration. AB - When the CO(2) concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3 phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO(2) in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO(2)) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO(2), providing further evidence that the cycle is well regulated. PMID- 16660067 TI - Asparaginase and asparagine transaminase in soybean leaves and root nodules. AB - Asparaginase activity ( 4 Glc 1 --> 3 Glc 1 --> 4 Glc ... has been isolated from several commercial preparations of Bacillus subtilis alpha-amylase including one purified by repeated crystallization. The beta-d-glucanase will not hydrolyze cellulose or laminarin. Gel filtration on a Bio-Gel P-200 column results in separation of the glucanase from the alpha-amylase. The enzyme is of the endo type as changes in the substrate viscosity appear long before the appearance of detectable reducing sugars. No evidence of product inhibition was revealed and appropriate substrates were converted to oligosaccharides, the quantity of which approaches theoretical yields. The products of the reaction were separated according to molecular size by use of Bio-Gel P-2 gel filtration and found to be consistent with the action pattern of the enzyme. Kinetic studies show that the enzyme has an optimum activity at pH 6.5, a V(max) of 13.9 mug glucose equivalent released/mug protein.hour, and an apparent Km of 3.4 mg of lichenan per ml. Potential application of this enzyme for the structural characterization of plant cell wall glucans is discussed. PMID- 16660081 TI - Characterization of starch breakdown in the intact spinach chloroplast. AB - Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with (14)C-starch photosynthetically from (14)CO(2). Starch breakdown was dependent upon inorganic phosphate and the (14)C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of (14)C from triose-phosphate to glycerate 3 phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate. PMID- 16660083 TI - Mesophyll cell protoplasts of potato: isolation, proliferation, and plant regeneration. AB - Mesophyll cell protoplasts were isolated from potato (Solanum tuberosum L. cv. Russet Burbank) leaves and induced to proliferate in culture. Protoplast division was observed only among preparations isolated from plants previously conditioned under short periods of low intensity illumination. Sustained growth and development of protoplast-derived calli (p-calli) occurred when they were maintained on defined media at 24 C under 500 lux lighting. Shoot bud development within p-calli was controlled by a number of factors including light, temperature, basic medium composition, nature and source of phytohormones, the continued presence of an osmoticum, low concentrations of a utilizable carbohydrate, and the developmental stage of the p-callus. PMID- 16660082 TI - Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit. AB - Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening. PMID- 16660084 TI - Indoleacetic Acid Levels in Phaseolus, Zea, and Pincus during Seed Germination. AB - The content of indoleacetic acid (IAA) was determined in dry and germinating seeds of French bean (Phaseolus vulgaris), sugar maize (Zea mays), and Scots pine (Pinus silvestris). IAA was found in both the dry and the soaked seeds of the three species examined. The amount of IAA per gram fresh weight was extremely different in the three species whereas the variation between different harvests of the same species was small. Maize contained the highest level of IAA and bean the smallest. The time of imbibition was of decisive importance for the level of IAA. In all three species the content of IAA increased considerably during the initial 4 hours of swelling. The highest level of IAA was found in seeds that had swelled for 24 to 48 hours, during which period the radicles began to emerge from the seed coat. Later, during the period of rapid root growth, the content of IAA declined. PMID- 16660085 TI - Seasonal allocation of photoassimilated carbon in douglas fir seedlings. AB - The uptake of CO(2) by Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings and the allocation of photoassimilated carbon among five vegetative tissues were closely related to seedling phenology. In May, newly flushing needles required 5.2% day(-1) of photoassimilated carbon relative to needle tissue carbon. As these needles matured, this carbon requirement declined to 1.95% day(-1) in August, to 0.94% day(-1) in November, and to 0.76% day(-1) in January. Other tissues of Douglas fir seedlings required different amounts of photoassimilated carbon for growth and metabolism. These data provide a strong link between daily CO(2) uptake and the regulation of carbon allocation by seasonal phenology.The data, obtained with long term labeling with (14)CO(2), were analyzed with a compartment model representing the dynamic flux of carbon through the seedlings. PMID- 16660086 TI - Effects of Indoleacetic Acid on the Quantity of Mitochondria, Microbodies, and Plastids in the Apical and Expanding Cells of Dark-grown Oat Coleoptiles. AB - We determined the number of mitochondria, microbodies, and plastids in dark-grown oat (Avena sativa) coleoptiles following incubation in indoleacetic acid (IAA) for a period of 60 minutes at 6-minute intervals. In the apical outer epidermis of coleoptiles, the mitochondria increased from 31.4 to 35 per cell section with a 6-minute incubation in IAA, and this trend persisted over the 60-minute incubation. Neither the microbodies, plastids, nor the dicytosomes (Gawlik and Miller 1974 Plant Physiol 54:217-221) responded to the hormone. The apical parenchyma showed no change in quantity of any of the organelles including the dictyosomes during IAA incubation. The quick response of mitochondria in the coleoptile tip could be interpreted as an association of this organelle with hormone transport, growth, or perhaps with gravity perception. In the subapical expansion region, IAA caused significant reductions of mitochondria, microbodies, and dictyosomes in the outer epidermis compared to the control, the timing of which preceded the IAA-induced elongation and of geotropism. The fast response of organelles in the various cells is probably a change in organelle volume rather than number. That microbodies show a response to the plant hormone in the permanently achlorophyllous epidermis indicates that these organelles, in addition to their peroxisomal functions in green leaves, also may have a growth regulation function. IAA treatment was without effect on the quantity of the various types of plastids (including the amyloplasts) in the different oat coleoptile cells. PMID- 16660087 TI - Gluconeogenesis from storage wax in the cotyledons of jojoba seedlings. AB - The cotyledons of jojoba (Simmondsia chinensis) seeds contained 50 to 60% of their weight as intracellular wax esters. During germination there was a gradual decrease in the wax content with a concomitant rise in soluble carbohydrates, suggesting that the wax played the role of a food reserve. Thin layer chromatography revealed that both the fatty alcohol and fatty acid were metabolized. The disappearance of wax was matched with an increase of catalase, a marker enzyme of the gluconeogenic process in other fatty seedlings. Subcellular organelles were isolated by sucrose gradient centrifugation from the cotyledons at the peak stage of germination. The enzymes of the beta oxidation of fatty acid and of the glyoxylate cycle were localized in the glyoxysomes but not in the mitochondria. The glyoxysomes had specific activities of individual enzymes similar to those of the castor bean glyoxysomes. An active alkaline lipase was detected in the wax bodies at the peak stage of germination but not in the ungerminated seeds. No lipase was detected in glyoxysomes or mitochondria. After the wax in the wax bodies had been extracted with diethyl ether, the organelle membrane was isolated and it still retained the alkaline lipase. The gluconeogenesis from wax in the jojoba seedling appears to be similar, but with modification, to that from triglyceride in other fatty seedlings. PMID- 16660088 TI - Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems. AB - A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of delta-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed. PMID- 16660089 TI - Sucrose Hydrolysis in Relation to Phloem Translocation in Beta vulgaris. AB - Asymmetrically labeled sucrose, (14)C(fructosyl)sucrose, was used to determine whether sucrose undergoes extracellular hydrolysis during phloem translocation in the sugar beet, Beta vulgaris. In addition, the metabolism of various sugars accumulated and translocated was determined in various regious of the plant. These processes were studied in detached regions as well as in the intact, translocating plant in the source leaf, along the translocation path, and in a rapidly growing sink leaf and storage beet. The data show that, unlike sucrose accumulation into the sink tissue of sugarcane, sucrose is neither hydrolzyed prior to phloem loading or during transit, nor is it extracellularly hydrolyzed during accumulation into sink leaves or the storage beet. PMID- 16660090 TI - Synthesis and Interconversion of Amino Acids in Developing Cotyledons of Pea (Pisum sativum L.). AB - Freshly isolated cotyledons from 10-day developing pea (Pisum sativum) seeds were fed radiolabeled precursors for 5 hours, and the specific radioactivity of the free and total protein amino acids was determined using a dansylation procedure. When the seven most abundant amino acids in phloem exudate of pea fruits (asparagine, serine, glutamine, homoserine, alanine, aspartate, glycine) were fed singly, their carbon was distributed widely among the aliphatic amino acids, proline and tryptophan; sporadic labeling of tyrosine and histidine also occurred. Feeding of glucose led to relatively greater labeling of aromatic amino acids including phenylalanine. The data support the involvement of known plant pathways in these interconversions. Labeling patterns were consistent with participation of the cyanoalanine pathway in the conversion of serine to homoserine, and with the synthesis of histidine from adenosine. All of the labeled amino acids were incorporated into protein. PMID- 16660091 TI - Influence of the level of nitrate nutrition on ion uptake and assimilation, organic Acid accumulation, and cation-anion balance in whole tomato plants. AB - Tomato plants (Lycopersicon esculentum L. var. Ailsa Craig) were grown in water culture in nutrient solution in a series of 10 increasing levels of nitrate nutrition. Using whole plant data derived from analytical and yield data of individual plant parts, the fate of anion charge arising from increased NO(3) assimilation was followed in its distribution between organic anion accumulation in the plant and OH(-) efflux into the nutrient solution as calculated by excess anion over cation uptake. With increasing NO(3) nutrition the bulk of the anion charge appeared as organic anion accumulation in the plants. OH(-) efflux at a maximum accounted for only 20% of the anion charge shift. The major organic anion accumulated in response to nitrate assimilation was malate. The increase in organic anion accumulation was paralleled by an increase in cation concentration (K(+), Ca(2+), Mg(2+), Na(+)). Total inorganic anion levels (NO(3) (-), SO(4) (2 ), H(2)PO(4) (-), Cl(-)) were relatively constant. The effect of increasing NO(3) nutrition in stimulating organic anion accumulation was much more pronounced in the tops than in the roots.It is suggested that increasing the level of NO(3) nutrition to tomato plants stimulates cation uptake and translocation as counter ions are required to accompany NO(3) (-) ions to the upper plant parts, the major site of NO(3) reduction. On NO(3) reduction, the resulting stoichiometric accumulation of organic anions is balanced by the cations originally accompanying NO(3) (-) ions. Organic anions and cations are largely retained in the upper plant parts. The results suggest that only a small fraction of the total K absorbed by the roots can be translocated downward from the leaves to the roots in the phloem sap. The possible extent of K recirculation is thus low. PMID- 16660092 TI - A polarographic study of glutamate synthase activity in isolated chloroplasts. AB - Illuminated pea (Pisum sativum) chloroplasts actively catalyzed (glutamine plus alpha-ketoglutarate)-dependent O(2) evolution (average of 12 preparations 10.6 mumole mg chlorophyll per hour). The reaction was specific for glutamine and alpha-ketoglutarate; concentrations of 0.2 mm alpha-ketoglutarate and 0.6 mm glutamine, respectively, effected half-maximum rates of O(2) evolution. The reaction was inhibited by 3-(3,4-dichlorophenyl)-1-1-dimethylurea and did not occur in the dark. After osmotic shock chloroplasts did not catalyze O(2) evolution. The reaction was inhibited by azaserine and glutamate but not by 10 mm ammonia, 2.5 mm methionine sulfoximine, or 5 mm amino-oxyacetate; addition of amino-oxyacetate together with aspartate inhibited O(2) evolution. Arsenate (3 mm) enhanced O(2) evolution. The highest molar ratio for O(2) evolved per mole of alpha-ketoglutarate supplied was 0.40; the corresponding values for glutamine in the absence and presence of 3 mm arsenate were 0.20 and 0.24, respectively. The (glutamine plus alpha-ketoglutarate)-dependent O(2) evolution is attributed to photosynthetically coupled glutamate synthase activity and the activity is sufficient to account for the assimilation of inorganic nitrogen. The low molar ratio for glutamine is discussed.Chloroplasts also catalyzed (aspartate plus alpha-ketoglutarate)-dependent O(2) evolution but this reaction was inhibited by 5 mm amino-oxyacetate and it was insensitive to azaserine and methionine sulfoximine. This reaction was attributed to transaminase and photosynthetically coupled malate dehydrogenase activities. PMID- 16660093 TI - Dormancy in Dioscorea: Gibberellin-Induced Inhibition or Promotion in Seed Germination of D. tokoro and D. tenuipes in Relation to Light Quality. AB - Effects of light and gibberellic acid (GA(3)) application on the germination of Dioscorea tokoro Makino and Dioscorea tenuipes Franch. et Savat. were observed. For complete germination, seeds of both species required prechilling in moist condition before incubation at a higher temperature. Red light irradiation during the incubation after the prechilling promoted germination; blue, green, or far red light markedly inhibited the germination of both species.Application of GA(3) induced complicated changes in the germination of both species in relation to light quality. In the germination of D. tokoro, GA(3) inhibited in the dark and red; however, it promoted germination in blue and far red light. GA(3) promoted germination of D. tenuipes in the dark and in blue, green, or far red light. These phenomena are explainable by assuming two counteractive reactions (germination-promoting and germination-inhibiting) which are both activated by applied GA(3). PMID- 16660094 TI - Selective Inhibition of K, Na, Cl, and PO(4) Uptake in Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Pathotoxin: Evidence for a Plasmalemma Target Site? AB - Pathotoxin preparations were obtained from either axenic culture filtrate of race T of Bipolaris maydis (Nisikado) Shoemaker (new culture media and toxin purification procedures are described) or extracts of maize leaves infected with the fungus. The toxins (10(-6) to 10(-8)m) caused inhibition of [(86)Rb]K(+) uptake in leaf discs and apical root segments of Zea mays L. cv W64A Texas (Tcms) and normal (N) cytoplasms. Significant inhibition was measurable as early as 5 min after adding toxin. In Tcms per cent inhibition was increased by increasing toxin concentration and time in toxin, by using solution at pH 5 rather than pH 7, by decreasing external KCl concentration over the range 50 to 0.1 mm (in the presence of 0.5 mm CaSO(4)), or by exposing leaf discs to light rather than dark during the uptake period in toxin. Root uptake of (22)Na(+) and (36)Cl(-) was inhibited to a lesser extent than K(+). Inhibition of (32)PO(4) (3-) uptake occurred after 40 min when cyclosis had ceased.When combined with data in the literature, our data indicate that the plasmalemma is the probable primary site of toxin action in N and Tcms maize. Comparison of the effects of toxin on K(+) uptake in N and Tcms maize suggests the existence of more than one mode of toxin action: a weak disruptive effect in N and Tcms, and in addition, specific membrane sites in Tcms involved in monovalent ion uptake.Six genotypes in N or Tcms cytoplasm which exhibited different degrees of disease susceptibility in the field showed a corresponding gradation of susceptibility to the toxin when a K(+) uptake bioassay was used. This correlation is strong evidence that the sites of toxin action affecting K(+) transport have characteristics closely related to cellular factors regulating susceptibility to fungal attack. PMID- 16660095 TI - Changes in the photosynthetic apparatus of maize in response to simulated natural temperature fluctuations. AB - The response of the photosynthetic apparatus to low temperature periods differed among three hybrids of maize (Zea mays L.) grown in a phytotron. Light-saturated photosynthetic rates, leaf chlorophyll content, and mesophyll cell photosynthetic unit density all declined with increasing duration of low temperature. No single metabolic or physiological parameter appeared to control the response of the three hybrids to low temperature stress. Among all temperature treatments, net photosynthetic rate on a leaf area basis was more closely correlated with leaf chlorophyll content than with any other measured parameter. Final shoot dry weight was most highly correlated with stomatal conductance to CO(2). PMID- 16660096 TI - Quantitative study of the importance of water permeability in plant cold hardiness. AB - The rate of ice formation was measured for Hedera helix L. cv. Thorndale (English ivy) bark exposed to -10 C. The cooling rate of bark exposed to -10 C was 31 C per minute. The water efflux rate required for ice formation to occur extracellularly was calculated from the rate of ice formation and the average cell diameter. The water potential difference driving the efflux of water to sites of extracellular ice was calculated from the sample temperature, osmotic water potential, and fraction of water frozen at a given freezing temperature. From the water efflux rate and water potential difference, the resistance of the barrier controlling movement of intracellular water to sites of extracellular ice was calculated. Comparison of the resistance of this barrier to water movement with the resistance of the cell membrane revealed that the membrane represented only 0.5% of the barrier resistance. Thus, membrane resistance can have little influence on the rate of water efflux and ice formation when bark is cooled at a rate of 31 C per minute. If ice formation occurred at the same rate in ivy bark as it occurred in a 10 mm MnCl(2) solution, the membrane resistance would still have represented only 1% of the resistance of the barrier to ice formation. Therefore, at a cooling rate of 31 C/minute, heat removal plays a large part in determining the rate of ice formation. At slower cooling rates experienced under natural freezing conditions the ability to remove heat would play an even larger role. It is concluded that under natural freezing conditions membrane resistance does not limit water efflux. PMID- 16660097 TI - Enzymic assay of 10 to 10 moles of sucrose in plant tissues. AB - Procedures are described for measuring sucrose in plant extracts or freeze-dried tissue in the range between 10(-7) and 10(-14) moles. The method is based on the destruction of pre-existing glucose and fructose, followed by the hydrolysis of sucrose and reduction of NADP(+) by a series of coupled enzymic reactions. Depending on the sensitivity required, the NADPH is determined directly with a spectrophotometer or a fluorometer, or is amplified as much as 30,000 times before fluorometric assay. The procedures suggested for the macro level are simpler than current methods, and those suggested for microanalysis are several orders of magnitude more sensitive.With this technique, single palisade parenchyma cells and single spongy parenchyma cells of Vicia faba leaflets were each found to contain about 2.2 pmoles of sucrose. PMID- 16660098 TI - Characterization of photosynthetic rhythms in marine dinoflagellates: I. Pigmentation, photosynthetic capacity and respiration. AB - Circadian rhythms in photosynthesis were defined for the first time in the dinoflagellates Glenodinium sp. (M. Bernard strain) and Ceratium furca Ehrenberg (B. Meeson strain) and compared with that in Gonyaulax polyedra Stein. All three phytoplankton species had photosynthetic rhythms with daily amplitudes ranging from 3 to 5 and maxima displayed about midday. The photosynthetic pigment content and absorption properties of the cells were constant over the circadian cycle. Diurnal periodicities in respiration never accounted for more than 30% of the photosynthetic rhythm and did not persist under constant conditions. There was sufficient similarity between the circadian rhythms of these three dinoflagellates to suggest the mechanism of regulation may be the same for each of them. PMID- 16660099 TI - Characterization of Photosynthetic Rhythms in Marine Dinoflagellates: II. Photosynthesis-Irradiance Curves and in Vivo Chlorophyll a Fluorescence. AB - Using data from light-dark cultures of Gonyaulax polyedra entrained to a 24-hour cycle, whole cell absorption curves and photosynthesis-irradiance curves were constructed for various circadian times. While whole cell absorbance and half saturation constants of photosynthesis showed no statistical difference that could be directly related to the photosynthetic rhythm, the initial slope of the photosynthesis-irradiance curve was a time-dependent parameter which altered in direct proportion to the change in photosynthetic capacity. The results indicated a temporal change in the relative quantum yield of photosynthesis, and the circadian rhythmicity of light-limited photosynthesis was established under constant conditions. Circadian rhythmicity was detected in room temperature chlorophyll fluorescence yield. Low temperature fluorescence kinetics also showed fluctuations. The results suggest that regulation of photosynthesis by the biological clock of Gonyaulax may be mediated through the membrane-bound light reactions and a partial explanation of the underlying mechanism is proposed. PMID- 16660100 TI - Freezing injury in onion bulb cells: I. Evaluation of the conductivity method and analysis of ion and sugar efflux from injured cells. AB - Onion (Allium cepa L.) bulbs were frozen to -4 and -11 C and kept frozen for up to 12 days. After slow thawing, a 2.5-cm square from a bulb scale was transferred to 25 ml deionized H(2)O. After shaking for standard times, measurements were made on the effusate and on the effused cells. The results obtained were as follows.Even when the scale tissue was completely infiltrated, and when up to 85% of the ions had diffused out, all of the cells were still alive, as revealed by cytoplasmic streaming and ability to plasmolyze. The osmotic concentration of the cell sap, as measured plasmolytically, decreased in parallel to the rise in conductivity of the effusate. The K(+) content of the effusate, plus its assumed counterion, accounted for only 20% of the total solutes, but for 100% of the conductivity. A large part of the nonelectrolytes in the remaining 80% of the solutes was sugars.The increased cell injury and infiltration in the -11 C treatment, relative to the -4 C and control (unfrozen) treatments, were paralleled by increases in conductivity, K(+) content, sugar content, and pH of the effusate. In spite of the 100% infiltration of the tissue and the large increase in conductivity of the effusate following freezing, no increase in permeability of the cells to water could be detected.The above observations may indicate that freezing or thawing involves a disruption of the active transport system before the cells reveal any injury microscopically. PMID- 16660101 TI - Freezing Injury in Onion Bulb Cells: II. Post-thawing Injury or Recovery. AB - Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (-4 C) or a severe freeze (-11 C). They were then thawed slowly over ice. During 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repaired in the moderately frozen bulbs. This was shown by the following post-thawing changes.Infiltration of the intercellular spaces increased from 80 to 90% to 100% after the severe freeze, and decreased from 30 to 50% to zero after the moderate freeze. All of the cells were alive immediately after thawing whether the freeze was moderate or severe. Corresponding to the infiltration results 7 to 12 days later, many to most were dead following the severe freeze, all were alive following the moderate freeze.The conductivity of the effusate from pieces of bulb tissue increased after the severe freezing, and decreased after the moderate freezing. The concentration of K(+), total solutes, and sugars in the effusate paralleled the conductivity changes. Neither the pH of the effusate nor the permeability of the cells (as long as cells were living) to water was changed following either the severe or the moderate freezes. Some treatments of the thawed tissue following the severe freeze halted the progress of injury.The above results indicate that the semipermeable properties of the cell are uninjured but that the ion and sugar transport mechanism is damaged by freezing. Most likely the primary injury is to the active transport mechanism involved in their transport. It must be concluded that the final injury following freezing and thawing cannot be evaluated from the degree of infiltration or the conductivity of the effusate immediately after thawing, since injury may progress or recede following the thawing. PMID- 16660102 TI - Methionine-induced Ethylene Production by Penicillium digitatum. AB - Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [(14)C]methionine.Following induction by the fungus, filtrates of induced shake cultures also evolved ethylene in increasing amounts by both enzymic and monenzymic reactions. Tracer experiments indicated that the ethylene released by the filtrate was derived from a fungal metabolite of methionine and not directly from methionine. PMID- 16660103 TI - Uptake and Utilization of Sugar Phosphates by Anabaena flos-aquae. AB - The effect of various sugar phosphates on CO(2) fixation in Anabaena flos-aquae was investigated and found to be very similar to that found for isolated spinach chloroplasts. One exception, glucose 6-phosphate, has a stimulatory effect on CO(2) fixation in Anabaena but not in isolated chloroplasts.Further examination of the role of glucose 6-phosphate metabolism in Anabaena indicates that: (a) this sugar phosphate can be taken up; (b) its uptake is greater in the light than the dark; (c) turnover of glucose 6-phosphate is inhibited in the light; and (d) glucose 6-phosphate can support dark CO(2) fixation. These results are discussed with reference to photosynthesis-related control of glucose 6-phosphate metabolism and the role of glucose 6-phosphate as a source for reducing equivalents and ATP in blue-green algae. PMID- 16660104 TI - Significance of photosynthetic and respiratory exchanges in the carbon economy of the developing pea fruit. AB - The nutritional economy of the developing fruit of Pisum sativum L. (cv. Greenfeast) was studied in terms of intake of translocate, incorporation of C and N into dry matter, transpiration, and CO(2) exchanges of the fruit with its external and internal atmospheres. The environmental conditions were 12-hr days (22 C, 850 mueinsteins m(-2) sec(-1) at fruit level); 12-hr nights of 15 C.Between 6 and 30 days after anthesis, pod photosynthesis resulted in small gains of CO(2) from the external atmosphere, and assimilated most of the CO(2) respired by the fruit during the day. From then until maturity (40 days) the fruit lost CO(2) during the day. Night losses of CO(2) increased with fruit age.The gas cavity of the fruit contained 0.15 to 1.5% (v/v) CO(2). Lower levels were maintained in the day than at night. CO(2) levels were influenced by fruit age, radiant flux, and temperature. Labeled CO(2) injected into the gas cavity was fixed by the pod but not by seeds in the light, and by neither pod nor seeds in darkness. Dark-to-light or light-to-dark transfer of a fruit promoted rapid changes in CO(2) and O(2) levels of the gas space, consistent with a shift in the assimilation-respiration balance of the pod.The fruit transpired 27.6 cm(3) H(2)O per gram dry matter accumulated. Daytime ventilation was greatest 12 to 15 days after anthesis and declined as pod photosynthesis became increasingly involved in the retrieval of CO(2) respired by pod and seeds. Most, 69% by weight, of the translocate from the parent plant was converted to dry matter of seeds; nearly half, 45%, to useful seed reserves (sugar plus starch-protein-oil, 45:20:1). Illumination resulted in a fruit requiring 16% less translocate than if laying down an equal amount of dry matter in darkness. PMID- 16660105 TI - Ontogenetic Interactions between Photosynthesis and Symbiotic Nitrogen Fixation in Legumes. AB - Photosynthetic data collected from Pisum sativum L. and Phaseolus vulgaris L. plants at different stages of development were related to symbiotic N(2) fixation in the root nodules. The net carbon exchange rate of each leaf varied directly with carboxylation efficiency and inversely with the CO(2) compensation point. Net carbon exchange of the lowest leaves reputed to supply fixed carbon to root nodules declined in parallel with H(2) evolution from root nodules. The decrease in H(2) evolution also coincided with the onset of flowering but preceded the peak in N(2) fixation activity measured by acetylene-dependent ethylene production. A result of these changes was that the relative efficiency of N(2) fixation in peas increased to 0.7 from an initial value of 0.4. The data reveal that attempts to identify photosynthetic contributions of leaves to root nodules will require careful timing and suggest that the relative efficiency of N(2) fixation may be influenced by source-sink relationships. PMID- 16660106 TI - Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AB - The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules. PMID- 16660107 TI - Effects of temperature on the hill reaction and photophosphorylation in isolated cactus chloroplasts. AB - Chloroplasts isolated from Opuntia polyacantha Haw. (Cactaceae) are capable of noncyclic electron transport and ATP synthesis. Hill reaction rates, measured by O(2) evolution or by ferricyanide reduction, increase with increasing temperature to approximately 40 C. The temperature optimum of NADP reduction is 42 C while the optimum for noncyclic photophosphorylation is 35 C. NADP-linked phosphorylation exhibits a higher coupling ratio (P/e(2)) than ferricyanide linked photophosphorylation. The temperature optima for photochemical energy production correlate with photosynthetic properties of Crassulacean acid metabolism (CAM) plants and are discussed in relation to the operation of CAM at high tissue temperature. PMID- 16660108 TI - Heme Synthesis in Soybean Root Nodules: I. On the Role of Bacteroid delta Aminolevulinic Acid Synthase and delta-Aminolevulinic Acid Dehydrase in the Synthesis of the Heme of Leghemoglobin. AB - During nodulation of soybean (Glycine max) by Rhizobium japonicum, variations in the activities of two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (ALAS) and delta-aminolevulinic acid dehydrase (ALAD) are described. delta-Aminolevulinic acid synthase activity is found in the bacteroid fraction of nodules, but is not detected in the plant fraction. Bacteroid ALAS activity parallels heme accumulation during nodule development. delta-Aminolevulinic acid dehydrase activity is found in both bacteroid and plant cytosol fractions. Bacteroid ALAD activity is constant or increases during nodulation while plant ALAD activity falls.Bacteroid ALAD activity is found in effective, not in inefficient nodules. Plant ALAD activity is found in both effective and inefficient nodules. Plant ALAD activity falls during development of both types of root nodules.These results support the contention that it is the bacteroid ALAS and ALAD activities, not those of the plant, that are directly involved in formation of leghemoglobin heme, suggesting that the bacteroid may be solely responsible for formation of leghemoglobin heme in the nodule symbiosis. PMID- 16660109 TI - Effects of ethephon, ethylene, and 2,4-dichlorophenoxyacetic Acid on asexual embryogenesis in vitro. AB - Asexual embryogenesis in Daucus carota L. ;Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 mul/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution. PMID- 16660110 TI - Messenger RNA-controlled Increase of Phenylalanine Ammonia-Lyase Activity in Parsley: Light-Independent Induction by Dilution of Cell Suspension Cultures into Water. AB - A specific antiserum, raised against purified phenylalanine ammonialyase from irradiated cell suspension cultures of parsley (Petroselinum hortense Hoffm.), was used to compare the enzyme species induced either by dilution or by irradiation of the cell suspensions, to investigate the effect of dilution on the rate of synthesis of the enzyme protein in vivo, and to analyze the changes in specific activity of polyribosomal mRNA for the enzyme subunits in vitro. The mRNA activity in vitro was measured by translation of the polyribosomal RNA in a rabbit reticulocyte lysate.The results of immunodiffusion assays indicated that dilution and irradiation of the cell suspensions induced immunologically identical molecular species of phenylalanine ammonia-lyase. Dilution resulted in a large but short term increase in the rate of synthesis of the enzyme. Highest rates of synthesis were reached after 5 to 6 hours, and were followed by a rapid decline. The curves obtained for the changes in the rate of enzyme synthesis, both in vivo and in vitro, coincided with the curve for changes in the rate of appearance of catalytically active enzyme, as calculated from the observed changes in enzyme activity. These results suggested that the changes in enzyme activity were caused by corresponding changes in the amount of enzyme-specific polyribosomal mRNA.The dilution of cell cultures had no significant effect on the total rate of incorporation of radioactivity into protein in vivo or on the general template activity of the polyribosomal RNA in vitro throughout at least 25 hours. PMID- 16660111 TI - Effects of some organic solvents on ethylene evolution from young cotton bolls. AB - The presence of promoter(s) of ethylene biosynthesis in young cotton (Gossypium hirsutum L.) fruits (bolls) was demonstrated by injection of an aqueous extract from bolls into other bolls and measurement of a 3-fold increase in rate of ethylene evolution. Injection of methionine did not affect rate of ethylene production, indicating that the promoter extracted from bolls was not methionine. Injection of the ethoxy analog of rhizobitoxine inhibited ethylene production, indicating that methionine is a precursor of ethylene in cotton bolls. Injection of organic solvents altered membrane permeability, as indicated by decreased resistance to electric current at 1,000 Hz, and stimulated ethylene evolution. The less polar solvents caused large increases in ethylene evolution, major loss of resistance, and visible evidence of membrane damage. The results support the hypothesis that membrane integrity affects rate of ethylene biosynthesis. PMID- 16660112 TI - Two effects of electrical fields on chloroplasts. AB - An electrical field across a suspension of Chenopodium chloroplasts stimulates the emission of delayed light during the time the field is on. This stimulation can be used to calculate the distance over which the electron moves in the untrapping process that gives the delayed light. An electrical field applied at the time of illumination gives a polarization to the suspension of chloroplasts that lasts for some seconds. This polarization is a new way to study delayed light and fluorescence from chloroplasts. PMID- 16660113 TI - Light-mediated Oxygen Uptake Measured in Wheat Etioplasts. AB - An in vitro O(2) assay was used to measure early response of wheat (Triticum aestivum L.) etioplasts to light. A transient photoinducible O(2) uptake occurred when dark-grown etioplasts were initially exposed to light. The rate of inducible O(2) consumption was dependent on both the intensity of light and the quantity of organelle protein present. Higher light intensities resulted in greater O(2) utilization per minute, and a greater quantity of organelle protein in the sample resulted in an increased rate of O(2) uptake under the same light intensity conditions. Experiments with various plant tissues as well as with mitochondrial respiratory inhibitors indicated that etioplasts are the organelles responsible for the photoinduced O(2) uptake phenomenon. A preliminary action spectrum study revealed that wavelengths 640 to 680 nm resulted in maximum O(2) uptake. This indicated the presence of an etioplast red light receptor pigment which induces O(2) uptake in etioplasts. PMID- 16660114 TI - Responses of enzymically isolated aleurone cells of oat to gibberellin a(3). AB - Oat (Avena sativa L.) aleurone layer cells (spheroplasts) were isolated by maceration of the aleurone layer with a mixture of commercially available cellulase and pectinase. About 20% of the cells present in intact layers were released as spheroplasts and 79 +/- 9% of the spheroplast population was viable as judged by methylene blue staining. The spheroplasts became disorganized in solutions containing less than 0.4 md-mannitol. When the spheroplasts were incubated for 48 hours, total activities of acid p-nitrophenyl phosphatase and acid proteinase increased and protein levels decreased. These changes were not effected by gibberellin A(3). Isolated aleurone layers incubated under the same conditions as the spheroplasts showed reduced responses to gibberellin A(3). It is concluded that the necessary presence of an osmoticum limits the value of spheroplasts as a system for studying the mechanism of action of gibberellin A(3) in the aleurone cell. PMID- 16660115 TI - Chemical inhibition of the glycolate pathway in soybean leaf cells. AB - Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in alpha-hydroxypyridinemethanesulfonic acid increased (14)CO(2) incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of (14)CO(2) into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of (14)C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of (14)CO(2) into glycolate by BHB-treated cells or glycine by INH treated cells was determined by the O(2)/CO(2) ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O(2), but these compounds increased the O(2) inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration. PMID- 16660116 TI - Amino Acid interactions in the regulation of nitrate reductase induction in cotton root tips. AB - Glycine, asparagine, and glutamine inhibited the induction by nitrate of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.). This inhibition was partially or entirely prevented when the inhibitor was applied in combination with any of several other amino acids. Studies of (14)C-labeled amino acid uptake showed that, in most cases, the apparent antagonism resulted simply from competition for uptake. However, certain antagonists did not curtail uptake. The most effective of these were leucine (against all three inhibitors), and isoleucine and valine (against asparagine or glutamine, but not glycine). These results show that interactions among amino acids in the regulation of nitrate reductase induction result from at least two mechanisms, one acting on uptake of inhibitory amino acids, and the other involving true antagonism. PMID- 16660117 TI - Succinoxidase Activity of Avocado Fruit Mitochondria in Relation to Temperature and Chilling Injury throughout the Climacteric Cycle. AB - Mitochondria were isolated from ;Fuerte' avocado fruit (Persea americana Mill.) at four different stages of the respiratory climacteric. Preclimacteric fruit had the highest rate of succinate oxidation and the postclimacteric mitochondria the lowest. Subsequently, successive additions of ADP increased the respiratory control ratio.Arrhenius plots of succinate oxidation of intact mitochondria from climacteric rise and climacteric peak fruit showed two transition temperatures, while only one was observed in preclimacteric fruit. The low temperature phase transition was at about 9 C, while the high one was at 20 C. In postclimacteric fruit, the low temperature transition decreased to between 5 and 2 C. The state 3 rate of succinate oxidation was highest for mitochondria from preclimacteric fruit and decreased for each later stage. The state 4 rates for preclimacteric and climacteric rise were the same, while both the climacteric peak and postclimacteric rates were about 40% lower than the preclimacteric O(2) uptake.The results indicate continuous changes in the mitochondrial membrane of the electron transport chain throughout the climacteric cycle. The change in the membrane influencing the phosphorylation system is greatest between climacteric rise and peak stages. Mitochondrial membranes of postclimacteric fruit are presumed to change from flexible disordered to solid ordered phase at a lower temperature than those of other climacteric stages. PMID- 16660118 TI - Influence of Ethylene on Indole-3-acetic Acid Concentration in Etiolated Pea Epicotyl Tissue. AB - Indole-3-acetic acid levels are diminished about 50% in 5- to 6-day-old epicotyls of etiolated pea (Pisum sativum L.) seedlings treated with 10 to 36mul/l ethylene for 18 to 24 hr. PMID- 16660119 TI - Isolation and Purification of an alpha-Mannosidase from Coleoptiles of Avena sativa. AB - An alpha-mannosidase has been purified from the coleoptiles of Avena sativa L. var. Segrehavre. The enzyme, which is tightly associated with the cell wall, was solubilized with 3 m LiCl. The purification involves precipitation with (NH(4))(2)SO(4), gel filtration, ion exchange chromatography, and isoelectric focusing. The enzyme appears homogeneous when chromatographed on disc gels and on isoelectric focusing gels. The enzyme runs as a single protein of constant specific activity when chromatographed on Sephadex G-200. The estimated molecular weight of the enzyme is 630,000. The enzyme appears to have no metal ion cofactor requirement and is insensitive to p-chloromercuribenzoate. The pH optimum for the enzyme with p-nitrophenyl-alpha-d-mannoside as the substrate is 4.5 and the K(m) is 3.2 mm. The enzyme may have some carbohydrate associated with it as indicated by a positive periodate-Schiff reaction on disc gels. PMID- 16660120 TI - Dissociation of polysome aggregates by protease k. AB - Apparent large size-classes of zein-synthesizing polysomes from developing kernels of Zea mays L. were converted to smaller polysomes after treatment with Protease K. The reduction in polysome size was not a result of ribonuclease activity, inasmuch as the enzyme did not affect the free polysomes or the size of the mRNA from the membrane-bound polysomes. High concentrations of MgCl(2) in polysome buffer inhibited ribonuclease activity and appeared to cause protein interaction between nascent zein polypeptides. Although Protease K inhibited the polysome's capacity for protein synthesis, it was a useful reagent for determining if polysomes were aggregated by protein. PMID- 16660121 TI - Role of lectins in plant-microorganism interactions: I. Binding of soybean lectin to rhizobia. AB - Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ((3)H-SBL) and repurified by affinity chromatography. FITC SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 x 10(6). While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth.Quantitative binding studies with (3)H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 x 10(7)m(-1). Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates. PMID- 16660122 TI - Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: II. Interdependency of Glycolate Synthesis upon pH and Calvin Cycle Intermediate Concentration in the Absence of Carbon Dioxide Photoassimilation. AB - The light-dependent synthesis of glycolate derived from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate was studied in the intact spinach (Spinacia oleracea) chloroplasts in the absence of CO(2). Glycolate yield increased with an elevation of O(2), pH, and the concentration of the phosphorylated compound supplied. No pH optimum was observed as the pH was increased from 7.4 to 8.5. The average maximal rate of glycolate synthesis was 50 mumoles per milligram chlorophyll per hour while the highest rate observed was 92 with 2.5 mm fructose 1,6-diphosphate in 100% O(2). The highest yields of glycolate synthesized from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate were 0.14, 0.24, and 0.30, respectively, on a molar basis. PMID- 16660123 TI - Subcellular Distributions of Isoenzymes in Fruits of a Normal Cultivar of Tomato and of the rin Mutant at Two Stages of Development. AB - Fruits of tomato (Lycopersicon esculentum Mill.) cv. Rutgers and of a nearly isogenic stock containing the ripening inhibitor gene rin harvested at green (66% mature) and ripe (107% mature) stages were studied for the subcellular distribution of isoenzymes using isoelectric focusing. The enzymes studied were peroxidases, esterases, phosphatases, phosphorylase, malate dehydrogenases, and IAA oxidases. During ripening of normal fruit the activities in the supernatant fraction of all of these enzymes, except malate dehydrogenase, decreased. In the particulate fractions some enzymes decreased while others increased in activity. The rin gene inhibited only some of the changes which occurred during ripening of normal fruit. It is postulated that changes in the degree to which enzymes are bound to membranes comprise one of the mechanisms by which the activities of enzymes are controlled in tomato pericarp, and that these membranes remain intact during ripening. PMID- 16660124 TI - Ice adhesions in relation to freeze stress. AB - In freezing, competitive interaction between ice and hydrophilic plant substances causes an energy of adhesion to develop through the interstitial liquid. The thermodynamic basis for the adhesion energy is discussed, with estimates of the energies involved. In this research, effects of adhesion energy were observed microscopically in conjunction with energies of crystallization and frost desiccation. The complex character of ice in intact crown tissue of winter barley (Hordeum vulgare L.) and the problems of sectioning frozen tissue without producing artifacts led to an alternative study of single barley cells in a mesh of ice and cell wall polymers. Adhesions between ice, cell wall polymers, and the plasmalemma form a complexly interacting system in which the pattern of crystallization is a major factor in determination of stress and injury. PMID- 16660125 TI - Polarographic study of ammonia assimilation by isolated chloroplasts. AB - Illuminated pea (Pisum sativum) chloroplasts catalyze (ammonia plus alpha ketoglutarate [alpha-KG])-dependent O(2) evolution at rates which are commensurate with other estimates of the flux of assimilated nitrogen (mean of eight determinations, 8.3 mumole per mg chlorophyll per hour, sd 2.4). The reaction was usually initiated with 1 mm ammonia after preincubating chloroplasts in the presence of alpha-KG, ADP, pyrophosphate, and MgCl(2).Progressive increases in ammonia concentration gave V(max)/2 at 0.2 mm (approximately) and V(max) at about 1 mm. Higher concentrations were inhibitory; at 7 mm the rate was again about V(max)/2. The highest ratio of O(2) evolved per mol of ammonia supplied was 0.36.The (ammonia plus alpha-KG)-dependent reaction was inhibited by methionine sulfoximine, azaserine, and aspartate in the presence of amino oxyacetate but not by amino-oxyacetate alone and not by l-glutamate. The rate of O(2) evolution in the presence of 1 mm ammonia and 2.5 mm alpha-KG was increased only slightly by addition of 5 mm glutamine. Similarly, the rate of O(2) evolution in the presence of 5 mm glutamine and 2.5 mm alpha-KG was increased only slightly by addition of 1 mm ammonia.The results are attributed to the incorporation of ammonia via glutamine synthetase and reductive transamination of the glutamine formed by photosynthetically coupled glutamate synthase using alpha KG as the amino acceptor. Several lines of evidence rule out the possibility that photosynthetically coupled glutamate dehydrogenase is involved. PMID- 16660126 TI - Auxin-induced H Secretion in Helianthus and Its Implications. AB - We have examined the ability of Helianthus hypocotyl segments as well as segments from a variety of other species to elongate in response to H(+) and to secrete H(+) in response to auxin and fusicoccin. In all cases a positive response was obtained when the cuticular barrier was abraded with carborundum. Removal of the cuticular barrier by "peeling" prevented detection of both auxin-induced elongation and H(+) secretion. Fusicoccin-induced growth and acid secretion are not prevented by peeling. These results suggest considerable tissue selectivity with respect to auxin action but considerably less specificity with respect to fusicoccin. It seems likely that in many dicots auxin-enhanced proton secretion and elongation are controlled by the epidermis and/or closely associated cell layers. The data presented in this paper provide further support for the acid growth theory of auxin action. PMID- 16660127 TI - Occurrence of alpha-amylase in the axis of germinating peas. AB - alpha-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing beta-amylase) using three different methods: the hydrolysis of beta-limit dextrin, the change in absorption spectra for the iodine starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in alpha-amylase activity during germination. The increase in total amylase activity (primarily beta-amylase) paralleled germination; the accumulation of alpha-amylase activity was not initiated for an additional day. The increased alpha-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions. PMID- 16660128 TI - Envelope Permeability to Possible Precursors of Carotenoid Biosynthesis during Chloroplast-Chromoplast Transformation. AB - During the transformation of chloroplasts to chromoplasts in Capsicum annuum L., the permeability of the envelope membranes to possible precursors of carotenoid biosynthesis (acetate, mevalonate, citrate) was tested. The plastids were isolated by gel filtration, and the uptake of labeled compounds was measured by a filtering centrifugation technique, using silicone oil. The different ripening stages were characterized by the ratio of chlorophyll to carotenoid content. The chloroplast membranes were shown to be impermeable to all of the metabolites tested. During the transformation there was a sharp increase of membrane permeability. In the mature chromoplast, the permeability to mevalonate and acetate again decreased to about 20% of the maximum value and reached zero for citrate. The results give evidence that during the transformation of chloroplasts to chromoplasts, precursors for carotenoid biosynthesis are translocated from extraplastidic sites into the plastids, there being possibly incorporated into carotenoids. PMID- 16660129 TI - Effects of Water and Turgor Potential on Malate Efflux from Leaf Slices of Kalanchoe daigremontiana. AB - Malate efflux from leaf cells of the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perrier was studied using leaf slices submerged in experimental solutions. Leaves were harvested at the end of the dark phase and therefore contained high malate levels. Water potentials of solutions were varied between 0 and -5 bar using mannitol (a slowly permeating solute) and ethylene glycol (a rapidly permeating solute), respectively. Mannitol solutions of water potentials down to -5 bar considerably reduced malate efflux. The slowly permeating solute mannitol reduces both water potential and turgor potential of the cells. The water potential of a mannitol solution of -5 bar is just above plasmolyzing concentration. Malate efflux in ethylene glycol at -5 bar was only slightly smaller than at 0 bar, and much higher than in mannitol at -5 bar. Tissues in rapidly permeating ethylene glycol would have turgor potentials similar to tissues in 0.1 mm CaSO(4). The results demonstrate that malate efflux depends on turgor potential rather than on water potential of the cells. PMID- 16660130 TI - Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum. AB - The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an "alternate" respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O(2) concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered. PMID- 16660131 TI - Effects of Light and Temperature on the Association between Zea mays and Spirillum lipoferum. AB - Zea mays was grown on a low N nutrient solution under 16 conditions of light and temperature in a crossed-gradient room in an attempt to determine whether or not variation in climatic conditions influences N(2) fixation by the association between maize and Spirillum lipoferum. Temperatures were 28, 32, 36, and 40 C and 10 C lower at night; light intensities were 500, 1,250, 2,400, and 3,000 ft-c. Plants harvested after 94 days showed no significant benefit from association with S. lipoferum either in dry weight production or in total N content; variations in temperature and light had only a small influence on N(2) fixation under the conditions tested. Measurements of total N, together with designated assumptions, indicated that less than the equivalent of 0.5 kilogram of N was fixed/hectare during the entire growing period by the maize-S. lipoferum association. Rates of C(2)H(2) reduction by replicate root samples generally were low and variable and did not correlate with the measurements of total N. PMID- 16660132 TI - Synthesis of the Small Subunit of Ribulose-1,5-bisphosphate Carboxylase by Soluble Fraction Polyribosomes of Pea Leaves. AB - The products of amino acid incorporation by pea (Pisum sativum L.) leaf soluble fraction polyribosomes in the wheat germ system were examined by two-dimensional electrophoresis and fluorography.There are two isoelectric variants of the small subunit of ribulose bisphosphate carboxylase in this organism, and the more alkaline of these is consistently labeled in the cell-free protein-synthesizing system. The more acid variant is also labeled, but often less extensively. A minority of leaf polyribosomes are recovered from low speed sedimentable fractions. While these appear also to synthesize the small subunits, the patterns of labeling do not indicate a preferential synthesis of these polypeptides by the sedimentable fraction polyribosomes.In the same experiments, labeling of the large subunit spots was sharply below background; these results confirm a cytoplasmic site of synthesis for small subunits of ribulose bisphosphate carboxylase. PMID- 16660133 TI - Influence of antimycin a and uncouplers on anaerobic photosynthesis in isolated chloroplasts. AB - Anaerobiosis depresses the light- and bicarbonate-saturated rates of O(2) evolution in intact spinach (Spinacia oleracea) chloroplasts by as much as 3-fold from those observed under aerobic conditions. These lower rates are accelerated 2 fold or more by the addition of 1 mum antimycin A or by low concentrations of the uncouplers 0.3 mm NH(4)Cl or 0.25 mum carbonyl cyanide m-chlorophenylhydrazone. Oxaloacetate and glycerate 3-phosphate reduction rates are also increased by antimycin A or an uncoupler under anaerobic conditions. At intermediate light intensities, the rate accelerations by either antimycin A or uncoupler are inversely proportional to the adenosine 5'-triphosphate demand of the reduction process for the acceptors HCO(3) (-), glycerate 3-phosphate, and oxaloacetate. The acceleration of bicarbonate-supported O(2) evolution may also be produced by adding an adenosine 5'-triphosphate sink (ribose 5-phosphate) to anaerobic chloroplasts. The above results suggest that a proton gradient back pressure resulting from antimycin A-sensitive cyclic electron flow is responsible for the depression of light-saturated photosynthesis under anaerobiosis. PMID- 16660134 TI - Factors affecting the extraction of intact ribonucleic Acid from plant tissues containing interfering phenolic compounds. AB - Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions. PMID- 16660135 TI - Occurrence and properties of polygalacturonase in Avena and other plants. AB - Polygalacturonase activity has been detected in a number of plants including seedlings of Phaseolus vulgaris, Zea mays, Avena sativa, and Pisum sativum. Particular emphasis was placed on characterizing the enzyme from oat seedlings. This enzyme is solubilized by 0.2 m NaCl, and its activity is highest near the apical tips of oat coleoptiles. It has a pH optimum between 5 and 5.5 and is activated by Ca(2+), with an optimal concentration of 0.4 mm. Cd(2+) also activates the enzyme but less effectively than Ca(2+). The rate of attack is maximal for substrates with chain lengths of about 20 units and slowest for digalacturonate. The oat enzyme hydrolyzes galacturonans by removing galacturonic acid units from the nonreducing ends and progressively shortens the substrate chains. PMID- 16660136 TI - Polyribosome Formation in Relation to Cytokinin-induced Cell Division in Suspension Cultures of Glycine max [L.] Merr. AB - We have investigated the relationship between cell proliferation and protein synthetic capacity in a cytokinin-requiring strain of cultured soybean cells (Glycine max [L.] Merr. cv. Sodifuri, of cotyledonary origin) in suspension culture. When transferred to a defined medium lacking cytokinin, very little cell division or cell enlargement took place over the course of a 6-day culture period. Cells transferred to medium of the same composition, but containing 0.5 mum zeatin, exhibited rapid initial growth, with maximum mitotic activity occurring after 24 hours in culture, and a doubling of the cell population within the first 36 hours of the culture period. The polyribosomal RNA content of the cells decreased over the course of the first 24 hours of the growth cycle while the polyribosome to monoribosome (P/M) ratio increased. The increase in the P/M ratio was greater in the cytokinin-treated cells. This apparent relationship between cytokinin-induced cell proliferation and polyribosome formation was examined further. Polyribosome formation was stimulated when zeatin was added directly to cell populations which had been cultured for 24 hours in medium lacking a cytokinin. Transfer to fresh medium alone also stimulated polyribosome formation, whether this medium contained a cytokinin or not. The magnitude of transfer-induced polyribosome formation depended upon the initial cell density (number of cells/ml of medium). Regardless of the initial cell density and independent of the P/M ratios attained, the cytokinin-treated cell populations divided while the cytokinin-deprived cell populations did not. In vivo labeling with [(35)S]methionine and slab gel electrophoretic separation of sodium dodecyl sulfate derivatives of the labeled polypeptides demonstrated qualitative changes in the spectrum of proteins synthesized by the cytokinin-treated cells. These qualitative changes were independent of the cell density (and hence, independent of the P/M ratio) but they preceded cytokinin-induced cell division. PMID- 16660137 TI - Polarity of Production of Polyphenols and Development of Various Enzyme Activities in Cut-injured Sweet Potato Root Tissue. AB - Investigation of polyphenol production in cut-injured sweet potato (Ipomoea batatas Lam. cv. Kokei 14) roots by histochemical and quantitative methods showed that polyphenols were produced in striking amounts in the proximal side of the tissue pieces (2 cm thick), but only in small amounts in cells of the distal side. In response to cut injury, formation of the enzymes related to polyphenol biosynthesis, phenylalanine ammonia-lyase and trans-cinnamic acid 4-hydroxylase, was also pronounced in the proximal side of the tissue pieces and slight in the distal side. The similar polarity was observed in the development of activities of various enzymes, such as NADPH-cytochrome c oxidoreductase, acid invertase, peroxidase, o-diphenol oxidase, and cytochrome c-O(2) oxidoreductase. Acropetal development of polyphenol contents and of various enzyme activities may be related to the acropetal movement of indoleacetic acid (IAA) in roots of various plants. Treatment of the distal surface of tissue pieces with IAA or 2,4 dichlorophenoxyacetic acid caused polyphenol production but treatment with gibberellic acid, abscisic acid, kinetin, or ethylene had little effect. The results suggest that IAA may play a role in the metabolic response to cut injury. PMID- 16660138 TI - Phloem loading in squash. AB - Squash (Cucurbita pepo L. var. melopepo torticalis, Bailey) leaves were supplied with (14)C-sucrose, then specific radioactivities of the glucose and galactose moieties of translocated stachyose were determined. In every case, the specific radioactivity of the galactose moiety was greater than that of the glucose moiety. It is concluded that the stachyose was not synthesized at either the phloem-loading site or subsequent to phloem loading, but rather in cells that were not a part of the translocation system, possibly the mesophyll cells. PMID- 16660139 TI - Stabilization of Oat Leaf Protoplasts through Polyamine-mediated Inhibition of Senescence. AB - Protoplasts isolated from Avena sativa L. leaves undergo progressive senescence when incubated aseptically in 0.6 m mannitol with or without added nutrients. This senescence is manifested by morphological deterioration and ultimate lysis of protoplasts, by a decrease in incorporation of [(3)H]uridine and [(3)H]leucine into macromolecules, and by a sharp increase in ribonuclease activity.The presence in the incubation medium of l-arginine, l-lysine, certain polyamines related to these amino acids (cadaverine, putrescine, spermidine), Ca(2+), or streptomycin stabilizes the protoplasts. Protoplasts incubated with 10 mml arginine or l-lysine show an initial inhibition of [(3)H]uridine incorporation, but with time, incorporation is restored to levels greater than in control protoplasts. The rise in ribonuclease activity of protoplasts is completely inhibited if the protoplasts are incubated with 10 mml-arginine. Greater incorporation of [(3)H]uridine into RNA of aging protoplasts is also maintained by appropriate concentration of cadaverine, putrescine, spermidine, Ca(2+), or streptomycin in the incubation medium; the same concentrations of these substances stabilize the protoplasts against additional lysis. PMID- 16660140 TI - Mode of action of gibberellic Acid and light on lettuce seed germination. AB - The seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) germinate in darkness at 25 C when treated by gibberellic acid (GA(3)) for 1 hour following 2 hours of imbibition. The time of GA(3) application influences the rate and the final percentage of seeds that germinate. In contrast, red light illumination given at different times affects only the rate and not the final germination percentage. The early process(es) of germination initiated by GA(3) or light treatment can be arrested by subjecting the treated seeds to a nongerminative temperature of 35 C. The results suggest differences in the mode of action of light and GA(3) during germination. They indicate that different kinds of processes are involved in the biochemical control of germination. PMID- 16660141 TI - Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went. AB - Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin. PMID- 16660142 TI - Development of Sulfate Uptake Capacity and ATP-Sulfurylase Activity during Root Elongation in Maize. AB - Sulfate uptake capacity and ATP-sulfurylase activity were determined in maize roots (Zea mays L. var. XL 363 and mutant XL 363 o2) at increasing root length. The pattern of uptake showed a close similarity to that of ATP-sulfurylase, both activities reaching the maximum level at 9 and 10 cm root length in the XL 363 and XL 363 o2 hybrids, respectively. In addition to the shift of the maximum, opaque-2 mutation caused an enhancement of the two activities at root length below and above the activity peak. The kinetic parameter of uptake, Km, showed a maximum at 3 to 4 and a minimum at 7 to 8 cm. The isoenzyme pattern of ATP sulfurylase was the same in the two hybrids and did not change with root elongation. A common regulatory mechanism is postulated for uptake and activation of sulfate. The kinetic behavior is interpreted as an index of flexibility of the transport system toward different nutrient status of the environment. PMID- 16660143 TI - Specificity of Auxin-binding Sites on Maize Coleoptile Membranes as Possible Receptor Sites for Auxin Action. AB - Dissociation coefficients of auxin-binding sites on maize (Zea mays L.) coleoptile membranes were measured, for 48 auxins and related ring compounds, by competitive displacement of (14)C-naphthaleneacetic acid from the binding sites. The sites bind with high affinity several ring compounds with acidic side chains 2 to 4 carbons long, and much more weakly bind neutral ring compounds and phenols related to these active acids, most phenoxyalkylcarboxylic acids, and arylcarboxylic acids except benzoic acid, which scarcely binds, and triiodobenzoic acids, which bind strongly. Specificity of the binding is narrowed in the presence of a low molecular weight "supernatant factor" that occurs in maize and other tissues. Activity of many of the analogs as auxin agonists or antagonists in the cell elongation response was determined with maize coleoptiles. These activities on the whole roughly parallel the affinities of the binding sites for the same compounds, especially affinities measured in the presence of supernatant factor, but there are some quantitative discrepancies, especially among phenoxyalkylcarboxylic acids. In view of several factors that can cause receptor affinity and biological activity values to diverge quantitatively among analogs, the findings appear to support the presumption that the auxin-binding sites may be receptors for auxin action. PMID- 16660144 TI - Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation. AB - Incubation of KCN-Hg-NH(2)OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2 fold greater than the rates obtained without incubation. Partial oxidation of p phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N'N'-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport. PMID- 16660145 TI - Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation. AB - A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4 dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p phenylenediamine or N,N,N',N'-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH(2)OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport. PMID- 16660146 TI - Glutamate synthase: a possible role in nitrogen metabolism of the developing maize endosperm. AB - Glutamate synthase activity was demonstrated in the developing endosperm of maize (Zea mays L.). The enzyme shows specificity for glutamine and alpha-ketoglutarate as amino donor and acceptor, respectively. Both NADH and NADPH function as electron donors although lower activities were often, but not always, obtained with NADPH. The apparent K(m) values for glutamine, alpha-ketoglutarate, and NADH were 1.35 mm, 0.57 mm, and 7 mum, respectively.The pattern of activity during endosperm development revealed a well defined peak coinciding with the period of most active N accumulation. Activity in general was related to the rates of accumulation throughout development. Maximum glutamate synthase activity was the order of 56 nmoles of glutamate formed per minute per endosperm compared with a rate of N accumulation of 9.5 nmoles per minute.It is suggested that glutamate synthase plays a key role in the N nutrition of the maize endosperm providing a means whereby N transported in the form of glutamine is made available for the synthesis of other seed protein amino acids via transaminase reactions. Transaminase activity involving glutamate, the product of the glutamate synthase reaction, was also demonstrated. PMID- 16660147 TI - Measurement of ozone injury by determination of leaf chlorophyll concentration. AB - A simple, rapid procedure is described for evaluating ozone injury to leaves of Phaseolus vulgaris L. cv. Pinto. Leaf chlorophyll is extracted with ethanol and analyzed spectrophotometrically; the concentration is expressed on the basis of leaf dry weight.The per cent chlorophyll reduction of ozone-injured leaves was highly correlated with the per cent visible necrosis and chlorosis (r = 0.96). The variability in injury estimates with chlorophyll analysis was slightly less than with visual evaluation. An evaluation of chlorophyll a and b concentrations separately showed that the chlorophyll a/b ratio decreased with increasing amounts of injury. Chlorophyll determinations made for leaves harvested at intervals after an ozone treatment indicated that maximum chlorophyll reduction had occurred by 4 days.This procedure for measuring ozone injury should be useful in eliminating the human bias associated with visible estimates of injury. PMID- 16660148 TI - Thermal Energy Exchange Model and Water Loss of a Barrel Cactus, Ferocactus acanthodes. AB - The influences of various diurnal stomatal opening patterns, spines, and ribs on the stem surface temperature and water economy of a CAM succulent, the barrel cactus Ferocactus acanthodes, were examined using an energy budget model. To incorporate energy exchanges by shortwave and longwave irradiation, latent heat, conduction, and convection as well as the heat storage in the massive stem, the plant was subdivided into over 100 internal and external regions in the model. This enabled the average surface temperature to be predicted within 1 C of the measured temperature for both winter and summer days.Reducing the stem water vapor conductance from the values observed in the field to zero caused the average daily stem surface temperature to increase only 0.7 C for a winter day and 0.3 C for a summer day. Thus, latent heat loss does not substantially reduce stem temperature. Although the surface temperatures averaged 18 C warmer for the summer day than for the winter day for a plant 41 cm tall, the temperature dependence of stomatal opening caused the simulated nighttime water loss rates to be about the same for the 2 days.Spines moderated the amplitude of the diurnal temperature changes of the stem surface, since the daily variation was 17 C for the winter day and 25 C for the summer day with spines compared with 23 C and 41 C, respectively, in their simulated absence. Ribs reduced the daytime temperature rise by providing 54% more area for convective heat loss than for a smooth circumscribing surface. In a simulation where both spines and ribs were eliminated, the daytime average surface temperature rose by 5 C. PMID- 16660149 TI - beta-d-Glucan of Avena Coleoptile Cell Walls. AB - A specific glucanase was used to liberate a noncellulosic beta-d-glucan from isolated cell walls of Avena sativa coleoptile tissue. Cell walls of this tissue contain as much as 7 to 9 mg of glucan/100 mg of dry wall. Because of the specific action pattern of the enzyme, a linkage sequence of.. 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc.. is indicated and the predominance of trisaccharide and tetrasaccharide as hydrolytic products suggests a rather regular repeating pattern in the polysaccharide. The trisaccharide and the tetrasaccharide are tentatively identified as 3-O-beta-cellobiosyl-d-glucose and 3-O-beta cellotriosyl-d-glucose, respectively. Recovery of these oligosaccharides following glucanase treatment of native wall material was feasible only after wall-bound glucosidases were inactivated. In the absence of enzyme inactivation the released fragments were recovered as glucose. The beta-d-glucan was not extracted from walls by either hot water or protease treatment.Cell walls prepared from auxin-treated Avena coleoptile segments yielded less glucan than did segments incubated in buffer suggesting an auxin effect on the quantity of this wall component. No IAA-induced change in the ratio of the trisaccharide and tetrasaccharide could be detected, suggesting no shift in the 1,3 to 1,4 linkage ratio. While the enzyme acts directly on the beta-d-glucan, no elongation response was apparent when Avena sections were treated with the purified glucanase. The presence of the glucan was not associated with any wound response which could be attributed to the preparation of coleoptile segments. The relationship of glucan metabolism to auxin growth responses is discussed. PMID- 16660150 TI - Phytochrome involvement in stimulation and alleviation of inhibition of plant growth by malformin. AB - Stimulation of stem elongation on green cuttings of Phaseolus aureus by malformin occurred only in red light and was specifically reversible by subsequent treatment with far red radiation. Inhibition of stem elongation of etiolated cuttings by malformin in the dark was alleviated by red light and was repeatedly reversible with far red irradiation. A direct or indirect effect of malformin on phytochrome action was suggested. PMID- 16660151 TI - Isolation and oxidative properties of intact mitochondria isolated from spinach leaves. AB - A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracea L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, alpha-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the preparations. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors.Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited. PMID- 16660152 TI - Localization of Cinnamic Acid 4-Monooxygenase and the Membrane-bound Enzyme System for Dhurrin Biosynthesis in Sorghum Seedlings. AB - The localization of three monooxygenase (hydroxylase) enzyme systems which occur in dark-grown seedlings of Sorghum bicolor has been studied. Cinnamic acid 4 hydroxylase (CAH) (trans-cinnamate 4-monooxygenase, EC 1.14.13.11), which has been increasingly utilized in plants as a marker for the endoplasmic reticulum, migrated with that fraction in continuous and discontinuous sucrose gradients. When 10 mm MgCl(2) was used to shift the density banding of the marker enzyme, NADPH cytochrome c reductase, from 1.12 to 1.17 g/cm(3), the CAH activity was displaced as well.The membrane-bound enzyme system involved in the biosynthesis of the cyanogenic glucoside dhurrin was also shown to be closely associated with the endoplasmic reticulum. This system contains hydroxylases capable of hydroxylating tyrosine to form N-hydroxytyrosine and hydroxylating p hydroxyphenylacetonitrile to form p-hydroxy-(S)-mandelonitrile. PMID- 16660153 TI - Effects of lanthanum and ethylenediaminetetraacetate on leaf movements of mimosa. AB - Lanthanum and ethylenediaminetetraacetate (EDTA) profoundly affect the rapid leaf movements of Mimosa pudica L. Lanthanum, which mimics calcium but does not penetrate the plasmalemma, inhibits the closing response but does not affect reopening. A low concentration of EDTA retards the reopening process while a higher EDTA concentration prevents the closing movement. There is evidence that the EDTA effects result from chelation of calcium ions rather than chelation of other cations. These results are discussed with regard to the role of calcium in leaf movements. PMID- 16660154 TI - Development of Photosystem I and Photosystem II Activities in Leaves of Light grown Maize (Zea mays). AB - To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O(2)-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O(2)-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O(2)-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts. PMID- 16660155 TI - A miniature stem thermocouple hygrometer. AB - An unprotected chromel-constantan thermocouple was mounted in a cavity (4 x 2 x 1 mm) with rounded corners in a chrome-plated brass block (10 x 6 x 4 mm). When installed against a soybean (Glycine max [L.] Merr.) xylem face, sealed with caulking gum, and insulated with polyurethane foam and aluminum foil, even rapidly changing stem water potentials could be followed accurately. Temperature gradients can be a problem. PMID- 16660156 TI - Biological Activity of O-beta-d-Glucopyranosylzeatin. AB - Concentrations of 10(-8) to 10(-5)m O-beta-d-glucopyranosylzeatin are less active than zeatin and zeatin riboside in the soybean (Glycine max L.) callus bioassay. At a concentration of 10(-4)m the glucoside was, however, more active or alternatively less toxic than similar concentrations of zeatin and zeatin riboside. Applied zeatin-O-glucoside is readily metabolized by soybean callus and both zeatin and zeatin riboside could be extracted from callus grown on basal medium containing the O-glucoside. PMID- 16660157 TI - Hydrogen reactions of nodulated leguminous plants: I. Effect of rhizobial strain and plant age. AB - The ATP-dependent evolution of H(2) catalyzed by nitrogenase and the hydrogenase catalyzed oxidation of H(2) have been implicated as factors influencing the efficiency of energy utilization in the N(2) fixation process. The effects of rhizobial strain and plant age on the H(2)-evolving and H(2)-utilizing activity of leguminous root nodules are described in this manuscript. Two classes of legume-Rhizobium combinations were observed in studies with soybeans (Glycine max L. Merr.) and cowpeas (Vigna unguiculata L. Walp.). One group evolved H(2) in air; the other group did not exhibit net evolution of H(2). The latter group metabolized H(2) formed within the nodule through the action of a hydrogenase. The capacity to oxidize H(2) was strongly linked to the strain of Rhizobium used to inoculate cowpeas and soybeans. Although the magnitude of H(2) evolution in air changed during vegetative growth of a given symbiont, the ratio of H(2) evolved in air to total nitrogenase activity was not appreciably altered during this period. No consistent difference in nitrogenase activity as measured by the C(2)H(2) reduction assay was observed between symbionts with an active hydrogenase and those that apparently lack the enzyme and evolve H(2). The effects of the two reactions of H(2) on total N(2) fixation and yield must now be established. PMID- 16660158 TI - Cell-free Synthesis of Pea Seed Proteins. AB - Both polysomes and polysomal RNA, isolated from cotyledons of ripening pea (Pisum sativum) seeds and supplemented respectively with wheat germ S-100 and S-30 fractions, were used to program the cell-free synthesis of polypeptides. The relationship of these polypeptide products to seed storage proteins has been investigated. When fractionated on sucrose density gradients the translation products did not coincide with native storage proteins, nor were they exactly coincident with the subunits of storage proteins on dissociating gels. Treatment with antiserum prepared against storage proteins precipitated only a very small proportion of these products. Nonetheless, tryptic peptide mapping showed that a significant proportion (up to 65%) of the in vitro products from cell-free systems were related to the storage proteins. Alternative interpretations of these results are that either the translatable mRNAs for storage proteins make up a small proportion of the total template isolated from pea cotyledon polysomes, or that storage protein polypeptides are made in significant amounts in vitro but lack major antigenic determinants which in vivo may be acquired during chain completion or post-translational modification. PMID- 16660159 TI - Water Flow in Beta vulgaris Storage Tissue. AB - The relative magnitudes of the hydraulic resistances, water capacities, and water potential equilibration time constants for the single cell, for the apoplast, and for the symplast in higher plant tissue are assessed. Swelling of beetroot (Beta vulgaris, var. ;Detroit Red') storage tissue sections in pure water is measured using a displacement transducer. This method of measurement avoids the difficulty of solute diffusion in the apoplast. Theoretical analysis of the experimental results shows that the main path of water flow into the tissue is the apoplast rather than the symplast, that the main resistance to water flow into the cells is usually the cell membrane rather than the apoplast, but that in some cases the apoplast resistance and water capacity can contribute significantly to the water potential equilibration time constant of the tissue. PMID- 16660160 TI - Fructokinase (Fraction IV) of Pea Seeds. AB - A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed. PMID- 16660161 TI - Cysteinyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties. AB - l-Cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus was purified approximately 300-fold and was free of contaminating aminoacyl-tRNA synthetases. Optimum assay conditions were determined and substrate specificity and inhibitor properties were investigated using the ATP-PPi exchange reaction. The Km values for l-cysteine, ATP, and PPi were 6.20 x 10(-5)m, 1.15 x 10(-3)m, and 1 x 10( 3)m, respectively. Both l-selenocysteine (Km = 5 x 10(-5)m) and alpha-l aminobutyric acid (Km = 1 x 10(-2)m) acted as alternative substrates of the purified cysteinyl-tRNA synthetase. The enzyme was sensitive to sulfhydryl group reagents; it was inhibited by sulfide, 0-acetylserine, and reduced glutathione. PMID- 16660162 TI - Mitochondria of Isolated Plant Cells (Acer pseudoplatanus L.): II. Copper Deficiency Effects on Cytochrome C Oxidase and Oxygen Uptake. AB - The effects of copper deficiency on cell culture growth, cell respiration, mitochondrial oxidative properties, and electron transport chain have been studied with suspension-cultured sycamore cells (Acer pseudoplatanus L.). Within the range of the copper concentration studied (0.1-25 mug/1 of culture medium), the mean rate of cell division is independent of copper concentration. An initial copper concentration lower than 2 mug/1 limited the maximum density of population reached at the stationary phase of growth.On a protein basis, the uncoupled O(2) uptake rates were about the same for normal and copper-deficient cells. In contrast, the half-maximal inhibition of O(2) uptake rate was obtained at greater KCN concentration in the normal cells (20 muM) compared to copper-deficient cells (2 muM). Similar results were obtained with the normal and copper-deficient sycamore cell mitochondria.In the copper-deficient mitochondria, the concentration of the cytochrome aa(3) was less than 0.02 nmol/mg mitochondrial protein or 1/20 of the normal rate. The b- and c-type cytochrome content was invariant with copper depletion. It appeared that cytochrome aa(3) is present in large excess in normal cells. This work also indicated that cytochrome c is a very mobile molecule. PMID- 16660163 TI - Asparagine synthetase in corn roots. AB - The level of asparagine synthetase is low in 10-mm root tips from corn seedings (Zea mays W64 x W182F) but relatively high in mature root sections taken 20 to 35 mm from the tip. When root tips are excised there is a marked increase in asparagine synthetase over a 5-hour period. In mature root sections, on the other hand, the asparagine synthetase activity declines over the same 5-hour period. The increase in the root tip is sensitive to cordycepin, 6-methylpurine, and cycloheximide, which indicates that both RNA and protein synthesis are involved in the formation of asparagine synthetase in the root tip sections. The glutamine analogue azaserine also inhibits formation of the enzyme in root tips, as does glucose. The increase in the root tip is not sensitive to asparagine. Additions of glucose or asparagine have no effect on enzyme activity in extracts. When cycloheximide, azaserine, or glucose is added to the mature root sections there is no effect on recovered enzyme activity. PMID- 16660164 TI - Comparison of three methods for measuring electrical resistances of plant cell membranes. AB - The reliability of two different membrane resistance-measuring methods that use a single intracellular microelectrode was tested against a conventional method that uses two intracellular microelectrodes. The first single-electrode method used a single square current pulse and required a constant microelectrode resistance. This method was unreliable because the electrode resistance changed markedly on cell penetration and changed with time within the cell. The second method used a high frequency square wave for injecting current into the cell and depended upon the membrane having a much longer RC (resistance x capacitance)-time constant than the microelectrode. The resistance values obtained by this latter method were usually different from membrane resistances obtained at the same time on the same cells using two intracellular microelectrodes. Therefore, neither single intracellular microelectrode method was as reliable as the conventional method. All tests were with coleoptile cells of Avena sativa var. Victory. PMID- 16660165 TI - Responses of adenine nucleotides in germinating soybean embryonic axes to exogenously applied adenine and adenosine. AB - The ATP content of soybean (Glycine max [L.] Merr. cv. Kent) axes incubated for 3 hours in 1 mm solutions of adenine and adenosine increased over 100% and 75%, respectively, over axes incubated in water. The increase in ATP was primarily due to the conversion of these purines to nucleotides via the nucleotide salvage pathway. The ATP formed was in a metabolically active pool because label from adenine was incorporated into acid-insoluble material. Adenine also increased the levels of GTP, UTP, and CTP, but not to the extent of the ATP level. PMID- 16660166 TI - pH Dependence of Photosynthesis and Photorespiration in Soybean Leaf Cells. AB - The effect of pH on the kinetics of photosynthesis, O(2) inhibition of photosynthesis, and photorespiration was examined with mesophyll cells isolated from soybean (Glycine max [L.] Merr.) leaves. At constant, subsaturating bicarbonate concentration (0.5 mm), O(2) inhibition of photosynthesis increased with increasing pH because high pH shifts the CO(2)-bicarbonate equilibrium toward bicarbonate, thereby reducing the CO(2) concentration. At constant, substrating CO(2) concentrations, cell photorespiration decreased with increasing pH. This was indicated by decreases in the CO(2) compensation concentration, O(2) inhibition of photosynthesis, and glycine synthesis. Km(CO(2)) values for isolated cell photosynthesis and in vitro ribulose-1, 5-diphosphate carboxylase activity decreased with increasing pH, while the Ki(O(2)) for both systems was similar at all pH values. The responses to pH of the corresponding kinetic constants of cell photosynthesis and in vitro RuDP carboxylase with respect to CO(2) and O(2) were identical. This provides additional evidence that the relative rates of photosynthesis and photorespiration in C(3) plants are determined by the kinetic properties of RuDP carboxylase. PMID- 16660167 TI - Isolation and some properties of ribulose-1, 5-bisphosphate carboxylase-oxygenase from red kidney bean primary leaves. AB - Purification of ribulose-1,5-bisphosphate carboxylase from primary leaves of Phaseolus vulgaris var. Red Kidney with ammonium sulfate precipitation, ion exchange chromatography, and gel filtration resulted in the complete loss of detectable oxygenase activity and the retention of a low velocity and a high K(m) form of both the carboxylase and oxygenase. The polyethylene glycol-6000-purified ribulose-1, 5-bisphosphate oxygenase displayed a broad pH optimum (7.9-9.4) and a high K(m) for O(2) and ribulose 1,5-bisphosphate (0.90 mm and 0.25 mm, respectively). Initiation of the oxygenase reaction with protein rather than ribulose 1,5-bisphosphate resulted in reduced activity. The enzymes prepared by the two purification procedures were electrophoretically different.Etiolated primary leaf tissue exhibited low rates of both carboxylase and oxygenase. Similar developmental kinetic activity was observed for both reactions during greening. Photosynthetic (14)CO(2) fixation was inhibited 95% by 100% N(2) gas during the first 24 hours of greening, but the inhibition was rapidly overcome by 48 to 72 hours of light exposure. PMID- 16660168 TI - Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L: Involvement of Lipid linked Intermediates. AB - Particulate preparations from developing cotyledons of Pisum sativum L. cv. Burpeeana catalyze glycosyl transfer from UDP-[(14)C]N-acetylglucosamine and GDP [(14)C]mannose. Radioactivity is transferred to lipid components soluble in chloroform-methanol (2:1) and chloroform-methanol-water (1:1:0.3) and into a water-insoluble and lipid-free residue.The chloroform-methanol-soluble component formed from GDP-[(14)C]mannose appears to be a mannosyl lipid, whereas the chloroform-methanol-water-soluble fraction is probably a mixed oligosaccharide lipid containing N-acetylglucosamine and mannose residues. The chloroform methanol-soluble component formed from UDP-[(14)C]N-acetylglucosamine appears to be N,N'-diacetylchitibiosyl lipid, which may be incorporated with mannose to form the chloroform-methanol-water-soluble mixed oligosaccharide lipid.The oligosaccharide lipid appears to function as a precursor for the transfer of the oligosaccharide to the peptide moiety in the formation of the glycoproteins. The bulk of the radioactivity, arising from UDP-[(14)C]N-acetylglucosamine, incorporated into the insoluble residue, is associated with glycoprotein. In contrast only a small percentage of radioactivity in the insoluble residue, arising from GDP-[(14)C]mannose incorporation, appears to be associated with glycoprotein. The majority of the radioactivity found in the residue fraction labeled from GDP-[(14)C]mannose appears to be associated with oligomannosyl residues. PMID- 16660169 TI - Reevaluation of the Effect of Calcium Ions on Auxin-induced Elongation. AB - The mechanism by which calcium ions inhibit cell elongation has been reinvestigated. Growth-inhibiting levels of calcium, when applied to isolated walls (in vitro treatment) do not decrease cell wall extensibility as measured by the Instron technique. Thus, the hypothesis that calcium inhibits growth by forming wall-stiffening calcium bridges must be abandoned. Treatment of living auxin-treated sections with calcium (in vivo treatment) does cause a decrease in the subsequently measured wall extensibility, but this decline appears to be simply a consequence of the growth inhibition rather than its cause. Growth inhibiting levels of calcium do not appreciably reduce the rate of auxin-enhanced H(+) excretion. Pretreatment with calcium does not reduce the capacity of walls to undergo acid-activated wall loosening in the absence of calcium. High concentrations of CaCl(2) (0.02 m) cause an initial elastic shrinkage of Avena sections comparable to that caused by the same osmolarity of mannitol, but the subsequent growth inhibition is too great to be explained by an osmotic inhibition. Calcium ions do inhibit H(+)-induced extension of frozen-thawed sections under tension. The growth-inhibitory effects of calcium, then, may be ascribed to a direct inhibition exerted by calcium ions on the H(+)-induced wall loosening process. PMID- 16660170 TI - Relative insensitivity of mitochondria in hardened and nonhardened rye coleoptile cells to freezing in situ. AB - Mitochondria were isolated from excised coleoptiles of hardened and nonhardened winter rye (Secale cereale L. cv. Puma) seedlings which had been frozen extracellularly to different temperatures. No significant differences in the respiratory functions (ADP/O and respiratory control) were observed between mitochondria isolated from nonlethally and lethally frozen cells of both the hardened and nonhardened rye. These results suggest that mitochondria in situ can retain their normal function even after the cell was killed by the dehydrative stresses of extracellular freezing. Presumably, a different level of sensitivity to freezing stresses exists between mitochondrial and other membranous elements in the cell. PMID- 16660171 TI - Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Mitochondrial Respiration and Several Dehydrogenases during Imbibition and Germination. AB - The influence of low temperature on soybean (Glycine max [L.] Merr. cv. Wells) energy transduction via mitochondrial respiration and dehydrogenases was investigated in this study during imbibition and germination. Mitochondria were isolated from embryonic axes of seeds treated at 10 and 23 C (control) by submergence in H(2)O for 6 hours and maintenance for an additional 42 hours in a moist environment. Arrhenius plots of initial respiration rates revealed that those from cold-treated axes had respiratory control (RC) ratios of near 1.0 above an inflection in the plot at 8 C. Arrhenius plots of control axes mitochondrial respiration showed RC ratios of 2.8 above and 5.0 below an inflection temperature of 12.5 C. Energies of activation for mitochondrial respiration between 20 and 30 C for the cold and control treatments were 7.8 and 15.6 kcal/mole, respectively. These data indicate possible differences in mitochondrial membranes, degree of mitochondrial integrity, and mitochondrial enzyme complement between the two treatments.Glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6P-DH), and NADP-isocitrate dehydrogenase (NADP-ICDH) were assayed from whole seeds and axes (after germination) during the 48 hours of temperature treatments. Activity of these dehydrogenases decreased during the first 6 hours with the exception of MDH. After germination at 23 C (48 hours) all five dehydrogenases increased in activity. Arrhenius plots of cotyledon dehydrogenase activities indicated that one inflection temperature between 6 and 18 C was present for each enzyme assayed. Differences were seen in Arrhenius plots of axes dehydrogenase activities with the two temperature treatments in the cases of GDH and MDH from mitochondrial pellets and with differences in enzyme extraction media. These data suggest that the temperature treatments yield differences in mitochondrial enzyme complement. There were no detectable inflection temperatures for the activities of G6P-DH and ADH extracted from axes. Arrhenius plots of NADP-ICDH activity indicated extreme cold sensitivity. The slopes of the plots for axes NADP-ICDH were very similar to those for mitochondrial respiration (23 C treatment) suggesting that this enzyme may limit mitochondrial respiration at low temperature in soybean tissues grown at moderate temperatures. PMID- 16660172 TI - Involvement of phaseolotoxin in halo blight of beans: transport and conversion to functional toxin. AB - Phaseolotoxin ([N(delta)-phosphosulfamyl]ornithylalanylhomoarginine) is produced by Pseudomonas phaseolicola (Burkh.) Dows. in liquid culture. When phaseolotoxin was applied to leaves of bean (Phaseolus vulgaris L.) at 0.1 to 1 nmoles/g fresh weight of leaf by a prick-assay procedure, the characteristic "halo" symptom of bean halo blight disease developed after 24 to 48 hours. At higher concentrations (10-100 nmoles/g fresh weight) the systemic symptoms, which are commonly a feature of diseased plants, also developed after 24 to 48 hours.When applied to bean leaves, phaseolotoxin was rapidly broken down by the sequential removal of homoarginine and alanine. N(delta)-Phosphosulfamylornithine was the major product formed, although phosphosulfamate and unreacted phaseolotoxin were also present. When P. phaseolicola infected bean plants, very little phaseolotoxin was detected within the plant, but the amount of N(delta)-phosphosulfamylornithine formed was sufficient to account for the observed chlorosis, the ornithine accumulation, and the systemic symptoms. N(delta)-Phosphosulfamylornithine therefore seemed to be the main functional phytotoxin of bean halo blight disease.When (35)S phaseolotoxin was applied to primary leaves, (35)S (assumed to be a mixture of phaseolotoxin, N(delta)-phosphosulfamylornithine, and phosphosulfamate) was actively loaded into the fine veins of the leaf and moved through the plant in the vascular system at a speed greater than 3 cm/hour, particularly toward the apical and lateral buds and the root tips. Certain factors which affect pholem transport (arsenate, cold) affected toxin movement and the expression of systemic symptoms. Autoradiography suggested that the (35)S was transported in the phloem.A model for the involvement of phaseolotoxin in halo blight disease is presented. PMID- 16660173 TI - Regulation of lipid synthesis in soybeans by two benzoic Acid herbicides. AB - The effects of 3-nitro-2,5-dichlorobenzoic acid (dinoben) and 3-amino-2,4 dichlorobenzoic acid (chloramben) on lipid formation and on the incorporation of various substrates into lipids by intact seeds and subcellular fractions of germinating soybean (Glycine max [L.] Merr. ;Amsoy') were studied. Dinoben (20 mug/ml) inhibited synthesis of total lipids 67%, neutral lipids 73%, glycolipids 51%, and phospholipids 39% in germinating seeds. When polar lipids were analyzed further, inhibition of individual lipid classes was also observed. Chloramben (20 mug/ml) stimulated total lipid synthesis 25%. With the exception of the mitochondrial fraction where malonate thiokinase was absent, dinoben inhibited up to 99% the incorporation of acetate and malonate into lipids, but did not inhibit acetyl-CoA and malonyl-CoA incorporation. Chloramben stimulated the incorporation of all substrates tested into lipids by all fractions except the mitochondrial fraction when malonate was the substrate. When dinoben and chloramben were used in combinations, chloramben did not reverse the inhibitory effect of dinoben.It is concluded that the dinoben inhibitory effect is specific and is associated with the acetate and malonate thiokinase systems. The chloramben effect is stimulatory to either acetyl-CoA carboxylase or fatty acid synthetase or both. PMID- 16660174 TI - Specific inhibition of phototropism in corn seedlings. AB - Geotropism was used as a control for the specificity of potential inhibitors of phototropism by the coleoptiles of corn (Zea mays) seedlings. The compounds tested fall into three categories showing: (a) no inhibition of either phototropism or geotropism (KCl); (b) nonspecific inhibition of both phototropism and geotropism (KCN); and (c) specific inhibition of phototropism (KI, NaN(3), and phenylacetic acid). Simultaneous irradiation of coleoptiles with phototropically inert light in addition to the phototropically active blue light also results in an inhibition of phototropism. Since azide, iodide, and phenylacetic acid are known to interact with flavins while a simultaneous irradiation with a phototropically inert light may depopulate the first triplet state of flavins, these data support the hypothesis that the photoreceptor pigment for phototropism in corn is a flavin. PMID- 16660175 TI - Ammonia regulation of carbon metabolism in photosynthesizing leaf discs. AB - Alfalfa (Medicago sativa L., var. El Unico) leaf discs, floating on buffer containing NH(4)Cl and photosynthesizing with (14)CO(2), produced more labeled amino acid and less sucrose than did control discs (no added NH(4)Cl). The level of pyruvate increased and that of phosphoenolpyruvate decreased. These and other changes in levels of labeled compounds led us to conclude that pyruvate kinase was activated by ammonia, resulting in increased transfer of photosynthetically incorporated carbon to synthesis of amino acid skeletons at the expense of sucrose synthesis. Carbon flow through enzymes catalyzing the anaplerotic reactions was apparently stimulated. PMID- 16660176 TI - Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening. AB - A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3. PMID- 16660177 TI - Translocation patterns in xanthium in relation to long day inhibition of flowering. AB - The nature of long day inhibition of flowering in the short day plant Xanthium strumarium L. was studied by correlating the flowering response with the translocation of (14)C-assimilates from induced leaves or parts thereof to the shoot tips.In contrast to an earlier study by Gibby and Salisbury (Plant Physiol 1971 47: 784-789) no inhibitory effect of an immature leaf in long day on the flower-promoting effect of an induced leaf was detected. When the stimulus moved in the basipetal direction, mature leaves in long day were inhibitory to flowering and at the same time reduced the amount of (14)C-photosynthate that accumulated in the receptor buds.Inhibition of flowering was observed when the apical half of a single leaf was induced and the basal half kept under long day conditions. Induction of a lateral leaf half, with the other half remaining in long day, resulted in a normal flowering response. More (14)C-photosynthate was translocated to the shoot tip from the basal than from the apical leaf half.Removal of the noninduced basal leaf half except for the midrib and major veins, cutting the basal half along the midrib, or keeping the basal half in darkness, resulted in normal flowering. In all three treatments the amounts of (14)C-assimilates that accumulated in the shoot tips also increased, presumably because competing export of nonlabeled assimilates from the basal leaf halves was diminished or eliminated.Autoradiography indicated that (14)C-assimilates produced in the apical half were channeled through the basal half in the major veins and midrib to the petiole without partitioning back into the mesophyll. Since the veins and midrib in the basal half were by themselves not inhibitory to flowering, it is unlikely that the floral stimulus was inactivated in the long day tissue. When the basal half was labeled with (14)CO(2), there was no indication in autoradiograms that (14)C-labeled assimilates moved in the blade in the apical direction.As a whole these results demonstrate that transmission of the floral stimulus and translocation of photosynthate are correlated, so that at least part of the long day inhibition in Xanthium can be explained in terms of translocation effects. However, the involvement of a transmissible inhibitor produced in long day tissue cannot be ruled out. PMID- 16660178 TI - Phospholipid turnover in soybean tissue cultures. AB - The degradation rates of phospholipids in soybean (Glycine max L. Merrill) suspension cultures were studied by pulse-chase experiments. The only chloroform soluble product of incorporation of radioactive choline was phosphatidylcholine, the bulk of which had a half-life of 36 hours. Ethanolamine was incorporated primarily into phosphatidylethanolamine, phosphatidylcholine at an intermediate level, and phosphatidylmonomethylethanolamine to a small extent. The phosphatidylethanolamine decayed in a triphasic fashion with half-lives of 12, 34, and 136 hours. Phosphatidylcholine in this case increased in radioactivity up to day 4 and thereafter declined with a 92-hour half-life. The radioactivity rose slightly to day 4 in phosphatidylmonomethylethanolamine after an initial rapid decline. When serine was used as a substrate, half-lives similar to those obtained with ethanolamine were obtained. Phosphatidylcholine contained the greatest amount of label, however, with phosphatidylethanolamine containing slightly less, and phosphatidylserine contained the least. Data also are presented for glycerol and acetate phospholipid product degradation. PMID- 16660179 TI - Utilization of net photosynthate for nitrogen fixation and protein production in an annual legume. AB - The economy of C and N in nodulated cowpea (Vigna unguiculata [L.] Walp.) was described in terms of fixation of CO(2) and N(2), respiratory losses of C, and the production of dry matter and protein.Net daytime gain of C by the shoot (net photosynthesis) rose to a maximum at flowering and then declined sharply due to abscission of leaves. Maximum N fixation occurred 10 days prior to maximum net photosynthesis. Shedding of nodules reduced fixation to zero by midfruiting. Fifty per cent of the plant's N and 37% of its net photosynthate were assimilated before flowering; 39% of plant N was incorporated into seed dry matter.Respiration of nodules and roots utilized 24% of the C from net photosynthate assimilated over the growth cycle; night respiration of shoots, 20%; dry matter production in seeds, 17%; and dry matter production in other plant parts, 39%. The proportion of net photosynthate translocated to the nodulated root decreased from 41 to 14% during growth. Developing fruits were major competitors for translocate. Nodules consumed 9% of the C from the plant's total net photosynthate, 43% of which was respired, 6% made into dry matter, and 51% returned to the shoot with N fixation products.For every 1 g N fixed, net photosynthate equivalent to 6.8 g carbohydrate was consumed by nodules, 25.7 g carbohydrate by the nodulated root. Translocate was used most efficiently for N fixation in late vegetative growth when nodules were most active and their carbohydrate supply still adequate.During vegetative growth and early flowering (0 to 78 days after sowing) cowpea consumed 17.2 g net photosynthate (as carbohydrate) for every gram of protein synthesized in its shoot. The comparable conversion in seed production was 32.5 g net photosynthate/g seed protein or 6.6 g/g seed dry matter. PMID- 16660180 TI - Role of bacterial lipopolysaccharide in attachment of agrobacterium to moss. AB - Gametophore induction in moss by Agrobacterium tumefaciens was inhibited by addition of lipopolysaccharide (LPS) from A. tumefaciens. The LPS did not affect bacterial viability or appear to bind to bacterial cells. LPS from nonbinding Agrobacterium radiobacter was not effective in reducing gametophore formation. A. tumefaciens LPS, if added 24 hours after addition of viable bacterial cells, had no effect in reducing gametophore formation. The polysaccharide portion of the LPS was identified as the binding component necessary for attachment of agrobacteria for induction of gametophores in moss and tumors in higher plants. PMID- 16660181 TI - Isolation of envelope membranes from bundle sheath chloroplasts of maize. AB - Bundle sheath strands were isolated from maize (Zea mays L.) leaves treated with preparations of cellulase, hemicellulase, and pectinase. A three-phase discontinuous gradient yielded two fractions of envelope membranes from bundle sheath chloroplasts. Buoyant densities were 1.06 and 1.09 g cm(-3). The lighter fraction contained membrane vesicles under light microscopy, but centrifugation produced a pellet that was too small and unstable for purposes of electron microscopy. The heavier fraction contained single and double membrane vesicles and was studied further. Enzymic, chemical, light microscopic, and electron microscopic examination showed less than 2% contamination by stromal contents, no contamination by microbial, microsomal, or mitochondrial membranes, and possible low levels of lamellar membrane contamination. Yields of 0.5 mg of envelope membrane protein were obtained from 56-g leaf sections. The Mg(2+)-dependent nonlatent ATPase activity, a marker enzyme for chloroplast envelope membranes, was 40 mumoles Pi released hr(-1) mg protein(-1), a value similar to that obtained with pure mesophyll chloroplast envelope membranes from other plants. PMID- 16660182 TI - Isolation of bacteria, transforming bacteria, and bacteroids from soybean nodules. AB - Postnuclei supernatant of soybean (Glycine max cv. Chippewa 64) nodule homogenate was fractionated by stepwise sucrose density gradient centrifugation into supernatant, endoplasmic reticulum and mitochondria, and three distinct bands with 1.22, 1.25, and 1.27 g/cm(3) of peak density. Based on their enzymic activities, composition of electron transport components, and ultrastructural characteristics, the lightest band appears to be the mature bacteroids; the intermediate band the transforming bacteria; and the heaviest, the bacteria. The isolation procedure separates nodule symbionts into different functional and developmental fractions, and it may be a valuable tool for studies involving development, regulation, and senescence of bacteroids in the nodule. PMID- 16660183 TI - Maintenance of High Photosynthetic Rates in Mesophyll Cells Isolated from Papaver somniferum. AB - The establishment and maintenance of high rates of photosynthetic CO(2) incorporation in mesophyll cells of Papaver somniferum (opium poppy) depend on a regime of dark and light periods immediately following isolation, as well as carefully adjusted conditions of isolation. Analysis of the incorporation pattern of (14)CO(2) by the isolated cells indicates an initial "stress-response" period of approximately 20 hours characterized by increased respiratory-type metabolism and diminished photosynthesis. Under the favorable regime, this period is followed by rapid recovery and the reinstatement of a metabolic state strikingly similar to that of intact leaves in which the initial rate of CO(2) incorporation is between 110 and 175 mumoles CO(2) fixed per mg chlorophyll per hour. The photosynthetic viability of these cells can be maintained for up to 80 hours. PMID- 16660184 TI - Photosynthetic Pod Wall of Pea (Pisum sativum L.): Distribution of Carbon Dioxide fixing Enzymes in Relation to Pod Structure. AB - The pod wall of pea (Pisum sativum L.) was shown to contain two distinct photosynthetic layers. The outer, comprising chlorenchyma of the mesocarp, captured CO(2) from the outside atmosphere; the inner, a chloroplast-containing epidermis lining the pod gas cavity, was involved in photoassimilation of the CO(2) released from respiring seeds.Structural features of the pod included the thick cuticle and stomata of the outer epidermis, the inward projecting veinlets of the vascular network in the mesocarp, the sparsity of air spaces, the fiber and parenchyma layers of the endocarp, and the abundant chloroplasts, thin cuticle, and rounded outer contours of cells of the inner epidermis.The inner epidermis showed high specific activities of ribulose 1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), contained up to 20% of the pod's chlorophyll, and was capable of fixing 66% of the CO(2) released during the photoperiod to the pod gas space by the seeds of a fully grown fruit.The in vitro carboxylation capacity of the pod exceeded the estimated gross photosynthesis of the fruit for all but the last few days of development. Chlorophyll content and carboxylation activity declined more markedly in the outer photosynthetic layers than in the inner epidermis.The ratio of activities of RuDP carboxylase to PEP carboxylase in pod extracts varied from 2.4:1 to 12:1 as against 48:1 to 156:1 in extracts of leaves.Structural and physiological properties of the pod were related to its capacity to conserve respired CO(2) and provide photosynthate to developing seeds. PMID- 16660185 TI - High resolution of peroxidase-indoleacetic Acid oxidase isoenzymes from horseradish by isoelectric focusing. AB - Several improved techniques for isoelectric focusing of isoenzymes in polyacrylamide gel slabs were developed. Using these techniques, three commercial sources of horseradish peroxidase were each examined with three commercial sources of carrier ampholytes to determine their respective isoenzyme profiles.A much higher degree of isoenzyme resolution was obtained than reported previously. A composite of all of the various tests gave a total of 42 peroxidase isoenzymes. Commercial sources of horseradish peroxidase showed many similarities in their isoenzyme patterns, but their differences were sufficient to recognize each source easily. Isoenzyme patterns for all three sources spanned the entire pH range with all three sources of wide range carrier ampholytes. Only a few isoenzymes stained darkly. Most isoenzymes stained moderately to lightly, but all were well resolved. Gels stained for indoleacetic acid oxidase activity showed the same pattern as gels stained for peroxidase activity. This was true for all three commerial sources of the enzyme. The isoenzyme patterns obtained in each run were entirely reproducible, and linear pH gradients were obtained in all cases. Limitations in the pH range of the wide range carrier ampholytes relative to the isoelectric points of the extreme anodic and cathodic isoenzymes led to the adoption of a modified definition of focusing time. In addition, the labeling of isoenzymes commonly used in electrophoresis was adapted for labeling isoenzymes resolved by isoelectric focusing. PMID- 16660186 TI - Localization of the Ethylene-synthesizing System in Apple Tissue. AB - Apple (Malus sp.) slices gradually lost the ability to synthesize ethylene when incubated with a mixture of enzymes that digest cell walls. The released protoplasts did not produce ethylene. The release of protoplasts was faster from climacteric fruit slices than from preclimacteric tissue. In protoplast suspension culture, as new cell wall was deposited (as judged by the intensity of fluorescence of regenerating protoplasts stained with Calcofluor White and the incorporation of labeled myo-inositol into their ethanol-insoluble residue), ethylene synthesis was gradually regained. Restored ethylene synthesis reached a maximum after 80 hours in protoplasts from preclimacteric fruit and in 120 hours in those from climacteric tissue. Addition of methionine (1 mm) to the culture medium was essential for appreciable synthesis of ethylene; and this synthesis was inhibited by the aminoethoxy analogue of rhizobitoxine and by propyl gallate, inhibitors of ethylene synthesis in higher plants. We suggest that the ethylene synthesizing enzyme system is highly structured in the apple cell and is localized in a cell wall-cell membrane complex. PMID- 16660187 TI - Preparation and Fractionation of Rhizobium Bacteroids by Zone Sedimentation through Sucrose Gradients. AB - Zone sedimentation through sucrose gradients was used for preparing Rhizobium bacteroids from lupin nodules and for separating them into slowly and rapidly sedimenting fractions.The purity of the bacteroids was established by electron microscopy and by enzyme assays, and they were shown to contain a CO-insensitive cytochrome c oxidase.Bacteroids sedimented more rapidly than broth-cultured Rhizobium bacteria, and bacteroids from old nodules sedimented more rapidly than bacteroids from young nodules.THERE APPEAR TO BE AT LEAST TWO FORMS OF BACTEROID IN OLD NODULES: slowly sedimenting bacteroids with moderate colony-forming ability resembling the bacteroids found in young nodules, and rapidly sedimenting bacteroids with much lower colony-forming ability. PMID- 16660188 TI - Effect of Photoperiod on Stomatal Opening in Vicia faba. AB - Stomatal apertures in darkness and subsequent average opening rates in light were measured in Vicia faba leaf discs throughout the nyctoperiods for plants grown on three light:dark cycles (8:16, 12:12, and 16:8). The time course of opening in darkness depended on the specific light:dark cycle with the maximum aperture always occurring at the time the lights normally went on. The light-induced opening rate was also maximum at the end of the nyctoperiod. PMID- 16660189 TI - Phycomyces: An Increase in Mechanical Extensibility during the Avoidance Growth Response. AB - The sporangiophore of Phycomyces shows a transient response to a double barrier, the avoidance growth response. Tensile tests conducted on the stage IV sporangiophore demonstrate that an increase in mechanical extensibility occurs about a minute after a double barrier stimulus. This change in mechanical extensibility is similar to the one that occurs after a light stimulus. We have concluded that the avoidance stimulus occurs somewhere on the same pathway between the photoreceptor mechanism and the final growth response. PMID- 16660190 TI - Cysteine transport into cultured tobacco cells. AB - Cysteine transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured on liquid B-5 medium was examined.Transport was linear with time or amount of tissue and had a pH optimum of 4.5. Cysteine transport over a wide concentration range was biphasic. The isotherm, for descriptive convenience, was divided into two segments both of which obeyed Michaelis-Menten kinetics. The Km for high affinity transport was in the range 1.7 x 10(-5)m(+/-0.17) while the Km for low affinity transport was in the range 3.5 x 10(-4)m(+/-0.13). Maximum velocities were 3 to 6 nmoles/g fresh weight/minute and 13 to 16 nmoles/g fresh weight/minute, respectively.Azide and 2,4-dinitrophenol caused more than 90% inhibition of net transport by either system. N,N'-Dicyclohexylcarbodiimide was not inhibitory while the inhibition by carbonylcyanide m-chlorophenylhydrazone was dependent on the cysteine concentration. Only those compounds that were inhibitory to transport caused significant efflux of labeled material from preloaded cells.Tobacco cells that had been preincubated in iodoacetamide or N ethylmaleimide did not transport cysteine while similar treatments with dithiothreitol were only slightly inhibitory or had no effect on transport.Transport by either system was, to some extent, inhibited by all other tested amino acids and analogs. Alanine, methionine, and S-methyl cysteine were most effective in inhibiting cysteine transport. Both alanine and methionine were competitive inhibitors of cysteine transport by either system with inhibition constants that were similar to the Km for the particular system. PMID- 16660191 TI - Root and nodule respiration in relation to acetylene reduction in intact nodulated peas. AB - Inoculated pea plants (Pisum sativum L.) were grown with N-free nutrients in a controlled environment room and rates of respiratory CO(2) evolution and C(2)H(2) reduction by the intact nodulated roots were determined. Experiments followed changes related to diurnal cycles, light and dark treatments, partial defoliation, aging of plants and NH(4)NO(3) addition. In all experiments, changes in C(2)H(2) reduction were associated with parallel changes in the respiration rate, although in all but the defoliation experiment there was a basal level of respiration which was independent of the rate of C(2)H(2) reduction. In conditions which affected growth or plant size as well as C(2)H(2) reduction, respiration changed by an average of 0.42 mg CO(2) (mumol C(2)H(2) reduced)(-1). However, some treatments decreased C(2)H(2) reduction without greatly changing the growth and in these conditions respiration was decreased by an average of 0.27 mg CO(2) (mumol C(2)H(2) reduced)(-1). While this value may also include some respiration associated with other processes, it is proposed that it more closely estimates respiration directly associated with energy utilization for acetylene reduction; whereas the higher value includes respiration related to maintenance and growth processes as well. PMID- 16660192 TI - Respiration and the energy requirement for nitrogen fixation in nodulated pea roots. AB - Pisum sativum L. cv. Trapper plants were inoculated and grown in a controlled environment on N-free nutrient solution. After 4 weeks N was supplied to treatment plants as NH(4)NO(3), KNO(3), or NH(4)Cl and rates of C(2)H(2) reduction, root + nodule respiration, and leaf photosynthesis were determined 1 week later. The increase in respiration per unit of C(2)H(2) reduction was not affected by either the form of N added or the light conditions during growth, although the basal respiration rate with no C(2)H(2) reduction increased with irradiance level. The mean regression coefficient from plots of respiration versus C(2)H(2) reduction was 0.23 + 0.04 (P [unk] .01) mg of CO(2) (mumol of C(2)H(2) reduced)(-1) which was very similar to the value for the coefficient of respiration associated with nitrogenase activity estimated by subtracting growth and maintenance respiration. Since the rate of N accumulation in N-free nutrient conditions was proportional to the rate of C(2)H(2) reduction, it appears that the method gives a true estimate of the energy requirements for N fixation which for these conditions was equivalent to 17 grams of carbohydrate consumed per gram of N fixed. PMID- 16660193 TI - Effects of chill stress on prompt and delayed chlorophyll fluorescence from leaves. AB - This paper describes the utilization of a portable solid state device for the simultaneous measurement of prompt and delayed fluorescence transients in leaves from a variety of species subjected to temperature lowering. The induction transients of the two phenomena were not identical as the peak in prompt fluorescence yield always preceded that of delayed fluorescence. Temperature lowering delayed the occurrence of peak fluorescence, increased prompt fluorescence yield, decreased delayed fluorescence yield, and caused the occurrence of a new, more rapid delayed fluorescence transient. Leaves from all species had qualitatively the same type of induction curves although the response to temperature differed between species. The delayed fluorescence yield of chill sensitive species was reduced to a greater extent than that of chill-insensitive species. Cold hardening leaf material did not greatly change the fluorescence response to temperature lowering. Arrhenius plots showed a linear relationship between delayed fluorescence yield and temperature. There were no breaks that would suggest membrane lipid phase changes. The data indicate that thylakoid membranes of chill-sensitive species are less capable of maintaining a light induced high energy state at low temperatures than are thylakoid membranes of chill-resistant species. PMID- 16660194 TI - Auxin Transport Inhibitors: III. Chemical Requirements of a Class of Auxin Transport Inhibitors. AB - The structural requirements of a proposed class of auxin transport inhibitors have been shown to be very similar to those required to inhibit the cress (Lepidium sativum) root geotropic response. A 2-carboxyphenyl group separated by a conjugated system of atoms from a second aromatic ring appears to be necessary for a molecule to have high activity. PMID- 16660195 TI - Interference by a phenylacetate pathway in isotopic assays for phenylalanine ammonia-lyase in leaf extracts. AB - Particulate and soluble fractions from leaves of Sorghum, Spinacia (spinach), and Coleus, capable of metabolizing l-phenylalanine to cinnamate or to caffeate, are also able to convert l-and d-phenylalanine to phenylacetate. Since cinnamate and phenylacetate are not effectively separated in commonly used chromatographic solvents, some of the isotropic assays used for phenylalanine ammonia-lyase are rendered ambiguous by the interference of this second pathway. Therefore, a "double decker," two-dimensional paper chromatographic method was designed to separate cinnamate and phenylacetate. This was combined with the use of phenylalanine labeled randomly or just in either the carbon 1 or 2 position of the side chain. PMID- 16660196 TI - Action Spectra for Phycobiliprotein Synthesis in a Chromatically Adapting Cyanophyte, Fremyella diplosiphon. AB - The action spectra for phycocyanin production by the cyanophyte Fremyella diplosiphon shows maxima at 463 and 641 nm. The action spectrum for phycoerythrin production includes maxima at 387 and 550 nm. The maxima are based on a relative response rate well within the linear ascending portion of the dose response curves; the positions of the maxima are independent of the relative response rates chosen for reference over a 3-fold range although the comparative effectiveness of light at pairs of wavelengths varies with the standard used for comparison. These action spectra differ from those reported previously for Tolypothrix tenuis by Fujita and Hattori (Plant Cell Physiol. 3: 209-220) and by Diakoff and Scheibe (Plant Physiol. 51: 382-385) in that blue light strongly promotes phycobiliprotein synthesis in F. diphosiphon but has been reported to have little or no effect on T. tenuis.These data suggest that the photoreceptor or photoreceptors which regulate phycobiliprotein metabolism in F. diplosiphon may be either one or two metalloporphyrin complexes or double pigment systems consisting of a red or green light-absorbing photoreceptor which can be activated directly or by energy transferred from a blue light-absorbing receptor. PMID- 16660197 TI - Inhibition of chloroplast electron transport reactions by trifluralin and diallate. AB - The herbicides trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N, N-dipropyl p-toluidine) and diallate (S-[2,3-dichloroallyl] diisopropylthiocarbamate) inhibit electron transport, ATP synthesis, and cytochrome f reduction by isolated spinach (Spinacia oleracea L.) chloroplasts. Both compounds inhibit noncyclic electron transport from H(2)O to ferricyanide more than 90% in coupled chloroplasts at concentrations less than 50 mum. Neither herbicide inhibits electron transport in assays utilizing only photosystem I activity, and the photosystem II reaction elicited by addition of oxidized p-phenylenediamine or 2,5-dimethylquinone is only partially inhibited by herbicide concentrations which block electron flow from H(2)O to ferricyanide. Inhibition of ATP synthesis parallels inhibition of electron flow in all noncyclic assay systems, and cyclic ATP synthesis catalyzed by either diaminodurene or phenazine metho-sulfate is susceptible to inhibition by both herbicides. These results indicate that trifluralin and diallate both inhibit electron flow in isolated chloroplasts at a point in the electron transport chain between the two photosystems. PMID- 16660198 TI - Development of Photosystem II Activity in Chlamydomonas reinhardi Mutants: Insertion of Photosystem II Units into Inactive Preexisting Membranes versus Continuous Formation of New Photosynthetic Membranes. AB - In a previous work the development of photosystem II activity during the greening process of the y-1 mutant of Chlamydomonas reinhardi was studied (Cahen, Malkin, Shochat, Ohad. Plant Physiol 58: 257-267). Measurements of quantum yield, maximal rate of electron transfer, flash yield, and fluorescence induction indicated that photosystem II development consists of two partially overlapping phases: (a) reorganization and integration of preexisting units; and (b) addition of newly formed units to the growing membranes.In the present work an attempt was made to study these two processes independently. This was achieved using two experimental systems: (a) cells of the y-1 mutant, greened in the presence of chloramphenicol (CAP), whose activity is repaired under conditions which prevent further chlorophyll synthesis and membrane growth (dark or light with addition of cycloheximide [CHI]); (b) greening cells of a pale mutant derived from y-1 in which chloroplast membranes are formed gradually during several cell divisions.In both systems different parameters of 2,6-dichlorophenolindophenol reduction, as well as fluorescence, were measured. The results are interpreted using a kinetic model reported previously. It is concluded that during repair of photosynthetic activity in inactive membranes which were formed previously in the presence of chloramphenicol, photosystem II reaction centers are formed and integrated in preexisting membranes. In the pale green there is complete de novo synthesis of units of constant composition which are added during formation of new chloroplast membranes. PMID- 16660199 TI - Stomatal and nonstomatal regulation of water use in cotton, corn, and sorghum. AB - Stomata of corn (Zea mays L.) and sorghum (Sorghum bicolor L.) responded to changes in leaf water potential during the vegetative growth phase. During reproductive growth, leaf resistances were minimal and stomata were no longer sensitive to bulk leaf water status even when leaf water potentials approached 27 bars. Stomata of corn, cotton (Gossypium hirsutum L.), and sorghum appear to respond to changes in the humidity deficit between the leaf and air and in this manner, regulated transpirational flux to some degree. Distinct differences in water transport efficiency were observed in the three species. Under nonlimiting soil water conditions, sorghum exhibited the greatest efficiency of water transport while under limiting soil moisture conditions, cotton appeared most efficient. Corn was the least efficient with respect to nonstomatal regulation of water use. Differences in drought tolerance among the three species are partially dependent on stomatal regulation of water loss, but efficiency of the water transport system may be more related to drought adaptation. This is particularly important since stomata of all three species did not respond to bulk leaf water status during a large portion of the growing season. PMID- 16660200 TI - Evidence that root pressure flow is required for calcium transport to head leaves of cabbage. AB - Young cabbage plants (Brassica oleracea L. var. capitata) that were exposed to an atmosphere at 50% relative humidity transpired freely and accumulated significant quantities of (45)Ca in the leaves. Plants that were enclosed by plastic bags to stop transpiration from all leaves exhibited guttation with the development of root pressure and also accumulated significant quantities of (45)Ca in the leaves. (45)Ca accumulation increased in the leaves and tended to decrease in roots and stems with increasing quantities of water transpired or guttated by the plant. When plants were only partially enclosed so that some leaves were covered and the remainder exposed, only the exposed leaves that were transpiring accumulated significant quantities of (45)Ca. The covered leaves of partially enclosed plants exhibited no guttation and accumulated little (45)Ca with no measurable (45)Ca at the margins of the leaves. The results demonstrate that root pressure flow is required to transport adequate amounts of Ca to those tissues in plants that are not undergoing transpirational water loss. PMID- 16660201 TI - Contribution of a Cyanide-insensitive Alternate Respiratory System to Increases in Formamide Hydro-lyase Activity and to Growth in Stemphylium loti in Vitro. AB - Stemphylium loti, a pathogen of a cyanogenic plant, possesses a cyanide insensitive alternate respiratory pathway. In the absence of cytochrome inhibitors, the alternate system had only a minor role in respiration. When S. loti was grown in medium amended with antimycin to block the cytochrome chain, the alternate system accounted for the total oxygen consumption associated with respiration.The contribution of the alternate respiratory system to increases in formamide hydro-lyase (FHL) activity and to growth in S. loti in vitro was assessed. FHL, induced by cyanide, converts cyanide to nontoxic formamide and is partially responsible for the tolerance of S. loti to high concentrations of cyanide in vitro. When the cytochromes were blocked and the cyanide-insensitive respiratory pathway accounted for 100% of the oxygen uptake associated with respiration, FHL activity, but not changes in dry weight, was positively correlated with activity of the alternate pathway. As the alternate pathway activity decreased with increasing concentrations of salicylhydroxamic acid, the level of FHL activity correspondingly decreased. The alternate respiratory system may provide for increases in FHL activity but not for growth. S. loti appears to have two mechanisms for cyanide tolerance in vitro: cyanide-insensitive respiration and FHL activity. The initial activity of FHL for detoxification of cyanide may depend on the alternate respiratory pathway when the cytochromes of the electron transport chain are blocked. PMID- 16660202 TI - HCO(3) Influx Across the Plasmalemma of Chara corallina: Divalent Cation Requirement. AB - An absolute requirement for divalent cations is reported for H(14)CO(3) (-) influx in Chara corallina. Effective substitution of eluted Ca(2+) by Mg(2+) and Sr(2+) was observed, but Mn(2+) was completely ineffective in restoring H(14)CO(3) (-) transport activity. Similarly, La(3+) could not substitute for Ca(2+) in this system. Low concentrations of ethylenediaminetetraacetate (0.01 to 0.06 mm) significantly enhanced the rate at which H(14)CO(3) (-) transport capacity was lost.Examination of the response of OH(-) efflux, during Ca(2+)-free treatment, indicated that the cellular control over OH(-) efflux remained unaffected until membrane integrity became severely affected. This conclusion was supported by the response of OH(-) efflux to 10 mm K(+). Therefore, assimilation of H(14)CO(3) (-) is not rate-limited by an effect of Ca(2+) elution on the OH(-) transport system. Kinetic experiments indicated that Ca(2+) removal from the membrane resulted in noncompetitive inhibition of H(14)CO(3) (-) assimilation; the apparent Michaelis constant remained unaltered over a wide range of conditions. An hypothesis is presented which suggests that membrane integrity is necessary for HCO(3) (-) transport to occur, but Ca(2+) (Mg(2+), Sr(2+)), per se, must be bound to the transport complex before activity is established. PMID- 16660203 TI - Effect of Light Intensity on Efficiency of Carbon Dioxide and Nitrogen Reduction in Pisum sativum L. AB - Photosynthetic efficiency, primary productivity, and N(2) reduction were determined in peas (Pisum sativum L. var. Alaska) grown at light intensities ranging from severely limiting to saturating. Plants grown under higher light intensities showed greater carboxylation and light capture potential and higher rates of net C exchange. Uptake of N(2), computed from measured C(2)H(2) reduction and H(2) evolution rates, also increased with growth light intensity, while the previously proposed relative efficiency of N(2) fixation, based on these same parameters, declined. The plot of N/C ratios (total nitrogen content/plant dry weight) increased hyperbolically with light intensity, and the plot of N(2)/CO(2) uptake ratios (N(2) uptake rate/net CO(2) uptake rate) increased linearly. Both plots extrapolated to the light compensation point. The data indicate that the relative efficiency of N(2) fixation is not necessarily correlated with maximum plant productivity and that evaluation of a plant's capacity to reduce N(2) is related directly to concurrent CO(2) reduction. A measure of whole plant N(2) fixation efficiency based on the N(2)/CO(2) uptake ratio is proposed. PMID- 16660204 TI - Light versus Dark Carbon Metabolism in Cherry Tomato Fruits: I. Occurrence of Photosynthesis. Study of the Intermediates. AB - The photosynthetic properties of the internal and peripheral tissues of the cherry tomato fruit (Lycopersicum esculentum var. cerasiforme Dun A. Gray) were investigated. Whole fruit and their isolated tissues evolve large amounts of CO(2) in darkness. In the light, this evolution decreases but nevertheless remains a net evolution; 3-(3,4-dichlorophenyl)-1,1-dimethylurea abolishes the effects of light.Incorporation of (14)CO(2) by leaves and fruit tissues demonstrates that the outer region of the fruit has the highest photosynthetic efficiency on a chlorophyll basis; the internal fruit tissue, richer in chlorophyll, has a much lower efficiency. The identification of intermediates following short term incubations with (14)CO(2) shows that in darkness the fruit accumulates the majority of label in malate. In the light, leaf tissue exhibits a pattern of incorporation characteristic of C-3 metabolism, whereas fruit tissue exhibits a decreased labeling of malate with a concomitant appearance of label in Calvin cycle intermediates. This is in agreement with the levels and types of carboxylating activities demonstrated in vitro; especially noteworthy is the very low ribulose diphosphate carboxylase activity in the internal fruit tissue.The photosynthetic potential, phosphoenolpyruvate carboxylase activity, and quantities of malate accumulated by fruit tissues are parallel to their chlorophyll content during growth and maturation. PMID- 16660205 TI - Light versus Dark Carbon Metabolism in Cherry Tomato Fruits: II. Relationship Between Malate Metabolism and Photosynthetic Activity. AB - The possible relationship between malate metabolism and photosynthetic activity in green tomato fruit tissues (Lycopersicum esculentum var. cerasiforme Dun A. Gray) was investigated. Initial experiments consisted of vacuum-infiltrating (14)C-3 or (14)C-4-malate into isolated tissues in darkness and then incubating the tissues under photosynthetic conditions. Other experiments involved a short pulse with (14)C-bicarbonate in darkness to label the malate pool(s), followed by a chase in the light in the presence of nonradioactive bicarbonate. Both series of experiments were followed by the separation and identification of labeled metabolic intermediates.Label initially in carbon atoms 3 and 4 of malate, corresponding also to C-3 of pyruvate and CO(2) after malate decarboxylation, was recovered as citrate + isocitrate, sugars and starch following incubations of tissues in the light. These data demonstrate that the reductive pentose phosphate cycle utilizes CO(2) furnished by malate metabolism due to the operation of the citric acid cycle and perhaps also to malic enzyme activity. Some synthesis of sugars and starch from C-3 of malate was observed in darkness or in the light 3 (3,4-dichlorophenyl)-1,1-dimethyl which could be due to gluconeogenesis. Pulse chase experiments indicated a rapidly turning over malate pool. PMID- 16660206 TI - In vitro formation and development of floral buds on tobacco stem explants: effects of kinetin and other factors. AB - Stem segments were excised from plants of Wisconsin 38 tobacco (Nicotiana tabacum L.) in three regions differing in their distance below the inflorescence. They were cultured in vitro in 8- or 16-hr days. After 8 weeks, floral and vegetative buds were counted, and extent of floral development was assessed. Kinetin at 10( 5)m inhibited formation and development of floral buds regardless of indoleacetic acid concentration. Supplied at this concentration with adequate auxin, kinetin stimulated vegetative bud formation and may have caused floral bud abortion. Indoleacetic acid (>/= 10(-6)m) inhibited vegetative and floral bud formation when supplied with low kinetin concentration (/= 10(-6)m), it inhibited floral bud formation and stimulated vegetative bud formation. More floral buds were formed in 16-hr days than in 8-hr days. Few formed on explants other than those derived from the region nearest the inflorescence regardless of other treatment. PMID- 16660207 TI - Floral induction of vegetative plants supplied a purified fraction of deoxyribonucleic Acid from stems of flowering plants. AB - It has been found that floral induced stems of flowering tobacco (Nicotiana tabacum cv. Wis. 38) plants contain large amounts of rapidly renaturing DNA, whereas noninduced stems of vegetative plants contain only small amounts. In addition, it has been shown that the striking qualitative difference in DNA between stems of flowering and vegetative plants mimics the over-all quantitative difference in DNA content (on a fresh weight basis). Therefore, the extra DNA in stems of flowering plants seems, at least in part, to represent preferential synthesis of rapidly renaturing DNA.Rapidly renatured DNA (flowering plants) has been purified (cesium chloride gradients) from heated-cooled DNA solution and under noninductive conditions has been tested for floral activity. It has been found that when rapidly renatured DNA in buffer solution is supplied to axillary vegetative buds of vegetative plants and then the axillary buds are defoliated every 4th day for 12 days, the treated buds change into flower buds. On the other hand, control axillary buds supplied buffer solution alone remain vegetative.In stem segments from flowering plants, the concept, discussed in previous reports, that indole-3-acetic acid may modify in vitro bud expression by directly affecting DNA synthesis has been reviewed. On the basis of this report, the concept is elaborated by proposing here that indole-3-acetic acid may act partially in bud expression by directly suppressing synthesis of rapidly renaturing DNA. PMID- 16660208 TI - Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene. AB - If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5'-S-methyl-5'-thioadenosine (MTA) would be the fragment nucleoside. When [Me-(14)C] or [(35)S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by (l)-2-amino-4-(2' aminoethoxy)-trans-3-butenoic acid, the conversion of [(35)S] or [Me(14)C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [(35)S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.When [(35)S]MTA was fed to climacteric or preclimacteric apple tissue, radioactivity was efficiently incorporated into MTR and methionine. However, when [(35)S]MTR was administered, radioactivity was efficiently incorporated into methionine but not MTA. This suggests that the sulfur of MTA is incorporated into methionine via MTR. A dual label experiment with [(35)S, Me-(3)H]MTA indicates that the CH(3)S group of MTA was transferred as a unit to form methionine.A scheme is presented for the production of ethylene from methionine, the first step being the activation of methionine by ATP to give SAM. SAM is fragmented to give ethylene, MTA, and other products. MTA is then hydrolyzed to MTR which donates its methylthio group to a four-carbon acceptor to reform methionine. PMID- 16660209 TI - Phospholipid Biosynthesis and Fatty Acid Content in Relation to Chilling Injury during Germination of Seeds. AB - The proportion of labeled (14)C-glycerol incorporated into phospholipids and the fatty acid composition of three phospholipids in germinating seeds and seedlings of chilling-sensitive lima beans (Phaseolus lunatus L.) and chilling-resistant broad beans (Vicia faba L.) and peas (Pisum sativum L.) at 10 and 25 C were determined. During the imbibition of seeds (first 24 hours), lima beans were sensitive to chilling injury at 10 C and a higher proportion of label was incorporated into phosphatidylethanolamine and phosphatidylglycerol than in broad beans and peas. Broad beans and peas incorporated a higher proportion of label into phosphatidylcholine. The oleic acid content of phosphatidylcholine was higher and linolenic acid content was lower in peas and broad beans than in lima beans at 10 and 25 C. The unsaturated to saturated fatty acid ratio was much higher for the chilling-resistant seeds than for the chilling-sensitive ones. In the seedling stage, the proportion of label incorporated into the four major phospholipids was similar in the three species regardless of temperature treatment. The fatty acid content of the phospholipids examined was not different in the three species in the seedling stage. PMID- 16660210 TI - Oxygen Concentration in Isolated Chloroplasts during Photosynthesis. AB - The O(2) concentration in intact and osmotically disrupted isolated spinach (Spinacia oleracea, L.) chloroplasts during photosynthesis was estimated. The chloroplasts were allowed to reduce 3-phosphoglycerate, CO(2), or ferricyanide in light until the rate of O(2) production was linear. When the light was turned off O(2) evolution from the chloroplasts continued for a few seconds. This prolonged O(2) evolution is due to an O(2) surplus inside the chloroplasts which equilibrates with that in the medium. From this surplus the O(2) concentration inside the chloroplasts at the moment when the light had been switched off was calculated. In all experiments the O(2) concentration inside the photosynthesizing chloroplasts was higher than that outside, but was dependent upon the O(2) concentration of the chloroplast medium. At low external O(2) concentration (30 mum) the ratio of the internal to the external O(2) concentration was about 5, whereas at concentrations corresponding to those in airsaturated water this ratio was close to 1. With osmotically broken chloroplasts this ratio was 1.2 at 30 mum O(2) and almost 1 from 150 mum onward. When the O(2) surplus found in broken chloroplasts during photosynthesis was related to the volume of the thylakoids, a ratio of about 2.3 was observed. PMID- 16660211 TI - Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus). AB - Germinating nasturtium pollen (Tropaeolum majus) is shown to excrete an enzyme(s) which hydrolyzes all types of monomers from biosynthetically labeled cutin and p nitrophenyl esters, which are model substrates for fungal cutinases. The pollen cutinase showed an optimum pH near 6.5 and was inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethyl maleimide but not by diisopropyl-fluorophosphate, an "active serine"-directed reagent indicating that the pollen enzyme is an "-SH cutinase" unlike the fungal enzyme which is a serine cutinase. Excretion of the pollen cutinase into the extracellular fluid was complete within 4 to 6 hours at 30 C. Since actinomycin D and cycloheximide showed little effect on the level of cutinase excreted, it appears that cutinase is an enzyme synthesized prior to germination. Release of cutinase into the medium did not require germination. Electron microscopy revealed the presence of a continuous cutin layer on mature stigma with extensive folds, which are proposed to play a role similar to that played by the cellular papillae found in the stigma of other plants. Chemical analysis of stigma cutin by depolymerization and combined gas-liquid chromatography and mass spectrometry showed that this cutin consists of mainly the C(16) family of acids. The major (70%) components were dihydroxy C(16) acids which consisted of 10,16- (64%), 9,16- (16%), 8,16- (12%), and 7,16- (8%) dihydroxy plamitic acid. Deuterium-labeling studies showed the presence of 16-oxo-9-hydroxy C(16) acid and 16-oxo-10-hydroxy C(16) acid in this cutin. The biochemical and ultrastructural studies indicate that the pollen tube may gain entry into stigma using cutinase excreted by the pollen. PMID- 16660212 TI - Histochemical Evidence for the Occurrence of Oligomycin-sensitive Plasmalemma ATPase in Corn Roots. AB - A cytochemical study has been made on the localization of ATPase activity in corn (Zea mays L.) roots. Light microscopy shows washing for 4 hours to increase the general ATPase activity in the peripheral layers of the root cortex; oligomycin and N,N-dicyclohexylcarbodiimide inhibit this activity, oligomycin being more effective. Ultrastructural studies of ATPase location show oligomycin treatment to inhibit both mitochondrial and plasmalemma ATPase, but only in the epidermis and outer cortex. Studies with lipid-soluble dyes indicate that oligomycin might not penetrate very deeply into root tissue in the time span of these experiments. It is suggested that the strong inhibition of ion absorption by oligomycin without a corresponding decline in ATP content is probably due to inhibition of ion absorption in the peripheral cell layers, thus limiting the supply of ion for symplastic transport to the uninhibited tissues. PMID- 16660213 TI - Cytochemical Localization of K-stimulated Adenosine Triphosphatase Activity in Xylem Parenchyma Cells of Barley Roots. AB - ATPase activity in xylem parenchyma cells of barley (Hordeum vulgare L.) roots was demonstrated cytochemically with a lead precipitation reaction. The methodical parameters of this cytochemical test were optimized for distinction between ATPase-specific and nonspecific precipitates. Optimum conditions were prefixation in 1% glutaraldehyde for 1 hour and incubation for 2 hours in a medium containing 2 mm each of ATP, Ca(2+), and Pb(2+) at pH 7 and 25 C. Problems of cytochemical localizations are discussed.ATPase activity occurred mainly at the plasmalemma, the endoplasmic reticulum nuclear envelope, and outer mitochondrial membranes of xylem parenchyma cells. The tonoplast of these cells showed only little ATPase activity. High K(+) concentrations stimulated ATPase activity, particularly at the plasmalemma. Diethylstilbestrol prevented the formation of ATPase-specific precipitates. The cytochemical demonstration of a K(+)-stimulated ATPase at the plasmalemma of xylem parenchyma cells is discussed in relation to the possible role of this membrane in ion transport to the vessels. PMID- 16660214 TI - Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold hardened Wheat Coleoptiles. AB - Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of (14)CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 muM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-(14)C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-(14)C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold hardened and unhardened coleoptiles. PMID- 16660215 TI - Temperature-dependent Changes in the Polysomal Population of Senescent (Ripening) Pear Fruit. AB - A loss and recovery of polysomes coincident with the temperature-dependent interruption and resumption of pear fruit (Pyrus communis, L.) senescence establishes a close correlation between the presence of protein-synthesizing machinery and the progression of senescence and ripening. PMID- 16660216 TI - Activation of endogenous respiration and anion transport in corn mitochondria by acidification of the medium. AB - Acidification of the suspending medium of corn mitochondria (Zea mays L., WF9 x Mo17) from pH 7.5 to pH 6.8 to 6.4 initiates osmotic swelling with the transportable anions citrate, sulfate, and phosphate. Swelling becomes pronounced with a combination of citrate plus sulfate or phosphate. Acidification proves to activate endogenous respiration, which is essentially zero at pH 7.5. The endogenous respiration transports citrate (in the presence of sulfate or phosphate) which then contributes to respiration and the accelerated osmotic swelling. Mersalyl will inhibit the swelling and antimycin inhibits the endogenous respiration. Magnesium appears to reduce the permeability of the membranes under the acid conditions. PMID- 16660217 TI - No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds. AB - alpha-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10(-6)m gibberellic acid (GA(3)) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10(-2)m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified alpha-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of alpha-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10(-4)m 5-FU prior to the addition of GA(3) resulted in decreased thermal stability of GA(3)-induced alpha-amylase and who interpreted this as evidence that the mRNA for alpha-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified alpha-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of alpha-amylase mRNA synthesis. PMID- 16660218 TI - Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: III. Polysaccharidic Origin of Labeled Glucose. AB - On the basis of solubility, hydrolysis by glucoamylase (EC 3.2.1.3), and monomeric composition, starch appears to be the major glucose-containing, hot water-soluble polysaccharide that is labeled when germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen is grown in the presence of myo-[2 (3)H]inositol, d-[R5,S5-(3)H]xylose, or l-[1-(14)C]arabinose. PMID- 16660219 TI - Indoleacetaldehyde in cucumber seedlings. AB - The presence of indoleacetaldehyde in cucumber (Cucumis sativus L.) cotyledons was demonstrated by thin layer chromatographic R(F) values in three solvent systems, by the formation and hydrolysis of a bisulfite adduct, and by chemical reduction to indoleethanol and oxidation to indoleacetic acid. Bioassays indicated a minimum indoleacetaldehyde content in etiolated cotyledons of 0.7 mug per kg fresh weight. Tissue samples from all parts of both green and etiolated cucumber seedlings reduced exogenously supplied indoleacetaldehyde to indoleethanol. PMID- 16660220 TI - Cucumber seedling indoleacetaldehyde oxidase. AB - Extracts of light-grown Cucumis sativus L. seedlings catalyzed the oxidation of indole-3-acetaldehyde to indole-3-acetic acid. No added cofactors were required. Inhibitor studies indicated that the enzyme is a metalloflavoprotein. While indole-3-aldehyde, benzaldehyde, and phenylacetaldehyde partially inhibited the oxidation of indole-3-acetaldehyde, suggesting that they may serve as alternative substrates, it is proposed that indoleacetaldehyde is the major substrate in vivo. 2,4-Dichlorophenoxyacetic acid strongly inhibited the indoleacetaldehyde oxidase activity, and it is proposed that this enzyme may be subject in vivo to feedback inhibition by indole-3-acetic acid. The enzyme was activated by brief heating or by treatment with mercaptoethanol. PMID- 16660221 TI - Inhibition of Ethylene Production in Penicillium digitatum. AB - Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and alpha-ketoglutarate as ethylene precursors, was inhibited by methionine, methionine sulfoxide, methionine sulfone, and methionine sulfoximine. Rhizobitoxine did not affect ethylene production but its ethoxy and methoxy analogues were effective inhibitors of ethylene production; its saturated methoxy analogue and kainic acid stimulated ethylene production. Tracer studies showed that the inhibitors blocked the conversion of [(3)H]glutamate into [(3)H]ethylene.In shake cultures of this fungus, which utilize methionine as the ethylene precursor, rhizobitoxine and its unsaturated analogues all inhibited, while the saturated methoxy analogue stimulated ethylene production. In both types of cultures inhibition was irreversible and was diminished by increasing concentrations of the ethylene precursor. The inhibitory activity or lack of it by rhizobitoxine and its analogues appears to be a function of their structural resemblance to glutamate and methionine as well as of their size and stereoconfiguration. These data suggest similarities between the ethylene-forming system in the fungus and in higher plants despite differences in precursors under some cultural conditions. PMID- 16660222 TI - Change in the proportion of two aspartokinases in carrot root tissue in response to in vitro culture. AB - Two isofunctional aspartokinases (EC 2.7.2.4) exist in fresh root tissue of carrot (Daucus carota, cv. Oogata sanzun). The threoninesensitive portion constitutes about 70% of the activity; the lysinesensitive, less than 20%. Culture of slices of carrot tissue for 3 days reversed the ratio as the lysine sensitive activity preferentially increased. Inhibition by threonine and lysine was additive in both enzyme preparations from fresh and cultured tissues. The activities were resolved into two distinct fractions of different sensitivity to threonine and lysine by DEAE-Sephadex A-50 column chromatography. PMID- 16660223 TI - Seasonal Variation of Glutathione and Glutathione Reductase in Needles of Picea abies. AB - In spruce (Picea abies) needles glutathione and glutathione reductase show a periodic seasonal variation with significantly increased levels during the winter. It is proposed that glutathione and glutathione reductase play an important role for the winter hardiness of leaves from evergreen plants. PMID- 16660224 TI - Osmotic adjustment in leaves of sorghum in response to water deficits. AB - The relationships among the total water potential, osmotic potential, turgor potential, and relative water content were determined for leaves of sorghum (Sorghum bicolor [L.] Moench cvs. ;RS 610' and ;Shallu') with three different histories of water stress. Plants were adequately watered (control), or the soil was allowed to dry slowly until the predawn leaf water potential reached either 0.4 megapascal (MPa) (treatment A) or -1.6 MPa (treatment B). Severe soil and plant water deficits developed sooner after cessation of watering in ;Shallu' than in ;RS 610', but no significant differences in osmotic adjustment or tissue water relations were observed between the two cultivars. In both cultivars, the stress treatments altered the relationship between leaf water potential and relative water content, resulting in the previously stressed plants maintaining higher tissue water contents than control plants at the same leaf water potential. The osmotic potential at full turgor in the control sorghum was -0.7 MPa: stress pretreatment significantly lowered the osmotic potential to -1.1 and 1.6 MPa in stress treatments A and B, respectively. As a result of this osmotic adjustment, leaf turgor potentials at a given value of leaf water potential exceeded those of the control plants by 0.15 to 0.30 MPa in treatment A and by 0.5 to 0.65 MPa in treatment B. However, zero turgor potential occurred at approximately the same value of relative water content (94%) irrespective of previous stress history. From the relationship between turgor potential and relative water content there was an approximate doubling of the volumetric elastic modulus, i.e. a halving of tissue elasticity, as a result of stress preconditioning. The influence of stress preconditioning on the moisture release curve is discussed. PMID- 16660225 TI - Control of Flowering in the Grapevine (Vitis vinifera L.): Formation of Inflorescences in Vitro by Isolated Tendrils. AB - Tendrils produced from shoot tips of grapevine (Vitis vinifera L.) cultured in vitro on Nitsch's medium developed into inflorescences when 5 to 10 mum benzyladenine (BA) or 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine (PBA) were applied directly to the tendril tips. Inflorescences did not form on tendrils if the cytokinins were supplied in the agar. Tendrils cultured in agitated liquid medium containing BA, PBA, or zeatin riboside showed profuse branching and tendrils were transformed into inflorescences. Calyx and corolla (calyptra) stamens and pistils developed normally in the presence of both zeatin riboside and PBA, but micro- and macrosporogenesis were absent.Inflorescences were formed by tendrils from five cultivars (Muscat of Alexandria, Shiraz, Carbernet Sauvignon, Wortley Hall, and Sultana syn. Thomson Seedless) and also on tendrils from 12- to 15-week-old-seedlings. PMID- 16660226 TI - Albumin storage proteins in the protein bodies of castor bean. AB - Of the total protein in the protein bodies of castor bean (Ricinus communis L.), approximately 40% is represented by a group of closely related albumins localized in the matrix of the organelle. This group of albumins has a sedimentation value of 2S and is resolved into several proteins of molecular weight around 12,000 daltons by sodium dodecyl sulfate-acrylamide gel electrophoresis. It has a high content of glutamate/glutamine and undergoes rapid degradation during the early stage of germination. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins, in addition to globulins, glutelins, and prolamines, are important storage proteins in seeds. PMID- 16660227 TI - Water potential in excised leaf tissue: comparison of a commercial dew point hygrometer and a thermocouple psychrometer on soybean, wheat, and barley. AB - Leaf water potential (Psi(leaf)) determinations were made on excised leaf samples using a commercial dew point hygrometer (Wescor Inc., Logan, Utah) and a thermocouple psychrometer operated in the isopiestic mode. With soybean leaves (Glycine max L.), there was good agreement between instruments; equilibration times were 2 to 3 hours. With cereals (Triticum aestivum L. and Hordeum vulgare L.), agreement between instruments was poor for moderately wilted leaves when 7 mm-diameter punches were used in the hygrometer and 20-mm slices were used in the psychrometer, because the Psi(leaf) values from the dew point hygrometer were too high. Agreement was improved by replacing the 7-mm punch samples in the hygrometer by 13-mm slices, which had a lower cut edge to volume ratio. Equilibration times for cereals were normally 6 to 8 hours. Spuriously high Psi(leaf) values obtained with 7-mm leaf punches may be associated with the ion release and reabsorption that occur upon tissue excision; such errors evidently depend both on the species and on tissue water status. PMID- 16660228 TI - Dependence of phytochrome action in seeds on membrane organization. AB - Germination of Amaranthus retroflexus L. seeds imbibed at 40 C is enhanced by establishing the active form of phytochrome before a reduction in temperature to <32 C. The half-time for effectiveness of the lower temperature is about 8 min at 15 C. Isolated membrane fragments of A. retroflexus seeds associated with the fluorescent probe 1,8-anilino-naphthalene sulfonate (ANS) increase in structural order as the temperature is lowered through the 32 C region. The germination response is decreased by the membrane-disruptive substances tris, octonoate, and ethanol. The results show that phytochrome activity is associated with an organized membrane. By using ANS with membrane fragments from Setaria faberi Herrm. seeds, leakage of amino acid was found to be enhanced at temperatures >32 C by a transition in the plasmalemma. PMID- 16660229 TI - Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi: Evidence for de Novo Synthesis and for Loss of Functional Enzyme Molecules. AB - Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-(3)H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [(15)N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [(14)N]nitrate and 100% H(2)O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation. PMID- 16660230 TI - Glycoprotein Synthesis in Plants: II. Structure of the Mannolipid Intermediate. AB - This paper reports the purification and structural determination of the mannolipid shown previously (Ericson and Delmer 1977 Plant Physiol 59: 341-347) to serve as an intermediate in glycoprotein synthesis in cotyledons of Phaseolus vulgaris. The mannolipid was purified by chromatography in organic solvents on diethylaminoethyl-cellulose, followed by repeated steps of deacylation and rechromatography on Sephadex LH-20. Binding and elution behavior on diethylaminoethyl-cellulose was consistent with the presence of a monophosphate residue. Lability of the mannolipid to mild acid treatment as well as its resistance to hot phenol treatment or catalytic hydrogenation are consistent with the structure of a polyprenol having a saturated alpha-residue. After methanolysis, the chloroform-methanol-soluble portion of the mannolipid was analyzed by mass spectrometry. The fragmentation pattern obtained was nearly identical to that obtained from standard dolichol-phosphate. An intense ion at m/e 69 represented the omega-terminal isoprenoid residue, and repeating fragments separated by 68 mass units were observed up to m/e of > 1,200. All evidence supports the conclusion that the mannolipid is dolichol-monophosphate-mannose and thus provides further support for the concept that the processes involved in the glycosylation of protein in higher plants are similar to those known to occur in the animal kingdom. PMID- 16660231 TI - Rapid release of protease inhibitors from soybeans: immunochemical quantitation and parallels with lectins. AB - Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 mug; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 mug, respectively; lyophilisate of water in which roots grew for 20 days, 5 mug; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 mug, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors. PMID- 16660232 TI - Proteolytic activation of a galactosyl transferase involved in osmotic regulation. AB - Osmotic regulation in the flagellate Ochromonas malhamensis Pringsheim is mainly mediated by changes in the pool size of alpha-galactosyl-(1 --> 1)-glycerol (isofloridoside). Isofloridoside phosphate synthase, a regulated key enzyme responsible for the formation of isofloridoside phosphate, appears to exist as an inactive proenzyme which can be activated by incubation of crude cell extracts with endogenous or exogenous proteases. PMID- 16660233 TI - Purification and Properties of an Elicitor of Castor Bean Phytoalexin from Culture Filtrates of the Fungus Rhizopus stolonifer. AB - Evidence has been obtained for the presence in filtrates of 3-day-old cultures of the fungus Rhizopus stolonifer grown on potato-dextrose medium of both high molecular weight and low molecular weight elicitors of the production of the phytoalexin casbene in cell-free extracts of castor bean (Ricinus communis L.) seedlings. The high molecular weight elicitor activity was purified by means of gel filtration chromatography. Both protein and carbohydrate are associated with the most purified fraction containing elicitor activity. The elicitor is inactivated by treatments at 60 C or higher temperatures for 15 minutes. The molecular weight of the purified elicitor was estimated from gel filtration chromatography in 10 mm Na-phosphate (pH 7) to be 30,000 +/- 5,000. Treatments of the purified elicitor fraction with either sodium periodate or the nonspecific protease preparation, pronase, substantially reduced its activity as an elicitor of casbene production. On the basis of these properties it is concluded that the elicitor is most likely a protein and may be a glycoprotein. It is estimated that 2 x 10(-8)m elicitor gives about a 14-fold increase in casbene synthetase activity in extracts of treated split seedlings in comparison with controls. This corresponds to about 50% of the maximal activity obtainable in this assay system developed to measure elicitor activity. PMID- 16660234 TI - Serinol phosphate as an intermediate in serinol formation in sugarcane. AB - A novel compound, serinol phosphate, was identified in sugarcane (Saccharum officinarum) clone 51NG97. It was produced by an enzyme-mediated transamination of dihydroxyacetone phosphate with either alanine, glutamate, aspartate, or glutamine serving equally well as an amino donor. Some detectable phosphatase activity was present in crude leaf enzyme preparation that hydrolyzed serinol phosphate. A proposal for a pathway of the biosynthesis of serinol in sugarcane was formulated.Serinol can serve as an "activator" of toxin production in attenuated cultures of the sugarcane pathogen Helminthosporium sacchari and it is present in susceptible clone 51NG97. Resistant clone H50-7209 does not possess serinol and likewise no dihydroxyacetone phosphate transaminase activity could be demonstrated in enzyme preparations of this clone. The concept of toxin activation in attenuated fungus cultures is briefly discussed relative to disease resistance and susceptibility. PMID- 16660235 TI - Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana. AB - The synthesis of H(2)O-soluble and NaOH-hydrolyzable bound forms of indole-3 acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous (14)C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in (14)C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana. PMID- 16660236 TI - Amino Acid recycling in relation to protein turnover. AB - Methods of measuring amino acid recycling in Lemna minor are described. The extent to which the recycling of individual amino acids may underestimate protein turnover has been measured for a number of amino acids. The methods have been used to study the relationship between protein turnover and amino acid recycling during nitrogen starvation. It is concluded that following the removal of nitrate from the environment, protein turnover is enhanced, the partitioning of amino acids between protein synthesis and amino acid metabolism is relatively constant, but the total amount of amino acids recycling is increased. PMID- 16660237 TI - Absence of pfr destruction in the modulation of phenylalanine ammonia-lyase synthesis of mustard cotyledons. AB - The relationship between the amount of active phytochrome (Pfr) produced by 5 minute light pulses and the rate of subsequent enzyme accumulation (phenylalanine ammonia-lyase, EC 4.3.1.5) of mustard (Sinapis alba L.) cotyledons was investigated. The response rapidly adjusts to changes of the Pfr level produced by light pulses of different wavelengths. Regardless of total phytochrome levels in the cotyledons, response adjustments to new photostationary states (phi(lambda)) are correlated with phi(alpha) values. On the other hand, the kinetics of enzyme accumulation shows no influence of Pfr destruction as determined spectrophotometrically (tau((1/2)) = 45 min) in the same organ (see Schafer et al. 1973 Photochem Photobiol 18: 331-334). It is concluded that the phytochrome molecules involved in the regulation of this response by light pulses comprise a small fraction of the total phytochrome of the cotyledons. In contrast to bulk phytochrome this fraction appears to be not subject to Pfr destruction. PMID- 16660238 TI - Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells. AB - The occurrence of photorespiration in soybean (Glycine max [L.] Merr.) leaf cells was demonstrated by the presence of an O(2)-dependent CO(2) compensation concentration, a nonlinear time course for photosynthetic (14)CO(2) uptake at low CO(2) and high O(2) concentrations, and an O(2) stimulation of glycine and serine synthesis which was reversed by high CO(2) concentration. The compensation concentration was a linear function of O(2) concentration and increased as temperature increased. At atmospheric CO(2) concentration, 21% O(2) inhibited photosynthesis at 25 C by 27%. Oxygen inhibition of photosynthesis was competitive with respect to CO(2) and increased with increasing temperature. The Km (CO(2)) of photosynthesis was also temperature-dependent, increasing from 12 mum CO(2) at 15 C to 38 mum at 35 C. In contrast, the Ki (O(2)) was similar at all temperatures. Oxygen inhibition of photosynthesis was independent of irradiance except at 10 mm bicarbonate and 100% O(2), where inhibition decreased with increasing irradiance up to the point of light saturation of photosynthesis. Concomitant with increasing O(2) inhibition of photosynthesis was an increased incorporation of carbon into glycine and serine, intermediates of the photorespiratory pathway, and a decreased incorporation into starch. The effects of CO(2) and O(2) concentration and temperature on soybean cell photosynthesis and photorespiration provide further evidence that these processes are regulated by the kinetic properties of ribulose-1,5-diphosphate carboxylase with respect to CO(2) and O(2). PMID- 16660239 TI - Inactivity of Oxidation Products of Indole-3-acetic Acid on Ethylene Production in Mung Bean Hypocotyls. AB - The suggestion that indole-3-acetic acid (IAA)-stimulated ethylene production is associated with oxidative degradation of IAA and is mediated by 3 methyleneoxindole (MOI) has been tested in mung bean (Phaseolus aureus Roxb.) hypocotyl segments. While IAA actively stimulated ethylene production, MOI and indole-3-aldehyde, the major products of IAA oxidation, were inactive. Tissues treated with a mixture of intermediates of IAA oxidation, obtained from a 1-hour incubation of IAA with peroxidase, failed to stimulate ethylene production. Furthermore, chlorogenic acid and p-coumaric acid, which are known to interfere with the enzymic oxidation of IAA to MOI, had no effect on IAA-stimulated ethylene production. Other oxidation products of IAA, including oxindole-3-acetic acid, indole-3-carboxylic acid, (2-sulfoindole)-3-acetic acid, and dioxindole-3 acetic acid, were all inactive. 1-Naphthaleneacetic acid was as active as IAA in stimulating ethylene production but was decarboxylated at a much lower rate than IAA, suggesting that oxidative decarboxylation of auxins is not linked to ethylene production. These results demonstrate that IAA-stimulated ethylene production in mung bean hypocotyl tissue is not mediated by MOI or other associated oxidative products of IAA. PMID- 16660240 TI - Regions of differential cell elongation and mitosis, and root meristem morphology in different tissues of geotropically stimulated maize root apices. AB - We examined cell length, mitosis, and root meristem "cuticle" in different tissues of geostimulated, red light-exposed primary roots of corn (Zea Mays, Wisconsin hybrid 64A x 22R). The examination was done at 15-minute intervals for a period of 240 minutes. Differences in cell elongation between the upper and lower sides were most prominent between 1.5 and 2.5 mm from the root meristem; the outer cortex had the greatest elongation growth, and the upper cells showed a significant increase in length compared to the lower. A differential mitosis was also found, with the lower tissue being greater. We infer that the mitotic activity is indicative of cell division, and this division occurs strictly in the first 1.5 mm of the root meristem. The combined effect of differential cell elongation and cell division results in the localization of the geotropic curvature in the 1.5- to 2.5-mm region from the root meristem. Mitosis that occurs primarily in the cortex and stele were asynchronous; the peak of cortical division preceded that of the stele. Both peaks occurred before the peak of geotropism. A densely stained layer separates the cap from the root meristem. This layer is thinner at the apex of the root meristem. The area of the thin region increased with time and peaked at 180 minutes after geostimulation, which was coincidental with the peak of the geotropic response. PMID- 16660241 TI - Differential cytokinin structure-activity relationships in phaseolus. AB - The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N(6) side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N(6)-(Delta(2)-isopentenyl)adenine and N(6)-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis zeatin, and N(6)-isopentyladenine was 100-fold more active than N(6)-(Delta(2) isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism. PMID- 16660242 TI - Shading Influence on the Sterol Balance of Nicotiana tabacum L. AB - Tobacco plants (Nicotiana tabacum L.) were grown in the field and the apex was removed at the 42-day stage. Shading screens were set up which produced 0, 26, 67, and 90% shade. Plants were grown an additional 25 days before leaves from top, middle, and bottom stalk positions were harvested. Each leaf group was analyzed for free sterol, steryl ester, steryl glycoside, and acylsteryl glycoside. The free sterol content was lowest in top leaves and highest in bottom leaves; however, the top leaves had more steryl ester than the bottom leaves. Leaf position had no effect on steryl glycosides and acylsteryl glycosides. Shading did not influence the level of any sterol class; but in general, shading increased stigmasterol and decreased sitosterol. This trend was observed for all sterol classes, and the free sterols showed the largest and most consistent change. The younger top leaves showed a greater response than the older bottom leaves, but bottom leaves always had more stigmasterol than sitosterol even without shade. PMID- 16660243 TI - Chlorophyll fluorescence assay for ozone injury in intact plants. AB - A chlorophyll fluorescence induction (Kautsky effect) assay predicted ozone induced injury in bean leaves (Phaseolus vulgaris) at least 20 hours before any visible sign of leaf necrosis. The extent of injury, which could be predicted during exposure to ozone, depended on concentration, exposure time, and leaf development stage. Much more injury occurred in light than in darkness and long exposures to lower ozone concentrations were more injurious than brief exposures to higher ones. The first detectable effect was on the photosynthetic water splitting enzyme systems, followed by inhibition of electron transport between the photosystems. The fluorescence assay provides a simple, rapid, nondestructive method for observing effects of ozone on plants. PMID- 16660244 TI - Correlation between Oxygen Availability, Energy Charge, and Protein Synthesis in Squash Cotyledons Isolated from Germinating Seeds. AB - The influence of O(2) availability on the rate of protein synthesis, the levels of RNA and of adenylates, and the value of the energy charge in squash (Cucurbita maxima) cotyledons isolated from seeds germinated for 15 or 28 hours at different O(2) concentration (3% or 20% O(2)) has been investigated.The rate of protein synthesis is five times lower in cotyledons maintained in 3% O(2) than in those maintained in 20% O(2). Also net RNA synthesis is almost blocked in 3% O(2), while in 20% O(2) it proceeds almost linearly for 48 hours.The different RNA contents cannot explain the different rates of protein synthesis.The results of shift experiments (cotyledons shifted from 20% to 3% O(2) or vice versa) show that the rate of protein synthesis is strictly correlated with actual O(2) availability and is largely independent of the one in the previous period. O(2) controls the development of the adenylate pool and particularly the increase of ATP level. Thus, both the adenylate pool and the values of the energy charge ratio are lower in cotyledons grown in 3% than in 20% O(2).The shifts of O(2) availability induce rapid changes of ATP, ADP, and AMP levels and thus of the values of the energy charge, which are about 0.7 at 3% O(2) and higher than 0.8 at 20% O(2), independent of previous O(2) availability.The rate of protein synthesis appears to be largely independent of the levels of the single nucleotides and better correlated to the energy charge values. PMID- 16660245 TI - Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: I. Conversion to Hexoses. AB - The myo-inositol oxidation pathway was investigated in regard to its role as a source of carbon for products of hexose monophosphate metabolism in germinated pollen of Lilium longiflorum Thunb., cv. Ace. myo-[2-(14)]Inositol and d-[1 (14)C]glucuronate had similar distributions of radioactivity, contributing about three times more label to polysaccharide-bound glucose than myo-[2-(3)H]inositol. In the course of glucogenesis label from the latter appeared as tritiated water in the medium. This exchange could be enhanced by supplying d-[5R,5S-(3)H]xylose instead of myo-[2-(3)H]inositol. When the former was administered, [(3)H]glucose was the only labeled sugar residue found in polysaccharide products. The soluble constituents of d-[5R,5S-(3)H]xylose-labeled pollen contained no traces of labeled xylose despite massive uptake and utilization.l-[1-(14)C]- and l-[5 (14)C]Arabinose produced similar labeling patterns in germinated pollen including incorporation of arabinosyl units into pollen tube polysaccharides and substantial glucogenesis which led to utilization of arabinose for respiration and further incorporation of labeled glucosyl units into pollen tube polysaccharides.d-[5-(3)H]Galacturonate was rapidly taken up by germinated pollen but slowly utilized, without conversion to other sugars, for incorporation into pollen tube polysaccharides. l-[6-(14)C]Gulonate was not taken up by pollen.Results strongly support a scheme of conversion from myo-inositol to hexose monophosphate and subsequent products of glucose metabolism that involves the myo-inositol oxidation pathway. PMID- 16660246 TI - Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates. AB - myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5 (14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5 (14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2 (3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis. PMID- 16660247 TI - Transverse viscoelastic extension in nitella: I. Relationship to growth rate. AB - Transverse viscoelastic extensibility was measured directly in isolated walls of Nitella internode cells. Cell walls extended transversely exhibit a yield point which is approximately twice the yield point in the longitudinal direction. Walls from young, growing cells are four to seven times more extensible longitudinally than transversely, while walls from mature, nongrowing cells are only two times more extensible longitudinally. Although longitudinal extensibility decreases drastically with the decrease in the growth rate, lateral extensibility is constant through development. There is a discrepancy between the lateral growth rate and transverse creep, since the lateral growth rate is not constant. However, the degree of wall anisotropy observed is consistent with the view that the transversely oriented cellulose microfibrils act as a "reinforcing filler" in Nitella cell walls. PMID- 16660248 TI - Isolation of polyribosomes and messenger RNA active in in vitro synthesis of soybean seed proteins. AB - Polyribosome preparations containing low proportions of monosomes to polyribosomes have been isolated from developing seeds of Glycine max L. Merrill using a high pH-high KCl buffer. The polyribosomes were functional in in vitro protein synthesis reactions using wheat germ 23,000g supernatant preparations. Results of experiments using aurintricarboxylic acid indicated that most or all of the amino acid incorporation in vitro resulted from the completion of nascent polypeptides associated with the isolated polyribosmes. RNA purified from polyribosome preparations by affinity chromatography on oligo(dT)-cellulose was also active in vitro, and had different Mg and K requirements for translation than did the polyribosomes. Translation of oligo(dT)-cellulose-purified mRNA was inhibited by the addition of 7-methylguanosine 5'-phosphate, suggesting that soybean mRNAs are "capped" at their 5' ends. Some, but not all, of the products of these reactions were identical in electrophoretic mobility to radioactive polypeptides of storage proteins produced in soybean cotyledons grown in culture. PMID- 16660249 TI - Preformed Messenger RNAs and Early Wheat Embryo Germination. AB - Wheat (Triticum aestivum L.) embryo homogenates have been fractionated into three cell fractions from which RNA was extracted and assayed for mRNA content by in vitro translation and by [(3)H]polyuridylic acid hybridization. In dry embryos the preformed mRNAs are distributed equally between a rapidly sedimenting "pellet" fraction and a cytoplasmic "ribosomal/subribosomal" fraction. During germination 25 to 40% of the total mRNA becomes polyribosomal. The remaining 60 to 75% is retained in the pellet and ribosomal/subribosomal fractions.To compare the nucleotide sequences of the different mRNA fractions, cDNAs were transcribed from polyribosomal (A+) RNA of 40-minute imbibed embryos and from total A(+) RNA of dry embryos, and the ability of these cDNAs to hybridize with the more prevalent classes of mRNA from the different cell fractions was analyzed. The results suggest that there is no significant difference between the preformed mRNAs that move into polyribosomes and those remaining in the nonpolyribosomal fractions. In addition there appears to be no difference between the mRNAs of polyribosomes from embryos germinated for 45 minutes and 5 hours. Between 5 hours and 2 days, however, there is a considerable change in the mRNA composition of the embryos. We conclude for the prevalent classes of message, that the preformed mRNAs of the wheat embryo are not involved in temporal regulation of early development but that they function primarily to allow a rapid resumption of growth upon exposure of the embryo to water. PMID- 16660250 TI - Regulation of the Photosynthesis Rhythm in Euglena gracilis: I. Carbonic Anhydrase and Glyceraldehyde-3-Phosphate Dehydrogenase Do Not Regulate the Photosynthesis Rhythm. AB - A circadian rhythm of O(2) evolution has been found in Euglena gracilis, Klebs strain Z. The rhythm persists for at least 5 days in constant dim light and temperature, but damps out in constant bright light. The phase of this rhythm can be shifted by a pulse of bright light and the period length is not changed over a 10 C span of growth temperature.The O(2) evolution rhythm is found in both logarithmic and stationary phase cultures, but CO(2) uptake is clearly rhythmic only in stationary phase cultures.The activity of glyceraldehyde-3-phosphate dehydrogenase was not rhythmic as previously reported (Walther and Edmunds [1973] Plant Physiol. 51: 250-258). Carbonic anhydrase activity was rhythmic when the cultures were maintained under a light-dark cycle with the highest enzyme activity coinciding with the fastest rate of O(2) evolution. However, the rhythm in carbonic anhydrase activity disappeared under constant conditions. Changes in the activities of these two enzymes are therefore not responsible for the rhythmic changes in photosynthetic capacity. PMID- 16660251 TI - Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum. AB - Large numbers of vacuoles (10(6)-10(7)) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 mumoles of cyanogenic glucoside dhurrin per 10(7) protoplasts or 10(7) vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum. PMID- 16660252 TI - Pressure probe technique for measuring water relations of cells in higher plants. AB - A new method is described for continuously measuring cell turgor pressure (P), hydraulic conductivity (L(p)), and volumetric elastic modulus (epsilon) in higher plant cells, using a pressure probe. This technique permits volume changes, DeltaV, and turgor pressure changes, DeltaP, to be determined with an accuracy of 10(-5) to 10(-6) mul and 3 to 5.10(-2) bar, respectively.The main principle of the new method is the same as the pressure probe developed by Zimmermann and Steudle in which pressure is transmitted to a pressure transducer by means of an oil-filled capillary introduced into the cell. In order to use the pressure probe for small tissue cells, the effective compressible volume of the apparatus has to be sufficiently small in comparison to the volume of the cell itself. This is achieved by accurately fixing the oil/cell sap boundary in the very tip of the microcapillary by means of an electronic feedback mechanism, so that the effective volume of the apparatus is reduced to about 2 to 10% of the cell volume. In this way also, errors arising from compressibility of the apparatus and temperature fluctuations can be excluded.Measurements on tissues cells of Capsicum annuum fruits yield epsilon values of 2 to 25 bar. Furthermore, epsilon can be shown to be a function of both cell turgor pressure and cell volume; epsilon increases with increasing turgor pressure and is higher in larger cells. PMID- 16660253 TI - Membrane Potential in Phaeoceros laevis: Effects of Anoxia, External Ions, Light, and Inhibitors. AB - Gametophyte cells of Phaeoceros laevis (L.) Prosk. have vacuole electric potentials (PDs) of about -175 millivolts; the steady PD is not affected by light but small transient PDs result after changing from light to darkness or darkness to light. The PD is more negative than the Nernst potentials for any of the permeating ions. Changes in the concentration of any one of the external ions between 0.1 and 10 mm have only a very small effect on the PD. Increases in external pH cause the PD to depolarize by a few millivolts. Azide, 2,4 dinitrophenol, and NH(4)Cl each cause rapid and reversible depressions of the PD; the effects of these agents are similar in magnitude in the light and in the dark. Anoxia depolarizes the PD by about 30 millivolts in the light and by about 60 millivolts in the dark. Ouabain and 3-(3,4-dichlorophenyl)-1,1-dimethylurea have no effects on the PD. It is concluded that the membrane potential is controlled by an electrogenic efflux pump, possibly for H(+). It is also concluded that the source of energy for the pump is respiration and not photosynthesis. PMID- 16660254 TI - Suberization: inhibition by washing and stimulation by abscisic Acid in potato disks and tissue culture. AB - Wounding of potato (Solanum tuberosum L.) tubers results in suberization, apparently triggered by the release of some chemical factor(s) at the cut surface. Suberization, as measured by diffusion resistance of the tissue surface to water vapor, was inhibited by mm concentrations of indoleacetic acid, unaffected by mm concentrations of traumatic acid, severely inhibited at mum concentrations of cytokinin, but stimulated by abscisic acid (ABA) at 10(-4)m. Thorough washing of potato disks up to 3 to 4 days after cutting resulted in severe inhibition of suberization as measured both by diffusion resistance and by the amount of the octadecene diol generated by hydrogenolysis (LiAlH(4)) of the tissue. Disks washed after 4 days did not show any inhibition of suberization. High performance liquid chromatographic analysis of the wash from fresh potato disks showed that about 14 ng of ABA was released into the wash per g of tissue. The amount of ABA released increased with time up to 4 to 6 hours of washing. The maximal amount of ABA was washed out after aging for 24 hours and after 2 days of aging ABA could no longer be found in the surface wash of the disks. Addition of ABA to the media of potato tissue cultures resulted in suberin formation whereas control cultures contained little suberin. The effect of ABA on suberization in the tissue cultures was shown to be linearly concentration-dependent up to 10( 4)m and a linear increase in suberin formation was seen up to about 8 days of culture growth on the media containing 10(-4)m ABA. From these results it is proposed that during the early phase of wound-healing ABA plays a role in triggering a chain of biochemical processes which eventually (in about 3 to 4 days) result in the formation of a suberization-inducing factor, responsible for the induction of the enzymes involved in suberin biosynthesis. PMID- 16660255 TI - Plasma Membrane-associated Adenosine Triphosphatase Activity of Isolated Cortex and Stele from Corn Roots. AB - The plasma membrane fractions from separated cortex and stele of primary roots of corn (Zea mays L. WF9 x M14) contained cation ATPase activity at similar levels but with somewhat different properties. ATPase activity from cortex was optimum at pH 6.5, showed a simple Michaelis-Menten saturation with increasing ATP.Mg, and showed complex kinetic data for K(+) stimulation similar in character to the kinetic data for K(+)-ATPase and K(+) influx in primary roots. The results for cortex indicate that homogenates of primary roots are dominated by membranes from cortical cells.ATPase activity from stele was optimum at pH 6.5 and showed another maximum at pH 9. At pH 6.5, activity from stele had properties similar to that from cortex except that the kinetics of K(+) stimulation closely approached that expected for a Michaelis-Menten enzyme. At pH 9, the enzyme activity from stele was inhibited by 5 mug/ml oligomycin, suggesting that a significant portion of the activity was of mitochondrial origin. Sucrose density gradient analysis indicated some contamination of mitochondrial membranes in the plasma membrane fraction from stele. The results for stele are consistent with the view that stelar parenchyma cells are not deficient in ion pumps. PMID- 16660256 TI - Role of Cation and Anion Uptake in Salt-stimulated Elongation of Lettuce Hypocotyl Sections. AB - The role of cation and anion uptake in salt-stimulated growth of light-grown, GA(3)-treated lettuce (Lactuca sativa L.) hypocotyl sections was investigated. Potassium chloride (10 mm) causes a 2-fold increase in the growth rate of GA(3) treated hypocotyl sections without affecting the growth rate of sections incubated in the absence of GA(3). Salt uptake is the same in both treatments, and furthermore the uptake of cation and anion is stoichiometric during the first 24 hours under all incubation conditions. The importance of the anion for cation uptake is demonstrated in experiments with benzenesulfonate(-) and iminodiacetate(2-). When K(+) and Na(+) are supplied only as the benzenesulfonate and iminodiacetate salts, growth and cation uptake are markedly reduced compared to KCl and NaCl. Calculation of the osmotic potential of salt-treated sections based on measurement of K(+) and Cl(-) uptake suggests that the observed increase in tissue osmolality is a result of salt uptake. Similarly, uptake of ions can account for the shift in water potential when sections are incubated in 10 mm KCl. We conclude that the change in growth rate of light-grown, GA(3)-treated sections caused by the addition of KCl or NaCl to the incubation medium results solely from decreased water potential of the tissue due to ion uptake. PMID- 16660257 TI - Light-dependent Emission of Hydrogen Sulfide from Plants. AB - With the aid of a sulfur-specific flame photometric detector, an emission of volatile sulfur was detected from leaves of cucumber (Cucumis sativus L.), squash and pumpkin (Cucurbita pepo L.), cantaloupe (Cucumis melo L.), corn (Zea mays L.), soybean (Glycine max [L.] Merr.) and cotton (Gossypium hirsutum L.). The emission was studied in detail in squash and pumpkin. It occurred following treatment of the roots of plants with sulfate and was markedly higher from either detached leaves treated via the cut petiole, or whole plants treated via mechanically injured roots. Bisulfite elicited higher rates of emission than sulfate. The emission was completely light-dependent and increased with light intensity. The rate of emission rose to a maximum and then declined steadily toward zero in the course of a few hours. However, emission resumed after reinjury of roots, an increase in light intensity, an increase in sulfur anion concentration, or a dark period of several hours.The emission was identified as H(2)S by the following criteria: it had the odor of H(2)S; it was not trapped by distilled H(2)O, but was trapped by acidic CdCl(2) resulting in the formation of a yellow precipitate, CdS; it was also trapped by base and the contents of the trap formed methylene blue when reacted with N,N-dimethyl-p-phenylenediamine and Fe(3+).H(2)S emission is not the cause of leaf injury by SO(2), since bisulfite produced SO(2) injury symptoms in dim light when H(2)S emission was low, while sulfate did not produce injury symptoms in bright light when H(2)S emission was high.The maximum rates of emission observed, about 8 nmol min(-1) g fresh weight( 1), are about the activity that would be expected for the sulfur assimilation pathway of a normal leaf. H(2)S emission may be a means by which the plant can rid itself of excess inorganic sulfur when HS(-) acceptors are not available in sufficient quantity. PMID- 16660258 TI - 2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins. AB - In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4 dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [gamma-(32)P]adenosine 5'-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F(1) is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F(1) but its identity is unknown. Urea acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins. PMID- 16660259 TI - Growth and epinasty of marigold plants maintained from emergence on horizontal clinostats. AB - Dry weight, leaf number, and leaf size of marigold plants (Tagetes patula) grown from emergence for 18 days on horizontal clinostats rotating at 15 revolutions per hour (rph), were similar to those of plants grown for the same period on vertically oriented clinostats rotating at 15 rph. The horizontally grown plants exhibited some epinasty which disappeared when plants were placed upright for 24 hours. Vertically grown plants when placed on horizontal clinostats for 24 hours exhibited more epinasty than plants grown from emergence on horizontal clinostats.Data are provided to demonstrate that leaves undergo movement (bending) during each rotation cycle that leads to the development of a leaf curvature that is oriented away from the direction of rotation. The results of this study suggest that epinasty of plants placed on horizontal clinostats could be due to uncontrolled movement of plants during rotation rather than controlled by gravity nullification. The usefulness of horizontal clinostats for gravity nullification or simulating weightlessness on plants is questioned. PMID- 16660260 TI - Time-dependent Changes in the Auxin Sensitivity of Coleoptile Segments: Apparent Sensory Adaptation. AB - When segments are excised from corn (Zea mays L.) coleoptiles they exhibit a very low rate of elongation for about 3.5 hours. A strong increase in growth rate (the spontaneous growth response) then occurs and persists for many hours. During the latent period preceding the spontaneous growth response there is an apparent increase with time in the sensitivity of the segments to indoleacetic acid (IAA). This increase in sensitivity is expressed as a 2- to 3-fold increase in the magnitude of the growth response to low levels of IAA and a 3-fold decrease in the latent period of the response during the first 3 hours following excision. A similar increase in sensitivity to low levels of IAA is noted if application of IAA is timed from the point of termination of a previous exposure to the hormone. Since the increase in responsiveness to low levels of IAA is not paralleled by an increase in the rate of uptake of the hormone, the data may be interpreted as evidence for a type of time-dependent sensory adaptation to auxin. The IAA dose response relationship also changes with time, and there is indirect evidence that an auxin-dependent inhibitor may influence the expression of the apparent sensory adaptation to auxin. PMID- 16660261 TI - Model for Short Term Movements in Stapelia variegata Linn. AB - Recently it was demonstrated that holographic interferometry could be used to quantify minute displacements of a mature Stapelia variegata Linn. The previously unobtainable measurements revealed that the short term response of the stapelia was regular enough so that the dynamics could be evaluated through the use of a highly damped mass, spring, dashpot model. Such a model is derived explicitly and the relationship between movements of the entire plant and the dynamics of individual cells or groups of cells is shown. PMID- 16660262 TI - Induction of "pore" formation in plant cell membranes by toluene. AB - Treatment with aqueous toluene-ethanol has been shown to induce "pore" formation in plant cell membranes. The evidence is as follows: [List: see text]While the principal experimental material was roots of Atriplex nummularia Lindl., the fact that similar results were also observed with leaves of Pisum sativum L. and with the alga Chlorella pyrenoidosa Chik. suggests that the phenomenon is general.Although the phenomenon of pore induction is qualitatively similar to that in microorganisms, the pores induced appear to be smaller. It is proposed that induced leakage could be the basis for the development of simple and rapid methods for plant biochemical studies. PMID- 16660263 TI - Amylopectin degradation in pea chloroplast extracts. AB - Phosphorolysis rather than phosphorylation of amylolysis products was found to be the major pathway of sugar phosphate formation from amylopectin by pea (Pisum sativum L.) chloroplast stromal proteins. The K(m) for inorganic phosphate incorporation was 2.5 mm, and ATP did not stimulate amylopectin-dependent phosphate incorporation. Arsenate (10 mm) inhibited phosphate incorporation into glucose monophosphates up to 46% and phosphoglucomutase activity 96%, resulting in glucose 1-phosphate accumulation as a product of amylopectin degradation. The intracellular distribution of enzymes of starch utilization was determined. Phosphorylase, phosphoglucomutase, and hexokinase were found in the chloroplast and cytoplasm, while beta-amylase was restricted to the cytoplasm. Maltase was not detectable; maltose phosphorylase was active in the chloroplast. PMID- 16660264 TI - Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts. AB - Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K(m) values for GSSG and NADPH were 23 mum and 4.9 mum, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.Illuminated sonicated pea chloroplasts, in the presence of catalytic amounts of NADPH, catalyzed GSSG-dependent O(2) evolution (mean of 10 determinations, 10.4 mumol per mg chlorophyll per hour, sd 1.4) with the concomitant production of GSH. The molar ratio of GSH produced to O(2) evolved was 3.8 and the highest ratios for O(2) evolved to GSSG added were 0.46 and 0.44. The K(m) value for GSSG was 26 mum. GSH inhibited the reaction. The reaction was attributed to photosynthetically coupled glutathione reductase.Ruptured chloroplasts, in the presence of catalytic amounts of GSSG and NADPH, did not catalyze sustained O(2) evolution in the presence of substrate amounts of hydrogen peroxide, dehydroascorbate, l-cystine, sulfite, or sulfate. PMID- 16660265 TI - Regulation of Phytoalexin Synthesis in Jackbean Callus Cultures: Stimulation of Phenylalanine Ammonia-Lyase and o-Methyltransferase. AB - Jackbean, Canavalia ensiformis (L.), callus tissues synthesized the phytoalexin, medicarpin (3-hydroxy-9-methoxypterocarpan), when treated with spore suspensions of Pithomyces chartarum (Berk. and Curt.) M. B. Ellis, a nonpathogen of jackbean. Medicarpin was isolated from treated callus tissue and identified by its ultraviolet and mass spectra. The minimum spore concentration found to elicit medicarpin synthesis after 26 hours was 1 x 10(5) spores/ml; levels of medicarpin in callus tissue increased linearly up to 1 x 10(7) spores/ml, indicating that the recognition sites for presumed elicitors were not saturated. Medicarpin was first detected in callus treated with 1 x 10(7) spores/ml, 6 to 12 hours after application, and the concentration reached a maximum at 48 hours, slowly declining thereafter to 72 hours. In callus treated with 3.15 mm HgCl(2), medicarpin concentrations were also maximum by 48 hours. Phenylalanine ammonia lyase (EC 4.3.1.5) activity increased 2-fold in spore-treated callus after 36 hours. Isoliquiritigenin, daidzein, and genistein o-methyltransferase (EC 2.1.1.6) activities were increased 3- to 4-fold in treated callus. Caffeic acid and naringenin were more efficient substrates for o-methyltransferase activity than the other flavonoids or apigenin, but there was no increase in these o methyltransferase activities in spore-treated callus. The phytoalexin response in this callus tissue culture system compares well with natural plant systems and should be an excellent system for investigating regulation of phytoalexin synthesis. PMID- 16660266 TI - Estimation of whole-plant resistance to gaseous exchange independent of leaf temperature measurement. AB - For studies into the uptake of mercury vapor by wheat (Triticum aestivum), a simple theory and plant chamber were employed to estimate total leaf resistance of whole plants to water vapor exchange. The estimates were independent of leaf temperature, for which mean values were indirectly determined. The approach involved the measurement, at steady-state conditions, of the net change in water vapor flux per unit of leaf surface (Deltaq(v)) in response to a small induced change in absolute humidity (DeltaC(a)). Assuming that total leaf resistance (r(l)) was constant and that change in leaf temperature (T(l)) was negligible, total leaf resistance was calculated from the equation, [Formula: see text]While the assumptions concerning r(l) and T(l) may or may not be correct, evidence is presented which indicates that such assumptions did not significantly alter estimates of r(l) from their true values for changes in ambient relative humidity ranging from 0.011 to 0.074. Total leaf resistance of groups of whole plants estimated in this manner did not differ for ambient temperatures of 17, 25, and 33 C. Mean values of r(l) ranged from 83 sec cm(-1) in darkness to 2.4 sec cm(-1) at an illumination of 12.8 klux. PMID- 16660267 TI - Effect of glycidate, an inhibitor of glycolate synthesis in leaves, on the activity of some enzymes of the glycolate pathway. AB - Under conditions where glycolate synthesis was inhibited at least 50% in tobacco (Nicotiana tabacum L.) leaf discs treated with glycidate (2,3-epoxypropionate), the ribulose diphosphate carboxylase activity in extracts and the inhibition of the activity by 100% oxygen were unaffected by the glycidate treatment. [1 (14)C]Glycidate was readily taken into leaf discs and was bound to leaf proteins, but the binding occurred preferentially with proteins of molecular weight lower than ribulose diphosphate carboxylase. Glycidate added to the isolated enzyme did not inhibit ribulose diphosphate carboxylase activity or affect its inhibition by 100% O(2). Thus, glycidate did not inhibit glycolate synthesis by a direct effect on ribulose diphosphate carboxylase/oxygenase.NADH-glyoxylate reductase and phosphoglycolate phosphatase activities were also unaffected in extracts of leaf discs supplied with glycidate, but NADPH-glyoxylate reductase was inhibited about 35% in such extracts. Addition of 10 mm glycidate to isolated NADPH-glyoxylate reductase inhibited the activity about 25% after 15 minutes of reaction.The small inhibition of NADPH-glyoxylate reductase by glycidate may help to explain the increase in glyoxylate concentration found in glycidate-treated leaf discs. Increasing the glyoxylate pool size in leaf discs has been shown to effectively block glycolate synthesis and photorespiration and increase net photosynthesis. Thus, the similar effects brought about by glycidate in leaf discs can be attributed to indirect effects of metabolic regulation. PMID- 16660268 TI - Inhibition of glutamate:glyoxylate aminotransferase activity in tobacco leaves and callus by glycidate, an inhibitor of photorespiration. AB - The effect of glycidate (2,3-epoxypropionate), an inhibitor of glycolate synthesis and photorespiration in leaf tissue, was studied on glutamate:glyoxylate and serine:glyoxylate aminotransferases and glycine decarboxylase activities in particulate preparations obtained from tobacco (Nicotiana tabacum L.) callus and leaves. Glycidate specifically and effectively inhibited glutamate:glyoxylate aminotransferase. The inhibition was dependent on glycidate concentration and, to a lesser extent, on substrate concentration. The enzyme was not protected by either substrate. Even with saturating substrate concentrations the glycidate inhibition was only partially reversed. Under the in vitro assay conditions, glycidate inhibition of the aminotransferase was reversible. Glutamate:glyoxylate aminotransferase is the only enzyme of the glycolate pathway thus far examined which is severely inhibited by glycidate. However, in leaf discs, pretreatment with glycidate decreased both glutamate:glyoxylate and serine:glyoxylate aminotransferase activities suggesting binding by glycidate in vivo.Glycidate increased the pool sizes of both glutamate and glyoxylate in leaf discs. It has been shown that increases in concentration of either of these metabolites decrease photorespiration and glycolate synthesis and increase net photosynthesis. It is proposed that glycidate inhibits photorespiration indirectly by increasing the internal concentrations of glutamate and glyoxylate, as a consequence of the inhibition of glutamate:glyoxylate aminotransferase activity. PMID- 16660269 TI - Biosynthesis of dolichyl phosphate: characterization and site of synthesis in algae. AB - This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system.A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an alpha-saturated polyprenyl phosphate.The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [(14)C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C(90) to C(105), C(95) and C(100) being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed.The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate. PMID- 16660270 TI - Pentose phosphate metabolism of potato tuber discs as influenced by prior storage temperature. AB - Tubers of Solanum tuberosum L. var. Russet Burbank, Kennebec, and Targhee were stored at 95% relative humidity and at 1.7, 4.4, 5.8, 7.2 or 10 C. Rates of O(2) consumption were measured periodically by removing individual tubers from storage, excising discs of pith parenchyma tissue, and incubating the discs in a Gilson respirometer at 30 C. With all three varieties, data suggest a greater involvement of pentose phosphate metabolism with tissues from tubers stored at 1.7 C as compared to those from higher temperatures. Discs from tubers stored at 1.7 C had higher respiratory quotient values (CO(2)/O(2)), were substantially less sensitive to the inhibitory effects of iodoacetic acid, and were somewhat more sensitive to the inhibitory effects of salicylhydroxamic acid than the discs from tubers stored at higher temperatures. Using [(14)C]glucose labeled in the 1 or the 6 carbon, mean C(1) to C(6) ratios in the Gilson after 3.5 hours of aging were 4.8:1 with discs from tubers stored at 1.7 C and 2.5:1 with discs from tubers stored at 7.2 C. Prior storage history is important in metabolic studies using potato tubers. PMID- 16660271 TI - Indole-3-acetic Acid Levels of Plant Tissue as Determined by a New High Performance Liquid Chromatographic Method. AB - A method for the analysis of indole-3-acetic acid (IAA) in plant extracts has been developed based on high performance liquid chromatography separation of IAA on a microparticulate strong anion exchange column followed by quantitation with two selective detectors: an electrochemical, carbon paste amperometric detector and/or a fluorescence detector. The detection limit for IAA is less than 1 nanogram with the fluorescence detector and less than 50 picograms with the electrochemical detector.The IAA levels are reported for various tissues of wheat, pinto beans, soybeans, cotton, and corn. PMID- 16660272 TI - Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm. AB - Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K(m) for acetyl-CoA of 10 mum and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K(i) 110 mum) and glycolate (K(i) 150 mum) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating (35)S labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [(35)S]methionine-labeled endosperm tissue. PMID- 16660273 TI - Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum. AB - At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [(35)S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2-day-old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, (35)S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction.Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [(35)S]methionine or (14)C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes.Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes. PMID- 16660274 TI - Effect of Ethephon, Indole Butyric Acid, and Treatment Solution pH on Rooting and on Ethylene Levels within Mung Bean Cuttings. AB - Light-grown mung bean (Phaseolus aureus Roxb.) cuttings were treated with buffered and nonbuffered solutions of Ethephon, indole butyric acid (IBA), and the combination of both. Ethephon treatment resulted in increased tissue ethylene levels with increasing solution pH, but had no effect on rooting. IBA treatment had no effect on tissue ethylene levels, but strongly promoted rooting. Combinations of Ethephon and IBA had no effect on rooting of mung bean cuttings beyond that obtained by IBA alone. PMID- 16660275 TI - Evidence that the lipid carrier for N-acetylglucosamine is different from that for mannose in mung beans and cotton fibers. AB - Cell-free enzyme particles from mung beans (Phaseolus aureus) or cotton (Gossypium hirsutum L.) fibers catalyze the incorporation of mannose from GDP [(14)C]mannose and N-acetylglucosamine from UDP-[(3)H]-N-acetylglucosamine into polyprenyl-type lipids. These lipids have been synthesized and purified and the lipid moieties compared to each other as well as to dolichyl phosphate and to lipids isolated from similar mannoseand N-acetylglucosamine-containing lipids from liver and aorta.The following lines of evidence indicate that in plants, the lipid carrier for N-acetylglucosamine is different from the lipid carrier for mannose: [List: see text]We propose that the apparent difference in the lipid carrier for these two sugars may be a point of control of glycoprotein synthesis. PMID- 16660276 TI - Ultraviolet-B Radiation-induced Inhibition of Leaf Expansion and Promotion of Anthocyanin Production: Lack of Involvement of the Low Irradiance Phytochrome System. AB - Leaf discs from expanding leaves of Rumex patientia L. were exposed to 7 hours of visible plus different levels of ultraviolet radiation in the 290 to 315 nm waveband (UV-B) and then placed in darkness. Leaf disc expansion was reduced and anthocyanin production was increased in discs exposed to moderate or high levels of UV-B radiation when compared to control discs. The possibility that the inhibition of leaf expansion by UV-B radiation might be at least partially phytochrome-mediated was examined by giving discs brief red or far red irradiation following exposure to UV-B radiation. Brief red radiation (R) following treatment with moderate or high UV-B radiation did not alter the pattern of growth or anthocyanin production compared to discs placed in darkness following UV-B treatment. However, a posttreatment with far red radiation (FR) reduced the growth of discs subjected previously to either moderate UV-B or no UV B irradiation to the level of growth of discs given high UV-B. FR posttreatment also decreased anthocyanin production in discs in moderate and high UV-B treatments. Effects of FR and UV-B radiation apparently do not involve the same mechanism. This was demonstrated by experiments in which FR following the UV-B treatments was in turn followed by R, which reversed the effects of the FR but did not alter the growth inhibition or increased anthocyanin production induced by moderate or high levels of UV-B radiation. PMID- 16660277 TI - Kinetics of C-photosynthate translocation in morning glory vines. AB - The movement of (14)C-photosynthate in morning glory (Ipomea nil Roth, cu. Scarlet O'Hara) vines 2 to 5 meters long was followed by labeling a lone mature leaf with (14)CO(2) and monitoring the arrival rate of tracer at expanding sink leaves on branches along the stem. To a first approximation, the kinetic behavior of the translocation profiles resembled that which would be expected from movement at a single velocity ("plug flow") without tracer loss from the translocation stream. There was no consistent indication of a velocity gradient along the vine length. The profile moved along the vine as a distinct asymmetrical peak which changes shape only slowly. The spatial distribution of tracer along the vine reasonably matched that predicted on the basis of the arrival kinetics at a sink, assuming plug flow with no tracer loss. These observations are in marked contrast to the kinetic behavior of any mechanism describable by diffusion equations.However, a progressive change in profile shape (a symmetrical widening) was observed, indicating a range of translocation velocities. A minimum of at least two factors must have contributed to the observed velocity gradient: the exchange of (14)C between sieve elements and companion cells (demonstrated by microautoradiography) and the range of velocities in the several hundred sieve tubes which carried the translocation stream. Possible effects of these two factors on profile spreading were investigated by means of numerical models. The models are necessarily incomplete, due principally to uncertainties about the exchange rate between sieve elements and companion cells and the degree of functional connectivity between sieve tubes of different conductivities. However, most of the observed profile spreading may be reasonably attributed to the combined effects of those two factors.The mass average velocity of translocation (calculated from the mean times of (14)C arrival at successive sink leaves) was about 75% of the maximum velocity (calculated from the times of initial detection at the same sink leaves), which was usually between 0.6 and 1 cm min(-1). Owing to tracer exchange between sieve elements and companion cells, the mass average velocity of tracer in the sieve tubes was probably closer to 86% of the maximum velocity, a figure which agreed with a predicted velocity distribution based on calculated sieve tube conductivities and the size distribution of functional sieve tubes. PMID- 16660278 TI - Source pool kinetics for C-photosynthate translocation in morning glory and soybean. AB - The kinetic behavior of translocation profiles indicates that their shape is determined largely by the rate at which tracer enters the sieve tubes in the source leaf. Confirmation of this relationship was sought by investigating the kinetics of (14)C in the immediate source pool for translocated sucrose in soybean (Glycine max L., cv. Bragg) and morning glory (Ipomea nil Roth, cv. Scarlet O'Hara) leaves. Quantitative microautoradiography was used to follow the water-soluble (14)C contents of the companion cells in minor veins after pulse labeling with (14)CO(2). In both morning glory and soybean, the observed kinetics in the companion cells matched reasonably well those expected from the shape of the translocation profiles.Marked compartmentation of sucrose was evident in soybean leaves in that the specific radioactivity of total leaf sucrose was greatest immediately after labeling and quickly declined, whereas labeling in the companion cells was low at first and did not reach a maximum for about 35 minutes. In morning glory leaves, the kinetics of sucrose specific radioactivity and of companion cell-labeling more closely paralleled one another. PMID- 16660279 TI - Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts. AB - Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-d ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-beta-d ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9 beta-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2 butenylamino)-9-beta-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2 butenylamino)-2-methylthio-9-beta-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation. PMID- 16660280 TI - Does the Induction of Flowering by Photoperiod Change the Polarity or Other Characteristics of Indole-3-acetic Acid Transport in Petioles for the Short Day Plant, Xanthium? AB - To test the hypothesis that photoinduction acts by changing the ability of the plant to transport hormones, rather than by changing the ability of organs to synthesize them, the transport of carboxy-labeled indole-3-acetic acid was measured in the short day plant Xanthium pensylvanicum. Plants grown under noninductive conditions were matched for developmental stage, then assigned by a mathematically random method to either short day or noninductive conditions of "short day + light break." After the plants had been subjected to one to seven cycles, the movement of auxin was followed through sections cut from the middle of petioles of various ages. Photoinduction, even with as many as seven cycles, had no effect on auxin movement in either the basipetal or acropetal direction. Auxin movement in vegetative Xanthium was similar to that in Coleus and Phaseolus: strongly polar in a basipetal direction through younger petioles, but with polarity declining with increasing petiole age and concomitant decreasing elongation. PMID- 16660281 TI - In Situ Estimates of Variable Plant Resistance to Water Flux in Ilex opaca Ait. AB - Xylem pressure potentials and stomatal diffusion resistances were measured in the field in Ilex opaca Ait. during days which differed in temperature and vapor pressure deficit. Water flux into leaves was calculated by combining the field data with laboratory determinations of the relation between tissue water deficit and water potential. Estimates of apparent plant resistance were then calculated from fluxes and differences between soil water potential and xylem tension. The resistance depended strongly on water flux, dropping by a factor of over 7 from low to high water flow rates. This extends the generality of variable plant resistances measured in controlled environment studies to I. opaca as it occurs naturally in the field. The relation of apparent plant resistance to water flux as estimated in this study can be useful in simulation models which calculate water uptake to leaves as a flux driven by a difference in soil and leaf water potentials across a resistance between the bulk soil and the leaf. PMID- 16660282 TI - De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls. AB - Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin. PMID- 16660283 TI - Distribution of incorporated, synthetic cytokinins in ribosomal RNA preparations from tobacco callus. AB - The distribution of incorporated synthetic cytokinins (N(6)-[8 (14)C]benzyladenine ([8-(14)C]bzl(6)Ade) and N(6)[8-(14)C]furfuryladenine ([8 (14)C]fr(6)Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-(14)C]bzl(6)Ade or [8 (14)C]fr(6)Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8 (14)C]bzl(6)A or [8-(14)C]fr(6)A in the rRNA peak after the fractionations. [8 (14)C]fr(6)A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.The distribution of [8-(14)C]fr(6)A moieties in mono- and oligonucleotides derived from combined treatment of [8-(14)C]fr(6)Ade-labeled rRNA with ribonuclease T(1) and pancreatic ribonuclease A was measured by fractionating the digest on a DEAE-cellulose column in the presence of 7 m urea and determining the [8-(14)C]fr(6)A content in each fraction. The [8-(14)C]fr(6)A content in the oligonucleotides varied from 46 to 210 mumol/mol of adenosine (A). The mol ratio of [8-(14)C]fr(6)A to A was three to four times greater for oligonucleotides containing uridine or cytidine ([A](n)Up or [A](n)Cp) than for those containing quanosine ([A](n)Gp). PMID- 16660284 TI - Cation pretreatment effects on nitrate uptake, xylem exudate, and malate levels in wheat seedlings. AB - Week-old wheat seedlings absorbed at least 40% NO(3) (-) from NaNO(3) when preloaded with K(+) than when preloaded with Na(+) or Ca(2+). Cultures of Triticum vulgare L. cv. Arthur were grown for 5 days on 0.2 mm CaSO(4), pretreated for 48 hours with either 1 mm CaSO(4), K(2)SO(4), or Na(2)SO(4), and then transferred to 1 mm NaNO(3). All solutions contained 0.2 mm CaSO(4). Shoots of K(+)-preloaded plants accumulated three times more NO(3) (-) than shoots of the other two treatments. Initially, the K(+)-preloaded plants contained 10-fold more malate than either Na(+)- or Ca(2+)-preloaded seedlings. During the 48-hour treatment with NaNO(3), malate in both roots and shoots of the K(+)-preloaded seedlings decreased. Seedlings preloaded with K(+) reduced 25% more NO(3) (-) than those preloaded with either Na(+) or Ca(2+). These experiments indicate that K(+) enhanced NO(3) (-) uptake and reduction even though the absorption of K(+) and NO(3) (-) were separated in time. Xylem exudate of K(+)-pretreated plants contained roughly equivalent concentrations of K(+) and NO(3) (-), but exudate from Na(+) and Ca(2+)-pretreated plants contained two to four times more NO(3) ( ) than K(+). Therefore K(+) is not an obligatory counterion for NO(3) (-) transport in xylem. PMID- 16660285 TI - Responses of succulents to plant water stress. AB - Experiments were performed to test the hypothesis that succulents "shift" their method of photosynthetic metabolism in response to environmental change. Our data showed that there were at least three different responses of succulents to plant water status. When plant water status of Portulacaria afra (L.) Jacq. was lowered either by withholding water or by irrigating with 2% NaCl, a change from C(3) photosynthesis to Crassulacean acid metabolism (CAM) occurred. Fluctuation of titratable acidity and nocturnal CO(2) uptake was induced in the stressed plants. Stressed Peperomia obtusifolia A. Dietr. plants showed a change from C(3) photosynthesis to internal cycling of CO(2). Acid fluctuation commenced in response to stress but exogenous CO(2) uptake did not occur. Zygocactus truncatus Haworth plants showed a pattern of acid fluctuation and nocturnal CO(2) uptake typical of CAM even when well irrigated. The cacti converted from CAM to an internal CO(2) cycle similar to Peperomia when plants were water-stressed. Reverse phase gas exchange in succulents results in low water loss to carbon gain. Water is conserved and low levels of metabolic activity are maintained during drought periods by complete stomatal closure and continual fluctuation of organic acids. PMID- 16660286 TI - Quaking and Gas Exchange in Leaves of Cottonwood (Populus deltoides, Marsh.). AB - Cottonwood (Populus deltoides, Marsh.) leaves are amphistomatous and have an adaptation in their petiole which allows them to oscillate in wind. A possible function of these oscillations in enhancing gas exchange was studied.Cottonwood leaves were found to oscillate in the presence of wind velocities frequently encountered in nature. A pressure differential across the leaf was shown to result in bulk flow of air through that leaf. Oscillating a cottonwood leaf at frequencies found to occur in nature was found to increase the rate of O(2) flux through the leaf. The measured changes in boundary layer resistances during oscillations were found to be insufficient to account for the increased O(2) flux. Thus, the bulk flow of air through an oscillating cottonwood leaf results in a decreased total resistance which is typically 25% less than that of a still leaf. PMID- 16660287 TI - Effects of Various Rates of Freezing on the Metabolism of a Drought-tolerant Plant, the Moss Tortula ruralis. AB - The response of the drought-tolerant moss Tortula ruralis ([Hedw.] Gaertn., Meyer, Scherb.) to freezing and thawing at controlled rates has been studied. Slow freezing (at 3 C per hour to -30 C) of hydrated T. ruralis leads to only temporary, reversible changes in metabolism. These changes can be considered to result from desiccation due to extracellular ice formation. In contrast, rapid freezing in liquid N(2) and thawing in 20 C water leads to deterioration in all aspects of metabolism studied: ribosome, protein, and ATP levels decrease, and in vivo and in vitro protein synthetic activity is lost rapidly. Such changes probably result from intracellular ice formation. Following freezing and thawing at an intermediate rate (60 C per hour), only ATP levels and in vivo protein synthesis are reduced. The protein-synthesizing apparatus (the polyribosomes) remains intact and active in an in vitro protein-synthesizing system even 24 hours after one 60 C per hour freeze-thaw cycle. These metabolic responses are discussed in terms of the two-factor hypothesis of Mazur et al. (1972 Exp. Cell Res. 71: 345-355). PMID- 16660288 TI - Development and properties of a wax ester hydrolase in the cotyledons of jojoba seedlings. AB - The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent K(m) value for N-methylindoxylmyristate was 93 muM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax). PMID- 16660289 TI - Regulation of Sulfate Assimilation in Plants: 7. Cysteine Inactivation of Adenosine 5'-Phosphosulfate Sulfotransferase in Lemna minor L. AB - When 0.5 mm cysteine is added to cultures of Lemna minor L. growing with sulfate as the sole sulfur source, there is a rapid 80% loss of extractable adenosine 5' phosphosulfate sulfotransferase. This loss is accompanied by an inhibition of sulfate uptake; however, lack of sulfate is not responsible for the decreasing adenosine 5'-phosphosulfate sulfotransferase activity.Cultivation with cysteine causes an increase in the cyst(e)ine pool of L. minor. This fact taken together with the observed inactivation of adenosine 5'-phosphosulfate sulfotransferase in crude extracts by cysteine suggests that the cysteine pool is involved in the in vivo regulation of the enzyme.The activity of adenosine 5'-phosphosulfate sulfotransferase is restored within 24 hours after transfer to a culture medium without cysteine. This restoration is partially blocked by 6-methyl purine and actinomycin D and completely by cycloheximide.Cycloheximide added to cultures of L. minor L. causes a loss of extractable APSTase comparable to the one obtained with cysteine. This loss may be in part due to cysteine, since cycloheximide causes a pronounced increase in the cysteine pool of L. minor. PMID- 16660290 TI - Shoot, Stolon, and Tuber Formation on Potato (Solanum tuberosum L.) Cuttings in Response to Photoperiod. AB - The intensity of "tuberization stimulus" in potato shoots (Solanum tuberosum L.) can be assessed from cuttings containing one or more leaves. Cuttings maintained in a mist chamber under long days will form tubers from underground buds if prior to taking the cutting the leaves received sufficient exposure to photoperiods less than the critical photoperiod. The greatest tendency to tuberize was found in cuttings that consisted of a single, fully expanded leaf and its subtended bud. Grafts showed that genetical differences in critical photoperiod resided in properties of the leaf. Short days before cutting tended to shift growth from above ground buds of two-node cuttings to below ground buds, even if the number of short days was insufficient for tuber induction. As few as 6 short days reduced growth of shoots at the upper bud and increased underground growth of shoots and stolons. PMID- 16660291 TI - Partial characterization of steryl ester biosynthesis in spinach leaves. AB - Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4 (14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm. PMID- 16660292 TI - Subcellular distribution of steryl ester biosynthesis in spinach leaves. AB - Higher steryl ester biosynthetic activities were obtained with Triton X-100 phosphatidylcholine-cholesterol mixed micelles than with Tween 80 phosphatidylcholine-cholesterol mixed micelles when incubated with spinach leaf (Spinacia oleracea L.) acetone powder preparations. The best incorporation of [4 (14)C]cholesterol into [4-(14)C]cholesteryl ester was obtained with a Triton X 100-phosphatidylcholine-cholesterol (10:1:1, w/w) mixed micelle system. This mixed micelle system, however, required 1,2-dipalmitin and fatty acid-free bovine albumin for optimal activity. The reaction exhibited a diglyceride specificity since the dipalmitin requirement could be replaced with neither 1-monopalmitin nor tripalmitin. Significant amounts of steryl ester biosynthetic activity were detected in the chloroplast (1,000g pellet), mitochondrial (3,000g pellet), and microsomal (20,000g and 88,000g pellet) fractions. Little activity was detected in the water-soluble (88,000g supernatant) fraction. The highest specific activity occurred in the 88,000g pellet. The 88,000g supernatant contained a heatstable, water-soluble substance that was required for optimal steryl ester biosynthesis in all of the pellet fractions. This factor was not lost during extensive dialysis but was destroyed by ashing, indicating that it was large and organic. Silver nitrate thin layer chromatography indicated that 60% of the biosynthesized steryl esters contained saturated fatty acids in the absence of 1,2-dipalmitin and that 83% contained saturated fatty acids in the presence of 1,2-dipalmitin. PMID- 16660293 TI - Presence of Chloride Reduces Malate Production in Epidermis during Stomatal Opening. AB - When stomata of isolated epidermis of Vicia faba are allowed to open in the presence of K(+) and iminodiacetate (an impermeant zwitterion), malate is formed in the epidermis; the increases in malate content follow a nearly linear relationship with stomatal aperture. Stomata of leaf sections of V. faba floated on water during opening also exhibit this relationship. When isolated epidermis is offered KCI, this relationship is not observed and less malate is detected at comparable stomatal apertures. The data indicate that Cl(-), if present at concentrations >/= 10(-5) eq liter(-1), can partially satisfy the anion requirement of guard cells of V. faba during stomatal opening. Discrepancies between earlier reports on the relative roles Cl(-) and malate play as counterions for K(+) in guard cells of V. faba could now be explained as resulting from variations in the availability of Cl(-) to guard cells. PMID- 16660294 TI - On the mechanism of aging in soybean seeds. AB - Changes in seeds of soybeans (Glycine max [L.] Merr. var. Wayne) which occur during accelerated aging (41 C, 100% relative humidity) showed subsequent loss of vigor, a decline in early respiratory activity, increased leakage of electrolytes, losses of as much as 10% dry weight from imbibing cotyledons, and a decrease in the swelling response of the imbibing system (seed plus H(2)O). Each of these changes with aging is interpreted as resulting from deteriorative changes in membranes. PMID- 16660295 TI - Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro. AB - Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction ("binding fraction") which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP(+) causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD(+) and NADP(+) can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of "surplus" nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme. PMID- 16660296 TI - Low temperature spectral properties of subchloroplast fractions purified from spinach. AB - Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F(I), F(II), and F(III), corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at -196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F(II) (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at -196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F(I), F(II), and F(III) at -196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F(I) showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F(II) and F(III) in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F(I) and F(II) caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at -196 C with an increase in the yield of fluorescence especially in the case of F(I). PMID- 16660297 TI - Source and sink leaf metabolism in relation to Phloem translocation: carbon partitioning and enzymology. AB - The import-export transition in sugar beet leaves (Beta vulgaris) occurred at 40 to 50% leaf expansion and was characterized by loss in assimilate import and increase in photosynthesis. The metabolism and partitioning of assimilated and translocated C were determined during leaf development and related to the translocation status of the leaf. The import stage was characterized by C derived from either (14)C-translocate or (14)C-photosynthate being incorporated into protein and structural carbohydrates. Marked changes in the C partitioning were temporally correlated with the import-export conversion. Exporting leaves did not hydrolyze accumulated sucrose and the C derived from CO(2) fixation was preferentially incorporated into sucrose. Both source and sink leaves contained similar levels of acid invertase and sucrose synthetase activities (sucrose hydrolysis) while sucrose phosphate synthetase (sucrose synthesis) was detected only in exporting leaves. The results are discussed in terms of intracellular compartmentation of sucrose and sucrose-metabolizing enzymes in source and sink leaves. PMID- 16660298 TI - Effects of Mechanical Stimulation on Avena Coleoptile Segment Elongation in a High Resolution, Continuous Growth-recording System. AB - The effects of an abrasive mechanical stimulation of the inner epidermal surfaces of excised Avena coleoptile segments were examined in relation to growth in the presence and absence of exogenously supplied indole-3-acetic acid. Mechanical stimulation of this nature, provided immediately following excision, was found to elicit a small, transient increase in endogenous growth rate which contributed to a larger initial rapid growth response (previously referred to as a tactile response). These results, contrary to the earlier reports, suggest that the inner epidermal mechanical or tactile stimulation does not account for the entire initial rapid growth response. Preliminary experiments indicate that an alternative form of mechanical stimulation (segment excision) may contribute to that portion of initial rapid growth which is not attributable to inner epidermal abrasion.Following its initial growth-enhancing effect, inner epidermal stimulation had either no effect or in some cases appeared inhibitory to endogenous growth. Growth in response to exogenous auxin was appreciably inhibited by this form of mechanical stimulation. PMID- 16660299 TI - Purification and properties of sucrose synthase from maize kernels. AB - Sucrose synthase was purified from 22-day-old maize (Zea mays L.) kernels to homogeneity by the successive steps of ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and affinity chromatography on a UDP hexanol-amino-agarose column. The degree of purification is 42-fold and the yield is over 80%. Polyacrylamide gel electrophoretic techniques, sedimentation velocity, and gel filtration studies revealed that the enzyme has identical subunits and could assume tetrameric, octameric, and other higher aggregated forms which are dependent on the ionic species and ionic strength of the solution. All of the enzyme forms exhibit catalytic activity but show differences in their specific activities. In most cases, the tetramer is the predominant form and has the highest specific activity. It is thus concluded that the tetramer could be the native form of the enzyme. The subunit protein has a molecular weight of 88,000 and a blocked NH(2) terminus which is not available to Edman degradation. Some general properties and the amino acid composition of the enzyme are also reported. PMID- 16660300 TI - Effects of Sink Removal on Photosynthesis and Senescence in Leaves of Soybean (Glycine max L.) Plants. AB - Photosynthetic rate, ribulose 1,5-bisphosphate carboxylase activity, specific leaf weight, and leaf concentrations of carbohydrates, proteins, chlorophyll, and inorganic phosphate were determined periodically from midbloom until maturity in leaves of soybean plants (Glycine max L., var. Hodgson) from which reproductive and vegetative sinks had been removed 32 hours before measurement, or continuously since midbloom.Leaf photosynthesis, measured in the top of the canopy, was partially inhibited by both sink removal treatments. This inhibition was of constant magnitude from midbloom until maturity.Leaf photosynthesis in the top of the canopy declined from midbloom until maturity in the control as well as in the desinked plants. The decline in photosynthesis was gradual at first, but later became more abrupt. The photosynthetic decline was equally evident in the yellowing leaves of control plants and in the dark green leaves of the continuously desinked plants.Neither the inhibition of photosynthesis by sink removal nor the decline in photosynthetic rate with time was clearly related to any of the measured traits. PMID- 16660301 TI - Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation. AB - The interaction between the ATP-dependent evolution of H(2) catalyzed by nitrogenase and the oxidation of H(2) via a hydrogenase has been postulated to influence the efficiency of the N(2)-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H(2) in air and the H(2) produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N(2) fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H(2) in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C(2)H(2) reduction and O(2) consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different.When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N(2) fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H(2)-evolving strain 176A27 produced increases in plant dry weight and total N(2) fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N(2) fixation for nodulated legumes capable of reutilizing the H(2) evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H(2)-recycling process in N(2)-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase. PMID- 16660302 TI - Effects of Irradiance during Growth on Adaptive Photosynthetic Characteristics of Velvetleaf and Cotton. AB - We grew velvetleaf (Abutilon theophrasti Medic.) and cotton (Gossypium hirsutum L. var. Stoneville 213) at three irradiances and determined the photosynthetic responses of single leaves to a range of six irradiances from 90 to 2000 mueinsteins m(-2)sec(-1). In air containing 21% O(2), velvetleaf and cotton grown at 750 mueinsteins m(-2)sec(-1) had maximum photosynthetic rates of 18.4 and 21.9 mg of CO(2) dm(-2)hr(-1), respectively. Maximum rates for leaves grown at 320 and 90 mueinsteins m(-2)sec(-1) were 15.3 and 10.3 mg of CO(2) dm(-2)hr(-1) in velvetleaf and 12 and 6.7 mg of CO(2) dm(-2)hr(-1) in cotton, respectively. In 1 O(2), maximum photosynthetic rates were 1.5 to 2.3 times the rates in air containing 21% O(2), and plants grown at medium and high irradiance did not differ in rate. In both species, stomatal conductance was not significantly affected by growth irradiance. The differences in maximum photosynthetic rates were associated with differences in mesophyll conductance. Mesophyll conductance increased with growth irradiance and correlated positively with mesophyll thickness or volume per unit leaf area, chlorophyll content per unit area, and photosynthetic unit density per unit area. Thus, quantitative changes in the photosynthetic apparatus help account for photosynthetic adaptation to irradiance in both species. Net assimilation rates calculated for whole plants by mathematical growth analysis were closely correlated with single-leaf photosynthetic rates. PMID- 16660303 TI - Photosynthetic Acclimation to Temperature in the Desert Shrub, Larrea divaricata: I. Carbon Dioxide Exchange Characteristics of Intact Leaves. AB - Larrea divaricata, a desert evergreen shrub, has a remarkable ability to adjust its photosynthetic temperature response characteristics to changing temperature conditions. In its native habitat on the floor of Death Valley, California, plants of this C(3) species when provided with adequate water are able to maintain a relatively high and constant photosynthetic activity throughout the year even though the mean daily maximum temperature varies by nearly 30 C from winter to summer. The temperature dependence of light-saturated net photosynthesis varies in concert with these seasonal temperature changes whereas the photosynthetic rate at the respective optimum temperatures shows little change.Experiments on plants of the same age, grown at day/night temperatures of 20/15, 35/25, and 45/33 C with the same conditions of day length and other environmental factors, showed a similar photosynthetic acclimation response as observed in nature. An analysis was made of a number of factors that potentially can contribute to the observed changes in the temperature dependence of net CO(2) uptake at normal CO(2) and O(2) levels. These included stomatal conductance, respiration, O(2) inhibition of photosynthesis, and nonstomatal limitations of CO(2) diffusive transport. None of these factors, separately or taken together, can account for the observed acclimation responses. Measurements under high saturating CO(2) concentrations provide additional evidence that the observed adaptive responses are primarily the result of changes in intrinsic characteristics of the photosynthetic machinery at the cellular or subcellular levels. Two apparently separate effects of the growth temperature regime can be distinguished: one involves an increased capacity for photosynthesis at low, rate limiting temperatures with decreased growth temperature, and the other an increased thermal stability of key components of the photosynthetic apparatus with increased growth temperature. PMID- 16660304 TI - Photosynthetic Acclimation to Temperature in the Desert Shrub, Larrea divaricata: II. Light-harvesting Efficiency and Electron Transport. AB - The response of photosynthetic electron transport and light-harvesting efficiency to high temperatures was studied in the desert shrub Larrea divaricata Cav. Plants were grown at day/night temperatures of 20/15, 32/25, or 45/33 C in rough approximation of natural seasonal temperature variations. The process of acclimation to high temperatures involves an enhancement of the stability of the interactions between the light-harvesting pigments and the photosystem reaction centers. As temperature is increased, the heat-induced dissociation of these complexes results in a decrease in the quantum yield of electron transport at limiting light intensity, followed by a loss of electron transport activity at rate-saturating light intensity. The decreased quantum yield can be attributed to a block of excitation energy transfer from chlorophyll b to chlorophyll a, and changes in the distribution of the excitation energy between photosystems II and I. The block of excitation energy transfer is characterized by a loss of the effectiveness of 480 nm light (absorbed primarily by chlorophyll b) to drive protochemical processes, as well as fluorescence emission by chlorophyll b. PMID- 16660305 TI - Cellulase activity and fruit softening in avocado. AB - Cellulase activity in detached avocado (Persea americana Mill.) fruits was found to be directly correlated with ripening processes such as climacteric rise of respiration, ethylene evolutin, and softening. This activity in the pericarp could be induced by ethylene treatment, and the more mature the fruit-the faster and the greater was the response. Only a very low cellulase activity could be detected in hard avocado fruit right after harvest. Cellulase activity was highest at the distal end of the fruit, lower in the midsection, and lowest at the proximal end. The enzyme is heat-labile and appeared to have activity of an endocellulase nature mainly. Electron micrographs of cell walls from hard and soft fruits are presented. PMID- 16660306 TI - Cytoplasm-specific Effects of Helminthosporium maydis Race T Toxin on Survival of Corn Mesophyll Protoplasts. AB - High yields of mesophyll protoplasts were obtained from leaves of corn (Zea mays L., inbred W64A). Many protoplasts survived a week in the dark in a simple osmoticum. Culture filtrate from Helminthosporium maydis race T at dilutions of 1:10,000 to 1:20,000 destroyed protoplasts with Texas male-sterile (T) cytoplasm. Substantial damage to protoplasts with nonmale-sterile (N) cytoplasm occurred only at a 1:20 dilution. High concentrations of partially purified H. maydis race T (HMT) toxin (32.5-130 mug dry weight/ml) did not reduce survival of protoplasts with N cytoplasm or C or S male-sterile cytoplasms after 6 days of exposure. Protoplasts with T or TRf (fertility restored) cytoplasm collapsed within 1 to 3 days after treatment with 0.13 mug of HMT toxin/ml, which was one-fifth the level causing 50% inhibition of T cytoplasm seedling root growth. Protoplasts with T cytoplasm which were washed after 30 minutes or more of exposure to HMT toxin also collapsed within a few days. Cultured W64A T protoplasts and freshly isolated protoplasts from inbreds C103 and Mo17 with T cytoplasm were less sensitive to HMT toxin than freshly isolated W64A T protoplasts. Toxin-treated protoplasts survived longer in the light than in the dark. The sensitivity and specificity of the system described will facilitate physiological, ultrastructural, and genetic studies of toxin action. PMID- 16660307 TI - Movement of Indole-3-acetic Acid and Tryptophan-derived Indole-3-acetic Acid from the Endosperm to the Shoot of Zea mays L. AB - The structures and the concentrations of all of the indolylic compounds that occur in the endosperm of the seeds of corn (Zea mays L.) are known. Thus, it should be possible to determine which, if any, of the indolylic compounds of the endosperm can be transported to the seedling in significant amounts and thus help identify the seed-auxin precursor of Cholodny (1935. Planta 23:289-312) and Skoog (1937. J. Gen. Physiol. 20:311-334). Of interest is the transport of tryptophan, indole-3-acetic acid (IAA), and the esters of IAA, which comprise 95% of the IAA compounds of the seed. We have shown that: (a) IAA can move from the endosperm to the shoot; (b) the rate of movement of IAA from endosperm to shoot is that of simple diffusion; (c) 98% of the transported IAA is converted into compounds other than IAA, or IAA esters, en route; (d) some of the IAA that has moved into the shoot has been esterified; (e) labeled tryptophan applied to the endosperm can be found as labeled IAA in the shoot; and (f) with certain assumptions concerning IAA turnover, the rate of movement of IAA and tryptophan-derived IAA from the endosperm to shoot is inadequate for shoot growth or to maintain IAA levels in the shoot. PMID- 16660308 TI - Uptake of mercury vapor by wheat: an assimilation model. AB - Using a whole-plant chamber and (203)Hg-labeled mercury, a quantitative study was made of the effect of environmental parameters on the uptake, by wheat (Triticum aestivum), of metallic mercury vapor, an atmospheric pollutant. Factors were examined in relation to their influence on components of the gas-assimilation model, [Formula: see text]where U(Hg) is the rate of mercury uptake per unit leaf surface, C(a)' is the ambient mercury vapor concentration, C(l)' is the mercury concentration at immobilization sites within the plant (assumed to be zero), r(l.Hg) is the total leaf resistance to mercury vapor exchange, and r(m.hg) is a residual term to account for unexplained physical and biochemical resistances to mercury vapor uptake.Essentially all mercury vapor uptake was confined to the leaves. r(l.hg) was particularly influenced by illumination (0 to 12.8 klux), but unaffected by ambient temperature (17 to 33 C) and mercury vapor concentration (0 to 40 mug m(-3)). The principal limitation to mercury vapor uptake was r(m.hg), which was linearly related to leaf temperature, but unaffected by mercury vapor concentration and illumination, except for apparent high values in darkness.Knowing C(a)' and estimating r(l.hg) and r(m.hg) from experimental data, mercury vapor uptake by wheat in light was accurately predicted for several durations of exposure using the above model. PMID- 16660309 TI - Ultraviolet Radiation-stimulated Efflux of 86-Rubidium from Cultured Tobacco Cells. AB - Ultraviolet (254 nm) radiation stimulated the efflux of (86)Rb(+) from liquid cultured tobacco (Nicotiana tabacum) cells; it did not stimulate the movement of mannitol or 2-deoxyglucose. These results indicate that the efflux of (86)Rb(+) is not to a generalized disruption of membrane structure. PMID- 16660310 TI - Thermoperiodism in cocklebur seed germination. AB - Germination potential in nondormant, upper cocklebur (Xanthium pensylvanicum Wallr.) seeds, which were incapable of germinating under constant temperatures below 25 C in air, was increased by exposure to diurnally alternating temperatures. The cocklebur seeds failed to respond to the temperature fluctuations in the beginning of water imbibition, and their responsiveness appeared only after aerobic presoaking for a limited period or after anaerobic pretreatment for 1 to 3 days.Maximal germination was obtained after exposure to a thermoperiodic regime of 8 hours at 23 C and 16 hours at 8 C. A process occurring during the high temperature phase was aerobic and had to precede the inductive low temperature phase, its effect increasing with temperature. Critical minimum length of the inductive low temperature phase changed with the duration of a preceding anaerobiosis, for instance about 4 hours after 1 day anaerobiosis, but about 2 hours after 2 days. Percentage of subsequent germination was in proportion to the number of thermoperiodic cycles. A process of the inductive low temperature phase was not perturbed by inserting a brief higher temperature period into its phase; indeed, such insertion rather increased germination potential when performed in the earlier parts of the inductive low temperature phase. The effect of the low temperature survived for 13 to 17 hours during the higher temperature period. PMID- 16660311 TI - Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants. AB - A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl(2) for maximum activity but was inhibited by MnCl(2); its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K(m) values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 mum and 11 mum; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5'-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5'-phosphate and 5-fluorouridine 5'-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides. PMID- 16660312 TI - Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: I. Characterization of the Response. AB - A rapidly induced, transitory increase in the rate of ethylene synthesis occurred in wounded tissue excised from actively growing regions of etiolated barley, cucumber, maize, oat, pea, tomato, and wheat seedlings. Cutting intact stems or excising 9-mm segments of tissue from near the apex of 7-day-old etiolated Pisum sativum L., cv. Alaska seedlings induced a remarkably consistent pattern of ethylene production. At 25 C, wound-induced ethylene production by segments excised 9 mm below the apical hook increased linearly after a lag of 26 minutes from 2.7 nanoliters per g per hour to the first maxium of 11.3 nanoliters per g per hour at 56 minutes. The rate of production then decreased to a minimum at 90 minutes, increased to a lower second maximum at 131 minutes, and subsequently declined over a period of about 100 minutes to about 4 nanoliters per g per hour. Removal of endogenous ethylene, before the wound response commenced, had no effect on the kinetics of ethylene production. Tissue containing large amounts of dissolved ethylene released it as an exponential decay with no lag period. Rapidly induced wound ethylene is synthesized by the tissue and is not merely the result of facilitated diffusion of ethylene already present in the tissue through the newly exposed cut surfaces. Previously wounded apical sections did not exhibit a second response when rewounded. No significant correlation was found between wound-induced ethylene synthesis and either CO(2) or ethane production. PMID- 16660313 TI - Subcellular Localization of Glycosyl Transferases Involved in Glycoprotein Biosynthesis in the Cotyledons of Pisum sativum L. AB - Subcellular membrane fractions from 21-day-old pea (Pisum sativum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of lipid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.Other mannosyl transferases are found at a density of 1.123 g/cc and 1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis. PMID- 16660314 TI - Physicochemical characterization of mitochondrial DNA from soybean. AB - Mitochondrial DNA (mtDNA) of soybean (Glycine max L.) was isolated and its buoyant density was contrasted with that of nuclear (nDNA) and chloroplast (ctDNA) DNA. Each of the three DNAs banded at a single, characteristic buoyant density when centrifuged to equilibrium in a CsCl gradient. Buoyant densities were 1.694 g/cm(3) for nDNA and 1.706 g/cm(3) for mtDNA. These values correspond to G-C contents of 34.7 and 46.9%, respectively. Covalently closed, circular mtDNA molecules were isolated from soybean hypocotyls by ethidium bromide-cesium chloride density gradient centrifugation. Considerable variation in mtDNA circle size was observed by electron microscopy. There were seven apparent size classes with mean lengths of 5.9 mum (class 1), 10 mum (class 2), 12.9 mum (class 3), 16.6 mum (class 4), 20.4 mum (class 5), 24.5 mum (class 6), and 29.9 mum (class 7). In addition, minicircles were observed in all preparations. Partially denatured, circular mtDNA molecules with at least one representative from six of the seven observed size classes were mapped. In class 4, there appear to be at least three distinct denaturation patterns, indicating heterogeneity within this class. It is proposed that the mitochondrial genome of soybean is distributed among the different size circular molecules, several copies of the genome are contained within these classes and that the majority of the various size molecules may be a result of recombination events between circular molecules. PMID- 16660315 TI - Free flow electrophoresis of chloroplasts. AB - Highly purified intact chloroplasts were isolated from spinach (Spinacia oleracea L.) leaves by free flow electrophoresis. Morphological and biochemical studies showed that the fraction enriched in intact chloroplasts has a higher protein to chlorophyll ratio and a higher linolenic acid content than the broken organelles of the other fraction. The intact chloroplasts prepared by electrophoresis retained their capacity for CO(2) fixation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that this fraction was rich in stroma and lamellae proteins. Free flow electrophoresis, which separates organelles and molecules according to their surface charges, is a good technique for producing purified chloroplasts with complete physiological activities. PMID- 16660316 TI - Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoe. AB - When intact Kalanchoe plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose 1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoe. PMID- 16660317 TI - Relationship between Chloroplast Membrane Fatty Acid Composition and Photosynthetic Response to a Chilling Temperature in Four Alfalfa Cultivars. AB - The photosynthetic responses of four alfalfa (Medicago sativa L.) cultivars to 10 and 22 C air temperatures were examined and the relationship between the photosynthetic response at 10 C and the fatty acid composition of the chloroplast membranes was determined. Chilling-resistant cultivars exhibited moderate reductions in photosynthesis at 10 C, compared to 22 C, and contained a significantly greater percentage of polyunsaturated fatty acids in the chloroplast membrane and a greater double bond index than the chilling-sensitive cultivars. The chilling-sensitive cultivars exhibited severe reductions in photosynthesis at 10 C, compared to 22 C. The reduction in photosynthesis at 10 C is shown to be negatively correlated (r = -0.94) with the double bond index of the chloroplast membranes of the cultivars observed.The results support the hypothesis that reduced photosynthesis due to chilling temperatures is influenced by the unsaturated fatty acid composition of the chloroplast membrane which affect temperature-induced phase changes in chloroplast membrane lipids. PMID- 16660318 TI - Release of Malate from Epidermal Strips during Stomatal Closure. AB - Isolated epidermal strips of Vicia faba and Commelina communis release malate into their bathing medium when stomata close. This release was largest (about 0.6 of the initial malate content) when epidermal strips of C. communis were floated on 10(-5) M (+/-)-abscisic acid. PMID- 16660319 TI - Malate synthesis by dark carbon dioxide fixation in leaves. AB - The rates of dark CO(2) fixation and the label distribution in malate following dark (14)CO(2) fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoe diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark (14)CO(2) fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO(2) at rates of 1.4, 3.4, 0.23, and 1.0 mumoles of CO(2)/mg of chlorophyll. hour, respectively. Net CO(2) fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO(2) for the duration of the 23-hour experiment.A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the beta-carboxyl (C(4)) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C(4) as much as 15 to 20%.The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO(2) fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoe plantlets. In addition, Kalanchoe leaves fix (14)CO(2) more rapidly than Kalanchoe plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls. PMID- 16660320 TI - Influence of Excision and Aging upon K Influx into Barley Roots: Recovery or Enhancement? AB - The influx of K(+) from (86)Rb-labeled solutions in the concentration range 0.008 to 0.2 mm into roots of intact plants and excised roots of barley plants (Hordeum vulgare [L.]) previously grown in 5 mm CaSO(4) (low K(+) roots) or 0.5 mm CaSO(4) plus 5 mm KCl (high K(+) roots) was measured. A consistent observation of these experiments was a substantial reduction of influx (usually by about 50%) following excision. The possible leakage of K(+) into the medium and subsequent dilution of specific activity of labeled solutions was eliminated as an explanation for influx reduction in excised low K(+) roots. Reduction of transpirational rates was also without effect upon influx into low K(+) roots. Excision followed by 2 hours aging in 0.5 mm CaSO(4) solution revealed that influx values recovered within the 2 hours to the values obtained in intact roots. It is concluded that much of the literature which describes the enhancement of ion uptake following excision actually describes excision damage followed by recovery. PMID- 16660321 TI - Effect of Growth Temperature on the Fatty Acid Composition of the Leaf Lipids in Atriplex lentiformis (Torr.) Wats. AB - Plants of Atriplex lentiformis had more saturated leaf lipids when grown at 43 C day/30 C night as compared to 23/18 C temperatures. In monogalactosyl diglyceride, the major change was the presence of hexadecatrienoic acid (16:3) at low but not high growth temperatures. In other lipids investigated, the major change was a decrease in linolenic acid (18:3) and increases in the more saturated fatty acids at high growth temperatures. Growth temperatures had little effect on the relative proportions of the galacto- and sulfolipids in the leaf. The increased lipid saturation is correlated with the greater thermostability of the photosynthetic apparatus at high growth temperatures in A. lentiformis but any cause and effect relationship is uncertain. PMID- 16660322 TI - HCO(3) Influx across the Plasmalemma of Chara corallina: Physiological and Biophysical Influence of 10 mm K. AB - The effect of 10 mm K(+) on the HCO(3) (-) influx in Chara corallina has been used to distinguish a Ca(2+)-dependent membrane integrity site from the HCO(3) ( ) transport site which is also Ca(2+)-dependent (Lucas and Dainty, Plant Physiology 1977 60: 862-867).In the presence of 0.2 mm Ca(2+) at pH 9, 10 mm K(+) inhibits the HCO(3) (-) influx and depolarizes the membrane potential. Inhibition of the HCO(3) (-) influx may be prevented by raising the Ca(2+) concentration in the solution or by addition of Mg(2+), Sr(2+), or Mn(2+). Protection is also afforded by 20 mm Na(+) and Cs(+) but not by Rb(+) which acts as a weak analog of K(+) in producing inhibition of the HCO(3) (-) influx and depolarization of the membrane potential. With the exception of Na(+), ions which prevent inhibition of the influx also prevent depolarization of the membrane potential.Once inhibited by 10 mm K(+), HCO(3) (-) transport cannot be restored by addition of Ca(2+), Mg(2+), or Sr(2+) in less than 3 hours. Addition of Mn(2+), however, results in a progressive restoration of the proportion of cells with influxes equal to the control value. Mn(2+) also produces an increase in the membrane potential with a sharp hyperpolarization occurring at a threshold of about-180 mv. This sudden recovery of the HCO(3) (-) influx in individual cells contrasts with the gradual recovery observed when the K(+) concentration is reduced to 0.2 mm. Since Mn(2+) cannot substitute for Ca(2+) at the HCO(3) (-) transport site, restoration of HCO(3) (-) transport by Mn(2+) involving the membrane integrity site, as evidenced by the effect on the membrane potential and resistance, is clearly separate. The other divalent cations were able to restore HCO(3) (-) transport if applied at a concentration of 2 mm for 14 hours.The OH(-) efflux is also inhibited by 10 mm KCl. It is postulated that voltage dependency of the OH(-) or HCO(3) (-) transport systems may account for the observed effects of 10 mm K(+) on the HCO(3) (-) influx. PMID- 16660323 TI - Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.). AB - The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles. PMID- 16660324 TI - Proteases of Senescing Oat Leaves: II. Reaction to Substrates and Inhibitors. AB - Two proteases isolated from senescent oat (Avena sativa) leaves have been subjected to further study. One of these, an acid protease active at pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by iodoacetamide (IAc). The other, active at pH 6.6, is inhibited by both PMSF and IAc. These results, together with previously reported evidence that mercaptoethanol stimulates the activity of only the neutral protease, are taken to indicate that the acid protease is probably of the serine type, whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are inhibited by irradiation in the presence of rose bengal, a selective histidine modification reagent. The acid protease was completely unaffected by chelators, but data on the neutral protease were equivocal.All protein substrates tested were attacked by both enzymes, though at strikingly different rates. Characterization of the digestion products, with denatured hemoglobin as substrate, indicated that the acidic enzyme is an endoprotease, while the neutral one is an exoprotease. Evidence is presented that these proteases undergo autolysis in vitro. PMID- 16660325 TI - Effects of root anaerobiosis on ethylene production, epinasty, and growth of tomato plants. AB - Experiments were performed to determine the source(s) of ethylene-causing epinasty in flooded tomato plants (Lycopersicon esculentum Mill.). Simultaneous measurements were made of ethylene synthesized by the roots and shoots of tomato plants exposed to either aerobic or anaerobic atmospheres in the root zone. When the root zone was made anaerobic by a flowing stream of N(2) gas, petiole epinasty and accelerated ethylene synthesis by the shoots were observed. In soil grown plants, ethylene synthesis by the root-soil complex increased under anaerobic conditions; but when grown in inert media under the same conditions, ethylene synthesis by roots remained constant or declined during the period of rapid epinastic growth by the petioles. Other characteristic symptoms of flooding, e.g. reduced growth and chlorosis, were also observed in plants with anaerobic roots. Pretreatment of plants with AgNO(3), an inhibitor of ethylene action, completely prevented epinasty, demonstrating that ethylene is the agent responsible for waterlogging symptoms. These results indicate that deprivation of O(2) to the roots is the primary effect of soil flooding, and that this is sufficient to cause increased ethylene synthesis in the shoot. The basis of the observed root-shoot communication is unknown, but root-synthesized hormones or specific ethylene-promoting factors may be involved. PMID- 16660326 TI - Resistance Analysis of Nocturnal Carbon Dioxide Uptake by a Crassulacean Acid Metabolism Succulent, Agave deserti. AB - Nocturnal CO(2) uptake by a Crassulacean acid metabolism succulent, Agave deserti Engelm. (Agavaceae), was measured so that the resistance properties of the mesophyll chlorenchyma cells and their CO(2) concentrations could be determined. Two equivalents of acidity were produced at night per mole of CO(2) taken up. The nocturnal CO(2) uptake became light-saturated at 3.5 mEinsteins cm(-2) of photosynthetically active radiation (400-700 nm) incident during the preceding day; at least 46 Einsteins were required per mole of CO(2) fixed. Variations in the daytime leaf temperature between 20 and 37 C had little effect on nocturnal CO(2) uptake. After the first few hours in the dark, the leaf liquid phase CO(2) resistance (r(liq) (CO(2) )) and the CO(2) concentration in the chlorenchyma cells (c(i) (CO(2) )) both increased, the latter usually reaching the ambient external CO(2) level at the end of the dark period. Increasing the leaf surface temperature above 15 C at night markedly increased the stomatal resistance, r(liq) (CO(2) ), and c(i) (CO(2) ).The minimum r(liq) (CO(2) ) at night was about 1.6 seconds cm(-1). Based on the ratio of chlorenchyma surface area to total leaf surface area of 82, this r(liq) (CO(2) ) corresponded to a minimum cellular resistance of approximately 130 seconds cm(-1), comparable to values for mesophyll cells of C(3) plants. The contribution of the carboxylation reaction and/or other biochemical steps to r(liq) (CO(2) ) may increase appreciably as the nighttime temperature shifts a few degrees from the optimum or after a few hours in the dark, both of which caused large increases in r(liq) (CO(2) ). This necessitates a large internal leaf area for CO(2) diffusion into the chlorenchyma to support moderate nocturnal CO(2) uptake rates by these succulent leaves. PMID- 16660327 TI - DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts: Fate of Single stranded Bacteriophage fd DNA. AB - Binding and uptake of exogenous DNA by nuclei isolated from Glycine max L. Merr were studied using (3)H-labeled single-stranded DNA of bacteriophage fd. A comparison of single-stranded with double-stranded DNA for binding and uptake by nuclei was also made.Isolated nuclei were incubated with (3)H-labeled single stranded bacteriophage fd DNA. Poly-l-lysine or DEAE-dextran at 0.1 and 1 mug/ml stimulated DNA binding. On the other hand, poly-l-lysine at 1 and 5 mug/ml increased DNA uptake but DEAE-dextran did not. Ten to 20 mm Ca and Mg ions were required for DNA uptake. EDTA (1-20 mm) did not differ from the control or the low levels of Ca or Mg ions. These observations on single-stranded fd DNA differed from those obtained with double-stranded DNA of Salmonella typhimurium. Kinetics of single-stranded DNA binding and uptake also deviated from that of double-stranded DNA.Analyses of nuclei-bound DNA by sucrose density gradient and CsCI density gradient centrifugation revealed extensive DNA degradation during a 45-minute incubation period. Poly-l-lysine protected against rapid degradation of bound DNA. DEAE-dextran enhanced DNA binding, but bound DNA was cleaved into much smaller polymers than those detected in control experiments or in the presence of poly-l-lysine. Sucrose density gradient and CsCI density gradient centrifugation analyses on DNA taken up by nuclei also showed extensive DNA degradation. Poly-l lysine slightly inhibited DNA degradation, but DEAE-dextran appeared to enhance degradation of incorporated DNA. Moreover, nonbound DNA in the incubation medium was completely degraded within a 20-minute incubation period. PMID- 16660328 TI - Influence of Varied Phosphorus Supply on Growth and Xylem Sap Cytokinin Level of Sycamore (Platanus occidentalis L.) Seedlings. AB - Effects of varying levels of phosphorus (P) on sycamore (Platanus occidentalis L.) growth and on the endogenous cytokinin levels in the xylem sap were tested. The seedlings were grown in aerated nutrient solutions in the greenhouse. Photoperiod was held constant at 16 hours. Four P concentrations, 0.02, 0.10, 0.50, and 2 mm, were used. There were no P deficiency symptoms even at the lowest P concentration. The maximum growth occurred in seedlings grown in 0.10 mm P. The greatest dry matter yield as well as stem height, stem diameter, and leaf area were produced at 0.10 or 0.50 mm P. The amount of cytokinin in xylem sap for each harvest during P nutrition followed closely the dry matter values, stem height, stem diameter, and leaf area. PMID- 16660329 TI - Chilling Stress to Soybeans during Imhibition. AB - Embryos, excised from seed coats of soybeans (Glycine max Merr. cv. ;Wayne'), leak profusely during the first minutes of imbibition. A discontinuity of temperature/leakage patterns occurs between 10 and 15 C; as embryos imbibe at 10 C or lower, disproportionately more solutes leak out per unit of water imbibed. Short periods of imbibition at or below 12 to 14 C reduce embryo germination and axis elongation; injury results from imbibition at 2 C for as little as 5 minutes. Humidifying embryos to 35 to 50% moisture before imbibition reduced leakage during imbibition and imparted some resistance to imbibitional chilling injury.The period of profuse leakage is interpreted as a time of membrane reorganization. Imposing a low temperature during this period prolongs the rapid leakage, suggesting delayed or faulty membrane reorganization. Reduced cold sensitivity of embryos with an initial 35 to 50% moisture content is presumed to be due to at least partial membrane reorganization in the embryo before imbibition. These data collectively are taken to indicate that low temperature interferes with normal membrane reorganization during imbibition, probably by modifying the physical state of membrane phospholipids, and that the consequent abnormal organization of membranes is a basic cause of low temperature injury. PMID- 16660330 TI - Light-induced Changes of the Carotenoid Levels in Chloroplast Envelopes. AB - The carotenoid content of thylakoids and envelopes isolated from dark-or light treated spinach (Spinacia oleracea L.) chloroplasts was compared. In thylakoids, light induced a decrease of violaxanthin parallel with a stoichiometric increase of zeaxanthin due to violaxanthin deepoxidation. In envelopes, violaxanthin was also decreased and the relative decrease was similar to thylakoids, but zeaxanthin increase was small resulting in an over-all decrease of the amount of envelope carotenoids. When violaxanthin deepoxidation in thylakoids was partly inhibited by 10 nm nigericin, violaxanthin decrease in the envelope was inhibited to a similar degree.These observations together with the absence of deepoxidase activity in isolated envelopes plus added stroma proteins suggest that light induced violaxanthin decrease in the envelope is not caused by an envelope or stroma deepoxidase but results from violaxanthin exchange between envelope and thylakoids. PMID- 16660331 TI - Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: I. The Auxin Hypothesis. AB - Red light-inhibited mesocotyl elongation, which occurs in intact Zea mays L. seedlings, was studied in excised segments which included the coleoptile (or parts therefrom) and apical centimeter of the mesocotyl. Experiments took into account, first, the ability of the segments to regenerate auxin supply sites, and, second, that auxin uptake can be greatly reduced if there is no cut surface, apical to the elongating cells, to act as a port of entry. In all cases, auxin completely reversed the inhibition of elongation by light. The results support the hypothesis that light regulates mesocotyl elongation by controlling auxin supply from the coleoptile. Sucrose concentration had no effect on auxin reversal of light-inhibited elongation, but relatively high concentrations of gibberellic acid (10 mum) could substitute for auxin in this system. PMID- 16660332 TI - Biochemical Induction of Fruiting in Schizophyllum: Isolation and Preliminary Purification of an Inducing Substance from Agaricus bisporus Mushrooms. AB - A diffusible substance was isolated from Agaricus bisporus mushrooms that induced haploid fruiting body formation in Schizophyllum commune. An efficient isolation procedure was developed. The fruiting-inducing substance was shown to be present in all stages of Agaricus mushroom development and there was no preferential accumulation of the activity in gill, cap, or stipe tissues. Some chemical and physical properties of the inducing factor are described. PMID- 16660333 TI - Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture. AB - Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose. PMID- 16660334 TI - Carrier-mediated Uptake of Arginine and Urea by Volvox carteri f. nagariensis. AB - Volvox carteri f. nagariensis takes up arginine via a high affinity, highly specific carrier, whereas carriers for neutral and acidic amino acids cannot be detected (even in nitrogen-starved cultures). Exogenous arginine is accumulated against a steep concentration gradient and is incorporated into protein with high efficiency, but it is not catabolized to any significant extent and will not serve as a nitrogen source adequate to support growth. Urea is also taken up by a saturable carrier, but several lines of evidence indicate that the arginine and urea carriers are distinct and different. Preexposure to arginine suppresses arginine uptake while stimulating urea uptake. The K(i) values observed for reciprocal, competitive inhibition of uptake by arginine and urea are orders of magnitude different from the respective K(m) values for uptake. The two uptake systems show entirely different patterns of sensitivity to inhibition by structural analogs. Finally, the V(max) values for arginine and urea uptake fluctuate independently (but in a regular pattern) during the asexual life cycle. The fluctuations of urea uptake activity are of considerable magnitude and appear to be linked to key phases of the developmental program. PMID- 16660335 TI - Carrier-mediated Uptake of Arginine and Urea by Chlamydomonas reinhardtii. AB - Chlamydomonas reinhardtii possesses a high affinity, highly specific carrier involved in uptake of exogenous arginine. Carrier-mediated uptake of other amino acids cannot be detected, even in cultures maintained on amino acids as a nitrogen source or starved for nitrogen. This fact may contribute to the difficulty of isolating strains auxotrophic for amino acids other than arginine; conventional selection media may not supply adequate quantities of amino acids to permit growth of auxotrophs. A urea carrier is also present in C. reinhardtii but is readily distinguished from the arginine carrier on the basis of kinetic properties and sensitivity to a range of structural analogs. Ammonia appears to play a major role in regulating (depressing) activity of the arginine uptake system. Activity of the urea uptake system is elevated in nitrogen-starved cultures and elevated even further in the presence of urea or arginine. Extensive, independent fluctuations in the two uptake systems observed in semisynchronous cultures suggest that both are subject to modulation by a complex set of interacting endogenous and exogenous factors. PMID- 16660336 TI - Corn Leaf Isoperoxidase Reaction to Mechanical Injury and Infection with Helminthosporium maydis: Effects of Cycloheximide. AB - Mechanical injury or infection with Helminthosporium maydis race T or O enhanced peroxidase activity in leaves of two corn inbreds which differ in their susceptibility to the fungal race T. Increases in activity were found in the soluble fraction extracted from tissues with 20 mm phosphate buffer (pH 6), and in the ionically bound fraction extracted from wall debris with 0.6 to 1 m NaCl; the covalently bound wall peroxidase fraction was unaffected. Mechanical injury and infection with either race enhanced the same distinctive cathodic isoforms present in the soluble fraction or in both the soluble and ionically bound fractions.During the first 20 to 22 hours after inoculation no differences were found in the enzyme enhancement in relation either to the corn line or fungal race. During the second day, the weaker the infection symptoms, the greater was the peroxidase enhancement. The race T-infected susceptible inbred showed no increase or a decrease in the enzyme activity, whereas in most cases both inbreds infected with race O exhibited a further peroxidase enhancement.Pretreatment of healthy leaves or leaf discs with cycloheximide at concentrations which did not cause a loss of turgor had no effect on peroxidase enhancement. However, in tissues inoculated with either the race T or O, cycloheximide, at concentrations which did not affect the mechanical injury-induced increase in peroxidase activity, did inhibit the enzyme enhancement and did cause a loss of turgor.The observed changes in isoperoxidase activities (a) confirm the assumption that the infection-induced enhancement of this enzyme results from a nonspecific response to injury; (b) support the postulation that the often observed small or no increases in peroxidase activity to compatible hosts are a consequence of metabolic disorders rather than the cause of a lower resistance; and (c) indicate that cycloheximide as a protein synthesis inhibitor may increase the compatibility between the host and pathogen, enhance metabolic disorders in inoculated tissues, and in consequence interfere with the nonspecific cell response to injury.Actinomycin D did not affect the mechanical injury- or infection-induced peroxidase enhancement. Neither did ethylene at 5 to 80 mul/l have a significant effect on the enzyme activity in healthy, injured, or infected tissues. PMID- 16660337 TI - Oligomycin-sensitive ATPase of Submitochondrial Particles from Corn. AB - To test the hypothesis (Plant Physiology 59: 155-157) that monocotyledons contain a unique oligomycin-insensitive ATPase, we prepared submitochondrial particles and a soluble fraction from sonicated corn mitochondria (Zea mays L. cv. Earliking). Although the ATPase activity of the whole sonicate was relatively insensitive to oligomycin, the corn submitochondrial particles possessed an ATPase activity that was nearly completely inhibited by oligomycin, and was activated by trypsin. This ATPase is similar to that from other sources (plants, animals, and microorganisms). The soluble fraction also contained an active ATPase, which was inhibited by azide and stimulated by sodium chloride and trypsin. The soluble fraction differed from other F(1)-ATPases in that it was cold-stable. PMID- 16660338 TI - Metabolism of Ammonium Ion and Glutamate in Relation to Nitrogen Supply and Utilization during Grain Development in Barley. AB - Changes in the activity of a number of enzymes concerned with amino acid synthesis and metabolism were recorded for the endosperm, testa pericarp, and embryo of developing barley (Hordeum distichum L.) grains. Both glutamate pyruvate transaminase and glutamate-oxaloacetate transaminase activities were present in all tissues and at all ages examined. Glutamate dehydrogenase activity was largely confined to endosperm while glutamine synthetase activity was mainly in the testa pericarp.Ammonium ion concentration was maximal in endosperm by 20 days after anthesis. Glutamate concentration varied in endosperm and was in the range of 3.5 to 8.5 mm between 20 and 45 days after anthesis. Significant levels of ammonium ion and glutamate were also present in the testa pericarp over the major part of the developmental period. PMID- 16660339 TI - RNA synthesis in whole cells and protoplasts of centaurea: a comparison. AB - Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20 minute pulse with [(3)H]adenosine. After a 4-hour pulse, however, poly(A) containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A) containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells. PMID- 16660340 TI - Auxin receptors of maize coleoptile membranes do not have ATPase activity. AB - Membrane-localized auxin-binding sites from coleoptiles and primary leaves of Zea mays L. which may be auxin receptors can be fully solubilized by 1 to 1.5 mg of Triton X-100 per mg of membrane protein (about 1 mg per gram of original tissue fresh weight), while 70% of the basal (Mg(2+))-ATPase and 85% of the K(+) stimulated (Mg(2+))-ATPase (pH 6) remain pelletable. Gel exclusion chromatography on Bio-Gel A-1.5m indicates that the solubilized receptors occur as detergent protein micelles of about 90,000 daltons equivalent molecular weight. Solubilized ATPase activities occur (a) as very large particles excluded from the gel, and (b) as particles of a size substantially smaller than the particles that exhibit auxin binding. The auxin-binding receptor therefore appears not to be an ATPase. PMID- 16660341 TI - Translocation of C in Macrocystis pyrifera (Giant Kelp). AB - The pattern of import and export of (14)C-labeled assimilates in Macrocystis pyrifera (L.) C. A. Agardh in southern California was studied by labeling single blades on fronds, in situ, with [(14)C]NaHCO(3) for 24 hours. The pattern was found to be similar to that known in dicotyledons: actively growing tissue imported and did not export. As a blade reached maturity it began to export, at first only acropetally to the apex which formed it, later also down the frond to sporophylls and frond initials at the base of the frond, and into the apical regions of juvenile fronds; finally there was a phase of declining export, late in the life of the blade, when transport was only downward. Young fronds imported from older fronds until they were approximately 3 meters long, by which time they had developed mature, upward exporting blades. No translocation was found from a younger frond to an older frond, nor was there transport upward from a blade on a frond lacking the apical region. PMID- 16660342 TI - Further Studies on Oxalic Acid Biosynthesis in Oxalate-accumulating Plants. AB - l-Ascorbic acid functions as a precursor of oxalic acid in several oxalate accumulating plants. The present study extends this observation to include Rumex crispus L. (curly dock), Amaranthus retroflexus L. (red root pigweed), Chenopodium album L. (lamb's-quarters), Beta vulgaris L. (sugar beet), Halogeton glomeratus M. Bieb. (halogeton), and Rheum rhabarbarum L. (rhubarb). Several species with low oxalate content are also examined.When l-[1-(14)C]ascorbic acid is supplied to young seedlings of R. crispus or H. glomeratus, a major portion of the (14)C is released over a 24-hour period as (14)CO(2) and only a small portion is recovered as [(14)C]oxalate, unlike cuttings from 2- or 4-month-old plants which retain a large part of the (14)C as [(14)C]oxalic acid and release very little (14)CO(2). Support for an intermediate role of oxalate in the release of (14)CO(2) from l-[1-(14)C]ascorbic acid is seen in the rapid release of (14)CO(2) by R. crispus and H. glomeratus seedlings labeled with [(14)C]oxalic acid.The common origin of oxalic acid carbon in the C1 and C2 fragment from l-ascorbic acid is demonstrated by comparison of (14)C content of oxalic acid in several oxalate-accumulators after cuttings or seedlings are supplied equal amounts of l [1-(14)C]- or l-[UL-(14)C]ascorbic acid. Theoretically, l-[1-(14)C]ascorbic acid will produce labeled oxalic acid containing three times as much (14)C as l-[UL (14)C]ascorbic acid when equal amounts of label are provided. Experimentally, a ratio of 2.7 +/- 0.5 is obtained in duplicate experiments with six different species. PMID- 16660343 TI - Membrane transport of sugars and amino acids in isolated protoplasts. AB - A method has been developed for observing membrane transport in isolated protoplasts. Transport of sugars and amino acids has been studied in protoplasts isolated from the mesophyll of Pisum sativum L. That uptake was not due to passive diffusion through damaged membranes was demonstrated by supplying simultaneously two sugar stereoisomers, the one (3)H-labeled and the other (14)C labeled. The protoplast membranes were sufficiently functional to discriminate strongly between these stereoisomers.To characterize transport the nonmetabolized glucose analogue 3-O-methyl glucose (MeG) and amino acid analogue alpha aminoisobutyric acid (AIB) were employed. When uptake was compared per unit of protein as between leaf strips and protoplasts prepared from the same tissue, it was estimated that the protoplasts had retained approximately 40 to 50% of the uptake ability of the whole cells. Uptake of neither MeG nor AIB by protoplasts was linear with time, but the tendency to flatten was more marked for AIB. Addition of Mg-ATP to buffered medium significantly promoted AIB uptake, an effect not ascribable to either chelation or pH. Transport of both MeG and AIB was markedly pH-dependent, uptake falling with rise in pH.The stimulatory effect of Mg-ATP and the pH dependence confirm that uptake was not due to a diffusional inward "leak" but involved membrane function.This work demonstrates the feasibility of using isolated protoplasts for membrane transport studies. The potential advantages of using protoplasts for such studies are pointed out. PMID- 16660344 TI - Light Penetration and Light-induced Seed Germination in Soil. AB - Light penetration through a Drummer silty clay loam and a Broomfield sand was measured spectrophotometrically and biologically. The spectrophotometric measurements showed that less than 1% of the incident light penetrated 2.2 millimeters at any wavelength between 350 and 780 nanometers for ped sizes up to 1 millimeter. Biological measurements with light-sensitive lettuce (Lactuca sativa) seeds in soil showed that an exposure to light equivalent to about 1 sunny day induced some germination of seeds which were 2 millimeters below the surface, but did not affect seeds 6 millimeters below the surface. PMID- 16660345 TI - Light modulation of enzyme activity: activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange. AB - Light and dark modulation experiments with pea (Pisum sativum L.) chloroplast stromal fractions pretreated with dithiothreitol (to reduce protein disulfide bonds) or with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (to block sulfhydryl groups) suggest that light modulation involves thiol-disulfide exchange on the modulatable stromal enzyme protein. Light-dependent reduction of DTNB involves a photosynthetic electron transport chain component located on the reducing side of photosystem I prior to ferredoxin; DTNB may be acting as a light effect mediator substitute. The thylakoid-bound light effect mediator system, then, in its light activated reduced form probably catalyzes thiol-disulfide exchange reactions on stromal enzymes. PMID- 16660346 TI - Photosynthetic characteristics of photoautotrophically grown tobacco callus cells. AB - Haploid callus cells of tobacco (Nicotiana tabacum) were grown photoautotrophically on a solid agar medium in the absence of sucrose in Petri plates in an atmosphere of 1% or 3% CO(2) in air. The averages of dry weight increases for four to five consecutive passages were 2.3- to 3.6-fold per 3-week passage for different subclones. Photosynthetic (14)CO(2) assimilation was maximum at about 1% CO(2) with half-maximal rates obtained at 0.2% CO(2). At saturating CO(2) concentration the average rate of CO(2) fixation was about 5 mumole per gram fresh weight per hour or about 125 mumole per mg of chlorophyll per hour.The existence of an active photorespiratory system in these tissues was established in a number of independent ways. The photosynthetic rate in 0.18% CO(2) was inhibited 38 to 50% in 100% O(2) compared with 21% O(2). Glycolate accumulated at a constant rate in the presence of 5 mm alpha-hydroxy-2 pyridinemethanesulfonic acid for 20 minutes in light. This rate was rapid relative to the photosynthetic rate. Glycolate synthesis was three times faster in autotrophic than in heterotrophic cells. [1-(14)C]Glycolate was rapidly metabolized and the products included (14)CO(2), [(14)C]glycine, and [(14)C]serine, thus demonstrating an active glycolate pathway. Photorespiration was demonstrated directly by measurement of an O(2)-dependent release of (14)CO(2) in the light from callus that fixed (14)CO(2) for about 22 hours. Autotrophic growth in 60% O(2) and 0.03% CO(2) was slowed and ceased entirely after two or three passages, while heterotrophic growth was unaffected by 60% O(2) in the atmosphere.The method of growing autotrophic callus which has an active photorespiratory system should facilitate the selection and analysis of photosynthetic mutants in which photorespiration is regulated. PMID- 16660347 TI - Purification and Kinetics of Higher Plant NADH:Nitrate Reductase. AB - Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150 fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 mumol of NADH oxidized/minute * milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 mumol of NADH oxidized/minute * milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site. PMID- 16660348 TI - Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism. AB - Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of beta-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown. PMID- 16660349 TI - Relative Requirements for Magnesium of Protein and Chlorophyll Synthesis in Euglena gracilis. AB - The relationships among Mg, growth, chlorophyll synthesis, and cytoplasmic polysome content were studied in Euglena gracilis grown in different levels of the metal. At all levels of magnesium from 20 to 1,600 mumolar, both protein and chlorophyll are formed with exponential kinetics. The apparent rates of synthesis and final yields of both components are greater at higher levels of Mg, but the rate of chlorophyll synthesis always exceeds the rate of protein formation; i.e. the most severely deficient cells contain proportionally more chlorophyll than the sufficient cells. Cytoplasmic polysomes isolated from Mg-deficient Euglena are indistinguishable from those isolated from control cells. We conclude that decreased rates of protein synthesis occur prior to and possibly are causal to decreased rates of chlorophyll synthesis, but that the mechanism of this inhibition remains unclear. PMID- 16660350 TI - Plant protoplast agglutination by lectins. AB - Concanavalin A, soybean (Glycine max L.) lectin, castor bean (Ricinus communis L.) lectin, and peanut (Arachis hypogaea L.) lectin were able to agglutinate protoplasts prepared from a variety of plant species. The seven other lectins tried were unable to agglutinate those protoplasts tested. Protoplasts prepared from 11 species were used. The lectins examined were not able to differentiate among protoplasts of different species. PMID- 16660351 TI - Uptake of glycylglycine by the scutellum of germinating barley grain. AB - The scutella separated from germinating barley grains (Hordeum vulgare L. cv. Himalaya) took up the dipeptide [(14)C]glycylglycine (Gly-Gly) rapidly from incubation media. The pH optimum of the process was about 4.5, and the rate of uptake conformed to Michaelis-Menten kinetics with an apparent K(m) of 2.3 mm and V(max) of 41 mumole gram(-1) hour(-1). The uptake was strongly inhibited by dinitrophenol and cyanide and by lack of O(2).After incubation of the scutella with Gly-Gly, no intact Gly-Gly was detectable in the scutella but the level of free glycine increased. The poorly hydrolyzed "model" dipeptide glycylsarcosine, which is actively taken up and accumulated by the scutella, was a competitive inhibitor of the uptake of Gly-Gly and completely inhibited the uptake at infinitely high inhibitor concentration. This suggests that Gly-Gly is taken up by the same mechanism as glycylsarcosine as an intact dipeptide (without hydrolysis in the membrane) and is hydrolyzed to free glycine by the abundant peptidases of the scutella.The uptake of Gly-Gly was not affected by glycine or leucine, but was strongly inhibited by all of the 10 dipeptides tested for inhibition. The three dipeptides tested for uptake, Ala-Gly, Pro-Gly, and Gly Pro, were all taken up by the scutella. Thus, the uptake mechanism for the dipeptides seems to be rather nonspecific with respect to the side chains of the amino acids. The high rates of the uptake suggest that this process has an essential role in the mobilization of reserve proteins in the germinating grain. PMID- 16660352 TI - Incidence of ripening and chilling injury on the oxidative activities and Fatty Acid compositions of the mitochondria from mango fruits. AB - The succinate oxidation capacities of mitochondria isolated from mango fruits (Mangifera indica L.) stored at 4, 8, 12, and 20 C were investigated during storage. In normally ripening fruits (at 12 and 20 C) the oxidative capacities increased during the first 10 days and then decreased slowly. At lower temperatures (4 and 8 C), the fruits showed chilling injury symptoms, after about 10 days of storage and the succinate oxidation capacities of mitochondria decreased progressively. Plots of succinate oxidation capacities as against storage temperature showed a marked discontinuity between 12 and 8 C, only when chilling injury was observed on fruits stored at low temperature.The variations of mitochondrial fatty acid composition during the storage of fruits at different temperatures were also investigated. A marked decrease of the molar ratio palmitoleic acid/palmitic acid, the predominant fatty acids in mitochondrial lipids, was observed to accompany both the succinate oxidation decrease and the induction of chilling injury. PMID- 16660353 TI - Phase Behavior of Chloroplast and Microsomal Membranes during Leaf Senescence. AB - Wide angle x-ray diffraction of chloroplast and microsomal membranes from primary leaves of Phaseolus vulgaris has revealed that for both types of membrane, portions of the lipid become crystalline as the tissue senesces. For young leaves the transition temperature is about 23 C for microsomes and below -30 C for chloroplast membranes, indicating that at physiological temperature the lipid is entirely liquid-crystalline. Between 2 and 3 weeks after planting the transition temperature rises to 38 C for microsomes, but for chloroplasts this increase to a point above physiological temperature does not occur until between 3 and 4 weeks. Thereafter the transition temperature continues to rise for both types of membrane with advancing senescence, although the rate of increase is greater for chloroplasts than for microsomes. The appearance at physiological temperature of gel phase lipid in the microsomes coincides temporally with the initiation of a decline in total protein in the tissue, and the incidence of crystallinity in chloroplasts coincides with loss of chlorophyll. This change in phase behavior cannot be attributed to an alteration in fatty acid composition, but for both membrane systems it correlates with an increase of about 4-fold in the sterol to phospholipid ratio. PMID- 16660354 TI - Quantitative Assessment of Ultrastructural Changes in Primary Roots of Corn (Zea mays L.) after Geotropic Stimulation: I. Root Cap. AB - The root cap is the site of gravity perception. In the study of caps of primary roots of corn (Zea mays L.), we compared the ultrastructure of geotropically responding roots that had received a 661 nm (red) irradiation (60 second) with nonresponding dark control roots kept in the dark, at comparable times following geotropic stimulation for a total of 150 minutes. The outstanding differences in the light-exposed root caps at the ultrastructural level were: (a) significantly more Golgi apparatus (dictyosomes) were found in the top than in the bottom of red-exposed cells; a random distribution is seen in the dark control cells; (b) the nucleus preferred the top in a greater number of the red-exposed cells; (c) the pattern of mitochondria localization was identical in both treatments, a greater preference for the top; however, the number of mitochondria was reduced in the bottom of red-treated cap cells as compared to the control cells. A lowering in number in the bottom of the red-treated cells was noted also in the dictyosomes; and (d) in a small percentage of cells that showed a preferential distribution of endoplasmic reticulum (ER), more red-exposed cells than controls, during the period 30 to 135 minutes after stimulation, had less ER in the top; however, a majority of the cells in both treatments showed no preferred position for ER distribution. Commonalities in ultra-structural behavior also existed between the red- and dark-treated root cap cells: (a) sedimentation of amyloplasts, with no difference in total number between treatments; and (b) a close association between amyloplasts and ER in both groups.Polarization of organelles occurred in both the geotropically responding and nonresponding roots. The differences in dictyosome and nuclear localization, and dictyosome and mitochondrial number could be correlated with the tropic response in the red exposed roots and no response in the dark roots, which in turn could be related to the reported hormonal events in the geotropism of roots. PMID- 16660355 TI - Quantitative Assessment of Ultrastructural Changes in Primary Roots of Corn (Zea mays L.) after Geotropic Stimulation: II. Curving and Noncurving Zones of the Root Proper. AB - In outer cortical cells of corn (Zea mays L.) roots we made ultrastructural comparisons between the geotropically responding (661 nm irradiated) and nonresponding (dark control) roots in both the curving and noncurving regions of the root. In the control treatment, Golgi apparatus (dictyosomes) and mitochondria exhibited centrifugal distribution (taking the stele as the center) in both regions of the roots (the organelles localized in the top of the cells in the upper tissues, and in the bottom of the cells in the lower tissues). In the geotropically responding roots, the distribution patterns were the same as those of the controls. However, in the zone of curvature the dictyosomes (but not the mitochondria) were randomly distributed in the cells of the upper tissues. This change in pattern of dictyosome distribution could be related to the change in cell elongation of upper cells. PMID- 16660356 TI - Metabolism of [5-h]proline by barley leaves and its use in measuring the effects of water stress on proline oxidation. AB - The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H(2)O as a measure of proline oxidation. When [5-(3)H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H(2)O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and gamma aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the (3)H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring (3)H(2)O recovery from [5-(3)H]proline were only slightly lower than rates estimated by incorporation of (14)C into oxidized products and loss of (14)C from total proline. Therefore, (3)H(2)O recovery from [5-(3)H]proline is useful in assessing the effects of stress on proline metabolism.Water stress inhibited proline oxidation, as reported previously. In addition, a reconversion of proline oxidation products to proline occurred in stressed leaves. This observation probably indicates a breakdown in cellular compartmentation of proline synthesis and proline oxidation. PMID- 16660357 TI - Relationship between Leaf Water Status and Endogenous Ethylene in Detached Leaves. AB - The pattern of changes in the internal concentration of ethylene in response to water stress was investigated in species with leaves that do abscise and leaves that do not abscise. When leaves which abscise were detached and exposed to dry air for up to 6 hours, a continuous increase of internal ethylene was observed. In water-stressed leaves which do not abscise only a transient rise in ethylene occurred. The peak, which was attained after 30 to 120 minutes, depending on the species studied, was followed by a sharp decline to the initial level. The principal site of ethylene production in response to a short period of water stress was in the blades rather than the petioles in both types of leaves. The internal ethylene level in leaves was reduced by pretreatment with the ethoxy analog of rhizobitoxine (an inhibitor of ethylene biosynthesis) or by maintaining the leaves under subatmospheric pressure. The results obtained by these methods showed that ethylene was not involved in the mechanism of stomatal movement in either turgid or in stressed leaves. Also, the increase in leaf abscisic acid content and the depletion of gibberellins induced by water stress were not related to the internal concentration of ethylene in the detached leaf. The different patterns of drought-induced ethylene production observed in the blades of leaves which exhibit abscission compared with those which do not exhibit abscission may indicate the involvement of ethylene in a primary event in the process of leaf abscission induced by water stress. PMID- 16660358 TI - Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae). AB - An enzyme catalyzing the conversion of mu- to kappa-carrageenan has been demonstrated in both haploid and diploid plants of Chondrus crispus. It acts at the polymer level producing 3,6-anhydro-d-galactose with the stoichiometric release of sulfate. Two-thirds of the recoverable enzyme was associated with the 15,000g pellet most of which could be solubilized by passage through a Ribi Cell Fractionator. The enzyme precipitated between 2.65 and 4.24 m (NH(4))(2)SO(4) and was partly purified on DEAE-cellulose columns. This sulfohydrolase has a pH optimum near 6.5 and is inhibited by molybdate, phosphate, sulfate, tungstate, cysteine, ATP, GTP, UDP, and by lambda-carrageenan. No activator was found. The enzyme showed a similar affinity for several preparations of mu-carrageenan and for the kappa-carrageenase-resistant fraction from kappa-carrageenan thus confirming that the latter is a biosynthetically unfinished molecule.A comparable extract from Gigartina stellata gave a higher specific activity for the sulfohydrolase, but was otherwise quite similar to the Chondrus enzyme. PMID- 16660359 TI - Cyanide-resistant Respiration in Freshly Cut Potato Slices. AB - Treating intact white potato (Solanum tuberosum L.) tuber with ethylene in air or O(2) made it possible to obtain freshly cut slices which exhibit cyanide resistant respiration. The cyanide-resistant path requires induction in whole tubers. The data also indicate that high O(2) concentration is necessary for the full development of cyanide-resistant respiration. PMID- 16660360 TI - Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System. AB - NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3 P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin. PMID- 16660361 TI - Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium. AB - The metabolic fate of l-[4-(14)C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Her. cv. Prince Rupert) under conditions comparable to data from l-[1-(14)C]ascorbic acid and l [6-(14)C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-(14)C]ascorbic acid and l-[1-(14)C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4 (14)C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6 (14)C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+) tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+) tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices. PMID- 16660362 TI - Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: II. Oxygen and Temperature Dependency. AB - Wound-induced ethylene synthesis by subapical stem sections of etiolated Pisum sativum L., cv. Alaska seedlings, as described by Saltveit and Dilley (Plant Physiol 1978 61: 447-450), was half-saturated at 3.6% (v/v) O(2) and saturated at about 10% O(2). Corresponding values for CO(2) production during the same period were 1.1% and 10% O(2), respectively. Anaerobiosis stopped all ethylene evolution and delayed the characteristic pattern of wound ethylene synthesis. Exposing tissue to 3.5% CO(2) in air in a flow-through system reduced wound ethylene synthesis by 30%. Enhancing gas diffusivity by reducing the total pressure to 130 mm Hg almost doubled the rate of wound ethylene synthesis and this effect was negated by exposure to 250 mul liter(-1) propylene. Applied ethylene or propylene stopped wound ethylene synthesis during the period of application, but unlike N(2), no lag period was observed upon flushing with air. It is concluded that the characteristic pattern of wound-induced ethylene synthesis resulted from negative feedback control by endogenous ethylene.No wound ethylene was produced for 2 hours after excision at 10 or 38 C. Low temperatures prolonged the lag period, but did not prevent induction of the wound response, since tissue held for 2 hours at 10 C produced wound ethylene immediately when warmed to 30 C. In contrast, temperatures above 36 C prevented induction of wound ethylene synthesis, since tissue cooled to 30 C after 1 hour at 40 C required 2 hours before ethylene production returned to normal levels. The activation energy between 15 and 36 C was 12.1 mole kilocalories degree(-1). PMID- 16660363 TI - Carbon isotope fractionation by ribulose-1,5-bisophosphate carboxylase from various organisms. AB - Carbon isotope fractionation by structurally and catalytically distinct ribulose 1,5-bisphosphate carboxylases from one eucaryotic and four procaryotic organisms has been measured under nitrogen. The average fractionation for 40 experiments was -34.1 per thousand with respect to the delta(13)C of the dissolved CO(2) used, although average fractionations for each enzyme varied slightly: spinach carboxylase, -36.5 per thousand; Hydrogenomonas eutropha, -38.7 per thousand; Agmenellum quadruplicatum, -32.2 per thousand; Rhodospirillum rubrum, -32.1 per thousand; Rhodopseudomonas sphaeroides peak I carboxylase, -31.4 per thousand; and R. sphaeroides peak II carboxylase, -28.3 per thousand. The carbon isotope fractionation value was largely independent of method of enzyme preparation, purity, or reaction temperature, but in the case of spinach ribulose-1,5 bisphosphate carboxylase fractionation, changing the metal cofactor used for enzyme activation had a distinct effect on the fractionation value. The fractionation value of -36.5 per thousand with Mg(2+) as activator shifted to 29.9 per thousand with Ni(2+) as activator and to -41.7 per thousand with Mn(2+) as activator. These dramatic metal effects on carbon isotope fractionation may be useful in examining the catalytic site of the enzyme. PMID- 16660364 TI - Localization of an uptake hydrogenase in anabaena. AB - Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H(2) uptake was detected (0.60 1.44 mumoles/[mg of chlorophyll a per hour]) in all three strains when grown with N(2) as the sole source of nitrogen. H(2) uptake (in vivo and in vitro) was severely suppressed in cultures grown on NH(4) (+) and lacking heterocysts. H(2) uptake in cell-free extracts could be readily measured with a methyl viologen ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N(2)/CO(2), vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase. PMID- 16660365 TI - Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts. AB - Chloroplasts were prepared from peas (Pisum sativum) in glucose-phosphate medium. In the presence of dl-glyceraldehyde, they catalyzed nitrite-dependent O(2) evolution (mean of 13 preparations, 17.5 mumole per mg chlorophyll per hour, sd 3.64). The optimum concentration of nitrite was 0.5 mm; 0.12 mm nitrite supported V(max)/2. The reaction was accompanied by the consumption of nitrite; 55 to 80% of the nitrite-N consumed was recovered as ammonia. In short experiments (less than 10 minutes) the O(2) to nitrite ratio approached 1.5, but thereafter decreased. There was no nitrite-dependent O(2) evolution with chloroplasts from plants grown without added nitrate but such chloroplasts could assimilate ammonia at about the usual rate. The results are consistent with the reduction of nitrite to ammonia involving nitrate-induced nitrite reductase and a reductant generated by the chloroplast electron transport chain.In the presence of ADP, pyrophosphate, and MgCl(2) the O(2) to nitrite ratio was typically 0.5 to 0.6 and the recovery of nitrite-N as ammonia about 60%. Under these conditions, alpha ketoglutarate increased the O(2) to nitrite ratio (0.9-1.35) and the recovery of nitrite-N as ammonia decreased to 27%. These data and the results of nitrite plus ammonia addition experiments (with and without alpha-ketoglutarate) are attributed to incorporation of nitrite-N into glutamate via the chloroplast enzymes nitrite reductase, glutamine synthetase, and glutamate synthetase. PMID- 16660366 TI - Developmental biochemistry of cotton seed embryogenesis and germination: x. Nitrogen flow from arginine to asparagine in germination. AB - The enzymic basis for the flow of nitrogen from arginine to asparagine during the first 3 days of germination has been measured in extracts from cotton (Gossypium hirsutum) cotyledons. Evidence that asparagine synthetase regulates asparagine accumulation in germination (for transport to the axis) is presented. Further, evidence that the bulk of the nitrogen passed from one generation to the next in dicots is through an asparagine cycle involving the following sequence asparagine --> arginine --> storage protein --> arginine --> asparagine is discussed. PMID- 16660367 TI - Metabolic Pathway of alpha-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition. AB - The uptake and metabolism of alpha-[5-(14)C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated (14)C appeared in organic acids and much less in amino acids, while in the control leaves, the (14)C contents of organic and amino acids were equal. In P deficient leaves, after longer incubation times the (14)C content of organic acids and amino acids increased, while that of CO(2) and residue fractions remained low. In full nutrient leaves the (14)C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO(2). In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO(2) release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO(2) liberation. PMID- 16660368 TI - Lateral Movement of Cations in corn Leaves. AB - Migration patterns of nutrient elements, viz. (59)Fe, (54)Mn, (65)Zn, and (86)Rb, supplied to young corn (Zea mays) leaves were studied using a modified chromatogram scanner. It was found that the isotopes supplied to one-half of the leaf did not migrate to the other side across the midrib, but moved generally toward the base of the applied part of the leaf. PMID- 16660369 TI - Phosphorus contamination in polyethylene glycol. AB - Concentrations of Fe, Mn, Cu, Zn, Ca, Mg, K, and P were examined in untreated and ion exchange resin-treated solutions of polyethylene glycol, molecular weight 3000 to 3700, polyethylene glycol (PEG 4000). Relatively high levels of P were found in untreated PEF-4000 solutions. The concentration of contaminating P in solutions prepared from untreated PEG 4000, even at high water potentials (-1 to 3 bars), was greater than what is usually found in soil solution. Occurrence of significant amounts of P in untreated PEG could introduce problems in experiments where (32)P and PEG are used together and where phosphate interactions may occur. PMID- 16660370 TI - Action Spectrum for Photosporogenesis in Botrytis cinerea Pers. ex Fr. AB - An action spectrum for photoinduced conidium formation in the fungus Botrytis cinerea Pers. ex Fr. was determined by exposing colonies to monochromatic radiation obtained from a diffraction grating monochromator. Wavelengths longer than 355 nm were ineffective even after exposures of 8 hours at intensities of 4,068 to 8,276 erg per cm(2) per second. Colonies were exposed at 22.5 C from 226 to 355 nm at about 5 nm intervals. Three prominent peaks of effectiveness occurred at about 231, 268, and 283 nm, and there was also a minor peak at about 303 nm. The most effective wavelength for inducing sporulation was 231 nm; it was 25% more effective than the second most effective wavelength (283 nm). PMID- 16660371 TI - Factors Involved in in Vitro Stabilization of Nitrate Reductase from Cotton (Gossypium hirsutum L.) Cotyledons. AB - Experiments were conducted to determine if pretreatment of cotton (Gossypium hirsutum L.) plants resulted in differential in vitro stabilities of nitrate reductase (NR) activity. Although NR activity declines markedly during the second half of the daily light period, in vitro NR stability is not modified by time of harvest. Phenylmethylsulfonylfluoride, iodoacetamide, and N-ethylmaleimide do not influence in vitro NR stability, suggesting that serine or sulfhydryl proteases are not responsible for in vitro lability of NR from cotton cotyledons.Imposition of water stress or artificial extension of the dark period lead to significant reductions in NR activity, but do not change in vitro NR stability.Dilution of a crude extract leads to increasing lability of NR; hence the marked instability of NR cannot be attributed to an inactivator which follows simple enzyme kinetics. Since in vitro NR activity is much more stable in presence of both NADH and NO(3) (-), substrate availability must be considered as a possible factor influencing in vivo NR stability. PMID- 16660372 TI - Cyclic adenosine 3':5'-monophosphate in axenic rye grass endosperm cell cultures. AB - Cyclic adenosine 3':5'-monophosphate (cAMP) was extensively purified from rye grass (Lolium multiflorum) endosperm cells grown in axenic suspension culture. The cAMP was purified by neutral alumina and anion and cation exchange chromatography. The cAMP was quantitated by means of a radiochemical saturation assay using a beef heart cAMP-binding protein and also by an assay involving activation of beef heart protein kinase. The cAMP levels found (corrected for recovery of tracer cyclic 3',5'-[8-(3)H]AMP included from the point of sample extraction) ranged from 2 to 12 pmol/g fresh weight. The material purified from rye grass cultures was indistinguishable from authentic cAMP with respect to chromatography in two cellulose thin layer systems, behavior on dilution in both the saturation and protein kinase activation assays, and rates of degradation by a mammalian cAMP phosphodiesterase. The cAMP from rye grass cultures was completely degraded by a mammalian cAMP phosphodiesterase, and 1-methyl-3 isobutylxanthine inhibited such degradation. The protein kinase activation and saturation assays gave essentially the same values for the cAMP content of axenic rye grass culture extracts. Material satisfying the above criteria for identity with cAMP was also isolated from the culture medium. The increase observed in medium cAMP levels during culture growth provides evidence for the synthesis and secretion of cAMP by rye grass endosperm cells in suspension culture. PMID- 16660373 TI - Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase. AB - When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K(+)-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose. PMID- 16660374 TI - Sites of absorption and translocation of iron in barley roots: tracer and microautoradiographic studies. AB - Absorption and translocation of labeled Fe were measured at various locations along the length of intact seminal axes and lateral roots of iron-sufficient (+Fe) and iron-stressed (-Fe) barley (Hordeum vulgare) plants. In seminal axes of +Fe plants, rates of translocation were very much higher in a zone 1 to 4 cm from the root tip than elsewhere in the root. Lateral roots of high rates of translocation were also restricted to a narrow band of maturing or recently matured cells. In -Fe plants the patterns of uptake and translocation were essentially the same as in +Fe plants but the rates were 7- to 10-fold higher. The amount of labeled Fe bound to the root itself was not increased by Fe stress and its distribution along the root seemed inversely related to the ability to translocate Fe.Microautoradiographic studies showed that most of the iron bound to roots was held in an extracellular peripheral band in which iron seemed to be precipitated. This process may be assisted by microbial colonies but did not depend on them since it was seen, although to a lesser extent, in sterile roots. In zones from which iron was translocated there was evidence that internal root tissues became labeled readily, but as translocation declined with distance from the root tip, radial penetration of Fe appeared to become progressively less. The results are discussed in relation to possible changes in the pH or redox potential of the surface of the root. PMID- 16660375 TI - Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves. AB - The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (-)-menthyl acetate, and leaf discs converted exogenous (-)-[G (3)H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (-)-[G-(3)H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K(m) and velocity for (-)-menthol were 0.3 mm and 16 nmol/hr. mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint. PMID- 16660376 TI - Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana. AB - The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [(14)C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required alpha ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5 phosphate seemed not to increase the enzyme activity in tumor plants.The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined. PMID- 16660377 TI - Nitrate Reductase and Soluble Cytochrome c Reductase(s) in Higher Plants. AB - The 4S cytochrome c (Cyt c) reductase activity of several plant species was markedly stimulated by cyanide and ferrocyanide but those of the 8S nitrate reductase component and other particulate components of the maize (Zea mays L.) scutellum by comparison, were increased only slightly. The effect of cyanide and ferrocyanide was not due to elimination of cytochrome oxidase interference but resulted from the stimulation of NADH-dependent reduction of Cyt c. A 4S Cyt c reductase component which could be isolated by ammonium sulfate fractionation and diethyl-aminoethyl-cellulose chromatography was found to be stimulated markedly by cyanide and ferrocyanide. The remaining 4S Cyt c reductase, which was insensitive to cyanide and ferrocyanide, was also fractionated with ammonium sulfate into two components. One of these, like the 8S Cyt c reductase, was sensitive to a protease from the maize roots which is relatively specific for nitrate reductase. This 4S Cyt c reductase species could be a subunit of nitrate reductase. PMID- 16660378 TI - Hydrogen (h(2)) evolution by rhizobia after synergetic culture with soybean cell suspensions. AB - Rhizobium japonicum cells were grown in liquid suspension cultures and separated from soybean plant cells by two to three bacterial membrane filters. Under these conditions, the plant cells elaborated materials into the medium which aided in the expression of a major rhizobial phenotype, namely, nitrogenase activity (acetylene reduction). The evolution of H(2) was also measured and this activity relative to acetylene reduction, was influenced by: (a) O(2); (b) the quantity of conditioned plant medium; and (c) ammonia. It is concluded that plant substances are of major importance in the H(2) evolution and nitrogenase activities of free living rhizobia in suspension cultures. PMID- 16660379 TI - Glucose Metabolism and Retention of Glucose Metabolites in the Wheat Embryo during Early Germination. AB - There is a lag period in the growth of excised wheat (Triticum vulgare Host.) embryos extending from 0.5 hour to 4.5 hours of their imbibition in water. During this time there is a sharp increase in the embryos' capability to retain several amino and organic acids, to synthesize cell wall components and starch, and to take up glucose. Their capability to metabolize glucose also increases by 30%. Elevation of the ATP content of 1-hour embryos by incubation in 3 mm adenosine is not sufficient to bring about these changes. These changes may be a part of a metabolic adjustment in the embryos which increases their growth potential. PMID- 16660380 TI - Mechanisms of resistance to sulfur dioxide in the Cucurbitaceae. AB - The relative resistance of four cultivars of the Cucurbitaceae (Cucumis sativus L. cv. National Pickling, and inbred line SC 25; Cucurbita pepo L. cv. Prolific Straightneck Squash, and cv. Small Sugar Pumpkin) to SO(2) was determined. According to plots of the degree of exposure to SO(2) (which depends on the SO(2) concentration and the duration of the exposure), there is an 8-fold difference in resistance to this toxic gas among these cultivars. However, if the degree of injury is plotted as a function of the amount of SO(2) absorbed, all four cultivars appear similarly sensitive to the gas. We conclude that the principal reason for special and varietal differences in resistance among these cultivars is the relative rate of absorption of the gas. The densities of stomata on the upper and lower surfaces of leaves did not differ sufficiently between cultivars to account for the differences in absorption rates. It remains to be determined whether the differences in rate of SO(2) absorption reflect differences in stomatal activity.Resistance of individual leaves changes with position on the plant axis (age of the leaf). There exists a gradient of decreasing resistance from the apex downward. This resistance gradient cannot be accounted for by differences in rates of SO(2) absorption. We infer the existence of a biochemically based, developmentally controlled resistance mechanism which functions after SO(2) has entered the leaf. Biochemical comparisons of old and young leaves with such differences in resistance should be helpful in determining the biochemistry of SO(2) toxicity. PMID- 16660381 TI - Properties of chloroplasts isolated from siphonous algae: effects of osmotic shock and detergent treatment on intactness. AB - Chloroplasts isolated from the siphonous green alga Caulerpa simpliciuscula (Turner) C.Ag. were shown to be resistant to dissolution by the nonionic detergent Teric-10 at concentrations as high as 0.3% (v/v) when treated at 0 C. There was little release of stromal enzymes under these conditions. These chloroplasts were disrupted by osmotic shock as shown by measurement of the release of both glucose-6-phosphate dehydrogenase and NADP-dependent glutamate dehydrogenase into the suspending medium. Ribulose-1,5-bisphosphate carboxylase, an accepted marker for chloroplast stromal protein in higher plants, was largely retained in the disrupted chloroplast following the osmotic shock. This is considered to be due to the location of a significant proportion of enzyme within the pyrenoid, which protects it from dissolution and causes it to behave as though it were an insoluble protein. PMID- 16660382 TI - Effect of light on ethylene production and hypocotyl growth of soybean seedlings. AB - The apical 1-cm hypocotyl of dark-grown ;Clark' soybean (Glycine max [L.] Merr.) seedlings produced ethylene at rates of 7 to 11 nanoliters per hour per gram when attached to the cotyledons. Such physiologically active rates occurred prior to the deceleration of hypocotyl elongation caused by the temperature of 25 C.Daily exposure of the etiolated seedlings to red light promoted hypocotyl elongation and prevented its lateral swelling. Red light treatment also caused a 45% decrease in ethylene production. Far red irradiation following the red treatment reversed the red effects, suggesting that the ethylene intervenes as a regulator in the phytochrome control of ;Clark' soybean hypocotyl growth at 25 C. PMID- 16660383 TI - Role of carbohydrates in proline accumulation in wilted barley leaves. AB - The effect of wilting on proline synthesis, proline oxidation, and protein synthesis-all of which contribute to proline accumulation-was determined in nonstarved barley (Hordeum vulgare L.) leaves. Nonstarved leaves were from plants previously in the light for 24 hours and starved leaves were from plants previously in the dark for 48 hours. Wilted leaves from nonstarved plants accumulated proline at the rate of about 1 mumole per hour per gram of fresh weight whereas wilted leaves from starved plants accumulated very little proline. Wilting caused a 40-fold stimulation of proline synthesis from glutamate in nonstarved leaves but had very little effect in starved leaves. Proline oxidation and protein synthesis, on the other hand, were inhibited by wilting in both nonstarved and starved leaves. Thus, the role of carbohydrates in proline accumulation is to supply precursors for the stimulated proline synthesis. These results further indicate that the main metabolic response causing proline to accumulate in wilted barley leaves is the stimulation of proline synthesis from glutamate. The difference between these results and those obtained with beans is discussed.Wilting caused an increased conversion of glutamate to other products. In nonstarved leaves, conversion to organic acids as well as to proline was increased. In starved leaves, wilting caused an increase in the conversion of glutamate to glutamine, aspartate, asparagine, and organic acids. PMID- 16660384 TI - Role of Lectins in Plant-Microorganism Interactions: II. Distribution of Soybean Lectin in Tissues of Glycine max (L.) Merr. AB - Three different assay procedures have been used to quantitate the levels of soybean (Glycine max [L.] Merr.) lectin in various tissues of soybean plants. The assays used were a standard hemagglutination assay, a radioimmunoassay, and an isotope dilution assay. Most of the lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was present in all of the tissues of young seedlings, but decreased as the plants matured and was not detectable in plants older than 2 to 3 weeks. Soybean lectin isolated from seeds of several soybean varieties were identical when compared by several methods. PMID- 16660385 TI - Essential Sulfhydryl Group in the Transport-catalyzing Protein of the Hexose Proton Cotransport System of Chlorella. AB - The polyene antibiotic nystatin transforms the sugar-proton contransport system of Chlorella to a mere facilitated diffusion system. This experimental condition was used to test the sugar-translocating unit of the active uptake system for possible essential sulfhydryl groups. It could be shown that the catalyzed translocation of sugar is sensitive to the sulfhydryl-reactive compound N ethylmaleimide. Sugar flow by passive leak as induced by the detergent Triton X 100 is not affected by sulfhydryl reagents. These results show that the sugar translocating carrier protein possesses a sulfhydryl group, which is essential for its function. PMID- 16660386 TI - Organic Acid Metabolism by Isolated Rhizobium japonicum Bacteroids. AB - Rhizobium japonicum bacteroids isolated from soybean (Glycine max L.) nodules oxidized (14)C-labeled succinate, pyruvate, and acetate in a manner consistent with operation of the tricarboxylic acid cycle and a partial glyoxylate cycle. Substrate carbon was incorporated into all major cellular components (cell wall + membrane, nucleic acids, and protein). PMID- 16660387 TI - 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species. AB - [2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species. PMID- 16660388 TI - Decomposition of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one in Aqueous Solutions. AB - Cyclic hydroxamic acids present in some species of Gramineae have been reported to be important in resistance of these plants to fungi and insects. Since the nonglucosylated forms of these acids are unstable in aqueous solution, in vitro methods for the measurement of their antibiotic properties have been difficult. Kinetics of the decomposition of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H) one (DIMBOA), the major hydroxamate in corn (Zea mays L.) extracts, were studied in buffered aqueous solutions from pH 5 to 7.5 at temperatures from 20 to 80 C. Kinetics were apparently first order under all conditions tested; energies of activation (24 to 28 kcal/mol) were nearly pH-independent. DIMBOA decomposed rapidly (half-life = 5.3 hours at 28 C, pH 6.75) relative to the time required for many procedures which have been used to demonstrate the biological activity of DIMBOA. The rate of disappearance of inhibitory activity of DIMBOA toward Erwinia carotovora was indistinguishable from the rate of decomposition of DIMBOA. Contrary to reports, yields of 6-methoxy-2-benzoxazolinone (MBOA) were not quantitative. Gas-liquid chromatography analytical procedures were developed for quantitation of trimethylsilyl and acetyl derivatives of MBOA. As measured by ultraviolet spectroscopy and/or gas-liquid chromatography, conversion of DIMBOA to MBOA ranged from 40 to 75% of theoretical in aqueous buffers, bacterial growth medium, and ethyl acetate extracts of corn tissue resuspended in buffer. Yields varied with temperature, pH, and constituents in the medium. PMID- 16660389 TI - Factors That Influence the Activity of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin 3(4H)-one on Erwinia Species in Growth Assays. AB - Factors affecting the inhibitory activity of 2,4-dihydroxy-7-methoxy-2H-1,4 benzoxazin-3(4H)-one (DIMBOA) against Erwinia carotovora, a nonpathogen of Zea mays L., and against a maize pathovar of Erwinia chrysanthemi (ECZ) were examined. Most experiments were performed with DIMBOA dissolved in a bacterial growth medium containing 10 g/liter of sucrose, inorganic salts, and 1 g/liter of casamino acids at pH 6.75. When temperature and pH were held constant, inhibition of E. carotovora varied linearly with the logarithm of the initial cell population. By altering temperatures, assays with constant pH and initial cell populations were performed under conditions of varying DIMBOA stability. When E. carotovora was grown at 24, 28, 32, and 36 C in the presence of 0.1 to 0.5 mm DIMBOA, the inhibition of bacterial growth was maintained long after DIMBOA had decomposed in the medium to levels which, if added initially, would not have been inhibitory. When assays were performed at pH 5.5, the pH of aqueous maize extracts, E. carotovora was more inhibited than at pH 6.75; however, ECZ was substantially less inhibited at the lower pH. PMID- 16660390 TI - Metabolic and Ultrastructural Changes in Winter Wheat during Ice Encasement Under Field Conditions. AB - The effect of ice encasement on the physiological, metabolic, and ultrastructural properties of winter wheat (Triticum aestivum L.) grown under field conditions was examined by artificially encasing winter wheat in ice during early winter. Cold hardiness and survival of ice-encased seedlings declined less rapidly in Kharkov, a cold-hardy cultivar than in Fredrick, a less hardy cultivar. Ethanol did not accumulate in non-iced seedlings, but increased rapidly upon application of an ice sheet. Lactic acid accumulated in both cultivars during late autumn, prior to ice encasement, and elevated levels of lactic acid were maintained throughout the winter in seedlings from both iced and non-iced plots. The rate of O(2) consumption of shoot tissue of seedlings from non-iced plots remained relatively constant throughout the winter, but declined rapidly in seedlings from ice encased plots. Major ultrastructural changes did not occur in shoot apex cells of non-iced winter wheat seedlings during cold hardening under field conditions. However, the imposition of an ice cover in early January resulted in a proliferation of the endoplasmic reticulum membrane system of the cells, frequently resulting in the formation of concentric whorls of membranes, often enclosing cytoplasmic organelles. Electrondense areas within the cytoplasm which appeared to be associated with the expanded endoplasmic reticulum were also frequently observed. PMID- 16660391 TI - Microviscosity of plasmalemmas in rose petals as affected by age and environmental factors. AB - The microviscosity of the plasmalemma of protoplasts isolated from rose (Rosa hyb. cv. Golden Wave) petals was measured by fluorescence depolarization. The plasmalemma's microviscosity was found to increase in petals which were allowed to age on cut flowers or after isolation as well as in isolated protoplasts aged in an aqueous medium. Increasing the temperature of the cut flowers or the isolated protoplasts enhanced the increase of the microviscosity of the protoplast plasmalemma. The mole ratio of free sterol to phospholipid was greater in protoplasts isolated from old flowers or in protoplasts aged after isolation than in protoplasts isolated from younger flowers. Microviscosity was greatest when protoplasts were aged at pH 4.4 and in the presence of Ca(2+). Artificial alterations of the sterol to phospholipid ratio in the protoplasts, induced by treatment with liposomes, caused similar changes in their measured microviscosity.These findings strongly suggest that the increase in the petal plasmalemma microviscosity with age is associated with an increase in the sterol to phospholipid ratio which results, at least partially, from the activity of endogenous phospholipases. PMID- 16660392 TI - Phototactic Responses of Cell Population to Repeated Pulses of Yellow Light in a Phytoflagellate Cryptomonas sp. AB - Positive phototaxis in cell populations of a phytoflagellate Cryptomonas sp. was recorded photoelectrically when the duration and intensity of repeated pulses of monochromatic yellow light (570 nm) interspersed with darkness were varied. Irrespective of the duration of the light pulses, phototactic responses to repeated pulses were as great as those to continuous irradiation and were linearly dependent on the logarithm of total incident light energy when the dark interval was shorter than 60 milliseconds. Under these conditions, reciprocity between duration and intensity held well. In contrast, when the dark interval exceeded 250 milliseconds, the responses were remarkably reduced regardless of light duration and were not affected by increasing the intensity of actinic light pulses.The present results clearly indicate that continuous stimulation with actinic light is not essential for the maximum effect, but that the length of dark interval is crucial in phototactic response. PMID- 16660393 TI - Glycoprotein Synthesis in Plants: III. Interaction between UDP-N Acetylglucosamine and GDP-Mannose as Substrates. AB - This report presents evidence that enzymes present in crude extracts prepared from developing cotyledons of Phaseolus vulgaris can catalyze the transfer of radioactivity from UDP-N-[(14)C]acetylglucosamine into a chitobiosyl-lipid, lipid oligosaccharide, and glycoprotein. Kinetic evidence supports the concept that the N-acetylglucosamine-containing lipids are precursors to the glycoprotein. Evidence is also presented which shows an interaction between GDP-mannose and UDP N-acetylglucosamine when used as substrates for the synthesis of lipid oligosaccharide and glycoprotein. Kinetic evidence, as well as isolation and characterization of the oligosaccharides released from lipid by mild acid hydrolyses, support the conclusion that mannose and N-acetylglucosamine are contained in the same oligosaccharide and that N-acetylglucosamine is present at the reducing end of the oligosaccharide. Ninety-eight per cent of the radioactivity which is incorporated from UDP-N-[(14)C]acetylglucosamine into the insoluble residue is solubilized by protease treatment. The glycopeptide released is quite similar in size and composition to the glycopeptide released by proteolytic digestion of vicilin, the major storage protein of Phaseolus vulgaris. PMID- 16660394 TI - Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants. AB - The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions. PMID- 16660395 TI - Lipid molecular species composition in developing soybean cotyledons. AB - The fatty acid composition of triglyceride and phospholipids in developing soybean cotyledons (Glycine max L., var. "Harosoy 63") was analyzed at several stages of growth between 30 and 70 days after flowering. Changes observed in fatty acid composition within each lipid class were related to the levels of lipid molecular species present in the oil. Thirteen molecular species of triglyceride were identified in developing cotyledons, however three of these groups: trilinolenic, dilinolenic-monolinoleic, and linolenic-linoleic-oleic triglycerides, were not found in the mature seed. In immature cotyledons, trioleic and trilinoleic triglycerides accounted for 50% of the structures found; the level of these molecules decreased to 24.9% in the mature seed. The dilinoleic-monolinolenic triglycerides increased from 0.4 to 23.4% during cotyledon development. Changes in triglyceride composition were compared to the levels of molecular species for each phospholipid class. Dilinoleic and monosaturated monolinoleic phospholipid species were dominant in all phospholipid classes throughout development. PMID- 16660396 TI - Control of Thylakoid Growth in Phaseolus vulgaris. AB - An attempt was made to answer whether the extent of thylakoid growth in Phaseolus vulgaris is controlled by a feedback inhibition mechanism, operating after insertion of all of the necessary components of the mature thylakoid, in the right amounts and ratio, or by parameters independent of the developmental stage of the membrane. This was done by following the growth of thylakoids, as monitored by the rate of chlorophyll accumulation and the rate of thylakoid protein synthesis, in etiolated plants exposed either directly to continuous light (transformation of prolamellar body to mature thylakoid) or first to periodic light and then to continuous light (transformation of prolamellar body to primary thylakoids and then to mature thylakoids). It was found that prolonged etiolation has no effect on the rate of thylakoid synthesis in continuous light. However, prolonged preexposure to periodic light diminishes drastically the rate of new thylakoid synthesis in continuous light. Since the thylakoids formed in the latter case are far from being complete, it seems that thylakoid growth can stop long before all of the necessary components are incorporated. Parameters independent of the developmental stage and composition of the membrane, therefore, seem to control membrane growth. PMID- 16660397 TI - Studies on Genetic Male-Sterile Soybeans: I. Distribution of Plant Carbohydrate and Nitrogen during Development. AB - Soybeans (Glycine max [L.] Merr. cv. NC 69-2774) were used to study the nonstructural carbohydrate and nitrogen content of plant tissues, and nitrogenase activity throughout the development of male-sterile and male-fertile plants. Male sterile plants set approximately 85% fewer pods plus seed than the male-fertile siblings and retained green leaves until a killing frost at 145 days after emergence. Reduced pod set caused increased carbohydrate accumulation in the leaf and root systems of male-sterile plants. Total carbohydrate in roots of male sterile plants increased from 1.7 to 7.6 times that in the male-fertile roots. A high proportion (60 to 70%) of the male-sterile root carbohydrate was starch. Apparently, root starch was not metabolized by the male-sterile plants. Late in plant development per cent nitrogen was higher in the male-sterile soybean tissues. However, no difference was found in the ability of the nodulated root systems from either genotype to fix nitrogen. PMID- 16660398 TI - Effect of Dim Light on the y-1 Mutant of Chlamydomonas reinhardtii. AB - The y-1 mutant of Chlamydomonas reinhardtii tends to die or revert to wild type when grown in the dark for a long period of time. A small amount of white light (0.5 lux) enables the y-1 mutant to grow indefinitely in a "near dark" condition. Under this condition, the y-1 mutant is physiologically and ultrastructurally similar to the dark-grown y-1 yet remains genetically stable. PMID- 16660399 TI - Development and Distribution of Dolichos biflorus Lectin as Measured by Radioimmunoassay. AB - A radioimmunoassay, capable of detecting the Dolichos biflorus lectin at concentrations as low as 400 ng/ml, was developed and used to follow the distribution of this lectin in the plant during its life cycle.The lectin was first detected in the seeds of the plant 27 days after flowering and rapidly attained the high level of lectin present in the mature seed. The lectin content of the plant is highest in the seeds and cotyledons and decreases as the storage materials of the cotyledons decrease.A low but measurable amount of material that reacts with antibodies to the seed lectin was detected in the leaves, stems, and pods of the plant. This material gives a precipitin band of only partial identity to the seed lectin when tested in immunodiffusion against antiserum to the seed lectin.No lectin was detected by the radioimmunoassay in the roots of the plant at any stage of development. PMID- 16660400 TI - Environmental Parameters Affecting Dark Response of Rice Seedlings (Oryza sativa L.) to Triacontanol. AB - Triacontanol applied to IR-8 rice (Oryza sativa L.) seedlings in nutrient solution caused an increase in dry weight during a 6-hour dark period. This increase was altered by atmospheric CO(2) and O(2) concentrations. The largest growth response occurred from 200 to 350 muliters/liter CO(2) with 5% O(2). The treated seedlings did not fix atmospheric CO(2) in the dark, and the immediate products of photosynthesis were not involved in the dry weight increase. The growth response was characterized by an increase in soluble and insoluble Kjeldahl-N, and soluble carbohydrates. The response curve for dry weight increase was a linear function of log presentation time of triacontanol. The response exhibited an apparent K(dose) of 25 minutes in 10 mug/liter triacontanol in the dark and 18 minutes in the light. Concentrations of 50 mug/liter and higher inhibited growth. PMID- 16660401 TI - Effect of triacontanol on plant cell cultures in vitro. AB - Triacontanol [CH(3)(CH(2))(28)CH(2)OH] increased growth in vitro of cell cultures of haploid tobacco (Nicotiana tabacum). The fresh weight of cell cultures of tomato (Lycopersicon esculentum), potato (Solanum tuberosum), bean (Phaseolus vulgaris), and barley (Hordeum vulgare x H. jubatum) was also increased. The increase in growth of tobacco callus seems to have been due to an increase in cell number. Another long chain alcohol, octocosanol [CH(3)(CH(2))(26)CH(2)OH], did not increase the growth of tobacco cell cultures. PMID- 16660402 TI - Effects of low temperature on respiration and uptake of rubidium ions by excised barley and corn roots. AB - The effect of temperature upon ion uptake and respiration was investigated with excised roots of corn (Zea mays) and barley (Hordeum vulgare). A strong inhibition (Q(10) = 5 to 8) of ion uptake was observed at temperatures below 10 C. At higher temperatures more normal temperature dependencies (Q(10) = 1.3 to 2) were obtained. When the data were plotted according to the Arrhenius relationship, two different activation energies were indicated above and below 10 C. Other studies have related such changes with temperature in activation energy of processes to changes in membrane properties induced by temperature. These results suggest that such phase transitions may affect ion uptake processes. If so, then differences among species in their capacity to maintain normal root function at low soil temperature and to resist low temperature stress may be related to differences in the physical properties of cellular membranes. PMID- 16660403 TI - Plant Nucleases: V. Survey of Corn Ribonuclease II Isoenzymes. AB - Corn (Zea mays L.) ribonuclease II of the root microsomal fraction was isolated from 11 inbreds and seven hybrids. Polyacrylamide gel electrophoresis revealed a total of five bands of activity among corn lines tested. Most inbreds had only one isoenzyme, but three had two isoenzymes. The hybrids tested contained all of the isoenzymes found in the parental inbreds. PMID- 16660404 TI - On the Resistance to Transpiration of the Sites of Evaporation within the Leaf. AB - The rates of transpiration from the upper and lower surfaces of leaves of Gossypium hirsutum, Xanthium strumarium, and Zea mays were compared with the rates at which helium diffused across those leaves. There was no evidence for effects of CO(2) concentration or rate of evaporation on the resistance to water loss from the evaporating surface ("resistance of the mesophyll wall to transpiration") and no evidence for any significant wall resistance in turgid tissues. The possible existence of a wall resistance was also tested in leaves of Commelina communis and Tulipa gesneriana whose epidermis could be easily peeled. Only when an epidermis was removed from a leaf, evaporation from the mesophyll tissue declined. We conclude that under conditions relevant to studies of stomatal behavior, the water vapor pressure at the sites of evaporation is equal to the saturation vapor pressure. PMID- 16660405 TI - Comparison of Light-dependent Oxygen Uptake, Protochlorophyll(ide)-650 Photoconversion, and Chlorophyll Disappearance in Wheat Etioplasts. AB - Red light exposures given to dark-grown wheat seedlings (Triticum aestivum L.) prior to etioplast isolation reduced the ability of these organelles to consume O(2). The same preharvest red light exposures also decreased protochlorophyll(ide) content of etioplasts. In addition, regeneration of both O(2) uptake rates as well as protochlorophyll(ide) levels followed a parallel time course. These similarities suggested that photoconversion of protochlorophyll(ide)-650 to chlorophyll(ide) may mediate some process with O(2) as the electron acceptor. This process appears to involve photooxidation of nonphotoconvertible protochlorophyll(ide) as well as of newly formed chlorophyll(ide). This hypothesis is further supported by the observations that: (a) the in vitro light induced O(2) uptake phenomenon was observed in solubilized protochlorophyll(ide) holochrome preparations; and (b) photoinduced O(2) uptake was reduced to zero rate by light exposure time equivalent to that required for chlorophyll(ide) and nonphotoconvertible protochlorophyll(ide) destruction. PMID- 16660406 TI - Subcellular localization of isocitrate lyase in nongreen tissue culture cells. AB - Density gradient centrifugation and electron microscopy were used to establish that isocitrate lyase present in Rosa cv. Paul's Scarlet cells was located in the mitochondria and not other membrane fractions. The enzyme may be important in glycine and serine synthesis. A comparison between the enzymic activity of isocitrate lyase and the amount of glycine and serine synthesized during logarithmic growth indicated that the activity was great enough to account for all of the carbon entering these amino acids during that stage of growth. PMID- 16660407 TI - Studies on the metabolism of lipid molecular species in immature soybean cotyledons. AB - Metabolism of lipid molecular species in soybean cotyledons (Glycine max [L.] Merr. var. "Harosoy 63") was determined from incorporation studies with radioactive acetate and glycerol. Lipid synthetic activity was highest in immature cotyledons at 30 days after flowering. Distinct differences in labeling patterns of molecular species within lipid classes demonstrated that selective utilization of diglyceride intermediates occurred in complex lipid biosynthesis in soybean. The phospholipid molecular species in this tissue that displayed the highest turnover rates had the following acyl combinations: saturate-linoleic and dioleic in phosphatidic acid; saturate-oleic in phosphatidylinositol and phosphatidylethanolamine; dioleic in phosphatidylcholine; oleic-dilinoleic in N acylphosphatidylethanolamine. Saturate-dilinoleic, oleic-dilinoleic, trioleic, and trilinoleic structures were rapidly synthesized species of triglyceride in immature soybean cotyledons. PMID- 16660408 TI - Comparative Investigation of the Action of Several Chlorosis-inducing Herbicides on the Biogenesis of Chloroplasts and Leaf Microbodies. AB - Seedlings of Triticum aestivum L. and Secale cereale L. were grown in the presence of six different (five having different chemical structures) chlorosis inducing herbicides: aminotriazole and its derivative SDR 5175, haloxidine, Sandoz 6706, fluometuron, and EMD-IT 5914. Concentrations were applied which allowed the leaves to grow normally and to reach normal total amino nitrogen contents but evoked a complete chlorosis (less than 6% chlorophyll). The effects of the herbicides on the accumulation of several chloroplast constituents and on peroxisomal and mitochondrial marker enzyme activities were compared. Wheat and rye, in general, gave very similar results, wheat being more sensitive to unspecific inhibitory effects.In dark-grown plants, the herbicides had no or only minor effects on the rRNA pattern and on enzyme activities of the leaves. In the light, all herbicides applied prevented the accumulation of carotenoids and of chloroplastic rRNA. Consequently, ribulose-1,5-bisphosphate carboxylase activity was virtually absent. After all herbicide treatments in light, the leaves contained only rather low catalase activity. In the presence of aminotriazole and haloxidine, the chloroplast-specific NADP-glyceraldehyde-3-phosphate dehydrogenase and the peroxisomal enzymes glycolate oxidase and hydroxypyruvate reductase had high or even normal activities, as in untreated leaves. In leaves treated with Sandoz 6706, fluometuron, or EMDIT 5914, the activities of the latter three enzymes were, in parallel, only very low. Some herbicides interfered with enzyme activities in vitro, particularly with those of catalase and of glycolate oxidase. Among mitochondrial enzymes, cytochrome c oxidase activity was either unaffected or lower, while fumarase had considerably higher activities in the herbicide-treated, as compared to untreated leaves. The specific effects on peroxisomal enzymes cannot be explained by the hypothesis of herbicide-induced photodestructions in carotene-deficient plastids. Alternative explanations for the genesis of the chlorosis are discussed. PMID- 16660409 TI - Isolation and Characterization of a Chromatin-associated Protein Kinase from Soybean. AB - A chromatin-associated casein-type protein kinase has been purified 500-fold from soybean (Glycine max, var. Wayne) tissue. The enzyme can be completely dissociated from isolated chromatin in 250 millimolar (NH(4))(2)SO(4). After purification, the kinase preparation is stable for at least 6 months at 0 C. The enzyme will phosphorylate casein, phosvitin, and denatured chromatin proteins, but not histones. Only ATP will serve as a phosphate donor with an apparent K(m) of 8 micromolar. Five millimolar Mg(2+) is required for maximal activity, but Mn(2+) will support phosphorylation at a lower level. The average molecular weight as determined by sucrose gradient sedimentation and gel filtration is approximately 55,000. Under conditions of low ionic strength [less than 250 millimolar (NH(4))(2)SO(4)] soybean casein kinase forms higher molecular weight aggregates with other chromosomal proteins in the preparation. The enzyme activity is not affected by cyclic AMP. Casein kinase shows a broad optimum between 7 and 8 and the isoelectric point is approximately 9. Preliminary data indicate that soybean casein kinase will not phosphorylate soybean RNA polymerases I or II, nor does it have any obvious effect on in vitro chromatin transcription by endogenous RNA polymerases. PMID- 16660410 TI - Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs. AB - The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) or autoclaved strains of B6 or B-48. Inhibition by B-48 or autoclaved B6 could still be obtained when these cells were added up to a maximum of 10 minutes after the addition of viable B6. Maximum inhibition occurred when these cells were added 10 minutes prior to the addition of B6. There was no inhibition observed when equal cell counts of B6 were added along with a Gram-positive bacterium or yeast cell, while inhibition was observed when these B6 cells were added simultaneously with other Gram negative cells. These results suggest that a physical, specific bacterial attachment that occurs within 10 minutes is necessary for tumor formation on potato discs. PMID- 16660411 TI - Inhibition of epicuticular wax deposition on cabbage by ethofumesate. AB - The weight of epicuticular wax on the surface of cabbage (Brassica oleracea var. Capitata ;Market Prize') leaves was reduced by soil treatments of ethofumesate (2 ethoxy-2,3-dihydro-3,3-dimethyl-5-benzofuranyl methanesulfonate) and EPTC (S ethyl dipropylthiocarbamate). Separation of epicuticular wax into major components by gas-liquid chromatography indicated that ethofumesate decreased the deposition of n-nonocosane and n-nonocosan-15-one on cabbage leaves but increased the deposition of a minor component, the long chain waxy esters. EPTC was less inhibitory to n-nonocosan-15-one deposition than was ethofumesate. EPTC did not increase long chain waxy ester deposition. Scanning electron micrographs revealed that ethofumesate almost totally eliminated the epicuticular wax on cabbage leaves while EPTC only diminished it. Cuticular transpiration was increased by ethofumesate but not by EPTC. Ethofumesate appears to be a more potent inhibitor of epicuticular wax deposition than EPTC. PMID- 16660412 TI - Isoenzyme of pyruvate kinase in proplastids from developing castor bean endosperm. AB - Proplastids from developing castor bean (Ricinus communis) endosperm have a pyruvate kinase activity which is extremely unstable on isolation from the organelle. It can be stabilized by 20 mm 2-mercaptoethanol in 20% ethylene glycol. In contrast the soluble pyruvate kinase is stable at 60 C for 10 minutes. The two activities have different pH optima. The soluble and the proplastid activities are eluted from a diethylaminoethyl-Sephadex A-25 sievorptive column at different ionic strengths. PMID- 16660413 TI - Evidence that the castor bean allergens are the albumin storage proteins in the protein bodies of castor bean. AB - The well characterized castor bean (Ricinus communis L.) allergens were identified as the low molecular weight albumin storage proteins in the matrix of the protein bodies in the endosperm. The methods of identification involved molecular weight estimation, amino acid composition, stability at 100 degrees C, solubility in various solvents, gel electrophoresis, and immunological techniques. The finding explains the wide distribution of allergens in various seeds. PMID- 16660414 TI - Alkylguanidine inhibition of ion absorption in oat roots. AB - The effect of various alkylguanidines on ion absorption and energy metabolism in oat (Avena sativa cv. Goodfield) roots has been investigated. Of several alkylguanidines tested, octylguanidine was the most effective inhibitor of both K(+) and Cl(-) absorption by excised roots. At 225 mum octylguanidine, the transport of both ions was inhibited within 60 seconds and to a similar extent. Octylguanidine inhibited mitochondrial oxidative phosphorylation and mitochondrial adenosine 5'-triphosphatase (ATPase). The plasma membrane ATPase was also inhibited if the membranes were diluted and pretreated with Triton X 100.Concentrations of octylguanidine giving half-maximal inhibition of K(+) influx, mitochondrial ATPase, oxidative phosphorylation, and plasma membrane ATPase were 50, 50, 100, and 275 mum, respectively. With increasing chain length, alkylguanidines (225 mum) became progressively more inhibitory to K(+) absorption and to the mitochondrial ATPase. Shorter chain guanidines slightly inhibited the plasma membrane ATPase, however, these compounds produced a slight stimulation in oxidative phosphorylation.Conditions of Triton treatment that were important in the elimination of permeability barriers of plasma membrane vesicles to ATP, Mg(2+), KCl, and octylguanidine were: concentration of Triton during pretreatment and in the assay media, concentrations of sucrose and plasma membrane during Triton treatment, and temperature of Triton treatment.Inhibition by octylguanidine of K(+) and Cl(-) absorption by excised oat roots may be due to an inhibition of either the plasma membrane ATPase or mitochondrial oxidative phosphorylation. The isolated plasma membrane did not appear to be permeable to octylguanidine since the plasma membrane ATPase was inhibited only after treating the membrane with Triton. This result indicates that the primary site of action of octylguanidine in excised root is more likely to be the plasma membrane ATPase than mitochondrial oxidative phosphorylation. PMID- 16660415 TI - Isoenzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinis communis L. AB - Two isoenzymes each of hexose-P isomerase, aldolase and 6-P-gluconate dehydrogenase have been found in the endosperm of developing castor beans (Ricinus communis L.). One isoenzyme for each activity is present in the proplastid fraction. Only one form of glucose-6-P dehydrogenase was found. It is suggested that the partition of an enzyme activity between cytosol and plastid is regulated by the synthesis of isoenzymes which are subcellular site specific. In addition, this report describes the use of diethylaminoethyl-Sephadex A-25 sievorptive chromatography for the preparation of plant enzymes. PMID- 16660416 TI - Changes in Membrane Permeability of Winter Wheat Cells following Freeze-Thaw Injury as Determined by Nuclear Magnetic Resonance. AB - Nuclear magnetic resonance (NMR) relaxation times were studied in acclimated and nonacclimated Kharkov winter wheat (Triticum aestivum L.) crowns and acclimated cell aggregates to determine if membrane permeability was altered by freezing. The NMR water signal decay consisted of two exponential components: a short one arising from extracellular water, and a long one arising from intracellular water. A slow freezethaw treatment of nonacclimated and 1-week acclimated crowns decreased the long relaxation time, suggesting membrane injury. Similar results were obtained for nonacclimated and acclimated crowns killed directly in liquid N(2).A significant increase in plasma membrane permeability to Mn(2+) was observed in acclimated freeze-killed crowns and cell aggregates. Freezing injury to plant tissue appears to be a membrane-related phenomenon, but more extensive injury occurs to nonacclimated and acclimated tissue with a high water content (cell aggregates) compared to acclimated tissue with a low water content (crowns). PMID- 16660417 TI - Association between Elemental Content and Fruit Ripening in rin and Normal Tomatoes. AB - Analysis of Ca and other inorganic ions in the pericarp of rin, a nonripening mutant, and normal tomato (Lycopersicon esculentum Mill) fruits revealed significant differences in their accumulations at advanced stages of fruit development. During early stages of fruit development, soluble Ca was higher in Rutgers and there were no detectable changes in the accumulation patterns of the other inorganic ions. In the mutant rin, bound Ca continued to increase with age and it was twice as high as compared to earlier stages. In the normal tomato, bound Ca decreased about 3-fold at later stages of development. Mg and Mn also showed some changes similar to Ca. K continued to increase with age and the mutant rin had lower levels than Rutgers throughout development. Other ions such as P, Zn, Cu, and Co were similar in the mutant and normal fruits. These results are interpreted as indicating that high levels of bound divalent cations in the mutant rin may be associated with an altered membrane and cell wall and play a role in fruit ripening. PMID- 16660418 TI - Inhibition of ethylene production in fruit slices by a rhizobitoxine analog and free radical scavengers. AB - The rhizobitoxine analog, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid (Ro), which effectively inhibits ethylene production in apple (Malus domestica Borkh.) and other tissues at concentrations at about 68 micromolar, inhibited ethylene production by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) fruit slices but only by about 15% in pink and ripe tomato tissue slices. Ethylene production in climacteric-rise and postclimacteric avocado slices was likewise relatively insensitive to 68 micromolar Ro. At 340 micromolar Ro, inhibition of ethylene production increased up to 50% in pink tomato slices, whereas 680 micromolar Ro was required to inhibit ethylene production by 30% in avocado slices. Incorporation of (14)C from [(14)C]methionine into ethylene in green and pink tomato tissues was inhibited by Ro to about the same extent as inhibition of total ethylene production. Results thus far are inconclusive as to the mechanism of Ro resistance in tomato and avocado tissues. At 1 millimolar, free radical scavengers such as benzoate, propyl gallate, nordihydroguaiaretic acid, and to a lesser extent, eugenol, inhibited ethylene production in both Ro sensitive (green tomato and apple) tissues and Ro-resistant (pink tomato and avocado) tissues. Therefore, free radical steps are suggested in the ethylene forming systems. PMID- 16660419 TI - Effects of Protein Synthesis Inhibitors on ent-Kaurene Biosynthesis during Photomorphogenesis of Etiolated Pea Seedlings. AB - Excised shoot tips from 10-day-old etiolated pea (Pisum sativum L. cv. Alaska) seedlings were incubated in solutions of chloramphenicol, cycloheximide, and lincomycin at different concentrations during periods of 0, 4, 8, and 12 hours of irradiation with high intensity white light. Enzyme extracts were prepared from the whole shoot tips and compared with extracts from nontreated shoot tips for their capacity to synthesize ent-kaurene from mevalonate. In control samples, kaurene synthesis increased during the first 8 hours of irradiation and decreased after 12 hours. Chlorophyll content increased steadily up to 12 hours of irradiation. Chloramphenicol and cycloheximide reduced both kaurene synthesis and chlorophyll formation to a similar extent during all periods of irradiation, the reduction being greatest after 8 hours of irradiation. Lincomycin, a specific inhibitor of the formation of chloroplast ribosomes in detached pea shoot tips, did not significantly affect kaurene synthesis activity but strongly inhibited chlorophyll formation. It is tentatively concluded that the increase in kaurene synthesis activity during normal photomorphogenesis in pea seedlings is due to photoinduction of de novo synthesis of one or more proteins involved in the biosynthetic pathway from mevalonate to kaurene. PMID- 16660420 TI - Rapid metabolism of propylene by pea seedlings. AB - Propylene uptake by intact pea seedlings (Pisum sativum L. cv. Alaska) was easily detected using standard gas chromatographic techniques suggesting rapid metabolism. Comparative studies with highly purified (14)C(3)H(6) and (14)C(2)H(4) under aseptic conditions verified that propylene was rapidly metabolized and indicated that some aspects of its metabolism were similar to that of ethylene since (14)C(3)H(6), like (14)C(2)H(4) (Beyer, Nature 1975, 255: 144-147), was oxidized to (14)CO(2) and incorporated into water-soluble tissue metabolites. However, (14)C(2)H(6) was metabolized at a substantially faster rate and unlike (14)C(2)H(4) the rate of (14)C(3)H(6) tissue incorporation exceeded its rate of oxidation to (14)CO(2). In addition the neutral (14)C-metabolites derived from (14)C(3)H(6) were chromatographically distinct from those formed from (14)C(2)H(4). PMID- 16660421 TI - C(2)H(4) metabolism in morning glory flowers. AB - Flowers of Ipomoea tricolor Cav. (cv. Heavenly Blue) were cut at various stages of development and evaluated for their ability to metabolize ethylene. Freshly cut buds or flowers were treated in glass containers for 8 hours with 6 mul/liter of highly purified (14)C(2)H(4). Following removal of dissolved (14)C(2)H(4), radioactivity was determined for the different flower tissues and trappd CO(2). (14)C(2)H(4) oxidation to (14)CO(2) and tissue incorporation occurred at very low to nondetectable levels 2 to 3 days prior to flower opening. About 1 day prior to full bloom, just at the time when mature buds become responsive to ethylene (Kende and Hanson, Plant Physiol 1976, 57: 523-527), there was a dramatic increase in the capacity of the buds to oxidize (14)C(2)H(4) to (14)CO(2). This activity continued to increase until the flower was fully opened reaching a peak activity of 2,500 dpm per three flowers per 8 hours. It then declined as the flower closed and rapidly senesced. A similar but smaller peak occurred in tissue incorporation and it was followed by a second peak during late flower senescence. This first peak in tissue incorporation and the dramatic peak in ethylene oxidation slightly preceded a large peak of natural ethylene production which accompanied flower senescence. The ethylene metabolism observed was clearly dependent on cellular metabolism and did not involve microorganisms since heat killing destroyed this activity and badly contaminated heat-killed flowers were unable to metabolize ethylene. PMID- 16660422 TI - Sulfur deprivation and nitrogen metabolism in maize seedlings. AB - The objective of this experiment was to elucidate the manner in which N metabolism is influenced by S nutrition. Maize (Zea mays L.) seedlings supplied with Hoagland solution minus SO(4) (2-) exhibited S deficiency symptoms 12 days after emergence. Prior to development of these symptoms, a decline in leaf blade nitrate reductase (NR, EC 1.6.6.1) activity was observed in S-deprived seedlings compared to normal seedlings. Twelve days after emergence, in vitro NR activity was diminished 50% compared to normal seedlings. Glutamine synthetase (EC 6.3.1.2) and NAD-glutamate dehydrogenase (EC 1.4.1.2) activities were less severely affected (19 and 13%, respectively, at day 12). NADP-glutamate dehydrogenase (EC 1.4.1.4) activity and leaf blade fresh weight were not altered by S deprivation. Concentrations of soluble protein and chlorophyll (a and b) in leaf blades were reduced 18 and 25%, respectively, at day 12. A significantly higher concentration of NO(3) (-)-N was observed for leaf blade and stem (culms, leaf sheaths, and unfurled leaves) fractions (46 and 31%, respectively) in S deprived plants. In contrast to the other parameters measured, NR activity in S deprived seedlings could be readily restored to the normal level by addition of SO(4) (2-). The apparent preferential effect of S deprivation on NR activity could be causally related to the observed changes in NO(3) (-)-N and soluble protein concentration. PMID- 16660423 TI - Metabolism of myo-Inositol by Germinating Lilium longiflorum Pollen. AB - Lilium Iongiflorum pollen tubes absorbed myo-[2-(3)H]inositol produced labeled metabolites which were separated into acid-soluble and -insoluble fractions. The soluble fraction contained labeled myo-inositol, d-glucuronic acid, myo-inositol 1-phosphate, and at least three other unidentified compounds. The acid-insoluble fraction contained considerable chloroformsoluble radioactivity and a labeled residue. Labeled myo-inositol was also absorbed by germinating pollen prior to the time of pollen tube initiation; however, there was a marked reduction in amounts of myo-inositol 1-phosphate and glucuronic acid produced by this pollen in comparison with growing pollen tubes. PMID- 16660424 TI - Properties of pyruvate kinase from soybean nodule cytosol. AB - The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N(2) fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K(+) and NH(4) (+) concentrations. In the presence of physiological concentrations of K(+) and high phosphoenolpyruvate (PEP) concentrations, NH(4) (+) inhibited enzyme activity. Comparisons of kinetic parameters (V(max) and apparent K(a)) for NH(4) (+) and K(+) with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K(a) (monovalent cation) and K(m) (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH(4) (+). PMID- 16660425 TI - Kinetics and Apparent K(m) of Oxygen Cycle under Conditions of Limiting Carbon Dioxide Fixation. AB - A mass spectrometer with a membrane inlet was used to monitor light-driven O(2) evolution, O(2) uptake, and CO(2) uptake in suspensions of algae (Scenedesmus obliquus). We observed the following. (a) The rate of O(2) uptake, which, in the presence of iodoacetamide, replaces the uptake of CO(2), showed a distinct plateau (V(max)) beyond approximately 30% O(2) and was half-maximal at approximately 8% O(2). We concluded that this light-driven O(2) uptake process, which does not involve carbon compounds, is saturated at lower O(2) concentrations than are photorespiration and glycolate formation. (b) In the absence of inhibitor, O(2) evolution was relatively unaffected by the presence or absence of CO(2). During the course of CO(2) depletion, electron flow to CO(2) was replaced by an equivalent flow to O(2). (c) There was a distinct delay between the cessation of CO(2) uptake and the increase in O(2) uptake. We ascribe this delay to the transient utilization of another electron acceptor-possibly bicarbonate or another bound form of CO(2). PMID- 16660426 TI - Protein Synthesis and Accumulation in Bean Cotyledons during Growth. AB - Analysis of total protein, of specific proteins by gel electrophoresis and immunoelectrophoresis, and of protein synthetic activity in vitro confirmed that intense protein synthesis and accumulation occurred as the French bean (Phaseolus vulgaris L). seed grew from 12 to 20 millimeters. These techniques showed that there was no globulin-1 (G1) fraction (requiring high salt for solubility) present in 6-millimeter seeds, and only very small amounts were synthesized in seeds less than 9 millimeters long. The 7- to 9-millimeter stages represent a 2 day transition period over which genetic information for the G1 protein becomes actively expressed, accounting for at least 50% of all protein synthesized in this tissue during the following 14 days. At maturity, the electrophoretic analysis confirmed that G1 globulin was the major storage protein, representing some 50% of the dry seed protein. Cell-free protein synthesis assays, including immunoprecipitation of the in vitro products, clearly showed G1 polypeptides to be among the polysome-directed products. PMID- 16660427 TI - Phototropic Response to Vectorial Light in Leaves of Lavatera cretica L. AB - The mechanism by which the mature leaf of certain plants reorients its lamina to face the sun throughout the day was studied in Lavatera cretica L. The photoreceptor for this response differs fundamentally from the one involved in the phototropic growth response, by sensing light as a vector, rather than as a difference in luminous flux. The photoreceptor is located in the veins, which radiate in the plane of the lamina from the pulvinus situated at the junction between the lamina and petiole. The integrated response to the messages from the different veins takes place by differential turgor changes in a motor tissue surrounding the central vascular cylinder of the pulvinus, in which the veins coalesce. The differential turgor in the different segments of the motor tissue determines the orientation of the lamina. The photoreceptor reacts only to a parallel light beam striking the vein obliquely (from above). When half of the lamina is shaded, the leaf does not reorient in response to perpendicular illumination and its reorientation in response to an oblique beam is slower and partial, to a greater extend when the half-leaf is centrifugally illuminated than when it is centripetally illuminated. Application of 2,3,5 tri-iodobenzoic acid to the base of the veins in the shaded half-leaf eliminated all restrictions from the response to centrifugal illumination and totally inhibited the response to centripetal illumination. The results are consistent with a hypothesis that centrifugally illuminated veins generate turgor in their associated motor tissue in the pulvinus by activating K(+) uptake, while centripetally illuminated veins cause loss of turgor in their associated motor tissue by deactivating K(+) uptake, which leads to passive leakage of K(+). When the entire lamina is exposed to oblique illumination, the centrifugally illuminated half and the centripetally illuminated half cooperate in the full response. Shaded parts of the lamina apparently interfere with the response by supplying their associated motor tissue with auxin, which presumably causes in it an active export of protons and concomitant uptake of K(+), thereby establishing a static "dark turgor" in it. PMID- 16660428 TI - Evaluation of the light/dark C assay of photorespiration: tobacco leaf disk studies with glycidate and glyoxylate. AB - Preincubation of illuminated tobacco (Nicotiana tabacum L.) leaf disks in glycidate (2,3-epoxypropionate) or glyoxylate inhibited photorespiration by about 40% as determined by the ratio of (14)CO(2) evolved into CO(2)-free air in light and in darkness. However, under identical preincubation conditions used for the light/dark (14)C assays, the compounds failed to reduce photorespiration or stimulate net photosynthesis in tobacco leaf disks based on other CO(2) exchange parameters, including the CO(2) compensation concentration in 21% O(2), the inhibitory effect of 21% O(2) on net photosynthesis in 360 microliters per liter of CO(2) and the rate of net photosynthetic (14)CO(2) uptake in air.The effects of both glycidate and glyoxylate on the (14)C assay are inconsistent with other measures of photorespiratory CO(2) exchange in tobacco leaf disks, and thus these data question the validity of the light to dark ratio of (14)CO(2) efflux as an assay for relative rates of photorespiration (Zelitch 1968, Plant Physiol 43: 1829-1837). The results of this study specifically indicate that neither glycidate nor glyoxylate reduces photorespiration or stimulates net photosynthesis by tobacco leaf disks under physiological conditions of pO(2) and pCO(2), contrary to previous reports. PMID- 16660429 TI - Evidence for amino Acid-h co-transport in oat coleoptiles. AB - Microelectrode and tracer techniques were used to test for possible amino acid H(+) co-transport in coleoptiles of Avena sativa L. cv. "Garry." The amino acid analogue alpha-aminoisobutyric acid (AIB) caused transient depolarization of the membrane potential. The absolute magnitude of the maximum depolarization was affected by the same factors that affected AIB transport. Both increased with higher concentrations of AIB, increased with higher acidities in the medium, and were enhanced by indoleacetic acid (which hyperpolarized the membrane potential). AIB transport was reduced as K(+) concentrations in the medium were increased and by the metabolic inhibitor NaN(3), both of which reduce membrane potentials. Our data fit an amino acid-H(+) co-transport model in which transport is controlled by both the membrane potential and proton concentration components of the chemical potential difference of protons across the coleoptile cell membrane. PMID- 16660430 TI - Tissue Slice and Particulate beta-Glucan Synthetase Activities from Pisum Epicotyls. AB - beta-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg(2+) optimum, apparent K(m)) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various beta-glucanases all show that both beta-1,3 and beta-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. beta-1,4 Linkages predominate at low substrate (5 mum) concentration. Kinetic data indicate that the capacity to synthesize beta-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate. PMID- 16660431 TI - Soluble Factors in Pisum Extracts Which Moderate Pisum beta-Glucan Synthetase Activity. AB - Homogenates of growing regions of the pea (Pisum sativum L.) epicotyl contain soluble factors (130,000g supernatant) which alter pea beta-glucan synthetase activity, as assayed using the substrate UDP-glucose and either particulate fractions or tissue slices as source of enzyme. A heat-stable dialyzable component is present which enhances as much as 3-fold the synthesis of alkali soluble and -insoluble products from millimolar levels of substrate. A heat labile nondialyzable component is also present which suppresses synthesis. This component dominates (the net effect of total crude extract) when low (mum) levels of substrate are employed. Methylation analysis shows that both components primarily affect the proportion of beta-1,4 rather than beta-1,3 linkages which are synthesized. The enhancing factor increases V(max) of the synthetase system and only activates in the presence of high levels of substrate. The suppressing factor appears to inactivate the synthetase, since losses of product or substrate are not significant during brief incubation with extract, the factor acts progressively with time with a pH optimum, and it destroys activity during preincubation with particles or slices. It co-precipitates with a protease (gelatinase) at between 20% and 40%-saturated (NH(4))(2)SO(4), and it co fractionates with a major component of total protease on Sephadex gel columns (G 200) with an elution volume corresponding to molecular weight 65,000. The concentrations of these factors are such that they could be natural moderators of synthetase activity in vivo if the two were ever brought in contact, and the inactivator could account for the lability of beta1,4-glucan synthetase which occurs upon tissue homogenization. PMID- 16660432 TI - Comparison of chorismate mutase isozyme patterns in selected plants. AB - A wide variety of plants have been assayed to determine if they contain three isozymes of chorismate mutase (EC 5.4.99.5) as does alfalfa (Medicago sativa L.) or two isozymes, as does mung bean (Phaseolus aureus). The isozymes were separated by disc electrophoresis. All anthophyta with the exception of some closely related Leguminosae contained three isozymes of chorismate mutase. The one coniferophyta (a pine), and pterophyta (a fern) and one microphyllophyta (a Selaginella) assayed contained two isozymes of chorismate mutase. All plants assayed contained measurable chorismate mutase levels and at least two isozymes of chorismate mutase. PMID- 16660433 TI - Fermentative Metabolism of Hydrogen-evolving Chlamydomonas moewusii. AB - The anaerobic metabolism of Chlamydomonas moewusii under both light (160 lux) and dark conditions has been examined using manometric and enzymic techniques. During anaerobiosis starch is broken down to glycerol, acetate, ethanol, CO(2), and H(2). The release of CO(2) and H(2) comes to an end when the starch pool is depleted.There are only slight differences in the ratio of the end products of fermentation between light and dark metabolism. In the light, glycerol production is diminished and H(2) evolution is enhanced, whereas the production rate of all other end products generally does not change. PMID- 16660434 TI - Effects of inhibitors of catalase on photosynthesis and on catalase activity in unwashed preparations of intact chloroplasts. AB - The catalase activity of unwashed preparations containing intact spinach (Spinacia oleracea L.) chloroplasts is inhibited both by cyanide and by azide at concentrations which also cause inhibition of photosynthetic CO(2)- dependent O(2) evolution.Aminotriazole can also be used to inhibit this contaminant catalase, and in this case inhibition of catalase can be achieved at aminotriazole concentrations which have little effect on the rate of photosynthetic CO(2) fixation. Aminotriazole may be used as a specific inhibitor of catalase in order to demonstrate inhibition of photosynthesis by added H(2)O(2).It is therefore concluded that inhibition of photosynthesis by cyanide and azide does not necessarily result from inhibition of catalase in the chloroplast preparation, and that intact chloroplasts do not produce inhibitory concentrations of H(2)O(2) under the best experimental conditions for CO(2) fixation. PMID- 16660435 TI - Purification and Characterization of the Photoreducible b-type Cytochrome from Dictyostelium discoideum. AB - The photoreducible cytochrome (Cyt) b from Dictyostelium discoideum was purified by differential precipitation with ammonium sulfate and chromatography over Sephadex G-100, diethylaminoethyl-cellulose, and calcium phosphate. The purified Cyt is composed of a single subunit of 15,000 daltons including a noncovalently bound protohaem, and exhibits in the reduced form alpha absorption bands at 555.5 and 560 nm at room temperature and 551 and 558.5 nm at 77 K. This Cyt is similar in some respects to Cyt b(557.5) from complex II of beef heart mitochondria, and to Cyt b(555) from the microsomal fraction of mung bean seedlings. Photoreduction by blue light of the purified Cyt b requires the addition of flavin; flavoprotein isolated from D. discoideum was the most active of four flavoproteins tested in catalyzing the photoreduction while diaphorase and l-amino-acid oxidase were inactive. PMID- 16660436 TI - Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana. AB - The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l glutamate:NAD(+) oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K(m) values for alpha ketoglutarate, NADH, NH(4) (+), NAD(+), and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the K(m) for alpha-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K(m) values for NADH and NAD(+) were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between alpha-ketoglutarate and an epsilon amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH(4) (+)-inducible glutamate dehydrogenase in this organism. PMID- 16660437 TI - Influence of the Irradiance on Carbohydrate Content and Rooting of Cuttings of Pine Seedlings (Pinus sylvestris L.). AB - Seedlings of Pinus sylvestris L. were grown for 6 weeks at an irradiance of either 8 or 40 watts per square meter in a controlled environment room. Cuttings from these plants were rooted in tap water for 75 days at either 8 or 40 watts per square meter. The photoperiod was 17 hours.During the first 30 days of the rooting period quantitative changes in carbohydrates were recorded in cuttings from the different treatments. The carbohydrate contents of the cuttings were mainly regulated by the irradiance during the stock plant stage and generally a higher carbohydrate level was found in cuttings from stock plants grown at 40 watts per square meter.The irradiance during the rooting period had only minor effects on the time course of root formation, whereas the irradiance during the stock plant stage did influence the subsequent root formation. Cuttings from stock plants grown at 8 watts per square meter rooted faster and with higher frequency than those from stock plants grown at 40 watts per square meter. These results are discussed in relation to the mentioned irradiance effects on carbohydrate content. PMID- 16660438 TI - Initial Organic Products of Fixation of [N]Dinitrogen by Root Nodules of Soybean (Glycine max). AB - When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate [(13)N]N(2), the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; (13)N in alanine becoms detectable at 1 minute of fixation and increases rapidly between 1 and 2 minutes. After 15 minutes of fixation, the major (13)N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas [(13)N]glutamine comprises only a small fraction of organic (13)N, and very little (13)N is detected in asparagine. The fixation of [(13)N]N(2) into organic products was inhibited more than 99% by C(2)H(2) (10%, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules. PMID- 16660439 TI - Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos. AB - The seeds of Cucurbita maxima contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globoid crystals. If glutaraldehyde fixation was followed by water washes or ethanol dehydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globoid crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generally an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis. PMID- 16660440 TI - Pyrimidine Metabolism in Lemna minor: I. Functional Compartmentation of Chloroplast Pyrimidine Metabolism in a Higher Plant. AB - Cytidine deoxyriboside (Cdr), uridine deoxyriboside (Udr), and guanosine deoxyriboside (Gdr), induce quantitative bleaching of the fronds of Lemna minor (duckweed) during growth in continuous light on photoheterotrophic medium. Cdr induced bleaching is not accompanied by a reduction in frond multiplication rate, but Udr- and Gdr-induced bleaching is. Bleaching by Cdr is fully prevented by thymidine (Tdr), cytidine (Cr), or uridine (Ur), but not by orotic acid (OA) which itself inhibits growth. Bleaching by Udr is not antagonized by Tdr, Cdr, Cr, Ur, or OA. The ability of Cdr to induce phenocopies of chlorophyll-deficient mutants in the absence of effect on growth rate is interpreted as indicating a functional compartmentation of pyrimidine metabolism between chloroplast and whole cell. On the assumption that Cdr induces bleaching by regulating the biosynthesis of deoxynucleoside triphosphates, and in analogy with the antagonism of fluorodeoxyuridine effects on growth by Tdr, Cr, or Ur, the suggestion is made that deoxycytidine is converted to thymidylate by a step other than that utilizing thymidylate synthetase. PMID- 16660441 TI - Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain. AB - Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K(+)-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots. PMID- 16660442 TI - Development and Repair of Photosystem II Activity in Normal and Chloramphenicol treated Euglena gracilis Cells. AB - Photosystem II activity, development, and organization were studied in membranes from dark-adapted Euglena gracilis Klebs var. Z Pringsheim cells during a modulated greening process of light-dark-light cycles. The results obtained from measurements of overlapping partial photosystem II (PSII) reactions (fluorescence induction parameters, quantum yield, flash yield, and maximal rate of H(2)O --> 2,6-dichlorophenolindophenol [DCIP] and 1,5-diphenylcarbazide [DPC] --> DCIP reactions) during these cycles indicate the formation of active PSII units in the dark. The necessity for proteins from the chloroplast translational machinery for this formation is evidenced by the inhibition of synthesis of the PSII units in chloramphenicol-treated cells. The effect of this drug, both during the dark and second light periods, can be summarized as follows: (a) disruption of the electron transfer connection to the plastoquinone pool, or decrease in the pool size; (b) loss of excitation energy transfer efficiency in the second light period; (c) impairment of the O(2) evolution appratus, as shown by comparison of the efficiency of DCP and H(2)O as electron donors.These conclusions are based on the use of a previously developed method of measurement and analysis of data (Cahen et al. 1976 Plant Physiol 58: 257-267). PMID- 16660443 TI - Two Biosynthetic Pathways to delta-Aminolevulinic Acid in a Pigment Mutant of the Green Alga, Scenedesmus obliquus. AB - Pigment mutant C-2A' of the unicellular green alga Scenedesmus obliquus develops only traces of chlorophyll and has no detectable amount of delta-aminolevulinic acid (ALA) when grown in the dark. In light it develops ALA and in the presence of levulinic acid (LA), a competitive inhibitor of ALA dehydratase, it accumulates 0.18 mmoles of ALA per 10 microliters of packed cell volume per 12 hours. This amount could be increased up to 15 times by feeding precursors and cofactors.Incubation with [U-(14)C]glutamate, [1-(14)C]glutamate, and [2 (14)C]glycine yielded significantly labeled ALA, whereas [1-(14)C]glycine did not label the ALA specifically. Thus, two pathways using either glycine/succinyl coenzyme A or incorporating the whole C-5-skeleton of glutamate into ALA are present in this alga. The efficiency of the glycine/succinyl-coenzyme A pathway seems to be three times higher than that of the glutamate pathway. Incubation with [5-(14)C]2-ketoglutarate, which can serve both pathways as a precursor, resulted in radioactivity of ALA as high as the sum of both labeling with [1 (14)C]glutamate and [2-(14)C]glycine.Since the newly synthesized chlorophyll was radioactive regardless of labeled substrate employed, both pathways culminate in chlorophyll formation. PMID- 16660444 TI - Physiological Effects of Surface Waxes: I. Light Reflectance for Glaucous and Nonglaucous Picea pungens. AB - Foliage reflectance was studied on glaucous and nonglaucous foliage of blue spruce (Picea pungens Engel.). Current-year and 1-year-old glaucous and nonglaucous foliage from mature trees and seedling glaucous and nonglaucous foliage had similar reflectance patterns in the 350 to 800 nanometer region. The highest reflectance was in the 750 to 800 nanometer region and the lowest reflectance was in the 670 nanometer region. Glaucous foliage had a higher percentage of light reflectance at all of the wavelengths of light. The largest difference of reflectance between glaucous and nonglaucous foliage was in the 350 nanometer region with a general decline in the difference to the smallest difference at the 800 nanometer region. PMID- 16660445 TI - Phytochrome-induced Increase of Fluorescein Translocation in Mung Bean Hypocotyls. AB - Moderate doses of red (660 nanometer) irradiation cause a rapid increase in the translocation of fluorescein in dark-grown mung bean hypocotyl (Vigna radiata L.) segments. The increase fails to appear following large doses of red (660 nanometers) irradiation. The red induced increase is prevented by a subsequent far red (730 nanometer) irradiation. Reversibility suggests the participation of phytochrome in the process. The increase in translocation is attributed to the generation of a positive electrostatic charge in the plasma membrane by some action of phytochrome on membrane molecules. PMID- 16660446 TI - Host-Pathogen Interactions: XIV. Isolation and Partial Characterization of an Elicitor from Yeast Extract. AB - An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi. PMID- 16660447 TI - Kinetic determination of the genome size of the pea. AB - Renaturation of pea (Pisum sativum) DNA has been used to estimate the size of the pea genome and the fraction of pea DNA containing repeated DNA sequences. Pea DNA renaturation and single copy tracer renaturation indicate that the size of the pea genome is 0.5 picograms. More than 70% of pea DNA sequences are repeated from 100 to 5,000 times. PMID- 16660448 TI - Temperature and photocontrol of onoclea spore germination. AB - Germination of Onoclea sensibilis L. spores is controlled by light and temperature. Temperatures of 30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minimum exposure of 16 to 24 hours at 30 C, suggesting that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonadditive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturating dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Dose response curves for red light induction of germination after varying times of imbibition at 25 C show no concomitant loss of sensitivity of the spores to red irradiation. This suggests that the mechanism and/or pathway of thermoinduction of germination differs from that of photoinduction. The loss of thermosensitivity as a result of presoaking at 25 C can be prevented if the spores are imbibed at 25 C in osmotic agents such as 0.3 molar mannitol or 0.1 gram per liter of polyethylene glycol 400 or in 0.08% dimethylsulfoxide or 10 micrograms per milliliter of herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(alpha,alpha,alpha trifluoro-m-tolyl-3-(2H)pyridazinone). The latter two substances are hypothesized to act upon membranes. These results suggest that the degree of hydration and possibly changes in membrane properties play a role in the change in sensitivity of Onoclea spores to temperature. PMID- 16660449 TI - Association of Potassium and Some Other Monovalent Cations with Occurrence of Polyphosphate Bodies in Chlorella pyrenoidosa. AB - Phosphate-starved Chlorella pyrenoidosa cells formed polyphosphate bodies (PB) upon transfer into nutrient solutions containing phosphate and potassium, or another monovalent cation, such as Na(+), NH(4) (+), Li(+), or Rb(+). The phenomenon was studied by chemical analyses, light microscopy, and electron microscopy.When the P-starved cells were transferred into a complete nutrient solution containing 100 micromolar P, they accumulated large quantities of P and K within several hours. The accumulation was accompanied by a corresponding appearance of PB in the cells. The absence of K from the medium prevented appreciable P accumulation and PB formation, but omitting Ca or Mg did not.The P starved cells exposed to a simple solution of at least 20 micromolar H(3)PO(4) and 100 micromolar KHCO(3) responded in a similar manner as the cells exposed to the complete nutrient solution. However, the PB appeared structurally different.It is proposed that monovalent cations are essential for PB formation in C. pyrenoidosa. K is suggested to be a major component of PB formed in K sufficient media. PMID- 16660450 TI - Interdependence of Nitrogen Nutrition and Photosynthesis in Pisum sativum L: I. Effect of Combined Nitrogen on Symbiotic Nitrogen Fixation and Photosynthesis. AB - Photosynthesis, primary productivity, N content, and N(2) fixation were determined as a function of applied NH(4) (+) in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH(4) (+) was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH(4) (+), and total N content increased between 0 and 2 millimolar NH(4) (+) but did not change significantly with higher NH(4) (+) applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH(4) (+) concentration (16 millimolar). Symbiotic N(2) fixation by inoculated plants was maximal in peas grown with 2 millimolar NH(4) (+); and apparent relative efficiency of N(2) fixation, calculated from C(2)H(2) reduction and H(2) evolution, was maximal in the 2 to 4 millimolar NH(4) (+) concentration range. The capacity to fix N(2) through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH(4) (+) concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH(4) (+) concentration uninoculated plants had greater CER, N content, and dry weight. PMID- 16660451 TI - Interdependence of Nitrogen Nutrition and Photosynthesis in Pisum sativum L: II. Host Plant Response to Nitrogen Fixation by Rhizobium Strains. AB - Physiological responses to infection by strains of Rhizobium leguminosarum which differed in their capacity to reduce N(2) were determined in 26-day-old pea plants (Pisum sativum L. cv. Alaska) grown under uniform environmental conditions in the absence of combined N. The highest N(2) reduction rates, calculated from H(2) evolution and C(2)H(2)-dependent C(2)H(4) production measurements, were approximately 6-fold greater than the lowest. Higher N(2) fixation rates were associated with greater CO(2) exchange rates (R(2) = 0.92) and carboxylation efficiency (R(2) = 0.99). Increases in the apparent relative efficiency of N(2) fixation [1-(H(2) evolved in air/C(2)H(2) reduced)] (acteroid efficiency) were associated with increases in whole-plant N(2) fixation efficiency (N(2)/CO(2) reduction ratio) (R(2) = 0.95). Whole-plant dry weight and total N content were related by regression analysis (R(2) = 0.98); both parameters were enhanced by increased N(2) fixation in a manner analogous to previously reported increases caused by greater external applications of NH(4) (+). These data reveal that photosynthetic parameters in genetically uniform host plants grown under identical environmental conditions are affected by N(2) fixation characteristics of the rhizobial symbiont. The measured efficiencies of micro- and macrosymbiont are directly related under uniform environments. PMID- 16660452 TI - Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants. AB - Some studies on the effects of xanthine oxidase inhibitor allopurinol [4 hydroxypyrazolo(3,4-d)pyrimidine] on allantoin metabolism of soybean plants (Glycine max cv. Tamanishiki) are reported. Soybean seedlings, aseptically germinated for 96 hours on agar containing 1 millimolar allopurinol, contained only slight amounts of allantoin, allantoic acid, and urea as compared with controls. Analysis of purines and pyrimidines of the allopurinol-treated seedlings showed marked accumulation of xanthine both in the cotyledons and seedling axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyledons had fallen. For nodulated plants, allopurinol caused a significant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a striking accumulation of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated seedlings. Allopurinol at a concentration of 50 micromolar strongly inhibited xanthine oxidase prepared from soybean nodules.The results suggested that the main pathway of allantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules. PMID- 16660453 TI - A Simple and Automatic Leaflet Movement-monitoring System. AB - A simple and automatic leaflet movement-monitoring system using phototransistors is described for Albizzia julibrissin. PMID- 16660454 TI - Effects of irradiance on relative growth rates, net assimilation rates, and leaf area partitioning in cotton and three associated weeds. AB - Cotton (Gossypium hirsutum L. var. ;Stoneville 213'), velvetleaf (Abutilon theophrasti Medic.), redroot pigweed (Amaranthus retroflexus L.), and hemp sesbania (Sesbania exaltata [Raf.] Cory) were grown in a controlled environment room at 31/25 C day/night temperature and three irradiances: 90, 320, and 750 mueinsteins meter(-2) second(-1). From total dry weights and leaf areas determined at intervals during the first exponential phase of growth, we used mathematical growth analysis techniques to calculate net assimilation rates (NAR), relative growth rates (R(w)), relative leaf area expansion rates (R(a)), leaf area partition coefficients (LAP), and leaf area ratios (LAR). In all four species, R(w), R(a), and NAR decreased with decreasing growth irradiance, while LAP and LAR increased. Within each species, R(w) was positively correlated with NAR but negatively correlated with LAP and LAR. In comparisons among the four species within each growth irradiance, R(w) was positively correlated with LAP. We discuss the relationship between LAP and LAR and show that LAP = (R(a)/R(w)) (LAR). PMID- 16660455 TI - Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination: II. Glyoxysomal Enzyme Development in Embryos. AB - The sequence of glyoxysomal enzyme development was investigated in cotyledons of cotton (Gossypium hirsutum L. cv. Deltapine 16) embryos from 16 to 70 days after anthesis (DAA). Catalase, malate dehydrogenase, and citrate condensing enzyme activities were barely detectable prior to 22 DAA, but showed dramatic increases from 22 to 50 DAA. Development of malate synthase activity, however, was delayed during this period, rising to peak activity from 45 to 50 DAA (just prior to desiccation) in the absence of any detectable isocitrate lyase activity. Substantial activities of all of these enzymes (except isocitrate lyase) persisted in the dry seeds. Isopycnic centrifugations on sucrose gradients demonstrated that the enzymes were compartmentalized within particles increasing in buoyant density with time of development (1.226 to 1.245 grams per cubic centimeter from 22 to 50 DAA). Of particular significance were the observations in 22-day embryos of smooth surfaced membrane dilations of rough endoplasmic reticulum having cytochemical catalase reactivity, and the demonstrations of catalase activities in microsomal fractions isolated throughout the 16- to 50-DAA period. Our data do not allow determination of the mechanism(s) for enzyme activation and/or addition to previously existing or newly formed microbodies, but do show that development and acquisition of enzyme activities within glyoxysomes occur sequentially and thus are not regulated in concert as previously thought. PMID- 16660456 TI - Water permeability of chlorella cell membranes by nuclear magnetic resonance: measured diffusion coefficients and relaxation times. AB - Measurement by two nuclear magnetic resonance (NMR) techniques of the mean residence time tau(a) of water molecules inside Chlorella vulgaris (Beijerinck) var. "viridis" (Chodot) is reported. The first is the Conlon and Outhred (1972 Biochim Biophys Acta 288: 354-361) technique in which extracellular water is doped with paramagnetic Mn(2+) ions. Some complications in application of this technique are identified as being caused by the affinity of Chlorella cell walls for Mn(2+) ions which shortens the NMR relaxation times of intra- and extracellular water. The second is based upon observations of effects of diffusion on the spin echo of intra- and extracellular water. Echo attenuation of intracellular water is distinguished from that of extracellular water by the extent to which diffusive motion is restricted. Intracellular water, being restricted to the cell volume, suffers less echo attenuation. From the dependence of echo amplitude upon gradient strength at several values of echo time, the mean residence time of intracellular water can be determined. From the mean residence time of intracellular water, the diffusional water permeability coefficient of the Chlorella membrane is calculated to be 2.1 +/- 0.4 x 10(-3) cm sec(-1). PMID- 16660457 TI - Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays. AB - An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-(14)C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 x 10(-8) molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-(14)C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin. PMID- 16660458 TI - Dual Mechanisms in Polyamine-mediated Control of Ribonuclease Activity in Oat Leaf Protoplasts. AB - Dibasic amino acids and polyamines added to oat (Avena sativa L.) leaf protoplast isolation media decrease the RNase activity of extracted protoplasts relative to controls. This effect, which is manifested even when the added polyamine is removed by exhaustive dialysis prior to assay, is due to a prevention of the rise in RNase activity which usually follows protoplast isolation. Polyamines, but not dibasic amino acids, also decrease RNase activity in vitro. This in vitro effect seems to result from electrovalent attachment of the polyamine to the RNA, because the greater the net positive charge on the polyamine, the greater is its inhibitory effect in vitro. The activity of dibasic amino acids when added during protoplast isolation probably results from their conversion to polyamines. PMID- 16660459 TI - Rapid Production of Auxin-induced Ethylene. AB - The time course of auxin-induced ethylene production was determined in mesocotyl segments of etiolated sorghum (Sorghum bicolor L. Moench) seedlings. The latent period between addition of auxin and a detectable rise in ethylene release was 15 to 20 minutes in four different genotypes. This may indicate that the initial effect of auxin on ethylene production is too rapid to involve synthesis of an ethylene-producing enzyme. The technique devised for these experiments involves placing tissue segments end to end in a glass tube, and it allows simultaneous determination of growth and ethylene production. PMID- 16660460 TI - Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis. AB - Either NO(3) (-) (16 millimolar) or NH(4) (+) (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO(3) ( ) or NH(4) (+) was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO(3) (-) or NH(4) (+). The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs. PMID- 16660461 TI - Oxidation of proline by plant mitochondria. AB - Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O(2) uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O(2), but not NAD. The major ninhydrin-positive product formed via Delta(1)-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO(2), organic acids, and aspartate were recovered after [(14)C]proline feeding experiments. These results demonstrate the mitochondrial association and O(2) dependence of plant proline metabolism. PMID- 16660462 TI - In Vivo Synthesis of Crown Gall-specific Agrobacterium tumefaciens-directed Derivatives of Basic Amino Acids. AB - Several kinds of primary sunflower (Helianthus annuus) crown gall tissues were established in tissue culture and then labeled in vivo with either [(14)C]arginine, [(14)C]histidine, [(3)H]lysine, or [(3)H]ornithine. Crown gall tissues incited by Agrobacterium tumefaciens strains that utilize octopine as a sole source of carbon or nitrogen for growth synthesized the four members of the N(2)-(1-carboxyethyl)-amino acid family: octopine, histopine, lysopine, and octopinic acid. Those tissues incited by A. tumefaciens strains that utilize nopaline synthesized nopaline and two new compounds, a lysine and an ornithine derivative (ornaline). A normal tissue culture, a habituated tissue culture, and a crown gall culture from a strain of the bacteria unable to utilize either octopine or nopaline did not synthesize any of the amino acid derivatives. We could not detect any other crown gall-specific derivatives of the four basic amino acids. PMID- 16660463 TI - Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene. AB - Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction. PMID- 16660464 TI - Isolation and Characterization of Carotenoid-rich Lipid Globules from Peridinium foliaceum. AB - Carotenoid-rich oil globules were isolated from the cytoplasm of the binucleate dinoflagellate, Peridinium foliaceum. These orange globules were collected from ruptured cells by ultracentrifugation on a sucrose density gradient, and checked for purity by electron microscopy. The osmiophilic globules were assayed for lipid (including pigment) and protein content. The lipid to protein ratio was 1.39:1, with a calculated density of the globules of 1.05 grams per cubic centimeter. The lipids were composed of hydrocarbon, wax ester (phytyl ester), triglyceride, and polar (no phospholipid) fractions. The biochemical composition indicated that the globules function as a reservoir of energy-rich components in the cell. Microspectrophotometric observations were consistent with pigment analyses which demonstrated that the globules were carotenoid-rich. In addition to beta-carotene, gamma-carotene, and canthaxanthin, the carotenogenic precursors: phytoene, phytofluence, zeta-carotene and beta-zeacarotene were isolated from the globules. Corrected fluorescence maxima of phytoene and phytofluene in hexane were recorded at 340 and 490 nanometers, respectively. Carotenes constituted 3.3% of the total oil globule lipid. The possibility of an extraplastidic carotenogenic enzyme system in P. foliaceum is discussed. PMID- 16660465 TI - Isolation of intact plastids from protoplasts from castor bean endosperm. AB - Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose "Onozuka" R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield. PMID- 16660466 TI - Hydrolases in vacuoles from castor bean endosperm. AB - Vacuoles were prepared from endosperm tissue of 4-day-old castor bean seedlings (Ricinus communis var. Hale) and purified on a stepped sucrose gradient. It was shown by assays of marker enzymes that there was only trace contamination of the final preparation by other organelles (mitochondria, glyoxysomes, nuclei, spherosomes, and plastids) and by cytoplasmic components. Hydrolytic enzymes (acid protease, carboxypeptidase, phosphodiesterase, RNAase, phytase and beta glucosidase) were present in the isolated vacuoles in amounts indicating a primarily vacuolar localization in vivo. The vacuoles also contained storage protein and high concentrations of sucrose. The over-all results indicate that the vacuoles from castor bean endosperm are the site of hydrolysis of the constituents of the protein bodies and are a temporary storage compartment for the sucrose produced from fat and protein reserves. PMID- 16660467 TI - Longitudinal profiles of carbon dioxide fixation capacities in marine macroalgae. AB - Fucus serratus L., Fucus spiralis L., and Fucus vesiculosus L. (Fucales, Phaeophyceae) as well as Laminaria digitata (Huds.) Lamour., Laminaria hyperborea (Gunn.) Fosl., and Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyceae) have been investigated for the distribution of enzymic CO(2) fixation capacities via phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (PEP-CK) and via ribulose-1,5 bisphosphate carboxylase (EC 4.1.1.39) (RubP-C) in different regions of the thalli. The maximum of PEP-CK activity is found to be confined to the growing regions of the algae, while the activity of RubP-C achieves its highest values in the entirely differentiated parts of the fronds. These findings are confirmed by the results of photosynthetic and light-independent (dark) carbon assimilation as determined by in vivo(14)CO(2) fixation. The physiological significance of these differential patterns of carboxylation patterns is discussed with respect to the ontogenetic stage and the chemical constitution of the different thallus parts. PMID- 16660468 TI - Carbon assimilation and translocation in soybean leaves at different stages of development. AB - Carbon assimilation, translocation, and associated biochemical characteristics of the second trifoliolate leaf (numbered acropetally) of chamber-grown soybean, Glycine max (L.) Merr., plants were studied at selected stages of leaf development during the period from 10 to 25 days postemergence. Leaves of uniform age were selected on the basis of leaf plastochron index (LPI).The test leaf reached full expansion (A(max)) and maximum CO(2) exchange rates on a leaf area basis at 17 days postemergence (LPI 4.1). Maximum carbon exchange rates per unit dry weight of lamina were attained several days earlier and declined as specific leaf weight increased. Chlorophyll and soluble protein continued to increase beyond the attainment of A(max), but were not accompanied by further increases in photosynthetic rates.Much of the fixed carbon in leaves is partitioned between starch and sucrose. Starch content of leaves as a percentage of dry weight at the end of an 11-hour photoperiod was taken as an indication of the potential energy reserve accumulated by the leaf. Starch levels were the same regardless of leaf age during the period from 0.3 A(max) to 7 days after attaining A(max). Respiratory and synthetic activity of leaves decreased considerably during the same period, suggesting that starch accumulation is not entirely controlled by the energy demands of the leaf.Sucrose content increased steadily during leaf expansion and was accompanied by corresponding increases in sucrose phosphate synthetase (EC 2.4.1.14) activity and translocation rates. Sucrose phosphate synthetase may have an important regulatory role in photosynthate partitioning and translocation. PMID- 16660469 TI - Comparison of photosynthetic activities of spinach chloroplasts with those of corn mesophyll and corn bundle sheath tissue. AB - Bundle sheath and mesophyll chloroplasts from Zea mays showed comparable rates of O(2) evolution, which amounted to about half of the rate observed in spinach (Spinacia oleracea) chloroplasts.Ratios of 4.5, 4.6, and 6.2 Mn(2+) atoms per 400 chlorophylls were observed in mesophyll, bundle sheath, and spinach chloroplasts, respectively. These ratios roughly correspond to the observed O(2) evolution rates.Rates of electron transport from water to methylviologen (photosystem I and II) in both types of corn chloroplasts were about one-third that in spinach. Compared to spinach, transport rates from reduced diaminodurene to methylviologen (photosystem I) were about one-third and greater than one-half in mesophyll and bundle sheath material, respectively.In both types of corn chloroplasts, electron flow from photosystem II to P700 was abnormal. This observation, together with the low rates of all activities, suggests that damage occurred during isolation. Such damage may limit the quantitative significance of observations made with these materials (including the following data).Measurements of flash yields of O(2) evolution or O(2) uptake showed that the size of the photosynthetic unit was the same in photosystems I and II and in all three types of chloroplasts (about 400 chlorophylls per equivalent).Similarity of the photochemical cross-section of the two photosystems in the three preparations was also found in optical experiments: that is the half-times of the fluorescence rise in the presence of 3 (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (photosystem II) and of the photooxidation of P700 (photosystem I).The ratio of P700 to chlorophyll appeared to be about 2-fold higher in bundle sheath chloroplasts than in the other materials (1/200 versus 1/400). PMID- 16660470 TI - Effects of gibberellic Acid and gold light on germination, enzyme activities, and amino Acid pool size in a dwarf strain of watermelon. AB - Gibberellic acid (GA(3)) promotes and continuous gold light inhibits germination of seeds of a dwarf strain (WB-2) of watermelon [Citrullus lanatus (Thunb.) Matsu. and Nakai]. Osmotic inhibition of germination with mannitol in light-grown seeds of WB-2 was only slightly reversed by GA(3) at the concentrations used, whereas, GA(3) substantially relieved osmotic inhibition in dark-grown seeds.The effects of GA(3) and gold light on development of catalase and invertase activities and on levels of free amino acids in germinating seeds of WB-2 were examined. Light depressed development of catalase and invertase activity. Levels of free amino acids increased more slowly in embryonic axes of light- than dark incubated seeds, but in cotyledons higher levels of amino acids were maintained in light-grown seeds. GA(3) accelerated the development of catalase activity in whole embryos and invertase activity in embryonic axes, but did not significantly affect invertase activity in cotyledons during germination. GA(3) had little effect on amino acid pools in cotyledons and embryonic axes. PMID- 16660471 TI - Water Relations, Stomatal Behavior, and Root Conductivity of Red Osier Dogwood during Acclimation to Freezing Temperatures. AB - Red osier dogwood (Cornus stolonifera Michx.) was artificially acclimated by exposing plants to 8-hour short days (SD) and low (15/5 C) temperatures for 54 to 63 days. Several factors including transpiration rate, stomatal resistance, and root conductivity were correlated so that the rate of water loss in acclimating plants was higher during the first 30 to 40 days of the acclimation sequence. Six days after transferring plants to SD conditions, the stomatal resistance (r(8)) decreased significantly below the r(8) of the 16-hour long day (LD) control plants at the same temperature. Transpiration rate increased by approximately 20 to 30% in the plants transferred to SD. After the initially higher transpiration rate and greater stomatal opening, the stomates closed tightly during the last 2 weeks of acclimation and the transpiration rate of the SD plants dropped to well below the LD control plants. By the end of the acclimation sequence, root conductivity to water uptake was two to three times lower in the SD plants. Leaf xylem water potentials were similar or slightly lower in the plants kept under SD conditions during the first 5 to 7 weeks of the acclimation sequence. During the last 10 to 15 days of acclimation when the stomates closed, SD leaf water potential rose significantly above the plants in the LD conditions. During acclimation, stem water content decreased by 40 to 50%. Changes in tissue hydration can be indirectly related to plant hardiness and may be affected by alteration of stomatal resistance, transpiration rate, and root conductivity during acclimation. PMID- 16660472 TI - Role of Lectins in Plant-Microorganism Interactions: III. Influence of Rhizosphere/Rhizoplane Culture Conditions on the Soybean Lectin-binding Properties of Rhizobia. AB - The influence of rhizosphere/rhizoplane culture conditions on the ability of various rhizobia to bind soybean seed lectin (SBL) was examined. Eleven strains of the soybean symbiont, Rhizobium japonicum, and six strains of various heterologous Rhizobium species were cultured in root exudate of soybean (Glycine max [L.] Merr.) and in association with roots of soybean seedlings which were growing either hydroponically or in montmorillonite clay soil amendment (Turface). All 11 of the R. japonicum strains developed biochemically specific receptors for the lectin when cultured under these conditions, whereas six of the 11 did not develop such receptors when cultured in synthetic salts medium. Two cowpea strains also developed receptors for SBL. The other four heterologous strains of rhizobia gave no evidence of biochemically specific SBL binding in either synthetic salts media or rhizosphere/rhizoplane cultures. These results demonstrate that the environment provided by plant roots is an important factor in the development of specific lectin receptors on the cell surface of R. japonicum. PMID- 16660473 TI - Gradient of Growth, Spontaneous Changes in Growth Rate and Response to Auxin of Excised Hypocotyl Segments of Phaseolus aureus. AB - Spontaneous growth was studied in excised mung bean (Phaseolus aureus Roxb.) hypocotyl segments. Measurements were made with a growth-recording apparatus using displacement transducers on single 5- to 6-millimeter samples excised from the growth zone immediately below the hook.Even for a given zone and under controlled experimental conditions, there are differences in the spontaneous growth of individual explants. Nevertheless, in every case, two phases of endogenous acceleration are found at 15 to 20 minutes, and 120 to 150 minutes after excision. Accelerations were separated by steady growth phases. Knowledge of the spontaneous growth curve appears important for the choice of the time of application of experimental stimuli. Auxin was added at various times after excision (0 to 6 hours). The classical biphasic response to auxin was obtained when the hormone was added during a steady phase of growth. However, the response was difficult to interpret when the hormone was added during an acceleration phase.Spontaneous and indoleacetic acid-induced growth were studied along the hypocotyl. Spontaneous growth rate and growth potential revealed by indoleacetic acid changed markedly along the growth gradient. The nature of spontaneous changes according to experimental time and state of differentiation of the cells is discussed. PMID- 16660474 TI - Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age. AB - The metabolism of [1-(14)C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10(-6) to 10(-5) molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4 D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram. PMID- 16660475 TI - Availability of Chloride Affects the Balance between Potassium Chloride and Potassium Malate in Guard Cells of Vicia faba L. AB - Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0.1 equivalent of K(+) per liter in the absence or presence of Cl(-). In the absence of Cl(-), iminodiacetate, a presumed impermeant zwitterion, served as anion. With no Cl(-) in the medium, 91% of the K(+) imported into the guard cells during stomatal opening was neutralized by malate production; import of Cl(-) (presumably from the rest of the epidermal tissue) contributed 6%. In the presence of Cl(-), 50% of the necessary negative charges were provided by malate synthesis, 45% by Cl(-) import. Stomatal opening was not obviously affected by the chloride concentration in the incubation medium, but malate production declined roughly linearly with the logarithm of [Cl(-)] between 10(-5) and 10(-1) equivalent per liter. PMID- 16660476 TI - Activity of Thylakoid-bound Ribosomes in Pea Chloroplasts. AB - Pea (Pisum sativum) chloroplast thylakoid membranes were prepared by washing in hypotonic buffers. These membranes contained bound ribosomes which were active in protein synthesis when supplemented with soluble components from a strain of Escherichia coli low in ribonuclease. After dissolving the membranes by Triton and purification of the ribosomes, sucrose density gradient profiles indicated the presence of polysomal material as well as monomeric ribosomes. Most of the products of protein synthesis remained associated with the thylakoid membranes even after ribosomes were removed completely by high salt concentrations in the absence of Mg(2+). Of the newly formed products, 50% could be digested by pronase, while the remainder were protected by their association with the thylakoid membranes. The products are likely to be a mixture of intrinsic and extrinsic membrane proteins, with only the former completely protected by the membranes from attack by proteases. PMID- 16660477 TI - Cadmium alteration of root physiology and potassium ion fluxes. AB - Segments of oat (Avena sativa L.) roots which had been exposed to 1 millimolar CdSO(4) in quarter-strength Hoagland No. 1 solution exhibited decreased respiratory rates, ATP levels, membrane-bound ATPase activity, and reduced K(+) fluxes. Respiration and ATP levels were decreased after a 2-hour treatment with 1 millimolar CdSO(4) to 65 and 75%, respectively, of control rates. A membrane bound, Mg(2+)-dependent, K(+)-stimulated acid ATPase was rapidly inhibited to 12% of control activity in the presence of 1 millimolar CdSO(4). Potassium uptake into root segments was inhibited to 80% of control values after 30 minutes in the presence of CdSO(4). A 2-hour pretreatment of root segments with CdSO(4) inhibited K(+) uptake to 15% of control values. Cytoplasmic K(+) efflux was inhibited with 1 millimolar CdSO(4).The rates and the degree of Cd(2+) inhibition of the parameters listed above suggest that one of the first sites of Cd(2+) action is the plasmalemma K(+) carrier (ATPase) in oat roots. PMID- 16660478 TI - Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts. AB - Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein synthesizing activity as measured by the incorporation of [(14)C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP(2)) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [(14)C]leucine into RuP(2) carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B. PMID- 16660479 TI - Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots. AB - A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K(+) -stimulated ATPase activity were extracted with 1 m NaClO(4), and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500 fold purification over the activity present in the microsomes was obtained. The K(+) -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K(+) -stimulated activity with ATP, GTP, UTP, CTP, ADP, alpha + beta-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca(2+) inhibit and alkylguanidines stimulate the K(+) -ATPase while bound to microsomes, they have no effect on the purified enzyme. PMID- 16660480 TI - Fatty Acid synthesis in endosperm of young castor bean seedlings. AB - Enzyme assays on organelles isolated from the endosperm of germinating castor bean (Ricinus communis) by sucrose density gradient centrifugation showed that fatty acid synthesis from [(14)C]malonyl-CoA was localized exclusively in the plastids. The optimum pH was 7.7 and the products was mainly free palmitic and oleic acids. Both NADH and NADPH were required as reductants for maximum activity. Acetyl-CoA, and acyl-carrier protein from Escherichia coli increased the rate of fatty acid synthesis, while low O(2) levels suppressed synthesis. In the absence of NADPH or at low O(2) concentration, stearic acid became a major product at the expense of oleic acid. Fatty acid synthesis activity was highest during the first 3 days of germination, preceding the maximum development of mitochondria and glyoxysomes. It is proposed that the plastids are the source of fatty acids incorporated into the membranes of developing organelles. PMID- 16660481 TI - Studies on molybdenum absorption and transport in bean and rice. AB - The patterns of molybdenum (MoO(4) (2-)) absorption and transport were investigated in intact bean (Phaseolus vulgaris L.) and rice (Oryza sativa L. cv. I.R.8) plants. The mobility of MoO(4) (2-) absorbed by roots and by leaves was compared with that of a freely mobile element, Rb(+). Although MoO(4) (2-) absorption by bean roots was nearly as high as that of Rb(+), its transport to the shoot was considerably less. When MoO(4) (2-) was fed to one of the primary leaves, most of it was transported to the stem and root. Evidence obtained here showed that MoO(4) (2-) was mobile. Experiments with intact rice seedlings revealed large differences in the absorption and transport of MoO(4) (2-) between the plants grown in CaSO(4) and those in Hoagland solution. Molybdate uptake by excised rice roots was suggested to be an active process since it was greatly inhibited by a metabolic inhibitor. The presence of Mn(2+), Zn(2+), Cu(2+), CI( ), or SO(4) (2-) in the absorption medium reduced MoO(4) (2-) uptake which was markedly enhanced by the presence of Fe(2+). PMID- 16660482 TI - Characterization of protoheme levels in etiolated and greening plant tissues. AB - The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326). PMID- 16660483 TI - A comparison of photosynthetic characteristics of encelia species possessing glabrous and pubescent leaves. AB - Measurements of the dependence of photosynthesis on light, CO(2), and temperature are reported for two species of Encelia (Compositae) which differ in leaf pubescence and in geographical distribution. Encelia californica is glabrous and occurs in relatively mild, but arid habitats and Encelia farinosa is heavily pubescent and occurs in hot, arid habitats. Both species possess the C(3) photosynthetic pathway. Under high irradiances and normal atmospheric conditions the two species have high photosynthetic rates, exceeding 3 nanomoles of CO(2) per square centimeter per second (48 milligrams of CO(2) per square decimeter per hour) and complete light saturation does not occur by full noon sunlight. The high photosynthetic capacity is related to a high efficiency of utilization of intercellular CO(2) combined with high stomatal conductance. Leaf estimates of total soluble protein and fraction I protein are higher in these species than in most plants, although the proportion of fraction I protein is not higher. Both E. californica and E. farinosa attain a maximum rate of photosynthesis between 25 and 30 C, despite the fact that the two species grow in very different thermal habitats. Neither E. californica nor E. farinosa shows significant acclimation in the temperature dependence of photosynthesis when grown under different temperature regimes. The presence of leaf hairs which reduce leaf absorptance and consequently leaf temperature plays an important part in the ability of E. farinosa to survive in its native high temperature environment. When the effects of pubescence are taken into account, there are few if any significant differences in the photosynthetic characteristics of the two species. PMID- 16660484 TI - Effect of growth temperature on chloroplast structure and activity in barley. AB - Seedlings of barley (Hordeum vulgare L. cv. Abyssinian) were grown at constant temperature and light intensity and the properties and structure of chloroplasts in the primary leaf were examined. Seventeen growth temperatures ranging from 2 to 37 C were employed. Three major effects of the growth temperature were seen. (a) At very low and high growth temperatures chloroplast biogenesis was inhibited. This occurred in plants grown at temperatures above 32 C while growth at 2 C resulted in a mixed population of pale yellow, pale green, and green plants. (b) Chloroplasts were produced at all other temperatures tested but growth temperatures within a few degrees of those inhibitory to chloroplast development resulted in chloroplasts with abnormal properties and structure. Chloroplasts in the green plants grown at 2 and 5 C showed a number of structural peculiarities, including a characteristic crimping of granal thylakoids. Photoreductive activity, measured using ferricyanide as the Hill oxidant in the presence of gramicidin D, was high, but this activity in chloroplasts isolated from plants grown at 2 C showed thermal inactivation at temperatures 5 degrees lower than was the case with plants grown at higher temperatures. High growth temperatures (30 to 32 C) yielded chloroplasts with reduced photoreductive activity and a tendency toward the formation of large grana and disorientation of the lamellar systems with respect to one another. Chloroplasts of the most affected plants (grown at 32 C) frequently contained a very large elongated granum, with narrow intrathylakoid spaces. (c) Photoreductive activity was not constant at intermediate growth temperatures but steadily declined with decreasing growth temperatures between 27 and 11 C. Some alterations in chloroplast structure were also observed.The changes in chloroplast activity and structure indicate that acclimation to temperature takes place over the entire temperature range in which chloroplast development is permitted. PMID- 16660485 TI - NADH-Nitrate Reductase Inhibitor from Soybean Leaves. AB - A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO(3). The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V(max).The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators. PMID- 16660486 TI - Oxygen effects on photosynthesis and C metabolism in desert plants. AB - The effect of 1% and 21% O(2) upon (14)CO(2) assimilation by desert plants exposed for 10 to 90 seconds has been studied. The plants studied can be divided into three groups with respect to O(2). The C(3) plants display the usual Warburg effect. No changes could be observed in the intensity of photosynthesis as a function of O(2) content in another group of plants (showing signs of Crassulacean acid metabolism). In still another group of plants (C(4) plants) the stimulating effect of O(2) on photosynthesis could be detected. In C(3) plants, O(2) inhibits the processing of carbon through the Calvin cycle intermediates. The involvement of carbon in the glycolate pathway fails to explain completely the inhibiting effect of O(2) on photosynthesis. It is assumed that O(2) inhibits the enzymes of the Calvin cycle. In C(4) plants O(2) stimulates the incorporation of (14)C into malate and aspartate. The incorporation of (14)C into the intermediates of the Calvin cycle in C(4) plants is inhibited much like that in typical C(3) plants. PMID- 16660487 TI - Effects of Sulfite on Metabolism in Isolated Mesophyll Cells from Papaver somniferum. AB - Exposure (30 minutes) of leaf-free mesophyll cells from the C-3 plant, Papaver somniferum, to concentrations of sulfite (SO(2) + HSO(3) (-) + SO(3) (-)) up to 20 millimolar stimulated the rate of CO(2) incorporation as much as 30%. The sulfite rapidly affects the metabolism of newly incorporated CO(2). Ammonia incorporation into glutamine and subsequent transamination reactions were stimulated during the short term exposure periods while glycolate metabolism apparently was inhibited by bisulfite at two points in the pathway. The results further indicate that glycolate is the major precursor of glycine in these cells. Prolonged periods of exposure (24 hours) to sulfite had somewhat different effects on carbon metabolism: the high concentrations (10 to 20 millimolar) severely inhibited all aspects of cellular metabolism while lower concentrations (1 millimolar) appeared to inhibit ammonia incorporation but stimulated synthesis of sucrose and starch. PMID- 16660488 TI - Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries. AB - [U-(14)C]Sucrose, myo-[U-(14)C]inositol, [6-(14)C]- and [U-(14)C]glucuronate, UDP [U-(14)C]glucuronate, [U-(14)C]gluconate, and l-[1-(14)C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of (14)C into several metabolites was studied.[U-(14)C]Sucrose was the most effective precursor of cellulose in young grape berries and of glucose and fructose in mature berries. On the other hand, UDP-[U-(14)C]glucuronate was the best precursor of pectic substance, followed by [(14)C]glucuronate and myo-[U-(14)C]inositol. l-[1-(14)C]Ascorbic acid was the most effective precursor of tartaric acid. In young berries, [U-(14)C]sucrose and [U-(14)C]gluconate also produced labeled tartaric acid, the latter a somewhat better precursor in the 3 weeks following flowering. The remaining test compounds were only poor sources of (14)C for tartaric acid although all three, glucuronate, UDP-glucuronate, and myo-inositol, were utilized by the grape berry for pectin biosynthesis.These results strongly indicate that tartaric acid is synthesized by a C-1 oxidation mechanism of hexose in young grape berries. PMID- 16660489 TI - Malate and Dihydroxyacetone Phosphate-dependent Nitrate Reduction in Spinach Leaf Protoplasts. AB - Isolated spinach (Spinacia oleracea L. var. Bloomsdale) leaf protoplasts reduced nitrate at rates of 9 micromoles per milligram chlorophyll per hour in light with a 3- to 4-fold stimulation in the presence of HCO(3) (-). A similar stimulation of nitrate reduction in the absence of CO(2) fixation was obtained by the addition of malate, oxaloacetate (OAA), phospho-3-glyceric acid (PGA), or dihydroxyacetone phosphate (DHAP). Stimulation by malate and DHAP was light independent, while the PGA and OAA effect was light-dependent. Nitrate reduction was found to be coupled to the cytoplasmic oxidation of DHAP or malate. The PGA/DHAP and OAA/malate shuttle across the chloroplast envelope has been demonstrated to support CO(2) fixation and/or nitrate reduction. The leaf protoplasts readily assimilated nitrate into amino-N in a stoichiometric relationship. PMID- 16660490 TI - Endogenous gibberellin and abscisic Acid content as related to senescence of detached lettuce leaves. AB - Levels of gibberillins (GAs) and of abscisic acid (ABA) in attached leaves of romaine lettuce (Lactuca sativa L.) declined as the leaf became older. The time course of changes in hormone levels, determined in detached lettuce leaves kept in darkness, revealed that a sharp decline in GAs accompanied by a moderate rise in ABA occurred before the onset of chlorophyll degradation. As senescence advanced, no GAs could be detected and a considerable rise of ABA was observed. A similar sequence of hormonal modifications, but more pronounced, was observed in the course of accelerated senescence induced by either Ethephon or water stress. When kinetin or GA(3) was applied to detached leaves, the loss of chlorophyll and the rise in ABA were reduced. Bound GAs were detected in senescent leaves. They were not found in the kinetin-treated leaves, which contained a relatively high level of free GAs. The results suggest that senescence in detached romaine lettuce leaves is connected with a depletion of free GAs and cytokinins, which is thereafter followed by a great surge in ABA. PMID- 16660491 TI - Growth, Phosphate Pools, and Phosphate Mobilization of Salt-stressed Sesame and Pepper. AB - The growth and phosphate mobilization of control and salt-stressed sesame (Sesamum indicum L.) and pepper (Capsicum annuum L.) plants were examined to ascertain whether or not translocation limits growth of salt-stressed plants. Plants were grown in a complete nutrient solution with and without excess salt. One-half of the control and salt-stressed plants were later transferred to phosphate-free culture solution ("-P" plants). Measurements of growth and phosphate pools in leaves indicated that with or without salinity "-P" plants utilized their phosphate reserves to support growth for a time at rates equaling those of plants supplied with phosphate. The results indicate that mobilization was not limiting for growth of salt-stressed plants.Defoliation experiments were performed at a developmental stage when the import of assimilates by the youngest expanding leaves could be changed by removing certain source or sink leaves. These experiments also indicated that phloem transport was not limiting for leaf growth on salt-stressed plants. PMID- 16660492 TI - Relative Contribution of Cytochrome-mediated and Cyanide-resistant Electron Transport in Fresh and Aged Potato Slices. AB - The respiration of fresh potato (Solanum tuberosum, var. Russet Burbank) slices is predominantly cyanide-sensitive whether in the presence or absence of uncoupler. By contrast, the wound-induced respiration which develops in thin slices with aging is cyanide-resistant, and in the presence of cyanide, sensitive to chlorobenzhydroxamic acid, a selective inhibitor of the cyanide-resistant respiration. Titration of the alternate path in coupled slices with chlorobenzhydroxamic acid, in the presence and absence of cyanide, shows that the contribution of the cyanide-resistant pathway to the wound-induced respiration is zero. Similar titrations with uncoupled slices reveal that the alternate path is engaged and utilized extensively.The maximal capacity of the cytochrome path (V(cyt)) has been estimated in fresh and aged slices in the presence of the uncoupler carbonyl-cyanide m-chlorophenyl hydrazone. It has been found that V(cyt) of aged slices is but 30 to 40% higher than that of fresh slices. The results suggest that the bulk of the wound-induced respiration is mediated through the cytochrome pathway which exists in fresh slices in suppressed form, and which is fully expressed by slice aging. The engagement of the alternate path by uncouplers in aged slices is attributed to an increase in substrate mobilization, with the result that the electron transport capacity of the cytochrome chain is exceeded. PMID- 16660493 TI - Antimycin-insensitive Cytochrome-mediated Respiration in Fresh and Aged Potato Slices. AB - The effect of antimycin A on the respiration of fresh potato (Solanum tuberosum var. Russet Burbank) slices has been determined in the presence and absence of m chlorobenzhydroxamic acid (CLAM). Two antimycin-binding sites are indicated. At low concentrations antimycin alone inhibits respiration only slightly. When CLAM and low antimycin are added together, respiration is sharply inhibited, as in response to cyanide. High antimycin alone is as inhibitory as cyanide. The branch point to the alternate path is intact in fresh slices, as is the hydroxamate sensitive component. The full alternate path is inoperative, however, as indicated by the sensitivity to cyanide. The data suggest an alternate path loop which bypasses the high affinity antimycin site and returns electrons to the cytochrome path. Antimycin at high concentrations prevents articulation of the loop with the cytochrome path.The respiration of aged slices is not only markedly resistant to antimycin at high concentrations, but quite insensitive to CLAM in the presence of antimycin. A model is proposed which involves parallel paths within complex III of the cytochrome path, with one path bearing the high affinity, and the other the low affinity antimycin site. With slice aging the antimycin affinity of the latter site is even further reduced, providing a relatively antimycin-insensitive bypass to both the high affinity antimycin sensitive cytochrome path, and the CLAM-sensitive alternate path. The alternate path loop in fresh slices is presumed to feed into the low affinity antimycin sensitive arm of the cytochrome path. PMID- 16660494 TI - Cyanide-resistant Respiration in Fresh and Aged Sweet Potato Slices. AB - The respiration of fresh sweet potato (Ipomoea batatas) slices is resistant to, and often stimulated by, cyanide and antimycin A. m-Chlorobenzhydroxamic acid (CLAM), a selective inhibitor of the alternate path, inhibits respiration in the presence of cyanide and has a limited inhibitory effect in the presence of antimycin A. Thus, a partial bypass of the antimycinsensitive site is indicated. Respiration rises 2-fold at best with slice aging, the increment being cytochrome mediated. The cyanide-resistant pathway contributes neither to coupled fresh slice respiration nor to the induced respiration in the absence of inhibitors of the cytochrome path. In the presence of uncoupler, however, the alternate path is engaged both in fresh and aged slices. V(cyt), the maximal capacity of the cytochrome path, remains essentially the same with slice aging, whereas V(alt) decreases from 20 to 60 per cent. The induced respiration is readily accommodated by the potential cytochrome path capacity of fresh slices, which is realized on aging. Accordingly, there is no need to invoke mitochondrial proliferation in explanation of the development of the induced respiration. The engagement of the alternate path in response to uncoupler reflects substrate mobilization to a degree that substrate oxidation exceeds the electron transport capacity of the cytochrome path.Fresh slices do not utilize exogenous substrates, whereas aged slices do so readily. Cerulenin, a specific inhibitor of fatty acid synthesis, prevents the development of the induced respiration as well as the capacity to oxidize exogenous substrates. It is suggested that lipid, and ultimately membrane, biosynthesis is central to the development of the induced respiration and the ability to use exogenous substrates, much as in potato. PMID- 16660495 TI - Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits. AB - The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.The maximal capacities of the cytochrome and alternate paths, V(cyt) and V(alt), respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V(alt) decreases slightly with ripening.The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged. PMID- 16660496 TI - Synthesis and deposition of zein in protein bodies of maize endosperm. AB - The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [(14)C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained. PMID- 16660497 TI - Induction of resistance to dark abscission by malformin in white light. AB - When cuttings or seedlings of Phaseolus aureus were treated proximally with malformin for 2 days in continuous white light, resistance to subsequent leaf abscission in the dark resulted. The amount of resistance diminished as the concentration of malformin decreased from 10 to 0.1 micromolar. Resistance to dark abscission persisted for 7 days in continuous light. Little resistance was obtained when cuttings were taken from seedlings grown under low irradiance and short photoperiods, but resistance gradually increased as the photoperiod increased. Resistance to dark abscission induced by malformin in light differs from inhibition of abscission by indoleacetic acid because when malformin is applied in the dark it stimulates abscission after distal or proximal application. Malformin induces resistance only in conjunction with light treatment. PMID- 16660498 TI - Ethylene and senescence in petals of tradescantia. AB - Flowers of Tradescantia (clone O2) which are ephemeral, produce ethylene during senescence with the maximum rates occurring during the initial period of fading. Senescing isolated petals produce ethylene in a similar manner, exhibit a loss of membrane semipermeability, and exogenous ethylene hastens the onset as well as the subsequent rate of this loss. The aminoethoxy analog of 0.1 millimolar rhizobitoxine completely inhibits ethylene production by isolated petals but only partially the loss of membrane semipermeability. Isolated petals acquire a sensitivity to ethylene as they mature, becoming fully sensitive on the day of anthesis. PMID- 16660499 TI - Inhibition of deoxyribonuclease activity in the medium surrounding plant protoplasts. AB - After 1 hour, exogenous deoxyribonucleic acid was degraded within a culture medium at 25 C (pH 6) containing protoplasts of Daucus carota L. var. sativa. Low temperature incubation (1 C) or the addition of 45 millimolar sodium citrate to the medium eliminated DNase activity for at least 4.5 hours. This DNase activity was not reduced at pH 7 or 9, nor by addition of 200 millimolar adenosine 5' triphosphate.Techniques were developed to ensure high protoplast plating efficiencies and high regenerative capabilities after low temperature treatment and the addition of sodium citrate to the medium. Results indicated citrate concentrations to 45 mm and 1 C temperatures revealed little or no effect on protoplast regeneration capacities. Protoplast viability was 90 to 95% at the time of plating as determined by phenosafranin staining and an estimated 50 to 60% of these undergo cell division in the solid agar medium. PMID- 16660500 TI - Hormones and young leaves control development of cotyledonary buds in tomato seedlings. AB - Hormonal and plant factors regulating the development of the inhibited cotyledonary buds of Lycopersicon esculentum Mill. cv. ;Fireball' seedlings were studied. Excision of the immature plumular leaves of 5- to 20- millimeter length significantly stimulated bud development after 2 to 4 days, but excision of leaves exceeding 20-millimeter length was without effect. Apical application of 20 microliters of 5 millimolar abscisic acid significantly promoted development of the cotyledonary buds after 6 days. A subapical ring of 0.1 millimolar concentration of 2,3,5-triiodobenzoic acid (TIBA) in lanolin significantly promoted cotyledonary bud development after 11 days. Twenty microliters of 0.1 millimolar 6-benzylaminopurine (BAP) applied directly to the cotyledonary bud loci significantly promoted bud development, but 1 micromolar gibberellin A(4/7) was ineffective. Application of 0.1 millimolar BAP in lanolin to the petiole or hypocotyl was ineffective. However, application of 0.1 millimolar TIBA as a ring around the petioles of the cotyledons or 1-centimeter on the hypocotyl below the cotyledons significantly promoted cotyledonary bud development. PMID- 16660501 TI - Evidence for a Functional myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen. AB - Addition of myo-inositol to pentaerythritol-based germination media repressed the conversion of d-[1-(14)C]glucose to labeled uronosyl and pentosyl units of tube wall pectic substance in lily pollen (Lilium longiflorum Thunb.). Conversion of d [1-(14)C]glucose to labeled glucosyl, galactosyl, and rhamnosyl units was unaffected. The reverse experiment, addition of d-glucose to pentaerythritol based media, failed to affect the conversion of myo-[2-(3)H]inositol to uronosyl and pentosyl units although the flow of label into products of myo-inositol linked glucogenesis was blocked. Results of these experiments are discussed in terms of a functional myo-inositol oxidation pathway.d-[1-(14)C]Glucose-labeled pollen tubes contain a labeled, 70% ethyl alcohol-soluble, acidic compound whose formation is blocked by the myo-inositol antagonist, 2-O,C-methylene-myo-inositol (Chen et al. 1977 Plant Physiol. 59: 658). This compound has been identified as l malic acid. PMID- 16660502 TI - Light-mediated Activation of Nitrate Reductase in Synchronous Chlorella. AB - The mechanism underlying the sharp increase in activity of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris forma tertia (strain 211 8k) during the first hour of the 7 hours/5 hours light/dark cycle was investigated. Using the method of density labeling and isopycnic centrifugation, it could be demonstrated that this rapid increase in activity is based on light-mediated activation rather than de novo synthesis of the enzyme. The problematic nature of cycloheximide specificity and models of nitrate reductase activation are discussed. PMID- 16660503 TI - 6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L. AB - The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl(2) stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6 phosphogluconate were 12 and 9.6 micromolar and for NADP(+) were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development. PMID- 16660504 TI - Fructokinase (Fraction III) of Pea Seeds. AB - A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K(m) values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K(m) for Mg adenosine 5'-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg(2+) was essential for activity but the enzyme was inhibited by excess Mg(2+) or ATP. Mg adenosine 5'-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K(+), Rb(+), and NH(4) (+) were the most effective. The possible role of fructokinase (fraction III) is discussed. PMID- 16660505 TI - Abscission of citrus leaf explants: no correlation with naphthaleneacetic Acid conjugation in the abscission zone. AB - The role of alpha-naphthaleneacetic acid (NAA) in the control of abscission in Citrus (Citrus sinensis L. Osbeck) leaf explants and its conjugation were studied in non-aged and 24-hour-aged explants. Dipping non-aged explants in 1.5 micromolar NAA for 15 minutes immediately after excision did not delay abscission whereas 150 micromolar NAA effectively delayed it. As incubation time was prolonged up to 24 hours after excision, the delaying effect of both concentrations gradually increased. In general, both concentrations did not delay abscission when applied to 24-hour-aged explants held for an additional period of up to 24 hours. The uptake and conjugation of (14)C-NAA to glucose and aspartic acid were similar in petiole, abscission zone, and leaf blade of non-aged and aged tissues, for all NAA concentrations. No correlation was established between the kinetics of abscission and the rate of conjugation in the abscission zone. PMID- 16660506 TI - Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen. AB - Glutamine is the first major organic product of assimilation of (13)NH(4) (+) by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of (13)NO(3) (-) by tobacco cells cultured on nitrate. The percentage of organic (13)N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. (13)NO(3) (-), used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of (13)N in glutamine, and a concomitant increase in the percentage of (13)N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of (13)N from (13)NH(4) (+) into glutamine more extensively than it inhibits the incorporation of (13)N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when (13)NH(4) (+) is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of (13)NH(4) (+) is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of (13)N from (13)NO(3) (-) into glutamate more strongly than it inhibits the incorporation of (13)N into glutamine, suggesting that the assimilation of (13)NH(4) (+) derived from (13)NO(3) (-) may be mediated solely by the glutamine synthetase-glutamate synthase pathway. PMID- 16660507 TI - Betaine Accumulation and [C]Formate Metabolism in Water-stressed Barley Leaves. AB - Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of -19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline-remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.When [methyl-(14)C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more (14)C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [(14)C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more (14)C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [(14)C]formate for about 18 hours in darkness, betaine was always the principal (14)C-labeled soluble metabolite. This (14)C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of (14)C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [(14)C]formate.These results are consistent with a net de novo synthesis of betaine from 1- and 2 carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product. PMID- 16660508 TI - Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates. AB - Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O(2) evolution of about 100 to 150 micromoles of O(2) per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO(3) with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O(2) per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5' diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5'-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO(3) and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed. PMID- 16660509 TI - Regulation of chloroplast photosynthetic activity by exogenous magnesium. AB - Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl(2), added in the dark, inhibited CO(2)-dependent O(2) evolution by Hordeum vulgare L. and Spinacia oleracea L. (C(3) plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O(2) evolution by Digitaria sanguinalis L. (C(4) plant) mesophyll chloroplasts from 80 to 100%. When Mg(2+) was added in the light, O(2) evolution was reduced only slightly. O(2) evolution in the presence of phosphoglycerate was less sensitive to Mg(2+) inhibition than was CO(2)-dependent O(2) evolution.Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg(2+) blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3 phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg(2+) inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes. PMID- 16660510 TI - Changes in Chloroplast Membrane Lipids during Adaptation of Barley to Extreme Salinity. AB - During adaptation of barley (Hordeum vulgare L.) seedlings to extremely high concentrations of sodium chloride in the root space, the content of galactolipids of chloroplast membranes decreased considerably. Alterations in membrane lipids were due to the high concentration of ions rather than to the increase in the water potential. Sodium chloride was accumulated in the leaf cells and affected lipid-synthesizing enzymes such as galactosyl transferase and acylase which are attached to the chloroplast envelope. The return of salt-adapted barley seedlings to a nutrient solution with low salt concentration resulted in a reversal of the observed changes. It is suggested that the decrease in content of galactolipids in biomembranes is one of the factors causing increased salt resistance in barley plants which are adapted to extreme salinity. PMID- 16660511 TI - Photolability of Photosynthesis in Two Separate Mutants of Scenedesmus obliquus: Preferential Inactivation of Photosystem I. AB - Two separate mutants of the green alga, Scenedesmus obliquus, are described in which photosynthesis is sensitive to moderate intensities of white light (100 mw cm(-2)). Heterotrophic cultures of both mutants lose photosynthetic activity when exposed to white light; the site of at least the initial phase of this inactivation is within photosystem I. Although all whole cell and cell-free reactions typical of photosystem I examined are inhibited by irradiation, the principal component of photosystem I affected is P-700. In light-sensitive-4 the inactivation of P-700 activity is restored during the subsequent dark period. This recovery is prevented by various antibiotics and by anaerobic conditions. In light-sensitive-41 P-700 activity is recovered only after a complete cell division and new growth. Irradiation periods which inhibit photosynthesis in both mutants are without effects upon the activity or presence of ferredoxin, ferredoxin-NADP(+) oxidoreductase, plastocyanin, cytochrome f(552), cytochrome b 562 or cytochrome b-559.Prolonged irradiation of cells of light-sensitive-41 causes the disappearance of photosystem II activity, alpha-tocopherol, and plastoquinone. Some decrease of both the chlorophylls and carotenoids occurs but there is no preferential deletion of any particular carotenoid. PMID- 16660512 TI - Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: I. Ornithine Carbamoyltransferase: Isolation and Some Properties. AB - Ornithine carbamoyltransferase (OCT) activity was detected in extracts from mature leaves, fruit, germinating seeds, and seedlings of Vitis vinifera L. Michaelis-Menten constants for OCT were 3.5 millimolar for carbamyl phosphate and 5.5 millimolar for l-ornithine. Concentrations of l-ornithine greater than 10 millimolar slightly inhibited the enzyme, whereas carbamyl phosphate at concentrations greater than the optimal (about 10 millimolar) did not affect OCT activity. l-Citrulline formation was linear with incubation period for the first 25 minutes and with increasing amounts of enzyme up to an equivalent of about 200 milligrams of fresh tissue. The optimum pH for in vitro OCT activity was between 8.4 and 8.8, and the optimum incubation temperature was 38 C. PMID- 16660513 TI - Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: II. Arginosuccinate Synthetase and Lyase. AB - Arginosuccinate (ASA) synthetase and lyase activities were detected in extracts from Vitis vinifera L. cv. Chenin blanc mature leaves and seedlings. Optimum reaction conditions for ASA synthetase were 10 millimolar l-citrulline, 7.5 millimolar l-aspartate, 3 to 4 millimolar ATP, 12 millimolar Mg(2+) (pH 7.5 to 8.0), enzyme extract up to equivalent of about 200 milligrams of fresh tissue, and incubation temperature of 38 to 40 C. Optimum reaction conditions for ASA lyase were 4 millimolar ASA-K salt (pH 7.3 to 7.8), amount of extract up to equivalent of about 180 milligrams of fresh tissue, and incubation temperature of 38 to 40 C. PMID- 16660514 TI - Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: III. In Vivo and In Vitro Studies of Arginase. AB - The presence of arginase (EC 3.5.3.7) in various tissues from Vitis vinifera L. cultivars was demonstrated by both in vivo and in vitro enzyme assays. Initial velocities determined by the two methods were in close agreement. Optimum conditions for maximum enzyme activity were 25 to 30 millimolar l-arginine, about 1 millimolar Mn(2+) (pH 9.4 to 9.8), and incubation temperature of 37 to 38 C. l Arginine hydrolysis was linear with increasing sliced fresh tissue up to 500 milligrams for in vivo assay, and with enzyme extract equivalent up to about 200 milligrams of fresh tissue for in vitro. Similarly, l-arginine hydrolysis was linear with incubation time for the first 45 minutes for in vivo assay and for the first 20 minutes for in vitro. PMID- 16660515 TI - Identification of an acyl donor in steryl ester biosynthesis by enzyme preparations from spinach leaves. AB - A pathway for steryl ester biosynthesis in acetone powder preparations from spinach (Spinacia oleracea L.) leaves has been elucidated; free sterol and 1,2 diglyceride were the substrates. Although animals synthesize cholesteryl esters by three distinct biosynthetic pathways, none of these pathways utilizes 1,2 diglyceride as an acyl donor. Phosphatidylcholine, phosphatidic acid, triglyceride, 1,3-diglyceride, 1-monoglyceride, free fatty acid, and fatty acyl CoA were not acyl donors for spinach leaf steryl ester biosynthesis in our assay system. The unstable 2 isomer of monoglyceride was not tested. It is possible that 1,2-diglyceride and 2-monoglyceride were both acyl donors for spinach leaf steryl ester biosynthesis. Acyl-labeled phosphatidylcholine and acyl-labeled phosphatidylethanolamine were rapidly degraded by acetone powder preparations to 1,2-diglyceride via phosphatidic acid. The 1,2-diglycerides were slowly metabolized to monoglycerides, triglycerides, free fatty acids, and steryl esters. The monoglycerides were rapidly degraded to free fatty acids and glycerol. PMID- 16660516 TI - Pattern of cell division and wound vessel member differentiation in coleus pith explants. AB - When pith parenchyma explants are taken from Coleus blumei plants and cultured on an agar medium containing sucrose and indoleacetic acid wound vessel members differentiate in 10 days. The time course of wound xylem appearance and an auxin requirement suggest that this uncomplicated system is responding in a manner comparable to wounded Coleus plants and cultured stem segments.Histological examination and cell size comparisons confirm that parenchyma cells divide before differentiating. When colchicine is used to prevent mitosis no tracheary elements differentiate. Following the time course of this cytodifferentiation histologically shows that xylem differentiates from cells that are the products of several cell divisions. PMID- 16660517 TI - Oxygen exchange in the pericarp green layer of immature cereal grains. AB - Rates of oxygen exchange in light and dark were recorded for immature detached barley spikelets and wheat florets both before and after successive removal of the husk (palea and lemma), transparent layer of the pericarp, and green layer of the pericarp. Results were compared with those for the mutant barley Albino lemma which has a pericarp lacking chlorophyll. There was no net oxygen evolution in the intact spikelets of Albino lemma when incubated in the light. Removal of the husk increased the rate of measured oxygen uptake in both light and dark. With normal barley and wheat, net oxygen evolution in the light was observed in intact spikelets and florets, as well as after husk removal and after both husk and transparent layer removal. Additional removal of the green layer of the pericarp resulted in a dramatic changeover from oxygen evolution in the light to oxygen uptake. The results suggest that some of the oxygen generated by pericarp photosynthesis remains within the grain. PMID- 16660518 TI - Characterization of the enzyme responsible for nopaline and ornaline synthesis in sunflower crown gall tissues. AB - Extracts prepared from sunflower (Helianthus annuus L.) crown gall tissues induced by Agrobacterium tumefaciens strains C58 and T37 (nopaline utilizers) catalyze the synthesis of nopaline and ornaline. These compounds are not synthesized in extracts of crown gall tissues induced by strains B6, 15955 (octopine utilizers), and AT1 (utilizes neither octopine nor nopaline) or in extracts of habituated sunflower callus. Both synthetic activities require NADPH, alpha-ketoglutarate, and either arginine or ornithine; histidine and lysine will not substitute. Incorporation of arginine or ornithine into product is inhibited by the other substrate but not by histidine or lysine. On the basis of inhibition and K(m) data, both activities appear to be catalyzed by one enzyme and the same enzyme is apparently present in crown gall tissues induced by strains C58 and T37. PMID- 16660519 TI - Chemical Cross-linking of Neighboring Thylakoid Membrane Polypeptides. AB - Cross-linking between protein components of whole spinach (Spinacia oleracea var. Nobel) thylakoids and of photosystem I- and II-enriched thylakoid fractions has been produced by reaction with the bifunctional imidoester dimethyl-3,3' dithiobispropionimidate dihydrochloride as well as by the oxidation of intrinsic sulfydryl groups with an orthophenanthrolinecupric ion complex. The mixture of membrane proteins and their cross-linked products has been analyzed by two dimensional sodium dodecyl sulfate electrophoresis, with a reductive cleavage step of the cross-linkages before the second dimension. Cross-linked aggregates up to a molecular weight of about 130 kilodaltons (kD) were analyzed, and it was inferred that the polypeptides appearing together in the same aggregates were neighbors within the membrane.In thylakoids as well as in isolated photosystem fractions, oligomers were formed by cross-linking polypeptides of the 60 to 90 kD range, among them the polypeptides of the chlorophyll-protein complex I. Polypeptides of 46, 19, and 12 kD were cross-linked to these complexes. Polypeptides of 25 and 22 kD, which are related to the chlorophyll-protein complex II, were cross-linked in thylakoids as well as in photosystem II fractions, suggesting that in the membrane these molecules are close together. In photosystem II fractions an oligomer having a molecular weight of about 60 kD was formed by cross-linking several polypeptides of different molecular weights: 40, 25, and 22 kD.Our cross-linking experiments show that protein interactions in the thylakoid membrane occurred mainly among the polypeptides of the two chlorophyll protein complexes, thus suggesting an oligomeric nature of these apoproteins. PMID- 16660520 TI - Bacitracin Inhibits the Synthesis of Lipid-linked Saccharides and Glycoproteins in Plants. AB - The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[(14)C]mannose into mannosyl phosphoryl-dolichol and of N-acetylglucosamine from UDP-[(3)H]N-acetylglucosamine into N-acetylglucosamine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mm bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-[(14)C]glucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of [(14)C]mannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mm bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%. PMID- 16660521 TI - Synthesis of beta-(1-->3)-Glucan from Extracellular Uridine Diphosphate Glucose as a Wound Response in Suspension-cultured Soybean Cells. AB - Soybean (Glycine max) suspension-cultured cells were incubated with 600 micromolar uridine diphosphate [(14)C]glucose, and the incorporation into alkali insoluble material was studied. When the cells were kept in suspension by shaking on a linear shaker, the incorporation was very low. The incorporation was stimulated 30-fold when the cells were continually resuspended by stirring with a narrow glass rod. The stirring procedure was shown to damage some of the cells, and the incorporation appeared to be a wound response. The alkali-insoluble material formed was a beta-(1-->3)-glucan, and it was synthesized from uridine diphosphate glucose which did not penetrate through the plasma membrane of intact cells. The synthetase activity was probably induced by the stirring procedure. No evidence for cellulose synthesis from extracellular uridine diphosphate glucose was obtained. PMID- 16660522 TI - Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules. AB - Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 muM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent K(m) (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 x 10(-5) M, which is compared to 3 x 10(-3) M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent K(m) estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID- 16660523 TI - Stomatal Response to Light of Solanum pennellii, Lycopersicon esculentum, and a Graft-induced Chimera. AB - To learn how species differences in stomatal behavior are regulated, the response of epidermal and leaf diffusive resistance to light was investigated in Lycopersicon esculentum Mill., Solanum pennellii Corr., and a periclinal chimera having an S. pennellii epidermis and an L. esculentum mesophyll that was produced from a graft of the two species. S. pennellii has about 23% fewer stomata per square millimeter than does L. esculentum, and the two species have contrasting stomatal sensitivities to light. The abaxial stomata of L. esculentum open in dimmer light and to a greater extent than the adaxial stomata. The abaxial and adaxial stomata of S. pennellii respond similarly to light incident on the adaxial epidermis and are less open at all quantum flux densities than comparable stomata of L. esculentum. The patterns of response to light of the abaxial and adaxial stomata of the chimera were practically identical to those of L. esculentum, and quite unlike those of S. pennellii. Thus, the pattern of stomatal light response in the chimera was regulated by the L. esculentum mesophyll. The reduction in stomatal frequency of the chimera, which was regulated by the epidermis of S. pennellii, contributed to the 40% difference in leaf diffusive resistance between the plants in moderate light. PMID- 16660524 TI - Regulation of glucose metabolism and cell wall synthesis in Avena stem segments by gibberellic Acid. AB - Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent beta-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only beta-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only beta-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity. PMID- 16660525 TI - Enhancement of ethylene formation by selenoamino acids. AB - Selenomethionine and selenoethionine enhanced ethylene production in senescing flower tissue of Ipomoea tricolor Cav. and in auxin-treated pea (Pisum sativum L.) stem sections. This enhancement was fully inhibited by the aminoethoxy analog of rhizobitoxine. Methionine did not have a comparable promotive effect, and ethionine partly inhibited ethylene production. When [(14)C]methionine was applied to flower or pea stem tissue followed by treatment with unlabeled selenomethionine or selenoethionine, the specific radioactivity of the ethylene evolved was considerably reduced. The dilution of the specific radioactivity of ethylene by selenomethionine, and in pea stem sections also by selenoethionine, was greater than the dilution by nonradioactive methionine at the same concentration. These results indicate that both selenoamino acids serve as precursors of ethylene and that they are converted to ethylene more efficiently than is methionine. PMID- 16660526 TI - Application of the chemiosmotic hypothesis to ion transport across the root. AB - The evidence on how ions accumulated in the root symplasm are released to the xylem vessels is examined. It is suggested that Mitchell's chemiosmotic hypothesis as applied to ion transport might account for the process. A model based on this hypothesis shows the symplasm as an osmotic unit connecting two isolated solutions, but with no significant difference in proton motive force across the unit. If it is assumed that the resistance to transport by plasmalemma uniports and antiports differs in the cortical and stelar ends of the symplasm, the model will provide for an influx of ions to the xylem. The reported properties of isolated steles suggests that the porters (carriers) do have properties in accord with the model. PMID- 16660527 TI - Gain of the feedback loop involving carbon dioxide and stomata: theory and measurement. AB - The physiological and physical components of the feedback loop involving intercellular CO(2) concentration (c(i)) and stomata are identified. The loop gain (G) is a measure of the degree of homeostasis in a negative feedback loop [the expression 1/(1-G) represents the fraction to which feedback reduces a perturbance]. Estimates are given for the effects of G on responses of stomata and c(i) to changes in ambient CO(2) concentration, light intensity, and perturbations in the water relations of a leaf. At normal ambient CO(2) concentration, the gain of the loop involving stomatal conductance and c(i) was found to be -2.2 in field-grown Zea mays, -3.6 if plants of this species were grown in a growth chamber, and zero in well watered Xanthium strumarium in the vegetative state. PMID- 16660528 TI - Effect of abscisic Acid on the gain of the feedback loop involving carbon dioxide and stomata. AB - Gains of the feedback loops involving intercellular CO(2) concentration on one hand, and CO(2) assimilation and stomata on the other (= assimilation loop with gain [G(A)] and conductance loop with gain [G(g)]) were determined in detached leaves of Amaranthus powelli S. Wats., Avena sativa L., Gossypium hirsutum L., Xanthium strumarium L., and Zea mays in the absence and presence of 10(-5)m (+/-) abscisic acid (ABA) in the transpiration stream. Determinations were made for an ambient CO(2) concentration of 300 microliters per liter. In the absence of ABA, stomata were insensitive to CO(2) (G(g) between 0.00 and -0.02) in A. sativa, G. hirsutum, and X. strumarium, sensitive in A powelli (G(g) = -0.46), and very sensitive in Z. mays (G(g) = -3.6). Addition of ABA increased the absolute values of the gain of the conductance loop in A. powelli (G(g) = -2.0), G. hirsutum (G(g) = -0.31), and X. strumarium (G(g) = -1.14). Stomata closed completely in A. sativa. In Z. mays, G(g) decreased after application of ABA to a value of -0.86, but stomatal sensitivity to CO(2) increased for intercellular CO(2) concentrations < 100 microliters per liter. The gain of the assimilation loop increased after application of ABA in all cases, from values between 0.0 (A. powelli) and -0.21 (Z. mays) in the absence of ABA to values between -0.19 (A. powelli) and -0.43 (Z. mays) in the presence of ABA. In none of the species examined did ABA affect the photosynthetic capacity of the leaves.The application of ABA caused stomatal narrowing which affected transpiration more than the assimilation of CO(2). In the case of A. powelli the transpiration ratio decreased without a concomitant reduction of the assimilation rate. PMID- 16660529 TI - Structure of Plant Cell Walls: VIII. A New Pectic Polysaccharide. AB - This paper describes the isolation and characterization of rhamnogalacturonan II, a hitherto unobserved component of the primary cell walls of dicotyledonous plants. Rhamnogalacturonan II constitutes 3 to 4% of the primary cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells. Rhamnogalacturonan II is a very complex polysaccharide yielding, upon hydrolysis, 10 different monosaccharides including the rarely observed sugars apiose, 2-O-methylxylose, and 2-O-methylfucose. In addition, rhamnogalacturonan II is characterized by the rarely observed glycosyl interconnections of 2-linked glucuronosyl, 3,4-linked fucosyl, and 3-linked rhamnosyl residues. These glycosyl linkages have never previously been detected in primary sycamore cell walls. Evidence is presented which suggests that polysaccharides similar to rhamnogalacturonan II are present in the primary cell walls of the three other dicotyledonous plants examined. PMID- 16660530 TI - Growth-induced Water Potentials in Plant Cells and Tissues. AB - A physical analysis of water movement through elongating soybean (Glycine max L. Merr.) hypocotyls was made to determine why significant water potentials persist in growing tissues even though the external water potentials were zero and transpiration is virtually zero. The analysis was based on a water transport theory modified for growth and assumed that water for growing cells would move through and along the cells in proportion to the conductivity of the various pathways.Water potentials calculated for individual cells were nearly in local equilibrium with the water potentials of the immediate cell surroundings during growth. However, water potentials calculated for growing tissue were 1.2 to 3.3 bars below the water potential of the vascular supply in those cells farthest from the xylem. Only cells closest to the xylem had water potentials close to that of the vascular supply. Gradients in water potential were steepest close to the xylem because all of the growth-sustaining water had to move through this part of the tissue. Average water potentials calculated for the entire growing region were -0.9 to -2.2 bars depending on the tissue diffusivity.For comparison with the calculations, average water potentials were measured in elongating soybean hypocotyls using isopiestic thermocouple psychrometers for intact and excised tissue. In plants having virtually no transpiration and growing in Vermiculite with a water potential of -0.1 bar, rapidly growing hypocotyl tissue had water potentials of -1.7 to -2.1 bars when intact and -2.5 bars when excised. In mature, nongrowing hypocotyl tissue, average water potentials were -0.4 bar regardless of whether the tissue was intact or excised.The close correspondence between predicted and measured water potentials in growing tissue indicates that significant gradients in water potential are required to move growth-associated water through and around cells over macroscopic distances. The presence of such gradients during growth indicates that cells must have different cell wall and/or osmotic properties at different positions in the tissue in order for organized growth to occur. The mathematical development used in this study represents the philosophy that would have to be followed for the application of contemporary growth theory when significant tissue water potential gradients are present. PMID- 16660531 TI - Polyamine Metabolism in Embryogenic Cells of Daucus carota: I. Changes in Intracellular Content and Rates of Synthesis. AB - Changes in the metabolism of polyamines, which seem to be involved in transcription and translation in animal systems, have been studied in cultured cells of Daucus carota (carrot) undergoing embryogenesis. Putrescine levels were elevated by as much as 2-fold over the control within 24 hours after transfer of the cells to embryogenic medium. Spermidine levels were elevated also but spermine levels appeared to be lower in embryogenic cells. Embryogenic cells incorporated [(14)C]arginine into putrescine at two times the rate of control cells. These changes suggest that polyamines may be involved in cellular differentiation during embryogenesis. PMID- 16660532 TI - Isolation and preliminary characterization of a casein kinase from cauliflower nuclei. AB - A casein-type protein kinase has been isolated from cauliflower (Brassica cauliflora Gars.) nuclei and purified to a specific activity of 23,000 units/milligram of protein (1 unit is defined as the transfer of 1 picomole of (32)Pi from gamma-[(32)P]ATP to substrate per minute at 28 C). The enzyme has a molecular weight of approximately 39,000 as judged by sucrose density gradient sedimentation. The casein kinase requires ATP as the phosphate donor and will phosphorylate casein and phosvitin, but not histones. The enzyme activity is not affected by cAMP or cGMP. The casein kinase appears to be analogous to casein kinases described in other plant and animal systems. PMID- 16660533 TI - Physiology of Tuberization in Solanum tuberosum L: cis-Zeatin Riboside in the Potato Plant: Its Identification and Changes in Endogenous Levels as Influenced by Temperature and Photoperiod. AB - Using high pressure liquid chromatography, the cucumber cotyledon bioassay, and mass spectrometry a cytokinin isolated from Solanum tuberosum L. cv. Katahdin plant tissues has been identified as cis-zeatin riboside. Zeatin riboside (ZR) levels in plants grown under inducing conditions (28 C day and 13 C night with a 10-hour photoperiod) were significantly higher than those in plants grown under noninducing conditions (30 C day and 28 C night with an 18-hour photoperiod). The highest level of ZR was noted in below-ground tissue after 4 days exposure to inducing conditions, with tuber initiation observed after 8 days. A companion study conducted to determine the effect of ZR on in vitro tuberization of noninduced rhizomes revealed that after 1 month in culture, controls exhibited 0% tuberization, while ZR treatments of 0.3 and 3.0 milligrams per liter showed 39 and 75% tuberization, respectively. PMID- 16660534 TI - Simultaneous measurement of nitrogen fixation estimated by acetylene-ethylene assay and nitrate absorption by soybeans. AB - An apparatus was designed for simultaneous measurement of rates of N(2) fixation estimated by C(2)H(2)-C(2)H(4) assay (N(2)[C(2)H(2)] fixation) and NO(3) (-) absorption by roots of intact, nodulated soybeans (Glycine max [L.] Merr.). The principal design features include: (a) a gas-tight mist chamber in which nodulated roots can be exposed simultaneously to C(2)H(2) in the gas phase and to a liquid phase containing NO(3) (-) sprayed in a fine mist; and (b) provision for sampling the gas phase for C(2)H(4) determination, and the liquid phase for NO(3) (-) determination.We studied NO(3) (-) absorption by soybeans as affected by nodulation, NO(3) (-) concentration during assay, and previous N nutrition during growth in nutrient solution culture in controlled environment chambers. It was established that 0.5 mm NO(3) (-) nearly saturated the NO(3) (-) absorption system of both nodulated and unnodulated soybeans when the concentration dependence of NO(3) (-) absorption rate was measured just after flowering began. Nitrate absorption rates were measured after development of N stress in unnodulated plants, and during recovery from N stress in nodulated plants. The results suggested that the lower capacity for NO(3) (-) absorption of nodulated plants was a consequence of N stress during the period of nodule growth and development.Nitrogen [C(2)H(2)] fixation rates were compared in intact plants assayed in the mist chamber and in excised roots assayed in both the mist chamber and in glass jars. Excised roots had a lower N(2)[C(2)H(2)] fixation rate than intact plants. The decline observed during the first hour after shoot removal was more pronounced for glass jar-assayed excised roots than for mist chamber-assayed excised roots.We discuss the advantages of our method for assessing the capability of a nodulated legume to acquire nitrogen through both N(2) fixation and absorption and assimilation of NO(3) (-). PMID- 16660535 TI - Effect of gibberellin a(3) on the endoplasmic reticulum and on the formation of glyoxysomes in the endosperm of germinating castor bean. AB - Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to a range of concentrations of gibberellin A(3) (GA(3)). Treatments for 20 hours with GA(3) concentrations of 0.5 muM or higher resulted in increased levels of NADH cytochrome c reductase, phosphorylcholine glyceride transferase, and malate synthase in endoplasmic reticulum (ER) isolated from endosperm on linear sucrose gradients. GA(3) treatment also resulted in increased RNA associated with ER. Malate synthase and catalase in crude homogenates were enhanced by 1 to 100 muM GA(3) concentrations. Isocitrate lyase, citrate synthase, malate synthase, catalase, and glycolate oxidase in isolated glyoxysomes were enhanced by 60, 20, 18, 40, and 28%, respectively, over controls. Treatment with abscisic acid led to decreased levels of glyoxysomal enzymes and reduced glyoxysomal protein. The effect of GA(3) and abscisic acid on the specific activities of glyoxysomes of different densities suggests that GA(3) influences enzyme levels and glyoxysome assembly. PMID- 16660536 TI - Effect of light and ontogenetic stage on sink strength in bean leaves. AB - Light (about 3,000 foot-candles) neither increased nor decreased the sink strength of young, rapidly expanding leaves of Phaseolus vulgaris L. cv. Black Valentine, as measured by the comparative rates of import of (14)C-labeled photosynthates by sink leaves in the light versus dark in short term experiments. Although irradiated sink leaves accumulated more (14)C activity, the difference was fully accounted for by photosynthetic reabsorption of respiratory CO(2) derived from substrates translocated to the sink leaves.Maximum sink strength was attained when the sink leaf reached 7 to 8 cm(2) in area (9 to 10% of its fully expanded size). Thereafter sink strength declined rapidly and asymptotically to a near zero value at about 45% final area. During this period, however, the rapid decline in translocation was offset by a rapid rise in the photosynthetic rate of the sink leaf, maintaining a near constant relative rate of dry weight increase until the sink leaf had expanded to about 17% of its final area. Although the increasing photosynthetic capacity was associated with a decreasing import capacity, suggesting that the rate of translocation to the sink leaf was controlled by the developing capacity of the sink leaf for photosynthesis, it was not possible to vary the total (true) translocation rate to the sink leaf by varying the photosynthetic rate of the sink leaf in short term light-dark experiments. Despite a high ratio of source to sink in these experiments, no evidence accrued that translocation into young bean leaves was ever sink-limited. PMID- 16660537 TI - Development and Localization of Carboxypeptidase Activity in Embryo-less Barley Half-kernels. AB - Distal half-kernels of barley (Hordeum vulgare L.), after imbibition for 1 day, produced a carboxypeptidase that was active on N-carbobenzoxy-l-phenylalanyl-l alanine and on N-carbobenzoxy-l-phenylalanyl-l-phenylalanine. For the ensuing 2 days, activity increased linearly and thereafter increased at reduced rates. Electrofocusing of an imbibed half-kernel homogenate produced coincident peaks of activity on both substrates. Experiments with dissected imbibed (3 days) half kernels showed that the enzyme arose in the aleurone layer. Enzyme production was inhibited by 6-methylpurine, cordycepin, cycloheximide, and p fluorophenylalanine, and activity was inhibited by phenylmethylsulfonylfluoride. The enzyme did not hydrolyze endopeptidase substrates over a range of pH.Gibberellic acid accelerated the rate of release from the aleurone, but was not essential for release and did not appreciably affect the ultimate amount of carboxypeptidase produced. In these respects, the carboxypeptidase appears to be unique among the known hydrolases produced by barley aleurone tissue. PMID- 16660538 TI - Azolla-Anabaena Relationships: VII. Distribution of Ammonia-assimilating Enzymes, Protein, and Chlorophyll between Host and Symbiont. AB - The N(2)-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free living isolate of the Azolla endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N(2) fixation by the symbiont. PMID- 16660539 TI - 2,4-Dichlorophenoxyacetic Acid Inhibits the Outer Membrane NADH Dehydrogenase of Plant Mitochondria. AB - The NADH dehydrogenase of potato (Solanum tuberosum) and mung bean (Phaseolus aureus) outer mitochondrial membranes is specifically inhibited by both 2,4 dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids but not by the natural auxin indole-3-acetic acid. PMID- 16660540 TI - Confounding of alternate respiration by lipoxygenase activity. AB - The initial burst of respiratory activity (Q(o) (2)) of imbibing soybean (Glycine max [L.] Merr. var. Wayne) seed tissue is cyanide-insensitive, and sensitive to salicylhydroxamate: presumptive evidence for the presence of alternate respiration. The initial O(2) consumption is also highly sensitive to propyl gallate. Soybean lipoxygenase exhibits similar characteristics of insensitivity to cyanide and sensitivity to salicylhydroxamate and to propyl gallate. The initial burst of respiration is enhanced by the addition of linoleic acid, a lipoxygenase substrate. These results indicate that the conventional tests for alternate respiration in plant tissues can be confounded by lipoxygenase; they also suggest that propyl gallate can be used to assess the possible participation of lipoxygenase in the O(2) uptake by plant tissues. PMID- 16660541 TI - Effects of ethylene and carbon dioxide on the germination of osmotically inhibited lettuce seed. AB - Lettuce seeds (Lactuca sativa L.) used in this study germinated 98% at 25 C in light or dark. Their germination was completely inhibited by 0.20 m NaCl, 0.35 m mannitol, or polyethylene glycol 6000 (-7 bars) under continuous light when germination tests were made in Petri dishes. Approximately 50% germination occurred in sealed flasks due to endogenously produced C(2)H(4) and CO(2). Removal of either or both gases prevented germination. In the presence of endogenous CO(2), addition of C(2)H(4) (0.5 to 16 microliters/liter) stimulated 95 to 100% germination (after 5 days) only in the light, but the rate of germination was dependent on C(2)H(4) concentration. At 16 microliters/liter C(2)H(4), full germination occurred within 72 hours. Addition of up to 3.2% CO(2) had no adverse effect on the C(2)H(4) action. Higher concentrations or the complete absence of CO(2) reduced both rate and total germination. CO(2) alone was ineffective.Under these osmotic conditions the promotive effect of C(2)H(4) was under the control of phytochrome. PMID- 16660542 TI - Exchange Properties of Isolated Cell Walls of Lemna minor L. AB - From our theoretical treatment which is an extension of the classical Donnan theory, we have estimated the rational selectivity coefficients of the carboxylic groups of the walls during exchanges of divalent ions against monovalent ones (i.e. calcium and potassium or calcium and sodium ions). These coefficients express the interactions between the different ions, those between the counter ions and the ionized groups of the wall, and the influence of water. These quantitative values are consistent with the great affinity of the carboxylic groups for the calcium ions. They vary with the experimental conditions, showing a purely physicochemical mechanism of "regulation" of the exchanges in the cells walls of Lemna minor L. PMID- 16660543 TI - Intercellular Correlations: Relation between DNA Synthesis and Cell Division in Early Stages of in Vitro Bud Neoformation. AB - As well as showing the existence, during the first stages of in vitro bud neoformation, of cell populations in a tissue composed of a single cell layer (stem epidermis of Torenia fournieri Lind), some new physiological characteristics of mitosis are defined. Most of the cells which divide during organogenesis synthesize their DNA between 20 and 48 hours of culture. On an epidermal strip (10 x 2.5 millimeters composed of about 5,500 cells) 20% of the original cells enter the S-phase. The first division takes place at the 20-hour stage after the entry into the S-phase of a cell population of about 25 cells. Almost none of the cells of this population divide. The greatest percentage of divisions occurs in cells which synthesize DNA near the 48-hour stage. The relation [Formula: see text] has a value of about 25 at the beginning of cell division (20 hours) and falls to a value of about 1.4 for cells which synthesize DNA near the 48-hour stage. A hypothesis of the existence of a mitotic stimulant in the epidermis is put forward; this stimulant, at first weak, increases progressively. PMID- 16660544 TI - Carbon Dioxide Fixation in the Carbon Economy of Developing Seeds of Lupinus albus (L.). AB - The effects of CO(2) concentration and illumination on net gas exchange and the pathway of (14)CO(2) fixation in detached seeds from developing fruits of Lupinus albus (L.) have been studied.Increasing the CO(2) concentration in the surrounding atmosphere (from 0.03 to 3.0% [v/v] in air) decreased CO(2) efflux by detached seeds either exposed to the light flux equivalent to that transmitted by the pod wall (500 to 600 micro-Einsteins per square meter per second) in full sunlight or held in darkness. Above 1% CO(2) detached seeds made a net gain of CO(2) in the light (up to 0.4 milligrams of CO(2) fixed per gram fresh weight per hour) but (14)CO(2) injected into the gas space of intact fruits (containing around 1.5% CO(2) naturally) was fixed mainly by the pod and little by the seeds.Throughout development seeds contained ribulose-1,5-bisphosphate carboxylase activity (EC 4.1.1.39), especially in the embryo (up to 99 micromoles of CO(2) fixed per gram fresh weight per hour) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) in both testa (up to 280 micromoles of CO(2) fixed per gram fresh weight per hour) and embryo (up to 355 micromoles of CO(2) fixed per gram fresh weight per hour).In kinetic experiments the most significant early formed product of (14)CO(2) fixation in both light and dark was malate but in the light phosphoglyceric acid and sugar phosphates were also rapidly labeled. (14)CO(2) fixation in the light was linked to the synthesis of sugars and amino acids but in the dark labeled sugars were not formed. PMID- 16660545 TI - Sucrose in the free space of translocating maize leaf bundles. AB - Following exposure of portions of mature maize (Zea mays L.) leaf strips to (14)CO(2), xylem exudate from the leaf strips contained [(14)C]sucrose. Sucrose was the only sugar in the xylem exudate which was obtained from the cut surface of the leaf strips by reducing the external pressure. The sucrose found in the xylem exudate apparently was obtained from the free space of the vascular bundles, its concentration amounting up to 0.25%. When [(14)C]glucose or [(14)C]fructose was supplied in the dark to one end of a maize leaf strip, each was taken up by the xylem, and transported to the opposite end. Xylem exudate from such leaf strips contained (14)C-labeled sucrose in addition to the (14)C labeled hexose. The results of this study support the view that sucrose is loaded into the companion cell-sieve tube complexes from the apoplast of the vascular bundles in the maize leaf. PMID- 16660546 TI - Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.). AB - The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.Labeling studies using (14)CO(2) and (15)N(2) and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis. PMID- 16660547 TI - Etioplast Development in Dark-grown Leaves of Zea mays L. AB - The ultrastructure of etioplasts and the acyl lipid and the fatty acid composition of sequential 2-centimeter sections cut from the base (youngest) to the top (oldest) of nonilluminated 5-day-old etiolated leaves of Zea mays L., and the acyl lipid and fatty acid composition of the etioplasts isolated from them have been investigated. There is a 2.5-fold increase in the size of the plastids from the base to the tip of the leaf, and an increase both in the size of the prolamellar body and in the length of lamellae attached to it. The etioplasts in the bundle sheath and mesophyll cells of the older, but not the younger leaf tissue, are morphologically distinct. The monogalactosyl and digalactosyldiglycerides, phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were the only detectable acyl lipids in the isolated etioplast fractions. Together with phosphatidylethanolamine these were also the major acyl lipids in the whole leaf sections. With increasing age of the leaf tissue, increases occurred in two of the major plastid lipids, monogalactosyldiglyceride and phosphatidylglycerol, while the levels of essentially nonplastid lipids remained constant or declined slightly. The monogalactosyldiglyceride to digalactosyldiglyceride ratio increased from 0.4 to 1.1 in the tissue sections of increasing age and from 0.7 to 1.2 in the etioplasts isolated from them. Similarly, the galactolipid to phospholipid ratio increased from 0.8 to 1.4 in the tissue and from 0.5 to 4.5 in the isolated plastids. In the latter, the proportions of phosphatidylglycerol (as a per cent of total phospholipid) increased from 20 to 41% with increasing age of plastids.Linolenic acid was the major fatty acid in the total lipid of each of the etioplast fractions, but it was only the major fatty acid in the total lipid of the oldest leaf tissue. Its proportion in both total lipid extracts and individual lipids increased with age. The trans Delta(3) hexadecenoic acid was absent from all lipids. The protochlorophyllide content of the tissue increased with age. The results are discussed in relation to the use of illuminated etiolated leaves for studying chloroplast development. PMID- 16660548 TI - Subunit structure and composition of oat seed globulin. AB - Oat (Avena sativa L.) seed globulin was extracted from ground caryopses with 1 m NaCl, 0.05 m Tris(hydroxymethyl)aminoethane (pH 8.5) at room temperature. The globulin had a sedimentation constant of 12.1, and a molecular weight of 322,000, as determined by analytical ultracentrifugation. The globulin could be separated into two major subunits by sodiumdodecyl sulfate polyacrylamide gel electrophoresis. Molecular weights of the subunits were 21,700 (alpha) and 31,700 (beta), and they were present in equimolar amounts. A subunit model of 6alpha and 6beta per molecule of globulin is proposed. Amino acid analysis indicated that the alpha subunit contained more basic amino acids and aspartic acid/asparagine but less glutamic acid/glutamine and glycine than the beta subunit. PMID- 16660549 TI - Photosynthetic and Carbohydrate Metabolism in Isolated Leaf Cells of Digitaria pentzii. AB - Mesophyll cells and bundle sheath strands were isolated rapidly from leaves of the C(4) species Digitaria pentzii Stent. (slenderstem digitgrass) by a chopping and differential filtration technique. Rates of CO(2) fixation in the light by mesophyll and bundle sheath cells without added exogenous substrates were 6.3 and 54.2 micromoles of CO(2) per milligram of chlorophyll per hour, respectively. The addition of pyruvate or phosphoenolpyruvate to the mesophyll cells increased the rates to 15.2 and 824.6 micromoles of CO(2) per milligram of chlorophyll per hour, respectively. The addition of ribose 5-phosphate increased the rate for bundle sheath cells to 106.8 micromoles of CO(2) per milligram of chlorophyll per hour. These rates are comparable to those reported for cells isolated by other methods. The K(m)(HCO(3) (-)) for mesophyll cells was 0.9 mm; for bundle sheath cells it was 1.3 mm at low, and 40 mm at higher HCO(3) (-) concentrations. After 2 hours of photosynthesis by mesophyll cells in (14)CO(2) and phosphoenolpyruvate, 88% of the incorporated (14)C was found in organic acids and 0.8% in carbohydrates; for bundle sheath cells incubated in ribose 5-phosphate and ATP, more than 58% of incorporated (14)C was found in carbohydrates, mainly starch, and 32% in organic acids. These findings, together with the stimulation of CO(2) fixation by phosphoenolpyruvate for mesophyll cells and by ribose 5 phosphate plus ATP for bundle sheath cells, and the location of phosphoenolpyruvate and ribulose bisphosphate carboxylases in mesophyll and bundle sheath cells, respectively, are in accord with the scheme of C(4) photosynthesis which places the Calvin cycle in the bundle sheath and C(4) acid formation in mesophyll cells.Starch and reducing sugars were present in both mesophyll and bundle sheath cells following a period of photosynthesis by whole leaves. However, when isolated cells were exposed to (14)CO(2) in the light, even with appropriate exogenous substrates, only bundle sheath cells accumulated appreciable amounts of labeled carbohydrates. Incubation of mesophyll cells in the light with ATP and either pyruvate and inorganic phosphate, or phosphoenolpyruvate, or 3-phosphoglycerate resulted in large increases in total carbohydrates. The 3-phosphoglycerate treatment produced the greatest increase. These results could not be explained on the basis of increased CO(2) fixation. They suggest that mesophyll cells are able to metabolize exogenously supplied 3 carbon compounds to carbohydrates, despite the apparent inability of these cells to utilize CO(2) for this purpose, and support the view that in the whole leaf 3 phosphoglycerate is transported from bundle sheath to mesophyll cells, where it is reduced to carbohydrate.Sucrose and sucrose-phosphate synthetases and invertase were localized mainly in bundle sheath cells. ADP-Glucose starch synthetase and amylase were present mainly in bundle sheath cells whereas starch phosphorylase was present mainly in mesophyll cells. PMID- 16660550 TI - Studies of Delta-Aminolevulinic Acid Dehydrase from Skeletonema costatum, a Marine Plankton Diatom. AB - The accumulation of delta-aminolevulinic acid and activities of delta aminolevulinic acid dehydrase were examined in the marine diatom, Skeletonema costatum, grown in the presence of levulinic acid. Levulinic acid concentrations greater than 10 mm affect growth and morphology, and inhibit chlorophyll synthesis. The algae recover from the effects of levulinic acid after 48 hours of exposure. The recovery is characterized by increased cellular cholorphyll content, decreased delta-aminolevulinic acid accumulation, decreased 3-(3,4 dichlorophenyl)-1, 1-dimethylurea-enhanced in vivo fluorescence, and the induction of a levulinic acid-activated delta-aminolevulinic acid dehydrase which does not follow Michaelis-Menten kinetics. The data indicate that levulinic acid blocks may be ineffective in vivo, and that delta-aminolevulinic acid is metabolized to amino and dicarboxylic acids. delta-Aminolevulinic acid dehydrase activities are used to estimate the capacity for chlorophyll synthesis. Results suggest this diatom may be capable of rapid chlorophyll turnover, which would allow the plant to light-shade adapt on the time scales appropriate to vertical mixing rates in the sea. PMID- 16660551 TI - Modification of disease resistance of tobacco callus tissues by cytokinins. AB - The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a "hypersensitive" reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype. PMID- 16660552 TI - Development of Bacteroids in Alfalfa (Medicago sativa) Nodules. AB - The morphology, acetylene reduction capability, and nucleic acid content of bacteroids in different regions of alfalfa (Medicago sativa var. Buffalo) nodules were studied by electron microscopy, gas chromatography, and laser flow microfluorometry, respectively. Bacteroids in the nodule tips were small (1 to 2.5 micrometers in length), had low nucleic acid content, and contained distinct central nucleoids. These bacteroids were comparatively inactive in acetylene reduction in situ. Bacteroids in the middle regions of alfalfa nodules were greatly enlarged (5 to 7 micrometers in length), had relatively high nucleic acid content, and did not possess central nucleoids. The bacteroids were very active in acetylene reduction. Bacteroids in the basal nodule region also were enlarged and without distinct nucleoid regions, but had relatively low nucleic acid content and low in situ acetylene-reducing activity. PMID- 16660553 TI - Amino Acid composition of germinating cotton seeds. AB - Total and free amino acid composition of germinating cotton seeds (Gossypium hirsutum L.) was determined. The germinating seeds were separated into cotyledon and developing axis fractions daily and the composition of each tissue was summed to get the whole seed composition. By separating the developing seeds into these two tissue fractions, and determining total and free amino acids, a balance sheet was developed for each amino acid. This technique allowed changes in distribution with time of each amino acid to be followed in each tissue. Data for total content and amount in protein of each amino acid are presented. Asparagine increased in the whole seed, and most of this increase was found in the free pool of the developing axis. Other amino acids (e.g. arginine, glutamic acid) increased in the free pool but showed an over-all decrease, indicating that they were being metabolized. Amino acid contents of storage and nonstorage protein isolates were determined. PMID- 16660554 TI - Regulatory isoenzymes of aspartate kinase and the control of lysine and threonine biosynthesis in carrot cell suspension culture. AB - Aspartate kinase (EC 2.7.2.4) from carrot (Daucus carota L.) cell suspension culture has been partially resolved into lysine-sensitive and threonine-sensitive components by gel filtration chromatography. The yield of lysine-sensitive aspartate kinase changed independently of the yield of the threonine-sensitive activity during the 4-week growth cycle of the culture, and this provides additional evidence for the existence of two independently regulated isoenzymes. Exogenously supplied lysine and threonine specifically inhibited the in vivo formation of lysine and threonine, respectively, from radioactive acetate. PMID- 16660555 TI - Regulation of Glyoxysomal Enzymes during Germination of Cucumber: I. Developmental Changes in Cotyledonary Protein, RNA, and Enzyme Activities during Germination. AB - Developmental patterns of glyoxylate cycle and photosynthetic activities have been correlated with electrophoretic profiles of cotyledonary RNA and protein in both light- and dark-grown cucumber seedlings (Cucumis sativus L.) Cytoplasmic rRNA increases 10-fold between days 0 and 5, and the steepest increase coincides with the most rapid rise in activities of the glyoxysomal enzymes, isocitrate lyase and malate synthase. Chloroplast rRNA and ribulose bisphosphate (RuBP) carboxylase begin rising at day 3, followed about a day later by increases in glyoxylate reductase activity and chlorophyll content. Of these phototrophic indicators, only chlorophyll requires light for its initial appearance. Sodium dodecyl sulfate gel electrophoresis of total and soluble cotyledonary protein showed several developmental patterns, including: (a) progressive disappearance of storage protein present initially in particulate form; (b appearance and subsequent disappearance of a family of polypeptides identified by molecular weight, developmental profile, and density gradient centrifugation as subunits of glyoxysomal enzymes; and (c) appearance and progressive increase (in both light- and dark-grown cotyledons) of the large and small subunits of RuBP carboxylase, as well as other polypeptides presumably of chloroplast and peroxisomal origin. PMID- 16660556 TI - Partitioning of Sugar between Growth and Nitrate Reduction in Cotton Roots. AB - The level of endogenous sugars was inversely related to nitrate availability in young cotton (Gossypium hirsutum L.) plants, with high nitrate causing a greater decline in sugar content of roots than of shoots. High nitrate (low sugar) plants also displayed relatively more shoot growth and less root growth than low nitrate (high sugar) plants. These data are consistent with the theory that roots are poor competitors for sugar, and that sugar supply is a major factor limiting root growth in vivo.The effects of endogenous sugar level on root growth and on nitrate reductase activity in the root were different. When root sugar level was experimentally controlled by varying nitrate concentration in the nutrient solution, root growth was less sensitive than nitrate reductase activity to sugar deficiency. Also, in sterile root tips cultured on media containing a wide range of sucrose concentrations, growth rate was considerably less sensitive to endogenous sugar deficiency than was nitrate assimilation rate. Similarly, in plants which were detopped or girdled, nitrate reductase activity in the roots declined more rapidly than did root sugars, especially glucose and fructose. These results suggest that when sugar is deficient, cotton roots preferentially use it for growth at the expense of nitrate reduction. PMID- 16660557 TI - Stimulation of cadmium uptake in relation to the cadmium content of plants. AB - The time course of cadmium uptake by the roots of intact tomato plants (Lycopersicon esculentum Mill.) was measured in a nutrient solution with a micromolar cadmium concentration until all cadmium in the medium was exhausted. Exhaustion taking a few hours, cadmium was repeatedly added to the nutrient solution. The initial rate of cadmium uptake was computed for each cadmium addition. This rate sharply increased and ultimately leveled off, the maximum value being about three times higher than the value measured after the first cadmium addition. The stimulating effect of cadmium was associated with an inhibitory effect at higher levels of cadmium concentrations. An increase in the net cadmium influx with time could not be explained by the binding of heavy metal to a fixed number of organic compounds. Conceivably, the production of binding sites could be increased and cadmium might play a part in controlling the rate of sites production. PMID- 16660558 TI - Variation in Plastid Number: Effect on Chloroplast and Nuclear Deoxyribonucleic Acid Complement in the Unicellular Alga Olisthodiscus luteus. AB - Changes in the physiological state of the multiplastidic alga Olisthodiscus luteus result in a shift in chloroplast complement from 33 to 21 plastids. The effect of this induced change in organelle complement on nuclear and chloroplast DNA levels has been analyzed. Data suggest that the absolute amount of chloroplast and nuclear DNA found within a cell remains constant but that the amount of chloroplast DNA per plastid is inversely proportional to the number of chloroplasts to which that DNA must be distributed. PMID- 16660559 TI - Nickel in plants: I. Uptake kinetics using intact soybean seedlings. AB - The absorption of Ni(2+) by 21-day-old soybean plants (Glycine max cv. Williams) was investigated with respect to its concentration dependence, transport kinetics, and interactions with various nutrient cations. Nickel absorption, measured as a function of concentration (0.02 to 100 mum), demonstrated the presence of multiple absorption isotherms. Each of the three isotherms conforms to Michaelis-Menten kinetics; kinetic constants are reported for uptake by the intact plant and for transfer from root to shoot tissues. The absorption of Ni(2+) by the intact plant and its transfer from root to shoot were inhibited by the presence of Cu(2+), Zn(2+), Fe(2+), and Co(2+). Competition kinetic studies showed Cu(2+) and Zn(2+) to inhibit Ni(2+) absorption competitively, suggesting that Ni(2+), Cu(2+), and Zn(2+) are absorbed using the same carrier site. Calculated K(m) and K(i) constants for Ni(2+) in the presence and absence of Cu(2+) were 6.1 and 9.2 mum, respectively, whereas K(m) and K(i) constants were calculated to be 6.7 and 24.4 mum, respectively, for Ni(2+) in the presence and absence of Zn(2+). The mechanism of inhibition of Ni(2+) in the presence of Fe(2+) and Co(2+) was not resolved by classical kinetic relationships. PMID- 16660560 TI - Nickel in Plants: II. Distribution and Chemical Form in Soybean Plants. AB - The gross tissue distribution, intracellular fate, and chemical behavior of Ni(2+) in soybean plants (Glycine max cv. Williams) were investigated. Following root absorption, Ni was highly mobile in the plant, with leaves being the major sink in the shoots for Ni during vegetative growth. A senescence >70% of the Ni present in the shoot was remobilized to seeds. Fractionation of root and leaf tissues showed >90% of the Ni to be associated with the soluble fraction of tissues; ultrafiltration of the solubles showed >77% of the Ni to be associated with the 10,000 to 500 molecular weight components of both roots and leaves. Chemical characterization of the soluble components (10,000 to 500 and >500 molecular weight) by thin layer chromatography and electrophoresis resolved a number of Ni-containing organic complexes. Major Ni-containing components formed in the root are transported in the xylem stream, and undergo partial modification on deposition in leaves. Nickel accumulated in seeds is primarily associated with the cotyledons. Chemical fractionation of cotyledon components showed 80% of the Ni to be associated with the soluble whey fraction, while 70% of this fraction was composed of Ni-containing components with molecular weight <10,000. PMID- 16660561 TI - Studies on the Specificity and Site of Action of alpha-Cyclopropyl-alpha-[p methoxyphenyl]-5-pyrimidine Methyl Alcohol (Ancymidol), a Plant Growth Regulator. AB - alpha-Cyclopropyl-alpha-[p-methoxyphenyl]-5-pyrimidine methyl alcohol (ancymidol) is an inhibitor of ent-kaur-16-ene oxidation in microsomal preparations from the liquid endosperm of immature Marah macrocarpus seeds. The K(i) for this inhibitor is about 2 x 10(-9)m. Ancymidol also blocks ent-kaur-16-en-19-ol and ent-kaur-16 en-19-al oxidation by the same preparations with a similar efficiency, but does not significantly inhibit ent-kaur-16-en-19-oic acid oxidation. Ancymidol appears to be specific for this series of oxidations in higher plant tissues. It does not inhibit the oxidation of kaurene nor kaurenoic acid in rat liver microsomes and has no significant effect on the oxidation of cinnamic acid in microsomal preparations from Sorghum bicolor seedlings. Ancymidol also does not inhibit kaurene oxidation in vitro nor in vivo in cultures of the fungus Fusarium moniliforme. The presence of ancymidol did not significantly alter the activities of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, or NADH-cytochrome b(5) reductase. The addition of ancymidol to suspensions of oxidized M. macrocarpus endosperm led to a difference spectrum with an absorption maximum at 427 nm and a minimum at 410 nm. PMID- 16660562 TI - Light-induced, Dark-reversible Absorbance Changes in Roots, Other Organs, and Cell-free Preparations. AB - Irradiation of maize (Zea mays) roots and coleoptiles with visible light causes dark-reversible absorbance changes in these organs. There is an increase in absorbance near 440 nanometers and smaller increases below 410 nanometers and about 595 nanometers. Decreases in absorbance are observed at about 420 nanometers and minor ones at 537 and 575 nanometers. These responses are also observed in cell-free preparations from roots and coleoptiles if dithionite, NADPH, or NADH is added prior to illumination. The dose curve for these effects has a distinct maximum at 420 nanometers and a minor one at 575 nanometers. Difference spectra and dose response curves indicate that heme compounds such as cytochromes or, more probably, peroxidase complexes are the photoreceptive and chemical reacting molecules. Siroheme-containing proteins may also be taken into consideration.The light-induced absorbance changes have half-lives of more than 200 seconds and 100 seconds in roots of maize and soybean, respectively. Two reactions, each with first order kinetics, appear to be superimposed. The respective rate constants for maize roots are about 0.004 and 0.04 seconds(-1). The generation of the effect has a much shorter half-life dependent on light intensity and wavelength. Little deviations from first order kinetics were detected. Rate constants for corn roots range between 0.05 and 0.01 seconds( 1).Apart from the problem in which hemoproteins are involved, there is the problem of correlating the reaction of the photoreactive and chemically reacting molecules to macroscopic responses such as phototropism. PMID- 16660563 TI - An Inborn Error of Potassium Metabolism in the Tomato, Lycopersicon esculentum. AB - A mutant of the tomato (Lycopersicon esculentum Mill.) was obtained through treatment of the seed with ethyl methane sulfonate. Its chief distinguishing characteristic is the requirement for an extraordinarily high K concentration in the medium (20 mm) if it is to develop without pronounced K deficiency symptoms, while the wild type grows without any such symptoms at 0.1 to 0.2 mm K. The lesion of the K-inefficient mutant does not lie in its inability to absorb and translocate K. PMID- 16660564 TI - Analysis of the Components Released from Potato Tuber Tissues during Maceration by Pectolytic Enzymes. AB - Endo-pectin lyase and endo-polygalacturonase of Aspergillus japonicus attack the middle lamella of plant tissue and cause tissue maceration. Galacturonides, neutral sugars, and proteins were released from potato tuber tissues during maceration by both purified enzymes. These three components accounted for 92% of the soluble products. The neutral sugars released were rhamnose, arabinose, and galactose with a molar ratio of 1:3:15. They were covalently linked to galacturonides. Over 85% of the galacturonides released by the enzymes were short chain products, which indicated that a large portion of the main chain of pectic substances is a homogalacturonan. The results of chromatography on columns of Sephadex G-100 and DEAE-cellulose suggested that a protein component may be attached to pectic substances. This protein did not contain hydroxyproline and, therefore, was different from the cell wall structural glycoprotein. PMID- 16660565 TI - Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data. AB - Density-labeling with 10 mm K(15)NO(3)/70% (2)H(2)O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis. PMID- 16660566 TI - Biosynthesis of the Diterpene Phytoalexin Casbene: Partial Purification and Characterization of Casbene Synthetase from Ricinis communis. AB - Casbene synthetase from 67-hour seedlings of Ricinis communis L. which had been treated with Rhizopus stolonifer spores was purified 700-fold by a combination of ammonium sulfate fractionation, QAE A-50 Sephadex chromatography, and G-100 Sephadex chromatography. Polyacrylamide disc gel electrophoresis revealed that the purified fraction was heterogeneous. No casbene synthetase was detected in extracts of seedlings which had not been exposed to the fungal spores; maximum activity was obtained from seedlings 14 hours after exposure to spores.The partially purified enzyme exhibited a broad pH optimum from pH 7.5 to 9.0 with half-maximal activity at pH 6.0 and 9.8. Chromatography on a calibrated Sephadex G-100 column indicated a molecular weight of 53,000 +/- 3,000 for casbene synthetase. Concentrations of Mg(2+) above 5 mm gave maximal stimulation of the activity. Mn(2+) was much less effective and was inhibitory at concentrations above 0.2 mm. The K(m) for geranyl-geranyl pyrophosphate was estimated as 1.9 mum. The activity was inhibited 50% by 2.5 mm N-ethylmaleimide; 10 mm iodoacetamide was not inhibitory. N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and the growth retardant 2'isopropyl-4'-(trimethylammonium chloride) 5'-methylphenyl piperidine-1-carboxylate (Amo-1618) were ineffective inhibitors of casbene synthetase, but the growth retardant tributyl-2, 4-dichlorobenzyl phosphonium chloride (Phosphon D) at a concentration of 5 mum inhibited the activity by 55%. PMID- 16660567 TI - Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings. AB - When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K(m) at 38 C of 1.8 x 10(-7)m. The K(m) for casein was 1.1 x 10(-6)m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence. PMID- 16660568 TI - Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules. AB - A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O(2)). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H(2). The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H(2) uptake efficiency of intact nodules. PMID- 16660569 TI - Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza. AB - The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their "inseparability." PMID- 16660570 TI - Method for overcoming the antiethylene effects of ag. AB - A technique is described for eliminating the antiethylene effects of the Ag(+) ion in the intact pea plant (Pisum sativum). The technique is based on the ability of the ethylene mimic, acetylene, to negate the antiethylene effect of Ag(+), presumably through salt formation, and subsequently to induce the ethylene response. PMID- 16660571 TI - Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves: Cellular Distribution of Enzymes and Energy Sources for Nitrate Reduction. AB - The localization of enzymes responsible for nitrate assimilation and the generation of NADH for nitrate reduction were studied in corn (Zea mays L.) leaf blades. The techniques used effectively separated mesophyll and bundle sheath cells as judged by microscopic observations, enzymic assays, chlorophyll a/b ratios and photochemical activities. Nitrate reductase, nitrite reductase, and the nitrate content of leaf blades were localized primarily in the mesophyll cells, although some nitrite reductase was found in the bundle sheath cells. Glutamine synthetase, NAD-malate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glutamate dehydrogenase were found in both types of cells, however, more NADP-glutamate dehydrogenase was found in the bundle sheath cells than in the mesophyll cells. These data indicate that the mesophyll cells are the major site for nitrate assimilation in the leaf blade because they contained an ample supply of nitrate and the enzymes considered essential for the assimilation of nitrate into amino acids. Because the specific activity of nitrate reductase was severalfold lower than the other enzymes involved in nitrate assimilation, nitrate reduction is indicated as the rate-limiting step in situ. A sequence of reactions is proposed for nitrate assimilation in the mesophyll cells of corn leaves as related to the C-4 pathway of photosynthesis. PMID- 16660572 TI - Phytostat for the growth of lemna in semicontinuous culture with low sulfate. AB - An apparatus is described by means of which Lemna perpusilla 6746 was grown photoautotrophically at a series of constant concentrations of inorganic sulfate, as low as 0.26 mum. Theoretical considerations relevant to this system are discussed and examples of the operation of the apparatus are presented. The data obtained were used to calculate sulfate uptake by the plant colonies as a function of sulfate concentration. The apparatus should be useful for the production of relatively large quantities of Lemna, grown under highly uniform conditions, with or without limitation of one or more nutrient(s). Other small vegetatively reproducing aquatic plants could be similarly studied. Uptake studies could be carried out on a variety of plant materials. PMID- 16660573 TI - Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations. AB - Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 mum, was labeled to radioisotopic equilibrium with (35)SO(4) (2-). Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. gamma Glutamylcyst(e)ine, erythro- and threo-thiothreonine, and S-methylcysteine were not detected. No volatile (35)S compounds were formed during plant growth at 1,000 mum sulfate, nor were significant amounts of (35)S compounds excreted into the medium.The amount of each component present in colonies grown over the 3,000 fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5- to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna, and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration.Procedures for the analysis of S adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented. PMID- 16660574 TI - Nuclear magnetic resonance relaxation times and plasmalemma water exchange in ivy bark. AB - Measurement of nuclear magnetic resonance (NMR) relaxation times (transverse [T(2)] and longitudinal [T(1)]) for Hedera helix L. cv. Thorndale (ivy) bark water indicates the presence of at least two populations of water with different relaxation characteristics. One population of water with short T(2) and T(1) was found to be composed of both hydration water and extracellular free water. The second population of water with long T(2) and T(1) was identified as intracellular bulk water.NMR relaxation of extracellular water protons is controlled by cell wall surface effects, possibly due to binding of paramagnetic cations by the cell walls. NMR relaxation of intracellular water protons is controlled by both water exchange to the extracellular environment and chemical exchange with a population of protons that is chemically shifted from that of the bulk water. The relaxation time of intracellular water is not measurably affected, either by intracellular paramagnetic ions or by increased viscosity of intracellular water. Manganese flux into the cells occurs at 1.7 x 10(-15) moles cm(-2) seconds(-1) and is independent of extracellular Mn(2+) concentration in the range 5 to 20 mm.The intracellular-extracellular water exchange time of ivy bark was found to be predominantly limited by membrane water permeability. A diffusional water permeability coefficient (P(d)) of approximately 3 x 10(-2) cm seconds(-1) was calculated for ivy cell membranes at 20 C. PMID- 16660575 TI - Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination. AB - The free amino acid concentrations in cotyledons and axes of soybean (Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine. PMID- 16660576 TI - Enzymic and substrate basis for the anaplerotic step in guard cells. AB - From the maximum rate of malate accumulation in Vicia faba L. guard cells during stomatal opening the maximum rate of organic anion synthesis is calculated to be 200 millimoles per kilogram dry weight per hour. A minimum estimate for the phosphoenolpyruvate (PEP) carboxylase-catalyzed reaction in guard cells is 650 millimoles per kilogram dry weight per hour which is significantly higher than in any other leaf tissue. The apparent K(mpep) of the guard cell enzyme is 60 mum at pH 8.7, but is probably higher at lower pH. The concentration of PEP in guard cells was 270mum (=2.2 x 10(-15) moles/guard cell pair) during anion synthesis. These results support the possibility that the carboxylation of PEP is the anaplerotic step in guard cells. PMID- 16660577 TI - Activity of the Electrogenic Pump in Chara corallina as Inferred from Measurements of the Membrane Potential, Conductance, and Potassium Permeability. AB - The effects of various inhibitors on the membrane potential, resistance, and K(+) permeability of Chara corallina were measured, providing evidence that there is an electrogenic pump in the membrane. It was found that: (a) 5.0 mum carbonyl cyanide m-chlorophenyl hydrazone depolarizes the membrane potential and increases the membrane resistance. This inhibition is faster in the dark than in the light but the extent of inhibition is the same in both cases. (b) Fifty mum dicyclohexylcarbodiimide increases the resistance and the K(+) permeability and depolarizes the membrane to a diffusion potential mainly controlled by K(+). (c) Forty mum diethylstilbestrol and 0.1 mm 2,4-dinitrophenol increase the resistance and depolarize the potential to a value given by the Goldman diffusion equation. (d) Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea and darkness (at pH 6) cause the membrane resistance to increase but neither has a large effect on the potential. 3-(3,4-dichlorophenyl)-1,1-Dimethylurea increases K(+) permeability while darkness decreases it.In all cases, the increase in resistance is interpreted as an inhibition of conductance through the electrogenic pump. As a consequence of this inhibition, the electrogenic component of the membrane potential is reduced, depolarizing the membrane. The electrogenic pump may be an H(+)-ATPase in the plasmalemma.5,5-Dimethyloxazolidine-2,4-dione at 5.0 mm decreases the membrane resistance, by lowering the internal pH providing more substrate for the pump. La(3+) decreased cation permeability and depolarized the membrane but, since it had little effect on the membrane resistance, it probably does not affect the electrogenic pump. PMID- 16660578 TI - Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth. AB - The effects of N source (6 mm nitrogen as NO(3) (-) or urea) and tungstate (0, 100, 200, 300, and 400 mum Na(2) WO(4)) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO(3) (-) content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO(3) (-)-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO(3) (-)-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 mum tungstate levels, acetylene reduction activity of nodulated roots of NO(3) (-)-grown plants exceeded the activity of comparable urea-grown plants.Addition of tungstate to the nutrient solution decreased plant growth, regardless of the N source, although the effect was more pronounced with NO(3) ( ) nutrition. The increased nodulation and decreased nitrate reductase activity noted with plants grown in the presence of tungstate and a high (6 mm) external supply of NO(3) (-) suggests that NO(3) (-) does not directly inhibit nodulation but rather affects nodulation indirectly through subsequent metabolism of NO(3) ( ). PMID- 16660579 TI - Comparison of the uptake of nitrate and ammonium by rice seedlings: influences of light, temperature, oxygen concentration, exogenous sucrose, and metabolic inhibitors. AB - The uptake of nitrate and ammonium by rice (Oryza sativa) seedlings was compared under various conditions. Nitrate uptake showed a 1-hour lag phase and then a rapid absorption phase, whereas ammonium uptake showed passive absorption during the first hour, then a shoulder of absorption, followed by a rapid metabolism dependent absorption phase. Light did not affect the uptake of nitrate or ammonium. The uptake of nitrate and ammonium was markedly suppressed by removal of the endosperm. After removal of the endosperm, the uptake was restored by exogenous supply of 30 mm sucrose. No appreciable nitrate uptake was detected at temperatures below 15 C, whereas appreciable ammonium uptake occurred at 15 C, although lowered passive absorption and complete inhibition of the rapid metabolism-dependent absorption phase were observed at 5 C.Nitrate uptake was decreased by aeration and also by bubbling the mixture with O(2). It was greatly depressed by bubbling the mixture with N(2). These results suggest that there is a low optimal O(2) concentration for nitrate uptake. In contrast, ammonium uptake was increased by aeration, not influenced by bubbling with O(2), and slightly decreased by bubbling the mixture with N(2).Nitrate uptake was almost entirely inhibited by addition of cycloheximide, KCN, or arsenate. These compounds had scarcely any effect on the passive absorption of ammonium, but entirely depressed its successive metabolism-dependent absorption. Ammonium uptake occurred before nitrate uptake in solution containing both nitrate and ammonium. PMID- 16660580 TI - Leaf Conductance in Relation to Assimilation in Eucalyptus pauciflora Sieb. ex Spreng: Influence of Irradiance and Partial Pressure of Carbon Dioxide. AB - Rates of assimilation and transpiration in Eucalyptus pauciflora Sieb. ex Spreng were measured at various ambient partial pressures of CO(2) and various irradiances and were used to estimate leaf conductance and intercellular partial pressure of CO(2). The responses of leaf conductance and rate of assimilation to change in intercellular partial pressure of CO(2) were expressed in terms of feedback. They are small in the sense that their combined effect was to reduce disturbances in intercellular partial pressure of CO(2) by 30% only. The magnitude of the feedback had no influence on the system as affected by irradiance, because the direct responses of conductance and rate of assimilation to changes in irradiance in the range 0.25 to 2 millieinsteins per meter per second were such that intercellular partial pressure was maintained almost constant. PMID- 16660581 TI - Lipid and Surface Wax Synthesis in Water-stressed Cotton Leaves. AB - The incorporation of [2-(14)C]malonate and [1-(14)C]acetate into internal lipid and surface wax by cotton leaves (Gossypium hirsutum L. ;Deltapine') having water potentials of -8 to -15 bars (controls) and -19 to -32 bars (water-stressed) was compared. Lipid from stressed leaves contained a mean of 57% more radioactivity than corresponding controls for five experiments. Acetyl coenzyme A carboxylase was not limiting to fatty acid synthesis in water-stressed cotton leaves at the water potential levels tested, whereas fatty acid synthetase was stimulated. In four of six experiments, wax from stressed leaves contained a mean of 38% less radioactivity than nonstressed leaves when incubated 24 hours after rehydration. Evidence is presented to show that after a suitable period of rehydration, previously stressed cotton leaves produce more wax than leaves prior to stressing. PMID- 16660582 TI - Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis. AB - A mutant strain, Y(9), of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y(9) cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y(9) cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y(9). The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed. PMID- 16660583 TI - Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium. AB - The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [(14)C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride treated plants did not differ significantly in the amount of [(14)C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [(14)C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable (14)C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated (14)C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments. PMID- 16660584 TI - Diurnal oscillation of amylolytic activity in spinach chloroplasts. AB - Chloroplasts isolated from spinach (Spinacia oleracea L., cv. vital(R)) plants grown under controlled light/dark and temperature regimes, contained the phosphorolytic and amylolytic pathways for starch breakdown. The latter consists at least of alpha- and beta-amylase and maltase. Only low amylolytic activity was observed in chloroplasts isolated during the light phase. In chloroplasts prepared during the dark phase, this activity was almost twice as high. These diurnal oscillations of the amylolytic activity were maintained when the plants were kept in prolonged darkness or continuous light. The amylolytic system exhibited a sharp pH optimum between 5.8 and 6.0. Phosphorylase activity, when assayed with saturating concentrations of inorganic phosphate, did not show diurnal fluctuations. PMID- 16660585 TI - Inhibition of photorespiration and increase of net photosynthesis in isolated maize bundle sheath cells treated with glutamate or aspartate. AB - Net photosynthetic (14)CO(2) fixation by isolated maize (Zea mays) bundle sheath strands was stimulated 20 to 35% by the inclusion of l-glutamate or l-aspartate in the reaction mixture. Maximal stimulation occurred at a 7.5 mm concentration of either amino acid. Since the photosynthetic rate and the glutamate-dependent stimulation in the rate were equally sensitive to a photosynthetic electron transport inhibitor, 3-(p-chlorophenyl)-1,1-dimethylurea, it was concluded that glutamate did not stimulate CO(2) fixation by supplying needed NADPH (NADH) through glutamate dehydrogenase. Treatment of the bundle sheath strands with glutamate inhibited glycolate synthesis by 59%. Photorespiration in this tissue, measured as the O(2) inhibition of CO(2) fixation (the Warburg effect), was inhibited by treatment with glutamate. The stimulation in net photosynthetic CO(2) fixation probably results from the decrease in photorespiratory CO(2) loss. This metabolic regulation of the rate of glycolate synthesis and photorespiration observed with isolated bundle sheath strands could account for the inability to detect rapid photorespiration in the mature intact maize leaf. PMID- 16660586 TI - Effect of l-Canavanine on Nitrate Reductase in Corn Roots. AB - l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 mum canavanine was effective in root tips whereas only 50 mum was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO(3) resulted in a loss of NRA. NO(3) (-) protected nitrate reductase from this inactivation in both root tip and mature root sections. PMID- 16660587 TI - Plant chemiluminescence. AB - Light production by plants was confirmed by measuring chemiluminescence from root and stem tissue of peas (Pisum sativum), beans (Phaseolus vulgaris), and corn (Zea mays) in a modified scintillation spectrophotometer. Chemiluminescence was inhibited by treating pea roots with boiling ethanol or by placing them in a N(2) gas phase. Chemiluminescence was increased by an O(2) gas phase or by the addition of luminol. NaN(3) and NaCN blocked both in vitro and in vivo chemiluminescence.It is postulated that the source of light is the hydrogen peroxide-peroxidase enzyme system. It is known that this system is responsible for chemiluminescence in leukocytes and it seems likely that a similar system occurs in plants. PMID- 16660588 TI - Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures. AB - Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to -3.9 C resulted in 80% lysis of the protoplasts. At -3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl >/= Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle. PMID- 16660589 TI - Inhibition of glucuronokinase by substrate analogs. AB - Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a K(i) value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl alpha-glucuronoside, methyl beta-glucuronoside, beta-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The beta glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective. PMID- 16660590 TI - Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: III. Induction and Transmission of the Response. AB - Increased ethylene synthesis was rapidly induced throughout the apical meristematic region of etiolated seedlings of Pisum sativum L., cv. Alaska by cuts made 1 centimeter from the apical hook. The wound signal was transmitted at about 2 millimeters per minute. Accumulation of substance(s) at the cut surfaces of excised sections, as the result of interrupted translocation, did not initiate or significantly contribute to wound-induced ethylene synthesis, nor was the cut surface the site of enhanced ethylene synthesis. Cutting subapical sections into shorter pieces showed that cells less than 2 millimeters from a cut surface produced about 30% less ethylene than cells greater than 2 millimeters from a cut surface. PMID- 16660591 TI - Distribution of alpha-Galactosidase in Cucurbita pepo. AB - The distribution of alpha-galactosidase (alpha-d-galactoside galactohydrolase [EC 3.2.1.22]) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series ([alpha-1-6-0-galactopyranosyl](n) sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of alpha-galactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total alpha-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl oligosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl oligosaccharides between the phloem and surrounding tissues in this plant. PMID- 16660592 TI - Light activation of ribulose bisphosphate carboxylase: purification and properties of the enzyme in tobacco. AB - The development of methods of preparation of long wavelength ultraviolet light capable of activating ribulose bisphosphate carboxylase is reported. This preparation was obtained from tobacco (Nicotiana tabacum) leaves which had reached about one-half maximum leaf weight. It was prepared at low ionic strength by use of mixed anion and cation exchange resins and buffers containing dimethylsulfoxide. The preparation is greatly enriched in fraction I protein to the point of apparent homogeneity. When assayed in the presence of saturating ribulose bisphosphate and sodium bicarbonate, the rate of carbon fixation is a linear function of long wavelength ultraviolet irradiation in the range of 20,000 to 30,000 ergs per square centimeter per second. Glutathione (5 mm) inhibits light activation without affecting activity in the dark. Copper sulfate inhibits both light and dark activity, but is slightly less effective in the presence of ultraviolet light. Sucrose inhibition of carboxylation is only readily apparent in the absence of ultraviolet light. Ammonium sulfate precipitation followed by solubilization in buffers containing dimethylsulfoxide plus heat treatment promotes ultraviolet light activation. PMID- 16660593 TI - Comparison and evaluation methods for the removal of ethylene and other hydrocarbons from air for biological studies. AB - A random sampling analysis of laboratory air and of air from commercially available cylinders indicated that they contain appreciable amounts of low molecular weight hydrocarbons, viz. methane, ethane, and ethylene, as contaminants. These impurities could lead to erroneous conclusions in studies of plant growth and metabolism. Different methods for removal of these contaminants were compared and evaluated in the present investigation for their suitability in plant studies. Most of the methods currently being used were found inadequate. The use of metal catalysts at high temperature, adapted from gas analysis techniques, provides an inexpensive and efficient method for removing hydrocarbons from air in both closed and continuous flow systems. PMID- 16660594 TI - Lead toxicity and phosphate deficiency in chlamydomonas. AB - The addition of lead salts to phosphate-containing Chlamydomonas reinhardtii media caused precipitation of Pb(3)(PO(4))(2), effectively removing phosphate from solution. The effect of Pb(2+) on growth of Chlamydomonas in liquid cultures depended strictly on the ratio of the equivalents of Pb(2+) to phosphate present. When the amount of Pb(2+) approached equivalency with phosphate, cell growth was initially slow as cells adhered to the surface of the precipitated Pb(3)(PO(4))(2). Later, cells grew at a normal rate, spread throughout the solution, and reached the same densities obtained in the absence of Pb(2+). Cells did not survive when the amount of Pb(2+) in the culture exceeded the equivalents of phosphate.Elemental analysis showed that in the presence of equivalent Pb(2+) and phosphate, considerable Pb(2+) remained in solution. The concentration of dissolved Pb(2+) did not vary significantly when the amount of Pb(2+) added to the culture was increased slightly, from an amount which permitted growth to an amount which completely prevented growth. The concentration of phosphate was decreased to an undetectable level when the amount of Pb(2+) approached equivalency with phosphate.In the presence of the chelating agent nitrilotriacetic acid, higher concentrations of Pb(2+) remained in phosphate containing media. The chelated Pb(2+) did not retard the growth of Chlamydomonas.It appears that Pb(2+) is not toxic to Chlamydomonas, but kills cells by depriving them of phosphate. PMID- 16660595 TI - Response of Membrane-bound Mg-activated ATPase of Tobacco Leaves to Tobacco Mosaic Virus. AB - Infectious material was formed at an early stage, and migrated into the mesophyll from the epidermis of tobacco leaves (Nicotiana tabacum cv. Samsun NN) during the period of 1 to 3 hours after inoculation with tobacco mosaic virus (TMV). The activity of membrane-bound Mg(2+)-activated ATPase from the mesophyll was stimulated two to four times within 30 minutes after inoculation with 1.0 microgram per milliliter of TMV. Maximum TMV stimulation of membrane-bound Mg(2+) activated ATPase activity in epidermis and mesophyll was observed at 0.5 and 3.0 hours after inoculation, respectively. This stimulation was also observed with ultraviolet irradiated TMV (only RNA was destroyed), whereas, the stimulation was not observed with heat-irradiated TMV (both coat and RNA were destroyed). Stimulation equal to that of TMV was observed by inoculation with cucumber green mottle mosaic virus and to a lesser extent with cucumber mosaic virus.These results illustrate that the stimulus resulting from inoculation with TMV transfers to underlying cells faster than the migration of TMV particles. This stimulus might be closely correlated to the structure of virus, but not to the infectivity of virus. PMID- 16660596 TI - Kinetics of Membrane Transport during Chloroplast Development. AB - In the course of plastid development there are changes in the permeability of the envelope membranes. An investigation of the kinetics of transport with largely uncontaminated and intact etioplast/etiochloroplast preparations from greening Avena sativa laminae demonstrates: (a) that etioplasts already possess specific translocators for the transporation of orthophosphate, dihydroxyacetone phosphate, 3-phosphoglycerate ("phosphate translocator"), and dicarboxylic acids ("dicarboxylate translocator"); (b) that changes in the rates of uptake during development are mainly due to changes in velocity for specific transport and not due to changes in the affinity for transport (K(m)) or nonspecific permeation. The very low competitive inhibition of transport of orthophosphate by dihydroxyacetone phosphate and 3-phosphoglycerate, observed for developmental stages corresponding to up to 3 hours of illumination of etiolated tissue, is discussed with respect to the possibility of an early phosphate transport mechanism that is different from the phosphate translocator of more developed plastids. PMID- 16660597 TI - Osmotically induced removal of water from fungal cells as determined by a spin probe technique. AB - Effects of physical environment on plasma membrane semipermeability and osmotic induction of changes in aqueous cytoplasmic volume were studied in vegetative and spore cells of a plant pathogenic fungus, Fusarium sulphureum. A direct method, employing a spin probe molecule that partitioned between intracellular aqueous and hydrophobic phases, allowed measurement of reversible water movement out of macroconidial cells and chlamydospores exposed to solutions of high osmolarity. Equilibrium distribution of the spin probe between intracellular aqueous and lipid phases was more rapid than movement of water in and out of the cells. The extent of water removal was exponentially dependent on osmotic strength. Some cells became irreversibly permeable to divalent cations on treatment with sodium chloride above 1.5 osmolar but addition of sucrose to the suspension medium at equivalent osmolar concentrations caused water removal without adversely affecting the viability. Sucrose also protected the plasma membrane against damage during freeze-drying. Induction of plasma membrane damage by osmotic shock or freeze-drying permitted rapid permeation of nickel ions. Neither slow equilibration of intracellular components with divalent paramagnetic cations nor partial permeability of damaged plasma membranes to these ions was observed. PMID- 16660598 TI - Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination. AB - The endosperm of castor bean seeds (Ricinus communis L.) contains two -SH dependent aminopeptidases, one hydrolyzing l-leucine-beta-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-beta-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an -SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two -SH dependent enzymes hydrolyzing the model substrate alpha-N-benzoyl-dl-arginine beta-naphthylamide (BANA). The carboxypeptidase is active on a variety of N carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6.The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. PMID- 16660599 TI - Isolation and Characterization of Factors in Sweet Potato Root Which Agglutinate Germinated Spores of Ceratocystis fimbriata, Black Rot Fungus. AB - A factor which agglutinates the germinated spores of Ceratocystis fimbriata was isolated from the sweet potato root. The factor is a glycoprotein with a molecular weight of 1.6 x 10(6) daltons and required divalent cations such as Ca(2+), Mn(2+), Ni(2+), and Mg(2+) for activity. The activity of the factor was pH-dependent. The factor also agglutinated rabbit erythrocytes and is classified as a phytohemagglutinin or lectin. The factor agglutinated germinated spores of seven strains of C. fimbriata to almost the same degree. The factor showed differential agglutinating activity toward the strains in the presence of unidentified low molecular weight factor(s) in the sweet potato root. These results support our earlier suggestion that the spore-agglutinating factors in host plants function as the determinants of specificity in some host-parasite interactions. PMID- 16660600 TI - Regulation of Glyoxysomal Enzymes during Germination of Cucumber: 2. Isolation and Immunological Detection of Isocitrate Lyase and Catalase. AB - The glyoxysomal enzymes isocitrate lyase and catalase have been isolated from etiolated cucumber (Cucumis sativus) cotyledons. The enzymes co-purified through polyethyleneimine precipitation and (NH(4))(2)SO(4) precipitation, and were resolved by gel filtration on Sepharose 6B followed by chromatography on diethylaminoethyl-cellulose (isocitrate lyase) or hydroxylapatite (catalase). Purity of the isolated enzymes was assessed by sodium dodecyl sulfate polyacrylamide electrophoresis, isoelectric focusing, and immunoelectrophoresis. Antibodies raised to both enzymes in rabbits and in tumor-bearing mice were shown to be monospecific by immunoelectrophoresis against total homogenate protein. Isocitrate lyase and catalase represent about 0.56% and 0.1%, respectively, of total extractable cotyledonary protein. Both enzymes appear to be present in a single form. Molecular weights of the native enzymes and its subunits are 225,000 and 54,500 for catalase, and 325,000 and 63,500 for isocitrate lyase. The pH optimum for isocitrate lyase is about 6.75 in morpholinopropane sulfonic acid buffer, but varies significantly with buffer used. The K(m) for d-isocitrate is 39 micromolar. A double antibody technique (rabbit anti-isocitrate lyase followed by (125)I-labeled goat anti-rabbit immunoglobulin G) has been used to visualize isocitrate lyase subunit protein on sodium dodecyl sulfate-polyacrylamide with high specificity and sensitivity. PMID- 16660601 TI - Regulation of ribosomal RNA accumulation by auxin in artichoke tissue. AB - Artichoke (Helianthus tuberosus L.) tuber tissue cultured in the presence of the auxin 2,4-dichlorophenoxyacetic acid accumulates ribosomal RNA at a rate of 0.135 micrograms per hour per explant whereas there is little accumulation in nontreated tissue. The addition of auxin enhanced the transcription of the 2.5 x 10(6) precursor 3.5-fold and increased the rate of processing 1.8-fold. The major effect of auxin, however, was a vast increase in the rate of processing of the 1.39 x 10(6) precursor to the 1.3 x 10(6) mature ribosomal RNA. The incorporation of label into the 0.7 x 10(6) mature ribosomal RNA of treated tissue was in 10 fold excess over the control after a 30-minute pulse and remained so throughout the remainder of the labeling period. This level, however, was not reached for the complementary 1.3 mature RNA until 3 hours of continuous labeling, decreasing from a initial value of 40-fold excess. A complication in the processing of ribosomal RNA is the apparent increase in the stability of the 0.7 x 10(6) mature RNA with auxin treatment. PMID- 16660602 TI - Membrane-associated Glycosyl Transferases in Cotyledons of Pisum sativum: Differential Effects of Magnesium and Manganese Ions. AB - In crude particulate fractions isolated from pea (Pisum sativum) cotyledons, the transfer of radioactivity from GDP-[(14)C]mannose to glycolipid appears to be preferentially stimulated by Mn(2+) while the transfer to lipid-free residue is enhanced by Mg(2+). In contrast, the transfer of radioactivity from UDP-N-acetyl [(14)C]glucosamine to glycolipid shows preferential stimulation by Mg(2+) while the transfer to lipid-free residue prefers Mn(2+). These results are accounted for by the differential stimulation by Mg(2+) and Mn(2+) of glycosyl transferases associated with subcellular membranes which were separated by isopycnic sucrose density centrifugation. PMID- 16660603 TI - Irradiation-enhanced Phytochrome Pelletability: Requirement for Phosphorylative Energy in Vivo. AB - Short, high intensity pulses of red and far red light are used to study, at room temperature, the kinetics of the in vivo dark reaction responsible for irradiation-enhanced phytochrome pelletability. The t((1/2)) for this reaction is 2 seconds at 25 C in both Avena shoots and Zea mays coleoptiles. This is the most rapid phytochrome-far red-absorbing form (Pfr)-mediated cellular response thus far reported. Anoxia, KCN, NaN(3) and carbonyl cyanide p trifluoromethoxyphenylhydrazone reduce the rate (but not the final extent) of the reaction by more than an order of magnitude. The rate of the reaction under these conditions is strongly correlated with the inhibitor-induced reductions in cellular ATP levels. Likewise, recovery in ATP levels upon withdrawal of the inhibitors is accompanied by a parallel recovery in the rate of the reaction. Cytochalasin B blocks cytoplasmic streaming without diminishing the pelletability response. Colchicine is likewise without effect. These data suggest a requirement for phosphorylative energy in one or more of the Pfr-dependent intracellular events leading to enhanced phytochrome pelletability. The possibility that this event might represent an ATP-dependent modification of the pigment protein itself in the Pfr form is discussed. PMID- 16660604 TI - Seasonal Variation in the Hormone Content of Willow: II. Effect of Photoperiod on Growth and Abscisic Acid Content of Trees under Field Conditions. AB - Levels of abscisic acid were followed in the xylem sap, mature leaves, and apices of field-grown willow (Salix viminalis L.) during the summer months, under natural and artificially extended photoperiods. Although the long day treatment prevented the general onset of dormancy, the plants grown under natural daylengths showed lower concentration of abscisic acid than those kept under long days. PMID- 16660605 TI - Depolarization of the Electrogenic Transmembrane Electropotential of Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Toxin, Azide, Cyanide, Dodecyl Succinic Acid, or Cold Temperature. AB - The transmembrane electrical potential of root cells of Zea mays L. cv. W64A in a modified 1x Higinbotham solution was partially depolarized by semipurified toxin obtained from Bipolaris (Helminthosporium) maydis race T. At a given toxin concentration depolarization of Texas cytoplasm cells was much greater than for normal cytoplasm cells. This observation correlated directly to the differential host susceptibility to the fungus. The time course and magnitude of depolarization were dependent on toxin concentration; at high concentration the electropotential difference change was rapid. Cortex cells depolarized more slowly than epidermal cells indicating that the toxin slowly permeated intercellular regions. Toxin concentrations which affected electropotential difference were of the same magnitude as those required to inhibit root growth, ion uptake, and mitochondrial processes.Azide, cyanide, and cold temperature (5 C) gave the same partial depolarization as did the toxin. Dodecyl succinic acid caused complete depolarization. These and other data indicate that one of the primary actions of the toxin is to inhibit electrogenic ion pumps in the plasmalemma. PMID- 16660606 TI - Role of potassium and malate in nitrate uptake and translocation by wheat seedlings. AB - Wheat seedlings (Triticum vulgare) treated with 1 mm KNO(3) or NaNO(3), in the presence of 0.2 mm CaSO(4), were compared during a 48-hour period with respect to nitrate uptake, translocation, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlings treated with KNO(3) absorbed and accumulated more nitrate, had higher nitrate reductase levels in leaves but less in roots, accumulated 17 times more malate in leaves, and accumulated more of the accompanying cation than seedlings treated with NaNO(3). Within seedlings of each treatment, changes in nitrate reductase activity and malate accumulation were parallel in leaves and in roots. Despite the great difference in malate accumulation, leaves of the KNO(3)-treated seedlings had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO(3)-treated seedlings. NADP-malic enzyme levels increased only slightly in leaves and roots of both KNO(3)- and NaNO(3)-treated seedlings. The effects of K(+) and Na(+) on all of these parameters can best be explained by their effects on nitrate translocation, which in turn affects the other parameters. In a separate experiment, we confirmed that phosphoenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO(3) treatment, and increased only slightly in the KCl control. PMID- 16660607 TI - Time course and spatial distribution of phenylalanine ammonia-lyase and peroxidase activity in wounded potato tuber tissue. AB - The time course and spatial distribution of wound-induced activities of phenylalanine ammonia-lyase and peroxidase were determined to establish correlations between molecular and cellular aspects of the wound-induced pattern of cell differentiation in potato (Solanum tuberosum L.) tissue. A high correlation between peroxidase activity and suberization was observed. Peroxidase activity increased for several days after wounding. Peroxidase content of suberizing cells was more than 10 times higher than that of the immediately adjacent dividing cells. Suberizing and dividing cells contained different isoperoxidases. Neither time course nor spatial distribution of the wound-induced activity of phenylalanine ammonia-lyase was directly correlated with the wound induced pattern of cell differentiation. PMID- 16660608 TI - Simultaneous separation of acidic and basic isoperoxidases in wounded potato tissue by acrylamide gel electrophoresis. AB - Preparation and use of a newly developed pH 4.3 horizontal thin layer acrylamide gel which permits the simultaneous separation of acidic and basic isoperoxidases in up to 30 samples is described. Use of cytochrome c, horseradish peroxidase, and a purified potato isoperoxidase as internal standards for a range in isoelectric points of peroxidases from pH 3 to 11 is introduced to facilitate comparison of results obtained with different materials and different methods. Distribution of tissue-specific isoperoxidases in different cell layers of wounded potato (Solanum tuberosum L.) tissue is shown and their purification described. Evidence for the in vitro degradation of basic potato isoperoxidases resulting in more acidic forms similar to isoperoxidases occurring in wounded potato tissue is presented. The significance of this observation for the postulated differential function of different isoperoxidases is discussed. PMID- 16660609 TI - 5-Oxoprolinase (l-Pyroglutamate Hydrolase) in Higher Plants: Partial Purification and Characterization of the Wheat Germ Enzyme. AB - 5-Oxoprolinase has been found to be widely distributed in higher plants. This enzyme catalyzes the ATP-dependent hydrolysis of 5-oxo-l-proline (l-pyrollidone carboxylate, l-pyroglutamate) to glutamate. The enzyme has been purified almost 60 fold from wheat germ (Triticum aestivum L). This enzyme requires a divalent cation, either Mn(2+) or Mg(2+), and a combination of both appears to be the most effective. There is also an absolute requirement for a monovalent cation best fulfilled by either NH(4) (+) or K(+). The K(m) for ATP is 0.4 mm and for 5-oxo-l proline is 14 mum. A small amount of activity is observed when other purine nucleotides such as ITP and GTP replace ATP. The substitution of the pyrimidine nucleotides CTP and UTP for ATP yield almost completely inactive preparations. The enzyme appears to have an active sulfhydryl group since there is an increase in activity in the presence of dithioerythritol. Preincubation with reagents such as N-ethylmaleimide or iodoacetamide lead to complete inactivation. The presence of this enzyme leads to the speculation of the possible presence of a gamma glutamyl cycle in higher plants. PMID- 16660610 TI - Participation of Photosynthesis in Floral Induction of the Long Day Plant Anagallis arvensis L. AB - The saturating photon flux density (400 to 700 nanometers) for induction of flowering of the long day plant Anagallis arvensis L. was 1,900 micromoles per square meter per second (6,000 foot-candles) when an 8-hour daylength was extended to 24 hours by a single period of supplementary irradiation. The saturating photon flux density for photosynthetic CO(2) uptake during the same single supplementary light period was lower, at about 1,000 to 650 micromoles per square meter per second (3,000 to 2,000 foot-candles).The per cent flowering and mean number of floral buds per plant were significantly reduced when the light extension treatment was given in CO(2)-free air, and glucose (10 kilograms per cubic meter in water) relieved this effect. Glucose solution also significantly increased flowering of plants given supplementary light treatment in atmospheric air under a photon flux density of 80 micromoles per square meter per second. Increasing the CO(2) concentration to 1.27 grams per cubic meter of CO(2) in air during the supplementary light period did not increase flowering.It is concluded that high photon flux densities promote flowering of Anagallis through both increased photosynthesis and the photomorphogenic action of high irradiance. PMID- 16660611 TI - Role of Indole-3-acetic Acid and Gibberellin in the Control of Internodal Elongation in Avena Stem Segments: Long Term Growth Kinetics. AB - Exogenous application of indoleacetic acid results in a significant suppression of the linear growth that is promoted by exogenous gibberellic acid in Avena stem segments in a fashion similar to that previously noted in Avena leaf base segments (van Overbeek and Dowding, 1961, Fourth International Conference Plant Growth Regulation). Treatment with the auxin transport inhibitors, methyl-2 chloro-9-hydroxyfluorene-(9)-carboxylate (CFM) or 2,3,5-triiodobenzoic acid (TIBA), alone promotes elongation growth of the stem segments over that of control growth. This effect is interpreted as being due to the interference in the transport of native indoleacetic acid by CFM and TIBA, thus removing the inhibitory effect of native indoleacetic acid on gibberellin-promoted growth in the internodal intercalary meristem. This results in a greater promotion of internodal growth by native gibberellins. In the presence of (2-chloroethyl) trimethylammonium chloride (CCC), the growth-promoting effects of CFM and TIBA are decreased, and the antiauxin, PCIB (4-chloro-phenoxyisobutyric acid), has no growth-promoting effects whatsoever. These results indicate that the CFM and TIBA promoted growth require the continuous presence of gibberellins. They further support the view that native indoleacetic acid acts as a growth suppressor hormone in its regulation of gibberellin-promoted internodal extension in Avena shoots. PMID- 16660612 TI - A Pod Leakage Technique for Phloem Translocation Studies in Soybean (Glycine max [L.] Merr.). AB - Radioactive photosynthetic assimilates, translocated to a soybean (Glycine max [L.] Merr. ;Fiskeby V') pod can be measured directly by excising the stylar tip of the pod under 20 mm ethylenediaminetetraacetate solution (pH 7.0) and allowing the material to leak into the solution. Pods at the source node received approximately 50% of the (14)C exported from the source leaf to the pod and leaked approximately 1 to 3% of this into the solution. More than 90% of the (14)C that leaked from the pods was found in the neutral fraction and, of this, about 93% was in sucrose. Fifteen amino acids were identified in the leakage including: alanine, arginine, asparagine, gamma-aminobutyric acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tyrosine, and valine. The majority of the (14)C in the basic fraction was found in serine ( approximately 30%) and asparagine ( approximately 23%). The inorganic ions K, Ca, P, Mg, Zn, and Fe were found in the leakage component. Nitrate was not detectable in the collected leakage solution. The absence of NO(3) (-) and the large proportion of the label in sucrose suggest a possible phloem origin for most of the material. The technique provides an uncomplicated, reproducible means of analyzing the material translocated into and through the soybean pod, as well as following the time course of label arrival at the pod. PMID- 16660613 TI - Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings. AB - The growth of the mung bean (Vigna radiata) seedling is accompanied by the biosynthesis and accumulation of the endopeptidase vicilin peptidohydrolase and the catabolism of the reserve proteins in the cotyledons. If the axis is removed from the dry seeds and the cotyledons incubated on moist sand the accumulation of vicilin peptidohydrolase is reduced by 77% and the catabolism of reserve proteins slowed to 25% of the rate in intact seedlings. The cotyledons and the cotyledon exudate are rich in asparagine and this amino acid accounts for more than half of the reduced nitrogen exported from the cotyledons. Glutamine synthetase and asparagine synthetase, two key enzymes in the pathway of asparagine synthesis, are under temporal control in the cotyledons. Their activities increase 3.5- and 10-fold, respectively, then decline again. These increases in enzyme activity occur to the same extent in excised cotyledons and are prevented when the cotyledons are incubated in 5 micromolar cycloheximide. The results indicate that the axis may control certain key metabolic events in the cotyledons, such as the synthesis of vicilin peptidohydrolase, while many other anabolic activities may not depend on a growing axis. PMID- 16660614 TI - Effect of ethylene and carbon dioxide on potato metabolism: stimulation of tuber and mitochondrial respiration, and inducement of the alternative path. AB - The respiration of potato tubers (Solanum tuberosum var. Russet Burbank) which have been kept at room temperature for 10 days is stimulated upon subsequent treatment with C(2)H(4) (10 microliters per liter) and O(2). The respiratory rise reaches a peak in 24 to 30 hours and thereafter declines. Coincident with the rise in tuber respiration is an increase in the respiratory rates of fresh slices and isolated mitochondria. Slices and mitochondria from C(2)H(4)- and O(2) treated tubers also display substantial resistance to CN, and the resistant respiration is inhibited by hydroxamates.The longer the tubers are stored after harvest, the less effective is C(2)H(4) in causing CN resistance in slices and mitochondria from treated tubers. Addition of 10% CO(2) to the C(2)H(4)-O(2) mixture, however, causes extensive CN resistance to develop, even in slices and mitochondria from old tubers. The results show that C(2)H(4), O(2), and CO(2) act synergistically to induce alternative path development in potatoes. PMID- 16660615 TI - Cold lability of pyruvate, orthophosphate dikinase in the maize leaf. AB - Cold lability of pyruvate, orthophosphate dikinase was investigated using a homogeneous, purified enzyme preparation from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. Its stability was markedly reduced below about 10 C and the rate of cold inactivation followed first order kinetics at a concentration lower than about 0.1 milligram of enzyme per milliliter. Cold inactivation was little affected by pH in the range which gives good stability for the enzyme at warm temperatures and the enzyme activity was protected strongly by inclusion of substrates (pyruvate and phosphoenolpyruvate) and polyols such as sucrose, sorbitol, and glycerol. Loss of catalytic activity was accompanied by an apparent dissociation of a tetrameric form of the enzyme (9S form) into a new, more slowly sedimenting (5.1S) component. Inclusion of pyruvate at 4 mM in the cold-treated enzyme had no effect on the sedimentation value. A sharp change in activation energy of the dikinase-catalyzed reaction was observed near 12 C and its break point appears to be close to the generally accepted critical low temperature limit for the growth of maize plants. PMID- 16660616 TI - Chemical changes in cutin obtained from cuticles isolated by the zinc chloride hydrochloric Acid method. AB - The composition of Malus pumila Mill. cutin from cuticles isolated using ZnCl(2) HCl reagent has been compared to that isolated by an oxalate-enzymic method. Molecular changes were observed and included the loss of unsaturated components and hydroxyl groups. Such changes may alter the results of physiochemical investigations of cuticles. PMID- 16660617 TI - Interactions of low temperature, water stress, and short days in the induction of stem frost hardiness in red osier dogwood. AB - The induction of stem frost hardiness by low temperature, water stress, short days, and their combinations in 2- and 4-month-old growing dogwoods (Cornus stolonifera) were investigated. When plants were subjected to more than one factor, the increased hardiness was the sum of the effects of the individual factors involved. No interactions among these factors on hardiness were observed during a 3-week treatment. Results indicate that low temperature, water stress, and short days initially trigger independent frost-hardening mechanisms. Plant ages significantly influenced the change in low temperature-induced frost hardiness, but not the water stress or short day-induced frost hardiness. PMID- 16660618 TI - Kinetin Reversal of NaCl Effects. AB - Leaf discs of Nicotiana rustica L. were floated on NaCl in the presence of kinetin or abscisic acid. On the 5th day (14)CO(2) fixation, [(3)H]leucine incorporation, stomatal conductance, and chlorophyll content were determined. Kinetin either partially or completely reversed the inhibitory effects of NaCl while ABA had no effect. PMID- 16660619 TI - Capping structures at the 5'-terminus of polyadenylated ribonucleic Acid in Avena coleoptiles. AB - Evidence is presented for the occurrence of 5'-terminal capping structures in the polyadenylated RNA of oat (Avena sativa) coleoptiles. These structures are composed of an inverted terminal nucleoside containing the modified base 7 methylguanine which is joined 5' to 5' with a second (penultimate) nucleoside by means of three phosphate groups in two pyrophosphate linkages. The penultimate nucleoside is joined to the remainder of the RNA molecule by a conventional 3',5' phosphodiester bond. A significant difference between the cap structures of oat coleoptile RNA and those of previously described higher eucaryotic cellular mRNAs is the lack of ribose methylations in the penultimate nucleosides of the plant RNA. PMID- 16660620 TI - Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L. AB - The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis. PMID- 16660621 TI - Influence of cobalt on soybean hypocotyl growth and its ethylene evolution. AB - Development of dark-grown "Clark" soybean (Glycine max [L.] Merr.) seedlings is abnormal at 25 C but normal at 20 and 30 C. At 25 C, hypocotyls swell and fail to elongate normally; lateral root formation and seedling ethylene evolution are enhanced.Co(2+) promoted hypocotyl elongation of etiolated "Clark" soybean seedlings by 28% when grown at 25 C. The same growth-promoting concentration reduced hypocotyl thickness and primary root elongation by 28 and 43%, respectively. Co(2+) inhibited ethylene production both of intact seedlings and of apical 1-centimeter hypocotyl segments with attached epicotyls and cotyledons by 65 and 60%, respectively. These results suggest that Co(2+) exerts its effects on the hypocotyl growth by inhibiting ethylene production, and also confirm our previous conclusion that abnormal ethylene production at 25 C is responsible for the inhibition of hypocotyl elongation and for its swelling. PMID- 16660622 TI - Determination of Indole-3-acetic Acid in Douglas Fir Using a Deuterated Analog and Selected Ion Monitoring: Comparison of Microquantities in Seedling and Adult Tree. AB - Indole-3-acetic acid (IAA) content in shoot tips of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) trees and seedlings was determined by combined gas chromatography-mass spectrometry using a deuterated analog (d(2)-IAA) as an internal standard and the technique of selected ion monitoring. Ratios of the peak heights of the deuterated analog internal standard to endogenous IAA revealed a slightly higher content of IAA in seedlings compared with the shoot tips collected in June. The relatively high level of IAA (2.9 micrograms per gram fresh weight) in seedlings is discussed in relation to in vitro propagation of this species. PMID- 16660623 TI - Characteristics of sugar uptake in hypocotyls of cotton. AB - Uptake of sucrose and hexoses by cotton (Gossypium hirsutum L.) hypocotyl segments from free space was shown to be an active, carrier-mediated process. Separate carriers existed for hexoses and sucrose. Accumulated sugars appeared in both soluble and insoluble fractions of the tissue. At optimum temperature and pH, sucrose uptake rate versus concentration was fit by a rectangular hyperbola with V(max) of 14 micromoles per gram fresh weight per hour and K(m) of 8 mm. Sucrose was the principal sugar found in the free space in vivo, and invertase activity was essentially absent from that space except after aging. PMID- 16660624 TI - Regulation of sugar uptake in hypocotyls of cotton. AB - Uptake of sucrose and hexoses by hypocotyl segments of cotton (Gossypium hirsutum L.) was shown to be dependent upon sugar level in the tissue. The effect was not related to total sugar level inasmuch as a portion of previously accumulated sugar was without influence on uptake. That portion was presumed to be compartmentalized, most likely in vacuoles. Growth regulators modified the uptake pattern apparently through alterations in secondary metabolism. Uptake and incorporation were inhibited by rotenone and stimulated by light. The light effect was blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A general model was presented for assimilate flux within sink tissues with free space as the conduit from phloem to carrier site. PMID- 16660625 TI - Spectral characterization of five chlorophyll-protein complexes. AB - Sodium dodecyl sulfate-solubilized chloroplast internal membranes of higher plants (cowpea [Vigna unguiculata L. Walp], chinese cabbage [Brassica chinensi L.], and tobacco [Nicotiana tabacum L.]) are resolved by polyacrylamide gel electrophoresis into two chlorophyll a- and three chlorophyll a,b-proteins. A small portion (about 15%) of the membrane chlorophyll migrates as a component of high electrophoretic mobility and presumably consists of detergent-complexed, protein-free pigment.One of the chlorophyll a-proteins is qualitatively similar to the P(700) chlorophyll a-protein but contains a much larger proportion of total chlorophyll (about 30%) than previously reported. The second chlorophyll a protein is a recently discovered component of the membrane and accounts for about 7% of the total chlorophyll. The absorption and fluorescence emission spectra of these two chlorophyll a-proteins differ.The three chlorophyll a,b-proteins are components of the chloroplast membrane chlorophyll a,b-light-harvesting complex which was previously resolved as a single chlorophyll-protein band. The two additional chlorophyll a,b-proteins observed in our work probably represent larger aggregates contained within that membrane complex which are preserved under the solubilization and electrophoretic conditions used here. PMID- 16660626 TI - Cytokinin biosynthesis in cultured rootless tobacco plants. AB - Biosynthesis of cytokinin in shoots was examined by growing rootless tobacco (Nicotiana tabacum) plants in vitro. The rootless plants were originated by culturing tobacco callus on a high cytokinin-low auxin medium to induce the formation of plantlets which were then grown on medium without exogenous cytokinin and auxin. The rootless plants supplied with [(14)C]adenine synthesized ethanol-ethyl acetate-water-soluble radioactive components, portions of which had the same chromatographic and electrophoretic mobilities as N(6)-(Delta(2) isopentenyl)adenine, N(6)-(Delta(2)-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl 2-butenylamino)purine and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-d ribofuranosylpurine. The total amount of these four major cytokinins was estimated to be present at a concentration of 14 to 23 nanomoles per kilogram of rootless plant. These data indicate that adenine serves as a precursor of the purine moiety of cytokinin molecules and that the cytokinin biosynthetic sites are also located in the shoot in addition to the presumed root sites. PMID- 16660627 TI - Developmental Differences in Embryos of High and Low Protein Wheat Seeds during Germination. AB - Developmental patterns of embryos from high and low protein wheat (Triticum aestivum) grain produced under varied fertilizer conditions were compared. High protein grain produced seedlings 25% heavier with 25% more total RNA, 30% more DNA, 40% more amino acids, 60% more ribosomes, and 80% more soluble protein content than that of low protein seed. Consistently higher glutamine synthetase and alpha-amylase and lower acid phosphatase activities were observed in high protein seeds, though the isozyme pattern of alpha-amylase was not different in the two kinds of seeds. The high total ribosomes and particularly, polysome content observed in high protein seeds may be responsible for the rapid growth and high yield of these seeds. PMID- 16660628 TI - Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ. AB - As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N(6)-(Delta(2) isopentenyl)adenine, N(6)-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2 hydroxyethyl piperazine-N'-ethanesulfonic acid) and 37 C the K(m) for N(6) (Delta(2)-isopentenyl)adenine was 57.1 micromolar; N(6)-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the V(max) for N(6)-(Delta(2) isopentenyl)adenine, N(6)-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N(6)-(Delta(2)-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism. PMID- 16660629 TI - Adenine Nucleotide Changes during Cold Acclimation of Winter Rape Plants. AB - During the first stage of hardening of winter rape plants (Brassica napus L. var. oleifera L., cv. Gorczanski), marked increase of ATP content in leaves was observed. Lowering the temperature from 5 to 0 C (the second stage of hardening) had no further effect on ATP content. In roots, not capable of hardening, pronounced decrease of ATP content was noted after prolonged exposure to cold. It was found that increased ATP content and higher energy charge in cold-treated leaves were due to light and dark processes. PMID- 16660630 TI - Studies on Cation-induced Thylakoid Membrane Stacking, Fluorescence Yield, and Photochemical Efficiency. AB - Trypsin digestion of photosynthetic membranes isolated from spinach (Spinacia oleracea L.) leaves eliminates the cation stimulation of chlorophyll fluorescence. High concentrations of cations protect the fluorescence yield against trypsin digestion, and the cation specificity for this protection closely resembles that required for the stimulation of fluorescence by cations. Trypsin digestion reverses cation-induced thylakoid stacking, and the time course of this effect seems to parallel that of the reversal of cation fluorescence. High concentrations of cations protect thylakoid stacking and cation-stimulated fluorescence alike. The cation stimulation of photosytem II photochemistry remains intact after trypsinization has reversed both cation-induced thylakoid stacking and fluorescence yield. It is concluded that cation-stimulated fluorescence yield, and not the cation stimulation of photosystem II photochemistry, is associated with thylakoid membrane stacking. PMID- 16660631 TI - Citrus tissue culture: stimulation of fruit explant cultures with orange juice. AB - In vitro growth of explant (juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice (10% v/v optimum) to a basal medium containing Murashige and Skoog salts, 50 grams per liter sucrose, 100 milligrams per liter myo-inositol, 5 milligrams per liter thiamine.HCl, 2 milligrams per liter 2,4 dichlorophenoxyacetic acid and 0.5 milligrams per liter kinetin. In analyzing this effect of orange juice on citron explant cultures, we failed to obtain increased yields by addition of appropriate concentrations of citric acid to the basal medium but obtained growth stimulation when the medium was supplemented with juice from an "acidless" orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid is involved. Addition of the inorganic ash corresponding to 10% (v/v) orange juice to the basal medium had no effect on yields. Similarly, the stimulatory effect of orange juice could not be explained based on its content of sucrose or of organic growth factors already present in the basal medium. PMID- 16660632 TI - Effect of High Cation Concentrations on Photosystem II Activities. AB - The effects of wide concentration ranges of NaCl, KCl, and MgCl(2) on ferricyanide reduction and the fluorescence induction curve of isolated spinach (Spinacia oleracea) chloroplasts were investigated. Concentrations of the monovalent salts above 100 mm and MgCl(2) above 25 mm produced a decrease in the rate of ferricyanide reduction by thylakoids uncoupled with 2.5 mm NH(4)Cl which cannot be attributed to changes in the primary photochemical capacity of photosystem II. Salt-induced decreases in the effective concentration of the secondary electron acceptor of photosystem II, plastoquinone, reduce the capacity for secondary photochemistry of photosystem II and this could contribute to the reduction in ferricyanide reduction by uncoupled thylakoids at high salinities. The rate of ferricyanide reduction by coupled thylakoids is little affected by salinity changes, indicating that the rate-limiting phosphorylation mechanism in electron flow from water to ferricyanide in coupled thylakoids is salt-tolerant, whereas the rate-limiting reaction in uncoupled ferricyanide reduction is considerably affected by salinity changes. Salt-induced changes in the fluorescence induction curve are interpreted in terms of changes in the rate constants for excitation decay by radiationless transitions, exciton transfer from photosystem II chlorophylls to other associated chlorophyll species, and photochemistry. PMID- 16660633 TI - Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx. AB - Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from -3 C to -40 or -50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate-requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O(2)-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes. PMID- 16660634 TI - Freezing of water in red-osier dogwood stems in relation to cold hardiness. AB - Studies of stem water in red-osier dogwood (Cornus stolonifera Michx.) using nuclear magnetic resonance spectroscopy indicated that most freezing occurs at temperatures above -30 C in cold-hardy and tender stems. Hardy and tender stems had about the same amount of unfrozen water at -40 C (0.28 gram of water per gram dry weight). When hardy stems were slowly cooled below -20 C, the temperature below which little additional freezing occurs, they survived direct immersion in liquid N(2) (-196 C). Fully hardy samples not slowly precooled to at least -15 C did not survive direct immersion in liquid N(2). The results support the hypothesis that cooling rate is an unimportant factor in tissue survival at and below temperatures where there is little freezable water. PMID- 16660635 TI - Induction of swelling in pea internode tissue by ethylene. AB - Ethylene inhibits polar cell expansion of etiolated pea (Pisum sativum) internode tissue and results in a radial cell expansion, swelling. A special gas-tight growth chamber was developed to monitor continuously the induction of cell expansion inhibition of excised internode segments in the presence of ethylene. Following a complex induction period which lasts about 3 hours, a low, but steady state growth rate is achieved. Ethylene removal experiments indicate that the gas induces an irreversible change in the cell expansion mode. If the tissue was transferred to an ethylene-free chamber after the steady-state rate had been achieved, this rate continued in the absence of the gas. If the gas was removed during an intermediate phase, that intermediate rate of growth continued after removal. In other experiments, segments were incubated with intact apical hooks and the induction period was much shorter than with isolated segments. Changes in imino acid metabolism have been correlated with the induction of swelling. Although total proline and hydroxyproline levels were not affected by ethylene treatment, incorporation of [(14)C]proline into a wall-associated protein was inhibited during the period when swelling occurred. These results suggest that ethylene is affecting proline pool sizes. PMID- 16660636 TI - Within-day Changes in the Polyribosome Content and in Synthesis of Proteins in Leaves of Capsicum annuum L. AB - Capsicum annuum cv. California Wonder was grown in controlled environment with a 12-hour photoperiod. The polyribosome content of leaves varied from 60 to 72% of total ribosomes with the highest level occurring in the middle of the photoperiod and the lowest in the middle of the dark period. The variation was accounted for by changes in the content of large polyribosomes (hexamers and larger). There was no indication of an immediate effect on polyribosome content of light-on or light off.The synthesis of proteins at two times in the 24-hour cycle was compared using a dual isotope technique. Statistically significant results were obtained that suggested that protein(s) with molecular weights of 26,000 daltons were preferentially synthesized in the photoperiod compared to the dark period. No evidence was found for the differential synthesis of proteins within the photoperiod. PMID- 16660638 TI - Host-Pathogen Interactions: XV. Fungal Glucans Which Elicit Phytoalexin Accumulation in Soybean Also Elicit the Accumulation of Phytoalexins in Other Plants. AB - A beta-glucan isolated from the mycelial walls of Phytophthora megasperma var. sojae and a glucan purified from yeast extract stimulate the accumulation of phytoalexins in red kidney bean, Phaseolus vulgaris, and stimulate the accumulation of the phytoalexin, rishitin, in potato tubers, Solanum tuberosum. These glucans have previously been shown to be potent elicitors of glyceollin accumulation in soybean, Glycine max.Treatment of kidney bean cotyledons with the glucan elicitors resulted in the accumulation of at least five fungistatic compounds. These compounds migrate during thin layer chromatography identically to the fungistatic compounds which accumulate in kidney beans which have been inoculated with Colletotrichum lindemuthianum, a fungal pathogen of kidney beans.Potatoes accumulate as much as 29 micrograms of rishitin per gram fresh weight following exposure to the glucan from Phytophthora megasperma var. sojae and as much as 19.5 micrograms of rishitin per gram fresh weight following exposure to yeast glucan. Potatoes accumulated 28 micrograms of rishitin per gram fresh weight following inoculation with live Phytophthora megasperma var. sojae. PMID- 16660637 TI - Host-Symbiont Interactions: III. Purification and Partial Characterization of Rhizobium Lipopolysaccharides. AB - The lipopolysaccharides of three strains each of Rhizobium leguminosarum, R. phaseoli, and trifolii have been purified and partially characterized. The last step in the purification procedure is gel filtration column chromatography using Sepharose 4B with an elution buffer consisting of ethylenediaminetetraacetic acid and triethylamine. Each of the lipopolysaccharides reported in this paper elutes as a symmetrical peak in the partially included volume of this Sepharose 4B column. The ratio of 2-keto-3-deoxyoctonate acid (a sugar which is characteristic of lipopolysaccharides) to hexose is constant throughout the carbohydrate containing peaks as they elute from the Sepharose 4B. The compositions and immunodominant structures of the purified lipopolysaccharides vary as much among strains of a single Rhizobium species as among the different species of Rhizobium. There is no obvious correlation between the nodulation group to which a Rhizobium belongs and the chemical composition or immunochemistry of the Rhizobium's lipopolysaccharide. There is extensive crosslysis by phage of strains of R. trifolii, R. phaseoli, and R. leguminosarum. This suggests that the receptors for these cross-lysing phage reside either in nonlipopolysaccharide structures or in common structures within the lipopolysaccharide which are not detected by compositional or immunochemical analysis. PMID- 16660639 TI - Phytochrome destruction: apparent inhibition by ethylene. AB - Phytochrome destruction begins immediately following actinic irradiation of 4-day old, dark-grown oat (Avena sativa L., cv. Garry) shoots grown in open containers. When grown in closed containers, otherwise identical oat shoots exhibit a delay of about 40 minutes between irradiation and the onset of destruction. This delay can be attributed to accumulation of ethylene by several criteria, including elimination of the delay by mercuric perchlorate. These data provide an explanation for otherwise contradictory observations concerning the presence of a delay prior to the onset of destruction. PMID- 16660640 TI - A sensitive technique for the rapid measurement of carbon dioxide concentrations. AB - A method has been developed to measure concentrations of CO(2) in gases rapidly. A gas sample is injected into a flowing carrier gas that passes through an infrared CO(2) analyzer. A strip chart recorded peak response is obtained which is proportional to the CO(2) concentration. A resolution of better than 2 microliters of CO(2) per liter of gas was obtained. Seven to 10 seconds were required for sample analysis once the sample was obtained. Sorghum bicolor plant respiration was determined at different temperatures by measuring CO(2) using this system and by using a conventional system. The correlation between techniques was 0.996, and about the same variation occurred within each method. This technique greatly increased the efficiency of the infrared CO(2) analyzer in our laboratory for use in plant respiration and photosynthetic studies. PMID- 16660641 TI - Use of lipophilic cations to measure the membrane potential of oat leaf protoplasts. AB - Uptake of the lipophilic cation triphenylmethylphosphonium into mesophyll protoplasts of oat (Avena sativa L. cv. "Garry") approaches equilibrium at 3 to 4 hours. The resulting external and internal concentrations are then used with the Nernst equation to obtain a membrane potential of -62 millivolts, inside negative. Potentials calculated in this manner are depolarized by adding 2 mm sodium azide and 50 mum carbonyl cyanide m-chlorophenylhydrazone as well as by increasing the external proton and potassium concentrations. The depolarizations are qualitatively similar to those seen when oat mesoyphll cells are measured in situ with microelectrodes. It is concluded that due to the lack of turgor and fragility of protoplasts, estimations of their membrane potential may be made more reliably, under some conditions, with lipophilic cations than with microelectrodes. PMID- 16660642 TI - Thymidine phosphorylation in wheat: analysis of phosphate transfer from ATP to thymidine. AB - Extracts of wheat (Triticum vulgare Vill. [Triticum aestivum L.] var. Lemhi) seedlings contain thymidine-phosphorylating activity with ATP, ADP, or AMP and nucleotide hydrolase activity (ATP --> --> AMP). The synthesis of [(32)P]dTMP exclusively from [alpha-(32)P]ATP with none detectable from [gamma-(32)P]ATP demonstrates the absence of thymidine kinase and the presence of nucleoside phosphotransferase as the only observable thymidine-phosphorylating enzyme. PMID- 16660643 TI - Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes. AB - Endogenous phospholipase D and phosphatidic acid phosphatase activities were demonstrated in membrane fractions isolated from soybean (Glycine max L.) hypocotyls. Phospholipase D activity was distributed widely among different membrane fractions while phosphatidic acid phosphatase was found predominantly in membranes equilibrating in lower sucrose densities. Phospholipase D action was unaffected by ethylenediaminetetraacetic acid, sodium salt or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid but was prevented by a mixture of 4% choline and 4% ethanolamine. Phosphatidic acid phosphatase was inhibited by 10 millimolar glycerol 1-phosphate or by homogenization media prepared with coconut milk as a solvent instead of water. Under fully protected conditions the phospholipid composition of soybean membrane fractions remained unchanged for at least 1 hour at 20 C. Membranes prepared under protected conditions had low phosphatidic acid contents and the phospholipid compositions closely resembled those of corresponding animal membranes. PMID- 16660644 TI - Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco. AB - The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic (14)CO(2) fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O(2) level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O(2) concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO(2) per gram fresh weight per hour (a 60% increase) when the O(2) level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO(2) per gram fresh weight per hour (a 22% increase). The decrease in the O(2) sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O(2) concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO(2) fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect. PMID- 16660645 TI - Role of calcium in serine transport into tobacco cells. AB - The transport of serine into tobacco (Nicotiana tabacum L. var. Xanthi) cells grown in liquid medium was studied. Serine transport was maximal below pH 4.0. A time-dependent stimulation of transport was observed when cells were incubated in medium containing 0.5 mm Ca(2+). Maximum transport rates were achieved after 6 hours preincubation in Ca(2+). The following three distinct roles of Ca(2+) in serine transport were demonstrated: time-dependent stimulation of transport rate, maintenance of high transport rates, and retention of transported material. Stimulation occurred in the presence of either Ca(2+) or Mg(2+) and was inhibited by either La(3+) or K(+). Removal of Ca(2+) from the transport medium caused a rapid decline in the rate of serine uptake. This decline was prevented by addition of La(3+) after Ca(2+) removal. Cells transferred to medium lacking Ca(2+) lost substantial amounts of transported serine, this loss was significantly reduced by either La(3+) or K(+).Cells placed in (45)Ca(2+) rapidly bound more than 3 micromoles of Ca(2+)/gram fresh weight, which was exchangeable within 10 minutes with medium Ca(2+). Seventy-five per cent of the (45)Ca(2+) transported into the cells in 4 hours could be exchanged with medium Ca(2+) in the same period. The amount of net Ca(2+) transport into tobacco cells is insignificant relative to the total exchangeable Ca(2+).It is proposed that serine transport into tobacco cells involves H(+) cotransport and that the stimulation by Ca(2+) is due to an increase in the proton-motive force. PMID- 16660646 TI - Effect of Plant Growth Regulators on Calcium-stimulated Serine Transport into Tobacco Cells. AB - The transport of serine into tobacco cells (Nicotiana tabacum L.) cultured in liquid medium was examined. Transport was inhibited approximately 50% by 2,4 dichlorophenoxyacetic acid, indoleacetic acid, alpha-naphthalene acetic acid, and kinetin at a concentration of 10 micrograms per milliliter. Transport was not inhibited by 2,6-dichlorophenoxyacetic acid and inhibited less than 25% by p chlorophenoxyacetic acid at this concentration. Removal of 2,4 dichlorophenoxyacetic acid from the transport medium resulted in an alleviation of inhibition. Gibberellic acid at concentrations from 2 to 20 micrograms per milliliter stimulated transport.It was previously shown that inhibition of transport by La(3+) was due to removal of Ca(2+) from surface sites and inhibition of Ca(2+) uptake by cells. None of the growth regulators tested had any significant effect on Ca(2+) binding and/or transport.A contributing factor to the low transport rates in the absence of Ca(2+) is the increased rate of serine efflux. None of the growth regulators tested had any significant effect on the rate of serine efflux. PMID- 16660647 TI - Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants. AB - Mass spectrometric techniques were used to trace the incorporation of [(18)O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C(3) plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [(18)O]oxygen at the CO(2) compensation point were heavily labeled with (18)O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of (18)O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [(18)O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of (18)O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.The carboxyl oxygens of 3-phosphoglycerate also became labeled with (18)O in 20- and 40-minute feedings with [(18)O]oxygen to intact leaves at the CO(2) compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 +/- 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO(2) compensation point.Oxygen uptake sufficient to account for about half of the rate of (18)O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO(2) compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo. PMID- 16660648 TI - Binding Specificity and Possible Analytical Applications of the Cytokinin-binding Antibody, Anti-N-Benzyladenosine. AB - Antibodies elicited in rabbits by immunization with an N(6)-benzyladenosine bovine serum albumin conjugate bound N(6)-benzyladenosine specifically. The affinity constants and specific binding site concentrations for a number of cytokinins and related compounds were estimated by nonlinear least squares analysis of direct or competitive ultrafiltration data. The antisera contained 230 to 330 nanomoles of cytokinin binding sites per gram protein. Affinity constants were 8.8 x 10(8) molar(-1) for 6-benzylaminopurine, 8.4 x 10(7) molar( 1) for kinetin, 9.1 x 10(7) molar(-1) for 6-(3-methyl-2-butenylamino)purine, 6.6 x 10(6) molar(-1) for 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine, and 2.0 x 10(4) molar(-1) for 6-methylaminopurine. Affinity constants were below the limit of detectability (<10(4) molar(-1)) for benzylamine, adenine, and other adenine derivatives without an N(6)-side chain. The N(6)-substituent was thus immunodominant, but the purine moiety was also necessary for binding affinity. The antibodies were immobilized on cyanogen bromide-activated Sepharose with 95% retention of binding capacity and without apparent change in affinity constants. Columns of the immobilized antibody retained 64% of the [(3)H]6-(3-methyl-2 butenylam-ino)purine from 2 nanomolar solutions and readily trapped [(14)C]6 benzylaminopurine that had been added to crude extracts of cabbage. Aqueous 10% pyridine adjusted to pH 7.3 with formic acid effectively eluted bound cytokinins from gel columns without loss of binding capacity of the immobilized antibody. PMID- 16660649 TI - Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants. AB - Triton X-100 solubilized chloroplast DNA but not nuclear DNA from a mixture of chloroplasts and nuclei. The buoyant density of chloroplast DNA was different from that of the satellite DNA in all of the species examined (Phaseolus coccineus, Cucumis sativus, Cucumis melo, Antirrhinum majus, Vicia faba, Oenothera fruiticosa youngii). Chloroplast DNA constituted between 4.3% and 0.25% of the total leaf DNA in these species, and was present as 5 to 20 copies in each chloroplast. PMID- 16660650 TI - Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase. AB - CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn(2+) for activity. Maximal activity was at 1.5 millimolar MnCl(2). A K(m) of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride. PMID- 16660652 TI - Nitrate Utilization by the Diatom Skeletonema costatum: I. Kinetics of Nitrate Uptake. AB - Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased. PMID- 16660651 TI - Photooxidative Damage in Photosynthetic Activities of Chromatium vinosum. AB - The capacity of photosynthetic CO(2) fixation in the anaerobic purple-sulfur bacterium, Chromatium vinosum is markedly impaired by strong illumination (9 x 10(4) lux) in the presence of 100% O(2). In the absence of HCO(3) (-), decline in activity occurred gradually, with about 40% of the initial activity remaining after a 1-hour incubation. The addition of 50 millimolar HCO(3) (-) to the incubation medium resulted in a measurable delay (about 30 minutes) of the inactivation process. Ribulose-1,5-bisphosphate carboxylase activity and light dependent O(2) uptake (electron flow) or crude extracts prepared after pretreatment of the bacterial cells with O(2) and light were not affected but the photophosphorylation capacity of either bacterial cells or chromatophores was drastically reduced. The inhibition of photophos-phorylation in the chromatophore preparations was significantly reduced by the addition of either an O(2) (-) scavenger, Tiron, or an (1)O(2) scavenger, alpha-tocopherol. These results suggest that the active O(2) species, O(2) (-) or (1)O(2), might take part in the observed inactivation.The pretreatment of the bacteria with O(2) and light inhibited CO(2) assimilation through the Calvin-Benson cycle, while relatively stimulating the formation of aspartate and glutamate. It also inhibited the conversion of glycolate to glycine, resulting in a sustained extracellular excretion of glycolate. The inactivation of photosynthetic CO(2) fixation by intact cells was enhanced by low temperature, KCN, or methylviologen addition during the pretreatment with O(2) and light. The mechanism(s) of O(2)-dependent photoinactivation of photosynthetic activities in Chromatium are discussed in relation to the possible role of photorespiration as a means of producing CO(2) in the photosynthetic system. PMID- 16660653 TI - Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake. AB - Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions. PMID- 16660654 TI - Purification and Characterization of RNA Polymerase from Fremyella diplosiphon. AB - We have purified and characterized a DNA-dependent RNA polymerase from the blue green alga Fremyella diplosiphon. This enzyme, purified by gel filtration, DEAE cellulose chromatography, and glycerol gradient centrifugation, is comprised of five polypeptide subunits. Their masses are 161,000, 134,000, 91,000, 72,000, and 41,000 daltons. Preparative electrophoresis of the purified enzyme on nondenaturing gels separates the 41,000-dalton polypeptide from the rest of the enzyme. The enzyme is extremely labile in the presence of a variety of salts of strong acids and bases; the purification procedure was devised to avoid exposure to such compounds. PMID- 16660655 TI - Mathematical analysis of the dependence of cell potential on external potassium in corn roots. AB - The K(+) dependence of normal (psi) and diffusion (psi(D)) potentials in corn roots [Zea mays L., hybrid (A619 x Oh43) x A632] was determined experimentally and analyzed with respect to the parameter xi [defined as exp (F psi/RT)]. In the presence of 10 micromolar carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), psi behaved as expected of a diffusion potential. Based upon the assumptions (a) that FCCP did not change any term of the Goldman-Hodgkin-Katz equation, and (b) that total potential was functionally the algebraic sum of psi(D) and psi(P) (the deviation from psi(D) due to an electrogenic system), psi(P) was found to be a complex function of external potassium and to have a minimum value of 0.69 millimolar K ion activity outside the cell. Analysis of psi allowed us to develop an equation which predicts a complicated K(+) dependence of psi such as that found by Mertz and Higinbotham (Membrane Transport in Plants and Plant Organelles. Springer-Verlag 1974). PMID- 16660656 TI - Purine nucleotide metabolism of germinating soybean embryonic axes. AB - Isolated soybean (Glycine max L. cv. Kent) embyronic axes metabolized [(14)C]glycine to ATP within the 1 hour of imbibition. Radioactivity was not detected in GTP until the 3rd hour. Throughout most of the first 24 hours of germination about 10 to 26 times as much label from [(14)C]glycine appears in ATP as GTP. About five times as much [(14)C]hypoxanthine and [(14)C]inosine was converted into GTP as into ATP in embryonic axes. Two independent pools of IMP appear to be used in purine nucleotide synthesis of soybean axes. PMID- 16660657 TI - Light-Dark Regulation of Starch Metabolism in Chloroplasts: I. Levels of Metabolites in Chloroplasts and Medium during Light-Dark Transition. AB - In Spinacia oleracea the kinetics of CO(2) fixation, of starch formation, and of changes in the levels of metabolites in chloroplasts and the surrounding medium has been investigated during light-dark and dark-light transitions with isolated intact chloroplasts.The internal level of orthophosphate stays constant throughout a light-dark-light cycle. The concentration of 3-phosphoglycerate in the chloroplasts is about 4 millimolar in the light and decreases in the dark within 3 minutes to about 1.6 millimolar. The level of the hexose monophosphates shows a reverse trend, increasing from about 2.2 millimolar in the light to 6 millimolar in darkness. In the subsequent light period both compounds reach their original levels within 2 minutes. The chloroplastic concentrations of dihydroxyacetone phosphate, of the pentose monophosphates, and of the hexose- and heptose bisphosphates remain constant at about 0.4 millimolar throughout the light-dark-light cycle.In the medium, the concentration of 3-phosphoglycerate increases and dihydroxyacetone phosphate decreases in the dark phase: this is due to an exchange of internal 3-phosphoglycerate for external dihydroxyacetone phosphate. Part of the reimported dihydroxyacetone phosphate is converted into hexose monophosphates via aldolase and fructose bisphosphatase during the first minutes of darkness. Due to the observed exchange transport reactions, the large difference between the transenvelope concentration gradients of 3 phosphoglycerate, dihydroxyacetone phosphate, and orthophosphate which exist in the light, is completely abolished after 2 to 3 minutes in the dark.The kinetics and the magnitudes of the changes of metabolite concentrations during the light dark-light cycle are compared to the kinetics of starch formation, and their relevance for a possible light-dark regulation of starch synthesis is discussed. PMID- 16660658 TI - Light-Dark Regulation of Starch Metabolism in Chloroplasts: II. Effect of Chloroplastic Metabolite Levels on the Formation of ADP-Glucose by Chloroplast Extracts. AB - The rate of ADP-glucose formation from [(14)C]glucose 6-phosphate and ATP by the soluble fraction of lysed chloroplasts is studied as a function of the levels of metabolites (3-phosphoglycerate, orthophosphate, hexose monophosphate, and ATP) as determined in whole chloroplasts of Spinacia oleracea in light and dark.A change in 3-phosphoglycerate concentration (from 4 to 1.4 millimolar, as in whole chloroplasts during light-dark transition) decreases the rate of ADP-glucose formation 6- to 7-fold. An increase in hexose monophosphate concentration from 2 to 6 millimolar, which occurs at the same time in whole chloroplasts, stimulates ADP-glucose formation only slightly.At constant levels of orthophosphate (4 millimolar) and 3-phosphoglycerate (4 millimolar), a change in ATP concentration from 0.2 to 1 millimolar causes an immediate 4- to 5-fold increase in the rate of ADP-glucose formation. Another significant stimulation of ADP-glucose formation (about 4- to 6-fold) is obtained after addition of dithiothreitol at high concentrations (50 millimolar). A simultaneous increase in the concentrations of 3-phosphoglycerate, ATP, and dithiothreitol, with orthophosphate and Mg(2+) being constant at 4 and 5 millimolar, respectively, causes a 130-fold increase in the rate of ADP-glucose formation (from 0.042 to 5.49 microgram atoms carbon per milligram chlorophyll per hour).The role of these and other factors is discussed with respect to light-dark regulation of starch formation in intact chloroplasts. PMID- 16660659 TI - Reversible effects of green and orange-red radiation on plant cell elongation. AB - The differential cell elongation of cress (Lepidium sativum) roots that results in geotropic bending can be decreased by green radiation with an action spectrum peaking at 550 nm. This decrease can be negated by prior or simultaneous irradiation by orange-red light with an action spectrum peaking at 620 nm. The green radiation appears to be effective during the cell elongation phase of geotropic response. PMID- 16660660 TI - Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening. AB - Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato beta-galactosidase. PMID- 16660661 TI - Cyanide-insensitive, Salicylhydroxamic Acid-sensitive Processes in Potentiation of Light-requiring Lettuce Seeds. AB - Phytochrome-mediated germination of Lactuca sativa L. cv. Waldmann's Green seeds was inhibited strongly by 10 millimolar salicylhydroxamic acid (SHAM), but only slightly delayed by the same level of KCN. SHAM was most effective if applied within the 8-hour potentiation period (release from dormancy) following red light treatment, but much less effective with completely potentiated seeds. SHAM at 3 millimolar actually hastened completion of potentiation, whereas concentrations of 6.6 millimolar or higher retarded the process. A temporary upsurge of O(2) consumption was particularly evident during the period of most rapid potentiation (3 hours after red light), especially in the seed sections containing the embryonic axis. The embryonic axis obtained from dormant seeds also contained most of the SHAM-sensitive O(2) uptake. However, 8 hours of potentiation caused loss of SHAM sensitivity from axes and a simultaneous gain of SHAM sensitivity by cotyledons. Concomitant with this increased sensitivity to SHAM, O(2) uptake by cotyledonary tissues lost some sensitivity to KCN. Red light-stimulated metabolic processes leading to germination were blocked more effectively by SHAM than by KCN, but O(2) consumption by both dormant and nondormant seeds was much less sensitive to 10 millimolar SHAM than to the same concentration of KCN. This apparent contradiction between effects of SHAM on potentiation and O(2) uptake may be a result of: (a) compensatory electron flow through the cytochrome pathway at the expense of the alternate pathway; (b) a functional site of action of SHAM that differs from the organized, energy-coupled respiratory system; or (c) a combination of these possibilities. PMID- 16660662 TI - Changes in the Strength of Lettuce Endosperm during Germination. AB - The forces required to puncture intact lettuce (Lactuca sativa) seed and pericarp, endosperm and embryo were measured by the Instron Universal Testing Machine. It required about 0.6 newton to puncture the endosperm in seeds imbibed in the dark at 6, 12 and 24 hours. Endosperm of seeds imbibed in the light or in dark with gibberellic acid required about 4.2 newtons at 6 and at 12 hours and only about 0.15 newton at 24 hours. Forces required to puncture embryo at all treatments and times remained constant at about 0.3 newton. Changes in the strength of the endosperm do not appear to be related directly to protrusion of the radicle. PMID- 16660663 TI - Endogenous Plant Growth Substances in Developing Fruit of Prunus cerasus L: VII. Isolation of Gibberellin A(32). AB - A polar gibberellin active in the dwarf rice (Oryza sativa L.) seedling and barley (Hordeum vulgare L.) half-seed assays was extracted from immature sour cherry (Prunus cerasus L.) seed and identified as gibberellin A(32) by gas-liquid chromatography and selected ion monitoring. PMID- 16660664 TI - Photosynthetic electron transport in isolated maize bundle sheath cells. AB - Fragments of bundle sheath strands, free of mesophyll cells and showing a chlorophyll a/b ratio of 6.0 to 6.6 were prepared from Zea mays by a mechanical method. They were unable to photoreduce ferricyanide but were able to photoreduce the membrane-permeant 2,5-dimethylquinone at a rate of 250 to 420 microequivalents per hour per mg chlorophyll (mueq/hr . mg Chl) at 21 C. In the presence of the catalase inhibitor KCN, methylviologen catalyzed a Mehler reaction at a rate of 120 to 180 mueq/hr . mg Chl. This was increased to 200 to 350 mueq/hr . mg Chl when the uncoupler methylamine was added. The rate of endogenous pseudocyclic electron flow, detected as a Mehler reaction, was also considerable (100 to 150 mueq/hr . mg Chl with methylamine). Diaminodurene supported a high rate of photosystem I-mediated electron flow to methylviologen (400 to 750 mueq/hr . mg Chl).When the tissue fragments were illuminated in a weakly buffered suspension, a reversible rise in the medium pH was observed which apparently originated from H(+) translocation in the thylakoids. The kinetics of the pH changes was rather slow (t((1/2)) > 15 seconds for pH rise; > 30 seconds for dark decay) but the extent of H(+) uptake was substantial (0.1 to 0.3 mueq/mg Chl). All of the electron transport reactions tested, including partial reactions which involve only photosystem I or photosystem II, invariably supported H(+) uptake. This suggests that two sites of energy conservation are associated with the photosynthetic chain in the bundle sheath chloroplasts (as in spinach chloroplasts) and that both of these sites are functional in vivo. The pH changes observed in the absence of exogenous electron carriers were abolished by 3-(3,4 dichlorophenyl)-1,1-dimethylurea or by anaerobiosis, indicating that the underlying endogenous electron transport was strictly a pseudocyclic reaction. There was no evidence of endogenous cyclic electron flow which might contribute to the energy metabolism of the bundle sheath cells. PMID- 16660665 TI - Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments. AB - Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [(86)Rb(+)] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10(-7) molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [(86)Rb(+)] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [(86)Rb(+)] flux is not a general feature of phytochrome action. PMID- 16660666 TI - Quantitation of 1,4-Benzoxazin-3-ones in Maize by Gas-Liquid Chromatography. AB - A gas-liquid chromatographic (GLC) procedure is reported for the quantitation of the trimethylsilyl (TMS) derivatives of substituted 2-hydroxy-2H-1,4-benzoxazin 3(4H)-ones (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one[HBOA]; 2-hydroxy-7-methoxy-2H 1,4-benzoxazin-3(4H)-one[HMBOA];2,4- dihydroxy-2H-1,4-benzoxazin-3(4H) one[DIBOA]; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[DIMBOA]; and 2,4 dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one[DIM (2)BOA]) found in maize (Zea mays L.) extracts. Derivatized samples were chromatographed on columns with liquid phases of 2% DC-11 and 3% OV-17 and detected by flame ionization. Internal standards were methyl palmitate and methyl stearate on DC-11 and methyl behenate on OV-17. Detector response was linear to at least 5 nanomoles for TMS(2)-HBOA and TMS(2)-DIBOA and to 19 nanomoles for TMS(2)-DIMBOA. Standard errors of 2% or less were obtained when four replicate samples were analyzed. For each of the 15 maize lines examined, the amount of DIMBOA determined by GLC was directly proportional to the amount of ferric chloride-reactive material determined colorimetrically. PMID- 16660667 TI - Ethylene and Ethane Production from Sulfur Dioxide-injured Plants. AB - After alfalfa (Medicago sativa) seedlings were exposed to approximately 0.7 microliter per liter SO(2) for 8 hours, elevated ethylene and ethane production was observed. Ethylene production peaked about 6 hours and returned to control levels by about 24 hours following the fumigation, while ethane production peaked about 36 hours and was still above control levels 48 hours after the fumigation. Light had an opposite effect upon the production of the two gases: ethane production rates were higher from plants held in light, whereas ethylene production rates were higher from those held in the dark. Peak ethylene and ethane production rates from SO(2)-treated plants were about 10 and 4 to 5 times greater, respectively, than those of the control plants. Ethylene appeared to be formed primarily from stressed yet viable leaves and ethane from visibly damaged leaves. The different time courses and light requirements for ethylene and ethane production suggest that these two gases were formed via different mechanisms. Light appears to have a dual role. It enhances SO(2)-induced cellular damage and plays a role for repairs. PMID- 16660668 TI - Rapid Increase in UDP-Glucose during Early Wheat Embryo Germination. AB - The cellular content of UDP-glucose in isolated wheat (Triticum aestivum L.) embryo increases 8-fold during the first 40 minutes of imbibition. An additional 3-fold increase in the amount of UDP-glucose was observed in the next 5 hours of germination. This communication also describes a unique, quantitative method to achieve a high sensitivity in a direct determination of UDP-glucose with Na [(32)P]pyrophosphate and UDP-glucose pyrophosphorylase. The sensitivity of the assay for UDP-glucose is 10 picomoles. PMID- 16660669 TI - Effect of Ethylene and Oxygen on the Development of Cyanide-resistant Respiration in Whole Plant Mitochondria. AB - Mitochondria from whole potatoes (Solanum tuberosum) ordinarily fail to oxidize respiratory substrates and to consume molecular O(2) in the presence of cyanide. Mitochondrial preparations obtained from tubers previously held for 24 hours in ethylene (10 microliters per liter) in air are only partially inhibited by cyanide. Application of ethylene in 100% O(2) led to an additional increase in the resistance of the mitochondrial respiration to cyanide. The resistance to cyanide was accompanied by a decrease in the respiratory control but no change in oxidative phosphorylation as shown by the measurement of ATP synthesis.The development of resistance to cyanide following the application of ethylene appears to require whole tubers and may represent an inductive process. PMID- 16660670 TI - Movement of Kinetin and Gibberellic Acid in Leaf Petioles during Water Stress induced Abscission in Cotton. AB - Movement of [(14)C]kinetin and [(14)C]gibberellic acid was examined in cotton (Gossypium hirsutum L.) cotyledonary petiole sections independent of label uptake or exit from the tissue. Sections 20 millimeters in length were taken from well watered, stressed, and poststressed plants. Transport capacity was determined using a pulse-chase technique. Movement of both kinetin and gibberellic acid was found to be nonpolar with a velocity of 1 millimeter per hour or less, suggesting passive diffusion. Neither water stress nor anaerobic conditions during transport of labeled material affected the transport capacity of the petioles.Results suggested strong kinetin binding but weak gibberellic acid binding in the tissue sections. Apparent binding of both growth regulators was unaltered by the experimental conditions. Movement of these two growth regulators within cotton cotyledonary petioles plays a minor role in the stress-induced, foliar abscission process. PMID- 16660671 TI - Responses of Atriplex spongiosa and Suaeda monoica to Salinity. AB - The growth and tissue water, K(+), Na(+), Cl(-), proline and glycinebetaine contents of the shoots and roots of two Chenopodiaceae, Atriplex spongiosa and Suaeda monoica have been measured over a range of external NaCl salinities. Both species showed some fresh weight response to low salinity mainly due to increased succulence. S. monoica showed both a greater increase in succulence (at low salinities) and tolerance of high salinities than A. spongiosa. Both species had high affinities for Na(+) and maintained constant but low shoot K(+) contents with increasing salinity. These trends were more marked with S. monoica in which Na(+) stimulated the accumulation of K(+) in roots. An association between high leaf Na(+) accumulation, high osmotic pressure, succulence, and a positive growth response at low salinities was noted. Proline accumulation was observed in shoot tissues with suboptimal water contents. High glycinebetaine contents were found in the shoots of both species. These correlated closely with the sap osmotic pressure and it is suggested that glycinebetaine is the major cytoplasmic osmoticum (with K(+) salts) in these species at high salinities. Na(+) salts may be preferentially utilized as vacuolar osmotica. PMID- 16660672 TI - Movement to the xylem exudate of rubidium accumulated in the apex of corn roots. AB - Apical segments of roots of corn (Zea mays, L.) were excised and mounted in experimental salt solutions containing (86)RbCl and CaCl(2). Xylem exudates were collected hourly beginning at the 21st hour. At the 24th hour, experimental solutions were replaced with solutions of other chlorides (nonlabeled) of Ca(2+) +/- some monovalent cation.Following replacement of the labeled solutions, the rate of (86)Rb(+) release to the exudates from root segments declined by about the same proportional amount regardless of the initial steady-state output rate or the kinds of salts used to replace the RbCl. The content of (86)Rb(+) in the roots also declined with time, but to a relatively less degree. Loss of accumulated (86)Rb(+) to the various external solutions following replacement was less than 6% in 6 hours. It is concluded that movement of accumulated Rb(+) to the exudate is dependent on the concentration of Rb(+) in the root but is largely independent of exchange between accumulated ions and other ions in transit across the root. PMID- 16660673 TI - Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism. AB - The relationship between ethylene action and metabolism was investigated in the etiolated pea seedling (Pisum sativum L. cv. Alaska) by inhibiting ethylene action with Ag(+), high CO(2), and low O(2) and then determining if ethylene metabolism was inhibited in a similar manner. Ag(+) (100 milligrams per liter) was clearly the most potent antiethylene treatment. Ag(+) pretreatment inhibited the growth retarding action of 0.2 microliters per liter ethylene by 48% and it also inhibited the incorporation of 0.2 microliters per liter (14)C(2)H(4) into pea tips by the same amount. As the ethylene concentration was increased from 0.2 to 30 microliters per liter, the effectiveness of Ag(+) in reducing ethylene action and metabolism declined in a similar fashion. Although Ag(+) significantly inhibited the incorporation of (14)C(2)H(4) into tissue metabolites, the oxidation of (14)C(2)H(4) to (14)CO(2) was unaffected in the same tissue.CO(2) (7%) inhibited ethylene-induced growth retardation but its effectiveness diminished at a greater rate than that of Ag(+) with increasing ethylene concentration. High CO(2) had just the opposite effect of Ag(+) since it inhibited (14)C(2)H(4) oxidation to (14)CO(2) without affecting tissue incorporation. In contrast to Ag(+), CO(2) did not inhibit ethylene action and metabolism to exactly the same extent, and the inhibition of metabolism did not rapidly decline with increasing (14)C(2)H(4) concentration. However, high CO(2) did alter the ratio of (14)C(2)H(4) tissue incorporation to (14)CO(2) production in a manner consistent with changes in ethylene effectiveness.Lowering the O(2) concentration to 5% reduced ethylene-induced growth retardation from 70 to 58% at 0.22 microliters per liter and inhibited (14)C(2)H(4) (0.25 microliters per liter) tissue incorporation and oxidation to (14)CO(2) by 26 and 45%, respectively. However, in contrast to Ag(+) and high CO(2) which slightly promoted growth in ethylene-free air, low O(2) reduced pea seedling growth under these conditions thereby severely limiting its usefulness as a specific antiethylene treatment.Collectively these data suggest that the metabolism of ethylene may be related to its action. PMID- 16660674 TI - Composition and Function of Thylakoid Membranes from Grana-rich and Grana deficient Chloroplast Mutants of Barley. AB - Chlorophyll-deficient barley (Hordeum vulgare) mutants were studied that had chlorophyll a/b ratios either higher or lower than the wild type. Mutants with high ratios (>5.2) had a reduced proportion of their photosynthetic lamellae appressed into grana ("grana-deficient" mutants) compared with wild type (chlorophyll a/b = 3.2), while the majority of lamellae in the chloroplasts with low chlorophyll a/b ratios (2.0-2.4) were organized into grana ("grana-rich" mutants).All mutants catalyzed photosystem I and photosystem II electron transport, were tightly coupled as evidenced by increased rates of electron transport in the presence of methylamine, and were able to generate a light dependent transmembrane proton gradient. Differences were evident in rates of electron transport per mole of chlorophyll. The mutants having high chlorophyll a/b ratios catalyzed 15- to 50-fold higher rates of ferricyanide photoreduction than the mutants having low chlorophyll a/b ratios, and 5- to 7-fold higher than the wild type.Low temperature absorption spectra of chloroplast fragments showed that the grana-deficient mutant with a high a/b ratio had a chlorophyll spectrum characteristic of a PSI preparation while mutants with the low ratio had a spectrum typical of a PSII preparation.The temperature fluorescence emission spectra of thylakoid membrane fragments from the two types of mutants were also strikingly different from one another, as were the electrophoretic patterns of the thylakoid polypeptides. PMID- 16660675 TI - Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics. AB - An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K(+) ((86)Rb), (36)Cl(-), H(2) (32)PO(4) (-) and (45)Ca(2+) from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K(+), Cl(-), and H(2)PO(4) (-) measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N(2) gas. In contrast, Ca(2+) influx was not tightly coupled to respiratory energy production. The influx of K(+) was highest between pH 6.5 and 7.5 whereas the influx of H(2)PO(4) (-) and Cl(-) was greatest between pH 4.5 and 5.5. Influx of K(+) and Cl(-) was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K(+) influx by more than 200% but had no effect on the influx of either Cl(-) or H(2)PO(4) (-). Apparent H(+) efflux, as measured by decrease in solution pH, was enhanced by K(+), stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport. PMID- 16660676 TI - Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: II. Selectivity and Kinetics. AB - Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca(2+), transport was selective for K(+) ((86)Rb) over Na(+). (36)Cl(-) transport was inhibited by Br(-) or I(-) but not by H(2)PO(4) (-). The kinetic data for short term (30 minutes) K(+) influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl(-) and H(2) (32)PO(4) (-) over the same concentration range were different from those observed for K(+), and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl(-) transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast. PMID- 16660677 TI - Synthesis of a possible precursor of alpha-amylase in wheat aleurone cells. AB - alpha-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific lambda-globulin for alpha-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic alpha amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of alpha-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of alpha-amylase in aleurone cells. PMID- 16660678 TI - Functional Comparison of the Photosystem II Center-Antenna Complex of a Phycocyanin-less Mutant of Cyanidium caldarium with That of Chlorella pyrenoidosa. AB - The photosystem II (PSII) antenna of a phycocyanin-less mutant (III-C) of Cyanidium caldarium was studied by means of O(2) activation and fluorescence induction measurements and compared to that of the green alga Chlorella pyrenoidosa.In Cyanidium this antenna contains only about 40 (Chl) a molecules per center yet shows PSII energy transfer as efficient as in Chlorella with 240 Chl a + b per center. In Cyanidium, PSII energy transfer occurs among at least four centers which are located close to each other. Cyanidium and Chlorella show the same PSII action and low temperature emission spectra, in the Chl a region despite the gross differences in antenna size.It is proposed that the PSII center antenna complex found in Cyanidium is a core unit of the center-antenna complex of green algae; the only major difference between them is the addition of light harvesting Chl a/b in the latter. It is further proposed that this core is the site of energy transfer between PSII centers. PMID- 16660679 TI - Effect of water regime on carbon isotope composition of lichens. AB - delta(13)C values of the lichens Ramalina duriaei and Teloschistes villosus collected in their natural habitat were repeatedly measured during 2 years. Results show variations in the stable carbon isotope ratios ((13)C/(12)C). Such variations are correlated to the seasonal rainfall, i.e. low values of delta(13)C of the lichens during the winter and high values of delta(13)C during the dry summer. Relatively low delta(13)C values were obtained also in laboratory experiments with lichens grown under controlled humid conditions and in lichens collected from humid habitats.The variations in carbon isotopes were associated with quantitative metabolic changes. Under humid conditions an increase was obtained in the total amount of the extracted water-soluble fraction of the plant tissues as well as in the relative content of soluble carbohydrates.Analysis of the possible factors which may cause such variations indicates that the quantity of precipitation and the exposure time to high humidity were the main environmental factors causing seasonal variations in the delta(13)C values of the lichens. Such variations are dependent mainly on enzymic reactions and are probably less influenced by purely physicochemical processes. In view of the data presented here the balance between carboxylation and decarboxylation reactions seems to be the major factor for the observed seasonal differences. Winter accumulation of (12)C enriched components causes an over-all decrease in delta(13)C. During the summer, those storage materials are respired with concomitant increase in the delta(13)C of the residual plant material. PMID- 16660680 TI - Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures. AB - Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth. PMID- 16660681 TI - Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta. AB - Arundinella hirta L. is a C(4) plant having an unusual C(4) leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C(4) species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C(4) pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells. PMID- 16660682 TI - Effect of Bentazon, a Hill Reaction Inhibitor, on Symbiotic Nitrogen-fixing Capability and Apparent Photosynthesis. AB - Symbiotic associations of bean plants (Phaseolus vulgaris L. cv. Blue Lake) and Rhizobium phaseoli strain 127K17 were treated with the Hill reaction inhibitor bentazon (3-isopropyl-1 H-2,1,3-benzothiadiazin-4-(3H)-one-2,2-dioxide). Plants receiving foliar and root treatments of 1.8 kilograms per hectare bentazon were assayed at 6 hour intervals for N(2)-fixing capacity by measuring C(2)H(2) dependent C(2)H(4) production and H(2) evolution and for CO(2) exchange rates. In foliar treated plants greatest measured inhibition of CO(2) exchange rates and N(2)-fixing capacity occurred 6 and 12 hours after treatment, respectively. In root-treated plants maximum inhibition of both processes was delayed by 6 hours, and was less severe than in foliar treated plants. Nitrogen-fixing capacity and CO(2) exchange rate recovered to control levels in all plants. Application of higher rates of bentazon resulted in greater inhibition of CO(2) exchange rate and N(2)-fixing capacity. Inhibition of the two processes was positively correlated (r = 0.985). The results indicate that inhibition of N(2)-fixing capacity was not caused by bentazon directly, but indirectly through limiting the availability of photosynthate to support root nodule activity. PMID- 16660683 TI - Intracellular location of carbonate dehydratase (carbonic anhydrase) in leaf tissue. AB - Two proteins which have carbonate dehydratase (carbonic anhydrase, EC 4.2.1.1) activity were shown to be in the chloroplasts and in the cytosol of leaves of Brassica chinensis, Spinacia oleracea, and in variegated leaves of Tradescantia albiflora and Hedera canariensis. The chloroplastic enzyme is smaller than the one in the cytosol, as it runs farther on gradient polyacrylamide gels. It was separated from the other by isolation of chloroplasts of Brassica and Spinacia on sucrose density gradients; approximately half of the total activity was in the chloroplasts. PMID- 16660684 TI - Effect of red light on geotropism in pea epicotyls. AB - Dose response curves were determined for phytochrome phototransformation and for a phytochrome-controlled decrease in geotropic curvature in epicotyls of dark grown Pisum sativum L. cv. Alaska. Ten times as much light was required to produce a spectrophotometrically detectable transformation of phytochrome as was required to produce a significant change in the geotropic response. The red light energy required for a 50% phytochrome transformation caused a 90% change in the physiological response. PMID- 16660685 TI - Correction of thermal gradient errors in stem thermocouple hygrometers. AB - Stem thermocouple hygrometers were subjected to transient and stable thermal gradients while in contact with reference solutions of NaCl. Both dew point and psychrometric voltages were directly related to zero offset voltages, the latter reflecting the size of the thermal gradient. Although slopes were affected by absolute temperature, they were not affected by water potential. One hygrometer required a correction of 1.75 bars water potential per microvolt of zero offset, a value that was constant from 20 to 30 C. PMID- 16660686 TI - Biosynthesis of Photosystem II Reaction Centers, Antenna and Plastoquinone Pool in Greening Cells of Cyanidium caldarium Mutant III-C. AB - Dark-grown etiolated cells of Cyanidium caldarium mutant III-C lacking >/=99% of their normal chlorophyll content and inactive for photosynthesis were greened in continuous light. Measurements of oxygen evolution and fluorescence kinetics indicate that during greening: (a) the photosystem II (PSII) antenna containing between 30 and 40 chlorophyll a per center undergoes little change in size from 5% of the centers synthesized per cell to fully active cells; (b) energy transfer between PSII centers appears very early in the greening process; (c) the plastoquinone pool size per PSII center (about 14 equivalents) does not vary during greening and has already attained full size after synthesis of only 13% of the full complement of centers.The PSII centers, antenna chlorophyll and plastoquinone are integrated into the membrane with constant stoichiometry throughout at least 90% of the greening process. These components are integrated such that in regions of the membrane containing active PSII centers, their density in the membrane hardly changes over the same period. The center-antenna complex and possibly the plastoquinone pool as well are organized into unit structures containing 40 chlorophyll a and 7 plastoquinones, respectively. Energy transfer between centers in PSII appears following aggregation of the center antenna units. PMID- 16660687 TI - Energy Transfer from the Phycobilisomes to Photosystem II Reaction Centers in Wild Type Cyanidium caldarium. AB - Nonsaturating light at 600 or 436 nanometers was used to excite specifically phycocyanin or chlorophyll a, respectively, both of which participate in light capture in photosystem II of Cyanidium caldarium. The ratio of absorption of light by phycocyanin to chlorophyll in photosystem II in this organism is >20 at 600 nanometers and Rb(+) > (Na(+) = Li(+)). Calcium and Mg(2+) ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO(4) (3-) > NO(3) (-) > Cl(-). The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca(2+) ions, or to effects on K(+) uptake. A mixture of Na(+) and K(+) in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na(+) plus K(+). The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action. PMID- 16660710 TI - Analysis of Variability in the Amaranthus Bioassay for Cytokinins: Effects of Water Stress on Benzyladenine- and Fusicoccin-dependent Responses. AB - The use of only the upper part of the hypocotyl and the cotyledons in the Amaranthus tricolor bioassay for cytokinins, instead of the whole seedling, was found to reduce the endogenous response and give a higher benzyladenine-dependent response. There is no marked difference in the uptake or metabolism of benzyladenine in whole seedlings compared with that in excised cotyledons.Analysis of variability in the bioassay showed that water availability to the cut seedlings and to whole seedlings is a major factor in the amounts of betacyanin accumulated during the subsequent induction period. The increase in the amount of betacyanin accumulated in response to benzyladenine, following conditions of water stress, is not correlated with differences in benzyladenine uptake. Endogenous production and fusicoccin stimulation is also increased following water loss by cut seedlings. Possible explanations for this stress induction may be found in responses of active transport to changes in turgor pressure.Although pretreatment of the roots of seedlings with mannitol stimulated subsequent induction by excised cotyledons, the presence of mannitol during the induction period inhibited the accumulation of betacyanin. This inhibition is not due to any effect on benzyladenine uptake. The susceptibility of amino acid uptake and polysome profiles to water stress suggests that the inhibition of betacyanin synthesis, a process dependent on protein synthesis, may be due to inhibition either of precursor (tyrosine) uptake or of the synthesis or activity of some enzyme in the pathway. PMID- 16660711 TI - Analysis of variability in the amaranthus betacyanin assay for cytokinins: effects of "aging" excised cotyledons. AB - "Aging" of excised cotyledons plus the top part of the hypocotyl of Amaranthus tricolor seedlings was carried out by washing in distilled H(2)O for varying periods. This led to increased betacyanin accumulation during the subsequent 24 hour induction period in the presence of tyrosine and Na(+) + K(+) phosphate. Endogenous accumulation as well as that dependent on added benzyladenine and on added fusicoccin was stimulated. This stimulation could not be due to a carryover of a wound-induced burst of ethylene since 2-chloroethylphosphonic acid (Ethrel) was shown to be extremely inhibitory to betacyanin synthesis if present during the induction process. It is possible that a wound-induced burst of ethylene could give rise to increased betacyanin synthesis as an after effect. The procedure for obtaining good induction with the most reproducible results is described. PMID- 16660712 TI - Temperature-dependent Expression of Betacyanin Synthesis in Amaranthus Seedlings. AB - Two phenomena related to temperature effects have been observed during the induction of betacyanin synthesis by a cytokinin (benzyladenine) in Amaranthus tricolor seedlings. One is a total inhibition of betacyanin accumulation at a temperature (39 C) at which seedling growth is unimpaired, and where there is still adequate uptake of benzyladenine. The other is the apparent induction of a higher potential for subsequent betacyanin synthesis following pretreatment of the seedlings at an elevated temperature. PMID- 16660713 TI - Role of Ethylene in Abscisic Acid-induced Callus Formation in Citrus Bud Cultures. AB - The induction of callus formation in cultured buds of Shamouti orange (Citrus sinensis [L.] Osbeck) by abscisic acid (ABA) is a multiphasic process. (Altman, and Goren 1974 Physiol Plant 32: 55.) A study of the mediation by ethylene on this effect of ABA was undertaken. It was found that: (a) ethylene and (2 chloroethyl) phosphonic acid, as well as ABA, induced callus formation; (b) callus induction is best attained when explants are exposed to ethylene during the 1st day after excision; and (c) ABA-induced callus formation is inhibited by rhizobitoxine analog, an inhibitor of ethylene biosynthesis. It is concluded that the effect of ABA on callus formation is mediated via ethylene. PMID- 16660714 TI - Borate Exchanges of Lemna minor L. as Studied with the Help of the Enriched Stable Isotopes and of a (n,alpha) Nuclear Reaction. AB - Despite the lack of a convenient radioisotope of boron, it is possible to measure unidirectional fluxes of borate between cellular systems and their external medium. It was accomplished by using the two purified stable isotopes ((10)B and (11)B), with (10)B specifically detected by a (n,alpha) nuclear reaction. The method was applied to compartmental analysis of borate with intact plants of Lemna minor L. Four compartments were suggested. Three of them apparently correspond to the three classical ones: free space (including easily dissociable borate monoesters), cytoplasm, and vacuole. The fourth one was interpreted as corresponding to very stable borate diesters in the cell walls. The method allows the determination of the borate capacities of the various compartments and of the borate unidirectional fluxes between the different compartments, at borate flux equilibrium. Other physicochemical data (mono and diester mass action constants, turn over numbers) were evaluated. The results are consistent with what is known of pure substances. PMID- 16660715 TI - A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration. AB - When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H(2)O(2) became "available" during the oxidation of [1 (14)C]glycolate was sufficient to account for the breakdown of the intermediate [1-(14)C]glyoxylate to formate (C(1) unit) and (14)CO(2). Under aerobic conditions formate production closely paralleled (14)CO(2) release from [1 (14)C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3 fold when the temperature was raised from 25 to 35 C, or when the rate of H(2)O(2) production was increased artificially by addition of an active preparation of fungal glucose oxidase.When [(14)C]formate was added to these preparations it was oxidized directly to (14)CO(2) by the peroxidatic action of peroxisomal catalase; however, the breakdown of formate was slow relative to the rate of formate production. For example, when [(14)C]formate was generated from [2-(14)C]glycolate it was not readily oxidized to (14)CO(2) in these organelles. Because the activity of formate-NAD(+) dehydrogenase in cell-free leaf extracts was low compared with that of formyl tetrahydrofolate synthetase it is suggested that most of the formate produced during glycolate oxidation could be metabolized via the one carbon pool and not oxidized directly to CO(2).At 25 C the rate of release of (14)CO(2) from [2-(14)C]glycolate in leaf discs was 40 to 50% of the rate from [1-(14)C]glycolate. Isonicotinyl hydrazide inhibited (14)CO(2) release from both [1-(14)C]- and [2-(14)C]glycolate; but this inhibitor was more effective in blocking (14)CO(2) release from [2-(14)C]glycolate. It is argued that the oxidation of the methylene carbon group of glycolate does not occur as a direct consequence of formate (C(1) unit) breakdown, but is a product of the further metabolism of formate and glycine, possibly, via serine. PMID- 16660716 TI - 8-[C]Benzylaminopurine Translocation in Phaseolus vulgaris. AB - [8-(14)C]Benzylaminopurine (BA) translocation was studied in whole plants of Phaseolus vulgaris L. under three different light regimes (continuous light, 8 hour light + 16-hour dark, dark). Applications were made to the apex, to a cotyledonary leaf, or to the root system. Results showed that no BA basipetal translocation occurred, however BA is easily absorbed by the root system and is translocated acropetally.The amount of BA absorbed and its acropetal translocation rate depend on the light regime. The hypothesis of a passive cytokinin transport through the xylem, regulated by the transpiration stream, is discussed. PMID- 16660717 TI - Aspects of 8-[C]Benzylaminopurine Metabolism in Phaseolus vulgaris. AB - Phaseolus vulgaris L. plants were supplied through the root with [8 (14)C]benzylaminopurine ([(14)C]BA). Collections of root, apex, and leaves were made 8 and 48 hours after labeling; ethanolic extracts of tissues were purified and subjected to thin layer chromatography on silica gel and/or cellulose powder.From [(14)C]BA at least two different metabolites originate, the BA riboside and ribotide. The balance among [(14)C]BA, the riboside, and the ribotide changed in time. The [(14)C]BA riboside was detected as the only labeled compound in the xylem sap collected from the stem. The biological function of BA riboside and ribotide is discussed. PMID- 16660718 TI - Identification of Cultivar Differences in Seed Polypeptide Composition of Peanuts (Arachis hypogaea L.) by Two-Dimensional Polyacrylamide Gel Electrophoresis. AB - Seed polypeptides from several cultivars of peanut (Arachis hypogaea L.) have been compared by means of a two-dimensional polyacrylamide gel electrophoresis. Protein was extracted from the defatted peanut meal by homogenizing in 5 millimolar K(2)CO(3)-9.5 molar urea. After addition of Nonidet P-40 (2%, v/v) and dithiothreitol (0.5%, w/v) the solution was centrifuged at 25,000 g. This procedure led to solubilization of more than 95% of the total protein. The clear supernatant fraction was then subjected to two-dimensional polyacrylamide gel electrophoresis, employing isoelectric focusing in the first dimension and electrophoresis in presence of sodium dodecyl sulfate in the second. After examining several cultivars, it was possible to construct a composite map to include all of the polypeptide species found among all of the cultivars examined. At least 74 major and between 100 and 125 minor components were detectable by Coomassie blue staining. The majority of these had isoelectric points between pH 4.4 and 8.0, and molecular weights between 16,000 and 75,000. Several different cultivars have been compared using this method and it has been shown that considerable variation exists among the major polypeptides present. The method should prove valuable for analyzing different genotypes and selecting varieties with a particular storage protein make-up, as well as for following compositional changes that occur during seed development and germination. PMID- 16660719 TI - Composition of globoid crystals from embryo protein bodies in five species of cucurbita. AB - Previous energy-dispersive x-ray analysis studies of globoid crystal composition in seed protein bodies gave an indication that there might be a correlation between seed size and the type of elements stored in globoid crystals. This possibility was tested by conducting energy-dispersive x-ray analysis studies of P, K, Mg, and Ca levels in globoid crystals of four embryo regions (radicle, stem, cotyledon center palisade mesophyll, cotyledon center spongy mesophyII) in each of five different Cucurbita species (C. mixta, C. moschata, C. foetidissima, C. pepo, and C. andreana). The species were chosen to provide a range of seed size and weight. Globoid crystals from all embryo regions in all five species contained P, K, and Mg. Some variations in the levels of these elements did occur but there was no consistent pattern with regard to area of the seed or with regard to seed size. Calcium distribution showed significant variations. In species with large seeds (C. mixta, C. moschata) Ca was mainly found in globoid crystals in the radicle. Globoid crystals in species with small seeds (C. foetidissima, C. pepo, C. andreana) contained Ca in all embryo regions tested. The results of this study support the concept that Ca distribution in globoid crystals can be correlated with seed weight. PMID- 16660720 TI - Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize. AB - Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts. PMID- 16660721 TI - Pyrophosphatase, Peroxidase and Polyphenoloxidase Activities during Leaf Development and Senescence. AB - Inorganic pyrophosphatase, peroxidase, and polyphenoloxidase activities were studied as the function of leaf insertion level in eight monocotyledonous and eight dicotyledonous species. Alkaline inorganic pyrophosphatase shows a declining activity toward the end of senescence whereas no regular drift in either peroxidase or polyphenoloxidase activities was noticed during senescence of attached leaves. In the primary leaves of rice, peroxidase and polyphenoloxidase activities were high in the senescent leaves and there exists a correlation between chlorophyll content and peroxidase activity though not with polyphenoloxidase activity. Upon detachment leaves exhibit increasing peroxidase and polyphenoloxidase activities with time. The distribution of the enzyme activities during senescence of attached leaves is suggested to be species specific, and an increase in peroxidase and polyphenoloxidase activities cannot be taken as an indicator of leaf senescence. PMID- 16660722 TI - Shifting the phase of the circadian rhythm in bioluminescence in gonyaulax with vanillic Acid. AB - Exposure for 4 hours to vanillic acid (4-hydroxy 3-methoxy benzoic acid) caused large delay phase shifts (5 to 6 hours) in the circadian rhythm of bioluminescence in Gonyaulax polyedra, when assayed at either 10 to 14 circadian time or 22 to 02 circadian time in constant light and temperature, provided that the pH of the medium was 7.1 or lower. Corresponding changes in the pH with acetic acid did not shift phase. Vanillic acid caused detectable depolarization of the membranes of Gonyaulax, as demonstrated with the cyanine dye fluorescence technique. PMID- 16660723 TI - Light-dependent Induction of Polyunsaturated Fatty Acid Biosynthesis in Greening Cucumber Cotyledons. AB - Greening cucumber (Cucumis sativus L.) cotyledons exhibited dramatic increases in the ability to desaturate exogenously added [1-(14)C]oleic acid and [1 (14)C]linoleic acid within 2 to 3 hours of illumination. These increases were effectively inhibited by 10 micrograms per milliliter cycloheximide. Oleate desaturation remained at a high level in constant light for 5 to 6 days after induction and then declined by about 50%; when returned to the dark, the tissue showed a sharp decrease in conversion of [(14)C]oleate to [(14)C]linoleate. Linoleate desaturation reached a maximum about 15 hours after induction and declined immediately thereafter while the tissue still was in the light; after induction had peaked return of the tissue to the dark showed a dramatic fall of linoleate desaturation. The changes in desaturation were correlated with the conversion of the principal fatty acid in the etiolated cotyledons, linoleate, to alpha-linolenate, and with the assembly of the chlorophyll-containing photosynthetic membranes. The incorporation of [1-(14)C]acetate into lipids showed no significant light stimulation. The role of light in the regulation of certain aspects of plant metabolism during development is discussed. PMID- 16660724 TI - Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium. AB - The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells. PMID- 16660725 TI - Polyamine Metabolism in Embryogenic Cells of Daucus carota: II. Changes in Arginine Decarboxylase Activity. AB - Embryogenic cultured cells of Daucus carota have been shown to synthesize putrescine from exogenously supplied [(14)C]arginine at twice the rate of control nonembryogenic cells. In the present paper, the activity of arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19), an important enzyme in the synthesis of putrescine, was assayed and also found to be elevated by as much as 2-fold in embryogenic cells. This difference between embryogenic and nonembryogenic cells was observed as early as 6 hours after the induction of embryogenesis and appeared not to result from the presence of a diffusible inhibitor or activator. It seemed to be dependent upon concomitant RNA and protein synthesis, as judged using 6-methyl-purine and cycloheximide. After cycloheximide addition to the culture medium, arginine decarboxylase activity declined with a half-time of about 30 minutes in both embryogenic and nonembryogenic cells. It is suggested that elevated arginine decarboxylase activity is involved in the mechanism leading to elevated putrescine levels in these cells and hence may play a role in the embryogenic process. PMID- 16660726 TI - In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities. AB - NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH(2))-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes-methylviologen-nitrate reductase and NADH-ferricyanide reductase-also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH(2)-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH(2)-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t((1/2)) [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH(2) do not donate electrons to the same site of the nitrate reductase protein. PMID- 16660727 TI - In Vitro Synthesis of a Precursor to the Methionine-rich Polypeptide of the Zein Fraction of Corn. AB - The messenger ribonucleic acid fraction isolated from a protein bodyenriched fraction of developing corn (Zea mays L.) endosperm stimulated the incorporation of radioactive amino acids into at least five polypeptides when added to a wheat germ extract capable of protein synthesis. Of these, the two major polypeptides formed with messenger from freshly frozen corn were identified as precursors to zein A and B, the two major polypeptides of the prolamine fraction of corn meal (21,600 and 19,600 molecular weight). The identification was based on the relative incorporations of radioactive leucine, lysine, and methionine, and the susceptibility of the zein A precursor, but not the zein B precursor to cleavage with cyanogen bromide. Using extracts from stored frozen corn, a third polypeptide of 14,500 molecular weight was identified as a major in vitro product. It was preferentially labeled with methionine and slightly larger than a similar peptide in the zein fraction of corn meal. Two other polypeptides of still lower molecular weight could be detected above the background of probably incomplete polypeptides. PMID- 16660728 TI - Effects of Controlled Atmospheres on Production of Sesquiterpenoid Stress Metabolites by White Potato Tuber: Possible Involvement of Cyanide-resistant Respiration. AB - Levels of katahdinone (solavetivone), lubimin, rishitin, and phytuberin, sesquiterpenoid stress metabolites of white potato (Solanum tuberosum), were monitored in tuber slices which were challenged with an extract of Phytophthora infestans and incubated under controlled atmospheres. A mixture of ethylene in air enhanced stress metabolite production. This enhancement was amplified by higher partial pressures of oxygen. Stress metabolite production was inhibited by salicylhydroxamic acid. These results suggest the involvement of cyanide resistant respiration in the production of potato stress metabolites, compounds which may serve as phytoalexins. PMID- 16660729 TI - Seasonal Changes in the Structure and Function of Mitochondrial Membranes of Artichoke Tubers: A Requisite for Surviving Low Temperatures during Dormancy. AB - The temperature limits of the order-disorder transition, and the Arrhenius activation energy of succinate oxidase activity for mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers were determined from the initiation to the termination of dormancy. The temperature limits for the transition at the initiation of dormancy were 25 and 3 C. These changed to 9 and -5 C at mid dormancy and returned to 25 and 2 C at the termination of dormancy. The Arrhenius activation energy measured in the temperature range above the transition was 35 kilojoules per mole at middormancy and decreased to 17 kilojoules per mole at the termination of dormancy when sprouting was evident. The coincidence of the changes in membrane structure and function with dormancy suggests that artichokes possess a mechanism for regulating membrane lipid structure so that cellular integrity of tuber tissue is maintained even when the tubers are exposed to low temperatures. PMID- 16660730 TI - A simple theory regarding ambimobility of xenobiotics with special reference to the nematicide, oxamyl. AB - A theory is presented to explain the phloem mobility of certain systemic xenobiotics that are not weak acids. It is shown that there is a theoretically optimum permeability that permits optimum circulation through the symplasm and apoplast (including the phloem and xylem) of Solanum tuberosum plants. The optimum permeability is large enough to permit substantial passive permeation into sieve cells in the source leaf and yet is small enough to permit phloem transport with some retention. The optimum permeability is a function of the velocity of sap flow in sieve tubes, the radius of the sieve tube, the over-all length of the plant, and the length of the carbohydrate and xenobiotic sources. It is argued that the nematicide, oxamyl, is near the optimum permeability under some experimental conditions. It is shown that depending on the strength of the carbohydrate sink in roots or growth points and depending on the permeability of the xenobiotic, there can be passive accumulation of xenobiotics in the sieve tubes in the carbohydrate sink regions. PMID- 16660731 TI - Ultrastructural Comparison of Cyanidium caldarium Wild Type and III-C Mutant Lacking Phycobilisomes. AB - Cyanidium caldarium wild type and III-C mutant lacking phycobilisomes were compared with respect to the ultrastructural organization of particles on the freeze-fractured thylakoid membrane.In the wild type, the particles on the exoplasmic fracture face were arranged in the same manner as that reported for the phycobilisomes on the membrane surface. The phycobilisomes constitute the major part of the photosystem II antenna and their absence in the III-C mutant was accompanied by a completely different arrangement of the particles on the exoplasmic fracture face.The density of these particles was almost two times higher in the mutant than in the wild type while that of the particles on the protoplasmic fracture face was about the same.The relative densities of the particles on the exoplasmic fracture face in the two organisms was consistent with the 2-fold higher photosystem II to photosystem I ratio in the mutant compared to the wild type as determined by measurements of the field indicating absorbance changes.These particles were 100 Angstroms in both organisms.It is concluded that the particles on the exoplasmic fracture face in Cyanidium are probably substructural units of the particles observed on the same surface in higher plants and green algae and attributed to photosystem II. PMID- 16660732 TI - In Vitro Binding of Agrobacterium tumefaciens to Plant Cells from Suspension Culture. AB - In vitro binding experiments were carried out using (32)P-labeled cells of the virulent Agrobacterium tumefaciens strain B6 and Datura innoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca(2+) and Mg(2+) reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperature-dependent. Escherichia coli, Salmonella typhimurium, Rhizobium japonicum, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Datura cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimolar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-l-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2- to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiographic analysis showed that bacteria were associated with the cell wall, and that often binding of bacteria was localized. Electron micrographs clearly illustrated a tight association of virulent A. tumefaciens strain B6 cells to the Datura cell wall. PMID- 16660733 TI - Relationship of Temperature to Stomatal Aperture and Potassium Accumulation in Guard Cells of Vicia faba. AB - Epidermal strips of Vicia faba were floated on 10 millimolar KCl at various temperatures and for several time periods. The diameter of the stomatal aperture was determined microscopically and K(+) content was estimated and expressed as the per cent of the guard cell stained. Stomatal opening was associated with increased K(+) in guard cells, but the quantitative association was modified both by time and temperature. At low temperatures (0-20 C) there was a prolonged Spannungsphase while at higher temperatures (30-45 C) motorphase was exhibited. During the motorphase there was a rapid opening of the stomates which was highly correlated with K(+) influx. At treatment periods of 360 minutes and temperatures higher than 25 C there appeared to be a maintenance phase during which K(+) concentration of the guard cells decreased without an equivalent decrease in aperture. PMID- 16660734 TI - Interaction of Chloroplasts with Inhibitors: Location of Carotenoid Synthesis and Inhibition during Chloroplast Development. AB - The inhibitor SAN 6706 [4-chloro-5-(dimethylamino)-2-(alpha,alpha,alpha, trifluoro- m-tolyl-3(2H)-pyridazinone] has been used to study the synthesis of carotenes and xanthophylls during the conversion of etioplasts to chloroplasts in developing barley (Hordeum vulgare) shoots. SAN 6706 inhibits carotenoid synthesis and causes an accumulation of phytoene, but it is also a potent inhibitor of chloroplast electron transport. When developing barley is treated with SAN 6706, carotenoid synthesis is inhibited but total photosynthesis is unaffected. The ability of SAN 6706 to inhibit carotenoid synthesis becomes progressively less if etiolated shoots are illuminated for increasing lengths of time before treatment. During the greening of treated barley shoots only light induced beta-carotene synthesis is immediately inhibited; xanthophyll synthesis is not affected until after about 8 hours. The hypothesis that SAN 6706 cannot enter the chloroplast but inhibits carotenoid synthesis from the cytoplasm is discussed, and the question as to whether there are not two separate groups of enzymes for the synthesis of carotenes and xanthophylls is considered. PMID- 16660735 TI - Effect of carbon dioxide on photorespiration. AB - The isotopic CO(2) technique for measuring photorespiration was shown to be a valid technique for measuring the unidirectional inward and outward fluxes of CO(2) from a sunflower (Helianthus annuus L.) leaf in the light. The rate of photorespiration was decreased little as the CO(2) concentration was increased from 20 to 1,150 microliters per liter. This finding contradicts the widely held assumption that photorespiration is suppressed at high CO(2) concentrations. Some discussion regarding this apparent conflict is presented. PMID- 16660736 TI - DNA content of free living rhizobia and bacteroids of various Rhizobium-legume associations. AB - The DNA content of bacteroids from 22 different Rhizobium-legume associations was compared to that of the corresponding free living Rhizobium species using laser flow microfluorometry. In all 18 effective associations, the bacteroids had either similar or higher DNA content than the free living rhizobia. Bacteroid populations isolated from effective clover (Trifolium repens) and alfalfa (Medicago sativa) nodules had an average DNA content of >1.5-fold higher than free living R. trifolii and R. meliloti. These populations also contained a significant number of bacteroids with more than 3-fold the DNA content of the free living rhizobia. Populations isolated from effective nodules of winged beans (Psophocarpus tetragonolobus), peas (Pisum sativum), and mung beans (Phaseolus aureus) had an average DNA content of 1.1- to 1.5-fold higher than free living R. "cowpeas" and R. leguminosarum. Bacteroids from nodules of lupins (Lupinus angustifolius and L. minaretta), kidney beans (Phaseolus vulgaris), and soybeans (Glycine max), however, had similar DNA content to the free living forms. Two of the four associations which formed ineffective nodules contained bacteroids with lower DNA content than the free living rhizobia. The other two associations contained bacteroids with slightly higher or similar DNA content to the free living rhizobia. Nodules of the ineffective associations also did not contain leghemoglobin. PMID- 16660737 TI - Photosynthetic rates of citronella and lemongrass. AB - Ten selections of citronella (Cymbopogon nardus [L.] Rendle) were grown at 32/27, 27/21, or 15/10 C day/night temperatures, and plants from three populations of lemongrass (Cymbopogon citratus [D.C.] Stapf from Japan or Sri Lanka and Cymbopogon flexuosus [D.C.] Stapf from India) were grown at 8- or 15-hour photoperiods. Net photosynthetic rates of mature leaves were measured in a controlled environment at 25 C and 260 microeinsteins per meter(2) per second. Rates declined with increasing leaf age, and from the tip to the base of the leaf blade. Rates for citronella leaves grown at 15/10 C were extremely low for all selections. Highest rates of net photosynthesis were recorded for four selections grown at 27/21 C and for two selections grown at 32/27 C. Lemongrass grown at 8 hour photoperiod had higher photosynthetic rates than that grown at 15-hour photoperiod. PMID- 16660738 TI - Importance of glutamate synthase in glutamate synthesis by soybean cell suspension cultures. AB - The specific activities of glutamate synthase|EC 2.6.1.53, l-glutamine: alpha ketoglutarate amino transferase (NADPH-oxidising)| and glutamine synthetase|EC 6.3.1.2, l-glutamate: ammonia ligase (ADP-forming)| extracted from soybean (Glycine max L.) cells grown in modified B5 medium were found to vary significantly in response to variations in the nitrogen content of the medium. The changes seen in specific activity levels could be correlated with similar patterns seen in the growth of the cells, in response to changes in the nitrogen content of the medium. By contrast, the specific activity of glutamate dehydrogenase|EC 1.4.1.2, l-glutamate: NAD(+) oxidoreductase (deaminating)|, was relatively low and invariant. Glutamate synthase was extracted from cells grown under optimal conditions, partially purified, and shown to have many properties in common with preparations of this enzyme extracted from other plant sources. Glutamate synthase was purified to homogeneity, using affinity chromatography on blue Sepharose. PMID- 16660739 TI - Analysis of Endosperm Sugars in a Sweet Corn Inbred (Illinois 677a) Which Contains the Sugary Enhancer (se) Gene and Comparison of se with Other Corn Genotypes. AB - The endosperm sugars of a new corn (Zea mays L.) mutant, sugary enhancer (se), were analyzed by gas-liquid chromatography and were compared with sugars of other genotypes. Illinois 677a, a sugary (su) inbred containing the se gene, was high in sucrose and was distinguished from all of the other genotypes by its high maltose content. During kernel development, the maltose content of IL677a increased to 3.28% dry weight at 40 days postpollination and remained high at the dry mature stage, whereas ;Silver Queen,' a high quality sugary (su) hybrid not possessing the se gene, showed no such trend in maltose accumulation. Sucrose, fructose, and glucose decreased during kernel development in ;Silver Queen' and IL677a from 19 days postpollination until the dry mature stage. The slow drying characteristic and the reduced starch content previously reported for maturing seeds of IL677a may be related to the maltose accumulation reported here. PMID- 16660740 TI - Seasonal Patterns of Nitrate Reductase and Nitrogenase Activities in Phaseolus vulgaris L. AB - The patterns of nitrate reductase activity (NRA) in the leaves (in vivo assay) and root nodule nitrogenase activity (C(2)H(2) reduction) were investigated throughout the season in field-grown Phaseolus vulgaris plants.Maximal NRA (per g fresh weight) occurred at early stages of leaf development but total activity (per leaf) was maximal when the leaf reached full size. In mature plants, most NRA was associated with the upper leaves. Nitrogenase activity was initiated about 2 weeks after sowing, reached a maximum at flowering (5 weeks after sowing) and declined rapidly thereafter. Nitrogenase activity followed the pattern of nodule development. After flowering, P. vulgaris was apparently able to take up and assimilate NO(-) (3) as evidenced by the increase in NO(-) (3) content of the stem and the high levels of NRA in the leaves. Total plant NRA was maximal after flowering and addition of NH(4)NO(3) to the soil at flowering resulted in even higher levels of NRA through most of the pod-filling period, thus resulting in higher seed yields (59% over control).It is proposed that P. vulgaris can benefit from both N(2) fixation and NO(-) (3) assimilation and that nitrate reductase plays an important role in the assimilation of nitrogen after flowering. PMID- 16660741 TI - Structure of Plant Cell Walls: IX. Purification and Partial Characterization of a Wall-degrading Endo-Arabanase and an Arabinosidase from Bacillus subtilis. AB - Wild type Bacillus subtilis, when grown on beet araban, secretes into its culture medium an endo-arabanase and two arabinosidases. An alternate procedure to one previously described (Kaji A, T Saheki 1975 Biochim Biophys Acta 410: 354-360) has been developed for the purification of the endo-arabanase. The purified endo arabanase is shown to be homogeneous by sodium dodecyl sulfate-urea disc gel electrophoresis (molecular weight approximately 32,000) and by isoelectric focusing (pI = 9.3). The endo-arabanase, acting on a branched araban substrate, has maximal activity at pH 6.0 and preferentially cleaves 5-linked arabinosyl residues. One of the arabinosidases (molecular weight approximately 65,000, pI = 5.3) has been purified to the point that it contains only one quantitatively minor contaminant, as shown by sodium dodecyl sulfate-urea disc gel electrophoresis and isoelectric focusing. The purified arabinosidase, acting on p nitrophenyl-alpha-l-arabinofuranoside, has maximal activity at pH 6.5, and, when acting on a branched araban substrate, preferentially attacks nonreducing terminal arabinosyl residues linked to the 2 or 3 position of other arabinosyl residues. Neither of the two purified enzymes is capable of hydrolyzing a variety of carbohydrate substrates which lack arabinosidic linkages. The purified endo arabinase is shown to be capable of releasing arabinosyl oligomers from the walls of suspension-cultured sycamore cells, thereby suggesting its usefulness as a probe in studying the structure of the araban component of primary cell walls. PMID- 16660742 TI - Composition of the aliphatic components of suberin of the endodermal fraction from the first internode of etiolated sorghum seedlings. AB - The stems from etiolated seedlings of Sorghum bicolor were separated into epidermal and endodermal fractions by manually removing the stele from the cortex. The epidermal fraction was shown to contain a lipid polymer whose monomeric composition was characteristic of cutin with dihydroxyhexadecanoic acid as a major component (25%). The endodermal fraction contained a lipid polymer whose monomeric composition was characteristic of suberin: hexadecanoic acid 12%, octadecenol 6%, octadecenoic acid 23%, omega-hydroxyhexadecanoic acid 17%, hexadecanedioc acid 8%, omega-hydroxyoctadecenoic acid 8%, and octadecenedioic acid 12%. This endodermal polymer is thought to be the suberin component of the Casparian band. PMID- 16660743 TI - Comparison of the Molecular Weights of Proteins Synthesized by Isolated Chloroplasts with Those Which Appear during Greening in Zea mays. AB - The proteins of prolamellar bodies of etioplasts and of thylakoid membranes of greening and mature chloroplasts from Zea mays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three classes of proteins were distinguished: those present in etioplasts and disappearing during greening, those absent in etioplasts and appearing during greening, and those present in both etioplasts and chloroplasts. The largest number of proteins belonged to this last class.The molecular weights of chloroplast thylakoid proteins were compared to the molecular weights of the membrane-associated proteins synthesized by isolated, mature chloroplasts. Thirteen of the 15 to 20 membrane-bound proteins made by isolated chloroplasts corresponded in size to proteins present in chloroplasts. Most of the 13 are present in both etioplasts and chloroplasts although a few were the same size as proteins which increase during greening. Production of most of the membrane proteins made in the plastids is not stringently regulated by light in vivo. The polypeptide subunits of the light harvesting pigment-protein complex, the most abundant proteins of the chloroplast thylakoids, were absent from etioplasts. They were not synthesized by isolated chloroplasts. PMID- 16660744 TI - Short term phytochrome control of oat coleoptile and pea epicotyl growth. AB - Continuous recordings of the effect of light on oat (Avena sativa L. cv. Victory) coleoptile and pea (Pisum sativum L. cv. Alaska) epicotyl growth were made. Using a single excised coleoptile 10 minutes of red light was found to promote growth after a latent period of 46 minutes. The stimulation was transient and was not far red-reversible. Blue and far red light also promoted growth with similar kinetics. The action of continuous red or far red light was similar to that of 10 minute light. The growth of the intact pea third internode (as well as excised segments) was strongly inhibited by red light, with a latent period of 80 minutes. This effect was far red-reversible, and far red and blue light caused only a slight inhibition of growth. PMID- 16660745 TI - Phytochrome Control of Cell Wall-bound Hydroxyproline Content in Etiolated Pea Epicotyls. AB - The red light inhibition of growth of the intact pea (Pisum sativum L. cv. Alaska) third internode was correlated with an increase in the content of cell wall-bound hydroxyproline. These changes were detected 3 hours after irradiation, and possibly at 1 hour. Far red light reversed the effects of red light. The iron chelator alpha,alpha'-dipyridyl reversed the red light effects on both growth and hydroxyproline content. Using segments incubated in vitro, no phytochrome mediated change in hydroxyproline content could be observed, perhaps because of an overwhelming wounding response. If plants were irradiated in situ and grown for 8 hours before excision and incubation of segments, some enhancement of hydroxylation by red light was detectable both colorimetrically and radioisotopically. The red light inhibition of segment growth was reversed by alpha,alpha'-dipyridyl. These results are examined in reference to the role of extensin in normal and induced growth cessation. PMID- 16660746 TI - Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation. AB - Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-(14)C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1 (14)C]- and [6-(14)C]glucose, while with [U-(14)C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the "carbon skeletons" for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.A comparison of (14)CO(2) release from nodules supplied with [1-(14)C]- and [6-(14)C]glucose indicated that the oxidative pentose phosphate pathway accounted for less than 6% of glucose metabolism. Several enzymes of the oxidative pentose phosphate and glycolytic pathways were assayed in vitro using the 12,000g supernatant fraction from nodule homogenates. In all cases, the specific activities were adequate to account for the calculated in vivo fluxes.Three out of four diverse treatments that inhibited nodule nitrogen fixation also inhibited nodule CO(2) fixation, and in the case of the fourth treatment, replacement of N(2) with He, it was shown that the normal entry of label from exogenous (14)CO(2) into the nodule amino acid pool was strongly inhibited. PMID- 16660747 TI - Osmotic regulation: physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase. AB - When cells of Poterioochromonas malhamensis Peterfi are exposed to media of increased osmotic strength, both the internal pool of isofloridoside, and activity in homogenates of isofloridoside-phosphate synthase increase, proportional to the degree of osmotic stress. During the first few minutes of exposure of cells to higher osmolalities, an early relatively small increase in enzyme activity was observed. At the same time a progressive activation of the enzyme in homogenates was noted, providing bovine serum albumin had been omitted from the homogenizing buffer. This in vitro activation was also proportional to the degree of prior osmotic stress, was more pronounced in the presence of fluoride, and was inhibited strongly by adding bovine serum albumin or other proteins. Since earlier work had demonstrated activation of the synthase by adding exogenous proteases, it is likely that this in vitro activation was due to protease activity in the homogenate. The presumed protease must have acquired activity in the cells in response to osmotic stress, and is likely to be responsible for the observed in vivo activation of this biosynthetic enzyme.Between 60 and 90 minutes after increasing the medium osmolarity the isofloridoside pool in cells approached a high steady-state level. About this time it was observed that isofloridoside-phosphate synthase activity passed transiently through a much higher level than before, and a higher molecular weight form of the active enzyme could be observed on gel filtration chromatography. PMID- 16660748 TI - Freeze Preservation of Somatic Embryos and Clonal Plantlets of Carrot (Daucus carota L). AB - Cell suspensions of carrot (Daucus carota L.) can be cryopreserved by slow freezing (about 2 C per minute) in medium containing dimethylsulfoxide as a cryoprotectant. After storage in liquid nitrogen and thawing they demonstrate a high viability and are able to resume growth. Such a method entirely fails to preserve clonal plantlets; somatic embryos cease organized development at the time of freezing and recover growth only by secondary embryogenesis. Modification of the procedure, involving the removal of superficial moisture from cryoprotectant-treated embryos and plantlets and enclosing them in a foil envelope before freezing, greatly improves their survival potential. The use of dimethylsulfoxide at levels between 2.5 and 20% (v/v) and freezing at rates between 1 and 5 C per minute yielded viable preparations under appropriate thawing conditions. In general, treatments which increased tissue dehydration before or during freezing were most successful when followed by relatively slow thawing. Conversely where dehydration to a lesser degree was achieved, more rapid thawing was advantageous. Postthawing washing or inoculation into liquid media was inhibitory to recovery. On semisolid regrowth medium, somatic embryos resumed normal development, whereas in plantlets the root and shoot meristem regions gave rise to new growth. In both cases, inclusion of activated charcoal in the medium promoted organized growth. PMID- 16660750 TI - Effect of water vapor pressure on the thermal decomposition of 2 chloroethylphosphonic Acid. AB - Decomposition of 2-chloroethylphosphonic acid (Ethephon) was studied in dried films at various water vapor pressures (0.6 to 86.9 millimeters Hg, 3.2 to 93.9% relative humidity) and temperatures (20, 30, 40, and 50 C) at pH 6.3 and 7.0. The rate of decomposition could be determined equally well by [(14)C]Ethephon or ethylene measurements. The rate increases at increasing water vapor pressures at a constant temperature and pH, up to an optimum. The optimum vapor pressure for decomposition approximately doubles for each 10 C increase. The activation energy for the decomposition reaction in water vapor pressures of 3.2 to 12 millimeters Hg is 8.7 and 14.3 kilocalories per mole at pH 6.3 and 7.0, respectively.Decomposition of Ethephon is inhibited above an optimum vapor pressure. The inhibition is stronger at lower temperatures and at pH 6.3 than at pH 7.0. The rate of decomposition and the inhibition observed at a low temperature (20 C) was found to be similar on various surfaces, including olive leaves.Failure to induce olive (Olea europaea L.) fruit abscission under certain environmental conditions can be readily attributed to rapid breakdown of Ethephon at elevated temperatures and low relative humidities. PMID- 16660749 TI - Isolation and enzymic characterization of euglena proplastids. AB - Organelles were isolated from dark-grown Euglena gracilis Klebs by sucrose density gradient centrifugation. Plastids, identified by triosephosphate isomerase and NADP glyoxylate reductase were present at an equilibrium density of 1.24 grams per cubic centimeter clearly separated from mitochondria at an equilibrium density of 1.22 grams per cubic centimeter. Assay for choline phosphotransferase and glucose-6-phosphatase showed that endoplasmic reticulum membranes were present at a density of 1.12 grams per cubic centimeter. The plastid fraction contained phosphofructokinase, pyruvate kinase, triosephosphate isomerase and aldolase indicating the operation of a glycolytic pathway. During regreening pyruvate kinase and phosphofructokinase in the developing proplastid decreased, neither enzyme being present in the mature chloroplast. However, plastids were present in the photosynthetic cell as shown by a peak of glycolysis enzymes at an equilibrium density of 1.24 grams per cubic centimeter.The integrity of isolated plastids was demonstrated by their capacity for protein synthesis. Plastids isolated from dark-grown cells rapidly incorporated [(35)S]methionine into protein with an absolute dependence on added ATP. The large subunit of ribulose diphosphate carboxylase was the major polypeptide synthesized by these isolated plastids. PMID- 16660751 TI - Allantoin and Allantoic Acid in Tissues and Stem Exudate from Field-grown Soybean Plants. AB - Samples of stem exudate and plant tissue collected from field-grown soybean (Glycine max [L.] Merr.) plants were analyzed for allantoin and allantoic acid. Nitrogen in nitrate plus amino acids exceeded ureide N concentration in stem exudate prior to flowering. During all of reproductive development (from about 40 days after planting until maturity), ureide N concentration was two to six times greater than amino acid plus nitrate N concentration. Allantoin and allantoic acid, not asparagine, are the principal forms of nitrogen transported from nodulated roots to shoots of the soybean plant. During pod and seed development ureide N comprised as high as 2.3, 37.7, and 15.8% of total N in leaf blades, stems + petioles, and fruits, respectively. The concentration of ureide in stems and fruits declined to nearly zero at maturity.Maximum ureide concentration of exudate collected from soybean nodules was 5.3 milligrams N per milliliter (94 micromoles allantoin per milliliter). This result supports evidence published by others that the site of allantoin synthesis is the nodule. PMID- 16660752 TI - Effects of the herbicide san 9789 on photomorphogenic responses. AB - The herbicide, 4-chloro-5-(methylamino)-2-(alpha,alpha,alpha-trifluoro-m-tolyl)- 3(2H)-pyridazinone (San 9789), an inhibitor that prevents both carotenoid and chlorophyll accumulation and normal chloroplast development in white light, does not affect the physiological effectiveness of phytochrome in dark-and light-grown plants. Red/far red reversibility of growth inhibition, stimulation of anthocyanin synthesis, and stimulation of phenylalanine ammonia-lyase synthesis are not significantly different in plants grown with and without San 9789. Despite the complete absence of photosynthesis, flowering could be induced in the long day plant Hordeum vulgare L. when sucrose was provided to the leaves. Since the nonphotochemical reactions of phytochrome also are not affected by the herbicide, San 9789 may be used as a tool to study the phytochrome system spectrophotometrically in plants grown for relatively long periods under high intensity white light. PMID- 16660753 TI - Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening. AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5 bisphosphate carboxylase decreased steadily during fruit development from 23 +/- 8 nmoles per minute per milligram protein at the mature green stage to 13.4 +/- 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio of carboxylase to oxygenase activity, which was about 10. Catalase activity reached a maximum during the climacteric, simultaneously with increased ethylene and CO(2) formation. Glycolate oxidase activity decreased during early stages of development and was barely detectable at the climacteric. Hydroxypyruvate reductase, associated with serine formation by the glycerate pathway, increased in specific activity during early stages of tomato fruit ripening. In the fruit of the rin tomato mutant, which does not ripen normally, none of these changes in enzyme activity occurred. PMID- 16660754 TI - Substrate specificity for the synthesis of cyclic Fatty acids by a flaxseed extract. AB - 12-Oxo-cis-10,15-phytodienoic acid is an enzymic product obtained from incubations of (9, 12, 15)-linolenic acid with extracts of flaxseed (Linum usitatissimum L.). 13-l-Hydroperoxy-cis-9, 15-trans-11-octadecatrienoic acid, a product of lipoxygenase catalysis, was an intermediate in the enzymic synthesis of 12-oxo-cis-10, 15-phytodienoic acid from (9, 12, 15)-linolenic acid. Substrate specificity studies showed that n-3,6,9 unsaturation was an absolute requirement for conversion of polyunsaturated fatty acids into analogous products containing a cyclopentenone ring. Fatty acids with 18, 20, or 22 carbons that satisfied this requirement were effective substrates. The optimum activity of the enzyme from flaxseed was at pH 7.2. PMID- 16660755 TI - Cell Cycle-related and Endogenously Controlled Circadian Photosynthetic Rhythms in Euglena. AB - The data presented for three strains of Euglena gracilis corroborate previous reports of a diel rhythm in photosynthetic capacity in division-synchronized cultures of this alga and extend these studies to free running, dividing and nondividing (stationary) cultures maintained in either 24-hour or 40-minute cycles of light and darkness. During synchronous growth entrained by LD: 12,12 or free running under LD: 1/3,1/3, photosynthetic CO(2) fixation was rhythmic with a period (24.0 or about 30 hours) corresponding to the period of the cell division rhythm in the population. Furthermore, the rhythm in CO(2) fixation (per cell) found in nondividing cultures maintained in LD: 12,12 persisted in LD: 1/3,1/3 for weeks with a free running, circadian period of approximately 30 hours. An endogenous, circadian rhythm in cellular chlorophyll was found to exist, independently of cell division, under both light regimens and in each individual experiment; this observation could reflect changes in the functional role of the pigment. In cultures maintained in LD: 1/3,1/3, the phase relationship between the rhythm of photosynthetic capacity and that of chlorophyll content varied, suggesting the possibility of desynchronization among circadian rhythms in a multioscillator, unicellular organism. PMID- 16660756 TI - Effects of filipin and steroids on phytochrome pelletability. AB - Red light given to dark-grown etiolated leaves of Hordeum vulgare L. in vivo or to crude homogenates increases the phytochrome content of the 20,000 g pellet on centrifugation. The steroids cholesterol and stigmasterol inhibit this red light induced phytochrome pelletability. Filipin (a polyene antibiotic, which is known to combine with steroids) inhibits red light-induced phytochrome pelletability. Filipin and steroids at the appropriate concentration applied together prevent the inhibition caused by either when applied alone. These results suggest that phytochrome may bind to a steroid component of membranes. The phospholipid phosphatidyl choline dipalmitoyl has no effect on red light-induced phytochrome pelletability. Preliminary evidence demonstrates a direct association of soluble phytochrome in its active form and steroids. The physiological significance of red light-induced pelletability and the primary mechanism of phytochrome action are discussed in terms of a hypothetical steroid-binding site. PMID- 16660757 TI - Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems. AB - Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V(max) for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals. PMID- 16660758 TI - Isolation and Partial Characterization of Transfer RNAs from Astragalus bisulcatus. AB - A procedure has been developed for the isolation of transfer RNA from the selenium accumulator plant Astragalus bisulcatus. This material appears free of interfering phenolic compounds, has a high guanosine to cytidine ratio, shows a major and modified nucleoside composition characteristic of plant transfer RNAs, and exhibits chromatographic and electrophoretic properties similar to transfer RNAs from other well studied bacterial and plant systems. RNAs isolated from A. bisulcatus seedlings incubated in the presence of (75)Se indicate some incorporation of radioactivity into the transfer RNAs, but at extremely low levels. The transfer RNAs were active in accepting amino acids, although their over-all levels of activity appeared low when compared with those from a homologous Escherichia coli aminoacylation reaction system. PMID- 16660759 TI - Proline Accumulation in Water-stressed Barley Leaves in Relation to Translocation and the Nitrogen Budget. AB - Mobilization of N from leaves of barley (Hordeum vulgare L.) during water stress, and the role of proline as a mobilized species, were examined in plants at the three-leaf stage. The plants responded to water stress by withdrawing about 25% of the total reduced N from the leaf blades via phloem translocation. Most of this N loss was during the first 2 days while translocation of (14)C photosynthate out of the stressed blade still remained active. Free proline accumulation in the blade was initially slow, and became more rapid during the 2nd day of stress. Although a major free amino acid, proline accounted for only about 5% of the total N (soluble + insoluble) retained in severely stressed blades. When the translocation pathway in water-stressed leaves was interrupted just below the blade by a heat girdle, a cold jacket, or by blade excision, N loss from the blade was prevented and proline began to accumulate rapidly on 1st day of stress. Little free proline accumulated in the blades until after the ability to translocate was lost. Proline was, however, probably not a major species of N translocated during stress, because proline N accumulation in heat girdled stressed leaves was five times slower than the rate of total N export from intact blades. PMID- 16660760 TI - Effect of Growth Temperature on the Lipid and Fatty Acid Composition, and the Dependence on Temperature of Light-induced Redox Reactions of Cytochrome f and of Light Energy Redistribution in the Thermophilic Blue-Green Alga Synechococcus lividus. AB - The thermophilic blue-green alga Synechococcus lividus was grown at 55 and 38 C. Arrhenius plots of the transient reduction of cytochrome during actinic illumination with light that excited both pigment systems revealed breaks near 43 and 26 C for cells grown at 55 C. In cells grown at 38 C these breaks occurred near 37 and 28 C, respectively. The shift from pigment state 1 to state 2 measured by fluorescence transients also showed characteristic breaks in the Arrhenius plots at 44 C for cells grown at 55 C and at 37 to 38 C and possibly at 25 C for cells grown at 38 C. The break points in the Arrhenius plots for the state shift as well as for the cytochrome f reduction are discussed in relation to phase transitions of thylakoid membrane lipids as studied by the temperature dependence of chlorophyll a fluorescence.The variations of fatty acid composition with growth temperature was also studied. When the growth temperature was lowered from 55 to 38 C, the amount of the saturated fatty acid 18:0 in the negatively charged lipids sulfoquinovosyl diglyceride and phosphatidyl glycerol decreased while the unsaturated fatty acids 18:1 and 16:1 increased. In mono- and digalactosyl diglycerides the saturated fatty acids 18:0 and 16:0 decreased and the unsaturated fatty acid 16:1 increased. In general there was an increase in the more fluid lipids in all of the lipid classes when the cells were grown at the lower temperature. PMID- 16660761 TI - Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves. AB - Spinach (Spinacia oleracea L.) leaf discs accumulated free proline when exposed to polyethylene glycol solutions of water potential less than -10 bars. At -20 bars, the accumulation was 11 micromoles per gram original fresh weight in a 24 hour period.When the leaf organelles were separated on a sucrose gradient, a proline oxidase was detected in the mitochondrial fraction. Isolated mitochondria were used for the study of the properties of the enzyme which was assayed by both oxygen uptake measurement and reduction of 2,6-dichlorophenol-indophenol in the presence of phenazine methosulfate. There was a stoichiometry of one-half mole of oxygen uptake per mole of Delta(1)-pyrroline-5-carboxylate production in the enzymic reaction. The enzyme had an optimal activity at pH 8.0 to 8.5 and an apparent K(m) value of 0.028 molar for proline. MgCl(2) and flavin adenine dinucleotide were required for maximal activity. Addition of sucrose, mannitol, or polyethylene glycol to reduce the water potential of the reaction mixture to as low as -20 bars resulted in little inhibition. The enzyme preparation was unable to reduce NAD to NADH, and NAD did not inhibit the enzyme activity. The enzyme preparation reduced cytochrome c in the presence of KCN. Triton X-100 at low concentration strongly inhibited the enzyme activity. The enzyme was apparently linked to the mitochondrial electron transport system. The in vitro activity of the enzyme under optimal assay conditions was high enough to prevent proline accumulation under water stress condition; presumably this activity was restrained in vivo. PMID- 16660762 TI - Identification of Traumatin, a Wound Hormone, as 12-Oxo-trans-10-dodecenoic Acid. AB - 12-Oxo-trans-10-dodecenoic acid (trans-10-ODA) is an oxidation product of polyunsaturated fatty acids in plant tissues. The structural similarity of trans 10-ODA and traumatic acid, a compound considered to be a wound hormone, suggested that trans-10-ODA might be a precursor of traumatic acid. Both trans-10-ODA and traumatic acid were active in the Wehnelt bean assay. The results were more consistent with trans-10-ODA than with traumatic acid. Cucumber (Cucumis sativus L. var. National Pickling) hypocotyls also showed a growth increase following treatment with trans-10-ODA, which suggested that trans-10-ODA has a more general influence on plant development than previously ascribed to traumatic acid.Runner beans (Phaseolus vulgaris L. var. Kentucky Wonder) were analyzed for the presence of endogenous trans-10-ODA and traumatic acid. These are the beans from which traumatic acid was originally isolated in 1939. They contained trans-10-ODA but no traumatic acid. Young beans were a better source of trans-10-ODA than older beans and an increase in the esterified form of trans-10-ODA with age may have been due to a conversion of the free acid to the esterified form. The amount of endogenous trans-10-ODA increased when bean pod tissue was sliced and wounded. Rapid stirring and the presence of oxygen increased autooxidation of trans-10-ODA to traumatic acid in runner beans, which indicated that the compound identified as traumatic acid is formed by autooxidation of trans-10-ODA and that trans-10 ODA is a natural compound with growth-regulating properties.Enzyme extracts of runner beans synthesized trans-10-ODA from linoleic acid. No enzymic synthesis of traumatic acid was observed even when cofactors were added to the reaction mixture. This confirmed the conclusion that traumatic acid is formed by autooxidation of trans-10-ODA. PMID- 16660763 TI - Ontogeny of Photosynthetic Performance in Fragaria virginiana under Changing Light Regimes. AB - Apparent photosynthesis and dark respiration were followed during development in four light environments of leaves of Fragaria virginiana Duchesne. Leaf expansion was completed more rapidly the higher the growth photon flux density and leaves senesced more quickly in high light. Maximum photosynthetic capacity coincided with the completion of blade expansion and declined quickly thereafter. Leaves were transferred from high to low and low to high photon flux densities at several stages during expansion. Leaf photosynthetic performance and anatomy were subsequently analyzed. Leaf anatomy and apparent photosynthesis per unit dry weight can be modified during expansion to reflect the predominant light conditions. Adaptive potential is greatest early in blade expansion and decreases as expansion is completed. PMID- 16660764 TI - Effects of Light and 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea on Levels of ATP in Lemna paucicostata 6746 and a Photosynthetic Mutant with Abnormal Flowering Responses. AB - The effects of light, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and ammonium ion on pool sizes of ATP were studied in Lemna paucicostata 6746 (wild type) and a photosynthetic mutant (strain 1073) with abnormal flowering responses. Wild type fronds were capable of endogenous and phenazine methosulfate catalyzed cyclic photophosphorylation. The endogenous cyclic photophosphorylation was inhibited by DCMU. The mutant fronds showed little endogenous but appreciable rates of phenazine methosulfate-catalyzed cyclic photophosphorylation. Treatment with DCMU during prolonged exposure to light did not result in elevated levels of ATP. Ammonium ion in the medium did not inhibit light-induced increases in pool sizes of ATP. It is concluded that the previously reported effects on flowering of DCMU, the photosynthetic mutation or ammonium ion, were not due to altered pool sizes of ATP. PMID- 16660765 TI - Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L. AB - Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. ;Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in ;Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit. PMID- 16660766 TI - Linkage Analysis of Hydroxyproline-poor Glycoprotein from Phaseolus vulgaris. AB - Hydroxyproline-poor glycoprotein contains a single polypeptide chain with lysine at the N-terminus. Removal of carbohydrate attached to serine by alkali treatment produces two polypeptide fractions. Labeling with (35)S indicates that most serine residues having a carbohydrate substituent removed by alkali occur on the polypeptide fraction of lower molecular weight. Following alkali treatment, two additional N-terminal amino acids, proline and glycine, were detected suggesting that alkali treatment also cleaves peptide bonds. Methylation analysis of native and degraded glycoproteins, extracted 24, 27, and 36 hours after wounding, demonstrates the following structural features of carbohydrate attached to serine. Arabinose may be (1 --> 2)-, (1 --> 3)-, or (1 --> 5)-linked, glucose occurs as a chain of beta-(1 --> 4)-linked residues, and galactose occurs as a nonreducing terminal unit. PMID- 16660767 TI - Metalloenzyme inhibitor from kidney beans: partial purification and characterization. AB - Inhibitory activity directed against metalloenzymes has been highly purified from extracts of red kidney beans (Phaseolus vulgaris). The inhibitor is a substance of small molecular weight and appears to be a chelator of Zn(2+). One milligram of the preparation inhibited 23 milligrams carboxypeptidase A. The inhibitor also strongly inhibited carboxypeptidase B and alkaline phosphatase and could activate phosphoglucomutase that had previously been inactivated with Zn(2+). The isoelectric point of the inhibitor is 4.7. The inhibitor activity was abolished by preincubation with Zn(2+), Ni(2+), Co(2+), or Cu(2+). The mechanism of inhibition of carboxypeptidases and alkaline phosphatase by the bean inhibitor is apparently due to the complexing and complete removal of Zn(2+) from the enzymes. PMID- 16660768 TI - Changes in Pool Sizes of Free Amino Acids and Amides in Leaves and Plastids of Zea mays during Leaf Development. AB - The concentrations of free amino acids and amides within isolated maize (Zea mays L.) plastids were determined and compared with concentrations in the leaf tissue. The concentrations were different for each individual amino acid and varied between 1 and 10 millimolar. At five different developmental stages concentrations in the plastids were greater than those in the intact leaf tissue. During development, from the proplastid stage to the mature chloroplast, the amount of each amino acid per plastid remained relatively constant, but there were decreases in concentrations of plastid amino acids resulting from the developmental increase in plastid volume. In proplastids, the free amino acids were present in greater concentrations than those previously found to inhibit partially amino acid-synthesizing enzymes located in chloroplasts. In the chloroplasts, the molarities of the free amino acids were within the range known to inhibit amino acid-synthesizing enzymes. PMID- 16660769 TI - Partial Characterization of a Potassium-stimulated Adenosine Triphosphatase from the Plasma Membrane of Meristematic and Mature Soybean Root Tissue. AB - A K(+)-stimulated adenosine triphosphatase was partially characterized in plasma membrane from meristematic and mature soybean root tissue. The substrate concentrations required for maximum enzyme activity (3 millimolar Mg.ATP) and pH optimum (6.5) were similar for both systems. Enrichment studies, performed to ensure that the membrane vesicle preparations were comparable, indicated similar purity levels at selected steps during purification. Phospholipid and sterol analyses further substantiated their similarity.Enzyme studies revealed significantly greater ATPase activity, per unit membrane protein, in the meristematic region. Mixing experiments indicated that the lower level of activity associated with vesicles from mature tissue was not due to endogenous inhibitor(s). PMID- 16660770 TI - Glutamine Synthetase/Glutamine: alpha-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula. AB - The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways. Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 mumoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):alpha ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 mumoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low K(m) for ammonia which has been described previously in these chloroplasts provides the second potential pathway. PMID- 16660771 TI - Derepression and Repression of Lysine-sensitive Aspartokinase during in Vitro Culture of Carrot Root Tissue. AB - The increment of lysine-sensitive aspartokinase (EC 2.7.2.4) activity during in vitro culture of carrot (Daucus carota, cv. Oogata sanzun) root tissue was explained in terms of derepression caused by an earlier decrease in the endogenous level of lysine, a possible end product repressor. Tissue content of free lysine decreased to about one-third of the initial level after 1 day of culture and no lysine was detected in the 2nd day. Inclusion of lysine (0.1 to 1.0 millimolar) in the culture medium resulted in a specific suppression of increase in lysine-sensitive aspartokinase activity without affecting the increase in threonine-sensitive aspartokinase activity. PMID- 16660772 TI - Amino Acid levels in carrot cell suspension culture: no correlation with aspartate kinase isoenzyme levels. AB - Free and protein amino acids were analyzed during the growth cycle of a suspension culture of carrot (Daucus carota L.) in which there is a 10-fold increase in a lysine-sensitive isoenzyme of aspartate kinase during the early part of the growth cycle. There is little change in either total amino acids or in the amino acids derived from aspartate between 3 and 24 days of culture. It is estimated that the demand for net synthesis of aspartate-derived amino acids is decreased as the growth rate declines and that there is no correlation between the amount of extractable lysine-sensitive aspartate kinase and the amounts of amino acids being synthesized. PMID- 16660773 TI - Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid. AB - Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction. PMID- 16660774 TI - Citrate and succinate uptake by potato mitochondria. AB - The uptake of [(14)C]citrate and [(14)C]succinate was studied in potato mitochondria (Solanum tuberosum var. Russet Burbank) using cellulose pore filtration and was found to occur by the same mechanisms as described for mammalian mitochondria. Potato mitochondria, in the absence of respiration, have a very low capacity for uptake by exchange with endogenous anions, taking up only 2.4 nanomoles citrate and 2.0 nanomoles succinate per milligram protein. Maximum citrate uptake of over 17 nanomoles per milligram protein occurs in the presence of inorganic phosphate, a dicarboxylic acid, and an external energy source (NADH), conditions where net anion accumulation proceeds, mediated by the interlinking of the inorganic phosphate, dicarboxylate, and tricarboxylate carriers. Maximum succinate uptake in the absence of respiratory inhibitors requires only added inorganic phosphate.Compounds which inhibit respiration (antimycin), the exchange carriers (mersalyl and benzylmalonate), or the establishment of the membrane proton motive force (uncouplers) reduce substrate accumulation. A potent inhibitor of the citrate carrier in animal mitochondria, 1,2,3-benzenetricarboxylic acid, does not inhibit citrate uptake in potato mitochondria. Citrate uptake is reduced by concurrent ADP phosphorylation and this reduction is sensitive to oligomycin. The initiation of state 3 after a 3 minute substrate state results in a reduction of the steady-state of citrate uptake by approximately 50%. Accumulation of succinate initially is inhibited by increasing sucrose concentration in the reaction medium from 50 to 400 millimolar.Limited substrate uptake is one of the factors responsible for the often observed depressed initial state 3 respiration rates in many mitochondrial preparations. Since nonlimiting levels of substrate in the matrix cannot be attained by energy-independent exchange, a dependence on respiration for adequate uptake results. Substrate limitation therefore occurs in the matrix for the period of time needed for energy-dependent accumulation of nonlimiting levels. PMID- 16660775 TI - Effects of cryoprotectants in combination on the survival of frozen sugarcane cells. AB - Cryoprotection of suspension cultures of sugarcane cells (Saccharum sp.) during freezing to various temperatures was tested using glucose, dimethylsulfoxide, and ethylene glycol at various concentrations, alone and in combinations. Viability of the thawed cells was assessed by triphenyltetrazolium chloride reduction, cell growth, and microscopic examination. Enhanced cryoprotection-as much as a doubling in viability value-was achieved by employing glucose and dimethylsulfoxide in mixtures, as compared with the lesser cryoprotective effect of either compound alone, at 1.9 molar total concentration in all cases; the mixture was most effective at a concentration of about 1.9 molar, with a molar ratio of the two components of about 1:3, respectively. Much of the increase in viability value arose from a decrease in toxic effect that came about through mixing the cryoprotective agents. Binary mixtures containing ethylene glycol and either glucose or dimethylsulfoxide were less effective and more toxic than comparable glucose-dimethylsulfoxide mixtures. Use of the optimized latter mixture allowed freezing of these tropical cells to -23 C with little decrease in survival, or to -40 C, still with the capability for delayed growth. PMID- 16660776 TI - Effects of disalicylidenepropanediamines on photosynthetic electron transport of isolated spinach chloroplasts. AB - The effects of disalicylidenepropanediamine (DSPD) and disulfo disalicylidenepropanediamine (sulfo-DSPD) on the photosynthetic electron transport of isolated chloroplasts have been reexamined.Our data suggest that DSPD, but not sulfo-DSPD, is an effective inhibitor of electron transport between photosystem II and photosystem I before or at plastocyanin. Furthermore, both DSPD and sulfo-DSPD block electron transport at the site of ferredoxin.Under certain conditions DSPD and even more so sulfo-DSPD function as autooxidizable electron acceptors.Finally it is shown that DSPD can cause an inhibition of photophosphorylation.According to our results the use of DSPD as a specific inhibitor of ferredoxin-dependent reactions has to be questioned. PMID- 16660777 TI - Role of auxin and gibberellin in differentiation of primary Phloem fibers. AB - The hypothesis that auxin and gibberellic acid (GA(3)) control the differentiation of primary phloem fibers is confirmed for the stem of Coleus blumei Benth. Indoleacetic acid (IAA) alone sufficed to cause the differentiation of a few primary phloem fibers. In long term experiments auxin induced a considerable number of fibers in mature internodes. GA(3) by itself did not exert any effect on fiber differentiation. Combinatiosn of IAA with GA(3) completely replaced the role of the leaves in primary phloem fiber differentiation qualitatively and quantitatively. Although the combined effect of the two growth hormones diminished considerably with increasing distance from the source of induction, auxin with GA(3) or IAA alone induced fibers in a few internodes below the application site. When various combinations of both hormones were applied, high concentrations of IAA stimulated rapid differentiation of fibers with thick secondary walls, while high levels of GA(3) resulted in long fibers with thin walls. The size of the primary phloem fibers correlated with the dimensions of the differentiating internode, thereby providing evidence that both growth regulators figure in the control of stem extension. High IAA/low GA(3) concentrations have an inhibitory effect on internode elongation, whereas low IAA/high GA(3) concentrations promote maximal stem elongation. PMID- 16660778 TI - Phycobilisomes from blue-green and red algae: isolation criteria and dissociation characteristics. AB - A general procedure for the isolation of functionally intact phycobilisomes was devised, based on modifications of previously used procedures. It has been successful with numerous species of red and blue-green algae (Anabaena variabilis, Anacystis nidulans, Agmenellum quadruplicatum, Fremyella diplosiphon, Glaucosphaera vacuolata, Griffithsia pacifica, Nemalion multifidum, Nostoc sp., Phormidium persicinum, Porphyridium cruentum, P. sordidum, P. aerugineum, Rhodosorus marinus). Isolation was carried out in 0.75 molar K-phosphate (pH 6.8 to 7.0) at 20 to 23 C on sucrose step gradients. Lower temperature (4 to 10 C) was usually unfavorable resulting in uncoupling of energy transfer and partial dissociation of the phycobilisomes, sometimes with complete loss of allophycocyanin. Intact phycobilisomes were characterized by fluorescence emission peaks of 670 to 675 nanometers at room temperature, and 678 to 685 nanometers at liquid nitrogen temperature. Uncoupling and subsequent dissociation of phycobilisomes, in lowered ionic conditions, varied with the species and the degree of dissociation but occurred preferentially between phycocyanin and allophycocyanin, or between phycocyanin and phycoerythrin. PMID- 16660779 TI - Amino Acid Uptake into Cultivated Mesophyll Cells from Asparagus officinalis L. AB - The uptake of threonine, aspartic acid, and isoleucine into cultivated asparagus cells was examined under culture conditions.Transport was linear with time and with the number of cells present in the assay. The upake of the three amino acids obeyed Michaelis-Menten kinetics with apparent affinities in the range of 0.1 to 0.02 millimolar. Azide and 2,4-dinitrophenol caused inhibition of threonine uptake. Reciprocal competitive inhibitions were found between threonine, aspartic acid, and isoleucine. Methionine and lysine competitively inhibited the uptake of these three amino acids. These results and the comparison of apparent affinities and inhibition constants are in favor of only one uptake system for threonine, aspartic acid, isoleucine, methionine, and lysine with different affinities for the different amino acids. All other amino acids with the exception of histidine, arginine, asparagine, and hydroxyproline inhibited threonine uptake. It is most likely that at least one component of the uptake system is shared by all amino acids. PMID- 16660780 TI - A Nuclear Magnetic Resonance Study of Water in Cold-acclimating Cereals. AB - Continuous wave nuclear magnetic resonance (NMR) studies indicated that the line width of the water absorption peak (Deltav(1/2)) from crowns of winter and spring wheat (Triticum aestivum L.) increased during cold acclimation. There was a negative correlation between Deltav(1/2) and crown water content, and both of these parameters were correlated with the lowest survival temperature at which 50% or more of the crowns were not killed by freezing (LT(50)). Regression analyses indicated that Deltav(1/2) and water content account for similar variability in LT(50). Slow dehydration of unacclimated winter wheat crowns by artificial means resulted in similarly correlated changes in water content and Deltav(1/2). Rapid dehydration of unacclimated crowns reduced water content but did not influence Deltav(1/2). The incubation of unacclimated winter wheat crowns in a sucrose medium reduced water content and increased Deltav(1/2). The increase in Deltav(1/2) appears to be dependent in part on a reduction in water content and an increase in solutes.Longitudinal (T(1)) and transverse (T(2)) relaxation times of water protons in cereals at different stages of cold acclimation were measured using pulse NMR methods. The T(1) and T(2) signals each demonstrated the existence of two populations of water, one with a short and one with a long relaxation time. During the first 3 weeks of acclimation, the long T(2) decreased significantly in winter-hardy cereals, and did not change in a spring wheat until the 5th week of hardening. There was no change in the long T(1) until the 3rd week of hardening for the winter cereals and until the 7th week of hardening for the spring wheat. No simple relationship could be established between T(1) or T(2) and cold hardiness. Neither continuous wave or pulsed NMR spectroscopy can be used as a diagnostic tool in predicting the cold hardiness of winter wheats. An increase in Deltav(1/2) or a reduction in relaxation times does not provide evidence for ordering of the bulk of the cell water. PMID- 16660781 TI - Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter. AB - Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [(35)S]SO(4) (2-) accumulation. Maximum uptake is secured in the presence of Mg(2+) and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [(35)S]SO(4) (2-) uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter. PMID- 16660782 TI - Methionine metabolism and ethylene formation in etiolated pea stem sections. AB - Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of l-[U-(14)C]methionine and, on the following morning, on 0.1 millimolar indoleacetic acid to induce ethylene formation. Following the overnight incubation, over 70% of the radioactivity in the soluble fraction was shown to be associated with S-methylmethionine (SMM). The specific radioactivity of the ethylene evolved closely paralleled that of carbon atoms 3 and 4 of methionine extracted from the tissue and was always higher than that determined for carbon atoms 3 and 4 of extracted SMM.Overnight incubation of pea stem sections on 1 millimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 millimolar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 millimolar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in l-[U (14)C]methionine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly. PMID- 16660783 TI - Indirect Action of Benzyladenine and Other Chemicals on Flowering of Pharbitis nil Chois: Action by Interference with Assimilate Translocation from Induced Cotyledons. AB - Benzyladenine (BA) brushed on the cotyledons of 4-day-old seedlings of Pharbitis nil Chois. markedly stimulates flowering. Greates response is obtained for concentrations between 44 and 440 micromolar. The action of BA is on processes in the cotyledon as shown by the response to its site of application, to the dosage applied and to the requirement for its application prior to the dark period. There was little or no effect of BA treatment on either the time measurement processes of photoperiodic induction or on the generation of floral stimulus. Transport of photosynthetic assimilate from the cotyledons to the shoot apex was altered.When only one of the pair of cotyledons was treated with BA it exported less (14)C-labeled assimilate to the shoot apex and there was a compensatory increase in assimilate outflow from the other cotyledon. When BA was applied to a cotyledon exposed to an inductive dark period, flowering was inhibited in association with the reduced export of assimilate. Conversely, when BA was applied to the noninduced cotyledon, flowering was promoted in association with an enhanced export of assimilate from the induced leaf. Clearly, cytokinins can have an indirect effect on photoperiodic induction by altering assimilate and, hence, floral stimulus translocation to the shoot apex.Two other chemicals which were previously considered as specific inhibitors of processes of floral induction in the cotyledon [Tris(2-diethylaminoethyl)phosphate trihydrochloride (SK&F 7997-A(3)) and cycloheximide] acted in the same manner as BA. Inhibitory effects of an illuminated cotyledon on flowering of Pharbitis were also shown to be mediated by interference of assimilate flow with transport of the stimulus for flowering. PMID- 16660784 TI - Photosynthetic carbon metabolism in seagrasses C-labeling evidence for the c(3) pathway. AB - The delta(13)C values of several seagrasses were considerably less negative than those of terrestrial C(3) plants and tended toward those of terrestrial C(4) plants. However, for Thalassia hemprichii (Ehrenb.) Aschers and Halophila spinulosa (R. Br.) Aschers, phosphoglycerate and other C(3) cycle intermediates predominated among the early labeled products of photosynthesis in (14)C-labeled seawater (more than 90% at the earliest times) and the labeling pattern at longer times was brought about by the operation of the C(3) pathway. Malate and aspartate together accounted for only a minor fraction of the total fixed label at all times and the kinetic data of this labeling were not at all consistent with these compounds being early intermediates in seagrass photosynthesis. Pulse chase (14)C-labeling studies further substantiated these conclusions. Significant labeling of photorespiratory intermediates was observed in all experiments. The kinetics of total fixation of label during some steady-state and pulse-chase experiments suggested that there may be an intermediate pool of inorganic carbon of variable size closely associated with the leaves, either externally or internally. Such a pool may be one cause for the C(4)-like carbon isotope ratios of seagrasses. PMID- 16660785 TI - Transverse Viscoelastic Extension in Nitella: II. Effects of Acid and Ions. AB - The transverse viscoelastic extension of isolated Nitella cell walls is stimulated by acid pH and by Mg(2+) and K(+) ions. In the presence of 1 millimolar citrate-phosphate buffer the threshold pH in the transverse direction is 3.5, compared to 4.5 in the longitudinal direction. The relative amounts of extension stimulated by acid are comparable in the two directions at their respective thresholds. Longitudinal and transverse Mg(2+) ion-induced extensibility begins at 10 millimolar and reaches a plateau between 10 and 100 millimolar. The threshold for K(+) ion enhancement is near 10 millimolar in the longitudinal direction and 50 millimolar in the transverse direction. Maximum stimulation by K(+) is obtained at 250 millimolar. At their respective maxima, Mg(2+) and K(+) induce equal amounts of extension. However, the relative amount of extension induced by ions is significantly less in the transverse than in the longitudinal direction. Ions and acids appear to affect different sites in the wall, inasmuch as neither treatment abolishes the effect of the other. Walls from rapidly growing cells are more sensitive to stimulation than nongrowing cells in the longitudinal direction but not in the transverse direction. PMID- 16660786 TI - Chloroplast DNA Sequence Homologies among Vascular Plants. AB - The extent of sequence conservation in the chloroplast genome of higher plants has been investigated. Supercoiled chloroplast DNA, prepared from pea seedlings, was labeled in vitro and used as a probe in reassociation experiments with a high concentration of total DNAs extracted from several angiosperms, gymnosperms, and lower vascular plants. In each case the probe reassociation was accelerated, demonstrating that some chloroplast sequences have been highly conserved throughout the evolution of vascular plants. Only among the flowering plants were distinct levels of cross-reaction with the pea chloroplast probe evident; broad bean and barley exhibited the highest and lowest levels, respectively. With the hydroxylapatite assay these levels decreased with a decrease in probe fragment length (from 1,860 to 735 bases), indicating that many conserved sequences in the chloroplast genome are separated by divergent sequences on a rather fine scale. Despite differences observed in levels of homology with the hydroxylapatite assay, S1 nuclease analysis of heteroduplexes showed that outside of the pea family the extent of sequence relatedness between the probe and various heterologous DNAs is approximately the same: 30%. In our interpretation, the fundamental changes in the chloroplast genome during angiosperm evolution involved the rearrangement of this 30% with respect to the more rapidly changing sequences of the genome. These rearrangements may have been more extensive in dicotyledons than in monocotyledons. We have estimated the amount of conserved and divergent DNA interspersed between one another.From the reassociation experiments, determinations were made of the percentage of chloroplast DNA in total DNA extracts from different higher plants; this value remained relatively constant when compared with the large variation in the diploid genome size of the plants. PMID- 16660787 TI - Galactolipid Synthesis in Vicia faba Leaves: III. Site(s) of Galactosyl Transferase Activity. AB - Leaves of Vicia faba were fed (14)CO(2) in light for periods of up to 6 hours. At intervals, leaf samples were homogenized and separated into fractions which contained "broken" and "intact" chloroplasts, and three other high speed centrifugal fractions containing other cell membranes and chloroplast envelopes. Analyses of the radioactive labeling of galactose from the galactolipids in these fractions and in purified chloroplast envelopes indicated that the major site of galactosyl transferase enzyme activity was in the chloroplast envelope. The data suggest that in time much of the radioactive galactolipid was transferred from the envelope to the thylakoid-containing fractions. The major site of galactolipid synthesis appears to be in the envelope but there is some evidence of another site in the thylakoids. PMID- 16660788 TI - Galactolipid Synthesis in Vicia faba Leaves: IV. Site(s) of Fatty Acid Incorporation into the Major Glycerolipids. AB - The fatty acids of the major glycerolipids of Vicia faba leaves were analyzed immediately following (14)CO(2) feeding. The leaves were fractionated into chloroplast and cytoplasmic fractions and the location of radioactivity in the fatty acids determined. The results indicate that the major site of incorporation of fatty acids is in the phospholipids. Phosphatidylcholine contained the highest level of radioactivity in the cytoplasmic fraction, whereas phosphatidylglycerol contained radioactivity in both the chloroplast and cytoplasmic fractions. The galactolipids contained very little radioactivity in comparison, this radioactivity being confined to high speed centrifugal fractions believed to contain the envelopes of the chloroplast. Our results suggest that phosphatidylcholine is a major site of incorporation of fatty acids (mainly in oleic acid) in the cytoplasm, whereas phosphatidylglycerol is also a site of incorporation involving both oleic and palmitic acids, inside and outside the chloroplast. PMID- 16660789 TI - Effect of ethylene on plasma membrane density in kidney bean abscission zones. AB - Plasma membranes from abscission zones of kidney bean seedlings were isolated on sucrose density gradients using cellulase and KCl-stimulated ATPase as marker enzymes. Following treatment of the seedling explants with 50 microliters per liter ethylene, an increase in the buoyant density of the plasma membrane fraction from 1.165 to 1.175 grams per cubic centimeter was observed. Such a change is consistent with the loss of lipids from the senescent cells of the abscission zone. PMID- 16660790 TI - Characterization of the Destruction of Phytochrome in the Red-absorbing Form. AB - Both the red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome undergo destruction, defined as the loss of photoreversibly detectable chromoprotein following actinic irradiation of dark-grown tissue, in 4-day-old etiolated oat seedlings. Pr and Pfr destruction follow the same time course, exhibit the same time delay after actinic irradiation when the plants are grown in sealed containers, result in a loss of antigenically detectable phytochrome, as determined by radial immunodiffusion assay, equal to the loss of spectrophotometrically detectable phytochrome, and have the same sensitivity to 2 mercaptoethanol and azide. We suggest that Pr destruction is a consequence of the same mechanism that is responsible for Pfr destruction. PMID- 16660791 TI - Binding of ColE1-kan Plasmid DNA by Tobacco Protoplasts: Nonexpression of Plasmid Gene. AB - Protoplasts prepared from cultured tobacco cells were treated with ColE1-kan plasmid DNA, a hybrid of ColE1 and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColE1-kan in tobacco tissue. PMID- 16660792 TI - ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties. AB - ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day old seedlings. A strict requirement for coenzyme A and adenosine 5'-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (-) Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5'-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis. PMID- 16660793 TI - Two light sources differentially affected ferric iron reduction and growth of cotton. AB - In growth chambers, low pressure sodium (LPS) plus incandescent (Inc) lamps and fluorescent cool-white (FCW) plus Inc lamps were used to determine their effects on growth of cotton (Gossypium hirsutum L.) and on the reduction of Fe(3+) to Fe(2+). Cotton plants grown under LPS + Inc light developed chlorosis and grew poorly, whereas plants grown under FCW + Inc lights were green. The chlorophyll concentration and top and root weights of cotton grown under LPS + Inc were lower than those under FCW + Inc. In solution, FCW + Inc lamps reduced about eight times more Fe(3+) to Fe(2+) than did LPS + Inc lamps. Fe(3+) is transported to plant tops as Fe(3+) citrate and if we assume that FCW + Inc light reduces Fe(3+) to Fe(2+) in plant foliage as it did in the solutions, then reduction of Fe(3+) by the light environment will make Fe(2+) in the tops more available for biochemical reactions. PMID- 16660794 TI - Indole-3-acetic Acid Oxidase from Peas: I. Occurrence and Distribution of Peroxidative and Nonperoxidative Forms. AB - Tissues of etiolated pea seedlings variety Alaska were examined for the presence of peroxidative and nonperoxidative forms of indoleacetic acid (IAA) oxidase. Enzymes were extracted in a sequence involving acetone powder preparation from pea tissues, buffer extraction of the powder, ammonium sulfate precipitation, dialysis, lyophilization, and acrylamide gel electrophoresis. Electrophoretically separable proteins were assayed for IAA oxidase activity with the Salkowski test, and peroxidase activity was based on the color reaction with benzidine and H(2)O(2). Each tissue examined contained several nonperoxidative IAA oxidases. No tissue contained more than three peroxidative IAA oxidases, whereas the plumule hooks (a tissue with a high IAA oxidase activity) contained no detectable peroxidases. The results indicate that nonperoxidative IAA oxidases might play a major role in the regulation of IAA content in pea seedlings. PMID- 16660795 TI - Salinity effects on leaf anatomy: consequences for photosynthesis. AB - Increasing salinity led to substantially higher ratios of mesophyll surface area to leaf area (A(mes)/A) for Phaseolus vulgaris and Gossypium hirsutum and a smaller increase for Atriplex patula, a salt-tolerant species. The increase in internal surface for CO(2) absorption did not lead to higher CO(2) uptake rates, since the CO(2) resistance expressed on the basis of mesophyll cell wall area (r(cell)) increased even more with salinity. The differences among species in the sensitivity of photosynthesis to salinity in part reflect the different A(mes)/A and r(cell) responses. PMID- 16660796 TI - Photocontrol of the germination of onoclea spores: v. Analysis of germination processes by means of temperature. AB - The physiological nature of photoinduced germination of Onoclea sensibilis L. spores was investigated by temporarily applying a range of temperatures, particularly 40 C, before and after short light treatment. Controls were germinated at 25 C.The preinduction phase, during which photosensitivity is maximally developed in the dark, is sensitive to temperature. Treatment at 40 C for 8 or more hours reduces the developed photosensitivity to a minimal level, but the inhibition by 40 C treatment is reversed slowly after subsequent incubation at 25 C in the dark. The postinduction phase, in which dark processes lead to stain uptake and eventually to visible protrusion, is also sensitive to temperature. Inhibition by 40 C occurs shortly after photoinduction, but disappears 6 or more hours after irradiation. Postinduction spores whose germination is inhibited by 40 C treatment recover the ability to germinate after subsequent incubation at 25 C plus a second light treatment. The inhibition and recovery take place faster in postinduction spores than in preinduction spores. In addition, escape from 40 C inhibition is found in the postinduction phase but not in the preinduction phase. Temperatures lower than 25 C exert slow inhibition of both pre- and postinduction processes, and 30 to 35 C act to stimulate germination.In comparison with our earlier work with anaerobiosis and cycloheximide, the postinduction step inhibited by 40 C can be located shortly after the step inhibited by anaerobiosis but before the cycloheximide sensitive step. PMID- 16660797 TI - Plasmalemma Voltage Noise in Chara corallina. AB - Voltage noise analysis is applied to plasmalemma ion transport in Chara corallina. There is a component in the noise power spectrum that is probably associated with current fluctuations within passive transport channels, and another component that may be associated either with fluctuations in the number of open channels, or with active transport. The data allow the calculation of time constants that may be attributable to molecular level events in these transport processes. PMID- 16660798 TI - Sorbitol-6-phosphate dehydrogenase from loquat fruit. AB - Sorbitol-6-phosphate dehydrogenase was found in flesh tissue of mature fruit of the loquat (Eriobotrya japonica Lindl. var. Tanaka). The enzyme was purified about 30-fold from the crude extract of the fruit, and was demonstrated to catalyze sorbitol-6-phosphate + NADP right harpoon over left harpoon glucose-6 phosphate + NADPH. The optimal pH values for sorbitol 6-phosphate oxidation and glucose 6-phosphate reduction were 9.8 and 9.1, respectively. PMID- 16660799 TI - Variants of soybean cells which can grow in suspension with maltose as a carbon energy source. AB - Suspension cultures of soybean line SB-1 have been grown using maltose as an carbon-energy source. The very slow growth in medium containing maltose has been used to select rapidly growing variants. These appear to arise as a series of sequential genetic changes (mutations?). These variant strains are stable when grown in sucrose medium for 100 generations and appear to be able to transport maltose actively into the cell. PMID- 16660800 TI - Protoplasts surviving freezing to -196 C and osmotic dehydration in 5 molar salt solutions prepared from the bark of winter black locust trees. AB - Free protoplasts were prepared from the living bark tissue of the trunk of summer and winter black locust trees by enzymic digestion of thin slices of the tissue for 3 hours in a medium containing 2% Onozuka cellulase, 2% Rhozyme pectinase, and 2% Driselase in mannitol solutions using 0.4 molar mannitol for summer tissue and 1.0 molar mannitol for winter tissues. Cleaned suspensions of protoplasts and also thin slices of tissue with cells intact were frozen to temperatures of -10 C, -20 C, -30 C, -40 C and liquid nitrogen in sucrose and balanced salt solutions. Similar suspensions of protoplasts were also subjected to strong osmotic dehydration (plasmorrhysis) in a series of balanced salt solutions of increasing molarity. Tests for survival showed that protoplasts retain the same properties of either extreme susceptibility or extreme resistance to injury by freezing or osmotic dehydration as the cells from which they are prepared. Winter protoplasts showed capability for tolerating freezing to -196 C and plasmorrhysis in 5 molar salt solutions. These results indicate that protoplasts are a valid and useful system for investigating the properties of the protoplasm and surface membranes associated with the seasonal development of extreme hardiness in the cells of woody plants. PMID- 16660801 TI - Absence of Lipid Oxidation during Accelerated Aging of Soybean Seeds. AB - When seeds of soybean were subjected to accelerated aging, the amount of total lipid which was extracted from the whole seed increased with "age," whereas the extractable phospholipid decreased slightly. This small decline primarily reflected changes in the amounts of phosphatidylcholine and phosphatidylethanolamine. The levels of unsaturated fatty acids in the whole seed and in the seed axis showed no decline during aging. Similarly, the fatty acids in a polar lipid extract from the whole seed showed little change in unsaturation. These results suggest that oxidation of seed lipids may be unrelated to the process of seed aging. PMID- 16660802 TI - Modeling the transport and utilization of carbon and nitrogen in a nodulated legume. AB - An empirical modeling technique was developed for depicting quantitatively the transport and partitioning of photosynthetically fixed C and symbiotically fixed N during 10-day intervals of a 40-day period in the growth of nodulated plants of white lupin (Lupinus albus L. cv. Ultra). Model construction utilized data for C and N consumption of plant parts and C:N weight ratios of the xylem and phloem fluids serving specific plant organs. Formulas were derived from calculating the net transport of C and N between plant parts in xylem and phloem. The models provided quantitative information on the dependence of growing organs on xylem and phloem for their supply of C and N, the cycling of N through leaflets and of C through nodules, the extent of direct incorporation of fixed N into growing nodules, and the involvement of N from shoot translocate in the nutrition of the nodulated root. Stem plus petioles abstracted considerably more N from xylem than expected from their transpirational activity. Xylem to phloem transfer of recently fixed N in mature stem and petioles was substantiated by the models, being depicted as a device for dispensing N to growing parts of the shoot extra to that attracted transpirationally in xylem or received as translocate from leaflets. PMID- 16660803 TI - Intracellular Localization of Some Key Enzymes of Crassulacean Acid Metabolism in Sedum praealtum. AB - The intracellular locations of six key enzymes of Crassulacean acid metabolism were determined using enzymically isolated mesophyll protoplasts of Sedum praealtum D.C. Data from isopycnic sucrose density gradient centrifugation established the chloroplastic location of pyruvate Pi dikinase, the mitochondrial location of NAD-linked malic enzyme, and exclusively nonparticulate (not associated with chloroplasts, peroxisomes, or mitochondria) locations of phosphoenolpyruvate carboxylase, NADP-linked malic enzyme, enolase, and phosphoglycerate mutase. The consequences of this enzyme distribution with respect to compartmentalization of the pathway and the transport of metabolites in Crassulacean acid metabolism are discussed. PMID- 16660804 TI - Phloem loading of sucrose: involvement of membrane ATPase and proton transport. AB - p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H(+) efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H(+)/K(+) exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase. PMID- 16660805 TI - Salt Requirement for Crassulacean Acid Metabolism in the Annual Succulent, Mesembryanthemum crystallinum. AB - In experiments with the facultative Crassulacean acid metabolism (CAM) species, Mesembryanthemum crystallinum, only plants which received high levels of inorganic salts fixed substantial amounts of CO(2) by the CAM pathway. Equivalent osmolarities of polyethylene glycol 6000 did not yield any CAM fixation. Plant water potential and turgor pressure had no detectable influence on the amount of CAM fixation. These observations rule out the possibility that the inorganic ions were acting as osmotic agents.Carbon dioxide and water exchange analysis showed that when water supply was not limiting, salt-deprived plants sustained higher reductive pentose phosphate cycle carbon fixation rates than salt-treated plants. Under water stress conditions, salt-deprived plants using only the reductive pentose phosphate cycle pathway assimilated less carbon and were less efficient in their water use than salt-treated plants using predominately the CAM pathway. These results support the hypothesis that the ability to use the CAM pathway reduces the capacity for reductive pentose phosphate cycle fixation but permits higher productivity in water-limited environments. PMID- 16660806 TI - Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts. AB - Millimolar concentrations of Mg(2+) inhibited CO(2)-dependent O(2) evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O(2) evolution by Mg(2+) and reduced the Mg(2+) inhibition of enzyme activation by light. Fructose 1,6 diphosphate and ribulose 5-phosphate also prevented the inhibition of O(2) evolution by Mg(2+) whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg(2+) inhibition of O(2) evolution shortened the lag phase of CO(2)-dependent O(2) evolution in the absence of M(2+). Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O(2) evolution and the inhibition of O(2) evolution by Mg(2+). The results suggested that Mg(2+) inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites. PMID- 16660807 TI - Possible role of volatile Fatty acids and abscisic Acid in the dormancy of oats. AB - Species of Avena differ markedly in their levels of pre- and post-harvest dormancy. These species offer the opportunity of determining if dormancy is related to the endogenous level of growth inhibitor. Germinability in two species of differing levels of dormancy, common oat Avena sativa L., and wild oat Avena fatua L. was assessed as were the contents of abscisic acid and volatile fatty acids of chain length C(6)-C(10). In A. sativa which did not possess postharvest dormancy there was no correlation between germination and inhibitor levels but in A. fatua the relationship between the content of fatty acid and dormancy was good. The loss of these fatty acids in dry storage by evaporation could explain after ripening. PMID- 16660808 TI - Effect of Daylength on Phenol Metabolism in the Leaves of Salvia occidentalis. AB - A method of phenol determination in plant leaves has been developed which is based on the in situ oxidation of these compounds in an atmosphere containing ammonia, followed by difference spectrophotometry. The development of the phenol pattern has been studied in each separate leaf of a Salvia occidentalis plant grown in short and in long days. During the light period the phenol content (mainly chlorogenic acid and isochlorogenic acids) increases in proportion to the length of this period, whereas during the subsequent dark period the phenol content decreases. This decrease does not continue during the second part of a dark period if that period is interrupted by a light break with red light. Instead a small increase is observed. This effect of red light can be reversed with far red light. It is argued that a correlation with flower induction in this short day plant can be construed if it is assumed that the continuous presence of certain o-dihydroxyphenols in the cytoplasm of leaf cells inhibits the synthesis or the transport of a flowering hormone. PMID- 16660809 TI - Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm. AB - Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3-day-old castor bean seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with molecular weights ranging from 10,000 to over 100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15 to 20% of the total translational products.Attempts to reconstitute rough endoplasmic reticulum by the addition of washed castor bean microsomal membranes to the translational system were unsuccessful, these membranes severely inhibiting protein synthesis. Canine pancreatic microsomes could be added to endosperm messenger RNA-dependent reticulocyte lysates at relatively high concentrations while still allowing significant protein synthesis. PMID- 16660810 TI - Abscisic Acid and the Photoperiodic Induction of Dormancy in Salix viminalis L. AB - A series of growth room experiments was carried out aiming to establish the role of abscisic acid on dormancy of Salix viminalis L. The inhibitor content and abscisic acid levels of extracts from roots, sap, leaves, and apical tissues of willow were measured using biological assay and gas-liquid chromatography.No evidence was obtained that photoperiodically mediated dormancy is associated with changes in abscisic acid levels or beta-inhibitor activity. PMID- 16660811 TI - Characteristics of the Flash-induced 515 Nanometer Absorbance Change of Intact Isolated Chloroplasts. AB - In intact (type A) chloroplasts isolated from mesophyll protoplasts of maize (Zea mays L. convar. KSC 360) the flash-induced 515 nanometer absorbance change was much higher than in conventionally prepared (types B and C) chloroplasts. The 515 nanometer signal of type A chloroplasts exhibited a biphasic rise: the initial very fast rise (rise time <<1 millisecond) was followed by a slow increase of absorbance (rise time 10 to 20 milliseconds). With decreasing degree of envelope retention the slow phase disappeared. Thus the biphasic rise of the flash-induced 515 nanometer absorbance change can be regarded as an attribute of intact chloroplasts.The 515 nanometer signal of intact chloroplasts was studied at various pH values of the external medium, with various dark intervals between the flashes and at different temperatures. The absorbance change was probed with electron transport inhibitors and ionophores. The data show that the fast phase of the absorbance increase was similar in chloroplasts isolated from protoplasts and in conventional chloroplast preparations. The slow rise, which has not been hitherto recognized in isolated chloroplasts, can be due to a contribution of the proton pump to the electric field which is generated across the thylakoid membranes. PMID- 16660812 TI - Properties and intramitochondrial localization of serine hydroxymethyltransferase in leaves of higher plants. AB - The activity of serine hydroxymethyltransferase in mitochondria isolated from spinach leaves was absolutely dependent on tetrahydrofolate; pyridoxal phosphate has no effect on the activity. The stability of this activity in the isolated mitochondria was dependent on the presence of sulfhydryl compounds. It was apparently more stable at pH 7.0 to 7.5 than at higher pH even though the pH optimum of serine hydroxymethyltransferase was 8.5 for both the mitochondrial and cytoplasmic fractions. Distribution studies have indicated that serine hydroxymethyltransferase was predominantly located in the mitochondria. The activity of serine hydroxymethyltransferase was observed to be co-compartmented with glycine decarboxylation and malate dehydrogenase behind the mitochondrial inner membrane. This activity could be solubilized by KCl from osmotically ruptured mitochondrial membrane fractions but substantial activity (35 to 40%) was still retained with the membrane fractions at 0.3 m KCl. This suggests that the glycine decarboxylation-serine hydroxymethyltransferase complex may be closely bound to the internal surface of the mitochondrial inner membrane.The relationship of this integrated enzyme complex to CO(2) evolution and serine synthesis during photorespiration and the physiological role of the dicarboxylate shuttle were discussed. PMID- 16660813 TI - Dark Carbon Dioxide Fixation under Aerobic and Anaerobic Conditions in Maize Leaves after Preillumination in the Absence of Oxygen: Ribulose 1,5-Bisphosphate Can Serve as a Primary Acceptor of Carbon Dioxide. AB - When dark (14)CO(2) fixation in maize leaves was carried out under anaerobic conditions after preillumination in the absence of O(2), the (14)C incorporation in aspartic acid was transient; its maximum level was very low compared with that of malic acid. The addition of 5% O(2) during the dark fixation period increased the total uptake of (14)CO(2) and the (14)C incorporation into aspartic acid.A study of the intramolecular distribution of radioactivity showed that 71 to 76% of the (14)C was located in the C(4) (beta-carboxyl) of malate and aspartate and the remainder in the C(1). This intramolecular labeling pattern did not change during the 5- to 60-second dark (14)CO(2) fixation period and was scarcely altered by the presence of O(2). Three degradation techniques led to similar data.The significance of these results is discussed taking into account the known possible carboxylation pathways. It is concluded that ribulose 1,5-bisphosphate can be a primary acceptor of CO(2) when maize leaves are preilluminated in the absence of O(2). PMID- 16660814 TI - A comparison of glutamate synthase obtained from maize endosperms and roots. AB - Glutamate synthase (EC 2.6.1.53) has been examined in developing endosperms and roots of maize. KCl is required for maximum activity in each tissue. The effect with KCl is seen with buffer strength of 25 to 100 millimolar in the assay. The optimum concentration for the enzyme from endosperm is 20 millimolar and for the enzyme from root tissue the saturating concentration is about 20 millimolar. In root material the enzyme is labile but activity can be restored if KCl is added to the assay. Divalent cations such as Mg(2+) or Mn(2+) also activate the enzyme to some extent.In each case NADH or NADPH can serve as reductant. The reaction is insensitive to alpha-aminooxyacetate, but is inhibited by glutamate, the glutamate analogs methionine sulfoximine and methionine sulfone, and by the glutamine analogs azaserine and albizziin. PMID- 16660815 TI - Assay of chilling injury in wild and domestic tomatoes based on photosystem activity of the chilled leaves. AB - Tomato leaves were detached and stored at 0 C for various periods of time. Chloroplasts were isolated from the leaves and their photoreductive activities were determined. Comparisons were made between two altitudinal forms of the wild tomato Lycopersicon hirsutum Humb. and Bonpl. (a tropical lowlands form and a highlands form adapted to growth at 3,100 meters), and two cultivars of the domestic tomato L. esculentum Mill. In each case the capacity of the isolated chloroplasts to photoreduce ferricyanide declined linearly with time of storage of the leaves at 0 C, but not at 10 C. This injury developed more slowly in the high altitudinal form of the wild tomato compared with the low altitudinal form and the two domestic cultivars indicating an enhanced resistance toward chilling injury in the tomato from 3,100 meters. Chloroplast activity declined in green tomato fruit held at 0 C, at about the same rate as in the chilled leaves.Measurements of photochemical activities in the isolated chloroplasts and in vivo measurements of cytochrome-554 photooxidation in chilled leaves showed that the site of action of the chilling effect was water donation to photosystem II.The chilling-induced impairment of photoreductive activity in chloroplasts provides a useful assay for detecting and measuring differences in the susceptibility of plants to chilling injury. PMID- 16660816 TI - Influence of plant sterols on the phase properties of phospholipid bilayers. AB - The effects of stigmasterol, sitosterol, campesterol, and cholesterol on the phase properties of dipalmitoylphosphatidylcholine bilayers have been compared by differential scanning calorimetry and x-ray diffraction. The sterols were equally effective at progressively reducing the cooperativity and the enthalpy of the dipalmitoylphosphatidylcholine phase transition as their concentrations in the bilayer were increased. Moreover, both differential scanning calorimetry and x ray diffraction indicated that the dipalmitoylphosphatidylcholine transition was eliminated by each of the sterols when they were present at a concentration of 33 mole%. This indicates that the interaction between phospholipid and both plant and animal sterols is stoichiometric, each sterol associating with two phospholipid molecules. At concentrations above 33 mole% the sterols were no longer completely solvated by the phospholipid, and sterol-sterol interaction resulted. Cholesterol, even at concentrations as high as 50 mole%, did not disrupt the lamellar structure of the bilayer. When these high concentrations of plant sterols were intercalated into the phospholipid, crystallinity, which presumably derives from sterol-sterol interaction, was detectable in the bilayer by x-ray diffraction. This observation is consistent with previous reports to the effect that the C(17) chains of the plant sterols render them less soluble in phospholipid than is cholesterol. It is clear that this solvation difference is of insufficient magnitude to affect the stoichiometry of dipalmitoylphosphatidylcholine-sterol interaction, but it could well account for the less effective modulation of lipid bilayer permeability exhibited by plant sterols in comparison with cholesterol. PMID- 16660817 TI - Reduction of Adenosine Triphosphate Levels in Susceptible Maize Mesophyll Protoplasts by Helminthosporium maydis Race T Toxin. AB - Helminthosporium maydis race T (HMT) toxin caused a reduction in the steady-state ATP levels when leaf mesophyll protoplasts isolated from maize containing Texas male-sterile (T) but not male-fertile (N) cytoplasm were incubated in the dark. At a toxin concentration 10 times the mean effectived dose for inhibition of root growth, the ATP levels began to fall in 30 to 90 seconds, fell by 50% in about 4 minutes, and reached 23% of the original levels in 100 minutes. This is faster than any previously observed response of whole cells or tissues to HMT toxin. In protoplasts incubated in the light, ATP levels were 25% higher than in the dark and were either unaffected or only slightly diminished by toxin. 3-(3,4 Dichlorophenyl)-1, 1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, overcame the effect of light on both toxin-treated and control protoplasts. Oligomycin, an inhibitor of mitochondrial ATP synthesis, mimicked the effects of toxin in the dark, in the light, and in the light plus DCMU, but it was not specific for T cytoplasm. During the first 24 hours of culture, ATP levels in control protoplasts increased in both the light and dark. In the dark, ATP was not detectable after 24-hour incubation in the presence of toxin, whereas in the light a substantial amount of ATP remained. Our results are compatible with the hypothesis that mitochondria in vivo are inhibited by HMT toxin. Other responses of cells and tissues to toxin can be explained in terms of reduced ATP levels. PMID- 16660818 TI - Ethylene production by callus and suspension cells from cortex tissue of postclimacteric apples. AB - Cortex tissue from postclimacteric ;Golden Delicious' apples (Malus domestica, Borkh.) stored at 0 C for 9 months after harvest were induced to form callus in vitro. Cell suspension cultures were subsequently formed from calli. Of five media tested, only the medium of Schenk and Hildebrandt (Can J Bot 1972, 50: 192) and that of Uchimiya and Murashige (Plant Physiol 1974, 54: 936) allowed callus formation. During growth both the callus and cell cultures produced ethylene in a pattern which showed a rapid rise and then a fall as the culture grew. (14)C Labeled methionine was converted to labeled ethylene by the cell suspension cultures, which also could be inhibited from producing ethylene by a rhizobitoxine analog or free radical scavengers. Ethylene production in these cultures, like that in intact fruit tissue slices, could be stimulated by IAA or suppressed by N(6)-(gamma,gamma-dimethylallyl) adenosine and GA(3). PMID- 16660819 TI - Variation in nitrogenase and hydrogenase activity of alaska pea root nodules. AB - Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H(2) and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H(2) and was not inhibited by the presence of 0.10 atmosphere C(2)H(2), which prevented H(2) evolution from nitrogenase. Total H(2) production from nitogenase was estimated as net H(2) evolution in air plus H(2) exchange in 0.10 atmosphere C(2)H(2). Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H(2) exchange and H(2) uptake activity of hydrogenase, differences observed in H(2) exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H(2) production by nitrogenase. Total H(2) production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H(2) production declined, but the potential proportion of nitrogenase-produced H(2) recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H(2) had significantly higher rates of N(2) reduction than those which did not exhibit hydrogenase activity. PMID- 16660820 TI - Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum. AB - A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C(4) plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O(2) evolution by chloroplasts isolated from protoplasts.Chloroplasts were isolated from protoplasts by several passages of the protoplasts through a 20-micrometer nylon mesh. Mesophyll chloroplasts were judged approximately 90 to 95% intact and bundle sheath chloroplasts 80 to 90% intact based on retention of chloroplast marker enzymes and the ferricyanide test for intactness. It was necessary to include 10 millimolar MgCl(2) in media for osmotically shocking the chloroplasts in order to obtain maximum and linear rates of ferricyanide-dependent O(2) evolution.Chloroplasts isolated from mesophyll protoplast preparations had low rates of light-dependent O(2) evolution in the presence of 10 millimolar NaHCO(3) (0.13 micromoles per milligram chlorophyll per minute) in comparison to bundle sheath chloroplasts (1 to 2.5 micromoles per milligram chlorophyll per minute). The mesophyll chloroplasts catalyze high rates of 3-phosphoglycerate-dependent O(2) evolution (2 to 4 micromoles per milligram chlorophyll per minute). Orthophosphate but not phosphoenolpyruvate inhibited the 3-phosphoglycerate-dependent O(2) evolution by the mesophyll chloroplasts. Rates of O(2) evolution were much higher with mesophyll than with bundle sheath chloroplasts in the presence of pyruvate plus oxaloacetate. The results are discussed in relation to the proposed function of these chloroplasts during C(4) photosynthesis. PMID- 16660821 TI - Sucrose translocation and storage in the sugar beet. AB - Several physiological processes were studied during sugar beet root development to determine the cellular events that are temporally correlated with sucrose storage. The prestorage stage was characterized by a marked increase in root fresh weight and a low sucrose to glucose ratio. Carbon derived from (14)C sucrose accumulation was partitioned into protein and structural carbohydrate fractions and their amino acid, organic acid, and hexose precursors. The immature root contained high soluble acid invertase activity (V(max) 20 micromoles per hour per milligram protein; K(m) 2 to 3 millimolar) which disappeared prior to sucrose storage. Sucrose storage was characterized by carbon derived from (14)C sucrose uptake being partitioned into the sucrose fraction with little evidence of further metabolism. The onset of storage was accompanied by the appearance of sucrose synthetase activity (V(max) 12 micromoles per hour per milligram protein; K(m) 7 millimolar). Neither sucrose phosphate synthetase nor alkaline invertase activities were detected during beet development. Intact sugar beet plants (containing a 100-gram beet) exported 70% of the translocate to the beet, greater than 90% of which was retained as sucrose with little subsequent conversions. PMID- 16660822 TI - Control of Leaf and Stem Growth in Light-grown Pea Seedlings by Two High Irradiance Responses. AB - The control exerted by light on leaf and stem growth in light-grown Alaska pea seedlings was studied during the main photoperiod. Two high irradiance responses were observed. The action spectrum for one had a single sharp peak at 600 nanometers. The action spectrum for the other showed a broad peak between 440 and 470 nanometers. These two light responses must be activated simultaneously for any inhibition of stem growth or promotion of leaf growth. Both action spectra may be explained in terms of the high irradiance response of phytochrome. PMID- 16660823 TI - Factors Influencing the Development of Cyanide-resistant Respiration in Potato Tissue. AB - Ethylene, cyanide gas, and volatalized ethanol, acetaldehyde, and acetic acid were applied in a continuous flow to whole potato tubers. Freshly cut slices were obtained periodically during the treatment, and showed a progressive development of a cyanide-resistant respiration. The application of the employed volatiles in 100% O(2) accelerated the onset and the magnitude of the cyanide-resistant respiration.These results show that, similar to ethylene and cyanide, the application of ethanol, acetaldehyde, or acetic acid can also lead to the development of cyanide-resistant respiration in whole potato tubers, and that this type of respiration is retained in freshly cut slices. PMID- 16660824 TI - Photocontrol of Anthocyanin Synthesis: VII. Factors Affecting the Spectral Sensitivity of Anthocyanin Synthesis in Young Seedlings. AB - Light-dependent anthocyanin synthesis is a typical high irradiance response (HIR) of plant photomorphogenesis. The spectral sensitivity of this response in young seedlings of cabbage and tomato is strongly affected by the length and mode of application of the light treatments. This observation suggests that the different experimental conditions, used in different action spectroscopy studies, might have been responsible, at least in part, for some of the reported differences in the characteristics of the HIR action spectra of different response-system combinations. In both cabbage and tomato, the values of the far red/blue, far red/red, and blue/red action ratios increase with increasing durations of the light treatments; this finding is in agreement with hypotheses of K. M. Hartmann (1966, 1967) and E. Schafer (1975) for phytochrome action in the HIR. The similarity in the trend of change of the values of the action ratios suggests the possibility that the photomorphogenic pigment system, involved in the photoregulation of anthocyanin synthesis, may be the same in cabbage and tomato, even though there are some differences in the spectral sensitivity of the response between the two species. PMID- 16660825 TI - Calcium distribution in globoid crystals of cucurbita cotyledon protein bodies. AB - Energy-dispersive x-ray analysis was used to investigate the location of globoid crystals with relatively high Ca levels within cotyledons of Cucurbita maxima, Cucurbita mixta, and Cucurbita andreana. The small globoid crystals in both upper and lower epidermal cells commonly contained Ca. Ca was present in globoid crystals of all provascular regions with the exception of the very small provascular regions of C. maxima. In C. maxima and C. mixta cotyledons, some cases were observed where Ca was found in the globoid crystals of the first layer of mesophyll cells surrounding the provascular region, but in general Ca was absent from globoid crystals of palisade and spongy mesophyll cells. In C. andreana, globoid crystals of palisade and spongy mesophyll cells commonly contained at least some Ca. Cell position and cell type are factors affecting the Ca content of globoid crystals in protein bodies. PMID- 16660826 TI - Enzymic Fractionation of the Stable Carbon Isotopes of Carbon Dioxide by Ribulose 1,5-bisphosphate Carboxylase. AB - The enzymic fractionation of the stable carbon isotopes of CO(2) (Deltaco(2)) was determined using a purified preparation of ribulose-1,5-bisphosphate (RuBP) carboxylase isolated from cotton (a C(3) plant) leaves. The bicarbonate concentration in the reaction mixture saturated the enzyme and furnished an infinite pool of (12)CO(2) and (13)CO(2) for enzyme fractionation. The RuBP was 96 to 98% pure. The phosphoglycerate synthesized in the reaction mixtures was purified free of RuBP, phosphoglycolate, and other phosphate esters by column chromatography on Dowex 1-Cl(-) resin. The average Deltaco(2) value of -27.1% was determined from five separate experiments. A discussion of the isotope fractionation associated with photosynthetic CO(2) fixation in plants shows that the enzymic fractionation of stable carbon isotopes of CO(2) by RuBP carboxylase is of major importance in determining the delta(13)C values of C(3) plants. PMID- 16660827 TI - Analysis and Characterization of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU) resistant Euglena: I. Growth, Metabolic and Ultrastructural Modifications during Adaptation to Different Doses of DCMU. AB - Cultures of Euglena gracilis Klebs strain Z Pringsheim were grown photoorganotrophically in the presence of different concentrations of 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) in the range of 0.05 to 250 micromolar. Cultures were serially transferred and various metabolic parameters were followed for 10 weeks. A process of adaptation occurred which was divided operationally into three phases. A phase of ultrastructural disorganization occurred, succeeded by a recovery phase; their intensity and duration were functions of the dose of DCMU. A stable adaptation phase then ensued. This phase was observed in all cultures except that exposed to the highest DCMU concentration. Adapted cells from all of the DCMU cultures contained twice the protein and half the paramylon of the control cells and thus utilized the carbon source to accumulate cellular reserves with only half the efficiency of controls. DCMU affected cellular metabolism as well as photosynthesis.The energy charge remained at high levels throughout adaptation, although the size of the adenylate pool was half that of controls at the disorganized phase. At this stage the ultrastructure of chloroplasts and mitochondria was considerably modified. The progressive changes of the parameters studied appeared to affect all of the cells in a given culture. PMID- 16660828 TI - Analysis and Characterization of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU) resistant Euglena: II. Modifications Affecting Photosynthesis during Adaptation to Different Doses of DCMU. AB - When grown in medium containing dl-lactate at 27 C in the light, Euglena gracilis Z populations underwent modifications of the pigment system in response to 0.05 to 250 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).Chlorophyll content dropped dramatically, the only remaining form being Chl a(673). Light driven O(2) evolution was no longer detectable for the two highest DCMU concentrations tested. The energy-capture cross-section of detectable photosystem II units remained unchanged, although intersystem energy transfer no longer occurred. Euglena at this stage had chloroplast membranes destacked and swollen. A recovery phase then occurred, marked by enhanced photosynthetic properties. The initial forms of chlorophyll which were accumulated were highly efficient for O(2) evolution. The newly formed photosystem II antennae were connected and of small size. Finally, the third phase involved the recovery of photosynthetic capacity similar to that of the controls as the thylakoids regained their normal structures.Since these modifications occurred in the entire population and DCMU resistance persisted through successive cell generations, these adapted Euglena were considered to be a variant of the Z strain, designated ZR. PMID- 16660829 TI - Cytokinin Activity in Lupinus albus L: IV. Distribution in Seeds. AB - Endogenous levels of cytokinin activity were examined in Lupinus albus L. seed at intervals of 2 weeks after anthesis using the soybean callus bioassay. High levels of cytokinin activity per gram seed material were present in the seeds at 2, 4, and 6 weeks after anthesis. The cytokinin activity per gram seed material was low at 8 and 10 weeks after anthesis. Cytokinin activity associated with each seed was greatest at 6 weeks after anthesis. The majority of the activity in the seeds at 4, 6, and 8 weeks after anthesis was in the endosperm. Cytokinin activity was also detected in the testas and embryos at 4, 6, 8, and 10 weeks, and the suspensors at 4 weeks. Column chromatography of extracts of the different seed fractions on Sephadex LH-20 indicated that the cytokinins present coeluted with zeatin, zeatin riboside, and the glucoside cytokinins. It is suggested that cytokinins are accumulated in the seeds and are stored in the endosperm mainly in the form of ribosides and glucosides of zeatin. The reduction in cytokinin activity in the seed coincides with the reduction in endosperm volume and embryo growth and suggests that these compounds are utilized during the course of seed maturation. PMID- 16660830 TI - Role of Potassium in Carbon Dioxide Assimilation in Medicago sativa L. AB - Alfalfa was grown hydroponically in 0, 0.6, and 4.8 millimolar K in order to determine the influence of tissue level of K on photosynthesis, dark respiration, photorespiration, stomatal and mesophyll resistance to CO(2), photosystem I and II activity, and synthesis and activity of ribulose 1,5-bisphosphate carboxylase (RuBPc).A severe (0.0 millimolar) and mild (0.6 millimolar) K deficiency, compared to plants grown at 4.8 millimolar K, produced a significant decrease in photosynthesis and photorespiration, but an increase in dark respiration. Both deficient K levels increased hydrophyllic resistance to CO(2), but only the severe deficiency increased stomatal resistance.Photosystem I and II activity of isolated chloroplasts was not affected by K deficiency. The apparent activity of a crude RuBPc preparation was significantly reduced in severely deficient plants. Activity of the enzyme could not be restored to normal rates by the addition of K to the reaction medium.The specific activity of RuBPc isolated from severely K deficient and K-sufficient leaflets was not significantly different, suggesting that K does not function in RuBPc activity. Incorporation of [(14)C]leucine into RuBPc, as a measure of synthesis, by K-deficient leaflets was reduced to 15% of K sufficient leaflets. The addition of K to the reaction medium stimulated [(14)C]leucine incorporation into RuBPc and 10 millimolar KNO(3) increased incorporation to 80% of K-sufficient leaflets. Actinomycin D and cycloheximide suppressed the K-stimulated incorporation of [(14)C]leucine into RuBPc, suggesting that the K-stimulated synthesis of RuBPc most likely represents de novo synthesis. PMID- 16660831 TI - Effect of glyphosate on auxin transport in corn and cotton tissues. AB - Basipetal auxin transport in 6-day-old dark-grown corn coleoptiles was severely inhibited by increasing levels of glyphosate applied during the transport period.The velocity of basipetal transport of [(14)C]indoleacetic acid in hypocotyls from 7-day-old cotton seedlings was significantly reduced when sublethal doses of glyphosate [N-(phosphonomethyl)glycine] were applied to the cotyledonary leaves 24 hours before transport measurement. Simultaneous application of glyphosate and indoleacetic acid during transport measurement had no effect on basipetal transport in cotton hypocotyl sections.Slowing of transport was inversely proportional to the dosage applied to both species. PMID- 16660832 TI - Effects of glycidate on carbon dioxide fixation with isolated spinach chloroplasts. AB - Glycidate (2,3-epoxypropionate) stimulated CO(2) fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate.Glycolate formation was not inhibited by glycidate, which is in agreement with the observation that preactivated ribulose-1,5 bisphosphate oxygenase is not inhibited by this compound. Furthermore, glycidate had no effect on electron transport (NADP reduction) and photophosphorylation, nor was a change in the coupling ratio observed. PMID- 16660833 TI - Environmental and genotypic effects on the respiration associated with symbiotic nitrogen fixation in peas. AB - Estimated values for the respiration associated with symbiotic nitrogen fixation in Pisum sativum L. were independent of irradiance, temperature, plant age, and CO(2) concentration, despite large variation in the total rates of C(2)H(2) reduction and root + nodule respiration. Similar values were also found in Phaseolus vulgaris L., Vicia faba L. and Glycine max (L.) Merr. Among all combinations of four Pisum cultivars with four Rhizobium leguminosarum inoculants only the plant genotype significantly affected the fixation-linked respiration, although both plant and bacterial types significantly influenced the total rate of C(2)H(2) reduction. On the basis of measured rates of H(2) evolution and C(2)H(2) reduction, or total nitrogen gain in the same system, the least respiration per unit of ammonia produced symbiotically was estimated as 4.8 to 6.9 moles CO(2) (mole NH(3))(-1) in Laxton's Progress and the greatest as 9.3 to 13.3 moles CO(2) (mole NH(3))(-1) in an Indian cultivar, as compared to a theoretical minimum respiration requirement of 4.7 moles CO(2) (mole NH(3))(-1) in peas. PMID- 16660834 TI - A Comparison of Acid-induced Cell Wall Loosening in Valonia ventricosa and in Oat Coleoptiles. AB - The acid-induced loosening of cell walls of Valonia ventricosa has been compared to that of frozen-thawed oat coleoptiles. The two acid extension responses are similar in regard to the shape of the pH response curve and the increase in plastic compliance induced by acid treatment. In both systems the acid response can be inhibited by Ca(2+) and in both the removal of the protons leads to a rapid termination of wall loosening. The two responses differ in several significant ways, however. The acid-induced extension of Valonia walls is more rapid than that of coleoptile walls, but of smaller total magnitude. Acid-induced loosening can occur in Valonia without the wall being under tension, but not in coleoptiles. The acid-induced extension of Valonia walls is not inhibited by 8 molar urea, whereas the response in oat coleoptiles is completely inhibited by this treatment. Ethylenediaminetetraacetate (EDTA) can cause wall loosening in Valonia comparable to that produced by low pH, whereas in coleoptiles EDTA causes a much smaller response. These results with Valonia are consistent with a mechanism of acid-induced wall loosening in which a central role is played by the displacement of Ca(2+) from the wall, while the larger part of acid-induced wall loosening in oat coleoptiles appears to be via a different mechanism. PMID- 16660835 TI - Isoenzymes of pyruvate kinase in etioplasts and chloroplasts. AB - Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localized in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity. PMID- 16660836 TI - Events Surrounding the Early Development of Euglena Chloroplasts: 14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells. AB - Lack of a suitable assay has thwarted attempts to measure cytochrome c-552 in dark-grown wild type cells of Euglena gracilis var. bacillaris in mutants and in other situations where the concentrations are low. Purification methods are described based on electrofocusing which provide a cytochrome c-552 preparation homogeneous enough to elicit a single reactive antibody in rabbits; this antibody is then used as a specific and sensitive assay for cytochrome c-552. Dark-grown cells of wild type and of mutants O(1)BS, O(2)BX, G(1)BU and P(1)BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 x 10(-11) micromoles of cytochrome c-552 per cell, 10 to 150 times less than light-grown cells. Light-grown cells of these mutants and of wild type show a ratio of chlorophyll to cytochrome of about 300 (mole to mole). Cytochrome c-552 is undetectable in dark-grown Y(1)BXD, Y(3)BUD, and W(34)ZUD which cannot carry plastid development beyond the proplastid in light; the light-grown cells of these mutants have levels of cytochrome similar to or lower than dark-grown wild type cells. Cytochrome c-552 is undetectable in light- and dark-grown mutants in which plastid DNA is undetectable (such as Y(2)BUL, W(3)BUL, W(8)BHL, and W(10)BSmL) consistent with the view, but not proving, that this molecule may be coded, at least in part, in plastid DNA. During light-induced chloroplast development in resting cells, cytochrome c-552 formation behaves in all respects like chlorophyll except that the dark-grown cells contain low amounts of the cytochrome c-552 but lack chlorophyll. Thus, both cytochrome c-552 and chlorophyll show the same lag period even when the length is changed by nutritional manipulation; preillumination largely eliminates the lag in the formation of both molecules, cycloheximide and streptomycin both inhibit the biosynthesis of chlorophyll and cytochrome c-552 in the same manner, and the formation of both during chloroplast development is strictly light-dependent. It is shown that chloroplasts isolated from Euglena by methods thought to give intact organelles, lack 95% of the cytochrome c-552; this and the loss of similar molecules may explain why these isolated chloroplasts are not photosynthetically active. PMID- 16660837 TI - Solubility of gases and the temperature dependency of whole leaf affinities for carbon dioxide and oxygen: an alternative perspective. AB - An analysis of the kinetics of simultaneous photosynthesis and photorespiration at the end of a diffusion path is applied to observed net photosynthetic rate as a function of O(2) and CO(2) concentrations. The data of Ku and Edwards (Plant Physiol. 59: 991-999, 1977) from wheat (Triticum aestivum L.) are analyzed in detail. Ku and Edwards, using an analysis that ignored diffusion resistance between the intercellular air space and fixation site, the competitive effect of CO(2) on photorespiration, and the actual concentrations of gases at the fixation site, concluded that: (a) the affinity coefficient of the leaf for CO(2) was approximately 3.5 to 5 micromolar; (b) this affinity coefficient is independent of temperature between 25 and 35 C; (c) the effect of O(2) was independent of temperature over this range; and (d) competition between CO(2) and O(2) is responsible for the major share of CO(2) loss from photosynthesis due to photorespiration. They suggest that using gas concentrations calculated as equilibium values in the liquid phase is very important in reaching these conclusions. By applying a more complete analysis to their data which includes diffusion in the cell, it is concluded that: (a) the affinity coefficient of the leaf for CO(2) is 0.1 to 1.1 micromolar; (b) the temperature dependence of this affinity coefficient cannot be determined from existing data, but there is no evidence to refute independent temperature effect on the two functions of ribulose-1,5-bisphosphate carboxylase-oxygenase being important in the regulation of whole leaf net photosynthesis; and (c) the competitive interplay of CO(2) and O(2) at ribulose-1,5-bisphosphate carboxylase may under certain conditions lead to a stimulation of fixation by the Calvin cycle because of photorespiration. These conclusions are reached whether CO(2) and O(2) are expressed as dissolved concentrations or as gas concentrations in the intercellular air space. The relative merits of these two expressions of concentration are discussed. PMID- 16660838 TI - Emission of ethylene and ethane by leaf tissue exposed to injurious concentrations of sulfur dioxide or bisulfite ion. AB - Leaf tissues injured with SO(2) gas or bisulfite ion in solution emit ethylene and ethane. The amounts of these gases produced by the tissues depend on the degree of exposure to SO(2) or bisulfite. The amount of ethylene produced in response to SO(2) fumigation correlates positively with SO(2) exposure (0 to 5.5 microliters per liter for 16 hours), SO(2) absorbed, and the amount of visible injury sustained by the leaf tissues. Ethane production is correlated positively with the injury resulting from treatment with bisulfite ion. The rate of emission of ethane from leaf discs of cucurbit cultivars as a result of exposure to bisulfite solutions is in agreement with the order and the degree of their resistance to injury by SO(2). Thus, exposure to bisulfite and the subsequent release of ethane can be used to determine the relative resistance of different species and cultivars to SO(2) gas.A rapid, simple, objective assay for SO(2) resistance based on ethane emission is described. This assay should preferentially detect SO(2) resistance which does not depend on stomatal behavior. The screening of several other cucurbits with this assay showed a 24 fold difference between the most and the least sensitive plants tested. PMID- 16660839 TI - Ethylene production by apple protoplasts. AB - Freshly prepared protoplasts from apple tissue that produced ethylene were obtained. Ethylene production was inhibited by osmotic shock, 0.01% Triton X-100, and aminoethoxyvinyl glycine. Protoplasts as well as the ethylene system were not greatly affected by protease treatment. PMID- 16660840 TI - Differential Ion Stimulation of Plasmalemma Adenosine Triphosphatase from Leaf Epidermis and Mesophyll of Nicotiana rustica L. AB - An ATPase preparation, presumed to be associated with plasma membrane due to the coincidence on isopycnic gradients of cellulase and beta-glucan synthetase at high substrate, has been isolated from the epidermal and mesophyll of tobacco leaf. The ATPase from both tissues was found to prefer ATP over other nucleotides. The pH optimum was 7.0 in the presence of 3 millimolar MgCl(2) and pH 6.5 in the presence of 3 millimolar MgCl(2) and 100 millimolar KCl. Monovalent ion stimulation patterns of the ATPases from these tissues were found to differ and ion accumulation patterns in these tissues reflect this difference: mesophyll accumulated roughly equal amounts of Na(+) and Rb(+) and its plasma membrane ATPase is also equally stimulated by these ions; on the other hand, epidermal ATPase preparations showed a stronger stimulation by Rb(+) than Na(+) and this tissue was found to accumulate Rb(+) in preference to Na(+). Abscisic acid and fusicoccin affected both ATPase activity and ion uptake, the former inhibiting and the latter stimulating these parameters. These data support the hypothesis that the epidermal plasmalemma ATPase is involved in stomatal opening. PMID- 16660841 TI - Effects of ouabain and low temperature on the sodium efflux pump in excised corn roots. AB - Ouabain (0.05 millimolar) and low temperature (4 C) both caused the tissue Na(+) content of excised 5-day-old corn roots to increase, indicating that there is an inhibition of the Na(+) efflux pump. Na(+) efflux was measured utilizing three different methods. Each method gave similar results in terms of rate and ouabain sensitivity. With one of these methods, the compartmental efflux method, it was demonstrated that rates for Na(+) efflux increase as the external Na(+) concentration is increased; e.g. the efflux rates are 0.529, 1.78, and 3.64 microequivalents per gram fresh weight per hour for external NaCl concentrations of 1, 10, and 30 millimolar, respectively. The data indicate that the Na(+) efflux pump is located in the plasmalemma of root cells.Na(+) efflux was stimulated for 30 to 60 minutes after the introduction of ouabain. This was followed in 60 to 90 minutes by an inhibition of Na(+) efflux. The Na(+) efflux rate returned to the original level on the removal of ouabain.The transport of Na(+) to the xylem vessels was stimulated by ouabain which most likely is a consequence of the ouabain-induced increase in cytoplasmic Na(+) content.Ouabain (0.05 millimolar) had little or no effect on K(+) and Cl(-) contents, and this implies the lack of an effect of ouabain on K(+) and Cl(-) fluxes. Ouabain at a concentration of 0.01 millimolar had no effect on Na(+) flux or on tissue ion content. With 0.5 millimolar ouabain the tissue contents of K(+), Na(+), and Cl( ) were greatly reduced.Evidence is presented indicating that ouabain has no effect on Na(+) efflux in pea roots. PMID- 16660842 TI - Effects of Variations in Daylength and Temperature on Net Rates of Photosynthesis, Dark Respiration, and Excretion by Isochrysis galbana Parke. AB - The effects of variable daylength and temperature on net rates of photosynthesis, dark respiration, and excretion of a unicellular marine haptophyte, Isochrysis galbana Parke, were examined and related to division rates. Six combinations of daylength (18:6, 12:12, 6:18 light:dark, LD) and temperature (20, 25 C) were used. Daily rates of net photosynthesis were closely correlated to division rates, suggesting a direct relationship, and were maximal when cells were grown at 12:12 LD at both temperatures and 18:6 LD at 20 C. A daylength of 6 hours decreased daily rates by decreasing the time for carbon uptake. Further, cells grown with this daylength had maximal chlorophyll a contents, suggesting a physiological adaptation by photosynthetic units to short light periods. A photoperiod of 18:6 LD at 25 C decreased daily rates of net photosynthesis by reducing the hourly rate of net photosynthesis via an unidentified mechanism. The importance of rates of net dark respiration in controlling daily net photosynthesis was small, with carbon lost during dark periods varying between 4 and 14% of that gained during light periods. Also, the influence of net excretion was small, varying between 1.0 and 5.5% of daily net photosynthesis. PMID- 16660843 TI - Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter. AB - Evidence is presented that K(+) uptake in corn root segments is coupled to an electrogenic H(+)/K(+) -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH(-)/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K(+) uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH(-)/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K(+) uptake. Collapse of the proton gradient across the membrane by (p trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K(+) uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K(+) and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K(+) uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H(+)/K(+) -exchanging ATPase to an OH(-)/Pi antiporter in corn root tissue. PMID- 16660844 TI - Low root temperature effects on soybean nitrogen metabolism and photosynthesis. AB - The influences of low root temperature on soybeans (Glycine max [L.] Merr. cv. Wells) were studied by germinating and maintaining plants at root temperatures of 13 and 20 C through maturity. At 42 days from the beginning of imbibition, 13 and 20 C plants were switched to 20 and 13 C, respectively. Plants were harvested after 63 days. Control plants (13 C) did not nodulate, whereas those switched to 20 C did and at harvest had C(2)H(2) reduction rates of 0.2 micromoles per minute per plant. Rates of C(2)H(2) reduction decreased rapidly in plants switched from 20 to 13 C; however, after 2 days, rates recovered to original levels (0.8 micromoles per minute per plant) and then began a slow decline until harvest. Arrhenius plots of C(2)H(2) reduction by whole plants indicated a large increase in the energy of activation below the inflection at 15 C. Highest C(2)H(2) reduction rates (1.6 micromoles per minute per plant) were at 58 days for the 20 C control. Root respiration rates followed much the same pattern as C(2)H(2) reduction in the 20 C control and transferred plants. At harvest, roots from 13 C treated plants had the highest activities for malate dehydrogenase, glutamate oxaloacetate transaminase, and phosphoenolpyruvate carboxylase. Roots from transferred plants had intermediate activities and those from the 20 C treatment the lowest activities. Newly formed nodules from plants switched from 13 to 20 C had much higher glutamate dehydrogenase than glutamine synthetase activity.Photosynthetic rates on a leaf area basis were about three times as high in the 20 C control as compared to 13 C control plants. Photosynthetic rates of plants switched from 20 to 13 C decreased to less than half the original rate within 2 days. Photosynthetic rates of plants switched from 13 to 20 C recovered to rates near those of the 20 C control plants within 2 weeks. All leaf enzymes assayed at harvest, with the exception of nitrate reductase, were highest in activity in the 20 C control plants. PMID- 16660845 TI - Phospholipid metabolism in plant mitochondria: submitochondrial sites of synthesis. AB - Intact mitochondria from the endosperm of castor bean were isolated on linear sucrose gradients. These mitochondria were ruptured and the membranes separated on discontinuous sucrose gradients into outer membrane, intact inner membrane, and ruptured inner membrane fractions. Each membrane fraction was examined for its capacity to synthesize phosphatidylglycerol, CDP-diglyceride, phosphatidylcholine via methylation, and phosphatidic acid. The syntheses of phosphatidylglycerol, CDP-diglyceride, and phosphatidylcholine were localized exclusively in the inner mitochondrial membrane fractions while phosphatidic acid synthesis occurred in both the inner and outer mitochondrial membranes. PMID- 16660846 TI - Starch and its component ratio in developing cotton leaves. AB - During cotton leaf development, starch accumulation was characterized by an initial rise to a maximum at the second to the fourth leaf from the apex. Then, starch content progressively decreased with leaf age. Starch accumulation was inversely related to the ratio of amylopectin to amylose. Differences between leaves in this ratio resulted from variations in both amylose and amylopectin levels. Fluctuations in amylose levels were more extreme than those of amylopectin.During the diurnal cycle, amylopectin was accumulated more than amylose in both young and old leaves during the day. During the night, amylopectin was degraded more than amylose in young leaves and vice versa in older leaves. The rate of change of the amylopectin to amylose ratio during the day was consistently higher than that during the night. PMID- 16660847 TI - Long Distance Transport in Macrocystis integrifolia: II. Tracer Experiments with C and P. AB - Discs from mature regions of Macrocystis blades picked up significantly more [(32)P]phosphate from the ambient medium than similar discs from young meristematic regions, and this uptake was higher in light than in darkness. Double-labeling experiments with NaH(14)CO(3) and [(32)P]phosphate, using intact fronds as well as cut frond segments, indicated that (32)P was translocated from mature blades to sink regions at velocities of 25 to 45 centimeters per hour, velocities comparable to (14)C translocation velocity in the same material. There was a slight delay in transport of (32)P which may be due to a delay in loading or to a high metabolism of (32)P in the transporting channels. Histoautoradiography of stipe segments in the translocation pathway indicated that transport of label occurred in the peripheral parts of medulla. An analysis of (32)P-labeled compounds in the fed blade and in the sieve tube sap, collected from basal cut ends of stipes, indicated major differences in labeling patterns. In the blade, a high proportion of (32)P was recovered as inorganic phosphate and relatively small amounts were found in hexose mono- and diphosphates, UDPG and ATP. In the sieve tube sap, however, only a small amount of (32)P was present as inorganic phosphate, a large proportion was found in hexose mono- and diphosphates, and appreciable amounts were present in ATP and UDPG. PMID- 16660848 TI - Influence of Cotyledons upon alpha-Amylase Activity in Pea Embryonic Axes. AB - alpha-Amylase activity remained relatively low in the axes of intact etiolated pea seedlings; the activity was predominantly confined to the epicotyl. Starch accumulated slightly. When the cotyledons were removed and the axes cultured on medium containing no carbon source, the starch reserve in the axes disappeared within a few days. This was accompanied by a 10- to 15-fold increase in alpha amylase activity, in the absence of additional epicotyl growth. The phenonemon was observed for axes throughout early growth, although the relative accumulation of alpha-amylase activity in cultured axes was less for older seedlings. This change was attributed to a reduced response by nongrowing tissues. There was no corresponding change in beta-amylase activity. These observations, described for several varieties of peas, demonstrate the control of cotyledons upon the utilization of stored reserves within the axis, with alpha-amylase as a key enzyme. PMID- 16660849 TI - Involvement of Nonhistone Chromosomal Proteins in Transcriptional Activity of Chromatin during Wheat Germination. AB - To clarify how the transcriptionally inactive chromatin of dormant wheat seed embryos becomes active during germination, we studied two kinds of chromatin associated proteins: histones and nonhistone proteins found in wheat germ.Two major nonhistone proteins were solubilized from purified germ chromatin with 5 molar urea, and were separated from histones and chromosomal RNA by BioRex-70 resin and diethylaminoethyl-cellulose chromatography, respectively. We found that purified 5 molar urea-soluble nonhistone proteins, including the two major nonhistone proteins, had no effect on the transcription of native wheat DNA.Two kinds of chromatins were reconstituted by gradient dialysis from a mixture of DNA, germ histones, and nonhistone proteins derived from germs or germinated seedlings. Reconstituted chromatins had a 1.4- to 1.6-fold higher protein content than DNA content, protein components similar to native chromatin, and had only about 17 to 25% and 37% the transcriptional activities of native seedling and germ chromatins, respectively. Using the transcriptional activity of chromatin reconstituted from DNA and histones alone as a standard, one kind of reconstituted chromatin containing nonhistone proteins of germ was only about one fourth as active as the standard. Another with nonhistone proteins from seedlings was about 1.3 to 1.5 times more active than the standard.The ratio of histones to DNA content is approximately 1.3 during germination, but the proportion of histone H1 to the total histones is reduced. PMID- 16660850 TI - Tissue Distributions of Dhurrin and of Enzymes Involved in Its Metabolism in Leaves of Sorghum bicolor. AB - The tissue distributions of dhurrin [p-hydroxy-(S)-mandelonitrile-beta-d glucoside] and of enzymes involved in its metabolism have been investigated in leaf blades of light-grown Sorghum bicolor seedlings. Enzymic digestion of these leaves using cellulase has enabled preparations of epidermal and mesophyll protoplasts and bundle sheath strands to be isolated with only minor cross contamination. Dhurrin was located entirely in the epidermal layers of the leaf blade, whereas the two enzymes responsible for its catabolism, namely dhurrin beta-glucosidase and hydroxynitrile lyase, resided almost exclusively in the mesophyll tissue. The final enzyme of dhurrin biosynthesis, uridine diphosphate glucose:p-hydroxymandelonitrile glucosyltransferase, was found in both mesophyll (32% of the total activity of the leaf blade) and epidermal (68%) tissues. The bundle sheath strands did not contain significant amounts of dhurrin or of these enzymes. It was concluded that the separation of dhurrin and its catabolic enzymes in different tissues prevents its large scale hydrolysis under normal physiological conditions. The well documented production of HCN (cyanogenesis), which occurs rapidly on crushing Sorghum leaves, would be expected to proceed when the contents of the ruptured epidermal and mesophyll cells are allowed to mix. PMID- 16660851 TI - Relationships between Stomatal Behavior and Internal Carbon Dioxide Concentration in Crassulacean Acid Metabolism Plants. AB - Measurements of internal gas phase CO(2) concentration, stomatal resistance, and acid content were made in Crassulacean acid metabolism plants growing under natural conditions. High CO(2) concentrations, sometimes in excess of 2%, were observed during the day in a range of taxonomically widely separated plants (Opuntia ficus-indica L., Opuntia basilaris Engelm. and Bigel., Agave desertii Engelm., Yucca schidigera Roezl. ex Ortiges, Ananas comosus [L.] Merr., Aloe vera L., Cattleya sp. and Phalanopsis sp.) and below ambient air concentrations were observed at night.Stomatal resistance was always high when CO(2) concentration was high and experiments in which attempts were made to manipulate internal CO(2) concentrations gave data consistent with stomatal behavior in Crassulacean acid metabolism being controlled by internal CO(2) concentration. Exogenous CO(2) applied in darkness at a concentration similar to those observed in the light caused stomatal resistance to increase.In pads of Opuntia basilaris Engelm. and Bigel. subjected to severe water stress internal gas phase CO(2) concentrations exhibited fluctuations opposite in phase to fluctuations in acid content. Stomatal resistance remained high and the opening response to low CO(2) concentration was almost entirely eliminated. PMID- 16660852 TI - Time course of photosynthetic response to changes in incident light energy. AB - The response of whole leaf photosynthetic rate in Fragaria virginiana to sudden changes in photosynthetically active radiation (PAR) is described. Two components of the response, consisting of a time lag and time constant, are estimated under varying PAR changes for plants grown under two different light regimes. Both the time lag and time constant are found to vary with PAR but not with growth light regime. A model of Thornley for leaf photosynthetic response is refuted and an alternative form is discussed. PMID- 16660853 TI - Possible Involvement of the Alternative Respiration System in the Ethylene stimulated Germination of Cocklebur Seeds. AB - Respiration of nondormant upper cocklebur (Xanthium pensylvanicum Wallr.) seeds was enhanced by exogenous C(2)H(4), proportionally to the concentration of C(2)H(4) and the duration of presoaking of the seeds. Benzohydroxamic acid (BHM) and salicylhydroxamic acid (SHM), inhibitors of alternative respiration, inhibited both the germination of nondormant lower cocklebur seeds and the respiration of the upper seeds presoaked for periods of 12 to 30 hours. Both the growth and respiration of axial and cotyledonary tissues were also inhibited by BHM. Moreover, BHM inhibited both the C(2)H(4)-induced germination of the upper seeds and their C(2)H(4)-stimulated respiration; the inhibition occurred only with concomitant addition of C(2)H(4) and BHM. The respiration of seeds with a secondary dormancy induced by presoaking for prolonged periods was markedly stimulated by C(2)H(4) but not suppressed by BHM. It was suggested that the alternative respiration system may be involved in the normal germination process of cocklebur seeds, secondary dormancy may result from its inactivation, and C(2)H(4) may exert its germination-promoting action by stimulating the alternative respiration. The effects of BHM and SHM can suggest but not prove the involvement of the alternative respiration in seed germination. PMID- 16660854 TI - Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: V. Effect of Abscisic Acid on Indoleacetic Acid Metabolism and Ethylene Evolution. AB - The wilty tomato mutant flacca, the normal cultivar Lycopersicon esculentum Mill. Rheinlands Ruhm, and abscisic acid-induced phenotypic revertants were compared with respect to ethylene evolution, activity of tryptophan aminotransferase, and [1-(14)C]indoleacetic acid decarboxylation.The level of ethylene evolution was higher in flacca plants than in the normal cultivar. Ethylene evolution was reduced to the wild type level in abscisic acid-induced phenotypic revertants and to a lesser extent in mutant plants grown under humid conditions. Leaf epinasty, which characterized flacca plants in the present experiments, did not appear in absciscic acid-treated mutant plants, but did appear under high humidity. Tryptophan aminotransferase activity, similar to ethylene evolution, was higher in flacca plants and was reduced to the normal level by abscisic acid treatment. Indoleacetic acid decarboxylation was similar in mutant and normal plants, but was increased by abscisic acid treatment. The relationships among ethylene, auxin, and the morphological symptoms which characterize the mutant are discussed. PMID- 16660855 TI - Spatial Distribution of Circadian Clock Phase in Aging Cultures of Neurospora crassa. AB - Neurospora crassa has been utilized extensively in the study of circadian clocks. Previously, the clock in this organism has been monitored by observing the morphological and biochemical changes occurring at the growing front of cultures grown on solid medium. A method has been developed for assaying the clock in regions of the culture behind the growing front, where no apparent morphological changes occur during the circadian cycle. Using this assay with Petri dish cultures that were 2 to 7 days old, the presence of a functional circadian clock not only at the growing front but in all other regions of the culture as well was demonstrated. Furthermore, the entire culture is not in the same phase, but shows a gradient of phases which is a function of the length of time the clock in a given part of the culture has been free-running. This gradient may be the result of a somewhat longer period of the oscillator behind the growing front compared to that at the growing front. The phase differences within a single culture of interconnected mycelium demonstrate the absence of total internal synchronization between adjacent regions of the hyphae under these conditions. PMID- 16660856 TI - Temperature Interactions with Growth Regulators and Endogenous Gibberellin-like Activity during Seedstalk Elongation in Carrots. AB - Stecklings (roots) of three cultivars of carrots (Daucus carota L.) were vernalized 10 weeks at 5 C and subsequently grown at each of three greenhouse night/day temperature regimes: high (27/32 C), medium (21/27 C), and low (15/21 C). Floral differentiation occurred first in the easy bolting cv. Scarlet Nantes, intermediate in cv. Danvers 126, and last in cv. Royal Chantenay. Stem elongation arising from the subapical meristematic region always preceded floral differentiation. Extractable gibberellin-like activity in carrot stem apices increased from harvest during the 10-week vernalization period, then remained constant even though floral differentiation and stem elongation occurred during an additional 20-week cold storage period. Low temperature had both an inductive and a direct effect on reproductive development depending on length of low temperature exposure.After 10 weeks vernalization at 5 C, high greenhouse temperature severely reduced ultimate seedstalk height and the endogenous gibberellinlike activity decreased rapidly during the first 3 weeks in the greenhouse. At the low greenhouse temperature, activity remained fairly constant during the 10-week sampling period. Changes in endogenous gibberellinlike activity were related with stem elongation, but not with floral initiation. Exogenous gibberellic acid (GA(3)) applied following vernalization prevented the inhibitory effect of high greenhouse temperature on seedstalk elongation and resulted in seedstalk heights comparable to untreated controls grown at the low greenhouse temperature. Exogenous applications of succinic acid-2,2 dimethylhydrazide and chlormequat reduced seedstalk height of carrot plants grown at the medium and low greenhouse temperatures to that of untreated controls grown at high temperature. Exogenous growth regulators and greenhouse temperature affected seedstalk elongation, but did not affect the number of plants that flowered. PMID- 16660857 TI - Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: II. Kinetic and Spectral Studies. AB - Red light induces two distinct inhibition responses in mesocotyls of etiolated corn seedlings. A light dose of 10 nanoeinsteins per square centimeter is saturating for the more sensitive response, whereas doses above 1,000 nanoeinsteins per square centimeter are required to exceed the threshold sensitivity of the less sensitive one. The sensitive response can be detected within 20 minutes of the onset of illumination whereas the other response does not become apparent until more than 4 hours after the beginning of irradiation. The reciprocity law is valid for the first response, but probably not for the second. An action spectrum for the first response shows two maxima, one at 640 nanometers and the other between 660 and 670 nanometers, with a pronounced minimum near 650 nanometers. The effects both of 640 and 665 nanometers of light were reversible by far red light, but doses of far red required for full reversibility were almost three orders of magnitude greater than the doses of red required either to saturate the initial inhibition or to reverse the effect of far red light. The results suggest that corn may contain a red-absorbing pigment other than phytochrome which in some way interacts with phytochrome in the inhibition of mesocotyl elongation by red light. PMID- 16660858 TI - Malate synthase activity in cotton and other ungerminated oilseeds: a survey. AB - Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute.Malate synthase activity was measurable in all seed types examined, whereas isocitrate lyase could not be detected in any of the seeds. We suggest that synthesis of malate synthase during seed development is universal among oilseeds in the absence of glyoxylate-cycle-associated isocitrate lyase activity. PMID- 16660859 TI - Susceptibility of plants to vascular disruption by macromolecules. AB - The xylem of alfalfa (Medicago sativa L.) was found to be susceptible to vascular obstruction by picomole quantities of dextrans. Not all parts of the xylem were equally susceptible to this plugging. The quantity of dextran of 2 x 10(6) molecular weight required to stop vascular flow was 8 picomoles in petiole junctions and 0.4 picomole in leaflet veins. Vascular flow through stems was greatly reduced but not stopped, even by over 150 picomoles of the dextran. The ability of dextrans to interfere with vascular conductance was directly correlated with their molecular weight. Dextrans of molecular weight less than 250,000 had little ability to stop vascular flow. PMID- 16660860 TI - Uptake and Utilization of Xylem-borne Amino Compounds by Shoot Organs of a Legume. AB - Amino compounds representative of the major N solutes of xylem sap were pulse-fed (10 to 20 minutes) singly in (14)C-labeled form to cut transpiring shoots of white lupin (Lupinus albus L.). (14)C distribution was studied by autoradiography and radioassays of phloem sap, leaflet tissues, and shoot parts harvested at intervals after labeling. Primary distribution of N by xylem was simulated using a 20-minute labeling pulse followed by a 30-minute chase in unlabeled xylem sap. Shoots fed (14)C-labeled asparagine, glutamine, valine, serine, or arginine showed intense labeling of leaflet veins and marked retention (35 to 78%) of (14)C by stem + petioles. Shoots fed (14)C-labeled aspartic acid or glutamic acid showed heaviest (14)C accumulation in interveinal regions of leaflets and low uptake (11 to 20%) of (14)C by stem + petioles. Departing leaf traces were major sites of uptake of all amino compounds, and the implications of this were evaluated. Fruits acquired only 1 to 5% of the fed label directly from xylem, but more than doubled their intake during the period 30 to 160 minutes after feeding through receipt of (14)C transferred from xylem to phloem in stem and leaves. (14)C-Labeled asparagine and valine transferred directly from xylem to phloem, but the (14)C of (14)C-labeled aspartic acid and arginine appeared in phloem mainly as metabolic products of the fed compound. The labeling of the soluble pool of leaflets reflected these differences. The significance of heterogeneity in distribution and metabolism of xylem amino compounds in the shoot was discussed. PMID- 16660861 TI - Transport of organic solutes in Phloem and xylem of a nodulated legume. AB - Collections of xylem exudate of root stumps or detached nodules, and of phloem bleeding sap from stems, petioles, and fruits were made from variously aged plants of Lupinus albus L. relying on nodules for their N supply. Sucrose was the major organic solute of phloem, asparagine, glutamine, serine, aspartic acid, valine, lysine, isoleucine, and leucine, the principal N solutes of both xylem and phloem. Xylem sap exhibited higher relative proportions of asparagine, glutamine and aspartic acid than phloem sap, but lower proportions of other amino acids. Phloem sap of petioles was less concentrated in asparagine and glutamine but richer in sucrose than was phloem sap of stem and fruit, suggesting that sucrose was unloaded from phloem and amides added to phloem as translocate passed through stems to sinks of the plant. Evidence was obtained of loading of histidine, lysine, threonine, serine, leucine and valine onto phloem of stems but the amounts involved were small compared with amides. Analyses of petiole phloem sap from different age groups of leaves indicated ontogenetic changes and effects of position on a shoot on relative rates of export of sucrose and N solutes. Diurnal fluctuations were demonstrated in relative rates of loading of sucrose and N solutes onto phloem of leaves. Daily variations in the ability of stem tissue to load N onto phloem streams were of lesser amplitude than, or out of phase with fluctuations in translocation of N from leaves. Data were related to recent information on C and N transport in the species. PMID- 16660862 TI - Hysteresis in the responses of membrane potential, membrane resistance, and growth rate to cyclic temperature change. AB - Measurements of electrical potential, membrane resistance, and elongation rate have been carried out on the developing pollen tube of Oenothera drummondii.The plasmalemma potential was observed to be -138 millivolts +/- 19 (sd). Approximately 70% of this potential was apparently due to the operation of an electrogenic pump(s). KCN rapidly and reversibly depolarized the potential to about -40 millivolts. Lowering the temperature from 20 to 4 C brought about similar rapid depolarization. The effects of KCN and of temperature were in no degree additive. KCN had only a small effect on membrane resistance. In contrast, the latter was markedly increased by lowering the temperature.When the pollen tubes were submitted to cyclic temperature changes striking hysteresis effects were observed in the response of all three parameters, membrane potential, resistance, and growth rate. The hysteresis pattern for potential differed from that for resistance but resembled that for growth rate (measured simultaneously on the same pollen tube). The correlation coefficient between potential and growth rate was very high.The probable relevance to our results of the hysteresis phenomena associated with "clustering" and phase transition in lipids is pointed out. Attention is also drawn to the possible significance of the large difference between the electric potentials at the start of the cooling and heating paths, respectively. PMID- 16660863 TI - Cysteinyl-tRNA Synthetase from Astragalus Species. AB - l-Cysteinyl-tRNA synthetases (EC 6.1.1.16) from four Astragalus species were partially purified. The substrate specificities of the cysteinyl-tRNA synthetase from three selenium accumulator species (A. crotalariae, A bisulcatus, and A. racemosus) were compared with those from two nonaccumulator species (A. lentigenosus and Phaseolus aureus). All species had similar K(m) values for cysteine, selenocysteine, and alpha-aminobutyric acid except A. bisulcatus which failed to use selenocysteine as a substrate and which had a K(m) for cysteine four times greater than the K(m) values for other species. PMID- 16660864 TI - Effects of red, far red, and blue light on enhancement of nitrate reductase activity and on nitrate uptake in etiolated rice seedlings. AB - The effects of red (R), far red (FR), or blue light (B) on the enhancement of nitrate reductase (NR) activity and on nitrate uptake in etiolated rice seedlings were examined. On 5-minute illumination followed by 12-hour dark, R caused marked increase of NR activity, but FR and B caused only slight increase. Illumination with 560 ergs per square centimeter per second of R for 5 minutes caused maximal increase. The effect of R was almost completely counteracted by subsequent illumination with 2,000 ergs per square centimeter per second of FR for 10 minutes, indicating that NR induction was mediated by phytochrome. Exogenous supply of inducer nitrate was not required during the 5-minute illumination and the R-FR cycles, if the seedlings were transferred to nitrate solution at the beginning of the dark incubation. NR activity in the shoots was found high when shoots were illuminated but was low when only roots were illuminated. On continuous illumination for 12 hours, B had more effect on NR increase than R.Nitrate uptake during 6-hour dark was not increased by exposure to R, FR, or B for 5 minutes at the beginning. On continuous illumination for 6 hours, R slightly increased nitrate uptake, whereas FR and B had no effect. PMID- 16660865 TI - Photoconversion of Photochlorophyllide in the y-1 Mutant of Chlamydomonas reinhardtii. AB - Dark-grown y-1 mutant cells of Chlamydomonas reinhardtii accumulate protochlorophyllide (Pchlide) in both 635 nanometers (P635) and 650 nanometers (P650) forms. Plastids in these cells lack the normal thylakoid membrane structure except some remnants of membrane vesicles. Using difference spectrophotometry, P635 is shown to be photoconverted to chlorophyllide at 672 nanometers (C672) and P650 is photoconverted to C688 followed by a rapid shift to C672 (Shibata shift) and regeneration of P650. Some of the Pchlide is not photoconverted despite repeated illumination. Although P650 is destroyed by freezing and thawing, it is not transformed into P635. Freezing and thawing treatment also made Pchlide no longer photoactive. PMID- 16660866 TI - Phototropic Responses of Vaucheria geminata to Intermittent Blue Light Stimuli. AB - Phototropic responses of a tip-growing coenocytic alga Vaucheria geminata to intermittent blue light pulses were analyzed. Curvatures caused by repeated light pulses separated by dark intervals of various lengths were as large as those obtained by continuous light of the same incident energy, unless the length of the dark interval exceeded a critical value of about 30 to 40 seconds. If the dark interval was longer than the critical length, bending no longer took place. Another response to intermittent irradiation was found. In a narrow range of light pulse and dark interval lengths, i.e. light pulses longer than 10 milliseconds and dark intervals shorter than a value ranging from 15 to 150 milliseconds, intermittent irradiation was more effective than continuous irradiation of the same total energy. In this region, the maximum response to repeated pulses was equal to the curvature caused by a continuous irradiation of the same total elapsed time. It is evident that in this condition the dark interval is not perceived and the alga responds as if it were in light. The results suggest that two different dark reactions may be involved in the phototropic response of this algal cell. PMID- 16660867 TI - Temperature Regulation of Growth and Endogenous Abscisic Acid-like Content of Tulipa gesneriana L. AB - The ontogenetic changes of dry matter and abscisic acid (ABA)-like content in the component organs of Tulipa gesneriana L. ;Paul Richter' and ;Golden Melody' under two temperature storage regimes were determined. The organ dry matter and ABA showed marked differences during 13 and 5 C dry storage and during subsequent growth at 13 C. Scale dry matter of both cultivars declined sharply when grown at 13 C. The basalplate of the cultivars showed an initial gain in dry matter, but declined subsequently. The shoot of both cultivars stored at 13 C exhibited greater dry matter gain than at 5 C. In contrast, the bulblets of the cultivars at 5 C showed a much higher rate of dry matter accumulation than at 13 C.An inhibitory substance extracted from tulip bulb organs co-chromatographed with authentic ABA and had identical thin layer chromatographic R(F) values of ABA in five solvent systems. The total ABA content per bulb increased 3-fold in ;Golden Melody' and 2- to 4-fold in ;Paul Richter' during the course of the temperature treatments. ABA was low in the scales and shoot, but it was high in the basalplate, bulblets, and roots. It is suggested that the probable ABA biosynthetic sites of tulip bulb are the developing bulblets, basalplate, and roots. PMID- 16660868 TI - Titration of Isolated Cell Walls of Lemna minor L. AB - A theoretical model has been built to bypass the equation of titration of the cell wall. This equation, which is an extension of the Henderson-Hasselbach equation, underlines the importance of the exchange constant, the ionic strength as well as the rate of neutralization. The model is restricted to the case when the ionization degree is equal to the neutralization degree. The shape of the titration curve is shown to be strongly dependent on the valency of the base used.Experimental results have shown that isolated cell walls bear at least two kinds of sites. The first sites which are titrated after a short time of equilibration are attributed to polyuronic acids (capacity: 0.3 milliequivalents per gram fresh cell walls). The second sites, are obtained after a long time of equilibration (capacity: 1.2 to 1.3 milliequivalents per gram, fresh cell walls). Titrations have been performed with different bases [KOH, NaOH, and Ca(OH)(2)] and under different ionic strengths.The results obtained with NaOH and KOH do not exhibit any difference of selectivity. Conversely, the sites have a much bigger affinity for the Ca(2+) ions than for the monovalent ones. The apparent pKa of the uronic acids was estimated to lie between 3.0 and 3.4; this is consistent with the values obtained with polyuronic acid solutions. PMID- 16660869 TI - Hydrolytic enzymes in the central vacuole of plant cells. AB - The hydrolase content of vacuoles isolated from protoplasts of suspension cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.THE INTRACELLULAR ACTIVITIES OF THE FOLLOWING ACID HYDROLASES WERE PRIMARILY LOCALIZED IN THE VACUOLE OF TOBACCO CELLS: alpha mannosidase, beta-N-acetylglucosaminidase, beta-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter (24% sucrose, w/w). PMID- 16660870 TI - Role of polygalacturonase in bean leaf abscission. AB - The role of polygalacturonase in leaf abscission was studied in explants of Phaseolus vulgaris L. cv. Red Kidney. Bean polygalacturonase was partially characterized and comparisons were made between the bean enzyme and previously reported higher plant polygalacturonases. Polygalacturonase isolated from bean leaf abscission zones has a pH optimum between 4.5 and 5.0 and hydrolyzed polygalacturonides in an exo-fashion. Activity was found to be higher with a deesterified substrate than with an esterified pectin. No correlation between polygalacturonase activity and abscission was observed. Activity remained virtually constant over the course of abscission in explants aged either in air or in ethylene. The enzyme was primarily localized in the abscission zone, however, indicating a possible involvement in the abscission process. A theoretical model which could explain the relationship between polygalacturonase and bean leaf abscission is discussed. PMID- 16660871 TI - Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen. AB - O(2) irreversibly inactivates hydrogenase from Chlamydomonas reinhardi. The mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of O(2) (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres(-1) minute(-1) (1.4 x 10(5) molar(-1) minute(-1)). At levels above 0.01 atmosphere O(2), the increased numbers of O(2) molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one O(2) molecule and resulting in lowered rates of inactivation.CO is a reversible inhibitor of hydrogenase acting competitively against H(2). The K(i) for CO is 0.0010 atmosphere. CO antagonizes O(2) inactivation. In a period when complete inactivation by O(2) would usually occur, the presence of CO greatly reduces the inactivation rate.After 3 hours of adaptation in whole cells, the presence of H(2) lowers the rate of deadaptation of hydrogenase. Inasmuch as H(2) promotes increased O(2) uptake the cellular concentration of O(2) is likely to be lower. After 48 hours of adaptation O(2) uptake is reduced even when H(2) is present and the pattern of deadaptation under O(2) with and without H(2) and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of cell-free hydrogenase by O(2) may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells. PMID- 16660872 TI - Estimation of potassium recirculation in tomato plants by comparison of the rates of potassium and calcium accumulation in the tops with their fluxes in the xylem stream. AB - A procedure to estimate the extent of K recirculation in plants is proposed. This is based on the ratio of the upward fluxes of K to Ca in the xylem sap from root to shoot with the ratio of K to Ca accumulation in plant tops.In a preliminary investigation the factors influencing the K to Ca ratio in the xylem sap were considered. Tomato plants were grown at three levels of K nutrition and harvested at different times during the 24-hour day period. It was shown that the K to Ca ratio in xylem sap changed dramatically depending on the time of sap collection after decapitation, the values falling from over 2 to less than unity over the 4 hour period of collection. Diurnal effects on exudation were less marked but also of significance. The level of K nutrition was of little importance. It is suggested that a representative xylem sap from tomato plants can best be obtained from samples taken between 15 and 60 minutes after decapitation.In a second experiment K recirculation was estimated. At nine harvesting stages over a 24 hour period the K to Ca ratio in the xylem sap was invariably higher than the K to Ca ratio of accumulation in the tops over the same period. From this information it was calculated that about 20% of the upward flux of K in the xylem stream resulted from recirculated K. PMID- 16660873 TI - Helminthosporium maydis Race T Toxin Induces Leakage of NAD from T Cytoplasm Corn Mitochondria. AB - The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD(+)-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD(+). Added NAD(+) partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD(+) linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD(+) and other coenzymes. PMID- 16660874 TI - Disappearance of porphobilinogen deaminase activity in leaves before the onset of senescence. AB - The activity of porphobilinogen deaminase was measured in young and senescent or mature leaves of pepper (Capsicum annuum), and poinsettia (Euphorbia pulcherrima). Whereas high activity was found in the crude extracts of the young leaves, almost no activity was found in the extracts of senescent or mature leaves. The decrease in deaminase activity was not due to the presence of an isolatable inhibitor. By purifying the crude enzyme extracts from leaves of different ages on DEAE-cellulose columns it was shown that the decrease in deaminase activity was due to a real decrease in the amount of enzyme. Fruiting also decreased porphobilinogen deaminase activity. Several kinetic constants of the C. annuum deaminase were determined. PMID- 16660875 TI - beta-1,3-Glucan in Developing Cotton Fibers: Structure, Localization, and Relationship of Synthesis to That of Secondary Wall Cellulose. AB - Evidence is presented for the existence of a noncellulosic beta-1,3-glucan in cotton fibers. The glucan can be isolated as distinct fractions of varying solubility. When fibers are homogenized rigorously in aqueous buffer, part of the total beta-1,3-glucan is found as a soluble polymer in homogenates freed of cell walls. The proportion of total beta-1,3-glucan which is found as the soluble polymer varies somewhat as a function of fiber age. The insoluble fraction of the beta-1,3-glucan remains associated with the cell wall fraction. Of this cell wall beta-1,3-glucan, a variable portion can be solubilized by treatment of walls with hot water, a further portion can be solubilized by alkaline extraction of the walls, and 17 to 29% of the glucan remains associated with cellulose even after alkaline extraction. A portion of this glucan can also be removed from the cell walls of intact cotton fibers by digestion with an endo-beta-1,3-glucanase. The glucan fraction which can be isolated as a soluble polymer in homogenates freed of cell walls is not associated with membranous material, and we propose that it represents glucan which is also extracellular but not tightly associated with the cell wall. Enzyme digestion studies indicate that all of the cotton fiber glucan is beta-linked, and methylation analyses and enzyme studies both show that the predominant linkage in the glucan is 1 --> 3. The possibility of some minor branching at C-6 can also be deduced from the methylation analyses. The timing of deposition of the beta-1,3-glucan during fiber development coincides closely with the onset of secondary wall cellulose synthesis. Kinetic studies performed with ovules and fibers cultured in vitro show that incorporation of radioactivity from [(14)C]glucose into beta-1,3-glucan is linear with respect to time almost from the start of the labeling period; however, a lag is observed before incorporation into cellulose becomes linear with time, suggesting that these two different glucans are not polymerized directly from the same substrate pool. Pulse-chase experiments indicate that neither the beta-1,3-glucan nor cellulose exhibits significant turnover after synthesis. PMID- 16660876 TI - Carbohydrate Levels and Photoassimilate Export from Leaves of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc. AB - Exposure of white bean seedlings to phytotoxic burdens of Co, Ni, or Zn reduced the export of (14)C-photoassimilates from the nearly fully expanded unifoliate leaves. Little (14)C reached the major sink areas, the young trifoliate leaves and the root tips, of seedlings exposed to metal. The unifoliate leaves accumulated sucrose, reducing sugars, and starch. These effects were evident within 1 or 2 days. PMID- 16660877 TI - Callose Deposition and Photoassimilate Export in Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc. AB - Callose accumulated on sieve plates of phloem of white bean seedlings exposed to excess Co, Ni, or Zn. The callose deposits ranged in thickness and were most pronounced in midribs of unifoliate leaves and their subtending petioles. Lesser callose deposits were found in stems. Although translocation of (14)C was reduced drastically in seedlings exposed to excess metal, no correlation was found between translocated (14)C and the amount of callose in the petioles. It is concluded that the inhibition of phloem translocation in seedlings exposed to excess metal is due to effects other than callose deposition. PMID- 16660878 TI - Stickiness to Glass: Circadian Changes in the Cell Surface of Chlamydomonas reinhardi. AB - Conditions were found in which Chlamydomonas reinhardi exhibits a circadian alteration of its cell surface, measured as ability to stick to glass. Under these same conditions the cells also show circadian rhythms of cell division and release of daughter cells. The three rhythmic phenomena were shown to have typical properties of rhythms controlled by the biological clock. The rhythm of stickiness was used to demonstrate that in a mixed culture containing two cell populations with natural periods differing by 2 to 3 hours, the cells did not mutally entrain each other and that this rhythm could be successfully applied in an enrichment procedure for mutants of the biological clock. Stickiness was shown to be independent of growth and motility of the cells and unaffected by red or far red illimination. Minimally sticking cells did not affect the sticking of maximally sticking cells in a mixed culture; nor was there a progressive increase in stickiness shown at the minimum from one cycle to the next in a pure culture. These results indicate that sticking probably is not mediated by long lived adhesive material or enzymes excreted into the medium. Several tests of the sensitivity of stickiness to replacement of the growth medium by distilled water or water containing various compounds suggest that ions might play an important role in the sticking reaction. PMID- 16660879 TI - Characterization of the Light-induced Transient States of the Chlorophyll Proteins 668 and 743 from Atriplex rosea. AB - The light-induced transient states of chlorophyll-protein 668 (Cp668) and its photoconverted from Cp743 were investigated using flash photolysis. Short lived transient species induced by a short flash were detected in both Cp668 and Cp743. The Cp668 transient had a half decay time of 2.0 milliseconds and showed a broad absorption band at 460 nanometers. The Cp743 transient had a half decay time of only 0.6 millisecond and had a major absorption peak at 410 nanometers in addition, to a broad absorption band around 530 nanometers. Both transient signals were quenched by oxygen. Cp668 had a temperature-dependent delayed fluorescence at room temperature with a half-life of 2.0 milliseconds, the same as the life-time of the absorption transient. This suggests that the transient species observed was a triplet state of chlorophyll.The light-induced transients of both Cp668 and Cp743 were formed with high efficiency. A very low quantum efficiency was found for the photoconversion of Cp668 to Cp743 suggesting another intermediate in the conversion sequence. The photoconversion reaction requires oxygen suggesting that an intermediate in the reaction sequence might be superoxide or singlet oxygen. PMID- 16660880 TI - Analysis of the Distribution of Potassium-stimulated Adenosine Triphosphatase Activity in Soybean Root. AB - The distribution of activity of a potassium-stimulated ATPase associated with the plasma membrane was determined in 4-day-old soybean roots. Changes in protein based specific activity of the enzyme coincided with developmental changes in the root. Activity was low in the region of the root cap, increased to a maximum in the meristematic region, decreased to a minimum as cell elongation proceeded, and then increased as lateral root development began. Analysis of the data indicated that fluctuations in enzyme activity can be described by a three-phase system which can be approximated by a linear-linear-linear piece-wise regression curve. The need for constructing a biological model to describe plasma membrane development is suggested. PMID- 16660881 TI - An electron microscope comparison of plasma membrane vesicles from meristematic and mature soybean root tissue. AB - Plasma membrane vesicles were isolated from homogenates of meristematic and mature soybean root tissue by differential sucrose gradient centrifugation. Vesicles were positively identified by the phosphotungstic acid-chromic acid procedure (PACP). The two preparations were comparable in size class distribution, mitochondrial contamination, and per cent plasma membrane vesicles present. Purity levels were estimated to be greater than 75%. The specificity of PACP was observed for a variety of cell types from both regions. Some variability in PACP staining was offset by careful modulation of the stain protocol and was found to be independent of developmental stage in subcellular fractions. Patchy or discontinuous staining, observed in both intact tissue and in subcellular fractions from both regions, was found to be a function of stain time. PMID- 16660882 TI - Developmental expression of genetically defined peptidases in maize. AB - The activities of genetically defined amino- and endopeptidases of maize were compared in pericarp, endosperm, and embryonic tissue of the maize kernel from 5 days postpollination until harvest. Activities were highest in the immature stages and declined as drying progressed. The expression of some of the peptidase genes contributed by the pollen parent was examined during early endosperm development in an F(1) cross. The paternally contributed peptidase variants could first be detected 7 days after pollination.During germination and early seedling growth the peptidase activities continued to decline in the endosperm but maintained high levels in the scutellum. The maize peptidases probably have no direct role in hydrolysis of endosperm storage proteins during germination and growth.The peptidase isozymes were associated with the soluble cytoplasmic fraction of the maize scutellum during early growth. Only one tissue specificity was found for the peptidases. Occurrence of a particular aminopeptidase isozyme, AMP2, is limited to endosperm tissue during kernel maturation. PMID- 16660883 TI - Influence of light and ambient carbon dioxide concentration on nitrate assimilation by intact barley seedlings. AB - The influence of light, dark, and ambient CO(2) on nitrate assimilation in 8- to 9-day-old barley seedlings was studied. To develop the photosynthetic apparatus fully, the seedlings were grown in nitrogen-free Hoagland solution for 5 days in darkness followed by 3 days in continuous light.The seedlings reduced nitrate and nitrite in both light and dark, although more slowly in darkness. The slower nitrate reduction in darkness was not due to decreased uptake, since the steady state internal concentration of nitrate was doubled. The faster nitrate reduction in light was attributed to recent products of photosynthetic CO(2) fixation supplying reducing energy, possibly by shuttle reactions between chloroplasts and cytoplasm. In carbohydrate-deficient tissue, it appeared that recently fixed photosynthate could supply all of the energy required for nitrate reduction. When sufficient metabolites were present in the green tissue, light was not obligatory for the reduction of nitrate and nitrite. PMID- 16660884 TI - Are the in vitro effects of disalicylidenepropanediamine mediated by salicylaldehyde? AB - Disalicylidenepropanediamine (DSPD) has been used widely to investigate photosynthesis using in vitro systems. Evidence is presented which shows that DSPD hydrolyzes rapidly in aqueous solution at pH 7.8 to salicylaldehyde and 1,3 diaminopropane. The effects attributed to DSPD could have been caused by salicylaldehyde. PMID- 16660885 TI - Isolation of Protoplasts and Chloroplasts from Flag Leaves of Triticum aestivum L. AB - Intact protoplasts and chloroplasts have been isolated from mature flag leaves of wheat (Triticum aestivum L.). Both showed high rates of photosynthesis, the best of which equaled those observed in the parent tissue (greater than 150 micromoles O(2) per milligram chlorophyll per hour). The presence of ethylenediaminetetraacetate and an alkaline medium (pH 8.4) were required in the isolation and assay for the achievement of maximum rates of photosynthesis by chloroplasts. Photosynthesis by isolated chloroplasts was inhibited at very low concentrations of external orthophosphate. PMID- 16660886 TI - Lithium Inhibition of the Thigmomorphogenetic Response in Bryonia dioica. AB - Pretreatment of young Bryonia dioica plants with lithium prevents the inhibition of elongation due to rubbing. Lithium treatment also suppresses the appearance of a specific cathodic isoperoxidase characteristic of rubbed plants. PMID- 16660887 TI - Inhibition of Polar Indole-3-acetic Acid Transport by Cycloheximide. AB - Cycloheximide inhibits polar indoleacetic acid transport in midrib tissues of leaves of citrus (Citrus sinensis [L.] Osbeck) and poplar (Populus deltoides Bartr.) as measured by the donor-receiver agar cylinder technique. It appears that the mechanism of auxin transport inhibition by cycloheximide consists in arresting protein synthesis and not in the disruption of energy flow. The interpretation of the data takes into account the involvement of either a carrier protein or auxin-induced proton excretion in auxin transport. PMID- 16660888 TI - Electron Spin Resonance of 2,2,6,6-Tetramethylpiperidine-1-Oxyl (TEMPO)-labeled Plant Leaves. AB - Temperature dependence for partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) between aqueous and lipid components of whole leaf tissue was measured. TEMPO is an electron spin resonance nitroxide label that has been used in model systems to detect membrane phase separations. Measurements were made on chilling sensitive tomato leaves, frost-sensitive potato leaves, and frost-hardy and supercooled wheat leaves. The results suggest a membrane phase separation at 11 C in tomato, 3 C in potato, and -11 C in wheat. PMID- 16660889 TI - Pyrimidine Metabolism in Lemna minor: II. Specific Inhibition of Plastid Replication in a Higher Plant Cytidine Deoxyriboside. AB - Short term photoheterotrophic growth of Lemna minor in the presence of 100 micrograms per milliliter (440 micromolar) cytidine deoxyriboside bleaches the fronds in the absence of effect upon growth rate (Frick 1978 Plant Physiol 61: 989-992). Serial sections of mesophyll cells of green and bleached fronds were compared in the light microscope. The number of plastids in cells of comparable size was reduced during bleaching by more than 50%. The total and average plastid profile area per section in the series was reduced on the order of 70%, and the summed plastid profile area over the entire cell was reduced about 90%. Thus, cytidine deoxyri-boside-bleached mesophyll cells contained fewer identifiable plastids (50%) of smaller volume (10-30%) than the green control cells. PMID- 16660890 TI - Reversal by Light of Ethylene-induced Inhibition of Spore Germination in the Sensitive Fern Onoclea sensibilis: An Action Spectrum. AB - Spores of the fern Onoclea sensibilis L. normally germinate to produce two cells of unequal size. The larger cell divides to produce the familiar heart-shaped prothallus. The smaller cell elongates and differentiates into the rhizoid but normally does not divide again. Onoclea spores germinate in complete darkness. Dark germination can be completely inhibited by ethylene gas (10 microliters per liter is saturating). This inhibition can be reversed by light. Broad band colored light studies were designed to determine which area of the spectrum was most effective in overcoming ethylene inhibition. White light treatment resulted in 17% germination. Blue light treatment resulted in 1% germination. Red light treatment resulted in 15% germination. Red light, therefore, was most effective and accounted for most of the effects of white light. A detailed action spectrum was constructed using narrow band interference filters in the wavelength range from 350 to 764 nanometers. The action spectrum has only one major peak at 711 nanometers. PMID- 16660891 TI - Factors Affecting Crown Gall Tumorigenesis in Tuber Slices of Jerusalem Artichoke (Helianthus tuberosus, L.). AB - Agrobacterium tumefaciens can induce tumors on thin slices which are excised from Jerusalem artichoke (Helianthus tuberosus) tubers and grown in culture on medium containing minerals and a carbon source. A comparative study was made of the kinetics of cell division in slices under three conditions: (a) slices which were untreated and showed only spontaneous (wound-induced) cell divisions; (b) slices treated with indoleacetic acid at several concentrations; and (c) slices treated with virulent or avirulent bacteria. The earliest spontaneous cell divisions were completed (as detected by the appearance of new daughter cell pairs) by about 3 hours. These cells divide only once. In indoleacetic acid-treated tissue, more cells divide, with the first cell pairs being detected slightly earlier than in slices not subjected to the hormone. The number of cells which divide is roughly proportional to auxin concentration. Tissue treated with virulent bacteria showed only the pattern of spontaneous cell division until about 72 hours, after which another burst of cell division commenced and continued indefinitely. The bacteria induced growths produced the unusual amino acids which are characteristic of crown gall tumors. The percentage of slices with tumors was sharply reduced if certain avirulent A. tumefaciens strains were applied prior to virulent strains. PMID- 16660892 TI - Long Distance Transport in Macrocystis integrifolia: I. Translocation of C labeled Assimilates. AB - Long distance transport of (14)C-labeled photoassimilate was studied in Macrocystis integrifolia Bory. Movement of label followed the source-sink relationship; mature blades closer to the holdfast with young 2 degrees and 3 degrees fronds transported mostly to the base, those closer to the frond apex transported mostly to the apex, and those in intermediate positions transported both acropetally and basipetally. The velocity of movement of (14)C as computed both from study of intact fronds and exudate was in the range of 35 to 72 centimeters per hour and these estimates are on the low side. The composition of the translocate as determined from intact fronds was the same as that determined from exudate analysis; furthermore, this composition was nearly identical with that of the photosynthate (40 to 50% mannitol and 40 to 50% amino acids). From these data we conclude that the exudate represents the sieve tube sap and that there is little if any selectivity exercised in the loading and translocation of photoassimilate. An analysis of translocated label in the growing apex is presented and indicates that the synthesis of polymeric compounds such as laminaran, alginate, cellulose, lipids, and "protein" occurs in situ from the transported mannitol and amino acids. Detailed data on chemical composition of sieve tube sap from M. integrifolia and M. pyrifera (L.) C.A. Agardh are given and compared with the sieve tube sap from higher plants. Finally, we show that stipe segments, 60 to 100 centimeters long with three to six attached blades, are useful for translocation studies in Macrocystis. PMID- 16660893 TI - Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest. AB - Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.Protease activity increased in nodules from harvested plants, reached a maximum at day 7 after harvest, and then declined to a level almost equal to the control by day 22 after harvest. Soluble protein and leghemoglobin decreased in nodules from harvested plants in an inverse relationship to protease activity.Nitrate reductase activity of nodules from harvested plants increased significantly within 24 hours and was inversely associated with acetylene reduction. The difference in nitrate reductase between nodules from harvested plants and control plants became less evident as shoot regrowth occurred and as acetylene reduction increased in the harvested plants.No massive loss of nodules occurred after harvest as evidenced by little net change in nodule fresh weight. There was, however, a rapid localized senescence which occurred in nodules of harvested plants. Histology of nodules from harvested plants showed that they degenerated at the proximal end after harvest. Starch in the nodule was depleted by 10 days after harvest. The meristem and vascular bundles of nodules from harvested plants remained intact. The senescent nodules began to regrow and fix nitrogen after shoot growth resumed. PMID- 16660894 TI - Legume Lectins: I. Immunological Cross-Reactions between the Enzymic Lectin from Mung Beans and other Well Characterized Legume Lectins. AB - A number of well characterized legume lectins including the enzymic lectin from Vigna radiata were examined for immunological relatedness. The immunological cross-reactions observed indicate that most of the legume lectins, including Vigna lectin, are evolutionarily closely related proteins. The possibility that these proteins are homologs with enzymic functions is discussed. PMID- 16660895 TI - Effect of ethanol, acetaldehyde, acetic Acid, and ethylene on changes in respiration and respiratory metabolites in potato tubers. AB - Ethanol, acetaldehyde, and acetic acid, when applied in a volatile state in air to potato tubers, led to a climacteric-like upsurge in respiration. The respiratory upsurge was markedly enhanced when the volatiles were applied in 100% O(2).Ethanol induced a decline in the level of 2-phosphoglyceric acid and phosphoenolpyruvate while leading to the accumulation of tricarboxylic acid cycle intermediates including isocitrate and alpha-ketoglutarate. The action of these compounds was similar to, but independent of, the action of ethylene. PMID- 16660896 TI - Stomatal response of populus clones to light intensity and vapor pressure deficit. AB - Responses of stomata of clones of Populus candicans Ait. x P. berolinensis Dipp. and Populus deltoides Bartr. x P. caudina (Ten.) Bugala to two levels of light intensity and vapor pressure deficit were studied in controlled environments. Significant stomatal responses to light and vapor pressure deficit were observed. Interactive effects of low light intensity and high vapor pressure deficit elicited greater stomatal closure than was obtained under low light or high vapor pressure deficit alone, indicating adaptation for increased water use efficiency under conditions unfavorable for photosynthesis relative to transpiration. Adaxial stomata of both clones were more sensitive than abaxial stomata to changing vapor pressure deficit and light intensity. Stomatal response to vapor pressure deficit appeared to be independent of bulk leaf water status. Stomata of P. candicans x P. berolinensis were more sensitive than stomata of P. deltoides x P. caudina to a change in vapor pressure deficit and less sensitive to a change in light intensity. The sensitivity of stomata of P. candicans x P. berolinensis to vapor pressure deficit may be related to drought resistance in its parentage (P. berolinensis). PMID- 16660897 TI - Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization. AB - The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.On the basis of valinomycin-induced swelling in potassium salts, suggestions were obtained that chloride, sulfate, and phosphate anions permeated more rapidly than acetate anions. Sulfhydryl reagents increased the rate of valinomycin-induced swelling in potassium phosphate, but had no effect on swelling in potassium acetate.Tributyltin induced a low rate of mitochondrial swelling in KCl in the absence of substrates, which indicated the presence of a low activity (Na(+)) K(+)/H(+) antiporter in the membrane. In the presence of NADH, rapid swelling, followed by a contraction, was observed upon addition of tributyltin. Swelling was insensitive to uncouplers, whereas contraction was uncoupler-sensitive. O(2) uptake (state 4) was greatest (3- to 4-fold stimulation) during the contraction phase, which indicated that the observed contraction was coupled to the pH gradient formed during electron transport. The results suggested that energization increased the activity of the (Na(+)) K(+)/H(+) antiporter in the inner mitochondrial membrane. PMID- 16660898 TI - Toxicity of Anaerobic Metabolites Accumulating in Winter Wheat Seedlings during Ice Encasement. AB - Ice encasement damages cold-hardened winter wheat without major disruption of cellular organelles. CO(2) accumulates during total ice encasement to higher levels in Kharkov than in less hardy Fredrick wheat. Partial ice encasement and exposure to a nitrogen atmosphere at -1 C allows greater CO(2) accumulation but neither treatment is as damaging as total ice encasement. Lactic acid accumulates to low levels only during the 1st day of encasement and thereafter remains constant. Exposure of plants to a combination of 50% CO(2) and 5% ethanol reduces survival, with a cultivar difference similar to that found in ice-encased plants. Plants in CO(2) and ethanol show a proliferation of membranes and nuclear condensation similar to that in cells of ice-encased plants. Permeability increases markedly in the presence of CO(2) and ethanol, to levels similar to or greater than those of iced plants. Ethanol alone does not increase permeability but in combination with CO(2) raises permeability of the less hardy Fredrick, although not of Kharkov, but reduces survival of both cultivars. A comparison of the endogenous levels of ethanol, CO(2), and lactic acid at the 50% kill point of plants due to ice encasement or due to externally supplied metabolite indicates that only CO(2) accumulates to independently toxic levels. Permeability and ultrastructural evidence suggest that CO(2) and ethanol in combination are the agents reducing plant viability during ice encasement. PMID- 16660899 TI - Changes in Chloroplast DNA Levels during Development of Pea (Pisum sativum). AB - Determinations were made of the percentage of chloroplast DNA (ct DNA) in total cell DNA isolated from shoots of pea at different stages of development. Labeled pea ct DNA was reassociated with a high concentration of total DNA; the percentage of ct DNA was estimated by comparing the rate of reassociation of this reaction with that of a model reaction containing a known concentration of unlabeled ct DNA. The maximum change in ct DNA content was from 1.3% of total DNA in young shoots to 7.3% in fully greened shoots. Analyses were also performed on DNA from embryos, etiolated tissue, roots, and leaves. The first leaf set to develop in pea was excised over a growth period of 8 days during which leaf length increased from 4 to 12 millimeters. Young leaves contained about 8% ct DNA; in fully greened leaves the level of ct DNA approached 12%, equivalent to as many as 9,575 copies of ct DNA per cell. Root tissue contained only 0.4% ct DNA. PMID- 16660900 TI - Respiration in Relation to Adenosine Triphosphate Content during Desiccation and Rehydration of a Desiccation-tolerant and a Desiccation-intolerant Moss. AB - O(2) consumption by the desiccation-tolerant moss Tortula ruralis and the desiccation-intolerant Cratoneuron filicinum increased markedly during the latter stages of desiccation. ATP content of the mosses during desiccation was not correlated with O(2) consumption, but was influenced by the rate at which the mosses lost water. The more rapid the water loss, the more ATP that was present in the dry mosses. The pattern of O(2) consumption on rehydration also was influenced by the previous rate of desiccation. After rapid desiccation of T. ruralis O(2) consumption upon rehydration was considerably elevated, and for up to 24 hours. After very slow desiccation the elevation was small and brief. Normal O(2) consumption did not occur in C. filicinum after rapid desiccation, but did so within a few hours of rehydration after slower speeds of drying. ATP levels in T. ruralis returned to normal within 5 to 10 minutes of rehydration. In C. filicinum, increases in ATP were closely correlated with O(2) consumption. These observations are considered to be related to differential damage caused to mitochondria and to cellular integrity by different speeds of water loss. The desiccation-tolerant moss appears to be able to repair the severe damage imposed by rapid desiccation whereas the desiccation-intolerant moss cannot. PMID- 16660901 TI - Effects of Octylguanidine on Cell Permeability and Other Protoplasmic Properties of Allium cepa Epidermal Cells. AB - Effects of octylguanidine (OG) were studied on the permeability of cells of the adaxial epidermis of Allium cepa bulb scales to water and methyl urea and on the protoplast surface. Interference of OG with the Ca(2+) and Al(3+) action on the cell surface was also investigated.Permeability of the cell membrane for water and methyl urea increased nearly three times in presence of OG. The effect of OG on cell permeability depended on its direct contact with the protoplast surface.The effect of OG on the interaction between the protoplast surface and the cell wall (wall attachment) was marked and rapid; OG (225 micromolar) decreased the time for protoplast detachment in hypertonic solutions from 420 to 120 seconds. The plasmolyzed protoplasts were immediately rounded off while the controls without OG remained heavily concave.A considerable increase in protoplast detachment time and a decrease in rounding percentage were found when cells were plasmolyzed after pretreatment with AlCl(3) (0.05 molar for 2 minutes). This effect was partially reversed by KCl which was further enhanced by addition of OG.Penetration of OG into the mesoplasm was manifested only after 10 to 15 minutes. Vacuolization and swelling of the protoplasm, fragmentation of the protoplast, and aggregation of the spherosomes, however, were observed only 30 minutes after transfer. No evidence for penetration of OG into the vacuole was found.The results support earlier suggestions that OG acts primarily on the protoplast surface by interacting with membrane proteins as well as with phospholipids. In several aspects, OG acts on the cell surface similarly to a surfactant. PMID- 16660902 TI - Salt-induced Inhibition of Phosphate Transport and Release of Membrane Proteins from Barley Roots. AB - Osmotic shock with sequential 30-minute treatments in ice-cold saline solutions and H(2)O released proteins from excised barley roots and inhibited the subsequent uptake of orthophosphate (Pi). The amount of protein released increased sharply at NaCl concentrations above 0.05 molar, approximately the threshold concentration above which Pi uptake was increasingly suppressed. About 60% of the nearly 100 micrograms of protein per gram fresh weight of roots that was eluted in 0.16 molar NaCl treatments apparently had no function in Pi transport, since it was eluted at NaCl concentrations (10 milligrams per milliliter). 3' Nucleotidase and the glycolytic enzymes (aldolase, hexokinase, phosphofructokinase, phosphoglucoisomerase, pyruvate kinase, and triose phosphate isomerase) showed no significant dependence on the cell density. Alternatively, if the NR and peroxidase activities were determined as a function of time in batch cultures, their levels were maximal 60 to 70 and 320 hours after subculture, respectively, the corresponding cell densities being 1 to 2 and 23 milligrams per milliliter. The relationship between cell density and NR and peroxidase activities is the same, whether these enzymes are measured in batch cultures during a growth cycle or in the cells cultured at different initial inoculum densities for a constant time. Conventionally enzymic changes have been correlated with growth phases; however, it is felt that the pattern of enzymic activities can also be interpreted as cell-density-dependent.In moss protonema, the dependence of cNPDE, IAA oxidase, and peroxidase on cell density may play an important role in modulating the endogenous levels of IAA and cAMP, both of which regulate the differentiation of specific cell types (Johri and Desai 1973 Nature New Biol 245: 223-224; and Handa and Johri 1976 Nature 259: 480-482). PMID- 16660906 TI - Photosynthesis in the Higher Plant Vicia faba: V. Role of Malate as a Precursor of the Tricarboxylic Acid Cycle. AB - In the higher plant Vicia faba, anomalous labeling patterns in the organic acids and related amino acids of the tricarboxylic acid cycle which result from photosynthetic (14)CO(2) fixation (in conjunction with an enzyme localization pattern unique to plant mitochondria) suggest that the tricarboxylic acid cycle functions primarily as a pathway leading to glutamic acid biosynthesis during autotrophic growth. The distribution of isotope in citrate indicates little recycling of oxaloacetate for the resynthesis of citrate. Rather, malate appears to provide both the C(2) and C(4) fragments for the synthesis of citrate, and [(3)H]formate and (14)CO(2)-labeling patterns implicate serine as the ultimate C(3) precursor of malate. PMID- 16660907 TI - Synchronization of somatic embryogenesis in a carrot cell suspension culture. AB - Synchronization of somatic embryogenesis was achieved in a carrot (Daucus carota L. cv. "Kurodagosun") suspension culture by sieving the initial heterogeneous cell population, by density gradient centrifugation in Ficoll solutions, and by subsequent repeated centrifugations at a low speed (50g) for a short time (5 seconds), followed by transferring the cell clusters obtained, which were composed of 3 to 10 cells, to a medium containing zeatin (0.1 micromolar) but no auxin. The frequency of embryo formation reached more than 90%, and synchrony of the embryogenetic process was observed at least in the early stages of the process. The system established in the present work provides a useful system for biochemical research into the mechanisms of somatic embryogenesis. PMID- 16660908 TI - Theoretical and experimental exclusion of errors in the determination of the elasticity and water transport parameters of plant cells by the pressure probe technique. AB - The volumetric elastic modulus of the cell wall and the hydraulic conductivity of the cell membranes were measured on ligatured compartments of different sizes of Chara corallina internodes using the pressure probe technique. The ratio between intact cell surface area and the area of puncture in the cell wall and membrane introduced by the microcapillary of the pressure probe was varied over a large range by inserting microcapillaries of widely varying diameters in different sized compartments. The relationship of the elastic modulus and the hydraulic conductivity to turgor pressure was independent of the ratio of intact cell surface area to the area of injury. The increase in the hydraulic conductivity below 2 bar turgor pressure and the volume dependence of the elastic modulus were shown to be the same as those observed in intact nonligatured cells. Theoretical considerations of the possible influence of injury of the cell wall and cell membrane around the inserted microcapillary on the measurement of the water transport and cell wall parameters do not explain the experimental findings. Thus, mechanical artifacts, if at all present, are too small to account for the observed dependence of the hydraulic conductivity and the elastic modulus on turgor pressure. The pressure probe technique thus represents an accurate method for measuring water transport parameters in both giant algal cells and in tissue cells of higher plants. PMID- 16660909 TI - Photosynthetic Adaptation of Solanum dulcamara L. to Sun and Shade Environments: II. Physiological Characterization of Phenotypic Response to Environment. AB - Photosynthetic and growth properties of Solanum dulcamara L. were studied under controlled environments. The 200 experimentally tested plants were clonal replicates of five field-collected individuals, three from fully exposed habitats and two from deeply shaded habitats. After 4 weeks of growth in one of eight environmental treatments, each plant was measured for leaf adaxial and abaxial conductance to water vapor, specific leaf weight, chlorophyll per square decimeter of leaf, photosynthetic unit size, light-saturated photosynthetic rate, total leaf area, and total leaf, stem, and root dry weights. Changes in light level influenced photosynthesis and growth of each plant more than changes in water availability or temperature. It is strongly suggested that the primary adaptive response of the tested individuals to changes in levels of light involves the regulation of leaf thickness. PMID- 16660910 TI - Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm. AB - The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO(2) occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development. PMID- 16660911 TI - Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings. AB - Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.Studies with radioactive acetate, glycerol, and orthophosphate indicate that the three major phospholipids are also synthesized in the cotyledons. Incorporation of glycerol and acetate into phospholipids of cotyledons is relatively constant throughout seedling growth, while the incorporation of [(32)P]orthophosphate steadily declines from a high value 24 hours after the start of imbibition. The newly synthesized phospholipids become associated with membranous organelles, especially the endoplasmic reticulum, and have an in situ half-life of 2 to 2.5 days.Determination of the activities of two enzymes involved in phospholipid biosynthesis (phosphorylcholine-glyceride transferase and CDP-diglyceride-inositol transferase) shows that the enzymes have their highest activities 12 hours after the start of imbibition. High activities for both enzymes were found in cotyledons of beans incubated at 1 C, indicating that the enzymes may preexist in the dry seeds.The experiments demonstrate that cotyledons start synthesizing new phospholipids immediately after imbibition, but that the rate of phospholipid catabolism far exceeds the rate of synthesis long before the cotyledons start to senesce. PMID- 16660912 TI - Abscisic Acid localization and metabolism in barley aleurone layers. AB - Aleurone layers of Hordeum vulgare, cv. ;Himalaya' took up [(14)C]-abscisic acid (ABA) when incubated for various times. Radioactivity accumulated with time in a low speed, DNA-containing pellet accounting for 1.6 to 2.3% of the radioactivity recovered in subcellular fractions at 18 hours. Thin layer chromatography of ethanolic or methanolic extracts of the cytosol, which contained greater than 95% of the radioactivity taken up by layers, revealed that labeled ABA was metabolized to phaseic acid (PA) and 4'-dihydrophaseic acid (DPA) and three polar metabolites Mx(1), Mx(2), and Mx(3). ABA was not metabolized by endosperm, incubated under conditions used for layers, indicating that metabolism was tissue specific. Layers metabolized [(3)H]DPA to Mx(1) and Mx(2). ABA, PA, and DPA methyl ester and epi-DPA-methyl ester inhibited synthesis of alpha-amylase by layers incubated for either 37 or 48 hours. These layers converted the methyl DPA and epi-methyl-DPA esters to their respective acids. DPA did not inhibit Lactuca sativa germination or root and coleoptile elongation of germinating Hordeum vulgare seeds, or coleoptile elongation of germinating Zea mays seeds. PMID- 16660913 TI - Chlorophyll Turnover in Skeletonema costatum, a Marine Plankton Diatom. AB - [(3)H]- and delta-[(14)C]Aminolevulinic acids were incorporated into the chlorophylls of Skeletonema costatum, a marine plankton diatom. In the stationary phase of growth, the tetrapyrrole-based pigments reached steady-state labeling after 10 hours. Under conditions of exponential cell division and chlorophyll accumulation, (3)H was rapidly lost from the labeled chlorophylls and was replaced with (14)C derived from delta-[4-(14)C]aminolevulinic acid. The kinetics of isotope dilution suggests recycling of tetrapyrrole precursors and/or two pigment pools, containing both chlorophyll a and chlorophyllide c, one which turns over rapidly (10 hours) and another which turns over more slowly (100 hours). Calculation of turnover times varied from 3 to 10 hours for chlorophyll a and from 7 to 26 hours for chlorophyllide c. The data suggest the dynamics of chlorophyll metabolism in S. costatum and explain the diatom's ability to undergo light-shade adaptation within a generation time. PMID- 16660914 TI - Characterization of the Phosphate-mediated Control of Ethylene Production by Penicillium digitatum. AB - Characterization of the phosphate effect on ethylene production by Penicillium digitatum is reported. A low level of phosphate (0.001 millimolar) was about 200 to 500 times as effective as a high phosphate level (100 millimolar) in stimulating ethylene production and the stimulation was readily reversed by addition of phosphate. This phosphate effect did not operate in static cultures. The precursor of ethylene in the stimulated low phosphate system was glutamate but not alpha-ketoglutarate, which is a precursor in static systems. Actinomycin D and cycloheximide effectively inhibited the low phosphate/high ethylene producing system. Alkaline phosphatase and protein kinase activities were higher in low than in high phosphate systems. We suggest that phosphate level regulates ethylene production by P. digitatum and that the regulation involves a phosphorylation or dephosphorylation reaction of some enzyme system associated with ethylene production. Phosphate-mediated control of ethylene production may also involve the transcriptional and translational machinery of the fungal cell. P. digitatum apparently can produce widely different levels of ethylene by different pathways, depending on culture conditions under which it is grown. PMID- 16660915 TI - Direct evidence for a sugar transport mechanism in isolated vacuoles. AB - Sugar transport has been directly observed in isolated higher plant vacuoles for the first time. The latter were released from protoplasts isolated from the mesophyll of Pisum sativum L.Uptake of l-glucose by the vacuoles was very slight in comparison with that of the d-glucose analog 3-O-methyl glucose (MeG), indicating, first, that a highly selective sugar uptake mechanism is seated in the tonoplast; and, second, that the mechanism was functioning in the isolated vacuoles.MeG uptake was markedly sensitive to the pH of the medium, falling as the external pH rose. Addition of MgATP to buffered medium strongly promoted MeG uptake by vacuoles, but not by the protoplasts from which they were released. Treatment with the proton ionophore SF(6847) drastically reduced uptake by the vacuoles, but had a lesser effect on uptake by the protoplasts. The inhibitory effect of SF(6847) on uptake by the vacuoles was countered to a substantial degree by the addition of MgATP.The influence of pH, the stimulatory effect of ATP, and the ATP-reversible inhibition by SF(6847) all strengthen the conclusion that the observed sugar uptake reflected membrane function and was not due to a diffusional inward leak through damaged membranes.The results are discussed in the light of currently held concepts regarding the driving force for sugar transport. PMID- 16660916 TI - Inhibition of Soybean Cell Growth by the Adsorption of Rhizobium japonicum. AB - Soybean cells in suspension culture were inhibited in their growth by mixed culture with Rhizobium japonicum 5033. Rhizobium cells had the ability to adsorb on the surface of soybean cells. Cell envelope prepared from Rhizobium by sonic oscillation inhibited the growth of soybean cells. The growth-inhibiting activity of the cell envelope was depressed by beta-glucosidase, KIO(4), urea, sodium cholate, and Triton X-100, but was stable on heating at 120 C for 15 minutes. Adsorption of the cell envelope on soybean cells was depressed by only beta glucosidase. The sodium cholate-soluble fraction of the cell envelope had the growth-inhibiting activity. Results in this paper suggest that components of the Rhizobium cell surface cause the inhibition of soybean cell growth after the adsorption of the Rhizobium cell to the soybean cell. PMID- 16660917 TI - Detection and characterization of sorbitol dehydrogenase from apple callus tissue. AB - Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2 methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity. PMID- 16660918 TI - Investigations of vacuoles isolated from tobacco: I. Quantitation of nicotine. AB - Nicotine was shown to be associated with mature vacuoles isolated from protoplasts of Nicotiana rustica. The vacuolar preparations also contained high levels of acid phosphatase, ATPase, and approximately 30% of the soluble protoplastic protein. The contamination of the vacuolar isolate by chlorophyll, succinate dehydrogenase, and NADPH cytochrome c reductase (markers for chloroplasts, mitochondria, and endoplasmic reticulum) was low. The enzymic activity associated with the vacuoles was not due to the exogenously supplied digestive enzymes used in the preparation of the protoplast. The relatively easy isolation of tobacco vacuoles makes this an excellent system for biochemical investigations of the vacuole. PMID- 16660919 TI - Guard cell starch concentration quantitatively related to stomatal aperture. AB - Using quantitative histochemical techniques, the carbohydrate levels of guard cells from open and closed stomatal apparatus of Vicia faba L. were compared. To minimize experimental error, all comparisons were between leaflets of the same pair. Stomata on one leaflet were caused to open by light and reduced CO(2). The other leaflet, which was in darkness, had closed stomata. In one experiment, data were also collected on palisade parenchyma, spongy parenchyma, and epidermal cells.Guard cell starch concentration was higher in the leaflets with closed stomata than in open stomata by 72 +/- 16 millimoles per kilogram dry weight (anhydroglucosyl equivalents) (N = 117, P < 0.02). Variation in guard cell starch concentration from one part of a leaflet to another was small. The data are consistent with the hypothesis that starch degradation provides the carbon skeletons for anion synthesis in guard cells during stomatal opening.Sucrose concentration was higher in guard cells when stomata were open than when they were closed in all three experiments (average difference = 45 +/- 7 millimoles per kilogram dry weight [N = 59, P < 0.01]). The variability of sucrose concentration within test leaflets prevented an unequivocal interpretation of these results. When all data are considered, it appears that soluble sugars increase in guard cells when stomata of Vicia faba open. PMID- 16660920 TI - Effect of temperature on water and ion transport in soybean and broccoli systems. AB - Steady-state flow rates and exudate osmotic potentials were measured from complete root systems from warm- (28/23 C) or cold-(17/11 C) grown soybean or broccoli (Brassica oleracea) plants at various pressures or different temperatures.In warm-grown soybean roots systems, a break occurred at 14.7 C in the Arrhenius plot of total flow at constant pressure. When plants were grown at lower temperatures, the break point shifted to 8 C. Broccoli, a chilling resistant species, showed no break for the temperature range used, but cooler growth temperatures decreased the activation energy for water flow through the root system from 18 kilocalories per mole to 9 kilocalories per mole. In both broccoli and soybean, cold-grown plants had lower exudate potentials and greater flow rates at low hydrostatic pressures than the warm-grown plants.These observations indicate that the rate-limiting site for passive water transport is a membrane which may be modified as the plant acclimates to varying growth temperatures. An additional part of the acclimation process is an increase in activity of root ion pumps. PMID- 16660921 TI - Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. AB - Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. gamma-Methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents. PMID- 16660922 TI - Rhizoid Differentiation in Spirogyra: III. Intracellular Localization of Phytochrome. AB - Localization of phytochrome which mediates rhizoid differentiation in Spirogyra was investigated. The red-absorbing form of phytochrome (Pr) seems to be distributed all over the cell periphery which remained in the centripetal end part after the centrifugation, as rhizoids formed equally well with red spotlight irradiation of three different parts of an end cell, i.e. distal end, middle, and proximal end, and with irradiation of centrifugal and centripetal end parts of a centrifuged end cell. The Pr distribution was confirmed with an experiment using far red irradiation over the entire cell, centrifugation, and red spotlight irradiation. The Pr-phytochrome molecules appeared to be mobile because no dichroic orientation was shown with polarized red spotlight irradiation. On the contrary, it is suggested that far red-absorbing form of phytochrome molecules are evacuated from the centripetal end part by the centrifugation in an experiment involving red irradiation over the entire cell-centrifugation-far red spot irradiation. Rhizoid formation was repressed markedly by far red irradiation of the centrifugal end part but not of the centripetal end part. PMID- 16660923 TI - Partitioning of C-photosynthate, and long distance translocation of amino acids in preflowering and flowering, nodulated and nonnodulated soybeans. AB - The influence of stage of development (preflowering versus flowering) in nodulated and nonnodulated soybeans (Glycine max [L.] Merr. cv. Wells) on partitioning of (14)C into assimilates following exposure of a soybean leaf to (14)CO(2) by both steady-state and pulse-labeling techniques was studied. Blades on the second fully expanded leaf from the stem apex were exposed to (14)CO(2). Radioactive assimilates were extracted from source leaf blades, petioles, and stems (both the path up and path down from source leaf), were separated into neutral (sugars), basic (amino acids), and acidic (organic acids, sugar phosphates) fractions by ion exchange chromatography. The basic fraction was further resolved using thin layer chromatography and the percentage of radioactivity recovered in each amino acid was determined.The distribution of radioactivity in the neutral, basic, and acidic fractions of the source leaf blades was significantly different from that of the transport path (petiole and stems). About 70% of the radioactivity in source leaf blades was recovered in the neutral fraction, whereas about 90% of the recovered radioactivity in the path was in the neutral fraction. (14)C-Aminoacids constituted 8 to 17% and 2 to 7% of the recovered radioactivity in source leaves and paths, respectively. Recovered (14)C in organic acids ranged from 13 to 20% and 2 to 7% in source leaves and paths, respectively. Partitioning of (14)C-assimilates among the neutral, basic, and acidic fractions was not affected by the presence of nodules or flowers. However, when steady-state labeling was compared to pulse labeling, a significantly lower percentage of (14)C was recovered in the neutral fraction with a concomitant increase in the basic fraction. Asparagine-arginine, serine, glutamate, gamma-aminobutyrate-alanine, and aspartate accounted for 69 to 85% of the recovered radioactivity in the basic fraction from the various treatments. [(14)C]Serine was significantly higher in pulse-labeling experiments, whereas glutamtate and proline were higher with steady-state labeling. [(14)C]Serine was significantly higher in nonnodulated plants than in nodulated plants, whereas gamma-aminobutyrate-alanine was significantly higher in preflowering plants as compared to flowering plants. PMID- 16660924 TI - Regulation of the Photosynthesis Rhythm in Euglena gracilis: II. Involvement of Electron Flow through Both Photosystems. AB - Rhythmic changes in the light reactions of Euglena gracilis have been found which help to explain the basic reactions effected in the circadian rhythm of O(2) evolution. Diurnal changes in the slope of light intensity plots indicated that the maximal rate of photosynthesis changed throughout the circadian cycle. No evidence was obtained consistent with the premise that changes in chlorophyll content, as measured by total chlorophyll or chlorophyll a/b ratio, or photosynthetic unit size are responsible for this rhythim.The rate of light induced electron flow through the entire electron chain (H(2)O to methyl viologen) was rhythmic both in whole cells and in isolated chloroplasts, and the highest rate of electron flow coincided with the highest rate of O(2) evolution. The individual activities of photosystem I (reduced from 2,6-dichlorophenol indophenol to methyl viologen) and photosystem II (H(2)O to 2,6-dichlorophenol indophenol) did not, however, change significantly with time of day, suggesting that the coordination of the two photosystems may be the site of circadian control. Evidence consistent with this concept was obtained from studies of low temperature emission from systems I and II following preillumination with system I or II light. PMID- 16660925 TI - Correlation of Adenosine Triphosphate Levels in Chara corallina with the Activity of the Electrogenic Pump. AB - The effect of a number of inhibitors on the ATP level in single cells of Chara corallina has been measured using the luciferin-luciferase assay. The uncouplers of phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone and 2,4 dinitrophenol, and the ATPase inhibitors, dicyclohexyl-carbodimide and diethylstilbestrol, all caused a marked reduction of the ATP level. These inhibitors also produced a large increase in the membrane resistance and a depolarization of the membrane potential to the diffusion potential. This is consistent with the plasmalemma containing an ATP-dependent electrogenic pump that provides the primary conductance through the membrane.Ethyl-3-(3 dimethylaminopropyl)carbodiimide, which depolarizes the membrane potential, has no effect on the ATP level and does not increase the membrane resistance. This inhibitor apparently does not enter the cell but may act by affecting the permeability of the membrane. Neither darkness nor 3-(3,4-dichlorophenyl)-1,1 dimethylurea lowers the ATP level and, while neither has much effect on the membrane potential, both cause a similar increase in resistance in comparison with the control in the light. The weak acid, 5,5-dimethyloxazolidine-2,4-dione, and the weak base, NH(3), do not affect the ATP level significantly but have effects on the electrogenic pump that are consistent with their postulated effects on the cytoplasmic pH, if H(+) is the substrate for the pump. PMID- 16660926 TI - Metabolism of Monoterpenes: Demonstration of (+)-Neomenthyl-beta-d-Glucoside as a Major Metabolite of (-)-Menthone in Peppermint (Mentha Piperita). AB - (-)-Menthone, the major monoterpene component of the essential oil of maturing peppermint (Mentha piperita L.) leaves (6 micromoles per leaf) is rapidly metabolized at the onset of flowering with a concomitant rise in the level of (-) menthol (to about 2 micromoles per leaf). Exogenous (-)-[G-(3)H]menthone is converted into (-)-[(3)H]menthol as the major steam-volatile product in leaf discs in flowering peppermint (10% of incorporated tracer); however, the major portion of the incorporated tracer (86%) resided in the nonvolatile metabolites of (-)-[G-(3)H]menthone. Acid hydrolysis of the nonvolatile material released over half of the radioactivity to the steamvolatile fraction, and the major component of this fraction was identified as (+)-neomenthol by radiochromatographic analysis and by synthesis of crystalline derivatives, thus suggesting the presence of a neomenthyl glycoside. Thin layer chromatography, ion exchange chromatography, and gel permeation chromatography on Bio-Gel P-2 allowed the purification of the putative neomenthyl glycoside, and these results suggested that the glycoside contained a single, neutral sugar residue. Hydrolysis of the purified glycoside, followed by reduction of the resulting sugar moiety with NaB(3)H(4), generated a single labeled product that was subsequently identified as glucitol by radio gas-liquid chromatography of both the hexatrimethylsilyl ether and hexaacetate derivative, and by crystallization to constant specific radioactivity of both the alditol and the corresponding hexabenzoate. These results, along with studies on the hydrolysis of the glycoside by specific glycosidases, strongly suggest that (+)-neomenthyl-beta-d glucoside is a major metabolite of (-)-menthone in flowering peppermint. This is the first report on the occurrence of a neomenthyl glycoside, and the first evidence implicating glycosylation as an early step in monoterpene catabolism. PMID- 16660927 TI - Conversion of l- and d-Phenylalanine to Phenylacetate via Phenylpyruvate in Sorghum Leaf Extracts. AB - The incorporation of dl-[(14)C]phenylalanine into phenylacetate reported previously in leaf enzyme preparations has been found to be catalyzed by two separate enzyme activities leading to phenylpyruvate, one using the l-, the other the d-isomer. Since both reactions occur anaerobically and are increased by the addition of pyridoxal phosphate and alpha-ketoglutarate, two transaminase (aminotransferase) activities appear to be involved. The activities of the l- and d-dependent forms are approximately equal in a crude particulate fraction, but range from 1:1 to 3:1 in a soluble supernatant fraction. Nonisotopic as well as isotopic assays to separate d- and l-activities are described. PMID- 16660928 TI - Isolation and Oxidative Properties of Intact Mitochondria from the Leaves of Sedum praealtum: A Crassulacean Acid Metabolism Plant. AB - A procedure is described for preparing intact mitochondria from leaves of Sedum praealtum D.C., a plant showing Crassulacean acid metabolism. These mitochondria oxidized malate, pyruvate, alpha-ketoglutarate, succinate, NADH, NADPH, and isocitrate with good respiratory control and ADP/O ratios better than those observed in mitochondria from other photosynthetic tissues.Malate oxidation was very resistant to inhibition by rotenone. Glycine oxidation was very slow with poor respiratory control and was resistant to rotenone inhibition. Antimycin A completely inhibited the oxidation of both NADH and NADPH. The oxidation of isocitrate, malate, succinate, and alpha-ketoglutarate was partially inhibited by antimycin A and cyanide. Overall rates of substrate oxidation were slow on a protein basis, but purification of the mitochondrial preparations on a linear sucrose gradient removed a large amount of nonmitochondrial protein. The original mitochondrial preparations contained little glycolate oxidase activity, and most of this activity was removed by the sucrose gradient. PMID- 16660929 TI - Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves. AB - The subcellular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chloroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADP-Glucose pyrophosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosylase) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction. Chromatography of a chloroplast extract on diethylaminoethyl-cellulose resolves the R- and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amylopectin as a substrate indicates an endolytic activity but displays properties unlike the typical alpha-amylase as isolated from endosperm tissue. PMID- 16660930 TI - A Transglucosylase from Sclerotinia libertiana. AB - A Sclerotinia enzyme preparation with predominant exo-beta-(1-->3)-glucanase activity has the capacity to mediate the formation of tetrasaccharide from 3-O beta-cellobiosyl-d-glucose or cellotriose, and a pentasaccharide from 3-O-beta cellotriosyl-d-glucose or cellotetraose. Transglucosylation is not observed when the enzyme is incubated in the presence of laminaritriose, laminaritetraose, or cellobiose. Substrate specificity of the reaction therefore resembles certain features of exo-beta-(1-->4)-glucanases. The optimum pH of the activity is 5.5 and the reaction is inhibited by nojirimycin but not by glucono-1,5-lactone. In contrast to the Sclerotinia glucanase, a Basidiomycete exo-beta-(1-->3)-glucanase has no apparent transglucosylase activity. The results indicate that a transglucosylase may have been an undetected constituent in exo-beta-(1-->3) glucanase preparations used for promoting growth in auxin-depleted tissues. PMID- 16660931 TI - Ultrastructural changes in the cell walls of ripening apple and pear fruit. AB - Ultrastructural changes in the cell walls of "Calville de San Sauveur" apples (Malus sylvestris Mill) and "Spadona" pear (Pyrus communis L.) fruit were followed during ripening. In apple, structural alterations in cell walls became apparent at advanced stages of softening and showed predominantly dissolution of the middle lamella. In pears softening was also associated with the dissolution of the middle lamella, and in addition a gradual disintegration of fibrillar material throughout the cell wall. In fully ripe fruit almost all of the fibrillar arrangement in the cell wall was lost. Application of enzyme solutions containing polygalacturonase and cellulase to tissue discs from firm pear fruit led to ultrastructural changes observed in naturally ripening pears. In apple polygalacturonase alone was sufficient to dissolve the middle lamella region of the cell walls, as was also found to occur in naturally ripening fruit. In both apple and pear the cell wall areas containing plasmodesmata maintained their structural integrity throughout the ripening process. At advanced stages of ripening vesicles appeared in the vicinity of plasmodesmata. PMID- 16660932 TI - Distribution of a Fatty Acid cyclase enzyme system in plants. AB - Extracts from tissues of 24 plant species were tested for the enzyme that catalyzes the conversion of 13-l-hydroperoxy-cis-9,15-trans-11-octadecatrienoic acid to the cyclic fatty acid 12-oxo-cis-10,15-phytodienoic acid. The enzyme was detected in 15 of the 24 tissues examined, and was demonstrated in seedlings, leaves, and fruits. PMID- 16660933 TI - Ion fluxes and phytochrome protons in mung bean hypocotyl segments: I. Fluxes of potassium. AB - K(+) [(86)Rb(+)] uptake by Phaseolus aureus Roxb. hypocotyl segments cut immediately below the hook is inhibited by the active form of phytochrome (Pfr). Short load-short wash experiments indicate that the inhibition of uptake occurs across the plasmalemma. A maximal inhibition of short term uptake occurs in 10 to 50 millimolar KCI. Low temperature had only a small effect on influx and the inhibition of influx from 50 millimolar KCI. A consideration of the electrochemical gradient for K(+) suggests that passive K(+) fluxes may predominate under these conditions. Red light induces small depolarizations of membrane potential in subhook cells. Far red light antagonizes this effect. Pfr inhibits efflux of K(+)[(86)Rb(+)] from subhook segments. This effect is also relatively insensitive to low temperature. This inhibition of efflux may reflect inhibition of a K(+) -K(+) exchange process, or reduced passive permeability of the plasmalemma to K(+). In contrast, Pfr enhances short term uptake of K(+)[(86)Rb(+)] in apical hypocotyl hook segments of Phaseolus aureus Roxb. Short load-short wash experiments indicate that fluxes across the plasmalemma are modified by Pfr. A maximal enhancement of short term influx occurs in 50 millimolar KCI. Influx and the red light enhancement of influx from 50 millimolar KCI are relatively insensitive to low temperature. Pfr also enhances efflux of K(+)[(86)Rb(+)] from preloaded apical hook segments. This increased influx may reflect enhancement of a K(+) -K(+) exchange process or increased passive permeability of the plasmalemma to K(+). PMID- 16660934 TI - Ion Fluxes and Phytochrome Protons in Mung Bean Hypocotyl Segments: II. Fluxes of Chloride, Protons, and Orthophosphate in Apical and Subhook Segments. AB - The active form of phytochrome (Pfr) decreased CI(-) uptake by subhypocotyl hook segments of Phaseolus aureus Roxb. and increased uptake by apical segments. Pfr had similar effects on Pi [(32)Pi] uptake. Modulations of Pi [(32)Pi] uptake were detectable 10 minutes following photoconversion. Pfr may modulate Pi influx across the plasmalemma. Pfr inhibited H(+) extrusion by subhook segments and enhanced extrusion by apical hook segments. No rapid effects on H(+) extrusion were found. Phytochrome may regulate a K(+) -H(+) exchange process. The differential responses of the two regions of the hypocotyl are discussed with respect to Pfr-mediated changes in growth and development. PMID- 16660935 TI - Metabolism of Tritiated Gibberellin A(20) in Immature Seeds of Dwarf Pea, cv. Meteor. AB - Tritium-labeled gibberellin A(20) ([(3)H]GA(20)) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of [(3)H]GA(20), the three in highest yield were GA(29), an alpha,beta-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA(20) --> GA(29) --> compound B --> the ketone was indicated. The amount of [(3)H]GA(29) in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [(3)H]ketone in the seed increased with time to its highest level at maturity. It is suggested that compound B and the ketone represent the major pathway of catabolism of GA(29), a 2beta-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation. PMID- 16660936 TI - Hydrogen Peroxide-mediated Oxidation of Indole-3-acetic Acid by Tomato Peroxidase and Molecular Oxygen. AB - The oxidation of indole-3-acetic acid by anionic tomato peroxidase was found to be negligible unless reaction mixtures were supplemented with H(2)O(2). The addition of H(2)O(2) to reaction mixtures initiated a period of rapid indole-3 acetic acid oxidation and O(2) uptake; this phase ended and O(2) uptake fell to a low level when the H(2)O(2) was exhausted. The stoichiometry of the reaction, which is highly dependent on enzyme concentration and pH, suggests that H(2)O(2) initiates a sequence of reactions in which indole-3-acetic acid is oxidized. PMID- 16660937 TI - A Constitutive Enzyme System for Glucose Transport by Chlorella sorokiniana. AB - It was found that the transport system for glucose (as measured by deoxyglucose uptake) in the high temperature strain of Chlorella (strain 07-11-05 or C. sorokiniana) was constitutive and the rate of uptake did not increase upon incubation of autotrophically grown cells with either deoxyglucose or glucose. The uptake obeyed Michaelis-Menten type kinetics with a concentration of 200 micromolar for half-saturation. The maximum rate of uptake was nearly 10 times faster per cell (at 38 C) than that reported for any other Chlorella. This rapid accumulation of deoxyglucose causes the passive efflux to become significant compared to the pump-driven influx and nonlinear uptake appears even after only 3 to 4 minutes. PMID- 16660938 TI - Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent. AB - Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP(+)-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP(+) isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K(i):17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds. PMID- 16660939 TI - Ribosome-thylakoid association in peas: influence of anoxia. AB - Isolated pea chloroplast thylakoids ordinarily have ribosomes attached which survive sequential washes. Extensive in vivo loss of these thylakoidbound ribosomes occurred if the pea plants were placed in the dark without O(2) for 2 or more hours. This loss was indicated from measurements of both the total thylakoid-bound RNA levels, and the capacity for amino acid incorporation into proteins on the addition of soluble enzymes for protein synthesis. Stroma ribosome profiles lost any indication of polysome structure due to the same anoxic treatment in vivo. The return of ribosomes to the thylakoids when plants were placed in the light in air occurred over an 8-hour time course. This return was prevented by lincomycin, spectinomycin, and chloramphenicol, indicating a requirement for protein synthesis steps in the stroma at some point in the reassociation process. PMID- 16660940 TI - Nicotine Biosynthetic Enzyme Activities in Nicotiana tabacum L. Genotypes with Different Alkaloid Levels. AB - Young plants of five Nicotiana tabacum L. genotypes were examined for activity of nicotine biosynthetic enzymes. Genotypes near isogenic except at two loci each with two alleles controlling nicotine level were used in a comparison of the four homozygous allelic combinations producing high, high intermediate, low intermediate, and low nicotine levels in a "Burley 21" background. Putrescine N methyltransferase (EC 2.1.1.53) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) activities in root tissue of these four genotypes were proportional to leaf nicotine level, whereas N-methylputrescine oxidase activity in root tissue differed in proportion and ranking. Quinolinic acid phosphoribosyltransferase activities in leaf tissue were lower than in roots, but no differences were found among the four genotypes. The homozygous recessive alleles at either locus affect levels of all three enzyme activities examined in roots. Each locus seems to be involved in regulation of nicotine metabolism, but whether directly as a regulatory locus or indirectly through the metabolic product of a structural locus is not known.No difference was observed between enzymic oxidation of putrescine and N-methylputrescine by leaf and root extracts of Burley 21 (a high nicotine, low nornicotine genotype) and a high nornicotine cultivar, "Robinson Medium Broadleaf." Putrescine was utilized as a substrate to a greater extent than N-methylputrescine by leaf extracts compared with root extracts of both cultivars. It was concluded that genetic differences in levels of nicotine and nornicotine were not due to differences in enzymic oxidation of these two precursors during alkaloid biosynthesis. PMID- 16660941 TI - Freezing injury and phospholipid degradation in vivo in woody plant cells: I. Subcellular localization of phospholipase d in living bark tissues of the black locust tree (robinia pseudoacacia L.). AB - The subcellular localization of phospholipase D in homogenates of living bark tissues of the black locust tree (Robinia pseudoacacia L.) was examined and found in both soluble and particulate fractions. At least some of the soluble enzyme was considered to be compartmentalized in vacuoles. Considerable amounts of phospholipase D seemed to be tightly bound on several membranes such as endoplasmic reticulum, tonoplast, and a membrane associated with potassium stimulated ATPase (pH 6.1). The mitochondrial fraction banding at the 40 to 43% (w/w) sucrose layer, however, had the lowest specific activity. The soluble and the particulate phospholipase D were considered to be similar in nature. It is possible that the particulate enzyme, as a part, may be derived from the coexisting nonvesiculated materials visualized in the electron micrograph of each membrane fraction. An involvement of the soluble or the presumed membrane-bound phospholipase D in phospholipid degradation in vivo during freezing at sublethal temperatures was discussed with special reference to freezing injury of plant cells. PMID- 16660942 TI - Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: II. Regulatory Effects of Divalent Cations on Activity of Membrane-bound Phospholipase D. AB - Activity of membrane-bound phospholipase D in microsomes from bark tissues of black locust tree (Robina pseudoacacia L.) was demonstrated to be regulated by a competitive binding of divalent cations. Binding of Ca(2+) at high concentrations (1 to 50 millimolar) modified the pH activity profile, shifting the optimum pH by 0.5 unit toward neutral and increasing the activity in the neutral pH. Mg(2+), on the other hand, inhibited the reaction of membrane-bound phospholipase D without added Ca(2+), and competitively inhibited the Ca(2+) stimulation. The regulatory effects of those ions were dependent on pH. Reduction in pH resulted in a decrease in the apparent dissociation constant for Ca(2+) and an increase in that for Mg(2+). From Lineweaver-Burk double reciprocal plots of Ca(2+) and the initial velocity, it was suggested that the binding of Ca(2+) in the higher concentration resulted in nearly the same conformational change of enzyme as reduction in pH. Mg(2+), on the other hand, counteracted those effects of Ca(2+) and lower pH on the enzyme conformation in such a manner as to inactivate. The membrane-bound phospholipase D because more sensitive to Ca(2+) and less sensitive to Mg(2+) as the hardiness of the tissues decreased. This fact may indicate that some qualitative changes in membranes are involved in the hardiness changes and also in the susceptibility of phospholipid to degradation by phospholipase D in plant cells. PMID- 16660943 TI - Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: III. Effects of Freezing on Activity of Membrane-bound Phospholipase D in Microsome enriched Membranes. AB - Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca(2+) and a complete loss of the regulatory action of Mg(2+) in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca(2+). Both factors would stimulate drastically the membrane bound phospholipase D, causing degradation of membrane phospholipids. PMID- 16660944 TI - Photosynthesis in Grass Species Differing in Carbon Dioxide Fixation Pathways: II. A Search for Species with Intermediate Gas Exchange and Anatomical Characteristics. AB - Thirty-three grass species were examined in two experiments in an attempt to locate plants with photosynthetic responses to O(2), CO(2) compensation concentrations, and leaf anatomy intermediate to those of C(3) and C(4) species. Species examined included seven from the Laxa group in the Panicum genus, one of which, P. milioides Nees ex Trin., has been reported earlier to have intermediate characteristics. The species with O(2)-sensitive photosynthesis typical of C(3) plants showed more than 37% increase in apparent photosynthesis at 2% O(2) compared to 21% O(2) at 25 C and 335 microliters per liter CO(2), whereas in Panicum milioides, P. schenckii Hack., and P. decipiens Nees ex Trin., members of the Laxa group of Panicum, increases ranged from 25 to 30%. The remainder of the species did not respond to O(2). Species with O(2) responses characteristic of C(3) plants exhibited CO(2) compensation concentrations of 44 microliters per liter or higher at 21% O(2) and 25 to 27.5 C and species characterized as O(2) insensitive had values of microliters per liter or less. The CO(2) compensation concentration (capital GHE, Cyrillic) values of P. milioides, P. schenckii, and P. decipiens ranged from 10.3 to 23.3 microliters per liter. Other species of the Laxa group of Panicum exhibited O(2) response and capital GHE, Cyrillic values of either C(3) (P. laxum Sw., P. hylaeicum Mez., and P. rivulare Trin.) or C(4) (P. prionitis Griseb.) plants. Leaves of species with O(2) response and CO(2) compensation values typical of C(3) plants had poorly developed or nearly empty bundle sheath cells, and much larger distances and mesophyll cell numbers between veins than did the O(2)-insensitive ones. Vein spacings in P. milioides, P. schenckii, and P. decipiens ranged from 0.18 to 0.28 millimeter and mesophyll cell number between veins from 5.2 to 7.8. While these vein spacings are closer than those of most C(3) grasses, two O(2)-sensitive species of Dactylis had vein spacings similar to these Panicums and veins in Glyceria striata, another O(2) sensitive plant, were separated by only four mesophyll cells and 0.12 millimeter. Bundle sheath cells of the three intermediate Panicums contained greater quantities of organelles than are typical for C(3) grasses. PMID- 16660945 TI - A possible mechanism of ammonium ion regulation of photosynthetic carbon flow in higher plants. AB - Addition of NH(4) (+) to the photosynthesizing leaf cells of Dolichos lab lab L. var. Lignosis Prain and leaf discs of Vigna sinensis L. savi ex Hassk caused a significant increase in the flow of photosynthetic carbon toward amino acids with a concomitant decrease toward sugars without affecting the over-all photosynthetic rate. Similar diversion of photosynthetic carbon away from sugars was also observed in the photosynthesizing isolated chloroplasts of V. sinensis, but the latter differed in that they accumulated organic acids rather than amino acids. In an effort to understand the mechanism of NH(4) (+)-mediated regulation, the specific and total activities of NAD(P)-glutamate dehydrogenase, glutamine synthetase, pyruvate kinase, alkaline fructose 1,6-bisphosphatase, and NAD(P) glyceraldehyde-3-phosphate dehydrogenase of the cells of D. lab lab were checked but none was affected by the added ammonium salts even after prolonged incubation. At certain concentrations, ammonium ions abolished the light activation of NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose 1,6-bisphosphatase in isolated chloroplasts from dark-adapted Vigna leaves without interfering with the basal dark activity of these enzymes. Based on these observations, a possible mechanism of action of NH(4) (+) in regulating the photosynthetic carbon flow is postulated. PMID- 16660946 TI - Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase. AB - Properties of phosphoenolpyruvate carboxylase in guard cells dissected from frozen-dried Vicia faba L. leaflets were studied using quantitative histochemical techniques. Control experiments with palisade cells and whole leaflet extract proved that the single cell approach was valid. Most characteristics of enzyme activity in guard cells were identical to those in the leaflet extract. The activities were highly dependent on temperature, with maximum activity at 25 to 35 C. Half-maximum activity (with 1 millimolar phosphoenolpyruvate [PEP]) was observed at 0.1 millimolar Mg(2+). Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent K(m(PEP)) was 0.15 millimolar at pH 7.7; with guard cells, the values were 1.49, 0.5 to 0.8, and 0.24 millimolar in three successive experiments. Additional experiments showed that apparent K(m(PEP)) of guard cell activity from plants within a single growth lot was reproducible and did not change during stomatal opening. Mixed extract experiments proved that soluble compounds were not responsible for the difference observed between leaflet and guard cell activities. The differences in apparent K(m(PEP)) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed. PMID- 16660947 TI - Effects of certain herbicides and their combinations on nitrate and nitrite reduction. AB - A study was made concerning the effect of various herbicides, when used alone or in combination, on nitrite accumulation in excised leaves of wheat (Triticum aestivum L., var. ;Centurk'). Treatment of leaves with photosynthetic inhibitor herbicides, known to interfere with the transfer of light energy, caused accumulation of nitrite under illuminated, aerobic conditions. When certain other herbicides, which do not interfere with the photosynthetic process, were applied to leaves and incubated under dark, aerobic conditions, nitrite accumulations were enhanced over those treated with photosynthetic inhibitors or the controls. The combination of photosynthetic inhibitor herbicides and certain other "nonphotosynthetic inhibitor" herbicides caused relatively large amounts of nitrite to accumulate in light or in darkness. Nitrite accumulation occurs when nitrate and nitrite reduction are not in balance. The proposed actions of the herbicides used in this study are discussed. This discussion provides a rationale for the accumulation of nitrite by the herbicide-treated leaves. PMID- 16660948 TI - Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: I. General Characteristics and Effect of Inhibitors. AB - Corn mitochondria show respiration-linked net accumulation of [(3)H]ADP in the presence of phosphate and magnesium, especially if the formation of ATP is blocked with oligomycin. Inhibition of ADP-ATP exchange by carboxyatractyloside also activates ADP accumulation, and addition of carboxyatractyloside or palmitoyl-coenzyme A to oligomycin-blocked mitochondria produces additional ADP uptake. With carboxyatractyloside the accumulated ADP is phosphorylated to ATP. With oligomycin, only a little ATP is formed. Millimolar concentrations of ADP are required for maximum uptake, and the K(m) (3.77 millimolar) for ADP translocation is independent of whether oligomycin or carboxyatractyloside is used. This is not true for ADP concentrations in the 0.05 to 0.25 millimolar range. Accumulated [(3)H]ADP rapidly exchanges with unlabeled AMP, ADP, or ATP, but not with other diphosphate nucleotides or 2 millimolar substrate anions. [(3)H]AMP is not accumulated, but [(3)H]ATP is accumulated to about one-half the extent of [(3)H]ADP. Tricarboxylate substrates inhibit ADP net uptake, and inhibition by citrate is competitive with K(i) = 10 millimolar. The evidence suggests the presence of a pathway, carboxyatractyloside-insensitive and different from the translocase, which operates to maintain adenine nucleotides in the matrix. PMID- 16660949 TI - Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: II. Phosphate and Divalent Cation Requirement. AB - The requirement for phosphate and Mg(2+) in energy-linked [(3)H] ADP accumulation by corn mitochondria has been studied. Arsenate will fully substitute for phosphate; sulfate partially substitutes; acetate, bicarbonate, and pyrophosphate are ineffective. Phosphate is also taken up by the mitochondria, but the ADP/Pi ratio varies widely with experimental treatments. ADP does not exchange with endogenous labeled phosphate, although Pi/(32)Pi exchange occurs.Mg(2+) is also accumulated during ADP uptake. Mg(2+) can be substituted with varying efficiency by other divalent cations, but not monovalent cations. Effective cations typically increase phosphate uptake, particularly Ca(2+) . Ca(2+) -activated ADP accumulation is insensitive to carboxyatractyloside over a wide range of Ca(2+) concentrations. When Ca(2+) is substituted for Mg(2+) it is not necessary to block ATP formation to secure high levels of ADP accumulation, since Ca(2+) will divert energy from ATP formation into ion uptake.It is suggested that the transport mechanism may carry out a concerted transport of ADP and phosphate with bound divalent cation. The phosphate transporter may be involved, or alternatively a special mechanism for trivalent anion transport may exist which acts cooperatively with the phosphate transporter. PMID- 16660950 TI - Regulation of Photosynthetic Activity in the Primary Leaves of Bean (Phaseolus vulgaris L.) by Materials Moving in the Water-conducting System. AB - Endogenous factors which determine the photosynthetic capacity of the leaf were studied in the fully expanded, primary leaves of young seedings of bean (cv. Bulgarian). Following removal of the shoot above the primary leaf node and excision of all axillary buds, the primary leaves increased in area and thickness, in chlorophyll content, in levels of soluble protein, and in the specific activity of ribulose-1,5-bisphosphate carboxylase. Plants in which phloem continuity was disrupted by heat-girdling of the stem, between the shoot above the primary leaf node and the organs below, did not exhibit similar increases, whereas the shoot above the girdle continued to grow for several days. Plants in which all developing trifoliate leaves were excised as soon as they became macroscopic exhibited an increase in their photosynthetic activity, area, and thickness, while their main stem and (leafless) branches made considerable growth. Transpiration from the primary leaves was the same in decapitated plants as in the heat-girdled ones, although in the latter it accounted for only about 30% of total transpiration.The photosynthetic capacity of the primary leaves is not controlled by source/sink relationship of photosynthates, but rather by the pattern of distribution of factors carried from the roots to the leaves in the transpiration stream. PMID- 16660951 TI - Influence of enol ether amino acids, inhibitors of ethylene biosynthesis, on aminoacyl transfer RNA synthetases and protein synthesis. AB - The analogs of rhizobitoxine, aminoethoxyvinylglycine (AVG) (l-2-amino-4-2' aminoethoxy-trans-3 butenoic acid) and methoxyvinylglycine (MVG) (l-2-amino-4 methoxy-trans-3-butenoic acid), that are potent inhibitors of ethylene biosynthesis at 0.1 millimolar also inhibited protein synthesis and charging of tRNA especially at 1 millimolar and higher concentrations. The saturated analog of MVG inhibited ethylene synthesis while the saturated analog of AVG did not. Both saturated AVG and MVG inhibit methionyl- and leucyl-amino acyl-tRNA synthetase. Because of the inhibition of amino acid metabolism in plant tissues by these rhizobitoxine analogs caution is advised in interpreting the results obtained with concentrations of compounds above 0.1 millimolar. PMID- 16660952 TI - Photosynthetic Enzyme Activities and Localization in Mollugo verticillata Populations Differing in the Levels of C(3) and C(4) Cycle Operation. AB - Ecotypic differences in the photosynthetic carbon metabolism of Mollugo verticillata were studied. Variations in C(3) and C(4) cycle activity are apparently due to differences in the activities of enzymes associated with each pathway. Compared to C(4) plants, the activities of C(4) pathway enzymes were generally lower in M. verticillata, with the exception of the decarboxylase enzyme, NAD malic enzyme. The combined total carboxylase enzyme activity of M. verticillata was greater than that of C(3) plants, possibly accounting for the high photosynthetic rates of this species. Unlike either C(3) or C(4) plants, ribulose bisphosphate carboxylase was present in both mesophyll and bundle sheath cell chloroplasts in M. verticillata. The localization of this enzyme in both cells in this plant, in conjunction with an efficient C(4) acid decarboxylation mechanism most likely localized in bundle sheath cell mitochondria, may account for intermediate photorespiration levels previously observed in this species. PMID- 16660953 TI - Effects of Substituted Pyridazinones (San 6706, San 9774, San 9785) on Glycerolipids and Their Associated Fatty Acids in the Leaves of Vicia faba and Hordeum vulgare. AB - The fatty acids of the major glycerolipids from the leaves of Vicia faba and Hordeum vulgare plants treated with three different concentrations of pyridazinone derivatives were analyzed. These compounds showed multiple effects on the levels of lipids and pigments. At low concentrations, the primary effect of San 9785 was on the level of linolenic acid (18:3) in the galactolipids of V. faba, whereas the effect of San 6706 was primarily on the trans-Delta(3) hexadecenoic acid (16:1) content in phosphatidylglycerol. At higher concentrations, the two compounds reduced the content of both fatty acids in the leaves. The results appear to indicate a differential effect of these herbicides on fatty acid accumulation and a difference in susceptibility of two fatty acids in the species examined. Electron microscopic studies revealed that two herbicides caused different abnormalities in V. faba chloroplast ultrastructure. PMID- 16660954 TI - Postharvest Variation in Cellulase, Polygalacturonase, and Pectinmethylesterase in Avocado (Persea americana Mill, cv. Fuerte) Fruits in Relation to Respiration and Ethylene Production. AB - Cellulase, polygalacturonase (PG), pectinmethylesterase (PME), respiration, and ethylene production were determined in single "Fuerte" avocado fruits from the day of harvest through the start of fruit breakdown. PME declined from its maximum value at the time of picking to a low level early in the climacteric. PG activity was not detectable in the preclimacteric stage, increased during the climacteric, and continued to increase during the postclimacteric phase to a level three times greater than when the fruit reached the edible soft stage. Cellulase activity was low in the preclimacteric fruit, started to increase just as respiration increased, and reached a level two times greater than at the edible soft stage. Cellulase activity started to increase 3 days before PG activity could be detected. Increased production of ethylene followed the increase in respiration and cellulase activity by about 1.5 days. These results indicate that a close relation exists between the rapid increase in the cell wall depolymerizing enzymes and the rise in respiration and ethylene production and refocused attention on the role of the cell wall and the associated plasma membrane in the early events of fruit ripening. PMID- 16660955 TI - Nitrogen Assimilation Pathways in Leaf Mesophyll and Bundle Sheath Cells of C(4) Photosynthesis Plants Formulated from Comparative Studies with Digitaria sanguinalis (L.) Scop. AB - Nitrogen assimilation in crabgrass Digitaria sanguinalis (L.) Scop., was studied by comparing leaf extracts with isolated mesophyll cell and bundle sheath strand extracts. The results show that both nitrate and nitrate reductase are localized in mesophyll cells; glutamine synthetase is nearly equally distributed in the mesophyll and bundle sheath; approximately 67% of the glutamate synthase activity is in the bundle sheath and 33% is in the mesophyll; and 80% of the glutamate dehydrogenase activity is in the bundle sheath, with the NADH-dependent form exhibiting a 2.5-fold higher activity than the NADPH-dependent form.Isolated crabgrass mesophyll cells reduce NO(2) (-) coupled to the photochemical production of O(2) but are inactive with NO(3) (-). The NO(2) (-) -dependent O(2) evolution is light-dependent; inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea; stimulated by photophosphorylation uncouplers; and exhibits a stoichiometry of O(2) evolved to NO(2) (-) reduced of 1.45 and 0.67 in coupled and uncoupled experiments, respectively. Isolated bundle sheath strands are inactive in O(2) evolution with NO(3) (-) or NO(2) (-).Based on these results, plus literature data, two schemes for crabgrass leaf nitrogen assimilation are presented, depending on whether the plant is using ammonium or nitrate as its nitrogen source. It is proposed that the increased nitrogen use efficiency in crabgrass and other C(4) plants is due partially to a "division of labor" between mesophyll and bundle sheath cells, where NO(3) (-) and NO(2) (-) reductase in mesophyll cells act as nitrogen reduction traps in an analogous fashion to phosphoenolpyruvate carboxylase acting as a CO(2) trap during C(4) photosynthesis. PMID- 16660956 TI - Cell Surfaces in Plant-Microorganism Interactions: I. A Structural Investigation of Cell Wall Hydroxyproline-rich Glycoproteins Which Accumulate in Fungus infected Plants. AB - Infection of muskmelon Cucumis melo seedlings by the fungus Colletotrichum lagenarium causes a 10-fold increase in the amount of cell wall hydroxyproline rich glycoprotein. Evidence for this increase was provided by studying two specific markers of this glycoprotein, namely hydroxyproline and glycosylated serine. The lability of the O-glycosidic linkage of wall-bound glycosylated serine in the presence of hydrazine, was used to determine the amount of serine which is glycosylated.A large increase in the hydroxyproline content of infected plants is shown, but the ratios of glycosylated serine to hydroxyproline are similar in healthy and infected plants. As far as these markers are concerned, the hydroxyproline-rich glycoproteins secreted into the wall as a result of the disease are similar to those of healthy plants. In addition, the extent of glycosylation of the wall serine, in both healthy and infected plants, decreases as the plant ages.Serine- and hydroxyproline-rich (glyco)peptides were also isolated after trypsinolysis of the wall. These (glyco)peptides include the galactosyl-containing pentapeptide, serine-hydroxyproline(4). This pentapeptide is characteristic of cell wall protein. PMID- 16660957 TI - Cell Surfaces in Plant-Microorganism Interactions: II. Evidence for the Accumulation of Hydroxyproline-rich Glycoproteins in the Cell Wall of Diseased Plants as a Defense Mechanism. AB - Enrichment of the cell wall in hydroxyproline-rich glycoprotein is involved in the defense of muskmelon (Cucumis melo) seedlings to Colletotrichum lagenarium, the causative agent of anthracnose. The extent to which this accumulation proceeds may be experimentally modified by treating plants with ethylene or growing them in the presence of free l-trans-hydroxyproline. It appears that the increase in the wall hydroxyproline-rich glycoprotein mediated through ethylene is paralleled by an increasing resistance of the host to the pathogen. Inversely, inhibiting the synthesis of this glycoprotein in diseased plants is strictly correlated to an accelerated and more intense colonization of the host by the pathogen.In both cases, the inverse relationship between the accumulation of hydroxyproline-rich glycoproteins and the ability of the pathogen to develop in the host has been checked by the quantification, in infected tissues, of glucosamine, a characteristic component of chitin-containing fungi. PMID- 16660958 TI - Phytochrome radioimmunoassay. AB - A phytochrome radioimmunoassay with a detection limit of about 2 nanograms has been developed. The radioimmunoassay does not suffer from the potential drawbacks of the commonly used spectral assay and requires less than 1 microliter of crude extract from dark-grown plants for quantitation of phytochrome. Measurement of phytochrome in crude extracts by radioimmunoassay gives values about 25% greater than those obtained by spectral assay. The amount of phytochrome detected in crude extracts of light-grown oats by radioimmunoassay is approximately 1% of that detected in comparable extracts from dark-grown oats. General interference by crude plant extracts with radioimmunoassays was also observed and corrected for. PMID- 16660959 TI - Phytochrome immunoaffinity purification. AB - We have developed a phytochrome immunoaffinity purification procedure that yields undegraded oat (Avena sativa L., cv. Garry) phytochrome of greater than 98% purity within 2 hours when starting with a brushite-purified preparation. Immunoaffinity-purified phytochrome, except for its greater purity, is indistinguishable from conventionally purified phytochrome by gel exclusion chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have also used the immunoaffinity technique to purify phytochrome from crude oat extracts, and from brushite-purified pea (Pisum sativum L., cv. Alaska) and rye (Secale cereale L., cv. Balbo) preparations. PMID- 16660960 TI - Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase. AB - Rabbit antiserum against beta-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with beta-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of beta-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-beta-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that beta-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of beta-amylase bound to protein bodies in cereal seeds. PMID- 16660961 TI - Bright light does not immediately stop the circadian clock of gonyaulax. AB - Circadian rhythms in acid-stimulated bioluminescence and cell division are observed for at least 16 days in bright continuous light (4.5 milliwatts per square centimeter or 20,000 lux). The photosynthesis rhythm also fails to stop immediately upon transfer of cell suspensions to bright light. After about 4 weeks under these conditions, all rhythms were observed to damp out. In cells transferred from bright light to continuous darkness, the rhythms were reset to about circadian hour 12 to 14, the phase of the beginning of a normal night. PMID- 16660962 TI - Importance to Gas Exchange of Mass Flow of Air through Leaves. AB - A reanalysis of results from a recent paper on the effect of oscillation on gas exchange through leaves of cottonwood (Populus deltoides, Marsh) is presented. Mass flow of air through the leaf cannot account for the observed increase in gas exchange during oscillation in that experiment. Consideration of various published data shows that in the field, mass flow will not constitute more than a few per cent of the total exchange for most agricultural crops, but may be a significant part of the exchange for the leaves of tall trees, as they can be exposed to high winds. PMID- 16660963 TI - Effect of temperature on respiration of a mesophilic and a thermophilic fungus. AB - The respiratory rates of mycelia of the mesophilic fungus, Aspergillus niger, and the thermophilic fungus, Thermomyces lanuginosus, were comparable at their respective temperature optima for growth. The respiratory rate of A. niger was independent of changes in temperature between 15 and 40 C. The respiratory rate of T. lanuginosus increased with increase in temperature between 25 and 55 C. PMID- 16660964 TI - Changes in Frost Hardiness of Stem Cortical Tissues of Cornus stolonifera Michx. after Recovery from Water Stress. AB - Moderate water stress increases frost hardiness in many woody plants but little attention has been given to changes in hardiness after recovery from water stress. Tests were carried out to examine how much water stress-induced frost hardiness remained when plants were rewatered under different day length regimes. Red osier dogwood plants (Cornus stolonifera Michx.) were water-stressed at normal growing temperatures in long day (LD) or short day (SD) conditions, exposed to 6 nights of freezing temperatures, and then returned to normal growing conditions with full water supply. Water-stressed plants gained an additional 8 to 10 C of hardiness. The amount of freeze-induced hardiness in both stressed and control plants was not significant (approximately 2 C) and was not affected by photoperiod. When plants were kept in or transferred to LD, they lost nearly all of their water stress-induced hardiness within 7 days after rewatering. Water stressed plants in SD lost the least amount of hardiness (5 C) when rewatered. In dogwood, water stress is an effective way to increase hardiness temporarily, but the photoperiod has a large effect on the retention of the acquired hardiness. PMID- 16660965 TI - Photosynthetic and Photorespiratory Characteristics of Mutants of Hordeum vulgare L. AB - The relationship between photosynthesis and photorespiration was determined in normal and 26 mutants of barley (Hordeum vulgare L. var. Himalaya). The rate of apparent photosynthesis ranged from 1 to 30 milligrams of CO(2) per square decimeter per hour. The variation in rate of photosynthesis was due, in some cases, to differences in chlorophyll content, in others to stomatal resistance, and in still others to unknown factors; but no single factor accounted for the variation. Photorespiratory activity, as determined by the (14)CO(2)/(12)CO(2) technique, CO(2) evolution into CO(2)-free air, and the response of photosynthesis to low and high O(2) concentrations, was positively and significantly correlated with photosynthesis. This supports the idea that the two processes are integrally and tightly coupled. There appears to be no competition between photosynthesis and photorespiration, and the probability of finding plants with high rates of photosynthesis and low rates of photorespiration measured under natural conditions, appears to be very low. PMID- 16660966 TI - A method for continuous measurement of export from a leaf. AB - Export of labeled material derived by continuous photosynthesis in (14)CO(2) was monitored with a Geiger-Muller detector positioned next to an exporting leaf blade. Rate of export of labeled material was calculated from the difference between rates of retention and net photosynthesis of labeled carbon for the observed leaf. Given certain conditions, including nearly constant distribution of labeled material among minor veins and various types of cells, count rate data for the source leaf can be converted to rate of export of carbon. Changes in counting efficiency resulting from changes in leaf water status can be corrected for with data from a transducer which measures leaf thickness.Export data agreed with data obtained by monitoring the arrival of (14)C in the sink region; isolated leaves gave values near zero for export. The method allows continuous nondestructive measurement of export of labeled carbon from a given leaf on an intact plant. The technique detects changes in export with a resolution of 10 to 20 minutes. PMID- 16660967 TI - Cadmium Uptake by Pinus resinosa Ait. Pollen and the Effect on Cation Release and Membrane Permeability. AB - Cadmium uptake by red pine (Pinus resinosa Ait.) pollen from a graded series of Cd(2+) solutions (0 to 2.88 microequivalents per 50 milligrams pollen) and its effect on membrane integrity were examined by atomic absorption spectroscopy. Uptake was strongly dependent on Cd(2+) concentration and was limited to adsorption and cation exchange in pollen walls during a 3-hour measurement period. Good correlation between measured Cd(2+) uptake and that predicted by the Langmuir and Freundlich isotherm equations indicated the adsorptive nature of Cd(2+) uptake. While substantial quantities of Ca(2+) and Mg(2+) were released by exchange mechanisms concurrent with Cd(2+) uptake, there was no evidence for leakage of cations due to membrane impairment as indicated by a poor correlation between Cd(2+) uptake and K(+) efflux. Virtually all Cd(2+) removed from solution was freely exchangeable with 0.5 millimolar CaCl(2) and demonstrated that Cd(2+) did not readily enter pine pollen but was adsorbed on the pollen wall. Ultraviolet transmission spectra of treatment solutions and analyses of phosphate and reducing sugar efflux also indicated that the potent toxicity of Cd(2+) to pollen germination and germ tube elongation was not the result of membrane damage. PMID- 16660968 TI - Enzymes of General Phenylpropanoid Metabolism and of Flavonoid Glycoside Biosynthesis in Parsley: Differential Inducibility by Light during the Growth of Cell Suspension Cultures. AB - Several enzymes of phenylpropanoid metabolism showed large changes in their inducibility by light during the growth cycle of cell suspension cultures from parsley (Petroselinum hortense Hoffm.). Two of the three enzymes of general phenylpropanoid metabolism (group I) and six of the approximately 13 enzymes of the flavone and flavonol glycoside pathways (group II) were investigated. Both enzymes of group I (phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase) were most efficiently induced at two different stages: first, soon after starting a new culture, and second, near the beginning of the stationary phase. In contrast, the enzymes of group II (acetyl-coenzyme A carboxylase, flavanone synthase, chalcone isomerase, UDP-apiose synthase, and at least one of two malonyltransferases) were maximally induced during exponential growth of the culture. This result supports the conclusions drawn from previous data that the two groups are regulated differentially and that the enzymes within each group are regulated in a coordinated manner. PMID- 16660969 TI - Rubidium (potassium) uptake by Arabidopsis: a comparison of uptake by cells in suspension culture and by roots of intact seedlings. AB - Experiments are reported in which the uptake of (86)Rb(+), used as an analog of K(+), into cultured cells of Arabidopsis thaliana is investigated. A single transport system is found with K(m) = 0.34 millimolar and V(max) = 14 nmoles per milligram of protein per hour. This system is blocked by the metabolic inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and by cold. At high concentrations of external K(+) (above 1 millimolar), a significant fraction of total uptake is energy-independent. No evidence is found for more than one energy dependent uptake system or for concentration-dependent modifications of a carrier as postulated in multiphasic transport models.Rb(+) uptake was also examined in cultured cells derived from an "osmotic mutant" of Arabidopsis. The system closely resembles that found in wild type cells with the exception that the Michaelis-Menten constants are higher: K(m) = 1 millimolar and V(max) = 32 nanomoles per milligram of protein per hour.The possibility that these results are artifacts associated with use of cultured cells was checked by examining (86)Rb(+) uptake by roots of intact seedlings of wild type Arabidopsis. A single energy-dependent transport system is found with K(m) = 0.42 millimolar which is not significantly different from the K(m) of cultured cells. There is also energy independent uptake at high external ion concentration. PMID- 16660970 TI - A circadian rhythm in oxygen uptake by samanea pulvini. AB - The rate of O(2) uptake by excised Samanea pulvini oscillates with a circadian rhythm during 52 hours of darkness. Rates of respiration increase during pulvinar opening and decrease prior to and during closure, consistent with the concept that opening requires a greater expenditure of energy. Externally supplied sucrose, necessary for perpetuation of the leaflet movement rhythm, has a small promotive effect on the rate of respiration. PMID- 16660971 TI - In vivo activity, purification, and characterization of shikimate kinase from sorghum. AB - Sorghum bicolor stems and shoots were observed to phosphorylate exogenously supplied shikimate, forming a product which could not be distinguished from shikimate 3-phosphate by anion exchange and thin layer chromatography. Upon treatment with phosphatase, this product gave rise to a compound which co chromatographed with shikimate.Shikimate kinase from stems was separated from phosphatase and ATPase and partially characterized. The stoichiometry of the reaction required equimolar quantities of ATP and shikimate to produce ADP and shikimate 3-phosphate. Maximal enzymic activity was observed near pH 9 in the presence of 11 millimolar MgCl(2). Graphs of reaction velocity as a function of substrate concentration were hyperbolic for both substrates and K(m) values of 0.2 and 0.11 millimolar were calculated for shikimate and ATP, respectively. Shikimate kinase was not inhibited by phenylalanine, tyrosine, or tryptophan, either alone or in combination. Slight inhibition was caused by p-coumarate and greater inhibition by caffeate. Inhibition was also observed in the presence of ADP and AMP, but the reaction velocity was not highly responsive to adenylate "energy charge" in experiments with ADP-ATP mixtures. PMID- 16660972 TI - Ligand Specificity of a High Affinity Cytokinin-binding Protein. AB - A soluble cytokinin-binding protein from wheat germ that has a high affinity for a range of purine cytokinins also interacts with a variety of nonpurine compounds that can affect cytokinin-modified processes in animal or plant cells or which bind to proteins known to interact with certain cytokinins. A variety of structurally disparate compounds which inhibit chloroplast photosystem II activity (including phenylurea, carbanilate, and alkylamino-2-chloro-sym-triazine compounds) displace kinetin from the protein in an apparently competitive fashion. However, various energy transfer inhibitors (including organotin compounds and N,N'-dicy-clohexylcarbodiimide) also inhibit kinetin binding to the protein. N(6),2-0'-Dibutyryl-3',5'-cyclic AMP and 1-methyl-3-isobutylxanthine (the effects of which on fibroblast morphology and motility can be mimicked by cytokinins) are inhibitors of kinetin binding to the protein. A variety of compounds that can have antimitotic effects (including 1-methyl-3 isobutylxanthine and certain alkylated cyclic nucleotide, carbanilate, and tryptamine compounds) displace kinetin from the protein. However, a variety of indole derivatives also displace kinetin from the cytokinin-binding protein, which in a qualitative sense has a broad ligand specificity. PMID- 16660973 TI - Reversal of alpha,alpha'-Dipyridyl-induced Porphyrin Synthesis in Etiolated and Greening Red Kidney Bean Leaves. AB - The chemical induction of porphyrin synthesis has been investigated in etiolated and greening leaves of Phaseolus vulgaris L. var. Red Kidney. When these leaves are incubated in darkness with solutions of transition metal ion chelators such as alpha,alpha'-dipyridyl, 1,10-phenanthroline, pyridine-2-aldoxime, or other related aromatic heterocyclic nitrogenous bases, they synthesize large amounts of protochlorophyllide and Mg protoporphyrins. Greening leaves produce more porphyrin than do etiolated leaves under such conditions. If the leaves are then transferred to 1 millimolar solutions of various transition metal salts such as Fe(2+), Zn(2+), or Co(2+) (but not Mn(2+) or Mg(2+)), Mg protoporphyrin (monomethyl ester) synthesis immediately ceases and the pigment(s) rapidly disappear(s); protochlorophyllide synthesis gradually diminishes during 4 to 8 hours of treatment. The loss in Mg protoporphyrin(s) can be accounted for by a simultaneous increase in protochlorophyllide in partially greened leaves but not in etiolated leaves. In the latter, the decline in Mg protoporphyrin(s) initiated by the application of Zn(2+) is retarded by low temperature and anaerobiosis but not by respiratory inhibitors. Cycloheximide inhibits the loss of Mg protoporphyrin(s) but does not affect their conversion to protochlorophyllide.THESE RESULTS INDICATE THAT: (a) greening leaves have a greater capacity to synthesize delta-aminolevulinic acid than do etiolated leaves; (b) alpha,alpha'-dipyridyl induction of porphyrin synthesis in etiolated and greening leaves can be blocked by application of certain transition metal salts; (c) in greening leaves the accumulated Mg protoporphyrin(s) are stoichiometrically converted to protochlorophyllide upon treatment with these salts whereas in etiolated leaves the accumulated Mg protoporphyrin(s) are labile and are not quantitatively converted to protochlorophyllide upon such treatment; (d) in etiolated leaves the accumulated Mg protoporphyrin(s) are destroyed via a light-independent, probably enzymic process which requires cytoplasmic protein synthesis. PMID- 16660974 TI - Identification of etioplast membranes in fractions from soybean hypocotyls. AB - Three independent methods, one cytological and two biochemical, were used to estimate contributions of plastids and plastid fragments to various membrane fractions. In thin sections viewed by electron microscopy, KMnO(4) selectively enhanced the images of plastid membranes in situ as well as in isolated fractions. The amounts of plastid fragments in isolated membrane fractions were determined by electron microscopic morphometry of fractions fixed with KMnO(4) in conjunction with analysis of galactolipids and carotenoids. Monogalactosyl and digalactosyl diglyceride contents were directly correlated with the amount of plastid membranes in the fractions identified by electron microscope morphometry. Amounts of carotenoids also correlated with plastid membranes except at very low levels where estimates based on carotenoids exceeded those based on morphometry. PMID- 16660975 TI - Chloroplast and Cytoplasmic Enzymes: VIII. Amino Acid Composition of the Pea Leaf Aldolases. AB - Pea leaf chloroplast aldolase contains seven more aspartyl (asx) residues and four fewer leucine and isoleucine residues than the cytoplasmic enzyme. The two forms are therefore primary isoenzymes, differing in amino acid sequence. PMID- 16660976 TI - Diterpene biosynthesis in maize seedlings in response to fungal infection. AB - A cell-free system which catalyzes the biosynthesis of terpene hydrocarbons when supplemented with mevalonate, Mn(2+), and ATP was prepared from the scutellum embryonic axis region of maize seedlings. The capacity of this system for the production of terpene hydrocarbons was enhanced 50- to 100-fold when the seedlings were exposed for 48 hours to the fungus Rhizopus stolonifer prior to tissue homogenization. The fungi Aspergillus niger, Fusarium moniliforme, and Verticillium albo-atrum also elicited this biosynthetic enhancement. The terpene hydrocarbon products were separable into six fractions by argentation thin layer chromatography. Radioactivity was contributed to five of these fractions when either geranylgeranyl pyrophosphate or copalyl pyrophosphate was supplied as substrate, suggesting that polycyclic diterpenoid hydrocarbons were the main products. Large scale biosynthetic reactions led to the acquisition of about 1 milligram of terpene hydrocarbon products plus some more polar terpenoid products. Analysis of the hydrocarbon products by gas chromatography and mass spectrometry led to the separation of six distinct diterpene hydrocarbons plus a fraction with a molecular weight of about 550. Three of the diterpene hydrocarbons were identified as kaur-16-ene, kaur-15-ene (isokaurene), and pimara 8(14),15-diene. None of the terpene hydrocarbon fractions tested displayed antifungal activity in the Cladosporium cucumerinum thin layer plate assay. PMID- 16660977 TI - Transport of nitrogen in the xylem of soybean plants. AB - Experiments were conducted to characterize the distribution of N compounds in the xylem sap of nodulated and nonnodulated soybean plants through development and to determine the effects of exogenous N on the distribution of N compounds in the xylem. Xylem sap was collected from nodulated and nonnodulated greenhouse-grown soybean plants (Glycine max [L.] Merr. "Ransom") from the vegetative phase to the pod-filling phase. The sum of the nitrogen in the amino acid, nitrate, ureide (allantoic acid and allantoin), and ammonium fractions of the sap from both types of plants agreed closely with total N as assayed by a Kjeldahl technique. Sap from nodulated plants supplied with N-free nutrient solution contained seasonal averages of 78 and 20% of the total N as ureide-N and amino acid-N, respectively. Sap from nonnodulated plants supplied with a 20 millimolar KNO(3) nutrient solution contained seasonal averages of 6, 36, and 58% of total N as ureide-N, amino acid-N, and nitrate-N, respectively. Allantoic acid was the predominant ureide in the xylem sap and asparagine was the predominant amino acid. When well nodulated plants were supplied with 20 millimolar KNO(3), beginning at 65 days, C(2)H(2) reduction (N(2) fixation) decreased relative to nontreated plants and there was a concomitant decrease in the ureide content of the sap. A positive correlation (r = 0.89) was found between the ureide levels in xylem sap and nodule dry weights when either exogenous nitrate-N or urea-N was supplied at 10 and 20 millimolar concentrations to inoculated plants. The results demonstrate that ureides play a dominant role in N transport in nodulated soybeans and that the synthesis of ureides is largely dependent upon nodulation and N(2) fixation. PMID- 16660978 TI - Studies of Rapidly Induced Wound Ethylene Synthesis by Excised Sections of Etiolated Pisum sativum L., cv. Alaska: IV. Requirement of a Water-soluble, Heat stable Factor. AB - The rate of wound ethylene synthesis was reduced by more than 85% when 9 millimeter subapical sections of etiolated 7-day-old Pisum sativum L., cv. Alaska seedlings were incubated in water during the 26-minute induction period prior to wound ethylene synthesis, but the rate of synthesis was unaffected if sections were incubated in water during the actual synthesis of wound ethylene. The characteristic timing of the wound response was unaffected by either treatment. The ability of various chemical solutions and aqueous plant extracts to alter the rate of wound ethylene synthesis was studied by first incubating subapical pea stem sections in solutions under anaerobic conditions (anaerobiosis delays the induction and synthesis of wound ethylene; Plant Physiol 61: 675-679), and then measuring wound ethylene synthesis after the tissue was transferred to air. Solutions of several reported precursors of ethylene synthesis, such as methionine, homoserine, or propanal, did not reverse the water-caused reduction of wound ethylene synthesis. A water-soluble, heat-stable factor in extracts from pea seedlings, and solutions of 23 nanomolar triacontanol, 10 micromolar kinetin, or 10 micromolar benzyladenine prevented the reduction of wound ethylene synthesis, but were ineffective if administered after an initial 15-minute anaerobic water incubation. This suggested that the active solutions may have only prevented the loss of some ephemeral, though necessary factor, rather than actually containing the substrate or inducer of wound ethylene synthesis. Attempts to isolate and characterize the active fraction from aqueous tissue extracts were unsuccessful. Free radical quenchers, inhibitors of protein synthesis, and rhizobitoxine, an inhibitor of ethylene synthesis from methionine, all reduced wound ethylene synthesis when administered in solutions which previously had maintained wound ethylene synthesis. PMID- 16660979 TI - Effect of Daylength on the Ability of Salicylic Acid to Induce Flowering in the Long-day Plant Lemna gibba G3 and the Short-day Plant Lemna paucicostata 6746. AB - When the long-day plant Lemna gibba L., strain G3 is grown on control medium the critical daylength is just under 10 hours. With 3.2 micromolar salicylic acid added to the medium substantial flower promotion is obtained on 9-, 10-, and 11 hour daylengths. On an 8-hour daylength salicylic acid treatment results in only a very small flowering response, and with daylengths less than 8 hours flowering is never obtained. Thus, salicylic acid treatment causes a shift in the critical daylength of about 2 hours, from just under 10 hours to just under 8 hours.Salicylic acid treatment also results in flower promotion in the short-day plant Lemna paucicostata Hegelm., strain 6746, and the optimal concentration of 1.8 to 3.2 micromolar agrees well with the results for L. gibba G3. In control medium the critical daylength for L. paucicostata 6746 is about 14 hours. When the plants are given 3.2 micromolar salicylic acid substantial flower promotion is obtained on daylengths of 13 and 14 hours, and the critical daylength is close to 15 hours. With daylengths longer than 15 hours, flowering is never obtained. These results are similar to those for L. gibba G3 since for both plants salicylic acid stimulates flowering by causing a shift in the critical daylength curve, but for L. paucicostata 6746 the shift is only about 1 hour and the critical daylength is extended rather than shortened. PMID- 16660980 TI - Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes. AB - Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars. PMID- 16660981 TI - Elongation Pathway for alpha-Linolenic Acid Synthesis in Spinach Leaves: A Reexamination. AB - Leaf slices from spinach exhibited considerable variation in their incorporation of [(14)C]bicarbonate and [1-(14)C]acetate into fatty acids. Reductive ozonolysis studies indicated that all the (14)C-labeled fatty acids were synthesized de novo; no in vivo evidence was found for the previously proposed elongation of hexadecatrienoate to alpha-linolenate. PMID- 16660982 TI - Cyanide-insensitive Respiration in Pea Cotyledons. AB - Mitochondria isolated by a zonal procedure from the cotyledons of germinating peas possessed a cyanide-resistant respiration. This respiration was virtually absent in mitochondria isolated during the first 24 hours of germination but thereafter increased gradually until the 6th or 7th day of seedling development. At this time between 15 and 20% of the succinate oxidation was not inhibited by cyanide. The activity of the cyanide-resistant respiration was also determined in the absence of cyanide. Relationships among mitochondrial structure, cyanide resistant respiration, and seedling development are discussed. PMID- 16660983 TI - Enhanced Dark Carbon Dioxide Fixation in Maize: Effect of the Oxygen Concentration during Preillumination on CO(2) Uptake and the Intramolecular Labeling Pattern of Malate and Aspartate. AB - The enhanced dark CO(2) uptake after a preillumination period under varying O(2) concentrations has been measured with maize, a C(4) plant. For comparison the same study has been conducted with tomato, a C(3) plant. Increasing the O(2) concentration during preillumination inhibits by 70% the subsequent dark CO(2) uptake in tomato but stimulates 2-fold this CO(2) uptake in maize. The O(2) enhancement of CO(2) uptake in maize is due to the enhancement of malate and aspartate synthesis. The percentages of radioactivity incorporated in the C-4 of malate and aspartate vary from 74 to 87% when O(2) concentration during preillumination is increased from 0 to 100%.An interpretation of these data led to the conclusion that malate and aspartate formed during the light-enhanced dark fixation result from two competing carboxylation reactions: a single carboxylation of phosphoenolpyruvate and a double carboxylation involving ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylases. This latter route of carbon flow prevails when preillumination is carried out anaerobically. It appears that increasing O(2) concentration stimulates phosphoenolpyruvate synthesis during preillumination and consequently favors the single carboxylation of phosphoenolpyruvate, leading to an increased percentage of radioactivity in the C-4 atom of malate and aspartate. PMID- 16660984 TI - In Vitro Stability of Nitrate Reductase from Wheat Leaves: II. Isolation of Factors from Crude Extract Which Affect Stability of Highly Purified Nitrate Reductase. AB - When a crude extract from 8-day-old wheat (Triticum aestivum L. cv. Olympic) leaves was fractionated by a combination of ammonium sulfate precipitation and Sephadex G-100 chromatography the presence of three factors which have a marked effect on the stability of highly purified nitrate reductase was revealed. Two of these factors (I and III) have a positive effect and the other factor (II) has a negative effect on stability. Factors I and III can each overcome the instability promoting effect of II; however, this was apparently not due to a direct effect on factor II.Both factors I and III have been subjected to further purification. Factor I can be separated into at least four fractions, each with stability promoting activity. Factor III appears to be a single factor.The in vitro activity and stability of nitrate reductase in crude extracts were found to vary diurnally. Stability and activity were highest 4 hours after the start of the light period and both were minimal 1 to 3 hours after the end of the light period. When crude extract was fractionated as described above and an assessment made of the relative amounts of I, II, and III, there appeared to be a distinct diurnal variation in their levels. Factors I and III were highest when in vitro nitrate reductase activity and stability were highest. Factor II was apparently out of phase in that maximum activity coincided with the time of minimum in vitro nitrate reductase activity and stability. PMID- 16660986 TI - Physiological control of exo- and endoproteolytic activities in germinating wheat and their relationship to storage protein hydrolysis. AB - The effects of gibberellic acid, abscisic acid, cycloheximide, actinomycin D, and cordycepin upon exo- and endoproteolytic activities and storage (gluten) protein hydrolysis in germinating wheat and in incubated embryoless wheat seeds have been studied. Early increases in endoproteolytic activity were insensitive to the addition of gibberellic acid and inhibitors of protein and RNA synthesis. Later increases in endoproteolytic activity were enhanced by gibberellic acid, strongly inhibited by abscisic acid and cycloheximide, and partially inhibited by actinomycin D and cordycepin. Increases in exoproteolytic activity were insensitive to the addition of gibberellic acid, abscisic acid, actinomycin D, and cordycepin but were inhibited in whole seeds when cycloheximide was added in the steeping medium. However, cycloheximide did not inhibit increases in exoproteolytic activity when added to embryoless seeds, to germinating whole seeds, or to seeds which had been stored at 4 C for extended periods of time. Comparison of the effects of gibberellic acid, abscisic acid, and inhibitors of protein and RNA synthesis upon storage protein hydrolysis and their effects upon proteolytic activity indicated that storage protein hydrolysis in germinating wheat is controlled by the rate of hormonally induced de novo synthesis of endoproteolytic enzymes. PMID- 16660985 TI - Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm. AB - In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [(14)C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [(14)C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.All of these reactions occur with the highest specific as well as total activity in the endoplasmic reticulum fraction. Whereas a membrane fraction lighter than the endoplasmic reticulum also contains a significant amount of glycosyl transfer activity, the enzymes seem to be present in very low amounts in glyoxysomes and mitochondria. The activities of the various reactions increase severalfold during the first days of germination, i.e. the time of rapid formation of glyoxysomes. The importance of these results in relation to intracellular protein segregation is discussed. PMID- 16660987 TI - Ozone alteration of membrane permeability in chlorella: I. Permeability of potassium ion as measured by rubidium tracer. AB - The addition of ozone to a suspension of Chlorella sorokiniana causes a rapid loss of K(+), as measured by efflux of (86)Rb from prelabeled cells. The efflux of the tracer is stimulated some 15 to 20 times over that of the control. For about 100 microliters per liter ozone, about 25 minutes (6 x 10(-8) moles O(3) delivered per minute) of exposure are required for a 50% depletion of the intracellular K(+). The stimulation of K(+) efflux is nearly linearly dependent upon the amount of ozone delivered into the solution. Following short pulses of ozone (lasting 1 to 5 minutes), efflux rates return to the control level but only after about 15 minutes.While influx of K(+) is ultimately inhibited by ozone, at low concentrations or for short exposure times the tracer influex is stimulated 100 to 200%. Ozone stimulation of an active pump mechanism is unlikely in view of a concomitant decrease in respiration. Thus, this influx may represent movement of K(+) along its electrochemical gradient. Assuming that influx and efflux are in steady-state according to the Goldman equation, it was calculated that the membrane potential for K(+) of -80 to -90 millivolts in control cells drops to 40 millivolts with ozone exposure and is accompanied by a calculated increased permeability to K(+) of 2- to 3-fold. PMID- 16660988 TI - Amino Acids Translocated from Turgid and Water-stressed Barley Leaves: I. Phloem Exudation Studies. AB - The phloem exudation technique of King and Zeevaart (Plant Physiol 1974 53: 96 103) was modified for use with barley plants, to investigate the effect of water stress upon amino acid translocation at seedling and grainfilled stages.Seedling leaves and flag leaves from unstressed and moderately water-stressed plants exuded (14)CO(2) assimilates, sugars, and amino acids when their sheaths were cut and immersed in a 5 millimolar solution of Na(2)EDTA (pH 7.0). By including PEG 6000 (-10 bars) in the Na(2)EDTA solution, leaves severed from moderately water stressed plants could be maintained in a wilted state. Such leaves produced about as much exudate as turgid leaves of unstressed plants.The following observations suggest a phloem origin for most of the exudate. Exudation was markedly stimulated by light and by CO(2) enrichment. The release of NO(3) (-) declined after cutting, and did not parallel exudation of (14)CO(2) assimilates, sugar, and amino acids. The relative quantities and specific radioactivities of sugars and amino acids in the exudate differed from those of sugars and amino acids extracted from sheath tissue.Major amino acids in exudate from unstressed seedling and flag leaves were glutamine, glutamate, serine, alanine, and aspartate; proline was virtually absent. Exudate from water-stressed leaves contained relatively more serine, and also some proline and gamma-aminobutyric acid. PMID- 16660989 TI - Amino Acids Translocated from Turgid and Water-stressed Barley Leaves : II. Studies with N and C. AB - Movement of labeled amino acids from leaf blades to sheaths was followed after supplying (13)NH(3) gas, (14)CO(2), or (14)C-amino-acids to attached blades of barley plants.Blades of turgid and wilted plants fed (13)NH(3) (at about 120 to 700 microliters per liter) had incorporated (13)N mainly into free glutamine and glutamate after 30 minutes, and turgid blades had exported 1 to 3% of the assimilated (13)N to the sheaths, mostly as glutamine and glutamate. Wilted blades exported less of the assimilated (13)N than turgid blades even though they exported (14)CO(2) assimilates as actively as turgid blades.When substrate amounts (about 0.28 micromole) of [(14)C]glutamate and [(14)C]proline were applied to turgid and wilted blades, these amino acids entered the phloem and were translocated at velocities similar to those for (14)CO(2) assimilates (about 0.2 centimeter per minute). Wilted blades metabolized tracer amounts of [(14)C]glutamate to glutamine and proline, and exported (14)C in the form of these three amino acids. Approximate calculations of mass transfer rates of glutamate, glutamine, and proline made for wilted blades indicated that glutamine and glutamate together carried 76 micrograms of N per day, whereas proline carried only about 9 micrograms of N per day. PMID- 16660990 TI - Fatty Acids and Circadian Rhythms in Phaseolus coccineus: Effects of Light, Temperature, and Chemicals. AB - Five major fatty acids, palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3), were identified in polar lipid extracts from pulvini of Samanea saman and Phaseolus coccineus. In P. coccineus their distribution varied quantitatively in the laminar pulvinus, petiolar pulvinus, petiole, stem, leaf and root. Short pulses of red light did not greatly affect the relative quantities of fatty acids in dark grown P. coccineus, but a 30 minute exposure of red light generally increased the degree of unsaturation by increasing linolenic acid and decreasing linoleic and palmitic acids.P. coccineus seeds were exposed to several substituted pyridazinones as well as cerulenin and dimethylethanolamine. The pyridazinones San 6706 and norflurazon altered fatty acid composition but also altered morphology and inhibited chlorophyll synthesis. Exposure to 10 C for 72 hours caused a small but significant increase in the degree of unsaturation of P. coccineus fatty acids but results were equivocal with S. saman. PMID- 16660991 TI - Diurnal growth trends, water potential, and osmotic adjustment of maize and sorghum leaves in the field. AB - The daily cycle of leaf elongation rate, water potential, and solute potential of maize and sorghum, as well as temperature, were monitored in the field. Major climatic features were high radiation and a minimum air temperature of about 12 C. Leaf elongation of both crops was slowest at night, presumably because of low temperature. Peak elongation rates were in daytime when leaf water potential (Psi) was low. Solute potential also decreased during daylight, thus permitting the maintenance of appreciable turgor pressure, a critical parameter for cell expansion.Leaf Psi versus relative water content (RWC) curves were developed by sampling detached leaves at intervals as they dried quickly in the laboratory. At a given RWC, Psi was lower in leaves at midday than early in the morning, which is evidence that the decrease in psi. at midday was caused by an increase in the amount of solute in the tissue. Estimates of psi. at 100% RWC were 4 bars lower at midday than early in the morning in both crops. Soluble sugars, mainly nonreducing, accounted for most of the observed psi. decrease in sorghum leaves. Shading the leaves from light eliminated most of the solute buildup. PMID- 16660992 TI - Sucrose Compartmentation in the Palisade Parenchyma of Vicia faba L. AB - Intracellular sucrose compartmentation in the palisade parenchyma of Vicia faba L. leaflets was investigated by comparing the specific radioactivity of photosynthetically labeled [(14)C]sucrose in samples enriched in vacuole to that in samples enriched in cytoplasm. Brief centrifugation of leaflet punches was used to sediment most of the palisade parenchyma cytoplasm in the adaxial ends of the cells. The punches were quick-frozen, freeze-substituted, and embedded in methacrylate. Samples enriched in cytoplasm or in vacuoles were obtained from paradermal sections. After pulse-labeling, the sucrose specific radioactivity in vacuole-enriched samples was fairly constant. Sucrose specific radioactivity in cytoplasm-enriched samples was about 2.5 times that in vacuole-enriched samples initially and declined thereafter. Earlier interpretation of intracellular sucrose compartmentation (Plant Physiol 1975 55: 704-711) had predicted larger specific activity differences (up to 20 times) between the cytoplasm and vacuole. The difference between the actual and predicted behavior is ascribed to the observed extent of cross-contamination in samples and, more importantly, to the confinement of sucrose to extrachloroplastic regions of the cytoplasm. PMID- 16660993 TI - Kinetics of phosphorus absorption by mycorrhizal and nonmycorrhizal tomato roots. AB - Kinetics of P absorption were investigated in mycorrhizal (Glomus fasciculatus) and nonmycorrhizal tomato (Lycopersicon esculentum) roots to determine why increased ion absorption by mycorrhizae occurs. Initial rates of absorption of (32)P were measured at 1 to 100 micromolar KH(2)PO(4) (pH 4.6). Absorption rates of mycorrhizae were about twice those of control roots. Augustinsson-Hofstee analysis yielded two linear phases; V(max) and K(m) were calculated for each phase. In the low phase (1 to 20 micromolar), V(max) values for the mycorrhizal and nonmycorrhizal roots were each 0.10 micromoles P per gram fresh weight per hour while K(m) values were 1.6 and 3.9 micromolar KH(2)PO(4), respectively. For the high phase (30 to 100 micromolar), V(max) values for mycorrhizal and nonmycorrhizal roots were 0.32 and 0.25 micromoles P per gram fresh weight per hour and K(m) values were 35 and 42 micromolar, respectively. These results indicate that at the lower phase concentrations, similar to those expected in most soil solutions, a major factor contributing to the increased uptake was an apparent greater affinity of the absorbing sites for H(2)PO(4) (-) (lower K(m)). PMID- 16660994 TI - A Cuvette Design for Measurement of Ethylene Production and Carbon Dioxide Exchange by Intact Shoots under Controlled Environmental Conditions. AB - A cuvette is described for simultaneous measurement of ethylene production and CO(2) fixation by intact shoots under controlled environmental conditions. This design overcomes potential problems associated with closed systems conventionally used for studies on ethylene production, allowing accurate determination of rates of ethylene production in plants exposed to different environmental conditions. PMID- 16660995 TI - Modulation of Chloroplast Fructose-1,6-bisphosphatase Activity by Light. AB - Inhibitor experiments indicate that light effect mediator(II) which is reductively activated by transfer of electrons from the photosynthetic electron transport system at or beyond ferredoxin, is involved in activation by light of fructose-1,6-bisphosphatase in the pea plant. Activation proceeds optimally when the pH is low and Mg(2+) is 10 millimolar. Modulation by light results in increases in maximal velocity, apparently as a result of changes in enzyme conformation. Pea leaf thylakoids are effective in modulating the activity of glyceraldehyde-3-phosphate dehydrogenase but not of fructose-1,6-bisphosphatase or glucose-6-phosphate dehydrogenase in Kalanchoe stromal extracts. There is apparently species specificity for modulation of some, but not all, of the modulatable enzymes. PMID- 16660996 TI - Water Relations of Cotton Plants under Nitrogen Deficiency: I. Dependence upon Leaf Structure. AB - Cotton plants (Gossypium hirsutum L.) grown on deficient levels of N exhibited many of the characteristics associated with drought resistance. In N-deficient plants, leaf areas and leaf epidermal cells were smaller than at the same nodes in high-N plants. N-deficient leaves lost only about half as much water per unit change in water potential as did high-N leaves. In addition, they maintained a greater relative water content than high-N leaves at any given potential. Osmotic potentials (determined from pressure-volume curves) were slightly lower in N deficient leaves. This difference in solute concentration was not from organic acids, which were almost unchanged. Sugar concentrations could account for only about 25% of the difference.Leaves of N-deficient plants contained considerably more dry matter per unit moisture. Most of this difference in dry weight was in the crude cell wall fraction. The pressure-volume curves and other indirect evidence strongly suggested that cell walls of N-deficient leaves were substantially more rigid than cell walls of high-N leaves. The effects of N deficiency on cell wall properties mimic the changes which occur during drought adaptation. PMID- 16660997 TI - Water Relations of Cotton Plants under Nitrogen Deficiency: II. Environmental Interactions on Stomata. AB - Nitrogen deficiency in cotton plants (Gossypium hirsutum L.) considerably increased the sensitivity of stomata to water stress. At air temperatures of 27, 35, and >/=40 C, threshold potentials for complete stomatal closure were -10, 15, and -26 bars in N-deficient plants and -20, -20, and -30 bars in high-N plants, respectively. This three-way interaction among N supply, water potential, and air temperature was similar to that exerted on leaf expansion. The effects of N supply on stomatal behavior could not be explained on the basis of either osmotic or structural considerations. Rather, effects of N deficiency on mesophyll and stomata were independent and divergent. Stomatal behavior may impart a stress avoidance type of drought resistance to N-deficient plants. PMID- 16660998 TI - Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves. AB - Light-grown 7-day-old wheat seedlings (Triticum aestivum, var. Maris Dove) showed an increase of 200% in plastids per cell between 1.7 and 4.5 centimeters from the leaf base. This increase was the result of divisions of young chloroplasts at various stages of development, and was well separated in distance, and therefore in time from the region of cell division in the basal meristem. [(3)H]Thymidine was incorporated into plastid DNA throughout the zone of plastid division, but not above it. PMID- 16660999 TI - Isolation and characterization of the protein body membrane of castor beans. AB - Intact protein bodies were isolated from dry castor bean seeds (Ricinus communis L.) after homogenization in nonaqueous medium. After repeated washing with glycerol to remove trapped lipid globules, the soluble matrix proteins were removed by the addition of aqueous buffer. The membrane remained attached to the insoluble protein crystalloids and was subsequently released by sonication. Purification of the membrane vesicles in a sucrose gradient produced a single band at a density of 1.21 grams per cubic centimeter. Treatment with 6 molar urea, 1 molar KCl, or 0.25 molar galactose had no effect on the equilibrium density of the membrane. Electron microscopy revealed a highly pure and uniform collection of membrane vesicles. No enzyme activity was specifically associated with the membrane. Sodium dodecyl sulfate gel electrophoresis of the protein body fractions showed that the membrane contained unique proteins, two of which were glycosylated. The membrane contained 153 nanomoles of phospholipid per milligram of protein. The composition of the phosphoglycerides was 51% ethanolamine, 41% choline, 8% inositol, and a trace of serine. PMID- 16661000 TI - Natural H Currents Traverse Growing Roots and Root Hairs of Barley (Hordeum vulgare L.). AB - With the aid of an extracellular vibrating electrode, natural electric fields were detected and measured in the medium near growing roots and root hairs of barley seedlings. An exploration of these fields indicates that both the root as a whole, as well as individual root hairs, drive large steady currents through themselves. Current consistently enters both the main elongation zone of the root as well as the growing tips of elongating root hairs; it leaves the surface of the root beneath the root hairs. These currents enter with a density of about 2 microamperes per square centimeter, leave with a density of about 0.5 to 1 microampere per square centimeter, and total about 30 nanoamperes.Responses of the natural fields to changes in the ionic composition of the medium as well as observations of the pH pattern in the medium near the roots (made with bromocresol purple) together indicate that much of the current consists of hydrogen ions. Altogether, H(+) ions seem to leak into growing cells or cell parts and to be pumped out of nongrowing ones. PMID- 16661001 TI - Temperature-sensitive Responses of Red Light-dependent Betacyanin Synthesis. AB - Amaranthus tricolor half-seedlings show a greater accumulation of betacyanin in response to a defined period of red light if given a pretreatment at an elevated temperature (40 C). Red light given before the shift to 40 C is ineffective. The maximum response is achieved after a 2-hour shift to 40 C and if the red light is given 1 hour after return to the germination temperature (25 C). The effect on red light induction of betacyanin synthesis and on fusicoccin induction by these conditions is similar, whereas the increase in cytokinin-dependent synthesis is greater. Both phytochrome-cytokinin synergism and fusicoccin-cytokinin synergism are changed in the same manner by this treatment. Phytochrome and fusicoccin responses are inhibited similarly by carbonyl-cyanide-p-trifluoromethoxy phenylhydrazone.If the seedlings are again returned to 40 C after the 25 C shift there is an inhibition of betacyanin synthesis. The interpretation of these results is that a shift to 40 C induces the formation of some component necessary for the full expression of phytochrome potential, that for the maximum formation or action of this component it is necessary for the seedlings to spend 1 to 2 hours at a lower temperature (for example 25 C) before red light is given and that for the subsequent processes in betacyanin synthesis an elevated temperature is inhibitory. PMID- 16661002 TI - Psychrophile C-phycocyanin. AB - C-Phycocyanin was purified from an alga isolated in the Antarctic in 1968 and grown since that time in our laboratory at 4 C. Certain of its spectroscopic and immunochemical properties were studied, along with detailed examinations of its chemical composition and quaternary structure. The hexamer-trimer equilibrium in the protein solution was studied as a function of pH, ionic strength, temperature, and susceptibility to certain small molecules. A major emphasis of the work was placed on a comparison of its protein-protein interactions with those of C-phycocyanins from both mesophilic and thermophilic algae. PMID- 16661003 TI - Effect of Exogenously Supplied Foliar Potassium on Phloem Loading in Beta vulgaris L. AB - The effect of foliar application of K(+) on processes associated with phloem loading was investigated in source leaves of sugar beet (Beta vulgaris L.). KCI was supplied exogenously at concentrations of up to 100 millimolar in the solution bathing the abraded upper epidermis of source leaves. K(+) added at concentrations below 30 millimolar generally promoted the rate of export of material derived from (14)CO(2) but not from exogenously applied [(14)C]sucrose. Paralleling promotion of export, the level of material derived from photosynthesis, which was released into the bathing solution, also increased in response to addition of K(+) to the free space. Net photosynthetic rate was not affected. K(+) at 5 and 15 millimolar concentrations did not stimulate uptake of [(14)C]sucrose into source leaf discs.The data suggest that the promotion of export rate by K(+) results primarily from an effect on the site regulating efflux of sucrose into the apoplast prior to loading into the minor veins, rather than on the loading site itself. A change in the level of sucrose in the free space appears to precede a change in export rate. PMID- 16661004 TI - Polygalacturonase and Cellulase Enzymes in the Normal Rutgers and Mutant rin Tomato Fruits and Their Relationship to the Respiratory Climacteric. AB - Cell wall enzymes at different stages of fruit development were compared between the normal Rutgers and the isogenic nonripening rin tomato. In Rutgers, a detectable increase in polygalacturonase (PG) activity was observed 6 days prior to the respiratory climacteric (43 days postanthesis). The maximum increase in PG activity occurred after C(2)H(2) and CO(2) production reached their peak. However, in the rin tomato, no change in PG activity was noted up to 100 days postanthesis. Cellulase activity increased in Rutgers fruits prior to the respiratory climacteric and continued to increase thereafter. Similar changes in cellulase activity were also observed in the nonclimacteric rin fruits. Short term ethylene treatment (2 days) of 36-day-old rin fruits increased cellulase activity, but had no effect on PG activity. Detectable changes in other parameters of ripening, such as chlorophyll loss and softening, also occurred prior to the respiratory climacteric. These results suggest that the failure of rin fruits to ripen is related to their low PG activity during maturity as compared with normal fruits. PMID- 16661005 TI - Measurement of ethylene binding in plant tissue. AB - Tobacco leaves were exposed to (14)C-labeled ethylene (3.7 x 10(-2) microliters per liter) in the presence and absence of unlabeled ethylene and other compounds. Most of the [(14)C]ethylene appears to be bound to displaceable sites. Lineweaver Burk plots for a one-half maximum response in a tobacco leaf respiration test gave a value of 0.3 microliter per liter for ethylene, 50 microliters per liter for propylene, and 266 microliters per liter for carbon monoxide. Scatchard plots for displacement of [(14)C]ethylene from the site gave 0.27 microliters per liter for ethylene, 42 microliters per liter for propylene, and 746 microliters per liter for carbon monoxide. At 2%, CO(2) displaces about 35% of the bound ethylene, but increasing the concentration to 10% does not displace the remaining [(14)C]ethylene. A value of 3.5 nanomolar was calculated for the concentration of ethylene-binding sites available to exogenous ethylene. This does not account for the sites occupied by endogenous ethylene, and the total number of binding sites is probably somewhat higher. Using tissue culture material, the system was shown to be stable to freezing and thawing; and the pi-acceptors, carbon monoxide, cyanide, n-butyl isocyanide, phosphorous trifluoride, and tetrafluoroethylene, were shown to compete with ethylene for binding. PMID- 16661006 TI - A Transformation-induced Alteration of Cellular Membranes in Crown Gall Tumor Cells. AB - Phospholipids were utilized as a membrane marker to test for transformation induced alteration of cellular membranes of cultured crown gall cells of Vinca rosea L. Fully transformed cells contained less than half the amount of phospholipids (7.8 micrograms lipid P per gram fresh weight) of normal V. rosea cells (21.4 micrograms lipid P per gram fresh weight). The normal V. rosea callus cells were not significantly different (P > 0.05) in phospholipid content from partially transformed crown gall cells (20.7 micrograms lipid P per gram fresh weight). Stimulation to rapid growth of the partially transformed cells by adding higher concentrations of inorganic salts and auxin did not significantly alter their phospholipid content (23.1 micrograms lipid P per gram fresh weight). These findings suggest that the transformation process is directly responsible for an alteration of the cellular membranes and that the membrane alteration cannot be attributed to secondary effects associated with the rapid growth of these neoplastic cells.Five classes of phospholipids were identified and quantified. Average percentage of total phospholipids contained in each class were as follows: phosphatidylcholine, 38.5 +/- 4.3; phosphatidylethanolamine, 22.2 +/- 0.8; phosphatidylinositol, 12.3 +/- 2.9; phosphatidylserine, 0.8 +/- 0.4; phosphatidic acid, 4.7 +/- 1.3. Several unidentified phospholipids were also quantified. The relative amounts of the various classes of phospholipid compounds in normal and crown gall cells of V. rosea were not significantly different. PMID- 16661007 TI - Energy-dependent Loading of Amino Acids and Sucrose into the Phloem of Soybean. AB - Radioactive sucrose, l-leucine, l-glutamate, and gamma-aminobutyrate were applied exogenously to abraded areas of soybean leaves. The three amino acids were translocated with similar velocities and mass transfer rates on a molar basis, although they were metabolized differently in the sink tissue. The concentration dependence of leucine translocation showed a triphasic saturation response, while sucrose translocation showed a biphasic saturation response to increasing concentration. Apparent K(m) and V(max) for leucine and sucrose loading in the phloem differed. Both leucine and sucrose translocation were inhibited by uncouplers, high K(+), and p-chloromercuribenzenesulfonic acid. Treatment with 0.8 m sorbitol had little effect on sucrose translocation but stimulated leucine translocation, indicating an apoplastic route of loading for leucine. No effect on mass transfer rates was observed when sucrose and amino acids were applied exogenously together. These data provide evidence that phloem loading of amino acids and sucrose is mediated by different and separate carriers, both being dependent on an energy-requiring mechanism. PMID- 16661008 TI - Growth and Specific Nodule Activity of Soybean during Application and Recovery of a Leaf Moisture Stress. AB - Soybean plants growing at day/night temperatures of 30/18, 26/18, and 22/18 C were subjected to a single drying and recovery cycle during an 18- to 19-day period in the early to midpod development stage. Leaf total electrochemical water potential was reduced to about -24 bars during the 4-day drying cycle at all temperatures, but recovered to control levels upon rewatering. The changes in dry matter accumulation in whole plants and plant parts, specific activity of nodules as measured by acetylene reduction, and levels of adenosine phosphates in nodules were measured periodically during stress and recovery.Vegetative and reproductive growth were about equally suppressed by the leaf moisture stress. Both rate of appearance and number of pods were reduced. However, a similar average weight per pod for both stressed and control plants at the conclusion of the recovery period suggests that individual pod development is not irreversibly affected by a single stress cycle and that yield potential is restricted by a decrease in number of pods or seed. Dry matter accumulation in plants and pods was unaffected by temperature.Specific nodule activity and energy charge of nodules declined concurrently with leaf moisture potential. Recovery of specific nodule activity following rewatering lagged behind recovery of leaf moisture potential, but energy charge of nodules recovered as rapidly as leaf moisture potential upon rewatering. Thus, the delayed recovery of specific nodule activity does not appear to be related to recovery of energy charge of the nodules. PMID- 16661009 TI - Relationships between Root Temperature and the Transport of Ammonium and Nitrate Ions by Italian and Perennial Ryegrass (Lolium multiflorum and Lolium perenne). AB - At root temperature below 14 C the absorption of (15)N from NH(4) (+) greatly exceeded that from NO(2) (-) by tillers of Lolium multiflorum and Lolium perenne under conditions where pH, external concentration, plant N status, and pretreatment temperature were varied. There was a marked increase in the temperature sensitivity of NO(3) (-) transport below 14 C, irrespective of the temperature at which plants were grown previously. A marked increase in the temperature sensitivity was also seen for NH(4) (+) transport, but this occurred at the lower temperature of 10 C. Pretreatment of roots at 8 C lowered this still further to 5 C. Above and below these transition temperatures the Q(10) values for NO(3) (-) and NH(4) (+) transport were similar. Thus, the increased absorption of NH(4) (+) relative to NO(3) (-) at low temperatures seems to be related primarily to the difference in transition temperatures.It seems possible that NO(3) (-) and NH(4) (+) are absorbed through separate regions of the cell membrane differing in lipid composition and phase transition temperatures. PMID- 16661010 TI - Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo. AB - The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [(14)C]NaHCO(3), [(14)C]carbamylaspartate, and [(14)C]orotic acid into uridine nucleotides; (b) the isolation of [(14)C]orotic acid when [(14)C]NaHCO(3) and [(14)C]carbamylaspartate were used as precursors; (c) the observation that 6 azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.Regulation of the activity of the orotate pathway by end product inhibition was demonstrated in the intact cells of excised roots by measuring the influence of added pyrimidine nucleosides on the incorporation of [(14)C]NaHCO(3) into uridine nucleotides. The addition of either uridine or cytidine inhibited the incorporation of [(14)C]NaHCO(3) into uridine nucleotides by about 80%. The observed inhibition was demonstrated to be readily reversible upon transfer of the roots to a nucleoside-free medium. Experiments employing various radiolabeled precursors indicated that one or both of the first two enzymes in the orotate pathway are the only site(s) of regulation of physiological importance. PMID- 16661011 TI - Development of Peptide Transport Activity in Barley Scutellum during Germination. AB - The scutella separated from nongerminated grains of barley (Hordeum vulgare L. cv. Pirkka) took up labeled glycylsarcosine (Gly-Sar), glycylglycine, and leucylleucine at a rate of about 2 nanomoles per scutellum per hour. About 55% of the uptake of Gly-Sar (2 millimolar) was due to nonmediated uptake, but the active component was similar to that operating in the scutella from germinating grains (pH optimum about 4.5, K(m) about 10 millimolar).When the grains were allowed to germinate aseptically on agar, the rate of uptake of Gly-Sar began to increase after a lag of about 5 hours. The increase continued until a maximum value of 70 nanomoles per scutellum per hour (35-fold increase) was attained after 3 days of germination. After this the activity began to decrease slowly. The uptake of glycylglycine and leucylleucine changed in a similar way. When embryo-containing halves of seeds or separated embryos were allowed to germinate for 12 hours on agar containing various inhibitors of RNA and protein synthesis, the increase in the peptide uptake activity was completely or partially inhibited. The increase was not affected by removal of the "embryo proper" or endosperm, or by additions of gibberellic or abscisic acid.Root tips and aleurone layers took up Gly-Sar very slowly; the rate of the mediated component of uptake was less than 1% of the uptake by scutella.The preferential localization of the peptide transport system in the scutellum and its rapid development at the beginning of germination strengthen the earlier conclusion that peptide transport plays an essential role in the mobilization of reserve proteins during germination. PMID- 16661013 TI - Loss of rubidium and potassium from barley roots on sudden chilling. AB - Sudden chilling of roots of barley pretreated in a labeled Rb(+) solution at 25 C causes a transient loss of label from the nonfree space. The half-time of the release is about 10 minutes. Within the first few hours of uptake the loss increases proportionally to the root Rb(+) content but varies between experiments as a fraction of this content: values from 4 to 20% were observed. The same phenomenon is found for K(+) labeled with (86)Rb(+). PMID- 16661012 TI - Distribution of Lectins in the Jumbo Virginia and Spanish Varieties of the Peanut, Arachis hypogaea L. AB - Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs. PMID- 16661014 TI - Incorporation of [S]Sulfate and [C]Bicarbonate into Karyotype-specific Polysaccharides of Chondrus crispus. AB - Gametophytic and sporophytic tissues of Chondrus crispus (Stackhouse) cultured in vitro were labeled with (35)S and (14)C. The major sulfated polysccharides isolated from the two karyotypes were characterized by KCI fractionation, immunoprecipitation, and infrared spectroscopy. Reproducibility of data has been demonstrated by an experiment using gametophytic T(4) strain with five replicates per time point. The rate of sulfate uptake was similar in haploid and diploid plants from a given area cultured for a similar time. Cultures from different sources cultured for different times showed different uptake and incorporation levels. Although sulfate uptake did not appear to be karyotype-related, the pattern of incorporation of (35)S and (14)C into polysaccharides was ploidy specific. PMID- 16661015 TI - Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves. AB - The activity of adenosine 5' triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, (35)SO(4) (2-) incorporation into adenosine 5' phosphosulfate, and ATP synthesis dependent upon adenosine 5' phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO(3) ( ) and CO(2) assimilation in leaves of C(4) plants. PMID- 16661016 TI - A Cytokinin-binding Protein from Wheat Germ: Isolation by Affinity Chromatography and Properties. AB - A cytokinin-binding protein has been isolated from wheat germ via ammonium sulfate precipitation, carboxymethyl Sephadex chromatography, and affinity chromatography on a column substituted with a derivative of kinetin riboside. On Sephadex G-200, the protein migrated with an apparent molecular weight of 122,000 daltons. The dissociation constant for kinetin was determined by equilibrium dialysis to be 1.2 micromolar; N(6)-benzylaminopurine and N(6)-(Delta(2) isopentenyl)adenine were also strongly bound. Little affinity was exhibited toward either cis-zeatin or trans-zeatin. PMID- 16661017 TI - Active cytokinins: photoaffinity labeling agents to detect binding. AB - FOUR SERIES OF AZIDOPURINES HAVE BEEN SYNTHESIZED AND TESTED FOR CYTOKININ ACTIVITY IN THE TOBACCO CALLUS BIOASSAY: 2- and 8-azido-N(6)-benzyladenines, N(6)-(Delta(2)-isopentenyl)adenines, and -zeatins, and N(6)-(2- and 4 azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8 azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 x 10(-5) micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 x 10(-6) micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 x 10(-5) micromolar, representing a sensitivity rarely attained.All of the azido compounds have been submitted to photolysis in aqueous ethanol, and the photoproducts have been detected and identified by low and high resolution mass spectrometry. They are rationalized as products of abstraction and insertion reactions of the intermediate nitrenes. The potential of the major released products as cytokinins was also assessed by bioassay. 2-Azido-N(6)-(Delta(2)-isopentenyl)adenine competed with [(14)C]kinetin for the cytokinin-binding protein isolated from wheat germ. When the azido compound was photolysed in the presence of this protein, its attachment effectively blocked the binding of [(14)C]kinetin. PMID- 16661018 TI - Effect of abscisic Acid on root hydraulic conductivity. AB - Reports of the effects of abscisic acid (ABA) on ion and water fluxes have been contradictory. Some of the confusion seems due to the interaction of ion and water transport across membranes. In these experiments root systems were subjected to hydrostatic pressures up to 5.0 bars to enable measurement of root conductance that was independent of measurement of osmotic potentials or ion fluxes.ABA between 5 x 10(-5) molar and 2 x 10(-4) molar resulted in a decrease in the conductance of the soybean root systems as compared with the controls. ABA treatment also eliminated the discontinuity in the Arrhenius plot of total flow versus reciprocal temperature at constant pressure. The results suggest that ABA acts at the membrane that is rate-limiting to water flow directly, or by altering metabolism that in turn affects the membrane. PMID- 16661019 TI - Effect of Fruits on Dormancy and Abscisic Acid Concentration in the Axillary Buds of Phaseolus vulgaris L. AB - The mechanism regulating the growth of adult plants in two determinate bean (Phaseolus vulgaris L.) cultivars was investigated. "Redkloud" plants flowered, formed fruits, and ceased shoot growth earlier than "Redkote" plants. Redkloud attained a smaller plant size, compared to Redkote, by imposing dormancy on axillary buds at an earlier age. In both cultivars, cessation of bud growth coincided with maximum combined fruit length per plant. Removal of fruits caused resumption of axillary bud growth within 4 to 5 days. The amount of new growth induced by fruit removal depended on the cultivar and plant age. In fully developed Redkloud plants, where shoot growth had already ceased, total leaf and shoot number per plant nearly doubled within 2 weeks following fruit removal. A much smaller response was observed in the still growing Redkote plants. Fruits, therefore, are assumed to play a major role in the regulation of shoot growth and total plant size through the control of axillary bud dormancy. It seems that smaller plant size, earlier maturity, and earlier senescence of Redkloud, compared to Redkote, were the result of earlier flowering, and accomplished in part through the growth-inhibiting action of fruits.The endogenous abscisic acid (ABA) concentration of axillary buds was higher in Redkloud than in Redkote. It increased with plant age in both cultivars. Five days after fruit removal the ABA level in bud tissue dropped to approximately 10 to 30% of the control level. When buds were treated with a solution of ABA containing 5 nanomoles of ABA per bud, growth was substantially inhibited. Fifteen days after ABA application the mean length of growing buds on intact and defruited plants was reduced by 40 and 62%, respectively, compared to the untreated controls. A role for ABA in axillary bud growth regulation was not firmly established, but these data suggest correlation between the growth potential of axillary buds and their ABA concentration. PMID- 16661020 TI - Effect of Older Fruits on Abortion and Abscisic Acid Concentration of Younger Fruits in Phaseolus vulgaris L. AB - In two cultivars of bean (Phaseolus vulgaris L., Redkloud and Redkote) the older fruits growing at the base of racemes aborted less frequently than the younger ones above them. When older fruits at the base of racemes were removed, the abortion rate of the younger ones was reduced and their abscisic acid (ABA) concentration was lowered. Thirteen days after fruit removal, 36 to 45% of the younger fruits remained viable on treated plants while less than 12% of the younger fruits were viable on control plants. On these intact controls the ABA concentration of young fruits was at least twice that of defruited plants. A similar difference was found when the ABA content was expressed on a per fruit basis, suggesting a direct regulatory influence of older fruits over the ABA content of younger fruits. PMID- 16661021 TI - Dimethylsulfoxide as a potential tool for analysis of compartmentation in living plant cells. AB - Data are presented which indicate that dimethylsulfoxide (DMSO) acts selectively on the plasma membrane of cultured tobacco cells, rendering it more permeable to small molecules, while having a far smaller effect on the permeability of the vacuolar membrane. The results which support this conclusion are: (a) DMSO (5 to 10%, by volume) causes complete release of [(14)C]tryptophan newly synthesized from [(14)C]indole while causing efflux of only about 20% of the total intracellular tryptophan pool; (b) similar concentrations of DMSO do not cause substantial release from these cells of phenolic compounds or preloaded neutral red, nor of beta-cyanin from fresh beet discs; (c) kinetic studies of release of tryptophan and neutral sugars and of efflux of (86)Rb(+) show that DMSO selectively promotes rapid release of a portion of the total pool, followed by a substantially slower release of the remaining pool; (d) when tobacco cell protoplasts are incubated in the presence of 7.5% (by volume) DMSO, rapid lysis is observed concomitant with the release of intact vacuoles. These data indicate that a procedure involving a brief treatment of intact plant cells or tissues with DMSO may be used to assess the distribution of metabolites between cytoplasmic and vacuolar compartments. PMID- 16661022 TI - Developmental changes in the activity of messenger RNA isolated from germinating castor bean endosperm. AB - The capacity of polyadenylated RNA from developing castor bean endosperm to program protein synthesis in a wheat germ cell-free translational system has been examined. Although the use of micrococcal nuclease-treated wheat germ extracts demonstrated a low but significant content of translatable mRNA in dry seeds, a large scale increase in total translational capacity was observed during germination. The cellular content of translatable mRNA peaked on the 4th day of germination and subsequently declined. It is concluded that protein synthesis in castor bean endosperm cells during germination is directed by newly transcribed mRNA. PMID- 16661023 TI - Metabolic and ultrastructural changes associated with flooding at low temperature in winter wheat and barley. AB - Cold-hardened winter wheat (Triticum aestivum L. cv. Fredrick) and winter barley (Hordeum vulgare L. cv. Dover) were exposed to total flooding at 2 C. Dover seedlings were damaged more quickly than Fredrick, and after 3 weeks of flooding the survival of Dover was reduced to 10% and Fredrick to about 50%. Tissue moisture was slightly greater in Dover than Fredrick throughout the 4-week flooding period. Carbon dioxide and ethanol accumulated throughout the 4-week flooding period in both cultivars. Lactic acid increased rapidly during the 1st week of flooding, and remained relatively constant during the remainder of the flooding period. Oxygen consumption of seedling shoot tissue after exposure to flooding declined abruptly after only 1 day of flooding, but recovered somewhat during the subsequent 2 weeks. The effect of flooding was more pronounced on the ultrastructure of Dover than Fredrick. Although proliferation of endoplasmic reticulum was observed in the early stages of flooding in both cultivars, the occurrence of distinct parallel arrays and concentric whorls of membranes was prevalent in the flooded barley. Severe ultrastructural damage to a large proportion of apical cells in both cultivars was observed after 2 to 3 weeks of flooding. PMID- 16661024 TI - In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor. AB - A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe(2+) may be the functional metal. The trypsin inhibitors N-alpha-p-tosyl-l lysine chloromethyl ketone and alpha-N-benzoyl-l-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase inactivating factor.The inactivating factor was more active toward the NADH nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive. PMID- 16661025 TI - Control of flowering in bougainvillea "san diego red.": metabolism of benzyladenine and the action of gibberellic Acid in relation to short day induction. AB - Benzyladenine (BA) and short day (SD) induction promote and gibberellic acid (GA) inhibits flowering in Bougainvillea "San Diego Red." GA is an overriding vegetative signal maintaining plants in a vegetative state even when BA is applied in SD conditions. SD promotes a more rapid conversion of BA to the ribotide and other "polar derivatives" (containing adenine derivatives). This effect of SD on BA metabolism is seen in root, stem, and apical bud tissues and is completely prevented by prior or simultaneous application of GA. GA treatment reduces the rate of polar derivative formation to that found in plants held in long days. The working hypothesis is that SD promotes flowering in Bougainvillea owing to reduced transport of gibberellins from leaves to roots and apical buds permitting metabolism of cytokinin, and perhaps other purine bases, to more polar forms that are more readily translocated and active in promoting reproductive development of the inflorescences axes. PMID- 16661026 TI - Rapid Changes in Translocation Patterns in Soybeans following Source-Sink Alterations. AB - The effects of source-sink alterations on the translocation patterns to soybean ("Fiskeby V") pods were studied using a pod leakage technique. The distribution of assimilates from a source leaf using double pulse experiments was followed at the pods at the source node and the node immediately below. Alterations were made by shading, illuminating, or excising two-thirds of the leaf area of the leaf at the node below. In control experiments both pulses exhibited identical time course patterns at both nodes. Shading the lower leaf during the first half of the experiment and illuminating during the second reduced the distribution of (14)C-assimilate to the lower node's pods from the source leaf by approximately 30 to 50% while having no effect at the source node. Illuminating the lower leaf during the first half of the experiment followed by excision of two-thirds of that leaf's area and shading increased the import from the source leaf by 4- to 33-fold relative to the control while reducing the distribution to the source node by up to 40%. The change in distribution pattern took place in less than 30 minutes with no apparent change in the source leaf net photosynthesis or in the rate of movement to the pods. The results indicate that any alterations in the source-sink balance will quickly produce a change in the distribution patterns to the pods. PMID- 16661027 TI - Photosynthetic oxygen reduction in isolated intact chloroplasts and cells in spinach. AB - The time course of light-induced O(2) exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopically labeled O(2) and a mass spectrometer. In dark-adapted chloroplasts and cells supplemented with saturating amounts of bicarbonate, O(2) evolution began immediately upon illumination. However, this initial rate of O(2) evolution was counterbalanced by a simultaneous increase in the rate of O(2) uptake, so that little net O(2) was evolved or consumed during the first approximately 1 minute of illumination. After this induction (lag) phase, the rate of O(2) evolution increased 3- to 4-fold while the rate of O(2) uptake diminished to a very low level. Inhibition of the Calvin cycle, e.g. with dl-glyceraldehyde or iodoacetamide, had negligible effects on the initial rate of O(2) evolution or O(2) uptake; both rates were sutained for several minutes, and about balanced so that no net O(2) was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of O(2) evolution and photoreduction were stimulated 40 to 50%.These results suggest that higher plant phostosynthetic preparations which retain the ability to reduce CO(2) also have a significant capacity to photoreduce O(2). With near-saturating light and sufficient CO(2), O(2) reduction appears to take place primarily via a direct interaction between O(2) and reduced electron transport carriers, and occurs principally when CO(2)-fixation reactions are suboptimal, e.g. during induction or in the presence of Calvin cycle inhibitors. The inherent maximum endogenous rate of O(2) reduction is approximately 25 to 50% of the maximum rate of noncyclic electron transport coupled to CO(2) fixation. Although the photoreduction of O(2) is coupled to ion transport and/or phosphorylation, this process does not appear to supply significant amounts of ATP directly during steady-state CO(2) fixation in strong light. PMID- 16661028 TI - Effect of Shoot Removal and Malate on the Activity of Nitrate Reductase Assayed in Vivo in Barley Roots (Hordeum vulgare cv. Midas). AB - There is a diurnal variation of nitrate reductase activity (NRA) measured in vivo in barley roots (Hordeum vulgare cv. Midas). In intact plants receiving a 16-hour photoperiod, NRA increases when the light is switched on, reaches a maximum value after 7 to 8 hours, and thereafter declines. Shoot removal (detopping) at the start of the photoperiod prevents the rise in NRA; detopping after 5 hours light leads to a rapid fall in NRA. The inclusion of 10 millimolar malate in the external medium causes a rise in NRA in plants detopped at the beginning of the photoperiod and thus seems to substitute partially for the illuminated shoot. Oxalate, fumarate, and tartrate did not have this effect. Preincubation of the roots of intact plants with 10 millimolar malate for 3 hours, prior to detopping, causes an increase in the flux of amino acids into the xylem sap of detopped roots. PMID- 16661029 TI - Posttranscriptional control of nitrate reductase of cultured tobacco cells by amino acids. AB - Using the inhibitor of RNA synthesis, 4,5',8-trimethylpsoralen plus near ultraviolet light, the half-life of the rate-limiting RNA species required for the induction of nitrate reductase in XD cells of tobacco was estimated to be 4 hours. Amino acids, the end product of nitrate assimilation, reduced the half life to 1 hour. It is suggested that amino acids exert a posttranscriptional control on the enzyme level. PMID- 16661030 TI - Light effects on development of an indeterminate plant. AB - The peanut (Arachis hypogaea L.) plant is indeterminate in growth habit and day neutral with respect to flower initiation. The Spanish-type cultivar used in this study begins flowering 3 to 4 weeks from planting under optimum environmental conditions. In this study, irradiance and photoperiod were used to alter the development of the peanut plant. Plants grown at low irradiance (300 microeinsteins per square meter per second) had the same number of leaves as the plants grown at high irradiance (500 microeinsteins per square meter per second), but they had a larger leaf surface area and were taller than plants grown at the high irradiance. However, flowering and other reproductive components (pegs, pods, and seeds) were reduced at low irradiance. Comparison of 8-, 12-, and 16 hour photoperiods at the high irradiance showed that the 16-hour photoperiod produced the largest amount of vegetative, but least amount of reproductive components. The plants grown at 8-hour photoperiod had one-third as much total leaflet area as plants grown at 16 hours, but six times more weight of mature seeds. The larger amount of photosynthetic surface (leaf area) did not result in more reproductive growth. The results indicate that the peanut plant may readily redistribute its available assimilates between vegetative and reproductive growth in response to irradiance and photoperiod. PMID- 16661031 TI - Thymidylate synthase activity from chlamydomonas cells and cultured tissues of Nicotiana, pinus, and daucus. AB - Prior preparation of N(5),N(10)-methylenetetrahydrofolate from dl tetrahydrofolate and [(14)C]formaldehyde resulted in an improved assay for thymidylate synthase. Although preparations from tobacco seedlings and cotton root tips (0.25 centimeter) were inconsistent with respect to enzyme activity, extracts from actively growing cell cultures of Chlamydomonas, Nicotiana hybrid callus, Pinus callus, and Daucus proembryonic cells contained significant levels of thymidylate synthase (12.3 to 23.8 nanomoles of thymidylate formed per milligram of protein per hour). PMID- 16661032 TI - Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants. AB - Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID- 16661033 TI - Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L. AB - Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N(2). Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments. PMID- 16661034 TI - Cyanide Inhibition of Acid-induced Growth in Avena Coleoptile Segments. AB - The comparative effects of metabolic inhibitors on acid- and auxininduced growth in oat (Avena sativa L. var. Victory) coleoptile segments have been examined. Acid (pH 4)-induced growth in both peeled and unpeeled segments is inhibited by 1 millimolar KCN when added at the time of acidification. KCN inhibits total acid induced growth by 59 and 76%, respectively, in peeled and nonpeeled segments during the first 60 minutes. The growth rate of cyanide-treated tissue drops to zero or near zero in both peeled and nonpeeled segments during this period. Cyanide inhibition of total acid-induced growth in peeled segments at pH 5 is even more severe, amounting to about 80% during the first 60 minutes. The possibility that inhibition by cyanide may be caused by some nonspecific effect of the inhibitor on a process other than respiration, e.g. turgor reduction due to membrane damage, has not been ruled out. Acid-induced growth is also inhibited by 3 millimolar sodium fluoride and by anoxia. In unpeeled segments total pH 4 induced growth is inhibited 73% by sodium fluoride and 38% by anoxia during the 1st hour. Possible corrections to the above inhibition percentages which may be necessary due to the sensitivity of basal growth to inhibitors are discussed. Cyanide was found to inhibit auxin-induced growth much more rapidly than acid induced growth. These data suggest that acid growth may be dependent on respiratory metabolism but to a lesser degree than is auxin-induced growth. If the acid growth theory of auxin action is correct, it appears that there may be two steps in the growth process which are dependent on respiratory metabolism: (a) auxin-induced proton pumping which is highly sensitive to respiratory inhibitors; and (b) acid-mediated wall loosening which is moderately and perhaps indirectly sensitive to respiratory inhibitors. PMID- 16661035 TI - l-Malate as an Essential Component of the Xylem Fluid of Corn Seedling Roots. AB - Corn seedling xylem exudate has a pH of 5.30 +/- 0.05 due to the presence of 10 millimolar malate which has a pK(a) of 5.13. This concentration of malate tends to buffer the xylem fluid at this pH. Exogenous treatment of corn seedling roots with CaCl(2) caused a concentration-dependent decrease in the pH of the xylem fluid as well as a decrease in the volume of fluid secreted into the xylem. Exogenous 50 millimolar CaCl(2) decreased exudate volume to 8% of control within 0.5 hour. Nitrate and malate deposition into the xylem was prevented by pretreatment in 5 millimolar CaCl(2); nitrate deposition was resumed shortly after resumption of malate deposition. Fifty millimolar l-tartrate, an inhibitor of the Cl(-), malate-activated ATPase (in vitro) of the tonoplast, also decreased exudate volume as well as slightly lowering exudate pH. The osmolality of the exudate was found to be constant at 70 +/- 11 milliosmomoles per kilogram in all treatments. Dixon plots (log of exudation rate versus pH of exudate) indicated a pK(a) of 5.11 for the exudation process which is very close to the pK(a) of l malate (5.13). In addition, a Dixon plot of the l-glutamine deposition (l glutamine being the major form of reduced nitrogen in the exudate) versus pH also indicated a pK(a) near 5.15.The pH optimum for glutamine transport into the xylem was 5.5. Deposition of glutamine into the xylem may be regulated by the xylem pH (5.30 +/- 0.05) which in turn may be regulated by the presence of 10 millimolar malate. It is proposed that the transport of glutamine into the xylem may provide the driving force for the exudation process. PMID- 16661036 TI - Effect of nuclear mutation in maize on photosynthetic activity and content of chlorophyll-protein complexes. AB - A number of new nuclear mutants have been isolated from maize by selection for high chlorophyll (Chl) fluorescence. These mutants show reduced rates of photosynthesis and/or are deficient in Chl. Electrophoretic examination of wild type thylakoid membranes revealed five Chl-protein complexes, two containing only Chl a and three containing Chl a and Chl b. A class of nonviable, photosystem I deficient mutants was found to be lacking one (A-1) of the two Chl a-protein complexes. A second class of nonviable, photosystem I-lacking mutants was found to be missing not only this A-1 complex but also one or more of the three Chl a and b-containing, light-harvesting Chl-protein complexes. Viable mutants were obtained which appeared to have lost just one of the Chl b-containing complexes, whereas a second class of viable mutants was missing all three of the Chl b complexes. The results confirm that the A-1 band is associated with the P700-Chl a-protein complex characterized previously. The data also indicate the existence of structurally different forms of the light-harvesting Chl a- and b-containing complexes. The results also show a lower molecular weight band (A-2) containing primarily Chl a and which appears to be required for viability. PMID- 16661037 TI - Ultrastructural and Enzymic Studies of Cell Membranes from Ice-encased and Noniced Winter Wheat Seedlings. AB - A marked increase in the amount of cisternal-like cytoplasmic membranes was observed after ice encasement of winter wheat (Triticum aestivum L.) seedlings. Linear sucrose gradients were employed to separate the various membrane components of the microsomal membrane fraction. NADH- and NADPH-cytochrome c reductase, two specific enzyme markers for plant endoplasmic reticulum (ER) were used to locate the ER in the linear gradients. The identity of the ER fraction was confirmed by determining the effect of EDTA and Mg(2+) in the preparative media on the distribution of NADH- and NADPH-cytochrome c reductase activity within the gradient. In the presence of EDTA which dissociates ribosomes from ER, peaks of activity for the two enzymes were observed at a density corresponding to that for "smooth" ER. When the media also contained an appropriate concentration of Mg(2+) to maintain the attachment of ribosomes to the ER, the peaks of activity for the enzymes shifted to a density corresponding to that for "rough" ER. NADH-cytochrome c reductase activity was similar for 24 C-grown and 2 C-grown iced seedlings, but significantly lower for 2 C noniced seedlings. No preferential increase in uptake of radioactive leucine or choline in the ER was observed during ice encasement. The accumulation of electron microscopically visible membrane arrays was not inhibited by the presence of protein synthesis inhibitors at concentrations which severely inhibited incorporation of [1 (14)C]leucine into membrane protein, but did not affect survival and growth of the seedlings. These observations indicate that the apparent proliferation of ER during ice encasement does not result from net membrane synthesis, but rather from reorganization of existing membrane elements within the cell. PMID- 16661038 TI - Respiratory changes with chilling injury of soybeans. AB - The leakage of solutes from cotyledons of soybeans (cv. Chippewa 64) was markedly stimulated by a chilling treatment (1 to 4 C) during the 1st minute of imbibition, but chilling after even 1 minute of water uptake resulted in little or no leakage increase. The respiratory rate of soybean particles was reduced more than 60% if a chilling treatment (15 minutes at 1 to 4 C) was given during the first minutes of imbibition, and little or no reduction was obtained if the chilling treatment was begun at 5 to 15 minutes after the start of imbibition. Using KCN as an inhibitor of cytochrome oxidase pathway of respiration and salicylhydroxamic acid as an inhibitor of the alternative pathway, it was found that the chilling injury involved a major reduction in the cytochrome pathway in whole axes and cotyledons and an engagement of the alternative pathway of respiration in cotyledon tissue. The suggestion is made that the chilling injury involves lesions resulting from temperature stress during the reorganization of membranes with water entry, and that both the leakage and the respiratory effects are consequences of these membrane lesions. PMID- 16661039 TI - Inhibition of glycine decarboxylation and serine formation in tobacco by glycine hydroxamate and its effect on photorespiratory carbon flow. AB - Glycine hydroxamate is a competitive inhibitor of glycine decarboxylation and serine formation (referred to as glycine decarboxylase activity) in particulate preparations obtained from both callus and leaf tissue of tobacco. In preparations from tobacco callus tissues, the K(i) for glycine hydroxamate was 0.24 +/- 0.03 millimolar and the K(m) for glycine was 5.0 +/- 0.5 millimolar. The inhibitor was chemically stable during assays of glycine decarboxylase activity, but reacted strongly when incubated with glyoxylate. Glycine hydroxamate blocked the conversion of glycine to serine and CO(2)in vivo when callus tissue incorporated and metabolized [1-(14)C]glycine, [1-(14)C]glycolate, or [1 (14)C]glyoxylate. The hydroxamate had no effect on glyoxylate aminotransferase activities in vivo, and the nonenzymic reaction between glycine hydroxamate and glyoxylate did not affect the flow of carbon in the glycolate pathway in vivo. Glycine hydroxamate is the first known reversible inhibitor of the photorespiratory conversion of glycine to serine and CO(2). PMID- 16661040 TI - A circuit analog model for studying quantitative water relations of plant tissues. AB - Using arrays of resistors and capacitors, a lumped circuit analog of plant tissue is developed. The circuit elements of the analog are identified in terms of physiological variables (hydraulic conductivities, water capacities, and cell dimensions) which can be measured in the laboratory. With the aid of a circuit simulation subroutine, the model was solved to predict water potential distributions as a function of position and time in plant tissues of three, six, and nine cells. Results presented for the six-cell case indicate that local equilibrium may or may not occur depending on the actual values of tissue hydraulic conductivities, water capacities, and the rate of change of water potential at the tissue boundaries. However, present measurements and estimates of tissue parameters suggest that local equilibrium is more the rule than the exception. Membrane resistance is an especially important parameter because it serves to isolate the vacuoles from the cell walls in addition to increasing the natural vacuole response time to changes in water potential.The proposed model should be useful in studying water transport processes in roots, stems, and leaves. Nonhomogeneity can be taken into account easily. Nonlinearity (changes in circuit parameter values with potential) which is known to occur in plant tissues could be incorporated also if the required information were available. PMID- 16661041 TI - Effects of Polyamines on Chlorophyll and Protein Content, Photochemical Activity, and Chloroplast Ultrastructure of Barley Leaf Discs during Senescence. AB - The polyamines putrescine, spermidine, and spermine prevent the loss of chlorophyll normally associated with senescence of excised leaf tissue maintained in darkness on water (control). Retention of chlorophyll in barley leaf discs was in the range of 90% 4 days after excision and placement on effective polyamine solutions. In contrast, the loss of soluble protein was hastened with 0.5 millimolar spermidine and spermine treatments but it was retarded by 0.5 millimolar putrescine.Photosystem I and II activities of chloroplasts from polyamine-treated leaf discs declined more rapidly as compared to the control. Chloroplast ultrastructural changes resulting from the polyamine treatments included the apparent destruction of the envelope, preservation of thylakoid membrane structure, and reduced accumulation of osmiophilic bodies. The influence of polyamines on senescence-related processes may be due to their cationic nature. PMID- 16661042 TI - Stabilization of Thylakoid Membranes by Spermine during Stress-induced Senescence of Barley Leaf Discs. AB - The effect of spermine on photochemical activity and polypeptide composition of chloroplasts from barley leaf discs during senescence in the dark was studied. Chloroplast membranes did not show photosystem II activity after spermine treatment when water was the electron donor, but in the presence of diphenylcarbazide, this activity was observed. The diphenylcarbazide-stimulated photoreduction of dichloroindophenol was 3-fold greater in leaf discs incubated for 72 hours in spermine than in water. Photosystem I activity was reduced by about 90% within the first 24 hours in the spermine-treated samples. This reduction, however, was not due to a decrease in the photosynthetic unit size. A preferential loss of polypeptides other than those associated with photosystem II was observed during senescence of the leaf discs in water, but this loss was reduced by spermine. Spermine treatment also prevented the appearance of several additional chlorophyll proteins found in the controls during senescence. The results have been interpreted on the basis of the interaction of spermine with thylakoid membranes resulting in stabilization of membrane function during senescence. PMID- 16661043 TI - Enzymology of l-Tyrosine Biosynthesis in Mung Bean (Vigna radiata [L.] Wilczek). AB - The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogenase and 4 hydroxyphenylpyruvate aminotransferase) and pretyrosine (prephenate aminotransferase and pretyrosine dehydrogenase) pathways of l-tyrosine biosynthesis were partially purified from mung bean (Vigna radiata [L.] Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorption, and gel-filtration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during all purification steps as well as after partial inactivation caused by p-hydroxymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by l-tyrosine at nonsaturating levels of substrate. The K(m) values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine.Two peaks of aromatic aminotransferase activity utilizing l-glutamate or l-aspartate as amino donors and 4 hydroxyphenylpyruvate, phenylpyruvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4 hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative. PMID- 16661044 TI - Photosynthesis and photorespiration in whole plants of wheat. AB - Wheat was cultivated in a small phytotronic chamber. (18)O(2) was used to measure the O(2) uptake by the plant, which was recorded simultaneously with the O(2) evolution, net CO(2) uptake, and transpiration. At normal atmospheric CO(2) concentration, photorespiration, measured as O(2) uptake, was as important as the net photosynthesis. The level of true O(2) evolution was independent of CO(2) concentration and stayed nearly equal to the sum of net CO(2) photosynthesis and O(2) uptake. We conclude that at a given light intensity, O(2) and CO(2) compete for the reducing power produced at constant rate by the light reactions of photosynthesis. PMID- 16661045 TI - Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions. AB - Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field. PMID- 16661046 TI - On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts. AB - With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O(2) evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented. PMID- 16661047 TI - Photosynthate Partitioning into Starch in Soybean Leaves: I. Effects of Photoperiod versus Photosynthetic Period Duration. AB - Photosynthesis, photosynthate partitioning into foliar starch, and translocation were investigated in soybean plants (Glycine max (L.) Merr. cv. Amsoy 71), grown under different photoperiods and photosynthetic periods to determine the controls of leaf starch accumulation. Starch accumulation rates in soybean leaves were inversely related to the length of the daily photosynthetic period under which the plants were grown. Photosynthetic period and not photoperiod per se appears to be the important factor. Plants grown in a 14-hour photosynthetic period partitioned approximately 60% of the daily foliar accumulation into starch whereas 7-hour plants partitioned about 90% of their daily foliar accumulation into starch. The difference in starch accumulation resulted from a change in photosynthate partitioning between starch and leaf residual dry weight. Residual dry weight is defined as leaf dry weight minus the weight of total nonstructural carbohydrates. Differences in photosynthate partitioning into starch were also associated with changes in photosynthetic and translocation rates, as well as with leaf and whole plant morphology. It is concluded that leaf starch accumulation is a programmed process and not simply the result of a limitation in translocation. PMID- 16661048 TI - Seasonal changes in the structure and function of mitochondrial membranes of artichoke tubers: acyl Fatty Acid composition and the effect of growth conditions. AB - Changes in the temperature response, fluidity, function and the acyl fatty acid composition, were determined for a mitochondria-rich membrane fraction from Jerusalem artichoke (Helianthus tuberosus L.) tubers during dormancy for a crop which matured in midsummer. The temperature of both the upper and lower limits of the membrane lipid transition decreased during dormancy from 26 C and 1 C to 4 C and -5 C, respectively. This was similar to the changes observed with crops maturing in late autumn. The order parameter of a spin label intercalated into the membrane lipids decreased from about 0.6 to 0.5 during dormancy and returned to the original value before sprouting, showing that membrane fluidity increased during dormancy. The activation energy of succinate oxidase of tuber mitochondria was generally high at middormancy when membrane lipids were more fluid and decreased as the membranes became more rigid at the end of dormancy. The fatty acid composition of the membrane lipids did not alter significantly during dormancy. The results indicate that neither decreasing day length nor low soil temperature during tuber maturation is essential for the initiation of the membrane changes necessary for tubers to avoid low temperature injury during dormancy. The increase in membrane fluidity during dormancy could not be accounted for by an increase in the proportion of unsaturated fatty acids in the membrane lipids. PMID- 16661049 TI - Oxidative phosphorylation in pea cotyledon submitochondrial particles. AB - Mitochondria and submitochondrial particles (SMP) from pea cotyledons were shown to catalyze oxidative phosphorylation as measured by (32)Pi uptake into phosphate esters. ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomycin. Experiments with the adenine nucleotide translocator inhibitor atractyloside indicated the SMP were inside-out. Mersalyl completely inhibited ATP synthesis by SMP, and a separate experiment indicated that mersalyl has a direct effect on the ATPase complex. The kinetics of ATP synthesis indicated a high affinity for phosphate (K(m) = 0.18 millimolar). ADP kinetics gave a biphasic curve with K(m) values of about 4.8 and 160 micromolar. O(2) uptake and ATP synthesis had a pH maximum of 7.6 while the ratio of micromoles phosphate esterified to microatoms O(2) taken up was highest at pH 7.2. Sodium chloride inhibited both ATP synthesis and O(2) uptake but stimulated the ATPase reaction. The SMP also catalyzed a slow ATP-phosphate exchange reaction. PMID- 16661050 TI - Nutritive Role of the Seedcoats during Embryo Development in Pisum sativum L. AB - Structural and metabolic features of the seedcoats of the developing pea seed indicate that events in the cells of the seedcoats are of major importance in controlling the development of the embryo. Information has been obtained on the distribution of N and P constituents of the seedcoats, embryo sac liquid, and the cotyledons of the embryo, in relation to the changes in the activities of several enzymes: aminopeptidases, beta-glucosidase, and acid phosphatase. The liquid contents of the embryo sac are considered to arise as a secretion from the tegmen. High concentrations of amino acids (about 0.3 molar), NH(4) (+) (about 0.1 molar), and orthophosphate (Pi) (up to 4 millimolar) were measured in this fluid. Since Pi was the only form of P present, the data confirm the possible function of some of the seedcoat acid phosphatase activity in the provision of Pi to the embryo. PMID- 16661051 TI - Relationships between Root System Water Transport Properties and Plant Size in Phaseolus. AB - Root system hydraulic conductivity (L(P)) was measured on Phaseolus plants of different ages and sizes. Data analysis showed that L(P) changed in a complex manner depending on plant size. As the plants increased in size, L(P) increased initially then gradually decreased followed by a final modest increase. Values for L(P) ranged between 0.8 x 10(-6) and 6.1 x 10(-6) centimeter per second per bar. Relationships between the root flow per unit leaf area at a pressure differential of 3 bars (QPL(3)), as well as the total root system conductance (L(R)), and plant size were also examined. Values for QPL(3) varied with plant size, somewhat like L(P). L(R) values continuously increased with plant size at rates which depended on the growth rate of the root surface area as well as L(P). Comparison of our data with the root conductivity constant (k(r)) of Taylor and Klepper (1975 Soil Sci, 120: 57-67) showed good agreement. The observations on Phaseolus were also confirmed for Glycine. Values for L(P) and k(r) of both species were within the same range. PMID- 16661052 TI - A Mechanism of Chlorosis Caused by 1,3-Dimethyl-4-(2,4-dichlorobenzoyl)-5 hydroxypyrazole, a Herbicidal Compound. AB - In organic solvents, 1,3-dimethyl-4-(2,4-dichlorobenzoyl)-5-hydroxypyrazole (DTP) converted chlorophyll a and b extracted from rice seedlings (Oryza sativa L. ;Kinmaze') into pheophytin a and b, respectively. On comparing the chlorophyll converting activity of DTP to those of acetic, glycolic, 2,4-dichlorobenzoic, monochloroacetic, 2,6-dichlorobenzoic, pyruvic, and dichloroacetic acids, it was demonstrated that DTP induced H(+) into chlorophyll specifically. 5 Hydroxypyrazoles, which seem to be dissociable, converted chlorophyll into pheophytin in vitro. These compounds also induced chlorosis in sedge seedlings (Cyperus serotinus Rottb.), when the seedlings were grown in media containing these compounds. However, 5-hydroxypyrazoles, which seem to be undissociable, and analogs having no hydroxy group caused neither the chlorophyll conversion in vitro nor chlorosis in the seedlings. Chlorosis in barnyardgrass seedlings (Echinochloa crus-galli Beauv. var. oryzicola Ohwi) induced by DTP was reversed by cultivating the seedlings in media containing DTP plus NaOH, KOH, NH(4)OH, Ca(OH)(2), sodium acetate, sodium pyruvate, sodium succinate, or sodium fumarate. Accumulation of the vinyl pheoporphyrin fraction in 4- day-old etiolated radish cotyledons (Raphanus sativus L. ;Minowase 2') was enhanced by incubating the cotyledons with delta-aminolevulinic acid in the dark. However, simultaneous treatment with delta-aminolevulinic acid and DTP reduced accumulation of the fraction and promoted formation of the uro, copro, and protoporphyrin fractions. These results suggest that DTP blocks the synthesis of protochlorophyllide in intact plants and induces consequent chlorosis, and the H(+) -donating activity of DTP might cause the reduction of protochlorophyllide biosynthesis. PMID- 16661053 TI - Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos. AB - The template activity of chromatin from winter wheat embryos gradually increased during germination and was regulated with some nonhistone proteins different from the two major ones, molecular weight 39k and 59k polypeptides, previously reported.To clarify chromosomal proteins which are involved in regulation of template activity of chromatin, we studied the quantitative and qualitative changes in chromosomal proteins. Differences in acid-soluble and acid-insoluble proteins between chromatins from wheat germ and embryos germinated for various times were visualized by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.Nonhistone proteins of 39k, 41k, and 50k molecular weights were specifically present in wheat germ and in 24- or 48-hour germinated wheat embryos, thereafter greatly reduced or finally disappeared. In contrast, nonhistone protein of 37k was absent in germ and in embryos germinated for 24 hours and appeared after 48 hours of germination. Thereafter it was present in abundant amounts in 96-hour germinated winter wheat embryos and in 72-hour germinated spring embryos, corresponding to 7 and 10% of total nonhistone proteins, respectively. Histone H1, especially H1d, was slightly reduced after 48 hour germination, as much as basic nonhistone proteins having electrophoretic mobilities between H1d and H2B. Further-more, similarity and diversity of chromosomal proteins between spring and winter wheat embryos are shown in this study. A subspecies of histone H1c of spring wheat had faster electrophoretic mobility than that of winter wheat. PMID- 16661054 TI - Control of Phycoerythrin Synthesis during Chromatic Adaptation. AB - Chromatic adaptation is the process by which blue-green algae alter the rates of biliprotein synthesis in response to changes in the color of available light. We have examined the control of phycoerythrin synthesis during the early stages of chromatic adaptation in Fremyella diplosiphon using fluorescence spectroscopy and (35)S-labeling of polypeptides. Phycoerythrin synthesis begins within 45 to 90 minutes after transfer of cells from red to green light, but is blocked by rifamycin. Transfer of cells from green to red light stops phycoerythrin synthesis with a t(1/2) = 45 minutes, as does the addition of rifamycin in green light. Transfer from green light to darkness slows but does not stop phycoerythrin synthesis. Gel electrophoresis of labeled polypeptides, both soluble and membrane-bound, shows that the synthesis of some polypeptides other than phycoerythrin are also affected by changes in light. These data suggest that chromatic adaptation involves gene regulation at the transcriptional level. PMID- 16661055 TI - Azolla-Anabaena Relationship: VIII. Photosynthetic Characterization of the Association and Individual Partners. AB - Photosynthesis in the Azolla-Anabaena association was characterized with respect to photorespiration, early products of photosynthesis, and action spectra. Photorespiration as evidenced by an O(2) inhibition of photosynthesis and an O(2) dependent CO(2) compensation concentration was found to occur in the association, and endophyte-free fronds, but not in the endophytic Anabaena. Analysis of the early products of photosynthesis indicated that both the fern and cyanobacterium fix CO(2) via the Calvin cycle. The isolated endophytic Anabaena did not release significant amounts of amino acids synthesized from recently fixed carbon. The action spectra for photosynthesis in the Azolla-Anabaena association indicated that the maximum quantum yield is between 650 and 670 nanometers, while in the endophyte the maximum is between 580 and 640 nanometers. Although the endophytic cyanobacterium is photosynthetically competent, any contribution it makes to photosynthesis in the intact association was not apparent in the action spectrum. PMID- 16661056 TI - Patterns of ehtylene production in senescing leaves. AB - Changes in the patterns of ethylene production, chlorophyll content, and respiration were studied in relation to the senescence of intact leaves and leaf discs. The primary leaves of pinto bean, which abscise readily during natural senescence, and tobacco and sugar beet leaves, which do not abscise, were used. A decrease in the rate of ethylene production and respiration, during the slow phase of chlorophyll degradation, was observed in leaf-blade discs cut from mature leaves and aged in the dark. During rapid chlorophyll loss both ethylene production and respiration increased and then decreased. These climacteric-like patterns were shown by leaf discs of all three species. Discs taken from leaves that had been senescing on the plant also showed a climacteric-like rise in ethylene production but not in respiration, which decreased continuously with leaf age. Climacteric-like patterns in the rise of ethylene and respiration for leaf discs were also shown by the petioles of both bean and tobacco leaves. This indicates that the rise of ethylene and respiration is characteristic of the general process of senescence in leaves and is not restricted to the abscission process. In contrast to the ethylene-forming systems in climacteric fruits and many flowers, the one in leaves declines sharply in the early stages of senescence. The subsequent rise of ethylene production appears to be associated with the rapid phase of chlorophyll breakdown, and may indicate the final stage of the senescence process during which ethylene could be actively involved in inducing leaf abscission. PMID- 16661057 TI - Ethylene as a regulator of senescence in tobacco leaf discs. AB - The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO(2), which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO(2) evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag(+) or enrichment of the atmosphere with 5 to 10% CO(2) reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag(+) was much more effective than CO(2) in retarding leaf senescence. Despite their senescence-retarding effect, Ag(+) and CO(2), which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag(+) was more effective than CO(2). The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence. PMID- 16661058 TI - Production and action of ethylene in senescing leaf discs: effect of indoleacetic Acid, kinetin, silver ion, and carbon dioxide. AB - Supraoptimal concentrations of indoleacetic acid (IAA) stimulated ethylene production, which in turn appeared to oppose the senescence-retarding effect of IAA in tobacco leaf discs. Kinetin acted synergistically with IAA in stimulating ethylene production, but it inhibited senescence. Silver ion and CO(2), which are believed to block ethylene binding to its receptor sites, delayed senescence in terms of chlorophyll loss and stimulated ethylene production. Both effects of Ag(+) were considerably greater than those of CO(2). IAA, kinetin, CO(2), and Ag(+), combined, acted to increase ethylene production further. Although this combination increased ethylene production about 160-fold over that of the control, it inhibited senescence. Treatment with 25 mul/l of ethylene in the presence of IAA enhanced chlorophyll loss in leaf discs and inhibited by about 90% the conversion of l-[3,4-(14)C] methionine to (14)C(2)H(4) suggesting autoinhibition of ethylene production.The results suggest that ethylene biosynthesis in leaves is controlled by hormones, especially auxin, and possibly the rate of ethylene production depends, via a feedback control system, on the rates of ethylene binding at its receptor sites. PMID- 16661059 TI - Flowering in bougainvillea: a function of assimilate supply and nutrient diversion. AB - Reproductive development, whether expressed as first node to flower or numbers of inflorescences developing, is promoted in direct relationship to leaf area and in inverse relationship to the numbers of axillary branches developing. Per cent soluble solids in the reproductive shoots vary with reproductive development. Cytokinin treatments promote inflorescence development and per cent soluble solids, further supporting a nutritional hypothesis in the control of flowering in Bougainvillea "San Diego Red." Gibberellin treatments inhibit reproductive development completely without significant lowering of per cent soluble solids, which is counter to expectations for a nutritional hypothesis. A closer examination of the reproductive axes, the tissues in which morphogenetic change occurs, must be made for the gibberellin-treated tissues. PMID- 16661060 TI - Senescence of Pear Fruit Cells Cultured in a Continuously Renewed, Auxin-deprived Medium. AB - Auxins and cytokinins support cell division in tissue and cell cultures. In cytokinin-independent pear (Pyrus communis) cells, omission of 2,4 dichlorophenoxyacetic acid (2,4-D) from the medium for two successive transfers leads to rapid cell lysis, unless the osmolarity is raised to 0.4 molar with mannitol. Use of this system (nutrients plus mannitol minus 2,4-D) for the study of cell senescence was explored both in batch culture and in a system designed to permit medium renewal without withdrawal of live cells.In both systems, an initial period (1-6 days) of limited increase in cell number is characterized by a continuous decrease in the respiratory activity and in protein and RNA synthesis to very low basal rates. In batch culture, cell death occurs after 13 to 15 days with little or no change in metabolic activity, or in protein and RNA synthesis. With renewal of cell medium, death is slightly delayed and is preceded by a burst in RNA synthesis followed by a notable increase in protein synthesis. Cycloheximide inhibition of protein synthesis is transient and its effect on cell longevity variable. Nonetheless, in all instances cell death is preceded by a burst in protein synthesis. Actinomycin D (1.6 micromolar) did not significantly affect protein synthesis but delayed RNA synthesis and cell death. The possible roles of auxin, osmoticum, and macromolecular synthesis in cellular senescence and death are discussed. PMID- 16661061 TI - Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA. AB - Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with (125)I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA. PMID- 16661062 TI - Control of Seed Germination by Abscisic Acid: I. Time Course of Action in Sinapis alba L. AB - The germination process of mustard seeds (Sinapis alba L.) has been characterized by the time courses of water uptake, rupturing of the seed coat (12 hours after sowing), onset of axis growth (18 hours after sowing), and the point of no return, where the seeds lose the ability to survive redesiccation (12 to 24 hours after sowing, depending on embryo part). Abscisic acid (ABA) reversibly arrests embryo development at the brink of radicle growth initiation, inhibiting the water uptake which accompanies embryo growth. Seeds which have been kept dormant by ABA for several days will, after removal of the hormone, rapidly take up water and continue the germination process. Seeds which have been preincubated in water lose the sensitivity to be arrested by ABA after about 12 hours after sowing. This escape from ABA-mediated dormancy is not due to an inactivation of the hormone but to a loss of competence to respond to ABA during the course of germination. The sensitivity to ABA can be restored in these seeds by redrying. It is concluded that a primary action of ABA in inhibiting seed germination is the control of water uptake of the embryo tissues rather than the control of DNA, RNA, or protein syntheses. PMID- 16661063 TI - Characterization of the 32,000 Dalton Chloroplast Membrane Protein: III. Probing Its Biological Function in Spirodela. AB - The rapidly turning over, photoinduced thylakoid protein, P-32000, is the main pulse-labeled membrane polypeptide in the chloroplasts of Spirodela oligorrhiza, yet little is known of its physiological function. Two hypotheses are tested: that P-32000 synthesis is necessary for thylakoid biogenesis; that it directly participates in photosynthesis. Spirodela cultures were dissected into expanding and fully mature tissue. Fronds from both developmental stages transcribed a 0.5 x 10(6) dalton RNA likely to be the message for P-32000. As to the protein itself, synthesis occurred in both types of tissue but was considerably enhanced in the fully mature state. Thus, a purely transient, developmental function for P 32000 during thylakoid biogenesis appears ruled out. Low concentrations of d threo-chloramphenicol severely suppressed P-32000 synthesis but not its turnover. As a result, fronds depleted in P-32000 were obtained. However, photoassimilation of CO(2) remained at 86% of normal in tissue > 80% depleted of P-32000. Thus, P 32000 did not appear to be rate-limiting, suggesting that it does not serve as a direct, integral part of the photosynthetic pathway. PMID- 16661064 TI - Isolation and purification of leaf starch components. AB - A procedure was developed for the separation and purification of amylose and amylopectin isolated from cotton leaves. Cotton leaves were homogenized in 0.02 molar phosphate buffer at pH 7.0 containing HgCl(2) plus toluene. Crude starch granules were collected by centrifugation and partially purified by treating with acetone and toluene. The starch granules were then dispersed in dimethylsulfoxide and precipitated with ethyl alcohol. The precipitate was suspended in boiling water. Amylose was separated from amylopectin and cell wall particles on a Sepharose 2B column and further purified with thymol and butanol. Amylopectin was then separated from the colloidal cell wall contaminants by its specific interaction with concanavalin A. Purities of starch components were verified by specific biochemical and enzymic tests in addition to their iodine-binding capacity. This procedure should also be suitable for purification of starch components from other plant sources. PMID- 16661065 TI - Sucrose uptake by sugar beet tap root tissue. AB - Sucrose uptake by discs of mature sugar beet root tissue incubated in [(14)C] sucrose exhibited nonsaturating kinetics over the concentration range of 1 to 500 millimolar. Uptake was inhibited by dinitrophenol, sodium cyanide, low O(2), and penetrating sulfhydryl inhibitors. ATPase inhibitors, sodium fluoride, and oligomycin reduced uptake by 20 and 40%, respectively. Uptake as asymmetrically labeled sucrose ([(14)C]glucose) occurred with approximately 80% retention of asymmetry, indicating a nonhydrolytic pathway. Uptake was against a concentration gradient and required metabolic energy.Glucose and fructose uptake exhibited typical saturation kinetics but rates of uptake were lower than that of sucrose, particularly at high concentration. Glucose strongly inhibited the uptake of sucrose and fructose but sucrose and fructose had little effect on the rate of glucose uptake. It is proposed that a major protion of the sucrose movement between its free space and vacuole occurs via a nonsaturating carrier at sites where the plasmalemma and tonoplast are appressed. PMID- 16661066 TI - Energy-linked Potassium Influx as Related to Cell Potential in Corn Roots. AB - Cell potentials and K(+) ((86)Rb) influx were determined for corn roots over a wide range of external K(+) activity (K degrees ) under control, anoxic, and uncoupled conditions. The data were analyzed using Goldman theory for the contribution of passive influx to total influx. For anoxic and uncoupled roots the K(+) influx shows the functional relationship with K degrees predicted with constant passive permeability, although K(+) permeability in uncoupled roots is about twice that of anoxic roots. In control roots the equation fails to describe K(+) influx at low K degrees , but does so at high K degrees , with a gradual transition over the region where the electrical potential becomes equal to the equilibrium potential for K(+) (psi = E(K)). In the low K degrees range, where net K(+) influx is energetically uphill, participation of an energy-linked K(+) carrier is indicated. In the high K degrees range, K(+) influx becomes passive down the electrical gradient established by the cell potential. Since the cell potential includes a substantial electrogenic component, anoxia or uncoupling reduces passive influx. PMID- 16661067 TI - Orthophosphate control of glucose-6-phosphate dehydrogenase light modulation in relation to the induction phase of chloroplast photosynthesis. AB - High concentrations of orthophosphate (Pi) inhibited CO(2)-dependent O(2) evolution and prevented the inactivation of glucose-6-P dehydrogenase by light in intact spinach and barley chloroplasts. Addition of glycerate-3-P to chloroplasts inhibited by Pi in the light, induced O(2) evolution and caused rapid inactivation of glucose-6-P dehydrogenase. The activity of phosphofructokinase detected in chloroplast preparations was not affected by light or by Pi.Dihydroxyacetone-P was a major product of chloroplast photosynthesis when optimum concentrations of Pi were used. Chloroplasts continued to form dihydroxyacetone-P at a slow rate in the presence of Pi at concentrations (2 to 4 millimolar) that gave complete inhibition of CO(2)-dependent O(2) evolution. Formation of dihydroxyacetone-P in the presence of 4 millimolar Pi was stimulated by light and either O(2) (150 micromolar) or sparker amounts of oxaloacetate.Conditions that favored dihydroxyacetone-P formation (high O(2) or low O(2) plus oxaloacetate) increased the optimum Pi concentration for CO(2) dependent O(2) evolution and stimulated O(2) evolution at high concentrations of Pi. The stimulation of O(2) evolution at superoptimal concentrations of Pi by O(2) or oxaloacetate was prevented by dithiothreitol.The results suggested that formation of pentose-P pathway intermediates via the oxidative pentose-P pathway may be limited by availability of NADP in the light but may occur at significant rates and thereby contribute to termination of the induction phase of O(2) evolution. PMID- 16661068 TI - Relative sensitivity of photosynthetic assimilation and translocation of carbon to water stress. AB - The relationship between photosynthesis and translocation rate changes as affected by water stress intensity and stage of plant development was evaluated in cotton and sorghum, representing a C(3) and a C(4) photosynthetic type, respectively. Photosynthetic rates were reduced as midday leaf water potentials declined from -14 to -27 bars in both species. Sorghum maintained higher photosynthesis and translocation rates compared to cotton at comparable leaf water potentials; however, the rate of change per bar decline in water potential was greater in sorghum than in cotton. Photosynthetic rates were reduced with increasing water stress prior to any significant change in translocation rates suggesting that photosynthesis is the more sensitive of the two processes. Severe water stress, corresponding to leaf water potentials of -27 bars, did not completely inhibit either photosynthesis or translocation. PMID- 16661069 TI - Azocoll-digesting Proteinases in Soybean Leaves: Characteristics and Changes during Leaf Maturation and Senescence. AB - Two different endopeptidases which digest the chromogenic substrate Azocoll were found in soybean leaves. Azocollase A has a molecular weight of 17,500 and a pI of 6.0. Azocollase B has a molecular weight of 52,000 and a pI of 9.0. Both digest Azocoll optimally at pH 9.0. Azocollase A is inhibited by 3 millimolar ethylenediamine tetraacetate (EDTA) and azocollase B by 100 micromolar parachloromercuribenzoate. Studies on whole plants grown in the greenhouse and in the field show that total azocollase activity gradually increased during leaf maturation when leaf protein and chlorophyll increased, and then declined again during leaf senescence. Young leaves which are still expanding contain mostly azocollase B and little azocollase A. Leaf maturation was associated with a dramatic increase in azocollase A (40- to 50-fold), while azocollase B activity increased more slowly. This increase in azocollase A occurred in the 2- to 3-week period following leaf expansion. Azocollase A, separated from other proteinases by gel filtration on Sephadex G-100, digested denatured leaf protein and casein, resulting in the release of free alpha-amino groups. Break-down of leaf proteins by autodigestion of extracts at pH 9.0 resulted in the release of free alpha amino groups and endopeptidic cleavage of polypeptides. However, polypeptide cleavage was not inhibited by parachloromercuribenzoate or EDTA indicating that the azocollases do not play a major role in the hydrolysis of leaf proteins in crude extracts. PMID- 16661070 TI - Enhancement of polyribosome formation and RNA synthesis of gibberellic Acid in wounded potato tuber tissue. AB - As part of a more detailed study on plant tumorigenesis, the action of gibberellic acid (GA(3)) in wounded potato tuber tissues as a model system has been evaluated. GA(3) stimulates total RNA synthesis in wounded tissues, the optimal concentration being 0.1 micromolar. The responsiveness of the tissue toward the hormone develops with time after wounding. Whereas freshly wounded tissue does not respond at all to the hormone, it becomes competent after about 6 hours, the competence being maximal after 1 day of wound healing.GA(3) enhances the formation of polyribosomes in wounded tissues and stimulates the synthesis of both ribosomal RNAs, transfer RNAs, 5S RNA, and a fraction, which in sucrose density gradients sediments between 18S rRNA and 5S RNA. This fraction contains presumptive mRNA.The hormone, then, is somehow recognized by wounded potato tissue in a time-specific way; the signal is transferred to the genome and triggers the synthesis of various RNA species. PMID- 16661071 TI - Gibberellic Acid Activates Chromatin-bound DNA-dependent RNA Polymerase in Wounded Potato Tuber Tissue. AB - Chromatin-bound DNA-dependent RNA polymerases react upon wounding of white potato tuber tissues with an increase in activity, which is additionally enhanced to 300% in the presence of 0.1 micromolar gibberellic acid (GA(3)). 2,4 Dichlorophenoxyacetic acid is only weakly effective and indoleacetic acid not at all. Wounding and treatment with GA(3) affect template availability of chromatin only slightly. The hormone has no effect on chromatin-bound RNA polymerases, if added in vitro.The enzymes from intact, wounded, and hormone-treated tissues possess similar characteristics: their activity is dependent on the presence of all four ribonucleotides and a divalent cation such as Mg(2+) or Mn(2+). However, the sensitivity of the enzymes from different preparations toward alpha-amanitin differs. Total RNA polymerase activity of chromatin was inhibited by alpha amanitin to about 44% in intact, to about 22% in wounded, and only 15% in GA(3) treated tissues. The relative activities of polymerases I and II were estimated by varying the (NH(4))(2)SO(4) and alpha-amanitin concentrations in the assay system. It is evident that GA(3) preferentially stimulates polymerase I and hence ribosomal RNA synthesis. RNA polymerase II is but slightly affected by GA(3). Nearest neighbor frequency analysis revealed that the RNA synthesized by the enzymes from the intact tuber is different from that of wounded or GA(3)-treated tissues. PMID- 16661072 TI - Photosynthate partitioning in soybean leaves at two irradiance levels: comparative responses of acclimated and unacclimated leaves. AB - High irradiance-acclimated soybean leaves had the same CO(2) exchange rates, but lower starch accumulation rates and correspondingly higher translocation rates than unacclimated leaves. Increased translocation rates were associated with increased sucrose phosphate synthetase (EC 2.4.1.14) activity. Foliar sucrose levels and adenosine diphosphate-glucose pyrophosphorylase (EC 2.7.7.9) activity were unaffected. Carbon assimilation, partitioning, and enzyme activity of unacclimated leaves were unaltered even after a second day's exposure to high irradiance. Results are consistent with the hypothesis that photosynthate partitioning between starch synthesis and sucrose translocation are controlled in part by the rate of sucrose synthesis. PMID- 16661073 TI - Measurement of ribulose 1,5-bisphosphate from spinach chloroplasts. AB - A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid. PMID- 16661074 TI - Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts. AB - The response of ribulose 1,5-bisphosphate levels and CO(2) fixation rates in isolated, intact spinach chloroplasts to pyrophosphate, triose phosphates, dl glyceraldehyde, O(2), catalase, and irradiance during photosynthesis has been studied. Within 1 minute in the light, a rapid accumulation of ribulose bisphosphate was measured in most preparations of intact chloroplasts, and this subsequently dropped as CO(2) fixation increased. Pyrophosphate, triose phosphates, and catalase increased CO(2) fixation and also the levels of ribulose bisphosphate. CO(2) fixation was inhibited by dl-glyceraldehyde and O(2) with corresponding decreases in ribulose bisphosphate. When the rate of photosynthesis decreased at limiting irradiances (low light), the level of ribulose bisphosphate in the chloroplast did not always decrease, suggesting that ribulose bisphosphate was not limiting CO(2) fixation under these conditions. When triose phosphates (fructose bisphosphate plus aldolase) were added to suspensions of chloroplasts at low irradiances, ribulose bisphosphate increased while CO(2) fixation decreased. These observations provide considerable evidence that high ribulose bisphosphate levels clearly are not solely sufficient to permit rapid rates of CO(2) fixation, but that factors other than ribulose bisphosphate concentration are overriding the control of photosynthesis.Isolated chloroplasts are capable of using carbon reserves to produce considerable ribulose bisphosphate. Upon illumination in the absence of CO(2) and O(2), intact chloroplasts produced up to 13 millimolar ribulose bisphosphate. PMID- 16661075 TI - Ribulose Bisphosphate Carboxylase and Proteolytic Activity in Wheat Leaves from Anthesis through Senescence. AB - Changes in ribulose bisphosphate carboxylase (RuBPCase) and proteolytic activity were followed in the flag leaf and second leaf of field-grown winter wheat (cv. Arthur). These changes were followed in relation to changes in leaf chlorophyll, protein, and photosynthesis, and seed development. Levels of RuBPCase were determined by rocket immunoelectrophoresis as described previously (Wittenbach 1978 Plant Physiol 62: 604-608). RuBPCase constituted 40 to 45% of the total soluble protein in the flag leaf and an even higher percentage of the soluble protein in the second leaf. This ratio remained unchanged until senescence when RuBPCase protein was apparently lost at a faster rate than total soluble protein. No change in the specific activity of RuBPCase on either a milligram protein or RuBPCase basis was observed until senescence. A close correlation existed among the various indices of senescence in the field, namely, the decline in chlorophyll, protein, photosynthesis, and RuBPCase activity. In addition, proteinase activity increased with the onset of senescence. These enzymes readily degraded RuBPCase, exhibiting a pH optimum of 4.8 to 5.0 and a temperature optimum of 50 C. Proteinase activity was modified by sulfydryl reagents suggesting the presence of sulfydryl groups at or near the active sites. PMID- 16661076 TI - Economy of Photosynthate Use in Nitrogen-fixing Legume Nodules: Observations on Two Contrasting Symbioses. AB - The economy of C use by root nodules was examined in two symbioses, Vigna unguiculata (L.) Walp. (cv. Caloona):Rhizobium CB756 and Lupinus albus L. (cv. Ultra):Rhizobium WU425 over a 2-week period in early vegetative growth. Plants were grown in minus N water culture with cuvettes attached to the nodulated zone of their primary roots for collection of evolved CO(2) and H(2). Increments in total plant N and in C and N of nodules, and C:N weight ratios of xylem and phloem exudates were studied by periodic sampling from the plant populations. Itemized budgets were constructed for the partitioning and utilization of C in the two species. For each milligram N fixed and assimilated by the cowpea association, 1.54 +/- 0.26 (standard error) milligrams C as CO(2) and negligible H(2) were evolved and 3.11 milligrams of translocated C utilized by the nodules. Comparable values for nodules of the lupin association were 3.64 +/- 0.28 milligrams C as CO(2), 0.22 +/- 0.05 milligrams H(2), and 6.58 milligrams C. More efficient use of C by cowpea nodules was due to a lesser requirement of C for synthesis of exported N compounds, a smaller allocation of C to nodule dry matter, and a lower evolution of CO(2). The activity of phosphoenolpyruvate carboxylase in nodule extracts and the rate of (14)CO(2) fixation by detached nodules were greater for the cowpea symbiosis (0.56 +/- 0.06 and 0.22 milligrams C as CO(2) fixed per gram fresh weight per hour, respectively) than for the lupin 0.06 +/- 0.02 and 0.01 milligrams C as CO(2) fixed per gram fresh weight per hour. The significance of the data was discussed in relation to current information on theoretical costs of nitrogenase functioning and associated nodule processes. PMID- 16661077 TI - Measurement of carbon dioxide compensation points of freshwater algae. AB - A technique is described for the measurement of total dissolved inorganic carbon by acid release as CO(2) followed by its conversion to methane and detection by flame ionization in a modified gas chromatograph. This method was used to determine the dissolved inorganic carbon concentration reached at compensation point when algae were allowed to photosynthesize in a closed system in a buffer at known pH, and the CO(2) compensation point was calculated from this concentration. The CO(2) compensation points of 16 freshwater algae were measured at acid and alkaline pH in air-saturated medium: at acid pH the CO(2) compensation points ranged from 4.8 to 41.5 microliters per liter while at alkaline pH they ranged from 0.2 to 7.2 microliters per liter. Removal of O(2) from the medium caused a slight lowering of compensation point at acid pH but had little effect at alkaline pH. These low, O(2)-insensitive compensation points are characteristic of C(4) plants. It is suggested that these low CO(2) compensation points are maintained by an active bicarbonate uptake by algae especially at alkaline pH. PMID- 16661078 TI - Diurnal variation in situ of photosynthetic capacity in ulva is caused by a dark reaction. AB - Ulva lactuca (sea lettuce) undergoes large diurnal oscillations of light saturated photosynthetic O(2) evolution in situ. Freshly collected samples from Great Harbor, Woods Hole, Massachusetts, had a maximum white light-saturated rate at noon that was 2.5-fold higher than the rate of matched samples collected at midnight. When kept under constant low level illumination, the cycle persisted for at least 36 hours, and after 2 weeks damped out to a constant level that was halfway between the minimum and maximum rates. The cyclic oscillations were apparent whether expressed on a weight or chlorophyll content basis, occurred in both lightly and heavily pigmented samples, and were not attributable to changes in chloroplast shading due to variations in chloroplast orientation within the frond cells. There were no cyclic variations in the initial slopes of the light saturation curves, in photosynthetic unit size, or in relative quantum efficiency. Measurement of the "fast" turnover time of photosynthesis by the delayed dual flash technique revealed no diurnal variations of this parameter. These results indicate that the cyclic variations in photosynthetic activity are modulated by a dark reaction at a step occurring after reduction of plastoquinone by electrons from photosystem II. PMID- 16661079 TI - Regulation of cytoplasmic and vacuolar volumes by plant cells in suspension culture. AB - Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite changes in the rate of cell division, cell size, rate of increase in fresh or dry weight, amount of cytoplasm per cell, the cellular concentration or fluxes of Na or K, and the rate of vacuolation. It is suggested that cytoplasmic volume is under the control of its own regulatory mechanism, which operates to give a constant exponential increase in cytoplasmic volume independently of most other observed cellular properties. PMID- 16661080 TI - Effect of vanadate on rhythmic leaflet movement in albizzia julibrissin. AB - Vanadate (Na(3)VO(4)) selectively and reversibly affects the rhythmic movement of Albizzia julibrissin leaflets. Leaflets floated on 1 millimolar vanadate open at the same rate or more rapidly than controls, but closure is inhibited. After 6 to 24 hours incubation, the inhibition can be reversed by a 24-to 48-hour period on water or control buffer. Recovery is complete in light-dark cycles, and it is almost complete under free-running conditions (prolonged darkness). Leaflets floated on 10 millimolar vanadate do not open in darkness, but they open at a reduced rate in light. Concentrations of 100 micromolar or less are ineffective. PMID- 16661081 TI - Effects of temperature perturbations on circadian conidiation in neurospora. AB - Studies on the circadian rhythm of conidiation in the bd strain of Neurospora crassa Shear and Dodge have shown that temperature step-up and step-down perturbations produce phase advances and delays, respectively. Pulse-up and pulse down treatments lead to both phase advances and delays. The resulting phase shifts can be very large, and few to no transients are observed.Small amplitude temperature cycles are capable of entraining the circadian rhythm, and holding bd at low temperatures appears to stop the circadian oscillator late in the subjective night (circadian time 2200). Aspects of the temperature responses that are somewhat unusual include the high sensivity, the lack of transients, and the phase at which the oscillator stops under low temperatures. PMID- 16661082 TI - Isolation and Characterization of Concanavalin A-labeled Plasma Membranes of Carrot Protoplasts. AB - The plasma membranes of protoplasts released from carrot suspension culture cells were labeled with [(14)C]acetyl-concanavalin A. After homogenization a single labeled membrane fraction was isolated in a continuous isopycnic Renografin gradient. The labeled membranes peaked at an apparent density of 1.14 grams per cubic centimeter between the Golgi fraction at a density of 1.11 grams per cubic centimeter as determined by latent IDPase activity and the mitochondria at a density of 1.16 grams per cubic centimeter as determined by the cytochrome c oxidase activity. This method provided a very discrete peak of putative plasma membrane. On discontinuous Renografin gradients a relatively pure fraction of labeled plasma membranes could be readily isolated at the 1.122 to 1.146 grams per cubic centimeter interface. The labeled fraction was enriched in both an ATPase (pH 6.5) and a glucan synthetase with a pH optimum of 6.5 whose activity was promoted by magnesium and cellobiose. Enzyme activities were not altered by the membrane label. PMID- 16661083 TI - Effects of pronase on isolated chloroplasts. AB - Subjecting isolated spinach chloroplasts to mild proteolysis (10-minute incubation at 20 C in 500 micrograms per milliliter pronase) caused chloroplast clumping but did not affect their integrity as measured by their ability to carry out light stimulated, glycerate-3-P-dependent O(2) evolution. Transmission electron microscopy revealed no detectable differences between the control and treated plastids. Mild proteolysis inactivated exogenously added pyruvate kinase and should be a useful technique in certain enzyme distribution studies. PMID- 16661084 TI - Kinetics and time dependence of the effect of far red light on the photoperiodic induction of flowering in wintex barley. AB - Flowering in the long day plant Hordeum vulgare L. var. Wintex barley was enhanced by the addition of far red light to the main light portion of the photoperiod. Far red energy was provided to produce quantum flux ratios (660/730 nm) and phytochrome photoequilibria (Pfr/total phytochrome) equivalent to those reported both beneath a leaf canopy and outside a canopy at twilight. The photoperiodic requirement for long days can be completely eliminated by the addition of far red light. However, both the effect of extending the photoperiod without far red and the addition of far red to 12-hour photoperiods were suboptimal. Maximal stimulation was achieved only when far red was added to continuous light. The duration of the period of maximal apex elongation rate, as well as the reduction of the time required for floral initiation, were saturated by three inductive cycles. When far red energy was provided intermittently during 3 days of continuous light, the ability to respond varied in a circadian manner. This enhancement of flowering by far red appears to be mediated by the "high irradiance response" of phytochrome. PMID- 16661085 TI - Purification and characterization of the carboxypeptidase isoinhibitors from potatoes. AB - Three zones of carboxypeptidase inhibitory activity were observed when heat stable extracts of potato tubers (cv. Russet Burbank) were chromatographed on carboxymethyl cellulose. The isoinhibitors found in these zones were denoted I, II, and III based upon their order of elution from this column. The predominant form (II) had previously been suggested to be a mixture of two polypeptides (IIa and IIb) differing in that IIa possessed an additional residue of glutamine (Hass et al. 1975 Biochemistry 14: 1334). These closely related isoinhibitors (IIa and IIb) were separated by equilibrium ion exchange chromatography and characterized. Isoinhibitor I was shown to be identical to II except for two replacements, Ser 30 --> Ala and Arg-32 --> Gly. These replacements had no significant effect on apparent K(i) values toward either carboxypeptidase A or B. Isoinhibitor III, which was identical to II except that it lacked the amino terminal pyrrolidone carboxylic acid and following glutamine residue, was also functionally indistinguishable from II in inhibition studies. It was concluded that at least two and possibly as many as five genes code for the various isoinhibitor species which are present in potato tubers. PMID- 16661086 TI - Distribution of carboxypeptidase isoinhibitors in the potato plant. AB - The total amount of carboxypeptidase inhibitor was estimated in extracts of the leaves, stems, and sprouts of Solanum tuberosum L. cv. Russet Burbank. Although the tuber contained the highest levels on a dry weight basis, inhibitor was also detected in the leaves, sprouts, and upper stems. The relative amounts of each of three carboxypeptidase isoinhibitor families were estimated in several plant tissues by purifying the mixture of isoinhibitors using immobilized carboxypeptidase and then resolving the individual families by polyacrylamide gel electrophoresis. These data demonstrated both that differences in distribution are found in the potato plant and that the three isoinhibitor families described previously (Hass GM, JE Derr, DJ Makus, CA Ryan 1979 Plant Physiol 64: 1022-1028) account for essentially all of the carboxypeptidase inhibitor activity in the tissues studied. PMID- 16661087 TI - Metabolism of l-Tyrosine to 4-Hydroxybenzaldehyde and 3-Bromo-4 Hydroxybenzaldehyde by Chloroplast-containing Fractions of Odonthalia floccosa (Esp.) Falk. AB - The biosynthesis of 4-hydroxybenzaldehyde and 3-bromo-4-hydroxybenzaldehyde from l-[U-(14)C]tyrosine has been demonstrated in chloroplast-containing fractions obtained by differential and isopycnic centrifugation from the marine red alga Odonthalia floccosa. Surfactant and high speed centrifugation studies indicate that the biosynthetic pathway involves a particulate enzyme system, possibly located on the thylakoid membranes. The following scheme, based upon identification of labeled (14)C-intermediates, is proposed for the formation of aldehydes: l-tyrosine --> 4-hydroxyphenylpyruvic acid --> 4-hydroxyphenylacetic acid --> 4-hydroxymandelic acid --> 4-hydroxybenzaldehyde --> 3-bromo-4 hydroxybenzaldehyde. PMID- 16661088 TI - Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels. AB - Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K(m) for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration. PMID- 16661089 TI - Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L: Sources of ATP for Glutamine Synthesis and Photoregulation of Tricarboxylic Acid Cycle Metabolism. AB - Metabolism of [(14)C]glutamate was studied in leaf discs of Vicia faba L. in light and in darkness. In white light glutamine was the main labeled product. In the dark label was principally in compounds closely associated with tricarboxylic acid cycle metabolism, predominantly aspartate. Entry of label from glutamate into tricarboxylic acid metabolism appeared to be at least partially by decarboxylation of glutamate to gamma-amino butyric acid, followed by conversion to succinate. 3-(3,4-dichlorophenyl)-1, 1-Dimethylurea inhibited light-enhanced synthesis of glutamine and caused reversion toward the dark pattern of metabolism. Methionine sulfoximine severely inhibited glutamine synthesis and caused accumulation of labeled malate.Monochromatic 650 nanometer light gave similar results to white light. Monochromatic light of 710 nanometers had a much smaller effect on glutamine synthesis but did significantly raise the ratio of labeled malate to aspartate. gamma-Amino [(14)C]butyric acid was metabolized entirely via tricarboxylic acid cycle metabolism in light or dark, and in the light the ratio of labeled malate to aspartate was raised.These results suggest that illuminated leaves metabolize glutamate to glutamine mainly in the chloroplasts. When chloroplastic glutamine synthesis fails to take place, either in darkness or in the presence of inhibitors, glutamate is apparently metabolized outside the chloroplast. Light lowers the NAD(+) to NADH ratio outside the chloroplast, consequently altering the equilibrium of the malate dehydrogenase reaction. Alteration of the malate to aspartate ratio by 710 nanometer light suggests that ATP generated by photosystem I-dependent cyclic photophosphorylation may affect extrachloroplastic NAD(+) to NADH ratios. PMID- 16661090 TI - Mechanism of decarboxylation of glycine and glycolate by isolated soybean cells. AB - Isolated soybean leaf mesophyll cells decarboxylated exogenously added [1 (14)C]glycolate and [1-(14)C]glycine in the dark. The rate of CO(2) release from glycine was inhibited over 90% by isonicotinic acid hydrazide and about 80% by KCN, two inhibitors of the glycine to serine plus CO(2) reaction. The release of CO(2) from glycolate was inhibited by less than 50% under the same conditions. This indicates that about 50% of the CO(2) released from glycolate occurred at a site other than the glycine to serine reaction. The sensitivity of this alternative site of CO(2) release to an inhibitor of glycolate oxidase (methyl-2 hydroxy-3-butynoate) but not an inhibitor of the glutamate:glyoxylate aminotransferase (2,3-epoxypropionate) indicates that this alternative (isonicotinic acid hydrazide insensitive) site of CO(2) release involved glyoxylate. Catalase inhibited this CO(2) release. Under the conditions used it is suggested that about half of the CO(2) released from glycolate occurred at the conversion of glycine to serine plus CO(2) while the remaining half of the CO(2) loss resulted from the direct oxidation of glyoxylate by H(2)O(2).The rate of glycine decarboxylation by the glycine to serine reaction was apparently controlled by the amount of NAD in the mitochondria. Mitochondrial electron transport inhibitors, KCN and actinomycin A, inhibited glycine decarboxylation while an uncoupler, 2,4-dinitrophenol, stimulated the reaction. Competition within the mitochondria between the enzymes of dark respiration and glycine decarboxylation for limiting NAD may force substantial amounts of the glycolate formed to be decarboxylated by the direct oxidation of glyoxylate. PMID- 16661091 TI - Influence of fusicoccin on the control of cell division by auxins. AB - An indirect stimulation of cell division by fusicoccin is demonstrated. The distribution of 2,4-dichlorophenoxyacetic acid molecules between Acer pseudoplatanus cells and their culture medium is strongly modified by a fusicoccin treatment, through the extracellular acidification induced by the toxin. As a consequence, a stimulation of cell division is observed when the intracellular auxin concentration is sufficiently increased to reach the threshold control level (Leguay JJ, J Guern 1977 Plant Physiol 60: 265 270).Independently of this indirect action on cell division, the number of cells per cluster is decreased and the volume of the cells increased in fusicoccintreated cells which show a typical enlargement response. PMID- 16661092 TI - Polarographic study of oxaloacetate reduction by isolated pea chloroplasts. AB - Suspensions of pea chloroplasts, prepared by differential centrifugation, catalyzed oxaloacetate-dependent O(2) evolution (mean rate of 29 determinations 10.9 micromoles per milligram of chlorophyll per hour, sd 3.2) with the concomitant production of malate. At optimum concentrations of oxaloacetate, both reactions were light-dependent, inhibited by 3-(3,4- dichlorophenyl)-1, 1 dimethylurea and oxalate, and enhanced 2.5- to 4-fold by 10 millimolar NH(4)Cl. At concentrations of oxaloacetate (<50 micromolar), 10 millimolar NH(4)Cl was inhibitory. The ratio of O(2) evolved to malate produced was 0.39 to 0.58. The ratio of O(2) evolved to oxaloacetate supplied was commensurate with the theoretical value of 0.5.Chloroplast suspensions contained both NAD- and NADP malate dehydrogenase activities. It was concluded from oxalate inhibition studies and the promotion of oxaloacetate-dependent O(2) evolution by shocked chloroplasts by NADPH (but not NADH) that the reaction was mediated via the NADP enzyme. PMID- 16661093 TI - Polarographic Study of Dicarboxylic-Acid-dependent Export of Reducing Equivalents from Illuminated Chloroplasts. AB - Isolated pea chloroplasts, prepared by differential centrifugation, catalyzed O(2) evolution in the light in the presence of 0.03 to 3 millimolar malate, 0.12 to 1.2 millimolar NAD, 4 millimolar pyruvate and exogenous NAD-malate dehydrogenase and lactate dehydrogenase. The reaction, which did not occur in the absence of any one of these factors, was accompanied by the consumption of pyruvate; the ratio of O(2) evolved to pyruvate consumed was <0.5. When 0.1 millimolar [(14)C]malate was supplied most of the (14)C label was recovered as malate. At low concentrations of malate (<0.1 millimolar), the ratio of O(2) evolved to malate supplied was greater than 0.5.Chloroplasts catalyzed oxaloacetate-dependent O(2) evolution in approximate agreement with the theoretical stoichiometry for the light-coupled reduction of oxaloacetate to malate. Subsequent addition of NAD, pyruvate, NAD-malate dehydrogenase, and lactate dehydrogenase caused immediate resumption of O(2) evolution with the concomitant reduction of pyruvate to lactate externally to the chloroplasts. Resumption of O(2) evolution did not occur in the absence of oxaloacetate. The results are consistent with a mechanism in which oxaloacetate/malate cycles continuously between the chloroplasts and the external solution and serves as a carrier for the transfer of light-generated reducing equivalents for pyruvate reduction outside the chloroplast. PMID- 16661094 TI - Comparative Enzymic Studies of Sucrose Metabolism in the Taproots and Fibrous Roots of Beta vulgaris L. AB - Comparative enzymic studies of sugar beet (Beta vulgaris L.) taproots and fibrous roots revealed differences in invertase (EC 3.2.1.26) and sucrose synthetase (EC 2.4.1.13) activity. Invertase activity of the two root forms differs with respect to specific activity, pH optimum, and enzyme solubility. Acid invertase (pH 4.5) in the taproot was restricted to the peripheral meristematic tissue which produces cells for both taproot and fibrous root growth. This finding supports the hypothesis that the enzyme regulates sucrose partitioning between the taproot and fibrous roots. A distinct alkaline invertase (pH 8.0) was detected in sucrose storage tissues of the taproot.The V(max) of taproot sucrose synthetase (sucrose cleavage reaction) was highest in the presence of UDP. However, the fibrous root enzyme had the highest V(max) with ADP as substrate. Differential nucleoside diphosphate substrate affinities may provide for compartmentation and separate regulation of sucrose cleavage and resultant hexose utilization in adjoining taproot and fibrous root tissues. PMID- 16661095 TI - Auxin-induced Ethylene Production and Its Inhibition by Aminoethyoxyvinylglycine and Cobalt Ion. AB - Auxin is known to stimulate greatly both C(2)H(4) production and the conversion of methionine to ethylene in vegetative tissues, while amino-ethoxyvinylglycine (AVG) or Co(2+) ion effectively block these processes. To identify the step in the ethylene biosynthetic pathway at which indoleacetic acid (IAA) and AVG exert their effects, [3-(14)C]methionine was administered to IAA or IAA-plus-AVG treated mung bean hypocotyls, and the conversion of methionine to S adenosylmethionine (SAM), 1-amino-cyclopropane-1-carboxylic acid (ACC), and C(2)H(4) was studied. The conversion of methionine to SAM was unaffected by treatment with IAA or IAA plus AVG, but active conversion of methionine to ACC was found only in tissues which were treated with IAA and which were actively producing ethylene. AVG treatment abolished both the conversion of methionine to ACC and ethylene production. These results suggest that in the ethylene biosynthetic pathway (methionine --> SAM --> ACC --> C(2)H(4)) IAA stimulates C(2)H(4) production by inducing the synthesis or activation of ACC synthase, which catalyzes the conversion of SAM to ACC. Indeed, ACC synthase activity was detected only in IAA-treated tissues and its activity was completely inhibited by AVG. This conclusion was supported by the observation that endogenous ACC accumulated after IAA treatment, and that this accumulation was completely eliminated by AVG treatment. The characteristics of Co(2+) inhibition of IAA dependent and ACC-dependent ethylene production were similar. The data indicate that Co(2+) exerts its effect by inhibiting the conversion of ACC to ethylene. This conclusion was further supported by the observation that when Co(2+) was administered to IAA-treated tissues, endogenous ACC accumulated while ethylene production declined. PMID- 16661096 TI - Assimilation and Transport of Nitrogen in Nonnodulated (NO(3)-grown) Lupinus albus L. AB - The response of nonnodulated white lupin (Lupinus albus L. cv. Ultra) plants to a range of NO(3) levels in the rooting medium was studied by in vitro assays of extracts of plant parts for NO(3) reductase (EC 1.6.6.1) activity, measurements of NO(3)-N in plant organs, and solute analyses of root bleeding (xylem) sap and phloem sap from stems and petioles. Plants were grown for 65 days with 5 millimolar NO(3) followed by 10 days with 1, 5, 15, or 30 millimolar NO(3). NO(3) reductase was substrate-induced in all tissues. Roots contained 76, 68, 62 and 31% of the total NO(3) reductase activity of plants fed with 1, 5, 15, and 30 millimolar NO(3), respectively. Stem, petioles, and leaflets contained virtually all of the NO(3) reductase activity of a shoot, the activity in extracts of fruits amounting to less than 0.3% of the total enzyme recovered from the plant. Xylem sap from NO(3)-grown nonnodulated plants contained the same organic solutes as from nodulated plants grown in the absence of combined N. Asparagine accounted for 50 to 70% and glutamine 10 to 20% of the xylem-borne N. The level of NO(3) in xylem sap amounted to 4, 13, 12, and 17% of the total xylem N at 1, 5, 15, and 30 millimolar NO(3), respectively. Xylem to phloem transfer of N appeared to be quantitatively important in supplying fruits and vegetative apices with reduced N, especially at low levels of applied NO(3). NO(3) failed to transfer in any quantity from xylem to phloem, representing less than 0.3% of the phloem-borne N at all levels of applied NO(3). Shoot organs were ineffective in storing NO(3). Even when NO(3) was supplied in great excess (30 millimolar level) it accounted for only 8% of the total N of stem and petioles, and only 2 and 1% of the N of leaflets and fruits, respectively. PMID- 16661097 TI - Economy of Carbon and Nitrogen in a Nodulated and Nonnodulated (NO(3)-grown) Legume. AB - Partitioning and utilization of assimilated C and N were compared in nonnodulated, NO(3)-fed and nodulated, N(2)-fed plants of white lupin (Lupinus albus L.). The NO(3) regime used (5 millimolar NO(3)) promoted closely similar rates of growth and N assimilation as in the symbiotic plants. Over 90% of the N absorbed by the NO(3)-fed plants was judged to be reduced in roots. Empirically based models of C and N flow demonstrated that patterns of incorporation of C and N into dry matter and exchange of C and N among plant parts were essentially similar in the two forms of nutrition. NO(3)-fed and N(2)-fed plants transported similar types and proportions of organic solutes in xylem and phloem. Withdrawal of NO(3) supply from NO(3)-fed plants led to substantial changes in assimilate partitioning, particularly in increased translocation of N from shoot to root. Nodulated plants showed a lower (57%) conversion of C or net photosynthate to dry matter than did NO(3)-fed plants (69%), and their stems were only half as effective as those of NO(3)-fed plants in xylem to phloem transfer of N supplied from the root. Below-ground parts of symbiotic plants consumed a larger share (58%) of the plants' net photosynthate than did NO(3)-fed roots (50%), thus reflecting a higher CO(2) loss per unit of N assimilated (10.2 milligrams C/milligram N) by the nodulated root than by the root of the NO(3)-fed plant (8.1 milligrams C/milligram N). Theoretical considerations indicated that the greater CO(2) output of the nodulated root involved a slightly greater expenditure for N(2) than for NO(3) assimilation, a small extra cost due to growth and maintenance of nodule tissue, and a considerably greater nonassimilatory component of respiration in root tissue of the symbiotic plant than in the root of the NO(3)-fed plant. PMID- 16661098 TI - Composition of Lipid-derived Polymers from Different Anatomical Regions of Several Plant Species. AB - The composition of the aliphatics of the protective cuticular polymers from different anatomical regions from several plant species was determined by combined gas-liquid chromatography and mass spectrometry of the depolymerization products derived from the polymers. The polymer from the aerial parts of Vicia faba showed similar composition; dihydroxypalmitic acid was the major (>85%) component of the cutin covering leaves, petioles, flower petals and stem with smaller amounts of palmitic acid and omega-hydroxy palmitic acid. On the other hand, the chief components of the polymer from the tap root were omega-hydroxy C(16:0) and C(18:1) acids and/or the corresponding dicarboxylic acids. The positional isomer composition of the dihydroxy C(16) acids was shown to be dependent upon anatomical location, developmental stage, and light. Apple cutin from rapidly expanding organs (flower petal and stigma) was shown to contain predominately C(16) family acids whereas the C(18) family dominated in cutin of slower growing organs (leaf and fruit). The composition of the aliphatic components of cutin found in the seed coats of pea, corn, barley, and lettuce was found to be similar to that of the cuticular polymer of the leaves in each species. PMID- 16661099 TI - Rapid growth responses of Avena coleoptile segments to lanthanum and other cations. AB - The rapid growth responses of oat (var. Victory) coleoptile segments treated with millimolar concentrations of the chlorides of La(3+), Ca(2+), K(+), and NH(4) (+), respectively, have been measured. La(3+) and Ca(2+) initially depressed the endogenous elongation rate. In the case of La(3+) a prolonged stimulatory effect on the rate of elongation was produced by concentrations of 50 millimolar down to 20 micromolar after an initial depression of elongation rate. The effect of K(+) was slightly stimulatory and showed a synergistic effect in combination with La(3+). NH(4) (+) produced an immediate rapid increase in elongation rate. La(3+) did not behave as a "super calcium" in its action upon the spontaneous growth response. The prolonged elongation of the La(3+)-treated segments exhibiting the spontaneous growth response is apparently a newly observed effect. These rapid growth responses are interpreted as an interaction between anionic lipid-protein complexes in the plasmalemma and the respective ions. PMID- 16661100 TI - Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L. AB - Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg(2+) and thiamine pyrophosphate.Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants. PMID- 16661101 TI - Polyribosomes from Pear Fruit: Changes during Ripening and Senescence. AB - Polysome profiles were examined from lyophilized peel tissue of ripening pear (Pyrus communis, L. var. Passe-Crassane). Messenger RNA chains bearing up to eight ribosomes (octamers) were resolved and exhibited the highest absorption peak when ribonuclease activity was eliminated during extraction. Neither normal ripening nor the increase of large polyribosomes that normally accompanies ripening and senescence of the fruit occurred when pretreatment at 0 C was omitted. Normal ripening and increase of large polyribosomes would, however, be initiated by an ethylene treatment. The size distribution of the polyribosomes remained essentially constant throughout a 4-month cold storage; there was, however, a large increase in ribosomes by the 12th week of storage. PMID- 16661102 TI - Stress and Protein Turnover in Lemna minor. AB - Transfer of fronds of Lemna minor L. to adverse growth conditions or stress situations causes a lowering of the growth rate and a loss of soluble protein per frond, the extent of the loss being dependent on the nature of the stress. The loss or protein is due to two factors: (a) a decrease in the rate constant of protein synthesis (ks); (b) an increase in the rate constant of protein degradation (kd). In plants adapted to the stresses, protein synthesis increases and the initially rapid rate of proteolysis is reduced. Addition of abscisic acid both lowers ks and increases kd, whereas benzyladenine seems to alleviate the effects of stress on protein content by decreasing kd rather than by altering ks. Based on the measurement of enzyme activities, stress-induced protein degradation appears to be a general phenomenon, affecting many soluble proteins. The adaptive significance of stress-induced proteolysis is discussed. PMID- 16661103 TI - Isolation and partial characterization of vacuoles from tobacco protoplasts. AB - Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5'-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted. PMID- 16661104 TI - Characterization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.). AB - The peripheral root cap cells of corn (cv. SX-17A) secrete a fucose-rich, high molecular weight, polysaccharide slime via the dictyosome pathway. To study the synthesis of this polysaccharide, a technique for isolating and assaying GDP fucose:polysaccharide fucosyl transferase activity was developed. Corn roots were excised from germinated seeds, incubated 12 hours at 10 C in water, and ground in 100 millimolar Tris or Pipes buffer (pH 7.0) with or without 0.5 molar sucrose. The membrane-bound enzyme was solubilized by sonication in the presence of 2 molar urea and 1.5% (v/v) Triton X-100 and assayed by monitoring the incorporation of GDP-[(14)C]fucose into endogenous acceptors. Optimum enzyme activity is expressed at pH 7.0 and 30 C in the presence of 0.8% (v/v) Triton X 100. The enzyme does not require divalent cations for activation and is inhibited by concentrations of MnCl(2) or MgCl(2) greater than 1 millimolar. Corn root cap slime will serve as an exogenous acceptor for the enzyme if it is first hydrolyzed in 5 millimolar trifluoroacetic acid for 60 minutes at 18 pounds per square inch, 121 C. This procedure prepares the acceptor by removing terminal fucose residues from the slime molecule. Kinetics of fucose release during hydrolysis of native slime and in vitro synthesized product suggests that the two polymers possess similar linkages to fucose. PMID- 16661105 TI - Intracellular Localization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.). AB - The intracellular site of synthesis of the fucose-rich polysaccharide slime secreted by corn roots was localized by monitoring the distribution of GDP fucose:polysaccharide fucosyl transferase activity in subcellular fractions of corn roots. Root tip sections were chopped in the presence of 0.56 molar sucrose and 100 millimolar Tris (pH 7.0). After a brief centrifugation, the homogenate was applied to a Sepharose 4B column (1.5 x 30 cm). The turbid, particulate portion of the supernatant fraction eluted at the void volume. Ninety per cent of the enzyme activity was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nanometer absorbance, electron microscope observation, and distributions of NADH-cytochrome c oxidoreductase and fucosyl transferase activities.TWO PEAKS OF FUCOSYL TRANSFERASE ACTIVITY WERE OBSERVED: one in fractions rich in smooth endoplasmic reticulum, and another in fractions rich in dictyosome cisternae and dictyosome vesicles. The data suggest that the synthesis of corn root slime begins in the endoplasmic reticulum and is completed in the dictyosomes prior to secretion; however, the possibility that more than one fucosyl transferase exists in corn roots is discusses. PMID- 16661106 TI - Diurnal Ion Fluctuations in the Mesophyll Tissue of the Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum. AB - Both laboratory- and field-grown Mesembryanthemum crystallinum plants exhibited large scale diurnal ion fluctuations. In mesophyll tissue, potassium and sodium levels varied in conjunction with acid levels while chloride levels varied in opposition. Thus, dark CO(2) fixation in this Crassulacean acid metabolism species seems analogous to the common plant process of malate synthesis to balance cation surplus. Sodium levels in the epidermis appeared to fluctuate in opposition to those in the mesophyll. It is proposed that inorganic cations cycle between mesophyll and epidermal tissue to balance malate accumulation and to produce stomatal opening in the dark. PMID- 16661107 TI - Glycerolipid and Fatty Acid Changes in Eastern White Pine Chloroplast Lamellae during the Onset of Winter. AB - Chloroplast lamellae of eastern white pine (Pinus strobus L.) were analyzed to determine changes in total glycerolipids, component glycerolipids, and glycerolipid fatty acids during the onset of winter hardiness. Samples were collected in September, November, and December when the average daily temperature varied between 23 and -10 C. Before November 2, phospholipids decreased 40 to 85%, glycolipids only 30%. Analysis of individual glycerolipids showed that glycerolipids containing 18:3 fatty acid were retained at the expense of glycerolipids esterified with saturated (16:0 and 18:0) and monounsaturated (18:1) fatty acids.Between mid-November and December, the total quantity of lamellar glycerolipids recovered to the September level. Increases in digalactosyl diglyceride and in 18:3 characterized the recovery period. High lamellar unsaturation achieved by mid-November appeared to be maintained during recovery through preferential incorporation of glycerolipids containing 18:3 (monogalactosyl diglyceride, digalactosyl diglyceride, phosphatidylglycerol, and phosphatidylcholine).These results suggest that eastern white pine chloroplasts maintain lamellar viscosity by increasing lamellar unsaturation and tolerate freeze desiccation by increasing the interfacial water-binding capacity of the lamellae. PMID- 16661108 TI - Net Photosynthesis and Early Growth Trends of a Dominant White Oak (Quercus alba L.). AB - Examination of the relationship between photosynthesis and growth of a dominant white oak (Quercus alba L.) tree has shown that most growth processes were either completed or well underway before the establishment of significant positive rates of net photosynthesis. Growth was initiated first in the root system (March 3), followed by stem cambial growth (March 26) and later by flower, leaf, and branch growth (April 10). During the period of rapid leaf and branch growth, root and cambial growth ceased and then resumed as the leaves approached maturity. The rapid rate of leaf maturation, the early appearance of positive rates of net photosynthesis in leaves (15% of final size) and the CO(2)-refixing capability of elongating branch tissue reduced the period of time that this white oak tree was dependent on stored reserves. Lower temperature optima and compensation points in developing leaves and stems indicated that the growth-temperature response was optimized for the lower seasonal temperatures observed during the spring. This temperature adaptation further reduced the time that this tree was dependent on stored reserves. PMID- 16661109 TI - A Strain of Rosa damascena Cultured Cells Resistant to Ultraviolet Light. AB - A strain of cultured cells of Rosa damascena Mill. which showed unusual resistance to damage by short wave (254 nanometers) ultraviolet radiation was isolated. The resistant cells were 2.2 to 2.8 times larger and had about twice the amount of DNA and more chromosomes than the parental, sensitive cells. The resistant cells also produced larger quantities of polyphenolic compounds, principally flavonoids, during the later phases of culture growth. At 10 days, resistant cells had 4 times more nonflavonoid polyphenolics and 14 times more flavonoids than parental cells. The resistance, which was also observed only in the later phases of culture growth, was best correlated with the production of polyphenolics, which apparently shielded ultraviolet-sensitive target molecules from damage. PMID- 16661110 TI - Cold storage of isolated class C chloroplasts: optimal conditions for stabilization of photosynthetic activities. AB - Preservation of photosynthetic activities (photophosphorylation, electron transport, fluorescence induction, 0.3-second delayed light emission) of isolated broken (class C) chloroplasts by low temperature storage was investigated under a wide range of conditions in order to optimize long time activity retention.The more labile functions (photophosphorylation and electron transport) required very low temperatures (below -79 C) and relatively high (above 20%, v/v) concentrations of cryoprotectives for satisfactory stabilization. Fluorescence induction and delayed light emission were less sensitive, especially during the 1st month of storage.Taking into account the effect of cryoprotectives on absolute activities prior to freezing, optimum activity retention was observed with a medium containing ethylene glycol (30%, v/v) and a storage temperature of 100 C or below. In this case, given fast thawing and high chloroplast concentration, practically 100% preservation of all of the photosynthetic activities investigated was obtained for at least 10 months, even with very simple freezing and storage procedures.The same optimal medium at somewhat higher temperatures (-79 C and to a lesser extent at -41 C) caused a dramatic uncoupling effect: photophosphorylation was inhibited in a few hours, while electron transport increased 3- to 5-fold. The enhanced electron transport was stable for almost a month and then declined sharply. This uncoupling effect was specific only to ethylene glycol. PMID- 16661111 TI - Swelling and contraction of potato mitochondria. AB - Mitochondria isolated from potato tubers fail to undergo passive osmotic swelling when suspended in isotonic Na(+) acetate or phosphate, in NaCl following addition of tripropyltin, or in Na(+) nitrate following addition of an uncoupler. Swelling under each of these conditions in mitochondria from other sources has been attributed to the inward movement of Na(+) on an endogenous Na(+)/H(+) exchanger. Such a monovalent cation/H(+) exchanger has also been implicated in respiration dependent cation extrusion and contraction of swollen mitochondria. Potato mitochondria swollen in chloride and nitrate salts extrude ions and contract when respiration is initiated. The contraction reaction is slower and less efficient than that in beef heart mitochondria, but like the latter, is sensitive to uncouplers and stimulated by nigericin, butacaine, and Mg(2+). These comparative studies suggest that a cation(+)/H(+) exchanger is present in potato tuber mitochondria, but that it functions exclusively as a cation-extruding mechanism. They further suggest that cation(+)/H(+) exchange activity is not identical in mitochondria from different sources and that these exchange components may have a directionality and regulatory features which differ with the metabolic needs of the source tissue. PMID- 16661112 TI - Transport of divalent cations: cation exchange capacity of intact xylem vessels. AB - The cation exchange capacity of the intact xylem vessels in cut shoots of papyrus (Cyperus papyrus spec.) has been determined. The cation exchange capacity is independent of the cation concentration in the transpiration stream, and is equal for Ca and Co. The high value of the cation exchange capacity (0.6 to 1 x 10(-7) equivalents per square centimeter vessel wall surface) leads to the hypothesis that the porous structure of the vessel wall, and not only the inner vessel wall surface, acts as a cation exchanger.Differences between anion ([(32)P]phosphate, [(45)Ca]EDTA(2-), [(115)Cd(m)]-EDTA(2-)), and cation ([(45)Ca](2+), [(115)Cd(m)](2+)) movement are explained in terms of transport with the transpiration flux or by exchange reactions. The competition between exchange sites and natural or synthetic ligands for the divalent cations is discussed. PMID- 16661113 TI - Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential. AB - The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (+/- standard deviation) for unplasmolyzed tobacco cells was -52 +/- 12 millivolts, for cells in 0.3 molar mannitol, -50 +/- 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, -49 +/- 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was -49 +/- 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells. PMID- 16661114 TI - Biochemical Specialization of Photosynthetic Cell Layers and Carbon Flow Paths in Suaeda monoica. AB - Suaeda monoica Frossk. ex J. F. Gmel is a C(4) plant with three different photosynthesizing cell layers. The outer chlorenchymatous layer shows a high activity of phosphoenolpyruvate (PEP) carboxylase but none of ribulose bisphosphate (RuBP) carboxylase. The electrophoretic protein band of RuBP carboxylase was missing in this layer. The second chlorenchymatous cells layer shows a very high activity of RuBP carboxylase and NAD malic enzyme and only traces of activity of PEP carboxylase. The third photosynthesizing cell type is comprised of the water tissue. It has moderate activities of RuBP carboxylase and PEP carboxylase. A model for carbon flow in Suaeda monoica leaves is proposed. PMID- 16661115 TI - Crystal morphology and carbon/carbon composition of solid oxalate in cacti. AB - Morphology, crystal structure, and carbon isotopic composition of calcium oxalate from representative species from the family Cactaceae were determined using scanning electron microscopy, x-ray diffraction, and isotope ratio mass spectrometry. Crystals from one species in the Opuntieae tribe of the Cactaceae were druses with acute points composed of the monohydrate form of calcium oxalate (whewellite). Crystals from three species in the Cereeae tribe were the dihydrate form of calcium oxalate (weddellite) forming druses made up of tetragonal and isodiametric crystallites. The oxalate was relatively enriched in (13)C isotope ( 7.3 to - 8.7 per thousand) compared with woody fibers (-13.3 to 14.1 per thousand) from the same plants. PMID- 16661116 TI - [C]Ethylene Metabolism during Leaf Abscission in Cotton. AB - Changes in (14)C(2)H(4) metabolism in the abscission zone were monitored during cotton (cv. Deltapine 16) leaf abscission. Rates of (14)C(2)H(4) oxidation to (14)CO(2) and tissue incorporation in abscission zone segments cut from the second true leaf of nonabscising leaves of intact plants were similar (about 200 disintegrations per minute per 0.1 gram dry weight per 5.5 hours) and relatively constant over a 5-day period. Deblading to induce abscission caused a dramatic rise in (14)C(2)H(4) oxidation, but tissue incorporation was not markedly affected. This rise occurred well before abscission, reaching a peak of 1,375 disintegrations per minute per 0.1 gram dry weight per 5.5 hours 2 days after deblading when abscission was 40%. The rate then gradually declined, but on day 5 when abscission reached completion, it was still nearly three times higher than in segments from nonabscising leaves. Application of 0.1 millimolar abscisic acid in lanolin to the debladed petiole ends increased the per cent abscission slightly and initially stimulated (14)C(2)H(4) oxidation. In contrast, naphthaleneacetic acid applied in a similar manner delayed and markedly inhibited both abscission and (14)C(2)H(4) oxidation.Petiole segments cut 1 centimeter from the abscission zone of intact second true leaves also incorporated and oxidized (14)C(2)H(4) to (14)CO(2) but at rates two and six times higher, respectively, than that of comparable adjacent abscission zone segments. However, in marked contrast to the abscission zone segments, no changes in oxidation were observed when the leaves were debladed to induce abscission.THESE RESULTS DEMONSTRATE THAT: (a) prior to abscission, the ethylene oxidation, but not the tissue incorporation pathway, rapidly increases in the abscission zone; (b) this increase does not occur in adjacent petiole tissue; and (c) changes in the rate of oxidation and per cent abscission brought about by hormone treatments parallel one another. The possible significance of these changes in ethylene metabolism is discussed with respect to the hypothesis that ethylene action and metabolism are directly related. PMID- 16661117 TI - Germination of soybean embryonic axes: nucleotide sugar metabolism and initiation of growth. AB - UDP-Sugars comprise the dominant class of nucleotide sugars in isolated soybean axes during early germination. While "dry" axes contain 1 nanomole per axis of UDP-sugars, further synthesis is initiated upon imbibition such that the concentration of total UDP-sugars reaches 8 nanomoles per axis or roughly 1 millimolar after 10 hours, when the axes begin to elongate. The GDP-sugars are essentially absent before imbibition, accumulate rapidly for 90 min to 173 picomoles per axis, then decrease somewhat, reattaining the earlier peak level shortly before growth begins. Meanwhile, the level of ADP-sugars is unchanged. These data indicate that the 10-hour lag period preceding axis growth does not result from a diminished ability to synthesize a major category of nucleotide sugars.Relative rates of synthesis of individual UDP- and GDP-sugars were determined by incorporation of [(3)H]uridine or [(3)H]guanosine. The distribution of label in the different classes of UDP-sugars and in the single class of GDP sugar was quantitatively similar when analyzed before, at the onset, or during early growth. It therefore seems unlikely that synthesis of a key nucleotide sugar controls the initiation of growth.The possible relevance of nucleotide sugars to growth is discussed and new methods for enzymic analysis of picomole levels of nucleotide sugars are described. PMID- 16661118 TI - Photoinhibition of intact attached leaves of c(3) plants illuminated in the absence of both carbon dioxide and of photorespiration. AB - When leaflets of bean and leaves of other species of C(3) plants are illuminated in the absence of CO(2) and at low O(2) partial pressure, the capacity for CO(2) assimilation at saturating light and its efficiency at low light intensities are inhibited. This photoinhibition is dependent on leaflet age and period of illumination. In young leaflets and following short exposure to these photoinhibitory conditions, some recovery of CO(2) assimilation capacity is observed immediately after treatment. Following substantial (70 to 80%) photoinhibition of CO(2) assimilation, recovery in fully expanded leaflets is observed only after 48 hours in normal air. The photoinhibition is largely prevented by providing CO(2) at partial pressures equivalent to the CO(2) compensation point, or by >210 millibars O(2) which permits internal CO(2) production by photorespiration. If leaflets are illuminated in 60 microbars CO(2) and 210 millibars O(2) (the CO(2) compensation point in air), no photoinhibition is observed. Electron transport processes and fluorescence emission associated with photosystem II are inhibited in chloroplast thylakoids isolated from leaflets after illumination in zero CO(2) and 10 millibars O(2). These studies support the hypothesis that CO(2) recycling through photorespiration is one means of effectively dissipating excess photochemical energy when CO(2) supply to illuminated leaves is limited. PMID- 16661119 TI - Responses of halophytes to high salinities and low water potentials. AB - The effects of nonsaline polyethylene glycol (PEG)-6000 and saline seawater solutions of comparable osmotic potential on the concentrations of organic solutes and inorganic ions in the tissues of halophytes (Plantago maritima L., Triglochin maritima L., Limonium vulgare Mill., Halimione portulacoides (L.) Aell) have been investigated. Studies were made to determine whether high salinities induce specific ion effects that are absent in plants grown in nonsaline solutions of comparable osmotic potential. Over-all, the responses of each species to the two different treatments (seawater or PEG) are similar; the accumulation of organic solutes (compatible osmotica) in tissues is primarily correlated with a decrease in the osmotic potential of culture solutions. Depending on the species, sorbitol, proline, reducing sugars, quaternary ammonium compounds, and alpha-amino nitrogen accumulate in tissues as the water potential of the tissues falls. Within a species there are differences in the concentrations of inorganic ions and organic solutes between roots and shoots of plants grown at high salinities or at high concentrations of PEG. PMID- 16661120 TI - Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L. AB - THE PRESENT STUDY COMPARES THE BINDING AND INHIBITORY ACTIVITY OF TWO PHOTOSYSTEM II INHIBITORS: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2 chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H(2)O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding ((14)C radiolabel assays) with binding constants (K) of 1.4 x 10(-8) molar and 4 x 10( 8) molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 x 10(-8) molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.We conclude that triazine resistance of both intact plants and isolated chloroplasts of Senecio vulgaris L. is based upon a minor modification of the protein in the photosystem II complex which is responsible for herbicide binding. This change results in a specific loss of atrazine (triazine)-binding capacity. PMID- 16661121 TI - Membrane potentials of vallisneria leaf cells and their relation to photosynthesis. AB - A study has been made of the effects of the inhibitors carbonylcyanide m chlorophenylhydrazone (CCCP), 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), and of anoxia on the light-sensitive membrane potential of Vallisneria leaf cells. The present results are compared with the known effects of these inhibitors on ion transport and photosynthesis (Prins 1974 Ph.D thesis). The membrane potential is composed of a diffusion potential plus an electrogenic component. The electrogenic potential is about -13 millivolts in the dark and -80 millivolts in the light. The inhibitory effect of DCMU and CCCP on the electrogenic mechanisms strongly depends on the light intensity used, the inhibition being less at a higher light intensity. This is of significance in view of the often conflicting results obtained with these inhibitors. With ion transport in Vallisneria the electrogenic pump derives its energy from phosphorylation; however, the process which causes the initial light-induced hyperpolarization and the process that keeps the membrane potential at a steady hyperpolarized state in the light have different energy requirements. The action of photosystem I alone is sufficient to induce the initial hyperpolarization. For continuous operation in the light the activity of photosystem II also is needed. PMID- 16661122 TI - Indoleacetaldehyde Reductase of Cucumis sativus L: KINETIC PROPERTIES AND ROLE IN AUXIN BIOSYNTHESIS. AB - Indoleacetaldehyde reductase catalyzes the conversion of indoleacetaldehyde to indole ethanol in extracts of Cucumis sativus L., with reduced pyridine nucleotide required as co-substrate. NADH and NADPH result in markedly different enzyme behavior, as reflected in reaction kinetics and in responses to inhibitors and activators. It is not yet clear whether there are two separate enzymes, one specific for NADH and the other for NADPH, or whether there is a single enzyme differentially influenced by the two co-substrates.In the presence of NADH, the indoleacetaldehyde reductase activity was inhibited by NaCl and displayed hyperbolic kinetics under all conditions tested. However, in the presence of NADPH the enzyme was activated by NaCl at concentrations up to 0.1 molar. Under certain conditions with NADPH as co-substrate, the enzyme showed kinetics sigmoidal with respect to indoleacetaldehyde concentration and was strongly inhibited by high concentrations of NADPH. It is possible that this substrate inhibition of the NADPH-linked indoleacetaldehyde reductase activity by NADPH, as well as the sigmoidicity with respect to indoleacetaldehyde concentration, may function in the regulation of auxin biosynthesis. PMID- 16661123 TI - Limiting Factors in Photosynthesis: I. USE OF IRON STRESS TO CONTROL PHOTOCHEMICAL CAPACITY IN VIVO. AB - The possibility of using Fe stress as an experimental tool in the study of limiting factors was explored. Results show that Fe stress decreased the chlorophyll (Chl) a, Chl b, carotene, and xanthophyll content of leaves of sugar beets (Beta vulgaris L.) and that the maximum rate of photosynthetic CO(2) uptake (P(max)) per unit area was linearly related to Chl (a + b) per unit area. Measurements of noncyclic ATP formation by isolated chloroplasts at light saturation indicate that photosynthetic electron transport capacity decreased concomitantly with pigment content under Fe stress.Iron stress decreased Chl per chloroplast but had no effect on the number of leaf cells per unit area, average leaf cell volume, number of chloroplasts per unit area, or leaf soluble protein per unit area. Average chloroplast volume, protein N per chloroplast, and ribulose bisphosphate carboxylase activity were diminished by Fe stress but to a lesser extent than Chl per chloroplast. The reduction in pigment concentration with Fe stress led to a relatively small decrease in light absorption, the fraction of incident light absorbed remaining high (49%) even at very low leaf Chl contents. There was no apparent change in the quantum yield of attached leaves at low irradiances, but at high irradiances, the capacity to convert absorbed light to chemical energy was greatly diminished in Fe-stressed leaves.THE RESULTS SUGGEST: (a) that P(max) per unit area are decreased linearly with Chl per unit area because of a decrease in photochemical capacity rather than a change in light absorption; and (b) that the effect of Fe stress may be sufficiently specific for it to be used as an experimental tool for the control and study of photochemical capacity in vivo. PMID- 16661124 TI - Limiting Factors in Photosynthesis: II. IRON STRESS DIMINISHES PHOTOCHEMICAL CAPACITY BY REDUCING THE NUMBER OF PHOTOSYNTHETIC UNITS. AB - It has been proposed that Fe stress may be used in the study of limiting factors in photosynthesis as an experimental means of varying photochemical capacity in vivo (Plant Physiol 1980 65: 114-120). In this paper the effect of Fe stress on photosynthetic unit number, size, and composition was investigated by measuring P(700), cytochrome (Cyt) f, chlorophyll (Chl) a, and Chl b in sugar beet leaves. The results show that when Fe stress reduced Chl per unit area by 80% (from 60 to 12 micrograms per square centimeter), it decreased the number of P(700) molecules per unit area by 88% and Cyt f per unit area by 86%; over the same range the Chl to P(700) ratio increased by 37% but there was no significant change in the Chl to Cyt f ratio. These data suggest that Fe stress decreases photochemical capacity and Chl per unit area by diminishing the number of photosynthetic units per unit leaf area.The ratio of Chl a to Chl b did not change with Fe stress. This suggests that the proportion of light-harvesting Chl a/b-protein complex within the photosynthetic unit remained constant. Electron microscopy of chloroplasts revealed that the decrease in the number of photosynthetic units which occurred during Fe stress was accompanied by a reduction in the number of granal and stromal lamellae per chloroplast and by a reduction in the number of thylakoids per granum. PMID- 16661125 TI - Biosynthesis of delta-Aminolevulinic Acid from Glutamate in Agmenellum quadruplicatum. AB - delta-Aminolevulinic acid accumulated in the culture medium when Agmenellum quadruplicatum strain PR-6 was incubated in the presence of levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, and specifically labeled glutamate and glycine. The delta-aminolevulinic acid was purified using Dowex 50W-X8 and cleaved by periodate to yield succinic acid and formaldehyde. The distribution of radioactivity in the two fragments suggested that in blue green algae the carbon skeleton of delta-aminolevulinic acid is derived directly from glutamate. However the possibility of the pathway of delta-aminolevulinic acid synthesis, from glycine and succinyl-coenzyme A also functioning in blue green algae was not eliminated as uptake of glycine was minimal. PMID- 16661126 TI - Calcium is Necessary for Motility in the Diatom Amphora coffeaeformis. AB - The marine diatom Amphora coffeaeformis required Ca(2+) and bicarbonate for motility. Movement was inhibited by the Ca(2+)-blocking agents ruthenium red and alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-alpha- aminopropyl]-3,4,5 trimethoxy phenyl acetonitrile and the metabolic energy uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. 3-(3',4-Dichlorophenyl)-1,1-Dimethyl urea was without effect on cells at a concentration that prevented O(2) production in the light. Although Sr(2+) could replace Ca(2+) in the attachment of cells to glass, it did not substitute for Ca(2+) in motility. PMID- 16661127 TI - Stimulation of Endomitotic DNA Synthesis and Cell Elongation by Gibberellic Acid in Epicotyls Grown from Gamma-irradiated Pea Seeds. AB - Large doses of gamma-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced.Application of gibberellic acid restores the endomitotic DNA synthesis and the cell elongation in epicotyls of irradiated seeds. The cortex cells reach again the 8 C DNA level in darkness.The results suggest that gamma-irradiation blocks endomitotic DNA synthesis and cell elongation by lowering the concentration of endogenous gibberellins. PMID- 16661128 TI - Importance of Aerodynamic Resistance to Water Use Efficiency in Three Conifers under Field Conditions. AB - The quantitative importance of aerodynamic resistance to H(2)O vapor and CO(2) exchange was determined for shoots from saplings of three conifers (Abies lasiocarpa [Hook] Nutt., Pinus contorta Dougl., Juniperus communis L.) under natural conditions in the field. A combination of relatively low stomatal resistances (<300 seconds per centimeter) and low wind speeds (<30 centimeters per second) led to substantial contributions of the aerodynamic resistance (R(wv) (a)) to water use efficiency (WUE = photosynthesis/transpiration) for all three species. For A. lasiocarpa, transpiration was calculated to be 44% less and photosynthesis 17% less due to the presence of R(wv) (a), which led to a predicted increase in WUE of 57% compared to the calculated WUE when R(wv) (a) was assumed negligible. Similar increases in WUE were computed for P. contorta (48%) with somewhat smaller values for J. communis (34%). These results are discussed in terms of the estimated importance of R(wv) (a) on water and photosynthetic relations of plants that have relatively low stomatal resistances and grow in microhabitats with low winds. PMID- 16661129 TI - Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS. AB - The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K(m) and V(max) of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg(2+), with complete inhibition by 2 micromolar HgCl(2). Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and alpha-naphthaleneacetic acid but not by beta-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl(2) and 200 millimolar mannitol) also blocked the auxin effect on invertase loss. PMID- 16661130 TI - Characteristics of Nitrate Reductase-inactivating Proteins Obtained from Corn Roots and Rice Cell Cultures. AB - Nitrate reductase (NR)-inactivating proteins from corn roots (Wf-9 x 38-11) and rice cell suspension cultures were tested against a partially purified NR obtained from corn leaves (W64A x W182E). The corn protein was purified 921-fold and the rice protein, 1,660-fold using standard purification procedures. Approximate molecular weight values were 75,000 for the corn protein, and 150,000 for the rice protein as determined by Sephadex G-100 gel filtration. The Sephadex treated proteins were characterized by electrophoresis on polyacrylamide gels. With a running pH of 9.4 the corn protein remained at the origin whereas the rice protein migrated with an R(F) value of 0.49. With a running pH of 4.0 the corn protein migrated with an R(F) value of 0.25. With the corn protein the activities of NR inactivation and hydrolysis of azocasein were detected in the same protein band. The rice protein, however, had no associated protease activity. From sodium dodecyl sulfate gel electrophoresis, there was one major protein band with an estimated molecular weight of 66,000 in corn protein. In rice protein four bands were observed with estimated molecular weights of 73,000, 66,000, 62,500, and 58,500, respectively.Both inactivators had an inhibitory effect on NADH-NR and NO(3) (-) induced NADH-cytochrome c reductase activities but they had less influence on the activities of FMNH(2)-NR and reduced methylviologen-NR. Inactivation of rice cell NR by rice inactivator was reversed by addition of NADH. Inactivation of corn leaf NR by rice inactivator was inhibited by the simultaneous addition of NADH, but rice inactivator-inactivated corn leaf NR could not be reactivated by NADH. PMID- 16661131 TI - Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris. AB - When nitrate reductase (NR) purified from Chlorella was incubated with NR inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R(F) value of 0.12 to an R(F) of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R(F) value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R(F) value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV NR activity or mobility suggesting that the rice protein binds to Chlorella NR. PMID- 16661132 TI - Cysteine metabolism in cultured tobacco cells. AB - Transported l-[(35)S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The (35)S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[(35)S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the (35)S label present as compounds other than cysteine.The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH(4) (+). Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K(m) to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria. PMID- 16661133 TI - Photosynthesis in Grass Species Differing in Carbon Dioxide Fixation Pathways: III. OXYGEN RESPONSE AND ENZYME ACTIVITIES OF SPECIES IN THE LAXA GROUP OF PANICUM. AB - Measurements of CO(2) exchange at varying O(2) concentrations in seven grass species of the Laxa group of Panicum and activities of five photosynthetic enzymes were compared to values obtained for these characters in a cool season C(3) grass, tall fescue (Festuca arundinacea Schreb.) and a C(4) grass, P. maximum Jacq. Plants were divided into three groups on the basis of the inhibition of apparent photosynthesis by 21% O(2.) Rates of apparent photosynthesis in P. prionitis Griseb. and P. maximum were virtually unaffected by changes in O(2) concentration. In another group consisting of P. hylaeicum Mez., P. rivulare Trin., P. laxum Sw., and tall fescue apparent photosynthesis was inhibited by 28.2 to 36.0% at 21% O(2.) An intermediate inhibition of 20.6 to 23.3% at 21% O(2) was exhibited by P. milioides Nees ex Trin., P. schenckii Hack., and P. decipiens Nees ex Trin. The CO(2) compensation concentration for P. prionitis and P. maximum was low ( ATPase. "Aged" or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO(3), which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or "aged" submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO(3) (-), but inhibited by Cl(-), indicating that Cl(-) stimulation is a distinguishing property of the pea mitochondrial ATPase complex. PMID- 16661175 TI - Nitrate uptake by roots as regulated by nitrate assimilation in the shoot of castor oil plants. AB - Ricinus communis was used to test the Ben Zioni-Dijkshoorn hypothesis that NO(3) uptake by roots can be regulated by NO(3) assimilation in the shoot. The rate of the anion charge from assimilated NO(3) (-) (and SO(4) (2-)) was followed in its distribution between organic acid anion accumulation and HCO(3) (-) efflux into the nutrient solution. In plants adequately supplied with NO(3) (-), HCO(3) (-) efflux accounted for between 56 and 63% of the anion charge. When the plants were subjected to a low NO(3) regime HCO(3) (-) excretion accounted for only 23% of the charge. A comparison of mature plants growing for a 10-day period at the two levels of NO(3) nutrition revealed that the uptake of NO(3) (-) at the higher level was increased 3-fold, whereas K uptake was unaltered. To trace ion movement within the plant, the ionic constituents of xylem and phloem sap were determined. In xylem sap these constituents were found to be predominantly K(+), Ca(2+), and NO(3) (-), whereas in the phloem sap they were mainly K(+) and organic acid anions. Results have been obtained which may be interpreted as providing direct evidence of NO(3) uptake by roots regulated by NO(3) reduction in the tops, the process being facilitated by the recirculation of K(+) in the plant. PMID- 16661176 TI - Effects of phaseic Acid and dihydrophaseic Acid on stomata and the photosynthetic apparatus. AB - Plant extracts containing phaseic acid (PA), as well as solutions of purified PA and dihydrophaseic acid (DPA) were applied to leaves, isolated mesophyll cells, and isolated epidermal strips. In Commelina communis, stomatal closure began 4 minutes after the addition of either 20 micromolar (+/-)-abscisic acid or 10 micromolar PA. Stomata closed less rapidly after treatment with 10 micromolar PA than after treatment with 10 micromolar (+/-)-abscisic acid in Amaranthus powelli, Hordeum vulgare, Xanthium strumarium, and Zea mays and did not respond at all to PA in Vicia faba. DPA (10 micromolar) did not cause stomatal closure in any species.Plant extracts containing PA reduced photosynthesis, as reported by Kriedemann et al. (Aust J Plant Physiol 2: 553-567, 1975). Subsequent experiments with PA purified by crystallization and with residues of solvents employed in the extraction of PA proved that it was not PA that impaired photosynthetic O(2) evolution or CO(2) uptake but unidentified contaminants of the allegedly pure solvents. PMID- 16661177 TI - Cryoprotection by glucose, sucrose, and raffinose to chloroplast thylakoids. AB - Differential cryoprotection is afforded to chloroplast thylakoids against freeze induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions. PMID- 16661178 TI - Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY. AB - Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro. PMID- 16661179 TI - Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO. AB - Nuclei free of RNase activity were isolated from 3-hour-imbibed wheat (var. Yamhill) embryos by centrifugation through a discontinuous gradient of Percoll. The maximum rate of RNA synthesis observed in these nuclei was approximately 5 picomoles [(3)H]UTP per milligram DNA per minute. Two monovalent cation optima were found when measured in the presence of 15 millimolar MgCl(2) or 2 millimolar MnCl(2); 15 and 75 millimolar (NH(4))(2)SO(4). At the high monovalent cation optimum, the rate of RNA synthesis was linear for the first 10 to 15 minutes of incubation and still increasing after 3 hours. RNA synthesized in vitro (30 minute pulse followed by a 30-minute chase) showed distinct 18 and 26S RNA peaks comprising 13 and 17% of the total radioactivity, respectively. The over-all pattern of RNA synthesized in vitro was similar to the in vivo pattern. Approximately 40 to 50% of the RNA synthesized was inhibited by alpha-amanitin at 4 micrograms per milliliter. The newly synthesized 6 to 10S RNA appeared to be selectively inhibited by alpha-amanitin. PMID- 16661180 TI - Pyruvate dehydrogenase complex from germinating castor bean endosperm. AB - Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA. PMID- 16661181 TI - Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives. AB - Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2 imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl] N,N'-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3 methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a(680), and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides. PMID- 16661182 TI - Xylem Transport of 1-Aminocyclopropane-1-carboxylic Acid, an Ethylene Precursor, in Waterlogged Tomato Plants. AB - Waterlogging is known to cause an increase in ethylene synthesis in the shoot which results in petiole epinasty. Evidence has suggested that a signal is synthesized in the anaerobic roots and transported to the shoot where it stimulates ethylene synthesis. Experimental data are presented showing that 1 aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, serves as the signal. Xylem sap was collected from detopped tomato plants (Lycopersicon esculentum Mill. cv. VFN8). ACC in the sap was quantitated by a sensitive and specific assay, and its tentative chemical identity verified by paper chromatography. ACC levels in both roots and xylem sap increased markedly in response to waterlogging or root anaerobiosis. The appearance of ACC in the xylem sap of flooded plants preceded both the increase in ethylene production and epinastic growth, which were closely correlated. Plants flooded and then drained showed a rapid, simultaneous drop in ACC flux and ethylene synthesis rate. ACC supplied through the cut stem of tomato shoots at concentrations comparable to those found in xylem sap caused epinasty and increased ethylene production. These data indicate that ACC is synthesized in the anaerobic root and transported to the shoot where it is readily converted to ethylene. PMID- 16661183 TI - Stress-induced Ethylene Production in the Ethylene-requiring Tomato Mutant Diageotropica. AB - Ethylene synthesis in vegetative tissues is thought to be controlled by indoleacetic acid (IAA). However, ethylene synthesis in the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) was much less sensitive to IAA than in the normal variety (VFN8). Yet, mechanical wounding stimulated ethylene production by the mutant. The dgt tomato provides an opportunity to study the regulation of stress ethylene independent of IAA effects. Waterlogging (i.e. anaerobic stress) stimulated production of the ethylene precursor, 1 aminocyclopropane-1-carboxylic acid (ACC), in the roots. The ACC was transported to the shoot where it was converted to ethylene. The dgt mutant efficiently utilized ACC for ethylene synthesis under aerobic conditions. The results confirm that the genetic lesion in dgt is located at a step prior to the formation of ACC. Furthermore, induction of ethylene synthesis by anaerobic or mechanical stresses in this mutant is independent of IAA action. PMID- 16661184 TI - C(4) Acid Metabolism and Dark CO(2) Fixation in a Submersed Aquatic Macrophyte (Hydrilla verticillata). AB - The CO(2) compensation point of the submersed aquatic macrophyte Hydrilla verticillata varied from high (above 50 microliters per liter) to low (10 to 25 microliters per liter) values, depending on the growth conditions. Plants from the lake in winter or after incubation in an 11 C/9-hour photoperiod had high values, whereas summer plants or those incubated in a 27 C/14-hour photoperiod had low values. The plants with low CO(2) compensation points exhibited dark (14)CO(2) fixation rates that were up to 30% of the light fixation rates. This fixation reduced respiratory CO(2) loss, but did not result in a net uptake of CO(2) at night. The low compensation point plants also showed diurnal fluctuations in titratable acid, such as occur in Crassulacean acid metabolism plants. However, dark fixation and diurnal acid fluctuations were negligible in Hydrilla plants with high CO(2) compensation points.Exposure of the low compensation point plants to 20 micromolar (14)CO(2) resulted in 60% of the (14)C being incorporated into malate and aspartate, with only 16% in sugar phosphates. At a high CO(2) level, the C(4) acid label was decreased. A pulse-chase study indicated that the (14)C in malate, but not aspartate, decreased after a long (270-second) chase period; thus, the C(4) acid turnover was much slower than in C(4) plants.Phosphoenolpyruvate carboxylase activity was high (330 micromoles per milligram chlorophyll per hour), as compared to ribulose bisphosphate carboxylase (20 to 25), in the plants with low compensation points. These plants also had a pyruvate, Pi dikinase activity in the leaves of 41 micromoles per milligram chlorophyll per hour, which suggests they are not C(3) plants. NAD- and NADP(+) malate dehydrogenase activities were 6136 and 24.5 micromoles per milligram chlorophyll per hour, respectively. Of the three decarboxylating enzymes assayed, the activities of NAD- and NADP(+)-malic enzyme were 104.2 and 23.7 micromoles per milligram chlorophyll per hour, while phosphoenolpyruvate carboxykinase was only 0.2.Low compensation point Hydrilla plants fix some CO(2) into C(4) acids, which can be decarboxylated for later refixation, presumably into the Calvin cycle. Refixation would be advantageous in summer lake environments where the CO(2) levels are high at night but low during the day. Hydrilla does not fit any of the present photosynthetic categories, and may have to be placed into a new group, together with other submersed aquatic macrophytes that have environmentally variable CO(2) compensation points. PMID- 16661185 TI - Silver uptake, distribution, and effect on calcium, phosphorus, and sulfur uptake. AB - Bean, corn, and tomato plants were grown in a nutrient solution labeled with (32)P, (45)Ca, or (35)S and varying concentrations of AgNO(3). Following a 6-hour treatment period, plants were harvested and analyzed. A low Ag(+) concentration (50 nanomolar) inhibited the shoot uptake of the ions investigated. In the roots, Ca uptake increased whereas P and S uptake decreased.Autoradiograms of bean and corn plants, using (110m)Ag, showed that Ag(+) was uniformly deposited in the bean shoot, but corn shoots had regions of high activity along the leaf margins and at the tips where guttation had occurred. Roots were heavily labeled and shoots (especially the new growth) continued to accumulate Ag(+) even after the intact plant was returned to Ag-free solution. Silver was believed to be phloem mobile since it was exported from a treated leaf. Bean plants removed one-half the Ag(+) from 4 liters of nutrient solution containing 50 nanomolar AgNO(3) within 1.5 hours, but took 16 hours for 20 liters of solution. PMID- 16661186 TI - Mechanism of monocarpic senescence in rice. AB - During grain formation stage (90 to 110 days), the youngest flag leaf of rice (Oryza sativa L. cv. Jaya) remained metabolically most active (as indicated by cellular constituents and enzyme activities) and the third leaf the least active. At the grain development stage (110 to 120 days) the above pattern of age-related senescence of the flag leaf completely changed and it senesced at a faster rate than the second leaf which remained metabolically active even up to grain maturation time (120 to 130 days), when both the flag and the third leaf partially senesced. Removal of any leaf temporarily arrested senescence of the remaining attached leaves, that of flag leaf did not hasten senescence of the second leaf, while that of either the second or the third accelerated senescence of the flag. Removal of the inflorescence after emergence or foliar treatment of intact plant with kinetin equally delayed senescence and produced an age-related, sequential mode of senescence or leaves. Both translocation and retention of (32)P by the flag leaf were maximum at the time of grain formation and that by the second leaf was maintained even up to grain maturation time. The induction of senescence of the flag leaf was preceded by a plentiful transport of (32)P to the grains. Kinetin treatment decreased the transport of (32)P, prolonged its duration, and almost equally involved all of the leaves in this process. The pattern of senescence of isolated leaf tips was similar to that of attached leaves. The level of endogenous abscisic acid-like substance(s) maintained a close linearity with the senescence behavior of the leaves of intact and defruited plants during aging, and the rise in abscisic acid in the flag leaf was also preceded by higher (32)P transport to the grains. PMID- 16661187 TI - Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways: IV. ANALYSIS OF REDUCED OXYGEN RESPONSE IN PANICUM MILIOIDES AND PANICUM SCHENCKII. AB - Reduced photorespiration has been reported in Panicum milioides on the basis of lower CO(2) compensation concentrations than in C(3) species, lower CO(2) evolution in the light, and less response of apparent photosynthesis to O(2) concentration. The lower response to O(2) in P. milioides could be due to reduced O(2) competition with CO(2) for reaction with ribulose 1,5-bisphosphate, to a reduced loss of CO(2), or to an initial fixation of CO(2) by phosphoenolpyruvate carboxylase. Experiments were carried out with Panicum maximum Jacq., a C(4) species having no apparent photorespiration; tall fescue (Festuca arundinacea Schreb.), a C(3) species; P. milioides Nees ex Trin.; and Panicum schenckii Hack. The latter two species are closely related and have low photorespiration rates. CO(2) exchange was measured at five CO(2) concentrations ranging from 0 to 260 microliters per liter at both 2 and 21% O(2). Mesophyll conductance or carboxylation efficiency was estimated by plotting substomatal CO(2) concentrations against apparent photosynthesis. In the C(4) species P. maximum, mesophyll conductance was 0.96 centimeters per second and was unaffected by O(2) concentration. At 21% O(2) mesophyll conductance of tall fescue was decreased 32% below the value at 2% O(2). Decreases in mesophyll conductance at 21% O(2) for P. milioides and P. schenckii were similar to that for tall fescue. On the other hand, loss of CO(2) in CO(2)-free air, estimated by extrapolating the CO(2) response curve to zero CO(2), was increased from 1.8 to 6.5 milligrams per square decimeter per hour in tall fescue as O(2) was raised from 2-21%. Loss of CO(2) was less than 1 milligram per square decimeter per hour for P. milioides and P. schenckii and was unaffected by O(2). The results suggest that the reduced O(2) response in P. milioides and P. schenckii is due to a lower loss of CO(2) in the light rather than less inhibition of carboxylation by O(2), since the decrease in carboxylation efficiency at 21% O(2) was similar for P. milioides, P. schenckii, and tall fescue. The inhibition of apparent photosynthesis by 21% O(2) in these three species at low light intensities was similar at 31 to 36% which also indicates similar O(2) effects on carboxylation. Apparent photosynthesis at high light intensity was inhibited less by 21% O(2) in P. milioides (16.8%) and P. schenckii (23.8%) than in tall fescue (28.4%). This lower inhibition in the Panicum species may have been due to a higher degree of recycling of photorespired CO(2) in these species than in tall fescue. PMID- 16661188 TI - Effects of Magnesium on Intact Chloroplasts: I. EVIDENCE FOR ACTIVATION OF (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE CHLOROPLAST ENVELOPE. AB - Exogenous Mg(2+) (2 millimolar) altered the stromal pH of intact spinach chloroplasts. Without added KCl in the medium, Mg(2+) decreased the stromal pH in the light by approximately 0.3 pH unit. External KCl (25 millimolar) largely prevented the acidification caused by Mg(2+). Effects on the stromal pH were not caused by changes in H(+) pumping across the thylakoid membrane because Mg(2+) had no effect on the light-induced quenching of atebrin fluorescence by intact chloroplasts. However, Mg(2+) affected H(+) fluxes across the envelope. Addition of Mg(2+) to intact chloroplasts in the dark caused a significant acidification of the medium that was dependent on the presence of K(+).External K(+) or Na(+) also prevented the inhibition of CO(2)-dependent O(2) evolution by Mg(2+), whereas choline chloride was less effective. The combination of Mg(2+) and K(+) stimulated O(2) evolution at suboptimal pH, inhibited O(2) evolution at optimal and superoptimal pH, and prevented the inhibition of photosynthesis caused by acetate. In the absence of added K(+), Mg(2+) was most inhibitory to O(2) evolution at suboptimal pH.The results suggested that Mg(2+) activated a reversible (Na(+))K(+)/H(+) exchange across the chloroplast envelope. It is postulated that changes in the stromal pH may explain the inhibition of photosynthesis caused by the presence of exogenous Mg(2+). PMID- 16661189 TI - Stimulation of phosphatidylethanolamine exchange by castor bean cytosol proteins. AB - Cytosol proteins prepared from castor bean endosperm (4-day-old) seedlings stimulate the exchange of [(3)H]phosphatidylethanolamine between liposomes and mitochondria. The acceleration of the exchange depends on the quantity of cytosol proteins, the time of incubation, and the respective amounts of liposomes and mitochondria. On a per seedling basis, the active proteins are essentially located in the endosperm, whereas the roots and the cotyledons are less rich in these proteins. PMID- 16661190 TI - Cellular distribution of polysome populations of castor bean endosperm. AB - The distribution of polysomes in the cells of the endosperm of very young (48 hours of germination) seedlings of castor bean (Ricinus communis var. Hale) has been examined. Seedlings exposed to 100 micromolar gibberellin A(3) for periods up to 24 hours showed increased numbers of polysomes associated with endomembranes. Cytosol-derived polysomes were generally of a shorter mean length than membrane-derived polysomes. Gibberellin did not lead significantly to increased numbers of cytosol-derived polysomes, nor did it lead to enhancement of any particular size class of polysomes. PMID- 16661191 TI - Biological Effects of Cytokinin Antagonists 7-(Pentylamino) and 7-(Benzylamino)-3 Methylpyrazolo(4,3-d)Pyrimidines on Suspension-cultured Tobacco Cells. AB - We have studied the biological effects of two structural analogs of cytokinins, 3 methyl-7-(pentylamino)pyrazolo(4,3-d)pyrimidine and 3-methyl-7 (benzylamino)pyrazolo(4,3-d)pyrimidine, on tobacco cell suspension cultures. These two cytokinin analogs are highly inhibitory to cytokinin-autonomous and cytokinin-requiring tobacco cells. The growth inhibitory effect is markedly antagonized by benzyladenine, but not by adenine. Cell suspensions of tobacco cells of a cytokinin-autonomous strain become cytokinin-dependent in the presence of 0.1 micromolar 3-methyl-7-(benzylamino)pyrazolo(4,3-d)pyrimidine. It is also demonstrated that growth inhibition of cell suspension cultures is accompanied by cell death and that only dividing cells are sensitive to this cytotoxic effect. PMID- 16661192 TI - Polyamine-induced DNA Synthesis and Mitosis in Oat Leaf Protoplasts. AB - Freshly isolated protoplasts from leaves of oat seedlings (var. Victory) which do not divide when cultured on a wide range of media are capable of incorporating tritiated leucine, uridine, and thymidine into trichloroacetic acid-insoluble macromolecules. Over 70% of the leucine and uridine incorporated over an 18-hour period are found in protein and RNA, respectively, as shown by hydrolysis of the macromolecular products with a specific protease or RNase. In contrast, little or none of the tritiated thymidine is incorporated into macromolecules hydrolyzable by DNase over an 18- to 96-hour period. Incorporation of thymidine into trichloroacetic acid-insoluble material declines sharply with increasing time of culture after 18 hours. However, addition of diamines or polyamines to the medium not only prevents the decline, but actually increases net thymidine incorporation, including a fraction going into DNA. A significant increase in mitoses and binucleate protoplasts is also observed in 72- to 168-hour cultures.The inability of oat leaf protoplasts to synthesize significant quantities of DNA suggests that they are arrested at the G(1) phase of the cell cycle. Treatment with polyamines appears to promote both DNA synthesis and the inception of mitotic activity in oat protoplasts, as in numerous animal and microbial cells. PMID- 16661193 TI - Effects of aminoethoxyvinylglycine and countereffects of ethylene on ripening of bartlett pear fruits. AB - Pear fruits (Pyrus communis L. var. Bartlett) were treated with solutions containing aminoethoxyvinylglycine (AVG) using a modified vacuum infiltration method that introduced 4.3 milliliters solution per 100 grams tissue. At concentrations of 1 millimolar, AVG strongly inhibited ethylene production and delayed for 5 days the respiratory climacteric and accompanying ripening changes in skin color and flesh firmness. AVG was less effective in inhibiting the ripening of more mature fruits. Fruit infiltrated with 5 millimolar AVG had not begun to ripen 12 days after initiation of ripening in the controls. When treated with ethylene the inhibited fruit exhibited a climacteric rise in respiration, softened, and became yellow. Treatment of the AVG infiltrated fruits with ethyelne for 24 hours resulted in no recovery in endogenous ethylene production, but in a stimulation of protein synthesis measured as a 200% increase in leucine incorporation by excised tissue and a 74% increase in the percentage of ribosomes present as polysomes. PMID- 16661194 TI - Occurrence of nicotinamide adenine dinucleotide phosphatelinked glyoxylate reductase in nonphotosynthetic xylem tissue of perennials. AB - Xylem extracts of poplar tree contained glyoxylate reductase specific for NADPH. By isoelectric focusing in the pH ranges 3.5 to 10 or 4 to 6, the enzyme exhibited a single peak of activity at pH 5.4. The enzyme showed essentially no activity toward hydroxypyruvate, pyruvate, or NADH. The reaction was optimal at pH 6.0 in phosphate buffer and the activity profile exhibited a sharp and narrow pH profile with half-maximal velocities at about pH 7.0. The K(m) of the enzyme for glyoxylate was 0.11 millimolar. The xylem tissue of poplar tree exhibited high levels of enzyme activity (30 micromoles per gram dry weight per hour) even in the wintering stage and a slight change in activity occurred in spring and fall at the time when metabolism transition occurs. PMID- 16661195 TI - Nutritional Regulation of Organelle Biogenesis in Euglena: REPRESSION OF CHLOROPHYLL AND NADP-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE SYNTHESIS. AB - Nitrogen deficiency and the presence of specific organic carbon sources prevent chloroplast development in Euglena. In exponentially growing cultures, chlorophyll levels were low and independent of the nitrogen content of the growth medium. Chlorophyll levels increased in stationary phase and the amount of chlorophyll formed was proportional to the initial nitrogen content of the growth medium; the greater the concentration of nitrogen, the greater the amount of chlorophyll synthesized during stationary phase. Washing experiments demonstrated that the major nutritional factor inhibiting chlorophyll synthesis in stationary phase cultures grown on medium containing a high carbon to nitrogen ratio was the absence of nitrogen rather than the presence of utilizable organic carbon.The light-induced synthesis of chlorophyll and of NADP-glyceraldehyde-3-phosphate dehydrogenase was inhibited when acetate or ethanol was added at the time of exposure of dark-grown resting cells to light. Malate addition, however, stimulated chlorophyll and enzyme synthesis. Both cell number and total cell protein increased after ethanol, acetate, or malate addition, indicating that the resting cells were not nitrogen-deficient. Ethanol and acetate specifically repress light-induced chlorophyll synthesis. NADP-glyceraldehyde-3-phosphate dehydrogenase synthesis was inhibited at a time, the first 24 hours of light exposure, when chlorophyll synthesis was unaffected by carbon addition. PMID- 16661196 TI - Xylary pH and Reduction Potential Levels of Iron-stressed Silver Maple (Acer saccharinum L.). AB - Xylary fluid pH and reduction potentials were measured on silver maple (Acer saccharinum L.) grown under Fe and pH stress. Although pH and reduction potential (millivolt/59.2) varied significantly in the nutrient solution, xylary pH and reduction potential remained constant. It was concluded that changes in the pH and reduction potential in the xylary fluid of silver maple are not responsible for iron chlorosis. PMID- 16661197 TI - Oxidation of Glycine via the Respiratory Chain in Mitochondria Prepared from Different Parts of Spinach. AB - Mitochondria were prepared from roots, stalks, leaves, and leaf veins of spinach. The mitochondrial preparations were examined for their ability to oxidize glycine via the respiratory chain. It is shown that the glycine-oxidizing capacity is restricted to photosynthetically active tissue. The activity is present in mitochondria from the green parts of the leaves, but not in mitochondria from roots, stalks, or leaf veins. PMID- 16661198 TI - Chlorophyll-Protein Complexes of the Cyanophyte, Nostoc sp. AB - Four chlorophyll-protein complexes have been resolved from the cyanophyte, Nostoc sp., by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis at 4 C. Complexes solubilized by SDS from Spinacia oleracea were run for comparison. As has been well documented, the P700-chlorophyll a-protein complex from the higher plant and blue-green algal samples are similar, and the light-harvesting pigment protein complex is present only in the former. Most noteworthy are two closely migrating chlorophyll proteins in Nostoc sp. which have approximately the same mobility as a single chlorophyll-protein band resolvable from spinach. The absorption maximum of the complex from spinach is at 667 nanometers, and those of the two complexes from Nostoc sp. are at 667 and 669 nanometers; the fluorescence emission maximum at -196 C is at 685 nanometers, and the 735 nanometer fluorescence peak, characteristic of the P700-chlorophyll a-protein complex, is absent. The apoproteins of these new complexes from Nostoc sp. and spinach are in the kilodalton range. It appears that at least one of these two chlorophyll protein complexes from Nostoc sp. compares with those recently described by others from higher plants and green algae as likely photosystem II complexes, perhaps containing P680, although no photochemical data are yet available. PMID- 16661199 TI - Evidence for HCO(3) Transport by the Blue-Green Alga (Cyanobacterium) Coccochloris peniocystis. AB - The possibility of HCO(3) (-) transport in the blue-green alga (cyanobacterium) Coccochloris peniocystis has been investigated. Coccochloris photosynthesized most rapidly in the pH range 8 to 10, where most of the inorganic C exists as HCO(3) (-). If photosynthesis used only CO(2) from the external solution the rate of photosynthesis would be limited by the rate of HCO(3) (-) dehydration to CO(2). Observed rates of photosynthesis at alkaline pH were as much as 48-fold higher than could be supported by spontaneous dehydration of HCO(3) (-) in the external solution. Assays for extracellular carbonic anhydrase were negative. The evidence strongly suggests that HCO(3) (-) was a direct C source for photosynthesis.Weakly buffered solutions became alkaline during photosynthesis with a one-to-one stoichiometry between OH(-) appearance in the medium and HCO(3) (-) initially added. Alkalization occurred only during photosynthesis and was blocked by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, diuron. It is suggested that HCO(3) (-) was transported into cells of Coccochloris in exchange for OH(-) produced as a result of HCO(3) (-) fixation in photosynthesis.The inorganic C concentration required to support a rate of photosynthesis of half the maximum rate (K(m)) was 6 micromolar at pH 8.0 or, in terms of available CO(2), a K(m) of 0.16 micromolar. This value is two orders of magnitude lower than reported K(m) values for the d-ribulose-1,5-bisphosphate carboxylase for blue-green algae. It is suggested that the putative HCO(3) (-) transport by Coccochloris serves to raise the CO(2) concentration around the carboxylase to levels high enough for effective fixation. PMID- 16661200 TI - Concomitant determination of folar nitrogen loss, net carbon dioxide uptake, and transpiration. AB - A closed system was designed for concomitant determination of net CO(2) uptake, transpiration, and foliar nitrogen (N) loss in soybeans (Glycine max [L.] Merr.). The CO(2) uptake was monitored by infrared analysis of system air removed periodically in aliquots. Leaf vapors were trapped in calibrated tubes at Dry Ice temperature, and transpiration rate was determined from the quantity of condensate. Subsequent pyrochemiluminescent analysis of this condensate revealed reduced N forms, although a [ill] percentage (4 to 15%) of oxidized forms was found.Maximum CO(2) uptake (22.3 milligrams per square decimeter per hour), transpiration (2.5 grams H(2)O per square decimeter per hour), and the total (4.9 micrograms per square decimeter per hour), reduced (4.1 micrograms per square decimeter per hour), and oxidized (0.7 microgram per square decimeter per hour) N loss rates were measured between 6.5 and 10.5 hours (30 C) of a simulated 13-hour photoperiod in leaves of V4 to V5 (three to four trifoliolate stage) soybeans. During a temperature study (20, 25, 30, and 35 C) with these plants and several leaf positions of older vegetative soybeans (V9 to V11), total and reduced N loss were maximum at 30 C (V4 to V5: 9.8 and 9.3 micrograms per square decimeter per hour; V9 to V11: 3.3 and 2.9 micrograms per square decimeter per hour); transpiration was maximal at 30 C in V4 to V5 plants (2.6 grams H(2)O per square decimeter per hour) and at 35 C in V9 to V11 plants (2.4 grams H(2)O per square decimeter per hour); and CO(2) uptake was maximal at 25 and 30 C during both sampling dates (V4 to V5: 22.7 to 23.5 milligrams CO(2) per square decimeter per hour: V9 to V11: 14.1 to 14.5 milligrams CO(2) per square decimeter per hour). At 30 and 35 C these parameters were highest in the youngest tissue of V9 to V11 plants.In V9 to V11 plants at all four temperatures, reduced N loss was correlated with total N loss (r = 0.99 at 20, 25, and 30 C and 0.97 at 35 C), and CO(2) uptake with transpiration (r = 0.47 at 20 C, 0.75 at 25 C, 0.85 at 30 C, and 0.81 at 35 C). Transpiration was correlated with both total and reduced N loss at 30 C (total N: r = 0.69; reduced N: r = 0.70) and 35 C (total N: r = 0.58; reduced N: r = 0.54). In addition, CO(2) uptake was directly related to total (r = 0.69 at 30 C and 0.56 at 35 C) and reduced (r = 0.67 at 30 C and 0.56 at 35 C) N loss at these two temperatures. PMID- 16661201 TI - Peroxide Levels and the Activities of Catalase, Peroxidase, and Indoleacetic Acid Oxidase during and after Chilling Cucumber Seedlings. AB - The activities of catalase, peroxidase, indoleacetic acid (IAA) oxidase and peroxide levels in cucumber plants during and after chilling were determined. During 96 hours at 5 C and 85% relative humidity, catalase activity declined, IAA oxidase activity increased, and peroxide concentrations increased. Peroxidase activity was not affected by chilling. When chilled plants were returned to 25 C to recover, enzyme activities and peroxide concentration were restored to their prechilling levels. The increase in peroxide and IAA oxidase activity may inactivate or destroy IAA and thus retard growth. PMID- 16661202 TI - Wounding Stimulates Cyanide-sensitive Respiration in the Highly Cyanide-resistant Leaves of Bryophyllum tubiflorum Harv. AB - The rate of respiration in sectioned leaves of Bryophyllum tubiflorum Harv. increases with decreasing section thickness. The rates of uninhibited respiration in 2- and 8-millimeter-thick sections are 74 and 46 microliters of O(2) per gram fresh weight of unruptured tissue per hour at 20 C, whereas the rate in the presence of cyanide is 31 microliters of O(2) in each case. The rates are unaffected by salicylhydroxamic acid, but cyanide and salicylhydroxamic acid together completely eliminate O(2) uptake. The capacity of the alternative respiratory pathway is thus initially high (estimated at 84% of the uninhibited respiratory rate in whole leaves) and remains constant but probably unexpressed subsequent to the rapid induction of wound respiration. PMID- 16661203 TI - Nodulation and Nitrogen Fixation in Cliffrose (Cowania mexicana var. stansburiana (Torr.) Jeps.). AB - Nodulation and acetylene reduction were demonstrated for cliffrose (Cowania mexicana var. stansburiana [Torr.] Jeps.) in a greenhouse trial. Nitrogen fixation was also verified with (15)N. Seedlings were grown in a soil known to nodulate both bitterbrush species (Purshia tridentata (Pursh) D.C. and P. glandulosa [Curran]) to which a suspension of crushed bitterbrush nodules had been added. Bitterbrush seedlings grew faster and reduced more acetylene than cliffrose seedlings planted in the same pots, but the specific activity of the nodules and the nodule mass to root mass ratios were approximately the same. PMID- 16661204 TI - Concentration and Metabolic Turnover of Indoles in Germinating Kernels of Zea mays L. AB - The amounts and rates of metabolic turnover of the indolylic compounds in germinating kernels of sweet corn were determined. Knowledge of pool size and rate of pool turnover has permitted: (a) identification of indole-3-acetyl-myo inositol as the major chemical form for transport of indole-3-acetic acid (IAA) from endosperm to shoot; (b) demonstration that the free IAA of the endosperm is turning over rapidly with a half-life of 3.2 hours; (c) identification of esters of IAA as the immediate precursors of IAA in the endosperm and shoot; (d) demonstration that neither tryptophan nor tryptamine is a major precursor of IAA for the seed or shoot; (e) identification of IAA-myo-inositol glycosides as precursors of IAA-myo-inositol.It is concluded that seedlings of Zea mays utilize esters of IAA, and not tryptophan or its derivatives, for the IAA requirements of the germinating seedling. PMID- 16661205 TI - Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L. AB - Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [(14)C]Indole-3 acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3 acetyl-myo-inositol is the first seed auxin precursor to be identified.Application of either [(14)C]IAA or (14)C-indole-3-acetyl-myo-inositol ester to the endosperm results in both free and esterified [(14)C]IAA in the seedling shoot. Esterification of free IAA and hydrolysis of indole-3-acetyl-myo inositol occurred in the shoot and not the endosperm yielding ratios of ester to free IAA which approximate the ratios of ester to free IAA normally found in corn shoot tissue. This proves, for the first time, that esterified IAA and free IAA are interconvertible in the growing shoot. Since free IAA may be limiting for plant growth, knowledge that the free hormone is in "equilibrium" with its conjugates suggests new methods for the control of plant growth. PMID- 16661206 TI - Response of leaf water potential, stomatal resistance, and leaf rolling to water stress. AB - Numerous studies have associated increased stomatal resistance with response to water deficit in cereals. However, consideration of change in leaf form seems to have been neglected. The response of adaxial and abaxial stomatal resistance and leaf rolling in rice to decreasing leaf water potential was investigated. Two rice cultivars were subjected to control and water stress treatments in a deep (1 meter) aerobic soil. Concurrent measurements of leaf water potential, stomatal resistance, and degree of leaf rolling were made through a 29-day period after cessation of irrigation. Kinandang Patong, an upland adapted cultivar, maintained higher dawn and midday leaf water potential than IR28, a hybrid selected in irrigated conditions. This was not explained by differences in leaf diffusive resistance or leaf rolling, and is assumed to result from a difference in root system extent.Stomatal resistance increased more on the abaxial than the adaxial leaf surface in both cultivars. This was associated with a change in leaf form or rolling inward of the upper leaf surface. Both responses, increased stomatal resistance and leaf rolling, were initiated in a similar leaf water potential range (-8 to -12 bars). Leaves of IR28 became fully rolled at leaf water potential of about -22 bars; however, total leaf diffusive resistance was only about 4 to 5 seconds per centimeter (conductance 0.25 to 0.2 centimeter per second) at that stage. Leaf diffusive resistance and degree of leaf rolling were linearly related to leaf water potential. Thus, leaf rolling in rice may be used as an estimate of the other two less obvious effects of water deficit. PMID- 16661207 TI - Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves. AB - Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD(+), oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO(3) (-) with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO(3) (-) reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (/=1 millimolar) inorganic phosphate, inclusion of NO(2) (-) as an electron acceptor, or bright illumination at low partial pressures of O(2). These inhibitions may be reversed by addition of uncoupling levels of NH(4)Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b(6) and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in DeltapH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO(3) (-)-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions.The rate of O(2) evolution is also decreased 3-fold when ambient CO(2) (0.63 millimolar HCO(3) (-) at pH 8.1) is used in place of saturating HCO(3) (-) and chloroplasts are illuminated aerobically with catalase and a low level (0.25 millimolar) of K(2)HPO(4). Only inhibitory effects are observed with additions of antimycin or NH(4)Cl. Under these conditions, excessive photophosphorylation or a large pH gradient does not limit the rate of photosynthesis. PMID- 16661229 TI - Gravity Functions of Circumnutation by Hypocotyls of Helianthus annuus in Simulated Hypogravity. AB - For more than a decade research on the botanical mechanism responsible for circumnutation has centered on whether or not these nearly ubiquitous oscillations can be attributed to a hunting process whereby the plant organ continuously responds to the gravity force and, by overshooting each stimulus, initiates a sustained oscillation or, driven by a not yet defined autogenic mechanism, performs oscillatory activities that require no external reinforcement to maintain the observed rhythms of differential growth.We explore here the effects of altered gravity force on parameters of circumnutation. Following our earlier publication on circumnutation in hypergravity we report here an exploration of circumnutation in hypogravity.Parameters of circumnutation are recorded as functions of the axially imposed gravity force. The same method was used (two-axes clinostat rotation) to produce sustained gravity forces referred to as hypergravity (1 < g), hypogravity (0 [unk] g < 1), and negative gravity (-1 < g < 0). In these three regions of the g-parameter nutational frequency and nutational amplitude were influenced in different ways.The results of our tests describe the gravity dependence of circumnutation over the full range of real or simulated gravity levels that are available in an earth laboratory. Our results demonstrated that nutational parameters are indeed gravity-dependent but are not inconsistent with the postulate that circumnutation can proceed in the absence of a significant gravity force. PMID- 16661230 TI - Abscisic Acid promotes both volume flow and ion release to the xylem in sunflower roots. AB - The effect of abscisic acid (ABA) on the exudation process in excised sunflower root was investigated. A promotion of both ion flux and volume exudation rate was observed. Cutting off the external supply of ions to the roots markedly increased the magnitude of the ABA effect. The promotive effect of ABA on exudation rate was extremely rapid (less than 6 minutes) and seemed to be biphasic. Reasons are given for relating the ABA effect to two separate actions, namely: (a) on water permeability; and (b) on the release of ions both from vacuoles to the cytoplasm and from symplasm to the xylem. Kinetin inhibited movement of ions to the xylem and its effect did not appear to be related to that of the ABA. PMID- 16661231 TI - Nutrient Influences on Leaf Photosynthesis: EFFECTS OF NITROGEN, PHOSPHORUS, AND POTASSIUM FOR GOSSYPIUM HIRSUTUM L. AB - The net rate of CO(2) uptake for leaves of Gossypium hirsutum L. was reduced when the plants were grown at low concentrations of NO(3) (-), PO(4) (2-), or K(+). The water vapor conductance was relatively constant for all nutrient levels, indicating little effect on stomatal response. Although leaves under nutrient stress tended to be lower in chlorophyll and thinner, the ratio of mesophyll surface area to leaf area did not change appreciably. Thus, the reduction in CO(2) uptake rate at low nutrient levels was due to a decrease in the CO(2) conductance expressed per unit mesophyll cell wall area (g(cell) (CO(2) )). The use of g(cell) (CO(2) ) and nutrient levels expressed per unit of mesophyll cell wall provides a new means of assessing nutrient effects on CO(2) uptake of leaves. PMID- 16661232 TI - Effect of Salinity upon Cell Membrane Potential in the Marine Halophyte, Salicornia bigelovii Torr. AB - The electrophysiology of root cells of the marine halophyte, Salicornia bigelovii Torr., has been investigated. Cellular concentrations of K(+), Cl(-), and Na(+) and resulting cell membrane potentials were determined as functions of time and exposure to dilutions of artificial seawater. Treatment of these data by the Nernst criterion suggests that Cl(-) is actively transported into these root cells, but that active transport need not be invoked to explain the accumulation of Na(+) at all salinities investigated nor for K(+) at moderate to high salinities. In low environmental salinity, the cell electropotential of Salicornia root cells was found to respond to inhibitors in a fashion similar to that observed in glycophytes; in high environmental salinity, root cell membrane potential appears to be insensitive to bathing salinity and m chlorocarbonylcyanide phenylhydrazone induces membrane hyperpolarization, in contrast to the response of glycophytes to such treatments. The fact that measured membrane potentials exceed diffusion potentials for Na(+), K(+), and Cl( ) and the observation of a rapid depolarization by CO in the dark suggests an electrogenic component in Salicornia root cell membrane potentials. PMID- 16661233 TI - Energization of the sugar transport mechanism in the plasmalemma of isolated mesophyll protoplasts. AB - The mechanism of 3-O-methyl-d-glucose transport through the plasmalemma has been investigated in protoplasts isolated from the mesophyll of Pisum sativum L. var. Dan.Analysis of the fluxes after 50 minutes of uptake showed that the gradual decrease in slope of the net uptake curve with time was not due to any decline in uptake capacity; it represented the approach to flux equilibrium of a small compartment of the protoplast, probably the cytoplasm.The energy of activation for initial flux into this compartment was 20 kilocalories per mole between 17 and 27 C. Very high discrimination was shown with regard to sugar isomers. Light strongly promoted flux (by a factor of 2.5 in the case of methyl glucose). Initial flux showed sharply contrasting inhibitor sensitivity in the light and the dark. Light uptake was sensitive to the proton conductor carbonyl cyanide m chlorophenylhydrazone (CCCP), but stable for at least the first 10 minutes to the ATPase inhibitors quercetin, rutin, and diethylstilbestrol, as well as to arsenate. Dark uptake, on the other hand, was stable to CCCP but was immediately depressed by quercetin, rutin, diethylstilbestrol, and arsenate.Protoplasts which received a light pretreatment before incubation in the dark took up methyl glucose at the accelerated light rate for the first 7 minutes. Moreover, the light pretreatment sensitized subsequent initial dark uptake to CCCP, and conferred on it the stability to ATPase inhibitors and arsenate characteristic of light uptake. After about 7 minutes the characteristic inhibitor responses of dark uptake were resumed.It is proposed that more than one mode of energy coupling for sugar transport may operate in these protoplasts. PMID- 16661234 TI - Some synthetic phytotoxins structurally related to rhynchosporoside. AB - The (2-O)alpha-d-glucopyranoside of 1,2-propanediol and [U-(14)C]glucose were used as substrates in a reaction with almond beta-glucosidase, which resulted in the production of some (2-O)alpha-d-oligoglucosides of 1,2-propanediol. As its substrate, the beta-glucosidase preferred the glucoside isomer that rotates plane polarized light to the right. Some of the glucosides produced in the enzymic reaction mixture possessed host selective toxin activity. It appears that the biological activity of the toxin is not dependent on the nature of the glycosidic linkage with the aglycone. PMID- 16661235 TI - Lipopolysaccharide Composition of the Wilt Pathogen, Pseudomonas solanacearum: CORRELATION WITH THE HYPERSENSITIVE RESPONSE IN TOBACCO. AB - In the induction of the hypersensitive response (HR) in tobacco by Pseudomonas solanacearum, the recognition between host and pathogen is thought to involve an interaction between plant lectins and bacterial lipopolysaccharide (LPS). The LPS of a series of strains of P. solanacearum were examined to determine if there are structural differences that might account for the ability or inability of these strains to induce the hypersensitive response. Analysis of the components of LPS by gas chromatography indicates a clear difference in sugar composition between the HR-inducing and non-HR-inducing strains, especially in terms of the percentage of glucose, xylose and rhamnose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows there are two distinct kinds of LPS, differing greatly in size, which correspond to rough and smooth LPS in other systems. In addition, a phage, CH154, was isolated which lyses non-HR-inducing bacteria and which is inactivated by LPS from these bacterial strains. Therefore, differences in LPS structure correlate strongly with host recognition of Pseudomonas solanacearum. PMID- 16661236 TI - Translocation: EFFLUX OF SUGARS ACROSS THE PLASMALEMMA OF MESOPHYLL PROTOPLASTS. AB - During photosynthesis by mesophyll protoplasts of wheat and tobacco, a linear efflux of sucrose and hexoses to the medium was observed, with the size of the intraprotoplast sugar pools remaining constant. Efflux of metabolites labeled by (14)CO(2) fixation was initially low because of dilution by internal pools, but increased exponentially with time. The results have significance both in terms of the mechanism of translocation and the use of isolated protoplasts in photosynthetic studies. PMID- 16661237 TI - Effect of cold on endogenous fucosyl transferase activity in corn roots. AB - Incubation of excised, 1-centimeter long root tips of corn in chilled distilled H(2)O increases the activity of extractable GDP fucose: polysaccharide fucosyl transferase. Enhancement of transferase activity is optimal at 10 C and incubation at this temperature for 8 hours causes a 3- to 5-fold stimulation of activity. Transfer of roots from 10 to 30 C reverses the enhancement of enzyme activity although reciprocal transfer of roots from 30 to 10 C does not stimulate the accumulation of transferase activity. PMID- 16661238 TI - Heterotrophic Carbon Dioxide Fixation Products of Euglena: EFFECTS OF AMMONIUM. AB - The metabolic products of heterotrophic (dark) CO(2) fixation by Euglena gracilis Klebs strain Z Pringsheim were separated and identified. They consisted of amino acids, phosphorylated compounds, tricarboxylic acid cycle intermediates, and nucleotides. Exposure of the cells to NH(4) (+) after a period of NH(4) (+) deprivation stimulated heterotrophic CO(2) fixation almost 4-fold, modifying the spectrum of the fixation products. In particular, the NH(4) (+) treatment stimulated fixation of CO(2) into glutamine, glycine, alanine, and serine. PMID- 16661239 TI - Photomorphogenically Defined Light and Resistance of Poa pratensis to Drechslera sorokiniana. AB - Photomorphogenic light definitions were derived by mathematical determination of the estimated phytochrome photoequilibrium for each light treatment spectrum. A wide range of photomorphogenic light treatments represented by spectra with estimated phytochrome photoequilibria of 0.45, 0.54, 0.60, 0.67, and 0.71 was utilized to determine the influence of photomorphogenically defined light on resistance of Poa pratensis L. to pathogenesis by Drechslera sorokiniana. Accurate resolution of D. sorokiniana leaf spot development required evaluation of separate leaf ages due to the sequential appearance, development, and senescence of P. pratensis leaves. Disease development (all light treatments) was greatest on leaf 4 (oldest, postmature) followed by leaf 1 (youngest, premature). Low levels of disease occurred on leaves 2 and 3 (mature). Photomorphogenic light defined by estimated phytochrome photoequilibria greater than 0.60 (natural light approximately 0.60) was most disease promotive on leaf 1. Conversely, photomorphogenic light defined by estimated phytochrome photoequilibria of less than 0.60 was most disease promotive on leaf 4. These responses indicate that inherent resistance or susceptibility expressed by P. pratensis to pathogenesis by D. sorokiniana is regulated in part by leaf age (developmental senescent stage) and by photomorphogenically defined light quality. A hypothesis is presented and discussed which integrates and speculates on these observations with respect to the literature. PMID- 16661240 TI - Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts. AB - The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO(2) fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO(2) fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5-phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions.The Pi optima of CO(2) fixation were studied when the rate of CO(2) fixation or the rate of Pi transport was varied by the application of specific inhibitors. For optimal performance the rates of CO(2) fixation and Pi transport have to be matched in such a way, that transport neither limits nor exceeds CO(2) fixation. This accounts for the large variation in the Pi optima for CO(2) fixation by different chloroplast preparations. PMID- 16661241 TI - Vein Loading in Seedlings of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc. AB - Vein loading in unifoliate leaves of white bean seedlings exposed to excess Co(2+), Ni(2+), or Zn(2+) for 1 to 4 days was studied by incubating leaf discs in [(14)C]sucrose. The discs from plants exposed to metal exhibited an increased total uptake of radiosucrose but reduced vein loading. Differences between treatments due to infiltration of disc margins were eliminated by analyzing 7 millimeter discs cut from the center of incubated 14-millimeter discs. Uptake of radiosucrose was greater particularly in discs from seedlings exposed to excess Ni(2+) and Zn(2+). The effect increased as exposure of the seedlings to metal increased up to 4 days. Autoradiographs showed vein loading in control leaf tissues with most of the radiosucrose accumulating in minor veins and little remaining in the mesophyll. In discs from metal-treated plants, most of the sucrose remained in the mesophyll without accumulating preferentially in the minor veins. This effect was evident within 24 hours of exposure to excess metal and intensified with longer exposures to metal. The inhibition of vein loading was also evident in situ. Both the preferential accumulation of sucrose into the minor veins of control tissues and the accumulation into mesophyll of metal exposed tissues were sensitive to 2,4-dinitrophenol and the blockage of sulfhydryl groups. It is concluded that the inhibition of vein loading contributes markedly to the observed toxicological effects of reduced photoassimilate export and of accumulation of carbohydrates in fully expanded leaves of bean seedlings exposed to excess metal ions. PMID- 16661242 TI - Photosynthetic Rates of Sporophytes and Gametophytes of the Fern, Todea barbara. AB - The photosynthetic rates of intact sporophytes or gametophytes of the fern Todea barbara grown in sterile culture were measured using an infrared gas analyzer. Sporophytes consisted of single whole plants with roots and leaves grown in tubes of agar. Gametophytes were grown as several plants covering the surface of the agar. Sporophytes had photosynthetic rates at light saturation of 8.50 microliters CO(2) per hour per milligram dry weight and 1,300 microliters CO(2) per hour per milligram chlorophyll, whereas rates for gametophytes were lower, 2.36 microliters CO(2) per hour per milligram dry weight and 236 microliters CO(2) per hour per milligram chlorophyll.O(2) inhibited the photosynthetic rates of both plants to a degree typical of C-3 plants. The light-saturated photosynthetic rates of sporophytes increased 42% and gametophytes increased 27% when the O(2) concentration was changed from 21 to 1%. PMID- 16661243 TI - Adenosine Phosphate and Nicotinamide Adenine Dinucleotide Pool Sizes during Shoot Initiation in Tobacco Callus. AB - Shoot-forming tobacco callus is found to have high levels of adenosine phosphates and NAD(+), and a low energy charge during meristemoid and shoot primordium formation. NADH levels are low and show little change during this period. There is a decline in the content of NADPH to nondetectable levels during the process, and a transient increase of NADP(+) is observed early in culture. These patterns are indicative of a shift to a more intensive rate of metabolism during meristemoid and shoot primordium formation and apparently reflect the requirement for energy and reducing power during organ initiation. PMID- 16661244 TI - Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS. AB - Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.The sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, p chloromercuriphenyl sulfonic acid, and mersalyl inhibited the oxidation of exogenous NADPH by the mitochondria whereas NADH oxidation was unaffected at similar concentrations of inhibitor. The data suggest that exogenous NADPH is oxidized by potato mitochondria via a dehydrogenase primarily situated on the outer face of the inner mitochondrial membrane that is neither the dehydrogenase involved with endogenous NADH oxidation nor with exogenous NADH oxidation. PMID- 16661245 TI - Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons. AB - The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5 to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO(3). Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO(3), subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO(3)-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active. PMID- 16661246 TI - Endoplasmic Reticulum of Mung Bean Cotyledons: ACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH. AB - Homogenates of mung bean cotyledons were subjected to equilibrium density centrifugation on linear sucrose gradients and the positions of the various organelles determined by assay of marker enzymes. Measurement of phospholipid distribution on such gradients showed that the major peak of phospholipid at a density of 1.11 to 1.13 grams per cubic centimeter coincided with the position of the endoplasmic reticulum (ER), confirming ultrastructural evidence that storage parenchyma cells are rich in ER. Germination and seedling growth were accompanied by a rapid decline in ER-associated phospholipid but a marked increase in the ER marker enzyme NADH cytochrome c reductase. Similar experiments with developing seeds indicated that the amount of ER-associated phospholipid increases during cotyledon expansion reaching a maximum during seed maturation. There was no subsequent decline during seed desiccation, instead ER-associated phospholipid levels were maintained in the dry seed until germination when catabolism was initiated 12 to 24 hours after the start of imbibition. This timing indicates that the observed ER breakdown is not an expression of the overall senescence of the cotyledons, but may represent the dismantling of the extensive rough ER used for reserve protein synthesis during cotyledon development. PMID- 16661247 TI - Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids. AB - Oxyleghemoglobin was used to supply low concentrations of O(2) to H(2)-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H(2) oxidation system of these bacteroids was capable of effectively utilizing O(2) at the low concentrations of O(2) expected to be found in soybean nodules. Apparent K(m) values of approximately 10 nanomolar O(2) have been calculated for the oxyhydrogen reaction. These values include the K(m) values for both H(2) oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H(2) additions stimulated C(2)H(2) reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H(2) oxidation system is capable of recycling all of the H(2) evolved during the N(2) fixation process. PMID- 16661248 TI - Organic Acids and Ionic Balance in Xylem Exudate of Wheat during Nitrate or Sulfate Absorption. AB - Experiments were designed to study the importance of organic acids as counterions for K(+) translocation in the xylem during excess cation uptake. A comparison was made of xylem exudate from wheat seedlings treated 72 hours with either 1.0 millimolar KNO(3) or 0.5 millimolar K(2)SO(4), both in the presence of 0.2 millimolar CaSO(4). Exudation from KNO(3) plants had twice the volume and twice the K(+) and Ca(2+) fluxes or rate of delivery to shoots, as K(2)SO(4) plants. Malate flux was 25% higher in K(2)SO(4) than in KNO(3) exudate. Malate was the principal anion accompanying K(+) or Ca(2+) in K(2)SO(4) treatment, while in the KNO(3) treatment, NO(3) (-) was the principal anion. The contribution of SO(4) (2 ) was negligible in both treatments. In a second experiment, exudate was collected every 4 hours during the daytime throughout a 72-hour treatment with KNO(3). Malate was the only anion present in exudate at first, just after the CaSO(4) pretreatment had ended. Malate concentration decreased and NO(3) (-) concentration increased with time and these concentrations were negatively correlated. By 62 hours, NO(3) (-) represented 80% of exudate anions. K(+) and NO(3) (-) concentrations in exudate were strongly correlated with K(+) and NO(3) (-) uptake, respectively. The first 36 hours of absorption from KNO(3) solution resembled the continuous absorption of K(2)SO(4), in that malate was the principal counterion for translocation of K(+). PMID- 16661249 TI - Measurement of tissue osmotic pressure. AB - Osmotic pressure measured by a modified pressure-volume method was compared with that of the mixed sap expressed from frozen and thawed tissue. The error in the latter technique averaged 11 and 16% (too dilute) for greenhouse and field leaves of Zea mays L. at all growth stages. These errors were not consistently calculated by a model of simple mixing between the matric and osmotic fractions, both of which decreased with plant age. Some other alternatives to the pressure volume method are discussed which are based on a more rapid estimate of the zero turgor point. PMID- 16661250 TI - Legume alpha-Galactosidases Which Have Hemagglutinin Properties. AB - Four legume species (four genera) were examined and found to contain hemagglutinins with properties similar to those which we have previously described for the enzymic hemagglutinin in Vigna radiata. Examination of extracts by gel filtration and ion exchange chromatography showed that an alpha galactosidase activity exactly co-purified with a hemagglutinin activity in each of the four species. The alpha-galactosidase activities in the four species were virtually identical to each other with respect to substrate and inhibitor specificity as well as kinetic behavior. Additionally the hemagglutinin activities in all four species displayed very similar carbohydrate specificities. The inhibitor specificities displayed by the enzymes and the hemagglutinins were qualitatively and quantitatively very nearly identical to each other. The remarkable similarities of these proteins, both to each other and to the previously described Vigna enzymic hemagglutinin, suggest that each of these plants may contain a homologue from a specific class of enzymic hemagglutinin. PMID- 16661251 TI - Identification of six endogenous gibberellins in spinach shoots. AB - Analysis of highly purified extracts from spinach shoots by combined gas chromatography-mass spectrometry has demonstrated the presence of six 13-C hydroxylated gibberellins (GAs): GA(53), GA(44), GA(19), GA(17), GA(20), and GA(29). The major GAs were GA(17), GA(19), and GA(20), whereas the other three GAs occurred in trace amounts. Structural considerations suggest that the six GAs identified in spinach are related in the following metabolic sequence: GA(53) --> GA(44) --> GA(19) --> GA(17) --> GA(20) --> GA(29). PMID- 16661252 TI - Variation in the Accumulation of Seed Storage Protein Among Genotypes of Phaseolus vulgaris (L.). AB - Differences in the accumulation of total seed protein and globulin-1 (G1) protein were detected among three inbred lines of common bean. Total protein accumulation ranged from 2.3 to 3.7 milligrams per cotyledon pair per day among lines. In all lines the dry weight and protein accumulation ceased and a loss of chlorophyll in the cotyledons occurred when the moisture content had fallen to 50% of fresh weight. G1 was first detected and rapid accumulation began 14 days after flowering in two lines, whereas in the cultivar Endogava zurundi namame, rapid accumulation was delayed until 20 days after flowering. Rates of G1 accumulation ranged from 1.0 to 1.8 milligrams G1 per cotyledon pair per day among lines. G1 accumulation ceased 6 days before the end of total protein accumulation in Sanilac. A steady rate of protein accumulation was observed in Sanilac, but pauses in the accumulation of G1 and of total protein were documented in Endogava zurundi namame. The rate of G1 accumulation preceding and following the pause in Endogava zurundi namame was 2.7 milligrams G1 per cotyledon pair per day, nearly double that of the other lines. PMID- 16661253 TI - Effect of Light on Glucose Utilization by Euglena gracilis. AB - The effect of light on glucose consumption by wild-type Euglena gracilis Z. and mutant cells has been studied. When dark- or light-grown wild-type cells are transferred from a medium containing sodium butyrate as the only carbon source to a glucose-containing medium, glucose consumption is blocked for 6 to 7 days when cultures are incubated under a light intensity of at least 600 lux. During this time cells multiply at the same rate as controls kept on media devoid of any utilizable organic carbon source. This light-induced inhibition of glucose consumption and of growth on glucose-containing medium is not related to photosynthesis since: (a) glucose consumption is inhibited by light intensities much lower than those required for high phototrophic growth; (b) the inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea does not overcome the inhibition of glucose consumption; and (c) nonphototrophic-growing mutants also show light-induced inhibition of glucose consumption and of growth on glucose-containing medium. This inhibition of growth by light might be explained by modification in the permeability of the cellular membrane. PMID- 16661254 TI - Soil Temperature Influences on Root Resistance of Pinus contorta Seedlings. AB - The influence of low temperature in the root zone on water uptake in lodgepole pine (Pinus contorta Dougl. ex Loud.) was studied under laboratory conditions. To remove soil hydraulic influences, two-year-old seedlings were transferred to solution cultures and maintained in temperature controlled water baths. Short term measurements of leaf conductance, leaf water potential and tritiated water movement were taken at root temperatures from 22 C down to 0 C. Root resistance was calculated to be 67% of total plant resistance at 7 C and 93% at 0 C. In addition an Arrhenius break was found in a plant resistance versus temperature plot, suggesting a significant change with temperature in the membrane pathway in the root water uptake system. PMID- 16661255 TI - Light Effects on the Synthesis of Ribulose-1,5-Bisphosphate Carboxylase in Lemna gibba L. G-3. AB - Placing light-grown Lemna gibba L. G-3 into the dark results in a changed pattern of protein synthesis. Although the amount of protein in the tissue and the over all rate of incorporation of [(35)S]methionine into protein does not significantly decline during four days of darkness, the rate of synthesis of three polypeptides declines dramatically. One of these polypeptides is the chlorophyll a/b-binding protein and the two others are the large and small subunits of ribulose-1,5-bisphosphate carboxylase. The changed rates of synthesis of the two subunits were examined after transitions of plants from light to dark and dark to light. The in vivo synthesis of both subunits, while declining to a low level during four days of darkness, increases rapidly upon returning the plants to white light. In addition, the level of poly(A) mRNA coding for the precursor polypeptide of the small subunit of the enzyme falls to a low level in the dark and increases rapidly in response to white light. The increase in translatable mRNA for the small subunit is rapid enough to account for a major part of the increased synthesis of this subunit. PMID- 16661256 TI - Glycosylation of pea cotyledon membranes. AB - Pea cotyledons were injected with d-[(14)C]mannose or d-[(14)C]-glucosamine and incubated for 1 to 1.5 hours. Cotyledons were homogenized and subcellular fractions were isolated by differential centrifugation followed by linear sucrose density gradient centrifugation.Radioactivity that was precipitated by trichloroacetic acid was associated most extensively with rough endoplasmic reticulum, Golgi membranes, a membrane with a density of 1.14 grams per cubic centimeter (possibly plasma membrane) and an unidentified subcellular component with a density of 1.22 grams per cubic centimeter. Lower levels of incorporation were observed in protein bodies and mitochondria.Isolated membrane fractions were lipid-extracted to determine which components of the membrane contained the label. Rough endoplasmic reticulum contained the most extensively labeled lipids which had similar properties to the lipid intermediates thought to be involved in glycoprotein assembly. The lipid free residues of the various membrane fractions contained radioactivity that was released by protease treatment. Acid hydrolysis of the residues indicated that most of the radioactivity was associated with mannose or glucosamine. It appears that various subcellular components of the pea cotyledon possess glycoproteins that contain mannose and glucosamine. PMID- 16661257 TI - Photoreversible calcium fluxes induced by phytochrome in oat coleoptile cells. AB - The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible DeltaA(725 - 800 nm) of >0.001 A centimeters(-1), recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells. PMID- 16661258 TI - Plastid Protease Activity and Prolamellar Body Transformation during Greening. AB - Two proteases active in and specific to oat etioplasts and up to 24-hour etiochloroplasts, only very slightly contaminated by other cellular compartments are described. The enzyme showed pH optima of 4.2 (acid) and 6.8 (neutral), temperature optima of 50 C and the highest level of enzyme activity was with prolamellar bodies (PLBs) as substrate. Both enzymes showed evidence of a sulfhydryl reagent requirement, particularly for the neutral enzyme. Levels of both proteases increased up to 4 hours of illumination of leaves, and then sharply decreased with the largest differences exhibited by the neutral protease. The pH values in the plastid stroma indicated that the neutral enzyme was likely to be the most important in PLB transformation. A comparison between plastid associated and extra-plastidic protease activities showed similar properties, except the affinity toward PLBs, which was much higher for plastid proteases (K(m): 0.2 and 1.1 milligrams protein per milliliter, respectively). PMID- 16661259 TI - Alternative Respiratory Pathway: ITS ROLE IN SEED RESPIRATION AND ITS INHIBITION BY PROPYL GALLATE. AB - Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O(2) uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O(2) uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles.The antioxidant propyl gallate was found to inhibit specifically alternative pathway electron transfer in isolated mung bean mitochondria. Half-maximal inhibition occurred with 2 to 5 micromolar propyl gallate. Kinetic analysis indicated that propyl gallate inhibition of the alternative pathway occurred at, or very near, the site of inhibition of the alternative pathway by salicylhydroxamic acid.A high level of lipoxygenase activity was found to be associated with washed mitochondria isolated from a variety of etiolated plant tissues. Most of this lipoxygenase activity could be eliminated from mung bean mitochondria if the mitochondria were purified on a discontinuous sucrose gradient. This indicated that the mitochondrial-associated activity was probably the result of nonspecific adsorption of lipoxygenase onto the mitochondrial membranes during isolation. PMID- 16661260 TI - Malate Decarboxylation by Kalanchoe daigremontiana Mitochondria and Its Role in Crassulacean Acid Metabolism. AB - Mitochondria isolated from Kalanchoe daigremontiana, a Crassulacean acid metabolism plant, decarboxylate added malate to pyruvate at rates of up to 100 micromoles per hour per milligram original chlorophyll in the presence of ADP. Omitting ADP reduces this rate by approximately 50%. Antimycin A inhibits malate decarboxylation and this inhibition could be relieved by addition of aspartate and alpha-ketoglutarate to the mitochondria. Increasing the pH of the external medium inhibited malate decarboxylation; a dramatic decrease in pyruvate production was observed between pH 7.2 and pH 7.4. It is suggested that cytoplasmic pH changes may regulate the contribution of mitochondria to malate decarboxylation in the light in vivo. PMID- 16661261 TI - Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings. AB - A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light. PMID- 16661262 TI - Photophosphorylation in isolated maize bundle sheath chloroplasts and cells. AB - Isolated maize bundle sheath chloroplasts showed substantial rates of noncyclic photophosphorylation. A typical rate of phosphorylation coupled to whole-chain electron transport (methylviologen or ferricyanide as acceptor) was 60 mumol per hour per milligram chlorophyll) with a coupling efficiency (P/e(2)) of 0.6. Partial electron transport reactions driven by photosystem I or II supported phosphorylation with P/e(2) values of 0.2 to 0.3. Thus, two sites of phosphorylation seem to be associated with the photosynthetic chain in much the same way as in spinach chloroplasts.Isolated bundle sheath cells were capable of photosynthetic electron transport with membrane permeant electron carriers (but not with ferricyanide) at rates which were similar to those found in isolated chloroplasts. ATP formation also occurred during electron transport when ADP and phosphate were present in the cell suspension. The rates of photophosphorylation reactions in cells were about 30 to 40% of those found in isolated chloroplasts (maximum rate in cells 80 mumoles ATP per hour per milligram chlorophyll with diaminodurene as electron carrier), with the exception of endogenous photophosphorylation (photophosphorylation without added electron carriers) whose rate was three to four times higher in cells than in chloroplasts. The endogenous photophosphorylation in cells appeared to be coupled to pseudo-cyclic, rather than cyclic, electron transport. It was accompanied by O(2) uptake (when the catalase inhibitor KCN was present), was sensitive to dichlorophenyldimethylurea and methylamine, but was totally insensitive to 20 micromolar antimycin which completely inhibited succinate-supported oxidative phosphorylation in the cells. The implications of these and other phenomena associated with photophosphorylation in bundle sheath cells are discussed. PMID- 16661263 TI - Dependence of Delayed Luminescence upon Adenosine Triphosphatase Activity in Chlorella. AB - Delayed luminescence and fluorescence yield after illumination by a short flash were measured in Chlorella pyrenoidosa in the presence of 3-(3,4-dichlorophenyl) 1,1-dimethylurea. Addition of tri-N-butyl-tin (TNBT), a specific inhibitor of ATPase, drastically increases the life-time of the reduced photosystem II primary acceptor Q(-) and decreases the intensity of delayed luminescence. This indicates a slowing of the charge recombination between the oxidized donor and reduced acceptor of photosystem II centers. No inhibition is observed in isolated chloroplasts when the membrane is permeable to ions, i.e. in the presence of Gramicidin D and KCl.It is suggested that there exists in dark-adapted algae a permanent proton gradient which stimulates the charge recombination process. This proton gradient results from the hydrolysis of a pool of ATP by membrane-bound ATPases and collapses after the addition of TNBT. The long lifetime of this proton gradient (several hours) indicates that the ATP probably comes from the mitochondria.The rate of the back reaction occurring from state S(3) (as defined by Kok, Forbush, and McGloin 1970 Photochem Photobiol 11: 457-475) is more dependent upon the pH gradient than for state S(2). PMID- 16661264 TI - Electron donation to photosystem I. AB - Electron donation to photosystem I was studied in highly resolved particles from spinach. Divalent cations increased the efficiency of electron donation from spinach plastocyanin to P700(+) through a decrease in the apparent K(m) for plastocyanin. Cytochrome f was not an efficient electron donor for P700(+) in the presence or absence of divalent cations. Cytochrome f photooxidation could be observed in the presence of both plastocyanin and divalent cations.The efficiencies of electron donors from eukaryotic and prokaryotic algae to P700(+) were also examined. Divalent cations enhanced the effectiveness of electron donors from eukaryotic organisms, while inhibiting electron donors from prokaryotic organisms. The prokaryotic electron donors were also much more efficient donors than were the electron donors from eukaryotic organisms. A correlation between the K(m) for the electron donor and its isoelectric point suggests that the net charge on the donor protein is a major determinant of the efficiency for electron donation. The data presented raise interesting questions with respect to the evolution of electron donation to photosystem I and the possibility of an additional electron carrier between plastocyanin and P700(+). PMID- 16661265 TI - Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: I. INFLUENCE OF EXOGENOUS SALTS ON OXALOACETATE REDUCTION. AB - Relatively high concentrations of monovalent salts (150 millimolar) stimulated light-saturated uncoupled rates of O(2) evolution linked to oxaloacetic acid (OAA) reduction by intact chloroplasts 2-to 3-fold. In contrast, monovalent salts partially inhibited light-saturated rates of O(2) evolution coupled to CO(2) fixation and uncoupled rates of nitrite reduction. In the presence of high salt concentration, light-saturated rates of electron transport were about equivalent for all three terminal electron acceptors. It is inferred that exogenous monovalent salts have at least two effects on photosynthetic electron transport, independent of photophosphorylation and CO(2) metabolism: a partial inhibitory effect common to OAA, NO(2) (-) and CO(2) reduction and a marked stimulatory effect unique to the photoreduction of OAA.The stimulation of electron transport to OAA was effected by certain exogenous monovalent salts (KCl or NaCl, but not LiCl). Divalent salts (MgCl(2) or CaCl(2)) and high osmotic strength were ineffective. The salt-induced stimulation was eliminated by low concentrations of phosphate or sulfate (>/= millimolar) and by higher concentrations of magnesium (>/=30 millimolar). These results suggest that the ion content of the medium (or cytosol) is potentially important in modulating photosynthetic electron transport events in intact chloroplasts. PMID- 16661266 TI - Mechanism of Linolenic Acid-induced Inhibition of Photosynthetic Electron Transport. AB - The effect of linolenic acid on photosynthetic electron transport reactions in chloroplasts has been localized at a site on the donor side of photosystem I and at two functionally distinct sites in photosystem II.In photosystem I, an increase in the electron transport rate occurs in the presence of 10 to 100 micromolar linolenic acid, followed by a decline in rate from 100 to 200 micromolar linolenic acid. The increase may result from an alteration of membrane structure that allows greater reactivity of the artificial donors 2,6 dichlorophenolindophenol (DPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine with plastocyanin. The decrease is due to loss of plastocyanin from the membrane since addition of purified plastocyanin to treated and washed chloroplasts leads to the reestablishment of photosystem I rates.In photosystem II, a reversible site and an irreversible site of inhibition have been located. At the irreversible site, there is a time-dependent loss of the loosely bound pool of Mn implicated in the water-splitting mechanism. At the reversible site, the photochemical charge separation is rapidly inhibited as evidenced by the high initial fluorescence yield upon illumination and the inhibition of artificial donor reactions in NH(2)OH-washed chloroplasts. When chloroplasts are washed after treatment with linolenic acid, the fluorescence returns to its original low value and there is a resumption of artificial donor activity from diphenylcarbazide --> DPIP. This reversible inhibition of the photoact is a unique characteristic of linolenic acid and suggests evidence for a new mode of inhibition of photosystem II. PMID- 16661267 TI - Effects of Chromatic Adaptation on the Photochemical Apparatus of Photosynthesis in Porphyridium cruentum. AB - Cells of Porphyridium cruentum were grown in different colors of light which would be absorbed primarily by chlorophyll (Chl) (red and blue light) or by the phycobilisomes (green or two intensities of cool-white fluorescent light), and samples of these cells were frozen to -196 C for measurements of absorption and fluorescence emission spectra. Cells grown in the high intensity white light had least of all of the photosynthetic pigments, a higher ratio of carotenoid/Chl, but essentially the same ratio of phycobilin to Chl as cells grown in the low intensity white light. The ratio of photosystem II (PSII) to photosystem I (PSI) pigments was affected by light quality; the ratios of phycobilin to Chl and of short wavelength (PSII) Chl to long wavelength (PSI) Chl were both greater in the cells grown in red or blue light.Light quality also exerted a strong influence on the structural and functional organization of the photochemical apparatus. Data on the relative optical cross-sections of PSI and PSII as a function of excitation wavelength indicate that cells grown in light absorbed primarily by the phycobilisomes package a large fraction of their Chl into PSI (PSI Chl/PSII Chl approximately 20), whereas cells grown in light absorbed by Chl distribute their Chl much more equitably (PSI Chl/PSII Chl approximately 1.5). In both types of cells the phycobilisomes transfer their excitation energy predominantly to PSII Chl with little or no direct energy transfer to PSI, but the yield of energy transfer from PSII to PSI is approximately twice as large for cells grown in the phycobilin wavelengths of light. These differences in functional organization and energy distribution account for the physiological expressions of chromatic adaptation. The effects of chromatic adaptation on O(2) evolution can be predicted from our calculations of energy distribution between PSI and PSII for cells grown in the different colors of light. PMID- 16661268 TI - Light-driven Uptake of Oxygen, Carbon Dioxide, and Bicarbonate by the Green Alga Scenedesmus. AB - Mass spectrometric techniques were used to study several aspects of the competition between O(2) and species of inorganic carbon for photosynthetically generated reducing power in the green alga, Scenedesmus.In contrast to wild type, no appreciable light-driven O(2) uptake was observed in a mutant lacking photosystem I. It is concluded that the carbon cycle-independent reduction of O(2) occurs at the expense of photosystem I-generated reducing equivalents.The commonly observed differences between CO(2)-grown and air-grown Scenedesmus with respect to CO(2) uptake and glycolate formation cannot be ascribed to differences in their capacity for light-driven O(2) uptake. There were no intrinsic differences found in O(2) uptake capacity between the two physiological types under conditions in which CO(2) was saturating or CO(2) uptake was inhibited. It was only under CO(2)-limited conditions that pronounced differences between the two physiological types were observed. This fact suggests that differences in O(2) metabolism and sensitivity between the two types really reflect differences in their capacity to assimilate inorganic carbon; in this respect they are analogous to C(3) and C(4) plants.The hypothesis that air-grown Scenedesmus can assimilate HCO(3) (-) by directly monitoring the time course of dissolved CO(2), O(2) uptake, and O(2) evolution in illuminated algal suspensions at alkaline pH was tested. Inasmuch as the measuring technique employed was fast compared to the nonenzymic equilibration of the inorganic carbon species, it was possible to determine the degree to which the CO(2) concentration deviated from equilibrium (with the other inorganic carbon species) during the course of illumination. The observed kinetics in air-grown and CO(2)-grown algae in the presence and absence of carbonic anhydrase, and a comparison of these kinetics with theoretical (computer-generated) time courses, support the idea that air-adapted algae are able to assimilate HCO(3) (-) actively at a high rate. The data suggest that these algae preferentially assimilate CO(2) and supply the balance of their needs by taking up HCO(3) (-). Since (unlike C(4) plants) these algae have no special CO(2) pump, and thus have a relatively low affinity for CO(2), HCO(3) (-) assimilation is the major carbon uptake process at alkaline pH even when the total CO(2) is present in millimolar concentrations. PMID- 16661269 TI - A Light-dependent Protein Kinase Activity of Chloroplasts. AB - A protein kinase activity from spinach chloroplasts, tightly associated with the thylakoid membranes, has been solubilized and partially characterized. This membrane-bound protein kinase is stimulated by light and electron transport activity through photosystem II appears to be required for stimulation.Electron transport inhibitors like 3,4-dichlorophenyl-1,1-dimethylurea, Tris, and NH(2)OH treatments, inhibit the light activation process. Furthermore, after Tris inhibition, the protein kinase activity is restored by washing the Tris-treated chloroplasts with dichlorophenol indophenol plus ascorbate. The protein kinase remains active in the dark after short illumination periods, suggesting that a product of electron transport may be involved in light activation.Two endogenous substrates of the protein kinase in thylakoid membranes are the N,N' dicyclohexylcarbodiimide-reactive proteolipid and the light-harvesting chlorophyll-protein complex. The membrane-bound protein kinase also phosphorylates externally added histone. PMID- 16661270 TI - Flash Inactivation of Oxygen Evolution: IDENTIFICATION OF S(2) AS THE TARGET OF INACTIVATION BY TRIS. AB - Brief saturating light flashes were used to probe the mechanism of inactivation of O(2) evolution by Tris in chloroplasts. Maximum inactivation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting O(2) evolving catalyst of photosystem II (S(2)-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: (a) hydroxylamine preequilibration caused a three-flash delay in the inactivation pattern; (b) the lifetimes of the Tris-inactivable and S(2)-states were similar; and (c) reagents accelerating S(2) deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II(s), as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH(3) was shown to be a competitive inhibitor of the Tris-induced inactivation. S(2)-dependent inactivation was inhibited by glutaraldehyde fixation of chloroplasts, possibly suggesting that inactivation proceeds via conformational changes of the S(2)-state. PMID- 16661271 TI - A method for enzymic extraction and the measurement of chloroplast RNA. AB - A method is described for measuring RNA associated with chloroplast thylakoid membranes. Washed thylakoids are incubated with ribonucleases A and T(1), under low Mg(2+) conditions, to release hydrolyzed RNA into solution. After removing the membranes by centrifugation, the mono- and oligonucleotides are adsorbed by Dowex 1-X2 in miniature columns made from Pasteur pipettes, and then eluted with 2 n HCl. RNA is estimated from the absorbance of the eluate at 260 nanometers, with corresponding values obtained by the orcinol reaction for pentose. Polyacrylamide gel electrophoresis patterns of extracted RNA indicate that our current procedures for preparation of thylakoids results in material containing variable and often significant levels of RNA from 80S ribosomes. Thus values for total RNA cannot be used as a valid estimate for the level of 70S ribosomes associated with these membranes, unless an additional procedure is used to estimate the per cent contamination by 80S ribosomes.Recoveries of digested RNA from the Dowex resin of 94 to 98% were obtained with 2 milliliters of HCl eluant, making possible the analysis of thylakoid samples with as little as 4 micrograms of RNA. The procedure involves small columns and only one centrifugation, so that it is useful for obtaining reliable measurements from multiple samples. PMID- 16661272 TI - On the quenching of the fluorescence yield in photosynthetic systems. AB - A modified matrix model describing transfer of excitation energy in the photosynthetic pigment system is discussed. In addition to the antenna pigments and reaction centers of the simple matrix model, a coupling complex is postulated mediating energy transfer between antenna and reaction centers. The values of the parameters describing the transfer properties of the coupling complex can be chosen in such a way that a number of recent unexplained measurements of fluorescence properties of various purple bacteria can be described. If such coupling complexes are present in oxygen evolving organisms, some of their properties must be different from those of purple bacteria. PMID- 16661273 TI - Influence of Hydrogen Peroxide upon Carbon Dioxide Photoassimilation in the Spinach Chloroplast: I. HYDROGEN PEROXIDE GENERATED BY BROKEN CHLOROPLASTS IN AN "INTACT" CHLOROPLAST PREPARATION IS A CAUSAL AGENT OF THE WARBURG EFFECT. AB - Photosynthesis and the Warburg effect (O(2) inhibition of photosynthesis) were evaluated in preparations of intact spinach chloroplasts enriched with varying amounts of lysed chloroplasts. Increasing the ratio of broken to intact plastids resulted in decreased rates of CO(2) assimilation.Hydrogen peroxide when added at 10 or more micromolar also inhibited photosynthesis in these preparations. Inhibition of the photosynthetic rate by both factors was eliminated by addition of catalase. These findings indicate that H(2)O(2) presumably generated by the broken chloroplasts was the causal agent of this inhibition.The Warburg effect also became more pronounced by increasing the level of broken to intact chloroplasts. This effect was examined as a function of added catalase, pH, and O(2) concentration. At 21% O(2) and 0.44 to 0.68 millimolar CO(2), catalase relieved the effect almost completely at pH 7.5, but at pH 8.3, the rate was restored only to about half or less of the control. At pH 7.6, 0.44 millimolar CO(2), and 100% O(2), the effect was only slightly overcome by catalase.A rise in glycolate synthesis by the isolated spinach chloroplast has been shown previously to be coupled to an increase in pH and O(2) (Kow, Robinson, Gibbs 1977 Plant Physiol 60: 492-495; Robinson, Gibbs, Cotler 1977 Plant Physiol 59: 530-534). At 21% O(2), glycolate synthesis was not affected by the addition of catalase at pH 7.5 or 8.3. It is proposed that at 21% O(2) and without some means of removing H(2)O(2), that portion of the Warburg effect attributed to glycolate synthesis has been overestimated at pH values in the order of 7.5. In contrast, that portion of the Warburg effect which was, at alkaline pH, insensitive to catalase represented the stress placed upon the photosynthetic carbon reduction cycle which resulted from an enhanced synthesis of glycolate. At 100% O(2) aeration and pH 7.5 to 8.5, the Warburg effect may also represent O(2)-mediated inhibition of a Calvin cycle enzyme within the intact plastid. PMID- 16661274 TI - Membrane Potential Changes Related to Active Transport of Glycine in Lemna gibba G1. AB - Accumulation of (14)C-labeled glycine and microelectrode techniques were employed to study glycine transport and the effect of glycine on the membrane potential (Deltapsi) in Lemna gibba G1. Evidence is presented that two processes, a passive uptake by diffusion and a carrier-mediated uptake, are involved in glycine transport into Lemna cells. At the onset of active glycine uptake the component of Deltapsi which depended on metabolism was decreased. The depolarized membrane repolarized in the presence of glycine. This glycine-induced depolarization followed a saturation curve with increasing glycine concentration which corresponded to carrier-mediated glycine influx kinetics. The transport of glycine was correlated with the metabolically dependent component of Deltapsi. It is suggested (a) that the transient change in Deltapsi reflects the operation of an H(+)-glycine cotransport system driven by an electrochemical H(+) gradient; and (b) that this system is energized by an active H(+) extrusion. Therefore the maximum depolarization of the membrane consequently depended on both the rate of glycine uptake and the activity of the proton extrusion pump. PMID- 16661275 TI - Cell Wall Metabolism in Ripening Fruit: I. CELL WALL CHANGES IN RIPENING ;BARTLETT' PEARS. AB - ;Bartlett' pear (Pyrus communis) fruits were picked at the mature, green stage and ripened at 20 C. Fruits at different stages of ripeness (based on flesh firmness) were homogenized, and the sugar and uronic acid contents of cell wall and soluble polysaccharides were determined. Substantial amounts of galacturonic acid and arabinose were lost from the wall fraction as the fruit ripened. Most of this cell wall material was recovered, in an 80% (volume/volume) ethanol insoluble form, from the soluble fraction of tissue homogenates. Structural analysis of ethanol-precipitable material indicates that it is an acidic (pectic) polymer-bearing side groups containing variously-linked arabinosyl residues. PMID- 16661276 TI - Cell Wall Metabolism in Ripening Fruit: II. CHANGES IN CARBOHYDRATE-DEGRADING ENZYMES IN RIPENING ;BARTLETT' PEARS. AB - Mature ;Bartlett' pear (Pyrus communis) fruits were ripened at 20 C. Fruits at different stages of ripeness were homogenized, and extracts of the low speed pellet (crude cell wall) were prepared. These extracts contained polygalacturonase, pectin esterase, and activity against seven p-nitrophenyl glycoside substrates. Polygalacturonase, alpha-galactosidase, and alpha mannosidase increased in activity as the fruit ripened. Cellulase and activities against pear wall xylan and arabinan were absent from the extracts. PMID- 16661277 TI - Localization of glycerate kinase and some enzymes for sucrose synthesis in c(3) and c(4) plants. AB - The localization of some key enzymes leading to sucrose synthesis in photosynthetic tissue of C(3) and C(4) species was investigated. These included UDP-glucose (UDPG) pyrophosphorylase, sucrose phosphate synthetase, and glycerate kinase. Whether glycerate kinase is localized exclusively in the chloroplast or partly outside the chloroplast could influence the fate of carbon flow to sucrose through the glycolate pathway.In the C(3) species wheat, intact chloroplasts were isolated from protoplasts. Following separation of the chloroplasts by differential centrifugation and by sucrose density gradient centrifugation, UDPG pyrophosphorylase, sucrose phosphate synthetase, and sucrose synthetase were found outside the chloroplast while glycerate kinase was localized in the chloroplast.In the C(4) species (maize, NADP-malic enzyme-type; Panicum miliaceum, NAD-malic enzyme type and Brachiaria erucaeformis, phosphoenolpyruvate carboxykinase type) the distribution of UDPG pyrophosphorylase and glycerate kinase between mesophyll and bundle sheath cells and their intracellular localization in mesophyll protoplasts was determined. Substantial activity of UDPG pyrophosphorylase was found in both mesophyll and bundle sheath cells while glycerate kinase was localized only in mesophyll cells. From C(4) mesophyll protoplasts, UDPG pyrophosphorylase was found to be cytoplasmic while glycerate kinase appears exclusively localized in the chloroplasts as determined by differential centrifugation and sucrose density gradients.It seems that in both C(3) and C(4) plants, the terminal steps of sucrose synthesis occur exclusively in the cytoplasm while carbon flow in the glycolate pathway to sucrose must occur through glycerate kinase in the chloroplasts. The localization of glycerate kinase in mesophyll cells of C(4) plants may have further implications for intercellular flow of carbon during metabolism in the glycolate pathway in these species. PMID- 16661278 TI - Reduction of auxin transport capacity with age and internal water deficits in cotton petioles. AB - Auxin transport was examined in leaf petioles taken from the upper, middle, and lower leaf canopy of large cotton plants. The ability of petioles to transport auxin decreased with age (position) of the leaves. Plant water deficit reduced transport regardless of age. These correlations support the view that reduced transport capacity of petioles plays a significant role in the induction of abscission of lower or older leaves during water deficits. PMID- 16661279 TI - Biological activities of indoleacetylamino acids and their use as auxins in tissue culture. AB - THE AUXIN ACTIVITIES OF A NUMBER OF INDOLEACETYLAMINO ACID CONJUGATES HAVE BEEN DETERMINED IN THREE TEST SYSTEMS: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-l-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-l-alanine and indoleacetylglycine. The other conjugates inhibit shoot formation weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-l alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid. PMID- 16661280 TI - beta-d-Glucan Hydrolase Activity in Zea Coleoptile Cell Walls. AB - Enzymes dissociated from corn (hybrid B73 x Mo17) seedling cell walls by solutions of high ionic strength possess the capacity to degrade Avena caryopsis glucan. Inhibitor studies disclosed that both endo- and exoenzyme activities were involved and that the reaction sequence paralleled the autolytic solubilization of beta-d-glucan in isolated cell walls.The salt-dissociated exoenzyme activity was strongly inhibited by HgCl(2) and to a lesser extent by parachloromercuribenzoate at a concentration of 100 micromolar. In the absence of these inhibitors, Avena caryopsis glucan was converted to monosaccharide, whereas in the presence of the mercurials, only endoenzyme activity was apparent and the glucan substrate was hydrolyzed yielding products with an average molecular size of 1.5 to 3.0 x 10(4) daltons. Endoenzyme hydrolysis of the caryopsis glucan could not be attributed to the participation of an enzyme specific for mixed linkage substrates.The autolytic capacity of isolated cell walls was similarly affected by inhibitors. In the presence of 100 micromolar HgCl(2), cell walls released from 60 to 80 micrograms per milligram dry weight as polymeric glucan during a 24-hour period. Monosaccharide accounted for less than 2% of the autolytically solubilized products. Analysis of the polymeric glucan product revealed a similarity in molecular size to the products obtained following treatment of Avena caryopsis glucan with salt-dissociated wall protein. The results suggest that among the salt-dissociated proteins are those responsible for the autolytic capacity of isolated cell walls. PMID- 16661281 TI - Utilization of Exogenous Inorganic Carbon Species in Photosynthesis by Chlorella pyrenoidosa. AB - The nature of the inorganic carbon utilized during photosynthesis by Chlorella pyrenoidosa was investigated using three experimental techniques (open gas analysis system with "artificial leaf" or "aqueous" chambers and O(2) electrode system) to measure carbon assimilation. Photosynthesis was studied as a function of pH and CO(2) concentration. The CO(2) concentration was inadequate to meet the requirements of photosynthesis only when HCO(3) (-) was added at high pH. Under all other conditions, the low and constant K(m) (CO(2)), in contrast to the highly variable K(m) (HCO(3) (-)), suggested that CO(2) was the major species utilized.Higher rates of photosynthesis were observed under limiting CO(2) conditions above pH 7.5 but rates of hydration of CO(2) were not consistent with the view that the stimulation in photosynthesis was supported by HCO(3) (-). In the same pH region lower rates of photosynthesis were observed under saturating CO(2) conditions. These conflicting changes seemed not to be related to pH but to some as yet undetermined effect of bicarbonate. No support was obtained for the view that the quantum efficiency was different under conditions where CO(2) was assimilated compared to conditions where CO(2) and HCO(3) (-) could be assimilated, although at saturating CO(2), lower maximal rates of photosynthesis were observed in the latter conditions. PMID- 16661282 TI - Photorespiration and Oxygen Inhibition of Photosynthesis in Chlorella pyrenoidosa. AB - The inhibition of photosynthesis by O(2) in air-grown Chlorella pyrenoidosa was investigated using three experimental techniques (artificial leaf, aqueous method, and O(2) electrode) to measure carbon assimilation. CO(2) response curves were determined under different O(2), pH, and temperature conditions. Regardless of the experimental technique and condition, O(2) inhibition was not evident until a concentration of 50% was reached; V(max) values were reduced whereas K(m) (CO(2)) values were unaffected by the increasing O(2) concentration. The response of photosynthesis to O(2) was independent of CO(2) and HCO(3) (-) concentrations as well as temperature. Relative rates of photosynthesis showed a 4 to 5% stimulation in 2% O(2), a 12% inhibition in 50% O(2), and a 24% inhibition in 100% O(2). The inhibition by 50% O(2) was still reversible after 20 minutes exposure whereas 100% O(2) caused irreversible inhibition after only 4 minutes.The O(2) inhibition is discussed in terms of the oxygenase reaction and a Mehler reaction supporting pseudocyclic electron flow. The results are inconsistent with the proposals that photorespiration exists in these algae and that a CO(2)-concentrating mechanism suppresses the O(2) inhibition of photosynthesis. PMID- 16661283 TI - A Comparison of Purified Host Specific Toxin from Helminthosporium maydis, Race T, and Its Acetate Derivative on Oxidation by Mitochondria from Susceptible and Resistant Plants. AB - NADH or succinate oxidation and malate oxidation were differentially affected in mitochondria from both susceptible and resistant corn by a purified and chemically characterized preparation of host-specific toxin from Bipolaris (Helminthosporium) maydis, race T. NADH and succinate oxidation by susceptible T corn mitochondria were stimulated 50 to 200% with apparent uncoupling from the cytochrome chain at approximately 10(-9)m toxin (5 to 20 ng/ml). Significant inhibition of malate oxidation was observed at slightly higher toxin concentrations, but oxidation was still coupled to ADP utilization. Inhibition of malate oxidation also was observed in N corn (resistant) and soybean mitochondria at approximately 1,000-fold greater concentrations, but stimulation of NADH and succinate oxidation was not found at any toxin concentration tested.A fully acetylated toxin derivative at approximately 1 microgram per milliliter also caused stimulation of NADH or succinate oxidation in T corn mitochondria, but not those of N corn or soybean mitochondria at 100 micrograms per milliliter. Malate oxidation was inhibited to the same extent by toxin acetate with mitochondria from T corn, N corn, and soybean. The blocking of hydroxyl groups in race T toxin by acetyl functions eliminated selectivity toward malate oxidation only. The data suggest that inhibition of malate oxidation is either a separate or secondary effect of selective action of toxin on T corn mitochondria, perhaps by interference with transport in or out of the matrix. Sensitivity of T, but not N, corn mitochondria to purified toxin decays within minutes after pellets are suspended in aqueous osmotica, with no obvious change in mitochondrial integrity. The action of race T toxin seems to involve a labile process, such as ion gradient(s), or an unstable structural conformation of T corn mitochondria. PMID- 16661284 TI - Day/Night Changes in the Sensitivity of Phosphoenolpyruvate Carboxylase to Malate during Crassulacean Acid Metabolism. AB - Phosphoenolpyruvate carboxylase (PEPC) was extracted from Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism, at frequent intervals during a 12-hour light/12-hour dark cycle. Inhibition of PEPC by malate was followed at pH 8.0 and 7.5, 1 minute after homogenization of leaves. PEPC was more sensitive to malate during the light than during the dark periods and inhibition by malate was more pronounced at pH 7.5 than 8.0. For example, PEPC was not or only slightly inhibited by 0.5 millimolar malate during the dark period at both pH values and the rates per milligram chlorophyll were about the same. During the light period, 0.5 millimolar malate resulted in a 20 to 30% reduction of PEPC activity at pH 8.0 and a 80 to 90% reduction at pH 7.5. These and other experiments, in which plants were kept in prolonged dark periods, indicate that the increase in sensitivity of PEPC to malate is correlated with the change from acidification to deacidification in the tissue. These interactions account for apparent changes in pH response of PEPC in crude extracts assayed at different times of the day/night cycle.The experiments suggest the existence of two distinct states of PEPC in vivo, which differ in their susceptibility to malate inhibition during a day/night cycle. The change in properties is discussed in relation to the regulation of PEPC during Crassulacean acid metabolism. PMID- 16661285 TI - Influence of light and temperature on monoterpene emission rates from slash pine. AB - There is a growing awareness of vegetation's role as a source of potentially reactive hydrocarbons that may serve as photochemical oxidant precursors. This study assessed the influence of light and temperature, independently, on monoterpene emissions from slash pine (Pinus elliottii Engelm.). Plants were preconditioned in a growth chamber, then transferred to an environmentally controlled gas exchange chamber. Samples of the chamber atmosphere were collected; the monoterpenes were concentrated cryogenically and measured by gas chromatography. Five monoterpenes (alpha-pinene, beta-pinene, myrcene, limonene, and beta-phellandrene) were present in the vapor phase surrounding the plants in sufficient quantity for reliable measurement. Light did not directly influence monoterpene emission rates since the emissions were similar in both the dark and at various light intensities. Monoterpene emission rates increased exponentially with temperature (i. e. emissions depend on temperature in a log-linear manner). The summed emissions of the five monoterpenes ranged from 3 to 21 micrograms C per gram dry weight per hour as temperature was increased from 20 to 46 C. Initially, emission rates from heat-stressed needles were similar to healthy needles, but rates decreased 11% per day. Daily carbon loss through monoterpene emissions accounted for approximately 0.4% of the carbon fixed during photosynthesis. PMID- 16661286 TI - Carbon dioxide fixation and related properties in sections of the developing green maize leaf. AB - Light and dark (14)CO(2) assimilation, pulse-chase ((14)CO(2) followed by (12)CO(2)) labeling experiments both in the light and in the dark, photorespiratory activity and some enzymes (ribulose 1,5-bisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, and NADP-malic enzyme) were followed in sections of 2.5 centimeters from the base (younger tissue) to the tip (oldest tissue) of the green maize leaf. Tissue was taken from the third leaf of 12- to 16-day-old plants consisting of sections 0 to 2.5 centimeters (base), 4.5 to 7.0 centimeters (center) and 9.0 to 11.5 centimeters (top) measured from the base. Some of these properties were also determined in the intact leaves of 4-day old maize plants.Electron microscopy indicated a Kranz anatomy in all sections. Differentiation into mesophyll granal chloroplasts and bundle sheath agranal chloroplasts had taken place only in the center and top pieces.All of the sections contained PEP carboxylase, RuBP carboxylase, and NADP-malic enzyme. The ratio of PEP:RuBP carboxylase increased from 3.03 (top) to 4.66 (base) whereas the PEP carboxylase:NADP-malic enzyme ratio rose from 2.87 (top) to 9.57 (base).Under conditions of light or dark, the majority of the newly incorporated (14)CO(2) was found in malate and aspartate in all sections and in 4-day-old leaves. The (14)C-labeling pattern typical of C(4) plants was present in the center and top sections and to a lesser extent in the 4-day-old leaves. In the base tissue, the percentage of radioactivity in malate and aspartate remained relatively constant both during photosynthesis and pulse-chase experiments. In contrast, radioactivity in glycerate-3-phosphate decreased with time coupled to an increase in sugar phosphates. To account for the isotopic pattern in the base tissue, parallel fixation by PEP carboxylase and RuBP carboxylase was proposed with the photosynthetic carbon reduction cycle functioning to some extent independently within the bundle sheath chloroplasts. The apparent lack of cooperation between the mesophyll and bundle sheath cells may have been due to inadequate levels of NADP-malic enzyme required for shuttling carbon as CO(2) from the PEP carboxylase products to the Calvin cycle. PMID- 16661287 TI - Salt-Induced Metabolic Changes in Dunaliella salina. AB - An increase of medium NaCl concentration induces Dunaliella cells to evolve O(2) photosynthetically even in the absence of CO(2). This NaCl-induced O(2) evolution may reflect the induced conversion of reserve carbohydrate to glycerol. The quantum yield for the NaCl-induced O(2) evolution, in the absence of CO(2), is 1.5-fold higher than that obtained for CO(2) fixation. Since the synthesis of glycerol from reserve carbohydrate in the absence of CO(2) requires only 0.5 ATP/NADPH, whereas photosynthesis requires at least 1.3 ATP/NADPH, it is concluded that the ATP/2e(-) ratio coupled to NADP reduction in Dunaliella is lower than required for CO(2) fixation. PMID- 16661288 TI - Chlorophyll proteins of photosystem I. AB - Data are presented which suggest the existence of a light-harvesting pigment protein complex which is functionally and structurally associated with photosystem I (PSI) reaction centers. These observations are based on techniques which allow isolation of PSI using minimal concentrations of Triton X-100. Properties of density and self aggregation allowed purification of a "native" PSI complex.The isolated PSI particles appear as 106 A spherical subunits when viewed by freeze fracture microscopy. When incorporated into phosphatidyl choline vesicles, the particles lose self-aggregation properties and disperse uniformly within the lipid membrane.The isolated PSI preparation contains 100 +/- 10 chlorophylls/P(700) (Chl a/b ratio greater than 18); this represents a recovery of 27% of the original chloroplast membrane Chl. These particles were enriched in Chl a forms absorbing at 701 to 710 nm. Chl fluorescence at room temperature exhibited a maximum at 690 nm with a pronounced shoulder at 710 nm. At 77 K, peak fluorescence emission was at 736 nm; in the presence of dithionite an additional fluorescence maximum at 695 nm was obtained at 77 K. This dual fluorescence emission peak for the PSI particles is evidence for at least two Chl populations within the PSI membrane subunit. The fluorescence emission observed at 695 nm was identified as arising from the core of PSI which contains 40 Chl/P(700) (PSI-40). This core complex, derived from native PSI particles, was enriched in Chl a absorbing at 680 and 690 nm and fluorescing with maximal emission at 694 nm at 77 K. PSI particles consisting of the PSI core complex plus 20 to 25 Chl antennae (65 Chl/P(700)) could also be derived from native PSI complexes. These preparations were enriched in Chl a forms absorbing at 697 nm and exhibited a 77 K fluorescence emission maximum at 722 nm.A comparison of native PSI particles which contain 110 Chl/P(700) (PSI-110) and PSI particles containing 65 Chl/P(700) (PSI-65) provides evidence for the existence of a peripheral Chl-protein complex tightly associated in the native PSI complex. The native PSI subunits contain polypeptides of 22,500 to 24,500 daltons which are not found in the PSI-65 or PSI 40 subfractions. It is suggested that these polypeptides function to bind 40 to 45 Chl per structural complex, including the Chl which emits fluorescence at 736 nm.A model for the organization of Chl forms is presented in which the native PSI membrane subunit consists of a reaction center core complex plus two regions of associated light-harvesting antennae. The presence of energy "sinks" within the antennae is discussed. PMID- 16661289 TI - A developmental study of photosystem I peripheral chlorophyll proteins. AB - An isolated "native" photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae ( approximately 45 Chl/P(700)) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association.Chloroplasts were isolated from a Chl b-less barley mutant and from partially-developed cucumber cotyledons (greened under intermittent illumination [ImL] chloroplasts) and were compared to control chloroplasts isolated from wild type barley and mature cucumber. Both the mutant and ImL chloroplasts exhibited a long wavelength fluorescence maximum at 724 nanometers at 77 K as compared to 735 to 738 nanometers emission maximum in the respective controls. Both the mutant and ImL chloroplasts were deficient in polypeptides of 21,500 to 24,500 daltons which were present in control membranes and in PSI fractions isolated from control membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range paralleled the appearance of PSI Chl species fluorescing at long wavelength ( approximately 735 nm).The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 Chl/P(700) (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus approximately 45 Chl/P(700). The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts. PMID- 16661290 TI - Ribulose-1,5-bisphosphate Carboxylase/Oxygenase and Polyphenol Oxidase in the Tobacco Mutant Su/su and Three Green Revertant Plants. AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was crystallized from a heterozygous tobacco (Nicotiana tabacum L.) aurea mutant (Su/su), its wild type sibling (su/su), and green revertant plants regenerated from green spots found on leaves of haploid Su plants. No differences were found in the specific activity or kinetic parameters of this enzyme, when comparing Su/su and su/su plants of the same age, which had been grown under identical conditions. The enzyme crystallized from revertant plants was also identical to the enzyme from wild-type plants with the exception of one clone, designated R2. R2 has a chromosome number approximately double that of the wild-type (87.0 +/- 11.1 versus 48). The enzyme from R2 had a lower V(max) for CO(2), although the K(m) values were identical to those for the enzyme from the wild-type plant. The enzyme from all mutant plants had identical isoelectric points, identical molecular weight as demonstrated by migration on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the same ratio of large to small subunits as the enzyme from the wild-type. The large subunit of the enzyme from tobacco leaves exhibited a different electrophoretic pattern than did the large subunit from spinach; there were two to three bands on SDS-polyacrylamide gels for the tobacco enzyme whereas the enzyme from spinach had only one species of large subunit.Total polyphenol oxidase activity was the same in leaves from the heterozygous mutant (Su/su) and wild-type (su/su) plants when correlated with developmental age as represented by morphology rather than by the chronological age of the plants. There was a marked increase in the soluble activity of this enzyme with increasing age of both plant types and also as a result of varying environmental conditions. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity correlated inversely with increases in the soluble activity of polyphenol oxidase in crude homogenates from which the carboxylase/oxygenase was crystallized over a generation of Su/su and su/su plants. Criteria are outlined for determining if differences in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase are caused by an effect of polyphenol oxidase activity and/or by some other extrinsic parameter. PMID- 16661291 TI - Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers. AB - The poly(A)-containing mRNA from barley aleurone layers pretreated with gibberellic acid has been purified by phenol-chloroform extraction and repeated oligo[d(pT)]-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitro translation systems with greater than 50% of the synthesized protein being alpha-amylase. The mRNA for alpha-amylase has been further purified by dimethylsulfoxide-formamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000. PMID- 16661292 TI - Biochemical and Cytological Analyses of RNA Synthesis in Kinetin-treated Pea Root Parenchyma. AB - Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinitiating DNA synthesis. Few cells synthesize RNA in the 1st hour of culture. In the presence of kinetin/auxin, the nuclear labeling index increases 2.5-fold as compared to control cultures. The RNA synthesis response has an apparent lag period of 2-4 hours as shown by double label ([(3)H]adenosine/[(14)C]adenosine) experiments. Qualitatively, the RNA synthesized at 4-6 hours sediments between 18S and 5S. The RNA synthesized at 14-16 hours and 24-26 hours is primarily ribosomal RNA when kinetin is present. In the absence of kinetin, no clear pattern of RNA synthesis emerges.The data are interpreted to mean that kinetin treatment elicits RNA synthesis in a small proportion of the population initially and this may involve messenger-like RNA. Later, more cells synthesize RNA and this is primarily rRNA. PMID- 16661293 TI - Starch depletion and sugars in developing cotton leaves. AB - Cotton plants (cv. Coker 100) were exposed to a 14-hour dark period. Starch degradation occurred with no accumulation of sugars due mainly to translocation. Considerable amounts of starch degradation products however were detected from leaves after phloem transport was blocked. A minor component (10 to 25% of total starch) with a linear structure, amylose, was preferentially degraded, whereas the major multiple-branched component (about 80%), amylopectin, showed an increasing resistance to degradation with leaf age. This relationship was also shown by the decreasing iodine-binding capacity of unit starch with increasing leaf age. The structural resistance of amylopectin to enzymic dark degradation was one of the barriers to starch dissolution in cotton. PMID- 16661294 TI - An apparent anomaly in peanut leaf conductance. AB - Conductance to gaseous transfer is normally considered to be greater from the abaxial than from the adaxial side of a leaf. Measurements of the conductance to water vapor of peanut leaves (Arachis hypogaea L.) under well watered and stress conditions in a controlled environment, however, indicated a 2-fold higher conductance from the adaxial side of the leaf than from the abaxial. Studies of conductance as light level was varied showed an increase in conductance from either surface with increasing light level, but conductance was always greater from the adaxial surface at any given light level. In contrast, measurements of soybean (Glycine max [L.] Merr.) and snapbean (Phaseolus vulgaris L.) leaf conductance showed an approximate 2-fold greater conductance from the abaxial surface than from the adaxial. Approximately the same number of stomata were present on both peanut leaf surfaces and stomatal size was similar. Electron microscopic examination of peanut leaves did not reveal any major structural differences between stomata on the two surfaces that would account for the differences in conductance. Light microscope studies of leaf sections revealed an extensive network of bundle sheaths with achloraplastic bundle sheath extensions; the lower epidermis was lined with a single layer of large achloraplastic parenchyma cells. Measurements of net photosynthesis made on upper and lower leaf surfaces collectively and individually indicated that two-thirds of the peanut leaf's total net photosynthesis can be attributed to diffusion of CO(2) through the adaxial leaf surface. Possibly the high photosynthetic efficiency of peanut cultivars as compared with certain other C(3) species is associated with the greater conductance of CO(2) through their upper leaf surfaces. PMID- 16661295 TI - Endogenous Cytokinins in Bougainvillea ;San Diego Red': I. OCCURRENCE OF CYTOKININ GLUCOSIDES IN THE ROOT SAP. AB - Using chromatographic, chemical, and enzymic techniques, 11 compounds capable of stimulating the division of soybean callus cells were tentatively identified in the root sap of Bougainvillea ;San Diego Red.' These cytokinin-like compounds included phosphorylated and glucosylated forms of zeatin, ribosylzeatin, and their dihydro derivatives. In addition, isopentenyl adenosine and isopentenyl adenine were apparently also present. The occurrence of glucosylated derivatives in the root sap of plants has not been substantiated previously. PMID- 16661296 TI - Metabolism of Oat Leaves during Senescence: VI. CHANGES IN ATP LEVELS. AB - The ATP content of 7-day-old Avena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. l-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, i.e. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling. PMID- 16661297 TI - Calcium Inhibition of Pyrophosphatase in Crude Plant Extracts: IMPLICATION OF SOLUBLE CALCIUM IN C(4) PHOTOSYNTHESIS. AB - Assays of alkaline pyrophosphatase activity in crude plant extracts are inhibited by soluble calcium coextracted with the enzyme from leaf tissues. Calcium concentrations in most extracts are high enough to interfere seriously with the assay.All C(4) plants examined kept their soluble calcium and interference with pyrophosphatase activity at very low levels, whereas the C(3) plants covered the whole range of soluble calcium concentration. The hypothesis is advanced that low soluble calcium might be a prerequisite for high activity of pyrophosphatase and the C(4) pathway. PMID- 16661298 TI - Movement of C-labeled Assimilates into Kernels of Zea mays L: III. AN ANATOMICAL EXAMINATION AND MICROAUTORADIOGRAPHIC STUDY OF ASSIMILATE TRANSFER. AB - Basal tissue of developing maize kernels was examined by light micros-copy and by scanning and transmission electron microscopy. Plasmodesmata occur in pedicel and endosperm parenchyma but were not seen between the placento-chalazal cells and basal endosperm transfer cells. A layer of noncellular material separates the transfer cells from the placentochalazal cells. Microautoradiography of (14)C labeled assimilates entering the kernels revealed that incoming sugars are not confined to the apoplast, but rather are present in the cytoplasm and vacuoles of pedicel and endosperm cells. No specific accumulation of radioactivity was seen in any particular tissue, although at later sampling times, a higher grain density in the pedicel than the endosperm indicated a general buildup of sugars in the pedicel. A possible model for sugar movement into developing kernels is discussed. PMID- 16661299 TI - Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES. AB - PLASMA MEMBRANE PREPARATIONS FROM SOYBEAN ROOT AND HYPOCOTYL CONTAINED THE FOLLOWING FREE STEROLS: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.Electron microscope studies indicated that all root plasma membrane preparations were equivalent in terms of relative purity. Hypocotyl membrane preparations contained significantly greater levels of contaminating membrane components.Root plasma membrane fractions were between 70 and 80% pure as determined by staining with the phosphotungstic acid-chromic acid procedure (PACP). Staining was most definitive for vesicles present in complete cross section. Electron micrographs showed that vesicles treated with concanavalin A (Con A)-ferritin were extensively labeled at the outer surface indicating the presence of mannosyl and/or glucosyl residues at the vesicle surface. Densities of ferritin were highest on vesicles present in oblique section. PACP and Con A ferritin were thus complementary with respect to topological specificity.The percentage of Con A-ferritin-labeled and/or PACP-stained vesicles in plasma membrane root preparations was greater than 80%. Con A did not bind in purified tonoplast preparations, and binding was reduced in regions of low PACP reactivity in a root membrane fraction containing a lowered proportion of plasma membrane. Con A specificity for the plasma membrane in subcellular membrane preparations is discussed. PMID- 16661300 TI - Light and the correlation of chloroplast development and coupling of phosphorylation to electron transport. AB - Coupling of phosphorylation to electron transport was examined by measuring the photosynthetic control ratio for broken wheat plastids isolated from seedlings at different greening stages. The photosynthetic control ratio progressively increased during greening and tight coupling was noted after granal stacking and thylakoid elongation. ADP impaired nonphosphorylating (state 2) electron transport rates of plastids at extremely early stages of greening and interfered with photosynthetic control measurements. Partially developed plastids exhibited low nonphosphorylating electron flow rates but did not exhibit high phosphorylating or uncoupled electron transport rates to the same extent as nearly developed plastids. Prolamellar body dispersal, primary thylakoid production, and the development of photosynthetic control were stimulated equally by 48 minutes of low irradiance, in cycles of 2 minutes every 2 hours, or by 9 hours of continuous light of moderate irradiance. Wheat plastids that greened for 6 hours in continuous light of moderate intensity did not exhibit photosynthetic control or much differentiation beyond the etioplast stage. It is concluded that plastid differentiation and the development of photosynthetic control early in greening under continuous light were limited by developmental time (dark time) rather than by either light intensity or duration. PMID- 16661301 TI - Stomatal closure and photosynthetic inhibition in soybean leaves induced by petiole girdling and pod removal. AB - The presence of strong sinks of photoassimilates is thought to stimulate photosynthesis by minimizing photosynthetic end product accumulation in leaves. This hypothesis was examined in soybeans (Glycine max [L] Merr.) with treatments designed to alter the phloem translocation of photoassimilates out of source leaves. Pod removal and petiole girdling resulted in 70 and 90% reductions, respectively, in leaf CO(2) exchange rate. Reductions of similar magnitude also were observed in stomatal diffusive conductivity.Time course data showed that CO(2) exchange rates as well as stomatal conductivities were significantly reduced 0.5 hour after girdling and 5 hours after pod removal, thus suggesting that stomatal closure was the cause of the reduced rates of photosynthesis.Mesophyll conductivities to CO(2), calculated from gas exchange data, were not affected by the treatments. Radiocarbon assimilation rates by leaf slices floated in KH(14)CO(3) solutions were not reduced by pod removal and were reduced only 10% by girdling. It was concluded that treatments which block or slow translocation from source leaves reduce their photosynthetic rate by inducing stomatal closure. PMID- 16661302 TI - The effect of glyoxylate on photosynthesis and photorespiration by isolated soybean mesophyll cells. AB - Incubating isolated soybean leaf mesophyll cells with glyoxylate increased the rates of CO(2) fixation by as much as 150%. In order to cause this stimulation, the glyoxylate must be presented to the cells before the NaHCO(3). Significant stimulation was observed 15 seconds after beginning the glyoxylate treatment. The glyoxylate-dependent stimulation was increased by high O(2) concentrations and decreased by high CO(2) concentrations. Glyoxylate treatment resulted in a 71% inhibition in the rate of CO(2) incorporation into glycolate and glycine. Glyoxylate may be stimulating net photosynthesis solely by decreasing photorespiration or it may be increasing the amount of CO(2) fixed by both decreasing photorespiration and increasing gross photosynthesis. Ribulose bisphosphate carboxylase, when preactivated and assayed in situ, was unaffected by the glyoxylate treatment. PMID- 16661303 TI - Pyrimidine Pathway in Boron-deficient Cotton Fiber. AB - Cotton ovules cultured in an insufficiency of boron (10 micromolar), showed inhibition of fiber growth by the ninth day in culture. Averaging data from eight to eleven days of culture under these conditions, total incorporation of [6 (14)C]orotic acid into fiber was inhibited by 59%. Inhibition was evident in all radioactively labeled pools, indicating that the effect may be at the membrane transport level or at an early stage of orotic acid metabolism. On a per cent basis, incorporation into RNA under boron deficiency was higher than under sufficiency. The effect is greater on the eighth day of culture, with a decreasing difference from controls up to the eleventh day. Conversely, the per cent incorporation into UDP-glucose was lower under boron deficiency than in controls, having a more or less constant value from 8 to 11 days of culture. Thus, a primary event of boron deficiency in cotton fiber culture is an alteration in the flow of metabolites through the pyrimidine synthesis pathway. PMID- 16661304 TI - Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea mays L. Leaves. AB - Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3 phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10(-5)m and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions. PMID- 16661305 TI - Distribution of Metabolites between Chloroplast and Cytoplasm during the Induction Phase of Photosynthesis in Leaf Protoplasts. AB - A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of (14)C-labeled products during photosynthesis. In the dark, CO(2) fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis. PMID- 16661306 TI - Lemna paucicostata Hegelm. 6746: DEVELOPMENT OF STANDARDIZED GROWTH CONDITIONS SUITABLE FOR BIOCHEMICAL EXPERIMENTATION. AB - Photoautotrophic and mixotrophic growth of Lemna paucicostata Hegelm. 6746 (formerly Lemna perpusilla Torr. 6746) was investigated to establish standardized conditions for biochemical studies. Optimal temperature for growth was 29 to 30 C. The medium used previously (Datko AH, Mudd SH, Giovanelli J 1977 J Biol Chem 252: 3436-3445) was modified by inclusion of NH(4)Cl, decreasing macronutrient and ethylenediamine tetraacetate concentration, increasing micronutrient concentration, and inclusion of bicarbonate (for photoautotrophic growth) or 2-(N morpholino)ethanesulfonic acid (for mixotrophic growth) buffers. Varying the sulfate concentration between 14 and 1 millimolar had no effect on growth. For photoautotrophic growth in the new medium (medium 4), the effects of CO(2) concentration, light intensity, and pH were measured. Under the optimal conditions, a multiplication rate (MR) of 300 to 315, equivalent to a doubling time of 23 to 24 hours was obtained. Addition of glutamine or asparagine did not increase this MR. For mixotrophic growth in low light, the effects of sucrose concentration and pH were determined. Under optimal conditions, MR was 210. A concentration of sucrose less than maximal for growth was chosen for the medium for experiments which will include (14)C-labeling of intermediates. MR under these conditions was 184. Growth was equally good in medium 4 and in half strength Hutner's medium when sulfate was high (0.4 to 1 millimolar), but better in medium 4 when sulfate was low (20 micromolar). Growth rates could be restored to normal in half-strength Hutner's with low sulfate by decreasing the molybdate concentration.By modifying medium 4 to contain very low amounts of sulfate, and by preconditioning medium and plants, it was shown that there was an increment in plant protein of approximately 2.5 micrograms per nanomole of added MgSO(4). Colonies undergoing sulfur limitation exhibited a slow growth rate and a high frond to colony ratio. Molybdate and selenate produced growth inhibition reversible by sulfate. Conditions were developed in which the plants could be maintained indefinitely in the presence of either molybdate or selenate in altered metabolic steady-states with lowered growth rates and protein per frond. PMID- 16661307 TI - Lemna paucicostata Hegelm. 6746: LIFE CYCLE AND CHARACTERIZATION OF THE COLONY TYPES IN A POPULATION. AB - The sequence of frond emergence and the intervals required for daughter colony separation have been determined for Lemna paucicostata Hegelm. 6746 growing under standardized conditions. After separation of a new mother colony, the first daughter colony is produced from the left meristematic pocket and separates after approximately 60 hours, the second daughter, produced from the right pocket, separates after a further 30 hours, and the third daughter, again from the left, after a further 40 hours. The pattern of alternating longer and shorter intervals for separation of daughters continues throughout the life of the mother colony.Protein contents of fronds and whole colonies were determined either by chemical methods or by labeling to isotopic equilibrium with (35)SO(4) (2-). The smallest fronds measured were 5 to 6% of the final area they would attain when fully expanded and contained 10% of the protein they would finally contain. Thus, most protein accumulation occurred during the phase of rapid frond expansion rather than in an earlier primordial stage. The specific rates of protein accumulation were 0.027 to 0.030 micrograms per microgram protein per hour during rapid frond expansion and relatively constant thereafter at 0.014 to 0.019 for whole colonies until at least four daughter colonies had separated.A substantial amount of (35)S is transferred directly from the mother frond to its attached daughter fronds. The results are consistent with the transfer of soluble compounds arising through turnover of protein in the mother frond.Tree diagrams and a mathematical treatment were utilized to relate the distribution of colony types and the over-all doubling time in a large population of Lemna colonies to the doubling times and modes of division of the individual colonies within that population. It was shown that a population with the growth properties described increases exponentially and that within a relatively few generations reaches an equilibrium population distribution which is independent of the initial proportions of the members of the population. The measured distribution of colony types producing odd-numbered as compared to those producing even-numbered daughters was the same as that predicted by the theoretical analyses. Once a mass culture of Lemna paucicostata has attained its equilibrium distribution of colony types, under our standard conditions the proportions of colonies in which the indicated daughter will be next to separate will be: 60%, first; 15%, second; 11%, third; 5%, fourth; 8% fifth or higher. Thus, about 91% of colonies will have produced no more than four daughter colonies. For at least this period the specific rate of protein accumulation is relatively constant and senescent changes in mother fronds are rare. In the equilibrium distribution, 39% of colonies will themselves be first daughters, 25% will be second daughters, 14% will be third daughters, 9% fourth, and 13% greater than fourth daughters. These combined results suggest that obtaining representative samples of colonies from such a population for biochemical studies should be relatively simple. PMID- 16661308 TI - Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L. AB - Developing pea cotyledons incorporate radioactivity in vivo from [(14)C]glucosamine and [(14)C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [(14)C]glucosamine and [(14)C]mannose is also incorporated into glycopeptides. Considerable redistribution of [(14)C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N acetylglucosamine to asparagine glycopeptide linkage has been isolated from [(14)C]glucosamine-labeled glycoproteins. PMID- 16661310 TI - Synthesis of Nitrate Reductase in Chlorella: I. EVIDENCE FOR AN INACTIVE PROTEIN PRECURSOR. AB - Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris was studied under inducing conditions, i.e. with cells grown on ammonia and then transferred to nitrate medium. Cycloheximide (but not chloramphenicol) completely inhibited synthesis of the enzyme, but only if it was added at the start (i.e. at the time of nitrate addition) of the induction period. Cycloheximide inhibition became less effective as induction by nitrate proceeded. Enzyme from small quantities of culture (1 to 3 milliliters of packed cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeled arginine into nitrate reductase was measured in the presence and absence of cycloheximide. Conditions were found under which the inhibitor completely blocked the incorporation of labeled amino acid, but only slightly decreased the increase in nitrate reductase activity. The results indicate that synthesis of nitrate reductase from amino acids proceeds by way of a protein precursor which is inactive enzymically. PMID- 16661309 TI - Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction. AB - The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity. PMID- 16661311 TI - Synthesis of Nitrate Reductase in Chlorella: II. EVIDENCE FOR SYNTHESIS IN AMMONIA-GROWN CELLS. AB - Antiserum was prepared against nitrate reductase (EC 1.6.6.1) purified to homogeneity from Chlorella vulgaris Beijerinck. Both crude antiserum and anti nitrate reductase antibodies prepared from it were used as re-agents to study the synthesis of nitrate reductase. Cell extracts from cultures which were grown with ammonia salts as the sole source of nitrogen contained almost no active enzyme. These extracts did contain material which binds to antibody and is thus immunologically related to purified nitrate reductase. The presence of this cross reacting material in cell extracts was detected by the ability of these extracts to (a) lower the titer of antisera; (b) form a biphasic precipitin curve with purified antibody; and (c) increase the peak height of a standard amount of purified nitrate reductase in rocket immunoelectrophoresis assay. These results suggest that ammonia-grown cells contain nitrate reductase precursor protein. PMID- 16661312 TI - Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase. AB - A single enzyme catalyzes the synthesis of all four N(2)-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N(2)-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (K(m)) differ, the ratio V/K(m) is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. PMID- 16661313 TI - ATP requirement for mg chelatase in developing chloroplasts. AB - The synthesis of Mg-protoporphyrin-IX from exogenous protoporphyrin-IX, in a crude plastid pellet extracted from greening cucumber cotyledons was found to require l-glutamate as a cofactor. It has now been shown that glutamate acts in the presence of contaminating mitochondria to provide an ATP regenerating system. With purified plastids, Mg chelatase is not stimulated by glutamate; instead, it requires a high concentration of ATP and is greatly stimulated by added phosphoenolpyruvate and pyruvate kinase. GTP, UTP, CTP, and ITP will not substitute for ATP. ADP in the absence of an ATP generating system is completely ineffective, whereas it is slightly inhibitory in the presence of 10 mm ATP. AMP is strongly inhibitory in the reaction; 50% inhibition is obtained at approximately 3.5 mm AMP in the presence of 10 mm ATP. PMID- 16661314 TI - Nitrogen Nutrition and Xylem Transport of Nitrogen in Ureide-producing Grain Legumes. AB - Xylem sap composition was examined in nodulated and nonnodulated cowpea (Vigna unguiculata [L.] Walp.) plants receiving a range of levels of NO(3) and in eight other ureide-forming legumes utilizing NO(3) or N(2) as sole source of nitrogen. A (15)N dilution technique determined the proportions of plant nitrogen derived from N(2) in the nodulated cowpeas fed NO(3). Xylem sap composition of NO(3)-fed, nodulated cowpea varied predictably with the relative extents to which N(2) and NO(3) were being utilized. The ratios of asparagine to glutamine (N/N) and of NO(3) to ureide (N/N) in xylem sap increased with increasing dependence on NO(3) whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO(3) (N/N) in xylem sap increased with increasing dependence on N(2) fixation. The amounts of NO(3) and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO(3) and the presence or absence of N(2) fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N(2) than when utilizing NO(3). In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.] Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap composition was discussed. PMID- 16661315 TI - Electrical Membrane Properties and Circadian Rhythm in Extensor Cells of the Laminar Pulvini of Phaseolus coccineus L. AB - Circadian changes of three parameters of electrical properties in extensor cells of the laminar pulvini of Phaseolus coccineus L. have been recorded by standard microelectrode techniques. In the low turgor state of the cells, the resting potential and the CN(-) (1 millimolar NaCN) induced depolarizations are large, while the K(+)-induced voltage changes are small, and vice versa in the high turgor state. These results demonstrate that during a circadian cycle the low turgor state of the extensor cells is correlated with the active state of an electrogenic pump. This correlation can be explained by electroosmosis. PMID- 16661316 TI - ATP Levels and their Effects on Plasmalemma Influxes of Potassium Chloride in Red Beet. AB - Tissue ATP concentrations in slices of red beet increase progressively with time for up to 7 days after cutting the root. ATP levels are higher in slices taken from stored roots than in slices from fresh roots. ATP is reduced during incubation in salt solutions.Plasmalemma influxes were measured by 6 minutes incubation with (42)K and/or (36)C1 under conditions in which cation and anion influxes are independent. Both fluxes showed an approximately linear correlation with ATP level when the latter was varied by addition of CN(-), carbonyl cyanide m-chlorophenylhydrazone, or oligomycin, except for Cl influx with oligomycin, where oligomycin had a greater effect on Cl influx than could be accounted for by the reduction in ATP alone.These results support the view that K(+) and Cl(-) influxes are both energized directly or indirectly by ATP, and in addition that the Cl pump may be directly inhibited by oligomycin. PMID- 16661317 TI - Effect of anoxia on ATP levels and ion transport rates in red beet. AB - The ATP content of disks of storage tissue of red beet is reduced under N(2) atmosphere to between 10 and 25% of its value in air. Plasmalemma fluxes of K(+) and Cl(-) are inhibited within 1 minute or less following removal of O(2), and the extent of this inhibition is entirely attributable to the depletion of ATP. The ATP content remains approximately constant or decreases slightly for up to 1 hour under N(2). On return to air, the ATP content recovers, reaching 75% of the aerobic level within 45 second. PMID- 16661318 TI - Kinetics of C-photosynthate uptake by developing soybean fruit. AB - By pulse-labeling field-grown soybean leaves for 60 seconds at midday with (14)CO(2) and then sequentially harvesting, dissecting, and extracting the radioactive fruit tissues (of pod and seeds), the route, uptake kinetics, and metabolic fate of (14)C-photosynthate as it was imported by 35- to 40-day-old pods were determined. As the [(14)C]sucrose pulse entered the pods, the seeds became radioactive immediately but a lag of nearly 30 minutes occurred before label could be detected in the pod wall pericarp.Import of the (14)C-pulse by the seeds was exclusively via the seed coats, where rapid unloading occurred. Maximum accumulation of label in the seed coat occurred in about 60 minutes at which time 59% of the total radioactivity in the fruit was in the three seed coats, whereas only 7% was in the cotyledons they enclosed. The photosynthate remained as [(14)C]sucrose as it passed through the seed coat, but appeared to be hydrolyzed relatively soon after uptake by the cotyledons. By 2.5 hours, 60% of the (14)C photosynthate pulse had passed into the cotyledons with only 27% remaining in the seed coats. Inasmuch as there is no vascular connection between maternal seed coat and the developing embryo, cotyledonary uptake of sucrose released from the inner seed coat surface may require specialized transport mechanisms. PMID- 16661319 TI - Effect of oxygen and temperature on the efficiency of photosynthetic carbon assimilation in two microscopic algae. AB - The CO(2) compensation points of Coccochloris peniocystis, a blue-green alga and Chlamydomonas reinhardtii, a green alga, were determined at pH 8.0 in a closed system by a gas chromatographic technique. The compensation point of Chlamydomonas increased markedly with temperature, rising from 0.79 microliter per liter CO(2) at 15 C to 2.5 microliters per liter CO(2) at 35 C. In contrast, the compensation point of Coccochloris at 20 C was 0.71 microliter per liter CO(2) and rose to only 0.95 microliter per liter CO(2) at 40 C.The compensation point of the green alga was significantly reduced at low O(2) concentrations (1 to 2%) when measured over the temperature range of 15 to 35 C. The compensation point of the blue-green alga, over the temperature range of 20 to 40 C, was unaffected by lowering the O(2) concentration.The whole cell CO(2) affinity of Chlamydomonas decreased substantially with increasing temperature at 21% O(2) whereas little change was observed over the same temperature regime when the CO(2) affinity was determined at O(2) concentrations of 1 to 2%. The CO(2) affinity of Coccochloris did not decrease significantly with either increasing temperature or O(2) concentration.These results suggest that while photorespiration is undetectable in Coccochloris some photorespiratory CO(2) release occurs in Chlamydomonas. PMID- 16661320 TI - Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity. AB - The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.Young lambsquarters plants were treated with atrazine by spraying the leaves at a rate which was lethal for susceptible plants after 5 or 6 days, but had little effect on the resistant biotype. Nitrite did not accumulate in either biotype, but remained present at the level of about 0.1 microgram nitrite N per gram fresh weight. The nitrate content of susceptible-type leaves did increase to two or three times the initial level, during the first four days after spraying. Usually the only visible effect on the plants during this time was a decreased growth rate. Twenty four hours after spraying the following activities had fallen to 25% or less of the activities of solvent-sprayed control plants: in vivo nitrite reductase, in vivo nitrate reductase, in vitro NADH-nitrate reductase, in vitro reduced flavin mononucleotidenitrate reductase, and in vitro NADH-diaphorase. In these atrazine treated plants, in vitro nitrite reductase activity with reducing potential supplied by methylviologen was not affected, nor were any of the above activities in leaves of atrazine-treated resistant plants. The abrupt fall in nitrate reductase represents an effect of atrazine not directly related to inhibition of photosynthesis. PMID- 16661321 TI - In Vitro Synthesis of the alpha and alpha' Subunits of the 7S Storage Proteins (Conglycinin) of Soybean Seeds. AB - Messenger RNAs (mRNAs), isolated from immature soybean (Glycine max L., Merr.) seeds, that bound to oligo(dT)-cellulose were fractionated by centrifugation in sucrose density gradients containing dimethyl sulfoxide. mRNAs with sedimentation values between 21S and 25S coded for the in vitro translation of polypeptides with electrophoretic mobilities similar to those of the alpha' and alpha subunits of the 7S seed storage protein. High pressure liquid chromatographic analyses of the trypsin-induced fragments ("column fingerprinting") verified that the polypeptides produced in vitro were closely related to authentic alpha' and alpha subunits.The fractions of RNA that coded for the in vitro synthesis of these subunits contained three major species of nonribosomal RNA (molecular weights of 1.1, 0.84, and 0.75 x 10(6)) sufficiently large to code for the synthesis of the alpha' and alpha subunits. PMID- 16661322 TI - Slow adaptive changes in urease levels of tobacco cells cultured on urea and other nitrogen sources. AB - Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4 fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 x 10( 5), then multiplied 19% more rapidly than low urease cells. This frequency is about 10(3) greater than that of other biochemical variants previously isolated from XD cells. The high urease activity gradually declined in cells transferred from urea to other nitrogen sources, but rose rapidly when such cells were returned to urea, indicating the existence within the cells of some form of record of their ancestors' growth on urea. The data indicate the existence of a mechanism for generation, at unusually high frequency, of metastable variants with high urease activity. This mechanism, coupled with enrichment for the variants' progeny by virtue of their higher multiplication rate on urea, can account for the observed slow increase in urease activity of the population. It is suggested that the molecular basis of the urease increase may be gene amplification, based on animal cell models. An alternative hypothesis, namely a specific response induced in all cells by urea and manifested as a very slow adaptive increase in urease, has not been ruled out. PMID- 16661323 TI - Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings. AB - Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [(14)C]linoleate formed from [(14)C]oleate but not [(14)C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings. PMID- 16661324 TI - Relationship between Chloroplast Development and ent-Kaurene Biosynthesis in Peas. AB - Treatment of etiolated pea (Pisum sativum (L. cv. Alaska) seedlings with 2' isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1 carboxylate (Amo-1618) prior to irradiation with white light inhibits photomorphogenesis and formation and stacking of thylakoid membranes in the chloroplasts, as well as (-)-kaur-16-ene (ent-kaurene)biosynthesis. Exogenous gibberellic acid also inhibits greening. A crudely determined action spectrum for the photoinduction of ent-kaurene biosynthesis shows two peaks, one in the blue region at 458 to 490 nanometers and another in the red region at 606 to 678 nanometers. The possible participation of phytochrome in the photoinduction of ent-kaurene biosynthesis is indicated by comparative effects of red, far red, and alternating red/far red irradiations on enhancement of enzyme activity. The activity of blue light as well as red shows a similarity of the photoinduction of ent-kaurene synthesis activity to the high irradiance responses, and indicates probable participation of a second photoreceptor. From these observations, it is concluded that photoinduction of ent-kaurene biosynthesis and chloroplast development in shoots are closely linked processes. PMID- 16661325 TI - Dark Opening of Stomates of Vicia faba in CO(2)-free Air: EFFECT OF TEMPERATURE ON STOMATAL APERTURE AND POTASSIUM ACCUMULATION. AB - Leaf epidermal strips of Vicia faba were floated on 10 millimolar KCl at temperatures of 10 to 45 C for several time periods in the dark. For the duration of the experiment, a jet of water-saturated, CO(2)-free air was passed over the strips. The width of the stomatal aperture was measured microscopically and the potassium content was estimated and expressed as the percentage of area of the guard cell pair stained with cobaltinitrite. Stomatal opening in the dark occurred at temperatures above 35 C. The high temperature aperture reduction previously observed at 45 C in the light was not observed in the dark. Potassium accumulation occurred at all temperatures except 10 C, with a maximum at 45 C, which appeared functionally different from that seen previously in the light. PMID- 16661326 TI - Characterization of an acyl-coenzyme a thioesterase associated with the envelope of spinach chloroplasts. AB - The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell. PMID- 16661327 TI - Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum. AB - Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO(2) fixation or CO(2) dependent O(2) evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO(2) fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO(2) fixation and CO(2)-dependent O(2) evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.A long lag period at optimum Pi concentration, no effect of high Pi levels on the lag phase, lack of severe inhibition of photosynthesis by high Pi concentrations, and the relatively low pH optimum for photosynthesis are characteristics unlike those previously reported with chloroplasts of C(3) plants. PMID- 16661328 TI - Interactions between Rhizobia and Lectins of Lentil, Pea, Broad Bean, and Jackbean. AB - A quantitative method was developed to measure the binding of fluorescent-labeled lentil (Lens esculenta Moench), pea (Pisum sativum L.), broad bean (Vicia faba L.), and jackbean (Canavalia ensiformis L., DC.) lectins to various Rhizobium strains. Lentil lectin bound to three of the five Rhizobium leguminosarum strains tested. The number of lentil lectin molecules bound per R. leguminosarum 128C53 cell was 2.1 x 10(4). Lentil lectin also bound to R. japonicum 61A133. Pea and broad bean lectins bound to only two of the five strains of R. leguminosarum, whereas concanavalin A (jackbean lectin) bound to all strains of R. leguminosarum, R. phaseoli, R. japonicum, and R. sp. tested. Since these four lectins have similar sugarbinding properties but different physical properties, the variation in bindings of these lectins to various Rhizobium strains indicates that binding of lectin to Rhizobium is determined not only by the sugar specificity of the lectin but also by its physical characteristics.The binding of lentil lectin and concanavalin A to R. leguminosarum 128C53 could be inhibited by glucose, fructose, and mannose. However, even at 150 millimolar glucose, about 15% of the binding remained. The binding of lentil lectin to R. japonicum 61A133 could be inhibited by glucose but not by galactose. It is concluded that the binding site of lentil lectin to R. japonicum is different from the binding site of soybean lectin to R. japonicum. PMID- 16661329 TI - Labeling and isolation of plasma membranes from corn leaf protoplasts. AB - A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [(125)I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg(2+)-dependent, K(+)-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions. PMID- 16661330 TI - Comparison of the Ability of Salicylic Acid and Ferricyanide to Induce Flowering in the Long-day Plant, Lemna Gibba G3. AB - Both salicylic acid and ferricyanide induce flowering in the long-day plant Lemna gibba L., strain G3 under 8- and 9-hour short days. In both cases the effect is daylength-dependent. Salicylic acid is ineffective on daylengths less than 8 hours and ferricyanide is ineffective on daylengths less than 5 hours. When both substances are given together a striking synergistic interaction is observed, and some flowering is obtained on daylengths as short as 3 hours. However, even with the optimal combinations the flower-inducing effect remains daylength dependent.Flowering of L. gibba G3 is inhibited under continuous light in both half-strength Hutner's medium (0.5 H), which contains 1.25 millimolar ammonium, and in ammonium-free half-strength Hutner's medium (NH(4) (+)-free 0.5 H). Salicylic acid is able to reverse the inhibition substantially in 0.5 H medium and to cause complete reversal in NH(4) (+)-free 0.5 H medium. By contrast, ferricyanide has no effect in 0.5 H medium and causes only a small reversal in NH(4) (+)-free 0.5 H medium. Flowering of L. gibba G3 is also inhibited under continuous light by copper. This inhibition is largely reversed by salicylic acid but ferricyanide has no effect. PMID- 16661331 TI - Role of hydration state and thiol-disulfide status in the control of thermal stability and protein synthesis in wheat embryo. AB - Reduced (GSH), oxidized (GSSG), and protein-bound (PSSG) glutathione were determined in dry and hydrated wheat embryos. Dry embryos contained about 0.6 mumoles per gram dry weight each of GSSG and PSSG, and these levels declined 5- to 10-fold within minutes after the onset of imbibition. GSH declined from about 8 to 2 mumoles per gram over a period of 90 minutes. Similar changes occurred when embryos were hydrated by storage at 100% relative humidity. The decline in glutathione levels was not reversed upon redrying hydrated embryos. About 40% of the cysteine residues of embryo protein was found to be in the disulfide form in both dry and imbibed embryos. The ability of wheat embryos to withstand heat shock was shown to correlate with water content but not GSSG content. Incorporation of [(35)S]methionine into protein was studied using a system based upon wheat embryo extract (S23). Incorporation rate was found to be sensitive to the nature of thiol added to the system and to be decreased by GSSG. S23 exhibited a substantial capacity to reduce GSSG and preparation of S23 having a GSSG content comparable to dry embryos required addition of large amounts of GSSG to the extraction buffer S23 prepared in this fashion exhibited a marked decrease in ability to support protein synthesis. These results suggest that the early decrease in GSSG during germination is necessary for optimal protein synthesis in wheat embryo. PMID- 16661332 TI - Ethylene Action and Loss of Membrane Integrity during Petal Senescence in Tradescantia. AB - Senescence of isolated petals of Tradescantia is accompanied by a large increase in membrane permeability, and application of ethylene hastens the onset of this increase. There is a 1- to 2.5-hour lag between ethylene application and the onset of anthocyanin efflux (an indicator of increased membrane permeability). Simultaneous application of 0.1 millimolar cordycepin or cycloheximide with ethylene abolishes the response to ethylene. Analysis of phospholipid levels in these petals during senescence has shown that the increase in membrane permeability is accompanied by a massive loss of phospholipids. Factors which enhance or retard the rate of anthocyanin efflux exert a corresponding effect on the rate of phospholipid loss. The composition of the phospholipid fraction remains unchanged during senescence. The activity of phospholipase D declines during senescence whereas that of acyl hydrolase remains essentially constant. PMID- 16661333 TI - Analysis of glycoconjugate saccharides in organelles isolated from castor bean endosperm. AB - The presence of lipid- and protein-bound sugars in the major organelle fractions isolated from germinating castor bean (Ricinus communis L.) endosperm has been established. Microsomes, glyoxysomes and mitochondria were subfractionated into a membrane fraction and a fraction containing peripheral membrane and soluble matrix proteins. The membranes were further subfractionated into monosaccharide lipid, oligosaccharide lipid and lipid-free protein components. The constituent sugars present in the prepared fractions were released and identified by gas liquid chromatography. While all derived protein fractions contained the N acetylglucosamine and mannose typically found in the inner core region attached to asparagine residues in many glycoproteins, some differences were noted in the organellar distribution of peripheral sugars such as fucose, arabinose, and xylose. PMID- 16661334 TI - Transverse Distribution of Phospholipids in Organelle Membranes from Ricinus communis L. var. Hale Endosperm: MITOCHONDRIA AND GLYOXYSOMES. AB - Phospholipase A(2) (Naja naja), the nonpenetrating dye trinitrobenzene sulfonate, and the penetrating dye dinitrofluorobenzene, were used to determine the transmembrane distributions of phospholipids of mitochondria and glyoxysomes isolated from endosperm tissue of castor bean (Ricinus communis L. var. Hale). These studies indicated that the phospholipid distributions were distinctly asymmetric in the accessible (reacted with the probes without total membrane disruption by detergents) pools of the glyoxysomal and inner mitochondrial membranes, but more nearly symmetric in the outer mitochondrial membrane. However, significant quantities of the phospholipids of the mitochondrial membranes were inaccessible to the probes used. An increased accessibility of the phospholipids of all membranes following Triton X-100 dispersion was found, and protein to phospholipid ratios in organelle membranes were found to correlate inversely with the accessibility of the phospholipids to the probes. The inaccessible phospholipids may be involved in lipid-protein interactions. PMID- 16661335 TI - Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function. AB - Density-labeling with 10 millimolar K(15)NO(3)/70% (2)H(2)O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the (1)H(2)O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage. PMID- 16661336 TI - Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles. AB - The application of neutral or acidic amino acids to oat coleptiles induced transient depolarizations of the membrane potentials. The depolarizations are considered to reflect H(+) -amino acid co-transport, and the spontaneous repolarizations are believed to be caused by subsequent electrogenic H(+) extrusion. The basic amino acids depolarized the cell membrane strongly, but the repolarizations were weak or absent. The depolarizations induced by the basic amino acids were weakly sensitive to manipulations of the extracellular and intracellular pH. The depolarizations induced by the other amino acids, in contrast, were more strongly affected by the pH changes. Several amino acids induced distinct but diminished depolarizations in the presence of 2,4 dinitrophenol or cyanide, but the repolarizations were generally eliminated. These experiments support the co-transport theory but suggest somewhat different mechanisms for the transport of the neutral, acidic, and basic amino acids. We suggest that the neutral amino acids are co-transported with a single H(+) and that accumulation depends upon both the DeltapH and the membrane potential components of the proton motive force. The acidic amino acids appear to be accumulated by a similar mechanism except that the transport of each molecule may be associated with a cation in addition to a single proton. The permanently protonated basic amino acids appear not to be co-transported with an additional proton. Accumulation would depend only on the membrane potential component of the proton motive force. PMID- 16661337 TI - Kinetics of N-(Delta-Isopentenyl)Adenosine Degradation in Tobacco Cells: EVIDENCE OF A REGULATORY MECHANISM UNDER THE CONTROL OF CYTOKININS. AB - Uptake and degradation of the cytokinin, N(6)-(Delta(2)-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N(6)(Delta(2)-isopentenyl)[8-(14)C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin. Consequently, only rates of N(6)-(Delta(2)-isopentenyl)adenosine degradation measured during the first 3 hours of incubation with [8-(14)C]-N-(6)(Delta(2) isopentenyl)adenosine are representative of the intrinsic in vivo cytokinin degradative activity of tobacco cells. Within these limits, it appears that cytokinin degradative activity is high in cytokinin-autonomous tobacco cells, as indicated by the half life of the supplied N(6)(Delta(2) isopentenyl adenosine (about 3 hours) when it is supplied at the physiological concentration of 0.2 micromolar. This cytokinin degradative activity appears to be under the control of cytokinins themselves because N(6)-(Delta(2)-isopentenyl)adenosine degradative activity is increased several-fold following a 3- to 4-hour delay after these cells have been exposed to a cytokinin. PMID- 16661338 TI - Temperature effects on soybean imbibition and leakage. AB - As a part of an analysis of the nature of chilling injury to seeds, measurements were made of the initial linear rates of water entry into and solute leakage out of cotyledons of soybean at various temperatures. Arrhenius plots were approximately linear for water entry into both living and dead cotyledons, with the slope (and activation energy) for entry into living cells being insignificantly higher than for dead cells, suggesting little effect of membrane barriers on water entry. The plots for solute leakage showed 10-fold lower leakage rates from living than from dead tissues; a reversal of slope in the Arrhenius plot at temperatures below 15 C reflected increasing leakage rates, interpreted as a quantitative disruption of membrane reorganization at the temperatures associated with chilling injury. PMID- 16661339 TI - Protoplast Fusion: EFFECT OF LOW TEMPERATURE ON THE MEMBRANE FLUIDITY OF CULTURED CELLS. AB - The relation between the composition of the phospholipid molecular species in a cell membrane and the velocity of protoplast fusion was studied using cells cultured at a low temperature (10 C). Cells cultured at a low temperature contained larger proportions of phospholipids of low phase transition point, the 1,2-dilinoleoyl-type, than those cultured at a normal temperature (25 C). When treated with polyethylene glycol 6000, protoplasts from cells cultured at 10 C fused and progressed to the fused sphere stage more rapidly than did those from cells cultured at 25 C. PMID- 16661340 TI - Photosynthesis, leaf resistances, and ribulose-1,5-bisphosphate carboxylase degradation in senescing barley leaves. AB - The relationship between loss of ribulose-1,5-bisphosphate carboxylase (RuBPCase) and the decline in photosynthesis during the senescence of barley primary leaves was assessed. Loss of RuBPCase accounted for about 85% of the decrease in soluble protein. RuBPCase was highly correlated with in vitro RuBPCase activity (r = 0.95) and gross photosynthesis (r = 0.96). However, the rate of photosynthesis per milligram RuBPCase increased during the early stages of leaf senescence. The concentration of nonreducing sugars was negatively correlated (1% level) with photosynthesis. Free alpha-amino N, in contrast to nonreducing sugars, declined markedly during senescence. A decrease in chlorophyll and an increase in in vitro protease activity was observed, but these changes did not appear to be closely related to the decline in photosynthesis and RuBPCase. Mesophyll resistance increased at the same rate that photosynthesis and RuBPCase declined. Stomatal resistance increased more rapidly than mesophyll resistance and accounted for about 24% of the total increase in resistance to CO(2) diffusion. The concentration of CO(2) in the intercellular air spaces decreased during the last stage of senescence. Although loss of RuBPCase probably is the primary event responsible for the decline in photosynthesis during leaf senescence, other factors such as in vivo regulation and stomatal aperture must also be considered. PMID- 16661341 TI - Biochemical studies on the iojap mutant of maize. AB - The white leaf tissue of seedlings of Zea mays L. affected by the recessive nuclear gene iojap shows no photosynthetic activity; it contains about 1.4% of carotenoid and less than 0.1% of chlorophyll a content of normal green tissue. Neither fraction I protein nor chloroplast adenosine triphosphatase (EC 3.6.1.4) (CF(1)) is detectable. This confirms earlier observations that plastids of white sectors of iojap maize do not contain ribosomes. About 40% of the activity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in green leaves could be found in white leaves indicating that the phosphoenolpyruvate carboxylase EC 4.1.1.31 is made on cytoplasmic ribosomes. The oxygen consumption of iojap-affected leaves is decreased. PMID- 16661342 TI - Effect of obstructed translocation on leaf abscisic Acid, and associated stomatal closure and photosynthesis decline. AB - Pod removal or petiole girdling, which causes obstruction of translocation, was found in our previous study to cause reduced rates of photosynthesis in soybean leaves due to stomatal closure. The purpose of this research was to determine the involvement of photoassimilate accumulation and leaf abscisic acid (ABA) levels in the mechanism of stomatal closure induced by such treatments.Leaf glucose and sucrose levels increased during the initial 12-hour period after depodding or petiole girdling. Starch, which represents a much larger pool of leaf carbohydrate, was not perceptibly increased above control levels during the 12 hour posttreatment period.When leaflets were exposed to nonphotosynthetic environments (shading or CO(2)-free air) for a 24-hour period after the translocation-obstructing treatments were applied and then returned to normal light or CO(2) concentration, stomatal diffusive conductivity was reduced 65% and 85% with depodding and girdling, respectively. These reductions were comparable to those previously observed without an intervening nonphotosynthetic exposure, thus indicating that photosynthate accumulations were not necessary for the observed response.Free and bound ABA (released on alkaline hydrolysis) were determined by gas liquid chromatography with electron capture detection following preparative high performance liquid chromatography. Free ABA in monitored leaves increased almost 10-fold 48 hours after complete depodding and 25-fold 24 hours after petiole girdling of such leaves. By 3 hours after treatment, in a time course study, free ABA had increased 2-fold above control values in depodded and 5-fold in girdled leaves. Leaf concentrations of bound ABA did not appear to be related to the treatment effects on stomata.Thus, the translocation-obstructing treatments cause an increased level of ABA by a mechanism not involving accumulation of photoassimilate. Increased leaf ABA levels, which were independent of water stress or leaf water potential, appear to be involved in the stomatal closure response. It is suggested that the mechanism of increased leaf ABA levels following translocation-obstruction may be due to an interference with normal translocation of ABA out of leaves. PMID- 16661343 TI - Alterations in source-sink patterns by modifications of source strength. AB - Bean plants, trimmed to a simplified "double source, double sink" translocation system (the paired primary leaves serving as the double source and the paired lateral leaflets of the immature first trifoliate leaf as the double sink) were used to study the magnitude and short-term time course of change in the allocation ratio (partition ratio) of assimilates translocated from the labeled primary leaf to its respective "near" and "far leaflet" sinks in response to an increase or decrease in the source strength of the opposite primary leaf (the "control" leaf). If the rates of net photosynthesis in the two primary leaves were similar, assimilates from the labeled source leaf partitioned to the leaflet sinks in the ratio of 5:1 or higher, the dominant sink being the leaflet "nearer" to the labeled source leaf. If the rate of net photosynthesis in the control leaf was increased substantially above that of the labeled source leaf, the rate of translocation from the labeled source to either the near leaflet sink or far leaflet sink remained unaffected, despite, presumably, a higher translocation rate from the control leaf, and hence a higher phloem pressure gradient (or increased cross-sectional area) in the transport pathway from the control leaf to the leaflet sinks. If the control leaf was excised, thus reducing the source leaf area by about a half, the translocation rate from the remaining source leaf rapidly doubled, the partition ratio becoming equal to unity. If the control leaf was darkened, the partition ratio adjusted to an intermediate value. Although export rates from the labeled source leaf were increased either by excising or darkening the control leaf, the rate of net photosynthesis in the labeled leaf remained constant. PMID- 16661344 TI - Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA. AB - The base composition of peanut (var. NC-17) DNA determined from thermal denaturation profiles showed an average guanine plus cystosine content of 34% which was in close approximation to 36% guanine plus cytosine calculated from the buoyant density. Buoyant density also indicated the absence of satellite DNA. The genome size, 2.0 x 10(9) base pairs, as determined by reassociation kinetics of the single copy DNA was close to the genome size determined by cytophotometry, 2.1 x 10(9) base pairs. Peanut DNA averaging 450 to 600 base pairs long, reassociated in phosphate buffer and fractionated by hydroxylapatite, indicated a DNA genome composition of 36% nonrepetitive or single copy DNA; reassociation in formamide and followed by optical methods indicated the repetitive DNA possesses highly repeated, intermediately repeated and rarely repeated components of DNA with DNA sequences repeated on the average about 38,000, 6,700, and 200 times each. Different criteria of reassociation in formamide revealed further subdivisions of these four separate components of DNA. The DNA of above mentioned NC-17 variety compared to Florigiant variety showed no differences in thermal denaturation profiles, buoyant density, or in genome size. PMID- 16661345 TI - Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L. AB - Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two "cap" analogues m(7) GTP and m(7) GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18 hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA. PMID- 16661346 TI - Direct measurement of sieve tube turgor pressure using severed aphid stylets. AB - Turgor pressure in individual sieve tubes was measured directly by gluing capillary micromanometers over exuding aphid stylets with cyanoac-rylate adhesive. Pressures of up to 10 bars were measured in sieve tubes of Salix babylonica, with an estimated accuracy of +/- 0.3 bars or better.For comparison with the direct measurements of sieve tube turgor, calculated values of turgor pressure were also obtained from the difference between leaf water potential and phloem exudate solute potential, estimated from its refractive index and sucrose content. In most cases the measured turgor pressure was greater than the calculated value. The discrepancy between the two values was most likely due to the presence of appreciable concentrations of potassium and amino acids in the phloem exudate. PMID- 16661347 TI - Conceptual Error in Determination of NAD-Malic Enzyme in Extracts Containing NAD Malic Dehydrogenase. AB - The typical procedure for determining NAD(+)-malic enzyme (EC 1.1.1.39) is to calculate the enzyme rate to be DeltaA(340)/Delta time after the endogenous NAD(+)-malic dehydrogenase (EC 1.1.1.37) catalyzed reaction has reached equilibrium. This ignores the equilibrium shift of oxaloacetic acid and NADH during the course of the NAD(+)-malic enzyme reaction and causes an error that varies depending on the reagent [malate], [NAD(+)], pH and final [NADH]. For a DeltaA(340) of 0.02, the error is about 80% and for a DeltaA(340) of 0.30, 20%. We develop this argument, give supportive data and present a simple method to circumvent the error. PMID- 16661348 TI - Effect of ATPase inhibitors on cell potential and k influx in corn roots. AB - Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Psi) and K(+) ((86)Rb) influx of corn root tissue over a wide range of K(+) activity. N,N'Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K(+) influx and depolarized Psi at low, but not at high, K(+) activity (K degrees ). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K(+) influx over a wide range of K degrees , with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Psi(p)) above 0.2 millimolar K degrees . Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K(+) influx and Psi(p) at low K degrees can be attributed to inhibition of the plasmalemma ATPase.It is concluded that by DCCD treatment, the energy-linked electrogenic system at high K degrees is separated from the energy-linked K(+) influx system at low K degrees . The results are analyzed in terms of electrical analogue models of the membrane. The presence of two, algebraically additive electrogenic components is indicated; one is better modeled as a current source (system I) and one as a voltage source (system II). No K(+) stimulation of system II is required to produce the observed K degrees dependence of Psi(p). PMID- 16661349 TI - Characteristics of Cold Acclimation and Deacclimation in Tuber-bearing Solanum Species. AB - The effect of temperatures on cold acclimation and deacclimation in foliage tissues was studied in Solanum commersonii (Oka 4583), a tuber-bearing potato. The threshold temperature for cold acclimation was about 12 C. In a temperature range of 2 to 12 C, the increase in hardiness was dependent on the acclimating temperature; the lower the acclimating temperature, the more hardiness achieved. A day/night temperature of 2 C, regardless of photoperiod, appeared to the optimum acclimating temperature for the Solanum species studied. A subfreezing temperature hardened plants less effectively. The maximum level of hardiness could be reached after 15 days of cold acclimation. However, it took only 1 day to deacclimate the hardened plants to a preacclimation level when plants were subjected to a warm regime from cold. The degree of deacclimation was dependent on the temperature of the warm regime.Based on cold tolerance and the capacity to acclimate to cold, tuber-bearing Solanum species could be grouped into five categories. Chilling injury was also observed in some of the tuber-bearing Solanum species. PMID- 16661350 TI - Effects of temperature, water potential, and light on germination responses of redroot pigweed seeds to ethylene. AB - The responses of redroot pigweed (Amaranthus retroflexus L.) seeds to nine ethylene concentrations between 0.5 and 50 microliters per liter were assessed at different temperatures and water potentials and in either continuous white light or darkness. Under all experimental treatments, the probit-transformed percentages increased linearly with the log of the ethylene concentration. In dormant seeds, the slope of the response line was unaffected by either light or water potential but increased with decreasing temperature. Conversely, the slope increased with increasing temperature in a partially afterripened seed lot.The ethylene response threshold for germination was little affected by temperature or light, ranging from 0.2 to 0.7 microliter per liter, but decreased to less than 0.1 microliter per liter at negative water potentials. Osmotic inhibition of germination at -4 bars was largely relieved by 1 microliter per liter ethylene. Such interactions between ethylene and other environmental conditions may play an important role in the course of germination of soil-borne seeds. PMID- 16661351 TI - RNA synthesis in germinating embryonic axes of soybean and wheat. AB - The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.Analysis of the ratio of radioactivity incorporated into the 18S and 26S rRNAs of the germinating embryos provided a further measure of the increased rates of RNA synthesis. With wheat embryos, the 26S/18S ratio increased from 1.0 at 1.5 hours to 1.5 at 3.5 hours, while with the soybean axes, distinct ribosomal patterns were obtained only after 4 hours and the 26S/18S rRNA ratio increased from 0.4 at 4 hours to 1.0 at 9 hours. The extent of methylation of the rRNA synthesized at 4 and 9 hours in the soybean axes was similar, indicating that the methylating capacity of the axes is probably not rate limiting to rRNA synthesis. In both seed embryo systems the level of UTP increased 2 to 3-fold during the lag phase of germination. With wheat embryos, the time course of the increase in UTP correlated approximately with the change in the rate of RNA synthesis. With the soybean axes, however, the increase in the rate of RNA synthesis occurred predominantly after the rise in the level of UTP. PMID- 16661352 TI - Forms of Dissolved Carbon Dioxide Required for Photosystem II Activity in Chloroplast Membranes. AB - High concentrations of both bicarbonate and formate inhibit photosynthetic O(2) evolution at pH 8.0. At this pH, only 2.4% of the total dissolved carbon dioxide exists as CO(2). At pH 7.3, where 11% of the total dissolved carbon dioxide exists as CO(2), HCO(3) (-) no longer inhibits. While formate still inhibits O(2) evolution at pH 7.3, its effect can be partially overcome if CO(2) is also present. The rate of binding of added (14)C-labeled inorganic carbon is nearly 10 fold more rapid when the internal pH of thylakoid membranes is at 6.0 than when it is at 7.8. These observations suggest that CO(2), not HCO(3) (-), is initially bound to the photosystem II reaction center and that the location of the binding site is on the inside thylakoid surface. However, additional data presented here suggest that, after binding, CO(2) is hydrated to HCO(3) (-) + H(+) in a pH dependent reaction. Two possible explanations of the "bicarbonate effect" are presented. PMID- 16661353 TI - Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts. AB - The metabolism of (14)C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [(14)C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-(14)C]diaminopimelic acid was readily converted into [(14)C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO(2) fixation. PMID- 16661354 TI - Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn. AB - Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208x and 66x less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins. PMID- 16661355 TI - Spherosomes of Castor Bean Endosperm: MEMBRANE COMPONENTS, FORMATION, AND DEGRADATION. AB - The membrane components of the castor bean spherosomes were characterized. The storage triacylglycerols of isolated spherosomes were extracted with diethyl ether, and the membrane was isolated by sucrose gradient centrifugation. It had an apparent equilibrium density of 1.12 grams per cubic centimeter, and possessed an antimycin A-insensitive NADH cytochrome c reductase and an acid lipase. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in roughly equal amounts were the major phospholipids. The membrane proteins were resolved into several major and minor protein bands of molecular weights ranging from 10,000 to 70,000 by acrylamide gel electrophoresis, and the protein pattern in the gel was different from those of the endoplasmic reticulum, mitochondrial, and glyoxysomal membranes.The varying amounts of spherosomal components in the seed were followed throughout seed maturation and germination. A striking similarity existed in the developmental pattern of each of the spherosomal components. This finding suggests that the spherosome is synthesized and degraded as one individual unit. The spherosomes isolated from maturing seeds exhibited rapid hydrolysis of the storage lipid in vitro, thus raising the problem of cellular control in preventing in vivo autolysis of the spherosomes during seed maturation. PMID- 16661356 TI - Effect of Light Intensity during Growth on Photoinhibition of Intact Attached Bean Leaflets. AB - In the study reported here, two different photoinhibitory phenomena were compared within a single plant species. Bean plants were grown in three different light intensities to simulate sun and shade environments. The effects of photoinhibitory treatments on in vivo CO(2) assimilation rates and in vitro chloroplast electron transport reactions were investigated and the extent to which carbon metabolism served to prevent photoinhibition was characterized. It was shown that the photoinhibition which follows exposure of intact leaflets of low light-grown bean plants to high light intensity in normal air is essentially similar to that which occurs when leaflets of plants grown in full sunlight are illuminated in the absence of CO(2) at low O(2) partial pressures. PMID- 16661357 TI - Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm. AB - Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK(c), is localized in the cytosol, and the other, PK(p), is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K(+) and Mg(2+). Both isozymes are inhibited by citrate, pyruvate, and ATP. PK(c) has a much broader pH profile than PK(p) and is also more stable. Both have the same K(m) (0.05 millimolar) for PEP, but PK(p) has a 10-fold higher K(m) (0.3 millimolar) for ADP than PK(c) (0.03 millimolar). PK(c) also has a higher affinity for alternate nucleotide substrates than PK(p). The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue. PMID- 16661358 TI - Thymidine Metabolism and the Measurement of the Rate of DNA Synthesis in Carrot Suspension Cultures: EVIDENCE FOR A DEGRADATION PATHWAY FOR THYMIDINE. AB - The kinetics of [(3)H]thymidine incorporation into the DNA of carrot suspension cultures were investigated. At a thymidine concentration of 0.1 micromolar, incorporation into DNA is not quantitative but ceases after only 14% of the thymidine has been incorporated. Thymidine incorporation into DNA is resumed following addition of a second aliquot of thymidine, which is consistent with substrate depletion. In vivo tracer experiments indicate that this may be due to a catabolic route for converting thymidine to beta-aminoisobutyric acid. Bearing these observations in mind, conditions for determining the rate of DNA synthesis using [(3)H]thymidine incorporation have been investigated. It is concluded that by increasing the thymidine concentration to 10 micromolar the assay period may be increased, by reducing the influence of the degradative pathway, and that cell density and incubation time are critical factors in establishing a valid measure of the rate of DNA synthesis using this method. PMID- 16661359 TI - Metabolism of hydrogen cyanide by higher plants. AB - A survey has been made of the occurrence and distribution of three enzymes which metabolize cyanide in a variety of higher plants including both cyanogenic and non-cyanogenic species. The enzymes investigated were beta-cyanoalanine synthase, rhodanese and formamide hydrolyase. beta-Cyanoalanine synthase was found to be present in every higher plant tested whereas rhodanese was found to occur far less commonly in plants. Formamide hydrolyase activity was not detected in any of the higher plants tested.In addition, quantitative analyses have been made of the potential hydrogen cyanide content of each plant investigated. A general trend was apparent between the hydrogen cyanide potential and cyanide metabolizing activity, in that the higher the hydrogen cyanide potential, in general, the higher the cyanide metabolizing activity. PMID- 16661360 TI - Allantoic Acid Synthesis in Soybean Root Nodule Cytosol via Xanthine Dehydrogenase. AB - Allantoin and allantoic acid are the major forms of nitrogen transported from soybean nodules to other parts of the plant. Neither the pathway or the site of ureide synthesis has been demonstrated in root nodules.Bacteroid and cytosol (plant portion) fractions were prepared and the purity of each fraction was determined with marker enzymes. A pathway for ureide synthesis by the cytosol fraction of soybean nodules was established by measuring allantoic acid or NADH production. Enzymes were found in the cytosol fraction which would synthesize allantoic acid from the product of de novo purine synthesis, inosine-5' monophosphate. Allantoic acid production by the nodule cytosol fraction with inosine-5'-monophosphate, inosine, xanthosine-5'-monophosphate, xanthosine, hypoxanthine, or xanthine as substrates was NAD(+)-dependent and blocked by allopurinol. Both bacteroid and cytosol fractions were capable of allantoic acid production with uric acid or allantoin as substrates. Allantoic acid synthesis from these two substrates was neither dependent on NAD(+) nor inhibited by allopurinol.These data suggest that the xanthine-oxidizing enzyme in the nodule is an NAD(+)-dependent xanthine dehydrogenase (EC 1.2.1.37) which is present only in the cytosol fraction. The NADH production by this enzyme plays a critical and energy-conserving role in the ureide synthetic pathway. Cytosol xanthine dehydrogenase activity was sufficient for the metabolism of fixed N since calculations showed similar rates of N(2) fixation and xanthine oxidation. PMID- 16661361 TI - Mitochondrial DNA from Oenothera berteriana: PURIFICATION AND PROPERTIES. AB - Mitochondrial DNA (mt-DNA) from Oenothera berteriana tissue culture cells was isolated and characterized with respect to buoyant density in CsCl, melting point, contour length, and restriction fragments.Because of the rather long purification procedure very few molecules retained their circularity. Only one distinct size class of molecules with a length of 100 kilobases was found. Restriction fragments were obtained with the enzymes, restrictionendonuclease I from Serratia marcescens, restrictionendonuclease III from Haemophilus influenzae, restrictionendonuclease I from Bacillus amyloliquefaciens H, and restrictionendonuclease I from Escherichia coli (Bohnert 1977 Exp Cell Res 106: 426-430); the added lengths of these fragments amounted to 180 to 190 kilobases. As in other higher plants, an intermolecular heterogeneity has to be postulated to explain the large number of restriction fragments. Unique to the mt-DNA from Oenothera berteriana, as compared to other higher plants, is the unusual high guanosine + cytosine content with 51% as determined by the buoyant density in CsCl of 1.710 grams/cubic centimeter and the melting point of 90 C. PMID- 16661362 TI - Effects of calcium on chlorophyll synthesis and stability in the early phase of greening in cucumber cotyledons. AB - The effects of calcium on chlorophyll accumulation and its stability in the early phase of greening in cucumber cotyledons were investigated. Chlorophyll accumulation was hardly affected by dark preincubation of cotyledons with 10 millimolar calcium solution, but was inhibited almost completely when 50 or 100 millimolar solution was used. On the other hand, 50 millimolar calcium inhibited delta-aminolevulinic acid formation in the light by only 75%. Calcium had little effect on the lag for initiation of protochlorophyllide(650) regeneration, but slowed down the rate of accumulation of protochlorophyllide(650). In calcium treated cotyledons, the chlorophyll formed by primary photoconversion was quickly decomposed in the dark. The present results show that calcium inhibited chlorophyll accumulation by inhibiting delta-aminolevulinic acid formation in the light and by stimulating the decomposition of newly formed chlorophyll, both effects being completely prevented by potassium. PMID- 16661363 TI - Use of High Performance Liquid Chromatography for the Determination of Endogenous Hormone Levels in Solanum tuberosum L. Subjected to Carbon Dioxide Enrichment of the Root Zone. AB - Changes in cytokinin, auxin, and abscisic acid levels were determined by high performance liquid chromatography following CO(2) enrichment of the root zone. Both trans zeatin and zeatin riboside significantly decreased in the roots 6 days after treatment with CO(2). In the leaves zeatin riboside increased whereas trans zeatin showed no significant difference from the control. Indoleacetic acid in the leaves increased dramatically for 6 days following CO(2) treatment; however, indoleacetic acid levels in the roots increased slightly after 2 days and remained higher than the control. Abscisic acid increased shortly after CO(2) treatments; after 6 days this difference was no longer evident. PMID- 16661364 TI - Inhibition of respiration and ion uptake by 2,3,5-triiodobenzoic Acid in excised barley roots. AB - The addition of 1 micromolar 2,3,5-triiodobenzoic acid (TIBA) to solutions containing KCl resulted in the inhibition of K and Cl uptake in excised barley roots. The effectiveness of TIBA as an inhibitor increased as the pH of the treatment solution decreased and approached the pK(a) of TIBA. A lag period of approximately 20 minutes existed prior to the onset of TIBA induced inhibition of ion uptake. Respiratory activity was also inhibited by TIBA. The data suggest that in this material, TIBA functions by entering the cytoplasm and inhibiting metabolism. Comparisons made on the effect of added Ca, showed that at pH 5.7 and higher, Ca had no effect on ion uptake whereas at lower pH values the presence of Ca enhanced uptake by offsetting the deleterious effects of H(+). PMID- 16661365 TI - Incorporation and Degradation of C and H-labeled Thymidine by Sugarcane Cells in Suspension Culture. AB - Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to beta-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2 (14)C]thymidine is lost as (14)CO(2), but the label of [(3)H]methylthymidine is found in the cell as [(3)H]beta-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID- 16661366 TI - A simple autoradiographic technique for studying diffusion of water into seeds. AB - Visual observations on rates and modes of water penetration into black bean seeds and into wheat and sorghum kernels during conditioning were accomplished by an autoradiographic procedure that eliminates a freezing microtome and liquid and stripping film emulsions. Seeds soaked in tritiated H(2)O were hand sectioned before freeze-quenching in liquid N(2) and subsequent block autoradiography on nuclear medicine film. PMID- 16661367 TI - Protoplasts as a means of studying chloroplast development in vitro. AB - Protoplasts obtained enzymically from etiolated primary leaves of oat were illuminated in vitro, and the process of etioplast chloroplast transformation followed. Chloroplast development proceeded up to 6 hours of incubation in the light (20 C). During this period, complete photosynthetic light and dark reactions were constituted, in addition to prolamellar body-degrading protease activity.In parallel, electron microscopic investigations showed a pronounced decrease in prolamellar body area from 89.5 square micrometers at 0 hours to 40.6 square micrometers at 6 hours, whereas the length of the thylakoid membranes (prothylakoids, thylakoids) increased from 21 micrometers at 0 hour to about 293 micrometers at 6 hours. This was accompanied by the formation of grana (bi- and polythylakoids).A comparison of plastid structure and function, developed within isolated protoplasts and those of organelles greened in intact leaves up to 6 hours, showed that there was only a slight lag in development of plastids illuminated in vitro to those illuminated in vivo. However, times of in vitro illumination longer than 6 hours resulted in signs of deterioration and the lack of further development, except for photosystem I activities. PMID- 16661368 TI - Carbon exchange rates of shoots required to utilize available acetylene reduction capacity in soybean and alfalfa root nodules. AB - The CO(2)-exchange rate required to make full use of available N(2)-fixation capacity, measured as acetylene reduction, was determined in soybean and alfalfa. Carbohydrates of root systems were depleted during a 40-hour dark treatment; then plants were exposed to a 24-hour light period during which different CO(2) exchange rates were maintained with various CO(2) concentrations. In three- and four-week-old soybeans and four-week-old alfalfa plants, acetylene-reduction capacity was used fully with CO(2)-exchange rates as low as 10 milligrams CO(2) per plant per hour. In six-week-old alfalfa plants, however, acetylene reduction rates increased linearly, and apparent N(2)-fixation capacity was not used fully when CO(2)-exchange rates were higher than 40 milligrams CO(2) per plant per hour. Under the conditions established, the energy cost of N(2) fixation, measured as Delta(respiration of roots + nodules)/Deltaacetylene reduction over dark-treatment values, was 0.453 milligrams CO(2) per micromole C(2)H(4) for all rates of acetylene reduction and for both ages of soybean and alfalfa plants. Thus, root-plus-nodule respiration was not promoted by higher rates of apparent photosynthesis after C(2)H(2)-reduction capacity became saturated, and all available capacity for apparent N(2) fixation had the same energy requirement. PMID- 16661369 TI - Co-purification of Pea and Bean Leaf Soluble Auxin-binding Proteins with Ribulose 1,5-Bisphosphate Carboxylase. AB - Soluble auxin-binding proteins (ABPs) were purified to constant specific activity from bean and pea leaves by a procedure involving (NH(4))(2)SO(4) fractionation, anion exchange chromatography and gel filtration. Pea and bean ABPs exactly co purify with ribulose-1,5-bisphosphate carboxylase (RuBPCase) in a variety of chromatographic separation procedures. The subunit compositions, electrophoretic purities and indole-3-acetic acid (IAA)-binding stoichiometries of the purified ABPs provide further evidence for the identity of RuBPCase and ABP. Pea ABP and bean ABP have dissociation constants for IAA of 0.8 and 1.3 micromolar, respectively, as determined by an (NH(4))(2)SO(4) precipitation assay for IAA binding to insolubilized ABP. IAA can bind to soluble bean and pea ABP (RuBPCase) as determined by equilibrium dialysis with affinities and stoichiometries similar to those determined for insolubilized ABP. PMID- 16661370 TI - Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein. AB - The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), alpha-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration. PMID- 16661371 TI - Plant Nucleases: VI. GENETIC AND DEVELOPMENTAL VARIABILITY IN RIBONUCLEASE ACTIVITY IN INBRED AND HYBRID CORN ENDOSPERMS. AB - The nuclease activity of developing corn endosperms was found to consist mainly of plant RNase I during the period of major deposition of dry weight. The RNase concentrations in most inbred lines and hybrids increased throughout development, but there were large differences among genotypes in the enzyme levels at all stages. Crosses were made among inbreds classified as containing high or low RNase levels. In most cases, the general patterns of enzyme levels during development of the hybrid endosperms were not changed greatly, or showed intermediate levels of activity compared to the inbred parents. When Oh43 was used as a maternal parent, two contrasting developmental patterns were produced by using two low RNase inbreds as pollen parents. There appear to be genetic controls not only on the gross RNase levels, but also on the timing of RNase synthesis and on its stability after the cells mature. Environmental influences on RNase levels in the endosperm were noted one year.At 18 days after pollination, the RNase levels in the endosperm crown were as much as 10 times higher than in the base. By 35 days after pollination, the enzyme levels were generally uniform; at 50 days, the basal tissue usually contained the highest levels. In some genotypes, however, the enzyme levels fell in the crown while they rose in the base. These changes suggest that RNase may be associated with developmental controls that operate as the different portions of the endosperm cease cell division and begin synthesis of starch and zein. PMID- 16661372 TI - Estimating Kinetic Parameters when the Amount of Enzyme Added to an Assay Is Not a Controlled Variable: NITROGENASE ACTIVITY OF INTACT LEGUMES . AB - Reliable estimates of Michaelis constants (K(m)) and inhibitor constants may be obtained, in the absence of control over the amount of enzyme being added to any assay system, provided the following constraints are met. Michaelis-Menten kinetics are obeyed. Two rate measurements must be made with the same sample of enzyme: at low and high substrate concentration for determining K(m) or minus and plus an inhibitor for determining inhibitor constants. The Michaelis constant may be calculated from the equation [Formula: see text] Inhibitor constants are derived graphically from Lineweaver-Burk or Dixon plots, once the K(m) has been calculated. The above technique has been applied to study of the acetylene reducing ability of intact legume plants. The apparent K(m) for acetylene reduction by nitrogenase in legume nodules is approximately 1/100 atmosphere in the absence of nitrogen and approximately 1/40 atmosphere in its presence. PMID- 16661373 TI - Carbon Dioxide and Flowering in Pharbitis nil Choisy. AB - The effects of photoperiod on floral and vegetative development of Pharbitis nil were modified by atmospheric CO(2) concentrations maintained during plant growth. Short day (SD) photoperiods caused rapid flowering in Pharbitis plants growing in 0.03 or 0.1% CO(2), while plants in long day (LD) conditions remained vegetative. At 1 or 5% CO(2), however, flower buds were developed under both the SD and LD photoperiods. Flowering was earliest in the plants exposed to SD at low CO(2) concentrations which formed floral buds at stem node 3 or 4. At high CO(2) concentrations, floral buds did not form until stem node 6 or 7. Both high CO(2) concentrations and LD photoperiods tended to enhance stem elongation and leaf formation.The occurrence of flowering under normally noninductive LD photoperiods at 1 or 5% CO(2) is readily explained in terms of higher photosynthetic rates. Plants grown at 0.03 or 1% CO(2) in either photoperiod tended to approach maximum photosynthesis between 0.1% and 0.5% CO(2). In addition, relative growth rates were not significantly increased by growth at 1 or 5% CO(2). Possible alternative mechanisms for the interactive effects of CO(2) and photoperiod are discussed. PMID- 16661374 TI - Purity of Chloroplasts Prepared from the Siphonous Green Alga, Caulerpa simpliciuscula, as Determined by Their Ultrastructure and Their Enzymic Content. AB - The ultrastructure and enzyme distribution in chloroplasts and other subcellular fractions isolated from the siphonous green alga, Caulerpa simpliciuscula, are described. The isolated chloroplasts were similar in appearance to those in the tissue from which they were derived, and in typical preparations 70% or more were intact. Chloroplasts which had lost their outer envelopes could be separated from intact plastids by centrifugation at low speeds through gradients of colloidal silica. Intact chloroplasts separated in this way retained their photosynthetic capacity and were impermeable to ferricyanide ions. The chloroplast preparations separated by differential centrifugation and refractionated using either discontinuous or continuous Percoll gradients contained non-chloroplast material. It was estimated that this amounted to a maximum of 10% of the mitochondrial population and 6% of cytoplasm extracted from the plant. The contaminating material surrounded the chloroplasts in a thin layer and was surrounded by a membrane. PMID- 16661375 TI - Asparagine formation in soybean nodules. AB - (15)NH(4) (+) and [(15)N](amide)-glutamine externally supplied to detached nodules from soybean plants (cv. Tamanishiki) were incorporated within nodule tissues by vacuum infiltration and metabolized to various nitrogen compounds during 60 minutes of incubation time. In the case of (15)NH(4) (+) - feeding, the (15)N abundance ratio was highest in the amide nitrogen of glutamine, followed by glutamate and the amide nitrogen of asparagine. In (15)N content (micrograms excess (15)N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. (15)NH(4) (+) was also appreciably assimilated into alanine.When [(15)N](amide)-glutamine was fed to detached nodules, (15)N was almost entirely in the amide nitrogen of asparagine. The result indicates the presence of active glutamine-linked asparagine synthesizing systems in soybean root nodules. PMID- 16661376 TI - Chloroplast Biogenesis 31: DETECTION OF AN INHIBITOR OF PROTOCHLOROPHYLL BIOSYNTHESIS IN CUCUMBER COTYLEDONS. AB - An extract from cucumber cotyledons was shown to cause an inhibition of protochlorophyll biosynthesis and accumulation. The extract inhibited the net synthesis of protochlorophyll as well as the incorporation of delta amino[(14)C]levulinic acid into protochlorophyllide and protochlorophyllide ester by excised cotyledons. The inhibition of delta-amino[(14)C]levulinic acid incorporation into the two protochlorophyll species was also observed in isolated etiochloroplasts before and after lysis of the plastids. The inhibition did not appear to involve the oxidation of the delta-aminolevulinic substrate or its translocation across the plastid membrane. Kinetic analysis of the rate of protochlorophyllide and protochlorophyllide ester biosynthesis in the presence and absence of the inhibitor suggested that the mode of inhibition of the two protochlorophyll species was different. PMID- 16661377 TI - Dynamic aspects and enhancement of leaf elongation in rice. AB - Some dynamic aspects of leaf elongation in rice were studied. Under both well watered and water-deficient conditions, leaf elongation rates were 15 to 30% greater during the day than during the night. Night temperatures below 27 C limited the rate of elongation at night but when night temperatures exceeded 27 C, night elongation rates exceeded rates during the day. The diurnal pattern of elongation was opposite to the pattern of bulk leaf turgor which was lower during the day than at night.Superimposed on the general diurnal pattern of leaf elongation were perturbations associated with the light/dark transitions. The rate of leaf elongation declined within minutes after illumination and remained low for 15 to 60 minutes, after which rapid rates ensued. The rate of leaf elongation was transiently accelerated within minutes after transition to dark and then declined to steady night rates after 30 to 60 minutes. Removal or covering of all subtending leaves eliminated these perturbations. Irrigation during the light-induced inhibition period did not influence leaf elongation rates of well watered plants but in stressed plants, high rates of elongation resumed immediately after irrigation.The rate of elongation was accelerated by hydrostatic pressure applied to roots of intact plants. The rate of leaf elongation increased with increasing pressure to about 5 bars and then showed no further increase with increasing pressure. This suggests that the rate of water uptake normally limits the rate of leaf elongation. The response to pressure could be altered by addition of an osmoticum to the root medium and elongation occurred only when the gradient of total water potential between the substrate and elongating leaf allowed water absorption. A model of leaf expansion based on water potential gradients is proposed to explain these observations. PMID- 16661378 TI - Screening for barley mutants with altered hormone sensitivity in their aleurone layers. AB - A method, based on the diffusion assay of alpha-amylase on agar plates, was developed to screen for barley (Himalaya) mutants with altered sensitivity to gibberellic acid (GA(3)) or abscisic acid (ABA) in their aleurone layers. The seeds produced by sodium azide-mutagenized barley were screened for their ability to synthesize and secrete alpha-amylase when treated with different combinations of hormones. Various GA(3)-insensitive or supersensitive, ABA-insensitive, temperature-dependent GA(3)-insensitive, and constitutive mutants have been identified. Several stable mutants with altered GA(3) sensitivity were recovered. Two of the homozygous GA(3)-insensitive mutants have been preliminarily characterized. The GA(3)-enhanced production of alpha-amylase and release of phosphatase are hampered in these mutants. However, they have normal stem height, and the uptake of GA(3) by their aleurone layers appears to be the same as that of wild-type barley. They are most likely regulatory mutants affecting both alpha amylase synthesis and phosphatase release. PMID- 16661379 TI - Composition of the Capsular and Extracellular Polysaccharides of Rhizobium japonicum: CHANGES WITH CULTURE AGE AND CORRELATIONS WITH BINDING OF SOYBEAN SEED LECTIN TO THE BACTERIA . AB - The chemical compositions of the capsular and extracellular polysaccharides of two strains of Rhizobium japonicum (311b 138 and 110) have been determined and correlated as a function of culture age with the ability of the bacteria from which they were obtained to bind soybean seed lectin.Each of the polysaccharides contains approximately constant amounts of mannosyl, glucosyl, and galacturonosyl residues in a molar ratio of 1:2:1. In addition they contain variable amounts of galactosyl and 4-O-methyl galactosyl residues. The total of galactose plus 4-O methyl galactose, however, is constant and equivalent to the amount of mannose, indicating that the 4-O-methyl galactose residues arise by methylation of galactose residues in the polysaccharides. In both strains the proportion of galactose to methyl galactose is considerably greater in the polysaccharide from bacteria which do bind lectin than in the polysaccharide from bacteria which do not bind lectin.In addition to the changes in polysaccharide composition, there is a reduction of about 50% in the percentage of cells which are encapsulated as the cultures mature from early to late log phase. Since only capsulated cells bind lectin, the combination of the change in capsular composition and loss of encapsulation is probably sufficient to account for the loss of lectin binding capacity during growth of cultures of Rhizobium japonicum 311b 138 and 110. PMID- 16661380 TI - Grain Protein Accumulation and the Relationship between Leaf Nitrate Reductase and Protease Activities during Grain Development in Maize (Zea mays L.): I. VARIATION BETWEEN GENOTYPES. AB - Four maize hybrids, two with high and two with low levels of postanthesis nitrate reductase activity were grown under field conditions. The characteristic enzyme patterns had been established in previous work. Nitrate reductase and proteases were measured in three representative leaves (ear leaf, fourth leaf above and fourth leaf below the ear) at intervals throughout the period of grain development. Concurrent with enzyme sampling, other plants were harvested and subdivided into top, middle and lower leaves, husks, stalks, and ear. Dry weights, nitrate, and reduced N were determined on all plant parts for each sampling. These data established the rate of N accumulation by the grain and depletion from the vegetative material and provide some insight into the relation between newly reduced and remobilized N and accumulation of grain N. Other plants were harvested at maturity for yield and harvest indices for dry weight and N.Nitrate reductase activity was higher in comparable leaves from the high than from the low nitrate reductase genotypes throughout the grain development period. There was no mathematical correlation between nitrate reductase activity and nitrate content of the leaves or stalks, however the high nitrate reductase genotypes maintained a higher amount of nitrate per plant (largely in the stalk) during the later stages of grain development. From the patterns of plant nitrate content it was deduced that the low nitrate reductase genotypes terminated nitrate absorption sooner than the high nitrate reductase types. Proteolytic activities (casein as substrate at pH 5.5 and 7.5) were higher and increased earlier in the low than in the high nitrate reductase genotypes. The "low nitrate reductase-high protease" genotypes had a higher percentage of grain N, and higher harvest index for N than did the "high nitrate reductase-low protease" genotypes. These results permit the tentative conclusions that: (a) redistribution of vegetative N accounted for more of the grain N in the low than in the high nitrate reductase genotypes; and (b) leaf protease activities are more closely related to the accumulation of grain N than leaf nitrate reductase activity. PMID- 16661381 TI - Comparison between Aging of Slices and Ethylene Treatment of Whole White Potato Tubers. AB - Cyanide-resistant O(2) consumption can be stimulated by either treating whole white potato tubers (Norchip) with ethylene, in the presence of 100% O(2), or aging slices obtained from untreated potato tubers. A comparison of alternative pathway activity elicited by either treatment was undertaken. The proportion of electrons flowing through the alternative path in the presence of intermediate concentrations of KCN and at various concentrations of salicylhydroxamic acid was identical in both cases. However, the respiration of slices from ethylene-treated tubers was in every case stimulated by KCN, whereas the aged slices never exhibited this phenomenon. Furthermore, the metabolism of d-[U-(14)C]glucose was several hundred times greater in aged slices than in fresh slices from C(2)H(4) treated tubers. These results, along with the respiratory kinetics of aged slices from ethylene-treated tubers, suggest that aged slices and fresh slices from ethylene-treated tubers are biochemically dissimilar. PMID- 16661382 TI - Induction of Secondary Dormancy in Chenopodium bonus-henricus L. Seeds by Osmotic and High Temperature Treatments and Its Prevention by Light and Growth Regulators. AB - Factors controlling the establishment and removal of secondary dormancy in Chenopodium bonus-henricus L. seeds were investigated. Unchilled seeds required light for germination. A moist-chilling treatment at 4 C for 28 to 30 days removed this primary dormancy. Chilled seeds now germinated in the dark. When chilled seeds were held in the dark in -8.6 bars polyethylene glycol 6000 solution at 15 C or in water at 29 C a secondary dormancy was induced which increased progressively with time as determined by subsequent germination. These seeds now failed to germinate under the condition (darkness) which previously allowed their germination. Continuous light or daily brief red light irradiations during prolonged imbibition in polyethylene glycol solution at 15 C or in water at 29 C prevented the establishment of the secondary dormancy and caused an advancement of subsequent germination. Far red irradiations immediately following red irradiation reestablished the secondary dormancy indicating phytochrome participation in "pregerminative" processes. The growth regulator combination, kinetin + ethephon + gibberellin A(4)+A(7) (GA(4+7)), and to a relatively lesser extent GA(4+7), was effective in preventing the establishment of the secondary dormancy and in advancing the germination or emergence time. Following the establishment of the secondary dormancy by osmotic or high temperature treatments the regulator combination was relatively more active than light or GA(4+7) in removing the dormancy. Prolonged dark treatment at 29 C seemed to induce changes that were partially independent of light or GA(4+7) control. The data presented here indicate that changes during germination preventing dark treatment determine whether the seed will germinate, show an advancement effect, or will become secondarily dormant. These changes appear to be modulated by light and hormones. PMID- 16661383 TI - Characterization of Three Photosystem II Mutants in Zea mays L. Lacking a 32,000 Dalton Lamellar Polypeptide. AB - Two fully blocked and one partially blocked photosystem II nuclear mutants have been selected in Zea mays. The fully blocked mutants lack photosystem II activity, variable fluorescence, the light-inducible C-550 signal, the high potential form of cytochrome b-559, and most or all of the low potential form of the cytochrome. The block in these mutants may primarily affect the reducing side of photosystem II, inasmuch as chloroplasts isolated from both mutants exhibit an elevated F695 fluorescence emission peak. The partially blocked mutant exhibits partial photosystem II activity and a reduction, but not the total loss of the variable fluorescence yield, the C-550 signal, and the high potential form of cytochrome b-559. Lamellae isolated from the fully blocked mutants are greatly deficient for a major lamellar polypeptide with an apparent molecular weight of 32,000 daltons, whereas lamellae from the partially blocked mutant show the partial loss of this same polypeptide, suggesting that the 32,000 dalton polypeptide is necessary for the proper function of photosystem II. PMID- 16661384 TI - In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes. AB - Vigna radiata polysomes efficiently incorporated [(75)Se]selenomethionine, [(14)C]methionine, and [(14)C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg(2+), 182 millimolar K(+), and pH 7.4. The rates of incorporation of [(75)Se]selenomethionine and [(14)C]methionine were similar when measured separately, but [(75)Se]selenomethionine incorporation was 35% less than [(14)C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [(75)Se]selenomethionine or [(35)S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity. PMID- 16661385 TI - Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts. AB - A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis. PMID- 16661386 TI - Density of Microbodies on Sucrose Gradients during Phytochrome-mediated Glyoxysome Peroxisome Transformation in Cotyledons of Mustard Seedlings. AB - The microbodies extracted from the cotyledons of mustard seedlings (Sinapis alba L.) form two bands (at 1.18 kilograms per liter together with the mitochondria, and at 1.24 kilograms per liter) on conventional isopycnic sedimentation density gradients. The artifactual co-banding of part of the microbodies with the mitochondria can be prevented by using flotation gradients. Using this procedure, a systematic investigation revealed no effect of seedling age and irradiation (far red or white light) on the density of the microbody population (1.242 +/- 0.002 kilograms per liter). Thus, although light, through phytochrome, induces conspicuous changes in their enzyme composition the microbodies appear as a homogeneous population of constant density on a sucrose gradient. PMID- 16661387 TI - Isolation of protoplasts and vacuoles from storage tissue of red beet. AB - A fast and efficient method is described for the isolation of protoplasts and vacuoles from storage tissue of Beta vulgaris L. The viability of the isolated protoplasts is indicated by the development within a few hours of plasma strands with active cyclosis as well as by transport activity.The effect of aging of the tissue on the yield and on the properties of the protoplasts is examined. Adding 1 millimolar dithiothreitol to the water during the aging of the tissue prevents a change of the cell wall structure which otherwise reduces the protoplast yield within 2 days to zero. When protoplasts are isolated from tissue after different times of aging, they show an increase in transport activity which parallels that in the intact tissue. PMID- 16661388 TI - Kinetics of C Distribution during Photosynthesis by Chloroplast Preparations Isolated from the Siphonous Alga Caulerpa simpliciuscula. AB - The kinetics of (14)C-labeling of compounds produced during photosynthesis by chloroplast preparations isolated from the green alga Caulerpa simpliciuscula were studied. After 10 minutes photosynthesis sucrose contained more (14)C than any other product, and continued to accumulate radioactivity during the whole hour of incubation. Glucose-6-phosphate and alanine also behaved as end products and continued to accumulate label during the period. In these organelles, glucose 6-phosphate replaced triose phosphate as the main compound exported from the chloroplast during shorter periods of photosynthesis. When either glucose-6 phosphate or 3-phosphoglycerate was supplied to the isolated chloroplasts, they were metabolized, but were not converted to either sucrose or alanine. It is proposed that many of the differences in metabolism which distinguish these algal chloroplasts from those isolated from higher plants are due to their isolation in the form of cytoplasts, i.e. chloroplasts surrounded by a thin layer of extrachloroplastic material which is membrane-bound. The restriction of diffusion of intermediates from the chloroplast by this cytoplast membrane appears to be at least as important as the rather small amount of cytoplasm present in determining the properties observed. PMID- 16661390 TI - Units of freezing of deep supercooled water in woody xylem. AB - The low temperature exotherms (LTE) of 1-year-old twigs of Haralson apple (Malus pumila Mill.), shagbark hickory (Carya ovata [Mill.] K. Koch), green ash (Fraxinus pennsylvanica Marsh), honey locust (Gleditsia triacanthos L.), American chestnut (Castanea dentata [Marsh] Borkh.), and red oak (Quercus rubra L.) were determined by differential thermal analysis (DTA). In one type of experiment freezing during a DTA experiment was halted for up to 2.5 hours after part of the supercooled water had frozen at temperatures between -25 and -42 C. Upon resumption of cooling the freezing started within 2 C of the stopping temperature. In a second type of experiment living and dead cells were microscopically observed in the same ray after partial freezing in the DTA apparatus. In another experiment, the LTE persisted even after tangential and radial sectioning of the twig to 0.13 millimeters. In a final experiment the LTE of a single multiseriate ray of red oak had the same shape as the LTE of wood with many uniseriate rays.These experiments confirm that the deep supercooled water in woody xylem or pith freezes in numerous independent events over a span of as much as 20 C. The units which freeze in an event are single cells or small groups of cells. Ice grows very slowly if at all from these units, and water moves very slowly from unfrozen cells to frozen ones. Deep supercooling of ray parenchyma does not require an intact ray. PMID- 16661389 TI - Activation of Glyceraldehyde-Phosphate Dehydrogenase (NADP) and Phosphoribulokinase in Phaseolus vulgaris Leaf Extracts Involves the Dissociation of Oligomers. AB - Phosphoribulokinase (EC 2.7.1.19, ATP: d-ribulose-5-phosphate-1 phosphotransferase) resembles the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, d-glyceraldehyde-3-phosphate: NADPH(+) oxidoreductase [phosphorylating]) of chloroplasts in that the activation of both of these enzymes involves the dissociation of oligomers (apparently tetrameric forms) with low catalytic activity to give protomers which possess higher catalytic activity. Gel filtration on Sepharose 6B has shown that the molecular weights of the oligomer and active protomer of phosphoribulokinase are, respectively, about 6.8 x 10(5) and 1.7 x 10(5), whereas the corresponding values for glyceraldehyde-3 phosphate dehydrogenase are 8.2 x 10(5) and 2.2 x 10(5). Activation of both enzymes occurs in response to either ATP, dithiothreitol, or cholate while the glyceraldehyde-3-phosphate dehydrogenase is also activated by NADPH. Activation/dissociation of these enzymes may involve conformational changes resulting from nucleotide binding, the reduction of sulfur bridges, and the cholate induced loosening of hydrophobic interactions. PMID- 16661391 TI - Plasmalemma Transport of OH in Chara corallina: III. FURTHER STUDIES ON TRANSPORT SUBSTRATE AND DIRECTIONALITY. AB - The identity of the plasmalemma-transported species that develops the alkaline bands of Chara corallina was investigated. The effect of fusicoccin on the rate of HCO(3) (-) assimilation, and on the time-dependent alkaline band pH buildup following low pH flushing, was found to be small, with no stimulatory effect. Computer simulation of the flushing experiments showed that in the experimental situation the alkaline band transport system was slowed down, rather than speeded up, by low pH flushing. A detailed theoretical examination of the maximum rate of proton production from water showed that measured alkaline band fluxes are too large to be explicable in terms of an H(+) influx system. The experimental and theoretical results indicate that the plasmalemma transport of OH(-) ions is responsible for the measured negative external electric potential and alkalinity flux which are associated with the alkaline band phenomenon. Consequently, HCO(3) (-) influx across the characean plasmalemma must be charge-balanced by the efflux of OH(-) ions. PMID- 16661392 TI - Isotopic Fractionation Associated With Symbiotic N(2) Fixation and Uptake of NO(3) by Plants. AB - Isotopic fractionation associated with N(2) fixation and NO(3) (-) uptake by plants are relevant to the accuracy of estimates of N(2) fixation based on differences in the natural abundance of (15)N between N(2) fixing and nonfixing plants. The isotope effect on N(2) fixation by soybeans (Glycine max [L] Merrill, variety Harosoy) and red clover (Trifolium pratense [L]) was determined from the difference in (15)N abundance between atmospheric N(2) and the total N of plants grown hydroponically with N-free nutrient solution. In soybeans the isotope effect was found to be +0.98 +/- 0.18 per thousand (beta = 0.99902). In clover the isotope effect was +1.88 +/- 0.14 per thousand (beta = 0.99812). The magnitude of these inverse isotope effects is small. However, they would lead to an underestimation of the amount of N(2) fixed, since the N of atmospheric origin which finally appears in the plant is made richer in (15)N by the inverse isotope effects than is atmospheric N(2), and, to that degree, is attributed to soil derived N in the calculation.Isotopic fractionation associated with NO(3) (-) uptake by plants does not have a critical effect on estimates of N(2) fixation which are based on natural abundance of (15)N since the (15)N abundance of soil derived N in plants is measured directly (i.e. after the N has undergone fractionation). Nevertheless, such fractionation is of some interest from the point of view of deciding upon the most appropriate sampling time. The isotope effect on NO(3) (-) uptake by a nonnodulating isoline of soybeans (variety Harosoy), marigold (Tagetes erecta [L]) and ryegrass (Lolium perenne [L]) was estimated from the difference between the (15)N abundance of the total N of plants grown hydroponically and that of NO(3) (-) supplied in the medium. It was found to be about -5 per thousand (beta = approximately 1.005). PMID- 16661393 TI - Distribution of N among plant parts of nodulating and nonnodulating isolines of soybeans. AB - Differences among plant parts in the natural abundance of (15)N are of interest from the point of view of developing a sampling strategy for using (15)N measurements to estimate the contribution of symbiotically fixed N to N(2) fixing plants, and because they reflect isotopic fractionation associated with degradation, transport, and resynthesis of N-bearing molecules. This paper reports such differences in nodulating and nonnodulating isolines of soybeans (Glycine max [L] (Merrill, variety Harosoy)) grown under several different conditions.Nodules were strikingly enriched in (15)N compared to other plant parts (by an average of 8.3 per thousand excess (15)N), and the enrichment increased with time during the growing season. (15)N was much more uniformly distributed among other plant parts. Although there were significant differences among other plant parts, the maximum deviation of the (15)N abundance of any plant part from that of the entire plant was about 2 per thousand (15)N excess. The (15)N abundance of the seed N was most representative of the whole plant.There were significant differences between isolines in the distribution of (15)N. The distribution of (15)N within plants also varied with experimental conditions. The implications of these results for estimation of N(2) fixation from measurements of the natural abundance of (15)N are discussed. PMID- 16661394 TI - Estimates of n(2) fixation based on differences in the natural abundance of N in nodulating and nonnodulating isolines of soybeans. AB - Estimates of the contribution of biologically fixed N to the total N of nodulating soybeans (Glycine max (L) Merrill, variety Harosoy) grown under a variety of conditions were made from: (a) differences in N yield between nodulating and nonnodulating isolines; and (b) differences in (15)N abundance between the two isolines. For plants grown in a greenhouse in nutrient-poor soil, both estimates showed a high level of N(2) fixation; from 58 to 89% N fixed by differences in N yield and from 51 to 95% by differences in (15)N abundance. Decreasing contributions of fixed N were estimated by both methods with increasing levels of added NO(3) (-). Results of field experiments carried out over two years on an unamended highly fertile midwestern soil showed a modest level of N(2) fixation by both methods (7.3 to 51% by differences in N yield, and 5.4 to 46% by differences in (15)N abundance). When the soil was amended with ground corn cobs, both methods showed higher contributions of fixed N. The two methods of estimating N(2) fixation gave similar results. Both appear to be semiquantitative and the standard errors of the estimates were about the same (6% on the average). PMID- 16661395 TI - Long Distance Transport in Macrocystis integrifolia: III. MOVEMENT OF THO. AB - Movement of THO and tritium-labeled photoassimilate was studied in intact fronds and frond cuttings of Macrocystis integrifolia following labeling of a mature blade by tritiated water. Both THO and tritium-labeled assimilate moved from the source blade to sink areas at velocities comparable to those recorded earlier for (14)C- and (32)P-labeled compounds. In intact fronds and frond cuttings, THO and tritium-labeled assimilate showed a declining gradient with increasing distance from the source. In the exudate collected from the basal cut end of the frond, there was a marked increase in radioactivity with time in the photoassimilate, but no such gradient was evident for THO. These results support the idea that, although both tritium-labeled assimilate and THO move in the sieve elements, THO is rapidly exchanged with water in the tissues surrounding the sieve elements. Finally, it is shown that THO is transported to the sink and there "unloaded"; indeed, it can move out of the plant itself. The data on velocity and directionality of transport as well as unloading of THO at the sink are discussed, along with computations on specific mass transfer, and favor the idea that Munch's pressure-flow hypothesis is applicable in Macrocystis for long distance translocation of photoassimilates. PMID- 16661396 TI - Development of Mitochondrial Activities in Pea Cotyledons during and following Germination of the Axis. AB - Development of mitochondrial activities in pea cotyledons during early times after the start of imbibition occurred in two phases. In the first phase (0 to 8 hours after the start of imbibition), succinate or NADH oxidation increased rapidly, while malate or alpha-ketoglutarate oxidation remained low. The latter activities developed only 8 to 12 hours after the start of imbibition (the second phase). Development in the first phase was induced by water uptake, but some development occurred even when the cotyledons were fully imbibed. The presence of the axis was required for the second phase of the development. It is suggested that mitochondrial development in the second phase is brought about by activation of the electron transfer path at a site between the oxidation of endogenous NADH and the reduction of ubiquinone. PMID- 16661397 TI - In vivo detection of membrane injury at freezing temperatures. AB - The release of hydrogen cyanide from Amelanchier alnifolia was monitored at 30 C and -10 C following lethal freezing at both slow and fast rates. Assuming that hydrogen cyanide release indicates membrane damage, it was concluded that during a fatal freeze-thaw cycle membrane damage occurred during cell contraction and, therefore, was not dependent upon membrane area expansion during thawing. PMID- 16661398 TI - Nitrate and Carbohydrate Effects on Nodulation and Nitrogen Fixation (Acetylene Reduction) Activity of Lentil (Lens esculenta Moench). AB - Lentils (Lens esculenta Moench, cv. Tekoas) grown in a nutrient solution containing 15 millimolar nitrate had 84% fewer nodules than lentils grown in nitrate-free nutrient solution. Nodules from the nitrate-grown plants weighed 71% less than nodules from the nitrate-free plants. Nitrate-grown plants also fixed much less nitrogen (measured by acetylene reduction) than the nitrate-free plants. When lentils were grown in a solution containing 15 millimolar nitrate and 75 millimolar fructose, glucose, or sucrose, however, the nitrogen fixation activity of their nodules was similar to that of nodules from nitrate-free plants. Leaves of lentils grown in the nitrate-sugar solutions had only about 7% as much nitrate reductase activity and accumulated only 10% as much nitrate as leaves from lentils grown in the nitrate solution alone. Roots of lentils grown in the nitrate-sugar solutions had similar nitrate reductase activity but accumulated only 17 to 25% as much nitrate as roots from lentils grown in the nitrate solution. The results indicate that the added sugars alleviated the inhibitory effects of nitrate on symbiotic nitrogen fixation not only by increasing the carbohydrate supply so lentils could support both nitrogen fixation and nitrate reduction but also by inhibiting the accumulation of nitrate and, hence, lowering nitrate reductase activity in the leaves. PMID- 16661399 TI - Metabolism of Specifically Labeled Glucose, Glucose 1-Phosphate, and Glucose 6 Phosphate via the Oxidative Pentose Phosphate Cycle in a Reconstituted Spinach Chloroplast System in Darkness and in the Light. AB - Using isolated spinach (hybrid 424) chloroplasts deprived of their envelopes (reconstituted chloroplast system), the metabolism of glucose, glucose 1 phosphate, and glucose 6-phosphate via the oxidative pentose phosphate cycle was analyzed. The activity of oxidative pentose phosphate cycle was monitored by continuous sampling of the CO(2) released during the decarboxylation process of 6 phosphogluconate.The rate of CO(2) released in the dark from [C-1-(14)C]glucose 6 phosphate was 4 to 6 micromoles per milligram chlorophyll per hour. A CO(2) release from the C-6 position of [C-6-(14)C]glucose 6-phosphate was hardly measurable within 60 minutes of incubation. Glucose 1-phosphate was readily converted to glucose 6-phosphate without externally added glucose bisphosphate and was metabolized via the oxidative pentose phosphate cycle. The phosphorylation of glucose to glucose 6-phosphate was mediated by hexokinase present in the reconstituted system. This step was rate-limiting for the over-all reaction of the oxidative pentose phosphate cycle with glucose being the substrate (0.5 micromoles per milligram chlorophyll per hour). Addition of hexokinase increased the rate of CO(2) release to 5 micromoles per milligram chlorophyll per hour.The flow of carbon through the oxidative pentose phosphate cycle was greatly reduced upon the addition of NADPH and ATP. Whereas NADPH inhibited the metabolism of [C-1-(14)C]glucose 6-phosphate via the oxidative pentose phosphate cycle, ATP stimulated carbon flow into the 3-phosphoglycerate, dihydroxyacetone phosphate, and bisphosphates pools via the glycolytic pathway mediated by phosphofructokinase. This regulatory phenomenon could also be demonstrated with the reconstituted system undergoing a dark-light-dark transition. Data are presented indicating the conditions under which glucose 6 phosphate or glucose 1-phosphate are metabolized via the oxidative pentose phosphate cycle and the glycolytic pathway. PMID- 16661400 TI - Role of the Cotyledons in the Phototropic Response of Lavatera cretica Seedlings. AB - Young seedlings of Lavatera cretica L. exhibit positive phototropism. The hypocotyl perceives unilateral illumination with blue light and curves towards the light source by unequal growth. In addition, the cotyledonary laminas perceive the vectorial component of unilateral illumination with blue light and reorient normal to the beam by creating a turgor differential in their pulvini. Excision of one cotyledon resulted in negative organotropic curvature of the hypocotyl, away from the remaining cotyledon. Illumination of the cotyledonary lamina did not participate in the phototropic curvature of the hypocotyl, so long as the lamina was free to reorient to face the beam. When the lamina was continuously exposed to vectorial photoexcitation, elongation of the hypocotyl on the side carrying the cotyledon could be enhanced, or inhibited, depending on the direction of the beam striking its lamina. PMID- 16661401 TI - Ethylene Production and Leaflet Abscission in Melia azedarach L. AB - Ethylene production or content was compared to leaflet abscission in detached, compound leaves of Melia azedarach L. In late autumn, when abscission was progressing from basal leaves upward, the oldest leaves both produced ethylene at the highest rates and abscised their leaflets first. When C(2)H(4) levels were measured in intercellular air removed immediately after leaves were harvested, C(2)H(4) levels were also highest in basal leaves and declined progressively in more apical leaves. Levels as high as 1.8 microliters C(2)H(4) liter(-1) air were observed. Earlier in the season groups of leaves demonstrated a pattern of sequential initiation of abscission from base to apex, but the peak rates of C(2)H(4) production followed an opposite trend, being highest in the youngest leaves. Peak rates of C(2)H(4) production occurred after the initiation of leaflet abscission and presumably are related to either the auxin content or a climacteric-like, autocatalytic phase of C(2)H(4) production not directly involved in the initiation of abscission. In these experiments, the early abscission of the older leaflets reflects their greater sensitivity to C(2)H(4), presumably due to lower auxin content. C(2)H(4) production rates in all experiments, with rare exceptions, exceeded 3 microliters per kilogram fresh weight per hour at least 24 hours before leaflet abscission reached 10%. This achieving of a threshold internal C(2)H(4) level is viewed as an initiating event in leaflet abscission. Hypobaric conditions, to facilitate the escape of endogenous C(2)H(4), delayed abscission compared to controls, and termination of hypobaric exposure allowed a normal progression of abscission as well as normal C(2)H(4) synthesis rates. All of the data indicate that C(2)H(4) initiates leaflet abscission in intact but detached leaves of Melia azedarach L. The seasonal patterns observed suggest that C(2)H(4), in concert with those hormones which govern sensitivity to C(2)H(4), regulate autumn leaf fall in this species. PMID- 16661402 TI - Purification and Some Properties of Cell Wall-bound Invertases from Sugar Beet Seedlings and Aged Slices of Mature Roots. AB - Cell wall-bound invertases (EC 3.2.1.26) from both sugar beet seedlings and aged slices of mature roots were purified to homogeneity separately with CM-cellulose chromatography and Bio-Gel P-150 gel filtrations. The enzymes behaved similarly throughout the purification procedures. The purified enzymes are identical as characterized by specific activity, gel electrophoretic mobility, K(m) for sucrose and raffinose (1.33 and 4.0 millimolar, respectively), mobility on Bio Gel P-150 (molecular weight 28,000), optimum pH (4.6 to 5.0), optimum temperature, and dependence on NaCl concentration for insolubilization by DNA. The results suggest that the enzymes may be encoded for by the same structural gene. PMID- 16661403 TI - Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways: V. RESPONSE OF PANICUM MAXIMUM, PANICUM MILIOIDES, AND TALL FESCUE (FESTUCA ARUNDINACEA) TO NITROGEN NUTRITION. AB - The response of apparent photosynthesis to N nutrition was studied in the C(3) grass, tall fescue (Festuca arundinacea Schreb.), in the C(4) species Panicum maximum Jacq., and in Panicum milioides Nees ex Trin., a species with characteristics intermediate between C(3) and C(4) photosynthetic types. Plants were grown in culture solution containing 1, 5, 50, and 200 milligrams N per liter. Apparent photosynthesis was measured on the youngest fully expanded leaves at 320 microliters of CO(2) per liter of air and 21% O(2). Leaf conductance was calculated from transpiration measurements, and CO(2) compensation concentrations were also estimated. Several leaf anatomical characteristics were studied on plastic-embedded material. Leaf N content was determined on leaves which were used in photosynthesis measurements.Apparent photosynthesis increased in a linear fashion with increases in leaf N content in all three species. The increase in apparent photosynthesis per unit increase in leaf N was over twice as great in P. maximum (9.7 milligrams CO(2) per square decimeter per hour for each increase of 1% leaf N) as in P. milioides and tall fescue. Apparent photosynthesis and its response to N level were similar in P. milioides and tall fescue, but at leaf N concentrations above 2%, apparent photosynthesis in these two species was only about one-half of that in P. maximum. Increases in apparent photosynthesis due to higher N levels were accompanied by increases in both leaf and mesophyll conductances in P. maximum, whereas in P. milioides and tall fescue only mesophyll conductance was significantly correlated with leaf N. Water use efficiency (apparent photosynthesis/transpiration) increased in a linear manner with increased leaf N in all three species but was more closely related to leaf N in P. milioides and tall fescue than in P. maximum. High N levels tended to increase leaf thickness and interveinal distance in all three species. The percentage of air space in leaf tissue and the mesophyll cell diameter were either not affected or changed only slightly by N nutrition with the exception that percentage of air space in tall fescue leaves rose from 17.3 to 24.8 with an increase in solution N level from 1 to 200 milligrams per liter. Although P. milioides has been shown to have photosynthetic and leaf anatomical characteristics intermediate to C(3) and C(4) species, its response to N was similar to that of the C(3) grass, tall fescue. PMID- 16661404 TI - Glycosidic Enzyme Activity in Pea Tissue and Pea-Fusarium solani Interactions. AB - Membrane barriers which prevent direct contact between Fusarium solani and pea endocarp tissue prevent fungal spores from inducing phytoalexin production. Conversely, preinduced host resistance responses are not readily transported from the plant across the membrane barrier to Fusarium macroconidia.Crude enzyme extracts from pea endocarp tissues partially degrade Fusarium solani f. sp. phaseoli cell walls. Activities of the glycosidic enzymes, chitinase, beta-1,3 glucanase, chitosanase, beta-D-N-acetylglucosaminidase, beta-D-N acetylgalactosaminidase, beta-D-glucosidase, alpha-D-glucosidase, and alpha-D mannosidase, were detected in pea endocarp tissue. If pods are challenged with Fusarium spores or chitosan, the chitinase activity of the infected tissue remains higher than water-treated pods 0.5 to 6 hours after treatment. The beta 1,3-glucanase activity increases within 6 hours in both inoculated and control tissue. Chitosanase activity was lower in tissue treated with Fusarium solani f. sp. pisi, f. sp. phaseoli or chitosan than in water-treated control tissue. Thus, the pea tissue contains glycosidic enzymes with the potential to degrade the major compounds of the Fusarium cell walls. PMID- 16661405 TI - Chitosan as a Component of Pea-Fusarium solani Interactions. AB - Chitosan, a polymer of beta-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection. PMID- 16661406 TI - Activation of nitrate reductase by extracts from corn scutella. AB - NADH-nitrate reductase (NR) from the primary leaves and root tips of corn seedlings (var. W64A x W182E) were activated by extracts from corn scutella. The activator extracted in potassium phosphate buffer (pH 7.5) or 80% (v/v) ethanol and fractionated by Dowex 1 (acetate) and Dowex 50 (H(+)) resins was recovered in the cationic fraction. The activator was not detected in extracts from shoots, roots, or endosperm of the seedlings. It activated the nitrate-induced cytochrome c reductase of NR complex but had slight inhibitory effects on the activities of FMNH(2)-NR and reduced methylviologen-NR. In addition the activator inhibited the activities of purified NR-inactivating proteins from corn roots (var. Wf9 x 38 11) and rice cell cultures. PMID- 16661407 TI - Production of Hexanal and Ethane by Phaeodactylum triconutum and Its Correlation to Fatty Acid Oxidation and Bleaching of Photosynthetic Pigments. AB - In a light-dependent reaction (3.5 kilolux) at pH 5, the evolution of hexanal, ethane, and ethylene has been established with cell suspensions of the diatom, Phaeodactylum tricornutum. During this process, chlorophyll and carotenoids are partially bleached. Addition of 25 millimolar alpha-linolenic acid or 12 millimolar docosahexaenoic acid yield total pigment destruction and enhancement of ethylene and ethane formation (by about 150 and 7,600%, respectively), whereas hexanal production decreases by 70%. Eicosapentaenoic acid, the major polyunsaturated fatty acid in diatoms, stimulates both ethane and hexanal formation (by about 1,400 and 130%, respectively), but reduces ethylene production (by about 60%). This competition suggests that the production of the volatile compounds is closely connected, although hexanal and ethylene obviously possess different unsaturated fatty acids as precursors. Both the kind of the fatty acids and their relative amounts seem to determine the pattern of the evolved hydrocarbons. The presence of 10 millimolar propylgallate inhibits the evolution of the volatile compounds by about 80%, indicating that radical formation might play a key role in this light-dependent cascade of reactions. PMID- 16661408 TI - Studies of the Mechanism of Enhancement of Phytochrome-dependent Lettuce Seed Germination by Prechilling. AB - Temperature and kinetic studies were performed to examine the mechanism by which prechilling stimulates phytochrome-dependent seed germination in lettuce, Lactuca sativa, L. cv. Grand Rapids. Imbibed seeds were given a short far red irradiation and one day of dark incubation at 20 C to establish very low levels of the far red-absorbing form of phytochrome-(Pfr). Germination was greatly stimulated by subsequent prechilling treatments when they were followed by a second short far red irradiation. Prechilling therefore increased germination sensitivity to the low, normally inhibitory Pfr levels established by far red irradiation. This sensitivity increased with lowered prechilling temperature to a maximum near 4 C. It was linearly dependent upon duration of prechilling at 4 C up to a near maximal response at 10 hours, and it decayed in a converse manner when seeds were returned to 20 C after 10 hours at 4 C. Prechilling also increased germination responses to subsequent periods of high levels of Pfr which were initiated by red and terminated by far red irradiations. High Pfr periods adequate to promote the germination of unchilled seeds produced sharp inflections at 18 C in the dependence of germination on prechilling temperature. Rates of phytochrome potentiation of germination were not affected by prechilling. The response to prechilling fit a mechanism involving homeoviscous adaptation of membrane lipids to temperature. PMID- 16661409 TI - Effect of NAD on Malate Oxidation in Intact Plant Mitochondria. AB - Potato tuber mitochondria oxidizing malate respond to NAD(+) addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD(+) in potato mitochondria, which prove to take up added NAD(+). This mechanism concentrates NAD(+) in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD(+)/NADH ratio. PMID- 16661410 TI - The Mechanism of Abscisic Acid-induced Proline Accumulation in Barley Leaves. AB - When leaf blades of fully expanded second leaves of barley (cv. Prior) were excised and incubated with the cut end in a 20 milligram per liter solution of abscisic acid, they accumulated proline at the rate of about 1 micromole per hour per gram fresh weight after a 3- to 4-hour lag. This accumulation occurred reproducibly only if leaves were pretreated by placing the cut end in a solution consisting of 50 millimolar sucrose and 1 millimolar glutamate. Treated leaves were taken from plants which had been in the light for 24 hours.Abscisic acid caused a stimulation of proline synthesis from glutamic acid. Proline oxidation rates were similar in leaves incubated in abscisic acid and in water even though the proline level in abscisic acid-treated leaves was 2.5 times the level in the water-treated controls. The incorporation of proline into protein was not affected by abscisic acid.These results are interpreted to indicate that the metabolic cause of abscisic acid-induced proline accumulation is a stimulation of proline synthesis from glutamic acid. Inhibition of the utilization of proline by oxidation and protein synthesis does not contribute to proline accumulation the way it does in drought-stressed leaves. PMID- 16661411 TI - Effect of temperature on electron transport activities of isolated chloroplasts. AB - Temperature-induced changes in electron transport activities of chloroplasts isolated from chilling-sensitive and chilling-resistant plants have been examined. Using methylviologen as electron acceptor, temperature-induced changes occurred in the photosystem II plus photosystem I activities of chloroplasts isolated from chilling-resistant spinach (Spinacia oleracea L.) and pea (Pisum sativum L. cv. Alaska) plants. The changes occurred at approximately 17 C for spinach and 15 C for pea. A temperature-induced change, at approximately 13 C, in photosystem I activity using methylviologen was also observed for pea chloroplasts. These results extend earlier work and indicate that temperature has a general effect on the functioning of thylakoid membranes.Chloroplasts isolated from chilling-sensitive bean plants (Phaseolus vulgaris L. cv. Blue Lake 141) show a temperature-induced change in ferricyanide reduction at approximately 12 C. These results with spinach, pea, and bean support the view that the presence of temperature-induced changes in chloroplast activity assayed in vitro is not correlated with chilling sensitivity. PMID- 16661412 TI - Membrane phospholipid phase separations in plants adapted to or acclimated to different thermal regimes. AB - The phase separation temperatures of total leaf phospholipids from warm and cool climate plants were determined in order to explore the relationship of lipid physical properties to a species' thermal habitat. The separation temperatures were determined by measuring the fluorescence intensity and fluorescence polarization of liposomes labeled with the polyene fatty acid probe trans parinaric acid. To focus on a single climatic region, Mojave Desert dicots (chiefly ephemeral annuals) were examined, with plants grown under identical conditions whenever possible. Winter active species showed lower phase separation temperatures than the summer active species. A group of warm climate annual grasses showed separation temperatures distinctly higher than those of a group of cool climate grasses, all grown from seed under the same conditions. Growth at low temperature seems correlated with (and may require) a low phase separation temperature. Winter active ephemerals appear genetically programmed to synthesize a mixture of phospholipids which will not phase separate in the usual growth conditions. When the lipids of desert perennials were examined in cool and warm seasons, there was a pronounced seasonal shift in the phase separation temperature, implying environmental influences on lipid physical properties. The relationship of these results to high and low temperature tolerance is discussed. PMID- 16661413 TI - A proposed mechanism for the stimulatory effect of bicarbonate ions on ATP synthesis in isolated chloroplasts. AB - The effect of bicarbonate ions on induction of Mg(2+)-ATPase activity, on the N ethylmaleimide inhibition of phosphorylation and on energy-dependent adenine nucleotide exchange has been examined with pea seedling chloroplasts. Incubation of chloroplasts with N-ethylmaleimide in the presence of 15 millimolar bicarbonate in the light results in enhanced inhibition of ATP synthesis when the preillumination pH is maintained between 7.0 and 7.5. Bicarbonate also enhances Mg(2+)-ATPase activity when it is included in the light-triggering stage at pH 7.0. The conditions (medium pH, bicarbonate concentration, etc.) for demonstrating the bicarbonate-induced enhancement of the N-ethylmaleimide inhibition and ATPase activity are similar to those required for the direct effect of bicarbonate on phosphorylation. Bicarbonate, under the same conditions, does not affect adenine nucleotide exchange (binding or release). It is concluded that the stimulatory effect of bicarbonate on ATP synthesis may be related to its ability to alter directly the conformation of the chloroplast coupling factor under conditions (suboptimal pH) where the enzyme shows minimal activity. PMID- 16661414 TI - Isolation and Identification of a Senescence-promoting Substance from Wormwood (Artemisia absinthium L.). AB - The senescence-promoting substance of wormwood (Artemisia absinthium L.) as detected by the oat (Avena sativa L. cv "Victory") leaf assay has been identified as (-)-methyl jasmonate, methyl (1S, 2R)-3-oxo-2-(2'-cis-pentenyl)-cyclopentane-1 acetate, by gas-liquid chromatography-mass spectrometry and optical rotatory dispersion. Its senescence-promoting effect was much stronger than that of abscisic acid, and even at such a low concentration as 1 to 2.5 micrograms per milliliter, it could completely eliminate the anti-senescence action of 2 micrograms per milliliter kinetin. Comparing the biological activity of the (-)- with the (+/-)-forms of methyl jasmonate, it seemed that only the (-)-form was biologically active. PMID- 16661415 TI - Diffusion from a Circular Stoma through a Boundary Layer: A FIELD-THEORETICAL ANALYSIS. AB - The case of diffusion of a gas from a single circular stoma through an unstirred boundary layer of finite thickness into a perfectly stirred atmosphere free of convective effects is examined theoretically, with the gas assumed to be at constant concentration across the stoma. The analysis employs a mathematical solution to an analogous problem in electrostatic physics previously obtained by Kuz'min (1972 Sov Phys Tech Phys 17: 473-476). The diffusion flux is shown to be no more than 1% greater than that into a perfectly unstirred atmosphere if the boundary layer is thicker than 40 times the stomatal radius. Under the conditions assumed, for realistic boundary-layer and stomatal dimensions, taking the diffusion flux through the boundary layer to be linear with the stomatal radius would usually involve no significant error. This result may indicate that the principal effect of wind velocity on mass exchange between leaf and atmosphere may be exerted through influencing convection outside the boundary layer rather than through determining the thickness of that layer. PMID- 16661416 TI - Effect of eucalyptus growth regulators on the water loss from plant leaves. AB - Three closely related growth regulators (G) that are present in some myrtaceous plants were examined for possible anti-transpirant effects. The bioassay material involved cuttings of mung bean and Eucalyptus rupicola. Stomatal resistance was determined by a diffusion porometer. Water loss was equated with water uptake by the cutting over a 24-hour period.In both bioassays, G reduced water loss. The reduced water loss was associated with stomatal closure. This anti-transpirant effect of G was five to ten times less than that of abscisic acid. The stomatal resistance to the diffusion of water vapor from mung bean leaves increased within 1 hour of application of G. Marked stomatal closure occurred after 6 hours when 5 to 7 micrograms of G had accumulated in the leaves.These results and earlier evidence, suggest that G growth regulators are involved in the water economy of Eucalyptus and perhaps other related genera. PMID- 16661417 TI - Inhibition of steryl glycoside biosynthesis by acyl coenzyme a and by digitonin. AB - ATP, GTP, CoA, Mg(2+), and Mn(2+) did not inhibit biosynthesis of steryl glycoside and acylated steryl glycoside when added singly to enzyme preparations from spinach leaves. The combination of ATP (but not GTP), CoA, and Mg(2+) or Mn(2+) caused marked inhibition, especially of steryl glycoside biosynthesis, when reaction mixture concentrations of the additions were 0.2 millimolar. Inhibition was attributed to acyl-CoA and could be reproduced by palmitoyl-CoA. The inhibition could be partially prevented by bovine serum albumin. The effects of palmitoyl-CoA were distinct at 10 micromolar, and 50% inhibition of biosynthesis was observed at 40 micromolar.Digitonin (0.6 millimolar) stopped steryl glycoside biosynthesis but permitted the conversion of steryl glycoside to acylated steryl glycoside, thus eliminating the possibility that acylated steryl glycoside is formed from sterol + an acyl-glucose donor. PMID- 16661418 TI - Amino Acid Transport in Suspension-cultured Plant Cells: II. CHARACTERIZATION OF l-LEUCINE UPTAKE. AB - l-Leucine (l-Leu) transport into suspension cultured Nicotiana tabacum L. cv. Wisconsin 38 cells has been investigated. Cells were batch-cultured and routinely assayed 3.5 to 4 days after subculturing. Uptake rates were measured over the concentration range of 10 micromolar to 150 millimolar. Kinetic analysis of the uptake rates indicated that uptake was multiphasic with three saturable phases and one unsaturable phase. The three saturable phases which occur in the concentration ranges of 10 to 40 micromolar, 50 to 100 micromolar, and 0.2 to 5.0 millimolar exhibited the following characteristics; (a) phases were energy dependent as shown by 84 to 94% inhibition of uptake rates by metabolic inhibitors; (b) phases exhibited broad pH optima between 3.0 and 5.5; (c) phases showed stereospecificity for l-Leu; (d) over a 12-hour incubation period, phases concentrated l-Leu 43, 90, and 10 times when the initial l-Leu concentration was 20 micromolar, 100 micromolar, and 1.0 millimolar, respectively; (e) phases had K(m) values of 17.6 micromolar, 60.1 micromolar, and 1.38 millimolar, respectively; and (f) in the temperature range of 17 to 27 C phases had Q(10) values of 2.1, 1.4, and 1.4, respectively. l-Leu uptake in the three saturable phases was inhibited by a 20-fold higher concentration of 18 other amino acids; phenylalanine, alanine, and methionine were the most effective inhibitors, whereas aspartic acid, asparagine, histidine, and arginine were the least effective. The nonsaturable phase which was responsible for increases in the uptake rate above 5.0 mm appeared to be primarily diffusional since it was minimally influenced by metabolic inhibitors and had a Q(10) of 1.3. PMID- 16661419 TI - Growth Responses and Adaptations of Fraxinus pennsylvanica Seedlings to Flooding. AB - Flooding induced several physiological and morphological changes in Fraxinus pennsylvanica seedlings, with stomatal closure among the earliest responses. Subsequent changes included: reduction in dry weight increment of roots, stems, and leaves; formation of hypertrophied lenticels and production of adventitious roots on submerged portions of the stem above the soil line; leaf necrosis; and leaf abscission. After 15 days of stomatal closure as a result of flooding, stomata began to reopen progressively until stomatal aperture was similar in flooded and unflooded plants. Adventitious roots began to form at about the time stomatal reopening began. As more adventitious roots formed, elongated, and branched, the stomata opened further. The formation of adventitious roots was an important adaptation for flooding tolerance as shown by the high efficiency of adventitious roots in absorption of water and in high correlation between the production of adventitious roots and stomatal reopening. PMID- 16661420 TI - Identification of the Low Molecular Weight Copper Protein from Copper-intoxicated Mung Bean Plants. AB - Mung bean plants (Wilczek) accumulate increasingly greater amounts of buffer extractable copper in both their shoots and roots when grown in liquid medium containing greater than 2 micrograms per milliliter copper (31.4 micromolar) as cupric sulfate. This increase in soluble copper is accompanied by an increase in the relative amount of low molecular weight (7,000 to 20,000) macromolecular bound copper and a decrease in the relative amount of high molecular weight (greater than 20,000) copper. The major low molecular weight copper protein has been isolated from copper-intoxicated mung bean plants by a combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. It was identified as mung bean plastocyanin on the basis of its molecular weight, optical behavior, and amino acid composition. No evidence was found for a low molecular weight copper-binding protein corresponding to mammalian thionein or chelatin. PMID- 16661421 TI - Endorhizal and Exorhizal Acetylene-reducing Activity in a Grass (Spartina alterniflora Loisel.)-Diazotroph Association. AB - Earlier studies indicated that bacteria responsible for nitrogenase activity of some grasses are located inside the roots. Those studies were conducted with excised roots in which a long, unexplained "lag phase" occurred before initiation of nitrogenase activity. When hydroponically maintained Spartina alterniflora Loisel. was incubated in a two-compartment system with acetylene, ethylene was produced following, at most, a 2-hour lag in both the upper (shoot) and lower (roots + water) phases. Ethylene production in the upper phase not attributable to leaf-associated acetylene-reducing activity or to diffusion of ethylene from around the roots is considered to represent "endorhizal acetylene-reducing activity," the internally produced ethylene diffusing into the upper phase via the lacunae. Ethylene produced in the lower phase is designated "exorhizal acetylene-reducing activity." The endorhizal acetylene-reducing activity, in comparison to exorhizal activity, was relatively insensitive to additions of HgCl(2), NH(4)Cl, or carbon sources to the lower phase. Post-lag acetylene reducing activity of roots excised from plants growing in soil responded to additions in a manner similar to that of endorhizal acetylene-reducing activity, whereas post-lag acetylene-reducing activity of rhizosphere soil responded in a manner similar to that of exorhizal acetylene-reducing activity. PMID- 16661422 TI - Biosynthesis of wound ethylene. AB - Untreated mung bean hypocotyls produced very little C(2)H(4) but, upon treatment with 10 millimolar Cu(2+) or 10 millimolar Cu(2+) + 10 millimolar Ca(2+), C(2)H(4) production increased 20- and 40-fold, respectively, within 6 hours. This increase in C(2)H(4) production was preceded and paralleled by an increase in 1 aminocyclopropanecarboxylic acid (ACC) content, but the level of S adenosylmethionine (SAM) was unaffected, suggesting that the conversion of SAM to ACC is a key reaction in the production of wound-induced C(2)H(4). This view was further supported by the observation that application of aminoethoxyvinylglycine, a known inhibitor of the conversion of SAM to ACC, eliminated the increases in ACC formation and in C(2)H(4) production. A significant increase in C(2)H(4) production was observed in the albedo tissue of orange in response to excision, and it was paralleled by an increase in ACC content. In columella tissue of unripe green tomato fruit, massive increases in the C(2)H(4) production rate (from 0 to 12 nanoliters per gram per hour), in ACC content (from 0.05 to 12 nmoles per gram), and in ACC synthase activity (from 0 to 6.4 units per milligram protein) occurred during the 9-hour incubation period following excision. Infiltration with 0.1 millimolar cycloheximide, an inhibitor of protein synthesis, completely blocked wound-induced C(2)H(4) production, ACC formation, and development of ACC synthase activity. These data indicate that wounding induces the synthesis of ACC synthase, which is the rate-controlling enzyme in the pathway of C(2)H(4) biosynthesis and, thereby, causes accumulation of ACC and increase in C(2)H(4) production. PMID- 16661423 TI - Inhibition of ethylene production by 2,4-dinitrophenol and high temperature. AB - 2,4-Dinitrophenol (DNP) and high temperature (35 to 40 C) are known to inhibit C(2)H(4) production in various plant tissues. The present study was made to determine the step in the C(2)H(4) biosynthetic pathway (methionine --> S adenosylmethionine [SAM] --> 1-aminocyclopropane-1-carboxylic acid [ACC] --> C(2)H(4)) at which these treatments exert their inhibitory effect. In mung bean hypocotyls the dose-inhibition curves for the effect of DNP on auxin-dependent C(2)H(4) production (in which auxin exerts its effect by stimulating the conversion of SAM to ACC) and on ACC-dependent C(2)H(4) production (in which ACC is directly utilized as precursor) were similar. It was concluded, therefore, that DNP at low concentrations (below 50 micromolar) exerted its effect primarily on the conversion of ACC to C(2)H(4), a step which is common to both systems. This view was further substantiated by quantitative analysis of the intermediates in the biosynthetic sequence. DNP exerted little influence on the content of SAM but caused a significant increase in the ACC content and marked inhibition in C(2)H(4) production, indicating that the conversion of ACC to C(2)H(4) is the crossover point. At higher concentrations (above 100 micromolar), DNP inhibited the conversion of methionine to ACC and to C(2)H(4), and this effect could be attributed to the inhibition of SAM synthesis.The optimal temperature for maximal C(2)H(4) production by apple tissue and mung bean hypocotyl is about 30 C. An increase in temperature to 35 C caused an accumulation of endogenous ACC, whereas C(2)H(4) production was greatly reduced. These results suggest that the conversion of ACC to C(2)H(4) is highly vulnerable to high temperature inhibition. PMID- 16661424 TI - Preparation of chloroplasts from euglena highly active in protein synthesis. AB - Chloroplasts can be obtained by gentle lysis or mild shear of spheroplasts of vitamin B(12)-deficient Euglena gracilis and then purified by isopycnic sedimentation on gradients of Ludox AM or Percoll. The chloroplasts appear compact and highly refractile by phase contrast or Hoffmann contrast microscopy. Upon incubation with [(3)H]leucine or [(35)S]methionine, the chloroplasts incorporate the amino acids into protein at rates that are 100-fold faster than we had previously observed with Euglena and up to 8-fold faster than with chloroplasts of spinach. Euglena chloroplasts prepared by the current procedure are thus qualitatively superior to those previously available from Euglena and at least as active in protein synthesis as chloroplasts from higher plants. PMID- 16661425 TI - Photomorphogenic Regulation of Chloroplast Replication in Euglena: ENHANCED LOSS OF CHLOROPLAST DNA IN RED LIGHT. AB - Chloroplast replication in Euglena gracilis is specifically inhibited by ultraviolet light and the effect is photoreactivable.The ability of irradiated cells to be photoreactivated is lost more rapidly if cells are incubated in red light than in darkness. A mutant, Y(9)ZNa1L, which lacks the red-blue photomorphogenic system regulating chloroplast synthesis does not show the red light-enhanced loss of photoreactivability. Another mutant, Y(11)P(27)ZD which has the red-blue system, but lacks the blue-light system also regulating chloroplast synthesis, shows the red-light effect. The red-light effect is seen in a mutant of photosynthetic electron transport, P(4)ZUL, which rules out a product of photosynthesis as a mediator of the effect. Inhibitors of protein synthesis on chloroplast ribosomes do not prevent the red-light-enhanced loss of chloroplast DNA. Chloroplast DNA is lost rapidly when UV-irradiated cells are incubated in red light, showing that the loss of photoreactivability is due to the loss of the substrate for photoreactivation, chloroplast DNA. Therefore, the red-blue photomorphogenic system is activating a chloroplast DNA-specific nuclease(s). A model is proposed for a light-mediated mechanism regulating the amount of chloroplast DNA: blue light would promote chloroplast DNA synthesis; red light would promote its degradation. The photomorphogenic systems regulating chloroplast synthesis might work by activating a chloroplast-specific modification-restriction mechanism. PMID- 16661426 TI - Oxygen exchange in leaves in the light. AB - Photosynthetic O(2) production and photorespiratory O(2) uptake were measured using isotopic techniques, in the C(3) species Hirschfeldia incana Lowe., Helianthus annuus L., and Phaseolus vulgaris L. At high CO(2) and normal O(2), O(2) production increased linearly with light intensity. At low O(2) or low CO(2), O(2) production was suppressed, indicating that increased concentrations of both O(2) and CO(2) can stimulate O(2) production. At the CO(2) compensation point, O(2) uptake equaled O(2) production over a wide range of O(2) concentrations. O(2) uptake increased with light intensity and O(2) concentration. At low light intensities, O(2) uptake was suppressed by increased CO(2) concentrations so that O(2) uptake at 1,000 microliters per liter CO(2) was 28 to 35% of the uptake at the CO(2) compensation point. At high light intensities, O(2) uptake was stimulated by low concentrations of CO(2) and suppressed by higher concentrations of CO(2). O(2) uptake at high light intensity and 1000 microliters per liter CO(2) was 75% or more of the rate of O(2) uptake at the compensation point. The response of O(2) uptake to light intensity extrapolated to zero in darkness, suggesting that O(2) uptake via dark respiration may be suppressed in the light. The response of O(2) uptake to O(2) concentration saturated at about 30% O(2) in high light and at a lower O(2) concentration in low light. O(2) uptake was also observed with the C(4) plant Amaranthus edulis; the rate of uptake at the CO(2) compensation point was 20% of that observed at the same light intensity with the C(3) species, and this rate was not influenced by the CO(2) concentration. The results are discussed and interpreted in terms of the ribulose-1,5-bisphosphate oxygenase reaction, the associated metabolism of the photorespiratory pathway, and direct photosynthetic reduction of O(2). PMID- 16661427 TI - Isolation and Characterization of Thiamin Pyrophosphotransferase from Glycine max Seedlings. AB - Thiamin:ATP pyrophosphotransferase (EC2.7.6.2) activity from soybean (Merr.) seedlings grown for 48 hours was determined by measuring the rate of [2 (14)C]thiamin incorporation into thiamin pyrophosphate. With partially purified (11-fold) enzyme, optimal activity occurred between pH 7.1 and 7.3, depending on the buffer system that was used. Assays were routinely conducted at a final pH of 8.1 in order to minimize interference from competing reactions. Enzyme activity required the presence of a divalent cation, and a number of nucleoside triphosphates proved to be active as pyrophosphate donors. Apparent K(m) values of 18.3 millimolar and 4.64 micromolar were obtained for Mg.ATP and thiamin, respectively. Among the compounds tested, pyrithiamin and thiamin pyrophosphate were most effective in inhibiting thiamin pyrophosphotransferase activity. Based on Sephadex G-100 gel filtration, soybean thiamin pyrophosphotransferase has a molecular weight of 49,000. PMID- 16661428 TI - Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination. AB - Thiamin pyrophosphotransferase activity was present in seedling extracts from several monocot and dicot species of agronomic as well as noncultivated plants. Changes in thiamin pyrophosphotransferase activity and thiamin pyrophosphate content were followed for 6 days in soybean (Merr.) seedlings. Maximum enzyme activity occurred 48 to 96 hours from imbibition. Thiamin pyrophosphate content peaked sharply at 36 hours and was preceded by increased thiamin pyrophosphotransferase activity. Addition of pyrithiamin, an inhibitor of in vitro thiamin pyrophosphotransferase activity, to the imbibition medium at various times inhibited subsequent fresh weight gains of soybean seedlings. These results indicated that, although not among the earliest phosphorylation events after initiation of water imbibition by soybean seeds, a substantial increase in thiamin pyrophosphate content did precede the onset of rapid seedling growth and development. Since both enzyme activity and thiamin appear to be available in unimbibed soybean seeds, ATP or other nucleoside triphosphate concentration may represent an important factor in modulating thiamin phosphorylation during early seedling development. PMID- 16661429 TI - Effect of Dehydration on Leakage and Membrane Structure in Lotus corniculatus L. Seeds. AB - Membrane damage as a result of dehydration was studied in Lotus corniculatus L. cv. Carroll seeds which had been pregerminated for 0, 12, and 24 hours prior to dehydration. During reimbibition, desiccation-tolerant (0- and 12-hour) seeds leaked relatively low quantities of all solutes (total electrolytes, potassium, phosphate, sugar, amino acid, and protein). Desiccation-sensitive (24-hour) seeds leaked higher levels, but evidence of selective permeability remained. Membrane damage was not manifested as a complete removal of the diffusion barrier, although its permeability properties were dramatically altered. Consequently, the plasmalemma was not ruptured or torn by the dehydration treatment, but a more subtle structural alteration occurred.The possibility that seed membranes form a hexagonal rather than a lamellar phase at moisture contents below 20% was investigated by x-ray diffraction. Phospholipids were extracted from desiccation tolerant (0-hour) and desiccation-sensitive (24-hour) seeds and hydrated to 5, 10, 20, and 40% water. This phospholipid-water system was examined using low-and wide-angle x-ray diffraction and was found to be exclusively lamellar, even at 5% water. Consequently, membrane damage and the leakage of cytoplasmic solutes from seeds cannot be explained by the formation of a hexagonal phase by membrane phospholipids. PMID- 16661430 TI - An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth. AB - A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed. PMID- 16661431 TI - The Synthesis of Guanosine 5'-Diphosphate l-Fucose from Guanosine 5'-Diphosphate 3,5-d-[H]Mannose Catalyzed by an Enzyme Extract from Fruits of the Flax. AB - An enzyme system from fruits of the flax plant is described that catalyzes the synthesis of the sugar nucleotide guanosine 5'-diphosphate l-fucose from guanosine 5'-diphosphate d-mannose with the intermediate formation of guanosine 5'-diphosphate 4-keto-6-deoxy-d-mannose. Tritium from-[(3)H]H(2)O was incorporated into the l-fucose portion of the sugar nucleotide in the course of the reaction, and tritium at the 3,5-carbons of the d-mannose moiety of GDP-d mannose was exchanged with protons in the medium. These results support a mechanism of synthesis analogous to that proposed for the formation of l-rhamnose and other 6-deoxy sugars. PMID- 16661432 TI - A proposed role of zein and glutelin as N sinks in maize. AB - Zea mays grown with high levels of N fertilizer transports more sucrose into kernels than with low N. Sucrose translocation was greatest in genotypes with the highest capacity to deposit nitrogenous compounds as zein and glutelin in the kernel. These two proteins combined contain about 80% of the total N in the kernel and about 60% of the total N in the plant at maturity. They appear to serve as a functional N sink for the deposition of nitrogenous compounds. As the N sink capacity increases with additional available N fertilizer, more sucrose is transported into the kernel, resulting in increased kernel weight and grain yield. Zein functions as a more dynamic N sink than glutelin because the synthesis of zein is readily manipulated by N fertilization and genetic means. Increases in N deposition in the normal endosperm induced by N fertilizer are confined primarily to zein. Early termination of zein accumulation in the opaque 2 mutant results in a reduction of sucrose movement into kernels. By using plants heterozygous for normal and opaque-2 in these studies, interplant variability was eliminated and the hypothesis relating the kernel N sink capacity to productivity was strengthened. PMID- 16661433 TI - Isolation and Characterization of Glycine Hydroxamate-resistant Cell Lines of Nicotiana tabacum. AB - Seven lines of haploid Nicotiana tabacum tissue culture selected for resistance to normally toxic levels of the glycine analog glycine hydroxamate, a competitive inhibitor of the glycine decarboxylase reaction, were investigated. The presence of glycine hydroxamate greatly increased the intracellular concentration of both glycine and alanine in wild type and resistant cell lines, suggesting that the inhibitor blocks both glycine- and alanine-utilizing reactions. All the resistant cell lines, whether grown in the presence or absence of glycine hydroxamate, had high intracellular concentrations of the 12 free amino acids which were analyzed, including glycine and serine. (These lines averaged 3.6 times the total amino acid content of wild-type cells in the absence of the inhibitor). The resistant cell lines were indistinguishable from wild-type cell lines in their metabolism of radioactively labeled glycine hydroxamate and glycine. Comparison of the metabolism of radioactively labeled alanine, glycolate, and glyoxylate in wild type and alpha resistant line also revealed no distinctive differences. Glycine decarboxylase activities were unaltered in the resistant cell lines. The cellular toxicity of glycine hydroxamate is considered in relation to (1) the competitive inhibition by glycine hydroxamate of the glycine- and alanine-utilizing enzymes and (2) the resultant imbalances caused by high intracellular concentrations of these amino acids. The significance of elevation of total free amino acid concentration in effecting resistance to the inhibitor is discussed.Plants were regenerated from 5 of these lines and callus cultures of explants were tested for glycine hydroxamate resistance. Plants from seedlings of two lines which retained the resistant characteristic in explanted callus did not have high amino acid levels in leaves. PMID- 16661434 TI - Betaine Synthesis from Radioactive Precursors in Attached, Water-stressed Barley Leaves. AB - In wilted barley leaves, betaine accumulates at about 200 nanomoles per 10 centimeters leaf per day. Results with (14)C-labeled precursors were qualitatively and quantitatively consistent with de novo synthesis of this betaine from serine via ethanolamine, choline, and betaine aldehyde and indicated that water stress may increase the activities of all steps in this pathway except the last.Doses (1 micromole) of each (14)C-labeled precursor were supplied as droplets to the tips of attached, 10-centimeter, second-leaf blades of turgid and wilted plants, and the incorporation of (14)C into betaine was followed. From the rates of betaine labeling, estimates were made of the potential capacities (nanomoles per 10 centimeters leaf per day) for the methylation and oxidation steps. Labeling of betaine from [(14)C]choline, [(14)C]ethanolamine, and [(14)C]serine was about 7- to 10-fold greater in leaves wilted for 2 days than in turgid leaves, whereas label from [(14)C]betaine aldehyde appeared in betaine at about the same rate in both turgid and wilted leaves. In leaves wilted for 2 days, the potential capacities for converting [(14)C]ethanolamine, [(14)C]choline, and [(14)C]betaine aldehyde to betaine all approached or exceeded the rate of betaine accumulation (about 200 nanomoles per 10 centimeters leaf per day); in turgid leaves, only the potential for converting betaine aldehyde to betaine exceeded this rate. The rate of conversion of [(14)C]ethanolamine to betaine increased 4-fold after 6 to 10 hours of wilting, which was soon enough to account for the onset of betaine accumulation. PMID- 16661435 TI - Spin-label Studies of Membranes in Rye Protoplasts during Extracellular Freezing. AB - Protoplasts isolated from epicotyls of nonhardened winter rye seedlings were spin labeled with the N-oxyl-4-4-dimethyloxazolidine derivatives of 5-ketostearic (5NS) and 16-ketostearic (16NS) acids. Spectra of the membrane-bound labels showed motional broadening with a rotational correlation time of 1.5 x 10(-8) second for 5NS and 1.5 x 10(-10) second for 16NS at 0 C. A procedure was developed to follow membrane changes in these protoplasts during extracellular freezing. With freezing, molecular motion of 5NS, but not of 16NS, spin probes was restricted. The increase in molecular order near the hydrated end of the membrane did not result from lowered temperatures inasmuch as no such change was observed in supercooled samples. These changes are probably due to dehydration of protoplast membranes during extracellular freezing. Similar results were obtained with multilayered egg yolk lecithin and are consistent with previous observations of changes in lecithin multibilayers during dehydration. Such alterations in membrane order might lead to irreversible membrane damage during extracellular freezing of plant cells. PMID- 16661436 TI - Partial characterization of fusicoccin binding to receptor sites on oat root membranes. AB - The possibility that fusicoccin (FC) binds to plasma membrane-associated ATPases of oat (cv. Victory) roots has been examined. Specific FC-binding in vitro is localized primarily on plasma membrane-enriched fractions. This FC-binding is greatly reduced by pretreatment of the membrane vesicles at temperatures above 45 C or with trypsin, and the same treatments cause the release of already bound FC. These results support the idea that the FC receptor is a protein located on the plasma membrane.Both active ATPases and FC-binding proteins were solubilized using 1% Triton X-100. When this material was fractionated using gel chromatography, the ATPase activity could be separated from the FC-binding proteins. The identity of the FC-binding proteins is discussed with regard to the extensive evidence which supports the involvement of plasma membrane-ATPase H(+)/K(+) pumps in FC-stimulated acidification and K(+) uptake. PMID- 16661437 TI - Histone Kinase from Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES. AB - A histone-type protein kinase (EC 2.7.1.37) has been partially purified (320 fold) from the crude extracts of soybean hypocotyls by means of a combination of gel filtration and anion exchange procedures. The purified enzyme fraction is devoid of the activities of phosphoprotein phosphatase (EC 3.1.3.16), histone protease, and casein (or phosvitin)-type kinase. The soybean histone kinase uses ATP to phosphorylate specifically lysine-rich histone H1 from either pea seedlings or calf thymus.The histone kinase requires free sulfhydryl group(s) for activity, but not stability. The pH optimum is around 9 to 10. The apparent K(m) values for histone H1 of pea seedlings and calf thymus are 0.4 and 0.9 micromolar, respectively. The K(m) values for ATP are 40 nanomolar with the optimal concentration of Mn(2+) (50 nanomolar) and 0.4 micromolar with that of Mg(2+) (5 millimolar). The estimated molecular weight of the kinase is 52,000 by gel filtration or 48,600 by sedimentation constant (3.2 S). cAMP does not alter the sedimentation velocity of the kinase. The enzyme activity is unaffected by cyclic nucleoside monophosphates and plant growth substances. Like arginine-rich histones, a variety of divalent cations and polycations (polyamines) are inhibitory.This cAMP-independent soybean histone kinase is not associated with the isolated ribosomes but shows highest specific activity in the nuclearchromatin fraction, suggesting that it may function in the regulation of histone H1 phosphorylation in the soybean hypocotyl. PMID- 16661438 TI - Phosphoprotein Phosphatase of Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES . AB - A soybean histone-type protein kinase was used to prepare (32)P-labeled histone H1 as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.1.3.16) from soybean hypocotyls. The phosphatase has been purified 169-fold by ammonium sulfate fractionation, ethanol precipitation, and chromatography on Sephadex G-150, DEAE-Sephadex A-25 and Sephadex G-100. The activity of the phosphoprotein phosphatase is distinct from that of acid and alkaline phosphatases (EC 3.1.3.1) as well as from that of nucleotidases. The final enzyme preparation does not contain histone protease activity, although it can be detected during the early stages of purification. The protease(s) apparently can attack phosphorylated histone H1, indicating that phosphorylation does not protect the protein against proteolytic degradation.The amounts of (32)P released from [(32)P]histone H1 are proportionally recovered as [(32)P]Pi, indicating that the dephosphorylation is due to the action of phosphoprotein phosphatase. The enzyme shows maximal activity at pH 7 to 8 and has a specific activity of 19 nanomoles of [(32)P]Pi released from [(32)P]histone H1 per minute per milligram of protein. The apparent K(m) for phosphohistone H1 is 4.0 +/- 0.4 micromolar. The estimated molecular weight of the enzyme is approximately 30,000 by gel filtration. The enzyme activity does not depend upon the addition of reducing agent and metal ion. Zn(2+), Co(2+), NaF, pyrophosphate, or ATP at 1 millimolar, however, inhibits the enzyme activity by about 70%. The enzyme activity is unaffected by cyclic nucleotides and plant growth substances but is inhibited by polyamines. All the phosphorylated histone species and protamine, not low molecular weight phosphoesters, act as competitive inhibitors for the dephosphorylation of [(32)P]histone H1.Besides its action on phosphohistone H1, the soybean enzyme also catalyzes the dephosphorylation of other phosphohistone species (H2A, H2B, H3, and H4), degraded phosphohistone H1, and possibly phosphorylated casein and phosvitin. All these results indicate that the enzyme is a nonspecific phosphoprotein phosphatase. PMID- 16661439 TI - Legume: alpha-GALACTOSIDASE FORMS DEVOID OF HEMAGGLUTININ ACTIVITY. AB - Twenty different legume species (20 genera) were examined for alpha-galactosidase and hemagglutinin activities. Although all of the species contained enzyme activity, only 13 of 20 contained hemagglutinin activities and none displayed a hemagglutinin activity comparable to the previously described alpha-galactosidase hemagglutinins.The alpha-galactosidase activities in the 20 species possessed remarkably similar kinetic behavior and carbohydrate specificities. All were inhibited by galactose, xylose, and inositol (very similar K(i) values from plant to plant) and had very similar K(m) values for the substrate, p-nitrophenyl alpha galactoside.Gel filtration analysis of extracts from nine species suggests that legume alpha-galactosidase activities may frequently reside in two molecular weight forms. However, all these species contained a large molecular weight enzyme activity with a size comparable to the alpha-galactosidase hemagglutinins.Immunochemical studies reveal that the alpha-galactosidases in these plants are immunologically related to an alpha-galactosidase-hemagglutinin and, therefore, are related to one another.These studies suggest that each of the legume species studied (and perhaps all members of this plant family) contain a homologue from a specific class of alpha-galactosidase. Although the previously described alpha-galactosidase-hemagglutinins appear to be members from this enzyme class, these proteins most frequently occur as forms devoid of hemagglutinin activity. PMID- 16661440 TI - The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants. AB - A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.The yield of vacuoles is approximately 44%. The major vacuole layer is < 7% contaminated by marker enzymes from the cytoplasm and other organelles but shows no contamination by chloroplasts. Isolated vacuoles were stable for >15 hours when left in Ficoll; however, dispersion into media of various osmotic concentrations resulted in decreased stability. Addition of mercaptobenzothiazole, CaCl(2), MgCl(2), bovine serum albumin, ethylenediaminetetraacetic acid, polyethylene glycol 600, and KH(2)PO(4) to the vacuole isolation media did not increase the stability of the isolated vacuoles.THIS TECHNIQUE WITH ONLY SLIGHT MODIFICATIONS HAS BEEN USED TO ISOLATE LEAF CELL VACUOLES FROM THE FOLLOWING CRASSULACEAN ACID METABOLISM PLANTS: pineapple, Kalanchoe fedtschenkoi, and Echeveria elegans. Spinach leaves also were used successfully. PMID- 16661441 TI - Steady-state Growth of the Marine Diatom Thalassiosira pseudonana: UNCOUPLED KINETICS OF NITRATE UPTAKE AND NITRITE PRODUCTION. AB - Seasonal studies of the vertical distribution of nitrate, nitrite, and phytoplankton in the oceans and studies using (15)N as a tracer of nitrate metabolism indicate that the reduction of nitrate by phytoplankton is a source of nitrite in the upper waters of the ocean. To better understand this process, the relationship between nitrate uptake and nitrite production has been examined with continuous cultures of the small marine diatom Thalassiosira pseudonana. In a turbidostat culture, the rates of nitrite production by T. pseudonana increase with light intensity. This process is only loosely coupled to rates of nitrate assimilation since the ratio of net nitrite production to total nitrate assimilation increases with increased rates of growth. In continuous cultures where steady-state concentrations of nitrate and nitrite were varied, T. pseudonana produced nitrite at rates which increased with increasing concentrations of nitrate. Again, the rates of nitrite production were uncoupled from rates of nitrate assimilation. The study was used to derive a mathematical description of nitrate and nitrite metabolism by T. pseudonana.The validity of this model was supported by the results of a study in which (15)N-labeled nitrite was introduced into the continuous culture, and the model was used to examine patterns in distribution of nitrite in the Antarctic Ocean and the Sargasso Sea. PMID- 16661442 TI - The Endoplasmic Reticulum of Mung Bean Cotyledons: ROLE IN THE ACCUMULATION OF HYDROLASES IN PROTEIN BODIES DURING SEEDLING GROWTH. AB - The subcellular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organelle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies. PMID- 16661443 TI - The influence of aliphatic alcohols on leaf senescence. AB - Because of the effects of ethanol used as a solvent in other experiments, the action of aliphatic alcohols on leaf senescence in the dark has been studied systematically. These compounds both maintain chlorophyll and prevent proteolysis in the dark, much as do the cytokinins and other senescence-delaying substances. The activity of the straight-chain alcohols increases in a log-linear fashion with increasing chain length up to 1-octanol. Introduction of a branch in the chain or of a second OH group greatly decreases, or in some cases annuls, the antisenescence activity. In all cases, the action on senescence is closely (although not always exactly) paralleled by opening of the stomata. Abscisic acid and exposure to high concentrations of osmoticum, both of which close the stomata, antagonize the action of the alcohols. Some interactions with other agents are noted. The effects are compared with reported effects on seed germination, on hemolysis and animal membranes, and especially on permeability to K(+) ions, and a tentative basis for the mechanism of action is advanced. PMID- 16661444 TI - Isolation of cytoplasmic enzymes from pollen. AB - The cytoplasmic isozyme of many cytoplasmic-organelle isozyme pairs, as well as other cytoplasmic enzymes in plants, can be readily obtained from pollen by soaking it in an appropriate buffer for 4 hours. Enzymes localized in subcellular organelles appear not to be released during the soaking period, although they are released if the pollen is crushed. The technique is a useful initial step in studies of subcellular localization of enzymes or for obtaining small quantities of cytoplasmic enzymes free of organellar contaminants. PMID- 16661445 TI - Partial Purification of an Ethylene-binding Component from Plant Tissue. AB - An ethylene binding component(s) has been partially purified from mung bean sprouts. Tissue was homogenized in 0.3 molar sucrose and 0.2 molar potassium phosphate buffer (pH 7.0). The homogenate was centrifuged, and resuspended fractions were assayed by incorporating them onto cellulose fibers (0.7 grams per milliliter). These were exposed to [(14)C]ethylene (3.7 x 10(-2) microliters per liter of 120 millicurie per millimole) in the presence or absence of 1000 microliters per liter unlabeled ethylene. The cellulose was transferred to separate containers and the [(14)C]ethylene was absorbed in mercury perchlorate and counted. Distribution of ethylene binding to various fractions was: 0 to 3,000g, 3%; 3,000 to 12,000g; 4%; 12,000 to 100,000g, 69%; cellular debris, 24%; 100,000g supernatant, 0%. Adjustment of the pH to 4.0 precipitates the ethylene binding component. Neutralization, addition of Triton X-100, and readjustment of the pH to 4.0 "solubilized" most of the binding component. Further purification was obtained by chromatography on CM-Sephadex in 10 millimolar potassium acetate buffer, (pH 5.0) containing 1% Triton X-100. Elution was with 200 millimolar potassium phosphate (pH 6.0) containing 1% Triton X-100. Upon treatment of the Triton "solubilized" component with cold acetone, over 90% of the binding capacity was lost. Extraction of the acetone-precipitated residue with 2% Triton X-100 restored some of the binding capacity which was found in the soluble fraction. The pH optimum for binding is 6.0. Passing the Triton X-100 extract of the acetone powder through Sepharose 6B provides considerable purification. The binding component moved ahead of most of the protein. PMID- 16661446 TI - Internal Inorganic Carbon Pool of Chlamydomonas reinhardtii: EVIDENCE FOR A CARBON DIOXIDE-CONCENTRATING MECHANISM. AB - The external inorganic carbon pool (CO(2) + HCO(3) (-)) was measured in both high and low CO(2)-grown cells of Chlamydomonas reinhardtii, using a silicone oil layer centrifugal filtering technique. The average internal pH values were measured for each cell type using [(14)C]dimethyloxazolidinedione, and the internal inorganic carbon pools were recalculated on a free CO(2) basis. These measurements indicated that low CO(2)-grown cells were able to concentrate CO(2) up to 40-fold in relation to the external medium. Low and high CO(2)-grown cells differed in their photosynthetic affinity for external CO(2). These differences could be most readily explained as being due to the relative CO(2)-concentrating capacity of each cell type. This physiological adaptation appeared to be based on changes in the abilities of the cells actively to accumulate inorganic carbon using an energy-dependent transport system.The energy dependence of CO(2) accumulation was investigated, using the inhibitors methyl viologen, 3-(3,4 dichlorophenyl)-1,1 dimethylurea, carbonyl cyanide trifluoromethoxyphenylhydrazone, and 3,5-di-tert-butyl-4-hydroxybenzylide nemalononitrile. It appears that the concentrating mechanism in both cell types may be dependent upon an energy supply linked to both phosphorylation in general and photophosphorylation. The treatment of low CO(2)-grown cells with the carbonic anhydrase inhibitor ethoxyzolamide decreased the apparent photosynthetic affinity for CO(2). This was correlated with a decrease in the transport of inorganic carbon into the cells.The nature of the CO(2)-concentrating mechanism, particularly with respect to a bicarbonate transport system, is discussed, and its possible occurrence in other algae is assessed. PMID- 16661447 TI - Biochemical Changes in Tuber-bearing Solanum Species in Relation to Frost Hardiness during Cold Acclimation. AB - Biochemical changes in potato leaves during cold acclimation have been examined and compared between a frost-tolerant S. acaule and a frost-susceptible S. tuberosum species. Changes were also examined in S. tuberosum, S. acaule, and S. commersonii species when they were hardened at different temperatures to varying hardiness levels.During three weeks of stepwise cold acclimation, S. acaule increased frost hardiness from -6.0 C (killing temperature) to -9.0 C, whereas frost hardiness of S. tuberosum remained unchanged at -3.0 C. Decreases in DNA content on a dry weight basis in both species suggest that matured leaf cells accumulated more dry matter during acclimation. The advantage of using DNA as a reference for comparing metabolite changes during cold acclimation is discussed.Under the stepwise acclimating conditions, both species showed the same trends for increasing total sugar and starch with an insignificant decrease in leaf water content. High levels of total RNA, rRNA, and total and soluble protein were observed in treated S. acaule plants as compared with controls, but not in S. tuberosum. Levels of total lipid and phospholipid also were high in treated S. acaule plants as compared with controls but decreased in S. tuberosum during acclimation.When S. tuberosum, S. acaule, and S. commersonii potatoes were cold treated at constant day/night temperatures of 10, 5, and 2 C with 14-hour daylength, each species responds differently in terms of frost hardiness increase upon subjecting plants to a low temperature. For instance, after 20 days at 2 C, a net frost hardiness of 3 and 7 C was observed in S. acaule and S. commersonii, respectively, whereas the frost hardiness in S. tuberosum remained unchanged. Also, various levels of frost hardiness can be achieved in a species by subjecting plants to different low temperature treatments. Under a warm regime of 20/15 C day/night temperatures (14-hour light), both S. acaule and S. commersonii can survive at -4.5 C or colder, whereas S. tuberosum can survive only at -2.5 C.Biochemical changes in the leaf tissue of these species were investigated at 5 day intervals during low temperature treatments. Increases in total sugar and starch were found in all three species during hardening, although S. tuberosum failed to harden. Soluble protein contents were increased in both S. acaule and S. commersonii but decreased in S. tuberosum. RNA contents change in a pattern similar to the soluble protein. Net increases of the soluble proteins were positively and significantly correlated with net increases of frost hardiness in S. acaule and S. commersonii. PMID- 16661448 TI - Uptake and subcellular compartmentation of gibberellin a(1) applied to leaves of barley and cowpea. AB - The uptake and subcellular accumulation of gibberellin A(1) (GA(1)) by leaves and protoplasts of barley (cv. Numar) and cowpea (cv. Blackeye pea No. 5) were investigated.Uptake of GA(1) by cowpea leaves is optimal at pH 5.8 and occurs by a saturable, probably carrier-mediated process having a half-maximal velocity at 10 to 20 micromolar. Uptake by both barley and cowpea leaves is inhibited by low temperature (+4 C) and the metabolic inhibitors 2,4-dinitrophenol and azide and is stimulated by ATP. Mesophyll protoplasts isolated from leaves fed radioactive GA(1) retain 20 to 80% of the radioactivity incorporated by excised leaves.The subcellular localization of the [(3)H]GA was determined by lysing protoplasts and separating subcellular organelles by density gradient centrifugation. Less than 5% of the incorporated [(3)H]GA was found associated with chloroplasts, mitochondria, nuclei, or other organelles or membranes with densities in sucrose gradients greater than 1.15 grams per cubic centimeter. Fifty to 100% of the [(3)H]GA was found in vacuoles. Isolated vacuoles were judged to be free of contamination by cytoplasm using phosphoenolpyruvate carboxylase as a marker enzyme. Osmotic breakage of vacuoles or protoplasts released > 95% of the [(3)H]GA, suggesting that GA is associated with the vacuolar sap rather than with the tonoplast membrane. PMID- 16661449 TI - Fractionation of Thylakoid Membranes with the Nonionic Detergent Octyl-beta-d glucopyranoside: RESOLUTION OF CHLOROPHYLL-PROTEIN COMPLEX II INTO TWO CHLOROPHYLL-PROTEIN COMPLEXES. AB - The detergent octyl-beta-d-glucopyranoside (30 millimolar in 2 millimolar Tris maleate, pH 7.0) preferentially extracts complexes containing protein and chlorophylls a plus b (CP) from spinach, leaving a residue highly enriched in CP I (P700-chlorophyll a protein). Use of the detergent results in a relatively gentle extraction since little free chlorophyll is formed and since sodium dodecyl sulfate-gel electrophoresis (on 10% acrylamide) of the extract also reveals the presence of two minor chlorophyll a complexes (apparent molecular weight, 47,000 and 43,000) instead of the usual single complex. The major complex preserved is CP 64, a chlorophyll a/b complex (apparent molecular weight, 64,000) which is an oligomer of another chlorophyll a/b complex, CP 27, the light harvesting complex (apparent molecular weight, 27,000). Dissociation of each complex reveals two polypeptides (molecular weight, 32,000 and 28,000) and limited proteolysis confirms that those of CP 64 have the same structure as those of CP 27. An additional chlorophyll a/b complex (apparent molecular weight, 29,000) is clearly separable from CP 27, and differs from it and CP 64 in having a higher chlorophyll a/b ratio and a single polypeptide (molecular weight, 29,000) which differs structurally from those of the other complexes. PMID- 16661450 TI - Evidence that Auxin-induced Growth of Soybean Hypocotyls Involves Proton Excretion. AB - The role of H(+) excretion in auxin-induced growth of soybean hypocotyl tissues has been investigated, using tissues whose cuticle was rendered permeable to protons or buffers by scarification (scrubbing). Indoleacetic acid induces both elongation and H(+) excretion after a lag of 10 to 12 minutes. Cycloheximide inhibits growth and causes the tissues to remove protons from the medium. Neutral buffers (pH 7.0) inhibit auxin-induced growth of scrubbed but not intact sections; the inhibition increases as the buffer strength is increased. Both live and frozen-thawed sections, in the absence of auxin, extend in response to exogenously supplied protons. Fusicoccin induces both elongation and H(+) excretion at rates greater than does auxin. These results indicate that H(+) excretion is involved in the initiation of auxin-induced elongation in soybean hypocotyl tissue. PMID- 16661451 TI - Partial Characterization of a Cadmium-binding Protein from the Roots of Cadmium treated Tomato. AB - A Cd-binding protein has been isolated from the roots of Cd-treated tomato plants cv. Rutgers. Almost all the Cd from a high-speed supernatant fraction was recovered in a 10,000-dalton fraction from a gel filtration column coincident with 250-nanometer absorbing material. DEAE-cellulose chromatography of this 10,000-dalton material yielded one major component, which eluted at 0.34 molar NaCl, had an absorption spectrum characteristic of metallothionein, and showed absorption changes upon acidification typical of metallothionein. Although the Cd binding protein did not behave like metallothioneins from animal sources during gel electrophoresis at pH 8.9, a single band containing Cd and staining with Coomassie brilliant blue could be detected following electrophoresis at pH 6.9. Synthesis of the Cd-binding protein appeared to be "induced" by treatment of the plants with Cd(2+). PMID- 16661452 TI - Method to Obtain a Chlorophyll-free Preparation of Intact Mitochondria from Spinach Leaves. AB - Mitochondria from green leaves of spinach have been prepared using a three-step procedure involving differential centrifugation, partition in an aqueous dextran polyethylene glycol two-phase system and Percoll gradient centrifugation. The mitochondrial fractions after the different steps of purification were compared. The final mitochondrial preparation was totally free from chloroplast material measured as chlorophyll content. The enrichment of mitochondria in relation to peroxisomes and microsomes was approximately 12 and 33 times, respectively, based on NAD:isocitrate dehydrogenase activity, glycolate oxidase activity, and NADPH:cytochrome c oxidoreductase activity. The apparent intactness of the inner and the outer mitochondrial membranes was higher than 90% as measured by latency of enzyme activities. The mitochondria showed high respiratory rates with respiratory control and the ADP/O ratios approached the theoretical limits. PMID- 16661453 TI - Photophosphorylation capacity of stable spheroplast preparations of anabaena. AB - Spheroplasts from Anabaena 7119 (formerly designated Nostoc muscorum) were prepared in the presence of serum albumin in 0.5 molar sucrose. Electron transport and photophosphorylation were preserved (> 70% of the maximum rate for 1 week). The pH profile of electron transport and photophosphorylation in the reactions H(2)O --> NADP, H(2)O --> methyl viologen, and H(2)O --> ferricyanide shows that uncoupling by ammonia is small throughout and increases slightly with higher pH. ADP + Pi increased NADP reduction from H(2)O by 2.5-fold. The ratios of ATP formed per electron pair transported ranged from 0.9 to 1.5. Effects of catalase and superoxide dismutase on the overall O(2) balance implicate pseudocyclic electron transport and phosphorylation. The quenching of 9 aminoacridine fluorescence indicates the formation of a Delta pH from 2 to 2.6 during illumination. This pH gradient is abolished by uncouplers; however, complete uncoupling is achieved only by 3-chlorocarbonyl cyanide phenylhydrazone or valinomycin + NH(4) (+). In the presence of NH(4) (+) alone, the membrane potential may act as the driving force for photophosphorylation.Increasing amounts of bovine serum albumin protected phosphorylation from uncoupling by silicomolybdic acid. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea-insensitive water oxidation by silicomolybdic acid provides evidence that the site for 3-(3,4 dichlorophenyl)-1, 1-dimethylurea action is on the acceptor and not donor side of photosystem II in the procaryote Anabaena. It is concluded that stable spheroplasts retain coupled electron transport approaching in vivo rates. PMID- 16661454 TI - Inhibition of Potato Tuber Invertase by an Endogenous Inhibitor: EFFECTS OF SALTS, pH, TEMPERATURE, AND SUGARS ON BINDING . AB - Binding between potato tuber invertase and its endogenous inhibitor followed second-order reaction kinetics. Binding rates were diminished by the presence of various inorganic salts, MgCl(2) being especially effective. This effect of MgCl(2) was used in binding rate studies by adding the salt with sucrose to reduce binding during assay of previously unbound activity. The optimal pH for binding was about 4.8, similar to the optimal pH for catalytic activity of invertase. The optimal temperature for binding was about 45 C, approximately 5 C less than the optimum for catalytic activity. Sucrose at concentrations as low as 2 millimolar slowed binding; reducing sugars had little or no effect on binding or on catalytic activity. PMID- 16661455 TI - Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide insensitive Electron Transport Pathway. AB - MALATE OXIDATION IN PLANT MITOCHONDRIA PROCEEDS THROUGH THE ACTIVITIES OF TWO ENZYMES: a malate dehydrogenase and a NAD(+)-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD(+) stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD(+) are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD(+)/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD(+)/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide sensitive cytochrome pathway and the cyanide-insensitive alternative pathway. PMID- 16661456 TI - Quantum Requirement for Photosynthesis in Sedum praealtum during Two Phases of Crassulacean Acid Metabolism. AB - The quantum requirement (QR) for photosynthesis in Sedum praealtum, a Crassulacean acid metabolism plant, was compared with that of wheat, a C(3) plant, and maize, a C(4) plant, at 30 C. During the deacidification phase in S. praealtum, approximately 16 moles quanta were absorbed per mole malate consumed. This is equivalent to 16 moles quanta per mole CO(2) fixed, assuming 1 mole CO(2) is assimilated per mole malate decarboxylated. This QR for Crassulacean acid metabolism is similar to that of the C(3) or C(4) plant under atmospheric conditions, even though there are considerable differences in the biochemistry of photosynthesis. During late-afternoon C(3)-like fixation of atmospheric CO(2) in S. praealtum, the QR was relatively high with values of 41 under 21% O(2) and 19 under 2% O(2). During the deacidification phase in S. praealtum, the relatively low QR can be accounted for by the repression of photorespiration and saturation of photosynthesis from the elevated CO(2) concentration in the leaves during malate decarboxylation. PMID- 16661457 TI - Effect of temperature on the synthesis and secretion of alpha-amylase in barley aleurone layers. AB - The effect of temperature on alpha-amylase synthesis and secretion from barley (c.v. Himalaya) half-seeds and aleurone layers is reported. Barley half-seeds incubated at 15 C in gibberellic acid (GA) concentrations of 0.5 and 5 micromolar for 16 hours do not release alpha-amylase. Similarly, isolated aleurone layers of barley do not release alpha-amylase when incubated for 2 or 4 hours at temperatures of 15 C or below following 12 hours incubation at 25 C at GA concentrations from 50 nanomolar to 50 micromolar. There is an interaction between temperature and GA concentration for the process of alpha-amylase release from aleurone layers; thus, with increasing GA concentration, there is an increase in the Q(10) of this process. A thermal gradient bar was used to resolve the temperature at which the rate of alpha-amylase release changes; thermal discontinuity was observed between 19 and 21 C. The time course of the response of aleurone tissue to temperature was determined using a continuous monitoring apparatus. Results show that the effect of low temperature is detectable within minutes, whereas recovery from exposure to low temperature is also rapid. Although temperature has a marked effect on the amount of alpha-amylase released from isolated aleurone layers, it does not significantly affect the accumulation of alpha-amylase within the tissue. At all GA concentrations above 0.5 nanomolar, the level of extractable alpha-amylase is unaffected by temperatures between 10 and 28 C. It is concluded that the effect of temperature on alpha-amylase production from barley aleurone layers is primarily on the process of enzyme secretion. PMID- 16661458 TI - Carbohydrates in Soybean Nodules: II. DISTRIBUTION OF COMPOUNDS IN SEEDLINGS DURING THE ONSET OF NITROGEN FIXATION. AB - During the first few days of nitrogen fixation activity by soybean (Glycine max (L.) Merr) root nodules, d-chiro-inositol, myo-inositol, sucrose, alpha,alpha trehalose, and maltose accumulate rapidly and reach concentrations several fold greater than concentrations in other plant organs. Concentrations of d-pinitol in nodules (>/=1.0 milligrams per gram fresh weight) were similar to concentrations in leaf blades. The concentration of fructose in nodules was lower than concentrations in other plant organs.Comparison of nonnodulated roots, nodulated roots (after removal of nodules), and nodules indicated that nodules may compete successfully with roots for carbohydrates, especially the disaccharides sucrose, alpha,alpha-trehalose, and maltose. Based on the isolation of protoplasts and bacteroids, it was tentatively concluded that the highest concentrations of cyclitols in soybean nodules are located in the infected region and that, inside infected cells, the highest concentrations of d-pinitol and myo-inositol are outside of bacteroids.Evidence for the identification of d-chiro-inositol and maltose in soybean nodules is presented. PMID- 16661459 TI - Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts. AB - A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease. PMID- 16661460 TI - Responses of superoxide dismutase and glutathione reductase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. AB - Responses of superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) activities were evaluated in leaf tissue from intact cotton plants (Cotton Branch 1697) which were exposed to 75% O(2), 350 microliters per liter CO(2) for 48 hours. Soluble protein was extracted from O(2)-treated and control tissue, and enzyme levels were determined. Superoxide dismutase activity in cotton leaf tissue was high (26 units per milligram protein) under normal conditions of 21% O(2), saturating light, and limiting CO(2), and neither qualitative nor quantitative differences in the cyanide-sensitive or -insensitive forms of the enzyme occurred in response to hyperoxic conditions. Glutathione reductase activity, however, was 2- to 3-fold higher in extracts from tissue exposed to 75% O(2). No increase in activity was observed for the peroxisomal enzymes, glycolate oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6). Results are consistent with an integrated pathway involving superoxide dismutase and glutathione reductase for protection of sensitive leaf components against detrimental effects of intermediate reduction products of O(2). PMID- 16661461 TI - Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: I. GRADIENT PURIFIED MITOCHONDRIA. AB - The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A- or cyanide-insensitive O(2) uptake. Further purification of this initial mitochondrial pellet using a linear Percoll (Pharmacia) density gradient separated the mitochondria into two bands of physiologically distinct activity. Based on the usual mitochondrial respiratory criteria of ADP/O and respiratory control values, these fractions were qualitatively similar to the crude pellet. However, we observed no antimycin A insensitive O(2) uptake in either gradient band. Antimycin A-insensitive O(2) consumption could be restored to the upper gradient band of mitochondria by the addition of linoleic acid. This activity was inhibited either by salicylhydroxamic acid or propyl gallate, a known lipoxygenase inhibitor. Likewise, addition of linoleic acid to the crude mitochondrial pellet elicited a 4- to 5-fold increase in O(2) uptake. This O(2) consumption was insensitive to antimycin A and cyanide but was inhibited by either propyl gallate or salicylhydroxamic acid. Electron microscopic examination revealed that only the lower gradient band contained contamination-free mitochondria, which, in turn, lacked ability to oxidize linoleic acid. Antimycin A-insensitive O(2) consumption in the differential centrifugation fraction from germinating durum wheat seedlings decreased over 64 hours of development. PMID- 16661462 TI - X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains. AB - Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain. PMID- 16661463 TI - Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability. AB - Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification. PMID- 16661464 TI - Evidence for Light-stimulated Synthesis of Phosphoenolpyruvate Carboxylase in Leaves of Maize. AB - Illumination (22,000 lumens per meter(2)) of etiolated maize plants for 80 hours brings about a 5-fold increase in phosphoenolpyruvate carboxylase activity per unit of protein. An increase in carboxylase protein and incorporation of [(35)S]methionine into the protein occurs simultaneously with the activity increase. In green plants, the level of phosphoenolpyruvate carboxylase protein and enzyme activity is dependent on the intensity of light during growth. These results are consistent with the conclusion that the activity increase results from light-stimulated de novo synthesis of phosphoenolypyruvate carboxylase protein. PMID- 16661465 TI - Pulse-labeling Studies on Protein Synthesis in Developing Pea Seeds and Evidence of a Precursor Form of Legumin Small Subunit. AB - Intact cotyledons were taken from pea seeds at various stages during seed development and pulse-labeled with (14)C-amino acids. Salt-soluble proteins then were extracted and fractionated on Na dodecyl sulfate-polyacrylamide gels. Storage proteins in these extracts were identified by their binding to immunoaffinity columns. The labeling studies showed that the synthesis of storage protein polypeptides accounts for a major part of total protein synthesis of developing cotyledons between 10 and 22 days after flowering. The distribution of the incorporated radioactivity between individual storage protein polypeptides varied with stage of development. For example, the synthesis of the 50 kilodalton complex of vicilin subunits dominated the early stages of protein accumulation but was a negligible proportion of the total incorporation in the later stages. On the other hand, the 75 kilodalton vicilin subunit was synthesized throughout this entire period. The major small subunit of legumin (20 kilodaltons) was not detected by either Coomassie blue staining or by 2-hour labeling during this period. It was found to arise during the desiccation phase of seed maturation from a long-lived precursor with a relative electrophoretic mobility equivalent to 19 kilodaltons. PMID- 16661466 TI - Soluble Sugars, Respiration, and Energy Charge during Aging of Excised Maize Root Tips. AB - Oxygen uptake and energy charge were monitored during aging of excised maize root tips and related to the soluble sugar content and exogenous sugar supply.Oxygen uptake declined immediately after excision to 50 to 30% of its initial value after 8 and 24 hours of aging at 25 C. There was also a sharp decline of the total sugar content (glucose, fructose, and sucrose). Starch content was very low at the time of excision and almost negligible 5 hours later. During the same period, the respiratory quotient declined from 1 to 0.75 and then remained stable.The addition of exogenous sugars induced a rapid rise of the respiratory rate which stabilized at a level correlated to the external sugar concentration. Addition of 0.2 molar glucose was necessary to restore the respiratory rate to the initial, also the maximum, level. These results indicate that metabolic activity of root tips is highly reliant on sugar import and carbohydrate reserves at the time of excision cannot compensate for the cessation of import. The control of respiration by substrate supply is in good agreement with the failure for dinitrophenol to stimulate oxygen uptake in aged sugar-depleted root tips.The energy charge remained constant at about 0.9, irrespective of the presence or absence of glucose and in spite of a large decline of respiratory activity in aged, sugar-depleted tissues. PMID- 16661467 TI - Changes in Wheat Leaf Polysomal Messenger RNA Populations during the Early Stages of Rust Infection: EFFECTS OF CHLORAMPHENICOL AND LINCOMYCIN ON CELL-FREE TRANSLATION BY POLYSOMES FROM HEALTHY AND INFECTED LEAVES. AB - Polysomes isolated from a susceptible variety of wheat leaves (cultivar W2691) and those inoculated with the wheat stem rust fungus (f. sp. tritici, race 126 ANZ-6, 7) were incubated in a cell-free protein-synthesizing system. Under these conditions, different size classes of polypeptides, ranging in molecular weight from 10,000 to 80,000, are radiolabeled. Using double-isotope labeling technique, we show that some discrete size classes of polypeptides are synthesized in significantly greater quantitites by polysomes from inoculated leaves compared to the corresponding size classes synthesized by polysomes from healthy leaves. These results confirm our previous observation that there are significant changes in the wheat leaf polysomal messenger RNA populations at 3 days after inoculation with the rust fungus.The effects of the organelle-specific inhibitors of protein synthesis, chloramphenicol and lincomycin, on in vitro polysomal messenger RNA translation were investigated. The polypeptides synthesized by polysomes from healthy and inoculated leaves in the presence of chloramphenicol were compared. The results show that, even in the presence of this antibiotic, the polysomes from inoculated leaves synthesize greater quantities of some size classes of polypeptides. These data indicate that changes in polysomal messenger RNA populations involve, at least in part, cytoplasmic messenger RNA. PMID- 16661468 TI - Phycomyces: MODIFICATION OF LIGHT-INDUCED SPIRAL GROWTH AFTER MECHANICAL CONDITIONING OF THE CELL WALL. AB - Mature stage IVb Phycomyces sporangiophores show left-hand spiral growth; that is, viewed from above, the sporangium rotates clockwise. It has been shown that mechanical conditioning (strain-hardening) of the cell wall by the Instron technique increases the ratio of rotation to the elongation growth rate compared to nonmechanically conditioned controls. It is reported that the addition of a saturating light stimulus to these sporangiophores causes a decrease in the ratio of rotation to elongation growth rate. This result is in agreement with the fibril slippage model, i.e. the counterclockwise rotation of stage IVa is a result of parallel fibrils lying in a right-handed spiral configuration slipping by one another. It is suggested that a light stimulus added to a mechanically conditioned stage IVb sporangiophore activates one or more cell wall-loosening enzymes which act by decreasing the number of intermolecular bonds between parallel fibrils causing fibril slippage, resulting in counterclockwise rotation. It is precisely this counterclockwise contribution that decreases the rotation to elongation growth ratio of mechanically conditioned and then light-stimulated stage IVb sporangiophores. PMID- 16661469 TI - Maintenance of High Photosynthetic Rates during the Accumulation of High Leaf Starch Levels in Sunflower and Soybean. AB - Sunflower (cv. "Mammoth Greystripe") and soybean (Merr. cv. "Amsoy 71") leaves were exposed to continuous light for at least 52 hours in an attempt to determine the relationship between leaf starch levels and photosynthetic rates. Immature rapidly expanding and relatively mature slowly expanding sunflower leaves were studied. After 52 hours continuous light, the rapidly expanding leaves accumulated high starch levels (3.3 milligrams per square centimeter, 43% of dry weight) with only about a 10% decline from the initial photosynthetic rate of 42 milligrams CO(2) per square decimeter per hour. Under the same conditions, the slowly expanding leaves accumulated less starch, but the photosynthetic rate declined 30%. Soybean leaves, which were slowly expanding, accumulated less starch than sunflower leaves (2.1 milligrams per square centimeter, 34% of dry weight), and their photosynthetic rates declined only about 10% after 54 hours continuous light.In sunflower and, to a lesser extent, in soybean, the accumulation of large amounts of leaf starch was not necessarily associated with an appreciable decline in photosynthetic rate. However, in sunflower, the stage of leaf maturity was a major determinant in the photosynthetic response to continuous, relatively high light with its associated starch accumulation. PMID- 16661470 TI - Loss of tomato cell wall galactan may involve reduced rate of synthesis. AB - Changes in the galactose content of the noncellulosic polysaccharides of tomato (Mill) fruit cell walls were analyzed under various conditions. On the plant, galactan decreased gradually during fruit growth. As normal fruits ripened, the loss of galactan increased sharply; this was not observed in attached rin fruits beyond the fully mature stage. The ability to produce new wall galactan in vitro was retained in mature fruit tissue but declined with ripening. Normal tomatoes ripening on the plant showed a transient increase in galactan content at the climacteric. It is suggested that the decline in wall galactan is partly due to reduced synthesis in senescing, normal fruits and in detached rin tomatoes. PMID- 16661471 TI - Phytochrome Modifies Blue-light-induced Electrical Changes in Corn Coleoptiles. AB - Unilateral blue light administered to corn coleoptile segments produces no alteration of transmembrane potential on the light side, and only a small and slow hyperpolarization on the dark side. Red light causes a 5-15 millivolt depolarization in cells on the light side causes and somewhat smaller effects on the dark side. Blue given after red causes a rapid hyperpolarization on both sides of the coleoptile. The effect of the potentiating red preirradiation is probably due to phytochrome, being largely abolished by far-red given after red, but before the blue light. The effect of prior red irradiation decays in the dark, showing a half-time of about 45 minutes at room temperature. This rapid cooperativity between phytochrome and the phototropic pigment may indicate a common locale, possibly in a membrane. PMID- 16661472 TI - Metabolism of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Soybean Root Callus : EVIDENCE FOR THE CONVERSION OF 2,4-D AMINO ACID CONJUGATES TO FREE 2,4-D. AB - An auxin-requiring soybean root callus metabolized [1-(14)C]-2,4 dichlorophenoxyacetic acid (2,4-D) to diethyl ether-soluble amino acid conjugates and water-soluble metabolites. The uptake in tissue varied with incubation time, concentration, and amount of tissue. Uptake was essentially complete (80%) after a 24-hour incubation and the percentage of free 2,4-D in the tissue fell to its lowest point at this time. At later times, the percentage of free 2,4-D increased and the percentage of amino acid conjugates decreased, whereas the percentage of water-soluble metabolites increased only slightly. Similar trends were seen if the tissue was incubated for 24 hours in radioactive 2,4-D, followed by incubation in media without 2,4-D for 24 hours. Inclusion of nonlabeled 2,4-D during the 24-hour chase period did not reduce amino acid conjugate disappearance but did reduce the percentage of free [1-(14)C]2,4-D. Thus, an external supply of 2,4-D does not directly prevent amino acid conjugate metabolism in this tissue. It is concluded that 2,4-D amino acid conjugates were actively metabolized by this tissue to free 2,4-D and water-soluble metabolites. PMID- 16661473 TI - Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VI. DIFFERENTIAL EFFECTS OF TEMPERATURE AND LIGHT INTENSITY ON PHOTORESPIRATION IN C(3), C(4), AND INTERMEDIATE SPECIES. AB - The effects of temperature and photosynthetically active radiation levels on photorespiration were investigated in Panicum milioides Nees ex Trin. and Panicum schenckii Hack., species known to have low photorespiration rates and other characteristics intermediate between C(3) and C(4) species. Comparisons were made with the C(3) grass species tall fescue (Festuca arundinacea Schreb.). An increase in temperature from 20 to 35 C raised photorespiration from 7.3 to 10.2 milligrams per square decimeter per hour in tall fescue, but the increase in P. schenckii was less than 1 milligram per square decimeter per hour. Increases in temperature caused much less change in CO(2) compensation concentration in P. milioides and P. schenckii than in tall fescue, values of 160 microliters per liter being obtained in tall fescue at 40 C compared to about 40 microliters per liter for P. milioides and P. schenckii. Photorespiration in P. schenckii increased by only about 1 milligram CO(2) per square decimeter per hour as the photosynthetically active radiation level was raised from 100 to 2,000 microEinsteins per square meter per second. Loss of CO(2) into CO(2)-free air actually decreased from 2.2 to 1.0 milligrams per square decimeter per hour as the radiation level was raised from 100 to 1,100 microEinsteins per square meter per second but tended to rise again at 2,000 microEinsteins per square meter per second. In contrast, photorespiration in tall fescue tripled with radiation level over the same range, reaching a maximum value of 7.2 milligrams per square decimeter per hour as determined by extrapolation of the CO(2) response curves to zero CO(2). The CO(2) compensation concentration in tall fescue was nearly insensitive to photosynthetically active radiation above 140 microEinsteins per square meter per second but, in P. milioides and P. schenckii, it decreased from values of 69 and 62 microliters per liter, respectively, to values of 21 and 16 as the radiation level was increased from 50 to 1075 microEinsteins per square meter per second. Interpolation of CO(2)-response curves showed that an increase in photosynthetically active radiation level from 100 to 2,000 microEinsteins per square meter per second reduced the CO(2) compensation value of P. schenckii from 38 to 19 microliters per liter. Data from these experiments indicate reduced photorespiration or a CO(2)-recycling mechanism in P. milioides and P. schenckii which causes apparent photorespiration to be nearly insensitive to temperature in the 20 to 35 C range and to decrease at high radiation intensities. PMID- 16661474 TI - Involvement of phospholipids in polyunsaturated Fatty Acid synthesis in developing soybean cotyledons. AB - Developing soybean (cv. Dare) cotyledons harvested at 30 days after flowering were pulse-labeled with [1-(14)C]oleoyl-CoA. The metabolic interrelation of radiolabeled unsaturated fatty acids between the major glycerolipid classes was determined at various time intervals. At chase time zero, [(14)C]oleic acid accounted for 99.2% of the total glycerolipid radioactivity, and phospholipids contained 92% of the total incorporated radioactivity. With time, phospholipids were metabolized in triacylglycerol biosynthesis and radioactivity was detected in polyunsaturated fatty acids. The hypothesis that phospholipids were metabolic intermediates in polyunsaturated fatty acid biosynthesis was tested by comparing the theoretical and the actual amount of radiolabeled oleic acid that was associated with triacylglycerol as a function of time. The radioactive oleic acid found in triacylglycerol at various intervals was derived from phospholipids via a diacylglycerol intermediate. Assuming no phospholipid desaturation, the potential or theoretical amounts of [(14)C]oleic acid that could be transferred to triacylglycerol from phospholipids was defined by a system of differential equations. The results demonstrated that the decline in [(14)C]oleic acid from phospholipid after long chase intervals was equal to the total amount of radioactive unsaturated fatty acids found in neutral lipids. The difference between the theoretical and actual amounts of [(14)C]oleic acid present in triacylglycerol after long time intervals was equal to the amount of radioactivity present in polyunsaturated fatty acids. Based upon those findings in soybeans, the desaturation of oleic acid associated with phospholipids was highly probable. PMID- 16661475 TI - Corn Root Protoplasts: ISOLATION AND GENERAL CHARACTERIZATION OF ION TRANSPORT . AB - A method was developed for the large scale and rapid isolation of intact viable corn root protoplasts. Pure and metabolically active protoplasts were collected using a flotation technique. Vital staining tests, light and electron microscopy, and measurements of basic metabolic processes indicated that the isolated protoplasts were metabolically active, and that the plasmalemma and other organelles were well preserved. The isolated protoplasts performed normal, active ion transport functions. Time course of K(+) and inorganic phosphate (H(2)PO(4) ( )) influx and the effects of external pH, carbonyl cyanide p trifluoromethoxyphenylhydrazone, fusicoccin, and diethylstilbestrol on K(+) and inorganic phosphate influx and net H(+) efflux in isolated protoplasts correlated well with data obtained on root segments. Data presented indicated that isolated protoplasts from roots can be used to gain additional insights into the mechanism of ion transport in plant cells. PMID- 16661476 TI - Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle. AB - Glyoxysomes isolated from germinating castor bean endosperm accumulate NADH by beta-oxidation of fatty acids. By utilizing the glutamate: oxaloacetate aminotransferase and malate dehydrogenase present in glyoxysomes and mitochondria, reducing equivalents could be transferred between the organelles by a malate-aspartate shuttle. The addition of aspartate plus alpha-ketoglutarate to purified glyoxysomes brought about a rapid oxidation of accumulated NADH, and the oxidation was prevented by aminooxyacetate, an inhibitor of aminotransferase activity. Citrate synthetase activity in purified glyoxysomes could be coupled readily to glutamate: oxaloacetate aminotransferase activity as a source of oxaloacetate, but coupling to malate dehydrogenase and malate resulted in low rates of citrate formation. Glyoxysomes purified in sucrose or Percoll gradients were permeable to low molecular weight compounds. No evidence was obtained for specific transport mechanisms for the proposed shuttle intermediates. The results support a revised model of gluconeogenic metabolism incorporating a malate aspartate shuttle in the glyoxysomal pathway. PMID- 16661477 TI - An Improved Method for Detecting Auxin-induced Hydrogen Ion Efflux from Corn Coleoptile Segments. AB - Conditions necessary to detect maximal auxin-induced H(+) secretion using a macroelectrode have been investigated using corn coleoptile segments. Auxin induced H(+) secretion is strongly dependent upon oxygenation or aeration when the tissue to volume ratio is high. Cuticle disruption or removal is also necessary to detect substantial auxin-induced H(+) secretion. The auxin-induced decrease in pH of the external medium is stronger when the hormone is applied to tissue in which the cuticle has been disrupted with an abrasive than when the hormone is applied to tissue from which the cuticle and epidermis have been removed by peeling. The lower detectable acidification of the external medium when using peeled segments appears to be due in part to the leakage of buffers into the medium and in part to the removal of the auxin-sensitive epidermal cells.The sensitivity of corn coleoptile segments to auxin, as measured by H(+) secretion, increases about 2-fold during the first 2 hours after excision. This change in apparent sensitivity to auxin as reflected by H(+) secretion is paralleled by a time-dependent change in the growth response to auxin. Under optimal conditions for detecting H(+) efflux (oxygenation, abrasion, hormone application 2 hours after excision), the latent period in auxin-induced H(+) efflux (about 7 or 8 minutes) is only half as great as the latent period in auxin induced growth (about 18 to 20 minutes). These observations are consistent with the acid growth hypothesis of auxin action. PMID- 16661478 TI - Effect of 1-Aminocyclopropane-1-Carboxylic Acid on the Production of Ethylene in Senescing Flowers of Ipomoea tricolor Cav. AB - Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to rib segments excised from flowers of Ipomoea tricolor Cav. resulted in the formation of C(2)H(4) in greater quantities than produced under natural conditions. The ability of ACC to enhance C(2)H(4) production was independent of the physiological age of the tissue and its capacity to synthesize C(2)H(4) without applied ACC. When ACC was fed to rib segments that had been treated with [(14)C]methionine, incorporation of radioactivity into C(2)H(4) was reduced by 80%. Aminoethoxyvinylglycine and aminooxyacetic acid inhibited C(2)H(4) production in rib segments of I. tricolor but had no effect on ACC-enhanced C(2)H(4) production. Protoplasts obtained from flower tissue of I. tricolor did not form C(2)H(4), even when incubated with methionine or selenomethionine. They produced C(2)H(4) upon incubation with ACC, however. ACC-dependent C(2)H(4) production in protoplasts was inhibited by n-propyl gallate, AgCl, CoCl(2), KCN, Na(2)S, and NaN(3). ACC-dependent C(2)H(4) synthesis in rib segments and protoplasts was dependent on O(2), the K(m) for O(2) being 1.0 to 1.4% (v/v). These results confirm the following pathway for C(2)H(4) biosynthesis in I. tricolor. methionine [selenomethionine] --> S-adenosylmethionine [selenoadenosylmethionine] --> ACC --> C(2)H(4). PMID- 16661479 TI - Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza. AB - Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size. PMID- 16661480 TI - Influence of Anaerobiosis on Chlorophyll Biosynthesis in Greening Oat Seedlings (Avena sativa L.). AB - The influence of anaerobiosis for 0.5 to 15 hours on the last steps of chlorophyll biosynthesis of etiolated oat seedlings was investigated. Phototransformation of protochlorophyllide to chlorophyllide is only slightly reduced and esterification of chlorophyllide is slightly increased by pretreatment under anaerobic conditions. Pretreated plants accumulate the geranylgeraniol ester of chlorophyllide rather than the phytol ester. Enzymic hydrogenation of the esterifying alcohol geranylgeraniol to phytol is presumably inhibited by anaerobiosis. PMID- 16661481 TI - A Differential Effect of Race T Toxin on Dark and Photosynthetic CO(2) Fixation by Thin Leaf Slices from Susceptible Corn. AB - The effect of purified host-specific toxin from Bipolaris (Helminthosporium) maydis, race T, on dark or light-dependent CO(2) fixation was studied with thin (1 x 8 mm) corn (Zea mays L.) leaf slices supplied H(14)CO(3) (-). At 5 to 30 nanograms per milliliter ( congruent with5 nanomolar), toxin significantly inhibited (20 to 40%) dark CO(2) fixation in susceptible (T) corn slices after either dark or light preincubations of 10-20 minutes. The same concentrations were effective to the same degree on photosynthesis, but the effect differed in that significant inhibition occurred after 25 minutes and only with light preincubation. Light preincubation without toxin did not shorten the time required for inhibition of photosynthesis after addition of toxin. Once photosynthetic inhibition was entrained, it was not reversed by subsequent periods of darkness. The results suggest the possibility that race T toxin affects two separate metabolic sites, and the data are discussed in view of currently held concepts of toxin action in susceptible tissue. PMID- 16661482 TI - Preparation and Characterization of a Highly Active Cell-free Protein synthesizing System from Dry Pea Primary Axes. AB - A highly active and reliable cell-free protein-synthesizing system from dry pea primary axes has been developed. The system has been optimized with respect to both overall activity and translational fidelity. Under optimal conditions, the system incorporates nearly 1,000 picomoles leucine per 50 microliters incubation mixture. At limiting tobacco mosaic virus RNA concentrations, its efficiency exceeds 1,000 picomoles leucine per picomole template molecules. The maximal size of the polypeptides coded for by tobacco mosaic virus RNA goes up to 180,000. A procedure has been worked out to reduce the endogenous template activity of the system. the dramatic increase of the activity of this pea cell-free system as compared to previous attempts can be explained by the use of Cl(-)-free buffers. PMID- 16661483 TI - Carrier Protein-mediated Transport of Neutral Amino Acids into Mung Bean Mitochondria. AB - The transport of neutral amino acids into mitochondria isolated from the hypocotyl of mung bean (Roxb.) was studied by the swelling technique. Isolated mitochondria swelled when added to an isosmotic solution of proline, serine, methionine, threonine, alanine, and glycine. The swelling was stereospecific in that it was faster in the l-amino acid than in the corresponding d-amino acid. Preincubation of the mitochondria with the sulfhydryl modifying reagents, p mercuribenzoate and mersalyl, resulted in an inhibition of the swelling caused by proline, serine, threonine, and glycine. The swelling induced by alanine was inhibited only by mersalyl, whereas that by methionine was inhibited only by p mercuribenzoate. In all cases, the inhibition caused by the sulfhydryl modifying reagents was readily reversible by the subsequent treatment of the mitochondria with dithiothreitol. N-Ethylmaleimide, another sulfhydryl-modifying reagent, did not cause any inhibition of the swelling. The findings indicate the existence of a protein mediated mechanism for the transport of neutral amino acids into plant mitochondria. PMID- 16661484 TI - Light-Shade Adaptation : TWO STRATEGIES IN MARINE PHYTOPLANKTON. AB - Using chlorophyll/P700 ratios, the size and number of photosynthetic units were estimated, as a function of light-shade adaptation in two species of marine phytoplankton: Skeletonema costatum, a diatom, and Dunaliella tertiolecta, a chlorophyte. In the diatom, light-shade adaptation is characterized primarily by changes in the size and not the number of P700 units, whereas in the chlorophyte, overall changes in chlorophyll content are related to changes in the number and not the size of P700 units. A correlation between the characteristics of P700 units and photosynthetic responses was not established. Both strategies of light shade adaptation effectively harvest and transfer light energy to reaction centers, however, the Skeletonema strategy is more effective at subsaturating intensities. The two strategies may represent an evolutionary divergence in photosynthetic adaptation to variations in light intensity. PMID- 16661485 TI - Thermotropic Properties of Thermophilic, Mesophilic, and Psychrophilic Blue-green Algae. AB - Thermotropic properties of blue-green algae grown at high, room, and low temperatures in H(2)O and D(2)O media were studied by highly sensitive differential scanning microcalorimetry. The thermograms of these organisms contain an endothermal peak in the temperature range of 50 to 70 C with an endothermal heat ranging from 0.14 to 1.91 joules per gram organism. The temperature at which the endothermal peak occurs is comparable with the thermal denaturation temperature of phycocyanin, the major biliprotein isolated from these algae. A good correlation can be found for the relative thermal stability of various organisms with that of the isolated biliproteins. The ability of these algae to resist thermal disruption is correlated with the thermal environments in which these algal cells grow. The thermal stability of normal algae is in the order of thermophile > mesophile > psychrophile. It was found that the deuterated mesophilic algae were less able to resist thermal disruption than ordinary mesophilic algae. PMID- 16661486 TI - Time Course of Induction of Cytochrome P-450, NADPH-Cytochrome c Reductase, and Cinnamic Acid Hydroxylase by Phenobarbital, Ethanol, Herbicides, and Manganese in Higher Plant Microsomes. AB - The mixed function oxidase trans-cinnamic acid 4-hydroxylase, cytochrome P-450, cytochrome b(5), and NADPH-cytochrome c (P-450) reductase were measured in microsomes from aging artichoke tuber slices exposed to manganese, ethanol, phenobarbital, and the herbicides Chloro-IPC, Dichlobenil, and Monuron. Although the microsomal hydroxylating complex is already induced by the slicing and aging process, 25 millimolar MnCl(2), 4 millimolar phenobarbital, and 300 millimolar ethanol caused a marked increase of hydroxylase activity and cytochrome P-450 content and shifted their time course. The herbicides, 200 micromolar Dichlobenil and 200 micromolar Monuron, were less effective. Chloro-IPC was slightly inhibitory. NADPH cytochrome c reductase was significantly increased only in phenobarbital-treated slices. Cytochrome b(5) was generally the least affected among the parameters being measured. The mechanisms by which these compounds increase cytochrome P-450 content and hydroxylase activity are not yet defined. PMID- 16661487 TI - Thioredoxin-like Activity of Thylakoid Membranes: THIOREDOXIN CATALYZING THE REDUCTIVE INACTIVATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE OCCURS IN BOTH SOLUBLE AND MEMBRANE-BOUND FORM. AB - The inactivation of pea leaf chloroplast glucose-6-phosphate dehydrogenase by dithiothreitol can be catalyzed by thioredoxin-like molecules that are present in chloroplasts. This thioredoxin activity occurs predominantly as a soluble species, but washed thylakoid membranes also exhibit some thioredoxin-like activity. The membrane-associated thioredoxin can be extracted by treatment with the detergent Triton X-100. The solubilized thioredoxin appears to have a molecular size similar to that of the soluble thioredoxin which catalyzes the same reaction. The thylakoid-bound activity constitutes only about 5% of the total chloroplast thioredoxin activity. The thioredoxin occurring in the membrane fraction cannot, however, be ascribed to the trapping of stroma since less than 0.1% of three stromal marker enzymes are found in the same thylakoid extract. PMID- 16661488 TI - Host recognition in the Rhizobium-soybean symbiosis. AB - Polar binding of Rhizobium japonicum to roots and root hairs of Glycine soja (L.) Sieb. and Zucc. is specifically inhibited by d-galactose and N-acetyl-d galactosamine, haptens of Glycine max seed lectin. A protein, immunologically cross-reactive with the G. max seed lectin, is present in G. soja seed extracts. Peptide mapping of the purified G. max and G. soja lectins indicates that the two are similar in structure. Soybean lectin can be localized on the surface of both G. max and G. soja roots by indirect immunolatex techniques. These observations indicate that the Rhizobium-binding lectin, previously isolated from seeds, also is present on the root surface-the site of the initial steps in the infection. This lectin is capable of binding Rhizobium japonicum to the root. PMID- 16661489 TI - Action Spectra for the Inhibition of Hypocotyl Growth by Continuous Irradiation in Light and Dark-Grown Sinapis alba L. Seedlings. AB - Action spectra for the inhibition by continuous (24-hour) irradiation of hypocotyl growth in 54-hour-old Sinapis alba L. seedlings were measured using seedlings which had had four different pretreatments. These seedlings were either (a) dark-grown with a high total phytochrome level, (b) dark-grown with a low total phytochrome level, (c) light-grown with chlorophyll, or (d) light-grown with no chlorophyll [treated with 4-chloro-5-(methylamino)-2-(alpha,alpha,alpha trifluoro-m-tolyl)-3(2H) -pyridazinone (San 9789)].The resulting action spectra show that the blue, red, and far-red (716 nm) wavebands are most inhibitory for dark-grown plants with high phytochrome content, whereas hypocotyl growth in dark grown plants with a low phytochrome content are only slightly inhibited by blue and far-red radiation. In light-grown plants, the effectiveness of blue and far red light almost disappears. The position of red light effectiveness in chlorophyll-containing plants is shifted to lower wavelengths compared with those containing no chlorophyll. PMID- 16661490 TI - Glutamine Synthetase in Rice: A COMPARATIVE STUDY OF THE ENZYMES FROM ROOTS AND LEAVES. AB - Chromatographic, kinetic, and regulatory properties of glutamine synthetase in rice were investigated. By DEAE-Sephacel column chromatography, two forms (glutamine synthetase 1 and glutamine synthetase 2) were identified in leaves and one form (glutamine synthetase R) was identified in roots. Purification on hydroxyapatite and gel electrophoresis showed that glutamine synthetase R was distinct from the leaf enzymes. The three isoforms were purified to similar specific activities and their properties were studied. Heat lability, pH optimum about 8, K(m) for l-glutamate of 20 millimolar, and inhibition by glucosamine 6 phosphate were the main characteristics of glutamine synthetase 2. Heat stability, pH optimum about 7.5, K(m) for l-glutamate of 2 millimolar, and no effect of glucosamine 6-phosphate differentiated glutamine synthetase 1 from glutamine synthetase 2. Glutamine synthetase R was also a labile protein but its kinetic and regulatory properties were quite similar to those of glutamine synthetase 1. These results clearly demonstrate the existence of three isoforms of glutamine synthetase in rice, two of which are located in the leaves and the third in the roots. PMID- 16661491 TI - Sequence of Chloroplast Degreening in Calamondin Fruit as Influenced by Ethylene and AgNO(3). AB - C(2)H(4) disrupts the internal membranes of the chloroplast and induces an increase in chlorophyllase activity in degreening calamondin [x Citrofortunella mitis (Blanco) Ingram and Moore] fruit. Whether the loss of chlorophyll in the peel is causally related to breakdown of the chloroplast and/or chlorophyllase activity is not readily apparent. Chlorophyllase levels were inversely related to chlorophyll content, but electron micrographs also showed that internal membranes of the chloroplasts were disrupted simultaneously with the decrease in chlorophyll content. Silver, a potent inhibitor of C(2)H(4)-mediated effects, retarded the loss of chlorophyll in calamondin rind, reduced the C(2)H(4)-induced increase in chlorophyllase level, and prevented the disruption of the chloroplast membranes. The results do not permit the proposal of a mechanism of C(2)H(4) metabolism in the degreening of calamondin fruit. PMID- 16661492 TI - Effect of butyl 2-hydroxy-3-butynoate on sunflower leaf photosynthesis and photorespiration. AB - Detached leaves and whole plants of sunflower were supplied with butyl 2-hydroxy 3-butynoate (BHB), a competitive inactivator of glycolate oxidase, to evaluate the possibility of inhibiting photorespiration and increasing photosynthetic efficiency. In all treatments in vivo and in vitro, BHB inhibited glycolate oxidase. With partially purified glycolate oxidase from spinach leaves, the apparent K(i) for BHB was 13.2 micromolar.Low concentrations of BHB neither decreased photorespiration nor increased net photosynthesis. At higher concentrations, either a proportional decrease in photosynthesis and photorespiration or an inhibition of net photosynthesis greater than photorespiration was observed. CO(2) evolution in BHB-treated leaves was O(2) sensitive and was derived from recent photosynthate. BHB inhibited photosynthesis in 2, 21, or 50% O(2) but the ratio of the rates of photosynthesis in these O(2) concentrations was the same as in control leaves. BHB treatment resulted in a stimulation of dark respiration.As photosynthesis, photorespiration, and dark respiration were all affected by BHB, the action of BHB on whole leaf metabolism appears to be complex. Substantial inhibition of photorespiration was accompanied by inhibition of photosynthesis and increases in photosynthesis were not observed. PMID- 16661493 TI - A Nitrate Reductase-less Variant Isolated from Suspension Cultures of Datura innoxia (Mill.). AB - A comparative study has been carried out of the growth of two lines of Datura innoxia (Mill.) cells, designated DI-6 and NR1, their resistance to chlorate, and their ability to assimilate nitrate in sterile culture. The NR1 cell line was isolated from DI-6 cultures by first growing the latter in a nitrate-based medium for 5 days and then transferring the cells to a medium containing 2 grams liter( 1) of casein hydrolysate as the sole N source and 49 millimolar KClO(3) for a 6 week incubation period. Cells which survived the chlorate treatment then were transferred to casein hydrolysate medium and have been cultured in the absence of chlorate for more than 18 months (NR1).DI-6 cells can grow in a nitrate-based medium, whereas NR1 cells can take up nitrate but cannot use it as a N source. The inability of NR1 to assimilate nitrate appears to be due to the lack of an active nitrate reductase in these cells. Through the use of a variety of electron donors and acceptors, the lack of nitrate reductase activity in NR1 cells was shown to be due to the absence of, or a defect in, that component of the enzyme which mediates the reduction of nitrate to nitrite.In other experiments, DI-6 and NR1 were grown on a solid medium containing casein hydrolysate (2 grams liter( 1)) as the sole N source. Under these culture conditions, neither cell line contained an active nitrate reductase. The growth on this medium was compared to that on the same medium containing chlorate at concentrations from 0 to 100 millimolar. DI-6 culture growth was inhibited by 70% at a chlorate concentration of 30 micromolar, whereas growth of NR1 was stimulated by more than 60% on the same medium and by 100% at a chlorate concentration of 30 millimolar. In the presence of 100 millimolar chlorate, the growth of both cell lines was completely inhibited. This clear difference between the response of DI-6 and NR1 cells to chlorate even in the absence of nitrate lends support to the observations by others that chlorate inhibits cells by a mechanism other than, or in addition to, its nitrate reductase-catalyzed conversion to chlorite.Nitrite reductase was induced by nitrate in NR1 cells as well as in DI-6. This observation is a further confirmation of the fact that nitrate, not nitrite, is the true inducer of the nitrate assimilatory pathway in higher plants. PMID- 16661494 TI - Development of Mitochondrial Activities in Pea Cotyledons: INFLUENCE OF DESICCATION DURING AND FOLLOWING GERMINATION OF THE AXIS. AB - Mitochondria in 4-hour imbibed and desiccated pea cotyledons develop in a similar manner upon rehydration to those in cotyledons hydrated only once. As a consequence of desiccation during imbibition, mitochondria revert to their original state as in the mature dry cotyledon, although limited damage occurs. This damage is more evident when the initial imbibition time before desiccation is longer. The presence of the axis does not protect cotyledonary mitochondria from damage, nor does it influence mitochondrial development upon rehydration of desiccated cotyledons. During the early hours after the start of imbibition mitochondrial development is dependent both upon hydration levels of the cotyledon and upon other metabolic processes, as shown by sensitivity to conditions that prevent ATP synthesis. PMID- 16661495 TI - Long Chain (C(20) and C(22)) Fatty Acid Biosynthesis in Developing Seeds of Tropaeolum majus: AN IN VIVO STUDY. AB - The storage triacylglycerols of nasturtium (Tropaeolum majus) seeds are composed principally of cis-11-eicosenoate and cis-13-docosenoate. To investigate the biosynthesis of these C(20) and C(22) fatty acids, developing seed tissue was incubated with various (14)C-labeled precursors. Incubation with [1-(14)C]acetate produced primarily cis-11-[1-(14)C]eicosenoate and cis-13-[1,3-(14)C]docosenoate in the triacylglycerol fraction, the odd-carbon [U-(14)C]oleate also formed from [(14)C] acetate was in the polar lipid fraction. Kinetic data showed that this oleate was not channeled into cis-11-eicosenoate nor cis-13-docosenoate over a 24 hour period. Under suitable conditions, nasturtium seed could also produce [(14)C]stearate, [(14)C]eicosenoate, and [(14)C]docosenoate from [1 (14)C]acetate. The results are discussed in terms of the number of pathways producing fatty acids. From pool size and other considerations, the results can be rationalized only in terms of different de novo systems for oleate biosythesis, one supplying oleate for incorporation into phospholipids and the other supplying oleate for chain elongation and subsequent esterification into triacylglycerols. Because of the probable heterogeneous nature of the seed tissue, it is not known if these two systems are operating in different cell types, in the same cell type at different stages of development, or in the same cell type concurrently. PMID- 16661496 TI - Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION. AB - The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C(20) and C(22) fatty acids, which contain an unusual Delta5 double bond. When [1-(14)C]acetate was incubated with developing seed slices, (14)C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13 docosenoate, and cis-5,cis-13-docosadienoate. The label from [(14)C]acetate and [(14)C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[(14)C]11-eicosenoate, cis-13-[(14)C]docosenoate, and cis-5,cis-13 [(14)C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C(20) and C(22) acyl species. The Delta5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-(14)C-labeled free fatty acids. Using [1-(14)C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate. PMID- 16661497 TI - Effect of Glyphosate on Intact Bean Plants (Phaseolus vulgaris L.) and Isolated Cells. AB - Whole bean (var. "Eastern Butterwax") plants and isolated cells were used to investigate possible mechanisms of action of glyphosate [N (phosphonomethyl)glycine]. Results showed that glyphosate was quickly absorbed by the whole plant but not by individual cells and that it caused a rapid reduction in leaf dry matter accumulation, leaf expansion, leaf angle, and stomatal aperture without affecting the water status of the plant. Glyphosate also caused a rapid reduction in cellular uptake of (86)Rb and (32)P which preceded its detrimental effects on photosynthesis, RNA and protein synthesis, and respiration of isolated cells. This reduction in ion absorption was not due to a loss of membrane integrity, decrease in energy supply or chelation of ions. It was concluded that glyphosate was directly inhibiting the ion absorption process of bean leaf cells. PMID- 16661498 TI - Polyunsaturated Fatty Acid Biosynthesis in Cotyledons from Germinating and Developing Cucumis sativus L. Seedlings. AB - Etiolated Cucumis sativus L. cotyledons preferentially catabolized exogenous [1 (14)C]oleic acid and [1-(14)C]linoleic acid with relatively little incorporation into complex lipids or desaturation of the (14)C-labeled fatty acids. Following a 16-hour exposure to light, the greening cotyledons efficiently desaturated the exogenous (14)C-labeled fatty acids. A small amount of oleate desaturation to linoleate was observed in etiolated tissue, but hardly any linoleate desaturation to alpha-linolenate was detected. Both oleate and linoleate desaturation showed diurnal variations with maxima at the end of light periods and minima at the end of dark periods. Illumination of etiolated tissue by flashing light, as opposed to continuous light, failed to stimulate either chlorophyll or alpha-linolenic acid biosynthesis, and both processes could be halted or reversed by 10 micrograms per milliliter cycloheximide. Production of polyunsaturated fatty acids from [1-(14)C]acetate, [1-(14)C]oleic acid, and [1-(14)C]linoleic acid, by greening cucumber cotyledons, was markedly affected by tissue integrity with finely chopped cotyledons having very little capacity for their synthesis and intact seedlings showing the highest rates. PMID- 16661499 TI - In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings. AB - Exogenous [1-(14)C]oleic acid and [1-(14)C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both (14)C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [(14)C] oleate and [1-(14)C]linoleate esterification and of [1-(14)C]oleate desaturation to [1-(14)C]linoleate. [1-(14)C]Linoleic acid was esterified more rapidly than [(14)C]oleic acid and its desaturation product, [1-(14)C]alpha-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine. PMID- 16661500 TI - Changes in the Levels of Abscisic Acid and Its Metabolites in Excised Leaf Blades of Xanthium strumarium during and after Water Stress. AB - The time course of abscisic acid (ABA) accumulation during water stress and of degradation following rehydration was investigated by analyzing the levels of ABA and its metabolites phaseic acid (PA) and alkalihydrolyzable conjugated ABA in excised leaf blades of Xanthium strumarium. Initial purification was by reverse phase, preparative, high performance liquid chromatography (HPLC) which did not require prior partitioning. ABA and PA were purified further by analytical HPLC with a muBondapak-NH(2) column, and quantified by GLC with an electron capture detector.The ABA content of stressed leaves increased for 4 to 5 hours and then leveled off due to a balance between synthesis and degradation. Since PA accumulated at a constant rate throughout the wilting period, it was concluded that the rate of ABA synthesis decreased after the first 4 to 5 hours stress. Conjugated ABA increased at a low rate during stress. This is interpreted to indicate that free ABA was converted to the conjugated form, rather than the reverse.Following rehydration of wilted leaves, the ABA level immediately ceased increasing; it remained constant for 1 hour and then declined rapidly to the prestress level over a 2- to 3-hour period with a concomitant rise in the PA level. In contrast to the rapid disappearance of ABA after relief of stress, the high PA content of rehydrated leaves declined only slowly. The level of conjugated ABA did not change following rehydration, indicating that conjugation of ABA was irreversible.Detached Xanthium leaves that were subjected to a wilting recovery-rewilting cycle in darkness, responded to the second wilting period by formation of the same amount of ABA as accumulated after the first stress period. PMID- 16661501 TI - Comparison of the levels of six endogenous gibberellins in roots and shoots of spinach in relation to photoperiod. AB - This communication describes the distribution of gibberellins (GAs) in roots and shoots of spinach in relation to photoperiod. From previous work (Metzger, Zeevaart 1980 Plant Physiol 65: 623-626) shoots were known to contain GA(53), GA(44), GA(19), GA(17), GA(20), and GA(29). We now show by combined gas chromatography-mass spectrometry that roots contain GA(44), GA(19), and GA(29). Trace amounts of GA(53) were detected by combined gas chromatography-selected ion current monitoring. Neither GA(17) nor GA(20) were detected in root extracts. Analysis by the d-5 corn bioassay also showed no effect of photoperiodic treatment on the levels of GA-like substances in root extracts. Both phloem and xylem exudates had patterns of GA-like activity similar to those found in shoots and roots, respectively. Moreover, foliar application of [(3)H]GA(20) resulted in the transport of label from the shoot to the roots. Over half of the label in the roots represented unmetabolized [(3)H]GA(20), indicating that part of the GA(20) in the phloem is transported to the roots. Consequently, if GA(20) is made in, or transported to the roots, it is rapidly metabolized in that organ. This is a clear indication that regulation of GA metabolism is greatly different in roots and shoots. PMID- 16661502 TI - Isolation of tonoplast vesicles from tobacco protoplasts. AB - Vacuoles were isolated from protoplasts of Nicotiana glutinosa by the method of Mettler and Leonard (Plant Physiol 1979 64: 1114-1120) with minor modifications so that the number of intact protoplasts contaminating the vacuole preparation was reduced to less than 1% (by number). Isopycnic centrifugation of a [(3)H]choline-labeled, sonicated vacuole preparation on linear 5 to 40% sucrose gradients indicated that tonoplast vesicles equilibrated at a density of about 1.12 grams per cubic centimeter. When tonoplast vesicles were isolated on discontinuous sucrose density gradients substrate specific ATPase activity was not found to be associated with this membrane fraction. These results are discussed in terms of the energetics of ion transport through the tonoplast membrane. PMID- 16661503 TI - Biochemical characterization of a catalase inhibitor from maize. AB - Some biochemical properties of the catalase inhibitor purified from maize scutella are described. The inhibitor is heat-labile and its activity is destroyed by trypsin, indicating that it is a protein. It does not appear to be a lectin nor does the inhibition involve proteolysis. The active inhibitor is a dimer with each subunit having a molecular weight of 5600 as determined by sodium dodecyl sulfate electrophoresis. A kinetic analysis performed in the presence of increasing levels of inhibitor gave unusual Lineweaver-Burk patterns. Possible explanations for these patterns are discussed. The inhibitor is active against all catalases tested from a wide variety of organisms. PMID- 16661504 TI - Effects of Boron Deficiency on Rubidium Uptake and Photosynthesis in the Diatom Cylindrotheca fusiformis. AB - Culturing the diatom Cylindrotheca fusiformis under boron-deficient conditions leads to changes in (86)Rb uptake and photosynthesis prior to any effect on the rate of cell division. The influx rate of (86)Rb into boron-deficient cells was 79% of the control rate after 5 to 5.5 hours culture. Despite lowered (86)Rb influx, however, boron-deficient diatoms accumulated more (86)Rb than did control cells; this was due to the deficient cells' lower efflux rate. After 24 hours culture, boron-deficient cells had accumulated 30% more (86)Rb than had control cells, while releasing (86)Rb at only one-half the control rate. Increased photosynthetic rates were another effect of boron deficiency during this early stage of culture. Prior to 20 hours boron-deficient culture, diatoms had photosynthetic rates 37% greater than those of control cells. Corresponding to the increase in photosynthesis, boron-deficient diatoms had 12% more carbohydrate than control cells after 16 hours culture. PMID- 16661505 TI - Further characterization of the in vitro binding of phytochrome to a membrane fraction enriched for mitochondria. AB - This study employs (125)I-labeled phytochrome ((125)I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. The binding of (125)I-Pfr to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of (125)I-Pr. However, (125)I-Pfr and (125)I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum (125)I-Pfr binding to membranes from light-exposed plants occurs within 30 seconds and is optimized in a reaction buffer containing 5 millimolar MgCl(2) at pH 6.8. Scatchard plots of the binding data for Pfr indicate a single high-affinity site with an affinity constant of 1.79 x 10(11) per molar. When optimal binding conditions are used, over 20% of the (125)I-P added is bound and a stoichiometry of about 100 molecules per mitochondrion is attained. When the specificity of binding is tested using competition experiments with a 15-fold excess of unlabeled phytochrome, (125)I-Pfr shows no specific binding to rat liver mitochondria. PMID- 16661506 TI - Modulation of a mitochondrial function by oat phytochrome in vitro. AB - Previous data in the literature have indicated that phytochrome could alter the rate of reduction of exogenously added NADP by a pea mitochondrial preparation in vitro. These results could not be duplicated using a mitochondrial preparation isolated from etiolated oat seedlings. Further experimentation demonstrated that the addition of Pr to the preparation, in combination with a far red light illumination, could significantly reduce the rate of oxidation of NADH by the external dehydrogenases of oat mitochondria. This response was characterized by a 15% decrease in reaction velocity at saturating substrate concentrations and a 2 fold increase in apparent K(m) as compared to values obtained after Pfr plus red light treatment. The response was photoreversible, the rate of oxidation of exogenous NADH being determined by the last light illumination given to the mitochondrial preparation. The interaction between phytochrome and the mitochondria was apparently occurring at the level of the inner mitochondrial membrane. A requirement for these results was that the mitochondria be isolated from plants that were illuminated with white or red light before extraction; mitochondria from unirradiated plants showed no dehydrogenase response to treatments with phytochrome plus actinic light. PMID- 16661507 TI - On the Gaseous Exchange of Ammonia between Leaves and the Environment: Determination of the Ammonia Compensation Point. AB - Whole shoots of Phaseolus vulgaris L. and other species were exposed to a range of partial pressures of gaseous ammonia in air and the resulting fluxes were measured. Net uptake is linear with partial pressure in the range 5 to 50 nanobars and is zero at a finite partial pressure, termed the ammonia compensation point. Below the compensation point, ammonia (or possibly other volatile amines) is evolved by the leaves. The compensation points in several species are near the low partial pressures found in unpolluted air and approximate to the K(m) of glutamine synthetase in vitro. In P. vulgaris L., the compensation point increases with temperature. PMID- 16661508 TI - Tocopherol and Organic Free Radical Levels in Soybean Seeds during Natural and Accelerated Aging. AB - Soybean seeds which had aged in long-term storage ("natural aging") or by exposure to high temperature and humidity ("accelerated aging") were analyzed for their tocopherol and organic free radical contents. Tocopherol levels remained unchanged during both types of aging. Three principal tocopherol homologues (alpha, gamma, delta) were present in fairly constant proportions throughout. Organic free radical levels were also remarkably stable, presumably due to the relatively immobile environment of the dry seed. These results, taken in conjunction with previous data on the stability of unsaturated fatty acids in soybean seeds, indicate that it is improbable that lipid peroxidation need play a significant role during natural or accelerated aging in this species. PMID- 16661509 TI - Evaluation of the Relative Ureide Content of Xylem Sap as an Indicator of N(2) Fixation in Soybeans: GREENHOUSE STUDIES. AB - The use of the relative ureide content of xylem sap [(ureide-N/total N) x 100] as an indicator of N(2) fixation in soybeans (Merr.) was examined under greenhouse conditions. Acetylene treatments to inhibit N(2) fixation were imposed upon the root systems of plants totally dependent upon N(2) fixation as their source of N and of plants dependent upon both N(2) fixation and uptake of exogenous nitrate. Significant decreases in the total N concentration of xylem sap from plants of the former type were observed, but no significant decrease was observed in the total N concentration of sap from the latter type of plants. In both types of plants, acetylene treatment caused significant decreases in the relative ureide content of xylem sap. The results provided further support for a link between the presence of ureides in the xylem and the occurrence of N(2) fixation in soybeans. The relative ureide content of xylem sap from plants totally dependent upon N(2) fixation was shown to be insensitive to changes in the exudation rate and total N concentration of xylem sap brought about by diurnal changes in environmental factors. There was little evidence of soybean cultivars or nodulating strains affecting the relative ureide content of xylem sap. ;Ransom' soybeans nodulated with Rhizobium japonicum strain USDA 110 were grown under conditions to obtain plants exhibiting a wide range of dependency upon N(2) fixation. The relative ureide content of xylem sap was shown to indicate reliably the N(2) fixation of these plants during vegetative growth using a (15)N method to measure N(2) fixation activity. The use of the relative ureide content of xylem sap for quantification of N(2) fixation in soybeans should be evaluated further. PMID- 16661510 TI - Replication of DNA by nuclei isolated from soybean suspension cultures. AB - DNA replication was studied in nuclei isolated from soybean cells grown in suspension culture. The isolation procedure involved the preparation of protoplasts, their lysis with a nonionic detergent and purification of nuclei. These nuclei synthesized low molecular weight DNA and joined these fragments into DNA of intermediate molecular weight. The characteristics of replication in isolated nuclei correlated well with those of the cells from which they were isolated, as shown by fluorodeoxyuridine synchronization and ultraviolet irradiation experiments. PMID- 16661511 TI - Characterization of alpha-Galactosidase from Cucumber Leaves. AB - Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000 dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively. PMID- 16661512 TI - Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.]. AB - The synthesis of (14)C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-(14)C]guanine or [8-(14)C]hypoxanthine, but not from [8-(14)C]adenine, was demonstrated in a cell-free extract from N(2)-fixing nodules of cowpea (Walp.). The (14)C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the (14)C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed. PMID- 16661513 TI - Metabolism of myo-[2-H]Inositol and scyllo-[R-H]Inositol in Ripening Wheat Kernels. AB - Injection of myo-[2-(3)H]inositol or scyllo-[R-(3)H]inositol into the peduncular cavity of wheat stalks about 2 to 4 weeks postanthesis led to rapid translocation into the spike and accumulation of label in developing kernels, especially the bran fraction. With myo-[2-(3)H]inositol, about 50 to 60% of the label was incorporated into high molecular weight cell wall substance in the region of the injection. That portion translocated to the kernels was utilized primarily for cell wall polysaccharide formation and phytate biosynthesis. A small amount was recovered as free myo-inositol and galactinol. When scyllo-[R-(3)H]inositol was supplied, most of the label was translocated into the developing kernels where it accumulated as free scyllo-inositol and O-alpha-d-galactopyranosyl-scyllo inositol in approximately equal amount. None of the label from scyllo-[R (3)H]inositol was utilized for either phytate biosynthesis or cell wall polysaccharide formation. PMID- 16661514 TI - Increase in Dry Weight and Total Nitrogen Content in Zea mays and Setaria italica Associated with Nitrogen-fixing Azospirillum spp. AB - The association between nitrogen-fixing bacteria from the genus Azospirillum and the grasses Zea mays and Setaria italica was investigated in sterilized Leonard jar assemblies. Nitrogen-fixing bacteria isolated from Cynodon dactylon roots in Israel and Azospirillum brasilense (Sp-7, Sp-80, and Cd) were examined. C(2)H(2) reduction activity was detected in systems containing 0.0 to 0.08 but not in those containing 0.16 gram per liter NH(4)NO(3). The organisms tested significantly increased plant dry weight (50-100%), total N content of leaves (50 100%) and C(2)H(4) production (300-1000 nanomoles C(2)H(4) per plant per hour). Highest C(2)H(2) reduction activities were obtained above 30 C and with high light intensities. Significant increases in S. italica dry weight (DW) and nitrogen (N) content were observed in sand (DW = 80%, N = 150%), sandy loam soil (DW = 80%, N = 75%) and loess (DW = 37%, N = 25%). The results obtained in this work clearly demonstrate the potential benefit of inoculating grasses with Azospirillum. PMID- 16661515 TI - Abscission of Citrus Leaf Explants: INTERRELATIONSHIPS OF ABSCISIC ACID, ETHYLENE, AND HYDROLYTIC ENZYMES. AB - The question whether abscisic acid (ABA) induces cellulase and polygalacturonase activity and, hence, abscission directly or whether its action is mediated by C(2)H(4) was studied in citrus (Osbeck var. Shamouti) leaf explants using aminoethoxyvinyl glycine (AVG), an inhibitor of C(2)H(4) biosynthesis. ABA in concentrations of 10 micromolar and higher induced C(2)H(4) production and accelerated abscission. AVG inhibited C(2)H(4) formation, activity of cellulase and polygalacturonase, and abscission in ABA-treated explants. AVG did not inhibit the increase in the activity of the cell-wall degrading enzymes or abscission in a saturating level of externally supplied C(2)H(4). This indicates that the effect of AVG resulted from inhibition of the formation of endogenous ethylene. The data indicate that in citrus leaf explants the induction of the activity of cellulase and polygalacturonase and abscission by ABA is mediated by C(2)H(4). PMID- 16661516 TI - Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: II. MECHANISM OF SALT-INDUCED INCREASE IN OXALOACETATE PHOTOREDUCTION. AB - The main focus of this study was to determine the mechanism by which certain exogenous monovalent salts stimulate rates of net O(2) evolution linked to oxaloacetate reduction in intact spinach chloroplasts. The influence of salts on the dicarboxylate translocator involved in the transport of oxaloacetate and on the activity and activation of the chloroplast enzyme NADP-malate dehydrogenase, which mediates electron transport to oxaloacetate, was examined. High concentrations of KCl (155 millimolar) increased the apparent K(m) for oxaloacetate but did not significantly alter the maximal velocity of uptake. Likewise, external salts (KCl, MgCl(2), or KH(2)PO(4)) had minimal effects on the magnitude of light activation of NADP-malate dehydrogenase. In contrast, measurements of chloroplast NADP-malate dehydrogenase activity (after release by osmotic shock) showed a marked dependence on salt concentration. Rates were stimulated approximately 2-fold by both monovalent (optimally 75 millimolar) and divalent (optimally 20 millimolar) salts. It was inferred that the salt-induced increase in net rates of O(2) evolution linked to oxaloacetate reduction is due, at least in part, to stimulation of NADP-malate dehydrogenase caused by monovalent cation permeability of the chloroplast inner envelope membrane. PMID- 16661517 TI - Identification of Selenocysteine in the Proteins of Selenate-grown Vigna radiata. AB - Selenocysteine, the selenium analog of cysteine, was identified in proteins of Vigna radiata (L.) Wilczak grown with selenate. To stabilize selenocysteine and prevent its breakdown, the carboxymethyl derivative was synthesized by the addition of iodoacetic acid to the protein extract from [(75)Se]selenate-grown plants. A (75)Se-labeled component of the carboxymethylated protein hydrolysate possessed chromatographic properties identical to those of a (14)C-labeled carboxymethylselenocysteine standard during paper and thin layer chromatography and during gel-exclusion, anion-exchange, and cation-exchange column chromatography. Detection of selenocysteine in proteins of a selenium-sensitive plant, and the possibility that the presence of this compound alters normal functions, provides an explanation for the toxic effects of selenium. PMID- 16661518 TI - Interaction between External and Internal Conditions in the Development of Photosynthetic Features in a Grass Leaf: I. REGIONAL RESPONSES ALONG A LEAF DURING AND AFTER LOW-LIGHT OR HIGH-LIGHT ACCLIMATION. AB - Morphological and functional features were compared along a developing third leaf and fully expanded leaf from high-light- and low-light-acclimated seedlings of Lolium multiflorum.The young leaf contains a gradient of differentiating tissue, ranging from meristematic cells at the leaf base to mature tissue at the tip; this gradient can be related to the maturation of a functional photosynthetic apparatus. Along the fully expanded leaf, a decreasing gradient from tip to base is maintained for functional characteristics (net maximum photosynthesis, chlorophyll content, and ribulose bisphosphate carboxylase activity) and for a number of structural parameters (number of mesophyll cells and their external surface area, number of chloroplasts and their envelope area), irrespective of the light regime. In contrast, a constancy in the absolute intrachloroplastic lamellar content per plastid was revealed whatever the position in the leaf or irradiance received. However, the relative membrane content was lower in high light chloroplasts due to their larger volume compared to low-light plastids (dilution effect).The longitudinal differences in functional and morphological characteristics are interpreted as the result of interaction between the internal gradient of differentiating tissue along a developing young leaf and the external light conditions during development. PMID- 16661519 TI - Interaction between External and Internal Conditions in the Development of Photosynthetic Features in a Grass Leaf: II. REVERSIBILITY OF LIGHT-INDUCED RESPONSES AS A FUNCTION OF DEVELOPMENTAL STAGES. AB - Lolium multiflorum plants were grown under a low- or high-light regime until third leaves had emerged to one-third their final length and then were transferred to a contrasting light regime. At this stage, the leaf possesses a tissue-age gradient from tip to base; thus, the reversibility of light-acclimated responses as a function of the degree of differentiation was analyzed in individual leaves.Regional responses in the apical, medial, and basal zones of leaves of transferred plants were analyzed before and after light transfer and compared to leaves from plants kept in constant light. Leaves transferred from low to high light showed rapid recovery and attained light-saturated rates and ribulose bisphosphate carboxylase activities equivalent to those of high-light controls. However, fresh or dry weight and chlorophyll content were intermediate between those for the two irradiances. In the reciprocal experiment, the apical leaf zone retained high-light characteristics for maximum photosynthesis, whereas all the other functional parameters adapted rapidly to values characteristic of their low-light counterparts (equivalent foliar zones).On the ultrastructural level, chloroplasts in the apical zone of transferred leaves surpassed their respective constant light controls in absolute membrane content and size. However, a shift to high light induced an increase in plastid volume and, in relative terms, the plastid membrane content was diluted. A shift to low-light treatment led to smaller membrane-dense plastids. The ultrastructural readaptation is realized through differential rates of increase in plastid volume and lamellar content. Proplastids (leaf base), or juvenile plastids having reached an intermediate developmental stage (leaf middle zone) at the time of transfer, took on characteristics of the latter light regime (equivalent to controls).These results provide evidence for rapid reacclimation processes under changing light regimes and suggest a capacity for regional light responses along the leaf. PMID- 16661520 TI - Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectrometric Assays. AB - Syntheses are described for tetra and pentadeutero indole-3-acetic acid (IAA) labeled in positions 4, 5, 6, 7 or 2, 4, 5, 6, 7 of the indole moiety. Polydeuterated IAA is proposed as an internal standard for gas chromatographic mass spectrometric analysis of IAA by selected ion monitoring. Nanogram amounts of IAA may be assayed by monitoring the base peak of IAA at m/z = 130 (134 for d(4)-IAA) and the molecular ion of the methyl ester of IAA at 189 (193 for d(4) IAA). Deuterium in positions 4, 5, 6, and 7 and, to only a slightly lesser extent, that in position 2 of IAA is retained during alkali treatment, thus permitting use of these compounds as internal standards for assay of IAA released by alkaline hydrolysis of ester and amide conjugates. The use of polydeutero internal standards separates the standards from the "isotope cluster" caused by the normal abundance of heavy isotopes and also permits use of reduced mass resolution, thus leading to a 10-fold increase in sensitivity.Tetradeutero IAA was used as an internal standard for determining free plus ester IAA in alkaline hydrolysates of Zea mays, and showed exact agreement between estimates based on the molecular ion of the methyl ester and those based upon base peak. Application of the method to measuring free IAA in the upper and lower halves of geotropically stimulated Zea shoots showed 61 +/- 4% of the free IAA to be on the lower side. PMID- 16661521 TI - Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds. AB - In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: alpha-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.CHANGES IN THE ACTIVITIES OF ALANINE: alpha-ketoglutarate aminotransferase (EC 2.6.1.2), glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.13) have also been measured, in cotyledons as well as seedcoats. On a fresh weight basis, the highest activities of asparaginase and both aminotransferases developed in the seedcoats, whereas the highest activities of the remaining enzymes developed in the cotyledons.The data indicate that the amide groups of imported asparagine and glutamine are metabolized differently, largely by asparaginase and glutamate synthase, respectively. The NH(4) (+) released by the action of asparaginase is evidently reassimilated in cotyledon cells by the joint action of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. The data emphasize the central importance of alpha-ketoglutarate-glutamate cycling in the redistribution of amino groups associated with the net synthesis of amino acids and reserve proteins. PMID- 16661522 TI - Characteristics and subcellular localization of phospholipase d and phosphatidic Acid phosphatase in mung bean cotyledons. AB - Mixed micelles of (32)P-labeled phosphatidylcholine or phosphatidic acid (PA) and the nonionic detergent octylphenol polyethylene oxide (NP-40 Nonidet) were used to assay the activities of phospholipase D and PA phosphatase in crude extracts of mung bean (Vigna radiata) cotyledons. Together these enzymes degrade phosphatidylcholine to free choline, inorganic phosphate, and sn-1,2 diacylglycerol. Both enzymes have pH optima around 5.0. The enzymes are present in fully imbibed cotyledons and increase in activity during seedling growth. Fractionation of cotyledon extracts on sucrose gradients showed that the cells contain two PA phosphatases. One enzyme with a pH optimum of 7.5 has the same distribution on sucrose gradient as the endoplasmic reticulum marker enzyme NADH cytochrome c reductase. The other, PA phosphatase, with a pH optimum of 5.0, was present in a protein body-rich fraction and in the load portion of the gradient. Fractionation of broken protoplasts on Ficoll gradients (a method which allows for the isolation of a high proportion of intact protein bodies) indicates that most of the cellular phospholipase D and PA phosphatase (pH 5.0) are associated with the protein bodies. Using column chromatography (DEAE-cellulose and Sephadex G-200), PA phosphatase (pH 5.0) was found to be a different enzyme from the major acid phosphatase in the cotyledons. Apparent molecular weights of phospholipase D and PA phosphatase were 150,000 and 37,000, respectively. The activity of phospholipase D was not affected by free choline, but was markedly inhibited by the choline analog and plant growth retardant isopropyl 4'-(trimethylammonium chloride-5'-methylphenyl piperidine-1-carboxylate (AMO 1618). The finding that these acid hydrolases are located in the protein bodies supports the conclusion that protein bodies form the general lytic compartment in the storage parenchyma cells. PMID- 16661523 TI - Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum. AB - The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Delta(5) to Delta(7) or to Delta(5,7). Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24alpha-ethyl = trans-Delta(22)-24alpha-ethyl > trans-Delta(22)-24beta-ethyl = 24alpha-E ethylidene = 24alpha-methyl > 24beta-methyl = trans-Delta(22)-24beta-methyl = 26 methyl = saturated C(7) side chain and C-20 R (17-alphaH, 20-alphaH, right-handed conformer) = cis-Delta(22)-C(7) side chain and C-20 R > saturated C(7) side chain and C-20 S (17-alphaH, 20-betaH, right-handed conformer) > no sterol = 29 hydroxyporiferasterol = 20alpha-hydroxycholesterol = 24xi-hydroxy-24 vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20alpha-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter. PMID- 16661524 TI - Transfer RNA-Peroxidase Interaction: INHIBITION OF INDOLE-3-ACETIC ACID OXIDATION. AB - Transfer RNA from wheat germ, yeast, and Escherichia coli inhibited the indoleacetic acid (IAA)-induced spectral change in horseradish peroxidase (EC 1.11.1.7) and the decarboxylation of IAA. The inhibition was limited to a delay after which the increase in A(427) and the decarboxylation of IAA resumed at the same rate as in the control; the duration of the inhibition was dependent on, but not proportional to, the concentration of tRNA. Alkaline hydrolysis destroyed the inhibitory activity of tRNA. The inhibition was completely abolished when the tRNA was added 30 seconds after IAA. Thus, the tRNA appears not to react with the enzyme intermediates formed during the reaction with IAA. The inhibition by tRNA was rapidly reversed by H(2)O(2) or additional IAA, but not by 2,4 dichlorophenol. Results suggest that the tRNA interferes with the initial reaction between IAA and the heme moiety of free peroxidase, thus preventing the formation of highly active enzyme intermediates essential for IAA degradation. PMID- 16661525 TI - Effects of pH and Ethephon on Betacyanin Leakage from Beet Root Discs. AB - Betacyanin leakage from beet root discs was found to increase with decreasing pH of the incubation medium. Although 10 millimolar Ca(2+) reduced pigment leakage at pH 3.5, it was ineffective at pH 2.3. Leakage was also stimulated by 100 micrograms per milliliter (2-chloroethyl)-phosphonic acid (pH 3.1), but when the solution was neutralized, this leakage was eliminated. Bubbling C(2)H(4) through a neutral medium containing beet discs had no effect on pigment leakage; it appears that the effect of Ethephon solutions on this process is a function of their low pH. PMID- 16661526 TI - Purification and Characterization of a Salt-extractable Hydroxyproline-rich Glycoprotein from Aerated Carrot Discs. AB - The salt-extractable hydroxyproline-rich glycoprotein (HRGP) of the cell wall of aerated carrot root discs has been studied by polyacrylamide gel electrophoresis. The predominant proline-labeled protein extractable from the cell wall is rich in hydroxyproline as shown by its specific loss of (3)H from proline labeled in position 4 and its shift in electrophoretic mobility after labeling in the presence of an inhibitor of hydroxyproline synthesis. Unlabeled HRGP can be identified by staining gels for carbohydrate. The HRGP has been purified by ion exchange chromatography and CsCl gradient centrifugation. The HRGP consists of about 50% protein and 50% carbohydrate with an overall molecular weight of 86,000. The amino acid composition of the protein portion consists of 50% hydroxyproline, 19% basic amino acids, 12% serine, and 10% tyrosine. This glycoprotein accumulates in a salt-extractable pool in the cell wall beginning between 10 and 20 hours of aeration and may also become incorporated into the nonextractable portion of the cell wall. PMID- 16661527 TI - Changes of Endogenous Gibberellin-like Substances with Sex Reversal of the Apical Inflorescence of Corn. AB - In developing apical meristems of corn, the level of acidic, ethyl acetate soluble gibberellin (GA)-like substances increased to a maximum of 108 micrograms GA(3)-equivalents per kilogram dry weight of tissue at inflorescence initiation, and then fell rapidly. At anthesis, only a trace (0.2 microgram per kilogram) of GA-like activity remained in the apical (male) inflorescences, whereas moderate activity (32 micrograms per kilogram), mostly of a nonpolar nature, was present in lateral, female, inflorescences.A sex reversal of the apical inflorescence, from male to female, was elicited by reducing the ambient light intensity. Higher levels of GA-like substances, particularly those eluting from a SiO(2) partition column in the nonpolar region, were observed at all harvests in the reverting meristems; levels increased to 180 micrograms per kilogram at inflorescence initiation, then dropped to 122 micrograms per kilogram in the apical (female), reverted meristems. This increase in endogenous GA-like activity with reversion to the female inflorescence is consistent with observations that (a) reversion can be obtained with exogenous application of GA(3) and (b) maleness is enhanced in GA-deficient mutants of maize. Endogenous GAs may thus play a key role in the control of sexuality of corn. PMID- 16661528 TI - Copper-mediated Lipid Peroxidation Processes in Photosynthetic Membranes. AB - The phytotoxic effect of Cu via the photosynthetic electron transport system was studied with isolated spinach chloroplasts. Cu(II) ions induce a light-driven peroxidation of membrane lipids leading to ethylene formation, the latter dominating over a concurrent ethane production. Seemingly, the hydroxyl radical originating from superoxide anion is the starting reactive O(2) species. Cu ions inhibit photosynthetic electron transport and apparently catalyze the formation of hydroxyl radical and Fenton-type reactions that result in destruction of unsaturated membrane fatty acids. The concept on the mode of action of Cu(II) and Cu(I) ions in lipid peroxidation as presented here suggests the influence of Cu on different reactions. Two sites are in the photosynthetic redox system; Cu participates in two Fenton-type reactions and in the conversion of ethyl radical to ethylene and ethane. PMID- 16661529 TI - Changes in Membrane Lipids of Roots Associated with Changes in Permeability: I. EFFECTS OF UNDISSOCIATED ORGANIC ACIDS. AB - Previous work has shown that undissociated forms of organic acids, such as formic, acetic, and propionic acids, increase the permeability of barley roots to ions. The work here was undertaken to test whether these undissociated acids affect the lipids from the root membranes in such a way as to account for the permeability increase. Relative amounts of the principal fatty acids from barley root membranes were measured as a function of organic acid concentration, pH, and time of treatment of barley roots under conditions similar to those of the previous studies.Undissociated formic, acetic, and propionic acids all rapidly increase the proportions of palmitic, stearic, and oleic acids and decrease proportions of linoleic and linolenic acids. Only the undissociated species are effective. The effects on the fatty acids from membrane lipids parallel effects on ion permeability. It is concluded that the increase in permeability produced by undissociated organic acid is due to changes in the lipids of barley root membranes. PMID- 16661530 TI - Regulation of Glutamine Synthetase by Light and during Nitrogen Deficiency in Synchronous Chlorella sorokiniana. AB - A method is described to achieve density labeling of proteins in unicellular algae by using (13)CO(2). This is a satisfactory procedure especially for work on nitrogen metabolism. The increase in activity of glutamine synthetase (EC 6.3.1.2.) and glutamate synthase (EC 1.4.7.1.) in Chlorella sorokiniana mediated by a dark/light shift and by nitrogen starvation were investigated. Using the method of density labeling and isopycnic centrifugation, we demonstrated that the increase in enzyme activity after a dark/light shift is based on activation rather than de novo synthesis. The increase in enzyme activity after transfer to nitrogen-deficient medium is based both on activation and de novo synthesis. PMID- 16661531 TI - Comparative resistance of the soil and the plant to water transport. AB - The resistances to liquid water transport in the soil and plant were determined directly and simultaneously from measurements of soil, root, and leaf water potentials and the flux of water through the soil-plant system to the sites of evaporation in the leaf. For soybean (Merr.) transporting water at a steady rate, water potential differences between soil and root were smaller than between root and leaf over the range of soil water potentials from -0.2 to -11 bars. As soil water was depleted, water flow through the soil and plant decreased to one-tenth the maximum rate, but both the soil resistance and plant resistance increased. The plant resistance remained larger than the soil resistance over the entire range of soil water availability. Previous suggestions that the soil is the major resistance have ignored the increase in plant resistance and/or assumed root densities that were too low. PMID- 16661532 TI - Inhibition by Catalase of Dark-mediated Glucose-6-Phosphate Dehydrogenase Activation in Pea Chloroplasts. AB - Dark activation of light-inactivated glucose-6-phosphate dehydrogenase was inhibited by catalase in a broken pea chloroplast system. Partially purified glucose-6-phosphate dehydrogenase from pea leaf chloroplasts can be inactivated in vitro by dithiothreitol and thioredoxin and reactivated by H(2)O(2). The in vitro activation by H(2)O(2) was not enhanced by horseradish peroxidase, and dark activation in the broken chloroplast system was only slightly inhibited by NaCN. These results indicate that the dark activation of glucose-6-phosphate dehydrogenase may involve oxidation by H(2)O(2) of SH groups on the enzyme which were reduced in the light by the light effect mediator system. PMID- 16661533 TI - Photosynthetic HCO(3) Utilization and OH Excretion in Aquatic Angiosperms: LIGHT INDUCED pH CHANGES AT THE LEAF SURFACE. AB - The utilization of HCO(3) (-) as carbon source for photosynthesis by aquatic angiosperms results in the production of 1 mole OH(-) for each mole CO(2) assimilated. The OH(-) ions are subsequently released to the medium. In several Potamogeton and Elodea species, the site of the HCO(3) (-) influx and OH(-) efflux are spatially separated. Described here are light- and dark-induced pH changes at the lower and upper epidermis of the leaves of Potamogeton lucens, Elodea densa, and Elodea canadensis.In the light, two phases could be discerned. During the first phase, the pH increased at both sides of the leaves. This pH increase apparently resulted from CO(2) fixation. During the second, so-called polar phase, the pH at the upper side increased further, but the pH at the lower side dropped below the pH of the ambient solution. The pH drop at the lower epidermis indicates that the K(+) influx exceeds the net CO(2) (HCO(3) (-) + CO(2)) influx slightly. This may result either from a proton pump driving an extra K(+) influx or from CO(2) diffusion from the cells into the outer medium previously taken up as HCO(3) (-). In the dark, a CO(2) gush was observed at both sides. During the polar phase, the upper side becomes electrically negative with respect to the lower side. Subsequent depolarization in the dark revealed that this potential difference consisted of a fast and a slow component. PMID- 16661534 TI - The Site of the Inhibition of the Shikimate Pathway by Glyphosate: I. INHIBITION BY GLYPHOSATE OF PHENYLPROPANOID SYNTHESIS IN BUCKWHEAT (FAGOPYRUM ESCULENTUM MOENCH) . AB - The nonselective herbicide glyphosate (n-[phosphonomethyl]glycine) inhibited the light-induced accumulation of phenylpropanoid substances (chlorogenic acid, procyanidin, rutin, anthocyanin) in etiolated buckwheat hypocotyls 90% at 1 millimolar. Structurally related compounds, such as n,n bis[phosphonomethyl]glycine, aminomethylphosphonate, methylglycine, and iminodiacetate, had little or no inhibiting effects. Of all amino acids tested, only l-phenylalanine reversed the inhibition, and partial reversal of anthocyanin synthesis was achieved with chorismate, phenylpyruvate, trans-cinnamate, p coumarate, and naringenin. Phenylalanine concentrations were reduced in glyphosate-treated hypocotyls, and glyphosate effectively reduced the high level of phenylalanine that was caused by the phenylalanine ammonia-lyase inhibitor l alpha-aminooxy-beta-phenylpropionate. Glyphosate had no significant effect on the time course of phenylalanine ammonia-lyase activity in hypocotyls incubated either in the dark or in the light. Under appropriate feeding conditions, glyphosate inhibited the incorporation of [(14)C]shikimate into all three aromatic amino acids, and radioactive shikimate accumulated in the tissue. The results lead to the conclusion that glyphosate interferes with the shikimate pathway at or prior to the formation of chorismate. PMID- 16661535 TI - The Site of the Inhibition of the Shikimate Pathway by Glyphosate: II. INTERFERENCE OF GLYPHOSATE WITH CHORISMATE FORMATION IN VIVO AND IN VITRO. AB - In the presence of the nonselective herbicide glyphosate (N [phosphonomethyl]glycine), buckwheat (Fagopyrum esculentum Moench) hypocotyls and cultured cells of Galium mollugo L. accumulate an organic acid, which was identified as shikimate by mass-spectroscopy of its methyl ester. After growth in 0.5 millimolar glyphosate for 10 days, G. mollugo cells contained shikimate in amounts of up to 10% of their dry weight. Synthesis of chorismate-derived anthraquinones in G. mollugo was blocked by glyphosate. Chorismate and o succinylbenzoate (an anthraquinone precursor) alleviated the inhibition. The conclusion drawn from these experiments, that glyphosate inhibits a step in the biosynthetic sequence from shikimate to chorismate, was substantiated by the finding that glyphosate is a powerful inhibitor of the conversion of shikimate to chorismate in cell-free extracts from Aerobacter aerogenes 62-1. PMID- 16661536 TI - Effects of divalent cations and polyethylene glycol on the membrane fluidity of protoplast. AB - Calcium is often used to stabilize membranes and enhance membrane fusion. We have used the fatty acid spin label, 5-nitroxy stearic acid to measure fluidity changes in the plasma membrane of carrot suspension culture cell protoplasts in response to divalent cations. Electron spin resonance spectra from spin-labeled protoplasts showed no membrane fluidity changes (as determined by the hyperfine splitting constant, 2A(max)) in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millimolar. Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 gauss in 2A(max) and evidence of exchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyethylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in the region of the 5-nitroxy stearic acid probe if added simultaneously with or following 10 millimolar Ca. Pretreatment with polyethylene glycol did, however, inhibit the Ca-induced phase separation. These data on a living system describe membrane structural changes under conditions similar to those used for protoplast fusion. PMID- 16661537 TI - Microheterogeneity of globulin-1 storage protein from French bean with isoelectrofocusing. AB - The major storage protein fraction, globulin-1 protein, of French bean (Phaseolus vulgaris L.) was analyzed by two-dimensional electrophoresis. The protein pattern suggested a more complex system for globulin-1 protein than the model of three polypeptides, alpha, beta, and gamma, differing in molecular weight. Isoelectrofocusing analyses of the individual proteins showed that each exhibited charge microheterogeneity over a similar pH range. Isoelectrofocusing banding patterns may help to understand the relationships between the globulin-1 polypeptide subunits. PMID- 16661538 TI - Ethylene Production by Chilled Cucumbers (Cucumis sativus L.). AB - Chilling at 2.5 C accelerated the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and C(2)H(4) production in cucumber fruit. Skin tissue contained higher levels of ACC and was more sensitive to chilling than was cortex tissue. Accumulation of ACC in chilled tissue was detected after 1 day of chilling and remained elevated even after C(2)H(4) production started to decline. These data suggest that ACC synthesis is readily stimulated by chilling, whereas the system that converts ACC to C(2)H(4) is vulnerable to chilling injury. Chilling-induced C(2)H(4) production was inhibited by amino-ethoxyvinylglycine, sodium benzoate, propyl gallate, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and cycloheximide. The utilization of methionine for ACC formation and chilling induced C(2)H(4) biosynthesis was established using l-[3,4-(14)C]methionine. Chilled tissue had a higher capacity to convert l-[3,4-(14)C]methionine to ACC and C(2)H(4) than did nonchilled tissue. PMID- 16661539 TI - Effect of Photoperiod on the Levels of Endogenous Gibberellins in Spinach as Measured by Combined Gas Chromatography-selected Ion Current Monitoring. AB - The changes in the levels of five endogenous gibberellins (GAs) in spinach in relation to photoperiodic treatment have been examined by combined gas chromatography-selected ion current monitoring. Long-day treatment caused a 5 fold decline in the level of GA(19) while the levels of GA(20) and GA(29) increased dramatically during the same period. In absolute terms, the level of GA(20) increased from 0.8 microgram per 100 grams dry weight in short days to 5.5 micrograms per 100 grams dry weight after 14 long days. The levels of GA(17) and GA(44) did not change significantly with long-day treatment. These results are consistent with the hypothesis that GA(19) is converted to GA(20) and that this conversion is under photoperiodic control. Since stem growth in spinach is correlated with an increase in the level of GA(20), one major aspect of photoperiodic control of stem growth might be the availability of GA(20) through regulation of the conversion of GA(19) to GA(20). PMID- 16661540 TI - A New Bacterial Agglutinin from Soybean: I. ISOLATION, PARTIAL PURIFICATION, AND CHARACTERIZATION. AB - A new bacterial agglutinin was isolated from seeds of the soybean cultivar Clark. Purification was carried out by ammonium sulfate precipitation and ion-exchange chromatography. The agglutinin is a heat-labile glycoprotein most active at pH 4.0. Addition of Ca(2+), Mn(2+) and Mg(2+) did not enhance the agglutinating activity of this glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate showed that the agglutinin is composed of two subunits of approximately 50,000 daltons each. In the undissociated state, it agglutinates Xanthomonas phaseoli var. sojensis, the causal agent of bacterial pustule disease of soybean, at concentrations as low as 10 micrograms protein per milliliter but has no hemagglutinating activity. The agglutinin could be distinguished from previously reported soybean lectins on the basis of solubility in ammonium sulfate, lack of hemagglutinating activity, molecular weight, hapten specificity, and immunological determinants. PMID- 16661541 TI - A New Bacterial Agglutinin from Soybean: II. EVIDENCE AGAINST A ROLE IN DETERMINING PATHOGEN SPECIFICITY. AB - The activity of a bacterial agglutinin from soybean seed [Glycine max (L.) Merrill cv. Clark] against two bacterial pathogens, Pseudomonas glycinea (causal agent of bacterial blight) and Xanthomonas phaseoli var. sojensis (causal agent of bacterial pustule) was determined. The agglutinin was active against several strains of X. phaseoli var. sojensis grown on nutrient agar, but there was no correlation between pathogenicity and agglutination. Agglutination was affected by the age of the bacterial cells and the growth medium used. None of seven strains of P. glycinea was agglutinated.Bacterial agglutination was inhibited by both purified lipopolysaccharide and extracellular polysaccharide from five strains of X. phaseoli var. sojensis. The lipopolysaccharides and extracellular polysaccharides from other species of bacteria were ineffective.Ultrastructural studies showed that an avirulent strain of X. phaseoli var. sojensis was attached to leaf mesophyll cell walls of the susceptible cultivar Clark by 34 hours after vacuum infiltration. Cells of this avirulent strain were enveloped by fibrillar and granular material at the mesophyll cell wall. In contrast, cells of a virulent strain were not attached or enveloped, and they remained free to multiply in the intercellular spaces. PMID- 16661542 TI - Growth-sustaining Water Potential Distributions in the Primary Corn Root: A NONCOMPARTMENTED CONTINUUM MODEL. AB - An equation is derived from transport theory to relate local growth rate to local water potential in an expanding tissue. For a noncompartmented continuum model, the relative elemental growth rate (L) equals the divergence of the tensor product of hydraulic conductivity (K) and the gradient of water potential, psi, i.e. L = big dn tri, open * [K . big dn tri, open psi]. This equation is solved numerically using published values of L and K to show the water potential distribution which can sustain the observed growth pattern in the primary root of Zea mays L. The water potential required to sustain growth decreases from the outside to the inside of the root, and the longitudinal profile shows most negative values near the location of the highest growth rate. A cell originally located near the apex experiences a loss and then a gain in water potential as it is displaced through the growth zone.THE APPROACH DIFFERS FROM PREVIOUS FORMULATIONS IN TWO RESPECTS: the assumption of spatial heterogeneity in growth rate, and the solution for spatial (site-specific) rather than material (cell specific) values of water potential. The role of air spaces and of components (wall and possibly cytoplasm) of the water-conducting pathway which do not accumulate water remains to be clarified; and, as in earlier work, the most uncertain aspects of the analysis are probably the values for hydraulic conductivity. PMID- 16661543 TI - Characterization of the spinach leaf phosphorylases. AB - The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V(max) for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K(m) for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K(m) values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V(max) values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K(m) values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K(m) value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity. PMID- 16661544 TI - Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES. AB - The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, beta-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca(2+) requirement or heat stability of seed endosperm alpha-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.The chloroplastic R-enzyme debranches amylopectin, beta-limit amylopectin, pullulan, and alpha-limit dextrins, but not rabbit liver glycogen. An increase in extinction coefficient and lambda(max) is detected when the debranched amylopectin and beta-limit amylopectin form a complex with I(2)-KI. Based on these properties, the chloroplastic R-enzyme is similar in enzymic activity to the R-enzyme observed in endosperm tissue. PMID- 16661545 TI - Regulation of Sulfate Assimilation in Tobacco Cells: EFFECT OF NITROGEN AND SULFUR NUTRITION ON SULFATE PERMEASE AND O-ACETYLSERINE SULFHYDRYLASE. AB - The effect of nitrogen and sulfur nutrition on sulfate permease and O acetylserine sulfhydrylase was studied in tobacco cells.Sulfate transport rates increased 10-fold in cells transferred to sulfur-deficient B-5 medium. The addition of either sulfate or l-cysteine reduced transport 95 and 80%, respectively. The pools of sulfate, cysteine, glutathione, and methionine declined in sulfur-starved cells. The addition of either sulfate or l-cysteine increased the pools of sulfur-containing compounds, but major quantitative differences were measured. Nitrogen-starved cells had low transport rates which were not increased by addition of nitrate/ammonia. The pools of sulfate, cysteine, and methionine were high in nitrogen-starved cells and remained high upon addition of a nitrogen source. The results show that sulfate transport is regulated by the intracellular sulfate pool.O-Acetylserine sulfhydrylase was not affected by sulfur nutrition. The extractable activity was high in B-5-grown cells, sulfur-deficient cells, and cells to which either sulfate or l-cysteine had been added. In contrast, the enzyme declined in cells transferred to nitrogen deficient medium and the amount of enzyme/g fresh weight increased 10-fold when nitrate/ammonia was added. The addition of nitrate/ammonia had no effect on the cysteine or methionine pools but increased the total amino acid pool. The amount of O-acetylserine was positively correlated with extractable enzyme activity. This enzyme is positively regulated by an effector (possibly O-acetylserine) which is high under conditions of net nitrate assimilation. PMID- 16661546 TI - Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet. AB - The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na(+) and K(+)) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets. PMID- 16661547 TI - Membrane Lipid Breakdown in Relation to the Wound-induced and Cyanide-resistant Respiration in Tissue Slices: A COMPARATIVE STUDY. AB - A study of a variety of bulky storage organs and fruits reveals that fresh slices fall into two categories with respect to their sensitivity to CN. Fresh slices in the first class are CN-sensitive, whereas slices of the second class are resistant to, and often stimulated by, CN. In tissue slices which are initially CN-sensitive, cutting initiates a burst of lipolytic activity. In CN-resistant fresh slices, there is no measurable lipid breakdown.Slicing evokes the wound respiration which is 5- to 10-fold that of the parent organ. Slice aging, in turn, evokes a further 2- to 3-fold respiratory increase, the wound-induced respiration, whether fresh slice respiration is CN-sensitive or -resistant. Estimation of the contribution by the cytochrome and alternative paths shows that the wound respiration in both groups is mediated by the cytochrome path. On the other hand, the wound-induced respiration in the first class is cytochrome path mediated, whereas, in some members of the second group, both pathways are utilized. Uncouplers of oxidative phosphorylation elicit a CN-sensitive increment in fresh slices as great or greater than the wound-induced respiration. Accordingly, de novo synthesis of mitochondria is ruled out as an explanation of the latter.The integrity of endomembranes, perhaps including mitochondrial membranes, is seemingly a prerequisite for the operation of the alternative path, that is, alternative path activity is lost concomitantly with membrane lipid breakdown. The development of the wound-induced respiration is not co-extensive with the development of the CN-resistant path in all tissue slices. The fundamental process of aging appears to involve activation of pre-existing respiratory capacity.Fresh slices from whatever source fail to utilize exogenous (14)C-labeled glucose, whereas aged slices do so readily. A transport lesion is indicated, the healing of which does not depend on the development of the wound induced respiration but does depend on fatty acid, and presumably membrane lipid, biosynthesis. PMID- 16661548 TI - Peptide Mapping Reveals Considerable Sequence Homology among the Three Polypeptide Subunits of G1 Storage Protein from French Bean Seed. AB - The major storage protein, G1 globulin, of bean (cv. Tendergreen) seeds was subjected to limited proteolysis with trypsin, chymotrypsin, papain, proteinase K, and protease V8 and to cleavage with cyanogen bromide and 2-(2 nitrophenylsulfanyl)-3-methyl-3'bromoindolenine. Mapping of peptides separated from each of the three G1 subunits by polyacrylamide gel electrophoresis revealed that many proteolytic cleavage sites were present at similar positions on the subunits. Evidence was adduced that the G1 subunits are homologous in amino acid sequence for about 61% of their length. The remaining region (possibly COOH terminal) of the subunits appears to be heterologous, with the alpha subunit bearing an additional methionine residue. PMID- 16661549 TI - Phytochrome Pelletability Induced by Irradiation in Vivo: TEST FOR IN VITRO BINDING OF ADDED [S]PHYTOCHROME. AB - Undegraded, highly purified [(35)S]phytochrome was immunoaffinity-purified either from dark control oat (cv. Garry) shoots or from etiolated oat shoots that were previously irradiated first with red and then with far-red light so that, if proper extraction conditions had been utilized, about 60% of the total phytochrome would have been pelletable. When [(35)S]phytochrome was added to extraction buffer immediately prior to homogenization of etiolated oat shoots, pelletability assays indicated that there was no preferential binding of [(35)S]phytochrome regardless of (a) whether it was purified from dark control or irradiated shoots, (b) whether it was added as phytochrome-red-absorbing form or phytochrome-far-red-absorbing form, or (c) whether it was added to dark control or red-irradiated shoots. Similarly, binding of [(35)S]phytochrome to resuspended pellets obtained from crude oat extracts was not specific for the source of [(35)S]phytochrome, for its form, or for the irradiation treatment given to intact shoots used to prepare the resuspended pellets. No evidence was obtained to support the hypothesis that phytochrome binds with specificity to particulate material in vitro under conditions used to assay for light-enhanced, in vivo induced phytochrome pelletability. PMID- 16661550 TI - Phytochrome Pelletability Induced by Irradiation in Vivo: MIXING EXPERIMENTS. AB - Samples of irradiated and control Avena sativa shoot tissue were homogenized together to determine whether, during homogenization phytochrome from irradiated tissue can bind to the particulate material simultaneously extracted from the control tissue. The level of phytochrome pelletability for such mixed tissue homogenizations is equal to: (a) the values obtained when the extracts from separate homogenizations of the two batches of tissue are mixed and then centrifuged; and (b) the arithmetic mean of the values obtained when the two batches of tissue are separately homogenized and separately tested for pelletability. This relationship is observed regardless of the ratio of control to irradiated tissue over the range from 4:6 to 9:1. These data indicate that the observed limit to the level of pelletability inducible in irradiated tissue (about 60%) does not result from a limited number of nonspecific particulate binding sites to which in vivo-modified phytochrome molecules have access at, or after, the moment of cell disruption. The possibility that pelletability may represent preservation of an association established in vivo is discussed. PMID- 16661551 TI - Protection of cellulose synthesis in detached cotton fibers by polyethylene glycol. AB - Detachment of the cotton fiber cell from the ovule results in loss of over 90% of the in vivo capacity for synthesis of [(14)C]cellulose from [(14)C]glucose. However, over 50% of the capacity for cellulose synthesis in the detached fiber population is protected when polyethylene glycol 4000 is present during detachment and incubation. Radioautography shows that approximately full capacity is restored in about half the fibers, whereas the other half of the population are incapable of cellulose synthesis from supplied glucose. The rate of cellulose synthesis in such fibers has a pH optimum of 6 and the optimum polyethylene glycol 4000 concentration is 0.06 molal (-9 bars). Cellulose synthesis in such detached fibers is synergistically stimulated by Ca(2+) and Mg(2+) and inhibited by K(+).Evidence is presented which indicates that the protection by polyethylene glycol 4000 is due to its ability to promote membrane resealing, which seems to be required for protecting cellulose synthesis in the detached fiber; however, the requirement for membrane integrity is not exclusively involved in the maintainence of an energy generating system for the synthesis. The possibility that a membrane potential may be required for maintaining an active cellulose synthesizing system is discussed. PMID- 16661552 TI - Carbon Dioxide and Water Vapor Exchange in the Crassulacean Acid Metabolism Plant Kalanchoe pinnata during a Prolonged Light Period: METABOLIC AND STOMATAL CONTROL OF CARBON METABOLISM. AB - Net CO(2) and water vapor exchange were studied in the Crassulacean acid metabolism plant Kalanchoe pinnata during a normal 12-hour light/12-hour dark cycle and during a prolonged light period. Leaf temperature and leaf-air vapor pressure difference were kept constant at 20 C and 9 to 10 millibar. There was a 25% increase in the rate of CO(2) fixation during the first 6 hours prolonged light without change in stomatal conductance. This was associated with a decrease in the intracellular partial pressure of CO(2), a decrease in the stimulation of net CO(2) uptake by 2% O(2), and a decrease in the CO(2) compensation point from 45 to 0 microbar. In the normal light period after deacidification, leaves showed a normal light dependence of CO(2) uptake but, in prolonged light, CO(2) uptake was scarcely light-dependent. The increase in titratable acidity in prolonged light was similar to that in the dark.The results suggest a change from C(3) photosynthetic CO(2) fixation in the second part of the 12-hour light period to a mixed metabolism in prolonged light with both ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase as primary carboxylating enzymes. PMID- 16661553 TI - Biochemistry of Fern Spore Germination: GLYOXYLATE AND GLYCOLATE CYCLE ACTIVITY IN ONOCLEA SENSIBILIS L. AB - In chlorophyll-containing spores of Onoclea sensibilis, depletion of lipid reserves during germination is correlated with increases in the activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. In Onoclea, the heterotrophic activity associated with lipid catabolism occurs at the same time that autotrophic activity is taking place. Increases in chlorophyll content and in the activity of glycolate oxidase were recorded during the earliest stages of spore germination. In this species, there is no temporal separation of heterotrophic and autotrophic reactions. Concurrent increases in glyoxylate and glycolate cycle activities appear to occur naturally. PMID- 16661554 TI - Efficiency of hydrogen photoproduction by chloroplast-bacterial hydrogenase systems. AB - A comparative study of H(2) photoproduction by chloroplasts and solubilized chlorophyll was performed in the presence of hydrogenase preparations of Clostridium butyricum. The photoproduction of H(2) by chloroplasts in the absence of exogenous electron donors, and with irreversibly oxidized dithiothreitol and cysteine, is thought to be limited by a cyclic transport of electrons wherein methylviologen short-circuits the electron transport in photosystem I. The efficiency of H(2) photoproduction by chloroplasts with ascorbate and NADPH is limited by a back reaction between light-reduced methylviologen and the oxidized electron donors. The use of a combination of electron donors (dithiothreitol and ascorbate), providing anaerobiosis without damage to chloroplasts, makes it possible to avoid consumption of reduced methylviologen for the reduction of oxidized electron donors and to exclude the short-circuiting of electron transfer. Under these conditions, photoproduction of H(2) was observed to occur with a rate of 350 to 400 micromoles H(2) per milligram chlorophyll per hour. In this case, the full electron-transferring capability of photosystem I (measured by irreversible photoreduction of methyl red or O(2)) is used to produce H(2). PMID- 16661555 TI - Diffusional Contribution to Carbon Isotope Fractionation during Dark CO(2) Fixation in CAM Plants. AB - A mathematical model is developed which can be used to predict in vivo carbon isotope fractionations associated with carbon fixation in plants in terms of diffusion, CO(2) hydration, and carboxylation components. This model also permits calculation of internal CO(2) concentration for comparison with results of gas exchange experiments. The isotope fractionations associated with carbon fixation in Kalanchoe daigremontiana and Bryophyllum tubiflorum have been measured by isolation of malic acid following dark fixation and enzymic determination of the isotopic composition of carbon-4 of this material. Corrections are made for residual malic acid, fumarase activity, and respiration. Comparison of these data with calculations from the model indicates that the rate of carbon fixation is limited principally by diffusion, rather than by carboxylation. Processes subsequent to the initial carboxylation also contribute to the over-all isotopic composition of the plant. PMID- 16661556 TI - Catabolism of adenine derivatives in leaves: study of the role of light on the in vivo activity of xanthine dehydrogenase. AB - The in vivo activity of xanthine dehydrogenase (E.C. 1.2.1.37) was followed in leaf discs excised from illuminated or darkened plants. In cotyledons of Pharbitis nil, 24 hours of darkness enhanced the in vivo activity of xanthine dehydrogenase which increased between 2 to 5-fold depending on the concentration of hypoxanthine of the solution where cotyledon discs were incubated. The same effect occurred in leaves of several other species, in plants with both high and low ureide content. However, the effect of light was not observed in leaves of Zea mays, Pennisetum americanum and Atriplex spongiosa, whereas, it appeared very clearly in other C(4) plants such as Sorghum sudanense and Portulaca oleracea. This enzymic activity in chlorophyll-deficient tobacco leaves was the same both for illuminated and darkened plants. In addition, the in vivo activity of xanthine dehydrogenase in roots of Pharbitis nil was not dependent upon the light conditions applied to leaves. In cotyledons of Pharbitis nil, the level of the in vivo activity of xanthine dehydrogenase was influenced by the energy of light and the duration of illumination. The supply of carbohydrates to darkened cotyledons had the same effect as light on the in vivo activity of xanthine dehydrogenase. It is proposed that the effect of light on the in vivo activity of xanthine dehydrogenase in leaves is mainly due to the production of photosynthates which changes the osmotic state of leaf tissue and thus modifies the level of the in vivo activity of xanthine dehydrogenase. PMID- 16661557 TI - On the Reason for the Different Photosynthetic Rates of Seedlings of Pinus silvestris and Betula verrucosa. AB - The growth and net photosynthetic properties of seedlings of Pinus silvestris L. and Betula verrucosa Ehrh., grown under identical conditions in a controlled environment chamber, were compared. The relative growth rate of birch was about twice that of pine. The rates of in situ net photosynthesis were 1.50 and 2.30 micromoles CO(2) meter(-2) second(-1) and the photosynthetic quantum yields under light-limiting conditions were 0.022 and 0.032 for pine and birch, respectively. The total leaf surface areas were used for calculating the CO(2) flux densities. The difference in the rates of in situ net photosynthesis depended equally on morphological and metabolic factors. It was assumed that a pronounced mutual shading and an unfavorable leaf inclination made the pine seedlings less efficient in absorbing the unidirectional light of the climate chamber than the broadleaved seedlings of birch. Both pine and birch were adapted to the growth conditions so the flux densities of absorbed quanta were rate-limiting for in situ net photosynthesis. It was concluded that the difference in the photosynthetic quantum yields (i.e. the linear slope of the photosynthetic light curve) of the two species defined the metabolically controlled part of the difference in the rate of in situ net photosynthesis. The quantum yield of pine was lower than that of birch and was partly explained by pine having a higher rate of photorespiration than birch. The remaining difference was most likely controlled by the properties of the chloroplast thylakoids, e.g. energy transfer efficiency between pigments, photosynthetic electron transport, or coupling between electron transport and photophosphorylation. PMID- 16661558 TI - Effect of Rapid Changes in Sink-Source Ratio on Export and Distribution of Products of Photosynthesis in Leaves of Beta vulgaris L. and Phaseolus vulgaris L. AB - Effects of increasing sink-source ratio on rate of translocation and net carbon exchange were studied by darkening all but one source leaf of Beta vulgaris L. or one primary leaf of Phaseolus vulgaris L. Rates of export of labeled material and patterns of its distribution among sinks were studied by means of GM detectors. Changes in export and import rates were compared with adjustments in starch, sucrose, and glucose levels in sugar beet source leaves before and during treatment.Sugar beet source leaf treatments which increased sink-source ratio had no sustained effect on rates of net carbon exchange, export, accumulation of starch, or the levels of sucrose and glucose on the day of treatment. However, the rate of import into a developing sugar beet leaf increased. Similar treatment of primary leaves of bean either had no effect on the rate of export of labeled material or caused up to a 20% increase. The increase in export was sufficient to account for the increase in import of labeled material into the far-sink leaflet. Nevertheless, distribution of labeled material exported to the sink regions changed; import of labeled material into the near-sink leaflet remained nearly unchanged and that into the roots decreased.The data indicate that rapid changes in sink-source ratio cause a redistribution of the products of photosynthesis but need not involve a change in the rate of export. PMID- 16661559 TI - Distribution of wheat germ agglutinin in young wheat plants. AB - A liquid phase, competition-binding radioimmunoassay for wheat germ agglutinin, with a detection limit of 10 nanograms, was developed in order to determine the distribution of this lectin in young wheat plants. Affinity columns for wheat germ agglutinin removed all antigenically detectable activity from crude extracts of wheat tissue; thus, the antigenic cross-reactivity detected by the assay possesses sugar-binding specificity similar to the wheat germ-derived lectin. The amount of lectin per dry grain is approximately 1 microgram, all associated with the embryo. At 34 days of growth, the level of lectin per plant was reduced by about 50%, with approximately one-third in the roots and two-thirds in the shoot. The data also indicate that actively growing regions of the plant (the bases of the leaves and rapidly growing adventitious roots) contain the highest levels of lectin. Half of the lectin associated with the roots could be solubilized by washing intact roots in buffer containing oligomers of N-acetylglucosamine, whereas the remainder is liberated only upon homogenization of the tissue. PMID- 16661560 TI - On the uptake, metabolism and retention of [h] gibberellin a(1) by barley aleurone layers at low temperatures. AB - Barley (c.v. Himalaya) aleurone layers were incubated in [(3)H]gibberellin A(1) (GA(1)) at low temperatures. At 3 and 4 C, (3)H-activity was steadily accumulated in aleurone layers, and this accumulation was correlated with significant [(3)H]GA(1) metabolism. At 1 and 1.5 C, metabolism could not be detected, and at these temperatures aleurone layers equilibrated with the [(3)H]GA(1) concentration in the incubation medium. At equilibrium, the total amount of (3)H activity per unit volume in the aleurone layers was higher than in the incubation medium. Aleurone layers incubated at 0.5 C for 72 hours with [(3)H]GA(1) in the presence of saturating levels of carrier GA(1) consistently retained lower levels of (3)H-activity than when incubated in [(3)H]GA(1) alone. The retention of [(3)H]GA(1) was unaffected by saturating levels of carrier GA(8). GA(1) retained by barley aleurone layers that were incubated at 0.5 C for 72 hours was able to induce alpha-amylase synthesis when aleurone layers were subsequently washed and transferred to a gibberellin-free medium at 25 C. PMID- 16661561 TI - In vivo Binding of Gibberellin A(1) in Dwarf Pea Epicotyls. AB - Binding of [(3)H]gibberellin A(1) (GA(1)) to extracts of dwarf pea epicotyls was investigated using sliced pea epicotyls (0.5-1.0 millimeter thick) that had been incubated in a solution containing [(3)H]GA(1) at 0 C for 3 days. Gel filtration of a 100,000g supernatant indicated binding to a high (HMW) and an intermediate molecular weight (IMW) fraction with estimated molecular weights of 6 x 10(5) daltons and 4 to 7 x 10(4) daltons, respectively. The bound (3)H-activity was [(3)H]GA(1) and not a metabolite as deduced by thin layer chromatography. The bound label did not sediment during centrifugation at 100,000g for 2 hours; also, binding was not disrupted after treatment of a combined HMW and IMW fraction with DNase, RNase, or phospholipase A or C, but it was disrupted by protease or heat treatment. These facts suggest that binding of [(3)H]GA(1) was occurring to a soluble protein(s). [(3)H]GA(1) bound to a combined HMW and IMW fraction was not susceptible to changes in pH, nor could it be exchanged with a variety of GAs tested under in vitro conditions. Under in vivo equilibrium conditions, biologically active GAs, such as GA(1), GA(3), GA(4), GA(5), GA(7), and keto GA(1), could reduce the level of [(3)H]GA(1) binding, whereas inactive GAs, such as iodo GA(1) methyl ester, GA(8), GA(13), GA(26), and non-GAs, such as (+/ )abscisic acid, had no effect. By varying the concentration of [(3)H]GA(1) in the incubation medium, the specific binding of [(3)H]GA(1) appeared to be due to two classes of binding sites having estimated K(d) of 6 x 10(-8) molar and 1.4 x 10( 6) molar. The concentrations of the two sites were estimated to be 0.45 picomole per gram and 4.04 picomoles per gram on a fresh weight and 0.1 picomole per milligram and 0.9 picomole per milligram on a soluble protein basis, respectively. PMID- 16661562 TI - Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean. AB - Soybeans grown with 2 millimolar NO(3) (-), which optimized apparent N(2) fixation by Rhizobium symbionts, showed significantly different rates of apparent photosynthesis and C(2)H(2) reduction during seedling development at two irradiances. Those physiological processes were lower for several weeks in plants grown at 1,500 microeinsteins per meter(2) per second than in those exposed to 700 microeinsteins per meter(2) per second. The irradiance-induced retardation was evident in short-term rates of apparent photosynthesis and N(2) fixation, as well as in measures of dry matter and total N accumulation. In spite of their previously inhibited development, plants grown at 1,500 microeinsteins per meter(2) per second were indistinguishable by day 28 from those exposed to 700 microeinsteins per meter(2) per second in terms of whole-shoot CO(2)-exchange rate; by day 35 they were identical in terms of whole-plant C(2)H(2)-reduction rate. On day 38 there was no significant difference in dry weight or N content between treatments. Shifting plants between irradiance treatments on day 21 showed that the higher irradiance also had a short-term inhibitory effect on C(2)H(2) reduction. The fact that 16 millimolar NO(3) (-) prevented the continuous exposure to 1,500 microeinsteins per meter(2) per second from inhibiting apparent photosynthesis suggested that seedlings grown on 2 millimolar NO(3) (-) with Rhizobium were N-limited. Although rates of apparent photosynthesis were similar by day 28, the additional week required to produce equal rates of apparent N(2) fixation between irradiance treatments showed that physiological adaptations of shoots, as well as photosynthesis per se, can affect root nodule activity. PMID- 16661563 TI - Effect of salts on the activity of carrot phosphofructokinase. AB - Phosphofructokinase (EC 2.7.1.11) from carrot roots was activated by a number of salts. Increase in salt concentration beyond the optimum generally led to a decrease in enzyme activity. Salts of the multivalent anions sulfate and phosphate were very effective activators and inhibitors. Potassium acetate and potassium succinate were also activators. Potassium tartrate and potassium citrate produced a small stimulation at low concentration but with further increase they became inhibitory. The results suggested that the salt effect was largely due to anions rather than cations. Salts such as NaCl, KCl, and in particular potassium phosphate, relieved the inhibition of carrot phosphofructokinase by phosphoenolpyruvate. KCl and potassium phosphate also reversed the inhibition of carrot phosphofructokinase by citrate. The possible significance of these observations in the regulation of glycolysis and carbohydrate metabolism, and in salt respiration is discussed. PMID- 16661564 TI - Economy of Carbon and Nitrogen in Nodulated and Nonnodulated (NO(3)-grown) Cowpea [Vigna unguiculata (L.) Walp.]. AB - The response of non-nodulated cowpea (Vigna unguiculata (L.) Walp. cv Caloona) to a wide range of NO(3) levels in the rooting medium was studied 40 days after sowing by in vitro assays of plant organs for NO(3) reductase (EC 1.6.6.1) and analyses of root bleeding (xylem) sap for nitrogenous solutes. Plants fed 1, 5, 10, 20, and 40 millimolar NO(3) showed, respectively, 64, 92, 94, and 91% of their total reductase activity in shoots and 34, 30, 66, 62, and 58% of the total N of their xylem sap as NO(3). These data, and the absence in the plants of significant pools of stored NO(3), indicated that shoots were major organs of NO(3) assimilation, especially at levels of NO(3) (10 to 40 millimolar) that maintained plant growth at near maximum rates. Partitioning and utilization of C and N were studied in nodulated, minus NO(3) plants and non-nodulated plants fed 10 or 20 millimolar NO(3), the levels of NO(3) which gave rates of growth and N assimilation closest to those of the symbiotic plants. The conversion of the C of net photosynthate to dry matter was similar in nodulated plants (67%) and NO(3) grown plants (64%), but greater proportions of photosynthate were translocated to below ground parts of nodulated plants (37%) than of NO(3)-fed plants (23 to 26%). Greater photosynthate consumption by nodulated roots was associated with proportionately greater root growth and respiration and 2-fold greater export of C in xylem than in the NO(3)-fed plants. Theoretical considerations suggest that the elevated CO(2) output of nodulated roots was due not only to CO(2) loss associated with nodule function, but also to a much greater nonassimilatory component of respiration in the supporting root of the nodulated plant compared to roots of the NO(3)-fed plants. Data are compared with previously published information from other legumes. PMID- 16661565 TI - Isolation and partial characterization of an Acid endoprotease present in dormant apple shoot bark. AB - A major protease present in dormant bark tissues of the apple (Malus domestica Borkh. cv. "Golden Delicious") was partially purified by hemoglobin-coupled Sepharose column chromatography. The protease active at pH 4.6 and at temperatures of 30 to 50 C was found to be sulfhydryl-dependent. Phenylmercuric acetate inhibited the enzyme approximately 50% at 2 millimolar, whereas phenylmethylsulfonyl fluoride inhibited less than 30% even at 10 millimolar. Substrate specificity and the separation of the enzyme reaction products indicated that the enzyme is likely to be an endoprotease. We suggest that the storage proteins in apple bark tissue undergo some modification prior to their eventual hydrolysis to amino acids, which requires a multi-enzyme system(s). Evidence is presented that the apple bark protein extracts enzymically release ninhydrin-positive compounds upon storage at 5 C. It is concluded that the activation of the sulfhydryl-dependent acid endoprotease is associated with the rapid metabolism of storage proteins which accompanies bud break upon regrowth. PMID- 16661566 TI - Promotion of sink activity of developing rose shoots by light. AB - Holding young rose shoots (Rosa hybrida cv. Marimba) in darkness while the rest of the plant was in light reduced the amount of (14)C assimilates recovered from the darkened shoot by half. Relative specific activity of the shoot tip grown in light was 13.5 times greater than that of the darkened one. The flower bud at the shoot tip degenerated in darkness and died. Shoots 2 to 3 centimeters long, after flower initiation, were most sensitive to the dark treatment. The degeneration is a gradual and reversible process in the first 8 days of darkness, followed by irreversible damage and atrophy. Darkening enhanced the ability of the young leaves to compete for the available assimilates over that of the darkened shoot tip. The enhancement of the mobilizing ability of the shoot tip by light is independent of photosynthesis since spraying with 3-(3,4-dichlorophenyl)-1,1 dimethylurea or holding shoots in a CO(2)-free atmosphere did not diminish the promoting effect of light on flower bud development or assimilate import. The possibility that light exerts its effect by photoproduction of ATP was also excluded inasmuch as no differences were found in ATP levels of shoot tips held in darkness and those held in light. PMID- 16661567 TI - Characterization of the light reaction in promoting the mobilizing ability of rose shoot tips. AB - Mixed fluorescent and incandescent light increased growth and sink strength of the uppermost young shoot of rose plants (Rosa hybrida cv. Marimba) in comparison to pure fluorescent light. This was manifested by increased apical dominance. Monochromatic low-energy red light, given by means of optic fibers for 24 hours to shoot tips that had been previously darkened for 5 days, increased the transport of (14)C-labeled assimilates to the intact tips and the uptake of [(14)C]sucrose by detached tips. Far-red had little or no effect, and blue was not effective at all in these reactions. Red light given directly to detached shoot tips, in vitro, increased the uptake of [(14)C]sucrose by the isolated tips. Adding far-red to the red greatly promoted the uptake, whereas blue and blue plus far-red were not active. The main character of the light reaction promoting sink activity in the shoot is that it is perceived by the shoot tip itself. It is operated by red light; far-red promotes the red effect but has little or no effect when alone. Light apparently promotes shoot sink activity by increasing the unloading process. PMID- 16661568 TI - Polygalacturonase Activity and Ethylene Synthesis during Cucumber Fruit Development and Maturation. AB - Polygalacturonase (EC 3.2.1.15) activity in seed cavity tissue from harvested cucumber fruit increased over 20-fold after the fruit had produced a transient burst of ethylene during maturation. This increase was observed in six cucumber cultivars and was present whether polygalacturonase activity was measured at pH 4.6 or 6.2. The seed cavity tissue pH decreased as polygalacturonase activity increased both in ripening fruit and in harvested immature fruit exposed to 10 microliters per liter ethylene in air. PMID- 16661569 TI - Phytochrome Responses to End-of-Day Irradiations in Light-grown Corn Grown in the Presence and Absence of Sandoz 9789. AB - Corn seedlings were grown in white light in the absence and presence of the chlorosis-inducing herbicide San 9789. The resulting green and achlorophyllous seedlings were used to investigate phytochrome-mediated responses to end-of-day far red irradiation and reversal of these responses by subsequent red irradiation. Mesocotyl and coleoptile elongation increased in response to end-of day far red irradiation, whereas the anthocyanin content of the coleoptiles was decreased. All three responses were reversible by red irradiation following the far red. Dose-response curves for far red induction and red reversal of these responses did not differ significantly for plants grown in the presence or absence of San 9789. Thus, San 9789 appears to affect neither phytochrome itself nor the response system involved. Chlorophyll screening likewise does not affect phytochrome relationships for these responses. PMID- 16661570 TI - Early Events in the Infection of Soybean (Glycine max L. Merr) by Rhizobium japonicum: I. LOCALIZATION OF INFECTIBLE ROOT CELLS. AB - The infectible cells of soybean roots appear to be located at any given time just above the zone of root elongation and just below the position of the smallest emergent root hairs. The location of infectible cells on the primary root at the time of inoculation was inferred from the position of subsequent nodule development, correcting for displacement of epidermal cells due to root elongation. Marks were made on the seedling growth pouches at the time of inoculation to indicate the position of the root tip and the zones of root hair development. Virtually all of the seedlings developed nodules on the primary root above the marks made at the root tips at the time of inoculation. None of the plants formed nodules on the root where mature root hairs were present at the time of inoculation. These results and profiles of nodulation frequency indicate that the location of infectible cells is developmentally restricted. When inoculations were delayed for intervals of 1 to 4 hours after marking the positions of the root tips, progressively fewer nodules were formed above the root tip marks, and the uppermost of these nodules were formed at progressively shorter distances above the marks. These results indicate that the infectibility of given host cells is a transient property that appears and then is lost within a few hours. The results also indicate that host responses leading to infection and nodulation are triggered or initiated in less than 2 hours after inoculation. The extent of nodulation above the root tip mark increased in proportion to the logarithm of the number of bacteria in the inoculum. PMID- 16661571 TI - Effect of CO(2), O(2), and Light on Photosynthesis and Photorespiration in Wheat. AB - Unidirectional O(2) fluxes were measured with (18)O(2) in a whole plant of wheat cultivated in a controlled environment. At 2 or 21% O(2), O(2) uptake was maximum at 60 microliters per liter CO(2). At lower CO(2) concentrations, it was strongly inhibited, as was photosynthetic O(2) evolution. At 2% O(2), there remained a substantial O(2) uptake, even at high CO(2) level; the O(2) evolution was inhibited at CO(2) concentrations under 330 microliters per liter. The O(2) uptake increased linearly with light intensity, starting from the level of dark respiration. No saturation was observed at high light intensities. No significant change in the gas-exchange patterns occurred during a long period of the plant life. An adaptation to low light intensities was observed after 3 hours illumination. These results are interpreted in relation to the functioning of the photosynthetic apparatus and point to a regulation by the electron acceptors and a specific action of CO(2). The behavior of the O(2) uptake and the study of the CO(2) compensation point seem to indicate the persistence of mitochondrial respiration during photosynthesis. PMID- 16661572 TI - Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots. AB - The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements. PMID- 16661573 TI - In Vivo Assay for the Synthesis of Hydroxyproline-rich Proteins. AB - Simple methods are described for following in vivo the rate of peptidylproline hydroxylation and for determining what proportion of the total proline incorporated into protein is hydroxylated. PMID- 16661574 TI - Mode of Dinitroaniline Herbicide Action: II. CHARACTERIZATION OF [C]ORYZALIN UPTAKE AND BINDING. AB - The intracellular binding of dinitroaniline herbicides was studied in order to analyze the mechanism of their colchicine-like action. When corn root apices (5 millimeters) are incubated in [(14)C]oryzalin (a dinitroaniline herbicide), the (14)C is taken up rapidly, reaching a plateau in about 4 hours, which corresponds to the minimum incubation time in oryzalin required to get maximum inhibition of elongation. At 4 hours, the [(14)C]oryzalin concentration inside the roots is 35 times higher than that in the incubation medium. Since this accumulation of [(14)C]oryzalin is not affected by 1 millimolar sodium azide and there is no metabolism of [(14)C]oryzalin under these conditions, the [(14)C]oryzalin must be accumulated (bound) in corn root apices by a process not requiring metabolic energy.Molecular sieve chromatography (Sephadex G-200) does not show any binding of [(14)C]oryzalin to any protein with molecular weight similar to the microtubule-subunit protein in rat brain or corn root extracts. However, a massive binding of [(14)C]oryzalin occurs in the insoluble fraction of corn root extracts. This binding is not localized in any particle size range, is not affected by sonication, is of high capacity, and is a loose (low affinity) association with the binding sites. These binding sites could be solubilized with membrane detergents, which suggests that oryzalin may bind to cellular membranes. Since boiling the homogenate does not affect [(14)C]oryzalin binding, oryzalin more likely binds to a lipid rather than a protein component of cellular membranes. PMID- 16661575 TI - Metabolic Activity and Energy Charge of Excised Maize Root Tips under Anoxia: CONTROL BY SOLUBLE SUGARS. AB - Energy charge and fermentative metabolism under anoxia were monitored in excised maize root tips after various times of aging in air and were related to their soluble sugar content. The energy charge value, which was 0.9 in air irrespective of the time of aging, dropped to a lower value within minutes of transfer to a nitrogen atmosphere. This value was dependent upon sugar content of the tissues which was itself a function of aging. The energy charge value after transfer to nitrogen was 0.6 in freshly excised tissue but only 0.2 in tissue aged for 4 hours. When aged tissues supplied with 0.2 molar glucose were transferred to nitrogen, the energy charge was 0.6, irrespective of the time of aging. When 0.2 molar glucose was added under nitrogen, energy charge rose to 0.6. This rise was faster in root tips aged for 8 hours than those aged for 24 hours.The rate of ethanol plus lactate production (representing 60 and 10%, respectively, of the total sugar consumption in anoxia) was closely correlated to the level of energy charge. It is concluded that, in anoxia, there is a quantitative relationship between the energy charge value and the level of metabolic activity via fermentative pathways. PMID- 16661576 TI - Effect of pH on the Activity of Some Respiratory Inhibitors. AB - Inhibition of respiration of cultured cells of Petunia hybrida by NaF, NaN(3), malonic acid, and salicylhydroxamic acid increased at low pH. This increase could be partially reversed by raising the pH of the medium. Uptake experiments show that the greater inhibition of malonic acid at low pH was not the result of greater uptake. The results suggest that the increase in inhibition at low pH could not be attributed to greater cell penetration. PMID- 16661577 TI - Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids. AB - The H(2)-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, (2)H(1)H exchange from a mixture of (2)H(2) and (1)H(2) in presence of (2)H(2)O and (1)H(2)O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H(2) oxidation reaction, there was no significant discrimination between (2)H(2) and (1)H(2), indicating that the initial H(2)-activation step in the over-all H(2) oxidation reaction is not rate-limiting. By use of improved methods, an apparent K(m) for H(2) of 0.05 micromolar was determined. The H(2) oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H(2) oxidation and stimulated O(2) uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H(2) to O(2). Partial pressures of H(2) at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO(2) evolution by low partial pressures of H(2) suggests that H(2) utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H(2) uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO(2) (initial concentration) in solution inhibited H(2) uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H(2) uptake by succinate, acetate, formate, and CO(2) in the metabolism of the H(2)-uptake positive strains of Rhizobium. PMID- 16661578 TI - Light-inducible Cytochrome Reduction in Membrane Preparations from Corn Coleoptiles: I. STABILIZATION AND SPECTRAL CHARACTERIZATION OF THE REACTION. AB - Conditions for obtaining reproducible light-induced reduction of a b-type cytochrome in membrane fractions from coleoptiles of dark-grown Zea mays L. include a glucose-glucose oxidase system that lowers O(2) tension and generates H(2)O(2), substrate amounts of ethylenediaminetetraacetic acid which, in some manner, facilitates photoreduction by both added flavin and the endogenous photoreceptor and a sample temperature below 10 C. Cytochrome reduction could be obtained by photoexcitation of either a tightly bound endogenous receptor, which is probably a flavin, or added riboflavin, flavin mononucleotide, or flavin adenine dinucleotide. The latter flavin was the least effective. The endogenous photoreceptor appears to be rather firmly bound to the membranes, suggesting that this association may also exist in vivo. When any of the above four photoreceptors or methylene blue were used to sensitize the reaction, a cytochrome with a reduced alpha-band near 560 nanometers and a Soret difference peak near 429 nanometers was the electron acceptor. This cytochrome could be clearly distinguished spectrally from other cytochromes that predominated in the membrane preparations. PMID- 16661579 TI - Solubilization of the receptor for N-1-naphthylphthalamic Acid. AB - A receptor protein for the auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), has been solubilized from corn coleoptile membranes using Triton X-100. [(3)H]NPA binding activity of the receptor was compared in soluble and membrane bound states. Both activities are abolished by treatment with trypsin. Differences between the two are observed in pH optima and rates of heat inactivation.At pH 5, the membrane-bound and solubilized receptors have similar affinity for NPA (K(d) about 10(-7) molar). Solubilization results in a 10-fold increase in affinity for the low-affinity auxin analogs, alpha- and beta naphthaleneacetic acid (K(d) about 10(-5) molar). There is no measurable competition by indoleacetic acid (K(d) > 10(-3) molar) for [(3)H]NPA binding to membranes, but there is competition with the solubilized receptor (K(d) for IAA about 10(-5) molar). There is no measurable competition with benzoic acid in either preparation. These observations of an affinity of auxin analogs for the solubilized NPA receptor raise the interesting possibility that the NPA binding site is one conformation of an auxin binding site involved in polar auxin transport. PMID- 16661580 TI - Oxygen effect on photosynthetic and glycolate pathways in young maize leaves. AB - To study the effect of O(2) on the photosynthetic and glycolate pathways, maize leaves were exposed to (14)CO(2) during steady-state photosynthesis in 21 or 1% O(2). At the two O(2) concentrations after a (14)CO(2) pulse (4 seconds) followed by a (12)CO(2) chase, there was a slight difference in CO(2) uptake and in the total amount of (14)C fixed, but there were marked changes in (14)C distribution especially in phosphoglycerate, ribulose bisphosphate, glycine, and serine. The kinetics of (14)C incorporation into glycine and serine indicated that the glycolate pathway is inhibited at low O(2) concentrations. In 1% O(2), labeling of glycine was reduced by 90% and that of serine was reduced by 70%, relative to the control in 21% O(2). A similar effect has been observed in C(3) plants, except that, in maize leaves, only 5 to 6% of the total (14)C fixed under 21% O(2) was found in glycolate pathway intermediates after 60 seconds chase. This figure is 20% in C(3) plants. Isonicotinyl hydrazide did not completely block the conversion of glycine to serine in 21% O(2), and the first carbon atom of serine was preferentially labeled during the first seconds of the chase. These results supported the hypothesis that the labeled serine not only derives from glycine but also could be formed from phosphoglycerate, labeled in the first carbon atom during the first seconds of photosynthesis.Another noticeable O(2) effect concerned differential labeling of phosphoglycerate and ribulose bisphosphate. Phosphoglycerate is more labeled than ribulose bisphosphate in air; the reverse is observed in 1% O(2). Changes in ribulose bisphosphate and phosphoglycerate pools exhibit similar trends. To understand the effect of O(2) on the distribution of (14)C in these two intermediates, it was postulated that, in air, there remains an oxygenase function which produces additional phosphoglycerate at the expense of ribulose bisphosphate. PMID- 16661581 TI - Procyanidins (Condensed Tannins) in Green Cell Suspension Cultures of Douglas Fir Compared with Those in Strawberry and Avocado Leaves by Means of C(18)-Reversed phase Chromatography. AB - The procyanidins (the most common type of proanthocyanidin or condensed tannin) from cell suspension cultures derived from cotyledons of Douglas Fir have been compared with those isolated from leaves of strawberry and avocado. Seventy per cent methanol (v/v) extracts from 100 milligrams fresh weight samples were analyzed by a combination of C(18)-reversed-phase columns with high-performance liquid chromatography, and normal phase paper chromatography. (-)-Epicatechin and its oligomers were generally retarded longer on C(18) columns than the corresponding units made of (+)-catechin when eluted with solvents made up of 5% acetic acid alone or mixed with methanol up to 15% (v/v). Douglas fir preparations contained the most complex set of procyanidins and consisted of oligomers of catechin and epicatechin, whereas strawberry and avocado contained mainly (+)-catechin and (-)-epicatechin derivatives, respectively. PMID- 16661582 TI - S-adenosylmethionine decarboxylase of corn seedlings. AB - S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been purified 500-fold in 30% yield from the extract of etiolated corn seedlings (cv. Golden Crossbantam Bell). This preparation had a molecular weight of approximately 25,000. The K(m) value was 5 micromolar for S-adenosylmethionine. Methylglyoxal bis(guanylhydrazone), hydroxylamine, and sulfhydryl reagents (such as p hydroxymercuriphenylsulfonate and N-ethylmaleimide) were effective inhibitors of this enzyme. Germination of corn seed was accompanied by a rapid increase in enzyme activity and maximum activity occurred in 5-day-old seedlings. PMID- 16661583 TI - Relationship between ATP Level and Activity of Fusicoccin-stimulated H/K-Exchange System in Plant Tissues. AB - The treatment with fusicoccin causes a slight but significant decrease (about 15%) in the ATP level in pea-internode and maize-coleoptile segments. This decrease is detectable within 15 minutes and is accompanied by a parallel increase in O(2) uptake. Sodium azide inhibits O(2) uptake and completely blocks the stimulation of O(2) uptake by fusicoccin in both pea and coleoptile segments. Benzohydroxamic acid does not affect either basal or fusicoccin-induced O(2) uptake in maize-coleoptile sections. The drop of ATP level induced by various treatments (sodium arsenate, 2-deoxyglucose, limiting O(2), and anaerobiosis) is accompanied by a parallel inhibition of K(+) uptake in maize coleoptiles treated with or without fusicoccin. These results are consistent with the hypothesis that ATP is the energy source for the fusicoccin-activated H(+)/K(+)-exchange system. PMID- 16661584 TI - Improved Procedure for the Estimation of Nanogram Quantities of Indole-3-acetic Acid in Plant Extracts using the Indolo-alpha-pyrone Fluorescence Method. AB - The indolo-alpha-pyrone fluorescence method of determining indole-3-acetic acid (IAA) is improved by adding butylated hydroxytoluene (BHT), an antioxidant, to samples: addition of BHT increases the fluorescence intensities and decreases their variability so that amounts of IAA as small as 0.1 to 1 nanogram become measurable. Interfering compounds, 4-chloroindole-3-acetic acid and 5 hydroxyindole-3-acetic acid, can be separated from IAA by thin-layer chromatography using polyamide as the solid support, and benzene-ethyl acetate acetic acid (70:25:5, v/v) as the developing solvent. Polyamide thin-layer chromatography is also superior in purifying IAA without significant loss or decomposition.A serious problem in extracting IAA from plant tissues is the considerable loss. Losses can result from decomposition of IAA when its solutions in organic solvents are evaporated to dryness, apparently due to some impurity in the solvents. Decomposition can be eliminated by simple treatments of the solvents, i.e. addition of BHT, washing with water, and passage through cotton wool. The latter is particularly effective in the case of ethyl acetate and ether. Loss of IAA by photodecomposition is also substantial even in dried samples. Ways to minimize the conversion of indole-3-pyruvic acid to IAA during extraction procedure are also described.A revised procedure of IAA extraction and determination is proposed. Applied to etiolated shoots of maize seedlings, it enables content to be estimated (18 nanograms per gram fresh weight) with over 90% recovery in terms of [(14)C]IAA added at the beginning of the extraction. PMID- 16661585 TI - Ethylene: indicator but not inducer of phytoalexin synthesis in soybean. AB - Cell wall preparations (elicitors) from Phytophthora megasperma var. sojae increase C(2)H(4) formation, phenylalanine ammonia lyase activity, and glyceollin accumulation in soybean cotyledons within about 1.5, 3, and 6 hours after treatment, respectively. The immediate precursor of C(2)H(4), 1-aminocyclopropane 1-carboxylic acid, stimulates C(2)H(4) formation like the elicitor within 1.5 hours after administration, whereas phenylalanine ammonia lyase activity and glyceollin concentration remain unchanged. Aminoethoxyvinylglycine, a specific inhibitor of C(2)H(4) formation in higher plants, inhibits elicitor-induced C(2)H(4) formation by about 95% but has no effects on phenylalanine ammonia lyase or glyceollin accumulation. It was concluded that C(2)H(4) is a signal accompanying the specific recognition process which finally leads to the induction of phytoalexin formation, but it is not functioning as a link or messenger in the induction sequence of glyceollin accumulation. PMID- 16661586 TI - Variations in K(m)(CO(2)) of Ribulose-1,5-bisphosphate Carboxylase among Grasses. AB - A survey of the K(m)(CO(2)) values of ribulose-1,5-bisphosphate carboxylase from 60 grass species shows that enzyme from C(3) grasses consistently exhibits lower K(m)(CO(2)) than does that from C(4) grasses. Systematically ordered variation in K(m)(CO(2)) of ribulose-1,5-bisphosphate carboxylases from C(3) and C(4) grasses is also apparent and, among C(4) grasses, this shows some correlation with C(4) types. PMID- 16661587 TI - Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds. AB - Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells. PMID- 16661588 TI - Model for Stress-induced Protein Degradation in Lemna minor. AB - Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID- 16661589 TI - Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.). AB - Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. ;Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10(-5) and 10(-6) molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10(-5) and 10(-6) molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin alpha (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system. PMID- 16661590 TI - Structure of Plant Cell Walls: X. RHAMNOGALACTURONAN I, A STRUCTURALLY COMPLEX PECTIC POLYSACCHARIDE IN THE WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS. AB - The purification and characterization of a pectic polymer, rhamnogalacturonan I, present in the primary cell walls of dicots is described. Rhamnogalacturonan I accounts for approximately 7% of the mass of the walls isolated from suspension cultured sycamore cells. As purified, rhamnogalacturonan I has a molecular weight of approximately 200,000 and is composed primarily of l-rhamnosyl, d galacturonosyl, l-arabinosyl, and d-galactosyl residues. The backbone of rhamnogalacturonan I is thought to be composed predominantly of d-galacturonosyl and l-rhamnosyl residues in a ratio of approximately 2:1. About half of the l rhamnosyl residues are 2-linked and are glycosidically attached to C(4) of a d galacturonosyl residue. The other half of the l-rhamnosyl residues are 2,4-linked and have a d-galacturonosyl residue glycosidically attached at C(2). Sidechains averaging 6 residues in length are attached to C(4) of the l-rhamnosyl residues. There are many different sidechains, containing variously linked l-arabinosyl, and/or d-galactosyl residues. PMID- 16661591 TI - Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS. AB - The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues. PMID- 16661592 TI - Utility of ethidium bromide in the extraction from whole plants of high molecular weight maize DNA. AB - A procedure is reported for the isolation of high molecular weight maize DNA from whole plant tissue. Nuclei are isolated in the presence of ethidium bromide from leaf, node, and tassel or endosperm tissues and the DNA is extracted and purified. The resulting DNA has a double-strand molecular weight of about 125 kilobase pairs and a single-strand molecular weight of about 125 kilobases. The DNA is cleavable by a number of common restriction endonucleases. PMID- 16661593 TI - Light Harvesting in Anacystis nidulans Studied in Pigment Mutants. AB - Spontaneous pigment mutants of Anacystis nidulans were self-selected for improved growth in far red light (> 650 nanometers). Questions were asked about those features of the light-harvesting mechanism which altered to give the mutants improved photosynthetic performance in far red. Answers were sought by comparing pigment and reaction center concentrations for the parent and six mutants grown in gold fluorescent and in far red light. Three significant results emerged. The ratio of reaction centers for photoreactions I and II (RC1/RC2) varied by a value of about 2.1 for all clones grown in gold and a value of about 1.1 for all clones grown in far red. Alteration of the ratio was not evident in any of the mutants.Phycobilisome alterations were evident as decreased phycocyanin content in all mutants. In three mutants, allophycocyanin became the major remaining phycobilisome component. Action spectra for photoreactions I and II allowed estimates of chlorophylls serving each of the two reaction centers. Ratios of chlorophylls to reaction centers within each photosystem were chlorophyll I/RC1 = 118 +/- 11 and chlorophyll II/RC2 = 52 +/- 9 for all seven clones grown in both gold and far red light. Remarkable constancy of these ratios, in spite of wide variation in cell material, supports an hypothesis that in A. nidulans there are two chlorophyll proteins, each bearing a reaction center and chlorophylls in fixed ratio. PMID- 16661594 TI - Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION. AB - The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H(2)O as electron donor for the photoreduction of NADP, (b) NADP replaced O(2) as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O(2) at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.The physiological mechanism for the activation of latent polyphenol oxidase remains an unanswered question. Present results suggest that activation could occur through either acidification or the release of free fatty acids. PMID- 16661595 TI - Water-relation Parameters of Individual Mesophyll Cells of the Crassulacean Acid Metabolism Plant Kalanchoe daigremontiana. AB - Water-relation parameters of leaf mesophyll cells of the CAM plant Kalanchoe daigremontiana have been determined directly in cells of tissue slices using the pressure-probe technique. Turgor pressures measured in cells of the second to fourth layer from the cut surface showed an average of 1.82 +/- 0.62 bar (mean +/ sd; n = 157 cells). This was lower than expected from measurements of the osmotic pressure of the cell sap. The half-time (T(1/2)) for water-flux equilibration of individual cells was 2.5 to 8.8 seconds. This is the fastest T(1/2) found so far for higher-plant cells. The calculated values of the hydraulic conductivity were in the range of 0.20 to 1.6 x 10(-5) centimeters second(-1) bar(-1), with an average of (0.69 +/- 0.46) x 10(-5) centimeters second(-1) bar(-1) (mean +/- sd; n = 8 cells). The T(1/2) values of water exchange of individual cells are consistent with the overall rates of water-flux equilibration measured for tissue slices.The volumetric elastic moduli (in) of individual cells were in the range 13 to 128 bar for turgor pressures between 0.0 and 3.4 bar; the average in value was 42.4 +/- 27.7 bar (mean +/- sd; n = 21 cells). This in value is similar to that observed for other higher-plant cells.The water-storage capacity of individual cells, calculated as C(c) = V/(in + pi(i)) (where V = cell volume and pi(i) = internal osmotic pressure) was 9.1 x 10(-9) cubic centimeters bar(-1) per cell, and the capacity for the tissue was 2.2 x 10(-2) cubic centimeters bar(-1) gram(-1) fresh weight. The significance of the water-relation parameters determined at the cellular level is discussed in terms of the water relations of whole leaves and the high water-use efficiency characteristic of CAM plants. PMID- 16661596 TI - Leaf Senescence and Abscisic Acid in Leaves of Field-grown Soybean. AB - Leaf senescence in field-grown soybean (Merrill) as defined by the period after full expansion, was studied by measuring abscisic acid (ABA), total soluble protein, and chlorophyll in leaves through the later part of the growing season. ABA concentrations increased significantly at the end of the season when leaves had started to turn yellow, well after total soluble protein and chlorophyll had started to decline. The results indicate that events occurring before leaf yellowing are more significant in evaluating leaf senescence since the yellowing condition and rise in ABA are effects of changes in physiological activity beginning when leaves are still green. PMID- 16661597 TI - Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts. AB - A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K(m) for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl(2) or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl(2). The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase. PMID- 16661598 TI - Zinc-inhibited Electron Transport of Photosynthesis in Isolated Barley Chloroplasts. AB - In isolated barley chloroplasts, the presence of 2 millimolar ZnSO(4) inhibits the electron transport activity of photosystem II, as measured by photoreduction of dichlorophenolindophenol, O(2) evolution, and chlorophyll a fluorescence. The inhibition of photosystem II activity can be restored by the addition of the electron donor hydroxylamine or diphenylcarbazide, but not by benzidine and MnCl(2). These observations suggest that Zn inhibits electron flow at the oxidizing side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide. No inhibition of photosystem I-dependent electron transport by 3 millimolar ZnSO(4) is observed. However, with concentrations of ZnSO(4) above 5 millimolar, photosystem I activity is partially inactivated. Washing Zn(2+)-treated chloroplasts partially restores the O(2) evolving activity. PMID- 16661599 TI - Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.): I. GENOTYPIC VARIATION. AB - The study presented here was an extension of a preceding field project concerned with changes in N metabolism of four maize hybrids during grain development. The objectives were to relate uptake, flux, and reduction of nitrate to accumulation of reduced N in growth-chamber-grown seedlings of the same four hybrids and to compare these results with those obtained in the field study.Hybrid D took up more nitrate than the other three hybrids, primarily because of a larger root system. The correlations between total N (nitrate plus reduced N plant(-1)) accumulated by harvest and root dry weight or shoot to root ratios were r = +0.97 and -0.90, respectively. Correlations with shoot dry weight were low. Although the larger root system indicates enhanced partitioning of photosynthate to the root of hybrid D, the observations made do not elucidate the role of photosynthate in increasing nitrate uptake. There was no genetic difference in partitioning of nitrate (per cent of total) among the plant parts; however, the hybrids differed in amounts of nitrate stored in stalks and midribs. Hybrids D and B accumulated more nitrate than A and C.Although two of the hybrids (A and C) with highest nitrate reductase activity had the lowest concentrations of nitrate in all plant parts, nitrate reductase activity was not correlated with accumulation of nitrate or reduced N for the four hybrids. Uptake and flux of nitrate were not numerically related to accumulation of reduced N for the four hybrids. Among the four hybrids, nitrate flux was not associated with level of leaf nitrate reductase activity. None of the individual parameters, as measured, would serve as an index for reduced N accumulation for these four hybrids. When the hybrid pairs were compared separately, it was evident that both rate of nitrate flux and level of nitrate reductase activity affect the accumulation of reduced N by the plant.Relative to the other hybrids, hybrid D that accumulated the most reduced N and nitrate as a 23-day-old seedling had the least reduced N in grain plus stover at maturity under field conditions. Hybrid C that had high nitrate reductase activity as a seedling had low nitrate reductase activity after anthesis under field conditions. These changes in metabolic activities with plant development and different environments illustrate the problems encountered in attempting to develop simple physiological or biochemical screening criteria useful in identifying superior cultivars at the seedling stage. PMID- 16661600 TI - Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.): II. EFFECT OF NUTRIENT NITRATE CONCENTRATION. AB - Two maize hybrids were grown under growth chamber conditions on solution or vermiculite medium that contained 2.5, 7.5, or 15 millimolar nitrate. The objectives were to determine: (a) the effect of nitrate supply on N metabolism and growth and (b) the interrelationship between nitrate uptake, flux, and reduction on the accumulation of reduced N and nitrate by the various plant parts and for the whole plant.Increases in nutrient nitrate concentration caused increases in (a) shoot, but not root, dry weight and (b) nitrate uptake, flux, and reduction and accumulation of nitrate and reduced N by the aerial plant parts of both hybrids. Increases in nitrate supply resulted in decreases in nitrate reductase activity and negligible increases in reduced N in the roots of both hybrids. At 2.5 and 15 millimolar, but not at 7.5 millimolar, external nitrate, hybrid B had higher rates of nitrate uptake and flux. Hybrid B also had lower nitrate reductase activity at all levels of external nitrate and accumulated less reduced N than did hybrid C, except when the plants were grown at 2.5 millimolar nitrate. Correlation values between nitrate uptake and flux were significant for each hybrid and for both hybrids, whereas correlations between nitrate reductase activity and flux and nitrate reductase activity and uptake were significant only for a given hybrid. The correlation values (pooling of all data) between (a) nitrate uptake, flux, or reduction and the accumulation of reduced N by the whole plant, and (b) flux x nitrate reductase activity and accumulation of reduced N were all positive and statistically significant. Because nitrate uptake, flux, and reduction (as measured) were all closely associated with the accumulation of reduced N by the plant, all three parameters should be measured in attempts to estimate the genetic potential of a maize genotype to accumulate reduced N. PMID- 16661601 TI - Auxin Transport Inhibitors: IV. EVIDENCE OF A COMMON MODE OF ACTION FOR A PROPOSED CLASS OF AUXIN TRANSPORT INHIBITORS: THE PHYTOTROPINS. AB - The more active members of a proposed class of auxin transport inhibitors have been shown to have the ability to inhibit the active movement of auxin at concentrations where they have little effect on auxin action and no significant auxin activity. They have also been shown to give rise to characteristic biphasic dose-response curves on cress root growth. Based on these physiological similarities and other common physiological properties, it is concluded that they may achieve their effects by a common mode of action which differs from that of other known auxin transport inhibitors. It is suggested that the name "phytotropins" be given to the class of auxin transport inhibitors now defined by a similar mode of action and common chemical properties. PMID- 16661602 TI - Effect of temperature on glycerol retention in the halotolerant algae dunaliella and asteromonas. AB - Algae of the genera Dunaliella and Asteromonas can maintain extremely high concentration gradients (>10(4)) of glycerol between the intracellular space and the medium. This unique ability is highly temperature-dependent. Treating the algae for several minutes at temperatures exceeding 60 C causes complete release of all the internally held glycerol; 50% release occurs around 50 C, but essentially none is released below 40 C. A similar behavior was observed in several species of Dunaliella, and one of Asteromonas and is independent of the salt concentration of the medium. The underlying mechanism may involve a temperature-dependent conformational transition of a component of the cellular membrane which is essential for glycerol impermeability. PMID- 16661603 TI - Apparent Bicarbonate Uptake and Possible Plasmalemma Proton Efflux in Chara corallina. AB - It is shown that the apparent uptake of bicarbonate by cells of Chara corallina could be the result of a proton efflux coupled to extracellular production of CO(2) from bicarbonate, with CO(2) being taken up by the cell. The theoretical results presented here show that the influx of CO(2) across the plasmalemma can be much greater than previously thought, if there is a large efflux of protons across the plasmalemma, and that, if this occurs, there would be a much steeper gradient of pH near the cell surface than previously thought possible. PMID- 16661604 TI - Influence of After-ripening on Phytochrome Control of Seed Germination in Two Varieties of Lettuce (Lactuca sativa L.). AB - The reversible photoreaction on seed germination of two varieties of lettuce differs remarkably not only with the variety but also with the germination temperature and the physiological conditions of seeds caused by after ripening.Grand Rapids showed reversible photoreaction at 25 C with 0 to 6 months and at 30 C with 5 to 6 months after-ripening. MSU 16 did not show any reversible photoreaction at 30 C with 0 to 6 months or at 25 C with 0 to 3 months after ripening although they were completely reversible with 4 to 6 months after ripening.These two varieties of lettuce seeds, however, showed reversible photoreaction at 20 C when they were sown immediately after harvest or after 1 and 2 months after-ripening. The photoreversion decreased after the 4-to 6-month stage. PMID- 16661605 TI - Increase in Internode Length of Phaseolus lunatus L. Caused by Inoculation with a Nitrate Reductase-deficient Strain of Rhizobium sp. AB - Dramatic differences in the height of lima beans (Phaseolus lunatus L.) treated with two different Rhizobium strains were studied. Lima beans were grown in Perlite in the greenhouse or in a minus-N culture solution in the growth chamber. The plants were inoculated with either Rhizobium sp. (lima bean) strain 127E15, which contains the constitutive nitrate reductase activity, or strain 127E14, which lacks that activity. For up to 3 weeks, no growth differences were observed in the plants inoculated with either strain. Five weeks after inoculation, however, those plants inoculated with strain 127E14 were significantly taller and had a larger number of leaves than those inoculated with strain 127E15. The difference in plant height was the result of increased internode elongation caused by inoculation with Rhizobium sp. 127E14. This response was observed with all lima bean cultivars tested, including Henderson, Fordhook, Allgreen, and Early Thorogreen. The growth difference occurred in plants cultured in the greenhouse or in the growth chamber.No differences were observed in fresh weights of nodules, roots, axes, leaves, or flowers. The number of nodules, pods, or flowers also did not differ. Acetylene reduction rates and total N content were equal in plants inoculated with strains 127E14 or 127E15.Inasmuch as only internode length and leaf number differed between Rhizobium treatments, the response appeared to be hormone-mediated. Application of gibberellic acid to the apex of plants inoculated with strain 127E15 caused an increase in plant height similar to that observed in untreated plants inoculated with strain 127E14. Conversely, the height of plants infected with strain 127E14 was decreased by application of gibberellin synthesis inhibitors to the root system. These data suggest that the increased growth caused by inoculation with strain 127E14 could be the result of increased gibberellin synthesis in lima bean nodules infected with that strain. Whether this response was related to the absence of nitrate reductase activity in strain 127E14 is unknown. PMID- 16661606 TI - Autodigestion in crude extracts of soybean leaves and isolated chloroplasts as a measure of proteolytic activity. AB - Two methods of measuring protein breakdown resulting from self-digestion during incubation in extracts of soybean leaves were examined. The release of free alpha amino-nitrogen was measured with ninhydrin, and the disappearance of the large subunit of ribulose bisphosphate carboxylase (RuBPcase) was followed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rates of protein breakdown were measured as a function of temperature, pH, and leaf developmental stage and in the presence of various proteinase inhibitors. These treatments had differential effects on apparent proteolysis, depending on the method used. Determination of the ratio of alpha-amino-nitrogen plus peptide bond-nitrogen to alpha-amino-nitrogen indicated that the ninhydrin method detected the activity of exopeptidases preferentially. The disappearance of the large subunit of RuBPCase as shown on gels was due primarily to the activity of endopeptidases. The sensitivity of the two types of proteolytic degradation to proteinase inhibitors differed.Determination of temporal changes in proteolytic activity during leaf development showed that total proteolytic activity, measured by either method, increased during leaf expansion and maturation and decreased during senescence. Incubation of intact isolated chloroplasts at 37 C resulted in the breakdown of the large subunit of RuBPcase, although the chloroplasts contained no measurable proteinase activity as determined by the release of alpha-amino-nitrogen during the incubation. No acid proteinase (pH 4.5) activity was detected in the chloroplasts when hemoglobin was used as a substrate. These results indicate that the proteinases which break down RuBPCase in isolated chloroplasts may not be detectable by conventional assay procedures. PMID- 16661607 TI - Hemoglobin-digesting Acid Proteinases in Soybean Leaves: CHARACTERISTICS AND CHANGES DURING LEAF MATURATION AND SENESCENCE. AB - Three proteinases which digest hemoglobin rapidly at acid pH (3.5 to 4.5) were identified in crude extracts of soybean (Merr.) leaves and separated by chromatography on DEAE-cellulose. All three enzymes were endopeptidases as judged by the ratio of alpha-amino-nitrogen plus peptide nitrogen over alpha-amino nitrogen in the trichloroacetic acid-soluble portion of hemoglobin digests. Proteinase I did not bind to diethylaminoethyl cellulose and was not inhibited by any of the proteinase inhibitors tested. Proteinase II was partially inhibited by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and p-chloromercuribenzoate. The inhibition by phenyl-methylsulfonyl fluoride can probably be accounted for by the presence of contaminating carboxypeptidase. Proteinase III was the most anionic of the three and required the presence of sulfhydryl reagents to prevent the irreversible loss of activity. All the proteinase preparations digested soy-bean ribulose bisphosphate carboxylase as shown by the disappearance of the large subunit of that protein, when partially digested preparations were subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. These experiments confirmed that the three proteinases were endopeptidases. All three proteinases were present throughout leaf development; proteinase I predominated in expanding leaves, whereas proteinase III became the predominant enzyme as the leaves matured. Senescence (yellowing) was associated with a decline in the activities of all three proteinases. PMID- 16661608 TI - Water Relations of Cotton Plants under Nitrogen Deficiency: III. STOMATAL CONDUCTANCE, PHOTOSYNTHESIS, AND ABSCISIC ACID ACCUMULATION DURING DROUGHT. AB - Nitrogen nutrition exerted a strong effect on stomatal sensitivity to water stress in cotton. In well-watered plants grown with 0.31 millimolar N in the nutrient solution, stomata closed at a water potential of -9 bars even though the wilting point was below -15 bars. For each doubling of nutrient N level, the water potential for stomatal closure decreased by about 2 bars. Elevated intercellular CO(2) concentrations caused only slight stomatal closure regardless of N nutrition. Exogenous abscisic acid (ABA) greatly increased stomatal sensitivity to elevated CO(2) concentrations.PLANTS SUBJECTED TO WATER STRESS GAVE THE FOLLOWING RESPONSES: (a) decreased stomatal conductance at ambient external CO(2) concentration; (b) increased stomatal sensitivity to elevated CO(2) concentrations; (c) decreased mesophyll conductance to CO(2); and (d) increased endogenous ABA content. All of these responses to stress occurred at a higher water potential in N-deficient plants than in normal plants. The results show that N nutrition and water stress interact to control ABA accumulation and the events regulated by that accumulation. PMID- 16661609 TI - Presence of Both Photosystems in Guard Cells of Vicia faba L: IMPLICATIONS FOR ENVIRONMENTAL SIGNAL PROCESSING. AB - A new procedure is reported for high-yield isolation of guard cell protoplasts from Vicia faba L. Delayed light emission and P(700) content plus absorption and fluorescence emission spectra of these protoplast extracts are reported. It is concluded that both photosystems are present. The presence of photosystem II and the absence of the reductive-step enzyme of the Calvin-Benson Cycle (Outlaw WH Jr, J Manchester, CH DiCamelli, DD Randall, B Rapp, GM Veith 1979 Proc Natl Acad Sci USA 76: 6371-6375) in a cell has no precedent in the literature. It is speculated that noncyclic photosynthetic electron flow is an environmental sensor which causes stomata to remain open in light. PMID- 16661610 TI - Photosynthesis, Growth, and Ultraviolet Irradiance Absorbance of Cucurbita pepo L. Leaves Exposed to Ultraviolet-B Radiation (280-315 nm). AB - Net photosynthesis, growth, and ultraviolet (UV) radiation absorbance were determined for the first leaf of Cucurbita pepo L. exposed to two levels of UV-B irradiation and a UV-B radiation-free control treatment. Absorbance by extracted flavonoid pigments and other UV-B radiation-absorbing compounds from the first leaves increased with time and level of UV-B radiation impinging on leaf surfaces. Although absorbance of UV-B radiation by extracted pigments increased substantially, UV-B radiation attenuation apparently was insufficient to protect completely the photosynthetic apparatus or leaf growth processes. Leaf expansion was repressed by daily exposure to 1365 Joules per meter per day of biologically effective UV-B radiation but not by exposure to 660 Joules per meter per day. Photosynthesis measured through ontogenesis of the first leaf was depressed by both UV-B radiation treatments. Repression of photosynthesis by UV-B radiation was especially evident during the ontogenetic period of maximum photosynthetic activity. PMID- 16661611 TI - Light or ethylene treatments induce transverse cell enlargement in etiolated maize mesocotyls. AB - Dark-grown maize seedlings (hybrid WF-9 x 38-11) exposed for 1 or more hours to white light and then returned to darkness developed mesocotyls with enlarged apical diameters. This swelling response was an all-or-none response, and the fraction of the seedling population that showed the response depended on seedling age at irradiation. Irradiation of the coleoptile alone was nearly as effective in causing this response as was irradiation of the nodal region of the epicotyl, but irradiation of the mesocotyl base was ineffective. Removal of the coleoptile prior to irradiation did not prevent the formation of the light-induced swelling. Exogenously applied C(2)H(4) (10 microliters per liter) for 24 hours in dark also induced swelling of the mesocotyl. The swelling induced in the intact seedlings was localized in the apical mesocotyl tissues with either light or C(2)H(4) treatment, and maximal response to both treatments occurred with 3- to 4-day-old seedlings. Swelling of the mesocotyl was the result of transverse cell enlargement, not increase of cell numbers. The evidence suggests that light and C(2)H(4) induce mesocotyl swelling in intact maize shoots by a common mechanism. PMID- 16661612 TI - Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus. AB - The oxyhydrogen reaction in the presence and absence of CO(2) was studied in H(2) adapted Scenedesmus obliquus by monitoring the initial rates of H(2), O(2), and (14)CO(2) uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere O(2), the pH(2) was varied from 0 to 1 atmosphere. Whereas the rate of O(2) uptake increased by only 30%, the rate of H(2) uptake increased severalfold over the range of pH(2) values. At 0.1 atmosphere H(2) and 0.02 atmosphere O(2), rates for H(2) and O(2) uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH(2) was changed from 0 to 1 atmosphere, the quotient H(2):O(2) changed from 0 to roughly 2. This change may reflect the competition between H(2) and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H(2) uptake. KCN inhibited equally respiration (O(2) uptake in the absence of H(2)) and the oxyhydrogen reaction in the presence and absence of CO(2). The uncoupler carbonyl cyanide p trifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO(2) has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast. PMID- 16661613 TI - Light induction of phosphoenolpyruvate carboxylase in etiolated maize leaf tissue. AB - An antibody for phosphoenolpyruvate carboxylase was used to isolate and to quantitate the enzyme from greening maize (cv. KOU 6) leaves. The increase in enzyme activity during greening was due to de novo synthesis, which was paralleled by increases in enzyme protein and incorporation of leucine. The light induced activity was due to one specific isoenzyme. The action spectrum for enzyme synthesis had red and blue peaks. PMID- 16661614 TI - Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves. AB - Aldose-6-phosphate reductase (alditol 6-phosphate:NADP 1-oxidoreductase) was isolated and characterized from mature apple leaves (Malus domestica cv. Starkrimson). The enzyme was purified 79-fold. The enzyme catalyzed the following reversible reaction: d-glucose 6-phosphate + NADPH + H(+) right arrow over left arrow d-sorbitol 6-phosphate + NADP(+). No activity was detected when NAD(+) was substituted for NADP(+) or when NADH was substituted for NADPH. The enzyme reduced d-galactose 6-phosphate at a higher rate than d-glucose 6-phosphate. d Mannose 6-phosphate and 2-deoxy-d-glucose 6-phosphate were reduced at low rates. d-Glucose 1-phosphate, d-fructose 6-phosphate, d-ribose 5-phosphate, d-glucose, and sorbitol did not serve as substrates. The pH optimum for both d-sorbitol 6 phosphate oxidation and d-glucose 6-phosphate reduction was 9.5. The K(m) values for d-sorbitol 6-phosphate oxidation and d-glucose 6-phosphate reduction were 3.9 and 20 millimolar, respectively. AgNO(3) (0.1 millimolar) and p chloromercuribenzoate (1.0 millimolar) completely inhibited the enzyme.Aldose-6 phosphate reductase activity was also detected in mature leaves from Golden Delicious and Antonovka apples (Malus domestica), Conference and Bartlett pears (Pyrus communis), Redhaven peach (Prunus persica), and Perfection apricot (Prunus armeniaca). This suggests that the enzyme has a wide distribution and plays an important role in sorbitol synthesis. PMID- 16661615 TI - Physical and morphological constraints on transport in nodules. AB - For active nodule nitrogen fixation, O(2), N(2), and carbohydrate must be transported throughout the nodule. No quantitative analysis of these transport processes in the nodules has been presented. By invoking several simplifying assumptions, a second-order differential equation for the various gradients and concentrations in the nodule was solved. Even though the nodule can only be approximated in this analysis, it indicates clearly that intercellular gas spaces must exist in nodules for adequate O(2) distribution. To preserve low O(2) concentrations and protect the nitrogenase, these gas spaces cannot be in direct contact with the ambient atmosphere. It is hypothesized that a gas barrier exists in the cortical region of the nodule to limit O(2) diffusion. This barrier would not substantially inhibit N(2) transport. Carbohydrate transport from the vascular tissue via diffusion in the liquid phase can adequately accommodate the requirements within the nodule. PMID- 16661616 TI - Auxin-regulated Wall Loosening and Sustained Growth in Elongation. AB - It is proposed that auxin regulates and coordinates both wall loosening and the supply of wall materials in elongation. The tenets of the proposal allowed testable predictions. It was determined that, if the cell walls of Glycine max L. var. Wayne hypocotyl segments are maintained in a loosened state (by excising the segments directly into pH 4 medium), exogenous auxin induced only the second response. It was also predicted and confirmed that elongating systems, e.g. pea epicotyl, with certain early auxin-induced growth kinetics (an initial high non steady-state rate followed immediately by a drop to a lower steady-state rate) would show a transient second response (in addition to the usual transient first response) when stimulated by pH 4 medium. Finally, it is pointed out that recent results which establish the existence of auxin-induced elongation-associated proteins support the proposition that auxin coordinates wall loosening and the supply of wall materials in elongation. PMID- 16661617 TI - Effect of sugars and amino acids on membrane potential in two clones of sugarcane. AB - Sugarcane (Saccharum officinarum L.) leaf parenchyma cells bathed in 1X solution maintained an average membrane potential of -135 millivolts in the dark. No difference in membrane potential was found between clones 51 NG 97 and H50 7209. An electrogenic pump appears to contribute to membrane potential in these cells. Sugars (25 millimolar) added externally caused the following membrane potential depolarizations (in millivolts) in clone 51 NG 97: glucose, 18 +/- 4; galactose, 24 +/- 7; 3-O-methylglucose, 10 +/- 4; sucrose, 22 +/- 3; fructose, 21 +/- 7; raffinose, 9 +/- 3; mannitol, 0; lactose, 0; melibiose, 0; and 1-O-methyl-alpha galactose, 0. Glycine (25 millimolar) and serine (10 millimolar) caused depolarizations of 47 +/- 7 and 23 +/- 2 millivolts, respectively. Depolarization shows saturation kinetics with respect to glucose concentration, with a K(m) of 3 to 6 millimolar. The metabolic inhibitors KCN and salicyl hydroxamic acid together caused depolarization of the membrane potential and greatly inhibited depolarization by 25 millimolar glucose and 25 millimolar raffinose. In a series of substitution experiments, glucose (25 millimolar) caused almost total inhibition of depolarization by raffinose, sucrose, and 3-O-methylglucose (all 25 millimolar), but only partial inhibition of depolarization to 25 millimolar glycine. Glycine (25 millimolar), also, only partially inhibited depolarization by 25 millimolar glucose. Total depolarization to 25 millimolar glycine and 25 millimolar glucose was comparable to the amount of depolarization of membrane potential caused by 1 millimolar KCN plus 1 millimolar salicyl hydroxamic acid. The results are consistent with a co-transport mechanism of membrane transport, with sugars and amino acids being transported by separate carrier systems. PMID- 16661618 TI - Purification and Properties of a Protein Which Binds Cytokinin-active 6 Substituted Purines. AB - A protein which binds 6-substituted purines of the cytokinin type with relatively high affinity has been extensively purified from wheat germ. Conventional chromatographic techniques, as well as an affinity matrix to which a cytokinin was covalently coupled, were used in the purification. The wheat germ cytokinin binding protein (CBF-1) has four unlike subunits and an apparent molecular weight of 183,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.CBF-1 is saturated at one cytokinin molecule per tetramer with a K(d) for 6-benzylaminopurine of 5 x 10(-7) molar. The protein exists both on the native wheat germ ribosome (1 molecule CBF-1 per 80S ribosome) and free in the cytosol with approximately three copies of the latter for each of the former. Data from affinity chromatography studies and cross-linking experiments strongly suggest that a specific binding site for CBF-1 occurs on the wheat germ ribosome. PMID- 16661619 TI - Co-transport of Potassium and Sugars across the Plasmalemma of Mesophyll Protoplasts. AB - Sugars (sucrose + hexoses) produced photosynthetically by isolated mesophyll protoplasts of wheat and tobacco were effluxed across the plasma membrane (3 to 10 micromoles hexose equivalents per milligram chlorophyll per hour). The efflux was sensitive to uncouplers and oligomycin which indicated a requirement for energy. A proton gradient was probably not coupled directly to the transport because changing the proton gradient across the plasma membrane by varying the pH of the medium or by adding sodium acetate had no significant effect on the rate of sugar release.A release of K(+) was associated with sugar efflux from the protoplasts. The molar ratio of K(+) to sugar varied between 1.5 and 2.5, depending on the species. Exogenous CKl, RbCl, and LiCl (50 millimolar each), but not NaCl or CsCl, significantly inhibited sugar efflux. Conditions that reduced sugar efflux (exogenous KCl, LiCl, mersalyl, or oligomycin) also reduced K(+) release and caused a time-dependent reduction in photosynthetic sucrose formation and increased amino acid and starch formation. Results obtained support the postulate that a K(+) symport is involved in the transport of sugar across the energized plasmalemma of photosynthetically active mesophyll cells. PMID- 16661620 TI - Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II. AB - The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1 dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO(2) photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight. PMID- 16661621 TI - Localization of Fungal Components in the Pea-Fusarium Interaction Detected Immunochemically with Anti-chitosan and Anti-fungal Cell Wall Antisera. AB - Antisera specific for purified cell walls of Fusarium solani f. sp. pisi and phaseoli and of shrimp shell chitosan were utilized as immunochemical probes to determine the location of fungal components in the pea-Fusarium interaction.Within 15 minutes after inoculation, fungal cell wall components appear to enter the plant cell and to accumulate inside the plant cell wall as fungal growth on the plant tissue is inhibited. The accumulation patterns of chitosan and all components containing hexosamine polymers resembled those of the fungal wall components.Chitosan is present on, and is released from, the outer surface of the fungal spore. Within 15 minutes after applying [(3)H]chitosan to the surface of the plant tissue, the label is readily detectable within the plant cytoplasm and conspicuously detectable within the plant nucleus. It is proposed that the potential for transport of chitosan between the spores of Fusarium solani and pea cells, in addition to its potential to inhibit fungal growth and elicit disease resistance responses, suggests chitosan has a major regulatory role in this host-parasite interaction. PMID- 16661622 TI - Chilling Susceptibility of the Blue-green Alga Anacystis nidulans: I. EFFECT OF GROWTH TEMPERATURE. AB - Effects of chilling treatment on the photosynthetic activities and the light absorption and fluorescence spectra were investigated in intact cells of the blue green alga Anacystis nidulans that were grown at different temperatures. When the algal cells grown at 38 C were treated at 0 C for 10 minutes, the photosynthesis and the Hill reaction with 1,4-benzoquinone were significantly inactivated and the light-absorption spectrum of carotenoids was modified. These parameters showed very similar temperature dependencies in the chilling susceptibility and the temperature regions critical for the susceptibility depended on the growth temperature. The midpoint values for the critical temperature regions were 4, 6, and 12 C in cells grown at 28, 33, and 38 C, respectively. It is proposed that a common mechanism would underlie the chilling susceptibility of the photosynthesis, the Hill reaction, and the carotenoid absorption spectrum. The decoupling of excitation transfer from allophycocyanin to chlorophyll a at the chilling temperatures occurred very slowly and is attributed to a somewhat different mechanism of the chilling susceptibility. PMID- 16661623 TI - Chilling Susceptibility of the Blue-green Alga Anacystis nidulans: II. STIMULATION OF THE PASSIVE PERMEABILITY OF CYTOPLASMIC MEMBRANE AT CHILLING TEMPERATURES. AB - Potassium ions and amino acids were found to leak from the cytoplasm to the outer medium when the blue-green alga, Anacystis nidulans, was exposed to the chilling temperatures. The leakage was marked below the critical temperature regions, the midpoint values for which were around 5 and 14 C in cells grown at 28 and 38 C, respectively. These temperature regions coincided with those critical for the susceptibility of the photosynthetic activities and the carotenoid absorption spectrum previously studied (Ono TA, N Murata 1981 Plant Physiol 67: 176 181).Potassium and magnesium ions in the cell suspension medium protected the algal cells from the chilling-induced damage of the Hill reaction with 1,4 benzoquinone. The activity of the Hill reaction which had been diminished by the first chilling treatment in a low salt medium was restored by the second chilling treatment of a high salt medium. The chilling susceptibility of the Hill reaction could be attributed to the leakage of cations from the cytoplasm due to increased permeability of the cytoplasmic membrane at the chilling temperatures.A mechanism is proposed to interpret the chilling susceptibility of A. nidulans: (a) at chilling temperatures, the bilayer lipids of the cytoplasmic membrane are in the phase separation state; (b) ions and solutes having low molecular weights leak from the cytoplasm to the outer medium when the lipids of the cytoplasmic membrane are in the phase separation state; (c) decreases in the intracellular concentrations of ions and solutes degrade the physiological activities of the cells. PMID- 16661624 TI - Hydroxyproline Enhancement as a Primary Event in the Successful Development of Erysiphe graminis in Wheat. AB - Host cell wall hydroxyproline enhancement was observed in the successful development of the parasite Erysiphe graminis DC. f. sp. tritici em Marchal (MS 1) on wheat (em Thell). Hydroxyproline enhancement, which was observed only in susceptible hosts, was detected as early as 25 hours after infection. This observation suggests that the increase in cell wall hydroxyproline is a primary event in the host-pathogen interaction of Erysiphe graminis in wheat. PMID- 16661625 TI - Partial purification and specificity of isofloridoside phosphatase. AB - A phosphatase has been partially purified from crude extracts of Poterioochromonas malhamensis. The enzyme appears to be specific for alpha galactosyl-(1 --> 1)-glycerol 3-phosphate as it is relatively inactive towards glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and sn-glycerol 3 phosphate. PMID- 16661626 TI - Organelle Membranes from Germinating Castor Bean Endosperm: II. ENZYMES, CYTOCHROMES, AND PERMEABILITY OF THE GLYOXYSOME MEMBRANE. AB - Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis.Enzyme activities involving the cofactors NADH or CoA were measurable in broken, but not in intact, glyoxysomes. Thus, it appears that cofactors for enzymes within the organelle cannot pass through the membrane. PMID- 16661627 TI - Response of Tomato Plants to Stressful Temperatures : INCREASE IN ABSCISIC ACID CONCENTRATIONS. AB - To investigate the abscisic acid (ABA) production of tomato (Mill.) plants in response to diurnal stressful temperatures, five-week old seedlings were exposed to day/night temperatures of 10/5, 15/10, 25/15, 35/25, or 45/35 C. The daylength was 16 hours with a light intensity of approximately 400 microeinsteins per meter per second. Plant tops were sampled at 12, 24, 48, and 72 hours. Free, alkaline hydrolyzable (conjugated), and total ABA quantities were measured using standard gas chromatographic techniques. All temperature regimes significantly increased both free and conjugated ABA levels over concentrations in control plants (25/15 C). The highest ABA levels were observed in plants exposed to the coolest temperature of 10/5 C. Since normal water potentials were obtained in plants of all treatments, the observed ABA response was not due to temperature-induced water stress. Therefore, temperature stress, like several other environmental stresses, induces the plant to produce high levels of ABA. Because of the similar involvement of ABA in temperature-induced and other environmental stresses, ABA may be a common mediator for all plant stresses. PMID- 16661628 TI - Partitioning of carbon and nitrogen and the nutrition of root and shoot apex in a nodulated legume. AB - Empirically based models depicting exchanges of C, N, and H(2)O in phloem and xylem among organs of nodulated white lupin (Lupinus albus cv Ultra) were constructed for the interval 51 to 58 days after sowing. Information was incorporated on the economy of C, N, and H(2)O in plant parts, the solute composition of transport fluids collected at selected sites on the plant, and the photosynthetic inputs, transpirational losses, and translocatory activities of different age groups of leaflets and stem + petiole segments of the shoot. Partitioning of C and N showed preferential transfer of N to the shoot apex, which imported 13 milligrams C per milligram N, compared with 54 milligrams C per milligram N for the nodulated root. Leaves translocated assimilates at a C:N weight ratio of 43 to 59, and older leaves serving the roots produced the translocate most rich in N relative to C. The shoot apex was enriched with N, additional to its intake from leaves, by direct uptake of xylem fluid (C:N ratio, 2.4) and receipt of nitrogenous solutes transferred from xylem to upward-moving phloem streams in upper regions of the stem. The models for flow of N and H(2)O indicated that xylem streams passing to leaves were substantially less rich in N than the adjacent stream moving through the body of the stem and that a progressive increase in concentration of N occurred within stem xylem elements from base to top of the shoot. This apparently resulted from an abstraction of N from xylem of departing leaf traces, possibly by xylem transfer cells, and a subsequent feedback of this N to xylem streams passing on up the shoot. Upper leaves and shoot apex, therefore, acquired more N from xylem per unit of H(2)O transpired than lower parts of the shoot. PMID- 16661629 TI - Synthesis, Storage, and Utilization of Amino Compounds in White Lupin (Lupinus albus L.). AB - Changes in total N and in free amino compounds were followed during growth of nodulated white lupin. Leaflets contained the greatest fraction of plant N but had lower proportions (1 to 4%) of their N in soluble amino form than stem + petioles (10 to 27%) and reproductive parts (15 to 33%). Mobilization of free amino compounds from plant parts to fruits contributed at most only 7% of the total N intake of fruits, compared with 50% in mobilization of other forms of N and 43% from fixation during fruiting. Asparagine was usually the most abundant free amino compound in plant parts, followed by glutamine and alanine. Valine, glycine, isoleucine, aspartic acid and gamma-aminobutyric acid comprised the bulk of the remaining soluble amino N. Composition of tissue pools of amino-N closely resembled that of xylem and phloem exudates. Data on N flow and utilization were combined with information on composition of transport fluids to quantify syntheses, exchanges, and consumptions of asparagine, glutamine, aspartic acid, and valine by organs of the 51- to 58-day plant. These amino compounds carried 56, 29, 5, and 2%, respectively, of the N exported from nodules and contributed in roughly commensurate proportions to transport exchanges and N increments of plant parts. There were, however, more than expected involvements of glutamine and valine in mobilization of N from lower leaves, of asparagine in xylem to phloem transfer, and of aspartic acid in cycling of N through the root, and there was a less than expected participation of aspartic acid in xylem to phloem transfer and in phloem translocation to the shoot apex. The significance of these differences is discussed. PMID- 16661630 TI - Chemiluminescence in Wounded Root Tissue : EVIDENCE FOR PEROXIDASE INVOLVEMENT. AB - Root and stem segments from soybean (Merrill cv. ;Bragg') showed an enhanced chemiluminescence upon mechanical injury. Roots emitted more light than did stems. Light emission was diminished by CN(-) and N(3) (-) but was not affected by rotenone and antimycin A. Catalase quenched chemiluminescence in wounded root segments as did ascorbic acid and hydroquinone. Superoxide dismutase addition resulted in a small diminution in light emission, but mannitol, an OH. scavenger, was without effect. The addition of H(2)O(2) to wounded root segments markedly elevated chemiluminescence in the presence of air as well as under N(2). It is concluded that peroxidases, found abundantly in roots, predominantly contribute to light emission in wounded plant tissue. PMID- 16661631 TI - Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: II. CHARACTERISTICS OF THE LATE FORMATION OF ACTIVE PHOTOSYSTEM II REACTION CENTERS DURING FIRST STAGES OF GREENING. AB - During light-induced greening of dark-grown, nondividing Euglena gracilis Z, there is a delay of about 10 hours in the formation of active photosystem II (PSII) reaction centers compared to chlorophyll synthesis. Experiments with greening under different light intensities rule out the possibility that this delay results from a late induction of active PSII reaction center formation when a definite amount of chlorophyll is attained in the early greened cells. Experiments on greening after preillumination show that this delay does not originate in a long, light-induced formation of specific synthesizing machinery for reaction center components. Experiments with greening in the presence of streptomycin show that, when this inhibitor of protein synthesis by chloroplastic ribosomes is added to dark-grown, preilluminated cells or to cells already greened for 24 hours, the formation of active PSII reaction centers is inhibited after a time which depends on the light intensity used for greening. Under very low light intensity (150 lux), the addition of streptomycin to 24-hour greened cells does not prevent further development of functional chloroplasts. These observations lead to the conclusion that streptomycin-insensitive chloro-plast development occurs due to syntheses of cytoplasmic origin and of light-induced pools of components synthesized early by chloroplastic ribo-somes. Conformational changes requiring time may allow the insertion of components necessary for the reorganization of PSII reaction centers in the developing thylakoid after synthesis. This hypothesis accounts for the observed delay in PSII reaction center formation compared to chlorophyll synthesis. PMID- 16661632 TI - Isolation of Functionally Intact Rhodoplasts from Griffithsia monilis (Ceramiaceae, Rhodophyta). AB - A procedure is described for isolating photosynthetically active rhodoplasts ("red algal chloroplasts") from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO(2) fixation and CO(2)-dependent O(2) evolution in the order of 200 micromoles per milligram chlorophyll a per hour when illuminated with red or green light and were approximately 80% intact. The response of the rate of photosynthesis to the inorganic phosphate and pyrophosphate concentrations in the medium was qualitatively similar to that previously reported for spinach chloroplasts. Osmotically shocked rhodoplasts evolved O(2) from ferricyanide in red, but not in green, light and were completely uncoupled. Rhodoplast envelope rupture appeared to be accompanied by phycobilisome loss from the thylakoids. PMID- 16661633 TI - Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: III. SEQUENCE OF EVENTS LEADING TO THE FORMATION OF FUNCTIONAL PHOTOSYSTEM II PHOTOSYNTHETIC UNITS. AB - Greening cells of Euglena were transferred back to darkness at different stages of chloroplast development in the presence or absence of specific inhibitors of protein synthesis. The analysis of chloroplast components showed that: (a) cycloheximide or streptomycin does not significantly inhibit the formation in darkness of active photosystem II (PSII) reaction centers if added after the lag phase for chloroplast development; (b) a limited number of active reaction centers are formed in the dark, sufficient to increase PSII reaction center to chlorophyll ratios to values close to those found in fully greened cells; (c) these dark-formed reaction centers appear to be inserted in already constituted and complete light-harvesting antennae. These results complement previous ones and lead us to propose a model for a sequential formation of PSII photosynthetic units during greening of Euglena, whereby conformational changes requiring time would allow already synthesized components of PSII reaction centers to be inserted or reorganized as active photochemical complexes in association with previously formed light-harvesting antennae. PMID- 16661634 TI - Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction. AB - The (K(+),Mg(2+))-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mol7) and stored in liquid N(2) without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg(2+), the enzyme was further activated by monovalent cations (K(+), NH(4) (+), Rb(+) >> Na(+), Cs(+), Li(+)). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K(m) of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K(+) approached simple Michaelis-Menten kinetics, with a K(m) of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K(+), and preferred Mn(2+) to Mg(2+). The results demonstrate that the (K(+),Mg(2+)) ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots. PMID- 16661635 TI - Water potential of aqueous polyethylene glycol. AB - Water potential (Psiomega) values were determined for aqueous colloids of four molecular sizes of polyethylene glycol (PEG) using freezing-point depression and vapor-pressure deficit methods. A significant third-order interaction exists between the method used to determine Psiomega, PEG molecular size, and concentration. At low PEG concentrations, freezing-point depression measurements result in higher (less negative) values for Psiomega than do vapor-pressure deficit measurements. The reverse is true at high concentrations. PEG in water does not behave according to van't Hoff's law. Psiomega is related to molality for a given PEG but not linearly. Moreover, Psiomega varies with the molecular size of the PEG. It is suggested that the Psiomega of PEG in water may be controlled primarily by the matric forces of ethylene oxide subunits of the PEG polymer. The term matricum is proposed for PEG in soil-plant-water relation studies. PMID- 16661636 TI - Increase in beta-N-Acetylglucosaminidase Activity during Germination of Cotton Seeds. AB - A marked increase in beta-acetylglucosaminidase (2-acetamido-2-deoxy-beta-d glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) activity was observed in the germinating cotyledon of cotton seeds. The enzyme was isolated from cotton seedlings and purified to study its physiological function in the germination of cotton seeds. The purification procedure involves ammonium sulfate fractionation, ion-exchange chromatography, gel filtrations, and concanavalin A-Sepharose 4B chromatography, and the purified beta-N-acetylglucosaminidase was shown to be homogeneous by disc electrophoresis. The molecular weight was estimated to be about 125,000 by gel filtration. The enzyme hydrolyzed both p-nitrophenyl-N acetyl-beta-d-glucosamine and p-nitrophenyl-N-acetyl-beta-d-galactosamine. When p nitrophenyl-N-acetyl-beta-d-glucosamine was used as substrate, K(m) and V(max) were 0.625 nanomolar and 228 moles per minute per milligram, respectively, and optimum activity was at pH 5.6. The enzyme liberated beta-linked N-acetyl glucosamine from chitin, ovalbumin, and pronase-digested wheat germ lectin. PMID- 16661637 TI - Inhibition of the Conversion of 1-Aminocyclopropane-1-carboxylic Acid to Ethylene by Structural Analogs, Inhibitors of Electron Transfer, Uncouplers of Oxidative Phosphorylation, and Free Radical Scavengers. AB - Cyclopropane carboxylic acid (CCA) at 1 to 5 millimolar, unlike related cyclopropane ring analogs of 1-aminocyclopropane-1-carboxylic acid (ACC) which were virtually ineffective, inhibited C(2)H(4) production, and this inhibition was nullified by ACC. Inhibition by CCA is not competitive with ACC since there is a decline, rather than an increase, in native endogenous ACC in the presence of CCA. Similarly, short-chain organic acids from acetic to butyric acid and alpha-aminoisobutyric acid inhibited C(2)H(4) production at 1 to 5 millimolar and lowered endogenous ACC levels. These inhibitions, like that of CCA, were overcome with ACC. Inhibitors of electron transfer and oxidative phosphorylation effectively inhibited ACC conversion to C(2)H(4) in pea and apple tissues. The most potent inhibitors were 2,4-dinitrophenol (DNP) and carbonyl cyanide m chlorophenylhydrazone (CCCP) which virtually eliminated ACC-stimulated C(2)H(4) production in both tissues. Still other inhibitors of the conversion of ACC to C(2)H(4) were putative free radical scavengers which reduced chemiluminescence in the free radical-activated luminol reaction. These inhibitor studies suggest the involvement of a free radical in the reaction sequence which converts ACC to C(2)H(4). Additionally, the potent inhibition of this reaction by uncouplers of oxidative phosphorylation (DNP and CCCP) suggest the involvement of ATP or the necessity for an intact membrane for C(2)H(4) production from ACC. In the latter case, CCCP may be acting as a proton ionophore to destroy the membrane integrity necessary for C(2)H(4) production. PMID- 16661638 TI - Some Characteristics of the System Converting 1-Aminocyclopropane-1-carboxylic Acid to Ethylene. AB - The rate of C(2)H(4) production in plant tissue appears to be limited by the level of endogenous 1-aminocyclopropane-1-carboxylic acid (ACC). Exogenous ACC stimulated C(2)H(4) production considerably in plant tissues, but this required 10 to 100 times the endogenous concentrations of ACC before significant increases in C(2)H(4) production were observed. This was partially due to poor penetration of ACC into the tissues. Conversion of ACC to C(2)H(4) was inhibited by free radical scavengers, reducing agents, and copper chelators, but not by inhibitors of pyridoxal phosphate-mediated reactions. The system for converting ACC to C(2)H(4) may be membrane-associated, for it did not survive treatment with surface-active agents and cold or osmotic shock reduced the capacity of the system to convert ACC to C(2)H(4). The reaction rate was sensitive to temperatures above 29 and below 12 C, which suggests that the system may be associated with membrane-bound lipoproteins. The data presented support the possibility that the conversion of exogenous ACC to C(2)H(4) proceeds via the natural physiological pathway. PMID- 16661639 TI - Accumulation of Maltose during Photosynthesis in Protoplasts Isolated from Spinach Leaves Treated with Mannose. AB - When mannose was included in the enzyme incubation medium during the preparation of protoplasts from leaves of spinach, maltose was an early product of protoplast photosynthesis and, after 12 minutes, accounted for up to 15% of the (14)C incorporated from (14)CO(2). Maltose was not detected in protoplasts prepared in the normal enzyme medium. Rapid separation of cytoplasm and chloroplasts following exposure to (14)CO(2) showed that maltose was present in both fractions. Direct measurements of [(14)C]maltose uptake indicated transport across the chloroplast envelope at rates similar to the transport of glucose. The source of maltose and site of its initial formation are discussed. PMID- 16661640 TI - Alpha-amylase synthesis in wheat kernels as influenced by the seed coat. AB - The effect of seed coat removal on the synthesis of alpha-amylase isoenzymes in wheat was investigated. The immature wheat endosperm-aleurone (seed coat and embryo detached) produced considerably less alpha-amylase activity than immature whole or de-embryonated wheat kernels, when incubated under identical conditions of 18.5 C and 99% humidity, in the presence or absence of gibberellic acid (GA(3)). The incubated endosperm-aleurone also exhibited unique alpha-amylase isoenzyme composition when compared to the isoenzyme compositions of incubated whole and de-embryonated immature and mature wheat kernels both in the presence or absence of GA(3). Subsequent studies indicated that the seed coat may contain factor(s) required for normal alpha-amylase isoenzyme synthesis. PMID- 16661641 TI - Regulation by Lipids of Plant Microsomal Enzymes: II. LIPID DEPENDENCE OF THE NADH-CYTOCHROME c REDUCTASE OF POTATO TUBERS. AB - Microsomal membranes from potato tubers were treated with a phospholipase C extracted from Bacillus cereus. A positive correlation could be observed between the hydrolysis of membranous phospholipids and the decrease of the NADH cytochrome c reductase activity. Addition of total lipid or phospholipid micelles to phospholipase C-treated microsomes partially restored the NADH-cytochrome c reductase activity, thus proving the lipid-dependence of this enzyme. PMID- 16661642 TI - Lipoxygenase, hydroperoxide isomerase, and hydroperoxide cyclase in young cotton seedlings. AB - Lipoxygenase was demonstrated in young cotton seedlings. It catalyzed the oxygenation of linoleic or linolenic acid, predominantly at carbon 13, and its molecular weight was estimated by gel filtration to be 100,000. Hydroperoxide isomerase was also present and converted hydroperoxylinoleic or hydroperoxylinolenic acid to alpha- or gamma-ketols. The enzyme utilized the 13 hydroperoxy isomer in preference to the 9 isomer and its molecular weight was estimated at 250,000 by gel filtration. In addition, hydroperoxide cyclase, which catalyzes the conversion of 13-hydroperoxylinolenic acid to 12-oxo-phytodienoic acid, was present. Hydroperoxide isomerase and hydroperoxide cyclase activities could not be separated by gel filtration and ion-exchange chromatography experiments, indicating the two enzyme activities may be associated with the same protein. The activities of all three enzymes were very low in the seed but increased immediately after germination, reached a maximum after 3 to 4 days, and then declined. The results suggest a role, as yet unknown, for these enzymes during early plant development. PMID- 16661643 TI - Chloroplast Biogenesis 34: SPECTROFLUOROMETRIC CHARACTERIZATION IN SITU OF THE PROTOCHLOROPHYLL SPECIES IN ETIOLATED TISSUES OF HIGHER PLANTS. AB - The fluorescence emission and excitation properties of protochlorophyll in etiolated cucumber (Cucumis sativus L.) cotyledons and primary bean (var. Red Kidney) leaves were characterized at 77 K. Contrary to previous studies, it appears that the short-wavelength protochlorophyll emission band consists of four fluorescent components, instead of only one nonphototransformable protochlorophyll. It was demonstrated that etiolated cucumber cotyledons synthesize and accumulate nontransformable protochlorophyll (E(440), F(630)) as well as short-wavelength phototransformable protochlorophyll (E(433), F(633)), (E(444), F(636)), and (E(445), F(640)). Long-wavelength phototransformable protochlorophyll (E(450), F(657)) is also formed. In this context, E refers to the Soret excitation maxima and F refers to the red emission maxima of the protochlorophylls.IN ETIOLATED BEAN LEAVES, THE CORRESPONDING SPECIES WERE: nontransformable protochlorophyll (E(440), F(630)), short-wavelength phototransformable protochlorophylls (E(441), F(633)), (E(442), F(636)), and (E(443), F(640)), and long-wavelength phototransformable protochlorophyll (E(447), F(657)). PMID- 16661644 TI - High-temperature Disruption of Guard Cells of Vicia faba: EFFECT ON STOMATAL APERTURE. AB - Increased variability in stomatal aperture at high temperatures can be attributed, in part, to the differential sensitivity of guard cells to thermal damage. Individual stomata become increasingly open at higher temperatures until guard cells are lethally damaged; at that temperature, apertures decrease. The extent of irreversible damage causing closure was estimated by K(+) uptake, neutral red accumulation, and visual scoring of chloroplasts.This study found that visual scoring of chloroplast disruption provided the best estimate of guard cell viability at high temperature. Removal of the damaged guard cells from the population sample resulted in a constant coefficient of variability for apertures over the temperature range 25 to 50 C. PMID- 16661645 TI - Light Modulation of Glyceraldehyde-3-phosphate Dehydrogenase and Glucose-6 phosphate Dehydrogenase by Photosynthetic Electron Flow in Pea Chloroplasts. AB - Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) and light inactivation of glucose-6-P dehydrogenase (EC 1.1.1.49) appear to be modulated within pea leaf chloroplasts by mediators which are reduced by photosynthetic electron flow from the photosystem I reaction center. Dichlorophenyl-1, 1-dimethylurea inhibition of this modulation can be completely reversed by ascorbate plus 2,6-dichlorophenolindophenol in broken chloroplasts, but not in intact chloroplasts. Intact chloroplasts are impermeable to 2,6 dichlorophenolindophenol at pH 7.5. Studies on the effect of light in reconstituted chloroplasts with photosystem I-enriched particles in the place of whole thylakoids revealed that photosystem I participates in the light modulation of NADP-linked glyceraldehyde-3-P dehydrogenase and of glucose-6-P dehydrogenase. PMID- 16661646 TI - Photosynthetic Response to Alkaline pH in Anabaena variabilis. AB - The rate of O(2) evolution and alkalization of the medium in low CO(2(-) ) grown Anabaena variabilis was observed as affected by the pH in the medium. Both rates are severely inhibited by pH values higher than 9.5, but the latter is more sensitive to this treatment. This finding, as well as the lag observed in alkalization of the medium, but not in O(2) evolution, following the addition of HCO(3) (-) indicates that the transport of HCO(3) and OH(-) (or H(+)) are not compulsorily coupled. The inhibition of photosynthesis by strongly alkaline pH is attributed to an alteration of the internal pH and, hence, the rate of carboxylation. This conclusion is supported by data showing that the rate of O(2) evolution is affected by pH more strongly at saturating [HCO(3) (-)] than at limiting [HCO(3) (-)]. Also, the rate of O(2) evolution at saturating light intensity is affected by pH more strongly than is the initial slope of the curve against light intensity or the rate of dark respiration. PMID- 16661647 TI - Precursor Forms of Pea Vicilin Subunits: MODIFICATION BY MICROSOMAL MEMBRANES DURING CELL-FREE TRANSLATION. AB - Polyribosomal RNA isolated from pea cotyledons at various developmental stages programmed the cell-free synthesis of polypeptides which were recognized by antibodies specific for pea storage proteins. There were quantitative and qualitative changes in the template activity during seed maturation. Most of the polysomal RNA was associated with the membrane fraction, and all of the template for storage protein occurred in this fraction. Using RNA from a stage of seed maturation at which the synthesis of the high-molecular weight vicilin polypeptides predominate, it was found that the major translation products, although antigenically recognizable as storage protein, did not coincide with the authentic vicillin polypeptides on denaturing polyacrylamide gels. The addition during translation of microsomal membranes from dog pancreas or pea cotyledons resulted in the appearance of new polypeptides which did coincide with some of the authentic vicilin polypeptides (in the apparent molecular weight regions of 75,000 and 50,000) and were antigenically recognizable as storage protein. Other translation products related to storage protein were not visibly altered in their electrophoretic mobility by the addition of membranes. Microsomal membranes treated with Triton X-100 were not effective in modifying the cell-free products. The modified vicilin polypeptides and at least two other translation products were protected from proteolytic degradation, suggesting that they were sequestered within microsomal vesicles. Thus, these storage protein components may be synthesized by a mechanism analogous to that described for membrane and secretory proteins (Blobel G, B Dobberstein 1975 J Cell Biol 67: 835-851). PMID- 16661648 TI - Effects of trypsin and cations on chloroplast membranes. AB - A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl(2)) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl(2)) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the "diffuse electrical layer" hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism. PMID- 16661649 TI - Transition of Lipid Synthesis from Chloroplasts to a Cytoplasmic System during Hardening in Chlorella ellipsoidea. AB - Chlorella ellipsoidea Gerneck (IAM C-27) was synchronously grown and cells at an intermediate stage in the ripening phase of the cell cycle were hardened at 3 C for 48 hours. At various times of hardening, the cells were pulse-labeled for 4 minutes with [(14)C]NaHCO(3) in the light or with [(14)C]glucose in the dark, and the incorporation rate of (14)C into total lipids was determined. A high incorporation rate of [(14)C]NaHCO(3) at zero time of hardening decreased after 6 hours. In the next 15 hours, a distinct increase was noted. This increase occurred prior to the development of frost hardiness. Cycloheximide completely inhibited both the increase and the development, and 3-(3,4-dichlorophenyl)-1,1 dimethylurea remarkably lowered the high incorporation rate at zero time. The incorporation rate of [(14)C]glucose increased along with hardiness in the dark. These results suggest that the major site of lipid synthesis shifts from chloroplasts to a cytoplasmic system during hardening of Chlorella. PMID- 16661650 TI - Purification and Characteristics of Sorbitol-6-phosphate Dehydrogenase from Loquat Leaves. AB - To study the role of sorbitol-6-phosphate dehydrogenase in sorbitol synthesis in leaves of Rosaceous plants, properties of the enzyme and its presence in several plants in the family was investigated. The activity of the enzyme, which catalyzes an NADP-dependent oxidation of the substrate to glucose-6-phosphate, was detected in leaves of Prunus mume, Prunus persica, Rhaphiolepsis indica, Sorbus aucuparia, Cydonia oblonga, Photinia glabra, Sorbaria kirilowii, and Spiraea thunbergii.The enzyme was purified about 60-fold from leaves of loquat (Eriobotrya japonica) using affinity chromatography with Blue Sepharose. Neither mannitol-1-phosphate nor fructose-6-phosphate served as substrate. Molecular weight of the enzyme was calculated to be 65,000 at pH 8.0 by gel filtration. Since sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a peptide of 33,000 daltons, the enzyme was assumed to be a dimer at pH 8.0 K(m) values for sorbitol-6-phosphate, glucose-6-phosphate, NADP, and NADPH were 2.22 millimolar, 11.6 millimolar, 13.5 micromolar, and 1.61 micromolar, respectively. Equilibrium constant for sorbitol-6-phosphate oxidation was 5.12 x 10(-10). Optimal pH for sorbitol-6-phosphate oxidation was 9.8. The enzyme showed its maximum activity within a broad pH range between 7 and 9 for glucose-6-phosphate reduction. The enzyme was more effective in the direction of glucose-6-phosphate reduction than in the reverse direction at neutral pH. Thus, it is suggested that the enzyme catalyzes sorbitol synthesis from glucose-6-phosphate during photosynthesis in leaves of Rosaceous plants. PMID- 16661651 TI - Function of Light in the Light-induced Geotropic Response in Zea Roots. AB - The light doses just above the threshold energy value for inducing the geotropic responsiveness in the roots of Zea mays L., cv. Golden Cross Bantam 70, caused a drastic rise in the NADPH level and a drop in the NADP level in 2-millimeter root tips. Some reducing agents lowered the threshold energy value up to about one third of the control. From these results, we deduce that light may exert two functions in the geotropic response of Zea primary roots, one being the photochemical transformation of a photoreceptor and the other being the induction of a reduction state in the tissue. PMID- 16661652 TI - Glycolate Excretion and the Oxygen to Carbon Dioxide Net Exchange Ratio during Photosynthesis in Chlamydomonas reinhardtii. AB - Chlamydomonas reinhardtii cells were grown in high (5% v/v) or low (0.03% v/v) CO(2) concentration in air. O(2) evolution, HCO(3) (-) assimilation, and glycolate excretion were measured in response to O(2) and CO(2) concentration. Both low- and high-CO(2)-grown cells excrete glycolate. In low-CO(2)-grown cells, however, glycolate excretion is observed only at much lower CO(2) concentrations in the medium, as compared with high-CO(2)-adapted cells. It is postulated that the activity of the CO(2)-concentrating mechanism in low-CO(2)-grown cells is responsible for the different dependence of glycolate excretion on external CO(2) concentration in low- versus high-CO(2)-adapted cells.The O(2)/CO(2) net exchange ratio is dependent on the CO(2) concentration in the medium and is linearly dependent on the fraction of glycolate excreted per CO(2) taken up. Glycolate excretion, however, is too low to account for the deviation of the O(2)/CO(2) net exchange ratio from unity. PMID- 16661653 TI - Abscisic Acid and photosynthesis in isolated leaf mesophyll cell. AB - Abscisic acid (AbA) treatments of concentrations of up to 135 micromolar did not inhibit photosynthesis in enzymatically isolated leaf mesophyll cells of Phaseolus vulgaris, Nicotiana tabacum, and Lycopersicum esculentum over periods of up to 5 hours. Thin slices of leaves preincubated in hypertonic solutions identical to those used to isolate cells were shown to synthesize AbA rapidly, although accumulation of AbA in the cells was low due to extensive release of the newly synthesized AbA into the medium. The levels of endogenously made AbA in leaf cells of Phaseolus vulgaris rose from a low of 0.27 micromolar to a high of 6.74 micromolar during 2 hours preincubation. Exogenously applied AbA can be taken up by the cells as was demonstrated using (14)[C]AbA. Thus, AbA applied at concentrations 19 times higher than endogenous levels does not change the rate of photosynthesis. PMID- 16661654 TI - A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5 bisphosphate Carboxylase and Oxygenase Activities. AB - A sensitive assay procedure is described for the simultaneous determination of ribulose-1,5-bisphosphate (RuBP) carboxylase and oxygenase activities. In this assay, [1-(3)H]RuBP is incubated with (14)CO(2) and O(2). Carboxylation rate is determined from (14)CO(2) incorporation and oxygenation rate is determined from [2-(3)H]glycolate-phosphate production. The assay was found to be suitable at all CO(2) and O(2) concentrations examined, which ranged from 0 to 300 micromolar CO(2) (20 millimolar NaHCO(3)) and 0 to 1.15 millimolar (100%) O(2). In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O(2)-glycolate phosphate-glycerate phosphate (1:1:1:1).The ratio of soybean (Merr.) RuBP carboxylase and oxygenase activities was measured at various CO(2) and O(2) concentrations and in the presence of several compounds which have been reported to alter differentially the rate of the two reactions. The ratio of the two activities was a linear function of the ratio of the CO(2) and O(2) concentrations. The substrate specificity factor V(c)K(o)/V(o)K(c), which determines relative carboxylase and oxygenase reaction rates as a function of the CO(2) and O(2) concentrations, was found to be 73 in the presence of Mg(2+). Of the several compounds which have been reported to alter differentially the two reactions, we found that only Mn(2+) substitution for Mg(2+) was effective. Compared to Mg(2+), Mn(2+) reduced the K(m)(O(2)) from 690 to 36 micromolar O(2) and reduced the specificity factor to about 4. The K(m)(CO(2)) was about 20 micromolar CO(2) in the presence of both Mg(2+) and Mn(2+). Comparison of reaction rates in the presence of activated and inactivated enzyme allowed a direct determination that both carboxylase and oxygenase activities are similarly activated by CO(2), with an activation equilibrium constant of about 1.3 millimolar NaHCO(3) (27 micromolar CO(2)) at pH 7.85 and 10 millimolar Mg(2+). PMID- 16661655 TI - Properties of Magnesium Chelatase in Greening Etioplasts: METAL ION SPECIFICITY AND EFFECT OF SUBSTRATE CONCENTRATIONS. AB - Evidence was obtained by means of low temperature spectrofluorimetry and high pressure liquid chromatography for the exclusive chelation of Mg(2+) into protoporphyrin IX by a plastid preparation extracted from greening cucumber cotyledons. Under the experimental conditions which were optimized for Mg(2+) chelation, Zn(2+) chelation was not detected. However, Zn(2+) chelation was observed when ATP was omitted and ZnCl(2) was added to the incubation mixture. The observed Zn(2+) chelation was partially nonenzymic. The requirements for the enzyme, Mg chelatase, are discussed in terms of the regulatory function of this enzyme. PMID- 16661657 TI - Photosynthate Partitioning into Starch in Soybean Leaves: II. IRRADIANCE LEVEL AND DAILY PHOTOSYNTHETIC PERIOD DURATION EFFECTS. AB - Two photosynthetic periods and photosynthetic photon flux densities (PPFD) were used to study the relationship between the rate of photosynthesis and starch accumulation in vegetative soybean leaves (Merr. cv Amsoy 71). Plants grown in short daily photosynthetic periods (7 hours) had higher rates of CO(2) fixation per unit leaf dry weight and of leaf starch accumulation than plants grown in long daily photosynthetic periods (14 hours) irrespective of PPFD. CO(2) fixation rates per unit leaf area were similar in 7-hour and 14-hour plants grown at low PPFD but were highest in 14-hour plants at the high PPFD. When single leaves of 14-hour plants were given 7-hour photosynthetic periods, their rates of starch accumulation remained unchanged. The programming of starch accumulation rate and possibly of photosynthetic rate by the length of the daily photosynthetic period is apparently a whole-plant, not an individual leaf, phenomenon. Programming of chloroplast starch accumulation rate by length of the daily photosynthetic and/or dark periods was independent of PPFD within the ranges used in this experiment. PMID- 16661656 TI - Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes. AB - The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [(14)C]ATP or [(14)C]oleic acid as labeled substrates, it can be shown that the envelope acyl CoA synthetase required both Mg(2+) and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [(14)C]ATP or with [(3)H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C(18) fatty acids.The results obtained suggest that the acyl-CoA thioesterase associated with envelope membranes could be an ultimate control to prevent the transport (outside of the chloroplast) or the insertion (into chloroplast lipids) of fatty acids with chains shorter than C(16). PMID- 16661658 TI - Phospholipid Metabolism in Plant Mitochondria: II. SUBMITOCHONDRIAL SITES OF SYNTHESIS OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLETHANOLAMINE. AB - CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were detected in mitochondrial fractions from castor bean (Ricinus communis) endosperm. These activities were not due to contamination of the fractions with endoplasmic reticulum. The enzymes were localized on both the inner and outer mitochondrial membranes.Only minor kinetic differences between the phosphatidylcholine-synthesizing activities of intact mitochondria and of the endoplasmic reticulum were found. The K(m) of the mitochondrial enzyme for CDP choline was about 2 units less than that for the enzyme of the endoplasmic reticulum (8.0 and 10.0 micromolar, respectively). The mitochondrial enzyme activity was maximal above 10 millimolar Mg(2+), whereas maximum endoplasmic reticulum activity was achieved by 4 millimolar. The endoplasmic reticulum enzyme was more stable at 37 C than was that of the mitochondria. The mitochondrial cholinephosphotransferase represented about 1 to 2% of the total activity isolated from castor bean endosperm. PMID- 16661659 TI - Simultaneous Phytochrome-controlled Promotion and Inhibition of Arginine Decarboxylase Activity in Buds and Epicotyls of Etiolated Peas. AB - The specific activity of arginine decarboxylase (ADC; l-arginine carboxylase; EC 4.1.1.19) rises steadily over an 8 hour experimental period in the growing buds and subapical epicotyl internodes of 6-day-old totally etiolated pea seedlings. Treatment with red light (R) completely annuls this rise in epicotyls but increases it in buds, thus paralleling the opposite effects of R on the growth of these two organs. Far red light (FR) reverses both effects of R on ADC and is, in turn, reversed by R, indicating phytochrome control. Effects in both organs are clearly seen within 2 hours. By 6 hours after R, the post-irradiation rise in ADC specific activity in buds is 3 times greater than that of the dark controls. Over the same period, ADC specific activity in epicotyls is inhibited by 56% relative to dark controls, reflecting zero net change after R and a continued rise in the dark. Cycloheximide inhibits the rise in ADC activity in both rapidly growing organs (epicotyls in dark and buds after R) but is without effect in both slower growing organs. Actinomycin D inhibits only in dark grown epicotyls, whereas chloramphenicol produces no inhibition in any system tested.ADC is the first enzyme to show a two-way, organ-specific response to phytochrome conversion from Pr to Pfr. This finding is discussed in relation to the growing evidence that polyamines formed from arginine may be important growth regulators in plants, as well as in microbial and animal cells. PMID- 16661660 TI - Isolation of a plant glycoprotein involved with control of intercellular recognition. AB - A recognition molecule was isolated from stigmas of S-allele genotype S(2)S(2) of Brassica oleracea var. capitata L. After Sephadex chromatography, it eluted as a single symmetrical peak during diethylaminoethane-cellulose chromatography. A high degree of purity was affirmed by: sedimentation as a single peak during ultracentrifugation through 5 to 20% sucrose gradients; elution as a single peak from Sephadex G-100; visualization as a single band which stains with Coomassie blue and periodic acid Schiff reagent after electrophoresis on polyacrylamide gels. Other criteria supporting the conclusion that it is a glycoprotein are: (a) the highly purified preparation is anthrone-positive and has a Lowry protein to anthrone-positive carbohydrate ratio of 1.3; (b) the preparation contains arabinose, galactose, glucose, and mannose, although it is not precipitated by concanavalin A; (c) the immunological properties of the molecule are lost following protease treatment, and it has a molecular weight of 90,000 by Sephadex gel-filtration analysis and 54,500 by velocity sedimentation analysis.In vitro pretreatment of S(2)S(2) pollen with the post-diethylaminoethane-purified S(2) glycoprotein prevented the S(2)S(2) pollen from germinating on three classes of compatible stigmas: (a) mature stigmas of genotypes S(3)S(3) and S(8)S(8), which are non-self genotypes; (b) immature stigmas of genotype S(2)S(2), where incompatibility is not expressed; and (c) mature stigmas with a recessive S(2) allele. Pretreatment of S(3)S(3) and S(8)S(8) pollen with the S(2) glycoprotein did not interfere with their germination. PMID- 16661661 TI - Effect of auxin on acropetal auxin transport in roots of corn. AB - Acropetal [(14)C]indoleacetic acid (IAA) transport was investigated in roots of corn. At least 40 to 50% of this movement is dependent on activities in the root apex. Selective excision of various populations of cells comprising the root apex, e.g. the root cap, quiescent center, or proximal meristem show that the proximal meristem is the critical region in the apex with regard to influencing IAA movement. The quiescent center has no influence and the root cap has only a minor effect. Excision and replacement of the proximal meristem with an exogenous supply of 10(-8) to 10(-9) molar IAA prevents the reduction in acropetal IAA transport which would normally occur in the absence of this meristem. Substituting 10(-9) molar IAA for the excised root cap brings about a significant increase in the amount of IAA moved acropetally, as compared to intact roots with the root cap still in place. From this and previous work, it is concluded that IAA synthesis occurring in the proximal meristem stimulates the movement of IAA from the basal to apical end of the root. PMID- 16661662 TI - Inhibitory effects of 2,3,5-triiodobenzoic Acid on ion absorption, respiration, and carbon metabolism in excised barley roots. AB - The ability of 2,3,5-triiodobenzoic acid (TIBA) to alter ion absorption, respiration, carbon metabolism, and the permeability of the cell membranes of excised barley roots has been examined. Roots pretreated in either H(2)O, KCl, or TIBA followed by treatment in KCl, TIBA, or KCl and TIBA demonstrated that inhibition of ion uptake due to TIBA was reversible. These studies also suggest that ions already accumulated within the vacuole remain sequestered after the addition of TIBA, whereas cytoplasmic ions leak out into the external medium. A 20-minute lag period was present prior to the onset of inhibition of O(2) consumption by TIBA. A b-type cytochrome from corn that is apparently associated with the plasmalemma and possibly involved in respiration or ion uptake, or both, was unaffected by TIBA. The addition of TIBA to treatment solutions resulted in the synthesis and accumulation of ethanol. Analysis of organic acids showed that only the malate concentration was affected by treatment with TIBA. A reduction of 26% was noted for malate in the presence of 2 micromolar TIBA. These combined results suggest that the inhibitory action of TIBA in barley roots involves an alteration of mitochondrial respiration and not a direct depolarization of the plasmalemma. PMID- 16661663 TI - Regulation of pyrimidine and arginine biosynthesis investigated by the use of phaseolotoxin and 5-Fluorouracil. AB - Purified phaseolotoxin inhibits the growth of carrot cells. Such inhibitions can be reversed completely by citrulline but not by arginine. This toxin inhibits ornithine transcarbamylase activity in vitro, which leads to an accumulation of ornithine and a decrease in arginine levels intracellularly. In carrot cells, 5 fluorouracil (5-FU) toxicity can be reduced by the addition of purified toxin and citrulline, or ornithine. The toxin also decreases the incorporation of [(14)C]uracil and [(14)C]5-FU into trichloroacetic acid precipitable material by 50%. Finally, a 5-FU-resistant line, F5 (Sung ZR, Jacques S 1980 Planta 148: 389 396), was found to be more sensitive to the toxin than were 5-FU-sensitive cells. One millimolar 5-FU roughly doubled the ability of F5 to tolerate phaseolotoxin. These results demonstrate a close regulation between the pyrimidine and arginine path-ways in carrots. PMID- 16661664 TI - Metal Complexation in Xylem Fluid : I. CHEMICAL COMPOSITION OF TOMATO AND SOYBEAN STEM EXUDATE. AB - Xylem fluid was analyzed for numerous solutes to characterize chemically the sap as a medium for forming and transporting metal complexes. The stem exudate was collected hourly for 8 hours from topped 31-day-old soybean (Glycine max L. Merr.) and 46-day-old tomato (Lycopersicon esculentum Mill.) plants grown in normal (0.5 micromolar) and Za-phytotoxic nutrient solutions. Soybean plants were grown in the normal and high-Zn solutions for 24 days; tomato plants were grown for 32 days. The exudate was analyzed for seven organic acids, 22 amino acids, eight inorganic solutes, apparent ionic strength, and pH. Significant changes in many solutes occurred over the 8-hour sampling period. These fluctuations depended on plant species, individual solute, and Zn treatment, and demonstrated that extrapolation of xylem-fluid analyses to whole-plant xylem sap is valid only for sap samples collected shortly after topping a plant. Exudate pH decreased over the 8-hour period for both species; exudate ionic strength increased for tomato and decreased for soybean. At the normal-Zn treatment (0 to 1 hour), the highest acid micromolar concentrations in soybean exudate were: asparagine, 2,583; citric, 1,706; malic, 890; and malonic, 264. Under the same conditions, the highest acid micromolar concentrations in tomato exudate were: maleic, 1,206; malic, 628; glutamine, 522; citric, 301; and asparagine, 242. Cysteine and methionine were above detection limits only in soybean exudate. Zinc phytotoxicity caused significant changes in many solutes. The analyses reported here provide a comprehensive data base for further studies on metal-complex equilibria in xylem fluid. PMID- 16661665 TI - Metal Complexation in Xylem Fluid : II. THEORETICAL EQUILIBRIUM MODEL AND COMPUTATIONAL COMPUTER PROGRAM. AB - Theoretical considerations of metal complex formation in aqueous solutions were used to develop a computer program (CHELATE) to calculate all equilibrium species (free metal ions, metal complexes, etc.) in any user-defined system, such as xylem fluid. Mass-balance equations were established to describe each free metal ion and each free ligand concentration as a function of solution pH, total metal or total ligand, hydrogen-association constants, and the stability constants of known metal complexes. A default data base can be altered by the user to define any desired system covered by the stored equilibrium data. The program can currently handle nine metal ions, 35 ligands, and 500 complex species. The validity of the program was confirmed by using experimental test systems in which free-metal ion activity measurements were made with ion-selective electrodes.Program CHELATE was used to calculate the distribution of six metals in 0- to 1-hour exudate from soybean (Glycine max L. Merr.) and tomato (Lycopersicon esculentum Mill.) plants grown in normal and Zn-phytotoxic nutrient solutions. The results indicated that Fe is bound by citric acid, and Cu is bound by several amino acids in the normal-Zn exudate. Most of the Cu in soybean exudate is bound to asparagine and histidine. In tomato, Cu is bound to histidine, glutamine, and asparagine. Zinc, Mn, Ca, and Mg are bound primarily by citric acid and malic acid in both species; the per cent bound for these metals is lower than that for Fe and Cu. Zinc phytotoxicity caused equilibrium concentration shifts and resulted in the formation of several additional metal complexes not found in the normal-Zn exudate. PMID- 16661666 TI - Metal Complexation in Xylem Fluid : III. ELECTROPHORETIC EVIDENCE. AB - The capacity of ligands in xylem fluid to form metal complexes was tested with a series of in vitro experiments using paper electrophoresis and radiographs. The xylem fluid was collected hourly for 8 hours from soybean (Glycine max L. Merr.) and tomato (Lycopersicon esculentum Mill.) plants grown in normal and Zn phytotoxic nutrient solutions. Metal complexation was assayed by anodic or reduced cathodic movement of radionuclides ((63)Ni, (65)Zn, (109)Cd, (54)Mn) that were presumed to have formed negatively charged complexes.Electrophoretic migration of Ni, Zn, Cd, and Mn added to xylem exudate and spotted on KCl- or KNO(3)-wetted paper showed that stable Ni, Zn, and Cd metal complexes were formed by exudate ligands. No anodic Mn complexes were observed in this test system. Solution pH, plant species, exudate collection time, and Zn phytotoxicity all affected the amount of metal complex formed in exudate. As the pH increased, there was increased anodic metal movement. Soybean exudate generally bound more of each metal than did tomato exudate. Metal binding usually decreased with increasing exudate collection time, and less metal was bound by the high-Zn exudate.Ni, Zn, Cd, and Mn in exudate added to exudate-wetted paper demonstrated the effect of ligand concentration on stable metal complex formation. Complexes for each metal were demonstratable with this method. Cathodic metal movement increased with time of exudate collection, and it was greater in the high-Zn exudate than in the normal-Zn exudate. A model study illustrated the effect of ligand concentration on metal complex stability in the electrophoretic field. Higher ligand (citric acid) concentrations increased the stability for all metals tested. PMID- 16661667 TI - Selenium Metabolism in Neptunia amplexicaulis. AB - ATP sulfurylase (EC 2.7.7.4), cysteinyl-tRNA synthetase (EC 6.1.1.16), and methionyl-tRNA synthetase (EC 6.1.1.10) from Neptunia amplexicaulis have been purified approximately 162-, 140- and 185-fold, respectively. Purified ATP sulfurylase in the presence of purified inorganic pyrophosphatase catalyzed the incorporation of sulfate into adenosine 5'-phosphosulfate; evidence of an analogous reaction with selenate is presented. Crude extracts catalyzed both the sulfate- and the adenosine 5'-phosphosulfate-dependent NADH oxidation in the adenosine 5'-phosphosulfate kinase assay of Burnell and Whatley (1977 Biochim Biophys Acta 481: 266-278), but an analogous reaction with selenate could not be detected. Both purified cysteinyl-tRNA synthetase and methionyl-tRNA synthetase used selenium-containing analogs as substrates in both the ATP-pyrophosphate exchange and the aminoacylation assays.It seems that selenium-containing amino acids are excluded from proteins by a mechanism(s) other than substrate discrimination at the amino acid activation stage of protein synthesis. PMID- 16661668 TI - Methionyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties. AB - l-Methionyl-tRNA synthetase (EC 6.1.1.10) from seeds of Phaseolus aureus has been purified approximately 290-fold. Optimum assay conditions were determined by using the ATP-pyrophosphate exchange assay and the aminoacylation assay. The enzyme catalyzes both selenomethionine- and selenoethionine-dependent ATP pyrophosphate exchange in addition to catalyzing the formation of selenomethionyl tRNA at a rate comparable to the rate of formation of methionyl-tRNA. Competition experiments were conducted to investigate further the substrate specificity of the purified enzyme. Two peaks of methionyl-tRNA synthetase were detected by using Sephadex G-200 gel filtration; the molecular weights of the two enzymes as determined by Sephadex G-200 column chromatography were 340,000 and 85,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a tetramer consisting of four identical monomers with molecular weights of 85,000. PMID- 16661669 TI - Comparative characterization of phosphoenolpyruvate carboxylase in c(3), c(4), and c(3)-c(4) intermediate panicum species. AB - Various properties of phosphoenolpyruvate carboxylases were compared in leaf preparations from C(3)-C(4) intermediate, C(3), and C(4)Panicum species. Values of V(max) in micromoles per milligram chlorophyll per hour at pH 8.3 were 57 to 75 for the enzyme from Panicum milioides, Panicum schenckii, and Panicum decipiens (all C(3)-C(4)). The values for Panicum laxum (C(3)) and Panicum prionitis (C(4)) were 20 to 40 and 952 to 1374, respectively. The V(max) values did not change at pH 7.3 except for the C(4) value, which increased about 24%. At pH 8.3, the phosphoenolpyruvate carboxylases from C(3) and C(3)-C(4) species had slightly higher K(m) HCO(3) (-) and lower K(') phosphoenolpyruvate values than did the C(4) enzyme. With each species at pH 7.3, all K(') phosphoenolpyruvate values were 2- to 4-fold greater.The enzyme from all species was inhibited 85 to 90% by 1 millimolar malate at rate-limiting phosphoenolpyruvate and Mg(2+) levels. With low levels of malate, 0.2 millimolar, the rate curve with respect to phosphoenolpyruvate was distinctly sigmoidal, and the inhibition was not eliminated at 5 millimolar phosphoenolpyruvate.Malate at 10 millimolar protected all phosphoenolpyruvate carboxylases from inactivation at 55 C at pH 5.5, but not at pH 8.3. Aspartate did not protect well. When incubated at 37 C at pH 8.3 without phosphoenolpyruvate, but with HCO(3) (-), the enzyme from several C(4) grasses lost 92 to 98% of the initial activity after 4 minutes, whereas the enzymes from C(3) and C(3)-C(4)Panicum species retained 60 to 70% of their activities. In contrast, 5 millimolar phosphoenolpyruvate stabilized the enzyme at 37 C in all plant extracts.The phosphoenolpyruvate carboxylase from C(3)-C(4) intermediate Panicum species has properties most similar to the enzyme from C(3)Panicum species. The higher leaf activity of the enzyme from the intermediate plants than from C(3) species is not due to any unusual property assayed other than a higher V(max.). PMID- 16661670 TI - Induction of reduced photorespiratory activity in submersed and amphibious aquatic macrophytes. AB - Incubation under water in a 30 C/14-hour or 12 C/10-hour photoperiod caused the CO(2) compensation points of 10 aquatic macrophytes to decrease below 25 or increase above 50 microliters CO(2) per liter, respectively. Submerged and aerial leaves of two amphibious angiosperms (Myriophyllum brasiliense and Proserpinaca palustris) maintained high compensation points when incubated in air but, when the submerged or aerial leaves of Proserpinaca were incubated under water, the compensation points dropped as low as 10. This suggests that, in addition to temperature and photoperiod, some factor associated with submergence regulates the compensation point of aquatic plants. In the high-compensation point plants, photorespiration, as a percentage of net photosynthesis, was equivalent to that in terrestrial C(3) plants. For Hydrilla verticillata, the decreasing CO(2) compensation points (110, 40, and 10) were associated with reduced photorespiration, as indicated by decreased O(2) inhibition, decreased rates of CO(2) evolution into CO(2)-free air, and increased net photosynthetic rates.The decrease in the CO(2) compensation points of Hydrilla, Egeria densa, and Cabomba caroliniana was accompanied by an increase in the activity of phosphoenolpyruvate, but not of ribulose bisphosphate, carboxylase. In Hydrilla, several C(4) enzymes also increased in activity to the following levels (micromoles per gram fresh weight per hour): pyruvate Pi dikinase (35), pyrophosphatase (716), adenylate kinase (525), NAD and NADP malate dehydrogenase (6565 and 30), NAD and NADP malic enzymes (239 and 44), and aspartate and alanine aminotransferases (357 and 85), whereas glycolate oxidase (6) and phosphoglycolate and phosphoglycerate phosphatases (76 and 32) showed no change. Glycolate dehydrogenase and phosphoenolpyruvate carboxykinase were undetectable. The reduced photorespiration in these plants may be due to increased CO(2) fixation via a C(4) acid pathway. However, for three Myriophyllum species, some other mechanism appears operative, as phosphoenolpyruvate carboxylase was not increased in the low compensation point state, and ribulose bisphosphate carboxylase remained the predominant carboxylation enzyme. PMID- 16661671 TI - Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination : IV. beta-OXIDATION. AB - Microbodies were isolated by zonal-rotor sucrose density gradient centrifugation from cotton (cv. DP 61) seeds at two distinct stages of embryogenesis (38 and 50 days after anthesis) and after 48 hours postgerminative growth. In all cases, beta-oxidation activity (palmitoyl-coenzyme A (CoA)-dependent reduction of acetylpyridine adenine dinucleotide or production of acetyl-CoA) and activities of the enzymes palmitate:CoA ligase, acyl-CoA oxidase, enoyl hydratase, 3 hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, plus catalase, were localized exclusively in the microbody fractions, i.e. none of the activities were associated with mitochondria. Acyl-CoA dehydrogenase activity could not be detected in any of the gradient fractions or in homogenates.Glyoxysomes isolated from cotyledons of 48-hour-germinated seeds were capable of beta-oxidation of acyl-CoAs of various chain lengths and degrees of unsaturation and were the sole site of 3-cis-2-trans enoyl-CoA isomerase activity. Direct measurement of the isomerase is the first demonstration of an enzyme required for unsaturated fatty acid catabolism in a higher plant. Palmitoyl-carnitine was not oxidized by any of the organelle fractions.Subfractionation of glyoxysomes by osmotic shock revealed that none of the beta-oxidation enzymes were tightly membrane-associated. PMID- 16661672 TI - Retardation of Senescence in Red Clover Leaf Discs by a New Antiozonant, N-[2-(2 Oxo-1-imidazolidinyl)ethyl]-N'-phenylurea. AB - Dark-induced senescence in leaf discs from O(3)-sensitive red clover trifoliates (Trifolium pratense L. cv. ;Pennscott') was markedly retarded by treatment with N [2-(2-oxo-1-imidazolidinyl)ethyl-N'-phenylurea (EDU). EDU also protects against acute and chronic foliar O(3) injury when sprayed on intact leaves or supplied to the plants through soil application. Senescence retardation was measured by time dependent analyses of chlorophyll, protein, and RNA in discs floated on aqueous EDU solutions ranging from 0 to 500 micrograms per milliliter EDU. Chlorophyll degradation, total protein, and nucleic acids were followed over 10-day test periods.EDU at 500 micrograms per milliliter (50 milligrams per pot), a concentration known to provide optimal protection to intact leaves against O(3) injury, was most effective in preventing chlorosis and in maintaining high concentrations of protein and RNA in the discs. In discs treated with 500 micrograms per milliliter EDU 90% of the chlorophyll was retained after 10 days in the dark. In contrast, lower concentrations (0, 125, and 250 micrograms per milliliter) showed the complete loss of chlorophyll or an intermediate retardation. The intermediate concentrations were similarly less effective in maintaining protein and RNA levels in the dark stressed leaf discs. It is suggested that EDU retards senescence and mitigates O(3) injury through the induction of specific free radical scavenging enzymes and in sustaining RNA and protein synthesis. PMID- 16661673 TI - Stabilization of Photosystem II (O(2) Evolution) of Spinach Chloroplasts by Radiation-induced Immobilization. AB - Spinach chloroplasts were immobilized with vinyl monomers by radiation-induced polymerization at low temperature and stored in buffer containing bovine serum albumin. The lifetime of O(2) evolution activity in photosystem II was prolonged remarkably in immobilized chloroplasts. Thermostability of immobilized chloroplasts stored in buffer containing bovine serum albumin was far better than that of immobilized chloroplasts in pure buffer and that of intact chloroplasts. When immobilized chloroplasts were stored in buffer including polyethylene glycol, the lifetime of O(2) evolution activity was longer than for those stored in buffer containing bovine serum albumin. PMID- 16661674 TI - Phytochrome Control of Maize Coleoptile Section Elongation : A RAPID LOSS OF PHOTOREVERSIBILITY. AB - A rapid loss of far red light (FR) reversibility of red-light (R) stimulated elongation of maize coleoptile sections was observed. Reversal was not possible when the interval between R and FR treatment was greater than 45 seconds. Most of the R-stimulated elongation occurred during the first 15 hours after irradiation.Exogenous gibberellic acid did not alter the time course of R/FR reversibility loss. These results are interpreted as indicating independence of R and gibberellic acid-stimulated elongation in this system. PMID- 16661675 TI - Studies in Wild Oat Seed Dormancy: I. THE ROLE OF ETHYLENE IN DORMANCY BREAKAGE AND GERMINATION OF WILD OAT SEEDS (AVENA FATUA L.). AB - Seed of Avena fatua were shown to exhibit a characteristic loss of dormancy during dry storage at 25 C, whereas similar seed stored at 5 C maintained dormancy. 2-Chloroethylphosphonic acid was shown to increase germination of partly dormant seed imbibed under certain temperature regimes; a similar effect could not be established for fully dormant or fully nondormant seed. Using gas liquid chromatography, natural ethylene levels were followed during imbibition of fully dormant and nondormant seed. A large peak in production was observed in the period prior to radicle emergence in the case of the nondormant seed. Measurements of ethylene production taken at 15 C, following periods of after ripening in moist soil at either 5 or 25 C, indicated that endogenous production was unlikely to be a main cause of dormancy breakage in this species. The possibility that endogenous ethylene could play a role in natural dormancy breakage in aged seeds is discussed. The practical possibilities of 2 chloroethylphosphonic acid as a dormancy breaking agent in a field situation are outlined. PMID- 16661676 TI - Control of Pyrimidine Biosynthesis in Synchronously Dividing Cells of Helianthus tuberosus. AB - Factors with potential for regulating pyrimidine biosynthesis in plant tissue have been explored in quiescent cells of Helianthus tuberosus induced to divide by auxin addition. Investigations confined to the first highly synchronous cell cycle of the tuber explants revealed that the relative activity of asparate carbamoyltransferase (ACTase) to ornithinecarbamoyltransferase (OCTase) (enzymes competing for carbamoyl phosphate for the pyrimidine and arginine pathways, respectively) changes from 0.5 in quiescent cells to 3.0 by the end of the first cell cycle. This was interpreted as a change in the state of cell function from accumulation of storage arginine to cell division with a concomitant demand for pyrimidine nucleotides for nucleic acid synthesis. The rise in ACTase activity began at the same time as the initiation of DNA synthesis and was dependent on continued DNA synthesis. OCTase activity declined whether or not auxin was added to the medium, whereas ACTase activity was observed to decline only in the absence of DNA synthesis.The low cellular concentration of the shared substrate, carbamoyl phosphate (2 micromolar), favored utilization of this substrate by the pyrimidine pathway over the arginine pathway because of the low K(m) (0.08 80 micromolar) for this substrate by ACTase compared to that for OCTase (9.0 millimolar). Unexpectedly, the total concentration of the feedback inhibitor for the pyrimidine pathway, UMP, was found to have more than doubled in dividing tissue at a time when pyrimidine nucleotide demand had increased. It is concluded that compartmentation decreased UMP in the vicinity of ACTase and/or that the extra UMP stabilizes newly synthesized ACTase in preparation for an even greater demand for nucleic acid synthesis in the second and subsequent cell cycles. PMID- 16661677 TI - Uptake and Concentration of Alkylamines by a Marine Diatom: EFFECTS OF H AND K AND IMPLICATIONS FOR THE TRANSPORT AND ACCUMULATION OF WEAK BASES. AB - Methylamine, ethylamine, and dimethylamine (10 micromolar) are taken up and concentrated 600 to 6,000-fold by Cyclotella cryptica. Methylamine is concentrated most strongly, and its accumulation and retention are relatively insensitive to external pH but strongly inhibited by 30 millimolar external K(+). Accumulation and retention of ethyl- and dimethylamine, on the other hand, are strongly affected by external pH and less sensitive to external [K(+)]. Intracellular pH, as estimated from neutral red staining and quenching of 9 aminoacridine fluorescence, was between 4 and 5, with the central vacuole being the major acidic compartment. The accumulation of ethyl- and dimethylamine could result from diffusion of the uncharged amine across the membrane(s) and passive equilibration of the charged form (R-NH(3) (+)) inside and outside the cell. Differences in the accumulation ratio and the ion dependence for methylamine uptake relative to ethyl- and dimethylamine uptake suggests that a different mechanism is responsible for the concentration of the simpler amine. PMID- 16661678 TI - Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT. AB - Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells. PMID- 16661679 TI - Amino Acid Transport into Cultured Tobacco Cells: II. EFFECT OF CALCIUM. AB - The effects of calcium ions on lysine transport into cultured Wisconsin-38 tobacco cells were examined. In the presence of calcium, lysine was transported at a relatively low rate for 30 to 40 minutes followed by a period of increasing rates and subsequent stabilization at a higher rate after 2 to 3 hours. In the absence of calcium, transport was uniformly low.Time-dependent stimulation of lysine transport rate was observed after the cells had been preincubated in calcium-containing media. Similar treatments also resulted in the stimulated uptake of a variety of other amino acids, organic compounds, and sulfate. The stimulation of lysine uptake was apparently not due to nutrient starvation.Lysine transport was not stimulated in a time-dependent fashion by K(+), La(3+), Mg(2+), or Mn(2+). Cells with stimulated transport rates continued to exhibit high rates when washed with calcium-containing media followed by transport in calcium containing media. All other cation wash treatments were inhibitory, although magnesium treatments resulted in partial preservation of stimulated transport rates. Cycloheximide inhibited the calcium/time-dependent stimulation of lysine transport and caused the stimulated rate to decay.The initial experimental treatments or the culture conditions may represent some form of shock that alters the membrane transport mechanism, thus reducing transport. The observed calcium/time-dependent stimulation may require protein synthesis and represents damage repair. PMID- 16661680 TI - Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis. AB - The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O(2) and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and alpha-ketoglutarate) in CO(2)-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO(2)-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO(2)-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source. PMID- 16661681 TI - Host-Symbiont Interactions : V. THE STRUCTURE OF ACIDIC EXTRACELLULAR POLYSACCHARIDES SECRETED BY RHIZOBIUM LEGUMINOSARUM AND RHIZOBIUM TRIFOLII. AB - The sequence of the glycosyl residues and the anomeric configurations of the glycosyl linkages of the acidic polysaccharides secreted by Rhizobium leguminosarum 128c53, Rhizobium leguminosarum 128c63, Rhizobium trifolii NA30, and Rhizobium trifolii 0403 have been determined. All four polysaccharides were found to have the following glycosyl repeating-unit structure, where galactosyl is Gal, glucosyl is Glc, glucuronosyl is GlcA, and pyruvyl is Pyr: [Formula: see text] Each of the glycosyl residues of these polysaccharides was determined to be in the d configuration and in the pyranose ring form. These results add support to the proposal that R. leguminosarum and R. trifolii have a particularly close genetic relationship. The significance of these results with regard to the possible function of these polysaccharides in the nodulation process is discussed. PMID- 16661682 TI - Production and characterization of antibodies and establishment of a radioimmunoassay for ribosylzeatin. AB - An antibody directed towards ribosyl-trans-zeatin has been produced and characterized. The antiserum was produced in rabbits using ribosyl-zeatin-bovine serum albumin as an immunogen. A radioimmunoassay which employed this antiserum and a tritiated antigen was established. As little as 10 picomoles ribosyl-trans zeatin could be detected. The specificity of the antiserum was measured in the radioimmunoassay by using nonradioactive nucleosides as competitive inhibitors. Changes in position N(6) were more effective in decreasing antibody recognition than changes in position 2. Of particular interest was the interaction of the isomer ribosyl-cis-zeatin. This compound was significantly less active as an inhibitor than ribosyl-trans-zeatin, demonstrating that the antibody was sensitive to minor changes in the structure of the antigen.The use of this antibody and the radioimmunoassay for ribosylzeatin provides a rapid method for the detection of ribosylzeatin, as well as offering the potential for immunoadsorbent columns which would be useful in the purification of macromolecules, such as tRNA, which contain the ribosylzeatin moiety. PMID- 16661683 TI - Assay and Characteristics of Circadian Rhythmicity in Liquid Cultures of Neurospora crassa. AB - Previous work on circadian rhythms of Neurospora crassa has been done almost exclusively with cultures expressing rhythmic conidiation and growing on solid agar medium. Such conditions severely restrict the kinds of biochemical experiments that can be carried out. We have now developed systems which allow indirect assay of circadian rhythmicity in liquid culture. Neurospora was grown in glucose and acetate liquid media under conditions which result in a range of growth rates and morphologies. Liquid media were inoculated with conidia and the cultures were grown in constant light for 33 or 48 hours, by which time floating mycelial pads had formed. Experimental pieces of mycelium then were cut and placed in fresh new liquid medium. As controls, other pieces of mycelium were cut and put directly on solid agar medium in race tubes. All cultures were transferred to constant darkness at this time. This light-to-dark transition set the phase of the circadian clock of both the liquid and solid cultures. At various times after the light-to-dark transition, the mycelial pieces in the liquid were transferred in the dark to solid medium in race tubes, where they grew normally and conidiated rhythmically. Comparison of the phase of the rhythm in these race tubes to the controls demonstrated that, under appropriate conditions, the circadian clock of the liquid cultures functions normally for at least two cycles in constant conditions. Using these culture systems, a significantly greater variety of biochemical studies of circadian rhythmicity in Neurospora is now possible. PMID- 16661684 TI - Eyespot Disease of Sugarcane : INDUCTION OF HOST-SPECIFIC TOXIN AND ITS INTERACTION WITH LEAF CELLS. AB - Helminthosporium sacchari produces a toxin which is responsible for the symptoms of eyespot disease in Saccharum officinarum. A rapid and highly repeatable bioassay based on increase in conductivity of tissue leachates showed that the interaction of toxin with sugarcane obeys Michaelis-Menten hyperbolic saturation kinetics. There was no evidence for positive or negative cooperation interaction. Resistant and susceptible cultivars of sugar cane had distinctive conductivity characteristics. Co-cultures of H. sacchari and suspension cultures of sugarcane gave up to a 4,000-fold increase in toxin production. PMID- 16661685 TI - Simulation of the effects of leaf senescence on membranes by treatment with paraquat. AB - Chloroplast and microsomal membranes from the primary leaf of bean acquired increasing proportions of gel phase lipid as the tissue senesced. The lipid-phase transition temperature for microsomes rose from about 25 to 43 C and that for chloroplasts rose from below -30 C to about 52 C within 5 weeks of planting. This was accompanied by large increases (2- to 4-fold) in the sterol to phospholipid ratio of the membranes, which reflected breakdown of phospholipid. Changes in fatty acid saturation were of insufficient magnitude to account for the rise in transition temperature. All of these senescence-related changes in chloroplast and microsomal membranes were also induced by treating young, 2-week-old-plants with 10 milligrams per liter paraquat. Within 48 hours of treatment, the transition temperature rose from 25 to 57 C for microsomes and from below -30 to 24 C for chloroplasts. The membranes sustained only small changes in fatty acid saturation, comparable to those incurred during natural senescence, and there was a selective loss of phospholipid, resulting in augmented sterol to phospholipid ratios. Malondialdehyde, a product of lipid peroxidation, rose by 2- to 3-fold in both senescing and paraquat-treated leaves. Paraquat is known to form cation redicals that react with O(2) to produce O(2) (-) and has been implicated as an agent of lipid peroxidation. Accordingly, these observations suggest that membrane deterioration during natural senescence may be due in part to free radical damage. PMID- 16661686 TI - Wound-induced Ethylene Formation in Albedo Tissue of Citrus Fruit. AB - Excised albedo tissue of citrus fruit (Citrus unshiu and Citrus hassaku) produced ethylene at an increasing rate in response to wounding and aging. The application of 1-aminocyclopropane-1-carboxylic acid (ACC) enhanced ethylene production in both the fresh and aged tissues, but this increase was greater in the aged tissue than in the fresh tissue. ACC content was very low in fresh tissue but increased greatly in aging tissue, paralleling the rise in ethylene production. Aminoethoxyvinylglycine (AVG) strongly inhibited ethylene production in the aged tissue. In the presence of ACC, however, ethylene production was not inhibited by AVG. These results suggest that ACC is an intermediate in the pathway of ethylene biosynthesis in the albedo tissue and that both steps of ACC formation and ACC conversion to ethylene are enhanced by wounding and aging. Inhibitors of protein synthesis, cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, strongly inhibited ethylene production in the albedo tissue, implying that protein synthesis is required to maintain the continuous evolution of ethylene. The stimulation of ethylene production by ACC was reduced by the addition of l-methionine, whereas d-methionine had very little inhibitory effect. Ethylene production in the albedo tissue was also inhibited by the addition of n propyl gallate and 3,5-dibromo-4-hydroxybenzoic acid. PMID- 16661687 TI - Ethanol and acetaldehyde in imbibing soybean seeds in relation to deterioration. AB - Deterioration as evidenced by decline in germination or seedling growth of soybean (cv. Essex) seeds during accelerated aging treatments at 41 C and 100% relative humidity is accompanied by increased levels of acetaldehyde and ethanol in imbibing embryonic axes and seeds. These increases become more pronounced with duration of the aging treatment. A similar inverse relationship between levels of acetaldehyde and ethanol and deterioration was observed when seeds were "naturally" aged for several years. During imbibition of low-vigor, accelerated aged seeds at 25 C, acetaldehyde and ethanol increased from near trace amounts in dry tissues to maximum levels at 4 hours. Increases in acetaldehyde and ethanol during imbibition were less in high- than in low-vigor seeds. Increases were also less pronounced in low-vigor seeds when water uptake injury was avoided by osmotically decreasing water uptake rate with 30% polyethylene glycol. Embryonic axes from deteriorated seeds were characterized by low rates of O(2) uptake and high respiratory quotients relative to the unaged controls. Anaerobic conditions and respiratory inhibitors, such as sodium azide, increased acetaldehyde and ethanol in unaged seeds to levels similar to those in accelerated-aged seeds after 2 hours imbibition. It is suggested that, during aging, an imbalance between tricarboxylic and glycolytic activities, present during early imbibition to some degree even in vigorous unaged seeds, becomes more pronounced and leads to accumulation of ethanol and acetaldehyde. PMID- 16661688 TI - Movement of C-compounds from Maternal Tissue into Maize Seeds Grown in Vitro. AB - Uptake from nutrient media into the cob and translocation of various (14)C compounds from maternal tissue (cob) into developing maize seeds was examined by using caryopsis cultures. Based on relative (14)C concentrations in the cob and the endosperm, it was concluded that the relative efficiencies of movement of amino acids (leucine, phenylalanine, proline), vitamins (thiamine HCl, nicotinic acid), and nucleic acid bases (adenine, thymine) from the cob to the endosperm were 11 to 250 times lower than that of sucrose. Thiamine was unique in that it was concentrated in the embryo at a level that was almost 10 times higher than in the endosperm. The absence of auxotrophic mutants requiring an organic supplement in higher plants (other than thiamine auxotrophs) may be explained by inadequate translocation of these essential metabolites into the mutant zygotes (embryos) to enable their development to mature seeds. PMID- 16661689 TI - Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VII. CHROMOSOME NUMBERS, METAPHASE I CHROMOSOME BEHAVIOR, AND MODE OF REPRODUCTION OF PHOTOSYNTHETICALLY DISTINCT PANICUM SPECIES. AB - Panicum species of the Laxa group were investigated in a series of published reports and were found to possess C(4), C(3), and intermediate photosynthetic characteristics. Taxonomic and other relationships among these plants, however, are not clear. It was the objective of this investigation to document chromosome number, metaphase I chromosome behavior, and mode of reproduction, including abnormalities in the embryo sac, for these species.Chromosome counts showed a basic number (x) of 10 and ploidy levels of diploid (2n = 2x = 20), tetraploid (2n = 4x = 40), and hexaploid (2n = 6x = 60) in this group of Panicum. One diploid and one tetraploid accession of the C(4) species, Panicum prionitis Griseb., were obtained. Of the intermediate species, Panicum milioides Nees ex Trin. was diploid, Panicum schenckii Hack. was hexaploid, and Panicum decipiens Nees, ex Trin. was found to possess two ploidy levels, one accession being diploid and the other accession being hexaploid. All the C(3) species, which included two accessions of Panicum laxum Sw., three accessions of Panicum hylaeicum Mez., and one accession of Panicum rivulare Trin., were tetraploid.Meiosis was regular with primarily bivalent pairing at metaphase I in all species except the tetraploid accession of P. prionitis which possessed from 4 to 10 tetravalents. Stainable pollen was high in all species, ranging from 70 to 99%. Embryo sac analyses showed a single sac in all plants except the tetraploid accession of P. prionitis, which was found to possess an additional sac at anthesis. An additional sac was also observed in some ovaries of the P. schenckii accession. Self-pollinated seed set was high in all accessions except the diploid accession of P. prionitis and one accession of P. laxum where no seed was set under bagged conditions.This information establishes, within the limits of this collection, a base for future studies on genetic, taxonomic, photosynthetic, and evolutionary relationships among these plants. Possession of the same basic chromosome number, regular meiotic pairing, a high degree of stainable pollen, and good seed set in most of the plants studied indicate possible success in making hybrids for a genetic study of photosynthetic pathways in Panicum. PMID- 16661690 TI - Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ. AB - A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH(4))(2)SO(4) precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000. PMID- 16661691 TI - Sulfate Transport in Cultured Tobacco Cells : EFFECTS OF CALCIUM AND SULFATE CONCENTRATION. AB - Sulfate transport by tobacco cells (var. Xanthi) cultured in liquid medium was investigated. Monophasic uptake was observed over a sulfate concentration range from 0.01 to 10 millimolar, and the K(m) was 20 micromolar. A time-dependent stimulation of transport was observed when cells were incubated in medium containing 0.5 millimolar Ca(2+). Calcium stimulation was dependent on the culture cycle and was maximal during the early exponential phase. It was not observed in sulfur-deficient cells with high transport rates and was relatively small in sulfate-loaded cells with low transport rates. A kinetic analysis showed that Ca(2+) increased the maximum rate of transport without affecting the K(m). PMID- 16661692 TI - Role of the testa in preventing cellular rupture during imbibition of legume seeds. AB - Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing. PMID- 16661693 TI - Distribution of Foliar-applied Boron Measured by Spark-source Mass Spectrometry and Laser-probe Mass Spectrography. AB - The distribution of foliar-applied boron ([(10)B]boric acid) in radish (Raphanus sativus L.) was studied using for analysis of the stable isotopes a technique allowing a high sensitivity: spark-source mass spectrometry. Boron was recovered in the nontreated aerial parts and in the roots; however, the greatest fraction was in the treated leaf. It was possible with a laser-probe mass spectrograph to show that boron was not superficially located in the treated area but was present in tissues at all levels of depth considered. PMID- 16661694 TI - Ethylene Effects in Pea Stem Tissue : EVIDENCE OF MICROTUBULE MEDIATION. AB - The marked effects of ethylene on pea stem growth have been investigated. Low temperature and colchicine, both known microtubule depolymerization agents, reverse the effects of ethylene in straight growth tests. Low temperature (6 C) also profoundly reduces the effects of gas in terms of swelling, hook curvature, and horizontal nutation. Deuterium oxide, an agent capable of rigidifying microtubular structure, mimics the effects of ethylene. Electron microscopy shows that microtubule orientation is strikingly altered by ethylene. These findings indicate that some of the ethylene responses may be due to a stabilizing effect on microtubules in plant cells. PMID- 16661695 TI - Role of Glutamate-oxaloacetate Transaminase and Malate Dehydrogenase in the Regeneration of NAD for Glycine Oxidation by Spinach leaf Mitochondria. AB - During glycine oxidation by spinach leaf mitochondria, oxygen consumption showed a strong and transient inhibition upon addition of oxaloacetate or aspartate plus alpha-ketoglutarate. During the course of the inhibition, aspartate and alpha ketoglutarate were stoichiometrically transformed into malate and glutamate.It is concluded that oxaloacetate formed by transamination is reduced by the malate dehydrogenase, which allows the regeneration of NAD(+) for glycine oxidation and, thus, by-passes the respiratory chain. Efficiency of a malate-glutamate/aspartate alpha-ketoglutarate shuttle upon illumination and under in vivo conditions is discussed. PMID- 16661696 TI - Slow Passive Diffusion of Orthophosphate between Intact Isolated Chloroplasts and Suspending Medium. AB - Isolated spinach chloroplasts purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extrachloroplastic contaminations, and to retain a high rate of CO(2)-dependent O(2) evolution.When suspended in a medium which avoided rupture of the envelope, intact purified chloroplasts progressively lost their phosphate content by passive diffusion. This led to a slow decrease in the uptake of labeled 3 phosphoglyceric acid or orthophosphate (Pi) and in the rate of CO(2)-dependent O(2) evolution by isolated chloroplasts. Under these conditions, there was a good correlation between the rate of CO(2)-dependent O(2) evolution and the concentration of Pi in the stroma space. Addition of Pi to the suspending medium at a final concentration of 10 millimolar, which counterpoised the slow efflux of Pi from the chloroplasts, slowed considerably the decrease in the rate of CO(2) dependent O(2) evolution. PMID- 16661697 TI - Stable Hydrogen Isotope Fractionations during Autotrophic and Mixotrophic Growth of Microalgae. AB - Isotope effects, studied with precision isotope ratio mass spectrometry, have been used to locate critical steps in the H metabolism of plants. By manipulating the growth conditions of versatile microalgae, the discrimination of H isotopes between water in the growth medium and the organically bonded H in carbohydrates from these microalgae was -100 to -120 per thousand and was regulated by both the light and the dark reactions of photosynthesis. Photosynthetic electron transport discriminated against the heavy isotope of H and formed a pool of reductant available for biosynthesis that was enriched in the light isotope. Growth in red or white light activated phosphoglyceric acid reduction and H isotope discrimination, when H was fixed into organic matter. An additional fractionation of -30 to -60 per thousand occurred during the biosynthesis of proteins and lipids and was associated with glycolysis. This fractionation paralleled the isotope effect seen in carbohydrate metabolism, indicating that H metabolism in photosynthesis was coupled with that in dark biosynthetic reactions via the pool of reductant, probably NADPH. PMID- 16661698 TI - Plasma Membrane Alterations in Callus Tissues of Tuber-bearing Solanum Species during Cold Acclimation. AB - Plasma membrane alterations in two tuber-bearing potato species during a 20-day cold acclimation period were investigated. Leaf-callus tissues of the frost resistant Solanum acaule Hawkes ;Oka 3878' and the frost-susceptible, commonly grown Solanum tuberosum ;Red Pontiac,' were used. The former is a species that can be hardened after subjecting to the low temperature, and the latter does not harden. Samples for the electron microscopy were prepared from callus cultures after hardening at 2 C in the dark for 0, 5, 10, 15, and 20 days. After 20 days acclimation, S. acaule increased in frost hardiness from -6 to - 9 C (killing temperature), whereas frost hardiness of S. tuberosum remained unchanged (killed at -3 C). Actually, after 15 days acclimation, a -9 C frost hardiness level in S. acaule callus cultures had been achieved.Membrane protein particle aggregation was monitored using freeze-fracture electron microscopy. Protein particles were aggregated in S. acaule up to 10 days after the initiation of acclimation treatment and then redistributed almost to the level of control after 15 days. No such changes were observed for S. tuberosum under similar experimental conditions. The change in protein particle aggregation pattern in S. acaule is interpreted as indicating the presence of an adaptive fluidity control mechanism in that species. PMID- 16661699 TI - Osmoregulation in Cotton in Response to Water Stress : I. ALTERATIONS IN PHOTOSYNTHESIS, LEAF CONDUCTANCE, TRANSLOCATION, AND ULTRASTRUCTURE. AB - Cotton plants subjected to a series of water deficits exhibited stress adaptation in the form of osmoregulation when plants were subjected to a subsequent drying cycle. After adaptation, the leaf water potential coinciding with zero turgor was considerably lower than in plants that had never experienced a water stress. The relationship between leaf turgor and leaf water potential depended on leaf age.Nonstomatal factors severely limited photosynthesis in adapted plants at high leaf water potential. Nonetheless, adapted plants maintained photosynthesis to a much lower leaf water potential than did control plants, in part because of increased stomatal conductance at low leaf water potentials. Furthermore, adapted plants continued to translocate recently derived photosynthate to lower leaf water potentials, compared with control plants.Stress preconditioning modified cellular ultrastructure. Chloroplasts of fully turgid adapted leaves contained extremely large starch granules, seemed swollen, and had some breakdown of thylakoid membrane structure. In addition, cells of adapted leaves appeared to have smaller vacuoles and greater nonosmotic cell volume than did control plants. PMID- 16661700 TI - Osmoregulation in Cotton in Response to Water Stress : II. LEAF CARBOHYDRATE STATUS IN RELATION TO OSMOTIC ADJUSTMENT. AB - Diurnal changes in tissue water potential components, photosynthesis, and specific leaf carbohydrates were examined in water stress-adapted and nonadapted cotton plants. Adapted plants exhibited lower daily minimum leaf water potentials and maintained turgor to lower leaf water potentials than nonadapted plants. Because of this turgor maintenance, photosynthesis continued in adapted plants at leaf water potentials that inhibited photosynthesis in nonadapted plants. Adapted plants exhibited lower rates of photosynthesis than did nonadapted plants when leaves were fully turgid. The inhibition was not due to stomatal restriction of CO(2) diffusion because leaf conductances of nonadapted and adapted leaves were similar at high leaf water potentials.Adapted plants had more glucose than nonadapted plants, the pattern of glucose accumulation depending on leaf age. Sucrose accumulation in response to decreasing water potential also depended on leaf age. Adapted young leaves exported sucrose, whereas nonadapted leaves of the same age accumulated sucrose at the same leaf water potential. Older leaves of both adapted and nonadapted plants accumulated sucrose as plants became stressed during the day.Old, fully expanded leaves from adapted plants contained up to 5 times more starch than did nonadapted leaves, although the kinetics of starch accumulation and degradation were similar in adapted and nonadapted leaves. In young leaves, adaptation did not affect starch accumulation.When adapted plants were destarched by 80 hours darkness, they became "de-adapted" plants. In nonadapted and de-adapted plants, photosynthesis, leaf conductance, and leaf turgor responded identically to declining leaf water potentials. The data implicate starch in the regulation at cellular nonosmotic volume and, thus, osmotic adjustment. PMID- 16661701 TI - Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5' NUCLEOTIDASES FROM WHEAT GERM CYTOSOL. AB - Two forms (F-I and F-II) of 5'-nucleotidases (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N(6)-(Delta(2) isopentenyl)adenosine 5'-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5'-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K(m) values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5'-phosphates, AMP being the preferred substrate. N(6)-(Delta(2)-isopentenyl)Adenosine 5' monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3'-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5'-nucleotidases may play an important role in regulating levels of "active cytokinin" in plant cells. PMID- 16661702 TI - Proteinases and enzyme stability in crude extracts of castor bean endosperm. AB - The stability of catalase, fumarase, and isocitrate lyase from deliberately broken organelles in crude extracts from endosperm tissue of castor bean seedlings has been examined. These enzymes are relatively stable at 2 C in extracts from endosperm of 2-day seedlings, but rapid losses of activity occur in extracts from older seedlings. These losses are shown to be brought about by the thiol-proteinase present in the extracts. The inclusion of 35% glycerol prevented the loss of catalase, fumarase, and isocitrate lysase activity, and various inhibitors of proteinases afforded limited protection. The most striking protectant was leupeptin, an inhibitor of serine and thiol-proteinases. Leupeptin completely inhibited the loss of activity of the three enzymes in crude extracts and improved yields when included in the grinding medium. PMID- 16661703 TI - Cyanide-sensitive and Cyanide-resistant Respiration in the Germination of Cocklebur Seeds. AB - Interrelation between the CN-sensitive cytochrome path and the CN-resistant, benzohydroxamic acid (BHM)-sensitive, or n-propylgallate (nPG)-sensitive alternative path in seed respiration during germination was examined using the nondormant upper and lower seeds of Xanthium pensylvanicum Wallr. The operation of both paths was required not only for normal germination of the lower seed but also for KCN- or NaN(3)-induced germination of both. From the sensitivity to BHM of the germination response, it became obvious that the alternative path exerts its physiological activity as soon as it develops during the early period of water imbibition. Pretreatments with KCN and NaN(3) for promoting germination, strikingly decreased only the engagement of the cytochrome path in the subsequent respiration without affecting that of the alternative path. Nevertheless, no germination occurred without the operation of the cytochrome path. This suggested that excess operation of the cytochrome path is detrimental to germination, being maximal following the BHM-sensitive phase.The alternative and cytochrome paths operated in a proportion of 1 to 5 in a period just before the lower seeds started to germinate. However, there was little difference between the upper seeds of relatively low germination potential and the lower seeds of relatively high potential with respect to both the developmental pattern of the alternative path and the balance of the alternative and cytochrome paths. The higher germination potential of the lower seeds may be related to their high capacities for the alternative path. PMID- 16661704 TI - Ammonia accumulation and inhibition of photosynthesis in methionine sulfoximine treated spinach. AB - Ammonia accumulation and photosynthetic rate inhibition took place when spinach leaf tissue was supplied with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. This effect was observed in the absence of significant inorganic nitrogen reduction or an exogenous source of ammonia. Both the time lag prior to the initial photosynthetic rate decrease and the rate of that decrease depend on the O(2) and MSO concentrations supplied to the leaf tissue. However, the total rate of ammonia accumulation was similar at both 20% and 2.2% O(2). The decline in photosynthetic rate was not caused by stomatal closure but may be a result of ammonia toxicity. The data point out the importance of glutamine synthetase in preventing the poisoning of leaf metabolism by ammonia generated internally through processes not involved in net nitrogen assimilation. The rapidity of the action of MSO in suppressing photosynthesis was unexpected and should not be overlooked in interpreting data from other experiments involving that inhibitor. MSO shows promise as a tool for investigating C-N flow, particularly during photorespiration. PMID- 16661705 TI - Mobilization of respiratory metabolism in potato tubers by carbon dioxide. AB - Applying high concentrations of CO(2) to whole potato tubers stimulated a rapid and pronounced respiratory gas exchange, which persisted for a prolonged time. The upsurge in respiration was proportional to the applied CO(2) concentrations and was further augmented by high O(2) levels. Tests using whole potatoes, or potato tissue slices from tubers previously treated with CO(2), indicated that the rapid CO(2)-induced respiration is sensitive to cyanide during the first 24 hours of CO(2) application. The respiratory rise cannot be attributed to the emergence of a cyanide-resistant alternative electron transport pathway, although prolonged applications of CO(2), up to 72 hours, led to a gradual development of the pathway. CO(2)-stimulated respiration was accompanied by a pronounced decline in the content of starch and glucose 6-phosphate, suggesting an active utilization of respiratory substrates. The ATP content in the CO(2)-treated potatoes increased markedly, resembling similar increases in tissues undergoing respiratory upsurge. PMID- 16661706 TI - A Nonaqueous Procedure for Isolating Starch Granules with Associated Metabolites from Maize (Zea mays L.) Endosperm. AB - A nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol was developed for the isolation from maize of starch granules with associated metabolites. In this procedure, immature endosperm tissue was quickly frozen at -156 C, freeze dried, homogenized in cold glycerol, filtered through Miracloth, and centrifuged through a higher density medium of 3-chloro-1,2-propanediol. The procedure was used to isolate starch granules from the endosperm of normal and the mutant amylose-extender dull waxy. Starch and water-soluble polysaccharide recovery was high with low cytoplasmic (RNA) and nuclear (DNA) contamination.Electron microscopic examination of the isolated starch granules failed to demonstrate the presence of the amyloplast's membrane. However, based on an examination of fresh, freeze-dried, and rehydrated freeze-dried normal endosperm, it is suggested that the amyloplast membrane and enclosed stroma metabolites were dried onto the surface of the starch granules during the freeze-drying procedure. Chemical analysis of the glycerol-propanediol isolated granules showed the presence of alcohol-soluble sugars, inorganic phosphate, and phosphate-containing compounds. These soluble metabolites may represent amyloplast stroma metabolites which became bound to the starch granules during freeze-drying. Thus, this isolation procedure should be useful when metabolites closely associated with starch granules in situ are to be evaluated. PMID- 16661707 TI - Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm. AB - Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2 propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1 phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1 phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm. PMID- 16661708 TI - Similarity of Ribulose-1,5-bisphosphate Carboxylases of Isogenic Diploid and Tetraploid Ryegrass (Lolium perenne L.) Cultivars. AB - Partially purified ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was isolated from diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) using two separate methods. The apparent K(m) (CO(2)) values for the enzymes prepared by either method did not differ significantly between diploid and tetraploid when assayed by two separate techniques. The unpurified enzymes from freshly lysed (and fully functional) protoplasts of both diploid and tetraploid cultivars gave virtually identical apparent K(m) (CO(2)) values. There was no indication of large differences in affinity for CO(2) of illuminated intact protoplasts from the two cultivars. PMID- 16661709 TI - Transfer ribonucleic Acid modification and its relationship to tumorous and nontumorous plant growth. AB - Detailed analyses of tRNA hydrolysates from four tissue types of Nicotiana tabacum, pith from intact plants, pith growing in culture, habituated tissue in culture, and crown gall tumor tissue in culture, revealed significant qualitative and quantitative differences in the pattern of methylation. Although pith from intact plants and pith growing in culture possessed seven different methylated nucleosides, only two were found in habituated and tumorous tissues in culture. Four of the five compounds accounting for the difference were tentatively identified as methylated guanosines. Evaluation of results in terms of several parameters, including growth rate, the tumorous state, habituation, tissue culture, and potential for differentiation, indicate that the extent of tRNA methylation may be correlated with the potential for differentiation of a particular tissue. PMID- 16661710 TI - Stomatal Response and Leaf Injury of Pisum sativum L. with SO(2) and O(3) Exposures : I. INFLUENCE OF POLLUTANT LEVEL AND LEAF MATURITY. AB - Plants of Pisum sativum L. ;Alsweet' were grown under a controlled environment and exposed to SO(2) and O(3) to determine whether changes in stomatal aperture during exposure were related to subsequent leaf injury. Stomata consistently closed with injurious levels of SO(2) and O(3). Measurements with diffusion porometers demonstrated approximately 75 and 25% lower conductance with SO(2) and O(3) exposures, respectively, compared to the conductance of control plants. Stomata also showed a closing response with noninjurious levels of SO(2) but an opening response with noninjurious levels of O(3). Stomata closed to the same degree with combinations of SO(2) plus O(3) as with SO(2) alone. Stomata of expanding leaves closed more during pollutant exposures than stomata of expanded leaves. The abaxial and adaxial stomata both exhibited closure with SO(2) and combinations of SO(2) plus O(3), but abaxial stomata tended to close and adaxial stomata tended to open with exposure to O(3) alone.The changes in stomatal aperture were not closely correlated with the amount of leaf injury produced by different pollutant levels. Stomata closed, not only with exposure to pollutant levels that caused severe necrosis, but also with levels that caused only a trace of injury. There was no evidence of a reduced amount of closure or even stomatal opening with combinations of SO(2) and O(3) compared to plants exposed to the pollutants alone to explain the large amount of injury to plants exposed to pollutant combinations. PMID- 16661711 TI - Stomatal Response and Leaf Injury of Pisum sativum L. with SO(2) and O(3) Exposures : II. INFLUENCE OF MOISTURE STRESS AND TIME OF EXPOSURE. AB - Stomatal response during exposure to SO(2) and O(3) and subsequent leaf injury were examined in plants of Pisum sativum L. ;Alsweet' grown in a peat-vermiculite medium in controlled environment chambers. Plants developing under moisture stress, induced by drying the medium to 50% of field capacity, exhibited greater stomatal closure during exposures and less than one-fourth the necrosis compared to plants developing in a medium maintained at field capacity. Plants under moisture stress had only a slightly more negative plant water potential ( approximately -4.0 bars) than at field capacity ( approximately -3.4 bars). Plants exposed to pollutants for 2 hours near the beginning or end of a 16-hour light period had greater stomatal closure during exposures and less leaf necrosis than plants exposed during the middle of the light period. PMID- 16661712 TI - Induction of glyconeogenic enzymes by gibberellin a(3) in endosperm of castor bean seedlings. AB - Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to gibberellin A(3) (GA(3)) (100 micromolar) for periods up to 20 hours. Endosperm homogenates were fractionated on linear sucrose gradients and enzymes in mitochondria, glyoxysome, and cytosol fractions were assayed. Gibberellin treatment resulted in increases in the activities of enzymes in all three compartments. There were also enzymes in all three compartments which were not affected by exogenous applications of GA(3). The isozymes of l-asparate-alpha-ketoglutarate aminotransferase in both mitochondria and glyoxysomes were induced coordinately, whereas the isozymes of citrate synthase and malate dehydrogenase were not. All gluconeogenic enzymes in glyoxysomes are induced by GA(3). With the exception of the mitochondrial malate dehydrogenase isozyme, all enzymes of the tricarboxylic acid cycle believed to participate in glyconeogenesis were increased. The cytosolic enzymes malate dehydrogenase, phosphoenolpyruvate carboxykinase, and fructose bisphosphatase were induced, but the levels of pyruvate kinase and enolase were not affected by GA(3) treatment. PMID- 16661713 TI - Coupling of chlorophyll metabolism with submembrane chloroplast particles, isolated with digitonin and gel electrophoresis. AB - An unusual set of submembrane particles is obtained from digitonintreated barley chloroplasts as five gel-electrophoretic zones. Four of them are photochemically active, whereas the most mobile fifth zone has essential traits of the light harvesting complexes. All of the particles contain the well-known chlorophyll protein complexes and represent an intermediate level of membrane organization. When isolated from plants fed delta-aminolevulinate in the dark, the fifth zone is characterized by a high level of protochlorophyllide, which is also present to a lesser extent in all the other zones. When [(14)C]aminolevulinate was fed in the dark, followed by exposing the plants to light, the same pattern of the distribution was observed for [(14)C]chlorophyll a. Thus, particles of all the types are involved in chlorophyll formation and the fifth zone is the most distinct in this respect. Its material seems to originate from the most intensely developing areas of the metabolically heterogeneous chloroplast membrane system. PMID- 16661714 TI - Involvement of Protons as a Substrate for the Sucrose Carrier during Phloem Loading in Vicia faba Leaves. AB - The effect of pH on uptake of exogenous sucrose by broadbean (Vicia faba L.) leaf discs without the lower epidermis has been investigated at various sucrose concentrations. The concentration dependence of sucrose uptake showed a biphasic saturation response. At high sucrose concentrations (>20 millimolar), sucrose uptake showed no pH dependence. At low sugar concentrations (<5 millimolar), plots of 1/V against 1/H(+) give straight lines which all intercept at the same point at the left of the ordinal axis. Calculations show that these data agree well with two-substrate kinetics for the carrier, the substrates being the protons and the sucrose molecules. Our results provide further evidence that protonation/deprotonation processes of the carrier are involved in phloem loading, especially for low sucrose concentrations of the apoplast. PMID- 16661715 TI - Photosynthesis-deficient Mutants of Chlamydomonas reinhardii with Associated Light-sensitive Phenotypes. AB - A series of non-photoautotrophic mutants of Chlamydomonas reinhardii was isolated by replica-plating mutagenized cells which had been grown in the dark. Many of these acetate-requiring mutants are photosensitive, showing poor growth on acetate medium in the light, but normal growth in the dark. Biochemical characterization showed that the photosensitive mutants all had specific lesions in photosynthesis or photosynthetic pigment accumulation. The acetate-requiring mutants which were not photosensitive were all able to fix CO(2). Among the light sensitive mutants are 15 which show uniparental inheritance. These include six with specific lesions in photosystem II and one with an altered large subunit of ribulose-1,5-bisphosphate carboxylase. Since these two classes of uniparental mutants have been rare or not previously reported, it seems likely that photosensitivity is an important factor which limited their detection in previous mutant isolation experiments. PMID- 16661716 TI - Photosystem II Photosynthetic Unit Sizes from Fluorescence Induction in Leaves : CORRELATION TO PHOTOSYNTHETIC CAPACITY. AB - The use of fluorescence induction measurements in leaves infiltrated with 3-(3,4 dichlorophenyl)-1,1-dimethylurea has been evaluated as a routine method for estimation of the concentration of the reaction centers of photosystem II relative to total chlorophyll in a wide variety of plant species. The procedure is based on a simple theory that takes into account the attenuation of light in passing through the leaf and the linear dependence of the fluorescence induction time from different parts of the leaf on the inverse of the local light intensity. A formula to calculate the reaction center concentration of photosystem II was obtained. The effect of the light attenuation is accounted for by a correction factor which could become practically insignificant by an optimal choice of the excitation and emission wavelengths and the geometry of the photodetector with respect to the sample. Estimation of quantum yields for primary photochemistry and influence of light scattering were considered. The results demonstrate the effect of the above factors under various circumstances and are in agreement, to a first approximation, with the theory.THE UTILITY OF THE METHOD IS DEMONSTRATED BY A DETAILED STUDY OF FOUR DESERT PLANT SPECIES: estimation of reaction center concentrations of both photosystem I (by estimation of P700) and photosystem II (by the fluorescence induction method) were made and were compared to the rates of CO(2) fixation. There was a good quantitative correlation between the photosynthetic rates and the concentration of photosystem II reaction centers (expressed as per chlorophyll or per unit area of the leaf), but no such correlation was found with photosystem I reaction centers.The ratio of total chlorophyll per reaction centers II varied in the range of about 200 to 800 in different species, but there was no variation of this parameter in any single species. PMID- 16661717 TI - Photosynthetic units of sun and shade plants. AB - A computer analysis of fluorescence induction curves of leaves treated with 3 (3,4-dichlorophenyl)-1,1 dimethylurea was done for several species. These measurements gave the ratios of the total chlorophyll to photosystem II reaction centers. This communication is a preliminary survey of sun and shade plants and demonstrates a significant variation in this ratio. In the sun plants, the photosynthetic unit sizes (chlorophyll reaction centers) varied between 220 to 480. The shade plants gave numbers mostly in the range between 630 to 940. The computer analysis of the fluorescence data also gave the connectivity parameter of energy transfer between photosynthetic units of photosystem II which varied between 0.2 and 0.5 but did not show any obvious correlation to the photosynthetic unit size. PMID- 16661718 TI - Rapid Suppression of Growth by Blue Light: OCCURRENCE, TIME COURSE, AND GENERAL CHARACTERISTICS. AB - The inhibition of stem elongation in dark-grown seedlings by blue light was studied with marking techniques and with a high-resolution, growth-measuring apparatus. Blue light rapidly suppresses growth in a variety of cultivated species. In some species, the inhibition persists only during the period of irradiation, after which time growth quickly returns to the high dark rate, whereas, in other species, the light response has an additional long-term component which lasts for at least several hours in the dark. The long-term inhibition may be mediated by phytochrome, whereas the rapid, short-term component is specific to a blue-light receptor.The rapid inhibition of growth in cucumber (Cucumis sativus L.) requires high-energy blue irradiation, which is perceived directly by the growing region of the hypocotyl and inhibits all regions below the hook to the same extent. Detailed investigation of the kinetics of the inhibition in cucumber and in sunflower (Helianthus annuus L.) shows that, after a short lag period (20 to 30 seconds in cucumber, 60 to 70 seconds in sunflower), the growth rate declines in an exponential fashion to a lower rate, with a half-time of 15 to 25 seconds in cucumber and 90 to 150 seconds in sunflower. Excision of the hypocotyl greatly reduces the sensitivity of the growth rate to blue-light inhibition. Because of the rapid kinetics, the blue light photoreceptor cannot affect cell enlargement by altering the supply of growth hormone or the sensitivity to hormones but probably operates more directly either on the biochemical process which loosens cell walls or on cell turgor. PMID- 16661719 TI - Vacuolar Deposition of Ascorbate-derived Oxalic Acid in Barley. AB - l-[1-(14)C]Ascorbic acid was supplied to detached barley seedlings to determine the subcellular location of oxalic acid, one of its metabolic products. Intact vacuoles isolated from protoplasts of labeled leaves contained [(14)C]oxalic acid which accounted for about 70% of the intraprotoplast soluble oxalic acid. Tracer labeled oxalate accounted for 36 and 72% of the (14)C associated with leaf vacuoles of seedlings labeled for 22 and 96 hours, respectively. PMID- 16661720 TI - Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: II. GRADIENT PURIFIED MITOCHONDRIA LACK CYANIDE-INSENSITIVE RESPIRATION. AB - Enriched mitochondrial fractions isolated from durum wheat seedlings via differential centrifugation exhibited classical cyanide- or antimycin A insensitive O(2) uptake which was inhibited by either salicylhydroxamic acid or propyl gallate. Further purification of this fraction using Percoll density gradients resulted in two discrete bands which were essentially homogeneous mitochondrial populations, as verified by electron microscopy. Respiratory O(2) uptake in these two fractions was completely inhibited by cyanide or antimycin A. Addition of linoleic acid to a third-step gradient band, which was shown to contain virtually no mitochondria, resulted in demonstrable cyanide-insensitive O(2) uptake. This O(2) consumption was completely inhibited by propyl gallate or salicylhydroxamic acid, two known lipoxygenase inhibitors. In contrast, addition of linoleic acid to the two purified mitochondrial fractions did not stimulate O(2) uptake. These data indicate that lipoxygenase oxygenation, the enzyme physically separable from the mitochondria, is responsible for the cyanide insensitive component of O(2) uptake that was observed in subcellular fractions isolated from etiolated wheat seedlings. PMID- 16661721 TI - CO(2) Exchange and Dinitrogen Fixation of Subterranean Clover in Response to Light Level. AB - Small swards of nodulated subterranean clover plants were grown in pots to a common dry weight under controlled conditions. The rooting medium was a porous calcined clay. All mineral nutrients except nitrogen were supplied daily in solution. Pots then were placed in an assimilation chamber for 3 days for the measurement of net CO(2) exchange at light levels ranging from 0.1 to 2.0 millieinsteins per square meter per second. N(2)-fixation (acetylene reduction) of each pot was measured subsequently. H(2)-evolution and N(2)-fixation were measured for similar treatments in separate experiments using smaller pots.CO(2) assimilation rate and N(2)-fixation rate responded similarly to increase in light level so that N(2)-fixation was found to be a linear function of both the daily (12-hour) net CO(2)-uptake and of the growth rate (24 hours) of the sward over a wide range. N-concentration of the plants was independent of the light level under which they were grown. The rate of N(2)-fixation per unit weight of nodule varied over a wide range depending on the number of nodules present and on the demand for nitrogen as determined by the growth rate of the sward. It is concluded that the amount of assimilate available to the whole plant is a major factor determining the rate of N(2)-fixation by the whole plant, although it may not be important in determining specific rates of nodule activity. N(2)-fixation per unit weight of nodule therefore is considered to have limited utility for comparative purposes.The relative efficiency of N(2)-fixation was found to be about 0.7 and independent of the level of the incident light after growth at two contrasting light levels, 0.4 and 1.0 millieinsteins per square meter per second. PMID- 16661722 TI - A comparison of 2,4-dichlorophenoxyacetic Acid metabolism in cultured soybean cells and in embryogenic carrot cells. AB - The removal or reduction in concentration of auxin is often a successful method for obtaining morphogenesis in cell cultures of higher plants, such as carrot, but not for soybean. For this reason, the metabolism of one auxin, 2,4 dichlorophenoxyacetic acid (2,4-D), was compared in both carrot and soybean cells. Whereas soybean cells conjugated a high percentage of their 2,4-D to amino acids, carrot cells contained primarily free 2,4-D. Moreover, after long-term exposure to 2,4-D, carrot cells released much more 2,4-D upon transfer to 2,4-D free (embryogenic) medium than did soybean cells. It appears that the retention of 2,4-D by soybean cells might interfere with subsequent morphogenesis. Because no impairment of 2,4-D efflux was found with short-term exposure to radiolabeled 2,4-D, it was concluded that 2,4-D retention in soybean cells might be due to a time-dependent, metabolic process. The conjugation of 2,4-D to amino acids was shown to be one such time-dependent process. Additionally, the release of 2,4-D from the cells was shown to be due primarily to a loss of free 2,4-D and not 2,4 D-amino acid conjugates. It seems that the greater retention of 2,4-D by soybean cells upon transfer to 2,4-D-free medium is due to greater formation of 2,4-D amino acid conjugates. PMID- 16661723 TI - Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum. AB - Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentum. Phycobilisome-thylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had O(2) evolution rates (about 450 micromoles O(2) per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles O(2) per hour per milligram chlorophyll). Energy transfer to photosystem II chlorophyll was evident from a high F695 nanometer (-196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closely spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 x 10(-6) micrograms phycoerythrin and 1.3 x 10(-6) micrograms chlorophyll was found to contain 5 to 7 x 10(5) phycobilisomes on a thylakoid area of 1.1 to 1.6 x 10(3) square micrometers. PMID- 16661724 TI - Osmoregulation in the Halotolerant Alga Asteromonas gracilis. AB - Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol. PMID- 16661725 TI - Subcellular Localization of Dhurrin beta-Glucosidase and Hydroxynitrile Lyase in the Mesophyll Cells of Sorghum Leaf Blades. AB - Studies with purified mesophyll and epidermal protoplasts and bundle sheath strands have shown that the cyanogenic glucoside dhurrin (p-hydroxy-(S) mandelonitrile-beta-d-glucoside) is localized in the epidermis of sorghum leaves whereas the enzymes involved in its degradation (dhurrin beta-glucosidase and hydroxynitrile lyase) are localized in the mesophyll tissue (Kojima M, JE Poulton, SS Thayer, EE Conn 1979 Plant Physiol 63: 1022-1028). The subcellular localization of these enzymes has now been examined using linear 30 to 55% (w/w) sucrose gradients by fractionation of mesophyll protoplast components. The hydroxynitrile lyase is found in the supernatant fractions suggesting a cytoplasmic (soluble cytoplasm, microsomal or vacuolar location). The dhurrin beta-glucosidase (dhurrinase) is particulate and mostly chloroplast-associated. The dhurrinase activity peak has a shoulder of activity more dense than that of the intact chloroplasts. This shoulder does not coincide with markers of any other cell fraction.In studies of chloroplasts isolated from ruptured mesophyll protoplasts by differential, low-speed centrifugation, the dhurrinase partitions in the same manner as the chloroplast marker triose phosphate dehydrogenase. Chloroplast localization of the beta-glucosidase has also been shown in histochemical studies using 6-bromo-2-naphthyl-beta-d-glucoside substrate coupled with fast Blue B. PMID- 16661726 TI - Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis. AB - Euglena chloroplast polypeptides are resolved by an adaptation of the two dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when (35)S-labeled chloroplast polypeptides are separated by the present two dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography. PMID- 16661727 TI - Parameters influencing the liposome-mediated insertion of fluorescein diacetate into plant protoplasts. AB - Maximum uptake of liposome-encapsulated fluorescein diacetate by Daucus carota protoplasts was observed when 6 x 10(6) protoplasts per milliliter were incubated with 2.4 x 10(7) liposomes per milliliter for 1 hour. In the case of Nicotiana glutinosa protoplasts, optimum ratio of protoplasts to liposomes was 1:10, where 2.3 x 10(5) protoplasts per milliliter were provided. Neutral and positive liposomes were found to be efficient vehicles to transfer their contents into plant protoplasts. When protoplasts treated with liposomes were cultured in a synthetic medium for 1 week, 20% resumed cell divisions. PMID- 16661728 TI - Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings. AB - Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis. PMID- 16661729 TI - Properties of Rhizopus stolonifer Polygalacturonase, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings. AB - Some properties of the polygalacturonase-elicitor from the filtrates of Rhizopus stolonifer cultures have been examined in an attempt to understand its mode of action as an elicitor of casbene synthetase activity in castor bean seedlings. Both the polygalacturonase activity and the elicitor activity are heat-labile with similar heat-sensitivity profiles. Also, the catalytic activity of the enzyme is lost on treatment with sodium periodate, as had been shown previously for the elicitor activity. The pH optimum of the enzyme activity with polygalacturonic acid as the substrate is 4.9. Exposures of germinating castor bean seedlings to the elicitor for short-term periods of 1 to 10 minutes followed by washing and incubation in sterile, distilled water are partially effective in elicitation in comparison with the continuous exposure of the seedlings over 11 hours to the same amount of the elicitor. The initial rate of reaction catalyzed by the enzyme is about 3 times faster with polygalacturonic acid as a substrate than with partially (50%) methylated polygalacturonic acid (pectin). The K(m) value of the enzyme for polygalacturonic acid is about 4.2 millimolar in terms of monomeric units and about 0.07 millimolar in terms of polymer concentration. Examination of the types of products formed by the action of the enzyme suggests that it is an endo-hydrolase. The amino acid composition of this enzyme is similar to those of other extracellular fungal proteins reported. The carbohydrate moiety of the glycoprotein polygalacturonase-elicitor is composed of 92% mannose and 8% glucosamine by gas chromatography-mass spectrometry analysis. The linkage group analysis of the carbohydrate moiety showed that mannosyl residues which are 1,2-linked comprise about 70% of the total glycosyl residues and demonstrated the presence of some 1,3,6- and 1,2,6-linked branching mannosyl residues. PMID- 16661730 TI - Acyl lipids, pigments, and gramine in developing leaves of barley and its virescens mutant. AB - Changes in acyl lipids and pigments during leaf development in a virescens barley mutant (M) and the normal (N) were studied. Apical 3-cm leaf segments were extracted with chloroform-methanol, the extracts were purified on Sephadex G-25 columns, and the polar lipids were separated on two-dimensional-thin layer chromatography silica gel plates. The pigment remaining on the Sephadex column was identified as flavonoids and a zone on the TLC plates which did not correspond to the usual standards was identified as gramine. Quantification of acyl lipids by either polar head group analysis or fatty acid analysis using heptadecanoate as an internal standard gave similar results. The per cent of the total lipid extract quantified for the M between 4 and 8 days ranged from 46 to 65% and that for the N ranged from 60 to 68%. Of these, acyl lipids represented 37 to 48% in the M and 43 to 50% in the N. By 8 days, mono- and digalacto syldiglyceride (MG and DG) accounted for 45 and 25% of the total acyl lipid of both the M and N. For the period of study here, this represented a 4-fold increase in MG and a 2.5-fold increase in DG in the M but only a 1.8-fold increase for MG and DG in the N. These increases were closely correlated with the increases in chlorophyll. Chlorophyll increased sharply between 4 and 6 days for the N, whereas, in the M, it rose from 7 to 50% relative to the normal by 8 days. The proportions of the various fatty acids were unique for the lipid classes. The only major quantitative change for a fatty acid was for hexadecanoate in phosphatidylglycerol which increased from 5% at 4 days to 25 to 30% by 8 days. Relative to the N, the carotenoid content of the M increased from 14 to 50% between 4 and 8 days. In both the M and N, the increase in beta-carotene and chlorophyll were closely correlated. PMID- 16661731 TI - Acetyl coenzyme a carboxylase activity in developing seedlings and chloroplasts of barley and its virescens mutant. AB - Acetyl coenzyme A (CoA) carboxylase activity of whole tissue homogenates and chloroplast preparations was analyzed as the acetyl-CoA-dependent incorporation of [(14)C]bicarbonate into an acid-stable product. The absolute requirement for ATP and MgCl(2), the complete inhibition with avidin, and end-product analysis were consistent with the presence of acetyl-CoA carboxylase activity. Little difference was found between the mutant and normal tissue homogenates from the 1- to 3-day growth stages, during which period both showed a 3-fold increase. However, by 4 days, the activity of the mutant exceeded that of the normal. Fractionation studies showed that the enzyme was a soluble protein present in the stromal fraction of chloroplasts. The biotin content was also highest in the stroma, although it was found in the lamellar fraction as well. For both the mutant and the normal, the highest acetyl-CoA carboxylase activities were obtained in the stromal preparations from 4-day seedlings (54 and 31 nmoles per milligram protein per minute for the mutant and the normal, respectively) with a progressive decline by 6 and 8 days. The difference between the mutant and the normal was not due to the accumulation of an inhibitor in the normal. PMID- 16661732 TI - Ammonia Assimilation in Alnus glutinosa and Glycine max: SHORT-TERM STUDIES USING [N]AMMONIUM. AB - The pattern of assimilation of NH(4) (+) by Alnus glutinosa, a N(2)-fixing, nonleguminous angiosperm, was examined. Detached nodules, roots, and nodulated roots of intact plants were exposed to (13)NH(4) (+) for up to 15 minutes. Glutamine was the most highly labeled compound at all times; the only other compound labeled significantly was glutamate. Similar results were obtained after incubating soybean (L. merr) nodules and roots with (13)NH(4) (+). These observations and the results of pulse-labeling and inhibitor studies with nodules of Alnus were distinctly different from those predicted for the assimilation of NH(4) (+) via glutamine synthetase and glutamate synthase and suggest that glutamate dehydrogenase may play a major role in the assimilation of exogenously supplied NH(4) (+). PMID- 16661733 TI - Photorespiration-deficient Mutants of Arabidopsis thaliana Lacking Mitochondrial Serine Transhydroxymethylase Activity. AB - Three allelic mutants of Arabidopsis thaliana which lack mitochondrial serine transhydroxymethylase activity due to a recessive nuclear mutation have been characterized. The mutants were shown to be deficient both in glycine decarboxylation and in the conversion of glycine to serine. Glycine accumulated as an end product of photosynthesis in the mutants, largely at the expense of serine, starch, and sucrose formation. The mutants photorespired CO(2) at low rates in the light, but this evolution of photorespiratory CO(2) was abolished by provision of exogenous NH(3). Exogenous NH(3) was required by the mutants for continued synthesis of glycine under photorespiratory conditions. These and related results with wild-type Arabidopsis suggested that glycine decarboxylation is the sole site of photorespiratory CO(2) release in wild-type plants but that depletion of the amino donors required for glyoxylate amination may lead to CO(2) release from direct decarboxylation of glyoxylate. Photosynthetic CO(2) fixation was inhibited in the mutants under atmospheric conditions which promote photorespiration but could be partially restored by exogenous NH(3). The magnitude of the NH(3) stimulation of photosynthesis indicated that the increase was due to the suppression of glyoxylate decarboxylation. The normal growth of the mutants under nonphotorespiratory atmospheric conditions indicates that mitochondrial serine transhydroxymethylase is not required in C(3) plants for any function unrelated to photorespiration. PMID- 16661734 TI - Recent studies of the enzymic synthesis of ricinoleic Acid by developing castor beans. AB - Oleate Delta(12)-hydroxylase activity was measured in extracts of developing castor bean seeds. Most of the hydroxylase activity is associated with microsomes. However, when microsomes are washed, the activity is completely lost. Some (50%) of the activity can be restored by addition of the 100,000g supernatant to the washed microsomes. Supernatant extracts (100,000g) of developing safflower seeds are able to restore all (100%) of the hydroxylase activity to the washed castor bean microsomes. In addition, purified mammalian catalase can restore some (25%) of the activity to the microsomes but is not as effective as either castor bean or safflower 100,000g supernatants. The K(m) of the hydroxylase for oxygen is 4 micromolar. Inasmuch as the activity was not inhibited by high concentrations of either carbon monoxide or cyanide, neither the involvement of cytochrome P450 nor other cytochrome systems is suggested. The enzyme system was not saturated by oleoyl-CoA, even at concentrations as high as 200 micromolar. When [(14)C]oleoyl-CoA is supplied as a substrate, the acyl component is rapidly transferred to phosphatidylcholine (PC). Hydroxylation may occur on PC or on a lipid which receives its acyl component from PC. However, exogeneously added 2-[1-(14)C]oleoyl-PC was hydroxylated at a much lower rate than was [1-(14)C]oleoyl-CoA added as the primary substrate. PMID- 16661735 TI - Interpreting Plant Responses to Clinostating: I. MECHANICAL STRESSES AND ETHYLENE. AB - The severe epinasty and other symptoms developed by clinostated leafy plants could be responses to gravity compensation and/or the mechanical stresses of leaf flopping. Epinasty in cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) is delayed by inhibitors of ethylene synthesis and action (aminoethoxyvinylglycine and Ag(+)), confirming the role of ethylene in clinostat epinasty. To test the possibility that clinostat mechanical stresses (leaf flopping) cause ethylene production and, thus, epinasty, vertical plants were stressed with constant, gentle, horizontal, or vertical shaking or with a quick, back-and-forth rotation (twisting). Clinostat leaf flopping was closely approximated but with a minimum of gravity compensation, by turning plants so their stems were horizontal, rotating them quickly about the stem axis, and then returning them to the vertical, repeating the treatment every four minutes (clinostat rotation time). None of these mechanical stresses produced significant epinasties, but vigorous hand-shaking (120 seconds per day) generated minor epinasties, as did Ag(+) applied daily (concentrations high enough to cause leaf browning). Plants gently inverted every 20 minutes developed epinasty at about the same rate and to about the same extent as clinostated plants, but plants inverted every 20 minutes and immediately returned to the upright position did not become epinastic. It is concluded that clinostat epinasty is probably caused by disturbances in the gravity perception mechanism, rather than by leaf flopping. PMID- 16661736 TI - Gravitropism in Higher Plant Shoots: I. A ROLE FOR ETHYLENE. AB - It has long been known that applied ethylene can redirect the gravitropic response, but only occasionally has it been suggested that ethylene normally plays a role in gravitropism. Two inhibitors of ethylene synthesis [Co(2+) and aminoethoxyvinylglycine (AVG)] and two inhibitors of ethylene action (Ag(+) and CO(2)) were shown to delay the gravitropic response of cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) stems. Gentle shaking on a mechanical shaker does not inhibit the gravitropic response, but vigorous hand shaking for 120 seconds delays the response somewhat. AVG and Ag(+) further delay the response of mechanically stimulated plants. AVG delays the response of defoliated and of decapitated plants. Plants laid on their side and restricted so that they cannot bend upward store both bending energy and gravitropic stimulus; they bend immediately when released from restriction (stored energy) and continue to bend for some hours after (stored stimulus). AVG retards the storage of bending energy but not of stimulus. In gravitropism, graviperception may first stimulate ethylene evolution, which may then influence bending directly, or responses involving ethylene could be more indirect. PMID- 16661737 TI - Carbon Dioxide Fixation in Soybean Roots and Nodules: I. CHARACTERIZATION AND COMPARISON WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING EARLY NODULE DEVELOPMENT. AB - These studies demonstrate that soybean (Merr) roots and nodules possess an active system for fixing CO(2). The maximum rates of CO(2) fixation observed for roots and nodules of intact plants were 120 and 110 nanomoles CO(2) fixed per milligram dry weight per hour, respectively. Results of labeling studies suggest a primary role for phosphoenolpyruvate carboxylase in CO(2) assimilation in these tissues. After pulse-labeling with (14)CO(2) for 2 minutes, 70% of the total radioactivity was lost within 18 minutes via respiration and/or translocation out of nodules. During the vegetative stages of growth of soybeans grown symbiotically, CO(2) fixation in nodules increased at the onset of N(2) fixation but declined to a lower level prior to the decrease in N(2) fixation. This decrease coincided with a decrease in the transport of amino acids, especially asparagine, and an increase in the export of ureides. These findings are consistent with a dual role for CO(2) fixation, providing substrates for energy-yielding metabolism and supplying carbon skeletons for NH(4) (+) assimilation and amino acid biosynthesis. PMID- 16661738 TI - Corn Agmatine Iminohydrolase: PURIFICATION AND PROPERTIES. AB - Agmatine iminohydrolase (EC 3.5.3.12) was purified 7,300-fold from extracts of corn shoots by chromatographic separations on diethylaminoethyl-cellulose, Sephadex G-100, and agmatine-affinity column. The enzyme was homogeneous by the criteria of analytical gel electrophoresis. Molecular weight estimated by Bio-Gel P-200 was 85,000, and the enzyme seems to be a dimer with identical subunits (molecular weight, 43,000). The isoelectric point determined by gel electrofocusing was 4.7. The optimal pH and temperature for activity were 6.5 and 60 C, respectively. The activation energy was 10.9 kilocalories per mole. High specificity exists for agmatine, the K(m) value for agmatine was 1.9 x 10(-4) molar, and the enzyme was present in the cytosol. The enzyme was sensitive to Cu(2+) and Zn(2+) and also was inhibited by p-hydroxymercuribenzoate and arcain. PMID- 16661739 TI - Inhibition of 2,4-dichlorophenoxyacetic Acid conjugation to amino acids by treatment of cultured soybean cells with cytokinins. AB - Kinetin, and all other cytokinins tested, inhibited the conjugation of [(14)C]2,4 dichlorophenoxyacetic acid (2, 4-D) to amino acids when supplied simultaneously with the 2,4-D to cultured soybean cells. Upon transfer to hormone-free medium, the cytokinin-treated cells released more of their [(14)C]2,4-D than did the control cells. Initial exposure to low 2,4-D and high kinetin levels resulted in the greatest release of 2,4-D upon subsequent transfer. The observed alteration in 2,4-D metabolism did not seem to be correlated with growth rate. Appropriate treatment of soybean cells with kinetin resulted in 2,4-D metabolism that resembled the 2,4-D metabolism of embryogenic carrot cells. However, no new morphological structures were observed in these soybean cultures, indicating that other factors are related to the failure of soybean cells to regenerate in culture. PMID- 16661740 TI - Phytochrome Action during Prechilling Induced Germination of Betula papyrifera Marsh. AB - Seeds of paper birch (Betula papyrifera Marsh.) were induced to germinate by prechilling at 3 C or by red light. The light requirement was mediated by phytochrome and the action of phytochrome during prechilling was investigated. Red irradiation (R) prior to prechilling markedly enhanced the effectiveness of the prechilling treatment in inducing subsequent germination at 18 C. Reversal of this enhancement by far-red irradiation (FR) was more effective when FR was supplied after a 1-week prechill treatment than after a 2-week treatment. The R enhancement effect exhibited a sharp drop as prechilling temperature was increased from 5 to 7 C. This decline is consistent with a membrane phase transition at about 7 C where Pfr action is diminished by a loss in sensitivity of its receptor sites. Although phytochrome action was observed during prechilling treatments, the seeds failed to germinate at prechilling temperatures. Therefore, it was concluded that while potentiation of germination by Pfr occurred during prechilling, some other reaction(s) leading to radicle protrusion requires higher temperatures. In one seed source loss of germination potential was observed with protracted storage at 3 C. This was prevented by R supplied during the prechilling treatment. Taken collectively the data suggest that action of phytochrome during prechilling is accentuated in these seeds by two factors: (a) an increase in the sensitivity (or number) of Pfr receptor sites; and (b) preservation of Pfr by deferment of thermal reversion. PMID- 16661741 TI - Effect of Temperature and BASF 13 338 on the Lipid Composition and Respiration of Wheat Roots. AB - The fatty acid composition of wheat seedling roots changed in response to temperature. As temperature declined, the level of linolenic acid increased and the level of linoleic acid decreased. The distribution of phospholipid classes was not influenced by temperature. Phosphatidyl choline and phosphatidyl ethanolamine were the predominant phospholipids isolated and comprised 85% of the total lipid phosphorus. Smaller quantities of phosphatidyl glycerol, phosphatidyl inositol, phosphatidic acid, and phosphatidyl serine were isolated. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine were the same and temperature affected the fatty acid composition of both phospholipids in the same manner.Growth in the presence of the substituted pyridazinone, BASF 13 338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), reduced the level of linolenic acid and increased the level of linoleic acid in the phosphatidyl choline, phosphatidyl ethanolamine, and total polar lipid fractions. BASF 13 338 did not affect the levels of palmitate, stearate, and oleate or the distribution of phospholipid classes.Respiration rates of wheat root tips were measured over a range of temperatures. The respiration rate declined as the temperature decreased. Neither the temperature at which the tissue was grown nor BASF 13 338 treatment influenced the ability of root tips to respire at any temperature from 4 to 30 C. The results indicated that the relative proportion of linolenic acid to linoleic acid did not influence the plants ability to grow and respire over the range of temperatures tested. PMID- 16661742 TI - Accumulation of Cyanophycin Granules as a Result of Phosphate Limitation in Agmenellum quadruplicatum. AB - Phosphate-limited growth of the blue-green alga Agmenellum quadruplicatum resulted in the accumulation of cyanophycin granule polypeptide (CGP), which is a 1:1 co-polymer of aspartic acid and arginine. The progressive accumulation of CGP began after depletion of phosphate from the medium. CGP increased in concentration much faster than the increase in cell number. Electron microscopy indicated that both the number of cyanophycin granules per cell section and the diameter of each granule increased as phosphate starvation progressed. A marked decrease in the electron density of the inter-thylakoidal areas took place concurrently with the accumulation of CGP. At the same time a progessive decrease in the pigment concentration of cells and in the rate of nitrate uptake was observed. Thirty-two hours after phosphate depletion from the medium up to 28% of total cellular nitrogen was found in CGP. PMID- 16661743 TI - Osmotic Adjustment of Cultured Tobacco Cells (Nicotiana tabacum var. Samsum) Grown on Sodium Chloride. AB - Tobacco cell cultures (var. Samsum) were grown on increasing levels of NaCl to select variants for increased salt tolerance. The osmotic adjustment of NaCl adapted and nonadapted cell lines was studied. Both cell lines were grown on modified Linsmaier and Skoog medium with or without NaCl. Few differences were found in the response of adapted and nonadapted lines to NaCl.The concentrations of sugars, Na(+), Cl(-), and NO(3) (-) were identical in the cells and medium. Potassium and amino acids were accumulated by the cells. All of the above solutes accounted for 80 to 90% of the osmotic potential for both cell lines when grown on basal medium with or without NaCl. The osmotic potential of growing cells was always 1 to 3 bars more negative than that of the medium. During the first 10 days culture, the cells hydrolyzed the 117 millimolar sucrose present in the fresh media, and the media became more negative by 3 bars. Growing cells absorbed and metabolized the sugars, NH(4) (+), and NO(3) (-) during the next 25 days, and the osmotic potential of the media and cells became less negative. The addition of 130 millimolar NaCl made the media and cells osmotically more negative by 6 bars throughout the growth cycle, as compared with cells growing on basal medium.The efflux of cellular solutes during distilled H(2)O washes was resolved into two components. The fast component (0.6 to 1.7 minutes half-time) included solutes of the free space and cytoplasm, whereas the slow component (1.6 to 4.9 hours half-time) represented the vacuolar solutes. Sodium and Cl(-) were present in the vacuole. No differences were observed in the solute efflux between the adapted and nonadapted cell lines. PMID- 16661744 TI - The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves. AB - Application of levulinic acid (LA), a competitive inhibitor of delta aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [(14)C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of (14)CO(2) evolution from labeled precursors, including amino and organic acids. Inhibition of (14)CO(2) evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-(14)C]ALA or [1-(14)C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of (32)Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed. PMID- 16661745 TI - Phytochrome control of two low-irradiance responses in etiolated oat seedlings. AB - Light-induced coleoptile stimulation and mesocotyl suppression in etiolated Avena sativa (cv. Lodi) has been quantitated. Etiolated seedlings showed the greatest response to light when they were illuminated 48 to 56 hours after imbibition. Two low-irradiance photoresponses for each tissue have been described. Red light was 10 times more effective than green and 1,000 times more effective than far red light in evoking these responses. The first response, which resulted in a 45% mesocotyl suppression and 30% coleoptile stimulation, had a threshold at 10(-14) einsteins per square centimeter and was saturated at 3.0 x 10(-12) einsteins per square centimeter of red light. This very low-irradiance response could be induced by red, green, or far red light and was not photoreversible. Reciprocity failed if the duration of the red illumination exceeded 10 minutes. The low irradiance response which resulted in 80% mesocotyl suppression and 60% coleoptile stimulation, had a threshold at 10(-10) einsteins per square centimeter and was saturated at 3.0 x 10(-8) einsteins per square centimeter of red light. A complete low-irradiance response could be induced by either red or green light but not by far red light. This response could be reversed by a far red dose 30 times greater than that of the initial red dose for both coleoptiles and mesocotyls. Reciprocity failed if the duration of the red illumination exceeded 170 minutes. Both of these responses can be explained by the action of phytochrome. PMID- 16661746 TI - Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants. AB - Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction. PMID- 16661747 TI - Differential Light Responses of Photosynthesis by Triazine-resistant and Triazine susceptible Senecio vulgaris Biotypes. AB - Studies were conducted to determine a physiological basis for competitive differences between Senecio vulgaris L. biotypes which are either resistant or susceptible to triazine herbicides. Net carbon fixation of intact leaves of mature plants was higher at all light intensities in the susceptible biotype than in the resistant biotype. Quantum yields measured under identical conditions for each biotype were 20% lower in the resistant than in the susceptible biotype. Oxygen evolution in continuous light measured in stroma-free chloroplasts was also higher at all light intensities in the susceptible biotype than in the resistant biotype. Oxygen evolution in response to flashing light was measured in stroma-free chloroplasts of both biotypes. The steady-state yield per flash of resistant chloroplasts was less than 20% that of susceptible chloroplasts. Susceptible chloroplasts displayed oscillations in oxygen yield per flash typically observed in normal chloroplasts, whereas the pattern of oscillations in resistant chloroplasts was noticeably damped. It is suggested that modification of the herbicide binding site which confers s-triazine resistance may also affect the oxidizing side of photosystem II, making photochemical electron transport much less efficient. This alteration has resulted in a lowered capacity for net carbon fixation and lower quantum yields in whole plants of the resistant type. PMID- 16661748 TI - Osmoregulation in the Avena coleoptile in relation to auxin and growth. AB - A study has been made of the effects of auxin and growth on the ability of Avena coleoptile sections to osmoregulate, i.e. to take up solutes so as to maintain their osmotic concentration, turgor pressure, and growth rate. The high auxin induced growth rate of Avena coleoptiles is maintained when cells are provided sucrose, glucose, NaCl, or KCl as a source of absorbable solutes, but not when 2 deoxy-d-glucose or 3-O-methyl-d-glucose is used. In the absence of auxin, cells take up solutes from a 2% sucrose solution and the osmotic concentration increases. The rate of solute uptake is even greater in the presence of auxin or fusicoccin, but the osmotic concentration rises only slightly because of the water taken up during growth. Solute uptake is not stimulated by auxin when growth is inhibited osmotically or by calcium ions. Solute uptake appears to have two components: a basal rate, independent of auxin or growth, and an additional uptake which is proportional to growth. Osmoregulation of sections may be limited by the rate of entry of solutes into the tissue rather than by their rate of uptake into the cells. PMID- 16661749 TI - Specificity of the carboxypeptidase inhibitor from potatoes. AB - Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K(i) about 10 micromolar) for a carboxypeptidase G(1) from an acinetobacterium was found in this study. PMID- 16661750 TI - Genetic control of chloroplast pigment development in soybeans as a function of leaf and plant maturity. AB - Changes in the major chloroplast pigments of pigment-deficient genotypes of soybeans were studied as a function of leaf age and plant age. The trends of pigment development are plotted as a function of relative leaf age at two periods of plant development. Comparisons are made between aging leaves from different nodes and leaves of different ages from the same node. In addition, trends of pigment development are expressed as a ratio of pigment-deficient/normal genotypes for five different genotypes and seven sampling periods. Those genotypes that exhibit a lag in production of pigments during leaf development show a similar lag in overall plant pigment development. PMID- 16661751 TI - Photosynthesis in Fescue: I. HIGH RATE OF ELECTRON TRANSPORT AND PHOSPHORYLATION IN CHLOROPLASTS OF HEXAPLOID PLANTS. AB - Chloroplasts isolated from tall fescue, Festuca arundinacea Schreb., showed high rates of electron transport, comparable to rates observed for spinach chloroplasts.Chloroplasts were well coupled and rates of electron transport from water to methyl viologen (photosystem II and I) were increased two to five times when ADP and inorganic phosphate or methylamine (uncoupler) were added to the reaction mixture. Ratios of P:2e for photosystem II plus I were found to be near 1.2. Electron transport rates from water to p-phenylenediamine or 2,6 dichlorobenzoquinone (photosystem II) were over 300 micromoles O(2) per hour per milligram chlorophyll, while P:2e ratios were found to be over 0.5. The highest rates of electron transport were found in electron flow from diaminodurene to methyl viologen (photosystem I) and P:2e ratios remained near 0.5.Light intensity saturation curves for photosystem II and I, as well as the photosystems independently, resembled curves for spinach, with saturation of electron transport in photosystem I and photosystem II separately occurring at 35% of the available light intensity (6000 microeinsteins per square meter per second). Photosystem II and I in sequence were saturated at about half this light intensity. PMID- 16661752 TI - Rapid Response of Suspension-cultured Parsley Cells to the Elicitor from Phytophthora megasperma var. sojae: INDUCTION OF THE ENZYMES OF GENERAL PHENYLPROPANOID METABOLISM. AB - Large and rapid increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, occurred in suspension-cultured parsley cells (Petroselinum hortense) treated with an elicitor preparation from Phytophthora megasperma var. sojae. Highest enzyme activities were obtained with an elicitor concentration similar to that required for maximal phenylalanine ammonialyase induction in cell suspension cultures of soybean, a natural host of the fungal pathogen.The changes in phenylalanine ammonia-lyase activity in parsley cells were caused by corresponding changes in the mRNA activity for this enzyme. Phenylalanine ammonia lyase mRNA activity increased much faster and transiently reached a much higher level in elicitor-treated than in irradiated cell cultures. In contrast to irradiation, treatment of the cells with the elicitor did not induce the enzymes of the flavonoid glycoside pathway, as demonstrated for acetyl-CoA carboxylase and chalcone synthase. Induction of these enzymes by light was abolished by simultaneous application of the elicitor. PMID- 16661753 TI - Abscisic Acid Translocation and Metabolism in Soybeans following Depodding and Petiole Girdling Treatments. AB - It was found earlier that depodding and girdling treatments which obstruct translocation, result in increased leaf AbA levels and partial stomatal closure. In the present work (+/-) [2-(14)C]abscisic acid (AbA) was introduced into leaves and the mass, and radioactivity of AbA and AbA-metabolites were analyzed following translocation obstruction to determine whether the increased AbA was due to higher rates of synthesis, or lower rates of catabolism or export. The (+/ ) [2-(14)C]AbA was introduced into soybean (Merr.) leaves by injection into the petiole region. AbA and AbA-metabolites (phaseic acid [PA], dihydrophaseic acid [DPA], AbA-conjugate, and an unknown metabolite) were separated with preparative high performance liquid chromatography. Methyl esters of AbA (free and that released after hydrolysis of AbA-conjugate), PA and DPA were determined with gas chromatography using electron capture detection.The level of AbA in leaf blades increased after girdling or depodding as was found previously. Accompanying this was an increase in PA in girdled leaves; but no discernible trends in the levels of DPA and AbA-conjugate were evident. The (+/-) [2-(14)C]AbA specific radioactivities declined similarly for all treatments, indicating that these treatments did not increase the rate of AbA synthesis.Export of injected (+/-)[2 (14)C]AbA from leaves was substantial. After girdling or depodding, this export was obstructed, as evidenced by the lack of decline in leaf blade radioactivity or lack of increase in pod radioactivity following these treatments. The higher AbA levels, which were observed in leaf blades of girdled and depodded plants, could be attributed largely to the translocation obstruction. PMID- 16661754 TI - Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation. AB - Isolated corn mitochondria (Zea mays cv. B73 x Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and Delta(1)-pyrroline-5 carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.Both proline and Delta(1)-pyrroline-5-carboxylic acid supported oxygen uptake in isolated mitochondria. Proline dependent oxygen uptake was relatively independent of pH with a maximum rate at pH 7.2. In contrast, Delta(1)-pyrroline 5-carboxylic acid-dependent oxygen uptake was sensitive to pH with an optimum at pH 6.1. The oxidation of proline and Delta(1)-pyrroline-5-carboxylic acid was inhibited by 10 micromolar rotenone. This indicates that electrons from these substrates enter the respiratory chain prior to at least one of the rotenone sensitive iron-sulfur proteins. Both substrates yielded ADP:O ratios of around 1.9 as compared to malate plus pyruvate (2.1), succinate (1.3), and exogenous NADH (1.2). PMID- 16661755 TI - The Peptide pools of germinating barley grains: relation to hydrolysis and transport of storage proteins. AB - A quantitative procedure for purifying small peptides from plant tissues, involving both ion-exchange and gel-exclusion chromatography, is described. Peptides were quantified and characterized by using the fluorescence reagents dansyl chloride and fluorescamine. Large pools of small peptides and amino acids have been identified in both the endosperm and embryo of germinating barley grains. The peptide pool of the endosperm increases during the first 3 days of germination, subsequently decreasing, an observation compatible with a role for peptides as intermediates in the breakdown of the storage proteins and their transfer to the embryo. The amino acid composition of these peptides indicates that all the major classes of storage protein contribute to the pool. The concentration of peptides produced in the endosperm during germination is sufficient for the efficient operation of the peptide transport system of the scutellar membrane characterized previously (Higgins and Payne, Planta 136: 71 76, 1977; Planta 138: 211-215 and 217-221, 1978). Data presented here indicate that peptides play at least as important a role as amino acids in the transfer of stored nitrogen from the endosperm to the embryo during germination. PMID- 16661756 TI - Sugar Uptake in Lily Pollen : A PROTON SYMPORT. AB - The data presented here are consistent with a proton-sugar co-transport in germinated pollen of Lilium longiflorum Thunb. Optimal uptake occurs at pH 5.0. A K(m) of 1.7 to 1.8 millimolar is obtained from the initial rate of pH change induced by sucrose uptake as well as from uptake of [U-(14)C]-sucrose. The energy of activation is - 11 kilocalories mole(-1). The effect of several inhibitors and sugar competitors on [U-(14)C]sucrose and d-[U-(14)C] glucose uptake is given. The possibility of hydrolysis of sucrose prior to its transport into the pollen tube has been considered and reasons for choosing a sucrose-type uptake are presented. The possible in vivo significance of this co-transport process during pollen germination is discussed. Germinated pollen has features to recommend it as an experimental system of choice for studies of sugar uptake. PMID- 16661757 TI - Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1. AB - High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = -220 millivolts). In plants maintaining a low pd (-110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H(+) cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H(+) -extrusion pump. PMID- 16661758 TI - Reduction of Hydraulic Conductivity during Inhibition of Exudation from Excised Maize and Barley Roots. AB - The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is shown to reduce the hydraulic conductivity of barley, maize, mung bean, and onion roots. In barley and maize, the reduction in exudation from excised roots is partly due to the reduction in the permeability of the root to water (I(p)), but it can be inferred that the rate of salt release to the xylem, is also inhibited. The action of CCCP on L(p) is suggested to be mainly in blocking the symplasmic pathway at the plasmodesmata. PMID- 16661759 TI - An Analysis of the Relationship between Respiration and the Transmembrane Potential in Corn Roots. AB - The effects of cyanide, anoxia, and temperatures varying from 2 to 42 C on the cell membrane electropotential difference (PD) of washed and freshly excised corn roots have been determined. Respiration rates of freshly excised root segments in response to cyanide and to varying temperatures were also measured. The cell membrane PD of roots which had been washed for 12 to 15 hours was almost insensitive to cyanide and anoxia but sensitive to low temperature. In contrast, the cell membrane PD of freshly excised roots was reversibly depolarized by all three treatments, cyanide depolarized from -117 to -86 millivolts and the sequential imposition of anoxia further lowered the PD to -69 millivolts. Anoxia applied first depolarized maximally and the PD was not further lowered by sequential cyanide treatment. Arrhenius plot analysis of the temperature response of respiration showed an apparent transition at 13 C with an activation energy of 20.0 kilocalories per mole below and 8.8 kilocalories per mole above the transition temperatures. The energy of activation for repolarization of PD is much higher; 53.4 kilocalories per mole below 7 to 8 C and 25.4 kilocalories per mole above this apparent transition. The energy requirement for polarization of the cell membrane PD was calculated based on the temperature responses of the cell membrane PD and respiration. It was estimated that 3.5% of the energy output from respiration at 22 C is required for cell polarization. It is unlikely that ion transport is limited by energy availability below the 8 C transition in this chill sensitive species. PMID- 16661760 TI - Temperature dependence of the concentration kinetics of absorption of phosphate and potassium in corn roots. AB - The effect of temperature on respiration and kinetics of H(2)PO(4) (-) and K(+) uptake in corn roots was determined in the range of 2 to 42 C. The response of uptake to temperature, determined from Q(10) and activation energy (Ea) data, for the anion and the cation differ significantly, especially in the range of uptake mechanism (Mech.) I. At 2.5 micromolar the Ea for K(+) uptake below the 13 C transition is 29.3 kilocalories per mole. As the K(+) concentration is increased, Ea declines and at 0.25 millimolar is 21.6 kilocalories per mole. Accompanying this change in Ea is a shifting of the apparent transition temperature from 13 to 17 C. Above the temperature transition the Ea's for K(+) uptake in the Mech. I range are quite low (3.0) and this value is unchanged by increases of K(+) concentration to 0.25 millimolar. In the range of Mech. II above 1 millimolar K(+) the temperature transitions are not seen and plots become linear. The Ea's show an increasing trend from 4.7 at 1 millimolar to 6.1 at 50 millimolar. The uptake of H(2)PO(4) (-) is much more temperature sensitive having a constant Ea at concentrations in the Mech. I range below the 13 C temperature transition. The Arrhenius plots reveal a second transition at 22 C and the Ea for this segment is 21.0. Above the second transition the Ea remains high (10.0) and is constant in the range of Mech. I. In the range of Mech. II there is a concentration dependent decline in Ea for H(2)PO(4) (-) uptake (22.7 at 1.0 millimolar to 1.0 at 50 millimolar). There is no definable low temperature transition at these concentrations. Ion uptake is found to be much more sensitive to low temperature than respiration in this chill-sensitive species. The data suggest that the low temperature reduction of ion transport is more closely related to restriction of function of active transport systems than to either respiration or membrane permeability. PMID- 16661761 TI - Relationship of Camphor Biosynthesis to Leaf Development in Sage (Salvia officinalis). AB - The camphor content of sage (Salvia officinalis L.) leaves increases as the leaves expand, and the increase is roughly proportional to the number of filled peltate oil glands which appear on the leaf surface during the expansion process. (14)CO(2) is more rapidly incorporated into camphor and its direct progenitors in expanding leaves than in mature leaves, and direct in vitro measurement of the key enzymes involved in the conversion of geranyl pyrophosphate to camphor indicates that these enzymes, including the probable rate-limiting cyclization step, are at the highest levels during the period of maximum leaf expansion. These results clearly demonstrate that immature sage leaves synthesize and accumulate camphor most rapidly. PMID- 16661762 TI - Electrical Properties of the Plasmalemma and Tonoplast in Valonia ventricosa. AB - Studies were made on the electric potentials of the plasmalemma (E(co)) and tonoplast (E(vc)) in small cells (1-3 mm diameter) of Valonia ventricosa. To measure E(co), microelectrodes with long tapers were inserted into the vacuole with the path of electrode entry off-center. The microelectrode then was pushed across the vacuole and into the cytoplasm on the opposite side of the cell. A reference electrode was placed in the artificial seawater bathing the cell. A similar method was used to measure E(vc) except that the reference electrode was placed in the vacuole.Both E(co) and E(vc) were influenced by light. In the light, E(co) was -70 millivolts and it changed to -60 millivolts in the dark (cytoplasm-negative to outside). For E(vc), the potentials were +86 millivolts in the light and +69 millivolts in the dark (vacuole-positive to cytoplasm). The vacuole potential (E(vo)) was demonstrated to be the algebraic sum of E(co) and E(vc). For example, in the light, the sum of the means (+/-se) for E(co) (= -70 +/- 1) and E(vc) (= +86 +/- 5) is +16 millivolts, which is comparable to the measured E(vo) of +17 +/- 2 millivolts. In the dark, the sum of E(co) (= -60 +/- 3) and E(vc) (+69 +/- 6) is +9 millivolts and the measured value of E(vo) is +9 +/- 4 millivolts.The external K(+) concentration had a controlling effect on both E(co) and the direct current resistance of the plasmalemma, which suggests that E(co) is largely a K(+) diffusion potential. The tonoplast electrical properties were affected only slightly by external K(+).The data presented are indicative of a K(+) electrogenic influx pump in the tonoplast. It is also considered possible that H(+) might be electrogenically pumped from the cytoplasm both into the vacuole and to the cell exterior. PMID- 16661763 TI - Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots. AB - The block in the electrogenic H(+) efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H(+) efflux is inhibition of K(+) influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H(+) efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N' dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.The results are discussed in terms of the presumed mode of action of fusicoccin on the plasmalemma ATPase. Inhibition of protein synthesis appears to inactivate the proton channel of the ATPase, possibly as the indirect result of disrupted metabolism. Fusicoccin reactivates or bypasses the blocked channel. PMID- 16661764 TI - In vitro auxin binding to cellular membranes of cucumber fruits. AB - Specific binding of 1-naphthaleneacetic acid (NAA) to crude membrane preparations from cucumber (Cucumis sativus L.) was demonstrated. This in vitro binding had a pH optimum of 3.75 and an equilibrium dissociation constant of 10 to 20 micromolar with 1250 picomoles binding sites per gram fresh weight. The NAA binding sites were pronase sensitive. The supernatant from the fruit partially inhibited the in vitro NAA binding to fruit membranes. NAA, 2-naphthoxyacetic acid, 3-indoleacetic acid, 2-4-dichlorophenoxyacetic acid, and 2,3,5 triiodobenzoic acid, which are reported to be very good inducers of parthenocarpy in cucumber, showed a high degree of specific binding to cucumber fruit membranes. In comparison, 2-naphthaleneacetic acid and indolepropionic acid, which are reported to be very weak auxins in corn coleoptile, pea stem, and strawberry fruit growth bioassays, did not bind efficiently to cucumber fruit membranes. In vitro binding studies with fruit membranes suggest that auxin stimulated fruit growth may be mediated by membrane-associated, auxin-binding protein(s). PMID- 16661765 TI - Regulation of Proteinase Inhibitor Synthesis in Tomato Leaves : IN VITRO SYNTHESIS OF INHIBITORS I AND II WITH mRNA FROM EXCISED LEAVES INDUCED WITH PIIF (PROTEINASE INHIBITOR INDUCING FACTOR). AB - Messenger RNA was isolated from young excised tomato leaves, induced to accumulate proteinase Inhibitors I and II with the proteinase inhibitor inducing factor (PIIF), and translated in vitro in a rabbit reticulocyte lysate system. Translatable messenger RNAs specific for Inhibitors I and II were present in PIIF induced leaves but were not present without PIIF induction. The nascent in vitro synthesized inhibitors migrated with an apparent molecular weight 2,000 to 3,000 daltons larger than that of the two inhibitors isolated from leaves. The molecular weights of the preinhibitors were identical whether translated from mRNA from PIIF-induced leaves or translated from mRNA isolated from wounded leaves. Incubation of excised PIIF-induced plants in CO(2)-free air doubled the rate of in vivo synthesis of Inhibitor I over that in normal air (Ryan CA 1977 Biochem Biophys Res Commun 77: 1004-1008) but did not affect the rate of in vivo Inhibitor II accumulation. The rate of incorporation of (35)SO(4) (2-) into soluble proteins was 70% less when leaves were incubated in CO(2)-free air rather than normal air. Messenger RNA isolated from PIIF-induced plants incubated in the presence or absence of CO(2) was translated in vitro. The amount of in vitro translatable mRNA present for each inhibitor (per microgram total mRNA) was the same in leaves incubated in either atmosphere. Therefore, the increased rate of synthesis and accumulation of Inhibitor I in a CO(2)-free atmosphere does not appear to result from an increased level of mRNA but appears to be controlled at a posttranscriptional level. PMID- 16661766 TI - Measurement of the sieve tube membrane potential. AB - A procedure is described for the measurement of the sieve tube membrane potential in the phloem of bark strips from Salix exigua Nutt. Measurements were made by inserting a measuring microelectrode into sap exuding from severed stylets of the willow aphid, Tuberolachnus salignus. Data taken from 20 bark strips gave an average potential of -155 +/- 9 millivolts. Evidence is presented for an electrogenic component of the sieve tube membrane potential. The occurrence of a saturable sucrose-induced membrane depolarization is consistent with the concept of sugar accumulation by a sucrose/H(+) co-transport mechanism. PMID- 16661767 TI - Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA. AB - Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form "pearl chains" which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells. PMID- 16661768 TI - Inhibition of photosynthetic carbon metabolism in isolated chloroplasts by iodoacetol phosphate. AB - Carbon dioxide-dependent and 3-phosphoglycerate (PGA)-dependent O(2) evolution by isolated chloroplasts of wheat is inhibited by micromolar levels of iodoacetol phosphate (IAP). Loss of the activity is time-dependent and a higher concentration of PGA increases the half-time for inhibition (e.g. at 40 micromolar IAP the half-time is about 0.5 minutes at 1 millimolar PGA compared to 1.5 minutes at 10 millimolar PGA). A marked inhibition of NADP glyceraldehyde-3-P dehydrogenase was observed when chloroplasts were pretreated with micromolar levels of IAP, osmotically shocked, and several stromal enzymes assayed.Extraction of enzymes from wheat protoplasts and treatment with IAP showed that nanomolar levels of the compound completely inhibited NAD and NADP glyceraldehyde-3-P dehydrogenase and the half-time for inactivation at 5 nanomolar IAP was about 1 minute. The inhibitory effect of IAP was not reversed by passing the enzyme extract through a column of Sephadex G-25. The concentration of IAP required for inhibition of the chloroplastic triose phosphate isomerase is about three orders of magnitude higher than that with glyceraldehyde-3-P dehydrogenase. Micromolar levels of IAP had no effect on ribulose-1,5-bisphosphate carboxylase. Inhibition of chloroplast photosynthesis and of glyceraldehyde-3-P dehydrogenase in protoplast extracts with IAP follows pseudo first-order kinetics.Pretreatment of chloroplasts with IAP did not inactivate the phosphate translocator of the chloroplast envelope. Iodoacetol phosphate may enter the chloroplasts through the phosphate translocator since a high concentration of IAP (0.5 millimolar) competitively inhibits uptake of (32)Pi. Iodoacetol phosphate had no effect on ferricyanide-dependent O(2) evolution with isolated chloroplasts. Also, IAP had no effect on light-dependent fixation of CO(2) through the carboxylation phase of the C(4) pathway with protoplast extracts of crabgrass mesophyll cells. The site of IAP inhibition of photosynthesis with wheat chloroplasts is suggested to be through the inactivation of glyceraldehyde-3-P dehydrogenase. PMID- 16661769 TI - Methylammonium Transport in Phaseolus vulgaris Leaf Slices. AB - Methylammonium (as a nonmetabolized analog of ammonium) transport was studied in leaf slices of Phaseolus vulgaris L. var. ;Hawkesbury Wonder.' The relationship of influx to external pH (6.0-10.5) shows that the influx at low external pH is a larger fraction of that at high external pH than would be expected from the pK(alpha) of methylammonium and the assumption that only CH(3)NH(2) is entering the cells. The relationship between methylammonium influx and external methylammonium concentration shows some evidence of saturation; this is a function of the transport system rather than of the (limited) methylammonium metabolism in the cells. The "equilibrium" concentration ratio for methylammonium between leaf slices and bathing medium is far higher than can be explained by the transport of CH(3)NH(2) alone and the pH of the compartments involved. These three lines of evidence strongly suggest that there is an influx of CH(3)NH(3) (+), possibly by a uniporter driven by the electrical potential of the cytoplasm with respect to the medium, as has been shown for other plant cells. Competitive inhibition of methylammonium influx by ammonium suggests that there is also an ammonium transport system. The significance of this for the recycling of N within the plant and for exchange of gaseous NH(3) between leaves and the atmosphere is discussed. PMID- 16661770 TI - Electrochemical aging responses in pisum: cellular adaptations or recovery from injury? AB - Following excision, etiolated epicotyl segments of Pisum sativum L. cv. Alaska exhibit a marked hyperpolarization of membrane potential which is followed by a linear accumulation of K(+) when segments are incubated in Higinbotham nutrient solution. Segments aged for several hours and then reexcised display only a slight depolarization of membrane potential and no delay in ion accumulation; thus, recovery from injury appears an unlikely explanation for these responses. Substances originating in either the plumule or the cotyledons do not seem to be directly involved in these "aging" responses. However, locally produced substances, such as ethylene, or substances originating in the roots have not been eliminated as causative factors. Cold temperatures and cycloheximide prolong the lag in K(+) accumulation indicating a metabolic explanation for the induced K(+) accumulation. However, similar specific activities of plasma membrane-bound ATPase were found in isolates from fresh and aged epicotyl segments. Reactivation of an ion transport mechanism, perhaps responsible for the osmotic control of growth in immature cells, is suggested as a possible explanation for the pattern of ion accumulation characteristic of excised pea epicotyl tissue. PMID- 16661771 TI - Electrogenic sucrose transport in developing soybean cotyledons. AB - Addition of sucrose to a solution bathing an excised developing soybean cotyledon causes a transient depolarization of the membrane potential, as measured using standard electrophysiological techniques. The magnitude of the depolarization is dependent on the concentration of both sucrose and protons in a manner which suggests carrier mediation; this process has an apparent K(m) for sucrose of about 10 millimolar. Agents interfering with the generation or maintenance of a proton electrochemical gradient eliminate these depolarizations. Electrogenic sugar transport is sensitive to sulfhydryl-modifying reagents; their effect appears to be through a direct interaction with the carrier protein and/or with the process establishing the proton electrochemical gradient across the plasma membrane. p-Chloromercuribenzene sulfonate appears to be a selective inhibitor of the carrier-mediated process itself. PMID- 16661772 TI - Metabolism of Glycolate in Isolated Spinach Leaf Peroxisomes : KINETICS OF GLYOXYLATE, OXALATE, CARBON DIOXIDE, AND GLYCINE FORMATION. AB - The flow of glyoxylate derived from glycolate into various metabolic routes in the peroxisomes during photorespiration was assessed. Isolated spinach leaf peroxisomes were fed [(14)C] glycolate in the absence or presence of exogenous glutamate, and the formation of radioactive glyoxylate, CO(2), glycine, oxalate, and formate was monitored at time intervals. In the absence of glutamate, 80% of the glycolate was consumed within 2 hours and concomitantly glyoxylate accumulated; CO(2), oxalate, and formate each accounted for less than 5% of the consumed glycolate. In the presence of equal concentration of glutamate, glycolate was metabolized at a similar rate, and glycine together with some glyoxylate accumulated; CO(2), oxalate, and formate each accounted for an even lesser percentage of the consumed glycolate. CO(2) and oxalate were not produced in significant amounts even in the absence of glutamate, unless glycolate had been consumed completely and glyoxylate had accumulated for a prolonged period. These in vitro findings are discussed in relation to the extent of CO(2) and oxalate generated in leaf peroxisomes during photorespiration. PMID- 16661773 TI - Effects of High Atmospheric CO(2) and Sink Size on Rates of Photosynthesis of a Soybean Cultivar. AB - The effect of sink strength on photosynthetic rates under conditions of long-term exposure to high CO(2) has been investigated in soybean. Soybean plants (Merr. cv. Fiskeby V) were grown in growth chambers containing 350 microliters CO(2) per liter air until pod set. At that time, plants were trimmed to three trifoliolate leaves and either 21 pods (high sink treatment) or 6 pods (low sink treatment). Trimmed plants were either left in 350 microliters CO(2) per liter of air or placed in 1000 microliters CO(2) per liter of air (high CO(2) treatment) until pod maturity. Whole plant net photosynthetic rates of all plants were measured twice weekly, both at 350 microliters CO(2) per liter of air and 1000 microliters CO(2) per liter of air. Plants were also harvested at this time for dry weight measurements. Photosynthetic rates of high sink plants at both measurement CO(2) concentrations were consistently higher than those of low sink plants, and those of plants given the 350 microliter CO(2) per liter of air treatment were higher at both measurement CO(2) concentrations than those of plants given the 1000 microliters CO(2) per liter of air treatment. When plants were measured under treatment CO(2) levels, however, rates were higher in 1,000 microliter plants than 350 microliter CO(2) plants. Dry weights of all plant parts were higher in the 1,000 microliters CO(2) per liter air treatment than in the 350 microliters CO(2) per liter air treatment, and were higher in the low sink than in the high sink treatments. PMID- 16661774 TI - Mechanism of specific inhibition of phototropism by phenylacetic Acid in corn seedling. AB - Using geotropism as a control for phototropism, compounds similar to phenylacetic acid that photoreact with flavins and/or have auxin-like activity were examined for their ability to specifically inhibit phototropism in corn seedlings using geotropism as a control. Results using indole-3-acetic acid, napthalene-1-acetic acid, naphthalene-2-acetic acid, phenylacetic acid, and beta-phenylpyruvic acid suggest that such compounds will specifically inhibit phototropism primarily because of their photoreactivity with flavins and not their auxin activity. For example, strong auxins, indole-3-acetic acid and naphthalene-1-acetic acid, affected both tropic responses at all concentrations tested whereas weak auxins, phenylacetic acid and naphthalene-2-acetic acid, exhibited specific inhibition. In addition, the in vivo concentration of phenylacetic acid required to induce specificity was well below that required to stimulate coleoptile growth. Estimates of the percentage of photoreceptor pigment inactivated by phenylacetic acid (>10%) suggest that phenylacetic acid could be used to photoaffinity label the flavoprotein involved in corn seedling phototropism. PMID- 16661775 TI - Morphology and ultrastructure of maternal seed tissues of soybean in relation to the import of photosynthate. AB - Cytological observations were made on developing seeds of soybean (Glycine max (L.) Merr. "Amsoy 71") using scanning and transmission electron microscopy and light microscopy. Attention was focused on the maternal tissues of the seed coat and embryo sac. An hypothesis of photosynthate import, unloading, and movement to the embryo is presented based on the results of these studies. PMID- 16661776 TI - Characterization of a Gibberellin-Insensitive Dwarf Wheat, D6899 : EVIDENCE FOR A REGULATORY STEP COMMON TO MANY DIVERSE RESPONSES TO GIBBERELLINS. AB - The effects of gibberellin in two wheat varieties, Nainari 60 and D6899, have been studied. D6899 is a dwarf wheat having a single locus mutation, the Rht 3 gene ("Tom Thumb" gene), which is located on chromosome 4A. When compared with the standard height wheat variety, Nainari 60, D6899 does not have a slowdown in cellular metabolism such as respiration rate, protein content, rate of protein synthesis, and uptake of amino acids. The content of ATP is even higher in D6899. However, all of the gibberellin-mediated physiological processes that we have studied, including leaf elongation, synthesis and release of hydrolytic enzymes, and secretion of phosphate ions and reducing sugars in aleurone layers, are retarded in D6899. D6899 and Nainari 60 have essentially the same uptake and metabolism of gibberellin and their levels of endogenous inhibitors such as abscisic acid do not differ drastically. The dosage-response curve of the gibberellin-mediated alpha-amylase production indicates that a rate-limiting step, which is common to many of the diverse gibberellin responses, is partially blocked in D6899 wheat. PMID- 16661777 TI - Release of a Soluble Phytoalexin Elicitor from Mycelial Walls of Phytophthora megasperma var. sojae by Soybean Tissues. AB - A soluble elicitor of glyceollin accumulation was released from insoluble mycelial walls of Phytophthora megasperma var. sojae after incubation with soybean cotyledon tissue for as little as 2 minutes. Various enzymic and chemical treatments of the released elicitor indicated that the activity resided in a carbohydrate moiety, and gel filtration disclosed the presence of at least two active molecular species. Cell-free extracts from soybean cotyledons or hypocotyls also released soluble elicitors from fungal cell walls that were similar to those released by living cotyledon tissue. These results may suggest that contact of fungal pathogens with host tissues is required to release fungal wall elicitors which then initiate phytoalexin accumulation in the plant. PMID- 16661778 TI - Reductive pentose phosphate cycle and oxidative carbohydrate metabolic activities in pea chloroplast stroma extracts. AB - Oxidative and reductive carbohydrate metabolism was studied in reaction mixtures based on chlorophyll-free stromal extracts from chloroplasts of Pisum sativum. A new assay system for the reductive pentose phosphate cycle was characterized.When provided with ATP, an enzymic ATP-regenerating system and reduced pyridine nucleotide, substantial rates of CO(2) fixation and pyridine nucleotide oxidation were observed following the addition of millimolar concentrations of reductive pentose phosphate cycle intermediates. The reduced pyridine nucleotide requirement could be met either by NADPH, or by NADH plus the added enzymes NAD(+)-glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase. When the assay system was primed with small amounts of reductive pentose phosphate cycle intermediates, lower rates of pyridine nucleotide oxidation were observed, but turnover of the complete cycle was demonstrated. Autocatalytic effects were not evident. The optimum pH and Mg concentrations for cycle turnover were similar to those believed to exist in the stroma of intact chloroplasts in the light.Oxidative carbohydrate metabolism was studied by supplying oxidized pyridine nucleotide and measuring its rate of reduction in the presence of sugar phosphates. Glycolytic activity, estimated as the rate of fructose-6-phosphate entry to the phosphofructokinase reaction was 2.7 micromoles per milligram chlorophyll per hour when fructose-6-phosphate was provided as substrate. Evidence based on glucose-6-phosphate and ribose-5-phosphate-dependent NADP(+) reduction showed that the oxidative pentose phosphate cycle was also active. Apparent oxidative pentose phosphate cycle turnover in the presence of ribose-5 phosphate, estimated as the rate of glucose-6-phosphate entry to the glucose-6 phosphate dehydrogenase reaction, was 1.7 micromoles per milligram chlorophyll per hour.It was concluded that under the defined conditions, reductive pentose phosphate cycle activity could be measured without interference from oxidative carbohydrate metabolism in this experimental system. PMID- 16661779 TI - Partial purification and characterization of a blue light-sensitive cytochrome flavin complex from corn membranes. AB - A membrane fraction which contains a blue light-sensitive flavin-cytochrome complex (Brain et al. 1977 Plant Physiol 59:948) has been partially purified by sucrose and Renografin gradient centrifugations. Assays for marker enzymes show that this membrane fraction is distinct from endoplasmic reticulum, golgi, and mitochondria. This membrane fraction co-sedimented with glucan synthetase II activity, a proposed marker for plasma membrane in higher plants. The purified membrane fraction shows virtually identical light minus dark and dithionite reduced minus oxidized difference spectra with difference bands near 427 and 557 nanometers, suggesting that contamination by other cytochrome-containing membrane fractions is not significant. The photoactivity can be completely solubilized by 0.1% Triton X-100, leaving the bulk of the membrane undissolved. The kinetics for cytochrome photoreduction are not significantly affected by solubilization, indicating that both flavin and cytochrome could be associated with the same protein moiety. PMID- 16661780 TI - Some aspects of the control of root growth and georeaction: the involvement of indoleacetic Acid and abscisic Acid. AB - Apical segments of roots of Zea mays L. cv. Orla and cv. Anjou show a strong georeaction during 7 hours geostimulation. This is abolished by detipping the segments and restored by replacing the tips upon the apical cut surfaces. After exodiffusion of endogenous indoleacetic acid (IAA) the retipped segments showed a significantly lower geocurvature. Application of low concentrations of IAA to the basal cut surface of root segments from which endogenous IAA had not been allowed to exodiffuse increased the geocurvature of retipped Orla segments but decreased geocurvature of Anjou segments. At appropriate concentration basally applied IAA restored the georeaction capacity of root segments from which the endogenous auxin had exodiffused. The implications of the interaction between exogenous and endogenous IAA in the control of root georeaction are discussed with special reference to the normal role of endogenous IAA in the regulation of root georeaction and the variation in endogenous IAA content of roots of different cultivars of maize. The probability that the normal control of root growth and georeaction involves concomitant actions in the elongation zone of IAA moving preferentially in the acropetal direction and basipetally transported growth inhibitors (such as abscisic acid) produced in the cap cells is stressed. PMID- 16661781 TI - Exclusion of selenium from proteins of selenium-tolerant astragalus species. AB - Protein fractions from three selenium-tolerant and three selenium-sensitive Astragalus species, grown in the presence of [(75)Se]selenate, were analyzed for their selenium content. Though tolerant species are known to accumulate considerably more selenium than do sensitive plants, protein fractions from the three selenium accumulators were found to contain significantly less selenium (0.46 to 0.57 picomoles selenium per milligram protein) than did protein fractions from the three nonaccumulators (4.17 to 5.02 picomoles selenium per milligram protein). Under similar conditions, seedlings of Vigna radiata (L.) Wilczek had taken up selenium (6.31 picomoles selenium per milligram protein) at levels comparable to those observed in the proteins of the nonaccumulator Astragali. These results establish that the ability to tolerate and to circumvent the toxic effects of selenium, characteristic of the accumulator species of Astragalus, is associated with a reduced incorporation of this element into protein. PMID- 16661782 TI - Selenium toxicity: aminoacylation and Peptide bond formation with selenomethionine. AB - Selenomethionine and methionine were compared as substrates for in vitro aminoacylation, ribosome binding, and peptide bond formation with preparations from wheat germ. Selenomethionine paralleled methionine in all steps of the translation process except peptide bond formation. Peptide bond formation with the initiating species of tRNA(Met) demonstrated that selenomethionyl-tRNA(Met) was less effective as a substrate than was methionyl-tRNA(f) (Met). Participation of selenomethionine in the initiation process of translation could be expected to reduce the overall rate of protein synthesis and might aid in explaining selenium toxicity in selenium-sensitive plants. PMID- 16661783 TI - In vitro incorporation of selenomethionine into protein by astragalus polysomes. AB - Selenium-accumulator plants synthesize selenium compounds that differ from those produced by nonaccumulators. To determine if there are any subcellular differences between accumulators and nonaccumulators in the use of selenomethionine in vitro, polysomes from Astragalus crotalariae (accumulator) and Astragalus lentiginosis (nonaccumulator) were translated in the presence of selenomethionine. Polysomes from both species efficiently used selenomethionine in vitro during the translation process. Inasmuch as no differences in the incorporation of selenomethionine into protein were observed between polysomes from the two types of Astragalus, it can be inferred that in accumulators there exists a mechanism that either prevents synthesis of selenomethionine or modifies this selenocompound to a derivative that cannot be incorporated into protein. PMID- 16661784 TI - Widespread distribution of some minor chlorophyll-protein complexes in some plants and algae. AB - The use of the non-ionic detergent octyl beta-d-glucoside has allowed the demonstration in spinach of a chlorophyll a+b-protein complex of apparent molecular weight 29 kilodaltons (Camm and Green, 1980, Plant Physiol 66: 428 432). Complexes analogous to this one also can be demonstrated in three grasses, in dicots of the Chenopodiaceae, Fabaceae, and Asteraceae, and in the siphonaceous green alga Acetabularia mediterranea. These complexes are clearly distinguishable from the light harvesting complex on the basis of the chlorophyll a/b ratio, apparent molecular weight, and polypeptide composition. In addition, most plants surveyed contained two, not one, minor chlorophyll a complexes as well as the chlorophyll a complex of Photosystem I. PMID- 16661785 TI - Role of malate synthase in citric Acid synthesis by maturing cotton embryos: a proposal. AB - Cotton embryos from 34 to 54 days after anthesis were analyzed for organic acids, and enzymes associated with organic acid metabolism. During this developmental period, embryos accumulated citrate. Malate synthase activity appeared at 46 days after anthesis and increased rapidly to 54 days. Of other enzymes examined, only citrate synthase activity increased during this period. As isocitrate lyase activity was absent from cotton embryos during maturation, an alternative source of glyoxylate would be required for in vivo malate synthase activity. Of several metabolic sources tested, glycine was converted to glyoxylate via a transamination reaction.Halves of 50-day (mature) cotton embryos incorporated radioactivity from [1-(14)C]acetate, [1-(14)C]glyoxylate, and [1-(14)C]glycine into organic acids. Embryo halves incubated with [(14)C]glyoxylate plus [(3)H]acetate synthesized double-labeled malate and citrate. Radioactive citrate isolated from 50-day cotton embryos incubated with [1-(14)C]acetate was degraded; label was distributed as follows: 55% in C(1), 33% in C(5), and 12% in C(6). Taken together, these data strongly suggest participation of malate synthase in citrate production in vivo.Separation of organelles by sucrose density gradient sedimentation revealed that malate synthase, malate dehydrogenase, and citrate synthase were compartmentalized together only in the peroxisome fraction (1.24 grams per milliliter). Peroxisomes isolated from 50-day embryos, when incubated with glyoxylate and [(3)H]acetyl-CoA, synthesized labeled malate and citrate, but only radioactive citrate accumulated. Incubations with glycine plus alpha ketoglutarate, in place of glyoxylate, also resulted in synthesis of radioactive citrate.A metabolic scheme illustrating the participation of cotton embryo peroxisomes in citrate synthesis is proposed. This scheme suggests a function for plant peroxisomes not previously elucidated. The ontogenetic and metabolic relationship between these organelles and glyoxysomes active in gluconeogenesis during postgerminative growth remains to be examined. PMID- 16661786 TI - Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells. AB - Soybean cells in suspension culture incorporate [(3)H]mannose into dolichyl phosphoryl-mannose and into lipid-linked oligosaccharides as well as into extracellular and cell wall macromolecules. Tunicamycin completely inhibited the formation of lipid-linked oligosaccharides at a concentration of 5 to 10 micrograms per milliliter, but it had no effect on the formation of dolichyl phosphoryl-mannose. Tunicamycin did inhibit the incorporation of [(3)H]mannose into cell wall components and extracellular macromolecules, but even at 20 micrograms per milliliter of antibiotic there was still about 30% incorporation of mannose. The radioactivity in these macromolecules was localized in mannose (70%), rhamnose (20%), galactose (8%), and fucose (2%) in the absence of antibiotic. But when tunicamycin was added, very little radioactive mannose was found in cell wall or extracellular components. The incorporation of [(3)H]leucine into membrane components and [(14)C]proline into cell wall components by these suspension cultures was unaffected by tunicamycin. However, tunicamycin did inhibit the appearance of leucine-labeled extracellular macromolecules, probably because it prevented their secretion. PMID- 16661787 TI - Photophosphorylation Associated with Photosystem II: IV. KINETIC ANALYSES OF PHOTOSYSTEM II CYCLIC PHOTOPHOSPHORYLATION ACTIVITY: EVIDENCE FOR TWO CYCLIC REACTIONS. AB - Photosystem II-dependent cyclic photophosphorylation activity produced by addition of p-phenylenediamines to KCN-Hg-NH(2)OH-inhibited chloroplasts is the product of two separate reactions when a proton/electron donor is the catalyst. The activity observed with an electron donor as catalyst consists of a single reaction. One of the cyclic reactions, evoked by low (900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time.Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present. PMID- 16661806 TI - Evidence of a Low Stromal Mg Concentration in Intact Chloroplasts in the Dark: I. STUDIES WITH THE IONOPHORE A23187. AB - The loss of Mg(2+) upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 millimolar MgCl(2) with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 millimolar MgCl(2) if the chloroplasts were in a K(+) -gluconate medium. Measurements of stromal pH in the dark indicated that, in the K(+) -gluconate medium, the stromal pH is nearly the same as that of the medium, whereas in the sorbitol medium it is much more acidic as reported previously. These observations suggest that the free Mg(2+) concentration in the stroma in the dark is between 1 and 3 millimolar. Other experiments on the inihibition by A23187 of CO(2) fixation in the light and in a system capable of catalyzing CO(2) fixation in the dark, and on the Mg(2+) binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mg(2+) concentration changes occur in the stroma that are important in the light-dark regulation of CO(2) fixation. PMID- 16661807 TI - Stomatal Conductance and Sulfur Uptake of Five Clones of Populus tremuloides Exposed to Sulfur Dioxide. AB - Plants of five clones of Populus tremuloides Michx. were exposed to 0, 0.2 or 0.5 microliter per liter SO(2) for 8 hours in controlled environment chambers. In the absence of the pollutant, two pollution-resistant clones maintained consistently lower daytime diffusive conductance (LDC) than did a highly susceptible clone or two moderately resistant clones. Differences in LDC among the latter three clones were not significant. At 0.2 microliter per liter SO(2), LDC decreased in the susceptible clone after 8 hours fumigation while the LDC of the other clones was not affected. Fumigation with 0.5 microliter per liter SO(2) decreased LDC of all five clones during the fumigation. Rates of recovery following fumigation varied with the clone, but the LDC of all clones had returned to control values by the beginning of the night following fumigation. Night LDC was higher in the susceptible clone than in the other clones. Fumigation for 16 hours (14 hours day + 2 hours night) with 0.4 microliter per liter SO(2) decreased night LDC by half. Sulfur uptake studies generally confirmed the results of the conductance measurements. The results show that stomatal conductance is important in determining relative susceptibility of the clones to pollution stress. PMID- 16661808 TI - Effect of xenon on the excited States of phototropic receptor flavin in corn seedlings. AB - The chemically inert, water-soluble heavy atom gas, xenon, at millimolar concentrations specifically quenches the triplet excited state of flavin in solution without quenching the flavin singlet excited state. The preferential quenching of the flavin triplet over the singlet excited state by Xe has been established by showing that the flavin triplet-sensitized photooxidation of NADH is inhibited while the fluorescence intensity and lifetime of flavin are not affected by Xe.The phototropic response of corn seedlings in an atmosphere of 90% Xe and 10% O(2) was compared with that in the 90% N(2) plus 10% O(2) mixture. No significant inhibition of phototropism and geotropism by Xe was observed, suggesting that a flavin singlet state is more likely involved than the triplet state in the primary photoprocess of phototropism in corn. PMID- 16661809 TI - Interaction between Light Intensity and NaCl Salinity and Their Effects on Growth, CO(2) Assimilation, and Photosynthate Conversion in Young Broad Beans. AB - Seedings of Vicia faba were grown for four weeks at two different light intensities (55 and 105 watts per square meter) in a saline (50 millimolar NaCl) and nonsaline nutrient solution. NaCl salinity depressed growth and restricted protein formation, CO(2) assimilation, and especially the incorporation of photosynthates into the lipid fraction. Conversion of photosynthates in leaves was much more affected by salinity than was photosynthate turnover in roots. The detrimental effect of NaCl salinity on growth, protein formation, and CO(2) assimilation was greater under low than under high light conditions. Plants of the high light intensity treatment were more capable of excluding Na(+) and Cl(-) and accumulating nutrient cation species (Ca(2+), K(+), Mg(2+)) than plants grown under low light intensity. It is suggested that the improved ionic status provided better conditions for protein synthesis, CO(2) assimilation, and especially for the conversion of photosynthates into lipids. PMID- 16661810 TI - Studies with 4,6-dioxoheptanoic Acid on etiolated and greening barley leaves. AB - 4,6-Dioxoheptanoic acid (DA), an inhibitor of 5-aminolevulinic acid (ALA) dehydratase (EC 4.3.1.24), causes ALA to accumulate at the expense of chlorophyll when applied to greening leaves of Hordeum vulgare L. var. Larker. Preincubating etiolated leaves with DA in darkness eliminates the lag phase in ALA accumulation during a subsequent exposure to illumination. More than 50% of the DA taken up during a 2-hour incubation disappeared during a subsequent 4-hour incubation. These results suggest that barley leaves can metabolize DA, and the products of this metabolism may enhance the capacity for ALA synthesis. PMID- 16661811 TI - Relationship between Thylakoid Membrane Fluidity and the Functioning of Pea Chloroplasts : EFFECT OF CHOLESTERYL HEMISUCCINATE. AB - Cholesteryl hemisuccinate has been incorporated into pea chloroplast thylakoids to investigate the relationship between fluidity and functioning of this membrane system. Levels of sterol which increased the apparent viscosity of the membrane, estimated by fluorescence polarization measurements using the lipophilic probe, 1,6-diphenyl-1,3,5 hexatriene, affected several photosynthetic processes. A decrease in fluidity was accompanied by an inhibition of dark limiting steps associated with electron transfer between photosystems two and one (PSII and PSI) as observed by the oxidation of the primary acceptor of PSII and by electron flow to ferricyanide. Also, treatment with cholesteryl hemisuccinate inhibited the saltinduced rise in chlorophyll fluorescence and changed the ionic conductivity of the membrane as judged by measurements of the decay of the lightinduced proton gradient. The results are discussed in terms of the effect of fluidity changes on the lateral diffusion of plastoquinone and chlorophyll protein complexes in the lipid matrix of the membrane. PMID- 16661812 TI - Sterol Composition and Accumulation in Glycine max (L.) Merr Leaves under Different Filtered Sunlight Conditions. AB - Soybean plants (Merr) were grown in the field in three plots. Sixteen days after sowing, two plots were covered with blue and red polyvinylchloride filters (0.45 millimeter thick) and one remained uncovered as control. Leaves of all plots were analyzed for total, free, esterified, and glycosidic sterols at two successive stages of plant growth (flowering and podripening).During the growth, total sterols increased in the control sample and under red and blue polyvinylchloride filters. Although free sterol contents were always the highest, the esterified sterols were mainly responsible for this increase. Red and blue polyvinylchloride filters caused a general decrease in the amounts of sterol classes but, during the growth, they caused the largest and most consistent changes. These filters particularly increased sitosterol and decreased stigmasterol. The changes in relative amounts of stigmasterol and sitosterol in soybean leaves might represent an interconversion between these two sterols. There seems to be a general increase-decrease relationship due to light quality. PMID- 16661813 TI - White Light Effects on the mRNA for the Light-Harvesting Chlorophyll a/b-Protein in Lemna gibba L. G-3. AB - Translation products of poly(A) mRNA isolated from Lemna gibba L. G-3 include a major polypeptide of 32,000 daltons which is immunoprecipitated by antiserum to chlorophyll a/b-protein from Chlamydomonas. This 32,000 dalton polypeptide represents a precursor to the light-harvesting chlorophyll a/b-protein of molecular weight 28,000 found in the thylakoid membranes of Lemna gibba. The amount of this translatable mRNA decreases relative to other translatable mRNAs when green plants grown in continuous white light are placed in darkness. This decrease occurs rapidly. The most rapid decline occurs during the first day; after 4 days of darkness, only a low level of this mRNA can be detected by in vitro translation. When the plants are returned to white light there is an increase in the relative level of this mRNA which can be easily detected within two hours. The in vivo synthesis of this protein has been assayed under the different light conditions. The light effects on the in vivo synthesis of the chlorophyll a/b-protein reflect the light effects on the translatable mRNA for the polypeptide. The results indicate that light induced changes in the synthesis, processing, or degradation of chlorophyll a/b-protein mRNA could account for the light-induced changes observed in the effective synthesis rates for the chlorophyll a/b-protein in vivo. PMID- 16661814 TI - Euploidy in Ricinus: EUPLOIDY EFFECTS ON PHOTOSYNTHETIC ACTIVITY AND CONTENT OF CHLOROPHYLL-PROTEINS. AB - The effects of nuclear genome duplication on the chlorophyll-protein content and photochemical activity of chloroplasts, and photosynthetic rates in leaf tissue, have been evaluated in haploid, diploid, and tetraploid individuals of the castor bean, Ricinus communis L. Analysis of this euploid series revealed that both photosystem II (2,6-dichlorophenolindophenol reduction) and photosystem I oxygen uptake (N,N,N',N'-tetramethyl-p-phenylenediamine to methyl viologen) decrease in plastids isolated from cells with increasingly larger nuclear complement sizes. Photosynthetic O(2)-evolution and (14)CO(2)-fixation rates in leaf tissue from haploid, diploid, and tetraploid individuals were also found to decrease with the increase in size of the nuclear genome. Six chlorophyll-protein complexes, in addition to a zone of detergent complexed free pigment, were resolved from sodium dodecyl sulfate-solubilized thylakoid membranes from cells of all three ploidy levels. In addition to the P700-chlorophyll a-protein complex and the light harvesting chlorophyll a/b-protein complex, four minor complexes were revealed, two containing only chlorophyll a and two containing both chlorophyll a and b. The relative distribution of chlorophyll among the resolved chlorophyll-protein complexes and free pigment was found to be similar for all three ploidy levels. PMID- 16661815 TI - Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber. AB - Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study. PMID- 16661816 TI - Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum. AB - The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR 6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.The rise in glutamine synthetase activity corresponded with the rapid decline in the nitrogen and c-phycocyanin content of the cells. Prior to nitrogen starvation, the nitrogen content of the cells was 140, 90, and 83 micrograms nitrogen per milligram dry weight for ammonia, urea, and nitrate grown cells, respectively. During nitrogen starvation where glutamine synthetase activity was highest, the nitrogen content of cells had declined to 35 to 40 micrograms nitrogen per milligram dry weight of cells. At the same time, the c-phycocyanin content of cells dropped by 95%. PMID- 16661817 TI - Utilization of indole analogs by carrot and tobacco cell tryptophan synthase in vivo and in vitro. AB - Twenty-three indole analogs were used to inhibit the growth of carrot and tobacco suspension cultures. The addition of tryptophan or indole partially reversed the inhibition of both cell lines only for 4-fluoroindole, 5-fluoroindole, and 6 fluoroindole. Inhibition of tobacco cell growth by 5-aminoindole, 5 methoxyindole, 6-methoxyindole, or 7-methoxyindole was also partially reversed. Previously selected carrot and tobacco lines, which have high free tryptophan levels, grew in the presence of the analogs for which reversal was noted in all cases except 5-aminoindole and also in some other cases. Growth inhibition caused by all 10 tryptophan analogs studied was partially reversible by tryptophan or indole and the high tryptophan lines were also able to grow in the presence of concentrations inhibitory to the wild type lines.Tryptophan synthase activity from both species could utilize most of the 2 or 3 position unsubstituted indole analogs to produce the corresponding tryptophan analogs. Using carrot cell extracts, the identity of the correct product was confirmed by automated amino acid analysis for 4-fluoroindole, 5-fluoroindole, and 5-hydroxyindole. The analogs most rapidly utilized by carrot tryptophan synthase were 6-methoxyindole and the 4-, 5-, or 6-fluoroindoles. When 4-fluoroindole, 5-hydroxyindole, 5 methoxyindole, or 5-methylindole were incubated with carrot cells the corresponding tryptophan analog was formed in all cases as identified by amino acid analysis.These results indicate that cultured carrot and tobacco cells can convert certain nontoxic indole analogs into toxic tryptophan analogs. Using 5 fluoroindole and 6-fluoroindole, attempts were made to isolate tryptophan auxotrophs lacking tryptophan synthase from diploid carrot and tobacco lines. However, only 5-methyltryptophan-resistant lines, which presumably accumulate high levels of free tryptophan, were recovered. PMID- 16661818 TI - Betaine accumulation and betaine-aldehyde dehydrogenase in spinach leaves. AB - Spinach leaf discs accumulated betaine when exposed to a mannitol solution of -20 bars. The accumulation was 12 micromoles per gram original fresh weight in a 24 hour period.Betaine-aldehyde dehydrogenase (EC 1.2.1.8) was assayed in various subcellular fractions prepared from spinach leaves, and it was found only in the soluble fraction. This cytosolic enzyme was purified 175-fold, and its properties were studied. The enzyme was relatively specific for betaine aldehyde as the substrate with an apparent K(m) value of 2.08 x 10(-4) molar. It also exerted activity on other aldehyde analogs tested, but with lower V(max) and higher K(m) values. The enzyme was relatively specific for nicotinamide adenine dinucleotide as the coenzyme, having an apparent K(m) value of 9.46 x 10(-6) molar; lower activities were observed when nicotinamide adenine dinucleotide phosphate or 3 acetyl pyridine adenine dinucleotide were tested as electron acceptors. The activity was enhanced by dithiothreitol and inhibited by p-chloromercuribenzoate, and the inhibition by p-chloromercuribenzoate was partially reversed by the subsequent addition of dithiothreitol. The activity was inhibited by high concentrations of NaCl and, to a lesser extent, proline. The equilibrium of the enzymic reaction was strongly in favor of betaine formation.The in vitro activity of the enzyme under optimal assay conditions was high enough to account for the amount of betaine accumulated under water stress conditions. The enzyme activity was the same in unstressed leaves and in leaves that had been water stressed for 24 hours. PMID- 16661819 TI - Citrus Tissue Culture : AUXINS IN RELATION TO ABSCISSION IN EXCISED PISTILS. AB - An in vitro bioassay for chemicals that affect Citrus abscission was used to identify three inhibitors of stylar abscission in lemon pistil explants incubated on defined nutrient media. The three inhibitors (picloram, 4-chlorophenoxyacetic acid, and 3,5,6-trichloropyridine-2-oxyacetic acid) are all auxins, and the most potent of them (i.e. picloram) was found to be at least 10 times more active in the bioassay than 2,4-dichlorophenoxyacetic acid. Picloram (2 micromolar) also was shown to be effective in inhibiting stylar abscission in pistil explants from other Citrus cultivars such as mandarin, Valencia, and Washington navel oranges and grapefruit. To study the physiology of auxins active as abscission inhibitors versus inactive auxins in lemon pistils, the transport and metabolism of [1 (14)C]-2,4-dichlorophenoxyacetic acid was compared with that of [2-(14)C]indole-3 acetic acid, which is without effect in the bioassay over the range from 0.1-100 micromolar. Insignificant quantities of labeled indole-3-acetic acid and/or labeled derivatives were found to reach the presumptive zone of stylar abscission under the test conditions. Labeled 2,4-dichlorophenoxyacetic acid and/or labeled derivatives also were transported slowly through pistils, but some radioactivity could be detected in the stylar abscission zone as early as 24 hours after the start of incubation. Extensive conversion of [2-(14)C]indole-3-acetic acid to labeled compounds tentatively considered to be glycoside and cellulosic glucan derivatives was found with the use of solvent extraction methodology. A significantly smaller percentage of the radioactivity in pistils incubated on [1 (14)C]-2,4-dichlorophenoxyacetic acid was found in fractions corresponding to these derivatives. Both transport and metabolism appear to be important factors affecting the activity of auxins as abscission inhibitors in the bioassay. PMID- 16661820 TI - Physiological Control of Chloride Transport in Chara corallina: I. EFFECTS OF LOW TEMPERATURE, CELL TURGOR PRESSURE, AND ANIONS. AB - The rate of Cl(-) transport at the plasma membrane of the freshwater alga Chara corallina is investigated with respect to possible in vivo controls acting in addition to the two well established ones of cytoplasmic Cl(-) and cytoplasmic pH. In contrast with results from many other plant tissues, halides appear to be the only anions capable of inhibiting Cl(-) transport, either from the outside or inside surfaces of the plasma membrane. Cell turgor pressure was also investigated. It was found that neither the influx of Cl(-) nor that of K(+) or HCO(2) (-) is sensitive to turgor. Internal osmotic pressure is also insensitive to turgor, a situation contrasting with that in closely related brackish water charophytes.After temperature downshift (from 20-4 C) Cl(-) transport displays a slow, time-dependent rise. Return of cells from 4 C to 20 C results in a large stimulation of Cl(-) influx in comparison with cells maintained at 20 C throughout. This stimulation persists for several hours and is also apparent (to a reduced extent) in cells which have had cytoplasmic composition controlled by intracellular perfusion. The stimulation therefore arises, in part, from a change in plasma membrane properties. The results are discussed with respect to recent work on membrane fluidity as a function of temperature. PMID- 16661821 TI - Evidence for Mediated HCO(3) Transport in Isolated Pea Mesophyll Protoplasts. AB - The kinetics of (14)C fixation, and inorganic C (C(inorg)) accumulation, have been followed in isolated pea mesophyll protoplasts. NaH(14)CO(3) was supplied to the protoplasts in media the pH of which was varied between 7 and 8.When (14)CO(2) fixation was plotted against the calculated concentration of free CO(2) in the media, the apparent K(m) for CO(2) was observed to rise as external pH increased. The V(max) did not alter significantly. Similarly, when C(inorg) uptake, either in the light or in the dark, was plotted against external CO(2) concentration the slope of the curves was steeper at higher external pH.Investigation of the time course of uptake showed that internal C(inorg) concentration rose throughout the experimental period, and that in the light it surpassed the external C(inorg) concentration after about 3 minutes. Irradiation of protoplasts previously taking up (14)C(inorg) in the dark brought about a sharp increase in the rate of (14)C(inorg) accumulation which was sustained for at least 20 minutes.Estimates of internal pH based on the distribution of labeled 5,5-dimethyloxazoladine-2,4-dione (DMO) between protoplast and medium suggested that internal pH altered relatively little with change in external pH. The values for internal pH as calculated from C(inorg) distribution were always higher than those calculated from DMO distribution, i.e. the internal C(inorg) concentration was higher than would be predicted on the assumption of passive distribution in accordance with pH.Addition of carbonic anhydrase to the external solution was without effect either on rate of (14)CO(2) fixation or C(inorg) accumulation.Various possible interpretations of the results are considered. It is concluded that the most reasonable explanation, consistent with all the data, is that HCO(3) (-) ions can cross the protoplast membranes, and that their passage is mediated by a transfer mechanism. PMID- 16661822 TI - Electrical potentials in stomatal complexes. AB - Guard cells of several species, but predominantly Commelina communis, were impaled by micropipette electrodes and potential differences measured that occurred between cell compartments and the flowing bathing medium. The wall developed a Donnan potential that was between -60 and -70 millivolt in 30 millimolar KCl at pH 7. The density of the fixed charges ranged from 0.3 to 0.5 molar; its dependence on pH was almost identical with the titration curve of authentic polygalacturonic acid. The vacuolar potential of guard cells of Commelina communis L., Zea mays L., Nicotiana glauca Graham, Allium cepa L., and Vicia faba L. was between -40 and -50 millivolt in 30 millimolar KCl when stomata were open and about -30 millivolt when stomata were closed. The vacuolar potential of guard cells of C. communis was almost linearly related to stomatal aperture and responded to changes in the ionic strength in the bathing medium in a Nernstian manner. No specificity for any alkali ion (except Li(+)), ammonium, or choline appeared. Lithium caused hyperpolarization. Calcium in concentrations between 1 and 100 millimolar in the medium led to stomatal closure, also caused hyperpolarization, and triggered transient oscillations in the intracellular potential. Gradients in the electrical potential existed across stomatal complexes with open pores. When stomata closed, these gradients almost disappeared or slightly reverted; all epidermal cells were then at potentials near -30 millivolt in 30 millimolar KCl. PMID- 16661823 TI - Resistance to acetohydroxamate acquired by slow adaptive increases in urease in cultured tobacco cells. AB - Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner 1980 Plant Phvsiol 65: 995-1003). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to about 4 days for 1U cells, while 1U and 12U cells double in 2 days on nitrate. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty per cent inhibition of growth by AHA occurred at 20 micromolar in 1U cells growing on urea and at 165 micromolar in 12U cells growing on urea, but at 600 micromolar for either 1U or 12U cells growing on nitrate. When 12U cells were grown on urea with 100 micromolar AHA, extractable urease activity decreased 80% within 2.5 hours and remained at this level for 2 weeks; the doubling time increased to 3.7 days, and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre AHA level by incubation at 30 C after extraction and separation from free AHA. AHA inhibited incorporation of (15)N from [(15)N]urea into Kjeldahl nitrogen in the cells, in spite of the increased intracellular urea. These results indicate that AHA acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease. PMID- 16661824 TI - Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases. AB - Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions. PMID- 16661825 TI - Protonation and light synergistically convert plasmalemma sugar carrier system in mesophyll protoplasts to its fully activated form. AB - The course of sugar fluxes into and out of protoplasts isolated from the mesophyll of Pisum sativum L. has been followed over brief time intervals (minutes). Light strongly stimulated net sugar influx at pH 8 as well as at pH 5.5. The proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited initial influx in the light, both at pH 8.0 and at pH 5.5. CCCP was without effect in the dark at either pH. All these results applied both to sucrose and to the nonmetabolizable glucose analog 3-O-methyl-d-glucose.When protoplasts at pH 5.5 were transferred from light to darkness, "stored" light driving force maintained uptake in the dark at the full light rate for the first 7 minutes. At pH 8, however, even 4 minutes after transfer to dark, uptake was well below the light rate. Initial uptake rates over a range of external concentrations were derived from progress curves obtained in the light and in the dark, both at pH 5.5 and at 7.7. When initial rate was plotted against concentration, simple Michaelis-Menten kinetics were observed only under the condition pH 5.5, light. In the dark at both pH values, and in the light at pH 7.7, complex curves with intermediate plateaus were obtained, strongly resembling curves reported for systems where mixed negative and positive cooperativity is operating.The same "K(m) for protons" was observed in the dark and in the light (10(-7) molar). Switching protoplasts in the dark from pH 8 to 5.5 failed to drive sugar transport by imposed protonmotive force, as judged by lack of sensitivity to CCCP. Switching protoplasts which had taken up sugar in the dark at pH 5.5 to pH 7 induced net efflux of sugar. Flux analysis showed that this effect was entirely due to the prompt fall in influx.It is concluded from the kinetic experiments that protonation alone is not sufficient to convert the sugar transport system to its fully activated high affinity form. A further light dependent factor which acts synergistically with protonation is required. PMID- 16661826 TI - Variations in Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Plants. AB - Studies of ribulose-1,5-bisphosphate (RuBP) carboxylase from taxonomically diverse plants show that the enzyme from C(3) and crassulacean acid metabolism pathway species exhibits lower K(m)(CO(2)) values (12-25 micromolar) than does that from C(4) species (28-34 micromolar). RuBP carboxylase from aquatic angiosperms, an aquatic bryophyte, fresh water and marine algae has yielded consistently high K(m)(CO(2)) values (30-70 micromolar), similar in range to that of the enzyme from C(4) terrestrial plants. This variation in K(m)(CO(2)) is discussed in relation to the correlation between the existence of CO(2) concentrating mechanisms for photosynthesis and the affinity of the enzyme for CO(2). The K(m)(RuBP) of the enzyme from various sources ranges from 10 to 136 micromolar; mean +/- sd = 36 +/- 20 micromolar. This variation in K(m)(RuBP) does not correlate with different photosynthetic pathways, but shows taxonomic patterns. Among the dicotyledons, the enzyme from crassinucellate species exhibits lower K(m)(RuBP) (18 +/- 4 micromolar) than does that from tenuinucellate species (25 +/- 7 micromolar). Among the Poaceae, RuBP carboxylase from Triticeae, chloridoids, andropogonoids, Microlaena, and Tetrarrhena has yielded lower K(m)(RuBP) values (29 +/- 11 micromolar) than has that from other members of the grass family (46 +/- 10 micromolar). PMID- 16661827 TI - Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES. AB - The effect of O(2) and pH on the in vitro synthesis of (14)C-labeled ureides from [8-(14)C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O(2) concentrations and low pH, ureide synthesis was inhibited and the (14)C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O(2) concentrations and high pH, also stimulated ureide synthesis, and the (14)C label was incorporated principally into allantoin. The overall response of the system to O(2) concentration and pH indicated that the per cent distribution of total (14)C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8 (14)C]hypoxanthine was also inhibited. PMID- 16661828 TI - Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT. AB - Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O(2) evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II. PMID- 16661829 TI - Characterization of Kanamycin-Resistant Cell Lines of Nicotiana tabacum. AB - Two kanamycin-resistant variants of Nicotiana tabacum were derived by culturing seedling leaf sections on a shoot-inducing medium containing kanamycin. The variants displayed a higher resistance to the structurally related antibiotic streptomycin than to kanamycin. The resistance phenotype was expressed when the tissue was cultured either as callus or as a differentiating tissue and was stably maintained in the absence of kanamycin. Plants regenerated from the variant lines had morphologically abnormal flowers and produced few viable seeds. These resistant lines are potentially useful for protoplast fusion or genetic modification experiments requiring selectable phenotypic markers. PMID- 16661830 TI - Isolation of a kaurene synthetase inhibitor from castor bean seedlings and cell suspension cultures. AB - Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (10(4)-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible. PMID- 16661831 TI - alpha-Galactoside Binding Proteins from Plant Membranes: Isolation, Characterization, and Relation to Helminthosporoside Binding Proteins of Sugarcane. AB - alpha-Galactoside binding proteins were isolated from cellular membranes of mint and tobacco as well as two clones of sugarcane which differ in their sensitivity to helminthosporoside, a toxic galactoside. Sodium trichloroacetate was used to disrupt membranes after which the proteins were purified using a melibiose Sepharose-6B affinity column. Proteins from mint, tobacco, and susceptible sugarcane had equal electrophoretic mobilities, whereas resistant sugarcane protein migrated more slowly. Pretreatment of the proteins with fluorescamine caused them to migrate with the tracking dye. Each of the proteins had molecular weights of about 100,000 and each was shown to be oligomeric. Gel filtration revealed that aqueous solutions of these membrane proteins contained a mixture of size species which included a high molecular weight multimer and lower molecular weight oligomers. The relative abundance of the oligomers was dependent upon protein concentration: the lower concentrations yielded higher relative amounts of oligomers (Kenfield and Strobel 1980 Biochim Biophys Acta 600: 705-712). Also, the binding activity of these receptors was inversely proportional to protein concentration. At low protein concentration (4 micrograms per milliliter), the K(d)'s of each of the proteins for galactinol, raffinose, and helminthosporoside was about 10 micromolar. At high protein concentrations (100 micrograms per milliliter), mint and resistant sugarcane proteins failed to bind alpha galactosyl ligands, whereas proteins from tobacco and susceptible sugarcane exhibited a markedly decreased binding activity compared to that at lower protein concentrations. Binding proteins from susceptible sugarcane were mixed with receptors from either resistant sugarcane or mint at low protein concentrations, then assayed for binding activity. Such mixtures showed a concentration-dependent decrease in binding activity analogous to the activity of homogeneous protein solutions. Bovine serum albumin, a nonsubunit protein, had no effect on the binding activity of the protein from susceptible sugarcane. Thus, receptors from diverse plants can associate in vitro and form functional oligomers. The amino acid composition of each of the binding proteins was similar but not identical. The significance of these results is discussed in regard to regulation of carbohydrate transport and sensitivity to phytotoxins. PMID- 16661832 TI - Cytokinin-Active Ribonucleosides in Phaseolus RNA: I. IDENTIFICATION IN tRNA FROM ETIOLATED PHASEOLUS VULGARIS L. SEEDLINGS. AB - The cytokinin-active ribonucleosides present in tRNA from etiolated Phaseolus vulgaris L. seedlings have been isolated and identified as cis-ribosylzeatin, 2 methylthio-ribosylzeatin, and N(6)-(Delta(2)-isopentenyl)-adenosine. The structures of the compounds were established on the basis of their chromatographic properties and the mass spectra of their permethylated and perdeuteromethylated derivatives. Cis-ribosylzeatin was the major cytokinin active constituent of tRNA from this source. PMID- 16661833 TI - Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS. AB - The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA(Cys), tRNA(Trp), tRNA(Tyr), a major peak of tRNA(Phe), and a large fraction of tRNA(Leu) were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA(Ser) species and with minor tRNA(Leu) species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected. PMID- 16661834 TI - Ferricyanide Reduction in Photosystem II of Spinach Chloroplasts. AB - Ferricyanide can be reduced in Photosystem II of spinach chloroplasts at 2 separate sites, both of which are sensitive to 3-(3,4-dichlorophenyl)-1,1 dimethylurea, but only one of which is sensitive to dibromothymoquinone. Data presented in this paper emphasize ferricyanide site II of Photosystem II, which is sensitive to thiol inhibition and may reflect a cyclic pathway around Photosystem II. Ferricyanide reduction sites 1 and 2 also differ from each other in fractions isolated from discontinuous sucrose gradients, from fragmented chloroplasts, and upon trypsin treatment. Sucrose density gradient centrifugation shows that ferricyanide reduction site 1 activity at pH 6 decreases from 30 to 50% in various isolated fractions, while the dibromothymoquinone-insensitive activity at pH 8 (site 2) is stimulated from 15 to 35%.Fragmentation of chloroplasts also stimulates ferricyanide site II activity, but trypsin treatment destroys ferricyanide reduction site II completely in 6 minutes. Ferricyanide reduction site 1 still retains 50% activity at this point. The meaning of these differences is discussed in terms of the physical location of these two sites on the thylakoid membrane. PMID- 16661835 TI - Crown-gall tumors possess tumor-specific antigenic sites on their cell walls. AB - Rabbits were injected with cell walls obtained from crown-gall tumor tissue or the corresponding cell walls from normal potato tissue. The serum obtained from rabbits 53 days after they were injected with tumor cell walls contained immunoglobins that reacted with both tumor and normal cell walls as well as with the cells from the inciting strain of Agrobacterium tumefaciens. When this serum was repeatedly absorbed against normal cell walls and the cells of the inciting strain of Agrobacterium tumefaciens, only tumor-specific immunoglobins remained. These immunoglobins did not react with cell walls obtained from meristematic (nontumorous) potato tissue. Yet this same serum reacted with crown-gall tumor cell walls obtained from turnip and carrot discs. PMID- 16661836 TI - Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN. AB - Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO(3) (-) at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO(3) (-)-N per hectare and harvested plants treated with 40 kilograms NO(3) (-)-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N(2) fixing capacity were accompanied by similar changes in levels of nodule soluble protein.After an initial lag of 24 hours, specific activities of alfalfa nodule glutamine synthetase, NADH-glutamate synthase, and NAD-glutamate dehydrogenase (oxidative amination) decreased similar to but less rapidly than acetylene reduction activity. Increased specific activities of these nodule enzymes occurred as acetylene reduction activity increased and shoot growth resumed. The observed rates of glutamine synthetase and glutamate synthase were sufficient to assimilate ammonia produced via symbiotic N(2) fixation. Nodule NADH-dependent glutamate dehydrogenase (reductive amination) specific activity was not associated with changes in acetylene reduction activity.The data indicate that host plant glutamine synthetase and NADH-glutamate synthase function to assimilate symbiotically fixed N and that NADH-dependent glutamate dehydrogenase may function in ammonia assimilation during senescence in alfalfa nodules. PMID- 16661837 TI - Ethylene Evolution following Treatment with 1-Aminocyclopropane-1-carboxylic Acid and Ethephon in an in Vitro Olive Shoot System in Relation to Leaf Abscission. AB - 1-Aminocyclopropane-1-carboxylic acid (ACC) supplied via the cut base of detached olive shoots caused a burst of ethylene from leaves, but other cyclopropanes tested did not exhibit this effect. Ethephon (ET) and another ethylene-releasing compound caused a prolonged increase in ethylene evolution. ACC had only a very limited effect on leaf abscission regardless of concentration, whereas shoots placed with cut bases in ET for 60 to 80 minutes exhibited 100% leaf abscission within 90 hours. Shoots with inflorescences treated with ET just prior to anthesis began to wilt in vitro within 20 to 30 hours and failed to exhibit leaf abscission. At earlier stages of development, ET induced more leaf abscission on reproductive shoots than on vegetative shoots. It is suggested that the duration of ethylene evolution from the leaves governs their potential for abscission and that bursts of ethylene evolution even though large in amount may not induce abscission. PMID- 16661838 TI - Structural and Immunochemical Studies on the Phytotoxic Peptidorhamnomannan of Ceratocystis ulmi. AB - A wilt-inducing peptidorhamnomannan produced by Ceratocystis ulmi, the causative agent in Dutch Elm disease, has been subjected to additional chemical and physical characterization. Gel filtration, reductive beta elimination, hydrofluoric acid deglycosylation, and ultracentrifugation experiments provide evidence that the wilt-inducing polymer is polydisperse with a molecular weight range of approximately 105,000 to 120,000. The carbohydrate portion of each molecule is composed of small percentages of mannose, mannobiose, mannotriose, and a tetra- or pentasaccharide composed of mannose and rhamnose plus a major component consisting of two or three long rhamnomannan chains each with a molecular weight range of 32,000 to 34,000. All saccharide units are attached via O-glycosidic linkages to a protein with a molecular weight of approximately 35,000.Rabbit antibodies directed against both C. ulmi and the purified peptidorhamnomannan have been prepared. Their possible use in evaluating the role of the polymer in the disease is discussed. PMID- 16661839 TI - Measurement of Protein Degradation in Leaves of Zea mays Using [H]Acetic Anhydride and Tritiated Water. AB - The rate of protein degradation in Zea mays leaves has been estimated by using tritiated water and [(3)H]acetic anhydride as the labeling agents. Both methods circumvent many of the problems usually associated with measuring protein degradation in plants. The half-life of ribulose-1,5-bisphosphate carboxylase protein in second leaves of 13-day-old seedlings under continuous light was found to be 7.8 +/- 0.9 days by the tritiated water technique and 6.5 +/- 0.8 days by the [(3)H]acetic anhydride method. The half-lives determined under a 14-hour light, 10-hour-dark photoperiod are 6.2 +/- 0.8 days with tritiated water and 5.4 +/- 0.4 days with [(3)H]acetic anhydride. Whereas the values obtained by the two methods do not differ significantly, the use of either method for the determination of protein half-life can be recommended. PMID- 16661840 TI - Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase. AB - Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH(4))(2)SO(4) was stable when frozen as small beads in liquid nitrogen and stored at -80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO(2) and Mg(2+) activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.The purified enzyme contained about 2 gram-atoms iron per mole enzyme that could not be removed by chelating agents. When the enzyme was incubated with 100 millimolar dithiothreitol and exposed to O(2), a purple dithiothreitol-iron complex was formed which could be removed by dialysis. The activities of ribulose-1,5-bisphosphate carboxylase and oxygenase were not altered by reducing the iron content to 0.7 mole per mole enzyme by treatment with dithiothreitol followed by exhaustive dialysis against iron free buffer. PMID- 16661841 TI - Factors affecting the induction of secondary dormancy in lettuce. AB - The relationship between the temperature at which germination of 50% of the seeds is inhibited in the light (GT(50) Light) and secondary dormancy was investigated in three cultivars of Lactuca sativa L. Seeds were incubated for varying periods under non-germinating conditions and subsequent germination in response to red light (R) was determined over a wide range of temperatures. Dark incubation at 32 C reduced the GT(50) Light of cv. New York but did not affect germination at temperatures below 24 C. Dark, 32 C incubation had no effect on the GT(50) Light of cv. Great Lakes. In cv. Grand Rapids, dark incubation at 15, 24, 32, or 35 C initially reduced the GT(50) Light. However, longer incubations induced a secondary dormancy, i.e., the seeds became unable to germinate at all temperatures in response to R given after the high temperature incubation. A single exposure to R at the beginning of a 32 C incubation slowed the induction of secondary dormancy. Repeated exposures to R prevented the induction of secondary dormancy, but did not prevent a decline in the GT(50) Light. GA(3) mimicked the effect of repeated R.The differences in the germination behavior of the three cultivars suggest that there may be qualitative differences in the germination mechanism of these cultivars. This research demonstrates the significance of monitoring germination at a range of temperatures to avoid misinterpretation of the data. PMID- 16661842 TI - Twilight effect: initiating dark measurement in photoperiodism of xanthium. AB - Six experiments studied the effects of low levels of red and far-red light upon the initiation of measurement of the dark period in the photoperiodic induction of flowering in Xanthium strumarium L. (cocklebur), a short-day plant, and compared effects with those of comparable light treatments applied for 2 hours during the middle of a 16-hour inductive dark period. Red light, or red plus far red, at levels that inhibit flowering when applied during the middle of the inductive dark period, either had no effect on the initiation of dark measurement (i.e., were perceived as darkness), or they delayed the initiation of dark measurement by various times up to the full interval of exposure (2 hours). Far red light alone had virtually no effect either at the beginning or in the middle of the dark period. These results confirm that time measurement in the photoperiodic response of short-day Xanthium plants is not simply the time required for metabolic dark conversion of phytochrome. Results also suggest that the pigment system (phytochrome?) and/or responses to it may be significantly different as they function during twilight (initiation of dark measurement), and as they function during a light break several hours later. Possible mechanisms by which cocklebur plants detect the change from light to darkness are discussed.COMPARING EXPERIMENTAL RESULTS WITH SPECTRAL LIGHT MEASUREMENTS DURING TWILIGHT AND WITH MEASUREMENTS OF LIGHT FROM THE FULL MOON LED TO TWO CONCLUSIONS: First, light levels pass from values perceived by the plant as full light to values perceived as complete darkness in only about 5.5 to 11.5 minutes, although twilight as perceived by the human eye lasts well over 30 minutes. Second, cocklebur plants probably do not respond to light from the full moon, even when most sensitive, 7 to 9 hours after the beginning of darkness. PMID- 16661843 TI - Light-dependent reduction of dehydroascorbate by ruptured pea chloroplasts. AB - Glutathione dehydrogenase (EC 1.8.5.1) was partially purified from pea shoots. The pH optimum was 7.6. The K(m) values for GSH and dehydroascorbate were 4.4 and 0.44 millimolar, respectively. The enzyme was inhibited by iodoacetate and CuSO(4) but not significantly by ZnCl(2) or NaN(3). Part of the total enzyme activity was associated with isolated chloroplasts.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H) and substrate concentrations of GSH or GSSG, catalyzed (dehydroascorbate plus glutathione) dependent O(2) evolution with the concomitant reduction of dehydroascorbate to ascorbate. Oxidation of ascorbate by ascorbate oxidase activity associated with the chloroplasts was relatively insignificant. ZnCl(2) inhibited (dehydroascorbate plus glutathione)-dependent O(2) evolution but not ascorbate formation. The reaction was attributed to light-dependent reduction of GSSG (involving glutathione reductase) coupled to the reduction of dehydroascorbate (involving glutathione dehydrogenase). Light-dependent reduction of GSSG appears to be the rate-limiting step in this reaction sequence at physiological concentrations of GSH. PMID- 16661844 TI - Flowering in Xanthium strumarium: INITIATION AND DEVELOPMENT OF FEMALE INFLORESCENCE AND SEX EXPRESSION. AB - Vegetative plants of Xanthium strumarium L. grown in long days were induced to flower by exposure to one or several 16-hour dark periods. The distribution of male and female inflorescences on the flowering shoot was described, and a scoring system was designed to assess the development of the female inflorescences. The time of movement of the floral stimulus out of the induced leaf and the timing of action of high temperature were shown to be similar for both the apical male and lateral female inflorescences.Strong photoperiodic induction of the plants favored female sex expression, while maleness was enhanced by exogenous gibberellic acid. The problem of the control of sex expression in Xanthium is discussed in relation to the distribution pattern of male and female inflorescences on the flowering shoot and to the state of the meristem at the time of the arrival of the floral stimulus. PMID- 16661845 TI - Light requirement for induction and continuous accumulation of an ammonium inducible NADP-specific glutamate dehydrogenase in chlorella. AB - The ammonium-inducible NADP-specific glutamate dehydrogenase of Chlorella sorokiniana was shown to require light for both its induction by ammonia in uninduced cells, and its continuous accumulation in fully induced cells. Addition of ammonia to uninduced cells in the light resulted in a 35-minute induction lag followed by linear and coincident increases in enzyme activity and antigen. Enzyme activity was not induced in the dark; however, transfer of these cells to the light resulted in an immediate increase in enzyme activity and antigen. The absence of an induction lag suggested that mRNA sequences and/or an enzyme precursor with different antigenic properties than the active holoenzyme accumulated in cells in the dark in ammonium medium. When fully induced cells were transferred to the dark, the activity of the enzyme quickly ceased to accumulate. In contrast to the NADP-specific isozyme, the cells also contain a constitutive NAD-specific isozyme which was shown to accumulate in cells in the dark in either ammonium or nitrate medium. PMID- 16661846 TI - Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans. AB - Two isoenzymes each of phosphoglucomutase, hexose phosphate isomerase, aldolase, fructose diphosphatase, phosphofructokinase, and 6-phosphogluconate dehydrogenase have been separated by DEAE-cellulose column chromatography of extracts from endosperm of germinating castor beans (Ricinus communis cv. Hale). One of each of the enzymes is localized in the cytosol and the other is confined to plastids. Developmental studies of these isoenzymes were carried out to clarify their roles in the endosperm. In extracts from ungerminated seeds the activities of marker enzymes of mitochondria (fumarase), plastids (ribulose bisphosphate carboxylase), and glyoxysomes (catalase) were low, but phosphoglucomutase, hexose phosphate isomerase, aldolase, and 6-phosphogluconate dehydrogenase were present in relatively high activity. The total amounts of these enzymes increased 3- to 4 fold during the first 5 days of growth. The activities of isoenzymes in the plastids rose in parallel with that of ribulose bisphosphate carboxylase to reach a maximum at day 4, and like the carboxylase they declined sharply thereafter. The activities of the cytosolic isoenzymes peaked at day 5. These changes are consistent with the roles previously proposed for the sequences present in plastid and cytosol. PMID- 16661847 TI - Effect of temperature on proton efflux from isolated chloroplast thylakoids. AB - Temperature-induced changes in the decay of the light-induced proton gradient of chloroplast thylakoids isolated from chilling-resistant and chilling-sensitive plants have been examined. In the presence of N-methylphenazonium methosulfate, the thylakoids isolated from chilling-resistant barley (cv. Kanby) and pea (cv. Alaska) and chilling-sensitive mung bean (cv. Berken) plants showed temperature induced changes at approximately 8.6, 13.3, and 14.0 C, respectively. Barley thylakoids assayed in the presence of sodium thiocyanate also showed a change at 8.6 C, whereas with no addition or upon the inclusion of both N-methylphenazonium methosulfate and sodium thiocyanate the change occurred at approximately 11.5 C.Temperature-induced changes in electron transport activities occurred at temperatures approximating those for proton efflux for each of the three plants. These results indicate that temperature has a general effect on thylakoid membranes and that chloroplast thylakoids from chilling-resistant and chilling sensitive plants have their proton permeability properties affected similarly by temperature. PMID- 16661848 TI - An Examination of the Leaf Quaking Adaptation and Stomatal Distribution in Populus tremuloides Michx. AB - The leaves of quaking aspen (Populus tremuloides Michx.) have a flattened petiole that allows them to quake (oscillate and roll) under low wind velocities. It was hypothesized that this adaptation might enable the plant to respond to windy conditions that would increase transpirational losses. No effects of wind with or without leaf quaking on stomatal resistance were observed under controlled conditions in the field. If wind and leaf quaking affect stomatal resistance, such effects must be small in comparison to those caused by other factors such as leaf water potential and ambient humidity.Aspen leaves are hypostomatal with stomata evenly distributed over the abaxial surface. This observation casts serious doubt on the hypothesis that quaking is an adaptation to increase bulk air flow through amphistomatal leaves. PMID- 16661849 TI - In vivo phytochrome difference spectrum from dark grown gametophytes of anemia phyllitidis L. Sw. Treated with norflurazon. AB - An in vivo phytochrome difference spectrum of dark grown Anemia phyllitidis L. Sw. gametophytes has been measured. The spectral characteristics estimated from the difference spectrum were as follows: red maximum at 662 nanometers, far red maximum at 737 nanometers, isosbestic point at 695 nanometers, and a shoulder from 620 to 630 nanometers. To diminish the influence of chlorophyll on phytochrome measurements in dark grown gametophytes, total chlorophyll content could be reduced by about 65% by treatment with 1 micromolar Norflurazon. In gametophytes grown in red light, the inhibition caused by 1 micromolar Norflurazon was 97.5%, based on g spores. Total carotenoid content of dark grown gametophytes was not appreciably different from that already present in dry spores. Herbicide treatment reduced this level to about 60%. Gametophytes grown in red light showed a nearly 7-fold increase in total carotenoids compared with that of the dark control. In treated light grown protonemata, no carotenoid accumulation took place, and the level remaining corresponded with that found in treated dark grown protonemata. PMID- 16661850 TI - Cytokinin-induced wall extensibility in excised cotyledons of radish and cucumber. AB - The mechanism of cytokinin-induced cell expansion in cotyledons excised from dark grown seedlings of radish (Raphanus sativus L.) and cucumber (Cucumus sativus L.) was studied. Cotyledons were incubated in dim light with or without 17 micromolar zeatin for periods up to 3 days. Fresh weights and osmotic potentials were measured daily. Cell wall extensibility properties were measured before and after the growth period. Also, experiments in which radish cotyledons were grown in mannitol solutions of various concentrations were performed. Comparisons of growth rates and increases of tissue osmotic potentials (toward zero) during growth without mannitol indicate that wall extensibility increased during the growth period and that this extensibility was enhanced by zeatin.Extensibility values derived from growth rates in mannitol provided indirect evidence of zeatin increased wall extensibility. These conclusions were verified by direct measurements of plasticity with an Instron extensiometer. Thus, growth stimulation of excised cotyledons by cytokinins apparently involves wall loosening, in addition to previously demonstrated increases of K(+) absorption and formation of reducing sugars. PMID- 16661851 TI - Uptake of benzyladenine by excised watermelon cotyledons. AB - The uptake of 8-[(14)C]N(6)-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.There seems to be a correlation between the level of radioactivity (i.e. of BA + derivatives) present in the cotyledons and the magnitude of hormonal responses that are observed four days after uptake. This relationship holds regardless of whether a given level of radioactivity has been reached after a short period of uptake or after a long period of uptake followed by washing. PMID- 16661852 TI - Deep undercooling of tissue water and winter hardiness limitations in timberline flora. AB - Deep undercooled tissue water, which froze near -40 C, was found in winter collected stem and leaf tissue of the dominant timberline tree species of the Colorado Rocky Mountains, Engelmann spruce (Picea engelmannii (Parry) Engelm.) and subalpine fir (Abies lasiocarpa (Hook.) Nutt.), and in numerous other woody species in and below the subalpine vegetation zone. Previous work on numerous woody plants indicates that deep undercooling in xylem makes probable a -40 C winter hardiness limit in stem tissue. Visual injury determinations and electrolyte loss measurements on stem tissue revealed injury near -40 C associated with the freezing of the deep undercooled stem tissue water. These results suggest that the winter hardiness limit of this woody flora is near -40 C. The relevance of deep undercooling in relation to timberline, the upper elevational limit of the subalpine forest, is discussed. PMID- 16661853 TI - Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots. AB - The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K(m) of 0.3 millimolar in the presence of NADPH, whereas the NADH eluted enzyme has a nitrate K(m) of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases. PMID- 16661854 TI - Direct and indirect measurements of Phloem turgor pressure in white ash. AB - Direct determinations and indirect calculations of phloem turgor pressure were compared in white ash (Fraxinus americana L.). Direct measurements of trunk phloem turgor were made using a modified Hammel-type phloem needle connected to a pressure transducer. Turgor at the site of the direct measurements was calculated from the osmotic potential of the phloem sap and from the water potential of the xylem. It was assumed that the water potentials of the phloem and xylem were close to equilibrium at any one trunk location, at least under certain conditions. The water potential of the xylem was determined from the osmotic potential of xylem sap and from the xylem tension of previously bagged leaves, measured with a pressure chamber. The xylem tension of bagged leaves on a branch adjacent to the site of the direct measurements was considered equivalent to the xylem tension of the trunk at that point. While both the direct and indirect measurements of phloem turgor showed clear diurnal changes, the directly measured pressures were consistently lower than the calculated values. It is not clear at present whether the discrepancy between the two values lies primarily in the calculated or in the measured pressures, and thus, the results from both methods as described here must be regarded as estimates of true phloem turgor. PMID- 16661856 TI - Nitrate Accumulation, Assimilation, and Transport by Decapitated Corn Roots : EFFECTS OF PRIOR NITRATE NUTRITION. AB - The effects of accumulated [(14)N]nitrate and its utilization in decapitated, 5 day-old dark-grown corn roots on influx, accumulation, xylem deposition, and reduction of concurrently absorbed nitrate during an 18-hour exposure to 0.5 millimolar K(15)NO(3) nutrient solution were examined. A 20-hour pretreatment in 15.0 millimolar K(14)NO(3) high nitrate (HN) resulted in a 2-fold greater tissue nitrate level than pretreatment in 0.5 millimolar K(14)NO(3) low nitrate (LN). Upon transfer to the 0.5 millimolar K(15)NO(3) solution, the net nitrate uptake rate in HN roots after 2 hours was 52% of the LN rate, but increased to 93% at the end of the uptake period. Despite an enhanced [(14)N]nitrate efflux from HN roots to the uptake solution, the efflux differences between the two pretreatments did not compensate for the decrease in net nitrate uptake. The [(15)N]nitrate influx rate was initially restricted by 33% in the HN roots compared to LN roots, but it had decreased to 7% by the end of the 18-hour uptake period. At this time, the total tissue nitrate levels were similar for both pretreatments. The rate of accumulation of [(15)N]nitrate in the tissue was relatively constant for both pretreatments, but was 25% less in HN roots. Of the previously accumulated [(14)N]nitrate, 52 and 46% remained after 18 hours in the LN and HN roots, respectively. The [(14)N]nitrate decline for HN roots was initially more rapid than in the LN roots which was linear over time. Xylem transport and efflux more than accounted for the decline in [(14)N]nitrate of LN roots and all but 4% in the HN roots which was attributed to reduction. Compartmentation of the previously accumulated nitrate was evident from the higher atom per cent (15)N of xylem nitrate compared to that of the tissue nitrate of both LN and HN roots. During the first 2 hours, xylem transport of [(14)N]nitrate by the HN roots was 49% greater than for LN roots, while [(15)N]nitrate transport was 9% less in HN roots compared to LN roots. Even though the reduction of [(15)N]nitrate in HN roots was 31% less than LN roots during the first 2 hours, [(15)N]nitrate was reduced more rapidly than the previously accumulated [(14)N]nitrate. After the first 4 hours, the relative partitioning of absorbed [(15)N]nitrate between accumulation, reduction, and translocation was similar regardless of pretreatment. PMID- 16661855 TI - Protein and nitrate content of lemna sp. As a function of developmental stage and incubation temperature. AB - Lemna protein per frond and per root increases with developmental stage until plants are at least two generations old. Protein per frond, per root, and per unit dry weight is greater in plants grown at 23.9 C than at 18.3 C. More protein is found in fronds than in roots, and more nitrate occurs in roots than in fronds. Nitrate per root increases with developmental stage and is higher (per root) in plants grown at 23.9 C than in those grown at 18.3 C. The distribution of generations within a growing population is constant for at least eight doubling times. Whether populations multiply slowly at 15.6 C or more rapidly at 23.9 C, fronds which have not yet produced progeny form 62% of the population; fronds which are one generation old form 24% of the population; and fronds which are two generations old form 9% of the population. PMID- 16661857 TI - Carbon metabolism in two species of pereskia (cactaceae). AB - The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor (14)CO(2) and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO(2) uptake pattern indicating C(3) carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO(2) recycling ("idling") that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO(2) uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested. PMID- 16661858 TI - Inhibition of nitrogen fixation in alfalfa by arsenate, heavy metals, fluoride, and simulated Acid rain. AB - The acute effects of aqueous solutions of As, Cd, Cu, Pb, F, and Zn ions at concentrations from 0.01 to 100 micrograms per milliliter and solutions adjusted to pH 2 to 6 with nitric or sulfuric acid were studied with respect to acetylene reduction, net photosynthesis, respiration rate, and chlorophyll content in Vernal alfalfa (Medicago sativa L. cv. Vernal). The effects of the various treatments on acetylene reduction varied from no demonstrable effect by any concentration of F(-) and 42% inhibition by 100 micrograms Pb(2+) per milliliter, to 100% inhibition by 10 micrograms Cd(2+) per milliliter and 100 micrograms per milliliter As, Cu(2+), and Zn(2+) ions. Zn(2+) showed statistically significant inhibition of activity at 0.1 micrograms per milliliter. Acid treatments were not inhibitory above pH 2, at which pH nitric acid inhibited acetylene reduction activity more than did sulfuric acid. The inhibition of acetylene reduction by these ions was Zn(2+) > Cd(2+) > Cu(2+) > AsO(3) (-) > Pb(2+) > F(-). The sensitivity of acetylene reduction to the ions was roughly equal to the sensitivity of photosynthesis, respiration, and chlorophyll content when Pb(2+) was applied, but was 1,000 times more sensitive to Zn(2+). The relationship of the data to field conditions and industrial pollution is discussed. PMID- 16661859 TI - Temperature-induced leakage from chilling-sensitive and chilling-resistant plants. AB - Leakage rates were determined from leaf cells loaded with rubidium and [(3)H]leucine. There was a differential response between leucine and rubidium leakage depending upon the species used. The rate of leucine leakage shows a small decline below 5 C for two altitudinal variants of Lycopersicon hirsutum Humb. and Bonpl., whereas Lycopersicon esculentum L. showed a marked increase below 5 C. Rubidium showed a marked increase in leakage rate below 10 C with the altitudinal variants, with only a slight increase for the L. esculentum species. A rough relationship existed between rubidium leakage rate at 1 C and the altitude of origin of the L. hirsutum race, the low altitudinal forms having higher leakage rates than the higher altitudinal variants. The L. esculentum lines show a rubidium leakage response similar to that of the high altitude L. hirsutum variants. Higher leakage rates were obtained if the calcium concentration in the medium was less than 1 millimolar and upon addition of metabolic poisons and detergents.The results are consistent with the view that chilling injury causes changes in the membrane and that cell leakage is an early symptom of this change in some species. Some chilling-sensitive species have increased leakage within 1 hour of exposure to chilling temperature. PMID- 16661860 TI - Studies in Wild Oat Seed Dormancy: II. ACTIVITIES OF PENTOSE PHOSPHATE PATHWAY DEHYDROGENASES. AB - A selected strain of wild oat (Avena fatua L.) seed has been shown to lose dormancy rapidly during moist soil incubation at 25 C, whereas seed kept similarly at 5 C maintained a high level of dormancy.The activities of cytosolic dehydrogenase enzymes of the pentose phosphate pathway were assayed throughout a period of moist soil incubation at these two temperatures. A distinction was made between extractable dehydrogenases from the embryo and the endosperm regions of the caryopsis.Dehydrogenase activities monitored in seeds incubated at 25 C gradually increased over the course of the investigation. The largest increases in activity occurred during incubation at 5 C, the situation in which dormancy is maintained. No obvious connection could be found between dormancy breakage and increased activity of the pentose phosphate pathway dehydrogenases. PMID- 16661861 TI - Alteration of Coding Properties of Polysome-Associated Messenger RNA in Potato Tuber Slices during Aging. AB - Kinetics of polysome formation, translational capacity, and coding properties of polysome-associated messenger RNA were investigated in potato tuber tissue discs during aging. Polysome content rapidly increased immediately after slicing from 14% of total ribosomes in freshly sliced discs to 55% within 12 hours of aging. The amount of polysomal RNA also increased 5-fold during this period. Translational capacity of polysome-associated messenger RNA increased in parallel with the increase in content of polysomal RNA of the tissue discs when measured in a wheat germ cell-free system. Analysis of the in vitro translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the majority of polypeptides coded by the messenger RNA did not vary greatly during the period of rapid polysome formation. Three types of messenger RNA were found to change in amount during that period: those which appeared only after aging, those which disappeared during aging, and those which disappeared early but reappeared later in the aging period. PMID- 16661862 TI - pH-Dependent Interactions between Pea Cell Wall Polymers Possibly Involved in Wall Deposition and Growth. AB - In an effort to detect a pH-dependent release of polymers such as xyloglucans, thought to be involved in auxin-induced cell wall expansion during growth, radioactively labeled cell walls from pea stem tissue were incubated at different pH values, and changes in water-soluble, ethanol- or trichloroacetic acid insoluble components were determined. This revealed the occurrence, at neutral pH, of a time- and pH-dependent binding of soluble pectin, in the walls, to a heat-labile, presumably protein, wall component, yielding a trichloroacetic acid insoluble pectin-protein complex. This reaction, which can also be observed between polymers in water extracts of cell walls, is inhibited at low pH and by Ca(2+), and appears to be of a physical, possibly lectin-like, nature. Progressive binding of pectin or of the pectin-protein complex to the insoluble wall structure is also observed. These reactions may be involved in wall assembly during its deposition, and may participate in, or be analogous to pH-dependent physical interactions that participate in, wall extension during cell growth. PMID- 16661863 TI - Anaerobic Metabolism in Germinating Seeds of Echinochloa crus-galli (Barnyard Grass) : METABOLITE AND ENZYME STUDIES. AB - Echinochloa crus-galli, a problem weed in rice fields, has the rare ability to germinate and to grow in a totally oxygen-free environment. After 7 days growth in the light or dark under N(2), E. crus-galli var. oryzicola produces a 2- to 3 centimeter nonpigmented shoot.Ethanol, malate, and lactate were measured in seeds germinated under N(2) and air, and compared with changes in the activities of alcohol dehydrogenase, malate dehydrogenase, NADP-malic enzyme, and phosphoenolpyruvate carboxylase. During a 7-day time course, ethanol levels increased 661 micromoles per gram dry weight under N(2), with no increase under air. Alcohol dehydrogenase activity increased 5.5 micromoles per gram dry weight per minute in N(2) compared to 1.0 in air. Corresponding increases for lactate were 7.9 micromoles per gram dry weight under N(2) and 2.7 under air, and for malate, 5.3 micromoles per gram dry weight under N(2) and 0.4 under air.Although 85% of the ethanol produced by the seedlings was found in the external solution under N(2), the seeds still contained 90 times more ethanol under anaerobic conditions than under air. No phosphoenolpyruvate carboxykinase activity was detected and phosphoenolpyruvate carboxylase activity was 10 times less under N(2) than air. Malic enzyme activity increased 5-fold under anaerobic conditions, comparable to the change under aerobic conditions. Proposed adaptive mechanisms of flood-tolerant species to anaerobiosis are discussed. PMID- 16661864 TI - Effects of Abscisic Acid on the Hydraulic Conductance of and the Total Ion Transport through Phaseolus Root Systems. AB - The response of solute and volume fluxes of Phaseolus root systems to applied AbA was observed under conditions of applied pressure which were used to enhance the volume flow. The growth regulator elicited three separate responses: a transient release of solutes to the xylem which was responsible for an initial increase in volume flux; a long term increase in the total ion flux; and a long term decrease in the hydraulic conductance of the root systems. The exact response was highly dependent on the magnitude of the pre-AbA volume flux density, the relative contributions of osmotic and pressure-induced flow, and the applied dosage.Calculations suggested that the volume flux in naturally exuding root systems is relatively insensitive to changes in the conductance. PMID- 16661865 TI - Phase-sequence of redroot pigweed seed germination responses to ethylene and other stimuli. AB - Phase-sequence studies showed that light, ethylene, and high temperature each enhanced germination of redroot pigweed (Amaranthus retroflexus L.) seeds when given during the first 24 hours of seed imbibition. Responses were maximal during the first 12 hours. After 48 hours all three stimuli given together caused 75% germination but each alone was ineffective. The main influence of water potential on seed germination occurred at about 24 hours, but the influence of CO(2) extended into the second and third days. Germination was reduced by water stress (-4 bars) or CO(2)-free air, but ethylene reversed the reduction even when administered after several days incubation. This suggested that environmental and hormonal factors affected redroot pigweed seeds at two distinct stages in the sequence of germination events. PMID- 16661866 TI - Phosphatidylinositol synthesis by a mn-dependent exchange enzyme in castor bean endosperm. AB - myo-Inositol is incorporated into phosphatidylinositol by an exchange reaction associated with the endoplasmic reticulum fraction isolated from post-germination castor bean endosperm. The reaction requires Mn(2+), has a pH optimum of 8.0, an apparent K(m) for myo-inositol of 26 micromolar, and is stimulated about 15-fold by certain cytidine derivatives. The cytidine derivatives appear to be converted to CMP, which may be the only active stimulator. These optimal exchange reaction conditions, both with and without CMP, differ from those for cytidine-5' diphosphodiglyceride: myo-inositol transferase (EC 2.7.8), so the exchange does not appear to be a reversal of the transferase. This conclusion is augmented by the low rates of CDP-diglyceride formation from cytidine derivatives when compared to the high rate of myo-inositol incorporation into phosphatidylinositol in the presence of the same cytidine derivatives and identical reaction conditions. PMID- 16661867 TI - Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XII. PURIFICATION AND PROPERTIES OF PRINCIPAL STORAGE PROTEINS. AB - The two principal storage proteins of the cottonseed have been purified and partially characterized. They have apparent molecular weights of 52,000 and 48,000 and are both comprised of sets of isoelectric isomers. Both sets have nearly identical solubility properties, ultraviolet absorption spectra, and contain similar proportions of amino acid residues. Furthermore, both sets share antigenic determinants suggesting their origin from a common ancestral gene. However, only the 52,000 set appears to be glycosylated. PMID- 16661868 TI - Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XIII. REGULATION OF BIOSYNTHESIS OF PRINCIPAL STORAGE PROTEINS. AB - The synthesis of the principal cottonseed storage proteins during embryogenesis has been followed by analyses of stained protein gels and of fluorographs of protein synthesized in vivo and from purified RNA in vitro in the wheat germ system. The kinetics of in vivo labeling as well as immunochemical cross reactivity indicate that the 52- and 48-kilodalton mature storage protein sets are derived from 70- and 67-kilodalton precursor protein sets that are abundant proteins in embryonic cotyledons and disappear in late embryogenesis. Identification of the initial translation products of the storage protein mRNA has not been clearly established although products of apparent molecular weights of 69,000 and 60,000 are the likely storage protein precursors.Storage protein synthesis falls off markedly in late embryogenesis simultaneously with the loss of a superabundant class of mRNAs (shown by cDNA:RNA reassociation) that are presumed to be those for the storage proteins. The synthesis of these proteins ceases abruptly when immature embryos are removed from the boll and allowed to germinate precociously or when this precocious germination is prevented by incubation in abscisic acid. Thus, abscisic acid is not implicated in the expression of the storage protein genes.A scheme involving co-translational processing into vesicles, glycosylation, and slow in situ cleavage to produce the mature storage proteins is proposed. PMID- 16661869 TI - Differences in Endogenous Levels of Gibberellin-Like Substances in Nodules of Phaseolus lunatus L. Plants Inoculated with Two Rhizobium Strains. AB - Lima bean plants (Phaseolus lunatus L.) inoculated with Rhizobium sp. strain 127E14, which lacks constitutive nitrate reductase activity, were significantly taller after 4 weeks of age than plants inoculated with strain 127E15, which contains constitutive nitrate reductase activity. Plants inoculated with either strain responded to application of 5 micrograms gibberellic acid per plant with rapid internode elongation; plants inoculated with strain 127E15 became less responsive to gibberellic acid from 3 to 5 weeks of age, while plants inoculated with strain 127E14 did not. The height of plants inoculated with strain 127E14 was reduced by 20% with application of gibberellin biosynthesis inhibitors to the roots, while height of plants inoculated with strain 127E15 was unaffected.Plants inoculated with strain 127E14 developed nodules containing 4 to 50 times greater quantities of extractable gibberellin-like substances than the nodules of plants inoculated with strain 127E15. Plants inoculated with strain 127E14 had nodules containing larger quantities of gibberellin-like substances in both the acidic and basic ethyl acetate fractions, and these differences were observed before the onset of rapid growth responsible for the difference in plant height.The internode elongation in plants infected with strain 127E14, its reversal by gibberellin biosynthesis inhibitors, and the accumulation of large quantities of gibberellin-like substances in nodules infected with strain 127E14 support the hypothesis that root nodules may contribute gibberellins to the host. PMID- 16661870 TI - Carbon Dioxide Exchange in Lichens : RELATIONSHIP BETWEEN NET PHOTOSYNTHETIC RATE AND CO(2) CONCENTRATION. AB - The relationship between net photosynthesis and CO(2) concentration was investigated for four species of lichen using an infrared gas analyzer operating in a closed loop system. All species showed a linear relationship at low CO(2) levels (100 microliters per liter) with CO(2) saturation levels being in excess of 400 microliters per liter. Detailed studies of Sticta latifrons showed a strong influence of thallus water content which resulted in the net photosynthetic response at high water contents still being nearly linear at 1000 microliters per liter CO(2). Very low CO(2) compensation values (5 microliters per liter) were obtained under some conditions but the value varied between thalli and with thallus water content. The results differ from previous studies which reported low CO(2) saturation levels (200 microliters per liter) and no apparent effect of water content. It is suggested that some of these differences may result from the use of a discrete sampling injection infrared gas analyzer system in the earlier studies and an assessment is made of the influence of nonsaturating CO(2) levels, lack of cuvette ventilation, and data presentation for this technique. PMID- 16661871 TI - Uptake and Release of Abscisic Acid by Isolated Photoautotrophic Mesophyll Cells, Depending on pH Gradients. AB - Uptake and release of abscisic acid (AbA) by isolated mesophyll cells of Papaver somniferum is characterized by the following observations: (a) Uptake rate is a linear function of the external AbA concentration in the range from 10(-6) to 5 x 10(-5) molar, and decreases with increasing pH. At any pH, uptake rate is linearly related to the concentration of undissociated abscisic acid, calculated from the pK = 4.7 according to the Henderson-Hasselbalch equation. At low external pH (5.0), AbA accumulation in the cells is about 10-fold. (b) Uptake of AbA is completely inhibited by salts such as KNO(2) or sodium acetate, which decrease the pH gradient between medium and cells. KCN or m-chlorocarbonylcyanide phenylhydrazone inhibits AbA uptake only after longer incubation periods (20-40 minutes). (c) Uptake rate as well as equilibrium concentration is significantly higher in light than in darkness. (d) At low external pH, release of AbA from preloaded cells is strongly stimulated by KNO(2). It is concluded that AbA is distributed between leaf cells and free space according to pH gradients, with the undissociated abscisic acid being the main penetrating species. Uptake and release occur via diffusion, without participation of a carrier. PMID- 16661872 TI - Host-Pathogen Interactions: XVI. PURIFICATION AND CHARACTERIZATION OF A beta GLUCOSYL HYDROLASE/TRANSFERASE PRESENT IN THE WALLS OF SOYBEAN CELLS. AB - The fact that fungal glucans will stimulate soybeans to accumulate phytoalexins prompted an investigation of soybean cell beta-1,3-glucanases and beta glucosidases, as well as the ability of these enzymes to hydrolyze the fungal glucans. Several beta-1,3-glucanases and beta-glucosidases can be solubilized from the walls of suspension-cultured soybean cells by treatment with 1.0 molar sodium acetate buffer. An enzyme, which has been termed beta-glucosylase I, is the dominant beta-1,3-glucanase in the cell wall extracts. Utilizing CM-Sephadex chromatography, hydroxylapatite chromatography, and affinity chromatography, beta glucosylase I has been purified 71-fold, with 39% recovery, from the mixture of cell wall enzymes. The affinity chromatography column material was prepared by covalently attaching p-aminophenyl-1-beta-d-glucopyranoside, an analog of a beta glucosylase I substrate, to Sepharose. beta-Glucosylase I, purified by this procedure, yields a single band on isoelectric focusing gels (pH 8.9). However, the purified beta-glucosylase I yields a darkly-staining protein band at an apparent molecular weight of 69,000 and several lightly-staining protein bands in sodium dodecyl sulfate polyacrylamide gels. Additional purification procedures fail to remove these lightly-staining protein bands.beta-Glucosylase I will hydrolyze the beta-glucan substrates, laminarin (3-linked) and lichenan (3- and 4 linked), and therefore, possesses beta-glucanase activity. Studies of the progressive hydrolysis of laminarin by beta-glucosylase I demonstrate that the enzyme hydrolyzes polysaccharide substrates in an exo manner. beta-Glucosylase I will also hydrolyze a variety of low molecular weight beta-glucosides including various beta-linked diglucosides. Thus, beta-glucosylase I also possesses beta glucosidase activity.Several lines of evidence are presented that the beta glucanase and the beta-glucosidase activities exhibited by purified beta glucosylase I preparations are catalyzed by the same enzyme. This evidence includes inhibition studies which indicate that the beta-glucanase and the beta glucosidase activities of beta-glucosylase I are catalyzed at the same active site. beta-Glucosylase I will also catalyze glucosyl transfer. This catalytic activity is responsible for the observed ability of the enzyme to synthesize di- and trisaccharides from laminarin. The disaccharides formed by beta-glucosylase I catalyzed transglucosylation are the beta-anomers of the 6-, 4-, 3-, and 2-linked diglucosides in the relative proportions of 10:1:1:1. The ability of beta glucosylase I to catalyze glucosyl transfer indicates that beta-glucosylase I is biochemically more similar to previously studied beta-glucosidases than to beta glucanases. This conclusion is supported by the observation that beta-glucosylase I is strongly inhibited by 1,5-d-gluconolactone, an inhibitor of beta glucosidases but not of beta-glucanases. PMID- 16661873 TI - Host-Pathogen Interactions : XVII. HYDROLYSIS OF BIOLOGICALLY ACTIVE FUNGAL GLUCANS BY ENZYMES ISOLATED FROM SOYBEAN CELLS. AB - The ability of beta-glucosylase I, a soybean cell wall beta-glucosyl hydrolase, to degrade elicitors of phytoalexin accumulation was studied. Extensive beta glucosylase I treatment of the glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae results in hydrolysis of 77% of the glucosidic bonds of the elicitor and destruction of 94% of its activity. Soybean cell walls contain some additional factor, probably one or more additional enzymes, which can assist beta-glucosylase I in hydrolyzing the glucan elicitor. This was demonstrated by the more rapid hydrolysis of the glucan elicitor by a mixture of soybean cell wall enzymes (containing beta-glucosylase I). In a single treatment, the mixture of cell wall enzymes hydrolyzed 91% of the glucosidic bonds and destroyed 85% of the activity of the elicitor. The enzymes from soybean cell walls will also hydrolyze elicitor-active oligoglucosides prepared from the mycelial walls of Phytophthora megasperma var. sojae. The active oligoglucosides are more susceptible than the glucan elicitor to hydrolysis by these enzymes. The mixture of cell wall enzymes or beta-glucosylase I, by itself, hydrolyzes more than 96% of the glucosidic bonds and destroys more than 99% of the activity of the oligoglucoside elicitor. Two possible advantages for the existence of these enzymes in the walls of soybean cells are discussed. PMID- 16661874 TI - Reaction of Mixed Valence State Cytochrome Oxidase with Oxygen in Plant Mitochondria: A STUDY BY LOW TEMPERATURE FLASH PHOTOLYSIS AND RAPID WAVELENGTH SCANNING OPTICAL SPECTROMETRY. AB - The reaction of mixed valence state cytochrome oxidase (Cu(A) (2+)a(3+) . Cu(B) (+)a(3) (2+)) with O(2) at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The "pure" difference spectra of the two species, I(M) and II(M), relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I(M) is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II(M) by an alpha band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O(2) (-) --> Cu(B) (2+) and O(2) (-) --> a(3) (2+) charge transfer transitions which would imply that O(2) (-) forms a bridging ligand between Cu(B) and the iron atom of cytochrome a(3) in species II(M). The kinetics of the reaction and the spectral characteristics of species I(M) and II(M) obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system. PMID- 16661875 TI - Adenylate and nicotinamide nucleotides in developing soybean seeds during seed fill. AB - Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity.Of the oxidized (NAD, NADP) and reduced (NADH, NADPH) nicotinamide nucleotide forms, NAD was the most abundant. Levels as high as 17.5 micrograms per seed were observed during the mid-seed filling stages. NADP was found almost exclusively in the reduced form with a NADP: NADPH ratio of less than 0.35, whereas the reverse was noted for NAD which was found mainly in the oxidized form with a NAD:NADH ratio in the range of 5 to 25. NADP was detected in low concentrations compared to the other adenylate and nicotinamide nucleotides. The nicotinamide redox charge defined as (NADH + NADPH)/(NAD + NADH) + (NADP + NADPH) was calculated to express the state of the energy balance between the oxidized and reduced nicotinamide nucleotide forms. The nicotinamide redox charge varied between 0.15 and 0.30 during seed development and was significantly lower than that found for the adenylate energy charge. PMID- 16661876 TI - Light-Induced Proton Gradient Formation in Intact Cells of Dunaliella salina. AB - A light-induced proton gradient (DeltapH) increase as exhibited by an increase of 9-aminoacridine fluorescence quenching is demonstrated between the external medium and the interior of the halophytic green alga Dunaliella salina. The formation and maintenance of the DeltapH is sensitive to electron transport inhibitors and to uncouplers. It is inhibited by p-chloromercuribenzenesulfonic acid (50% inhibition at 3 micromolar), which does not affect photosynthetic O(2) evolution. It is concluded that the observed DeltapH is located across the plasmalemma or the chloroplast envelope. The formation and maintenance of the light-induced proton gradient requires the presence of Na(+). Substitution of NaCl by KCl or glycerol results in inhibition of the DeltapH formation. The proton gradient is also sensitive to ATPase and energy transfer inhibitors. It is suggested that a Na(+)/H(+) pump mechanism may be involved in the formation of the proton gradient in intact Dunaliella cells. PMID- 16661877 TI - Alternative path mediated chloride absorption in cyanide-resistant tissues. AB - The ability of two cyanide-resistant tissues-aged potato slices and fresh preclimacteric banana slices-to take up chloride in the presence of cyanide has been established. Extensive inhibition of chloride uptake by cyanide and chlorbenzhydroxamate together indicates that chloride absorption in the presence of cyanide is supported by respiration mediated by the alternative path. The partial inhibition of respiration-dependent chloride uptake by chlorbenzhydroxamate alone is independent of its effect on the alternative oxidase, and points to inhibition of the transport process per se. PMID- 16661878 TI - Kinetics of Adaptation to Osmotic Stress in Lentil (Lens culinaris Med.) Roots. AB - When intact roots of lentil (Lens culinaris Med.) are subjected to severe osmotic stress by treatment with a solution of low water potential, they immediately begin to shrink. Within 10 to 15 minutes, shrinkage ceases, and within 20 minutes, the roots resume growth. The time lag between application of osmoticum and resumption of growth varies from about 10 to 30 minutes over the range of external water potentials of -2 to -12.4 bars. For external water potentials as low as -8.7 bars the new steady rate of growth in the presence of osmoticum is approximately equal to that prevailing before application of osmoticum. For external water potentials between -8.7 and -13 bars growth resumes, but the new rate is less than that prior to addition of osmoticum. Measurements of changes in the internal solute content during adaptation show that the solute content of the root increases but that the magnitude of the increase is, by itself, insufficient to account for the resumption of rapid growth. PMID- 16661879 TI - Influence of cold acclimation on membrane injury in frozen plant tissue. AB - Cold-acclimated twigs of Amelanchier alnifolia Nutt. released less HCN at -4.5 C than nonacclimated twigs following slow freezing to -25 C or rapid freezing to 78 C. Cold-acclimated twigs frozen slowly to -25 C released more HCN than cold acclimated twigs frozen only to -4.5 C. Cold-acclimated twigs frozen slowly to 25 C and then rapidly to -78 C released less HCN at -4.5 C than cold-acclimated twigs frozen rapidly to -78 C. In general, K(+) efflux and the inability to reduce triphenyl tetrazolium chloride following freezing and thawing paralleled HCN release at -4.5 C. Because low K(+) efflux and high triphenyl tetrazolium chloride reduction are known to depend upon membrane integrity, the increased K(+) efflux and the decreased triphenyl tetrazolium chloride reduction following freezing and thawing provide indirect evidence that HCN release at -4.5 C is a measure of membrane damage in frozen cells. PMID- 16661880 TI - Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast. AB - Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO(2). Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O(2) is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO(2) required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO(2), the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3 phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.In contrast, incubation of purified ribulose bisphosphate carboxylase with ribulose bisphosphate irreversibly inhibited activation, especially in the absence of CO(2). Addition of the same amount of ribulose bisphosphate to lysed chloroplasts did cause some deactivation of the carboxylase in the extract, but full activation returned when the ribulose bisphosphate was consumed. The ribulose bisphosphate carboxylase in the chloroplast is not irreversibly inhibited by high levels of ribulose bisphosphate. PMID- 16661881 TI - Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : I. DETERMINATION OF THE ROLE OF METHIONINE AS THE PRECURSOR OF ETHYLENE. AB - The hypersensitive reaction of Samsun NN tobacco leaves to tobacco mosaic virus (TMV) was accompanied by a large increase in ethylene production, just before necrotic local lesions became visible. Normal and virus-induced ethylene production were both largely inhibited by 0.1 millimolar aminoethoxyvinylglycine indicating that methionine is a main ethylene precursor.The contribution of methionine to ethylene production was estimated by labeling leaves with l-[U (14)C]methionine and comparing the specific activities of methionine within and ethylene produced by the leaf. When taken up through the petiole, methionine was largely retained in the veins, leading to production of ethylene with a far higher specific activity in the veins than in the interveinal tissue. After TMV infection, ethylene production increased only in the interveinal tissue, resulting in a decrease in specific activity of the ethylene produced. In the interveinal tissue, the specific radioactivity of the ethylene was lower than expected if methionine were the only precursor. After labeling by vacuum infiltration, the specific activities of the ethylene produced by water- and TMV inoculated leaves were both identical and in accordance with the specific radioactivity of methionine. Inasmuch as the content of 1-aminocyclopropane-1 carboxylic acid was increased severalfold two days after TMV infection, methionine can be considered to be the only ethylene precursor in healthy and in TMV-infected tobacco leaves.The increase in ethylene production after TMV infection was not accompanied by an increased concentration of free methionine within the leaf. Compartmentation of methionine does not appear to be a regulating factor since labeled methionine supplied to the leaf by vacuum infiltration is equilibrated very rapidly with any methionine pool within the leaf cells. PMID- 16661882 TI - Cycloheximide resistance in carrot culture: a differentiated function. AB - Cultured carrot cells grow as unorganized callus tissue in medium containing auxin. Upon removal of the auxin from the medium, they grow in an organized manner and differentiate into embryos. In the normal cell line, W001C, the callus growth can be inhibited by cycloheximide, but the embryonic growth cannot. A variant cell line, WCH105, whose callus growth is resistant to cycloheximide, was isolated. The mechanism of cycloheximide resistance in embryos of both lines and in WCH105 callus was found to be cycloheximide inactivation. In addition to auxin, bromodeoxyuridine can also promote callus growth in carrot culture. Callus cultures maintained by bromodeoxyuridine behave the same as do those maintained by auxin. WCH105 callus is resistant, whereas W001C callus is sensitive to cycloheximide inhibition. Except for the onset of embryogenesis, cycloheximide inactivation is expressed throughout the embryo developmental stages up to the plantlets. These results suggest that cycloheximide inactivation is a function expressed in the differentiated, but not in the undifferentiated, tissues. PMID- 16661883 TI - Limitations on the Utilization of Glycolate by Chlamydomonas reinhardtii. AB - Growth and shorter term incorporation measurements with both wild type Chlamydomonas reinhardtii and a mutant (F-60, lacking phosphori-bulokinase activity) indicate that the rate of glycolate utilization is always relatively low. Growth support with external glycolate is restricted to cells with full photosynthetic capacity. A high concentration of glycolate is required for optimal growth support and incorporation of [(14)C]glycolate. Glycolate incorporation is low at pH 3.8 even with the relatively free permeability. The F 60 mutant can take up only small quantities of glycolate in spite of photosynthetic electron transport and photophosphorylation competencies. This requirement for photosynthetic carbon metabolism indicates a significant difference in the glycolate pathway of this alga. No growth condition significantly increases glycolate incorporation rates. There is no evidence that one of the primary enzymes, glycolate dehydrogenase, is limiting utilization; measurements of glycolate uptake and excretion do not always correlate with its activity. Since the maximal utilization rate of glycolate is low, control of glycolate formation is important in preventing the loss of this fixed carbon from the algal cell. PMID- 16661884 TI - Separation and measurement of direct and indirect effects of light on stomata. AB - Conductance for water vapor, assimilation of CO(2), and intercellular CO(2) concentration of leaves of five species were determined at various irradiances and ambient CO(2) concentrations. Conductance and assimilation were then plotted as functions of irradiance and intercellular CO(2) concentration. The slopes of these curves allowed us to estimate infinitesimal changes in conductance (and assimilation) that occurred when irradiance changed and intercellular CO(2) concentration was constant, and when CO(2) concentration changed and irradiance was constant. On leaves of Xanthium strumarium L., Gossypium hirsutum L., Phaseolus vulgaris L., and Perilla frutescens (L.), Britt., the stomatal response to light was determined to be mainly a direct response to light and to a small extent only a response to changes in intercellular CO(2) concentration. This was also true for stomata of Zea mays L., except at irradiances < 150 watts per square meter, when stomata responded primarily to the depletion of the intercellular spaces of CO(2) which in turn was caused by changes in the assimilation of CO(2).Stomata responded to light even in leaves whose net exchange of CO(2) was reduced to zero through application of the inhibitor of photosynthetic electron transport, cyanazine (2-chloro-4[1-cyano-1 methylethylamino]-6-ethylamino-S-triazine). When leaves were inverted and irradiated on the abaxial surface, conductance decreased in the shaded and increased in the illuminated epidermis, indicating that the photoreceptor pigment(s) involved are located in the epidermis (presumably in the guard cells). In leaves of X. strumarium, the direct effect of light on conductance is primarily a response to blue light.Stomatal responses to CO(2) and to light opposed each other. In X. strumarium, stomatal opening in response to light was strongest in CO(2) free air and saturated at lower irradiances than in CO(2) containing air. Conversely, stomatal closure in response to CO(2) was strongest in darkness and it decreased as irradiance increased. In X. strumarium, P. vulgaris, and P. frutescens, an irradiance of 300 watts per square meter was sufficient to eliminate the stomatal response to CO(2) altogether. Application of abscisic acid, or an increase in vapor pressure deficit, or a decrease in leaf temperature reduced the stomatal conductance at light saturation, but when the data were normalized with respect to the conductance at the highest irradiance, the various curves were congruent. PMID- 16661885 TI - Hypocotyl Growth and Peroxidases of Bidens pilosus: EFFECT OF COTYLEDONARY PRICKINGS AND LITHIUM PRETREATMENT. AB - Pricking one cotyledon of young Bidens pilosus plants induces rapid inhibition of hypocotyl growth, essentially in its middle portion. Analysis of soluble peroxidases indicates rapid changes (increase of activity) in basic isoenzymes followed by more progressive enhancement of the acidic ones. Pretreatment of the plants with lithium prevents the inhibition of elongation due to pricking as well as the peroxidase changes. The phenomenon is similar to the previously described thigmomorphogenetic process in Bryonia dioica. PMID- 16661886 TI - A split-root technique for measuring root water potential. AB - Water encounters various resistances in moving along a path of decreasing potential energy from the soil through the plant to the atmosphere. The reported relative magnitudes of these pathway resistances vary widely and often these results are conflicting. One reason for such inconsistency is the difficulty in measuring the potential drop across various segments of the soil-plant-atmosphere continuum. The measurement of water potentials at the soil-root interface and in the root xylem of a transpiring plant remains a challenging problem.In the divided root experiment reported here, the measured water potential of an enclosed, nonabsorbing branch of the root system of young corn (Bonanza) plants to infer the water potential of the remaining roots growing in soil was used. The selected root branch of the seedling was grown in a specially constructed Teflon test tube into which a screen-enclosed thermocouple psychrometer was inserted and sealed to monitor the root's water potential. The root and its surrounding atmosphere were assumed to be in vapor equilibrium. PMID- 16661887 TI - Heterotrophic n(2) fixation and distribution of newly fixed nitrogen in a rice flooded soil system. AB - Rice (Oryza sativa L.) plants growing in pots of flooded soil were exposed to a (15)N(2)-enriched atmosphere for 3 to 13 days in a gas-tight chamber. The floodwater and soil surface were shaded with a black cloth to reduce the activity of phototrophic N(2)-fixing micro-organisms. The highest (15)N enrichments were consistently observed in the roots, although the total quantity of (15)N incorporated into the soil was much greater. The rate of (15)N incorporation into roots was much higher at the heading than at the tillering stage of growth. Definite enrichments were also found in the basal node and in the lower outer leaf sheath fractions after 3 days of exposure at the heading stage. Thirteen days was the shortest time period in which definite (15)N enrichment was observed in the leaves and panicle. When plants were exposed to (15)N(2) for 13 days just before heading and then allowed to mature in a normal atmosphere, 11.3% of the total (15)N in the system was found in the panicles, 2.3% in the roots, and 80.7% in the subsurface soil. These results provide direct evidence of heterotrophic N(2) fixation associated with rice roots and the flooded soil and demonstrate that part of the newly fixed N is available to the plant. PMID- 16661888 TI - Strain-dependent temperature-sensitive phase in crown gall tumorigenesis. AB - The effect of high temperature treatments on the early stages of crown gall tumorigenesis in sunflowers was investigated. Treatments of 32 C initiated at various times during the first ten days after infection had a similar effect on tumors induced by Agrobacterium tumefaciens strains B(6) and C58. Tumor growth was sensitive to 32 C until 60 to 72 hours after infection and was stimulated by 32 C after that time. Therefore, the "inception phase" for both C58 and B(6) induced tumors ends between 60 to 72 hours after infection. In contrast, B(6) and C58 tumors varied in their response to 37 C treatments during the first 168 hours after infection. Both C58 and B(6) tumors were sensitive to 37 C during the first 72 hours; however, B(6) tumors became resistant to 37 C after 96 hours, whereas C58-induced tumors remained sensitive until 144 to 168 hours after infection.The growth rate of C58 and B(6) tumors in plants moved to 32 C at 90 hours after infection was the same. B(6) tumors in plants moved to 37 C at 90 hours after infection also continued to grow rapidly, whereas C58 tumors in plants moved to 37 C at 90 hours exhibited a complete cessation of tumor growth. The 37 C temperature-sensitive period specific to C58 tumors ceases by 168 hours after infection. This indicates that the temperature-sensitive functions are no longer required after 168 hours. The possible significance of this work with regard to the presence of a temperature-sensitive plasmid, pTiC58, in strain C58 and the mechanism of tumor-inducing plasmid DNA maintenance in incipient tumor cells is discussed. PMID- 16661889 TI - Wound-induced membrane lipid breakdown in potato tuber. AB - Freshly cut slices of potato tuber show an extensive loss of membrane lipid components which may be as great as 35% for phospholipids and 30% for glycolipids, in less than 15 minutes at 3 C. Phosphatidyl-choline, phosphatidyl ethanolamine and mono- and di-galactosyl diglycerides comprise the bulk of the lipids that are degraded. Concomitantly, there is a pronounced loss of linoleic and linolenic acids. Whereas degradative events elicited by slicing proceed to a depth of at least 10 millimeters from the surface, phospholipid biosynthesis, as well as the development of the wound induced respiration and cyanide resistance on aging, are restricted to the superficial 1 millimeter. PMID- 16661890 TI - Effect of salt on auxin-induced acidification and growth by pea internode sections. AB - The capacity of excised internode sections of pea to grow and secrete protons in response to indoleacetic acid (IAA) and Ca(2+) and K(+) treatments was examined. By incubating unpeeled and unabraded sections in rapidly flowing solutions, it was shown that acidification of the external medium in the presence or absence of IAA is dependent on the presence of Ca(2+) and K(+). Similar results were obtained when unpeeled and unabraded sections were incubated in dishes with shaking. When peeled or abraded sections were incubated with shaking in IAA, H(+) release was also dependent on the presence of Ca(2+) and K(+). The release of H(+) from sections incubated in Ca(2+) and K(+) is not caused by displacement of H(+) from binding sites in the cell wall. Rather, the release of protons from sections is temperature dependent, and it is concluded that this is a metabolically linked process. Although Ca(2+) and K(+) are essential for the release of H(+) from isolated stem sections of peas, these cations do not influence elongation. Despite the large increase in proton release induced by Ca(2+) and K(+) either in the presence or absence of auxin, growth in the presence of these ions was never greater than it was in their absence. Furthermore, cations do not affect the neutral sugar or uronic acid composition of the solution which can be centrifuged from isolated sections. As is the case for growth, an increase in the neutral sugar and uronide composition of the cell wall solution is dependent only on IAA. It is concluded that IAA-induced growth of pea stem sections is independent of the secretion of protons. PMID- 16661891 TI - Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules. AB - The intracellular location of enzymes involved in the synthesis of the ureides, allantoin and allantoic acid, was investigated in nodules of Glycine max L. Merr. Cellular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 nanomoles per min per gram fresh weight) was totally soluble, whereas approximately 15% of the total uricase and catalase activities (1 and 2000 micromoles per minute per gram fresh weight, respectively) was in the fraction containing intact peroxisomes. Allantoinase activity (680 nanomoles per minute per gram fresh weight) was associated with the microsomal fraction, which apparently originates from the endoplasmic reticulum. PMID- 16661892 TI - Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves. AB - The subcellular location of NADP(+)-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP(+)-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP(+)-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP(+)-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K(m) values of 11 micromolar (NADP(+)), 35 micromolar (isocitrate), 78 micromolar (Mn(2+)), 0.3 millimolar (Mg(2+)) and showed optimum activity at pH 8 to 8.5 with Mn(2+) and 8.8 to 9.2 with Mg(2+). The NADP(+)-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP(+)-isocitrate dehydrogenase activity in the chloroplasts might help supply alpha-ketoglutarate for glutamate synthase action. PMID- 16661893 TI - Inhibition of seagrass photosynthesis by ultraviolet-B radiation. AB - Effects of ultraviolet-B radiation on the photosynthesis of seagrasses (Halophila engelmanni Aschers, Halodule wrightii Aschers, and Syringodium filiforme Kutz) were examined. The intrinsic tolerance of each seagrass to ultraviolet-B, the presence and effectiveness of photorepair mechanisms to ultraviolet-B-induced photosynthetic inhibition, and the role of epiphytic growth as a shield from ultraviolet-B were investigated.Halodule was found to possess the greatest photosynthetic tolerance for ultraviolet-B. Photosynthesis in Syringodium was slightly more sensitive to ultraviolet-B while Halophila showed relatively little photosynthetic tolerance. Evidence for a photorepair mechanism was found only in Halodule. This mechanism effectively attenuated photosynthetic inhibition induced by ultraviolet-B dose rates and dosages in excess of natural conditions. Syringodium appeared to rely primarily on a thick epidermal cell layer to reduce photosynthetic damage. Halophila seemed to have no morphological or photorepair capabilities to deal with ultraviolet-B. This species appeared to rely on epiphytic and detrital shielding and the shade provided by other seagrasses to reduce ultraviolet-B irradiation to tolerable levels. The presence of epiphytes on leaf surfaces was found to reduce the extent of photosynthetic inhibition from ultraviolet-B exposure in all species.Observations obtained in this study seem to suggest the possibility of anthocyanin and/or other flavonoid synthesis as an adaptation to long term ultraviolet-B irradiation by these species. In addition, Halophila appears to obtain an increased photosynthetic tolerance to ultraviolet B as an indirect benefit of chloroplast clumping to avoid photo-oxidation by intense levels of photosynthetically active radiation. PMID- 16661894 TI - Enzyme development and glyoxysome characterization in cotyledons of cotton seeds. AB - Unimbibed, mature cotton seeds (cv. Deltapine 61) were found to possess activity for all gluconeogenesis-related enzymes examined, except for isocitrate lyase activity. This indicates that transcription and translation of most enzymes needed for postgerminative growth takes place during seed maturation. This is in contrast with the generalization that "germination" enzymes are synthesized de novo from previously untranslated mRNAs conserved in dry seeds. All enzyme activities increased 3-fold or greater following imbibition, and most remained constant after reaching their peak. Notable exceptions were activities for three beta oxidation enzymes and fructose bisphosphatase, which decreased precipitously after peaking with other enzyme activities.Standard sucrose gradient procedures with swing-out rotors were not useful for isolating cotton glyoxysomes. Satisfactory and reproducible results ultimately were obtained with sucrose gradients constructed in a Beckman JCF-Z zonal rotor. Specific activities of glyoxysomal enzymes were 2- to 3-fold lower than those reported for other oil seeds, except malate dehydrogenase which was 10-fold lower. Electron microscopy revealed that protein body fragments were the primary contaminant of glyoxysome fractions. Glyoxysomes were subfractionated by osmotic shock treatments to evaluate sub-organelle localization of constituent enzymes, several of which have not been examined in other oil seeds. PMID- 16661895 TI - The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds. AB - Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F --> E --> C --> A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase. PMID- 16661896 TI - Identification of a dihydrophaseic Acid aldopyranoside from soybean tissue. AB - A previously unidentified abscisic acid metabolite has been isolated and characterized. (+/-)-[2-(14)C]Abscisic acid was incubated in intact soybean leaves and pods; the radiolabeled metabolite was purified by high performance liquid chromatography with on-line scintillation spectrometry detection. Gas chromatography-mass spectrometry was used to obtain spectra of the acetylated and methyl esterified derivatives. The data were consistent with a proposed dihydrophaseic acid-aldopyranoside identity. Conjugation through the 4'-hydroxyl of dihydrophaseic acid is suggested. PMID- 16661897 TI - Validation of a radioimmunoassay for indole-3-acetic Acid using gas chromatography-selected ion monitoring-mass spectrometry. AB - A radioimmunoassay for indole-3-acetic acid (IAA) has been validated by comparison with a physico-chemical assay utilizing gas chromatography-selected ion monitoring-mass spectrometry and 4,5,6,7-tetradeutero-indole-3-acetic acid as an internal standard. The radioimmunoassay provided a reliable estimate of the free IAA content of etiolated corn shoots. However, base hydrolysis of extracts for determination of ester IAA released substances which interfered with the radioimmunoassay. Interference was detected by internal controls and by lack of agreement with the mass spectral method. Interfering compounds could be removed from extracts by chromatography on diethylaminoethyl- and hydroxypropylated (lipophilic) Sephadex G-25. Following such purification the radioimmunoassay estimate of the total (free + ester) IAA content of etiolated corn shoots agreed with the mass spectral method within 2% on the average. PMID- 16661898 TI - Metabolism of Monoterpenes : EVIDENCE FOR COMPARTMENTATION OF l-MENTHONE METABOLISM IN PEPPERMINT (MENTHA PIPERITA) LEAVES. AB - Previous studies have shown that the monoterpene ketone l-[G-(3)H]-menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaf discs of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-beta-d-glucoside (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). The metabolic disposition of the epimeric reduction products of the ketone, which is a major constituent of peppermint oil, is highly specific, in that little neomenthyl acetate and little menthyl glucoside are formed. However, when l-[3 (3)H]menthol and d-[3-(3)H]neomenthol are separately administered to leaf discs, both menthyl and neomenthyl acetates and menthyl and neomenthyl glucosides are formed with nearly equal facility, suggesting that the metabolic specificity observed with the ketone precursor was not a function of the specificity of the transglucosylase or transacetylase but rather a result of compartmentation of each stereospecific dehydrogenase with the appropriate transferase. A UDP glucose:monoterpenol glucosyltransferse, which utilized d-neomenthol or l-menthol as glucose acceptor, was demonstrated in the 105,000g supernatant of a peppermint leaf homogenate, and the enzyme was partially purified and characterized. Co purification of the acceptor-mediated activities, and differential activation and inhibition studies, provided strong evidence that the same UDP-glucose-dependent enzyme could transfer glucose to either l-menthol or d-neomenthol. Determination of K(m) and V for the epimeric monoterpenols provided nearly identical values. The acetylcoenzyme A:monoterpenol acetyltransferase previously isolated from peppermint extracts (Croteau, Hooper 1978 Plant Physiol 61: 737-742) was re examined using l-[3-(3)H]menthol and d-[3-(3)H]neomenthol as acetyl acceptors, and the K(m) and V for both epimers were, again, very similar. These results demonstrate that the specific in vivo conversion of l-menthone to l-menthyl acetate and d-neomenthyl-beta-d-glucoside cannot be attributed to the selectivity of the transferases, and they clearly indicate that the metabolic specificity observed is a result of compartmentation effects. PMID- 16661899 TI - Mechanisms of passive potassium influx in corn mitochondria. AB - Corn mitochondria in 100 millimolar KCl show accelerated passive swelling upon addition of uncoupler. This unusual response has been compared with swelling produced by valinomycin, tripropyltin, and nigericin. It is concluded that the driving force for swelling lies with the chloride gradient and a high P(Cl):P(K) ratio, the chloride influx creating a negative membrane potential. The action of uncoupler is to facilitate K(+) influx via the endogenous H(+)/K(+) antiporter. The antiporter is active over the pH range 6 to 8, is not sensitive to Mg(2+) concentration, and is not inactivated by aging. It is not clear why corn mitochondria show this exceptional activity of the H(+)/K(+) antiporter in K(+) influx. It is speculated that during isolation the antiporter may be exposed or activated, and that it contributes to cyclic K(+) transport and high State 4 respiration rates. PMID- 16661900 TI - Conversion of phenylalanine into tyrosine by portulaca callus. AB - The incorporation of [(14)C]phenylalanine and [1,6-(14)C]shikimic acid into tyrosine was investigated in the callus of Portulaca grandiflora, var. JR (L.). By inhibiting phenylalanine with 1-alpha-aminooxy-beta-phenyl-propionic acid and tyrosinase with 1-cysteine-HCl and hydrazine-hydrate, the possible synthesis of tyrosine from phenylalanine was demonstrated. Tetrahydropterine sulfate was an effective activator of this pathway and tyrosine accumulation via 4-hydroxy prephenic acid is regulated by feedback inhibition. l-alpha-Amminooxy-beta phenylpropionic acid inhibits both phenylalanine ammonia-lyase and the production of phenylalanine from prephenic acid. PMID- 16661901 TI - Distribution of iron-containing superoxide dismutase in vascular plants. AB - Superoxide dismutases (EC 1.15.1.1) in vascular plants representing different evolutionary levels were characterized using polyacrylamide gel electrophoresis. The three forms of the enzyme were distinguished from each other based on the following criteria: a) the Cu-Zn enzyme is sensitive to cyanide wherease the Fe and Mn enzymes are not; and b) the Cu-Zn and Fe enzymes are inhibited by H(2)O(2) whereas the Mn enzyme is H(2)O(2)-resistant. Of the 43 plant families investigated, the Fe-containing superoxide dismutase was found in three families: Gingkoaceae, Nymphaceae, and Cruciferae. PMID- 16661902 TI - Light-Induced Release of Bound Glucose-6-phosphate Dehydrogenase to the Stroma in Pea Chloroplasts. AB - Light affects the partitioning of glucose-6-phosphate dehydrogenase between thylakoids and stroma in the chloroplast. Illumination of intact chloroplasts changes the ratio between bound and free enzyme from approximately 1:1 to 1:2. Treatment with NADPH, inorganic phosphate, or high pH also results in release of the enzyme from isolated thylakoids. PMID- 16661903 TI - Two Kinds of Protein Glycosylation in a Cell-Free Preparation from Developing Cotyledons of Phaseolus vulgaris. AB - Membrane preparations from developing cotyledons of red kidney bean (Phaseolus vulgaris L.) transferred radioactive mannose from GDP-mannose (U-[(14)C]mannose) to endogenous acceptor proteins. The transfer was inhibited by the antibiotic tunicamycin, suggesting the involvement of lipidoligosaccharide intermediates typical of the pathway for glycosylation of asparagine residues. This was supported by the similarity of the linkage types of radioactive mannose in lipid oligosaccharide and glycoprotein products; both contained labeled 2-linked mannose, 3,6-linked and terminal mannose typical of glycoprotein "core" oligosaccharides. As expected for "core" glycosylation, the transfer of labeled N acetylglucosamine (GlcNAc) from UDP-GlcNAc (6-[(3)H]GLcNAc) to 4-linkage in endogenous glycoproteins could also be demonstrated. However, most of the radioactive GlcNAc was incorporated into terminal linkage, in a reaction insensitive to tunicamycin. The proteins receiving "core" oligosaccharide in vitro were heterogeneous in size, in contrast to those receiving most of the GlcNAc (which chiefly comprised the seed reserve-proteins phaseolin and phytohemagglutinin). It is suggested that following "core" glycosylation, single GlcNAc residues are attached terminally to the oligosaccharides of these seed proteins, without the involvement of lipid-linked intermediates. Phaseolin from mature seeds does not possess a significant amount of terminal GlcNAc and so it is possible that these residues are subsequently removed in a processing event. PMID- 16661904 TI - Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2). AB - Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO(2) fixation for more than 60 hours. The incorporation of (14)CO(2) under saturating CO(2) conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for gamma-aminobutyric acid. The measurements of specific radio-activities and of the approaches to (14)C "saturation" of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulatory effects in incorporation of (14)C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginine, and histidine. No effect or slight decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, (14)C labeling decreased, but specific radioactivity increased. The production of labeled gamma aminobutyric acid was virtually stopped by ammonia.The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. The data on the effects of added ammonia on total labeling, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis. PMID- 16661905 TI - Enzyme Regulation in C(4) Photosynthesis : PURIFICATION AND PROPERTIES OF THIOREDOXIN-LINKED NADP-MALATE DEHYDROGENASE FROM CORN LEAVES. AB - NADP-malate dehydrogenase, a light-modulated enzyme of C(4) photosynthesis, was purified to homogeneity from leaves of corn. The pure enzyme was activated by thioredoxin m that was reduced either photochemically (with ferredoxin and ferredoxin-thioredoxin reductase) or chemically (with dithiothreitol). Unactivated corn leaf NADP-malate dehydrogenase had a molecular weight of 50,000 to 60,000 and was chromophorefree. The enzyme appeared to have a high content of serine and glycine and to contain both S-S and SH groups. Consequently, NADP malate dehydrogenase seems to be capable of undergoing reversible oxidation/reduction during its photoregulation. PMID- 16661906 TI - Reevaluation of anaerobic nitrite production as an index for the measurement of metabolic pool of nitrate. AB - The use of anaerobic nitrite production as an index for the measurement of metabolic pool of nitrate was reevaluated using primary leaves of 7-day-old barley and 10-day-old soybean seedlings. The seedlings were grown in nutrient solutions containing 5 to 15 millimolar nitrate. The nitrate-free in vivo assay system of nitrate reductase was used for measuring the production of nitrite. Both the duration and extent of nitrite production were dependent on the level of endogenous nitrate in the tissue. At cessation of nitrite production, 30 to 50% of the endogenous nitrate was reduced to nitrite. Nitrate from the tissue leaked continuously into the surrounding medium so that, at cessation of nitrite production, nitrate supply from the tissue was exhausted. The cessation of nitrite production, therefore, may have been caused by the depletion of endogenous nitrate from the tissue. It is concluded that anaerobic nitrite production is not a valid index for the measurement of the size of the metabolic pool of nitrate. PMID- 16661907 TI - Nitrogen Stress and Apparent Photosynthesis in Symbiotically Grown Pisum sativum L. AB - Pea plants (Pisum sativum L. cv. Alaska) were inoculated individually with one of 15 Rhizobium leguminosarum strains and grown under uniform environmental conditions in the absence of combined N. Differences in effectiveness of the Rhizobium strains produced plants with differing rates of whole plant apparent N(2) fixation and total N content at the same morphological stage of development. Plants were analyzed to determine interactions between N(2) fixation, N allocation, apparent photosynthesis, and growth. Total leaf N increased linearly with total N(2) fixation (R(2) = 0.994). The proportion of total N allocated to leaves, the per cent N content of individual leaves, and the photosynthetic efficiency of individual leaves showed a curvilinear response with increasing plant N content. Differences in allocation patterns of leaf N between plants with low and high N content resulted in differences in the relationship between total N content and plant dry weight. Results from this study show that N(2) fixation interacts with leaf photosynthetic efficiency and plant growth in a manner that is dependent upon the allocation of symbiotically fixed N. PMID- 16661908 TI - Effect of gibberellic Acid on silica content and distribution in sugarcane. AB - The effect of gibberellic acid on the content and distribution of silicon in the stem, leaf sheath, and leaf lamina of sugarcane was analyzed in relation to the effect of gibberellic acid on stem growth. Silicon content was measured by neutron activation analysis, and its distribution was followed by scanning electron microscopy and X-ray analysis.Foliarly applied gibberellic acid increased stem length and fresh weight and decreased silicon content. Gibberellic acid treatments had little or no effect on growth or silicon content of leaf laminae or sheaths. The close correlation between increase in growth of an internode in response to gibberellic acid and the decrease in silicon content of that internode indicated a dilution effect of growth on the amount of silicon rather than a direct effect of gibberellic acid on silicon deposition. This conclusion was supported by scanning electrom microscopy, X-ray map photos, and counts of silica cells per unit of epidermis area. PMID- 16661909 TI - Biogenesis of Mitochondria in Imbibed Peanut Cotyledons : II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA. AB - THERE ARE TWO TYPES OF MITOCHONDRIA PRESENT IN IMBIBED PEANUT COTYLEDONS: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known. PMID- 16661910 TI - Light-induced conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide phosphate in higher plant leaves. AB - Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.Sucrose density gradient centrifugation of wheat protoplast extracts and differential centrifugation of protoplast extracts from various plants showed that most of the NAD kinase was localized in the chloroplasts. Therefore, the conversion of NAD to NADP is considered to occur in the chloroplasts. However, with extracts of maize mesophyll protoplasts, the enzyme was localized in the extrachloroplast fraction. The NAD kinase was activated some 30% by illumination of leaves or protoplasts of pea and wheat after preincubation in the dark.These results suggest that, in general, the light-induced conversion of NAD to NADP occurs in the chloroplast and is catalyzed by photoactivated NAD kinase using photochemically produced ATP. PMID- 16661911 TI - Impairment of photosynthesis by chilling-temperatures in tomato. AB - Chilling of attached tomato leaves (cv. Rutgers) in the dark for 16 hours at 1 C decreased both photosynthesis and transpiration. To separate the effects of chilling on stomatal CO(2) conductance from more direct effects of chilling on the chloroplasts' activities, measurements of photosynthesis and transpiration were made at atmospheric and saturating CO(2) levels. At atmospheric CO(2), the inhibition of photosynthesis was approximately 60%, of which about 35% was attributable to the impairment of chloroplast function and about 25% was attributable to decreased stomatal conductance. However, the affinity of the photosynthetic apparatus for CO(2) was not changed by chilling, since the dependence of the relative rate of photosynthesis on the intercellular CO(2) concentration was unaltered. The apparent quantum requirement for CO(2) reduction also was identical in chilled and unchilled plants. This observation contradicts the widely held notion that the chilling-induced inhibition of photosynthesis is caused by an impairment of the water oxidation mechanism. The impairment of chloroplast activity was not a consequence of an unfavorable water status within the leaf, since chilling caused only a small drop (1 bar) in water potential. A small loss of chlorophyll resulted as a secondary effect of chilling, but this loss of chlorophyll was eliminated as a cause of the inhibition of photosynthesis.No recovery of chloroplast activity occurred during the subsequent light period after chilling. The recovery seemed to be inhibited by light or to require both a light period and a dark period or to occur after a considerable lag period. After a period of both light and dark, restoration of stomatal conductance occurred more slowly than did the recovery of chloroplast activity. PMID- 16661912 TI - Crassulacean Acid Metabolism in the Epiphyte Tillandsia usneoides L. (Spanish Moss) : RESPONSES OF CO(2) EXCHANGE TO CONTROLLED ENVIRONMENTAL CONDITIONS. AB - Patterns of CO(2) exchange in Spanish moss under various experimental conditions were measured using an infrared gas analysis system. Plants were collected from a study site in North Carolina and placed in a gas exchange chamber for several days of continuous measurements. No substantial seasonal effects on CO(2) exchange were observed. High rates of nocturnal CO(2) uptake were observed under day/night temperature regimes of 25/10, 25/15, 25/20, 30/20, and 35/20 C; however, daytime temperatures of 40 C eliminated nighttime CO(2) uptake and a nighttime temperature of 5 C eliminated nocturnal CO(2) uptake, regardless of day temperature. Constant chamber conditions also inhibited nocturnal CO(2) uptake. Constant high relative humidity (RH) slightly stimulated CO(2) uptake while low nighttime RH reduced nocturnal CO(2) uptake.Reductions in daytime irradiance to approximately 25% full sunlight had no effect on CO(2) exchange. Continuous darkness resulted in continuous CO(2) loss by the plants, but a CO(2) exchange pattern similar to normal day/night conditions was observed under constant illumination. High tissue water content inhibited CO(2) uptake. Wetting of the tissue at any time of day or night resulted in net CO(2) loss. Abrupt increases in temperature or decreases in RH resulted in sharp decreases in net CO(2) uptake.The results indicate that Spanish moss is tolerant of a wide range of temperatures, irradiances, and water contents. They also indicate that high nighttime RH is a prerequisite for high rates of CO(2) uptake. PMID- 16661913 TI - Effects of Temperature, Nitrogen Fertilization, and Plant Age on Nitrogen Fixation by Setaria italica Inoculated with Azospirillum brasilense (strain cd). AB - The association between the nitrogen-fixing bacterium Azospirillum brasilense (strain cd) and the grass Setaria italica was studied under different environmental and soil conditions. Highest acetylene reduction rates in intact plants were observed at the booting stage of Setaria (2350 nmol ethylene produced hour(-1) plant(-1)) at 27 C. Higher temperatures, up to 32 C, enhanced ethylene reduction. Significant increases in shoot dry weight, panicle weight, and length were obtained in inoculated plants fertilized with suboptimal NH(4)NO(3) levels. The increase in nitrogen content of plants inoculated with A. brasilense was shown to be due to N(2) fixation. This was demonstrated by growing plants in washed quartz sand with no combined nitrogen. The bacteria also increased branching and development of roots. It was concluded that inoculation of Setaria with A. brasilense may lead both to increases in plant yield and saving of nitrogen fertilizer. PMID- 16661914 TI - In vitro gibberellin a(4) binding to extracts of cucumber hypocotyls. AB - Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]-gibberellin(4) (GA(4)) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [(3)H]GA(4) to macromolecular components. [(3)H]GA(4) binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [(3)H]GA(4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3)H]GA(4) was due to a single class of binding sites having an estimated K(d) of 10(-7) molar and a concentration of 0.8 x 10(-12) moles gram(-1) fresh weight or 0.4 x 10(-12) moles milligram(-1) soluble protein. PMID- 16661915 TI - The role of ethylene in the senescence of oat leaves. AB - The evolution of ethylene, both from the endogenous source and from added 1 aminocyclopropane-1-carboxylic acid (ACC), has been followed in close relationship with the senescent loss of chlorophyll from seedling oat leaves. In white light, where chlorophyll loss is slow, the ethylene evolution increases slowly at first, but when the loss of chlorophyll becomes more rapid, ethylene evolution accelerates. CoCl(2) inhibits this increase and correspondingly maintains the chlorophyll content, with an optimum concentration of 10 micromolar. The rapid rate of chlorophyll loss in the dark is slightly decreased by 3-aminoethoxyvinyl glycine (AVG), by cobalt, and slightly stimulated by ACC. The slower chlorophyll loss in white light, however, is almost completely inhibited by silver ions, greatly decreased by cobalt and by AVG, and strongly increased by ACC. Since the chlorophyll loss is accompanied by proteolysis, it represents true senescence. Chlorophyll loss in light is also strongly antagonized by CO(2), 1% CO(2) giving almost 50% chlorophyll maintenance in controls, while in the presence of added ACC or ethylene gas, the chlorophyll loss is 50% reversed by about 3% CO(2). The ethylene system in leaves is thus more sensitive to CO(2) than that in fruits. Indoleacetic acid also clearly decreases the effect of ACC. It is shown that kinetin, CO(2), Ag(+), and indoleacetic acid, all of which oppose the effect of ethylene, nevertheless increase the evolution of ethylene by the leaves, and it is suggested that ethylene evolution may, in many instances, mean that its hormonal metabolism is being prevented.Abscisic acid somewhat increases ethylene evolution also, but its action in promoting senescence in light is antagonized only partially by Ag(+), Co(2+), or AVG. For this and a number of other reasons it is concluded that ethylene and abscisic acid both independently control leaf senescence in the light. PMID- 16661916 TI - Nocturnal Accumulation of Malic Acid Occurs in Mesophyll Tissue without Proton Transport to Epidermal Tissue in the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum: EVIDENCE AGAINST A PREVIOUS HYPOTHESIS. AB - The inducible Crassulacean acid metabolism plant, Mesembryanthemum crystallinum, accumulates malic acid, i.e. equivalent amounts of malate anions and protons in the mesophyll cells at night. Levels of malate and titratable acidity are low in the epidermal tissue and do not change significantly during the day/night cycle. This result is in contrast to a recent report (Bloom 1979 Plant Physiol 64: 919 923) that the synthesis of malic acid during dark CO(2) fixation is associated with an equivalent exchange of inorganic cations from epidermal tissue with protons in the mesophyll cells. PMID- 16661917 TI - Host-Pathogen Interactions : XVIII. ISOLATION AND BIOLOGICAL ACTIVITY OF GLYCINOL, A PTEROCARPAN PHYTOALEXIN SYNTHESIZED BY SOYBEANS. AB - A previously unrecognized phytoalexin has been isolated from soybean cotyledons that had been infected with bacteria or exposed to ultraviolet light. The phytoalexin has been purified to homogeneity by silica gel flash chromatography and high pressure liquid chromatography. It has been structurally characterized by its ultraviolet, circular dichroism and nuclear magnetic resonance spectra, polarimetry, and its mass spectrometric fragmentation pattern. The phytoalexin, (6aS,11aS)-3,6a,9-trihydroxypterocarpan, is a compound that had previously been detected in CuCl(2)-treated soybeans and is structurally related to the previously identified soybean phytoalexins glycerollins I to IV. It is proposed that the trivial name glycinol be used for this phytoalexin. Glycinol is a broad spectrum antibiotic capable of prolonging the lag phase of growth of all six bacteria examined, namely Erwinia carotovora, Pseudomonas glycinea (races 1 and 3), Escherichia coli, Xanthomonas phaseoli, and Bacillus subtilis. Glycinol also inhibits the growth of the fungi Phytophthora megasperma f. sp. glycinea (race 1), Saccharomyces cerevisiae, and Cladosporium cucumerinum. Glycinol is a static agent against the six bacterial species listed above and against S. cerevisiae, and appears to be static against the other fungi examined. As with other phytoalexins, there is no correlation between the pathogenicity of a microorganism and its sensitivity to glycinol. PMID- 16661918 TI - Uncouplers stimulate photosynthesis in intact chloroplasts by enhancing light activation of enzymes regulated by the ferredoxin-thioredoxin system. AB - Some uncouplers stimulate CO(2)-dependent O(2) evolution by intact spinach chloroplasts at pH 8.6. This effect is not due to alkalinization of the stroma. The stimulation is observed only when photosynthesis has been partly inhibited by the presence of H(2)O(2), generated in a Mehler-type reaction by the broken chloroplasts which always contaminate the intact chloroplast preparations. The addition of methyl viologen increases the Mehler-type reaction and results in greater inhibition of photosynthesis. The addition of excess catalase stimulates photosynthesis by preventing accumulation of H(2)O(2). The uncouplers stimulate photosynthesis primarily by enhancing the light-activation of enzymes that are regulated by the ferredoxin-thioredoxin system, and this effect results from the influence of the uncouplers on the redox poising of the ferredoxin in the intact chloroplasts. PMID- 16661919 TI - Physical Aspects of Fruit Growth : THEORETICAL ANALYSIS OF DISTRIBUTION OF SURFACE GROWTH FORCES IN FRUIT IN RELATION TO CRACKING AND SPLITTING. AB - The theory of shells has been applied to some aspects of the physics of fruit growth. Four form and structural attributes are identified which may intensify mechanical stress in the skin of a growing fruit and alter the distribution of that stress. One is a radius-related factor introduced by deviation of shape from that of a sphere and the other three are related to attachment of a fruit to a plant and to provision of a vascular system: core diameter, core tensile strength, and structure of the core/skin interface. The last factor also applies in principle to a hole which may be introduced for example at the style canal. These factors either alone or in combination can cause stresses far in excess of those predicted for a spherical shell of similar volume and wall thickness. They are considered in relation to their effect on fruit morphogenesis and the occurrence of disorders such as rainfall-induced splitting and cracking. PMID- 16661920 TI - Effect of polar lipids on ATPase activity of membrane preparations from germinating radish seeds. AB - Membrane preparation (sedimenting between 13,000(g) and 80,000(g)) of germinating radish seeds (Raphanus sativus L.) was active in hydrolyzing ATP and, to a lesser extent, a variety of other phosphorylated compounds. Dicyclohexylcarbodiimide (DCCD) and diethylstilbestrol significantly inhibited the ATPase activity (40%) while their effect on hydrolysis of other phosphorylated compounds was much less.The sucrose density gradient analysis of the membrane preparation showed that the position of the DCCD-sensitive K(+)-dependent ATPase was similar to that found for plasma membrane of other plant material.Cholate treatment of membrane preparation removes almost all phospholipids, and ATPase activity is barely detectable. However, the addition of polar lipids completely restores the ATPase activity but does not restore general phosphatase activity.The ATPase of the polar lipids restored cholate preparation, showed a high sensitivity to DCCD and diethylstilbestrol (up to 90% inhibition), a complete dependence on Mg(2+), and a strong dependence on K(+) at low concentration; the pH optimum of ATPase was close to 6.5, and the K(m) for ATP-Mg was 0.51 millimolar. ATPase activity was much greater when polar lipids from 24-hour-germinated seeds were added. PMID- 16661921 TI - Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings. AB - Fluidity of membrane lipids of shoot and root tissue and of chloroplasts from young wheat seedlings of contrasting freezing tolerance was investigated by measuring the motion and order parameters after spin labeling. A striking similarity was observed in membrane lipid fluidity of the five cultivars grown at 22 C. After cold hardening by growth at 2 C, a small change in membrane lipid fluidity was observed, but this was not correlated with the development of freezing tolerance, and there was no alteration in the transition temperature of membrane lipids. The results show that neither changes in membrane lipid fluidity nor transition temperature are a necessary feature of cold acclimation in wheat. PMID- 16661922 TI - Peptidohydrolases of Soybean Root Nodules : IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS. AB - Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-l-Leu-beta-naphthylamide, while the second hydrolyzed n-benzoyl-l-Arg-beta-naphthylamine (Bz-l-Arg-betaNA), n-benzoyl-l-Arg p-nitroanilide (Bz-l-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-l-Arg-betaNA, Bz-l-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-beta naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-beta-naphthylamides. The major activity of two of the aminopeptidases was directed toward l-Leu- and l-Met-beta naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-beta-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima. PMID- 16661924 TI - Physiological Control of Chloride Transport in Chara corallina: II. THE ROLE OF CHLORIDE AS A VACUOLAR OSMOTICUM. AB - The extent to which Cl(-) is replaceable as the major anionic constituent of the vacuole of Chara corallina was investigated. It was found that external Cl(-) is not essential in order for nongrowing cells to increase internal osmotic pressure. After growth of cells in low (9 micromolar) Cl(-), the vacuolar Cl(-) concentration is one-half that of cells grown at normal external Cl(-) concentration (850 micromolar). In contrast, both internal osmotic pressure and total concentration of the major cations, K(+) and Na(+), in the same cells were found to be only slightly sensitive to the external Cl(-) concentration. Thus, it is proposed that, at limiting external Cl(-) concentration, the cell is able to transport or synthesize another anion for vacuolar use rather than utilize a neutral solute.Although the total vacuolar (K(+) + Na(+)) concentration is relatively insensitive to low Cl(-) conditions, a large increase in K(+) was recorded. This increase in K(+) was offset by a correspondingly large fall in Na(+). This is unrelated to any mechanistic dependence of Na(+) on Cl(-) for entry into the cell but may indicate a control system acting on vacuolar sequestration of both Na(+) and Cl(-). Cells grown in low Cl(-) display an ability to take up Cl(-), at enhanced rates, from the medium. The enhancement of Cl(-) influx is maintained for several hours after external Cl(-) is raised to a high level and indicates the existence of a control on Cl(-) influx acting in addition to that of cytoplasmic Cl(-) concentration, which has a shorter decay time.The results are discussed in relation to similar work on higher plants. PMID- 16661923 TI - Disulfiram inhibition of the alternative respiratory pathway in plant mitochondria. AB - Disulfiram (tetraethylthiuram disulfide) was found to be a potent and selective inhibitor of the alternative respiratory path of plant mitochondria. The onset of inhibition by disulfiram takes several minutes and the inhibition is not readily reversed by washing, nor by metal ions. By contrast, thiols such as dithiothreitol not only reverse, but also prevent, disulfiram inhibition. Inhibition by disulfiram and by hydroxamic acids are not mutually exclusive. Structural analogs of disulfiram are far less potent inhibitors, with the exception of bisethyl xanthogen. Inhibition is due to disulfiram, per se, and not to its reduction product, diethyldithiocarbamate, a powerful chelator. Accordingly, the inhibitory effect of disulfiram is considered to involve the formation of mixed disulfides with one or more sulfhydryl groups in the alternative path. Disulfiram does not act as an electron sink diverting electron flow from oxygen.Disulfiram inhibition was observed only with isolated mitochondria or submitochondrial particles. In intact cells or tissues either a failure to absorb disulfiram, or its dissipation in the cytosol, precludes inhibition. In vitro, bovine serum albumin reduces disulfiram inhibition by complexing free inhibitor.The binding of (35)S-disulfiram by cyanide-resistant mitochondria displays the same kinetics as disulfiram inhibition. A comparison was made of (35)S-disulfiram binding by cyanide-sensitive and cyanide-resistant potato mitochondria. Cyanide-resistant mitochondria were obtained from ethylene treated potato tubers. Incorporation of label proved essentially the same in both types of mitochondria, suggesting that the disulfiram-sensitive component of the alternative path is present in untreated potato tubers, and is not induced by ethylene. PMID- 16661925 TI - Properties of the Photosynthetic System and DNA of Cyanophora paradoxa Cyanelles. AB - The cyanelle from the photosynthetic biflagellate protist Cyanophora paradoxa has been studied in terms of its photosynthetic properties. Structurally, the cyanelle resembles unicellular cyanobacteria. The cyanelle is readily released from the host cell by means of the French press. The isolated cyanelle shows typical photosystem I and photosystem II activities as well as phenazine methosulfate-mediated photophosphorylation. The kinetic parameters K(m) and V(max) were determined for CO(2) fixation in the cyanelle and cells of C. paradoxa and compared to a cyanobacterium. The determined values were not much different, although the cyanobacterium had a significantly greater rate of CO(2) fixation, and the cyanelle was least active in this regard. Photosystem I chlorophyll-protein complex is readily isolated from the thylakoid membrane. In all these respects, the photosynthetic apparatus of the cyanelle resembles that of cyanobacteria. No nitrogen fixation activity was observed. Attempts to regenerate the isolated cyanelle were not successful, but in some cases, an unidentified cyanobacterium grew up in standing cultures of C. paradoxa cyanelles. Buoyant density data indicate that the strain of C. paradoxa we have investigated differs from that employed by others, since our strain shows a value of 1.716 grams per cubic centimeter and others report values of 1.695 and 1.691. PMID- 16661926 TI - Phloem Transport and the Regulation of Growth of Sorghum bicolor (Moench) at Low Temperature. AB - Leaf expansion in Sorghum bicolor (Moench) was severely retarded by low night temperatures (5 C). However, this was not reflected in the early measurements of relative growth rate, indicating that the response was not associated with a deterioration of the photosynthetic system. For plants grown at 30/25 C (day/night) and subsequently held at an ambient temperature of 30 C, phloem transport, as measured either by the movement of (14)C-photosynthate through a zone of controlled temperature or by accumulation of dry matter distal to this zone, was inhibited by temperatures below 10 C. The speed of movement of (32)P through the temperature controlled zone was more sensitive to temperature with reductions apparent below 20 C. Although there was some recovery in the movement of (32)P following 3 days equilibration at low temperature (1 to 10 C), the new values (approximately 100 centimeters per hour) were still only about one-third of those obtained in the high temperature controls. For plants held at an ambient temperature of 21 C, which is well below the optimum for growth, translocation was only inhibited by temperatures below 5 C. Although low temperature may reduce the carrying capacity of the phloem of S. bicolor, this is unlikely to be an important factor in regulating the growth of the plants at low temperatures. PMID- 16661927 TI - A model for predicting ionic equilibrium concentrations in cell walls. AB - Purified cell walls were prepared from roots of Horse bean (Vicia faba L., var. minor) and Yellow Lupine (Lupinus luteus L.). Two methods were used: (a) grinding and (b) lysis of the endocellular contents by Triton X-100. The Ca(2+), Mg(2+), and K(+) contents were determined after incubation in various solutions in such a manner that the measurements gave access to the undisturbed equilibrium contents. The results were used to test a model which describes the ionic atmosphere in the cell walls as a result of simultaneous electrostatic interactions between free ions (Donnan effect) and specific association equilibria, including acido-basic ones. This model correctly accounted for the whole set of experimental results and predicted the values of the unmeasurable local concentrations and pH. PMID- 16661928 TI - Sensing of volume changes by poterioochromonas involves a ca-regulated system which controls activation of isofloridoside-phosphate synthase. AB - In Poterioochromonas, osmotically induced shrinkage is reversed by an accumulation of isofloridoside. In crude extracts, the isofloridosidephosphate synthase is activated by an enzyme system, the activity of which is increased at reduced cell volume and decreases again when cells reswell. The synthase activating enzyme system also spontaneously gains activity in cell homogenates. This process is affected by the presence of ethylene glycol-bis-(2-aminoethyl ether)-N,N-tetraacetate or ethylenediaminetetraacetate in the homogenization buffer and can also be irreversibly initiated by addition of Ca(2+) ions to homogenates. Pretreatment of the cells with the Ca(2+)-ionophore A 23187 also enhances spontaneous operation of the activation system in homogenates. Addition of the calmodulin-binding drugs trifluoperazine, fluphenazine, or chlorpromazine to homogenates promotes the activation process. The results suggest that detection of volume changes involves a Ca(2+)-controlled and possibly calmodulin mediated reaction sequence located in membranes. This mechanism might involve zymogens and, after stimulation by cell shrinkage, appears to produce or activate a specific protease capable of activating the isofloridosidephosphate synthase. PMID- 16661929 TI - Glycine metabolism and oxalacetate transport by pea leaf mitochondria. AB - Isolated pea leaf mitochondria oxidatively decarboxylate added glycine. This decarboxylation could be linked to the respiratory chain (in which case it was coupled to three phosphorylations) or to mitochondrial malate dehydrogenase when oxalacetate was supplied. Decarboxylation rates measured as O(2) uptake, or CO(2) and NH(3) release were adequate to account for whole leaf photorespiration. Oxalacetate-supported glycine decarboxylation, measured by linking malate efflux to added malic enzyme, yielded rates considerably less than the electron transport rates. Butylmalonate inhibited malate efflux but not oxalacetate entry; phthalonate inhibited oxalacetate entry but had little effect on malate or alpha ketoglutarate oxidation. It is suggested that oxalacetate and malate transport are catalyzed by separate carrier systems of the mitochondrial membrane. PMID- 16661930 TI - Nutritional Regulation of Organelle Biogenesis in Euglena: INDUCTION OF MICROBODIES. AB - Exposure of dark grown resting Euglena to ethanol produced a transient increase in the specific activity of the glyoxysomal enzyme malate synthase. Enzyme specific activity increased during the first 24 hours of ethanol treatment and then declined. Light exposure or malate addition failed to increase enzyme specific activity. The increase and decrease in enzyme specific activity represented changes in the amount of active enzyme. In both wild type cells and the plastidless mutant W(3)BUL, enzyme levels were always higher in the dark than in the light.The specific activity of the peroxisomal enzyme glycolate dehydrogenase began to increase 24 hours after dark grown resting Euglena were exposed to light. Ethanol, but not malate, prevented the increase and promoted a decrease in glycolate dehydrogenase levels. Cycloheximide produced a decline in enzyme levels similar to the decline produced by ethanol addition. Glycolate dehydrogenase was present in the plastidless mutant W(3)BUL indicating that it is coded in the nucleus and synthesized on cytoplasmic ribosomes. Streptomycin, a specific inhibitor of chloroplast protein synthesis and 3-(3,4-dichlorophenyl) 1,1-dimethylurea, an inhibitor of photosynthetic CO(2) fixation, inhibited the photoinduction of glycolate dehydrogenase while having no effect on the photoinduction of NADP dependent glyceraldehyde-3-phosphate dehydrogenase, another light induced, nuclear coded, cytoplasmically synthesized enzyme. Taken together, these results suggest that microbodies are continually synthesized in resting Euglena and their enzyme complement is determined through substrate induction of glyoxysomal and peroxisomal enzymes. PMID- 16661931 TI - Inhibition of anion transport in corn root protoplasts. AB - The effects of several amino-reactive disulfonic stilbene derivatives and N-(4 azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl(-), SO(4) (2-), and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4'-isothiocyano-2,2'-stilbenedisulfonic acid, 4,4'-diisothiocyano 2,2'-stilbenedisulfonic acid, 4,4'-diamino-2,2'-stilbenedisulfonic acid, and NAP taurine inhibited Cl(-) and SO(4) (2-) but not Pi and K(+) uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl(-) or SO(4) (2-) uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl(-) and SO(4) (2-) transport inhibitors. PMID- 16661932 TI - Effects of Environmental pH on the Internal pH of Chlorella pyrenoidosa, Scenedesmus quadricauda, and Euglena mutabilis. AB - The effect of external pH on two laboratory-cultured acid-intolerant species (Chlorella pyrenoidosa Chick and Scenedesmus quadricauda Turp. Breb.) and one acid-tolerant species from a natural population (Euglena mutabilis Schmitz) was examined by measuring internal pH. These measurements were made with the weak acid (14)C-dimethyloxazolidine-2,4-dione after cells had been incubated for 2 and 6 hours at external pH levels from 3.0 to 8.0. Photosynthetic and respiration rates of the three species were also measured over the range of external pH levels.All three species regulated their internal pH levels over the 6-hour incubation time. C. pyrenoidosa and S. quadricauda had internal pH levels around 7.0, regardless of external pH. E. mutabilis had a wider internal pH range, from 5.0 at low external pH to 8.0 at high external pH. External pH had no effect on either photosynthetic or respiration rates. Statistical comparisons showed that there was a significant difference between the acid-intolerant and acid-tolerant species with regard to the level of internal pH maintained and the response of internal pH to external pH. PMID- 16661933 TI - Purification of Phytochrome by Affinity Chromatography on Agarose-Immobilized Cibacron Blue 3GA. AB - The binding of phytochrome to Cibacron Blue 3GA was utilized to develop a new affinity purification procedure for phytochrome. Brushite-purified phytochrome from rye (Secale cereale c.v. Cougar) was bound to agarose-immobilized blue dye in 0.1 molar potassium phosphate (pH 7.8), contaminating proteins washed out with 0.5 molar KCl, and homogeneous phytochrome eluted with 10 millimolar flavin mononucleate. Ninety-five per cent of the phytochrome applied bound, and 60 to 65% was eluted, giving a 25 to 30% yield for the complete one-day procedure. Affinity-purified rye phytochrome was identical to conventionally purified phytochrome in its behavior on sodium dodecyl sulfate gels, in gel exclusion chromatography, in sedimentation in sucrose density gradients and in its spectral properties. PMID- 16661934 TI - Noncovalent Intermolecular Forces in Phycobilisomes of Porphyridium cruentum. AB - Using sensitized fluorescence as a measure of intactness of phycobilisomes isolated from Porphyridium cruentum, the effects of various environmental perturbations on phycobilisome integrity were investigated. The rate of phycobilisome dissociation in 0.75 ionic strength sodium salts proceeds in the order: SCN(-) > NO(3) (-) > Cl(-) > C(6)H(5)O(7) (3-) > SO(4) (2-) > PO(4) (3-), as predicted from the lyotropic series of anions and their effects on hydrophobic interactions in proteins. Similarly, increasing temperature (to 30 C) and pH values approaching the isoelectric points of the biliproteins stabilize phycobilisomes. Deuterium substitution at exchangeable sites on the phycobiliproteins decreases the rate of phycobilisome dissociation, while substitution at nonexchangeable sites increases rates of dissociation. It is concluded that hydrophobic intermolecular interactions are the most important forces in maintaining the phycobilisome structure. Dispersion forces also seem to contribute to phycobilisome stabilization. The adverse effects of electrostatic repulsion must not be ignored; however, it seems that the requirement of phycobilisomes of high salt concentrations is not simply countershielding of charges on the proteins. PMID- 16661935 TI - Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts. AB - Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca(2+) in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed. PMID- 16661936 TI - Correlations between Potassium Uptake and Hydrogen Efflux in Barley Varieties : A POTENTIAL SCREENING METHOD FOR THE ISOLATION OF NUTRIENT EFFICIENT LINES. AB - Rates of hydrogen ion secretion and potassium ((86)Rb) absorption by intact roots of twenty-four barley varieties were measured in solutions containing K(2)SO(4) (1 x 10(-4) to 1 x 10(-3) molar) plus 5 x 10(-4) molar CaSO(4), at initial pH values in the range 5.3 to 5.5. Fluxes of H(+) and K(+) were strongly correlated in short-term experiments (up to 15 minutes) as well as in long-term experiments (lasting 24 hours). The observed correlations provide the basis for a preliminary screening method, designed to segregate varieties with high rates of potassium uptake by the use of an acid-base indicator (methyl red). PMID- 16661937 TI - Association of Formation and Release of Cyclic AMP with Glucose Depletion and Onset of Chlorophyll Synthesis in Poterioochromonas malhamensis. AB - Depletion of glucose from the culture medium by Poterioochromonas malhamensis results in cessation of growth and accumulation of cyclic adenosine 3':5' monophosphate (cAMP), followed by formation of chlorophyll and an increase in extracellular cAMP. Readdition of glucose to the culture medium causes P. malhamensis to release its intracellular cAMP into the medium. These results suggest that formation of the photosynthetic apparatus in P. malhamensis may be repressed by glucose, and that high cAMP conveys the regulatory information that the glucose supply is inadequate. This pattern is reminiscent of cAMP-mediated escape from catabolite repression in bacteria. PMID- 16661938 TI - Nitrate Assimilation and Crassulacean Acid Metabolism in Leaves of Kalanchoe fedtschenkoi Variety Marginata. AB - The enzymes necessary to assimilate ammonia either via glutamine synthetase and glutamate synthase or via the glutamate dehydrogenase pathways are present in both green and white leaf tissues of Kalanchoe fedtschenkoi. Nitrate reductase activity develops to a maximum in a Crassulacean acid metabolism (CAM) plant canopy before either ribulose 1,5-bisphosphate carboxylase, or phosphoenolpyruvate carboxylase, or CAM. Nitrate reductase also is activated each morning and is inactivated late in the day as in other plants. However, there does not appear to be any direct relationship between nitrate reductase activity and the level of acid, its daily pattern or the amplitude of CAM. Though nitrate reductase is activated maximally each day by light, in Kalanchoe leaves for six days the activity followed a precise daily pattern independent of continuous light or dark. PMID- 16661939 TI - Azido auxins: synthesis and biological activity of fluorescent photoaffinity labeling agents. AB - Three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4-N(3)-IAA, 5-N(3) IAA, and 6-N(3)-IAA) have been synthesized for use as fluorescent photoaffinity labeling agents. The pK(a) values of these compounds (4-N(3)-IAA, 4.67; 5-N(3) IAA, 4.65; 6-N(3)-IAA, 4.66; all +/- 0.04) are experimentally indistinguishable from the pK(a) of 3-indoleacetic acid (IAA, 4.69 +/- 0.04). The auxin activity of these IAA derivatives has been determined in several systems. In soybean, pea, and corn straight growth assays, all three analogs induce growth comparable to that caused by IAA. In the tobacco pith assay, all three analogs elicit a maximum increase in fresh weight at least 40 to 50% of that caused by IAA. Optimal growth is attained in the tobacco pith assay at slightly higher concentrations of 4-N(3) IAA and 6-N(3)-IAA (30 micromolar) than required for IAA (10 micromolar); however, maximal growth is achieved at a slightly lower concentration of 5-N(3) IAA (3 micromolar). The N(3)-IAAs, like IAA, are transported basipetally through tobacco pith tissue. PMID- 16661940 TI - Role of indoleacetic Acid and abscisic Acid in the correlative control by fruits of axillary bud development and leaf senescence. AB - When fully filled pods of bean plants were deseeded, the rate of axillary bud growth and the chlorophyll content of leaves were increased. Application of 0.1% indoleacetic acid (IAA) in lanolin on the deseeded pods caused abscission of axillary buds, inhibited growth of the remaining buds, and decreased leaf chlorophyll content. The response of bud development to fruit-applied IAA was concentration dependent between 0.001 and 0.1% IAA (representing from 2 to 200 micrograms IAA per fruit) resulting in greater growth inhibition at higher IAA concentrations.When plants were defruited so that the number of fruits per plant was adjusted to 0, 6, 12, or 18, a dosage effect of fruits on photosynthesis was observed. Removal of all fruits caused a rise in the CO(2)-exchange rate (CER). With increasing fruit dosage, plants showed leaf senescence of increasing intensity and a corresponding decline in CER. In contrast to the effect of fruit applied IAA on leaves and buds, it delayed the senescence of treated fruits. When axillary buds were treated directly with aqueous solutions of IAA, no growth inhibition occurred.Abscisic acid (AbA) applied on deseeded pods, up to a concentration of 0.1% AbA in lanolin, failed to inhibit axillary bud development or to cause leaf senescence.The results support the hypothesis that the correlative control of axillary bud development and leaf senescence by fruits involves the participation of both IAA and AbA. IAA, released by the seeds, may play the role of the correlative signal that moves from the fruit to the target organ, where it stimulates the synthesis or accumulation of AbA. AbA, in turn, may be responsible for the inhibition of axillary bud development and the enhancement of leaf senescence. PMID- 16661941 TI - Phycoerythrins of the Red Alga Callithamnion: VARIATION IN PHYCOERYTHROBILIN AND PHYCOUROBILIN CONTENT. AB - Phycoerythrins of several species of the higher red alga Callithamnion show virtually identical spectra, typical of R-phycoerythrins, with absorption maxima at 565, 539, and 497 nanometers. One species, Callithamnion roseum, produces a phycoerythrin lacking the peak at 539 nanometers. Comparison of a "typical" R phycoerythrin from Callithamnion byssoides with the "atypical" phycoerythrin of C. roseum shows that both proteins carry 35 bilins per native molecule of 240,000 daltons; however, C. byssoides phycoerythrin carries 27.6 phycoerythrobilin and 7.3 phycourobilin groups, whereas C. roseum phycoerythrin carries 24.1 phycoerythrobilin and 10.9 phycourobilin groups. These differences in the relative amounts of the bilin prosthetic groups account in large measure for the differences between the absorption spectra of the native proteins. The ratio of phycoerythrobilin to phycourobilin in C. roseum phycoerythrin can be modulated by varying the light intensity during growth.Data on the physical, immunological and spectroscopic properties of Callithamnion phycoerythrins indicate that the variation in the relative number of the two bilin prosthetic groups does not affect significantly the conformation of the biliprotein. PMID- 16661942 TI - Utilization of aldehydes and alcohols by soybean bacteroids. AB - Aldehydes, alcohols and acids were tested for their ability to support acetylene reduction and oxygen consumption by Rhizobium japonicum bacteroids isolated from soybean nodules. Several alcohols and aldehydes increased acetylene reduction and oxygen uptake. This is consistent with the concept that the plant nodule cytosol can metabolize carbohydrate via anaerobic fermentative pathways. PMID- 16661943 TI - Polyamine oxidase in oat leaves: a cell wall-localized enzyme. AB - The localization and activity of polyamine oxidase (PAO; EC 1.5.3.3), was investigated in leaves and protoplasts of oat seedlings. Activity of the enzyme is highest with spermine as substrate; spermidine is also oxidized, but putrescine and cadaverine are unaffected by the enzyme. Protoplasts isolated following digestion of leaves with cellulase in hypertonic osmoticum showed no PAO activity, and about 80% of the total leaf PAO activity could be accounted for in the cell wall debris. Histochemical localization experiments showed intense PAO activity in guard cells and in vascular elements whose walls are not digested by cellulase. When protoplasts were cultured in a medium suitable for regeneration of cell wall, PAO activity could be detected as the cellulose wall developed. Thus, PAO appears to be localized in cell walls.Since applied spermine and spermidine prevent senescence of detached leaves, PAO activity was investigated during leaf senescence. The specific activity of PAO declines with increasing age of attached leaves and with increasing senescence of excised leaves incubated in darkness. This decline in enzyme activity, which parallels the decreases in chlorophyll and protein content used as measures of leaf senescence, suggests that the enzyme is not involved in the control of senescence of oat leaves. PMID- 16661944 TI - Enzymic and protein character of tonoplast from hippeastrum vacuoles. AB - The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg(2+)-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, alpha-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K(2)HPO(4) or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels. PMID- 16661945 TI - Photosynthetic and Respiratory Activity in Anacystis nidulans Adapted to Osmotic Stress. AB - Anacystis nidulans cultures growing photoautotrophically in a minimal medium were exposed to different concentrations of NaCl, up to 0.4 molar. Initially, a marked decrease in photosynthetic activity took place, which was in direct relation to the salt concentration and which was not associated with a change in endogenous respiratory activity. After a period of exposure to salinity, a process of adaptation became apparent, being manifested in a partial reversal of the decline in photosynthesis and a marked increase in endogenous respiration. PMID- 16661946 TI - Two types of ventilated porometers compared on broadleaf and coniferous species. AB - Two ventilated porometers (diffusion and steady-state) were compared on four broadleaf and five coniferous species. The diffusion porometer gave consistently lower conductance values for both types of species, reflecting a direct stomatal response to low chamber humidity. At high conductance values, the porometers produced a linear and nearly equal response, but the diffusion porometer was less sensitive at low conductance values. This was due to lower air flow (20% of the velocity in the steady-state porometer) and water vapor sorption (by its acrylic plastic chamber). The broadleaf species had less variation (R(2) = 0.81) than did the coniferous species (R(2) = 0.61), but, with the latter, there was better correspondence between the two porometers, possibly due to sampling technique. Conductance values were clustered by species. PMID- 16661947 TI - Reevaluation of the cyanide resistance of seed germination. AB - Although high levels of KCN (53 micromoles per gram fresh weight of seed, corresponding to 3.2 millimolar) failed to block germination of lettuce seeds incubated in covered Petri dishes, the same levels totally blocked germination in sealed dishes. Inhibition was reversed by removing the seal. Placement of KCN remote from seeds also blocked germination in closed systems. Cyanide effectiveness was enhanced by acidifying the KCN solution but negated by the presence of a trap containing strong alkali. Low levels of aqueous HCN (2.6 micromoles HCN per gram, corresponding to 0.16 millimolar) injected into sealed dishes gave maximal inhibition of germination, suggesting that the effectiveness of KCN was due to formation of HCN in KCN solutions. Studies with nine additional crop species generally supported the interpretation that cyanide inhibition of germination has been underestimated in the past due to escape of volatile HCN from open systems. PMID- 16661948 TI - In vitro promotion by auxins of divalent ion release from soybean membranes. AB - Release of divalent ions from membrane pellets of soybean hypocotyls was promoted by the natural auxin, indole-3-acetic acid, and the synthetic auxin, 2,4 dichlorophenoxyacetic acid. The calcium release occurred at auxin concentrations as low as 1 nanomolar, and maximum release was observed at 1 micromolar. Hormone concentrations greater than 1 micromolar showed reduced effectiveness in releasing membrane-associated calcium. 2,3-Dichlorophenoxyacetic acid, a weak auxin analog of 2,4-dichlorophenoxyacetic acid, did not promote calcium release. In some experiments, the analog actually promoted calcium association with the membranes. Red blood cells treated in a similar manner to soybean hypocotyl membranes did not release calcium in response to indole-3-acetic acid. The release phenomenon was hormone specific but not ion specific. Auxin released manganese from membranes in a manner similar to that of calcium. The calcium release, following auxin treatment, is accompanied by a decrease in membrane associated sites for calcium binding. PMID- 16661949 TI - Protein synthesis and phospholipids in soybean axes in response to imbibitional chilling. AB - The responses of two cultivars of soybean (Merr.) to a chilling treatment (4 C for first hour of imbibition) were compared. The germination of cv. Biloxi was unaffected by the treatment, while the germination of cv. Fiskeby was reduced. The phospholipid fatty acids of dry axes of the two cultivars were very similar, and, thus, could not be correlated with their responses to chilling. The fatty acid composition of chilling-tolerant Biloxi did not change over a subsequent 23 hour warm incubation, but there was a marked reduction in the unsaturated fatty acids of chilling-sensitive Fiskeby after 12 hours, which may be a symptom of deterioration. Protein synthesis in both cultivars was reduced by the chilling treatment. Redrying of Biloxi axes up to 18 hours after the onset of imbibition had no effect on their germination upon rehydration. Germination of Fiskeby axes was reduced by redrying after 8 hours of imbibition. After 7 months of dry storage of intact seeds, the sensitivity of the axes to chilling was retested. Biloxi axes had become chilling-sensitive, while the germination of Fiskeby axes was reduced to zero by the chilling treatment. A hypothesis is presented that imbibitional chilling sensitivity is an indication of reduced vigor, axes with a high vigor can tolerate the stress, while those without cannot. PMID- 16661950 TI - Characterization of ATPase activity associated with corn leaf plasma membranes. AB - A Mg(2+)-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg(2+)-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N' dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A K(m) of 0.28 millimolar Mg(2+)-ATP was determined for the K(+)-ATPase, and the principal effect of potassium was on the V(max) for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme.The cation-stimulated ATPase of corn leaves is biochemically similar to other plant plasma membrane enzymes. Thus, the ATPase can serve as a reliable qualitative plasma membrane marker providing its activity is well characterized. PMID- 16661951 TI - Effects of Powdery Mildew and Water Stress on CO(2) Exchange in Uninfected Leaves of Barley. AB - Net photosynthesis is stimulated in third seedling leaves of barley plants whose lower two leaves are heavily infected by Erysiphe graminis f.sp. hordei Marchal. Stimulation is greater in water-stressed than in well-watered plants. In stressed, but not in well-watered plants, stimulation is associated with the maintenance of high leaf water potential and high leaf conductance. A small part of the changes in net photosynthesis is attributable to changes in respiratory metabolism in the third leaf, and other possible causes are discussed. PMID- 16661952 TI - Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : I. EFFECT OF AUXIN. AB - The metabolism of polysaccharides by pea stem segments treated with and without auxin was investigated using a centrifugation technique for removing solution from the free space of the cell wall. Glucose is the predominant sugar in both the ethanol-soluble and ethanol-insoluble fractions of the cell wall solution extracted with water. In the water-soluble, ethanol-insoluble polysaccharides, arabinose, xylose, galactose, and glucose make up 9.5, 23.8, 23.9, and 39.9%, respectively, of the neutral sugars, while rhamnose, fucose, and mannose are present at concentrations between 0.5 and 2.0%.Auxin treatment enhances the levels of xylose and glucose in ethanol-insoluble polysaccharides relative to controls, and this difference can be detected within 30 minutes of auxin treatment. Cellulose-binding experiments show that the enhanced levels of xylose and glucose are in a polymer having the cellulose-binding properties of xyloglucan. (3)H-glucose labeling experiments confirm the auxin-enhanced metabolism of the xyloglucan fraction; however, increased labeling of arabinose is also observed in auxin-treated sections. Auxin treatment also causes a marked increase in the level of uronic acids centrifuged from pea internode sections. Thus, after 3 hours of incubation in indoleacetic acid, the level of uronic acids in the ethanol-insoluble polysaccharides which can be recovered by centrifugation is increased 2- to 3-fold over sections incubated in water. These auxin-enhanced changes in xylose, glucose, and uronic acids are correlated with enhanced rates of section growth.Incubation of excised pea internode sections in acidic buffers also enhances the rate of xyloglucan and polyuronide metabolism. This acid enhanced metabolism of xyloglucan and polyuronide is inhibited by low temperature, suggesting that it is enzyme-mediated.Extraction of the cell wall solution with CaCl(2) increases the yield of all neutral sugars. Arabinose and mannose are increased 4- and 3-fold, respectively, and xylose and glucose by about 20%, while galactose levels are 40% higher in cell wall solution extracted with CaCl(2) than in that extracted with water. Although calcium increases the amount of neutral sugars extracted, it does not affect the auxin-induced changes in neutral sugars. Extraction of the cell wall solution with ethyleneglycol-bis (beta-aminoethyl ether)-N,N'tetraacetic acid enhances the yield of uronic acids and also increases the difference due to auxin treatment. PMID- 16661953 TI - Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : II. EFFECT OF ETHYLENE. AB - The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H(2)O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H(2)O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl(2) results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl(2) increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides. PMID- 16661954 TI - Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid. AB - Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H(+) excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H(+) excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H(+) in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O(2) uptake and before the increased rate of Rb(+) uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H(+) excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H(+) excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H(+) excretion by IAA and FC. PMID- 16661955 TI - Membrane-mediated decrease in root exudation responsible for phorphorus inhibition of vesicular-arbuscular mycorrhiza formation. AB - The mechanism responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation in sudangrass (Sorghum vulgare Pers.) was investigated in a phosphorus-deficient sandy soil (0.5 micrograms phosphorus per gram soil) amended with increasing levels of phosphorus as superphosphate (0, 28, 56, 228 micrograms per gram soil). The root phosphorus content of 4-week-old plants was correlated with the amount of phosphorus added to the soil. Root exudation of amino acids and reducing sugars was greater for plants grown in phosphorus-deficient soil than for those grown in the phosphorus-treated soils. The increase in exudation corresponded with changes in membrane permeability of phosphorus-deficient roots, as measured by K(+) ((86)Rb) efflux, rather than with changes in root content of reducing sugars and amino acids. The roots of phosphorus-deficient plants inoculated at 4 weeks with Glomus fasciculatus were 88% infected after 9 weeks as compared to less than 25% infection in phosphorus-sufficient roots; these differences were correlated with root exudation at the time of inoculation. For plants grown in phosphorus-deficient soil, infection by vesicular-arbuscular mycorrhizae increased root phosphorus which resulted in a decrease in root membrane permeability and exudation compared to nonmycorrhizal plants. It is proposed that, under low phosphorus nutrition, increased root membrane permeability leads to net loss of metabolites at sufficient levels to sustain the germination and growth of the mycorrhizal fungus during pre- and postinfection. Subsequently, mycorrhizal infection leads to improvement of root phosphorus nutrition and a reduction in membrane-mediated loss of root metabolites. PMID- 16661956 TI - Influence of Turgor Pressure Manipulation on Plasmalemma Transport of HCO(3) and OH in Chara corallina. AB - A modified version of the osmotic shock technique was used to investigate HCO(3) (-) and OH(-) transport in the alga Chara corallina. Cell turgor was brought close to zero and then restored. When turgor was reduced to near the plasmolytic point using an osmoticum, little effect was observed on H(14)CO(3) (-) assimilation and OH(-) transport. However, when turgor was recovered in these cells, there was a large reduction in HCO(3) (-) and OH(-) transport activity. In contrast, when cells were air-dried to zero turgor, and rewetted to restore turgor, no significant effect on OH(-) transport was observed.The effect of plasmolysis was also studied. When Chara cells were plasmolyzed, in most cases, all OH(-) transport activity was lost and acidification was observed along the cell surface.None of the above treatments had any long term effect on cyclosis. These results are discussed with respect to present concepts of turgorinduced changes in membrane functions. PMID- 16661957 TI - Amino Acid transport in germinating castor bean seedlings. AB - During germination and early growth of the castor bean (Ricinus communis) nitrogenous constituents from the endosperm are transferred via the cotyledons to the growing embryo. Exudate collected from the cut hypocotyl of 4-day seedlings contained 120 millimolar soluble amino nitrogen and glutamine was the predominant amino acid present, comprising 35 to 40% of the total amino nitrogen. To determine the nature of nitrogen transfer, the endosperm and hypocotyl were removed and glutamine uptake by the excised cotyledons was investigated. Uptake was linear for at least 2 hours and the cotyledons actively accumulated glutamine against a concentration gradient. The uptake was sensitive to respiratory inhibitors and uncouplers and efflux of glutamine from the excised cotyledons was negligible. Transport was specific for the l-isomer. Other neutral amino acids were transported at similar rates to glutamine. Except for histidine, the acidic and basic amino acids were transported at lower rates than the neutral amino acids. For glutamine transport, the K(m) was 11 to 12 millimolar and the V(max) was 60 to 70 micromoles per gram fresh weight per hour. Glutamine uptake was diminished in the presence of other amino acids and the extent of inhibition was greatest for those amino acids which were themselves rapidly transported into the cotyledons. The transport of amino acids, on a per seedling basis, was greatest for cotyledons from 4-to 6-day seedlings, when transfer of nitrogen from the endosperm is also maximal. It is concluded that the castor bean cotyledons are highly active absorptive organs transporting both sucrose and amino acids from the surrounding endosperm at high rates. PMID- 16661958 TI - Changes in Chlorophyll a and b Content in Dark-Incubated Cotyledons Excised from Illuminated Seedlings: THE EFFECT OF CALCIUM. AB - Cucumber seedlings were illuminated for various time periods, cotyledons excised, placed in the dark, and changes in chlorophyll a and b content monitored. During the dark periods, chlorophyll b content decreased while chlorophyll a did not. When the illumination time was lengthened, the percentage of chlorophyll b decomposition from initial levels decreased. Ca(2+) at 50 millimolar prevented the decrease in chlorophyll b and caused a decrease in chlorophyll a. The effect of Ca(2+) decreased with increased illumination time. Cycloheximide and chloramphenicol inhibited chlorophyll b decrease, but did not induce chlorophyll a decrease.When Ca(2+) was applied to cotyledons, excised from 4 hour illuminated seedlings and preincubated with water in the dark until chlorophyll b content had decreased to a low level, chlorophyll b began to accumulate in the dark. Cycloheximide and chloramphenicol had no such effect. Ca(2+) also induced an accumulation of chlorophyll b in cotyledons excised from seedlings treated with periodic light (2 minutes light followed by 98 minutes dark) during dark incubation. Our results indicate that there is unstable chlorophyll present in the early phase of greening, and that Ca(2+) induces chlorophyll b synthesis from chlorophyll a in the dark. PMID- 16661959 TI - Stress-induced osmotic adjustment in growing regions of barley leaves. AB - Young barley seedlings were stressed using nutrient solutions containing NaCl or polyethylene glycol and measurements were made of leaf growth, water potential, osmotic potential and turgor values of both growing (basal) and nongrowing (blade) tissues. Rapid growth responses similar to those noted for corn (Plant Physiology 48: 631-636) were obtained using either NaCl or polyethylene glycol treatments by which exposure of seedlings to solutions with water potential values of -3 to -11 bars effected an immediate cessation of leaf elongation with growth resumption after several minutes or hours. Latent periods were increased and growth resumption rates were decreased as water potential values of nutrient solutions were lowered. In unstressed transpiring seedlings, water potential and osmotic potential values of leaf basal tissues were usually -6 to -8 bars, and 12 to -14 bars, respectively. These tissues began to adjust osmotically when exposed to any of the osmotic solutions, and hourly reductions of 1 to 2 bars in both water potential and osmotic potential values usually occurred for the first 2 to 4 hours, but reduction rates thereafter were lower. When seedlings were exposed to solutions with water potential values lower than those of the leaf basal tissues, growth resumed about the time water potential values of those tissues fell to that of the nutrient solution. After 1 to 3 days of seedling exposure to solutions with different water potential values, cumulative leaf elongation was reduced as the water potential values of the root medium were lowered. Reductions in water potential and osmotic potential values of tissues in leaf basal regions paralleled growth reductions, but turgor value was largely unaffected by stress. In contrast, water potential, osmotic potential, and turgor values of leaf blades were usually changed slightly regardless of the degree and duration of stress, and blade water potential values were always higher than water potential values of the basally located cells. It is hypothesized that blades have high water potential values and are generally unresponsive to stress because water in most of the mesophyll cells in this area does not exchange readily with water present in the transpiration stream. PMID- 16661960 TI - Resolution and Properties of Two High Affinity Cyclic Adenosine 3':5' monophosphate-Binding Proteins from Wheat Germ. AB - A high affinity cAMP-binding protein (cABP II) was purified to homogeneity from wheat germ. The apparent molecular weight of cABP II, as determined from gel exclusion chromatography, is 5.2 x 10(5) (at low ionic strength) and 2.8 x 10(5) (at high ionic strength). One polypeptide subunit (molecular weight, 80,000) was resolved by polyacrylamide gel electrophoresis of cABP II under subunit dissociating conditions. The purification protocol employed resolves cABP II from a distinct, less acidic cAMP-binding protein (cABP I). The K(d) values for cAMP are about 10(-6) molar and 10(-7) molar for cABP II and cABP I, respectively. The cAMP-binding sites of cABP I and cABP II have a marked adenine-analog specificity, binding adenine, adenosine, adenine-derived nucleosides and nucleotides and a variety of adenine derivatives having cytokinin activity. While cABP II is phosphorylated in reactions catalyzed by endogenous protein kinases, there is no evidence for modulation of these cABP II-protein kinase interactions by cAMP. PMID- 16661961 TI - Lipid composition of chloroplast membranes from weed biotypes differentially sensitive to triazine herbicides. AB - Chloroplasts were isolated from triazine-sensitive and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.), common lambsquarter (Chenopodium album L.), and redroot pigweed (Amaranthus retroflexus L.). Chloroplast lipids were extracted and analyzed for differences among sensitive and resistant biotypes. The distribution of lipid between major lipid classes differed in chloroplasts from resistant and susceptible biotypes. Chloroplasts from resistant biotypes contained higher proportions of monogalactosyl diglyceride and phosphatidyl ethanolamine and lower proportions of digalactosyl diglyceride and phosphatidyl choline than did chloroplasts from susceptible biotypes. Monogalactosyl diglyceride and phosphatidyl ethanolamine were also quantitatively higher in membranes of resistant versus susceptible biotypes. The major lipid classes of resistant chloroplast membranes contained lipids comparatively richer in unsaturated fatty acids with the exceptions of digalactosyl diglyceride from all three biotypes and phosphatidyl ethanolamine from common groundsel. Results correlated changes in triazine sensitivity with qualitative and quantitative differences in the lipid composition of chloroplast membranes. PMID- 16661962 TI - 4-methyleneglutamine in peanut plants: dynamics of formation, levels, and turnover in relation to other free amino acids. AB - Neither 4-methyleneglutamine nor 4-methyleneglutamic acid were found in free or bound form in ungerminated peanut seeds (Arachis hypogaea L.). Both, however, were formed soon after germination; whereas, 4-methyleneglutamic acid appeared slightly before 4-methyleneglutamine, the former remained at a low concentration while the level of 4-methyleneglutamine rose rapidly between 2 and 10 days of germination and declined slowly thereafter. Free proline and glutamine followed a pattern similar to 4-methyleneglutamine; on the other hand, asparagine increased for at least 20 days but other free amino acids remained at relatively low, constant levels. In mature peanut plants, 4-methyleneglutamine occurred in all parts except developing pods, was virtually the only free amino acid in xylem sap, and constituted about 70% of the total soluble nitrogen of sap. In contrast, 4-methyleneglutamic acid was found only in leaves and stems in highly variable amounts.Levels of 4-methyleneglutamine were largely independent of the processes of nodulation and nitrogen fixation. This amide was degraded much more slowly in nitrogen-deficient plants than were any of the other free amino acids and did not become labeled when a photosynthesizing plant was exposed to (14)CO(2) for 24 hours. The results indicate that 4-methyleneglutamine is the principle carrier of nitrogen in the xylem of peanut plants but its carbon skeleton appears to turn over very slowly. PMID- 16661963 TI - Biosynthesis of stress ethylene induced by water deficit. AB - Wheat leaves normally produced very little ethylene, but following a water deficit stress which caused a loss of 9% initial fresh weight, ethylene production increased more than 30-fold within 4 hours and declined rapidly thereafter. The changes in ethylene production were paralleled by an increase and subsequent decrease in 1-aminocyclopropanecarboxylic acid (ACC) content. The level of S-adenosylmethionine was unaffected, suggesting that the conversion of S adenosylmethionine to ACC is a key reaction in the production of water stress induced ethylene. This view was further supported by the observation that application of ACC to nonstressed leaf tissue caused a 70-fold increase in ethylene production, while aminoethoxyvinylglycine, a known inhibitor of the conversion of S-adenosylmethionine to ACC, inhibited ACC accumulation as well as the surge in ethylene production if the inhibitor was applied prior to the stress treatment. Cycloheximide, an inhibitor of protein synthesis, effectively blocked both ethylene production and ACC formation, suggesting that water stress induces de novo synthesis of ACC synthase, which is the rate-controlling enzyme in the pathway of ethylene biosynthesis. PMID- 16661964 TI - Stimulation of plasmalemma adenosine triphosphatase from oat roots by inorganic and organic cations: concentration-dependence, selectivity, and sites. AB - K(+)-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 millimolar KCl. The phases obeyed Michaelis-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 millimolar. Stimulation by alkali cations was in the order K(+) > Rb(+) > Na(+) > Cs(+) > Li(+) or in a closely related sequence. The specificity reflected differences in V(max), not in affinity (K(m) (-1)). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 millimolar appeared monophasic rather than biphasic. Substitution on the quaternary nitrogen of the amino alcohols decreased their effectiveness, as did extension and branching of the chain. Stimulation was maximal at about pH 7 both for K(+) and choline.The kinetics of K(+) stimulation are multiphasic, not cooperative, as was also found for uptake. The ATPase is also stimulated by organic cations, but the difference in kinetics indicates the existence of separate sites for stimulation and transition. PMID- 16661965 TI - Nitrate Reduction in Roots as Affected by the Presence of Potassium and by Flux of Nitrate through the Roots. AB - Dark-grown, detopped corn seedlings (cv. Pioneer 3369A) were exposed to treatment solutions containing Ca(NO(3))(2), NaNO(3), or KNO(3); KNO(3) plus 50 or 100 millimolar sorbitol; and KNO(3) at root temperatures of 30, 22, or 16 C. In all experiments, the accelerated phase of NO(3) (-) transport had previously been induced by prior exposure to NO(3) (-) for 10 hours. The experimental system allowed direct measurements of net NO(3) (-) uptake and translocation, and calculation of NO(3) (-) reduction in the root. The presence of K(+) resulted in small increases in NO(3) (-) uptake, but appreciably stimulated NO(3) (-) translocation out of the root. Enhanced translocation was associated with a marked decrease in the proportion of absorbed NO(3) (-) that was reduced in the root. When translocation was slowed by osmoticum or by low root temperatures, a greater proportion of absorbed NO(3) (-) was reduced in the presence of K(+). Results support the proposition that NO(3) (-) reduction in the root is reciprocally related to the rate of NO(3) (-) transport through the root symplasm. PMID- 16661966 TI - The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts : I. INTERACTION OF ADENINE NUCLEOTIDES AND ENERGY TRANSFER INHIBITORS WITH THE COUPLING FACTOR COMPLEX. AB - The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e(-) = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg(2+) ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C(3) and C(4) mesophyll chloroplasts. PMID- 16661967 TI - A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities. AB - A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-alpha-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack. PMID- 16661968 TI - Homocysteine Biosynthesis in Green Plants: Physiological Importance of the Transsulfuration Pathway in Lemna paucicostata. AB - To permit an assessment of the relative contributions of the transsulfuration and the direct sulfhydration pathways for homocysteine biosynthesis, the time course of incorporation of (35)S from (35)SO(4) (2-) into various sulfur-containing compounds in Lemna paucicostata has been determined. Plants were grown with either low (4.5 micromolar) or ample (1,000 micromolar) sulfate in the medium. At the shortest labeling times, (35)S-cystathionine was the predominant (35)S containing organic sulfur compound. The flux of sulfur into cystathionine was sufficient to sustain the known rate of methionine biosynthesis. It was calculated that transsulfuration accounted for at least 90 and 85% of the total homocysteine synthesis in low and ample sulfate-grown plants, respectively (and may have accounted for 100%). No marked rise in the (35)S-soluble cysteine:(35)S homocysteine ratio was observed even at the shortest labeling times, but it is argued that this may be due to (a) the observed compartmentation of soluble cysteine, and (b) the impracticality of using labeling times shorter than 17 seconds. Additional evidence supporting the importance of transsulfuration in Lemna is briefly described. PMID- 16661969 TI - Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes. AB - The association of K(+)-stimulated, Mg(2+)-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg(2+)-ATPase activity to stimulation by K(+) and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg(2+)-ATPase activity was stimulated up to 700% by addition of K(+). Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg(2+). Ca(2+) was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO(3) and about 40% by diethylstilbestrol. PMID- 16661970 TI - Oxidative Phosphorylation in Germinating Lettuce Seeds (Lactuca sativa) during the First Hours of Imbibition. AB - Experiments with lettuce seeds during the first hours of imbibition showed that oxygen is necessary to sustain high adenine nucleotide ratios and consequently, energy charge values are higher than 0.8 as is usually the case in normally metabolizing tissues.The energy charge value (0.2) of dry seeds soaked in aerated water increased to normal values (0.8) within 30 minutes. The energy charge value of seeds imbibed under cyanide or nitrogen stayed at low values, about 0.3 for 30 minutes. Nitrogen and cyanide treatment of seeds imbibed in aerated water produced a decrease of energy charge to low values within 3 minutes. During the first minutes of imbibition, the oxygen uptake is cyanide-sensitive. The effect of the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone was not as clear-cut. However, results were obtained which agree with the occurrence of oxidative phosphorylation during the first hours of imbibition. These results indicate that a normal cytochromic pathway synthesizes ATP during the first minutes and hours following the imbibition of lettuce seeds. PMID- 16661971 TI - Effect of anoxia on energy charge and protein synthesis in rice embryo. AB - Energy charge, adenine nucleotide levels, and protein synthesis were studied during the transfer of rice seedlings from air to anoxia. Within minutes, the energy charge value dropped from 0.90 in air to 0.50 in the seed and 0.60 in the coleoptile after the transfer to a nitrogen atmosphere, and then increased to a value of 0.80 during the subsequent hours. The sum of nucleotides also dropped to 60% of the value in air in the seeds and to 30% in the coleoptiles. However, during the anaerobic growth of coleoptiles, a considerable increase in the nucleotide pool occurred.The incorporation of amino acids into proteins was measured at different stages in anoxic treatment. In rice embryo, we observed a considerable protein synthesis correlated with a high value of energy charge under anoxia. The analysis of labeled proteins by two-dimensional polyacrylamide gel electrophoresis showed a modified pattern of polypeptides synthesized during anoxic treatment. Some of these proteins were intensively labeled and appeared to be induced by anaerobic treatment.Our data indicate that high metabolic activity occurs in rice embryo under anoxia, which can be correlated with a high energy charge value. These phenomena may be part of the mechanisms which permit the adaptation of rice embryos to anaerobiosis. PMID- 16661972 TI - Isolation and Characterization of Chloroplast DNA from the Marine Chromophyte, Olisthodiscus luteus: Electron Microscopic Visualization of Isomeric Molecular Forms. AB - Chloroplast DNA (ctDNA) from the marine chromophytic alga, Olisthodiscus luteus, has been isolated using a whole cell lysis method followed by CsCl-Hoechst 33258 dye gradient centrifugation. This DNA, which has a buoyant density of 1.691 grams per cubic centimeter was identified as plastidic in origin by enrichment experiments. Inclusion of the nuclease inhibitor aurintricarboxylic acid in all lysis buffers was mandatory for isolation of high molecular weight DNA. Long linear molecules (40 to 48 micrometers) with considerable internal organization comprised the majority of the ctDNA isolated, whereas supertwisted ctDNA and open circular molecules averaging 46 micrometers were occasionally present. Also observed in this study were folded ctDNA molecules with electron dense centers ("rosettes") and plastid DNA molecules which have a tightly wound "key-ring" center. The ctDNA of Olisthodiscus has a contour length that is median to the size range reported for chlorophytic plants.A minor component of the total cellular DNA, which originates from a DNase insensitive cellular structure, has a buoyant density of 1.694 grams per cubic centimeter. This DNA consists predominantly of linear molecules, but open circles 11.5 micrometers in length and rare 22-micrometer dimers were also present.This study represents the first analysis of the extranuclear DNA of a chromophytic alga. PMID- 16661973 TI - Kinetics of ATP synthesis in pea cotyledon submitochondrial particles. AB - A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two K(m) values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low K(m) for ADP was involved in ATP synthesis. alpha,beta Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent K(m) of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the K(m) of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The K(m) of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis. PMID- 16661974 TI - Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3 Phosphate Acyltransferases from Chloroplasts. AB - Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.The enriched enzyme fractions possessed a specificity for glycerol 3 phosphate as acyl acceptor and did not use dihydroxyacetone phosphate. Besides acyl-CoA, acyl-acyl carrier protein also can function as acyl donor. With acyl CoA as acyl donor, the enzyme shows a high positional specificity, since the predominant product is 1-acylglycerol 3-phosphate.Different acyl-CoAs, when offered separately, were all accepted as substrates, whereas incubations with mixtures of palmitoyl-, stearoyl-, and oleoyl-CoA demonstrated a preference for oleic acid. The acyltransferase from spinach displays higher selectivity than does the enzyme from pea and, therefore, may be responsible for the preferred esterification of oleic acid at the C-1 position in chloroplast lipids and the exclusion of palmitic acid from this position as observed during in vivo labeling experiments. PMID- 16661975 TI - Optical and Potentiometric Study of the b- and c-Type Cytochromes in Mushroom Agaricus bisporus Lge Mitochondria. AB - Differential spectrometry revealed two species for the b-type, as well as for the c-type, cytochromes in mitochondria from Agaricus bisporus Lge. The two b-type components are denoted according to their peak position in the alpha region at room temperature, i.e. b(560) and b(566). The b(556) component present in all the studied higher plant mitochondria was not detected in the system. At 293 K, the c type cytochromes exhibit a common alpha band with a maximum at 550 nanometers. This band is split at 77 K, with peak positions at 547 nanometers (cytochrome c) and 552 nanometers (cytochrome c(1)).Redox titrations, analyzed according to a new method (Denis, Neau, Blein 1980 Bioelectrochem Bioenerg 7: 757-773), also yielded the resolution of two species of b-type and c-type cytochromes. The b components have midpoint potential values at pH 7.2 and 20 C of E(m1) = 40 +/- 10 millivolts and E(m2) = 175 +/- 10 millivolts. Their calculated individual difference spectra exhibit alpha bands with identical maxima at 561 nanometers, in contrast with optical observations. The midpoint potential values of the resolved c components are 210 +/- 10 millivolts and 292 +/- 5 millivolts at pH 7.2 and 20 C. Their corresponding calculated difference spectra have their alpha band maxima at 553 and 551 nanometers. These two species have been identified respectively with cytochrome c(1) and cytochrome c. The ratio of the maximum optical contributions, c/c(1), is in the range 4/1 to 5/1, in contrast with other results related to yeast or higher plant mitochondria where it amounts to 2/1 or even 1.3/1. PMID- 16661976 TI - Low proton conductance of plant cuticles and its relevance to the Acid-growth theory. AB - Evidence obtained on the relation between the pH of the medium and the growth of intact stem sections is compatible with the acid-growth theory only if the proton conductance of the cuticle is so low that the cuticle is an effective barrier to the entry or exit of protons from the tissue. By measuring the rate at which protons cross frozen-thawed epidermal strips of sunflower (Helianthus annuus L.) and soybean hypocotyls (Glycine max Morr.) and enzymically isolated cuticles of Berberis aquifolium Persh. and tomato (Lycopersicum esculentum Mill.) fruit, we have now demonstrated the low proton conductance of the cuticular layer. Unless the conductance is enhanced by abrasion of the cuticle or by removal of the cuticular waxes, proton movement into and out of a tissue across the cuticle will be significant only over long time periods. PMID- 16661977 TI - Uptake and distribution of N-phosphonomethylglycine in sugar beet plants. AB - Glyphosate (N-phosphonomethylglycine) was readily transported in sugar beet plants (Beta vulgaris L., Klein E type, monogerm). Concentrations in sink leaves reached 2.5 to 13.7 micromolar in 10 hours from a 15 millimolar solution supplied to one mature leaf. Distribution of glyphosate followed that of [(3)H]sucrose used as a marker for materials transported by phloem, indicating that this is the primary means for distribution of glyphosate. Possible mechanisms of entry into the sieve tubes were evaluated using isolated leaf discs. Concentration dependence of uptake and kinetics of exodiffusion from tissue indicate a passive, nonfacilitated mechanism. Uptake was not affected by pH, eliminating the passive, weak acid mechanism. Permeability of the plasmalemma to glyphosate was calculated as 1.7 x 10(-10) meters per second. This characteristic would allow slow entry and exit from the phloem, and together with other physiological parameters of the plant, is postulated to allow accumulation and transport in the phloem. PMID- 16661978 TI - Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis. AB - The distribution of ions, particularly K and Na, was studied in roots of barley seedlings grown on various ionic solutions. Analyses were made by means of electron probe x-ray microanalysis using frozen, fractured bulk specimens. By this technique, it was demonstrated that there can be variability in the ratio K/Na measured in the vacuoles of cortical cells, with this ratio often being lower in epidermal cells of the root than in the inner cortex. A sharp difference in the K/Na ratio was also found between cells of the endodermis and those of the adjacent cortex, and generally higher ratios of K/Na occurred in the stele than in the cortex. Estimation of the concentrations in the cytoplasm was at the limit of resolution of this technique, but it can be shown that the K/Na ratio in the cytoplasm was higher than that in the vacuole. In low salt roots, the K concentration in the cytoplasm was higher than that in the vacuoles. The results with the x-ray microprobe confirm other measurements based on flux analysis or analysis of small samples of the root. PMID- 16661979 TI - Effects of Light and of Fusarium solani on Synthesis and Activity of Phenylalanine Ammonia-Lyase in Peas. AB - Phenylalanine ammonia-lyase was purified from peas, and a specific antiserum against the enzyme was produced in rabbits. The antiserum was used to study the first 8 hours of the phenylalanine ammonia-lyase activity response in two different organs of the pea from different developmental stages and in response to two different stimuli. Etiolated seedlings were pulse-labeled with l [(35)S]methionine after either no light exposure or after specific periods of irradiation with blue light. Immature pods were pulse labeled with mixed l [(3)H]amino acids after specific time periods following inoculation of the pod endocarp surfaces with macroconidia of Fusarium solani. Immunoprecipitates isolated from extracts of each group were analyzed with sodium dodecyl sulfate disc gel electrophoresis and were found to contain a radioactive protein with an electrophoretic mobility identical to that of the phenylalanine ammonia-lyase subunit (M(r) 81,000). The radioactivity contained in the subunit band was interpreted as being due to de novo synthesis of the enzyme. The net rate of phenylalanine ammonia-lyase labeling, found to be initially low in both tissue types, rose dramatically, peaking at approximately a six- to ten-fold greater level at 4 hours after the beginning of the stimulus. Thereafter, the rate of labeling declined slowly. Inoculation with F. solani f. sp. pisi, a true pathogen of peas, caused a fifty per cent greater rate of peak labeling than did inoculation with a nonpathogen, F. solani f. sp. phaseoli. The time profile of the changing rate of labeling correlates with the changing activity level of the enzyme which peaks at 12 hours after the onset of the stimulus. The data presented favor a model which explains the changing activity of phenylalanine ammonia-lyase as being due to a changing rate of synthesis or degradation (or both) of the enzyme rather than due to the activation of a preformed zymogen. PMID- 16661980 TI - Vacuolar contents of fruit subepidermal cells from vitis species. AB - Enzymic treatment of mature, anthocyanin-containing grape berry sub-epidermal tissues from DeChaunac grapes released intact protoplasts. Filtration of the protoplast suspension through glass wool under mild suction resulted in the release of vacuoles. The total and individual contents of anthocyanins, flavonol glycosides, hydroxycinnamic acid esters, sugars, organic acids, and cations were determined in both tissue and vacuole preparations. A method for pH determination in intact vacuoles is reported. Based on the qualitative and quantitative anthocyanin composition, the average pH of the vacuoles was determined to be 2.7 (sd +/- 0.17). The data suggest that anthocyanins are present in fruit subepidermal tissues in a noncomplexed form. PMID- 16661981 TI - Sucrose uptake by developing soybean cotyledons. AB - Sucrose uptake by excised developing soybean cotyledons shows a biphasic dependence on sucrose concentration. At concentrations less than about 50 millimolar external sucrose, uptake can be described as a carrier-mediated process, with a K(m) of 8 millimolar. At higher external sucrose concentrations, a linear dependence becomes apparent, which suggests the participation of a nonsaturable component in total uptake. Sucrose absorption is dependent on the presence of an electrochemical potential gradient for protons since agents interfering with the generation or maintenance of this gradient (NaN(3) or carbonylcyanide-m-chlorophenyl hydrazone) decrease sucrose transport to a level at or below that predicted from the operation of the noncarrier-mediated process alone. The saturable component of sucrose uptake is also sensitive to the sulfhydryl-modifying compounds N-ethylmaleimide and p-chloro mercuribenzenesulfonate. The thiol-reducing agent diethioerythritol reverses fully the p-chloro-mercuri-benzenesulfonate inhibition, but not that of N-ethyl maleim de. Sucrose transport is sensitive to external pH, being decreased at high pH(0). Since sucrose-induced depolarization of the membrane potential and carrier mediated sucrose influx show similar pH-dependence, inhibitor sensitivity, and values of K(m) for sucrose, a sucrose/proton contransport process appears to operate in developing soybean cotyledon cells. Measurement of free space and intracellular sucrose concentrations in vivo suggests that the carrier-mediated process is fully saturated and that sucrose transport may be limiting for sucrose accumulation by the developing seed. PMID- 16661982 TI - Photoinhibition and P700 in the Marine Diatom Amphora sp. AB - The marine diatom Amphora sp. was grown at a light intensity of 7.0 x 10(15) quanta centimeter(-2) second(-1). Light saturation of photosynthesis for these cells was between 6.0 and 7.0 x 10(16) quanta centimeter(-2) second(-1). At light intensities greater than saturation, photosynthetic (14)CO(2) fixation was depressed, while P700 unit size (chlorophyll a concentration/P700 activity) increased and number of P700 units per cell decreased. After a 1-hour exposure of Amphora sp. to a photoinhibitory light intensity of 2.45 x 10(17) quanta centimeter(-2) second(-1), there was a 45 to 50% decrease in the rate of (14)CO(2) fixation relative to the rate at the culture light intensity. There also was a 25% increase in P700 unit size and a 30% reduction in the number of P700 units per cell but no change in total chlorophyll a concentration. Following this period of photoinhibition, the cells were returned to a light regime similar to that in the original culture conditions. Within 1 hour, both number of P700 units per cell and P700 unit size returned to levels similar to those of cells which were kept at the culture light intensity. The rates of photosynthesis did not recover as rapidly, requiring 2 to 3 hours to return to the rate for the nonphotoinhibited cells. Our results indicate that a decrease in P700 activity (with a resultant increase in P700 unit size) may be partially responsible for the photoinhibition of algal photosynthetic carbon dioxide fixation. PMID- 16661983 TI - Formate oxidation and oxygen reduction by leaf mitochondria. AB - Mitochondria isolated from the leaves of several plant species were investigated for the presence of NAD-linked formate dehydrogenase. The NADH produced was oxidized by the electron transport sequence and was coupled to ATP synthesis. The amounts of formate dehydrogenase, and, thereby, the capacity for formate dependent O(2) uptake, varied greatly among species. While no activity was detectable in mitochondria from soybean leaves, the rate of formate oxidation by spinach mitochondria was about one-half the rate of malate oxidation. In spinach, only mitochondria from green tissues oxidized formate. These last two observations raise questions as to the role of this reaction and the possible sources of the formate metabolized. PMID- 16661984 TI - Proton Fluxes Associated with Sugar Uptake in Vicia faba Leaf Tissues. AB - Vicia faba leaf fragments bring the pH of their incubation medium to about 4.7, whatever the initial pH value. At this pH, addition of 20 millimolar sucrose causes a transient (20 to 40 minutes) alkalinization (0.05 to 0.10 pH unit) of the medium. The alkalinization is not observed in the presence of p chloromercuribenzenesulfonic acid which blocks the sucrose carrier involved in phloem loading without affecting the ATPase (Delrot, Despeghel, Bonnemain 1980 Planta 149: 144-148). Addition of 20 millimolar glucose, fructose, or 3-O methylglucose induces weaker alkalinization than sucrose. Sequential additions of sugars show that: (a) sucrose- and hexose-induced proton fluxes are nearly saturated at 20 millimolar sugar (b) there is no competition between sucrose and hexoses for inducing proton influxes whereas (c) glucose and 3-O-methylglucose are competing for a common system.Autoradiographs performed under the conditions used for the observation of proton fluxes show a slight accumulation of [(14)C]sucrose into the veins within 2 minutes of uptake, whereas [(14)C]glucose and 3-O-methyl [(14)C]glucose are localized in the mesophyll. These data support the proton-sucrose cotransport hypothesis of phloem loading and show that mesophyll cells are able to take up hexoses by symport with protons.The apparent sucrose/proton stoichiometry is constant below 5 millimolar sucrose (about 1.9 sucrose per proton taken up) but increases up to 6 sucrose per proton, between 5 and 15 millimolar sucrose. This confirms our previous study indicating that above 5 millimolar sucrose, a system which exhibits little pH dependence is involved in the uptake.Simultaneous measurements of H(+) and K(+) fluxes indicate that sucrose uptake is accompanied by a reduction of K(+) uptake rate, thus suggesting that sucrose and K(+) uptake can compete in dissipating the protonmotive force. PMID- 16661985 TI - Light and dark reactions of the uptake hydrogenase in anabaena 7120. AB - Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole filaments or isolated heterocysts that contained nitrogenase consumed H(2) in the presence of C(2)H(2) or N(2) in a light-dependent reaction. If nitrogenase was inactivated by O(2) shock, filaments catalyzed H(2) uptake to an unidentified endogenous acceptor in the light. Addition of NO(3) (-) or NO(2) (-) enhanced these rates. Isolated heterocysts consumed H(2) in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H(2) uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanide-sensitive oxyhydrogen reaction that was inactivated by O(2) at O(2) concentrations above 2%. Light-dependent H(2) uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of O(2), light-dependent H(2) uptake occurred simultaneously with the oxyhydrogen reaction. The pH optima for both types of H(2) uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena. PMID- 16661986 TI - Physiological reactions of the reversible hydrogenase from anabaena 7120. AB - The reversible hydrogenase from Anabaena 7120 appeared when O(2) was continuously removed from a growing culture. Activity increased further when cells were incubated under argon in the dark or in the light plus 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Hydrogenase existed in an inactive state during periods of O(2) evolution. It could be reductively activated by exposure to reduced methyl viologen or by dark, anaerobic incubation. Hydrogenase-containing cells evolved H(2) slowly during dark anaerobic incubations, and the rate of H(2) evolution was increased by illumination with low intensity light. Light enhancement of H(2) evolution was of short duration and was eliminated by the ferredoxin antagonist disalicylidene diaminopropane. Physiological acceptors that supported H(2) uptake included NO(3) (-), NO(2) (-), and HSO(3) (-), and light had a slight influence on the rate of H(2) uptake with these acceptors. Low levels of O(2) supported H(2) uptake, but higher concentrations of O(2) inactivated the hydrogenase. Hydrogen uptake with HCO(3) (-) as acceptor was the most rapid reaction measured, and it was strictly light-dependent. It occurred only at low light intensities, and higher light intensities restored normal O(2)-evolving photosynthesis. It is suggested that hydrogenase is present to capture exogenous H(2) as a source of reducing equivalents during growth in anaerobic environments. PMID- 16661987 TI - Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa. AB - The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar ;M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO(3) once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N(2). During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments. PMID- 16661988 TI - RNA, proteins and polyamines during tube growth in germinating apple pollen. AB - Variations of RNA, protein, and free- and trichloroacetic acid-soluble bound polyamine levels were determined during tube growth in germinating Malus domestica Borkh. cv. Starkrimson pollen.During rehydration of pollen no marked differences were observed, whereas, during germination, RNA, proteins, and polyamines showed parallel decreases. At the same time, there was synthesis of RNA and polyamines as indicated by use of labeled precursors. The data indicate that during germination: (a) the genes for rRNA, tRNA, and probably mRNA are active; (b) the enzymes involved in polyamine biosynthesis are very active. High levels of free arginine during the first 15 minutes were observed, probably in response to a demand for this precursor in polyamine biosynthesis. Moreover, profiles of the variations in the specific activities of RNA and polyamines showed similar patterns. The results indicate that biosynthesis of RNA and polyamines precedes tube emergence. The possible role of these compounds, which are known to be released into the medium in the progamic phase of the fertilization processes, is considered. PMID- 16661989 TI - Localization of DNA in Mature and Young Wheat Chloroplasts Using the Fluorescent Probe 4'-6-Diamidino-2-phenylindole. AB - The spatial organization of chloroplast DNA in developing and dividing wheat chloroplasts was studied in the light microscope using the fluorescent probe 4'-6 diamidino-2-phenylindole, which binds specifically to DNA.The DNA of wheat chloroplasts was localized at the periphery of the plastid, frequently in a discrete band. No relocalization of the DNA was observed during plastid replication. This peripheral location of the DNA was shown to differ from the central random location of DNA in tobacco and spinach chloroplasts. PMID- 16661990 TI - Cytokinins from the Moss Physcomitrella patens. AB - Gametophore-over-producing mutants of the moss, Physcomitrella patens, when grown in liquid culture export high levels of cytokinin into their culture medium. The cytokinin produced by these mutants is postulated to account for their peculiar phenotype, that of mosses treated with exogenous cytokinin. N(6)-(Delta(2) isopentenyl)adenine, the major cytokinin, has been identified previously in two of these mutants (Wang, Cove, Beutelmann, Hartmann 1980 Phytochemistry 19: 1103 1105) and now in additional representatives. A second cytokinin, zeatin, has been identified by its chromatographic behavior and mass spectrum including chemical ionization mass spectrometry of its permethyl derivative. PMID- 16661991 TI - Cytokinin Biosynthesis in Mutants of the Moss Physcomitrella patens. AB - Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-(14)C]adenine to N(6)-[(14)C](Delta(2) isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N(6)-(Delta(2)-isopentenyl)adenine and N(6)-(Delta(2) isopentenyl)adenosine monophosphate. PMID- 16661992 TI - Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa. AB - The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-(14)C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [(14)C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [(14)C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [(14)C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [(14)C]geranylgeranyl pyrophosphate from [1-(14)C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino 1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway. PMID- 16661993 TI - Use of Phloem exudate technique in the study of amino Acid transport in pea plants. AB - The phloem exudation technique using ethylenediaminetetraacetic acid (EDTA) was evaluated in studies of amino acid translocation in Pisum sativum L. seedlings. Exudation of phloem sap from cut petioles of fully expanded leaves was enhanced by EDTA (20 millimolar disodium salt [pH 7.0]). Amino acids (mainly asparagine, homoserine, glutamate, and also aspartate and serine) were present in petiole exudates from EDTA-treated leaves at levels which were commonly 5- to 10-fold (or more) higher compared with water-treated controls. Exudation was greater from darkened leaves, and the pattern of amino acids was markedly different from the more uniform mixture leaking from water-treated controls.After feeding (14)C labeled amino acids to the leaf blade, distribution of radioactivity in components of the exudate differed from that of the leaf tissue, suggesting selectivity of amino acid loading. [(14)C]Asparagine was converted to 2 hydroxysuccinamic acid and to other amino acids by the leaf, but was recovered in exudate mainly as asparagine (60%) and aspartate (30%). Similarly, in the exudate, 65 to 70% of the label from [(14)C]-aspartate was in amino acids, although in the leaf tissue 50% was in the organic acid fraction and only 11% remained as aspartate. Metabolism of asparagine and aspartate was essentially the same in intact leaf blades as in EDTA-treated leaves. Despite the possibility of EDTA damage in the petiole, phloem loading of amino acids appeared to be relatively unimpaired. Although the amount of labeled material appearing in the exudate is less than the amount translocated in the intact plant, the technique is useful in the study of amino acid transport. PMID- 16661994 TI - Physiological rates of starch breakdown in isolated intact spinach chloroplasts. AB - Starch breakdown with rates above 10 muatom carbon per mg chlorophyll per hour has been monitored in spinach chloroplasts and compares favorably with the rates in whole leaves. Intact starch-loaded chloroplasts were prepared from protoplasts to avoid rupture during mechanical homogenization and rapid centrifugation. Particular attention was paid to the identification of all the products of starch degradation and to measuring the actual rates of their accumulation. The products of starch breakdown included triose phosphate, 3-phosphoglycerate, CO(2), glucose, and some maltose. Comparison of the rates of metabolism of added glucose and of the conversion of starch to phosphorylated intermediates showed that starch phosphorolysis was the major pathway leading to phosphorylated endproducts. From the results, the relative contribution of phosphorolysis and hydrolysis to starch breakdown and the contribution of glycolysis and the oxidative pentose phosphate cycle can be estimated. Phosphate has a large influence on the metabolism of the chloroplast in the dark. PMID- 16661995 TI - Photosynthesis of Lipids from CO(2) in Spinacia oleracea. AB - Young expanding spinach leaves exposed to (14)CO(2) under physiological conditions for up to 20 minutes assimilated CO(2) into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the (14)C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO(2)in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H(14)CO(3) into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor. PMID- 16661996 TI - Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans. AB - The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA. PMID- 16661997 TI - Isolation of a Factor from Apple that Agglutinates Erwinia amylovora. AB - Extracts prepared from apple seeds contain a factor (AF) capable of agglutinating cells of Erwinia amylovora. In drop agglutination tests, AF is more active in agglutinating an avirulent, acapsular strain of E. amylovora than a virulent, capsular strain. AF precipitates in agar plates with a receptor derived from boiled cells of avirulent acapsular strain and, therefore, can be located during fractionation by rocket electrophoresis. AF was heat-stable and had a pH optimum for agglutination near congruent with3.6 pH. The agglutination activity was not affected by the presence of Mg(2+), Ca(2+), or EDTA. AF was separated into two fractions (AF I and AF II) by elution from a Bio-Gel P-100 column. The precipitin and agglutination activities associated with AF II were found to be present in a positively charged molecule which was sensitive to treatment with protease and trypsin and, hence, presumably resides in a protein. The approximate molecular weight of AF II was determined to be 12,600 daltons. Besides precipitating the receptor derived from cells of avirulent acapsular strain, AF II was capable of precipitating extracellular polysaccharide from cultures of virulent capsular strain, sodium polygalacturonate, and carboxymethylcellulose. These three polymers also inhibited the agglutination activity associated with AF II. AF II could be replaced by poly-l-lysines in both the precipitin and agglutination assays. In addition, in antigen absorption experiments, poly-l-lysines were found to remove the receptors for AF II from the boiled extracts of avirulent acapsular strain. Based on these observations, it is proposed that the activity of AF II resides in a highly positively charged protein which causes agglutination of bacterial cells by interacting on a charge-charge basis with negatively charged components on the surface of the bacterial cells. PMID- 16661998 TI - In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins. AB - Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane associated 3'-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products. PMID- 16661999 TI - Carbohydrate metabolism of cactus in a desert environment. AB - The concentration of glucan, mucilage, soluble carbohydrates, and malic acid were determined in Opuntia bigelovii Engelm. during a 23-week period. The experiment began during the dry summer by irrigation to stimulate Crassulacean acid metabolism and was followed by 13 weeks of drought. After the 13-week drought period, the plants were irrigated throughout a 10-week period until late December. The maximum level of malic acid determined each day at dawn decreased throughout the drought period and increased after irrigation. High levels of malic acid occurring at dawn are indicative of active Crassulacean acid metabolism. Soluble carbohydrates also decreased during drought and increased after irrigation. Both glucan and mucilage increased slightly for about 9 weeks during the drought period and then began to decrease. Irrigation was accompanied by a further decrease in concentration of glucan and mucilage. Since both glucan and mucilage changed in a similar manner and since their concentrations in the tissue are correlated, it is hypothesized that both function as storage carbohydrates. Whereas glucan is the nocturnal substrate for malic acid synthesis, there are no data to support or refute a similar hypothesis for mucilage. PMID- 16662000 TI - Characterization of a molecular modification of phytochrome that is associated with its conversion to the far-red-absorbing form. AB - Phytochrome that has been photoinduced to pellet by irradiation of intact oat (cv. Garry) shoots and recovered from a pellet obtained by centrifugation of crude extracts exhibits modified behavior when compared to soluble phytochrome isolated from shoots that had never been irradiated. This modified behavior includes retarded mobility during sodium dodecyl sulfate polyacrylamide gel electrophoresis (Boeshore ML, LH Pratt 1980 Plant Physiol 66: 500-504). The electrophoretic mobility of several different kinds of phytochrome preparations were examined to study how this modification might arise.Phytochrome that was extracted in the pelletable condition from red-, far-red-irradiated tissue, but without added divalent cation so that it did not pellet, did not exhibit an altered electrophoretic mobility. Hence, this modification of phytochrome is not required for the expression in vitro of pelletability induced in vivo. Phytochrome that was extracted in the pelletable condition and in the far-red absorbing form, but without added divalent cation so that it did not pellet, and phytochrome in the far-red-absorbing form that remained in the supernatant after collection of pellets containing pelleted phytochrome both electrophoresed with reduced mobility. Thus, this modification does not arise as a consequence of phytochrome having been pelleted. Differential sensitivity of phytochrome to different handling conditions also is not the cause of this modification since the far-red-absorbing form of phytochrome, which was extracted in the pelletable condition but by the same protocol used to extract soluble phytochrome, also exhibited reduced mobility. Furthermore, the reduced electrophoretic rate is not due to a simple differential lability of the far-red-absorbing form of phytochrome to extraction conditions, since partially purified soluble phytochrome that was exposed in the far-red-absorbing form to the isolation and extraction conditions used for preparation of soluble phytochrome did not exhibit the alteration.The data are instead consistent with the more complex interpretation that phytochrome is modified in vitro if two conditions are met: (a) that phytochrome is extracted in the far-red-absorbing form or is converted to the far-red-absorbing form in the crude extract soon after extraction and (b), that phytochrome remains in the far-red-absorbing form in the crude extract for at least a brief period.The possibility that the phytochrome modification studied here might have arisen because of a change in carbohydrate content was tested by periodic acid Schiff staining of sodium dodecyl sulfate polyacrylamide gels. No carbohydrate was detected in any of the phytochrome preparations that were examined. This inability to detect carbohydrate is in direct contrast to the report of Roux et al. (1975 Physiol Plant 35: 85-90). PMID- 16662001 TI - Role of carotenoids in the phototropic response of corn seedlings. AB - The herbicide, 4 chloro-5-(methylamino)-2-(alpha,alpha,alpha,-trifluoro-m-tolyl) 3 (2H)-pyridazinone (SAN 9789), which blocks the synthesis in higher plants of colored carotenoids but not of flavins, was used to examine the involvement of carotenoids in corn seedling phototropism. It was concluded that "bulk" carotenoids are not the photoreceptor pigment based on the results that increasing concentrations of SAN 9789 (up to 100 micromolar) did not alter the phototropic sensitivity to 380 nanometers light (using geotropism as a control) and did not increase the threshold intensities of fluence response curves for both 380 and 450 nanometers light even though carotenoid content was reduced to 1 to 2% of normal. SAN 9789 treatment, however, did reduce seedling sensitivity toward 450 nanometers light indicating that carotenoids are involved in phototropism. Carotenoids, which are located mainly in the primary leaves, may act in phototropism as an internal screen, enhancing the light intensity gradient across the seedling and thus contributing to the ability of the seedling to perceive light direction. These results indicate that the action spectra for phototropic responses can be significantly affected by the absorbance of screening pigments in vivo thus altering its shape from the in vitro absorption spectrum of the photoreceptor pigment. PMID- 16662002 TI - Catabolism of [1-C]levulinic Acid by etiolated and greening barley leaves. AB - Levulinic acid (LA), a competitive inhibitor of delta-aminolevulinic acid (ALA) dehydratase (EC 4.2.1.24), has been used extensively in the study of ALA formation during greening. When [1-(14)C]LA is administered to etiolated barley (Hordeum vulgare L. var. Larker) shoots in darkness, (14)CO(2) is evolved. This process is accelerated when such tissues are incubated with 2 millimolar ALA or placed under continuous illumination. Label from the C-1 of LA becomes incorporated into organic acids, amino acids, sugars, lipids, and proteins during a 4-hour incubation in darkness or in the light. This metabolism is discussed in relation to the use of LA as a tool in the study of chlorophyll synthesis in higher plants. PMID- 16662003 TI - Effect of pH, Mg, CO(2) and Mercurials on the Circular Dichroism, Thermal Stability and Light Scattering of Ribulose 1,5-Bisphosphate Carboxylases from Alfalfa, Spinach and Tobacco. AB - Circular dichroism, differential scanning calorimetry and light-scattering measurements of ribulose 1,5-bisphosphate carboxylase (E.C. 4.1.1.39) from alfalfa, spinach and tobacco show: a) The conformation and thermal stability of the native carboxylases are sensitive to changes in pH and to activation of the enzyme with Mg(2+) and CO(2). The helical content, denaturation temperature (T(d)) and specific enthalpy of denaturation (Deltaq) decreased with increase in pH. Addition of Mg(2+) and CO(2) at pH 9 increased T(d) by 4 to 5 C; at pH 7.5 the changes in T(d) were smaller. b) Addition of mercurials produced changes in conformation and thermal stability. The decrease in helical content of the enzymes with increase in pH was enhanced by the addition of p chloromercuribenzoate. At pH 9, addition of p-chloromercuribenzoate or of 1-(3 (chloromercuri)-2-methoxypropyl)urea decreased T(d) by 11.4 to 20.2 C and Deltaq by 2.1 to 2.8 calories per gram. c) The spinach carboxylase undergoes the largest and the tobacco the smallest changes in conformation and thermal stability upon change in pH or treatment with mercurials. d) The calorimetric data suggest that the large and small subunits are heat denatured independently but at the same temperature. e) Light scattering measurements at pH 9 of p-chloromercuribenzoate treated tobacco enzyme showed that there is no dissociation into subunits upon heating to temperatures greater than T(d). A ;ball and string' model for the carboxylase molecule is proposed to reconcile independence of subunit denaturation with apparent strong interactions between subunits. PMID- 16662004 TI - Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves. AB - In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [(14)C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [(14)C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [(14)C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.The following experiments, in which tracer amounts of [(14)C]ethanolamine or [(14)C]formate were supplied to wilted barley leaves, implicated phosphoryl and phosphatidyl bases as intermediates in the methylation steps between ethanolamine and phosphatidylcholine. Label from both [(14)C]ethanolamine and [(14)C]formate entered phosphorylmonomethylethanolamine and phosphorylcholine very rapidly; these phosphoryl bases were the most heavily labeled products at 15 to 30 minutes after label addition and lost label rapidly as the fed (14)C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from [(14)C]ethanolamine and [(14)C]formate at early times; the corresponding free bases and nucleotide bases were not. Addition of a trapping pool of phosphorylcholine reduced [(14)C]ethanolamine conversion to both phosphatidylcholine and betaine, and resulted in accumulation of label in the trap.A computer model of the synthesis of betaine via phosphatidylcholine was developed from (14)C kinetic data. The model indicates that about 20% of the total leaf phosphatidylcholine behaves as an intermediate in betaine biosynthesis and that a marked decrease (>/=2-fold) in the half-life of this metabolically active phosphatidylcholine fraction accompanies wilting. Dual labeling experiments with [(14)C]choline and [(3)H]glycerol confirmed that the half-life of the choline portion of phosphatidylcholine falls by a factor of about 2 in wilted leaves. PMID- 16662005 TI - Cycloheximide stimulation of cyanide-resistant respiration in suspension cultures of senescent pear fruit cells. AB - Pear fruit cells undergoing a period of senescence in auxin-deprived media develop a substantial cyanide resistant respiration in response to the addition of 0.7 to 3.5 micromolar cycloheximide. The inhibitor does not affect overall cellular repiratory activity and titrations with salicylhydroxamic acid reveal that only a minor portion, about 10%, of the alternate pathway is utilized by the cycloheximide-treated senescent cells. The alternate respiratory pathway appears to be of mitochondrial origin but is not induced by chloramphenicol. PMID- 16662006 TI - Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean. AB - All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD(+) and NADP(+). The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH(4)Cl and 1.5 at 12.5 millimolar NH(4)Cl. K(m) values for NH(4) (+) and NAD(P)H are reduced at low salt concentrations. The low K(m) values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations. PMID- 16662007 TI - Effects of carbon dioxide on ethylene production and action in intact sunflower plants. AB - A continuous flow system was used to study the interactions between carbon dioxide and ethylene in intact sunflower (Helianthus annuus L.) plants. An increase in the concentration of carbon dioxide above the ambient level (0.033%) in the atmosphere surrounding the plants increased the rate of ethylene production, and a decrease in carbon dioxide concentration resulted in a decrease in the rate of ethylene production. The change in the rate of ethylene production was evident within the first 15 minutes of the carbon dioxide treatment. Continuous treatment with carbon dioxide was required to maintain increased rate of ethylene production. The rate of carbon dioxide fixation increased in response to high carbon dioxide treatment up to 1.0%. Further increases in carbon dioxide concentration had no additional effect on carbon dioxide fixation. Carbon dioxide concentrations higher than 0.11% induced hyponasty of the leaves whereas treatment with 1 microliter per liter ethylene induced epinasty of the leaves. PMID- 16662008 TI - Cadmium distribution and chemical fate in soybean plants. AB - The distribution and chemical behavior of Cd(2+) in tissues and its chemical form in xylem water of soybean plants (cv. Williams) were investigated. Following root absorption, Cd is strongly retained by roots, with only 2% of the accumulated Cd being transported to leaves; as much as 8% was transported to seeds during seed filling. In vivo xylem exudates contained two anionic Cd complexes in addition to inorganic forms of Cd. Once accumulated in root and leaf tissues, Cd rapidly equilibrated between the insoluble, soluble, and organelle fractions. Of the solubles, which contain 50% of the Cd, >50% was associated with components of >10,000 molecular weight, and <8% was associated with <500 molecular weight components. Cadmium accumulated in soybean seeds was primarily associated with cotyledons. Fractionation of seeds showed the soy proteinate and soy whey to contain 32 and 50% of the accumulated Cd, respectively. PMID- 16662009 TI - Effect of nitrate in the rooting medium on carbohydrate composition of soybean nodules. AB - Nodulated soybean plants (Glycine max [L.] Merr) were grown in sand culture. Carbohydrate composition of nodules, roots, and leaf blades was determined and related to the effects of nitrate in nutrient solution on nodule growth and on nitrogenase activity of nodules.When plants were grown without nitrate for 6 weeks and then supplied with 150 milligrams NO(3)-N/liter for 4 or 7 days, there was an 80% decline in acetylene reduction activity of nodulated roots, relative to the 0-N control. The 80% decline in nitrogenase activity was accompanied by a decline in nodule glucose concentration of about 0.15 milligram per gram fresh weight and an increase in nodule sucrose concentration of about 0.23 milligram per gram fresh weight.Plants were grown with 0, 30, or 100 milligrams NO(3) N/liter for 5 or 6 weeks to study long-term effects of nitrate on nodule growth. The 100-N treatment reduced nodule weight/plant by 70% but reduced the sum of sucrose + glucose + fructose concentration in nodules by only 12%. The ratios of [sucrose] in nodules/[sucrose] in roots and [fructose] in nodules/ [fructose] in roots increased slightly in response to nitrate, indicating that nitrate affects sugar concentration in roots more than sugar concentration in nodules.The effect of nitrate on glucose concentration of nodules was consistently negative. However, if it is assumed that sucrose, glucose, and fructose are equivalent in their ability to support nodule functions, then the overall results are not consistent with the hypothesis that nitrate inhibits nodule growth and activity by reducing the accumulation of carbohydrate in nodules. PMID- 16662010 TI - Organelle-specific isozymes of citrate synthase in the endosperm of developing ricinus seedlings. AB - Chromatographic analysis of organelle-associated citrate synthase activity revealed distinct mitochondrial and glyoxysomal forms of the enzyme. The chromatographic elution patterns on hydroxylapatite, carboxymethylcellulose and DEAE-cellulose of citrate synthase from the endosperm of 4.5-day-old castor bean seedlings revealed significant differences for mitochondrial and glyoxysomal activities of the enzyme. The endoplasmic reticulum-associated citrate synthase activity eluted from DEAE-cellulose in a pattern that was identical to that of the glyoxysomal activity. The same kinds of organelle specific isozyme elution patterns were observed with young, developing seedlings. Gibberellic acid treatment of young seedlings increased total recoverable citrate synthase activity from endosperm tissue but did not modify the organelle specific isozyme relationships. PMID- 16662011 TI - Effects of leupeptin on proteinase and germination of castor beans. AB - Leupeptin, a tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloidstorage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH proteinase is intimately involved in the mobilization of storage proteins. PMID- 16662012 TI - Involvement of ethylene in chlorophyll degradation in peel of citrus fruits. AB - The effect of ethylene on chlorophyll degradation in the peel of Robinson tangerine (X Citrus reticulata Blanco) and calamondin (X Citrofortunellamitis [Blanco] Ingram and Moore) fruits was studied. The chlorophyll degrading system in the peel of these two citrus species was not self-sustaining but required ethylene to function. Chlorophyll degradation ceased immediately when fruit were removed from ethylene and held in ethylene-free air at 0.2 atmospheric pressure. However, at atmospheric pressure, chlorophyll degradation continued for 24 hours in the absence of exogenous ethylene. Although chlorophyllase levels were negatively correlated with chlorophyll content in the peel (r = -0.981; P < 0.01), the level of chlorophyllase activity did not change when fruit were removed from ethylene, even though chlorophyll degradation had stopped. From these observations, it was concluded that ethylene is necessary for chlorophyll degradation in the two species of citrus studied, but its primary role is not solely for the induction of chlorophyllase activity. PMID- 16662013 TI - Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin. AB - Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-(14)C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH(3) content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-(14)C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of (14)CO(2) followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low. PMID- 16662014 TI - Differential compartmentation of gibberellin a(1) and its metabolites in vacuoles of cowpea and barley leaves. AB - The metabolism and efflux of gibberellin A(1) (GA(1)) taken up by leaves of cowpea (Vigna sinensis cv. Blackeye pea No. 5), as well as the distribution of GA(1) metabolites in the protoplasts and vacuoles of cowpea and barley (Hordeum vulgare L. cv. Numar), were studied.GA(1) is metabolized rapidly in cowpea leaf discs to products tentatively identified as gibberellin A(8) (GA(8)) and gibberellin A(8) glucoside (GA(8)-glu). After labeling leaf discs with [(3)H]GA(1) for 1 hour, the release of radioactivity from the leaf was followed. Over a 12-hour period, the level of radioisotope in the tissue declined to about 35% of the original, after which no further release was observed. At this time, almost all of the radioactivity remaining in the leaf was GA(8)-glu, while most of the radioactivity which had been released was unmetabolized GA(1).Mesophyll protoplasts and vacuoles were isolated from cowpea and barley leaves previously fed [(3)H]GA(1). These protoplasts retain the ability to metabolize GA(1), indicating that neither the leaf structure nor the cell wall is necessary for this metabolism. A higher proportion of GA(8)-glu was found in the vacuoles relative to the entire protoplasts. The results obtained suggest that GA(1) metabolites are preferentially compartmentalized in the vacuoles relative to GA(1). PMID- 16662015 TI - Foliar Fatty Acids and Sterols of Soybean Field Fumigated with SO(2). AB - Sixty-day-old soybean plants were exposed in the field to 78.7 parts per one hundred million of SO(2) in an open-air fumigation system for 20 days. Leaves from the top one-fourth and bottom one-fourth of the plants were analyzed for chlorophyll, free fatty acids, fatty acid esters, polar lipid fatty acids, and sterols. Fumigated plants had a lower chlorophyll, free fatty acid, and polar lipid content, but a higher fatty acid ester content. Of the individual fatty acids, linoleic and linolenic acid increased with SO(2) fumigation while palmitic acid decreased. SO(2) fumigations had only a minor effect on leaf sterols. In general, the lower, more mature leaves showed a greater response to SO(2) exposure. PMID- 16662016 TI - Annual Variation in Sterol Levels in Leaves of Taraxacum officinale Weber. AB - Sterol levels in dandelion (Taraxacum officinale Weber) leaves were monitored over a period of 19 months. Sitosterol was the most abundant free sterol, followed by stigmasterol, then campesterol. Cholesterol could not be detected. With the exception of stigmasterol and campesterol, esters were present in greater quantities than were free forms, with 4,4-dimethyl sterol esters being the most abundant type. Glycosides occurred only sporadically. Free 4-demethyl sterols were maximal during the winter months; levels correlated negatively with sunshine and temperature, but proportions did not alter significantly. Sitosterol ester and cycloartenol ester (but not others) showed the opposite response, with levels correlating positively with sunshine and temperature. Relative amounts of 4-demethyl sterol esters remained reasonably constant, but those of cycloartenol ester and 24-methylene cycloartanol ester varied on an annual basis and were negatively correlated with each other. PMID- 16662017 TI - Interaction of ethylene and a cytokinin in promoting hypocotyl elongation in a dwarf strain of watermelon. AB - Experiments were conducted to study the interaction of ethylene and the cytokinin N(6)-benzyladenine (BA) in promoting hypocotyl elongation in a dwarf strain of watermelon (Citrullus lanatus [Thunb] Matsu. and Nakai). Optimum promotion of hypocotyl elongation is elicited by an apical treatment with 0.2 microgram BA. At dosages above 0.3 microgram per apex, BA-enhancement of elongation is reduced concomitant with stimulation of ethylene production and lateral expansion of hypocotyls. Application of the ethylene biogenesis inhibitor, aminoethoxyvinylglycine, at dosages from 0.3 to 10 micrograms per apex inhibited BA-induced ethylene production. In seedlings treated with 0.2 microgram BA, 10 micrograms aminoethoxyvinylglycine per apex reduced ethylene production to about one-third of control levels and reduced BA stimulation of hypocotyl elongation by 74%. Exposure of watermelon seedlings to 60 +/- 10 nanoliters per liter of ethylene in a flowing system nearly eliminated aminoethoxyvinylglycine inhibition of BA-promoted growth. The results suggest that physiological levels of internal ethylene are required for cytokinin promotion of hypocotyl elongation in watermelon. PMID- 16662019 TI - Ozone Sensitivity in Sweet Corn (Zea mays L.) Plants: A Possible Relationship to Water Balance. AB - Stomatal characteristics affecting gas exchange were compared in two sweet corn hybrids (var. Bonanza and Monarch Advance) which differ in foliar ozone sensitivity. No significant differences were observed in stomatal frequencies, guard cell lengths, or conductances to water vapor diffusion.When plant water status was compared, leaf-water potentials for the ozone-resistant cultivar, Bonanza, were lower. A comparison of the relationship between soil- and leaf water potential indicated that the leaf-water potential of Bonanza was more sensitive to declines in soil-water potential. Additionally, a comparison of stomatal conductance to water vapor diffusion as soil moisture declined and following root detachment indicated that stomata of Bonanza were, likewise, more sensitive to increasing water stress. Data suggest that these differences are attributable to a greater shoot-to-root fresh weight ratio and higher resistance to water movement in the water-conducting tissues for Bonanza.Our observations suggest that root and water delivery system characteristics play a major contributory role in the determination of foliar ozone sensitivity in this species. PMID- 16662018 TI - Effect of vanadate on bean leaf movement, stomatal conductance, barley leaf unrolling, respiration, and phosphatase activity. AB - Vanadate (Na(3) VO(4)) inhibits leaf movement and stomatal conductance of Phaseolus vulgaris L. cv. Carlos Favorit in light-dark cycles as well as photomorphogenetic leaf unrolling of Hordeum vulgare L. cv. Rupal. Inhibition was 50% by 10 to 100 micromolar vanadate and 100% by millimolar vanadate. Leaf unrolling was also inhibited by oligomycin and diethylstilbestrol.Millimolar vanadate is required to affect respiration of bean plants and barley leaves, while even this concentration has no effect on respiration of isolated pulvinal tissue.A nonspecific phosphatase activity extracted from both bean pulvini and barley leaves was found in all of four fractions of a differential centrifugation procedure (i.e. cell walls + nuclei, mitochondria, microsomes, super-natant), but more than 90% of the specific and total activity was located in the supernatant. The phosphatase activities of all fractions were inhibited by concentrations of vanadate similar to those which inhibited leaf movement, stomatal conductance of bean, and unrolling of barley leaves. PMID- 16662020 TI - Ploidy effects on anatomy and gas exchange of tall fescue leaves. AB - A growth chamber study was designed to interpret differences in CO(2) exchange rate (CER) and leaf diffusive resistance among 4X, 6X, 8X, and 10X ploidy levels of tall fescue (Festuca arundinacea, Schreb). Mesophyll cell size, stomatal density, number of major and minor veins, and bundle cap size of leaf blades were evaluated. Diffusive resistance tended to decrease and CER to increase with increasing ploidy level. Mean stomatal density decreased from 43.6 per square millimeter to 30.6 per square millimeter as ploidy level increased from 4X to 8X. The 10X ploidy level exhibited the highest stomatal density, 47.2 per square millimeter. Major veins decreased from a mean of 14.2 to 10.2, and minor veins increased from a mean of 4.2 to 6.6, per leaf blade as ploidy increased from 4X to 10X. Total number of veins decreased significantly from a mean of 18.4 to 15.7 as ploidy increased from 4X to 8X.Length and width of mesophyll cells tended to increase as ploidy increased from 4X to 8X, but then decreased again at 10X. The number of cells in the bundle cap showed no trend among ploidy levels. Estimated volume of mesophyll cells increased six times between the 4X and the 6X level while chromosome number of nuclear DNA per cell increased only 50%. However, increases in estimated cell volume were proportional to chromosome number as ploidy increased from 6X to 8X. The relationship between cell volume and chromosome number at 10X was intermediate between that at 4X and 6X or 8X.Differences in stomatal density and diffusive resistance did not fully account for the ploidy effect on CER. Further mesophyll cell volume was positively related to CER, a factor contrary to earlier experiments. PMID- 16662021 TI - Photosynthesis in Polyploid Tall Fescue : II. PHOTOSYNTHESIS AND RIBULOSE-1, 5 BISPHOSPHATE CARBOXYLASE OF POLYPLOID TALL FESCUE. AB - Net photosynthesis on a leaf area and leaf weight basis increased significantly with ploidy in a 4X, 6X, 8X and 10X allopolyploid series of tail fescue (Festuca arundinacea Schreb.). Total protein did not increase significantly with ploidy. Rocket immunoelectrophoresis was used to quantitate ribulose-1, 5-bisphosphate carboxylase (RuBPCase) protein. RuBPCase content, expressed on both a concentration basis and as a percentage of total protein increased significantly with ploidy in both field and greenhouse experiments. The range of RuBPCase content was 16 to 73% of total protein and 2.8 and 6.5 mg/ml of extract. Specific activity of RuBPCase did not increase significantly with ploidy. Chlorophyll concentration increased as a quadratic function of ploidy, with the mean for 8X genotypes representing maximal chlorophyll content. Evidence is presented that increasing concentrations of RuBPCase are associated with higher net photosynthesis rates in tall fescue. This suggests that RuBPCase may represent a marker for increased net photosynthesis. RuBPCase was extracted in a partially active state or inhibited state and must be fully activated by Mg(2+) and HCO(3) (-) to measure maximal activities. Polyploidization appeared to increase selectively the allocation of total protein for synthesis of RuBPCase; however, there was also a range for carboxylase content among the genotypes within a given ploidy level. PMID- 16662022 TI - Effect of Sulfhydryl Reagents on the Biophysical Properties of the Plasmalemma of Chara corallina. AB - The administration of the sulfhydryl reagent N-ethyl-maleimide (NEM) to internodal cells of Chara corallina caused alterations in the biophysical properties of the plasmalemma, as measured with electrophysiological and radioactive tracer techniques. The membrane potential depolarized to, or near, the calculated Nernst potential for potassium (E(K)) after 30 seconds' exposure to 0.1 millimolar NEM. During this time, the ATP level did not decrease below the control value, and the specific membrane resistance did not increase; only upon further exposure to NEM did the resistance approach the value observed in the dark. In the depolarized state, the membrane potential responded to changes in the external potassium concentration in the manner of a K(+)-electrode, but it retained it's relative insensitivity to external sodium.THESE RESULTS ARE INTERPRETED IN THE FOLLOWING MANNER: NEM causes a) an increase in the membrane permeability to K(+) (i.e. an increase in K(+) conductance); and b) perturbation of the electrogenic transport system(s) of the plasma membrane. The latter effect is manifested in a manner that is not consistent with an inhibition of ATP catalysis by a voltage-dependent ATPase possessing conductance. The nonpermeant sulfhydryl modifier, p-chloromercuribenzenesulfonic acid, appeared to affect membrane properties in a similar, but reversible, way. PMID- 16662023 TI - Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin. AB - Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P 40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines. PMID- 16662024 TI - Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L. AB - Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 x 10(5) daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with beta-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed. PMID- 16662025 TI - Iron Uptake and Translocation by Macrocystis pyrifera. AB - Parameters of iron uptake have been determined for blade tissue of Macrocystis pyrifera (L.) C. Ag. These include the effects of iron concentration, light, various inhibitors, and blade type. All experiments were conducted in the defined artificial seawater Aquil. Iron uptake is light independent, energy dependent, and dependent on the reduction from Fe(3+) to Fe(2+). Iron is concentrated in the sieve tube exudate; exudate analysis revealed the presence of other micronutrients. Iron and other micronutrient translocation is discussed. PMID- 16662026 TI - Correlation between Changes in Mitochondrial Membranes of Artichoke Tubers and Their Hardening and Dormancy. AB - Spin labeling studies using mitochondrial membranes of Jerusalem artichoke (Helianthus tuberosus L.) showed that the decrease during winter in the temperatures of the upper and lower lipid transitions correlated with the development of freezing hardiness of the tubers. The killing temperature for tuber tissue reached a minimum of -12 C, about 5 C degrees lower than the lower transition. Freeze-hardiness decreased when the lower transition increased at the time of sprouting.Low temperature storage was not required to induce freeze hardiness or to lower the transitions. These changes occurred in tubers under field conditions and at a constant growth temperature of 25 +/- 1 C. In both conditions, tuber dormancy preceded the mitochondrial membrane changes.In young nondormant tubers, cessation of growth by storage at 0 or 4 C and the induction of dormancy with abscisic acid led to decreased temperatures of the upper and lower transitions.Changes in Arrhenius activation energy of succinate oxidase were correlated with the seasonal changes in the upper and lower transitions confirming that these were indeed a reflection of altered thermal responses of mitochondrial membranes, which might be part of a general mechanism in this plant of insuring membrane integrity during the freeze-thaw process. In contrast with chilling injury, there is no precise correlation between the temperature of the lower transition and the temperature below which freezing injury occurs. PMID- 16662027 TI - Pyrophosphorylases in Solanum tuberosum: II. CATALYTIC PROPERTIES AND REGULATION OF ADP-GLUCOSE AND UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITIES IN POTATOES. AB - Pyrophosphorylytic kinetic constants (S(0.5), V(max)) of partially purified UDP glucose- and ADP-glucose pyrophosphorylases from potato tubers were determined in the presence of various intermediary metabolites. The S(0.5) of UDP-glucose pyrophosphorylase for UDP-glucose (0.17 millimolar) or pyrophosphate (0.30 millimolar) and the V(max) were not influenced by high concentrations (2 millimolar) of these substances. The most efficient activator of ADP-glucose pyrophosphorylase was 3-P-glycerate (A(0.5) = 4.5 x 10(-6) molar). The S(0.5) for ADP-glucose and pyrophosphate was increased 3.5-fold (0.83 to 0.24 millimolar) and 1.8-fold (0.18 to 0.10 millimolar), respectively, with 0.1 millimolar 3-P glycerate while the V(max) was increased nearly 4-fold. The magnitude of 3-P glycerate stimulation was dependent upon the integrity of key sulfhydryl groups ( SH) and pH. Oxidation or blockage of -SH groups resulted in a marked reduction of enzyme activity. Stimulations of 3.1-, 2.9-, 4.8-, and 9.5-fold were observed at pH 7.5, 8.0, 8.5, and 9.0, respectively, in the presence of 3-P-glycerate (2 millimolar). The most potent inhibitor of ADP-glucose pyrophosphorylase was orthophosphate (I(0.5) = 8.8 x 10(-5). molar). This inhibition was reversed with 3-P-glycerate (1.2 x 10(-4) molar), resulting in an increased I(0.5) value of 1.5 x 10(-3) molar. Likewise, orthophosphate (7.5 x 10(-4) molar) caused a decrease in the activation efficiency of 3-P-glycerate (A(0.5) from 4.5 x 10(-6) molar to 6.7 x 10(-5) molar). The significance of 3-P-glycerate activation and orthophosphate inhibition in the regulation of alpha-glucan biosynthesis in Solanum tuberosum is discussed. PMID- 16662028 TI - Partial purification and characterization of endoproteinases from senescing barley leaves. AB - Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP(1)) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP(2)) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP(2) contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [(14)C]ribulose-1,5-bisphosphate carboxylase ([(14)C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [(14)C]RuBPCase biphasically. A third endoproteinase (EP(3)), not detected by standard proteolytic assays, was observed when [(14)C]RuBPCase was the substrate. PMID- 16662029 TI - Compartmentation of Sulfur Metabolites in Tobacco Cells : USE OF EFFLUX ANALYSIS. AB - The distribution of sulfur-containing metabolites in cultured tobacco cells was determined by analyzing efflux kinetics. Transported sulfate rapidly labeled the cytoplasmic pools of sulfate (1 hour) and sulfur amino acids (6 hours). Excess sulfate and amino acids were transported into the vacuole. The size and distribution of the amino acid pool was not affected by increasing the sulfate content of the cells.Transported cysteine was rapidly degraded to sulfate. The cytoplasmic pools of sulfate and amino acids were completely labeled within 6 hours, whereas the vacuolar pools were labeled more slowly. The intracellular cysteine pool was elevated by decreasing the pH of the transport medium. In all experiments, between 60 and 90% of the label was present in the vacuole. PMID- 16662030 TI - Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts. AB - Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4 (2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation. PMID- 16662031 TI - Desaturation of Fatty acids associated with monogalactosyl diacylglycerol: the effects of san 6706 and san 9785. AB - The effects of two substituted pyridazinone herbicides, San 6706 and San 9785, on photosynthesis, dark respiration, and fatty acid metabolism were studied in mature leaf tissue of Vicia faba.Both San 6706 and San 9785 inhibited photosynthesis within 2 hours after initial exposure of leaf tissue to the chemicals although San 9785 was more effective in inhibiting photosynthesis than San 6706. Neither San 6706 nor San 9785 had any marked effect on dark respiration.Kinetic studies using (14)CO(2) indicated that synthesis of 18:3 esterified to monogalactosyl diacylglycerol (MGDG) was not completely inhibited by San 9785 up to 48 hours after feeding. Radioactivity was observed to accumulate in MGDG (digalactosyl diacylglycerol [DGDG] and sulpholipid [SL]) 18:2 at the expense of 18:3 but the specific radioactivity of MGDG 18:3 continued to increase throughout the experiment indicating only partial inhibition of MGDG 18:3 synthesis. No significant differences were observed in the metabolism of other fatty acids. The metabolism of fatty acids from leaf tissue was not affected by treatment with San 6706.The data indicate that there are at least two sites for 18:2 desaturation to form 18:3, one associated with MGDG in the chloroplast, which is inhibited by San 9785, and one or more sites not inhibited by San 9785. The fatty acid specific radioactivity data support the hypothesis that there is vectorial transfer of fatty acids from phosphatidyl choline to MGDG to produce the large quantities of MGDG 18:3 found in higher plant tissue. PMID- 16662032 TI - Metabolism of linoleic Acid by barley lipoxygenase and hydroperoxide isomerase. AB - The oxidation of linoleic acid in incubation mixtures containing extracts of barley lipoxygenase and hydroperoxide isomerase, and the production of these enzymes in quiescent and germinated barley, were investigated. The ratio of 9 hydroperoxylinoleic acid to 13-hydroperoxylinoleic acid was higher for incubation mixtures containing extracts of quiescent barley than for mixtures containing extracts of germinated barley; production of 13-hydroperoxylinoleic acid from germinated barley exceeded that of quiescent barley. Hydroperoxy metabolites of linoleic acid were converted to 9-hydroxy-10-oxo-cis-12-octadecenoic acid, 13 hydroxy-10-oxo-trans-11-octadecenoic acid, and small amounts of 11-hydroxy-12,13 epoxy-cis-9-octadecenoic acid and 11-hydroxy-9,10-epoxy-cis-13-octadecenoic acid whether quiescent or germinated barley was the enzyme source; a fifth product, 13 hydroxy-12-oxo-cis-9-octadecenoic acid was formed only when germinated barley was the enzyme source.Lipoxygenase was readily extracted by buffer, but hydroperoxide isomerase was bound in a catalytically active state to the insoluble barley grist and was efficiently extracted only when Triton X-100 was included in the extraction buffer. Hydroperoxide isomerase was localized in the embryo of quiescent barley, but it was present in the embryo, acrospire, and in small but concentrated amounts in the rootlet of germinating barley. The levels of both lipoxygenase and hydroperoxide isomerase increased through the thirteenth day of germination. PMID- 16662033 TI - Characterization of Carrot Cell Wall Protein : I. EFFECT OF alpha, alpha' DIPYRIDYL ON CELL WALL PROTEIN SYNTHESIS AND SECRETION IN INCUBATED CARROT DISCS. AB - A single glycoprotein accounts for the majority of radioactivity secreted to the cell wall when incubated carrot (Daucus carota) discs are labeled with radioactive proline or arabinose. The ferrous chelator alpha,alpha'-dipyridyl prevents the synthesis of this protein. A new proline-labeled protein is made in the presence of alpha,alpha'-dipyridyl and is secreted to the cell wall. The protein has little, if any, carbohydrate attached to it and has a molecular weight of 55,000 daltons. This protein appears to be the nonhydroxylated, nonglycosylated form of the major cell wall glycoprotein. alpha,alpha'-Dipyridyl does not prevent proline label from becoming tightly (presumably covalently) bound to the cell wall, providing further evidence that hydroxylation and arabinosylation are not required for the covalent attachment of proteins to the cell wall. Messenger RNA extracted from incubated carrot discs produces a product which electrophoreses similarly to the protein made in the presence of alpha,alpha'-dipyridyl. The possible use of the carrot disc system to study gene structure and regulation is discussed. PMID- 16662034 TI - Characterization of Carrot Cell Wall Protein : II. IMMUNOLOGICAL STUDY OF CELL WALL PROTEIN. AB - Antiserum was raised against a synthetic nona-peptide which was predicted to have considerable homology with the unhydroxylated, unglycosylated precursor of cell wall proteins from several plants. The antiserum is able to recognize the major cell wall protein of incubated carrot (Daucus carota) root discs which is produced when the discs are treated with a proline hydroxylase inhibitor and then labeled with radioactive proline. This technique has potential applications in studying cell wall biosynthesis and its regulatory control mechanisms. PMID- 16662035 TI - Effects of growth irradiance levels on the ratio of reaction centers in two species of marine phytoplankton. AB - Cells of two species of single-celled marine algae, the diatom Skeletonema costatum (Greve), Cleve, and the chlorophyte Dunaliella tertiolecta Butcher, were cultured in white light of high (500-600 microeinsteins per square meter per second) and low (30 microeinsteins per square meter per second) intensity. For both algal species, cells grown at low light levels contained more chlorophyll a and had a lower ratio of chlorophyll a to chlorophylls b or c than did cells grown at high light levels. When photosynthetic unit sizes were measured on the basis of either oxygen flash yields or P(700) photooxidation, different results were obtained with the different species. In the chlorophyte, the cellular content of photosystem I (PSI) and photosystem II (PSII) reaction centers increased in tandem as chlorophyll a content increased so that photosynthetic unit sizes changed only slightly and the ratio PSI:PSII reaction centers remained constant at about 1.1. In the diatom, as the chlorophyll content of the cells increased, the number of PSI reaction centers decreased and the number of PSII reaction centers increased so that the ratio of PSI:PSII reaction centers decreased from about unity to 0.44. In neither organism did photosynthetic capacity correlate with changes in cellular content of PSI or PSII reaction centers. The results are discussed in relationship to the physical and biological significance of the photosynthetic unit concept. PMID- 16662036 TI - Contributions of Respiratory and Photosynthetic Pathways during Growth of a Facultative Photoautotrophic Cyanobacterium, Aphanocapsa 6714. AB - Comparison of the growth parameters and photosynthetic capacities of cells of Aphanocapsa 6714 under various growth conditions led to the following conclusions: (a), no enzymic regulation of CO(2)/glucose assimilation takes place in this strain; (b), functioning of photodependent phosphorylating pathways turns off oxidative ATP synthesis; (c), no efficient regulation of pigment synthesis exists in these cells; (d), most modulations of photosynthetic activities probably occur through structural modifications of the photosynthetic membranes (a small proportion of the pigments might appear as a nonintegrated pool in the cell and be sensitive to synthesis regulation); and (e), photosystem II activity would be dependent on light intensity in a discontinuous way, the consequence of this property being the appearance of two successive exponential phases during phototrophic growth in adequate light conditions. PMID- 16662037 TI - Hydroxyproline Glycosides in Secretory Arabinogalactan-Protein of Phaseolus vulgaris L. AB - The carbohydrate moiety of secretory arabinogalactan protein in bean seedlings (Phaseolus vulgaris L. cv. Prelude) is attached to the peptide backbone through hydroxyproline, serine, and threonine. Hydroxyproline-linked side chains, consisting of arabinose, galactose, glucose, and rhamnose, comprise the major part of the sugar residues. These hydroxyproline glycosides differ from those in non-extractable cell wall protein but show similarities with those in wall protein of the alga Chlamydomonas. PMID- 16662038 TI - Proton efflux from corn roots induced by tripropyltin. AB - Tripropyltin restores medium acidification by washed corn root tissue in which electrogenic H(+) efflux has been blocked by ATPase inhibitors or injury. However, the restored H(+) efflux is not electrogenic and will not drive K(+) influx, and, by itself, tripropyltin is inhibitory to K(+) influx. Tripropyltin elicits a 5-fold increase in endogenous chloride efflux, and Cl(-)/OH(-) exchange can, thus, account for the observed acidification of the medium. This explanation cannot be applied equally to the acidification produced by the K(+)/H(+) exchanging ionophore nigericin. PMID- 16662039 TI - Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by N(2) Incorporation. AB - The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of (15)N(2) incorporation. After addition of carrier NH(4) (+) to digested cultures, those exposed to (15)N(2) (25 atom%) had a (15)N content of 3.16 atom% compared to 0.354 atom% (15)N in the controls. PMID- 16662040 TI - Diurnal Changes in Metabolite Levels and Crassulacean Acid Metabolism in Kalanchoe daigremontiana Leaves. AB - Diurnal changes in levels of selected metabolites associated with glycolysis, the C(3) cycle, C(4)-organic acids, and storage carbohydrates were analyzed in active Kalanchoe daigremontiana Crassulacean acid metabolism leaves. Three metabolic transition periods occurred each day. During the first two hours of light, nearly all of the metabolite pools underwent transient changes. Beginning at daylight, stomata opened transiently and closed again within 30 minutes; malate synthesis continued for about 1 hour into the light; C(3) photosynthesis began within 30 minutes; and net quantities of starch and glucan began to accumulate after 2 hours, continuing linearly throughout the rest of the day.THE SECOND TRANSITION OCCURRED IN MIDAFTERNOON: stomata reopened; malate decarboxylation nearly terminated; and the assimilation of ambient CO(2) occurred primarily via the C(3) cycle. The third transition occurred at dark: stomata transiently closed before opening again; the C(3) cycle stopped; malate synthesis started in about 1 hour; starch and glucan degradation began within 1 hour; and the bulk of carbon flow was through glycolysis leading to the synthesis and accumulation of malate throughout the night. At night, the levels of metabolites involved in acidification and glycolysis (except for phosphoenolpyruvate) generally accumulated. Phosphoenolpyruvate levels peaked near midday and were minimal at night. The ribulose 1,5-bisphosphate pool was depleted at night, while sedoheptulose 1,7-bisphosphate, fructose 1,6-bisphosphate, glucose 6-phosphate, and fructose 6-phosphate accumulated. PMID- 16662041 TI - Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts. AB - The photosystem II core complex (TSF-IIa) is composed of polypeptides of molecular weight 54-, 47-, 42-, and 30 kilodaltons (kD) and cytochrome b-559. After treatment with trypsin or alpha-chymotrypsin for 20 hours, the TSF-IIa particles still retained their photochemical activity and the light-induced cytochrome b-559 signal, although all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex from 250,000 to 100,000 daltons as determined by gel filtration, and also decreased the protein to chlorophyll ratio by 40%. Chlorophyll a appeared to be associated with the 47- and 42 kD polypeptides. Proteolysis of the complex produced a single chlorophyll a band with a slightly higher electrophoretic mobility. This band was not equivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), but did not completely abolish it.It was concluded that although the polypeptides of the photosystem II core complex were cleaved by the proteases, many of the peptide fragments do not dissociate from the complex, particularly the chlorophyll a containing portions, leaving the complex photochemically active. This supports the concept that chlorophyll has a structural function in chlorophyll-protein complexes. PMID- 16662042 TI - Characterization and quantitation of concanavalin a binding by plasma membrane enriched fractions from soybean root. AB - The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritiated ((3)H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of (3)H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10(15) molecules of (3)H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of alpha-methyl mannoside.A major peak of (3)H-Con A binding was also observed in fractions recovered from the 25/30% interface of a complex discontinuous sucrose density gradient when membranes were isolated in the absence of Mg(2+). When high Mg(2+) was present in the isolation and gradient media, the peak was shifted to a fraction recovered from the 34/38% sucrose interface. These results suggest that Con A binding sites are also present on membranes of the endoplasmic reticulum. The amount of Con A bound by endoplasmic reticulum membranes was at least twice the amount bound by membranes in plasma membrane enriched fractions when binding was compared on a per unit membrane protein basis. In contrast, mitochondrial inner membranes, which equilibrate at the same density as plasma membranes, had little ability to bind the lectin. PMID- 16662043 TI - Metabolism of cytokinin: deribosylation of cytokinin ribonucleoside by adenosine nucleosidase from wheat germ cells. AB - Adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) which catalyzes the deribosylation of N(6)-(Delta(2)-isopentenyl)adenosine and adenosine to form the corresponding bases was partially purified from wheat germ. This enzyme (molecular weight 59,000 +/- 3,000) deribosylates the ribonucleosides at an optimum pH of 4.7 K(m) values for the cytokinin nucleoside and adenosine are 2.38 and 1.43 micromolar, respectively, in 50 millimolar Tris-citrate buffer (pH 4.7) at 30 C. The presence of adenosine and other cytokinin nucleosides inhibited the hydrolysis of N(6)-(Delta(2)-isopentenyl)adenosine but this reaction was insensitive to guanosine, uridine, or 3'-deoxyadenosine. It is hypothesized that an adequate level of "active cytokinin" in plant cells may be provided through the deribosylation of cytokinin riboside in concert with other cytokinin metabolic enzymes. PMID- 16662044 TI - Phytochrome control of maize coleoptile section elongation: the role of cell wall extensibility. AB - Maize (Zea mays) coleoptile section cell wall extensibility was found to be stimulated by red light. This stimulation was largely removed by simultaneous or immediately subsequent far-red treatment. Qualitatively similar patterns of response occurred at 0 C and 20 C. Plastic extensibility responded more than elastic extensibility after red light treatment. Red-induced extensibility increases were detectable by 20 minutes after irradiation, and extensibility continued to increase up to at least 1 hour after irradiation. The kinetics of escape from far-red reversibility indicate that the initial events leading to this phenomenon are among the fastest known phytochrome responses. PMID- 16662045 TI - Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts. AB - Reverse-phase evaporation lipid vesicles (REV) liposomes, consisting of phosphatidyl choline and stearylamine in 1:3 molar ratio, encapsulated approximately 30% of exogenously supplied recombinant DNA vector, pBR322. The DNA sequestered in REV liposomes was highly tolerant to DNase.A two-step procedure was developed, which involves encapsulation of DNA with liposomes using one-tenth phosphate-buffered saline-0.5 molar mannitol, followed by incubation of liposome DNA with protoplasts in phosphate buffer-0.5 molar mannitol.About 11% of liposome encapsulated DNA was transferred into protoplasts, whereas 6% uptake was observed in the control. Although some degradation of incorporated DNA occurred inside protoplasts, 50% of the total radioactivity resolved by 0.8% agarose gel was associated with pBR322 forms in 5-hour incubation. After 20-hour incubation, open circular DNA disappeared completely and maintenance of covalently closed circular DNA was confirmed. PMID- 16662046 TI - Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO(2) Release. AB - Mechanically isolated soybean leaf cells metabolized added glycolate by two mechanisms, the direct oxidation of glyoxylate and the decarboxylation of glycine. The rate of glyoxylate oxidation was dependent on the cellular glyoxylate concentration and was linear between 0.58 and 2.66 micromoles glyoxylate per milligram chlorophyll. The rate extrapolated to zero at a concentration of zero. The concentration and, therefore, the rate of oxidation of glyoxylate could be decreased by adding glutamate or serine to the cells. These substrates were amino donors for the transamination of glyoxylate to glycine. In the presence of these amino acids more CO(2) was released from added glycolate via the glycine decarboxylation reaction and less by the direct oxidation of glyoxylate.Leaves from soybean plants of various ages grown under different nitrogen regimes had glyoxylate concentrations of about 80 to 100 nanomoles per milligram chlorophyll. Using the isolated cells as a model to determine the relationships between the glyoxylate concentration and rate of its decarboxylation indicated that about 2.5% of the photorespiratory CO(2) would arise from this reaction. This percentage would not be expected to vary greatly with growth conditions. PMID- 16662047 TI - Proanthocyanidins and potential precursors in needles of douglas fir and in cell suspension cultures derived from seedling shoot tissues. AB - Proanthocyanidins and their potential precursors have been analyzed by paper chromatography and C(18) reversed phase columns with high performance liquid chromatography. Total proanthocyanidins on a dry weight basis in cell suspension cultures derived from seedlings of Douglas fir (Pseudotsuga menziesii) were equal to or greater than those found in mature needles of randomly selected outdoor grown trees. The major monomer and dimer were catechin and epicatechin-catechin, respectively. Although only procyanidins were detectable in cell suspension cultures, mature needles of outdoor-grown trees contained prodelphinidins as well. Immature needles (flush growth) of the same trees contained only trace amounts of prodelphinidins. Eriodictyol-7-glucoside and dihydroquercetin-3' glucoside were present in all tissues examined. The amount of eriodictyol-7 glucoside was strongly correlated with total proanthocyanidins in immature needles of flush growth (r = 0.89, p = 0.001). The most complex pattern of flavonoids was found in flush growth needles, which contained in addition to the above, naringenin-7-glucoside and five to six flavone glycosides. Chlorogenic acid was detected only in seedlings and in flush growth needles. PMID- 16662048 TI - A circadian rhythm in the rate of light-induced electron flow in three leguminous species. AB - Three legume species, Pisum sativum L., Glycine max (L.), and Phaseolus vulgaris L., were grown in light-dark cycles and then maintained in constant dim light. During the constant conditions, chloroplasts were isolated throughout the day and assayed for various light-reaction activities. Similar results were found for all three species. The rate of whole-chain, light-induced electron flow (H(2)O to methyl viologen) was rhythmic over a 24-hour period provided an uncoupler of photophosphorylation was present. Chloroplasts varied in their response to uncouplers on a 24-hour basis and the per cent stimulation of electron flow was rhythmic. Neither PSII activity (H(2)O to DCPIP or light-induced pH changes in the presence of K(3)Fe(CN)(6)), PSI activity (DCPIPH(2) ascorbate to methyl viologen) or the rate of oxidation of hydroquinone (TMQH(2) to methyl viologen) could be identified as a rate-limiting step for the rate of electron flow. The capability to photophosphorylate as measured by a photosynthetic control assay was also constant with time. A rhythm in oxygen evolution was also observed with leaf mesophyll cell suspensions isolated from Pisum. The possible involvement of dynamic changes in the composition or configuration of the thylakoid membrane is discussed. PMID- 16662049 TI - High Levels of Malic Enzyme Activities in Vicia faba L. Epidermal Tissue. AB - Specific activities of NADP-malic enzyme, NAD-malic enzyme, phosphoenolpyruvate carboxykinase and pyruvate, orthophosphate dikinase in various cells of Vicia faba L. leaflets were determined. Expressed on dry weight, chlorophyll or protein basis, the averages for NADP- and NAD-malic enzyme specific activities were higher in guard cells than in photosynthetic parenchyma cells. Malic enzyme specific activities were also high in epidermal cells. Phosphoenolypyruvate carboxykinase activity was not detected in Vicia leaf extracts or guard cells; the assay techniques were validated by mixed Vicia-Brachiaria leaf extraction and assays on nanogram samples of Brachiaria bundlesheath cells. It was inferred from these data that guard cell malate depletion is by decarboxylation to pyruvate in the epidermal layer, but how the various epidermal cells interact remains obscure.Pyruvate, orthophosphate dikinase activity could not be demonstrated unequivocally in Vicia leaf extracts, Vicia guard cell protoplast extracts, or in Vicia guard cells. The assay techniques were validated by mixed Vicia-Kochia leaf extraction and assays on nanogram samples of Kochia mesophyll cells. How (or if) pyruvate is phosphorylated by epidermal tissue for entry into gluconeogenesis is unknown. PMID- 16662050 TI - Nitrate Assimilation during Vegetative Regrowth of Alfalfa. AB - Dry matter accumulation, nitrate reductase activity of various organs, nitrate accumulation, nitrogen derived from nitrate, and nitrogen content were studied during 17 days of vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.). Seedlings were grown in the glasshouse and treated with 0, 40, and 80 kilograms N per hectare applied as K(15)NO(3) to determine whether reduced nitrogenase activity after shoot harvest limited vegetative regrowth. The role of nodules in reducing NO(3) (-) during this period of low nitrogenase activity was also investigated.Applied nitrogen had no effect on shoot dry matter accumulation during the regrowth cycle. This observation suggests that reduced nitrogenase activity after shoot removal does not limit vegetative regrowth. Nitrate reductase activity was present in all tissues and ranked leaves > nodules > stems > roots. Root nitrate reductase did not respond to applied K(15)NO(3), while leaf, stem, and nodule nitrate reductase increased significantly. Significant increases in total plant nitrate reductase activity were highly correlated with shoot regrowth. Nodules incorporated N from (15)NO(3) (-), and calculations showed that 1.5% of the total plant N formed during regrowth could have been derived through nodule nitrate reductase. Nitrate accumulated in tissues of all plants, including those grown in the absence of added K(15)NO(3). Accumulation of NO(3) (-) in plant tissues ranked leaves > stems > roots. Analysis of the percentage of (15)N showed that plants grown in either 40 or 80 kilograms N per hectare derived 65 to 75% of the N that accumulated during regrowth from NO(3) ( ). Approximately 23 milligrams of N . plant(-1) accumulated during the regrowth period. Leaves accounted for 80% of this net increase, while stems accounted for 20%. There was no net change in root N.These results indicate that, although alfalfa will efficiently utilize low levels of applied N in lieu of nitrogen fixation, N fertilization of seedling alfalfa after shoot removal will be of little value to increase yields. These data also indicate that nodules reduce NO(3) (-), but their contribution to total plant N during regrowth is relatively small. PMID- 16662051 TI - Oxidation of proline by mitochondria isolated from water-stressed maize shoots. AB - Proline oxidation and coupled phosphorylation were measured in mitochondria after isolation from shoots of water-stressed, etiolated maize (Zea mays L.) seedlings. Both state III and state IV rates of proline oxidation decreased as a logarithmic function of increased seedling water stress between -5 and -10 bars. Proline oxidation rates decreased 62% (state III) and 58% (state IV) as seedling water potentials were decreased from -5 to -10 bars. By comparison, oxidation of succinate, exogenous NADH, or malate + pyruvate decreased only 10 to 15% in this stress range. These decreases were a linear function of increased stress and were comparable to oxidation rates of mitochondria subjected to varying in vitro osmotic potentials. Osmotically induced in vitro stress reduced proline oxidation rates linearly with more negative osmotic potentials, a decrease that was similar to the responses of the other substrates to more negative osmotic potentials. Some decrease in coupling, with all substrates as determined by ADP/O ratios, was observed under osmotic stress. Mitochondria were also isolated from shoot tissue that had been stressed and then rewatered. On a percentage basis, the recovery of proline oxidation was greater than that of the other substrates.The decreases in the proline oxidase activity of mitochondria after only slight stress indicate a mitochondrial sensitivity to water stress at significantly less negative water potentials than previously reported for measurements of maize membrane permeability and respiratory activity. PMID- 16662052 TI - Differences in steady-state net ammonium and nitrate influx by cold- and warm adapted barley varieties. AB - A flowing nutrient culture system permitted relatively rapid determination of the steady-state net nitrogen influx by an intact barley (Hardeum vulgare L. cv Kombar and Olli) plant. Ion-selective electrodes monitored the depletion of ammonium and nitrate from a nutrient solution after a single pass through a root cuvette. Influx at concentrations as low as 4 micromolar was measured. Standard errors for a sample size of three plants were typically less than 10% of the mean.When grown under identical conditions, a variety of barley bred for cold soils had higher nitrogen influx rates at low concentrations and low temperatures than one bred for warm soils, whereas the one bred for warm soils had higher influx rates at high concentrations and high temperatures. Ammonium was more readily absorbed than nitrate by both varieties at all concentrations and temperatures tested. Ammonium and nitrate influx in both varieties were equally inhibited by low temperatures. PMID- 16662053 TI - In Vivo Nitrite Reduction in Leaf Tissue of Phaseolus vulgaris L. AB - Experiments were performed to establish a procedure for in vivo measurement of nitrite utilization by leaf tissue of bean (Phaseolus vulgaris L. cv. Top Crop).To measure light-dependent nitrite disappearance, a single disc of leaf tissue was exposed to light for 1 hour at 30 C while immersed in incubation medium (approximately 0.11 milliliter per square centimeter of leaf area) in the bottom of a tall-form glass beaker. The incubation medium was 100 millimolar phosphate buffer (pH 7.5) with added wetting agent and nitrite. The wetting agent combination of 1% 1-propanol plus 0.05% Neutronyx-600 was used in some experiments for compatibility with established in vivo nitrate reductase (NR) assays; however, 0.05% Neutronyx-600 alone was found to be a suitable substitute. Parallel assays run in the dark on related tissue are recommended as a means to determine the amount of nitrite synthesized within the tissue by the NR system. Adding the results of the two assays gives an estimate of total nitrite utilization by the leaf tissue. It was found that 20 millimolar nitrite in the incubation medium was the most suitable level of external nitrite for promoting light-dependent nitrite disappearance. This was also found to reduce, sometimes to zero, the rate of synthesis of nitrite by NR. NR activity declined steadily with advancing age. Except for very young tissue, the rate of nitrite disappearance was independent of age. Nitrite disappearance was completely blocked by diuron. PMID- 16662054 TI - Photosynthetic Characteristics of Portulaca grandiflora, a Succulent C(4) Dicot : CELLULAR COMPARTMENTATION OF ENZYMES AND ACID METABOLISM. AB - The succulent, cylindrical leaves of the C(4) dicot Portulaca grandiflora possess three distinct green cell types: bundle sheath cells (BSC) in radial arrangement around the vascular bundles; mesophyll cells (MC) in an outer layer adjacent to the BSC; and water storage cells (WSC) in the leaf center. Unlike typical Kranz leaf anatomy, the MC do not surround the bundle sheath tissue but occur only in the area between the bundle sheath and the epidermis. Intercellular localization of photosynthetic enzymes was characterized using protoplasts isolated enzymatically from all three green cell types.Like other C(4) plants, P. grandiflora has ribulose 1,5-bisphosphate carboxylase and the decarboxylating enzyme, NADP(+)-malic enzyme, in the BSC. Unlike other C(4) plants, however, phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, and NADP(+)-malate dehydrogenase of the C(4) pathway were present in all three green cell types, indicating that all are capable of fixing CO(2) via phosphoenolpyruvate carboxylase and regenerating phosphoenolpyruvate. Other enzymes were about equally distributed between MC and BSC similar to other C(4) plants. The enzyme profile of the WSC was similar to that of the MC but with reduced activity in most enzymes, except mitochondrion-associated enzymes.Intracellular localization of enzymes was studied in organelles partitioned by differential centrifugation using mechanically ruptured mesophyll and bundle sheath protoplasts. Phosphoenolpyruvate carboxylase was a cytosolic enzyme in both cells; whereas, ribulose 1,5-bisphosphate carboxylase and NADP(+)-malic enzyme were exclusively compartmentalized in the bundle sheath chloroplasts. NADP(+)-malate dehydrogenase, pyruvate, Pi dikinase, aspartate aminotransferase, 3 phosphoglycerate kinase, and NADP(+)-triose-P dehydrogenase were predominantly localized in the chloroplasts while alanine aminotransferase and NAD(+)-malate dehydrogenase were mainly present in the cytosol of both cell types. Based on enzyme localization, a scheme of C(4) photosynthesis in P. grandiflora is proposed.Well-watered plants of P. grandiflora exhibit a diurnal fluctuation of total titratable acidity, with an amplitude of 61 and 54 microequivalent per gram fresh weight for the leaves and stems, respectively. These changes were in parallel with changes in malic acid concentration in these tissues. Under severe drought conditions, diurnal changes in both titratable acidity and malic acid concentration in both leaves and stems were much reduced. However, another C(4) dicot Amaranthus graecizans (nonsucculent) did not show any diurnal acid fluctuation under the same conditions. These results confirm the suggestion made by Koch and Kennedy (Plant Physiol. 65: 193-197, 1980) that succulent C(4) dicots can exhibit an acid metabolism similar to Crassulacean acid metabolism plants in certain environments. PMID- 16662055 TI - Vacuole/Extravacuole distribution of soluble protease in hippeastrum petal and triticum leaf protoplasts. AB - The subcellular distribution of soluble protease in anthesis-stage, anthocyanin containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using (125)I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with (125)I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates. PMID- 16662056 TI - Correction of flow resistances of plants measured from covered and exposed leaves. AB - The difference in water potential between an enclosed nontranspiring leaf and an adjacent exposed transpiring leaf, and the transpiration rate of a similarly exposed leaf, were used to calculate the change in hydraulic resistance of sorghum (Sorghum bicolor [L.] Moench) and sunflower (Helianthus annuus L.) leaves throughout the day and at various rates of transpiration. Since cotton (Gossypium hirsutum L.) leaves enclosed in aluminum foil alone had enclosed leaf water potentials about 0.06 megapascals lower than similar leaves enclosed in a polyethylene bag shielded with aluminum foil, the sorghum and sunflower leaves were enclosed in polyethylene bags shielded with aluminum foil. Enclosing the exposed leaf in a plastic sheath just prior to excision led to the water potential measured by the pressure chamber technique being 0.3 to 0.4 megapascals higher at rapid transpiration rates than in exposed leaves not sheathed just prior to excision. This error, previously shown to arise from rapid water loss after excision, led to an overestimation of the leaf hydraulic resistance in both species. Correction of the error reduced the resistance by 40 to 90% in irrigated sorghum and by about 40% in irrigated and unirrigated sunflower. After correction, the hydraulic resistances were still flow-dependent, but the dependency was markedly reduced in sorghum. PMID- 16662057 TI - Mechanism of glycolate transport in spinach leaf chloroplasts. AB - The incorporation of (14)CO(2) into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to (14)CO(2) (NaH(14)CO(3), 1 millimolar) in the light was determined as a function of O(2) concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K(m) (O(2)) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic (14)CO(2) fixation under an O(2) atmosphere.Glycolate transport into and out of chloroplasts was studied using the silicon oil centrifugation method. Both uptake and loss of glycolate were found to be rapid, with small temperature dependencies between 0 degrees C and 25 degrees C (Q(10) = 1.1). The reaction rate as a function of the concentration of glycolate up to 30 millimolar was linear in both directions.The effect of external pH on the reaction rate in both directions was also examined. Glycolate penetrates rapidly, even at pH 8, showing a surprisingly high permeation of the glycolate anion. This rate was about 30 micromoles per milligram Chl per hour at 0 degrees C, and the initial concentration of glycolate of 10 millimolar with a pH range of 7 to 8. The observed rate is comparable to the reported value for glycolate synthesis in chloroplasts under photorespiratory conditions. The uptake of glycolate into chloroplasts was accelerated below pH 7, while the rate of excretion was considerably lowered. It is, thus, suggested that undissociated glycolic acid penetrates the chloroplast envelopes more rapidly than does the anion. PMID- 16662058 TI - Thioredoxin/Glutaredoxin System of Chlorella: CHLORELLA ADENOSINE 5' PHOSPHOSULFATE SULFOTRANSFERASE CANNOT USE THIOREDOXIN OR GLUTAREDOXIN AS COFACTORS. AB - Using the thioredoxin/glutaredoxin-dependent adenosine 3'-phosphate 5' phosphosulfate reductase coupled assay system, the Chlorella thioredoxin/glutaredoxin system has been partially purified and characterized. A NADPH-thioredoxin reductase and two thioredoxin/glutaredoxin activities, designated as Chlorella thioredoxin/glutaredoxin protein I and II (CPI and CPII), were found in crude extracts of Chlorella. Similar to their counterparts from Escherichia coli, both CPI and CPII are heat-stable low molecular proteins of approximately 14,000. While CPI (but not CPII) is a substrate for its homologous NADPH-thioredoxin reductase as well as for E. coli NADPH-thioredoxin reductase, CPII is better than CPI as a substrate for reduction by the glutathione system. Based on these properties, CPI and CPII may be classified as Chlorella thioredoxin and Chlorella glutaredoxin, respectively. The Chlorella NADPH thioredoxin reductase (M(r) = 72,000, with two 36,000-dalton subunits) resembles E. coli-thioredoxin reductase in size. Besides Chlorella thioredoxin, the Chlorella thioredoxin reductase will also use E. coli thioredoxin, but not glutaredoxin, as a substrate. Although a thioredoxin-like protein has been implicated in higher plant light-dependent sulfate reaction, neither Chlorella thioredoxin nor glutaredoxin can stimulate the thiol-dependent adenosine 5' phosphosulfate-sulfotransferase reaction. Furthermore, Chlorella thioredoxin and glutaredoxin, in conjunction with an appropriate reductase system, cannot replace the thiol requirement of Chlorella adenosine 5'-phosphosulfate-sulfotransferase. The exact physiological roles and subcellular localization of the Chlorella thioredoxin and glutaredoxin systems remain to be determined. PMID- 16662059 TI - Metabolic Changes in Gladiolus Cormels During the Break of Dormancy: The Role of Dark CO(2) Fixation. AB - Dark CO(2) fixation in Gladiolus X gandavensis Van Houtte cormels increases during the break of dormancy by low-temperature storage or by cytokinins. The in vitro activities of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in preparations from dormant and nondormant cormels were compared with dark fixation rates in vivo. The distribution of (14)C-label in the carboxylation products in dormant, nondormant, water-imbibed, and benzyladenine- and abscisic acid-treated cormels was compared by pulse-chase experiments. Dormant cormels have more label in malate and less in citrate and amino acids. Malate utilization in dormant cormels is slower than in nondormant ones. Citrate and glutamine accumulate in dormant cormels in inactive pools. Benzyladenine induces in dormant cormels changes similar to cold storage. Dark fixation is among the first reactions which are activated during the break of dormancy by both benzyl adenine and cold storage. PMID- 16662060 TI - Photosynthesis in Fescue : III. RATES OF ELECTRON TRANSPORT IN A POLYPLOID SERIES OF TALL FESCUE PLANTS. AB - Photosystem I electron transport activity has been found to be considerably higher in a decaploid tall fescue (Festuca arundinacea Schreb.) genotype as compared to a common hexaploid genotype. The decaploid genotype also displayed a higher photosystem whole chain (Photosystem II plus Photosystem I uncoupled) activity, suggesting a connection between polyploidy and increased electron transport activity. However, when a polyploidy series of tall fescue, ranging from diploid to decaploid with several different genetic isolates at each ploidy level, was examined in natural growth conditions, no effect of increasing genome content on electron transport and photophosphorylation was found. These results suggest that a gene component of one of the genomes involved may be responsible for the increased activity rather than simply the total chromosome content. PMID- 16662061 TI - Enzymes of Purine Biosynthesis and Catabolism in Glycine max: I. COMPARISON OF ACTIVITIES WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING NODULE DEVELOPMENT. AB - During the period examined from 12 to 63 days after planting, the ureides, allantoin and allantoic acid, were the predominant nitrogenous solutes in the xylem exudate of soybeans (Glycine max [L.]) growing solely on symbiotically fixed nitrogen, accounting for approximately 60% and greater than 95% of the total nitrogen in the xylem exudate before and after the onset of active nitrogen fixation, respectively. For plants between 18 and 49 days of age, the apparent rate of ureide export estimated from concentrations of ureides in xylem exudate collected over a period of one hour was closely related to the rate of nitrogen fixation estimated from measurements of C(2)H(2) reduction by nodulated root systems. After this time, the apparent rate of ureide export per plant continued to increase, reaching a maximum value at day 63 of 12 micromoles per plant per hour, even though the rate of C(2)H(2) reduction per plant declined approximately four-fold. The most probable pathway for the biosynthesis of ureides involves the catabolism of purines. The levels of phosphoribosylpyrophosphate (PRPP) synthetase, which catalyzes the formation of the PRPP required for purine synthesis, increased in parallel with the rates of nitrogen fixation (C(2)H(2)) from day 18 reaching a maximum value of 13.9 micromoles per plant per hour at day 49, and then both activities declined rapidly. During the period of active nitrogen fixation the ratio of PRPP synthesis estimated from measurements of PRPP synthetase activity in cell-free extracts to the apparent rate of ureide export was between 1 and 2. The activities of the enzymes of purine catabolism, xanthine dehydrogenase, uricase, and allantoinase, increased in parallel with the increases in nodule mass and the export of ureides with maximum activities of 13, 119, and 79 micromoles per plant per hour, corresponding with apparent rates of ureide export in the range of 9.5 to 11.9 micromoles per plant per hour. These results demonstrate that there is a close association between nitrogen fixation, PRPP synthetase activity, and ureide export in soybeans and support the proposal that recently-fixed nitrogen is utilized in the de novo synthesis of purines which are subsequently catabolized to produce the ureides. PMID- 16662062 TI - Hexokinase II of Pea Seeds. AB - A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K(m), 48 micromolar) for glucose and a relatively low affinity (K(m), 10 millimolar) for fructose. The K(m) for MgATP was 86 micromolar. Mg(2+) was required for activity, but excess Mg(2+) was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al(3+) was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed. PMID- 16662063 TI - Properties of Kaurene Synthetase from Marah macrocarpus Endosperm: Evidence for the Participation of Separate but Interacting Enzymes. AB - Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two step sequence catalyzed by kaurene synthetase; the first step (A activity) involves the conversion of geranylgeranyl pyrophosphate into the intermediate ent-trans labda-8(17), 13-dien-15-yl pyrophosphate (copalyl pyrophosphate) which is further cyclized to ent-kaurene in the second step (B activity). The resolution of enzyme fractions which catalyze each step independent of the other has been accomplished for the first time by means of QAE Sephadex A-50 chromatography and polyacrylamide gel electrophoresis of kaurene synthetase preparations from endosperm tissue of immature seed of Marah macrocarpus. Molecular weights for the A and B enzymes were each estimated as approximately 82,000 by means of gel filtration chromatography and sedimentation velocity determinations.Experiments in which [(14)C]geranylgeranyl pyrophosphate and [(3)H]copalyl pyrophosphate were incubated simultaneously with kaurene synthetase preparations demonstrated that copalyl pyrophosphate derived from [(14)C]geranylgeranyl pyrophosphate is more readily converted to kaurene than is exogenously added [(3)H]copalyl pyrophosphate. This implies that copalyl pyrophosphate derived from the catalytic site of the A enzyme is preferentially channelled to the B enzyme catalytic site for conversion to ent-kaurene, rather than freely equilibrating with a pool of copalyl pyrophosphate in the medium. Experiments in which the rates of the overall AB and independent B activities of kaurene synthetase preparations were measured as a function of total protein concentration further suggest that overall AB activity is catalyzed by an AB enzyme complex which is in equilibrium with free A and B enzymes. A model is proposed for M. macrocarpus kaurene synthetase in which separate but interacting A and B enzymes must associate for the efficient production of ent-kaurene from geranylgeranyl pyrophosphate. PMID- 16662064 TI - Water Relations of Leaf Epidermal Cells of Tradescantia virginiana. AB - Water-relation parameters (cell turgor pressure [P], volumetric elastic modulus [epsilon] and hydraulic conductivity [Lp]) of individual leaf epidermal cells of Tradescantia virginiana have been determined with the pressure-probe technique. Turgor was 4.5 +/- 2.1 [41] bar (mean +/- sd; in brackets the number of cells) and ranged from 0.9 to 9.6 bar. By vacuum infiltration with nutrient solution, it was raised to 7.5 +/- 1.5 [5] bar (range: 5.3-8.8 bar). There was a large variability in the absolute value of epsilon of individual cells. epsilon ranged from 40 to 360 bar; mean +/- sd: 135 +/- 83 bar; n = 50 cells. epsilon values of individual cells seemed to be rather independent of changes in cell turgor. A critical assessment of the errors incurred in determining epsilon by the technique is included. The half-times of water exchange of individual cells ranged from 1 to 35 seconds, which gave values of 0.2 to 11 x 10(-6) centimeters per second per bar for Lp (mean +/- sd: 3.1 +/- 2.3 x 10(-6) centimeters per second per bar; n = 39 cells). The large range in Lp and epsilon is believed to be due to the difficulties in determining the effective surface area of water exchange of the cells. Lp is not influenced by active salt pumping driven by respiration energy inasmuch as it was not altered by 0.1 millimolar KCN. The temperature dependence of Lp (T((1/2))) was measured for the first time in individual higher-plant cells. Lp increased by a factor of 2 to 4, when the temperature was increased by 10 C. The activation energy of water exchange was found to be between 50 and 186 kilojoules per mole. Within the large range of variation it was found that T((1/2)), Lp, and epsilon did not change under various experimental conditions (intact and excised tissue, water content and turgidity, age, etc.). Similar results were obtained for the epidermal cells of Tradescantia andersoniana. The measurements suggest that the entire epidermis would respond very rapidly (i.e. with a half-time of 1 to 30 s) to a demand for water from the stomata. PMID- 16662065 TI - Transient susceptibility of root cells in four common legumes to nodulation by rhizobia. AB - Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation. PMID- 16662066 TI - The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage. AB - In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-(14)C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.Protein synthesis is similar for 4 hours in both healthy and infected protoplasts. Accumulation of labeled spermidine stops after 2 hours in healthy protoplasts but continues in the infected protoplasts. Much of the newly synthesized protein and spermidine is present in the easily sedimentable fraction of the readily disrupted protoplasts.Disrupted and diluted protoplasts have a decreased ability to metabolize methionine to protein and spermidine. The residual synthetic activity is essentially entirely in the easily sedimentable fraction. However, this fraction is unable to synthesize spermine, an activity found in protoplasts and disrupted protoplasts. Disrupted protoplasts contain spermidine synthase (EC 2.5.1.16) and about a quarter of this activity is present in a low-speed sedimentable fraction containing the chloroplasts. The protoplast system is suitable for an analysis of polyamine synthesis in turnip yellow mosaic virus infection and appears particularly suitable for study of the distribution of the enzymes involved. PMID- 16662067 TI - Effects of freezing on spinach leaf mitochondria and thylakoids in situ and in vitro. AB - The sensitivity of spinach (Spinacia oleracea L.) leaf mitochondria and chloroplast membranes to subzero temperature stress was compared after freezing of the membrane systems in situ and in vitro. Respiratory and photosynthetic activities were measured polarographically.When leaves were frozen under controlled conditions for 2 hours to various minimum temperatures and mitochondria and chloroplasts isolated after thawing, the membrane systems showed a nearly simultaneous inactivation of respiratory and photosynthetic activities between -5 to -7 C. At that temperature range in both membrane systems phosphorylation became only slightly more affected than electron transport, i.e. after freezing in situ conspicuous uncoupling of phosphorylation from electron transport was not observed.In contrast, mitochondria and thylakoids isolated from the same preparation of intact leaves under comparable conditions using NaCl as osmoticum exhibited differences in sensitivity towards freezing for 2 to 4 hours at -25 C in vitro. In the absence of cryoprotectants, photophosphorylation of isolated thylakoids became completely uncoupled from electron transport which was increased several-fold compared with the unfrozen controls. Inactivation of respiratory functions of isolated mitochondria followed the same pattern as observed after freezing in situ. In the presence of sucrose for protection of thylakoids significantly higher concentrations of the cryoprotectant were necessary than for preservation of mitochondria. Thus, under the conditions used in this study chloroplast membranes proved to be more sensitive to freezing in vitro than mitochondria. PMID- 16662068 TI - Host-Pathogen Interactions : XIX. THE ENDOGENOUS ELICITOR, A FRAGMENT OF A PLANT CELL WALL POLYSACCHARIDE THAT ELICITS PHYTOALEXIN ACCUMULATION IN SOYBEANS. AB - An elicitor of phytoalexin accumulation (endogenous elicitor) is solubilized from purified cell walls of soybean (Glycine max [L.] Merr., cv. Wayne) by extracting the walls with hot water or by subjecting the walls to partial acid hydrolysis. The endogenous elicitor obtained from soybean cell walls binds to an anion exchange resin. The elicitor-active material released from the resin contains oligosaccharides rich in galacturonic acid; small amounts of rhamnose and xylose are also present. The preponderance of galacturonic acid in the elicitor-active fragments suggests that the elicitor is, in fact, a fragment of a pectic polysaccharide. This possibility is supported by the observation that treatment of the wall fragments with a highly purified endopolygalacturonase destroys their ability to elicit phytoalexin accumulation. This observation, together with other evidence presented in this paper, suggests that galacturonic acid is an essential constituent of the elicitor-active wall fragments. Endogenous elicitors were also solubilized by partial hydrolysis from cell walls of suspension-cultured tobacco, sycamore, and wheat cells. PMID- 16662069 TI - Effect of Light Quality on Stomatal Opening in Leaves of Xanthium strumarium L. AB - Flux response curves were determined at 16 wavelengths of light for the conductance for water vapor of the lower epidermis of detached leaves of Xanthium strumarium L. An action spectrum of stomatal opening resulted in which blue light (wavelengths between 430 and 460 nanometers) was nearly ten times more effective than red light (wavelengths between 630 and 680 nanometers) in producing a conductance of 15 centimoles per square meter per second. Stomata responded only slightly to green light. An action spectrum of stomatal responses to red light corresponded to that of CO(2) assimilation; the inhibitors of photosynthetic electron transport, cyanazine (2-chloro-4[1-cyano-1-methylethylamino]-6 ethylamino-s-triazine) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, eliminated the response to red light. This indicates that light absorption by chlorophyll is the cause of stomatal sensitivity to red light. Determination of flux response curves on leaves in the normal position (upper epidermis facing the light) or in the inverted position (lower epidermis facing the light) led to the conclusion that the photoreceptors for blue as well as for red light are located on or near the surfaces of the leaves; presumably they are in the guard cells themselves. PMID- 16662071 TI - The effects of ear removal on senescence and metabolism of maize. AB - Ears were removed from field grown maize (Zea mays L.) to determine the effects on senescence and metabolism and to clarify conflicting literature reports pertaining to these effects. Ears were removed at three days after anthesis and comparisons were made of changes in metabolism between eared and earless plants until grain of the eared plants matured as judged by black layer formation.The initial visual symptom following ear removal was the development of reddish colored leaves. As judged by leaf yellowing, the removal of ears not only initiated an earlier onset but enhanced the rate of senescence. With this exception, the visual patterns of senescence were similar for earless and eared plants. Other characteristics associated with ear removal were: (a) marked decrease in dry weight and reduced N accumulation by the whole plant; (b) progressive, parallel decreases in leaf reduced N, nitrate reductase activity, and chlorophyll; (c) increases in carbohydrate content of both the leaf and stalk and of reduced N in the stalk. These changes indicate that ear removal reduced photosynthesis and nitrate reduction by approximately equal proportions and that the stalk serves as an alternate sink for both carbohydrate and nitrogen.The remobilization of nitrogen from the leaf was not dependent on the presence of an ear. A logical reason for the more rapid loss of nitrogen from the leaf of the earless plants appears to be the cessation of nitrate uptake and/or flux of nitrate to the leaves.From these results and from related experiments we tentatively conclude that the loss of nitrogen from the leaf is a major cause of death of the intact maize plant. PMID- 16662070 TI - Transfer of oligosaccharide to protein from a lipid intermediate in plants. AB - A lipid-bound oligosaccharide was isolated from pea (Pisum sativum) cotyledons incubated with [(14)C]mannose. The oligosaccharide moiety appeared to be identical with the one obtained from rat liver, known to contain three glucoses, nine mannoses, and two N-acetylglucosamines, and to be involved in protein glycosylation.Enzymes obtained from soya (Glycine max) roots and developing pea cotyledons were found to catalyze the transfer of oligosaccharide from the lipid intermediate to endogenous protein. The enzymes require Mn(2+) and detergent for activity. Evidence is presented indicating that the lipid-bound oligosaccharide with three glucoses is transferred faster than that with less. Some of the peripheral mannoses could be removed without affecting the rate of transfer.The protein-bound oligosaccharide, formed by incubation of whole cotyledons or by transfer with the enzyme preparation, could be released by protease and endo-beta N-acetylglucosaminidase treatment, as expected for an asparagine-bound high mannose oligosaccharide. PMID- 16662072 TI - Availability of reduced N and carbohydrates for ear development of maize. AB - Changes in dry weights, reduced N, nitrate, and nitrate reductase activity of various plant parts of the above ground vegetation (stover) and ears of field grown maize were measured at intervals between anthesis and grain maturity. Nonstructural carbohydrate contents were also measured in some instances. Changes in dry weight and reduced N content were used to approximate net in situ photosynthetic and nitrate assimilation activities and to determine whether the availability of photosynthate or reduced N was limiting grain production.Of the five hybrids studied, all showed extensive remobilization (loss) of reduced N from the stover during grain development. This loss of stover N was initiated by 18 to 21 days after anthesis. Most of this loss of N (about 70%) was from the leaves. In contrast, three of the five hybrids had more vegetative dry weight at grain maturity than at anthesis, while the loss of stover dry weight by the other two hybrids was negligible. By 42 days after anthesis when the bulk of the ear weight had been acquired, the average gain in stover dry weight for the five hybrids was 12% while the loss of stover reduced N was 28%. Where measured, the increase in stover dry weight was largely due to deposition of carbohydrates in the stalk. These results show that the photosynthetic capacity was adequate while nitrate reduction capacity was inadequate for ear demands. The changes in the rate of accumulation of dry weight and reduced N by the ear indicated that the rate of supply of reduced N to the ear could have limited ear development for one of the five hybrids. The dry weight and carbohydrate (where measured) accumulation in the vegetation during the first 42 days after anthesis infers that the rate of supply of photosynthate to the ear was probably not a limiting factor for any of the five hybrids.The maximum remobilization of stover N during grain development was 1.8 g N plant(-1) for the genotypes examined, while the amount of reduced N accumulated by the grain varied from 1 to 5 g plant(-1). The amount of newly reduced N (nitrate reduced after anthesis) provided from 48 to 72% of the total N accumulated by the ear. The relative amounts of newly reduced N and remobilized N vary with genotype and environment. With respect to insuring high productivity, it was concluded that there is more flexibility in the system (genotype and environment) for increasing the supply of newly reduced N than in enhancing the remobilization of vegetative N. PMID- 16662073 TI - Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria. AB - NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica oleracea L., var. botrytis). The NAD-linked activity is accompanied by a minor NADP-linked activity. Some contaminant NADP malic enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations. NAD-dependent malic enzyme has been purified 38 fold by ammonium sulfate fractionation and gel permeation chromatography, to a specific activity up to 2 micromoles per minute per milligram.The nature of the activating effect of coenzyme A and dithiothreitol has been investigated. Both compounds act by decreasing the apparent Michaelis constants for l-malate and NAD(+) (and NADP(+)), V(max) remaining approximately constant. However, enzyme fully activated by dithiothreitol can still be stimulated up to 2.4-fold by coenzyme A treatment.Velocity versus substrate responses show hyperbolic kinetics under present assay conditions (pH 7.5, 2 millimolar Mn(2+)), but biphasic kinetics have been observed with enzyme purified in the presence of 10 millimolar dithiothreitol, suggesting enzyme heterogeneity with respect to an activated state. This condition is reverted to linearity by treatment with coenzyme A. K(m) values do not vary with changing concentrations of the second substrate. Enzyme molecular weight is 400,000 in the completely activated state and 200,000 in the ;inactivated' state; intermediate forms are also found. All coenzyme A derivatives tested are effective activators, showing activation constants lower than for coenzyme A itself. The concentration dependence of the activation is sigmoidal. PMID- 16662074 TI - Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L. AB - Rapid, sensitive, and selective methods for the determination of the (15)N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [(15)N]H(4) (+) assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate (15)N abundance with an accuracy of +/-1 atom% excess (15)N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of (15)N labeled precursors. PMID- 16662075 TI - Carbon and nitrogen limitations on soybean seedling development. AB - Carbon and nitrogen limitations on symbiotically grown soybean seedlings (Glycine max [L.] Merr.) were assessed by providing 0.0, 1.0, or 8.0 millimolar NH(4)NO(3) and 320 or 1,000 microliters CO(2)/liter for 22 days after planting. Maximum development of the Rhizobium-soybean symbiosis, as determined by acetylene reduction, was measured in the presence of 1.0 millimolar NH(4)NO(3) under both levels of CO(2). Raising NH(4)NO(3) from 0.0 to 8.0 millimolar under 320 microliters CO(2)/liter increased plant dry weight by 251% and Kjeldahl N content by 287% at 22 days after planting. Increasing NH(4)NO(3) from 1.0 to 8.0 millimolar under 320 microliters CO(2)/liter increased total dry weight and Kjeldahl N by 100 and 168%, respectively, on day 22. Raising CO(2) from 320 to 1,000 microliters CO(2)/liter during the same period had no significant effect on Kjeldahl N content of plants grown with 0.0 or 1.0 millimolar NH(4)NO(3). The maximum CO(2) treatment effects were observed in plants supplied with 8.0 millimolar NH(4)NO(3), where dry weight and Kjeldahl N content were increased 64% and 20%, respectively. An increase in shoot CO(2)-exchange rate associated with the CO(2)-enrichment treatment was reflected in a significant increase in leaf dry weight and starch content for plants grown with 1,000 microliters CO(2)/liter under all combined N treatments. These data show directly that seedling growth in symbiotically grown soybeans was limited primarily by N availability. The failure of the CO(2)-enrichment treatment to increase total plant N significantly in Rhizobium-dependent plants indicates that root nodule development and functioning in such plants was not limited by photosynthate production. PMID- 16662076 TI - Influence of 6-Benzylaminopurine on Fruit-Set and Seed Development in Two Soybean, Glycine max (L.) Merr. Genotypes. AB - The influence of 6-benzylaminopurine (BA) on the premature abscission of developing soybean, Glycine max (L.) Merr. fruits of 2 genotypes was studied. BA was applied during the critical period of fruit-setting. The tested concentration range of BA was from 1 micromolar to 5 millimolar; 2 millimolar was optimal. Spray application of 2 millimolar BA to terminal inflorescences at the R(3) developmental stage of field-grown soybeans significantly increased fruit-set and seed yield of the Shore genotype during three growing seasons. In contrast, the Essex genotype gave significant responses two out of three seasons. The response of Shore was generally more pronounced than that of Essex. The apical fruits on the inflorescences gave the greatest response to BA. Seed weight increase was apparent 3-4 weeks after BA treatment. PMID- 16662077 TI - Organic Acid contents of soybean: age and source of nitrogen. AB - The organic acid content of soybean (Glycine max v. Hodgson) root, stem, and leaf tissue was followed for 33 days after germination. Malonate was the predominant acid in leaf and root tissue, whereas fumarate was predominant in the stem. The malonate concentrations of the stem and root showed similar variations with time, but the leaf response was quite different. In nodules from 33-day-old plants, malonate was the predominant acid. Malonate levels in root and nodule tissue of 33-day-old plants were depressed in response to the addition of either nitrate or ammonia. Nodule tissue had a higher malonate concentration on nitrate nitrogen than it did on ammonium nitrogen, whereas root tissue had the higher malonate concentration on ammonium nitrogen. Analysis of organic acid concentrations of roots as a function of age and distance from the root tip in young soybean seedlings revealed a zone consistently high in malate. The malonate level in the entire root rose dramatically in tissue of age 96 to 120 hours. PMID- 16662078 TI - Biosynthesis of malonate in roots of soybean seedlings. AB - Many plants accumulate malonate, but it was shown earlier that malonate does not accumulate as a deadend product of metabolism in soybean (Glycine max v. Hodgson tissues. The metabolism of malonate in the soybean plant at the whole tissue and enzymic level was followed, and the pathway of malonate biosynthesis in young soybean root tissue was shown to be via acetyl-coenzyme A carboxylase. PMID- 16662079 TI - Purification of Spinach Leaf ADPglucose Pyrophosphorylase. AB - ADPglucose pyrophosphorylase from spinach leaves has been purified to homogeneity by hydrophobic chromatography carried out in 1 molar phosphate buffer. After polyacrylamide gel electrophoresis, the preparation showed only one protein staining band that coincided with a single activity stain. The enzyme appears to be composed of two subunits with molecular weights of 44,000 and 48,000, respectively, as determined by SDS polyacrylamide gel electrophoresis. Thus ADPglucose pyrophosphorylase of spinach appears to be comprised of subunits which are similar in size to the subunits of ADPglucose pyrophosphorylase isolated from bacterial sources. In contrast, a subunit molecular weight of 96,000 has been reported for the maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). The purified enzyme retains similar allosteric and catalytic properties as reported previously and is more sensitive to phosphate inhibition under "dark"-simulated conditions than under "light" simulated conditions. PMID- 16662080 TI - The effect of rotenone on respiration in pea cotyledon mitochondria. AB - Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of alpha-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria. PMID- 16662081 TI - Measuring water potential (activity) from free water to oven dryness. AB - Water activities (potentials) in plant materials were measured over the range from free water to oven dryness with a Spanner thermocouple psychrometer. In a two-step procedure, water was first condensed on the thermocouple junction for several minutes. The sample was then inserted under the wet thermocouple and the maximum psychrometric cooling was measured in about 10 seconds. Calibration was with saturated salt slurries of known water activities. Psychrometric cooling was a nearly linear function of the water activity and of the negative log of the water potential. The psychrometric cooling to water activity relationship agreed with wetbulb temperature depression to relative humidity relationships given in tables. Water activities of wheat grains and leaves decreased sharply in a curvilinear fashion as their water contents decreased. Some problems of the procedure are discussed. PMID- 16662082 TI - Solute leakage resulting from leaf desiccation. AB - The leakage of solutes from foliar tissue is utilized as a dynamic measure of apparent changes in membrane integrity in response to desiccation. It is found that rehydrating leaf discs of cowpea (Vigna sinensis [L.] Endl.) show increasing leakiness in proportion to the extent of prior desiccation, whereas Selaginella lepidophylla Spring., a resurrection plant, does not. The elevated leakage rate of cowpea after desiccation recovers with time, and the passage of time in the stressed condition results in reduced subsequent leakiness. These characteristics are interpreted as suggesting that the leakage of solute reflects the condition of cellular membranes, and that desiccation stress leads to lesions in the membranes. The kinetics of solute leakage is suggested as a simple means of following changes in membrane lesions and associated features of membrane repair and hardening. PMID- 16662083 TI - Immunological approach to structural comparisons of assimilatory nitrate reductases. AB - Homogeneous squash cotyledon reduced nicotinamide-adenine dinucleotide (NADH):nitrate reductase (NR) was isolated using blue-Sepharose and polyacrylamide gel electrophoresis. Gel slices containing NR were pulverized and injected into a previously unimmunized rabbit. This process was repeated weekly and antiserum to NR was obtained after four weeks. Analysis of the antiserum by Ouchterlony double diffusion using a blue-Sepharose preparation of NR resulted in a single precipitin band while immunoelectrophoresis revealed two minor contaminants. The antiserum was found to inhibit the NR reaction and the partial reactions to different degrees. When the NADH:NR and the reduced methyl viologen:NR activities were inhibited 90% by specifically diluted antiserum, the reduction of cytochrome c was inhibited 50%, and the reduction of ferricyanide was inhibited only 30%. Antiserum was also used to compare the cross reactivities of NR from squash cotyledons, spinach, corn, and soybean leaves, Chlorella vulgaris, and Neurospora crassa. These tests revealed a high degree of similarity between NADH:NR from the squash and spinach, while NADH:NR from corn and soybean and the NAD(P)H:NR from soybean were less closely related to the squash NADH:NR. The green algal (C. vulgaris) NADH:NR and the fungal (N. crassa) NADPH:NR were very low in cross reactivity and are apparently quite different from squash NADH:NR in antigenicity. Antiserum to N. crassa NADPH:NR failed to give a positive Ouchterlony result with higher plant or C. vulgaris NADH:NR, but this antiserum did inhibit the activity of squash NR. Thus, it can be concluded from these immunological comparisons that all seven forms of assimilatory NR studied here have antigenic determinants in common and are probably derived from a common ancestor. Although these assimilatory NR have similar catalytic characteristics, they appear to have diverged to a great degree in their structural features. PMID- 16662084 TI - Effects of carbon dioxide and oxygen on the regulation of photosynthetic carbon metabolism by ammonia in spinach mesophyll cells. AB - Photosynthetic carbon metabolism of isolated spinach mesophyll cells was characterized under conditions favoring photorespiratory (PR; 0.04% CO(2) and 20% O(2)) and nonphotorespiratory (NPR; 0.2% CO(2) and 2% O(2)) metabolism, as well as intermediate conditions. Comparisons were made between the metabolic effects of extracellularly supplied NH(4) (+) and intracellular NH(4) (+), produced primarily via PR metabolism. The metabolic effects of (14)CO(2) fixation under PR conditions were similar to perturbations of photosynthetic metabolism brought about by externally supplied NH(4) (+); both increased labeling and intracellular concentrations of glutamine at the expense of glutamate and increased anaplerotic synthesis through alpha-ketoglutarate. The metabolic effects of added NH(4) (+) during NPR fixation were greater than those during PR fixation, presumably due to lower initial NH(4) (+) levels during NPR fixation. During PR fixation, addition of ammonia caused decreased pools and labeling of glutamate and serine and increased glycolate, glyoxylate, and glycine labeling. The glycolate pathway was thus affected by increased rates of carbon flow and decreased glutamate availability for glyoxylate transamination, resulting in increased usage of serine for transamination. Sucrose labeling decreased with NH(4) (+) addition only during PR fixation, suggesting that higher photosynthetic rates under NPR conditions can accommodate the increased drain of carbon toward amino acid synthesis while maintaining sucrose synthesis. PMID- 16662085 TI - In Vivo Determination of Parameters of Nitrate Utilization in Wheat (Triticum aestivum L.) Seedlings Grown with Low Concentration of Nitrate in the Nutrient Solution. AB - Six genotypes of winter wheat (Triticum aestivum L.) differing in grain protein concentration were grown on a nutrient solution containing low concentrations of NO(3) (-) (2 millimolar). Total NO(3) (-) uptake varied between genotypes but was not related to grain protein content. An in vivo nitrate reductase assay was used to determine the affinity of the enzyme for NO(3) (-), and large phenotypic variations were observed. In vivo estimations of the concentration and size of the metabolic pool were variable. However, the three genotypes with the higher ratios of metabolic pool size to leaf total NO(3) (-) concentration were the high protein varieties. It is proposed that a high affinity of nitrate reductase for nitrate might be a biochemical marker for the capacity of the plant to continue assimilating NO(3) (-) for a longer period during the last stage of growth.The potential use of such physiological criteria as markers is discussed, in particular with respect to breeding programs for the development of plants with efficient nitrogen utilization. PMID- 16662086 TI - Temperature Compensation of Circadian Period Length in Clock Mutants of Neurospora crassa. AB - Temperature compensation of circadian period length in 12 clock mutants of Neurospora crassa has been examined at temperatures between 16 and 34 degrees C. In the wild-type strain, below 30 degrees C (the "breakpoint" temperature), the clock is well-compensated (Q(10) = 1), while above 30 degrees C, the clock is less well-compensated (Q(10) = 1.3). For mutants at the frq locus, mutations that shorten the circadian period length (frq-1, frq-2, frq-4, and frq-6) do not alter this temperature compensation response. In long period frq mutants (frq-3, frq-7, frq-8), however, the breakpoint temperature is lowered, and the longer the period length of the mutants the lower the breakpoint temperature. Long period mutants at other loci exhibit other types of alterations in temperature compensation-e.g. chr is well-compensated even above 30 degrees C, while prd-3 has a Q(10) significantly less than 1 below 30 degrees C. Prd-4, a short period mutant, has several breakpoint temperatures. Among four double mutants examined, the only unusual interaction between the individual mutations occurred with chr prd, which had an unusually low Q(10) value of 0.86 below 27 degrees C. There was no correlation between circadian period length and growth rate. These strains should be useful tools to test models for the temperature compensation mechanism. PMID- 16662087 TI - Light Requirement for AgNO(3) Inhibition of Ethrel-Induced Leaf Abscission from Cuttings of Vigna radiata. AB - To obtain information regarding the antiethylene properties and binding site of Ag(+), studies were initiated to define conditions under which Ag(+) does or does not inhibit ethylene action. AgNO(3), applied as a leaf spray, inhibited 2 chloroethylphosphonic acid (Ethrel)-induced leaf abscission from green cuttings of Vigna radiata in white light but lost considerable activity in the dark. In the absence of Ethrel, AgNO(3) stimulated abscission in the dark. When cuttings were dark-aged for 24 hours prior to treatment with AgNO(3) and aged for an additional 24 hours in the dark after treatment, good inhibition of subsequent Ethrel-induced abscission was restored by returning the cuttings to light. However, when dark aging was preceded by far-red irradiation, considerably less inhibition of Ethrel-induced abscission was restored in the light. AgNO(3) was completely inactive on cuttings aged in the dark and treated with Ethrel in the dark. Light is required for the antiethylene activity of AgNO(3) with regard to leaf abscission of Vigna. PMID- 16662088 TI - Effects of O(2) Tension and Temperature during Light Uptake of CO(2) on the Dark Release of CO(2) by Excised C(3) and C(4) Leaves. AB - A study was conducted on a C(4) (Panicum maximum) and a C(3) (Panicum bisulcatum) species to determine the nature of the dark release of (14)CO(2) with respect to its responses to changes in temperature and O(2) tension during light CO(2) uptake of (14)CO(2).The total one minute uptake of (14)CO(2) increased with increased temperatures up to 30 degrees C for both species at all O(2) tensions.The total release of (14)CO(2) during the first four minutes of dark by the C(4) species was 5 to 10 times greater than the C(3) species; however, there was no consistent pattern in the C(4) species as a function of temperature or O(2) tension. In contrast, the dark release by the C(3) species was similar to that of light uptake as a function of temperature or O(2) tension. The percentage of the one-minute (14)CO(2) fixation that was released as (14)CO(2) during the first four minutes of dark increased with O(2) tension for the three highest temperatures (20, 30, and 38 degrees C) and remained, though reduced, after a 10 minute dark chase in (12)CO(2) for the C(3) species.IT WAS CONCLUDED THAT: (a) the source of the dark CO(2) release in C(3) species is different from that of C(4) species; (b) the dark CO(2) release in C(3) species likely is derived from intermediates of photorespiration and is similar to the postillumination burst of CO(2); and (c) the source(s) of the dark CO(2) release in C(4) species is not photorespiration. PMID- 16662089 TI - Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE. AB - Addition of exogenous Mg(2+) (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K(+). Addition of K(+) (10-50 millimolar) reversed both stromal acidification and K(+) efflux from the chloroplast caused by Mg(2+). These data suggested that Mg(2+) induced reversible H(+)/K(+) fluxes across the chloroplast envelope. Ca(2+) and Mn(2+) (2 millimolar) were as effective as 4 millimolar Mg(2+) in causing K(+) efflux from chloroplasts and inhibition of O(2) evolution. In contrast, 10 millimolar Ba(2+) induced only a small amount of inhibition. The lack of strong inhibition by Ba(2+) indicated that the effects of divalent cations such as Mg(2+) cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg(2+) was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O(2) evolution (43%) induced by Mg(2+).A variety of ATPase inhibitors were tested for effects on Mg(2+)-induced H(+)/K(+) fluxes. Oligomycin was the only ATPase inhibitor which specifically inhibited photosynthesis in the presence of Mg(2+) + K(+), but had little or no effect in the absence of these cations. In the presence of oligomycin, much higher concentrations (50 millimolar) of exogenous K(+) were required to reverse Mg(2+)-induced acidification and inhibition of O(2) evolution than in its absence. Oligomycin (in the absence of divalent cations) increased the inhibition of photosynthesis caused by sodium acetate, which acts by causing stromal acidification. In addition, the chloroplast envelope ATPase was inhibited partially (45%) by oligomycin. These results suggested that H(+) fluxes across the chloroplast envelope are regulated by two mechanisms: (a) an active, oligomycin-sensitive H(+) efflux and (b) a reversible, Mg(2+)-dependent, oligomycin-insensitive H(+)/K(+) exchange. PMID- 16662090 TI - Modulation of PSI and PSII Organization During Loss and Repair of Photosynthetic Activity in a Temperature Sensitive Mutant of Chlorella pyrenoidosa. AB - Photosynthetic activity and organization of chlorophyll(Chl)-protein complexes in a temperature sensitive mutant of Chlorella pyrenoidosa have been investigated. The mutant is practically indistinguishable from wild type cells when grown at 25 C. However, mutant cells grown at 33 C do not synthesize Chl and lose their ability to evolve O(2). O(2) evolution and Chl synthesis are restored upon incubation of the 33 C grown cells at 25 C in absence of cell division (repair).Based on polarographic measurements of photosynthetic activities, variable fluorescence, 77 K fluorescence emission, excitation spectra, analysis of Chl-protein complexes, membrane polypeptide pattern and radioactive labeling using sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques during growth at 33 C and/or under repair conditions, it is concluded that: a, polypeptides of chloroplastic translation required for H(2)O-splitting activity are absent from membranes of 33 C grown cells. Their synthesis and/or assembly during the repair process is light-dependent. b, Polypeptides required for the formation of photosystem II and photosytem I reaction centers continue to be formed during growth at 33 C in absence of Chl synthesis. These can be assembled into functional units following Chl synthesis and energization of the membranes during the repair process. c, The Chl-protein complex serving as an antenna of photosystem I is disorganized, and the Chl is used for the formation of functional reaction centers of photosystem I during growth at 33 C.These results show that Chl-protein complexes can be dissociated in vivo and reassembled in a different way; and formation of Chl-protein complexes can occur stepwise from previously synthesized and newly formed components including both polypeptides and Chl. PMID- 16662091 TI - Hormonal Control of Mitotic Development in Tobacco Protoplasts: TWO-DIMENSIONAL DISTRIBUTION OF NEWLY-SYNTHESIZED PROTEINS. AB - Two-dimensional separation of proteins newly synthesized by tobacco mesophyll protoplasts cultivated in vitro allows us to detect, reproducibly, 257 spots. The pattern is extremely stable throughout the three days of culture, the intensity of only 24 spots varying during this time. The absence of cytokinin (N(6) benzyladenine) in the culture medium prohibits entry into S phase but does not modify the pattern, indicating that none of the observed proteins is specifically synthesized in S, G(2), or M phases. The presence of 2,4-dichlorophenoxyacetic acid is necessary for the mitotic development of protoplasts. It induces the appearance of one protein, increases the level of another, and reduces that of eight others. All proteins sensitive to auxin belong to the group of proteins the levels of which vary during culture. PMID- 16662092 TI - Rapid Growth and Apparent Total Nitrogen Increases in Rice and Corn Plants following Applications of Triacontanol. AB - Triacontanol (TRIA) increased fresh and dry weight and total reducible nitrogen (total N) of rice (Oryza sativa L.) seedlings within 40 minutes. Increases in total N in the supernatants from homogenates of corn (Zea mays L.) and rice leaves treated with TRIA for one minute before grinding occurred within 30 and 80 minutes, respectively. The source for the increase was investigated utilizing atmospheric substitution and enrichment and depletion studies with (15)N. The increase in total N in seedlings was shown to be independent of method of N analysis and the presence of nitrate in the plants. Automated Kjeldahl determinations showing apparent increases in N composition due to TRIA were shown to be correlated with hand Kjeldahl, elemental analysis, and chemiluminescent analysis in three independent laboratories. TRIA did not alter the nitrate uptake or endogenous levels of nitrate in corn and rice seedlings. Enrichment experiments revealed that the total N increases in rice seedlings, in vivo, and in supernatants of corn leaf homogenates, in vitro, are not due to atmospheric N(2). TRIA increased the soluble N pools of the plants, specifically the free amino acid and soluble protein fractions. No differences in depletion or enrichment of (15)N incorporated into soluble and insoluble N fractions of rice seedlings could be detected on an atom per cent (15)N basis. The apparent short term total N increases cannot be explained by current knowledge of major N assimilation pathways. TRIA may stimulate a change in the chemical composition of the seedlings, resulting in interference with standard methods of N analysis. PMID- 16662093 TI - Apical control of branch movements in white pine: biological aspects. AB - Two-year-old branches on control trees (Pinus strobus L.) were compared through a season with branches on trees stem-girdled just above, or below, the branch whorl. All branches first sagged down for 20 days and then moved up for 40 days. Then, control branches reversed and moved back down while branches in both girdle treatments continued to move up. Movement reversal correlated with cessation of both elongation and diameter growth in control branches. Diameter growth continued in branches of girdled trees. Control branches continued to stiffen even after diameter growth stopped. Differences in movements due to girdling are from compression wood formed after cessation of branch elongation. Apical control stops cambial activity and compression wood formation in branches after branch elongation ceases, allowing photosynthate produced in the branch to move to the stem. Control branches bend down from increasing self-weight after cambial activity ceases. PMID- 16662094 TI - Demonstration of auxin binding to strawberry fruit membranes. AB - Presence of specific auxin-binding sites in strawberry fruit (Fragaria ananassa Duch. cv. Ozark Beauty) membranes has been demonstrated. These 1 naphthaleneacetic acid (NAA)-binding sites in the 80,000g to 120,000g fraction of the strawberry fruit membrane were pronase sensitive with an estimated equilibrium dissociation constant for NAA of 1.1 x 10(-6) molar. The minimum concentration of NAA required to stimulate strawberry fruit growth was at least one order of magnitude higher than the minimum concentration of NAA required to stimulate corn coleoptile elongation. This was consistent with the higher equilibrium dissociation constant (lower affinity) for auxin binding to strawberry fruit membranes than to corn coleoptiles. Twelve auxin analogs, ranging from very strong to weak auxins, were tested for abilities to stimulate in situ strawberry fruit growth and to bind (displace or compete with NAA) to strawberry fruit membranes. The observed positive correlation (r = 0.74) between the in vitro binding to the 80,000 to 120,000 membrane fraction and the in situ biological activity of these analogs indicated that the NAA-binding sites in strawberry fruit membranes may represent physiologically relevant auxin receptors. PMID- 16662095 TI - Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species. AB - High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5' diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I(50) = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I(50) = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I(50) = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg(2+) and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour.A heat-stable proteinaceous factor was identified in desalted crude leaf extracts that increased ATP sensitivity of the partially purified enzyme. Apparently, during purification a dissociable factor, that is required for maximum sensitivity to low concentrations (<1 millimolar) of Mg:ATP, is lost from the enzyme. PMID- 16662096 TI - Reversal of glyphosate inhibition of carrot cell culture growth by glycolytic intermediates and organic and amino acids. AB - Various cytokinins and purines were ineffective in reversing glyphosate (0.25 millimolar)-induced growth inhibition of carrot (Daucus carota L.) cell suspension cultures. Aspartate was particularly effective in reversing glyphosate inhibition, but asparagine and various combinations of lysine, methionine, threonine, and homoserine (eventual products of aspartate metabolism) were not effective. When organic acids of the tricarboxylic acid cycle were added to the medium, particularly good reversal of inhibition could be obtained with alpha ketoglutarate, succinate, and malate. Citrate provided only moderate reversal but the reversal given by glutamate was comparable to that of aspartate and the more effective tricarboxylic acid cycle intermediates. Pyruvate was somewhat toxic to cells when added early in the cell cycle but was most effective at reversing glyphosate inhibition when added at this time. If pyruvate addition was delayed, it was less toxic but was also a less effective reversing agent for glyphosate inhibition.All of the effective reversing agents for glyphosate inhibition found in this study can serve either directly or indirectly as carbon skeletons for respiration and ammonia assimilation and have previously been shown to be effective detoxifying agents for ammonia in cell culture systems. The results of this study suggest that glyphosate inhibition of growth in this system may be due to depletion of respiratory substrate which may eventually result in ammonia accumulation. PMID- 16662097 TI - Subcellular Localization of IAA Oxidase in Peas. AB - Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.The enzymic properties of IAA oxidase in the floating pea epicotyl segments are similar to those previously reported in the literature. Other experiments indicate that approximately 70% of the activity is at the cut surfaces of the tissue. In addition, up to 50% of the IAA oxidase activity could be pelleted in a membranous fraction when the released enzyme was centrifuged at 10,000(g). Higher centrifugal forces reduced the proportion of the enzyme in the pellet, suggesting that vesicles containing IAA oxidase rupture at these forces.Subcellular localization of IAA oxidase was accomplished by use of sucrose density gradient centrifugation and fractionation. It was found that the enzyme is associated most closely with Golgi and also to a lesser degree with the lysosomes and endoplasmic reticulum. It is suggested that little if any IAA oxidase is freely soluble in the cytoplasm and is therefore unlikely to have a role in controlling normal growth via IAA levels. The enzyme may, however, have a role in diseased or damaged tissue. PMID- 16662098 TI - Comparative studies on the sodium, potassium, and chloride relations of a wild halophytic and a domestic salt-sensitive tomato species. AB - In long-term experiments with differentially salinized nutrient solutions, plants of Lycopersicon esculentum Mill cv. Walter failed at Na(+) concentrations of 200 millimolar or more but tolerated K(+) concentrations of that magnitude. The behavior of the wild, salt-tolerant Lycopersicon cheesmanii (Hook) C. H. Mull., accession number 1401, was diametrically different; it tolerated Na(+) at 200 millimolar, but K(+) at the same concentration proved toxic to it.Short-term comparative studies on the absorption and translocation of Na(+), K(+), and Cl(-) of the two species were carried out using radioactive tracers with excised roots and whole plants. These studies showed that, under high salt conditions (50-100 millimolar NaCl), the tolerant 1401 freely accumulated Na(+) in the shoot, while the salt-sensitive cultivar excluded it from the leaves, where it has been shown to be toxic.In experiments where K(+) was limiting, the salt-tolerant species could partially substitute Na(+) for K(+). Sodium stimulated growth even when K(+) was present at adequate concentrations. The domestic cultivar could not substitute Na(+) for K(+) and showed no similar growth stimulation when Na(+) was added in the presence of adequate K(+). The salt-tolerant 1401 was more efficient in K(+) absorption than was the domestic cultivar at both low and moderate ambient K(+) concentrations.The two species differed little in their chloride relations. PMID- 16662099 TI - Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM. AB - Scutellar tissues were dissected from germinating rice seeds and the incorporation of [(35)S]methionine into the alpha-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti alpha-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify alpha-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that alpha-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of alpha-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of alpha-amylase. PMID- 16662100 TI - Endoplasmic Reticulum Formation during Germination of Wheat Seeds : A QUANTITATIVE ELECTRON MICROSCOPE STUDY. AB - This study demonstrates germination-induced ultrastructural changes in wheat (Triticum aestivum L. cv Arthur) aleurone cells. Seeds imbided for 4 hours in water contained endoplasmic reticulum (ER) or ER-like membranes as vesicles or as short segments of membrane associated with the spherosomes on the periphery of aleurone grains. Aleurone cells incubated between 8 and 10 hours contained abundant ER membranes mainly associated with the nuclear envelope and, to a lesser extent, with the spherosomes surrounding the aleurone grain. The membranes located on the periphery of the nucleus occurred as regions of stacked cisternae. When aleurone cells were analyzed by morphometry, the increase in ER during incubation was found to be greater than 2-fold. During the same incubation period, other organelles did not change significantly. The early increase in ER was not affected by gibberellin incubation. Thus, the rapid proliferation of ER observed during the early stages of germination in aleurone cells of wheat is not likely to be controlled directly by gibberellin. PMID- 16662101 TI - Evidence for Sulfhydryl Involvement in Regulation of Phytoalexin Accumulation in Trifolium repens Callus Tissue Cultures. AB - White clover (Trifolium repens L.) callus tissue cultures accumulated the phytoalexin medicarpin after treatment with sulfhydryl reagents. After 24-hour exposures to sulfhydryl reagents, maximum obtainable levels of medicarpin, determined by high performance liquid chromatography analysis, were found with 50 millimolar N-ethyl maleimide, 25 millimolar HgCl(2), 2 millimolar p chloromercuribenzoic acid, and 0.5 millimolar iodoacetamide. Increased medicarpin levels were also observed in callus treated with p-chloromercuribenzene sulfonic acid, but the highest concentration tested (11.8 millimolar) did not produce the maximum response. After sulfhydryl treatment, medicarpin levels were unchanged for 4 to 6 hours, but steadily increased thereafter with maximum accumulation occurring by 48 to 50 hours for p-chloromercuribenzoic acid, p chloromercuribenzene sulfonic acid, and HgCl(2) treated callus. Medicarpin levels did not increase in iodoacetamide-treated callus until 8 hours after sulfhydryl exposure, and medicarpin levels were still increasing linearly after 50 hours. Three other metabolic inhibitors, KCN, NaF, and Na(3)AsO(4), did not exhibit elicitor activity, indicating cell death was not a factor in the response. Pretreatment of callus with 20 millimolar dithiothreitol followed by 40 millimolar N-ethyl maleimide did not produce the phytoalexin response. Preincubation with dithiothreitol also prevented elicitor activity of HgCl(2) and p-chloromercuribenzene sulfonic acid. These results suggested that dithiothreitol pretreatment somehow prevented sulfhydryl groups within the cell from reacting with the test compounds. These experiments established that the integrity of sulfhydryl groups is important in regulating phytoalexin accumulation in callus cells. PMID- 16662102 TI - Interamino Acid Inhibition of Transport in Higher Plants : EVIDENCE FOR TWO TRANSPORT CHANNELS WITH ASCERTAINABLE AFFINITIES FOR AMINO ACIDS. AB - Data from published experiments were analyzed to determine the number and specificities of amino acid transport channels in cells of higher plants. Each experiment measured the uptake of a labeled amino acid in the presence of unlabeled amino acids, used one at a time, in the incubating medium. The observed interamino acid inhibitions can be accounted for by two transport channels, each with characteristic affinities that were computed from the observed interamino acid inhibitions. The first channel is a general transport system with the following relative affinities for the amino acids: methionine 75, alanine 75, phenylalanine 64, tyrosine 64, leucine 63, cysteine 58, serine 57, glycine 56, tryptophan 54, glutamine 51, threonine 49, valine 44, isoleucine 44, glutamic acid 44, proline 43, histidine 33, lysine 32, asparagine 22, arginine 22, aspartic acid 18. The second channel is a basic amino acid tranport system with relative affinities for arginine, lysine, and histidine of 66, 39, and 21, respectively. The affinities for the other acids in the second channel are lower. Despite considerable diversity in the species, tissues, and solute concentrations employed in the experiments, multiple regression equations (Y = alpha + beta(1)X(1) + betaX(2), in which Y is the observed transport inhibition and X(1) and X(2) are the relative transport affinities of the two channels) account for 50 to 99% of the variance in all but six experiments, five of which employed unusually high solute concentrations. PMID- 16662103 TI - Evidence for Receptor Function of Auxin Binding Sites in Maize : RED LIGHT INHIBITION OF MESOCOTYL ELONGATION AND AUXIN BINDING. AB - When 3- to 4-day-old dark-grown maize (Zea mays L. WF9 x Bear 38) seedlings are given red light, auxin-binding activity localized on endoplasmic reticulum membranes of the mesocotyl begins to decrease after 4 hours; by 9 hours, it falls to 50 to 60% of that in dark controls, on either a fresh weight or total particulate protein basis. Endoplasmic reticulum-localized NADH:cytochrome c reductase activity decreases in parallel. Loss of binding is due to decrease in number of sites, with no change in their affinity for auxin (K(d) 0.2 micromolar for naphthalene-1-acetic acid). Elongation of mesocotyl segments in response to auxin decreases with a similar time course. Elongation of segments from irradiated plants shows the same apparent affinity for auxin as that of the dark controls. Auxin-binding activity and elongation response also decrease in parallel down the length of the mesocotyl. These observations are consistent with a role of endoplasmic reticulum-localized auxin binding sites as receptors for auxin action in cell elongation. PMID- 16662104 TI - Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis). AB - Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID- 16662105 TI - Mobilization of metabolites from leaves to grains as the cause of monocarpic senescence in rice. AB - The pattern of senescence was studied by following the changes in chlorophyll and protein in the leaves and by measuring (32)P retention and export from source to sink during development of the rice plant (Oryza sativa L. cv. Jaya) subjected to different manipulative treatments. With the advance of reproductive development, the chronological sequence of leaf senescence was changed, so that the flag and the third leaf senesced earlier than did the second leaf. In presence of the daughter shoot of defruited plants, senescence was delayed in all three leaves of the mother plant, as compared to the same leaves of intact plants. Senescence of all three leaves was further delayed when both panicle and daughter shoots were removed from the plant. The above manipulative treatments caused the initial sequential pattern of senescence of leaves to persist. Removal of both panicle and daughter shoots caused little export of (32)P between leaves. In the presence of daughter shoots of defruited plants, export of (32)P was maximum from leaves of the mother plant to the nearest daughter shoots. This led to earlier senescence of such mother plant leaves than that of plants from which both panicle and daughter shoots were removed. The pattern of senescence and export of (32)P in the flag and the second leaf of the daughter shoot was essentially the same as that of the intact plant. Based on these findings, it was concluded that mobilization of metabolites from source to sink is the primary cause of monocarpic senescence in rice. PMID- 16662106 TI - Characterization of the inhibition of k absorption in oat roots by salicylic Acid. AB - The phenolic compounds salicylic acid (o-hydroxybenzoic acid) and ferulic acid (4 hydroxy-3-methoxycinnamic acid) inhibited K(+) ((86)Rb(+)) absorption in excised oat (Avena sativa L. cv. Goodfield) root tissue. Salicylic acid was the most inhibitory. The degree of inhibition was both concentration- and pH-dependent. With decreasing pH, the inhibitory effect of the phenolic increased. During the early stages of incubation, the time required to inhibit K(+) absorption was also pH- and concentration-dependent. At pH 4.0, 5x10(-4) molar salicylic acid inhibited K(+) absorption about 60% within 1 minute; whereas, at pH 6.5, this concentration affected absorption only after 10 to 15 minutes. However, at 5 x 10(-3) molar and pH 6.5, salicylic acid was inhibitory within 1 minute. The capacity of the tissue to recover following a 1-hour treatment in 5 x 10(-4) molar salicylic acid ranged from no recovery at pH 4.5 to complete recovery at pH 7.5. The absorption of salicylic acid was pH-dependent, also. As pH decreased, more of the phenolic compound was absorbed by the tissue. The increased absorption of the compound at low pH most likely contributed to apparent tissue damage at pH 4.5 and might have accounted for the lack of recovery of K(+) absorption as pH decreased.Under the proper conditions of pH and concentration, phenolic acids such as salicylic acid could significantly affect mineral absorption by plants in the field. PMID- 16662107 TI - Isolation and Characterization of Vacuoles from Melilotus alba Mesophyll. AB - Methods for the preparation of protoplasts and vacuoles from mesophyll tissues of sweet clover (Melilotus alba Desr.) are described. Vacuoles are obtained using a new procedure which involves lysis of the plasmalemma during a brief centrifugation of protoplasts through a diethylaminoethyl dextran layer. This method combines the release of vacuoles and their purification in one step. The contamination of vacuole preparations was found to be low, as judged by enzymic markers and microscopic inspection. The method described is rapid and gives a good yield of vacuoles without causing changes in osmotic pressure. Several hydrolases were found to be located in vacuoles from sweet clover, which were also examined for their amino acid content. PMID- 16662108 TI - Subcellular Localization of 2-(beta-d-Glucosyloxy)-Cinnamic Acids and the Related beta-glucosidase in Leaves of Melilotus alba Desr. AB - The distribution of the glucosides of trans- and cis-2-hydroxy cinnamic acid and of the beta-glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts.The specific activities of the beta-glucosidase in the two tissues were also similar; thus, most of the enzyme is contained in mesophyll tissue. However, the amount of enzyme in mesophyll protoplast extracts amounts to only 1 to 2% of the activity present in leaf homogenates when chlorophyll was the basis for comparison. (This small amount of coumarin-beta-glucosidase present in protoplasts is not associated with chlorophyll-containing fractions.) In contrast, 90% of the uridine diphosphate glucose-o-coumaric acid glucosyl transferase activity present in leaf homogenate was recoverable in protoplasts prepared from intact leaves. Such results indicate that most of the coumarin-beta glucosidase in M. alba leaves is located in the extracytoplasmic space. Only a small fraction (7%) of this extra cytoplasmic beta-glucosidase was associated with individual cells or cell clusters isolated from clover leaves. PMID- 16662109 TI - Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species. AB - The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content. PMID- 16662110 TI - Trehalose Toxicity in Cuscuta reflexa: CORRELATION WITH LOW TREHALASE ACTIVITY. AB - A toxic effect of alpha,alpha-trehalose in an angiospermic plant, Cuscuta reflexa (dodder), is described. This disaccharide and its analogs, 2-aminotrehalose and 4 aminotrehalose, induced a rapid blackening of the terminal region of the vine which is involved in elongation growth. From the results of in vitro growth of several angiospermic plants and determination of trehalase activity in them, it is concluded that the toxic effect of trehalose in Cuscuta is because of the very low trehalase activity in the vine. As a result, trehalose accumulates in the vine and interferes with some process closely associated with growth. The growth potential of Lemna (a duckweed) in a medium containing trehalose as the carbon source was irreversibly lost upon addition of trehalosamine, an inhibitor of trehalase activity. It is concluded that, if allowed to accumulate within the tissue, trehalose may be potentially toxic or inhibitory to higher plants in general. The presence of trehalase activity in plants, where its substrate has not been found to occur, is envisaged to relieve the plant from the toxic effects of trehalose which it may encounter in soil or during association with fungi or insects. PMID- 16662111 TI - Phycobilisome Structure of Porphyridium cruentum: POLYPEPTIDE COMPOSITION. AB - Purified phycobilisomes of Porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the alpha and beta subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the gamma subunit of phycoerythrin. Another colored polypeptide had a molecular weight of 95,000 and the characteristics of long wavelength-emitting allophycocyanin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin. In addition, the core fraction was enriched in a colored 95,000 dalton polypeptide. Inasmuch as a polypeptide with the same molecular weight is found in thylakoid membranes (free of phycobilisomes), it is suggested that this polypeptide is involved in anchoring phycobilisomes to thylakoid membranes. PMID- 16662112 TI - Hydroponic growth and the nondestructive assay for dinitrogen fixation. AB - Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK(a) 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day. PMID- 16662113 TI - Ethylene Inhibition of Phytochrome-Induced Germination in Potentilla norvegica L. Seeds. AB - Germination of Potentilla norvegica L. (rough cinquefoil) seeds stimulated by fluorescent irradiations of nearly 24 hours was inhibited by ethylene at <1 microliter per liter. Sensitivity to ethylene inhibition was highest during and immediately after the irradiation. By delaying ethylene treatment until about a day after the light potentiation, seeds escaped the inhibition. Ethylene inhibition may be readily reversed upon release of the gas and reirradiation of the seeds. Imbibition of seeds at 10 or 15 degrees C, or at high temperatures of 35 and 40 degrees C, partially prevented subsequent inhibition by ethylene. Alternating temperatures during germination nearly overcame the inhibition from 1 microliter per liter ethylene, but not higher doses. With brief red-irradiation and alternating temperatures, 0.1 microliter per liter ethylene promoted germination about 2-fold. These data suggest that ethylene may loosely associate on a site required for phytochrome action. The effect of temperature that opposed the inhibition may be to deny the association of ethylene with the site. Loose association is supported by the reversal of inhibition by gas release and increased temperature during germination. A blocking effect was shown by the failure of phytochrome to act when ethylene was present. PMID- 16662114 TI - Synthesis, salvage, and catabolism of uridine nucleotides in boron-deficient squash roots. AB - Previous work has provided evidence that plants may require boron to maintain adequate levels of pyrimidine nucleotides, suggesting that the state of boron deficiency may actually be one of pyrimidine starvation. Since the availability of pyrimidine nucleotides is influenced by their rates of synthesis, salvage, and catabolism, we compared these activities in the terminal 3 centimeters of roots excised from boron-deficient and -sufficient squash plants (Cucurbita pepo L.). Transferring 5-day-old squash plants to a boron-deficient nutrient solution resulted in cessation of root elongation within 18 hours. However, withholding boron for up to 30 hours did not result in either impaired de novo pyrimidine biosynthesis or a change in the sensitivity of the de novo pathway to regulation by end product inhibition. Boron deprivation had no significant effect on pyrimidine salvage or catabolism. These results provide evidence that boron deficient plants are not starved for uridine nucleotides collectively. Whether a particular pyrimidine nucleotide or derivative is limiting during boron deprivation remains to be examined. PMID- 16662115 TI - Effects of plant age and extraction conditions on the properties of homoserine dehydrogenase isolated from maize seedlings. AB - Homoserine dehydrogenase (EC 1.1.1.3) was extracted from shoots of etiolated seedlings of Zea mays L. which had been grown for periods ranging from three to thirteen days. Both the amount of enzyme extracted and its regulatory properties, as measured by the sensitivity of the enzyme to inhibition by the feedback modulator, l-threonine, were found to be a function of seedling age and extraction conditions. Equivalent amounts of enzyme with similar properties could be isolated from young seedlings under a variety of conditions. Extraction media containing comparatively low concentrations of the buffer component and a high concentration of dithioerythritol were found to be required for optimum extraction of the enzyme from shoots of seedlings grown longer than four days and from leaves of light-grown plants. In the absence of dithioerythritol, diminished regulatory control was observed to be a direct function of seedling age. Evidence of rapid desensitization of the enzyme during extraction was obtained from experiments in which dithioerythritol was added to extracts prepared in the absence of a thiol compound. Therefore, previous observations of growth-dependent desensitization in a number of plants could be due to incomplete extraction or to changes in cellular factors which inactivate and/or alter the enzyme. Whether the enzyme itself becomes increasingly susceptible to alteration during seedling growth remains to be established. PMID- 16662116 TI - Quantitative estimates of the distribution of homoserine dehydrogenase isozymes in maize tissues. AB - The low molecular weight threonine-resistant (class I) and the higher molecular weight threonine-sensitive (class II/III) isozymes of homoserine dehydrogenase (EC 1.1.1.3) isolated from Zea mays L. were shown to differ in stability during incubations in the presence of urea. Class II/III was inactivated by urea in a time- and concentration-dependent manner, with complete inactivation occurring within 24 hours at 5 degrees C in 4.0 m urea. Under identical conditions, neither the activity nor the properties of class I were affected. Therefore, it was possible to estimate the amounts and properties of both maize isozymes in crude mixtures by measurements of enzyme activity before and after treatment with urea.The relative amounts of the two isozymes proved to be tissue-specific. When shoots of etiolated seedlings were extracted under optimum conditions, the resultant preparations contained about 16% class I and 84% class II/III. This distribution of isozymes, as well as the regulatory properties of class II/III, were constant during growth of the seedlings between 4 and 13 days. Enzyme preparations isolated from shoots of light-grown plants contained higher proportions of class I. The two isozymes were not uniformly distributed within leaves, as the basal meristematic region contained high levels of II/III and small amounts of I. During leaf maturation, the amount of II/III declined while the level of I remained constant or increased slightly. As a result, nearly half of the enzyme extracted from leaf tips was class I. The synthesis of specific members of the aspartate family of amino acids might be expected to differ when the ratio of threonine-sensitive to threonine-resistant homoserine dehydrogenase is altered. However, additional information on the subcellular localization and the catalytic characteristics of the two enzymes is required for evaluation of this possibility. PMID- 16662117 TI - Metabolism of trans-Aconitic Acid in Maize : I. PURIFICATION OF TWO MOLECULAR FORMS OF CITRATE DEHYDRASE. AB - Trans-aconitate synthesis via citrate dehydrase was determined in crude extracts of maize (Zea mays L.) coleoptiles. Two molecular forms of this enzyme were purified by substrate-specific elution from DEAE-cellulose, ammonium sulfate precipitation, and gel filtration. Each molecular form migrates as a single band in isoelectric focusing. Gel filtration and sodium dodecyl sulfate electrophoresis provided evidence that one enzyme form is composed of four 80,000 dalton subunits while the other is composed of two 60,000-dalton subunits. There was no evidence of proteolytic conversion of the large to the small molecular weight form when the former was incubated with either the 15,000(g) supernatant or with proteases. The data indicate that the two molecular forms of citrate dehydrase are isozymes. PMID- 16662118 TI - Temperature and Aging Effects on Leaf Membranes of a Cold Hardy Perennial, Fragaria virginiana. AB - The lipid composition of leaves of wild strawberry (Fragaria virginiana Duchesne) was analyzed throughout an annual growth cycle in the field. Cellular hardiness to temperature stress was assessed concomitantly by a solute leakage technique. Leaves were shown to be very sensitive to an applied temperature of -5 degrees C during the summer months but insensitive to a 35 degrees C treatment. This general pattern was also seen in young overwintering leaves but was reversed after a period of low-temperature hardening of these same leaves. Associated with cold hardening of the overwintering leaves was a twofold increase in the phospholipid content of the leaf membranes with a proportionately smaller increase in free sterols. The large increase in phospholipids presumably is due primarily to the proliferation of a sterol-poor membrane fraction, probably the endoplasmic reticulum. These quantitative changes in membrane material may be important in increasing freezing tolerance in the overwintering leaf cells by enhancing the overall capacity of the cell for plasma membrane and tonoplast extension through vesicle fusion using components from this endomembrane pool. Analysis of electron micrographs of hardened leaf cells showed an increase in vesiculated smooth endoplasmic reticulum and tonoplast membrane over nonhardened leaf cells, the latter resulting in an enhanced tonoplast surface area to vacuolar volume ratio. During this same period, no changes in the fatty acid or free sterol composition were detectable, suggesting that regulation of membrane fluidity via these components is not required for cold acclimation in this species. During aging and senescence of both the overwintering and the summer leaves, the cellular membranes remained functionally intact but became progressively more vulnerable to temperature stress. Free sterol content increased during this time. This feature may be related to the inability of the older leaves to withstand environmental stress. Increasing sensitivity of the cellular membranes to stress may, in turn, be causally related to the actual onset of senescence in these leaves, thus explaining why only the older leaves senesce when the plant is challenged by periodic environmental stress. PMID- 16662120 TI - Evidence That IAA Conjugates Are Slow-Release Sources of Free IAA in Plant Tissues. AB - Evidence that indoleacetic acid (IAA) conjugates are metabolized via enzyme catalyzed hydrolysis to free IAA and that their biological activities are related to the rates at which they are hydrolyzed by the tissue is presented. These conclusions are based on the following observations. Slow but continuous decarboxylation of the IAA moiety of IAA-l-alanine and IAA-glycine occurs when these conjugates are applied to pea (Pisum sativum L. cv. Alaska) stem segments. Inasmuch as IAA conjugates are protected from peroxidase-catalyzed oxidative decarboxylation, the conjugates are probably hydrolyzed and the freed IAA then further metabolized. Free IAA and IAA-l-alanine are converted, by pea stem tissue, into the same metabolites. The metabolism is enzymic, since conjugates of IAA with the d-isomers of the amino acids are inactive. Ethylene production induced by IAA-l-alanine and by IAA-glycine is correlated with their hydrolysis, as indicated by their decarboxylation and with the appearance or nonappearance of IAA metabolites in the tissues. PMID- 16662119 TI - Slow Transients in the Activity of the NAD Malic Enzyme from Crassula. AB - The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or tau ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoe daigremontiana has a similar lag.The lag is greatest in freshly prepared enzyme and diminishes with storage at -70 degrees C, but the activity of the enzyme also diminishes with storage.The lag is inversely proportional to the concentration of enzyme, both in the assay and in storage prior to assay. The lag is also inversely proportional to the concentration of malate used in the assay, which poses particular problems because the lag with low malate concentrations may be so long that activity begins to be lost before the steady-state rate is reached.Various buffer ions produce different lags, but the lag with all buffers is longer than in the absence of buffer. The effectors CoA and SO(4) (2-) in the assay substantially decrease the lag. The lag is shorter with Mn(2+) as the required divalent cation than when Mg(2+) is used.The response of enzyme activity to pH shows that the intrinsic activity is greater with magnesium than with manganese, although the rate actually attained is lower with Mg(2+) because the pK values for the response to pH are closer together when that cation is used. The enzyme has a higher optimum pH and a broader response to pH when Mn(2+) is used. The change in lag with pH follows the general pattern of activity with longer lags at intermediate pH values.Preincubation of the enzyme with various reaction components and effectors reduces the lag, with NADH being the most effective. The presence of NADH in the assay is much more effective, but none of the treatments tried will completely eliminate the lag of freshly prepared enzyme. PMID- 16662121 TI - Ripening behavior of wild tomato species. AB - Nine wild tomato species were surveyed for variability in ripening characteristics. External signs of ripening, age of fruit at ripening, and ethylene production patterns were compared. Ethylene production was monitored using an ethylene-free air stream system and gas chromatography. Based on these ripening characteristics, the fruits fell into three general categories: those that change color when they ripen, green-fruited species that abscise prior to ripening, and green-fruited species that ripen on the vine.The fruits that change color, Lycopersicon esculentum var. cerasiforme, Lycopersicon pimpinellifolium and Lycopersicon cheesmanii, exhibited a peak of ethylene production similar to the cultivated tomato; there were differences, however, in the timing and magnitude of the ethylene production. Peak levels of ethylene production are correlated with age at maturity. For the two species that abscise prior to ripening, Lycopersicon chilense and Lycopersicon peruvianum, ability to produce ethylene varied with stage of maturity. The two species differed from each other in time of endogenous ethylene production relative to abscission, suggesting differences in the control mechanisms regulating their ripening. For two of the green-fruited species that ripen on the vine, Lycopersicon chmielewskii and Lycopersicon parviflorum, ethylene production was correlated to fruit softening. For Lycopersicon hirsutum and Solanum pennellii, however, ethylene production was not correlated with external ripening changes, making questionable the role of ethylene as the ripening hormone in these fruits. PMID- 16662122 TI - Kinetics of toluene-induced leakage of low molecular weight solutes from excised sorghum tissues. AB - The relationship between toluene concentration and the rate of leakage of solutes from toluene-treated roots and leaves of Sorghum bicolor, L. Moench, was studied to determine the effect of toluene on plant cell membranes. A threshold concentration of 0.2% toluene was needed to induce leakage. Maximal leakage rates were obtained with 0.5% toluene. Low molecular weight solutes, such as amino acids, sugars, and inorganic ions, leaked from treated tissue, while macromolecules, such as protein were retained. The rates at which the low molecular weight solutes diffused from treated cells decreased with increasing molecular weight. At 25 degrees C, treatment of roots and leaves with 0.5% toluene resulted in the quasi-quantitative leakage of solutes within 180 minutes. At 1 degrees C, roots and leaves differed in their response to toluene. The rates of leakage from roots at 1 degrees C were much lower and the total amounts much smaller than at 25 degrees C, while in leaves the difference between the two temperatures was very small.The procedure of treating tissues with 0.5% toluene for 180 minutes at 25 degrees C proved to be a rapid and simple technique for quantitative extraction of water-soluble, low molecular weight solutes from plant cells into the extracting medium while macromolecular constituents are retained inside the cells. PMID- 16662123 TI - Analysis of the dynamic and steady-state responses of growth rate and turgor pressure to changes in cell parameters. AB - The physical analysis of plant cell enlargment is extended to show the dependence of turgor pressure and growth rate under steady-state conditions on the parameters which govern cell wall extension and water transport in growing cells and tissues, and to show the dynamic responses of turgor and growth rate to instantaneous changes in one of these parameters. The analysis is based on the fact that growth requires simultaneous water uptake and irreversible wall expansion. It shows that when a growing cell is perturbed from its steady-state growth rate, it will approach the steady-state rate with exponential kinetics. The half-time of the transient adjustment depends on the biophysical parameters governing both water transport and irreversible wall expansion. When wall extensibility is small compared to hydraulic conductance, the growth rate is controlled by the yielding properties of the cell wall, while the half-time for changes in growth rate is controlled by the water transport parameters. The reverse situation occurs when hydraulic conductance is lower than wall extensibility. The analysis also shows explicitly that turgor pressure is tightly coupled with growth rate when growth is controlled by both water transport and wall yielding parameters.In growing tissue where the resistance to water flow is distributed throughout the tissue, the physical analysis is more complicated because gradients in water potential (and hence turgor pressure) are required to sustain high growth rates. However, the analysis of growth in such tissues shows that the turgor and time-course relations are similar to that in single cells. These turgor and time-course relations provide experimentally useful ways for determining (a) whether growth is limited by water uptake, and (b) whether an agent which alters the growth rate does so by affecting the water transport or wall yielding properties or both. PMID- 16662124 TI - Rapid Suppression of Growth by Blue Light : BIOPHYSICAL MECHANISM OF ACTION. AB - The mechanism of the rapid inhibition of hypocotyl elongation by blue light was investigated in cucumber (Cucumis sativus L.) and sunflower (Helianthus annuus L.) seedlings by measuring the changes in turgor during the response. A special device, based on the resonance frequency principle, was built which permitted simultaneous and continuous measurements of both tissue rigidity (turgor) and growth rate on a single intact hypocotyl. The large decrease in growth rate following blue irradiation was consistently accompanied by a small increase in resonance frequency. This result indicates that blue light inhibits growth by decreasing the yielding properties of the cell walls, resulting in a slight rise in turgor because of the coupling between growth rate and turgor.The nature of the blue-light inhibition was further studied by measuring the influence of light dose and temperature on the time course of inhibition (lag-time, half-time of inhibition, and amount of inhibition) with the aid of a microcomputer-based system for measuring growth rate and for controlling light duration and energy. The light dose has no influence on either the lag-time or the half-time of inhibition, but strongly affects the amount of inhibition. In contrast, a 10 degrees C drop in temperature (from 30 to 20 degrees C) lengthened the lag-time of the blue-light response, but did not significantly affect the half-time or the per cent inhibition by blue light. The half-time for changes in hypocotyl length (induced by applying a hydrostatic pressure to the roots or to the cut end of seedlings with roots excised) was found to be the same as the half-time of the blue-light inhibition (15 to 25 seconds in cucumber; 90 to 150 seconds in sunflower). These results support the idea that blue light, after a fixed lag period, induces an immediate decrease in the yielding properties of the cell walls. The growth rate subsequently decreases with a half-time that depends on the time required for cell turgor pressures to reach their new steady-state values. PMID- 16662125 TI - Growth, water content, and solute accumulation of two tobacco cell lines cultured on sodium chloride, dextran, and polyethylene glycol. AB - Simulated drought tolerance was compared for an NaCl-adapted and a nonadapted cell line of tobacco (Nicotiana tabacum var. Samsum) to determine the relationship of salt and drought tolerances. The osmotic adjustment and growth of these two lines was followed when cultured on solid media which contained isosmotic concentrations of NaCl, KCl, polyethylene glycol (PEG) or dextran. One line was adapted to growth on 130 millimolar NaCl, but the other was not.The growth of NaCl-adapted and nonadapted cell lines was equally inhibited (61 per cent of control) by 130 millimolar NaCl. Growth inhibition was greater on PEG or dextran than on NaCl. Growth ceased on the second passage of dextran for the nonadapted cells, while the NaCl-adapted cells grew slowly for four passages on dextran. Water contents for both cell lines were 95 per cent on NaCl or KCl and 70 to 88 per cent on PEG 1540 or 4000 or dextran after the second passage on these media.On dextran or PEG 4000, 46 to 89 per cent of the cellular osmotic potential was produced by the solutes initially present in the medium. Similarly, on NaCl, almost 100 per cent was attributable to solutes in the medium. It was concluded that cells grown on the nonpenetrating solutes had a more negative osmotic potential than those grown in the absence of added solute due to partial dehydration, greater uptake of external ions, and possibly the production of unidentified osmotica. Adjustment to growth on penetrating solutes may have enabled the adapted line to overcome the osmotic stress produced by nonpenetrating dextran. PMID- 16662126 TI - Phytotropins: III. NAPHTHYLPHTHALAMIC ACID BINDING SITES ON MAIZE COLEOPTILE MEMBRANES AS POSSIBLE RECEPTOR SITES FOR PHYTOTROPIN ACTION. AB - Certain members of the phytotropin class of auxin transport inhibitors are shown to bind with high affinity to the known naphthylphthalamic acid binding sites in maize (Zea mays) coleoptiles. The binding site is, thus, a phytotropin binding site. In general, the degree of binding correlates with the phytotropin structure activity rules and with physiological activities of model compounds. It is argued that the binding site may be a receptor, and it also may be the receptor involved in the control of the auxin transport process. The possibility is raised that the binding sites may be intrinsic receptors for endoanalog(s) of the phytotropins. PMID- 16662127 TI - An in vitro control mechanism for potato stress metabolite biosynthesis. AB - Ethylene/oxygen (E/O(2)) elevates sesquiterpenoid stress metabolite (SSM) levels in potato (Solanum tuberosum L.) tuber tissue which is reacting hypersensitively. To determine whether E/O(2) retards SSM turnover, a measured amount of rishitin was applied to tuber tissue which was then incubated in air or E/O(2), and rishitin disappearance was monitored. No difference in the rate of rishitin disappearance was detected between air and E/O(2) incubations. However, tissue treated with rishitin and incubated in E/O(2) accumulated intermediates of the katahdinone and phytuberin pathways. This was not the case in rishitin-air treatments. These results suggest the dual involvement of ethylene and SSM intermediates in the regulation of the biosynthesis of SSM, compounds which may serve as phytoalexins. PMID- 16662128 TI - Kinetic Complexity, Homogeneity, and Copy Number of Chloroplast DNA from the Marine Alga Olisthodiscus luteus. AB - The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar(-1) and second(-1) (Cot((1/2)) 0.24 molar second) under conditions equivalent to 180 millimolar Na(+) and 60 degrees C. Given the 92 x 10(5) dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples. PMID- 16662129 TI - Temperature Effects on the Activity of the Alternative Respiratory Pathway in Chill-Sensitive Cucumis sativus. AB - After 48 hours at 2 degrees C, hypocotyls from chill-sensitive Cucumis sativus seedlings showed a burst of O(2) uptake. The alternative pathway became engaged to close to 45% full capacity during this postchilling respiratory burst. However, it only accounted for up to 50% of this increased respiratory O(2) uptake. By 24 hours after chilling, when the seedlings were fully recovered from visible symptoms of chilling injury, the flux through the alternative pathway was back to the level (about 10%) found before chilling. Blocking chilling-induced ethylene production with aminoethoxyvinylglycine had no effect on this increased utilization of the alternative pathway.The direct effects of temperature on respiration rates and the effects of inhibitors suggested that there was a rapid increase in alternative pathway activity and decrease in the cytochrome pathway activity. The possibility that the alternative pathway represents a compensatory mechanism for the more labile cytochrome pathway is discussed. PMID- 16662130 TI - The Azolla-Anabaena azollae Relationship : XI. PHYCOBILIPROTEINS IN THE ACTION SPECTRUM FOR NITROGENASE-CATALYZED ACETYLENE REDUCTION. AB - Visible absorption spectra are presented for the Azolla caroliniana Willd. Anabaena azollae Strass. association and the individual partners. Although absorption by the phycobiliproteins of the endophytic cyanobacterium clearly complements the absorption by the fern pigments, their contribution to the absorption spectrum of the association is effectively concealed by the preponderance of the Azolla pigments. Action spectra for nitrogenase-catalyzed C(2)H(2) reduction in both the Azolla-Anabaena association and the endophytic Anabaena demonstrate that quanta absorbed by the phycobiliproteins is as effective as that absorbed by chlorophyll a in driving this photosystem I-linked process. Under anaerobic conditions, the inhibition of photosystem II activity by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron did not selectively decrease the relative quantum yields in the region of phycobiliprotein absorption. At the well below saturating light intensities used for the action spectra studies, the absolute rates of C(2)H(2) reduction were increased uniformly via respiratory linked processes under aerobic conditions. The occurrence of phycobiliproteins in heterocysts of the endophytic Anabaena was demonstrated using fluorescence microscopy of intact filaments. Fluorescence micrographs of Anabaena cylindrica filaments are presented for comparison. PMID- 16662131 TI - Stimulation of Photosystem I Electron Transport by High Concentration of 3-(3,4 Dichlorophenyl)-1,1-dimethyl Urea in Uncoupled Chloroplasts. AB - The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol --> methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate limitation imposed by energy coupling was not affected. Using N,N,N',N' tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts. PMID- 16662132 TI - Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves. AB - Studies were conducted to quantitate the evolution of nitrogen oxides (NO((x))) from soybean [Glycine max (L.) Merr.] leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N(2) and argon) consistently resulted in greater NO((x)) evolution than did aerobic gas purging (air and O(2)). The evolution of NO((x)) was dependent on gas flow rate and on NO(2) (-) formation in the assay medium; although a threshold level of NO(2) (-) appeared to exist beyond which the rate of NO((x)) evolution did not increase further.The loss of NO((x)) from in vivo NR assays under gas purging explains partially, but not stoichiometrically, the decrease in NO(2) (-) accumulation in in vivo NR assay medium with young soybean leaves. The lack of stoichiometry between NO((x)) evolution and apparent NO(2) (-) loss suggests that other mechanisms are also involved in loss of NO(2) (-) or inhibition of formation of NO(2) (-) during anaerobic and aerobic incubation conditions imposed on the in vivo NR assay of soybean. The mechanism of NO((x)) evolution under the assay conditions imposed and the relevance of this phenomenon to intact plants remains unclear. PMID- 16662133 TI - Rapid electric responses of oats to phytochrome show membrane processes unrelated to pelletability. AB - The electric potential difference changes observed on etiolated oat coleoptiles in response to phytochrome transformation have been further studied using contacts on the coleoptile surface. Results are given, at 0.4 second resolution, for the first 1.5 minutes after saturating flashes of light each lasting 1 second.Responses to initial red (662 nanometers), to far red (about 700 nanometers and above) 10 minutes later, and to second red 10 minutes later still, all have time courses that are approximately Gaussian sigmoid in shape. The response to far red is of opposite sign to the response to red. Approximate magnitudes of the three changes, 1 minute after the light flash, are +6 millivolts for red, -10 millivolts for far red, +3.5 millivolts for the second red. It is argued that the observations reflect a hyperpolarization of the plasmalemma of coleoptile cells following red light and depolarization following far red. The response to red is not produced by a change in membrane permeability to K(+). The mechanism could include a change to Na(+) or Cl(-) permeability or a modulation of an electrogenic pump: enhanced H(+) extrusion, Ca(2+) extrusion, or Cl(-) uptake. The response to far red could be produced by the reverse of one of those changes.The Gaussian curve is fitted to the data to determine the time at which the responses begin. Each response begins 4.5 seconds after the start of the flash of light. These delays are not related to the time course of phytochrome pelletability or redistribution in the cell. The delays may be due to some interaction of the transformed phytochrome with the plasma-lemma. Alternatively, the transformed phytochrome may interact quickly with some other structure which initiates a signal that takes 4.5 seconds to reach the plasmalemma. PMID- 16662134 TI - Photorespiration in Air and High CO(2)-Grown Chlorella pyrenoidosa. AB - Oxygen inhibition of photosynthesis and CO(2) evolution during photorespiration were compared in high CO(2)-grown and air-grown Chlorella pyrenoidosa, using the artificial leaf technique at pH 5.0. High CO(2) cells, in contrast to air-grown cells, exhibited a marked inhibition of photosynthesis by O(2), which appeared to be competitive and similar in magnitude to that in higher C(3) plants. With increasing time after transfer to air, the photosynthetic rate in high CO(2) cells increased while the O(2) effect declined. Photorespiration, measured as the difference between (14)CO(2) and (12)CO(2) uptake, was much greater and sensitive to O(2) in high CO(2) cells. Some CO(2) evolution was also present in air-grown algae; however, it did not appear to be sensitive to O(2). True photosynthesis was not affected by O(2) in either case. The data indicate that the difference between high CO(2) and air-grown algae could be attributed to the magnitude of CO(2) evolution. This conclusion is discussed with reference to the oxygenase reaction and the control of photorespiration in algae. PMID- 16662135 TI - Mutual Antagonism of Sulfur Dioxide and Abscisic Acid in Their Effect on Stomatal Aperture in Broad Bean (Vicia faba L.) Epidermal Strips. AB - Abscisic acid (ABA) was found to counteract the stomatal opening in Vicia faba L. caused by SO(2). The antagonism between SO(2) and ABA was mutual, and their combined effect depended upon which compound was in the greatest concentration. Stomatal apertures were monitored in detached epidermal strips floated in the light on aqueous solutions of SO(2) (sulfurous acid) and/or ABA in 0.01 molar sodium citrate buffer (pH 5.8). Low concentrations of sulfurous acid (10(-10) to 10(-7) molar) increased stomatal aperture, but concentrations greater than 10(-5) molar decreased it. A progressive decrease in aperture size occurred as ABA was increased from 10(-10) to 10(-5) molar.No evidence was found for a direct chemical reaction between the buffered sulfurous acid and ABA (exogenous or endogenous). Extractable, endogenous ABA in the strips remained relatively constant after exposure to several different concentrations of sulfurous acid. A technique for quantitating ABA from methanolic extracts of small samples of epidermis (20 milligrams dry weight) using reverse phase high performance liquid chromatography is described. PMID- 16662136 TI - Growth and metabolic activity of lemon juice vesicle explants in vitro. AB - Actively growing juice-vesicle explants, established from small lemon (Citrus limon L. Burm. f. var. ;Eureka') fruits, were cultured in vitro on defined media. The juice vesicles continued to enlarge in culture for several months, without callus proliferation, and their initial growth was promoted by indoleacetic acid, gibberellic acid, and N(6)-benzyladenine.The metabolic competence of the explants, and its relevance to whole fruit development, was further investigated. Juice vesicles continued to accumulate protein on all media, for at least 16 days, but sugars did not change much during culture. Acid invertase increased dramatically during the first days of culture, and its specific activity was markedly promoted by indoleacetic acid, and to a lesser degree by gibberellic acid. Total peroxidase of juice vesicles increased progressively up to the 11th day of culture, and its specific activity was promoted by all three hormones, especially by N(6)-benzyladenine. Explant growth was accompanied by appearance of several cathodic and anodic isoperoxidases.The results suggest that the study of this unique juice vesicle system may contribute to a better understanding of fruit development. PMID- 16662137 TI - Promotion by gibberellic Acid of polyamine biosynthesis in internodes of light grown dwarf peas. AB - When gibberellic acid (GA(3); 5-35 micrograms per milliliter) is sprayed on 9-day old light-grown dwarf Progress pea (Pisum sativum) seedlings, it causes a marked increase in the activity of arginine decarboxylase (ADC; EC 4.1.1.9) in the fourth internodes. The titer of putrescine and spermidine, polyamines produced indirectly as a result of ADC action, also rises markedly, paralleling the effect of GA(3) on internode growth. Ammonium (5-hydroxycarvacryl) trimethyl chloride piperidine carboxylate (AMO-1618; 100-200 micrograms per milliliter) causes changes in the reverse direction for enzyme activity, polyamine content, and growth. GA(3) also reverses the red-light-induced inhibition of ADC activity in etiolated Alaska pea epicotyls; this is additional evidence for gibberellin-light interaction in the control of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17), an alternate source of putrescine arising from arginine, is not increased by GA(3) or by AMO-1618.The results support the hypothesis that ADC and polyamine content are important regulators of plant growth. PMID- 16662138 TI - Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS. AB - Corn (Zea mays, L.), bean (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), spinach (Spinacia oleracea L.), and sugarbeet (Beta vulgaris L.) grown under iron deficiency, and Potamogeton pectinatus L, and Potamogeton nodosus Poir. grown under oxygen deficiency, contained less chlorophyll than the controls, but accumulated Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester. No significant accumulation of these intermediates was detected in the controls or in the tissue of plants stressed by S, Mg, N deficiency, or by prolonged dark treatment. Treatment of normal plant tissue with delta-aminolevulinic acid in the dark resulted in the accumulation of protochlorophyllide. If this treatment was carried out under conditions of iron or oxygen deficiency, less protochlorophyllide was formed, but a significant amount of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester accumulated.These results are consistent with the presence of an O(2), Fe-requiring step between Mg protoporphyrin IX monomethyl ester and protochlorophyllide. PMID- 16662139 TI - Purification and regulatory properties of mung bean (vigna radiata L.) serine hydroxymethyltransferase. AB - Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C(1) fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto.The enzyme preparation was extremely stable in the presence of folate or l-serine. Pyridoxal 5' phosphate, ethylenediaminetetraacetate and 2-mercaptoethanol prevented the inactivation of the enzyme during purification. The enzyme functioned optimally at pH 8.5 and had two temperature maxima at 35 and 55 degrees C. The K(m) values for serine were 1.25 and 68 millimolar, corresponding to V(max) values of 1.8 and 5.4 micromoles of HCHO formed per minute per milligram protein, respectively. The K(0.5) value for l-tetrahydrofolate (H(4)folate) was 0.98 millimolar. Glycine, the product of the reaction and d-cycloserine, a structural analog of d-alanine, were linear competitive inhibitors with respect to l-serine with K(i) values of 2.30 and 2.02 millimolar, respectively. Dichloromethotrexate, a substrate analog of H(4)folate was a competitive inhibitor when H(4)folate was the varied substrate. Results presented in this paper suggested that pyridoxal 5'-phosphate may not be essential for catalysis.The sigmoid saturation pattern of H(4)folate (n(H) = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (n(H) = 1.0) and the increase in sigmoidicity by NAD(+) and adenine nucleotides, negative allosteric effectors (n(H) = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) serine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, dl-alpha-methylserine and dl-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the n(H) value from 2.0 for H(4)folate to 1.5 in presence of l-serine.These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes. PMID- 16662140 TI - Effects of Iron and Oxygen on Chlorophyll Biosynthesis : II. OBSERVATIONS ON THE BIOSYNTHETIC PATHWAY IN ISOLATED ETIOCHLOROPLASTS. AB - The conversion of l-glutamate to delta-aminolevulinate, in preparations of cucumber etiochloroplasts incubated in vitro, was inhibited by protoheme IX and Mg-protoporphyrin IX. Mg-protoporphyrin IX was destroyed in the presence of air and plastids; this breakdown was accelerated by S-adenosyl methionine. Mg protoporphyrin IX was also converted to protochlorophyllide in vitro. This conversion exhibited an absolute requirement for atmospheric oxygen and was strongly stimulated by S-adenosyl methionine and by darkness.Based on these results, and on the results of the preceding paper (Spiller, Castelfranco, Castelfranco 1981 Plant Physiol 68: 107-111), a comprehensive hypothesis for the role of O(2) and Fe in chlorophyll biosynthesis is formulated. PMID- 16662141 TI - A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species. AB - Conversion of [(14)C]galactose (Gal) 1-P, UDP-[(14)C]Gal, or UDP-[(14)C]glucose to [(14)C]sucrose was observed when cell-free homogenates of cucumber (Cucumis sativus L.) fruit peduncles were incubated with individual (14)C-labeled substrates, appropriate cofactors, and fructose. The sucrose product was labeled only in the glucose moiety. Conversion of [(14)C]Gal-1-P to [(14)C]sucrose was catalyzed by extracts of peduncles from all other stachyose transporting species tested, as well as green bean (a sucrose transporter) but was not catalyzed by peduncle extracts from three other sucrose transporting species. In cucumber, the ability of extracts to form [(14)C]sucrose from [(14)C]Gal-1-P was greater when peduncles were harvested from growing fruit than from unpollinated ovaries. [(14)C]Sucrose formation from [(14)C]Gal-1-P was inhibited by Mg . PPi, Mg . UDP, UMP, and sucrose. alpha-Galactosidase, galactokinase, UDP-gal pyrophosphorylase, UDP-Gal-4'-epimerase, UDP-glucose pyrophosphorylase, and sucrose synthase activities were detected in peduncle extracts. Neither sucrose phosphate synthetase nor hexose-1-P uridyltransferase were detected. Peduncle tissue contained a small pool of free galactose. These results suggest a potential pathway for the metabolism of galactose moieties hydrolyzed from stachyose, the major sugar transported by cucumber plants. PMID- 16662142 TI - Degradation of isolated tomato cell walls by purified polygalacturonase in vitro. AB - Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.Similar cell wall-bound protein extracts from ripe Nr fruit were not as effective and those from ripe rin fruit gave no cell wall degradation at all in vitro. This was correlated with the absence of polygalacturonase in rin and low activity of Nr extracts.Purified polygalacturonase was capable of in vitro cell wall degradation and it seems that this enzyme can account for the cell wall degradation observed with the total cell wall-bound protein extracts from ripe fruit. PMID- 16662144 TI - Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.). AB - In excised pro(1-1) mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [(14)C]proline, [(14)C]glutamic acid, [(14)C]glutamine, and [(14)C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO(2) production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro(1-1) mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro(1 1) mutant proline catabolism prevails over proline synthesis. PMID- 16662143 TI - Chilling-Susceptibility of the Blue-Green Alga Anacystis nidulans: III. LIPID PHASE OF CYTOPLASMIC MEMBRANE. AB - The lipid phase of cytoplasmic membrane was studied by freeze-fracture electron microscopy in the chilling-susceptible blue-green alga, Anacystis nidulans. At growth temperatures, intramembrane particles were distributed at random in the fracture faces of cytoplasmic membrane, whereas, at chilling temperatures, the fracture faces were composed of particle-free and particle-containing regions. These findings indicate that lipids of the cytoplasmic membrane were in the liquid-crystalline state at the growth temperatures and in the phase-separation state at the chilling temperatures. Temperatures for the onset of phase separation were 5 and 16 degrees C in cells grown at 28 and 38 degrees C, respectively.In comparison, another blue-green alga, Anabaena variabilis, which is not susceptible to chilling, was also examined by the freeze-fracture electron microscopy. The intramembrane particles were distributed at random in the fracture faces of cytoplasmic membrane at the growth, as well as at the chilling, temperatures.The results in this and previous studies suggest that the chilling susceptibility of A. nidulans is a result of irreversible leakage of ions from the cytoplasm when the lipids of cytoplasmic membrane are in the phase-separation state at low temperatures. PMID- 16662145 TI - Protein Synthesis and Photosynthetic Recovery in the Resurrection Plant, Selaginella lepidophylla. AB - The effects of inhibition of protein synthesis on whole-plant CO(2) exchange and on protein synthesis during hydration of the resurrection plant Selaginella lepidophylla (Hook and Grev.) were examined. Both chloramphenicol and cycloheximide inhibited the redevelopment of photosynthetic capacity which normally occurs within 24 hours of hydration in the light. The inhibitory effect of chloramphenicol was greater than that of cycloheximide. The onset of chloramphenicol inhibition of net photosynthesis occurred only after 12 hours of hydration. Cycloheximide stimulated net CO(2) influx early after rehydration and inhibited net CO(2) influx after 14 hours of hydration. Total protein synthesis, as measured by l-[(35)S]methionine incorporation, increased through 24 hours of hydration. Based upon the results obtained with the protein synthesis inhibitors, most protein synthesis within the first 12 hours of hydration was cytoplasm directed, whereas the rate of organelle-directed protein synthesis remained low until 12 hours of hydration and increased rapidly thereafter. These data suggest that both organelle- and cytoplasm-directed protein synthesis are necessary for full photosynthetic recovery during rehydration of S. lepidophylla. PMID- 16662146 TI - Effects of Powdery Mildew Infection on the Efficiency of CO(2) Fixation and Light Utilization by Sugar Beet Leaves. AB - Sugar beet leaves (Beta vulgaris L.) infected with powdery mildew (Erysiphe polygoni D.C.) show declining rates of net photosynthesis as the disease develops; relative to healthy controls, reductions of 35, 70, and 75% were observed at 9, 16, and 22 days after inoculation, respectively. A leaf gas exchange procedure in which an air stream flowed through the leaf showed that mesophyll conductance declined in parallel with photosynthesis in mildew-infected leaves. Viscous flow conductance of diseased leaves also declined over the same period suggesting that stomatal aperture was reduced. From the magnitude and time course of disease effects on stomatal aperture and mesophyll conductance, it appears that the effects at the mesophyll level were primarily responsible for mediating the decline in net photosynthesis. Changes in mesophyll conductance were closely correlated with reduced activity of ribulose-1,5-bisphosphate carboxylase on a leaf area basis. This decrease could be attributed to a reduction in the concentration of the enzyme, a reduction which was greater than the reduction in total soluble protein. The quantum efficiency of light use was also decreased by the disease. Mildew-infected leaves had quantum yields that were reduced, relative to healthy leaves, by 17 and 22% at 14 and 18 days after inoculation, respectively. PMID- 16662147 TI - Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds. AB - Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination. PMID- 16662148 TI - Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans. AB - CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W 1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C. PMID- 16662149 TI - In Vitro Synthesis and Processing of Wheat alpha-Amylase : TRANSLATION OF GIBBERELLIC ACID-INDUCED WHEAT ALEURONE LAYER RNA BY WHEAT GERM AND XENOPUS LAEVIS OOCYTE SYSTEMS. AB - Wheat (Triticum aestivum) RNA was used to program synthesis of the alpha-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as alpha-amylase by immunoprecipitation with rabbit anti-alpha-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic alpha-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of alpha-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of alpha-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the alpha-amylase protein to a form which co-migrated with authentic alpha-amylase purified from malted wheat and with the protein synthesized in oocytes. PMID- 16662150 TI - Collection and Identification of Allelopathic Compounds from the Undisturbed Root System of Bigalta Limpograss (Hemarthria altissima). AB - Collection of allelopathic chemicals from the undisturbed plant root system is difficult because of their low concentrations and the high level of contaminants in growth media such as soil. A new approach for the continuous trapping of quantities of extracellular chemicals from donor plants is described. Bigalta limpograss (Hemarthria altissima), a tropical forage with allelopathic activities, was established in sand culture. Nutrient solution was circulated continuously through the root system and a column containing XAD-4 resin. Extracellular hydrophobic metabolites were selectively adsorbed by the resin, while inorganic nutrients were recycled to sustain plant growth. Columns were eluted with methanol and the eluate separated into neutral, acidic, and basic fractions. Bioassays of trapped root exudates using lettuce seed combined with paper and thin layer chromatography showed that the inhibitors were mainly phenolic compounds. The active neutral fraction was methylated and analyzed by gas chromatography-mass spectrometry. Twelve compounds were identified, with two additional compounds tentatively identified. 3-Hydroxyhydrocinnamic, benzoic, phenylacetic, and hydrocinnamic acids were the major rhizospheric compounds with known growth regulatory activities.Inasmuch as the root system was undisturbed and the recovery of exudates was highly efficient compared to conventional solvent extraction methods, this trapping system should be useful for a wide range of studies that relate to the chemistry of the rhizosphere. PMID- 16662151 TI - Isolation and Characterization of an Iron-Containing Superoxide Dismutase From Water Lily, Nuphar luteum. AB - A cyanide-insensitive superoxide dismutase (EC 1.15.1.1) was purified from leaves of the water lily Nuphar luteum (L.) Sibth. and Smith Subsp. macrophyllum (Small) Beal. The enzyme had a molecular weight of 46,000 and was composed of two equally sized subunits. Metal analysis showed the protein to contain about 1 gram atom of iron per dimer. The iron-containing superoxide dismutase was sensitive to H(2)O(2) as well as to azide. Antibody to the protein did not cross-react with iron superoxide dismutase isolated from the eucaryote Brassica or with algal extracts. PMID- 16662152 TI - Relative Content of NO(3) and Reduced N in Xylem Exudate as an Indicator of Root Reduction of Concurrently Absorbed NO(3). AB - It is unclear if the relative content of NO(3) (-) and reduced N in xylem exudate provides an accurate estimate of the percentage reduction of concurrently absorbed NO(3) (-) in the root. Experiments were conducted to determine whether NO(3) (-) and reduced N in xylem exudate of vegetative, nonnodulated soybean plants (Glycine max [L.] Merr., ;Ransom') originated from exogenous recently absorbed (15)NO(3) (-) or from endogenous (14)N pools. Plants either were decapitated and exposed to (15)NO(3) (-) solutions for 2 hours or were decapitated for the final 20 minutes of a 50-minute exposure to (15)NO(3) (-) in the dark and in the light. Considerable amounts of (14)NO(3) (-) and reduced (14)N were transported into the xylem, but almost all of the (15)N was present as (15)NO(3) (-). Dissimilar changes in transport of (14)NO(3) (-), reduced (14)N and (15)NO(3) (-) during the 2 hours of sap collection resulted in large variability over time in the percentage of total N in the exudate which was reduced N. Over a 20-minute period the rate of (15)N transport into the xylem of decapitated plants was only 21 to 36% of the (15)N delivered to the shoot of intact plants. Based on the proportion of total (15)N which was found as reduced (15)N in exudate and in intact plants in the dark, it was estimated that 5 to 17% of concurrently absorbed (15)NO(3) (-) was reduced in the root. This was much less than the 38 to 59% which would have been predicted from the relative content of total NO(3) (-) and total reduced N in the xylem exudate. PMID- 16662153 TI - Ion balance, uptake, and transport processes in n(2)-fixing and nitrate- and urea dependent soybean plants. AB - The objective of this study was to examine the influence of N(2) fixation and NO(3) (-)-N and urea-N assimilation on ion balance, uptake, and transport processes in soybean (Glycine max L. Merr.).Inoculated plants were grown in Perlite supplied daily with nutrient solutions which contained zero-N, 10 and 20 millimolar NO(3) (-)-N, and 10 and 20 millimolar urea-N, and they were sampled 41, 76, and 151 days after transplanting. Total uptake of inorganic cations and anions was determined by analysis of tissue for K(+), Ca(2+), Mg(2+), Na(+), total N from NO(3) (-), total S, H(2)PO(4) (-), and Cl(-). Differences in total inorganic cations (C) and inorganic anions (A) in plant tissue were used to estimate total carboxylate content.Internal OH(-) generation resulting from excess cation uptake (net H(+) excretion) by the roots accounted for more than 89% of the carboxylate accumulation in N(2)- and urea-fed plants, while OH(-) generation resulting from SO(4) (2-) reduction accounted for less than 11%. Shoots contained over 89% of the total plant carboxylate content. Malate balanced about 75% of the excess inorganic cationic charge of the xylem sap; allantoate and aspartate balanced most of the remaining charge. These results indicate that carboxylates (primarily malate) are synthesized in roots of N(2)- and urea-fed plants and transported to the shoots in the xylem to maintain charge balance. The high malate concentration resulted in the C/N weight ratio of xylem sap from N(2) fed plants being >2.0, even though 83% of the N was transported as allantoin and allantoic acid which have a C/N ratio of 1.0. The data emphasize that C and N content of N compounds should not be the sole basis for calculating the C/N weight ratio of xylem sap.The C-to-A uptake ratio for plants supplied 10 millimolar NO(3) (-) ranged from 1.24 to 1.57 during development, indicating that internal OH(-) was generated both by excess cation uptake and by NO(3) (-) and SO(4) (2-) reduction. The C-to-A uptake ratio for 20 millimolar NO(3) (-) -fed plants ranged from 0.86 to 0.96 during development, indicating a small net OH(-) efflux from the roots for support of excess anion uptake. On a seasonal basis, only 15% of the OH(-) generated during NO(3) (-) and SO(4) (2-) reduction was associated with OH(-) efflux (excess anion uptake), while 85% was associated with carboxylate accumulation. The malate concentration in xylem sap from plants supplied 20 millimolar NO(3) (-) was only one-third that of N(2)- and urea-fed plants; however, it did balance 75% of the excess inorganic cationic charge. Potassium, recycling to accommodate excess anion uptake by 20 millimolar NO(3) fed plants, was calculated to involve at most 17% of the total K(+) absorbed during the 41- to 76-day growth interval. PMID- 16662155 TI - ATP-Dependent Calcium Transport in Plasmalemma Preparations from Soybean Hypocotyls : EFFECT OF HORMONE TREATMENTS. AB - A plasmalemma-enriched membrane preparation from etiolated soybean (Glycine max L., cv. Wayne) hypocotyls possesses an active ATP-dependent calcium pump which leads to calcium accumulation when assayed by the methods of Gross and Marme (1978, Proc Natl Acad Sci USA 75: 1232-1236). Two-hour treatment of segments from the elongating zone of the hypocotyl with growth-promoting concentrations of indoleacetic acid gives up to 100 percent increase in the calcium transport activity. Conversely, similar pretreatment with zeatin or other cytokinins is inhibitory. In the meristematic and maturing zones of the hypocotyl, zeatin has the opposite effect of promoting calcium transport activity. One facet of cell growth regulation may lie with hormonally mediated changes in efflux pumping of calcium. PMID- 16662154 TI - Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation : REGULATION BY PRIMER, ADENYLATES, FERREDOXIN, AND PYRIDINE NUCLEOTIDES. AB - A particulate preparation (MgP) capable of photosynthetic CO(2) assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl(2) buffered with Tricine at pH 8.5. This CO(2) assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO(2) fixation by MgP were similar to those found with whole chloroplasts.Glycerate-3-phosphate (PGA), fructose-1, 6-bisphosphate (FBP), and ribose-5-phosphate (R5P) but not fructose-6-P (F6P) functioned as primers. Concentrations of PGA and FBP but not of R5P higher than 2 millimolar were inhibitory to CO(2) fixation. A lag of CO(2) fixation was observed with PGA and FBP but not with R5P. This lag as well as inhibition by NADP, ADP, and ATP in the FBP-primed preparation was eliminated by an equimolar mixture of FBP plus F6P indicating FBPase as the sensitive site. NADP, ADP, and ATP also blocked CO(2) fixation by the PGA-fortified preparation but inhibition was even more sensitive than that observed when FBP was added. Inhibition by AMP in the PGA and FBP primed preparations was not affected by the addition of F6P. When R5P was the starting primer, inhibition of CO(2) fixation was relatively insensitive to the adenylates and NADP. In contrast to the parent whole chloroplast, CO(2) fixation by MgP was insensitive to high (5 millimolar) inorganic phosphate. Depending upon the ferredoxin concentration, NAD was as effective as NADP in supporting CO(2) fixation. PMID- 16662156 TI - Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves. AB - The in vivo oxidation of the C(4) and C(5) of 5-aminolevulinic acid (ALA) to CO(2) has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of (14)CO(2) evolution from leaves fed [4-(14)C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of (14)CO(2) evolution from leaves fed [5-(14)C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C(4) is oxidized to CO(2) via the tricarboxylic acid cycle to a greater extent than is the C(5). PMID- 16662157 TI - Enhanced Incorporation of Tritium into Glycolate during Photosynthesis by Tobacco Leaf Tissue in the Presence of Tritiated Water. AB - Tobacco (Nicotiana tabacum var. Havana Seed) leaf discs were allowed to photosynthesize for 3 to 20 minutes in the presence of (14)CO(2) and (3)H(2)O. Several metabolites of the Calvin cycle and photorespiratory pathway were isolated and purified and the (3)H:(14)C values measured. Glycolate had a 5- to 10-fold higher (3)H:(14)C than the Calvin cycle intermediate 3-phosphoglyceric acid, or its end product sucrose. The glycolate oxidase inhibitor alpha-hydroxy-2 pyridinemethanesulfonic acid caused glycolate to accumulate in the tissue and lowered the (3)H:(14)C in glycolate to a value similar to that in 3 phosphoglyceric acid. Phosphoglycolate, a possible precursor of glycolate arising from the Calvin cycle, exhibited a (3)H:(14)C value similar to 3-phosphoglyceric acid under all conditions. The finding of a (3)H enrichment in glycolate suggests that another source of glycolate, possibly the reduction of glyoxylate, exists in leaf tissue. Analyses of incorporation of (3)H into the pro-2R and pro-2S hydrogens of glycolate, in the presence and absence of alpha-hydroxy-2 pyridinemethanesulfonic acid, suggest an alternative source of glycolate. Biochemical mechanisms to account for (3)H enrichment into glycolate are evaluated. PMID- 16662158 TI - Altered glycine decarboxylation inhibition in isonicotinic Acid hydrazide resistant mutant callus lines and in regenerated plants and seed progeny. AB - Isonicotinic acid hydrazide (INH), an inhibitor of the photorespiratory pathway blocking the conversion of glycine to serine and CO(2), has been used as a selective agent to obtain INH-resistant tobacco (Nicotiana tabacum) callus cells. Of 22 cell lines that were INH-resistant, none were different from wild-type cells in their ability to take up [(3)H]INH or to oxidize INH to isonicotinic acid. In 7 of the 22 cell lines, INH resistance was associated with decreased inhibition of NAD-dependent glycine decarboxylation activity in isolated mitochondrial preparations. In the cell line that was most extensively investigated (I 24), this biochemical phenotype (exhibiting a 3-fold higher K(i) with INH) was observed in leaf mitochondria of regenerated plants and of plants produced from them by self-fertilization. After crosses between resistant and sensitive plants, the decreased inhibition of glycine decarboxylation was observed among F(2) and backcross progeny only in those plants previously identified as INH-resistant by callus growth tests. In contrast, in siblings identified as INH-sensitive, glycine decarboxylation was inhibited by INH at the wild-type level. This demonstration of the transfer of an altered enzyme property from callus to regenerated plants and through seed progeny fulfills an important requirement for the use of somatic cell genetics to produce biochemical mutants of higher plants. PMID- 16662159 TI - Changes in two ribonuclease isozymes during rust infection of flax cotyledons. AB - A ribonuclease fraction previously purified from flax by gel filtration was further resolved into two components by hydroxyl apatite chromatography. These were homogeneous with respect to electrophoresis and isoelectric focusing. Both enzymes are of RNase I type but differ in substrate specificity, kinetic properties, pH response, and isoelectric point.The two RNase isozymes show consistent properties when extracted from variety Bison (susceptible) or variety Bombay (resistant) with or without infection with race 3 of flax rust. The relative amounts of these isozymes change markedly during infection. These observations provide an explanation for the apparent qualitative changes in RNase noted previously. Differences between susceptible and resistant reactions in the early stages of disease are discussed. PMID- 16662161 TI - Octopine Accumulation Early in Crown Gall Development is Progressive. AB - A purification of octopine from crown gall tissue was developed to quantitate conversion of precursor [(3)H]arginine into [(3)H]octopine. Plant wound tissue which was sterile or infected with an avirulent strain of Agrobacterium tumefaciens did not accumulate detectable quantities of octopine, consistent with opine synthesis not being induced by wounding or infection. Octopine was only recovered from tissue infected with virulent tumor-inducing strains of A. tumefaciens. In every case tested, the morphological appearance of tumors preceded the accumulation of octopine by at least 1 week, and in some instances 3 weeks. Thus, what was necessary and sufficient for the expression of plant hormones (auxin and cytokinin) required for tumor growth was not sufficient for the accumulation of octopine. The possible nature of the temporal difference in the expression of hormone autotrophy and octopine synthesis is discussed. PMID- 16662160 TI - Interaction of Ferredoxin-NADP Oxidoreductase with the Thylakoid Membrane. AB - The binding of ferredoxin-NADP reductase to spinach chloroplast membranes was studied by washing the membranes with different media. Release of the enzyme from the thylakoids was greater in 0.75 millimolar EDTA but was not complete inasmuch as 20% the activity remained membrane-bound after three washes.A Scatchard plot of binding experiments suggests the presence of one type of binding site and a stoichiometry of 3 to 4 nanomoles of reductase per micromole of chlorophyll was calculated. Rebinding has a nonspecific requirement for cations. Their effectiveness increased with their valency. Rebinding of purified enzyme to depleted membranes resulted in a stimulation of its diaphorase activity.It is suggested that binding of ferredoxin-NADP reductase to thylakoid membranes is dependent upon neutralization of negative charges. PMID- 16662162 TI - Redistribution of Tritium during Germination of Grain Harvested from myo-[2 H]Inositol- and scyllo-[R-H]Inositol-Labeled Wheat. AB - Wheat kernels from myo-[2-(3)H]inositol- or scyllo-[R-(3)H]inositol-labeled plants (Sasaki and Loewus 1980 Plant Physiol 66: 740-745) were used to study redistribution of (3)H into growing regions during germination. Most of the labeled 1-alpha-galactinol (or the analogous scyllo-inositol galactoside) was hydrolyzed within 1 day. Water-soluble phytate was dephosphorylated within 3 days. A large reserve of bound phytate continued to release myo-inositol over several days. Translocation of free myo-inositol to growing regions provided substrate for the myo-inositol oxidation pathway and incorporation of (3)H into new cell wall polysaccharides.Cell wall polysaccharides in the kernel were degraded during germination. The labeled residues were translocated to growing regions and reutilized for new cell wall formation. Pentosyl residues accounted for most of this label.Free scyllo-inositol followed a path of translocation from kernel to seedling similar to that of myo-inositol. Unlike myo-inositol, it did not furnish substrate for the myo-inositol oxidation pathway but accumulated as free scyllo-inositol in the seedling.The fate of phytate-derived myo-inositol during germination of wheat is discussed in relation to a recent scheme of phytate metabolism proposed by De and Biswas (1979 J Biol Chem 254: 8717-8719) for germinating mung bean seedlings. PMID- 16662163 TI - Reduction in Sink-Mobilizing Ability following Periods of High Carbon Flux. AB - Sink tissues may play a significant role in determining photosynthetic rates through their ability to mobilize assimilates. The objective in this study was to determine if the mobilizing ability of taproot sink tissues of sugarbeet (Beta vulgaris) could become limiting when assimilate supply was maintained at a high level for an extended period of time. Assimilate supply was either enhanced by CO(2) enrichment or reduced by shading.Field-grown sugarbeet plants were exposed to ambient CO(2) and one of five photosynthetically active radiation (PAR) durations: 10-hours PAR; 6-hours PAR; 3-hours PAR; 1-hour PAR; and continuous 80% shade conditions or 1,000 microliter per liter CO(2) and 10-hour PAR. Taproots were harvested at 1600 hours on the day following the initiation of the treatments. The sucrose-uptake capacity of excised tissue discs was determined in 30 millimolar morpholinopropane sulfonic acid (pH 7.0) containing 40 millimolar [(14)C]sucrose.Rates of sucrose uptake were inversely related to the supply of photosynthate during the preceding light period. CO(2) enrichment reduced uptake capacity relative to the control. In contrast, reducing the duration of PAR increased uptake over the control. Leaf starch accumulation was correlated with reduced uptake capacity. The results indicate that, under the conditions employed here, the mobilizing ability of sinks may limit carbon flux from source and to sink during periods of high photosynthetic rates. PMID- 16662164 TI - Ozone Inhibition of Photosynthesis in Chlorella sorokiniana. AB - Exposure of Chlorella sorokiniana (07-11-05) to ozone inhibits photosynthesis. In this study, the effects of ozone on O(2) evolution and fluorescence yields are used to characterize this inhibition. At an ozone dose of about 3 micromoles delivered to 2 x 10(9) cells, the photosynthetic rate of the cells is inhibited 50%, as indicated by a decrease in bicarbonate-stimulated O(2) evolution (control rate, 1.4 +/- 0.3 x 10(-15) moles per cell per minute).Normal patterns of chlorophyll fluorescence are also altered. Upon continuous exposure to ozone (3.5 x 10(-7) moles O(3) per minute), three stages of change in relative fluorescence yields are observed: (a) a rise in variable yield with no corresponding change in nonvariable yield (after 1-2 minutes), which was interpreted to be a shift in the energy flow pathway; (b) a decline in variable yield with a slight rise in nonvariable yield (requiring 3-5 minutes), interpreted to be a blockage in the CO(2) fixation pathways; and (c) complete blockage of variable yield with a concurrent decline in nonvariable yield (8-10 minutes), interpreted to be a destruction of the pigment system. The timing of each stage depended upon the ozone concentration and its delivery rate to the cell suspension. These results are compared with ozone-induced decline in photosynthesis and leaf water potential changes reported for other plant systems.Evidence is also presented to suggest that ozone effects on the photosynthetic processes are attributable to ionic imbalances brought about by ozone interaction with the plasmalemma rather than a direct effect on the chloroplast. PMID- 16662165 TI - A Novel Enolic beta-Ketoaldehyde Phytotoxin Produced by Stemphylium botryosum f. sp. lycopersici : PARTIAL CHEMICAL AND BIOLOGICAL CHARACTERIZATION. AB - A new phytotoxin, stemphyloxin I, C(21)H(32)O(5), was isolated from cultures of the pathogenic fungus Stemphylium botryosum f. sp. lycopersici. The toxin is a tricyclic compound possessing a most unusual beta-ketoaldehyde group. Injection of stemphyloxin I into a tomato leaflet caused unlimited necrotic spots and a loss of turgor, which at higher toxin concentration wilted the whole compound leaf. Visible symptoms could be observed at a toxin concentration as low as 2.7 micromolar. Stemphyloxin I is a nonspecific toxin. It exhibits a differential toxicity towards various plants, tomato and eggplant being the most sensitive. Incorporation of [(14)C]amino acids into proteins of exponentially growing tomato cell suspension was completely suppressed in the presence of 1 micromolar toxin. The toxin showed no significant difference in its inhibitory activity against green and white tomato cell cultures. The methoxy derivative of stemphyloxin I, in which the beta-ketoaldehyde group is exclusively modified, showed a reduction of approximately 50 times in its inhibitory activity as compared to the toxin. The diacetate derivative conferred the same activity as stemphyloxin I. PMID- 16662166 TI - Ribosomal RNA synthesis in soybean suspension cultures growing in different media. AB - The transcription of ribosomal RNA has been studied in suspension tissue cultures of soybean Glycine max L. Merr cv. Mandarin cells (SB-1). A large precursor molecule was synthesized which contains RNA homologous to the 25 and 18S cistrons. Transcription was from one strand and appeared to start adjacent to the 18S cistron and to proceed through the 18S DNA, a <0.6-kilobase transcribed spacer and the 25S cistron. A nontranscribed spacer region was identified. When cells grew rapidly in sucrose (24 hours doubling time) they contained 7 times as many ribosomes as when they grew slowly in maltose (200 hours doubling time). Upon transfer from maltose to sucrose, cells began to accumulate ribosomes at a rapid rate (80-fold more rapid synthesis than in maltose medium) within 2 hours at 33 degrees C. The 2-hour lag is due in large part to a longer processing time during which newly synthesized RNA is packaged into ribosomes. Therefore, the increase in transcription rate may occur within a few minutes of the transfer to sucrose. PMID- 16662167 TI - Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : II. TIME COURSE OF LEVELS OF INTERMEDIATES AND IN VIVO CONVERSION RATES. AB - Ethylene production was stimulated severalfold during the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV). Exogenous methionine or S adenosylmethionine (SAM) did not increase ethylene evolution from healthy or TMV infected leaf discs, although both precursors were directly available for ethylene production. This indicates that ethylene production is not controlled at the level of methionine concentration or availability, nor at the level of SAM production or concentration. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) stimulated ethylene production considerably. Thus, ethylene production is primarily limited at the level of ACC production.The regulation of ethylene production during the hypersensitive reaction to TMV was further studied by determining the time course of the concentrations of methionine, SAM, and ACC, as well as the course of their in vivo conversion rates. Endogenous concentrations of methionine and SAM remained unaffected until late in infection. On the contrary, the peak in ethylene production near the time of local lesion development was preceded by a large increase in ACC production. As a result of this increase, ACC accumulated in the leaf tissue. Only after local lesions became visible, the capacity to convert ACC into ethylene increased severalfold, associated with a sharp decrease in ACC content and a large increase in ethylene production.Ethylene production in tobacco leaves reacting hypersensitively to TMV is thus regulated at the level of both the production of ACC and its conversion to ethylene. PMID- 16662168 TI - Amino Acid Transport in Suspension-Cultured Plant Cells : III. COMMON CARRIER SYSTEM FOR THE UPTAKE OF l-ARGININE, l-ASPARTIC ACID, l-HISTIDINE, l-LEUCINE, AND l-PHENYLALANINE. AB - The transport of l-arginine (l-Arg), l-aspartic acid (l-Asp), l-histidine (l His), and l-phenylalanine (l-Phe) has been investigated in suspension cultures of Nicotiana tabacum cv. Wisconsin 38 cells. Uptake of these amino acids is pH- and energy-dependent, concentrative (except for l-Asp), stereospecific, dependent upon culture growth phase, and, apparently, carrier-mediated. A 1,000-fold higher concentration of l-leucine (l-Leu) can inhibit all of the energy-dependent uptake of l-Arg, l-Asp, l-His, and l-Phe. A 1,000-fold higher concentration of l-Arg or l-Phe can inhibit all of the energy-dependent uptake of l-Leu. l-Asp and l-His cannot inhibit all of the energy-dependent l-Leu uptake. However, evidence is presented which indicates that l-Leu enters the cell via the carrier system which also transports l-Asp and l-His. These inhibition data are consistent with the hypothesis that all of these amino acids are transported by a single carrier system. PMID- 16662169 TI - Twenty-four-hour induction of freezing and drought tolerance in plumules of winter rye seedlings by desiccation stress at room temperature in the dark. AB - Exposure of seedlings of winter rye (Secale cereale L., cv. Puma) for 2 weeks or 24 hours to desiccation stress (40% relative humidity) at room temperature (21 degrees C) in the dark induced degrees of freezing and drought tolerance in the plumules comparable to those produced by cold conditioning for 2 weeks at 3 degrees C. The induction was associated with repression of growth and could not be produced in plumules excised from the seedlings indicating a requirement for translocation of nutrients from the endosperm. Rapid increase in osmotic pressure, soluble proteins, and phospholipids in plumules in association with the development of freezing and drought tolerance and the requirement of endosperm suggested diversion of nutrient from use in extension growth, to use in augmentation of protoplasm in plumule cells. Since cold acclimation slowed or arrested growth and is associated with augmentation of protoplasm, it is suggested that the common element in the induction of freezing tolerance by cold and drought is the necessity for producing a condition of augmented protoplasm and membranes in cells thus reinforcing a similar conclusion reached from seasonal studies on woody plants. PMID- 16662170 TI - Correlation between Cold- and Drought-Induced Frost Hardiness in Winter Wheat and Rye Varieties. AB - Exposure of six wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar to 40% relative humidity for 24 hours induced the same degree of freezing tolerance in seedling epicotyls as did cold conditioning for 4 weeks at 2 degrees C.Frost hardiness varietal relationships were the same in desiccation stressed and cold-hardened seedlings. Drought stress could, therefore, be used as a rapid and simple method for inducing frost hardiness in seedling shoots in replacement of cold conditioning. PMID- 16662171 TI - Measurement of photorespiration in algae. AB - The rates of true and apparent photosynthesis of two unicellular green algae, one diatom and four blue-green algae were measured in buffer at pH 8.0 at subsaturating concentrations of dissolved inorganic carbon (13-27 micromolar). Initial rates of depletion from the medium of inorganic carbon and (14)C activity caused by the algae in a closed system were measured by gas chromatography and by liquid scintillation counting, respectively. The rate of photorespiration was calculated as the difference between the rates of apparent and true photosynthesis. The three eucaryotic algae and two blue-green algae had photorespiratory rates of 10 to 28% that of true photosynthesis at air levels of O(2). Reduction of the O(2) level to 2% caused a 52 to 91% reduction in photorespiratory rate. Two other blue-green algae displayed low photorespiratory rates, 2.4 to 6.2% that of true photosynthesis at air levels of O(2), and reduction of the O(2) concentration had no effect on these rates. PMID- 16662172 TI - On the Participation of Phosphoribulokinase in the Light Regulation of CO(2) Fixation. AB - CO(2) fixation by a suspension of isolated spinach chloroplasts was terminated by turning off the light, and changes of metabolite levels in the chloroplast stroma and the surrounding medium were assayed. Whereas CO(2) fixation comes to a total stop within 15 seconds, a conversion of triose phosphates to heptose, hexose, and pentose monophosphates is found to occur for 1 to 2 minutes afterwards. It seems from these data that an inactivation of fructose and sedoheptulose bisphosphatases proceeds with a lag period. In contrast, the conversion of pentose monophosphates to ribulose 1,5-bisphosphate is inhibited immediately after the stop of illumination. As the stromal level of freely available ATP was not depleted under this condition, these data demonstrate that ribulose 5 phosphate kinase was very rapidly inactivated after darkening of the chloroplasts. Essentially, the same effect is also observed when CO(2) fixation is partially inhibited by addition of moderate concentrations of m-chlorocarbonyl phenylhydrazone, partially uncoupling photophosphorylation. It appears from these results, that the activity of ribulose 5-phosphate kinase is not only regulated by light through the mediation of reduced carriers like thioredoxin but also by alternative parameters, e.g. stromal metabolite levels. PMID- 16662173 TI - Cytoplasmic Male Sterility in Barley : VIII. LIPOXYGENASE ACTIVITY AND ANTHER AMINO NITROGEN IN THE msm1-Rfm1a SYSTEM. AB - The lipoxygenase (LOX) activity was determined in almost isogenic types of barley (Hordeum vulgare L.): normal cv. Adorra, cytoplasmic male sterile (msm1), and msm1 barley with restored fertility, heterozygous for the Rfm1a restorer gene. The LOX activity was lowest in male steriles in the leaf tissue studied at the anthesis stage. The LOX activity in developing anthers was higher than in leaf tissue, and decreased during degeneration of the sterile anthers.On polyacrylamide gel electrophoresis slabs, the LOX of anther homogenates moved in a complex which evidently carried some lipid and pigment, too. The LOX zones showed pseudoisoenzymic movement, i.e. a gradual increase in mobility dependent on the age of the anthers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs, the LOX zones contained three polypeptides. When sporopollenin production ends in the fertile anthers, a fourth polypeptide (molecular weight 91,200) appears in their LOX zones. This ;late' polypeptide is missing from steriles, and is suggested as being associated with the termination of sporopollenin production in the tapetum of fertiles.Sterile anthers were found to be almost devoid of soluble NH(2)-N, which supports the idea of their starvation. This starvation can reasonably be held responsible for the absence of late proteins (e.g. the 91,200 dalton polypeptide), and is reinforced by the uncontrolled production of sporopollenin. PMID- 16662174 TI - Localization of ribulose bisphosphate carboxylase in the guard cells by an indirect, immunofluorescence technique. AB - Ribulose bisphosphate carboxylase, a key enzyme in the photosynthetic carboxylation process, has been localized through an indirect immunofluorescent technique in the guard cells of some of the 41 species of plants examined. This sample includes 17 families of both dicotyledons and monocotyledons, one gymnosperm, and one pteridophyte. Plants were selected to represent all of the three major photosynthetic categories, namely C(3), C(4), and Crassulacean acid metabolism. Antibodies raised against tobacco (Nicotiana tabacum L.) ribulose bisphosphate carboxylase were used for this immunofluorescent study. A good degree of fluorescence was observed in the guard cells of seven out of 21 species exhibiting Crassulacean acid metabolism. C(3) plants exhibited a very low degree (almost negligible) of fluorescence, while the C(4) species did not exhibit any fluorescence. PMID- 16662175 TI - Inhibition of shoot geotropism by neutral buffers. AB - Submerged hypocotyl sections from Helianthus have been used to test the effect of neutral buffers on shoot geotropism. When hypocotyls have been abraded, it is found that increasing the molarity (0.25 to 20 mm) of pH 6.8 K-phosphate buffer, as well as other buffering systems, results in a strong inhibition of geotropic curvature. Buffer strength has no such effect on the curvature of nonabraded segments. One possible explanation for these data is that asymmetric shoot growth following geostimulation may require the establishment of a proton gradient across the cell walls of the shoot. When neutral buffers have access to the wall space (i.e. in abraded segments), they may prevent the establishment of such a gradient. PMID- 16662176 TI - Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH. AB - Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish. PMID- 16662177 TI - A comparison of intrinsic endoplasmic reticulum membrane proteins in maturing seeds and germinated seedlings of castor bean. AB - The intrinsic membrane proteins of the endoplasmic reticulum from endosperm of maturing and germinated seedlings of castor bean (Ricinus communis) were studied. Preparations were simultaneously subjected to two-dimensional polyacrylamide gel electrophoresis. At least 30 separate proteins were distinguished by staining the gels with Coomassie R-250. The characteristic protein profiles obtained from 0.2 m KCl-washed membranes of each endoplasmic reticulum source are highly reproducible. Of these proteins, three to six that were present in maturing seed were found also in germinating seedlings. In general, the majority of membrane proteins from the endoplasmic reticulum of maturing seed were of a higher molecular weight than those from germinated seedlings. PMID- 16662178 TI - Simultaneous consideration of tissue and substrate potassium concentration in k uptake kinetics: a model. AB - The importance of the simultaneous consideration of tissue and substrate concentration in the estimation of ion uptake is discussed. An elaboration of the model of ion uptake, originally proposed by E. Epstein, is developed. The modified model takes both tissue and substrate ion concentration into account and uses true constants in the estimation of uptake. A test case-K(+) uptake by a barley cultivar-has been presented to show the working of the model. The relevance of the modified model is also pointed out. PMID- 16662179 TI - Minimizing Nitrate Reduction during Kjeldahl Digestion of Plant Tissue Extracts and Stem Exudates : APPLICATION TO N STUDIES. AB - From 10 to 60% of the nitrate present in plant tissue extracts and stem exudates of corn (Zea mays L.) was found to be reduced during Kjeldahl digestion, even in the absence of added reducing agents. This reduction is of particular concern in [(15)N]nitrate assimilation studies, because it results in an overestimate of nitrate reduction. To overcome this problem, a method was developed for removing nitrate prior to Kjeldahl digestion, thereby preventing nitrate reduction. The procedure utilizes hydrogen peroxide for partial oxidation of organic matter in order to minimize the nitration of organic compounds. The free nitrates are then volatilized as nitric acid from concentrated sulfuric acid at 95 degrees C. When the proposed method was used as a pretreatment to Kjeldahl digestion, less than 0.5% of the applied nitrate was recovered in the reduced nitrogen fraction of plant tissue extracts and stem exudates. PMID- 16662180 TI - Characteristics of teratomas regenerated in vitro from octopine-type crown gall. AB - Crown galls induced by infection of tobacco plants with Agrobacterium tumefaciens strain C58-Cl(pTiB6S3) were excised and cultured in vitro. After about one year of culture on medium-lacking phytohormones, two noncloned lines spontaneously formed shoots. Leaf explants from shoots of tumor-line T5 were capable of growing on hormone-free medium, and the resulting mixture of organized and unorganized tissue synthesized octopine. Detached leaves from T5 shoots also synthesized octopine. These results establish that shoots from this octopine-type tumor contain transformed cells and are true crown-gall teratomas. PMID- 16662181 TI - Glutamine Synthetase of Germinating Peanuts : PROPERTIES OF TWO CHROMATOGRAPHICALLY DISTINCT FORMS AND THEIR ACTIVITY TOWARD 4-METHYLENEGLUTAMIC ACID. AB - Glutamine synthetase activity, extracted from an acetone powder of 7-day germinated peanuts (Arachis hypogaea L.), was precipitated by ammonium sulfate (40-60% saturation) and further purified by gel filtration and calcium phosphate gel treatment. When it was adsorbed to and subsequently eluted from a column of diethylaminoethyl-cellulose, two peaks of activity (designated glutamine synthetase 1 and 2) were obtained which were enriched 150- and 20-fold, respectively, over the initial extract. Glutamine synthetase 1 was present in ungerminated seeds and in the cotyledons during germination; glutamine synthetase 2 appeared during germination and was found largely in the developing plant. Compared with glutamine synthetase 2, glutamine synthetase 1 appeared to have a slightly smaller molecular weight and was more stable to heat and storage. The catalytic properties of the two forms were essentially the same. Whereas neither form catalyzed gamma-glutamyltransferase activity with 4-methyleneglutamine, both glutamine synthetases 1 and 2 catalyzed an ATP- and NH(4) (+)-dependent conversion of [(14)C]-4-methyleneglutamic acid to [(14)C]-4-methyleneglutamine, but the K(m) value for 4-methyleneglutamic acid was 10-fold greater and the V(max) only one-fourth that measured with l-glutamic acid. This is the first report of glutamine synthetase activity with 4-methyleneglutamic acid as substrate, although the level of this activity does not appear adequate to account for the rapid synthesis of 4-methyleneglutamine observed in germinating peanuts. PMID- 16662182 TI - Temperature and oxygen effects on C-photosynthate unloading and accumulation in developing soybean seeds. AB - The environmental sensitivity of the processes associated with the import of photosynthate by developing soybean seeds was investigated within intact fruit and with excised, immature embryos. Intact pods of field-grown (Glycine max [L.] Merr.) Amsoy 71 soybeans were subjected to localized regimes of 0, 21, or 100% O(2) and 15, 25, or 35 degrees C during pulsechase translocation experiments and, 2.5 hours later, the uptake and distribution of (14)C-photosynthate among dissected fruit tissues determined. In other experiments, excised embryos were incubated in [(14)C]sucrose solutions under various experimental conditions to separate the effects of these treatments on accumulation by the embryos from those which may operate on phloem unloading in the maternal seedcoat.Import of (14)C-photosynthate by intact soybean fruit was both temperature- and O(2) dependent. This dependency was shown to occur only within the seeds; import by the pod walls was essentially insensitive to fruit temperature or O(2) treatments. The embryos of anaerobic fruit were completely unlabeled, regardless of fruit temperature. But under anaerobic in vitro incubation conditions, uptake of [(14)C]sucrose in excised embryos was only 30% less than that in aerobic in vitro conditions. The data suggest that, within intact fruit, anoxia prevented sucrose efflux from the seed coat phloem and any subsequent uptake by the embryo. The demonstrated energy dependence of phloem unloading may reflect requirements for membrane integrity or energy metabolism in the companion cell-sieve element complex, consistent with a facilitated unloading process.Collectively, these data characterize the environmental sensitivity of photosynthate import in developing soybean fruit. They imply that environmental regulation of import may occur at both the embryo level and at the phloem terminals within the seed coat. PMID- 16662183 TI - Measuring chlorophyll a and C-labeled photosynthate in aquatic angiosperms by the use of a tissue solubilizer. AB - A compound that quantitatively correlated with chlorophyll a could be measured fluorometrically in the extracts of leaves of three aquatic angiosperms (Myriophyllum heterophyllum Michx., Potamogeton crispus L., Elodea canadensis Michx.) treated with the tissue solubilizer BTS-450. Fluorescent characteristics of the solubilized plant tissues were stable for several weeks in the dark at temperatures up to 60 degrees C but rapidly degraded in sunlight or when acidified. (14)C-Labeled photosynthate, which had been fixed by leaf discs during 1- to 10-hour exposure to H(14)CO(3), was also readily extracted by the tissue solubilizer. Solubilizer extraction can, therefore, be used to determine both chlorophyll a content and (14)C incorporation rates in the same leaf sample. The method is practical, because no grinding is required, the fluorescent characteristics of the extracts are stable, and analyses can be performed with very little plant material (about 3 milligrams). PMID- 16662184 TI - Hydrolysis of Ribulose-1,5-bisphosphate Carboxylase by Endoproteinases from Senescing Barley Leaves. AB - The hydrolysis of (14)C-labeled ribulose-1,5-bisphosphate carboxylase (RuBPCase) by two partially purified endoproteinases from senescing barley (Hordeum vulgare v. Numar) leaves is described. The major thiol proteinase, EP(1), exhibits biphasic kinetics which appear to be caused by a region of the large subunit of RuBPCase that is highly sensitive to attack by EP(1). This proteinase further hydrolyzes both the large and small subunit to smaller peptides. A second proteinase, EP(2), appears to convert the small subunit of RuBPCase rapidly to a 13.7-kilodalton fragment during initial stages of hydrolysis and then to degrade both this fragment and the large subunit. The presence of a third endoproteinase, EP(3), was discovered when [(14)C]RuBPCase, which appeared to be homogeneous by sodium dodecyl sulfate polyacrylamide electrophoresis, seemed to undergo very low but significant rates of "autolysis." The large molecular weight fragments produced by EP(3) were different from those of EP(1) and EP(2). PMID- 16662185 TI - Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose. AB - Changes in the concentrations of NH(4) (+) and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO(3) (-) as a nitrogen source, the concentrations of NH(4) (+) and amides increased to 4.0 x 10(-1) and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH(4) (+) was added to the NO(3) (-) base starting medium, the concentration of NH(4) (+) in the cells was increased to 7.0 x 10(-1) micromoles per gram fresh weight.A comparison between the concentration of NH(4) (+) in the cells (4.3 x 10(-4) molar) with the K(m) values for glutamate dehydrogenase (5 x 10(-3) molar) and glutamine synthetase (1.7 x 10(-5) molar) showed that the endogenous NH(4) (+) would have to be concentrated 10-fold in compartments possessing glutamate dehydrogenase in order for the substrate to reach one-half the saturation level for that enzyme.The influence of protein synthesis on the level of NH(4) (+) and amides was examined by blocking protein synthesis with cycloheximide or puromycin and measuring changes in NH(4) (+) and amide concentration over the subsequent 4 hours. The level of both NH(4) (+) and amides showed substantial increases when protein synthesis was blocked. Ammonium accumulated to concentrations surpassing those reported by other authors to be toxic to plants. The reduction of NO(3) (-) did not appear to be influenced by the blockage of protein synthesis. PMID- 16662186 TI - Nucleotide Sequence Homology Exists between the Chloroplast and Nuclear Ribosomal DNAs of Euglena gracilis. AB - The nuclear and chloroplast ribosomal DNAs from Euglena were shown to have specific regions of nucleotide sequence homology. The regions of homology were identified by hybridization of restriction endonuclease DNA fragments of cloned chloroplast and nuclear ribosomal DNAs to one another. The regions of homology between these two ribosomal DNAs were in that part of the genes that code for the 3' end of the small rRNAs (16S and 19S) and near or at the DNA sequences coding for the 5S RNAs. The nucleotide sequence homology between these regions was estimated to be approximately 94% by the melting point depression of a hybrid formed between the two ribosomal DNAs. PMID- 16662187 TI - Pyruvate orthophosphate dikinase from the immature grains of cereal grasses. AB - Pyruvate orthophosphate dikinase has been identified in the green grains of eight cereal grasses, most of which are classified as C(3) plants. The wheat (Triticum aestivum L. cv. Lerma Rojo) grain enzyme was further investigated: activity was low in very young grains, increased to a maximum at about 25 days after anthesis, then returned to a low level as the grain matured. It appeared to be located in the aleurone layer. A procedure was developed for obtaining partially purified preparations of pyruvate orthophosphate dikinase from the ears of wheat, oat (Avena sativa L.), barley (Hordeum distichum L.), and rye (Secale cereale L.). These preparations were suitable for measuring activities in both the forward and reverse reaction directions. The affinities of these enzymes for the six substrates (pyruvate, orthophosphate, and ATP in the forward reaction; phosphoenolpyruvate, pyrophosphate, and AMP in the reverse reaction) were determined and found to be similar to the reported affinities of the enzyme from the leaves of the C(4) plant Zea mays. A possible role for pyruvate orthophosphate dikinase in cereal grains is considered briefly. PMID- 16662188 TI - Oxidation of reduced pyridine nucleotide by a system using ascorbate and hydrogen peroxide from plants and algae. AB - A NAD(P)H oxidizing system (NAAP) was detected and partially purified from leaves of spinach and Sedum praealtum, seeds and leaves of pea and cells of green and red algae which oxidized NAD(P)H in the presence of ascorbate and H(2)O(2).The partially-purified spinach system had substrate K(m) values of 5 micromolar for NADH, 50 micromolar for H(2)O(2), and 300 micromolar for l-ascorbic acid at the pH optimum of 6.8. NADH was a better electron donor than NADPH. Among other electron donors, isoascorbic acid had considerable activity but hydroquinone and resorcinol had only weak activities. The enzyme was inhibited by cyanide, alpha,alpha'-dipyridyl, and mono-and di-thiol reagents. Inhibition by thiol reagents was partially restored by Fe(2+) as was enzymic activity lost following dialysis against buffer.Subcellular localization studies with spinach and S. praealtum leaves indicated that a portion of the cell's NAAP was in the chloroplast fraction. Photosynthetic conditions resulted in a decrease in this activity solubilized from spinach and S. praealtum chloroplasts. The presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or Fe(2+) in the incubation medium eliminated the light-mediated inhibition of NAAP.NAAP may function in the recycling of NAD(P)H generated in the dark within the chloroplast. Inasmuch as all preparations of NAAP contained ascorbate peroxidase activity, the data do not rule out the possibility that NAAP is the same protein as ascorbate peroxidase or, alternatively, a combination of ascorbate peroxidase and some other enzyme. PMID- 16662189 TI - Enhancement of Phloem Exudation from Fraxinus uhdei Wenz. (Evergreen Ash) using Ethylenediaminetetraacetic Acid. AB - Ethylenediaminetetraacetic acid (EDTA) enhanced the exudation of (14)C-labeled assimilates from excised leaflets and whole plant specimens of Fraxinus uhdei Wenz. A 2 millimolar EDTA concentration was found to be most effective in promoting exudation from excised leaflets, while 10 millimolar EDTA was most effective in whole plants experiments. Exudation rate reached a maximum after 24 hours in both experiments. The continuous presence of EDTA throughout the treatment period was required for maximum exudation from excised leaflets. Stachyose, raffinose, verbascose, and sucrose were the principal compounds found to occur in exudate samples. These compounds are typically transported in sieve elements of various Fraxinus species suggesting the exudate was of phloem origin. Electron microscope studies of petiolule sieve plate pores from excised leaflets showed substantially less callose appearing after treatment with EDTA than after H(2)O treatment. It is suggested that EDTA enhances phloem exudation by inhibiting or reducing callose formation in sieve plate pores. The exudation enhancement technique described for whole plant specimens is suggested as a useful means of collecting phloem sap and studying translocation in woody plants. PMID- 16662190 TI - Aggregated Forms of Malate and Citrate Synthase are Localized in Endoplasmic Reticulum of Endosperm of Germinating Castor Bean. AB - The endosperm of 3-day germinated seedlings of Ricinus communis was homogenized in the presence or absence of Mg(2+). When the Mg(2+) -containing homogenate was fractionated on linear, 20 to 40% sucrose gradients, the endoplasmic reticulum (ER) reached equilibrium at a density of 1.146 grams per cubic centimeter. Absence of Mg(2+) in the grinding medium resulted in displacement of the ER in the gradient from a density of 1.146 to 1.138 grams per cubic centimeter. At either density, the activities of both malate and citrate synthase were found to overlap the activity of NADH-cytochrome c reductase (an ER marker) in the gradient. Furthermore, this overlap of activities was observed whether the gradients were centrifuged for 3 or 19 hours. An analysis of sedimentation characteristics of the solubilized enzymes revealed that they exist, predominantly, as a 5.2S (s(20,w) x 10(-13)) form (malate synthase) and a 6.8S form (citrate synthase) in the glyoxysomes and cytosol. When the two enzymes were released from the ER, they appeared as aggregate forms of 70S and 55S, respectively. These results support the conclusion that the synthases are associated with the ER. PMID- 16662191 TI - Sulfur dioxide inhibition of translocation in bean plants. AB - Exposure of the source leaf of bean (Phaseolus vulgaris L. cv. Black Valentine) for 2 hours to 2.9 microliters per liter SO(2) inhibited the net photosynthetic rate an average of 75% and, simultaneously, the translocation rate an average of 45%. Calculations indicated that the experimentally determined translocation rates from SO(2)-stressed leaves were lower than were the rates expected on the basis of the observed reductions in photosynthesis. It is inferred that, under SO(2) stress, the phloem-loading system becomes a major limiting step in controlling the translocation rate.Following removal of SO(2), photosynthesis recovered quite rapidly (to about 60% of its preexposure rate within 2 hours), but the translocation rate failed to increase during this time interval. This delayed response of translocation to removal of SO(2) does not appear to be due to an injury effect of SO(2), inasmuch as a similar effect was obtained by exposing the source leaf to a short (2-hour) interval of dark. PMID- 16662193 TI - Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves. AB - Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole. PMID- 16662192 TI - Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue. AB - This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4 hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway. PMID- 16662194 TI - Photoperiodic control of gibberellin metabolism in spinach. AB - [(3)H]GA(20) applied to spinach plants (Spinacia oleracea L.) was metabolized to several products. Two of these were identified by combined gasliquid chromatography-radio counting as [(3)H]GA(29) and [(3)H]3-epi-GA(1). Inasmuch as both GA(20) and GA(29) are endogenous gibberellins in spinach (Metzger, Zeevaart 1980 Plant Physiol 65: 623-626), it was concluded that the conversion of GA(20) to GA(29) is a natural process. However, 3-epi-GA(1) was not detected in extracts of spinach shoots analyzed by combined gas chromatography-mass spectrometry. This indicates that the conversion of exogenous [(3)H]GA(20) to [(3)H]3-epi-GA(1) may be an artifact.Long-day pretreatment of spinach shoots caused a 2-fold increase in the rate of [(3)H]GA(20) metabolism over the rate of metabolism in plants maintained under short-day conditions. Furthermore, [(3)H]GA(29) accumulated more rapidly under long than under short days, whereas photoperiodic treatment had no effect on the accumulation of [(3)H]3-epi-GA(1). Thus, the long-day-induced increase in the level of endogenous GA(29) in spinach shoots (Metzger, Zeevaart 1980 Plant Physiol 66: 844-846) appears to be the result of an increased capability to convert GA(20) to GA(29). PMID- 16662195 TI - Osmoregulation in the Avena Coleoptile : CONTROL OF SOLUTE UPTAKE IN PEELED SECTIONS. AB - Peeled Avena sativa coleoptile sections (i.e. sections from which the epidermis has been removed) have been used to study the control of solute uptake under conditions where the uptake is not limited by the cuticular barrier. In the presence of 2% sucrose, auxin enhances the rate at which the total osmotic solutes increase, but this appears to be a response to the increased growth rate, inasmuch as the auxin effect is eliminated when growth is inhibited osmotically. When sections are incubated in sucrose or in 20 millimolar NaCl, the osmotic concentration increases until a plateau is reached after 8 to 24 hours. Auxin has no effect on the initial rate of increase in osmotic concentration but causes the osmotic concentration to reach a plateau earlier and at a lower osmotic conentration value. This difference in steady-state osmotic concentration is, in part, a response to auxin itself, as it persists when auxin-induced growth is inhibited osmotically. The upper limit for osmotic concentration does not appear to be determined by the turgor pressure, inasmuch as a combination of sucrose and NaCl gave a higher plateau osmotic concentration than did either solute alone. We suggest that the rate of solute uptake is determined by the availability of absorbable solutes and by the surface area exposed to the solutes. Each absorbable solute reaches a maximum internal concentration independent of other absorbable solutes; the steady-state osmotic concentration is simply the sum of these individual internal concentrations. PMID- 16662196 TI - Senescence and the Fluidity of Rose Petal Membranes : RELATIONSHIP TO PHOSPHOLIPID METABOLISM. AB - In previous work, senescence of rose petal cells has been shown to be accompanied by a gradual decrease of membrane fluidity, as measured by a fluorescence polarization technique. Concomitantly, an increase in the free sterol-to phospholipid ratio was found. Both observations were verified in this study. Further, experiments carried out on whole tissue and isolated protoplasts during senescence revealed that there was no quantitative change in the level of free sterols. The content of phospholipids decreased without any significant change in their composition. Results from experiments measuring the incorporation of [(32)P]orthophosphate indicated a reduced capacity for phospholipid synthesis in senescent cells. Both young and old tissue showed phospholipase A and D activity, the former increasing with age.It was concluded that the fluidity of rose petal membranes decreases with age as a result of a decrease in phospholipid content, brought about by both reduced synthesis and enhanced degradation. Evidence supporting the view that the phenomena observed are related specifically to changes in the plasmalemma is discussed. PMID- 16662197 TI - Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C(3) Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism. AB - Mesembryanthemum crystallinum, a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCl concentrations of 20 and 400 millimolar in the rooting medium. Plants from the low salinity treatment showed exclusively C(3)-photosynthetic net CO(2) fixation, whereas plants exposed to the high salinity level exhibited net CO(2) dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracellular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cells. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein.Experiments established the extraorganellar (cytoplasmic) location of phosphoenolpyruvate carboxylase, enolase, phosphoglyceromutase, and NADP-malic enzyme; the mitochondrial location of NAD-malic enzyme; and the chloroplastic location of pyruvate, Pi dikinase. NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphohexose isomerase, and phosphoglycerate kinase were associated with both cytoplasm and chloroplasts. NADP-dependent malate dehydrogenase activity was found in both the chloroplastic and extrachloroplastic fractions; the activity in the chloroplast showed an optimum at pH 8.0 and was dependent upon preincubation of enzyme with dithiothreitol. The extrachloroplastic activity showed an optimum at pH 6.5 and was independent of pretreatment with dithiothreitol. Protoplast extracts of M. crystallinum performing CAM exhibited higher activities (expressed per mg chlorophyll per min) of phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malic enzyme, NAD-malic enzyme, NADP-malate dehydrogenase, enolase, phosphoglyceromutase, NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphohexose isomerase than protoplast extracts from M. crystallinum not exhibiting CAM. The increase in total activity of the latter three enzymes following exposure of plants to 400 millimolar NaCl and the development of CAM was due to specific increases in the levels of activity in the cytoplasm. PMID- 16662198 TI - [N]NMR determination of asparagine and glutamine nitrogen utilization for synthesis of storage protein in developing cotyledons of soybean in culture. AB - Solid-state [(15)N]NMR was used to measure the use of the amide and amino nitrogens of glutamine and asparagine for synthesis of storage protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No major discrimination in the incorporation of the amide or amino nitrogens of glutamine into protein is apparent, but the same nitrogens of asparagine are used with a degree of specificity. During the first seven days in culture with asparagine as the sole nitrogen source, the amino nitrogen donates approximately twice as much nitrogen to protein as does the amide nitrogen. The use of the amide nitrogen increases with longer periods of culture. The reduced use of the amide nitrogen was confirmed by its early appearance as ammonium in the culture medium. The amide nitrogen of asparagine was found at all times to be an essential precursor for protein because of its appearance in protein in residues whose nitrogens were not supplied by the amino nitrogen. In addition, methionine sulfoximine inhibited growth completely on asparagine, indicating that some ammonium assimilation is essential for storage protein synthesis. These results indicate that in a developing cotyledon, a transaminase reaction is of major importance in the utilization of asparagine for synthesis of storage protein and that, at least in the early stages of cotyledon development, reduced activities of ammonium assimilating enzymes in the cotyledon tissue or in other tissues of the seed or pod may be a limiting factor in the use of asparagine-amide nitrogen. PMID- 16662199 TI - N- and [C]NMR determination of utilization of glycine for synthesis of storage protein in the presence of glutamine in developing cotyledons of soybean. AB - Solid-state (15)N- and [(13)C] NMR have been used to measure quantitatively the utilization of glycine in the presence of glutamine for the synthesis of storage protein in immature cotyledons of soybean (Glycine max L. cv. Elf) in culture. The presence of an equal molar amount of glycine in the medium causes a decrease in the use of glutamine-amide nitrogen. Glycine nitrogen is incorporated extensively into peptide bonds (in amounts greater than what would be expected if it appeared solely in glycine residues), but is used sparingly for synthesis of histidine ring residues, guanidino nitrogen residues of arginine, and lysine residues. The modest use of glycine carbon in protein synthesis does not parallel the use of glycine nitrogen. PMID- 16662200 TI - Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe. AB - Although intact fruits of unripe cantaloupe (Cucumis melo L.) produce very little ethylene, a massive increase in ethylene production occurred in response to excision. The evidence indicates that this wound ethylene is produced from methionine via 1-aminocyclopropanecarboxylic acid (ACC) as in ripening fruits. Excision induced an increase in both ACC synthase and the enzyme converting ACC to ethylene. Ethylene further increased the activity of the enzyme system converting ACC to ethylene. The induction by ethylene required a minimum exposure of 1 hour; longer exposure had increasingly larger effect. The response was saturated at approximately 3 microliters per liter ethylene and was inhibited by Ag(+). Neither ethylene nor ACC had a promotive or inhibitory effect on ACC synthase beyond the effect attributable to wounding. PMID- 16662201 TI - Effect of IAA on Growth and Soluble Cell Wall Polysaccharides Centrifuged from Pine Hypocotyl Sections. AB - Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment. PMID- 16662202 TI - Effect of Atmospheric CO(2) Enrichment on Growth, Nonstructural Carbohydrate Content, and Root Nodule Activity in Soybean. AB - The objective of this study was to determine whether the supply of current photosynthate was limiting root nodule activity. Both short-term (36 hours) and long-term (16 days) periods of CO(2) enrichment were imposed on vegetative, growth chamber-grown soybean plants (Glycine max. [L.] Merr. cv. ;Clay') to increase the supply of current photosynthate and to observe the effects on photosynthate partitioning in the plants, plant growth, and root nodule activity.Neither total nor specific nodule activities were increased during exposure to short-term (36 hours) CO(2) enrichment. Dry weight of the leaves increased after 12, 24, and 36 hours of CO(2) enrichment and dry weight of the stems plus petioles increased after 36 hours of CO(2) enrichment. Dry weights of the roots and nodules were not altered by short-term CO(2) enrichment. Short-term CO(2) enrichment increased the total nonstructural carbohydrates in the leaves and stems plus petioles, but not in the roots and nodules. Analyses of the separate pools of carbohydrate reserves indicated that the majority of the additional carbohydrate provided by short-term CO(2) enrichment was stored as leaf starch with relatively little being partitioned to the roots and nodules.Long-term CO(2) enrichment (16 days) did not enhance specific nodule activity. Shoot, root, and nodule dry weights were increased 109, 34%, and 56% respectively. Total nodule activity per plant was significantly enhanced only after 16 days of treatment and was related to increased nodule mass. These results indicate that the increased total nodule activity in response to CO(2) enrichment is a consequence of a general growth response of the plant.Results of both studies indicate that nodule activity was not directly limited by current photosynthesis but rather by the partitioning and utilization of photosynthate in the plant. PMID- 16662203 TI - Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl. AB - In vitro translation products of polyadenylated RNA from untreated and auxin treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment. PMID- 16662204 TI - Auxin- and ethylene-induced changes in the population of translatable messenger RNA in Basal sections and intact soybean hypocotyl. AB - In vitro translation products of polyadenylated RNA from untreated and auxin treated basal sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. Within one hour of 2,4-dichlorophenoxyacetic acid treatment, the translatable messenger RNAs for at least twelve in vitro translation products are modulated upward. In vitro translation products of polyadenylated RNA from untreated, auxin-treated and Ethephon-treated intact soybean hypocotyl were also analyzed. Within two hours of treatment with either 2,4-dichlorophenoxyacetic acid or Ethephon, the translatable messenger RNAs for a group of high molecular weight in vitro translation products are modulated upward. There is a particular set of translatable messenger RNA, encoding in vitro translation products in the 24,000 to 32,000 molecular weight range, that is specifically modulated upward by auxin treatment in intact soybean hypocotyl and in hypocotyl sections. PMID- 16662205 TI - Photosynthetic response of seagrasses to ultraviolet-a radiation and the influence of visible light intensity. AB - Inhibition of photosynthesis by ultraviolet-A radiation (UV-A, 315-380 nanometers) was examined in three marine angiosperms: Halophila engelmannii Aschers, Halodule wrightii Aschers, and Syringodium filiforme Kutz. Sensitivity to UV-A and photosensitization to UV-A by photosynthetically active radiation (PAR, 380-700 nanometers) were characterized.Net photosynthesis by Halodule and Syringodium was unaffected by UV-A irradiation in the absence of PAR. Irradiation of Syringodium by a combined beam of UV-A and PAR resulted in photosynthetic inhibition. The depression of net photosynthesis was found to be a function of PAR intensity at a fixed level of UV-A irradiation. Inhibition of photosynthesis in Halodule by the combined beam was minimal and suggests adaptation to environmental irradiation levels.Halophila was the only species examined, subject to photosynthetic inhibition by UV-A in the absence of PAR. Irradiation with PAR intensities characteristic to Halophila in the natural system as the combined beam, appeared to negate the inhibition. Increasing the PAR component of the combined beam above environmental norms resulted in photosynthetic inhibition greater than that observed for UV-A alone. PMID- 16662206 TI - Sources of ammonium in oat leaves treated with tabtoxin or methionine sulfoximine. AB - Excised 7-day-old oat (Avena sativa L. cv. Jaycee) leaves were incubated in media containing 7.1 millimolar KNO(3) and 0.15 millimolar tabtoxin or 1 millimolar methionine sulfoximine (MSO) to investigate the sources of the observed ammonium accumulated. Tabtoxin and MSO are known inhibitors of glutamine synthetase, the first enzyme in the primary pathway of ammonium assimilation. During a 4- to 6 hour incubation, there was little net change in protein or total amino acid concentration. Alanine, aspartate/asparagine, and glutamate/glutamine decreased markedly under these treatments, whereas several other amino acids increased. Exogenous (15)N from K(15)NO(3) was taken up and incorporated into the nitrate and ammonium fractions of leaves treated with tabtoxin or MSO. This result and the high in vitro activities of nitrate reductase indicated that reduction of nitrate was one source of the accumulated ammonium. Leaves incubated under 2% O(2) to reduce photorespiration accumulated only about 13% as much ammonium as did those under normal atmospheres. We conclude that most of the tabtoxin- or MSO induced ammonium came from photo-respiration, and the remainder was from nitrate reduction. PMID- 16662207 TI - Fruit age and changes in abscisic Acid content, ethylene production, and abscission rate of cotton fruits. AB - The relationships of fruit age, abscisic acid (ABA) concentration, ethylene evolution, and abscission rates were studied in an effort to determine why cotton (Gossypium hirsutum L., cv. Deltapine 16) fruits rarely abscise more than 15 days after anthesis. Because abscission of cotton fruits is increased by conditions that limit photosynthesis, greenhouse-grown plants with fruits of various ages were placed in dim light for 3 days to induce high rates of fruit abscission. Abscission rates, ABA concentrations, and ethylene evolution rates were determined for fruits of various ages. Almost all of the young fruits abscised, but abscission rate declined with age until almost no abscission was observed in fruits that were 15 or more days past anthesis.Dim light increased the ABA concentrations of fruits that were 6 to 11 days old but did not increase ABA concentrations in fruits that were younger or older. The concentration of ABA declined with fruit age from peak values at 4 and 6 days after anthesis. Dim light also increased ethylene evolution from fruits up to 10 days old but had little effect on ethylene production or abscission of fruits more than 11 days old. Ethylene evolution declined with fruit age from peak values at 4 and 6 days after anthesis. Fruits of various ages (from plants not exposed to dim light) were sliced to induce high rates of wound ethylene production. The results indicated that the capacity for ethylene production declined with fruit age, parallel with a decline in abscission rate. Decreases in ABA concentration and ethylene evolution with fruit age indicate that change in the capacity to synthesize these hormones, especially in response to stress, is one cause of the decline in abscission rates as cotton fruits become older. PMID- 16662208 TI - Restricted nitrate influx and reduction in corn seedlings exposed to ammonium. AB - The effect of ambient ammonium (0.5 millimolar [(14)NH(4)](2)SO(4)) added to a nutrient solution containing 1.0 millimolar K(15)NO(3), 99 atom per cent (15)N, upon [(15)N]nitrate assimilation and utilization of previously accumulated [(14)N]nitrate was investigated. Corn seedlings, 5-day-old dark-grown decapitated (experiment I) and 10-day-old light-grown intact (experiment II), which had previously been grown on K(14)NO(3) nutrient solution, were used. In both experiments, the presence of ambient ammonium decreased [(15)N]nitrate influx (20% after 6 hours) without significantly affecting the efflux of previously accumulated [(14)N]nitrate. In experiment I, relative reduction of [(15)N]nitrate (reduction as a percentage of influx) was inhibited more than was [(15)N]nitrate influx. Nevertheless, in experiment I, where all reduction could be assigned to the root system, the absolute inhibition of reduction during the 12 hours (13 micromoles/root) was less than the absolute inhibition in influx (24 micromoles/root). The data suggest that the influence of ammonium on [(15)N]nitrate influx could not be totally accounted for by the decrease in the potential driving force which resulted from restricted reduction; an additional impact on the influx process is indicated. Reduction of [(15)N]nitrate in experiment II after 6 hours accounted for 30 and 18% of the tissue excess (15)N in the control and ammonium treatments, respectively. Relative distribution of (15)N between roots and exudate (experiment I), or between roots and shoots (experiment II) was not affected by ammonium. On the other hand, the accumulation of [(15)N]nitrate in roots, shoots, and xylem exudate was enhanced by ammonium treatment compared to the control, whereas the accumulation of reduced (15)N was inhibited. PMID- 16662210 TI - Callose induction in cowpea by uridine diphosphate glucose and calcium phosphate boric Acid treatments. AB - Calcium phosphate-boric acid treatments and UDP-glucose both elicited aniline blue fluorescent, periodic acid-Schiff's reagent-resistant, deposits in association with the cell walls of cowpea (Vigna sinensis [Torner] Savi cv. Early Ramshorn) tissue. Those deposits induced by calcium phosphateboric acid treatment ultrastructurally resembled the "wound callose" commonly triggered by cell damage; they were formed in seemingly intact cells of stems and leaves and their formation was associated with an increase in the surface density of rough endoplasmic reticulum in the cell cytoplasm. In contrast, UDP-glucose induced a more rapid accumulation of aniline blue fluorescent material, but only at the cut edges of stem slices. Comparative light and electron microscopy indicated that the material was incorporated into the walls of the damaged cells, even when such cells were devoid of organized cytoplasm. These results indicate a difference in the mode and site of synthesis between wound callose and that elicited by exogenous UDP-glucose. They support the hypothesis that externally supplied UDP glucose cannot be utilized by intact cells. PMID- 16662209 TI - Immunopurification and initial characterization of dicotyledonous phytochrome. AB - Antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated zucchini squash (Cucurbita pepo L., cv. Black Beauty). The antiserum was prepared by injecting into a rabbit immunoprecipitates between zucchini phytochrome and specific antiserum against undegraded oat (Avena sativa L., cv. Garry) phytochrome. Specific antiphytochrome immunoglobulins were purified from this crude serum by an affinity column consisting of conventionally purified undegraded pea phytochrome covalently linked to cyanogen bromide activated agarose. These purified immunoglobulins were also linked to cyanogen bromide-activated agarose and were used to immunopurify zucchini, pea (Pisum sativum L., cv. Alaska), and lettuce (Lactuca sativa L., cv. Grand Rapids) phytochrome. All three dicotyledonous phytochromes exhibited a monomer size near 120,000 daltons by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. Absorbance spectra of immunopurified zucchini phytochrome indicated that the ratio of visible to ultraviolet absorbance for purified zucchini phytochrome is lower than that observed for oat phytochrome. The isoelectric point of zucchini phytochrome, which was observed to be heterogeneous by this criterion, was found to be in the range of 6.5 to 7.0, higher than that observed for oat phytochrome. The electrophoretic mobility of zucchini phytochrome was found to be similar to that observed for oat and pea phytochrome under conditions that were nondenaturing and did not involve any molecular sieving effect. The amino acid analysis of zucchini phytochrome is similar to that reported previously for oat and rye (Secale cereale L., cv. Balbo) phytochrome. PMID- 16662211 TI - Effect of photoinhibitory treatments on the activity of light-activated enzymes of c(3) and c(4) photosynthetic carbon metabolism. AB - Exposure of maize leaves to a 3-hour photoinhibitory treatment (photon flux rate of 2,000 microeinsteins meter(-2) second(-1), CO(2)-free air) resulted in lower activities of the light-activated enzymes NADP malate dehydrogenase, pyruvate, Pi dikinase, and ribulose-5-phosphate kinase. The activities could be recovered partially either by incubating enzyme extracts with dithiothreitol or by illuminating the treated leaf in air. Several enzymes which are not light activated were not affected by the treatment. Ribulose-5-phosphate kinase activity was also reduced when bean plants grown in low light were subjected to a similar photoinhibitory treatment.It is suggested that, although the reactivation of these enzymes may be correlated with the short term increase of CO(2) uptake capacity observed when photoinhibited leaves are returned to illumination in air, inactivation of these enzymes does not contribute significantly to the long term in vivo expression of photoinhibition observed after 2 to 4 hours.The results provide an example of partial inactivation of light-activated enzymes under illumination equivalent to full sunlight. PMID- 16662212 TI - Effects of kaempferol on the oxidative properties of intact plant mitochondria. AB - The effects of kaempferol on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls were investigated. Kaempferol inhibited the state 3 oxidation rate of malate, NADH, and succinate, but was without effect on the ascorbate tetramethyl p-phenylenediamine oxidation rate. The inhibition was almost the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 180 micromolar kaempferol was added during state 4, the tight coupling of succinate or NADH oxidation was not released. The results obtained indicate that kaempferol inhibits the mitochondrial electron flow at, or just after, the flavoprotein site. PMID- 16662213 TI - A comparative evaluation of the level of concanavalin a binding by enriched plasma membrane fractions from developing soybean roots. AB - The Concanavalin A (Con A) binding capacity of plasma membranes isolated from meristematic and mature regions of four-day-old soybean (Glycine max L. Merr. cv. Wells) roots was compared. Con A binding was studied using a radiochemical assay with tritiated ((3)H)-Con A and by an electron microscope technique using Con A ferritin (Con A-F). In both cases, plasma membranes isolated from meristematic tissue bound significantly more Con A than did corresponding membranes from mature tissue. The relative difference in reactivity, as determined by the two procedures, was approximately 49% ((3)H-Con A) and 46% (Con A-F). In contrast, K(m) values, determined from (3)H-Con A binding curves, were approximately the same, indicating that receptor sites on plasma membranes from both sources were qualitatively similar.Since Con A specifically interacts with saccharide containing portions of membrane glycoproteins and/or glycolipids, the data suggests that there is a decrease in the concentration of those components in plasma membranes during development or that there are qualitative changes in the structure of their oligosaccharide side chains. PMID- 16662214 TI - Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs. AB - The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine. PMID- 16662215 TI - Hormonal control of tobacco crown gall tumor morphology. AB - The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus. PMID- 16662216 TI - Biosynthesis, deposition, and partial characterization of potato suberin phenolics. AB - Alkaline nitrobenzene oxidation of the polymeric materials from wound-healed potato (Solanum tuberosum L. var. White Rose) tuber tissue liberated p hydroxybenzaldehyde, vanillin, and minor amounts of syringaldehyde as determined by gas chromatography/mass spectrometry. The aromatic aldehydes were derived only from periderm. The amounts of aromatic aldehydes liberated were used as a measure of the deposition of phenolic suberin components. Phenolic deposition began after about 2 days of wound healing; after 8 days the amounts of p-hydroxybenzaldehyde released by nitrobenzene oxidation leveled off at 5 milligrams per gram dry weight and after 12 days vanillin liberation reached a maximum at 7.5 milligrams per gram dry weight. The time course of deposition of the phenolic polymeric material is analogous to that reported for the deposition of the aliphatic components of suberin and therefore these results are consistent with the proposed structure of suberin. Experiments with radiolabeled l-phenylalanine and cinnamic acid indicated that exogenous phenylalanine was less efficient than cinnamic acid as a precursor of suberin phenolics. Nitrobenzene oxidation of radiolabeled suberin preparations gave three major labeled fractions: a diethyl ether-soluble fraction containing aromatic aldehydes ( approximately 20%), an ethyl acetate-soluble fraction containing unknown compounds ( approximately 15%), and a condensed phenolic fraction ( approximately 10%). Thin-layer and gas-liquid chromatographic analysis of the ether fraction showed that the major labeled components were vanillin and p-hydroxybenzaldehyde. The condensed tannin fraction revealed the presence of several labeled macromolecular phenolic fractions. Elution profiles of the condensed tannin fraction from tissues suberized for different periods of time were essentially identical, suggesting qualitative similarity of deposition and polymerization of suberin phenolics throughout the duration of wound healing. Chlorogenic acid accumulation in wound healing potato tuber discs was measured by high-performance liquid chromatography. The level of this compound reached 130 micrograms per disk after 11 days and did not decline even after the deposition of suberin ceased, revealing no precursor role for this acid in suberization. PMID- 16662217 TI - Accumulation of beta-Carboline Alkaloids and Serotonin by Cell Cultures of Peganum harmala L: I. CORRELATION BETWEEN PLANTS AND CELL CULTURES AND INFLUENCE OF MEDIUM CONSTITUENTS. AB - A number of cell cultures of Peganum harmala were initiated to check for a correlation between the harman alkaloid content of seedlings and cell lines derived therefrom. Despite a poor correlation between callus or suspension culture lines and parent plants, the mean alkaloid contents of strains derived from seedlings with higher alkaloid yields were nevertheless higher than the mean contents of strains derived from low yield plants. Generally, alkaloid accumulation decreased with the numbers of transfers. By permanent visual selection for fluorescent areas of the calluses, however, a mean content of 0.1% harman alkaloids and 0.1% serotonin could be maintained, which was 10 times higher than in unselected callus cultures.The effects of medium constituents on harman alkaloid and serotonin accumulation were measured for a low yielding, faster growing suspension culture strain and a slowly growing, but high yielding cell line. This led to the development of a production medium without 2,4 dichlorophenoxyacetic acid and phosphate, and with Ca, Mg, and nitrate as sole macronutrients. When this production medium was used, the accumulation of harman alkaloids and serotonin was increased from 0.1 to 1% in the low yielding cell line and from 1 to 2% in the high yielding strain. PMID- 16662218 TI - Relation of polyamine synthesis and titer to aging and senescence in oat leaves. AB - Polyamine biosynthesis in senescing leaves of Avena sativa L. was measured by determining the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). Polyamine content was also estimated by thin layer chromatography and high performance liquid chromatography. Arginine decarboxylase activity decreases progressively in aging attached first leaves and in senescing excised leaves in the dark. Conversely, it increases during light exposure of excised leaves, which retards senescence. Ornithine decarboxylase activity is high and constant in the attached leaf, irrespective of age; it decreases in excised leaves kept in the dark and in the light, irrespective of senescence. S-Adenosyl-l-methionine decarboxylase shows no correlation with age or senescence. Levels of putrescine, diaminopropane, agmatine, and spermidine are high in young leaves and decline with age. The best single indicator of senescence is usually spermidine, which decreases in excised leaves incubated in the dark, but increases in such leaves with time of light exposure. Spermidine generally has a reciprocal relationship with putrescine, indicating that spermidine synthase, which converts putrescine to spermidine, may exert important physiological control. These data support the view that polyamines play an important role in the regulation of plant development. PMID- 16662219 TI - Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers. AB - The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally. PMID- 16662220 TI - Water Stress Effects on Nitrogen Assimilation and Growth of Trifolium subterraneum L. Using Dinitrogen or Ammonium Nitrate. AB - The relative effects of water stress on growth parameters of subterranean clover (Trifolium subterraneum L. cv. Woogenellup) dependent on either N(2) or 8 millimolar NH(4)NO(3) for N were examined. Whole-plant carbon exchange rate (CER), acetylene reduction (AR), dry matter production, and Kjeldahl N accumulation were measured on uniform, intact swards of clover that were maintained under adequately watered conditions or were subjected to three cycles of water stress (leaf water potential 650 nm) light, Anacystis nidulans accumulates protochlorophyllide to concentrations about one-tenth of the chlorophyll. From whole cell fluorescence spectra, protochlorophyll(ide) was identified also in another blue-green, and in a red, alga grown in far-red light. PMID- 16662247 TI - Niacin Biosynthesis in Seedlings of Zea mays. AB - Evidence obtained from incubation of corn (Zea mays cv. Golden Bantam) seedlings in dl-[benzene ring-U-(14)C]tryptophan, l-[5-(3)H]tryptophan, l-[U (14)C]aspartate and [U-(14)C]glycerol indicates that niacin is synthesized in these plants via oxidative degradation of tryptophan. Aspartate and glycerol do not appear to be precursors of niacin in corn seedlings. PMID- 16662248 TI - Effect of ethylene on the gibberellic Acid-enhanced synthesis and release of amylase by isolated barley aleurone layers. AB - Methods were developed and extended to enable the incubation of isolated barley (Hordeum vulgare cv. Himalaya) aleurone layers under carefully controlled conditions for studies on effects of ethylene on amylase synthesis and release. When layers in medium containing gibberellic acid were exposed to ethylene, the synthesis and release of amylase were altered relative to layers maintained in an ethylene-free environment. These ethylene effects were detected at the smallest concentration used, 0.041 nl/ml, indicating a very low threshold value. During the initial 24 h, ethylene accelerated both the appearance of total amylase activity, and the release of this activity from the aleurone layers. On the other hand, ethylene reduced the total amount of amylase activity that was recovered from samples after 48 and 72 h.Ethylene did not stimulate the release of amylase from membrane-bound structures within the aleurone layers, and did not interact with the enzyme directly.The isoelectric patterns of amylase activity and proteins released from control and ethylene-treated aleurone layers in 24 h were identical. Therefore, ethylene promoted only quantitative differences in amylase synthesis rather than qualitative differences. PMID- 16662249 TI - Modes of ethylene action in the release of amylase from barley aleurone layers. AB - The development of xylanase activity by isolated barley (Hordeum vulgare cv. Himalaya) aleurone layers exposed to gibberellic acid was enhanced by ethylene, whereas the rate of glucanase synthesis was unaffected by ethylene. The elevated xylanase activity expressed in ethylene-treated tissue may be responsible for enhanced release of amylase in response to ethylene.The redistribution of ionic species in response to gibberellic acid and ethylene was explored. The release of calcium was much more sensitive to ethylene than the release of any of the other ions investigated (potassium, magnesium, and phosphate). Ethylene induced a 101% increase in the release of calcium from gibberellic acid-treated aleurone layers. The possible relevance of this observation to the ethylene-enhanced release of amylase is discussed. PMID- 16662250 TI - Effects of light quantity and quality on the decarboxylation of malic Acid in crassulacean Acid metabolism photosynthesis. AB - The rate of malic acid consumption in the Crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana Hamet et Perrier was found to be more rapid than the rate of photosynthetic oxygen evolution under all levels of irradiation by white light. This accounts for the accumulation of carbon dioxide in CAM tissues in the light.Action spectra of malate consumption and photosynthetic oxygen evolution in Kalanchoe were similar. Experiments using monochromatic photosynthetically active light in addition to a range of narrow waveband irradiations demonstrated that malic acid consumption in the experiments from which the action spectrum of acid consumption was constructed was not limited by the rate of photosynthesis. These data indicate that light involved in the promotion of malate consumption in CAM is absorbed by the same pigments that absorb the light which powers photosynthesis. PMID- 16662251 TI - Isolation of a wheat cell line with altered membrane properties. AB - A spontaneous dimethylsulfoxide (DMSO)-tolerant cell line was isolated from a cell culture of wheat (Triticum monococcum L.). The tolerant cells were able to grow in the presence of 4% DMSO. Cells formed from protoplasts of the tolerant line required DMSO for division in culture medium of high osmotic value.Fatty acid composition and the molar ratio of phospholipids/sterols suggest a more ordered membrane structure in the tolerant line. Accordingly, a lower K(+) influx rate was detected in the tolerant cells in comparison with the original line. These characteristics were maintained after 6 months' cultivation of the cells in DMSO-free growth medium. This suggested that genetic changes could be responsible for differences between the two cell lines. PMID- 16662252 TI - Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves. AB - Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, alpha mannosidase, and beta-N-acetylglucosamidase were found in the vacuole to varying degrees, but no beta-glucosidase was localized in the vacuole. PMID- 16662253 TI - Oxidation of c-Type Cytochromes by the Membrane-Bound Cytochrome Oxidase (Cytochrome aa(3)) of Blue-Green Algae. AB - Respiratory particles containing an aa(3)-type cytochrome oxidase were prepared from Anacystis nidulans, Synechocystis 6714, Synechococcus lividus, Anabaena variabilis, Nostoc sp. strain MAC, Nostoc muscorum, and Mastigocladus laminosus. Oxidation of c-type cytochromes by membrane preparations of the different blue green algae was observed using purified cytochromes from horse heart, Candida krusei, tuna, Saccharomyces oviformis, Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas palustris, Rhodocyclus purpureus, Paracoccus denitrificans, Anacystis nidulans, Anabaena variabilis, Euglena gracilis, and Scenedesmus obliquus. Rapid oxidations were consistently observed with the mitochondrial c-type cytochromes (horse heart cytochrome c reacts most rapidly) and with cytochromes c(2) from Rhodopseudomonas palustris and Rhodocyclus purpureus; in contrast, the cytochrome c(2) from Rhodospirillum rubrum and the plastidic cytochromes from E. gracilis and Scendesmus obliquus were inactive with all membrane preparations. All reactions were inhibited by low concentrations of KCN, NaN(3), and CO, and they were activated by Tween 80, thus indicating participation of the terminal oxidase. The results are discussed in view of the spectral similarities between the terminal oxidase of blue-green algae and the mitochondrial aa(3)-type cytochrome oxidase of plants and other eukaryotes. PMID- 16662254 TI - Physical Aspects of Fruit Growth: STRESS DISTRIBUTION AROUND LENTICELS. AB - The skin around a lenticel on a soft fruit has been modelled as a thin elastic plate with a rigid circular inclusion and applied tensile loads at the edges. A solution for the stress distribution in the skin has then been found using the linear theory of elasticity. From that solution the severity of the stress concentration and the location and form of initial cuticular failure have been deduced, the latter two being in broad agreement with observed crack initiation in the cuticle of grapes. PMID- 16662255 TI - Stimulation of ammonia and 2-oxoglutarate-dependent o(2) evolution in isolated chloroplasts by dicarboxylates and the role of the chloroplast in photorespiratory nitrogen recycling. AB - Intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves showed a light-dependent O(2) evolution (5.5 +/- 0.75 micromoles per milligram chlorophyll per hour) when supplied with ammonia and 2-oxoglutarate. This (ammonia, 2 oxoglutarate)-dependent O(2) evolution was stimulated 2- to 4-fold by the dicarboxylates, malate, succinate, fumarate, glutarate, and l-tartarate. Evolution of O(2) in the presence of malate was dependent on the presence of both 2-oxoglutarate and NH(4)Cl; malate with only either 2-oxoglutarate and NH(4)Cl alone did not support O(2) evolution. Furthermore, in the presence of malate, the amount of O(2) evolved was solely dependent on the amount of NH(4)Cl or 2 oxoglutarate added and malate did not affect the ratio of O(2) evolved to NH(4)Cl or 2-oxoglutarate consumed. Studies with inhibitors (2-(3,4-dichlorophenyl)-1,1 dimethyl urea, methionine sulfoximine, and azaserine) indicated that the above activity was directly linked to glutamine synthetase and glutamate synthase activity in the chloroplast and was not caused by the metabolism of malate. The V(max)/2 of (ammonia, 2-oxoglutarate)-dependent O(2) evolution was reached at 32 micromolar NH(4)Cl and 6 millimolar (approximately) 2-oxoglutarate in the absence of malate, and at 22 micromolar NH(4)Cl and 73 micromolar 2-oxoglutarate when malate (3 millimolar) was present.Intact chloroplasts isolated from pea (Pisum sativum) leaves also showed a stimulation of (ammonia, 2-oxoglutarate)-dependent O(2) evolution by malate. However glutamine was required for this activity even though glutamine with only either NH(4)Cl or 2-oxoglutarate did not respond to malate stimulation.The measured rates of (ammonia, 2-oxoglutarate)-dependent O(2) evolution in isolated spinach chloroplasts in the presence of malate were about 19.5 +/- 4.5 micromoles O(2) evolved per milligram chlorophyll per hour. This is adequate to sustain photorespiratory NH(3) recycling and the refixation of NH(3) arising from NO(3) under ambient conditions in the light. The role of the chloroplast in photorespiratory NH(3) recycling and the nature of the associated transport of 2-oxoglutarate into the chloroplast is discussed. PMID- 16662256 TI - Phase Shift in the Circadian Rhythm of Floral Promotion by Far Red Energy in Hordeum vulgare L. AB - Eight-day-old barley seedlings (Hordeum vulgare L. cv. Wintex) were pretreated with a single 24-hour daylight fluorescent photoperiod that was supplemented with sufficient far-red energy (FR) to produce a relative red (R)/FR ratio of 0.5. These plants undergo floral initiation about a week after they are returned to 12 hour daylight fluorescent photoperiods (R/FR ratio, 5.5), but floral development does not begin for an additional 2 weeks. Addition of FR light to a subsequent 12 hour photoperiod decreases the lag period between initiation and development by 10 days without affecting the rate of development. Extending the photoperiod to 24 hours has the same effect on the lag period, but this treatment also increases the rate of development. FR present during the second half of this 24-hour photoperiod only further increases the rate of development. Thus, the presence of FR during the first half of the photoperiod appears to affect the time of onset of floral development, while its presence during the second half of the photoperiod affects the rate of this development.When a 6-hour pulse of FR was given at various times during a 72- to 96-hour continuous daylight fluorescent period, the response varied rhythmically and was maximal during the second half of each 24-hour cycle. When one 6-hour FR pulse, given at a point of maximal response, is followed by a second 6-hour FR pulse at various times relative to the first, the phase of this rhythm is advanced by about 12 hours. This suggests that FR has two separate but concomitant effects. It causes (a) earlier and/or more rapid flowering and (b) it alters the phase of the endogenous circadian rhythm that regulates the ability of the plant to respond. PMID- 16662257 TI - Catabolism of porphobilinogen by etiolated barley leaves. AB - When [2,4-(14)C]porphobilinogen (PBG) or [2 (aminomethyl),5-(14)C]PBG is administered to etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness, label becomes incorporated into CO(2), organic and amino acids, sugars, lipids, and proteins during a 4-hour incubation. Less than 1% of the label, however, is incorporated into porphyrins. The rate of (14)CO(2) evolution from leaves fed [2,4-(14)C]PBG is strongly inhibited by anaerobiosis but is unaffected by aminooxyacetic acid, while the rate of (14)CO(2) evolution from [2(aminomethyl),5-(14)C]PBG is strongly inhibited by aminooxyacetic acid but is not affected by anaerobiosis.THESE RESULTS SUGGEST THAT: (a) exogenous PBG is taken up and metabolized by etiolated barley leaves; (b) PBG is not metabolized exclusively to porphyrins but can be converted to a variety of intermediary metabolites; (c) this metabolism involves reactions which are partially dependent upon O(2) and pyridoxal phosphate. PMID- 16662258 TI - Synthesis and movement of abscisic Acid in water-stressed cotton leaves. AB - Synthesis and movement of abscisic acid (ABA) into the apoplast of water-stressed cotton (Gossypium hirsutum L.) leaves were examined using pressure dehydration techniques. The exudates of leaves dehydrated in a pressure chamber contained ABA. The level of ABA in the exudates was insensitive to the leaf water potential when dehydration occurred over a 3-hour period. When leaves were rapidly dehydrated in the pressure chamber and held at a balance pressure coincident with the point of zero turgor, ABA accumulated in the leaf tissue and then in the apoplast, but only after 2 to 3 hours of zero turgor. Slow dehydration of leaves by equilibration over varying mannitol concentrations resulted in some accumulation of ABA prior to the point of zero turgor, but ABA accumulated in the tissue and apoplast most rapidly after the onset of zero turgor.When intact plants were allowed to dehydrate, stomata of leaves attached to the plant began closing as the leaf water potential decreased to -12 bars. Leaves began accumulating ABA at about -14 bars, but accumulation of ABA in the apoplast did not occur until the leaf water potential reached -16 bars. Although the apoplastic fraction of ABA should represent an "active" pool of ABA readily accessible to the guard cells, the data suggest that there may be stomatal closure prior to accumulation of ABA in the apoplast. A role for the small amounts of apoplastic ABA not dependent upon water stress is proposed for this initial stressinduced stomatal response. PMID- 16662259 TI - Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.). AB - Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO(3) (-) or NH(4) (+). Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH(4) (+)-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.Enzymological data indicate that nodules of these ineffective alfalfa genotypes are uniquely differentiated plant organs. Decreasing or constant plant GS and GOGAT specific activities in ineffective nodules in response to applied N suggest that factors in addition to N supply are involved in the induction of high levels of plant ammonia assimilating enzymes in nodules. Genotype differences observed for nodule enzyme specific activities support the concept that ineffectiveness may be expressed in different ways within the nodule. Senescence was evident in ineffective nodules of N-treated plants of both genotypes, indicating that nodule senescence induced by applied N may not be closely linked to symbiotic effectiveness in alfalfa. Data for ammonia-assimilating enzymes in roots suggest the GS/GOGAT pathway operates only at low levels of soil N and that GDH functions to detoxify high levels of soil NH(4) (+). PMID- 16662260 TI - Effects of Membrane ATPase Inhibitors on Light-Induced Phase Shifting of the Circadian Clock in Neurospora crassa. AB - Effects of several membrane ATPase inhibitors on light-induced phase shifting of the circadian conidiation rhythm in Neurospora crassa were examined using mycelial discs in liquid culture. Suppression of phase shifting by the inhibitors was strongly dependent on the pH of the liquid medium in which the discs were cultured during the time from light-dark transition (beginning of free-run) to light irradiation. When discs were cultured in pH 6.7 medium, azide, the inhibitors of plasma membrane ATPase (diethylstilbestrol and N, N' dicyclohexylcarbodiimide), and ethanol completely suppressed the effect of light on the clock. In contrast, mycelial discs cultured in pH 5.7 medium were fully phase-shifted by light in the presence of the same and even higher concentrations of the chemicals. However, sensitivity to light of the discs cultured in relatively acidic medium was eight times higher than that of the discs cultured at neutral pH. Oligomycin and venturicidin, inhibitors of mitochondrial ATPase, did not suppress phase shifting by light at either pH. PMID- 16662261 TI - Effects of cryogenic treatment on plantlet production from frozen and unfrozen date palm callus. AB - Embryogenic date palm (Phoenix dactylifera L. var. Medjool) callus cultures were treated with a cryoprotective mixture of polyethylene glycol (Carbowax 6000), glucose, and dimethylsulfoxide (10%/8%/10%, w/v); treated with the mixture, frozen to -196 degrees C, and then thawed; or left untreated. Growth subsequent to treatment was measured as fresh weight increase and as the number of embryos produced during 18 weeks of culture. The growth of calli that were frozen and thawed, compared to the other treatments, was greatly inhibited during the first 9 weeks of culture. This inhibition disappeared in subcultured tissue. In all treatments, cultures initiated plantlets after 9 weeks. Enzyme polymorphism, for five gene-associated enzyme systems including alcohol dehydrogenase, esterase, peroxidase, phosphoglucomutase, and phosphoglucoisomerase, was analyzed in leaves of regenerated plantlets by using starch gel electrophoresis for separation. Isozyme patterns were similar for all treatments. PMID- 16662262 TI - An alpha-Galactosidase with Hemagglutinin Properties from Soybean Seeds. AB - Soybean (Glycine max L.) seeds contain a galactose-binding protein which displays two activities: (a) an alpha-galactosidase activity and (b) a hemagglutinin activity. The alpha-galactosidase-hemagglutinin was purified to homogeneity by conventional protein purification procedures and also by affinity chromatography. This protein can be easily separated from soybean agglutinin, the N-acetyl-d galactosamine-specific lectin in soybean. Further, these two agglutinins show no immunological relatedness. The alpha-galactosidase-hemagglutinin can be reversibly converted by pH changes from a tetrameric form which displays both enzymic and hemagglutinin activities to a monomeric form which displays enzymic activity only. Although both the monomeric and tetrameric forms are enzymically active, they display different pH optima and carbohydrate specificities. PMID- 16662263 TI - Factors Influencing beta-Glucan Synthesis by Particulate Enzymes from Suspension Cultured Lolium multiflorum Endosperm Cells. AB - Particulate enzymes from suspension-cultured ryegrass (Lolium multiflorum Lam.) endosperm cells incorporated glucosyl residues from UDP-glucose and GDP-glucose into beta-glucans. Three types of beta-glucans were produced from UDP-glucose: 1,3-beta-glucan; 1,4-beta-glucan; and mixed-linkage 1,3;1,4-beta-glucan. As in other systems, relatively more 1,4-beta-glucan was produced from a low (10 micromolar) UDP-glucose concentration, and relatively more 1,3-beta-glucan was produced from a high (1 millimolar) UDP-glucose concentration. However, in ryegrass, 1,3;1,4-beta-glucan represented a major proportion of the products at both low and high UDP-glucose concentrations. The arrangement of linkages in the 1,3;1,4-beta-glucan was different at the two concentrations; at the low UDP glucose concentration, more sequences of three consecutive 1,4-linkages were produced.The effects of pH, temperature, and metal ion concentrations on incorporation were dependent on the UDP-glucose concentration. At the low UDP glucose concentration, incorporation into all three types of beta-glucan increased with increasing pH. At the high UDP-glucose concentration, 1,3-beta glucan was the major product at pH 7 and below; 1,4-beta-glucan synthesis was optimal at pH 8; and synthesis of 1,3;1,4-beta-glucan was greatest above pH 8.With 10 micromolar GDP-glucose as substrate, 1,4-beta-glucan, but no 1,3;1,4 beta-glucan, was produced. Incorporation from either UDP-glucose or GDP-glucose was not influenced by the presence of the other. PMID- 16662264 TI - Ent-Kaurene Biosynthesis in Extracts of Helianthus annuus L. Seedlings. AB - Kaurene synthetase B activity (conversion of copalyl pyrophosphate to ent kaurene) is readily detectable in crude cell-free extracts of 3- to 4-day old dark-grown sunflower (Helianthus annuus cv. Mammoth) seedlings, whereas little or no kaurene synthetase AB activity (conversion of geranylgeranyl pyrophosphate to ent-kaurene) can be found in these extracts under comparable assay conditions. A low amount of AB activity is evident only if an extensively dialyzed extract is used in low concentrations as the enzyme source. One factor which may contribute to the low apparent levels of AB activity is the presence of inhibitory factors in the crude sunflower extract since these extracts can be shown to act as a potent inhibitor of Marah macrocarpus endosperm kaurene synthetase AB activity. Heat treatment (100 degrees C) or dialysis of the sunflower extract reduces the amount of its inhibitory activity. Also, it was observed that low concentrations of extensively dialyzed sunflower extracts act to stimulate M. macrocarpus AB activity. There is no evidence for the presence of an inhibitory factor for M. macrocarpus kaurene synthetase B activity in sunflower extracts. However, there does appear to be present in the crude preparation of sunflower extract a dialyzable factor(s) that impedes its own B activity. There is little information to date on the nature of these inhibitory and stimulatory factors for kaurene synthetase activity or their possible roles in physiological regulation. The possible presence of such factors should be considered, however, when attempting to evaluate kaurene synthetase activities in extracts of vegetative plants. PMID- 16662265 TI - Chlorophyll a Fluorescence Transients in Mesophyll and Guard Cells : MODULATION OF GUARD CELL PHOTOPHOSPHORYLATION BY CO(2). AB - Chlorophyll fluorescence transients from mesophyll and guard cell chloroplasts of variegated leaves from Chlorophytum comosum were compared using high resolution fluorescence spectroscopy. Like their mesophyll counterparts, guard cell chloroplasts showed the OPS fluorescence transient indicating the operation of the linear electron transport and the possible generation of NADPH in these organelles. They also showed a slow fluorescence yield decrease, equivalent to the MT transition in mesophyll, suggesting the formation of the high energy state and photophosphorylation. Unlike the mesophyll chloroplasts, the fluorescence from guard cell chloroplasts lacked the increment of the SM transition, indicating that the two types of chloroplasts have some metabolic differences. The presence of CO(2) (supplied as bicarbonate, pH 6.7) specifically inhibited the MT-equivalent transition while its absence accelerated it. These observations constitute the first specific evidence of a guard cell chloroplast response to CO(2). Control of photosynthetic ATP levels in the guard cell cytoplasm by CO(2) may provide a mechanism regulating the availability of high energy equivalents at the guard cell plasmalemma, thus affecting stomatal opening. PMID- 16662266 TI - Energy Coupling in H-Amino Acid Cotransport : ATP DEPENDENCE OF THE SPONTANEOUS ELECTRICAL REPOLARIZATION OF THE CELL MEMBRANES IN OAT COLEOPTILES. AB - Experiments were undertaken in order to test the mechanism of energy coupling for amino acid uptake proposed in the cotransport hypothesis. According to the hypothesis an electrochemical potential difference in H(+) is established by active H(+) extrusion. That potential difference then drives the cotransport of H(+) and amino acids into the cells. Application of amino acids to oat (Avena sativa var. Victory) coleoptiles induced transient depolarizations of the cell membrane electrical potentials considered to reflect the joint uptake of H(+) and amino acids followed by an enhanced H(+) extrusion. In the presence of KCN, cysteine induced strong depolarizations, but the rate of repolarization depended linearly upon the cyanide-adjusted ATP level of the tissue. At an ATP level 44% of normal, the membrane potential was 74% of normal, but the repolarization after cysteine-induced depolarization was practically nil. Sudden transitions from room temperature to temperatures below 15 degrees C induced sharp depolarizations of the membrane which then repolarized within 3 min; the ATP content of the tissues was unaffected. Cysteine and alanine induced strong depolarizations at temperatures between 5 and 25 degrees C, and the Q(10) for the rate of depolarization was 1.5 for cysteine and 1.6 for alanine. The Q(10) for the rate of repolarization was 3.0 for cysteine and 2.0 for alanine. These experiments support the prevailing view that the depolarizations are caused by the passive joint influx of H(+) and amino acids and that the repolarizations depend upon the ATP-dependent extrusion of H(+). PMID- 16662267 TI - Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA. AB - Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; alpha-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter. PMID- 16662268 TI - A Direct Confirmation of the Standard Method of Estimating Intercellular Partial Pressure of CO(2). AB - The partial pressure of CO(2) inside leaves of several species was measured directly. Small gas exchange chambers were clamped above and below the same section of an amphistomatous leaf. A flowing gas stream through one chamber allowed normal CO(2) and water vapor exchange. The other chamber was in a closed circuit consisting of the chamber, an infrared gas analyzer, and a peristaltic pump. The CO(2) in the closed system rapidly reached a steady pressure which it is believed was identical to the CO(2) pressure inside the leaf, because there was no flux of CO(2) across the epidermis. This measured partial pressure was in close agreement with that estimated from a consideration of the fluxes of CO(2) and vapor at the other surface. PMID- 16662269 TI - Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L. AB - To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [(3)H]GA(20) and [(3)H]GA(1) were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [(3)H]GA(20) was metabolized to a compound chromatographically identical to 3-epi-GA(1). [(3)H]GA(1) was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA(8). [(3)H]3-epi-GA(1) applied to plants under LD was metabolized much more slowly than was [(3)H]GA(1), and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [(3)H]GA(20) and [(3)H]GA(1). It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA(20) is the precursor of 3-epi-GA(1), but there is at present no evidence indicating the precursor of GA(1). PMID- 16662270 TI - Effect of Benzyladenine Treatment Duration on delta-Aminolevulinic Acid Accumulation in the Dark, Chlorophyll Lag Phase Abolition, and Long-Term Chlorophyll Production in Excised Cotyledons of Dark-Grown Cucumber Seedlings. AB - Cotyledons excised from dark-grown cucumber (Cucumis sativus L. cv. Aonagajibai) seedlings were incubated in the dark with the cytokinin benzyladenine for different time periods. Then, various greening parameters were examined, including protochlorophyll(ide) to chlorophyll(ide) photoconversion and delta aminolevulinic acid accumulations in the dark, both triggered by a 5-minute red light pulse.Benzyladenine rapidly stimulates delta-aminolevulinic acid accumulation, protochlorophyll(ide) regeneration, and lag-phase abolition. The red-light-pulse-induced protochlorophyll(ide) to chlorophyll(ide) photoconversion does not appear to be affected by benzyladenine treatment. Long-term chlorophyll production in the light is stimulated after longer incubations with the cytokinin.Rates of delta-aminolevulinic acid accumulation in the dark were similar to chlorophyll production rates in the light (maximum rates of 20 and 19 to 23 nanomoles per gram fresh weight per hour, respectively, in mole equivalents). The data strongly suggest that benzyladenine abolishes the lag phase by triggering delta-aminolevulinic acid synthesis potentiation and that the cytokinin has other stimulatory effects on chlorophyll production after longer incubations. PMID- 16662271 TI - Pigmentation and Soluble Peroxidase Isozyme Patterns of Leaves of Pedilanthus tithymaloides L. variegatus as a Result of Daily Temperature Differences. AB - In a constant environment with a narrow (less than 8 degrees C) daily temperature difference (deltat), leaves of Pedilanthus tithymaloides L. variegatus usually appeared green. After at least two days of deltat > 10 degrees C, new leaves had become green-white, and a red pigmentation appeared and increased if high deltat conditions were maintained. If plants were returned to a narrow deltat, new green leaves reappeared. Electrophoretic patterns of soluble peroxidase isozymes changed during the color changes. Three groups of electrophoretic bands occurred, and each was related to a characteristic tissue. The development of red color was correlated with the appearance of the group with the fastest electrophoretic mobility and the highest peroxidase activity. PMID- 16662272 TI - Far-Red Reversal of Red Light Effect during Long-Night Induction of Potato (Solanum tuberosum L.) Tuberization. AB - The hypothesis that phytochrome is involved in the regulation of potato (Solanum tuberosum L.) tuberization was tested. When 5 minutes of red light were given in the middle of the 16-hour dark period to which whole plants were exposed daily for 14 days before making cuttings, the percentage of tuberization on cuttings decreased. The effect of red light was significantly reversed by 2 minutes of far red light given immediately after the red in each of two separate experiments. This supports the hypothesis that phytochrome is at least indirectly involved.Longer exposures to red light were not always as effective as a 5-minute exposure in reducing tuberization and were not reversible by far-red light. PMID- 16662273 TI - An improved bioassay for cytokinins using cucumber cotyledons. AB - The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5 day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N(6)-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins. PMID- 16662274 TI - Glycoprotein Biosynthesis in Chlamydomonas: I. IN VITRO INCORPORATION OF GALACTOSE FROM UDP-[C]GALACTOSE INTO MEMBRANE-BOUND PROTEIN. AB - A crude membrane fraction from Chlamydomonas reinhardii was found to catalyze d galactose transfer from UDP-galactose to endogenous proteins. Highest incorporation rates were achieved by incubation at 25 degrees C and pH 7.5 in the presence of 10 millimolar Fe(2+). Hydrolytic studies on the labeled polymer revealed that radioactivity was attached to protein via an alkali-stable and acid labile linkage. Identification of galactose as the only labeled sugar in the acid hydrolysate and results of a tentative estimation of the molecular weight of the charged alkaline degradation product indicate that monomeric galactose units are transferred to form an O-glycosidic bond with peptidyl hydroxyproline. No indications were found for a similar linkage to serine which, in contrast to the hydroxyproline-O-glycoside linkage, is acid-stable but is cleaved by beta elimination. Chromatography of the sodium dodecyl sulfate-solubilized polymer on Sepharose-6B demonstrated that galactosyl residues are mainly associated with proteins which are of considerably higher molecular weight than are the majority of sodium dodecyl sulfate-denatured membrane proteins in this fraction. PMID- 16662275 TI - Bud Induction with Cytokinin : A LOCAL RESPONSE TO LOCAL APPLICATION. AB - A portion of the surface of detached Graptopetalum paraquayense E. Walther leaves can be used to assay small amounts of reagents in lanolin for their ability to induce shoots only at the site of application. The cytokinins benzyladenine, kinetin, and 6-(gamma,gamma-dimethylallylamino)purine (DMAAP) were tested, and DMAAP was most effective in bud induction at concentrations below 1%. The higher the hormone concentration, the sooner the appearance of leaf primordia and the higher the ultimate yield of buds. Leaves treated with DMAAP for 2 days developed buds as rapidly as those with longer treatments. PMID- 16662276 TI - Autoinhibition of Ethylene Production in Citrus Peel Discs : SUPPRESSION OF 1 AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHESIS. AB - Wound ethylene formation induced in flavede tissue of citrus fruit (Citrus paradisi MacFad. cv. Ruby Red) by slicing was almost completely inhibited by exogenous ethylene. The inhibition lasted for at least 6 hours after removal of exogenous ethylene and was then gradually relieved. The extent of inhibition was dependent upon the concentration of ethylene (1 to 10 microliters/liter) and the duration of treatment. The increase in wound ethylene production in control discs was paralleled by an increase in 1-aminocyclopropane-1-carboxylic acid (AAC) content, whereas in ethylene-treated discs there was little increase in ACC content. Application of ACC completely restored ethylene production in ethylene pretreated discs, indicating that the conversion of ACC to ethylene is not impaired by the presence of ethylene. Thus, autoinhibition of ethylene synthesis was exerted by reducing the availability of ACC. Ethylene treatment resulted in a decrease in extractable ACC synthase activity, but this decrease was too small to account for the marked inhibition of ACC formation. The data indicate that autoinhibition of ethylene production in citrus flavede discs results from suppression of ACC formation through repression of the synthesis of ACC synthase and inhibition of its activity. PMID- 16662277 TI - Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves. AB - The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by (14)CO(2) assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total (14)CO(2) fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = -0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.Four nodulated soybean cultivars were grown to maturity in a greenhouse. Fully expanded leaves at the top of the canopy were sampled during vegetative growth (45 days), at flowering (79 days), and at mid-podfill (120 days). In general, activities of SPS and uridine-5' diphosphatase were highest during vegetative growth, and they decreased during reproductive development, whereas activity of sucrose synthase and leaf starch content tended to increase. Leaf starch was negatively correlated with levels of SPS (r = -0.71). The results support the postulate that sucrose-P synthetase is a key control point regulating the photosynthetic formation of sucrose, and, hence, starch. PMID- 16662278 TI - Rapid increase in deep supercooling of xylem parenchyma. AB - Malus pumila Mill. twigs were collected from September through December and stored at 5 degrees C until the low temperature exotherms of the xylem were determined by differential thermal analysis. During the differential thermal analysis, cooling was interrupted, and temperatures of 5 to -18 degrees C were held for 0.4 to 10 hours before cooling to -50 degrees C was resumed. Control twigs were cooled to -50 degrees C without interruption. Holding the twigs at 1.3 to -5 degrees C shifted the start of the low temperature exotherm from about -20 to -30 degrees C. Slightly higher (2.6 degrees C) and lower (-10 degrees C) temperatures were occasionally effective. The shift began within 20 to 30 minutes and increased progressively to 150 minutes. The acclimation was reversibly inhibited by N(2) atmosphere. PMID- 16662279 TI - Analysis of polyamines in higher plants by high performance liquid chromatography. AB - A sensitive (0.01-1 nmol) method has been developed for the analysis of polyamines in higher plant extracts based on high performance liquid chromatography (HPLC) of their benzoyl derivatives (Redmond, Tseng 1979 J Chromatogr 170: 479-481). Putrescine, cadaverine, agmatine, spermidine, spermine, and the less common polyamines nor-spermidine and homospermidine can be completely resolved by reverse phase HPLC, isocratic elution with methanol:water (64%, v/v) through a 5-mum C(18) column, and detection at 254 nm. The method can be directly applied to crude plant extracts, and it is not subject to interference by carbohydrates and phenolics. A good quantitative correlation was found between HPLC analysis of benzoylpolyamines and thin layer chromatography of their dansyl derivatives. With the HPLC method, polyamine titers have been reproducibly estimated for various organs of amaranth, Lemna, oat, pea, Pharbitis, and potato. The analyses correlate well with results of thin layer chromatography determinations. PMID- 16662280 TI - Comparative Effects of Ancymidol and Its Analogs on Growth of Peas and Ent Kaurene Oxidation in Cell-Free Extracts of Immature Marah macrocarpus Endosperm. AB - The plant growth retardant alpha-cyclopropyl-alpha-(4-methyoxyphenyl)-5 pyrimidine methyl alcohol (ancymidol) and a series of analogs of this substance in which one or more of the substituents were varied were tested for their comparative biological activity. The compounds were tested as inhibitors of internode elongation in peas and as inhibitors of the oxidation of ent-kaurene catalyzed by microsomal preparations from the liquid endosperm of Marah macrocarpus seeds. The relative effectiveness of a substance was generally the same as an inhibitor of the two processes. Ancymidol was the most effective. Substitution of the alcohol group of ancymidol by either methoxy or hydrogen groups reduced the activity only slightly. Substitution of the cyclopropyl group by an isopropyl moiety also had little effect on the activity. However, substitution of the cyclopropyl group with a phenyl or other aryl substituent greatly reduced the effectiveness of the analog as an inhibitor. Replacement of the 4-methoxyphenyl substituent with a similar substituent such as 4-chlorophenyl had little effect on activity, but replacement with a 2-methoxyphenyl group greatly reduced activity. Analogs in which the pyrimidyl moiety of ancymidol was modified were inactive in whole plants, but moderately active in the cell-free ent-kaurene oxidation system. The application of gibberellic acid can overcome the growth inhibitions due to treatment of the test plants with 10(-5)m or lower concentrations of the inhibitors. However, the inhibitory effects of 10(-4)m or higher concentrations of inhibitors on test plants were not overcome by the applications of exogenous gibberellic acid. These results support the idea that the effects of low concentrations of these substances on plant growth are primarily a consequence of their ability to inhibit ent-kaurene oxidation and gibberellin biosynthesis. Other modes of inhibition may operate at higher inhibitor concentrations. PMID- 16662281 TI - Comparative Effects of Substituted Pyrimidines on Growth and Gibberellin Biosynthesis in Gibberella fujikuroi. AB - The fungicide alpha-(2,4-dichlorophenyl)-alpha-phenyl-5-pyrimidine methyl alcohol (triarimol) and four other structural analogs of this substance, in which one or more of the substituents were varied, were tested for their comparative effects on growth and gibberellin biosynthesis in the fungus Gibberella fujikuroi. Each of the five analogs tested was capable of inhibiting growth as measured by dry weight in 5-day-old cultures. Three of them [alpha-(2-chlorophenyl)-alpha-(4 chlorophenyl)-5-pyrimidine methyl alcohol, fenarimol; alpha-(2-chlorophenyl) alpha-(4-fluorophenyl)-5-pyrimidine methyl alcohol, nuarimol; and triarimol] were effective at appreciably lower concentrations than the other two [alpha-(4 chlorophenyl)-alpha-(1-methylethyl)-5-pyrimidine methyl alcohol, experimental compound EL 509; and alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidine methyl alcohol, ancymidol].All five substances also inhibited gibberellin production as measured by gibberellin content of fungus filtrates. The relative effectiveness of the compounds as inhibitors of growth and gibberellin production were similar. These analogs were also shown to inhibit ent-kaurene oxidation by microsomal preparations from fungal mycelia. Thus, the site of inhibition of gibberellin biosynthesis may be the same for the fungus as the one affected by this group of substances in higher plant tissues.The structure-activity relationships between the analogs are opposite to those observed in higher plant tissues. The fungicides fenarimol, nuarimol, and triarimol, which were most effective in inhibiting growth and gibberellin biosynthesis in the fungus, were much less effective than EL 509 and ancymidol in inhibiting growth and gibberellin biosynthesis in higher plants. These results indicate that the ent kaurene oxidase systems from the two sources have somewhat different molecular characteristics, and thus, interact differently with this group of substances. PMID- 16662283 TI - Stability and Synthesis of Phospholipids during Desiccation and Rehydration of a Desiccation-Tolerant and a Desiccation-Intolerant Moss. AB - The fatty acid composition of the phospholipids from the desiccation-tolerant moss Tortula ruralis (Hedw.) Gaertn, Meyer and Scherb and the desiccation intolerant moss Cratoneuron filicinum has been determined. No changes in composition occur in either moss as a consequence of rapid drying, but, after slow drying, there is a decline in some unsaturated fatty acids. Upon rehydration of T. ruralis after slow drying, these acids decline further; however, within 105 minutes, they regain the same levels as those in undesiccated controls. A smaller and more transient decline occurs after rapid desiccation. Most phospholipid unsaturated fatty acids decrease during rehydration of C. filicinum, and their levels are not recovered. After both rapid and slow drying of T. ruralis, acetate and glycerol are incorporated into the phospholipid fraction, although de novo synthesis, alone, might not account for the increase in unsaturated fatty acids upon rehydration. Very little acetate or glycerol is incorporated during rehydration of C. filicinum. Loss of unsaturated fatty acids from the phospholipids of T. ruralis does not appear to be associated with increased lipoxygenase activity. Furthermore, there is little correlation between the extent of peroxidation of fatty acids due to desiccation and changes in the phospholipid fraction. PMID- 16662282 TI - Dissociation, reassociation, and purification of plastid and cytosolic phosphoglucose isomerase isozymes. AB - The plastid and cytosolic isozymes of the dimeric enzyme phosphoglucose isomerase (EC 5.3.1.9) from spinach (Spinacia oleracea) and cauliflower (Brassica oleracea) were purified to apparent homogeneity. The isozymes from sunflower (Helianthus annuus) and Clarkia xantiana were partially purified. When subunits from two electrophoretically distinguishable cytosolic isozymes, either from the same or from different species, were dissociated and allowed to reassociate in each other's presence, an active hybrid enzyme, consisting of one subunit of each type, was formed in addition to the two original homodimers. Active hybrid enzymes were also formed by dissociation and reassociation of plastid isozymes. Hybrid molecules were not produced between the plastid and cytosolic subunits, suggesting that they are not able to bind with each other. Additional differences between the plastid and cytosolic isozymes are described. PMID- 16662284 TI - Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry. AB - Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O(2) partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O(2) microelectrode measurements which showed that the O(2) partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O(2) uptake as a function of temperature and partial pressure of O(2) were consistent with diffusion limitation of O(2) uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O(2) regulation in legume and Parasponia nodules is very similar and differs from O(2) regulation in actionorhizal nodules. PMID- 16662285 TI - A Reanalysis of the Two-Component Phloem Loading System in Beta vulgaris. AB - Kinetic analysis of [(14)C]sucrose loading into sugar beet leaf discs revealed the presence of two transport components. At low exogenous sucrose concentrations, a saturable component, which exhibited Michaelis-Menten characteristics, was the main mode of transport. At concentrations greater than 50 millimolar, phloem loading was dominated by a linear component which appeared to operate as a first order kinetic transport process. Over the exogenous sucrose concentrations employed, influx could be described by the equation v = V(max)S/(S + K(m)) + kS. Influx via both processes was strongly pH-dependent. Evidence is presented that the linear component was not explicable in terms of simple diffusion, or exchange diffusion, into either mesophyll or minor vein phloem tissue. Extensive metabolic conversion of sucrose was not a factor contributing to influx at high external sucrose concentrations. At present, it is believed that both components operate in parallel at the membrane bounding the sieve element-companion cell complex. The saturable component is identified with sucrose-H(+) cotransport. While the significance of the linear component has been established, its nature remains to be elucidated. PMID- 16662286 TI - Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide. AB - The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H(2)O(2) could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H(2)O(2)-generating enzyme. This increase in PGA formation due to H(2)O(2) was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O(2) and by the NAD(P)H-oxidizing system.It was concluded that the oxidation of NAD(P)H by a system using ascorbate and H(2)O(2) can serve as a means of recycling NAD(P)H but that another reaction involving ascorbate and NAD(P)H may also function in the spinach chloroplast. PMID- 16662287 TI - Phycomyces: discovery of the aiming error in the avoidance response. AB - It has been determined experimentally that, when a stage IVb Phycomycea sporangiophore is subjected to an asymmetric avoidance stimulus (a single barrier), it uses its own net rotation to convert the spatial stimulus to a temporal one. This finding explains the sporangiophore's nonadaptation to a single-barrier stimulus and adaptation to a double-barrier stimulus. PMID- 16662288 TI - Dinitrogen fixation in male-sterile soybeans. AB - Partial male-sterile (ms(4)/ms(4)) soybeans (Glycine max L. Merr.) and their fertile isoline (Ms(4)/Ms(4)) were grown in adjoining field plots. From 62 until 92 days after emergence, the nitrogenase activity, assayed by acetylene reduction, of the average male-sterile plant was approximately twice that of the average fertile plant. At approximately 100 days after emergence, the assayable nitrogenase activity of the fertile plants fell to zero, whereas the nitrogenase of the partial male-sterile plants continued to be active for two additional weeks. Thus, this male-sterile plant seems to fix dinitrogen both at a higher rate and over a longer duration than does its fertile isoline. PMID- 16662289 TI - Partial characterization of the in vitro activation of inactive pyruvate, pi dikinase from darkened maize leaves. AB - In vitro activation of dark-inactivated pyruvate, orthophosphate dikinase extracted from maize (Zea mays L. cv. Golden Cross Bantam T51) leaves was examined. The inactive form of the enzyme and orthophosphate behaved kinetically as substrates for the reaction, which was catalyzed by an activating factor. This factor was bound by Blue Dextran Sepharose 4B and could be eluted by KCl at a concentration of 0.5m. The molecular weight of the maize leaf activating factor was about 88,000. Cibacron Blue 3G-A, a reactive moiety of Blue Dextran, inhibited the factor competitively with respect to the concentration of the inactive dikinase with a K(i) of 4.6 micromolar. Adenosine diphosphate and pyrophosphate were also found to be competitive inhibitors of activation, with respect to the inactive dikinase, giving K(i) values of 90 and 140 micromolar, respectively. Adenosine, other nucleotide diphosphates, and dinucleotides gave little or no inhibition of activation. These results suggest the association of a nucleotide, presumably nucleotide diphosphate, with the inactive form of pyruvate, orthophosphate dikinase. PMID- 16662290 TI - Transmission of the Monocarpic Senescence Signal via the Xylem in Soybean. AB - During monocarpic senescence in soybean (Glycine max [L.] Merrill cv. Anoka) there is a remobilization of nitrogen from the leaves to the seeds, and it has been hypothesized that this loss of nitrogen from the leaves induces foliar yellowing. The phloem in a small segment of the petiole between the pods and the target leaf can be inactivated with a jet of steam. When a plant is depodded except for a single pod cluster in the center of the plant, the pod cluster induces yellowing of the nearest leaf even if the petiole contains a zone of dead phloem, whereas most of the rest of the plant remains green. The nitrogen content of these leaves with a dead phloem zone in their petioles does not decrease greatly, even though the leaves turn yellow. A similar treatment of a single leaf on a fully depodded plant (leaves stay green) does not cause that leaf to turn yellow. Since nutrients would have to be withdrawn from the leaves via the phloem, the pods do not induce yellowing by pulling nutrients out of the leaf and must be able to exert their influence via the xylem. PMID- 16662291 TI - Crassulacean Acid Metabolism in the Succulent C(4) Dicot, Portulaca oleracea L Under Natural Environmental Conditions. AB - Crassulacean acid metabolism (CAM) was examined under natural environmental conditions in the succulent C(4) dicot Portulaca oleracea L. Two groups of plants were monitored; one was watered daily (well watered), while the other received water once every 3 to 4 weeks to produce a psi of -8 bars (drought stressed). Gas exchange, transpiration rate, and titratable acidity were measured for 24-hour periods during the growing season. CAM activity was greatest in drought-stressed plants during late August which had 13 hour days and day/night temperatures of 35/15 degrees C. Under these conditions net CO(2) uptake occurred slowly throughout the night. Diurnal fluctuations of titratable acidity took place in both leaves and stems with amplitudes of 17 and 47 microequivalents per gram fresh weight, respectively. Transpiration data indicated greater opening of stomata during the night than the day. CAM was less pronounced in drought stressed P. oleracea plants in July and September; neither dark CO(2) uptake nor positive carbon balance occurred during the July measurements. In contrast, well watered plants appeared to rely on C(4) photosynthesis throughout the season, although some acid fluctuations occurred in stems of these plants during September.To determine the fate of the CO(2) assimilated at night in drought stressed Portulaca plants, exposure to (14)CO(2) during the night followed by 9 hours of ambient air in the light. Malate was the predominant compound labeled during the night, with some citrate and aspartate. No (14)CO(2) release was detected during the following day and by midafternoon the majority of the label was found in the insoluble fraction (predominantly starch). These results substantiate our earlier work with growth-chamber-grown plants and show that limited CAM activity can occur in the succulent C(4) dicot Portulaca oleracea L. under certain natural environmental conditions. PMID- 16662292 TI - Dark Stomatal Movement in Sunflowers in Response to Illumination under Nitrogen. AB - Experiments were performed on intact sunflowers (Helianthus annuus) placed in a specially designed experimental chamber which allows instantaneous modifications of the atmospheric composition without changing any other conditions. After one night in normal conditions, the plant was illuminated under pure nitrogen atmosphere; the opening stomatal movement, measured as a transpiration rate variation, was inhibited. After an anoxia time period not exceeding one hour, the light was turned off and normal air restored. The stomatal movement was no longer inhibited, and a transient increase in the transpiration rate, referred to here as the postillumination transpiration peak (PITP), was observed.The quantity of transpired water during the PITP can be related to the total incident light energy supplied during the light-nitrogen period. Furthermore, the addition of a dark-nitrogen period between the light-nitrogen and dark-air periods caused the PITP to decrease. The PITP is almost suppressed after a 20-minute dark-nitrogen period.It is shown that the PITP does not result from a hydropassive mechanism but is metabolically controlled. Moreover, it seems that the PITP is not due to a CO(2)-suppression effect during the light-nitrogen period.The results are interpreted in terms of stomatal mechanism. The metabolites leading to PITP originate from the reducing equivalents created during the light-nitrogen period. They could be synthesized at the beginning of the PITP period (darkness under normal air) or during the dark-nitrogen plus CO(2) period between the light nitrogen and PITP periods. The results obtained are related to the first steps of classical photoactive stomatal opening. PMID- 16662293 TI - Stimulation of h(2)s emission from pumpkin leaves by inhibition of glutathione synthesis. AB - The effect of inhibitors of glutathione (GSH) synthesis, namely gamma-methyl glutamic acid, d-glutamic acid, cystamine, methionine-S-sulfoximine (MSX), buthionine-S-sulfoximine, and GSH itself, on the emission of H(2)S was investigated. All these compounds stimulated H(2)S emission from pumpkin (Cucurbita pepo L. cv Small Sugar Pumpkin) leaf discs in response to sulfate. MSX and GSH were the most effective compounds, stimulating H(2)S emission from leaf discs of mature pumpkin leaves by about 80% in response to sulfate. Both inhibitors did not appreciably enhance H(2)S emission in response to l-cysteine and inhibited H(2)S emission in response to sulfite.Treatment with MSX or GSH enhanced the uptake of sulfate by pumpkin leaf discs, but did not affect the incorporation of sulfate into reduced sulfur compounds. Inhibition of GSH synthesis by MSX or GSH caused an increase in the pool size of cysteine, and, simultaneously, reduced the incorporation of labeled sulfate into cysteine. The incorporation of labeled sulfate into the sulfite and sulfide pools of the cells are stimulated under these conditions.These observations are consistent with the idea that inhibition of GSH synthesis leads to an elevated cysteine pool that inhibits further cysteine synthesis. The H(2)S emitted under these conditions appears to arise from diversion of a precursor of the sulfur moiety of l cysteine. Therefore, stimulation of H(2)S emission in response to sulfate upon inhibition of GSH synthesis may reflect a role of H(2)S emission in keeping the cysteine concentration below a critical level. PMID- 16662294 TI - Control of Leaf Expansion by Nitrogen Nutrition in Sunflower Plants : ROLE OF HYDRAULIC CONDUCTIVITY AND TURGOR. AB - Nitrogen nutrition strongly affected the growth rate of young sunflower (Helianthus annuus L.) leaves. When plants were grown from seed on either of two levels of N availability, a 33% decrease in tissue N of expanding leaves was associated with a 75% overall inhibition of leaf growth. Almost all of the growth inhibition resulted from a depression of the daytime growth rate. Measurements of pressure-induced water flux through roots showed that N deficiency decreased root hydraulic conductivity by about half. Thus, N deficiency lowered the steady-state water potential of expanding leaves during the daytime when transpiration was occurring. As a result, N-deficient leaves were unable to maintain adequate turgor for growth in the daytime. N deficiency also decreased the hydraulic conductivity for water movement into expanding leaf cells in the absence of transpiration, but growth inhibition at night was much less than in the daytime. N nutrition had no detectable effects on plastic extensibility or the threshold turgor for growth. PMID- 16662295 TI - Photosynthetic Metabolism of Aspartate in Mesophyll and Bundle Sheath Cells Isolated from Digitaria sanguinalis (L.) Scop., a NADP-Malic Enzyme C(4) Plant. AB - Mesophyll cells and bundle sheath strands isolated from leaves of the C(4) plant Digitaria sanguinalis (L.) Scop. are capable of utilizing aspartate as a Hill oxidant. The resulting O(2) evolution upon illumination depends on the presence of 2-oxoglutarate, is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and is stimulated by methylamine. The rate of aspartate-dependent O(2) evolution with mesophyll cells was similar to those with phosphoenolpyruvate + CO(2) or with oxalacetate. Amino-oxyacetate, an inhibitor of aspartate aminotransferase, inhibited the aspartate-dependent O(2) evolution. Aspartate aminotransferase and NADP(+) -malate dehydrogenase are located in the mesophyll chloroplasts. These data suggest that aspartate is converted to oxalacetate via aspartate aminotransferase in the chloroplasts of mesophyll cells and that oxalacetate is subsequently reduced to malate, which is coupled to the photochemical evolution of O(2). This suggestion is further verified by the inhibition of phosphoenolpyruvate-dependent (14)CO(2) fixation by aspartate + 2-oxoglutarate, which presumably acts as oxalacetate and competes with phosphoenolpyruvate + CO(2) for NADPH. dl-Glyceraldehyde inhibited aspartate-dependent O(2) evolution in the bundle sheath strands but not in the mesophyll cells. The data indicate that aspartate may be converted to malate in both mesophyll and bundle sheath cells. In NADP(+) -malic enzyme species, aspartate may exist as a C(4) dicarboxylic acid reservoir which can contribute to the C(4) cycle through its conversion to malate. PMID- 16662296 TI - Potassium Channels in Chara corallina: CONTROL AND INTERACTION WITH THE ELECTROGENIC H PUMP. AB - Plasmalemma electrical properties were used to investigate K(+) transport and its control in internodal cells of Chara corallina Klein ex Willd., em R.D.W. Cell exposure to solutions containing 10 mm KCl caused the potential, normally -250 millivolts (average), to depolarize in two steps. The first step was a 21 millivolt depolarization that lasted from 1 to 40 minutes. The second step started with an action potential and left the membrane potential at -91 millivolts, with a 10-fold reduction in resistance. We suggest that the second step was caused by the opening of K(+) -channels in the membrane. This lowered the resistance and provided a current pathway that partially short-circuited the electrogenic pump. Although largely short-circuited, the electrogenic pump was still operating as indicated by: (a) the depolarized potential of -91 millivolts was more negative than Ek (=-42 millivolts in 10 mm K(+)); (b) a large net K(+) uptake occurred while the cell was depolarized; (c) both the electrogenic pump inhibitor, diethylstilbestrol, and the sulfhydryl-reagent N-ethylmaleimide (which increased the passive membrane permeability) further depolarized the potential in 10 mm KCl.A two-phase recovery back to normal cell potentials occurred upon lowering the K(+) concentration from 10 to 0.2 mm. The first phase was an apparent Nernst potential response to the change in external K(+) concentration. The second phase was a sudden hyperpolarization accompanied by a large increase in membrane resistance. We attribute the second phase to the closing of K(+) channels and the removal of the associated short-circuiting effect on the electrogenic pump, thereby allowing the membrane to hyperpolarize. Further experiments indicated that the K(+) -channel required Ca(2+) for normal closure, but other ions could substitute, including: Na(+), tetraethylammonium, and 2,4,6 triaminopyrimidine. Apparently, K(+) -channel conductance is determined by competition between Ca(2+) and K(+) for a control (gating?) binding site. PMID- 16662297 TI - Ethylene Production and Leaflet Abscission of Three Peanut Genotypes Infected with Cercospora arachidicola Hori. AB - Ethylene can induce abscission of leaves and other plant organs. Increased ethylene production by plant tissues can occur after invasion by microorganisms. The fungus Cercospora arachidicola Hori, attacks peanut leaflets and causes defoliation. Our objective was to determine if ethylene was involved in this defoliation. Leaves of three peanut, Arachis sp., genotypes were inoculated with C. arachidicola. Two genotypes, ;Tamnut 74' and PI 109839, produced ethylene and were defoliated. The third genotype, PI 276233, a wild species, did not produce ethylene above control levels and was not defoliated. Increase in ethylene production by Tamnut 74 and PI 109839 coincided with appearance of disease symptoms. Tamnut 74 produced the most ethylene, but PI 109839 was equally defoliated. Thus, less overall ethylene production did not necessarily indicate a more resistant genotype in this system unless ethylene production remained at control levels, as it did for PI 276233. Ethylene sufficient to initiate abscission could have been produced by the seventh day after inoculation when it was similar for both Tamnut 74 and PI 109839, but 3 to 4 times control amounts. This occurred before the rapid increase in ethylene production and before disease symptoms were visible. Silver ion, a potent inhibitor of ethylene action, was sprayed at three concentrations on intact Tamnut 74 plants. All rates reduced abscission and 150 mg/liter Ag(I) decreased abscission to below 10%. The data indicate that ethylene produced by peanut leaves in response to C. arachidicola infection initiates abscission and that ethylene action can be blocked by Ag(I) in such a host-pathogen interaction. PMID- 16662298 TI - Leucine transport in cells isolated from cold-hardened and nonhardened winter rye. AB - The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25 degrees C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5 degrees C efflux from the cells was inhibited.The apparent K(m) and V(max) for leucine uptake for both types of cells were of the order of 20 to 60 micromolar and 0.5 to 1.3 nanomoles per minute per 10(6) cells. The pH and temperature optima for both types of cells were 5 and 25 degrees C, respectively. The leucine transport system for these cells was relatively specific for amino acids lacking either bulky or charged groups on the amino acid side chains.Arrhenius plots for leucine uptake by hardened and nonhardened cells showed discontinuities at 13 degrees C, and the energies of activation were similar. The results suggests that biochemical changes which occur in rye cells upon cold hardening did not result in an observable perturbation of the properties of the leucine transport system. PMID- 16662299 TI - Evidence for a Cl-Stimulated MgATPase Proton Pump in Oat Root Membranes. AB - The possibility that plant membrane-bound MgATPases may act as electrogenic proton pumps has been investigated. Using an oat (Avena sativa L. cv. Victory) root membrane preparation which is partially enriched in tightly sealed vesicles, we have shown that MgATP stimulates the uptake of the membrane-permeable anion [(14)C]SCN(-) by the vesicles; this indicates that an electrical potential (interior positive) is generated across the membrane. Both Cl(-) ions and the proton ionophore trifluoromethoxy(carbonyl-cyanide)phenylhydrazone inhibit the MgATP-driven [(14)C]SCN(-) uptake, presumably by collapsing the MgATP-generated membrane potential. The uptake of the pH gradient probe [(14)C]imidazole into the vesicles is also greatly stimulated by MgATP, indicating the presence of a transmembrane proton gradient (interior acid). MgATP-driven [(14)C]imidazole uptake is temperature sensitive, Cl(-)-stimulated, substrate specific for MgATP, sensitive to the MgATPase inhibitors vanadate and N,N'-dicyclohexylcarbodiimide, and completely eliminated by trifluoromethoxy(carboxyl-cyanide)phenylhydrazone. The mitochondrial ATPase inhibitor oligomycin has little effect on the MgATPase activity and on the MgATP-dependent [(14)C]SCN(-) and [(14)C]imidazole uptake. These data indicate that a class of oat root membrane-bound MgATPases, stimulated primarily by Cl ions, is capable of using the free energy of ATP-hydrolysis to generate an apparent electrochemical proton gradient in vitro. PMID- 16662300 TI - Inducible cadmium binding complexes of cabbage and tobacco. AB - Cadmium complexes with apparent molecular weights of 10,000 were observed in aqueous extracts of Cd-treated cabbage (Brassica capitata L., cv. red danish) and tobacco (hybrid of Nicotiana glauca and N. langsdorffii) plants. The amount of complex (as Cd) recovered was found to be dependent on the concentration of the metal in the growth medium and the total time of exposure of plants to the metal. Induction of the complex at moderate levels of (112)Cd exposure was monitored after labeling the complex with (109)Cd in vitro. The constitutive nature of the ligand of the complex in cabbage and tobacco leaves was suggested when control plant extracts were exposed to (109)Cd. Such extracts contained (109)Cd, which eluted from Sephadex G-50 in the region of Cd complex. Simultaneous labeling with (112)Cd and (35)S or (32)P indicated that the complex contained sulfur but probably not phosphorus. The amount of (35)S which eluted coincident with (112)Cd complex increased during complex induction. No evidence was found for the presence of 10,000 molecular weight Cd complex in stem exudates (vascular sap) of Cd-treated plants.The results obtained are consistent with the presence in these tissues of a ligand which is both inducible and constitutive and binds Cd in mercaptide bonds. All of these properties, and others reported earlier, are characteristic of Cd-metallothionein formed in animals. PMID- 16662301 TI - Effect of 2,4-dichlorophenoxyacetic Acid on glucosylation of scopoletin to scopolin in tobacco tissue culture. AB - 2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated the formation of scopoletin and scopolin in tobacco (Nicotiana tabacum L. ;Bright Yellow') cell culture. It especially stimulated the uptake of scopoletin from culture medium into the cells and the glucosylation of scopoletin to its monoglucoside, scopolin. This phenomenon is peculiar to 2,4-D, in contrast to other plant hormones. 2,4-D (1 mug/ml) stimulated the glucosylation of scopoletin to scopolin by enhancing UDP glucose:scopoletin glucosyltransferase (SGTase) activity. The enhancement of SGTase activity caused by treatment with 2,4-D was observed when the syntheses of RNA and protein were inhibited by either actinomycin-D and/or cycloheximide. However, the stimulatory effect of 2,4-D was inhibited by treatment with dinitrophenol. Furthermore, SGTase with or without treatment by 2,4-D in vivo for 24 hours, was isolated from cultured tobacco cells. The enzymes were purified about 200-fold by precipitation with (NH(4))(2)SO(4) and chromatography with Sephadex G-100, DEAE-cellulose, and hydroxyapatite. The specific activity of 2,4 D-treated SGTase was 10 times higher than that of untreated SGTase even in the purified fraction, which showed one protein band under electrophoresis. These results suggest that the enhancement of SGTase activity by 2,4-D is due to the energy-dependent activation of the enzyme already present, but not due to the de novo synthesis of the enzyme. PMID- 16662302 TI - Light-induced increase in the number and activity of ribosomes bound to pea chloroplast thylakoids in vivo. AB - Within 8 to 10 minutes of illumination, chloroplast thylakoids of pea (Pisum sativum) became enriched 30 to 100% in ribosomes bound by nascent chains. Following (or, in some experiments, coincident with) this apprarent redistribution was a 25 to 65% increase in the total bound ribosome population, which was then maintained at this higher level during the normal light period. On transfer of plants to darkness, the bound ribosome population decreased to the lower dark level. White, blue (400 to 520 nanometers), and orange (545 to 690 nanometers) light were all effective in producing an increase in the bound ribosome population. The level of bound ribosomes in the oldest leaves of 16-day old plants was 15-fold less than in the still-maturing leaf but was still increased by illumination.In vivo experiments with chloramphenicol and lincomycin indicated a requirement for protein synthesis by the 70S ribosomes both for the light-induced shift to the population bound by nascent chains and for the increase in the total thylakoid-bound population. When thylakoids from plants in darkness or exposed to light for increasing periods were incubated in an Eschericia coli cell-free protein synthesizing system, 15 minutes of prior illumination in vivo produced a 60% increase in [(3)H]leucine incorporation. This stimulation preceded the increase in total bound ribosomes but corresponded in time to observed increases in the ribosomes bound by nascent chains.A light intensity of 100 micromoles per meter(2) per second, but not 25 micromoles per meter(2) per second, caused a significant increase in bound ribosomes over a 30 minute period. Strong inhibition in vivo by 3',4'-dichlorophenyl-1, 1 dimethylurea suggests that noncyclic electron flow is essential for light-induced ribosome redistribution. PMID- 16662303 TI - Isozymes of the glycolytic enzymes in endosperm from developing castor oil seeds. AB - Ion filtration chromatography on diethylaminoethyl-Sephadex A-25 has been used to separate two isozymes each of triose phosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase, glycerate 3-phosphate kinase, enolase, and phosphoglycerate mutase from homogenates of developing castor oil (Ricinus communis L. cv. Baker 296) seeds. Crude plastid fractions, prepared by differential centrifugation, were enriched in one of the isozymes, whereas the cytosolic fractions were enriched in the other. These data (and data published previously) indicate that plastids from developing castor oil seeds have a complete glycolytic pathway and are capable of conversion of hexose phosphate to pyruvate for fatty acid synthesis. The enzymes of this pathway in the plastids are isozymes of the corresponding enzymes located in the cytosol. PMID- 16662304 TI - Mineral reserves in castor beans: the dry seed. AB - Elemental composition and distribution of the mineral reserves in the endosperm and embryo tissues of Ricinus communis cultivars Hale and Zanzibarensis were investigated. Energy dispersive x-ray analysis was used to determine the elemental composition of the globoid crystals, while atomic absorption spectrometry allowed quantification of the elements, particularly Ca, in various seed regions. No major differences were found between the two cultivars with regard to the elemental distribution in globoid crystals. While the majority of globoid crystals contained P, K, and Mg, the occasional one also contained Ca. In extremely rare instances, Fe was detected in globoid crystals. Ca-containing globoid crystals were more common in provascular cell protein bodies in the stem and radicle. Polarized light microscopy, micro-incineration, and acid solubility tests demonstrated the presence of calcium oxalate crystals in the innermost testa which adheres to the endosperm and is often mistakenly identified as endosperm. Atomic absorption spectrometry revealed that most of the calcium present in castor bean seeds is localized in the testa. On a perseed-region basis, the much larger endosperm contains more Ca than does the embryo. However, on a unit-weight basis, the radicle-plus-stem regions contain considerably more Ca than does the cotyledon or endosperm, an observation that is consistent with the observed distribution pattern for Ca-containing globoid crystals. PMID- 16662305 TI - Wheat Storage Proteins : ISOLATION AND CHARACTERIZATION OF THE GLIADIN MESSENGER RNAs. AB - A total RNA extract was prepared from developing wheat seeds using guanidine-HCl to eliminate endogenous RNase activity. The RNA preparation, substantially free of protein, carbohydrate and DNA, was chromatographed on either a poly uridylic acid-agarose or poly guanylic acid-agarose column to yield a gliadin-enriched mRNA fraction. Only slight differences were observed for the products synthesized in a wheat germ cell-free translation system when either poly adenylic acid enriched or cytosine-rich RNA was used as a template. These results are consistent with the high proline content of the gliadins and indicate that a large proportion of the mRNA activity in these RNA preparations is directed toward gliadin synthesis. After a second affinity chromatography step, the gliadin-enriched mRNA fraction was fractionated by two cycles on sucrose-density gradient centrifugation under denaturing conditions. The RNA sedimented as a broad band with a peak at 14S and a shoulder at the 11S region of the sucrose gradient. RNA from the peak 14S fraction translated predominantly the two major gliadin polypeptides which had molecular weights of 34,000 and 36,000. Analysis of the 14S RNA by methylmercury hydroxide-agarose gel electrophoresis revealed the presence of a predominant RNA species with a molecular size of 415,000 (1,200 nucleotides). PMID- 16662306 TI - Ethylene, Ethane, Acetaldehyde, and Ethanol Production By Plants under Stress. AB - Red pine (Pinus resinosa Ait.) and paper birch (Betula papyrifera Marsh.) seedlings exposed to sulfur dioxide produced acetaldehyde and ethanol, and exhibited increased production of ethylene and ethane. Gas chromatographic measurement of head space gas from incubation tubes containing leaves or seedlings was a simple method of simultaneously measuring all four compounds. Increased ethylene production had two phases, a moderate increase from the beginning of the stress period and a large increase just prior to appearance of leaf lesions. Ethane production in SO(2)-stressed plants did not increase until lesions appeared. Acetaldehyde and ethanol production began within 6 hours at 0.3 microliter per liter SO(2) and 24 hours at 0.1 microliter per liter SO(2) and continued throughout a 6-day fumigation. Production of acetaldehyde and ethanol continued when plants were removed to clean air for up to 2 days. A higher concentration of SO(2) (0.5 microliter per liter) induced acetaldehyde and ethanol production within 2 hours of the start of fumigation of birch and pine seedlings. A number of other stresses, including water deficit, freezing, and ozone exposure induced production of acetaldehyde and ethanol. Production of these compounds was not due to hypoxia, as the O(2) partial pressure in the incubation vessels did not decline. Increasing the O(2) partial pressure to 300 millimeters Hg did not affect production of these compounds. Production of ethylene, acetaldehyde, and ethanol declined when more than 80% of the leaf area became necrotic, while ethane production was linearly related to the percentage of necrosis. A number of woody and herbaceous plant species produced acetaldehyde and ethanol in response to freezing stress, while others did not. Measurement of these four compounds simultaneously in the gas phase may be a valuable method for monitoring plant stress, particularly air pollution stress. PMID- 16662307 TI - Glutamate synthase from rice leaves. AB - Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) from rice leaves (Oryza sativa L. cv Delta) was purified 206-fold with a final specific activity of 35.9 mumoles glutamate formed per min per milligram protein by a procedure including ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephacryl S-300 gel filtration, and ferredoxin-Sepharose affinity chromatography. The purified enzyme yielded a single protein band on polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was estimated to be 224,000 daltons by Sepharose 6B gel filtration. Electrophoresis of the dissociated enzyme in sodium dodecyl sulfate-polyacrylamide gel gave a single protein band which corresponds to the subunit molecular weight of 115,000 daltons. Thus, it is concluded that the glutamate synthase is composed of two polypeptidic chains exhibiting the same molecular weight. Spectrophotometric analysis indicated that the enzyme is free of iron-sulfide and flavin. The pH optimum was 7.3. The enzyme had a negative cooperativity (Hill number of 0.70) for glutamine, and its K(m) value increased from 270 to 570 mum at a glutamine concentration higher than 800 mum. K(m) values for alpha-ketoglutarate and ferredoxin were 330 and 5.5 mum, respectively. Asparagine and oxaloacetate could not be substituted for glutamine and alpha ketoglutarate, respectively. Enzyme activity was not detected with pyridine nucleotides as electron donors. Azaserine and several divalent cations were potent inhibitors. The purified enzyme was stabilized by dithiothreitol. PMID- 16662308 TI - Metabolism of Tryptophan, Indole-3-acetic Acid, and Related Compounds in Parasitic Plants from the Genus Orobanche. AB - Metabolic reactions involving the aliphatic side chain of tryptophan were studied in the holoparasitic dicotyledonous plants Orobanche gracilis Sm., O. lutea Baumg., and O. ramosa L. Unlike known autotrophic plants, the parasite metabolized l-tryptophan directly to indole-3-carboxaldehyde, which was further converted to indole-3-methanol and indole-3-carboxylic acid. Independently, these metabolites were also formed from d-tryptophan, tryptamine, indole-3-lactic acid, and indole-3-acetic acid. As in autotrophic plants, tryptophan and tryptamine were also converted, via indole-3-acetaldehyde, to indole-3-acetic acid, indole-3 ethanol, and its glucoside. The branch of tryptophan metabolism relevant to auxin biogenesis and catabolism is, therefore, not rudimentary in Orobanche but even more complex than in autotrophic higher plants. PMID- 16662309 TI - Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene. AB - The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function. PMID- 16662310 TI - Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE. AB - Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented. PMID- 16662311 TI - Seasonally Induced Changes in Acyl Lipids and Fatty Acids of Chloroplast Thylakoids of Pinus silvestris: A CORRELATION BETWEEN THE LEVEL OF UNSATURATION OF MONOGALACTOSYLDIGLYCERIDE AND THE RATE OF ELECTRON TRANSPORT. AB - Current-year needles were sampled regularly from an approximately 20-year-old natural stand of Pinus silvestris. Chloroplast thylakoids were isolated. The electron transport capacities of photosystem II + photosystem I, as well as of the partial photoreactions, were measured. The amounts and the fatty acid compositions of monogalactosyldiglyceride, digalactosyldiglyceride, and sulfolipid of the thylakoids were analyzed. The fatty acid composition of the phospholipids (total) was also determined.There was a 2-fold increase in the content of the galactolipids and the sulfolipid in the thylakoid preparations during the autumn. Except for monogalactosyldiglyceride that decreased in content during early winter, the content of the other two lipids remained high during the winter, until spring when their content fell toward a low summer level. The ratio of monogalactosyldiglyceride to digalactosyldiglyceride was lower in thylakoids isolated from frosthardened (autumn, winter) than in those isolated from unhardened (summer) needles.Only monogalactosyldiglyceride showed pronounced and significant seasonal variations in the molar ratio of unsaturated to saturated fatty acids. It was low in the winter, and it increased strongly during the spring. The highest ratio was observed in September, prior to the first night frosts, after which a steady decrease toward the low winter value occurred. This decrease is thought to be a reflection of membrane damages (photooxidation of membrane components, including polyunsaturated fatty acids), as earlier seen reflected in chlorophyll destruction, electron transport inhibition, and structural changes.There was very good correlation between the seasonal variations in the capacity for electron transport from water to NADP and the level of unsaturation of monogalactosyldiglyceride (r = 0.93). It has been shown earlier that the winter inhibition of electron transport preferentially occurs at the lipophilic electron transport carrier plastoquinone and that the free miscibility of plastoquinone and monogalactosyldiglyceride is prevented in saturated monogalactosyldiglyceride. Therefore, it is hypothesized that the decreased level of unsaturation of monogalactosyldiglyceride reduces the mobility of plastoquinone in the thylakoids, resulting in an inhibition of the electron transport by way of plastoquinone. PMID- 16662312 TI - Arginine and ornithine decarboxylases, the polyamine biosynthetic enzymes of mung bean seedlings. AB - General properties and relative activities of l-arginine decarboxylase (ADC) (EC 4.1.1.19) and l-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. alpha-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity.High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A comparison of several plant species indicates a wide range of ADC-to-ODC activity ratio.It is suggested that both ADC and ODC are active in plant tissues and that their relative contribution to putrescine biosynthesis is dependent upon the type of tissue and growth process. PMID- 16662313 TI - Nitrogen Isotope Fractionation Associated with Nitrate Reductase Activity and Uptake of NO(3) by Pearl Millet. AB - Nitrogen isotope fractionation by Pearl Millet (Pennisetum americanum L. and P. mollissimum L.) grown on nitrate was associated with nitrate reductase activity. Fractionation was evidenced at the step of nitrate reduction when the substrate to-enzyme ratio was high (possibly saturating for the active sites of the nitrate reductase enzyme), for instance in young seedlings having a low nitrate reductase activity or in seedlings grown on high nitrate concentration.When the substrate concentration was low (and, hence, the active sites of the enzyme were possibly not saturated), the isotopic discrimination could only be associated with the uptake of nitrate into the cell. In that case, isotopic fractionation was null. It is concluded that the uptake of nitrate does not discriminate among nitrogen isotopes. PMID- 16662314 TI - Polyribosomes from Pear Fruit: II. CHANGES OCCURRING IN PULP TISSUES DURING RIPENING AND SENESCENCE. AB - Detailed analyses of polysome profiles from lyophilized pulp tissues of pear fruits (Pyrus communis L. cv. Passe-Crassane) at different stages of ripening and senescence, and estimates of the amount of polysomal-associated mRNA, lead to the conclusions that during senescence (ripening), the ripening and the over-ripening processes can clearly be separated and respectively linked to the first and the second increase in the large mRNA species. Ethylene synthesis which occurs at the beginning of a normal ripening at 15 degrees C after a cold storage or an ethephon treatment is related to an increase in mRNA and ribosomal material found only in pulp tissues. Finally, we suggest that in the pear fruit, the sequence of events which occurs during senescence (ripening) is initiated by two systems which regulate ethylene biogenesis, and that the first system is efficient only at low temperatures (from 0 to 4 degrees C). PMID- 16662315 TI - Oligomycin effects on ATPase and photophosphorylation of pea chloroplast thylakoid membranes. AB - Oligomycin inhibited the membrane-bound, Ca(2+)-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg(2+)-dependent (light- and dithiothreitol (DTT) activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition.Sensitivity of isolated pea thylakoid Mg(2+)-dependent ATPase to oligomycin was found during summer months but not during the winter. This was traced to a seasonal variation in relative humidity with values of 20% or less during the winter, leading to lack of inhibition in the isolated chloroplasts. The effect of low relative humidity during growth could be reversed by placing plants for 17 to 20 hours in a chamber at 50% relative humidity. The optimum humidity range for sensitivity to oligomycin was between 40 and 60%.With chloroplasts potentially sensitive to oligomycin due to "permissive" growth conditions, the inhibition was modulated by a number of parameters during chloroplast activation or ATPase assay. Preincubation in high concentrations of KCl diminished oligomycin sensitivity, and this effect was reversed by valinomycin. The greatest sensitivity, as well as the highest ATPase rates, occurred only if thylakoids were activated by light and DTT at a relatively high temperature (35 to 40 degrees C). Controlling the levels of uncouplers and running the reaction in darkness rather than in the light, both of which diminished the degree of membrane energization, increased the sensitivity to oligomycin. It is possible that accessibility of the oligomycin binding site is diminished by membrane energization. These results are contrasted with those from studies of variability in sensitivity of yeast mitochondria to oligomycin. PMID- 16662316 TI - Origin of acetate in spinach leaf cell. AB - Mitochondria were isolated from spinach (Spinacia oleracea L.) leaves using a Percoll gradient step. The high purity of the organelle fraction is demonstrated by electron microscopy and biochemical parameters. In the matrix space of these mitochondria, a short-chain acyl-coenzyme A hydrolase is present that converts acetyl-coenzyme A to acetate and coenzyme A with reasonable rates (K(m), 150 micromolar; V(max), 140 nanomoles acetate formed milligram(1) protein hour(-1)). The enzyme is product inhibited by coenzyme A-sulfhydryl, other thiols are ineffective; however, the disulfides 5,5'-dithio-bis-(2-nitrobenzoate) and cystamine stimulate the hydrolysis. The possible role of this mitochondrial enzyme as a means of generating free acetate from pyruvate via acetyl-coenzyme A in the mitochondria of mature spinach leaves is discussed. It is suggested that free acetate moves rapidly from the mitochondrion to the chloroplast where acetyl coenzyme A synthetase, solely localized in this organelle, converts the metabolically inert free acetate to the highly active acetyl-coenzyme A. PMID- 16662317 TI - Genome Expression during Normal Leaf Development : I. CELLULAR AND CHLOROPLAST NUMBERS AND DNA, RNA, AND PROTEIN LEVELS IN TISSUES OF DIFFERENT AGES WITHIN A SEVEN-DAY-OLD WHEAT LEAF. AB - Changes in genome expression during normal cellular and plastid development in the first leaf of young (7-day-old) wheat (Triticum aestivum var. Maris Dove) were investigated by examining homogeneous populations of leaf cells and plastids of several developmental ages present in the same leaf. The cells were characterized over a period immediately following the last cell division. All of the leaf cells had cytoplasmic contents and nuclei, and between 44% (young tissue) and 54% (older tissue) of the leaf cells were mesophyll cells. Chloroplast development was complete 36 hours after the chloroplasts had ceased dividing. Extremely large changes occurred in cellular constituents over a very short period of leaf development. Maximum rates of accumulation of ribulose bisphosphate carboxylase per mesophyll cell (80 picograms/hour), chlorophyll per mesophyll cell (9 picograms/hour), and 70S ribosomes per mesophyll cell (19 x 10(5)/hour) were recorded.Total cellular DNA varied from 40 to 60 picograms/cell, reflecting the changes in nuclear and chloroplast DNA synthesis during different phases of cellular and chloroplast division. The period of maximum accumulation of protein, total RNA, and both 80S and 70S ribosomes occurred between 36 and 48 hours after the last cell division. Between 48 and 60 hours, 70S rRNA per cell and protein content per cell continued to increase as 80S rRNA per cell declined. Ribulose bisphosphate carboxylase per cell increased 20-fold between 15 and 60 hours. PMID- 16662318 TI - Photosynthesis and Respiration of Developing Populus tremuloides Internodes. AB - The photosynthetic and respiratory performance of developing internodes of Populus tremuloides was evaluated by infrared gas analysis. Anatomical and morphological transitions were related to metabolic activity. Photosynthetic rates ranged from 6.0 to 10.0 milligrams CO(2) per decimeter squared per hour in the youngest internodes to 2.5 to 3.8 milligrams CO(2) per decimeter squared per hour in internodes with fully developed bark tissues. Respiration exceeded the rate of photosynthesis on the average by a factor of two. Stem photosynthesis increased with temperature up to 40 degrees C and declined steeply between 40 and 50 degrees C. Stem respiration increased nearly linearly to temperatures as high as 50 degrees C. PMID- 16662319 TI - Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts. AB - Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O(2) per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH(3), which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH(3) reassimilation occurs outside the mitochondria. PMID- 16662320 TI - Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : I. FACTORS AFFECTING NET CO(2) UPTAKE IN INTACT LEAVES AND CONTRIBUTION FROM RIBULOSE-1,5 BISPHOSPHATE CARBOXYLASE MEASURED IN VIVO AND IN VITRO. AB - As part of an extensive analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, we have examined the response of leaf gas exchange and ribulose-1,5-bisphosphate (RuBP) carboxylase activity to temperature. Emphasis was placed on elucidating the specific processes which regulate the temperature response pattern. The inhibitory effects of above optimal temperatures on net CO(2) uptake were fully reversible up to 40 degrees C. Below 40 degrees C, temperature inhibition was primarily due to O(2) inhibition of photosynthesis, which reached a maximum of 65% at 45 degrees C. The response of stomatal conductance to temperature did not appear to have a significant role in determining the overall temperature response of photosynthesis. The intracellular conductance to CO(2) increased over the entire experimental temperature range, having a Q(10) of 1.2 to 1.4. Increases in the apparent Michaelis constant (K(c)) for RuBP carboxylase were observed in both in vitro and in vivo assays. The Q(10) values for the maximum velocity (V(max)) of CO(2) fixation by RuBP carboxylase in vivo was lower (1.3-1.6) than those calculated from in vitro assays (1.8-2.2). The results suggest that temperature dependent changes in enzyme capacity may have a role in above-optimum temperature limitations below 40 degrees C. At leaf temperatures above 40 degrees C, decreases in photosynthetic capacity were partially dependent on temperature induced irreversible reductions in the quantum yield for CO(2) uptake. PMID- 16662321 TI - Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : II. CONTRIBUTION FROM ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION. AB - As part of an analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, the response of electron transport and photophosphorylation to temperature in isolated chloroplast thylakoids has been examined. The response of the light reactions to temperature was found to depend strongly on the preincubation time especially at temperatures above 35 degrees C. Using methyl viologen as a noncyclic electron acceptor, coupled electron transport was found to be stable to 38 degrees C; however, uncoupled electron transport was inhibited above 38 degrees C. Photophosphorylation became unstable at lower temperatures, becoming progressively inhibited from 35 to 42 degrees C. The coupling ratio, ATP/2e(-), decreased continuously with temperature above 35 degrees C. Likewise, photosystem I electron transport was stable up to 48 degrees C, while cyclic photophosphorylation became inhibited above 35 degrees C. Net proton uptake was found to decrease with temperatures above 35 degrees C supporting the hypothesis that high temperature produces thermal uncoupling in these chloroplast thylakoids. Previously determined limitations of net photosynthesis in whole leaves in the temperature region from 35 to 40 degrees C may be due to thermal uncoupling that limits ATP and/or changes the stromal environment required for photosynthetic carbon reduction. Previously determined limitations to photosynthesis in whole leaves above 40 degrees C correlate with inhibition of photosynthetic electron transport at photosystem II along with the cessation of photophosphorylation. PMID- 16662322 TI - Effects of G, a Growth Regulator from Eucalyptus grandis, on Photosynthesis. AB - A growth regulator (G; 4-ethyl-1-hydroxy-4,8,8,10,10 pentamethyl-7,9-dioxo-2,3 dioxyabicyclo (4.4.0) decene-5) from Eucalyptus grandis (Maiden) reduced stomatal conductance and also photosynthetic capacity when fed through the transpiration stream of detached leaves. The concentration of G required for this effect was high (10(-4) molar), but the amount of G taken up (dose) was below the level which has previously been found in E. grandis leaves. Similar effects were observed in detached leaves of Xanthium strumarium L. though almost 10 times more G was required. G reduced CO(2)-dependent O(2) evolution from isolated cells of X. strumarium. In spinach (Spinacia oleracea L.) chloroplasts, electron transport through photosystem II was reduced by G. It is proposed that G affects stomatal conductance and photosynthesis by reducing photosystem II activity in both the guard cell chloroplasts and mesophyll cell chloroplasts. PMID- 16662323 TI - Regulation of H Excretion : EFFECTS OF OSMOTIC SHOCK. AB - Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H(+) excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H(+) uptake, but osmoregulation can still occur, and enhanced H(+) uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H(+) rather than affecting only H(+) or counter ion uptake.Plasmolysis alone does not inhibit fusicoccin-enhanced H(+) excretion, and the rehydration step must be rapid and in the cold for maximum inhibition. This suggests that the plasma membrane is perturbed, possibly due to release or rearrangement of membrane protein. Compared to corresponding osmolarities of sorbitol or NaCl, polyethylene glycol 4000 is much less effective during the plasmolysis step; the possibility is discussed that the ineffectiveness of polyethylene glycol 4000 is due to its preservation of plasmodesmata. PMID- 16662324 TI - Regulation of H Excretion : ROLE OF PROTEIN RELEASED BY OSMOTIC SHOCK. AB - When the protoplasts of peeled oat leaf segments (Avena sativa L.) expand after a brief plasmolysis (osmotic shock), fusicoccin-enhanced H(+) excretion is reduced and protein is released to the rehydration medium. This shock protein seems to arise from the cell surface, not from the interior of leaky cells or from broken cells, because (a) the protein differs quantitatively and qualitatively from protein of cell homogenates as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis; (b) peroxidase, phosphatase, and malate dehydrogenase activities, which are associated with the cell surface, are detected in the shock fluids; (c) the specific activities of enzymes in shock fluids are different than those of cell homogenates; (d) the amount of protein released is correlated with tissue mass, not number of cut surfaces and is not diminished by pre-washing the tissue.Some of the shock protein may arise from plasmodesmata; this suggestion is based on (a) the cell surface origin of the protein; (b) the presence in the shock fluid of NADPH-cytochrome c reductase activity, usually associated with the endoplasmic reticulum which traverses plasmodesmata; (c) on the release of smaller amounts of protein after plasmolysis with polyethylene glycol 4000, an osmoticum which may tend to preserve plasmodesmata.The amount of protein released by osmotic shock is correlated with the extent of inhibition of fusicoccin-enhanced H(+) excretion. A specific function for the shock protein is implied by the presence of a component which specifically binds fusicoccin. PMID- 16662325 TI - Estimation of Free, Conjugated, and Diffusible Indole-3-acetic Acid in Etiolated Maize Shoots by the Indolo-alpha-pyrone Fluorescence Method. AB - Procedures for estimating free indoleacetic acid (IAA extracted from tissue homogenates by aqueous acetone), conjugated IAA (extracted by aqueous acetone and hydrolyzed by 1 n KOH), and diffusible IAA (diffused from the excised tissue into water), in shoots of etiolated 3-day-old maize (Zea mays L. cv. GH 390) seedlings are described, the indolo-alpha-pyrone fluorescence method being used to assay IAA. The reliability of the procedure is shown by comparative IAA determinations of the extracts using the gas chromatography-mass spectrometry method in which the methyl ester, heptafluorobutyryl derivative of IAA is assayed using the selected-ion-monitoring technique with deuterated IAA as an internal standard. A 3-millimeter-long coleoptile tip, a coleoptile with its included leaves and nodal region (whole coleoptile), and a mesocotyl each contains 0.2, 1.7, and 1.5 nanograms of free IAA, respectively. The whole coleoptile and the mesocotyl contain slightly less conjugated IAA than their content of free IAA. IAA diffuses from the coleoptile tip at the rate of 1.0 nanograms per tip per hour; from the base of the whole coleoptile and a set of leaves excised from a coleoptile, IAA diffuses at the rate of 0.62 and 0.17 nanogram per plant part per hour, respectively. The data obtained support the classical assumption that the coleoptile tip produces IAA. It is also suggested that some IAA is decomposed during its downward transport in the coleoptile. PMID- 16662326 TI - Water Relations of Seagrasses: STATIONARY VOLUMETRIC ELASTIC MODULUS AND OSMOTIC PRESSURE OF THE LEAF CELLS OF HALOPHILA OVALIS, ZOSTERA CAPRICORNI, AND POSIDONIA AUSTRALIS. AB - The stationary volumetric elastic modulus (epsilon(s)) of the leaf cells of three seagrasses (Halophila ovalis (R.Br.) Hook, Zostera capricorni Aschers, and Posidonia australis Hook f.) was evaluated from estimates of epsilon(s) plus intracellular osmotic pressure (epsilon(s) + II(i)) and II(i). The estimates of (epsilon(s) + II(i)) were made using a linear displacement transducer to measure very small changes in thickness of leaf tissue produced by changes in external osmotic pressure (II(o)). epsilon(s) increases with increasing turgor pressure in each of the species and the maximum values of epsilon(s) are: 22 megapascals for H. ovalis, 17 megapascals for Z. capricorni, and 51 megapascals for P. australis.There is a hysteresis in thickness changes versus changes in II(o) which indicates a hysteresis in the relationship between volume and turgor pressure. The hysteresis results in epsilon(s) being different for swelling and for shrinking cells over the same range of II(o) and this may be important in other aspects of plant-water relations.A new design of an apparatus employing a linear displacement transducer for measuring very small changes in tissue thickness is described. The new design has the advantages of virtually frictionless movement and a precision of 0.05 micrometer. PMID- 16662327 TI - Effect of salinity on tomato fruit ripening. AB - Tomato (Lycopersicon esculentum Mill) plants from various cultivars growing on half-strength Hoagland solution were exposed at anthesis to 3 or 6 grams per liter NaCl. Salinity shortened the time of fruit development by 4 to 15%. Fruits of salt-treated plants were smaller and tasted better than did fruits of control plants. This result was obtained both for ripe fruits tested on the day of picking and for those picked at 100% development and allowed to ripen at room temperature for 9 days. Percentage of dry weight, total soluble solids, and titratable acidity; content of reducing sugars, Cl(-), Na(+), and various pericarp pigments; and electrical conductivity of the juice were higher in fruits of saline-treated plants than they were in those of control plants, while the pH was lower. Ethylene and CO(2) evolution rates during ripening; as well as the activities of pectin methyl esterase, polymethylgalacturonase, and polygalacturonase; were also higher in fruits of the saline-treated plants. The treatment with 6 grams per liter NaCl shortened the fruit shelf life considerably. PMID- 16662328 TI - Why is the substomatal chamber as large as it is? AB - The rate of CO(2) uptake by the mesophyll is examined as a function of the size of the substomatal chamber. Using the techniques of classical electric circuit analysis and a model in which the uptake is linear in the ambient CO(2) concentration, it is shown that the optimal chamber radius is several times larger than is the pore radius. This is somewhat larger than necessary for the reduction of transpirational water loss, and it offers an explanation for the otherwise inexplicably large size of the chamber. PMID- 16662329 TI - Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves. AB - The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment. All of the enzymes involved in these reactions are soluble when prepared from whole-leaf homogenates. Mechanical separation of epidermal fragments from the mesophyll, followed by preparation of the soluble enzyme fraction from each tissue, revealed that the neomenthol dehydrogenase and the glucosyl transferase resided specifically in the mesophyll layer, whereas the menthol dehydrogenase and substantial amounts of the acetyl transferase were located in the epidermis, presumably within the epidermal oil glands. These results suggest that the compartmentation of menthone metabolism in peppermint leaves is intercellular, not intracellular. PMID- 16662330 TI - Involvement of a Primary Electrogenic Pump in the Mechanism for HCO(3) Uptake by the Cyanobacterium Anabaena variabilis. AB - The response of the membrane potential to HCO(3) (-) supply has been studied in the cyanobacterium Anabaena variabilis strain M-3 under various conditions. Changes in potential were followed with the aid of the lipophilic cation tetraphenyl phosphonium bromide.Addition of HCO(3) (-) to CO(2)-depleted cells resulted in rapid hyperpolarization. The rate and extent of hyperpolarization were greater in low-CO(2)-adapted than in high-CO(2)-adapted cells. Addition of the electron acceptor p-nitrosodimethylaniline which resulted in O(2) evolution in CO(2)-depleted cells did not cause hyperpolarization. The hyperpolarization was not attributable to a change in pH or in ionic strength of the medium. Pretreatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented the hyperpolarization. KCN depolarized hyperpolarized cells. Addition of HCO(3) (-) also brought about immediate K(+) influx which was succeeded after about 2 minutes by K(+) efflux.TWO OF THE MODELS CONSIDERED WOULD BE CAPABLE OF EXPLAINING THESE AND PREVIOUS FINDINGS: (a) a primary electrogenic pump for transporting HCO(3) (-) ions; (b) proton-HCO(3) (-) contransport, the driving force for which is generated by a proton pump which is sensitive to the HCO(3) ( ) concentration. PMID- 16662331 TI - Glutamine Synthetase in Spinach Leaves : IMMUNOLOGICAL STUDIES AND IMMUNOCYTOCHEMICAL LOCALIZATION. AB - By polyacrylamide gel electrophoresis, DEAE Sephacel, and hydroxyapatite chromatography, one form of glutamine synthetase has been identified in spinach (Spinacia oleracea L. cv. Monstrueux de Viroflay) leaves. It is localized only inside the chloroplast. The enzyme was purified to homogeneity and specific antibodies against the protein were raised by immunization of rabbits. The intracellular localization of glutamine synthetase in spinach leaves was studied by indirect immunofluorescence microscopy on thin-sectioned spinach leaves. It has been demonstrated that the enzyme is specifically associated with the chloroplasts of parenchymatous cells. PMID- 16662332 TI - Lysine-ketoglutarate reductase activity in developing maize endosperm. AB - Lysine-ketoglutarate reductase activity was detected and characterized in the developing endosperm of maize (Zea mays L.). The enzyme showed specificity for its substrates: lysine, alpha-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was close to 7, and the K(m) for lysine and alpha-ketoglutarate were 5.2 and 1.8 millimolar, respectively. PMID- 16662333 TI - Cadmium-binding components in soybean plants. AB - Soybean (Glycine max L.) plants exposed to (109)Cd readily absorb the element. Differential centrifugation of leaf, stem, and root homogenates followed by radioassay showed that Cd was associated primarily with the 105,000g supernatant. Separation of this fraction by gel chromatography and subsequent analysis by radioassays revealed that (109)Cd was bound to macromolecules of >50,000, 13,800, and 2,280 molecular weights. The >50,000 and 2,280 molecular weight fractions probably are nonspecific binding of Cd to normal cell components. The 13,800 molecular weight (109)Cd-bound component was found to be inducible by cadmium. It had a high ultraviolet absorbance at 254 nm and a low absorbance at 280 nm at pH 8.6. PMID- 16662334 TI - Induction of HCO(3) Transporting Capability and High Photosynthetic Affinity to Inorganic Carbon by Low Concentration of CO(2) in Anabaena variabilis. AB - The apparent affinity of photosynthesis for inorganic carbon in Anabaena variabilis strain M-3 increased during the course of adaptation from high to low CO(2) concentration (5% and 0.03% v/v CO(2) in air, respectively). This was attributed to an increased ability of the cells to accumulate inorganic carbon during the course of adaptation to low CO(2) conditions. The release of phycobiliproteins was used to evaluate the sensitivity of the cells to lysozyme treatment followed by osmotic shock. High CO(2)-grown cells were more sensitive to this treatment than were low CO(2) ones. The efflux of inorganic carbon from cells preloaded with radioactive bicarbonate is faster in high than it is in low CO(2)-adapted cells. It is postulated that the cell wall or membrane components undergo changes during the course of adaptation to low CO(2) conditions. This is supported by electron micrographs showing differences in the cell wall appearance between high and low CO(2)-grown cells. The increasing ability to accumulate HCO(3) (-) and the lessened sensitivity to lysozyme during adaptation to low CO(2) conditions depends on protein synthesis. The increase in affinity for inorganic carbon during the adaptation to low CO(2) conditions is severely inhibited by the presence of spectinomycin. Incubation in the light significantly lessens the time required for the adaptation to low CO(2) conditions. PMID- 16662335 TI - Metabolism of Monoterpenes: Conversion of l-Menthone to l-Menthol and d Neomenthol by Stereospecific Dehydrogenases from Peppermint (Mentha piperita) Leaves. AB - The monoterpene ketone l-menthone is specifically converted to l-menthol and l menthyl acetate and to d-neomenthol and d-neomenthyl-beta-d-glucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham) leaves. The selectivity of product formation results from compartmentation of the menthol dehydrogenase with the acetyl transferase and that of the neomenthol dehydrogenase with the glucosyl transferase. Soluble enzyme preparations, but not particulate preparations, from mature peppermint leaves catalyzed the NADPH-dependent reduction of l-menthone to both epimeric alcohols, and the two dehydrogenases responsible for these stereospecific transformations were resolved by affinity chromatography on Matrex Gel Red A. Both enzymes have a molecular weight of approximately 35,000, possess a K(m) for NADPH of about 2 x 10(-5)m, are very sensitive to inhibition by thiol directed reagents, and are not readily reversible. The menthol dehydrogenase showed a pH optimum at 7.5, exhibited a K(m) for l-menthone of about 2.5 x 10( 4)m, and also reduced d-isomenthone to d-neoisomenthol. The neomenthol dehydrogenase showed a pH optimum at 7.6, exhibited a K(m) for l-menthone of about 2.2 x 10(-5)m, and also reduced d-isomenthone to d-isomenthol. These stereochemically distinct, but otherwise similar, enzymes are of key importance in determining the metabolic fate of menthone in peppermint, and they are probably typical of the class of dehydrogenases thought to be responsible for the metabolism of monoterpene ketones during plant development. PMID- 16662336 TI - Leaf conductance as a function of photosynthetic photon flux density and absolute humidity difference from leaf to air. AB - FOR AN ENTIRE SEASON OF STOMATAL ACTIVITY, LEAF OR NEEDLE CONDUCTANCE WAS OBSERVED ON FOUR SPECIES, EACH IN A DIFFERENT GENUS: Engelmann spruce (Picea engelmannii Parry ex Engelm.), subalpine fir (Abies lasiocarpa [Hook.] Nutt.), lodgepole pine (Pinus contorta var. latifolia Engelm.), and aspen (Populus tremuloides Michx.). Conductance in the natural environment was described for all species by photosynthetic photon flux density (PPFD) and absolute humidity difference from leaf to air (DAH), as follows: Conductance = b(1) ( radicalPPFD/ radicalDAH) + b(2) ( radicalPPFD/DAH) + b(3) ( radicalPPFD/DAH(2)). The only data not fitting this relationship were conifer data collected after freezing nights or aspen data collected during a short period in August when water stress occurred. In both cases, leaf conductance was reduced. It is proposed that PPFD and DAH are primary factors controlling stomatal function for plants growing in their native range; secondary factors, such as temperature and water stress, affect conductance intermittently, except when plants are growing outside their normal environmental conditions. PMID- 16662337 TI - Evaluation of season, temperature, and water stress effects on stomata using a leaf conductance model. AB - A model was developed earlier describing conductance for three conifers (Picea engelmannii Parry ex Engelm., Abies lasiocarpa [Hook.] Nutt., and Pinus contorta var. latifolia Engelm.) and one hardwood (Populus tremuloides Michx.) using only two terms, photosynthetic photon flux density (PPFD) and absolute humidity difference from leaf to air (DAH). Using residual analysis techniques (actual minus estimated conductance), it was determined that no seasonal or temperature effects existed that were not taken into account with PPFD and DAH. However, conductance was reduced on days following cold nights (below 4 degrees C) or, in aspen, when xylem pressure potential was below -20 bars (1 bar = 10(5) Pa). The following model takes these terms into account: Conductance = b(1) ( radicalPPFD/ radicalDAH) + b(2) ( radicalPPFD/DAH) + b(3) ( radicalPPFD/DAH(2)) + b(4)f(T(min)) + b(5)f(psi(threshold)), where the first three terms describe normal conductance, and the last two terms account for reductions in conductance caused by cold night temperatures or water stress. PMID- 16662338 TI - Kinetic characterization of spinach leaf sucrose-phosphate synthase. AB - The spinach (Spinacia oleracea) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturation curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At subsaturating concentrations of fructose-6 phosphate, 1,5 anhydroglucitol-6-phosphate had a stimulatory effect on enzyme activity, suggesting multiple and interacting fructose-6-phosphate sites on sucrose-phosphate synthase. The concentrations required for 50% of maximal activity were 3.0 millimolar and 1.3 millimolar, respectively, for fructose-6 phosphate and UDPglucose. The enzyme was not stimulated by divalent cations. Inorganic phosphate proved to be a potent inhibitor, particularly at low concentrations of substrate. Phosphate inhibition was competitive with UDPglucose, and its K(i) was determined to be 1.75 millimolar. Sucrose phosphate, the product of the reaction, was also shown to be a competitive inhibitor towards UDPglucose concentration and had K(i) of 0.4 millimolar. The kinetic results suggest that spinach leaf sucrose-phospahte synthase is a regulatory enzyme and that its activity is modulated by the concentrations of phosphate, fructose-6 phosphate, and UDPglucose occurring in the cytoplasm of the leaf cell. PMID- 16662339 TI - Potentiating effect of pure oxygen on the enhancement of respiration by ethylene in plant storage organs: a comparative study. AB - A number of fruits and bulky storage organs were studied with respect to the effect of pure O(2) on the extent and time-course of the respiratory rise induced by ethylene. In one group, of which potato (Solanum tuberosum var. Russet) and carrot (Daucus carota) are examples, the response to ethylene in O(2) is much greater than in air. In a second group, of which avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii Lambert var. Valery) are examples, air and O(2) are equally effective. When O(2)-responsive organs are peeled, air and O(2) synergize the ethylene response to the same extent in parsnip (Pastinaca sativa), whereas O(2) is more stimulatory than air in carrots. In the latter instance, carrot flesh is considered to contribute significantly to diffusion resistance. The release of CO(2), an ethylene antagonist, is recognized as another element in the response to peeling.The potentiating effect of O(2) is considered to be primarily on ethylene action in the development of the respiratory rise rather than on the respiration process per se. On the assumption that diffusion controls O(2) movement into bulky organs and the peel represents the major diffusion barrier, simple calculations indicate that the O(2) concentration in untreated organs in air readily sustains respiration. Furthermore, in ethylene-treated organs in pure O(2), the internal O(2) concentration is more than enough to maintain the high respiration rates. Skin conductivity to O(2) is the fundamental parameter differentiating O(2)-responsive from O(2)-nonresponsive fruits and bulky storage organs. The large preceding the earliest response to ethylene, as well as the magnitude of the ethylene-induced respiratory rise, is also controlled by permeability characteristics of the peel. PMID- 16662340 TI - Selective enhancement of alternative path capacity in plant storage organs in response to ethylene plus oxygen: a comparative study. AB - The respiration rise in bulky storage organs induced by ethylene plus pure O(2) is accompanied by an increase in the CN-resistant respiration, or alternative path. Whereas a lesser respiratory stimulation in response to ethylene is demonstrable in air and increased by peeling, ethylene-induced alternative path development depends on the synergistic effect of pure O(2), with or without peeling. The effect of ethylene plus O(2) is evident, whether untreated parent organs yield CN-sensitive or CN-resistant fresh slices. Alternative path capacity and maximal cytochrome oxidase-mediated electron transport have been separately estimated. Ethylene plus O(2) selectively enhances the alternative path. It is proposed that the gross rise in respiration evoked by ethylene is implemented by a system with an O(2) requirement much higher than that of cytochrome oxidase, while the ethylene-induced development of the alternative path depends on a system of still higher O(2) requirement. PMID- 16662341 TI - Inhibition of Photosynthetic Energy Conversion by Cupric Ion : EVIDENCE FOR Cu COUPLING FACTOR 1 INTERACTION. AB - This study describes a specific Cu(2+) and light-dependent inhibition of spinach (Spinacia oleracea L.) chloroplast reactions involving coupling factor 1 function. A primary effect is an inhibition of photophosphorylation induced by illumination of Class II chloroplasts with micromolar Cu(2+) and pyocyanine in the absence of ADP, Mg(2+), and HPO(4) (2-). The inhibition, which is dependent on free Cu(2+) as indicated by protection by ethylene diamine tetraacetic acid and dithiothreitol, requires illumination (electron flow) for establishment of the specific inhibition to be noted. Protection is also afforded by uncouplers and some partial protection is provided by micromolar concentrations of ADP and ATP. The data strongly suggest that Cu(2+) causes an O(2)-independent oxidation of sulfhydryl groups on coupling factor 1, which are essential to catalytic function. This conclusion is supported by the reduction of energy-dependent (3)H N-ethylmaleimide labeling of the gamma subunit of coupling factor 1 by the Cu(2+) light pretreatment. PMID- 16662342 TI - Host-Pathogen Interactions : XX. BIOLOGICAL VARIATION IN THE PROTECTION OF SOYBEANS FROM INFECTION BY PHYTOPHTHORA MEGASPERMA F. SP. GLYCINEA. AB - Phytophthora megasperma f.sp. glycinea, which causes soybean (Glycine max) root and stem rot, exists as several races which differ in their ability to infect a range of soybean cultivars. A glycoprotein-rich fraction (Fraction I) isolated from fungal culture fluid protects soybean seedlings from infection with compatible races. In an early study (13), seedlings were protected only by Fraction I purified from incompatible races. In 1979, seedlings were better protected by Fraction I isolated from incompatible races than by Fraction I isolated from compatible races. In 1980, seedlings were protected equally well by Fraction I from incompatible and compatible races. Materials similar in composition to Fraction I did not protect seedlings from infection. No cause could be identified for the apparent change, during the 3-year period, in the race specificity of the protection assay. Variability in the bioassay prohibited further purification or characterization of Fraction I components that protect seedlings from infection. PMID- 16662343 TI - Leaf water potential, stomatal resistance, and photosynthetic response to water stress in peach seedlings. AB - Individual groups of peach (Prunus persica [L.] Batsch) seedlings stressed to 17, -26 and -36 bars recovered to control levels within 1, 3, and 4 days, respectively. Stomatal resistance was significantly correlated with both leaf water potential and net photosynthesis. In seedlings stressed to -52 bars, leaf water potential and stomatal resistance recovered sooner than net photosynthesis, despite recovery of 0(2) evolution at a rate similar to leaf water potential. Therefore, some nonstomatal factor other than reduction in photochemical activity must be responsible for the lag in recovery of CO(2) assimilation following irrigation. PMID- 16662344 TI - Incorporation of [C]Phenylalanine into Flavan-3-ols and Procyanidins in Cell Suspension Cultures of Douglas Fir. AB - L-[(14)C]Phenylalanine, fed to cell suspension cultures of Douglas fir, (Pseudotsuga menziesii Franco) was incorporated simultaneously, but at different rates, into (+)-catechin, (-)-epicatechin, and procyanidins of increasing molecular weight. Asymmetric labeling of dimers and polymers was demonstrated, with more label appearing in the upper than in the lower or terminal unit. In addition, the total pool of free monomers was 10 to 30 times more highly labeled than was this lower, terminal unit of dimers and higher oligomers. Since the dimer, epicatechin-catechin, contained more label than catechin-catechin, it is concluded that the carbocation with the 2,3-cis stereochemistry of (-) epicatechin was formed more rapidly than was that of the 2,3-trans type of (+) catechin. PMID- 16662345 TI - Wound-Induced RNase Activity in Sweet Potato : EVIDENCE FOR REGULATION AT TRANSCRIPTION. AB - Upon wounding of sweet potato (Ipomea batatas, Lam. var. Puerto Rico) RNase activity increases rapidly following a 4-hour lag, peaks in 24 hours, and then declines. Cycloheximide inhibits induction indicating that increased activity is probably due to de novo synthesis. The half-time (t(0.5)) for RNase degradation in presence of cycloheximide (1.8 hours) is constant throughout the rise and decline in RNase activity. Induction is not affected by exogenous ethylene, but is dependent on production of endogenous ethylene. The following evidence is consistent with the hypothesis that the activity of RNase is regulated at transcription. (a) Wound induction of RNase is inhibited by actinomycin D (ACTD), cordycepin, or alpha-amanitin. (b) Addition of ACTD at 9, 12, or 24 hours causes an immediate decline in RNase. (c) Six measurements of the natural decline in RNase between 24 and 36 hours show a t(0.5) of 14.1 +/- 1 hour. (d) Use of proecdures for measurement of the t(0.5) for degradation of mRNA in bacteria and plants show a mean t(0.5) of 14 +/- 1 hour for degradation of RNase mRNA in presence of ACTD. From the fact that both the degradation of RNase in presence of ACTD and the natural decline in RNase have a t(0.5) that is very similar to the t(0.5) for degradation of RNase mRNA, it is postulated that the natural decline in sweet potato RNase is due to repression of the RNase gene as a result of which the rate of degradation of RNase follows the t(0.5) for degradation of RNase messenger. PMID- 16662346 TI - l-Canavanine Metabolism in Jack Bean, Canavalia ensiformis (L.) DC. (Leguminosae). AB - l-Canavanine, a highly toxic arginine antimetabolite, is the principal nonprotein amino acid of many leguminous plants. Labeled-precursor feeding studies, conducted primarily with [(14)C]carbamoyl phosphate, and utilization of the seedlings of jack bean, Canavalia ensiformis (L.) DC. (Leguminosae), have provided evidence for l-canavanine biosynthesis from l-canaline via O-ureido-l homoserine. This reaction pathway appears to constitute an important in vivo route of canavanine production. Canavanine cleavage to canaline may represent a degradative phase of canavanine metabolism distinct from the anabolic reactions described above. Thus, while these reactions of canavanine metabolism bear analogy to the mammalian Krebs-Henseleit ornithine-urea cycle, no evidence has been obtained at present for the reutilization of canaline in ureidohomoserine formation. PMID- 16662347 TI - Methionine biosynthesis in lemna: inhibitor studies. AB - A search was made for compounds that would inhibit methionine biosynthesis in Lemna paucicostata Hegelm. 6746. dl-Propargylglycine (0.15 micromolar) produced growth inhibition and morphological changes which were prevented by exogenous methionine. Also, dl-propargylglycine inhibits cystathionine gamma-synthase activity. l-Aminoethoxyvinylglycine (0.05 micromolar) produced growth inhibition and morphological changes partially preventable by exogenous methionine. l Aminoethoxyvinylglycine impairs the cleavage of cystathionine to homocysteine. Lysine and threonine, at concentrations which individually had little effect on growth or morphology of Lemna, together produced growth inhibition and morphological changes preventable by exogenous methionine. The resulting metabolic block prevented conversion of cysteine to cystathionine, presumably secondary to depletion of the supply of O-phosphohomoserine.Inhibition of Lemna growth resulted when the molybdate:sulfate ratio in the medium was increased to 20:1 or more. Such inhibition was prevented by lowering this ratio to 0.3 or less. A non-steady-state experiment (molybdate:sulfate, 20:1) showed that molybdate inhibited sulfate uptake, but it provided no evidence of a further impairment in the organification of sulfate. Molybdate-induced growth inhibition of Lemna was prevented by cystine but not by cystathionine or methionine. Cystathionine is not converted by Lemna to cysteine rapidly enough to sustain growth. PMID- 16662348 TI - Methionine Biosynthesis in Lemna: STUDIES ON THE REGULATION OF CYSTATHIONINE gamma-SYNTHASE, O-PHOSPHOHOMOSERINE SULFHYDRYLASE, AND O-ACETYLSERINE SULFHYDRYLASE. AB - Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine gamma synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine gamma-synthase, it was observed that (a) adding external methionine (2 mum) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 muml-aminoethoxyvinylglycine or with 36 mum lysine plus 4 mum threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine gamma-synthase activity, whereas O acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O phosphohomoserine sulfhydrylase.The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine gamma-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine gamma-synthase are probably activities of one enzyme that has low specificity for its sulfur containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of O-acetylserine sulfhydrylase provides an explanation in molecular terms for transsulfuration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis. PMID- 16662349 TI - Studies on the reconstitution of o(2)-evolution of chloroplasts. AB - Extraction of spinach (Spinacia oleracea L.) chloroplasts with cholate-asolectin in the absence of Mg(2+) results in the rapid and selective inactivation of O(2) evolution and a partial (30 to 40%) loss of photosystem II (PSII) donor activity without extraction of thylakoid bound Mn ( approximately 5 to 6 Mn per 400 Chlorophyll). Inclusion of ethylene glycol in the extractions inhibits loss of O(2) evolution and results in quantitative and qualitative differences in proteins solubilized but does not significantly inhibit the partial loss of PSII donor activity. Similarly, in two stage experiments (extraction followed by addition of organic solvent and solubilized thylakoid protein), O(2) evolution (V and V(max)) of extracted chloroplasts is enhanced approximately 2.5- to 8-fold. However, PSII donor activity remains unaffected. This reversal of cholate inactivation of O(2) evolution can be induced by solvents including ethanol, methanol, 2-propanol, and dimethyl sulfoxide. Such enhancements of O(2) evolution specifically required cholate-solubilized proteins, which are insensitive to NH(2)OH and are only moderately heat-labile. NH(2)OH extraction of chloroplasts prior to cholate-asolectin extraction abolishes reconstitutability of O(2) evolution. Thus, the protein(s) affecting reconstitution is unlike those of the O(2).Mn enzyme. The specific activity of the protein fraction effecting reconstitution of O(2) evolution is greatest in fractions depleted of the reported Mn-containing, 65-kilodalton, and the Fe-heme, 232-kilodalton (58 kilodalton monomer), proteins. Divalent ( approximately 3 millimolar) and monovalent ( approximately 30 millimolar) cations do not affect reconstitution of PSII donor activity but do affect reconstitution of O(2) evolution by decreasing the protein(s) concentration required for reconstitution of O(2) evolution in nonfractionated, cholate-asolectin extractions. The data indicate a reconstitution of the PSII segment linking the PSII secondary donor(s) to O(2) evolving centers. PMID- 16662350 TI - Beta-Amylases from Alfalfa (Medicago sativa L.) Roots. AB - Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or beta-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato beta-amylase, while that of bacterial alpha-amylase was considerably higher. The identification of maltose and beta-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all beta-amylases.The pH optimum for both beta-amylase fractions was 6.0. Both light and heavy beta-amylases showed normal Michaelis Menten kinetics, with soluble starch as substrate, and had respectively K(m) values of 5.9 and 6.8 milligrams starch per milliliter and V(max) of 640 and 130 international units per milligram protein. Arrhenius plots indicated that the energy of activation for the heavy beta-amylase remained relatively unchanged (12.7 to 13.0 kilocalories per mole) from 0 to 30 degrees C, whereas the energy of activation for the light amylase increased from 12.0 to about 28.0 kilocalories per mole at 8.7 degrees C as temperature was lowered. The light amylase was shown to be inhibited by maltose. PMID- 16662351 TI - Levels of oxygenated Fatty acids in young corn and sunflower plants. AB - Three oxygenated unsaturated fatty acids were investigated to determine whether they were present in seedlings of corn (Zea mays L. cv. NK PX443) and sunflower (Helianthus annuus L. cv. Sundak). The three compounds, 13-hydroxy-12-oxo-cis-9 octadecenoic acid (I), 13-hydroxy-12-oxo-cis,cis-9, 15-octadecadienoic acid (II), and 12-oxo-cis,cis-10, 15-phy-todienoic acid (III), were detected and estimated by gas chromatography-mass spectrometry selected ion monitoring of their trimethylsilyloxy, methyloxime derivatives with 20-carbon analogs added as internal standards. In corn, the concentration of III increased between 5 and 10 days, while I and II remained relatively constant. A higher concentration of II was observed in corn seedlings grown in the light than those grown in the dark. Wounding increased the levels of all three compounds. In sunflower seedlings, the concentrations of I, II, and III increased between 6 and 10 days. The intracellular concentration of III in 10-day-old light-grown seedlings was estimated to be 200 nm in corn and 40 nm in sunflower. PMID- 16662352 TI - Sources of Free IAA in the Mesocotyl of Etiolated Maize Seedlings. AB - Sources of free indole-3-acetic acid (IAA) for the mesocotyl of intact etiolized maize ((Zea mays L.) seedlings are evaluated. The coleoptile unit, which includes the primary leaves and the coleoptilar node, is the main source of free IAA for the mesocotyl. The seed and the roots are not immediate sources of IAA supply. Dependence of the apical growing region of the mesocotyl on the coleoptile unit as a source of free IAA is almost total. One-half or more of the supply of IAA comes from the coleoptile tip, the rest mainly from the primary leaves. Removal of the coleoptile tip results in inhibition of mesocotyl elongation. The hypothesis that growth of the mesocotyl is regulated by auxin supplied by the coleoptile is supported. Conjugated forms of IAA appear to play little part in regulating the levels of free IAA in the shoot. PMID- 16662353 TI - Inhibition of cyanide-resistant respiration in pea cotyledon mitochondria by chloroquine. AB - The action on mitochondrial respiration of a ubiquinone analog, chloroquine, has been studied using purified mitochondria from the cotyledons of germinating peas (Pisum sativum L. var. Homesteader). Chloroquine at 3 millimolar did not inhibit malate or succinate oxidation at pH 7.2, but it did inhibit malate (but not succinate) oxidation at pH 8.2. Cyanide-resistant respiration was also inhibited.The implications of these experiments on the role of ubiquinone in the cyanide-resistant respiratory pathway and on the location of the alternate oxidase are discussed. PMID- 16662354 TI - Ascorbate as a substrate for photoproduction of hydrogen by photosystem I of chloroplasts. AB - The photoproduction of hydrogen by 2-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibited chloroplasts from ascorbate under anaerobic conditions was studied in the pH range 5.0 to 7.5 using methyl viologen (MV), N,N,N',N'-tetramethyl-P phenylenediamine (TMPD), and excess hydrogenase from Desulfovibrio desulfuricans. (a) At neutral and basic pHs, the photoreduction of MV, which reacted back with photoxidized ascorbate (dehydroascorbate [DHASC]), and the rates of H(2) photoproduction were very low. The slow H(2) photoproduction was explained by the reversible reduction of MV by the photoproduced H(2) (catalyzed by hydrogenase) and its reoxidation by DHASC resulting in H(2) uptake. (b) At pH 5.2, relatively high initial rates of H(2) photoproduction were obtained, which were comparable to the rates of O(2) consumption at pH 5.2 by photosystem I (catalyzed by photoreduced MV). However, accumulation of photoreduced MV under anaerobic conditions was not detected. In the presence of high concentrations of protons, the H(2) uptake by DHASC was very slow because the equilibrium concentration of H(2)-reduced MV was very small, thus allowing H(2) evolution mediated by photoreduced MV to compete with the back reaction with DHASC. (c) The continuous accumulation of DHASC, which was generated together with H(2), gradually slowed the H(2) evolution until it stopped after about 3 hours. At high concentrations, DHASC was able to compete with the coupling of photoreduced MV to hydrogenase and H(2) evolution. (d) Dithiothreitol (DTT) reduced the DHASC and consequently competed with the back reaction of the photoreduced and H(2)-reduced MV with DHASC. DTT thus prolonged the time period of H(2) photoproduction from ascorbate and abolished the dependence of its rate on pH in the range of 5.2 to 7.5 (e) A study of H(2) uptake by chemically oxidized ascorbate (in the dark) showed that MV and hydrogenase were both required to catalyze electron transfer from H(2) to DHASC. TMPD prevented this H(2) consumption by DHASC (in a chloroplast reaction mixture containing MV and hydrogenase). Illumination restored the H(2) uptake presumably by generating reduced MV which activated the hydrogenase. PMID- 16662355 TI - Cyanide-insensitive respiration in relation to growth of a low-temperature basidiomycete. AB - The cyanogenic low-temperature basidiomycete (Coprinus psychromorbidus Redhead and Traquair), unlike other cyanide-tolerant fungi, does not detoxify cyanide via formamide hydro-lyase. Instead, tolerance apparently depends on cyanide insensitive respiration involving activity of the mitochondrial alternative oxidase. Respiration and growth of young mycelium that lacks alternative oxidase activity are blocked both by cyanide and 1 mum antimycin. When activity of the alternative oxidase is elicited in young mycelium by 0.05 mm cyanide, subsequent treatment with antimycin stimulates respiration and fails to halt growth. Older mycelium becomes tolerant coincidentally with the release of cyanide by the mycelium. Tolerant older mycelium in medium containing 0.05 to 1.0 mum antimycin grows at 30 to 45% of the control rate. Cyanide- and antimycin-tolerant growth and respiration are blocked by salicyl hydroxamic acid, an inhibitor of the alternative oxidase, and by rotenone, which inhibits ATP synthesis at site I. PMID- 16662356 TI - Effect of Ethylene on the Release of alpha-Amylase through Cell Walls of Barley Aleurone Layers. AB - A large portion of the gibberellic acid (GA(3))-induced alpha-amylase in isolated aleurone layers is transported into the incubation medium. In the presence of GA(3) and ethylene, an even larger portion of the enzyme is found in the medium. Employing an acid washing technique developed by Varner and Mense (Plant Physiol 1972 49:187-189), it was observed that ethylene significantly reduces the amount of alpha-amylase trapped by the thick cell walls of aleurone layers. However, the amount of enzyme remaining in the cell (within the boundary of plasma membrane) is not affected by ethylene. Ethylene has no observable effect on membrane formation as measured by the incorporation of [(32)P]orthophosphate into phospholipids. Because of these observations it is suggested that ethylene enhances the release of alpha-amylase, i.e. transport of alpha-amylase across cell walls, but not the secretion of alpha-amylase, i.e. transport of alpha amylase past the barrier of plasma membrane. The possible mechanism of this ethylene effect is discussed. PMID- 16662357 TI - Dehydration effects on imbibitional leakage from desiccation-sensitive seeds. AB - Changes in electrolyte leakage and viability in response to dehydration stress were examined in two species of seeds that do not survive desiccation. Leakage from silver maple (Acer saccharinum L.) seeds increased markedly as seed moisture contents decreased from 45 to 35% (fresh weight basis) and germination decreased from 97 to 5%, coincidentally. Time course curves of imbibitional leakage from areca palm (Chrysalido-carpus lutescens [Bory] Wendl.) embryos showed an increase in both initial leakage and steady-state leakage rates in response to dehydration from an original moisture content of 84 to as low as 53%. Absorbance at 530 nanometers of extracts from triphenyl tetrazolium chloride stained embryos of areca palm was used as a measure of viability. Absorbance decreased significantly in response to dehydration as embryo moisture content decreased from 80 to 30%. Collectively, the data suggest that membranes in the desiccation-sensitive seed tissues studied are damaged by dehydration below a critical moisture content, 40% in silver maple seed and 55% in areca palm embryos, and that the membrane damage contributes to loss of viability. PMID- 16662358 TI - Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells. AB - The effects of added glycine hydroxamate on the photosynthetic incorporation of (14)CO(2) into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO(2) and 20% O(2)) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO(2) and 2.7% O(2)). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO(2) and NH(4) (+). During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the "reverse" flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration. PMID- 16662359 TI - Effects of Cations and Abscisic Acid on Chlorophyll a Fluorescence in Guard Cells of Vicia faba. AB - The effects of cations and abscisic acid on chloroplast activity in guard cells of Vicia faba were investigated by analysis of the transient of chlorophyll a fluorescence. When epidermal strips containing guard cells as the only living cells were incubated in water and illuminated with strong light, chlorophyll a fluorescence rose rapidly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K(+) or Na(+), and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca(2+) suppressed the enhancement by Na(+) and, to a lesser extent, that by K(+). Abscisic acid also suppressed the enhancement by K(+) and Na(+). Since the fluorescence decline reflects the increase of intrathylakoid H(+) concentration necessary for photophosphorylation, the acceleration of the decline by K(+) (or Na(+) in the absence of Ca(2+)) implicates chloroplast activity in ion accumulation by guard cells in the light. The differential effects of phosphate and chloride suggest that chloroplast activity may be involved in malate formation in guard cells in the light. PMID- 16662360 TI - Complete turgor maintenance at low water potentials in the elongating region of maize leaves. AB - Leaf elongation rate, water potential, and osmotic potential were measured in the fifth leaf of maize (Zea mays L.) plants growing in soil from which water was withheld for varying times. Elongation occurred in the basal region, which was enclosed by other leaf sheaths. When water was withheld from the soil, leaf elongation decreased and eventually ceased even though enough solutes accumulated in the elongating region to maintain turgor virtually constant. In the exposed blade, however, turgor was lost and wilt symptoms developed. If the night was prolonged, the elongating region lost much of its ability to accumulate solute, which suggests that the accumulating solutes were of recent photosynthetic origin. Under these conditions, leaf elongation was restricted to higher water potentials than under the usual photoperiodic regime.The solute accumulation and turgor maintenance of the elongating region at low water potentials indicate that differences in water status and physiological behavior exist along grass leaves and that the water status of the elongating region cannot be inferred from measurements on the exposed blade. The increased sensitivity of leaf elongation to low water potentials in prolonged darkness indicates that accumulation of solute and maintenance of turgor play a role in maintaining leaf growth. However, the inhibition of elongation that occurred even when solute accumulation was sufficient to completely maintain turgor indicates that some factor other than photosynthate supply and turgor also affected growth and caused most of the losses in growth under dry conditions. PMID- 16662361 TI - Influence of calcium and magnesium on ethylene production by apple tissue slices. AB - The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca(2+), Mg(2+), or Ca(2+) plus Mg(2+) severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca(2+), Mg(2+), or Ca(2+) plus Mg(2+) virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1 acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca(2+), Mg(2+), and Ca(2+) plus Mg(2+). Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca(2+) in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane. PMID- 16662362 TI - Control by Phytochrome of Cytoplasmic Precursor rRNA Synthesis in the Cotyledons of Mustard Seedlings. AB - The influence of phytochrome (high irradiance reaction; operationally, continuous far red light) on the incorporation of [(3)H]uridine into the cytoplasmic 2.5 megadalton precursor rRNA in the cotyledons of mustard (Sinapis alba L.) seedlings has been investigated. After irradiating 36-hour-old etiolated seedlings with 12 hours of far red light, the rate of incorporation is stimulated about 2-fold, leading to 50% labeling of the precursor rRNA pool about 15 minutes after the tracer has reached the nucleotide precursor pool. In the dark control, there is a significantly smaller pool of precursor rRNA which is half-saturated with label only after about 27 minutes. Since neither the specific radioactivity of the UTP pool nor the processing of the precursor rRNA demonstrate a corresponding light-dependent change, it is concluded that phytochrome mediates an increase of the transcription of the rRNA genes. This gene activation accounts for the increased accumulation of mature cytoplasmic rRNA during the course of photomorphogenesis of the cotyledons. PMID- 16662363 TI - Physiology of Root-Associated Nitrogenase Activity in Oryza sativa. AB - An intact method for measuring immediately linear rates of acetylene reduction was used to investigate the relationship between temperature, pH, O(2) concentration, and light intensity with the rate of root-associated nitrogenase activity in rice (Oryza sativa L.). Nitrogenase activity varied over a temperature range of 10 to 50 degrees C and optimal rates of acetylene reduction were recorded at 35 degrees C. Nitrogenase activity was also influenced by the pH of the liquid surrounding the roots prior to assay. Maximal rates of acetylene reduction were recorded over a pH range from 5.8 to 7.5. Nitrogenase activity was significantly reduced by concentrations of O(2) 0.5% (v/v) or more when the intact plant assay method was used, and no optimum was detected. However, when the plant tops were removed and the cut ends sealed from the atmosphere for 4 hours, acetylene reduction rates were maximal at 0.25% O(2) (v/v). When plants were moved from sunlight (1,400 microeinsteins per square meter per second) to shade (9.6) root-associated nitrogenase activity at 35 degrees C significantly decreased 15 min later to one-fourth the rate and recovered upon return to sunlight. When the light intensity reaching the leaf canopy was progressively reduced from 1,050 to 54 microeinsteins per square meter per second the rate of root-associated nitrogenase activity decreased from 550 +/- 135 to 192 +/- 55 nanomoles ethylene per gram dry root per hour. The study suggests that the rate of root-associated nitrogenase activity in rice at constant temperature may well be mediated by variations in the concentration of O(2) resulting from changes in the rate of photosynthesis as well as variations in the rate of transport of photosynthate. PMID- 16662364 TI - Measurement and preservation of the in vivo activation of ribulose 1,5 bisphosphate carboxylase in leaf extracts. AB - Photosynthetic carbon fixation is regulated in the chloroplast by the amount of ribulose 1,5-bisphosphate carboxylase which is activated. The activated carboxylase was preserved in detached leaves (barley, maize, soybean, spinach, wheat) for 90 min when stored on ice. With leaf extracts stored at 2 degrees C, the amount of activated enzyme, representing that originally in the leaf, as well as the fully activated enzyme, formed by incubation of leaf extracts with Mg(2+) and bicarbonate, both slowly declined in activity. However, for each activity this decline was proportional such that the ratio (percent activation) appeared constant. No change was observed in activation of the enzyme during the brief time of leaf homogenization. Optimal conditions (Mg(2+), incubation time) for measurement of leaf activation of ribulose bisphosphate carboxylase vary depending on the plant.3-Phosphonoproprionate, a positive effector of the purified ribulose bisphosphate carboxylase and a metabolically inert analog of 2 phosphoglycolate, was used to examine what metabolic effectors might do to enzyme activation during leaf homogenization and preparation of the extract at 2 degrees C. Activation under these conditions was not altered by 3-phosphonoproprionate. When 3-phosphonoproprionate was brushed on attached leaves or taken up by the transpiration stream of detached leaves, a considerable increase in activation of the carboxylase was measured. PMID- 16662365 TI - Inhibition of cytokinin action and of heat/aging induced potential for cytokinin action by inhibitors of membrane synthesis and function. AB - Data to support the hypothesis that cytokinin action, in inducing the biosynthetic pathway involved in betacyanin synthesis in Amaranthus tri-color seedlings, is dependent on both membrane synthesis and function is presented. The experimental system included a pretreatment of heat shock (40 degrees C) and aging of cotyledon explants. This produced the conditions necessary for the full expression of cytokinin potential during the subsequent betacyanin induction. Cerulenin, an inhibitor of fatty acid and sterol synthesis, inhibited both the heat-induced potential for cytokinin action and the benzyladenine-dependent induction itself. This was also true of metyrapone, an inhibitor of hydroxylation reactions involving cytochrome P450. Gammexane, an inhibitor of phospholipid turnover, impaired the heat-induced process but not the benzyladenine-dependent betacyanin accumulation. This was also the case with 2-isopropyl-4-dimethylamino 5-methyl phenyl-1-piperidine carboxylate methyl chloride, an inhibitor of the cyclization steps in sterol and gibberellin synthesis. Filipin at 100 micrograms per milliliter inhibited both processes, particularly the heat-induced potential. The effect of various steroids and fatty acids on induction is recorded together with experiments aimed at using them to reverse some of the inhibitions. The effect of cerulenin on heat-induced potential was partially reversed by preparations of Amaranthus lipids. Some reversal of the filipin effects was obtained with beta-sitosterol and stigmasterol. It is concluded that menbrane synthesis is stimulated during the heat/aging pretreatment and during the induction and that some membrane function(s) is necessary for subsequent cytokinin action. PMID- 16662366 TI - Uptake and Metabolic Fate of Glucose, Arabinose, and Xylose by Zea mays Coleoptiles in Relation to Cell Wall Synthesis. AB - According to the acid-growth hypothesis, auxin-induced secretion of hydrogen ions activate "wall loosening" enzymes that change the rheological properties of the cell wall. The wall loosening process may yield monosaccharides by the enzymic cleavage of load-bearing polysaccharides. Our study was initiated to determine the metabolic fate of such sugars when released from the major hemicellulosic polysaccharides of the cell walls of Zea mays coleoptiles.Excised coleoptile sections accumulated radioactive glucose, arabinose, and xylose supplied in an incubation medium, and the radioactivity from these sugars was incorporated into polysaccharides. At least 50% of the radioactivity from glucose accumulated in the soluble neutral sugar fraction regardless of external concentrations. The distribution of radioactivity from xylose into all subcellular fractions was similar to that from glucose, indicating that xylose was converted to glucose before being used by the coleoptile. IAA increased the incorporation of glucose into cell wall polysaccharide and neutral sugar pools when the exogenous concentration was higher than 1 millimolar.Over 80% of the radioactivity from arabinose accumulated by the coleoptile sections was incorporated into soluble and noncellulosic polymers; IAA induced an increase in the incorporation of arabinose into noncellulosic polymers by 22%. Accumulation of radioactivity from arabinose into polysaccharide was enhanced by IAA at concentrations of exogenous arabinose up to 33 millimolar.IAA promoted the incorporation of both arabinose and glucose into cell wall polysaccharides even when elongation was inhibited by CaCl(2), indicating that the influence of IAA was not a consequence of the growth response. PMID- 16662367 TI - Elicitation of Casbene Synthetase Activity in Castor Bean : THE ROLE OF PECTIC FRAGMENTS OF THE PLANT CELL WALL IN ELICITATION BY A FUNGAL ENDOPOLYGALACTURONASE. AB - Endopolygalacturonase isolated from culture filtrates of the fungus Rhizopus stolonifer was shown previously to act as an elicitor of biosynthetic capacity for the antifungal agent, casbene, in castor bean (Ricinus communis L.) seedlings (S.-C. Lee, C.A. West 1981 Plant Physiology 67:633-639). Selective amidation of exposed carboxyl groups of the pure fungal endopolygalacturonase using intermediate activation with a water-soluble carbodiimide under mild conditions leads to inactivation of its enzymic activity. Tests of active and partially inactivated preparations of the enzyme reveal a close correlation between the levels of catalytic and elicitor activities. This suggests that the catalytic activity of the enzyme is necessary for its function as an elicitor. Treatment of the cell-free particulate fraction of homogenates of castor bean seedlings with the active fungal endopolygalacturonase results in the production of a heat stable, water-soluble component which is highly active as an elicitor of casbene synthetase activity. Several additional lines of evidence, including the susceptibility of the heat-stable elicitor fraction to partial inactivation following prolonged treatment with endopolygalacturonase, indicate that the heat stable elicitor is most likely a pectic fragment of the plant cell wall and that it is a required intermediate in the process of elicitation of casbene synthetase activity by the fungal endopolygalacturonase. PMID- 16662369 TI - Pyridine nucleotide specificity of barley nitrate reductase. AB - NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH. PMID- 16662368 TI - Extranuclear DNA of a Marine Chromophytic Alga : RESTRICTION ENDONUCLEASE ANALYSIS. AB - Two extranuclear DNA species have been isolated from the marine alga Olisthodiscus luteus. Rapid lysis of cells followed by the immediate addition of CsCl to the lysate was critical to the preservation of these satellite DNA species. Restriction endonuclease analysis demonstrates a molecular weight of 99 x 10(6) for chloroplast DNA and 23 x 10(6) for a second satellite species. The origin of the second satellite is not known. However, this smaller satellite DNA which originates from a nonnuclear, DNAse insensitive cellular component, displays no sequence homology with ctDNA by hybridization experiments. Constancy of restriction endonuclease fragment patterns of chloroplast and second satellite species during all phases of the growth cycle, whether cultures were maintained synchronously or asynchronously, was demonstrated. PMID- 16662370 TI - Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase. AB - A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K(m) values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K(m) values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.Unlike that of the wild-type, the nitrate reductase from nar1a did not bind to blue dextran-Sepharose. The nar1a enzyme did bind to Affi Gel Blue, but recoveries were low. The NADH and NADPH nitrate reductase activities of nar1a were not separated by affinity chromatography. The nitrate reductase in nar1a is a different enzyme than the wild-type NADH nitrate reductase and appears to be a NAD(P)H-bispecific enzyme. PMID- 16662371 TI - Visualization of a Spinach Plastid Transcriptionally Active DNA-Protein Complex in a Highly Condensed Structure. AB - A transcriptionally active DNA-protein complex isolated from spinach Spinacia oleracea plastids is visualized by electron microscopy in different conditions. This structure, after glutaraldehyde fixation, is highly condensed. DNA is supertwisted with proteins bound to it producing a beaded substructure. When glutaraldehyde fixation is omitted this structure is less condensed and DNA fibrils come out from a proteinous central body. The DNA-protein complex can be separated into two populations by CsCl centrifugation: one with a buoyant density of 1.570 grams per cubic centimeter and the other of 1.610 grams per cubic centimeter. By visualization of these two populations, it is concluded that proteins are either firmly bound to DNA in the central body, or more loosely bound to the DNA fibrils. These latter proteins could play a role in enzymic functions and/or in the supercoiling of DNA.The DNA from the DNA-protein complex possesses all fragments that belong to pure circular chloroplast DNA hydrolyzed by two restriction enzymes: Bam HI and Eco RI. Some molecules observed in a supercondensed form with a beaded substructure probably contain entire chloroplast DNA molecules.A hydrolysis test with microccocal nuclease gives no indication of the presence of ;nucleosome-like' structures. Thirty-six polypeptides with molecular weights ranging from 12,000 to 180,000 are present in the complex, and seven of them are highly soluble in 0.4 n H(2)SO(4); their molecular weights range from 14,000 to 46,000 as shown by two-dimensional gel electrophoresis.No linolenic acid can be detected in the preparation, indicating the absence of chloroplast membranes. PMID- 16662372 TI - Quantification of allelopathic potential of sorghum residues by novel indexing of richards' function fitted to cumulative cress seed germination curves. AB - The inhibitory activity of aqueous extracts of field-grown sorghum (Sorghum bicolor cv. Bird-a-boo) herbage and roots was quantitatively indexed by three aspects of cumulative cress (Lepidium sativum cv. Curlycress) seed germination: the germination onset; weighted mean rate; and final germination percentage. Extract potency was greatest for herbage collected four weeks after planting but declined sharply thereafter as the plants matured. About 91% of the inhibitory activity obtained from four-week-old herbage was in a low molecular weight fraction. Differential effects of herbage and root extracts on cress seed germination suggest that the nature and/or proportion of biologically active substances extractable from these plant parts is dissimilar. PMID- 16662373 TI - Electrophoretic Comparison of Mitochondrial Polypeptides from Maize Lines Susceptible and Resistant to Helminthosporium maydis Race T. AB - The mitochondrial polypeptides from maize lines susceptible and resistant to Helminthosporium maydis were studied by single- and two-dimensional gel electrophoresis. Approximately 120 polypeptides were detected by two-dimensional gel electrophoresis. However, it was not possible to detect qualitative differences between the mitochondrial polypeptides of the two inbreds. These observations are discussed with reference to the putative mechanism of action of the pathotoxin. PMID- 16662374 TI - Effect of carbon dioxide and light on ethylene production in intact sunflower plants. AB - High CO(2) concentration (0.5%) increased the rate of ethylene production, measured in a continuous flow system, in intact sunflower (Helianthus annuus L.) plants. However, the rate of ethylene production subsided to near control levels after approximately 24 hours. The effect of high CO(2) could only be observed in light. Although high CO(2) concentration had no effect on the rate of ethylene production in darkness, prolonged exposure (approximately 16 hours) of plants to high CO(2) in the dark prevented the increase in ethylene production when the plants were exposed to light and high CO(2). PMID- 16662375 TI - Transport, metabolism, and redistribution of xylem-borne amino acids in developing pea shoots. AB - Amino acid metabolism and transport was investigated in the leaves of 3-week-old nonnodulated seedlings of Pisum sativum L. Xylem sap entering the shoot contained nitrate (about 5 millimolar), and amino compounds (11 millimolar) of which 70% was asparagine plus glutamine; aspartate and homoserine were also present. Mature leaves showed stable nitrogen levels and incoming nitrogen was redistributed to growing leaves. Younger leaves, still enclosed in the stipules, showed negligible rates of transpiration, suggesting that most of their nitrogen must arrive in the phloem.(14)C-Labeled amides and amino acids were supplied to detached shoots through the xylem, and metabolism and redistribution were followed over 12 hours in light. Asparagine entering mature leaves was reexported directly to young leaves, with relatively little metabolic conversion. Substantial amounts of glutamine were converted to glutamate, which was exported (with unchanged amide) with little further conversion. The pattern of redistribution was confirmed when (14)C-labeled amino acids were applied directly to the under surface of mature leaves. Labeled compounds were found in the phloem exudate from treated leaves, and the composition resembled the pattern of labeling in the compounds arriving in the young developing leaves. PMID- 16662376 TI - A soybean seed urease-null produces urease in cell culture. AB - Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from ;wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize).No urease activity or only traces of urease activity were detected in cotyledons of developing or germinating Itachi seeds. In contrast, callus cultures induced from cotyledon, shoot tip, root, or root tip tissues of Itachi seedlings exhibited ureolytic activity equivalent to that of Prize cultures. Shoot tip cultures of both Prize and Itachi grew with urea as sole nitrogen source. Most or all of the ureolytic activity in crude extracts of Prize and Itachi suspension culture cells is seed-like urease in thermal stability, recognition by antibodies to the seed enzyme, hydroxyurea sensitivity, and nickel requirement for synthesis. It has been reported previously (Polacco, Havir 1979 J Biol Chem 254: 1707-1715; Polacco, Sparks, Jr, Havir 1979 Genet Eng 1: 241-259) that partially purified cell culture urease is identical to seed urease by immunological and electrophoretic criteria. These results suggest that urease is under different developmental controls in the seed and in cell culture.In both Prize and Itachi cultures, utilization of the ureide allantoin, unlike that of urea, is not dependent on nickel. This suggests that ureide catabolism does not require urease. PMID- 16662377 TI - A comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes. AB - Plasma membrane-associated ATPase obtained from cauliflower (Brassica oleraceae L.) florets isolated and assayed by several different procedures was stimulated 150 to 400% by K(+). In contrast, winter wheat (Triticum aestivum L. cv. Kharkov 22 MC) shoot and root ATPase obtained by the same methods exhibited only 10 to 25% stimulation by K(+). The level of K(+)-stimulation of the wheat enzyme was not significantly increased by purifying the crude microsomal membrane fraction using sucrose density gradients. ATPase associated with density gradient-purified cauliflower membranes was inhibited by Ca(2+), high ATP concentration in the presence of low Mg(2+), and by several metabolic inhibitors. In contrast, the wheat enzyme was largely unaffected by all of these treatments. The plasma membranes of intact wheat and cauliflower cells gave a positive reaction with the plasma membrane-specific, phosphotungstic acid-chromic acid stain (PACP). A high proportion of the cauliflower membrane vesicles in the putative plasma membrane enriched fraction stained with PACP, whereas only a small proportion of the wheat membrane vesicles reacted positively with PACP. These results indicate that a plasma membrane-enriched fraction has been isolated successfully from cauliflower floret tissue, but that none of the procedures used effectively separate plasma membranes from homogenates of wheat shoots and roots. PMID- 16662378 TI - Role of the colorless polypeptides in phycobilisome reconstitution from separated phycobiliproteins. AB - A phycoerythrin (PE) and phycocyanin (PC) mixture was separated from allophycocyanin on calcium phosphate chromatography from completely dissociated phycobilisomes of the blue-green alga, Nostoc sp. After dialysis of the PE-PC mixture in 0.75 m potassium phosphate, pH 7, which allows reassociation of the dissociated pigment-proteins, complexes of PE and PC in a 2:1 m ratio (PE/PC complex) as well as complexes predominantly of PC (PC/PE complex) were then separated by sedimentation on linear sucrose gradients. These complexes resemble the rods of intact phycobilisomes and transfer energy efficiently from PE to PC. They contain the Group II colorless polypeptides described by Tandeau de Marsac and Cohen-Bazire (1977 Proc Natl Acad Sci USA 74: 1635 61639). Phycobilisomes can be reconstituted by combining the allophycocyanin pool with (a) the PE-PC mixture, (b) the PE/PC complex, or (c) the PC/PE complex. Successful reconstitution is measured by absorption, fluorescence, circular dichroism, and electron microscopy. The major requirement for reconstitution is the 29 kilodalton colorless polypeptide. In its absence, no phycobilisomes are formed. It is the only colorless polypeptide common to both the PE/PC complex and the PC/PE complex, and appears to be the polypeptide responsible for rod attachment to the allophycocyanin. In addition, high phosphate concentrations and 20 degrees C temperatures are needed for reconstitution. PMID- 16662379 TI - A new method for the determination of hydraulic conductivity and cell volume of plant cells by pressure clamp. AB - Internodes of Chara corallina were used for experiments in which cell turgor pressure was clamped by means of the pressure probe technique. Essentially, the procedure consisted of a combination of volume and turgor pressure relaxations. This technique permits the determination of the cell volume by nonoptical means. The values obtained are in agreement with the ones determined by optical means. Furthermore, the hydraulic conductivity (L(p)) was determined from the initial slope of the volume relaxation; the values thus obtained are in agreement with those calculated from the half-times of pressure relaxations. The determination of L(p) from volume relaxation measurements has the advantage that the cell volume, the volumetric elastic modulus of the cell wall, and the internal osmotic pressure do not have to be known. Furthermore, the half-time of volume relaxation is longer than that of pressure relaxation, as shown by theory and experiment. This may be used to enhance the resolution of the relaxation measurement and, thus, to improve the accuracy of L(p) determinations for higher plant cells which exhibit a very fast pressure relaxation. PMID- 16662380 TI - Trehalose Toxicity in Cuscuta reflexa: SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING. AB - Trehalose, an alpha,alpha-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [(14)C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding. PMID- 16662381 TI - Adenine nucleotide content of corn roots as affected by injury and subsequent washing. AB - The adenine nucleotide content of the 2-centimeter segments excised from tray grown corn (Zea mays L., WF9 x Mo17) roots declines for the first hour after excision. Concomitant with the loss of adenine nucleotides is a decline in respiration and a leakage of K(+). With continued washing, these parameters partially or completely recover and increased phosphate influx develops. Increasing the wound effect by cutting 0.5-centimeter segments gives a more rapid and pronounced degradation of adenine nucleotides and slower recovery. Conversely, the mild injury caused by submerging intact roots induces less degradation and produces greater net adenine nucleotide synthesis during recovery; adding auxin to the washing medium produces a similar result. With all treatments, there is stabilization of energy charge at about 0.85.Brief submersion or rubbing of intact roots, as well as recutting washed and recovered root segments, will initiate the transient loss of adenine nucleotides but will not induce increased phosphate influx.It is suggested that the loss in adenine nucleotides may reflect homeostasis in energy charge via catabolism arising from membrane permeability changes. PMID- 16662382 TI - Fatty Acid Synthetase of Spinacia oleracea Leaves. AB - The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, beta-ketoacyl-ACP synthetase, beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.From these results, it was concluded that the fatty acid synthetase system of spinach leaves, as well as that of safflower seeds, was nonassociated and similar to the Escherichia coli system. PMID- 16662383 TI - Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts. AB - A system has been developed for the study of photosynthetic CO(2) fixation by isolated spinach chloroplasts at air levels of CO(2). Rates of CO(2) fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO(2) fixation, suggesting that the decline in CO(2) fixation may have been caused by deactivation of the enzyme.In contrast, the addition of 50 micromolar 3 phosphoglycerate or 50 micromolar glycerate doubled the initial rate of CO(2) fixation without increasing the activity of the carboxylase. Addition of 4 millimolar MgCl(2) plus 2 millimolar pyrophosphate had no effect on the initial rate of CO(2) fixation nor its decline but did stop deactivation of the chloroplast carboxylase during the decline.The decline in photosynthesis with isolated chloroplasts, where RuBP is high, may be due to deactivation of the chloroplast RuBP carboxylase; however, measurements of this parameter may not always indicate this. Care must be taken when interpreting measurements of the ;activated' RuBP carboxylase as more forms are measured than the enzyme . CO(2) . Mg(2+) . RuBP form directly involved in carboxylation or oxygenation. PMID- 16662384 TI - H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60. AB - Using manometric and enzymic techniques, H(2) and CO(2) evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.In the dark, starch was broken down, and H(2) and CO(2) was released. The uncoupler, carbonyl cyanide m fluorophenylhydrazone with an optimum concentration of 5 to 10 micromolar, increased the rate of CO(2) release and starch breakdown but depressed H(2) formation. It was suggested that carbonyl cyanide m-fluorophenylhydrazone increased the rate of starch breakdown by making the chloroplast membrane permeable to H(+), removing a rate-limiting step, and leading to an altered fermentative pattern.Photoevolution of H(2) and CO(2), but not starch breakdown, was stimulated by acetate. Maximum stimulation occurred at concentrations from 1 to 10 millimolar. Carbonyl cyanide m-fluorophenylhydrazone stimulated starch breakdown and CO(2) and H(2) release in the light, but not to the extent of acetate. Inasmuch as the uptake and subsequent metabolism of acetate required ATP, it was suggested that acetate, like carbonyl cyanide m fluorophenylhydrazone, stimulated H(2) photoproduction by removing ATP which limited the sequence of reactions. The contribution of photosystem II to the photoproduction of H(2), as judged from the effect of 10 micromolar 3-(3,4 dichlorophenyl)-1, 1-dimethylurea, was at least 80%.CO(2) photoevolution increased linearly with time, but H(2) photoevolution occurred in two phases: a rapid initial phase followed by a second slower phase. The rate of H(2) release increased hyperbolically with light intensity, but the rate of CO(2) production tended to level off and decrease with increasing light intensity, up to 145 watts per square meter. It was proposed that a changing CO(2) and H(2) ratio is the result of interaction between the carbon and hydrogen metabolism and the photosynthetic electron transport chain. PMID- 16662385 TI - Cytokinin modification of mitochondrial function. AB - 6-Benzylaminopurine, 6-(Delta(2)-isopentenylamino)purine, 6-furfurylaminopurine, rotenone, and antimycin A inhibited oxidation of NADH by mitochondrial sonicates or submitochondrial particles (but not by intact mitochondria) from pea (Pisum sativum L., cult. Alaska) stems and mung bean (Vigna radiata L. Wilczak) hypocotyls. The above purine cytokinins can interfere with electron transport from NADH to the cytochrome system in the inner mitochondrial membrane. Adenine did not inhibit oxidation by sonicated mitochondria, and zeatin was almost ineffective. Zeatin scarcely inhibited state 3 malate respiration by intact mitochondria, but the O-formyl and O-n-propionyl esters of zeatin and the O acetyl ester of 2-chlorozeatin were more active. Perhaps zeatin is ineffective because it does not get into the inner membranes of the isolated mitochondria, whereas the esters and other cytokinins mentioned above do. N-4-(2-chloropyridyl) N'-Phenylurea, which has cytokinin-like effects on plant growth and development, inhibited NADH oxidation by sonicated mitochondria. It also inhibited malate, succinate, and NADH oxidation by intact mitochondria; in contrast, the latter two oxidations were not decreased by purine cytokinins.The benzyl, isopentenyl, and furfuryl aminopurines inhibited malate (but not succinate) oxidation by rat liver mitochondria, if ADP was present. In the absence of ADP, these cytokinins promoted succinate (but not malate) oxidation. Zeatin and adenine had slight, if any, effect in either situation. PMID- 16662386 TI - Aggregation of plant protoplasts by artificial lipid vesicles. AB - Sonicated unilamellar lipid vesicles, consisting of egg lecithin, stearylamine, and cholesterol in 7:2:1 molar ratios, promoted the aggregation of tobacco (Nicotiana glutinosa) protoplasts with the aid of mono- or divalent cations.A reaction mixture containing liposomes (0.4 micromoles lipid per milliliter), 50 millimolar CaCl(2), 0.5 molar mannitol, and 5 x 10(5) protoplasts per milliliter resulted in approximately 25% protoplast aggregation. To achieve the maximum protoplast aggregation, 1.6 x 10(8) liposomes per protoplast per hour would be required.The kind of liposomes effective in protoplast aggregation were positively charged, small-size vesicles which were obtained either by 60-minute sonication or by membrane filtration in conjunction with sonication. PMID- 16662387 TI - Phenotypical Temperature Adaptation of Protein Synthesis in Wheat Seedlings : QUALITATIVE ASPECTS. INVOLVEMENT OF AMINOACID:tRNA-LIGASES. AB - Phenotypical temperature adaptation of protein synthesis in wheat (Triticum aestivum L.) seedlings is not affected by darkness (etiolation), by partial inhibition of protein biosynthesis (10(-3)m fluorophenylalanine), or by changing the amino acid precursor and the radioisotope ([(3)H]valine instead of [(14)C]leucine). The temperature coefficient (mu), as well as the optimum temperature of in vivo protein synthesis, increases with rising preadaptation temperature, as normally observed. Protein turnover studies revealed that only proteins with a short half-life time (t((1/2)) = 2 to 4 hours) are labeled to a measurable extent during the temperature adaptation experiments. A heat-labile protein has been detected and partially characterized by means of polyacrylamide gradient gels. Leucine:tRNA-ligase (EC 6.1 1.4) from heat-pretreated wheat seedlings exhibits enhanced thermal stability. In Arrhenius curves, the upper transition point shifts from 30 to 34 degrees C, depending on preadaptation temperature. Only the leucine:tRNA-ligase extracted from heat-adapted plants is stable when the enzyme extracts are subjected to a 34 degrees C heat treatment. PMID- 16662388 TI - Pathway of malic Acid synthesis in response to ion uptake in wheat and lupin roots: evidence from fixation of C and C. AB - Malate synthesis by CO(2) fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH(13)CO(3) as well as with NaH(14)CO(3). The distribution of (14)C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of (13)C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short term experiments (15, 30 seconds) showed that (14)C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-(13)C]-, [4-(13)C]-, and [1,4-(13)C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria. PMID- 16662389 TI - Solubilization and Characterization of an Acyl-Coenzyme A : O-LYSOPHOSPHOLIPID ACYLTRANSFERASE FROM THE MICROSOMES OF DEVELOPING SAFFLOWER SEEDS. AB - Acyl-CoA:O-lysophospholipid acyltransferase activity was measured in extracts of developing safflower (Carthamus tinctorius var. UC-1) seeds. Acyltransferase activity was present in all subcellular fractions. The microsomal acyltransferase activity was solubilized with either 30 mm-n-octylglucoside, 1% deoxycholate, or 100 mum lysophosphatidylcholine. The pH optimum of the lysophosphatidylcholine dependent acyltransferase activity was at 9.0. Among the various acyl-CoA's tested, oleoyl-CoA was the best substrate and with it the enzyme had a K(m) of 9.5 mum. Among the various acyl acceptors tested, lysophosphatidylcholine (oleoyl) gave the highest activity and the optimal concentration was 30 mum. Acyltransferase activity was stimulated by ethanol and inhibited by bovine serum albumin and divalent cations. PMID- 16662390 TI - Nitrate Reduction by Roots of Soybean (Glycine max [L.] Merr.) Seedlings. AB - Studies were conducted with 9 to 12 day-old soybean (Glycine max [L.] Merr. cv. Williams) seedlings to determine the contribution of roots to whole plant NO(3) ( ) reduction. Using an in vivo -NO(3) (-) nitrate reductase (NR) assay (no exogenous NO(3) (-) added to incubation medium) developed for roots, the roots accounted for approximately 30% of whole plant nitrate reductase activity (NRA) of plants grown on 15 mm NO(3) (-).Nitrogen analyses of xylem exudate showed that 53 to 66% of the total-N was as reduced-N, depending on the time of day of exudate collection. These observations supported enzyme data that suggested roots were contributing significantly to whole plant NO(3) (-) reduction. In short-term feeding studies using (15)N-NO(3) (-) significant and increasing atom percent (15)N excess was found in the reduced-N fraction of xylem exudate at 1.5 and 3 hours after feeding, respectively, which verified that roots were capable of reducing NO(3) (-).Estimated reduced-N accumulation by plants based on in vivo NO(3) (-) NR assays of all plant parts substantially over-estimated actual reduced-N accumulation by the plants. Thus, the in vivo NR assay cannot be used to accurately estimate reduced-N accumulation but still serves as a useful assay for relative differences in treatment conditions. PMID- 16662391 TI - Distribution of Free and Ester Indole-3-Acetic Acid in the Cortex and Stele of the Zea mays Mesocotyl. AB - The distribution of free and ester-linked indole-3-acetic acid (IAA) in the vascular stele and cortex-epidermis of the Zea mays mesocotyl was measured by gas chromatography-selected ion monitoring-mass spectrometry and by radioimmunoassay with good agreement between the two assay methods. On a per plant basis, 72% of the free IAA was found in the stele and 28% was in the cortex, whereas 80% of the ester IAA was in the cortex with 20% localized in the stele. On a fresh weight basis, the concentration of free IAA was 15 to 28 times higher in the stele than in the cortex, whereas the concentration of ester IAA was similar in the two tissues. The concentration of free IAA in the apical portion of the cortex was 3 times higher than in the basal portion, and this distribution correlated with the relative growth rates of the apical and basal portions of the mesocotyl. No changes in the longitudinal distribution of ester IAA were found in either the cortex or stele. PMID- 16662392 TI - Interaction between Mitochondrial Cytochromes and Linoleic Acid Hydroperoxide: POSSIBLE CONFUSION WITH LIPOXYGENASE AND ALTERNATIVE PATHWAY. AB - O(2) uptake by tissue extracts in the presence of linoleic acid is generally ascribed to lipoxygenase. Such an O(2) uptake can be observed not only with mitochondria of Solanum tuberosum L. and Arum maculatum L. and pure lipoxygenase but also with cytochrome c. However, the rate of oxidation is highly dependent on the procedure used to prepare the solutions of linoleic acid. Unless special care is taken to prevent contact between linoleic acid and O(2), it appears that linoleic acid hydroperoxide is readily formed. This derivative can be readily oxidized by mitochondria or cytochrome c. On the other hand, the use of a rapid and specific enzymic procedure to estimate the disappearance of linoleic acid demonstrates that linoleic acid itself is not consumed at any appreciable rate by mitochondria or cytochrome c, the true substrate being linoleic acid hydroperoxide. During the reaction, the heme nucleus of added cytochrome c or of mitochondrial cytochromes undergoes deep alterations. Therefore, caution should be exerted when equating an O(2) uptake observed in the presence of linoleic acid to a lipoxygenase activity. The same holds true for the similarity of reaction towards specific inhibitors between lipoxygenase and the cyanide-insensitive pathway oxidase. PMID- 16662393 TI - Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION. AB - Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply. PMID- 16662394 TI - Vacuoles from Sugarcane Suspension Cultures : II. CHARACTERIZATION OF SUGAR UPTAKE. AB - Vacuoles, isolated from sugarcane (Saccharum sp.) cells, took up 3-O methylglucose and sucrose and the evidence suggests specific transport systems for these sugars. There was no evidence of sugar efflux from preloaded vacuoles. Vacuoles in situ accumulated 3-O methylglucose, sucrose, glucose, and fructose, as shown by incubation of protoplasts with labeled sugar and subsequent analysis of vacuolar and cytoplasmic radio-activity. During the initial minutes of incubation, the amount and concentration of labeled sugar was higher in the cytoplasm than in the vacuole, but subsequently there was active uptake and accumulation into the vacuole. The rate of hexose transfer into the vacuole in situ approached that of hexose uptake by isolated vacuoles; however, the rate of sucrose uptake by isolated vacuoles was below the in situ rate. The site of sucrose synthesis was in the cytoplasm. PMID- 16662395 TI - Vacuoles from Sugarcane Suspension Cultures : III. PROTONMOTIVE POTENTIAL DIFFERENCE. AB - The electrochemical proton gradient across the tonoplast of isolated (Saccharum sp.) vacuoles and vacuoles in situ was measured. The isolated vacuoles show no significant protonmotive potential difference, the pH gradient of 0.8 (inside acid) was balanced by a membrane potential of about -80 mv (inside negative). From pH and uncoupler insensitivity and K(+) sensitivity, it was concluded that the experimentally caused K(+) gradient created the electric potential.Qualitatively different results were obtained on vacuoles in situ: the pH gradient is greater (1.3), the membrane potential positive inside. Uncoupler sensitivity is evidence for an enzyme system transducing protons inward as a cause for the considerable protonmotive force at the tonoplast in situ. PMID- 16662396 TI - Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX. AB - N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable delta-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 x 10(-6) molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and (59)Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX. PMID- 16662397 TI - Enzymes of amide and ureide biogenesis in developing soybean nodules. AB - Amide and ureide biogenic enzymes were measured in the plant fraction of soybean (Glycine max) nodules during the period 11 to 23 days after inoculation with Rhizobium japonicum (USDA 3I1b142). Enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, and aspartate aminotransferase, showed substantial increases in their specific activities over the time course. These increases paralleled the induction of nitrogenase activity in the bacteroid and leghemoglobin synthesis in the plant fraction. The specific activity of asparagine synthetase, however, showed a rapid decline after an initial increase in specific activity. Following the initial increases in the ammonia assimilatory enzymes, there was an increase in the activity of 5 phosphoribosylpyrophosphate amidotransferase, the enzyme which catalyzes the first committed step of de novo purine biosynthesis. This was followed by a dramatic increase in the purine oxidative enzymes, xanthine dehydrogenase and uricase. Smaller increases were observed in the activities of enzymes associated with the supply of metabolites to the purine biosynthetic pathway: phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene tetrahydrofolate dehydrogenase.The concentration of asparagine in the plant fraction decreased at the same time as the observed decrease in asparagine synthetase activity. This was followed by a recovery in plant fraction levels of asparagine in the presence of a continuing fall in the glutamine concentration and continued low asparagine synthetase activity.The data presented are consistent with initial assimilation of ammonia into glutamine and aspartate, which are metabolized by an elevation of endogenous purine biosynthetic enzymes, and then, by the induction of a specific group of purine oxidative enzymes, directed to allantoic acid production. PMID- 16662398 TI - Some Physiological Changes Occurring during the Senescence of Auxin-Deprived Pear Cells in Culture. AB - Part of the changes in the hormonal balance involved in plant senescence is due to an auxin limitation. Some of its physiological consequences are studied using pear (Pyrus communis L.) cells cultured in a continuously renewed medium in which 2,4-dichlorophenoxyacetic acid (2,4-D) was absent. In these conditions, an assessment was made of the absence of nutrient deficiency.In the period preceding cell death, the rate of respiration and ethylene production remain low, and no major changes were observed in the total protein and RNA content of the cells. Beginning around day 9, an important efflux of three amino acids (serine, threonine, and aspartic acid) occurs among which serine represents more than 52%. However, exogenous serine supplied to the medium fails to show any senescence promoting effect. At the same time, leucine uptake and incorporation sharply and simultaneously increased. The presence of 2,4-D inhibits both these phenomena and prevents cell death. It is proposed that auxin deprivation is responsible for unmasking a program of synthesis of new proteins involved in cell death. PMID- 16662399 TI - Regulation of Cytoplasmic and Vacuolar pH in Maize Root Tips under Different Experimental Conditions. AB - (31)P-Nuclear magnetic resonance spectra of perfused maize (Zea mays L., hybrid WW x Br 38) root tips, obtained at 10-minute intervals over 12 hours or longer, indicate that no cytoplasmic or vacuolar pH changes occur in these cells in the presence of 25 millimolar K(2)SO(4), which induces extrusion of 4 to 5 microequivalents H(+) per gram per hour. In contrast, hypoxia causes cytoplasmic acidification (0.3-0.6 pH unit) without a detectable change in vacuolar pH. The cytoplasm quickly returns to its original pH on reoxygenation. Dilute NH(4)OH increases the vacuolar pH more than it does the cytoplasmic pH; after NH(4)OH is removed, the vacuole recovers its original pH more slowly than does the cytoplasm. The results indicate that regulation of cytoplasmic pH and that of vacuolar pH in plant cells are separate processes. PMID- 16662400 TI - Senescence of Rice Leaves : VII. PROLINE ACCUMULATION IN SENESCING EXCISED LEAVES. AB - Proline content increased greatly in detached rice (Oryza sativa cv. Taichung Native 1) leaves during senescence. There was a slight but significant increase in proline level after one day of incubation, and, subsequently, proline accumulated relatively rapidly. By 4 days after excision, the level of proline had increased 30- to 50-fold, which is similar to the level seen in the water stressed detached rice leaves. It is unlikely that the proline accumulation in detached leaves is to be derived solely from protein hydrolysis, since the addition of l-glutamic acid increased the proline level during senescence. The proline analog, 3,4-dehydroproline, did not affect the level of proline during senescence. It seems that accumulation of proline may, at least in part, result from an increased rate of synthesis, possibly due to a disruption of the normal feedback inhibition of proline synthesis. Potassium cyanide and 2,4-dinitrophenol strongly inhibited proline accumulation, indicating that some energy compound(s) may participate in proline accumulation during senescence of excised rice leaves. PMID- 16662401 TI - Penetration of soybean root systems by abscisic Acid isomers. AB - The penetration of soybean (Glycine max L. cv. Ransom) root systems by exogenously applied isomers of abscisic acid was monitored by measuring the concentration of the chemical in the xylem exudate of root systems exposed to a three bar hydrostatic pressure difference. The cis-trans isomer penetrated more readily than the trans-trans isomer; however, up to 6 hours was needed to reach steady-state values. Exogenous abscisic acid also decreased volume flux through the root system and increased total carbon dioxide efflux from the vessel containing the root system. PMID- 16662402 TI - Abscisic Acid transport coefficients of phaseolus root systems. AB - Diffusive and convective transport coefficients of Phaseolus vulgaris L. cv. Ouray root systems for abscisic acid for (ABA) were measured. The convective coefficient (reflection coefficient or osmotic efficiency factor) sigma was determined to be 0.96 for ABA while the diffusive coefficient, omega, was found to be 1.44 x 10(-11) mole per square centimeter per second per bar. Steady-state concentrations of ABA in the root system exudates were not achieved until at least three hours after the applications suggesting either a slow saturation of binding sites or equilibration with tissues surrounding the xylem. PMID- 16662403 TI - Thermal behavior and lipid composition of cauliflower plasma membranes in relation to ATPase activity and chilling sensitivity. AB - A plasma-membrane fraction rich in ion-stimulated ATPase activity was isolated from cauliflower (Brassica oleracea L.) buds. The activity of the ATPase was dependent on Mg(2+) and stimulated 4-fold by K(+). The lipids of the membrane fraction contained 57% by weight of phospholipid, 16% glycolipid including sterol glycosides, and 27% neutral lipids. Sterols and sterol esters comprised 9% by weight of the total lipid fraction, and the m ratio of total sterol to phospholipid was 0.5. Fatty acid unsaturation of the membrane lipids was 75%. Arrhenius plots of the Mg(2+) and Mg(2+) + K(+) stimulated ATPase activity were biphasic with an increase in activation energy occurring below about 12 degrees C, a response typical of some membrane-associated enzymes of chilling-sensitive plants. No thermal transitions were detected in the membranes or membrane lipids between 0 and 30 degrees C using differential scanning calorimetry and electron spin resonance spectroscopy. This type of thermal behavior is typical of membranes of chilling-resistant plants. It was concluded that the low temperature increase in activation energy of the ion-stimulated, membrane-associated ATPase is an intrinsic property of the enzyme system and not the result of a transition in the bulk membrane lipid. PMID- 16662404 TI - Peroxidation of tobacco membrane lipids by the photosensitizing toxin, cercosporin. AB - Cercosporin, a nonspecific toxin from Cercospora species, is a photosensitizing compound which rapidly kills plant cells in the light. Cell death appears to be due to a cercosporin-mediated peroxidation of membrane lipids. Tobacco leaf discs treated with cercosporin showed a large increase in electrolyte leakage 1 to 2 minutes after irradiation with light. All tobacco protoplasts exposed to cercosporin in the light were damaged within 45 minutes. Chloroform:methanol extracts of toxin-treated suspension cultures gave positive reactions for lipid hydroperoxides in the thiobarbituric acid test. Cercosporin-treated leaf discs emitted high concentrations of ethane 12 to 24 hours after incubation in the light. Cercosporin also oxidized solutions of methyl linolenate as determined by the thiobarbituric acid assay and the emission of ethane. alpha-Tocopherol had an inhibitory effect on the cercosporin-mediated lipid peroxidation. PMID- 16662405 TI - Metabolism of l-Threonic Acid in Rumex x acutus L. and Pelargonium crispum (L.) L'Her. AB - l-Threonic acid is a natural constituent in leaves of Pelargonium crispum (L.) L'Her (lemon geranium) and Rumex x acutus L. (sorrel). In both species, l [(14)C]threonate is formed after feeding l-[U-(14)C]ascorbic acid to detached leaves. R. acutus leaves labeled with l-[4-(3)H]- or l-[6-(3)H]ascorbic acid produce l-[(3)H]threonate, in the first case internally labeled and in the second case confined to the hydroxymethyl group. These results are consistent with the formation of l-threonate from carbons three through six of l-ascorbic acid. Detached leaves of P. crispum oxidize l-[U-(14)C] threonate to l-[(14)C]tartrate whereas leaves of R. acutus produce negligible tartrate and the bulk of the (14)C appears in (14)CO(2), [(14)C]sucrose, and other products of carbohydrate metabolism. R. acutus leaves that are labeled with l-[U-(14)C]threonate release (14)CO(2) at linear rate until a limiting value of 25% of the total [U (14)C]threonate is metabolized. A small quantity of [(14)C]glycerate is also produced which suggests a process involving decarboxylation of l-[U (14)C]threonate. PMID- 16662406 TI - Low Temperature-Induced Alterations in the Chloroplast and Microsomal Membranes of Dunaliella salina. AB - The metabolic regulation of membrane lipid composition has been examined using the cell wall-less, unicellular green alga Dunaliella salina (UTEX 1644) as a model system. Low temperature stress was employed to initiate and study the regulatory response.When cultures growing logarithmically at 30 degrees C were chilled to 12 degrees C, cell division ceased for approximately 100 hours, and then the cells resumed logarithmic growth at a slower rate. The phospholipid, glycolipid and protein content, on a per cell basis, was, in each case, approximately 20% higher in cells grown at 12 degrees C. The volume of the 12 degrees C-acclimated cells was 2.8 times that of 30 degrees C-grown cells. The quantity of chloroplast membrane, as determined by morphometric analysis, was 20% greater, whereas the content of microsomal membrane material was more elevated, being approximately 2.8 times that of 30 degrees C-grown cells.Lipid compositional analyses were carried out on purified chloroplasts and microsomes isolated from Dunaliella grown at 30 and 12 degrees C and also from cells 12 and 60 hours following a shift from 30 to 12 degrees C. In both chloroplast and microsomal phospholipids fatty acid unsaturation increased during acclimation to low temperature. Generally, microsomal phospholipids responded more quickly and to a greater extent than did chloroplast phospholipids. Despite these alterations, little change in the relative proportions of phospholipid classes was observed in either cell fraction.In sharp contrast to the pattern of phospholipid change, chloroplast glycolipids responded to low temperature by significantly increasing the proportion of one specific class, digalactosyl diglycerides, relative to monogalactosyl diglycerides, while showing minimal change in fatty acid distribution within any given glycolipid class.The ease and rapidity with which Dunaliella cells can be manipulated with respect to environmental stress and isolation of intact cell organelles makes it particularly well suited for research on intermembrane lipid dynamics within the plant cell. PMID- 16662407 TI - Formulae for determination of chlorophyllous pigments extracted with n,n dimethylformamide. AB - The extraction of chlorophylls in higher plant tissue using N,N-dimethylformamide expedites the process and enables the determination of small samples with low pigment level.Absorption spectra of Chl a, Chl b, and Pchl and of their acidified derivatives, the phaeophytins, were recorded. Conversion of Chl b to its corresponding acidified product occurs much more slowly than that of Chl a and Pchl. When acidified, Pchl differs from Chl a and Chl b by the disappearance of the red band in the absorption spectrum. Specific extinction coefficients were determined and formulae for quantitative determination of pigments concentrations were developed. When concentrations of pigments are low, as in etiolated plant material, the absorption spectra of the chlorophylls can be distorted due to the presence of other substances simultaneously extracted; formulae for pigment determination under such circumstances were also derived. PMID- 16662408 TI - Transport of the herbicide 3-amino-1,2,4-triazole by cultured tobacco cells and leaf protoplasts. AB - Transport of the herbicide amitrole (3-amino-1,2,4-triazole) by suspension cultured cells and leaf protoplasts of Nicotiana tabacum cv. Wisconsin 38 has been investigated. Cells were batch-cultured and routinely assayed 3 days after subculturing. Uptake rate was pH-independent, energy independent, and culture growth phase-dependent, with growing cells exhibiting the highest rates. At a concentration of 0.2 millimolar amitrole, uptake rates yielded a Q(10) of 1.6 in the 18 to 28 degrees C temperature range. Amitrole was not concentrated over a 48 hour period and showed unsaturable kinetics over the concentration range of 0.01 to 50.0 millimolar. Uptake was not significantly influenced by a 100-fold higher concentration of several amino acids (l-Asp, gamma-amino-n-butyric acid, l-His, l Leu, l-Met, l-Trp), sucrose, glucose, fructose, and oxaloacetic acid. Uptake rate inhibition by malic acid and stimulation by NH(4)SCN were statistically significant. Amitrole was bound to cellular material, but uptake of amitrole by tobacco leaf protoplasts demonstrated that cell walls did not qualitatively influence uptake. These results indicate that amitrole enters the cells via simple diffusion. PMID- 16662409 TI - Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves. AB - Plastids at different stages of development were isolated from light-grown wheat (Triticum aestivum, var. Maris Dove) seedling leaves, and the average chloroplast DNA (cpDNA) per plastid at each developmental stage was measured directly. In the earliest stages of development, the number of plastids per cell and the amount of cpDNA per cell increased with cell age, but cpDNA per plastid remained constant at between 800 and 1,000 genome copies per plastid. After this phase, plastids per cell continued to increase, but cpDNA per plastid decreased. Subsequently, both plastids per cell and cpDNA per plastid remained constant as cell age increased, the final DNA content being approximately 300 genome copies per plastid. These results are related to previous reports of cpDNA changes during the development of dicotyledonous plants, and to theories about the regulation of chloroplast numbers per cell. PMID- 16662410 TI - Development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of barley and its virescens mutant. AB - The development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of greening wild-type barley (Hordeum vulgare L. cv. Gateway) and its virescens mutant were studied. The rate of chlorophyll accumulation per plastid was faster in the wild-type than in the mutant seedlings upon illumination after 6 days of etiolation, but was not different after 8 days. Although the protein content per plastid did not vary during greening, there was a change in the sodium dodecyl sulfate-polyacrylamide gel polypeptide profiles. High molecular weight proteins of 96,000 and 66,000 decreased whereas those at 34,000, 27,000 and 22,000 increased in relative quantity as a function of greening. The fully greened mutant seedlings were not deficient in the light-harvesting chlorophyll protein complex (LHC) or the reaction centers of photosystem I and photosystem II. Photosystem I-associated photochemical activities appeared within the first hour of plastid development and photosystem II associated activities and O(2) evolution within the next 6 hours. In all cases, the developmental rates per unit protein were slower in the mutant following 6 days of etiolation, but no differences between the two genotypes could be seen after 8 days due to a decrease in the developmental rate of the wild-type chloroplasts. An increase in photosynthetic unit size associated with plastid morphogenesis was faster in the wild-type seedlings after 6 days, but again the difference was negligible after 8 days. It was concluded that no single measured photochemical parameter is affected by this mutation, but rather, all aspects of chloroplast development are affected similarly by an overall reduction in the rate of chloroplast morphogenesis. This mutant, therefore, undergoes the normal pattern of proplastid to chloroplast development, but at a markedly reduced rate. PMID- 16662411 TI - Uptake and Degradation of Cyclic AMP by Chloronema Cells. AB - Suspension cultures of intact chloronema cells of the moss Funaria hygrometrica take up [(3)H]cAMP and degrade it rapidly. The increase in total radioactivity accumulated by the cells was linear up to 30 minutes. Initially, the major degradation products were 5'-AMP and adenosine, but later predominantly ADP and ATP. In spite of rapid degradation, the amount of extracellularly applied cAMP retained by the cells is about 4-fold higher than the maximum endogenous level of cAMP reported previously (Handa, Johri 1977 Plant Physiol 59: 490-496). The uptake showed a distinct dependence on the density of the culture. Cells at a lower cell density (1-2 milligrams per milliliter) accumulated 4 to 6 times more radioactivity than the cells at high density (>10 milligrams per milliliter). The cyclic nucleotide phosphodiesterase (cNPDE) activity of whole cells (18 milliunits per milligram protein) was comparable to that of protoplasts (23 milliunits per milligram protein), but about 4-fold lower than that of lysed protoplasts (80 milliunits per milligram protein), indicating an intracellular degradation of cAMP by chloronema cells. PMID- 16662412 TI - Light modulation of phosphofructokinase in pea leaf chloroplasts. AB - Chloroplastic phosphofructokinase, phosphorylase, phosphoglucomutase, and phosphoglucoisomerase in peas are light inactivated. The effect of light on phosphofructokinase is mimicked by dithiothreitol. DCMU, arsenite, and sulfite inhibit light modulation of the enzyme. No effect of light inactivation on the K(m) (fructose-6-P) or pH optima of phosphofructokinase was observed. PMID- 16662413 TI - Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts. AB - Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H(2)O(2)-dependent oxidation of ascorbate and ascorbate-dependent reduction of H(2)O(2). The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K(m) value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN(3), KCN, and 8-hydroxyquinoline but not ZnCl(2) or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H), 2 millimolar l-ascorbate, and substrate concentrations of oxidized or reduced glutathione, catalyzed O(2) evolution when H(2)O(2) was added. Since the reaction was not inhibited by 0.1 millimolar NaN(3) and did not occur in the dark, it was concluded that catalase was not involved. Light-plus H(2)O(2)-dependent O(2) evolution consisted of two distinct phases. The first phase was ascorbate-dependent and typically represented 10% of the total amount of O(2) evolved. The second phase was dependent on ascorbate and glutathione. The properties of the second phase were consistent with the operation of light coupled glutathione reductase sequentially coupled to glutathione dehydrogenase and ascorbate peroxidase. PMID- 16662414 TI - Sequestration of pea reserve proteins by rough microsomes. AB - Free polysomes, polysomes released from membranes, and rough microsomal vesicles isolated from developing cotyledons of Pisum sativum L. cv. Burpeeana were used to direct cell-free protein synthesis in a wheat germ system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the polypeptide products had molecular weights ranging from 12,000 to 74,000. Some of the polypeptides migrated during electrophoresis with the same mobility as polypeptides present in legumin and vicilin preparations. By the use of rabbit antibodies raised against pea reserve proteins it was established that polysomes released from membranes and rough microsomes directed the synthesis of polypeptides that were related to reserve proteins whereas free polysomes did not.Centrifugation studies indicated that the majority of the radioactivity incorporated by rough microsomes was specifically associated with the microsomes. The incorporated radioactivity of sedimented microsomes was not released by treatment with KCl and was resistant to proteolysis unless detergent was present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the sequestered translation products were related to pea reserve proteins. It is concluded that the reserve proteins of pea cotyledons are synthesized exclusively by membrane-bound polysomes and that they are sequestered within the rough endoplasmic reticulum. PMID- 16662415 TI - Photoactivation of the Water-Oxidation System in Isolated Intact Chloroplasts Prepared from Wheat Leaves Grown under Intermittent Flash Illumination. AB - Photoactivation of the latent oxygen-evolving system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination was investigated, and the following results were obtained: (a) The water-oxidation activity generated on illuminating the isolated intact chloroplasts was as high as that generated in intact leaves, indicating that all the machinery necessary for the activity generation is assembled within intact chloroplasts. (b) The generation of water-oxidation activity was accompanied by enhancement of the activity of diphenylcarbazide-oxidation, and both processes share the same photochemical reaction but with respective rate-limiting dark reactions of different efficiencies. (c) A23187, an ionophore for divalent cations, strongly inhibited the generation of water-oxidation activity but did not affect the activity once generated, which suggested that Mn atoms in the chloroplasts are susceptible to the ionophore before photoactivation but turn immune after photoactivation. (d) The generation of water-oxidation activity was not affected by the inhibitors of ATP formation and CO(2) fixation, but was inhibited by nitrite, methylviologen and phenylmercuric acetate which suppress or inhibit the reduction of ferredoxin in intact chloroplasts. It was inferred that some factor(s) probably present in stroma to be reduced by PSI photoreaction is involved in the process of photoactivation. PMID- 16662416 TI - Spin-Probe Studies during Freezing of Cells Isolated from Cold-Hardened and Nonhardened Winter Rye : MOLECULAR MECHANISM OF MEMBRANE FREEZING INJURY. AB - Mesophyll cells isolated from cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) were spin-labeled with the fatty-acid spin probe N-oxyl-4,4 dimethyloxazolidine 5-ketostearic acid. The probe was intercalated within the cellular membranes and changes in probe motion were followed during extracellular freezing of the cells. A correlation was observed between the lethal freezing temperatures (LT(50)) of the cells and the maximum hyperfine splitting value achieved by the incorporated probe. Rigid limit spectra indicated that a more ordered average packing was attained by membranes of hardened cells which survived freezing to lower temperatures.Nonhardened cells fixed with osmic acid at lethal freezing temperatures, in the frozen state, showed both conversion of normal bilayered cellular membrane ultrastructure to an amorphous state and condensation of cellular membranes to form densely packed multibilayered vesicles. Taken together with the spin-labeling data, these results suggest that at least one molecular mechanism of extracellular freezing injury involves the irreversible conversion of planar membrane bilayers to structures having less ordered packing and increased surface curvatures. PMID- 16662417 TI - Synthesis and accumulation of nitrite in soybean nodules supplied with nitrate. AB - Nodulated soybean plants (Glycine max [L.] Merr) were grown in sand culture without combined N or with a continuous supply of nitrate in nutrient solution. Moderate nitrate concentration (30 milligrams N per liter) had little effect on nodule weight/plant while high nitrate concentration (100 milligrams N per liter) depressed nodule weight/plant by 70 to 80% with harvests 30 to 60 days after planting and initiation of nitrate treatments.The effect of nitrate supply on ammonium, amino, and ureide nitrogen concentrations in nodules was small and inconsistent. In contrast, nitrate and nitrite concentrations in nodules were directly proportional to nitrate supply and inversely proportional to nodule weight/plant. Correlations between nitrate or nitrite concentration in nodules and nodule weight/plant were highly significant.Cytosol from soybean nodules was found to contain NADH-dependent nitrate reductase activity (typical activity was 0.1 micromole per milligram protein x hour). A Rhizobium japonicum mutant (derived from strain 61A76) lacking nitrate reductase was employed to show that the cytosol enzyme activity is of host origin. Growth of nodules formed by the mutant lacking nitrate reductase was inhibited by nitrate. These nodules did contain nitrite although concentrations of nitrite (about 0.3 microgram N per gram fresh weight) were low relative to nitrite concentrations (about 1.5 microgram N per gram fresh weight) in nodules formed by R. japonicum strain 61A76. The overall results support the idea that the depression of legume nodule growth by nitrate is directly related to the metabolism of nitrate in nodules. PMID- 16662418 TI - Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria. AB - Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination. PMID- 16662419 TI - Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L. AB - The respiration of dark-grown Nicotiana glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibited by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5. PMID- 16662420 TI - Superoxide Dismutase: A POSSIBLE PROTECTIVE ENZYME AGAINST OZONE INJURY IN SNAP BEANS (PHASEOLUS VULGARIS L.). AB - An experimental chemical N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea (EDU), is an effective protectant against acute and chronic foliar injury due to ozone (0(3)) when sprayed on intact leaves or supplied to the plants through soil application. An 0(3)-sensitive snap bean cultivar (Phaseolus vulgaris L. ;Bush Blue Lake 290') was systemically treated with EDU (0, 25, 50, and 100 milligrams per 15-centimeter diameter pot) to determine if EDU-induced or activated protective oxyradical and peroxyl scavenging enzymes. EDU-enhanced tolerance to O(3) injury always correlated with increases in superoxide dismutase (SOD) and catalase activities in the leaves. Peroxidase levels correlated more closely with foliar injury. Greater SOD levels in young leves compared to older leaves were associated with lower ozone sensitivities in these tissues.Polyacrylamide slab gel electrophoresis separations and specific determinations of SOD activity showed that EDU-treated plants possessed markedly greater SOD activity than non treated plants. Tolerant plant tissues may have enhanced enzyme scavenging capabilities for the protection against toxic oxyradicals. Experimental confirmation for the oxyradical theory for O(3) phytotoxicity and SOD involvement in the detoxification process are presented. PMID- 16662421 TI - Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex. AB - The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf(*)-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [(35)S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf(*)-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts. PMID- 16662422 TI - Pyrophosphorylases in Solanum tuberosum: III. PURIFICATION, PHYSICAL, AND CATALYTIC PROPERTIES OF ADPGLUCOSE PYROPHOSPHORYLASE IN POTATOES. AB - ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C(3) NH(2)). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg(2+) are reported. The enzyme V(max) is stimulated about 1.5- to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed. PMID- 16662423 TI - Polar lipid composition of a plastid ribosome-deficient barley mutant. AB - Green and white leaves of the barley mutant line ;albostrians' were compared for their polar lipid content and fatty acid composition. The mutant plastids of the white leaves have a double-layered envelope, but in contrast with the normal chloroplasts, lack 70 S ribosomes and thylakoids. In the green leaves, the amount of monogalactosyldiacylglycerol (MGDG) consistently exceeds the amount of digalactosyldiacylglycerol (DGDG) and the amount of galactolipids exceeds the amount of phospholipids. In contrast, in white leaves the amount of DGDG exceeds the amount of MGDG and the amount of phospholipids exceeds the amount of galactolipids. In white leaves, the galactolipid composition reflects the plastid envelope composition which is rich in DGDG, whereas in green leaves the galactolipid composition reflects the thylakoid composition which is rich in MGDG. These results demonstrate the likelihood that all the enzymes involved in galactolipid, sulfolipid and fatty acid synthesis are coded by the nuclear genome. PMID- 16662424 TI - Deposition of Matrix and Crystalloid Storage Proteins during Protein Body Development in the Endosperm of Ricinus communis L. cv. Hale Seeds. AB - Protein bodies within the endosperm of castor bean (Ricinus communis L. cv. Hale) seeds arise from numerous small vacuoles which progressively become filled with storage protein, of which the crystalloid proteins make up approximately 70%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the crystalloids are a family of at least four proteins which reduce to two complementary groups after 2-mercaptoethanol treatment. The matrix, which comprises the remainder, has two major components, the soluble albumins and the lectins. The lectins are the only glycoproteins within the mature protein body. Both cytochemical staining and SDS-PAGE indicate that the synthesis of the crystalloid and the majority of matrix proteins begins some 20 days after pollination. Additionally, the crystalloid proteins are synthesized concurrently, whereas there is temporal variation in the synthesis of matrix proteins. PMID- 16662425 TI - Relative abundance of ureides and nitrate in plant tissues of soybean as a quantitative assay of nitrogen fixation. AB - The relationship between the relative abundance of ureides ([ureide-N/ureide-N plus nitrate-N] x 100) in the shoot axis (stems plus petioles), nodulated roots and leaflets of "Bragg" soybean (Glycine max [L.] Merrill) and the symbiotic dependence of these plants was examined under glass-house conditions. Plants, inoculated with effective Rhizobium japonicum CB1809, were grown with their roots exposed continuously to a nutrient solution containing either 0, 1.5, 3.0, 6.0 or 12.0 millimolar NO(3)-N per liter. Nodulation and N(2)-acetylene fixation were correlated inversely with the level of nitrate. Seasonal acetylene reduction profiles for each of the nitrate treatments were integrated and the symbiotic dependence ([N(2) fixed per total plant N] x 100) determined using a conversion ratio of 1.5:1 (acetylene reduced:N(2) fixed), calculated from the zero NO(3) treatment. Examination of the nitrogenous solutes of the shoot axis and nodulated roots showed linear relationships between the relative abundance of ureides and the symbiotic dependence of the plants. Two standard curves, depicting these relationships during vegetative and reproductive growth, were drawn for each plant part. The overriding effect of plant age invalidated any attempt to develop a standard relationship for leaflets. Data from two diurnal studies suggested that relative ureides were insensitive to diurnal fluctuations, thus simplifying sampling procedures. Plant material could be stored at ambient temperatures (20 30 degrees C) for up to 24 h without affecting the relative concentration of ureides and nitrate. It is suggested that the shoot axis provides the most suitable target organ when using this technique as a quantitative assay for N(2) fixation because of ease of sampling of these tissues, especially with field grown plants. PMID- 16662426 TI - Metabolism of 2,4-dichlorophenoxyacetic Acid in 2,4-dichlorophenoxyacetic Acid resistant soybean callus tissue. AB - Three 2,4-dichlorophenoxyacetic acid (2,4-D) -resistant root callus tissue lines of Glycine max L. Merrill var. Acme were derived by culturing callus tissue 2 to 6 months on 40 milligrams per liter 2,4-D and designated 40R, 40B, and 40C. Tissue line 40R had a lower level of 2,4-D uptake in 2-week-old tissue which disappeared in 3.5-week-old tissue and less free 2,4-D following incubation for 24 hours with [1-(14)C]2,4-D. This tissue line accumulated more hydroxylated glycosides of 2,4-D than did nonresistant tissue. Tissue line 40B showed no difference in uptake of 2,4-D when compared to nonresistant tissue but it did contain less free 2,4-D and more hydroxylated glycosides. The metabolism of 2,4-D in the 40C tissue line did not differ significantly from nonresistant tissue although uptake was less. The 40R line reverted to the same 2,4-D sensitivity as Acme root callus following six transfers on 10 micromolar naphthaleneacetic acid medium. The altered 2,4-D uptake and metabolism characteristic of 40R were also lost. The levels of amino acid conjugates of 2,4-D in the resistant root callus tissue lines were either lower or not significantly different from the Acme tissue lines. Therefore, variations in uptake and metabolism of 2,4-D do not wholly explain the resistance of the derived tissue lines, and perhaps modification of the active site or compartmentation is involved. PMID- 16662427 TI - Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings. AB - The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols. PMID- 16662428 TI - A Thioredoxin-Mediated Activation of Glutamine Synthetase and Glutamate Synthase in Synchronous Chlorella sorokiniana. AB - The effects of thioredoxin, dithioerythrol, and mixtures of both on enzymes involved in N metabolism of Chlorella sorokiniana have been studied. Glutamine synthetase, inactivated in vivo, was activated 8-fold by thioredoxin and dithioerythrol. By the same treatment, the activity of glutamate synthase was stimulated nearly 4-fold. Thus, two key enzymes of N metabolism were shown to be regulated via thioredoxin. The enzymes of the nitrate reducing system, i.e. nitrate reductase and nitrite reductase, were not affected by thiols. From these results, a model of NO(3) (-) metabolism is put forward which considers the regulating effect of light. PMID- 16662429 TI - Recycling of 5'-methylthioadenosine-ribose carbon atoms into methionine in tomato tissue in relation to ethylene production. AB - The ribose moiety of 5'-methylthioadenosine (MTA) is metabolized to form the four carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-(14)C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C(2)H(4) and other unidentified compounds. However, when [U-(14)C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C(2)H(4), but not MTA. This suggests that C(2)H(4) formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O(2) dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue. PMID- 16662431 TI - Cytochrome P-450-Dependent omega-Hydroxylation of Lauric Acid by Microsomes from Pea Seedlings. AB - Microsomes from apical buds of pea (Pisum sativum L. var. Telephone a rames) seedlings hydroxylate lauric acid at the omega-position. This oxidation is catalyzed by a cytochrome P-450 enzyme which differs from laurate hydroxylases previously described in microorganisms and mammals by its strict substrate specificity and the ability of low NADH concentrations to support unusually high oxidation rates. The apparent K(m) for lauric acid was 20 micromolar. NADPH- and NADH-dependent laurate hydroxylation followed non-Michaelian kinetics with apparent K(m) values ranging from 0.2 to 28 micromolar for NADPH, and 0.2 to 318 micromolar for NADH. When induced by the photomorphogenic photoreceptor phytochrome, the time course for the enhancement of laurate omega-hydroxylase was totally different from that of the cinnamic acid 4-hydroxylase, providing evidence for the existence of multiple cytochrome P-450 species in pea microsomes. PMID- 16662430 TI - Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings. AB - Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP [(14)C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K(d)) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man(7)GlcNAc(2), Man(5)GlcNAc(2), Man(3)GlcNAc(2), Man(2)GlcNAc(2), and ManGlcNAc(2). These oligosaccharides were treated with endoglucosaminidase H and alpha- and beta-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB(3)H(4) reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manbeta-GlcNAc-GlcNAc; D, Manalpha1-->3Manbeta-GlcNAc-GlcNAc; C, Manalpha1- >2Manalpha1-->3Manbeta-GlcNAc-GlcNAc; B, Manalpha1-->2Manalpha1-->2Manalpha1--> 3(Manalpha1-->6)Manbeta-GlcNAc-GlcNAc; and A, Manalpha1-->2Manalpha1--> 2Manalpha1-->3(Manalpha1--> [Manalpha1-->6]Manalpha1-->6) Manbeta-GlcNAc-GlcNAc. The synthesis of the Man(7)GlcNAc(2) was greatly diminished when tunicamycin (10 mug/ml) was added to the incubation mixtures. PMID- 16662432 TI - Adaptive Changes in ATPase Activity in the Cells of Winter Wheat Seedlings during Cold Hardening. AB - A cytochemical study of ATPase activity in the cells of cold hardened and nonhardened winter wheat (Triticum aestivum L. cv. Nongke No. 1) seedlings was carried out by electron microscopic observation of lead phosphate precipitation. ATPase activity associated with various cellular organelles was altered during cold hardening. (a) At 22 degrees C, high plasmalemma ATPase activity was observed in both cold hardened and nonhardened tissues; at 5 degrees C, high activity of plasmalemma ATPase was observed in hardened tissues, but not in unhardened tissues. (b) In nonhardened tissues, tonoplast and vacuoles did not exhibit high ATPase activity at either 22 or 5 degrees C, while in hardened tissues high activity was observed at both temperatures. (c) At 5 degrees C, ATPase activity of nucleoli and chromatin was decreased in hardened tissues, but not in nonhardened tissues. It is suggested that adaptive changes in ATPase activity associated with a particular cellular organelle or membrane may be associated with the development of frost resistance of winter wheat seedlings. PMID- 16662433 TI - Relationships of leaf Fatty acids to cold hardening of citrus seedlings. AB - Three cultivars of citrus with different sensitivities to freezing temperatures (citron, Citrus medica L.; rough lemon, C. limon Burm. F; sour orange, C. aurantium L.) were cold hardened for 4 weeks. Lipids from leaves of hardened and control seedlings were fractionated and analyzed for fatty acids. The absolute amount of triglycerides and phospholipids increased in the leaves upon hardening. With hardening, total linoleic acid also increased 141% in citron, 210% in rough lemon, and 233% in sour orange. Specific increases in linoleic acid were found in triglycerides, in the four phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol), and in neutral lipids more polar than triglycerides. Trans-3-hexadecenoic acid was found only in phosphatidylglycerol. PMID- 16662434 TI - Effects of exogenous ethylene on ethylene production in citrus leaf tissue. AB - Exogenous ethylene stimulated ethylene production in intact citrus (Citrus sinensis L. Osbeck cv. "Washington Navel") leaves and leaf discs following a 24 hour exposure. Studies with leaf discs showed that ethylene production decreased when ethylene was removed by aeration. The extent of stimulation was dependent upon the concentration of exogenous ethylene (1-10 microliters per liter). Silver ion blocked the autocatalytic effect of ethylene at concentrations of 0.5 millimolar and lower, but increased ethylene production at higher concentrations. The stimulating effect of ethylene resulted from the enhancement of both 1 aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. Whereas autocatalysis was evident following 24 hours incubation, autoinhibition of wound- and mannitol-induced ethylene production was observed during the first 24-hour incubation. Ethylene treatment during this period resulted in a marked decrease in ACC levels and ethylene production rates. Furthermore, in leaf discs treated for 24 hours with ethylene, ethylene production rates increased greatly during the first 2 hours after removal of exogenous ethylene by aeration. This increase was eliminated if the discs were transferred to propylene instead of air, indicating that the autocatalytic effect of ethylene is counteracted by its autoinhibitory effect. It is suggested that autocatalysis involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to ethylene, whereas autoinhibition involves suppression of the activity of these two enzymes. PMID- 16662435 TI - Stimulation of ethylene production in citrus leaf discs by mannitol. AB - Wound ethylene formation induced in leaf tissue of citrus (Citrus sinensis [L.] Osbeck cv. "Washington Navel") by excision was significantly stimulated by mannitol after a lag period of about 6 hours. The extent of stimulation was dependent upon the concentration of mannitol (10 to 100 millimolar). This increased ethylene production was not simply due to osmotic effect or water stress as other osmoticums tested failed to exert such an effect. The stimulatory effect of mannitol resulted from both the enhancement of 1-aminocyclopropane-1 carboxylic acid (ACC) formation and the conversion of ACC to ethylene. The effect on the latter step was particularly pronounced in aged discs. The use of labeled mannitol showed that it was taken up by the leaf discs, utilized for respiration, and metabolized to sucrose, but no radioactivity was detected in the ethylene. PMID- 16662436 TI - Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation. AB - The biosynthetic mechanism of alpha-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmaze) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of alpha-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of alpha-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of alpha-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of alpha-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of alpha-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of alpha-amylase were found to consist of three isozymes. The in vitro translated precursor forms of alpha-amylase consisted of four multiple components. These results indicate that heterogeneity of alpha-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice alpha-amylase isozymes are encoded by multiple genes. PMID- 16662437 TI - Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis. AB - Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO(2) or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP(+) is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O(2) to H(2)O(2). Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations. PMID- 16662438 TI - Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.). AB - Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd(2+) at concentrations above 1 micromolar. Cd(2+)-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd(2+), which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca(2+), present during a 2-hour preincubation, reduced the effect of Cd(2+) on leakage and ACC conversion. This suggests that Cd(2+) exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed. PMID- 16662439 TI - Potassium nutrition and translocation in sugar beet. AB - The effect of increased net foliar K(+) accumulation on translocation of carbon was studied in sugar beet (Beta vulgaris, L. var. Klein E and US H20) plants. Net accumulation of recently absorbed K(+) was studied by observing arrival of (42)K(+) per unit area of leaf. Labeled K(+) was added to give an initial concentration at 2 or 10 millimolar K(+) in mineral nutrient solution. Because the newly arrived K(+) constitutes a small part of the total leaf K(+) in plants raised in 10 millimolar K(+), export of (42)K(+) by phloem was negligible over the 2- to 3-day period; consequently, accumulation is a measure of arrival in the xylem. In leaves from plants in 2 millimolar K(+), export by the phloem was estimated to be of the same order as import by the xylem; K(+) per area was observed to remain at a steady-state level. Increasing the supply of K(+) to 10 millimolar caused arrival in the xylem to increase 2- to 3-fold; K(+) per area increased gradually in the mature leaves. Neither net carbon exchange nor translocation of sugar increased in response to a faster rate of arrival of K(+) over a 6- to 8-hour period. In the absence of short-term effects, it is suggested that K(+)-promoted increase in synthetic metabolism may be the basis of the increased carbon assimilation and translocation in plants supplied with an above minimal level of K(+). PMID- 16662440 TI - Cytokinin structure-activity relationships and the metabolism of N-(delta isopentenyl)adenosine-8-C in phaseolus callus tissues. AB - The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N(6)-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern x Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N(6) isopentyladenine, N(6)-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N(6)-[Delta(2)-isopentenyl]adenine, N(6)-[Delta(2) isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure activity relationships were correlated with differences in the metabolism of (14)C-N(6)-(Delta(2)-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of (14)C-N(6)-(Delta(2)-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites. PMID- 16662441 TI - Photosynthetic o(2) exchange kinetics in isolated soybean cells. AB - Light-dependent O(2) exchange was measured in intact, isolated soybean (Glycine max. var. Williams) cells using isotopically labeled O(2) and a mass spectrometer. The dependence of O(2) exchange on O(2) and CO(2) was investigated at high light in coupled and uncoupled cells. With coupled cells at high O(2), O(2) evolution followed similar kinetics at high and low CO(2). Steady-state rates of O(2) uptake were insignificant at high CO(2), but progressively increased with decreasing CO(2). At low CO(2), steady-state rates of O(2) uptake were 50% to 70% of the maximum CO(2)-supported rates of O(2) evolution. These high rates of O(2) uptake exceeded the maximum rate of O(2) reduction determined in uncoupled cells, suggesting the occurrence of another light-induced O(2) uptake process (i.e. photorespiration).Rates of O(2) exchange in uncoupled cells were half-saturated at 7% to 8% O(2). Initial rates (during induction) of O(2) exchange in uninhibited cells were also half-saturated at 7% to 8% O(2). In contrast, steady-state rates of O(2) evolution and O(2) uptake (at low CO(2)) were half-saturated at 18% to 20% O(2). O(2) uptake was significantly suppressed in the presence of nitrate, suggesting that nitrate and/or nitrite can compete with O(2) for photoreductant.These results suggest that two mechanisms (O(2) reduction and photorespiration) are responsible for the light-dependent O(2) uptake observed in uninhibited cells under CO(2)-limiting conditions. The relative contribution of each process to the rate of O(2) uptake appears to be dependent on the O(2) level. At high O(2) concentrations (>/=40%), photorespiration is the major O(2)-consuming process. At lower (ambient) O(2) concentrations (/= 15). In vitro urease synthesis is directed by a large RNA (3,000-3,300 nucleotides). Urease synthesis occurs throughout the normal protein biosynthetic phase of the developing seed. Surprisingly, the activity/antigen ratios of urease increase throughout development. Urease appears to be in a more highly polymerized state in mature beans versus beans in early development.During the 55 days from pollination to maturity, urease specific antigen (antigen versus total seed protein) is greatest on the 20th day, representing 0.6% of total extractable protein. Its synthesis proceeds until the end of the protein biosynthetic phase, approximately day 40. In contrast, the appearance of urease enzyme activity lags that of antigen during early development (11-20 days) and plateaus in late development. Mixing experiments suggest no role for putative urease inhibitors or activators during development. However, several electrophoretically slow migrating forms are unique to the urease of mature beans. It is not known if these are more active species.An active urease species exhibits an RNAse sensitive cosedimentation with a heavy polyribosome class (n >/= 15). Polyadenylated RNA, size-fractionated to 3,000 to 3,300 bases, directed the synthesis in vitro of a major translational product electrophoretically and immunologically similar to the in vivo-synthesized urease subunit. PMID- 16662444 TI - Stereospecific conversion of 1-aminocyclopropanecarboxylic Acid to ethylene by plant tissues : conversion of stereoisomers of 1-amino-2 ethylcyclopropanecarboxylic Acid to 1-butene. AB - Inasmuch as the molecule of 1-aminocyclopropanecarboxylic acid (ACC) possesses reflective symmetry but lacks rotational symmetry, the two chemically alike methylene groups can be distinguished by a stereospecific enzyme. To determine whether ACC conversion to ethylene by plant tissues proceeds in a stereospecific fashion, the four stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid (AEC) were administered to postclimacteric apple (Malus sylvestris Mill., var. Golden Delicious), excised preclimacteric cantaloupe (Cucumis melo L., var. reticulatis Naud cv. PMR-45), and etiolated mung bean (Vigna radiata L., Wilczek, var. Berken) hypocotyls. In each case (1R,2S)-AEC was the preferred substrate yielding 1-butene. In contrast, all AEC isomers were converted equally well to butene by chemical oxidation using NaOCl. Both ACC and AEC appear to be substrates for the same enzyme since both reactions are inhibited in parallel by N(2) or Co(2+), both reactions are induced in parallel by excision, and when both substrates are present simultaneously each will act as an inhibitor with respect to the other. The aforementioned observations indicate that ACC is stereospecifically converted to ethylene. For AEC to be the most active precursor of 1-butene, the ethyl substituent should be trans to the carboxyl group and the pro-(S) methylene group should be unsubstituted. This observation leads to the suggestion that the enzyme interacts with amino, carboxyl, and pro-(S) methylene groups, a configuration corresponding to a l-amino acid. This view is consistent with the observation that the l-forms of alanine and methionine inhibit the conversion of ACC to ethylene more than the corresponding d-amino acids in the mung bean hypocotyl system. PMID- 16662445 TI - Effects of high-temperature stress on various biomembranes of leaf cells in situ and in vitro. AB - The sensitivity of photosynthetic and respiratory functions to supraoptimal temperature stress was compared after heating of leaves, protoplasts and membrane systems of spinach (Spinacia oleracea L. cv. Monatol) and lettuce (Valerianella locusta [L.] Betcke) in situ and in vitro.After heating of whole leaves or protoplasts, endogenous respiration was not or only slightly affected at temperatures which caused a marked decrease of photosynthesis. This was manifested when mitochondria and thylakoids were isolated from heat-treated leaves. In the presence of exogenous substrates, mitochondrial electron transport and phosphorylation were even somewhat stimulated compared to the controls.Inactivation of net CO(2) uptake of whole leaves following heat stress and of the photochemical activities of chloroplast membranes isolated from heat treated leaves of the same origin occurred nearly simultaneously. In protoplasts, photosynthesis was inactivated at temperatures far below those which caused drastic changes in the integrity of the tonoplast and the plasmalemma. This indicates that damage occurring within the chloroplasts rather than alterations in the compartmentation of the cell is responsible for the high sensitivity of photosynthesis to supraoptimal temperature stress.Mitochondria and thykaloids isolated from the same preparation of intact leaves under comparable conditions and subjected to heat treatment in vitro, however, were inactivated nearly in the same temperature range. Thus, mitochondria are much more stable within their cytoplasmic environment. PMID- 16662446 TI - Regulation by Lipids of Plant Microsomal Enzymes: III. Phospholipid Dependence of the Cytidine-Diphospho-Choline Phosphotransferase of Potato Microsomes. AB - Cytidine-diphospho-choline diacyl-glycerol phosphorylcholine phosphotransferase activity was demonstrated in potato (Solanum tuberosum L.) microsomes and the incorporation of cytidine-diphospho[(14)C]choline into phosphatidylcholine was characterized by the time course of (14)C incorporation and the effect of microsomal protein concentration on choline incorporation.Potato microsomes were progressively delipidated by treatments (2 min at 0 degrees C) with increasing amounts of phospholipase C from Bacillus cereus. A decrease in choline phosphotransferase activity was observed in parallel with the progressive hydrolysis of membrane phospholipids. A 70% (or more) phospholipid hydrolysis provoked the total inactivation of the enzyme.Adding back exogenous phospholipids (in the form of liposomes) to phospholipase C-treated membranes restored the enzymic activity. Restoration could be obtained with egg yolk phospholipids as well as with potato phospholipids. Restoration was time dependent and completed after 10 minutes; restoration was also dependent on the quantity of liposomes added to lipid-depleted membranes: the best restorations were obtained with 1 to 2.5 milligrams of phospholipid per mg of microsomal protein; higher phospholipid to protein ratios were less efficient or inhibitory.These results clearly demonstrate the phospholipid dependence of the cytidine-diphospho-choline phosphotransferase from potato microsomes. PMID- 16662447 TI - A simple method for the determination of resistance to gas diffusion in plant organs. AB - A simple method was developed for the determination of resistance coefficients for ethylene diffusion in plant tissues based on the kinetic analysis of the efflux of preloaded ethane gas. Efflux curves were analyzed to obtain first-order rate constants and resistance coefficients. Resistance coefficients determined by the ethane efflux and steady-state methods were found to agree well. Employing the ethane efflux method, it was shown that over 97% of gas exchange of tomato (Lycopersicum esculentum Mill., cv. ;Ace') fruits occurs through the stem scar. The resistances to diffusion of tomato skin and stem scar were found to be 280,000 and 300 seconds per centimeter, respectively; the combined resistance of intact tomato fruits was approximately 7,800 seconds per centimeter. The ethane efflux method was employed to show that plastic shrink-wrapping of English cucumbers (Cucumis sativus L. var anglicus Bailey) increased the resistance to ethane diffusion from 1.1 x 10(3) to 23 x 10(3) seconds per centimeter. PMID- 16662448 TI - Diurnal changes in the chilling sensitivity of seedlings. AB - Seedlings of tomato (Lycopersicon esculentum, Mill.) varied diurnally in their sensitivity to chilling temperatures. If chilled near the end of the dark period when they were most sensitive, the time taken to kill half of the seedlings was approximately 3 days, whereas in samples taken 4 hours after the onset of dark, a period of 6 days of chilling was required. Sensitivity dropped rapidly after the onset of the light period. This rhythm was exogenously controlled by the diurnal changes in light, rather than in the temperature. The susceptibility of predawn seedlings could be reduced by exposure to light, by water stress, or by abscisic acid applied to the leaves. However, the subsequent changes in sensitivity to chilling did not correlate with stomatal aperture. Six other chilling-sensitive species showed similar diurnal changes in their chilling sensitivity. PMID- 16662449 TI - Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids. AB - The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7 degrees C and -3 degrees C with membrane lipids from plants grown at 45 degrees C/32 degrees C (day/night) and 20 degrees C/15 degrees C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7 degrees C or -3 degrees C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2 degrees C and 33 degrees C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29 degrees C (or 33 degrees C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7 degrees C (or -3 degrees C) its completion. Because of the present results, however, this interpretation needs to be modified.Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes. PMID- 16662450 TI - delta-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity. AB - delta-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t(1/2) = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of 1 millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t(1/2) = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light- or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3 (3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic O(2) production. The decrease that occurs on dark-to-light transfer can be diminished by 12- or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular regions, which thereby supplants the necessity for ALA formation via the ALA synthase route. PMID- 16662451 TI - Isolation and Quantitation of beta-d-Glucopyranosyl Abscisate from Leaves of Xanthium and Spinach. AB - From previous work (Zeevaart 1980 Plant Physiol 66: 672-678) Xanthium leaves are known to contain a high level of alkali-hydrolyzable conjugated abscisic acid. This abscisic acid conjugate has been isolated and identified by mass spectrometry, nuclear magnetic resonance, and chemical and enzymic degradation techniques, as the glucosyl ester of abscisic acid, beta-d-glucopyranosyl abscisate. The glucosyl ester of abscisic acid was the only abscisic acid conjugate found in Xanthium leaves. It was also isolated from spinach leaves.An insignificant amount of the glucosyl ester of abscisic acid partitioned into diethyl ether, whereas 12% partitioned into ethyl acetate. Consequently, removal of abscisic acid by partitioning with ethyl acetate will result in considerable losses of the glucosyl ester of abscisic acid from the aqueous phase. Diethyl ether is, therefore, recommended for separation of abscisic acid and the glucosyl ester of abscisic acid by solvent partitioning.A method for quantitation of the glucosyl ester of abscisic acid as the tetraacetate derivative by gas-liquid chromatography with an electron capture detector was developed. The level of beta d-glycopyranosyl abscisate in Xanthium leaves increased from 3.6 nanomoles per gram fresh weight in turgid leaves to 22.9 nanomoles in leaves from plants subjected to seven wilting-recovery cycles. beta-d-glycopyranosyl abscisate in Xanthium leaves may be a stable end product of abscisic acid metabolism. PMID- 16662452 TI - Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins. AB - Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides. PMID- 16662453 TI - Effect of water stress on cotton leaves : I. An electron microscopic stereological study of the palisade cells. AB - Palisade cells from fully expanded leaves from irrigated and nonirrigated, field grown cotton (Gossypium hirsutum L. cv. Paymaster 266) were subjected to a microscopic examination to evaluate the effect of water stress on subcellular structures. The water potential difference between the two treatments was 13 bars at the time of sampling. The dimensions of the palisade cells and their density per unit leaf area were determined by light microscopy. Palisade cells from stressed plants had the same diameter, but were taller than their counterparts in irrigated plants. The density of the palisade cells was the same in both treatments as was the fractional volume of the intercellular space. It was concluded that the reduced leaf area observed in the stressed plants resulted primarily from a mitotic sensitivity to water stress. Further, expansion of palisade cells was not inhibited by the stress imposed in this study.Morphometric analysis of electron micrographs was used to evaluate the subcellular structure of palisade cells from nonstressed and stressed plants. The fractional volumes of cell walls, total cytoplasm, chloroplasts, starch granules, intrachloroplast bodies, mitochondria, peroxisomes, and central vacuoles were determined. The surface densities of grana and stroma lamellae, outer chloroplast membranes, mitochondrial cristae, endoplasmic reticulum and Golgi cisternae were also measured. The number of chloroplasts, mitochondria, and peroxisomes were determined. These data were expressed as actual volumes, areas, and numbers per palisade cell for each treatment. Palisade cells from stressed plants had thinner cell walls, larger central vacuoles and approximately the same amount of cytoplasm compared to cells from nonstressed plants. Within the cytoplasm, stressed plants had more but smaller chloroplasts with increased grana and stroma lamellae surfaces, larger mithchondria with reduced cristae surfaces, smaller peroxisomes and reduced membrane surfaces of endoplasmic reticulum and Golgi cisternae. PMID- 16662454 TI - Characterization of Chloroplasts Isolated from Triazine-Susceptible and Triazine Resistant Biotypes of Brassica campestris L. AB - Chloroplasts isolated from triazine-susceptible and triazine-resistant biotypes of Brassica campestris L. were analyzed for lipid composition, ultrastructure, and relative quantum requirements of photosynthesis. In general, phospholipids, but not glycolipids in chloroplasts from the triazine-resistant biotype had a higher linolenic acid concentration and lower levels of oleic and linoleic fatty acids, than chloroplasts from triazine-susceptible plants. Chloroplasts from the triazine-resistant biotype had a 1.6-fold higher concentration of t-Delta3 hexadecenoic acid with a concomitantly lower palmitic acid concentration in phosphatidylglycerol. Phosphatidylglycerol previously has been hypothesized to be a boundary lipid for photosystem II. Chloroplasts from the triazine-resistant biotype had a lower chlorophyll a/b ratio and exhibited increased grana stacking. Light-saturation curves revealed that the relative quantum requirement for whole chain electron transport at limiting light intensities was lower for the susceptible biotype than for the triazine-resistant biotype. Although the level of the chlorophyll a/b light-harvesting complex associated with photosystem II was greater in resistant biotypes, the increased levels of the light-harvesting complex did not increase the photosynthetic efficiency enough to overcome the rate limitation that is inherited concomitantly with the modification of the Striazine binding site. PMID- 16662455 TI - Cytohistochemical techniques for calcium localization and their application to diseased plants. AB - Lesion delimitation and resistance of old bean (Phaselous vulgaris L., cv. Red Kidney) plants to Rhizoctonia solani Kuhn have been suggested to result from increased calcium pectate formation in walls. Ultrastructural histochemistry was used to determine the site of calcium in tissues adjacent to lesions and in older bean hypocotyls. Hypocotyl lesion tissue and uninoculated control tissue were treated with ammonium oxalate or potassium pyroantimonate during fixation. Treatment with potassium pyroantimonate, but not with oxalate, resulted in granular deposits in cell walls of healthy and lesion tissue. Granules also occurred on the plasma membrane of cells adjacent to lesions and in organelles of damaged cells, but wall granule density was not increased. Cell walls from healthy 24-day-old plants had a greater granule density than those for 8-day-old plants. Wall granules were removed from thin sections with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Energy dispersive analysis of x-rays also suggested that potassium pyroantimonate localized calcium. Chemical analyses showed that some calcium was retained in tissues after fixation. The results suggest that there are different mechanisms for lesion delimitation and age-induced resistance. PMID- 16662456 TI - Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue. AB - Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6 bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient. PMID- 16662457 TI - Growth, Pigment Synthesis, and Ultrastructural Responses of Phaseolus vulgaris L. cv. Blue Lake to Intermittent and Flashing Light. AB - Growing bean plants (Phaseolus vulgaris L. cv. Blue Lake) on cycles of 1 minute light-1 minute dark or 5 minutes light-5 minutes dark, providing an integrated 12 hours light-12 hours dark per day for each set of plants, led to production after 21 days of new leaves low or lacking in chloroplast pigments. Subsequently, dry weight increase was sharply cut. Leaf area was affected by the light regimes after the second week of growth. By the fourth week, plants on the 1 minute light 1 minute dark cycle showed about one-half the leaf area of the controls. Shoot growth was favored over root growth to the greatest degree on the 1 minute light 1 minute dark regimes. Chlorophyll a/b ratios were close to 3.0 in all of the intermittent light regimes, but the total amounts of chlorophyll in milligrams per primary leaf were higher from day 9 to day 23 for the 12 hour light-12 hours dark controls than for other plants.Although they produced chlorophyll, the plants receiving 1 or 2 milliseconds per second of light continued to lose weight at the same rate as the dark controls; thus, it is assumed there was no net photosynthesis. Plants receiving flashing light allocated significantly more food reserves from the seed to roots than did dark controls. Total chlorophyll formation was significantly accelerated by 2 milliseconds per second light. With 1 millisecond per second light, it took 5 days longer to achieve the same level of chlorophyll. After the 18th day, there was a steady decline in chlorophyll, b degrading more rapidly than a.It is thought that several light-driven reactions are involved in the observed pigment synthesis, photosynthesis, food allocation, and growth of bean. Some of these reactions may be cyclic and others linear. Collectively, they must reach a harmonic point for normal metabolism and development to occur. Because time courses for each of these reactions are different, the intermittent and flashing light technique offers the possibility of individually studying some of the key light-driven reactions. PMID- 16662458 TI - Physiology of Movements in Stems of Seedling Pisum sativum L. cv. Alaska : I. Experimental Separation of Nutation from Gravitropism. AB - Gravitropism and nutation in the stems of dark-grown, seedling peas (Pisum sativum L. cv. Alaska) were recorded on time-lapse photographs made with photomorphogenetically inactive light. Although gravitropism and nutation have been connected by several different theories in the past, our experiments indicate that the two processes are in fact dissociable. The evidence is as follows: (a) Nutational patterns are asymmetric. There is much greater amplitude of oscillation in the plane parallel ( parallel) to the plane of the apical hook than in the plane perpendicular ( perpendicular), yet the average gravitropic response is equal in these two planes. (b) Brief red light irradiation given 16 to 24 hours before observation greatly increases the amplitude of nutation in the parallel-plane, but has no influence on the kinetics of gravitropic response. (c) An inhibitor of auxin transport, alpha-naphthylphthalamic acid, strongly inhibits nutation at 5 micromolar but affects gravitropism only at higher concentrations. (d) Nutation is also strongly inhibited by removal of the apical bud, but gravitropism is unaffected. (e) The period of nutation does not exhibit a constant relationship to the response time of gravitropism. The above evidence is inconsistent with theories that gravitropism is an asymmetrically modified nutation or, alternatively, that nutational oscillations result in a simple fashion from gravitropic overshoots. The evidence is consistent with, although not proof of, autonomous factors such as an endogenous rhythm of growth as the cause of nutation in pea stems. However, gravity and nutation do interact. Nutation in a population of seedlings can be synchronized and brought into phase by a single gravitropic induction. Furthermore, the response time and initial rate of gravitropic curvature depend to some extent on the phase of nutational curvature at which gravitropic induction is begun. PMID- 16662459 TI - Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis. AB - Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G(1), DNA synthesis, S, the second growth stage, G(2), mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4', 6-diamidino-2-phenylindole dichloride.Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G(q), an hypothesized side loop from G(1). All morphological stages, not only division, appear to be phased by the circadian clock. PMID- 16662460 TI - Photoperiodic Control of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy : The Effect of Skeleton and Continuous Light Photoperiods. AB - The control of night-break timing was studied in dark-grown seedlings of Pharbitis nil (Choisy cv. Violet) following a single continuous or skeleton photoperiod. There was a rhythmic response to a red (R) interruption of an inductive dark period, and the phasing of the rhythm was influenced by the preceding light treatment.Following a continuous white light photoperiod of 6 hours or less, the points of maximum inhibition of flowering were constant in real time. Following a continuous photoperiod of more than 6 hours, maximum inhibition occurred at 9 and 32.5 hours after the end of the light period. The amplitude of the rhythm during the second circadian cycle was much reduced following prolonged photoperiods.Following a skeleton photoperiod, the time of maximum sensitivity to a R interruption was always related to the second pulse of the skeleton, R(2), with the first point of maximum inhibition of flowering occurring after 12 to 18 hours and the second after 39 hours. Without a second R pulse, the time of maximum sensitivity to a R interruption was related to the initial R(1) pulse. A ;light-off' or dusk signal was not mimicked by a R pulse ending a skeleton photoperiod; such a pulse only generated a ;light-on' signal and initiated a new rhythm.It is concluded that the timing of sensitivity to a R interruption of an inductive dark period in Pharbitis nil is controlled by a single circadian rhythm initiated by a light-on signal. After 6 hours in continuous white light, the phase of this rhythm is determined by the transition to darkness. Following an extended photoperiod, the timing characteristics were those of an hourglass; this seemed to be due to an effect on the coupling or expression of a single circadian timer during the second and subsequent cycles, rather than to the operation of a different timing mechanism.In addition to the effects on timing, the photoperiod affected the magnitude of the flowering response. PMID- 16662461 TI - Metabolism of Exogenous Indoleacetic Acid to Its Amide Conjugates in Cucumis sativus L. AB - Incubation of hypocotyl segments of light-grown Cucumis sativus L. in 0.1 millimolar 3-indoleacetic acid for 16 hours led to the formation of indoleacetylaspartate and indoleacetylglutamate. There was no evidence for the formation of other conjugates of 3-indoleacetic acid with individual amino acids during the period from 4 to 48 hours of incubation. Indoleacetylglutamate reached its maximum concentration after about 4 hours of incubation and indoleacetylaspartate after about 8 hours. These levels remained unchanged for at least 40 hours. Indoleacetylaspartate caused small increases in cucumber hypocotyl segment growth at high concentrations, 1 millimolar being more effective than 0.1 millimolar. PMID- 16662462 TI - Influence of Giving Salicylic Acid for Different Time Periods on Flowering and Growth in the Long-Day Plant Lemna gibba G3. AB - When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH(4) (+)-free 0.5 H medium) growth is excellent, but flowering is severely inhibited and often is zero. Addition of 10 micromolar salicylic acid (SA) to NH(4) (+)-free 0.5 H medium quickly reverses this inhibition and leads to optimal flowering. The SA treatment also leads to a considerable reduction in the growth rate and increase in frond gibbosity. Removal of SA from the medium quickly leads to an increase in the growth rate and a large decrease in flowering. Thus, for maximal effectiveness SA must be present in the medium for the entire experiment, and the effect of SA is clearly not inductive. PMID- 16662463 TI - Uptake and Metabolism of [C]Salicylic Acid in Lemna gibba G3. AB - When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH(4) (+)-free 0.5 H medium) there is virtually no flowering, but addition of 10 micromolar salicylic acid (SA) to the medium results in substantial flowering. Using this system, the uptake and metabolism of [(14)C]SA in L. gibba G3 has been examined. SA uptake is rapid and linear for at least the first 24 hours. After 30 minutes, nearly 90% of the radioactivity in the plants is present as free SA. Part of this is rapidly converted to one or more bound forms of SA that appear either in the acidic butanol fraction or in the aqueous residue, and after 12 hours an equilibrium is reached between the free and bound forms of SA. When plants receive SA for 6 days and then are switched to control medium, both the free and bound SA remain nearly constant for at least 5 days. However, there is virtually no transfer of SA from mother fronds to daughter fronds, indicating that the SA is apparently sequestered within the cell. Cell fractionation studies show that nearly 95% of the SA remains in the supernatant even after a 2-hour centrifugation at 300,000 g. Thus, it is unlikely that SA is confined within a specific organelle, but rather is probably secreted into the vacuole. PMID- 16662464 TI - Cell division cycles and circadian clocks : phase-response curves for light perturbations in synchronous cultures of euglena. AB - The cell division rhythm in Euglena gracilis Klebs (Z strain) freeruns with a circadian period (30.2 +/- 1.8 hours for 156 monitored oscillations) in aerated, magnetically stirred, 8-liter, axenic batch cultures grown photoautotrophically at 25 degrees C in LD: 3,3, (7,500 lux, cool-white fluorescent) 6-hour light cycles from the moment of inoculation. Cell number was measured at 2-hour intervals with an automatic fraction collector and Coulter Electronic Particle Counter. At different circadian times throughout the 30-hour division cycle, 3 hour light perturbations were imposed on free-running cell populations by giving light during one of the intervals when dark would have fallen in the LD: 3,3 regimen. Using the onset of division as the phase reference point, the net steady state phase advance or delay (+/-Deltaphi) of the rhythm was determined after transients, if any, had subsided (usually in one or two days) relative to an unperturbed control culture. Both +Deltaphi and -Deltaphi were found, with maximum values of approximately +/-11 to 12 hours being obtained at circadian time (CT) 20 to 22 (the ;breakpoint'); little, if any phase shift occurred if the light signal was given between CT 6 and CT 12. The phase-resetting curve obtained by plotting new phase (phi') versus old phase (phi) was of the type 0 (;strong') variety. Light perturbations, no matter when imposed, engendered new phases which mapped to a relatively restricted portion (CT 6 to CT 13) of the circadian cycle.These data provide the first detailed phase-response curve for a circadian mitotic clock. The findings, therefore, not only further support the hypothesis that a circadian oscillator (perhaps exhibiting limit cycle behavior) can modulate cell division in eukaryotic cells, but also provide a useful basis for the dissection of the nature and extent of the coupling between cell division and circadian cycles. PMID- 16662465 TI - Cryostorage of cloned amino Acid analog-resistant carrot and tobacco suspension cultures. AB - Five clones were isolated from five different amino acid analog-resistant Daucus carota L. var. Sativa and Nicotiana tabacum L. cv. Xanthi cell lines. The individual clones were similar in their resistance to dl-5-methyltryptophan, S-(2 aminoethyl)-l-cysteine, or azetidine-2-carboxylic acid, and in their corresponding free amino acid levels.The cell suspensions were stored using a controlled freezing rate at -196 degrees C with concentrations up to 40% of the four cryoprotectants: mannitol, proline, dimethylsulfoxide, glycerol, and combinations of dimethyl-sulfoxide and glycerol. No less than 55% post-thaw viability, determined by phenosafranin dye exclusion, was obtained after storage using a cryoprotectant mixture of 10% glycerol and 10% dimethylsulfoxide. Growth of the cryostored cells could be obtained consistently only by using feeder plate methodology with this combination of cryoprotectants. Post-thaw viability and percentage of cells demonstrating growth, as estimated by growth kinetics, were found to be similar. This indicates that little selection occurred during the freezing and recovery process. In addition, the amino acid analog-resistant traits were unaltered following cryostorage.Suspension cultures of Datura innoxia Mill. were frozen similarly with maximum post-thaw viability of 38%, but subsequent growth was not obtained.Protoplasts of D. innoxia, tobacco and carrot were also cryostored using a mixture of 10% dimethylsulfoxide and 10% glycerol as cryoprotectants. Viabilities of no less than 40% were obtained, however, only the carrot protoplasts regenerated cell walls and underwent cell division. PMID- 16662466 TI - Comparison of photoperiodic sensitivity of green-leafed and red-leafed perilla. AB - The flowering responses of Perilla frutescens (L.) Britt. (green-leafed Perilla) and P. crispa (Thunb.) Tanaka (red-leafed Perilla) to different 24-hour photoperiodic regimes were compared quantitatively. Although both taxa are short day plants, P. frutescens was less strict in its short-day requirement, forming macroscopically visible inflorescence primordia and open flowers even in continuous light after 77 to 85 and 132 days, respectively, compared to 12 and 22 days in 8-hour days. P. crispa took only 4 days longer to reach the same stages in 8-hour short days, but exhibited a sharply defined critical day-length between 14 and 16 hours. Two-hour light-breaks in the middle of a 16-hour night were more effective in inhibiting floral development in P. crispa than in P. frutescens, for which they had a small effect compared to true long-days, particularly on the formation of inflorescence primordia. The number of short days needed to obtain the flowering in P. frutescens plants raised in continuous light decreased in a linear manner with the age of the plants upon transfer to short days. PMID- 16662467 TI - Effects of a triterpenoid saponin on spectral properties of undegraded pea phytochrome. AB - Soyasaponin I, a triterpenoid saponin isolated from etiolated pea (Pisum sativum cv. Alaska) shoots and identified as Pfr killer, was examined for its effects on spectral properties of undegraded pea phytochrome. When soyasaponin I in concentrations of 100 micromolar or lower was added to Pr in the dark, the spectrum of Pr was not significantly affected, whereas in the presence of 120 micromolar or higher concentrations the absorption maximum of Pr shifted from 666 to 658 nanometer with slight decrease of absorbance. After a brief exposure of the mixture to red light, the increase in absorbance at 666 nanometers that occurs in the dark was inhibited at 26 micromolar and higher soyasaponin I concentrations; the maximum effect being reached at about 180 micromolar. The decrease in absorbance at 724 nanometers in the dark after red light irradiation was somewhat inhibited by 60 micromolar and totally prevented by 410 micromolar soyasaponin I. When P(658) was irradiated with red light in the presence of 220 micromolar or higher soyasaponin I concentrations, a bleached form (P(bl)) was produced instead of Pfr. P(bl) showed no dark spectral changes, and the phototransformation of P(bl) to P(658) required a significantly high irradiance of far-red light. When the saponin was added to Pfr in the dark, none of the above-described spectral changes occurred, although the same effects were observed after the mixture was exposed briefly to far-red light followed by red light. PMID- 16662468 TI - Epinephrine, Propranolol, and the Sucrose-Ammonium Inhibition of Flowering in Lemna paucicostata 6746. AB - The sucrose-ammonium inhibition of flowering Lemna paucicostata 6746 in continuous blue light or in short days was partially overcome by epinephrine. This reversal was prevented by propranolol, an antagonist of epinephrine in animals. In ammonium-free medium, propranolol inhibited flowering, and this inhibition was completely overcome by epinephrine. Increased levels of Ca(2+), Pi and nitrate partially reversed the inhibition by propranolol. Concentrations of cAMP, adenine, and adenosine that partially overcame the sucrose-ammonium inhibition did not affect flowering in cultures treated with propranolol. The possibility is discussed that the effects on flowering of sucrose-ammonium, propranolol, and epinephrine were due to altered intracellular levels of cAMP or of a cAMP-like compound. PMID- 16662469 TI - Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds. AB - Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination. PMID- 16662470 TI - Nucleoside Diphosphate Levels in Taproots and Fibrous Roots of Beta vulgaris L. : Implifications for Control of Sucrose Allocation between Two Sinks. AB - Sucrose synthase (EC 2.4.1.13) of sugarbeet (Beta vulgaris L.) fibrous roots has a distinctly different order of preference for nucleoside diphosphate substrates than that of the taproot enzyme (Silvius, Snyder 1979 Plant Physiol 64: 1070 1073). Separation and quantitation of UDP, ADP, and GDP in root extracts by high pressure liquid chromatography revealed that UDP levels in taproot tissue were 5 to 10 times greater than those of fibrous root tissues. The lower fibrous root UDP levels were associated with significantly higher ADP and GDP levels in these roots as compared to the taproot. These differences are consistent with differences in the substrate affinity of sucrose synthase and suggest a regulatory role of the enzyme in the control of sucrose cleavage and utilization between the two root types. PMID- 16662471 TI - Calcium influx into corn roots as a result of cold shock. AB - Corn roots or washed corn root tissue exposed to cold shock absorb 20 to 24% more (45)Ca(2+) into a nonexchangeable phase than control roots. Addition to fusicoccin largely prevents this additional calcium influx. The results are discussed in relation to injury-induced changes in membrane permeability of root cell membranes. PMID- 16662472 TI - Role of boron in the far-red delay of nyctinastic closure of albizzia pinnules. AB - Boron has been found to be necessary for the delaying action of 710 nanometer irradiation on the nyctinastic closing of Albizzia julibrissin pinnules. It is effective only over a narrow micromolar range. In contrast, the delaying action of 470 nanometer irradiation on closure is inhibited by boron. The effect of boron on leaflet closing occurs rapidly. Boron is suggested to be involved in a phytochrome action that precedes and is required for a delaying effect on pinnule closing subsequently induced by an unknown far-red absorbing pigment. PMID- 16662473 TI - Source-sink relations in maize mutants with starch-deficient endosperms. AB - Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of (14)CO(2) were supplied to source leaf blades for 2-hour periods, followed by separation and identification of (14)C assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total (14)C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel (14)C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations. PMID- 16662474 TI - Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts. AB - A plasmalemma-bound NADH oxidation system (Lin 1982 Proc Natl Acad Sci USA 79: 3773-3776) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH stimulated O(2) consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which have been treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 versus 9.4 kilocalories per mole per degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5 to 25 degrees C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K(+) uptake into cell volume of the protoplast. However, the NADH-stimulated K(+) uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed one extra peptide band with approximately 42 kilodalton molecular weight. PMID- 16662475 TI - Differential effect of tungsten on the development of endogenous and nitrate induced nitrate reductase activities in soybean leaves. AB - The effect of tungsten on the development of endogenous and nitrate-induced NADH- and FMNH(2)-linked nitrate reductase activities in primary leaves of 10-day-old soybean (Glycine max [L.] Merr.) seedlings was studied. The seedlings were grown with or without exogenous nitrate. High levels of endogenous nitrate reductase activities developed in leaves of seedlings grown without nitrate. However, no endogenous nitrite reductase activity was detected in such seedlings. The FMNH(2) linked nitrate reductase activity was about 40% of NADH-linked activity. Tungsten had little or no effect on the development of endogenous NADH- and FMNH(2)-linked nitrate reductase activities, respectively. By contrast, in nitrate-grown seedlings, tungsten only inhibited the nitrate-induced portion of NADH-linked nitrate reductase activity, whereas the FMNH(2)-linked activity was inhibited completely. Tungsten had no effect on the development of nitrate-induced nitrite reductase activity. The complete inhibition of FMNH(2)-linked nitrate reductase activity by tungsten in nitrate-grown plants was apparently an artifact caused by the reduction of nitrite by nitrite reductase in the assay system. The results suggest that in soybean leaves either the endogenous nitrate reductase does not require molybdenum or the molybdenum present in the seed is preferentially utilized by the enzyme complex as compared to nitrate-induced nitrate reductase. PMID- 16662476 TI - ATP sulfurylase from higher plants : purification and preliminary kinetics studies on the cabbage leaf enzyme. AB - ATP sulfurylase was purified extensively from green cabbage (Brassica capitata L.) leaf. The enzyme appears to be an asymmetric dimer composed of 57,000 dalton subunits. Initial velocity and product inhibition studies of the forward and reverse reactions point to an obligately ordered kinetic mechanism with MgATP adding before MoO(4) (2-) (or SO(4) (2-)). and MgPPi leaving before AMP + MoO(4) (2-) (or adenosine-5'-phosphosulfate [APS]). The addition of excess purified fungal APS kinase to assay mixtures increased the rate of (35)SO(4) (2-) incorporation and MgPPi formation and extended the linearity of the forward reaction. This effect can be ascribed to the continual removal of APS, a potent product inhibitor of ATP sulfurylase. The specific activities of the enzyme in the APS synthesis, molybdolysis, MgATP synthesis, and sulfate-dependent [(32)P] MgPPi-MgATP exchange assays were 3.3, 38, 38, and 4.3 micromole product formed per minute per milligram protein, respectively. PMID- 16662477 TI - Uptake and effects of N-benzyladenine in excised watermelon cotyledons: influence of cotyledon age. AB - Some characteristics of uptake of [8-(14)C]N(6)-benzyladenine (BA) by watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either excised immediately after 24 hours inhibition (day 0) or cultured in the dark for 48 hours on moist filter paper (day 2) have been compared.The uptake of BA seems to be passive in cotyledons of both kinds. The initial rate of uptake is, however, much slower in day 2 cotyledons. This is probably due to a higher resistance of cell membranes to BA influx. When the day 2 cotyledons are frozen and thawed, so that the membrane barrier is abolished, the amount of BA taken up is the same as in day 0 cotyledons.In spite of the lower rate of uptake, the physiological effects of BA in day 2 cotyledons are as strong as in day 0 cotyledons and occur with a shorter lag time. Sensitivity to BA seems indeed to start 24 to 48 hours after excision. PMID- 16662478 TI - Nitrate Uptake into Barley (Hordeum vulgare) Plants : A New Approach Using ClO(3) as an Analog for NO(3). AB - Evidence is presented that chlorate is an extremely good analog for nitrate during nitrate uptake by intact barley (Hordeum vulgare cv. Fergus) roots. The depletion of ClO(3) (-) or NO(3) (-) from uptake media over 2 to 6 hours by seedlings was found to be dependent on combined NO(3) (-) plus ClO(3) (-) concentrations, and total anion uptake was equivalent at different NO(3) ( )/ClO(3) (-) ratios. After loading barley seedlings with (36)ClO(3) (-) for 6 hours, kinetic parameters were derived from the analysis of efflux of [(36)Cl] chlorate into unlabeled solution. On the basis of this analysis, the half times for exchange for the cytoplasmic and vacuolar phases were 17 minutes and 20 hours, respectively.Data pooled from a number of different experiments were used to calculate kinetic constants (K(m) and V(max)) for (36)ClO(3) (-) influx into barley roots at different external ClO(3) (-)/NO(3) (-) ratios, using short (10 minutes) influx times. There appeared to be no discrimination by the root cells between ClO(3) (-) and NO(3) (-). Lineweaver-Burk analysis of the interaction between nitrate and chlorate were characteristic of competitive inhibition at low nitrate concentrations (0-0.5 mm). At higher concentrations, in the range of >1 mm, similar interactions between these ions were evident. PMID- 16662479 TI - De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.). AB - Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N(2) per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N(2) per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate. PMID- 16662480 TI - Regulation of Glycine Decarboxylase and l-Serine Hydroxymethyltransferase Activities by Glyoxylate in Tobacco Leaf Mitochondrial Preparations. AB - Glyoxylate at a concentration of 10 millimolar caused 50% inhibition of decarboxylation of 20 millimolar [1-(14)C]glycine and accompanying synthesis of serine in a mitochondria-enriched preparation from tobacco (Nicotiana tabacum var. John Williams Broadleaf) leaves. None of the other compounds tested including formate, acetate, oxalate, aspartate, and glutamate appreciably affected activity. Occasional inhibition produced by glycolate may have resulted from residual glycolate oxidase in these preparations. Added glyoxylate was not converted to glycine in these preparations and about 98% of it could be recovered at the end of the reaction. Hence, the observed inhibition by glyoxylate did not result from dilution of radioactivity in the substrate.Glyoxylate also regulated synthesis of HCHO from l-serine catalyzed by l-serine hydroxymethyltransferase in mitochondrial preparations. Control of this enzyme activity by glyoxylate was complex and was characterized by enhancement of activity at low glyoxylate concentrations (less than 10 millimolar) and inhibition by concentrations generally above 10 millimolar. These results define potential sites of biochemical regulation of important steps in the pathway of photorespiratory carbon flow and are considered in the light of other observations of effects of exogenous glyoxylate on photorespiration in leaf tissue. PMID- 16662481 TI - Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool. AB - Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate. PMID- 16662482 TI - Use of the ureide technique to describe the nitrogen economy of field-grown soybeans. AB - Nitrogen fixation was estimated in ;Bragg,' ;Forrest,' and ;Bethel' soybean (Glycine max [L.] Merrill) from seven locations northwest of New South Wales, Australia, by relating ureide and nitrate contents of plant parts sampled at regular intervals during growth to standard curves derived under controlled nitrate regimes. Estimates were combined with data on crop growth and mineral N contents of soils to (a) determine the total requirements for N by the crops, (b) determine the contributions of N(2) fixation to crop growth, and (c) relate symbiotic dependence ([N(2) fixed/total plant N] x 100) of the crops to levels of mineral N in the soil at sowing. At two locations, Myall Vale and Glenara, levels of ureides in the shoot axes and roots of unnodulated seedlings were surprisingly high at the first time of sampling, perhaps reflecting effects of uptake of ammonium-N by the soybeans or breakdown and remobilization of cotyledonary protein. Ureides in plant parts declined significantly by the second (V5 to V7 growth stage) sampling. Subsequently, ureide contents increased whereas levels of nitrate in plant parts decreased. The relative abundance of ureides ([ureide N/ureide-N + nitrate-N] x 100) in the shoot axes and nodulated roots of both crops increased linearly from almost zero during mid-vegetative growth (V5 to V7) to virtually 100% during late reproductive growth (R4 to R5, Myall Vale and R6, Glenara). The data suggest a steady transition in soybeans at both locations from dependence upon mineral N for early growth to complete reliance upon fixed N during late reproductive growth. Estimates of seasonal N(2) fixation for soybeans at the seven locations ranged from 73 to 288 kilograms per hectare N (shoot axes ureides) and from 147 to 337 kilograms per hectare N ha (nodulated roots ureides). PMID- 16662483 TI - Localization of the Enzymes of Quinolizidine Alkaloid Biosynthesis in Leaf Chloroplasts of Lupinus polyphyllus. AB - Studies with purified chloroplasts of Lupinus polyphyllus LINDL. leaflets indicate that the first two enzymes of quinolizidine alkaloid biosynthesis, lysine decarboxylase and 17-oxosparteine synthase, are localized in the chloroplast stroma. Thus, both enzymes share the same subcellular compartment as the biosynthetic pathway of lysine, the precursor of quinolizidine alkaloids. The activity of diaminopimelate decarboxylase, the final enzyme in lysine biosynthesis, is about two to three orders of magnitude higher than that of the enzymes of alkaloid formation. PMID- 16662484 TI - Effect of BASF 13-338, a Substituted Pyridazinone, on Lipid Metabolism in Leaf Tissue of Spinach, Pea, Linseed, and Wheat. AB - A substituted pyridazinone (BASF 13-338) inhibited photosynthesis in spinach (Spinacia oleracea, Hybrid 102 Arthur Yates Ltd.) leaf discs and reduced the incorporation of [1-(14)C]acetate into trienoic acids of diacylgalactosylglycerol while causing radioactivity to accumulate in diacylgalac-tosylglycerol dienoic acids. Although BASF 13-338 inhibited photosynthesis in isolated spinach chloroplasts, it did not prevent dienoate desaturation. In discs, the labeling of fatty acids was affected by the inhibitor only in diacylgalactosylglycerol. Very little radioactivity was incorporated into trienes of phosphatidylcholine and the proportion of the label recovered in the fatty acids of phosphatidylcholine was not changed by BASF 13-338. The herbicides caused an increase in the proportion of the lipid (14)C incorporated into diacylgalactosylglycerol and a decrease in labeling of phosphatidylcholine, whereas the proportion of (14)C recovered in other lipids remained unchanged. Similar results were obtained with pea (Pisum sativum cv. Victory Freeze), linseed (Linum usitatissimum cv. Punjab), and wheat (Triticum aestivum cv. Karamu). With these species, a greater proportion of the label was incorporated into phosphatidylcholine and less into diacylgalactosylglycerol than with spinach. The data indicate that trienoate synthesis uses diacylgalactosylglycerol as substrate. BASF 13-338 appears to act at that step, and seems to cause in spinach a shift in polyenoate synthesis from the pathway involving microsomal phosphatidylcholine to the pathway operating inside the chloroplast. PMID- 16662485 TI - Cell Surfaces in Plant-Microorganism Interactions : III. In Vivo Effect of Ethylene on Hydroxyproline-Rich Glycoprotein Accumulation in the Cell Wall of Diseased Plants. AB - Ethylene production and cell wall hydroxyproline-rich glycoprotein (HRGP) biosynthesis are greatly enhanced in melon (Cucumin melo cv. Cantaloup charentais) seedlings infected with Colletotrichum lagenarium. Short-term experiments performed in the presence of specific inhibitors of the ethylene pathway from methionine, namely l-canaline and amino-ethoxyvinylglycine, indicate that under non-toxic conditions, both ethylene and [(14)C]hydroxyproline deposition in the cell wall of infected tissues are significantly lowered. On the contrary, treatment of healthy tissues with 1-aminocyclopropane 1-carboxylic acid, a natural precursor of ethylene, stimulates both the production of the hormone and the incorporation of [(14)C]hydroxyproline into cell wall proteins.The data provide the first evidence of the in vivo effect of ethylene on the cell wall hydroxyproline-rich glycoprotein biosynthesis in plants. PMID- 16662486 TI - Activation and Deactivation of H-ATPase in Intact Chloroplasts. AB - The light activation mechanism of the latent H(+)-ATPase was investigated in intact spinach (Spinacia oleracea, Hybrid 424) chloroplasts. The following observations were made. (a) Photosystem I electron acceptors such as methyl viologen, nitrite, oxaloacetate, etc., inhibit the light activation of the enzyme. (b) The electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU) fully inhibits the process. (c) Ascorbate plus diaminodurene or dithionite can restore light activation in DCMU-poisoned chloroplasts. (d) The activated state of the enzyme decays rather slowly (within a few minutes) after illumination of the intact chloroplasts. (e) The rate of dark decay is accelerated by oxidants (H(2)O(2) or ferricyanide) and slowed down by dithiothreitol.It is suggested that the physiological mechanism for regulation of the H(+)-ATPase involves oxidation and reduction reactions in a manner which resembles the regulation of the light-activated carbon cycle enzymes. PMID- 16662487 TI - Isolation and Characterization of Chloroplast DNA from Chlorella ellipsoidea. AB - A circular DNA molecule was isolated from chloroplasts of Chorella ellipsoidea. The DNA had a buoyant density of 1.695 grams per cubic centimeter (36% GC) and a contour length of 56 micrometers (175 kilobase pairs). The restriction endonuclease analysis gave the same size. Agarose gel electrophoretic patterns of chloroplast DNA digested by several restriction endonucleases were also presented. The digestion by the restriction enzymes, HpaII, MspI, SmaI, and XmaI revealed no appreciable methylation at CG sites in chloroplast DNA. PMID- 16662488 TI - Relationship between Phospholipid Breakdown and Freezing Injury in a Cell Wall Less Mutant of Chlamydomonas reinhardii. AB - The effects of freezing and thawing on a cell wall-less mutant (CW15+) of Chlamydomonas reinhardii were investigated by monitoring enzyme release, cell viability, cell ultrastructure, and lipid composition. Cells suspended in Euglena gracilis medium were extremely susceptible to freezing injury, the median lethal temperature in the presence of extracellular ice being -5.3 degrees C. Cell damage was associated with a release of intracellular enzymes and massive breakdown of cellular organization. Changes in phospholipid fatty acid composition consistent with either a peroxidation process or phospholipase A(2) activity were evident, but the time course of these changes showed clearly that alterations in phospholipid fatty acid composition were a secondary, pathological event and not the the primary cause of freeze-thaw injury in Chlamydomonas reinhardii CW15+. PMID- 16662489 TI - Electrical Changes in the Apical Cell of the Fern Gametophyte during Irradiation with Photomorphogenetically Active Light. AB - Electrophysiological procedures were used to evaluate cellular responses of fern (Onoclea sensibilis L.) gametophytes to photomorphogenetically active light. Red, far red, and blue light caused rapid changes in the membrane potential of the apical cell of the gametophyte filament; other cells in the filament were not similarly responsive. Measurements made with one electrode in the apical cell revealed that the membrane potential depolarized in red light and repolarized in far red light. Irradiation with blue light caused a hyperpolarization, the rapidity of which was dependent on a red light pretreatment. More refined measurements with one electrode in the tip of the apical cell and another in the base of the cell showed that both red and blue light treatments cause the apical cell to behave as a dipole. Because of the profound, long-term morphological changes that follow light irradiation in this organism, it was hoped to use it to elucidate the role that electrical parameters play in determining subsequent developmental events. PMID- 16662490 TI - Sorbitol metabolism and sink-source interconversions in developing apple leaves. AB - In apple (Malus domestica Borkh.) sorbitol is the primary product of photosynthesis, the major translocated form of carbon, and a common fruit constituent and storage compound. Previous work on sorbitol metabolism has revealed a NADPH-dependent aldose 6-phosphate reductase (A6PR) in green tissues, and a NAD-dependent sorbitol dehydrogenase in nongreen tissues. Results here show a decrease in sorbitol dehydrogenase activity and an increase in A6PR activity as leaves developing in the spring undergo the transition from sink to source. Sorbitol dehydrogenase activity reached a minimum as A6PR peaked. These changes were related to increases in leaf carbohydrate levels, especially sorbitol, and to increases in rates of net photosynthesis. Studies conducted in the autumn on senescing leaves also showed changes in enzyme activites, leaf carbohydrate levels, and photosynthesis. At this time, however, sorbitol dehydrogenase increased in specific activity, whereas A6PR activity, leaf carbohydrates, and photosynthetic rates all decreased substantially. Other experiments showed differences in the ability of young and mature leaves to metabolize sorbitol and in the distribution of sorbitol enzymes in leaves at transitional developmental stages. The results suggest that sorbitol metabolism in apple is tightly controlled and may be related to mechanisms regulating partitioning or source and sink activity. PMID- 16662491 TI - Studies on the aminotransferases participating in the glycolate metabolism of the alga mougeotia. AB - Microbodies from Mougeotia spec., Strain 168.80 contain aminotransferases for conversion of glyoxylate to glycine and serine to hydroxypyruvate. Formation of glycine is possible at highest rates with alanine and glutamate as amino donors, whereas for deamination of serine, pyruvate and glyoxylate are the most convenient substrates. A serine hydroxymethyl-transferase was found exclusively in the mitochondrial fraction. There are indications that this enzyme is bound to the mitochondrial membranes. The activities of all transferases are increased under culture conditions stimulating the synthesis of glycolate. PMID- 16662492 TI - Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds. AB - Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K(m) value computed for inosine is 65 micromolar.AMONG THE INOSINE ANALOGS TESTED, THE FOLLOWING NUCLEOSIDES ARE SUBSTRATES FOR THE LUPIN INOSINE NUCLEOSIDASE: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V(max)/K(m)) towards adenosine is 48 times lower than that towards inosine.In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawelkiewicz 1978 Planta 139: 245 247), the inosine nucleosidase is present in both lupin seeds and seedlings. PMID- 16662493 TI - Effect of butanedioic Acid mono (2,2-dimethylhydrazide) on the activity of membrane-bound succinate dehydrogenase. AB - Mitochondria isolated from hypocotyls of five-day-old bean (Phaseolus vulgaris L. ;Black Valentine') seedlings rapidly oxidized succinate, malate, and NADH. Oxidation rates, respiratory control, and ADP:O ratios obtained with saturating concentrations of all three substrates indicated that the mitochondria were tightly coupled. The mitochondrial preparation was then employed to investigate the respiration-inhibiting effects of butanedioic acid mono (2,2-dimethyl hydrazide) (daminozide) a plant growth retardant having structural similarity to an endogenous respiratory substrate (succinate). Daminozide markedly inhibited the activity of membrane-bound succinate dehydrogenase. Inhibition was of the competitive type (apparent K(i), 20.2 millimolar) with respect to succinate. Although not excluding other hypotheses, the results support an active role for daminozide in the suppression of respiration as an important metabolic site of its action as a plant growth regulator. PMID- 16662494 TI - Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization. AB - During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH(3) are discussed. PMID- 16662495 TI - Biological Properties of d-Amino Acid Conjugates of 2,4-D. AB - Some d-amino acid (glutamic acid, valine, or leucine) conjugates of 2,4 dichlorophenoxyacetic acid (2,4-D) at 10(-5) molar, stimulated elongation of Avena sativa L. var Mariner coleoptile sections and growth of soybean (Glycine max. L. var Amsoy) tissue as much as did the l-amino acid conjugates at 10(-6) molar. The d-methionine conjugate did not stimulate growth of soybean root callus tissue but did stimulate Avena elongation. The d-aspartic acid conjugate did not stimulate elongation of Avena coleoptiles but did stimulate growth of root callus tissue.Pretreatment of root callus tissue with 100 micromolar 2,4-D-(d) -or (l) glutamic acid for 18 hours prior to incubation for 6 hours in [1-(14)C]2,4-D resulted in a greater uptake of [1-(14)C]2,4-D than did pretreatment with 2,4-d. Qualitatively the metabolites were similar in all preincubation treatments. Preincubation with 2,4-D-(d)-glutamic acid also resulted in larger free [(14)C]2,4-D pool and a significant increase in water-soluble conjugates when compared with 2,4-D, or 2,4-D-(l)-glutamic acid preincubations. PMID- 16662496 TI - Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules. AB - A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation. PMID- 16662497 TI - Reversible light-activation of ribulose bisphosphate carboxylase/oxygenase in isolated barley protoplasts and chloroplasts. AB - The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO(3) to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO(2)-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO(2) and Mg(2+). In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO(2) levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO(2) demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO(2) fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light activation of the carboxylase in isolated barley protoplasts at physiologically significant CO(2) levels. PMID- 16662498 TI - Photocontrol of the Functional Coupling between Photosynthesis and Stomatal Conductance in the Intact Leaf : Blue Light and Par-Dependent Photosystems in Guard Cells. AB - The photocontrol of the functional coupling between photosynthesis and stomatal conductance in the leaf was investigated in gas exchange experiments using monochromatic light provided by lasers. Net photosynthesis and stomatal conductance were measured in attached leaves of Malva parviflora L. as a function of photon irradiance at 457.9 and 640.0 nanometers.Photosynthetic rates and quantum yields of photosynthesis were higher under red light than under blue, on an absorbed or incident basis.Stomatal conductance was higher under blue than under red light at all intensities. Based on a calculated apparent photon efficiency of conductance, blue and red light had similar effects on conductance at intensities higher than 0.02 millimoles per square meter per second, but blue light was several-fold more efficient at very low photon irradiances. Red light had no effect on conductance at photon irradiances below 0.02 millimoles per square meter per second. These observations support the hypothesis that stomatal conductance is modulated by two photosystems: a blue light-dependent one, driving stomatal opening at low light intensities and a photosynthetically active radiation (PAR)-dependent one operating at higher irradiances.When low intensity blue light was used to illuminate a leaf already irradiated with high intensity, 640 nanometers light, the leaf exhibited substantial increases in stomatal conductance. Net photosynthesis changed only slightly. Additional far-red light increased net photosynthesis without affecting stomatal conductance. These observations indicate that under conditions where the PAR-dependent system is driven by high intensity red light, the blue light-dependent system has an additive effect on stomatal conductance.The wavelength dependence of photosynthesis and stomatal conductance demonstrates that these processes are not obligatorily coupled and can be controlled by light, independent of prevailing levels of intercellular CO(2). The blue light-dependent system in the guard cells may function as a specific light sensor while the PAR-dependent system supplies a CO(2)-modulated energy source providing functional coupling between the guard cells and the photosynthesizing mesophyll. PMID- 16662499 TI - Physical Properties of Mitochondrial Lipids from Lycopersicon hirsutum. AB - Mitochondrial lipids from Lycopersicon hirsutum undergo a broad thermal transition beginning well below 0 degrees C and ending at approximately 25 degrees C. Differential thermal analysis of mitochondrial lipids isolated from ecotypes of L. hirsutum that differ in chilling sensitivity indicates that these lipid preparations have physically similar properties. This was confirmed by electron-spin-resonance experiments, although this technique failed to detect the broad transition detected by differential thermal analysis. No quantitative differences were observed between the percentages of individual lipid classes (based on polar head group) or between the fatty acid compositions of mitochondrial lipids from the two ecotypes investigated. These results suggest that the observed differences between the responses of these ecotypes to prolonged exposure to 5 degrees C may not be related to differences between the physical properties of their mitochondrial lipids. PMID- 16662500 TI - A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo. AB - A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5 bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO(2) for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO(2), irrespective of the atmospheric O(2) concentration. In this respect, the mutant is distinguished from previously described high-CO(2) requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism. PMID- 16662501 TI - Enzymes for acetaldehyde and ethanol formation in legume nodules. AB - Soybean (Glycine max L. var. Wilkin) nodules contain acetaldehyde and ethanol. The cytosol of soybean and other legume nodules contains pyruvic decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1). Some of the properties of these enzymes from soybean nodules are described. Their presence indicates that in the microaerobic nodule cytosol some carbohydrate is metabolized by fermentative pathways like those in the roots of flood-tolerant plants. PMID- 16662502 TI - Effects of the relative extrachloroplastic concentrations of inorganic phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate on the rate of starch synthesis in isolated spinach chloroplasts. AB - The effect of external inorganic phosphate (Pi) on starch synthesis in isolated spinach (Spinacia oleracea American Hybrid No. 424) chloroplasts in the presence of millimolar concentrations of 3-phosphoglycerate (PGA) and/or dihydroxyacetone phosphate (DAP) was examined. Whereas CO(2) fixation was relatively constant as the ratio of the external phosphate to the PGA + DAP varied from 1:3 to 3:1, starch synthesis varied from 17% to 2% of the CO(2) fixation rate. With DAP alone, maximal starch synthesis was about 10% of the CO(2) fixation rate. The data demonstrate that the Pi/(PGA + DAP) ratio in the cytoplasm of plant cells could serve to regulate the flow of newly fixed carbon into starch without alterations in the rate of CO(2) fixation. PMID- 16662503 TI - Dark metabolism of carbon monoxide in lettuce leaf discs. AB - In the dark, leaf tissue of crisphead lettuce (Lactuca sativa L.) metabolized (14)CO to (14)CO(2) and acid-stable products. Tissue incubated at 2.5 degrees C for 3.5 hours and 48 hours converted about 1% and 17%, respectively, of the applied (14)CO to (14)CO(2), and incorporated about 0.04% and 0.6% of the (14)C in acid-stable products. Examination of soluble acid-stable products from (14)CO and (14)CO(2)-treated leaf tissue revealed that the labeling patterns of both treatments were identical during the 3.5-hour and the 48-hour incubation periods. Malate, citrate, and aspartate together comprised 70% or more of the soluble radioactivity from both treatments. Incorporation of radioactivity from CO into soluble acid-stable products during a 3-hour incubation period at 2.5 degrees C was inhibited 90% by adding 3% nonradioactive CO(2). These results indicate that in head lettuce in the dark (14)CO is metabolized primarily to (14)CO(2) which is the precursor of acid-stable products. In leaf discs at 2.5 degrees C, the apparent K(m) for CO oxidation to CO(2) was 5.3 microliters per liter and the V(max) was 9.7 nanoliters per gram per hour. The mitochondrial fraction of the leaf homogenate was the most active fraction to oxidize CO to CO(2), and this activity was heat-labile and cyanide-sensitive. PMID- 16662504 TI - Interaction of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogens. AB - A hydroxyproline-rich glycoprotein was isolated from tobacco (Nicotiana tabacum L.) callus tissue cultures by an acidic-ethanol extraction procedure and purified to about 95% homogeneity by ion exchange chromatography on carboxymethyl cellulose. This glycoprotein agglutinated cells of an avirulent strain (B-1) of the bacterial pathogen Pseudomonas solanacearum but not its parental, virulent isolate (K-60). Bacterial lipopolysaccharide (from K-60 strain) inhibited this agglutination. The tobacco glycoprotein also agglutinated zoospores of both compatible and incompatible races of Phytophthora parasitica var. nicotianae. Although 34 potential haptens were tested, no low-molecular-weight carbohydrate that inhibited bacterial or fungal agglutination was found. The agglutination activity of the tobacco glycoprotein was sensitive to pronase and sodium periodate. The apparent molecular weight of the glycoprotein was 120,000. The protein moiety was basic (12% lysine and 5% histidine) and contained 38% hydroxyproline. The carbohydrate moiety comprised 26% (by weight) of the glycoprotein, and contained 87% arabinose, 8% galactose, and 5% glucose. The glycoprotein labeled with fluorescein isothiocyanate bound significantly better to the avirulent isolate (B-1) of P. solanacearum than to the virulent strain (K 60). Binding to the avirulent cells was inhibited by incubation in a higher ionic strength medium (e.g. 0.2 m NaCl). The labeled glycoprotein also bound to cystospores and mycelia of both races of P. parasitica var. nicotianae. This fungal-glycoprotein interaction was inhibited by the lipopolysaccharide from strain K-60 and by higher ionic strength conditions. PMID- 16662505 TI - Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems. AB - An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95 degrees C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl(2) increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3 degrees C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed. PMID- 16662506 TI - Identification of the leaf vacuole as a major nitrate storage pool. AB - Highly purified vacuoles were isolated from protoplasts derived from green barley (Hordeum vulgare var. Numar) leaves, in order to determine their role as a NO(3) (-) storage sink. alpha-Mannosidase and acid phosphatase activities were used as markers to identify vacuoles, alpha-mannosidase being the more suitable. Nitrate and alpha-mannosidase, which were released from vacuoles destroyed during lysis of protoplasts, moved at unequal rates in the density gradient used for vacuole isolation. Purified vacuoles retained less NO(3) (-) than alpha-mannosidase during a single washing. Empirically determined corrections were used to account for NO(3) (-) movement in estimating the percentage of total cellular nitrate found in the vacuole. Vacuoles from plants grown in the presence of NO(3) (-) contained 58% of the total cellular NO(3) (-) and therefore represent a major NO(3) (-) storage pool. PMID- 16662507 TI - Evidence of singlet oxygen participation in the chlorophyll-sensitized photooxidation of indoleacetic Acid. AB - Chlorophyll-sensitized photooxidation of indoleacetic acid (IAA)-with chlorophyll extracted from Pisum sativum L. cv. Alaska W.R.-was determined in the presence of deuterium oxide and known quenchers of singlet oxygen ((1)O(2)) to explore the involvement of (1)O(2) in the reaction. O(2) uptake was measured in light in a buffered aqueous micellar system containing Triton X-100, KCl, chlorophyll, and IAA. The rate of O(2) uptake was zero in darkness. The reaction was stimulated by deuterium oxide and inhibited by sodium azide indicating that (1)O(2) participated in IAA photooxidation. Both mannitol and superoxide dismutase failed to inhibit O(2) uptake suggesting that neither the hydroxyl radical nor the superoxide anion played a significant role in the reaction. PMID- 16662508 TI - Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma). AB - Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding beta-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of beta-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye. PMID- 16662509 TI - Effect of trypsin treatment of photosystem I particles on the electron donation to p700. AB - Proteolysis of photosystem I particles had no effect on P700 oxidation but did inhibit the rate of P700(+) reduction. The V(max) values were decreased for both dichlorophenol and plastocyanin, but the K(m) values were unaffected indicating that trypsin treatment altered electron transfer rather than the binding of the donor to the photosystem I complex. The salt dependence of P700(+) reduction was unaffected. The effects of P700(+) reduction were the same for the preparations of different workers (Shiozawa, Alberte, Thornber 1974 Arch Biochem Biophys 165: 388; and Bengis, Nelson 1975 J Biol Chem 250: 2783).In both cases, the 70 kilodalton, chlorophyll-containing polypeptide was digested confirming its role in transferring electrons from plastocyanin to P700. The fact that the preparation of Shiozawa et al. lacks subunit (III) but still used plastocyanin as the electron donor rules out a role for this subunit as "the plastocyanin binding protein." Subunit III was also digested in the Bengis and Nelson preparation. PMID- 16662510 TI - Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine. AB - Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H(2)S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H(2)S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H(2)S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H(2)S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H(2)S formation and its release occurred in response to l-cysteine. Feeding experiments with [(35)S]l-cysteine showed that most of the sulfur in H(2)S was derived from sulfur in the l-cysteine supplied and that the H(2)S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. (35)S labeled SO(3) (2-) and SO(4) (2-) were found in the tissue extract in addition to internal soluble S(2-). These findings suggest the existence of a sulfur cycle which converts l-cysteine to SO(4) (2-) through cysteine desulfhydration. PMID- 16662511 TI - Resistance to injury by sulfur dioxide : correlation with its reduction to, and emission of, hydrogen sulfide in Cucurbitaceae. AB - In Cucurbitaceae young leaves are resistant to injury from acute exposure to SO(2), whereas mature leaves are sensitive. After exposure of cucumber (Cucumis sativus L.) plants to SO(2) at injurious concentrations, illuminated leaves emit volatile sulfur, which is solely H(2)S. Young leaves emit H(2)S many times more rapidly than do mature leaves. Young leaves convert approximately 10% of absorbed [(35)S]SO(2) to emitted [(35)S]H(2)S, but mature leaves convert less than 2%. These results suggest that a high capability for the reduction of SO(2) to H(2)S and emission of the H(2)S is a part of the biochemical basis of the resistance of young leaves to SO(2). PMID- 16662512 TI - Direct Spectrophotometric Measurement of Photosystem I and Photosystem II Activities of Photosynthetic Membrane Preparations from Cyanophora paradoxa, Phormidium laminosum, and Spinach. AB - Vesicles prepared with the French press from membranes of cyanelles of Cyanophora paradoxa retain O(2) evolution activity with rates up to 500 micromoles 2,6 dichlorophenolindophenol reduced per hour per milligram chlorophyll. This activity is immediately lost when the vesicles are transferred from the sucrose phosphate-citrate preparation buffer into dilute phosphate buffer. Similar preparations from Phormidium laminosum, a thermophilic cyanobacterium retain activity under such conditions. Photosystem I activities of both cyanobacterial vesicle preparations were determined by direct spectrophotometric measurement of N,N,N',N'-tetramethyl-p-phenylenediamine photooxidation in the presence of anthraquinone-2-sulfonate. The rates so determined were compared with rates of O(2) taken up in the presence of methyl viologen or anthraquinone-2-sulfonate as electron acceptors. The predicted stoichiometry of two was observed for moles of N,N,N',N'-tetramethyl-p-phenylenediamine oxidized per mole of oxygen taken up. Anthraquinone-2-sulfonate was the better electron acceptor, and maximal rates of 943 micromoles per hour per milligram chlorophyll for O(2) uptake were observed for Phormidium laminosum preparations in the presence of superoxide dismutase. For purposes of comparison, spinach chloroplasts were assayed for similar activities. All preparations were readily assayed for photosystem I activity by the direct spectrophotometric method, which has advantages of simplicity and freedom from errors introduced by photoxidation of other substrates by photosystem I when O(2) uptake is measured. PMID- 16662513 TI - Interactions between Nitrogen Fixation, Mycorrhizal Colonization, and Host-Plant Growth in the Phaseolus-Rhizobium-Glomus Symbiosis. AB - Bean (Phaseolus vulgaris L. cv. Dwarf) roots were inoculated with Rhizobium phaseoli and colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum Gerd. and Trappe or left uncolonized as controls. The symbiotic associations were grown in an inert substrate using 0, 25, 50, 100, or 200 milligrams hydroxyapatite (HAP) (Ca(10)[PO(4)](6)[OH](2)) per pot as a P amendment. Plant and nodule dry weights and nodule activity increased for both VAM and control plants with increasing P availability, but values for VAM plants were significantly lower in all parameters than for controls. Inhibition of growth and of N(2) fixation in VAM plants was greatest at the lowest and highest P regimes. It was smallest at 50 milligrams HAP, where available P at harvest (7 weeks after planting) was 5 micrograms P per gram substrate. At this level of P availability, the association apparently benefited from increased P uptake by the fungal endophyte. Percent P values for shoots, roots, and nodules did not differ significantly (p > 0.05) between VAM and control plants. The extent of colonization, fungal biomass, and the fungus/association dry weight ratio increased several fold as HAP was increased from 0 to 200 milligrams. It is concluded that intersymbiont competition for P and photosynthate was the primary cause for the inhibition of growth, nodulation, and nodule activity in VAM plants. Impaired N(2) fixation resulted in N stress which contributed to inhibition of host plant growth at all levels of P availability. PMID- 16662514 TI - The epsilon Subunit of the Chloroplast Coupling Factor 1 from Euglena gracilis: A Possible Role in Controlling ATPase Activity. AB - The coupling factor from chloroplasts (CF(1)) of Euglena gracilis Z strain is an active ATPase in situ, and its activity cannot be increased by treatment with trypsin or heating as is the case with the CF(1) from other sources. The smallest subunit of CF(1), the epsilon subunit, is supposed to be involved in controlling the ATPase activity. We have devised a simple technique for rapid and large-scale isolation of this subunit. The epsilon subunit from Euglena CF(1), although having only a limited inhibitory effect on Euglena CF(1), drastically inhibited the ATPase activity of heat-activated spinach CF(1). The inhibition of spinach CF(1) could be reversed by passage through Sephadex G-50 or by a second heat activation. An antibody to the epsilon subunit of Euglena CF(1) cross-reacted only weakly with CF(1) from spinach, Sorghum, Kalanchoe, or Anacystis nidulans, but reacted well with whole Euglena CF(1) in addition to its epsilon subunit. The antibody increased the ATPase activity of Euglena and Anacystis CF(1) and of unactivated or partially activated spinach CF(1). The results suggest that the function of the epsilon subunit in Euglena CF(1) is similar to its function in CF(1) from other sources. The data also suggest that changes induced in spinach CF(1) by activation involves modifications in subunits other than the epsilon one. PMID- 16662515 TI - Emission of hydrogen sulfide from sulfur dioxide-fumigated pine trees. AB - Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO(2) in the field emit H(2)S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H(2)S is the only reduced sulfurous compound emitted from SO(2) fumigated leaves. The emission is light and SO(2) concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H(2)S several hours after the termination of prolonged SO(2) fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts. PMID- 16662516 TI - Mechanical properties of the rice panicle. AB - Curvature, bending moment, and second moment of stem cross-sectional area were evaluated from photographic data and used to compute flexural rigidity and Young's modulus in the panicle rachis of rice, Oryza sativa L. ;M-101.' Flexural rigidity C, and its components E, Young's modulus, and I, the moment of inertia of the area about the neutral axis, were evaluated 1.5 cm (tip), 9.5 cm (mid), and 16.5 cm (base) from the tip of the panicle rachis. In dynes per square centimeter, C increases from 1.1 x 10(3) near the tip to 1.09 x 10(4) in the middle to 5.35 x 10(4) in the basal region of the rachis. Of the components of C, the I changes have the larger effect, increasing from 2.12 x 10(-7) centimeters(4) near the tip to 8.21 x 10(-7) centimeters(4) in mid regions to 6.0 x 10(-6) centimeters(4) in the basal regions. Young's modulus increases from 4.8 x 10(9) dynes per square centimeter near the tip to 1.4 x 10(10) dynes per square centimeter in mid regions then falls to 7.4 x 10(9) dynes per square centimeter near the base of the main stem. Values of Young's modulus from Instron experiments were in satisfactory agreement with values calculated from the beam bending equation. Flexural rigidity in the curved region of the panicle proved independent of panicle load, indicating that the dissected panicle rachis behaves in some respects as a tapered loaded beam. PMID- 16662517 TI - N abundance of nodules as an indicator of N metabolism in n(2)-fixing plants. AB - This paper expands upon previous reports of (15)N elevation in nodules (compared to other tissues) of N(2)-fixing plants. N(2)-Fixing nodules of Glycine max (soybeans), Vigna unguiculata (cowpea), Phaseolus vulgaris (common bean), Phaseolus coccineus (scarlet runner bean), Prosopis glandulosa (mesquite), and Olneya tesota (desert ironwood) were enriched in (15)N. Nodules of Vicia faba (fava beans), Arachis hypogaea (peanut), Trifolium pratense (red clover), Pisum sativum (pea), Lathyrus sativus (grass pea), Medicago sativa (alfalfa), and Lupinus mutabilis (South American lupine) were not; nor were the nodules of nine species of N(2)-fixing nonlegumes. The nitrogen of ineffective nodules of soybeans and cowpeas was not enriched in (15)N. Thus, (15)N elevation in nodules of these plants depends on active N(2)-fixation. Results obtained so far on the generality of (15)N enrichment in N(2)-fixing nodules suggest that only the nodules of plants which actively fix N(2) and which transport allantoin or allantoic acid exhibit (15)N enrichment. PMID- 16662518 TI - The Effects of Cyanide and Azide on the Photoreduction of 3-Phosphoglycerate and Oxaloacetate by Wild Type and Two Reductive Pentose Phosphate Cycle Mutants of Chlamydomonas reinhardtii. AB - 3-Phosphoglycerate- and oxaloacetate-dependent O(2) photoevolution by permeabilized cell preparations (Pressates), prepared from wild type (Wt) and two reductive pentose phosphate cycle mutants of Chlamydomonas reinhardtii showed different sensitivities to the inhibitors sodium cyanide and sodium azide. NaCN (1.5 millimolar) severely inhibits both CO(2)- and 3-phosphoglycerate-dependent O(2) photoevolution by the Wt Pressate, but does not inhibit 3-phosphoglycerate dependent O(2) photoevolution by Pressates prepared from the mutants rcl-u-1-10 6C (which lacks ribulose, 1-5, bisphosphate carboxylase activity) and F60 (which lacks phosphoribulokinase activity). NaN(3) (0.5 millimolar) inhibits 3 phosphoglycerate-dependent O(2) photoevolution by the rcl-u-1-10-6C Pressate more severely than in the Pressates prepared from F60 and Wt. A higher concentration of NaN(3) (2.0 millimolar) severely inhibited oxaloacetate-dependent O(2) photoevolution by the rcl-u-1-10-6C, but not by the F60 Pressate. O(2) exchange dependent upon methyl viologen was not strongly inhibited by 2 millimolar NaN(3) in either of the mutant Pressates. The data suggests that the mutational lesions which resulted in decreased ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase activities effected changes in other photosynthetic reactions, either by direct interactions between component proteins or by causing changes in substrate or cofactor availability to the partial reactions. PMID- 16662519 TI - Metabolism and translocation of allantoin in ureide-producing grain legumes. AB - Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-(14)C], [1,3-(15)N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2 (14)C]allantoin to (14)CO(2). A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO(3) (-) grown plants.[(14)C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-(14)C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots. PMID- 16662520 TI - Organelle-bound malate dehydrogenase isoenzymes are synthesized as higher molecular weight precursors. AB - Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrullus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by mono-specific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a post-translational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons). PMID- 16662521 TI - Photosynthesis in submersed macrophytes of a temperate lake. AB - The photosynthetic carbon fixation pathways and levels of carbon-fixing enzymes of four dominant submersed macrophytes of Lawrence Lake, southern Michigan, were investigated during the main growth season (May to November). All four species (Scirpus subterminalis Torr., Najas flexilis (Willd.) Rostk. and Schmidt, Potamogeton praelongus Wulf., and Myriophyllum heterophyllum Michx.) were C(3) plants based on their patterns of (14)C pulse-chase incorporation. High levels of phosphoenolpyruvate carboxylase were also found in these species. These levels, as well as the ribulose 1,5-biphosphate carboxylase/phosphoenolpyruvate carboxylase ratio of the leaves, varied throughout the growing season and exhibited highest values in July. No shift in carbon fixation pathways, however, could be detected from July to October. The possible functions of phosphoenolypyruvate carboxylase in these plants, as well as the significance of C(3) metabolism in submersed plants of temperate lakes, are delineated. PMID- 16662522 TI - Different Rates of Metabolism of Soybean Proteinase Inhibitors during Germination. AB - During germination, the content of the major Bowman-Birk proteinase inhibitor (BB E) in the cotyledons of soybean (Glycine max [L.] Merrill cv. Fiskeby V) seeds decreases, becoming a minor form by the sixth day of germination. One of the three other minor species (BB-D) of this inhibitor in the dry seed increases to become the major form in six-day cotyledons. The other two minor species (BB-C and BB-F) also appear to decrease during germination, but at a slower rate compared to the original major inhibitor form BB-E. By 13 days of germination, no distinct Bowman-Birk inhibitor species can be discerned in the cotyledons. The content of the major Kunitz proteinase inhibitor (K-B) also decreases during germination, but much more slowly. One new form of the Kunitz inhibitor (K-A) becomes apparent by the sixth day. By the 13th day, the proportion of the main isoinhibitor to the late-appearing form is approximately two to one. This difference in the rates of disappearance and appearance of isoinhibitor species in the Bowman-Birk and Kunitz proteinase inhibitor classes suggests a differential metabolism of these two types of proteins and a possible difference in function in the soybean plant. PMID- 16662523 TI - Characterization of nigericin-stimulated ATPase from sealed microsomal vesicles of tobacco callus. AB - To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco (Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N' dicyclohexylcarbodiimide (10 mum), tributyltin (5 mum), and partially by vanadate (200 mum), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 mm) and oligomycin (5 mug/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH(4) (+) > K(+), Rb(+), Cs(+), Na(+), Li(+) > Tris(+). Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl(-) = acetate > SO(4) (2-) > benzene sulfonate > NO(3) (-). The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H(+) pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast. PMID- 16662524 TI - Induction of Flavonoid Synthesizing Enzymes by Light in Etiolated Pea (Pisum sativum cv. Midfreezer) Seedlings. AB - Etiolated pea (Pisum sativum cv. Midfreezer) seedlings respond to illumination with white light by changes in the activity of phenylpropanoid and flavonoid synthesizing enzymes. Unlike in cell cultures, changes in enzyme activity in pea seedlings are not concerted. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity peaked approximately 18 hours after onset of illumination. The phenylacetate path did not interfere with the measurement of phenylalanine ammonia-lyase activity. Activity of cinnamic acid 4-hydroxylase (EC 1.14.13.11) showed an early peak after 8 hours illumination, declined thereafter sharply, then gradually increased during the remainder of the experiment. Activities of chalcone synthase and UDP glucose:flavonol 3-O-glucosyltransferase (EC 2.4.1.91) increased steadily and reached a plateau after approximately 70 hours illumination time. Activity of 4 hydroxycinnamate:coenzyme A ligase (EC 6.2.1.12) remained relatively unchanged, whereas that of chalcone isomerase (EC 5.5.1.6) declined steadily during the course of the experiment. The relative in vitro enzyme activities suggest that the rate-limiting step for the phenylpropanoid path is the cinnamic acid 4 hydroxylase, that of the flavonoid pathway is the chalcone synthase. Integration of enzyme activity curves, however, show that only the curve deriving from phenylanine ammonia-lyase activity matches closely the production of the flavonol glycosides. PMID- 16662525 TI - Citrate transport in corn mitochondria. AB - Citrate uptake by corn mitochondria (Zea mays L. B73 x Mol9) was investigated by osmotic swelling and [(14)C]citrate accumulation. Uptake driven by passive influx, ammonium gradients, and respiration was followed. There was no requirement for phosphate and/or malate to secure citrate uptake, although under some conditions these additives were promotive. Inhibition of the phosphate and dicarboxylate carriers did not eliminate citrate uptake. Citrate(in)/malate(out) exchange occurs, but at a rate too slow to account for observed citrate uptake, and depletion of endogenous malate only reduced citrate uptake by 38%. It was concluded that citrate can be rapidly accumulated by a mechanism other than by exchange for dicarboxylates. The effect of uncoupler on respiration-driven [(14)C]citrate accumulation, and studies of passive swelling using ionophores and uncouplers indicated that the major avenue of citrate uptake is by H(+)/citrate co-transport with a pH optimum near 4.5. The in vivo role of this mechanism is not yet understood. PMID- 16662526 TI - Photosynthesis and Inorganic Carbon Usage by the Marine Cyanobacterium, Synechococcus sp. AB - The marine cyanobacterium, Synechococcus sp. Nageli (strain RRIMP N1) changes its affinity for external inorganic carbon used in photosynthesis, depending on the concentration of CO(2) provided during growth. The high affinity for CO(2) + HCO(3) (-) of air-grown cells (K((1/2)) < 80 nanomoles [pH 8.2]) would seem to be the result of the presence of an inducible mechanism which concentrates inorganic carbon (and thus CO(2)) within the cells. Silicone-oil centrifugation experiments indicate that the inorganic carbon concentration inside suitably induced cells may be in excess of 1,000-fold greater than that in the surrounding medium, and that this accumulation is dependent upon light energy. The quantum requirements for O(2) evolution appear to be some 2-fold greater for low CO(2)-grown cells, compared with high CO(2)-grown cells. This presumably is due to the diversion of greater amounts of light energy into inorganic carbon transport in these cells.A number of experimental approaches to the question of whether CO(2) or HCO(3) (-) is primarily utilized by the inorganic carbon transport system in these cells show that in fact both species are capable of acting as substrate. CO(2), however, is more readily taken up when provided at an equivalent concentration to HCO(3) (-). This discovery suggests that the mechanistic basis for the inorganic carbon concentrating system may not be a simple HCO(3) (-) pump as has been suggested. It is clear, however, that during steady-state photosynthesis in seawater equilibrated with air, HCO(3) (-) uptake into the cell is the primary source of internal inorganic carbon. PMID- 16662527 TI - Regulation of Sulfate Assimilation in Plants : XIII. Assimilatory Sulfate Reduction during Ontogenesis of Primary Leaves of Phaseolus vulgaris L. AB - The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of (35)SO(4) (2-) into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5' phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of (35)SO(4) (2-) into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5'-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO(2) and nitrate assimilation. PMID- 16662528 TI - Uptake of Benzyladenine by Tuber Slices of Jerusalem Artichoke (Helianthus tuberosus L.) over a Wide Concentration Range. AB - Uptake of the cytokinin benzyladenine by tuber slices of Jerusalem artichoke (Helianthus tuberosus L.) increased with the concentration of benzyladenine in the medium in the range 10(-8) to 10(-4) molar. The kinetics of uptake could be precisely represented by four phases of a single, multiphasic isotherm. K(m) and V(max) increased in a regular manner upon transition to higher phases. The fit to multiphasic kinetics was significantly better than the fit to cooperative kinetics or to the sum of four independent Michaells-Menten terms. PMID- 16662529 TI - The Influence of Gibberellic Acid and Temperature on the Growth Rate of Avena sativa Stem Segments. AB - A micro-growth measuring technique was used to determine the growth response of stem segments of Avena sativa cv. Avon to a variety of gibberellic acid (GA(3)) concentrations over a range of incubation temperatures. Growth rate varied with GA(3) concentration, the temperature at which the rate was measured, and the growth temperature of the plants prior to excision of the segments. The curves relating segment extension rates to temperature were affected by GA(3) such that the linear portion of the curve was shifted to higher rates as GA(3) concentration was increased. The results seem to be analogous to the GA(3) induced shifts of thermally induced phase transitions in glucose leakage from liposomes, observed earlier (Wood, Paleg 1974 Aust J Plant Physiol 1: 31-40). PMID- 16662530 TI - Participation of ornithine decarboxylase in early stages of tomato fruit development. AB - The apparent association of ornithine decarboxylase (ODC) with rapid cell proliferation in developing tomato (Lycopersicon esculentum Mill. cv. Pearson ms 35) fruits has been previously described. Further evidence is provided by the use of two ODC inhibitors, alpha-difluoromethylornithine (alpha-DFMO) and alpha methylornithine (alpha-MO). Fruit development was inhibited by these inhibitors if applied during the period of intensive cell division. When applied in vitro, the two inhibitors were shown to inhibit the activity of ODC but not that of arginine decarboxylase (ADC). When applied in vivo, alpha-DFMO, a catalytic irreversible inhibitor, caused 97.1% reduction of ODC activity in the dialyzed extract from the treated ovaries, while it had no effect on ADC. On the other hand, alpha-MO, a reversible inhibitor, did not reduce the activity of these two enzymes in the dialyzed extracts when applied in vivo. The dialysis procedure probably removed alpha-MO from the enzyme fraction. Putrescine, the product of both ODC and ADC, alleviated the inhibition of fruit development but did not restore ODC activity to the control level. These results suggest that in the young developing tomato fruit, ODC is the enzyme responsible for the synthesis of putrescine, which is essential for the early stages of fruit development. The reduced activity of ODC elicited by putrescine suggests a mechanism of feedback regulation by enzyme repression or release of an ODC anti-enzyme. PMID- 16662531 TI - Ornithine decarboxylase and arginine decarboxylase activities in meristematic tissues of tomato and potato plants. AB - Ornithine decarboxylase and arginine decarboxylase activities were measured in roots and buds of tomato (Lycopersicon esculentum Mill. cv. Pearson ms-35) and potato (Solanum tuberosum cv. Desire) plants. In both tomato and potato, the activity of ornithine decarboxylase was the highest at the root tip, decreasing proximally. The same was true for potato buds. In vegetative buds of tomato, the highest activity was found in the youngest leaves. The older the leaf, the lower was orithine decarboxylase activity. Arginine decarboxylase, on the other hand, did not display a similar gradient. These findings are in accordance with the suggestion that in tomato and potato elevated ornithine decarboxylase activity is associated with intense mitotic activity. PMID- 16662532 TI - Ethephon-Induced Gummosis in Sour Cherry (Prunus cerasus L.) : I. Effect on Xylem Function and Shoot Water Status. AB - Ethephon, (2-chloroethyl)phosphonic acid, was sprayed at concentrations up to 69.2 millimolar to enhance gum formation in 1-year-old shoots of mature Prunus cerasus L. cv Montmorency trees. Gum accumulation caused rupturing of the shoot periderm, followed by gum extrusion. Lower ethephon concentrations were required to induce gum formation in spring and early summer (1.7-3.5 millimolar) then in late summer and fall (13.8-69.2 millimolar). The number of functional vessels, shoot hydraulic conductance, and water potential of both leaf and internode tissue decreased as gum content of shoots increased. Nontreated control shoots also contained small quantities of gum. There was no difference in neutral sugar composition of gum exuded by the tree, obtained from aqueous shoot extracts, or flushed from the vessels of shoots, whether induced by ethephon or not. Severe decrease in shoot and leaf water potential was associated with shoot die-back. Recovery of xylem function may occur where gummosis is less severe. Discrepancy between measured and predicted hydraulic conductance increased as shoot gum content increased, suggesting that decrease in number of functional vessels alone was not sufficient to explain the effects of gum on loss of shoot hydraulic conductance. Increased gum content in those vessels remaining functional would increase vessel sap viscosity and further reduce hydraulic conductance. The viscosities necessary to account for discrepancy between measured and predicted hydraulic conductance were calculated. Gum concentration less than 1.0% (w/v) would produce these viscosities. PMID- 16662533 TI - Ethephon-Induced Gummosis in Sour Cherry (Prunus cerasus L.) : II. Flow Characteristics of Gum Solutions. AB - Flow of sour cherry (Prunus cerasus L. cv. Montmorency) gum solutions through a glass capillary was Newtonian for pressure gradients from 0 to 1.8 megapascals per meter, and hydraulic conductance was inversely proportional to solution viscosity in this range. However, flow became plastic at pressure gradients above 1.8 megapascals per meter, resulting in a decrease in solution viscosity. The magnitude of this effect diminished as gum concentration increased. Flow of water, a solution of the component sugar monomers of sour cherry gum, and sucrose solutions remained Newtonian over the entire pressure gradient range examined (0 4 megapascals per meter). Plastic flow of gum solutions in the vessels of intact sour cherry shoots is possible under pressure gradients induced by transpiration when high resistance to flow occurs over short distances. PMID- 16662534 TI - A Comparison of Soybean Agglutinin in Cultivars Resistant and Susceptible to Phytophthora megasperma var. sojae (Race 1). AB - The amount of soybean agglutinin (SBA) detectable by radioimmunoassay in seeds of resistant cultivars to Phytophthora megasperma var. sojae was approximately twice that of susceptible cultivars. SBA was preferentially released at earlier times (6-9 hours) and in higher amounts in the imbibate from resistant cultivars as compared to susceptible cultivars. The lectin in the imbibate was immunologically identical to the seed lectin, indicating little or no proteolysis had occurred, and was active in hemagglutination. Binding of fluorescein isothiocyanate-labeled SBA to mycelial cell walls could be abolished by adding N-acetyl galactosamine or galactose. Purified SBA at concentrations of 150 to 300 micrograms inhibited mycelial growth by 50%, and the imbibate from Govan (resistant) cultivar was more inhibitory than the imbibate from Shore (susceptible) cultivar. Removal of SBA from the imbibate by affinity chromatography abolished the inhibition of mycelial growth, but the inhibition could be recovered from the eluant containing lectin. PMID- 16662535 TI - Proline Oxidation in Corn Mitochondria : Involvement of NAD, Relationship to Ornithine Metabolism, and Sidedness on the Inner Membrane. AB - Proline-dependent oxygen uptake in corn mitochondria (Zea mays L. B73 x Mo17 or Mo17 x B73) occurs through a proline dehydrogenase (pH optimum around 7.2) bound to the matrix side of the inner mitochondrial membrane. Sidedness was established by determining the sensitivity of substrate-dependent ferricyanide reduction to antimycin and FCCP (P-trifluoromethoxycarbonylcyanide phenylhydrazone). Proline dehydrogenase activity did not involve nicotinamide adenine dinucleotide reduction, and thus electrons and protons from proline enter the respiratory chain directly. Delta(1)-Pyrroline-5-carboxylate (P5C) derived from proline was oxidized by a P5C dehydrogenase (pH optimum approximately 6.4). This enzyme was found to be similar to proline dehydrogenase in that it was bound to the matrix side of the inner membrane and fed electrons and protons directly into the respiratory chain.Ornithine-dependent oxygen uptake was measurable in corn mitochondria and resulted from an ornithine transaminase coupled with a P5C dehydrogenase. These enzymes existed as a complex bound to the matrix side of the inner membrane. P5C formed by ornithine transaminase was utilized directly by the associated P5C dehydrogenase and was not released into solution. Activity of this dehydrogenase involved the reduction of nicotinamide adenine dinucleotide. PMID- 16662536 TI - Properties of a Mixed Function Oxygenase Catalyzing Ipomeamarone 15-Hydroxylation in Microsomes from Cut-Injured and Ceratocystis fimbriata-Infected Sweet Potato Root Tissues. AB - Ipomeamarone 15-hydroxylase activity was found in a microsomal fraction from cut injured and Ceratocystis fimbriata-infected sweet potato (Ipomoea batatas Lam. cv. Norin No. 1) root tissues and its optimum pH was 8.0. The enzyme reaction required O(2) and NADPH. The K(m) values calculated for ipomeamarone and NADH were approximately 60 and 2 micromolar, respectively. NADPH alone had little effect on enzyme activity but activated the reaction in the presence of low concentrations of NADPH. Ipomeamarone 15-hydroxylase activity was strongly inhibited by p-chloromercuribenzoic acid and markedly suppressed by cytochrome c and p-benzoquinone. KCN was an activator rather than an inhibitor for the reaction. CO inhibited the activity strongly and its inhibition was partially reversed by light. CO difference spectra of the reduced microsomal fraction showed two absorption maxima at 423 and 453 nm; the latter maximum may be due to a cytochrome P-450. These results suggest that ipomeamarone 15-hydroxylase is a cytochrome P-450-dependent, mixed-function oxygenase.Ipomeamarone 15-hydroxylase activity was not found in fresh tissue of sweet potato roots. However, the activity appeared and increased markedly in response to cut-injury or infection by Ceratocystis fimbriata, and reached a maximum after 24 to 36 hours of incubation. The increase in activity in the latter case was 3- to 5-fold higher than in the former. The time course patterns of development and successive decline in ipomeamarone hydroxylase activities were similar to those for cinnamic acid 4-hydroxylase activity, which had been described as a cytochrome P-450 dependent, mixed-function oxygenase. However, little substrate competition was found between ipomeamarone 15-hydroxylase and cinnamic acid 4-hydroxylase in our preparations. PMID- 16662537 TI - Distribution of Lectins in Tissues, Derived Callus, and Roots of Psophocarpus tetragonolobus (Winged Bean). AB - The distribution of lectin in parental tissues, roots formed de novo from parental stem tissue, and derived callus cells of Psophocarpus tetragonolobus has been measured by hemagglutinating activity and radioimmunoassay. The antisera used for the radioimmunoassay was raised in rabbits to lectin isolated from seeds by affinity chromatography using insolubilized hog gastric mucin. The distribution of lectin in buffer extracts of the tissues (or cells) and the extracellular medium favors the tissues for in vitro grown roots, regardless of the culture conditions used. The lectin content of the extracellular medium is more significant for callus, regardless of its conditions of culture. The lectin activity of extracts of in vitro grown roots was higher than that of mature roots from whole plants. Differences in relative levels of lectin activity measured by hemagglutination and by radioimmunoassay, and differences in saccharide inhibition of hemagglutination, suggest the presence of multiple lectins in extracts of different tissues. PMID- 16662538 TI - Inhibition by adenine derivatives of the cyanide-insensitive electron transport pathway of plant mitochondria. AB - The effect of benzylaminopurine was studied on cyanide-resistant mitochondria isolated from aged slices of potato tuber (Solanum tuberosum L. var. Bintje). Benzylaminopurine specifically acted on the cyanide-resistant alternative pathway. In the case of succinate oxidation, it mimicked the action of salicylhydroxamic acid and restored a good oxidative phosphorylation. Kinetic analyses showed that inhibitions by benzylaminopurine, salicylhydroxamic acid, and disulfiram occurred at mutually exclusive sites on the alternative pathway. Cyanide-resistant malate oxidation was only partially inhibited by benzylaminopurine and this inhibition occurred for low concentrations of this compound. On the other hand, the oxidation of exogenous NADH remained unaffected.The effects of several adenine derivatives with or without cytokinin activity and that of a purine analog with anticytokinin activity were also studied. The variation in effectiveness to inhibit cyanide-resistant electron transport was: benzylaminopurine and 7-pentylamino-3-methylpyrazolo (4,3 d) pyrimidine (anticytokinin) > alpha-alpha' dimethyl-allyl-adenine > 6-benzoylamino 9-benzylpurine > kinetin > adenine. No correlation was observed between the ability to inhibit the alternative pathway and the biological activity of these compounds. Liposolubility appeared as a major factor for potential inhibitory effect on the alternative pathway. PMID- 16662540 TI - Inhibition of photosynthesis by ethylene-a stomatal effect. AB - Ethylene at hormonally significant levels inhibited net photosynthesis of the cultivated peanut (Arachis hypogaea L.) as measured by gas analysis. Upon the removal of ethylene, the inhibition was naturally overcome at the concentration exposure duration combinations tested. Increased length of exposure of 1 microliter of ethylene per liter of air up to 6 hours increased the degree of net photosynthesis inhibition (68% reduction after 6-hour exposure). Significantly greater inhibition of photosynthesis by ethylene was detected on peanut genotypes having higher photosynthetic efficiency. In contrast to peanut, hormonal concentrations of ethylene only moderately inhibited sweet potato, Jerusalem artichoke, and sunflower photosynthesis and was without effect on beans, peas, Irish potato, Mimosa pudica, and white clover. No inhibition could be found by ethylene on ribulose 1,5-biphosphate carboxylase activity in vitro. Photosynthesis was lowered at all CO(2) concentrations below ambient at an O(2) concentration of 1.5%, indicating that the action of ethylene was not affected by low O(2); concomitantly, an increase in the CO(2) compensation point occurred. Diffusion resistance measurements of leaf water vapor loss made on ethylene treated peanut leaves showed a measurable decrease in leaf conductance which correlated with net photosynthesis decrease. Ethylene influenced the conductance of abaxial stomata more so than adaxial. PMID- 16662539 TI - Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop. AB - The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase. PMID- 16662541 TI - Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs. AB - The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28 degrees C, is higher than that of mitochondria from tissue incubated at 8 degrees C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28 degrees C tissue than in mitochondria from 8 degrees C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28 degrees C tissue and 8 degrees C tissue.The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8 degrees C tissue, but also by lower values of respiration via the cytochrome pathway.A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37 degrees C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs. PMID- 16662542 TI - Microautoradiography of water-soluble compounds in plant tissue after freeze drying and pressure infiltration with epoxy resin. AB - It is difficult to retain and localize radioactive, water-soluble compounds within plant cells. Existing techniques retain water-soluble compounds with varying rates of efficiency and are limited to processing only a few samples at one time. We developed a modified pressure infiltration technique for the preparation of microautoradiographs of (14)C-labeled, water-soluble compounds in plant tissue. Samples from cottonwood (Populus deltoides Bartr. ex Marsh.) labeled with (14)C were excised, quick frozen in liquid N(2), freeze-dried at -50 degrees C, and pressure-infiltrated with epoxy resin without intermediate solvents or prolonged incubation times. The technique facilitates the mass processing of samples for microautoradiography, gives good cellular retention of labeled water-soluble compounds, and is highly reproducible. PMID- 16662543 TI - Dynamics of Galactolipids and Plastids in Nonphotosynthetic Cells of Glycine max Suspension Cultures : A Morphological and Biochemical Study. AB - The age-dependent interrelationship of galactolipids and plastids in heterotrophic cell suspension cultures of Glycine max (soybean) was studied with regard to aging of nonphotosynthetic cells. Cells were propagated in the dark and under illumination with white light, and were harvested at days 7 (end of logarithmic phase), 14, and 21 (extended stationary phase). Electron microscopy revealed in dark-grown cells a proliferating decay of the amyloplast-type plastids, which could be correlated to a decrease of galactolipids. This trend was dramatically reversed in irradiated cultures, where the plastids of day 21 cells appeared rejuvenated. A concomitant increase of galactolipid content in the cells was observed, yet chlorophyll synthesis and photosynthetic activity were not induced. The dynamics of galactolipid contents did not correlate with total lipid contents in dark-grown as well as in irradiated cultures. [(3)H]Galactose served as a radioactive probe for the subcellular localization of galactolipids by electron microscopic autoradiography. Apart from plastids, galactolipids may also be constituents of the plasma membrane. The results render the heterotrophic cell suspension culture a suitable model to study the impact of senescence on plastids of nonphotosynthetic cells. PMID- 16662544 TI - Influence of Nitrate and Ammonia on Photosynthetic Characteristics and Leaf Anatomy of Moricandia arvensis. AB - The leaf anatomy and certain photosynthetic properties of nitrate- and ammonia grown plants of Moricandia arvensis (L.) DC., a species previously reported to be a C(3)-C(4) intermediate, were investigated. Nitrate-grown plants had a high level of malate in the leaves while ammonia-grown plants had low levels of malate. In young leaves of nitrate-grown plants, there was a diurnal fluctuation of malate content, increasing during the day and decreasing during the night. Titratable acidity remained low in leaves of both nitrate- and ammonia-grown plants.In nitrate-grown plants, the activity of phosphoenolpyruvate (PEP) carboxylase was about 2-fold higher than in ammonia-grown plants, the latter having activity typical of C(3) species. Also, in nitrate-grown plants, the ratio of activities of ribulose 1,5-bisphosphate (RuBP) carboxylase/PEP carboxylase was lower than in ammonia-grown plants. Nitrate reductase activities were higher in nitrate- than in ammonia-grown plants and the greatest activity was found in younger leaves.With nitrate-grown plants, during a pulse-chase experiment the label in malate, as a percentage of the total labeled products, increased from about 7% after a 10-second pulse with (14)CO(2) up to 17% during a 5-minute chase with (12)CO(2). The pattern of (14)C labeling in various metabolites suggests the primary carboxylation is through RuBP carboxylase with a secondary carboxylation through PEP carboxylase. In similar experiments, with ammonia-grown plants, the percentage label in malate was only 0% to 4% with no increase in malate labeling during the chase period. The CO(2) compensation point was lower in nitrate-grown than ammonia-grown plants.There was no evidence of Kranz-like anatomy in either the nitrate or ammonia-grown plants. Mitochondria of bundle-sheath cells were strikingly positioned along the inner tangential wall. This might allow the chloroplasts of these cells to fix the mitochondrial photorespired CO(2) more effectively and contribute to the low CO(2) compensation point in the species. Chloroplasts of bundle-sheath cells and contiguous mesophyll cells were similar in size and structure in plants grown on different media, although chloroplast thylakoids and stromata of the ammonia-grown plants stained more intensely than those of nitrate-grown plants. In addition, irregular clusters of phytoferritin particles occurred in the chloroplasts of the ammonia-grown plants.The results indicate that the substantial activity of PEP carboxylase, incorporation of CO(2) into malate, the high malate content, and in part the relatively low CO(2) compensation point in Moricandia arvensis may be accounted for by metabolism of nitrate rather than by a state of C(3)-C(4) intermediacy. PMID- 16662545 TI - Isolation and identification of a new growth inhibitor, raphanusanin, from radish seedlings and its role in light inhibition of hypocotyl growth. AB - A neutral growth inhibitor, for which the name raphanusanin is proposed, has been isolated in crystalline form from light-exposed Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) seedlings and identified as a new compound, 3-methoxy-4-methylthio-2-piperithione by spectrometric analyses.Applied raphanusanin inhibited the hypocotyl growth of etiolated radish and lettuce seedlings at concentrations higher than 1.5 x 10(-6) molar.The endogenous raphanusanin contents in cotyledons and hypocotyls of radish seedlings increased more under red light, but decreased or maintained the initial level in the dark. Its content in roots showed almost no change between the light and dark materials. PMID- 16662546 TI - A Transient Burst of CO(2) from Geranium Leaves during Illumination at Various Light Intensities as a Measure of Photorespiration. AB - A transient CO(2) burst is exhibited by irradiated leaves of the C(3) plant geranium (Pelargonium X hortorum, Bailey) after the irradiance is quickly lowered. The light CO(2) burst appears to be related to photorespiration because of its irradiance dependency and its sensitivity to other environmental components such as CO(2) and O(2) concentration. The term post-lower-irradiance CO(2) burst or PLIB is used to describe the phenomenon. The PLIB appears to be a quantitative measurement of photorespiration with intact geranium leaves. The PLIB has been observed with intact leaves of other C(3) plants but not with C(4) leaves. Therefore, it is proposed that, after maximizing intact leaf photosynthetic rates and leaf chamber gas measuring conditions, photorespiration can be measured with intact C(3) leaves such as geranium as a transient post lower-irradiance CO(2) burst. PMID- 16662547 TI - Treatment of the thylakoid membrane with surfactants : assessment of effectiveness using the chlorophyll a absorption spectrum. AB - Treatment of higher plant (Nicotiana tabacum L. var. Samsun) chloroplast thylakoid membranes with surfactants results in a shift of the chlorophyll a absorption maximum in the red spectral region from its in vivo value of 678.5 nanometers to shorter wavelengths. The magnitude of this shift is correlated with membrane disruption, and is not necessarily due to the release of pigment from pigment-protein complexes present in the membrane. Membrane disruption has been measured by the amount of pigment in the supernatant fraction after centrifugation of surfactant treated membranes. For an equivalent amount of disruption, the extent of the blue-shift is influenced by the ionic nature of the surfactant: anionic surfactants cause small shifts, cationic surfactants cause the largest ( approximately 10 nanometers) shifts, and nonionic surfactants produce intermediate shifts. The wavelength of maximum absorbance of chlorophyll a in the red region is a convenient criterion for assessing the potential utility of different surfactants for studies on the structure, composition and function of higher plant thylakoid membranes. PMID- 16662548 TI - Functional Organization of the Chlorophyll-Containing Complexes of Chlamydomonas reinhardi: A Study of Their Formation and Interconnection with Reaction Centers in the Greening Process of the y-1 Mutant. AB - The stepwise synthesis and assembly of photosynthetic membrane components in the y-1 mutant of Chlamydomonas reinhardi have been previously demonstrated (Ohad 1975 In Membrane Biogenesis, Mitochondria, Chloroplasts and Bacteria, Plenum, pp 279-350). This experimental system was used here in order to investigate the process of formation and interconnection of the energy collecting chlorophylls with the reaction centers of both photosystems I and II. The following measurements were carried out: photosynthetic electron flow at various light intensities, including parts or the entire electron transfer chain; analysis of the kinetics of fluorescence emission at room temperature and fluorescence emission spectra at 77 K, and electrophoretic separation of membrane polypeptides and chlorophyll protein complexes. Based on the data obtained it is concluded that: (a) each photosystem (PSI and PSII) contains, in addition to the reaction center, an interconnecting antenna and a main or light harvesting antenna complex; (b) the formation of the light harvesting complex, interconnecting antenna, and reaction centers for each photosystem can occur independently. (c) the interconnecting antennae link the light harvesting complexes with the respective reaction centers. In their absence, energy transfer between the light harvesting chlorophylls and the reaction centers is inefficient. The formation of the interconnecting antennae and efficient assembly of photosystem components occur simultaneously with the de novo synthesis of chlorophyll and at least three polypeptides, one translated in the cytoplasm and two translated in the chloroplast. The synthesis of these polypeptides was found to be light dependent. PMID- 16662549 TI - 3-phosphoglycerate phosphatase activity in chloroplast preparations as a result of contamination by Acid phosphatase. AB - The presence of a nonspecific acid phosphatase which had high activity with 3 phosphoglycerate as substrate has recently been reported in Spinacia oleracea L. chloroplasts (Mulligan, Tolbert 1980 Plant Physiol 66: 1169-1173). The subcellular localization of this activity has been reinvestigated by differential centrifugation of spinach leaf homogenates. The fraction sedimenting at 1,200g comprised mostly intact chloroplasts and contained more than half the chlorophyll but only 5% of the 3-phosphoglycerate phosphatase activity present in the homogenate. The fraction of the homogenate pelleting at 5,000g contained broken chloroplasts and had considerable 3-phosphoglycerate phosphatase activity. Further purification of the 1,200g pellet fraction on a Percoll step gradient yielded greater than 95% intact chloroplasts, yet the phosphatase activity was reduced more than 15-fold on a chlorophyll basis by this purification.When the intact chloroplast and cytoplasmic fractions of mesophyll protoplasts were separated by silicone oil filtering centrifugation, the chloroplast fraction contained more than 90% of the chlorophyll but had less than 12% of the 3 phosphoglycerate phosphatase activity. By contrast, more than 60% of the 2 phosphoglycolate phosphatase was recovered in this chloroplast fraction supporting previous evidence that this phosphatase is localized in the chloroplast stroma.It is concluded that 3-phosphoglycerate phosphatase activity is not localized in the chloroplast but that the activity present in chloroplast preparations results from contamination by acid phosphatase, which either binds to the thylakoid membranes during preparation or is present as some other contaminant in the preparation. Inasmuch as the enzyme acts on a broad range of substrates its presence in chloroplast preparations, particularly when the percentage of intact chloroplasts is low, could produce artifacts in metabolic studies such as measurement of phosphorylation. PMID- 16662550 TI - Photosynthesis and inorganic carbon transport in isolated asparagus mesophyll cells. AB - The possibility of HCO(3) (-) transport into isolated leaf mesophyll cells of Asparagus sprengeri Regel has been investigated. Measurement of the inorganic carbon pool in these cells over an external pH range 6.2 to 8.0, using the silicone-fluid filtration technique, indicated that the pool was larger than predicted by passive (14)CO(2) distribution, suggesting that HCO(3) (-) as well as CO(2) crosses the plasmalemma. Intracellular pH values, calculated from the distribution of (14)CO(2) between the cells and the medium, were found to be higher (except at pH 8.0) than those previously determined by 5,5-dimethyl[2 (14)C]oxazolidine-2,4-dione distribution. It is suggested that the inorganic carbon accumulated above predicted concentrations may be bound to proteins and membranes and thus may not represent inorganic carbon actively accumulated by the cells, inasmuch as in a closed system at constant CO(2) concentration, the photosynthetic rates at pH 7.0 and 8.0 were 5 to 8 times lower than the maximum rate which could be supported by CO(2) arising from the spontaneous dehydration of HCO(3) (-). Furthermore, CO(2) compensation points of the cells in liquid media at 21% O(2) at pH 7.0 and 8.0, and the K((1/2)) CO(2) (CO(2) concentration supporting the half maximal rate of O(2) evolution) at 2% O(2) at pH 7.0 and 8.0 are not consistent with HCO(3) (-) transport. These results indicate that the principal inorganic carbon species crossing the plasmalemma in these cells is CO(2). PMID- 16662551 TI - Leaf Conductance during the Final Season of a Senescing Aspen Branch. AB - Leaf conductance, transpiration, and environmental conditions were measured on two aspen (Populus tremuloides Michx.) branches in a natural stand, using an automatic cuvette system. Fortuitously, leaves on one branch senesced about 10 days early, allowing comparison between a senescing branch and a normal branch. Terminal bud development was retarded on the senescent branch, and a portion of the branch eventually abscised about 20 centimeters from the end. Roughly 1% to 2% of the other branches on the study tree and adjacent trees of that clone also senesced and were dead the following spring.Although no visual symptoms of senescence were observed until September, stomatal behavior was atypical shortly after leaves were fully expanded. During July and August, leaf conductances under full sunlight were higher on the branch which senesced than on the branch which was normal, reaching values greater than 1.0 centimeters per second, and conductance was highly variable. PMID- 16662552 TI - Modulation of chloroplast phosphofructokinase by NADPH : a mechanism for linking light to the regulation of glycolysis. AB - Phosphofructokinase has been partially purified from spinach (Spinacia oleracea) chloroplasts and studied from the standpoint of light/dark regulation. At concentrations reported to occur physiologically, NADPH effected a sharp inhibition of the enzyme by: (a) lowering its affinity (increasing the apparent K(m)) for both of its substrates, ATP and fructose 6-phosphate; and (b) lowering its V(max). Inhibition by NADPH was independent of pH and was observed both at pH 7.9 (pH of chloroplast stroma in the light) and pH 7.0 (stromal pH in the dark). The results are consistent with the conclusion that NADPH provides a mechanism for linking light to the modulation of phosphofructokinase activity and thereby to the regulation of glycolysis in chloroplasts. PMID- 16662553 TI - Localization of spin labels in oat leaf protoplasts. AB - An assay based on light-mediated oxidation was used to determine whether specific spin labels were partitioned throughout the protoplast or retained in the plasmalemma of Avena sativa L. cv. Garry and Park. Many classes of spin label were tested, including phospholipids, fatty acid, fatty acid methyl ester, maleimide, iodoacetamide, short chain hydrocarbon, androstane, 2,2,6,6 tetramethyl-4-aminopiperidinooxyl (TEMPAMINE) and 2,2,6,6 tetramethylpiperidinooxyl (TEMPO). All except the phosphotidylcholine spin label were found to partition throughout the cell. The phosphotidylcholine spin label may have been selectively retained in the plasmalemma. PMID- 16662554 TI - Interaction of nitroxide spin labels with chloroplasts. AB - Chloroplasts isolated from oats eliminated the electron spin resonance (ESR) signals from spin labels in white light and partially restored them in far-red light. Only the white light-mediated reaction was blocked by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU). In contrast, oat (Avena sativa L. cv. Garry and Park) leaf mesophyll protoplasts oxidized the spin labels in both white and far-red light, with and without DCMU. Light had no obvious effect on spin label motion within chloroplast membranes. The results suggest that, in isolated chloroplasts, nitroxide spin labels may be reduced by photosystem I within the thylakoid bilayer resulting in loss of the ESR signals. The reduced forms may be reoxidized by an element of the photosynthetic electron transport chain which operates between the DCMU block and the photosystem I reaction center. In addition, a light-mediated destruction of the spin labels occurs in both chloroplasts and protoplasts. The reduced form of the nitroxide (i.e. the hydroxylamine) may be resistant to this destruction. PMID- 16662555 TI - Diurnal Pattern of Translocation and Carbohydrate Metabolism in Source Leaves of Beta vulgaris L. AB - Transitions in carbohydrate metabolism and translocation rate were studied for evidence of control of export by the sugar beet (Beta vulgaris L. Klein E.) source leaf. Steady-state labeling was carried out for two consecutive 14-hour light periods and various quantities related to translocation were measured throughout two 24-hour periods. Starch accumulation following illumination was delayed. Near the end of the light period, starch stopped accumulating, whereas photosynthesis rate and sucrose level remained unchanged. At the beginning of the dark period there was a 75-minute delay before starch was mobilized. The rate of import to the developing sink leaves at night was similar to that during the day, whereas export decreased considerably at night.Starch accumulation and degradation seemed to be initiated in response to the level of illumination. Cessation of starch accumulation before the end of the light period was initiated endogenously. Exogenous control appeared to be mediated by the level of sucrose in the source leaf while endogenous control seemed to be keyed to photoperiod or photosynthetic duration. PMID- 16662556 TI - Relationship between Net CO(2) Assimilation and Dry Weight Accumulation in Field Grown Tobacco. AB - To assess the variability of net photosynthetic CO(2) exchange per unit leaf area and to construct budgets for stands of field-grown tobacco (Nicotiana tabacum, Connecticut Broadleaf), a number of short-time measurements were made on all available leaf positions on two varieties using a hand-held transparent chamber for conducting gas exchange measurements on leaves. Measurements of net CO(2) exchange were carried out on 18 separate days during a 35-day period, beginning 22 days after the seedlings were transplanted to the field. Gas exchange assays on leaves were conducted under ambient conditions of temperature and light intensity at all times of day. Solar radiation was monitored throughout the period, and losses of respiratory CO(2) from stems, roots, and leaves (in the dark) were estimated. A simple model was proposed to relate daily total CO(2) input to irradiance and total leaf area. The total leaf area was assumed to be a function of day number. Dark respiratory losses accounted for 41% to 47% of total CO(2) assimilation. Analysis of variance indicated that the two varieties were not significantly different in whole plant rate of CO(2) fixation per unit of leaf area. CO(2) input was closely associated with leaf area within each variety. Throughout the experiment, the difference between the two varieties in total leaf area per plant was the largest single factor in determining net CO(2) inputs. The cumulative dry weight increase for each variety was similar to the prediction of net dry matter input obtained by gas exchange measurements, thus confirming the close relationship between total plant net CO(2) assimilation and dry weight yield. PMID- 16662557 TI - Trehalose Toxicity in Cuscuta reflexa: Cell Wall Synthesis Is Inhibited upon Trehalose Feeding. AB - alpha,alpha-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta reflexa Roxb. vine. The incorporation of radioactivity from [(14)C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confined to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A(3), which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxyglucose, which has been shown to be a potent inhibitor of cell wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of cell wall polysaccharides, the chief component of which was presumed to be cellulose. PMID- 16662558 TI - Insensitivity of Water-Oxidation and Photosystem II Activity in Tomato to Chilling Temperatures. AB - Chilling tomato plants (Lycopersicon esculentum Mill. cv. Rutgers and cv. Floramerica) in the dark resulted in a sizable inhibition in the rate of light- and CO(2)-saturated photosynthesis. However, at low light intensity, the inhibition disappeared and the absolute quantum yield of CO(2) reduction was diminished only slightly. The quantum yield of photosystem II (PSII) electron flow was 18% lower when measured in chloroplasts isolated from chilled leaves than in chloroplasts isolated from unchilled leaves. Even though the maximum rate of PSII turnover in these chloroplasts was 12% lower subsequent to chilling, it was in all cases two or more times that required to support the light- and CO(2) saturated rate of photosynthesis measured in the attached leaf. The concentration of active PSII centers in chloroplasts isolated from leaves either before or after chilling was determined by measurement of the products of water oxidation from a series of saturating flashes short enough to turnover the electron transport carriers only a single time. There was no significant change in the concentration of active PSII centers due to dark chilling.It was concluded that PSII activity and water oxidation capacity are not significantly impaired in tomato by chilling in the dark and therefore are not primary aspects of the inhibition of CO(2) reduction observed in attached leaves. PMID- 16662559 TI - Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4,-diol. AB - A NADPH-dependent reductase activity, capable of converting (+)-dihydroquercetin (2,3-trans) to its 3,4-diol (a leucocyanidin), has been demonstrated in crude, soluble protein extracts derived from cell suspension cultures of Douglas fir (Pseudotsuga menziessi). Neither NADH nor ascorbate substituted as the H-donor. Quantitative analyses were based on the production of cyanidin, the formation of an adduct with vanillin, and on absorbance at 280 nanometers. Nonenzymic reduction of (+)-dihydroquercetin with NaBH(4) produced two presumably isomeric flavan-3,4,-diols. One of these was similar to the enzymically produced diol, based on products isolated by chromatography on paper, on thin-layer cellulose and on C(18) reversed-phase columns (high performance liquid chromatography), and on the conversion of the diol to the all-trans dimer of (+)-catechin upon the addition of (+)-catechin. PMID- 16662560 TI - Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin. AB - Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated gamma' chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body.After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region. PMID- 16662561 TI - Nuclear buoyant density determination and the purification and characterization of wild-type neurospora nuclei using percoll density gradients. AB - A procedure has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be negligible in the nuclear preparations, as determined by electron microscopy and by following a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified in the Percoll gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was determined to be 1.08 grams per milliliter based on refractive index measurements. PMID- 16662562 TI - UV-Stimulated K Efflux from Rose Cells: Counterion and Inhibitor Studies. AB - Irradiation of a washed suspension of cultured rose (Rosa damascena var. Gloire de Guilan) cells with about 1,680 joules per square meter of short wave ultraviolet (UV) light (254 nanometers) caused K(+) to appear in the external medium. Short-term tracer ((86)Rb(+)) experiments confirmed the earlier suggestion (Wright, Murphy 1978 Plant Physiol 61: 434-436) that UV increases the efflux of K(+); there was also a small decrease in influx of K(+). There was a partial recovery of fluxes from the effects of UV radiation, but no net accumulation of K(+) within 16 to 18 hours after the irradiation. The K(+) appearing in the medium was matched by an equivalent amount of HCO(3) (-); it was suggested that HCO(3) (-) was the principal counterion for the K(+) flux induced by UV. Inhibitors of ATP synthesis (10(-5) molar carbonyl cyanide m-chlorophenyl hydrazone; 0.05 millimolar KCN plus 0.75 millimolar salicylhydroxamic acid) strongly reduced the UV-stimulated K(+) leakage, suggesting that the leakage was dependent in some way on ATP concentration inside the cells. The UV-induced K(+) leakage was also dependent on temperature and the presence of Ca(2+) in the external medium. PMID- 16662564 TI - Metabolism of trans-Aconitic Acid in Maize : II. Regulatory Properties of Two Compartmented Forms of Citrate Dehydrase. AB - Kinetics of two molecular forms of K-dependent citrate dehydrase in maize (Zea mays L.) are reported. The isozymes, designated CD I and CD II, were found to be compartmented in mitochondria and cytosol, respectively.CD I exhibited hyperbolic kinetics with respect to both citrate and potassium with K(m) 2.3 and 12 millimolar, respectively. Maximum velocity was 0.38 micromole of trans-aconitic acid per minute per milligram protein. The pH optimum was 7.2. trans-aconitic synthesis by CD I is regulated by both citrate concentration and pH.CD II exhibited hyperbolic kinetics with respect to citrate (K(m) 0.6 millimolar) but sigmoidal kinetics with respect to potassium. trans-aconitic acid synthesis by CD II is regulated by potassium. This may account for the positive correlation between leaf potassium and trans-aconitic acid in certain grasses (Clark 1968 Crop Sci 8: 165). PMID- 16662563 TI - Immobilized thylakoids in a cross-linked albumin matrix: effects of cations studied by electron microscopy, fluorescence emission, photoacoustic spectroscopy, and kinetic measurements. AB - Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O(2) evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O(2) production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na(+) (or K(+)), 1 mm Mg(2+), and 0.1 mm Mn(2+) are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F(735) to F(695) ratio increases and the PA(676) to PA(440) ratio decreases. PMID- 16662565 TI - Chloroplast alkaline fructose 1,6-bisphosphatase exists in a membrane-bound form. AB - An alkaline fructose 1,6-bisphosphatase activity associated with soybean (Glycine max cv Beeson) chloroplasts appears to be membrane-bound. The pH optimum of the membrane-associated activity corresponds to that found for activity associated with the stroma. Illumination of washed thylakoids results in an increase in alkaline fructose 1,6-bisphosphatase activity in the absence of any added stromal factors. Exposure to pH 8.0 results in a partial release of enzyme activity from the membrane. The activation status of the enzyme does not appear to alter its association with the membrane. PMID- 16662566 TI - Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes. AB - An action spectrum for anthocyanin formation in dark-grown broom sorghum (Sorghum bicolor Moench, cv Acme Broomcorn and cv Sekishokuzairai Fukuyama Broomcorn) seedlings was determined over the wavelength range from 260 to 735 nanometers. The action peaks were at 290, 650, 385, and 480 nanometers in descending order of height. The action of the 290-nanometer peak was not affected by subsequently given far red light, whereas those of the other three action peaks were nullified completely. The nullification of the 385-nanometer peak action by far red light was reversible. When an irradiation at these action peaks was followed by a phytochrome-saturating fluence of red light irradiation, the action of the 290 nanometer peak remained, whereas that of the 385-nanometer peak as well as those of the 650- and 480-nanometer peaks was masked by the action of the second irradiation. These findings suggested that the 290- and 385-nanometer action peaks involved different photoreceptors, the latter being phytochrome. The blue light-absorbing photoreceptor as reported to be a prerequisite for phytochrome action in milo sorghum was not found to exist in the broom sorghums.The action spectrum deprived of the involvement of phytochrome was determined in the ultraviolet region by irradiating with far red light following monochromatic ultraviolet light. The spectrum had a single intense peak at 290 nanometers and no action at all at wavelengths longer than 350 nanometers. PMID- 16662567 TI - pH in Vacuoles Isolated from Castor Bean Endosperm. AB - Vacuoles were prepared from germinating castor bean endosperm (Ricinus communis var Hale) and purified by filtration through a cotton layer under physiological osmolarity. The purity of vacuoles prepared by this method was comparable with that prepared by a sucrose step gradient centrifugation reported in a previous paper (Nishimura, Beevers 1978 Plant Physiol 62: 44-48). It was shown by assays of marker enzymes that the final preparation contained trace contamination of other organelles (glyoxysomes, mitochondria, and endoplasmic reticulum) and the cytosol. The isolated vacuoles were stained with neutral red, indicating that the intravacuolar pH is acidic. Intravacuolar pH of isolated vacuoles was determined by measuring the distribution of [(14)C]methylamine in the vacuoles and by directly measuring the pH of vacuolar extracts. The pH of isolated vacuolar extracts was 5.7 to 5.9. Similar values were obtained by the methylamine method and it was shown that intravacuolar pH increased as the pH of the medium was increased. PMID- 16662568 TI - Distribution of Secondary Plant Metabolites and Their Biosynthetic Enzymes in Pea (Pisum sativum L.) Leaves : Anthocyanins and Flavonol Glycosides. AB - Leaves of a novel strain of peas (Pisum sativum L.) were used to determine the distribution of secondary metabolites and their biosynthetic enzymes. Leaf epidermal layers in this strain are easily separated from the parenchyma. Anthocyanins and flavonol glycosides were localized in epidermal vacuoles only. Among the biosynthetic enzymes studied, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), S-adenosyl-1-methionine (SAM):caffeic acid and SAM:quercetin methyltransferases (o-dihydric phenol methyltransferase, EC 2.1.1.42) and a flavonoid 7-O-glucosyltransferase (EC 2.4.1.91) were chiefly localized in the parenchyma, whereas trans-cinnamate 4-monooxygenase (EC 1.14.13.11), hydroxycinnamate:CoA ligases (EC 6.2.1.12) and a flavonoid 3-O glucosyltransferase (EC 2.4.1.91) were found mainly in the epidermis. Flavanone (chalcone) synthase activity was found only in the epidermis, whereas chalcone isomerase (EC 5.5.1.6) was evenly distributed in epidermal and parenchyma tissues. PMID- 16662569 TI - Identification and Quantitative Analysis of Indole-3-Acetyl-l-Aspartate from Seeds of Glycine max L. AB - Indole-3-acetyl-l-aspartate (IAAsp) was isolated from seeds of Glycine max L. cv. Hark and its identity established by its chromatographic performance and its mass spectral fragmentation. Following acid hydrolysis, the aspartate moiety was shown to be the l-enantiomer by reverse phase high performance liquid chromatographic retention time of the bisethyl ester derivatized with 2,3,4,6-tetra-O-acetyl-beta d-glycopyranosyl isothiocyanate. Isotope dilution analysis using [(14)C]IAAsp as internal standard showed that soybean seed contained 10 mumol/kg IAAsp and this accounted for one-half of the total indoleacetic acid of the seed. PMID- 16662570 TI - Effect of exogenous and endogenous nitrate concentration on nitrate utilization by dwarf bean. AB - The effect of the exogenous and endogenous NO(3) (-) concentration on net uptake, influx, and efflux of NO(3) (-) and on nitrate reductase activity (NRA) in roots was studied in Phaseolus vulgaris L. cv. Witte Krombek. After exposure to NO(3) ( ), an apparent induction period of about 6 hours occurred regardless of the exogenous NO(3) (-) level. A double reciprocal plot of the net uptake rate of induced plants versus exogenous NO(3) (-) concentration yielded four distinct phases, each with simple Michaelis-Menten kinetics, and separated by sharp breaks at about 45, 80, and 480 micromoles per cubic decimeter.Influx was estimated as the accumulation of (15)N after 1 hour exposure to (15)NO(3) (-). The isotherms for influx and net uptake were similar and corresponded to those for alkali cations and Cl(-). Efflux of NO(3) (-) was a constant proportion of net uptake during initial NO(3) (-) supply and increased with exogenous NO(3) (-) concentration. No efflux occurred to a NO(3) (-)-free medium.The net uptake rate was negatively correlated with the NO(3) (-) content of roots. Nitrate efflux, but not influx, was influenced by endogenous NO(3) (-). Variations between experiments, e.g. in NO(3) (-) status, affected the values of K(m) and V(max) in the various concentration phases. The concentrations at which phase transitions occurred, however, were constant both for influx and net uptake. The findings corroborate the contention that separate sites are responsible for uptake and transitions between phases.Beyond 100 micromoles per cubic decimeter, root NRA was not affected by exogenous NO(3) (-) indicating that NO(3) (-) uptake was not coupled to root NRA, at least not at high concentrations. PMID- 16662571 TI - Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z. AB - Glycolate was excreted from the 5% CO(2)-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O(2) under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO(2) after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O(2) and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO(2) but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed. PMID- 16662572 TI - myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb. AB - A Mg(2+)-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and beta-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6 phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3 Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg(2+). At 4 millimolar, Co(2+), Fe(2+) or Mn(2+) are less effective. Substantial inhibition is obtained with 0.25 molar Li(+). With beta glycerophosphate as substrate the K(m) is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, beta-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.In higher plants this myo-inositol-1 phosphatase links myo-inositol biosynthesis to the myo-inositol oxidation pathway to produce an alternative path from d-glucose 6-phosphate to UDP-d-glucuronate that bypasses UDP-d-glucose dehydrogenase. myo-Inositol-1-phosphatase also furnishes free myo-inositol for reactions that lead to other cyclitols and cyclitol-containing compounds of biosynthetic and/or regulatory significance in plant growth and development. PMID- 16662573 TI - Active, Irreversible Accumulation of Extreme Levels of H(2)SO(4) in the Brown Alga, Desmarestia. AB - The brown algae Desmarestia ligulata var. ligulata (Lightf.) Lamour., and D. viridis (Mull.) Lamour., accumulate H(2)SO(4) until their average internal pH is 0.5 to 0.8. A related species, D. aculeata (L.) Lamour., does not accumulate acid. The H(2)SO(4) accumulation is accompanied by a reduction in the K(+) and Cl(-) content, presumedly to maintain osmotic balance. Measurements of the membrane potential and H(+) and SO(4) (2-) concentrations indicate that both ions are accumulated in the vacuole against their electrochemical potential gradients.The internal pH remains constant in all three species over the growing season, despite striking changes in the algal morphology. The pH is not affected by periods of darkness of up to 34 hours. Sulfate accumulated in the vacuoles appears to be trapped there since incubation of D. ligulata for up to 10 days in sulfate-free medium resulted in little loss of either vacuolar sulfate or H(+). Although the uptake of H(2)SO(4) into the vacuole must require energy, the maintenance of the vacuolar H(2)SO(4) may be due to the impermeability of the tonoplast, with little necessity for continued expenditure of energy. PMID- 16662574 TI - Abscisic Acid stimulation of suberization : induction of enzymes and deposition of polymeric components and associated waxes in tissue cultures of potato tuber. AB - Effect of abscisic acid (ABA) on suberization of potato (Solanum tuberosum var. Russet-Burbank) tuber tissue culture was studied by measuring deposition of suberin components and the level of certain key enzymes postulated to be involved in suberization. ABA treatment resulted in a 3-fold increase in the polymeric aliphatic components of suberin and a 4-fold increase in the polymeric aromatic components. Hydrocarbons and fatty alcohols, two components characteristic of waxes associated with potato suberin, increased 9- and 5-fold, respectively, as a result of ABA treatment. Thus, the deposition of the polymeric aliphatics and aromatics as well as waxes, all of which have been postulated to be components of suberized cell walls, was markedly stimulated by ABA. omega-Hydroxy-fatty acid dehydrogenase which showed a rather high initial level of activity increased only 60% due to ABA treatment. Phenylalanine ammonia-lyase activity reached a maximum at a 5-fold level after 4 days in the ABA medium, whereas the control showed only a 3-fold increase. ABA treatment also resulted in a dramatic (7-fold) increase in an isozyme of peroxidase which has been specifically associated with suberization. Thus, ABA appears to induce certain key enzymes which are most probably involved in suberization. PMID- 16662575 TI - Wound-induced resistance to cellulase in oat leaves. AB - Peeling the epidermis induces the development of resistance to cellulolytic digestion in the mesophyll cell wals of the first leaf of 1- to 3-week-old oat seedlings (Avena sativa var. Victory). Development of resistance occurs between 3 and 11 hours after the abaxial epidermis is peeled from the blade, and is inhibited by actinomycin D (20 micrograms per milliliter) or cycloheximide (1 microgram per milliliter). Other methods of wounding (cutting with a razor blade, stabbing with a dissection needle or brushing with diatomaceous silica) also induce resistance in cells near the wounds. Peeling similarly induces resistance to the digestion of mesophyll cell walls by Cellulysin (Calbiochem) in pea, corn, wheat, and barley. PMID- 16662576 TI - Ethylene as an effector of wound-induced resistance to cellulase in oat leaves. AB - Peeling the abaxial epidermis from oat leaves (Avena sativa var. Victory) induces the formation of wound ethylene and the development of resistance to cellulolytic digestion of mesophyll cell walls. Ethylene release begins between 1 and 2 hours after peeling in the light or dark. Aminoethoxyvinylglycine (AVG, 0.1 millimolar), CoCl(2) (1.0 millimolar), propyl gallate (PG, 1.0 millimolar) or aminooxyacetic acid (AOA, 1.0 millimolar) inhibits, whereas AgNO(3) stimulates wound ethylene formation. Incubation on inhibitors of ethylene biosynthesis (AVG, CoCl(2), PG, AOA) or action (AgNO(3), hypobaric pressure or the trapping of ethylene with HgClO(4)) also prevents the development of wound-induced resistance to enzymic cell wall digestion. 1-Aminocyclopropane-1-carboxylic acid (ACC, 1.0 millimolar) reverses AVG (0.1 millimolar) inhibition of the development of resistance. Exogenous ethylene partially induces the development of resistance in unwounded oat leaves.These results suggest that peeling of oat leaves induces ethylene biosynthesis, which in turn effects changes in the mesophyll cells resulting in the development of resistance to cellulolytic digestion. PMID- 16662577 TI - Oxygen-dependent aging of seeds. AB - When seeds of soybeans (Glycine max Amsoy var.) or safflower were stored under high O(2) concentrations, their per cent germination declined rapidly. For example, soybean seeds stored under 7.7 atmospheres O(2) pressure at 25 degrees C and 17% moisture lost all viability within 22 days, whereas under 7.7 atmospheres N(2), the per cent germination remained greater than 80%. Germination decreased continually in O(2) pressures ranging from 0 to 7.7 atmospheres. High levels of O(2), moisture, or temperature each acted independently to cause losses of germination, but when applied simultaneously, these factors acted synergistically. Soybean seeds were also aged under conditions of high temperature (44 degrees C) and humidity (100% RH), which have been routinely used to accelerate aging. Under these conditions, no O(2) dependence of seed death was observable.Increased lipid oxidation was not detected in seeds that had lost germination ability due to high O(2) treatment. Seeds of two safflower varieties that contained either high oleic or high linoleic fatty acid compositions were subjected to high O(2) treatment. Although the lipid of the high oleic variety is markedly more stable to oxidative degradation, we detected no significant difference in the O(2) tolerance of these seeds. PMID- 16662578 TI - Thylakoid polypeptides of light and dark aged chloroplasts. AB - Spinach (Spinacia oleracea) chloroplasts were aged at 4 degrees C under red light and in the dark. The electron transport activity was monitored together with the thylakoid polypeptide patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The light-induced decay of photosystem II (PSII) activity (half life, about 4 hours) was correlated with a decrease in polypeptides with apparent molecular weights of 36, 48, and 50 kilodaltons. There was very little decay of photosystem I (PSI) activity until after 8 hours illumination. Prior freezing of the chloroplasts enhanced the decrease in PSI activity which was correlated with chlorophyll-protein complex I (CPI) disappearance and an increase in a polypeptide with apparent molecular weight of 60 kilodalton. No variations were detected in the light-harvesting chlorophyll a/b protein. In the dark, the decay of PSII started at 4 to 6 hours and showed a half life of about 30 hours. PSI activity decay (half life about 6 days) occurred simultaneously with the disappearance of CPI. The use of bovine serum albumin (30 mg/mg of chlorophyll) in the light-induced decay experiments increased the stability of PSII more than 2-fold; in the dark experiments, the stability of both photosystems was also more than doubled and the stability of the CPI complex was considerably improved. Comparative electrophoresis of the purified proteins indicated no changes in the cytochrome f band or in the subunits of the ATPase coupling factor during the light-induced decay experiments. Heating of purified PSI particles prior to electrophoresis showed that the 60 kilodaltons polypeptide increased with the disappearance of CPI. PMID- 16662579 TI - An investigation into the roles of photosynthesis and respiration in h efflux from aerated suspensions of asparagus mesophyll cells. AB - Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H(+) efflux. Rates varied between 0.12 and 1.99 nanomoles H(+) per 10(6) cells per minute or 3 and 40 nanomoles H(+) per milligram chlorophyll per minute. The mean rate of H(+) efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H(+) efflux from illuminated cells. Bubbling with N(2) or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H(+) efflux in light or dark. In the absence of aeration, H(+) efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO(2)-free air, rates of H(+) efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO(2) fixation but not photosynthetic CO(2) fixation. It is suggested that H(+) efflux is dependent on respiration and dark CO(2) fixation, but independent of photosynthesis. PMID- 16662580 TI - Does ethylene play a role in the release of lateral buds (tillers) from apical dominance in oats? AB - The growth of lateral buds (tillers), which are undergoing release from apical dominance, was measured in upright and gravistimulated intact Avena sativa L. cv. ;Victory' (oat) shoots as well as in isolated Avena stem segments treated with kinetin and sucrose. During release, the tiller bud initially shows a slow rate of elongation accompanied by swelling. It is followed by a more rapid rate of elongation. Ethylene (C(2)H(4)) production in shoot segments containing a tiller bud was found to occur at the onset of tiller swelling during gravistimulation as well as during inflorescence emergence. Exogenous application of indoleacetic acid or C(2)H(4) inhibits kinetin-induced tiller bud swelling and elongation. However, stem segments pulsed for 24 hours in C(2)H(4) or the C(2)H(4) biosynthesis precursor, 1-amino-cyclopropane-1-carboxylic acid (ACC) and then transferred to kinetin and sucrose, showed a significant increase in swelling elongation as compared with segments maintained under the same conditions but without C(2)H(4) or ACC in the pulse. Segments pulsed for 24 hours with kinetin and sucrose plus the ACC biosynthesis inhibitor, aminoethoxyvinylglycine, or the C(2)H(4) action inhibitor, CO(2), then transferred to kinetin and sucrose medium, showed inhibition of tiller swelling during the pulse and of subsequent elongation. These results indicate that C(2)H(4) plays a role in promoting tiller swelling during the onset of tiller release from apical dominance and may act as a modulator hormone in promoting tiller elongation in the presence of cytokinin. PMID- 16662581 TI - Growth of Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) in Still Nutrient Medium. AB - Cell suspension cultures were established from the callus proliferation of leaf explants of 10- to 12-day-old seedlings of the peanut (Arachis hypogaea L. var. TMV-3). The cells could be cultivated in both agitated and still media, the latter promoting more of chlorophyll (Chl) synthesis. High Chl content (210-240 micrograms Chl per gram fresh weight), yield of free and pipetable cells, presence of all the pigments in the same ratio as that of the leaf tissue, and high rates of O(2) evolution (140-170 micromoles O(2) per milligram Chl per hour) were some of the desirable features of the still-grown cell cultures. However, considerable variations with regard to the above characters were observed between the cell cultures of different varieties of the peanut.O(2) evolution by the cultured cells was dependent on exogenous supply of HCO(3) (-). A well-developed photosynthetic apparatus as evidenced from photosystem I and photosystem II activities of the isolated chloroplasts and variable fluorescence measurements with the cell cultures was further documented by electron microscopic evidence of distinct granal stackings in chloroplasts and sodium dodecyl sulfate polyacrylamide gel separation of thylakoid membranes into P700 Chl a protein complex and light-harvesting Chl a/b complex. Evidence is presented for the relative increase in the Chl associated with P700 Chl a protein complex in contrast to the light-harvesting Chl a/b complex in the cultured cells as compared to intact leaf. PMID- 16662582 TI - Carbon Assimilation in Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) Grown in Still Nutrient Medium. AB - The relative transport of photosynthetic and dark carboxylation products in photoheterotrophic cells of Arachis hypogaea L. var. TMV-3 at varied phases of growth were determined. Despite the presence of an equally competent photosynthetic apparatus as determined from (14)CO(2) incorporation rates in the dark and light, pulse-chase experiments revealed little or no change in the radioactivity of the C(3) intermediates but rapid disappearance of label from the dark carbon assimilates (malate and other tricarboxylic acid cycle intermediates) with a simultaneous increase in the aminoacid pool in early log-phase (10 days old) cells. However, significant flow of carbon through the photosynthetic intermediates resulting in the accumulation of sugars occurred in the late log phase (34 days old) cells. Limitation of exogenous sugar in the nutrient milieu and depletion of reserve carbohydrates stored in starch of the chloroplasts of the cells were considered as the decisive factors in promoting transport of C(3) cycle intermediates through the reductive pentose phosphate pathway in photoheterotrophic cells. The observed drain of radioactivity even from the small amounts of tricarboxylic acid cycle intermediates synthesized during photosynthesis into glutamate indicated that the transport of carbon through the nonautotrophic pathway is not controlled by these factors. PMID- 16662583 TI - Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues. AB - Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes. PMID- 16662584 TI - Enzymological basis for herbicidal action of glyphosate. AB - The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn(2+) (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co(2+) for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co(2+) is a V(max) activator with respect to both substrates, glyphosate cannot act simply by Co(2+) chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5 enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target. PMID- 16662585 TI - Inhibition of oxygen evolution in chloroplasts by ferricyanide. AB - Preincubation of chloroplasts from pea leaves (Pisum sativum L. cv. Kelvedon) with 0.5 millimolar ferricyanide in the dark, caused a parallel inhibition of the rate of rise of the variable fluorescence and the rate of electron transport. Both reactions were inhibited to a similar extent by varying the time of preincubation, the concentration of ferricyanide during preincubation, and by raising the concentration of salts in the preincubation medium. Ferricyanide treatment of Tris-washed chloroplasts did not inhibit electron transport from the Photosystem II (PSII) electron donor 1,5-diphenylcarbazide to methylviologen. The inhibition of the variable fluorescence rise and of NADP reduction (caused by ferricyanide pretreatment) was bypassed by addition of the PSII electron donor couple hydroquinone/ascorbate. It was concluded that preincubation of chloroplasts with ferricyanide in the dark inhibited electron transport between water and PSII. PMID- 16662586 TI - Polyamines and root formation in mung bean hypocotyl cuttings : I. Effects of exogenous compounds and changes in endogenous polyamine content. AB - The effect of several polyamines (putrescine, spermidine, and spermine), their precursors (l-arginine and l-ornithine), and some analogs and metabolic inhibitors (l-canavanine, l-canaline, and methylglyoxal-bis [guanylhydrazone]) on root formation have been studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings.Exogenously applied polyamines did not promote adventitious root formation. Rooting was inhibited by l-canavanine and l-canaline, and this inhibition was reversed by the corresponding amino acids l-arginine and l ornithine. Methylglyoxal-bis (guanylhydrazone), an inhibitor of S adenosylmethionine decarboxylase and polyamine biosynthesis, was also found to inhibit root formation. All compounds at concentrations of >10(-4) molarity completely inhibited natural root formation, whereas at <10(-5) molarity only the indole-butyric acid-induced root formation was inhibited.Indole-butyric acid induced root formation was accompanied by a considerable increase in polyamine levels, more than 2-fold of the control. Whereas senescing (unrooted) cuttings evinced a rapid decline in polyamine content during 48 hours, indole-butyric acid treatment resulted in elevated levels of putrescine and increased putrescine to spermidine ratio. The changes in polyamines were dependent on indole-butyric acid concentration and were organ specific. PMID- 16662587 TI - Ethylene effects on amylase activity from isolated barley aleurone layers : possible modification by proteolytic enzymes. AB - The effect of protease inhibitors on the response of gibberellic acid-treated barley aleurone layers to ethylene was examined. In the absence of protease inhibitors, ethylene plus gibberellic acid initially increased the production of amylase activity relative to layers incubated with gibberellic acid alone. Exposure to ethylene plus gibberellic acid for 48 hours or longer, however, led to depressed levels of amylase activity compared to samples incubated with gibberellic acid in hydrocarbon-free air.The direct assay of proteolytic activity revealed a small increase in activity in response to ethylene. The significance of this response was probed further by including inhibitors of barley proteases in the incubation medium. When potassium bromate was introduced, ethylene did not cause any alteration in amylase activity compared to samples incubated in hydrocarbon-free air. However, in the presence of N-ethylmaleimide, ethylene treatment induced a 52% increase in amylase activity recovered from samples after 48 hours. These results suggest that proteases contribute to the loss of amylase activity in response to ethylene and thus alter the apparent effect of ethylene on amylase synthesis. The effect of protease inhibitors on other hydrolases is also discussed.During the incubation period, the pH of the medium declined significantly. However, ethylene had no effect on the extent of this decline. PMID- 16662588 TI - Inhibition of o(2) consumption resistant to cyanide and its development by N propyl gallate and salicylhydroxamic Acid. AB - Kinetics of inhibition of cyanide-insensitive O(2) uptake by n-propyl gallate (PG) and salicylhydroxamic acid (SHAM) were determined in fresh slices from ethylene-treated tubers of Solanum tuberosum ;Norchip' and with mitochondria and lipoxygenase (EC 1.13.11.12) isolated from these tubers. PG and SHAM appeared to be inhibiting at identical sites in mitochondria but at disparate sites in slices. The apparent K(I) for SHAM was similar in mitochondria and slices. However, the apparent K(I) for PG in mitochondria was about 40-fold lower than the K(I) for PG inhibition of lipoxygenase activity. The amount of lipoxygenase associated with mitochondria increased when tubers were treated with ethylene. PG, but not SHAM, inhibited aging-induced development of cyanide-insensitive respiration. The latter two phenomena are in accord with the hypothesis that lipid metabolism is required for the development of the alternative pathway. PMID- 16662589 TI - Effects of 2,4-dinitrophenol on membrane lipids of roots. AB - Previous work has shown that the undissociated form of 2,4-dinitrophenol (DNP) increases the permeability of barley (Hordeum vulgare var. trebi) roots to ions. The present studies were undertaken to determine whether the effects of undissociated DNP were directly on membrane lipids. Relative amounts of the principal fatty acids from the lipids of barley root membranes were assayed as a function of DNP concentration, pH, and time of treatment under conditions similar to the previous studies of DNP effects on permeability. Undissociated DNP increases the proportions of palmitic and oleic acids and decreases linoleic and linolenic acids with no changes in the amounts of total fatty acids. The effects are immediate, as are the effects on permeability. Only the undissociated DNP is effective. Anionic DNP has no effect, although it is the major species taken up by the roots both at pH 5 and pH 7. DNP has no effect on respiration at either pH, indicating that undissociated DNP effects are on the membranes and not a general metabolic effect. The close parallelism between the effects of DNP on the composition of membrane lipids and on permeability suggests that the increase in permeability produced by undissociated DNP is due to a direct effect on the root membranes. PMID- 16662590 TI - Phytochrome action on chlorophyll synthesis-a study of the escape from photoreversibility. AB - A brief red light pretreatment (pulse), operating through phytochrome, stimulates the synthesis of chlorophyll a and b in Sorghum vulgare shoots that are placed in continuous saturating white light. The red light effect is fully reversible by a far-red (756 nanometers) light pulse for 45 minutes. Thereafter, escape from reversibility is fast, being completed within 2 hours. It is shown here that physiologically active phytochrome (Pfr) is required continuously during these first 45 minutes if the onset of the loss of photoreversibility is to begin 45 minutes after the red light treatment. Thus, the initial action of Pfr consists of two distinct processes: the first process is to overcome the lag prior to escape from photoreversibility; the second process is the actual stimulation of chlorophyll synthesis by Pfr. The duration of the lag prior to escape from photoreversibility depends on the level of Pfr established by the light pulse. The duration increases with increasing Pfr levels from nondetectable to 45 minutes. Above approximately 15% Pfr (Pfr/P(lot) approximately 0.15), the duration of the lag prior to escape from photoreversibility remains constant at 45 minutes. PMID- 16662591 TI - Localization of phytochrome in oats by electron microscopy. AB - Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction. PMID- 16662592 TI - Intracellular Localization of Nitrate Reductase in Neurospora crassa. AB - Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall plasmalemma region and in the tonoplast membranes. PMID- 16662593 TI - Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles. AB - The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor. PMID- 16662594 TI - Anesthetic Effects on Secondary Dormancy and Phytochrome Responses in Setaria faberi Seeds. AB - Seeds of giant foxtail (Setaria faberi Herm.) entered secondary dormancy after pretreatment in H(2)O at 35 degrees C. Pretreatment in 0.1 m ethanol, or several other substances with anesthetic properties, prevented secondary dormancy induction. Pretreatment in 0.5 m ethanol inhibited germination in darkness, but germination could be stimulated by a red irradiation. Germination was initially insensitive to light. Two separate responses are indicated. The first, affected by a variety of substances and low (0.1 m or less) concentrations of ethanol, is related to anesthetic effects and prevention of secondary dormancy. The second, induction of response to red irradiation, is caused by 0.5 m ethanol and some closely related substances. The anesthetic effect is accomplished within the first 8 hours of imbibition while the phytochrome induction effect required treatment for more than 24 hours. Both responses were lost if the 35 degrees C imbibition began in H(2)O. Involvement of cell membranes is suggested in the prevention of secondary dormancy by anesthetics. PMID- 16662595 TI - Molecular morphology of cyanobacterial phycobilisomes. AB - Phycobilisomes were isolated from several cyanobacteria following cell lysis with Triton X-100. They were purified by phosphate precipitation and hydrophobic interaction chromatography. Their phycobiliprotein compositions were quantitatively determined by application of sets of simultaneous absorbance equations to gel chromatographic separations of the chromoproteins. Phycobilisomes purified from several cyanobacteria had characteristic elution times on agarose gel chromatography. Combining electron microscope observations of phycobilisome structure, phycobiliprotein composition, and agarose gel chromatography estimates of molecular weight permitted the calculation of many details of phycobilisome molecular structure. Complementary chromatic adaptation resulted in a change of phycobilisome composition and structure. The polypeptide compositions of phycobilisomes were examined by sodium dodecyl sulfate-agarose gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phycobilisomes were composed of phycobilipeptides derived from the constituent phycobiliproteins. Higher molecular-weight phycobilipeptide aggregates were also observed. The dominant forces responsible for the maintenance of phycobilisome structure are concluded to be hydrophobic interactions. PMID- 16662596 TI - Phytochrome Regulation of Flowering in the Long-Day Plant, Hyoscyamus niger. AB - A daylength extension with incandescent light is more effective in promoting flowering of long-day plants like Hyoscyamus niger than fluorescent light. A low phytochrome photoequilibrium (Pfr/P(tot)), attained by a far-red irradiation at the close of long days under fluorescent light, also promotes flowering. Moreover, if flower initiation processes are initiated by several long days, a low phytochrome photoequilibrium at the end of short, postinduction photoperiods also enhances flowering. The initiation phase of flowering requires Pfr to be present whereas the development phase proceeds more rapidly in the absence of Pfr. Spectral dependence studies, therefore, could be misinterpreted if the initiation and development stages are combined into a single audit of flowering. PMID- 16662597 TI - Effect of far-red and green irradiation on the nyctinastic closure of albizzia leaflets. AB - The nyctinastic closing of Albizzia julibrissin pinnules is delayed by exposure to far-red radiation at 710 and 730 nanometers, with the former more effective than the latter. Far-red radiation at 750 and 770 nanometers has no effect on the process. Red light at 660 nanometers, which by itself has no effect, delayed closure when given before or simultaneously with far-red radiation at 750 or 770 nanometers. Low doses of green light, on the other hand, prevented all far-red radiations from delaying closure when given together with one of them. Effectiveness peaks at 550 nanometers. Green light by itself has no effect on the closing process.From these and previous results, it is concluded that phytochrome is one of two photoreceptors in the process, that the other photoreceptor is an unknown pigment, and that the unknown photoreceptor requires some prior effect of the far-red-absorbing form of phytochrome before its action. Predictions are made of some of the properties of the unidentified pigment. PMID- 16662598 TI - Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium. AB - The ultrastructural changes occurring in the scutellar epithelium cells of rice seeds have been studied during germination and early seedling growth. During this time, several prominent structural changes occur, including (a) formation, development, and proliferation of organelles such as mitochondria, rough endoplasmic reticulum, free ribosomes, and Golgi apparatus; (b) folded structural modification of plasmamembranes in later stages; and (c) conspicuous decrease in lipid-storing spherosomes. Glyoxysome-like electron dense particles are detectable but their formation is much less prominent. It is conceivable that all these structural changes are related to the enhancement of the metabolic activities of the epithelial cells including the synthesis of hydrolytic enzymes such as alpha-amylase and their secretion into the endosperm tissues. Some enzyme activities characteristic of mitochondria and glyoxysomes have been determined using the crude scutellar extracts, and the results dealing with the low activities of the glyoxylate cycle enzymes and palmitoyl-coenzyme A oxidase appear to indicate that fatty acid breakdown is possibly via mitochondrial beta oxidation, although we reserve a definitive conclusion on the glyoxysomes being nonfunctional in fatty acid oxidation in rice seedlings. PMID- 16662599 TI - Comparative Phytochrome Immunochemistry as Assayed by Antisera against Both Monocotyledonous and Dicotyledonous Phytochrome. AB - Preparation and characterization of antisera against lettuce (Lactuca sativa L., cv. Grand Rapids) and pea (Pisum sativum L., cv. Alaska) phytochrome is described. These antisera, together with previously obtained antisera against zucchini (Cucurbita pepo L., cv. Black Beauty) and oat (Avena sativa L., cv. Garry) phytochrome, were used to compare by Ouchterlony double immunodiffusion phytochrome isolated from etiolated lettuce, pea, bean (Phaseolus vulgaris L., cv. Taylor Horticultural Bush), zucchini, oat and rye (Secale cereale L., cv. Balbo) seedlings. Cross reactivity between monocotyledonous phytochrome and antidicotyledonous-phytochrome serum and between dicotyledonous phytochrome and antimonocotyledonous-phytochrome serum was always weak or not perceptible by this assay. Among the four dicotyledonous phytochromes examined, pea and bean were the most similar immunochemically as anticipated. Pea and lettuce phytochrome somewhat unexpectedly also exhibited similar immunochemical reactivity. Zucchini phytochrome by contrast was immunochemically distinct from pea, bean, and lettuce phytochrome, although it did react with all three antidicotyledonous-phytochrome sera. Initial attempts to identify immunoglobulins that would recognize phytochrome regardless of its source indicated that they may exist. Such immunoglobulins are of interest because they might react with one or more determinants that could be part of an active site of phytochrome. These immunoglobulins, once isolated, could thus serve as a potential probe for the active site of phytochrome. PMID- 16662600 TI - Cluster bean-a ureide- or amide-producing legume? AB - Xylem sap of cluster bean (Cyamopsis tetragonoloba L. cv FS-277) and pigeonpea (Cajanus cajan cv UPAS-120) were analyzed for total nitrogen, amide nitrogen, and ureide nitrogen at flowering stage. Nitrogenase, uricase, and allantoinase were compared in nodules of cluster bean and pigeonpea. Xylem sap of cluster bean exhibited higher amounts of amides as compared to ureides, and the activities of uricase and allantoinase (ureide-producing enzymes) in nodules were also low, whereas the reverse was the case for pigeonpea. Based on these investigations, it has been concluded that cluster bean is an amide-producing legume rather than ureide-producing as had been reported earlier. PMID- 16662601 TI - 1-aminocyclopropane-1-carboxylic Acid concentrations in shoot-forming and non shoot-forming tobacco callus cultures. AB - Shoot-forming tobacco (Nicotiana tabacum var. Wisconsin 38) callus tissues contain significantly lower concentrations of the ethylene precursor 1 aminocyclopropane-1-carboxylic acid compared to non-shoot-forming callus tissues. This difference is evident 1 day after subculture to shoot-forming or non-shoot forming medium, and is maintained through the first week of growth. The lack of auxin in shoot-forming medium is the probable cause for this difference in ACC concentrations. PMID- 16662602 TI - Tissue Distributions of Chlorogenic Acid and of Enzymes Involved in Its Metabolism in Leaves of Sorghum bicolor. AB - The tissue distributions of cholorgenic acid, chlorogenic acid oxidase, and three other enzymes involved in the metabolism of this secondary (natural) product have been investigated in leaf-blades of light-grown seedlings of Sorghum bicolor. Cholorogenic acid was found only in epidermal and mesophyll protoplasts isolated from the leaf; 60% of the chlorogenic was contained in the epidermal fraction. Nearly all (90%) of the chlorogenic acid oxidase was found in the mesophyll protoplasts. The bundle-sheath strands, on the other hand, contained no chlorogenic acid and essentially none of the oxidase. Three other enzymes required for the synthesis of chlorogenic acid, but also for other plant products, were found in all three tissue fractions. PMID- 16662603 TI - Photoinhibition of Reaction Centers of Photosystems I and II in Intact Bryopsis Chloroplasts under Anaerobic Conditions. AB - Illumination of intact Bryopsis corticulans chloroplasts under anaerobic conditions induced a decline of chlorophyll fluorescence and photoinhibition of Photosystems I and II. The time course of the light-induced decline of chlorophyll fluorescence and the decreases of activities of reactions sensitized by Photosystems I and II were compared. Photosystem I activity decreased in parallel with the disappearance of active P700. The time course of the destruction of the reaction center of Photosystem II was similar to that of photoinhibition of 2,6-dichlorophenolindophenol-Hill reaction.It appears that the initial events in photoinhibition are the destruction of the reaction centers of Photosystems I and II and that the reaction centers that are inhibited become quenchers of chlorophyll fluorescence.Effects of inhibitors of electron transfer and of an electron donor to Photosystem I showed that photoinhibition was related to Photosystem I activity. PMID- 16662604 TI - In Vivo Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) Seedlings in Light and Darkness. AB - In vivo NO(3) (-) reduction in roots and shoots of intact barley (Hordeum vulgare L. var Numar) seedlings was estimated in light and darkness. Seedlings were placed in darkness for 24 hours to make them carbohydrate-deficient. During darkness, the leaves lost 75% of their soluble carbohydrates, whereas the roots lost only 15%. Detached leaves from these plants reduced only 7% of the NO(3) (-) absorbed in darkness. By contrast, detached roots from the seedlings reduced the same proportion of absorbed NO(3) (-), as did roots from normal light-grown plants. The rate of NO(3) (-) reduction in the roots accounted for that found in the intact dark-treated carbohydrate-deficient seedlings. The rates of NO(3) (-) reduction in roots of intact plants were the same for approximately 12 hours, both in light and darkness, after which the NO(3) (-) reduction rate in roots of plants placed in darkness slowly declined. In the dark, approximately 40% of the NO(3) (-) reduction occurred in the roots, whereas in light only 20% of the total NO(3) (-) reduction occurred in roots. A lesser proportion was reduced in roots because the leaves reduced more nitrate in light than in darkness. PMID- 16662605 TI - Site of Synthesis of NADPH: Protochlorophyllide Oxidoreductase in Rye (Secale cereale). AB - The site of synthesis of the plastid membrane-located enzyme, protochlorophyllide reductase, has been determined. Plastid ribosome-deficient and normal rye (Secale cereale L., cv Rheidol) plants were grown in darkness at 33 degrees C and 22 degrees C, respectively. Extracts from these plants were analyzed for the levels of different ribosomal RNAs and cytochrome f and the activity of a number of enzymes with well-established sites of synthesis. The results confirmed that the higher temperature had induced a specific inhibition of protein synthesis in the plastids. The activity and level of protochlorophyllide reductase was unaffected by growth at the higher temperature, suggesting it to be a cytoplasmically synthesized enzyme. PMID- 16662606 TI - Pigment-protein complexes of illuminated etiolated leaves. AB - Photoconversion of protochlorophyllide in etiolated leaves of Avena sativa L., var. Pennal or Peniarth and Phaseolus vulgare L., var. ;The Prince' results in the sequential appearance of spectrally distinct chlorophyllide complexes (Chlide 678, 684, and 672). This paper reports on the generation of similar forms in vitro, under controlled conditions, using well characterized etioplast membranes enriched in the enzyme protochlorophyllide reductase. Excess NADP(+) and NADPH stabilize complexes related to Chlide 678 and Chlide 684, respectively, whereas addition of exogenous Pchlide induces formation of a species related to Chlide 672. Evidence is provided to support the suggestion that Chlide 678 and Chlide 684 represent ternary complexes of the enzyme protochlorophyllide reductase, with Chlide and either NADP(+) (Chlide 678) or NADPH (Chlide 684). Chlide 672 is seen as ;free' pigment dissociated from the enzyme. The role of Pchlide in this dissociation, observed spectroscopically as the ;Shibata shift,' is discussed. PMID- 16662607 TI - Photosystem II Activity, Plastoquinone A Levels, and Fluorescence Characterization of a Virescens Mutant of Barley. AB - Chloroplasts isolated from seedlings of a virescens mutant of barley (Hordeum vulgare L cv Gateway) grown for 6 days under continuous illumination had lower levels of photosystem II activities on a chlorophyll basis than wild-type seedlings. After 8 days, however, the photosystem II rates of the mutant and wild type were approximately equal. Lower levels of the photosystem II activities in the mutant were correlated with a smaller functional plastoquinone pool size as determined by room temperature fluorescence induction. Higher levels of extractable plastoquinone A on a chlorophyll basis, however, were obtained from mutant chloroplasts. An increased room temperature fluorescence yield in the mutant was shown to be due to a higher level of initial fluorescence. A decreased sigmoidicity in the room temperature fluorescence induction transient in the presence of diuron and an increased 77 K fluorescence emission at 680 nanometers lead us to believe that a certain population of the light harvesting chlorophyll protein complex in the mutant membranes is unconnected to photo-system II reaction centers. Although photochemical activities of the mutant approach wild type values as the mutant develops, the population of dissociated light harvesting complexes does not appear to change. PMID- 16662608 TI - Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts. AB - Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2 degrees C and 8.5 at 20 degrees C, but at all pH values the rate of uptake was higher at 20 degrees C than at 2 degrees C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2 degrees C, the K(m) was 0.3 millimolar and the V(max) was 13 micromoles per milligram of chlorophyll per hour. At 20 degrees C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.Although there was some metabolism of glycerate by chloroplasts in the light, even at 2C, the actual concentration of glycerate in the stroma, determined by chromatography, was 2 to 4 times that in the incubation medium. NH(4)Cl stimulated glycerate uptake and decreased its metabolism by the chloroplasts resulting in stromal concentrations of glycerate up to 6 times higher than in the medium. A number of weak acids inhibited glycerate uptake, lactate and glyoxylate being the most effective. The inhibition was not competitive with respect to glycerate. Glycerate uptake was also inhibited by p chloromercuriphenyl sulfonate.Substrates of the dicarboxylate transporter did not inhibit glycerate uptake. Of the four substrates of the phosphate transporter tested, 3-phosphoglycerate and dihydroxyacetone phosphate inhibited glycerate uptake but orthophosphate and glyceraldehyde-3-phosphate had little effect. The inhibition by 3-phosphoglycerate was competitive with respect to glycerate, with a K(i) of 0.2 millimolar, suggesting that 3-phosphoglycerate binds to the same site as glycerate. However, glycerate only inhibited 3-phosphoglycerate uptake slightly and the inhibition was not competitive, indicating that glycerate does not bind to the phosphate transporter. Glycerate did not counter exchange with (32)P in chloroplasts which had been preloaded with (32)Pi, confirming that glycerate did not enter via the phosphate transporter.The results suggest that the chloroplast envelope contains a specific glycerate transporter, which differs from the previously reported transporters in that it is stimulated by light. PMID- 16662609 TI - Senescence in isolated carnation petals : effects of indoleacetic Acid and inhibitors of protein synthesis. AB - Indoleacetic acid induces senescence in isolated carnation (Dianthus caryophyllus, cv. White Sim) petals, increasing the duration and amount of ethylene production. This effect is inhibited by Actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, a translational inhibitor of protein synthesis. The ability of petals to respond to indoleacetic acid appears to be a function of physiological age. Indoleacetic acid is capable of enhancing ethylene evolution and senescence only in specific portions of the petal. PMID- 16662610 TI - Effects of Light and Nutrients on Leaf Size, CO(2) Exchange, and Anatomy in Wild Strawberry (Fragaria virginiana). AB - Plants of a single genotype of wild strawberry, Fragaria virginiana Duchesne, were grown with or without fertilizer in high (406 microeinsteins per square meter per second) and low (80 microeinsteins per square meter per second) light. High-light leaves were thicker than low-light leaves and had greater development of the mesophyll. Within a light level, high-nutrient leaves were thicker, but the proportions of leaf tissues did not change with nutrient level. Maximum net CO(2) exchange rate and leaf size were greatest in high-light, high-nutrient leaves and lowest in high-light, low-nutrient leaves. Changes in mesophyll cell volume largely accounted for differences in CO(2) exchange rate in low-light leaves, but not in high-light leaves.Leaf size in these experiments was apparently determined by nutrient and carbon supply. This may explain the observation that the largest leaves produced by wild strawberries in the field occur in high-light, mesic habitats, rather than in shady habitats. PMID- 16662611 TI - Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo. AB - The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed. PMID- 16662612 TI - Allophycocyanin I and the 95 Kilodalton Polypeptide : The Bridge between Phycobilisomes and Membranes. AB - Allophycocyanin was isolated from dissociated phycobilisomes from Nostoc sp. and was separated into allophycocyanin I, II, III, and B as described elsewhere. If the separation of the proteins following phycobilisome isolation is done in the presence of the protease inhibitor, phenylmethylsulfonylfluoride, associated with allophycocyanin I are two colored polypeptides of 95 kilodalton (kD) and 80 kD, belonging to the class of Group I polypeptides as defined by Tandeau de Marsac and Cohen-Bazire (Proc Natl Acad Sci USA 1977 74: 1635-1639). Allophycocyanin I has a fluorescence maximum of 680 nanometers as do intact phycobilisomes and has thus been suggested to be the final emitter of excitation energy in phycobilisomes. Thylakoid membranes washed in low ionic strength buffer containing phenylmethylsulfonylfluoride lose all biliproteins, but retain the 95 kD and 80 kD polypeptides. As suggested by Tandeau de Marsac and Cohen-Bazire, these are likely to be the polypeptides involved in binding the phycobilisome to the membrane. As these polypeptides are isolated with allophycocyanin I, structural evidence is provided for placing allophycocyanin I as the bridge between the phycobilisome and the membrane. These Group I polypeptides and the 29 kD polypeptide (involved in rod attachment to the APC core) are particularly susceptible to proteolytic breakdown. It is thought that in vivo the active protease may be selectively attacking these polypeptides to detach the phycobilisome from the membrane and release the phycoerythrin and phycocyanin containing rods from the allophycocyanin core for greater susceptibility of the biliproteins to protease attack. PMID- 16662613 TI - Isolation and Characterization of the Central Component of the Phycobilisome Core of Nostoc sp. AB - Freshly isolated allophycocyanin is recovered from linear sucrose gradients made in 0.75 molar potassium phosphate buffer (pH 7.0) in three sizes: 19s, 10.3s, and 5.5s. The largest aggregate is a complex of a 680 nm fluorescing allophycocyanin I in the form (alphabeta)(3)gamma, where gamma is the 95 kilodalton (kD) polypeptide, and two 660 nanometer fluorescing allophycocyanin II (alphabeta)(3) molecules; the complex, stabilized in high phosphate concentrations, fluoresces maximally at 675 nanometers. The 10.3s fraction is a hexamer of allophycocyanin of the 660 nanometer fluorescing type, perhaps attached through two polypeptides of 46 kD and 44 kD. The 5.5s component of the allophycocyanin pool is the usual trimeric form of allophycocyanin (alphabeta)(3). A similar 19s fraction is the major component of allophycocyanin I isolated under optimum conditions in the presence of the protease inhibitor, phenylmethylsulfonylfluoride. This 19s fraction is apparently a central component of the core of the phycobilisome with its 95 kD polypeptide the attachment point of the phycobilisome and membrane. The 95 kD polypeptide has both long wavelength absorption and fluorescence bands which seem to account for the long wavelength fluorescence properties of allophycocyanin I. PMID- 16662614 TI - Water Relations of Cotton Plants under Nitrogen Deficiency: V. Environmental Control of Abscisic Acid Accumulation and Stomatal Sensitivity to Abscisic Acid. AB - Suboptimal N nutrition increased the water potential for stomatal closure in water stressed cotton (Gossypium hirsutum L.) leaves. This increased sensitivity to water stress had two components, increased accumulation of abscisic acid (ABA) and increased apparent stomatal sensitivity to ABA. Low N increased the threshold water potentials for stomatal closure and ABA accumulation by about 4 bars and 2 bars, respectively. Low N also greatly increased stomatal response to low concentrations of exogenous ABA applied to excised leaves through the transpiration stream. In low N leaves, kinetin decreased stomatal response to ABA to the level observed with high N leaves. Kinetin by itself had little effect on stomata, nor did it alter stomatal response to ABA in high N leaves. The results suggest a cytokinin-ABA balance which is altered by suboptimal N nutrition to favor stomatal closure during stress.Ambient temperature and N nutrition interacted to alter stomatal response to water stress. Stress-induced ABA accumulation and apparent stomatal sensitivity to ABA were independently affected. The effects of each treatment, and their interaction, could be explained as the net result of changes in both accumulation and apparent sensitivity. Although the results document environmental control of stomatal response to ABA, either altered partitioning of ABA between active and inactive pools, or altered sensitivity of the guard cells, could account for the data. PMID- 16662615 TI - Cytokinesis, cell expansion, and the potential for cytokinin-autonomous growth in tobacco pith. AB - Pith tissue from Nicotiana tabacum L. cv ;Maryland Mammoth' or ;Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center. PMID- 16662616 TI - Phospholipid Motional Characteristics in a Dry Biological System : A P-Nuclear Magnetic Resonance Study of Hydrating Typha latifolia Pollen. AB - Analysis of the proton-decoupled (31)P-nuclear magnetic resonance (NMR) spectrum of fully hydrated Typha latifolia pollen revealed the presence of two main peaks: A broad asymmetrical component of a ;bilayer' lineshape and a much narrower symmetrical component originating from phosphorus compounds undergoing rapid isotropic motion. From (a) (31)P-NMR experiments on the hydrated total pollen phospholipids, (b) saturation transfer (31)P-NMR experiments, and (c) the fraction of lipid phosphate in the pollen, it can be concluded that the great majority of the endogenous phospholipids are arranged in extended bilayers in which the lipid phosphates undergo fast (tau(c) < 10(-6) second) long axis rotation. This bilayer arrangement of phospholipids was observed in the pollen down to hydration levels of at least 10.9% moisture content. At the lowest level of pollen hydration examined (5.2%) the (31)P-NMR spectrum had a solid state lineshape demonstrating that all the phosphorus-containing compounds (including the phospholipids) were virtually immobile. PMID- 16662617 TI - Rapid Changes in the Pattern of Electric Current around the Root Tip of Lepidium sativum L. following Gravistimulation. AB - Using a highly sensitive vibrating electrode, the pattern of naturally occurring electric currents around 1-day-old primary roots of Lepidium sativum L. growing vertically downward and the current pattern following gravistimulation of the root has been examined. A more or less symmetrical pattern of current was found around vertically oriented, downward growing roots. Current entered the root at the root cap, the meristem, and the beginning of the elongation zone and left the root along most of the elongation zone and in the root hair zone. After the root was tilted to a horizontal position, we observed current flowing acropetally at the upper side of the root cap and basipetally at the lower side within about 30 seconds in most cases. After a delay of several minutes, acropetally oriented current was also found flowing along the upper side of the meristematic zone. The apparent density of the acropetal current in the root cap region increased and then decreased with time. Gravitropic curvature was first visible approximately 10 minutes after tilting of the root to the horizontal position. Since the change in the pattern of current in the root cap region precedes bending of the root and is different for the upper and lower side, a close connection is suggested between the current and the transduction of information from the root cap to the elongation zone following graviperception in the cap. PMID- 16662618 TI - Bacterial ice nucleation: a factor in frost injury to plants. AB - Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Lohnis) Dye are sufficient to incite frost injury to sensitive plants at -5 degrees C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about -2 degrees C, whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at -5 degrees C are always active as ice nuclei at -5 degrees C. INA bacteria incited frost injury to all of the species of sensitive plants tested. PMID- 16662619 TI - Relationship between Ice Nucleation Frequency of Bacteria and Frost Injury. AB - Not every cell of a given bacterial isolate that has ice-nucleating properties can serve as an ice nucleus at any given time and temperature. The ratio between the number of ice nuclei and number of bacterial cells in a culture (i.e. nucleation frequency) was found to vary with incubation temperature, growth medium composition, culture age, and genotype. Optimal conditions for ice nucleus production in vitro included incubation of the bacterial cells at 20 to 24 degrees C on nutrient agar containing glycerol. The relationship between nucleation frequency and frost injury was examined by subjecting corn seedlings to -4 degrees C immediately after they were sprayed with bacterial suspensions with different nucleation frequencies and by following both ice nucleus concentration and bacterial population size on leaves of corn seedlings as a function of time after bacterial application. The amount of frost injury to growth chamber-grown corn seedlings at -4 degrees C was a function of the number of ice nuclei active at that temperature on the leaves. The number of ice nuclei, in turn, is the product of the nucleation frequency and population size of ice nucleation-active bacteria present on the leaves. PMID- 16662620 TI - Biosynthesis of storage proteins in developing rice seeds. AB - Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [(14)C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt soluble, but the mature glutelin subunits were almost salt insoluble.In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57 kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide. PMID- 16662621 TI - Photosynthetic Responses to Irradiance by the Grey Mangrove, Avicennia marina, Grown under Different Light Regimes. AB - Photosynthetic responses to irradiance by the grey mangrove, Avicennia marina (Forstk.) Vierh var. australasica (Walp.) Moldenke, were studied using seedlings grown under natural understory shade and exposed conditions as well as in the laboratory under high and low light regimes, i.e. 100% and 6% sunlight, respectively. Leaves in exposed locations were subjected to daylight quantum flux densities greater than 1,000 microeinsteins per square meter per second from 0900 to 1700 hours, whereas those in understory shade experienced only 30 to 120 microeinsteins per square meter per second, interrupted for brief periods by sunflecks ranging in quantum flux density from 800 to 1,500 microeinsteins per square meter per second. The low light regime was similar in light intensity to that of the understory environment, but lacked sunflecks.Leaves from the understory environment showed several properties of ;shade' leaves; i.e. they contained more chlorophyll on both a leaf area and fresh weight basis but had a lower specific weight and greater area than exposed leaves, and were enriched in chlorophyll b relative to a. However, there were no significant differences in either the gas exchange or leaf chlorophyll fluorescence characteristics of the two populations, both being typical of ;sun' leaves.Leaves grown in the laboratory under low and high light regimes had similar properties. However, light saturated assimilation rates in the leaves from the low light treatment were 20% less and became light saturated at a lower quantum flux density than those of leaves grown under the high light regime. The ecological significance of these results is discussed. PMID- 16662622 TI - High rates of protein synthesis by isolated chloroplasts. AB - Improvements are described in the preparation and in vitro conditions of an intact pea (Pisum sativum Progress No. 9) chloroplast system which provides high efficiency for translation of endogenous messenger RNA, using light as an energy source. High rates result in the incorporation into protein of up to 100 nanomoles tritiated leucine per milligram chlorophyll. These rates suggest extensive reinitiation, and repeated utilization of the messenger RNA that code for thylakoid proteins. Up to 39 radioactive thylakoid peptide bands were detected by fluorography after labeling with tritiated leucine. PMID- 16662623 TI - Localization of a proton-translocating ATPase on sucrose gradients. AB - Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing beta-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin. PMID- 16662624 TI - Light-induced h secretion and the relation to senescence of oat leaves. AB - When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H(+) secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H(+) pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H(+) ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H(+) secretion and acceleration of senescence, may suggest a regulatory role for H(+) secretion in leaf senescence. PMID- 16662625 TI - Changes in the Activities of Ribulose 1,5-Bisphosphate and Phosphoenolpyruvate Carboxylases in Submersed Aquatic Angiosperms during Aging. AB - Changes in the activities of ribulose-1,5-bisphosphate carboxylase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPcase) during aging and senescence of leaves of submersed aquatic angiosperms, Potamogeton pectinatus L., Vallisneria spiralis L., and Hydrilla verticillata (L.f.) Royle were studied. The activity of RuBPcase decreased with both aging and senescence in three species. PEPcase activity increased from young to mature leaves in all three plants and also in old leaves of Hydrilla. The ratio of RuBPcase to PEPcase activity was lowest in mature and highest in old leaves, and increased with aging of isolated mature leaves in Potamogeton and Vallisneria, but decreased markedly in Hydrilla with aging and senescence. Kinetin treatment (0.23 mm) increased RuBPcase activity in three species and PEPcase activity in Potamogeton and Vallisneria, but decreased PEPcase activity in Hydrilla. Treatments with 2-chloroethylphosphonic acid (Ethrel, 0.69 mm) and abscisic acid (ABA, 0.075 mm) showed almost an opposite trend. PMID- 16662626 TI - Role of peroxidase in lignification of tobacco cells : I. Oxidation of nicotinamide adenine dinucleotide and formation of hydrogen peroxide by cell wall peroxidases. AB - The two peroxidase isoenzyme groups (G(I) and G(III)) localized in the cell walls of tobacco (Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H(2)O(2) formation. Peroxidases of the G(III) group are slightly more active than those of the G(I) group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H(2)O(2) required the presence of Mn(2+) and a phenol as cofactors. The addition of H(2)O(2) to the reaction mixture accelerated subsequent NADH-dependent H(2)O(2) formation. In the presence of both cofactors or Mn(2+) alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H(2)O(2) accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H(2)O(2) formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88). PMID- 16662627 TI - Role of Peroxidase in Lignification of Tobacco Cells : II. Regulation by Phenolic Compounds. AB - Coniferyl alcohol is the primary substrate for peroxidase-mediated lignification, a process which depends on the generation of H(2)O(2) by NADH oxidation. We measured the concentrations of various phenols (synthetic and natural) at which maximal enhancement of NADH oxidation occurs. Coniferyl alcohol was found to stimulate NADH oxidation at a much lower concentration (0.01 mm) than other natural or synthetic phenols (1-100 mm). In addition, coniferyl alcohol prevented the conversion of active peroxidase into the inactive intermediate compound III which is usually formed in the presence of NADH-at equally low concentrations. This conversion was found to be a prerequisite for stimulation of NADH-oxidation, but it was not necessarily connected to stimulation.The oxidation of NADH and coniferyl alcohol (or guaiacol) can occur simultaneously, but there is a strong competitive interaction between these two substrates. At pH 5, the presence of NADH at concentrations 30 to 60 times lower than the phenols completely prevents their oxidation. The results are discussed in relation to the role of cell wall peroxidases in conversion of coniferyl alcohol to lignin and in formation of H(2)O(2). PMID- 16662628 TI - H Uptake and Extrusion by Nitella clavata. AB - Very high rates of H(+) extrusion by internodal cells of Nitella clavata Kutz were measured after acid loading at pH 4.6. The highest rate observed, 160 picomoles per square centimeter per second, was more than twice the rate of photosynthetic bicarbonate utilization under saturating light. These results are consistent with the recently proposed hypothesis that bicarbonate is not taken in directly but is protonated at the exterior surface; the CO(2) thereby formed diffuses preferentially into the cell because of the asymmetric concentration gradient.The H(+) taken up, about 150 nanomoles per square centimeter in 2 hours, was distributed in three fractions: 30% in the cell wall, 40% in the cytoplasm, and 30% in the vacuole. This was concluded from the kinetics of the H(+) release by intact cells and isolated walls, and from the pH decrease of the vacuolar sap.The cytoplasmic H(+) was extruded rapidly, with a half-time of about 2 minutes when the external pH was 5.7 or higher. The extrusion of the vacuolar H(+) only proceeded at a measurable rate when the [K(+)] in the medium was raised to 20 millimolar; the half-time was about 100 minutes. There was little H(+) extruded when the external pH was 5.0. PMID- 16662629 TI - Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power. AB - The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O(2) evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6 dichlorophenolindophenol or as O(2) uptake with methyl viologen as acceptor.Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p benzoquinone (photosystem II) from H(2)O and to methyl viologen (photosystem I) from 3,3'-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O(2) evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O(2) evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33 0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts. PMID- 16662630 TI - Alpha-amylase secretion by single barley aleurone layers. AB - The secretion of alpha-amylase from single isolated (Hordeum vulgare L. cv Himalaya) aleurone layers was studied in an automated flow-through apparatus. The apparatus, consisting of a modified sample analyzer linked to a chart recorder, automatically samples the flow-through medium at 1 minute intervals and assays for the presence of alpha-amylase. The release of alpha-amylase from aleurone layers begins after 5 to 6 hours of exposure to gibberellic acid and reaches a maximum rate after 10 to 12 hours. The release of alpha-amylase shows a marked dependence on Ca(2+), and in the absence of Ca(2+) it is only 20% of that in the presence of 10 millimolar Ca(2+). Withdrawal of Ca(2+) from the flow-through medium results in the immediate cessation of enzyme release and addition of Ca(2+) causes immediate resumption of the release process. The effect of Ca(2+) is concentration-dependent, being half-maximal at 1 millimolar Ca(2+) and saturated at 10 millimolar Ca(2+). Ruthenium red, which blocks Ca(2+) but not Mg(2+) efflux from barley aleurone layers, renders alpha-amylase release insensitive to Ca(2+) withdrawal. Inhibitors of respiratory metabolism cause a burst of alpha-amylase release which lasts for 0.5 to 5 hours. Following this phase of enhanced alpha-amylase release, the rate of release declines to zero. Pretreatment of aleurone layers with HCl prior to incubation in HCN also causes a burst of alpha-amylase release, indicating that the inhibitor is affecting the secretion of alpha-amylase and not its movement through the cell wall. The rapid inhibition of alpha-amylase release upon incubation of aleurone layers at low temperature (5 degrees C) or in 0.5 molar mannitol also indicates that enzyme release is dependent on a metabolically linked process and is not diffusion limited. This conclusion is supported by cytochemical observations which show that, although the cell wall matrix of aleurone layers undergoes extensive digestion after gibberellin treatment, the innermost part of the cell wall is not degraded and could influence enzyme release. PMID- 16662631 TI - A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells Cultivated in Liquid Medium. AB - (31)P nuclear magnetic resonance has been used to study the vacuolar and cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max cells grown as cell suspensions. The adaptation of this technique to plant cells grown in liquid medium is described with emphasis on the removal of Mn(2+) and phosphate from the extracellular medium and on providing the O(2) supply of the cells in the nuclear magnetic resonance tube and the various problems of calibration. Aerobic and anaerobic cells show large differences in their glucose 6-phosphate, their cytoplasmic inorganic phosphate pools, and their cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and vacuolar inorganic phosphate pools have been observed for the three cell strains studied. PMID- 16662632 TI - Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria. AB - Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination. PMID- 16662633 TI - Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme : A Possible Regulatory Mechanism. AB - NAD malic enzyme can exist in dimer, tetramer, or octamer form. Freshly prepared enzyme from Solanum tuberosum var. Chieftan exists predominantly as the octamer and during storage is progressively converted into lower molecular weight forms. High ionic strength favors dimer formation, whereas high concentrations of malate or citrate favor tetramer formation. The tetramer is the most active form, having a low K(m) for malate and a high V(max). The dimer, with its high K(m) and low V(max), is the least active form. Malate may regulate NAD malic enzyme by controlling its state of oligomerization. PMID- 16662634 TI - Accumulation of Free Fatty Acids during Hardening of Chlorella ellipsoidea. AB - Chlorella ellipsoidea Gerneck (IAM C-27) was synchronously grown, and cells at an intermediate stage in the ripening phase of the cell cycle were hardened at 3 degrees C for 48 hours. A nonpolar lipid which increased greatly during hardening was analyzed by gas-liquid chromatography. Palmitic, oleic, linoleic, and linolenic acids were the main components of the lipid. Electron micrographs revealed the appearance of lipid bodies in hardened cells. When formation of free fatty acids and lipid bodies was inhibited with cycloheximide, oligomycin, and 3 (3,4-dichlorophenyl)-1,1-dimethylurea, the development of a high level of hardiness was always inhibited. However, the converse results were not always realized. Cells hardened in the dark in the absence of glucose developed a measurable hardiness in spite of their failure to form free fatty acids. The appearance of lipid bodies was invariably accompanied by the formation of the fatty acids. In pulse-labeling with [(14)C]NaHCO(3) for 4 minutes at zero time and at the 12th hour of hardening, initial incorporation rates of (14)C into total lipids of whole cells and the cellular membrane fraction were significantly higher than that into free fatty acids. These results suggest that, although fatty acids are inserted into membrane lipids during hardening, the accumulation of free fatty acids and the appearance of lipid bodies per se are not involved in the development of frost hardiness. PMID- 16662635 TI - Efficiency of Nitrogen Assimilation by N(2)-Fixing and Nitrate-Grown Soybean Plants (Glycine max [L.] Merr.). AB - Nodulated and non-nodulated (not inoculated) soybeans (Glycine max [L.] Merr. cv Wells) were grown in controlled environments with N(2) or nonlimiting levels of NO(3) (-), respectively, serving as sole source of nitrogen. The efficiency of the N(2)-fixing plants was compared with that of the nitrate-supplied plants on the basis of both plant age and plant size. Efficiency evaluations of the plants were expressed as the ratio of moles of carbon respired by the whole plant to the moles of nitrogen incorporated into plant material.Continuous 24-hour CO(2) exchange measurements on shoot and root systems made at the beginning of flowering (28 days after planting) indicated that N(2)-fixing plants respired 8.28 moles of carbon per mole of N, fixed from dinitrogen, while nitrate-supplied plants respired only 4.99 moles of carbon per mole of nitrate reduced. Twenty-one day-old nitrate-supplied plants were even more efficient, respiring only 3.18 moles of carbon per mole of nitrate reduced. The decreased efficiency of the N(2) fixing plants was not due to plant size since, on a dry weight basis, the 28-day old N(2)-fixing plants were intermediate between the 28- and 21-day-old nitrate supplied plants.The calculated efficiencies were predominantly a reflection of root-system respiration. N(2)-fixing plants lost 25% of their daily net photosynthetic input of carbon through root-system respiration, compared with 16% for 28-day-old nitrate-supplied plants and 12% for 21-day-old nitrate-supplied plants. Shoot dark respiration was similar for all three plant groups, varying between 7.9% and 9.0% of the apparent photosynthate.The increased respiratory loss by the roots of the N(2)-fixing plants was not compensated for by increased net photosynthetic effectiveness. Canopy photosynthesis expressed on a leaf area basis was similar for 28-day-old N(2)-fixing plants (15.5 milligrams CO(2) square decimeter per hour) and 21-day-old nitrate-supplied plants (14.5 milligrams CO(2) square decimeter per hour). Both were similar in total canopy leaf area. The larger nitrate-supplied plants (28-day-old) had lower photosynthetic rates (12.5 milligrams CO(2) square decimeter per hour), presumably due to self-shading of the leaves.These data indicate that, during the early stages of plant development, dependence solely on N(2)-fixation is an expensive process compared to nitrate reduction in nitrate-supplied plants, since the N(2)-fixing plants retained 8% to 12% less of their photosynthate as dry matter. PMID- 16662636 TI - Interaction of carbon and nitrogen metabolism in the productivity of maize. AB - Five maize (Zea mays L.) hybrids, FS854, B73 x Mo17, B84 x Mo17, B73 x B77, and P3382, grown under field conditions, were sampled at intervals during the grain filling period. Plants were subdivided into stalks (including sheaths), leaves, and kernels. These parts were assayed for dry weight, reduced nitrogen, and extractable nonstructural carbohydrates. The duration and rates of net nitrate reduction and photosynthesis were approximated by the changes over time in the accumulation of reduced nitrogen and dry weight by the plant (total, above ground), respectively.Data on the accumulation of reduced nitrogen and dry weight by the plant show that decreases in nitrate reduction preceded (in time and extent for four of the hybrids and in extent for FS854) decreases or cessation of photosynthesis. FS854 continued to accumulate reduced nitrogen and dry matter throughout the grain-filling period.The patterns of change in stalk carbohydrate and reduced nitrogen during the early stages of ear development show the stalk serves as a storage reservoir and that these reserves were remobilized during the final stages of grain development. The marked increase and maintenance of dry weight and carbohydrate content of stalks until 34 days after anthesis, shows the capacity of the leaves to produce photosynthate through the first half of the grain-filling period exceeds the needs of the ear and/or the transport system. In contrast, stalk nitrogen content shows a slight increase up to 12 days after anthesis and decreases continually thereafter. Leaf nitrogen was lost continuously throughout grain development. The potential capacity of the plant to supply newly reduced nitrogen was inadequate to support initiation and early development of the kernels without remobilization of vegetative nitrogen. Of the two hybrids having delayed leaf senescence, FS854 with its initially higher concentration and content of reduced nitrogen in the stalk, initiated and developed a bigger ear than P3382, which had lower levels of stalk nitrogen.Three of the five hybrids had ;near linear' rates of accumulation of kernel dry weight, whereas none of the hybrids had linear rates of gain in kernel nitrogen. All hybrids had maximum or near maximum rates of gain of kernel nitrogen between 26 and 34 days after anthesis and a marked reduction (41-52%) of rates in the following sampling interval. These decreases are concurrent with decreases in rates of nitrate reduction (nitrogen accumulation) by the whole plant for four of the hybrids and with decreases in remobilization of nitrogen from the vegetation of FS854. Data for the ratio of rates of accumulation of dry weight/reduced nitrogen by the kernels versus time after anthesis, show that the accumulation of dry weight and reduced nitrogen are independent of each other. The variations in the ratio values appear best related to variations in the availability of nitrogen from the vegetation. PMID- 16662637 TI - Biosynthesis, translocation, and accumulation of betaine in sugar beet and its progenitors in relation to salinity. AB - Like other halophytic chenopods, sugar beet (Beta vulgaris L.) can accumulate high betaine levels in shoots and roots. N,N,N-trimethylglycine impedes sucrose crystallization and so lowers beet quality. The objective of this research was to examine the genetic variability and physiological significance of betaine accumulation in sugar beet and its relatives. Three cultivated genotypes of B. vulgaris and two genotypes of the wild progenitor B. maritima L. were grown with and without gradual salinization (final NaCl concentration = 150 millimolar). At 6 weeks old, all five genotypes had moderately high betaine levels in shoots and roots when unsalinized (averages for all genotypes: shoots = 108 micromoles per gram dry weight; roots = 99 micromoles per gram dry weight). Salinization raised betaine levels of shoots and roots 2- to 3-fold, but did not greatly depress shoot or root growth. The genotype WB-167-an annual B. maritima type-always had approximately 40% lower betaine levels in roots than the other four genotypes, although the betaine levels in the shoots were not atypically low.THE SITE AND PATHWAY OF BETAINE SYNTHESIS WERE INVESTIGATED IN YOUNG, SALINIZED SUGAR BEET PLANTS BY: (a) supplying 1 micromole [(14)C]ethanolamine to young leaf blades or to the taproot sink of intact plants; (b) supplying tracer [(14)C]formate to discs of leaf, hypocotyl, and taproot tissues in darkness. Conversion of both (14)C precursors to betaine was active only in leaf tissue. Very little (14)C appeared in the phospholipid phosphatidylcholine before betaine was heavily labeled; this was in marked contrast to the labeling patterns in salinized barley. Phosphorylcholine was a prominent early (14)C metabolite of both [(14)C]ethanolamine and [(14)C]formate in all tissues of sugar beet. Betaine translocation was examined in young plants of sugar beet and WB-167 by applying tracer [methyl-(14)C]betaine to a young expanded leaf and determining the distribution of (14)C after 3 days. In all cases, extensive (14)C translocation to young leaves and taproot sink occurred; neither in the fed leaf nor in sink organs were any (14)C metabolites of betaine detected. PMID- 16662638 TI - Biochemical characterization of canavalin, the major storage protein of jack bean. AB - The structure of canavalin, a jack bean (Canavalis ensiformis) protein homologous to phaseolin, the major seed storage protein of Phaseolus vulgaris, has been investigated by x-ray crystallography and found to be a hexamer composed of three identical pairs of similar but nonidentical subunits related by a perfect 3-fold axis and pseudo dyad axes (strict C(3) and pseudo D(3)). One member of each pair of subunits is derived from the amino terminal half of a precursor polypeptide of molecular weight 49,000 and the other from its carboxy terminal half. Thus, the crystallographic evidence indicates that the precursor polypeptide is a tandem duplicate and is structurally redundant (McPherson A. 1982 J Biol Chem 255: 10472). A number of physical and chemical properties of the protein in both the uncleaved and the cleaved form were investigated. These included the native molecular weights, amino acid analyses, number of exposed sulfhydryl groups, carbohydrate content, metal ion analysis, crystallization behavior, and the fate of the protein in developing seeds. It was also found that the purified precursor protein possesses a substantial level of alpha-d-mannosidase activity and seems to share a number of other physical and chemical properties with that enzyme. PMID- 16662639 TI - Isolation of spinach leaf peroxisomes in 0.25 molar sucrose solution by percoll density gradient centrifugation. AB - A procedure for isolating spinach (Spinacia oleracea L.) leaf peroxisomes in 0.25 molar sucrose solution by Percoll density gradient centrifugation followed by removal of the Percoll by washing and centrifugation was established. The preparation contains more than 90% peroxisomes as intact organelles with no detectable chlorophyll or cytochrome oxidase contamination. The peroxisomes are stable at 0 to 4 degrees C or 25 degrees C for at least 2 hours. PMID- 16662640 TI - Isolation and purification of intact peroxisomes from green leaf tissue. AB - Intact peroxisomes were prepared from green leaves of a number of C(3) species, both monocots and dicots. A protoplast extract from which chloroplasts have been removed by a 1-minute 10,000g centrifugation was applied to a step gradient of 5, 15, 30, and 45% Percoll containing 0.5 molar sucrose, 0.1% BSA, and 25 millimolar Hepes-KOH (pH 7.5). After centrifugation, a peroxisomal fraction with low contamination by chloroplastic and mitochondrial markers was recovered from the 30/45% Percoll interface. This fraction was passed through a Sepharose 2B column to remove the Percoll which resulted in a peroxisomal preparation exhibiting high intactness (estimated from enzyme latency) and stability. PMID- 16662641 TI - Taxonomic survey for the presence of ribulose-1,5-bisphosphate carboxylase activity in guard cells. AB - Guard cell pairs were dissected from freeze-dried leaves of plants representing 15 families, including monocots, dicots, and pteridophytes. All three major photosynthetic carbon pathways (C(2), C(4), and Crassulacean acid metabolism) were represented. These individual guard cell pairs were assayed quantitatively for ribulose-1,5-bisphosphate carboxylase specific activity. Assay sensitivity averaged 0.08 picomoles of ribulose-P(2) dependent P-glycerate formation (i.e. 100-fold more sensitive than required to detect the activity present in a single Vicia faba mesophyll cell). The calculated specific activities for guard cells and mesophyll cells averaged 4 and 472 millimoles per kilogram dry weight per hour, respectively. For all species surveyed, (a) the enzyme activity calculated for guard cells was below the detection limit of the assay, or (b) the specific activity (weight or cell basis) calculated for guard cells was less than 1% of the specific activity calculated for adjacent mesophyll cells. Based on this survey, the generalization is made that the photosynthetic carbon reduction pathway is absent, or virtually so, in guard cell chloroplasts. PMID- 16662643 TI - Desiccation of Axes of Phaseolus vulgaris during Development of a Switch from a Development Pattern of Protein Synthesis to a Germination Pattern. AB - Immature seeds of Phaseolus vulgaris removed from the pod at 32 days of development do not germinate unless first subjected to a desiccation treatment. This change from development to germination caused by premature drying is mirrored in the pattern of protein synthesis by the axes. Rehydrated axes from 32 day-developed seeds cease to synthesize proteins that are uniquely associated with development, but instead synthesize some proteins that are similar to those made in the germinating axes from mature dry seeds. Desiccation of 22-day developed seeds does not lead to their germination, nor does it cause a switch from a developmental to a germination mode of protein synthesis by the axes. It is proposed that desiccation plays a role in permanently suppressing developmental protein synthesis and in inducing germination protein synthesis. PMID- 16662642 TI - Inhibition by polyamines of macromolecular synthesis and its implication for ethylene production and senescence processes. AB - Applied diamines and polyamines inhibited the incorporation of radioactively labeled leucine and uridine into trichloroacetic acid-insoluble material in apple (Malus domestica Borkh, cv Golden Delicious) fruit tissue. The inhibitory effect was in general more pronounced with the higher molecular weight amines. Putrescine at 5 millimolar inhibited leucine incorporation by 37% and uridine by 44%. Spermidine and spermine at the same concentration inhibited uridine incorporation by 60%. The polyamines at concentrations between 0.1 and 1.0 millimolar inhibited leucine incorporation by 55 to 90%. The inhibitory effect of 0.1 to 10 millimolar polyamines on dark- and wound-induced senescence or ethylene production, is discussed in the light of interference with macromolecular synthesis. PMID- 16662644 TI - Relationship between Vital Staining and Subculture Growth during the Senescence of Plant Tissue Cultures. AB - The vital staining properties of rose cultures (Rosa cv Paul's Scarlet) of increasing age were compared with their ability to be subcultured. At 4-day intervals beginning on day 14, after cell division and expansion had stopped, cells were stained separately with Evans blue, fluorescein diacetate, and phenosafranine. The degree to which parent cultures stained with each of these dyes was compared to the dry weight of their subcultures harvested after 9 and 21 days of growth.Staining with either Evans blue or fluorescein diacetate was demonstrated to be a good means of establishing when senescing cells died. However, the staining properties of aging cultures did not correlate well with their ability to be subcultured, because an increasing proportion of the living cells appeared to lose their ability to divide as senescence progressed. PMID- 16662645 TI - Photosynthetic oxygen exchange in isolated cells and chloroplasts of c(3) plants. AB - Photosynthetic O(2)-production and photorespiratory O(2)-uptake were measured, using stable isotope techniques, in isolated intact leaf cells of the C(3) plant Xanthium strumarium L., and isolated intact chloroplasts of Spinacia oleracea L (var. Yates 102). Considerable light dependent O(2)-uptake was observed in both systems, a proportion of which could be suppressed by CO(2) (63% suppression in chloroplasts by 50 micromolar CO(2), 58% in cells by 100 micromolar CO(2) and 250 micromolar O(2)). At low O(2), O(2)-uptake was CO(2) insensitive. At high CO(2) up to 19% of total electron flow was to O(2) in cells and up to 14% in chloroplasts. O(2)-uptake showed inhibition by KCN (61% in cells, 35% in chloroplasts by 0.2 millimolar KCN). O(2)-uptake half saturated at 75 to 85 micromolar O(2) in cells and 50 to 65 micromolar O(2) in chloroplasts, at low CO(2). The results are discussed in terms of the RuP(2)-oxygenase reaction and direct photoreduction of O(2) via a Mehler reaction. PMID- 16662646 TI - Investigation of the Mechanism of Action of a Chlorosis-Inducing Toxin Produced by Pseudomonas phaseolicola. AB - A toxin that induced chlorotic haloes (typifying haloblight disease) on primary leaves of Phaseolus vulgaris L. (var. Canadian Wonder) was partially purified from culture filtrates of the causative agent Pseudomonas phaseolicola (Burkh.) Dowson. This material was used to investigate chlorosis induction. Haloes could only be induced in those bean leaves that were expanding and synthesizing chlorophyll (Chl); the toxin, therefore, does not promote Chl breakdown. Chl, carotene, and xanthophyll synthesis were inhibited in sections of greening barley (Hordeum vulgare L.) leaves, irrespective of the irradiance level. In parallel experiments, the toxin decreased the level of 5-aminolevulinic acid by amounts sufficient to account for toxin-inhibition of Chl synthesis. Electron microscopy revealed no difference between the transformation of etioplasts into chloroplasts in toxin-treated and control tissue, despite a 60% reduction in Chl in the former. The incorporation of [(14)C]acetate into lipid by greening barley leaf sections and by isolated Pisum sativum chloroplasts in the light and the dark was inhibited about 60% by the toxin. The distribution of radioactivity among the spectra of acyl residues was the same in the control and toxin-treated material. It is suggested that the toxin interferes with an early process common to the synthesis of different lipids, including Chl. PMID- 16662647 TI - Evidence for compartmentalization of conjugates of 2,4-dichlorophenoxyacetic Acid in soybean callus tissue. AB - Soybean Glycine max L. Merrill var. Amsoy 71 root callus tissue labeled with [1 (14)C]2,4-dichlorophenoxyacetic acid (2,4-D) which was subsequently incubated for 24 hours in the absence of 2,4-D, released considerable amounts of label into the media. These results led to an examination of the efflux of 2,4-D and 2,4-D metabolites during a 6-hour time period. Fifty% of the free 2,4-D was lost in 15 minutes and 99% in 6 hours. After 6 hours, only about 48% of the ether-soluble fraction (mainly the glutamic and aspartic conjugates) and about 33% of the aqueous-soluble fraction (mainly hydroxylated glycosides) effluxed from the tissue. Neutral red efflux from stained callus tissue was enhanced only 5% above the control by treatment with 7.5% dimethylsulfoxide (DMSO) and 50% with 20% DMSO. Similar soybean callus tissue preincubated with [1-(14)C]2,4-D and subsequently incubated with H(2)O, 7.5% DMSO, and 20% DMSO was examined for efflux of (14)C label. DMSO similarly enhanced the efflux of the ether and aqueous soluble conjugates.DMSO concentrations of less than 10% did not damage the vacuolar membranes which also has been reported with cultured tobacco cells (Delmer 1979 Plant Physiol 64: 623-629). From these data, it seems that the 2,4-D metabolites are located in a compartment of the cell and presumably the vacuole. PMID- 16662648 TI - Isocitrate lyase from flax : terminal residues, composition, active site, and catalysis. AB - The cleavage of D(s)-isocitrate catalyzed by isocitrate lyase from Linum usitatissimum results in the ordered release of succinate and glyoxylate. The glyoxylate analog 3-bromopyruvate irreversibly inactivates the flax enzyme in a process exhibiting saturation kinetics and protection by glyoxylate or isocitrate or the competitive inhibitor l-tartrate. Succinate provides considerably less protection. Results with 3-bromopyruvate suggest that this reagent modifies plant and prokaryotic isocitrate lyases differently. Treatment of the tetrameric 264,000-dalton flax enzyme with carboxypeptidase A results in a release of one histidine/subunit which is concordant with loss of activity. The only N-terminal residue is methionine. Treatment of flax enzyme with diethylpyrocarbonate at pH 6.5 selectively modifies two histidines per 67,000-dalton subunit. The reaction of one histidine residue is abolished by the binding of l-tartrate and the modification of one is coincident with inactivation. The carboxy-terminal and active-site modifications establish that one histidine residue/monomer is essential in the flax enzyme and considerably extend information heretofore available only for fungal and bacterial isocitrate lyase. PMID- 16662649 TI - pH Dependence and Kinetics of Glycolate Uptake by Intact Pea Chloroplasts. AB - Experiments in which [1-(14)C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C(3) plants. PMID- 16662650 TI - Characterization of the active sucrose transport system of immature soybean embryos. AB - Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [(14)C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35 degrees C, saturating illumination (pH 6), and 21% oxygen, the apparent K(m) for sucrose is approximately 8 millimolar and V(max) is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone. PMID- 16662651 TI - Malate oxidation, rotenone-resistance, and alternative path activity in plant mitochondria. AB - The effect of cyanide and rotenone on malate (pH 6.8), malate plus glutamate (pH 7.8), citrate, alpha-ketoglutarate, and succinate oxidation by cauliflower (Brassica oleracea L.) bud, sweet potato (Ipomoea batatis L.) tuber, and spinach (Spinacia oleracea and Kalanchoe daigremontiana leaf mitochondria was investigated. Cyanide inhibited all substrates equally with the exception of malate plus glutamate; in this case, inhibition of O(2) uptake was more severe due to an effect of cyanide on aspartate aminotransferase. Azide and antimycin A gave similar inhibitions with all substrates. Subsequent addition of NAD had no effect with any substrate. Providing that oxalacetate accumulation was prevented, rotenone inhibited all NAD-linked substrates equally and caused ADP:O ratios to decrease by one-third. Addition of succinate to mitochondria oxidizing malate stimulated oxygen uptake, but adding citrate and alpha-ketoglutarate did not. These results indicate that there is no direct link between malic enzyme and the rotenone- and cyanide-resistant respiratory pathways, and that there is no need to postulate separate compartmentation of malic enzyme and the other NAD-linked enzymes in the matrix. PMID- 16662652 TI - Rapid fractionation of wheat leaf protoplasts using membrane filtration : the determination of metabolite levels in the chloroplasts, cytosol, and mitochondria. AB - A technique is presented for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the mitochondrial matrix of wheat (Triticum aestivum, var ;Timmo') leaf protoplasts, in which membrane filtration is used to prepare fractions enriched in the different subcellular fractions within 0.1 seconds after disruption of the protoplasts. By closing a syringe, protoplasts are forced through a net and disrupted, diluting the cytosol into the medium and also releasing intact chloroplasts and mitochondria which can then be immediately removed on membrane filters placed behind the nylon net. By varying the membrane filters, different filtrates are obtained corresponding to (a) mainly cytosol, or (b) cytosol and mitochondria with only low levels of chloroplasts; alternatively, (c) the entire protoplast contents are obtained by omitting the filters. The filtrates are immediately split, half flowing into HClO(4) where they are immediately quenched for subsequent metabolite analyses; the other half flows into detergent and is used to monitor the exact distribution of marker enzymes in each individual fractionation. Using the measured distributions of metabolite and of marker enzymes in the three filtrates, the subcellular distribution of the metabolite can be algebraically calculated. The method is presented using ATP as an example.The quench time (0.1 second) made possible by membrane filtration is considerably faster than has been possible in the previously developed techniques using silicone oil centrifugation for chloroplasts (1 second) or mitochondria (1 minute). This rapid quench makes it possible to investigate subcellular pools which have a rapid turnover, like the adenine nucleotides. PMID- 16662653 TI - Adenine nucleotide levels in the cytosol, chloroplasts, and mitochondria of wheat leaf protoplasts. AB - Recently, a new method has been described, in which membrane filtration is used to allow the levels of adenine nucleotides in the chloroplast stroma, the cytosol, and the mitochondrial matrix to be measured. This method is now used to investigate the effect of illumination, of respiratory inhibitors, and of uncouplers on the distribution of ATP, ADP, and AMP in wheat (Triticum aestivum var. ;Timmo') leaf protoplasts. (a) The adenine nucleotides are apparently equilibrated by adenylate kinase in the stroma and the cytosol, but not in the mitochondrial matrix. (b) The ATP/ADP quotient in the cytosol is considerably higher than that in the mitochondrial matrix or the chloroplast stroma. (c) A large gradient exists between the ATP/ADP quotients in the cytosol and the mitochondrial matrix in the dark, with a very low ATP/ADP quotient in the mitochondria. This gradient is lowered by uncouplers or respiratory inhibitors showing that, as in animal tissues, it reflects the energization of the mitochondria. (d) In the dark, the stromal ATP/ADP is lower than in the light, and appears to be maintained, at least in part, by import from the cytosol. (e) The cytosolic ATP/ADP, however, actually decreases in the light. This contradicts the widespread assumption, that export of photosynthetically produced ATP from the chloroplast leads to an increase in the cytosolic ATP/ADP, which then inhibits oxidative phosphorylation in the mitochondria. (f) The mitochondrial ATP/ADP increases in the light, and the gradient between the cytosol and mitochondrial matrix falls. This is also difficult to understand in terms of an inhibition of oxidative phosphorylation in the light due to a lack of ADP in the cytosol. (g) The significance of the measured variations in the adenine nucleotide pools are discussed with respect to the diurnal carbohydrate metabolism in a leaf, and to the metabolic function of the chloroplast, the cytosol and the mitochondria. PMID- 16662654 TI - Chloroplast sulfhydryl groups and the light activation of fructose-1,6 bisphosphatase. AB - Studies of isolated intact spinach (Spinacia oleracea L.) chloroplasts reveal that most of the available sulfhydryl groups are associated with stromal protein as opposed to a thylakoid membrane fraction under non-denaturing conditions. Increases in sulfhydryl content of approximately 50% occurred with illumination and could be correlated kinetically with a reductive activation of fructose-1,6 bisphosphatase during CO(2)-assimilation. Inhibition of linear electron flow with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented light driven increases in both fructose-1,6-bisphosphatase activity and the relative sulfhydryl number. These results provide evidence for the operation of a reductive enzyme activating system in vivo. PMID- 16662655 TI - Phase Shifting of the Circadian Clock by Diethylstilbestrol and Related Compounds in Neurospora crassa. AB - Phase shifts of the circadian conidiation rhythm in Neurospora crassa were induced by 3-hour treatments of mycelia in liquid medium with diethylstilbestrol (DES), dienestrol (DIE), hexestrol (HEX), diethylstilbestroldipropionate (DESP), and dienestroldiacetate (DIEA). Over a 24-hour period beginning 24 hours after the transition from light to constant dark, maximum phase shifts occurred about 36 hours. DES was the most effective of the drugs tested, giving 10-hour phase advances at 20 micromolar. DIE and HEX caused similar phase shifts as DES at 40 micromolar. The two derivatives of the last, DESP and DIEA, were much less effective in shifting phase; only a few hours of phase advance result from treatments at 80 micromolar concentrations.The activity of isolated plasma membrane ATPase was inhibited by DES and partially by HEX, but not by DIE, DESP, or DIEA. O(2) consumption of the mycelia was inhibited equally by DES, DIE, and HEX, while DIEA and DESP had little effect. Phase-shifts by DES cannot be interpreted as evidence that plasma membrane ATPase is a component of the circadian clock. PMID- 16662656 TI - In Vitro Synthesis of the Chlorophyll Isocyclic Ring : Transformation of Magnesium-Protoporphyrin IX and Magnesium-Protoporphyrin IX Monomethyl Ester into Magnesium-2,4-Divinyl Pheoporphyrin A(5). AB - Developing chloroplasts of Cucumis sativus L., cv Beit Alpha which were incubated with either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester in darkness produced a partially phototransformable protochlorophyllide species that was tentatively identified as Mg-2,4-divinyl pheoporphyrin a(5). S Adenosylmethionine stimulated Mg-2,4-divinyl pheoporphyrin a(5) formation irrespective of the starting material used. In the case of Mg-protoporphyrin IX monomethyl ester, this stimulation was attributed to the need to remethylate substrate that had been hydrolyzed by an endogenous methylesterase which converts part of the added Mg-protoporphyrin IX monomethyl ester to Mg-protoporphyrin IX.NADP and NADPH stimulated the conversion of Mg-protoporphyrin IX to Mg-2,4 divinyl pheoporphyrin a(5). The conversion required oxygen and was half saturated at 50 micromolar dissolved O(2). The conversion was insensitive to inhibitors of iron-sulfur and heme-containing proteins, to Cu chelators, H(2)O(2), and peroxide scavengers. However, the conversion was extremely sensitive to phenazine methosulfate, methylene blue, and methyl viologen.A decrease of the plastids' ability to convert Mg-protoporphyrin IX to Mg-2,4-divinyl pheoporphyrin a(5) after lysis in 0.1 molar NaCl suggested a requirement for plastid integrity. PMID- 16662657 TI - Sulfite-induced lipid peroxidation in chloroplasts as determined by ethane production. AB - Ethane formation, as a measure of lipid peroxidation, was studied in spinach (Spinacia oleracea L.) chloroplasts exposed to sulfite. Ethane formation required sulfite and light, and occurred with concomitant oxidation of sulfite to sulfate. In the dark, both ethane formation and sulfite oxidation were inhibited. Ethane formation was stimulated by ferric or ferrous ions and inhibited by ethylenediamine tetraacetate. The photosynthetic electron transport modulators, 3 (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and phenazine methosulfate, inhibited both sulfite oxidation and ethane formation. Methyl viologen greatly stimulated ethane formation, but had little effect on sulfite oxidation. Methyl viologen, in the absence of sulfite, caused only a small amount of ethane formation in comparison to that produced with sulfite alone. Sulfite oxidation and ethane formation were effectively inhibited by the radical scavengers, 1,2 dihydroxybenzene-3,5-disulfonic acid and ascorbate. Ethanol, a hydroxyl radical scavenger, inhibited ethane formation only to a small degree; formate, which converts hydroxyl radical to superoxide radical, caused a small stimulation in both sulfite oxidation and ethane formation. Superoxide dismutase inhibited ethane formation by 50% when added at a concentration equivalent to that of the endogenous activity. Singlet oxygen did not appear to play a role in ethane formation, inasmuch as the singlet oxygen scavengers, sodium azide and 1,4 diazobicyclo-[2,2,2]-octane, were not inhibitory. These data are consistent with the view that O(2) is reduced by the photosynthetic electron transport system to superoxide anion, which in turn initiates the free radical oxidation of sulfite, and the free radicals produced during sulfite oxidation were responsible for the peroxidation of membrane lipids, resulting in the formation of ethane. PMID- 16662658 TI - A simple and sensitive DNA assay for plant extracts. AB - A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4',6'-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay. PMID- 16662659 TI - Loss of Hydrogen from Carbon 5 of d-Glucose during Conversion of d-[5-H,6 C]Glucose to l-Ascorbic Acid in Pelargonium crispum (L.) L'Her. AB - Conversion of d-[5-(3)H,6-(14)C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Her cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a (3)H:(14)C ratio of 4 to be compared with 9, the ratio in d-[5-(3)H,6-(14)C]glucose initially. Conversion of d-[6-(3)H,6-(14)C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a (3)H:(14)C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom. PMID- 16662660 TI - Nodule protein synthesis and nitrogenase activity of soybeans exposed to fixed nitrogen. AB - Nitrate or ammonium was added to soybean (Glycine max L. Merrill cv Corsoy) plants grown in plastic pouches 10 days after nodules first appeared. By the third day of treatment with 10 millimolar nitrate, nitrogenase specific activity (per unit nodule weight) had decreased to 15% to 25% of that of untreated plants. Longer incubations and higher concentrations of nitrate had no greater effect. In addition, exogenous nitrate or ammonium resulted in slower nodule growth and decreased total protein synthesis in both the bacterial and the plant portion of the nodule (as measured by incorporation of (35)S). Two-dimensional gel electrophoresis revealed that the nitrogenase components were not repressed or degraded relative to other bacteroid proteins. In the presence of an optimal carbon source, the nitrogenase specific activity of nodules detached from nitrate treated plants was equivalent to that of nodules from untreated plants. These results are consistent with models that propose decreased availability or utilization of photosynthate in root nodules when legumes are exposed to fixed nitrogen. PMID- 16662661 TI - Reorganization of the Photosystem II Unit in Developing Thylakoids of Higher Plants after Transfer to Darkness : Changes in Chlorophyll b, Light-Harvesting Chlorophyll Protein Content, and Grana Stacking. AB - A light-dependent reversible grana stacking-unstacking process, paralleled by a reorganization of thylakoid components, has been noticed in greening etiolated bean (Phaseolus vulgaris, var. red kidney) leaves upon transfer to darkness. The reorganization, based on biochemical and biophysical criteria, involves mainly the photosystem II (PSII) unit components: upon transfer to darkness, the light harvesting chlorophyll protein (LHCP), its 25 kilodalton polypeptide and chlorophyll b are decreased, while the CPa and its 42 kilodalton polypeptide are increased and new PSII units of smaller size are formed. This reorganization of components occurs only in thylakoids still in the process of development and not in those present in steady state conditions.It is proposed that this process does not reflect the turnover of the LHCP component per se, but a regulatory process operating during development, by which the ratio of light-harvesting to PSII reaction center components, determined by the environmental conditions, controls the photosynthetic rate. PMID- 16662662 TI - Hydrogen peroxide synthesis in isolated spinach chloroplast lamellae : an analysis of the mehler reaction in the presence of NADP reduction and ATP formation. AB - Light-dependent O(2) reduction concomitant with O(2) evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of O(2) to H(2)O(2) (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar O(2), O(2) photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn(2+), NADP, and the extent of coupling of phosphorylation to electron flow.In the presence of an uncoupling concentration of NH(4) (+), saturating light intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 millimolarity), and Mn(2+) (50-1000 micromolarity), the maxium rates of O(2) reduction were 13-25 micromoles/milligram chlorophyll per hour, while concomitant rates of O(2) evolution and NADP reduction were 69 to 96 and 134 to 192 micromoles/milligram chlorophyll per hour, respectively. Catalase did not affect the rate of NADPH or ATP formation but decreased the NADPH:O(2) ratios from 2.3-2.8 to 1.9-2.1 in the presence of rate-limiting as well as saturating concentrations of NADP.Photosynthetic electron flow at a rate of 31 micromoles O(2) evolved/milligram chlorophyll per hour was coupled to the synthesis of 91 micromoles ATP/milligram chlorophyll per hour, while the concomitant rate of O(2) reduction was 0.6 micromoles/milligram chlorophyll per hour and was calculated to be associated with an apparent ATP formation of only 2 micromoles/milligram chlorophyll per hour. Thus, electron flow from H(2)O to O(2) did not result in ATP formation significantly above that produced during NADP reduction. PMID- 16662663 TI - Some properties of the chloroplast envelope as revealed by electrophoretic mobility studies of intact chloroplasts. AB - The electrophoretic mobility of mature spinach (Spinacia oleracea L. var. Americana) chloroplasts sampled over a 7-month period was between -2.03 and -2.45 micrometers per second per volt per centimeter when suspended in a solution containing 1 millimolar CaCl(2). The surface charge density of EDTA-treated chloroplasts was calculated to be -7,400 electrostatic units per square centimeter representing, on the average, one electronic charge per 645 square Angstroms. Electrophoretic mobility increases during plastid maturation. Calcium, but not magnesium, generally stabilized the envelope of isolated plastids against small increases in surface charge that occur with time in the absence of calcium. Pronase caused a sharp, but temporary, decrease in the electrophoretic mobility of chloroplasts. This was interpreted as representing a transient binding of pronase to the envelope surface during proteolysis. No -SH groups were detected on the surface of the plastid envelope. Inasmuch as the isoelectric point of intact chloroplasts was found to be at pH 4.5, it is likely that the major part of the total surface charge results from the presence of exposed carboxyl groups of intrinsic envelope proteins that are not readily hydrolyzed by mild pronase treatment. PMID- 16662664 TI - Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule. AB - Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [(14)C]glycerol-3-phosphate and [(14)C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol 3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Delta(3)-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation. PMID- 16662665 TI - Effect of ethylene on [C]indole-3-acetic Acid metabolism in leaf tissues of woody plants. AB - The effect of ethylene on [(14)C]indole-3-acetic acid (IAA) metabolism was investigated in defoliation sensitive leaf tissues of citrus (Citrus sinensis) and resistant leaf tissues of eucalyptus (Eucalyptus camaldulensis). IAA metabolites were fractionated into 80% ethanol-soluble, H(2)O-soluble, NaOH soluble, and insoluble components. In citrus, pretreatment with 25 microliters per liter ethylene for 24 hours significantly increased the amount of ethanol- and H(2)O-extractable conjugates during the first hour of incubation in [(14)C]IAA and increased 3- to 4-fold the formation of NaOH-extractable conjugates during the entire 6-hour incubation period. However, induction of the IAA-aspartate conjugation system was inhibited by ethylene. In eucalyptus, ethylene pretreatment only slightly stimulated the formation of IAA metabolites. Increased formation of ethanol-extractable conjugates in ethylene-pretreated eucalyptus tissues was observed only after 6 hours of incubation. Chromatographic analysis indicated that the ethanol and H(2)O extracts of both species contained various low molecular weight conjugates, whereas in citrus leaf tissues high molecular weight conjugates accounted for most of the greater radioactivity detected in the NaOH extracts as a result of ethylene-pretreatment. It is suggested that ethylene may reduce the level of endogenous IAA in citrus leaf tissues by stimulating IAA conjugation. PMID- 16662666 TI - Measurements of membrane potentials in plant mitochondria with the safranine method. AB - The positively charged dye, safranine, has been used as an indicator of membrane potentials in mung bean (Phaseolus aureus) and Voodoo lily (Sauromatum guttatum) mitochondria under a variety of metabolic conditions. The spectral response of safranine has been calibrated with respect to a K(+) diffusion potential and was found to be linearly related to the developed potential within the range of 50 to 160 millivolts. Both respiration and ATP hydrolysis gave rise to a membrane potential of approximately 135 millivolts. Respiratory inhibitors such as cyanide and antimycin depolarized the potential, whereas rotenone has little effect. No potentials were developed during NADH supported cyanide insensitive respiration. It is concluded that safranine may be a useful spectrophotometric probe of the mitochondrial membrane potential. PMID- 16662667 TI - Nature of photooxidative events in leaves treated with chlorosis-inducing herbicides. AB - Leaves of rye seedlings (Secale cereale L.) grown in the presence of four chlorosis-inducing herbicides under a low light intensity of 10 lux formed chlorophyll. When segments of such dim-light-grown leaves were exposed to 30,000 lux at either 0 degrees C or 30 degrees C, treatments with aminotriazole or haloxidine (group 1) showed no or only minor changes of their chlorophyll contents. In treatments with San 6706 or difunon (group 2), however, rapid photodestruction of chlorophyll occurred both at 0 degrees C and at 30 degrees C and was accompanied by an increase of malondialdehyde that was not seen in the presence of group 1 herbicides. Unlike the in vivo behavior, virtually equal rates of chlorophyll breakdown were observed for aminotriazole and San 6706 treatments in suspensions of isolated chloroplasts from 10 lux-grown leaves after exposure to strong light. The free radical scavengers p-benzoquinone and hydroquinone and the d-penicillamine copper complex exerting superoxide dismutating activity effectively prevented photooxidation of chlorophyll in 10 lux-grown herbicide-treated leaf segments or even restored an accumulation of chlorophyll at 30,000 lux. Ascorbate and several singlet oxygen or hydroxyl radical scavengers had no protective effects. Deuterium oxide and H(2)O(2) did not enhance the degradation of chlorophyll. Superoxide dismutase activity was decreased in leaves bleached in the presence of group 2 herbicides. PMID- 16662668 TI - Isoxazolin-5-ones and Amino Acids in Root Exudates of Pea and Sweet Pea Seedlings. AB - Seeds of Pisum sativum L. cv Finale and Lathyrus odoratus L. cv Spencer were germinated aseptically in moistened sand in the dark. At several stages, the amino acid composition of the exudate and of the corresponding roots was analyzed. A number of common amino acids, including homoserine, were exuded by the growing seedling root in an early stage and were partly reabsorbed later. A number of uncommon amino acids, including several isoxazolin-5-one derivatives, uracil alanines, l-gamma-glutamyl-d-alanine, and alpha-aminoadipic acid were exuded at different rates. PMID- 16662669 TI - Modeling C and N transport to developing soybean fruits. AB - Xylem sap and phloem exudates from detached leaves and fruit tips were collected and analyzed during early pod-fill in nodulated soybeans (Glycine max (L.) Merr. cv Wilkin) grown without (-N) and with (+N) NH(4)NO(3). Ureides were the predominant from (91%) of N transported in the xylem of -N plants, while amides (45%) and nitrate (23%) accounted for most of the N in the xylem of +N plants. Amino acids (44%) and ureides (36%) were the major N forms exported in phloem from leaves in -N plants, but amides (63%) were most important in +N plants. Based on the composition of fruit tip phloem, ureides (55% and 33%) and amides (26% and 47%) accounted for the majority of N imported by fruits of -N and +N plants, respectively.C:N weight ratios were lowest in xylem exudate (1.37 and 1.32), highest in petiole phloem (24.5 and 26.0), and intermediate in fruit tip exudate (12.6 and 12.1) for the -N and +N treatments, respectively. The ratios were combined with data on fruit growth and respiration to construct a model of C and N transport to developing fruits. The model indicates xylem to phloem transfer provides 35% to 52% of fruit N. Results suggest the phloem entering fruits oversupplies their N requirement so that 13% of the N imported is exported from fruit in the xylem. PMID- 16662670 TI - Temperature-induced fluorescence changes : a screening method for frost tolerance of potato (solanum sp.). AB - Field-grown tuber-bearing potatoes were screened for frost tolerance in a late stage of development. Three different clones of Solanum tuberosum L. and two interspecific crosses between clones of S. tuberosum and the wild potato species S. demissum Lindl. were studied. Two different methods were used. (a) Temperature induced fluorescence changes of intact leaves were measured in freeze-thaw cycles between 20 degrees C and -10 degrees C. The variable fluorescence pattern was characterized in relation to frost tolerance. (b) Controlled freezings of plants in a climate chamber with successively increased low temperature stress, of 1 to 2 hours duration during the dark period. Freezing damages were classified visually.The short-term frost during the fluorescence measurement was compared with the long-term frost treatments in the climate chamber. The results of the two were identical to ranking of the different clones for frost tolerance. The temperature-induced fluorescence changes also monitored progressive damages to the chloroplast membranes when plants were exposed to successively lower temperatures in a controlled climate chamber freezing test. It was deduced from the fluorescence measurements that the freezing injury of potato occurs on the water splitting side of photosystem II. PMID- 16662671 TI - Growth and water relations of cultured tomato cells after adjustment to low external water potentials. AB - Cultured cells of tomato, Lycopersicon esculentum Mill. cv VFNT-cherry, have been selected for resistance to water stress (low water potential) imposed by the addition of polyethylene glycol to the culture medium. The ability of nonselected cells to grow in media with low water potentials changes dramatically with the age of the cells (with respect to days following inoculation) whereas there is little effect of the age of selected cells on growth over the same media water potentials. The increased resistance of selected cells has limited stability in the absence of stress, indicating that resistance is established by a slow reversible adaptive process.Increased resistance (growth) in the presence of water stress appears to result from considerable osmotic adjustment by the cells. Growth cycle-dependent changes in resistance of nonselected cells are correlated with osmotic potential changes which are associated with the normal cell growth pattern in culture. Lowered osmotic potential is maintained by selected cells throughout the entire growth cycle and may explain the growth cycle independence of growth of selected cells on polyethylene glycol-containing media. Osmotic adjustment of resistant cells at stationary phase can be as much as 40 bar. Turgor is maintained by resistant cells (as high as 21 bar) in media with low water potentials at least partly at the expense of cell expansion. PMID- 16662672 TI - Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves. AB - Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO(2)-free buffer at 21% O(2), showed a decrease (30-70%) in CO(2)-dependent O(2) evolution and (14)CO(2) uptake. This photoinhibition was observed only when the O(2) concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO(2) concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO(2) concentration was increased to 30 micromolar. Addition of small quantities of P glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose 1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O(2) evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO(2) concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O(2) evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O(2) dependent photoinhibition of light saturated CO(2) fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O(2) for photoinhibition of CO(2) fixation capacity in isolated chloroplasts may be explained by an effect of O(2) in allowing metabolic depletion of Calvin cycle intermediates. PMID- 16662673 TI - Manipulating the incorporation of [1-C]acetate into different leaf glycerolipids in several plant species. AB - During short term labeling of expanding leaves of seven plant species with [1 (14)C]acetate, 35 to 64% of the label incorporated into lipids was found in phosphatidylcholine and 5 to 24% in phosphatidylglycerol. In pumpkin, sunflower, broad bean, and maize, only 4 to 12% of the label was found in diacylgalactosylglycerol, but in tomato, parsley, and spinach, the proportion was 17 to 31%. The latter group was further distinguished by having diacylgalactosylglycerol containing C16:3.The proportions of total incorporated [1-(14)C]acetate entering the lipids could be manipulated in a predictable manner. Phosphatidylcholine labeling was depressed by treating intact leaves with glycerol or ethylene glycol monomethyl ether or by incubating leaf discs in vitro. An associated increase in phosphatidylglycerol labeling occurred within the first group of plants, whereas an increase in labeling of either diacylgalactosylglycerol, phosphatidylglycerol, or sulfolipid occurred within the second group. Treating intact leaves with glycerol or incubating leaf discs in vitro was shown to elevate cellular concentrations of sn-glycerol 3 phosphate.These results have been interpreted in terms of the two-pathway hypothesis for glycerolipid biosynthesis, in which it is proposed that phosphatidylcholine is synthesized via a different pathway (eukaryotic) to that for synthesis of phosphatidylglycerol (prokaryotic). Both pathways may contribute toward the synthesis of diacylgalactosylglycerol, with the contribution of each being assessed from the proportion of hexadecatrienoic acid found in the particular plant. PMID- 16662674 TI - Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum. AB - A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25 degrees C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 x 10(9) molar(-1).second(-1) at pH 7.8 and 25 degrees C. The enzyme was not sensitive to NaCN or to H(2)O(2), but was inhibited by N(3) (-). The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented. PMID- 16662675 TI - Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4' Diisothiocyano-2,2'-Disulfonic Acid Stilbene. AB - Chloride transport, presumably via a Cl(-)-2H(+) co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter(2) per second) was stimulated 4-fold by an 18-hour Cl(-) starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter(2) per second after a 60-minute pre-exposure to 0.5 millimolar 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter(2) per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl(-) influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl(-) to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl(-) restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl(-) influx by inhibiting the putative plasmalemma H(+)-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl(-), in contrast to the presence of Cl(-) during pretreatment of controls. The differential effect could result from competition between Cl(-) and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl(-) transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell. PMID- 16662676 TI - Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots. AB - The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K(+). K(+)-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K(+) transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate. PMID- 16662677 TI - Respiratory and Nitrogenase Activities of Soybean Nodules Formed by Hydrogen Uptake Negative (Hup) Mutant and Revertant Strains of Rhizobium japonicum Characterized by Protein Patterns. AB - Rates of respiratory CO(2) loss and nitrogenase activities of H(2) uptake negative mutant strains and H(2) uptake-positive revertant strains of Rhizobium japonicum have been investigated. Two-dimensional gel protein patterns of bacteroids formed by inoculation of soybeans (Glycine max L.) with these two strains show that they are closely related and revealed only one obvious difference between them. On the basis of molecular weight standards, it was concluded that the missing protein spot in the H(2) uptake-negative mutant strain could be caused by a failure of the mutant to synthesize hydrogenase. Nodules formed by the H(2) uptake-negative mutant strain evolved respiratory CO(2) at a rate of about 10% higher than that of nodules formed by the H(2) uptake-positive revertant strain. During short-term experiments employed, rates of both C(2)H(2) reduction and (15)N(2) fixation varied considerably among replicate samples and no statistically significant differences between mutant and revertant strains were observed. It was observed that increasing the partial pressure of O(2) over nodules significantly decreased the proportion of nitrogenase electrons allocated to H(+). PMID- 16662678 TI - Methionine Synthesis in Lemna: Inhibition of Cystathionine gamma-Synthase by Propargylglycine. AB - Propargylglycine, vinylglycine, and cysteine each cause irreversible inactivations of cystathionine gamma-synthase (and, in parallel, of O phosphohomoserine sulfhydrylase) activities in crude extracts of Lemna paucicostata. Inactivation by propargylglycine or vinylglycine is completely prevented by 40 millimolar O-phospho- or O-succinyl-l-homoserine; that by cysteine is only partially prevented. Propargylglycine (PAG), the most potent of these inhibitors, causes rapid and drastic inactivation of both activities in intact Lemna. Studies of plants growing in steady states in the presence of various concentrations (0-150 nanomolar) of PAG showed that 16% of control activity is necessary and sufficient to maintain normal rates of growth and methionine biosynthesis, and that 10% of control activity is essential for viability. Addition of either 2 micromolar methionine or 31 micromolar cystine to growth medium containing 150 nanomolar PAG permits growth at 75 to 100% of control rates when enzyme activity is less than 10% of control. Whereas methionine presumably rescues by directly providing the missing metabolite, cystine may rescue by enhancing substrate accumulation and thereby promoting flux through residual cystathionine gamma-synthase. The results indicate that the down regulation of cystathionine gamma-synthase to 15% of control which occurs when plants are grown in 2 micromolar methionine (Thompson, Datko, Mudd, Giovanelli Plant Physiol 69: 1077-1083), by itself, is not sufficient to reduce the rate of methionine biosynthesis. PMID- 16662679 TI - Hydroxyproline-rich bacterial agglutinin from potato : extraction, purification, and characterization. AB - A protein, extracted from Katahdin potato (Solanum tuberosum L. cv ;Katahdin') tubers and purified by ion exchange chromatography and gel filtration, agglutinates avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum, but only weakly agglutinates virulent strains. The agglutinin has very low hemagglutinating activity (in contrast to potato lectin) and is a glycoprotein containing about 61% carbohydrate. The carbohydrate moiety contains 91% (weight%) arabinose, 5% galactose, 3% glucose, and 1% glucosamine. The protein portion is rich in hydroxyproline (42%), lysine (16%), serine (9%), and proline (9%). The entire agglutinin has a molecular weight of 91,000 +/- 5,000 and is very basic (pI > 11). Shape estimations based on the concentration dependence of the sedimentation coefficient, the high viscosity ([eta] = 92.7), the frictional coefficient (f/f(o) = 2.15), and axial ratio (a/b = 25) indicate that the agglutinin is a prolate ellipsoid. PMID- 16662680 TI - Two-Dimensional Electrophoretic Mapping of Proteins of Bundle Sheath and Mesophyll Cells of the C(4) Grass Digitaria sanguinalis (L.) Scop. (Crabgrass). AB - Two-dimensional electrophoresis was performed on proteins of bundle sheath and mesophyll cells isolated from the C(4) grass Digitaria sanguinalis (L.) Scop. Two dimensional maps of these proteins were constructed and ribulose-1,5-biphosphate carboxylase and phosphoenolpyruvate carboxylase were identified. Of the total number of proteins found in both cell types, 36% were found only in bundle sheath cells, 17% only in mesophyll cells, and 47% in both cell types. By comparison, the distributions of 48 enzymes assayed in these cell types were 35%, 21%, and 44%, respectively.Protein patterns were also compared with C(4) plants exhibiting different decarboxylation pathways and, in both bundle sheath and mesophyll cells, proteins were found which were unique to each species. Bundle sheath proteins of one C(4) species were found to be more like bundle sheath proteins of another C(4) species than like mesophyll proteins of the same species. PMID- 16662681 TI - Characteristics and Activity Changes of Proteolytic Enzymes in Apple Leaves during Autumnal Senescence. AB - At least four different proteinases are present in senescing apple leaves (Malus domestica Borkh. cv. Golden Delicious) as determined by their pH optima, substrate specificity, and their reactivity to proteinase inhibitors. An enzyme active at pH 4.5 to 5.0 appears to be a sulfhydryl-dependent (iodoacetamide and phenylmercuric acetate-sensitive) endoproteinase, and degradation of the large subunit of ribulose bisphosphate carboxylase was observed only with this enzyme. It is tentatively concluded that this endoproteinase is responsible for the breakdown of ribulose bisphosphate carboxylase in vivo. However, the presence of more than one endoproteinase in apple leaves is suggested by the broad range of pH optima of the SH-dependent enzyme. Another enzyme active at pH 6.0 appears to be a carboxypeptidase, and was sensitive to phenylmethylsulfonylfluoride. This enzyme showed a strong hydrolytic activity against carbobenzoxyphenylalanylalanine. A sulfhydryl-dependent aminopeptidase and a second hydroxyl-dependent carboxypeptidase were active at pH 7.5Total autolytic activity (the sulfhydryl-dependent endoproteinase) as measured by the disappearance of proteins decreased during the period of protein decline. Evidence is presented that the measured proteinase activity can be dependent on assay methods and substrates. While the disappearance of protein measures most of endo-type activity, the ninhydrin assay appears to measure exo-type activity preferentially. PMID- 16662682 TI - Differences between Effects of Undissociated and Anionic 2,4-Dinitrophenol on Permeability of Barley Roots. AB - Effects of 2,4-dinitrophenol (DNP) and several other substituted phenols on permeability of barley roots (Hordeum vulgare var. Trebi) to ions were assayed as a function of pH and phenol concentration. Solutions containing 0.1 micromolar undissociated DNP increase the permeability of barley root cells to small ions such as K(+), Na(+), Ca(2+), and Cl(-) with no inhibition of respiration. Undissociated forms of the other phenols increase permeability also, but they are less effective than DNP. Only the undissociated DNP is effective. Anionic DNP does not increase permeability or inhibit ion uptake, although it is the major species accumulated by the roots, both at pH 5 and pH 7. At pH 7, in contrast to pH 5, 10 micromolar DNP has no effect on ion permeability of barley roots yet it uncouples oxidative phosphorylation of barley root mitochondria. This indicates that the all too common use of DNP as a test for active transport or involvement of ATP synthesis can be misleading. PMID- 16662683 TI - Preparation of Avocado Mitochondria Using Self-Generated Percoll Density Gradients and Changes in Buoyant Density during Ripening. AB - Mitochondria from avocado (Persea americana Mill, var. Fuerte and Hass) can be rapidly prepared at every stage of ripening using differential centrifugation and self-generated Percoll gradients. The procedure results in improved oxidative and phosphorylative properties, especially for mitochondria isolated from preclimacteric fruits.A gradual change in the buoyant density of avocado mitochondria takes place during ripening. Climacteric and postclimacteric avocado mitochondria have the same buoyant density as other plant mitochondria (potato, cauliflower), whereas mitochondria from preclimacteric fruit have a lower density. The transition in buoyant density occurs during the climacteric rise, and two populations of intact mitochondria (p = 1.060 and p = 1.075) can be separated at this stage. Evidence indicates that the difference in mitochondrial buoyant density between preclimacteric and postclimacteric mitochondria is likely due to interactions with soluble cytosolic components. PMID- 16662684 TI - Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle. AB - After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide insensitive respiration, especially with alpha-ketoglutarate and malate as substrates. Whereas alpha-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD(+), glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric. PMID- 16662685 TI - Isolation and transport properties of protoplasts from cortical cells of corn roots. AB - A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 x MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl(2), 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 x 10(3)/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.Cortical cell protoplasts exhibited energy-dependent uptake of K(+) ((86)Rb), H(2) (32)PO(4) (-), and (36)Cl(-) as well as net H(+) extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K(+) was highest between pH 7.5 and 8.0, whereas the influx of H(2)PO(4) (-) was greatest between pH 4.0 and 5.0. K(+) and H(2)PO(4) (-) influx and net H(+) efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl(-) was low, but not greatly different from that observed for other plant cells. K(+) flux was somewhat high, probably because the K(+) concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue. PMID- 16662686 TI - Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings. AB - N(6)-(Delta(2)-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N(6)-(Delta(2)-isopentenyl) adenosine antibodies retained tRNAs containing N(6) (Delta(2)-isopentenyl) adenosine and N(6)-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N(6)-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N(6)-(Delta(2)-isopentenyl) adenosine, and N(6)-(Delta(2)-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N(6)-(Delta(2)-isopentenyl) adenosine and N(6)-(4 hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNA(Phe) and tRNA(Tyr) contained cytokinins whereas tRNA(Ala) did not. PMID- 16662687 TI - Physiology of Movements in Stems of Seedling Pisum sativum L. cv Alaska : II. The Role of the Apical Hook and of Auxin in Nutation. AB - The relationship between the apical hook and stem nutation in etiolated Alaska pea (Pisum sativum L. cv Alaska) seedlings was explored. The hook and maximum nutational displacement have the same plane of symmetry, and both are affected by light acting through phytochrome. However, the two processes do not appear to be obligatorily coupled. Light effects on nutation involve at least two components, an increase in amplitude as well as an increase in frequency. These components can be separated from one another on the basis of developmental time course or red light fluence. Excision of the plumule, leaving the hook attached to the stem, inhibits photostimulated nutation. This inhibition can be overcome by application of indole-3-acetic acid to the remaining stem. If the hook is also excised, then nutation in the stem cannot be restored by indole-3-acetic acid. It is possible, although not yet proven, that the oscillatory process regulating nutation in the stem is itself localized in the hook and that rhythms in the transport of indole-3-acetic acid are involved. PMID- 16662688 TI - An assessment of phase transitions in soybean membranes. AB - Phase transitions were measured in vesicles of phospholipids, alone and in various combinations, and in pelleted mitochondrial membranes, using thermal (DSC) and optical methods. The objective was to consider their possible involvement in chilling injury of soybeans (Glycine max [L.] Merr. cv Wayne 1977). Saturated phospholipids showed clear transitions in the temperature range of 50 degrees C to near 0 degrees C. When mixtures of two phospholipids were examined, there was a marked lowering and broadening of the transition peaks, and a shift in the transition temperatures to intermediate temperatures. The unsaturated phospholipids that occur naturally in soybeans showed no detectable phase transitions in this temperature range, alone or in combinations. Examination of the polar lipids from soybean asolectin revealed no transitions in the biological temperature range; the additions of cations such as Ca(2+) and La(3+) did not evoke a detectable phase transition in them. Mitochondrial membrane pellets likewise showed no transitions. The application of these two direct methods of examination of membrane components without the addition of foreign agents did not support the suggested occurrence of a bulk phase transition which could be related to chilling injury in soybeans. PMID- 16662689 TI - Temperature effects on oxidative metabolism of dormant sugar pine seeds. AB - When dormant sugar pine (Pinus lambertiana L.) seeds were imbibed at 5 degrees C, they showed a rapid increase in O(2) uptake, ATP level, and moisture content during the first 4 days. This was followed by a plateau phase until 60 days, after which a second significant increase in all three features occurred as dormancy was broken. During the plateau phase, conventional CN-sensitive respiration accounted for 74 to 79% of the total O(2) uptake. When dormant sugar pine seeds were imbibed at and maintained at 25 degrees C, a different pattern occurred. Water uptake was much more rapid during the first 4 days and no second increase occurred after 60 days because the seeds did not break dormancy. There was an initial burst of O(2) uptake and ATP formation, but these both declined abruptly after 24 to 48 hours. Levels about half those of seeds at 5 degrees C were maintained through the rest of a 90-day period. CN-sensitive respiration declined during imbibition at 25 degrees C, and accounted for only 55 to 61% of the total O(2) uptake. The inability of dormant sugar pine seeds to germinate at temperatures above about 17 degrees C may therefore result from initial temperature effects on membrane properties, leading to reduced O(2) uptake, reduced cytochrome oxidase electron transport activity, and lowered ATP levels. PMID- 16662690 TI - Mechanism of photoactivation of electron transport in intact bryopsis chloroplasts. AB - The mechanism of photoactivation of photosystem I electron transport was studied in intact Bryopsis corticulans chloroplasts. The evidence from chemical and photochemical studies suggests that photoactivation is a consequence of a reduction of an electron transport component, presumably ferredoxin-NADP(+) reductase. O(2) does not act as a mediator of the process but rather acts as an electron acceptor after photoactivation has occurred. We suggest that the initial function of the chloroplasts in a transition from dark to light is to initiate pseudocyclic electron flow. PMID- 16662691 TI - Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato. AB - Eicosapentaenoic and arachidonic acids in extracts of Phytophthora infestans mycelium were identified as the most active elicitors of sesquiterpenoid phytoalexin accumulation in potato tuber slices. These fatty acids were found free or esterified in all fractions with elicitor activity including cell wall preparations. Yeast lipase released a major portion of eicosapentaenoic and arachidonic acids from lyophilized mycelium. Concentration response curves comparing the elicitor activity of the polyunsaturated fatty acids to a cell-free sonicate of P. infestans mycelium indicated that the elicitor activity of the sonicated mycelium exceeded that which would be obtained by the amount of eicosapentaenoic and arachidonic acids (free and esterified) present in the mycelium. Upon acid hydrolysis of lyophilized mycelium, elicitor activity was obtained only from the fatty acid fraction. However, the fatty acids accounted for only 21% of the activity of the unhydrolyzed mycelium and the residue did not enhance their activity. Centrifugation of the hydrolysate, obtained from lyophilized mycelium treated with 2n NaOH, 1 molarity NaBH(4) at 100 degrees C, yielded a supernatant fraction with little or no elicitor activity. Addition of this material to the fatty acids restored the activity to that which was present in the unhydrolyzed mycelium. The results indicate that the elicitor activity of the unsaturated fatty acids is enhanced by heat and base-stable factors in the mycelium. PMID- 16662692 TI - Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons. AB - Cotyledons of developing Phaseolus vulgaris L. cv Greensleeves seeds were labeled for 2 to 3 hours with (3)H-amino acids, and newly synthesized phytohemagglutinin (PHA) was isolated by affinity chromatography with thyroglobulin-Sepharose. The presence of 1% Tween in the homogenate increased the yield of radioactive PHA by 50 to 100%. Isopycnic sucrose gradients were used to show that this detergent released PHA was associated with the endoplasmic reticulum (ER), and pulse-chase experiments showed that the half-life of the PHA in the ER was 90 to 120 minutes. Since PHA is transiently associated with the ER and accumulates in protein bodies, we postulate that this rapidly turning over pool of PHA in the ER represents protein en route to the protein bodies. The PHA in the ER has the same sedimentation constant as mature PHA and is capable of agglutinating red blood cells. The observations substantiate earlier claims that plant cells contain membrane-bound lectins. However, they also indicate that these lectins are not necessarily functional components of the membranes with which they are associated, but may represent transport pools of lectin normally localized in other cellular compartments. PMID- 16662693 TI - Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system : on the use of light-induced cytochrome B reduction as a marker for the plasma membrane. AB - Presumptive plasma membrane fractions have been prepared from oat (Avena sativa L. cv. Brighton) roots and shoots, respectively, by partition of microsomal fractions in a dextran-polyethylene glycol two-phase system. The plasma membranes had a high affinity for the polyethylene glycol-rich upper phase, whereas membranes from mitochondria and other organelles partitioned in the dextran-rich lower phase or at the interface. Thus, relatively pure plasma membranes were obtained by only two partition steps, and within 3 hours from homogenization of the material.The plasma membranes from both organs were enriched in K(+) stimulated Mg(2+)-dependent ATPase and glucan synthetase II, two tentative markers for the plant plasma membrane. Silicotungstic acid, an indicative stain for the plasma membrane, stained the vesicles recovered from the upper phase, but failed to stain the membranes partitioning in the lower phase or at the interface.The plasma membranes were also enriched in a light-reducible b cytochrome. This b-cytochrome can be measured by its light-induced absorbance change and may serve as a marker for the plant plasma membrane. PMID- 16662694 TI - Sucrose and Glucose Uptake into Beta vulgaris Leaf Tissues : A Case for General (Apoplastic) Retrieval Systems. AB - Concentration curves for sugar and amino acid uptake by Beta vulgaris L. leaf tissues contained both a saturable and a linear component. Similarly shaped curves were obtained for influx of sucrose, glucose, and 3-O-methyl glucose by leaf discs, whole petiole slices, petiole segments containing pith tissue only, and petiole segments containing vascular bundles, although the tissues took up the various sugars via different proportions of saturable versus linear uptake. Two millimolar p-chloromercuribenzenesulfonic acid selectively inhibited the saturable component of sucrose uptake, but had almost no effect on the linear component. Uptake of glucose and 3-O-methyl glucose remained unaffected by p chloromercuribenzenesulfonic acid treatment. Anoxia was found to inhibit the linear component of both sucrose and 3-O-methyl glucose influx, while the saturable component remained unaffected. The linear component of sucrose uptake was also competitively inhibited by maltose, as well as being selectively promoted by certain exposures to 5 millimolar N-ethylmaleimide, 2 micrograms per milliliter cycloheximide, and high levels of mannitol acting as osmoticum. These results support the proposal that the linear component is due to a process more complex than simple, or exchange, diffusion. It would also appear that the linear transport component utilizes a separate energy source than does the saturable component of sucrose influx.Evidence for phloem loading from the apoplast was re examined with respect to the present findings. Saturable sucrose uptake by minor vein tissues may represent retrieval of solute from the free space, which could explain the ;apoplastic loading' phenomenon. PMID- 16662695 TI - Effects of aminoacetonitrile on net photosynthesis, ribulose-1,5-bisphosphate levels, and glycolate pathway intermediates. AB - The effects of aminoacetonitrile (a competitive inhibitor of glycine oxidation) on net photosynthesis, glycolate pathway intermediates, and ribulose-1,5 bisphosphate (RuBP) levels have been investigated at different O(2) and CO(2) concentrations with soybean (Glycine max)[L] Merr. cv Pioneer 1677) leaf discs floated on 25 millimolar aminoacetonitrile (AAN) for 50 minutes prior to assay.At 2% O(2) and 200 or 330 microliters per liter CO(2), the inhibitor had no effect on the rate of net photosynthesis and RuBP levels when compared with the control levels. At 11% to 60% O(2), AAN caused a decrease in net photosynthesis in addition to the inhibition by O(2). This extra inhibition ranged from 22% to 59% depending on the O(2) and CO(2) concentrations. The levels of RuBP, however, were 1.3 to 2.7 times higher than in the control plants at the same O(2) concentrations. At 40% O(2) and 200 microliters per liter CO(2), the inhibitor caused a 6-fold increase in glycine and more than 2-fold increase in glyoxylate levels, whereas those of glycolate decreased by approximately one-half.The decrease in net photosynthesis observed with AAN is not the result of the depletion of the RuBP pool due to the lack of recycling of carbon from the glycolate pathway to the Calvin cycle. The higher levels of RuBP caused by AAN in photorespiratory conditions, suggest that RuBP carboxylase was inhibited. Glyoxylate could be a possible candidate for the inhibition of the enzyme but what is known so far about its inhibitory properties in vitro may not fit the existing in vivo conditions. An alternative explanation for the inhibition is proposed. PMID- 16662696 TI - Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells. AB - Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 mug/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba(2+) > Ca(2+) > Sr(2+) > Mg(2+). Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca(2+) and Na polygalacturonate. PMID- 16662697 TI - Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii. AB - The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO(2) uptake in both light and dark. CO(2) uptake rates were a function of CO(2) levels in the medium. At 2,500 microliters CO(2) per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO(2) level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO(2) uptake rates. In the dark, CO(2) uptake rates were also a function of length of time in the night period. Measurements of dark CO(2) uptake showed that, at both 2,500 and 500 microliters CO(2) per liter, rates declined during the night period. At the higher CO(2) level, dark CO(2) uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO(2) per liter, net CO(2) uptake in the dark at 1800 h was replaced by net CO(2) evolution in the dark at 0600 h. At both CO(2) levels, the overnight decline in net CO(2) uptake was marked by periodic bursts of accelerated CO(2) uptake. CO(2) uptake in the light was similar at 1% and 21% O(2), and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO(2) uptake to the total carbon gain is complicated by the diurnal flux in CO(2) availability under field conditions. PMID- 16662698 TI - Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots. AB - The ATP phosphohydrolase (ATPase) activity of a corn (Zea mays L., WF9 x Mo17) root plasma membrane fraction was enriched almost 2-fold by selective extraction with 0.1% (w/v) deoxycholate. The detergent treatment solubilized about 30% of the total membrane protein and some ATP hydrolyzing activity that was not K(+) stimulated, but the major portion of the ATPase activity could be pelleted with membranes. The properties of the ATPase associated with the detergent-extracted plasma membrane fraction were similar to those for the ATPase of the untreated plasma membrane fraction with respect to substrate specificity, pH optimum, kinetics with MgATP, ion stimulation, and inhibitor sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only minor differences in protein composition resulting from the detergent treatment.The plasma membrane fraction from corn roots contained an endogenous protein kinase activity. This was shown by the time course of phosphate incorporation and by the labeling of a number of protein bands on SDS-polyacrylamide gel electrophoresis. The deoxycholate treatment removed measurable protein kinase activity and allowed the demonstration of a rapidly turning over covalent phosphorylated intermediate associated with the detergent-extracted plasma membrane fraction. The phosphorylated intermediate was present as a 100,000 dalton polypeptide and may represent the catalytic subunit of the plasma membrane K(+)-ATPase. PMID- 16662699 TI - Simultaneous oxidation of glycine and malate by pea leaf mitochondria. AB - Mitochondria isolated from pea leaves (Pisum sativum L.) readily oxidized malate and glycine as substrates. The addition of glycine to mitochondria oxidizing malate in state 3 diminished the rate of malate oxidation. When glycine was added to mitochondria oxidizing malate in state 4, however, the rate of malate oxidation was either unaffected or stimulated. The reason both glycine and malate can be metabolized in state 4 appears to be that malate only used part of the electron transport capacity available in these mitochondria in this state. The remaining electron transport capacity was used by glycine, thus allowing both substrates to be oxidized simultaneously. This can be explained by differential use of two NADH dehydrogenases by glycine and malate and an increase in alternate oxidase activity upon glycine addition. These results help explain why photorespiratory glycine oxidation and its associated demand for NAD do not inhibit citric acid cycle function in leaves. PMID- 16662700 TI - Evaluation of h secretion relative to zeatin-induced growth of detached cucumber cotyledons. AB - Cytokinins promote expansion of cotyledons detached from seedlings of more than a dozen species. The zeatin-enhanced expansion of cucumber (Cucumis sativus L. cv Marketer) cotyledons was investigated. In addition, whether acid secretion is involved in wall loosening accompanying such accelerated growth was evaluated. For cotyledons abraded with carborundum or cut into either eight or 18 pieces, we detected no zeatin-enhanced acidification of the growth medium during growth periods of 3 days. Measurements of pH values on each surface of zeatin-treated, abraded cotyledons after 3 days of growth also showed no detectable acidification caused by the hormone. Furthermore, with several buffers at pH values ranging from 5 to 8, growth of nonabraded, abraded, or cut cotyledons with or without zeatin was independent of external pH. However, experiments restricted to about 12 hours indicated that certain acidic buffers enhanced growth of cotyledons cut into 18 pieces. Lastly, concentrations of fusicoccin that caused growth promotion equal to that of zeatin initiated substantial acidification of the medium. Collectively, these data suggest that zeatin-induced expansion of detached cucumber cotyledons is independent of H(+) secretion. PMID- 16662701 TI - Properties of peach flower buds which facilitate supercooling. AB - Water in dormant peach (Prunus persica [L.] Batsch. var. ;Harbrite') flower buds deep supercooled. Both supercooling and the freezing of water within the bud axis and primordium as distinct components depended on the viability of the bud axis tissue. The viability of the primordium was not critical. Supercooling was prevented by wounding buds with a dissecting needle, indicating that bud structural features were important. Bud morphological features appeared to prevent the propagation of ice through the vascular tissue and into the primordium. In dormant buds, procambial cells had not yet differentiated into xylem vessel elements. Xylem continuity between the bud primordium and adjacent tissues did not appear to be established until buds had deacclimated. It was concluded that structural, morphological, and physiological features of the bud facilitated supercooling. PMID- 16662702 TI - Role of Endogenous Growth Regulators in Seed Dormancy of Avena fatua: I. Short Chain Fatty Acids. AB - The hypothesis that endogenous short chain fatty acids (C 6-C 10) are important in maintaining seeds of wild oat (Avena fatua L.) in the dormant state by acting as natural germination inhibitors (Berrie, Buller, Don, Parker, 1979 Plant Physiol 63: 758-764) was investigated. When germination of nondormant seeds was inhibited by treatment with short chain fatty acids, the seeds did not revert to a similar biochemical and physiological state as exhibited by dormant seeds. First, nonanoic acid-induced inhibition of seed germination was not reversed by hormone treatments which normally break dormancy in wild oat seeds. Second, nondormant seeds treated with short chain fatty acids maintained similar relative proportions of the pentose phosphate pathway and the Embden-Meyerhoff-Parnas pathway for respiratory glucose metabolism as that found in the nondormant controls. Seeds imbibed in the presence of nonanoic acid lost more amino acids and proteins into the imbibition solution than did the untreated controls, suggesting membrane damage had occurred. Inasmuch as increasing concentrations of nonanoic acid also progressively reduced the growth of the coleoptile and roots of intact seedlings until all growth ceased and no germination occurred, the inhibition of seed germination could be due to a nonspecific inhibition of growth of the embryo, perhaps because of disruption of membrane structure and function. Finally, no correlation between endogenous levels of short chain fatty acids in seeds or isolated embryonic axes and seed dormancy could be demonstrated. PMID- 16662703 TI - Correlation between the Lipid Composition and the Responsiveness of Avena sativa Stem Segments to Gibberellic Acid. AB - The lipid composition of Avena sativa stem segments was manipulated using BASF 13 338 (formerly Sandoz 9785) and growth temperature, in order to establish whether there were correlations between responsiveness of the tissue to gibberellic acid (GA(3)) and the presence, before hormone treatment, of specific lipid components. High correlations were obtained between GA(3)-induced growth and total phospholipid, individual phospholipids, and fatty acids (except for linolenic acid), total saturated fatty acids, stigmasterol content, and the unsaturated/saturated fatty acid ratio. It was concluded that, although the lipid composition, and particularly the total saturated fatty acid content, seem to be important contributory determinants of the GA(3)-induced growth response in this system, they may not be obligatory prerequisites, nor the only endogenous factors capable of influencing the response. However, the results are consistent with the hypothesis that membranes are involved in the hormonal mechanism and/or very early stages of the mode of GA(3) action in this tissue. PMID- 16662704 TI - delta-Aminolevulinic Acid Formation from gamma,delta-Dioxovaleric Acid in Extracts of Euglena gracilis. AB - gamma,delta-Dioxovaleric acid (DOVA) has been proposed as a precursor to heme and chlorophyll in plants and algae. DOVA transaminase activity was found in extracts of the unicellular green alga Euglena gracilis Klebs strain Z Pringsheim. Optimum conversion of DOVA to delta-aminolevulinic acid (ALA) occurred at pH 6.8. ALA formation was linear with time for at least 30 minutes at 37 degrees C and was proportional to amount of cell extract in the incubation mixture. Boiled cell extract was inactive. DOVA transaminase from either wild-type or aplastidic derivative strain W(14)ZNaIL ran as a single band in agarose gel permeation chromatography, with a calculated molecular weight of 98,000 +/- 3,000. l Glutamic acid was the most effective amino donor. d-Glutamic acid was inactive. K(m) values for l-glutamic acid and DOVA were 11 and 1.1 millimolar, respectively. Pyridoxal phosphate stimulated activity maximally at 30 micromolar, and (aminooxy)acetate was strongly inhibitory. Glyoxylic acid was a competitive inhibitor with respect to DOVA, with an inhibition constant of 0.62 millimolar. Wild-type and aplastidic cells vielded equal activity, 31 +/- 1 nanomoles ALA per 30 minutes per 10(7) cells, whether grown in light or dark. DOVA transaminase could not be separated from glyoxylate transaminase activity by agarose gel permeation or diethylaminoethyl-cellulose column chromatography. In all fractions, glyoxylate transaminase activity was at least 75 times greater than DOVA transaminase activity. DOVA transamination appears to be catalyzed by glyoxylate transaminase, and not to be of physiological significance with respect to chlorophyll synthesis in Euglena. PMID- 16662705 TI - Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress. AB - Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1 carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO(2+) prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co(2+) had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co(2+), conversion of both root- and petiole synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O(2) roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants. PMID- 16662706 TI - Stomatal behavior and water relations of waterlogged tomato plants. AB - The effects of waterlogging the soil on leaf water potential, leaf epidermal conductance, transpiration, root conductance to water flow, and petiole epinasty have been examined in the tomato (Lycopersicon esculentum Mill.). Stomatal conductance and transpiration are reduced by 30% to 40% after approximately 24 hours of soil flooding. This is not due to a transient water deficit, as leaf water potential is unchanged, even though root conductance is decreased by the stress. The stomatal response apparently prevents any reduction in leaf water potential. Experiments with varied time of flooding, root excision, and stem girdling provide indirect evidence for an influence of roots in maintaining stomatal opening potential. This root-effect cannot be entirely accounted for by alterations in source-sink relationships. Although 1-aminocyclopropane-1 carboxylic acid, the immediate precursor of ethylene, is transported from the roots to the shoots of waterlogged tomato plants, it has no direct effect on stomatal conductance. Ethylene-induced petiole epinasty develops coincident with partial stomatal closure in waterlogged plants. Leaf epinasty may have beneficial effects on plant water balance by reducing light interception. PMID- 16662707 TI - Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves. AB - Spinach leaf (Spinacia oleracea L.) discs infiltrated with [(15)N]glycine were incubated at 25 degrees C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of (15)N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [(15)N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of (15)N-label into the other amino acids was observed at longer incubation time. The per cent (15)N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of (15)N-labeling suggests that glutamine synthetase was involved in the initial refixation of (15)NH(3) derived from [(15)N]glycine oxidation in spinach leaf discs.The (15)N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the (15)N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of (15)N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH(3) during glycine oxidation in spinach leaves. PMID- 16662708 TI - Effect of the Intensity and Duration of Light at Various Temperatures on the Germination of Oldenlandia corymbosa L. Seeds. AB - At temperatures below 35 to 40 degrees C, fairly intense continuous white light (13 watts per square meter) inhibits germination of Oldenlandia corymbosa L. seeds, and the lower the temperature, the greater the inhibition. However, such lighting may enable seeds to germinate later in the dark; their degree of germinability depends both on the duration of lighting and on the temperature during lighting and after transfer to the dark. PMID- 16662709 TI - Characterization of lignin in situ by photoacoustic spectroscopy. AB - Photoacoustic spectroscopy is a recently developed nondestructive analytical technique that provides ultraviolet, visible, and infrared absorption spectra from intensely light scattering, solid, and/or optically opaque materials not suitable for conventional spectrophotometric analysis. In wood and other lignocellulosics, the principal ultraviolet absorption bands, in the absence of photosynthetic pigments, arise from the aromatic lignin component of the cell walls. Photoacoustic spectra of extracted lignin fragments (milled wood lignin) and synthetic lignin-like polymers contain a single major absorption band at 280 nanometers with an absorption tail extending beyond 400 nanometers. Photoacoustic spectra of pine, maple, and oak lignin in situ contain a broad primary absorption band at 300 nanometers and a longer wavelength shoulder around 370 nanometers. Wheat lignin in situ, on the other hand, exhibits two principle absorption peaks, at 280 nanometers and 320 nanometers. The presence of absorption bands at wavelengths greater than 300 nanometers in intact lignin could result from (a) interacting, nonconjugated chromophores, or (b) the presence of more highly conjugated structural components formed as the result of oxidation of the polymer. Evidence for the latter comes from the observation that, on the outer surface of senescent, field-dried wheat culms (stems), new absorption bands in the 350 to 400 nanometer region predominate. These new bands are less apparent on the outer surface of presenescent wheat culms and are virtually absent on the inner surface of either senescent or presenescent culms, suggesting that the appearance of longer wavelength absorption bands in senescent wheat is the result of accumulated photochemical modifications of the ligin polymer. These studies also demonstrate photoacoustic spectroscopy to be an important new tool for the investigation of insoluble plant components. PMID- 16662710 TI - Oleate desaturation in young winter wheat root tissue. AB - [1,2-(14)C]Acetate was incorporated into the lipids of young wheat (Triticum aestivum L. cv Kharkov 22 MC) root tissue, but predominantly into sterols. [1 (14)C]Ammonium oleate was initially incorporated mainly into phosphatidylcholine (PC), and later into triglycerides (TGs). Diglycerides (DGs) contained 16% of the lipid (14)C after 5 minutes and 8% after 40 minutes. The proportion of the label of each lipid group incorporated into linoleate during an 80-minute incubation increased at similar rates for each group, and was always highest in PC. Radioactivity was detected in PC-linoleate earlier than in linoleate of the other groups. During a prolonged incubation after a 15-minute pulse labeling, the percentage of the lipid (14)C incorporated into PC and DGs was high at the end of the pulse but decreased later, while that in TGs increased to 64% after 4 hours. The proportion of the label of each group recovered in linoleic acid peaked in all groups after 4 hours, except for the TGs where it increased slowly throughout the experiment. Only traces of radioactivity were detected in linolenate. The data are compatible with a pathway in which oleate is incorporated into PC, is desaturated to linoleate on PC, and where the linoleate-enriched DGs are transferred from PC to TGs. PMID- 16662711 TI - Influence of light on the heat sensitivity of the photosynthetic apparatus in isolated spinach chloroplasts. AB - The most heat-sensitive functions of chloroplasts in Spinacia oleracea L. including the stromal carboxylation reaction, the light-induced electrical field gradient across the thylakoid membrane, as well as the overall photosynthetic CO(2) fixation were less affected by heat if chloroplasts were heated in the light: 50% inactivation occurred around 35 degrees C in the dark and around 40 degrees C in the light. Relative low light intensities were sufficient to obtain optimal protection against heat. In contrast, the light-induced DeltapH across the thylakoid membrane, the photophosphorylation, and the photochemical activity of photosystem II which were less sensitive to heat in the dark (50% inactivation above 40 degrees C) were not protected by light. Photosystem II even was destabilized somewhat by light.The effect of light on the heat sensitivity of the water-splitting reaction was dependent on the pH in the medium. Protection by light only occurred at alkaline pH, in which case heat sensitivity was high (50% inactivation at 33 degrees C in the dark and at 38 degrees C in the light). Protection was prevented by uncouplers. At pH 6.8 when the heat sensitivity was low in any case (50% inactivation at 41 degrees C in the dark), light had no further protecting effect.Protection by light has been discussed in terms of light-induced transport of protons from the stroma to the thylakoid space and related ion fluxes. PMID- 16662712 TI - Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : site-specific inhibition of the photosynthetic carbon reduction cycle. AB - The effects of reduced osmotic potential on the photosynthetic carbon reduction cycle were investigated by monitoring photosynthetic processes of spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts exposed to increased assay medium sorbitol concentrations. CO(2) assimilation was found to be inhibited at 0.67 molar sorbitol by about 60% from control rates at 0.33 molar sorbitol. This level of stress inhibition was greater than that affecting the reductive phase of the cycle; glycerate 3-phosphate reduction was inhibited at 0.67 molar by 27 to 40%. Sorbitol (0.67 molar) inhibited the rate of O(2) evolution at saturating and limiting concentrations of NaHCO(3), and extended the lag phase of O(2) evolution. This indicated that factors which are rate-limiting to the photosynthetic process are adversely affected by reduced osmotic potential.Analysis of photosynthetic products following CO(2) fixation in 0.33 molar sorbitol and 0.67 molar sorbitol indicated that reduced osmotic potential facilitated increases in the levels of fructose 1,6-bisphosphate and triose phosphates with reductions in glucose 6-phosphate and fructose 6-phosphate, implicating fructose 1,6-bisphosphatase as a site of osmotic stress. Osmotic inhibition of the reductive portion (glycerate 3-phosphate to triose phosphate) of the photosynthetic carbon reduction cycle was partially attributed to feedback inhibition by the product, triose phosphate, on glycerate 3-phosphate reduction. A saturating concentration of ribose 5-phosphate partially overcame osmotic inhibition of CO(2)-supported O(2) evolution, indicating another but apparently less severe site of stress inhibition in the sequence of ribose 5-phosphate to glycerate 3-phosphate. PMID- 16662713 TI - Limiting Factors in Photosynthesis: III. Effects of Iron Nutrition on the Activities of Three Regulatory Enzymes of Photosynthetic Carbon Metabolism. AB - When Fe was withheld from sugar beets (Beta vulgaris L. CV F58-44H1), the activities of NADP-glyceraldehyde-3-phosphate dehydrogenase, fructose 1,6 bisphosphatase, and ribulose 5-phosphate kinase were not diminished, while chlorophyll per area was decreased by 75%. On resupplying Fe, chlorophyll per area increased to control levels within 5 days, whereas the activities of the three enzymes remained approximately constant. These results support the view advanced earlier (Terry 1980 Plant Physiol 65: 114-120) that the photosynthetic effects of Fe deprivation are mediated by changes in the lamellar components of chloroplasts and not by changes in stromal enzymes involved in photosynthetic carbon reduction. PMID- 16662714 TI - Effect of pod removal on leaf senescence in soybeans. AB - Depodding soybean (Glycine max [L] Merr. cv Wye) plants results in an apparent inhibition of senescence as indicated by leaf chlorophyll and soluble protein retention. However, leaf photosynthesis and ribulose bisphosphate carboxylase (Rubisco) levels begin to decline earlier in depodded than in control, podded plants. The initial decline in photosynthesis is correlated with a decrease in leaf transpiration, while the latter decline is associated with the loss of Rubisco. Total soluble protein remains high in depodded plants because several polypeptides, three in particular, increase in amounts sufficient to offset the loss of Rubisco. Thus, depodding appears to change the function of the leaf rather than simply delaying or preventing the decline in leaf function. Changes in specific leaf weight and starch content following depodding suggest that the leaf may be changing to a storage organ. PMID- 16662715 TI - Growth and Chromatic Adaptation of Nostoc sp. Strain MAC and the Pigment Mutant R MAC. AB - A spontaneous, stable, pigmentation mutant of Nostoc sp. strain MAC was isolated. Under various growth conditions, this mutant, R-MAC, had similar phycoerythrin contents (relative to allophycocyanin) but significantly lower phycocyanin contents (relative to allophycocyanin) than the parent strain. In saturating white light, the mutant grew more slowly than the parent strain. In nonsaturating red light, MAC grew with a shorter generation time than the mutant; however, R MAC grew more quickly in nonsaturating green light.When the parental and mutant strains were grown in green light, the phycoerythrin contents, relative to allophycocyanin, were significantly higher than the phycoerythrin contents of cells grown in red light. For both strains, the relative phycocyanin contents were only slightly higher for cells grown in red light than for cells grown in green light. These changes characterize both MAC and R-MAC as belonging to chromatic adaptation group II: phycoerythrin synthesis alone photocontrolled.A comparative analysis of the phycobilisomes, isolated from cultures of MAC and R MAC grown in both red and green light, was performed by polyacrylamide gel electrophoresis in the presence of 8.0 molar urea or sodium dodecyl sulfate. Consistent with the assignment of MAC and R-MAC to chromatic adaptation group II, no evidence for the synthesis of red light-inducible phycocyanin subunits was found in either strain. Phycobilisomes isolated from MAC and R-MAC contained linker polypeptides with relative molecular masses of 95, 34.5, 34, 32, and 29 kilodaltons. When grown in red light, phycobilisomes of the mutant R-MAC appeared to contain a slightly higher amount of the 32-kilodalton linker polypeptide than did the phycobilisomes isolated from the parental strain under the same conditions. The 34.5-kilodalton linker polypeptide was totally absent from phycobilisomes isolated from cells of either MAC or R-MAC grown in green light. PMID- 16662717 TI - Terpenoid Metabolism in Plastids : Localization of alpha-Tocopherol Synthesis in Capsicum Chromoplasts. AB - The synthesis of alpha-tocopherol from 2,3-dimethylphytylquinol and S-adenosyl-l methionine was achieved using Capsicum annuum fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:gamma-tocopherol methyl-transferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction. PMID- 16662716 TI - Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts. AB - Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes. PMID- 16662718 TI - Screening and selection of maize to enhance associative bacterial nitrogen fixation. AB - The ability of maize (corn, Zea mays L.) to support bacterial nitrogen fixation in or on maize roots has been increased, through screening and selection. Isotopic N fixed from (15)N(2) was found on the roots. The nitrogen-fixing association was found in germplasm from tropical maize, but this activity can be transferred to maize currently used in midwestern United States agriculture. PMID- 16662719 TI - Auxin Uptake and the Rapid Auxin-Induced Growth in Isolated Sections of Helianthus annuus. AB - When a sunflower (Helianthus annuus L.) hypocotyl section is placed in a solution of 10 micromolar 1-naphthylacetic acid, the majority of the auxin enters via the cut surfaces. However, it is the minority entering radially which causes the typical growth response. Auxin supplied only to the apical cut surface gives a weak, slower response. PMID- 16662720 TI - Endosperm and pericarp involvement in the supercooling of imbibed lettuce seeds. AB - Fully hydrated lettuce (Lactuca sativa L.) seeds showed dual freezing exotherms ( 9 and -18 degrees C), even after 10 hours imbibition. Only the -9 degrees C exotherm was observed in seeds imbibed for 20 hours, but without external nucleation, all water in the embryo supercooled. Results indicate that the endosperm acts as a barrier to ice propagation. Other experiments suggest that the pericarp may also protect the embryo under certain freezing conditions. PMID- 16662721 TI - Identification of Abscisic Acid in Tulipa gesneriana L. by Gas-Liquid Chromatography with Electron Capture and Combined Gas-Liquid Chromatography and Mass Spectrometry. AB - A major growth inhibitory substance of tulip bulbs (Tulipa gesneriana L. cv Paul Richter) has been unequivocally shown to be abscisic acid (ABA). The ABA methyl ester of the free ether-soluble acid fractions of tulip organs had the identical retention time on gas-liquid chromatography with electron capture detector as authentic ABA methyl ester. In addition, the mass spectra were the same. On a unit dry matter basis, the basalplate and floral shoot contained 3.6 and 2.6 times more ABA than the fleshy scales, respectively. PMID- 16662722 TI - Succinate dehydrogenase : a partial purification from mung bean hypocotyls and soybean cotyledons. AB - A procedure was developed for the partial purification of succinate dehydrogenase from mung bean (Vigna radiata L.) hypocotyls and soybean (Glycine max [L] Merr. v. Ransom) cotyledons. The procedure utilized a Triton X-100 extraction followed by ammonium sulfate precipitation. The final fraction was enriched in two polypeptides with approximate molecular weights of 67,000 and 30,000 daltons, exhibited a pH optima of 7.0 to 7.5, contained a b-type cytochrome, and exhibited the characteristic ferredoxin-type and high potential iron-sulfur protein-type electron paramagnetic resonance signals reported for the iron-sulfur centers of mammalian succinate dehydrogenase. Inhibition constants of 1.15 and 24.6 micromolar for oxaloacetate and malonate, respectively, were obtained. PMID- 16662723 TI - Profile of Basic Carbon Pathways in Guard Cells and Other Leaf Cells of Vicia faba L. AB - Guard cells and three other cell types from Vicia faba L. ;Longpod' leaflets were assayed for enzymes that catalyze one step in each of five major carbon pathways in green plants: the photosynthetic carbon reduction pathway (ribulose bisphosphate carboxylase, EC 4.1.1.39), the photosynthetic carbon oxidation pathway (hydroxypyruvate reductase, EC 1.1.1.81), glycolysis ([NAD] glyceraldehyde-P dehydrogenase, EC 1.2.1.12), the oxidative pentose-P pathway (6 P-gluconate dehydrogenase, EC 1.1.1.44), and the tricarboxylic acid pathway (fumarase, EC 4.2.1.2). Neither ribulose-bisphosphate carboxylase nor hydroxypyruvate reductase could be detected in guard cells or epidermal cells; high levels of these activities were present in mesophyll cells. The specific activity of fumarase (protein basis) was about 4-fold higher in guard cells than in epidermal, palisade parenchyma or spongy parenchyma cells. (NAD) glyceraldehyde-P and 6-P-gluconate dehydrogenases also were present at high protein specific activities in guard cells (2- to 4-fold that in meosphyll cells).It was concluded that the capacity for metabolite flux through the catabolic pathways is high in guard cells. In addition, other support is provided for the view that photoreduction of CO(2) by these guard cells is absent. PMID- 16662724 TI - Structure of Plant Cell Walls : XII. Identification of Seven Differently Linked Glycosyl Residues Attached to O-4 of the 2,4-Linked l-Rhamnosyl Residues of Rhamnogalacturonan I. AB - Seven differently linked glycosyl residues have been found to be glycosidically linked to O-4 of the branched 2,4-linked l-rhamnosyl residues contained in the rhamnosyl and galacturonosyl backbone of the cell wall pectic polysaccharide rhamnogalacturonan I. These seven glycosyl residues are, therefore, the first residues of at least seven different side chains attached to the rhamnogalacturonan backbone. These first side chain glycosyl residues are 5 linked l-arabinofuranosyl and terminal 3-, 4-, 6-, 2,6-, and 3,6-linked d galactopyranosyl residues. The existence of at least seven different side chains in rhamnogalacturonan I indicates that rhamnogalacturonan I is either an exceedingly complex polysaccharide or that rhamnogalacturonan I is a family of polysaccharides with similar or identical rhamnogalacturonan backbones substituted with different side chains. PMID- 16662725 TI - Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content. AB - Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg(2+) to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca(2+) to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca(2+) before exerting its antisenescence action. PMID- 16662726 TI - Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves : II. Inhibition of Ethylene Biosynthesis and Possible Mode of Action. AB - The effects of the polyamines spermidine and 1,3-diaminopropane on ethylene biosynthesis and chlorophyll (Chl) loss were studied in peeled leaves of oat (Avena sativa L., var. Victory) incubated in the dark. Peeling off the epidermal cells induces an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity, resulting in an enhanced ACC and ethylene formation. Both polyamines inhibit ethylene biosynthesis from methionine by inhibiting ACC synthase activity and, more effectively, the conversion of ACC to ethylene. They also inhibit Chl loss occurring between 24 and 48 h of dark incubation; but, as shown by inhibitor experiments, inhibition of Chl loss does not result from inhibition of ethylene formation. Ethylene production and Chl loss, both associated with senescence, require membrane integrity; thus, treatments which promote deterioration of membranes inhibit both processes. Ca(2+) in the incubation medium competitively reduces the polyamine-mediated inhibition of ACC conversion and Chl loss. The data suggest that polyamines initially attach to membranes, thereby inducing changes which, in turn, lead to inhibition of ethylene biosynthesis and retardation of senescence. PMID- 16662727 TI - Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina. AB - Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO(2) = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with (15)N(2) incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N(2)-fixing flora which may subsidize their nutritional demand for nitrogen. PMID- 16662728 TI - Genome Expression during Normal Leaf Development : 2. Direct Correlation between Ribulose Bisphosphate Carboxylase Content and Nuclear Ploidy in a Polyploid Series of Wheat. AB - The quantitative relationships between ribulose bisphosphate carboxylase, nuclear ploidy, and plastid DNA content were examined in the nonisogenic polyploid series Triticum monococcum (2x), Triticum dicoccum (4x), and Triticum aestivum (6x). Ribulose bisphosphate carboxylase per mesophyll cell increased in step with each increase in nuclear ploidy so the ratios of ribulose bisphosphate carboxylase per mesophyll cell (picograms) to nuclear DNA per mesophyll cell (picograms) were almost identical in the three species. Ribulose bisphosphate carboxylase per plastid was 14.1, 14.7, and 16.8 picograms in the 2x, 4x, and 6x ploidy levels, respectively. Plastid area in these three species decreased with increasing nuclear ploidy so the concentration of ribulose bisphosphate carboxylase in the plastoids was 60% higher in the hexaploid compared to the diploid species. DNA levels per plastid were 64 and 67 femtograms for the diploid and tetraploid species, respectively, but were 40% less in the plastids of the hexaploid species. These relationships are discussed in terms of cellular and plastid control of ribulose bisphosphate carboxylase content. PMID- 16662729 TI - Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants. AB - Corn (Zea mays L.) plants were assayed for nitrogenase activity (C(2)H(2) reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O(2) resulted in lower rates of C(2)H(2) reduction. The time course of C(2)H(2) reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn x teosinte) and indicated that a long preincubation at reduced O(2) is not absolutely required for early detection of nitrogenase activity. Isolation of N(2)-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N(2)-fixing bacteria. PMID- 16662730 TI - Metabolism of tritiated gibberellin a(20) in maize. AB - After the application of 2.36 Curies per millimole [2,3-(3)H]gibberellin A(20) (GA(20)) to 21-day-old maize (Zea mays L., hybrid CM7 x CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of (3)H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [(3)H]GA(1) on the basis of co-chromatography with standard [(3)H]GA(1) on SiO(2) partition, high resolution isocratic elution reverse phase C(18) high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [(3)H]GA(8) and C/D ring-rearranged [(3)H]GA(20), although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C(18) high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA(1) and GA(8), or with retention times where conjugates of GA(20) would be expected to elute. Conversion to [(3)H]GA(1) was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [(3)H]GA(20)-like metabolite was the major acidic product, while conversion to [(3)H]GA(1) was low. PMID- 16662731 TI - Ontogenetic variation of four cytokinins in soybean root pressure exudate. AB - Cytokinins exported from the root may be involved in the correlative control of plant development. To test this hypothesis in soybean ((Glycine max [L.] Merr. cv. McCall, cv Chippewa 64, and cv Hodgson 78), cytokinins were intercepted en route from the root to the shoot by collecting root pressure exudate from detopped roots. The quantities of four cytokinins in the exudate were studied throughout the development of plants grown in the field and in controlled environment chambers. Zeatin, zeatin riboside, and their dihydro derivatives, dihydrozeatin and dihydrozeatin riboside, were isolated and quantitated using high-performance liquid chromatography.Cytokinin fluxes (pmoles per plant per hour) were independent of exudate flux (grams per plant per hour). All fluxes are averages for a 6- or 8-h collection period. The ribosides accounted for the majority of the observed cytokinin transport. The fluxes of zeatin riboside and dihydrozeatin riboside increased from low levels during vegetative growth to maxima during late flowering or early pod formation. Before the seeds began rapid dry matter accumulation, zeatin riboside and dihydrozeatin riboside fluxes decreased and remained at low levels through maturation. The fluxes of zeatin and dihydrozeatin were low throughout development.No correlation was found between cytokinin fluxes and nodule dry weight or specific nodule activity (acetylene reduction).The timing of distinct peaks in zeatin riboside and dihydrozeatin riboside fluxes during flowering or pod formation suggests that cytokinins exported from the root may function in the regulation of reproductive growth in soybean. PMID- 16662732 TI - A Comparative Study of the Physiology of Symbioses Formed by Rhizobium japonicum with Glycine max, Vigna unguiculata, and Macroptilium atropurpurem. AB - Although Rhizobium japonicum nodulates Vigna unguiculata and Macroptilium atropurpurem, little is known about the physiology of these symbioses. In this study, strains of R. japonicum of varying effectiveness on soybean were examined. The nonhomologous hosts were nodulated by all the strains tested, but effectiveness was not related to that of the homologous host. On siratro, compared to soybean, many strains reversed their relative effectiveness ranking. Both siratro and cowpea produced more dry matter with standard cowpea rhizobia CB756 and 176A22 than with the strains of R. japonicum. Strains USDA33 and USDA74 were more effective with siratro and cowpea than with soybean. The strain USDA122 expressed high rates of hydrogenase activity in symbiosis with the cowpea as well as the soybean host. The strains USDA61 and USDA74 expressed low levels of hydrogenase activity in symbiosis with cowpea, but no activity was found with soybean. Our results indicate host influence for the expression of hydrogenase activity, and suggest the possibility of host influence of nitrogenase for the allocation of electrons to N(2) and H(+). PMID- 16662733 TI - Role of cytokinin in differentiation of secondary xylem fibers. AB - The differentiation of secondary xylem fibers was studied in cultured hypocotyl segments of Helianthus annuus L. It is shown that cytokinin is both a limiting and controlling factor in the early stages of fiber differentiation. In the absence of kinetin or zeatin, there was no fiber differentiation. However, cytokinin could induce fiber differentiation only in the presence of indoleacetic and gibberellic acids. First fibers were observed in the tissue after 12 days in culture, and their number increased linearly during the following 2 weeks. At low cytokinin levels, there was a positive correlation between cytokinin concentration in the medium and the number of fibers formed in the explants. A similar correlation was also found at low gibberellic acid concentrations. At high concentration, zeatin was more effective than kinetin. It seems that later stages of fiber differentiation can occur in the absence of cytokinin. It is proposed that the mechanism which controls and determines the early stages of fiber differentiation is based on an interaction of three major hormonal signals: indoleacetic acid plus gibberellic acid from the leaves with zeatin from the root apices. PMID- 16662734 TI - Estimation of osmotic parameters accompanying zeatin-induced growth of detached cucumber cotyledons. AB - Water potential (psi), the osmotic potential (psi(pi)), and the pressure potential (psi(p)) of detached cotyledons isolated from Cucumis sativus L. cv Marketer seedlings after 0, 1.5, and 3 days growth with and without zeatin were determined. From zero time to 3 days, cotyledons incubated without exogenous zeatin exhibited a slight decrease in psi (from -0.4 to -1.0 bars), while those grown with zeatin developed even more negative values (about -4 bars). Both groups showed rising psi(pi) values (decreases in solutes per unit volume), but this rise was more dramatic in those treated with zeatin. These data indicate that the capacity of zeatin-treated cotyledons to take up water more rapidly than controls and thus expand faster must be due to wall loosening, as reflected in psi(p) values which declined during 3 days from about +11 bars to about +1.4 bars.It was also found that freshly detached cotyledons or those grown without exogenous zeatin exhibited osmoregulation in polyethylene glycol (PEG) solutions. That is, while cotyledons initially lost H(2)O into certain PEG solutions, their psi values decreased over time and they began absorbing water after 1 to 4 hours. After 3 days growth, zeatin-treated cotyledons had lost most of this capacity of osmoregulate. It seems likely that osmoregulation in cotyledons not treated with zeatin is due to wall loosening rather than changes in psi(pi). Zeatin-treated cotyledons with already loosened walls may not have this option to deal with water stress and thus simply come to equilibrium with external PEG solutions. PMID- 16662735 TI - Regulation by amino acids of photorespiratory ammonia and glycolate release from ankistrodesmus in the presence of methionine sulfoximine. AB - Methionine sulfoximine induced release of ammonia from illuminated cells of Ankistrodesmus braunii (Naegeli) Brunnth, in normal air, but less in air enriched to 3% CO(2). In normal air, methionine sulfoximine also induced glycolate release. Addition of either glutamate, glycine, or serine suppressed glycolate release, whereas glutamate and glycine at the same time stimulated ammonia release. The results indicate that inhibition of glutamine synthetase and thereby inhibition of photorespiratory nitrogen cycling restricts the sink capacity for glycolate in the photorespiratory carbon cycle. An external supply of glutamate, glycine, or serine seems to stimulate glyoxylate transamination and thus partly restores the sink capacity. Calculations of total glycolate formation rates in air from glycolate and ammonia release rates in the presence of methionine sulfoximine and glutamate revealed values of approximately 20 micromoles glycolate per milligram chlorophyll per hour on the average. Similar calculations led to an estimated rate of photorespiratory ammonia release in air, in the absence of methionine sulfoximine, of about 10 micromoles per milligram chlorophyll per hour on the average, a value comparable to the primary nitrogen assimilation rate of 8 micromoles per milligram chlorophyll per hour. PMID- 16662736 TI - Biochemical Changes that Occur during Senescence of Wheat Leaves : I. Basis for the Reduction of Photosynthesis. AB - Changes in activities of photosynthetic enzymes and photochemical processes were followed with aging of vegetative and flag leaves of wheat (Triticum aestivum L. cv Roy). Activities of stromal enzymes began to decline prior to photochemical activities. In general, total soluble protein and the activities of ribulose-1,5 bisphosphate carboxylase and NADP-triose-phosphate dehydrogenase declined in parallel and at an earlier age than leaf chlorophyll (Chl), leaf photosynthesis, and photosynthetic electron transport activity. Leaves appeared to lose whole chloroplasts as opposed to a general degradation of all chloroplasts based on three lines of evidence: (a) electron transport activity calculated on an area basis declined much earlier than the same data expressed on a Chl basis; (b) Chl content per chloroplast was similar for mature and senescent tissue; and (c) the absorbance at 550 nanometers (light scattering) per unit of Chl remained essentially constant until the end of senescence. Chloroplasts did, however, undergo some modifications before they were lost (e.g. loss of stromal enzyme activities), but the reduction in leaf photosynthesis was apparently caused by a loss of whole chloroplasts. PMID- 16662737 TI - Characterization of the alpha-Amylases Synthesized by Aleurone Layers of Himalaya Barley in Response to Gibberellic Acid. AB - The gibberellic acid (GA(3))-induced alpha-amylases from the aleurone layers of Himalaya barley (Hordeum vulgare L. cv Himalaya) have been purified by cycloheptaamylose-Sepharose affinity chromatography and fractionated by DEAE cellulose chromatography. Four fractions (alpha-amylases 1-4) were obtained which fell into two groups (A and B) on the basis of a number of characteristics. Major differences in serological characteristics and in proteolytic fingerprints were found between group A (alpha-amylases 1 and 2) and group B (alpha-amylases 3 and 4). Also, the lag time for appearance of group B enzyme activity was longer than for group A, and the appearance of group B required higher GA(3) levels than group A. The components of each group behaved similarly, although differences in proteolytic fingerprints were detected.These results together with those from other studies indicate that GA(3) differentially controls the expression of two alpha-amylase genes or groups of genes giving rise to two groups of alpha amylases with many different properties. PMID- 16662738 TI - Sources, Fluxes, and Sinks of Nitrogen during Early Reproductive Growth of Maize (Zea mays L.). AB - A study was designed to (a) identify sources and sinks of N in the maize (Zea mays L.) shoot, by estimating net N fluxes for each of seven parts of the shoot, (b) determine effects of N entering the plant upon fluxes of N absorbed before reproductive growth, and (c) determine the effects of the opaque-2 gene on N fluxes in the maize shoot during early reproductive growth. Plants of a maize hybrid (Pioneer 3369A) and its opaque-2 counterpart (Pioneer L3369) were grown in a greenhouse using nutrient solution/sand culture, with NO(3) (-) as the N source during the vegetative growth phase. Beginning at the time of pollination, the same nutrient regime was continued, except that some plants received no N, and others received 3.75 millimolar (15)N as NO(3) (-)-N.Stalk and leaves were found to be primary N sources for the grain, while shank, husk, and cob acted first as N sinks, then as N sources during reproductive growth. Net fluxes of N for each plant part were estimated by calculating the first derivatives of regression equations used to fit data for N contents of each plant part as functions of time. All parts of the shoot were sinks for exogenous N (absorbed after pollination). Thirty-six days after pollination, the grain contained 60% endogenous N (absorbed before pollination) when 3.75 millimolar NO(3) (-)-N was supplied after pollination. Rates of total N influx to the grain were identical whether or not N was supplied in the nutrient solution during reproductive growth. At 36 days after pollination, less N had accumulated in the grain of the opaque-2 genotype, but otherwise there were no differences in N contents or dry weights of the shoots due to the opaque-2 gene. Absence of N from the rooting medium significantly affected N fluxes throughout the shoot during reproductive growth, but there were no detectable effects of the opaque-2 gene on N fluxes in parts of the plant other than the grain. PMID- 16662739 TI - Enantiomeric Form of myo-Inositol-1-Phosphate Produced by myo-Inositol-1 Phosphate Synthase and myo-Inositol Kinase in Higher Plants. AB - The product of myo-inositol-1-phosphate synthase, EC 5.5.1.4, from mature pollen of Lilium longiflorum Thunb., cv Ace (Easter lily) and that of myo-inositol kinase, EC 2.7.1.64, from wheat germ has been identified as 1l-myo-inositol-1 phosphate by gas chromatography of its trimethylsilyl-methyl phosphate derivative on a glass capillary column bearing a chiral phase. PMID- 16662740 TI - Biosynthesis of Indol-3-yl-acetyl-myo-inositol Arabinoside in Kernels of Zea mays L. AB - Extracts of immature kernels of Zea mays L. catalyzed the synthesis of indol-3-yl acetyl-myo-inositol arabinoside from indol-3-yl-acetyl-myo-inositol and UDP-[U (14)C]xylose. The product contained radioactivity which upon hydrolysis with trifluoroacetic acid cochromatographed with arabinose and not xylose. The amount of product from the reaction was proportional to the amount of indol-3-yl-acetyl myo-inositol added, and the product was positive to Ehmann's reagent for indoles. In addition, the product and authentic indol-3-yl-acetyl-myo-inositol arabinoside had the same R(F) or retention time in three chromatographic systems.By analogy to the wheat germ system, it is proposed that a UDP-d-xylose 4-epimerase is present in immature kernels of maize and that this enzyme catalyzes the conversion of UDP-d-xylose to UDP-l-arabinose, the probable sugar donor to indol 3-yl-acetyl-myo-inositol.The present data demonstrates the presence of a previously undescribed enzyme, UDP-arabinose:indol-3-yl-acetyl-myo-inositol arabinosyl transferase in maize kernels. This work together with our prior studies provides a pathway for the biosynthesis of all the low molecular weight esters of indol-3-yl-acetic acid in maize kernels. PMID- 16662741 TI - Spectral evidence for a component involved in hydrogen metabolism of soybean nodule bacteroids. AB - A component with a difference spectrum similar to that of b-type cytochromes which becomes reduced upon the addition of H(2) has been demonstrated in soybean nodule bacteroids. This electron carrier, referred to as component 559-H(2), is present in hydrogenase-positive strains of Rhizobium japonicum but has not been detected in mutants that lack hydrogenase activity or in hydrogenase-negative wild-type strains. A positive correlation between concentrations of component 559 H(2) and hydrogenase activities has been established. These results provide further evidence that component 559-H(2) is involved in H(2) metabolism in R. japonicum. PMID- 16662742 TI - Non-Mendelian Inheritance of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-Resistant Thylakoid Membrane Properties in Chlamydomonas. AB - A uniparentally inherited 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) resistant mutant of Chlamydomonas reinhardii, Dr2, which has a resistance mechanism of the type defined as ;primary,' has been isolated. In vitro Hill reactions catalyzed by isolated thylakoid membranes reveal a reduced apparent affinity of the thylakoids for DCMU. These changes in membrane properties quantitatively account for the resistance of mutant Dr2 to herbicide inhibition of growth. The properties of this mutant show that all of the Hill reaction inhibiting DCMU binding sites are under identical genetic control. Mutant Dr2 is a useful new uniparental genetic marker, since it has a novel phenotype and it may be possible to identify its altered gene product. The low cross-resistance of Dr2 to atrazine suggests that there may be considerable flexibility in exploiting induced herbicide resistance of crop plants for improving herbicide specificity.Four mendelian mutants in at least three loci all have resistance mechanisms in the class we define as ;secondary.' They are as sensitive as wild type to in vitro inhibition of the Hill reaction, and must acquire resistance in vivo by preventing the active form of the herbicide from reaching the sensitive site. PMID- 16662743 TI - Characterization of mitochondrial DNA in citrus. AB - Characterization of mitochondrial DNA from leaves of four species of the genus Citrus by electron microscopy shows the presence of circular molecules with a great size dispersion and absence of discrete size classes. Restriction endonuclease patterns confirm the heterogeneity of the molecules. PMID- 16662744 TI - Rhythms in glutamine synthetase activity, energy charge, and glutamine in sunflower roots. AB - Roots of sunflower plants (Helianthus annuus L. var. Mammoth Russian) subjected to L12:D12, L18:D6, and L12:D12 followed by continuous light all display rhythms of about 12 hours for glutamine synthetase (GS) activity (transferase reaction) with one peak in the ;light phase' and one in the ;dark phase.' Root energy charge (EC = ATP+(1/2)ADP/ATP+ADP+AMP) is directly correlated with GS, but the GS rhythm is better explained as the result of a rhythmic adenine nucleotide ratio (ATP/ADP+AMP) that regulates enzyme activity through allosteric modification. When L12:D12 plants are subjected to free-running conditions in continuous darkness, only diurnal rhythms for GS and EC, with peaks in the dark phase, remain. The 12-hour root rhythms for GS and EC appear to be composed of two alternating rhythms, one a diurnal, light-dependent, incompletely circadian light phase rhythm and the other a light-independent, circadian dark phase rhythm.Only glutamine, of the root amino acids, displays cyclical changes in concentration, maintaining under all conditions a 12-hour rhythm that is consistently synchronized with, but nearly always inversely correlated with, GS and EC rhythms. PMID- 16662745 TI - Fatty Acid Composition and Nitrate Uptake of Soybean Roots during Acclimation to Low Temperature. AB - Fatty acid composition of old and new roots was determined for soybeans (Glycine max [L.] Merr. cv Ransom) at root-zone temperatures of 14, 18, and 22 degrees C during a 26-day period. New roots had a greater concentration of polyunsaturated fatty acids than old roots. The ratio of polyunsaturated to saturated fatty acid concentration in new roots exposed to 14 and 18 degrees C peaked at 16 days and declined, while the corresponding ratio in old roots increased throughout the treatment period. Apparently the response of fatty acid composition in old and new roots to low temperature was mediated by tissue aging or differentiation. These findings were contrary to the concept that modifications in fatty acid composition remain constant at lower temperatures.The function of root tissues exposed to lower temperature was evaluated with respect to the ability of the root systems to absorb NO(3) (-). Over the relatively long periods of exposure, the ability of whole root systems to absorb NO(3) (-) was similar at cool and warm temperatures. The effect of cool temperature on functioning of roots appeared to involve reductions in the rates of initiation and differentiation of young root tissues rather than changes in membrane permeability related to alteration of fatty acid composition. PMID- 16662746 TI - Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots. AB - Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg(2+)-ATPase activity of the sealed vesicles was stimulated by Cl(-) and NH(4) (+) and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO(3) (-), was insensitive to K(+), and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg(2+), was stimulated by Cl(-), inhibited by NO(3) (-), was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg(2+) >/= Mn(2+) > Co(2+)) and were inhibited by high concentrations of Ca(2+). The similarity of the ionophore stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F(1)F(0)-ATPase and from those of the K(+)-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. PMID- 16662747 TI - Effect of Fusicoccin on Dark CO(2) Fixation by Vicia faba Guard Cell Protoplasts. AB - When Vicia faba guard cell protoplasts were treated with fusicoccin, dark (14)CO(2) fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark (14)CO(2) fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of (14)C among products and in the C-4 position of malate (initially more than 90% of [(14)C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of (14)CO(2) fixed and malate content.It was concluded that (a) fusicoccin causes an increase in the rate of dark (14)CO(2) fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark (14)CO(2) fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase. PMID- 16662748 TI - Ploidy effects in isogenic populations of alfalfa : I. Ribulose-1,5-bisphosphate carboxylase, soluble protein, chlorophyll, and DNA in leaves. AB - The influence of polyploidization on ribulose-1,5-bisphosphate carboxylase (RuBPCase), buffer-soluble protein (BSP), chlorophyll (Chl), and DNA was examined in fully expanded leaves of isogenic diploid-tetraploid (DDC 2X-4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). The concentration of RuBPCase in leaf extracts was determined by rocket immunoelectrophoresis. Activities of RuBPCase, expressed per milligram protein or per milligram Chl, and leaf tissue concentrations of RuBPCase, BSP, Chl, and DNA were similar between ploidy levels of the DDC 2X-4X set. Tetraploids and octoploids were similar in RuBPCase activities, expressed per milligram protein or per milligram Chl, and in leaf tissue concentrations of RuBPCase and DNA. Octoploids were significantly lower than tetraploids in concentrations of Chl and BSP.When compared on a per leaf basis, tetraploids were 80% higher in BSP and essentially double comparable diploids in fresh weight, RuBPCase, Chl, and DNA. The observation that leaves of the DDC tetraploid population contain twice as much DNA as comparable diploids suggests that leaves of both ploidy levels contain similar numbers of cells. Leaves of the octoploid population were 33% to 80% higher than corresponding tetraploids in BSP, fresh weight, RuBPCase, Chl, and DNA. Ratios of RuBPCase to DNA and Chl to DNA were similar across ploidy levels of both isogenic sets suggesting that cellular content of Chl and RuBPCase increases proportionately with the amount of DNA per cell. PMID- 16662749 TI - Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts. AB - Photosynthetically-active protoplasts isolated from isogenic sets of diploid tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO(2)-dependent O(2) evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy. PMID- 16662750 TI - Ploidy Effects in Isogenic Populations of Alfalfa : III. Chloroplast Thylakoid Bound Coupling Factor 1 in Protoplasts and Leaves. AB - The influence of polyploidization on chloroplast coupling factor 1 (CF1) was examined in leaves and leaf protoplasts from isogenic diploid-tetraploid (DDC 2X 4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). Alfalfa CF1 was purified to homogeneity and was found to contain five subunits with molecular weights of 57,900, 54,300, 38,700, 23,100, and 15,200. Rocket immunoelectrophoresis with anti-spinach CF1 gamma immunoglobulins was used to quantify CF1 from protoplasts and leaves. In the DDC 2X-4X set, fresh weight per cell and cellular content of CF1, chlorophyll (Chl), and DNA doubled with ploidy. Ratios of CF1 to Chl of leaf protoplasts and leaves were similar between diploids and tetraploids. In the IC 4X-8X set, octoploid protoplasts were 90% higher in Chl than were comparable tetraploids, whereas octoploids were 50 to 60% higher than tetraploids in fresh weight per cell and cellular content of CF1 and DNA. Concentrations of CF1 and Chl in leaves and ratios of CF1 to DNA in protoplasts were similar across ploidy levels of both isogenic sets. Therefore, cellular content of CF1 increases proportionately with the amount of DNA per cell or gene dosage. PMID- 16662751 TI - Light-Stimulated Burst of Carbon Dioxide Uptake following Nocturnal Acidification in the Crassulacean Acid Metabolism Plant Kalanchoe diagremontiana. AB - CO(2) exchange characteristics were studied during the light-stimulated burst of CO(2) uptake (MB) immediately following a period of nocturnal CO(2) fixation in the Crassulacean acid metabolism plant Kalanchoe daigremontiana. During the early parts of the MB, stimulation of net CO(2) uptake by low ambient O(2) concentration (1.5%) was small, and leaves showed the capacity for net CO(2) uptake at low ambient CO(2) partial pressure (30 microbars) and when the MB was interrupted by darkness. During the later phase of the MB, stimulation of net CO(2) uptake by 1.5% O(2) was increased, and net CO(2) loss was recorded both at 30 microbars CO(2) and during dark interruptions. These results suggest that CO(2) fixation during the MB occurs simultaneously via phosphoenolpyruvate carboxylase (predominant during the early phase of the MB) and via ribulose bisphosphate carboxylase (predominant during the later phase of the burst). The magnitude and duration of the MB was increased by a reduction in the length of the dark period and by low (15 degrees C) compared to high (30 degrees C) leaf temperatures. PMID- 16662752 TI - Potassium transport in corn roots : I. Resolution of kinetics into a saturable and linear component. AB - Influx isotherms were obtained for (86)Rb(+) uptake into 2-cm corn (Zea mays [A632 x (C3640 x Oh43)] root segments for both low- (0.2 millimolar CaSO(4)) and high-salt (0.2 millimolar CaSO(4) + 5 millimolar KCl) grown roots. Unlike the discontinuous curves usually presented for K(+) influx, our isotherms were smooth, nonsaturating curves that approached linearity at K(+) (Rb(+)) concentrations above 1 millimolar. The kinetics for K(+) transport could be resolved into saturable and linear components. The saturable components yielded K(m) values of 16 and 86 micromolar for low- and high-salt roots, respectively, while V(max) values were 5.62 and 1.85 moles per gram fresh weight per hour. Results of experiments with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS) indicated that the saturable and linear components were independent mechanisms of K(+) transport.Short-term NEM exposures (30 seconds to 5 minutes) selectively inhibited the saturable system, but had little effect on the linear component. Increasing NEM exposures resulted in further inhibition and subsequent abolition of the saturable component; the linear component exhibited limited NEM sensitivity. PCMBS elicited the same general inhibitory trends, although it was less effective as a saturable component inhibitor.The effects of NEM and PCMBS on K(+) efflux were also studied. Short NEM exposures had no effect on cytoplasmic efflux, while inhibiting vacuolar efflux significantly. From these data, it is unclear at which site(s) NEM is acting. A more complex response was obtained with PCMBS, where a monophasic efflux curve was observed. Analysis indicated that the vacuolar efflux was stimulated, while the cytoplasmic component was abolished.The nature of the linear component is discussed, and it is proposed that the mechanism may be more complex than simple facilitated diffusion. PMID- 16662753 TI - Subcellular Localization of a UDP-Glucose:Aldehyde Cyanohydrin beta-Glucosyl Transferase in Epidermal Plastids of Sorghum Leaf Blades. AB - Epidermal and mesophyll protoplasts, prepared from leaf blades of 6-day-old light grown Sorghum bicolor seedlings were separated by differential sedimentation and assayed for a number of enzymes. The epidermal protoplasts contained higher levels of NADPH-cytochrome c reductase (EC 1.6.2.4), triose phosphate isomerase (EC 5.3.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), and a UDP glucose:cyanohydrin beta-glucosyl transferase (EC 2.4.1.85), but lower levels of NADP(+) triosephosphate dehydrogenase (EC 1.2.1.13) than did mesophyll protoplasts. When protoplast preparations were lysed and applied to linear sucrose density gradients, triosephosphate isomerase was found to be present in epidermal plastids. A significant fraction (41%) of the glucosyl transferase activity was also associated with the epidermal plastids. PMID- 16662754 TI - Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles. AB - Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H(+)-transport as measured by [(14)C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP.Mg, is temperature sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m chlorophenylhydrazone. Of the divalent cations tested, Mn(2+) is almost as effective as Mg(2+), while Ca(2+) is ineffective. Excess divalent cations, particularly Ca(2+), reduces the pH gradient. H(+) transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with (36)Cl(-) indicate that ATP-driven H(+) transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, inhibit methylamine accumulation and (36)Cl(-) uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N'-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin. PMID- 16662755 TI - Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation. AB - Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [(14)C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [(14)C]SCN(-) uptake and (36)Cl(-) uptake activities coincided with [(14)C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker. PMID- 16662756 TI - Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts. AB - Photoassimilation of (14)CO(2) by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3 phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO(2) assimilation occurred.The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator. PMID- 16662757 TI - Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules. AB - Host plant glutamine synthetase (GS) has been purified 100-fold from N(2)-fixing alfalfa (Medicago sativa L.) nodules by a new procedure involving preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a final step. An SDS-polypeptide fraction corresponding to plant GS was identified and consisted of two major polypeptides of 40,000 to 45,000 molecular weight. Antibodies to the SDS-polypeptide fraction were raised in mice by intraperitoneal injection, and antisera were collected as ascitic fluid. Crude extracts of soluble protein from the plant fraction of nodules were resolved by SDS-PAGE and then subjected to electrophoresis in the second dimension into antibody containing agarose gel. A single immunochemically active protein species was observed using this crossed immunoelectrophoresis method, even though both major GS SDS-polypeptides were apparently resolved in the first (SDS-PAGE) dimension. Plant GS protein in crude nodule extracts was quantitated immunochemically by comparison with immunoprecipitin arcs of similarly treated amounts of pure antigen. Using this technique, it was determined that plant GS was present at 150 micrograms per gram fresh weight or 1.2% of total plant soluble protein in N(2) fixing alfalfa nodules.Results suggest that alfalfa nodule plant GS consists of two major subunit polypeptides, but only a single immunochemically active native protein was observed. The crossed immunoelectrophoresis procedure described here should be generally applicable for immunochemical detection of lower abundance components of crude plant extracts. PMID- 16662758 TI - Occurrence of nitrate reductase inhibitor in rice plants. AB - Nitrate reductase inhibitor is usually found in the roots of rice plants (Oryza sativa L. cv MR7), but it was also produced in the shoots of aging plants. The inhibitor was inducible in the shoot of rice seedlings by dark, minus-nitrate or plus-ammonium treatments. There appears to be a general involvement of the inhibitor in the control of nitrate assimilation in the plant. PMID- 16662759 TI - Membrane Lipid Physical Properties in Annuals Grown under Contrasting Thermal Regimes. AB - Trans-parinaric acid was used to determine the order/disorder transition temperatures of phospholipids extracted from leaves of warm- and cool-season annuals grown under contrasting thermal regimes. All species were capable of adjusting this property, although there was considerable variation in the extent of the adjustment. PMID- 16662760 TI - Synthesis of Oat Globulin Precursors : Analogy to Legume 11S Storage Protein Synthesisa. AB - The oat (Avena sativa L.) seed globulin was found to be synthesized in vitro as 60,000 to 64,000 dalton precursors. In vivo protein labeling yielded polypeptides of 58,000 to 62,000 daltons, suggesting cleavage of signal sequences from the precursors. Further cleavage is apparently required to separate the alpha and beta polypeptide sequences which are known to form disulfide-linked 53,000 to 58,000 dalton species in the (alphabeta)(6) holoprotein. The data are discussed with respect to analogous synthesis and processing of some legume 11S storage proteins. PMID- 16662761 TI - Sterling hendricks: a molecular plant physiologist. PMID- 16662762 TI - Localization of phytochrome in oats by electron microscopy. AB - Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction. PMID- 16662763 TI - Intracellular Localization of Nitrate Reductase in Neurospora crassa. AB - Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall plasmalemma region and in the tonoplast membranes. PMID- 16662764 TI - Energetics of Amino Acid Uptake by Vicia faba Leaf Tissues. AB - The uptake of [U-(14)C]threonine and of (alpha-(14)C]aminoisobutyrate (alpha-AIB) by Vicia faba leaf discs is strongly pH dependent (optimum: pH 4.0) and exhibits biphasic saturation kinetics. Kinetics of alpha-AIB uptake at different pH values indicate that acidic pH values decrease the K(m) of the carriers while the maximal velocity remains nearly unaffected. Similar results were obtained for both system 1 (from 0.5 to 5 millimolar) and system 2 (from 20 to 100 millimolar).After addition of amino acids to a medium containing leaf fragments, alkalinizations depending both on the amino acid added and on its concentration have been recorded.The effects of compounds which increase (fusicoccin) or decrease (uncouplers, ATPase inhibitors, high KCl concentrations) the protonmotive force were studied both on the acidification of the medium and on amino acid uptake by the tissues. There is a close relationship between the time required for the effect of these compounds on the acidification and that needed for inhibition of uptake.Studies with thiol inhibitors show that 0.1 millimolar N ethylmaleimide preferentially inhibits uptake by the mesophyll whereas 0.1 millimolar parachloromercuribenzenesulfonate affects rather uptake by the veins.New evidence was found which added to the electrophysiological data already supporting the occurrence of proton amino acid symport in leaf tissues, particularly in the veins. PMID- 16662765 TI - Measurement of CO(2) and H(2)O Vapor Exchange in Spinach Leaf Discs : Effects of Orthophosphate. AB - A leaf chamber has been designed which allows the measurement of both CO(2) and water vapor exchange in Spinacia oleracea leaf discs. The center of the disc lies within a cylindrical gas chamber and its margins are enclosed within a cavity through which water or various metabolites can be pumped. In saturating light and normal atmospheres, the leaf discs have a relatively low resistance to H(2)O vapor transfer (r(w) = 1.87 seconds per centimeter) and can support high rates of photosynthesis for several hours. The abaxial surface of a disc had a higher resistance to water vapor transfer (r(w) = 3.22 seconds per centimeter) than the adaxial (r(w) = 2.45 seconds per centimeter) despite having a higher stomatal frequency (abaxial, 105/square millimeter; adaxial, 58/square millimeter). In 2% O(2), the discs required an internal concentration of CO(2) of 115 microliters per liter to support one-half of the maximal velocity of apparent photosynthesis (average value, 66 milligrams CO(2) per square decimeter per hour). In 20% O(2), the comparable values are 156 microliters per liter and 56 milligrams CO(2) per square decimeter per hour. In air, apparent photosynthesis saturated at intensities (750 microeinsteins per square meter per second) well below that of daylight but, when the internal CO(2) was raised to 700 to 900 microliters per liter, photosynthesis was not saturated even at daylight intensities (2025 microeinsteins per square meter per second). The distribution of Prussian blue crystals, formed after ferrocyanide feeding, showed that water entered the disc via the vasculature. When 25-minute pulses of orthophosphate were provided in the feeding solution, there were concentration-dependent increases in both r(w) and r(m) leading to inhibition of photosynthesis. The orthophosphate-dependent inhibitions were reversible. PMID- 16662766 TI - Effects of mannose on photosynthetic gas exchange in spinach leaf discs. AB - When mannose is provided in the transpiration stream to spinach (Spinacia oleracea) leaf discs, a series of specific and nonspecific changes occur in CO(2) and H(2)O vapor exchange as a function of feeding time. The initial increases in apparent photosynthesis and transpiration are nonspecific effects due to osmotic changes leading to passive stomatal opening. The mannose-specific effects are: (a) time-dependent changes in the CO(2) concentration required for saturation; (b) complex kinetics of the inhibition of CO(2) assimilation dependent on CO(2) and O(2) concentrations and the duration of feeding (high CO(2) and low O(2) lead to rapid inhibitions of photosynthesis); (c) elimination of the capacity of 2% O(2) to stimulate photosynthesis; and (d) oscillations in the CO(2) exchange rate following transitions from 20% to 2% O(2). The mannose-specific effects are reversible by orthophosphate. The mannose-dependent changes in gas exchange are attributed to altered [ATP]/[ADP] ratios. PMID- 16662767 TI - Effect of inhibitors on ammonia-, 2-oxoglutarate-, and oxaloacetate-dependent o(2) evolution in illuminated chloroplasts. AB - The evolution of O(2) in spinach chloroplasts in the presence of oxaloacetate (OAA) was inhibited by a wide range of dicarboxylates. In contrast, (ammonia, 2 oxoglutarate)-dependent O(2) evolution was stimulated by malate, succinate, fumarate, glutarate, maleiate, and l-tartrate although OAA has little effect. This increase in O(2) evolution was accompanied by a similar increase in (14)C incorporation from [5-(14)C]oxoglutarate into amino acids which was sensitive to azaserine inhibition. Glutamate and aspartate inhibited (ammonia, 2-oxoglutarate) dependent O(2) evolution, but this inhibition was relieved by the addition of succinate, malate, or fumarate. OAA-dependent O(2) evolution also was inhibited by glutamate and aspartate, but succinate, malate, or fumarate had little effect on this inhibition. Phthalonate and n-butyl malonate inhibited (ammonia, 2 oxoglutarate)-dependent O(2) evolution competitively with respect to 2 oxoglutarate and uncompetitively with respect to malate. Both these inhibitors inhibited OAA-dependent O(2) evolution competitively. This evidence suggests that different mechanisms might be involved in the transport of OAA, 2-oxoglutarate, and malate into the chloroplasts. PMID- 16662768 TI - Cellular expansion at low temperature as a cause of membrane lesions. AB - Rates of solute leakage from excised discs of cucumber (Cucumis sativus L. cv Straight Eight) cotyledons were altered by temperature during plasmolysis in the manner of a simple diffusion phenomenon; the log of the leakage rate increased in proportion to the temperature. During deplasmolysis, however, leakage rates responded to temperature with a very different pattern: chilling conditions (below about 20 degrees C) caused large increases in leakage rates, indicating disruption of membrane integrity in the tissues. The time course of restoration of normal leakage rates after deplasmolysis/chilling damage indicated a rapid repair of the lesions. A similar sensitivity to low temperatures was found during rehydration after leaf desiccation, with low temperatures again causing high leakage rates. It is suggested that low temperatures interfere with membrane expansion, possibly by lowering elasticity and hindering the incorporation of lipid material into the expanding membrane. The expansion of tissues at low temperatures may cause lesions in cellular membranes, contributing to chilling injury. PMID- 16662769 TI - Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea. AB - The carbon and nitrogen economies of a single cultivar of cowpea (Vigna unguiculata (L.) Walp.cv Caloona) nodulated with either a high H(2)-evolving strain (176A27) or a low H(2)-evolving strain (CB756) of Rhizobium were compared. The two symbioses did not differ in total dry matter production, seed yield, nitrogen fixed, the spectrum of nitrogenous solutes produced by nodules for export, or the partitioning of net photosynthate within the plant throughout the growth cycle. Detailed examination of the carbon and nitrogen economy of the nodules, however, showed a significant difference between the symbioses. Nodules formed with CB756 lost less CO(2) in respiration compared to the higher H(2) evolving symbioses and this could have been largely responsible for a 36% better economy of carbon use in CB756 nodules during the period of maximum H(2) evolution (48-76 days) and over the whole growth period (20-90 days), a 16% economy. In terms of overall net photosynthate generated by the plant, these economies were equivalent to 5% and 2% of the carbon utilized in the two periods, respectively. From the differences in H(2) evolution and CO(2) production by nodules of the two symbioses, the cost of H(2) evolution was found to be 3.83+/ 0.6 millimoles CO(2)/millimoles H(2) for plants grown in sand culture and 1.69 +/ 0.48 millimoles CO(2)/millimoles H(2) for those in water culture. In both symbioses, the ratio of H(2) evolution to N(2) fixed varied markedly during ontogeny, indicating a significant variation in the relative efficiency and thus metabolic cost of N(2) fixation at different stages during development. PMID- 16662770 TI - Relation between the Light-Harvesting Chlorophyll a-Protein Complex LHCPa and Photosystem I in the Alga Chlamydobotrys stellata. AB - The light-harvesting chlorophyll protein system of the alga Chlamydobotrys stellata consists of an as yet uncharacterized algal chlorophyll a-protein, called LHCPa, and a common photosystem II-related chlorophyll a/b-protein, called LHCPb (Brandt, Kaiser-Jarry, Wiessner 1982 Biochim Biophys Acta 679: 404-409). For further characterization, this LHCPa was isolated from the organism by polyacrylamide isoelectrofocusing and reelectrophoresis. It contains only chlorophyll a and has only one apoprotein (32,000 daltons). When separated from autotrophically grown cells, its absorption peak is at 674 nm and its isoelectric point at 5.3. Photoheterotrophic cultivation of the algae shifts the absorption maximum of LHCPa to 679 nm and its isoelectric point to 4.8. This LHCPa is a component of photosystem I particles. In relation to the total chlorophyll a content, the amount of LHCPa is low in autotrophic algae, but increases under photoheterotrophic growth conditions, where the organisms do not have the ability to assimilate CO(2) photosynthetically. PMID- 16662771 TI - beta-Galactosidases in Ripening Tomatoes. AB - Tomatoes (Lycopersicon esculentum L.) contained a high level of beta galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of beta-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, beta-galactosidases I and III levels decreased but the beta-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K(m) value with p-nitrophenyl-beta-galactoside, and stability with respect to pH and temperature. beta-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of beta-1, 4-linked galactose. The ability of beta galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening. PMID- 16662772 TI - Expression of 5-Methyltryptophan Resistance in Plants Regenerated from Resistant Cell Lines of Datura innoxia. AB - Seventy-nine 5-methyltryptophan-resistant cell lines have been selected from haploid Datura innoxia Mill. cell cultures by plating suspensions in agar medium containing a growth inhibitory concentration of 5-methyltryptophan. Mutagen treatment increased the frequency of resistance. The eleven variants tested posses an altered anthranilate synthase less sensitive to feedback inhibition by tryptophan. All five of the variants which were analyzed for free amino acids contained elevated levels of free tryptophan (8 to 30 times the wild type level). None of the selected cell lines were auxin-autotrophic. Resistance to 5 methyltryptophan, altered anthranilate synthase, and high free tryptophan (4 to 44 times) were also expressed in leaves of plants regenerated from the variant lines and in cultures reinitiated from the resistant plants. These results show that the amino acid overproduction phenotype can be selected at the cellular level of organization and be expressed identically in whole plants regenerated from the selected cells. PMID- 16662773 TI - Relation of increased potassium nutrition to photosynthesis and translocation of carbon. AB - Effects of supplying K(+) at 2 or 10 millimolarity concentration on net carbon exchange and translocation of products of photosynthesis were studied in plants of Beta vulgaris L. (var. Klein E). Transport of K(+) into and out of leaves was studied with (42)K over a 3-day period. Increasing the K(+) supplied to the roots from 2 millimolarity, a level just sufficient to overcome obvious deficiency symptoms, to 10 millimolarity resulted in a gradual accumulation of K(+) per unit area and an increased export of K(+) to sink regions. No significant increase in net carbon exchange was observed in leaves that had accumulated a high level of K(+) per unit area. Initiation rate, total area, and total fresh weight of leaves of plants with K(+) supplied at 10 millimolarity was similar to that for leaves from plants at a 2 millimolarity level. Shoot/root ratio and dry weight accumulation, which are indicative of translocation and partitioning over the long term, were independent of K(+) supply in the 2 to 10 millimolarity range. Accumulation of K(+) by exporting leaves and its subsequent recirculation to sinks increased when K(+) supply was increased in this range but did not appear to affect carbon nutrition even after a long period. PMID- 16662774 TI - Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization. AB - Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate. PMID- 16662775 TI - Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants. AB - The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds. PMID- 16662776 TI - Phosphofructokinase activities in photosynthetic organisms : the occurrence of pyrophosphate-dependent 6-phosphofructokinase in plants and algae. AB - A pyrophosphate-dependent phosphofructokinase (PPi-PFK) activity is detectable in extracts of a wide variety of primitive and advanced plants, the Charalean algae, and in the photosynthetic bacterium, Rhodospirillum rubrum. Angiosperms with extractable PPi-PFK activities 4- to 70-fold higher than the respective ATP-PFK activities tend to be succulent and to exhibit CAM. Even though PPi-PFK activity is not detected in crude extracts of some well known CAM plants, e.g. plants in the Crassulaceae, gel filtration of the extract and/or inclusion of the PPi-PFK activator, fructose 2,6-bisphosphate, in the assay reveals that a PPi-PFK activity is present in these species. Fructose 2,6-bisphosphate likewise activates PPi-PFK activities in extracts of C(3) and C(4) plants. C(3) and C(4) plant PPi-PFK activities are roughly equivalent to ATP-PFK activities in the same species. PPi-PFK activity is also detected in some bryophytes, lower vascular plants, ferns, and gymnosperms. The Charophytes, advanced algae presumed to be similar to species ancestral to vascular plants, exhibit at least 4-fold higher PPi-PFK than ATP-PFK activities. R. rubrum also exhibits a much higher PPi-PFK activity than ATP-PFK activity. These data indicate that PPi-PFK may serve as an alternate enzyme to ATP-PFK in glycolysis in a wide range of photosynthetic organisms. PMID- 16662777 TI - Analysis of the Heterogeneity of the 40,000 Molecular Weight Tuber Glycoprotein of Potatoes by Immunological Methods and by NH(2)-Terminal Sequence Analysis. AB - Among the major soluble tuber proteins of potato (Solanum tuberosum L.) is a group of glycoproteins having apparent molecular weights of approximately 40,000. This group of proteins as purified by ion-exchange and affinity chromatography has been given the trivial name ;patatin.' Patatin exists in a number of charge forms which differ between potato cultivars and in some cases can also be resolved into a number of bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, by immunodiffusion and immunoelectrophoresis, it was found that the isoforms of patatin are immunologically identical both within a cultivar as well as between cultivars. A high degree of homology between the isoforms of patatin is also indicated by NH(2)-terminal amino acid sequence analysis. PMID- 16662778 TI - Induction and accumulation of major tuber proteins of potato in stems and petioles. AB - A family of immunologically identical glycoproteins with apparent molecular weights of approximately 40,000 are among the major tuber proteins of potato (Solanum tuberosum L.). These proteins, as purified by ion-exchange and affinity chromatography, have been given the trivial name ;patatin.' To determine if patatin can be used as a biochemical marker to study the process of tuberization, its amount was measured in a variety of tissues by rocket immunoelectrophoresis and by enzyme-linked immunosorbent assay (ELISA).Patatin comprises 40 to 45% of the soluble protein in tubers regardless of whether they are formed on underground stolons or from axillary buds of stem cuttings. Under normal conditions, patatin is present in only trace amounts, if at all, in leaves, stems, or roots of plants which are either actively forming tubers or which have been grown under long days to prevent tuberization. However, if tubers and axillary buds are removed, patatin can accumulate in stems and petioles. This accumulation occurred without any obvious tuber-like swelling and would occur even under long days. In all tissues containing large amounts of patatin, the other tuber proteins were also found as well as large amounts of starch. PMID- 16662779 TI - Volume regulation in poterioochromonas: involvement of calmodulin in the ca stimulated activation of isofloridoside-phosphate synthase. AB - In Poterioochromonas malhamensis Peterfi (syn. Ochromonas malhamensis Pringsheim) osmotically induced shrinkage is reversed by an accumulation of isofloridoside. Addition of Ca(2+) ions to homogenates from standard volume cells initiates an enzyme system for the activation of isofloridoside-phosphate synthase. This process is stimulated in the presence of Ca(2+) by calmodulin, isolated from the same alga or from bovine brain, and requires the presence of membranes. The stimulation observed when Ca(2+) is added without exogenous calmodulin is inhibited by the calmodulin-binding substance R 24571. These results show that the effect of Ca(2+) is mediated by calmodulin. The Ca(2+)/calmodulin-dependent activation is enhanced when fluoride or molybdate ions are present in the homogenization buffer. This might indicate the involvement of a phosphorylated compound in the activation mechanism. PMID- 16662780 TI - In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds. AB - alpha-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. alpha-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of alpha-amylase in V. mungo cotyledons.The rate of incorporation of the label from [(3)H]leucine into alpha-amylase and the ratios of dpm in alpha-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of alpha-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons alpha-amylase was synthesized and accumulated, because of low degrading activity during incubation. PMID- 16662781 TI - Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C(3) and C(4) Leaves : The Relationship between NH(3) Accumulation and Photorespiratory Activity. AB - Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH(3) in seedling leaves of C(3) (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C(4) (corn [Zea mays W6A x W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH(3) accumulation is higher in C(3) (about 17.8 micromoles per gram fresh weight per hour) than in C(4) (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH(3), the rate of NH(3) accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH(3) was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO(3) (-) during either the growth or experimental period. When O(2) levels in the air were reduced to 2%, MSX resulted in some accumulation of NH(3) (6.0 micromoles per gram fresh weight per hour). At these levels of NH(3), there was no significant inhibition of rates of CO(2) fixation. There was also a minor, but significant, accumulation of NH(3) in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH(3) in both C(3) and C(4) leaves. These results support the hypothesis that GS is important in the assimilation of NH(3) in leaves and that the glycine-serine conversion is a major source of that NH(3). PMID- 16662782 TI - Inhibition by calcium of senescence of detached cucumber cotyledons: effect on ethylene and hydroperoxide production. AB - The effect of Ca on senescence was followed in detached cucumber (Cucumis sativus L.) cotyledons floating on various solutions in the dark. Compared with those in water, cotyledons in 10(-4) molar CaCl(2) exhibited reduced chlorophyll loss and H(2)O(2) production, reduced and delayed ethylene production, and did not undergo a burst in CO(2) production. In contrast, Mg had little effect on cotyledon senescence, whereas K stimulated chlorophyll loss but did not increase H(2)O(2) accumulation of ethylene and CO(2) production. This reduction in the rate of senescence by Ca could also be achieved by increasing the endogenous levels of Ca in the cotyledons before excision, although the reduction was less than that with Ca in the external solution. The addition of H(2)O(2) to the solutions on which cotyledons were floated stimulated chlorophyll breakdown, but effects on ethylene and CO(2) were not consistent. PMID- 16662783 TI - Reduced Glutathione as an Effector of Phosphoenolpyruvate Carboxylase of the Crassulacean Acid Metabolism Plant Sedum praealtum D.C. AB - Reduced glutathione, but not mercaptoethanol or dithiothreitol, inhibits phosphoenolpyruvate carboxylase (PEPC) in desalted leaf extracts from Sedum praealtum D.C. The inhibition is more evident at low pH values (< 7.2) and becomes increasingly smaller at higher pH. In the presence of the inhibitor, the hyperbolic rate curve of night PEPC is transformed to sigmoid and the S(0.5) is increased. When the enzyme is extracted during the day, the rate curve is sigmoid and it is not changed by the inhibitor, though the S(0.5) is further increased. Oxidized glutathione is completely inactive. Levels of reduced glutathione in leaf tissue are distinctly higher in the light. A role of photosynthetically reduced glutathione in the regulation of PEPC in Crassulacean acid metabolism species appears probable. PMID- 16662784 TI - Purification and Characterization of a New Indole Oxygenase from the Leaves of Tecoma stans L. AB - A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to homogenity. The purified enzyme system catalyzes the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and 30 degrees C. Two moles of oxygen are consumed and one mole of anthranilic acid is formed for every mole of indole oxidized. Dialysis resulted in complete loss of the activity. The inactive enzyme could be reactivated by the addition of concentrated dialysate. The enzyme is not inhibited by copper-specific chelators, non-heme iron chelators or atebrin. It is not a cuproflavoprotein, unlike the other indole oxygenases and oxidases. PMID- 16662785 TI - Partial Sequence Analysis of the 5S to 18S rRNA Gene Region of the Maize Mitochondrial Genome. AB - The nucleotide sequence has been determined for a 664-base pair region of maize (Zea mays L.) mtDNA which contains the 3' end of the 18S rRNA gene, the 5S rRNA gene, and an intergenic region of 108 base pairs. Specific regions of the 18S rRNA gene show striking homology with the corresponding gene in Escherichia coli. PMID- 16662786 TI - Cytochromes of Rhizobium japonicum 61A76 Bacteroids from Soybean Nodules. AB - Bacteroids of Rhizobium japonicum 61A76 were isolated from nodules of field-grown soybean plants by sucrose density gradient fractionation. The major cytochromes, aa(3), b, c, and possibly o were present in the bacteroids throughout the active nitrogen-fixing life of the nodule. This is in contrast with previous reports using other R. japonicum strains in which cyotchromes aa(3) and o were not found. PMID- 16662787 TI - Identification and Metabolism of 1-(Malonylamino)cyclopropane-1-carboxylic Acid in Germinating Peanut Seeds. AB - Peanut seeds (Arachis hypogea L. Yue-you 551) contain 50 to 100 nanomoles per gram conjugated 1-aminocyclopropanecarboxylic acid (ACC). Based on paper chromatography, paper electrophoresis, and gas chromatography-mass spectrometry, it was verified that the major ACC conjugate was N-malonyl-ACC (MACC). Germinating peanut seeds converted [2-(14)C]ACC to ethylene 70 times more efficiently than N-malonyl-[2-(14)C]ACC; when ACC was administered, most of it was metabolized to MACC. Germinating peanut seeds produced ethylene and converted l-[3,4-(14)C]methionine to ethylene; this ethylene biosynthesis was inhibited by aminoethoxyvinylglycine. These data indicate that MACC occurs in peanut seeds but does not serve as the source of ethylene during germination; ethylene is, however, synthesized from methionine via ACC. PMID- 16662788 TI - Elevated riboflavin requirement for postphotoinductive events in sporulation of a trichoderma auxotroph. AB - A riboflavin auxotroph of Trichoderma required more riboflavin for blue-light induced conidiation than for growth. Colonies transferred after illumination from 0.2 micromolar (limiting conidiation, not growth) to 3.5 micromolar riboflavin (nonlimiting), responded the same as those grown at 3.5 micromolar. The additional riboflavin is therefore not required as a photoreceptor. The data do not rule out the hypothesis that cryptochrome(s) is/are flavin(s). PMID- 16662789 TI - Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography. AB - NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn(2+) column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons. PMID- 16662790 TI - Adaptation of the Cyanobacterium Anabaena variabilis to Low CO(2) Concentration in Their Environment. AB - The rate of adaptation of high CO(2) (5% v/v CO(2) in air)-grown Anabaena to a low level of CO(2) (0.05% v/v in air) was determined as a function of O(2) concentration. Exposure of cells to low (2.6%) O(2) concentration resulted in an extended lag in the adaptation to low CO(2) concentration. The rate of adaptation following the lag was not affected by the concentration of O(2). The length of the lag period is markedly affected by the O(2)/CO(2) concentration ratio, indicating that the signal for adaptation to low CO(2) may be related to the relative rate of ribulose-1,5-bisphosphate carboxylase/oxygenase activities, rather than to CO(2) concentration proper. This suggestion is supported by the observed accumulation of phosphoglycolate following transfer of cells from high to low CO(2) concentration. PMID- 16662791 TI - Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays. AB - A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth. PMID- 16662792 TI - Isolation of Mitochondria from Leaf Tissue of Panicum miliaceum, a NAD-Malic Enzyme Type C(4) Plant. AB - A mechanical isolation procedure was developed to study the respiratory properties of mitochondria from the mesophyll and bundle sheath tissue of Panicum miliaceum, a NAD-malic enzyme C(4) plant. A mesophyll fraction and a bundle sheath fraction were obtained from young leaves by differential mechanical treatment. The purity of both fractions was about 80%, based on analysis of the cross-contamination of ribulose bisphosphate carboxylase activity and phosphoenolpyruvate carboxylase activity.Mitochondria were isolated from the two fractions by differential centrifugation and Percoll density gradient centrifugation. The enrichment of mitochondria relative to chloroplast material was about 75-fold in both preparations.Both types of mitochondria oxidized NADH and succinate with respiratory control. Malate oxidation in mesophyll mitochondria was sensitive to KCN and showed good respiratory control. In bundle sheath mitochondria, malate oxidation was largely insensitive to KCN and showed no respiratory control. The oxidation was strongly inhibited by salicylhydroxamic acid, showing that the alternative oxidase was involved. The bundle sheath mitochondria of this type of C(4) species contribute to C(4) photosynthesis through decarboxylation of malate. Malate oxidation linked to an uncoupled, alternative pathway may allow decarboxylation to proceed without the restraints which might occur via coupled electron flow through the cytochrome chain. PMID- 16662793 TI - Effects of o(2) concentration on rice seedlings. AB - The ability of rice, wheat, and oat seedlings to germinate and grow as the O(2) concentration was lowered to zero was compared. The germination of rice was completely unaffected by O(2) supply, whereas that of oats and wheat was strongly retarded at levels below 5% O(2). In contrast to the coleoptiles of oats and wheat and to roots of all three species where growth was progressively diminished as the O(2) concentration was lowered, that of the rice coleoptile was progressively increased. However, the dry weight and content of protein, sugars, and cellulose were all depressed in the rice coleoptile in anoxia, and the levels of several respiratory enzymes, particularly those of mitochondria, were also much lower than those of the coleoptiles grown in air. In 1% O(2), the growth of the rice coleoptile was similar to that in air. The effect of ethanol concentration on germination and growth of rice was measured. Coleoptile growth was reduced when the ethanol concentration exceeded 40 millimolarity, and root growth was somewhat more sensitive. Coleoptiles of all three species grown in air were transferred to N(2), and ethanol accumulation was measured over 24 hours. The rate of ethanol accumulation in oats was close to that in rice, and in all three species the amounts of ethanol lost to the surrounding medium were those expected from simple diffusion from the tissue. The ability of the rice coleoptile to grow in anoxia is apparently not due to a particularly low rate of ethanol formation or to unusual ethanol tolerance. Any explanation of the success of rice in anoxia must encompass the much lower rate of ATP synthesis than that in air and account for the biochemical deficiencies of the coleoptile. PMID- 16662794 TI - Nuclear Suppressors of the Photosensitivity Associated with Defective Photosynthesis in Chlamydomonas reinhardii. AB - Several nuclear mutations were recovered that suppress the photosensitivity associated with the Chamydomonas reinhardii chloroplast mutant rcl-u-1-10-6C, which is defective in ribulose-1,5-bisphosphate carboxylase/oxygenase. Two of the suppressor mutations affect other components of photosynthesis. These results show that suppressors of photosensitivity are sufficiently common to permit the recovery of photosensitive, photosynthesis-deficient mutants in bright light, and indicate that photosynthesis-deficient mutants selected and maintained in the light may accumulate suppressors which can confuse the biochemical analysis of lesions in photosynthesis. One of the suppressor mutations inhibits photosystem II activity, indicating that photosensitivity can be mediated by partial reactions of the photosynthetic electron transport chain. PMID- 16662795 TI - Expression of Storage Protein Genes in Developing Wheat (Triticum aestivum L.) Seeds : Correlation of RNA Accumulation and Protein Synthesis. AB - Ribonucleic acid and protein synthesis in developing wheat kernels have been studied through in vivo labeling of wheat heads in culture. In INIA 66R wheat labeled with [5-(3)H]uridine for 24-hour periods between 9 and 33 days after flowering, the total rate of RNA accumulation in endosperm/testa pericarp tissues was highest in the youngest seeds, and declined with increasing seed age. In contrast, the rate of accumulation of poly(A)(+) RNA approximately doubled between 12 and 15 days after flowering, reached a maximum between 15 and 18 days, and declined to half the maximum rate by 24 days. Protein synthetic capacity, measured by in vitro translation of extracted seed RNA, increased in a developmental pattern similar to that of poly(A)(+) RNA accumulation, but remained near maximal through 24 days after flowering. Gliadins were prominent in the in vitro translation products. When seed protein was labeled in vivo with l [(3)H]leucine, extracted, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a significant change in the protein synthesis profile was apparent between 12 and 15 days after flowering, and was coincident with a marked increase in storage protein synthesis. Qualitatively similar characteristics were exhibited by the cultivar Cheyenne, although in a shorter developmental period. These results are consistent with a direct relation between levels of mRNA and rates of protein synthesis in developing wheat seeds, with a relatively long storage protein mRNA lifetime, and with control of storage protein gene expression primarily at the level of mRNA transcription. PMID- 16662796 TI - Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA. AB - It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S deficient plants indicated a reduction of 60 to 70% in synthesis of legumin related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA. PMID- 16662797 TI - Structural Features Required for Inhibition of Soybean Lipoxygenase-2 by Propyl Gallate : Evidence that Lipoxygenase Activity Is Distinct from the Alternative Pathway. AB - The ability of 19 structural analogs of propyl gallate to inhibit purified soybean seed (Glycine max [L.] Merr. var. Ransom) lipoxygenase-2 (EC 1.13.11.12) was determined. The results indicate that the o-dihydroxy and not the ester function of propyl gallate is essential for inhibition of lipoxygenase. Catechol thus represents the minimum inhibitory structure. Among those compounds possessing an o-dihydroxy function, the K(i)' for inhibition of lipoxygenase is directly related to the lipophilicity of the inhibitor as measured by the octanol water partition coefficient. The structural features of propyl gallate necessary for inhibition of lipoxygenase were found to differ from those required for inhibition of the plant mitochondrial alternative pathway. This further supports the concept that the alternative oxidase and lipoxygenase are functionally distinct species. PMID- 16662798 TI - Carbon dioxide effects on ethanol production, pyruvate decarboxylase, and alcohol dehydrogenase activities in anaerobic sweet potato roots. AB - The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O(2) and N(2) gases disappeared quickly and were replaced by CO(2). There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO(2) environment, followed by submergence and a N(2) environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO(2)-treated and 100% N(2)-treated roots. CO(2) atmospheres also resulted in higher pyruvate decarboxylase activity than did N(2) atmospheres. Concentrations of CO(2) were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO(2) concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase. PMID- 16662799 TI - Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves. AB - The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO(3) (-) and NO(2) (-) assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO(3) (-) or NO(2) (-) were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO(3) (-) reduction in both light and darkness, while photosynthetic inhibitors did not.The NO(2) (-) concentration in the control leaves was very low in both light and darkness; 98% or more of the NO(2) (-) formed from NO(3) (-) was further assimilated in control leaves. More NO(2) ( ) accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO(2) (-), 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO(2) (-) reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.NITRITE ASSIMILATION WAS MUCH MORE SENSITIVE TO UNCOUPLERS IN DARKNESS THAN IN LIGHT: in darkness, 74% or more of NO(2) (-) formed from NO(3) (-) was further assimilated, whereas in light, 95% or more of the NO(2) (-) was further assimilated. PMID- 16662800 TI - Metabolism of Oat Leaves during Senescence : VII. The Interaction of Carbon Dioxide and Other Atmospheric Gases with Light in Controlling Chlorophyll Loss and Senescence. AB - In air largely freed from CO(2), senescence of isolated oat (Avena sativa cv Victory) seedling leaves is no longer prevented by white light; instead, the leaves lose both chlorophyll and protein as rapidly as in the dark. Senescence in light is also accelerated in pure O(2), but it is greatly delayed in N(2); 100% N(2) preserves both protein and chlorophyll in light and in darkness. In light in air, most of the compounds tested that had previously been found to delay or inhibit senescence in darkness actually promote the loss of chlorophyll, but they do not promote proteolysis. Under these conditions, proteolysis can therefore be separated from chlorophyll loss. But in light minus CO(2), where chlorophyll loss is rapid in controls, two of these same reagents prevent the chlorophyll loss. Unlike the many reagents whose action in light is thus the opposite of that in darkness, abscisic acid, which promotes chlorophyll loss in the dark, also promotes it in light with or without CO(2). Kinetin, which prevents chlorophyll loss in the dark, also prevents it in light minus CO(2). In general, therefore, the responses to light minus CO(2) are similar to the responses to darkness, and (with the exception of abscisic acid and kinetin) opposite to the response to light in air. PMID- 16662801 TI - Nitrogen Redistribution during Grain Growth in Wheat (Triticum aestivum L.) : IV. Development of a Quantitative Model of the Translocation of Nitrogen to the Grain. AB - Translocation of nitrogen was measured in wheat (Triticum aestivium L. cv SUN 9E) plants grown without an exogenous supply of nitrogen from the time that the flagleaf began to emerge, and a model of nitrogen translocation was constructed to describe translocation on one day during the linear period of grain growth. Nitrogen for grain development was derived entirely by the redistribution of nitrogen from vegetative organs. Leaves contributed 40%, glumes 23%, stem 23%, and roots 16% of the nitrogen incorporated by the grains on the fifteenth day after anthesis. Less than 50% of the nitrogen exported from leaves was translocated directly to the grain via the phloem, the rest was translocated to the roots and was cycled in the roots and exported to the shoot in the transpiration stream. Nitrogen imported by leaves and glumes via the xylem was not accumulated in these organs but was transferred to the phloem for reexport from the organs. A large proportion (60%) of the nitrogen in the transpiration stream was cycled in the glumes. The glumes were also a major source of nitrogen for grain development. It was considered likely that this organ always plays an important role in nitrogen metabolism in wheat. PMID- 16662802 TI - Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO(2) Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica. AB - The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H(+) accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C(3) photosynthesis. The total nocturnal H(+) accumulation and CO(2) uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H(+) accumulation and 4 moles per square meter per day for CO(2) uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H(+) accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period.A minimum ratio of H(+) accumulated to CO(2) taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H(+)-to-CO(2) ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H(+) accumulation and CO(2) uptake decreased, but the H(+)-to-CO(2) ratio increased. A ratio of two H(+) per CO(2) is consistent with the H(+) production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus indica when CAM CO(2) uptake is strongly favored over respiratory activity. PMID- 16662803 TI - Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates. AB - GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of beta-1,4-glucans in vivo. PMID- 16662804 TI - Characterization of the Isozymes of alpha-Mannosidase Located in the Cell Wall, Protein Bodies, and Endoplasmic Reticulum of Phaseolus vulgaris Cotyledons. AB - Cotyledons of maturing Phaseolus vulgaris seeds contain three isozymes of alpha mannosidase which can be separated by isoelectrofocusing. They have isoelectric points of 5.3, 5.8, and 6.5 to 7.5 and were named I, II, and III in order of ascending pI. All three had an acid pH optimum (4.5) and required Zn(2+) for maximal activity. Isozymes I and II were present in the protein bodies. Together they accounted for 85% of the total activity. Isozyme III was essentially absent from isolated protoplasts but could be extracted from isolated cell walls. All three isozymes were also found to be associated with the endoplasmic reticulum, and the proportion of the total activity in this fraction decreased from 20% in immature cotyledons to 6% in mature cotyledons. The results are interpreted as evidence that newly synthesized alpha-mannosidase is sequestered in the lumen of the ER prior to its transport to the protein bodies or the cell wall. PMID- 16662805 TI - Enzymic mechanism of starch breakdown in germinating rice seeds : 15. Immunochemical study on multiple forms of amylase. AB - The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using beta-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major alpha-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of alpha-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the beta-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.Evidence showing that beta-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation. PMID- 16662806 TI - Hypobaric Control of Ethylene-Induced Leaf Senescence in Intact Plants of Phaseolus vulgaris L. AB - A controlled atmospheric-environment system (CAES) designed to sustain normal or hypobaric ambient growing conditions was developed, described, and evaluated for its effectiveness as a research tool capable of controlling ethylene-induced leaf senescence in intact plants of Phaseolus vulgaris L.Senescence was prematurely induced in primary leaves by treatment with 30 parts per million ethephon. Ethephon-derived endogenous ethylene reached peak levels within 6 hours at 26 degrees C. Total endogenous ethylene levels then temporarily stabilized at approximately 1.75 microliters per liter from 6 to 24 hours. Thereafter, a progressive rise in ethylene resulted from leaf tissue metabolism and release. Throughout the study, the endogenous ethylene content of ethephon-treated leaves was greater than that of nontreated leaves.Subjecting ethephon-treated leaves to atmospheres of 200 millibars, with O(2) and CO(2) compositions set to approximate normal atmospheric partial pressures, prevented chlorophyll loss. Alternately, subjecting ethephon-treated plants to 200 millibars of air only partially prevented chlorophyll loss. Hypobaric conditions (200 millibars), with O(2) and CO(2) at normal atmospheric availability, could be delayed until 48 hours after ethephon treatment and still prevent most leaf senescence. In conclusion, hypobaric conditions established and maintained within the CAES prevented ethylene-induced senescence (chlorosis) in intact plants, provided O(2) and CO(2) partial pressures were maintained at levels approximating normal ambient availability.An unexpected increase in endogenous ethylene was detected within nontreated control leaves 48 hours subsequent to relocation from winter greenhouse conditions (latitude, 42 degrees 00'' N) to the CAES operating at normal ambient pressure. The longer photoperiod and/or higher temperature utilized within the CAES are hypothesized to influence ethylene metabolism directly and growth-promotive processes (e.g. response thresholds) indirectly. PMID- 16662807 TI - Heat shock proteins in maize. AB - The pattern of protein synthesis in roots of 3-day-old maize seedlings (Zea mays L.) is rapidly and dramatically altered when the incubation temperature is raised from 25 to 40 degrees C. One-dimensional sodium dodecyl sulfate gels reveal that although synthesis of the proteins observed at 25 degrees C continues at 40 degrees C, a new set of ;heat shock proteins' (hsp) is induced within 20 minutes of the temperature transition. The hsp have molecular weights of 87, 85, 79, 78, 77, 72, 70, 27, 22, and 18 kilodaltons. The 10 hsp are visible on autoradiograms but not on stained gels, suggesting that the proteins do not accumulate to any great extent.The induction of the hsp is transitory. With prolonged high temperature treatment, the synthesis of hsp continues for 4 hours in excised roots and for 8 hours in the roots of intact seedlings before declining sharply. Coincident to the decline in synthesis of the 10 hsp is the gradual increase in intensity of three new polypeptides having molecular weights of 62, 49.5, and 19 kilodaltons. These proteins begin to appear about the time that synthesis of the other 10 hsp becomes maximal.Shifting the temperature back to 25 degrees C also causes a decline in synthesis of hsp, but this decline occurs more rapidly than that seen during prolonged heat shock. A decrease in hsp synthesis becomes apparent 2 hours after the roots are returned to 25 degrees C.Shifting the temperature from 25 to 45 degrees C results in a pattern of protein synthesis different from that observed after a shift to 40 degrees C. Normal protein synthesis continues, except four proteins, which are produced in small amounts at lower temperatures, show greatly enhanced synthesis at 45 degrees C. These proteins have apparent molecular weights of 83, 81, 68, and 65 kilodaltons. Also, the 10 hsp listed above are not synthesized. It is suggested that at least two distinct high-temperature responses are present in maize, which may reflect the metabolic changes generated at different elevated temperatures. PMID- 16662808 TI - A Comparison of the Surface Polysaccharides from Rhizobium leguminosarum 128C53 smrif with the Surface Polysaccharides from Its Exo Mutant. AB - The surface polysaccharides of Rhizobium leguminosarum 128C53 sm(r)rif(r) (parent) and its exo(-1) mutant were isolated and characterized. The parent carries out normal symbiosis with its host, pea, while the exo(-1) mutant does not nodulate the pea. The following observations were made. (a) The parent produces lipopolysaccharide (LPS), typical acidic extracellular polysaccharide (EPS), and three additional polysaccharides, PS1, PS2, and PS3. The PS1 and PS2 fractions are likely to be the capsular polysaccharide (CPS) and are identical in composition to the EPS. The PS3 fraction is a small-molecular-weight glucan. (b) The exo(-1) mutant produces LPS, EPS, and a PS3 fraction, but does not produce significant amounts of either PS1 or PS2. The LPS from the exo(-1) mutant appears to be identical to the parental LPS. Analysis of the EPS from exo(-1) shows that it consists of two polysaccharides. One polysaccharide is identical to the LPS and comprises 70% of the exo(-1) EPS. The second polysaccharide is identical to the exo(-1) PS3 and comprises 30% of the exo(-1) EPS. This result shows that the exo(-1) mutant does not produce any of the typical acidic parental EPS and that the major polysaccharide released into the media by the exo(-1) mutant is intact LPS. The exo(-1) mutant PS3 fraction was found to contain two polysaccharides, PS3-1 and PS3-2. The PS3-2 polysaccharide is identical to the parental PS3 described above. The PS3-1 polysaccharide has a composition similar to the polysaccharide portion of the LPS. This result suggests that the exo(-1) mutant produces LPS polysaccharide fragments. These LPS polysaccharide fragments are not produced by the parent strain. PMID- 16662809 TI - Specific Determination of alpha-Amylase Activity in Crude Plant Extracts Containing beta-Amylase. AB - The specific measurement of alpha-amylase activity in crude plant extracts is difficult because of the presence of beta-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70 degrees C for 20 min, HgCl(2) treatment, and the use of the alpha-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While beta amylase can liberate no color alone, over 10 International units per milliliter beta-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at beta-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate beta-amylase interference: (a) the dilution procedure, the serial dilution of samples until beta-amylase levels are below levels that interfere; (b) the beta-amylase saturation procedure, addition of exogenous beta-amylase to increase endogenous beta-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in beta amylase activity or inhibitory effects of the commercial beta-amylase. The beta amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as alpha-amylases in alfalfa and soybeans are heat labile. Whereas HgCl(2) proved to be a potent inhibitor of beta-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited alpha-amylase in barley malt. The reported alpha-amylase activities in crude enzyme extracts from a number of plant species are apparently the first specific measurements reported for any plant tissues other than germinating cereals. PMID- 16662810 TI - Mode of action of a herbicide : johnsongrass and methanearsonic Acid. AB - Johnsongrass (Sorghum halepense (L.) Pers.) is sensitive to methanearsonate, foliar application resulting in a topkill. Investigation of the pattern of photosynthesis by radioautography revealed an accumulation of malate in methanearsonate-treated leaves. Accumulation of malate was attributed to an inhibition of NADP(+)-malic enzyme which was found to be sensitive to sulfhydryl group reagents including arsenosomethane, CH(3)AsO. Methanearsonate was found to act as an oxidant in the Hill reaction using spinach chloroplasts, the photoproduct being a sulfhydryl group reagent.These results suggest that methanearsonate inhibits CO(2) release from malate in bundle sheath cells, depriving the plant of its source of carbon for sucrose production. The mechanism of inhibition of enzymes sensitive to sulfhydryl group reagents by arsenosomethane is addressed. PMID- 16662811 TI - Adaptation of Lemna paucicostata to Sublethal Methionine Deprivation. AB - During initial exposure to 40 nanomolar propargylglycine (PAG), Lemna paucicostata colonies undergo abnormal fragmentation and a lag in frond emergence, most severe at 24 to 48 hours. Thereafter, frond emergence resumes and the frond/colony ratio rises. Such ;adapted' plants withstand subculture into the same concentration of PAG without fragmentation or decreases in frond emergence, and display enhanced tolerance to higher concentrations. Adaptation is not dependent upon outgrowth of a few preexisting especially tolerant plants. Exogenous methionine prevents these events and overcomes the PAG-induced lag in frond emergence even after it is underway. These changes in frond emergence are not reflected in the rates of protein and wet weight accumulation which decrease by about 25% during the first 24 hours and continue unchanged thereafter. Cystathionine gamma-synthase activity rapidly decreases to 9% of control during the first 12 hours of exposure to 40 nanomolar PAG but thereafter climbs to 12% of control. Studies of the uptake and internal concentration of PAG during these events are reported.Exposure to a combination of 36 micromolar lysine plus 3 micromolar threonine is an alternative means to bring about sublethal methionine deprivation. Thus exposed, Lemna undergoes an analogous sequence of effects on morphology and growth which are preventable by exogenous methionine and which lead to an adapted state. Cystathionine gamma-synthase specific activity in plants adapted to 36 micromolar lysine plus 3 micromolar threonine is 1.8 times control. However, addition of PAG showed that under these conditions enzyme activity can be decreased to as little as 54% of control without affecting the growth rate. Together these results suggest that adaptation is related to methionine limitation and that the plants adjust, in part, by increasing the steady-state concentrations of cystathionine gamma-synthase and other enzymes in the methionine pathway. PMID- 16662812 TI - Light Activation of Purified Aconitase by Washed Thylakoid Membranes of Pea (Pisum sativum L.). AB - Purified aconitase, an iron-sulfur protein, from either beef heart mitochondria or pig heart can be activated fully by light when combined with washed thylakoid membranes from pea (Pisum sativum L.) chloroplasts. The light activation of the enzyme does not require any other additive or cofactor and is sensitive to 3-(3,4 dichlorophenyl)-1,1-dimethyl urea, 2,6-dichlorophenol-indophenol, ferricyanide, and methyl viologen, indicating that the photoelectron transport system of the thylakoid membranes, and in particular, photosystem I, is involved in the process of activation. Light activation of the enzyme is also markedly inhibited when the thylakoid membranes are treated with sulfite or arsenite, and abolished totally when the membranes are treated with Zwittergent, suggesting that the light effect mediator involved in the light modulation of chloroplastic enzymes mediates the activation of purified aconitase also. PMID- 16662813 TI - Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris. AB - Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors. PMID- 16662814 TI - Metabolism of Tritiated Gibberellins A(4) and A(9) in Norway Spruce, Picea abies (L.) Karst. : Effects of a Cultural Treatment Known to Enhance Flowering. AB - Shoots of mature grafted propagules of Picea abies (L.) Karst. metabolized [(3)H]gibberellin A(4) (GA(4)) to at least 14 acidic substances, two of which were tentatively identified by gas-liquid radiochromatography as GA(2) (possibly an artifact) and GA(34). [(3)H]GA(9) was converted into a number of metabolites, one of which was chromatographically similar to, but not identical with, GA(4). Metabolism was maximally 61 and 57% over 48 hours for GA(4) and GA(9), respectively, and was correlated with the rate of change (i.e. increase followed by decrease) in endogenous GA-like substances as shoot elongation progressed. Propagules covered with a clear plastic film, a treatment which promotes flowering, metabolized [(3)H]GA(4) more slowly than did control plants in the open. Inasmuch as a GA(4/7) mixture can also promote flowering in P. abies, the retarded metabolism of [(3)H]GA(4) may reflect the manner in which trees under plastic metabolize endogenous GA-like substances. If so, then the stimulating effect of this cultural treatment on flowering may come about through an increased level of endogenous, less polar GA-like substances. PMID- 16662815 TI - N-Methyl Mesoporphyrin IX Inhibits Phycocyanin, but Not Chlorophyll Synthesis in Cyanidium caldarium. AB - The ability of N-methyl mesoporphyrin IX (NMMP) to block heme synthesis by specifically inhibiting enzymic iron insertion into protoporphyrin IX was exploited to test whether heme is a precursor of the bilin chromophore of phycocyanin (PC). A strain of the unicellular rhodophyte Cyanidium caldarium which forms normal amounts of both chlorophyll (Chl) and PC in the dark was employed to avoid phototoxic effects of exogenous porphyrins. Relative Chl and PC content were assayed spectrophotometrically on whole cell suspensions.When cells were grown in the dark on a glucose-based heterotrophic medium at 42 degrees C, neither division rate nor Chl synthesis was affected by NMMP up to 3.0 micromolar and for as long as 72 hours. NMMP had a dose-dependent inhibitory effect on PC synthesis. PC to Chl absorbance ratios, relative to control cell values, were 100%, 89%, 86%, and 50% in cells grown for 48 hours with 0.3, 1.0, 3.0, and 10.0 micromolar NMMP, respectively. NMMP also caused the accumulation of intracellular protoporphyrin.The ability of NMMP to cause intracellular accumulation of protoporphyrin and to block PC synthesis specifically while allowing normal Chl formation is consistent with its action as a specific inhibitor of enzymic iron chelation, and supports the role of heme as a precursor to the phycobilins. PMID- 16662816 TI - Developmental changes in the potential for h(2)s emission in cucurbit plants. AB - Based on results obtained with leaf discs exposed to sulfate, leaves on cucurbit plants (Cucurbita pepo L. cv Small Sugar Pumpkin and Cucumis sativus cv Chipper) 1 to 2.5 weeks old have a low potential for H(2)S emission (less than 10 picomoles per min per cm(2) leaf area) in response to sulfate, whereas discs from most of the leaves on plants 3 to 4 weeks old emit H(2)S at a higher rate (50 to 150 picomoles per min per cm(2) leaf area). This difference is determined by the age of the plant, and is independent of the leaves' age or developmental stage. In response to l-cysteine, however, discs from leaves on cucurbit plants 1 to 2.5 weeks old emit H(2)S at higher rates (15 to 50 picomoles per min per cm(2) leaf area) than in response to sulfate. Furthermore, the potential for H(2)S emission in response to l-cysteine decreases with increasing age of the individual leaf. Thus, most of the potential for H(2)S emission in response to l-cysteine is developed during germination and the early growth of cucurbit plants, but most of the potential for H(2)S emission in response to sulfate arises later in the development of the plants.The developmental changes in the potential for H(2)S emission in response to l-cysteine in vivo are paralleled by changes in the cysteine desulfhydrase activity extractable from the leaves. This extractable activity, which is thought to be responsible for the generation of H(2)S by leaf tissue in response to l-cysteine, can be increased by preincubation of leaf discs in l-cysteine. Overt cysteine desulfhydrase activity is up to 2-fold higher, and latent cysteine desulfhydrase activity is up to 4-fold higher, in leaves on cucurbit plants 1 to 2.5 weeks old than in leaves on plants 3 to 4 weeks old. Thus, most of the cysteine desulfhydrase activity develops during the early period of growth of a cucurbit plant. Overt cysteine desulfhydrase activity passes through a maximum value during the development of each leaf; total as well as latent cysteine desulfhydrase activities, however, decline with increasing age of the individual leaf in much the same way as H(2)S emission in response to l cysteine declines. PMID- 16662817 TI - Mechanical properties of the plasma membrane of isolated plant protoplasts : mechanism of hyperosmotic and extracellular freezing injury. AB - The volume of isolated protoplasts of rye (Secale cereale L. cv Puma) in a suspending solution at constant concentration is shown to be negligibly changed by tensions in the plasma membrane which approach that tension necessary to lyse them. This allows a detailed investigation of the plasma membrane stress-strain relation by micropipette aspiration.Over periods less than a second, the membrane behaves as an elastic two-dimensional fluid with an area modulus of elasticity of 230 millinewtons per meter. Over longer periods, the stress-strain relation approaches a surface energy law-the resting tension is independent of area and has a value of the order 100 micronewtons per meter. Over longer periods the untensioned area, which is defined as the area that would be occupied by the molecules in the membrane at any given time if the tension were zero, increases with time under large imposed tensions and decreases under sufficiently small tension. It is proposed that these long term responses are the result of exchange of material between the plane of the membrane and a reservoir of membrane material. The irreversibility of large contractions in area is demonstrated directly, and the behavior of protoplasts during osmotically induced cycles of contraction and expansion is explained in terms of the membrane stress-strain relation. PMID- 16662818 TI - In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase. AB - Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO(2) (-) and NH(4) (+) under blue than under red light. Under similar conditions, but with NO(2) (-) as the only nitrogen source, the cells consumed NO(2) (-) and excreted NH(4) (+) at similar rates under blue and red light. In the presence of NO(3) (-) and air with 2% CO(2) (v/v), no excretion of NO(2) (-) and NH(4) (+) occurred and, moreover, if the bubbling air of the cells that were currently excreting NO(2) (-) and NH(4) (+) was enriched with 2% CO(2) (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO(3) (-) under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m(-2)) and low CO(2) tensions, cells growing on NO(3) (-) or NO(2) (-) may not have sufficient carbon skeletons to incorporate all the photogenerated NH(4) (+). Because these cells should have high levels of reducing power, they might use NO(3) (-) or, in its absence, NO(2) (-) as terminal electron acceptors. The excretion of the products of NO(2) (-) and NH(4) (+) to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO(3) (-) and possibly of the whole nitrogen metabolism in green algae. PMID- 16662819 TI - Assimilation of [N]Nitrate and [N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions. AB - Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [(15)N]nitrate and [(15)N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [(15)N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [(15)N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.In dark anaerobic conditions, the assimilation of [(15)N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [(15)N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [(15)N]nitrite assimilation. PMID- 16662820 TI - De novo synthesis of 3'-nucleotidase in germinating wheat embryo. AB - The enzyme 3'-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up (35)SO(4) between 40 and 46 hours of germination synthesized (35)S-labeled 3'-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3'-nucleotidase activity is a consequence of de novo synthesis of the enzyme. PMID- 16662821 TI - Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus. AB - Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation. PMID- 16662822 TI - Metabolism of Magnesium Protoporphyrin Monomethyl Ester in Chlamydomonas reinhardtii. AB - The y-1 mutant of Chlamydomonas reinhardtii is defective in the conversion of protochlorophyllide (Pchlide) to chlorophyllide in the dark. Aerobic delta aminolevulinic acid (ALA) feeding of y-1 cells causes protoporphyrin monomethyl ester (PME) to accumulate in addition to increased levels of Pchlide. y-1 cell homogenates are not capable of methylating protoporphyrin (PROTO) to form PME but can methylate magnesium protoporphyrin (MgP) to form magnesium protoporphyrin monomethyl ester (MgPME). Anaerobic ALA feeding of y-1 causes concomitant accumulation of PME and MgPME. y-1 cells treated with alpha,alpha'-dipyridyl (DP) accumulate MgPME but not PROTO or PME. A mutant strain (bme) of Chlamydomonas has been isolated which has very little chlorophyll and accumulates PME. bme Cell homogenates can methylate MgP but not PROTO. We propose that: (a) in Chlamydomonas, PME is the initial breakdown product of MgPME; (b) both the breakdown of MgPME to PME and the conversion of MgPME to Pchlide require O(2); (c) the breakdown of MgPME to PME appears to require Fe; and (d) the PME accumulated in the bme mutant is the result of an increased breakdown of MgPME. PMID- 16662823 TI - Amitrole Absorption by Bean (Phaseolus vulgaris L. cv ;Red Kidney') Roots : Mechanism of Absorption. AB - The mechanism of transport of the herbicide 3-amino-1,2,4-triazole (amitrole) into Phaseolus vulgaris roots appears to be passive, as judged by the effect of temperature (Q(10) = 1.3 between 15 and 25 degrees C) and the lack of sensitivity to metabolic inhibition afforded by 2,4-dinitrophenol and NaN(3). Amitrole absorption is a linear function of external concentration over several orders of magnitude and, thus, is not facilitated by a carrier mechanism. The absorption of amitrole is sensitive to external pH, being stimulated under acid conditions. This stimulation of amitrole absorption is seen at low ( Rb(+) > Na(+) > Li(+) but when the concentration was raised to 50 millimolarity, the sequence changed to K(+) >/= Na(+) >/= Rb(+) > Li. The activity was not synergistically stimulated by combinations of Na(+) and K(+). The enzyme was insensitive to NaN(3), oligomycin, ouabain, and sodium molybdate but sensitive to N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles. PMID- 16662830 TI - Cytokinins of the Developing Mango Fruit : Isolation, Identification, and Changes in Levels during Maturation. AB - The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N(6)-(Delta(2)-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 x 10(3) micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 x 10(2) micromolar) and naphthalene acetamide (3.1 x 10 micromolar) at the young fruit stage. PMID- 16662831 TI - Involvement of abscisic Acid in potato cold acclimation. AB - Upon exposure to 2 degrees C day/night (D/N), leaves of Solanum commersonii (Sc) began acclimating on the 4th day from a -5 degrees C (killing temperature) hardy level to -12 degrees C by the 15th day. Leaves of S. tuberosum L. (St) cv ;Red Pontiac' typically failed to acclimate and were always killed at -3 degrees C. Leaves of control (20/15 degrees C, D/N) and treated plants (2 degrees C, D/N) of St showed similar levels of free abscisic acid (ABA) during a 15-day sampling period. In treated Sc plants, however, free ABA contents increased 3-fold on the 4th day and then declined to their initial level thereafter. The increase was not observed in leaves of Sc control plants.Treated St plants showed a slightly higher content of leaf soluble protein than controls. In Sc, leaves of controls maintained relatively constant soluble proteins, but leaves of treated plants showed a distinct increase. This significant increase was initiated on the 4th day, peaked on the 5th day, and remained at a high level throughout the 15-day sampling period.Exogenously applied ABA induced frost hardiness in leaves of Sc plants whether plants were grown under a 20 degrees C or 2 degrees C temperature regime. When cycloheximide was added to the medium of stem-cultured plants at the beginning of 2 degrees C acclimation, or at the beginning of the ABA treatment in the 20 degrees C regime, it completely inhibited the development of frost hardiness. However, when cycloheximide was added to plants on the 5th day during 2 degrees C acclimation, the induction of frost hardiness was not inhibited. The role of ABA in triggering protein synthesis needed to induce frost hardiness is discussed. PMID- 16662832 TI - Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes. AB - The galactosylation steps in the biosynthesis of galactolipids involve two different enzymes; a UDP-Gal:diacylglycerol galactosyltransferase and a galactolipid:galactolipid galactosyltransferase. Previous localization studies have shown that in spinach these enzymes are located in the chloroplast envelope. Our results with peas (Pisum sativum var Laxton's Progress No. 9) confirm these results and extend the localization by providing evidence that the galactosyltransferases are in the outer membrane of the envelope. The specific activity of UDP-Gal:diacylglycerol galactosyltransferase in outer membrane preparations was 6 to 10 times greater than that exhibited by inner membrane preparations. In addition, using quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis, it was possible to show that the UDP Gal:diacylglycerol galactosyltransferase activity associated with inner membrane preparations could be accounted for by outer membrane contamination. It is concluded from these results that this enzyme is located predominantly, if not exclusively, in the outer membrane of the envelope. An analysis of the galactolipid products synthesized by the highly purified outer membrane showed that the galactolipid:galactolipid galactosyltransferase is also present, suggesting that this enzyme is also an outer membrane enzyme. The implication of these results is that the final assembly of galactolipids is carried out on the outer membrane of the chloroplast envelope. PMID- 16662833 TI - Diurnal changes in volume and specific tissue weight of crassulacean Acid metabolism plants. AB - The diurnal variations in volume and in specific weight were determined for green stems and leaves of Crassulacen acid metabolism (CAM) plants. Volume changes were measured by a water displacement method. Diurnal variations occurred in the volume of green CAM tissues. Their volume increased early in the light period reaching a maximum about mid-day, then the volume decreased to a minimum near midnight. The maximum volume increase each day was about 2.7% of the total volume. Control leaves of C(3) and C(4) plants exhibited reverse diurnal volume changes of 0.2 to 0.4%. The hypothesis is presented and supported that green CAM tissues should exhibit a diurnal increase in volume due to the increase of internal gas pressure from CO(2) and O(2) when their stomata are closed. Conversely, the volume should decrease when the gas pressure is decreased.The second hypothesis presented and supported was that the specific weight (milligrams of dry weight per square centimeter of green surface area) of green CAM tissues should increase at night due to the net fixation of CO(2). Green CAM tissues increased their specific weight at night in contrast to control C(3) and C(4) leaves which decreased their specific weight at night. With Kalanchoe daigremontiana leaves, the calculated increase in specific leaf weight at night based on estimates of carbohydrate available for net CO(2) fixation was near 6% and the measured increase in specific leaf weight was 6%.Diurnal measurements of CAM tissue water content were neither coincident nor reciprocal with their diurnal patterns of either volume or specific weight changes. PMID- 16662834 TI - Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp. AB - We have identified the function of the ;extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055 1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination. PMID- 16662835 TI - Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308. AB - The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1 dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I(50) value) in Aphanocapsa membranes for diuron was 6.8 x 10(-9) molar and the I(50) value for atrazine was 8.8 x 10(-8) molar. (14)C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 x 10(-8) molar for atrazine and 1.7 x 10(-7) molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [(14)C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide binding site. Experiments involving the photoaffinity label [(14)C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32 kilodalton protein in Aphanocapsa 6308 cell extracts. PMID- 16662836 TI - Reversible dark-induced senescence of soybean root nodules. AB - Nodule senescence was induced in intact soybean [Glycine max. (L.) Merr., cv Woodworth] plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in 12 to 16 days. Acetylene reduction activity returned to normal, and both total soluble protein and leghemoglobin were resynthesized while protease activity against azocasein decreased to the level of control nodules. The nodule population that had turned green after 8 days in the dark exhibited a progressive increase in red color starting nearest the exterior of the nodule, and after 16 days of recovery nodules were indistinguishable from control nodules maintained under a normal photoperiod. PMID- 16662837 TI - Changes in the Electrophoretic Patterns of the Soluble Proteins of Winter Wheat and Rye following Cold Acclimation and Desiccation Stress. AB - The degrees of freezing tolerance acquired by winter wheat (Triticum aestivium L.) and rye (Secale cereale L. cv Puma) were similar following a 4-week cold conditioning and a 24-hour desiccation stress. Soluble proteins were extracted from shoots of cold-conditioned or desiccation-stressed seedlings and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Quantitative changes in the electrophoretic patterns of the soluble proteins of the different cultivars grown in different environments were detected, but the changes were not equivalent following cold conditioning and desiccation stress. The abundance of two polypeptide bands showed a significant increase correlated to the degree of freezing tolerance and, hence, the polypeptides in these bands may play a role in the development of freezing tolerance. PMID- 16662838 TI - Relationship between the Kinetic Properties and the Small Subunit Composition of Nicotiana Ribulose-1, 5-bisphosphate Carboxylase. AB - Genetic variability in the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in several Nicotiana species has been characterized by isoelectric focusing patterns. This heritable variation provides an opportunity to examine the functional role of each of these subunits. In this study, specifically designed RuBPCase enzymes composed of identical large subunits but different small subunits were constructed in vivo by interspecific hybridization between the species N. sylvestris, N. tabacum, N. glauca, N. glutinosa, N. plumbaginifolia, and N. tomentosiformis. Small subunit polypeptides were combined to form a sequence of one, two, three, and four polypeptides with the large subunit of N. sylvestris. Kinetic properties of these hybrid enzymes were compared. No differences in the specific activity of either carboxylation or oxygenation nor in K(m) values for ribulose 1,5-bisphosphate, CO(2), or O(2) were detected among the RuBPCase enzymes from the various interspecific hybrids. Likewise, the ratio of carboxylation to oxygenation was constant. PMID- 16662839 TI - Structure of the Thylakoids and Envelope Membranes of the Cyanelles of Cyanophora paradoxa. AB - The cyanelles of Cyanophora paradoxa Korsch. are photosynthetically active obligate endosymbionts in which phycobiliproteins serve as the major accessory pigments. Freeze-fracture electron micrographs of thylakoids in isolated cyanelles reveal long parallel rows of particles covering most of the E-face, while a more random particle arrangement is evident in some areas. The center-to center spacing of particles within these rows is about 10 nanometers. Their mean diameter was measured at 9.4 nanometers. The particles on the P-face have a mean diameter of 7.2 nanometers. Thylakoids that retained nearly the full complement of phycobiliproteins (determined spectrophotometrically and by gel electrophoresis) were isolated from the cyanelles. In thin sections of these preparations, rows of disc-shaped phycobilisomes are evident on the surface of the thylakoids. The spacing of the rows of phycobilisomes corresponds to that of the rows of E-face particles (approximately 45 nanometers, center to center). The periodicity of the disc-shaped phycobilisomes within a row is 10 nanometers suggesting a one-to-one association between phycobilisomes and E-face particles.In addition, visualization of the protoplasmic surface (PS) of isolated thylakoids by freeze-etch electron microscopy shows that rows of disc-shaped phycobilisomes are aligned directly above rows of particles exhibiting two subunits, presumably the P-surface projections of the 10-nanometer intramembrane particles. These observations, together with earlier studies indicating that the 10-nanometer E-face particles probably represent photosystem II (PSII) complexes, suggest that phycobilisomes are positioned on the thylakoid surface in direct contact with PSII centers within the thylakoid membrane.The inner envelope membrane of the cyanelles, observed in freeze-fracture replicas, resembles cyanobacterial plasma membranes and is dissimilar to the chloroplast envelope membranes of red or green algae. The envelope of isolated cyanelles exhibits two additional layers: (a) a 5- to 7-nanometer-thick layer that lies adjacent to the inner membrane and which seems to correspond to the peptidoglycan layer of cyanobacteria; and (b) a layer external to the purported peptidoglycan layer that exhibits fracture faces similar to those of the lipopolysaccharide layer of gram negative bacteria. Our findings indicate that the supramolecular architecture of cyanelles differs only slightly from free-living cyanobacteria to which they are presumably related. PMID- 16662840 TI - Cytochemical and cytofluorometric evidence for guard cell photosystems. AB - Evidence for photosynthetic linear electron transport in guard cells was obtained with two sensitive methods of high spacial resolution. Light-dependent diaminobenzidine oxidation (an indicator of PSI) and DCMU-sensitive, light dependent thiocarbamyl nitroblue tetrazolium reduction (an indicator of PSII) were observed in guard cell plastids of Hordeum vulgare L. cv Himalaya using electron microscopic cytochemical procedures. DCMU-sensitive Chl a fluorescence induction (an indicator of PSII) was detected in individual guard cell pairs of Vicia faba L. cv Longpod using an ultramicrofluorometer. At least for these species, we conclude these results are proof for the presence of PSII in guard cell chloroplasts, which until now has been somewhat controversial. PMID- 16662841 TI - Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group. AB - Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C(3), C(4) or C(3)-C(4) intermediate were undertaken to examine features associated with C(3) and C(4) photosynthesis. The C(3) species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C(3)-C(4) species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C(3) species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C(3)-C(4) species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C(4) species Panicum prionitis Griseb. and the C(3)-C(4) species, but in C(3) species the organelles were similar in size or were smaller in the bundle sheath cells. The C(3)-C(4) species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C(3)-C(4) species. PMID- 16662842 TI - Abscisic Acid accumulation in cotton leaves in response to dehydration at high pressure. AB - Pressure-volume techniques were utilized to examine the control of abscisic acid (ABA) accumulation in dehydrated cotton (Gossypium hirsutum L. cv Tamcot SP 37) leaves. Leaves were rapidly dehydrated in a pressure chamber to a balance pressure coincident with the loss of cellular turgor, and then the pressure was either maintained at that level or released. Rapid accumulation of ABA began within two hours after the balance pressure was achieved, whether or not the high pressure potential of the cells was maintained by the externally imposed pressure. The results show that loss of pressure per se does not trigger ABA accumulation in dehydrated leaves. Rather, the stimulus may be related to cellular shrinkage and relaxation of the elastic wall. PMID- 16662843 TI - Gas Chromatography-Mass Spectroscopy Identification of 1-Aminocyclopropane-1 carboxylic Acid in Compressionwood Vascular Cambium of Pinus contorta Dougl. AB - Following cation and anion exchange chromatography, 1-aminocyclopropane-1 carboxylic acid (ACC) was converted to the 2,4-dinitrophenyl derivative and then purified by high-performance liquid chromatography (HPLC). After three HPLC steps, endogenous ACC was identified by GCMS in the vascular cambium on the lower side of Pinus contorta Dougl. ssp. latifolia branches in association with compressionwood differentiation, but ACC was not detected in the opposite wood cambial region on the upper sides of the same branches.The possibility that ACC and ethylene have physiological roles in cambial activity and compressionwood tracheid differentiation is discussed. PMID- 16662844 TI - Enclosure of mitochondria by chloroplasts. AB - In Panicum species of the Laxa group, some of which have characteristics intermediate to C(3) and C(4) photosynthesis species, some mitochondria in leaf bundle sheath cells are surrounded by chloroplasts when viewed in profile. Serial sectioning of leaves of one Laxa species, Panicum schenckii Hack, shows that these mitochondria are enclosed by chloroplasts. Complete enclosure rather than invagination also is indicated by absence of two concentric chloroplast membranes surrounding the mitochondrial profiles. PMID- 16662845 TI - On the Ratio of Photosynthetic Reaction Centers RC2/RC1 in Chlorella. AB - Chlorella pyrenoidosa (Emerson strain) was grown under high and under low irradiance. Measurements of the O(2) flash yield and the P700 absorption change gave estimates for the ratio of photoreaction centers RC2/RC1 at 1.2 to 1.5. A background light 1 did not increase flash yield. PMID- 16662847 TI - Light and stomatal metabolism : I. Possible involvement of light modulation of enzymes in stomatal movement. AB - New evidence is provided regarding the direct effect of light on stomatal opening in the epidermis of the pea (Pisum sativum L. var Little Marvel) leaf. Light modulates the activity of a number of key enzymes involved in stomatal metabolism. When isolated epidermal strips are illuminated, phosphoenolpyruvate carboxylase, NADP-malate dehydrogenase, and NADP-isocitrate dehydrogenase are activated; and aspartate aminotransferase is inactivated. Sulfhydryl compounds, dithiothreitol and glutathione, enhance stomatal opening in epidermal strips both in light or darkness while the sulfhydryl reagent N-ethylmaleimide inhibits, indicating the possible involvement of sulfhydryl groups in stomatal movements. Further, light treatment increases measureable thiol levels in the epidermis about 3-fold. These results suggest that light modulation of enzymes in the epidermis may play a significant role in the mechanism of stomatal movement. PMID- 16662846 TI - Comparison of the glyoxysomes and the glyoxysomal enzymes in maize lines with high or low oil content. AB - The developmental profile of the glyoxysomes and their component enzymes catalase, malate synthase, and isocitrate lyase were compared in the scutellum of two maize (Zea mays) lines, Illinois High Oil (IHO, approximately 20% lipid content) and Illinois Low Oil (ILO, less than 0.5% lipid content). The microbodies participate in the catabolism of the seed lipids and are responsible for leading the catabolic products (acetyl-Coenzyme A) into gluconeogenesis. The aim of this study was to determine whether changes in lipid content of the seed resulted in changes in the levels of the glyoxysomal enzymes. Enzyme activity measurements, immunological measurements (in the case of catalase), cell fractionation studies, and electron microscopic observations indicated that the IHO and ILO lines contain similar populations of glyoxysomes and exhibit similar catalase and malate synthase specific activities, despite the significant difference (40-fold) in their lipid content. Only the specific activity of isocitrate lyase was higher (2-fold higher) in the IHO seeds as compared to the ILO. PMID- 16662848 TI - Light and Stomatal Metabolism : II. Effects of Sulfite and Arsenite on Stomatal Opening and Light Modulation of Enzymes in Epidermis. AB - The effect of sulfite and arsenite on stomatal opening and light modulation of enzymes was examined in isolated epidermal strips of Pisum sativum L. var Little Marvel leaves. Sulfite or arsenite at 10 micromolar rapidly inhibited the stomatal opening process in light. Light activation of phosphoenolpyruvate carboxylase and NADP-malate dehydrogenase was completely diminished when the epidermal strips were incubated for 2 hours in light with either sulfite or arsenite at 10 micromolar. The data obtained suggest that the inhibition of stomatal opening by sulfite or arsenite in light might result from the inhibition of light modulation of key enzymes in guard cells. PMID- 16662849 TI - beta-1,3-Endoglucanase from Soybean Releases Elicitor-Active Carbohydrates from Fungus Cell Walls. AB - Two enzymes from soybean (Glycine max L. Merr. cv Harosoy 63) cotyledons released elicitor-active carbohydrates from cell walls of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea. They were identified as isoenzymes of beta-1,3-endoglucanase (EC 3.2.1.39) with isoelectric points of pH 8.7 and 10.5. The pI 10.5 enzyme was extracted in the greatest amount and was isolated as a homogeneous protein of about 33,000 daltons as determined by gel filtration and sodium dodecyl sulfategel electrophoresis. The purified enzymes hydrolyzed several beta-1,3-glucans in a strictly random manner, but degraded neither beta 1,6- nor beta-1,4-glucans. PMID- 16662850 TI - Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources. AB - Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean beta-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous beta-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase. PMID- 16662851 TI - Nitrogen Turnover and Assimilation during Regrowth in Trifolium subterraneum L. and Bromus mollis L. AB - Subterranean clover (Trifolium subterraneum L. cv Woogenellup) and soft chess grass (Bromus mollis L. cv Blando) were grown in monocultures with (15)NH(4)Cl added to the soil to study nitrogen movement during regrowth following shoot removal. Four clipping treatments were imposed. Essentially all available (15)N was assimilated from the soil prior to the first shoot harvest. Measurements of total reduced nitrogen and (15)N contained within that nitrogen fraction in roots, crowns, and shoots at each harvest showed large, significant (P Br(-) > NO(3) ( ) > SO(4) (2-), iminodiacetate, benzene sulfonate. Anion stimulation of H(+) pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl(-) by a H(+) pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K(+) in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. DeltapH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H(+) -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H(+) pumps at the plasma membrane and tonoplast of higher plant cells. PMID- 16662876 TI - Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution. AB - The effect of nitrogen form (NH(4)-N, NH(4)-N + NO(3) (-), NO(3) (-)) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO(3) (-) increases, and NH(4) (+) and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO(3) (-), NH(4)-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH(-) and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH(-) or by the synthesized organic anions rather than by nitrate itself. Addition of HCO(3) (-) to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO(3) ( ), like OH(-), increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO(3) (-), NH(4) N, and amide-N. PMID- 16662877 TI - Effects of aspartate and other compounds on glyphosate uptake and growth inhibition in cultured carrot cells. AB - The strong correlation between glyphosate uptake and growth inhibition of cultured carrot (Daucus carota L. cv Danvers) cells incubated in the presence of aspartate suggests that aspartate reverses glyphosate inhibition of growth primarily by reducing intracellular glyphosate concentration. Other compounds which reverse glyphosate inhibition of cell growth gave a range of effects on glyphosate uptake: succinate, alpha-ketoglutarate, glutamate, pyruvate, and malate at 10 millimolar and phenylalanine at 2 millimolar reduced uptake by 0, 8, 11, 16, 27, and 34%, respectively. These results suggest that more than one mechanism of reversal may operate in these cells.Glyphosate and aspartate produced only minor effects on intracellular ammonia, media pH, and cell viability. This suggests that ammonia toxicity may not be an important mechanism of action of glyphosate in this system. PMID- 16662878 TI - Properties of substantially chlorophyll-free pea leaf mitochondria prepared by sucrose density gradient separation. AB - Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, alpha-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca(2+), whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O(2) uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase. PMID- 16662879 TI - Release of protein from normal and mutant tomato cell walls. AB - The nitrogen content of cell wall preparations from normal tomato (cv Ailsa Craig) fruit remained constant during ripening, whereas salt-soluble protein increased throughout this process. Tomato polygalacturonase released about twice as much protein from the preparations as salts did, with a maximum at the orange stage of development. Polygalacturonase-solubilized protein from the tomato mutant ;ripening inhibitor' (rin) was less, and that from the mutant ;Never ripe' (Nr) cell walls was more than that from normal wall preparations. Release of protein by fungal cellulase was limited, but was increased by the addition of polygalacturonase from the same source. Salt-solubilized protein contained a range of enzymic activities but these were distributed between fewer multimolecular forms than is the case for whole cell preparations. The results suggest that metabolically active protein, removable by strong salt solutions, cellulase, or polygalacturonase, remains attached to the cell walls of tomato fruit until late in ripening. The unusual amounts of protein attached to the cell walls of mutant fruit appear to be a reflection of the absence of some or all of the isoenzymes of polygalacturonase that are associated with normal ripening. PMID- 16662880 TI - Gibberellins and heterosis in maize : I. Endogenous gibberellin-like substances. AB - Under controlled environment and/or field conditions, vegetative growth (height, internode length, leaf area, shoot dry weight, grain yield) was greater in an F(1) maize hybrid than in either parental inbred. Endogenous gibberellin (GA) like substances in apical meristem cylinders were also higher in the hybrid than in either inbred, both on a per plant and per gram dry weight basis. There were no apparent qualitative differences in GA-like substances, however. Levels of GA like substances in all genotypes were highest prior to tassel initiation. Chromatographic comparisons of the GA-like substances and authentic standards of GA native to maize on gradient-eluted SiO(2) partition and reverse-phase C(18) high-pressure liquid chromatography columns are described. No consistent differences in abscisic acid levels of the three genotypes were observed. This correlation of heterosis for endogenous GA-like substances with heterosis for growth suggests that amounts of endogenous GA may be related to hybrid vigor in maize. PMID- 16662881 TI - Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20). AB - Two maize inbreds, CM7 and CM49, and CM7 x CM49, their F(1) hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A(3) (GA(3)), and in their ability to metabolize GA(20), a native GA of maize. The leaf sheath elongation response to GA(3) was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA(3), whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA(3), but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA(3), but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA(3), but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA(3) were not at the expense of the root system. Rapid metabolism of [2,3-(3)H]GA(20) occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest growing inbred. Thus, rate of GA(20) metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize. PMID- 16662882 TI - Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation. AB - Detached roots and nodules of the N(2)-fixing species, Albus glutinosa (European black alder), actively assimilate CO(2). The maximum rates of dark CO(2) fixation observed for detached nodules and roots were 15 and 3 micromoles CO(2) fixed per gram dry weight per hour, respectively. The net incorporation of CO(2) in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO(2) fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO(2) fixation and N(2) fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total (14)C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N(2)-fixing nodules and roots of alder. PMID- 16662883 TI - Phosphoglycerate dehydrogenase from soybean nodules : partial purification and some kinetic properties. AB - Phosphoglycerate dehydrogenase (EC 1.1.1.95), an enzyme believed to be involved in the synthesis of serine, an intermediate in ureide biosynthesis, has been purified about 200-fold from nodules of soybean (Glycine max L. Merr. cv Amsoy 71). The reaction was reversible and exhibited a strong pH dependence with optima of 9.4 and 6.1 for the forward and reverse reactions. The K(m) values for the forward reaction were 0.25 millimolar for NAD(+) and 0.29 millimolar for d-3 phosphoglycerate at pH 9.4, while those for the reverse reaction were 12 mum for NADH and 0.15 millimolar for 3-phosphohydroxypyruvate at pH 7.5. NADPH functioned as an alternate reductant with a K(m) of 0.15 millimolar. Product inhibition for the reverse reaction was competitive for NAD(+) with respect to NADH and noncompetitive for phosphoglycerate with respect to phosphohydroxypyruvate. Phosphoglycerate dehydrogenase activity was dependent on inorganic ions and was not inhibited by serine. PMID- 16662884 TI - Partial Purification and Properties of an Alkaline alpha-Galactosidase from Mature Leaves of Cucurbita pepo. AB - A fourth molecular from of alpha-galactosidase, designated L(IV), an alkaline alpha-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165 fold. It was active over a narrow pH range with optimal hydrolysis of p nitrophenyl-alpha-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K(m) determinations in McIlvaine buffer (200 millimolar Na(2)-phosphate, 100 millimolar citric acid, pH 7.5) for p nitrophenyl-alpha-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L(IV) was partially inhibited by Ca(2+), Mg(2+), and Mn(2+), more so by Ni(2+), Zn(2+), and Co(2+), and highly so by Cu(2+), Ag(2+), Hg(2+) and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-alpha-d-galactoside or by myo inositol, but alpha-d-galactose was a strong inhibitor. As observed for most other forms of alpha-galactosidase, L(IV) only catalyzed the hydrolysis of glycosides possessing the alpha-d-galactose configuration at C(1), C(2), and C(4), and did not hydrolyze p-nitrophenyl-alpha-d-fucoside (alpha-d-galactose substituted at C(6)). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-alpha-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L(IV) in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L(IV)in vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L(IV) activity through coordination with some toxic cation introduced as a buffer contaminant. PMID- 16662885 TI - Effects of Age, Water Stress, and 1-Aminocyclopropane-1-carboxylic Acid on Leaflet Movement in Albizzia julibrissin. AB - The movement patterns of Albizzia julibrissin leaflets transferred from light to darkness differ for leaflets of different age: the older the leaflet, the slower and less completely it closes. Water stress, which enhances ethylene synthesis in other plants, and 1-aminocyclopropane-1-carboxylic acid (ACC), precursor of ethylene, both mimic the effect of aging by reducing the rate and extent of dark induced closure. Brief far-red compared to red irradiation before darkness does not appear to alter the closure of young leaflets, but far-red preirradiation retards the closure of middle-aged and old leaflets floating on water, and middle aged leaflets treated with ACC. A change in some membrane property and/or cell wall lignification are suggested as possible explanations for the alteration of leaflet movement. PMID- 16662886 TI - A New Mechanism for Adaptation to Changes in Light Intensity and Quality in the Red Alga Porphyra perforata: III. Fluorescence Transients in the Presence of 3 (3,4-Dichlorophenyl)-1,1-dimethylurea. AB - In the red alga Porphyra perforata, the level of chlorophyll fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased during illumination of the thallus. The results showed that: (a) this decay was related to the photooxidative activity of photosystem I; (b) Q, the primary electron acceptor of photosystem II, became oxidized during the decay of the fluorescence; (c) reagents which inhibit the back reaction of photosystem II inhibited the decay.From these results, it is suggested that, when conditions in the chloroplasts of this red alga become too oxidative, excess light energy can be converted to heat as a result of an accelerated back reaction of photosystem II. This may be one of the mechanisms by which this alga can cope with the high salt and high light conditions that can occur in its natural habitat. PMID- 16662887 TI - Variable Photosynthetic Metabolism in Leaves and Stems of Cissus quadrangularis L. AB - By measuring titratable acidity, gas exchange parameters, mesophyll succulence, and (13)C/(12)C ratios, we have shown that Cissus quadrangularis L. has C(3)-like leaves and stems with Crassulacean acid metabolism (CAM). In addition, the nonsucculent leaves show the diurnal fluctuations in organic acids termed recycling despite the fact that all CO(2) uptake and stomatal opening occurs during the day. Young succulent stems have more C(3) photosynthesis than older stems, but both have characteristics of CAM. The genus Cissus will be a fruitful group to study the physiology, ecology, and evolution of C(3) and CAM since species occur that exhibit characteristics of both photosynthetic pathways. PMID- 16662888 TI - Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach. AB - (14)CO(2) photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C(3) plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg(2+) were present in the assays, their influence upon CO(2) assimilation was also examined. Free Mg(2+) was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO(2) assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).PGA-supported O(2) evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O(2) evolution slowed as time passed, corresponding to the effect of ADP on CO(2) assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O(2) evolution became more severe with time. This did not mirror CO(2) assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O(2) evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O(2) evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO(2) assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.The appearance of label in the medium was measured when [U-(14)C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg(2+) complexes. The rate of back exchange was stimulated by the presence of Mg(2+). This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg(2+) complexes. A portion of the CO(2) assimilation and O(2) evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations. PMID- 16662889 TI - Iron nutrition-mediated chloroplast development. AB - Membrane development in chloroplasts was explored by resupplying iron to iron deficient sugar beet (Beta vulgaris L. cv F58-554H1) and monitoring changes in lamellar components during regreening. The synthesis of chlorophyll a, chlorophyll b, and Q, the first stable electron acceptor of photosystem II, exhibited a lag phase during the first 24 to 48 hours of resupply. In contrast, the per area amounts of P(700) and cytochrome f increased linearly over the first 48 hours. During the early regreening period, the Q to P(700) ratio was 2.6 and decreased to 0.7 after 96 hours of regreening. The rate of photosynthesis (net CO(2) uptake) per chlorophyll increased during the first 48 hours of resupply, then by 96 hours decreased to values typical of control plants. The results suggest that there was preferential synthesis of the measured photosystem I components during the first 24 to 48 hours, while from 48 to 96 hours there was rapid synthesis of all components. The iron nutrition-mediated chloroplast development system provides a useful experimental approach for studying biomembrane synthesis and structural-functional relations of the photosynthetic apparatus. PMID- 16662890 TI - C Tracer Evidence for Synthesis of Choline and Betaine via Phosphoryl Base Intermediates in Salinized Sugarbeet Leaves. AB - Like other chenopods, sugarbeets (Beta vulgaris L. cv Great Western D-2) accumulate glycine betaine when salinized; this may be an adaptive response to stress. The pathway of betaine synthesis in leaves of salinized (150-200 millimolar NaCl) sugarbeet plants was investigated by supplying [(14)C]formate, phosphoryl[(14)C]monomethylethanolamine ([(14)C][unk] MME) or phosphoryl[(14)C]choline ([(14)C][unk] choline) to leaf discs and following (14)C incorporation into prospective intermediates. The (14)C kinetic data were used to develop a computer model of the betaine pathway.When [(14)C]formate was fed, [unk] MME, phosphoryldimethylethanolamine ([unk] DME) and [unk] choline were the most prominent methylated products at short labeling times, after which (14)C appeared in free choline and in betaine. Phosphatidylcholine labeled more slowly than [unk] choline, choline, and betaine, and behaved as a minor end product. Very little (14)C entered the free methylethanolamines. When [(14)C][unk] MME was supplied, a small amount was hydrolyzed to the free base but the major fate was conversion to [unk] DME, [unk] choline, free choline, and betaine; label also accumulated slowly in phosphatidylcholine. Label from supplied [(14)C][unk] choline entered choline and betaine rapidly, while phosphatidylcholine labeled only slowly and to a small extent.These results are consistent with the pathway [unk] MME -->[unk] DME --> [unk] choline --> choline --> --> betaine, with a minor side branch leading from [unk] choline into phosphatidylcholine. This contrasts markedly (a) with the pathway of stress-induced choline and betaine synthesis in barley, in which phosphatidylcholine apparently acts as an intermediate (Hitz, Rhodes, Hanson 1981, Plant Physiol 68: 814-822); (b) with choline biogenesis in mammalian liver and microorganisms. Computer modeling of the experimental data pointed strongly to regulation at the [unk] choline --> choline step, and also indicated that the rate of [unk] choline synthesis is subject to feedback inhibition by [unk] choline. PMID- 16662891 TI - Translocation of [C]sucrose in wheat. AB - Flag leaves of wheat plants (Triticum aestivum L. em. Thell. cv ;Duke') were supplied with (14)C(glucosyl)sucrose. Translocated [(14)C]sucrose was isolated, then hydrolyzed. Label appeared in both the hexose moieties indicating that some randomization of label had occurred. However, near the radioactive front essentially all of the (14)C was in the glucose moiety, suggesting that randomization occurred after unloading, supporting the conclusion that sucrose was taken up intact by phloem and translocated unaltered. PMID- 16662892 TI - Intracellular distribution of free sugars in quiescent cottonseed. AB - Quiescent cottonseeds (Gossypium hirsutum L.) were fractionated non-aqueously to protein bodies and nonparticulate cytoplasm for analyses of sugar contents. Nonparticulate cytoplasm was more than 3-fold richer in total free sugars than were protein bodies, but based on the content of cytoplasm in each fraction, sugars were distributed almost equally in them. Thin-layer chromatography and high-pressure liquid chromatography revealed five sugars: sucrose, raffinose, tetrasaccharides I and II, and stachyose. Raffinose and stachyose were located mainly, and tetrasaccharide I totally, in nonparticulate cytoplasm compared to protein bodies; sucrose and tetrasaccharide II were distributed about equally in each fraction. PMID- 16662893 TI - Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide sensitive ATPase from pea cotyledon mitochondria. AB - The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F(0)F(1) preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis. PMID- 16662894 TI - Inhibition of coral and algal photosynthesis by ca-antagonist phenothiazine drugs. AB - The effects of various calcium ion antagonists and ion transport inhibitors on photosynthetic O(2) evolution of corals, isolated zooxanthellae, sea anemone tentacles, and Chlorococcum oleofaciens were measured. Only the phenothiazine drugs were effective at inhibiting photosynthesis. Trifluoperazine, a calcium ion antagonist drug, inhibited at low concentrations, with 10(-4) molar and 8 x 10( 6) molar completely abolishing photosynthesis in the intact corals and isolated zooxanthellae, respectively. Net photosynthetic O(2) evolution of C. oleofaciens was eliminated by concentrations of trifluoperazine as low as 2.8 x 10(-5) molar. PMID- 16662895 TI - Comparison of ethylenediaminetetraacetate-enhanced exudation from detached and translocation from attached bean leaves. AB - A technique for collection of phloem exudate from detached leaves using 20 millimolar EDTA (pH 7.0) has previously been developed (King, Zeevaart 1974 Plant Physiol 53: 96-103). It was the aim of the present study to determine the efficiency of this technique in relation to undisturbed export from attached leaves. Paired primary leaves of bean seedlings (Phaseolus vulgaris L. cv Montcalm) were used to minimize variations in plant material. Attached leaves, exposed to (14)CO(2) for 10 minutes with subsequent excision of one of the leaves and collection of the exudate over a 12-hour period, showed a 25% export of total assimilated (14)C from the attached versus 15% of total assimilated (14)C in the form of exudation from the detached ones. Leaf excision changed the labeling pattern within the leaf, increasing% total leaf (14)C-activity in the ethanolic fraction, while decreasing activity in the starch fraction, as compared to attached leaves. This was presumably caused by a lack of translocation from the detached leaves. Excision did not affect dark respiration. However, measurements of total nonstructural carbohydrates in leaf starch and neutral fractions indicated no significant differences between attached and leaves detached in EDTA. Thus, in terms of actual carbon export, and accompanying distribution of nonexported carbohydrate within the leaf, EDTA-enhanced exudation compares favorably with translocation from attached leaves. PMID- 16662896 TI - Regulation of Cell Shape in Euglena gracilis: I. Involvement of the Biological Clock, Respiration, Photosynthesis, and Cytoskeleton. AB - The alga Euglena gracilis Z. changes its shape two times per day when grown under the synchronizing effect of a daily light-dark cycle. At the beginning of the light period when photosynthetic capacity is low, the population of cells is largely spherical in shape. The mean cell length of the population increases to a maximum in the middle of the light period when photosynthetic capacity is greatest, and then decreases for the remainder of the 24-hour period. The population becomes spherical by the end of the 24-hour period when the cycle reinitiates. These changes are also observed under constant dim light conditions (up to 72 hours) and are therefore controlled by the biological clock and represent a circadian rhythm in cell shape. In constant dim light, the cell division rhythm is either arrested or slowed considerably, while the cell shape rhythm continues.The involvement of respiratory and photosynthetic pathways in the cell shape changes was investigated with energy pathway inhibitors. Antimycin A and NaN(3) both inhibited the round to long and long to round shape changes, indicating that the respiratory pathways are involved. DCMU and atrazine inhibited the round to long shape change but did not affect the long to round transition, indicating that light-induced electron flow is necessary only for the round to long shape change.The influence of the cell shape changes on the photosynthetic reactions was investigated by altering cell shape with the cytoskeletal inhibitors cytochalasin and colchicine. Both inhibitors blocked the round to long and long to round shape changes. Cytochalasin B was found to have minimal cytotoxic effects on the photosynthetic reactions, but colchicine significantly inhibited light-induced electron flow and the in vivo expression of the photosynthetic rhythm. PMID- 16662897 TI - Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids. AB - Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR(-) 108, NR(-) 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N(2)-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR(-) 108 and NR(-) 303 contained neither nitrate reductase nor nitrite reductase activities.Addition of 20 millimolar KNO(3) to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO(3) treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.The addition of either KNO(3) or KNO(2) to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules. PMID- 16662898 TI - Alcohol dehydrogenase and an inactivator from rice seedlings. AB - Alcohol dehydrogenase (ADH) was measured in the various organs of rice seedlings (Oryza sativa) growing in air. In extracts from ungerminated seeds, the ADH is stable, but in extracts from seedlings more than 2 days old the enzyme initially present loses activity in a time- and temperature-dependent fashion, due to the presence of an inactivating component which increases with age in roots and shoots. The inactivation can be prevented completely by dithiothreitol, and when this is included in the extraction medium the apparent loss of total ADH in roots and shoots with age is not observed. In seedlings grown in N(2), ADH levels in coleoptile extracts are higher than those in air, the enzyme is stable, and no inactivator can be detected. When seedlings grown for 5 days in air were transferred to N(2) for 3 days, ADH levels increased and there was a decline in inactivator activity. Transfer back to air after 1 day in N(2) led to loss of the accumulated ADH and increase in inactivator. These reciprocal changes and the fact that the inactivator is absent from coleoptiles of seedlings grown in N(2) appear to suggest a regulatory role for the inactivator in vivo. However, it is clear that high levels of inactivator and ADH can exist in cells of seedlings grown in air for long periods without loss of enzyme activity, and it is argued that they must normally be separately compartmented. PMID- 16662899 TI - Alcohol dehydrogenase inactivator from rice seedlings : properties and intracellular location. AB - The alcohol dehydrogenase (ADH) inactivator from aerobically grown rice (Oryza sativa) coleoptiles was shown to be associated with membranes which were recovered in sucrose gradients at peak density 1.13 grams per cubic centimeter. When Mg(2+) was included in the gradient, the inactivator was recovered at peak density 1.16 grams per cubic centimeter coinciding with the marker enzyme for endoplasmic reticulum, antimycin A-insensitive NADH cytochrome c reductase. ADH was recovered exclusively in cytosol fractions. The inactivator attacks ADH from several plant sources and from yeast. There was no evidence for proteolysis when pure yeast ADH was inactivated by the inactivator, but there was a loss of SH groups from ADH during inactivation which was restored after incubation with dithiothreitol under denaturing conditions. The inactivator did not attack other SH enzymes tested but did result in loss of SH groups from glutathione and dithiothreitol which prevent ADH inactivation. When O(2) was removed from the inactivator assay medium, the inactivation as well as the loss of SH groups from yeast ADH was significantly depressed. PMID- 16662900 TI - Quantitation of Chill-Induced Release of a Tubulin-Like Factor and Its Prevention by Abscisic Acid in Gossypium hirsutum L. AB - The degree of tubulin polymerization in cotton (Gossypium hirsutum L. cv Acala) cotyledonary tissue was estimated by radioimmunoassay which measured the amount of a tubulin-like factor. It was assumed that the release of this tubulin-like factor indicated depolymerization of microtubules. Exposure to chilling resulted in complete release of the tubulin-like factor. Pretreatment with abscisic acid in the light almost completely prevented the chill-induced release of the tubulin like factor. Addition of colchicine during the chilling period accelerated the release of the tubulin-like factor. Pretreatment with abscisic acid greatly reduced this effect of colchicine. It is concluded that the destruction of the microtubular network is involved in the development of chilling injury in cotton. Abscisic acid apparently decreased chilling injury by stabilization of the microtubular network. PMID- 16662901 TI - Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants. AB - The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0 degrees C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold hardened leaves tolerated extracellular freezing between -3.5 degrees and -8.5 degrees C. However, if ice initiation in the supercooled leaves occurred at any temperature below -2.6 degrees C, the leaves were lethally injured.To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above -1 degrees C. They had threshold temperatures of -0.7 degrees and 0.8 degrees C, respectively, for freezing in distilled H(2)O. Although freezing could be initiated in the Solanum leaves above -1 degrees C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between -2.5 degrees and -6.5 degrees using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant. PMID- 16662902 TI - Manipulation by tridemorph, a systemic fungicide, of the sterol composition of maize leaves and roots. AB - The roots of maize (Zea mays L. var LG11) seedlings were watered with a solution of Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide, for 3 to 4 weeks from the onset of germination. Very few big up tri, open(5)-sterols, the major sterols of the control, were detected in the treated plants, and 9beta,19-cyclopropyl sterols accumulated dramatically in both roots and leaves. big up tri, open(8)-sterols were also found when low concentrations of the drug were used. The time course of the accumulation of the new sterols has been studied in plants treated with various concentrations (1-20 milligrams per liter) of Tridemorph. We found that: (a) cycloeucalenol-obtusifoliol isomerase, an enzyme opening the cyclopropane ring of cyclopropyl sterols, was strongly inhibited by the drug; and (b) the drug diffused readily from the roots to the whole plant and reached its enzymic targets in most of the leaf cells. The data obtained offer an opportunity to evaluate the physiological and biochemical consequence of the almost complete replacement of big up tri, open(5)-sterols by cyclopropyl sterols in higher plant cells. PMID- 16662903 TI - Changes in tobacco cell membrane composition and structure caused by cercosporin. AB - Cercosporin, a toxin produced by Cercospora species, rapidly kills plant cells in the light. Previous work has shown that cercosporin treatment causes products of lipid peroxidation to be released. We have found that the unsaturated acyl chains of lipids in tobacco (Nicotiana tabacum) cell membranes are destroyed when cells are treated with cercosporin. Concomitant with this change in composition is a change in structure of the membranes as detected by two different fatty acid spin labels, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (denoted I[12,3]) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (denoted I[1,14]). Cercosporin causes the membranes to become more rigid at all temperatures tested and increases the membrane phase transformation temperature from 12.7 degrees C to 20.8 degrees C. PMID- 16662904 TI - Putrescine and Acid Stress : Induction of Arginine Decarboxylase Activity and Putrescine Accumulation by Low pH. AB - Incubation of peeled oat Avena sativa L. var Victory leaf segments on media of pH 5.0 or below leads to a rapid and massive increase in the titer of putrescine while incubation at pH values above 5.0 causes little or no change. The low pH effect is independent of the buffer system employed. Putrescine levels rise within 3 hours and reach their peak 8 to 9 hours after acidification. At this time, putrescine titer is eight times greater at pH 3.5 than at 6.0. None of the other polyamines shows a response to changes in external pH. The increase in putrescine is blocked by the addition of cycloheximide or dl-alpha difluoromethylarginine, a specific inhibitor of the putrescine biosynthetic enzyme, arginine decarboxylase. In one experiment, arginine decarboxylase activity was 110% greater at pH 4.0 than at 6.0 after a 4-hour incubation, although the average increase over many experiments was 47%. The activity of the other possible putrescine biosynthetic enzyme, ornithine decarboxylase, falls throughout the incubation period and is virtually equal at pH 4.0 and 6.0. PMID- 16662905 TI - Mechanism of Glyoxylate Decarboxylation in the Glycolate Pathway in Euglena gracilis Z : Participation of Mn-Dependent NADPH Oxidase in Chloroplasts. AB - The mechanism for decarboxylating the carboxyl carbon of glycolate was studied in Euglena gracilis Z, which liberates more than 70% of the carboxyl carbon as CO(2) during glycolate metabolism.In the isolated mitochondria of E. gracilis, glycolate was oxidized to glyoxylate, 25% of which was aminated to glycine, with the remaining unchanged in the presence of glutamate; in the absence of the amino donor, glyoxylate was not changed. Irrespective of the presence or absence of the amino donor, no decarboxylation took place in Euglena mitochondria. In Euglena chloroplasts glyoxylate was actively decarboxylated by the action of hydrogen peroxide generated by the reaction of a Mn(2+)-dependent NADPH oxidase. This enzyme was purified 120-fold over the crude extract and some of its properties studied. Oxidation of one molecule of NADPH was accompanied by the formation of one molecule of H(2)O(2); the NADPH oxidation was not attributable to the action of l-ascorbate peroxidase, the sole peroxidase present in E. gracilis. Specific participation of chloroplasts and mitochondria in glycolate metabolism is discussed. PMID- 16662906 TI - Pigment analysis of chloroplast pigment-protein complexes in wheat. AB - Pigment-protein complexes separated from wheat (Triticum aestivum L. selection ND96-25 by two gel electrophoresis techniques were analyzed by high-performance liquid chromatography for chlorophylls and carotenoids. The two techniques are compared, and pigment analyses are given for the major reaction centers and light harvesting complexes. Reaction centers contain mostly chlorophyll a, carotene, and lutein, whereas light-harvesting complexes contain chlorophyll a, chlorophyll b, lutein, and neoxanthin. The amounts of violaxanthin are variable. PMID- 16662907 TI - Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves. AB - The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine gamma-synthase are confined to the chloroplast. Cystathionine beta lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm. PMID- 16662908 TI - DNA Content of Beta vulgaris Chloroplasts during Leaf Cell Expansion. AB - During the growth of beet leaves from 2 to 3 to 25 to 30 centimeters, the leaf cells increase in size, the average number of chloroplasts per cell increases from 11 to 65 and the amount of chloroplast DNA per cell increases from 1100 to 1900 plastome copies. The average number of copies of the plastome per chloroplast decreases from 104 in 2 to 3-centimeter leaves to 29 in 25 to 30 centimeter leaves during a period when the chloroplasts undergo two to three rounds of division and increase diameter from 1.5 to 4.9 micrometers. This result is at variance with previously published studies of beet chloroplasts but agrees with the conclusions reached in more recent studies of pea and spinach and wheat leaf cell expansion. PMID- 16662909 TI - Stomatal sensitivity to carbon dioxide and humidity: a comparison of two c(3) and two c(4) grass species. AB - The sensitivity of stomatal conductance to changes of CO(2) concentration and leaf-air vapor pressure difference (VPD) was compared between two C(3) and two C(4) grass species. There was no evidence that stomata of the C(4) species were more sensitive to CO(2) than stomata of the C(3) species. The sensitivity of stomatal conductance to CO(2) change was linearly proportional to the magnitude of stomatal conductance, as determined by the VPD, the same slope fitting the data for all four species. Similarly, the sensitivity of stomatal conductance to VPD was linearly proportional to the magnitude of stomatal conductance. At small VPD, the ratio of intercellular to ambient CO(2) concentration, C(i)/C(a), was similar in all species (0.8-0.9) but declined with increasing VPD, so that, at large VPD, C(i)/C(a) was 0.7 and 0.5 (approximately) in C(3) and C(4) species, respectively. Transpiration efficiency (net CO(2) assimilation rate/transpiration rate) was larger in the C(4) species than in the C(3) species at current atmospheric CO(2) concentrations, but the relative increase due to high CO(2) was larger in the C(3) than in the C(4) species. PMID- 16662910 TI - Proteolytic Activity in Soybean Root Nodules : Activity in Host Cell Cytosol and Bacteroids throughout Physiological Development and Senescence. AB - Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis. PMID- 16662911 TI - Mechanisms of citrate transport and exchange in corn mitochondria. AB - Previous work (Birnberg, Jayroe, Hanson 1982 Plant Physiol 70: 511-516) demonstrated that corn mitochondria (Zea mays L.) can accumulate citrate by a malate- and phosphate-independent proton symporter. This uptake and symport of other ions were investigated. Passive swelling experiments indicated that corn mitochondria can accumulate several other anions by proton symport, but only isocitrate is taken up nearly as effectively as citrate. At the optimal pH (4.5), active uptake of carrier-free [(14)C]citrate in 50 micromolar mersalyl is inhibited by fourteen anions, but only the I(50) (the concentration of inhibitor required to reduce uptake of carrier-free [(14)C]citrate by 50%) values of citrate (0.08 millimolar) and d-and l-isocitrate (0.5 millimolar) are less than 4 millimolar. Isocitrate is a competitive inhibitor of citrate uptake and [(14)C]isocitrate is accumulated with a K(m) similar similar to its I(50). Valinomycin reduces net active citrate accumulation at pH 7.5, consistent with the relatively low V(max) for citrate uptake. At pH 4.5, mersalyl reduces the rate of citrate uptake without changing the affinity of the carrier for citrate. Thus, the corn mitochondria have a high-affinity, mersalyl-insensitive carrier selective for citrate that also transports isocitrate.The pH optimum for oxidation of both endogenous substrates and citrate is approximately pH 6.8, but citrate oxidation is low at all pH values and is poorly coupled to ATP synthesis. Under active conditions only, at pH 7.0, malate/citrate exchange occurs with 4 millimolar malate being sufficient to remove about half the matrix citrate. Therefore, in vivo both citrate uptake by proton symport and efflux by malate/citrate exchange should occur, with the net direction of citrate movement determined by the cytoplasmic pH, and citrate and malate concentrations; in most cases, net efflux is likely to be favored. PMID- 16662912 TI - An interpretation of some whole plant water transport phenomena. AB - A treatment of water flow into and through plants to the evaporating surface of the leaves is presented. The model is driven by evaporation from the cell wall matrix of the leaves. The adsorptive and pressure components of the cell wall matric potential are analyzed and the continuity between the pressure component and the liquid tension in the xylem established. Continuity of these potential components allows linking of a root transport function, driven by the tension in the xylem, to the leaf water potential. The root component of the overall model allows for the solvent-solute interactions characteristic of a membrane-bound system and discussion of the interactions of environmental variables such as root temperature and soil water potentials. A partition function is developed from data in the literature which describes how water absorbed by the plant might be divided between transpiration and leaf growth over a range of leaf water potentials.Relationships between the overall system conductance and the conductance coefficients of the various plant parts (roots, xylem, leaf matrix) are established and the influence of each of these discussed.The whole plant flow model coupled to the partition function is used to simulate several possible relationships between leaf water potential and transpiration rate. The effects of changing some of the partition function coefficients, as well as the root medium water potential on these simulations is illustrated.In addition to the general usefulness of the model and its ability to describe a wide range of situations, we conclude that the relationships used, dealing with bulk fluid flow, diffusion, and solute transport, are adequate to describe the system and that analogically based theoretical systems, such as the Ohm's law analogy, probably ought to be abandoned for this purpose. PMID- 16662913 TI - Role of sucrose-phosphate synthase in partitioning of carbon in leaves. AB - Variations in leaf starch accumulation were observed among four species (wheat [Triticum aestivum L.], soybean [Glycine max L. Merr.], tobacco [Nicotiana tabacum L.], and red beet [Beta vulgaris L.]), nine peanut (Arachis hypogea L.) cultivars, and two specific peanut genotypes grown under different nutritional regimes. Among the genotypes tested, the activity of sucrose phosphate synthase was correlated negatively with leaf sucrose content in seven of the nine peanut cultivars as well as the two peanut cultivars grown with different mineral nutrition. The peanut cultivars differed in the effect of 10 millimolar sucrose on sucrose phosphate synthase activity in leaf extracts. Enzyme activity in crude leaf extracts was inhibited by sucrose (10-42%) in four of the cultivars tested whereas five cultivars were not. Overall, the results suggest that a correlation exists between the activity of sucrose phosphate synthase and starch/sucrose levels in leaves. PMID- 16662914 TI - Changes in the Activities of Pyrrolooxygenases during the Germination of Wheat Grains. AB - Porphobilinogen oxygenase, skatole pyrrolooxygenase, and tryptophan pyrrolooxygenase were found in the different parts of germinating wheat (Triticum aestivum) grain seedlings. In the embryos of grains germinated for 24 hours, the activities of PBG oxygenase and skatole pyrrolooxygenase were inhibited by a labile inhibitor. Tryptophan pyrrolooxygenase activity was not inhibited. Embryos of grains germinated for 48 hours showed higher activities for the three enzymes. The latter were also present in the radicles and coleoptiles of 96-hour germinated wheat grains. A DEAE-cellulose analysis of a crude enzymic preparation from embryos allowed the separation of two molecular forms of the three pyrrolooxygenases. The more cationic forms of porphobilinogen oxygenase and skatole pyrrolooxygenase were associated with the inhibitor. This form of porphobilinogen oxygenase had allosteric kinetics while the more anionic form had Michaelis kinetics. Both forms of skatole pyrrolooxygenase had Michaelis kinetics. The activity of tryptophan pyrrolooxygenase was highest in seedling roots and was found to be inhibited in seedling young leaves. This enzyme oxidized tryptophanyl dipeptides, as well as a nonapeptide, to N-formylkynurenine containing peptides. The pyrrolooxygenase also oxidized the tryptophanyl residues of lysozyme, chymotrypsin, and trypsin. PMID- 16662915 TI - Promotion of fern rhizoid elongation by metal ions and the function of the spore coat as an ion reservoir. AB - The perine, or outer coat, of spores of the fern Onoclea sensibilis L. may be chemically removed by a brief treatment with dilute NaClO. Treated spores germinate normally on glass-redistilled H(2)O, but elongation of the rhizoid which is differentiated during germination is severely limited. Rhizoid elongation in perine-free spores, however, is normal when the spores are germinated on Knop's mineral medium or on single-salt solutions of Ca(2+), Mn(2+), or Mg(2+). In intact spores which retain their perine, rhizoid elongation is normal on distilled H(2)O, and the perine serves as a source of ions which are available to the spores and can sustain rhizoid elongation, even when the external medium is deficient. Electron micrographs show that there are structural differences in the rhizoid wall between perine-free spores germinated on distilled H(2)O or on nutrient solutions, and also a difference in the number of vesicles in the apical cytoplasm. Localization of Mg(2+) and Ca(2+) in the elongating rhizoid can be visualized with chlorotetracycline fluorescence. No concentration of these ions can be detected by this technique in the small rhizoid initial cell before cell elongation begins. PMID- 16662916 TI - Xylem and Phloem transport and the functional economy of carbon and nitrogen of a legume leaf. AB - Exchanges of CO(2) and changes in content of C and N were studied over the life of a leaf of Lupinus albus L. These data were combined with measurements of C:N weight ratios of xylem (upper stem tracheal) and phloem (petiole) sap to determine net fluxes of C and N between leaf and plant. Phase 1 of leaf development (first 11 days, leaf to one-third area) showed increasing net import of C and N, with phloem contributing 61% of the imported C and 18% of the N. (14)C feeding studies suggested the potential for simultaneous import and export through phloem over the period 9 to 12 days. Phase 2 (11-20 days, leaf attaining maximum area and net photosynthesis rate) exhibited net import through xylem and increasing export through phloem. Eighty-two% of xylem-delivered N was consumed in leaf growth, the remainder exported in phloem. Phase 3 (20-38 days) showed high but declining rates of photosynthesis, translocation, and net export of N. Phase 4 (38-66 days) exhibited substantial losses of N and declining photosynthesis and translocation of C. C:N ratio of xylem sap remained constant (2.3-2.6) during leaf life; petiole phloem sap C:N ratio varied from 25 to 135 over leaf development. The relationships between net photosynthesis and N import in xylem were: phase 1, 4.8 milligrams C per milligram N; phase 2, 24.7 milligrams C per milligram N; phase 3, 91.9 milligrams C per milligram N; and phase 4, 47.7 milligrams C per milligram N. PMID- 16662917 TI - Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf. AB - Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and gamma-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine. PMID- 16662918 TI - Hydrogenation of geranylgeraniol : two pathways exist in spinach chloroplasts. AB - The reduction of geranylgeranylpyrophosphate to phytylpyrophosphate in spinach chloroplasts is described for the first time. The reductase is localized in the chloroplast envelope. By contrast, the reduction of the geranylgeranyl moiety in Chl synthesis is catalyzed in the thylakoids (via Chl synthetase). NADPH functions as electron donor in both reactions. Chl synthetase is firmly bound to the thylakoid membranes, and very little activity is found in the stroma fraction. Chl synthetase in chloroplasts can use the pyrophosphate ester of either phytol, geranylgeraniol, or farnesol, phytylpyrophosphate being the preferred substrate. Exogenous Chlide exhibits no influence on Chl synthesis by chloroplast subfractions. PMID- 16662919 TI - Limiting Factors in Photosynthesis: IV. Iron Stress-Mediated Changes in Light Harvesting and Electron Transport Capacity and its Effects on Photosynthesis in Vivo. AB - Using iron stress to reduce the total amount of light-harvesting and electron transport components per unit leaf area, the influence of light-harvesting and electron transport capacity on photosynthesis in sugar beet (Beta vulgaris L. cv F58-554H1) leaves was explored by monitoring net CO(2) exchange rate (P) in relation to changes in the content of Chl.In most light/CO(2) environments, and especially those with high light (>/=1000 microeinsteins photosynthetically active radiation per square meter per second) and high CO(2) (>/=300 microliters CO(2) per liter air), P per area was positively correlated with changes in Chl (a + b) content (used here as an index of the total amount of light-harvesting and electron transport components). This positive correlation of P per area with Chl per area was obtained not only with Fe-deficient plants, but also over the normal range of variation in Chl contents found in healthy, Fe-sufficient plants. For example, light-saturated P per area at an ambient CO(2) concentration close to normal atmospheric levels (300 microliters CO(2) per liter air) increased by 36% with increase in Chl over the normal range, i.e. from 40 to 65 micrograms Chl per square centimeter. Iron deficiency-mediated changes in Chl content did not affect dark respiration rate or the CO(2) compensation point. The results suggest that P per area of sugar beet may be colimited by light-harvesting and electron transport capacity (per leaf area) even when CO(2) is limiting photosynthesis as occurs under field conditions. PMID- 16662920 TI - Quantitative changes in in vitro and in vivo protein synthesis in aging and rejuvenated soybean cotyledons. AB - Cotyledons of light-grown soybean (Glycine max L. var Wayne) seedlings were used as a model system to study the possibility that aging requires qualitative changes in protein synthesis. Cotyledons reached a final stage of senescence and then abscised about 22 days after imbibition. Cotyledon senescence was reversed at 20 days after germination by epicotyl removal. Thereafter, the cotyledons regained much of the chlorophyll, RNA, protein, and polyribosomes lost during aging.Total poly(A)mRNA was extracted from 4-, 12-, 20-day-old, and rejuvenated cotyledons and translated in a wheat germ system. Comparison of translation products on two-dimensional O'Farrell gels showed that many translation products increased in quantity during aging, while roughly half as many decreased. Rejuvenation returned the translation products to approximately 4-day-old levels in roughly half of those products which were diminished with age. Conversely, almost one-third of the products which had increased with age decreased with rejuvenation. None of the translation products were totally lost nor were newly synthesized products detected during aging. Therefore, aging in this system probably does not involve complete gene repression or depression. The observation that epicotyl removal causes a reversal in the levels of various proteins synthesized in vitro was corroborated by similar observations following in vivo labeling of cotyledon sections and analysis by SDS-polyacrylamide gel electrophoresis and fluorography. Densitometric scans of fluorograms revealed a gradual shift in profiles of both in vitro and in vivo translation products during aging. Rejuvenated cotyledon proteins had a profile resembling that of 4 day-old cotyledons. The overall level of [(35)S]methionine incorporation into protein in vivo declined gradually during aging but was restored to 4-day-old levels within 2 days after epicotyl removal. PMID- 16662921 TI - Isolation and characterization of infected and uninfected cells from soybean nodules : role of uninfected cells in ureide synthesis. AB - The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6 phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction. PMID- 16662922 TI - Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate. AB - The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K(m) values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH(4))(2)SO(4) fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3 butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O(2) concentration. Glycolate oxidation increased 3.6-fold when the O(2) atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O(2) concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate. PMID- 16662923 TI - Purification and Characterization of Griffonia simplicifolia Leaf Lectins. AB - Leaves from mature Griffonia simplicifolia plants were examined for the presence of leaf lectins possessing sugar binding specificities similar to the four known seed lectins (GS-I, GS-II, GS-III, GS-IV). Three (GS-I, -II, -IV) of the four known G. simplicifolia seed lectins were present in the leaves. Leaf G. simplicifolia lectins I and IV were similar to the respective seed lectins. Leaf GS-II, however, was composed of two types of subunits (M(r) = 33,000 and 19,000), whereas the seed lectin consists of only one type of subunit (M(r) 32,500). Seed and leaf GS-II lectins also had different isoelectric points. All leaf and seed lectins were similar with respect to their hemagglutination and glycoconjugate precipitation properties and all subunits contained covalently bound carbohydrate. Leaf GS-IV appeared slightly under-glycosylated compared to seed GS IV.The fate of GS-I and GS-II seed lectins in aging cotyledons was investigated. GS-I isolectins usually contain isolectin subtypes associated with each main isolectin. Upon inbibition and germination, these GS-I isolectin subtypes disappeared. Over time, GS-II lectin did not change its disc gel electrophoretic properties. PMID- 16662924 TI - Ammonia exchange and photorespiration in chlamydomonas. AB - Two hours after the addition of l-methionine-dl-sulfoximine to the cell suspension, glutamine synthetase activity was inhibited by more than 90% in air grown Chlamydomonas reinhardii. Cells continued to take up NH(3) from the medium provided that the concentration of dissolved CO(2) was high (equilibrated with 4% CO(2) in air). This NH(3) uptake, about 30% of the control, is discussed in terms of glutamate dehydrogenase activity. Without CO(2), or with a low CO(2) level, a NH(3) excretion was observed, the rate of which depended on the actual concentration of the dissolved CO(2). Experiments with (15)NH(3) demonstrated that no NH(3) uptake was masked by this excretion and inversely that no excretion occurred during the uptake in the conditions where it took place. Furthermore, the NH(3) excretion observed in the absence of CO(2) increased when O(2) concentration rose to 15% and was inhibited when 10 millimolar isonicotinic acid hydrazide was supplied to the algal suspension. Thus, NH(3) excretion in the presence of l-methionine-dl-sulfoximine seems to be related to a photorespiratory process inasmuch as it presents the same properties with regard to the O(2) and the isonicotinic acid hydrazide effects. These results favor the hypothesis that NH(3) produced in the medium originates from the glycine to serine reaction. On the other hand, partial inhibition (50%) of photosynthesis by l-methionine-dl sulfoximine was attributed to uncoupling between electron transfer and photophosphorylation due to NH(3) accumulation into the cell. PMID- 16662925 TI - Age Dependence of Photosynthesis in the Caribou Lichen Cladina stellaris. AB - The fruticose thallus of the lichen Cladina stellaris (Opiz.) Brodo can be subdivided into individual whorls of branches of known age. Photosynthesis declines steadily with age from a maximum rate of 0.76 milligram CO(2) per gram dry weight per hour in 1-year-old whorls to 0.02 milligram CO(2) per gram dry weight per hour after 15 years. Conversely, the dry biomass of the whorls increases up to age 9 years and then approximately levels off. Photosynthesis in whorls older than 15 years is less than 0.01 milligram per gram per hour. Progressive changes in thallus color with age are associated with the observed photosynthetic decline. Whorls aged 6 years and younger together account for 18% of thallus biomass but 50% of photosynthetic activity. The implications of these results for the idea that the lichen symbiosis results in truly integrated organisms with senescence phenomena akin to those in higher plants is discussed. PMID- 16662926 TI - Gramine Accumulation in Leaves of Barley Grown under High-Temperature Stress. AB - The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as ;Arimar' and CI 12020 accumulate gramine (N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as ;Proctor' do not. When grown at optimal temperatures (21 degrees C/16 degrees C, day/night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two- to three-leaf stage from 21 degrees C/16 degrees C to supra-optimal temperatures (>/=30 degrees C/25 degrees C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight).Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan -->--> AMI --> MAMI --> gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [(14)C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [(14)C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene (14)C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [(14)C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [(14)C]formate label into gramine and MAMI when supplied with AMI; the ratio [(14)C]gramine/[(14)C]MAMI fell with leaf age, suggesting that the two N methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific.In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures. PMID- 16662927 TI - Reduced osmotic potential effects on photosynthesis : identification of stromal acidification as a mediating factor. AB - Addition of sorbitol, which facilitated reductions in reaction medium osmotic potential from standard (0.33 molar sorbitol, -10 bars) isotonic conditions to a stress level of 0.67 molar sorbitol (-20 bars), inhibited the photosynthetic capacity of isolated spinach (Spinacia oleracea) chloroplasts. This inhibition, which ranged from 64 to 74% under otherwise standard reaction conditions, was dependent on reaction medium inorganic phosphate concentration, with the phosphate optimum for photosynthesis reduced to 0.05 millimolar at the low osmotic potential stress treatment from a value of 0.25 millimolar under control conditions.Stromal alkalating agents such as NH(4)Cl (0.75 millimolar) and KCl (35 millimolar) were also found to affect the degree of low osmotic potential inhibition of photosynthesis. Both agents doubled the rate of NaHCO(3)-supported O(2) evolution under the stress treatment, while hardly affecting the control rate at optimal concentrations. These agents also reduced the length of the lag phase of photosynthetic O(2) evolution under the stress treatment to a much greater degree. The rate-enhancement effect of these agents under the stress treatment was reversed by sodium acetate, which is known to facilitate stromal acidification.The reaction medium pH optimum for photosynthesis under the stress treatment was higher than under control conditions. In the presence of optimal NH(4)Cl, this shift was no longer evident.Internal pH measurements indicated that the stress treatment caused a 0.43 and 0.24 unit reduction in the stromal and intrathylakoid pH, respectively, under illumination. This osmotically induced acidification was not evident in the dark. The presence of 0.75 millimolar NH(4)Cl partially reversed the osmotically induced reduction in the illuminated stromal pH. It was concluded that stromal acidification is a mediating mechanism of the most severe site of low osmotic potential inhibition of the photosynthetic process. PMID- 16662928 TI - Inhibition of Glutamine Synthetase from Pea by Tabtoxinine-beta-lactam. AB - Tabtoxinine-beta-lactam, a hydrolytic product of tabtoxin produced by Pseudomonas syringae pv. tabaci, apparently inactivates pea seed glutamine synthetase. Inhibition of the enzyme's initial velocity is linear over a range of 0.5 to 5 millimolar tabtoxinine-beta-lactam in the presence of 10 millimolar glutamate. A method for the purification of glutamine synthetase from dried peas is presented which gives a 30% yield with a 2,000-fold increase in specific activity. A method for obtaining highly purified tabtoxinine-beta-lactam and tabtoxin in good yields is also presented. The authenticity and purity of tabtoxinine-beta-lactam and tabtoxin were verified by chromatography, biological activity, and (1)H and (13)C nuclear magnetic resonance spectroscopy. PMID- 16662929 TI - Host-Pathogen Interactions : XXII. A Galacturonic Acid Oligosaccharide from Plant Cell Walls Elicits Phytoalexins. AB - Elicitors of phytoalexin accumulation in soybean (Glycine max L. Merr., cv Wayne) cotyledons were released from soybean cell walls and from citrus pectin by partial acid hydrolysis. These two hydrolysates yielded nearly identical distributions of elicitor activity when fractionated on anion-exchange columns. Chromatography of the pectin elicitor on gel filtration and high-pressure anion exchange columns did not further purify the elicitor. Elicitor activity of the preparation was lost by treatment with either endo-alpha-1,4-polygalacturonase or pectate lyase. Glycosyl residue compositions of the purified elicitors from cell walls and pectin were both found to be approximately 98% galacturonosyl residues. Linkage analysis of the pectin elicitor showed that most, if not all, of the galacturonosyl residues were alpha-1,4-linked. The high-mass molecular ions detected by fast atom bombardment-mass spectrometry of the most active elicitor fractions from cell walls and pectin both corresponded precisely to a molecule composed of 12 galacturonosyl residues. These results suggest that dodeca-alpha 1,4-d-galacturonide is the active elicitor, but the possibility remains that the active component could be a slightly modified oligogalacturonide present, but not detected, in the purified fractions. PMID- 16662930 TI - Auxin requirements of sycamore cells in suspension culture. AB - Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase). PMID- 16662931 TI - Plant 5-methylthioribose kinase: properties of the partially purified enzyme from yellow lupin (lupinus luteus L.) seeds. AB - Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg(2+) and Mn(2+) acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The K(m) values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively.Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the gamma-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (K(i) = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues. PMID- 16662932 TI - Analysis of the role of the phosphate translocator and external metabolites in steady-state chloroplast photosynthesis. AB - The role of the phosphate translocator and the importance of the extrachloroplastic concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in steady-state photosynthesis is examined with a kinetic model. The steady-state stromal concentrations of these compounds are calculated as a function of the rate of the various partial reactions of photosynthesis, at various external concentrations which span those likely to occur in vivo. It is shown how the net transport requirements of the various reactions necessitate different adjustments in the stromal concentrations of these compounds, away from the equilibrium values expected in the absence of metabolism. Under most circumstances, the high exchange capacity of the phosphate translocator relative to the transport requirements of CO(2) fixation limits the extent of these displacements, but conditions when the phosphate translocator is limiting photosynthesis are observed and discussed. The model provides a basis for a more quantitative understanding of the role of the phosphate translocator and the external concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in photosynthesis. PMID- 16662933 TI - Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : I. Leaf Anatomy, Photosynthetic Responses to O(2) and CO(2), and Activities of Key Enzymes in the C(3) and C(4) Pathways. AB - Four species of the genus Flaveria, namely F. anomala, F. linearis, F. pubescens, and F. ramosissima, were identified as intermediate C(3)-C(4) plants based on leaf anatomy, photosynthetic CO(2) compensation point, O(2) inhibition of photosynthesis, and activities of C(4) enzymes. F. anomala and F. ramosissima exhibit a distinct Kranz-like leaf anatomy, similar to that of the C(4) species F. trinervia, while the other C(3)-C(4) intermediate Flaveria species possess a less differentiated Kranz-like leaf anatomy. Photosynthetic CO(2) compensation points of these intermediates at 30 degrees C were very low relative to those of C(3) plants, ranging from 7 to 14 microliters per liter. In contrast to C(3) plants, net photosynthesis by the intermediates was not sensitive to O(2) concentrations below 5% and decreased relatively slowly with increasing O(2) concentration. Under similar conditions, the percentage inhibition of photosynthesis by 21% O(2) varied from 20% to 25% in the intermediates compared with 28% in Lycopersicon esculentum, a typical C(3) species. The inhibition of carboxylation efficiency by 21% O(2) varied from 17% for F. ramosissima to 46% for F. anomala and were intermediate between the C(4) (2% for F. trinervia) and C(3) (53% for L. esculentum) values. The intermediate Flaveria species, especially F. ramosissima, have substantial activities of the C(4) enzymes, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malate dehydrogenase, indicating potential for C(4) photosynthesis. It appears that these Flaveria species may be true biochemical C(3)-C(4) intermediates. PMID- 16662934 TI - Mechanism of iron uptake by peanut plants : I. Fe reduction, chelate splitting, and release of phenolics. AB - Iron deficiency in peanuts (Arachis hypogeae L.) caused an increase in release of caffeic acid, a higher rate of Fe(III) reduction, and increased rates of both Fe(III) chelate splitting and iron uptake.Experiments on Fe(III) reduction by phenolics (in vitro experiments) and by roots of Fe-deficient peanuts exclude the direct involvement of released phenolics in Fe(III) reduction by roots: Fe(III) reduction by phenolics had a pH optimum higher than 8.0 and was strongly dependent on the concentration and the stability of the supplied Fe(III) chelates. In contrast, Fe(III) reduction by roots of Fe-deficient peanuts had a pH optimum of about 5.0 and was less dependent on the stability of the supplied Fe(III) chelates. Furthermore, the observed release of phenolics into nutrient solution would have to be at least 200 times higher to attain the reduction rates of roots of Fe-deficient peanuts. The results of these experiments support the idea of an enzymic reduction of Fe(III) on the plasmalemma of cortical cells of roots. PMID- 16662935 TI - Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2). AB - Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO(2) to activate the enzyme, changes in CO(2) between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO(2) levels and 21% O(2) or 1% or less O(2), the levels of ribulose bisphosphate were high and not limiting for CO(2) fixation. With high leaf ribulose bisphosphate, the K(act)(CO(2)) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO(2) and O(2), activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg(2+) and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg(2+) and pH most likely determine the ratio of inactive enzyme RuBP to active enzyme-CO(2)-Mg(2+)-RuBP forms. Higher irradiances favor more optimal Mg(2+) and pH, with greater activation of the carboxylase and increased photosynthesis. PMID- 16662936 TI - Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves. AB - (15)N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (Avena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The (15)N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of (15)N label in serine to (15)N label in glycine declined substantially. PMID- 16662937 TI - Ploidy Effects in Isogenic Populations of Alfalfa (Medicago sativa L.) : IV. Similarity in Physical and Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase. AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) was isolated from isogenic diploid-tetraploid and tetraploid-octoploid sets of alfalfa (Medicago sativa L.) leaves. Molecular weights of RuBPCase subunits were similar across ploidy levels of both isogenic sets with subunits of 52,000 and 14,000. Apparent K(m)(CO(2)) values and substrate specificity factors (V(c)K(o)/V(o)K(c)) of RuBPCase were similar across ploidy levels of both isogenic sets. These results indicate that ploidy had no effect on the kinetic properties of RuBPCase in alfalfa. PMID- 16662938 TI - Role of magnesium in the plasma membrane ATPase of red beet. AB - The phosphorylation technique was used to assess the role of Mg in the red beet (Beta vulgaris L.) plasma membrane ATPase. When an excess of ethylenediaminetetraacetate (Tris salt, pH 6.5) was added to phosphorylation reactions at steady-state, the phosphorylation level declined exponentially and the rate constant for dephosphorylation was similar to that observed when phosphorylation reactions were chased with unlabeled ATP. When KCl was included with the EDTA chase, a 2.4-fold increase in the turnover of the phosphoenzyme was observed. Thus, the formation of the phosphorylated intermediate but not its breakdown requires free Mg to be present. When an excess of unlabeled ATP containing MgSO(4) was added to plasma membranes incubated for 20 seconds with [gamma-(32)P]ATP in the absence of MgSO(4), a burst of phosphorylation was observed that declined exponentially. The rate constant for this decline was similar to that observed for phosphoenzyme turnover after initial labeling in the presence of MgSO(4). Extrapolation of this kinetic plot to zero time indicated that ATP binding can occur when MgSO(4) is absent. It is proposed that Mg has a specific role in the transphosphorylation reaction of the terminal phosphate group of ATP to the enzyme. PMID- 16662939 TI - Complete Release of Axillary Buds from Apical Dominance in Intact, Light-Grown Seedlings of Pisum sativum L. following a Single Application of Cytokinin. AB - Single applications of either 6-benzyladenine or zeatin to inhibited axillary buds of intact, light-grown seedlings of Pisum sativum L. cv Black-eyed Susan, resulted in the formation of rapidly elongating, leafy shoots. Similar treatment with kinetin or isopentenyladenine caused only limited but outgrowth which stopped 6 days after application. PMID- 16662940 TI - Growth Response of Halophila engelmanni Aschers. to Sulfide, Copper, and Organic Nitrogen in Marine Sediments. AB - Growth response of the seagrass Halophila engelmanni Aschers. was studied using defined culture techniques. When added to the rhizosphere, nutrient-seawater medium supported vigorous plant growth, if substantial Cu (10 times that in modified Hoagland solution) plus organic N were present. H(2)S up to 0.2 millimolar in the sediment was not inhibitory. A requirement for exogenous organic N, especially urea, is postulated for vigorous growth of Halophila. PMID- 16662941 TI - Sucrose uptake and compartmentation in sugar beet taproot tissue. AB - Active sucrose uptake by discs of mature sugar beet (Beta vulgaris L. cv GW-D2 and USH-20) root tissue shows a biphasic dependence on external sucrose. At concentrations up to 20 millimolar sucrose, the active uptake mechanism appears to approach saturation, with an apparent K(m) of 3.6 millimolar. At higher external sucrose concentrations, a linear dependence becomes obvious indicating the probable presence of a nonsaturable, metabolically dependent uptake component. Active transport was not observed at external sucrose concentrations that caused tissue plasmolysis. Passive sucrose uptake in unplasmolyzed tissue showed a linear dependence on external sucrose concentration. The mitochondrial and/or suspected vacuolar ATPase inhibitors oligomycin, diethylstilbestrol, and N,N-dicyclohexylcarbodiimide strongly inhibited active sucrose uptake, whereas the putative plasmalemma-specific ATPase inhibitor orthovanadate was without effect.Sucrose efflux patterns from root discs indicated three distinct sucrose compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole with the vacuole being the slowest releasing compartment. The sucrose contents and volumes of the compartments indicated that sucrose uptake into the vacuole was against a concentration gradient. Combined sucrose uptake/efflux analyses indicated that sucrose uptake into the vacuole is primarily an active transport process while transport into the cytoplasm is apparently passive, at least at external sucrose concentrations above 20 millimolar. We discuss the possibility that active sucrose uptake into the vacuoles of sugar beet storage cells is rate limited by passive sucrose transport to the active uptake site. PMID- 16662942 TI - Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants. AB - A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilizing an aqueous two-polymer phase system is outlined. Mainly, the plant used was Orchard grass (Dactylis glomerata L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-15 millimolar Tris-maleate (pH 7.3), and 30 millimolar NaCl. In this system, the plasma membranes and the other membranes were preferentially partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a single partition (i.e. about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase. The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mg(2+)-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a high specificity for Mg(2+) and ATP. KCl stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be applicable to a wide variety of plants and plant tissue including green leaves. PMID- 16662943 TI - Adaptation of ribonucleic Acid metabolism to anoxia in rice embryos. AB - Rice (Oryza sativa var. Cigalon) is a plant which can adapt to very stringent anoxic conditions. It has previously been shown that during the adaptation period the energy charge can be used as a marker for metabolic activity. We have studied RNA metabolism during this period and correlated it with changes in the energy charge. Uptake of labeled precursor, UTP-specific activity, and incorporation were measured. Immediately after transfer to anaerobic conditions, the UTP pool size is reduced and the overall rate of incorporation drops. During adaptation, the rate of incorporation increases and stabilizes at about half of its value in aerobic conditions. Analysis of RNA shows that rRNA and mRNA are synthesized and that the processing of ribosomal RNA precursor is altered. Polyribosomes are present throughout the adaptation period although their amount is reduced during the first hour of anoxia. Changes in poly(A) content were noticed, indicating that some mRNA are rapidly degraded. Taken together, the results show that the RNA metabolism can be modulated during adaptation to anoxia in a parallel manner with energy charge changes. PMID- 16662944 TI - Deep Undercooling in Woody Taxa Growing North of the -40 degrees C Isotherm. AB - The freezing of deep undercooled water in cold-hardened 3-year-old stems of 16 woody taxa was studied in mid-January by differential thermal analysis. The initiation temperature and the size of the low temperature exotherm (LTE) were compared for nonthawed, thawed, and freeze-killed stems. In general, the initiation temperature of the LTE for nonthawed stems occurred at a lower temperature than for thawed stems and freeze-killed stems. In some cases, no LTE was detected in nonthawed stems although a LTE was detected after thawing. The size of the LTE increased after thawing the stem and also after the stem was freeze killed. The LTE observed in one species disappeared upon exposure to continuous low sub-zero temperatures. Results suggest that undercooling which subsequently results in the LTE in woody stems is due to the cell wall and the plasma membrane. During periods of prolonged freezing, cellular water migrates from the cells which undercool to extracellular ice. This results in a concentration of cell solutes which lowers the homogeneous nucleation temperature of the cell sap. The cold hardiness of nonthawed and thawed stems was compared by a controlled freeze test. In general, thawing had little effect on the survival temperature whereas it had a marked effect on the initiation of the LTE. PMID- 16662945 TI - Changes in Intracellular pH Are Not Correlated with the Circadian Rhythm of Neurospora. AB - Intracellular pH (pH(i)) was measured during the circadian cycle of Neurospora. Internal pH of Neurospora cultures in liquid medium was assayed by the 5,5 dimethyl-2,4-oxazolidinedione method and gave values for pH(i) which were similar to those previously obtained by other workers using pH-microelectrodes with agar grown cultures. Cytoplasmic pH changed in liquid medium cultures, but these changes were not related to the circadian clock. Furthermore, treatments which raise or lower pH(i) do not phase-shift the circadian rhythm. These results indicate that pH(i) plays no specific role in regulating the circadian clock of Neurospora. PMID- 16662947 TI - Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase. AB - We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on (2)H(2)O as compared to ACC synthase from H(2)O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[(35)S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.Gel filtration on Sephadex G-100 gave a molecular weight estimate for ACC synthase of 57,000 +/- 1,500 daltons. Hydrophobic interaction chromatography on phenyl-Sepharose yielded a 60- to 70-fold purification of the enzyme. SDS-gel electrophoresis of this preparation indicated the presence of one intense band at 57,000 daltons and several less intense bands. Affinity chromatography was of limited usefulness in the purification of ACC synthase since the enzyme could not be eluted specifically from any of the affinity gels tried. Purification methods that involved pH changes led to a rapid loss of ACC synthase activity. ACC synthase was estimated to comprise less than 1% of the total protein in tomato pericarp tissue. PMID- 16662946 TI - Synthesis of oxalic Acid by enzymes from lettuce leaves. AB - A rapid purification of lactate dehydrogenase and glycolate oxidase from lettuce (Lactuca sativa) leaves is described. The kinetics of both enzymes are reported in relation to their possible roles in the production of oxalate. Lettuce lactate dehydrogenase behaves like mammalian dehydrogenase, catalyzing the dismutation of glyoxylate to glycolate and oxalate. A model is proposed in which glycolate oxidase in the peroxisomes and lactate dehydrogenase in the cytosol are involved in the production of oxalate. The effect of pH on the balance between oxalate and glycolate produced from glyoxylate suggests that in leaves lactate dehydrogenase may function as part of an oxalate-based biochemical, pH-stat. PMID- 16662948 TI - Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature. AB - Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25 degrees C, but not at 15 degrees C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15 degrees C and 25 degrees C.The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25 degrees C than at 15 degrees C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15 degrees C and 25 degrees C after the puncture force was reduced to between 0.3 and 0.4 newtons.Application of gibberellic acid(4+7) (100 microliters per liter) resulted in earlier germination at 15 degrees C and 25 degrees C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O(2) concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid(4+7), but only at 25 degrees C. At 15 degrees C, high O(2) concentrations slowed germination and endosperm strength decline. PMID- 16662949 TI - Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria. AB - The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K(i) values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K(i) of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation. PMID- 16662950 TI - Effect of the host legume on acetylene reduction and hydrogen evolution by Rhizobium nitrogenase. AB - The relative efficiency (RE) of N(2) fixation (RE = 1 - [H(2) evolved in air]/[acetylene reduced]) was investigated in a Rhizobium strain lacking uptake hydrogenase activity (Hup(-)). Variation in RE of such strains presumably reflects changes in the electron allocation coefficient of nitrogenase. By artificially extending the normal dark period of 24-day-old Pisum sativum L. cv ;Alaska' inoculated with the Hup(-)R. leguminosarum strain 3740, reproducible changes in RE were obtained. The RE showed no change during a normal 8-hour night, but a significant increase in RE was measured after 20 hours in the dark. Upon returning to the normal 550 microEinsteins per square meter per second light treatment, RE declined to previous levels within 2 hours. If, after the 20-hour dark treatment, plants were returned to 90 or 190 microEinsteins per square meter per second or maintained in the dark, RE did not decline significantly. The RE varied inversely with changes in soluble sugar content of root nodules. A similar pattern of changes in RE during an extended dark period and subsequent light treatment was measured in 28-day-old Alaska peas and in the Hup(-)R. trifolii strain 162X99 in symbiosis with Trifolium subterraneum L. cv ;Woogenellup.' These results suggest that Rhizobium cells may produce short-term alterations in the electron allocation coefficient of nitrogenase in response to physiological changes. The observed changes in the bacterial RE favored N(2) reduction over proton reduction when soluble sugars provided by the host plant declined. PMID- 16662951 TI - Photosynthesis in Tall Fescue : IV. Carbon Assimilation Pattern in two Genotypes of Tall Fescue Differing in Net Photosynthesis Rates. AB - We previously reported that the net photosynthetic rate of a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) was 32 to 41 versus 22 milligrams CO(2) per square decimeter per hour in a hexaploid genotype (V6-802) (Randall, Nelson, Asay Plant Physiol 59: 38-41). The high rate was later correlated with increases in total ribulose 1,5-bisphosphate carboxylase protein (17%) and activity (27%) (Joseph, Randall, Nelson Plant Physiol 68: 894-898). This report characterizes photosynthesis with respect to light saturation and early products of photosynthesis in an attempt to identify regulatory metabolic site(s) in these two genotypes. Analysis of the early products of photosynthesis indicated that both genotypes fixed CO(2) via the Calvin-Benson cycle with phosphoglyceric acid as the initial primary product. Both genotypes had similar (14)C-labeled intermediates. Sucrose was the primary sink of (14)CO(2) assimilation. After 10 min of (14)CO(2) assimilation with attached leaves, sucrose accounted for 89% (decaploid) and 81% (hexaploid) of the total (14)C incorporated. In 10 min, this amounted to 1.3 (decaploid) and 0.8 (hexaploid) mumol [(14)C]sucrose formed g fresh weight(-1) and reflected the observed differences in photosynthetic rates. There was limited labeling of starch (1%) and fructan (1%). Results of total nonstructural carbohydrates and P(i) analysis also demonstrated sucrose was the predominant carbohydrate in fescue leaves. Quantitative differences in sucrose and P(i) between the two genotypes may reflect changes in partitioning and this possibility is discussed. PMID- 16662952 TI - Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor. AB - The predominant storage protein of oat (Avena sativa L.) seeds is a saline soluble globulin with a mol wt of 320,000 which is composed of six large (M(r) = 35,000 to 40,000) and six small (M(r) = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH(2)-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)(+) RNA with anti oat globulin sera yielded M(r) = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M(r) = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides. PMID- 16662953 TI - Heterogeneity of pumpkin ribosomal DNA. AB - The ribosomal DNA (rDNA) of Cucurbita pepo L. has been found to consist of tandemly arrayed repeat units, most of which are 10 kilobases in length. Thirty six repeat units, cloned into the HindIII site of pACYC 177, fall into seven classes which differ from each other in length and/or nucleotide sequence. Most of the heterogeneity occurs in noncoding portions of the repeat unit although there is some nucleotide sequence variation in the coding portion as well. Heterogeneity of base modification was observed in genomic rDNA of which two examples are: (a) all of the repeat units have three BamHI sites, one of which is unavailable for restriction in about half of the units and (b) all of the CCGG sites except one are methylated at the internal cytidine in many of the units; a second site is unmethylated in some of the units and in a very few units a third site remains unmethylated. PMID- 16662954 TI - Photosynthesis and Stomatal Conductance of Potato Clones (Solanum tuberosum L.) : Comparative Differences in Diurnal Patterns, Response to Light Levels, and Assimilation through Upper and Lower Leaf Surfaces. AB - A few potato clones, such as A6948-4, had higher rates of photosynthesis in the field than the Russet Burbank and were able to maintain higher rates not only during mid-day but also in the early morning and late evening hours. In addition, they maintained higher carbon assimilation rates over a range of photosynthetic photon flux density from 400 to 2,000 microeinsteins per square meter per second.Stomatal conductance increased linearly as irradiance increased from 500 to 2,000 microeinsteins per square meter per second with all four potato clones that were examined. Obviously, comparative measurements of stomatal conductance or diffusive resistance with potato must be taken at a known and constant photosynthetic photon flux density.The upper (adaxial) leaf surface of some potato clones provided a surprising contribution to total carbon assimilation. Neither stomatal conductance, number of stomata per unit area, total area of the stomatal apparatus, nor chlorophyll content appear to account for differences in carbon assimilation rates among clones. PMID- 16662955 TI - Computer-Assisted Image Analysis of Tissues of Ethrel-Treated Pinus taeda Seedlings. AB - A microcomputer based video image analyzer is described and demonstrated in the analysis and comparison of tissue types of 6-month-old Ethrel-treated and control seedlings of loblolly pine (Pinus taeda L.). Two different programs were employed. ;Topographer I' sought, identified, and measured cell or resin canal lumen areas, and ;Area' measured tracheid length and silhouette areas. Ethrel treatment induced an increase in radial growth and a decrease in shoot elongation and needle growth. Bark production was stimulated by the same treatment. The tracheid lumens of Ethrel-treated plants were shorter and had a smaller cross sectional area, whereas the tracheid cell walls increased in thickness. The treatment also increased resin canal formation in the xylem. PMID- 16662956 TI - Characterization of leaf senescence and pod development in soybean explants. AB - Excised soybean (Glycine max [L.] Merrill) cv Anoka leaf discs tend to remain green even after the corresponding intact leaves have turned yello on fruiting plants. We have found that explants which include a leaf along with a stem segment (below the node) and one or more pods (maintained on distilled H(2)O) show similar but accelerated leaf yellowing and abscission compared with intact plants. In podded explants excised at pre-podfill, the leaves begin to yellow after 16 days, whereas those excised at late podfill begin to yellow after only 6 days. Although stomatal resistances remain low during the first light period after excision, they subsequently increase to levels above those in leaves of intact plants. Explants taken at mid to late podfill with one or more pods per node behave like intact plants in that pod load does not affect the time lag to leaf yellowing. Explant leaf yellowing and abscission are delayed by removal of the pods or seeds or by incubation in complete mineral nutrient solution or in 4.6 micromolar zeatin. Like chorophyll breakdown, protein loss is accelerated in the explants, but minerals or especially zeatin can retard the loss. Pods on explants show rates and patterns of color change (green to yellow to brown) similar to those of pods on intact plants. These changes start earlier in explants on water than in intact plants, but they can be delayed by adding zeatin. Seed dry weight increased in explants, almost as much as in intact plants. Explants appear to be good analogs of the corresponding parts of the intact plant, and they should prove useful for analyzing pod development and mechanisms of foliar senescence. Moreover, our data suggest that the flux of minerals and cytokinin from the roots could influence foliar senescence in soybeans, but increased stomatal resistance does not seem to cause foliar senescence. PMID- 16662957 TI - Changes of cyanide content and linamarase activity in wounded cassava roots. AB - When cassava (Manihot esculenta Crantz) root was cut into blocks and incubated under laboratory conditions, the blocks showed more widespread and more even symptoms of physiological deterioration than those under natural conditions. Thus, the tissue block system has potential for biochemical studies of natural deterioration of cassava root. The changes in cyanide content and linamarase (linamarin beta-d-glucoside glucohydrolase; EC 3.2.1.21) activity in various tissues during physiological deterioration were investigated. Total cyanide content increased in all parts of block tissue after 3-day incubation. The degree of increase in cyanide was most pronounced in white parenchymal tissue, 2 to 3 millimeters thick, next to the cortex (A-part tissue), where no physiological symptoms appeared. On the other hand, linamarase activity was decreased in all parts of block tissue after a 3-day incubation. A time course analysis of A-part tissue indicated a clear reciprocal relationship between changes in total cyanide and linamarase activity; total cyanide increased, while linamarase activity decreased. Free cyanide constituted a very small portion of the total cyanide and did not change markedly. PMID- 16662958 TI - Dynamics of imbibition by soybean embryos. AB - Measurements of the rate of imbibition by isolated cotyledons of soybean (Glycine max (L.) Merr cv Wayne) indicated that water uptake rates were slowed by low temperatures and by low initial moisture contents of the tissue. The role of water viscosity in the temperature effects on imbibition was examined, and a linear relation between imbibition rate and the reciprocal of viscosity was found only for seeds of very high initial moisture content. Adding solutes which lowered the surface tension, or increased the wetting ability of the water, yielded markedly increased rates of imbibition, especially for tissue of low initial moisture content. The data are interpreted as indicating a first component of water entry which is a wetting reaction influenced by the surface tension of the water, and a second component which resembles water flow through a porous matrix and is influenced by the water viscosity. We speculate that damage to soybeans during imbibition, such as in the case of chilling injury, may be particularly related to the initial wetting reaction rather than to the longer term imbibitional rate. PMID- 16662959 TI - Extraction and partial characterization of the glycine decarboxylase multienzyme complex from pea leaf mitochondria. AB - Glycine decarboxylase has been successfully solubilized from pea (Pisum sativum) leaf mitochondria as an acetone powder. The enzyme was dependent on added dithiothreitol and pyridoxal phosphate for maximal activity. The enzyme preparation could catalyze the exchange of CO(2) into the carboxyl carbon of glycine, the reverse of the glycine decarboxylase reaction by converting serine, NH(4) (+), and CO(2) into glycine, and (14)CO(2) release from [1-(14)C]glycine. The half-maximal concentrations for the glycine-bicarbonate exchange reaction were 1.7 millimolar glycine, 16 millimolar NaH(14)CO(2), and 0.006 millimolar pyridoxal phosphate. The enzyme (glycine-bicarbonate exchange reaction) was active in the assay conditions for 1 hour and could be stored for over 1 month. The enzymic mechanism appeared similar to that reported for the enzyme from animals and bacteria but some quantitative differences were noted. These included the tenacity of binding to the mitochondrial membrane, the concentration of pyridoxal phosphate needed for maximum activity, the requirement for dithiothreitol for maximum activity, and the total amount of activity present. Now that this enzyme has been solubilized, a more detailed understanding of this important step in photorespiration should be possible. PMID- 16662960 TI - 5-Aminolevulinate Synthesis in Permeabilized Filaments of the Blue-Green Alga Anabaena variabilis. AB - Filaments of the blue-green alga Anabaena variabilis permeabilized by dimethylsulfoxide (DMSO) produce increased amounts of 5-aminolevulinate in the presence of levulinic acid. The metabolic activity of the filaments remains unperturbed in the presence of up to 7.5% (v/v) DMSO. Studies utilizing DMSO permeabilized filaments confirm that 5-aminolevulinate is synthesized preferably from glutamate and, to a lesser extent, from alpha-ketoglutarate in this organism. PMID- 16662961 TI - Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells. AB - Compartmental analysis of (35)SO(4) (2-) exchange kinetics is used to obtain SO(4) (2-) fluxes and compartment contents in carrot (Daucus carota L.) storage root cells, where 2 to 5% of the SO(4) (2-) taken up is reduced to organic form. The necessary curve fitting is verified by (a) consistency between ;content versus time' and ;rate versus time' plots of washout data; (b) agreement between loading and washout kinetics; and (c) correct identification of the fastest exchange phase as being from extracellular spaces.Sulfate is actively transported up an electrochemical potential gradient at both plasmalemma and tonoplast. The plasmalemma influx is from 2 to 10 times higher than the tonoplast influx, is much greater than the SO(4) (2-) reduction rate, and would not limit the rate of either. This is consistent with the finding that the plasmalemma influx is not regulated by internal SO(4) (2-) or cysteine (Cram 1982 Plant Sci Lett, in press).Both SO(4) (2-) influxes rise with only limited saturation as the external SO(4) (2-) concentration increases up to 50 millimolarity. Both effluxes appear to be passive, with extensive recycling in the plasmalemma influx pump. SO(4) (2 ) permeability is about 10(-11) meter per second at both membranes.The high, nonlimiting fluxes of SO(4) (2-) at the plasmalemma relative to the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier 1981 Biochim Biophys Acta 644: 24-35) contrasts with SO(4) (2-) fluxes in bacteria and with Cl(-) fluxes in plant cells. Their implications for work on characteristics and regulation of SO(4) (2-) uptake in roots and tissue cultures are discussed. PMID- 16662962 TI - Measurement of CO(2) Dissolved in Aqueous Solutions Using a Modified Infrared Gas Analyzer System. AB - Total dissolved inorganic carbon (SigmaCO(2)) and aqueous carbon dioxide (H(2)CO(3) (*)) in nutrient solutions may be measured by the injection of small gas or liquid samples (1 microliter to 8 milliliters) into a gas stripping column connected in-line with an infrared gas analyzer. The measurement of SigmaCO(2) in solution requires sample acidification, while H(2)CO(3) (*) and gaseous CO(2) are measured without the addition of lactic acid. The standard curve for SigmaCO(2) was linear up to 300 nanomoles CO(2). Maximum sensitivity was approximately 300 picomoles. Measurements of H(2)CO(3) (*) were independent of pH. Consequently, SigmaCO(2) and H(2)CO(3) (*) could be used to calculate the pH, HCO(3) (-), and CO(3) (2-) values of nutrient solutions. Injection and complete analyses required from 0.8 to 2 minutes. PMID- 16662963 TI - Inhibition of cytokinin-regulated responses by calmodulin-binding compounds. AB - Two classes of compounds which bind to calmodulin in a calcium-dependent manner (neuroleptic drugs and local anaesthetics) were used to investigate the possible involvement of a calcium-dependent regulator protein in the action of the plant hormones cytokinins.The cytokinin-induced synthesis of betacyanin in Amaranthus tricolor seedlings was used as one test system. The calmodulin antagonists inhibited betacyanin synthesis with the following order of potency: fluphenazine > trifluoperazine = pimozide > chlorpromazine > dibucaine > penfluridol > haloperidol > tetracaine, over a concentration range (IC(50)) of 0.1 to 0.6 millimolar. Red light and fusicoccin increase betacyanin accumulation and are synergistic with cytokinins. These red light- and fusicoccin-dependent inductions were inhibited by lower concentrations of the drugs than cytokinin-dependent induction, and the order of potency of the drugs was not precisely the same. The results are compatible with the hypothesis that cytokinins may act, at least in part, by changing ion fluxes, with the additional involvement of a calmodulin.The second cytokinin-regulated response studied was growth in soybean callus culture. In this system, inhibition was observed with lower concentrations of drugs than in cytokinin-dependent betacyanin synthesis with an order of inhibitory potency of pimozide = trifluoperazine > penfluridol > chlorpromazine > haloperidol > tetracaine. The effective concentration range (IC(50)) was from 0.07 to 0.5 millimolar.Inhibition of betacyanin synthesis by 0.15 millimolar trifluoperazine and of soybean callus growth by 2 millimolar tetracaine were both reversible. PMID- 16662964 TI - Calmodulin-binding drugs affect responses to cytokinin, auxin, and gibberellic Acid. AB - Trifluoperazine, a phenothiazine tranquilizer, and tetracaine, a local anesthetic, have been found to inhibit a variety of plant hormone responses at concentrations compatible with their known inhibition of Ca(2+)-calmod-ulin dependent enzyme activities. Among these responses are cytokinin-dependent betacyanin synthesis and increase in fresh weight in Amaranthus tricolor cotyledons, auxin-dependent increase in length of wheat coleoptile segments and gibberellic acid-dependent induction of alpha-amylase synthesis in barley aleurone layers. The reversibility of some of these inhibitory effects has been demonstrated, indicating that, up to a point, a generalized membrane destruction can be ruled out. The evidence, taken in conjunction with numerous examples from the literature showing calcium involvement in the action of all of the plant hormones, support a unifying theory of hormone action. PMID- 16662965 TI - Glutamine Synthetases of Higher Plants : Evidence for a Specific Isoform Content Related to Their Possible Physiological Role and Their Compartmentation within the Leaf. AB - The chromatographic properties of glutamine synthetase isoforms have been investigated in a wide range of higher plant leaves and shoots using ion exchange chromatography. Different patterns of glutamine synthetase isoform content were observed. Among higher plants, four patterns or groups could be recognized. The first group is characterized by having only cytosolic glutamine synthetase, whereas the second group is distinguished by having only chloroplastic glutamine synthetase. The third group is characterized by cytosolic glutamine synthetase being a minor component of the total leaf glutamine synthetase activity. The fourth group is distinct from the other groups in having high cytosolic and chloroplast glutamine synthetase activity. Immunological studies have been undertaken on a few species from each group to identify unambiguously both cytosolic and chloroplastic glutamine synthetases. PMID- 16662966 TI - Growth temperature effects on thylakoid membrane lipid and protein content of pea chloroplasts. AB - The lipid composition and level of unsaturation of fatty acids has been determined for chloroplast thylakoid membranes isolated from Pisum sativum grown under cold (4 degrees /7 degrees C) or warm (14 degrees /17 degrees C) conditions. Both the relative amounts of lipid classes and degree of saturation were not greatly changed for the two growth conditions. In cold-grown plants, there was a slightly higher linolenic and lower linoleic acid content for the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol. In contrast to thylakoid membranes, a non-thylakoid leaf membrane fraction including the chloroplast envelope, had a higher overall level of fatty acid unsaturation in cold-grown plants due mainly to an increase in the linolenic acid content of MGDG, DGDG, phosphatidylglycerol, and phosphatidylcholine. The most clear cut change in the thylakoid membrane composition was the lipid to protein ratio which was higher in the cold-grown plants. PMID- 16662967 TI - Light-Induced Breakdown of NADPH-Protochlorophyllide Oxidoreductase In Vitro. AB - Light-induced loss of the enzyme protochlorophyllide reductase (EC 1.6.99.1.), already described as a characteristic of whole plants, has now been demonstrated in vitro using etioplast membrane preparations of Avena Sativa L. var Peniarth and Secale cereale L. var Rheidol. Some evidence is presented, based upon temperature, pH, and inhibitor sensitivity of the process, that loss of enzyme may be the result of proteolysis. The light-induced process can, in vitro, be largely prevented by addition of the substrates of the reductase, protochlorophyllide and NADPH. It is concluded that light causes the breakdown of the reductase in vivo and in vitro by producing ligand-free enzyme as a consequence of the photoconversion reaction. PMID- 16662968 TI - Sulfur Dioxide Flux into Leaves of Geranium carolinianum L. : Evidence for a Nonstomatal or Residual Resistance. AB - The concurrent exchange of SO(2) and H(2)O vapor between the atmosphere and foliage of Geranium carolinianum was investigated using a whole-plant gas exchange chamber. Total leaf flux of SO(2) was partitioned into leaf surface and internal fractions. The emission rate of SO(2)-induced H(2)S was measured to develop a net leaf budget for atmospherically derived sulfur. Stomatal resistance to SO(2) flux was estimated by two techniques: (a) R(s) (SO(2)') from SO(2) data using analog modeling techniques and (b) R(s) (SO(2) ) from analogy to H(2)O (i.e. 1.89 R(s) (H(2)o)).The emission of H(2)S was positively correlated with the rate of SO(2) flux into the leaf interior. An accounting of the simultaneous, bidirectional flux of gaseous sulfur compounds during pollutant exposure showed that sulfur accumulation in the leaf interior of G. carolinianum was 7 to 15% lower than that estimated solely from mass-balance calculations of SO(2) flux data (i.e. ignoring H(2)S emissions).The esimate of stomatal resistance to pollutant flux from the SO(2) data (R(s) (SO(2)')) was consistently less than the simultaneous estimate derived from analogy to H(2)O vapor (R(s) (SO(2) )). The resultant of R(s) (SO(2)') - R(s) (SO(2) ), which was always negative, is indicative of a residual resistance to SO(2) flux into the leaf interior. On a comparative basis, SO(2) molecules experienced less pathway resistance to diffusion than effluxing H(2)O molecules. It is proposed that the SO(2):H(2)O path length ratio is less than unity, as a consequence of the pollutant's high water solubility and unique chemical reactivity in solution. Thus, the diffusive paths for H(2)O and SO(2) in G. carolinianum are not completely synonymous. PMID- 16662969 TI - Gas Exchange, Stomatal Behavior, and deltaC Values of the flacca Tomato Mutant in Relation to Abscisic Acid. AB - The relationship between stomatal conductance and capacity for assimilation was investigated in flacca, a mutant of tomato (Lycopersicon esculentum Mill.) that has abnormal stomatal behavior and low abscisic acid (ABA) content. The assimilation capacity, determined by measuring assimilation rate as a function of intercellular CO(2) pressure, did not differ in leaves of flacca and its parent variety, Rheinlands Ruhm (RR). On the other hand, stomatal conductance of flacca leaves was greater than that of RR, and could be phenotypically reverted by spraying with 30 micromolar ABA. Stomatal conductance of flacca leaves was also reduced by increasing CO(2) pressure, increasing leaf to air vapor pressure difference, and decreasing quantum flux, irrespective of ABA treatment.The high conductance of flacca leaves resulted in a high intercellular CO(2) pressure. This allowed greater discrimination against (13)CO(2), as evidenced by more negative delta (13)C values for flacca as compared to RR. The delta (13)C values of both flacca and RR plants as influenced by ABA treatment were consistent with predictions based on gas exchange measurements, using a recent model of discrimination. PMID- 16662970 TI - Water Relations and Growth of the flacca Tomato Mutant in Relation to Abscisic Acid. AB - The flacca mutant in tomato (Lycopersicon esculentum Mill. cv Rheinlands Ruhm) was employed to examine the effects of a relatively constant diurnal water stress on leaf growth and water relations. As the mutant is deficient in abscisic acid (ABA) and can be phenotypically reverted to the wild type by applications of the growth substance, inferences can be made concerning the involvement of ABA in responses to water stress. Water potential and turgor were lower in leaves of flacca than of Rheinlands Ruhm, and were increased by ABA treatment. ABA decreased transpiration rates by causing stomatal closure and also increased the hydraulic conductance of the sprayed plants. Osmotic adjustment did not occur in flacca plants despite the daily leaf water deficits. Stem elongation was inhibited by ABA, but leaf growth was promoted. It is concluded that, in some cases, ABA may promote leaf growth via its effect on leaf water balance. PMID- 16662971 TI - Site of natural N enrichment of soybean nodules. AB - Soybean (Glycine max L. Merrill) nodules are usually more enriched in (15)N than other tissues. We show that both bacteroids and nodule cortex are considerably more enriched in (15)N than nodule cytosol, with bacteroids being slightly more enriched than the cortex. Hence, (15)N enrichment occurs in cells of both plant and bacterial origin. PMID- 16662972 TI - Leaf Cytosolic Fructose-1,6-bisphosphatase : A Potential Target Site in Low Temperature Stress. AB - Leaf cytosolic fructose-1,6-bisphosphatase (FBPase), partially purified from both spinach (Spinacia oleracea, var Hipack) and peas (Pisum sativum, var Progress No. 9), is reversibly inactivated by exposure to low temperature. Thus, even though assays were conducted at 22 degrees C, samples incubated at 0 to 12 degrees C had greatly reduced activity relative to controls maintained at 22 degrees C. Following incubation at 22 degrees C prior to assay, the inactivated samples regained their initial activity. Chloroplast FBPase, by contrast, was unaffected by low temperature treatment. This feature as well as lack of a response of cytosolic FBPase to thioredoxins f or c(f) and to chloroplast FBPase antibody indicate that the FBPase isozymes of leaves are different proteins. PMID- 16662973 TI - Long-lived and short-lived heat-shock proteins in tobacco mesophyll protoplasts. AB - We have studied modifications in the pattern of proteins synthesized by tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts when they are transferred from 25 degrees C to 40 degrees C. The synthesis of one group of proteins is practically unaffected by the heat shock. On the other hand, the synthesis of most other 25 degrees C proteins is greatly reduced, while specific heat-shock proteins appear: 17 stable, neutral, major proteins, which are synthesized throughout the culture period at the higher temperature and which correspond to those observed in other organisms, and two basic proteins with a short lifetime and which are synthesized only during the first 2 hours of heat shock. We suggest that these latter proteins are regulatory peptides which intervene in the inhibition of 25 degrees C syntheses. PMID- 16662974 TI - Nickel is not required for apourease synthesis in soybean seeds. AB - Soybeans (Glycine max L. Merr. cv ;Maple Presto') harvested from plants cultured in nickel-free medium had <0.005% the activity of nickel-sufficient beans and only 15% the activity of a urease-null variety, Itachi. However, whereas Itachi has no detectable urease protein, nickel-free beans of the variety Maple Presto exhibit normal or near normal levels of urease apoprotein. Thus, nickel isn't necessary for urease apoprotein synthesis. The apoprotein wasn't activated by nickel in vitro but, upon seed imbibition of nickel, urease was partially activated. This in vivo activation was not inhibited by cychoheximide. PMID- 16662975 TI - Photochemistry of 124 kilodalton Avena phytochrome in vitro. AB - The photochemical properties of purified 124 kilodalton (kD) Avena cv Garry phytochrome are examined and compared with those of the proteolytically degraded 118/114 kD species. The proportion of the chromoprotein in the far red absorbing form, Pfr, following saturating red irradiation is 0.86 for 124 kD phytochrome, substantially higher than the values of 0.79 determined here and 0.75 reported in the literature for 118/114 kD preparations. The ratio of the quantum yields for Pr to Pfr phototransformation and for Pfr to Pr phototransformation (phir/phifr) is 1.76 for the 124 kD molecule and 0.98 for the 118/114 kD species. Based on extinction coefficients determined using the Lowry assay as a measure of protein weight, the individual phototransformation quantum yields for 124 kD phytochrome are 0.17 for Pr --> Pfr (phir) and 0.10 for Pfr --> Pr (phifr). Comparison of these quantum yields with those of the 118/114 kD species (where phir = phifr = approximately 0.11) indicates that proteolytic degradation of the 124 kD molecule to the 118/114 kD species significantly affects only phir. Therefore, the lower proportion of Pfr at photoequilibrium observed for 118/114 kD preparations is explained mainly in terms of a reduced efficiency of Pr --> Pfr phototransformation. The absolute Pfr absorbance spectrum for 124 kD phytochrome obtained by correcting the measured spectrum for residual Pr exhibits a maximum at 730 nm and differs from previous absolute Pfr spectra for both ;120' kD and 60 kD phytochrome in that it lacks a shoulder in the red region of the spectrum. PMID- 16662976 TI - An in vivo technique for the study of Phloem unloading in seed coats of developing soybean seeds. AB - A technique has been developed which permits mechanistic studies of phloem unloading in developing seeds of soybean (Glycine max cv Clark) and other legumes. An opening is cut in the pod wall and the embryo surgically removed from the seedcoat without diminishing the capacity of that tissue for assimilate import, phloem unloading, or efflux. The sites of phloem unloading were accessible via the seedcoat apoplast and were challenged with inhibitors, solutes, buffers, etc., to characterize the unloading process.Unloading is stimulated by divalent metal chelators and diethylstilbestrol, and inhibited by metabolic uncouplers and sulfhydryl group modifiers. Solutes released from the seed coat had a carbon/nitrogen ratio of 31 milligrams carbon per milligram nitrogen; sucrose represented 90% of the carbon present and various nitrogenous solutes contributed the remaining 10%. Unloading could be maintained for up 8 hours at rates of 0.5 to 1.0 micromoles per hour, providing a valid, convenient in vivo technique for studies of phloem unloading and seed growth mechanisms. PMID- 16662977 TI - Light- and sugar-mediated control of direct de novo flower differentiation from tobacco thin cell layers. AB - The nature of organs neoformed from tobacco (Nicotiana tabacum cv Samsun) thin cell layers is influenced by the quantity of light supplied and on the sequence of this supply. It is observed that glucose exhibits similar effects. In the presence of glucose at 167 millimolar, continuous light of 50 watts per square meter is required for optimal flower differentiation in vitro. However, 50 watts per square meter irradiance limited to 6 days is sufficient to trigger flower formation in 80% of the explants provided that light is applied from day 6 to day 11 of culture. This critical phase may correspond to the initiation phase during which soluble sugars are mainly needed as carbon energy source rather than as osmoregulators. Under continuous or precise sequential sugar deprivations, either no organogenesis occurs, or abnormal structures or buds are formed. Therefore, light per se is not sufficient to induce flower differentiation. Conversely, a specific quantitative combination of glucose and sucrose almost substitutes for the light requirement for differentiation of anthers and pistils. These observations suggest that, during the sequence of events leading to flower differentiation, light acts on energy-dependent sugar uptake and metabolism and on the increase of reducing potential of chloroplasts. PMID- 16662978 TI - Development and Characterization of Ribulose-1,5-bisphosphate Carboxylase : Evidence Indicating a Lack of Carboxylase Function in CO(2) Fixation in Endosperm of Germinating Castor Bean Seedlings. AB - Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis Menten constants, K(m), for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO(2), and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg(2+) and HCO(3) (-). The preincubation of the carboxylase with 1 millimolar HCO(3) (-) and 5 millimolar MgCl(2) resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate.In studies of dark (14)CO(2) fixation by endosperm slices, [(14)C]malate and [(14)C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H(14)CO(3) (-). In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as (14)CO(2). The minimal incorporation of the label from (14)CO(2) into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO(2) fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO(2) fixation. PMID- 16662979 TI - Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance. AB - Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O(2).Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O(2). Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [(14)C]sucrose primarily to CO(2) and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [(14)C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O(2). Lipids were also labeled when seeds were supplied with [(14)C]ethanol or [(14)C]acetate. Labeling experiments using the above compounds plus [(14)C]NaHCO(3), showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O(2). In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O(2). PMID- 16662980 TI - Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides. AB - The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo. PMID- 16662981 TI - Enzymes of ureide synthesis in pea and soybean. AB - Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5'-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5'-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation. PMID- 16662982 TI - A Guide to Establishing Water Potential of Aqueous Two-Phase Solutions (Polyethylene Glycol plus Dextran) by Amendment with Mannitol. AB - A practical guide to calculating the mannitol (MAN) amendment required to achieve the desired water potential (Psi) of polyethylene glycol/dextran (PEG/DEX) aqueous two-phase systems for protoplast purification is presented. The empirically generated equation Psi = 305[PEG'](2)[MAN] + 0.74[PEG'][MAN]T - 103[PEG'][MAN] + 5.6[PEG'](2)T - 623[PEG'](2) - 0.25[PEG']T + 12.7[PEG'] - 0.078[MAN]T - 22.75[MAN]accurately predicts experimental Psi (in bars). [PEG'] indicates the presence of DEX where [DEX] = [PEG]/(0.6-0.4[PEG]). The equation is applicable for these ranges: [PEG'] from 0.047 to 0.13 gram per gram H(2)O; [MAN] from 0 to 0.7 molal; T from 4.5 to 40 degrees C. Actual Psi should differ from derived Psi by no more than 8% for the least negative values to 4% for the most negative values. The Psi for solutions of MAN, of PEG, and of DEX were also determined. Equations to fit data for each were generated. Analyses indicated a significant synergistic effect on Psi when MAN is added to PEG/DEX and, at certain concentrations, between PEG and DEX. PMID- 16662983 TI - Evaluation of the water potentials of solutions of polyethylene glycol 8000 both in the absence and presence of other solutes. AB - Published and additional data for polyethylene glycol 8000 (PEG), formerly PEG 6000, solution water potentials (Psi) are compared. Actual bars Psi over the concentration range of 0 to 0.8 gram PEG per gram H(2)O and temperature (T) range of 5 to 40 degrees C are best predicted (probably within +/- 5%) by this equation: Psi = 1.29[PEG](2)T - 140[PEG](2) - 4.0[PEG]. Although transformable through division by [PEG] to virial equation form, results indicate that the coefficients are not virial. Mannitol (MAN) interacts with PEG to produce Psi significantly lower than additive. Vapor pressure osmometer (VPO) data for MAN PEG synergism compared favorably with those from thermocouple hygrometry; and VPO data showing the interactions between PEG and four salts are presented. The synergism of MAN-PEG and of NaCl-PEG are related linearly to the concentration of solute added with PEG. PMID- 16662984 TI - Effect of cyanide in dark and light on the membrane potential and the ATP level of young and mature green tissues of higher plants. AB - The effect of CN(-) and N(2) on the electrical membrane potential (E(m)) was compared with that of CN(-) on the ATP levels in cotyledons of Gossypium hirsutum and in Lemna gibba L. In mature cotton tissue, CN(-) depolarized E(m) to the energy-independent diffusion potential (E(D)) in the dark. In the light E(m) recovered transiently. The same was observed in leaves of Nicotiana, Avena, Impatiens, Kalanchoe, and in Lemna. In contrast, in young cotton cotyledons and tobacco leaves and, to a large extent, in +sucrose-grown Lemna, E(m) was depolarized to E(D) also in the light in a similar way as in the dark.In Lemna grown without sucrose, the energy-dependent component of E(m) was only partially depolarized by CN(-) in dark or light. Cyanide plus salicylhydroxamic acid completely reduced E(m) to E(D), abolished respiration and photosynthesis, and severely diminished the ATP level. This suggests the operation of a CN(-) insensitive respiration in uninjured Lemna. The initial CN(-)-induced decay of the ATP level in cotton and Lemna was more rapid than the decay of E(m). CN(-) induced oscillations of the ATP level were followed by similar but slower oscillations of E(m). This supports the view of a general dependence of E(m) on ATP. Discrepancies between inhibitor-induced changes of E(m) and ATP levels are suggested to result from additional regulation of E(m) by the cytoplasmatic pH value.A comparison of E(D) in young and mature cotton cotyledons in the dark and in the light suggests that in growing young cotyledons the different effect of CN(-) in the light is due to a less effective photosynthesis together with high mitochondrial respiration. In Lemna and in mature cotton tissue, E(m) in the light is maintained by noncyclic photophosphorylation and photosystem II, which is only partly inhibited by CN(-), thus resulting in an incomplete depolarization and recovery of E(m). Complete inhibition of photosynthetic O(2) evolution and membrane depolarization by CN(-) plus salicylhydroxamic acid are suggested to result from photooxidation. PMID- 16662985 TI - Expression of Nitrate and Nitrite Reductase Activities under Various Forms of Nitrogen Nutrition in Phaseolus vulgaris L. AB - The main objectives of this work were to study the effect of different N sources on plant growth, N accumulation, and on the expression of nitrate reductase activity in Phaseolus vulgaris L. leaves. Plants were grown under greenhouse conditions (15 to 25 kilolux; 16/8 hour day/night cycles) in plastic pots filled with perlite: vermiculite (1:1) and watered daily with a minus N solution (N(2) plants) or supplemented with either KNO(3), (NH(4))(2)SO(4), or urea as combined N sources.Significant levels of nitrate reductase activity in trifoliolate leaves of N(2)-, NH(4) (+)-, urea-, or NO(3) (-)-dependent plants was demonstrated throughout this work. Leaves from the urea- or NH(4) (+)-grown plants accumulated NO(2) (-) in the dark but not in the light when NO(2) (-) was supplied by vacuum infiltration. These results indicated that the potential for reduction of NO(3) ( ) or NO(2) (-) was not impaired by growing the plants on NH(4) (+) or urea and, in addition, provided evidence for the occurrence of a non-nitrate-inducible nitrite reductase. The nitrate reductase activities associated with N(2)-, NH(4) (+)-, or urea-dependent plants are tentatively regarded as ;constitutive' to differentiate from the widely occurring NO(3) (-)-inducible nitrate reductase activity.Plants grown on NO(3) (-) or urea accumulated significantly larger amounts of reduced N and dry matter as compared to NH(4) (+)- and N(2)-dependent plants. Regardless of N treatment, or size of plants, about 50% of the N accumulated by the plant was allocated to the leaves. PMID- 16662986 TI - Variability of Reaction Kinetics for Ribulose-1,5-bisphosphate Carboxylase in a Barley Population. AB - The photosynthetic enzyme ribulose bisphosphate carboxylase-oxygenase [EC 4.1.1.39] (RuBPCase) plays a key role in the carbon reduction system of plants. In this study, we determined the kinetic variability of RuBPCase among 46 varieties of Hordeum vulgare L. at two ages. The V(max) CO(2) and K(m) CO(2) of RuBPCase was determined for each cultivar. Varietal differences were found in K(m) CO(2) and V(max) CO(2) for one and four genotypes, respectively. One variety exhibited atypical behavior in both K(m) and V(max). A comparison of varieties and age showed a significant interaction between these factors for K(m) but not for V(max). These data indicate the presence of kinetic variability in RuBPCase within the H. vulgare population and perhaps between plant ages. PMID- 16662987 TI - Subcellular localization of rice leaf aryl acylamidase activity. AB - The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in rice (Oryza sativa L. var Starbonnet) leaves was investigated. The enzyme hydrolyzes and detoxifies the herbicide propanil (3,4 dichloropropionanilide) thereby accounting for immunity of the rice plant to herbicidal action. Fractionation of mesophyll protoplasts by differential centrifugation yielded the highest specific activity of amidase in the crude mitochondrial fraction. Further separation of density gradients of the silica sol Percoll also indicated that this enzyme was mitochondrial. By the use of biochemical markers, the purified mitochondrial fraction was shown to be substantially free of contamination from nuclei, chloroplasts, golgi, and plasma membranes. Subfractionation of the purified mitochondria suggests that this enzyme is located on the outer membrane. PMID- 16662988 TI - Effect of photoperiod on the metabolism of deuterium-labeled gibberellin a(53) in spinach. AB - Application of gibberellin A(53) (GA(53)) to short-day (SD)-grown spinach (Spinacia oleracea L.) plants caused an increase in petiole length and leaf angle similar to that found in plants transferred to long days (LD). [(2)H] GA(53) was fed to plants in SD, LD, and in a SD to LD transition experiment, and the metabolites were identified by gas chromatography with selected ion monitoring. After 2, 4, or 6 SD, [(2)H]GA(53) was converted to [(2)H]GA(19) and [(2)H]GA(44). No other metabolites were detected. After 2 LD, only [(2)H] GA(20) was identified. In the transition experiment in which plants were given 4 SD followed by 2 LD, all three metabolites were found. The results demonstrate unequivocally that GA(19), GA(20), and GA(44) are metabolic products of GA(53), and strongly suggest that photoperiod regulates GA metabolism, in part, by controlling the conversion of GA(19) to GA(20). PMID- 16662989 TI - Gas Exchange and Phytoluminography of Single Red Kidney Bean Leaves during Periods of Induced Stomatal Oscillations: A Demonstration of an Integrated, Spatially Resolving Physiometric Technique. AB - This report examines the capabilities of a new approach to the study of gas exchange and electron transport properties of single, intact leaves. The method combines conventional aspects of analysis with an image intensification system that records the spatial distribution of delayed light emission (DLE) over single leaf surfaces. The combined system was used to investigate physiological perturbations induced by exposure of single leaves of Phaseolus vulgaris cv ;California Light Red' to a combination of SO(2) (0.5 microliters per liter) and ozone (0.1 microliters per liter). Exposure of one-half of a leaf to this combination induced DLE and stomatal oscillations, but only in the half of the leaf exposed to the combined gases. Examination of phytoluminographs taken during these oscillations revealed distinct leaf patches where the greatest changes in DLE intensity occurred. This phenomenon is interpreted to be evidence that control of stomatal activity of intact plant leaves occurs within discrete leaf areas defined within the vascular network. PMID- 16662990 TI - Differential Response in the Water Status of Immature and Mature Fronds of the Ostrich Fern (Matteuccia struthiopteris [L.] Todaro) to a Mild Water Stress. AB - Experiments were conducted in growth chambers to examine the effect of a mild water stress (-200 kilopascals polyethylene glycol) on frond elongation and water status of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Measurements were taken for two days, starting one day after the application of polyethylene glycol. Total water potential in control (well-watered) plants was always significantly higher in immature fronds than in mature fronds. The osmotic potential in mature fronds was always significantly lower (about 800 kilopascals) than in immature fronds in both control and stressed plants. In immature fronds, the stress decreased elongation and total water and pressure potentials, while in mature fronds it increased total water and pressure potentials. The decreases in total and pressure potentials in immature fronds were approximately equal to the increases in mature fronds. PMID- 16662991 TI - Evidence for a Relationship between H Excretion and Auxin in Shoot Gravitropism. AB - The role of auxin and protons in the gravitropic response of the sunflower (Helianthus annuus L. cv Sungold) hypocotyl has been investigated. No physiological asymmetry in acid-growth capacity could be detected between the upper and lower surfaces of gravistimulated hypocotyls. These data imply that neutral buffers inhibit shoot gravitropism by preventing the establishment of a lateral proton gradient along gravitropically stimulated hypocotyls. Indirect evidence that auxin is involved in the establishment and/or maintenance of such a gradient derives from the quantitative assessment of the effects of exogenous auxin, anti-auxins, and vanadate on gravicurvature. At low concentrations, exogenous auxin accelerated curvature; at high concentrations, curvature was prevented. Vanadate, an inhibitor of auxin-enhanced H(+) secretion, alpha-(p chlorophenoxy)isobutyric acid (PCIB), an anti-auxin, and 2,3,5-triiodobenzoic acid (TIBA), an auxin-transport inhibitor, prevented observable asymmetric proton excretion using a brom cresol purple agar technique and also inhibited gravicurvature. Vanadate, PCIB, and TIBA inhibition of gravicurvature could be reversed with acid treatment to the lower surface of a gravistimulated hypocotyl. Auxin treatment to the lower surface of a gravistimulated hypocotyl did not reverse vanadate-induced inhibition, but it did partially reverse PCIB- and TIBA induced inhibition. These results indicate a close relationship between the acid growth theory and the differential growth responses of the sunflower hypocotyl during gravitropism. PMID- 16662992 TI - Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. AB - Photosynthetically active chloroplasts retaining high rates of fatty acid synthesis from [1-(14)C]acetate were purified from leaves of both 16:3 (Solanum nodiflorum, Chenopodium album) and 18:3 plants (Amaranthus lividus, Pisum sativum). A comparison of lipids into which newly synthesized fatty acids were incorporated revealed that, in 18:3 chloroplasts, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) were significantly less active than in 16:3 chloroplasts. In contrast, labeling rates of MGD from UDP-[(14)C]gal were similar for both types of chloroplasts.The composition and positional distribution of labeled fatty acids within the glycerides synthesized by isolated 16:3 and 18:3 chloroplasts were similar and in each case only a C18/C16 diacylglycerol backbone was synthesized. In nodiflorum chloroplasts, C18:1/C16:0 MGD assembled de novo was completely desaturated to the C18:3/C16:3 stage.Whereas newly synthesized C18/C18 MGD could not be detected in any of these chloroplasts if incubated with [(14)C]acetate after isolation, chloroplasts isolated from acetate-labeled leaves contained MGD with labeled C18 fatty acids at both sn-1 and sn-2 positions. Taken together, these results provide further evidence on an organellar level for the operation of pro- and eucaryotic pathways in the biosynthesis of MGD in different groups of plants. PMID- 16662993 TI - Effects of KCN and Salicylhydroxamic Acid on the Root Respiration of Pea Seedlings. AB - Polarography, using cylindrical platinum electrodes, proved suitable for measuring changes in the internal apical O(2) concentration of the primary root of pea (Pisum sativum L. cv Meteor) effected by KCN and/or salicylhydroxamic acid (SHAM) in the bathing medium. An electrical rootaeration analog was used to help evaluate some of the results. Concentrations of KCN 1%). During the 14 day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO(2) incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O(2) evolution could be detected although at high pH and light intensity O(2) evolution was recorded. PMID- 16663020 TI - ABA Uptake in Source and Sink Tissues of Sugar Beet. AB - The mode of abscisic acid (ABA) uptake was studied in excised leaf and root tissue discs of sugar beet (Beta vulgaris L.). Discs were incubated in buffered medium that contained 1 mm CaCl(2) and [(14)C]ABA. The sensitivity of ABA uptake to metabolic inhibitors and temperature indicated that the ABA transport system had an energy-dependent component. Energy-dependent uptake was greater in leaf than in root tissue (70% and 50%, respectively). Energy-dependent uptake by both tissues and passive uptake by root tissues were highly pH dependent. Maximal uptake was observed at pH 5.5. Leaf tissue incubated in the dark showed a 50% reduction of uptake as compared with tissue under light. The decrease was due to reduced passive uptake.The results suggest that ABA moves across membranes as the undissociated lipophilic species. As a weak acid, ABA would dissociate and accumulate in the more alkaline compartment. Therefore, the distribution of ABA within the tissue is regulated by the pH differential between any two compartments. Although diffusion may be the predominant form of transport, the uptake of ABA is dependent on metabolic energy for the establishment of a pH gradient across the membrane. PMID- 16663022 TI - The role of ethylene in the growth response of submerged deep water rice. AB - We investigated the effect of partial submergence on internode elongation in a Bangladesh variety of floating or deep water rice (Oryza sativa L., cv. Habiganj Aman II). In plants which were at least 21 days old, 7 days of submergence led to a 3- to 5-fold increase in internodal length. During submergence, the ethylene concentration in the internodes increased from about 0.02 to 1 microliters per liter. Treatment of nonsubmerged plants with ethylene also stimulated internode elongation. When ethylene synthesis in partially submerged plants was blocked with aminooxyacetic acid and aminoethoxyvinylglycine, internode elongation was inhibited. This growth inhibition was reversed when ethylene biosynthesis was restored with 1-aminocyclopropane-1-carboxylic acid (ACC). Radio-labeling studies showed that ethylene in floating rice was synthesized from methionine via ACC. Internodal tissue from submerged plants had a much higher capacity to form ethylene than did internodal tissue from nonsubmerged plants. This increase in ethylene synthesis appeared to be due to enhanced ACC formation rather than to increased conversion of ACC to ethylene. Our results indicate that ethylene produced during submergence is required for the stimulation of growth in submerged floating rice plants. PMID- 16663021 TI - Transport of dicarboxylic acids in castor bean mitochondria. AB - Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of (14)C from [(14)C]malate or [(14)C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of (14)C from [(14)C]malate or [(14)C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K(m) values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4 dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [(14)C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [(14)C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4 degrees C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [(14)C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K(m) of 2.5 millimolar and V(max) of 250 nanomoles per minute per milligram protein at 4 degrees C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation. PMID- 16663023 TI - How does deep water rice solve its aeration problem. AB - In partially flooded deep water rice (Oryza sativa L. cv Habiganj Aman II), continuous air layers trapped between the hydrophobic, corrugated surface of the leaf blades and the surrounding water constitute the major path of aeration. The conduction of gases through the internal air spaces of the leaf is negligible compared to the conduction of gases through the external air layers. The total volume of the air layers on both sides of a leaf blade is about 45% of the volume of the leaf blade itself. The size of the air layers around submerged leaf blades of cereals not adapted to conditions of partial flooding, e.g. of oats, barley, and wheat, is considerably smaller than that of rice. Gases move through the air layers not only by diffusion but also by mass flow. In darkness, air is drawn down from the atmosphere through the air layers along a pressure gradient created by solubilization of respiratory CO(2) in the surrounding water. In light, photosynthetic O(2) is expelled through the air layers to the atmosphere because the solubility of O(2) in water is much lower than that of CO(2). Air layers greatly increase the rate of photosynthetic carbon fixation by enlarging the surface of the gas-liquid interface available for CO(2) uptake from the water. Air layers are vital for the survival of the partially submerged rice plant. When leaves are washed with a dilute solution of a surfactant (Triton X-100), no air layers are formed under water. Plants without air layers do not grow in response to submergence, and the submerged parts of the plant deteriorate as evident by rapid loss of chlorophyll and protein. Air layers provide a significant survival advantage even to completely submerged rice plants. PMID- 16663024 TI - Diurnal variations in leaf fluorescence induction kinetics: variable fluorescence in crassulacean Acid metabolism plants. AB - The variable fluorescence of leaves from Kalanchoe daigremontiana and pineapple, Ananas comosus, both CAM plants, was found to change over a 24-hour cycle and to exhibit high temperature-dependent maxima during the night period. The time course of the induced fluorescence was correlated with malic acid accumulation but not with other aspects of CAM such as with the nature of the decarboxylation pathway or with stomatal movements. The variable fluorescences of sunflower (Helianthus annuus L.) and corn (Zea mays L.) leaves were compared with the CAM plants diurnally; both plants also exhibit high fluorescence maxima during the night period. We conclude that the assembly of the photosystems in the light is a primary process in photosynthesis induction and may be influenced by other cellular metabolic processes, specifically in the case of CAM leaves by malic acid accumulation. PMID- 16663025 TI - Interaction of chloroplasts with inhibitors: effects of two diphenylether herbicides, fomesafen and nitrofluorfen, on electron transport, and some comparisons with dibromothymoquinone, diuron, and paraquat. AB - Several effects on pea (Pisum sativum L. var Onwards) chloroplasts of a new diphenylether herbicide, fomesafen (5-[2-chloro-4-trifluoromethyl-phenoxy]-N methanesulfonyl-2 -nitrobenzamide) have been compared with those of a herbicide of related structure, nitrofluorfen (2-chloro-1-[4-nitrophenoxy]-4 [trifluoromethyl]benzene). Although both compounds produce the same light dependent symptoms of desiccation and chlorosis indicative of a common primary mechanism of action, this study is concerned with a more broadly based investigation of different effects on the electron transport system. Comparisons have also been made with other compounds interacting with the chloroplast. Unlike nitrofluorfen, fomesafen has little effect as an inhibitor of electron flow or energy transfer. Both compounds have the ability to stimulate superoxide production through a functional electron transport system, and this involves specifically the p-nitro substituent. The stimulation, which is not likely to be an essential part of the primary herbicidal effect, is diminished under conditions that remove the coupling factor. Evidence suggests that both diphenylethers may be able to bind to the coupling factor, and kinetic studies reveal this for dibromothymoquinone as well. Such a binding site might be an important feature in allowing the primary effect of the diphenylether herbicides to be expressed. PMID- 16663026 TI - Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids. AB - In vivo CO(2) fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.Forage harvest and nitrate application reduced both in vivo and in vitro CO(2) fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.The results demonstrate that CO(2) fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO(2) fixation for attached and detached alfalfa nodules at low CO(2) concentrations (0.13-0.38% CO(2)) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO(2) fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa. PMID- 16663027 TI - Changes in Starch Formation and Activities of Sucrose Phosphate Synthase and Cytoplasmic Fructose-1,6-bisphosphatase in Response to Source-Sink Alterations. AB - Short term experiments were conducted with vegetative soybean plants (Glycine max L. Merr. ;Ransom' or ;Arksoy') to determine whether sourcesink manipulations, which rapidly changed the ;demand' for sucrose and partitioning of photosynthetically fixed carbon into starch, were associated with alterations in activities of sucrose-P synthase and/or cytoplasmic fructose-1,6-bisphosphatase in leaf extracts. When demand for sucrose from a particular source leaf was increased by defoliation of other source leaves, starch accumulation was restricted and activities of both enzymes were markedly enhanced. When demand for sucrose from source leaves was limited by excision, starch accumulation in the detached leaves was increased while activity of sucrose-P synthase declined sharply. The consistent responsiveness of sucrose-P synthase activity to changes in demand for sucrose supports the contention that regulation of sucrose-P synthase is an integral component of the system which controls sucrose biosynthesis and partitioning of carbon between starch and sucrose biosynthesis in the light. PMID- 16663028 TI - Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast. AB - A method for cellular fractionation of Chlamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for cell fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination. PMID- 16663029 TI - Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii. AB - Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (-), fix CO(2) at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO(2) fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO(2) fixation in the light.High concentrations of inorganic phosphate (>2 millimolar) inhibit CO(2) fixation. This inhibition is overcome by the addition of glycerate 3 phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg(2+) concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO(2) fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer. PMID- 16663030 TI - Developmental Changes in the Free Amino Acid Pool and Total Protein Amino Acids of Pea Cotyledons (Pisum sativum L.). AB - Changes in the levels of twenty-two free amino acids and in the amino acid composition of the total protein were measured throughout the development of cotyledons of a dwarf garden pea, Pisum sativum cv Greenfeast, grown in a constant environment. A sensitive double-isotope dansylation technique was used. Fresh weight, dry weight, and protein content were also followed. Twenty of the amino acids showed synchronous changes in levels, giving a developmental pattern containing four peaks; major peaks occurred very early and very late in development. The amino acid composition of the total protein, which was always very different from that of the free amino acid pool, showed early changes to one consistent with the final storage protein composition of the seed. These changes included a 50% drop in methionine content and a 70% rise in cysteine. While the maximum free methionine level occurred early in development, that of cysteine was late. PMID- 16663031 TI - Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport. AB - Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds. PMID- 16663032 TI - Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization. AB - The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.In growth chamber studies, the fully expanded first trifoliolate leaf from NO(3) (-)-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO(3) (-) in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO(3) (-)-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO(3) (-)-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO(3) (-), and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO((x))] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild type plants. It is concluded that: (a) the NR activity in leaves of NO(3) (-) grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO((x)) evolution and occurs only in physiologically young leaves. PMID- 16663033 TI - Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance. AB - Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean (Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO(3) (-)-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO(3) (-) in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR 4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days.Urea-grown LNR-2 seedlings lack both inducible and constitutive NR activity, and were resistant to four days of treatment with 0.5 mm ClO(3) (-). Urea-grown wild-type seedlings, having only constitutive NR activity, developed ClO(3) (-) toxicity symptoms and suffered decreases in unifoliolate leaf NR activity and chlorophyll concentration. This suggests that (a) the reduction of ClO(3) (-) to ClO(2) (-) by NR is the major cause of ClO(3) (-) toxicity in soybeans and (b) the constitutive NR is active in situ.Segregation of the F(2) of reciprocal crosses between the wild type and the mutants indicated that absence of constitutive NR activity was controlled by a single recessive nuclear gene. Evolution of NO((x)) gas was also absent in these mutants, and this was found to be inherited jointly with constitutive NR activity: in 346 segregants, no recombinants were found. Allelism tests between LNR-2 and LNR-3, and LNR-2 and LNR-4, indicated that the constitutive NR mutation was at the same locus in each mutant. PMID- 16663034 TI - Hemicellulosic polymers of cell walls of zea coleoptiles. AB - Hemicellulosic polymers comprised about 43% of the primary walls of Zea mays L. cv WF9 x Bear 38 coleoptiles; these polymers were separated by an alkali-gradient into three major fractions. Fraction 1 (GAX I) was solubilized from walls with 0.01 to 0.045 n KOH and consisted of novel glucuronoarabino(galacto)xylans. Nearly six of every seven residues of these xylans were substituted predominantly with single arabinosyl sidegroups. Fraction 2 (GAX II), material released by 0.45 to 0.8 n KOH, was also enriched with glucuronoarabinoxylan, but only two of every three xylose residues was substituted. This xylan was similar to those found in Zea and other Graminaceous species. Both of these xylan fractions contained uronic acid, terminal- and 4-linked galactosyl, and small amounts of 2-, 3-, 5-, and 3,5-linked arabinosyl units. Fraction 3 (MG-GAX) was released by 2.0 to 3.0 n KOH and consisted of about 60% mixed-linked glucan and about 40% glucuronoarabinoxylan. This fraction represented about half of the total hemicellulosic material of the primary walls of these coleoptiles.The molecular weight of the highly substituted GAX I was approximately 21 kilodaltons as determined by the ratio of reducing sugar to total sugar, but ultracentrifugation studies and gel chromatography on Sepharose 4B-200 indicated that GAX I formed larger aggregates of primarily 50 to 90 kilodaltons, whereas most of the GAX II and virtually all of the MG-GAX materials were excluded by Sepharose 4B; exclusion from the Sepharose was correlated with the presence of mixed-linked glucan. Only GAX II and MG-GAX material demonstrated any appreciable binding to cellulose in vitro. PMID- 16663035 TI - Effect of Lithium on Thigmomorphogenesis in Bryonia dioica Ethylene Production and Sensitivity. AB - Rubbing internodes of Bryonia dioica plants reduced their ethylene production but increased their capacity to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. These results were explained by the previously shown rubbing-induced decrease of indoleacetic acid, which controls the level of ACC synthase, and by the increase of membrane-associated peroxidases which would participate in the conversion of ACC-ethylene. Pretreatment of the plants with Li had no significant effect on control plants but counteracted the rubbing-induced decrease of ethylene production and diminished the capacity of the internodes to convert ACC to ethylene. Exogenously applied ethylene induced an increase of peroxidase activity similar to that caused by rubbing. Inasmuch as both effects were reduced by Li, it was concluded that Li inhibition of thigmomorphogenetic processes was essentially due to a Li inhibition of the effect of ethylene formed in response to mechanical stimuli. The decreased ethylene production and ACC conversion capacity in the presence of Li were explained by a cellular redistribution of peroxidases. PMID- 16663036 TI - Effect of Deseeding on the Indole-3-acetic Acid Content of Shoots and Roots of Zea mays Seedlings. AB - We wished to determine the effect of endosperm removal on the amounts of free and esterified indole-3-acetic acid (IAA) in young Zea mays seedlings. The increases of IAA derived from endosperm and from biosynthesis, but without correction for catabolic losses, were 0.9 picomole of free IAA per shoot per hour, and 1.1 picomoles per shoot per hour of ester IAA. After deseeding, free IAA in the shoot declines by 40% following kernel removal and total (free + ester) IAA declines at a rate of about 1 picomole per shoot per hour. A slight, but insignificant increase of ester IAA occurs following endosperm removal. In the primary roots, the decreases of free IAA and total (free + ester) IAA are accelerated by seed removal. Thus, the endosperm appears to be a major source of IAA for the shoot and root. PMID- 16663037 TI - Conversion of Helminthosporium sacchari Toxin to Toxoids by beta Galactofuranosidase from Helminthosporium. AB - Helminthosporium sacchari produces a host-selective toxin and structurally related nontoxic compounds, here referred to as ;toxoids.' Toxin and the three toxoids were each isolated to a high level of purity and were hydrolyzed under acidic conditions. The released galactose was measured by a galactose oxidase/peroxidase assay. Toxin was found to contain four units of galactose per molecule, as previously reported. Toxoids I, II, and III contained one, two, and three units of galactose, respectively. In cultures of the fungus, toxin concentration peaked at 3 weeks, followed by a rapid decline; as toxin levels fell, the total amount of toxoids increased. An enzyme with beta galactofuranosidase activity was found in small amounts in the cultures of H. sacchari; the enzyme converted toxin to the toxoids in vitro. beta Galactofuranosidase was previously known from very few micro-organisms; therefore, several pathogenic Helminthosporia and other fungi were tested for production. beta-Galactofuranosidase activity in culture filtrates and mycelia of H. victoriae, H. maydis, H. carbonum, and H. turcicum was much greater than in filtrates and mycelium of H. sacchari. More work is needed to determine the significance of enzyme production by these fungi. No beta-galactofuranosidase was evident from Fusarium oxysporum and Cladosporium cucumerinum. PMID- 16663038 TI - Membrane properties of isolated winter wheat cells in relation to icing stress. AB - Isolated cell preparations of winter wheat (Triticum aestivum L.) were utilized to examine the effect of ice encasement at -1 degrees C and exposure to ethanol on metabolic and biochemical properties of cells. Following icing and ethanol treatments, passive efflux of amino acids increased gradually with duration of exposure to the stress, and closely paralleled the decline in viability of cells. In contrast, uptake of (86)Rb declined much more rapidly than viability following exposure to icing or ethanol. Electron spin resonance spectroscopy studies revealed no significant change in molecular ordering within the cell membranes following icing or exposure to ethanol, whereas a small but significant increase in order was detected in the noniced controls. O(2) consumption by isolated cells declined only gradually due to icing and ethanol treatments, and remained relatively high even when cell viability was severely reduced. These results indicate that the plasma membrane is a primary site of injury during ice encasement and that damage to the ion transport system is the earliest manifestation of this injury. PMID- 16663039 TI - Relation between Respiration and Senescence in Oat Leaves. AB - The respiration of excised oat (Avena sativa cv Victory) leaves and their sensitivity to inhibitors was followed during senescence under varied conditions. The respiration rate, which in controls reaches its peak on the third day in darkness, is lowered at the time of fastest loss of chlorophyll (as reported earlier) by seven unrelated reagents that all delay dark senescence. When senescence is delayed by white light or by cytokinins, the respiratory rise is correspondingly delayed. Kinetin and l-serine, which act as antagonists on senescence, also act as antagonists on the respiratory rate. However, an exception to this close correspondence between senescence and the respiratory rise is offered by the lower aliphatic alcohols, which delay dark senescence and yet accelerate the onset of the respiratory rise.The respiration of freshly cut leaves is insensitive to KCN up to 8 millimolar, but sensitive to benzhydroxamate (BAM), 1 to 2 millimolar BAM causing 25% promotion and higher concentrations inhibiting. At the respiratory peak, however, part of the respiration becomes KCN sensitive. Low concentrations of alcohols in darkness, or 3-(3,4-dichlorophenyl) 1,1-dimethylurea, diuron, in light, also render part of the respiration KCN sensitive, but this sensitivity soon disappears again. Some 10 to 15% of the respiration is insensitive to both inhibitors. Thus, cyanide sensitivity comes and goes, while BAM sensitivity is always present. The current concept of the cyanide-resistant pathway as an overflow, therefore, does not fit well with behavior of these leaves. The respiratory rise in leaf senescence is similar to, but not identical with, the climacteric in ripening fruits and the aging phenomenon in tuber slices. PMID- 16663040 TI - Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm. AB - Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction. PMID- 16663041 TI - Effect of Heat Shock on the mRNA-Directed Disease Resistance Response of Peas. AB - Pea tissue ;heat shocked' for 2 hours at 40 degrees C accumulates mRNAs that code for a series of new proteins called heat shock proteins. A different messenger RNA population, which codes for a high level of 20 or more ;resistance proteins,' accumulates in pea tissue as it resists plant pathogenic fungi. Heat shock treatment applied prior to fungal inoculation prevents the high level accumulation of messenger RNA coding for the 20 resistance proteins and blocks disease resistance. If the resistance response is initiated before the heat shock treatment or after heat shock recovery, messenger RNA accumulates for the resistance proteins and disease resistance develops. PMID- 16663042 TI - Host-Pathogen Interactions : XXIII. The Mechanism of the Antibacterial Action of Glycinol, a Pterocarpan Phytoalexin Synthesized by Soybeans. AB - The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol at bacteriostatic concentrations (e.g. 50 micrograms per milliliter) inhibits the ability of Erwinia carotovora to incorporate [(3)H]leucine, [(3)H]thymidine, or [(3)H]uridine into biopolymers. Exposure of Escherichia coli membrane vesicles to glycinol at 20 micrograms per milliliter results in inhibition of respiration-linked transport of [(14)C]lactose and [(14)C]glycine into the vesicles when either d-lactate or succinate is supplied as the energy source. The ability of E. coli membrane vesicles to transport [(14)C]alpha-methyl glucoside, a vectorial phosphorylation-mediated process, is also inhibited by glycinol at 20 micrograms per milliliter. Furthermore, exposure of membrane vesicles to glycinol (50 micrograms per milliliter) at 20 degrees C results in the leakage of accumulated [(14)C]alpha-methyl glucoside-6-phosphate. The effects of the phytoalexins glyceollin, capsidiol, and coumestrol, and daidzein, a compound structurally related to glycinol but without antibiotic activity, upon the E. coli membrane vesicle respiration-linked transport of [(14)C]glycine and of [(14)C]alpha-methyl glucoside was also examined. Glyceollin and coumestrol (50 micrograms per milliliter), but not daidzein, inhibit both membrane-associated transport processes. These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits d-lactate dependent transport of [(14)C]glycine but not vectorial phosphorylation-mediated transport of [(14)C]alpha-methyl glucoside. Thus, capsidiol's mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined. PMID- 16663043 TI - H fluxes in excised samanea motor tissue : I. Promotion by light. AB - Previous investigators revealed that white light-promoted leaflet opening in Samanea saman (Jacq) Merrill depends upon K(+) uptake by extensor cells and efflux from flexor cells of the pulvinus, while dark-promoted closure depends upon K(+) fluxes in the opposite directions. We now monitored H(+) fluxes during pulvinar movement to test a model proposing coupled H(+)/K(+) fluxes. H(+) fluxes were monitored by measuring changes in the pH of a weakly buffered solution (initial pH = 5.5) bathing excised strips of extensor or flexor tissue. White light at hour 3 of the usual dark period promoted pulvinar opening, H(+) efflux from extensor cells and uptake by flexor cells, while darkness at hours 2 to 4 of the usual light period promoted pulvinar closure, H(+) uptake by extensor cells and efflux from flexor cells. The following conditions altered H(+) fluxes during dark-promoted closure. (a) Light reversed the directions of the fluxes in both extensor and flexor cells. (b) Anoxia increased the rate of H(+) uptake by extensor cells and promoted H(+) uptake (rather than efflux) by flexor cells, consistent with an outwardly directed H(+) pump. KCN showed similar effects initially, but they were transient. (c) Increase in external pH from 5.5 to 6.7 promoted H(+) efflux (rather than uptake) by extensor cells and increased the rate of H(+) efflux from flexor cells, presumably by decreasing the rate of inward diffusion. (d) Change in external K(+) did not alter H(+) fluxes by extensor cells, but removal of external K(+) decreased the rate of H(+) efflux from flexor cells by 70%. These observations support a model for coupled H(+)/K(+) fluxes in pulvinar cells during light-and dark-promoted leaflet movements. PMID- 16663044 TI - H Fluxes in Excised Samanea Motor Tissue : II. Rhythmic Properties. AB - Homogeneous groups of cells were excised at regular intervals from opposing (extensor and flexor) motor tissue of Samanea saman (Jacq) Merrill maintained in white light for 34 hours. H(+) fluxes between the tissue and bathing solution were then monitored during 30 minutes of darkness. Flux rates in both cell types vary with circadian rhythms. Flexor cells secrete H(+) to the medium during two thirds of the circadian cycle and take up H(+) during the remainder of the cycle, while extensor cells take up H(+) from the medium during the entire cycle. PMID- 16663045 TI - Effects of SO(2) on Stomatal Metabolism in Pisum sativum L. AB - Pea (Pisum sativum L. cv ;Little Marvel') plants were exposed to SO(2) for short term (3 hours) and long term (2 days) at 0.2 and at 0.5 microliter per liter (ppm) levels. The effect of this treatment on the activity of phosphoenolpyruvate carboxylase, NAD- and NADP-malate dehydrogenases, and alanine aminotransferase from epidermis and whole leaves was investigated. Short-term exposure to SO(2) at 0.2 or 0.5 ppm decreased the activity of the carboxylase and the dehydrogenases in the epidermis. In contrast, the activity of the same three enzymes increased in whole leaves with either short- or long-term exposure to SO(2). Alanine aminotransferase in epidermis or whole leaves was not much affected by short-term exposure, but the epidermal activity was decreased and whole leaf activity was increased with long-term exposure. SO(2) exposure which was initiated prior to illumination decreased the free thiol content of both epidermis and of whole leaf. Net photosynthesis was reversibly inhibited by long-term exposure to SO(2) at 0.5 ppm. No effect of 0.5 ppm SO(2) on stomatal conductance was detectable after 3 hours. Stomatal conductance appeared to decrease after longer exposure times (2 days) at 0.5 ppm. PMID- 16663046 TI - Isolation and characterization of oat globulin messenger RNA. AB - When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton ;putative' precursors of oat globulin was probably the RNA which was approximately 18S in size. PMID- 16663047 TI - A morphometric study on the effects of ethylene treatment in promoting abscission of tobacco flower pedicels. AB - The ultrastructural changes observed in ethylene-induced abscission of tobacco flower pedicels (Nicotiana tabacum L. ;Little Turkish') were studied by the techniques of morphometric analysis. The surface area of the membranes, relative volume of the organelles, and the number of organelles were determined for both ethylene-treated and control cells. In pedicels exposed to ethylene for 4.5 to 5 hours, abscission was evident within the separation zone. The most significant change in cell structure was observed in the surface area of the rough endoplasmic reticulum which more than doubled with ethylene treatment of the tissue. PMID- 16663048 TI - Paraveinal Mesophyll of Soybean Leaves in Relation to Assimilate Transfer and Compartmentation : III. Immunohistochemical Localization of Specific Glycopeptides in the Vacuole after Depodding. AB - Immunohistochemical staining was used to determine the cellular distribution of two glycosylated polypeptides (molecular weights of 27 and 29 kilodaltons) which are normally present at low levels in soybean (Glycine max var ;Wye') leaves but which markedly accumulate after depodding. These polypeptides, which comprise a substantial portion of the total leaf soluble protein of depodded plants, were exclusively located in the vacuoles of paraveinal mesophyll and associated bundle sheath cells. These results support the unique role of the soybean leaf paraveinal mesophyll in the transport and spatial compartmentation of nitrogen reserves in relation to seed filling. PMID- 16663049 TI - Influence of Water Stress on the Vacuole/Extravacuole Distribution of Proline in Protoplasts of Nicotiana rustica. AB - Tobacco (Nicotiana rustica) plants were stressed by addition of polyethylene glycol solution (-20 bar) to the growth medium. The proline contents and concentrations in total protoplasts, vacuoles, and extravacuolar fractions of these plants have been determined and compared with protoplasts and cell fractions of well-watered plants. As compared to the control, the stress treatment of intact plants results in a 7-fold increase of the proline content in the extravacuolar fraction while the vacuolar content was enriched only 2.6-fold. In protoplasts of control plants, a proline concentration ratio (extravacuole to vacuole) of 1 was measured. In protoplasts of stressed plants, this ratio was nearly 3. Thus, water stress seems to have an effect on a tonoplast-localized transport system for proline. PMID- 16663050 TI - On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil. AB - Dunaliella bardawil, a beta-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a beta-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of beta-carotene per cell. As a result, the beta-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and beta-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis ( approximately 600 micromoles O(2) evolved per milligram chlorophyll per hour).Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the beta carotene content per cell, and therefore the higher the beta-carotene-to chlorophyll ratio. The results are interpreted as indicating a protecting effect of beta-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell. PMID- 16663051 TI - Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration. AB - In spinach (Spinacia oleracea Hybrid 102 [New World seeds]) and wheat (Triticum aestivum L. cv Gabo) leaves, O(2) uptake rates in the dark were faster after the plants had been allowed to photosynthesize for a period of several hours. Alternative path activity also increased following a period of photosynthesis in these leaves. No such effects were observed with isolated mitochondria. In spinach and wheat leaves, the level of fructose plus glucose decreased during a period of darkness. In pea (Pisum sativum cv Alaska) leaves, the level of these sugars did not vary significantly during the day, and respiratory rates were also constant. In slices cut from wheat leaves harvested at the end of the night, addition of sugars increased the rate of respiration and engaged the previously latent alternative oxidase. In pea leaves, O(2) uptake in the first few minutes following illumination was faster than that observed before illumination, but declined during the next 15 to 20 minutes. Adding the alternative oxidase inhibitor salicylhydroxamic acid, or imposing high bicarbonate concentrations during the period of photosynthesis, prevented the rise in O(2) uptake rate during the immediate post illumination period.We conclude that the level of respiratory substrate in leaves determines their rate of O(2) uptake, and the degree to which the alternative path contributes to that O(2) uptake. PMID- 16663052 TI - Regulation of chloroplastic carbonic anhydrase : effect of magnesium. AB - It was previously reported that magnesium ion inhibited carbonic anhydrase (Bamberger and Avron 1975 Plant Physiol 56: 481-485). Studies with partially purified carbonic anhydrase from spinach (Spinacia oleracea L.) chloroplasts show that the effect was the result of the chloride counterion and not the magnesium ion. Enzyme activity was reduced 50% upon addition of 3 to 10 millimolar MgCl(2) or KCl while all additions of MgSO(4) between 0.3 and 10 millimolar were mildly stimulatory. PMID- 16663053 TI - Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : I. General Characteristics. AB - Xerosicyos danguyi H. Humb. (Cucurbitaceae) is an unusual leaf succulent endemic to Madagascar. Under well-watered conditions the plant exhibited Crassulacean acid metabolism (CAM), as characterized by large diurnal changes in titratable acidity, predominantly nighttime stomatal opening and CO(2) uptake, and high delta(13)C values. When plants were exposed to water stress for a minimum of a month, they shifted to a mode of carbon metabolism previously labeled CAM-idling. Under this mode of metabolism, the plants exhibited reduced stomatal opening, reduced CO(2) uptake, dampened diurnal fluctuations in titratable acidity, and no apparent changes in the delta(13)C values. Additionally, investigations showed that the stress hormones 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) and abscisic acid increased as much as 6-fold in the water-stressed plants. The results are discussed in relation to physiological significance and evolution of the CAM-idling mode of metabolism. PMID- 16663054 TI - Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : II. Enzymology. AB - Xerosicyos danguyi Humbert (Cucurbitaceae) is a leaf succulent endemic to Madagascar. Under well-watered conditions, the plant exhibited Crassulacean acid metabolism (CAM) but shifted to a dampened form of CAM, CAM-idling, when subjected to water stress. The purpose of this investigation was to examine the effects of a shift in carbon metabolism on phosphoenolpyruvate carboxylase and on NADP-malic enzyme in X. danguyi. Experiments were conducted to determine the diurnal patterns of enzyme activity and pH optima of both enzymes, as well as the approximate molecular mass, kinetic patterns, malate inhibition, and glucose-6 phosphate stimulation of phosphoenolpyruvate carboxylase. The two enzymes extracted from well-watered and water-stressed plants were similar in most parameters investigated; thus, CAM-idling appeared to be only a dampened form of CAM photosynthesis. PMID- 16663055 TI - Cooperation of cytoplasmic and plastidial translation in formation of the photosynthetic apparatus and its stage-specific efficiency. AB - In synchronized Euglena gracilis, strain Z, the synthesis of the apoproteins for the chlorophyll-protein-complexes CPI, CPa, and LHCP is light-dependent and takes place in the light period in a characteristic consecutive fashion: CPI at 1 to 2 hours, CPa at 7 to 12 hours, and LHCP at 8 to 12 hours. The syntheses sequence of the chlorophyll-protein-complexes coincides with the efficiency alterations of the photosynthetic apparatus of E. gracilis during the light period of the cell cycle. In particular, the synthesis onset of the photosystem II-related polypeptides CPa and LHCP aligns with the decrease of oxygen evolution at 6 hours of the light period and is discussed as reorganization process in the thylakoids. PMID- 16663056 TI - Dehydration Injury in Germinating Soybean (Glycine max L. Merr.) Seeds. AB - The sensitivity of soybean (Glycine max L. Merr. cv Maple Arrow) seeds to dehydration changed during germination. Seeds were tolerant of dehydration to 10% moisture if dried at 6 hours of imbibition, but were susceptible to dehydration injury if dried at 36 hours of imbibition. Dehydration injury appeared as loss of germination, slower growth rates of isolated axes, hypocotyl and root curling, and altered membrane permeability. Increased electrolyte leakage due to dehydration treatment was observed only from isolated axes but not from cotyledons, suggesting that cotyledons are more tolerant of dehydration. The transition from a dehydration-tolerant to a dehydration-susceptible state coincided with radicle elongation. However, the prevention of cell elongation by osmotic treatment in polyethylene glycol (-6 bars) or imbibition in 20 micrograms per milliliter cycloheximide did not prevent the loss of dehydration tolerance suggesting that neither cell elongation nor cytoplasmic protein synthesis was responsible for the change in sensitivity of soybean seeds to dehydration. Furthermore, the rate of dehydration or rate of rehydration did not alter the response to the dehydration stress. PMID- 16663057 TI - P(700) Chlorophyll a-Protein : Purification, Characterization, and Antibody Preparation. AB - The P(700) chlorophyll alpha-protein was purified by preparative sodium dodecyl sulfate (SDS) gel electrophoresis from SDS-solubilized barley (Hordeum vulgare L., cv Himalaya) chloroplast membranes. After elution from the gel in the presence of 0.05 to 0.1% Triton X-100, the recovered protein had a chlorophyll/P(700) ratio of 50 to 60/1 and contained no chlorophyll b or cytochromes. Analysis of the polypeptide composition of the chlorophyll-protein revealed a 58 to 62 kilodalton (kD) polypeptide component but no lower molecular weight polypeptides. The 58 to 62 kD component was further resolved into two distinct polypeptide bands which were subsequently mapped by partial cyanogen bromide digestion and Staphylococcus aureus proteolysis. Based on results from the mapping experiments and other data, we suggest that the two components are conformational variants of a single polypeptide. Measurement of the chlorophyll to protein ratio by quantitative amino acid analysis and consideration of the yield of P(700) in the protein isolate suggest that, contrary to previous models (Bengis and Nelson, 1975, 1977), P(700)in vivo is associated with a minimum of four subunits of approximately 60 kD.Antibodies raised against the photochemically active chlorophyll-protein complex from barley reacted specifically with the 58 to 62 kD apoprotein. The same preparative electrophoresis procedure was used to isolate photochemically active P(700) chlorophyll a-protein from soybean (Glycine max L.), tobacco (Nicotiana tobacum L.), petunia (Petunia x hybrida), tomato (Lycopersicum esculentum), and Chlamydomonas reinhardti. The isolated complex from all species exhibited identical polypeptide compositions and chlorophyll/P(700) ratios. Antibodies to the barley protein cross reacted with all species tested demonstrating the highly conserved structure of the apoprotein. PMID- 16663058 TI - Metabolism of C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules. AB - The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by (14)CO(2)-labeling studies and analysis of nodule enzymes. Plants were exposed to (14)CO(2) for 30 minutes, followed by (12)CO(2) for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in alpha,alpha-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids. PMID- 16663059 TI - Identification of enzymes that are effective for isolating protoplasts from grass leaves. AB - Cellulase C(1), cellulase Cx, and xylanase were isolated and purified from a cellulase preparation of Trichoderma viride as enzymes effective in the isolation of protoplasts from oat leaves. Pectin lyase which is specific for methyl galacturonide linkages was also found to be a useful enzyme for the isolation of protoplasts from the tissues. This suggested that pectic polysaccharides with a high degree of esterification may play an important role in cell walls of Gramineae. It was necessary to use the mixture of cellulase C(1), cellulase Cx, xylanase, and pectin lyase for the rapid isolation of protoplasts, while a small amount of protoplasts could be isolated from oat leaves by cellulase C(1) plus xylanase or cellulase C(1) plus pectin lyase. The mixture of four enzymes also was effective in the isolation of protoplasts from the leaves of wheat, barley, and corn. PMID- 16663060 TI - Clonal variation for tolerance to polyethylene glycol-induced water stress in cultured tomato cells. AB - Cell clones were isolated from a population of cultured tomato (Lycopersicon esculentum Mill cv VFNT-cherry) cells and their tolerance to polyethylene glycol (PEG)-induced water stress was measured. Considerable variation for tolerance among the clones was found. Tolerance differences between clones appeared to be spontaneous and were different from tolerance differences between adapted and unadapted cells. Unlike adapted (selected by exposure to PEG) cells, cell clones retained their relative tolerance for many generations in the absence of selection pressure, and tolerance of both relatively tolerant and intolerant clones was very dependent on growth cycle stage and inoculum density. Analysis of subclones isolated from relatively tolerant and intolerant parent clones revealed that each parent clone gives rise to progeny with tolerances near the mean tolerance of both parents. However, progeny populations of both tolerant and intolerant parents are enriched with individuals with phenotypes nearer the mean response of their respective parent populations. When exposed to PEG, relatively tolerant and intolerant clones alike become adapted to the level of PEG to which they are exposed, and have the same phenotypic level of tolerance. Thus, selection by exposure to stress is unable to discriminate (on the basis of growth) between the innately tolerant and intolerant cell types within the population. This is indicated also by the fact that clones isolated from a population of cells adjusted to growth on 25% PEG do not show an enriched frequency of tolerant phenotypes when grown in the absence of PEG compared to the nonselected normal cell population which has never been adjusted to growth on PEG. PMID- 16663061 TI - Partial Purification and Characterization of Cystine Lyase from Cabbage (Brassica oleracea var capitata). AB - Cabbage (Brassica oleracea var capitata) leaves were used as a source of cystine lyase. Diethylaminoethyl-cellulose chromatography resolved two peaks of activity, designated I and II.Cystine lyase I (molecular weight 145,000) and O-acetylserine sulfhydrylase (molecular weight 70,000) were resolved by Bio-Gel A-0.5M chromatography. This isozyme catalyzed an alpha,beta-elimination reaction with cystine, cysteine, O-acetylserine, and several S-substituted cysteines. The substrate specificity was similar to previously reported S-alkylcysteine lyases. The elution profiles during purification, and heat inactivation studies indicated that the above reactions were catalyzed by a single protein. The pH optimun with cystine and cysteine as substrate was 8.5 to 9.0, and the K(m) values were: cystine (0.3 mm), cysteine (0.3 mm), O-acetylserine (6 mm), and S-methylcysteine sulfoxide (1.8 mm).Cystine lyase II was resolved into three peaks (molecular weight greater than 500,000, 240,000, and 145,000) using Bio-Gel A-0.5M chromatography. This enzyme degraded l-cystine, l-cysteine, O-acetylserine, S methylcysteine sulfoxide, and djenkolic acid. The pH optimum with cystine and cysteine was 8.5 to 9.0, and the K(m) values were: cystine (0.3 mm), cysteine (0.3 mm), O-acetylserine (12.5 mm), and S-methylcysteine sulfoxide (3.7 mm). PMID- 16663062 TI - Subcellular localization of hexose kinases in pea stems: mitochondrial hexokinase. AB - The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K(m) 76 micromolar) and mannose (K(m) 71 micromolar) and a relatively low affinity for fructose (K(m) 15.7 millimolar). The K(m) for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al(3+) was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems. PMID- 16663063 TI - Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves. AB - Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.During the time proline was accumulating, [(14)C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [(14)C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses. PMID- 16663064 TI - Cell Wall Metabolism in Ripening Fruit : III. Purification of an Endo-beta-1,4 xylanase that Degrades a Structural Polysaccharide of Pear Fruit Cell Walls. AB - A beta-1,4-xylanase has been purified from the mixture of carbohydrate-degrading enzymes found in a commercial preparation from cultures of Trichoderma viride. Purification from the desalted enzyme mixture is accomplished either by preparative isoelectric focusing or in two-column chromatographic steps. The xylanase has maximal activity at pH 5.0 and a molecular weight of approximately 13,000 daltons. The enzyme loses activity when heated to above 45 degrees C. The xylanase degrades xylans from larch and pear cell walls in an apparently endo fashion. PMID- 16663065 TI - Dependence of photosynthetic rates on leaf density thickness in deciduous woody plants grown in sun and shade. AB - Comparisons of photosynthetic rates were made on leaves of ten species of woody dicotyledons grown in the field under full sun or under a canopy which transmitted approximately 18% of full light. Photosynthesis and dark respiration were measured and compared on various bases: area, chlorophyll, fresh weight of lamina, density thickness (fresh weight per unit area), and protein.Light saturated photosynthesis per unit area or unit chlorophyll was about 1.5 times greater in the sun leaves than in the shade leaves and essentially equal per unit fresh weight or unit protein. Sun leaves were thicker but the enzymes per unit fresh weight remained constant as thickness varied. Chlorophyll per unit area remained about constant; chlorophyll per unit fresh weight varied inversely with changes in leaf thickness. Thus, density thickness variation is important in photosynthetic adaptation to sun and shade. This is also shown by the relationship between light-saturated photosynthesis per unit area and density thickness. PMID- 16663066 TI - Growth characteristics, grain filling, and assimilate transport in a shrunken endosperm mutant of barley. AB - The reported inheritance pattern of the seg1 shrunken endosperm mutant of barley (Hordeum vulgare L. cv Betzes) suggests that some defective process in the maternal plant tissues, and not in the endosperm, prevents normal grain filling in the mutant. To identify the physiological mechanism of the mutation, we compared growth, carbon exchange, and assimilate transport of Betzes and seg1 plants. Betzes and seg1 plants did not differ in mean relative growth rate, mean net assimilation rate, or carbon exchange rate. The rate and duration of grain growth of seg1 was lower than Betzes on intact plants and on detached, cultured spikes. Increasing the supply of sucrose in culture media up to 300 mm sucrose did not eliminate differences between normal and mutant grain growth. Translocation of (14)C-labeled assimilates into seg1 grains ceased by 21 days after anthesis, and assimilates were diverted to lower plant parts. In contrast, assimilates were still entering Betzes grains at 29 days after anthesis. Evidence suggests that some maternal spike or grain tissue is affected by the mutation after the onset of grain filling. Identification of the specific seg1 defect may provide information about the cessation of normal grain filling. PMID- 16663067 TI - Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis. AB - A NaCl-tolerant cell line which was selected from ovular callus of ;Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na(+) and Cl(-) uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K(+) and Cl(-) accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl(-). (d) Removal of Ca(2+) from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt sensitive cells does not change. PMID- 16663068 TI - Investigations on the Mechanism of the Brassinosteroid Response: I. Indole-3 acetic Acid Metabolism and Transport. AB - A brassinosteroid treatment of light-grown first internode sections of Phaseolus vulgaris results in an increased bending response following unilateral indole-3 acetic acid (IAA) application. Reverse isotope dilution analysis shows that this increased response is not due to an increase in the concentration of applied IAA in the tissue or a change in the amount of IAA conjugated. Treatment with the brassinosteroid also does not affect the rate of IAA transport as measured using the agar block method. These results indicate that even though brassinosteroid potentiates auxin action, it does not have a direct effect on IAA uptake, metabolism, or cell to cell transport. PMID- 16663069 TI - Effect of supra-ambient oxygen on nitrogenase activity (c(2)h(2)) and root respiration of soybeans and isolated soybean bacteroids. AB - Isolated soybean (Glycine max [L.] Merr. cv Wilkin) bacteroids have O(2) dependent nitrogenase activity which is strongly inhibited by supraoptimal O(2) concentrations. Oxygen-inhibited nitrogenase activity is recovered by addition of 10 millimolar sodium succinate or by lowering the O(2) concentration.Brief treatment of roots of intact soybean plants with 1.0 atmosphere O(2) reduces nitrogenase activity (C(2)H(2)). There is a rapid partial recovery of activity within 2 to 3 hours, and a slower return to near normal levels by 36 hours. The drop and recovery of nitrogenase activity is accompanied by a parallel drop and increase in root respiration. There is a direct relationship between the change in respiration and the change in acetylene reduction following O(2) treatment. The O(2)-mediated changes in nitrogenase activity and root respiration are not affected by the planting medium. The ratio of the change in respiration to the change in nitrogenase activity was the same in 13 soybean cultivars. PMID- 16663070 TI - Root respiration associated with nitrogenase activity (c(2)h(2)) of soybean, and a comparison of estimates. AB - Root respiration associated with symbiotic fixation in soybean (Glycine max [L.] Merr.) was estimated by four methods.Averaged over the life of the plant, the root respires 5.8 milligrams C per milligram N accumulated from fixation. When nitrogenase (C(2)H(2)) activity and root respiration were decreased by treating roots briefly with 1.0 atmosphere O(2), the respiration associated with nitrogenase was estimated as 2.10 micromoles CO(2) per micromole C(2)H(4).When nitrogenase activity and respiration were decreased by addition of nitrate, the respiration associated with fixation was calculated as 2.90 micromoles CO(2) per micromole C(2)H(4). Removing nodules from roots decreased fixation and root respiration, and the ratio was 4.08 micromoles CO(2) per micromole C(2)H(4). When soybean plants were kept in prolonged darkness, then returned to light, the associated drop and recovery of respiration and nitrogenase activity had a ratio of 4.36 micromoles CO(2) per micromole C(2)H(2). PMID- 16663071 TI - Phytochrome-mediated cellular photomorphogenesis. AB - Red light-induced cell elongation and division in intact, etiolated oat (Avena sativa cv Lodi) seedlings have been assessed. The middle of coleoptile was especially responsive in the very low fluence range whereas the region immediately below the coleoptile tip and the two regions just above the coleoptilar node were more responsive than the entire organ in the low fluence range. These responses in the coleoptile are both the result of an increase in cell elongation. Coleoptile cell division is slightly inhibited in the very low and slightly stimulated by red light in the low fluence range.The one-sixth of the mesocotyl closest to the node is more suppressed in its growth than is any other region in the very low fluence range. However, the low fluence response involved the entire mesocotyl equally. In the apical one-sixth of the mesocotyl, a strong suppression of cell division and a weak suppression of cell elongation occurs. In the lower five regions of the mesocotyl, red light in both fluence ranges suppresses only cell elongation. Apparently, the difference between red light-induced oat growth stimulation and suppression primarily involves differences in the response of the cell elongation process. PMID- 16663072 TI - Endogenous levels of abscisic Acid and decanoic Acid in dutch iris bulbs and the influence of abscisic Acid and decanoic Acid on iris meristems cultured in vitro. AB - Abscisic acid (ABA) and decanoic acid inhibited shoot elongation and floral development of Dutch iris (Iris hollandica Hoog. cv Ideal) meristems cultured in vitro. No synergism with respect to inhibition of leaf growth between ABA and decanoic acid was observed. With monthly harvest dates, from July 10, 1981 to October 10, 1981, there was a progressive decrease in endogenous level of free ABA in ;Ideal' iris bulbs. Bulbs subjected to a full set of the usual preplanting storage conditions flowered, on average, 46 days after planting versus 194 days after planting for bulbs planted directly after harvest. ABA levels at harvest were 4- to 5-fold those after the preplanting storage treatment. In general, ABA levels did not correlate well with the length of time from planting until flowering of iris bulbs. Endogenous decanoic acid levels did not follow any pattern with respect to harvest date or postharvest treatment. After the postharvest high temperature treatment, there was about a 3-fold increase in nonscale decanoic acid concentration. Decanoic acid levels, in nonscale tissue, remained high after each of the other postharvest treatments. It is concluded that there is no good evidence to support the contention that either ABA or decanoic acid is directly involved in iris bulb dormancy. PMID- 16663073 TI - Phytochrome Control of the Expression of Two Nuclear Genes Encoding Chloroplast Proteins in Lemna gibba L. G-3. AB - Hybridization probes for two nuclear-coded chloroplast proteins of Lemna gibba L. G-3 have been constructed in order to investigate phytochrome regulation of specific sequences. The first probe is a cDNA clone encoding the small subunit of ribulose 1,5-bisphosphate carboxylase. This probe was isolated from a set of Lemna cDNA clones in the bacterial plasmid pBR322. The second probe is a subclone of a genomic clone encoding the light-harvesting chlorophyll a/b-protein. This clone was isolated from a set of genomic clones constructed in the lambda vector Charon 4 with L. gibba DNA fragments generated by partial EcoR1 digestion. The identity of these clones was confirmed by in vitro translation of RNA which hybridized to the cloned DNA. Plants grown under continuous white light contain high concentrations of both RNA sequences; however, when these plants are put into darkness the concentration of these RNAs decreases rapidly relative to the total amount of RNA. Plants grown in the dark with intermittent red light (2 minutes/8 hours) and put into complete darkness for 8 days also contain lower concentrations of the sequences in the total RNA. One minute of red light after this dark period results in a rapid increase in the levels of RNA hybridizing to the probes. The effect of red light can be reversed by far-red light. These experiments demonstrate that phytochrome action can rapidly influence either the rates of transcription or the rates of degradation of these mRNAs. PMID- 16663074 TI - Oxygen and hydrogen isotopes in fruit and vegetable juices. AB - (18)O/(16)O ratios from the juices of a number of fruits and vegetables were measured and found to be isotopically more enriched than the water in which they grew. Fast-growing high-water-content vegetables exhibited less enrichment than slower growing fruits such as apples, pears, and plums. (18)O/(16)O measurements were also made on the water from various sections of several plants, and the enrichment was found to occur in the following order: leaves > fruit > stem >/= ground water.D/H and (18)O/(16)O measurements were made on a series of grape juice samples and, when plotted against each other, gave a slope of 3.9, indicating that the physical process causing this enrichment was probably evaporation, i.e. evapotranspiration. PMID- 16663075 TI - Provisions of reductant for the hydroxypyruvate to glycerate conversion in leaf peroxisomes : a critical evaluation of the proposed malate/aspartate shuttle. AB - A series of experiments, with Secale cereale and Triticum aestivum var Argee, to evaluate critically the ability of a malate/aspartate shuttle to provide reducing equivalents to drive hydroxypyruvate reduction to glycerate led to the conclusion that the shuttle, as previously envisioned, does not supply NADH to the peroxisomal matrix. First, analysis of coupled malate dehydrogenase and glutamate oxaloacetate transaminase activities in the directions required for intraperoxisomal NADH generation indicated that the peroxisomal enzyme activities were insufficient to account for necessary rates of photorespiratory carbon flux. Second, although the peroxisomal isozyme of malate dehydrogenase comprised a substantial portion (40%) of total cellular activity, less than 7% of the cellular glutamate-oxaloacetate transmaminase activity was associated with the peroxisomes. Third, a peroxisomal extract was able to reduce added NAD only slowly upon addition of malate and glutamate. The rate of NAD reduction was greatly enhanced in the presence of exogenously added glutamateoxaloacetate transaminase. Finally, intact peroxisomes were unable to reduce hydroxypyruvate to glycerate when supplied with malate and glutamate in the absence of exogenously added pyridine nucleotides, although they readily reduced hydroxypyruvate when exogenous pyridine nucleotides were supplied. Three alternative mechanisms, which are in agreement with observed data and which could serve to supply the reducing power to the peroxisomal matrix, are discussed. PMID- 16663076 TI - Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes. AB - Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum. PMID- 16663077 TI - Interaction of Combined Nitrogen with the Expression of Root-Associated Nitrogenase Activity in Grasses and with the Development of N(2) Fixation in Soybean (Glycine max L. Merr.). AB - Soluble root N concentrations of corn, sorghum, pearl millet, rice, wild rice, and soybeans were determined and related to measurements of nitrogenase activity and changes in availability of combined N to plants. In corn, sorghum, and pearl millet, applications of fertilizer N increased soluble root N concentrations, but root-associated nitrogenase activity was negligible in control and treated plants. Applications of NH(4)NO(3) to rice increased the water soluble root N concentrations and inhibited root-associated nitrogenase activity. In wild rice, root-associated nitrogenase activity was absent during vegetative growth and developed at the reproductive growth stage. The soluble root N concentration decreased progressively as wild rice grew indicating that the availability of combined N in the root environment declined. Therefore, development of nitrogenase activity in wild rice is associated with the change in availability of combined N in the root environment. The development of nitrogenase activity in wild rice was probably not due to colonization of roots by N(2)-fixing bacteria because most probable numbers of recovery did not significantly vary throughout the plants' growth cycle. In field-grown soybeans with or without fertilizer N application, we also observed a relationship between a decrease in soluble root N concentration and the development of nitrogenase activity. PMID- 16663078 TI - Inhibition of phytoalexin synthesis in arachidonic Acid-stressed potato tissue by inhibitors of lipoxygenase and cyanide-resistant respiration. AB - Arachidonic acid-stressed potato tuber discs synthesized the phytoalexin rishitin. This synthesis was inhibited by salicylhydroxamic acid (SHAM), and to a lesser extent by tetraethylthiuram disulfide (disulfiram). Disulfiram was less effective apparently because it was inactivated in the tuber discs. Disulfiram and SHAM both inhibited cyanide-resistant respiration of whole potato discs and lipoxygenase extracted from these discs. When low disulfiram concentrations were used, the lipoxygenase inhibition was quickly overcome, again because the disulfiram apparently was inactivated by oxidation. PMID- 16663079 TI - Protein Changes during the Stratification of Malus domestica Borkh. Seed. AB - Apple seeds (Malus domestica Borkh. cv Golden Delicious) were stratified at 5 and 15 degrees C for various lengths, weighed, and soluble protein of axis and cotyledon tissue was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only seeds treated at 5 degrees C germinated; seeds treated at 15 degrees C did not germinate. Optimal germination required 63 days of stratification. Excised embryos required less stratification time for germination than intact seeds. When stratification was less than 35 days, the resulting seedlings from 5 degrees C stratified embryos were dwarfed and epinastic. After 63 days of stratification, axes from 5 and 15 degrees C treated intact seeds had increased in fresh weight by 72 and 28% (w/w), respectively. The dry weights of the axes did not change significantly and both fresh and dry weights of cotyledons remained unchanged during stratification. Total soluble protein in axes and cotyledons changed very little during stratification. However, axis polypeptide profiles changed. Most obvious was the occurrence of a new polypeptide and the increase of four other clearly identifiable polypeptides during 5 degrees C treatment. The levels of the five most predominant axis proteins decreased at the same time. We observed no changes in the profiles of soluble cotyledon proteins. Control seeds kept at -10 degrees C showed none of the reported changes. PMID- 16663080 TI - Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells : III. Subcellular Distribution of Prolyl Hydroxylase. AB - The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(l-proline) in the presence of oxygen, alpha-ketoglutarate, ferrous ion, and ascorbate. The K(m) for poly(l-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocollagen analog (Pro Pro-Gly)(5).4H(2)O.Fractionation of membranes from protoplast lysates on a discontinuous sucrose/sorbitol density gradient, followed by centrifugation on a linear sucrose gradient in the presence of Mg(2+), leads to a clear separation of a number of membrane components. The membrane components have been tentatively identified using marker enzymes and assayed for peptidyl prolyl hydroxylase. It is concluded that the ryegrass prolyl hydroxylase is enriched in Golgi-derived membranes, but that significant amounts are also located in other subcellular fractions, including the rough endoplasmic reticulum. PMID- 16663081 TI - Internode length in pisum: do the internode length genes effect growth in dark grown plants? AB - Internode length in light-grown peas (Pisum sativum L.) is controlled by the interaction of genes occupying at least five major loci, Le, La, Cry, Na, and Lm. The present work shows that the genes at all of the loci examined (Le, Cry, and Na) also exert an effect on internode length in plants grown in complete darkness. Preliminary results using pure lines were verified using either segregating progenies or near isogenic lines. The major cause of the differences was due to a change in the number of cells per internode rather than to an alteration of the cell length. Since the genes occupying at least two of these loci, Le and Na, have been shown to be directly involved with gibberellin metabolism, it appears that gibberellins are not only essential for elongation in the dark but are limiting for elongation in the nana (extremely short, na), dwarf (Na le), and tall (Na Le) phenotypes. These results are supported by the large inhibitory effects of AMO 1618 treatments on stem elongation in dwarf and tall lines grown in the dark and the fact that applied gibberellic acid could overcome this inhibition and greatly promote elongation in a gibberellin-deficient na line. It is clear that the internode length genes, and in particular the alleles at the Le locus, are not acting by simply controlling the sensitivity of the plant to light. PMID- 16663082 TI - Modification of Blue Light Photoresponses by Riboflavin Analogs in Neurospora crassa. AB - The effect of riboflavin analogs on blue light responses in a riboflavin mutant of Neurospora crassa was studied. The analogs 1-deazariboflavin and roseoflavin, which have red-shifted absorption, acted as photoreceptors for the photosuppression and phase shifting of circadian conidiation by 540 nm light, but were ineffective as photoreceptors for the induction of carotenoid synthesis. These results provide addtional evidence implicating a flavin photoreceptor for at least two blue light responses of Neurospora. PMID- 16663083 TI - Regulation of Sucrose Synthesis by Cytoplasmic Fructosebisphosphatase and Sucrose Phosphate Synthase during Photosynthesis in Varying Light and Carbon Dioxide. AB - The aim of this work was to investigate whether sucrose synthesis in the cytosol of leaf cells is regulated in response to the supply of energy and organic carbon from the chloroplast. Fluxes into sucrose and metabolite levels in wheat (Triticum aestivum var Timmo) leaf protoplasts were compared in a range of light intensities and CO(2) concentrations, showing that sucrose-phosphate synthase and the cytosolic fructose-1,6-bisphosphatase are inhibited in situ when the supply of trioseP from the chloroplasts decreases. Such a regulation might aid CO(2) fixation in limiting conditions by permitting stromal metabolites to be maintained at higher levels than would otherwise be possible. PMID- 16663084 TI - Water Relations, Diurnal Acidity Changes, and Productivity of a Cultivated Cactus, Opuntia ficus-indica. AB - Physiological responses of the Crassulacean acid metabolism (CAM) plant Opuntia ficus-indica (Cactaceae) were studied on a commercial plantation in central Chile. Young cladodes (flattened stems) and flower buds exhibited daytime stomatal opening, whereas mature cladodes and fruit exhibited the nocturnal stomatal opening characteristic of CAM plants. Severe water stress suppressed the nocturnal stomatal opening by mature cladodes, but their high water vapor conductance occurring near dawn was not affected. Nocturnal acidity increases were not as sensitive to water stress as was the nocturnal stomatal opening. The magnitude of the nocturnal acidity increases depended on the total daily photosynthetically active radiation (PAR), being 90% PAR-saturated at 27 moles per square meter per day for a mean nighttime air temperature of 5 degrees C and at 20 moles per square meter per day for 18 degrees C. Inasmuch as the PAR received on unshaded vertical surfaces averaged about 21 moles per square meter per day, nocturnal acidity increases by the cladodes were on the verge of being PAR-limited in the field. The net assimilation rate, which was positive throughout the year, annually averaged 3.4 grams per square meter per day for 1.0 and 2.0-year-old plants. Plants that were 5.4 years old had 7.2 square meters of cladode surface area (both sides) and an annual dry weight productivity of 13 megagrams (metric tons) per hectare per year when their ground cover was 32%. This substantial productivity for a CAM plant was accompanied by the highest nocturnal acidity increase so far observed in the field, 0.78 mole H(+) per square meter. PMID- 16663085 TI - Derepression of amino Acid-h cotransport in developing soybean embryos. AB - The uptake of the unnatural amino acid alpha-aminoisobutyric acid (AIB) and glutamine by developing soybean (Glycine max Merr. cv Chippewa 64) embryos was investigated. In freshly excised embryos, the accumulation ratio (cytoplasmic concentration/external concentration) of AIB did not exceed 1.0. After an 18-hour preincubation in nitrogen-free medium the accumulation ratio of AIB exceeded 4.5 at an external AIB concentration of 10 micromolar. This indicates the derepression of an active amino acid uptake mechanism operative at low external amino acid concentration. The presence of sucrose, NH(4)NO(3), or glutamine during a 21-hour preincubation prior to measuring glutamine uptake inhibited the enhancement of uptake by 43%, 51%, and 96%, respectively. The time course of the decline in free amino acids and the time course of enhancement of amino acid uptake was not consistent with enhanced uptake resulting from relief of transinhibition, but suggested instead the derepression of synthesis of new carriers. The time course of enhancement of amino acid uptake was paralleled by an increase in glutamine-induced depolarization of the membrane potential. The kinetics of glutamine uptake indicated the presence of a saturable and a nonsaturable component of uptake. The saturable component of uptake is attributed to a mechanism of amino acid-H(+) cotransport which is derepressed by nitrogen and/or carbon starvation. At physiological concentrations of amino acids, uptake through the saturable system in freshly excised embryos is negligible. Thus, uptake through the nonsaturable system is of primary importance in the nitrogen nutrition of developing soybean embryos. PMID- 16663086 TI - Germination of Echinochloa crus-galli (Barnyard Grass) Seeds under Anaerobic Conditions : Respiration and Response to Metabolic Inhibitors. AB - Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO(2) in Echinochloa and rice is lower and the peak rate of CO(2) evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO(2)] N(2)/[CO(2)] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N(2), but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed. PMID- 16663087 TI - Rate-Limiting Steps of Electron Transport in Chloroplasts during Ontogeny and Senescence of Barley. AB - Partial photochemical activities and concentrations of electron carriers were measured relative to chlorophyll in barley (Hordeum vulgare L.) thylakoids, isolated from primary leaves during ontogeny and senescence. Thylakoids from mature leaves generated somewhat higher quantum efficiencies than thylakoids from premature or senescing leaves; this phenomenon did not appear to be caused by any deficiency of water-splitting enzyme. Under conditions of saturating light, the noncyclic electron flux from water to the reducing side of photosystem I increased during leaf ontogeny, peaked at maturity, and declined during senescence. However, electron fluxes appeared to be limited at different steps before and after leaf maturity. Before leaf maturity, the rate-limiting step was located prior to the reoxidation of plastohydroquinone. After leaf maturity, the decline in noncyclic electron flux correlated with a decrease in the concentration of cytochromes f and b(6). This correlation, together with a consideration of mechanisms of entry and exit of electrons in 3-(3,4 dichlorophenyl)-1,1-dimethylurea-treated thylakoids, suggests that the cytochrome f/b(6)-containing complex, and not plastocyanin or P700, is the site of entry of electrons from the reduced forms of 2,6-dichlorophenolindophenol and diaminodurene. It is therefore proposed that in senescing leaves the cytochrome f/b(6)-containing complex limited electron transport by constraining the rate of reduction of cytochrome f by plastohydroquinone. PMID- 16663088 TI - Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber. AB - The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O(2) consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD(+) and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD(+) ratio was high, alpha-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD(+) ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and alpha-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD(+) ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD(+)-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites. PMID- 16663089 TI - An endogenous alpha-amylase inhibitor in barley kernels. AB - Barley (Hordeum distichum cv Klages) kernels were shown to contain a factor that converted malted barley alpha-amylase II to the alpha-amylase III form. After purification by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel, and gel-filtration on Bio Gel P60, the factor gave a single band of protein on isoelectric focusing. The purified factor inhibited hydrolysis of soluble starch by alpha-amylase II from malted barley and germinated wheat (Triticum aestivum cv Neepawa). However, alpha-amylase I from these cereals was not affected. The inhibitor was not dialyzable and was retained by a PM 10 ultrafiltration membrane suggesting a molecular weight greater than 10,000 daltons. Heat treatment of the inhibitor at 70 degrees C for 15 minutes at pH 5.5 and 8.0 resulted in considerable loss of inhibitory activity. PMID- 16663090 TI - Immunological characterization of maize starch branching enzymes. AB - Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties. PMID- 16663091 TI - Heat stress responses in cultured plant cells : development and comparison of viability tests. AB - The response of suspension-cultured pear (Pyrus communis cv Bartlett) cells to heat stress was studied using three viability tests: regrowth (culture growth during 10 days after stress); triphenyltetrazolium chloride reduction; and electrolyte leakage. Critical (50% injury) temperatures for a 20-minute exposure were 42 degrees , 52 degrees , and 56 degrees C, respectively, for these viability tests. Electrolyte leakage had the lowest temperature coefficient. Heat stress inhibition of triphenyltetrazolium chloride reducing capacity was much greater if the viability test was conducted 3 days, rather than immediately, after the stress treatment. Consistent with a major role for indirect metabolic strain in heat injury, treatment with 3.6 micromolar cycloheximide and heat stress (20 minutes at 43 degrees C) affected culture regrowth similarly. We conclude that the measurements of direct response are not adequate substitutes for regrowth tests in assessing heat injury to cultured plant cells. PMID- 16663092 TI - Lysine transport in two barley mutants with altered uptake of basic amino acids in the root. AB - Amino acid uptake was examined in two barley (Hordeum vulgare L.) mutants R906 and R4402 which had been selected as resistant to the lysine analog S-(2 aminoethyl)-cysteine. The mutants were found to be allelic by crossing and examination of F(1) and F(2) progeny. The mutant genes were designated aec1a and aec1b, respectively. The uptake of the basic amino acids lysine, arginine, and ornithine from 50 micromolar solutions was strongly decreased in roots of the mutants, whereas uptake of neutral and acidic amino acids was unaffected. The pattern of uptake of lysine over the range 10(-7) to 10(-2) molar was consistent with there being, principally, two uptake systems operating for basic amino acids in roots and that a low-concentration, high-affinity system is reduced or lacking in the mutants. The residual transport activity in the mutants had a different relative affinity for lysine and arginine to the wild-type system. Uptake of lysine by leaf slices was unimpaired in the mutants suggesting that the leaf uptake system is unaffected by the aec1 gene. PMID- 16663093 TI - Na-Dithionite Promotes Photosynthetic Sulfide Utilization by the Cyanobacterium Oscillatoria limnetica. AB - The light- and sulfide-dependent induction process leading to photosystem I mediated sulfide utilization by Oscillatoria limnetica, for either H(2) evolution or CO(2) photoassimilation, was studied. The identical dependence on pH of the lag length, the inhibition of leucine incorporation and final H(2)S concentration imply that the latter exerts a deleterious effect on nonadapted cells.Na dithionite (Na(2)S(2)O(4)), Na-sulfite (Na(2)SO(3)), or ethanol cannot serve as photosynthetic electron donors. However, when these compounds were added to the sulfide-containing system, the need for induction was eliminated. At pH 6.9, in the presence of 3.5 millimolar sulfide, these substances (at concentrations of 10 millimolar, 5 millimolar, or 0.4 molar, respectively) completely abolished the delay preceding sulfide-dependent H(2) evolution. It is suggested that all three compounds expose a site capable of directly accepting sulfide electrons.Only dithionite could adapt the cells to sulfide utilization on its own. Sulfite or ethanol acted only in the presence of sulfide. It is implied that this specific activity of dithionite is related to its characteristic low redox potential.Sulfide-dependent H(2) evolution was insensitive to 3-(3,4 dichlorophenyl)-1,1-dimethylurea, but was inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, in the presence as well as in the absence of dithionite. In both cases, therefore, the plastoquinone was implied in the electron transport from sulfide. PMID- 16663094 TI - Adaptation of the Cyanobacterium Microcystis aeruginosa to Light Intensity. AB - Light intensity adaptation (20 to 565 microeinsteins per square meter per second) of Microcystis aeruginosa (UV-027) was examined in turbidostat culture. Chlorophyll a and phycocyanin concentrations decreased with increasing light intensity while carotenoid, cellular carbon, and nitrogen contents did not vary. Variation in the number but not the size of photosynthetic units per cell, based on chlorophyll a/P(700) ratios, occurred on light intensity adaptation. Changes in the numbers of photosynthetic units partially dampened the effects of changes in light intensity on growth rates. PMID- 16663095 TI - Light-enhanced protein synthesis in gravitropically stimulated root caps of corn. AB - Light stimulates gravitropic bending (downward growth) in roots of many cultivars of corn (Zea mays). In this work, using the cultivar Merit, we show that light stimulates protein synthesis in the root cap, with protein levels increasing 1.3 to 1.6 times that recorded for tissues maintained in continuous dark. Light enhances protein levels both in intact caps (attached to the root) and in caps in culture. Protein synthesis is optimal in cultured caps when 1 nanomolar indole-3 acetic acid is included in the culture medium. If cap tissue is illuminated and subsequently returned to the dark, in the 2-hour period following illumination protein levels decline to that observed in dark controls. It is proposed that light-stimulated protein synthesis mediates in part downward bending in roots of these cultivars of corn. PMID- 16663096 TI - Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots. AB - When assayed in the presence of azide, NO(3) (-) was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 x M017) root microsomal membranes. The distribution of the NO(3) (-)-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H(+)-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 mum vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO(3) (-)-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H(+) from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K(+) > NH(4) (+) > Rb(+) > Cs(+) > Li(+) > Na(+) > choline(+)) whereas the NO(3) (-)-sensitive ATPase was stimulated by anions (Cl(-) > Br(-) > C(2)H(3)O(2) (-) > SO(4) (2-) > I(-) > HCO(3) (-) > SCN(-)). Both ATPases required divalent cations. However, the order of preference for the NO(3) (-)-sensitive ATPase (Mn(2+) > Mg(2+) > Co(2+) > Ca(2+) > Zn(2+)) differed from that of the vanadate-sensitive ATPase (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO(3) (-)-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO(3) (-)-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO(3) (-)-sensitive ATPase was stimulated by gramicidin and was associated with NO(3) (-)-inhibitable H(+) transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO(3) (-)-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO(3) (-) sensitivity is a better criterion by which to identify this ATPase than either Cl(-) stimulation or H(+) transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization. PMID- 16663097 TI - Posttranscriptional Regulation of Ribulose 1,5-bisphosphate Carboxylase Small Subunit Accumulation in Chlamydomonas reinhardtii. AB - The coordinated synthesis of the subunits of ribulose 1,5-bisphosphate carboxylase was examined by analysis of the chloroplast ribosome-deficient mutant of Chlamydomonas reinhardtii, ac20crl. The absence of the chloroplast-synthesized large subunit of this enzyme from cells of this strain is a direct consequence of the lack of chloroplast ribosomes. In contrast, the absence of the cytoplasmically-synthesized small subunit of ribulose 1,5-bisphosphate carboxylase from this mutant is not understood. To discern the cause of this absence, we have compared results of in vivo radioactive labeling experiments with those of cell-free translations of RNA from ac20crl. Protein products from these experiments were identified by one-and two-dimensional electrophoretic analyses. Neither subunit, revealed either as a stained band or by fluorography of proteins radioactively labeled in vivo for 2 hours, was detected in ac20crl. Cell-free translation of polyadenylated RNA obtained from ac20crl, however, revealed wild-type levels of mRNA for the precursor to the small subunit. This messenger was found to be associated with subribosomal RNP and polysomes. We conclude that the absence of the small subunit of ribulose 1,5-bisphosphate carboxylase from ac20crl is the result of a translational or posttranslational event. PMID- 16663098 TI - Effect of glyphosate on ethylene production in tobacco callus. AB - Glyphosate (N-phosphonomethylglycine) caused a significant decrease or a slight increase in ethylene production in tobacco callus (Nicotiana tabacum L.) depending on the concentration of indole-3-acetic acid (IAA) present in the medium. IAA stimulated ethylene production, but a pretreatment with glyphosate greatly reduced the IAA-induced ethylene production. Inasmuch as glyphosate treatment promoted the metabolism of IAA, the decrease in ethylene production induced by glyphosate is attributed to the rapid loss of free IAA in the treated tissue. PMID- 16663099 TI - Mass spectrometric analysis of cytokinins in plant tissues : v. Identification of the cytokinin complex of datura innoxia crown gall tissue. AB - The cytokinin complex of Datura innoxia Mill. crown gall tissue was purified by ion exchange, Sephadex LH-20 chromatography and reversed-phase high performance liquid chromatography. By gas chromatography-mass spectrometry using (2)H-labeled compounds, the following cytokinins were identified in the basic fraction eluting from a cation exchange column: zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, their corresponding O-glucosides, 7- and 9-glucosides of zeatin, 9-glucoside of dihydrozeatin, isopentenyladenine, and isopentenyladenosine. Zeatin riboside 5'-monophosphate was the major cytokinin nucleotide in the tissue. In addition, dihydrozeatin riboside and isopentenyladenosine were identified in the nucleotide fraction following enymic degradation. PMID- 16663100 TI - Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins. AB - A bioassay to measure the incorporation of [(14)C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [(14)C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [(14)C]leucine uptake. The effectiveness of the wall fragments in inhibiting [(14)C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities. PMID- 16663101 TI - Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana. AB - Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoe daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoe leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f(1) and f(2)) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoe leaf preparations. Kalanchoe ferredoxin-thioredoxin reductase was similar to that of C(3) and C(4) plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoe ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoe ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C(3), C(4), or CAM photosynthesis. PMID- 16663102 TI - Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on Thioredoxin-Linked Enzymes of KalanchoE daigremontiana. AB - Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) were identified and purified from the Crassulacean acid metabolism (CAM) plant, Kalanchoe daigremontiana. FBPase and SBPase showed respective molecular weights of 180,000 and 76,000, and exhibited immunological cross-reactivity with their counterparts from chloroplasts of C(3) (spinach) and C(4) (corn) plants. Based on Western blot analysis, FBPase was composed of four identical 45,000 dalton subunits and SBPase of two identical 38,000-dalton subunits. Immunological evidence, together with physical properties, indicated that both enzymes were of chloroplast origin.Kalanchoe FBPase and SBPase could be activated by thioredoxin f reduced chemically by dithiothreitol or photochemically by a reconstituted Kalanchoe ferredoxin/thioredoxin system. Both enzymes were activated synergistically by reduced thioredoxin f and thier respective substrates.Kalanchoe FBPase could be partially activated by Mg(2+) at concentrations greater than 10 millimolar; however, such activation was considerably less than that observed in the presence of reduced thioredoxin and Ca(2+), especially in the pH range between 7.8 and 8.3. In contrast to FBPase, Kalanchoe SBPase exhibited an absolute requirement for a dithiol such as reduced thioredoxin irrespective of Mg(2+) concentration. However, like FBPase, increased Mg(2+) concentrations enhanced the thioredoxin-linked activation of this enzyme.In conjunction with these studies, an NADP-linked malate dehydrogenase (NADP-MDH) was identified in cell-free preparations of Kalanchoe leaves which required reduced thioredoxin m for activity.These results indicate that Kalanchoe FBPase, SBPase, and NADP-MDH share physical and regulatory properties with their equivalents in C(3) and C(4) plants. In contrast to previous evidence, all three enzymes appear to have the capacity to be photoregulated in chloroplasts of CAM plants, thereby providing a means for the functional segregation of glucan synthesis and degradation. PMID- 16663103 TI - Nature of the rate-limiting step in the supply of inorganic carbon for photosynthesis in isolated asparagus mesophyll cells. AB - Accumulation of acid-stable and acid-labile C has been studied in cells isolated from cladophylls of Asparagus sprengeri regel, as a function of the concentrations of the various inorganic C (C(i)) species in the external medium.The rate of CO(2) fixation was higher when C(i) was supplied as CO(2) as opposed to HCO(3) (-), at the same concentration. Participation of HCO(3) (-) was indicated when the external CO(2) and HCO(3) (-) concentrations were chosen such that, owing to interconversion between C(i) species, the same CO(2) concentrations would be reached at a certain point in time, regardless of which species was supplied initially; at this point, a higher fixation rate was observed in the case of HCO(3) (-) supply.In the presence of carbonic anhydrase, the apparent affinity for C(i) was raised. This enzyme raised fixation rate even under steady-state conditions, but only at limiting external C(i) concentrations. The decrease in external CO(2) concentration was correlated with a decreasing internal C(i) level when CO(2) was the species supplied.When 10 mum CO(2) was supplied initially fixation rate was almost independent of pH. However, when HCO(3) (-) was supplied at concentrations calculated to yield 10 mum CO(2) at equilibrium, fixation rate rose with pH. Carbonic anhydrase raised the fixation rate over the entire pH range when HCO(3) (-) was the species supplied.It is concluded that CO(2) was the major C(i) species permeating from the medium to the carboxylating site. Further, our results bring clear evidence that formation of CO(2) from HCO(3) (-) in the unstirred layer, and the diffusion of both species in this layer, rate limit CO(2) fixation by these isolated cells. PMID- 16663104 TI - Purification and Properties of Flavonol-Ring B Glucosyltransferase from Chrysosplenium americanum. AB - A novel glucosyltransferase which catalyzed the transfer of glucose from UDP glucose to positions 2' and 5' of partially methylated flavonols was isolated from the shoots of Chrysosplenium americanum Schwein ex Hooker. It was purified 225-fold by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, hydroxyapatite, and polybuffer ion exchanger. This glucosyltransferase appeared to be a single polypeptide with an apparent molecular weight of 42,000 daltons, pH optimum of 7.5 to 8.0, and an isoelectric point of 5.1. It had low but similar K(m) values for the 2' and 5' positions of flavonol substrates and the cosubstrate UDP-glucose and was inhibited by both reaction products, the glucosides formed, and UDP.Glucosyltransferase activity was independent of divalent cations, was not inhibited by EDTA, but showed requirement for SH groups. The differential effect on enzyme activity of metal ions, especially cupric ion, and various SH group reagents seemed to indicate the involvement of two active sites in the glucosylation reaction; the site specific for 2' activity being more susceptible than that of the 5' activity. The substrate specificity expressed by this glucosyltransferase and the requirement of at least two para-oriented B-ring substituents (at 2' and 5') for activity support this view. PMID- 16663105 TI - Effect of 2-(3,4-Dichlorophenoxy)-triethylamine on the Synthensis of cis Polyisoprene in Guayule Plants (Parthenium argentatum Gray). AB - The application of 2-(3,4-dichlorophenoxy)-triethylamine to guayule (Parthenium argentatum Gray var 593) plants results in a 2-fold stimulation of rubber synthesis and a 1.5- to 3-fold increase in mevalonic acid kinase, isopentenyl pyrophosphate isomerase, and rubber transferase. The increase in these enzymic activities accounts in part for the chemical induction of rubber synthesis. PMID- 16663106 TI - Effects of Light Quality on Tiller Production in Lolium spp. AB - Variations in phytochrome status in grasses were provoked by end of day red and far-red treatments and above canopy irradiations. Lolium perenne L. and Lolium multiflorum Lam. plants developed more tillers when illuminated with higher red/far-red ratios. These results show that branching of grasses is controlled by phytochrome activity in a way similar to that in dicotyledon plants. PMID- 16663107 TI - Separation of galactolipid molecular species by high-performance liquid chromatography. AB - A technique for the separation, detection, and quantification of molecular species of monogalactosyldiglyceride and digalactosyldiglyceride is described. Use of the technique to analyze the molecular species composition of the galactolipids isolated from Dunaliella salina chloroplasts is presented. The results indicate that the respective compositions of the two lipids are quite different. This suggests that the enzymes involved in galactolipid metabolism are very specific with respect to acyl chain composition and pairing, or that extensive retailoring of constituent acyl chains occurs following formation of digalactosyldiglyceride from monogalactosyldiglyceride. PMID- 16663108 TI - Gas exchange in paphiopedilum: lack of chloroplasts in guard cells correlates with low stomatal conductance. AB - Net photosynthesis and stomatal conductance were measured in attached leaves of Paphiopedilum insigne. At 20 degrees C and a vapor-pressure deficit of 0.5 kilopascal, both net photosynthesis and stomatal conductance were light-saturated below 0.2 millimole per square meter per second, a response typical of shade plants. The absolute values of photosynthetic rate and conductance however were remarkably low, presumably reflecting an adaptation to the low-light, limited nutrient habitat characteristic of these orchids. The leaves also showed a vapor pressure deficit response, with net photosynthesis and conductance varying over a 2-fold range between 0.3 and 1.6 kilopascals.These results confirm that Paphiopedilum stomata are functional. The correlation between achlorophyllous guard cells and low conductance rates, however, singles them out as an exceptional biological system, exhibiting basic differences from typical stomata in higher plants. Available evidence showing that guard-cell chloroplasts are needed to sustain high conductance rates at moderate to high irradiances indicates that the genetic changes leading to the loss of chloroplast differentiation in Paphiopedilum guard cells were not deleterious because of the low conductance rates characteristic of this genus. PMID- 16663109 TI - N(2) fixation and h(2) evolution by six species of tropical leguminous trees. AB - The C(2)H(4)/(15)N(2) and H(2)/(15)N(2) ratios for six species of tropical leguminous trees are reported. C(2)H(4)/(15)N(2) ratios ranged from 2.4 to 4.7; values for the H(2)/(15)N(2) ratios were between 0.6 and 1.4. Relative efficiency values, based on C(2)H(2) reduction, (15)N incorporation, and H(2) evolution during (15)N incorporation varied between 0.68 and 0.84 for the six species. Overall, approximately 30% of the electron flow through nitrogenase was used for H(2) evolution. PMID- 16663110 TI - Effect of rhizobial strain and host plant on nitrogen isotopic fractionation in legumes. AB - Lotus pedunculatus L., Medicago sativa L., Macroptilium atropurpureum, Glycine max, and Trifolium repens L. were grown in a N-free medium and inoculated with one of ten Rhizobium strains. Dry matter, N content, and delta(15)N values were determined for various plant parts.Nodules, with the exception of those from lucerne, were enriched in (15)N relative to atmospheric N. Considerable variation was found in delta(15)N values of plant herbage (-4.5 to +0.8). The extent of isotopic discrimination was dependent on both the host plant and the infecting rhizobial strain. This further complicates, but does not invalidate, the use of small variations in the natural abundance of (15)N to estimate the contribution of symbiotically fixed N(2) to the N in legume herbage. Some other implications of the observed differences are also discussed. PMID- 16663111 TI - Anaerobiosis and Release from Dormancy in Apple Embryos: Leaching of (+/-) [C]Abscisic Acid and its Metabolites under Aerobic and Anaerobic Conditions. AB - An anaerobic treatment released Pyrus malus L. cv Golden Delicious embryos from their primary dormancy. It also suppressed the inhibitory effect induced by exogenous abscisic acid (ABA) on after-ripened embryos. For the study of ABA metabolism, a two-step culture method was developed. Embryos in primary dormancy were cultivated aerobically in the presence of [(14)C]ABA (first culture). Some were directly analyzed to evaluate metabolism of absorbed ABA. The remaining embryos were cultivated on moist cotton without ABA, either in aerobic or anaerobic conditions (second culture). The amounts of ABA and its metabolites were measured both in the embryos and the water-leachates. After the second culture, the embryos showed a spectacular decrease in ABA content, with no difference between anaerobic and aerobic cultures. The amount of ABA glucose ester increased slightly in aerobiosis but diminished markedly in anaerobiosis. Radioactivity of the butanol fraction, which corresponded to polar conjugates, decreased considerably in anaerobiosis, whereas it increased in aerobiosis.Analysis of the water-leachates indicated that, compared to aerobic conditions, anaerobiosis increased total leaching of radioactive materials (x 4.2) as well as leaching of ABA (x 1.4). In addition, anaerobiosis induced leaching of conjugates, such as ABA glucose ester and butanol-soluble metabolites. We concluded that the anaerobic treatment affects mainly membrane permeability. PMID- 16663112 TI - Host Plant Cultivar Effects on Hydrogen Evolution by Rhizobium leguminosarum. AB - The effect of host plant cultivar on H(2) evolution by root nodules was examined in symbioses between Pisum sativum L. and selected strains of Rhizobium leguminosarum. Hydrogen evolution from root nodules containing Rhizobium represents the sum of H(2) produced by the nitrogenase enzyme complex and H(2) oxidized by any uptake hydrogenase present in those bacterial cells. Relative efficiency (RE) calculated as RE = 1 - (H(2) evolved in air/C(2) H(2) reduced) did not vary significantly among ;Feltham First,' ;Alaska,' and ;JI1205' peas inoculated with R. leguminosarum strain 300, which lacks uptake hydrogenase activity (Hup(-)). That observation suggests that the three host cultivars had no effect on H(2) production by nitrogenase. However, RE of strain 128C53 was significantly (P 5%), and contained (100%) stromal enzyme activity ribulose-1,5-bisphosphate carboxylase. The chloroplast preparation is significantly contaminated by mitochondria, as determined by succinate dehydrogenase activity. Chloroplasts prepared from Wt cells retained CO(2)-dependent O(2) photoevolution at rates in excess of 60 micromoles per milligram chlorophyll per hour, an activity which is severely inhibited by the addition of 10 millimolar KH(2)PO(4). The chloroplasts are osmotically sensitive as determined by ferricyanide-dependent O(2) photoevolution. PMID- 16663134 TI - Evidence for a beta-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase. AB - A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO(4) at pH 6.5 and then treated with sodium [(3)H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110 degrees C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [(3)H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the beta-carboxyl side chain of an aspartic acid residue in the active site of the enzyme. PMID- 16663135 TI - Distribution of calmodulin-stimulated ca transport into membrane vesicles from green spinach leaves. AB - A microsomal fraction isolated from green spinach leaves exhibited a Mg(2+) and ATP-dependent (45)Ca(2+) uptake. Addition of 10 micromolar carbonyl cyanide m chlorophenylhydrazine had no effect. The cationophore A23187 (10 micromolar) induced the release of (45)Ca(2+) accumulated by membrane vesicles. Membranes prepared from lower epidermis showed the highest Ca(2+) accumulation activity. Microsomal fractions from petiole, lamina, and midrib were less active. The stimulation by bovine brain calmodulin was about 30% for the lower epidermis, 23.5% for midrib, and below 20% for petiole and lamina. PMID- 16663136 TI - A role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis in spinach leaves. AB - The subcellular distribution of fructose 2,6-bisphosphate in spinach (Spinacia oleracea) leaves was studied using nonaqueous fractionation, showing that all, or almost all, is located in the cytosol. The amount of fructose 2,6-bisphosphate present in leaves during the diurnal cycle was measured and compared to the accumulation of starch and sucrose, and the amounts of selected phosphorylated intermediates in the leaf. Upon illumination, the level of fructose 2,6 bisphosphate decreases, but prolonged illumination leads to an increase in the level to above that found in the dark, which accompanies the onset of rapid accumulation of starch in the leaf. PMID- 16663137 TI - Characterization of Solute Efflux from Dehydration Injured Soybean (Glycine max L. Merr) Seeds. AB - Soybean (Glycine max L. Merr) seeds lose their tolerance of dehydration between 6 and 36 hours of imbibition. Soybean axes and cotyledons were excised 6 hours (tolerant of dehydration) and 36 hours (susceptible) after commencing imbibition and subsequently dehydrated to 10% moisture. Kinetics of the efflux of potassium, phosphate, amino acid, sugar, protein, and total electrolytes were compared in the four treatments during rehydration. Only slight differences were observed in the kinetics of solute efflux between the two cotyledon treatments dehydrated at 6 and 36 hours suggesting that the cotyledons may retain their tolerance of dehydration at this stage of germination. Several symptoms of injury were observed in the axes dehydrated at 36 hours. An increase in the initial leakage of solutes during rehydration, as quantified by the y-intercept of the linear regression line for solute efflux between 2 and 8 hours suggests an increased incidence of cell rupture. An increase in the rate of solute efflux (slope of regression line between 2 and 8 hours) from fully rehydrated axes was observed in comparison to axes dehydrated at 6 hours. The Arrhenius activation energy for potassium, phosphate, and amino acid efflux decreased and for protein remained unchanged. Both observations indicate an increase in membrane permeability in dehydration-injured tissue. Increasing the H(+) concentration of the external solution increased K(+) efflux from both control and dehydrated/rehydrated samples, increased sugar efflux from axes at 6 hours imbibition but decreased sugar efflux from axes at 36 hours imbibition, indicating changes in membrane properties during germination. The dehydration treatment did not alter the pattern of the pH response of axes dehydrated at 6 or 36 hours but did increase the quantity of potassium and sugar efflux from dehydration injured axes. These results are interpreted as indicating that dehydration of soybean axes at 36 hours of imbibition increased both the incidence of cell rupture during rehydration and altered membrane permeability of the rehydrated tissue. PMID- 16663138 TI - Gradients of polyamines and their biosynthetic enzymes in coleoptiles and roots of corn. AB - The distribution of diamines, polyamines, and their biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase in roots and coleoptiles of corn (Zea mays var Golden Cross Bantam) seedlings have been determined. Putrescine content, expressed on either a fresh weight or protein basis, increases from the tip to the base in both roots and coleoptiles. In roots, this gradient is paralleled by an activity gradient of arginine and ornithine decarboxylases. Spermidine is distributed equally along the length of coleoptiles; in roots, this is true only on a protein basis. Free spermine is detectable only in the root tip, but a bound form is present throughout the root and coleoptile. The results are compared with gradients in protein and DNA content and discussed in relation to the possible cellular roles of polyamines. PMID- 16663139 TI - Differential leakage of intracellular substances from imbibing soybean seeds. AB - Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max [L.] Merr.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25 degrees C) of 3.0 and 15.8 kilocalories per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intracellular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells. PMID- 16663140 TI - Comparison of Photosynthetic Performance in Triazine-Resistant and Susceptible Biotypes of Amaranthus hybridus. AB - The rate of CO(2) reduction in the S-triazine-resistant biotype of smooth pigweed (Amaranthus hybridus L.) was lower at all levels of irradiance than the rate of CO(2) reduction in the susceptible biotype. The intent of this study was to determine whether or not the lower rates of CO(2) reduction are a direct consequence of the same factors which confer triazine resistance. The quantum yield of CO(2) reduction was 23 +/- 2% lower in the resistant biotype of pigweed and the resistant biotype of pigweed had about 25% fewer active photosystem II centers on both a chlorophyll and leaf area basis. This quantum inefficiency of the resistant biotype can be accounted for by a decrease in the equilibrium constant between the primary and secondary quinone acceptors of the photosystem II reaction centers which in turn would lead to a higher average level of reduced primary quinone acceptor in the resistant biotype. Thus, the photosystem II quantum inefficiency of the resistant biotype appears to be a direct consequence of those factors responsible for triazine resistance but a caveat to this conclusion is discussed. The effects of the quantum inefficiency of photosystem II on CO(2) reduction should be overcome at high light and therefore cannot account for the lower light-saturated rate of CO(2) reduction in the resistant biotype. Chloroplast lamellar membranes isolated from both triazine-resistant and triazine-susceptible pigweed support equivalent rates of whole chain electron transfer and these rates are sufficient to account for the rate of light saturated CO(2) reduction. This observation shows that the slower transfer of electrons from the primary to the secondary quinone acceptor of photosystem II, a trait which is characteristic of the resistant biotype, is nevertheless still more rapid than subsequent reactions of photosynthetic CO(2) reduction. Thus, it appears that the lower rate of light-saturated CO(2) reduction of the resistant biotype is not limited by electron transfer capacity and therefore is not a direct consequence of those factors which confer triazine resistance. PMID- 16663141 TI - Role of Osmotic Potential Gradients during Water Stress and Leaf Senescence in Fragaria virginiana. AB - The physiological basis underlying differences in sensitivity of different aged leaves to water stress was investigated in Fragaria virginiana Duchesne. Differential susceptibility of only older leaves to water stress in the field during summer months appeared related to gradients in leaf osmotic potential within the plant and by an age dependency in the ability of leaves to adjust osmotically when challenged by periodic water deficits. Under greenhouse conditions, older leaves senesced invariably during an imposed water stress while control leaves of comparable age and stressed younger leaves remained green. Osmotic potentials of intermediate aged and younger leaves became approximately 1 to 2 bars lower after a single cycle of imposed stress and up to 10 bars lower after two cycles of stress. Pronounced gradients in leaf osmotic potential within individual whole plants were observed following two cycles of water stress that were significantly different from control values. Osmotic adjustment was dependent on leaf age with the greatest capacity for adjustment in the intermediate aged leaves. Loss of osmotic adjustment was rapid upon rewatering with a half-life of 4 days. An irreversible component of adjustment was observed, amounting to about 10% (or 2 bars) of the maximally adjusted state. This irreversible component could be accounted for in part by significant changes in cell size and other anatomical alterations in the leaf that affect cellular osmotic volume, and, hence, cellular water relations. PMID- 16663142 TI - Osmotic Adjustment and the Development of Freezing Resistance in Fragaria virginiana. AB - Cold temperature acclimation in strawberry (Fragaria virginiana) leaves apparently involves the alteration of cellular osmotic properties. Alterations in leaf osmotic potential were closely correlated with alterations in soluble carbohydrate content of the leaf tissue and changing temperatures. Leaf starch content was inversely related to soluble carbohydrate levels, suggesting that starch is a partial source of osmoticum during osmotic adjustment associated with cold temperature stress. Free amino acid changes were more closely linked to senescence and growth processes while changes in ion content suggested a rapid mobilization of solutes at the onset of freezing temperatures. This was supported by changes in whole plant gradients in leaf osmotic potential before and after exposure to freezing temperatures. In terms of freezing resistance and the role of osmotic adjustment in the development of resistance, it was found that of all leaves undergoing osmotic adjustment only the younger leaves survived, suggesting an age-dependent component to freezing resistance in leaves. Freezing resistance appears to involve alterations in several cellular properties that act in concert to confer a hardy state of the tissue. Although osmotic adjustment may be an important component of the final combination of cellular properties, this study indicates that solute accumulation does not function alone to confer freezing resistance. PMID- 16663143 TI - Temperature dependence of the enzymic carboxylation and oxygenation of ribulose 1,5-bisphosphate in relation to effects of temperature on photosynthesis. AB - Carboxylase and oxygenase activities of ribulose bisphosphate carboxylase purified from wheat were measured over the temperature range 5 to 35 degrees C either at constant O(2) and CO(2) concentrations or where the O(2) and CO(2) simulated the concentrations in water equilibrated at each temperature with the same gaseous phase. At constant CO(2) (14 micromolar) and O(2) (0.34 millimolar), the oxygenase to carboxylase ratio remained constant at 0.21 between 5 and 25 degrees C but increased to 0.26 at 35 degrees C. At O(2) and CO(2) concentrations near those expected in water equilibrated with air (21% [v/v] O(2)) containing 300 mul/l CO(2) at the various temperatures, the ratio of oxygenase to carboxylase activity increased 2.2-fold between 15 and 35 degrees C. At CO(2) and O(2) concentrations expected in water in equilibrium with subatmospheric concentrations of CO(2) in air (21% [v/v] O(2), 210 mul/l CO(2)), the oxygenase to carboxylase ratio increased from 0.25 at 10 degrees C to 0.56 at 35 degrees C. Between 20 and 30 degrees C, the apparent Q(10) value for the oxygenase reaction was 1.78 and that for the carboxylase was 1.26. Hence, the different responses of photosynthesis and photorespiration to temperature are due more to changes in the relative solubilities of CO(2) and O(2) (the solubility ratio) than to changes in kinetic parameters of the reactions catalyzed by ribulose bisphosphate carboxylase. PMID- 16663144 TI - Synthesis and degradation of barley nitrate reductase. AB - Nitrate and light are known to modulate barley (Hordeum vulgare L.) nitrate reductase activity. The objective of this investigation was to determine whether barley nitrate reductase is regulated by enzyme synthesis and degradation or by an activation-inactivation mechanism. Barley seedling nitrate reductase protein (cross-reacting material) was determined by rocket immunoelectrophoresis and a qualitative immunochemical technique (western blot) during the induction and decay of nitrate reductase activity. Nitrate reductase cross-reacting material was not detected in root or shoot extracts from seedlings grown without nitrate. Low levels of nitrate reductase activity and cross-reacting material were observed in leaf extracts from plants grown on nitrate in the dark. Upon nitrate induction or transfer of nitrate-grown etiolated plants to the light, increases in nitrate reductase activity were positively correlated with increases in immunological cross-reactivity. Root and shoot nitrate reductase activity and cross-reacting material decreased when nitrate-induced seedlings were transferred to a nitrate-free nutrient solution or from light to darkness. These results indicate that barley nitrate reductase levels are regulated by de novo synthesis and protein degradation. PMID- 16663145 TI - Membrane-Associated NAD-Dependent Isocitrate Dehydrogenase in Potato Mitochondria. AB - The oxidation isotherms for citrate and isocitrate by potato (Solanum tuberosum var. Russet Burbank) mitochondria in the presence of NAD differ markedly. Citrate oxidation shows positively cooperative kinetics with a sigmoid isotherm, whereas isocitrate oxidation shows Michaelis-Menten kinetics at concentrations up to 3 millimolar, and cooperative kinetics thereafter up to 30 millimolar. In the absence of exogenous NAD, the isocitrate isotherm is sigmoid throughout. The dual isotherm for isocitrate oxidation in the presence of exogenous NAD reflects the operation of two forms of isocitrate dehydrogenase, one in the matrix and one associated with the inner mitochondrial membrane. Whereas in intact mitochondria the activity of the membrane-bound enzyme is insensitive to rotenone, and to butylmalonate, an inhibitor of organic acid transport, isocitrate oxidation by the soluble matrix enzyme is inhibited by both. The membrane-bound isocitrate dehydrogenase does not operate through the NADH dehydrogenase on the outer face of the inner mitochondrial membrane, and is thus considered to face inward. The regulatory potential of isocitrate dehydrogenase in potato mitochondria may be realized by the apportionment of the enzyme between its soluble and bound forms. PMID- 16663146 TI - Isolation and Characterization of Inner Membrane-Associated and Matrix NAD Specific Isocitrate Dehydrogenase in Potato Mitochondria. AB - The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms. PMID- 16663147 TI - Sources of sucrose translocated from illuminated sugar beet source leaves. AB - A search for source leaf sucrose pools that differed in their relation to export was carried out in photosynthesizing leaves of Beta vulgaris L. The time course of depletion of [(14)C]sucrose in a leaf in unlabeled CO(2) following steady state labeling provided evidence for two distinct sucrose pools. After the start of the light period, leaf blade sucrose remained constant although it exchanged between the two pools. Newly synthesized sucrose destined for export passed through one pool more rapidly than through the other. All of the leaf blade sucrose appeared to exchange with export sucrose. Modeling and regression analysis of [(14)C]sucrose data provided a means for estimating the size of the two pools. From 20 to 40% of the sucrose was calculated to be present in the pool that provided the less direct path to export; this was likely vacuolar sucrose. The remainder of the sucrose in the blade was probably in the cytoplasm and veins. Added amounts of leaf blade sucrose, produced in response to elevated CO(2), appeared to be stored mainly in the vacuolar compartment. PMID- 16663148 TI - Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning. AB - Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing (15)N-enriched organic matter. Seasonal N(2) fixation activity was determined by periodically assaying plants for reduction of C(2)H(2). N(2) fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N(2) fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of (15)N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N(2) fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.Gas samples were taken from soil columns several times during the growth cycle of the plants. H(2) was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H(2) consumption by soil bacteria. Estimation of N(2) fixation by acetylene reduction activity was closest to the direct (15)N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct (15)N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer. PMID- 16663149 TI - Effect of cold acclimation on intracellular ice formation in isolated protoplasts. AB - When cooled at rapid rates to temperatures between -10 and -30 degrees C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation-cooling to 15 or -20 degrees C at rates of 10 or 16 degrees C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at -5 degrees C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of -15 degrees C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of -42 degrees C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution. PMID- 16663150 TI - Maintenance of air in intercellular spaces of plants. AB - Although air-filled intercellular spaces are necessary and ubiquitous in higher plants, little attention has been paid to the possible mechanisms by which these spaces are kept from being flooded. The most likely mechanism is that the living plant cell may maintain a hydrophobic monolayer on the surfaces of adjacent intercellular spaces. The existence of ;apparent free space' in cell walls and the fact that detergent solutions do not enter the intercellular spaces argue against this hypothesis. It is concluded that the mechanism by which these important air spaces are maintained is still unknown. PMID- 16663151 TI - Cold Acclimation of Suspension Cultures of Pinus sylvestris in Response to Light and Temperature Treatments. AB - Suspension cultures of Pinus sylvestris L. (Provenance Sodra Ydre) were used to determine frost hardiness after manipulating daylengths and temperatures. Frost hardiness was determined with the triphenyltetrazolium chloride (TTC) reduction method. The cultures were able to acclimate and increase frost hardiness levels; both low temperature (2 degrees C) and short day (8 h) treatments were used at the same time, but increased survival temperatures were not achieved when only one type of stimulus was used. Inasmuch as intact seedlings can be partially acclimated by a single type of stimulus, the results indicate that the organization of the cells to tissues plays a role for the hardening process in vivo. PMID- 16663152 TI - Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants. AB - Positive phototropism of perithecial beaks in the fungus Neurospora crassa has been demonstrated. The effect was shown to be mediated by blue light. When mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. However, when wild type, albino-1, albino-2, or albino-3 strains were used as the protoperithecial parent, phototropism occurred.The results show that both photoinduced carotenogenesis and phototropism in N. crassa are controlled by the white collar-1 and white collar-2 loci. Thus, the sensory transduction pathways for the two photoresponses must have some steps in common. The results further support the proposal that the white collar strains are regulatory mutants blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains can carry out light induction but have the albino phenotype because they are each defective for a different enzyme in the carotenoid biosynthetic pathway. PMID- 16663153 TI - In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections. AB - The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which (14)CO(2) is collected following uptake of [1-(14)C]IAA by excised tissue sections. After a 30 minute equilibration period, the evolution of (14)CO(2) was found to follow an approximately linear course with respect to both time and tissue weight.Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the (14)CO(2) evolved per milligram tissue and the percent of [1-(14)C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-(14)C]IAA treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation. PMID- 16663154 TI - Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): I. Control of Net Uptake by NO(3) Efflux. AB - A computer-controlled multichannel data acquisition system was employed to obtain continuous measurements of net nitrate or chlorate uptake by roots of intact barley plants (Hordeum vulgare cv Betzes) using nitrate-specific electrodes. Plants, previously grown in solutions maintained at 10 or 200 micromolar NO(3) ( ) (low N or high N conditions, respectively), were provided with 200 micromolar NO(3) (-) or ClO(3) (-) during the uptake period. Initial rates of NO(3) (-) uptake were several times higher in low N plants than in high N plants. Within 10 min, uptake in the former plants declined to a new steady rate which was sustained for the remainder of the experiment. No such time-dependent changes were evident in the high N plants. Rates and patterns of net chlorate uptake exhibited almost identical dependence upon previous nitrate provision. NO(3) (-) ((36)ClO(3) (-)) influx, by contrast, appeared to be independent of NO(3) (-) pretreatment prior to influx determination. Nitrate efflux, estimated by several different methods, was strongly correlated with internal nitrate concentration of the roots. PMID- 16663155 TI - Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4). AB - The influence of NH(4) (+), in the external medium, on fluxes of NO(3) (-) and K(+) were investigated using barley (Hordeum vulgare cv Betzes) plants. NH(4) (+) was without effect on NO(3) (-) ((36)ClO(3) (-)) influx whereas inhibition of net uptake appeared to be a function of previous NO(3) (-) provision. Plants grown at 10 micromolar NO(3) (-) were sensitive to external NH(4) (+) when uptake was measured in 100 micromolar NO(3) (-). By contrast, NO(3) (-) uptake (from 100 micromolar NO(3) (-)) by plants previously grown at this concentration was not reduced by NH(4) (+) treatment. Plants pretreated for 2 days with 5 millimolar NO(3) (-) showed net efflux of NO(3) (-) when roots were transferred to 100 micromolar NO(3) (-). This efflux was stimulated in the presence of NH(4) (+). NH(4) (+) also stimulated NO(3) (-) efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO(3) (-) occur via effects upon efflux. By contrast to the situation for NO(3) (-), net K(+) uptake and influx of (36)Rb(+)-labeled K(+) was inhibited by NH(4) (+) regardless of the nutrient history of the plants. Inhibition of net K(+) uptake reached its maximum value within 2 minutes of NH(4) (+) addition. It is concluded that the latter ion exerts a direct effect upon K(+) influx. PMID- 16663156 TI - On the light dependence of Fatty Acid synthesis in spinach chloroplasts. AB - The capacity of intact chloroplasts to synthesize long chain fatty acids from acetate depends on the stroma pH in Spinacia oleracea, U. S. hybrid 424. The pH optimum is close to 8.5. Lowering of the stroma pH leads to a reduction of acetate incorporation but does not suffice to eliminate fatty acid synthesis completely. Chain elongation from palmitic to oleic acid shows the same pH dependence. Fatty acid synthesis is activated in the dark upon the simultaneous addition of dihydroxyacetone phosphate and orthophosphate supplying ATP and oxaloacetate for reoxidation of NADPH in the stroma. Under these conditions both dark fatty acid synthesis and synthesis of oleate from palmitate show the same pH dependence as in the light. Dark fatty acid synthesis is further stimulated by increasing the stromal Mg(2+) concentration with the ionophore A 23187. In contrast to CO(2) fixation, dark fatty acid synthesis is considerably reduced by dithiothreitol (DTT). This observation may be due to an acetyl-CoA deficiency, caused by a nonenzymic acylation of DTT, and a competition for ATP between DTT activated CO(2) fixation and fatty acid synthesis. Because d,l-glyceraldehyde as inhibitor of CO(2) fixation compensates the DTT effect on dark fatty acid synthesis, reducing equivalents may be involved in the light dependence of acetate activation. PMID- 16663157 TI - Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus: Inhibition by Cyanide or Antimycin and Salicylhydroxamic Acid or Propyl Gallate. AB - Gametangia of the aquatic phycomycete Allomyces macrogynus have a cyanide- and antimycin A-insensitive respiration, which is sensitive to salicylhydroxamic acid (alternative respiration). Propyl gallate is also an inhibitor of this alternative pathway, and propyl gallate is more efficient than hydroxamic acid. Gametangial respiration is insensitive to propyl gallate, but propyl gallate sensitivity is gradually established when the gametangia are titrated with cyanide. Carbonyl cyanide m-chlorophenyl hydrazone stimulates the cyanide sensitive respiration and engages the alternative sensitive respiration. Sodium azide inhibits both the alternative and the cyanide-sensitive respiration, but the cyanide-sensitive respiration is inhibited 10 times more efficiently than the alternative respiration. Rotenone inhibits the total respiration and the propyl gallate-insensitive respiration by 33% and the cyanide-insensitive respiration by 43%.The kinetic results reported here are discussed with respect to the models of de Troostembergh and Nyns (1977 Arch Int Physiol Biochem 85:404-406; 1978 Eur J Biochem 53:423-432) and of Bahr and Bonner (1973 J Biol Chem 248:3446-3450) for the partitioning of electrons between cyanide-insensitive and propyl gallate insensitive respiration. The results reported here do not agree with the model of de Troostembergh and Nyns. PMID- 16663158 TI - Photorespiratory glycine metabolism in corn leaf discs. AB - The total glycine pool in Zea mays L. Mo17xB73 leaf discs was measured after steady state photosynthesis in 50%, 21% and 1% O(2). The glycine pool was a function of O(2) concentration; it was largest in 50% O(2) and smallest in 1% O(2). Incubation of discs with methyl hydroxybutynoic acid in 21% O(2) in the light caused an accumulation of carbon in glycolate. This accumulation was O(2) sensitive, as subsequent photosynthetic periods in 50%, 21%, and 1% O(2) resulted in the largest glycolate pool in 50% O(2) and the smallest in 1% O(2). At the same time, the O(2)-dependent increase in the glycine pool was eliminated. After untreated leaf discs reached steady state photosynthesis in 21% O(2), measurements made subsequently in darkness, or in 1% O(2) in the light, showed that the glycine pool decreased. On the basis of these results, we conclude that a major portion of the total glycine pool in corn is an intermediate in the photorespiratory glycolate pathway. Considering both the rate of decay of the glycine pool in the dark and the rate of decay of the glycine pool after changing from 21% to 1% O(2), we conclude that this glycine pool is turning over slowly. PMID- 16663159 TI - Effect of pod removal on leaf photosynthesis and soluble protein composition of field-grown soybeans. AB - Well nodulated, field-grown soybeans (Glycine max [L.] Merr. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings. PMID- 16663160 TI - Purification and characterization of a soybean leaf storage glycoprotein. AB - Removing the pods from soybean (Glycine max [L.] Merr. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides ( approximately 27, 29, 54, and 80 kilodaltons) is enhanced, and these polypeptides accumulate to levels comprising over 50% of the soluble protein. Heat girdling the petiole also causes the accumulation of these polypeptides, suggesting that the signal for changing leaf function may be associated with inhibition of phloem transport. The 27 and 29 kilodalton polypeptides are glycosylated and have been purified to greater than 90% by (NH(4))(2)SO(4) fractionation, concanavilin A affinity, and gel filtration chromatography. These peptides appear to comprise a single protein. Mouse antiserum has been prepared against this glycoprotein and has been used to check for cross-reactivity with seed proteins and to quantitate changes with leaf development. No cross-reactivity was observed with seed soluble proteins from several stages of development. Quantitation showed the highest content in podded plants at, and shortly following, flowering, with levels subsequently declining in conjunction with seed growth. In depodded plants, the level of glycoprotein continued to increase following flowering and accounted for 45% of the soluble leaf protein by 4 weeks after depodding. PMID- 16663161 TI - Indole-3-acetic Acid (IAA) and IAA Conjugates Applied to Bean Stem Sections: IAA Content and the Growth Response. AB - High resolution growth recording techniques and reverse isotope dilution analysis were used to study the relationship between indole-3-acetic acid (IAA) concentration and curvature of excised bean (Phaseolus vulgaris L. cv Bush Burpee Stringless) first internode sections unilaterally treated with hormone. The maximum rate of curvature occurred rapidly (within 25 minutes) and was proportional to the log of the amount of applied IAA recovered in the tissue. The rate of curvature decreased after 30 minutes although little or no lateral migration of applied IAA occurred and tissue levels of IAA increased. The biologic activity of IAA-amino acid conjugates was found to be directly related to the amount of free IAA, resulting from their hydrolysis, which could be recovered from the tissue. PMID- 16663162 TI - Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain. AB - Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and-possibly to a lesser extent-by some other amino acids taken up from the endosperm. PMID- 16663163 TI - Tryptophan enhancement of somatic embryogenesis in rice. AB - Cereal embryos can produce two types of callus. One type, termed "embryogenic," consists of small meristematic-like cells and gives rise to many plants by somatic embryogenesis if placed on a suitable regeneration medium. The other is termed "nonembryogenic" and consists of long tubular cells which gives rise to few or no plants. High concentrations of tryptophan increased the formation of embryogenic callus in three rice cultivars (Oryza sativa L. Calrose 76, Pokkali, and IR 36) but not in four others (Mahsuri, Bg 400-1, H(4), and Giza 159). The best concentration of tryptophan for Pokkali and Calrose 76 was 100 micrograms per milliliter, and for IR 36, 50 micrograms per milliliter. Indoleacetic acid at 100 micrograms per milliliter promoted an effect similar to that of tryptophan on Calrose 76. The difference between japonica (Calrose 76, Giza 159) and indica (Pokkali, IR 36) varieties is not the causal factor for the difference in response to tryptophan. Kinetin does not appear to be a requirement for embryogenic callus formation in Calrose 76. Plant regeneration from Calrose 76 embryogenic callus occurred at low levels in media containing no hormones. 6 benzyladenine, or 2,3,5-triiodobenzoic acid but not indoleacetic acid at 0.1 to 0.5 micrograms per milliliter significantly increased regeneration. PMID- 16663164 TI - Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes. AB - Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o(2) uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O(2) for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine. PMID- 16663165 TI - Uptake and distribution of sodium and potassium by corn seedlings : I. Role of the mesocotyl in ;sodium exclusion'. AB - The distribution of sodium and potassium throughout corn (Zea mays L. [A632 x Crows 3640] x Oh 43) plants is not simply a matter of uptake by cortical cells and irreversible delivery to the xylem for upward transport. We show that sodium, but not potassium, accumulates in the mesocotyl of corn seedlings grown on NaCl medium. Upon transfer to NaCl-free medium, total sodium is reduced by export through the roots but remains at high levels within the mesocotyl. We report experiments which consider uptake from the xylem.Shoots excised at the seed were allowed to transpire solutions containing (22)Na and (42)K. Potassium uptake within the mesocotyl was very sensitive to concentration, increasing 27-fold between 1 and 10 millimolar. Sodium uptake was dependent upon the square root of the concentration suggesting active accumulation. At sodium concentrations below 1 millimolar, more than 80% of the sodium in the plant was retained in the mesocotyl. Both the uptake by and retention within the mesocotyl were dependent upon transpiration rate as well as concentration. We discuss the limitations of measuring uptake from a finite, depletable medium. The mesocotyl is a modified root with a cuticularized epidermis. We discuss the feasibility of using this ;plastic-coated root' as a model for root transport studies. PMID- 16663166 TI - Uptake and Distribution of Sodium and Potassium by Corn Seedlings : II. Ion Transport within the Mesocotyl. AB - In this paper, uptake and distribution of sodium and potassium within the mesocotyl are considered in excised, 8-day-old corn (Zea mays L.) seedlings supplied with label via the transpiration stream. The stele and cortex were dissected following uptake and analyzed separately. At equal concentrations, sodium uptake by the stele was much more rapid than potassium uptake, and sodium was preferentially retained within the stele. Transport of sodium to the cortex halted when the supply of ions in the transpiration stream was interrupted. Potassium would not substitute for sodium in restoring this transport but neither did it compete with sodium for transport to the cortex. In the presence of continued sodium supply, transport was temperature sensitive.By labeling first with (22)Na for 2 hours and subsequently with (24)Na for up to 21 hours, three sodium pools were identified within the stele. The first was rapidly transportable to the cortex. The second equilibrated rapidly with the first but was not itself directly available for transport. We postulate that these represent the stelar symplasm and apoplasm, respectively. A third pool was not transported and probably represents sequestration within the vacuoles of some cell type. Transport of label acquired during the initial 2 hours proceeded with a half-time of approximately 10 hours with 10 millimolar sodium present during the redistribution period, and with a half-time of approximately 30 hours at 1 millimolar sodium.A working model is presented which explains these characteristics and supplies approachable questions for subsequent study. PMID- 16663167 TI - Organization of Chlorophyll a in the Light-Harvesting Chlorophyll a/b Protein Complex as Shown by Circular Dichroism : Liquid Crystal-Like Domains. AB - The development of thylakoid stacking, accumulation of the light-harvesting chlorophyll a/b protein complex (LHCP), and the changes of circular dichroism (CD) which reflect the organization of chlorophyll molecules in greening thylakoids of bean Phaseolus vulgaris cv Red Kidney leaves were investigated.Chloroplasts formed under intermittent light contained large double sheets of membrane with extensive appression in addition to separate lamellae. Thylakoids of such chloroplasts were devoid of LHCP and exhibited a relatively small CD in the chlorophyll absorption region. Upon continuous illumination, the rearrangement of membranes to characteristic grana and the accumulation of the LHCP was accompanied by the gradual appearance of the very intense CD signal with peaks at 682 to 684 (+) and 665 to 672 nanometers (-). The magnitude of differential absorption was approximately 100 times larger than that of the chlorophyll a in solution. This suggests a superhelical liquid crystal-like organization for LHCP, a texture which can be altered by changes of the electric field in the photosynthetic membranes. PMID- 16663168 TI - Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor. AB - Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [(14)C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3 fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [(14)C]aspartate to asparagine, over 10 fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.Only traces of glutamine dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue. PMID- 16663169 TI - Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference. AB - Photosynthesis, stroma-pH, and internal K(+) and Cl(-) concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K(+), H(+), Cl(-)) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).Isolated intact chloroplasts are a Donnan system which accumulates cations (K(+) or added Tetraphenylphosphonium(+)) and excludes anions (Cl(-)) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium(+) distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K(+) or Na(+) and the release of protons all become very small. Light-induced K(+) uptake is not abolished by valinomycin suggesting that the K(+) uptake is not primarily active. Intact chloroplasts contain higher K(+) concentrations (112 157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO(2) fixation at high K(+) concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO(2) fixation in intact chloroplasts. PMID- 16663170 TI - Solubilization of the starch-granule-bound starch synthase of normal maize kernels. AB - The starch-granule-bound starch synthase from Zea mays has been solubilized with a recovery of between 50 and 84%. Chromatography of the solubilized enzyme on DEAE-Sepharose resolves two fractions of activity which may be distinguished by their response to citrate. Neither solubilized isoenzyme displays any significant activity with UDPglucose. PMID- 16663171 TI - Phospholipids and Lipid Acyl Hydrolase (Phospholipase) in Leaf Galls (Hymenoptera: Cynipidae of Black Oak [Quercus robor L.]). AB - Phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and cardiolipin are the major phospholipids in young leaves of black oak (Quercus robor L.). Except for phosphatidylcholine, young, developing cynipid-galls on black oak leaves, i.e. the insect-transformed tissues, contain less phospholipid than normal leaf tissues. Lipid acyl hydrolase activity determined by the cleavage of free fatty acids from a labeled phospholipid substrate is higher in the tissue extracts from galls than from leaves. The increase in enzyme activity and the altered phospholipid composition are discussed in relation to expected membrane modifications and transport phenomena in insect-transformed tissues. PMID- 16663172 TI - A transplasmamembrane electron transport system in maize roots. AB - Ferricyanide is actively reduced by intact maize (Zea mays L., var XL 342) roots. This reduction is salt and temperature dependent, is stimulated by fusicoccin, and is accompanied by decrease of external pH. In anaerobic conditions, ferricyanide partially restores fusicoccin-induced proton extrusion. A salt-, temperature-, and pH-dependent cyanide-insensitive NADH-ferricyanide oxidoreductase activity can be demonstrated in microsomes isolated from the same plant tissue. This evidence supports the hypothesis, as proposed by Craig and Crane (1982 Plant Physiol 67: S-558, S-835), that the ferricyanide reduction is carried out by a transmembrane NADH dehydrogenase. PMID- 16663173 TI - Ozone-Induced Reduction in Quantity of Ribulose-1,5-bisphosphate Carboxylase in Alfalfa Foliage. AB - The concentration of ribulose-1,5-bisphosphate carboxylase was measured in the foliage of ;Moapa 69,' ;Ladak,' and ;Vernal' cultivars of alfalfa (Medicago sativa L.) after plants were exposed to 0.25 or 0.30 microliters per liter ozone for 2 hours. The quantity of ribulose-1,5-bisphosphate carboxylase decreased 80%, 68%, and 36% in leaves which did not exhibit ozone symptoms and to undetectable levels, 99% and 79% in symptomatic foliage for each cultivar, respectively. PMID- 16663174 TI - Fructose 2,6-bisphosphate and the regulation of pyrophosphate-dependent phosphofructokinase activity in germinating pea seeds. AB - THE ACTIVITY OF PYROPHOSPHATE: d-fructose-6-phosphate-1-phosphotransferase (EC 2.7.1.90, PPi-PFK) in cotyledons and sprouts of germinating pea seeds (Pisum sativum cv Alaska or Green Arrow) increases rapidly during the first 2 to 3 days after imbibition and then declines to a lower activity. The reaction toward fructose 1,6-bisphosphate formation is activated greatly by fructose 2,6 bisphosphate (fru 2,6-P(2)); however, the sensitivity of the enzyme's activity to fru 2,6-P(2) activation changes during germination.The cotyledon enzyme was partially purified and exists in two forms apparently with different molecular weights. The large form shows little sensitivity to fru 2,6-P(2), while the small form shows a high sensitivity to this effector (K(a) = 15 nanomolar). Gel filtration experiments indicate that fru 2,6-P(2) is involved in converting the small form into the large form. We propose that the interconversion of two forms of the PPi-dependent PFK by fru 2,6-P(2) is one mechanism for regulating glycolysis during seed germination. PMID- 16663175 TI - Photosynthesis, Ribulose-1,5-bisphosphate Carboxylase, Electron Transport, and Ribulose 1,5-Bisphosphate of Virescent and Normal Green Wheat Leaves. AB - CO(2) gas exchange, ribulose-1,5-bisphosphate, and electron transport have been measured in leaves of a yellow-green mutant of wheat (Triticum durum var Cappelli) and its wild type strain grown in the field. All these parameters, expressed on leaf area basis, were similar in both genotypes except electron transport which was more than double in the wild type. These results, treated according to a recent photosynthesis model for C(3) plants, seem to indicate that the electron transport rate of mutant leaves is not sufficient to support the carboxylation derived through both the assimilation rate and the in vitro ribulose-1,5-bisphosphate carboxylase activity. It is suggested that under our experimental conditions photosynthetic electron transport is not the sole energy dependent determinant of ribulose-1,5-bisphosphate regeneration in the mutant. PMID- 16663176 TI - Effect of Flooding on Starch Accumulation in Chloroplasts of Sunflower (Helianthus annuus L.). AB - Chloroplasts in leaves of sunflower (Helianthus annuus L. cv hybrid 894) whose roots were flooded for 4 days showed an increase in the level of starch in chloroplasts when examined with the electron microscope. Starch determination showed significantly higher levels in leaves of flooded plants. Chloroplast and mitochondrial structure seemed otherwise normal. PMID- 16663177 TI - Proton Flux and Elongation in Primary Roots of Barley (Hordeum vulgare L.). AB - The elongation zone of the primary root of barley (Hordeum vulgare L.) has been reported to be markedly basic in pH, in apparent contradiction of the acid-growth theory. We determined simultaneously the location of the elongation zone and the basic zone in these roots and found them indeed to be the same. However, sections of barley root elongation zones were found to respond to acidic, basic, and neutral solutions as predicted by the acid-growth theory. PMID- 16663178 TI - Ultraviolet-Stimulated KHCO(3) Efflux from Rose Cells: Regulation of Cytoplasmic pH. AB - Suspension-cultured cells of Rosa damascena that have been irradiated with ultraviolet light (254 nanometers, 2.1 x 10(4) joules per square meter) rapidly lose K(+) and HCO(3) (-) ions to the medium. If the HCO(3) (-) is derived from respiratory CO(2) inside the cell, then loss of HCO(3) (-) should be accompanied by an acidification of the cytoplasm. Estimates of the pH of control and ultraviolet-irradiated cells by (31)P-nuclear magnetic resonance spectroscopy indicated that, following irradiation, the pH of both cytoplasm and vacuole dropped by 0.2 to 0.3 units. This change was not as great as was predicted from the observed HCO(3) (-) loss. Analysis of nitrogenous compounds in the cell suggested that reduction of nitrate and synthesis of gamma-aminobutyric acid absorbed some of the protons formed by the synthesis and dissociation of bicarbonate. PMID- 16663179 TI - Environmental Responses of the Post-lower Illumination CO(2) Burst as Related to Leaf Photorespiration. AB - As leaf irradiance is decreased in increments, a single transient CO(2) burst is exhibited by C(3) plant leaves. This post-lower illumination CO(2) burst (PLIB) is sensitive to changes in irradiance, to changes in the concentrations of O(2) and CO(2), and to temperature. Increasing O(2) concentrations above ambient produces a progressively larger PLIB while increasing CO(2) concentrations above ambient produces a progressively smaller PLIB. The PLIB, which exhibits many responses to environment common with other methods for measuring photorespiration and photosynthesis, is proposed as a measure of photorespiration in illuminated leaves of C(3) plants. Although the PLIB cannot be used as a quantitative measurement of photorespiration, we propose that the PLIB is a rapid, easy, relatively inexpensive, nondestructive method for evaluating photorespiration in intact illuminated C(3) leaves in air. PMID- 16663180 TI - Purification and Partial Characterization of a Genetically-Defined Superoxide Dismutase (SOD-1) Associated with Maize Chloroplasts. AB - The chloroplast-associated form of superoxide dismutase from maize (Zea mays L.) (SOD-1) has been purified by a stepwise procedure consisting of (NH(4))(2)SO(4) fractionation, G-100 Sephadex gel filtration, DEAE-Sephacel chromatography, and hydroxylapatite chromatography. This procedure resulted in a single band on sodium dodecyl sulfate-polyacrylamide gels indicating that the preparation is homogeneous. The holoenzyme molecular weight was estimated at 31,000 to 33,000 by gel filtration. The subunit molecular weight of this dimeric protein was estimated at 14,500 on sodium dodecyl sulfate-polyacrylamide gels. Studies involving amino acid composition analysis, immunological cross-reactivity, in vitro subunit hybridizations, and H(2)O(2) sensitivity indicate that SOD-1 differs significantly from SOD-2 and SOD-4, the other cupro-zinc forms of SOD from maize. The possible physiological role of SOD-1 within the chloroplast is discussed. PMID- 16663181 TI - Photosynthetic Membranes of Porphyridium cruentum: An Analysis of Chlorophyll Protein Complexes and Heme-Binding Proteins. AB - Three chlorophyll-protein complexes (CP I, CP III, CP IV) were electrophoretically separated from thylakoids of the eukaryotic red alga Porphyridium cruentum. CP I contained the primary photochemical reaction center of photosystem I as judged by its light-induced reversible absorbance change at 700 nanometers, by its fluorescence emission maximum at 720 nanometers (-196 degrees C), and by the molecular weight of its apoprotein (68,000 daltons). CP III and CP IV appeared to belong with photosystem II as suggested by the absence of light-reversible absorbance at 700 nanometers, by their fluorescence maximum at 690 nanometers (-196 degrees C), and by the presence of a chlorophyll-binding polypeptide with a molecular weight of about 52,000 daltons. CP IV when completely denatured had two additional polypeptides of about 40,000 and 48,000 daltons. All three chlorophyll-protein complexes contained carotenoids: the chlorophyll/carotenoid molar ratio of 15:1 for CP I, and 20:1 for CP III and CP IV. The thylakoid membranes of P. cruentum contained four cytochromes, detected by heme-dependent peroxidase activity, but there was no observed association with the electrophoretically separated chlorophyll-protein complexes. PMID- 16663182 TI - Uptake of l-Ascorbate by Intact Spinach Chloroplasts. AB - Uptake of l-[1-(14)C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital(r)) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K(m) of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20 degrees C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V(max) but did not change the K(m) for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator. PMID- 16663183 TI - Effect of altering the root-zone temperature on growth, translocation, carbon exchange rate, and leaf starch accumulation in the tomato. AB - Tomato seedlings (Lycopersicon esculentum Mill. cv Vendor) were grown hydroponically with their root systems maintained at a constant temperature for a 2-week period commencing with the appearance of the first true leaf. Based on fresh and dry weight and leaf area, the optimal root-zone temperature for seedling growth was 30 degrees C. The carbon exchange rate of the leaves was also found to increase with rising root-zone temperature up to 30 degrees C. However, a more complex relationship seems to exist between root-zone temperature and the accumulation of (14)C-labeled assimilates in the roots; inasmuch as there is no enhancement in this accumulation at the most growth promoting root-zone temperatures (22-30 degrees C). PMID- 16663184 TI - A Comparison of Pigment-Protein Complexes among Normal, Chlorophyll-Deficient and Senescent Soybean Genotypes. AB - Pigment-protein complexes were isolated from chloroplasts of normal green and several types of chlorophyll-deficient soybeans. The complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparisons were made between normal and chlorophyll-deficient genotypes of the relative amounts of chlorophyll associated with Photosystem I (PSI), Photosystem II (PSII), light harvesting, and free pigment complexes.Chlorophyll-deficient genotypes, compared to normal green genotypes, have fewer light-harvesting complexes and a higher ratio of PSII to PSI complexes. Chlorophyll associated with PSII in yellow genotypes is in relatively higher amounts in spite of the fact that these genotypes have much less grana stacking than normal green genotypes. Although PSII activity has been associated with appressed regions of grana in normal plants, our work shows that the association does not always hold true. PMID- 16663185 TI - Differential light induction of nitrate reductases in greening and photobleached soybean seedlings. AB - Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO(3) (-), tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO(3) (-) and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO(3) (-) and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO(3) (-). The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO(3) (-), further indicating that the isoform of nitrate reductase induced in norflurazon treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO(3) (-) requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO(3) (-) requirement for induction were quite different. K(m) values for NO(3) (-) were identical for both nitrate reductases. PMID- 16663186 TI - Metabolism of carbon and nitrogen by soybean seedlings in response to vegetative apex removal. AB - Short-term (31-hour diurnal) growth-chamber studies were conducted to determine the effects of removing the vegetative apex (meristem and developing trifoliolate leaves) on net photosynthesis (changes in plant dry weight), on distribution of metabolites among plant parts, and on nitrate metabolism and reduced-N accumulation by soybean [Glycine max (L.) Merr.] seedlings. Roots and stems served as alternate sinks for dry matter accumulation in the absence of the vegetative apex. Sugar concentration in roots increased (42%) within 4 hours of vegetative apex removal, and remained higher than for the controls during the 31 hour experimental period. Nitrate assimilation (nitrate reductase activity and total accumulation of reduced-N) was also enhanced in response to vegetative apex removal. Although dry matter accumulation was similar between treated and control plants (113 versus 116 milligrams per plant) over the 31-hour sampling period, more nitrate (1.31 versus 0.79 milligrams per plant) and more reduced-N (3.96 versus 3.45 milligrams per plant) accumulated in treated plants during the same interval. It was concluded that vegetative apex removal had little effect on overall net photosynthesis of soybean seedlings during the 31-hour treatment period, but did alter partitioning of photosynthate and enhanced uptake, transport, and reduction of nitrate. Implications are that uptake and metabolism of nitrate by soybeans may be limited by flux of carbohydrate to the roots, although hormonal effects due to vegetative apex removal cannot be ruled out. PMID- 16663187 TI - Leaf Starch Metabolism during the Growth of Pepper (Capsicum annuum) Plants. AB - Levels of starch and sugars, and the activities of amylase and starch phosphorylase were measured in expanding leaves harvested in early morning and early evening during the growth of pepper (Capsicum annuum L.; cv ;Bellboy') plants. The differences between starch levels 1 hour after dawn and 1 hour after dusk increased during the period of initial fruit expansion. This diurnal starch difference was strongly correlated with post-dusk amylase activities in leaves at both stages of expansion. There was also a strong correlation between levels of amylase in immature and those in mature leaves throughout the experiment. Phosphorylase activity showed no direct relationship to leaf starch levels, and there was no similarity between activities in immature and mature leaves. An increase in photosynthesis during plant development was observed which could account for the increased starch synthesis at initial fruit expansion. PMID- 16663188 TI - Quantitative Measurements of Phosphofructokinase in the Shoot Apical Meristem, Leaf Primordia, and Leaf Tissues of Dianthus chinensis L. AB - Phosphofructokinase (EC 2.7.1.11) activity in the apical meristem, leaf primordia, and leaf tissues (epidermis, mesophyll, xylem, phloem) of Dianthus chinensis L. was determined. These data show that activity on a dry weight per hour basis was about 1000 millimoles in the apical meristem and two youngest primordia, and declined to 150 millimoles in the mesophyll of the sixth leaf pair and to 80 millimoles in older leaves just expanded out of the bud. On a cell per hour basis, the activity increased from 75 femtomoles in cells of the apical meristem and leaf primordia to 1.5 picomoles in the mesophyll of the sixth leaf pair. However, on a cell volume (cubic micrometers) per hour basis, the opposite was true. The results indicate that (a) on a dry weight or unit cell volume basis the capacity for glycolysis declines during leaf development and that (b) within the apical meristem, incipient primordia are not associated with phosphofructokinase activity greater than adjacent areas of the meristem. In comparison to glucose-6-phosphate dehydrogenase activity previously measured in equivalent samples (Croxdale, Outlaw 1983 Planta 157: 289-297), the capacity of the glycolytic pathway is greater than the pentose phosphate pathway in the apical meristem and two youngest pair of primordia. By the time leaves are six plastochrons old, the capacity for carbohydrate oxidation by the hexose monophosphate shunt exceeds that by glycolysis. PMID- 16663189 TI - Abscisic Acid-induced freezing resistance in cultured plant cells. AB - The effect of abscisic acid (ABA) on the cold hardiness of cell suspension was investigated. Cell suspension cultures of winter wheat (Triticum aestivum L. cv Norstar), winter rye (Secale cereale L. cv Cougar), and bromegrass Bromo inermis Leyss treated with 7.5 x 10(-5) molar ABA for 4 days at 20 degrees C could tolerate -30 degrees C, whereas the control cultures tolerated only -7 to -8 degrees C. The optimum concentration for increasing the cold hardiness of the cultures was 7.5 x 10(-5) molar. The degree of cold hardiness and the rate of hardening obtained by ABA treatment was significantly higher than that induced by low temperature alone. Of ten species tested, ABA was only effective on those cultures which were capable of cold hardening upon exposure to low temperatures. The results suggest that ABA bypasses the cold requirement for hardening and also suggests that ABA triggers the genetic system(s) responsible for inducing the hardening process. PMID- 16663190 TI - Role of roots in regulating the growth rate and cytokinin content in leaves. AB - The role of roots in the enhancement of cytokinin content and leaf growth of Phaseolus vulgaris plants after decapitation and partial defoliation was investigated. Partial excision of the roots of plants which were decapitated above the primary leaf node resulted in a reduction of leaf growth and soluble proteins accumulation in the primary leaves. Roots excision was done at time of decapitation and repeated 8 days later. Endogenous cytokinins, known to be involved in enhancing shoot growth, accumulated in the leaves and stems of decapitated and partially defoliated plants. Lower levels of cytokinins were detected in the leaves of decapitated plants with only a partial root system. The level of cytokinins in the roots of decapitated plants was reduced by partial root excision. The growth and accumulation of cytokinins in leaves were, however, not totally suppressed by removing a large proportion of the roots. At the commencement of the experiment the stem had a higher cytokinin content than both the leaves and roots. This suggests that the stem could be an alternative source of cytokinins to the leaves. The cytokinin complement in the leaves of decapitated plants is not identical to that in the roots. It appears that cytokinins supplied by the roots are metabolized in the leaves, or that alternatively certain cytokinins are synthesized in the leaves themselves. PMID- 16663191 TI - Separation of monovinyl and divinyl protochlorophyllides and chlorophyllides from etiolated and phototransformed cucumber cotyledons. AB - A method was developed to separate the monovinyl and divinyl forms of protochlorophyllide and chlorophyllide by high pressure liquid chromatography using a silicic acid column coated with dodecyl residues and a moving phase containing the lipophilic cation, tetrabutyl ammonium. The solvent was 70% methyl alcohol containing varying amounts of methyl ethyl ketone. The separation was carried out at 0 degrees C. This method was used to test and confirm a previous report that, in cucumber cotyledons, divinyl protochlorophyllide is phototransformed to give divinyl chlorophyllide, which is biologically unstable and disappears rapidly in the dark. PMID- 16663192 TI - Appearance and Disappearance of Cyanide-Resistant Respiration in Vigna mungo Cotyledons during and following Germination of the Axis. AB - Mitochondrial preparations isolated from black gram (Vigna mungo L.) cotyledons exhibited cyanide-resistant respiration which was of mitochondrial origin. The appearance and the disappearance of this alternative respiration took place during and following imbibition. During the first 6 hours of imbibition, the respiration was completely inhibited by cyanide, but after this time the alternative respiration markedly developed, reaching a maximal cyanide-resistance 12 to 16 hours after the start of imbibition. Subsequently, the alternative respiration gradually disappeared. The actions of cycloheximide and chloramphenicol indicated that the appearance was dependent on cytoplasmic protein synthesis and that the disappearance depended on both cytoplasmic and mitochondrial protein synthesis. The alternative pathway contributed to state 4 respiration, but not to state 3 respiration, in mitochondria from 1-day-old cotyledons. On day 3, it contributed to neither state 3 nor state 4. PMID- 16663193 TI - Mode of action of plastic film in extending life of lemon and bell pepper fruits by alleviation of water stress. AB - The mechanism by which seal-packaging individual fruit in high density polyethylene film delays deterioration was investigated with lemon (Citrus limon [L.] Burm. f. cv Eureka) and bell pepper (Capsicum annuum L. cv Maor) fruits. Seal-packaging effects were due to the water-saturated atmosphere in the sealed enclosure around the fruit. Softening of fruit was highly correlated with declining water potential of fruit. Sealing drastically inhibited softening as well as changes in cell wall pectins. Sealing also delayed disintegration of membrane as shown by the inhibited leakage of amino acids, in particular, and electrolytes in general. All these effects of sealing were prevented or reduced by including hygroscopic CaCl(2) in the sealed enclosure which reduced the ambient humidity. Furthermore, some of these effects of sealing could be achieved also by maintaining nonsealed fruit in water-saturated atmosphere. Sealing effects could not be related to a possible ;modified atmosphere' mechanism in O(2), CO(2), or ethylene. This work supports the hypothesis that the mode of action of sealing in the polyethylene relates to the alleviation of water stress which exists in harvested fruit. PMID- 16663194 TI - Terpenoid Metabolism in Plastids : Isolation and Biochemical Characteristics of Capsicum annuum Chromoplasts. AB - A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl(2) in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids. PMID- 16663195 TI - Immunocytochemical and Cytochemical Localization of Photosystems I and II. AB - Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P(700) chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P(700) chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae. PMID- 16663196 TI - Potassium Transport in Corn Roots : II. The Significance of the Root Periphery. AB - The relative transport capabilities of the cells of the root periphery and cortex were investigated using a variety of experimental techniques. Brief (30 seconds to 1 minute) exposures with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS), dramatically reduced (86)Rb(+) (0.2 millimolar RbCl) uptake into 2 centimeter corn (Zea mays [A632 x (C3640 x Oh43)]) root segments. Autoradiographic localization studies with [(3)H]NEM and [(203)Hg]PCMBS demonstrated that, during short term exposures with either reagent, sulfhydryl binding occurred almost exclusively in the cells of the root periphery.Corn root cortical protoplasts were isolated, and exhibited significant K(+)((86)Rb(+)) influx. The kinetics for K(+) uptake were studied; the influx isotherms were smooth, nonsaturating curves that approached linearity at higher K(+)(Rb(+)) concentrations (above 1 millimolar K(+)). These kinetics were identical in shape to the complex kinetics previously observed for K(+) uptake in corn roots (Kochian, Lucas 1982 Plant Physiol 70: 1723-1731), and could be resolved into a saturable and a first order kinetic component.The existence of a hypodermal apoplastic barrier was investigated. The apoplastic, cell wall binding dye, Calcofluor White M2R, appeared to be excluded from the cortex by the hypodermis. However, experiments with damaged roots indicated that this result may be an artifact resulting from the binding of dye to the epidermal cell walls. Furthermore, [(203)Hg] PCMBS autoradiography demonstrated that the hypodermis was not a barrier to apoplastic movement of PCMBS.These results suggest that although cortical cells possess the capacity to absorb ions, K(+) influx at low concentrations is limited to the root periphery. Cortical cell uptake appears to be repressed under these conditions. At higher concentrations, cortical cells may function to absorb K(+). Such a model may involve regulation of cortical cell ion transport capacity. PMID- 16663197 TI - Expolygalacturonase from Suspension Cultures of Marchantia polymorpha: Its Presence and Involvement in Pectic Polysaccharide Degradation. AB - Polygalacturonase was isolated from cell suspension cultures of a thalloid liverwort, Marchantia polymorpha. The enzyme in the ;buffer-soluble' protein fraction was dialyzed at pH 5.2 and further purified 91-fold by a combination of chromatographic techniques including CM-Sephadex, Sephacryl S-200, DEAE-Sephadex, and Sephadex G-200. The purified enzyme had an optimum activity in the pH range at 3.6 to 3.8 and molecular weight of 76,000 daltons, and its activity was not stimulated by cations. The enzyme was identified as an exohydrolase from viscometric data and chromatographic analysis of the reaction products.The polysaccharides extracted from the Marchantia cell walls with 2% (w/v) Na hexametaphosphate solution were separated into two fractions, neutral polysaccharides (fraction P-N) and acidic polysaccharides (fraction P-A) by a DEAE-Sephadex column. The fraction P-N was not susceptible to the purified exopolygalacturonase, whereas fraction P-A was partially degraded. This resulted in hydrolysis of 19.5% of the glycosyl linkages of fraction P-A with the release of galacturonic acids. The specific activity of exopolygalacturonase increased during the growth cycle. PMID- 16663198 TI - Cytokinins in the Leaves of Ginkgo biloba: I. The Complex in Mature Leaves. AB - Cytokinin conjugates of zeatin, ribosylzeatin, and their respective dihydro derivatives tentatively have been identified as the major cytokinins present in mature Ginkgo biloba L. leaves. Ribosylzeatin was present in higher levels than zeatin and dihydrozeatin. No evidence could be found that 6-(2,3,4-trihydroxy-3 methylbutylamino)purine occurs as a metabolite in the mature leaves. From the available evidence, it is concluded that cytokinin conjugates are probably the major metabolites formed in the leaves of this deciduous gymnosperm. PMID- 16663199 TI - Shikimate Pathway Activity during Shoot Initiation in Tobacco Callus Cultures. AB - The activity of the shikimic acid pathway during shoot initiation in tobacco (Nicotiana tabacum L. Wisconsin 38) callus was examined. Enhancement of the activities of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase, shikimate kinase, chorismate mutase, and anthranilate synthase was observed during culture of tobacco callus under shootforming conditions in comparison to tissue cultured under non-organforming conditions. Confirmation of these findings was obtained by examining the incorporation of d-[(14)C]glucose into quinic and shikimic acids and of [(14)C]shikimic acid into tyrosine, phenylalanine, and tryptophan. PMID- 16663200 TI - Development of a radioimmunoassay for the soybean phytoalexin glyceollin I. AB - A radioimmunoassay for glyceollin I, the major phytoalexin produced by soybean (Glycine max [L.] Merr.), has been developed. Antibodies were raised in rabbits against a glyceollin I-bovine serum albumin conjugate. The antisera were used to establish a radioimmunoassay for glyceollin I using [(125)I]glyceollin I as the tracer. A logit plot of a standard concentration series yielded a straight line in the range of 1 to 100 picomoles (0.34-34 nanograms) of glyceollin I. The structurally related pterocarpan phytoalexins, glyceollins II and III, glyceollidin II and glycinol, which also accumulate in infected soybean tissue, show a low cross-reactivity in the radioimmunoassay (0.5-5% at 50% displacement of the tracer). Two related isoflavones present constitutively in soybean tissue, daidzein and genistein, have cross-reactivities of less than 0.84% and 1.1%, respectively. The radioimmunoassay permitted the quantitative determination of glyceollin I in 15-micrometer microtome sections of soybean hypocotyl tissue infected with zoospores of Phytophthora megasperma f. sp. glycinea. PMID- 16663201 TI - Photosynthesis and ion content of leaves and isolated chloroplasts of salt stressed spinach. AB - Spinach (Spinacia oleracea) plants were subjected to salt stress by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar. Plants were harvested 3 weeks after starting NaCl treatment. Fresh and dry weight of both shoots and roots was decreased more than 50% compared to control plants but the salt-stressed plants appeared healthy and were still actively growing. The salt-stressed plants had much thicker leaves. The salt-treated plants osmotically adjusted to maintain leaf turgor. Leaf K(+) was decreased but Na(+) and Cl(-) were greatly increased.The potential photosynthetic capacity of the leaves was measured at saturating CO(2) to overcome any stomatal limitation. Photosynthesis of salt-stressed plants varied only by about 10% from the controls when expressed on a leaf area or chlorophyll basis. The yield of variable chlorophyll a fluorescence from leaves was not affected by salt stress. Stomatal conductance decreased 70% in response to salt treatment.Uncoupled rates of electron transport by isolated intact chloroplasts and by thylakoids were only 10 to 20% below those for control plants. CO(2) dependent O(2) evolution was decreased by 20% in chloroplasts isolated from salt stressed plants. The concentration of K(+) in the chloroplast decreased by 50% in the salt-stressed plants, Na(+) increased by 70%, and Cl(-) increased by less than 20% despite large increases in leaf Na(+) and Cl(-).It is concluded that, for spinach, salt stress does not result in any major decrease in the photosynthetic potential of the leaf. Actual photosynthesis by the plant may be reduced by other factors such as decreased stomatal conductance and decreased leaf area. Effective compartmentation of ions within the cell may prevent the accumulation of inhibitory levels of Na(+) and Cl(-) in the chloroplast. PMID- 16663202 TI - Nutritional control of regreening and degreening in citrus peel segments. AB - A method for reversibly regreening and degreening citrus epicarp in vitro using peel segments was developed.Peel segments from mature degreened fruit promptly regreened when kept in light upon agar medium containing low (15 millimolar) concentrations of sucrose. Higher concentrations of sucrose inhibited this regreening, but NO(3) (-) and certain amino acids included in the media overcame the inhibition by sucrose. However, l-serine strongly inhibited regreening. In the presence of nitrogen, sucrose promoted regreening.Peel segments from green fruit remained green on media with low concentrations of sucrose and on media with high concentrations of sucrose and 60 millimolar KNO(3), but degreened in response to high concentrations of sucrose in the absence of nitrogen. Nitrate overcame the degreening effects of high sucrose concentrations in both light and dark. Peel segments were reversibly degreened and regreened by transferring the segments between appropriate media.Nitrate in the media markedly reduced the levels of endogenous sugars in the epicarp and increased endogenous amino acid levels. Sucrose in the media increased endogenous sugar levels and, in the presence of nitrate, increased endogenous amino acid levels. In the absence of nitrogen, high sucrose concentrations reduced endogenous amino acid concentrations. PMID- 16663203 TI - Organization and Function of Chlorophyll in Membranes of Cyanobacteria during Iron Starvation. AB - Cells of Anacystis nidulans strain R2 and of Synechococcus cedrorum were grown in an iron-deficient medium. Iron starvation induced several pronounced effects without influencing the viability of these cells. The phycocyanin and chlorophyll contents of these cells were depressed, and the absorption maxima of membrane bound chlorophyll was blue-shifted by 5 nanometers. Cells showed a dramatic increase in original and in maximal chlorophyll fluorescence when monitored at room temperature. Low temperature chlorophyll fluorescence revealed a loss in fluorescence at 696 and 716 nanometers; much of the remaining fluorescence emission was at 686 nanometers. These changes suggest an alteration of membrane composition and structure. This was documented by an electrophoretic analysis of iron-deficient membranes. The prominent findings were: (a) large chlorophyll protein complexes were not observed in iron-deficient membranes, although the chlorophyll-binding proteins were present; (b) the staining of acrylamide gels with 3,3',5,5'-tetramethylbenzidine plus peroxide indicated that iron deficiency led to a decrease in the quantity of cytochromes. These results support a structural model of the relation between fluorescence and chlorophyll organization in Anacystis. In addition, they suggest a method for studying cytochrome and chlorophyll protein assembly in these membranes. PMID- 16663204 TI - Intermediates in the recycling of 5-methylthioribose to methionine in fruits. AB - The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [(14)CH(3)]MTR was not metabolized in cell free extract from avocado fruit. Either [(14)CH(3)]MTR plus ATP or [(14)CH(3)]5-methylthioribose-1 phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as alpha-keto-gamma-methylthiobutyric acid (alpha-KMB) and alpha-hydroxy-gamma-methylthiobutyric acid (alpha-HMB) by chromatography in several solvent systems. [(35)S]alpha-KMB was found to be further metabolized to methionine and alpha-HMB by these extracts, whereas alpha-HMB was not. However, alpha-HMB inhibited the conversion of alpha-KMB to methionine. Both [U (14)C]alpha-KMB and [U-(14)C]methionine, but not [U-(14)C]alpha-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of alpha-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR --> MTR-1-P - > alpha-KMB --> methionine --> S-adenosylmethionine --> 1-aminocyclopropane-1 carboxylic acid --> ethylene. PMID- 16663205 TI - Localization of lipoxygenases 1 and 2 in germinating soybean seeds by an indirect immunofluorescence technique. AB - Lipoxygenases 1 and 2 were localized in etiolated germinating soybean seeds (Glycine max [L.]. Merr. var. Williams) by an indirect immunofluorescence staining technique. Sections of paraffin-embedded seedlings were stained with affinity-purified antibodies directed against lipoxygenase 1 or 2. The specificity of the immunofluorescence technique was examined by use of nonimmune serum or immunoglobulin G preparations after total adsorption with the appropriate lipoxygenase coupled to Sepharose 4B.After immunofluorescence staining with antilipoxygenase 1 or 2 IgG storage tissues of cotyledons fluoresce strongly the first days of germination. After 3 days, the abaxial hypodermis, the epidermis, and the vascular bundle sheaths show fluorescence, especially after incubation with antilipoxygenase 2 IgG. Fluorescence in cortex and pith of the hypocotyl migrates to the vascular cylinder during germination. In primary leaves, all tissues show fluorescence after 1 day of germination. In storage tissues of cotyledons, cytoplasm around the protein bodies fluoresces, whereas in other tissues protein bodies or other large cell organelles fluoresce.It is reasonable to suggest that lipoxygenase exerts its function in cells at the time that rigorous changes in metabolism take place, namely at the start of mobilization of reserves in storage tissues and start of biosynthesis of chloroplastids in several tissues. PMID- 16663206 TI - Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth. AB - A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO(2) requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO(2) concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O(2). In contrast to a CO(2) compensation concentration of near zero in wild type at all O(2) concentrations examined, the mutant exhibited a high, O(2)-stimulated CO(2) compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of (14)CO(2). At air levels of CO(2) and O(2), the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO(2) concentration. At ambient CO(2), wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO(2) limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO(2) concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO(2), creating a high internal CO(2) concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO(2) concentration. PMID- 16663207 TI - Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii. AB - A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO(2) concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO(2) response of photosynthesis indicated a reduced affinity for CO(2) in the mutant. At air levels of CO(2) (0.03-0.04%), O(2) inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O(2) at saturating CO(2) concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO(2) concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO(2) concentrating system in C. reinhardii photosynthesis. PMID- 16663208 TI - Maple sap uptake, exudation, and pressure changes correlated with freezing exotherms and thawing endotherms. AB - Sap flow rates and sap pressure changes were measured in dormant sugar maple trees (Acer saccharum Marsh.). In the forest, sap flow rates and pressure changes were measured from tap holes drilled into tree trunks in mature trees and sap flow rates were measured from the base of excised branches. Excised branches were also brought into the laboratory where air temperature could be carefully controlled in a refrigerated box and sap flow rates and sap pressures were measured from the cut base of the branches.Under both forest and laboratory conditions, sap uptake occurred as the wood temperature declined but much more rapid sap uptake correlated with the onset of the freezing exotherm. When sap pressures were measured under conditions of negligible volume displacement, the sap pressure rapidly fell to -60 to -80 kilopascals at the start of the freezing exotherm. The volume of water uptake and the rate of uptake depended on the rate of freezing. A slow freezing rate correlated with a large volume of water uptake, a fast freezing rate induced a smaller volume of water uptake. The volume of water uptake ranged from 0.02 to 0.055 grams water per gram dry weight of sapwood. The volume of water exuded after thawing was usually less than the volume of uptake so that after several freezing and thawing cycles the sapwood water content increased from 0.7 to 0.8 grams water per gram dry weight.These results are discussed in terms of a physical model of the mechanism of maple sap uptake and exudation first proposed by P. E. R. O'Malley. The proposed mechanism of sap uptake is by vapor distillation in air filled wood fiber lumina during the freezing of minor branches. Gravity and pressurized air bubbles (compressed during freezing) cause sap flow from the canopy down the tree after the thaw. PMID- 16663209 TI - A rapid regulatory response governing nodulation in soybean. AB - The number of nodules which develop on the primary root of soybean seedlings (Glycine max L. Merr) after inoculation with Rhizobium japonicum is substantially diminished in the region of the root developmentally 10 to 15 hours younger than the region maximally susceptible to nodulation at the time of inoculation. This rapid inhibition of nodulation has been investigated by inoculating soybean seedlings with rhizobia at two different times, 15 hours apart. Living R. japonicum cells, but not heterologous rhizobia or UV-killed cells of the homologous bacterium, were capable of eliciting the rapid inhibitory response. Nodulation responses to varying inoculum concentrations showed that bacterial dosages could be superoptimal, resulting in reduced nodulation and reduced inhibition of nodulation. When suspensions of R. japonicum were dripped uniformly onto the root surfaces, the degree of inhibition of nodulation in developmentally younger regions of the root was correlated with the number of nodules formed in the older and initially most susceptible region of the root. Nodulation in the developmentally younger region of the root, however, was affected very little if the first inoculum was restricted to contact with root cells in the region initially most susceptible to nodulation. The rapid regulatory response may be an important factor contributing to the clustering of nodules in the crown region of soybean roots in field-grown plants and the sparse nodulation commonly observed in younger regions of the root. PMID- 16663210 TI - Effects of low concentrations of o(3) on net photosynthesis, dark respiration, and chlorophyll contents in aging hybrid poplar leaves. AB - Chronic exposure of hybrid poplar (Populus deltoides x trichocarpa) plants to low concentrations of ozone had negative impact upon net photosynthetic capacity, dark respiration, and leaf chlorophyll contents. Exposure to as much as 0.20 microliters per liter O(3) had no immediate effect on net photosynthesis (P(n)), but chronic exposure to 0.125 or 0.085 microliters per liter had a number of gradual effects on CO(2) exchange. These included increased dark respiration and consequently increased light compensation points in very young leaves (4-6 days old); and decreased P(n), leaf chlorophyll a and b contents, light saturation points, and apparent quantum yields in leaves 10 to 70 days old. Decreased P(n) was partially due to accelerated aging in leaves exposed to O(3), and lightsaturated P(n) was linearly related to chlorophyll a + b contents. Differences in light-saturated P(n) between control and O(3)-treated leaves of the same age were mostly due to photosaturation in O(3)-treated leaves and to a much lesser extent to lowered apparent quantum yields. Also, since P(n) and dark respiration were most affected by O(3) at different leaf ages, distinct modes of action are suggested. The effects of leaf aging on CO(2) exchange were considerable, but typical of other species. However, careful monitoring of the interacting effects of leaf age and pollutant exposure was needed in order to characterize the impact of chronic O(3) exposure upon CO(2) exchange. PMID- 16663211 TI - Relationships between Carbon Assimilation, Partitioning, and Export in Leaves of Two Soybean Cultivars. AB - To evaluate leaf carbon balance during rapid pod-fill in soybean (Glycine max [L.] Merrill), measurements were made of CO(2) assimilation at mid-day and changes in specific leaf weight, starch, and sucrose concentrations over a 9-hour interval. Assimilate export was estimated from CO(2) assimilation and leaf dry matter accumulation. Chamber-grown ;Amsoy 71' and ;Wells' plants were subjected on the day of the measurements to one of six photosynthetic photon flux densities in order to vary CO(2) assimilation rates.Rate of accumulation of leaf dry matter and rate of export both increased as CO(2) assimilation rate increased in each cultivar.Starch concentrations were greater in Amsoy 71 than in Wells at all CO(2) assimilation rates. At low CO(2) assimilation rates, export rates in Amsoy 71 were maintained in excess of 1.0 milligram CH(2)O per square decimeter leaf area per hour at the expense of leaf reserves. In Wells, however, export rate continued to decline with decreasing CO(2) assimilation rate. The low leaf starch concentration in Wells at low CO(2) assimilation rates may have limited export by limiting carbon from starch remobilization.Both cultivars exhibited positive correlations between CO(2) assimilation rate and sucrose concentration, and between sucrose concentration and export rate. Carbon fixation and carbon partitioning both influenced export rate via effects on sucrose concentration. PMID- 16663212 TI - Invertase activity and the kinetin-stimulated enlargement of detached radish cotyledons. AB - Cytokinin treatment is known to promote expansion of light-grown excised radish (Raphanus sativus L. cv Crimson Giant) cotyledons. This expansion, at least in part, seems to be related to an increased accumulation of osmotically active reducing sugars. Kinetin treatment did not cause increased levels of isocitrate lyase activity over the controls, but stimulated increased levels of two invertase forms, designated types I and II. Type I was soluble and type II was insoluble after homogenization in 10 millimolar tris(hydroxymethyl)aminomethane HCl (pH 7.0). Both types were soluble after homogenization in 300 millimolar NaCl. At low salt concentration, type II was retained on a diethylamioethyl cellulose column and type I was not. Type II was then eluted from the column at high salt concentration. Types I and II exhibited pH optima of 5.3 and 4.3, Michaelis constants of 4.96 and 1.23 millimolar sucrose, and molecular weights of 65,000 and 57,000 daltons, respectively. The kinetin promotion of reducing sugar accumulation may be related to increased levels of the two invertase forms, but is probably not a result of direct cytokinin-stimulated glyoxysomal activity. PMID- 16663213 TI - A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts. AB - After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3 (3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg(2+) than by Ca(2+) ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca(2+) ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca(2+) selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg(2+) site on the 3-(3,4 dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca(2+) ions, implying that the selective Ca(2+) effect on photosystem II (selective Ca(2+) site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems. PMID- 16663214 TI - Translocation and Metabolism of Endosperm-Applied [2-C] Indoleacetic Acid in Etiolated Avena sativa L. Seedlings. AB - The role of free indole-3-acetic acid (IAA) in the endosperm of Avena sativa L. seedlings was investigated to determine its contribution to free IAA in the shoot. [2-(14)C]IAA was injected into the endosperm of darkgrown seedlings and the transport and metabolism of the [(14)C]-labeled compounds determined. It was concluded that translocation of free IAA directly from the endosperm is probably not a significant source of free IAA in the shoot, mainly because even small amounts of [(14)C]IAA introduced into the endosperm were rapidly metabolized. This suggested that, in Avena, free IAA does not normally exist in the liquid endosperm. PMID- 16663215 TI - Selective inhibition of proline hydroxylation by 3,4-dehydroproline. AB - The effect of proline analogs on peptidyl proline hydroxylation has been studied in vivo using aerated root slices of Daucus carota. One analog, 3,4 dehydroproline, acted at micromolar concentrations to rapidly and selectively inhibit peptidyl proline hydroxylation. A structurally altered hydroxyproline rich cell wall glycoprotein was synthesized and secreted by dehydroproline treated tissue. The capacity to hydroxylate proline recovered slowly following a short pulse treatment with the analog, with a halftime for recovery of about 24 hours. Recovery was not altered by supplying exogenous proline. Dehydroproline had little effect on the induction of nitrate reductase by nitrate, nor on wound induced increases in amino acid uptake and protein synthesis. In contrast, other proline analogs inhibit proline hydroxylation only at millimolar concentrations. It is hypothesized that dehydroproline acts as an enzyme-activated suicide inhibitor of prolyl hydroxylase. This analog should become a useful tool for elucidating the functional significance of hydroxyproline-rich glycoproteins. PMID- 16663216 TI - Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in Gonyaulax polyedra. AB - Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O(2) uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O(2) evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as (14)C fixation or O(2) evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax. PMID- 16663217 TI - Development of Photosynthetic Activity following Anaerobic Germination in Rice Mimic Grass (Echinochloa crus-galli var oryzicola). AB - Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24 hour midtreatment of air before light.Development of photosynthetic activity in etiolated shoots, determined by CO(2) gas exchange, (14)CO(2) uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development. PMID- 16663218 TI - Reversible conjugation of gibberellins in situ in maize. AB - Gibberellins [(3)H]GA(4) (1.33 Curies per millimole) and [(3)H]GA(20) (2.36 Curies per millimole) were injected into the shanks of maize (Zea mays L.) cobs during rapid grain filling and mature seeds were subsequently harvested. Extracts of mature, dry seeds from 1980 feeds yielded only 20 to 30% of the (3)H radioactivity in acidic, ethyl acetate-soluble form, and this was principally associated with the precursor, with lesser amounts of the major metabolite, [(3)H]GA(1) (putative identification based on sequential SiO(2) partition, and gradient-eluted reverse-phase C(18) high performance liquid chromatography [HPLC]). Most of the radioactivity in the dry seeds was associated with compounds having partition characteristics of, and co-chromatographing on, sequential SiO(2) partition and reverse-phase HPLC with glucosyl conjugates of the precursors (GA(4) or GA(20)) and their probable major metabolite (GA(1)). The majority of conjugate associated with the precursor GA(4) eluted coincidental with GA(4) glucoside. Subsequent acid or enzymic hydrolysis (beta-glucosidase or cellulase) yielded the free GAs, putative identification being based on isocratic HPLC of each (3)H-labeled conjugate --> hydrolysis --> isocratic HPLC of the (3)H labeled hydrolysate. Upon imbibition of the seeds, radioactivity associated with the conjugate fraction decreased; concomitantly, statistically significant increases in levels of free [(3)H]GA-like compounds were observed. Although the specific ratios of GA-like and GA-glucosyl conjugate-like substances varied substantially across years, hybrids, and even, in different plants from the same hybrid, this ;reversible conjugation' (i.e. apparent conjugation during seed maturation followed by release of the GA moiety during germination), was reproducible for [(3)H]GA(20) in seed from two maize hybrids produced over 2 years. PMID- 16663219 TI - Phytohormones, Rhizobium Mutants, and Nodulation in Legumes : III. Auxin Metabolism in Effective and Ineffective Pea Root Nodules. AB - High specific activity [(3)H]indole-3-acetic acid (IAA) was applied to the apical bud of intact pea (Pisum sativum L. cv Greenfeast) plants. Radioactivity was detected in all tissues after 24 hours. More radioactivity accumulated in the nodules than in the parent root on a fresh weight basis and more in effective (nitrogen-fixing) nodules than in ineffective nodules (which do not fix nitrogen).For most samples, thin layer chromatography revealed major peaks of radioactivity at the R(F) values of IAA and indole-3-acetylaspartic acid (IAAsp) and further evidence of the identity of these compounds was obtained by chromatography in other systems. Disintegrations per minute due to IAA per unit fresh weight were significantly greater for root than for nodule tissue, but were not significantly different for effective and ineffective nodules. Radioactivity due to IAAsp, expressed both on a percentage basis and per unit fresh weight, was significantly greater for nodule than for root tissue and significantly greater for the effective nodules than for the ineffective nodules. When [(3)H]IAA was applied to effective nodules, IAAsp was the dominant metabolite in the nodule. The data suggest that metabolism of auxins may be important for the persistence of a functional root nodule. PMID- 16663220 TI - Light-Harvesting System of the Red Alga Gracilaria tikvahiae: I. Biochemical Analyses of Pigment Mutations. AB - Wild type Gracilaria tikvahiae, a macrophytic red alga, and fourteen genetically characterized pigment mutants were analyzed for their biliprotein and chlorophyll contents. The same three biliproteins, phycoerythrin, phycocyanin, and allophycocyanin, which are found in the wild type are found in all the Mendelian and non-Mendelian mutants examined. Some mutants overproduce R-phycoerythrin while others possess only traces of phycobiliprotein; however, no phycoerythrin minus mutants were found. Two of the mutants are unique; one overproduces phycocyanin relative to allophycocyanin while the nuclear mutant obr synthesizes a phycoerythrin which is spectroscopically distinct from the R-phycoerythrin of the wild type. The phycoerythrin of obr lacks the typical absorption peak at 545 nanometers characteristic of R-phycoerythrin and possesses a phycoerythrobilin to phycourobilin chromophore ratio of 2.6 in contrast to a ratio of 4.2 found in the wild type. Such a lesion provides evidence for the role of nuclear genes in phycoerythrin synthesis. In addition, comparisons are made of the pigment compositions of the Gracilaria strains with those of Neoagardhiella bailyei, a macrophytic red alga which has a high phycoerythrin content, and Anacystis nidulans, a cyanobacterium which lacks phycoerythrin. The mutants described here should prove useful in the study of the genetic control of phycobiliprotein synthesis and phycobilisome structure and assembly. PMID- 16663221 TI - Light-Harvesting System of the Red Alga Gracilaria tikvahiae: II. Phycobilisome Characteristics of Pigment Mutants. AB - Phycobilisomes were isolated from wild type Gracilaria tikvahiae and a number of its genetically characterized Mendelian and non-Mendelian pigment mutants in which the principal lesions result in an increase or decrease in the accumulation of phycoerythrin. Both the size and phycoerythrin content of the phycobilisomes are proportional to the phycoerythrin content of the crude algal extracts. In most of the strains examined, the structure and function of the phycocyanin allophycocyanin phycobilisome cores are the same as in wild type. The phycobilisome architecture is derived from wild type by the addition or removal of phycoerythrin. The same pattern is observed for the phycobilisome of mos(2) which contains a large excess of phycocyanin that is not bound to the phycobilisome. The single exception is a yellow, non-Mendelian mutant, NMY-1, which makes functional phycobilisomes composed of phycoerythrin and allophycocyanin with almost no phycocyanin. Characterization of the ;linker' polypeptides of the phycobilisome indicates that a 29 kilodalton protein is required for the stable incorporation of phycocyanin into the phycobilisome. Evidence is provided for the requirement of nuclear and cytoplasmic genes in phycobilisome synthesis and assembly. The symmetry properties of the phycobilisome are considered and a structural model for the reaction center II phycobilisome organization is presented. PMID- 16663222 TI - Transport of purine and pyrimidine bases and nucleosides from endosperm to cotyledons in germinating castor bean seedlings. AB - During germination and early growth of castor bean (Ricinus communis), all cellular constituents of the endosperm are eventually transferred to the growing embryo. The present results bear on the transport of breakdown products of nucleic acids. The total content of nucleic acids and nucleotides declines rapidly between day 4 and day 8 of seedling development. Concomitant with this decline, a secretion of adenosine, guanosine, and adenine from excised endosperms into the incubation medium takes place, accompanying a much more extensive release of sucrose and amino acids. Release of nucleotides could not be detected. The rates of release were linear for at least 5 hours for all compounds measured, indicating that they were liberated due to a coordinated metabolism. Uptake studies with cotyledons removed from the seedling showed that these have the ability to absorb all the substances released from the endosperm. Besides sucrose and amino acids, both nucleosides and free purine and pyrimidine bases were taken up by the cotyledons with high efficiency. AMP was also transported whereas ATP was not. Kinetic analyses were carried out to estimate the maximal uptake capacities of the cotyledons. Rates of uptake were linear for at least 1 to 2 hours and saturation kinetics were observed for all substances investigated. It is concluded that nucleosides can serve best as transport metabolites of nucleic acids, inasmuch as they are taken up by the cotyledons with the highest efficiency, the V(max)/K(m) ratios being considerably higher than those found for free purine and pyrimidine bases. For both adenosine and adenine transport, the V(max) was about 2 micromoles per hour per gram fresh weight, and the K(m) values were 0.12 and 0.37 millimolar, respectively. The rates of metabolite release from the endosperm and the capacity of the absorption system in the cotyledons are shown to account for the observed rates of disappearance of nucleic acids from the endosperm and efficient transport to the growing embryo. PMID- 16663223 TI - Ionic Osmoregulation during Salt Adaptation of the Cyanobacterium Synechococcus 6311. AB - The mechanisms of salt adaptation were studied in the cyanobacterium Synechococcus 6311. Intracellular volumes and ion concentrations were measured before and after abrupt increases of external NaCl concentrations up to 0.6 molar NaCl. Equilibrium volumes, measured with a rapid and accurate electron spin resonance spin probe method, showed that at low NaCl concentrations the cells did not shrink as expected for an impermeable solute. However, when the NaCl concentration exceeded a critical value, volume losses occurred. These losses were not fully reversed by hypoosmotic treatment, suggesting membrane damage. The critical value of irreversible volume loss paralleled the increase in salinity during cell growth. Rapid mixing experiments showed that exposure of Synechococcus 6311 to non-damaging NaCl concentrations caused water extrusion from the cells; the volume decreases were time resolved to about 200 milliseconds. Subsequently, volumes increased rapidly as NaCl moved into the cells. Controls recovered their volumes within 15 seconds, while salt-adapted cells grown at 0.6 molar NaCl required 1 minute for volume equilibration. This decrease in the rate of cell volume recovery indicates that salt adaptation is accompanied by changes in cell membrane properties. Subsequent to these initial rapid volume changes, a more gradual sequence of ion movement and sugar accumulation was observed. Under conditions for photoautotrophic growth, significant Na(+) extrusion was observed 30 min after salt shock. Sucrose accumulation reached a maximum value after 16 hours and K(+) accumulation reached equilibrium after 40 hours. The final concentrations of K(+) and Na(+) and sucrose and glucose inside the 0.6 molar NaCl-grown cells indicate that the inorganic ions and organic ;compatible' solutes are the major osmotic species which account for the adaptation of Synechococcus 6311 to salt. PMID- 16663224 TI - Seed Dormancy in Red Rice : III. Response to Nitrite, Nitrate, and Ammonium Ions. AB - Sodium nitrite at 10 millimolar breaks dormancy of dehulled red rice (Oryza sativa). While germination is light independent, low pH conditions (pH 3) are required for maximum response. Water and buffer controls at pH 3 remain dormant. The response to nitrite occurs at 25 and 30 degrees C but is reduced at 20 degrees C, although nondormant seeds germinate readily at this temperature. The contact time for response to nitrite is less than 2 h at the start of imbibition. Seeds imbibed first in water show reduced germination when subsequently transferred to nitrite. Dehulled seeds show little or no response to nitrate and ammonium ions.Intact seeds remain dormant in the presence of nitrite or nitrate unless partially dry-afterripened. The pH dependence of nitrite sensitivity is reduced in intact, afterripening seeds. In highly dormant seeds, vacuum infiltration experiments suggest that the hull restricts uptake of nitrite. PMID- 16663225 TI - Orientation and integrity of plasma membrane vesicles obtained from carrot protoplasts. AB - Two fractions enriched in plasma membrane derived from suspension-cultured carrot (Daucus carota L.) cells were examined to determine if they differed from each other either in physical nature or in orientation. Parameters studied included the protein composition of purified membranes derived from trypsinized and nontrypsinized protoplasts as well as from trypsinized purified plasma membranes, the effect of inhibitors and membrane perturbants on ATPase activity, the binding of [acetyl-(14)C]concanavalin A to purified membrane fractions, and the competitive removal of [acetyl-(14)C]concanavalin A from purified membranes derived from [acetyl-(14)C]concanavalin A-labeled protoplasts. One fraction (at density of 1.102 grams per cubic centimeter on Renografin gradients) appears to be a mixed population of ;tightly' sealed vesicles with the majority being rightside-out vesicles of plasma membrane, and the other fraction (density 1.128 grams per cubic centimeter) apparently is a population of predominantly ;leaky' vesicles and/or nonvesicular fragments of plasma membrane, a large portion of which appear to be ;leaky' inside-out vesicles. In addition, it is shown that plasma membrane-enriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition. PMID- 16663226 TI - Effect of temperature on nitrogenase functioning in cowpea nodules. AB - Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30 degrees C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20 degrees C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30 degrees C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15 degrees C to a maximum around 35 degrees C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38 degrees C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35 degrees C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15 degrees C and 47 degrees C. PMID- 16663227 TI - Endogenous auxin and ethylene in pellia (bryophyta). AB - The occurrence of endogenous indole-3-acetic acid and ethylene in bryophyte tissue was tentatively demonstrated using gas chromatography, high performance liquid chromatography, and double-standard isotope dilution techniques. Rapidly elongating stalks (or setae) of Pellia epiphylla (L.) Corda sporophytes contain approximately 2.5 to 2.9 micrograms per gram fresh weight of putative free IAA. Ethylene released by setae increases during growth from 0.027 to 0.035 nanoliter per seta per hour. Application of 5 microliters per liter ethylene inhibits auxin stimulated elongation growth of this tissue, a result which suggests that both endogenously produced compounds act in tandem as natural growth modulators. PMID- 16663228 TI - Purification and separation of plant gibberellins from their precursors and glucosyl conjugates. AB - A procedure using two small preparative columns (in sequence) of C(18) reverse phase Bondapak B material with methanolic extracts of plant tissue (Pisum sativum L., Malus domestica Borkh., Pimpinella anisum L.) yields two fractions: (i) gibberellin (GA) precursors, and (ii) free GA/GA methyl esters (GA-Me)/GA glucosyl conjugates. The discrete separation of (iii) free GA/GA-Me from (iv) GA glucosyl conjugates is then accomplished by a combination of differential solvent solubility and SiO(2) partition chromatography. All fractions are almost pigment free, and appreciable dry weight purification was accomplished for the GA precursor and free GA/GA-Me fractions. Solvent volumes can be kept low, no buffer salts are introduced, and each fraction (i, iii, iv) can be subjected directly to preparative or analytical reverse phase C(18) high performance liquid chromatography without recourse to solvent partitioning, and often without further purification. PMID- 16663229 TI - Ethylene-induced lateral expansion in etiolated pea stems : kinetics, cell wall synthesis, and osmotic potential. AB - Treatment of etiolated pea (Pisum sativum L.) internode tissue with ethylene gas inhibits elongation and induces lateral expansion. Precise kinetics of the induction of this altered mode of growth of excised internode segments were recorded using a double laser optical monitoring device. Inhibition of elongation and promotion of lateral expansion began after about 1 hour of treatment and achieved a maximum by 3 hours. Similar induction kinetics were observed after treating internodes with colchicine and 2,6-dichlorobenzonitrile, an inhibitor of cellulose synthesis. In sealed flask experiments, ethylene had no detectable effect on incorporation of label from [(14)C]glucose into any of the classical pectin, hemicellulose, or cellulose wall fractions. Ethylene inhibited fresh weight increase (total cell expansion) of both excised internode segments (in sealed flasks) and intact seedlings. Ethylene treatment resulted in an increase in cell sap osmolality in those tissues (intact and excised) which are inhibited by the gas. A model for ethylene-induced inhibition of elongation and induction of lateral expansion is presented. PMID- 16663230 TI - Ethylene-induced lateral expansion in etiolated pea stems : the role of Acid secretion. AB - Ethylene-induced inhibition of elongation and promotion of lateral expansion in the stems of etiolated pea (Pisum sativum L. var Alaska) seedlings is not associated with any alteration of auxin-stimulated proton extrusion. Indeed, lateral expansion in response to ethylene apparently requires an acidified wall since it is prevented by strong neutral buffers and by the ATPase inhibitor orthovanadate. Ethylene treatment reduces the capacity of live and frozen-thawed sections to extend in the longitudinal direction in response to acid. The effect of ethylene on lateral acid growth capacity is more complicated. Ethylene-treated internodes do not exhibit acid-induced lateral expansion. Ethylene-treated segments which have been frozen-thawed do show an enhanced capacity to extend in the transverse direction at acid pH, but only when the inner tissues have been removed by coring. We conclude that two of the factors which control the directionality of expansion during ethylene treatment are a decrease in the sensitivity of the walls to acid longitudinally and an increase in the sensitivity of the outer cortical parenchyma walls to acid in the transverse direction. PMID- 16663231 TI - Isolation of ;Vacuoplasts' from Poterioochromonas malhamensis. AB - A method is reported for the isolation of ;vacuoplasts' from Poterioochromonas malhamensis. Vacuoplasts are separated mechanically by centrifugation on silica sol gradients. They consist of the leucosin storage vacuole, a portion of the plasma membrane, and some cytoplasmic components. This method is suited to give a high yield of vacuoplasts.Vacuoles are the largest membrane-bound organelles in plant cells. Only a few methods exist for their large scale isolation and purification in an intact and physiologically active state. The high shear forces used to disrupt cell walls during tissue fractionation usually also disrupt the fragile vacuoles. Intact vacuoles have been isolated from root storage tissue of Beta vulgaris L. (14) by a slicing procedure or by lysing enzymically prepared protoplasts from yeast (12). Vacuoles from higher plants have been isolated by osmotic lysis of protoplasts (18), treatment of protoplasts with polybases (6, 7), or centrifugation of protoplasts at high g forces against a solution of Ficoll (16).The wall-less flagellate P. malhamensis is a fresh water organism with two unequal flagellae, belonging to the order Chrysomonadina, a group whose members characteristically store oil and leucosin. Leucosin (= chrysolaminarin) is stored in a posterior vacuole, which may be so large as to almost fill the cell. Leucosin is a polysaccharide composed of beta- (1-->3)-linked d-glucose residues. It has a degree of polymerization of about 34, is water soluble, and probably has a slightly branched structure (1). PMID- 16663232 TI - Soybean adaptation to water stress at selected stages of growth. AB - Soybean (Glycine max [L.] Merr. cv Braxton) plants were grown in sandy soil with only natural rainfall (N) or with supplemental irrigation (I). Water-stressed plants grew more extensive root systems, whereas irrigated plants developed larger shoots and smaller root systems. Maximum stomatal apertures were observed at the beginning of each photoperiod. Partial stomatal closure occurred each afternoon, but stomata of I plants remained open longer than those of N plants. Significant reductions in net carbon fixation rate generally accompanied decreases in stomatal aperture, which coincided with periods of high temperature, low relative humidity, maximum solar radiation, and water stress. Leaf water potential decreased from morning to afternoon, with a greater decrease observed for N plants. Midafternoon stomatal closure did not occur in N plants with very large root systems following a heavy rain which saturated the soil profile. With smaller root systems and greater evaporative demand from larger shoots, the I plants continued to show midafternoon stress following the heavy rain. The large root systems of the N plants absorbed sufficient water to meet shoot evaporative demand for several days following the rain. Root soil system resistance apparently contributed to the afternoon water stress in the I plants. PMID- 16663233 TI - Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis. AB - Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., ;Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6 bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light. PMID- 16663234 TI - Light and Shade Effects on Abscission and C-Photoassimilate Partitioning among Reproductive Structures in Soybean. AB - Field experiments were conducted in 1981 and 1982 to study the effects of low irradiance supplemental light on soybean (Glycine max [L.] Merr. cv Evans) flower and pod abscission. Cool-white and red fluorescent lights illuminated the lower part of the soybean canopy during daylight hours for 3 weeks late in flowering. At the same time, flowers and young pods on half the plants were shaded with aluminum foil. Flowers were tagged at anthesis and monitored through abscission or pod maturity.Responses to red and white lights were similar. Supplemental light tended to reduce abscission and increase seed weight per node compared to natural light. Shading flowers and pods increased abscission and reduced seed weight per node. Number of flowers produced per node, individual seed weight, and seeds per pod were not affected by light or shade treatments.Further studies examined the effects of shading reproductive structures on their capacity to accumulate (14)C-photoassimilates. Individual leaves were pulse labeled with (14)CO(2) 1, 2, and 4 weeks post anthesis. Flowers and pods in the axil of the labeled leaf were covered with aluminum foil 0, 24, 72, and 120 hours before pulsing.Shading flowers and pods resulted in a 30% reduction in the relative amount of radiolabel accumulated from the source leaf. The reduction in (14)C accumulation due to shading was evident regardless of the length of the shading period and was most pronounced when the shades were applied early in reproductive development. We conclude that light perceived by soybean flowers and young pods has a role in regulating both their abscission and their capacity to accumulate photoassimilates. PMID- 16663235 TI - Comparison of Activities of the Host-Specific Toxin of Helminthosporium maydis, Race T, and a Synthetic C(41) Analog. AB - It previously had been proposed that the host-selective toxin of Helminthosporium maydis race T consists of a series of unusual linear (C(35) to C(45))polyketols, of equal toxicity on a weight or molar (10(-8)-10(-9)) basis. Previous laboratory synthesis of T-toxin analogs was limited to shorter (C(15) to C(26)) versions which possessed the requisite specificity for susceptible corn (Zea mays) but were less toxic on a weight or molar (10(-6)-10(-7)) basis. In the present study, a C(41) analog with four beta-ketol units spaced by CH(2) bridges as in native toxin has been synthesized. On a weight or molar basis, it is as effective as native toxin or its purified components in stimulating NADH oxidation of mitochondria from susceptible corn, thus providing firm evidence for the correctness of the proposed structures of T-toxin. Additional support derives from the observation that C(24) and C(26) analogs with -(CH(2))(4)- and (CH(2))(6)- bridges between ketol groups are not as effective in stimulating NADH oxidation as are C(23) and C(25) analogs with the -(CH(2))(3)- and -(CH(2))(5)- bridges of native T-toxin.It was calculated that a single molecule of the C(41) analog is at least 300 times more effective in stimulating mitochondrial oxidation than a molecule of the C(23) or C(25) analogs. This emphasizes the importance of chain length for toxicity, perhaps through perturbation of membrane functions of mitochondria and/or chloroplasts. PMID- 16663236 TI - Analysis of Indole-3-acetic Acid Metabolism in Zea mays Using Deuterium Oxide as a Tracer. AB - A method using deuterium oxide (D(2)O) as a tracer was used to study indole-3 acetic acid (IAA) metabolism in Zea mays seedlings. Seeds were imbibed and grown for 4 days in 30% D(2)O in the dark. IAA was then isolated from roots and shoots and analyzed for deuterium content by mass spectrometry. We found that a significant portion of the IAA isolated from plants had incorporated deuterium at nonexchangeable sites of the indole ring. This indicates that some of the IAA in the germinating seedling is made via de novo indole synthesis. Moreover, we found that the deuterium content of IAA was 2.6 times greater in shoots than in roots. These results indicate that at least some of the IAA in roots and shoots came from different biosynthetic pathways. It appears that the fraction of IAA produced via de novo indole synthesis is greater in shoots than in roots. PMID- 16663237 TI - Abscission: quantification of light control. AB - Low level red light treatments prevented dark-induced leaf abscission in mung bean (Vigna radiata L. Wilczek cv Jumbo) cuttings. The amount of inhibition depended upon the level and length of the light treatment. The red light inhibition could be reversed by a brief exposure of tissue to far-red light. PMID- 16663238 TI - Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex. AB - Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H(2)O to methyl viologen), while retaining activities associated with photosystem II (H(2)O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons. PMID- 16663239 TI - Lipase in the Lipid Bodies of Corn Scutella during Seedling Growth. AB - In the scutella of corn (Zea mays), lipase activity is absent in ungerminated seeds and increases during seedling growth. At the peak stage of lipolysis, about 50% of the lipase activity is recovered in the lipid body fraction after flotation centrifugation. The lipase is tightly bound to the lipid bodies, and resists solubilization by repeated washing with buffers or NaCl solutions. Isolated lipid bodies undergo autolysis of internal triacylglycerols, resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies have been extracted with diethyl ether, the lipase is recovered in the membrane fraction. The lipase has an optimal activity at pH 7.5 in the autolysis of lipid bodies, or on trilinolein or N-methylindoxylmyristate. Of the various acylglycerols examined, the enzyme is active only on acylglycerols of linoleic and oleic acids which are the major fatty acid constituents of corn oil. The activity is not greatly affected by NaCl, CaCl(2), or pretreatment of the enzyme with p-chloromercuribenzoate or mersalyl, and detergents abolish the activity. The enzyme hydrolyzes trilinolein completely to fatty acids; during the course of reaction, there is little accumulation of di- or mono-linolein. PMID- 16663240 TI - Factors which affect the amount of inorganic phosphate, phosphorylcholine, and phosphorylethanolamine in xylem exudate of tomato plants. AB - Phosphate in the xylem exudate of tomato (Lycopersicon esculentum) plants was 70 to 98% inorganic phosphate (Pi), 2 to 30% P-choline, and less than 1% P ethanolamine. Upon adding (32)Pi to the nutrient, Pi in xylem exudate had the same specific activity within 4 hours. P-choline and P-ethanolamine reached the same specific activity only after 96 hours. The amount of Pi in xylem exudate was dependent on Pi concentration in the nutrient and decreased from 1700 to 170 micromolar when Pi in the nutrient decreased from 50 to 2 micromolar. The flux of 0.4 nmoles organic phosphate per minute per gram fresh weight root into the xylem exudate was not affected by the Pi concentration in the nutrient solution unless it was below 1 micromolar. During 7 days of Pi starvation, Pi in the xylem exudate decreased from 1400 to 130 micromolar while concentrations of the two phosphate esters remained unchanged.The concentration of phosphate esters in the xylem exudate was increased by addition of choline or ethanolamine to the nutrient solution, but Pi remained unchanged. Upon adding [(14)C]choline to the nutrient, 10 times more [(14)C]P-choline than [(14)C]choline was in the xylem exudate and 85 to 90% of the ester phosphate was P-choline. When [(14)C]ethanolamine was added, [(14)C]P-ethanolamine and [(14)C]ethanolamine in the xylem sap were equal in amount. P-choline and P-ethanolamine accumulated in leaves of whole plants at the same time and the same proportion as observed for their flux into the xylem exudate. No relationship between the transport of P choline and Pi in the xylem was established. Rather, the amount of choline in xylem exudate and its incorporation into phosphatidylcholine in the leaf suggest that the root is a site of synthesis of P-choline and P-ethanolamine for phospholipid synthesis in tomato leaves. PMID- 16663241 TI - Differences in the physical properties of native and partially degraded phytochrome as probed by their differential sensitivity to permanganate oxidation. AB - The differential sensitivities to permanganate oxidation of the red and far-red forms of native phytochrome from Avena sativa L. cv Mulaga (isolated as Pfr from red-irradiated tissue) and of partially degraded phytochrome (isolated as Pr from nonirradiated tissue) were determined. The far-red absorbing form of partially degraded phytochrome was 5 times more sensitive than its red-absorbing form, while both the far-red and red forms of native phytochrome exhibited identical sensitivity. The present data obtained with partially degraded phytochrome are in apparent agreement with the data and model of Hahn, Kang, and Song (1980 Biochem Biophys Res Commun 97: 1317-1323). Their model suggests that the chromophore of the red-absorbing form of phytochrome is buried in a hydrophobic crevice in the protein, while that of the far-red form is exposed. The data obtained with native phytochrome, however, are at variance with their model. Our data obtained with native phytochrome suggests that the chromophore of the red and the far-red absorbing forms of native phytochrome both are in a relatively protected environment and that only following partial proteolytic degradation of the phytochrome does the chromophore of its far-red form become relatively more exposed. The protective influence of the labile peptide could either be direct, because of its close physical proximity to the chromophore, or indirect, resulting in an alteration in chromophore-protein interaction. PMID- 16663242 TI - Effects of soil flooding on leaf gas exchange of tomato plants. AB - Carbon dioxide and water vapor exchange of tomato (Lycopersicon esculentum Mill. cv Rheinlands Ruhm) leaves were measured before and after 24 h of soil flooding to characterize both stomatal and nonstomatal responses to the stress. Leaf epidermal conductance to water vapor decreased by 47% after flooding, accompanied by an increase in the sensitivity of stomata to changes in CO(2) concentration. Assimilation rates under ambient conditions fell by 27%, and the inhibition could not be overcome by elevated CO(2) partial pressures. Stomatal conductance limited the assimilation rate to approximately the same degree both before and after flooding. The reduction in photosynthetic capacity was not due to a decrease in apparent quantum yield or to an increase in photorespiration. The results were analyzed according to a recent model of photosynthesis, and possible mechanisms underlying the flooding effect are discussed. PMID- 16663243 TI - Involvement of plant growth substances in the alteration of leaf gas exchange of flooded tomato plants. AB - Ethylene, abscisic acid, and cytokinins were tested for their ability to either induce or prevent the changes which occur in gas exchange characteristics of tomato (Lycopersicon esculentum Mill. cv. Rheinlands Ruhm) leaves during short term soil flooding. Ethylene, which increases in the shoots of flooded plants, had no effect on stomatal conductance or photosynthetic capacity of drained plants. Abscisic acid, which also accumulates in the shoots of flooded plants, could reproduce the stomatal behavior of flooded plants when sprayed on the leaves of drained plants. However, photosynthetic capacity of drained plants was unaffected by abscisic acid sprays. Cytokinin export from the roots to the shoots declines in flooded plants. Spray applications of benzyladenine increased stomatal conductance in both flooded and drained plants. In addition, the decline in photosynthetic capacity during flooding was largely prevented by supplementary cytokinin applications. The possible involvement of these growth substances in modifying leaf gas exchange during flooding is discussed. PMID- 16663245 TI - Two photosynthetic mechanisms mediating the low photorespiratory state in submersed aquatic angiosperms. AB - The submersed angiosperms Myriophyllum spicatum L. and Hydrilla verticillata (L.f.) Royal exhibited different photosynthetic pulse-chase labeling patterns. In Hydrilla, over 50% of the (14)C was initially in malate and aspartate, but the fate of the malate depended upon the photorespiratory state of the plant. In low photorespiration Hydrilla, malate label decreased rapidly during an unlabeled chase, whereas labeling of sucrose and starch increased. In contrast, for high photorespiration Hydrilla, malate labeling continued to increase during a 2-hour chase. Thus, malate formation occurs in both photorespiratory states, but reduced photorespiration results when this malate is utilized in the light. Unlike Hydrilla, in low photorespiration Myriophyllum, (14)C incorporation was via the Calvin cycle, and less than 10% was in C(4) acids.Ethoxyzolamide, a carbonic anhydrase inhibitor and a repressor of the low photorespiratory state, increased the label in glycolate, glycine, and serine of Myriophyllum. Isonicotinic acid hydrazide increased glycine labeling of low photorespiration Myriophyllum from 14 to 25%, and from 12 to 48% with high photorespiration plants. Similar trends were observed with Hydrilla. Increasing O(2) increased the per cent [(14)C]glycine and the O(2) inhibition of photosynthesis in Myriophyllum. In low photorespiration Myriophyllum, glycine labeling and O(2) inhibition of photosynthesis were independent of the CO(2) level, but in high photorespiration plants the O(2) inhibition was competitively decreased by CO(2). Thus, in low but not high photorespiration plants, glycine labeling and O(2) inhibition appeared to be uncoupled from the external [O(2)]/[CO(2)] ratio.These data indicate that the low photorespiratory states of Hydrilla and Myriophyllum are mediated by different mechanisms, the former being C(4)-like, while the latter resembles that of low CO(2)-grown algae. Both may require carbonic anhydrase to enhance the use of inorganic carbon for reducing photorespiration. PMID- 16663244 TI - Newly Synthesized mRNA Is Translated during the Initial Imbibition Phase of Germinating Maize Embryo. AB - RNA synthesis is activated in the cells of the plant embryo very soon after the start of seed imbibition. We previously reported that mainly heterogeneous nuclear RNA is synthesized in the radicle of Zea mays embryo during the first hours of germination. The present study was undertaken in order to detect the time of appearance of the newly synthesized messenger RNA in the polysomes of germinating maize axes.Free polysomes were prepared from embryonic axes rehydrated for 2 hours in the presence of radioactively labeled uridine. These polysomes were shown to be labeled and to contain labeled particles sedimenting, after dissociation with EDTA, in the 10S to 40S region of a sucrose gradient. The labeled polysomal RNA migrates heterogeneously in a gel with a mean size corresponding to about 16S, and 60% of these molecules are polyadenylated.The data indicate that the newly synthesized RNA associated with the polysomes after 2 h of germination consists of messenger RNA molecules. Analysis of the polysomes prepared 0.5 and 1 h after the start of imbibition suggests that translation of the newly synthesized messenger RNA probably occurs within the 1st hour of imbibition of the isolated axis, thus well before the completion of the initial water uptake. PMID- 16663246 TI - A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation. AB - Soybean membrane preparations specifically bound [(14)C]mycolaminaran, a branched beta-1,3-glucan produced by Phytophthora sp. which elicits production of the phytoalexin glyceollin in soybean tissues. A Scatchard plot of the binding data disclosed the presence of a single affinity class of binding sites with a K(d) value of 11.5 micromolar for the glucan. To assess the physiologic importance of mycolaminaran binding in phytoalexin elicitation, several derivatives of mycolaminaran were prepared. Reduced mycolaminaran had slightly greater elicitor activity and binding affinity than the native substance, while periodinated mycolaminaran was virtually devoid of either elicitor activity orbinding capability. Phosphorylated mycolaminaran, on the other hand, gave values for both elicitor activity and membrane binding which were intermediate between the native and periodinated preparations. No other tested carbohydrates competed with the binding of [(14)C]mycolaminaran. Soybean membrane preparations contained beta-1,3 endoglucanase activity that degraded mycolaminaran and reduced both its efficiency as a phytoalexin elicitor and its membrane binding at temperatures above 0 degrees C. Once [(14)C]mycolaminaran bound to membranes, however, it was not appreciably susceptible to glucanase attack and could not be displaced with excess unlabeled ligand. Taken collectively, the observations suggest that the membrane binding sites are mycolaminaran-specific receptors which are physiologically involved in the initiation of phytoalexin production in soybean cotyledons. Because the binding of mycolaminaran to membranes was abolished by heat and proteolytic enzymes, the receptor is probably a protein(s) or glycoprotein(s). PMID- 16663247 TI - Differential Changes in the Amount of Protein Complexes in the Chloroplast Membrane during Senescence of Oat and Bean Leaves. AB - Antibodies against the individual subunits of protein complexes in the chloroplast membranes were used to follow the amounts of these polypeptides during foliar senescence. No change was found in the amount of polypeptides of photosystem I reaction center and the chloroplast coupling factor during senescence of oat (Avena sativa L.) and bean (Phaseolus vulgaris L.) leaves. A significant decrease in the amount of the different components of the cytochrome b(6)-f complex was detected. This change may account for the decrease in the rate of electron transport, which might be the rate limiting step of photosynthesis in senescing leaves. PMID- 16663248 TI - Autoanalytical procedure for the determination of allantoin and allantoic Acid in soybean tissue. AB - Ureide analyses of soybean (Glycine max L.) tissues were accomplished with a modified and simplified automated analysis used to determine allantoin concentration in rat urine. The length of the circuit and flow rates of the solutions were reduced, and NaOH was used for color development at room temperature. Keto-acids did not significantly interfere with the determinations of ureides except for glyoxylic acid in extracts of fresh soybean tissue. The interference caused by glyoxylic acid was avoided by adding phenylhydrazine HCl to the solution of NaOH used for alkaline hydrolysis of allantoin. PMID- 16663249 TI - Relationship between N(2)-Fixing Efficiency and Natural N Enrichment of Soybean Nodules. AB - Non-nodular tissue of soybean (Glycine max L. Merrill) plants grown hydroponically in the absence of added N have a (15)N abundance close to that of atmospheric N(2). In contrast, nodules are usually enriched in (15)N. In this paper, we report measurements of the (15)N abundance of foliar tissue and nodules of soybeans inoculated with 11 variably efficient strains of Rhizobum japonicum and grown hydroponically with no added N. The efficiency of the 11 symbioses varied over a wide range as judged by a 16-fold difference in N content. The degree of (15)N enrichment of nodules was closely correlated with N(2)-fixing efficiency (milligrams N fixed per milligram N in the nodules).These results confirm prior preliminary data based on six variably efficient R. japonicum strains. The strong correlation between (N)N enrichment of soybean nodules and N(2)-fixing efficiency is consistent with the hypothesis that new nodule tissue is synthesized from a pool of recently fixed N within the same nodule. PMID- 16663250 TI - C Nuclear Magnetic Resonance Studies of Crassulacean Acid Metabolism in Intact Leaves of Kalanchoe tubiflora. AB - (13)C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoe tubiflora was used to observe Crassulacean acid metabolism in vivo. (13)C signals from C-4 of malate were observed after overnight exposure of leaves to (13)CO(2). Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, (13)C signals were detected in all four carbons of malate in the previously labeled leaves. The (13)C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination. PMID- 16663251 TI - Ultrastructural and chemical evidence that the cell wall of green cotton fiber is suberized. AB - Green cotton (Gossypium hirsutum L.) fibers were shown by electron microscopy to have numerous thin concentric rings around the lumen of the cell. These rings possessed a lamellar fine structure characteristic of suberin. LiA1D(4) depolymerization and gas chromatography-mass spectrometry analysis showed the presence of a suberin polymer in the green cotton with the major aliphatic monomers being omega-hydroxydocosanoic acid (70%) and docosanedoic acid (25%). Ordinary white cotton was shown by chemical and ultrastructural examination to be encircled by a thin cuticular polymer containing less than 0.5% of the aliphatic components found in green cotton. PMID- 16663252 TI - Arginine catabolism in the cotyledons of developing and germinating pea seeds. AB - Arginine is the predominant free amino acid in the cotyledons of developing seeds of Pisum sativum L. cv Marzia. Breakdown of arginine was measured by injecting l [guanido-(14)C]arginine into detached cotyledons. Cotyledons of developing seeds showed a low rate of (14)CO(2) evolution whereas a much higher rate of (14)CO(2) evolution was measured from cotyledons of seeds 4 days after the onset of germination. The activities of the catabolic enzymes arginase, urease, and ornithine aminotransferase were measured throughout development and germination. Arginase and ornithine aminotransferase were present at an early stage of development. Urease activity appeared later as the seeds started to desiccate. During germination, all three enzymes were present. The different course of activity of these enzymes indicates that they are controlled separately.To explain the simultaneous presence of arginine and arginase without arginine degradation in the cotyledons of developing seeds, we propose a different intracellular localization of substrate and enzyme. In cotyledons of germinating pea seeds, urease has an enzymic function in arginine degradation. PMID- 16663253 TI - Nonstomatal Inhibition of Net CO(2) Uptake by (+/-) Abscisic Acid in Pharbitis nil. AB - (+/-) Abscisic acid (ABA) injected into petioles of attached transpiring leaves of Pharbitis nil Chois. cv violet reduced the photosynthetic capacity of the mesophyll of these leaves as well as the stomatal conductance to CO(2) diffusion. Greater than 75% of the injected ABA was recovered as ABA, suggesting that ABA rather than some metabolite thereof was the active compound. The nonstomatal effect of ABA increased from 30% reduction in photosynthesis at 0.25 micromolar ABA in the leaf blade to 90% reduction at 18 micromolar. Despite the effect of ABA on the nonstomatal component of leaf net CO(2) uptake, it was calculated that a substantial part of the reduction in leaf net CO(2) uptake (50-80%) could be accounted for by the effect of ABA on stomatal conductance. PMID- 16663254 TI - Simultaneous measurement of oxygen evolution and chlorophyll fluorescence from leaf pieces. AB - An apparatus is described which permits the simultaneous measurement of O(2) evolution and chlorophyll a fluorescence from illuminated discs or pieces of green leaves. O(2) is measured in the gas phase in a temperature-controlled chamber of approximately 5-milliliter capacity. Calibration is effected by injection of air through vents. Response time is approximately 1.5 seconds for O(2), and full scale deflection, in normal operating mode, is approximately 10 micromoles O(2). The apparatus may also be used to monitor fluorescence alone, in an open mode, in which gas is passed continuously through the chamber. PMID- 16663255 TI - Simultaneous measurement of oscillations in oxygen evolution and chlorophyll a fluorescence in leaf pieces. AB - In spinach (Spinacia oleracea) and barley (Hordeum vulgare) leaves, chlorophyll a fluorescence and O(2) evolution have been measured simultaneously following re illumination after a dark interval or when steady state photosynthesis has been perturbed by changes in the gas phase. In high CO(2) concentrations, both O(2) and fluorescence can display marked dampening oscillations that are antiparallel but slightly out of phase (a rise or fall in fluorescence anticipating a corresponding fall or rise in O(2) by about 10 to 15 seconds). Infrared gas analysis measurements showed that CO(2) uptake behaved like O(2) evolution both in the period of oscillation (about 1 minute) and in its relation to fluorescence. In the steady state, oscillations were initiated by increases in CO(2) or by increases or decreases in O(2). Oscillations in O(2) or CO(2) did not occur without associated oscillations in fluorescence and the latter were a sensitive indicator of the former. The relationship between such oscillations in photosynthetic carbon assimilation and chlorophyl a fluorescence is discussed in the context of the effect of ATP or NADPH consumption on known quenching mechanisms. PMID- 16663256 TI - Plant Disease and the Regulation of Enzymes Involved in Lignification: Increased Rate of De Novo Synthesis of the Three Tobacco O-Methyltransferases during the Hypersensitive Response to Infection by Tobacco Mosaic Virus. AB - The mechanism underlying the increase of activity of the three O methyltransferases of tobacco (Nicotiana tabacum) after infection by tobacco mosaic virus (TMV) has been investigated with a density-labeling method. The three O-methyltransferases from healthy or TMV-infected leaves fed with H(2)O or (2)H(2)O were purified by ion-exchange chromatography and their mean buoyant densities were calculated from their respective distribution profiles after centrifugation to equilibrium on RbCl gradients. Densities were corrected with respect to the mean buoyant density of a radioactive density marker prepared from tobacco leaves floated on a solution containing l-[(3)H]leucine and selected on a preparative gradient for its density close to those of the O-methyltransferases. The introduction of (2)H into the pool of amino acids from which the enzymic proteins were synthesized was monitored. By measurement of the labeling of beta galactosidase synthesized by bacteria from the plant amino acids, it was shown that infection did not alter the rate of labeling of the pool of amino acids. The buoyant-density values of the three O-methyltransferases were determined, and density-labeled enzymes from healthy and infected materials were compared. The largest density shifts from the native enzyme were measured for O methyltransferases from infected leaves. These results show that the increase in activity of the three enzymes after infection is due to the stimulation of the rate of de novo synthesis of enzyme proteins. PMID- 16663257 TI - Variation in Quantum Yield for CO(2) Uptake among C(3) and C(4) Plants. AB - The quantum yield for CO(2) uptake was measured on a number of C(3) and C(4) monocot and dicot species. Under normal atmospheric conditions (330 microliters per liter CO(2), 21% O(2)) and a leaf temperature of 30 degrees C, the average quantum yields (moles CO(2) per einstein) were as follows: 0.052 for C(3) dicots, 0.053 for C(3) grasses, 0.053 for NAD-malic enzyme type C(4) dicots, 0.060 for NAD-malic enzyme type C(4) grasses, 0.064 for phosphoenolpyruvate carboxykinase type C(4) grasses, 0.061 for NADP-malic enzyme C(4) dicots, and 0.065 for NADP malic enzyme type C(4) grasses. The quantum yield under normal atmospheric conditions was temperature dependent in C(3) species, but apparently not in C(4) species. Light and temperature conditions during growth appeared not to influence quantum yield. The significance of variation in the quantum yields of C(4) plants was discussed in terms of CO(2) leakage from the bundle sheath cells and suberization of apoplastic regions of the bundle sheath cells. PMID- 16663258 TI - Role of o-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate. AB - In the presence of excess sulfate, cysteine synthesis in pumpkin (Cucurbita pepo) leaves is not limited by sulfate reduction, but by the availability of O acetylserine. Feeding of O-acetylserine or its metabolic precursors S-acetyl coenzyme-A and coenzyme A to leaf discs enhanced the incorportion of [(35)S]sulfate into reduced sulfur compounds, mainly into cysteine, at the cost of lowered H(2)S emission; the uptake and reduction of sulfate is not affected by these treatments. beta-Fluoropyruvate, an inhibitor of the generation of S-acetyl coenzyme A via pyruvate dehydrogenase, stimulated H(2)S emission in response to sulfate. This stimulation is overcompensated by addition of O-acetylserine, S acetyl-coenzyme A, or coenzyme A. These results indicate that, in the presence of high amounts of sulfate, excess sulfur is reduced and emitted as H(2)S into the atmosphere. The H(2)S emitted seems to be produced by liberation from a precursor of cysteine rather than by cysteine desulfhydration. PMID- 16663259 TI - Interaction of indoleacetic Acid and gibberellic Acid in the short-term growth kinetics of oat stem segments. AB - Gibberellins and auxins are the only hormones known that promote growth in oat (Avena sativa) stem segments, but when applied together, indoleacetic acid inhibits gibberellic acid-induced growth appreciably. This study shows that in addition to this inhibitory role, indoleacetic acid shortens the response time of the tissue to gibberellic acid. PMID- 16663260 TI - Analysis of pea chloroplast inner and outer envelope membrane proteins by two dimensional gel electrophoresis and their comparison with stromal proteins. AB - Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles. PMID- 16663261 TI - Cell Walls of Phaseolus vulgaris Leaves Contain the Azocoll-Digesting Proteinase. AB - The leaves of Phaseolus vulgaris L. cv Greensleeves contain an endopeptidase with a pH optimum of 9.0 and an isoelectric point between 10.0 and 10.5. This endopeptidase is the only abundant Azocoll-digesting proteinase in the leaves. The activity of this enzyme is highest in immature leaves and declines as the leaf matures and senesces. Enzymically isolated protoplasts contain very little of this proteinase. The proteinase can be recovered readily from the extracellular fluid obtained by gentle centrifugation of leaf strips vacuum infiltrated with a buffered solution. These experiments indicate that the Azocoll digesting proteinase is located in the periplasmic space and/or the cell wall. PMID- 16663262 TI - Determination of C distribution in photosynthetic serine and phosphoglycerate from grape leaves. AB - Serine and phosphoglyceric acid are the classical marker intermediates of photorespiration and reductive carbon assimilation in C(3) plants. The present paper introduces a new and fast method for the determination of (14)C distribution in these compounds by selective elimination of C-3 (NaIO(4)) or C-1 (ninhydrin/ceric sulfate). Reproducibility of the procedure was found to be better than +/-1% upon degradation of [U-(14)C]serine and [U-(14)C]glycerate standards.Determination of labeling patterns in (dephosphorylated) phosphoglycerate and serine, extracted from grape leaf discs (Vitis vinifera L.) after administration of (14)CO(2) for 1 minute showed that (14)C distribution in serine changes considerably with changing assimilatory conditions. Interpretations of data basing on unusual labeling patterns of serine must therefore be accepted with caution. PMID- 16663263 TI - Pathway of Photosynthetic Malate Formation in Vitis vinifera, a C(3) Plant. AB - The time course of intramolecular isotope distribution in phosphoglyceric acid and serine was determined after exposure of grape leaf discs (Vitis vinifera L.) to (14)CO(2) (1000 microliters per liter) for variable metabolic periods, and the labeling patterns were compared with the respective isotope distribution in the C(1-3) fragment of malic acid. The results clearly support the classical concept of a close precursor-product relationship between photosynthetic phosphoglycerate and malic acid. Under the assimilatory conditions used in this study, there was no indication of an immediate carbon transfer from serine to malate as has been suggested for C(3) plants (Kent et al. 1974 Plant Physiol 53: 491-495) because of a coincident labeling of these compounds in Vicia faba. According to our data, previous evidence in favor of this hypothetical pathway is based largely on an unusual (14)C distribution in serine, due to an extreme suppression of photorespiration, as well as on arbitrary comparisons between compounds of divergent kinetic characteristics and consequently different degrees of metabolic label randomization. PMID- 16663264 TI - Isolation and Identification of Plasma Membrane from Light-Grown Winter Rye Seedlings (Secale cereale L. cv Puma). AB - An effective method for the isolation of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma) was established using a liquid two polymer phase separation. The conditions for the specific partition of plasma membrane into the polyethylene glycol-enriched upper phase were examined, including variations in the polymer concentration, buffer system, pH, and NaCl addition in the phase partition system. The most effective phase partition system for the isolation of plasma membrane from winter rye consisted of 5.6/5.6% (w/w) polyethylene glycol 4000/dextran T500 in 0.25 molar sucrose-10 millimolar potassium phosphate-30 millimolar NaCl (pH 7.8), repeated once. When the isolated plasma membrane was centrifuged on a linear sucrose density gradient, a single band was found at the 34% (w/w) sucrose layer (1.141 grams per cubic centimeter) which co-fractionated with the pH 6.5-ATPase.Identification of plasma membrane was performed by the combination of phosphotungstic acid-chromic acid stain and specific binding of N-1-naphthylphthalamic acid. Based on morphometrical observations after phosphotungstic acid-chromic acid stain, the isolated plasma membrane consisted mostly of vesicles of high purity. The isolated plasma membrane also showed extremely high specificity for N-1-naphthylphthalamic acidbinding, 10-fold higher than other membranes. It was also confirmed that there is a distinct difference in properties between plasma membrane and other membranes. The endomembranes such as from chloroplasts, mitochondria, and endoplasmic reticulum were observed to be highly sensitive to Zn(2+) ion and lower pH, which resulted in an abrupt aggregation of membranes. On the contrary, plasma membrane was very stable to these treatments and no aggregation was observed. These unique properties of isolated plasma membrane are generally observed in a wide variety of plant species and can be utilized for the assessment of the purity of preparations of isolated plasma membranes and for their identification. PMID- 16663265 TI - Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet. AB - Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-(14)C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs. PMID- 16663266 TI - Phase Shift in the Potassium Uptake Rhythm of the Duckweed Lemna gibba G3 Caused by an Azide Pulse. AB - A 6-hour application (6-hour pulse) of 1 millimolar azide significantly changed the phase of the potassium uptake rhythm of Lemna gibba G3. The phase response curve obtained was type 0 and very similar to that caused by a 6-hour pulse of low temperature (5 degrees C) or darkness. The magnitude of the phase shift and the type of the phase response curve depended on the concentration of azide. However, 6-hour pulses of 3 millimolar cyanide or 10 micromolar (3-(3,4 dichlorophenyl)-1,1-dimethylurea) failed to shift the phase of the rhythm, while these pulses lowered the rate of carbon dioxide uptake or release. Azide, even at 3 micromolar, selectively reduced the amplitude of the rhythm without inhibiting the mean level of potassium uptake. PMID- 16663267 TI - In situ field measurement of leaf water potential using thermocouple psychrometers. AB - Thermocouple psychrometers are the only instruments which can measure the in situ water potential of intact leaves, and which can possibly be used to monitor leaf water potential. Unfortunately, their usefulness is limited by a number of difficulties, among them fluctuating temperatures and temperature gradients within the psychrometer, sealing of the psychrometer chamber to the leaf, shading of the leaf by the psychrometer, and resistance to water vapor diffusion by the cuticle when the stomates are closed. Using Citrus jambhiri, we have tested several psychrometer design and operational modifications and showed that in situ psychrometric measurements compared favorably with simultaneous Scholander pressure chamber measurements on neighboring leaves when the latter were corrected for the osmotic potential. PMID- 16663268 TI - Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves. AB - Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C(18:0) and C(16:0) were synthesized in equal proportion. With the epidermal FAS system when either acetyl CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.The properties of malonyl CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K(m) values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that beta-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell. PMID- 16663269 TI - Reduction of N-acetyl methionine sulfoxide in plants. AB - An enzymic activity which catalyzes the reduction of N-acetyl-methionine sulfoxide to l-N-acetyl-methionine has been observed in a wide variety of plant tissues. Its activity depended on the presence of dithiotreithol in the incubation medium. l-Methionine-sulfoxide was essentially inactive as a substrate. Of all the physiological reductants tested, only thioredoxin partially replaced dithiothreithol. When fractions obtained by gradient centrifugation of gently disrupted barley protoplasts were assayed for the reductase, the activity was largely associated with chloroplasts although approximately 15% was found in the cytosolic compartment. The enzyme, isolated from spinach chloroplasts, had a broad pH optima between 7.0 and 8.0, and its K(m) for N-acetyl methionine sulfoxide is 0.4 millimolar. The possible participation of this ubiquitous enzyme in enzyme regulation is discussed. PMID- 16663270 TI - Comparison between a Stable NaCl-Selected Nicotiana Cell Line and the Wild Type : K, Na, and Proline Pools as a Function of Salinity. AB - An NaCl-resistant line has been developed from suspension-cultured tobacco cells (Nicotiana tabacum/gossii) by stepwise increases in the NaCl concentration in the medium. Resistance showed stability through at least 24 generations in the absence of added NaCl.Above an external NaCl concentration of 35 millimolar, proline concentration in the selected cells rose steeply with external NaCl, particularly so above 100 millimolar NaCl. Proline accumulation in the wild type was far slighter. Selected cells which had been grown for 24 generations in the absence of added NaCl accumulated proline strongly on re-exposure to NaCl medium, indicating stability of this character. Proline accumulation was fully reversible with a half-time of about 6 hours. When selected cells were transferred sequentially to lower and lower NaCl concentrations, their proline content fell to the level corresponding to the new NaCl concentration. The NaCl-selected cells responded to water stress (i.e. added mannitol) by accumulating markedly more proline than did the wild type.The addition of Ca(2+) to the growing and rinsing media minimized Na(+) and K(+) binding in the Donnan free space of cell walls and thus allowed assessment of intracellular Na(+) and K(+). In both cell types, internal Na(+) content rose steadily as a function of external NaCl concentration. In the course of 7 days in NaCl media, the wild type cells lost a considerable part of their K(+) content, the extent of the loss increasing with rise in external NaCl concentration. The selected cells, by contrast, lost no K(+) at external NaCl concentrations below 50 millimolar external NaCl, and at higher concentrations lost less than the wild type. PMID- 16663271 TI - Effects of SO(2) and O(3) on Allocation of C-Labeled Photosynthate in Phaseolus vulgaris. AB - A series of laboratory exposures of two varieties of bush bean (Phaseolus vulgaris L., var 274 and var 290) was conducted to determine the sensitivity of [(14)C]photosynthate allocation patterns to alteration by SO(2) and O(3). Experiments with the pollution-resistant 274 variety demonstrated short-term changes in both (14)C and biomass allocation to roots of (14)CO(2)-labeled plants but no significant effect on yield by up to 40 hours of exposure to SO(2) at 0.50 microliters per liter or 4 hours of O(3) at 0.40 microliters per liter. Subsequent experiments with the more sensitive 290 variety demonstrated significant alteration of photosynthesis, translocation, and partitioning of photosynthate between plant parts including developing pods. Significant increases in foliar retention of photosynthate (+40%) occurred after 8 hours of exposure to SO(2) at 0.75 microliters per liter (6.0 microliters per liter-hour) and 11 hours of exposure to O(3) at 0.30 microliters per liter-hour (3.3 microliters-hours). Time series sampling of labeled tissues after (14)CO(2) uptake showed that the disruption of translocation patterns was persistent for at least 1 week after exposures ceased. Subsequent longer-term exposures at lower concentrations of both O(3) (0.0, 0.10, 0.15, and 0.20 microliters per liter) and SO(2) (0.0, 0.20, and 0.40 microliters per liter) demonstrated that O(3) more effectively altered allocation than SO(2), that primary leaves were generally more sensitive than trifoliates, and that responses of trifoliate leaves varied with plant growth stage. Altered rates of allocation of photosynthate by leaves were generally associated with alterations of similar magnitude and opposite direction in developing pods. Collectively, these experiments suggest that allocation patterns can provide sensitive indices of incipient growth responses of pollution-stressed vegetation. PMID- 16663272 TI - Microsomal Membrane Changes during the Ripening of Apple Fruit. AB - The changes in leakage and viscosity of microsomal membranes from apples (Malus sylvestris cv Calville de San Sauveur) at different stages of ripening were examined. These changes were correlated with those in the lipid composition of the membranes, sterols, phospholipids, and fatty acids of the phospholipids. The greatest changes in membrane properties occurred as the fruit reached its climacteric and this corresponded with a change in the sterol:phospholipid ratio in the membranes. Changes were also found in fatty acid unsaturation level, but primarily in the postclimacteric stage of ripening. PMID- 16663273 TI - Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag. AB - Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. ;Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable. PMID- 16663274 TI - Effects of Salinity on Primary Processes of Photosynthesis in the Red Alga Porphyra perforata. AB - The effects of salinity on the primary processes of photosynthesis were studied in the red alga Porphyra perforata. The results show that there are at least three sites in the photosynthetic apparatus of this alga that were affected by increased salinity. The first site, photoactivation and dark-inactivation of electron flow on the reducing side of photosystem I, was completely inhibited at high salinity. The second site, electron flow on the oxidizing side (water side) of photosystem II, was inhibited as was the re-oxidation of Q in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The third site affected by high salinity was the transfer of light energy probably from pigment system II to I. High salinity reduced the amount of light energy that reached the reaction centers of photosystem II.These effects are discussed in relation to the mechanisms available to this plant to avoid photoinhibition when it is exposed to stresses such as high light and high salinity which are conditions that are commonly found in the intertidal habitat. PMID- 16663275 TI - Analysis of acetylene reduction rates of soybean nodules at low acetylene concentrations. AB - It has been previously proposed that acetylene reduction data at subsaturating acetylene concentrations could be interpreted by use of the Michaelis-Menten equation, based on the acetylene concentration external to the nodules. One difficulty of this view is that the assumption that the system is not diffusion limited is violated when studying intact nodules. The presence of a gas diffusion barrier in the nodule cortex leads to an alternate expression for the gas exchange rates at subsaturating gas concentrations. A theoretical comparison of the ;apparent' Michaelis-Menten model and diffusion model illustrated the difficulties observed in the former model of overestimating the Michaelis-Menten coefficient and yielding a correlation between the Michaelis-Menten coefficient and the maximum rate. On the other hand, use of a diffusion model resulted in (a) estimates of the Michaelis-Menten coefficient consistent with enzyme studies, (b) stability of the estimates of the Michaelis-Menten coefficient independent of treatment, and (c) a sensitivity of the diffusion barrier conductance to plant drought stress. It was concluded that all studies of nodule gas exchange need to consider possible effects caused by the presence of a diffusion barrier. PMID- 16663276 TI - Starch synthesis in shriveled and plump triticale seeds. AB - Seven lines of triticale (X Triticosecale Wittmack) with either shriveled or plump seed characteristics were planted in the field, and seed developmental changes in weight, starch content, the activity of starch biosynthetic and degradative enzymes, and ATP content were studied in three consecutive years in Oregon. Experimental results varied among genotypes and with growing environment, but overall indicated that: (a) amylase activity was higher in shriveled lines, but was not directly synchronized with the occurrence of shriveling at later stages of seed formation; (b) ADP-glucose starch synthase contributed to starch accumulation in triticale, but it appeared not to be associated with shriveledness as no stage-related changes were observed; and (c) ADP-glucose pyrophosphorylase activity was 2- to 3-fold higher in plump lines than that of shriveled lines, indicating that this enzyme may play an important role in the degree of plumpness or stach accumulation of triticale seeds. PMID- 16663277 TI - Photosynthetic rate control in cotton : stomatal and nonstomatal factors. AB - The relationship between single leaf photosynthesis and conductance was examined in cotton (Gossypium hirsutum L.) across a range of environmental conditions. The purpose of this research was to separate and define the degree of stomatal and nonstomatal limitations in the photosynthetic process of field-grown cotton.Photosynthetic rates were related to leaf conductance of upper canopy leaves in a curvilinear manner. Increases in leaf conductance of CO(2) in excess of 0.3 to 0.4 mole per square meter per second did not result in significant increases in gross or net photosynthetic rates. No tight coupling between environmental influences on photosynthetic rates and those affecting conductance levels was evident, since photosynthesis per unit leaf conductance did not remain constant. Slowly developing water stress caused greater reductions in photosynthesis than in leaf conductance, indicating nonstomatal limitations of photosynthesis.Increases in external CO(2) concentration to levels above ambient did not produce proportional increases in photosynthesis even though substomatal or intercellular CO(2) concentration increased. The lack of a linear increase in photosynthetic rate in response to increases in leaf conductance and in response to increases in external CO(2) concentration demonstrated that nonstomatal factors are major photosynthetic rate determinants of cotton under field conditions. PMID- 16663278 TI - Photosynthetic rate control in cotton : photorespiration. AB - The purpose of this research was to determine the magnitude of photorespiration in field-grown cotton (Gossypium hirsutum L.) as a function of environmental and plant-related factors. Photorespiration rates were estimated as the difference between measured gross and net photosynthetic rates.A linear increase in photorespiration was observed as air temperature increased from 22 to 40 degrees C at saturating photon flux density. At 22 degrees C, photorespiration was less than 15 per cent of net photosynthesis and very comparable to the dark respiration rate. At 40 degrees C, photorespiration represented about 50 per cent of net photosynthesis. Gross photosynthesis had a temperature optimum of 32 to 34 degrees C. Water stress, as indicated by Psi(L), did not alter the ratio of gross photosynthesis to net photosynthesis when the confounding effects of leaf temperature differences were accounted for in the data analyses. A reduction in both gross and net photosynthesis was apparent as Psi(L) declined from -2.0 megapascals indicating direct effects of water stress on the photosynthetic process. Photorespiration expressed as a proportion of net photosynthesis increased as water stress intensified.Cotton cultivars possessing a fruit load had significantly higher gross and net photosynthetic rates and lower photorespiration rates than did photoperiod-sensitive cotton strains without a fruit load. Within the fruiting types, which were genetically very similar, only minor differences were observed in the photorespiration:net photosynthesis ratios. However, in the photoperiod-sensitive strains, considerable genetic variability existed when photorespiration was expressed as a proportion of net photosynthesis. These results suggest that the kinetics of ribulose-1,5 bisphosphate carboxylase:oxygenase may be different and, thus, the possibility of genetically reducing photorespiration exists. PMID- 16663279 TI - Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare). AB - We report here that calmodulin isolated from the monocotyledon barley is indistinguishable by a variety of criteria from calmodulin isolated from the dicotyledon spinach. In contrast to previous reports, we find that barley (Hordeum vulgare) calmodulin has an amino acid composition similar to that of vertebrate and spinach calmodulins, including the presence of a single trimethyllysinyl residue, and that barley calmodulin quantitatively activates cyclic nucleotide phosphodiesterase. Furthermore, spinach and barley calmodulins are similar in terms of tryptic peptide maps and immunoreactivity with various antisera that differ in their molecular specificities for calmodulins. Limited amino acid sequence analysis demonstrates that the region around the single histidinyl and trimethyllysinyl residues is identical among barley, spinach, and vertebrate calmodulins and that barley calmodulin, like spinach calmodulin, has a novel glutamine residue at position 96. We conclude that calmodulin is highly conserved among higher plants and that detailed sequence analysis is required before significant differences, if any, can be assigned to barley or other higher plant calmodulins. These studies suggest that calmodulin's fundamental importance to the eukaryotic cell may have been established prior to the evolutionary emergence of higher plants. PMID- 16663280 TI - Far-red light-induced changes in intracellular potentials of spinach mesophyll cells: interaction with red light. AB - In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way. PMID- 16663281 TI - Volumetric components of seed imbibition. AB - Swelling parameters were determined for 15 seed species; the swelling quotients were found to range from less than 1.1 (castorbean) to 2.8 (mungbean) and swelling coefficients ranged from 0.09 (castorbean) to 0.42 (cowpea). Swelling leads to a lowering of specific gravity of those seeds with high initial specific gravity, and an elevation of those with low initial specific gravity. The extent of swelling bears a linear relationship with moisture content. Redrying to air dryness only partially restores the original volume, but oven drying completely restores it. Temperatures alter both the rate and the extent of swelling. Solutes in the imbibing solution alter the dynamics of the volume increases, indicating several types of influences; these include osmotic effects, salt effects, valence effects, pH effects, and lyotropic effects. It is suggested that deformation resulting from imbibitional swelling may contribute to the stresses experienced by seed tissues during hydration. PMID- 16663282 TI - Inhibition of photosynthesis by carbohydrates in wheat leaves. AB - The rate of net CO(2) assimilation of mature wheat (Triticum aestivum L.) leaves in ambient air (21% O(2), 340 microbars CO(2)) declined with time of illumination at temperatures lower than 25 degrees C, but not at higher temperatures, and the rate of decline increased when maintained in air with higher CO(2) concentration (700-825 microbars). In this latter case, the decline in the rate of net CO(2) assimilation also occurred at high temperatures. Stomatal conductance also declined with time in some cases and stomata became more sensitive to CO(2), but this was not the primary cause of the decrease in CO(2) assimilation because internal partial pressure of CO(2) remained constant. Treatments which reduced the rate of translocation (e.g. lower temperatures, chilling the base of the leaf) produced a marked decline in CO(2) assimilation of leaves in atmospheric and high CO(2) concentrations. The decreased net CO(2) assimilation was correlated with carbohydrate accumulation in each case, suggesting end product inhibition of photosynthesis. Analysis of CO(2) assimilation in high carbohydrate leaves as a function of intercellular CO(2) partial pressure showed reduction in the upper part of the curve. The initial slope of this curve, however, was not affected. Photosynthetic rates in the upper part of this curve generally recovered after a short period in darkness in which carbohydrates were removed from the leaf. The stimulation of net CO(2) assimilation by 2% O(2) (Warburg effect), and the apparent quantum yield, decreased after several hours of light. PMID- 16663283 TI - Partial purification and characterization of dihydrodipicolinic Acid reductase from maize. AB - Dihydrodipicolinic acid reductase, an enzyme which catalyzes the pyridine nucleotide-linked reduction of dihydrodipicolinic acid to tetrahydrodipicolinic acid in the biosynthetic pathway leading to l-lysine, has been partially purified from maize (Zea mays cv Pioneer 3145) kernels. The crude maize extract and the partially purified enzyme were assayed for dihydrodipicolinic acid reductase by their ability to restore the capability of crude extracts of a mutant Escherichia coli (CGSC 4549; defective in dihydrodipicolinic acid reductase) to synthesize diaminopimelic acid from aspartic acid and pyruvic acid.In a study of its properties, the Michaelis constant obtained for dihydrodipicolinic acid was 4.3 x 10(-4) and for NADPH the K(m) was 4.6 x 10(-5). The enzyme had a pH optimum close to 7 and was much more temperature labile than the bacterial enzyme. Its molecular weight was 8.0 x 10(4).Several compounds, viz., alpha-picolinic acid, l pipecolic acid, isophthalic acid, and isocinchomeronic acid, with structures similar to dihydrodipicolinic acid inhibited the reductase reaction. Dipicolinic acid, the most potent of these inhibitors, acted as a competitive inhibitor with a K(i) of 9.0 x 10(-4). The competitive inhibition of the reductase reaction by dipicolinic acid (oxidized dihydrodipicolinic acid) suggests that the substrate for this enzyme was in the ring rather than the open chain form. Oxidized pyridine nucleotide inhibited the activity slightly. PMID- 16663284 TI - Effects of abscisic Acid and ethylene on the gibberellic Acid-induced synthesis of alpha-amylase by isolated wheat aleurone layers. AB - Gibberellic acid-induced alpha-amylase synthesis in wheat aleurone layers (Triticum aestivum L. var Potam S-70) escaped from transcriptional control 30 h after addition of the hormone, as evidenced by the tissue's loss of susceptibility to cordycepin. Abscisic acid inhibited the accumulation of alpha amylase activity when added to the tissue during this cordycepin-insensitive phase of enzyme induction. alpha-Amylase synthesis was not restored by the addition of cordycepin, indicating that the response to abscisic acid was not dependent upon the continuous synthesis of a short lived RNA. When ethylene was added simultaneously or some time after abscisic acid, the accumulation of alpha amylase activity was sustained or quickly restored. The loss of susceptibility to cordycepin was completely prevented when aleurone layers were incubated with a combination of gibberellic and abscisic acids from the start of the induction period. This effect of abscisic acid was not reversed by ethylene. On the basis of these observations, it is suggested that abscisic acid inhibits both the transcription and translation of alpha-amylase mRNA, and that only the latter site of action is susceptible to reversal by ethylene.The rate of incorporation of [methyl-(14)C]choline into phospholipids was also inhibited by abscisic acid. Ethylene reversed this effect. The effects of abscisic acid and ethylene on phospholipid synthesis were not dependent upon the presence of gibberellic acid. No direct relationship was found between the control of alpha-amylase synthesis and membrane formation by abscisic acid and ethylene. PMID- 16663285 TI - Release of Calcium from Suspension-Cultured Glycine max Cells by Chitosan, Other Polycations, and Polyamines in Relation to Effects on Membrane Permeability. AB - Treatment with chitosan of suspension-cultured Glycine max cells labeled with (45)Ca(2+) caused a rapid release of calcium, which was complete much earlier than the chitosan-induced leakage of intracellular electrolytes and probably reflects calcium loss primarily from the cell wall and/or plasma membrane. A linear correlation was found between calcium release from chitosan-treated whole cells or isolated cell walls and the amount of chitosan bound. Other polycations (poly-l-lysine, histone, DEAE-dextran, and protamine sulfate), low molecular weight polyamines (spermine, spermidine, and putrescine) and polyanions (polygalacturonate and poly-l-aspartate, which act as chelating agents) also released calcium from whole cells and isolated cell walls; however, only the polycations increased membrane permeability. Poly-l-lysines of differing molecular weight showed a similar ability to release calcium, but their effect on membrane permeability increased with increasing molecular weight. The results suggest that the effect of polycations on permeability is not the direct result of calcium displacement from the cell surface but is probably due to cross linking of surface components. The order of effectiveness of inorganic cations in displacing calcium from whole cells and isolated cell walls was Ca(2+), Ba(2+), Sr(2+) > Mg(2+) > K(+), Na(+). PMID- 16663286 TI - Changes in [C]Atrazine Binding Associated with the Oxidation-Reduction State of the Secondary Quinone Acceptor of Photosystem II. AB - One hypothesis of triazine-type herbicide action in photosynthetic material is that the herbicide molecule competes with a secondary quinone acceptor, B, for a binding site at the reaction center of photosystem II. The binding affinity of B has been suggested to change with its level of reduction, being most strongly bound in its semiquinone form. To test this hypothesis, [(14)C]atrazine binding studies have been carried out under different photochemically induced levels of B reduction in Pisum sativum. It is found that herbicide binding is reduced in continuously illuminated samples compared to dark-adapted samples. Decreased binding of atrazine corresponds to an increase in the semiquinone form of B. With flash excitation, the herbicide binding oscillates with a cycle of two, being low on odd-numbered flashes when the amount of semiquinone form of B is greatest. Treatment with NH(2)OH was found to significantly decrease the strength of herbicide binding in the dark as well as stop the ability of p-benzoquinone to oxidize the semiquinone form of B. It is suggested that the mode of action of NH(2)OH is disruption of quinones or their environment on both the oxidizing and reducing sides of photosystem II. Herbicide binding was found to be unaltered under conditions when p-benzosemiquinone oxidation of the reduced primary acceptor, Q(-), is herbicide insensitive; weak herbicide binding cannot explain this herbicide insensitivity. It is concluded that the quinone-herbicide competition theory of herbicide action is correct. Also, since quinones are lipophilic the importance of the lipid composition of the thylakoid membrane in herbicide interactions is stressed. PMID- 16663287 TI - Spinach Leaf Intra and Extra Chloroplast Phosphorylase Activities during Growth. AB - The amino terminal sequence of the spinach (Spinacia oleracea L. cv Bloomsdale Long Standing) leaf cytoplasmic phosphorylase was determined and shown to have little similarity to the known sequence of the potato tuber phosphorylase. The antigenic reaction of spinach chloroplast phosphorylase and rabbit muscle phosphorylase a to antiserum prepared against spinach leaf cytoplasmic phosphorylase was tested. Neither phosphorylase gave a positive reaction when tested by immunodiffusion or neutralization of enzyme activity. The two spinach phosphorylases were assayed throughout the growth of the plant. Activity of cytoplasmic phosphorylase increased 4- to 8-fold at 30 to 35 days from sowing. Enzyme protein levels, as measured by antibody neutralization, increased by a similar amount. There was no corresponding increase in chloroplast phosphorylase activity. The chloroplast phosphorylase varied in parallel with the chloroplast enzyme ADPglucose pyrophosphorylase. Starch levels were high during the earlier stages of growth and then fell to a constant low level just before the increase in cytoplasmic phosphorylase. The results are discussed with respect to the relationship and functions of the two phosphorylases. PMID- 16663288 TI - Studies on Genetic Male-Sterile Soybeans : II. Effect of Nodulation on Photosynthesis and Carbon Partitioning in Leaves. AB - Soybean (Glycine max L. Merr.) germplasm, essentially isogenic except for loci controlling male sterility (ms(1)) and nodulation (rj(1)), were developed to study the effects of reproductive development and nitrogen source on certain aspects of photosynthesis. Plants were sampled from flowering (77 days after transplanting) until maturity (150 days after transplanting). With all four genotypes, net carbon exchange rates were highest at flowering and declined thereafter. Photosynthetic rates of the sterile genotypes (nodulated and non nodulated) declined more rapidly than the fertile genotypes, and after 105 days, both sterile genotypes maintained low but relatively constant carbon exchange rates (<3 milligrams CO(2)/gram fresh weight per hour). Photosynthetic rates and starch accumulation (difference between afternoon and morning levels) declined with time. The sterile genotypes attained the highest morning starch levels, which reflected reduced starch mobilization. After 92 days, the proportion of photosynthetically fixed carbon that was partitioning into starch (relative leaf starch accumulation) in the sterile genotypes increased dramatically. In contrast, relative leaf starch accumulation in the fertile genotypes remained relatively constant with time. Throughout the test period, all four genotypes maintained leaf sucrose levels between 5 and 15 micromoles glucose equivalents per gram fresh weight.The activities of sucrose phosphate synthase (SPS) in leaf extracts of the four genotypes declined from 77 to 147 days. Nodulated genotypes tended to maintain higher activities (leaf fresh weight basis) than did the non nodulated genotypes. In general, relative leaf starch accumulation was correlated negatively with the activity of SPS (normalized with leaf net carbon exchange rate) in leaf extracts for all four genotypes during early reproductive development, and for the fertile genotypes at all sampling dates. In contrast, leaf sucrose content was correlated positively with SPS activity during early reproductive development. These results suggested that a direct relation existed between the activity of SPS and starch/sucrose levels in soybean leaves. However, the interaction between these processes also may be influenced by other factors, particularly when leaf photosynthetic rates and plant demand for assimilates is low, as in the sterile genotypes. PMID- 16663289 TI - C(3) Photosynthesis and Crassulacean Acid Metabolism in a Kansas Rock Outcrop Succulent, Talinum calycinum Engelm. (Portulacaceae). AB - The potential for Crassulacean acid metabolism (CAM) was investigated in the sandstone outcrop succulent Talinum calycinum in central Kansas. Field studies revealed CAM-like diurnal acid fluctuations in these plants. These fluctuations persisted under all moisture and temperature regimes in the laboratory. Despite this CAM-like acid metabolism, simultaneous gravimetric determinations of day- and nighttime transpiration rates indicated the presence of a C(3) gas exchange pattern. Subsequent analyses of diurnal CO(2) and H(2)O exchange patterns under well-watered conditions and after 3, 5, and 7 days of drought confirmed these findings, though low rates of nocturnal CO(2) uptake were observed on the fifth night after continuous drought. Finally, the delta(13)C/(12)C value of this succulent, -27.8 per thousand, emphasizes the insignificance of any nocturnal CO(2) uptake in the lifelong accumulation of carbon in this species. Thus, it is proposed that T. calycinum is a C(3) plant with some CAM characteristics, including the ability to re-fix respiratory CO(2) at night under all moisture regimes, potentially resulting in a conservation of carbon, and occasionally to fix atmospheric CO(2) at night. These findings may prove to be common among rock outcrop succulents. PMID- 16663290 TI - Iron-sulfur centers and activities of the photosynthetic electron transport chain in iron-deficient cultures of the blue-green alga aphanocapsa. AB - Cultures of the blue-green alga, Aphanocapsa, were grown under iron-limiting conditions and changes in concentration of redox components of the photosynthetic electron transport chain, particularly iron-sulfur centers, were monitored by spectroscopic methods. A moderate iron depletion (1/10 of the normal concentration) had little effect on photosynthetic electron transport reactions and growth. Nevertheless, the amount of membrane-bound non-heme iron decreased sharply, and ferredoxin was nearly totally replaced by a flavin-containing protein, flavodoxin. Severe iron-deficiency (1/100 of the normal concentration) was accompanied by growth inhibition and decreased rates of photosynthetic electron flow. The Photosystem I reaction center was most affected by iron depletion as evidenced by a decrease in the amounts of iron-sulfur centers A, B, and X. However, formation of other redox proteins, even those that do not contain iron, was also inhibited by severe iron deficiency. PMID- 16663291 TI - Effects of Light and Elevated Atmospheric CO(2) on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves. AB - Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO(2) per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO(2) levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.Diurnal measurements of RuBPCase (expressed in micromoles CO(2) per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO(3) (-) and Mg(2+), but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO(3) (-) and Mg(2+)-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO(3) (-) and Mg(2+)-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO(2) and Mg(2+). PMID- 16663292 TI - Photoregulation of Phosphoenolpyruvate Carboxylase in Salsola soda L. and Other C(4) Plants. AB - Photoactivation of phosphoenolpyruvate carboxylase was found to occur in several, though not all, C(4) species examined; Salsola soda L. was used for a detailed study of this effect of light.Activity differences between light and darkness are maximized when glycerol (25% v/v) is included in the extraction medium and in the absence of mercaptoethanol. In plants grown in the growth chamber, the night-form of the enzyme, in addition to low activity, shows a positive cooperativity (with phosphoenolpyruvate), which is gradually abolished by light of increasing intensities. This allosteric behavior is absent in plants adapted to a high light environment. Activation and deactivation, under light and darkness respectively, are quite fast, suggesting post-translational regulation. The photoactivation appears to depend on photosynthetic electron flow, since it is saturated at high photon fluxes (around 1000 microeinsteins per square meter per second) and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. PMID- 16663293 TI - Photosynthetic/Photorespiratory Carbon Metabolism in the C(3)-C(4) Intermediate Species, Moricandia arvensis and Panicum milioides. AB - The distribution of (14)C in photosynthetic metabolites of two naturally occurring higher plants with reduced photorespiration, Moricandia arvensis and Panicum milioides, in pulse and pulse-chase (14)CO(2) incorporation experiments was similar to that for the C(3) species, M. foetida and Glycine max. After 6 seconds of (14)CO(2) incorporation, only about 6% of the total (14)C fixed was in malate and aspartate in both M. arvensis and P. milioides. The apparent turnover of the C(4) acids was very slow, and malate accumulated during the day in M. arvensis. Thus, C(4) acid metabolism by M. arvensis and P. milioides had no significant role in photosynthetic carbon assimilation under the conditions of our experiments (310 microliters CO(2) per liter, 21% O(2), 1100 or 1900 micromoles photon per square meter per second, 27 degrees C).After a 36-second chase period in air containing 270 microliters CO(2) per liter, about 20% of the total (14)C fixed was in glycine with M. arvensis, as compared to 15% with M. foetida, 14% with P. milioides, and 9% with G. max. After a 36-second chase period in 100 microliters CO(2) per liter, the percentage in glycine was about twice that at 270 microliters CO(2) per liter in the C(3) species and P. milioides, but only 20% more (14)C was in glycine in M. arvensis. These data suggest that either the photorespiratory glycine pool in M. arvensis is larger than in the other species examined or the apparent turnover rate of glycine and the flow of carbon into glycine during photorespiration are less in M. arvensis. An unusual glycine metabolism in M. arvensis may be linked to the mechanism of photorespiratory reduction in this crucifer. PMID- 16663294 TI - Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles. AB - Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles. PMID- 16663295 TI - Polyribosomes from aging apple and cherry fruit. AB - The sequence of events which occurs during the ripening of the Passe-Crassane pear fruit have been previously studied. In this work, we have investigated the ripening of another climacteric fruit (Pyrus malus L. cv Golden Delicious) and of a nonclimacteric fruit (Prunus avium L. cv Bigarreau Napoleon). We show that both climacteric fruits exhibit the same preclimacteric sequence of events. Differences exist, however, between the Golden Delicious apple and the Passe Crassane pear in that the protein synthesis capacity of the two fruits is not the same during the over-ripening period. On the other hand, a nonclimacteric fruit, the Bigarreau Napoleon cherry, does not show an increase in its protein synthesis capacity during the over-ripening period. PMID- 16663296 TI - Does light inhibit ethylene production in leaves? AB - The effect of light on the rate of ethylene production was monitored using two different techniques-leaf segments incubated in closed flasks versus intact plants in a flow-through open system. Three different plants were used, viz sunflower (Helianthus annuus), tomato (Lycopersicon esculentum), and soybean (Glycine max). Experiments were conducted both in the presence and absence of 1 aminocyclopropane-1-carboxylic acid (ACC).The results obtained indicate that, in all three species studied, light strongly inhibits ethylene production when cut leaf segments are incubated in the presence of ACC in closed flasks. When ethylene measurements are made with ACC-sprayed intact plants using a continuous flow system, the effect of light on ethylene production is only marginal. In leaf segments of sunflower and soybean incubated only in distilled H(2)O in closed flasks, light promotes ethylene production. In tomato, there is no difference between the rate of ethylene production between light and darkness under such conditions. When measurements are made with intact plants in a continuous flow system, the rate of ethylene production is almost identical in light and darkness, in the three plants studied.It is concluded that the effect of light on cut leaf segments incubated in the presence of ACC in closed flasks can be attributed to the techniques used for these measurements. Light has little effect on ethylene production by intact plants in an open system. PMID- 16663297 TI - Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation. AB - A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study. PMID- 16663298 TI - De Novo Purine Biosynthesis in Intact Cells of Cucurbita pepo. AB - The capacity of intact cells of roots excised from summer squash plants (Cucurbita pepo L. cv Early Prolific Straightneck) to synthesize purine nucleotides de novo was investigated. Evidence that purine nucleotides are synthesized de novo included: (a) demonstration of the incorporation of [1 (14)C]glycine, [2-(14)C]glycine, NaH(14)CO(3), and H(14)COONa into total adenine nucleotides; (b) observation that the addition of azaserine or aminopterin, known inhibitors of de novo purine synthesis in other organisms, blocked the incorporation of these precursors into adenine nucleotides; and (c) demonstration that the purine ring synthesized from these precursors was labeled in a manner consistent with the pathway for de novo purine biosynthesis found in microorganisms and animal tissues. Under optimal conditions, the activity of this pathway in roots excised from 2-day-old squash plants was 244 +/- 13 nanomoles (mean +/- standard error, n = 17) NaH(14)CO(3) incorporated into summation operatorAde (the sum of the adenine nucleotides, nucleoside and free base) per gram tissue during the 3-hour incubation period.The possible occurrence of alternative enzymic reactions for the first steps of de novo purine biosynthesis was also investigated. No conclusive evidence was obtained to support the operation of alternative enzymic reactions in the intact cell of C. pepo. PMID- 16663299 TI - Freezing injury and root development in winter cereals. AB - Upon exposure to 2 degrees C, the leaves and crowns of rye (Secale cereale L. cv ;Puma') and wheat (Triticum aestivum L. cv ;Norstar' and ;Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to -11 degrees C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of 20 degrees C while root growth was reduced at -15 degrees C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to -15 degrees C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to -11 degrees C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration. PMID- 16663300 TI - Short and long term effects of root and shoot chilling of ransom soybean. AB - The immediate short term effects on some physiological processes and the long term effects on morphology and reproductive development of root- and shoot chilled soybeans (Glycine max L. cv Ransom) were studied. Roots or shoots of 16- or 17-day-old plants were chilled at 10 degrees C for one week, and then rewarmed to 25 degrees C. Leaf elongation rate, net CO(2) uptake rate, and stomatal conductance decreased during root or shoot chilling. Root chilling had only temporary effects on water relations, while shoot chilling caused large changes in potentials during chilling. Most processes measured returned to control levels after two days of rewarming. Root-chilled plants harvested 90 days after emergence were similar in morphology and seed weight to controls. Shoot-chilled plants showed a large increase over controls in axillary branch growth, but an early abortion of flowers and a delayed resumption of flowering caused a 78% reduction in seed weight. Root chilling in this study was found to have little or no long term effect on the plants, while shoot chilling caused significant changes in vegetative morphology, and a delay in flowering and subsequent pod filling. PMID- 16663301 TI - Bicarbonate/CO(2)-Facilitated Conversion of 1-Amino-cyclopropane-1-carboxylic Acid to Ethylene in Model Systems and Intact Tissues. AB - Bicarbonate markedly enhances ethylene production from 1-aminocyclopropane-1 carboxylic acid (ACC) in model chemical systems where the conversion is free radical-mediated, in thylakoid membrane suspensions of Phaseolus vulgaris L. cv Kinghorn where the reaction is light-dependent, and in microsomal membrane suspensions and intact tissues where the reaction is enzymically mediated. In two model systems generating free radicals-the Fenton reaction and a reaction mixture containing xanthine/xanthine oxidase, NaHCO(3) (200 millimolar) increased the formation of ethylene from ACC by 84-fold and 54-fold, respectively. Isolated thylakoid membranes also proved capable of ACC-dependent ethylene production, but only upon illumination, and this too was enhanced by added NaHCO(3). As well, light-induced inhibition of ACC-dependent ethylene production by leaf discs was relieved by adding 200 millimolar NaHCO(3). Finally, NaHCO(3) (200 millimolar) augmented ACC-dependent ethylene production from young carnation flowers by about 4-fold, and the conversions of ACC to ethylene by microsomes isolated from carnation flowers and etiolated pea epicotyls were higher by 1900 and 62%, respectively, in the presence of 200 millimolar NaHCO(3).This increased production of ethylene appears not to be due to bicarbonate or CO(2)-induced release of the gas from putative receptor sites, since the addition of NaHCO(3) to sealed reaction mixtures after the ACC to ethylene conversion had been terminated had no effect. Spin-trapping studies have confirmed that bicarbonate does not facilitate the formation of free radicals thought to be involved in the conversion of ACC to ethylene. Nor did bicarbonate alter the physical properties of the membrane bilayer, which might indirectly modulate the activity of the membrane-associated enzyme capable of converting ACC to ethylene. Rather, bicarbonate appears to directly facilitate the conversion of ACC to ethylene, and the data are consistent with the view that CO(2) derived from bicarbonate is the active molecular species. PMID- 16663302 TI - Role of Endogenous Plant Growth Regulators in Seed Dormancy of Avena fatua: II. Gibberellins. AB - Gibberellin A(1) (GA(1)) was identified by combined gas chromatographymass spectrometry as the major biologically active gibberellin (GA) in seeds of wild oat (Avena fatua L.) regardless of the depth of dormany or stage of imbibition. Both unimbibed dormant and nondromant seeds contained similar amounts of GA(1) as estimated by the d5-maize bioassay. During imbibition, the level of GA(1) declined in both dormant and non-dormant seeds, although the decline was more rapid in dormant seeds. Only in imbibing nondormant seeds did the GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride (CCC), cause a reduction in the level of GA(1) from that observed in control seeds. These results are interpreted as an indication that while afterripening does not cause a direct change in the levels of GAs during dry storage, it does induce a greater capacity for GA biosynthesis during imbibition.Nondormant seeds imbibed in the presence of 50 millimolar CCC germinated equally as well as untreated seeds. When wild oat plants were fed CCC throughout the entire life cycle, viable seeds were produced that lacked detectable GA-like substances. These seeds afterripened at a slightly slower rate than the controls. Moreover, completely afterripened (nondormant) seeds from plants fed CCC continuously contained no detectable GA-like substances, and when these seeds germinated, dwarf seedlings were produced, indicating GA biosynthesis was inhibited during and after germination. In total, these results suggest that the increased capacity for GA biosynthesis observed in imbibing nondormant seeds is not a necessary prerequisite for germination. It is therefore possible that GA biosynthesis in imbibing nondormant seeds is one of many coordinated biochemical events that occur during germination rather than an initiator of the processes leading to germination. PMID- 16663303 TI - Effects of Thidiazuron on Cytokinin Autonomy and the Metabolism of N-(Delta Isopentenyl)[8-C]Adenosine in Callus Tissues of Phaseolus lunatus L. AB - The effects of a highly cytokinin-active urea derivative, N-phenyl-N'-1,2,3 thiadiazol-5-ylurea (Thidiazuron), and zeatin on cytokinin-autonomous growth and the metabolism of N(6)-(Delta(2)-isopentenyl)[8-(14)C]adenosine ([(14)C]i(6) Ado) were examined in callus tissues of two Phaseolus lunatus genotypes, cv Jackson Wonder and P.I. 260415. Tissues of cv Jackson Wonder maintained on any concentration of Thidiazuron became cytokinin autonomous, whereas only tissues exposed to suboptimal concentrations of zeatin displayed cytokinin-autonomous growth. Tissues of P.I. 260415 remained cytokinin dependent under all these conditions. The metabolism of [(14)C]i(6) Ado was similar for the two genotypes, but differed with the medium used. [(14)C]i(6) Ado was rapidly converted to N(6) (Delta(2)-isopentenyl)[8-(14)C]adenosine 5'-P ([(14)C]i(6) AMP) by tissues grown on zeatin-containing medium, whereas only traces of the nucleotide were formed in tissues grown on medium with Thidiazuron. Incubation with [(14)C] i(6) AMP of tissues grown in the presence of Thidiazuron resulted in rapid conversion to [(14)C]i(6) Ado, while [(14)C]i(6) AMP persisted in tissues maintained on zeatin. Thus, Thidiazuron appears to stimulate enzyme activity converting the ribonucleotide to ribonucleoside. Although the cytokininactive phenylureas and adenine derivatives differ in their effects on cytokinin autonomy as well as nucleotide formation, the two types of effects do not seem to be related. PMID- 16663304 TI - Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera x Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos. AB - Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera x Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4 degrees C, and subsequently incubated at 26 degrees C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H(2)O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO(2) partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H(2)O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4 degrees C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H(2)O-soluble fraction declined throughout chilling. Activity in the GA precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26 degrees C after chilling enhanced activity in the free GA and H(2)O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26 degrees C with (+/-) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H(2)O-soluble fractions.Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA(4) and GA(9) in ABA-treated, chilled embryos was also confirmed by GLC-MS. PMID- 16663305 TI - Ribonucleic Acid and protein metabolism in pea epicotyls : I. The aging process. AB - Aging of actively growing, etiolated pea Pisum sativum L. var Alaska plants was initiated by removing the plumules of plants in the third internode stage, and applying lanolin to the cut apices of otherwise intact plants. During the subsequent 4-day aging period, several degenerative events occurred in this apical 10-millimeter region. Ribosomal RNA and messenger RNA contents declined, polyribosomes disaggregated, and the protein synthesizing capacity of the polysomes decreased.Two-dimensional, silver-stained protein patterns revealed that aging altered the relative amounts of specific cellular proteins accumulated in vivo. In addition, polypeptide patterns generated by cell-free translation of total and polysomal RNA, isolated from unaged and aged tissues, showed major modifications. More than 200 spots could be resolved by two-dimensional gel fluorography of translation products using RNA from fresh tissues. Of these 200 spots, about eight appeared or increased when total RNA from aged tissue was used, and about 58 disappeared or declined. When polysomal RNA from aged tissue was used as template, about 12 spots appeared or increased, whereas about 64 disappeared or decreased. In general, the products which increased after aging were lower molecular weight and those that decreased were higher molecular weight. PMID- 16663306 TI - Ribonucleic Acid and Protein Metabolism in Pea Epicotyls : II. Response to Wounding in Aged Tissue. AB - Aged pea Pisum sativum L. var Alaska epicotyl tissue was wounded by excising the apical 10 or 20 millimeters and incubating the excised segments upright in buffer. Wounding induced a very rapid formation of polysomes which was accompanied by minor increases in ribosomes, mRNA, and poly(A) and by a doubling of the in vivo protein synthesizing capacity. This increase in protein synthesis in vivo was matched by a similar increase in polypeptide synthesis in vitro in wheat germ reactions primed by polysomes. However, in vitro reactions primed by total and polysomal RNA from wounded tissue were affected much less.Two dimensional gel patterns of silver-stained proteins accumulated in vivo were almost unchanged, even after 6 hours of wounding, since only two spots decreased in intensity and none increased. In contrast, two-dimensional gel fluorographs of polypeptides generated in vitro by both total RNA and polysomal RNA showed numerous changes within 3 hours of wounding. Of the more than 200 spots visualized by fluorography, 17 decreased and 26 increased when total RNA from wounded tissue was used; 15 decreased and 10 increased when polysomal RNA was used. Those polypeptides that decreased after wounding were generally of lower molecular weight; those which increased were of higher molecular weight.Although wounding must be affecting transcription insofar as different mRNAs must be present to encode different polypeptides, its major effect appears to be on translation, presumably through formation of ribosomes with greater translational efficiency. PMID- 16663307 TI - Ribonucleic Acid and Protein Metabolism in Pea Epicotyls : III. Response to Auxin in Aged Tissue. AB - Applications of auxin to the tips of intact aged pea Pisum sativum L. var Alaska epicotyls resulted in an increase in the content of polyribosomes and poly(A) and in the capacity of isolated polysomes to support protein synthesis in vitro. Few changes were seen in the two-dimensional gel patterns of silver-stained proteins accumulated (or degraded) in vivo even after 15 hours of auxin treatment. In contrast, substantial changes were evident in the two-dimensional gel fluorographs of polypeptides generated in vitro by total RNA and by polysomal RNA from tissue treated with auxin for only 6 hours. Of the 200 spots resolved by fluorography, total RNA from auxin-treated tissue generated 33 spots with increased intensity and 10 with decreased intensity; polysomal RNA yielded 33 spots which increased and only three that decreased. In general, the polypeptides that increased in intensity were higher molecular weight and those that decreased were lower molecular weight. These changes occurred prior to growth and might be prerequisite for the auxin-induced slow growth response seen in this aged tissue.Comparisons were made between the changes in RNA and protein metabolism occuring during aging and after wounding and auxin treatment of aged tissue. Aging causes a decline in poly(A), polysomes, and protein synthesizing capacity, whereas wounding and auxin treatment cause increases. Wounding appears to act primarily at the level of translation, whereas auxin has a greater effect on transcription. It is argued that the use of excised tissue to study auxin effects on RNA and protein metabolism should be avoided. PMID- 16663308 TI - Stimulation of h efflux and inhibition of photosynthesis by esters of carboxylic acids. AB - Suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the influence of various carboxyester compounds on rates of net H(+) efflux in the dark or light and photosynthetic O(2) production. Addition of 0.15 to 1.5 millimolar malathion, alpha-naphthyl acetate, phenyl acetate, or p-nitrophenyl acetate stimulated H(+) efflux and inhibited photosynthesis within 1 minute. In contrast, the more polar esters methyl acetoacetate or ethyl p-aminobenzoate had little or no effect on either of these two processes. A 0.15 millimolar concentration of alpha-naphthylacetate stimulated the normal rate of H(+) efflux, 0.77 nanomoles H(+) per 10(6) cells per minute by 750% and inhibited photosynthesis by 100%. The four active carboxyester compounds also stimulated H(+) efflux after the normal rate of H(+) efflux was eliminated with 0.01 milligrams per milliliter oligomycin or 100% N(2). Oligomycin reduced the ATP level by 70%. Incubation of cells with malathion, alpha-naphthyl acetate, or p-nitrophenyl acetate resulted in the generation of the respective hydrolysis products ethanol, alpha-naphthol, and p nitrophenol. It is proposed that inhibition of photosynthesis and stimulation of H(+) efflux result when nonpolar carboxyester compounds enter the cell and generate acidic carboxyl groups when hydrolyzed by esterase enzymes. PMID- 16663309 TI - Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress. AB - Osmotic adjustment was studied in cultured cells of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) adapted to different levels of external water potential ranging from -4 bar to -28 bar. The intracellular concentrations of reducing sugars, total free amino acids, proline, malate, citrate, quaternary ammonium compounds, K(+), NO(3) (-), Na(+), and Cl(-) increased with decreasing external water potential. At any given level of adaptation, the maximum contribution to osmotic potential was from reducing sugars followed by potassium ions. The sucrose levels in the cells were 3- to 8-fold lower than reducing sugar levels and did not increase beyond those observed in cells adapted to -16 bar water potential. Concentrations of total free amino acids were 4- to 5-fold higher in adapted cells. Soluble protein levels declined in the adapted cell lines, but the total reduced nitrogen was not significantly different after adaptation. Uptake of nitrogen (as NH(4) (+) or NO(3) (-)) from the media was similar for adapted and unadapted cells. Although the level of quaternary ammonium compounds was higher in the nonadapted cells than that of free proline, free proline increased as much as 500-fold compared to only a 2- to 3-fold increase observed for quaternary ammonium compounds. Although osmotic adjustment after adaptation was substantial (up to -36 bar), fresh weight (volume increase) was restricted by as much as 50% in the adapted cells. Altered metabolite partitioning was evidenced by an increase in the soluble sugars and soluble nitrogen in adapted cells which occurred at the expense of incorporation of sugar into cell walls and nitrogen into protein. Data indicate that the relative importance of a given solute to osmotic adjustment may change depending on the level of adaptation. PMID- 16663310 TI - Cadmium uptake kinetics in intact soybean plants. AB - The absorption characteristics of Cd(2+) by 10- to 12-day-old soybean plants (Glycine max cv Williams) were investigated with respect to influence of Cd concentration on adsorption to root surfaces, root absorption, transport kinetics and interaction with the nutrient cations Cu(2+), Fe(2+), Mn(2+), and Zn(2+). The fraction of nonexchangeable Cd bound to roots remained relatively constant at 20 to 25% of the absorbed fraction at solution concentration of 0.0025 to 0.5 micromolar, and increased to 45% at solution concentration in excess of 0.5 micromolar. The exchangeable fraction represented 1.4 to 32% of the absorbed fraction, and was concentration dependent. Using dinitrophenol as a metabolic inhibitor, the ;metabolically absorbed' fraction was shown to represent 75 to 80% of the absorbed fraction at concentration less than 0.5 micromolar, and decreased to 55% at 5 micromolar. At comparatively low Cd concentrations, 0.0025 to micromolar 0.3, root absorption exhibited two isotherms with K(2) values of 0.08 and 1.2 micromolar. Root absorption and transfer from root to shoot of Cd(2+) was inhibited by Cu(2+), Fe(2+), Mn(2+), and Zn(2+). Analyses of kinetic interaction of these nutrient cations with Cd(2+) indicated that Cu(2+), Fe(2+), Zn(2+), and possibly Mn(2+) inhibited Cd absorption competitively suggesting an involvement of a common transport site or process. PMID- 16663311 TI - Root absorption and transport behavior of technetium in soybean. AB - The absorption characteristics and mechanisms of pertechnetate (TcO(4) (-)) uptake by hydroponically grown soybean seedlings (Glycine max cv Williams) were determined. Absorption from 10 micromolar solutions was linear for at least 6 hours, with 30% of the absorbed TcO(4) (-) being transferred to the shoot. Evaluation of concentration-dependent absorption rates from solutions containing 0.02 to 10 micromolar TcO(4) (-) shows the presence of multiphasic absorption isotherms with calculated K(s) values of 0.09, 8.9, and 54 micromolar for intact seedlings. The uptake of TcO(4) (-) was inhibited by a 4-fold concentration excess of sulfate, phosphate, selenate, molybdate, and permanganate; no reduction was noted with borate, nitrate, tungstate, perrhenate, iodate, or vanadate. Analyses of the kinetics of interaction between TcO(4) (-) and inhibiting anions show permanganate to be a noncompetitive inhibitor, while sulfate, phosphate, and selenate, and molybdate exhibit characteristics of competitive inhibitors of TcO(4) (-) transport suggesting involvement of a common transport process. PMID- 16663312 TI - Pyruvate orthophosphate dikinase in wheat leaves. AB - Pyruvate orthophosphate dikinase (PPDK) was found in wheat (Triticum aestivum L. cv Cheyenne [CI 8885]) leaves both by activity assays and by the protein blot method. The specific activity of the wheat enzyme is comparable to that of PPDK from maize leaves. Of the total soluble protein in wheat leaves, about 0.05% was PPDK, comparable to the amount in the immature wheat seed and about 1/70th the amount found in mesophyll cells of maize. Immunoprecipitation of wheat PPDK with maize enzyme antiserum indicates partial identity, and the apparent subunit molecular weight is the same based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID- 16663313 TI - Light-induced production of singlet oxygen and superoxide by the fungal toxin, cercosporin. AB - Cercosporin, a toxin produced by members of the fungal genus Cercospora, is a photosensitizing compound which rapidly kills plant cells in the light. We have found that cercosporin, when activated by light in the presence of oxygen, is able to generate both singlet oxygen and superoxide ions. Cercosporin, when illuminated in the presence of O(2), reacted with cholesterol to form the 5alpha hydroperoxide of cholesterol which is only produced by reaction with singlet oxygen. Cercosporin, in the presence of light, O(2), and a reducing substrate, was also able to reduce p-nitro blue tetrazolium chloride, a compound readily reduced by superoxide. Superoxide dismutase, a scavenger of superoxide, inhibited this reaction. Production of both singlet oxygen and superoxide by cercosporin must be considered when studying the possible mechanisms of resistance to cercosporin. PMID- 16663314 TI - Isolation, purification, and partial characterization of formate dehydrogenase from soybean seed. AB - Soybean (Glycine soja var Beeson) formate dehydrogenase has been isolated, purified, and partially characterized by affinity chromatography. The enzyme is a dimer having a total molecular weight of 100,000 and a subunit weight of 47,000. It has activity over a broad pH range, is stable for months at 4 degrees C, and has K(m) values of 0.6 millimolar and 5.7 micromolar for formate and NAD, respectively. PMID- 16663315 TI - Oxygen fixation into hydroxyproline in etiolated maize seedlings : verification by tandem mass spectrometry. AB - Etiolated maize (Zea mays L.) seedlings were grown in the dark for 5 days in an atmosphere enriched with 10.0 atom% (18)O(2). Hydroxyproline was isolated from root and shoot tissues, purified, and methylated. It was not possible to determine (18)O incorporation into hydroxyproline by conventional mass spectrometry because the final product was not sufficiently pure. The final product was analyzed successfully by tandem mass spectrometry. The (18)O content of the hydroxyl oxygen atom was 10 +/- 0.7 atom%. This result demonstrates that the hydroxyl oxygen atom in hydroxyproline was derived exclusively from molecular oxygen. PMID- 16663316 TI - Regulation of ADPGlucose Synthesis in Guard Cells of Commelina communis. AB - The activator specificity of the ADPglucose pyrophosphorylase from Commelina communis guard cells is the same as observed for the mesophyll cell enzyme. 3 Phosphoglycerate was found to be the most effective activator. Fifty per cent of maximal stimulation was observed at about 100 micromolar. Inorganic phosphate was found to be a potent inhibitor giving 50% inhibition at 0.3 millimolar. These results are discussed with respect to regulation of starch synthesis in guard cells. PMID- 16663317 TI - Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis. AB - When protons are exchanging for bivalent cations (Cu(2+), Zn(2+), or Ca(2+)) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to approximately 3.4, which is the intrinsic pK value of the constitutive alpha-d galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared. PMID- 16663318 TI - Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves: Changes in Photosynthesis and Enzymes Involved in Carbon Assimilation from Leaf Development through Senescence. AB - Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 +/- 0.07 micromoles of CO(2) per minute per milligram at 25 degrees C) throughout the experimental period.Kinetic parameters related to CO(2) fixation were examined using the purified carboxylase. The K(m)(CO(2)) and V(max) values were 12 micromolar and 1.45 micromoles of CO(2) per minute per milligram, respectively (pH 8.2 and 25 degrees C). The in vitro specific activity calculated at the atomospheric CO(2) level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf. PMID- 16663319 TI - Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels. AB - An inhibitor of malted barley (Hordeum vulgare cv Conquest) alpha-amylase II was purified 125-fold from a crude extract of barley kernels by (NH(4))(2)SO(4) fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the alpha-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between alpha-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over alpha-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition. PMID- 16663320 TI - Ethylene production by auxin-deprived, suspension-cultured pear fruit cells in response to auxins, stress, or precursor. AB - Auxin-deprived, mannitol-supplemented, suspension-cultured pear (Pyrus communis L. Passe Crassane) fruit cells produce large quantities (20-40 nanoliters ethylene per 10(6) cells per hour) of ethylene in response to auxins, CuCl(2) or 1-amino-cyclopropane-1-carboxylic acid (ACC). Maximum rates of production are achieved about 12 hours after the addition of optimal amounts of indoleacetic acid (IAA), naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4 D), 4 to 5 hours after the addition of CuCl(2) and 1 to 2 hours after the addition of ACC. Supraoptimal concentrations of IAA result in a lag phase followed by a normal response. High concentrations of NAA and 2,4-D result in an early (4-5 hours) stress response and injury.Continuous protein and RNA synthesis are essential for elaboration of the full IAA response; only protein synthesis is necessary for the response to CuCl(2) and ACC. Based on polysomal states and rates of amino acid incorporation, CuCl(2) partially inhibits protein synthesis while nonetheless stimulating ethylene production. In general, ethylene production by the pear cells resembles that of other plant systems. Some differences may reflect the sensitivity of the cells and are discussed. The relatively high levels of ethylene produced and the experimental convenience of the cultured cells should make them especially suitable for further investigations of ethylene production and physiology. PMID- 16663321 TI - Role of pit membranes in macromolecule-induced wilt of plants. AB - Macromolecules present in low concentrations in xylem fluid of Medicago sativa L. var DuPuits will increase the resistance to xylem liquid flow. This increase in resistance was found to be reversible by backflushing the xylem. Autoradiography showed that very large molecules do not pass through pit membrane pores. A comparison of pit membrane pore sizes to molecule sizes suggests that increased resistance to xylem flow is a result of plugging pit membrane pores. It was also found that pit membranes located in two parts of the plant differ in the apparent diameter of their pores and, thus, in their susceptibility to plugging by macromolecules. Macromolecules in xylem fluid may result from hostparasite interactions and may play a significant role in the outcome of the interaction. PMID- 16663322 TI - Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis. AB - Addition of NAD(+) to purified potato (Solanum tuberosum L.) mitochondria respiring alpha-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O(2) uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl NAD(+) (NAP(4)-NAD(+)), an inhibitor of NAD(+) uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD(+)-stimulated malate-cytochrome c reductase activity, and reduction of added NAD(+) by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD(+) reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD(+) by storing on ice for 72 hours, was completely dependent on added NAD(+), and the effect of NAD(+) on these mitochondria was prevented by incubating them with NAP(4)-NAD(+). External NAD(+) reduction by these mitochondria was not affected by NAP(4)-NAD(+). We conclude that all effects of exogenous NAD(+) on plant mitochondrial respiration can be attributed to net uptake of the NAD(+) into the matrix space. PMID- 16663323 TI - Some Enzymic Activities in the Germinating Oil Palm (Elaeis guineensis) Seedling. AB - In germinating oil palm (Elaeis guineensis var D x P) seedling, an active lipase was present in the shoot but absent from both the kernel and the haustorium. It has an optimum pH of 6.2 and a smaller peak at pH 8.6. The shoot lipase was active against a number of mono-, di-, and triacylglycerols as well as the endogenous lipids present in the shoot, haustorium, and kernel. Activity against related substrates were in the order: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin > tripalmitin. The level of the enzyme in the seedling was highest at a relatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase and isocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyruvate carboxykinase and lauroyl-coenzyme A oxidase were located in the haustorium. The levels of the enzymes paralleled seedling development and were slightly higher in light-grown seedlings. Fatty acyl-coenzyme A synthetase activity was very low and was found in both the shoot and haustorium. PMID- 16663324 TI - The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies. AB - The metabolism of (14)C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C(16), C(18:1), C(18:2)) but the kernel contained about 68% C(12) and C(14) fatty acids. Haustorium fatty acids were intermediate between the two. [(14)C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO(2), and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C(16), C(18), and C(18:1) fatty acids but in the kernel about 18% to 32% of the (14)C-fatty acids were C(12) fatty acids.[(14)C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[(14)C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C(16) and C(18)) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[(14)C]laurin.When tri-[(14)C]laurin or [(14)C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[(14)C] laurin was hydrolyzed and partly metabolized to water-soluble substances. PMID- 16663325 TI - Photoreduction of oxygen in mesophyll chloroplasts of c(4) plants: a model system for studying an in vivo mehler reaction. AB - Mesophyll chloroplasts of three C(4) sub types, Panicum miliaceum (NAD-malic enzyme), Panicum maximum (PCK), and Zea mays (NADP-malic enzyme), were prepared from protoplast extracts and used to study the photoreduction of O(2). The processes of O(2) uptake and evolution in these preparations, which lack ribulose 1,5-bisphosphate carboxylase/oxygenase, were studied simultaneously using stable isotopes of O(2) and mass spectrometry. The responses of O(2) uptake to O(2) tension and addition of various substrates (3-phosphoglycerate, pyruvate, and oxaloacetate) were studied in detail. The addition of photosynthetic substrates differing in ATP to NADPH demands indicated that photoreduction of O(2) in these chloroplast preparations is linked to ATP production and strongly regulated by NADP(+) levels. The results clearly indicate that photoreduction of O(2) could be of physiological relevance in balancing the ATP to NADPH requirements of C(4) mesophyll chloroplasts. PMID- 16663326 TI - Estimation of the volumetric elastic modulus and membrane hydraulic conductivity of willow sieve tubes. AB - Severed aphid stylets were used to follow the kinetics of sieve tube turgor and osmotic pressure (pi) responses following step changes in water potential applied to the cambial surface of willow (Salix exigua Nutt.) bark strips. The kinetics of the turgor response were monitored with a pressure transducer. In separate experiments, the kinetics of the pi response were followed by freezing point determinations on stylet exudate. The sieve tube volumetric elastic modulus in the bark strips was about 21 bars, but may be higher in intact stems. The membrane hydraulic conductivity was about 5 x 10(-3) centimeters per second per bar; several factors make it difficult to estimate its value accurately. Differences in the turgor pressure (P) and pi responses, as well as the relatively more rapid initial turgor response to a water potential (psi) change, suggested a time-dependent component in sieve tube wall elasticity.Our observations were generally not supportive of the idea that sieve tubes might osmoregulate. However, the bark strip system may not be suitable for addressing that question.Separate measurements of psi, P, and pi demonstrate that the relationship predicted by the fundamental cell water potential equation, psi = P pi, is applicable within experimental error (+/- 0.4 bar) to sieve tube water relations. PMID- 16663327 TI - Purification and Characterization of an Iminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.). AB - Iminopeptidase (EC 3.4.11.5) was substantially purified from the primary leaves of 7-day-old wheat seedlings (Triticum aestivum L.). The purification procedure consisted of five steps: acid precipitation, molecular exclusion chromatography on Sephacryl S-200, Ultrogel AcA 44, Sepharose 2B and ion-exchange chromatography on DEAE-cellulose. Iminopeptidase isolated in this manner was only active against the beta-naphthylamides of proline and hydroxyproline. For each substrate, the pH optimum was 7.4 and activity was sensitive to sulfhydryl group inhibitors. The iminopeptidase hydrolyzed the dipeptides Pro-Leu, Pro-Gly, Hyp-Gly, and Pro-Tyr. Iminopeptidase activity against the dipeptide Pro-Gly was higher than against Hyp Gly. The molecular weight was estimated to be about 400,000. Evidence was obtained for the existence of endogenous inhibitors of iminopeptidase activity. PMID- 16663328 TI - Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides. AB - Cultured spinach (Spinacia oleracea L. cv Monstrous Viroflay) cells incorporated exogenous l-[(3)H]arabinose sequentially into beta-l-arabinopyranose-1-phosphate, uridine diphospho-beta-l-arabinopyranose, uridine diphospho-alpha-d-xylopyranose and (in some experiments) alpha-d-xylopyranose-1-phosphate. The amount of (3)H in each of these compounds reached a plateau after a few minutes, and could be rapidly chased with nonradioactive l-arabinose, demonstrating rapid turnover. After a few minutes' lag, incorporation of (3)H into the arabinofuranosyl, arabinopyranosyl, and xylopyranosyl residues of polysaccharides was linear with respect to time. The kinetics of labeling were compatible with UDP-beta-l arabinopyranose and UDP-alpha-d-xylopyranose being the immediate precursors of arabians (both the pyranose and the furanose residues) and xylans, respectively. No other radioactive nucleotides were formed; in particular, UDP-arabinofuranose was absent. There was no evidence for conversion of arabinopyranose to arabinofuranose within the polysaccharides, suggesting that this conversion occurs during polymer synthesis. The glycolipids detected showed too slow a turnover to be intermediates of pentosan synthesis. PMID- 16663329 TI - No influence of the embryonic axis on the development of diamine oxidase in pea cotyledons. AB - A reexamination has been made for the supposed regulation of pea (Pisum sativum cv Alaska) cotyledonary diamine oxidase (EC 1.4.3.6) activity by the embryonic axis. When dry cotyledons from which the embryo and testa have been removed surgically are imbibed by soaking in water, there is little increase of the enzyme activity during subsequent incubation on filter paper. However, if the dry cotyledons are imbibed and maintained on filter paper from the first, the increase of the enzyme activity is similar to that in the intact seedling. Thus, rapid imbibition of the isolated dry cotyledons is responsible for repression of enzyme development, and a role for the axis need not be invoked. PMID- 16663330 TI - Effect of water stress on the reduction of nitrate and nitrite by soybean nodules. AB - The effects of water stress on nitrate reductase and nitrite reductase activities in symbiotic nodules were examined in field-grown soybean plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme activity indicated that the nodule cytosol and bacteroids contained both nitrate reductase and nitrite reductase activities. The reduction of nitrate in bacteroids increased significantly as nodule water potential declined from -0.6 to -1.4 megapascals, and then decreased when -1.8 megapascals water potential was reached. On the contrary, the reduction of nitrate in nodule cytosol was inhibited as water stress progressed. Increases in water stress intensity also caused a significant inhibition in nitrite reductase activities of bacteroids and nodule cytosol within soybean nodules. The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions. PMID- 16663331 TI - Isolation of mesophyll protoplasts from mature leaves of soybeans. AB - A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO(3) (-) dependent photosynthesis; greater than 75 micromoles O(2) evolved per milligram chlorophyll per hour at 25 degrees C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves. PMID- 16663332 TI - A pathway for photosynthetic carbon flow to mannitol in celery leaves : activity and localization of key enzymes. AB - In the polyol producing plant, celery (Apium graveolens L.), mannitol is a major photosynthetic product and a form in which carbohydrate is translocated. Measurements of whole leaf extracts of celery indicated substantial activity of the following enzymes: mannose-6-P reductase, mannose-6-P isomerase, mannitol-1-P phosphatase, and nonreversible glyceraldehyde-3-P dehydrogenase. The activities of these enzymes were either undetectable or very low in the nonpolyol producing plants, Secale cereale L. (rye) and Vigna mungo (L.) Hepper (black gram).Mesophyll protoplasts were enzymically isolated from celery leaves, broken with a Yeda press and the intracellular localization of the above enzymes for mannitol synthesis studied following differential and/or sucrose density gradient centrifugation of the protoplast extract. These data suggested the enzymes involved in mannitol synthesis are exclusively localized in the cytoplasm. Ninety five to 100% of the activity of these enzymes, along with the cytoplasmic marker enzyme phosphoenolpyruvate carboxylase, was found in the cytosolic fraction.We propose the pathway of photosynthetic carbon flow from triose-P to mannitol in celery occurs via fructose-6-P, mannose-6-P, and mannitol-1-P; these final reactions being catalyzed by the cytoplasmic enzymes, mannose-6-P isomerase, NADPH-dependent mannose-6-P reductase, and mannitol-1-P phosphatase, respectively. The requirement for NADPH may be met via the cytoplasmically located NADP-linked nonreversible glyceraldehyde-3-P dehydrogenase. PMID- 16663333 TI - Gravity-Induced Polar Transport of Calcium across Root Tips of Maize. AB - Calcium movement across primary roots of maize (Zea mays, L.) was determined by application of (45)Ca(2+) to one side of the root and collection of radioactivity in an agar receiver block on the opposite side. Ca movement across the root tip was found to be at least 20 times greater than movement across the elongation zone. The rapid movement of Ca across the tip was severely inhibited in roots from which the root cap had been removed. Ca movement across the tip was also strongly retarded in roots pretreated with 2,4-dinitrophenol or potassium cyanide. Orientation of roots horizontally had no effect on Ca movement across the elongation zone but caused a strong asymmetry in the pattern of Ca movement across the tip. In gravistimulated roots, the movement of Ca from top to bottom increased while movement from bottom to top decreased. The data indicate that gravistimulation induces polar movement of Ca toward the lower side of the root cap. An earlier report (Lee, Mulkey, Evans 1983 Science 220: 1375-1376) from this laboratory showed that artificial establishment of calcium gradients at the root tip can cause gravitropic-like curvature. Together, the two studies indicate that Ca plays a key role in linking gravistimulation to the gravitropic growth response in roots. PMID- 16663334 TI - Translocation of carbohydrates and proline in young grapefruit trees at low temperatures. AB - Girdling and defoliation of fruit-bearing grapefruit (Citrus paradisi Macf.) branches inhibited the accumulation of soluble carbohydrates and proline in fruit tissues during low temperature treatment of trees. These treatments did not inhibit hydrolysis of sucrose to reducing sugars. Flavedo and albedo tissues responded similarly to low temperatures but little or no change occurred in the juice. Therefore, soluble carbohydrates and proline do not appear to interchange between different tissues of the fruit at low temperatures but instead are translocated into the fruit from other parts of the plant. Girdling fruit-bearing branches immediately after low temperature treatments inhibited the accumulation of sucrose in fruit tissues at dehardening temperatures. Also, proline levels decreased rapidly in fruit on girdled branches at dehardening temperatures. This rapid decrease suggests proline may serve as a source for respiratory energy in grapefruit during rapidly changing temperatures that favor active growth and during recovery of citrus from environmental stress. PMID- 16663335 TI - Role of ethylene in the senescence of detached rice leaves. AB - The role of ethylene in the senescence of detached rice leaves in relation to their changes in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production was studied. In freshly excised rice leaf segments, ACC level and ethylene production rates were very low. Following incubation, the rates of ethylene production increased and reached a maximum in 12 h, and subsequently declined. The rise of ethylene production was associated with a 20- to 30-fold increase in ACC level.Ethylene seems to be involved in the regulation of the senescence of detached rice leaves. This conclusion was based on the observations that (a) maximum ethylene production preceded chlorophyll degradation, (b) ACC application promoted chlorophyll degradation, (c) inhibitors of ethylene production and ethylene action retarded chlorophyll degradation, and (d) various treatments such as light, cycloheximide, alpha,alpha-dipyridyl, Ni(2+), and cold temperature, which retarded chlorophyll degradation, also inhibited ethylene production.Abscisic acid promoted senescence but significantly decreased ethylene production, whereas benzyladenine retarded senescence but promoted ethylene production. This is interpreted to indicate that abscisic acid treatment increased the tissue sensitivity to ethylene, whereas benzyladenine treatment decreased it. PMID- 16663336 TI - Electron Transport-Dependent Chlorophyll-a Fluorescence Quenching by O(2) in Various Algae and Higher Plants. AB - A comparison of chlorophyll-a fluorescence in brown algae (Macrocystis integrifolia, Fucus vesiculosis), green algae (Scenedesmus obliquus, Ulva sp.) and higher plants (bean, corn) show differences in the relative fluorescence intensities and induction time courses which characterize each type of plant. These differences are not reflected in either the maximum fluorescence emission in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (F(max)) or the nonvariable fluorescence (F(o)). Constancy of F(o) and F(max) suggests functional similarities of photosystem II and associated antennae pigments in the various classes of plants. The time course differences are observed only in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and appear, therefore, to be electron transport dependent. During induction, the peak in fluorescence (F(p)) is much lower in all of the algae studied than in the higher plants. Exogenous O(2) strongly quenches F(p) in all plants studied and our data indicate that the low F(p) in the algae can be partially accounted for by endogenous O(2) quenching. PMID- 16663337 TI - Evolution of o(2) in brown algal chloroplasts. AB - A METHOD IS DESCRIBED FOR THE ISOLATION OF PHOTOSYNTHETICALLY ACTIVE CHLOROPLASTS FROM FOUR SPECIES OF BROWN ALGAE: Fucus vesiculosis, Nereocystis luetkeana, Laminaria saccharina, and Macrocystis integrifolia. When compared to lettuce and spinach chloroplasts, the algal chloroplasts all showed lower activities for both photosystems II and I. Chloroplasts from all the plants produced H(2)O(2), with photosystem I functioning as the O(2) reductant in the light. In contrast to the green plants, however, brown algal chloroplasts strongly reduced O(2) under conditions where both photosystems II and I remain active. Relative variable fluorescence values were lower both in intact plants and chloroplasts of the brown algae than for either spinach or lettuce. It is suggested that although light harvesting activities appear similar in all the plants, details of electron transport in brown algae may differ from those of green plants. PMID- 16663338 TI - Mechanism of Short Term Fe Reduction by Roots : Evidence against the Role of Secreted Reductants. AB - The hypothesized role of secreted reducing compounds in Fe(III) reduction has been examined with Fe-deficient peanuts (Arachis hypogaea L. cv A124B). Experiments involved the exposure of roots to (a) different gas mixtures, (b) carbonyl cyanide m-chlorophenylhydrazone (CCCP), and (c) agents which impair membrane integrity.Removing roots from solution and exposing them to air or N(2) for 10 minutes did not result in any accumulation in the free space of compounds capable of increasing rates of Fe(III) reduction when roots were returned to solutions. On the contrary, exposing roots to N(2) decreased rates of Fe(III) reduction. CCCP also decreased rates of Fe(III) reduction.Acetic acid and ethylenediaminetetraacetic acid (disodium salt) (EDTA) impaired the integrity and function of the plasma membranes of roots of Fe-deficient peanuts. That is, in the presence of acetic acid or EDTA, there was an efflux of K(+) from the roots; K(+) ((86)Rb) uptake was also impaired. Acetic acid increased the efflux from the roots of compounds capable of reducing Fe(III). However, both acetic acid and EDTA caused rapid decreases in rates of Fe(III) reduction by the roots. In addition to peanuts, acetic acid also decreased rates of Fe(III) reduction by roots of Fe-deficient sunflowers (Helianthus annuus L. cv Sobrid) but not maize (Zea mays L. cv Garbo).These results suggest that, at least in the short term, the enhanced Fe(III) reduction by roots of Fe-deficient plants is not due to the secretion of reducing compounds. PMID- 16663339 TI - Early Events in Maize Seed Development : 1-Methyl-3-phenyl-5-(3 [trifluoromethyl]phenyl)-4-(1H)-Pyridinone Induction of Vivipary. AB - Preharvest sprouting or vivipary is induced in developing maize (Zea mays, inbred Tx 5855 and Va 35) seeds by fluridone, a pyridinone inhibitor of carotenoid biosynthesis. Fluridone has a maximal effect on vivipary at 11 days after pollination (DAP) and little effect at 13 DAP in the inbred maize line Tx 5855. Abscisic acid partially reversed the chemically induced vivipary. Though the precise mechanism of fluridone-induced vivipary is unknown, these results indicate that there are important developmental changes occurring at 11 DAP which reversibly commit the immature embryo to vivipary or dormancy. PMID- 16663340 TI - ATP-Dependent Proteolytic Activity from Spinach Leaves. AB - Spinach (Spinacia oleracea CV Bloomsdale Long Standing) leaf cytoplasmic starch phosphorylase and rabbit muscle phosphorylase a were inactivated by incubation with partially purified leaf extract in the presence of ATP and Mg(2+). The inactivating factor(s) were heat stable and susceptible to protease attack. Phosphorylase inactivation was prevented by incubation in the presence of p aminobenzamidine and phenylboronic acid, or prolonged treatment with phenylmethylsulfonyl fluoride or leupeptin for the ATP-stimulated inhibitory activity. Mg(2+) -dependent inactivation was prevented by incubation with leupeptin, phenylmethylsulfonyl fluoride, p-aminobenzamidine, or 5'-adenylate. ATP-mediated inactivation of phosphorylase was stimulated by Mg(2+) with a reduction in the apparent K(m) for ATP. Casein-degrading activities with the same properties of ATP and/or Mg(2+) stimulation, heat stability, and susceptibility to proteinase inhibitors were detected suggesting that phorphorylase inactivation was due to proteolysis. The activity was greatest at about the time of flowering and also appeared to depend on the light regime. PMID- 16663341 TI - Ribulose bisphosphate carboxylase synthesis in barley leaves: a developmental approach to the question of coordinated subunit synthesis. AB - The coordination of the synthesis of the large and small subunits of ribulose 1,5 bisphosphate carboxylase (RuBPCase) was studied in young light-grown barley (Hordeum vulgare L. var. UC566) leaves. Since a barley leaf is a continuum of different aged cells with the youngest cells at the base and the oldest at the tip, developmental changes could be investigated by comparing different leaf regions. The rate of total cytoplasmic protein synthesis increased to a maximum before the rate of total organelle protein synthesis. The different positions of the maxima suggested that the synthesis of the small RuBPCase subunit on cytoplasmic ribosomes and the large RuBPCase subunit on chloroplast ribosomes might not be coupled during barley leaf development. However, measurements of the amounts and rates of synthesis of the subunits showed that they were coupled. Although the amounts of the RuBPCase subunits increased from the younger to the older leaf regions, the subunits were present in an equimolar ratio. While the rates of synthesis of both subunits increased to a maximum in a midleaf region and then declined, the ratio of the rates remained constant. That the subunit amounts remained equimolar and the synthetic rates proportional while total RuBPCase synthesis was changing indicated that the synthesis of the subunits was closely coordinated during leaf development. A close coordination was also supported by the kinetics of the inhibition of subunit synthesis in the presence of cycloheximide. PMID- 16663342 TI - Are polyamines transported in etiolated peas? AB - To investigate the possible transport of polyamines and their precursor amino acids, (14)C-labeled putrescine, spermidine, arginine, or lysine were injected into cotyledons of 4-day etiolated pea (Pisum sativum L. cv Alaska) seedlings. After 4 hours the shoot, root, and cotyledons were homogenized and the extracted, dansylated polyamines separated by thin-layer chromatography. Little radioactivity was transported from the cotyledons when [(14)C]putrescine or [(14)C]spermidine were injected and of the radioactivity in the axis, none could be recovered as polyamines. Injection of [(14)C]arginine or [(14)C]lysine, on the other hand, led to a significant transport of radioactivity into the axis, of which a large fraction was present in the form of the diamines, putrescine or cadaverine, respectively. These results indicate that polyamines in the growing regions of etiolated pea seedlings probably arise from transport and conversion of amino acid precursors. PMID- 16663343 TI - Expression of zein in long term endosperm cultures of maize. AB - Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by poly-acrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue specific proteins are retained in prolonged culture. PMID- 16663344 TI - Separation of two types of electrogenic h-pumping ATPases from oat roots. AB - Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H(+)/K(+) exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN(-) distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl(-) and inhibited by NO(-) (3) or 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The distribution of Cl(-)-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H(+) pumping into vesicles as monitored by [(14)C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K(+). These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl(-) stimulated, H(+)-pumping ATPase may be enriched in vacuolar-type membranes; the H(+)-pumping ATPase that is stimulated by K(+) and inhibited by vanadate is most likely associated with plasma membrane-type vesicles. PMID- 16663345 TI - Lipid biosynthesis in developing mustard seed: formation of triacylglycerols from endogenous and exogenous Fatty acids. AB - Cotyledons of developing mustard (Sinapis alba L.) seed have been found to synthesize lipids containing the common plant fatty acids and very long-chain monounsaturated (icosenoic, erucic, and tetracosenic) and saturated (icosanoic, docosanoic, and tetracosanoic) fatty acids from various radioactive precursors. The in vivo pattern of labeling of acyl lipids, either from fatty acids synthesized ;endogenously' from radioactive acetate or malonate, or from radioactive fatty acids added ;exogenously', indicates the involvement of the following pathways in the biosynthesis of triacylglycerols. Palmitic, stearic, and oleic acid, synthesized in the acyl carrier protein-track, are channeled to the Coenzyme A (CoA)-track and converted to triacylglycerols via the glycerol-3 phosphate pathway. Pools of stearoyl-CoA and oleoyl-CoA are elongated to very long-chain saturated and monounsaturated acyl-CoA, respectively. Most of the very long-chain saturated acyl-CoAs acylate preformed diacylglycerols. Very long-chain monounsaturated acyl-CoAs are converted to triacylglycerols, partly via phosphatidic acids and diacylglycerols, and partly by acylation of preformed diacylglycerols. PMID- 16663346 TI - Effect of Water Stress, Seed Coat Restraint, and Abscisic Acid upon Different Germination Capabilities of Two Tomato Lines at Low Temperature. AB - Two tomato (Lycopersicon esculentum Mill.) lines with greatly different capabilities to germinate at 10 degrees C were compared with respect to sensitivity to experimental treatments which affect the water status of the embryo. Germination rates and final percentages could be drastically changed (at 25 degrees C) by (a) removing the mechanical constraint from the radicle tip, (b) imposing water stress by an osmoticum, (c) stress hardening of the seeds through osmotic pretreatment, and (d) inhibiting embryo expansion by abscisic acid (ABA). All treatments showed a similar difference in germination vigor between the two lines indicating that cold sensitivity is in fact a matter of water relations rather than of phase transitions in membrane lipids. Inhibition of germination by ABA was completely abolished by removing the mechanical constraint from the radicle tip. Osmotic stress of -3 bar which quantitatively replaced this constraint in inhibiting germination also restored the sensitivity to ABA. It is concluded that all these treatments act on the balance between the hydraulic extension force of the embryo radicle and the opposing force of the seed layers covering the radicle tip. The difference in cold sensitivity between the two seed lines resides either in the osmotic potential or the pressure potential of the germinating embryo. PMID- 16663347 TI - Activity in vivo and redox States in vitro of nitro- and chlorodiphenyl ether herbicide analogs. AB - Excised cucumber (Cucumis sativus L. cv 447 Wisconsin SMR 18) cotyledons were sensitive to acifluorfen-methyl (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2 nitrobenzoate) and MC-15608 (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2 chlorobenzoate). Injury was detected by monitoring efflux of 3-O-methyl-d-[U (14)C]glucose from herbicide-treated tissue after exposure to light. Efflux kinetics of 3-O-methyl-[(14)C]glucose from cotyledons treated with either acifluorfen-methyl (AFM) or MC-15608 were similar. Neither herbicide was active in darkness.Cyclic voltammetry was used to study redox behavior of AFM and MC 15608 in protolytic and aprotic solvents. In protic media reduction of AFM was chemically irreversible. Reduction of MC-15608 in protolytic solvent was not observed. In aprotic solvent, the electrode reaction of AFM was quasireversible. The voltammogram of MC-15608 in aprotic solvent was indicative of a multielectron, completely irreversible electrode reaction.Although the physiological data indicate that AFM and MC-15608 have similar effects in vivo, the redox behavior of the two compounds is quite different. These results suggest that mechanisms involving direct reduction and reoxidation of the diphenyl ether molecule are probably not the basis for the action of these herbicides. PMID- 16663348 TI - Interaction of Benzylaminopurine with Electron Transport in Plant Mitochondria during Malate Oxidation. AB - The effect of 6-benzylaminopurine (BA) was assayed on malate oxidation in mitochondria isolated from fresh and aged potato (Solanum tuberosum L.) slices. Depending on the experimental pH, two pathways for malate oxidation were selected. A pH of 7.7 favored the activity of malate dehydrogenase, which is connected with a rotenone-sensitive NADH dehydrogenase, whereas at pH 6.5 malic enzyme, linked to a rotenone-resistant NADH dehydrogenase, was more active.Experimental results indicate the existence of two sites of inhibition for BA. The first site is common with the site of inhibition of rotenone. The second site is on the classical cyanide-resistant alternative pathway, but is different from the site of salicylhydroxamic acid (SHAM) inhibition, as in succinate oxidation.Moreover, a distinct cyanide-resistant pathway, sensitive to SHAM but resistant to BA, is found to coexist with the well-known alternative pathway which is sensitive to SHAM and BA. This outlet of electrons can accommodate 10% of the total electron flow in mitochondria from fresh slices, and up to 30% in mitochondria from aged slices. PMID- 16663349 TI - Role of endoplasmic reticulum in biosynthesis of oat globulin precursors. AB - Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin alpha-beta dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced alpha-beta dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the alpha + beta subunits, were labeled in protein bodies.The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller alpha and beta subunits. PMID- 16663350 TI - Water relationships and incorporation of C assimilates in tubers of potato plants differing in potassium nutrition. AB - Potato (Solanum tuberosum L. cv Saturna) plants were grown in pots with varying supplies of K fertilizer (1.25, 5, and 10 grams K(2)O per 12 kilograms soil). Four weeks after midflower, plants were supplied with (14)CO(2) for 12 hours and osmotic and water potential in tubers were determined. Assimilation of (14)CO(2) increased from 504 (K(1)) to 1860 (K(5)) and 1922 kilobecquerels per plant per 12 hours (K(10)). In all treatments, about half of the (14)C was translocated to the tubers within 12 hours, although calculated turgor pressure in tubers which could be considered as a potential counter-pressure to phloem unloading increased from +5.3 (K(1)) to +5.9 (K(5)) and +6.0 bars (K(10)), respectively. Incorporation of (14)C per gram tuber dry weight as well as per gram tuber starch was significantly higher in K(5) than in K(1) and slightly increased even further in the K(10) treatment, where tuber pressure sap contained the highest K concentration (179 millimolar K). PMID- 16663351 TI - Metabolic Relations between Methylxanthines and Methyluric Acids in Coffea L. AB - Metabolism of purine alkaloids in the leaves of Coffea dewevrei De Wild et Durand var excelsa Chev, Coffea liberica Bull ex Hiern and Coffea abeokutae Cramer was studied by analyzing leaf discs collected during vegetative development and by feeding the following radioactive tracers: [(14)C]theobromine, [(14)C]caffeine, and [(14)C]theacrine (1,3,7,9-tetramethyluric acid). Their principal metabolites were quantitatively and qualitatively determined. All three species convert the precursors to the same radioactive products, and proceed through the same four maturity stages characterized by the alkaloid accumulation pattern and by a particular transformation potency: (stage 1) young plant accumulating caffeine, transforms theobromine to caffeine; (stage 2) caffeine is gradually replaced by theacrine, theobromine and caffeine are converted to theacrine; (stage 3) theacrine disappears whereas liberine (O(2), 1,9-thrimethyluric acid) accumulates, theacrine is metabolized to liberine; (stage 4) branched-out plant containing liberine but no theacrine, caffeine is converted rapidly to liberine via theacrine. Methylliberine (O(2),1,7,9-tetramethyluric acid), presumably the direct precursor of liberine, is occasionally found in low concentrations at stage 3 and 4.The collective term ;liberio-excelsoid' introduced by geneticists for the numerous races or species of Pachycoffea is in accordance with the phytochemical equality found in this work. PMID- 16663352 TI - l-Arginine and l-Canavanine Metabolism in Jack Bean, Canavalia ensiformis (L.) DC. and Soybean, Glycine max (L.) Merr. AB - Studies have been conducted with the arginase (l-arginine amidinohydrolase, EC 3.5.3.1) of two legumes: jack bean, Canavalia ensiformis (L.) DC., a l-canavanine containing plant and soybean, Glycine max, a canavanine-free species. Analyses of the arginase obtained from gradient-purified mitochondria of these legumes revealed that the arginine-dependent (ADA) and canavanine-dependent activities (CDA) were localized within this organelle.Kinetic analyses of affinity-purified mitochondrial arginase revealed an apparent K(m) of 7 to 8 millimolar for arginine with both the jack bean and soybean arginases. Comparable determinations with canavanine revealed an apparent K(m) of 38 millimolar with the jack bean enzyme; the affinity for this arginine analog with the soybean enzyme is so poor that product formation remained linear even with a canavanine concentration of 890 millimolar.A single macromolecule appears to be responsible for both the ADA and CDA of jack bean arginase. Ion-exchange chromatography of mitochondrial arginase revealed that the ADA and CDA eluted as a single, discrete peak from DEAE-cellulose. Analyses with arginine- and canavanine-linked Sepharose failed to reveal more than one enzyme. Both the ADA and CDA increased by nearly identical amounts following elution from arginine- and canavanine-linked cyanogen bromide activated sepharose. Neither ADA nor CDA increased preferentially over the other. PMID- 16663353 TI - Light effects in mycorrhizal soybeans. AB - Soybean (Glycine max. L. Merr.) plants were grown in an experiment with a 3 x 3 factorial design using different levels of light (170, 350, and 700 muE.m(-2).s( 1)) and P as factors. Plants were grown in a greenhouse in pot cultures using a soil low in plant-available P under three P regimes: no additional P, P added as KH(2)PO(4), or P uptake enhanced by colonization of the host plant with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe. Development of the VAM fungal endophyte and of plants under all three P regimes was depressed by limiting light. However, the growth response of VAM plants to increasing light relative to non-VAM plants in the absence of additional P increased while the response relative to non-VAM plants with additional P decreased slightly. The highly significant interaction between the factors (P < 0.001) of the experiment was due to differences in the magnitude and direction of simple effects of the factors. The implications of these differences in terms of source-sink relationships of the symbionts and the value of different non-VAM controls in interpreting VAM effects are discussed. PMID- 16663354 TI - Sensitivity of tonoplast-bound adenosine-triphosphatase from hevea to inhibitors. AB - The tonoplast-bound H(+)-translocating ATPase from Hevea latex was found to be insensitive to vanadate, diethylstilbestrol, and octylguanidine, which are specific inhibitors of the plasma membrane ATPase. The inhibitors of the mitochondrial ATPase, oligomycin and azide, and also rotenone and antimycin A, were all without effect. In contrast, trimethyltin chloride strongly inhibited the activity of Hevea tonoplast ATPase.Among the different carbodiimides tested, which strongly inhibit the Hevea tonoplast ATPase, N,N'-dicyclohexylcarbodiimide was the most inhibitory. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline was also an efficient inhibitor.This unique inhibitor sensitivity of the Hevea tonoplast H(+)-translocating ATPase suggests that this enzyme differs in its mode of operation from all other known H(+)-translocating ATPases. PMID- 16663355 TI - Estimation of Photorespiration Based on the Initial Rate of Postillumination CO(2) Release: I. A Nonsteady State Model for Measurement of CO(2) Exchange Transients. AB - Although open systems have been used for the study of transients in leaf CO(2) exchange such as the postillumination burst, these systems frequently do not permit reliable estimates of transient rates due to their nonsteady state nature. A nonsteady state mathematical approach is described which predicts changes in CO(2) concentration in the leaf chamber and infrared gas analyzer measuring cell as a function of leaf CO(2) exchange rate in Nicotiana tabacum vars John Williams Broadleaf and Havana Seed. With the aid of a computer, a numerical formula simulates the mixing and dilution which occurs as CO(2) passes through the finite volume of the measuring cell of the analyzer. The method is presented with special relevance to photorespiration as manifested by the postillumination burst of CO(2). The latter is suggested to decline with the first order kinetics following darkening of a C(3) leaf. This approach provides a basis for reliable estimation of the initial and, hence, maximal rate of CO(2) evolution during the postillumination burst under a variety of environmental conditions. PMID- 16663356 TI - Estimation of Photorespiration Based on the Initial Rate of Postillumination CO(2) Release: II. Effects of O(2), CO(2), and Temperature. AB - An open system associated with an infrared gas analyzer was employed to study transients in CO(2) exchange generated upon darkening preilluminated leaf discs of tobacco (Nicotiana tabacum vars John Williams Broadleaf and Havana Seed). An empirical formula presented previously enabled prediction of the analyzer response under nonsteady state conditions as a function of time and of the leaf CO(2) exchange rate. A computer was used to evaluate parameters of the leaf CO(2) release rate to provide an estimate of the initial rate of postillumination CO(2) evolution and to produce maximal agreement between predicted and observed analyzer responses. In 21% O(2), the decline in rate of CO(2) evolution upon darkening followed first order kinetics. Initial rates of CO(2) evolution following darkening were relatively independent of the prior ambient CO(2) concentrations. However, rates of photorespiration expressed as a fraction of net photosynthesis declined rapidly with increasing external CO(2) concentration at 21% O(2). Under normal atmospheric conditions, photorespiration was 45 to 50% of the net CO(2) fixation rate at 32 degrees C and high irradiance. The rapid initial CO(2) evolution observed upon darkening at 21% O(2) was absent in 3% O(2). Rates of photorespiration under normal atmospheric concentrations of CO(2) and O(2) as measured by the postillumination burst were highly dependent upon temperature (observed activation energy = 30.1 kilocalories per mole). The results are discussed with respect to previously published estimates of photorespiration in C(3) leaf tissue. PMID- 16663357 TI - Regulation of Spinach Leaf Sucrose Phosphate Synthase by Glucose-6-Phosphate, Inorganic Phosphate, and pH. AB - Sucrose phosphate synthase was partially purified from spinach leaves and the effects and interactions among glucose-6-P, inorganic phosphate (Pi), and pH were investigated. Glucose-6-P activated sucrose phosphate synthase and the concentration required for 50% of maximal activation increased as the concentration of fructose-6-P was decreased. Inorganic phosphate inhibited sucrose phosphate synthase activity and antagonized the activation by glucose-6 P. Inorganic phosphate caused a progressive increase in the concentration of glucose-6-P required for 50% maximal activation from 0.85 mm (minus Pi) to 9.9 mm (20 mm Pi). In the absence of glucose-6-P, Pi caused partial inhibition of sucrose phosphate synthase activity (about 65%). The concentration of Pi required for 50% maximal inhibition decreased with a change in pH from 6.5 to 7.5. When the effect of pH on Pi ionization was taken into account, it was found that per cent inhibition increased hyperbolically with increasing dibasic phosphate concentration independent of the pH. Sucrose phosphate synthase had a relatively broad pH optimum centered at pH 7.5. Inhibition by Pi was absent at pH 5.5, but became more pronounced at alkaline pH, whereas activation by glucose-6-P was observed over the entire pH range tested. The results suggested that glucose-6-P and Pi bind to sites distinct from the catalytic site, e.g. allosteric sites, and that the interactions of these effectors with pH and concentrations of substrate may be involved in the regulation of sucrose synthesis in vivo. PMID- 16663358 TI - Isolation-Inflicted Injury to Mitochondria from Fresh Pollen Gradually Overcome by an Active Strengthening during Germination. AB - Activities of segments of the electron transport pathway of mitochondria isolated from pollen of Typha latifolia L. during the course of germination in vitro were compared with those of mitochondria in intact grains. For this purpose, suitable inhibitors and artificial substrates were selected for their ability to penetrate through the exine, intine, and plasmalemma. In contrast to their counterparts in vivo, mitochondria isolated during the initial stages of germination exhibited low rates of electron transport, resulting from loss of NAD(+) and displacement of cytochrome c from its site of action. The phosphorylative capacity was also impaired. Great caution must be exercised therefore, before interpreting results obtained with isolated mitochondria.The gradually acquired resistance of mitochondria to injury during isolation as germination proceeds was shown to depend on an energy-requiring process and not solely on a rearrangement at the membrane level, or imbibitional differences. De novo syntheses of proteins or fatty acids were not required for the strengthening of mitochondria since cycloheximide, chloramphenicol, and cerulenin did not prevent this change. The nature of the energy-requiring process remains obscure. It is probable that strengthening of mitochondrial membranes during seed germination has been misinterpreted due to similar effects of isolational injury. PMID- 16663359 TI - Photosynthetic and Stomatal Responses of Two Mangrove Species, Aegiceras corniculatum and Avicennia marina, to Long Term Salinity and Humidity Conditions. AB - Gas exchange characteristics were studied in two mangrove species, Aegiceras corniculatum (L.) Blanco and Avicennia marina (Forstk.) Vierh. var australasica (Walp.) Moldenke, grown under a variety of salinity and humidity conditions. The assimilation rate was measured as a function of the intercellular CO(2) concentration [A(c(i)) curve]. The photosynthetic capacity decreased with increase in salinity from 50 to 500 millimolar NaCl, as shown by decline in both the initial linear slope and the upper plateau of the A(c(i)) curve, with A. corniculatum being the more sensitive species. The decline in photosynthetic capacity was enhanced by increase in the leaf to air vapor pressure difference from 6 to 24 millibars, but this treatment caused a decrease in only the upper plateau of the A(c(i)) curve. Stomatal conductance was such that the intercellular CO(2) concentration obtaining under normal atmospheric conditions occurred near the transition between the lower linear and upper plateau portions of the A(c(i)) curves. Thus, stomatal conductance and photosynthetic capacity together co-limited the assimilation rate, which declined with increasing salinity and decreasing humidity. The marginal water cost of carbon assimilation was similar in most treatments, despite variation in the water loss/carbon gain ratio. PMID- 16663360 TI - Carbon, hydrogen, and oxygen isotope ratios of cellulose from plants having intermediary photosynthetic modes. AB - Carbon and hydrogen isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from species of greenhouse plants having different photosynthetic modes were determined. When hydrogen isotope ratios are plotted against carbon isotope ratios, four clusters of points are discernible, each representing different photosynthetic modes: C(3) plants, C(4) plants, CAM plants, and C(3) plants that can shift to CAM or show the phenomenon referred to as CAM-cycling. The combination of oxygen and carbon isotope ratios does not distinguish among the different photosynthetic modes. Analysis of the carbon and hydrogen isotope ratios of cellulose nitrate should prove useful for screening different photosynthetic modes in field specimens that grew near one another. This method will be particularly useful for detection of plants which show CAM-cycling. PMID- 16663361 TI - Incorporation of Oxygen into Glycolate, Glycine, and Serine during Photorespiration in Maize Leaves. AB - Glycolate, glycine, and serine extracted from excised Zea mays L. leaves which had been allowed to photosynthesize in the presence of (18)O(2) were analyzed by gas chromatography-mass spectrometry. In each case, only one of the oxygen atoms of the carboxyl group had become labeled. The maximum enrichment observed in glycine and serine was attained after 5 minutes and 15 minutes of exposure to (18)O(2) at the CO(2) compensation point; the labeling was very high, reaching 70 to 73% of that in the applied O(2). Thus, it appears that all or nearly all of the glycine and serine are synthesized in maize leaves via fixation of O(2). In the presence of CO(2) (380 or 800 microliters per liter), (18)O-labeling was markedly slower.Glycolate enrichment was variable and much lower than that in glycine and serine. It is possible that there are additional pathways of glycolate synthesis which do not result in the incorporation of (18)O from molecular oxygen. An estimation of the metabolic flow of O(2) through the photorespiratory cycle was made. It appeared that less than 75% of the O(2) taken up by maize leaves is involved in this pathway. Therefore, other processes of O(2) metabolism must occur in the light. PMID- 16663362 TI - Terpenoid metabolism in plastids : sites of phytoene synthetase activity and synthesis in plant cells. AB - The biosynthesis of phytoene from prephytoene pyrophosphate has been localized exclusively in the plastid compartment of ruptured protoplasts derived from Triticum leaves and Capsicum fruits.The phytoene synthetase activity in Triticum leaves deficient in plastid ribosomes was comparable to those obtained in normal leaves. In addition, the stimulation of phytoene synthetase activity observed in green Capsicum fruit after 2-(4-chlorophenylthio)triethylamine hydrochloride treatment was not abolished by chlororamphenicol and lincomycin, in contrast to the inhibition observed after cycloheximide treatment.These data conclusively show that phytoene synthetase is localized exclusively in the plastid compartment in higher plants and that its synthesis is not performed on 70S ribosomes. PMID- 16663363 TI - Biological Activity of the Isomeric Forms of Helminthosporium sacchari Toxin and of Homologs Produced in Culture. AB - The effect of Helminthosporium sacchari (HS) toxin isomers and related, pathogen produced compounds on dark CO(2) fixation in HS-susceptible sugar cane leaf slices was investigated. HS toxin consists of a mixture of three isomeric bis-5-O (beta-galactofuranosyl)-beta-galactofuranosides (A, B, and C) differing in the position of one double bond in the sesquiterpene aglycone. Maximum inhibition of dark CO(2) fixation in susceptible sugar cane (CP52-68) occurred within 30 to 40 minutes, and amounts necessary to reach 50% inhibition values typically were approximately 1.7 micromolar for natural toxin mixture ( approximately 2:3:5 mixture of isomers A:B:C) and 4, 6, and 0.7 micromolar for isomers A, B, and C, respectively. Other fractions from cultures of the pathogen consist of comparable mixtures of sesquiterpene isomers but have only 1, 2, or 3 galactofuranose units (HS(1), HS(2), HS(3)) or two alpha-glucopyranose units as well as four beta galactofuranose units (HS(6)). The lower toxin homologs were not toxic to clone CP52-68, but protected sugar cane from the effects of toxin. Minimum ratios of protectant: toxin giving 95% protection were approximately 50:1, 6:1, and 12:1 for HS(1), HS(2), and HS(3), respectively. HS(2) and HS(3) protected when added up to 12 minutes after toxin as well as when added with or before toxin. Some common plant galactopyranosides were not toxic and did not protect at 500:1 molar excess. The sample of HS(6) was toxic at 500 micromolar, and did not protect against HS toxin. With the availability of purified, homogeneous preparations of HS toxin, homologs, and chemically modified or synthetic analogs, the dark CO(2) fixation assay should prove to be a useful tool for understanding the mode of action of HS toxin. PMID- 16663364 TI - Biochemistry of photosynthesis in species of triticum of differing ploidy. AB - Illuminated flag leaves of Triticum monococcum(2X), T. urartu(2X), T. dicoccum(4X), T. dicoccoides(4X), and T. aestivum(6X) were exposed to (14)CO(2) for 10 seconds and subsequently allowed to continue photosynthesis in the ambient air for periods of up to 2 minutes. The relative distribution of (14)C among water-soluble products in the leaves was similar for each species at each sampling time. After the 10-second pulse of (14)CO(2), radioactivity was mainly in phosphate esters with less than 5% in C(4) acids. Subsequently, radioactivity increased in sucrose, glycine, and serine at the expense of that in phosphate esters. By 2 minutes, between 18% and 29% of the (14)C was in glycine plus serine. The results suggest rapid photorespiration in all species and an absence of C(4) photosynthesis.d-Ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) was partly purified from seedling leaves of each of the five Triticum species. Each preparation was assayed for simultaneous carboxylase and oxygenase activities in 2.1 millimolar NaHCO(3) and 265 micromolar O(2) at pH 8.2 and 25 degrees C. The mean ratio of carboxylase to oxygenase activities was 6.11 +/- 0.16 (standard error); differences between values for different species were not statistically significant. The results do not explain the faster rates of photosynthesis per unit leaf area reported for diploid and tetraploid species of Triticum compared to the hexaploid. PMID- 16663365 TI - Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome. AB - Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome-red-absorbing form and phytochrome-far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action. PMID- 16663366 TI - Role of nitrogen assimilation in seed development of soybean. AB - A nondestructive acetylene reduction assay for nitrogenase activity of soybean (Glycine max L. Merr) field plots is presented. Plots consisted of 120 x 150 x 30 centimeter boxes containing 65 plants. The plants were grown in a medium grade sand under controlled nutrient, moisture, and root temperature conditions. Acetylene at a concentration of 10 milliliters per liter was circulated through manifolds in the chambers; equilibration required 5 minutes, and activity was linear with time. Optimum growth and assay environments resulted in activity of 70 micromoles ethylene per plant per hour. Plant development and yield were comparable to soil-grown companion plots.The well accepted hypothesis that developing seeds deprive the nodules of carbohydrate was not substantiated. The nondestructive acetylene reduction profile did not decline until 30 days after the onset of seed development (R-5). This result was consistent with reports from the literature which indicated that 60% of seasonal nitrogen was fixed after R-5. Further, a high correlation shown between integrated seasonal acetylene reduction and yield (r = 0.999) suggested a cooperative relationship between the roots and shoot. A reduction in source:sink ratio (60% defoliation) after R-5 had no effect on acetylene reduction. This showed that neither an increase in sink demand by the pods nor a carbon shortage during podfill decreased dinitrogen fixation. A conceptual model relating seed growth with carbon and nitrogen assimilation is proposed. PMID- 16663367 TI - In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis: I. Some Characteristics of Fatty Acid Synthesis. AB - In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH(4))(2)SO(4) fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution. PMID- 16663368 TI - Evidence for Host Genome Involvement in Cytokinin Metabolism by Male and Female Cells of Mercurialis annua Transformed by Strain 15,955 of Agrobacterium tumefaciens. AB - When male and female individuals of a dioecious species Mercurialis annua L. were inoculated with the same strain of Agrobacterium tumefaciens (15,955), the corresponding tumor tissues of each sex clearly differed in their endogenous cytokinin content; only the male tumors had a morphogenetic feminizing effect on male flowers.In male tumor tissues, zeatin (Z) in higher quantity than ribosyl zeatin (RZ) became the major metabolite in contrast with the general situation for crown-galls; the female tumor tissues were characterized by an increase of total endogenous cytokinins and by the appearance of some specific metabolites such as a methyl-thio-Z and several glycosylated Z derivatives that had not been detected in healthy apices.In both male and female tumor tissues, the cis form of RZ, present in healthy apices as 30% of trans-RZ form, was no longer detectable.Quantitative and qualitative differences characterize male and female tumor tissues (host genes expression) but since differences also appeared between healthy male and female apices and their corresponding tumor tissues (TDNA gene expression), it can be tentatively concluded that a complex interaction between host cytokinin genes and those of TDNA control the endogenous metabolism of tumor tissues. PMID- 16663369 TI - Biosynthesis of Protoheme and Heme a Precursors Solely from Glutamate in the Unicellular Red Alga Cyanidium caldarium. AB - Two biosynthetic routes to the heme, chlorophyll, and phycobilin precursor, delta aminolevulinic acid (ALA) are known: conversion of the intact five-carbon skeleton of glutamate, and ALA synthase-catalyzed condensation of glycine plus succinyl-coenzyme A. The existence and physiological roles of the two pathways in Cyanidium caldarium were assessed in vivo by determining the relative abilities of [2-(14)C]glycine and [1-(14)C]glutamate to label protoheme and heme a. Glutamate was incorporated to a much greater extent than glycine into both protoheme and heme a, even in cells that were unable to form chlorophyll and phycobilins. The small incorporation of glycine could be accounted for by transfer of label to intracellular glutamate pools, as determined from amino acid analysis. It thus appears that C. caldarium makes all tetrapyrroles, including mitochondrial hemes, solely from glutamate, and there is no contribution by ALA synthase in this organism. PMID- 16663370 TI - Acquisition of Thermotolerance in Soybean Seedlings : Synthesis and Accumulation of Heat Shock Proteins and their Cellular Localization. AB - When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28 degrees C up to 40 degrees C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45 degrees C followed by incubation at 28 degrees C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45 degrees C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40 degrees C or a brief (10-minute) pulse treatment at 45 degrees C followed by a 28 degrees C incubation provide protection (thermal tolerance) to a subsequent exposure at 45 degrees C. Maximum thermoprotection is achieved by a 2-hour 40 degrees C pretreatment or after 2 hours at 28 degrees C with a prior 10-minute 45 degrees C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSP-like proteins, and also provides thermoprotection to a 45 degrees C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions.During 40 degrees C HS, some HSPs become localized and stably associated with purified organelle fractions (e.g. nuclei, mitochondria, and ribosomes) while others do not. A chase at 28 degrees C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40 degrees C chase period. If the seedlings are subjected to a second HS after a 28 degrees C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions. The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40 degrees C to 45 degrees C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28 degrees C but become organelle-associated during a subsequent HS at 40 degrees C. PMID- 16663371 TI - Sugar uptake by cotton tissues: leaf disc versus cultured roots. AB - The tissue accumulation of sucrose, glucose, and fructose has been studied in cultured cotton (Gossypium hirsutum L.) roots and leaf discs. Sucrose uptake by both tissues from high apoplastic concentrations was independent of pH but has a slightly acidic pH optimum from low concentrations. Like other higher plant tissues, cotton root cells accumulate sucrose via a ;saturable,' inhibitor sensitive mechanism and a linear, inhibitor-resistant mechanism. The linear mechanism of sucrose uptake is not as pronounced in leaf disc data as it is in root data. Further, sucrose uptake by cotton leaf discs is more resistant than uptake by root cells to pH alterations, inhibitors, and monosaccharides in the uptake medium. The saturable phase of sucrose influx into cotton root is eliminated by glucose, fructose, and high pH. Sucrose influx into both tissues is not altered by osmotica up to 200 milliOsmolar. Sucrose accumulated by both tissues is rapidly converted to other chemical forms, especially in root tissue where only approximately 50% remains as neutral sugars 1 hour following the start of radiolable exposure. Although the entry of radiolabeled sucrose is faster in abraded leaf discs, they give the same response patterns to pH, inhibitors, and monosaccharide as do unabraded discs.The sucrose accumulation kinetics of cotton roots and leaf discs differ. These differences may be related to the physiological roles (source versus sink) of the two tissues in the intact plant. PMID- 16663372 TI - Acclimation of photosynthesis to low leaf water potentials. AB - Photosynthesis is reduced at low leaf water potentials (Psi(l)) but repeated water deficits can decrease this reduction, resulting in photosynthetic acclimation. The contribution of the stomata and the chloroplasts to this acclimation is unknown. We evaluated stomatal and chloroplast contributions when soil-grown sunflower (Helianthus annuus L.) plants were subjected to water deficit pretreatments for 2 weeks. The relationship between photosynthesis and Psi(l), determined from gas-exchange and isopiestic thermocouple psychometry, was shifted 3 to 4 bars towards lower Psi(l), in pretreated plants. Leaf diffusive resistance was similarly affected. Chloroplast activity, demonstrated in situ with measurements of quantum yield and the capacity to fix CO(2) at all partial pressures of CO(2), and in vitro by photosystem II activity of isolated organelles, was inhibited at low Psi(l) but less in pretreated plants than in control plants. The magnitude of this inhibition indicated that decreases in chloroplast activity contributed more than closure of stomata both to losses in photosynthesis and to the acclimation of photosynthesis to low Psi(l). PMID- 16663373 TI - A Novel Method for the Activation and Solubilization of Coupling Factor 1 from Chloroplasts of Euglena gracilis. AB - A method was found for the in situ activation of the latent Ca(2+)-ATPase of the coupling factor from chloroplasts of Euglena gracilis and its resultant solubilization. The activation causes the concomitant solubilization of the enzyme and facilitates easy and nearly complete extraction. The activation consists of lowering the pH of isolated chloroplasts for 2 minutes to pH 4.5 to 4.7 with acetic acid, followed by neutralization. Increases in activity as high as 18-fold can be obtained. The method does not appear to work with chloroplasts from other sources. PMID- 16663374 TI - Temperature dependence of energy-transducing functions and inhibitor sensitivity in chloroplasts. AB - A comparative analysis of the temperature dependence of energy-transducing reactions in spinach (Spinacia oleracea) chloroplasts and their sensitivity for uncouplers and energy-transfer inhibitors at different temperatures is presented. Arrhenius plots reveal two groups of transitions, around 19 degrees C and around 12 degrees C. Activities that show transitions around 19 degrees C include linear electron flow from water to ferricyanide, its coupled photophosphorylation, the dark-release of the fluorescent probe atebrin, and the slow component of the 515 nm (carotenoid) absorbance decay after a flash. The transitions around 12 degrees C are observed with pyocyanine-mediated cyclic photophosphorylation, light- and dithioerythritol-activated ATP hydrolysis, the dark-release of protons, and the fast 515 nm decay component. It is suggested that both groups of temperature transitions are determined by proton displacements in different domains of the exposed thylakoid membranes. The effects of various uncouplers and an energy transfer inhibitor are temperature dependent. Some uncouplers also show a different relative inhibition of proton uptake and ATP synthesis at lower temperatures. The efficiency of energy transduction (ATP/e(2)) varied with temperature and was optimal around 10 degrees C. PMID- 16663375 TI - Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester. AB - During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative lambda(max) at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S(8) configuration. No labeled elemental sulfur was detected when the cells were grown on [(35)S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella. PMID- 16663376 TI - Salt adaptation of the cyanobacterium synechococcus 6311 growing in a continuous culture (turbidostat). AB - Physiological aspects of salt-adaptation in the cyanobacterium Synechococcus 6311 growing in a continuous culture (turbidostat) were studied. The process of salt adaptation was completed within 3 days, as expressed by the specific growth rate of cells grown in the presence of 0.2 and 0.4 molar NaCl. An increase in photosynthetic activity during the adaptation period leads to the accumulation of soluble sugars, essential for osmoregulation in the salt-grown cells. Cells grown in the presence of 0.4 molar NaCl showed an initial inhibition in the rate of protein synthesis which was enhanced after the 1st day of salt stress. After adaptation, salt-grown cells showed an increase in phycobiliprotein synthesis and a higher phycobiliprotein to protein ratio. PMID- 16663377 TI - Reversion from light-induced inhibition of seed germination by respiratory inhibitors. AB - Treating of the dark-imbibed lettuce (Lactuca sativa L.) seeds prior to light irradiation with 1 millimolar KCN or NaN(3) in the dark for 3 hours prevented blue light and far-red light-induced inhibitions of phytochrome-mediated germination. Similarly, salicylhydroxamic acid (SHAM) at 10 millimolar counteracted the blue and far-red light inhibitions, the combined application of KCN and SHAM being more effective than KCN or SHAM alone in some experiments. These respiratory inhibitors slightly inhibited phytochrome-mediated lettuce seed germination. These results indicate that both CN-sensitive, conventional cytochrome oxidase and CN-resistant (SHAM-sensitive), alternative respiration may be involved in the light inhibition or that an appropriate balance of both may be necessary for the light inhibition. PMID- 16663378 TI - Pyruvate orthophosphate dikinase: intracellular site of synthesis in maize leaf cells. AB - Pyruvate orthophosphate dikinase is synthesized in non-green leaf cells of the maize mutant iojap. Since iojap plastids lack ribosomes, it is concluded that the site of synthesis of pyruvate orthophosphate dikinase in maize leaf cells is on ribosomes in the cytoplasm. PMID- 16663379 TI - Plant Desiccation and Protein Synthesis. IV. RNA Synthesis, Stability, and Recruitment of RNA into Protein Synthesis during Desiccation and Rehydration of the Desiccation-Tolerant Moss, Tortula ruralis. AB - Upon rehydration of desiccated Tortula ruralis, RNA synthesis is immediately resumed; this resumption is quicker in moss recovering from slow drying than from rapid drying. Newly synthesized RNA enters the protein synthetic complex almost immediately upon rehydration, reaching control steady state levels within 2 hours after slow drying and 6 hours after rapid drying. RNA synthesized in the 1st hour following the readdition of water enters into polysomes much earlier after slow drying than after rapid drying. The RNA components of the protein synthetic complex are stable to desiccation at either slow or rapid speeds, although more so following the former drying regime. Immediately upon rehydration, these conserved RNA are readily utilized for protein synthesis, and continue to be so at least 4 hours thereafter. Hence, the speed of desiccation affects the rate at which RNA is synthesized upon subsequent rehydration, as well as the mode of utilization of that RNA. PMID- 16663380 TI - Characterization of amino Acid efflux from isolated soybean cells. AB - Cells from reproductive soybean (Glycine max [L.] Merr.) plants were isolated using a mechanical-enzymic technique that produced a high yield of uniform, physiologically active cells. Cells were incubated in a pH 6.0 buffered solution and subjected to various treatments in order to determine the nature of net amino acid efflux. Total net amino acid (ninhydrinreactive substances) efflux was not affected by the following conditions: (a) darkness, (b) aeration, (c) K(+) concentrations of 0.1, 1.0, 10, or 100 millimolar and (d) pH 4, 5, 6, 7, or 8. The Q(10) for net amino acid efflux between 10 degrees C and 30 degrees C was 1.6. Thus, it seems that net amino acid efflux requires neither current photosynthetic energy nor a pH/ion concentration gradient. Amino acid analyses of the intra-and extracellular fractions over time showed that each amino acid was exported linearly for at least 210 minutes, but that export rate was not necessarily related to internal amino acid pools. Amino acids that were exported fastest were alanine, lysine, leucine, and glycine. Addition of the inhibitor p chloromercuriphenyl sulfonic acid, 3(3,4-dichlorophenyl)-1,1-dimethylurea, or carbonylcyanide p-trifluoromethoxyphenylhydrazone increased the rate of total amino acid efflux but had specific effects on the efflux of certain amino acids. For example, p-chloromercuriphenyl sulfonic acid greatly enhanced efflux of gamma aminobutyric acid, which is not normally exported rapidly even though a high concentration normally exists within cells. The data suggest that net amino acid efflux is a selective diffusional process. Because net efflux is the result of simultaneous efflux and influx, we propose that efflux is a facilitated diffusion process whereas influx involves energy-dependent carrier proteins. PMID- 16663381 TI - Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes. AB - Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO(2), 6% O(2), and 88% N(2) for 14 weeks at 12 degrees C, exhibited a temporal separation of certain biochemical events associated with ripening.The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22 degrees C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere. PMID- 16663382 TI - Effects of fusaric Acid on tomato root hair membrane potentials and ATP levels. AB - Using standard microelectrode techniques, we measured the effects of fusaric acid (FA) on the membrane potential of tomato (Lycopersicon esculentum Mill. cv New Yorker 870) incipient root hair cells. At pH 5.3, FA caused a hyerpolarization, the magnitude of which increased with FA concentrations from 0.05 to 0.50 millimolar. A depolarization followed, the rate and magnitude of which increased with the concentration of FA and exposure to FA. Partial repolarizations occurred after exposures to 1.0 millimolar FA for less than 8 to 10 minutes, after longer exposures to lower FA concentrations, or after longer exposures to 1.0 millimolar FA in a less concentrated nutrient solution. The amount of ATP in tomato root tips decreased by about 85% after incubation for 80 min in 1.0 millimolar FA.At pH 7.2 and 8.2, the depolarization caused by an 8-minute exposure to 1.0 millimolar FA was immediate and much more rapid than at pH 5.2 and 6.3, but its magnitude was not as great. At pH 6.3, 7.2, and 8.2, the depolarization was at least partially reversible. The data are consistent with FA having at least three effects that elicited changes in tomato root cell electrical potential differences between the cell's interior and the external solution. PMID- 16663383 TI - [C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley. AB - Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [(14)C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-(14)C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains. PMID- 16663384 TI - Stomatal Behavior and CO(2) Exchange Characteristics in Amphistomatous Leaves. AB - The possibility that differences in stomatal conductance between upper and lower surfaces of amphistomatous leaves are adaptations to differences in CO(2) exchange characteristics for the two surfaces was investigated. The ratio of upper to lower stomatal conductance was found to change little in response to light and humidity for well-watered sunflower (Helianthus annuus L.) plants. Stressing the plants (psi = -17 bars) and rewatering 1 day before gas exchange measurements reduced upper conductance more severely than lower in both indoor- and outdoor-grown plants, and caused small changes in conductance ratio with light and humidity. A similar pattern was found using outdoor grown sunflower and cocklebur (Xanthium strumarium L.) plants. Calculated intercellular CO(2) concentrations for upper and lower surfaces were always close to identical for a particular set of environmental conditions for both sunflower and cocklebur, indicating that no differences in CO(2) exchange characteristics exist between the two surfaces. By artificially creating a CO(2) gradient across the leaf, the resistance to CO(2) diffusion through the mesophyll was estimated and found to be so low that despite possible nonhomogeneity of the mesophyll, differences in CO(2) exchange characteristics for the two surfaces are unlikely. It is concluded that differences in conductance between upper and lower stomates are not adaptations to differences in CO(2) exchange characteristics. PMID- 16663385 TI - Host-Pathogen Interactions : XXV. Endopolygalacturonic Acid Lyase from Erwinia carotovora Elicits Phytoalexin Accumulation by Releasing Plant Cell Wall Fragments. AB - Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat labile elicitors were highly purified by cation-exchange chromatography on a CM Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two alpha-1,4-endopolygalacturonic acid lyases (EC 4.2.2.2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 x 10(-9) molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls. PMID- 16663386 TI - Perturbation of Chara Plasmalemma Transport Function by 2[4(2',4' Dichlorophenoxy)phenoxy]propionic Acid. AB - Electrophysiological measurements on internodal cells of Chara corallina Klein ex Willd., em. R.D.W. revealed that in the presence of (2-[4-(2',4' dichlorophenoxy)phenoxy]propionic acid) (diclofop) the membrane potential was very sensitive to the pH of the bathing medium. At pH 5.7, 100 micromolar diclofop caused a slow reduction in the electrogenic component of the membrane potential to the value of -123 +/- 5 millivolts. Membrane resistance initially decreased, recovered transiently, then stabilized at approximately 65% of the control value. At pH 7.0, the potential appeared to plateau around -200 millivolts before rapidly declining to -140 +/- 4 millivolts; removal of diclofop resulted in recovery of the electrogenic component. Diclofop reduced cytoplasmic ATP levels by 96.4% and 36.6% at pH 5.7 and 7.0, respectively. At pH 8.2, diclofop did not change the ATP concentration significantly, but induced a hyperpolarization of the membrane potential to near -250 millivolts, and also reduced or inhibited the dark-induced hyperpolarization; the light-induced depolarization was reduced to a lesser extent. DCMU applied in the light elicited the same response at the plasmalemma as placing cells in the dark. When K(+) channels were opened and cells depolarized with 10 millimolar K(+), diclofop induced a further depolarization of approximately 30 millivolts. Cells decoupled with HPO(4) (-2) were still sensitive to diclofop. Currents associated with OH(-) efflux and HCO(3) (-) influx, as measured with a vibrating probe technique, became spatially destabilized and reduced in magnitude in the presence of diclofop. After 60 minutes, most of the cell surface was engaged in a low level of OH(-) efflux activity. The results indicate that diclofop may be a proton ionophore at pH 7.0 and 5.7. At pH 8.2, diclofop may inhibit the operation of the H(+)-ATPase and OH(-) efflux systems associated with HCO(3) (-) transport by perturbing the control processes that integrate the two, without a reduction in ATP concentration. PMID- 16663387 TI - Photochemical Apparatus Organization in Anacystis nidulans (Cyanophyceae) : Effect of CO(2) Concentration during Cell Growth. AB - Anacystis nidulans cells grown under high (3%) CO(2) partial pressure have greater phycocyanin to chlorophyll ratio (Phc/Chl) relative to cells grown under low (0.2%) CO(2) tension (Eley (1971) Plant Cell Physiol 12: 311-316). Absorbance difference spectrophotometry of A. nidulans thylakoid membranes in the ultraviolet (DeltaA(320)) and red (DeltaA(700)) regions of the spectrum reveal photosystem II/photosystem I (PSII/PSI) reaction center ratio (RCII/RCI) changes that parallel those of Phc/Chl. For cells growing under 3% CO(2), the Phc/Chl ratio was 0.48 and RCII/RCI = 0.40. At 0.2% CO(2), Phc/Chl = 0.38 and RCII/RCI = 0.24. Excitation of intact cells at 620 nm sensitized RCII at a rate approximately 20 times faster than that of RCI, suggesting that Phc excitation is delivered to RCII only. In the presence of DCMU, excitation at 620 nm induced single exponential RCII photoconversion kinetics, suggesting a one-to-one structural-functional correspondance between phycobilisome and PSII complex in the thylakoid membrane. Therefore, phycobilisomes may serve as microscopic markers for the presence of PSII in the photosynthetic membrane of A. nidulans. Neither the size of individual phycobilisomes nor the Chl light-harvesting antenna of PSI changed under the two different CO(2) tensions during cell growth. Our results are compatible with the hypothesis that, at low CO(2) concentrations, the greater relative amounts of PSI present may facilitate greater rates of ATP synthesis via cyclic electron flow. The additional ATP may be required for the active uptake of CO(2) under such conditions. PMID- 16663388 TI - Photosynthetic and Stomatal Responses of the Grey Mangrove, Avicennia marina, to Transient Salinity Conditions. AB - Measurements of gas exchange characteristics were made on intact, attached leaves of hydroponically grown seedlings of Avicennia marina (Forstk.) Vierh. var australasica (Walp.) Moldenke as the NaCl concentration of the culture solution was varied by step changes of 50 millimolar NaCl every 2nd day from 50 to 500 to 50 millimolar NaCl. The CO(2) assimilation rate, stomatal conductance, intercellular CO(2) concentration, and evaporation rate decreased at salinities above 250 millimolar NaCl and recovered substantially upon return to the original salinity.The assimilation rate was measured as a function of the intercellular CO(2) concentration [A(c(i)) curve]. The lower linear portion of this curve was insensitive to variation in salinity, whereas the upper nonlinear portion declined with increasing salinity, indicating a reduction in the capacity for CO(2) assimilation which recovered upon return to the original salinity. Stomatal conductance changed such that the intercellular CO(2) concentration measured under normal atmospheric conditions occurred in the transition between the lower, linear and upper nonlinear portions of the A(c(i)) curve. Thus, stomatal conductance and photosynthetic capacity together co-limited the assimilation rate. The changes in gas exchange characteristics were such that water loss was minimal relative to carbon gain. PMID- 16663389 TI - Effect of salinity on nodule formation by soybean. AB - A split-root growth system was employed to evaluate the effect of NaCl on nodule formation by soybean (Glycine max L. Merr. cv Davis). By applying the salinity stress and rhizobial inoculum to only one-half the root system, the effects of salinity on shoot growth were eliminated in the nodulation process. Rhizobium colonization of inoculated root surfaces was not affected by the salt treatments (0.0, 26.6, 53.2, and 79.9 millimolar NaCl). While shoot dry weight remained unaffected by the treatments, total shoot N declined from 1.26 grams N per pot at 0.0 millimolar NaCl to 0.44 grams N per pot at 79.9 millimolar NaCl. The concentration of N in the shoot decreased from 3.75% N (0.0 millimolar NaCl) to 1.26% N at 79.9 millimolar NaCl. The decrease in shoot N was attributed to a sharp reduction in nodule number and dry weight. Nodule number and weight were reduced by approximately 50% at 26.6 millimolar NaCl, and by more than 90% at 53.2 and 79.9 millimolar NaCl. Nodule development, as evidenced by the average weight of a nodule, was not as greatly affected by salt as was nodule number. Total nitrogenase activity (C(2)H(2) reduction) decreased proportionally in relation to nodule number and dry weight. Specific nitrogenase activity, however, was less affected by salinity and was not depressed significantly until 79.9 millimolar NaCl. In a second experiment, isolates of Rhizobium japonicum from nodules formed at 79.9 millimolar NaCl did not increase nodulation of roots under salt stress compared to nodule isolates from normal media (0.0 millimolar NaCl). Salt was applied (53.2 millimolar NaCl) to half root systems at 0, 4, 12, and 96 hours from inoculation in a third experiment. By delaying the application of salt for 12 hours, an increase in nodule number, nodule weight, and shoot N was observed. Nodule formation in the 12- and 96-hour treatments was, however, lower than the control. The early steps in nodule initiation are, therefore, extremely sensitive to even low concentrations of NaCl. The sensitivity is not related to rhizobial survival and is probably due to the salt sensitivity of root infection sites. PMID- 16663390 TI - Temperature-Induced Change in the Water Relations of Abies amabilis (Dougl.) Forbes. AB - Water conductance through Abies amabilis seedlings was measured while the roots were exposed to temperatures from 15 to 0.25 degrees C. Before conductance was measured, the seedlings were preconditioned for 3 months at either a high temperature (23 degrees C) or a low temperature (3 degrees C). For both groups of seedlings, conductance decreased as root temperature decreased. Conductance was lowest at 0.25 degrees C. In addition, preconditioning at 3 degrees C for 3 months significantly lowered conductance to water at all root temperatures. Under the same environmental conditions, seedlings preconditioned at 3 degrees C had less than 25% of the transpirational water loss of seedlings preconditioned at high temperature. A decrease in leaf osmotic potential also resulted from low temperature preconditioning. In trees growing in the subalpine forest, which is the natural habitat of Abies amabilis, both decreased leaf conductance to water vapor and lower osmotic potentials were evident in winter. Since in winter the temperature of the soil in the subalpine zone remains less than 1 degrees C for many months, lowered leaf conductance and decreased osmotic potentials appear to be mechanisms which aid in preventing desiccation damage. PMID- 16663391 TI - Amelioration of chilling-induced water stress by abscisic Acid-induced changes in root hydraulic conductance. AB - Pretreatment of soybean (Glycine max L. var Ransom) root systems with abscisic acid (ABA) ameliorates the deleterious effect of low temperatures on root hydraulic conductance. ABA treatment of root systems subsequently chilled to 10 degrees C with shoots at 25 degrees C resulted in higher leaf water potentials and lower stomatal resistances. If the root systems are left at 25 degrees C, ABA causes stomatal closure. Membrane alterations are suggested as a mechanism for the ABA action in plant response to chilling stress. PMID- 16663392 TI - Host recognition in the Rhizobium-soybean symbiosis: detection of a protein factor in soybean root exudate which is involved in the nodulation process. AB - The mechanism of host-symbiont recognition in the soybean-Rhizobium symbiosis was investigated utilizing mutants of R. japonicum defective in nodulation. Soybeans were grown in clear plastic growth pouches allowing the identification of the area on the root most susceptible to Rhizobium nodulation; the area between the root tip (RT) and smallest emergent root hair (SERH). The location of nodules in relation to this developing zone is an indication of the rate of nodule initiation. Nodules were scored as to the distance from the RT mark made at the time of inoculation. Seventy-eight per cent of the plants nodulate above the RT mark when inoculated with the wild type R. japonicum strain 3I1b110 with the average distance of the uppermost nodule being approximately 2 millimeters above the RT mark. These data indicate that the wild type strain initiates nodulation rapidly within the RT-SERH zone following inoculation. However, inoculation with the slow-to-nodulate mutant strain HS111 resulted in 100% of the plants nodulating only below the RT mark with the average distance of the uppermost nodule being approximately 56 millimeters below the RT mark. Thus, mutant strain HS111 is defective in the ability to rapidly initiate infection leading to nodulation within the RT-SERH zone. The location of the nodules suggest that stain HS111 must ;adapt' to the root environment before nodulation can occur. To test this, strain HS111 was incubated in soybean root exudate prior to inoculation. In this case, 68% of the plants nodulated above the RT mark with the average distance of the uppermost nodule being approximately 1 millimeter below the RT mark. Experiments indicated that the change in nodule initiation by strain HS111 brought about by incubation in soybean root exudate was due to a phenotypic, rather than a genotypic change. The half-time of root exudate incubation for strain HS111 necessary for optimal nodulation enhancement was less than 6 hours. Heat sensitivity and trypsin sensitivity of the nodulation enhancement factor(s) in soybean root exudate indicate a protein was involved in the reversal of the delay in nodulation by mutant strain HS111. PMID- 16663393 TI - Influence of Iron Deprivation on the Membrane Composition of Anacystis nidulans. AB - Cultures of the cyanobacterium Anacystis nidulans were grown under iron-deficient conditions and then restored by the addition of iron. Membrane proteins from iron deficient and iron-restored cells were analyzed by lithium dodecyl sulfate polyacrylamide gradient gel electrophoresis. The incorporation of [(35)S]sulfate into membrane proteins and lactoperoxidase-catalyzed (125)I iodination were used to monitor the rates of polypeptide biosynthesis and surface exposure of membrane proteins, respectively. These polypeptide profiles revealed major differences in the membrane composition of iron-deficient and normal cells. Iron deficiency caused a decrease in the amount of certain important membrane proteins, reflecting a decreased rate of biosynthesis of these peptides. Several photosystem II peptides also showed an increase in surface exposure after iron stress. In addition, iron deficiency led to the synthesis of proteins at 34 and 52 kilodaltons which were not present in normal cells. When iron was restored to a deficient culture, a metabolic sequence was initiated within the first 12 h after the addition of iron which led to phenotypically normal cells. Pulse labeling with [(35)S]sulfate during this period demonstrated that iron addition initiates a coordinated pattern of synthesis that leads to the assembly of normal membranes. PMID- 16663394 TI - Psychrometric Field Measurement of Water Potential Changes following Leaf Excision. AB - In situ measurement of sudden leaf water potential changes has not been performed under field conditions. A laboratory investigation involving the measurement of leaf water potential prior to and 2 to 200 minutes after excision of citrus leaves (Citrus jambhiri) showed good linear correlation (r = 0.99) between in situ leaf psychrometer and Scholander pressure chamber measurements. Following this, a field investigation was conducted which involved psychrometric measurement prior to petiole excision and 1 minute after excision. Simultaneous pressure chamber measurements were performed on neighboring leaves prior to the time of excision and then on the psychrometer leaf about 2 minutes after excision. These data indicate that within the first 2 minutes after excision, psychrometer and pressure chamber measurements were linearly correlated (r = 0.97). Under high evaporative demand conditions, the rate of water potential decrease was between 250 and 700 kilopascals in the first minute after excision. These results show that the thermocouple psychrometer can be used as a dynamic and nondestructive field technique for monitoring leaf water potential. PMID- 16663395 TI - Enhancement by ethylene of cellulysin-induced ethylene production by tobacco leaf discs. AB - Cellulysin-induced ethylene production in tobacco (Nicotiana tabacum L.) leaf discs was enhanced several-fold by prior exposure of the leaf tissue to ethylene. This enhancement in the response of the tissue to Cellulysin increased rapidly during 4 and 8 hours of pretreatment with ethylene and resulted from greater conversion of methionine to ethylene. On treatment with Cellulysin, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) in leaf discs not pretreated with ethylene markedly increased while that of the ethylene-pretreated tissue was only slightly higher than in the tissue incubated in the absence of Cellulysin. Ethylene-treated tissue, however, converted ACC to ethylene at a faster rate than air controls. These data indicate that ethylene stimulates Cellulysin-induced ethylene production by stimulating the conversion of ACC to ethylene. Data are also presented on a possible relation of this phenomenon to ethylene produced by the tobacco leaf upon interaction with its pathogen, Alternaria alternata. PMID- 16663396 TI - Microsomal Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella. AB - A detailed analysis of the low temperature-induced alterations of Dunaliella salina (UTEX 1644) microsomal membrane lipids was carried out. Microsomal membranes were isolated from cells grown at 30 degrees C, from cells shifted to 12 degrees C for 12 hours, and from cells acclimated to 12 degrees C. Fatty acid analyses of the major lipid classes demonstrated significant changes in the fatty acid composition of phosphatidylcholinemine (PE) and phosphatidylglycerol (PG) but not phosphatidylcholine (PC) during the initial 12 hours at low temperature. These changes did not entail enhanced desaturation of linoleic acid. Subsequent to 12 hours, the proportions of linolenic acid increased in all phospholipids.Molecular species analyses of the phospholipids demonstrated that the most immediate changes following a shift to low temperature were limited to several molecular species of PE and PG. The changes observed in PE included a decrease in C(30) species and concomitant increases in C(34) and C(36) species. Compositional changes associated with PG entailed the emergence of a new molecular species (18:1/18:1) not found at 30 degrees C. The retailoring of molecular species resulted in an increase in the number of species having two unsaturated acyl chains and did not reflect a simple enhancement of desaturase activity as suggested by the fatty acid analysis. We conclude that the initial alterations in response to low temperature stress involve discrete changes in certain molecular species. These and further alterations of molecular species following acclimation to low temperature would appear to augment increases in acyl chain desaturation as a means of modifying membrane properties in response to low temperature stress. PMID- 16663397 TI - Chloroplast Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella. AB - The alterations in chloroplast phospholipid acyl chain composition and phospholipid molecular species composition of Dunaliella salina (UTEX 1644) were monitored during acclimation to low temperature. Chlorophyll fluorescence yield, an indicator of chloroplast membrane stability, was used as a physical means of following the acclimation process.Minor alterations in phospholipid acyl chain composition were evident within 36 hours of shifting the cells from 30 to 12 degrees C. Between 36 and 60 hours, pronounced changes in the acyl chain composition of phosphatidylglycerol (PG) were observed. Changes in the acyl chain composition of phosphatidylcholine (PC) did not occur until sometime after 60 hours.Alterations in the phospholipid molecular species during acclimation were also examined. The pattern of change observed in PC molecular species, namely a decrease in species having one saturated chain (16:0) paired with a C(18) acyl chain and a concomitant increase in species having two unsaturated C(18) acyl chains, suggests that molecular species changes augment fatty acid compositional changes as a mean of adapting to low temperature. The molecular species of PG were found to change abruptly between 36 and 60 hours following a shift to low temperature. During this time, a dramatic alteration in the threshold temperature of thermal denaturation of the photosynthetic apparatus, as measured by chlorophyll fluorescence, also occurred. Lipid compositional changes other than those associated with PG were negligible during this time. This strongly suggests that a correlation exists between the molecular species composition of PG and the thermal stability of the photosynthetic membrane. PMID- 16663398 TI - Occurrence of Isoenzymes of Glutamine Synthetase in the Alga Chlorella kessleri. AB - Two forms (GS(1) and GS(2)) of glutamine synthetase have been isolated, separated by ion exchange chromatography, and partly characterized from cells of the green alga Chlorella kessleri. Both forms are present in cells grown autotrophically or heterotrophically on various nitrogen sources, but under all nutritional conditions GS(1) was found to be the major isoenzyme present (60-80%). The activity of both isoenzymes was greatest in cells grown under nitrogen-limiting conditions. Both isoenzymes have molecular weights in the range 340 to 350,000 daltons. GS(1) was found to have a greater thermostability than GS(2): GS(1) was stable at 30 degrees C while GS(2) lost 95% of its activity in 30 minutes. GS(1) was much less sensitive to thiol reactive reagents than GS(2). PMID- 16663399 TI - Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism. AB - The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0(2) uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited big up tri, open(1)-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.The effect of T4C on proline metabolism in detached green barley leaves was investigated. T4C inhibited proline oxidation in turgid leaves, increasing the proline content of these leaves slightly. In wilted leaves (that are synthesizing proline rapidly), T4C inhibited proline synthesis, which resulted in a decrease in the proline content of the leaves. big up tri, open(1) pyrrolidine-5-carboxylic acid reductase (the last enzyme in proline synthesis) was not inhibited by T4C, and thus T4C's influence is prior to that step of the synthetic pathway. T4C had no influence on the incorporation of proline into protein. PMID- 16663400 TI - Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts. AB - Recent experiments show that exogenous NADH increases the O(2) consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V(max) of the mechanism I (saturable) component of K(+) uptake, while K(m) was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K(+) influx and H(+) efflux, while NADH-stimulated K(+) uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N'-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H(+) and K(+) fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K(+) influx and a more than 3-fold increase of H(+) efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K(+) uptake. PMID- 16663401 TI - Prevention of Action of Far-Red-Absorbing Phytochrome in Rumex crispus L. Seeds by Ethanol. AB - Phytochrome-enhanced germination of curled dock (Rumex crispus L.) seeds is further stimulated by pretreatments in solutions of 0.5 to 2 molar methanol and 0.03 to >/= 0.3 molar 2-propanol during a 2-day 20 degrees C imbibition. Similar pretreatments in 0.1 molar ethanol, acetaldehyde, and n-propanol inhibit phytochrome-enhanced germination. If exposure to ethanol is delayed until 16 hours after a red irradiation, seeds escape the ethanol inhibition indicating a mechanism other than toxicity. The rate of escape from ethanol inhibition roughly parallels the escape from phytochrome control in seeds held in water only, indicating possible ethanol effects on phytochrome. It was found that ethanol pretreatment prevents the far-red absorbing form of phytochrome (Pfr) from acting but does not accelerate dark decay or prevent transformation. Ethanol inhibition may be prevented if ethanol pretreatment is at 10 degrees C instead of 20 degrees C, or may be overcome by transferring ethanol-pretreated seeds to 10 degrees C in water. Similarly, ethanol inhibition can be overcome by a 2-hour 40 degrees C temperature shift concluding the pretreatment. It is proposed that the ethanol causes perturbations at a membrane which prevent Pfr from acting. PMID- 16663402 TI - Effect of nitrogen source on ureides in soybean. AB - In field-grown soybeans (Glycine max L. Merr. cv Harosoy), the percentage of N in the xylem as ureides increased with increasing N(2) fixation. During a 9-week collection period, the ureide content varied from 9.0 to 69.2% of the xylary N. Between 9 and 11 weeks (early pod fill), there was a good correlation (r = 0.93) between C(2)H(2) reduction and the per cent N in xylem as ureides. The per cent N as ureides, however, does not always indicate the reliance of the plant on symbiotic N(2) fixation. This ureide content also depended on the level of NO(3) (-) available to the roots. Non-nodulated soybeans given from 0 to 200 kilogram N per hectare produced xylem sap which averaged from 31.8% to 9.0% N, respectively, in the xylem as ureides over the 9-week period.Feeding of (15)N(2), (15)NH(4), or (15)NO(3) to greenhouse-grown soybeans indicated substantial differences in the initial distribution of N by the xylem stream, but the ultimate distribution of N between plant parts and grain did not vary with available N or percentage of xylary N as ureides. Amino acids, not ureides, were the major source of N in the phloem. The soybeans maintained a similar composition in phloem irrespective of the xylem sap constituents, with N derived from N(2), NH(4), or NO(3) being equally accessible to the phloem stream. PMID- 16663403 TI - Influence of elevated carbon dioxide on water relations of soybeans. AB - Soybean (Glycine max L. Merrill cv ;Bragg') plants were grown in pots at six elevated atmospheric CO(2) concentrations and two watering regimes in open top field chambers to characterize leaf xylem potential, stomatal resistance and conductance, transpiration, and carbohydrate contents of the leaves in response to CO(2) enrichment and water stress conditions. Groups of plants at each CO(2) concentration were subjected to water stress by withholding irrigation for 4 days during the pod-filling stage.Under well watered conditions, the stomatal conductance of the plants decreased with increasing CO(2) concentration. Therefore, although leaf area per plant was greater in the high CO(2) treatments, the rate of water loss per plant decreased with CO(2) enrichment. After 4 days without irrigation, plants in lower CO(2) treatments showed greater leaf tissue damage, lower leaf water potential, and higher stomatal resistance than high CO(2) plants. Stomatal closure occurred at lower leaf water potentials for the low CO(2) grown plants than the high CO(2) grown plants. Significantly greater starch concentrations were found in leaves of high CO(2) plants, and the reductions in leaf starch and increases in soluble sugars due to water stress were greater for low CO(2) plants. The results showed that even though greater growth was observed at high atmospheric CO(2) concentrations, lower rates of water use delayed and, thereby, prevented the onset of severe water stress under conditions of low moisture availability. PMID- 16663404 TI - Phytohormones, Rhizobium mutants, and nodulation in legumes : v. Cytokinin metabolism in effective and ineffective pea root nodules. AB - [(3)H]Zeatin riboside was supplied to intact pea (Pisum sativum) plants either onto the leaves or onto the root nodules. When applied directly to nodules, approximately 70% of recovered radioactivity remained in the nodules, approximately 15% was detected in the root system, and 15% was in the shoot. However, when supplied to the leaves, little (3)H was transported, with approximately 0.05% of recovered radioactivity being found in the root system and nodules. On a fresh weight basis, nodules accumulated more (3)H than the parent root. In both types of studies, metabolites with an intact zeatin moiety were detected in root nodules.In all experiments, two-dimensional thin layer chromatography revealed that little (3)H remained as zeatin riboside in root or nodule tissue at the end of the labeling period. Nodules metabolized [(3)H]zeatin riboside to the following cytokinins/cytokinin metabolites: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, lupinic acid, nucleotides of adenine and zeatin, and the dihydro derivatives of many of these compounds.Although a few small differences were observed, there were no major differences between root and nodule tissue in their metabolism of [(3)H] zeatin riboside. Furthermore, any differences between effective and ineffective nodules were generally minor. PMID- 16663405 TI - Reinforced Polyproline II Conformation in a Hydroxyproline-Rich Cell Wall Glycoprotein from Carrot Root. AB - The salt-extractable hydroxyproline-rich cell wall glycoprotein from carrot (Daucus carota L.) roots is composed of 35% (w/w) protein, 3% (w/w) galactose, and 62% (w/w) arabinose. The arabinose is attached to hydroxyproline as tetra- and trisaccharides. The circular dichroism of the glycoprotein shows that it is completely in the polyproline II conformation. After deglycosylation of the glycoprotein, the polyproline II conformation of the peptide backbone was lost. This indicates that the carbohydrate reinforces the polyproline II conformation. PMID- 16663406 TI - Osmoregulation or turgor regulation in chara? AB - Chara corallina Klein ex Willd. wm. R.D.W. (= C. australis R. Br.), a fresh water alga, maintains a constant internal osmotic pressure when external osmotic pressure is increased. This results in a decrease in turgor pressure. Chara osmoregulates effectively in the presence of high CaCl(2) and raffinose, but is less efficient in response to increased NaCl. Decreasing external pH from 7 to 5 results in a decrease in turgor, but increasing it to values as high as 9 has no effect. Increasing the daily amount of light from 0.5 to 24 hours has no effect on turgor. PMID- 16663407 TI - Magnesium deficiency results in increased suberization in endodermis and hypodermis of corn roots. AB - The composition of the aliphatic components of suberin in the stele and cortex of young corn (Zea mays L.) roots was determined by combined gas-liquid chromatography/mass spectrometry of the LiAlD(4) depolymerization products. omega Hydroxy acids were shown to be the major class of the aliphatic components of both the hypodermal (35%) and endodermal (28%) polymeric materials with the dominant chain length being C(24) in the former and C(16) in the latter. Nitrobenzene oxidation of the roots generated p-hydroxybenzaldehyde and vanillin with much less syringaldehyde. Electron microscopic examination of the hypodermal and endodermal cell walls from roots of corn plants grown in a Mg(2+) -deficient (0.03 millimolar) nutrient solution showed that these walls were more heavily suberized than the analogous walls of roots from plants grown in normal (2 millimolar) Mg(2+) levels. Analysis of the LiAlD(4) depolymerization products of the suberin polymers from these roots showed that the roots grown in low Mg(2+) had 3.5 times as much aliphatic suberin monomers on a weight basis as the roots from plants grown in nutrient with normal Mg(2+) levels. Roots from plants grown in Mg(2+) -deficient nutrient solution released 3.8 times the amount of aromatic aldehydes upon nitrobenzene oxidation as that released from normal roots. As the degree of Mg(2+) deficiency of the nutrient solution was increased, there was an increase in the aliphatic and aromatic components characteristic of suberin. Thus, both ultrastructural and chemical evidence strongly suggested that Mg(2+) deficiency resulted in increased suberization of the cell walls of both hypodermis and endodermis of Zea mays roots. The roots from Mg(2+) -deficient plants also had a higher amount of peroxidase activity when compared to control roots. PMID- 16663408 TI - Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons. AB - As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5 fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons. PMID- 16663409 TI - Calcium Inhibits Phase Shifting of the Circadian Conidiation Rhythm of Neurospora crassa by the Calcium Ionophore A23187. AB - Effects of the calcium ionophore, A23187, and antimycin A on the circadian conidiation rhythm of Neurospora crassa were examined. A23187 at a concentration of 1 mum in medium not containing divalent cations delayed the phase by 10 hours at CT 10 and advanced it by 5 hours at CT 14 (CT 12 corresponds to the time that discs are transferred from light to dark). This phase shifting was completely inhibited by addition of 0.1 millimolar CaCl(2) but not by MgCl(2) at any concentrations examined.Antimycin A inhibited respiration by 90% at a concentration of 0.2 micrograms per milliliter and lowered the ATP content by 85%. Antimycin A alone caused small phase advances but in combination with A23187 resulted in a large phase delay at CT 10. This phase shifting was not reversed by addition of CaCl(2) lower than 10 millimolar. PMID- 16663410 TI - Is there a role for the apex in shoot geotropism? AB - Experiments with horizontal etiolated sunflower (Helianthus annuus L.) seedlings supported centrally such that both apical and basal ends are free to react to geostimulus, revealed that the apical end commences curvature 1 to 2 hours earlier than the basal end. The later curvature in the basal region is a consequence of the absence of growth in the initial period rather than merely slower growth. A comparison of zonal growth rates in a vertical and a horizontal seedling confirmed that geostimulus induces a renewal of growth in a region where growth had ceased. Removing the apical half of the hypocotyl showed that the curvature resulting from this growth initiation in the basal region is dependent on attachment to the apical region. Evidence that this dependence is unlikely to be due to energy deficiency is adduced. The prior response of the apical end to geostimulus and the apically dependent later initiation of new growth in the basal region are compatible with the delay inherent in message transport from apex to base and are considered as evidence for apical involvement in the totality of the seedling's georesponse. PMID- 16663411 TI - Azido auxins : photolysis in solution and covalent binding to soybean. AB - The potential of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic (4 N(3)IAA, 5-N(3)IAA, and 6-N(3)IAA), as photoaffinity labeling agents for the detection and isolation of auxin receptors was assessed by irradiating these compounds at 365 nm on TLC plates, in solution, and in contact with soybean (Glycine max L. Merr. var. Wayne) hypocotyl. Photolysis on TLC plates produces immobile spots, indicating extremely polar or covalent binding of the photoproducts to the plates. On irradiation in buffer or in buffer containing sucrose, all three compounds decompose at rates that are first order in N(3)IAA to give fluorescent solutions. Photolysis through a Pyrex filter is slower than that through quartz, but the filter prevents tissue damage and allows a given dose of irradiation to photolyze all three N(3)IAAs to the same extent. The effects of photolysis of these compounds in vivo were evaluated with a straight growth assay using etiolated soybean hypocotyl segments. According to this assay, the photoproducts of the N(3)IAAs possess little auxin activity. Irradiation of soybean hypocotyl tissue after 1-hour exposure to 4-N(3)IAA in the dark causes the tissue to grow during 12 hours as much as tissue that is continuously exposed to 4-N(3)IAA in the dark for this period, suggesting that, on photolysis, this auxin analog binds irreversibly to an auxinsensitive site. Although the fluorescence intensity of the photolyzed N(3)IAAs is weak enough to require another method of detecting the bound analog under physiological conditions, the evidence for covalent binding of the N(3)IAAs on photolysis implies that these compounds will be satisfactory photoaffinity labeling agents. PMID- 16663412 TI - Azido auxins: quantitative binding data in maize. AB - The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N(3)IAA), and of the photoproducts of 5-N(3)IAA to the naphthalene-1-acetic acid (NAA) binding sites of Zea mays L. WF9 x BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4- and 5-N(3)IAA bind to these sites with affinities similar to that of IAA, while 6-N(3)IAA and the photoproducts of 5-N(3)IAA bind less tightly. This binding is fully reversible in the dark. Binding of 5-N(3)IAA becomes covalent and irreversible upon UV irradiation, as evidenced by a 30% loss in NAA binding at sites pretreated with 5-N(3)IAA and UV irradiation, then washed extensively. IAA or NAA, included with this 5-N(3)IAA pretreatment, can protect the sites from blockage, whereas benzoic acid and tryptophan are unable to protect the site, indicating that 5-N(3)IAA specifically labels the auxin sites. PMID- 16663413 TI - IAA-Induced Growth Responses of Decapitated Corn Seedlings: Indications of Two Apparent Adaptations with a Possible Role in Gravitropism. AB - The vertical growth responses of corn seedlings (Zea mays L. Mo17 x B73) were determined over an 8-hour period. When seedlings were decapitated 3 millimeters from the coleoptile's tip and supplied with indole-3-acetic acid (IAA) in 1.5% agar blocks, the response was dependent both on time and IAA concentration. The dose-response curves changed in shape and magnitude depending on the total time of IAA application. High concentrations (>3.2 x 10(-6) molar) initially produced high relative growth rates that decreased back to the intact rate (0.03 millimeter per hour per millimeter) after 3 hours. Low concentrations (<1.0 x 10( 6) molar), or agar blocks without IAA, resulted in a rapid decrease from the intact rate to a level that stabilized at 0.01 millimeter per hour per millimeter until the growth rate began to recover after 3 to 4 hours. Intermediate concentrations produced responses similar to that of the intact organ, though some features of these responses were unique.The coleoptile curvature in response to gravity depended upon whether the coleoptiles were intact, decapitated, or decapitated and supplied with IAA. Coleoptiles decapitated and not supplied wth IAA showed little or no curvature for 3 hours after decapitation. By this time an adaptation, evoked by the low IAA level, had developed and the coleoptiles began to curve steadily. When 1.0 or 3.2 x 10(-6) molar IAA was supplied, curvature was initiated within the first 30 minutes and reached a maximum rate before decreasing and stopping after 3 to 4 hours. The sequence of events in response to these concentrations was similar to the intact sequence but the curvature rate was reduced to one-third to one-half. A model for the autotropic response involving an auxin concentration-dependent, growth-modulating mechanism capable of two modes of adaptation is described. PMID- 16663414 TI - Interrelationship of Gene Expression, Polysome Prevalence, and Respiration during Ripening of Ethylene and/or Cyanide-Treated Avocado Fruit. AB - Upon initiation of ripening in avocado fruit (Persea americana Mill. cv Hass) with 10 microliters/liter ethylene, polysome prevalence and associated poly(A)(+) mRNA increase approximately 3-fold early in the respiratory climacteric and drop off to preclimacteric levels at the peak of the respiratory climacteric. The increase in poly(A)(+) mRNA on polysomes early in the respiratory climacteric constitutes a generic increase in constitutive mRNAs. New gene expression associated with ripening is minimal but evident after 10 hours of ethylene treatment and continues to increase relative to constitutive gene expression throughout the climacteric. The respiratory climacteric can be temporally separated into two phases. The first phase is associated with a general increase in protein synthesis, whereas the second phase reflects new gene expression and accumulation of corresponding proteins which may be responsible for softening and other ripening characteristics. A major new message on polysomes that arises concomitantly with the respiratory climacteric codes for an in vitro translation product of 53 kilodaltons which is immunoprecipitated by antiserum against avocado fruit cellulase.Cyanide at 500 microliters/liter fails to affect the change in polysome prevalance or new gene expression associated with the ethylene evoked climacteric in avocado fruit. Treatment of fruit with 500 microliters/liter cyanide alone initiates a respiratory increase within 4 hours, ethylene biosynthesis within 18 hours, and new gene expression akin to that educed by ethylene within 20 hours of exposure to cyanide. PMID- 16663415 TI - Comparison of water potentials measured by in situ psychrometry and pressure chamber in morphologically different species. AB - Leaf water potentials measured by in situ psychrometry were compared with leaf water potentials measured by the pressure chamber technique at various values of water potential in Helianthus annuus, Helianthus nuttallii, Vigna unguiculata, Nerium oleander, Pistacia vera, and Corylus avellana. In V. unguiculata, the leaf water potentials measured by the in situ psychrometer oscillated at the same periodicity as, and proportional to, the leaf conductance. In all species, potentials measured by in situ psychrometers operating in the psychrometric mode were linearly correlated with potentials measured with the pressure chamber. However, the in situ psychrometers underestimated the leaf water potential in the two Helianthus species at low water potentials and overestimated the water potential in P. vera, N. oleander, and C. avellana. The underestimation in the two Helianthus species at low water potentials resulted from differences in water potential across the leaf. The overestimation in P. vera, N. oleander, and C. avellana was considered to arise from low epidermal conductances in these species even after abrasion of the cuticle. Pressure-volume studies with Lycopersicon esculentum showed that less water was expressed from distal than proximal leaflets when the whole leaf was slowly pressurized. The implication of this for water relations characteristics obtained by pressure-volume techniques is discussed. We conclude that in situ psychrometers are suitable for following dynamic changes in leaf water potential, but should be used with caution on leaves with low epidermal conductances. PMID- 16663416 TI - Occurrence of 4-cl-indoleacetic Acid in broad beans and correlation of its levels with seed development. AB - Seeds and leaves of Vicia faba L. contain considerable amounts of 4-Cl indoleacetic acid as determined by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. The unchlorinated auxin could not be detected in either leaves or seeds at different stages of development. The developmental stages of fruits and seeds were characterized by the rate of straight growth of pods, the rate of fresh weight increase, and the development of the endosperm. The peak of 4-Cl-indoleacetic acid content per seed coincides with the maximum rate of water accumulation in the seeds. PMID- 16663417 TI - A debranching enzyme deficiency in endosperms of the sugary-1 mutants of maize. AB - Many of the sugary-1 mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme [alpha-(1, 6) glucosidase] activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing alpha-1,6 branch points into the linear alpha-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one of these peaks of activity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (0 to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant. PMID- 16663418 TI - Role of amides, amino acids, and ureides in the nutrition of developing soybean seeds. AB - The various nitrogenous solutes important to embryo development in symbiotic soybean plants were determined during the midpodfilling stage. Glutamine was the principal form of nitrogen, contributing 55% of the embryo nitrogen requirement. Asparagine was the second most important, contributing 20%. The ureides allantoin and allantoic acid directly contributed only insignificantly to the total nitrogen requirement of the embryo. These conclusions were based upon analyses of tissue extracts, translocation studies of radiolabeled solutes, analysis of in vivo seed coat exudate collected from the freespace of attached, surgically altered seeds, and the in vitro culture of isolated immature soybean embryos. PMID- 16663419 TI - Auxin concentration/growth relationship for Avena coleoptile sections from seedlings grown in complete darkness. AB - A biphasic auxin dose-response curve has been obtained for indole-acetic acid (IAA)-stimulated growth of subapical sections of coleoptiles from totally dark grown oats (Avena sativa L. cv Lodi). The curve for growth at 6 h is composed of a log-linear phase and a modified bell-shaped phase separated by a plateau. The curve is log-linear from 0.003 to 0.4 micromolar IAA when sections are incubated in pH 5.9 buffer. The plateau of IAA concentration-neutral growth is seen from 0.4 to 4.0 micromolar IAA. Further increase in growth occurs from 4.0 to 10 micromolar IAA. Changing the pH of the buffer from 5.9 to 5.5 or 6.2 changes the shape of the curve, shifting the plateau to lower IAA concentration, or abolishing it, respectively. The synthetic auxin 2,4-dichlorophenoxyacetic acid also shows a biphasic dose-response curve, but the synthetic auxin 1-naphthalene acetic acid does not. The plateau is not affected by the auxin-transport inhibitor 2,3,5-triiodobenzoic acid. The plateau is eliminated by taking sections from coleoptiles grown under continuous dim red light. We advance a model to account for these results based on two modes of auxin uptake into the cell: carrier-mediated uptake and uptake via chemiosmotic diffusion. PMID- 16663420 TI - Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences. AB - We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein. PMID- 16663421 TI - Use of zinc ions to study thylakoid protein phosphorylation and the state 1-state 2 transition in vitro. AB - At ATP concentrations less than 0.2 millimolar, zinc ions cause a marked stimulation of endogenous protein phosphorylation in thylakoid membranes isolated from tobacco (Nicotiana tabacum L. cv Turkish Samsun), pea (Pisum sativum L. cv Feltham First) and spinach (Spinacia oleracea L. cv Northland). The greatest stimulatory effect was observed at Zn(2+) concentrations of 1 to 2 millimolar; higher concentrations were inhibitory. The stimulatory effect of Zn(2+) was independent of Mg(2+) concentration from 1 to 5 millimolar and thus does not appear to be due to the formation of a Zn(2+) -ATP complex. Phosphorylation of histones IIA, an exogenous protein substrate, was inhibited by 2 millimolar Zn(2+). At low levels of ATP, Zn(2+) not only stimulates general endogenous protein phosphorylation, but also the phosphorylation of the apoproteins of the light-harvesting chlorophyll a/b-protein complex. However, under these conditions Zn(2+) inhibits the ATP-induced quenching of photosystem II fluorescence and the increase in the ratio of photosystem I to photosystem II fluorescence which are both characteristic of the State 1-State 2 transition. These results suggest that phosphorylation of the light-harvesting chlorophyll a/b-protein complex may not directly bring about the State 1-State 2 transition. PMID- 16663422 TI - Is the cytosolic pi concentration a limiting factor for plant cell respiration? AB - The substrate-dependent O(2) uptake by sycamore (Acer pseudoplatanus L.) cell mitochondria in the presence of ADP and limiting Pi concentrations has been measured. The Pi concentration for half-maximum O(2) uptake rate was found to be in the range 20 to 50 micromolar for all the substrates tested. (31)P NMR of intact sycamore cells indicated that the Pi concentration in the cytoplasm was in the range 5 to 6 millimolar, approximately 100-fold higher than the Pi concentration required for maximum O(2) uptake rates by isolated mitochondria. When sycamore cells were transferred to a culture medium devoid of Pi, the cytoplasmic Pi concentration decreased from 6 to less than 3 millimolar, but the intact cell respiration remained practically constant for at least 4 days. These results strongly suggest that, in vivo, the respiration rate of sycamore cells is not limited by the quantity of Pi supplied to the mitochondria. PMID- 16663423 TI - Differential Senescence of Maize Hybrids following Ear Removal : I. Whole Plant. AB - Visual senescence symptoms and associated changes in constituent contents of three field-grown maize (Zea mays L.) hybrids (Pioneer brand 3382, B73 x Mo17, and Farm Service brand 854) were compared in response to ear removal. Whole plants were harvested at eight intervals during the grain-filling period, and analyzed for dry matter, total N and nitrate N, phosphorus, sugars, and starch.Upper leaves of earless P3382 and B73 x Mo17 showed reddish discoloration by 25 days after anthesis (DAA) and all leaves had lost most of their chlorophyll by 40 DAA. In striking contrast, leaves of earless FS854 plants remained green and similar in appearance to eared controls throughout the grain-filling period.For all hybrids, ear removal led to a decrease in dry weight, reduced N, total N, and phosphorus contents of the total plant, and an increase in carbohydrate content of the leaves and stalks, relative to respective controls. Although changes in carbohydrate and N contents, which previously had been associated with senescence, were observed for all earless hybrids, these changes were followed by accelerated senescence and early death only for P3382 and B73 x Mo17. By 30 DAA, earless P3382 and B73 x Mo17 plants ceased to accumulate dry weight, total N, and phosphorus, indicating a termination of major metabolic activities. In contrast, earless FS854 plants retained a portion of these metabolic activities until 58 DAA, indicating a role for roots in determining rate of senescence development. Thus, the course of senescence was more accurately reflected by measurements of metabolic activities than by measurements of metabolite contents at any given time. These results show that the ear per se does not dictate the rate or completion of the senescence process, and implicated an association between the continued accumulation of N and associated root activities with the delayed senescence pattern of the earless FS854 plants. It is evident that studies involving control of senescence among species must also consider genotypic influences within species. PMID- 16663424 TI - Differential Senescence of Maize Hybrids following Ear Removal : II. Selected Leaf. AB - In conjunction with a study of the effects of ear removal on the senescence of whole maize (Zea mays L.) plants, visual symptoms and associated changes in constituent contents and activities of a selected leaf (first leaf above the ear) were determined. Leaves were sampled from field-grown eared and earless Pioneer brand 3382, B73 x Mo17, and Farm Services brand 854 maize hybrids at nine times during the grainfilling period.VISUAL SYMPTOMS INDICATED THE FOLLOWING SEQUENCE AND RATE OF SENESCENCE: earless B73 x Mo17 > earless P3382 >> eared B73 x Mo17 >> eared P3382 eared FS854. All earless hybrids showed increases in leaf dry weight and sugar content; however, the increases were transitory for P3382 and B73 x Mo17, but continuous throughout the grain-filling period for FS854, indicative of continued photosynthetic activity of the latter. All earless hybrids exhibited similar and transitory starch accumulation patterns. Thus, FS854 was an exception to the concept that carbohydrate accumulation accelerates leaf senescence. Ear removal resulted in accelerated losses of reduced N, phosphoenolpyruvate and ribulose bisphosphate carboxylases, phosphorus, chlorophyll, nitrate reductase activity, and moisture for P3382 and B73 x Mo17 plants. In contrast, the loss of all components (except phosphorus) was similar for the selected leaf of earless and eared FS854.Although the loss of nitrate reductase activity, reduced N, and carboxylating enzymes accurately reflected the development of senescence of the selected leaf, the rate of net loss of reduced N and carboxylating enzymes appeared to be regulated. We deduced that the rate of flux of N into the leaf was a factor in regulating the differing rates of senescence observed for the six treatments; however, we cannot rule out the possibility of concurrent influence of growth regulators or other metabolites. PMID- 16663425 TI - Spinach pyruvate kinase isoforms : partial purification and regulatory properties. AB - Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K(m) values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K(i) for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K(a) of 0.05 millimolar, and glutamate is an inhibitor with a K(i) of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation. PMID- 16663426 TI - Aging of cylinders excised from pulp tissues of the ;golden delicious' apple. AB - Aging cylinders excised from ;Golden Delicious' apple (Pyrus malus L.) pulp, like the intact fruit, exhibit some characteristic phenomena such as rise in respiration (climacteric), ethylene synthesis, enzymic changes, and increase in ribosomes and mRNA. Aging of cylinders of pulp tissues may offer a useful physiological tool for the study of maturation and senescence. PMID- 16663427 TI - Polyamine Biosynthetic Enzymes in the Cell Cycle of Chlorella: Correlation between Ornithine Decarboxylase and DNA Synthesis at Different Light Intensities. AB - During the life cycle of Chlorella vulgaris Beijerinck var vulgaris fa. vulgaris growing synchronously, the specific activity of ornithine decarboxylase peaked at the 2nd hour of the cycle, whereas that of arginine decarboxylase changed only slightly, increasing towards the end of the cycle. The endogenous level of putrescine and spermidine on a per cell basis increased gradually up to the 8th hour of the cycle, and declined thereafter. Thus, the peak of ornithine decarboxylase activity and the polyamine increase preceded both DNA replication (which took place between the 6th and 8th hours of the cycle) and autospore release (which started at the 8th hour). A 2-fold increase in the light intensity caused doubling of the DNA content, resulting in doubling of the number of autospores per mother cell. It also brought about a 2-fold increase in the specific activity of ornithine decarboxylase and polyamine content, the peaks being at the same hour of the cycle under high and low light intensities. The increase in cell number and polyamine content in a Chlorella culture grown under high light intensity was inhibited by alpha-difluoromethyl ornithine, a specific inhibitor of ornithine decarboxylase, this inhibition being partially reversed by putrescine.It is suggested that in C. vulgaris the sequence of events which relates polyamine biosynthesis to cell division is as follows: increased ornithine decarboxylase activity, accumulation of polyamines, DNA replication, and autospore release. PMID- 16663428 TI - Endosperm Protein Synthesis and l-[S]Methionine Incorporation in Maize Kernels Cultured In Vitro. AB - This study was conducted to examine protein synthesis and l-[(35)S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[(35)S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing patterns for endosperm proteins were similar for field and cultured kernels throughout development. By 15 days, over 70% of the l [(35)S]methionine taken up was present in endosperm proteins. Label incorporation visualized by fluorography generally followed the protein intensity of the stained gels. The high methionine content, low molecular weight zeins (i.e. 15 and 9 kilodaltons) were highly labeled. All of the radioactivity in hydrolyzed zein samples was recovered in the methionine peak indicating minimal conversion to l-[(35)S]cysteine. The procedure described here is suitable for long term culture and labeling experiments in which continued kernel development is required. PMID- 16663429 TI - Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm. AB - Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500 cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6 bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed. PMID- 16663430 TI - Short term acclimation of spinach to high temperatures: effect on chlorophyll fluorescence at 293 and 77 Kelvin in intact leaves. AB - Using intact leaves of Spinacia oleracea (L.), reversible temperature-induced changes in chlorophyll fluorescence emitted at room temperature and at 77K were studied. Interpretation of fluorescence at 77K was largely facilitated by developing a new method to minimize reabsorption artifacts (;diluted leaf powder'). Leaves of plants grown at 15 to 20 degrees C were exposed for several hours to different temperatures. Upon incubation at 35 degrees C in the dark or in the light, the following changes in 77K fluorescence occurred with a half-time of less than 1 hour: (a) the initial fluorescence (F(0)) of photosystem I increased by 15%, while that one of photosystem II somewhat decreased; (b) although variable fluorescence declined in both photosystems, the decrease in photosystem II (40%) was more severe; (c) the changes were less significant after 480-nanometer excitation light was replaced by 430-nanometer light. The data were interpreted in terms of a reversible, temperature-induced change in thylakoid structure and related change in the distribution of the absorbed energy in favor of photosystem I, at the expense of photosystem II excitation, probably accompanied by an increase in the rate of thermal deactivation of excited states. The considerable decrease in the variable part of room temperature fluorescence gives rise to the suggestion that this transition has lowered the reduction level of plastoquinone, i.e. has increased electron flow through photosystem I, relative to photosystem II. Possible physiological and mechanistic analogies between this temperature-induced state transition and the light-dependent state 1 state 2 regulation has been discussed. PMID- 16663431 TI - Hormone-mediated regulative action of the sunflower shoot apex on growth and cation level in the cotyledons: an additional manifestation of apical control. AB - Decapitation of the shoot apex of seedlings of Helianthus annuus Lin. above the cotyledonary node brought about promotion of growth in the cotyledons. Potassium level in the cotyledons of decapitated plants was higher, and that of sodium lower, than in those of intact plants. IAA applied to the cut stem surface imitated the effects of the apex. Application of kinetin to the cotyledons antagonized the apex or the auxin in their influence on growth and cation level. Labeled IAA applied to the cut stem surface penetrated into the cotyledons in significant amounts. It was concluded that growth and monovalent cation level in the cotyledons are regulated by auxin released from the shoot apex and that at least part of the auxin effect is exerted directly in the cotyledons. A function of the apex as a sink for cytokinins may also be involved in the control mechanism. PMID- 16663432 TI - Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase. AB - Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO(2) (1200 microliters per liter) than in ambient CO(2) (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO(2). Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO(2). The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO(2) compared with 50% in the ambient atmosphere. While CO(2) enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO(2). PMID- 16663433 TI - Physiological responses to salinity in selected lines of wheat. AB - Two selections of bread wheat, Triticum aestivum L., differing in their relative salt resistance, were grown in salinized solution culture, and relative growth rates, osmotic adjustment, ion accumulation, and photosynthesis were monitored to study the responses of the plants to salinity.Differences in water relations were minimal and were only apparent for 3 days following salinization. The lines differed substantially in their relative growth rates and photosynthetic responses for several weeks following salinization, despite full osmotic adjustment. Concentrations of major cations and Cl(-) in the plant organs were remarkably similar in both lines, indicative of minimal differences in gross ion absorption and translocation.The authors interpret these results to suggest that the major difference between these two lines of wheat was their response to specific ion effects, at the level of the organ, tissue, cell, and subcellular entities. Superior compartmentation of toxic ions by the more salt-tolerant line, presumably in the vacuole, might have enabled it to maintain its cytoplasmic metabolic apparatus in a stabler and more nearly normal state than the sensitive line was able to do; a measure of true cytoplasmic toleration of salt may also be a factor. PMID- 16663434 TI - Guard Cell Starch Biosynthesis Regulated by Effectors of ADP-Glucose Pyrophosphorylase. AB - ADP-glucose pyrophosphorylase catalyzes the regulated step of starch bioynthesis in mesophyll chloroplasts. This enzyme is activated by a high ratio of the concentrations of 3-P-glycerate to inorganic phosphate (Pi) in light. In contrast, starch in guard cell chloroplasts is degraded when stomata open, which usually occurs in light. We have investigated the biochemical causes for this contrasting phenomenon.Vicia faba L. leaflets were sampled in darkness and after various periods of illumination. The samples were quick-frozen and freeze-dried. Guard cells and other cells were dissected out, weighed, and assayed for ADP glucose pyrophosphorylase activity, 3-P-glycerate, and Pi. In the pyrophosphorolytic direction, ADP-glucose pyrophosphorylase specific activity in guard cells was 2.7 moles per kilogram protein per hour, which was comparable to the values obtained for palisade and spongy cells. The specific activity in epidermal cells was 4-fold lower. Under our assay conditions, the guard cell enzyme activity was 5-fold higher in the presence of 3-P-glycerate and 5-fold lower with Pi (i.e. similar to the results obtained with extracts of fresh leaflet). During three minutes of illumination, 3-P-glycerate concentration in palisade cells increased 2.5-fold to 10 millimoles per kilogram dry mass. The concentration of 3-P-glycerate in guard cells was 20-fold lower and unaffected by illumination. The concentration of Pi was approximately 17 millimoles per kilogram dry mass in palisade cells, but was 10-fold higher in guard cells. These overall cellular Pi concentrations were unaffected by illumination. We conclude that starch biosynthesis in guard cells is not activated by light because of the low and constant 3-P-glycerate concentration there. We interpret this last to be a consequence of the absence of the photosynthetic carbon reduction pathway in chloroplasts of these cells. PMID- 16663435 TI - Histological Compartmentation of Phosphate in Vicia faba L. Leaflet : Possible Significance to Stomatal Functioning. AB - Guard cells, epidermal cells, palisade parenchyma cells, and spongy parenchyma cells of Vicia faba L. leaflet were analyzed for inorganic phosphate and phosphorus. On a molar basis, cells in the epidermal layer contained about 15 fold more inorganic phosphate than mesophyll cells did. Although a metabolic role for this asymmetric distribution cannot be defined unequivocally, we note that high epidermal inorganic phosphate would buffer against pH changes in the epidermis during stomatal movements. PMID- 16663436 TI - Sink to source translocation in soybean. AB - The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [(14)C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [(14)C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue. PMID- 16663437 TI - Low temperature induction of hormonal sensitivity in genotypically gibberellic Acid-insensitive aleurone tissue. AB - An exposure of genetically gibberellic acid-insensitive isolated wheat aleurone tissue/deembryonated seeds to low temperature for 20 hours prior to addition of exogenous gibberellic acid results in a significant increase in sensitivity to gibberellic acid. The results may reflect a low temperature-induced increase in hormone receptor sites and could have important implications for elucidating the nature of the primary site of hormone action. PMID- 16663438 TI - Kaurene Synthetase Activity in Helianthus annuus L. : Increases in Enzyme Activity after Storage of Seedlings in Liquid Nitrogen. AB - In previous studies, the conversion of geranylgeranyl pyrophosphate to ent kaurene (kaurene synthetase AB activity) could not be detected readily in crude extracts of sunflower (Helianthus annuus L.) seedlings (Shen-Miller, West 1982 Plant Physiol 69: 637-641). These investigations also revealed the presence of inhibitors for Marah macrocarpus kaurene synthetase AB activity in crude extracts of sunflower seedlings. It has now been found that crude extracts prepared from intact sunflower seedlings stored in liquid N(2) for several days have greatly enhanced AB activity in comparison with frozen, but not stored, controls. The levels of activity for the conversion of copalyl pyrophosphate to ent-kaurene (kaurene synthetase B activity) are affected only slightly by storage of intact seedlings in liquid N(2). Extracts from intact seedlings that had been stored in liquid N(2) also showed less inhibitory activity for Marah macrocarpus endosperm kaurene synthetase AB activity. PMID- 16663439 TI - Vacuolar localization of ethylene-induced chitinase in bean leaves. AB - The localization of ethylene-induced endochitinase was studied in bean (Phaseolus vulgaris L. cv Saxa) leaves. The specific activity of chitinase in mesophyll protoplasts isolated from the leaves was as high as in tissue homogenates, indicating that most of the enzyme was located intracellularly. Vacuoles isolated and purified from the protoplasts were found to contain most of the intracellular chitinase activity. PMID- 16663440 TI - Evidence for control of carbon partitioning by fructose 2,6-bisphosphate in spinach leaves. AB - Excision of spinach (Spinacia oleracea L.) leaves had no effect on photosynthetic rates, but altered normal carbon partitioning to favor increased formation of starch and decreased formation of sucrose. The changes were evident within 2 hours after excision. Concurrently, leaf fructose-2,6-bisphosphate content increased about 5-fold (from 0.1 to 0.5 nanomoles per gram fresh weight). The activities of sucrose-P synthase and cytoplasmic fructose 1,6-bisphosphatase in leaf extracts remained constant during the time period tested. It is postulated that the rise in fructose 2,6-bisphosphate was responsible for the change in carbon partitioning. PMID- 16663441 TI - Multiple Subunit Composition of Chloroplastic Glutamine Synthetase of Nicotiana tabacum L. AB - Chloroplastic glutamine synthetase from tobacco leaves (Nicotiana tabacum L. var Xanthi) was purified to homogeneity. By using sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance liquid chromatography, a single subunit was identified with a molecular weight of 45,000 daltons. However the native protein seems to be composed of four different subunits which can be separated by isoelectrofocusing. It is suggested that different genes with eventual posttranslational and/or posttranscriptional modifications may control the synthesis of the chloroplastic glutamine synthetase. PMID- 16663442 TI - Effective Absorption Cross-Sections in Porphyridium cruentum: Implications for Energy Transfer between Phycobilisomes and Photosystem II Reaction Centers. AB - Effective absorption cross-sections for O(2) production by Porphyridium cruentum were measured at 546 and 596 nanometers. Although all photosystem II reaction centers are energetically coupled to phycobilisomes, any single phycobilisome acts as antenna for several photosystem II reaction centers. The cross-section measured in state I was 50% larger than that measured in state II. PMID- 16663443 TI - Quantitation of oat globulin by radioimmunoassay. AB - A radioimmunoassay was used to determine the globulin content of developing oat (Avena sativa L. var Coker 6622) seeds. The globulin content increased from 0.32 to 2.37 milligrams per seed from 2 to 21 days post anthesis. The amount of globulin did not increase from 21 to 30 days post anthesis. When the total protein of seeds harvested 21, 24, and 30 days post anthesis was measured by micro-Kjeldahl analysis, it was determined to be 3.15 milligrams per seed for each sampling date. When the amount of globulin per seed was compared to this value, the relative proportion of globulin to total seed protein was approximately 75%. PMID- 16663444 TI - Starch Degradation in Synchronously Grown Chlamydomonas reinhardtii and Characterization of the Amylase. AB - The activities of amylase and phosphorylase were monitored during the 12-hour light/dark synchronous cell cycle of autotrophically grown Chlamydomonas reinhardtii 11-32/90. The activity of amylase increased from 7.3 to 42 micromole reducing equivalents per 10(9) cells per hour while phosphorylase increased from 43 to 214 micromole glucose 1-phosphate released per 10(9) cells per hour between the midlight and middark periods. Cellular fractionation indicated that both enzymes were localized solely within the chloroplast. The pH optima for amylase and phosphorylase were 6.7 to 7.6 and 6.0 to 7.4, respectively. The amylase is a heat-labile alpha-amylase which is insensitive to ethylenetetraaecetate but inhibited by N-ethylmaleimide. PMID- 16663445 TI - Determination of Solute Permeability in Chara Internodes by a Turgor Minimum Method : Effects of External pH. AB - The analysis of Sha'afi et al. (Sha'afi, Rich, Mickulecky, Solomon 1970 J Gen Physiol 55: 427-450) for determining solute permeability in red blood cells has been modified and applied to turgid plant cells. Following the addition of permeating solute to the external medium, a biphasic response of cell turgor can be measured with the pressure probe in isolated internodes of Chara corallina. After an initial decrease in turgor due to water flow (water phase), turgor increases due to the uptake of the solute (solute phase) until the original turgor is reattained. From the pressure/time course in the neighborhood of the minimum turgor, the permeability of the osmotic solute can be determined. The data obtained by the minimum method for rapidly permeating (ethanol, methanol) and slowly permeating (formamide, dimethylformamide) solutes are similar to those calculated from the half-time of pressure changes during the solute phase and to data obtained by other workers using radioactive tracers. The methods employing the pressure probe were applied to examine the effect of high pH (up to pH 11) on the membrane permeability. There appeared to be no effect of high pH on the permeability coefficients, reflection coefficients, and hydraulic conductivity. PMID- 16663446 TI - Atrazine, bromacil, and diuron resistance in chlamydomonas: a single non mendelian genetic locus controls the structure of the thylakoid binding site. AB - A series of Chlamydomonas reinhardii mutants were selected for resistance to the herbicides atrazine, bromacil, and diuron. Four of these have reduced herbicide binding to the thylakoid membranes and show the non-Mendelian inheritance pattern characteristic of chloroplast genes. These mutants show a variety of selective alterations in binding of the three herbicides. These changes account for the observed patterns of in vivo cross-resistance. Analyses of chloroplast gene recombination indicate that these four mutations are in the same gene. Overall, the results suggest that this gene codes for a protein component of the herbicide binding site. One of the mutants has slow phototrophic growth and altered electron transport as has been observed in atrazine-resistant higher plant varieties, but the others are normal in these respects. The slow growth characteristic of this mutant seems to be the consequence of the same mutation which confers herbicide resistance.The mutants isolated also include a large number which achieve resistance by some secondary mechanism. These are all nuclear gene mutations, and represent numerous loci. They also show a variety of patterns of cross-resistance, but the mechanisms behind them have not yet been investigated. PMID- 16663447 TI - Effects of Ultraviolet-B Irradiance on Soybean : V. The Dependence of Plant Sensitivity on the Photosynthetic Photon Flux Density during and after Leaf Expansion. AB - Soybeans (Glycine max [L.] Merr. cv Essex) were grown in a green-house, and the first trifoliate leaf was either allowed to expand under a high photosynthetic photon flux density (PPFD) (1.4 millimoles per square meter per second) or a low PPFD (0.8 millimoles per square meter per second). After full leaf expansion, plants from each treatment were placed into a factorial design experiment with two levels of ultraviolet-B (UV-B) radiation (0 and 80 milliwatts per square meter biologically effective UV-B) and two levels of concomitant PPFD (0.8 and 1.4 millimoles per square meter per second) resulting in a total of eight treatments. Measurements of net photosynthesis and the associated diffusion conductances, ribulose-1,5-bisphosphate carboxylase activity, chlorophyll and flavonoid concentrations, and leaf anatomy were examined for all treatments. Leaves expanded in the high PPFD were unaffected by UV-B radiation while those expanded in the low PPFD were sensitive to UV-B-induced damage. Likewise, plants which were UV-B irradiated concomitantly with the high PPFD were resistant to UV B damage, while plants irradiated under the low PPFD were sensitive. The results of this study indicate that both anatomical/morphological and physiological/biochemical factors contribute toward plant sensitivity to UV-B radiation. PMID- 16663448 TI - Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity. AB - The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities. PMID- 16663449 TI - Characterization of Radish (Raphanus sativus) Storage Proteins. AB - Radish (Raphanus sativus cv Rond rose a bout blanc Vilmorin) seeds, as other cruciferae oil seeds, contain two major types of storage protein aggregates which can be separated by gel filtration into 12 and 1.7 Svedberg fractions. These two fractions have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, and two bidimensional gel electrophoresis systems. These results were compared with those obtained with rapeseed storage proteins. Radish 12 Svedberg particles are made of a series of nine major polypeptides ranging from 33 to 30 kilodaltons. These polypeptides present charge heterogeneity. The 12 Svedberg particle is made of six subunits approximately 55 kilodaltons. Each subunit is a couple of two polypeptides linked by a disulfide bridge. The 1.7 Svedberg particle has a simpler composition. It is made of two polypeptides of 10 and 12 kilodaltons and smaller peptides of approximately 7 kilodaltons. Twelve and 1.7 Svedberg particles also differ in their amino acid composition, the 1.7 Svedberg being particularly rich in glutamic acid and proline. Its components are basic. The organization of the rapeseed storage protein is similar but more complex. PMID- 16663450 TI - Degradation of the Major Storage Protein of Phaseolus vulgaris during Germination : Role of Endogenous Proteases and Protease Inhibitors. AB - Cotyledons from Phaseolus vulgaris L. (var. Improved Tendergreen) were tested for their activity on alpha-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) and azocasein during a germination periood of 10 days. Both activities increased throughout germination when activity was expressed on the basis of dry weight or protein. That these two activities were most likely due to the action of different enzymes was indicated by the fact that (a) optimal pH for the hydrolysis of BAPNA and azocasein was 8.2 and 5.5, respectively, and (b) the digestion of azocasein was considerably enhanced by mercaptoethanol and partially inhibited by thiol protease inhibitors, N-ethylmaleimide, and E-64, whereas these same regents caused little change in activity toward BAPNA. The three subunits of the major storage protein, G1, disappeared during germination and were accompanied by the accumulation of lower molecular weight products. The breakdown of G1 by extracts of the germinated beans could be demonstrated in vitro at pH 5 to 6. This activity was enhanced by mercaptoethanol and completely abolished by N ethylmalemide, leupeptin, and E-64. It is concluded that a thiol protease with an acid pH optimum is primarily responsible for the disappearance of the major storage protein during germination. Although an inhibitor of the plant thiol protease, papain, is present in the mature bean and decreases during germination, its role in the control of the breakdown of the storage protein remains to be elucidated. PMID- 16663451 TI - Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume. AB - The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N(2). Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K(+) content and high ratio of Mg(2+) to Ca(2+). Fruit cryopuncture sap became labeled with (14)C following feeding of [(14)C]urea to leaves or adjacent walls of the fruit, of (14)CO(2) to the pod gas space, and of [(14)C] asparagine or [(14)C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of (14)C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of (14)C distribution in phloem sap suggested that [(14)C]allantoin was metabolized to a greater extent in its passage to the fruit than was [(14)C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO(3) fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO(3)-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of (15)NO(3) to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant. PMID- 16663452 TI - Proteins Produced during Salt Stress in Tobacco Cell Culture. AB - The protein pattern of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that have become adapted to a medium containing 10 grams NaCl per liter was compared to that of unadapted cells on one-dimensional sodium dodecyl sulfate gels. Two protein bands (32,000 and 20,000 daltons) were much more abundant in the salt-adapted cells, and one protein (26,000 daltons) was unique to the salt cells. This protein pattern did not change during the growth cycle of the cells. When salt-adapted cells are transferred to control medium, their ability to grow in the salt-containing medium returns to that of control cells after one passage in the control medium (Hasegawa, Bressan, Handa 1980 Plant Cell Physiol 21: 1347). Within this time the levels of the 32,000 and 20,000 dalton proteins also return to that of the control cells, but the 26,000 dalton protein does not disappear until after at least two passages in control medium. Amino acid analyses of these three proteins revealed that they all contain some hydroxyproline. PMID- 16663453 TI - Invertases of Lilium Pollen : Characterization and Activity during In Vitro Germination. AB - Two different forms of invertase are found in pollen of lily (Lilium auratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partially purified and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K(m) of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are beta-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germination. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations. PMID- 16663454 TI - Biosynthesis of Storage Proteins in Ripening Agrostemma githago L. Seeds. AB - The synthesis of storage proteins in ripening Agrostemma githago seeds was studied by in vivo pulse and pulse-chase experiments with labeled amino acids and labeled glucosamine. It was found that storage proteins were not synthesized directly, but via cleavage of several large precursor proteins. Two disulfide linked proteins of 38 and 25 kilodaltons were synthesized via a single large precursor protein. This precursor protein contained internal disulfide bridges, at least one of which is involved in holding the subunit structure together following cleavage of the precursor. A similar mode of biosynthesis was noted for two other disulfide-linked proteins of 36 and 22 kilodaltons. The half-life of the precursors was about 2 hours. This mode of processing is analogous to the synthesis of legumin in legumes and globulin in oats. A third pair of disulfide bonded proteins (41 and 23 kilodaltons) was synthesized from a precursor protein in several steps. These included a legumin-like cleavage, whereafter the subunits remained disulfide-bonded. Then, from the largest subunit, a part was cleaved off, probably a storage protein of 17 kilodaltons. This 17-kilodalton protein was not disulfide-bonded to the 41 and 23-kilodalton complex. The first processing step was fast, the second slow: The half-lives of the precursors were about 3 and 10 hours, respectively. Finally, a group of 16- and 17-kilodalton proteins was synthesized by cleavage of large precursor proteins, likely in two steps. After cleavage, the proteins were not disulfide-bonded. The half-life of the precursors was short, less than 1 hour. In addition, for the 38-, 23-, and one of the 17 kilodalton proteins, a small decrease of relative molecular weight was observed as a last processing step. This was likely due to deglycosylation. PMID- 16663455 TI - Nonphysiological binding of ethylene by plants. AB - Ethylene binding to seedling tissue of Vicia faba, Phaseolus vulgaris, Glycine max, and Triticum aestivum was demonstrated by determining transit time required for ethylene to move through a glass tube filled with seedling tissue. Transit time for ethylene was greater than that for methane indicating that these tissues had an affinity for ethylene. However, the following observations suggest that the binding was not physiological. Inhibitors of ethylene action such as Ag(+) ions and CO(2) did not decrease binding. Mushrooms which have no known sites of ethylene action also demonstrated ethylene binding. The binding of acetylene, propylene, ethylene, propane, and ethane more closely followed their solubility in water than any known physiological activity. PMID- 16663456 TI - cis-Polyisoprene Synthesis in Guayule Plants (Parthenium argentatum Gray) Exposed to Low, Nonfreezing Temperatures. AB - Exposure of guayule plants (Parthenium argentatum Gray) to 6 months of a night temperature of 7 degrees C results in a 2-fold stimulation of cis-polyisoprene (rubber) formation over that of control plants exposed to 21 to 24 degrees C night temperature. Control and cold-treated plants contained 2.18% and 5.69% rubber, respectively. Examination of the stem apices by transmission electron microscopy showed extensive formation of rubber particles in the cold-treated plants compared to the control plants. The rubber particles in guayule are formed in the cytoplasm and fuse to form large globular deposits. The surface area of the rubber particles and globules range from 4 x 10(-6) to 2.9 x 10(-3) square micrometers. The deposition of rubber in the cytoplasm of the cortical parenchyma cells differs from rubber deposition in the vacuoles of laticifers of Asclepias syriaca. Electron micrographs of stem cortical parenchyma in control plants show mature cells with large central vacuoles, thin layers of parietal cytoplasm, and smaller numbers of rubber particles. Radioactive acetate and mevalonate are incorporated into rubber at a faster rate in stem slices from cold-treated plants compared to slices from control plants. A faster rate of these reactions may account for the increase in rubber synthesis in the cold-treated plants. PMID- 16663457 TI - H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase. AB - Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO(3) (-)-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO(3) (-)-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H(+) transport, with the transport process showing the same differential sensitivity to NO(3) (-) and vanadate as the ATPase activity.Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO(3), stimulated by anions (Cl(-) > Br(-) > acetate > HCO(3) (-) > SO(4) (2-)), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H(+) transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H(+) ATPase and further indicates the presence of a higher density, vanadate sensitive, H(+)-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin. PMID- 16663458 TI - H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase. AB - The H(+)/ATP stoichiometry was determined for an anion-sensitive H(+)-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A(340)) or by monitoring the release of (32)P from [gamma-(32)P]ATP. Inasmuch as this anion-sensitive H(+)-ATPase is strongly inhibited by NO(3) (-), initial rates of H(+) influx and ATP hydrolysis were measured in the absence and presence of NO(3) (-) to account for ATPase activity not involved in H(+) transport. The NO(3) (-)-sensitive activities were calculated and used to estimate the ratio of H(+) transported to ATP hydrolyzed. These measurements resulted in an estimate of the H(+)/ATP stoichiometry of 1.96 +/- 0.14 suggesting that the actual stoichiometry is 2 H(+) transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H(+) across the tonoplast measured in vivo, our result suggests that the H(+)-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply. PMID- 16663459 TI - Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles. AB - Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg(2+). The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl(-), but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N'-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO(3) (-); but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl(-), inhibited by NO(3) ( ), and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H(+) transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H(+) pumping ATPase of red beets originated from the tonoplast. Tonoplast H(+)-ATPases with similar properties appear to be widely distributed in higher plants and fungi. PMID- 16663460 TI - Isotope ratios of cellulose from plants having different photosynthetic pathways. AB - Hydrogen and carbon isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from C(3), C(4), and Crassulacean acid metabolism (CAM) plants were determined for plants growing within a small area in Val Verde County, Texas. Plants having CAM had distinctly higher deuterium/hydrogen (D/H) ratios than plants having C(3) and C(4) metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C(4) plants had many D/H ratios similar to those of C(3) plants, so that hydrogen isotope ratios cannot be used to distinguish between these two photosynthetic modes. Portulaca mundula, which may have a modified photosynthetic mode between C(4) and CAM, had a hydrogen isotope ratio between those of the C(4) and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C(4) and CAM plants occurs. Thus, oxygen isotope ratios are not useful in distinguishing between these metabolic modes. A plot of hydrogen isotope ratios versus oxygen isotope ratios for this sample set shows considerable overlap between oxygen isotope ratios of the different photosynthetic modes without a concomitant overlap in the hydrogen isotope ratios of CAM and the other two photosynthetic modes. This observation is consistent with the hypothesis that higher D/H ratios in CAM plants relative to C(3) and C(4) plants are due to isotopic fractionations occurring during biochemical reactions. PMID- 16663461 TI - Structure of the arabinogalactan from zea shoots. AB - The structure of the arabinogalactan obtained from the buffer-homogenate of Zea mays L. (hybrid B73 x Mo17) shoots has been studied. The purified polysaccharide was investigated by methylation analysis before and after controlled acid hydrolysis. Arabinogalactan-1 consists of arabinose, galactose, xylose, uronic acid, and glucose in the molar ratio of 37.1:55.8:3.0:4.1:trace, and arabinogalactan-2 consists of the same sugars in the ratio of 35.4:53.9:1.6:9.2:trace. A trace of protein was detected in arabinogalactan-1 and about 0.2% was present in 2. About 20% of the galactose residues in arabinogalactan-1 constitute a (1 --> 3)-linked galactan chain and approximately 60% constitute a (1 --> 6)-linked galactan sequence. About 15% of the galactose residues in arabinogalactan-1 are substituted by galactose in the 3- and 6 positions, thereby constituting branch points of the galactan framework. The remainder (5%) of the galactose residues in arabinogalactan-1 are located at nonreducing terminal positions. About 85% of the (1 --> 6)-galactosyl sequence is substituted, mostly by single arabinose residues. Nonreducing terminal glucuronic acid is attached to C-6 of galactose residues. The basic structure of arabinogalactan-2 is similar to that of arabinogalactan-1. PMID- 16663462 TI - Purification, Characterization, and Fractionation of the delta-Aminolevulinic Acid Synthesizing Enzymes from Light-Grown Chlamydomonas reinhardtii Cells. AB - The synthesis of delta-aminolevulinate from glutamate by Chlamydomonas reinhardtii membrane-free cell homogenates requires Mg(2+), ATP, and NADPH as cofactors. The pH optimum is about 8.3. When analyzed by a Fractogel TSK gel filtration column the delta-aminolevulinate synthesizing enzymes, including glutamate-1-semialdehyde aminotransferase, elute with an apparent molecular weight of about 45,000. The enzymes obtained from the gel filtration column were separated into three fractions by affinity column chromatography. One fraction binds to heme-Sepharose, one to Blue Sepharose, while the enzyme converting the putative glutamate-1-semialdehyde to delta-aminolevulinic acid is retained by neither column. All three fractions are necessary for the conversion of glutamate to delta-aminolevulinate. The delta-aminolevulinate synthesizing enzymes from Chlamydomonas are sensitive to inhibition by heme but not sensitive to inhibition by protoporphyrin. PMID- 16663463 TI - Effects of exogenous methionine on storage protein composition of soybean cotyledons cultured in vitro. AB - Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental l-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S beta-subunit gene. Concomitant with the absence of the beta-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of beta subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression. PMID- 16663464 TI - Storage Protein Composition of Soybean Cotyledons Grown In Vitro in Media of Various Sulfate Concentrations in the Presence and Absence of Exogenous l Methionine. AB - Immature soybean (Glycine max L. Merrill cv Provar) cotyledons were grown aseptically for 6 days in complete culture medium with zero, deficient (17 micromolar), sufficient (1.5 millimolar), or supraoptimal (7.5 millimolar) levels of sulfate. Some cotyledons at each sulfate concentration were supplemented with l-methionine. No sulfate or 17 micromolar sulfate were inadequate for growth and protein accumulation, but all the major subunits of the 7S and 11S storage protein fractions were detected. The ratio of 11S to 7S proteins was <1.0. Addition of 8.4 millimolar methionine overcame the restriction of cotyledon growth and protein accumulation in the sulfate-deficient media, and the ratio of 11S to 7S proteins was significantly increased. The amino acid compositions of the 7S and 11S fractions from sulfate-sufficient cotyledons and from sulfate deficient cotyledons were not significantly different. There was no difference in fresh weight or total protein accumulation in cotyledons grown in 1.5 millimolar or 7.5 millimolar sulfate. At 7.5 millimolars sulfate, the 11S to 7S ratio was significantly increased, and the amount of beta-subunit in the 7S fraction decreased. At all sulfate levels supplemented with methionine, the 11S to 7S ratio was greater than 1, and no beta-subunit was detected in the 7S fraction. Supplemental methionine in media of any sulfate concentration increased growth and protein methionine content to a greater extent than high (7.5 millimolar) sulfate only. Adding supraoptimal sulfate is not equivalent to supplementing with methionine. Results of this study of in vitro growth are compared to results of studies of seed development on intact plants supplied with various sulfur concentrations. PMID- 16663465 TI - Carbohydrate metabolism in leaf meristems of tall fescue : I. Relationship to genetically altered leaf elongation rates. AB - The physiological bases for genetic differences in leaf growth rates were examined in two genotypes of tall fescue (Festuca arundinacea Schreb.) selected for a 50% difference in leaf elongation rate. Genotypes had similar dark respiration rates and concentrations of carbohydrate fractions in the leaf meristem and in each daily growth segment above the meristem. Dark respiration rates and concentrations of nonreducing sugars, fructans, and takadiastase soluble carbohydrates were highest in leaf intercalary meristems and declined acropetally with tissue age. Concentrations of reducing sugars were 1.0% of dry weight in leaf meristems, 3.7% of dry weight in tissue adjacent to the meristem, then decreased progressively with distance from the meristem. Glucose, fructose, and myo-inositol comprised over 90% of the monosaccharides present in leaf meristems. Soluble protein concentration was 9.7 milligrams per gram fresh weight in leaf meristems, 5.5 milligrams per gram in tissues immediately above the meristem and, thereafter, increased linearly with distance from the meristem.Leaf meristems of the genotype exhibiting rapid leaf elongation contained 30% more soluble protein than those of the genotype selected for slow leaf elongation. The 4-fold difference in size of the leaf meristem appeared to be more important in influencing leaf elongation than were other characteristics examined. PMID- 16663466 TI - Carbohydrate Metabolism in Leaf Meristems of Tall Fescue : II. Relationship to Leaf Elongation Rates Modified by Nitrogen Fertilization. AB - Our objective was to examine alterations in carbohydrate status of leaf meristems that are associated with nitrogen-induced changes in leaf elongation rates of tall fescue (Festuca arundinacea Schreb.). Dark respiration rates, concentrations of nonstructural carbohydrates, and soluble proteins were measured in leaf intercalary meristems and adjacent segments of elongating leaves. The two genotypes used differed by 43% in leaf elongation rate. Application of high nitrogen (336 kilograms per hectare) resulted in 140% higher leaf elongation rate when compared to plants receiving low nitrogen (22 kilograms per hectare). Leaf meristems of plants receiving high and low nitrogen had dark respiration rates of 5.4 and 2.9 microliters O(2) consumed per milligram structural dry weight per hour, respectively. Concentrations of soluble proteins were lower while concentrations of fructan tended to be slightly higher in leaf meristems of low nitrogen plants when compared to high-nitrogen plants. Concentrations of reducing sugars, nonreducing sugars, and takadiastase-soluble carbohydrate of leaf meristems were not affected by nitrogen treatment. Total nonstructural carbohydrates of leaf meristems averaged 44 and 39% of dry weight for low- and high-nitrogen plants, respectively. Within the leaf meristem, approximately 74 and 34% of the pool of total nonstructural carbohydrate could be consumed per day in high- and low-nitrogen plants, respectively, assuming no carbohydrate import to the meristem occurred. Plants were able to maintain high concentrations of nonstructural carbohydrates in leaf meristems despite a 3-fold range in leaf elongation rates, suggesting that carbohydrate synthesis and transport to leaf intercalary meristems may not limit leaf growth of these genotypes. PMID- 16663467 TI - Phytochrome intermediates and action spectra for light perception by dry seeds. AB - It has previously been demonstrated that far-red irradiation of dry Lactuca sativa L. seeds results in inhibition of subsequent germination. Although red has no effect on dry seeds, a red irradiation following a farred irradiation reverses the effect of far-red. This phenomenon is most noticeable in seeds with artificially raised levels of phytochrome in the far-red absorbing form. Qualitatively similar results have been found for the seeds of Plantago major L., Sinapis arvensis L., and Bromus sterilis L. Action spectra studies on Plantago seeds show that the action peaks for promotion and inhibition of germination of hydrated seeds are at 660 and 730 nanometers, respectively. The action spectrum for inhibition of subsequent germination following irradiation of dry seeds is qualitatively and quantitatively similar to that for hydrated seeds, with an action peak at 730 nanometers, indicating absorption by phytochrome in the far red absorbing form. However, the action spectrum for the reversal of this far-red effect on dry seeds has a broad peak at 680 nanometers and subsidiary peaks at 650 and 600 nanometers. It is proposed that this effect is due to light absorption by the phytochrome intermediate complex meta-Fa, and that the action spectrum reflects the in vivo absorption properties of this intermediate. PMID- 16663468 TI - Asparagine biosynthesis in soybean nodules. AB - Asparagine biosynthesis in soybean (Glycine max [L.] Merr.) nodules has been difficult to demonstrate due to the poor conversion of suspected immediate precursors to asparagine and the instability of the key enzyme asparagine synthetase. The present study was designed to explore the effects of two ammonium assimilation inhibitors on the metabolism of (14)CO(2) to [(14)C]asparagine and to demonstrate the existence in nodules of the enzyme asparagine synthetase. When detached nodules were incubated in (14)CO(2), radioactivity in asparagine (as a percentage of amino acid cpm) increased 10-fold over 4 hours. Vacuum infiltration of 10 mm methionine sulfoximine or 10 mm azaserine prior to (14)CO(2) incubations decreased both the rate of dark fixation and the radioactivity in the amino acid fraction. These inhibitors also decreased the recovery of label in aspartate and asparagine. These results, plus the sequence of labeling of metabolites from (14)CO(2), are consistent with a glutamine-dependent synthesis of asparagine from aspartate with oxalacetate as a precursor to aspartate.An enzyme catalyzing the ATP- and glutamine-dependent amidation of aspartic acid to form asparagine was isolated from soybean nodules. High levels of sulfhydryl protectants were required and the inclusion of glycerol and substrates in the extraction buffer helped to stabilize the enzyme. Enzyme activity in taproot nodules increased between 38 and 41 days after planting and peaked soon after flower initiation (45 days). The activity then declined to basal levels by 70 days. On a total enzyme activity basis, there was 170-fold more asparagine synthetase activity in the infected zone of the nodule than in the cortex, and 205-fold more activity in the cytosol than the bacteroid fraction. The enzyme has a broad pH maximum around pH 8.25, and the apparent K(m) values for the substrates aspartate, MgATP, and glutamine are 1.24 mm, 0.076 mm, and 0.16 mm, respectively. Ammonium ion can replace glutamine as the nitrogen donor, but the K(m) value of the enzyme for ammonium ion is 40-fold higher than that for glutamine. PMID- 16663469 TI - Solubilization and partial purification of ATPase from a rose cell plasma membrane fraction. AB - The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction of suspension cultured cells of rose (Rosa damascena) by two different solubilization procedures. Solubilization with 30 mm octyl-beta-d-glucopyranoside followed by precipitation with ammonium sulfate increased the specific activity of the enzyme about 6-fold. Solubilization with 1% cholate removed all but 1% of the phospholipids and resulted in an almost total loss of ATPase activity. The subsequent addition of polar lipids restored >90% of the ATPase activity with a doubling in specific activity. Fractionation of the cholate-solubilized ATPase activity on a Sephadex G-150 column resulted in 88% of the ATPase activity being recovered in two discrete, approximately equal peaks. Both ATPase activities were similar to plasma membrane ATPase activities in pH optimum, substrate specificity, ion stimulation, and inhibitor sensitivity. Assays of marker enzymes for Golgi apparatus, endoplasmic reticulum, and mitochondria revealed only a low contamination (<7%) from other membranes in the plasma membrane-enriched preparations. Lacking an unequivocal marker for the tonoplast, intact vacuoles were isolated, and their membrane density and ATPase activity were characterized and shown not to correspond to those of the putative plasma membrane preparation. These results suggest that there are two forms of ATPase separable by size in the plasma membrane of rose. PMID- 16663470 TI - Photoinactivation of Detergent-Solubilized Plasma Membrane ATPase from Rosa damascena: Action Spectra. AB - The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs(+) protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins. PMID- 16663471 TI - Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein. AB - During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [(3)H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat. PMID- 16663472 TI - Changes in Cytokinins and Gibberellin-Like Substances in Pinus radiata Buds during Lateral Shoot Initiation and the Characterization of Ribosyl Zeatin and a Novel Ribosyl Zeatin Glycoside. AB - Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of "short shoots" (needle fascicles) to "long shoots" (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.Permethyl derivatives of the two highly active CK fractions were examined by capillary gas chromatography-mass spectrometry after separation by C(18) reverse phase high performance liquid chromatography. The mass spectra indicated the presence of: 9-beta-d-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2 enylamino)purine (zeatin riboside) and 9-[hexosyl(probably glucosyl)-beta-d ribofuranosyl]-6-(4-hydroxy-3-methyl-but-2- enylamino)purine (a glycoside of zeatin riboside in which the glycosyl moiety is attached directly to the ribosyl moiety at an unknown position). PMID- 16663473 TI - Measurement of mitochondrial ATPase activity in maize root tips by saturation transfer p nuclear magnetic resonance. AB - We show that saturation transfer (31)P nuclear magnetic resonance can be used to measure the activity of the mitochondrial ATPase of maize (Zea mays L. hybrid WW x Br38) root tips in vivo. Unidirectional rates of ATP synthesis were determined in the steady state (i.e. ATP and cytoplasmic orthophosphate constant) under various conditions. These measurements, and determinations of oxygen consumption, give a P/O ratio (measured in the living tissue) close to 3. In succinate-fed root tips the P/O ratio is approximately 2. Cyanide inhibits the rate of ATP synthesis by two-thirds (P/O ratio approximately 1), with an effective inhibitor constant of approximately 35 micromolars. We show that the alternative electron pathway cannot make ATP, and does not normally operate in this tissue. This method of studying plant mitochondrial metabolism avoids potential artifacts encountered in studies using isolated and purified mitochondria. The method also allows, for the first time, direct and simultaneous examination of the relationship between the rate of ATP synthesis and levels of metabolites such as ATP, and derived parameters such as phosphorylation potential. PMID- 16663474 TI - Characterization of cultured tobacco cell lines resistant to ethionine, a methionine analog. AB - Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110x), threonine (18x), and lysine (5x). In vitro enzymic analysis of lysine sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K(m) values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression. PMID- 16663475 TI - Propylamine transferases in chinese cabbage leaves. AB - We have found spermidine synthase and spermine synthase activities in extracts of leaves of Chinese cabbage (Brassica pekinensis var. Pak Choy) and have developed an assay of the former in crude extracts. The method is based on the transfer of the propylamine moiety of decarboxylated S-adenosylmethionine to labeled putrescine, followed by ion-exchange separation of the labeled amine substrate and product, which are then converted to the 5-dimethylamino-1-napthalene sulfonyl (dansyl) derivatives and further purified and identified by thin layer chromatography. The specific radioactivity of putrescine present in the reaction mixture is determined, as is the radioactivity present in dansyl spermidine. The enzyme is also present in extracts of spinach leaves.Spermidine synthase has been purified about 160-fold from Chinese cabbage leaves. After partial purification, a rapid coupled enzymic assay has been used to study various properties of the enzyme. The plant enzyme shows maximum activity at pH 8.8 in glycine-NaOH buffer and has a molecular weight of 81,000. The K(m) values for decarboxylated S adenosylmethionine and putrescine are 6.7 and 32 micromolar, respectively. The enzyme activity is inhibited strongly by dicyclohexylamine, cyclohexylamine, and S-adenosyl-3-thio-1, 8-diaminoctane. Of these, dicyclohexylamine is the most potent inhibitor with an I(50) at 0.24 micromolar. PMID- 16663476 TI - Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I. AB - A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G 75. Immunological assays using antibodies to the P(700)-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes. PMID- 16663477 TI - Spore Germination and Rhizoid Differentiation in Onoclea sensibilis: A Two Dimensional Electrophoretic Analysis of the Extant Soluble Proteins. AB - Following a geometrically asymmetrical cell division during germination of spores of the fern Onoclea sensibilis L., the small cell differentiates into a rhizoid and the large cell divides to form the protonema. Using silver-staining of two dimensional gels, we have examined the soluble proteins of spores during germination and of separated rhizoid protoplasts and protonemal cells. Of over 500 polypeptides followed, nearly 25% increased or decreased in prominence during spore germination and the initial phases of rhizoid elongation. Soluble proteins from purified protoplasts of young rhizoids were quantitatively different from those of protonemal cells and germinated spores. Nine polypeptides which appeared after cell division were substantially more prominent in rhizoid protoplasts than in whole germinated spores and have been putatively designated rhizoid-specific polypeptides. The differences in the soluble protein composition of young rhizoids and protonemal cells probably reflect the differential organelle distribution between the two cells as well as differential net protein synthesis in the cytoplasms of the two cells. PMID- 16663478 TI - Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration. AB - The frequency of incorporation of the cytokinin N(6)-[p-(3)H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 x 10(4) conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration. PMID- 16663479 TI - Incorporation of N-Benzyladenine into Messenger Poly(A)-RNA of Tobacco Cells Incubated at Stimulatory or Cytostatic Cytokinin Concentration. AB - Poly(A)-RNA was purified from Nicotiana tabacum cell suspension cultures grown in the presence of N(6)-benzyladenine (BA). Cells were incubated with concentrations of 0.4 micromolar BA, optimal for cell division (OPT) or 10 micromolar BA, a cytostatic concentration (OVD), or without cytokinin (CTL) as a control. After 55 hours, total RNA was extracted from the cells and poly(A)-RNA was purified by oligo(dT)-cellulose binding. Similar yields of poly(A)-RNA were obtained for OPT or CTL cell samples; the compared recovery from the OVD cell samples was reduced by more than half. Poly(A)-RNA extracted from OPT or OVD cells contained BA nucleotide, inserted at the respective frequencies of 78 and 506 micromoles per mole conventional nucleotide. BA was inserted into the transcript as well as into the poly(A) segments. No qualitative or quantitative difference was observed between the in vitro translation activities of poly(A)-RNA extracted from OPT or CTL cells. The electrophoretic analysis of translation products from OVD mRNA showed a deficiency in proteins of molecular weight over 50 kilodaltons. This deficiency may be explained by a change in the coding properties of OVD mRNA rather than by a deficiency of the high molecular weight components in the mRNA population. PMID- 16663480 TI - A Seed Storage Protein with Possible Self-Affinity through Lectin-Like Binding. AB - The primary storage protein of oat (Avena sativa L.) seeds, globulin, was shown to have a specific carbohydrate-binding activity. The globulin was capable of hemagglutinating rabbit red blood cells and this hemagglutination was inhibited by the beta-glucan, laminarin, as well as by carbohydrate which had been cleaved from the native globulin. Globulin with carbohydrate-binding activity was isolated from cell wall preparations and from defatted flour. The lectin activity apparently resides in the alpha-subunit of the globulin and has affinity for the carbohydrate which is O-glycosidically linked to the globulin. A portion of this carbohydrate is attached to the beta-subunit. Two affinity columns were synthesized utilizing laminarin and the carbohydrate from the native globulin as ligands. The hemagglutinating activity bound to both of these columns. The activity was specifically eluted from the globulin-carbohydrate affinity column with carbohydrate cleaved from native globulin by an alkali-catalyzed beta elimination. The possible roles of this unique self-binding capacity are discussed. PMID- 16663481 TI - Activation state of ribulose bisphosphate carboxylase in soybean leaves. AB - Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg(2+) and CO(2) (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO(3) (5 millimolar saturating) and Mg(2+) (5 millimolar optimal) for 5 minutes at 25 degrees C or 30 minutes at 0 degrees C before assay. Initial activity was about 70% of total activity. K(act) (Mg(2+)) and K(act) (CO(2)) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg(2+) in the leaf extract was sufficient to support considerable catalytic activity. While Mg(2+) was essential for both activation and catalysis, Mg(2+) levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg(2+) was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0 degrees C. Initial activity quickly deactivated at 25 degrees C in the absence of high CO(2). Total activity slowly declined irreversibly upon storage of leaf homogenate at 25 degrees C. PMID- 16663482 TI - Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells: I. Polysaccharides Associated with Cellulose. AB - Cell walls of suspension-cultured cells of Rosa glauca were fractionated by two different extraction procedures. The first involved a stepwise fractionation scheme based on alkaline extraction. The second took advantage of the powerful cellulose solvent system N-methylmorpholine N-oxide/dimethyl sulfoxide which is capable of solubilizing whole cell walls. From the analytical composition of each solubilized fraction and of the corresponding residues, the fate of each type of cell wall polysaccharide constituent was followed at each step of the extraction scheme and the mode of action of the extractant was interpreted. Although the two fractionation procedures were very different, they yielded very similar cellulosic complex residues and extracts, thus delimiting two blocks of polysaccharides in the cell wall. The cellulose residues still comprised uronic acid-containing polysaccharides and hemicelluloses in association with cellulose. Graded acid hydrolysis provided evidence for the central role of a homogalacturonan core interconnecting xyloglucans and arabinogalactans. A tentative model showing the possible interaction existing between the constituent polysaccharides still associated to cellulose after alkaline extraction is presented. Hydrogen bonding between xyloglucan and cellulose is confirmed, and glycosidic linkages between xyloglucans and pectic polymers are suggested. PMID- 16663483 TI - Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells: II. Multiple Forms of Xyloglucans. AB - Xyloglucans, characteristic hemicellulosic polysaccharides of plant primary walls, have been isolated from Rosa glauca suspension-cultured cells. The cell wall material was fractionated by two sequences of extraction based on solubilization of the hemicelluloses in alkaline and organic solvent systems, respectively. In both cases, only a part (about 50%) of the total xyloglucan could be extracted, the rest remaining tightly associated with cellulose and necessitating the use of acid to be solubilized. Purification of xyloglucans was effected by formation of a gel in appropriate mixtures of dimethyl sulfoxide and water. Further fractionation could be achieved on a cellulose column eluted with chaotropic solvents. This demonstrated the heterogeneity of xyloglucans in the primary cell walls. Analytical data show that all fractions are constituted with the same sugars: l-arabinose, l-fucose, d-galactose, d-xylose, and d-glucose, but their relative proportions differ, particularly the ratio of glucose to xylose which varies from 1.2 to 2 within the different xyloglucans. The structure of these hemicelluloses was established by methylation analysis and shown to consist of a (1 --> 4)-linked glucan backbone which carries substituents on the O-6 of glucose. Here again, the multiple forms of xyloglucans was suggested by the various patterns of substitutions found on the different fractions. The configuration of the linkages were established by (13)C nuclear magnetic resonance spectroscopy and shown to be beta for the glucan backbone, alpha for the xylosyl and fucosyl substituents, and beta for the galactosyl substituents. These configurations agree with the specific rotation of the xyloglucan. PMID- 16663484 TI - In Vivo Nitrogenase Regulation by Ammonium and Methylamine and the Effect of MSX on Ammonium Transport in Anabaena flos-aquae. AB - Ammonium suppresses nitrogenase activity in Anabaena flos-aquae (Lyng) Breb. at all pH values tested. l-Methionine-dl-sulfoximine at 1 millimolar totally inhibited glutamine synthetase, and 10 micromolar partially inhibited. Both concentrations protected nitrogenase activity from ammonium-induced suppression at pH 7.1 and 8.1. At pH 9.3 and 10.2, methionine sulfoximine did not alleviate the suppression of nitrogenase by ammonium. This pH-dependent protection of nitrogenase activity is a result of the noncompetitive inhibition of the ammonium transporter by methionine sulfoximine. At pH 7.1 and 8.2, ammonium is protonated and methionine sulfoximine inhibits its entry into the cell. At pH 9.3 and 10.2, unprotonated ammonia is abundant and may enter the cell independent of the transport system. The effects of ammonium are closely mimicked by the ammonium analog methylamine. These results suggest that ammonium per se is an important in vivo regulator of nitrogen fixation and its function can be mimicked by methylamine. Previous studies employing methionine sulfoximine may have to be re evaluated in light of the inhibitory effects of methionine sulfoximine on the ammonium transporter. PMID- 16663485 TI - Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.). AB - Glycine decarboxylation and serine synthesis were studied in pea (Pisum sativum L.) leaf discs, in metabolically active intact chloroplasts, and in mitochondria isolated both partially by differential centrifugation (i.e. ;crude') and by further purification on a Percoll gradient. Glycolate, glyoxylate, and formate reduced glycine decarboxylase activity ((14)CO(2) and NH(3) release) in the crude green-colored mitochondrial fractions, and in the leaf discs without markedly altering serine synthesis from [1-(14)C]glycine. Glycolate acted because it was converted to glyoxylate which behaves as a noncompetitive inhibitor (K(i) = 5.1 +/- 0.5 millimolar) on the mitochondrial glycine decarboxylation reaction in both crude and Percoll-purified mitochondria. In contrast, formate facilitates glycine to serine conversion by a route which does not involve glycine breakdown in the crude mitochondrial fraction and leaf discs. Formate does not alter the conversion of two molecules of glycine to one CO(2), one NH(3), and one serine molecule in the Percoll-purified mitochondria. In chloroplasts which were unable to break glycine down to CO(2) and NH(3), serine was labeled equally from [(14)C]formate and [1-(14)C]glycine. The maximum rate of serine synthesis observed in chloroplasts is similar to that in isolated metabolically active mitochondria. Formate does not appear to be able to substitute for the one-carbon unit produced during mitochondrial glycine breakdown but can facilitate serine synthesis from glycine in a chloroplast reaction which is probably a secondary one in vivo. PMID- 16663486 TI - ADPglucose Pyrophosphorylase from the CAM Plants Hoya carnosa and Xerosicyos danguyi. AB - ADPglucose pyrophosphorylase from the Crassulacean acid metabolism plants Hoya carnosa and Xerosicyos danguyi were partially purified to study their regulatory and kinetic properties. The molecular weight of the native enzymes from both plants was determined to be about 209,000. The enzyme from both plants was found to be activated by glycerate 3-phosphate and inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg(2+) are reported. The significance of the activation by glycerate 3-phosphate and inhibition by inorganic phosphate of ADPglucose synthesis catalyzed by the H. carnosa and X, danguyi enzymes is discussed. ADPglucose synthesized by the above enzymes was found to be the most effective donor of the glucosyl portion to alpha-glucan primer in the starch synthase reaction observed in CAM plants. PMID- 16663487 TI - Variation in photosynthetic pigments and plastoquinone contents in sugar beet chloroplasts with changes in leaf copper content. AB - The changes in the contents of chlorophyll (Chl) a, Chl b, carotenoid, and plastoquinone in sugar beet (Beta vulgaris) chloroplasts were investigated during Cu deficiency and resupply. With the onset of Cu deficiency, extrachloroplastic Cu decreased more rapidly than chloroplast Cu, which eventually accounted for all of the Cu present in the leaf. The ultrastructure of Cu-deficient chloroplasts did not differ from the control except in very young, severely deficient leaves. During Cu depletion and resupply, Chl a, Chl b, carotenoid, and plastoquinone contents changed concomitantly. Because of the concurrent changes in the contents of terpenoid-containing pigments and plastoquinone with Cu depletion and resupply, we suggest that Cu might be involved in the regulation of the early steps of terpenoid biosynthesis prior to the formation of geranyl-geranyl pyrophosphate. PMID- 16663488 TI - Uronide Deposition Rates in the Primary Root of Zea mays. AB - The spatial distribution of the rate of deposition of uronic acids in the elongation zone of Zea mays L. Crow WF9 x Mo 17 was determined using the continuity equation with experimentally determined values for uronide density and growth velocity. In spatial terms, the uronide deposition rate has a maximum of 0.4 micrograms per millimeter per hour at s = 3.5 mm (i.e., at the location 3.5 mm from the root tip) and decreases to 0.1 mg mm(-1) h(-1) by s = 10 mm. In terms of a material tissue element, a tissue segment located initially from s = 2.0 to s = 2.1 mm has 0.14 mug of uronic acids and increases in both length and uronic acid content until it is 0.9 mm long and has 0.7 mug of uronide when its center is at s = 10 mm. Simulations of radioactive labeling experiments show that 15 min is the appropriate time scale for pulse determinations of deposition rate profiles in a rapidly growing corn root. PMID- 16663489 TI - Accumulation of Plastoquinone A during Low Temperature Growth of Winter Rye. AB - Chloroplasts isolated from rye (Secale cereale L. cv Puma) grown at 5 degrees C (RH) accumulated 260% more plastoquinone A (PQA) per plastid than chloroplasts isolated from rye grown at 20 degrees C (RNH). The number of plastoglobuli increased by 270% in RH chloroplasts compared with RNH plastids. When RH plastids were lysed and washed, the number of plastoglobuli associated with thylakoid membranes decreased significantly, yet the PQA levels remained high. Room temperature fluorescence induction indicated that (a) there is no change in the size of the PQA pool immediately available for photochemistry in RNH and RH thylakoids and (b) there is a pool of oxidized PQA present in RNH and RH thylakoids which is not available for photochemistry. The accumulated PQA in RH thylakoids may reflect an increased nonphotochemical function such as regulation of thylakoid protein phosphorylation or protection against photoinhibition. PMID- 16663490 TI - Regulation of k influx in barley : effects of low temperature. AB - Influx and accumulation of K(+) in barley (Hordeum vulgare L. cv Fergus) roots were measured at two temperatures (10 degrees C and 20 degrees C) in plants which had been grown with roots and shoots at 20 degrees C (HT plants), with roots and shoots at 10 degrees C (LT plants), and with roots at 10 degrees C and shoots at 20 degrees C (DT plants). Under conditions where K(+) was in limited supply during the prior growth period, K(+) influx and accumulation were consistently higher in roots of DT and LT plants than in those of HT plants. Thus, it would appear that this low temperature response is not limited specifically to conditions in which temperature differentials are maintained between roots and shoots. Nevertheless, it was generally the case that increases of influx were larger in DT and LT plants so that the temperature differentials may intensify the low temperature response. When K(+) influx was examined over a wide range of root [K(+)], it was seen that the characteristic reduction of influx associated with increased internal [K(+)] was substantially greater in HT than DT or LT plants. Transfer of plants grown under HT conditions to DT or LT regimes led to both short-term and long-term adjustments of influx. The former became apparent within 6 hours of exposure to the new conditions and decayed within minutes of transfer back to 20 degrees C. The long-term adjustments were only apparent after prolonged exposure (days) to the lower root temperature and these did not decay as rapidly. Regardless of shoot temperature, the transfer of roots from 20 degrees C to 10 degrees C caused a gradual increase of root [K(+)] so that 4 days later LT and DT roots contained, respectively, 53.3 and 49.83 micromoles per gram compared to 17.82 micromoles per gram for roots maintained at 20 degrees C. PMID- 16663491 TI - Freeze-fracture ultrastructure of thylakoid membranes in chloroplasts from manganese-deficient plants. AB - Leaves from spinach (Spinacia oleracea L. cv Hybrid 102) plants grown in Mn deficient nutrient solution were characterized by chlorosis, lowered chlorophyll a/b ratio and reduced electron transport. There were characteristic changes in room temperature fluorescence induction kinetics with increased initial yield (F(o)) and decreased variable fluorescence (F(v)). The fluorescence yield after the maximum fell rapidly to a level below F(o). The shape of the rise from F(o) to the maximum was altered and the size of photosystem II units increased, as measured by half-rise time of F(v) in the presence of 3-(3,4-dichlorophenyl)-1,1 dimethylurea. The Mn-deficient leaves were harvested before necrosis, when thin section electron microscopy revealed no disorganization of the thylakoid system. Thylakoid membranes were examined by freeze-fracture electron microscopy. The effect of Mn-deficiency was the specific loss of three-quarters of the particles from the endoplasmic fracture face of appressed thylakoids (EFs). Mn-deficient leaves were restored to near normal 2 days after application of exogenous Mn to the nutrient solution. It is concluded that the loss of most, but not all, functional photosystem II reaction centers from grana, with no alteration in light-harvesting complex or photosystem I, is responsible for the fluorescence and functional properties observed. The response of thylakoids to Mn deficiency shows that there is a fundamental difference in composition and function of stacked and unstacked endoplasmic fracture particles. The stacked endoplasmic fracture particle probably contains, in close association, the photosystem II reaction center and also the Mn-containing polypeptide, the 3-(3,4 dichlorophenyl)-1,1-dimethylurea-binding protein, and all electron transport components in between. PMID- 16663492 TI - A Highly Active Oxygen-Evolving Photosystem II Preparation from the Cyanobacterium Anacystis nidulans. AB - A highly active O(2)-evolving Photosystem (PS)-II fraction has been isolated from the cyanobacterium, Anacystis nidulans R2, using an isolation buffer containing high concentrations of sucrose and salts and subsequent solubilization of the thylakoid membranes with the detergent Triton X-100. The isolated fraction had very high PSII activity (2500 micromoles O(2) per milligram chlorophyll per hour) and was largely depleted of PSI activity. Fluorescence emission spectra (77 K) and polypeptide analysis indicated that this preparation is highly enriched in PSII, but almost completely devoid of Cyt b(6)-f and PSI complexes. PMID- 16663493 TI - Ability of Pollen to Germinate prior to Anthesis and Effect of Desiccation on Germination. AB - The ability of pollen to germinate prior to anthesis was tested using Easter lily (Lilium longiflorum L.) and corn (Zea mays L.). Lily pollen normally dries to a low moisture content between anthesis and pollination while corn does not. The corn pollen germinated well (about 73%) when removed from anthers 1 day before anthesis and placed on culture medium. The lily pollen germinated poorly (0 to 5%) when harvested one to six days before anthesis. However, the lily pollen harvested one or two days before anthesis gave greatly improved germination (about 55%) after it was dried to a low moisture content. The results indicate that an internal control prevents premature germination of lily pollen and that drying is the final stage of pollen maturation. A different sort of regulatory mechanism must operate to prevent premature germination of corn pollen. PMID- 16663494 TI - Synthesis of the crystalloid protein complex in vivo in the endosperm of developing castor bean seeds. AB - The synthesis of subunit polypeptides of the crystalloid protein complex has been examined in endosperm from developing castor bean (Ricinus communis L. cv Hale) seeds. Pulse-label and -chase studies in vivo have shown that synthesis initially involves the formation of high molecular weight precursors (50 to 60 kilodaltons) comprising peptide-linked acidic and basic polypeptides. Precursor processing involves the posttranslational cleavage of the peptide bond to yield authentic and polypeptides. This processing has a half-life of 35 to 40 minutes and is preceded by a 45- to 60-minute lag period. Both precursor and subunit polypeptides are shown to exhibit similar molecular weight and pI heterogeneity, and this is suggested to be due to the expression of a multigene family. PMID- 16663495 TI - Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica. AB - Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline. PMID- 16663496 TI - Temperature-dependent phase behavior of phosphatidylglycerols from chilling sensitive and chilling-resistant plants. AB - Seven major lipid classes were isolated from leaves of chilling-sensitive and chilling-resistant plants, and the temperature-dependent phase behaviors of their aqueous dispersions were studied by a fluorescence polarization method using trans-parinaric acid and its methyl ester. Phosphatidylglycerols from the chilling-sensitive plants went from the liquid crystalline state into the phase separation state at about 30 degrees C in 100 mm NaCl and at about 40 degrees C in 5 mm MgCl(2). In contrast, phosphatidylglycerols from the chilling-resistant plants went into the phase separation state at a much lower temperature. The other classes of lipids remained in the liquid crystalline state at all temperatures between 5 degrees C and 40 degrees C regardless of the chilling sensitivity of the plants, except sulfoquinovosyl diacylglycerol from sponge cucumber in which phase separation seemed to begin at about 15 degrees C. Compositions and positional distributions of fatty acids of the lipids suggest that the phosphatidylglycerols from the chilling-sensitive plants, but no other lipids, contained large proportions of molecular species which undergo phase transition at room temperature or above. The thermotropic phase behaviors and the fatty acid compositions suggest that, among the major lipid classes from leaves of the chilling-sensitive plants, only phosphatidylglycerol can induce a phase transition. Since a major part of this lipid in leaves originates from the chloroplasts, phase transition probably occurs in the chloroplast membranes. PMID- 16663497 TI - Isolation and Partial Purification of Cadmium-Binding Protein from Roots of the Grass Agrostis gigantea. AB - A cadmium-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat-stable proteins were chromatographed on the anion exchanger QAE Sephadex A-25. The major cadmium fraction was purified further by gel filtration on Sephadex G-75 in 1 molar KCl buffer. The resulting protein preparation was light brown, had an apparent molecular weight of 3700, contained 29% cysteine and close to 4 gram atoms cadmium/mole. The cadmium:cysteine ratio was 1:2.7. Spectroscopic measurements indicated cadmium-thiolate coordination. The roots produced the metallothionein-like protein when they were exposed to cadmium for 7 days. PMID- 16663498 TI - Enzymes of sucrose breakdown in soybean nodules: alkaline invertase. AB - The specific activities of acid and alkaline invertases (beta-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-alpha-d glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6 phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6 phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.Soybean nodule alkaline invertase was partially purified and shown to be a beta-d-fructofuranosidase which was specific for sucrose. The pH optimum was 7.6 and the K(m) for sucrose was 10 millimolar. Fructose was a competitive inhibitor. Tris was a noncompetitive inhibitor and the enzyme was very sensitive to inhibition by heavy metals. PMID- 16663499 TI - Inactivation by phenylglyoxal of the specific binding of 1-naphthyl acetic Acid with membrane-bound auxin binding sites from maize coleoptiles. AB - The specific binding of 1-[(3)H]naphthyl acetic acid (NAA) to membrane-bound binding sites from maize (Zea mays cv INRA 258) coleoptiles is inactivated by phenylglyoxal. The inactivation obeys pseudo first-order kinetics. The rate of inactivation is proportional to phenylglyoxal concentration. Under conditions at which significant binding occurs, NAA, R and S-1-naphthyl 2-propionic acids protect the auxin binding site against inactivation by phenylglyoxal. Scatchard analysis shows that the inhibition of binding corresponds to a decrease in the concentration of sites but not in the affinity. The results of the present chemical modification study indicate that at least one arginyl residue is involved in the positively charged recognition site of the carboxylate anion of NAA. PMID- 16663500 TI - Differences in water relations and physiological characteristics in leaves of wheat associated with leaf position on the plant. AB - The seasonal change in leaf water potential and its components, stomatal resistance, specific leaf weight, photosynthesis rate, the activities of ribulose 1,5-bisphosphate carboxylase and nitrate reductase, and soluble proteins were measured in flag leaves (ninth from base in position), seventh and fifth leaves of wheat Triticum aestivum L. cv Kalyansona. Flag leaves had a lower water and solute potential and lower or equal turgor pressure than seventh and fifth leaves. These differences were found to be independent of environment. The rate of photosynthesis and nitrate reductase activity were always lower in fifth and seventh leaves than in flag leaf. The photosynthetic efficiency in flag leaves appeared to be associated with lower stomatal resistance and higher specific leaf weight. The relations between leaf water potential and relative water content showed a change with leaf position. This change possibly allows flag leaf to maintain its functional efficiency despite its lower water potential. PMID- 16663501 TI - Ultrasonic Acoustic Emissions from the Sapwood of Thuja occidentalis Measured inside a Pressure Bomb. AB - An improved method of counting acoustic emission (AE) events from water-stressed stems of cedar (Thuja occidentalis L.) is presented. Amplified AEs are analyzed on a real time basis by a microcomputer. The instrumentation counts AE events in a fashion nearly analogous to scintillation counting of radioactive materials.The technique was applied to measuring ultrasonic AEs from the stems of cedar inside a pressure bomb. The shoots were originally fully hydrated. When the shoots are dehydrated in the bomb by application of an overpressure very few AEs were detected. When the bomb pressure is reduced after dehydration of the shoot, AE events could be detected. We conclude that ultrasonic AEs are caused by cavitation events (= structural breakdown of water columns in the tracheids of cedar) and not by the breaking of cellulose fibers in the wood. PMID- 16663502 TI - Characterization of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson. AB - The nature and sequence of metabolic events during phase II (early morning) Crassulacean acid metabolism in Opuntia erinacea var columbiana (Griffiths) L. Benson were characterized. Gas exchange measurements under 2 and 21% O(2) revealed increased O(2) inhibition of CO(2) fixation with progression of phase II. Malate and titratable acidity patterns indicated continued synthesis of C(4) acids for at least 30 minutes into the light period. Potential activities of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme exhibited little change during phase II, while light activation of NADP-malate dehydrogenase, pyruvate, orthophosphate dikinase, and ribulose-1,5-bisphosphate carboxylase was apparent. Short-term (14)CO(2) fixation experiments showed that the per cent of (14)C incorporated into C(4) acids decreased while incorporation into other metabolites increased with time. PEPC exhibited increased sensitivity to 2 millimolar malate, and the K(i)(malate) for PEPC decreased markedly with time. Sensitivity of PEPC to malate inhibition was considerably greater at pH 7.5 than at 8.0. The results indicate that decarboxylation and synthesis of malate occur simultaneously during the early morning period, and that phase II acid metabolism is not limited by CO(2) diffusion through stomata. With progression of phase II, CO(2) fixation by PEPC decreases while fixation by ribulose-1,5-bisphosphate carboxylase increases. PMID- 16663503 TI - Role of seagrass photosynthesis in root aerobic processes. AB - The role of shoot photosynthesis as a means of supporting aerobic respiration in the roots of the seagrass Zostera marina was examined. O(2) was transported rapidly (10-15 minutes) from the shoots to the root-rhizome tissues upon shoot illumination. The highest rates of transport were in shoots possessing the greatest biomass and leaf area. The rates of O(2) transport do not support a simple gas phase diffusion mechanism. O(2) transport to the root-rhizome system supported aerobic root respiration and in many cases exceeded respiratory requirements leading to O(2) release from the subterranean tissue. Release of O(2) can support aerobic processes in reducing sediments typical of Z. marina habitats. Since the root-rhizome respiration is supported primarily under shoot photosynthetic conditions, then the daily period of photosynthesis determines the diurnal period of root aerobiosis. PMID- 16663504 TI - Chloroplast ultrastructure, chlorophyll fluorescence, and pigment composition in chilling-stressed soybeans. AB - Shoots of 16-day-old soybeans (Glycine max L. Merr. cv Ransom) were chilled to 10 degrees C for 7 days and monitored for visible signs of damage, ultrastructural changes, perturbations in fluorescence of chlorophyll (Chl), and quantitative changes in Chl a and b and associated pigments. Precautions were taken to prevent the confounding effects of water stress. A technique for the separation of lutein and zeaxanthin was developed utilizing a step gradient with the high performance liquid chromatograph. Visible losses in Chl were detectable within the first day of chilling, and regreening did not occur until the shoots were returned to 25 degrees C. Ultrastructurally, unstacking of chloroplast grana occurred, and the envelope membranes developed protrusions. Furthermore, the lipids were altered to the point that the membranes were poorly stabilized by a glutaraldehyde/osmium double-fixation procedure. Chl fluorescence rates were greatly reduced within 2 hours after chilling began and returned to normal only after rewarming. The rapid loss of Chl that occurred during chilling was accompanied by the appearance of zeaxanthin and a decline in violaxanthin. Apparently a zeaxanthin-violaxanthin epoxidation/de-epoxidation cycle was operating. When only the roots were chilled, no substantial changes were detected in ultrastructure, fluorescence rates, or pigment levels. PMID- 16663505 TI - Tetranitromethane oxidation of phytochrome chromophore as a function of spectral form and molecular weight. AB - Tetranitromethane bleaches Avena phytochrome. The phytochrome (far-red absorbing form; Pfr) chromophore of 124 kilodalton (kD) phytochrome is oxidized 8 times more rapidly than the red absorbing form (Pr). Proteolysis of the 124 kD molecule to the extensively studied mixture of 118 and 114 kD polypeptides increases the rate of oxidation of Pfr 5-fold without affecting the rate of Pr oxidation. As a result, the Pfr form of 118/114 kD preparations is oxidized at a rate 40 times greater than the Pr form. Further proteolytic degradation of the chromoprotein to 60 kD results in an additional increase in the oxidation rates of both Pr and Pfr. These differences in reactivity to tetranitromethane indicate that the chromophore of Pfr is either intrinsically more chemically reactive and/or physically more accessible than the Pr chromophore and that the reactivity/accessibility of both spectral forms is increased by proteolysis. The enhanced reactivity of the Pfr chromophore after proteolytic cleavage of the 6 to 10 kD polypeptide segment(s) from the 124 kD species is further evidence that these segment(s) affect the environment of the native photoreceptor. PMID- 16663506 TI - Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5 bisphosphate Carboxylase and the Relationship to Photosynthesis. AB - The in vitro ribulose-1,5-bisphosphate (RuBP) carboxylase activity per unit of leaf nitrogen was found to be 30% greater in Triticum aestivum than in T. monococcum. This was due to a higher specific activity of the enzyme from T. aestivum, as the amount of RuBP carboxylase protein per unit of total leaf nitrogen did not differ between the genotypes. The occurrence of higher specific activity of RuBP carboxylase is shown to correlate with possession of the large subunit derived from the B genome of wheat.Despite the greater RuBP carboxylase activity per unit of leaf nitrogen in T. aestivum, the initial slopes of curves relating rate of CO(2) assimilation to intercellular p(CO(2)) are similar in T. aestivum and T. monococcum for the same nitrogen content per unit leaf area. The similarity of the initial slopes is the result of a greater resistance to CO(2) transfer between the intercellular spaces and the site of carboxylation in T. aestivum than in T. monococcum. PMID- 16663507 TI - Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1) ATPase from the Halotolerant Alga Dunaliella bardawil. AB - The isolation of the chloroplast ATP synthase complex (CF(0)-CF(1)) and of CF(1) from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil alpha subunit migrates ahead of the beta subunit and epsilon-migrates ahead of subunit II of CF(0) when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF(1) isolated from D. bardawil resembles the CF(1) isolated from Chladmydomonas reinhardi in that a reversible, Mg(2+)-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P(i) exchange catalyzed by D. bardawil CF(0) CF(1) proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg(2+)-ATPase of the D. bardawil CF(1) is more resistant to glycerol inhibition than the octylglucoside-activated, Mg(2+)-ATPase of spinach CF(1) or the ethanol-activated, Mg(2+)-dependent ATPase of the C. reinhardi CF(1). Both cyclic photophosphorylation and ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) are more sensitive to high concentrations of NaCl than is the spinach complex. PMID- 16663508 TI - Properties of Lectins in the Root and Seed of Lotononis bainesii. AB - A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin. PMID- 16663509 TI - Role of Lectins in the Specific Recognition of Rhizobium by Lotononis bainesii. AB - Fluorescein isothiocyanate (FITC)-labeled lectin purified from the root of Lotononis bainesii Baker was bound by cells of five out of seven L. bainesii nodulating strains of Rhizobium under culture conditions. With the exception of a strain of Rhizobium leguminosarum, strains of noninfective rhizobia failed to bind the root lectin under these conditions. The two nonlectin binding L. bainesii-specific strains did not bind root lectin on the L. bainesii rhizoplane although this was observed with three other L. bainesii-nodulating strains. A single Rhizobium japonicum strain bound root lectin on the L. bainesii rhizoplane. There was no evidence of an interaction between the L. bainesii seed lectin and the Rhizobium strains tested.Root lectin-specific FITC-labeled antibodies were bound to the tips of developing root hairs and lateral growth points of more mature root hairs of L. bainesii seedlings. The damaged edges of severed root hairs always bound FITC-labeled root lectin antibody. Seed lectin specific FITC-labeled antibodies were not bound to the roots of L. bainesii. The preemergent root hair region of L. bainesii was most susceptible to infection by rhizobia but nodules also emerged in the developing and mature root hair regions. Lectin exposed at growth points on L. bainesii root hairs may provide a favorable site for host plant recognition of infective strains of Rhizobium. PMID- 16663510 TI - Tolerance of photosynthesis to high temperature in desert plants. AB - Winter- and summertime-active desert annual species were grown at different temperatures to assess their capacity for photosynthetic acclimation. Thermal stability of photosynthesis was determined from responses of chlorophyll fluorescence to increased temperature. Photosynthesis in winter ephemerals grown at 28 degrees C/21 degrees C became unstable close to 41 degrees C in contrast to the summer annuals which were stable up to about 46 degrees C. Growth at higher temperature (43 degrees C/32 degrees C) resulted in increases in thermal stability of 5 to 7 degrees C for the winter annuals and 3 to 4 degrees C for the summer annuals, showing that temperature can provide the primary stimulus for acclimation of the photosynthetic apparatus. The magnitude of these changes was very similar to the range of field values observed for the respective floras, indicating that the thermal acclimation response under field conditions was qualitatively similar to that occurring under controlled growth conditions. Perennial species, co-existing with these annuals in the desert, were on average more thermostable. The cacti were exceptionally heat stable, the threshold for fluorescence increase averaging 55 degrees C. PMID- 16663511 TI - Variations in the Specific Activity of Ribulose-1,5-bisphosphate Carboxylase between Species Utilizing Differing Photosynthetic Pathways. AB - The in vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase) (micromoles CO(2) fixed per minute per milligram enzyme) from a number of C(3) and C(4) species and one green alga were measured. RuBPCases from species which utilize the C(4) pathway have a specific activity approximately 2-fold higher than those from C(3) species. RuBPCase from Chlamydomonas reinhardtii has a specific activity similar to the C(4) enzyme. Higher specific activity forms of RuBPCase are associated with a decreased enzyme affinity for CO(2) (increased K(m)[CO(2)]). A small but significant difference in the specific activity of RuBPCase from two C(4) decarboxylation types was also observed. The relationship between enzymic properties and the presence or absence of a CO(2) concentrating mechanism is discussed. PMID- 16663512 TI - De Novo Maltotriose Biosynthesis from the Reducing End by Spinacia oleracea L. Chloroplasts. AB - The distribution of (14)C in the various glucose residues of maltotriose was studied as a function of time of photosynthesis of isolated chloroplasts of spinach (Spinacia oleracea L.) using (14)CO(2). The distribution of label showed that the reducing-end glucose residue was labeled first and the label subsequently distributed to the second and third glucose residues at approximately equal rates.A mechanism for the distribution of label and the synthesis of maltotriose from the reducing end is presented. The mechanism has postulated to be the same as that for the maltose synthase recently described by Schilling. Maltose biosynthesis from alpha-d-glucose-1-phosphate was characterized as involving two glucosyl intermediates by a double displacement mechanism with inversion of configuration. The mode of enzymic action by which maltosyl intermediates were transferred to glucosyl intermediates was consistent with the fractional distribution of radioactivity found in each glucose unit of maltotriose during short term photosynthesis experiments. PMID- 16663513 TI - Soybean leaf urease: a seed enzyme? AB - The soybean (Glycine max L. [Merrill]) var Itachi has 0.2 to 0.3% the urease activity found in developing embryos of a normal line, Prize. The hydroxyurea sensitivity and pH preference of this basal seed urease indicate that it represents a unique enzyme rather than an unusually low level of the normal seed urease. Itachi's seed urease is less sensitive to hydroxyurea inhibition (65-80% inhibition) than Prize seed urease (85-95% inhibition) and is more active at pH 6.1 and 8.8 than at 7.4, whereas the normal seed urease is least active at pH 8.8. Both properties of the basal seed urease are in agreement with the behavior of the leaf urease in extracts of Prize and Itachi leaves.Neither the leaf urease nor the Itachi seed urease is immuneprecipitated by affinity-purified seed urease antibodies. However, when antibody is in excess, Staphylococcus aureus (Cowan) cell walls containing protein A can precipitate soluble antibody-urease complexes (47-68% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% of Prize seed urease is precipitated. At a 100-fold dilution of antibody, 60% of Prize seed urease is still antibody-complexed while the antibody recognition of the leaf or Itachi seed urease is reduced to 2 to 24%.The cell culture urease also resembles leaf urease by the criteria of pH preference, hydroxyurea sensitivity, and recognition by seed urease antibodies. In the presence of cycloheximide, nickel stimulates cell culture urease levels (14- or 35-fold depending on assay pH) indicating that cell cultures make a preponderance of apourease under nickel-limiting conditions.Inasmuch as the ureases of leaf, cell culture, and Itachi seeds are more closely related to each other than they are to the abundant (Prize) seed urease, suggests that the three tissues either contain an identical urease or related tissue-specific isozymes. This second form of urease may have an assimilatory role since it is found in both leaf and seed sink tissues and is required for urea assimilation in cell culture (Polacco 1977 Plant Physiol 59: 827-830). PMID- 16663514 TI - Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn. AB - The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca(2+) transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive (45)Ca(2+) transport into mitochondria of corn roots. Ca(2+) uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca(2+) uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca(2+) transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate driven Ca(2+) transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca(2+) uptake. However, ATP-dependent Ca(2+) uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([(14)C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn. PMID- 16663515 TI - Abscisic Acid metabolism by source and sink tissues of sugar beet. AB - The fate of exogenously applied, labeled abscisic acid (+/-)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10(-6) molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues. PMID- 16663516 TI - Imbibitional chilling injury in pollen: involvement of the respiratory chain. AB - Chilling injury is sustained by dry pollen of Typha latifolia L. upon hydration in germination medium at 0 degrees C. This injury is evidenced as poor germination, low vigor, and depressed respiration. Isolated mitochondria showed multiple sites of impaired electron transport. Besides losses of cytochrome (Cyt) c and NAD(+), the activities of membrane-bound enzyme complexes such as Cyt oxidase, NADH-duroquinone oxidoreductase, succinate-duroquinone oxidoreductase, and malate-duroquinone oxidoreductase were severely affected.Similarly, as in isolated mitochondria, in situ tests of mitochondrial activity showed that Cyt c was partially lost from its site of action. Re-addition of the lost Cyt c to the grains restored the N,N,N',N'-tetramethyl p-phenylenediamine dihydrochloride plus ascorbate-mediated electron transport from Cyt c to O(2), but did not significantly accelerate the overall O(2) uptake. Electron flow to duroquinone in the injured grains was low, indicating that lesions at the substrate side of ubiquinone determine the rate of O(2) consumption. Leakage of NAD(+), and also of adenylate phosphates and Krebs cycle substrates out of the injured grains, was considerable.Increasing the initial moisture content of the grains strongly enhanced their resistance to cold hydration. Below 17% moisture content (fresh weight basis), the decrease in vigor closely matched the loss of NAD(+) and adenosine phosphates. Vitality was irreversibly lost by cold hydration below 10 to 12% initial moisture content.Injury to dry pollen was prevented by imbibition at 27 degrees C. Decrease of vigor and increased leakage, however, started below 20 degrees C, and complete loss of vitality occurred below 10 degrees C.These results are interpreted as evidence that loss of membrane integrity is the primary cause of imbibitional chilling injury. PMID- 16663517 TI - Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine. AB - The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with (15)N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas. PMID- 16663518 TI - Purification and comparative characterization of an enolase from spinach. AB - An enolase has been purified to apparent homogeneity, as measured by gel electrophoresis, some 400-fold from spinach (Spinacia oleracea). This is the first plant enolase that has been purified to homogeneity. At moderate ionic strengths, the 5,5-dithio-bis-2-(nitrobenzoate) (DTNB)-or parachloromercuribenzoate-reacted enzyme elutes from a Bio-Gel P-200 column with somewhat greater volumes than the yeast enzyme (M(r) = 93,000) indicating a greater size. Its elution volume from Ultrogel in 50% ammonium sulfate, however, suggests it exists as an active monomer (M(r) = 47,000). Sodium dodecyl sulfate gel electrophoresis indicates the subunit molecular weight is 50,000 +/- 3,000, like that of yeast enolase.The enzyme contains 23 +/- 4 half-cystines per mole of subunit. Titrations with DTNB in guanidine hydrochloride or nondenaturing media indicate that most of these, if not all, are in the reduced state. Reaction of one or more of the sulfhydryls with DTNB or parachloromercuribenzoate stabilizes the enzyme.The kinetic parameters of the reaction catalyzed by spinach enolase, as well as the inhibitions by transition metal ions and fluoride, are similar to those properties of the yeast and rabbit muscle enzymes. PMID- 16663519 TI - Regulation of alpha-amylase activity in bean stem tissues. AB - alpha-Amylase activity was assayed in 1-centimeter sections taken from bean (Phaseolus vulgaris var. Kentucky Wonder) hypocotyls and epicotyls at measured distances from the cotyledons. The activity was low throughout the hypocotyl for the first 7 days. An increase was first observed with etiolated hypocotyls in the basal region, becoming higher in the more central regions by 14 to 17 days. By 21 days the activity was highest in the upper region, but had decreased in the lower regions. A comparable pattern was observed for the epicotyl from etiolated seedlings, the activity increasing first in the region closest to the cotyledons. These increases were associated with loss of cells from the pith in the hypocotyl and epicotyl of both dark- and light-grown plants. Since the changes were observed in tissues virtually devoid of starch, it is hypothesized that the control mechanism is related to the cellular disassembly associated with the mobilization of materials released during senescence rather than to a regulation by the enzyme's substrate or products. PMID- 16663520 TI - Peroxidase Release Induced by Ozone in Sedum album Leaves: Involvement of Ca. AB - The effect of ozone was studied on the peroxidase activity from various compartments of Sedum album leaves (epidermis, intercellular fluid, residual cell material, and total cell material). The greatest increase following a 2-hour ozone exposure (0.4 microliters O(3) per liter) was observed in extracellular peroxidases. Most of the main bands of peroxidase activity separated by isoelectric focusing exhibited an increase upon exposure to ozone. Incubation experiments with isolated peeled or unpeeled leaves showed that leaves from ozone treated plants release much more peroxidases in the medium than untreated leaves. The withdrawal of Ca(2+) ions reduced the level of extracellular peroxidase activity either in whole plants or in incubation experiments. This reduction and the activation obtained after addition of Ca(2+) resulted from a direct requirement of Ca(2+) by the enzyme and from an effect of Ca(2+) on peroxidase secretion. The ionophore A23187 promoted an increase of extracellular peroxidase activity only in untreated plants. The release of peroxidases by untreated and ozone-treated leaves is considerably lowered by metabolic inhibitors (3-(3,4 dichlorophenyl)-1,1-dimethylurea and sodium azide) and by puromycin. PMID- 16663521 TI - Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts. AB - Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H(2)O(2). The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H(2)O(2), and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN(3). Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H(2)O(2). These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts. PMID- 16663522 TI - Purification and properties of spinach leaf debranching enzyme. AB - Starch debranching enzyme was purified from intact spinach (Spinacia oleracea L. cv Vital) chloroplasts and from a spinach leaf extract using affinity chromatography on Sepharose 6B-bound cycloheptaamylose (Schardinger beta dextrin). The enzyme from both sources was homogeneous upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spinach leaf debranching enzyme appears to consist of a single polypeptide chain, since the molecular weight of the native protein (110,000 daltons) was not changed by treatment with sodium dodecyl sulfate. Only one spinach leaf debranching enzyme band could be detected after electrophoresis of a leaf extract on amylopectin-containing polyacrylamide gel, the retardation factor of which coincided with that of the single band seen with the chloroplast enzyme. The purified enzyme exhibited strong pullulanase activity, the specific activity being 69 units per milligram protein with pullulan and 22 units per milligram protein with amylopectin. Cycloheptaamylose is a potent competitive inhibitor of spinach leaf debranching enzyme. The pH optimum of the enzyme was found to be 5.5. The purified enzyme is rather unstable at both 20 degrees and 0 degrees C. Part of the activity lost under storage or at a suboptimal pH could immediately be restored by the addition of thiols. The reactivatable protein, being of the same molecular weight as the native enzyme, exhibited a somewhat altered electrophoretic mobility resulting in one or two minor bands on a zymogram. PMID- 16663523 TI - Xylem development in prunus flower buds and the relationship to deep supercooling. AB - Xylem development in eight Prunus species was examined and the relationship to deep supercooling assessed. Dormant buds of six species, P. armeniaca, P. avium, P. cerasus, P. persica, P. salicina, and P. sargentii deep supercooled. Xylem vessel elements were not observed within the dormant floral primordia of these species. Instead, discrete bundles containing procambial cells were observed. Vascular differentiation resumed and xylem continuity was established during the time that the capacity to deep supercool was lost. In P. serotina and P. virginiana, two species which do not supercool, xylem vessels ran the length of the inflorescence and presumably provided a conduit for the spread of ice into the bud. The results support the hypothesis that the lack of xylem continuity is an important feature of buds which deep supercool. PMID- 16663524 TI - Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum. AB - The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C(4) plant, 96 to 100% of adenosine 5' phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C(3) species, ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5' phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5'-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C(4) plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants. PMID- 16663525 TI - Enhancement of Ethylene Release from Leaf Tissue during Glycolate Decarboxylation : A Possible Role for Photorespiration. AB - When leaf discs of Xanthium strumarium L. and Salvia splendens L. are incubated in sealed flasks in the light, more C(2)H(4) gas is released in the presence of added CO(2) (30-200 millimolar NaHCO(3)) than without CO(2). In Salvia, the maximum rate of C(2)H(4) release occurs when sufficient CO(2) (above 125 millimolar NaHCO(3)) is added to saturate photosynthesis confirming previous studies. The maximum rate of C(2)H(4) release from illuminated discs is similar to the rate in the dark with or without CO(2) in both species. Glycolate enhances a CO(2)-dependent C(2)H(4) evolution from illuminated leaf discs. However, the maximum rate of C(2)H(4) release with glycolate is the same as that observed with saturating CO(2). When photosynthesis is inhibited by darkness or by 3-(3,4 dichlorophenyl)-1,1-dimethylurea, glycolate has no effect.Studies with [2,3 (14)C]-1-aminocyclopropane-1-carboxylic acid (ACC) show that the pattern of C(2)H(4) release and the specific activity of the (14)C(2)H(4) in the presence and absence of glycolate is similar to that described above, indicating that glycolate does not alter uptake of the exogenously supplied precursor (ACC) or stimulate C(2)H(4) release from an endogenous source at appreciable rates. Glycolate oxidase in vitro generates H(2)O(2) which stimulates a slow breakdown of ACC to C(2)H(4), but since exogenous glycolate is oxidized to CO(2) in both the light and the dark it is argued that the glycolate-dependent increase in C(2)H(4) release from illuminated leaf discs is not mediated directly by the action of enzymes of glycolate catabolism. The effects of glycolate and CO(2) are not easily explained by changes in stomatal resistance. The data support the view that glycolate decarboxylation at subsaturating levels of CO(2) in the light stimulates C(2)H(4) release by raising the CO(2) level in the tissue. PMID- 16663526 TI - Kaempferol glycosides and enzymes of flavonol biosynthesis in leaves of a soybean strain with low photosynthetic rates. AB - Soybean (Glycine max L.) strains which accumulate kaempferol 3-(2(G) glucosylgentiobioside) in their leaves fix CO(2) at rates significantly lower than those lacking this compound (Buttery, Buzzell 1976 Crop Sci 16: 547-550), and kaempferol aglycone is a well known inhibitor of photosynthesis in vitro. However, since neither kaempferol nor any of its glycosides could be detected in mesophyll cells isolated from mature soybean leaves we suspect that kaempferol 3 (2(G)-glucosylgentiobioside) has no direct inhibitory effect on photosynthesis. The most rapid stage of flavonoid accumulation, and the highest level of activity for several enzymes of phenolic biosynthesis, occurs in leaflets 2.5 to 3 centimeters long. Mesophyll cells isolated from these leaflets contain about 70% of the whole leaf activity for shikimate dehydrogenase, 24% of the 4 coumarate:CoA ligase activity, 35% of the activity for chalcone-flavanone isomerase, but no demonstrable activity for phenylalanine ammonialyase. Our results suggest a highly tissue-specific pattern of secondary phenolic metabolism in soybean leaves. PMID- 16663527 TI - Transpiration- and growth-induced water potentials in maize. AB - Recent evidence from leaves and stems indicates that gradients in water potential (psi(w)) necessary for water movement through growing tissues are larger than previously assumed. Because growth is sensitive to tissue psi(w) and the behavior of these gradients has not been investigated in transpiring plants, we examined the water status of all the growing and mature vegetative tissues of maize (Zea mays L.) during high and low rates of transpiration. The psi(w) measured in the mature regions of the plant responded primarily to transpiration, while the psi(w) in the growing regions was affected both by transpiration and growth. The transpiration-induced potentials of the mature tissue formed a gradient of decreasing psi(w) along the transpiration stream while the growth-induced potentials formed a gradient of decreasing psi(w) from the transpiration stream to the expanding cells in the growing tissue. The growth-induced gradient in psi(w) within the leaf remained fairly constant as the xylem psi(w) decreased during the day and was associated with a decreased osmotic potential (psi(s)) of the growing region (osmotic adjustment). The growth-induced gradient in psi(w) was not caused by excision of the tissue because intact maize stems exhibited a similar psi(w). These observations support the concept that large gradients in psi(w) are required to maintain water flow to expanding cells within all the vegetative tissues and suggest that the maintenance of a favorable gradient in psi(w) for cell enlargement may be an important role for osmotic adjustment. PMID- 16663528 TI - Rates and products of long-chain Fatty Acid synthesis from [1-C]acetate in chloroplasts isolated from leaves of 16:3 and 18:3 plants. AB - Chloroplasts highly active in the synthesis of long-chain fatty acids from [1 (14)C]acetate were prepared from leaves of Solanum nodiflorum, Chenopodium quinoa, Carthamus tinctorius, and Pisum sativum. These preparations were used to test whether the various additions to incubation media found to stimulate the synthesis of particular lipid classes in vitro by Spinacia oleracea chloroplasts were applicable generally. Chloroplasts from 18:3 plants incorporated a greater proportion of radioactivity into unesterified fatty acids under control conditions than did those from 16:3 plants. Supplying exogenous sn-glycerol 3 phosphate or Triton X-100 to chloroplasts increased the synthesis of glycerolipids in all cases and accentuated the capacity of chloroplasts from 18:3 plants to accumulate phosphatidic acid rather than the diacylglycerol accumulated by chloroplasts from 16:3 plants. The UDP-galactose-dependent synthesis of labeled diacylgalactosylglycerol was much less active in incubations of chloroplasts from 18:3 plants also containing sn-glycerol 3-phosphate and Triton X-100 compared with similar incubations from 16:3 plants. Exogenous CoA stimulated total fatty acid synthesis in all chloroplast preparations and the further addition of ATP diverted radioactivity from the unesterified fatty acid to acyl-CoA. The results have been discussed in terms of the two pathway hypothesis for lipid synthesis in leaves. PMID- 16663529 TI - Photocontrol of Hypocotyl Elongation in De-Etiolated Cucumis sativus L. : Long Term, Fluence Rate-Dependent Responses to Blue Light. AB - Hypocotyl growth in Cucumis sativus L. cv Ridge Greenline is inhibited by increasing blue light (B) fluence rate in a near log linear fashion once a low fluence threshold is exceeded. Deviation from log linearity at the highest fluence rate used here is due to light perceived by the cotyledons and this effect is assigned to phytochrome. This response can be removed by Norflurazon treatment, without affecting the rest of the fluence response curve.There is also some activation of phytochrome by lower fluence rates of B, an effect which contributes to the overall inhibition of growth. Responses to photostationary state and cycling rate indicate, however, that B does not primarily act via phytochrome, but through a specific blue light photoreceptor. PMID- 16663530 TI - Endoplasmic reticulum as a site of phenylpropanoid and flavonoid metabolism in hippeastrum. AB - The nature of bound forms of enzymes of phenylpropanoid and flavonoid metabolism have been investigated in Hippeastrum CV Dutch Red Hybrid. Particulate components of petal homogenates were fractionated on sucrose gradients and the EDTA shift method was employed to characterize membranes of the endoplasmic reticulum. In magnesiumcontaining gradients, a portion of phenylalanine ammonia lyase, chalcone synthase, glucosyl transferase, and all of the trans-cinnamate 4-monooxygenase and NADH Cytochrome c reductase (the last an endoplasmic reticulum marker) were associated with membranes equilibrating at 1.18 specific gravity. In gradients lacking magnesium and containing EDTA, the above activities-except chalcone synthase, which was lost-and protein were diminished at 1.18 specific gravity and enhanced at lower densities characteristic of membranes of the smooth endoplasmic reticulum. These results are consistent with the contention that endoplasmic reticulum is a site of phenylpropanoid and flavonoid metabolism in Hippeastrum. PMID- 16663531 TI - Naturally occurring protein crystals in the potato : inhibitor of papain, chymopapain, and ficin. AB - Protein crystals isolated from potato tubers were found to consist of a proteinase inhibitor active against the cysteine proteinases papain, chymopapain, and ficin. The molecular weight as determined by gel filtration at pH 4.3 or by gel electrophoresis in the presence of dodecylsulfate was 80 kilodaltons. When the inhibitor was evaluated at pH 8.4 in a linear concentration (4-30% polyacrylamide) under nondenaturing conditions, it appeared as two bands of approximately 320 to 350 kilodaltons indicating that the inhibitor forms tetrameric aggregates in neutral or weakly alkaline media, while the monomeric form predominates under acidic conditions. Gel filtration in the presence of varying amounts of papain suggested that the monomer combines with four papain molecules. The inhibitor contains no cystine. PMID- 16663532 TI - Electric Currents around Growing Trichoderma Hyphae, before and after Photoinduction of Conidiation. AB - Electric currents were measured around Trichoderma harzianum (Rifai) hyphae using an extracellular vibrating electrode. A steady current enters growing hyphal tips and along the side of the apical millimeter. In addition, outward currents were detected at about one-ninth of the locations tested, 60 to 150 minutes after illumination but not in dark controls. This sporadic, localized outward current pattern might be an early biophysical response to blue light. PMID- 16663533 TI - Plant Desiccation and Protein Synthesis : V. Stability of Poly (A) and Poly (A) RNA during Desiccation and Their Synthesis upon Rehydration in the Desiccation Tolerant Moss Tortula ruralis and the Intolerant Moss Cratoneuron filicinum. AB - Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)(-) RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)(+) RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)(-) RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)(+) RNA in rapid- and slow dried moss closely reflect the state of the protein synthetic complex in these dried samples.Poly(A)(-) RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)(+) RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)(+) RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)(+) RNA for protein synthesis. The rate of synthesis of poly(A)(+) RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)(+) RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance. PMID- 16663534 TI - Plant Desiccation and Protein Synthesis : VI. Changes in Protein Synthesis Elicited by Desiccation of the Moss Tortula ruralis are Effected at the Translational Level. AB - Upon rehydration of the moss Tortula ruralis following desiccation at a rapid or slow rate, there is increasing utilization of newly synthesized-poly(A)(+) RNA for protein synthesis. Initially, poly(A)(+) RNA conserved in the dry moss is associated with polysomes, but by 2 hours of rehydration there is an overwhelming recruitment of newly synthesized poly(A)(+) RNA, at the expense of conserved messages. In rehydrated moss, there is a marked synthesis in vivo of new proteins, which are separable by two-dimensional electrophoresis, and identifiable by fluorography. These new proteins, termed rehydration proteins, are synthesized after both rapid and slow desiccation, but their synthesis persists longer after rapid desiccation. The protein patterns obtained following in vitro translation of bulk RNA from hydrated, desiccated, and rehydrated moss were qualitatively identical. Thus the differences in protein patterns observed in vivo must result from preferential selection of specific mRNAs from the same pool, which is indicative of control of protein synthesis at the translational level. The implications of these observations in relation to the response of the moss to drying in its natural environment are discussed. PMID- 16663535 TI - Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts. AB - Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. delta-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of delta-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of delta-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, delta aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power. PMID- 16663536 TI - Accumulation and transport of abscisic Acid and its metabolites in ricinus and xanthium. AB - When intact plants of Xanthium strumarium L. were water stressed, the youngest leaves accumulated the highest levels of abscisic acid (ABA). On the other hand, when leaves of different ages were detached and then stressed, the capacity to produce ABA was highest in the mature leaves. Radioactive ABA was transported from mature leaves to the shoot tips and young leaves, as well as to the roots, as evidenced by the presence of radioactive ABA and phaseic acid in the xylem exudate coming from the roots. Thus, ABA was recirculated in the plant, moving down the stem in the phloem and back up in the transpiration stream to the mature leaves. Phloem exudate collected by the use of the EDTA technique had a high concentration of ABA and phaseic acid which increased several-fold after water stress. The high ABA levels in immature leaves and apical buds are, therefore, mainly due to import from older leaves, rather than to in situ synthesis.In Ricinus communis L. cv gibsonii labeled ABA was rapidly exported from a mature leaf as measured by the appearance of radioactive ABA in the phloem collected from incisions in the stem below the treated leaf. After 2 h, small amounts of radioactive phaseic acid and dihydrophaseic acid were also present in the phloem exudate. The glucosyl ester of abscisic acid, beta-d-glucopyranosyl abscisate, was hydrolyzed before entering the phloem and moved as free ABA. It is concluded that the glucosyl ester of ABA itself cannot cross cell membranes and is, therefore, not transported. PMID- 16663537 TI - Characterization of the increased lysophospholipase activity in gibberellic Acid treated barley aleurone layers. AB - A lysophospholipase (LPL) activity appears in the aleurone of barley (Hordeum vulgare L. cv Himalaya) half seeds during imbibition on moist agar. Secretion of LPL by half seeds is promoted by GA(3); the increase in secretory rate is almost linear from 10(-10) to 10(-6) molar GA(3). LPL activity is likewise promoted in isolated aleurone layers by GA(3). Its secretion into the incubation medium requires the continued presence of GA(3) and commences after a 10 to 14 hour lag period when 10 millimolar Ca(2+) is present. In the absence of Ca(2+), the lag period remains unchanged but attainment of the maximum secretory rate is delayed. Ca(2+) alone has very little effect either on LPL activity accumulated in the aleurone layer or in the surrounding medium. However, 50 millimolar Ca(2+) together with GA(3) dramatically increase the level of secreted activity and of total (accumulated and secreted) activity.The metabolic inhibitors cycloheximide and actinomycin D inhibit the accumulation of LPL activity in the aleurone and also the secreted activity. Actinomycin D added after the lag period results in a much lower inhibition. The increase in LPL activity in response to GA(3) occurs as a result of de novo synthesis; LPL activity from barley half seeds incubated in 80% D(2)O in the presence of GA(3) undergoes a shift to higher density compared with the activity from similar controls incubated in H(2)O. The characteristics of the GA(3) enhancement of LPL activity are compared specifically with alpha-amylase and generally with other GA(3)-controlled hydrolases. PMID- 16663538 TI - Heat stress responses in cultured plant cells : effect of culture handling and age. AB - The pipetting of pear (Pyrus communis cv Bartlett) suspension cultures was followed by a substantial but transient decrease in heat sensitivity. During a culture cycle, pear cells were most sensitive to heat at day 3, which coincided with the period of most active cell division. To minimize serious artifacts, the influence of culture handling and age on parameters such as heat sensitivity must be standardized. PMID- 16663539 TI - Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures. AB - Chenopodium rubrum cells were grown in suspension as a photoautotrophic culture with a 16 hour day. Cell growth had three phases: a 3-day lag, a 3-week logarithmic phase, and a 10-day stationary phase. Chlorophyll content increased steadily during log phase and reached a level of 0.5 to 0.6 mg Chl g(-1) fresh weight. Soluble protein of the cells increased more rapidly from day 4 to day 12 than during midlog phase. Initially, ammonium was taken up in preference to nitrate. However, during the second two weeks of growth, ammonium and nitrate were taken up simultaneously; this period of growth was the time of highest rates of N uptake by the cultured cells. Glutamine synthetase had a high specific activity (17 mumol.hour(-1) mg(-1) protein) in day 1 cells, and this level was sustained until midlog phase when it increased by 20%. Methyl viologen-dependent glutamate synthase specific activity increased rapidly in lag phase cells (day 4 = 10 mumol.hour(-1) mg(-1) protein), but decreased by day 9 to about 50% of the peak and remained constant. NADH:nitrate reductase specific activity increased rapidly in lag phase cells and reached a plateau that lasted from day 4 to 14 (1 mumol.hour(-1) mg(-1) protein). Methyl viologen-dependent nitrite reductase specific activity was high when assayed on day 5 and increased to a maximum on day 15 to 16 (12 mumol.hour(-1) mg(-1) protein). NADPH- and NADH-dependent glutamate dehydrogenase specific activities remained rather constant throughout the growth cycle. The cells appeared to have developed photosynthetic competence and to have leaf-like activities of nitrogen assimilation enzymes. PMID- 16663540 TI - Effect of plant hormones on sucrose uptake by sugar beet root tissue discs. AB - Abscisic acid (ABA), auxins, cytokinins, gibberellic acid, alone or in combination were tested for their effects on short-term sucrose uptake in sugar beet (Beta vulgaris cv USH-20) roots. The effect of ABA on active sucrose uptake varied from no effect to the more generally observed 1.4-to 3.0-fold stimulation. A racemic mixture of ABA and its trans isomer were more stimulatory than ABA alone. Pretreating and/or simultaneously treating the tissue with K(+) or IAA prevented the ABA response while cytokinins and gibberellic acid did not. While the variable sensitivities of beet root to ABA may somehow be related to the auxin and alkali cation status of the tissue, tissue sensitivity to ABA was not correlated with ABA uptake, accumulation, or metabolic patterns. In contrast to ABA, indoleacetic acid (IAA) and other auxins strongly inhibited active sucrose uptake in beet roots. Cytokinins enhanced the auxin-induced inhibition of sucrose uptake but ABA and gibberellic acid did not modify or counteract the auxin effect. Trans-zeatin, benzyladenine, kinetin, and gibberellins had no effect on active sucrose uptake. None of the hormones or hormone mixtures tested had any significant effect on passive sucrose uptake. The effects of IAA and ABA on sucrose uptake were detectable within 1 h suggesting a rather close relationship between the physiological activities of IAA and ABA and the operation of the active transport system. PMID- 16663541 TI - Permeability of chloroplast envelopes to mg: effects on protein synthesis. AB - When suspended in media lacking free Mg(2+), chloroplasts from young pea plants (Pisum sativum CV Progress No. 9) lose 25 to 75% of their stromal Mg(2+) content to the medium, without breakage. This effect amounts for the inhibition of protein synthesis in the dark by ATP in excess of the Mg(2+) provided, since free ATP chelates Mg(2+). The rate of loss is from 1 to 4.5 microgram-atoms Mg(2+)/milligram Chl/hour; and depleted chloroplasts take up Mg(2+) from the medium at even faster rates, to a total amount not much more than that present originally (0.8 to 1.8 microgram-atoms/milligram Chl with an average of 1.33 +/- 0.32 mug-atoms/mg Chl). Leakage is completely prevented by 0.25 to 0.40 millimolar external Mg(2+). Addition of Mg(2+) at a level sufficient to prevent leakage from intact chloroplasts results in approximately 20% stimulation in light-driven protein synthesis. PMID- 16663542 TI - Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts. AB - The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23 degrees C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5 degrees C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [(14)C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and (14)CO(2) bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO(2) in the chloroplast. PMID- 16663543 TI - Chloroplast Structure and Starch Grain Accumulation in Leaves That Received Different Red and Far-Red Levels during Development. AB - An important step in understanding influence of growth environment on carbon metabolism in plants is to gain a better understanding of effects of light quality on the photosynthetic system. Electron microscopy was used to study chloroplast ultrastructure in developing and fully expanded leaves of tobacco (Nicotiana tabacum L. cv Burley 21). Brief exposures to red or far-red light at the end of each day during growth under controlled environments influenced granum size, granum number and starch grain accumulation in chloroplasts, and the concentration of sugars in leaf lamina. Far-red-treated leaves had chloroplasts with more but smaller grana than did red-treated leaves. Red light at the end of the photosynthetic period resulted in more and larger starch grains in the chloroplasts and a lower concentration of sugars in leaves. Chloroplast ultrastructure and starch grain accumulation patterns that were initiated in the expanding leaves were also evident in the fully expanded leaves that received the treatment during development. It appears that the phytochrome system in the developing leaves sensed the light environment and initiated events which influenced chloroplast development and partitioning of photosynthate to adapt the plant for better survival under those environmental conditions. PMID- 16663544 TI - Ontogenetic Changes in the Transport of Indol-3yl-acetic Acid into Maize Roots from the Shoot and Caryopsis. AB - The quantities of endogenous indol-3yl-acetic acid (IAA) in endosperms and scutella of 6-day-old maize seedlings (Zea mays L. cv Giant White Horsetooth) were determined by a fluorimetric method. Endosperms were found to contain 33.4 nanograms IAA per plant, and scutella 7.5 nanograms IAA per plant. [5-(3)H]IAA applied to endosperms of 6-day-old seedlings moved into the roots and radioactivity accumulated at the apex of the primary root within 8 hours. Two to 7-day-old seedlings were treated simultaneously with [5-(3)H]IAA in the endosperm and [2-(14)C] IAA on the shoot apex. The patterns of transport into the root were found to change during ontogeny: in successively older plants, transport from the shoot into the roots increased relative to transport from the endosperm into the roots. The auxin required for the growth of maize roots could, therefore, partially be contributed by the shoot and endosperm. Ontogenetic changes in the relative importance of these two supplies could be of significance for the integration of growth and development between shoot and root. PMID- 16663545 TI - Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid. AB - Polyamine metabolism and its relation to the induction of alpha-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA(3)) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA(3) (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of alpha amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA(3). Kinetic studies show that both applied [U-(14)C]ornithine and [U-(14)C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA(3). Additionally, added GA(3) does not affect the uptake and turnover of [1,4-(14)C]Put, nor does it affect the conversion of Put --> Spd or Spd --> Spm. Treatment of the aleurone layers with GA(3) for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-(14)C] adenosine into ATP. The lower the level of Spd the less alpha-amylase formation is induced by added GA(3). The reduction of GA(3)-induced alpha-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA(3), with respect to induction of alpha-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction. PMID- 16663546 TI - Reduction of ferric leghemoglobin in soybean root nodules. AB - Reduction of ferric leghemoglobin to ferrous leghemoglobin in soybean nodules (Glycine max [L.] Merr. cv Woodworth) was studied using a spectrophotometer equipped with an in-cell space diffuse reflectance accessory. Nodule slices prepared and scanned under nitrogen gas showed a ferrous leghemoglobin absorption spectrum. Nodule slices equilibrated with 100% O(2) or air exhibited two absorption bands characteristic of oxygenated leghemoglobin. The addition of CO shifted those bands to CO leghemoglobin absorption bands. Potassium ferricyanide was not effective in oxidizing ferrous to ferric leghemoglobin in nodule slices. However, ferric leghemoglobin was formed by treating the nodule slices with hydroxylamine, and this was confirmed by complexing the ferric leghemoglobin to acetate, fluoride, or nicotinic acid. The diminution of ferric leghemoglobin was monitored as a function of time, and in the presence of nicotinic acid, the conversion of ferric to ferrous leghemoglobin was monitored by the appearance of ferrous leghemoglobin nicotinate complex as a function of time. Ferric leghemoglobin reduction was also confirmed by direct transmission spectrophotometry. The evidence presented here suggests that ferrileghemoglobin reduction occurs in nodule slices. PMID- 16663547 TI - Characteristics of galacturonic Acid oligomers as elicitors of casbene synthetase activity in castor bean seedlings. AB - Partial digestion of polygalacturonic acid with polygalacturonase isolated from Rhizopus stolonifer produces a mixture of alpha-1,4-d-galacturonide oligomers which act to elicit casbene synthetase activity in castor bean (Ricinus communis L). These oligomers were separated by anion exchange chromatography on DEAE Sephadex A-25 into discrete sizes and their degrees of polymerization were analyzed by fast atom bombardment mass spectrometry. A minimum degree of polymerization of nine units appears to be required for elicitor activity; trideca-alpha-1,4-d-galacturonide was the most active of the oligomers tested. Methyl-esterification of the carboxylate groups greatly diminishes the elicitor activity of the oligomers, a finding which suggests a requirement for the polyanionic character of the oligomers for full activity. The fact that a number of other polyanionic polymers tested as casbene synthetase elicitors did not show significant activity indicates that structural features other than the polyanionic character are also necessary for activity. PMID- 16663548 TI - Photosystem electron-transport capacity and light-harvesting antenna size in maize chloroplasts. AB - Spectrophotometric and kinetic measurements were applied to yield photosystem (PS) stoichiometries and the functional antenna size of PSI, PSII(alpha), and PSII(beta) in Zea mays chloroplasts in situ. Concentrations of PSII and PSI reaction centers were determined from the amplitude of the light-induced absorbance change at 320 and 700 nm, which reflect the photoreduction of the primary electron acceptor Q of PSII and the photooxidation of the reaction center P700 of PSI, respectively. Determination of the functional chlorophyll antenna size (N) for each photosystem was obtained from the measurement of the rate of light absorption by the respective reaction center. Under the experimental conditions employed, the rate of light absorption by each reaction center was directly proportional to the number of light-harvesting chlorophyll molecules associated with the respective photosystem. We determined N(P700) = 195, N(alpha) = 230, N(beta) = 50 for the number of chlorophyll molecules in the light harvesting antenna of PSI, PSII(alpha), and PSII(beta), respectively. The above values were used to estimate the PSII/PSI electron-transport capacity ratio (C) in maize chloroplasts. In mesophyll chloroplasts C > 1.4, indicating that, under green actinic excitation when Chl a and Chl b molecules absorb nearly equal amounts of excitation, PSII has a capacity to turn over electrons faster than PSI. In bundle sheath chloroplasts C < 1, suggesting that such chloroplasts are not optimally poised for linear electron transport and reductant generation. PMID- 16663549 TI - Phytotoxicity of Air Pollutants: Evidence for the Photodetoxification of SO(2) but Not O(3). AB - Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum flacca Mill. (tomato) were used to evaluate the phytotoxicity of SO(2) and O(3) in the light and dark. Plants were grown in controlled environment chambers and exposed to SO(2) or O(3) in the light or dark at the same environmental conditions at which they were grown. The pea plants were treated with fusicoccin to ensure open stomata in the dark; the stomata of the tomato mutant remained open in the dark. Both species exhibited 64% to 80% less foliar necrosis following exposure to SO(2) (0.5 to 1.0 microliter per liter for 2 hours) in the light than in the dark. The decrease in SO(2) injury for light versus dark exposed plants was greater in fully expanded than expanding leaves. Both species exhibited 30% greater foliar necrosis following exposure to O(3) (0.2 microliter per liter for 2 hours) in the light than dark. The increase in O(3) injury in the light versus dark was similar for leaves at all stages of expansion. Leaf conductance to water vapor was 7% to 11% and 23% higher in the light than dark for fusicoccin-treated peas and tomato plants, respectively, indicating greater foliar uptake of both pollutants in the light than dark. Thus, the decreased SO(2) toxicity in the light was not associated with pollutant uptake, but rather the metabolism of SO(2). In contrast, the increased toxicity of O(3) in the light was at least in part associated with increased uptake or could not be separated from it. PMID- 16663550 TI - Characterization of the Electrogenicity of Soybean (Glycine max L.) Roots : ATP Dependence and Effect of ATPase Inhibitors. AB - The ATP-dependence of the membrane potential of soybean (Glycine max L. cv Williams '79) roots was determined by correlating the decrease in ATP levels with the decrease in the membrane potential caused by carbonyl cyanide m chlorophenylhydrazone (CCCP) or cyanide. The effects of the ATPase inhibitors, vanadate and fluoride, were also examined in an attempt to establish the role of a plasma membrane localized electrogenic proton pumping ATPase in generating the membrane potential.The membrane potential of intact seedling roots bathed in artificial pond water was -167 +/- 27 millivolts (n = 130); the ATP level was 161 +/- 27 nanomoles per gram fresh weight (n = 102) when measured using rapid acid homogenization of the intact roots. Depolarization to the diffusion potential ( 100 millivolts) occurred at about 90 nanomoles ATP per gram fresh weight, 50 to 70% of the control ATP level.Cyanide-induced ATP loss and membrane potential depolarization occurred at similar rates, but CCCP-induced ATP loss was slower than the rate of depolarization. CCCP may initially depolarize the membrane potential by increasing the proton permeability of the plasma membrane and short circuiting the putative electrogenic proton pump. Vanadate and NaF caused a relatively slight depolarization of the membrane potential. Both inhibited microsomal ATPase activity substantially at the concentrations causing the depolarization. PMID- 16663551 TI - Osmotic stress-induced polyamine accumulation in cereal leaves : I. Physiological parameters of the response. AB - Putrescine and spermidine accumulate in cereal cells and protoplasts exposed to various osmotica (sorbitol, mannitol, proline, betaine, or sucrose). The response is fast (1-2 hour lag), massive (50- to 60-fold increase in putrescine), and is not due to release of putrescine from a bound form or to conversion from spermidine. It rather involves the activation of the biosynthetic pathway mediated by arginine decarboxylase (ADC; EC 4.1.1.19) (Flores and Galston 1982 Science 217: 1259). Polyamine accumulation and the rise in ADC activity in osmotically stressed tissue are prevented by ADC inhibitors (alpha difluoromethylarginine, d-arginine, and l-canavanine) but are not affected by alpha-difluoromethylornithine and methylornithine, inhibitors of the alternative putrescine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17). Putrescine accumulation by oat and corn leaves is maximal in solutions only slightly hyperosmotic (0.4 molar). The stress response, which declines with leaf age, is completely prevented by cycloheximide (10 to 50 micrograms per milliliter) when added during the first hour of exposure to osmoticum, and partially by transcription inhibitors (cordycepin, Actinomycin D, 5 to 20 micrograms per milliliter). Oat seedlings allowed to wilt by withholding water also show a rise in polyamine titer and ADC activity. This response is not readily reversible upon rewatering. PMID- 16663552 TI - Osmotic Stress-Induced Polyamine Accumulation in Cereal Leaves : II. Relation to Amino Acid Pools. AB - Arginine decarboxylase activity increases 2- to 3-fold in osmotically stressed oat leaves in both light and dark, but putrescine accumulation in the dark is only one-third to one-half of that in light-stressed leaves. If arginine or ornithine are supplied to dark-stressed leaves, putrescine rises to levels comparable to those obtained by incubation under light. Thus, precursor amino acid availability is limiting to the stress response. Amino acid levels change rapidly upon osmotic treatment; notably, glutamic acid decreases with a corresponding rise in glutamine. Difluoromethylarginine (0.01-0.1 millimolar), the enzyme-activated irreversible inhibitor of arginine decarboxylase, prevents the stress-induced putrescine rise, as well as the incorporation of label from [(14)C]arginine, with the expected accumulation of free arginine, but has no effect on the rest of the amino acid pool. The use of specific inhibitors such as alpha-difluoromethylarginine is suggested as probes for the physiological significance of stress responses by plant cells. PMID- 16663553 TI - Bound water in soybean seed and its relation to respiration and imbibitional damage. AB - In an attempt to understand the initial stage of seed imbibition-the wetting stage-we have examined water binding in dry soybean cotyledon tissue using water sorption isotherm curves. The sorption isotherms show three levels of water affinity: a region of strongly bound water at moisture contents below 8%, a region of weakly bound water at moisture contents between 8 and 24%, and a region of very loosely bound water at contents greater than 24%. The enthalpies of the water binding for the three sectors were -6 to -12.5, about -2.5, and about -0.5 kilocalories per mole water, respectively.The degree of physiological activity in the tissue reflects the level of water binding. O(2) consumption is first detectable in the second region of water affinity (8-24% water), and increases dramatically with increasing water content above about 24%. Damage due to imbibing water is greatest when initial seed moisure contents are in the region of strongest water binding. Damage is lessened and finally absent when seed moisture contents are increased to the second and then to the third level of water affinity. PMID- 16663554 TI - Isolation and Some Properties of an Aminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.). AB - The isolation and characterization of the AP1 form of aminopeptidase (EC 3.4.11.) previously identified (Waters, Dalling 1979 Aust J Plant Physiol 6: 595-606) in the primary leaves of wheat (Triticum aestivum L. cv Egret) seedlings is reported. The enzyme shows a high preference for a substrate which contains an aromatic side chain, whether this be either a synthetic beta-naphthylamide or a peptide substrate. Maximum activity with both types of substrates occurred around pH 7.6. The stability of AP1 was reduced by exposure to high pH and by incubation at temperatures above 20 degrees C in the absence of substrate. AP1 was inhibited by the metal chelators bathocuproine and bathophenanthroline and the sulfhydryl group inhibitors p-chloromercuribenzoate and N-ethylmaleimide. The molecular weight, estimated by gel filtration, was 57,000. The K(m) value for activity against the synthetic substrate Phe-beta-NA (0.20 millimolar) was slightly lower than that for Phe-Phe (0.50 millimolar) although the enzyme activity against peptide substrates was considerably greater than with Phe-beta-NA. PMID- 16663555 TI - Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side. AB - In a split-root system of soybeans (Glycine max L. Merr), inoculation of one half side suppressed subsequent development of nodules on the opposite side. At zero time, the first side of the split-root system of soybeans received Rhizobium japonicum strain USDA 138 as the primary inoculum. At selected time intervals, the second side was inoculated with the secondary inoculum, a mixture of R. japonicum strain USDA 138 and strain USDA 110. In a short-day season, nodulation by the secondary inoculum was inhibited 100% when inoculation was delayed 10 days. Nodulation on the second side was significantly suppressed when the secondary inoculum was delayed for only 96 hours. In a long-day season, nodule suppression on the second side was highly significant, but not always 100%. Nodule suppression on the second side was not related to the appearance of nodules or nitrogenase activity on the side of split-roots which were inoculated at zero time. When the experiments were done under different light intensities, nodule suppression was significantly more pronounced in the shaded treatments. PMID- 16663556 TI - Phloem Unloading of Amino Acids at the Site of Attachment of Cuscuta europaea. AB - By washing out (14)C-solutes or (3)H-solutes in 0.5 mm CaSO(4) during a period of 5 to 6 hours, the release of amino acids by excised stem segments of broad bean (Vicia faba L. cv Witkiem) was studied. Three hours after pulse labeling with l valine, l-asparagine, or alpha-aminoisobutyric acid (AIB), hollow stem segments were excised from the plant and incubated in a washout solution.In experiments with valine and asparagine, stem segments parasitized by Cuscuta europaea released a higher percentage of labeled solutes into the bathing medium than control segments. This can be ascribed to enhanced phloem unloading at the site of attachment of Cuscuta. At low temperature (0 degrees C) and after addition of p-chloromercuribenzene sulfonate to the bathing medium, parasitized segments did not release an enhanced percentage of labeled solutes, in comparison with control segments. These data suggest a metabolic control of the phenomenon of enhanced phloem unloading of amino acids. In experiments with AIB, an enhanced release of labeled solutes could not clearly be observed, but at the site of attachment of Cuscuta an accumulation of labeled solutes was measured. Accumulation of AIB in parenchyma cells, before the start of a washout experiment, will tend to obscure the phenomenon of enhanced phloem unloading. PMID- 16663557 TI - Conditions Required for the Rapid Activation In Vitro of the Chloroplast Fructose 1,6-bisphosphatase. AB - Conditions required for the reductive activation of purified, spinach chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11) have been determined in vitro. Full reductive activation was observed only when fructose-1,6-bisphosphate and Mg(2+) were present at the same time as the reducing agent (dithiothreitol). Reduction in the absence either of fructose-1,6-bisphosphate or of Mg(2+) slowly and irreversibly inactivated the enzyme. The concentration of fructose-1,6 bisphosphate that must be present during reduction for maximum activation depends upon the divalent cation present: it is highest with Mg(2+), lower with Ca(2+), and lowest when both Mg(2+) and Ca(2+) are present. A scheme for the reductive activation and inactivation of the enzyme is presented. PMID- 16663558 TI - Effect of sulfhydryl reagents on k efflux from rose cells: relationship to ultraviolet-stimulated efflux. AB - N-Ethylmaleimide causes a rapid efflux of K(+) from suspension-cultured cells of Rosa damascena. This efflux shows many characteristics of the ultraviolet-induced efflux of K(+), including the appearance of HCO(3) (-) together with the K(+) and inhibition by respiratory inhibitors. Cysteine inhibits the ultraviolet-induced efflux of K(+). These results are interpreted to mean that ultraviolet induces K(+) efflux through an alteration of sulfhydryl residues. PMID- 16663559 TI - Light-induced transformation of amyloplasts into chloroplasts in potato tubers. AB - The transformation of amyloplast into chloroplasts in potato (Solanum tuberosum L.) tuber tissue can be induced by light. Excised potato tuber discs illuminated with white light of 3000 lux began to synthesize chlorophyll after a lag period of 1 day, and continued to synthesize chlorophyll for 3 weeks. In this paper we present evidence, based on ultracentrifugal sedimentation and immunoprecipitation, that the light-mediated synthesis of Ribulose-1,5 bisphosphate carboxylase began 1 day after illumination with white light. When illuminated the chloroplasts isolated from light-grown potato tuber tissue incorporated [(35)S]methionine into polypeptides, one of which has been identified as the large subunit of Ribulose-1,5-bisphosphate carboxylase. These chloroplasts are functional as determined by O(2) evolution in the Hill reaction. PMID- 16663560 TI - Reevaluation of the role of bicarbonate and formate in the regulation of photosynthetic electron flow in broken chloroplasts. AB - The stimulation of the Hill reaction in CO(2)-depleted broken chloroplasts (Pisum sativum L. cv Rondo) by the total amount of dissolved CO(2) and HCO(3) (-) (bicarbonate(*)) was measured at several formate concentrations. Formate appears to be a competitive inhibitor of the bicarbonate(*) stimulation of electron flow. From these experiments we have obtained a reactivation constant (K(r)) of 78 +/- 31 micromolar NaHCO(3) and an inhibition constant (K(i)) of 2.0 +/- 0.7 millimolar HCOONa at pH 6.5. In the absence of formate, significant electron flow was measured at a bicarbonate(*) concentration well below K(r), suggesting that electron flow from Q, the primary electron acceptor of photosystem II, to plastoquinone can proceed when no bicarbonate(*) is bound to the regulatory site at the Q(B)-protein. If so, bicarbonate(*) stimulation of electron flow is mainly a diminution of the inhibition of electron flow by formate. In view of the results, it is proposed that regulation of linear electron flow by bicarbonate(*) and formate is a mechanism that could link cell metabolism to photosynthetic electron flow. PMID- 16663561 TI - In Vivo Natural-Abundance C Nuclear Magnetic Resonance Studies of Living Ectomycorrhizal Fungi : Observation of Fatty Acids in Cenococcum graniforme and Hebeloma crustuliniforme. AB - Natural-abundance (13)C nuclear magnetic resonance spectroscopy has been used to study intact mycelia of the ectomycorrhizal fungi Cenococcum graniforme (Ascomycetes) and Hebeloma crustuliniforme (Basidiomycetes). A number of sharp resonances are observed in living fungi. These signals primarily arise from fatty acyl chains and carbohydrate nuclei. The spectra are interpreted in terms of relative concentrations of the major fatty acids present in the fungal triglycerides. The small line width of fatty acids (mainly oleic, linoleic, and palmitic acids) resonances and spin-lattice relaxation time are indicative of fast rotational reorientations and are consequently thought to arise from fatty acyl chains in fat droplets. We were able to locate the site of lipids accumulation within mycelia using light microscopy and histological staining. Many lipid droplets were observed in mycelia of both species.These results suggest that fatty acids droplets could be involved in carbon storage and metabolism from ectomycorrhizal fungi. PMID- 16663562 TI - Parachloromercuribenzenesulfonic Acid : a potential tool for differential labeling of the sucrose transporter. AB - Vicia faba leaf discs without epidermis were pretreated with parachloromercuribenzenesulfonic acid (PCMBS), rinsed and incubated on [(14)C]sucrose (1 or 40 millimolar). Those sucrose concentrations were chosen as representative of the apparent uptake system 1 (1 millimolar) and system 2 (40 millimolar) previously characterized. Pretreatment with 0.5 millimolar PCMBS for 20 minutes inhibited system 1 and system 2 by about 70%.Addition of unlabeled sucrose during PCMBS-pretreatment protected the carrier(s) from the inhibition, whereas glucose, fructose, and sucrose analogs were unable to afford protection. At 1 millimolar [(14)C]sucrose, the protection resulted in a small but consistent reduction of normal inhibition (from 63 to 45%) for sucrose concentrations of 50 millimolar and more during pretreatment. Contrarily, at 40 millimolar [(14)C]sucrose, the protection increased linearly with the sucrose concentration in the pretreatment medium, and complete prevention of inhibition was reached for 250 millimolar sucrose.The protection was not due to exchange diffusion and was located in the veins. Michaelian kinetics indicated that PCMBS and sucrose compete with each other at the active site of the carrier.Among 14 compounds tested (sugars, amino-acids, hormones, (32)P), sucrose uptake was by far the most sensitive to PCMBS. Sucrose preferentially protected its carrier(s) from inhibition. Treatment with 20 millimolar cysteine or 20 millimolar dithioerythreitol reversed inhibition by PCMBS pretreatment. PMID- 16663563 TI - 6-azauracil-resistant variants of cultured plant cells lack uracil phosphoribosyltransferase activity. AB - 6-Azauracil-resistant variants of Haplopappus gracilis (Nutt.) Gray and Datura innoxia Mill. lack activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme that catalyzes the conversion of uracil and 6-azauracil to uridine 5'-monophosphate and 6-azauridine-5'-monophosphate, respectively. Resistant cells are competent to take up uracil from their growth medium but do not convert it into a form that can be used for macromolecular synthesis. In extracts from resistant cells, orotate monophosphate decarboxylase, a target enzyme of 6 azauridine monophosphate, is fully sensitive to the phosphorylated analog. These results strongly suggest that uracil phosphoribosyltransferase is the major pathway of pyrimidine salvage in cells of these species and that loss of this enzyme activity confers on the variants resistance to 6-azauracil. PMID- 16663564 TI - Incorporation of oxygen into abscisic Acid and phaseic Acid from molecular oxygen. AB - Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% (18)O(2) and 80% N(2) indicates that one atom of (18)O is incorporated in the 6'-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing (18)O(2) indicates that one atom of (18)O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1'-, 4'-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1'- and 4'-positions of abscisic acid which is converted to abscisic acid under conditions of water stress. PMID- 16663565 TI - Rapid Accumulation of gamma-Aminobutyric Acid and Alanine in Soybean Leaves in Response to an Abrupt Transfer to Lower Temperature, Darkness, or Mechanical Manipulation. AB - Soybean (Glycine max [L.] Merr) leaves contain a low level (0.05 micromole per gram fresh weight) of gamma-aminobutyric acid (Gaba) but the concentration of this non-protein amino acid increased to 1 to 2 micromoles per gram fresh weight within 5 minutes after transfer of plants or detached leaves from 33 degrees C to 22 degrees C or lower temperatures. A parallel decrease occurred in the concentration of glutamate. Accumulation of Gaba was also triggered by mechanical damage to the soybean leaves, but in plants subjected to a gradual reduction in temperature (2 degrees C per minute) only a small increase in Gaba occurred. A rapid increase in the concentration of alanine and decrease in glycine occurred upon transfer of the soybean plants to darkness and was not influenced by temperature. When plants were returned to normal growing conditions, all changes in amino acid concentrations were fully reversed in 1 hour.In soybean leaf discs incubated with [(14)C]glutamate, a rapid accumulation of [(14)C]Gaba was detected, and glutamate decarboxylase activity of the soybean leaf considerably exceeded (>30-fold) that of Gaba pyruvate transaminase. Part of the transaminase was localized in the mitochondria, but glutamate decarboxylase was not associated with any organelle or membrane component of the leaf cell. We consider that Gaba accumulation results from some change in intracellular compartmentation of the cell triggered by low temperature shock or mechanical damage. The accumulation of alanine due to a light-dark transition could be accounted for by transamination. [(14)C]Alanine formation was demonstrated when soybean leaf extracts were incubated with glutamate, aspartate, or serine and [(14)C]pyruvate.The changes in amino acid concentrations described for soybean leaves were demonstrated for all the vegetative tissues of the soybean plant and at variable rates in the leaves of a range of plant species. The response in detached tomato (Lycopersicon esculentum Mill.) leaves was of a similar magnitude to soybean. Thus, precautions are necessary to minimize changes in amino acid composition induced by manipulation and extraction of plant material. PMID- 16663566 TI - Maintenance of Normal or Supranormal Protein Accumulation in Developing Ovules of Glycine max L. Merr. during PEG-Induced Water Stress. AB - Protein accumulation in developing ovules of hydroponically grown soybean (Glycine max L. Merr.) plants was unaffected or enhanced by polyethylene glycol induced water stress. The cultivar Wayne and the experimental variety ;9656' were severely stressed by inclusion of PEG-6000 in the nutrient solution to water potentials as low as -20 bar. Leaves rapidly yellowed and abscised under these conditions. Fresh and dry weight of ;Wayne' ovules was reduced by severe stress, but protein content was unaffected. Ovules of 9656 were more resistant to severe stress: fresh weight and dry weight were unaffected by stress and protein content increased. Moderately stressed Wayne ovules behaved like severely stressed 9656 ovules: seed fresh weight and dry weight were unaffected and protein content increased. However, protein content did not increase if the plants were defoliated. No changes in seed protein quality were observed with stress, based on polypeptide banding patterns after one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. PMID- 16663567 TI - Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos. AB - The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and gamma aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo. PMID- 16663568 TI - Source/Sink relations of abscising and nonabscising soybean flowers. AB - The partitioning of recently fixed (14)C to setting and abscising flowers within the axillary raceme of ;Clark' isoline E1t soybeans (Glycine max L. Merr) was examined as a function of time after anthesis of individual flowers. In such racemes, the first four flowers showed a 17% abscission while the next four flowers showed 47% abscission.Source/sink relations of flowers I-IV (normally setting) were compared to those of flowers V-VIII (normally abscising) by pulse labeling source leaves with (14)CO(2) and determining the radioactivity of individual flowers after a 4-hour chase period. The relative specific activity (RSA;% disintegrations per minute per% dry weight), sink strength (% disintegrations per minute), and its components, sink size (milligrams dry weight) and sink intensity (% disintegrations per milligram dry weight) were then calculated as a function of days after anthesis.Sink intensity (i.e. the competitive ability to accumulate photoassimilate per unit mass) was very high prior to anthesis of both setting and abscising flowers. Sink intensity then became very low for the first 3 days following anthesis after which it recovered in normally setting flowers, but failed to recover in normally abscising flowers. It is concluded that soybean reproductive abscission is determined at or very near the day of anthesis. PMID- 16663569 TI - Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples. AB - Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1 aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO(2) production. The results with ripened, AVG-treated apples indicate (a) a dose response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 mul/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO(2); (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene.Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO(2), but not by 1.25% CO(2). However, each of these CO(2) concentrations reduced the rate of increase of ACC synthase activity. PMID- 16663570 TI - Deep Supercooling in Most Tissues of Wintering Sasa senanensis and Its Mechanism in Leaf Blade Tissues. AB - Cold hardiness of leaf blades, leaf sheaths, culms, rhizomes, and leaf buds in wintering Sasa senanensis (Fr. et Sav.) Rehder, a dwarf bamboo, was studied paying special attention to the types of resistance mechanisms which were determined with differential thermal analysis. Coincidence of LT(25) (lethal temperature at which 25% of the tissues are injured) with the initiation temperature of LTE (low temperature exotherm) suggested that all of these tissues described above owe their cold hardiness mechanism mostly to deep supercooling. Deep supercooling in leaf blades was also substantiated with microscopic observations, suggesting that the units of supercooling were minute tissues compartmentalized by longitudinal and cross veins. It was also shown that cooling rates and storage of shoots at -5 degrees C for 1 to 5 days in the ice-inoculated state did not greatly affect the supercooling ability of leaf blades. Sasa senanensis seemed to exhibit a unique strategy against prolonged subzero temperature, and its leaves would be a good system for the study on mechanisms of deep undercooling in plants. PMID- 16663571 TI - Changes in Levels of alpha-Amylase Components in Barley Tissues during Germination and Early Seedling Growth. AB - Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18 degrees C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for alpha-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same alpha-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar alpha-amylase was excreted into the endosperm compared to aleurone synthesized alpha-amylase. Aleurone cells appeared to synthesize appreciably more alpha amylase than did scutellar tissue. PMID- 16663572 TI - Studies of sugars and sorbitol in developing corn kernels. AB - Sugars and sorbitol were determined on corn (Zea mays L.) kernels harvested at various developmental stages, using sugary (su), sugary-sugary enhancer (su se), and starchy (Su) cultivars. In all cultivars tested, the sorbitol content increased from trace amounts in unpollinated ovules to a maximum at about the time that rapid starch synthesis was proceeding. Thereafter, sorbitol and sugars decreased continuously to the mature dry stage. Sorbitol in the su se kernels was higher than that of other cultivars from 28 days postpollination onwards; sucrose and maltose were higher from 21 days onwards. [(14)C]Sorbitol was recovered from kernel base, pedicel, and endosperm of IL677a (su se) kernels after allowing a flag leaf to fix (14)CO(2) photosynthetically. No [(14)C]sorbitol was detected in the shank of the ear, and none was detected by the gas chromatograph. [(14)C]Sucrose was the predominant labeled substance recovered from the kernel base, pedicel, and endosperm tissues during the 10-h chase period, as well as from the shank of the ear, and nonradioactive sucrose was the predominant ethanol soluble compound detected by the gas chromatograph. Hence, sorbitol appears not to be translocated from corn leaves as it is in certain woody plants of the rose family. The altered sugar profile of su se kernels may be related to reduced starch synthesis, but the biochemical mechanism is not yet known. PMID- 16663573 TI - Possible roles of calcium and calmodulin in the biosynthesis and secretion of alpha-amylase in rice seed scutellar epithelium. AB - The scutellar epithelial cells of rice (Oryza sativa L. cv Kimmaze) seeds actively secrete alpha-amylase in an early stage of germination. Employing an in vivo system of freshly dissected scutellar tissues, effect of Ca(2+) on the biosynthesis and the secretion of alpha-amylase have been studied. The maximum biosynthetic rate was saturated at about 0.5 mm external Ca(2+) concentrations, whereas the secretion continued to increase to concentrations above 10 mm Ca(2+). In the presence of 1 mm Ca(2+), 0.01 mum A-23187 significantly increased both the biosynthesis and the secretion of alpha-amylase.A cation-specific requirement for Ca(2+) was apparent, since both biosynthesis and extracellular secretion of alpha amylase were inhibited by 0.1 mm EGTA but were increased above basal rate only with Ca(2+) and Sr(2+); K(+), Mg(2+), and Ba(2+) being ineffective.La(3+) and ruthenium red (selective inhibitors of [Ca(2+) + Mg(2+)]-ATPase) were found to profoundly inhibit the secretion of alpha-amylase. A calmodulin antagonist, W-7, also inhibited the secretion of alpha-amylase at concentrations where the enzyme synthesis was not much affected. Overall data indicate that Ca(2+) movement and secretion of alpha-amylase are tightly linked and it is likely that they are regulated by the cytoplasmic Ca(2+) concentration under possible control by calmodulin. PMID- 16663574 TI - Fermentative Metabolism of Chlamydomonas reinhardtii: I. Analysis of Fermentative Products from Starch in Dark and Light. AB - The anaerobic starch breakdown into end-products in the green alga Chlamydomonas reinhardtii F-60 has been investigated in the dark and in the light. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonyl cyanide-p trifluoromethoxyphenyl hydrazone (FCCP) on the fermentation in the light have also been investigated.Anaerobic starch breakdown rate (13.1 +/- 3.5 micromoles C per milligram chlorophyll per hour) is increased 2-fold by FCCP in the dark. Light (100 watts per square meter) decreases up to 4-fold the dark rate, an inhibition reversed by FCCP. Stimulation of starch breakdown by the proton ionophore FCCP points to a pH-controlled rate-limiting step in the dark, while inhibition by light, and its reversal by FCCP, indicates a control by energy charge in the light.In the dark, formate, acetate, and ethanol are formed in the ratios of 2.07:1.07:0.91, and account for roughly 100% of the C from the starch. H(2) production is 0.43 mole per mole glucose in the starch. Glycerol, d-lactate, and CO(2) have been detected in minor amounts.In the light, with DCMU and FCCP present, acetate is produced in a 1:1 ratio to formate, and H(2) evolution is 2.13 moles per mole glucose. When FCCP only is present, acetate production is lower, and CO(2) and H(2) evolution is 1.60 and 4.73 moles per mole glucose, respectively.When DCMU alone is present, CO(2) and H(2) photoevolution is higher than in the dark. Without DCMU, CO(2) and H(2) evolution is about 100% higher than in its presence. In both conditions, acetate is not formed. In all conditions in the light, ethanol is a minor product. Formate production is least affected by light.The stoichiometry in the dark indicates that starch is degraded via the glycolytic pathway, and pyruvate is broken down into acetyl-CoA and formate. Acetyl-CoA is further dissimilated into acetate and ethanol. In the light, acetate is produced only in the presence of FCCP and, when photophosphorylation is possible, it is used in unidentified reactions. Ethanol formation is inhibited by the light in all conditions. PMID- 16663575 TI - Cytosolic NADPH is the electron donor for extracellular fe reduction in iron deficient bean roots. AB - Pyridine nucleotides were determined in lateral roots of iron-deficient and iron sufficient Phaseolus vulgaris L. cv Prelude. In iron-deficient plants, total NADP per gram fresh weight and the NADPH/NADP(+) ratio were twice the values found in iron-sufficient plants. The NADPH/NADP(+) ratio in iron-deficient plants was considerably lowered after a 2 minute incubation in 1 millimolar ferricyanide. Total NAD was not influenced by growth conditions and was mainly present in oxidized form.These results indicate that NADPH is the electron donor for the high Fe(III) reduction activity found in iron-deficient roots, a process that is part of the Fe-uptake mechanism. PMID- 16663576 TI - Evidence for the occurrence of, and possible physiological role for, cyanobacterial calmodulin. AB - Phosphate uptake by the blue-green alga Oscillatoria limnetica Lemmerman is stimulated by micromolar concentrations of Ca(2+). The calmodulin antagonists 4 (3-[2(trifluoromethyl)phenylthiazin-10-yl]propyl)-1-piperazine ethanol-HCl and its monofluoro-analog inhibit orthophosphate uptake of Oscillatoria limnetica by over 97% implying involvement of calmodulin in this process. A calmodulin-like protein was quantitated in cell-free extracts from O. limnetica by radioimmunoassay. PMID- 16663577 TI - Characterization of oat vicilin-like polypeptides. AB - The 7S and 3S globulin fractions were extracted and characterized from Avena sativa L. seeds which showed similar solubility characteristics and holoprotein size to those of the vicilin fraction in legumes. These holoproteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectrofocussing, and two-dimensional electrophoresis. Among the components of the oat reserve globulins, the 7S fraction was capable of binding to concanavalin A-Sepharose, thus indicating it to be a glycoprotein. This pattern of the glycosylation of the reserve proteins in oat resembles that observed in certain legume seeds such as Pisum sativum and Glycine max. The results support the notion that the protein components of cereal and legume globulins may be homologous. PMID- 16663578 TI - Ratio of cut surface area to leaf sample volume for water potential measurements by thermocouple psychrometers. AB - Evaporative losses from the cut edge of leaf samples are of considerable importance in measurements of leaf water potential using thermocouple psychrometers. The ratio of cut surface area to leaf sample volume (area to volume ratio) has been used to give an estimate of possible effects of evaporative loss in relation to sample size. A wide range of sample sizes with different area to volume ratios has been used. Our results using Glycine max L. Merr. cv Bragg indicate that leaf samples with area to volume values less than 0.2 square millimeter per cubic millimeter give psychrometric leaf water potential measurements that compare favorably with pressure chamber measurements. PMID- 16663579 TI - Protein synthesis patterns : relevance of old and new messenger RNA in germinating maize embryos. AB - The proteins synthesized during the first hours of seed imbibition were studied in axes and scutellum of maize embryos separately. Increase in fresh weight was followed in the embryonic axes through the germination period. Pulse labeling experiments with (14)C-amino acids were carried out at two stages of development: 0 to 6 and 18 to 24 hours in the presence and absence of alpha-amanitin. The proteins were analyzed by two-dimensional gel electrophoresis and fluorography. Results showed a major pattern of proteins common to both tissues, axes and scutellum (;house keeping' proteins), besides the specific proteins synthesized by each tissue. In the axes, the changes in proteins observed between the periods of 0 to 6 and 18 to 24 hours of development seem to be due both to newly synthesized mRNA as well as to delayed translation of stored mRNA species. PMID- 16663580 TI - Helminthosporium maydis T Toxin Increased Membrane Permeability to Ca in Susceptible Corn Mitochondria. AB - Though Helminthosporium maydis race T (HmT) toxin decreased active Ca(2+) uptake into mitochondria isolated from susceptible (T) but not resistant (N) corn (Kimber, Sze, 1984 Plant Physiol 74: 804-809 the mode of toxin action is not understood. This study shows that HmT toxin or A23187 (a Ca(2+) ionophore) dissipated a Ca(2+) gradient in T mitochondria. However, HmT toxin had no effect on Ca(2+) gradients in N mitochondria or microsomal vesicles from T or N corn. The results suggest that HmT toxin increased membrane permeability to Ca(2+) in mitochondria of T corn specifically. PMID- 16663581 TI - Chlorophyll-Protein Complexes of a Photosystem II Mutant of Maize : Evidence that Chlorophyll-Protein a-2 and a Chlorophyll-Protein Complex Derived from a Photosystem I Antennae System Comigrate on Polyacrylamide Gels. AB - Use of the octyl beta-d-glucopyranoside solubilization procedure of Camm and Green (1980 Plant Physiol 66: 428-432) reveals that thylakoid membranes of a photosystem (PS) II-deficient maize (Zea mays L.) mutant lack two chlorophyll protein (CP) complexes associated with PSII, i.e. CPa-1 and CPa-2. In contrast, when lithium dodecyl sulfate is used to solubilize the membranes of the mutant prior to electrophoretic separation, a CP complex is observed which has a mobility similar to that of CPa-2. Comparison of spectral characteristics and polypeptide composition of the green bands in this region taken from samples of the mutant, normal sibling control plants and from PSII preparations indicate that the CP complex observed in the mutant represents a portion of a light harvesting complex of PSI (Mullet et al. 1980 Plant Physiol 65: 814-822). The green band observed in normal maize samples can contain both the CPa-2 complex as well as the CP complex derived from the PSI antennae system. PMID- 16663582 TI - Nitrate deficiency shortens the circadian period in gonyaulax. AB - The circadian rhythms in bioluminescence and photosynthesis in Gonyaulax polyedra suspended in unsupplemented sea water have been compared to the same rhythms in f/2, an enriched seawater medium. Cells suspended in sea water for 2 days in continuous light (450 microwatts per square centimeter) showed significantly shorter circadian periods and lower amplitudes than did cells in f/2 medium (a period of 22.2 hours as compared to 23.5 hours). Both period and amplitude changes could be completely reversed by the addition of nitrate at one-fourth or more of the concentration in f/2 medium (0.88 millimolar). The addition to autoclaved seawater of phosphate, vitamins, minerals, or soil extract in concentrations present in f/2 medium had no effect. Thus, the shortening of the circadian period is the consequence of reduced nitrogen supply. Since both the rhythms in bioluminescence and photosynthesis showed similarly shortened circadian periods and lower amplitudes, it is probable that the depletion of nitrate directly affects the circadian clock. PMID- 16663583 TI - Synthesis of the psbA Gene Product in Euglena: In Organello and In Vitro. AB - To identify the psbA gene product of Euglena gracilis, we compared products translated in organello and in vitro. The most prominently labeled membrane protein of isolated Euglena plastids migrates as a band at 28 kilodaltons. An apparent precursor appears at 30 kilodaltons under conditions which inhibit the synthesis of cytoplasmically synthesized proteins. Translation of the 14S mRNA selected by hybridization with the Sephacryl S-500-immobilized psbA gene, however, yields products of approximately 37- and 41-kilodaltons. In organello, no significant label migrates to this region of the gel. We interpret these data to indicate that the primary translation product of Euglena psbA gene is larger than that of higher plants, but the mature, processed polypeptide is smaller. PMID- 16663584 TI - Mefluidide protection of severely chilled crop plants. AB - Mefluidide, the common name of N-(2,4-dimethyl-5-[([trifluoromethyl] sulfonyl)amino]phenyl)acetamide, is capable of protecting chilling sensitive plants such as cucumber (Cucumis sativus L.) and corn (Zea mays L.) from chilling injury. The applied concentrations that protect plants from stress are species specific. Applied above a threshold concentration, it has no protective effect. Regardless of its immediate potential for agriculture, it appears at least to be a powerful tool for the biologist in the study of the protection from temperature stress mechanism. PMID- 16663585 TI - Synthesis and degradation of nitrite reductase in pea leaves. AB - We have shown in a previous publication (Gupta, Beevers 1983 J Exp Bot 34: 1455 1462) that the level of extractable nitrite reductase activity in pea (Pisum sativum cv Burpeeana) leaves is subject to environmental perturbations. In the current study, we have used rocket immunoelectrophoresis to quantitate the level of nitrite reductase protein in extracts from pea plants subjected to various environmental treatments. The level of nitrite reductase cross-reacting material closely followed the level of assayable nitrite reductase activity. The environmental conditions which enhanced the level of extractable nitrite reductase activity resulted in an increased level of nitrite reductase cross reacting material in the extracts. In contrast, environmental conditions which resulted in a decrease in the level of extractable nitrite reductase activity produced a decline in cross-reacting material. These results indicate that the environmentally induced modulation of extractable nitrite reductase activity involves alteration of enzyme level and is not mediated by a reversible activation-inactivation of the existing enzyme. PMID- 16663586 TI - Induction of the De Novo Formation of the Photosystem I-Related Light-Harvesting Chlorophyll Protein Complex LHCPa by Photoheterotrophic Nutrition. AB - The green alga Chlamydobotrys stellata contains in addition to the normal light harvesting chlorophyll protein complex LHCPb a special LHCPa which is free of chlorophyll b and connected only to photosystem I (Brandt, Zufall, Wiessner 1983 Plant Physiol 71: 128-131). The kinetics of these two LHCP forms were analyzed during the transition in nutrition of the alga from autotrophy to photoheterotrophy, e.g. the replacement of CO(2) by acetate as carbon source. As shown by incorporation experiments with [(14)C]acetate, this change in nutrition leads to an increased synthesis of LHCPa, whereas the synthesis of the photosystem II-related LHCPb decreases. The increase of the LHCPa synthesis starts immediately after the onset of photoheterotrophic nutrition together with the synthesis of the chlorophyll protein complex CPI. There is no interchange of complex components between LHCPa and LHCPb during the depletion of the latter. The formation of LHCPa is discussed with respect to the regulation of gene expression. PMID- 16663587 TI - Effects of gibberellic Acid on endogenous indole-3-acetic Acid and indoleacetyl aspartic Acid levels in a dwarf pea. AB - Two-week-old dwarf peas (Pisum sativum cv Little Marvel) were sprayed with gibberellic acid (GA(3)), and after 3 or 4 days the upper stem and young leaf samples were analyzed for indole-3-acetic acid (IAA) and indole-3-acetyl aspartic acid by an isotope dilution high performance liquid chromatography method. GA(3) increased IAA levels as much as 8-fold and decreased indole-3-acetyl aspartic acid levels. PMID- 16663588 TI - Endogenous indole-3-acetic Acid in the stem of tobacco in relation to flower neoformation as measured by mass spectroscopic assay. AB - The contents of free indole-3-acetic acid (IAA) and alkali-labile, conjugated IAA were measured in relation to a ;floral gradient' present in epidermis and subepidermis tissues of flowering plants of Nicotiana tabacum by capillary gas chromatographic spectrometric analysis by selected ion monitoring (GC-SIM-MS) using 2,4,5,6,7-penta deutero IA ((2)H(5)-IAA) as an internal standard. In floral axes, floral branches and stems with floral branches, free IAA levels (dry weight) were 387, 253, and 417 nanograms, and bound IAA levels were 99, 1089, and 268 nanograms. In vegetative tissue of the first plus second internodes (measured from top), and of the 11th to 13th internodes, free IAA levels were 826 and 500 nanograms, and bound IAA levels were 1421 and 286 nanograms, respectively. Since flower-forming ability of excised cells from the epidermis and subepidermis shows a gradient in an in vitro system, but levels of IAA in these tissues do not, there thus appears to be no correlation between flower-forming ability (in vitro) and endogenous IAA levels (at the time of excision) in tobacco stem tissues. PMID- 16663589 TI - Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems. AB - Photosynthate partitioning was examined in seedings of sour orange (Citrus aurantium L.) and Carrizo citrange (Poncirus trifoliata [L.] Raf. x C. sinensis [L.] Osbeck) grown with split root systems inoculated on one side with vesicular arbuscular mycorrhizal fungus (Glomus intraradices Schenck and Smith). Source sink relations were studied without mitigating differences in mineral content or physiological age that can occur in separate plant comparisons, because phosphorus was evenly distributed between leaves on opposite sides of the seedlings. Above-ground portions of each plant were exposed to (14)CO(2) for 8.5 minutes and ambient air for 2 hours, followed by extraction and identification of labeled assimilates. Mycorrhizal halves of root systems accumulated 66 and 68% of the (14)C-labeled photosynthates translocated to roots of sour orange and ;Carrizo' citrange, respectively, as well as an average of 77% greater disintegrations per minute per gram fresh weight. Distribution of (14)C-labeled assimilates was independent of phosphorus effects on photosynthate partitioning in leaves and did not reflect fresh or dry weights of roots or degree of mycorrhizal dependency of the species. Differences in radioactivity between mycorrhizal and nonmycorrhizal root halves after 2 hours indicated at least 3 to 5% of the whole plant (14)C-labeled photosynthates were allocated to mycorrhizae related events on one side and that twice this amount, or 6 to 10%, might be expected if the entire root system was infected. PMID- 16663590 TI - Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase. AB - A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86 fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K(m) values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N(2)-fixing alfalfa nodules. PMID- 16663591 TI - Phosphorylation of membrane-located proteins of soybean in vitro and response to auxin. AB - Isolated membranes of soybean incorporate (32)P from gamma-[(32)P]ATP in vitro. The incorporation was rapid and did not require added calcium. When displayed on 10% sodium dodecyl sulfate-polyacrylamide gels, several protein bands were revealed. An apparent auxin (2,4-dichlorophenoxyacetic acid) stimulation of (32)P incorporation into material from membrane vesicles insoluble in trichloroacetic acid-perchloric acid may be reflected partly in enhanced incorporation into protein bands with apparent molecular weights of 45,000 and 50,000. Additionally, a low molecular weight component was sometimes observed where incorporation was stimulated 2- to 3-fold by auxin. However, protein-bound radioactivity represented only a small fraction of the total radioactivity of the acid insoluble material. Other labeled constituents, not retained on the gels, may contribute to the apparent, rapid (10 s or less) auxin response of the isolated membranes. Stimulation of incorporation into the low molecular weight component was given by diglyceride plus calcium, constituents known to augment protein kinase activities in other systems. PMID- 16663592 TI - Use of Na-Nuclear Magnetic Resonance To Follow Sodium Uptake and Efflux in NaCl Adapted and Nonadapted Millet (Panicum miliaceum) Suspensions. AB - Cellular Na(+) transport was followed in vivo by (23)Na nuclear magnetic resonance (NMR) using anionic dysprosium-based shift reagents to resolve internal and external (23)Na(+) resonances. Proso millet (Panicum miliaceum) cell suspensions adapted for rapid growth on 130 mm NaCl had biphasic (23)Na efflux kinetics when shifted to low Na(+) medium, while nonadapted cells had little measurable Na(+) efflux after preloading with (23)NaCl. Uptake of (23)Na was also observed using (23)Na NMR. The resonance frequency of the external Na(+) dysprosium (III) triphosphate, relative to that of the (23)Na in the cells, was sensitive to pH, permitting the pH of the external medium to be followed during the course of in vivo experiments. PMID- 16663593 TI - Quantitation and purification of quaternary ammonium compounds from halophyte tissue. AB - A simple and sensitive spectrophotometric assay for quaternary ammonium compounds (QACs) based on Dragendorff's reagent is described. Although not specific for a particular QAC, the assay allows for rapid survey of tissue for QAC content. A separation method for QACs in halophyte tissue containing high levels of cations was developed using ion exchange resins and ninhydrin to remove the amino acid fraction. PMID- 16663594 TI - Ethylene Contamination of CO(2) Cylinders: Effects on Plant Growth in CO(2) Enrichment Studies. AB - Ethylene concentrations of CO(2) cylinders from different sources ranged from <0.1 up to 475 microliters per liter. The effect of the higher ethylene concentrations on growth of species in CO(2) enrichment studies was examined. Though maize plants showed no effect on C(2)H(4), growth of tomato, rice, mung bean, and phalaris was substantially reduced by ambient concentrations of ethylene in the range of 0.02 to 0.06 microliters per liter. PMID- 16663595 TI - Electrophoretic transfer as a technique for the detection and identification of plant amylolytic enzymes in polyacrylamide gels. AB - An electrophoretic transfer technique was developed for the specific identification of isozymes of starch debranching enzyme, alpha-amylase, and beta amylase. Amylolytic enzymes are separated by native polyacrylamide slab gel electrophoresis and proteins in gels are electrophoretically transferred through starch-containing polyacrylamide gels. Each amylolytic enzyme degrades starch in the transfer gel to its characteristic limit dextrin as it moves through the gel. Various limit dextrins in the starch gel are identified by their characteristic color development in KI/I solution. Isozymes of starch debranching enzyme, alpha amylase, and beta-amylase can be easily identified in the same gel. PMID- 16663596 TI - Protein and Lipid Compositions of Isolated Plasma Membranes from Orchard Grass (Dactylis glomerata L.) and Changes during Cold Acclimation. AB - The chemical composition of plasma membrane fractions isolated from orchard grass seedlings (Dactylis glomerata L.) was analyzed and compared with endomembranes. The plasma membrane is characterized by an enrichment of sterols and a lower degree of unsaturation of phospholipids. Steryl glycosides were found to be one of the lipid components of the plasma membrane, but steryl esters and galactolipids were barely detectable. Diphosphatidyl glycerol was characteristically detected in the mitochondrial membrane, but not in the plasma membrane fraction. Plasma mambrane fraction was also characterized by its ;lower fluidity' in comparison with the endomembranes. This may be due to the large amount of sterols and the lower degree of phospholipid unsaturation in plasma membranes.Electrophoretic comparison of polypeptides was also made between different membranes. The distribution patterns of polypeptides revealed on one- and two-dimensional SDS-slab gels were characteristic for those membranes. The presence of glycopeptide complements was a useful criterion for distinguishing plasma membrane from other membranes. The plasma membrane and the ER + Golgi membranes were enriched in glycopeptides. However, a marked difference was revealed in the total number and the molecular weights of the peptides.During cold acclimation of orchard grass seedlings, the degree of fatty acid unsaturation increased only slightly in plasma membrane, unlike in endomembranes. The change in sterols-to-phospholipids ratio in plasma membrane was also slight. On the other hand, the phospholipid-to-protein ratio increased significantly in the plasma membrane as cold hardiness increased. A significant change in the polypeptide complements of plasma membrane was also demonstrated during cold acclimation. PMID- 16663597 TI - Studies on Freezing Injury of Plant Cells: I. Relation between Thermotropic Properties of Isolated Plasma Membrane Vesicles and Freezing Injury. AB - The thermotropic transition of plasma membrane of Dactylis glomerata was studied by using fluorescence polarization of embedded fluorophore, 1,6-diphenyl-1,3,5 hexatriene. Under the presence of 35% ethylene glycol, reversible thermotropic transitions were observed in isolated plasma membrane vesicles in nearly the same temperature range as the temperature of freezing injury to cells. In liposomes prepared from isolated plasma membranes, however, the thermotropic transitions occurred at much lower temperatures in comparison with those of intact membrane vesicles. Following treatment with pronase, the thermotropic transition also shifted downward.Thus, the thermotropic properties of plasma membranes appeared to be dependent on the membrane proteins. In vitro freezing of the isolated plasma membrane vesicles without addition of any cryoprotectant, such as sorbitol, resulted in an irreversible alteration both in the fluorescence anisotropy values and the temperatures for the thermotropic transition, suggesting an irreversible alteration in the membrane structure, presumably changes in lipid-protein interactions and protein conformation. PMID- 16663598 TI - Utilization of a response-surface technique in the study of plant responses to ozone and sulfur dioxide mixtures. AB - A second order rotatable design was used to obtain polynomial equations describing the effects of combinations of sulfur dioxide (SO(2)) and ozone (O(3)) on foliar injury and plant growth. The response surfaces derived from these equations were displayed as contour or isometric (3-dimensional) plots. The contour plots aided in the interpretation of the pollutant interactions and were judged easier to use than the isometric plots. Plants of ;Grand Rapids' lettuce (Lactuca sativa L.), ;Cherry Belle' radish (Raphanus sativus L.), and ;Alsweet' pea (Pisum sativum L.) were grown in a controlled environment chamber and exposed to seven combinations of SO(2) and O(3). Injury was evaluated based on visible chlorosis and necrosis and growth was evaluated as leaf area and dry weight. Covariate measurements were used to increase precision. Radish and pea had greater injury, in general, that did lettuce; all three species were sensitive to O(3), and pea was most sensitive and radish least sensitive to SO(2). Leaf injury responses were relatively more affected by the pollutants than were plant growth responses in radish and pea but not in lettuce. In radish, hypocotyl growth was more sensitive to the pollutants than was leaf growth. PMID- 16663599 TI - Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation. AB - The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.Rates of CO(2) evolution and H(2) evolution from intact, nodulated root systems under Ar:O(2) atmospheres decreased in parallel when plants were maintained in an extended dark period. While H(2) evolution approached zero after 36 hours of darkness at 22 degrees C, CO(2) evolution rate remained at 38 degrees of the rate measured in light. Of the remaining CO(2) evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO(2) (per gram dry weight nodule) per hour at 22 degrees C and 30 degrees C, respectively.The cost of N(2) fixation in terms of CO(2) evolved per electron pair utilized by nitrogenase was estimated from the slope of H(2) evolution rate versus CO(2) evolution rate. The cost was 2 moles CO(2) evolved per mole H(2) evolved and was independent of temperature.In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H(2) evolution. PMID- 16663600 TI - Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density. AB - Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4 dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [(14)C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed. PMID- 16663601 TI - Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation. AB - The effects of methionine sulfoximine and ammonium chloride on [(14)C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U(14)C]glutamate in the light and in the absence of inhibitor or NH(4)Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO(2) became slightly radioactive. Ammonium chloride (10 mm) increased formation of [(14)C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and (14)CO(2). Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH(3) to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the (14)C from [U (14)C]glutamate to be incorporated into malate and succinate, whereas most of the (14)C from [1-(14)C]glutamate was metabolized to CO(2) and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through "dark" fixation of CO(2) released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-(14)C] glutamate without inhibitor or NH(4)Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and (14)CO(2). Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine. PMID- 16663602 TI - Effect of Endosperm Removal on 7 Normal NaOH-Labile Indole-3-acetic Acid Conjugates in Shoots and Roots of Zea mays Seedlings. AB - The pool of amide-linked indole-3-acetic acid (amide IAA) in the shoot of growing etiolated seedlings of Zea mays increases between the 3rd and 5th day of germination to equal the amount of free IAA and two-thirds the amount of ester IAA. Deseeding the germinant changes the pool size of free and amide IAA in a manner suggestive of conversion of endogenous free IAA to amide IAA. Deseeding also caused an almost total disappearance of amide IAA from the root, demonstrating that the pool of amide IAA is not inert and can be actively metabolized in young Z. mays seedlings. PMID- 16663603 TI - Glyoxylate transamination in intact leaf peroxisomes. AB - Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective alpha-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed. PMID- 16663604 TI - Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells. AB - The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells. PMID- 16663605 TI - Source of endoproteolytic activity associated with purified ribulose bisphosphate carboxylase. AB - The association of endoproteolytic activity with purified ribulose bisphosphate carboxylase (RuBPCase) from barley (Hordeum vulgare var Numar) leaves was investigated. RuBPCase purified by chromatography on agarose gel and diethylaminoethyl-cellulose was free of associated endoproteolytic activity. The addition of leupeptin and casein to the extraction buffer completely eliminated proteolysis during initial extraction of RuBPCase. Endoproteolytic activity previously found associated with RuBPCase was identified as being due to contamination of endoproteinases from broken vacuoles. PMID- 16663606 TI - Sequence of key events in shoot gravitropism. AB - It has recently been shown that asymmetric acid efflux is closely correlated with the gravitropic curvature of plant shoots and roots. The research reported here addresses whether auxin (IAA) redistribution in shoots is the cause or result of asymmetric acid efflux.When abraded sunflower (Helianthus annuus cv Mammoth) hypocotyls are submerged in 20 millimolar neutral buffer, gravicurvature is greatly retarded relative to 0.2 millimolar controls. Nevertheless, in both buffer systems there is a similar redistribution of [(3)H]IAA toward the lower surface of gravistimulated sunflower hypocotyls. These results suggest that graviperception initiates IAA redistribution, which in turn results in auxin induced asymmetric H(+) efflux across the shoot. This interpretation is reinforced by data showing the effects of removal of the epidermal layers (peeling), osmotic shock, and morphactin treatment on gravicurvature and [(3)H]IAA redistribution. Peeling and osmotic shock inhibit gravicurvature but not redistribution. Morphactin inhibits both processes but does not inhibit hypocotyl straight growth. PMID- 16663607 TI - Limiting Factors in Photosynthesis: V. Photochemical Energy Supply Colimits Photosynthesis at Low Values of Intercellular CO(2) Concentration. AB - Although there is now some agreement with the view that the supply of photochemical energy may influence photosynthetic rate (P) at high CO(2) pressures, it is less clear whether this limitation extends to P at low CO(2). This was investigated by measuring P per area as a function of the intercellular CO(2) concentration (C(i)) at different levels of photochemical energy supply. Changes in the latter were obtained experimentally by varying the level of irradiance to normal (Fe-sufficient) leaves of Beta vulgaris L. cv F58-554H1, and by varying photosynthetic electron transport capacity using leaves from Fe deficient and Fe-sufficient plants. P and C(i) were determined for attached sugar beet leaves using open flow gas exchange. The results suggest that P/area was colimited by the supply of photochemical energy at very low as well as high values of C(i). Using the procedure developed by Perchorowicz et al. (Plant Physiol 1982 69:1165-1168), we investigated the effect of irradiance on ribulose bisphosphate carboxylase (RuBPCase) activation. The ratio of initial extractable activity to total inducible RuBPCase activity increased from 0.25 to 0.90 as leaf irradiance increased from 100 to 1500 microeinsteins photosynthetically active radiation per square meter per second. These data suggest that colimitation by photochemical energy supply at low C(i) may be mediated via effects on RuBPCase activation. PMID- 16663608 TI - Abscission: characterization of light control. AB - Exposure of mung bean (Vigna radiata [L.] Wilczek cv Jumbo) cuttings to low level red light inhibits dark-induced leaf abscission. A 12-hour daily light requirement for maximum inhibition of abscission was equally effective as a continuous red light treatment or shorter light-dark cycles. Transfer of cuttings from dark to light stopped the abscission process at the time of transfer. The available evidence suggests a light receptor located in the leaves with an abscission inhibitor translocated from lighted leaf to the abscission zone. PMID- 16663609 TI - Rhizobium infection and nodule development in soybean are affected by exposure of the cotyledons to light. AB - The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root. PMID- 16663610 TI - Kok effect and the quantum yield of photosynthesis : light partially inhibits dark respiration. AB - The linear response of photosynthesis to light at low photon flux densities is known to change abruptly in the vicinity of the light compensation point so that the quantum yield seems to decrease as radiation increases. We studied this ;Kok effect' in attached sunflower (Helianthus annuus L. cv IS894) leaves using gas exchange techniques. The effect was present even though respiration was constant in the dark. It was observed at a similar photon flux density (7 to 11 micromole photons per square meter per second absorbed photosynthetically active radiation) despite a wide range of light compensation points as well as rates of photosynthesis. The effect was not apparent when photorespiration was inhibited at low pO(2) (1 kilopascal), but this result was complicated because dark respiration was quite O(2)-sensitive and was partially suppressed under these conditions. The Kok effect was observed at saturating pCO(2) and, therefore, could not be explained by a change in photorespiration. Instead, the magnitude of the effect varied as dark respiration varied in a single leaf, and was minimized when dark respiration was minimized, indicating that a partial suppression of dark respiration by light is responsible. Quantum yields measured at photon flux densities between 0 and 7 to 11 micromole photons per square meter per second, therefore, represent the combined yields of photosynthesis and of the suppression of a component of dark respiration by light. This leads to an overestimate of the quantum yield of photosynthesis. In view of these results, quantum yields of photosynthesis must be measured (a) when respiration is constant in the dark, and (b) when dark respiration has been inhibited either at low pO(2) to eliminate most of the light-induced suppression of dark respiration or at photon flux densities above that required to saturate the light-induced suppression of dark respiration. Significant errors in quantum yields of photosynthesis can result in leaves exhibiting this respiratory behavior if these principles are not followed. PMID- 16663611 TI - Salinity Effects on Water Potential Components and Bulk Elastic Modulus of Alternanthera philoxeroides (Mart.) Griseb. AB - Pressure volume curves for Alternanthera philoxeroides (Mart.) Griseb. (alligator weed) grown in 0 to 400 millimolar NaCl were used to determine water potential (Psi), osmotic potential (psi(s)), turgor potential (psi(p)) and the bulk elastic modulus (epsilon) of shoots at different tissue water contents. Values of psi(s) decreased with increasing salinity and tissue Psi was always lower than rhizosphere Psi. The relationship between psi(p) and tissue water content changed because epsilon increased with salinity. As a result, salt-stressed plants had larger ranges of positive turgor but smaller ranges of tissue water content over which psi(p) was positive. To our knowledge, this is the first report of such a salinity effect on epsilon in higher plants. These increases in epsilon with salinity provided a mechanism by which a large difference between plant Psi and rhizosphere Psi, the driving force for water uptake, could be produced with relatively little water loss by the plant. A time-course study of response after salinization to 400 millimolar NaCl showed Psi was constant within 1 day, psi(s) and psi(p) continued to change for 2 to 4 days, and epsilon continued to change for 4 to 12 days. Changes in epsilon modified the capacity of alligator weed to maintain a positive water balance and consideration of such changes in other species of higher plants should improve our understanding of salt stress. PMID- 16663612 TI - Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme. AB - The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N(2) and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0 degrees C and for several months at -80 degrees C. The enzyme was purified using (NH(4))(2)SO(4) fractionation, brushite-hydroxyl-apatite chromatography and blue sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO(3). About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO(2) (-) per minute per milligram protein). A sequential elution with NADH followed by KNO(3) (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO(3) (-)-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed. PMID- 16663613 TI - Mechanism of seed priming in circumventing thermodormancy in lettuce. AB - Lettuce (Lactuca sativa L. cv Minetto) seeds were primed in aerated solutions of 1% K(3)PO(4) or water at 15 degrees C in the dark for various periods of time to determine the manner by which seed priming bypasses thermodormancy. Seeds which were not primed did not germinate at 35 degrees C, whereas those which were primed for 20 h in 1% K(3)PO(4) or distilled H(2)O had up to 86% germination. The rate of water uptake and respiration during priming were similar regardless of soak solution. Cell elongation occurred in both water and 1% K(3)PO(4), 4 to 6 h prior to cell division. Both processes commenced sooner in water than K(3)PO(4). Radicle protrusion (germination) occurred in the priming solution at 21 h in water and 27 h in 1% K(3)PO(4).Respiration, radicle protrusion and cell division consistently occurred sooner in primed (redried) seeds compared to nonprimed seeds when they were imbibed at 25 degrees C. Cell division and elongation commenced after 10 h imbibition in primed (redried) seeds imbibed at 35 degrees C. Neither process occurred in nonprimed seeds. Respiratory rates were higher in both primed and nonprimed seeds imbibed at 35 degrees C compared to those imbibed at 25 degrees C, although radicle protrusion did not occur in nonprimed seeds which were imbibed at 35 degrees C. It is apparent that cell elongation and division are inhibited during high temperature imbibition in nonprimed lettuce seeds. Seed priming appears to lead to the irreversible initiation of cell elongation, thus overcoming thermodormancy. PMID- 16663614 TI - Inhibition of 2,4-dichlorophenoxyacetic Acid-stimulated elongation of pea stem segments by a xyloglucan oligosaccharide. AB - Xyloglucan, isolated from the soluble extracellular polysaccharides of suspension cultured sycamore (Acer pseudoplatanus) cells, was digested with an endo-beta-1,4 glucanase purified from the culture fluid of Trichoderma viride. A nonasaccharide rich Bio-Gel P-2 fraction of this digest inhibited 2,4-dichlorophenoxyacetic-acid stimulated elongation of etiolated pea stem segments. The inhibitory activity of this oligosaccharide fraction exhibited a well-defined concentration optimum between 10(-2) and 10(-1) micrograms per milliliter. Another fraction of the same xyloglucan digest, rich in a structurally related heptasaccharide, did not, at similar concentrations, significantly inhibit the elongation. PMID- 16663615 TI - Nonosmotic Effects of Polyethylene Glycols upon Sodium Transport and Sodium Potassium Selectivity by Rice Roots. AB - Addition of polyethylene glycol (PEG) as an osmotic agent (at -230 kilopascals) dramatically lessened the toxicity of NaCl (at 50 moles per cubic meter) to rice (Oryza sativa L.) seedlings. This was explained by a reduction in the uptake of NaCl. This reduction was much greater than could be accounted for by the lowered transpiration rate resulting from the solute potential changes due to the PEG.Low concentrations of PEG (-33 kilopascals and less) had no effect upon transpiration rate but reduced sodium uptake (from 10-50 moles per cubic meter NaCl) by up to 80%. PEG (at -33 kilopascals) also reduced chloride uptake but had no effect upon the uptake of potassium from low (0.5-2.0 moles per cubic meter) external concentrations. However, the increased uptake of potassium occurring between 2 and 10 moles per cubic meter external concentration was abolished by PEG. Similar concentrations of mannitol had no effect upon sodium uptake in rice. PEG, in similar conditions, had much less effect upon sodium uptake by the more salt resistant species, barley.(22)Na studies showed that PEG reduced the transport of sodium from root to shoot, but had a long half time for maximal effect (several days).(14)C-labeled PEG was shown to bind to microsomal membranes isolated from rice roots; it is suggested that this is due to multipoint attachment of the complex ions of PEG which exist in aqueous solutions. It is argued that this reduces passive membrane permeability, which accounts for the large effect of PEG on sodium influx in rice and the different effects on sodium influx and (carrier dependent) potassium influx. PMID- 16663616 TI - Calorimetry of nitrogenase-mediated reductions in detached soybean nodules. AB - Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N(2) fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made.There was major heat output by nodules for all of the nitrogenase substrates tested (H(+), N(2), N(2)O, and C(2)H(2)) further establishing the in vivo energy inefficiency of biological N(2) fixation. Exposure to a short burst of 100% O(2) partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H(+) reductions was 171 +/- 6 kilocalories per mole H(2) evolved in an Ar-O(2) atmosphere, that for N(2) fixation was 784 +/- 26 kilocalories per mole H(2) evolved and N(2) fixed, and that for C(2)H(2) reduction was 250 +/- 12 kilocalories/mole C(2)H(4) formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a DeltaH' of -200 kilocalories per mole 2e(-) is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N(2) fixation cost of 9.5 grams glucose per gram N(2) fixed or 3.8 grams C per gram N(2), which is in close agreement with earlier calculations based on nodular CO(2) production. PMID- 16663617 TI - Effects of pod removal on metabolism and senescence of nodulating and nonnodulating soybean isolines: I. Metabolic constituents. AB - Field studies were conducted in 1981 and 1982 to ascertain the effects of pod removal on senescence of nodulating and nonnodulating isolines of soybean (Glycine max [L.] Merr. cv Harosoy) plants. Specifically, the test hypothesis was that nodules act as a nitrogen source and a carbohydrate sink which would in turn prevent or delay senescence in the absence of pods. Senescence was judged by changes in metabolite levels, in dry matter accumulation, and by visual observation.For both nodulated and nonnodulated plants, pod removal had no effect on the magnitude or rate of dry matter and reduced-N accumulation by whole plants. Phosphorus accumulation was significantly less in both nodulated- and nonnodulated-depodded plants, compared with respective control plants with pods. These data suggested a role for pods in phosphorus uptake. Accumulation of dry matter, reduced N, and phosphorus ceased at approximately the same time for all treatments.Pod removal did affect partitioning of plant constitments, with leaves and stems of depodded plants serving as a major alternate sink for accumulation of dry matter, reduced N, phosphorus, and nonstructural carbohydrates (primarily starch). While depodded plants eventually lost a significant amount of leaves, leaf drop was delayed relative to plants with pods; and depodded plants still retained some green leaves at 2 weeks past grain maturity of control (podded) plants.The results indicated that senescence patterns of soybean plants were the same for nodulated and nonnodulated plants, and that pods did not control the initiation of senescence, but rather altered the partitioning of plant constituents and the visual manifestations of senescence. PMID- 16663618 TI - Effects of Pod Removal on Metabolism and Senescence of Nodulating and Nonnodulating Soybean Isolines: II. Enzymes and Chlorophyll. AB - The objectives of this work were to determine the effect of sink strength (presence or absence of pods) and nitrogen source (nodulating versus nonnodulating plants) on enzymic activities, chlorophyll concentration, and senescence of soybean (Glycine max [L.] Merr. cv Harosoy) isolines. A 2-year (1981-1982) field study was conducted.For both nodulated and nonnodulated plants, ribulose bisphosphate carboxylase (RuBPCase) activity of upper-canopy leaves was decreased by pod removal in both years, while chlorophyll concentration was decreased in 1981 only. Nonnodulated plants had lower RuBPCase activity in 1981 and lower chlorophyll concentration in both years compared with nodulated plants. In both years, and for all treatments, RuBPCase activity and chlorophyll began to decline at about the same time, but the rate of decline was less for depodded than for podded plants. Leaves in the middle and lower parts of the canopy had similar RuBPCase activity and chlorophyll concentration trends as upper-canopy leaves for all treatments.Profiles of nitrate reductase activity (NRA) were similar for all treatments in both 1981 and 1982. Acetylene reduction profiles were similar for nodulated-podded and nodulated-depodded plants. The peak and decline in NRA profiles preceded the peak and decline in acetylene reduction profiles. The rate of decline in acetylene reduction activity was less for depodded plants, especially in 1982, but activities reached zero by the final sampling time. Thus, nodule senescence was not prevented by pod removal.Based on seasonal profiles of RuBPCase activity, chlorophyll, NRA, and acetylene reduction activity, the initiation of senescence appeared to occur at the same approximate time for all treatments and, thus, did not depend on the presence or absence of pods or nodules. The hypothesis that nodules act as a nitrogen source and carbohydrate sink to delay senescence in the absence of pods was not correct. PMID- 16663619 TI - Use of polyethylene glycol in isolation and assay of stable, enzymically active starch granules from developing wheat endosperms. AB - A procedure using polyethylene glycol (PEG), molecular weight 1000, was developed for the isolation of starch granules from wheat endosperm. Immature endosperm tissue was cut repeatedly in 300 millimolar PEG 1000 and filtered through Miracloth. Centrifugation separated a pellet from a supernatant with inhibitory activity. The pellet contained several enzyme activities, including soluble and bound components of starch synthase, starch phosphorylase, and sucrose synthase activities. The starch phosphorylase activity was unaffected by several washings with 300 millimolar PEG 1000 but was lost when the granules were washed once without PEG or washed with sucrose, glycerol, or sorbitol (up to 30%, w/v). The fraction of starch synthase, remaining on the granules after a wash without PEG (the ;bound' activity) was not affected by the addition of 30% sorbitol to the wash buffer. This fraction became larger with grain development (0.2-0.7).To obtain high activity, PEG was required not only during isolation of granules but also in the assay of both starch phosphorylase and starch synthase giving optimum activity at 225 to 255 millimolar. PEG reduced the requirement for glycogen as primer with soluble starch synthase. However, the ;bound' starch synthase activity was unaffected by PEG. PEG of different size were compared by their effects in the assay of starch granules: with increase in molecular size, the same effect was obtained at ever lower polymer concentration (w/v) down to a limit.Treatment of granules with Triton X-100 did not affect their starch synthase activity, but it removed the capacity to incorporate label from UDP [(14)C]G into non-starch polymers.It is concluded that PEG, like some other active compounds (ethanol Na(3)-citrate, and Ficoll) could mediate enzyme-primer interaction by exclusion. PMID- 16663620 TI - Involvement of Calcium and Calmodulin in Membrane Deterioration during Senescence of Pea Foliage. AB - The prospect that Ca(2+) promotes senescence by activating calmodulin has been examined using cut pea (Pisum sativum co Alaska) foliage as a model system. Senescence was induced by severing 17-day-old plants from their roots and maintaining them in aqueous test solutions in the dark for an additional 4 days. Treatment of the foliage with the Ca(2+) ionophore (A23187) during the senescence induction period promoted a lateral phase separation of the bulk lipids in microsomal membranes indicating that internalization of Ca(2+) facilitates membrane deterioration. In addition, microsomal membranes from ionophore-treated tissue displayed an increased capacity to convert 1-aminocyclopropane-1 carboxylic acid to ethylene and an increased propensity to produce the superoxide anion (O(2) (tau)). Treatment of the tissue with fluphenazine during the senescence-induction period, which prevents binding of the Ca:Calmodulin complex to enzymes, delayed membrane deterioration as measured by these criteria. It also proved possible to simulate these in situ effects of the Ca(2+) ionophore on ethylene production and O(2) (tau) formation by treating microsomal membranes isolated from young tissue with phospholipase A(2) in the presence of Ca(2+) and calmodulin, and these effects of phospholipase A(2) and Ca:calmodulin were inhibited by calmodulin antagonists. The observations collectively suggest that internalized Ca(2+) promotes senescence by activating calmodulin, which in turn mediates the action of phospholipase A(2) on membranes. PMID- 16663621 TI - Photosynthetic electron transport in guard cells of diverse species. AB - Guard cells of plants representing 18 species were assayed qualitatively for potential to conduct photosynthetic linear electron transport. These plants included C(3) pteridophytes, C(3) and C(4) monocots, and C(3), C(4), and Crassulacean acid metabolism dicots. By use of a microfluorospectrophotometer, guard cell samples in epidermal peels were isolated optically. Chlorophyll fluorescence was monitored from the onset of excitation light. For guard cells of all these species, fluorescence intensity increased during illumination. When samples were preincubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, however, there was a more rapid increase in fluorescence. These results indicate that all tested guard cells conduct photosynthetic electron transport through the reaction center of photosystem II. PMID- 16663622 TI - Influence of Osmotic Adjustment on Leaf Rolling and Tissue Death in Rice (Oryza sativa L.). AB - Osmotic adjustment, measured by the lowering of the osmotic potential at full turgor, and its influence on leaf rolling and leaf death was assessed in the lowland rice (Oryza sativa L.) cultivar IR36 in both the greenhouse and field. The degree of osmotic adjustment varied with the degree and duration of stress, but was usually 0.5 to 0.6 megapascal (maximally 0.8 to 0.9 megapascal) under severe stress conditions. In leaves in which osmotic adjustment was 0.5 to 0.6 megapascal, leaf rolling and leaf death occurred at lower leaf water potentials in adjusted than in nonadjusted leaves. We conclude that osmotic adjustment aids in the drought resistance of rice by delaying leaf rolling, thereby maintaining gas exchange, and by delaying leaf death. PMID- 16663623 TI - Comparative effects of ethylene and cyanide on respiration, polysome prevalence, and gene expression in carrot roots. AB - Treatment of carrot roots (Daucus carota L.) with 10 microliters per liter ethylene in O(2) evokes a three- to four-fold increase in polysome prevalence and associated poly(A)(+) RNA. The increase in polysome prevalence is attended by a similar change in CO(2) evolution. The increase in polysomal poly(A)(+) mRNA constitutes primarily a generic increase in constitutive mRNAs as assayed by in vitro translation. However, changes in the relative abundance of several in vitro translatable ethylene specific mRNAs do occur.Cyanide, at concentrations which inhibit cytochrome oxidase, initiates a respiratory rise very similar in kinetics and magnitude to that evoked by ethylene. Moreover, the combined treatment with cyanide and ethylene evokes a respiratory response resembling that caused by ethylene or cyanide alone. Nevertheless, cyanide, in the presence of ethylene, significantly inhibits the increase in polysome prevalence and new gene expression associated with ethylene treatment of carrot roots. Separation of in vitro translation products by one-dimensional and two-dimensional gel electrophoresis shows that several new in vitro translation products appear in cyanide-treated carrots different from those evoked by ethylene. Engagement of the less energy efficient alternative electron transport path by cyanide may be responsible for inhibition of the normal ethylene associated increase in polysome prevalence and new gene expression. The implications of these results on regulation of respiratory metabolism are discussed and compared with the results for similar experiments with avocado fruit (Tucker and Laties 1984 Plant Physiol 74: 307-315) in which cyanide does not inhibit an ethylene educed increase in polysome prevalence and change in gene expression. PMID- 16663624 TI - Auxin-regulated polypeptide changes at different stages of strawberry fruit development. AB - The pattern of polypeptides at different stages of strawberry (Fragaria ananassa Duch. cv Ozark Beauty) fruit development was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An 81,000-dalton polypeptide appeared between 5 and 10 days after pollination. Polypeptides with molecular weights of 76,000 and 37,000 daltons were formed after 10 days. The control exerted by auxin in the stage-specific formation of polypeptides was investigated by stopping fruit growth after removing the achenes and reinitiating fruit growth by the application of a synthetic auxin, alpha-naphthaleneacetic acid (NAA). When the achenes were removed from the 5- and 10-day-old fruits, the fruits failed to grow, the 81,000 dalton polypeptide was not formed between 5 and 10 days, and the 76,000- and 37,000-dalton polypeptides were not formed between 10 and 20 days. Application of NAA to fruits deprived of auxin by removal of achenes resulted in the resumption of growth and also in the appearance of these polypeptides. Removal of achenes of the 5- or 10-day-old fruits and growing them without auxin resulted in the formation of 52,000- and 57,000-dalton polypeptides. These two polypeptides were not formed when NAA was applied to fruits after removal of achenes. Supply of NAA to auxin-deprived fruits 5 days after removal of achenes resulted in resumption of growth and also in the disappearance of these two polypeptides, pointing out their possible relation to the inhibition of fruit growth. PMID- 16663625 TI - Primary Action of Indole-3-acetic Acid in Crown Gall Tumors: Increase of Solute Uptake. AB - Exogenously added indole-3-acetic acid at a concentration of 100 micromolars stimulates d-glucose uptake (or 3-O-methyl-d-glucose uptake) by 25% in crown gall tumors induced on potato tuber tissue by Agrobacterium tumefaciens strain C 58. The titration of the endogenous IAA with the auxin antagonist 2-naphthaleneacetic acid at 100 micromolars reduces d-glucose uptake by about 80%. The apparent inhibition constant K(i) is 21 micromolars. Other auxin antagonists like 1 naphthoxyacetic acid and 2-(p-chlorophenoxy)-2-methylpropionic acid show similar effects. The uptake of the amino acids leucine, methionine, tryptophan, lysine, and aspartic acid is also inhibited by 2-naphthaleneacetic acid to similar degrees. The auxins 1-naphthaleneacetic acid and 2-naphthoxyacetic acid at concentrations between 10 and 100 micromolars inhibit solute uptake only slightly (inhibition less than 20%). The impact of the results on the postulated role of indole-3-acetic acid as a modifier of the electrochemical proton gradient across the plasmalemma in crown gall tumor tissue is discussed. PMID- 16663626 TI - Photobiology of diagravitropic maize roots. AB - Light-induced modification of gravitropism in etiolated roots of Zea mays cv Bear x W38 is a low fluence response mediated by phytochrome. This cultivar has a threshold of 10(-6) mol m(-2) and becomes saturated with 10(-2) mol m(-2) of red light. The maximum light-mediated response of 32 degrees downward from horizontal occurs in roots 10 to 30 millimeters in length, 120 to 165 minutes after irradiation. Reciprocity is valid from 2 to at least 9,000 seconds and the response can be about 90% reversed by far red light. Photoreversibility is lost (;escape' occurs) about 20 minutes after red irradiation but appears to be regained 60 to 80 minutes later. A red light-induced (or synchronized) nutation in the apparent curvature rather than unusual escape characteristics may explain these results. PMID- 16663627 TI - Photosynthetic response and adaptation to high temperature in desert plants : a comparison of gas exchange and fluorescence methods for studies of thermal tolerance. AB - The temperature threshold for the onset of irreversible loss of photosynthetic capacity of leaves was examined in studies of net CO(2) exchange and by chlorophyll fluorescence techniques. Close agreement was found between the temperature threshold for a dramatic increase in the fluorescence of chlorophyll from intact leaves and the leaf temperature at which the capacity for photosynthetic CO(2) fixation (measured at rate saturating light intensity by infrared gas analysis) began to be temperature unstable (i.e. decline with time of exposure to a constant temperature). This decline in CO(2) uptake was not a result of a stomatal response yielding a reduced intercellular CO(2) concentration at high temperature, and it is interpreted as an indication of progressive damage to some essential component(s) of the leaf. The temperature dependent change in chlorophyll fluorescence apparently also indicated the onset of this damage. The fluorescence assay could be conducted with discs of leaves collected from remote locations and kept moist while they were transported to a central location, allowing assessment of the high temperature tolerance of leaves which developed under natural field conditions. These assays were verified using a mobile laboratory to study gas exchange of attached leaves in situ. The high temperature sensitivity of leaves of plants growing under natural conditions were similar to those of the same species grown in controlled environments of similar thermal regimes. High temperature in controlled environment studies brought about acclimation responses which increased the threshold for high temperature damage as measured by gas exchange. Studies of fluorescence versus temperature confirmed that this method could be used to quantify these responses, and permitted the kinetics of the acclimation response to be examined. Gas exchange studies, while providing similar estimates of thermal stability, required more time, more elaborate instrumentation, and are particularly difficult to conduct with field plants growing in situ. PMID- 16663628 TI - Regulation of the shikimate pathway of carrot cells in suspension culture. AB - The activity of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino heptulosonate 7-phosphate synthase, is demonstrated in extracts of Daucus carota cells grown in suspension culture. Maximum specific enzyme activity is found midway through the logarithmic growth of the culture; cells in lag and stationary phases of growth have lower enzyme levels. The enzyme is activated by tyrosine and tryptophan. The extent of activation varies during cell growth. PMID- 16663629 TI - Hydraulic conductance as a factor limiting leaf expansion of phosphorus-deficient cotton plants. AB - Suboptimal levels of phosphorus (P) strongly inhibited leaf expansion in young cotton (Gossypium hirsutum L.) plants during the daytime, but had little effect at night. The effect of P was primarily on cell expansion. Compared to plants grown on high P, plants grown on low P had lower leaf water potentials and transpiration rates, and greater diurnal fluctuations in leaf water potential. Hydraulic conductances of excised root systems and of intact transpiring plants were determined from curves relating water flow rate per unit root length to the pressure differential across the roots. Both techniques showed that low P significantly decreased root hydraulic conductance. The effects of P nutrition on hydraulic conductance preceded effects on leaf area. Differences in total root length, shoot dry weight, and root dry weight all occurred well after the onset of differences in leaf expansion. The data strongly indicate that low P limits leaf expansion by decreasing the hydraulic conductance of the root system. PMID- 16663630 TI - Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : III. Responses of Protoplasts and Intact Chloroplasts. AB - Protoplasts and intact chloroplasts isolated from Agropyron smithii Rybd. were utilized in an effort to determine the limiting factor(s) for photosynthesis at supraoptimal temperatures. Saturated CO(2)-dependent O(2) evolution had a temperature optimum of 35 degrees C for both protoplasts and intact chloroplasts. A sharp decline in activity was observed as assay temperature was increased above 35 degrees C, and at 45 degrees C only 20% of the maximal rate remained. The temperature optimum for 3-phosphoglycerate reduction by intact chloroplasts was 35 degrees C. Above this temperature, 3-phosphoglycerate reduction was more stable than CO(2)-dependent O(2) evolution. Reduction of nitrite in coupled intact chloroplasts had a temperature optimum of 40 degrees C with only slight variation in activity between 35 degrees C and 45 degrees C. Reduction of nitrite in uncoupled chloroplasts had a temperature optimum of 40 degrees C, but increasing the assay temperature to 45 degrees C resulted in a complete loss of activity. Reduction of p-benzoquinone by protoplasts and intact chloroplasts had a temperature optimum of 32 degrees C when measured in the presence of dibromothymoquinone. This photosystem II activity exhibited a strong inhibition of O(2) evolution as assay temperature increased above the optimum. It is concluded that, below the temperature optimum, ATP and reductant were not limiting photosynthesis in these systems or intact leaves. Above the temperature optimum, photosynthesis in these systems is limited in part by the phosphorylation potential of the stromal compartment and not by the available reductant. PMID- 16663631 TI - Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution. AB - A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions. PMID- 16663632 TI - Pyruvate orthophosphate dikinase of c(3) seeds and leaves as compared to the enzyme from maize. AB - Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C(3) plants (wheat, pea, green bean, plum, and castor bean), in some C(3) leaves (tobacco, spinach, sunflower, and wheat), and in C(4) (maize) kernels. The enzyme in the C(3) plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C(3) seeds and leaves is much less than in C(4) leaves. PPDK is present in kernels of the C(4) plant, Zea mays in amounts comparable to those in C(4) leaves.Regulatory properties of the enzyme from C(3) tissues (wheat) are similar to those of the enzyme from C(4) leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves).Following incorporation of (14)CO(2) by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development. PMID- 16663633 TI - Pyruvate orthophosphate dikinase gene expression in developing wheat seeds. AB - The amount of pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) protein in wheat (Triticum aestivum L. var Cheyenne) grains was determined at different stages of development by the protein blot method. The variation in PPDK protein with time in developing wheat grains was similar to that of the enzyme's activity reported by Meyer et al. (1982 Plant Physiol 69: 7-10). The variation in levels of PPDK mRNA with seed development was determined by analysis of polypeptides immunoprecipitated by anti-PPDK serum from in vitro translation products of extracted seed RNA. This mRNA variation was similar to that of the in vivo enzyme levels and the correlation is consistent with the regulation of PPDK gene expression by the level of its mRNA.The highest level of PPDK in developing wheat seeds occurs later than the highest levels of both ribulose bisphosphate carboxylase (EC 4.1.1.39) and of chlorophyll, which are located in the green pericarp tissue. PPDK was located in both endosperm and pericarp tissue of the seeds. The tissue location and developmental profile of seed PPDK are consistent with a metabolic role of providing phosphoenolpyruvate as a substrate for recapturing respiratory CO(2) in the seed, and possibly for amino acid interconversions during development. PMID- 16663634 TI - Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods. AB - Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12 h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3 PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6 bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any ;feedback' inhibition of PS. The higher 7 hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates. PMID- 16663635 TI - Activation of Coupling Factor 1 from Euglena gracilis Chloroplasts : Conditions for Optimal Activation and Their Possible Physiological Significance. AB - The recently described method for the activation of the Ca(2+)-ATPase of coupling factor 1 from chloroplasts (CF(1)) of Euglena gracilis by low pH occurs optimally in high concentrations of NaCl, and is unaffected by the acid used to lower the pH to 4.5. Activation is inhibited by light, and this effect can be reversed by the presence of NADP(+), ADP + inorganic phosphate, or an uncoupler. There appears to be no difference between the activities in the soluble and the particulate phases, and they seem to represent the same enzyme. The response of the activation process to light and to effectors of electron transport and phosphorylation indicates a possible physiological role for the acid activation of Euglena CF(1). PMID- 16663636 TI - Effects of rotenoids on isolated plant mitochondria. AB - The effects of several rotenoids have been studied on potato (Solanum tuberosum L.) tuber and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls mitochondria. The selective inhibition of mitochondrial complex I is characterized by several tests: (a) no effect can be observed on exogenous NADH or succinate oxidation; (b) malate oxidation is inhibited at pH 7.5; (c) one third decrease of ADP/O ratio appears during malate oxidation at pH 6.5 or during alpha-ketoglutarate, citrate, or pyruvate oxidation at a pH about 7; (d) during malate oxidation at pH 6.5, a transient inhibition appears which can be maintained by addition of exogenous oxaloacetate; (e) in potato mitochondria, the inhibition of malate oxidation disappears at pH 6.5 when NAD(+) is added. Then, a one-third decrease of the ADP/O ratio can be measured.Such a selective inhibition of complex I is obtained with deguelin, tephrosin, elliptone, OH-12 rotenone, and almost all the rotenoids extracted from Derris roots. The presence of the rings A, B, C, D, E seems to be necessary for the selective inhibition. Opening of the E ring and hydroxylation of the 9 position (rot-2'-enoic acid) give a rotenoid derivative with multisite inhibitory activities on flavoproteins, which are quite comparable to those of common flavonoids such as kaempferol (Ravanel et al. 1982 Plant Physiol 69: 375-378). PMID- 16663637 TI - Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.). AB - Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially. PMID- 16663638 TI - Lack of ATP requirement for light stimulation of glycerate transport into intact isolated chloroplasts. AB - Light increased the initial rate and the extent of glycerate uptake by intact isolated chloroplasts. Half-maximum stimulation occurred with 10 to 20 watts per square meter of red light. Preillumination of chloroplasts enhanced uptake in a subsequent dark period. The light effect was abolished by DCMU and also by uncoupling agents such as nigericin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone.Arsenate and phlorizin only inhibited glycerate uptake to the extent that metabolism in the chloroplast was decreased by insufficient ATP. The concentration of glycerate accumulated in the chloroplast stroma was not significantly decreased. Chloroplasts isolated from young pea shoots (Pisum sativum, L. cv Massey Gem) were depleted of ATP by incubation with inorganic pyrophosphate or with ATP analogs. These treatments also decreased metabolism of glycerate but the actual concentration of glycerate accumulated in the chloroplast stroma was not decreased.The results indicate that glycerate uptake is driven by ion gradients established across the chloroplast envelope in the light. ATP is not involved in the transport of glycerate into chloroplasts, being required only for the subsequent metabolism of glycerate in the chloroplast stroma. It is proposed that glycerate transport may be coupled to the proton gradient established in the light across the chloroplast envelope. PMID- 16663639 TI - Tissue specificity of the heat-shock response in maize. AB - The tissue specificity of the heat-shock response in maize was investigated. The ability to synthesize heat shock proteins (hsp) at 40 degrees C, as well as the intensity and duration of that synthesis, was analyzed in coleoptiles, scutella, green and etiolated leaves, suspension-cultured cells, germinating pollen grains, and primary root sections at different stages of development. One-dimensional sodium dodecyl sulfate gel electrophoresis of extracted proteins revealed that most of the tissues synthesized the typical set of 10 hsp, but that the exact characteristics of the response depended upon the tissue type. While elongating portions of the primary root exhibited a strong heat shock response, the more mature portions showed a reduced ability to synthesize hsp. Leaves, whether green or etiolated, excised or intact, constitutively synthesized a low level of hsp at 25 degrees C, and high levels could be induced at 40 degrees C. Suspension cultures of Black Mexican sweet corn synthesized, besides the typical set of hsp, two additional polypeptides. In contrast to all the other tissues, germinating pollen grains could not be induced to synthesize the typical set of hsp but did synthesize two new polypeptides of 92 and 56 kD molecular weight.The heat shock response was transient for most of the tissues which synthesized the standard set of hsp. Hsp synthesis was detected up to 2 to 3 hours, but not at 10 hours of continuous 40 degrees C treatment. The exception was suspension cultured cells, in which hsp synthesis showed only a slight reduction after 10 hours at 40 degrees C. Tissue-specific differences in the heat-shock response suggest that there are differences in the way a given tissue is able to adapt to high temperature.We have confirmed the previous suggestion that maize hsp do not accumulate in substantial quantities. Using two-dimensional gel analysis, hsp could be detected by autoradiography but not by sensitive silver staining techniques. PMID- 16663640 TI - Modification of cytokinins by cauliflower microsomal enzymes. AB - N(6)-(Delta(2)-isopentenyl)Adenine and N(6)-(Delta(2)-isopentenyl)adenosine were hydroxylated, respectively, to 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-trans-2-butenylamino) -9-beta-ribofuranosylpurine in the presence of NADPH and the microsomal fraction from cauliflowers (Brassica oleracea L.). The hydroxylating reaction was completely inhibited by 10 millimolars metyrapone and partially inactivated by 10-minute treatment of the microsomal preparation with ethylene. The cytokinins were also dealkylated by the microsomal enzymes and formed adenine from cytokinin base and adenosine from cytokinin nucleoside. These results suggest that plant cytochrome P-450 is involved in the conversion of one type of cytokinin to another, and in the modification of cytokinin molecules. PMID- 16663641 TI - Photoregulation of Anthocyanin Synthesis : VIII. Effect of Light Pretreatments. AB - A comparative study of the spectral sensitivity of anthocyanin production in dark grown and light-pretreated systems was carried out in Brassica oleracea L., Lycopersicon esculentum Mill., Secale cereale L. and Spirodela polyrrhiza L. Light pretreatments bring about an enhancement of the inductive, red-far red reversible response in all systems, a decrease of the continuous irradiation response in cabbage, rye, and tomato seedlings, and an enhancement of the continuous irradiation response in cabbage leaf disks. Light pretreatments also bring about a marked change in the spectral sensitivity of the continuous irradiation response. The different effect of light pretreatments on the photosensitivity of the response to short and long wavelength irradiations suggests that two photoreceptors, phytochrome and cryptochrome, may be involved in the photoregulation of anthocyanin production. PMID- 16663642 TI - Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq. AB - The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of (14)CO(2) uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO(2) uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO(2) uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days. PMID- 16663643 TI - Biosynthesis of jasmonic Acid by several plant species. AB - Six plant species metabolized (18)O-labeled 12-oxo-cis,cis-10,15-phytodienoic acid (12-oxo-PDA) to short chain cyclic fatty acids. The plant species were corn (Zea mays L.), eggplant (Solanum melongena L.), flax (Linum usitatissimum L.), oat (Avena sativa L.), sunflower (Helianthus annuus L.), and wheat (Triticum aestivum L.). Among the products was jasmonic acid, a natural plant constituent with growth-regulating properties. The pathway is the same as the one recently reported by us for jasmonic acid synthesis in Vicia faba L. pericarp. First, the ring double bond of 12-oxo-PDA is saturated; then beta-oxidation enzymes remove six carbons from the carboxyl side chain of the ring. Substrate specificity studies indicated that neither the stereochemistry of the side chain at carbon 13 of 12-oxo-PDA nor the presence of the double bond at carbon 15 was crucial for either enzyme step. The presence of enzymes which convert 12-oxo-PDA to jasmonic acid in several plant species indicates that this may be a general metabolic pathway in plants. PMID- 16663644 TI - Ethylene-Induced Chlorosis in the Pathogenesis of Bipolaris sorokiniana Leaf Spot of Poa pratensis. AB - Endogenous ethylene of Poa pratensis leaves infected by Bipolaris sorokiniana was evaluated as a factor in leaf chlorosis during pathogenesis. Detectable increases in endogenous ethylene of leaves of intact plants under normal ambient pressure occurred 12 hours after inoculation and was maximum at 48 hours; from 48 to 96 hours the ethylene progressively decreased. Necrotic lesions surrounded by chlorotic halos occurred on infected leaves between 24 and 48 hours. Midvein chlorosis interconnecting individual lesions and complete chlorosis of all tissues not directly affected by the lesions occurred between 72 and 96 hours, after maximum production of ethylene at 48 hours. The chlorophyll loss in infected leaves by 96 hours was 44% compared with controls.Subjecting inoculated leaves of intact plants to a controlled atmospheric-environmental system with an atmospheric pressure of 233 millibars and O(2) and CO(2) partial pressures adjusted to approximately that of normal ambient pressure during infection and disease development prevented most midvein chlorosis and complete chlorosis, but did not prevent necrotic lesion or chlorotic halo development. Under the hypobaric conditions, chlorophyll loss during disease development was reduced to 22% compared with controls at 96 hours. The observations suggest that ethylene may function late in pathogenesis of this host-pathogen interaction and is responsible for much of the chlorophyll loss after its maximum production at 48 hours. PMID- 16663645 TI - Sulfate Uptake and Its Regulation in Lemna paucicostata Hegelm. 6746. AB - The results of studies of SO(4) (2-) uptake by Lemna paucicostata are most simply interpreted by the hypothesis that at least two components are involved, one saturating and one linear, ;nonsaturating.' The saturating component has a low K(m) and high specificity for SO(4) (2-). Uptake by the nonsaturating component is less affected by pH and temperature than is that of the saturating system. SO(4) (2-) efflux is not quantitatively important in Lemna under standard conditions (20 micromolar SO(4) (2-)) (Datko AH, SH Mudd 1980 Plant Physiol 65: 906-912). 55% of newly taken up (35)SO(4) (2-) enters a slowly turning over compartment (vacuole?); 45% remains in a compartment (cytoplasm?) in which it is rapidly metabolized to organic compounds.Growth in increased concentrations of SO(4) (2-) or cystine, but not methionine, down-regulates the saturating, but not the nonsaturating, system. Growth in limiting SO(4) (2-) up-regulates the saturating system. Overall, a 500-fold change was observed. Reciprocal inhibition experiments demonstrated that molybdate and SO(4) (2-) are taken up by a common mechanism, but growth in molybdate failed to up-regulate SO(4) (2-) uptake. Regulation by growth in SO(4) (2-) or cystine did not markedly affect uptake of phosphate or of several organic compounds.The saturating system contributes 99% of SO(4) (2-) uptake under standard conditions, providing sufficient SO(4) (2-) so it is not limiting. In nature the same system likely contributes at least 65 to 70%. PMID- 16663646 TI - Responses of Sulfur-Containing Compounds in Lemna paucicostata Hegelm. 6746 to Changes in Availability of Sulfur Sources. AB - The steady state concentrations of S-containing compounds formed in Lemna paucicostata Hegelm. 6746 in response to variations in source and concentrations of sulfur were measured. Neither growth rates nor protein accumulation were markedly affected by the various growth conditions. Ignoring complications due to possible compartmentation, the results are consistent with internal pools of both SO(4) (2-) and cyst(e)ine (or products of their metabolism), but not methionine, being effectors of regulation of high affinity SO(4) (2-) uptake. As SO(4) (2-) in the growth medium was increased to 10 mm, down-regulation of high affinity SO(4) (2-) uptake was more than compensated for by unregulated uptake via the "non-saturating" uptake system. Tissue inorganic SO(4) (2-) accumulated but formation of reduced sulfur remained constant. Some conversion of l-cystine sulfur to SO(4) (2-) occurred. Presence of l-cystine in the medium (a) down regulated high affinity SO(4) (2-) uptake and (b) decreased the rate of SO(4) (2 ) organification. The net results were decreased (7 mum l-cystine) or normal (14 mum l-cystine) total tissue SO(4) (2-) and dose-dependent accumulation of soluble cyst(e)ine and glutathione, but not of soluble methionine. l-Methionine was not metabolized to cyst(e)ine or its products. Presence of l-methionine in the medium led to increased total tissue sulfur, accounted for almost wholly by manyfold increases in soluble methionine, AdoMet, and S-methylmethionine sulfonium. Soluble cyst(e)ine increased slightly. PMID- 16663647 TI - Comparisons of Peptide hydrolase activities in cereals. AB - Carboxypeptidase activity (hydrolysis of N-carbobenzoxy-l-phenylalanyl-l-alanine) is high in a number of temperate zone cereals, originating in Asia Minor (wheat, barley, oats, wild oats, rye, triticale) compared to other cereals originating in central America or Asia (maize, sorghum, rice). However, endopeptidase activity (hydrolysis of azocasein or hemoglobin) is relatively much higher in the latter group. Comparison of trichloroacetic acid (TCA)-soluble products derived from the hydrolysis of hemoglobin showed that carboxyterminal amino acids (histidine, arginine, and tyrosine), are released when extracts from wheat and barley endosperms are used. With extracts from corn endosperms, much more TCA-soluble ultraviolet- absorbing material is released, but very little is released as free amino acids within the first 2 hours and the expected C-terminal amino acids of hemoglobin are not detected in significant amounts. These results suggest that the method of hydrolysis of the storage proteins may be significantly different in these two classes of cereals. PMID- 16663648 TI - Effect of N-source on soybean leaf sucrose phosphate synthase, starch formation, and whole plant growth. AB - Soybeans (Glycine max L. Merr. cv Tracy and Ransom) were grown under N(2) dependent or NO(3) (-)-supplied conditions, and the partitioning of photosynthate and dry matter was characterized. Although no treatment effects on photosynthetic rates were observed, NO(3) (-)-supplied plants in both cultivars had lower starch accumulation rates than N(2)-dependent plants. Leaf extracts of NO(3) (-) supplied plants had higher activities of sucrose phosphate synthase (SPS) and cytoplasmic fructose-1,6-bisphosphatase (FBPase) than N(2)-dependent plants. The variation in starch accumulation was correlated negatively with the activity of SPS, but not the activity of FBPase, UDP-glucose pyrophosphorylase, or ADP glucose pyrophosphorylase. These results suggested that starch accumulation is biochemically controlled, in part, by the activity of SPS. Leaf starch content at the beginning of the photoperiod was lower in NO(3) (-)-supplied plants than N(2) dependent plants in both cultivars which suggested that net starch utilization as well as accumulation was affected by N source.Total dry matter accumulation and dry matter distribution was affected by N source in both cultivars, but the cultivars differed in how dry matter was partitioned between the shoot and root as well as within the shoot. The activity of SPS was correlated positively with total dry matter accumulation which suggested that SPS activity is related to plant growth rate. The results suggested that photosynthate partitioning is an important but not an exclusive factor which determines whole plant dry matter distribution. PMID- 16663649 TI - Stilbene Synthase and Chalcone Synthase : Two Different Constitutive Enzymes in Cultured Cells of Picea excelsa. AB - Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3',4',5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4'-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4',5 trihydroxystilbene (resveratrol).Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID- 16663650 TI - Rhythmicity in ethylene production in cotton seedlings. AB - Cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under a photoperiod of 12 hour darkness and 12 hour light showed daily oscillations in ethylene evolution. The rate of ethylene evolution began to increase toward the end of the dark period and reached a maximum rate during the first third of the light period, then it declined and remained low until shortly before the end of the dark period. The oscillations in ethylene evolution occurred in young, mature, and old cotyledons (7 to 21 day old). These oscillations in ethylene evolution seemed to be endogenously controlled since they continued even when the photoperiod was inverted. Moreover, in continuous light the oscillations in ethylene evolution persisted, but with shorter intervals between the maximal points of ethylene evolution. In continuous darkness the oscillations in ethylene evolution disappeared. The conversion of [3,4-(14)C]methionine into [(14)C] ethylene followed the oscillations in ethylene evolution in the regular as well as the inverted photoperiod. On the other hand, the conversion of applied 1 aminocyclopropane-1-carboxylic acid into ethylene did not follow the oscillations in ethylene evolution, but was affected directly by the light conditions. Always, light decreased and darkness increased the conversion of applied 1 aminocyclopropane-1-carboxylic acid into ethylene. It is concluded that in the biosynthetic pathway of ethylene the conversion of 1-aminocyclopropane-1 carboxylic acid into ethylene is directly affected by light while an earlier step is controlled by an endogenous rhythm. PMID- 16663651 TI - Pollination-Induced Corolla Wilting in Petunia hybrida Rapid Transfer through the Style of a Wilting-Inducing Substance. AB - Pollination or wounding of the stigma of Petunia hybrida flowers led to the generation of a wilting factor and its transfer to the corolla within 4 hours. This was concluded from the effects of time course removal of whole styles. In this 4-hour period, pollen tubes traversed only a fraction of the total distance to the ovaries. Both pollination and wounding of the stigma immediately resulted in an increase of ethylene evolution. Accelerated wilting, however, occured only when treated styles remained connected with the ovaries, and not when they were detached and left in the flower. A wilting factor was found in eluates collected from the ovarian end of the styles, only in the case of previous pollination or wounding. In such eluates, the level of the ethylene precursor 1-amino cyclopropane-1-carboxylic acid was below detection.These observations suggest a material nature of the wilting factor in Petunia flowers, which rapidly passes through the style to the corolla, but which is different from 1-aminocyclopropane 1-carboxylic acid. PMID- 16663652 TI - Divergent transport mechanisms for pyrimidine nucleosides in petunia pollen. AB - Petunia hybrida pollen exhibits divergent transport mechanisms for pyrimidine nucleosides. Uridine and cytidine show all the properties of being actively transported, a nucleoside transport mechanism not hitherto reported in plant cells. Contrasting with this, thymidine transport has the properties of a nonactive, carrier-mediated system. Reasons for these different mechanisms are considered to lie in the high demand for uridine and cytidine, obtained perhaps from stylar tissue, for the biosynthetic reactions of the pollen tube, while thymidine demand is lower due to the absence of DNA replication in germinating Petunia pollen. PMID- 16663653 TI - A rapid reverse phase evaporation method for the reconstitution of uncharged thylakoid membrane lipids that resist hydration. AB - Comparison of several lipid reconstitution methods showed that they were not equally efficient at transferring the predominant thylakoid lipid, monogalactosyldiglyceride (MG), to the aqueous phase. We report a reverse phase evaporation method that employs Freon 11 as a lipid solvent and is capable of successfully hydrating MG in spinach (Spinacia oleracea L.) at room temperature within minutes. Using this method it is possible to force an equal weight mixture of MG and digalactosyldiglyceride into small bilayer vesicles without the formation of inverted micellar ;lipidic particles' in the membranes. PMID- 16663654 TI - Oxalate metabolism by tobacco leaf discs. AB - The turnover rate of oxalate in leaf discs of Nicotiana tabacum, var Havana Seed, during photosynthesis was estimated to be 1 to 2 micromoles per gram fresh weight per hour. Radioactivity from the enzymic oxidation of [(14)C]oxalate rapidly appeared in neutral sugars (mainly sucrose), organic acids (mainly malate), and amino acids. Only 5% of the radioactivity was released to the atmosphere as (14)CO(2), and no formate or formaldehyde could be detected. The metabolism of oxalate was not increased by raising the O(2) concentration from 1% to 21% to 60%, nor was the formation of [(14)C]oxalate from [2-(14)C]glyoxylate changed under the same conditions as was previously observed in vitro (Havir 1983 Plant Physiol 71: 874-878). While oxalate is not an inert end product of the glycolate pathway, it contributes little to the formation of photorespiratory CO(2). PMID- 16663655 TI - A rapid, sensitive method for quantitating subunits in purified ribulose bisphosphate carboxylase preparations. AB - Studies of the interactions of the large and small subunits of ribulose bisphosphate carboxylase require a knowledge of the concentrations of the subunits present in various preparations and their ratio. Since existing sodium dodecyl sulfate-gel electrophoresis procedures proved quantitatively unreliable, a technique based on high performance-gel filtration was developed. The latter is most reliable, takes only about 30 minutes to perform, and detects a minimum of 0.25 micrograms of each subunit. PMID- 16663656 TI - Aspartate Carbamyltransferase : Site of End-Product Inhibition of the Orotate Pathway in Intact Cells of Cucurbita pepo. AB - Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH(14)CO(3), but not [(14)C]carbamylaspartate or [(14)C]orotic acid, into uridine nucleotides (SigmaUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH(14)CO(3) into SigmaUMP by 80% but did not inhibit the incorporation of NaH(14)CO(3) into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH(14)CO(3) into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH(14)CO(3) into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides. PMID- 16663657 TI - Glycolate metabolism is under nitrogen control in chlorella. AB - The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate. PMID- 16663658 TI - Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties. AB - Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP(+) to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP(+). The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K(m) of NADP(+) being 2.5-fold higher for pI 4.3 form. Free ATP (K(m) = 0.64 millimolar) and ADP (K(m) = 1.13 millimolar) act as competitive inhibitors with respect to NADP(+) in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition. PMID- 16663659 TI - Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves. AB - The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [(15)N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no (15)N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [(15)N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH(3) accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of (15)N into glutamine-amide, NH(3), and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system. PMID- 16663660 TI - Enzymes of beta-Oxidation in Different Types of Algal Microbodies. AB - The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the beta oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H(2)O(2)-forming oxidase rather than a dehydrogenase.The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae. PMID- 16663661 TI - Tunicamycin Prevents Cellulose Microfibril Formation in Oocystis solitaria. AB - The effect of tunicamycin (TM) on the development of the cell wall in Oocystis solitaria has been investigated. It was found that 10 micromolar TM completely stops the assembly of new microfibrils as observed at the ultrastructural level. During cell wall formation, freeze fracture replicas of the E-face of the plasma membrane reveal two major substructures: the terminal complexes (TC), paired and unpaired, and the microfibril imprints extending from unpaired TCs. In cells treated for 3 hours or longer with TM, the TCs are no longer visible, whereas microfibril imprints are still present. Because of the reported highly selective mode of action of TM, our results implicate a role for lipid-intermediates in cellulose synthesis in O. solitaria. It is assumed that TM prevents the formation of a glycoprotein which probably is a fundamental part of the TCs and may act as a primer for the assembly of the microfibrils. PMID- 16663662 TI - Light-Induced Nuclear Synthesis of Spinach Chloroplast Fructose-1,6 bisphosphatase. AB - Etiolated spinach (Spinacia oleracea L. var Winter Giant) seedlings show a residual photosynthetic fructose-1,6-bisphosphatase activity, which sharply rises under illumination. This increase in activity is due to a light-induced de novo synthesis, as it has been demonstrated by enzyme labeling experiments with (2)H(2)O and [(35)S]methionine. The rise of bisphosphatase activity under illumination is strongly inhibited by cycloheximide, but not by the 70S ribosome inhibitor lincocin, which shows the nuclear origin of this chloroplastic enzyme. PMID- 16663663 TI - Measurement of subcellular metabolite levels in leaves by fractionation of freeze stopped material in nonaqueous media. AB - This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the vacuole of spinach (Spinacia oleracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane, and fractionated by density gradient centrifugation. Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated.As a first application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About three times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole falls from a maximum of 45 millimolars at the end of the day to an almost undetectable value of approximately 1 millimolar at the end of the night. PMID- 16663664 TI - Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis. AB - A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO(2) concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6 bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate. PMID- 16663665 TI - Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : II. Partitioning between Sucrose and Starch. AB - The role of fructose 2,6 bisphosphate in partitioning of photosynthate between sucrose and starch has been studied in spinach (Spinacia oleracea U.S. hybrid 424). Spinach leaf material was pretreated to alter the sucrose content, so that the rate of starch synthesis could be varied. The level of fructose 2,6 bisphosphate and other metabolites was then related to the accumulation of sucrose and the rate of starch synthesis. The results show that fructose 2,6 bisphosphate is involved in a sequence of events which provide a fine control of sucrose synthesis so that more photosynthate is diverted into starch in conditions when sucrose has accumulated to high levels in the leaf tissue. (a) As sucrose levels in the leaf rise, there is an accumulation of triose phosphates and hexose phosphates, implying an inhibition of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase. (b) In these conditions, fructose 2,6 bisphosphate increases. (c) The increased fructose 2,6-bisphosphate can be accounted for by the increased fructose 6-phosphate in the leaf. (d) Fructose 2,6 bisphosphate inhibits the cytosolic fructose 1,6-bisphosphatase so more photosynthate is retained in the chloroplast, and converted to starch. PMID- 16663666 TI - Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : III. Properties of the Cytosolic Fructose 1,6-Bisphosphatase. AB - The cytosolic fructose 1,6-bisphosphatase from spinach (Spinacia oleracea U.S. hybrid 424) leaves has been partially purified and its response to fructose 2,6 bisphosphate, AMP, and fructose 1,6-bisphosphate studied, using concentrations present in the cytosol during photosynthesis. In the presence of fructose 2,6 bisphosphate, the substrate saturation kinetics for fructose 1,6-bisphosphate are sigmoidal, with half-maximal activity being attained in 0.1 to 1 millimolar concentration range. The inhibition is enhanced by AMP. Using these results, and information published elsewhere on metabolite concentrations, it is discussed how fructose 1,6-bisphosphatase activity will vary in vivo in response to alterations in the availability of triose phosphate and AMP, and the accumulation of the product, fructose 6-phosphate. PMID- 16663667 TI - Control of lactate dehydrogenase, lactate glycolysis, and alpha-amylase by o(2) deficit in barley aleurone layers. AB - After 4 days in an atmosphere of N(2), aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of alpha-amylases when incubated with gibberellic acid (GA(3)) in air. However, layers did not produce alpha-amylase when GA(3) was supplied under N(2), apparently because alpha-amylase mRNA failed to accumulate.When an 8-hour pulse of [U-(14)C]glucose was supplied under N(2) to freshly prepared aleurone layers, both [(14)C]lactate and [(14)C]ethanol accumulated; the [(14)C]lactate/[(14)C]ethanol ratio was about 0.3. Prior incubation of layers for 1 day under N(2) changed this ratio to about 0.8, indicating an increase in the relative importance of the lactate branch of glycolysis.l(+)Lactate dehydrogenase (LDH) activity was low in freshly prepared aleurone layers and increased 10-fold during 2 days under N(2), whereas alcohol dehydrogenase activity (ADH) was high initially and rose by 60%. The responses of LDH and ADH activities to O(2) tension were dissimilar; when layers were incubated in various O(2)/N(2) mixtures, LDH activity peaked at 2 to 5% O(2) whereas ADH activity was highest at 0% O(2). The LDH activity was resolved into several enzymically active bands by native polyacrylamide gel electrophoresis.We conclude that barley aleurone layers are highly adapted to O(2) deficiency, that they possess an inducible LDH system as well as an ADH system, and we infer that the LDH and ADH systems are independently regulated. PMID- 16663668 TI - Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers. AB - Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O(2) deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O(2) levels were reduced below 5%, and were most intense at 0% O(2).In vivo(35)S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O(2), synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O(2), the synthesis of all three ADH peptides increased markedly.Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O(2) deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O(2), and about 100-fold after 1 day at 0% O(2).We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O(2), that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features. PMID- 16663669 TI - A highly sensitive, flow through h(2) gas analyzer for use in nitrogen fixation studies. AB - Studies of H(2) evolution by N(2) fixing systems are frequently limited by an inability to accurately measure H(2) gas concentrations of less than about 10 microliters per liter. In this study, a H(2) gas analyzer is described which is able to accurately and reproducibly detect up to 100 times lower H(2) concentrations than most thermal conductivity gas chromatographs or other conventional instruments used for the measurement of H(2) gas. This high level of sensitivity (maximum of about 0.02 microliter per liter H(2) per millivolt output) and the ability to continuously monitor H(2) concentration directly in a flowing gas stream, makes this instrument well suited for use in an open gas exchange system.Since the sensor used in the instrument was also sensitive to other combustible gases, it was necessary to demonstrate that H(2) was the only combustible gas produced by the N(2) fixing system being studied. When an air stream was passed through a pot containing nodulated soybean (Glycine max L.) roots, gas chromatographic analysis of the effluent gas stream revealed that H(2) was the only combustible gas present. These results were supported by other studies in which no combustible gases were detected in the effluent gas stream from soybean roots nodulated with USDA 110, a Rhizobium strain which displays active uptake hydrogenase activity. PMID- 16663670 TI - Effects of vanadate on the plasma membrane ATPase of red beet and corn. AB - The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K(i) for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K(+), and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K(m) and V(max) of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats. PMID- 16663671 TI - Isolation and Identification of the Phenols of Paul's Scarlet Rose Stems and Stem Derived Suspension Cultures. AB - The phenols of Paul's Scarlet rose stems and stem-derived cell cultures have been analyzed using C(18)-reversed-phase high performance liquid chromatography.Rose stems were found to contain gallic acid, (+)catechin, (-)epicatechin, the dimers (-)epicatechin-(+)catechin and (+)catechin-(+)catechin, a polymeric procyanidin, ferulic acid, and several gallotannins. In contrast, a cell suspension of Paul's Scarlet rose which has been maintained in culture for over 25 years contained only low levels of gallic acid and (-)epicatechin-(+)catechin. The phenol content of a second rose cell line which was started from the same initial isolate in 1957, but which was maintained in a laboratory other than our own was quantitatively and qualitatively similar to the cell line kept in our laboratory for the last 20 years. A third cell line which we started 6 months ago contained a wide variety of phenols, most of which were in common with those of rose stems.Selective subculturing of smaller cell clumps of our oldest cell line failed to enhance either the quantities or the diversity of phenols which accumulated in these cultured cells. Possible reasons for the failure of selective subculturing to enhance phenol levels in this long-established cell line are discussed. PMID- 16663672 TI - Pea xyloglucan and cellulose : I. Macromolecular organization. AB - A macromolecular complex composed of xyloglucan and cellulose was obtained from elongating regions of etiolated pea (Pisum sativum L. var. Alaska) stems. Xyloglucan could be solubilized by extraction of this complex with 24% KOH-0.1% NaBH(4) or by extended treatment with endo-1,4-beta-glucanase. The polysaccharide was homogeneous by ultracentrifugal analysis and gel filtration on Sepharose CL 6B, molecular weight 330,000. The structure of pea xyloglucan was examined by fragmentation analysis of enzymic hydrolysates, methylation analysis, and precipitation tests with fucose- or galactose-binding lectins. The polysaccharide was composed of equal amounts of two subunits, a nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and a heptasaccharide (glucose/xylose, 4:3), which appeared to be distributed at random, but primarily in alternating sequence. The xyloglucan:cellulose complex was examined by light microscopy using iodine staining, by radioautography after labeling with [(3)H]fucose, by fluorescence microscopy using a fluorescein-lectin (fucose-binding) as probe, and by electron microscopy after shadowing. The techniques all demonstrated that the macromolecule was present in files of cell shapes, referred to here as cell-wall ;ghosts,' in which xyloglucan was localized both on and between the cellulose microfibrils. Since the average chain length of pea xyloglucan was many times the diameter of cellulose microfibrils, it could introduce cross-links by binding to adjacent fibrils and thereby contribute rigidity to the wall. PMID- 16663673 TI - Pea Xyloglucan and Cellulose : II. Hydrolysis by Pea Endo-1,4-beta-Glucanases. AB - Two auxin-induced endo-1,4-beta-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K(m) values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V(max) values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-beta-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-beta-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-beta-glucanase in growing tissue. PMID- 16663674 TI - Solubilization and Reconstitution of Pisatin Demethylase, a Cytochrome P-450 from the Pathogenic Fungus Nectria haematococca. AB - Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tested was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2',5'-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase. PMID- 16663675 TI - Reserve carbohydrate in maize stem : [C]glucose and [C]sucrose uptake characteristics. AB - Maize (Zea mays L.) stem is thought to function alternately as a net importing and net exporting organ during ontogeny, depending on whole plant photosynthetic source and sink status. The [(14)C]sucrose and [(14)C]glucose uptake capacity of stem tissues was investigated to increase our understanding of the transport factors which may influence sink status.Uptake from solutions containing up to 200 millimolar radiolabeled sugar showed that d-glucose uptake consisted of saturable and nonsaturable components, while sucrose uptake was primarily nonsaturable during the kernel-fill stages. l-Glucose uptake lacked the saturable component but both d and l isomers apparently had similar slopes for the nonsaturable component. Uptake was sensitive to inhibitors and temperature, and was increased slightly by lowered pH.The seasonal chronology for saturable uptake by isolated vascular bundles and associated pith revealed highest rates between anthesis and early kernel growth, corresponding with the stage when net sugar accumulation rates were highest. For isolated pith, the rates increased at the final stages of plant development.The rate of labeled l-glucose movement from vascular bundles into pith in isolated stem segments was greater at the silking stage than at later developmental stages, suggesting a lower resistance to diffusive transport from vascular bundles into pith at silking. Studies with stem plus ear explants showed that the capability for sugar transport from pith to vascular bundles and for phloem loading and export from the stem region was present throughout the developmental period from early kernel fill (milk) to late kernel fill (dent). PMID- 16663676 TI - Dependency of Nitrate Reduction on Soluble Carbohydrates in Primary Leaves of Barley under Aerobic Conditions. AB - Nitrate reduction was studied as a function of carbohydrate concentration in detached primary leaves of barley (Hordeum vulgare L. cv Numar) seedlings under aerobic conditions in light and darkness. Seedlings were grown either in continuous light for 8 days or under a regimen of 16-hour light and 8-hour dark for 8 to 15 days. Leaves of 8-day-old seedlings grown in continuous light accumulated 4 times more carbohydrates than leaves of plants grown under a light and dark regimen. When detached leaves from these seedlings were supplied with NO(3) (-) in darkness, those with the higher levels of carbohydrates reduced a greater proportion of the NO(3) (-) that was taken up. In darkness, added glucose increased the percentage of NO(3) (-) reduced up to 2.6-fold depending on the endogenous carbohydrate status of the leaves. Both NO(3) (-) reduction and carbohydrate content of the leaves increased with age. Fructose and sucrose also increased NO(3) (-) reduction in darkness to the same extent as glucose. Krebs cycle intermediates, citrate and succinate, did not increase NO(3) (-) reduction, whereas malate slightly stimulated it in darkness.In light, 73 to 90% of the NO(3) (-) taken up was reduced by the detached leaves; therefore, an exogenous supply of glucose had little additional effect on NO(3) (-) reduction. The results indicate that in darkness the rate of NO(3) (-) reduction in primary leaves of barley depends upon the availability of carbohydrates. PMID- 16663677 TI - A transmissible plant shoot factor promotes uptake hydrogenase activity in Rhizobium symbionts. AB - Shoot/root grafting studies showed organ and host cultivar effects on net H(2) evolution from Pisum sativum L. root nodules. Net H(2) evolution from those nodules represents the sum of H(2) formed by Rhizobium nitrogenase and H(2) oxidized by any uptake hydrogenase present in the bacteria. Grafts between pea cultivars ;JI1205' or ;Alaska' and ;Feltham First' in symbioses with R. leguminosarum 128C53 showed that shoots of both JI1205 and Alaska increased H(2) uptake significantly (P 50 node) vegetative phase in short days. A representative of this class, response type G, was used as a receptor in short days for donors of other flowering response types. The qualitative and quantitative flowering response of G receptors depended on the genotype of the donor. Donors containing sn hr induced the earliest development, followed by sn Hr and Sn hr donors. The Lf and E loci in foliar donors apparently did not affect flowering of G. Five-leaved > single-leaved > cotyledonary donors in effecting a flowering response in G, in part due to the longer life of the foliar donors. The responses of G to the various donors were generally consistent with the proposed roles of Lf, E, and Sn, but the role of Hr in these grafts was unclear. PMID- 16663679 TI - Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle. AB - Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26 degrees C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42 degrees C. At 34 and 38 degrees C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42 degrees C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38 degrees C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38 degrees C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau. PMID- 16663680 TI - Kinetic Variance of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Isolated from Diverse Taxonomic Sources : II. Analysis by Two Dual Label Methods. AB - Two dual label methods were used to investigate kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39). In addition to using [1-(14)C,5-(3)H]RuBP (method 1), we describe here the detailed assay with (14)CO(2) and [5-(3)H]RuBP (method 2), which generates [(3)H,(14)C]3 phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (v(c)/v(o)) is calculated from (3)H/(14)C ratios of substrates and products. v(c)/v(o) was found to be a linear function of [CO(2)]/[O(2)], constant over a 4-minute assay interval, and invariant of the degree of enzyme activity. Accurately measurable v(c)/v(o) ratios range from approximately 0.3 to 6. The K(m) and V(max) of both enzymes may be determined as a composite constant, V(c)K(o)/V(o)K(c). By method 2, the directly compared, relative values at 40 micromolar CO(2) and 1240 micromolar O(2) were: Spinacia oleracea (74), Chlorella pyrenoidosa (31), Plectonema boryanum (32), and Rhodospirillum rubrum (8). With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. oleracea was 69 +/- 3. PMID- 16663681 TI - Influence of Sulfur Nutrition on Developmental Patterns of Some Major Pea Seed Proteins and Their mRNAs. AB - In addition to the marked reduction in legumin synthesis and legumin mRNA levels reported earlier (Chandler, Higgins, Randall, Spencer 1983 Plant Physiol 71: 47 54), pulse labeling of S-deficient Pisum sativum L. seeds showed that a high relative level of total vicilin (vicilin plus convicilin) synthesis was maintained throughout the entire phase of protein accumulation, whereas in nondeficient seeds vicilin synthesis is largely confined to the first half of this phase. Fractionation of pulse-labeled proteins on Na-dodecylsulfate polyacrylamide gels showed that the synthesis of the M(r) 50,000 family of vicilin polypeptides was increased and greatly extended in S-deficient seeds whereas that of convicilin was slightly reduced. Other changes apparent from pulse-labeling experiments include a depression, to different degrees, in the synthesis of three major albumin polypeptides.The level of the mRNAs for seven major seed proteins was followed throughout development of control and sulfur deficient seeds. In all cases, the changes in each mRNA closely reflected the pattern of synthesis of its corresponding polypeptide seen by pulse labeling. S deficient seeds showed an elevated level of M(r) 50,000 vicilin mRNA which remained high throughout seed formation, whereas legumin mRNA levels were greatly reduced at all stages of development.When S-deficient plants were given an adequate supply of sulfate midway through seed development, there was a shift toward the protein synthesis profile characteristic of healthy plants. The synthesis of legumin and two albumins rapidly increased and the synthesis of M(r) 50,000 vicilin declined more slowly. Similar responses were seen in detached, S deficient seeds supplied directly with adequate sulfate. PMID- 16663682 TI - Resolution and Reconstitution of Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase, the Enzyme System Responsible for the Formation of the Chlorophyll Isocyclic Ring. AB - Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, the system responsible for the formation of the chlorophyll isocyclic ring in developing cucumber (Cucumis sativus L. cv Beit Alpha) chloroplasts, was resolved into two enzymic components: a high-speed supernatant and a membrane pellet. This reconstituted enzyme system required reduced pyridine nucleotide for activity. PMID- 16663683 TI - Localization of Mg-Chelatase and Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Activities within Isolated, Developing Cucumber Chloroplasts. AB - Magnesium chelatase and magnesium protoporphyrin IX monomethyl ester (oxidative) cyclase activities were both sensitive to inhibition by p-chloromercuribenzoate in intact, developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts. Magnesium chelatase was also sensitive to the membrane-impermeable mercurial p chloromercuribenzene sulfonate (PCMBS), while cyclase activity was only slightly sensitive. When the plastids were pretreated with PCMBS, triosephosphate dehydrogenase activity was inhibited very slightly, indicating that PCMBS does not readily penetrate through the chloroplast envelope. These results suggest that magnesium chelatase is located in the chloroplast envelope, while the cyclase is located deeper within the chloroplast. PMID- 16663684 TI - Partitioning of Nitrogen among Ribulose-1,5-bisphosphate Carboxylase/Oxygenase, Phosphoenolpyruvate Carboxylase, and Pyruvate Orthophosphate Dikinase as Related to Biomass Productivity in Maize Seedlings. AB - Maize (Zea mays L. cv Golden Cross Bantam T51) seedlings were grown under full sunlight or 50% sunlight in a temperature-controlled glasshouse at the temperatures of near optimum (30/25 degrees C) and suboptimum (17/13 degrees C) with seven levels of nitrate-N (0.4 to 12 millimolars). The contents of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPD), and ribulose-1,5-P(2) carboxylase/oxygenase (RuBisCO) were immunochemically determined for each treatment with rabbit antibodies raised against the respective maize leaf proteins (anti-PEPC and anti-PPD) or spinach leaf protein (anti-RuBisCO). The content of each enzymic protein increased with increasing N and raised under reduced temperature. The positive effect of light intensity on their contents was evident only at near optimal temperature. The relative increase in PEPC and PPD content with increasing N was significantly greater than that of RuBisCO irrespective of growth conditions. These enzymic proteins comprised about 8, 6, and 35% of total soluble protein, respectively, at near optimal growth condition. In contrast to significant increase in the proportion of soluble protein allocated to PEPC and PPD seen under certain conditions, the proportion allocated to RuBisCO decreased reciprocally with an increased biomass yield by N supply.These results indicated that the levels of PEPC and PPD parallel to maize biomass more tightly than that of RuBisCO at least under near optimal growth condition. PMID- 16663685 TI - Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation. AB - A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers. PMID- 16663686 TI - Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. AB - Several proteases, i.e., pronase, a mixture of trypsin and chymotrypsin, and thermolysin were screened as potential surface probes of isolated intact pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Of these, only thermolysin met the criteria of a suitable probe. Thermolysin destroyed outer envelope polypeptides, but did not affect inner envelope polypeptides, envelope permeability properties or such chloroplast activities as metabolite transport and O(2) evolution. PMID- 16663687 TI - Physiological Studies on Pea Tendrils : XIV. Effects of Mechanical Perturbation, Light, and 2-Deoxy-d-Glucose on Callose Deposition and Tendril Coiling. AB - When excised tendrils of pea (Pisum sativum L. cv Alaska) are mechanically perturbed there is an immediate and transient increase in callose deposition in the sieve cells. Mechanical perturbation (MP) results in a coiling response in light-grown tendrils and in dark-adapted tendrils, provided, in the latter case, that they receive adequate illumination within a limited period of time after MP. In nonperturbed tendrils the number of callose deposits decreases to some minimum with increasing time in the dark, and their ability to coil in the dark in response to MP diminishes with time in the dark. The transient increase of callose deposition due to MP, however, occurs whether or not tendrils are dark adapted, and whether they receive light or are retained in the dark after MP. This indicates that if callose is directly involved in tendril coiling, then it exerts its effect on the sensory perception of the mechanical stimulus. In the present investigation, there is never tendril coiling without the transient increase in callose, and the time after MP at which the peak of callose deposition occurs precedes the time of the peak amount of coiling.An inhibitor of callose formation, 2-deoxy-d-glucose (DDG), is equally effective at inhibiting tendril coiling and MP-induced callose deposition, indicating, within the limitations of the specificity of DDG, that callose deposition may be required in order for the coiling response to occur. Alternatively, DDG may prevent the availability of some other factor necessary for tendril coiling. PMID- 16663688 TI - Photosynthesis in Encelia farinosa Gray in Response to Decreasing Leaf Water Potential. AB - Photosynthetic responses of intact leaves of the desert shrub Encelia farinosa were measured during a long term drought cycle in order to understand the responses of stomatal and nonstomatal components to water stress. Photosynthetic rate at high irradiance and leaf conductance to water vapor both decreased linearly with declining leaf water potential. The intercellular CO(2) concentration (c(i)) remained fairly constant as a function of leaf water potential in plants subjected to a slow drought cycle of 25 days, but decreased in plants exposed to a 12-day drought cycle. With increasing water stress, the slope of the dependence of photosynthesis on c(i) (carboxylation efficiency) decreased, the maximum photosynthetic rates at high c(i) became saturated at lower values, and water use efficiency increased. Both the carboxylation efficiency and photosynthetic rates were positively correlated with leaf nitrogen content. Associated with lower leaf conductances, the calculated stomatal limitation to photosynthesis increased with water stress. However, because of simultaneous changes in the dependence of photosynthesis on c(i) with water stress, increased leaf conductance alone in water-stressed leaves would not result in an increase in photosynthetic rates to prestressed levels. Both active osmotic adjustment and changes in specific leaf mass occurred during the drought cycle. In response to increased water stress, leaf specific mass increased. However, the increases in specific leaf mass were associated with the production of a reflective pubescence and there were no changes in specific mass of the photosynthetic tissues. The significance of these responses for carbon gain and water loss under arid conditions are discussed. PMID- 16663689 TI - Relationships between Respiration Rate and Adenylate and Carbohydrate Pools of the Soybean Fruit. AB - Relationships between respiration rate and adenylate and carbohydrate pools of the soybean (Glycine max L. Merrill) fruit during rapid seed growth were evaluated. Plants at mid pod-fill were subjected to different concentrations of CO(2) to alter the amount of photosynthate produced and, thus, available to the fruit. Respiration rate of the intact fruits was measured, along with glucose, sucrose, and starch concentrations, adenylate energy charge (AEC), and total adenylate pool (SigmaAdN) in the pod wall, seed coat, and cotyledons. The concentration of sucrose remained relatively constant in the pod wall (1.0 milligram per 100 milligrams dry weight), seed coat (6.5 milligrams per 100 milligrams dry weight), and cotyledons (4.5 milligrams per 100 milligrams dry weight) at moderate and high respiration rates. Furthermore, AEC remained relatively constant in the pod wall (0.55), seed coat (0.24), and cotyledons (0.44) during changes in respiration rate. This suggests that the amount of assimilate transported to the fruit, and its flux through the sucrose pools of the fruit parts, were important in the regulation of the respiration rate of the fruit. The average SigmaAdN in the seed coat (1300 picomoles per milligram dry weight) was significantly greater than in the cotyledons (750 picomoles per milligram dry weight) and pod wall (300 picomoles per milligram dry weight). In addition, the SigmaAdN in the seed coat and cotyledons increased with increasing respiration rate of the fruit. The high SigmaAdN in the seed coat and its increase with increases in respiration rate of the fruit suggest that an energy requiring process is involved in the movement of sucrose through the seed coat. PMID- 16663690 TI - In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium, Anabaena Variabilis: II. Acyl Transfer and Complex Lipid Formation. AB - In vitro fatty acid transfer to form complex lipids was observed in crude cell extracts of Anabaena variabilis using [1-(14)C]palmitoyl-acyl carrier protein, [1 (14)C]stearoyl-acyl carrier protein, and [1-(14)C]oleoyl-acyl carrier protein substrates. The data indicated that there was a rapid transfer of the fatty acids into the complex lipids. The greatest amount of radioactivity was observed in the monogalactosyl diacylglycerol fractions and there appeared to be a preference for the transfer of stearate over palmitate. The exogenously added lysophospholipids, (lysophosphatidylglycerol, lysophosphatidylcholine) and 2-monopalmitin acted as acceptors in acyl transfer. Addition of the hypolipidemic drug, WY14643, inhibited the fast acyl transfer reaction and showed that the first product of acyl transfer was diglyceride followed by monogalactosyl diacylglycerol. Thioesters of Coenzyme A do not seem to be involved in these reactions. PMID- 16663691 TI - Purification of Hydrogenase from Chlamydomonas reinhardtii. AB - A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H(2) produced per milligram protein per minute. The molecular weight was determined to be 4.5 x 10(4) by gel filtration and 4.75 x 10(4) by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H(2) production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55 degrees C. The K(m) of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag. PMID- 16663692 TI - Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain. AB - alpha-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20 degrees C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl(2), the alpha-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of alpha-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of alpha-amylase activity in the starchy endosperm in a whole grain.Scutella separated after 1 day of germination released small amounts of alpha-amylase activity into 10 millimolar CaCl(2). This release was linear for at least 1 hour and did not occur at 0 degrees C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of alpha-amylase activity in the starchy endosperm in a whole grain.Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total alpha-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity. PMID- 16663693 TI - Suberin production by isolated tomato fruit protoplasts. AB - The multilamellar wall secreted by protoplasts isolated from locule tissue of tomato (Lycopersicon esculentum L.) fruit was purified, and an extract was obtained after depolymerization with BF(3)-methanol. Analysis of this extract using thin layer chromatography demonstrated the presence of fatty acid methyl esters, fatty alcohols, dicarboxylic acid dimethyl esters, and omega-hydroxy acid methyl esters. These components were quantified using an Iatroscan thin layer chromatography-flame ionization detection system. The different chain lengths in each group were identified and quantified using gas chromatography. The results clearly indicated the presence of suberin. PMID- 16663694 TI - Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation. AB - The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below -30 degrees C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below -25 degrees C. There was a close association between the per cent unfrozen water at -40 degrees C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at -40 degrees C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone. PMID- 16663695 TI - Cryopreservation of Alkaloid-Producing Cell Cultures of Periwinkle (Catharanthus roseus). AB - A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5 degrees C per minute to -40 degrees C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40 degrees C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation. PMID- 16663696 TI - Novel Phycoerythrins in Marine Synechococcus spp. : Characterization and Evolutionary and Ecological Implications. AB - Four clones of the marine, unicellular, cyanobacteria Synechococcus spp., were examined for the spectral and biochemical features of their phycoerythrins (PE) and their photosynthetic characteristics. Two spectral types of PE which are distinct from known PEs were found. One PE type possessed absorption maxima at 500 and 545 nm and a fluorescence emission at 560 nm. Upon denaturation in acid urea, two chromophore absorption maxima were obtained, one corresponding to phycourobilin (A(max) 500 nm) and one at 558 nm, ascribed to a phycoerythrobilin like chromophore. The ratio of phycoerythrobilin-like to phycourobilin chromophores was 4.9:1.3. This PE possessed two subunits of M(r)s of 17.0 and 19.5 kD for the alpha and beta subunits, respectively. The other PE possessed a single symmetrical absorption at 551 nm and a fluorescence emission at 570 nm. This phycobiliprotein showed a single chromophore absorption band (A(max) 558 nm) and yielded two polypeptides, an alpha of 17.5 kD and a beta subunit of 20.8 kD. Both PEs showed a (alpha, beta)(n) structure. The presence of phycoerythrobilin like chromophores (A(max) 558 nm) appears to be diagnostic of this marine cyanobacterial group. The features of these PEs combined with additional biochemical data, suggest a possible evolutionary link between the PE-containing marine Synechococcus group and the red algal chloroplast. When the Synechococcus clones were grown under low light intensity the PE-containing clones showed higher photosynthetic performance, larger photosynthetic units sizes, reaction center I to II ratios near unity, and steeper initial slopes of photosynthesis versus irradiance curves than a non-PE-containing clone. These findings demonstrate the high photosynthetic efficiency of PE-containing clones in low light environments common to middepth neritic and oceanic habitats. PMID- 16663697 TI - Enzymic dissociation of zea shoot cell wall polysaccharides : I. Preliminary characterization of the water-insoluble fraction of zea shoot cell walls. AB - The water-insoluble fraction, obtained after successive treatment of an insoluble fraction of a buffer-homogenate of Zea mays L. hybrid B73 x Mo 17 shoots with 3 molar LiCl and hot water, was treated with alpha-amylase to remove starch. This fraction has been subjected to sugar composition analysis and the major glycosidic linkages were determined. The results suggest that the water-insoluble fraction is mainly composed of arabinoxylan, (1 --> 3), (1 --> 4-)-beta-d-glucan, xyloglucan, (1 --> 4)-galactan, polygalacturonic acid, and cellulose. The insoluble fraction also contained ferulic acid (about 440 micrograms per 50 milligrams dry weight), which was released from the fraction by treatment with aqueous NaOH.Novo Ban 120 from Bacillus subtilis was found to be an appropriate enzyme source for selective dissociation of Zea shoot wall polysaccharides. PMID- 16663698 TI - Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides : II. Dissociation of (1 --> 3),(1 --> 4)-beta-d-Glucan by Purified (1 --> 3),(1 --> 4)-beta-d-Glucan 4 Glucanohydrolase from Bacillus subtilis. AB - The water-insoluble cell wall fraction obtained after successive treatments of an insoluble fraction of a buffer-homogenate of Zea shoots with 3 molar LiCl, hot water and alpha-amylase, was subjected to dissociation by enzymes. Treatment of the water-insoluble fraction with the purified Bacillus subtilis (1 --> 3),(1 --> 4)-beta-d-glucan 4-glucanohydrolase dissociated cell wall fragments (5.1% of the water-insoluble fraction) which were characterized in detail. Structural analyses of the fragment oligosaccharides revealed blocks of more than three contiguous (1 --> 4)-linkages and regions consisting of two or more contiguous (1 --> 3) linkages. On the basis of these results, we propose that the Zea shoot beta-d glucan may be represented as a folded structure. Furthermore, the presence of contiguous (1 --> 3)-linkages in the beta-d-glucan offers potential sites for enzyme attack and may account for the generation of specific products released by the endogenous endo-beta-d-glucanase in autolysis and/or polymer modification process in Zea shoot cell walls. PMID- 16663699 TI - Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides : III. Purification and Partial Characterization of an Endo-(1 --> 4)-beta-d-Xylanase from a Bacillus subtilis Enzyme Preparation. AB - An endo-(1 --> 4)-beta-xylanase [(1 --> 4)-beta-d-xylan 4-xylanohydrolase, EC 3.2.1.8)] has been isolated from a commercial preparation of Bacillus subtilis alpha-amylase (Novo Ban 120). The purified xylanase exhibited an optimum activity at pH 5.5, and maximum activity at a temperature of 50 degrees C. The enzyme digests a (1 --> 4)-beta-d-xylan from larch yielding 4-linked xylobiose and xylotriose as predominant products but does not hydrolyze 4-linked xylotriose and xylobiose. The enzyme also digests a (1 --> 3), (1 --> 4)-beta-d-mixed linkage sequence xylan from Rhodymenia into 4-linked xylobiose and xlyotriose, and 3,4 mixed linked oligosaccharides having a degree of polymerization of four or more. It is concluded that this enzyme is capable of hydrolyzing sequences of four or more (1 --> 4)-beta-d-linked xylose residues. PMID- 16663700 TI - Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides : IV. Dissociation of Glucuronoarabinoxylan by Purified Endo-(1 --> 4)-beta-Xylanase from Bacillus subtilis. AB - The structure of a glucuronoarabinoxylan in Zea mays L. (hybrid B73 x Mo17) shoot cell walls has been studied. The water-insoluble fraction of Zea shoot cell walls, pretreated with purified Bacillus subtilis (1 --> 3), (1 --> 4)-beta-d glucan 4-glucanohydrolase, was treated with purified B. subtilis endo-(1 --> 4) beta-xylanase. Carbohydrates (2.6% of the waterinsoluble fraction of Zea shoot cells walls) derived from the enzyme treatment consisted of glucuronoarabinoxylan fragments with molecular weights which varied from a few hundred to over 2.0 x 10(5) daltons. Structural analyses of the fragments suggested that the glucuronoarabinoxylan had a xylan backbone which contained (1 --> 4)-beta-d xylopyranosyl residues, with about 60 to 70% substitution at the C-2 or C-3 position with arabinose, glucuronic acid, and other substituents. Furthermore, the glucuronoarabinoxylan contained a phenolic component which appeared to be primarily ferulic acid bonded to carbohydrate, probably by an ester linkage. The amount of ferulic acid was approximately 3 micrograms per 100 micrograms of carbohydrate. PMID- 16663701 TI - Theoretical and experimental errors for in situ measurements of plant water potential. AB - Errors in psychrometrically determined values of leaf water potential caused by tissue resistance to water vapor exchange and by lack of thermal equilibrium were evaluated using commercial in situ psychrometers (Wescor Inc., Logan, UT) on leaves of Tradescantia virginiana (L.). Theoretical errors in the dewpoint method of operation for these sensors were demonstrated. After correction for these errors, in situ measurements of leaf water potential indicated substantial errors caused by tissue resistance to water vapor exchange (4 to 6% reduction in apparent water potential per second of cooling time used) resulting from humidity depletions in the psychrometer chamber during the Peltier condensation process. These errors were avoided by use of a modified procedure for dewpoint measurement. Large changes in apparent water potential were caused by leaf and psychrometer exposure to moderate levels of irradiance. These changes were correlated with relatively small shifts in psychrometer zero offsets (-0.6 to 1.0 megapascals per microvolt), indicating substantial errors caused by nonisothermal conditions between the leaf and the psychrometer. Explicit correction for these errors is not possible with the current psychrometer design. PMID- 16663702 TI - Stimulation of Mitochondrial Calcium Uptake by Light during Growth of Corn Shoots. AB - Ca(2+) uptake in mitochondrial fractions, isolated on Percoll discontinuous density gradients, from light- and dark-grown corn (Zea mays L. var W64A x W182E) shoots was characterized by dual wavelength spectroscopy and the Ca(2+)-sensitive dye murexide. In light-grown seedlings, the rate of mitochondrial Ca(2+) uptake was about 40 nanomoles per minute per milligram of mitochondrial protein. A portion of the Ca(2+) uptake required an exogenous supply of ATP (65%) while the remaining 35% was the respiratory substrate-dependent reaction. Ruthenium red (2 micromolar) completely inhibited both ATP- and substrate-dependent reactions. There was no detectable Ca(2+) efflux from the mitochondria with the inhibitor. When the mitochondrial fraction was prepared from the dark-grown shoots, the rate of uptake, in particular the ATP-dependent reaction, was greatly reduced. The dark treatment caused a reduction in mitochondrial Ca content which is largely due to the reduction of Ca associated with the mitochondrial membrane rather than to a reduction of Ca in the soluble matrix. PMID- 16663703 TI - Heat Stress Responses in Cultured Plant Cells : Heat Tolerance Induced by Heat Shock versus Elevated Growing Temperature. AB - Using cultured pear (Pyrus communis cv Bartlett) cells, heat tolerance induced by heat shock was compared to that developed during growth at high temperature. After growth at 22 degrees C, cells exposed to 38 degrees C for 20 minutes (heat shock) showed maximum increased tolerance within 6 hours. Cells grown at 30 degrees C developed maximum heat tolerance after 5 to 6 days; this maximum was well below that induced by heat shock. Heat shock-induced tolerance was fully retained at 22 degrees C for 2 days and was only partly lost after 4 days. However, pear cells acclimated at 30 degrees C lost all acquired heat tolerance 1 to 2 days after transfer to 22 degrees C. In addition, cells which had been heat acclimated by growth at 30 degrees C showed an additional increase in heat tolerance in response to 39 degrees C heat shock. The most striking difference between heat shock and high growth temperature effects on heat tolerance was revealed when tolerance was determined using viability tests based on different cell functions. Growth at 30 degrees C produced a general hardening, i.e. increased heat tolerance was observed with all three viability tests. In contrast, significantly increased tolerance of heat-shocked cells was observed only with the culture regrowth test. The two types of treatment evoke different mechanisms of heat acclimation. PMID- 16663704 TI - Synthesis and Turnover of the Chloroplast Coupling Factor 1 in Chlamydomonas reinhardi. AB - Studies on the biosynthesis of the chloroplast coupling factor 1 (CF(1)) in Chlamydomonas reinhardi have been initiated. The ratio of CF(1) to chlorophyll in the cell was shown to be independent of the density of the culture. No turnover of assembled CF(1) could be detected, thus suggesting that CF(1) was synthesized at a rate equivalent to that of net chlorophyll synthesis. A lag of between 5 to 7 minutes in the incorporation of radioactive precursor sulfate into assembled CF(1) was measureable. This puts an upper limit on the pool size of any precursor to the assembled CF(1) complex. The pool size is estimated to be equivalent to 1% of the total CF(1) in the cell. PMID- 16663705 TI - Structural characterization of a higher plant calmodulin : spinacia oleracea. AB - Calmodulin is a eukaryotic calcium binding protein which has several calcium dependent in vitro activities. Presented in this report is a structural characterization of calmodulin from spinach leaves (Spinacia oleracea). Spinach calmodulin may be representative of higher plant calmodulins in general since calmodulin from the monocotyledon barley (Hordeum vulgare) is indistinguishable by a variety of physical, chemical, and functional criteria (Schleicher, Lukas, Watterson 1983 Plant Physiol 73: 666-670). Spinach calmodulin is homologous to bovine brain calmodulin with only 13 identified amino acid sequence differences, excluding a blocked NH(2)-terminal tripeptide whose sequence has not been elucidated. Two extended regions of sequence identity are in the NH(2)-terminal half of the molecule, while nine of the 13 identified differences are in the COOH terminal half of the molecule. Two of the changes, a cysteine at residue 26 and a glutamine at residue 96, require a minimum of two base changes in the nucleotide codons. Both of these changes occur in the proposed calcium binding loops of the molecule. Five additional amino acid differences found in spinach calmodulin had not been observed previously in a calmodulin. As described in an accompanying report (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798), these limited number of amino acid sequence variations appear to result in differential effects on the activation of calmodulin-dependent enzymes by plant and vertebrate calmodulins. PMID- 16663706 TI - Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins. AB - In the preceding paper (Lukas, Iverson, Schleicher, Watterson 1984 Plant Physiol 75: 788-795), we reported that the amino acid sequence of spinach calmodulin has at least 13 amino acid sequence differences from vertebrate calmodulin. In the present study, we investigated the effect of these amino acid sequence substitutions on the enzyme activator properties of vertebrate and plant calmodulins. Calmodulins from spinach and the green alga Chlamydomonas reinhardtii activate chicken gizzard myosin light chain kinase in a manner similar but not identical to chicken calmodulin. In contrast, these calmodulins have very different NAD kinase activator properties. The concentration required for half-maximal activation of pea seedling NAD kinase by spinach calmodulin (3-4 nanomolar) is lower than the corresponding concentrations of chicken (20 nanomolar) and Chlamydomonas (40 nanomolar) calmodulins. However, the maximum level of activation obtained with Chlamydomonas calmodulin is 4- to 6-fold higher than spinach or chicken calmodulin. These data indicate that the limited structural heterogeneity among calmodulins have differential effects on their biochemical activities. PMID- 16663707 TI - Influence of Applied NaCl on Crassulacean Acid Metabolism and Ionic Levels in a Cactus, Cereus validus. AB - To determine possible physiological responses to salinity, seedlings of Cereus validus Haworth, a cactus from Salinas Grandes, Argentina, were treated with up to 600 millimolar NaCl for up to 16 days when they were about 9 months old and 100 millimeters tall. Salt stress decreased stem biomass, e.g. it was 19.7 grams for controls and 11.4 grams for plants treated with 400 millimolar NaCl for 14 days. Nocturnal CO(2) uptake in these obligate Crassulacean acid metabolism (CAM) plants was inhibited 67% upon treatment with 400 millimolar NaCl for 14 days (controls, 181 millimoles CO(2) per square meter), while nocturnal accumulation of malate was inhibited 49% (controls, 230 millimoles malate per square meter). The larger accumulation of malate as compared to uptake of atmospheric CO(2) suggests that internal CO(2) recycling occurred during the dark period. Such recycling was lower in the controls ( approximately 20%) than in the NaCl-treated plants ( approximately 50%). The nocturnal increase in malate and titratable acidity depended on the total daily photosynthetically active radiation available; measurements suggest a quantum requirment of 26 photons per malate. As NaCl in the medium was increased to 600 millimolar in daily increments of 50 millimolar, Na and Cl concentrations in the roots increased from about 7 to 100 millimolar, but K concentration in the cell sap remained near 26 millimolar. Concomitantly, concentrations of Na and Cl in the shoots increased from 8 to 17 millimolar and from 1 to 7 millimolar, respectively, while the K concentration increased about 16 to 60 millimolar. In plants maintained for 14 days at 500 millimolar NaCl, the root levels of Na and Cl increased to 260 millimolar, the shoot levels were about 60 millimolar, and the stem bases began to become necrotic. Such Na retention in the roots together with the special possibilities of carbon reutilization given by CAM are apparently survival mechanisms for the temporarily saline conditions experienced in its natural habitat. PMID- 16663708 TI - Day-Night Variations in Malate Concentration, Osmotic Pressure, and Hydrostatic Pressure in Cereus validus. AB - Malate concentration and stem osmotic pressure concomitantly increase during nighttime CO(2) fixation and then decrease during the daytime in the obligate Crassulacean acid metabolism (CAM) plant, Cereus validus (Cactaceae). Changes in malate osmotic pressure calculated using the Van't Hoff relation match the changes in stem osmotic pressure, indicating that changes in malate level affected the water relations of the succulent stems. In contrast to stem osmotic pressure, stem water potential showed little day-night changes, suggesting that changes in cellular hydrostatic pressure occurred. This was corroborated by direct measurements of hydrostatic pressure using the Julich pressure probe where a small oil-filled micropipette is inserted directly into chlorenchyma cells, which indicated a 4-fold increase in hydrostatic pressure from dusk to dawn. A transient increase of hydrostatic pressure at the beginning of the dark period was correlated with a short period of stomatal closing between afternoon and nighttime CO(2) fixation, suggesting that the rather complex hydrostatic pressure patterns could be explained by an interplay between the effects of transpiration and malate levels. A second CAM plant, Agave deserti, showed similar day-night changes in hydrostatic pressure in its succulent leaves. It is concluded that, in addition to the inverted stomatal rhythm, the oscillations of malate markedly affect osmotic pressures and hence water relations of CAM plants. PMID- 16663709 TI - Elongation of the First Internode of the Pea Seedling during Etiolation : Interaction of Environment and Genotype. AB - In the present study, 40 lines of peas, Pisum sativum L., from the Weibullsholm collection were examined for elongation response of the first internode to growth in soil, sand, or in darkness in air. The results obtained establish that appearance of the first (lowest) node of the epicotyl at the surface of soil was usual in only a few of the lines tested (L25, L1088, L1570). By contrast, appearance of the first node at the surface of sand as a result of enhancement of first internode extension occurred in 80% of the lines tested. In many lines, extension in sand or soil was more than in darkness in air, a surprising observation given that the known response of the pea epicotyl to pressure is to produce the extension inhibitor, ethylene. Results of two crosses, one of tallxdwarf and one of tallxtall, point to the conclusion that the Le/le locus exercises only partial control over first internode extension and that other, as yet unknown, loci are implicated. PMID- 16663710 TI - Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity. AB - The contribution of the alternative pathway in root respiration of Pisum sativum L. cv Rondo, Plantago lanceolata L., and Plantago major L. ssp major was determined by titration with salicylhydroxamate (SHAM) in the absence and presence of cyanide. SHAM completely inhibited the cyanide-resistant component of root respiration at 5 to 10 millimolar with an apparent K(i) of 600 micromolar. In contrast, SHAM enhanced pea root respiration by 30% at most, at concentrations below 15 millimolar. An unknown oxidase appeared to be responsible for this stimulation. Its maximum activity in the presence of low SHAM concentrations (1-5 millimolar) was 40% of control respiration rate in pea roots, since 25 millimolar SHAM resulted in 10% inhibition. In plantain roots, the maximum activity was found to be 15%. This hydroxamate-activated oxidase was distinct from the cytochrome path by its resistance to antimycin. The results of titrations with cyanide and antimycin indicated that high SHAM concentrations (up to 25 millimolar) block the hydroxamate-activated oxidase, but do not affect the cytochrome path and, therefore, are a reliable tool for estimating the activity of the alternative path in vivo. A considerable fraction of root respiration was mediated by the alternative path in plantain (45%) and pea (15%), in the latter because of the saturation of the cytochrome path. PMID- 16663711 TI - Involvement of Plasma Membrane Alterations in Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma). AB - Using plasma membranes with high purity isolated from winter rye seedlings (Secale cereale L. cv Puma) by an aqueous two-polymer phase partition technique, lipid and protein changes were determined during the development of cold hardiness.The degree of fatty acid unsaturation and proportions of phospholipid classes changed only slightly during cold acclimation. A small change, however, was observed in sterol composition; beta-sitosterol increased gradually with a corresponding decrease in campesterol plus stigmasterol during cold acclimation. The phospholipid to protein ratio increased proportionally to the increase in cold hardiness. During the period of acclimation, the sterol to phospholipid ratio declined and was inversely related to the development of cold hardiness.A distinct difference between polypeptide components of nonacclimated and acclimated plasma membrane was observed on two-dimensional sodium dodecyl sulfate slab polyacrylamide gels. A change was also detected in glycopeptides. These results suggest that the plasma membrane is in a dynamic state during cold acclimation, changing in response to the development of cold hardiness. PMID- 16663712 TI - Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants. AB - The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I(50) values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron. PMID- 16663713 TI - Optimal conditions for translation by thylakoid-bound polysomes from pea chloroplasts. AB - Polysomes bound to washed thylakoids from pea Pisum sativum cv Progress No. 9 chloroplasts are capable of protein synthesis when supplemented with amino acids, ATP and a regenerating system, GTP, and soluble factors required for translation. The extent of protein synthesis in previous reports, however, was quite low when compared to in organello translation. By systematic testing of parameters in the isolation of thylakoids and reaction mixture components we have been able to establish more optimal conditions. Incorporation of 2 to 10 nanomoles of leucine per milligram chlorophyll in a 20-minute reaction period is now possible, representing a 10- to 60-fold increase over amounts previously reported. Autoradiographs of solubilized, electrophoresed membranes show about 30 discrete labeled polypeptides which remain associated with the thylakoid membranes. PMID- 16663714 TI - Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris. AB - Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK(a) values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO(-)CH(2)NH(2) (+)CH(2)PO(3) (2-), and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K(i) = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K(i)' = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate]. PMID- 16663715 TI - Is modulation of the rate of proton pumping a key event in osmoregulation? AB - The net uptake of 3-O-methylglucose into leaf segments obtained from Senecio mikanioides Otto, and net proton efflux from the segments, were both promoted when the osmotic potential of the medium was decreased by addition of mannitol, sorbitol, or polyethylene glycol (optimal osmolarity, 0.3 Osmolar for mannitol and sorbitol). The effect was not due to promotion of ;aging', since the antibiotic cerulenin suppressed aging without reducing the size of the mannitol stimulation; further, mannitol did not accelerate aging. Neither was the effect ascribable to diminished efflux (i.e. reduced ;leak' because: first, visualization of the unidirectional sugar fluxes by double labeling indicated that the effect of added osmoticum was to promote influx rather than to reduce efflux; second, compartment analysis did not suggest any effect of mannitol on the rate constants for efflux from either the slowly equilibrating or more rapidly equilibrating compartment. The effect was not specific to poly-ols since it was also obtained with betaine and choline chloride. Since methyl glucose is not taken up into the phloem it could not be ascribed to a turgor effect on phloem loading. We conclude that the effect may reflect osmoregulation. As the sugar flux is probably driven by protonmotive force, it is likely that the effects on proton flux and on sugar flux are related. We suggest that the plasmalemma-sited proton pump is sensitive to the hydrostatic pressure gradient across the plasmalemma-cell wall complex, and functions both as detector and as effector in osmoregulation. PMID- 16663716 TI - Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Photoregulation. AB - The preparation of a rabbit antibody to ribulose-1,5-bisphosphate carboxylase (RuBPCase) from Euglena gracilis and its use to quantitate RuBPCase in dark- and light-grown cells and during light-induced chloroplast development (greening) are described. Light-grown Euglena have at least 36 times more RuBPCase than dark grown Euglena. Light is required for both the initiation and continued increase in net synthesis of RuBPCase over the dark level: brief illumination 12 hours before exposure to continuous light eliminates the lags in the accumulation and increase in activity of RuBPCase (as well as in chlorophyll accumulation); net synthesis is blocked in greening cells returned to the dark or exposed to 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Streptomycin or cycloheximide prevents RuBPCase accumulation when added at the beginning of greening but only partially blocks accumulation when added after 25 hours of greening. After 24 hours of greening, the activity of RuBPCase per milligram chlorophyll continues to increase slowly while concentration of the enzyme per milligram chlorophyll remains constant. This increased activity may be due to activation of the enzyme as well as to net synthesis. PMID- 16663717 TI - Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Nutritional Regulation. AB - The accumulation of ribulose-1,5-bisphosphate carboxylase (RuBPCase) in resting Euglena gracilis strain Z during greening is photo-regulated (Freyssinet, Eichholz, Buetow 1984 Plant Physiol 75: 850-857. Greening resting cells are not photosynthetically competent for about the first 24 hours in the light. Therefore, substrates for a net synthesis of the enzyme must come from endogenous constituents. During this time, degradation of endogenous paramylum (carbohydrate) reserves provides the main source of substrates. By about 24 hours of greening, resting cells are photosynthetically competent and RuBPCase accumulation becomes highly sensitive to 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Therefore, from about 24 hours of greening onward, substrates (and/or energy) for RuBPCase synthesis are provided by photosynthesis. Ethanol, a nutritional substrate ordinarily used constitutively by Euglena for growth, inhibits RuBPCase accumulation when added to the resting medium in the light. The alcohol exerts this negative regulatory effect by limiting the availability of substrates needed for a net synthesis of the enzyme. PMID- 16663718 TI - Measurement of the pyrophosphate content of plant tissues. AB - Pyrophosphate (PPi) was measured in pea (Pisum sativum L.) and corn (Zea mays L.) tissues by using an enzymic method based on PPi-dependent phosphofructokinase (PPi-PFK). Different organs of pea and corn seedlings were extracted to determine if PPi is present in sufficient amounts to serve as a substrate for the PPi-PFK activity in these tissues. The amount of PPi is at least 14% to 70% that of the ATP content in shoots and roots of peas and corn; and, for various plant tissues, ranges from 5 to 39 nanomoles of PPi per gram fresh tissue weight. We conclude that PPi is available as a substrate for the glycolytic function of PPi-PFK in plants. Furthermore, the presence of substrate amounts of PPi in plant tissues implies that plant energetics also must be evaluated in terms of PPi as an energy source and phosphate donor. PMID- 16663719 TI - Analysis of the Complexity and Diversity of mRNAs from Pollen and Shoots of Tradescantia. AB - The mRNAs of the mature pollen grain of Tradescantia paludosa at anesthesia and of vegetative shoots have been compared by analyzing the kinetics of hybridization between homologous and heterologous reactions of cDNA to poly(A)RNA in excess. The mRNAs in pollen can be divided into three abundance classes with complexities of 5.2 x 10(4), 1.6 x 10(6), and 2.1 x 10(7) nucleotides. The three classes are made up of sequences that constitute 15, 60, and 24% of the mRNAs and each sequence is present on an average at 26,000, 3,400, and 100 copies, respectively, per pollen grain. About 20,000 different genes are expressed in pollen as compared to about 30,000 in vegetative shoots. Estimates have been made of pollen mRNA sequences shared with those of shoot tissue and of shoot sequences common to those in pollen. PMID- 16663720 TI - Leakage of Dhurrin and p-Hydroxybenzaldehyde from Young Sorghum Shoots Immersed in Various Solvents. AB - Spectral scanning was used to provide estimates of the leakage of the cyanogenic glucoside, dhurrin (p-hydroxy-[S]-mandelonitrile-beta-d-glucoside), and its metabolite, p-hydroxybenzaldehyde (p-HB), from young light-grown shoots of Atlas sorghum (Sorghum bicolor [L.] Moench) when these shoots were immersed in water, toluene, chloroform or mixtures of water and toluene or water and chloroform. Minimal leakage of dhurrin and virtually no leakage of p-HB occurred with water as the solvent. The 0.5% concentration (v/v) of both toluene and chloroform was more effective than either the 1.0 or 2.0% concentrations in effecting leakage of the two solutes. With either 0.5% toluene or 0.5% chloroform as the solvent, 80 to 90% of the total dhurrin was extracted from shoots in a 3-hour period. Breakdown of dhurrin during extraction was much more extensive with 0.5% chloroform than with 0.5% toluene. Some loss of p-HB occurred during 3- or 6-hour extractions in the water-organic solvent mixtures; spectral and chromatographic evidence suggested partial conversion of p-HB to p-hydroxybenzoic acid. With undiluted toluene or chloroform as solvents, extracts contained appreciable amounts of free p-HB but essentially no dhurrin. These solvents were less effective than the water-organic solvent mixtures in extracting the solutes from the shoot issue. PMID- 16663721 TI - Sulfate Assimilation in C(4) Plants: Intercellular and Intracellular Location of ATP Sulfurylase, Cysteine Synthase, and Cystathionine beta-Lyase in Maize Leaves. AB - The activity of ATP sulfurylase, cysteine synthase, and cystathionine beta-lyase was measured in crude leaf extracts, bundle sheath strands, and mesophyll and bundle sheath chloroplasts to determine the location of sulfate assimilation of C(4) plant leaves. Almost all the ATP sulfurylase activity was located in the bundle sheath chloroplasts while cysteine synthase and cystathionine beta-lyase activity was located, in different proportions, in both chloroplast types.A new spectrophotometric assay for measuring ATP sulfurylase activity is also described. PMID- 16663722 TI - A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus. AB - The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M 1 callus cultures. The relative O(2) uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures. PMID- 16663723 TI - Factors permitting prolonged translation by isolated pea chloroplasts. AB - The following parameters were found to prolong the time-course of translation by isolated pea (Pisum sativum, cv Progress No. 9) chloroplasts: addition of other amino acids (an effect synergistic with sufficient free Mg(2+)), use of lower light intensities, and additions of inorganic phosphate and ATP. In a chloroplast system which includes these parameters, active translation usually extends to almost an hour. The total amount of leucine incorporated is routinely 60 to 100 nanomoles/milligram chlorophyll and often 200 nanomoles/milligram chlorophyll. Accurate estimation of the amount of amino acid incorporated depends on supplying the labeled amino acid at a concentration sufficient to overcome isotope dilution effects from endogenous pools. Approximately 39 thylakoid and 60 stroma polypeptides were visible on autoradiographs after labeling with [(35)S]methionine. Label in a few of the polypeptide bands was increased or decreased by specific changes in the reaction conditions. Due to the long period of activity and the large number of labeled products, this chloroplast system should be useful for future studies of chloroplast translation. PMID- 16663724 TI - Factors Affecting the Emission of Monoterpenes from Red Pine (Pinus densiflora). AB - The mechanism of monoterpene emission from Pinus densiflora was studied using an environmentally controlled gas cabinet. It was found that monoterpene emission rate increases exponentially with temperature and is also influenced by light. These observations were explained reasonably by a mechanism whereby monoterpene emission rate depends on the monoterpene amount in the leaf oil and its saturated vapor pressure. PMID- 16663725 TI - A high proportion of hybridomas raised to a plant extract secrete antibody to arabinose or galactose. AB - A high proportion of hybridomas, obtained from mice immunized with style extracts prepared from mature flowers of an ornamental tobacco, Nicotiana alata, secrete antibody to arabinogalactan protein (AGP). The specificity of the antibodies secreted by three cloned cell lines is primarily directed to beta-d galactopyranose and alpha-l-arabinofuranose; antibodies from two cell lines preferentially bind beta-d-galactopyranose residues and antibodies from the other cell line preferentially bind alpha-l-arabinofuranose. As AGPs are components of most plant tissues and exudates, it is likely that attempts to raise monoclonal antibodies to other plant extracts will result in hybridomas producing antibodies to AGPs. PMID- 16663726 TI - Determination of compartmented metabolite pools by a combination of rapid fractionation of oat mesophyll protoplasts and enzymic cycling. AB - In vivo pool sizes of a range of metabolites have been determined in subcellular fractions of darkened and illuminated mesophyll protoplasts of Avena sativa L. These estimations were made by combining a method of rapid protoplast fractionation with enzymic cycling techniques. Results are given for reduced and oxidized pyridine nucleotides, triose phosphates, 3-phosphoglycerate, inorganic phosphate, aspartate, malate, oxaloacetate, glutamate, 2-oxoglutarate, and citrate, from chloroplasts, mitochondria, and a fraction representing the remainder of the protoplast. The results indicate distinct differences of compartmented levels of certain metabolites between darkened and illuminated protoplasts. PMID- 16663727 TI - Short-Term Effects of CO(2) on Gas Exchange of Leaves of Bigtooth Aspen (Populus grandidentata) in the Field. AB - The short term effects of increased levels of CO(2) on gas exchange of leaves of bigtooth aspen (Populus grandidentata Michx.) were studied at the University of Michigan Biological Station, Pellston, MI. Leaf gas exchange was measured in situ in the upper half of the canopy, 12 to 14 meters above ground. In 1900 microliters per liter CO(2), maximum CO(2) exchange rate (CER) in saturating light was increased by 151% relative to CER in 320 microliters per liter CO(2). The temperature optimum for CER shifted from 25 degrees C in 320 microliters per liter CO(2) to 37 degrees C in 1900 microliters per liter CO(2). In saturating light, increasing CO(2) level over the range 60 to 1900 microliters per liter increased CER, decreased stomatal conductance, and increased leaf water use efficiency. The initial slope of the CO(2) response curve of CER was not significantly different at 20 and 30 degrees C leaf temperatures, although the slope did decline significantly during leaf senescence. In 1900 microliters per liter CO(2), CER increased with increasing light. The light saturation point and maximum CER were higher in 30 degrees C than in 20 degrees C, although there was little effect of temperature in low light. The experimental results are consistent with patterns seen in laboratory studies of other C(3) species and define the parameters required by some models of aspen CER in the field. PMID- 16663728 TI - Auxin-induced regulation of protein synthesis in tobacco mesophyll protoplasts cultivated in vitro: I. Characteristics of auxin-sensitive proteins. AB - The presence of auxin (2,4-D), in the culture medium of tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G(0) to phase G(1) of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4 dichlorophenoxyacetic acid affects the synthesis of 11.Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium.Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis.These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle. PMID- 16663729 TI - Auxin-Induced Regulation of Protein Synthesis in Tobacco Mesophyll Protoplasts Cultivated In Vitro: II. Time Course and Level of Auxin Control. AB - When protoplasts, previously cultivated in a medium lacking auxin, are transferred to complete medium, proteins whose synthesis is stimulated by the hormone become detectable after about 30 minutes and reach a constant level 2 to 4 hours after the beginning of hormonal treatment. In contrast, proteins whose level of synthesis is reduced by auxin, are only affected after 6 hours of treatment.Short radioactive labelings in deficient medium followed by chases in complete medium show that auxin does not interfere with posttranslational processes.Analysis of in vitro translation products of protoplast RNA shows that the time courses of auxin effects on protein synthesis and mRNA accumulation are perfectly superimposable. This allows us to exclude the possibility that auxin affects the translation process, but indicates that this hormone acts by regulating the concentration of the auxin-sensitive protein mRNAs. PMID- 16663730 TI - C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development. AB - Partitioning and translocation of (14)C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of (14)CO(2) for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total (14)C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered (14)C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the (14)C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The (14)C-organic acids comprised from 3% to 14% of the soluble (14)C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. (14)C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while (14)C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of (14)C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that (14)C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling. PMID- 16663731 TI - Salinity Effects on Photosynthesis and Growth in Alternanthera philoxeroides (Mart.) Griseb. AB - Alternanthera philoxeroides, alligator weed, was grown at five different NaCl concentrations to determine the effect of salinity on factors related to the net rate of CO(2) uptake (P(n)). Over the range of 0 to 400 millimolar NaCl, P(n) declined 51%. Stomatal conductance declined in parallel with P(n) and as a result there was no reduction in intercellular CO(2) concentration and therefore no reduction in the amount of CO(2) available for photosynthesis. The CO(2) compensation point did not change with salt stress. Increases in leaf thickness tended to compensate slightly for the negative effects of salinity on leaf cell metabolism, at least in relation to P(n). On a mesophyll cell area basis, soluble protein was relatively constant in leaves developed at 100 to 400 millimolar NaCl while total chlorophyll decreased at all salinities. Dry weight production and P(n) were closely correlated in alligator weed grown at different salinities. Plants produced less leaf area per unit dry weight as salinity increased, which may aid in water conservation. PMID- 16663732 TI - Relationship between Auxin and Amino Acid Metabolism of Tobacco Protoplast Derived Cells. AB - Single amino acids were found to be highly toxic to protoplast-derived cells of tobacco (Nicotiana tabacum cv Xanthi) cultured at low density in a culture medium containing a low naphthaleneacetic acid concentration (0.05 micromolar). The cytotoxicities of alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, proline, and valine were reduced when the naphthaleneacetic acid concentration of the culture medium was increased to 1 micromolar. This selective modification of amino acid toxicity by naphthaleneacetic acid could not be correlated with modifications of uptake rates or incorporation of these amino acids into protein or amino acid-auxin conjugates. A mutant clone resistant to high naphthaleneacetic acid concentrations and affected in root morphogenesis did not display, at the cellular level, the naphthaleneacetic acidmediated modification of amino acid cytotoxicity. PMID- 16663733 TI - Effects of Temperature and CO(2) Enrichment on Carbon Translocation of Plants of the C(4) Grass Species Echinochloa crus-galli (L.) Beauv. from Cool and Warm Environments. AB - Plants of Echinochloa crus-galli from Quebec and Mississippi were grown under two thermoperiods (28 degrees C/22 degrees C, 21 degrees C/15 degrees C) and two atmospheric CO(2) concentrations (350 and 675 microliters per liter) to examine possible differential responses of northern and southern populations of this C(4) grass species. Translocation was monitored using radioactive tracing with short lived (11)C. CO(2) enrichment induced a decrease in the size of the export pool in plants of both populations. Other parameters did not strongly respond to elevated CO(2). Low temperature reduced translocation drastically for plants from Mississippi in normal CO(2) concentration, but this reduction was ameliorated at high CO(2). Overall, plants from Quebec had a higher (11)C activity in leaf phloem and a higher percentage of (11)C exported, whereas these northern plants had lower turnover time and smaller pool size than plants from the southern population. PMID- 16663734 TI - Studies on Genetic Male-Sterile Soybeans : III. The Initiation of Monocarpic Senescence. AB - Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male-sterility (ms(1)) and nodulation (rj(1)) was utilized to investigate the effects of reproductive tissue development and nitrogen source on the initiation of monocarpic senescence. The experimental genotypes (Ms(1)Rj(1), Ms(1)rj(1), ms(1)Rj(1), and ms(1)rj(1), were selected from a cross between N69-2774 and N59 5259, and were inbred to the F(5) generation. Green-house-grown plants were collected during the period of flowering (77 days after transplanting) until maturity (147 days after transplanting). Leaf tissues from the respective genotypes were analyzed at the various harvest dates for RNA, phenolic, and chlorophyll concentrations; acid protease activity; polypeptide banding patterns of chloroplast thylakoids; and chloroplastic ultrastructure.Regardless of nitrogen source, total chlorophyll concentrations declined between 77 and 119 days after transplanting, resulting in a 40% loss of chlorophyll per square centimeter in all genotypes. Leaf chlorophyll levels continued to decline at a constant rate in male-fertile genotypes, but remained at a constant level (26 micrograms chlorophyll per square centimeter) in male-sterile genotypes, for the remainder of the study. With increased leaf age, a gradual disruption of thylakoid structures was observed, particularly in chloroplasts from the male fertile genotypes. Chloroplasts from the male-sterile genotypes appeared to lose starch grains but increased their number of chloroplastic lipid bodies with leaf aging. These data suggest that monocarpic senescence in soybeans was initiated at or before flowering. Although reproductive tissue development probably augmented the process, the response attributed to seed formation was not apparent until the mid-pod fill stage (119 days after transplanting). All genotypes had similar changes in other cellular components that are recognized as indicators of plant senescence regardless of whether the plants produced seed. PMID- 16663735 TI - Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO(3)-Limited Chemostats. AB - Synechococcus leopoliensis was grown in HCO(3) (-)-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO(2)). The O(2) to CO(2) ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO(3) (-) rather than CO(2) for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO(3) (-)-limited cells and also had a reduced capacity for HCO(3) (-) transport. HCO(3) (-)-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO(2) grown cells. However, the low affinity cells of the present study were growing at CO(2) concentrations less than air saturation. This suggests that supranormal levels of CO(2) not required to induce the physiological changes usually ascribed to high CO(2) cells. PMID- 16663736 TI - Removal by a Trace of Sodium of the Period Lengthening of the Potassium Uptake Rhythm Due to Lithium in Lemna gibba G3. AB - The effect of Li(+) on the period of the K(+) uptake rhythm in the flow medium culture of the duckweed (Lemna gibba G3) was investigated under various ionic conditions. In the presence of Li(+) at 0.2 millimolar or higher concentrations, the period was longer than the normal level of 25.4 hours by 2 hours. Li(+) also lowered the amplitude of the rhythm. Although Na(+) itself did not change any parameter of the rhythm, simultaneous application of Na(+) at a very low concentration (20 micromolar) almost completely removed the effects of 0.5 millimolar Li(+) on both the period and the amplitude. However, divalent cations (Ca(2+) and Mg(2+)) or Rb(+) did not remove the Li(+) action on the period. The effect of Li(+) and its removal by Na(+) corresponded to intracellular Li(+) and Na(+) levels. The period was prolonged when the duckweed contained more Li(+) than 5 micromoles/gram fresh weight. But the Li(+) effect was cancelled when the in vivo Na(+) level was greater than one-fifth that of Li(+), even if the Li(+) level exceeded over 5 micromoles/gram fresh weight. PMID- 16663737 TI - Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States. AB - Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to -20 degrees C) by (31)P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (-400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration. PMID- 16663738 TI - Effect of Photoperiod on Photosynthate Partitioning and Diurnal Rhythms in Sucrose Phosphate Synthase Activity in Leaves of Soybean (Glycine max L. [Merr.]) and Tobacco (Nicotiana tabacum L.). AB - Studies were conducted to identify the existence of diurnal rhythms in sucrose phosphate synthase (SPS) activity in leaves of three soybean (Glycine max L. [Merr.]) and two tobacco (Nicotiana tabacum L.) cultivars and the effect of photoperiod (15 versus 7 hours) on carbohydrate partitioning and the rhythm in enzyme activity. Acclimation of all the genotypes tested to a short day (7 hours) photoperiod resulted in increased rates of starch accumulation, whereas rates of translocation, foliar sucrose concentrations, and activities of SPS were decreased relative to plants acclimated to long days (15 hours). Under the long day photoperiod, two of the three soybean cultivars (;Ransom' and ;Jupiter') and one of the two tobacco cultivars (;22NF') studied exhibited a significant diurnal rhythm in SPS activity. With the soybean cultivars, acclimation to short days reduced the activity of SPS (leaf fresh weight basis) and tended to dampen the amplitude of the rhythm. With the tobacco cultivars, photoperiod affected the shape of the SPS-activity rhythm. The mean values for SPS activity (calculated from observations made during the light period) were correlated positively with translocation rates and were correlated negatively with starch accumulation rates. Overall, the results support the postulate that SPS activity is closely associated with starch/sucrose levels in leaves, and that acclimation to changes in photoperiod may be associated with changes in the activity of SPS. PMID- 16663739 TI - Promotion of Flowering in Brassica campestris L. cv Ceres by Sucrose. AB - Flower initiation of the quantitative long-day plant Brassica campestris cv Ceres was earlier and at a lower final leaf number when sucrose was added to the medium in which plants were grown in sterile culture. The optimal concentration of sucrose was 40 to 80 millimolar. This flower-promoting effect of sucrose was not osmotic, as mannitol, sodium chloride, and polyethylene glycol were not effective at equal osmotic potentials.Seedlings grown heterotrophically after treatment with 4-chloro-5-(dimethylamino)-2-phenyl-3-(2H)-pyridazinone to prevent chlorophyll accumulation were also induced to form flower primordia earlier as the sucrose concentration in the medium was increased up to 80 millimolar. Inclusion of 4 millimolar sodium nitrate in the culture medium of green plants did not reduce the flower-promoting effects of sucrose but delayed initiation in plants grown without added sucrose.Removal of CO(2) during a single main or supplementary light period, or both, greatly reduced flower initiation. It is concluded that sucrose may be an important controlling factor determining floral initiation in Brassica. PMID- 16663740 TI - Does pollination induce corolla abscission of cyclamen flowers by promoting ethylene production? AB - Very low ethylene production rates were measured in nonpollinated Cyclamen persicum Mill flowers, and no change in production was observed during the whole life span of the flower until death. Normal senescence was accompanied by a gradual discoloration and loss of turgor followed by wilting. Pollination induced a dramatic increase in ethylene evolution, culminating in a peak 4 days after pollination, and abscission of the corolla on that day. Silver-thiosulfate, an inhibitor of ethylene action, had no effect on longevity of unpollinated flowers, but completely nullified the effect of pollination on corolla abscission. Exposing unpollinated flowers to very high ethylene concentrations (50 microliters per liter) for 48 hours did not promote corolla abscission or senescence. 1-Aminocyclopropane-1-carboxylic acid, the immediate precursor of ethylene, increased ethylene production by unpollinated flowers more than 100 fold, but did not promote corolla abscission. 1-Aminocyclopropane-1-carboxylic acid did enhance corolla abscission of pollinated flowers. It is concluded that the main effect of pollination in inducing corolla abscission of cyclamen is by rendering the tissue sensitive to ethylene, apart from the promotion of ethylene production. PMID- 16663741 TI - Effect of phosphorus and zinc nutrition on soybean seed phytic Acid and zinc. AB - The relationships between nutrient P and Zn levels and the phytic acid, P, and Zn concentrations in soybean (Glycine max L. Merr. cv ;Williams 79') seed were studied. Phytic acid increased linearly from 4.2 to 19.2 milligrams per gram as nutrient P treatment was varied from 2.0 to 50 milligrams per liter and Zn was held constant at 0.05 milligrams per liter. Leaf P concentration during seed development was found to be closely related to the concentrations of seed P and phytic acid. Leaf and seed Zn concentrations both responded positively to increasing nutrient Zn treatment. The effects of P treatment on plant and seed P and phytic acid were largely independent of the effects of Zn treatment on leaf and seed Zn. Phytic acid to Zn molar ratios ranging from 3.6 to 33.8 were observed.The effects of nutrient P treatments on the concentrations of phytic acid, seed P, and leaf P were also studied in the P-sensitive (gene np) cultivars ;Harosoy' and ;Clark' and their respective P-tolerant (gene Np) near-isogenic lines L66-704 and L63-1677. In general, the positive relationships observed among nutrient P, leaf P, seed P, and phytic acid concentrations were similar to those observed in the studies with Williams 79. When fertilized with low or moderate nutrient P (2.5 and 25.0 milligrams P per liter, respectively) no significant differences in any parameter were observed between Harosoy or Clark and their respective P-tolerant isolines. When fertilized with high nutrient P (100 milligrams P per liter), Harosoy seed had a significantly higher concentration of phytic acid (30 milligrams per gram) than did seed of its P-tolerant near isogenic line L66-704 (24.2 milligrams per gram phytic acid), whereas no significant difference was observed between Clark and its P-tolerant near isogenic line L63-1677 (22.8 and 21.6 milligrams per gram, respectively). Variation in the phytic acid concentrations in the mature seed of the cultivars and isolines more closely paralleled leaf P concentrations observed during seed development (49 days after flowering), than those observed at the onset of seed development (14 days after flowering). Electrophoresis and ion-exchange chromatography revealed that partially phosphorylated intermediates do not appear when phytic acid accumulation is greatly reduced by limiting the nutrient P or when accumulation is greatly accelerated by excess P. PMID- 16663742 TI - Effect of ammonium on nitrate utilization by roots of dwarf bean. AB - The effect of exogenous NH(4) (+) on NO(3) (-) uptake and in vivo NO(3) (-) reductase activity (NRA) in roots of Phaseolus vulgaris L. cv Witte Krombek was studied before, during, and after the apparent induction of root NRA and NO(3) ( ) uptake. Pretreatment with NH(4)Cl (0.15-50 millimolar) affected neither the time pattern nor the steady state rate of NO(3) (-) uptake.When NH(4) (+) was given at the start of NO(3) (-) nutrition, the time pattern of NO(3) (-) uptake was the same as in plants receiving no NH(4) (+). After 6 hours, however, the NO(3) (-) uptake rate (NUR) and root NRA were inhibited by NH(4) (+) to a maximum of 45% and 60%, respectively.The response of the NUR of NO(3) (-)-induced plants depended on the NH(4)Cl concentration. Below 1 millimolar NH(4) (+), the NUR declined immediately and some restoration occurred in the second hour. In the third hour, the NUR became constant. In contrast, NH(4) (+) at 2 millimolar and above caused a rapid and transient stimulation of NO(3) (-) uptake, followed again by a decrease in the first, a recovery in the second, and a steady state in the third hour. Maximal inhibition of steady state NUR was 50%. With NO(3) (-) induced plants, root NRA responded less and more slowly to NH(4) (+) than did NUR.Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, relieved the NH(4) (+) inhibition of the NUR of NO(3) (-)-induced plants. We conclude that repression of the NUR by NH(4) (+) depends on NH(4) (+) assimilation. The repression by NH(4) (+) was least at the lowest and highest NH(4) (+) levels tested (0.04 and 25 millimolar). PMID- 16663743 TI - Purine synthesis and catabolism in soybean seedlings : the biogenesis of ureides. AB - The ureides, allantoin and allantoic acid, are the major nitrogenous substances transported within the xylem of N(2)-fixing soybeans (Glycine max L. Merr. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobium or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [(14)C]serine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3- and 4-day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [(14)C]serine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings. PMID- 16663744 TI - Azido auxins : photoaffinity labeling of auxin-binding proteins in maize coleoptile with tritiated 5-azidoindole-3-acetic Acid. AB - Tritiated 5-azidoindole-3-acetic acid (5-N(3)-[7-(3)H]IAA), a photoaffinity labeling agent, was used to photolabel proteins of a crude microsomal preparation from maize (Zea mays L., Bear Hybrid, WF9 x BR38) coleoptile. Approximately 50% of the bound radioactivity was solubilized in 5 molar urea containing Triton X 100, and the extract was fractionated using a variety of techniques. High performance liquid chromatography demonstrated that, although many membrane proteins incorporated tritiated label, only a few showed reduced incorporation in the presence of excess indole-3-acetic acid. By contrast, no detectable reduction in incorporation was observed in the presence of excess naphthalene-1-acetic acid. Results from isoelectric focusing gel electrophoresis indicate that the proteins that showed reduced incorporation of photolyzed 5-N(3)-[7-(3)H]IAA in the presence of IAA fell into two main groups: one which focuses between pH 5.2 and 5.7 (pI 4.8-5.3) and another around pH 6.2 (pI 5.8). In sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins migrated as four bands with apparent molecular weights of 60, 49, 45, and 37 kilodaltons. The auxin-transport inhibitor, 2,3,5-triiodobenzoic acid, competes for the labeling by 5-N(3)-[7 (3)H]IAA, suggesting that some of these proteins may be involved in auxin transport. PMID- 16663745 TI - Quantitation of cytokinins in biological samples using antibodies against zeatin riboside. AB - The cross-reactivity of antibodies elicited in rabbits against zeatin riboside, to a wide range of naturally occurring cytokinins, was examined. As well as to zeatin riboside, the antisera cross-reacted to a considerable extent with zeatin, lupinic acid, zeatin-9-glucoside, zeatin riboside 5'-monophosphate and to a much lesser, but measurable extent, with dihydrozeatin riboside and dihydrozeatin. Chromatographic methods were devised which allowed separation of all these cross reactive compounds. Four biological samples, extracts of immature Zea mays kernels, immature seeds of Lupinus luteus, and Datura innoxia crown gall tumor tissue, and a sample of Agrobacterium tumefaciens culture supernatant, were purified by these chromatographic methods, using [(3)H]zeatin riboside as a recovery marker, and at each stage of the purification process, were subjected to radioimmunoassay over a range of dilutions. At each stage of sample purification, sample dilution curves were found to be parallel to the standard curve. Sample cytokinin levels estimated by radioimmunoassay were in close agreement to those available in the literature for similar samples assayed by alternative methods. However, in some samples, unknown cross-reacting compounds were detected. PMID- 16663746 TI - Mode of action of abscisic Acid in barley aleurone layers : abscisic Acid induces its own conversion to phaseic Acid. AB - As a part of our effort to study the mode of action of abscisic acid (ABA) and its metabolites during seed germination, we have investigated the regulation of ABA metabolism in barley (Hordeum vulgare) aleurone layers and a few other plant tissues. The rate of conversion of [(3)H]ABA to [(3)H]phaseic acid (PA), the first stable metabolite of ABA, is enhanced by 2- to 5-fold in barley aleurone layers when the tissue is pretreated with ABA. However, the conversion of [(3)H]PA to [(3)H] dihydrophaseic acid (DPA), the next metabolite after PA, is not enhanced by pretreatment with either ABA or PA. The ABA enhancement of its own metabolism in barley aleurone layers is detectable with a pretreatment of ABA ranging from 10(-3) to 10(-4) molar. This apparent self-induction of ABA conversion to PA can be observed after the barley aleurone layers have been treated with 10(-5) molar ABA for as short as 2 hours, and is inhibited by the transcription inhibitor, cordycepin (3'-deoxyadenosine), or the translation inhibitor, cycloheximide. The self-induction of ABA conversion to PA also occurs in wheat aleurone layers, but not in other plant tissues that have been investigated, including corn root tips, barley embryos, barley, and soybean leaf discs. It is probably a phenomenon unique to the aleurone layers of some cereal grains. In view of the recent observations that ABA is able to induce new proteins in barley aleurone layers, we suggest that some of these ABA-induced proteins are involved in the conversion from ABA to PA in this tissue. PMID- 16663747 TI - Cell Surfaces in Plant-Microorganism Interactions : IV. Fungal Glycopeptides Which Elicit the Synthesis of Ethylene in Plants. AB - The production of ethylene by melon (Cucumis melo cv Cantaloup charentais) tissues is stimulated during incubation in the presence of fungal glycopeptides extracted from Colletotrichum lagenarium, a pathogen of melon. These glycopeptides, called elicitors of ethylene, are found in the mycelium, the cell wall, and the culture filtrate. Elicitation of ethylene is a relatively early phenomenon and lasts for several hours. Upon purification of the crude elicitor extract by gel filtration and ion exchange chromatography, three elicitors were isolated. The three elicitors contained amino acid, sugar, and phosphate residues, and they have a decreased activity after partial chemical degradation of their sugar moiety.Elicitation of ethylene is not fungal species specific. Elicitors of phytoalexins, obtained from three Phytophtora species, enhanced ethylene biosynthesis in melon tissues. PMID- 16663748 TI - Behavior of the Plasma Membrane of Isolated Protoplasts during a Freeze-Thaw Cycle. AB - Cryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves (Secale cereale L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT(50)) (-5 degrees C) was expansion-induced lysis of the plasma membrane during warming and thawing of the suspending medium when the decreasing osmolality resulted in osmotic expansion of the protoplasts. When cooled to temperatures below the LT(50), the predominant form of injury was loss of osmotic responsiveness following cooling so that the protoplasts were osmotically inactive during warming. Only a low incidence (<10%) of expansion-induced lysis was observed in protoplasts isolated from acclimated (ACC) leaves, and the predominant form of injury following cooling to the LT(50) (-25 degrees C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI(50)) of NA protoplasts osmotically expanded from isotonic solutions was 1122 +/- 172 square micrometers. Similar values were obtained when the protoplasts were osmotically expanded from hypertonic solutions. The TSAI determined from cryomicroscopic measurements of individual NA protoplasts was similar to the TSAI(50) values obtained from osmotic manipulation. The TSAI(50) of ACC protoplasts expanded from isotonic solutions (2145 +/- 235 square micrometers) was approximately double that of NA protoplasts and increased following osmotic contraction. Osmotic contractions were readily reversible upon return to isotonic solutions. During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts whereas exocytotic extrusions formed on the surface of ACC protoplasts. During osmotic expansion following thawing of the suspending medium, the endocytotic vesicles remained in the cytoplasm of NA protoplasts and the protoplasts lysed before their original volume and surface area were regained. In contrast, the exocytotic extrusions were drawn back into the surface of ACC protoplasts as the protoplasts regained their original volume and surface area. PMID- 16663749 TI - Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons. AB - Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus vulgaris. The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H(2)O(2) only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (O(2) (-), H(2)O(2), OH) are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo. PMID- 16663750 TI - Enzymes of the Poly-beta-Hydroxybutyrate and Citric Acid Cycles of Rhizobium japonicum Bacteroids. AB - The activities of several enzymes of the citric acid and poly-beta hydroxybutyrate cycles were measured in Rhizobium japonicum 3I1B-143 bacteroids which had been isolated from soybean nodules by sucrose gradient centrifugation. During the period of developing nitrogenase activity, the specific activity of fumarase, hydroxybutyrate dehydrogenase, beta-ketothiolase, and pyruvate dehydrogenase complex increased whereas acetoacetate-succinyl-CoA transferase and isocitrate dehydrogenase decreased. Malate dehydrogenase activity remained constant. The amount of available acetyl-CoA, based on pyruvate dehydrogenase activity, should be sufficient to support both metabolic cycles concurrently. The temporal relationship between nitrogenase activity and poly-beta-hydroxybutyrate accumulation has been reexamined. PMID- 16663751 TI - Polyproline II Confirmation in the Protein Component of Arabinogalactan-Protein from Lolium multiflorum. AB - Circular dichroism spectra of the arabinogalactan-protein from suspension cultured Lolium multiflorum (ryegrass) endosperm cells demonstrate the presence of polyproline II conformation in the protein moiety of the proteoglycan. Subject to a number of theoretical and practical constraints of the method, it can be estimated that at least 30% of the protein component is in this conformation. PMID- 16663752 TI - Introduction of xylem differentiation in lactuca by ethylene. AB - Evidence was obtained to support the hypothesis that ethylene is involved in xylem differentiation in primary pith explants of Lactuca sativa L. cv Romaine cultured in vitro. Xylem elements differentiated when explants were supplied indole-3-acetic acid (IAA) in combination with either the ethylene biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene-releasing agent 2-chloroethylphosphonic acid (CEPA), or kinetin. In contrast, no xylem elements differentiated in the presence of IAA, kinetin, ACC, or CEPA alone, or when kinetin was supplied together with ACC or CEPA. These results show that ethylene will substitute qualitatively for cytokinin during auxin-induced xylogenesis, and suggest that both ethylene and auxin are required for xylem differentiation in Lactuca. PMID- 16663753 TI - Effect of shortened photosynthetic period on C-assimilate translocation and partitioning in reproductive soyeans. AB - Starch accumulation rate in leaves of vegetative soybeans is inversely related to the length of the daily photosynthetic period. However, it is not known whether a similar response would be observed during reproductive growth. Soybeans (Glycine max L. Merr. cv Amsoy 71) were grown to three stages of reproductive growth (beginning seed, mid seed-fill, and late seed-fill) under 12-hour daylengths, and then shifted to 6-hour photosynthetic periods (12-hour photoperiods) for 4 days. One and 4 days after treatment, a mid-canopy leaf was pulsed with (14)CO(2), and sampled for radiolabeled starch and water-soluble compounds at 0.5, 1, 3, 9, and 21 hours after labeling.Plants exposed to the 6-hour photosynthetic periods at the beginning seed stage retained and incorporated significantly more label as starch than did those given 12-hour photosynthetic periods. However, plants exposed to the shortened photosynthetic periods at the late seed-fill stage partitioned less label into starch. Plants exposed at mid seed-fill gave a variable response.Shortened photosynthetic periods resulted in preferential partitioning of recently fixed carbon to the seed at the expense of the pod wall. The results of these experiments suggest that the increased sink demand present during late reproductive growth may be of greater importance in control of leaf starch accumulation than is the length of the daily photosynthetic period. PMID- 16663754 TI - Alteration of C-assimilate partitioning in leaves of soybeans having increased reproductive loads at one node. AB - The objectives of this study were to determine if the partitioning of recently fixed carbon between starch and water-soluble compounds could be altered by increasing the pod load in the leaf axil, and if the presence of source leaves acropetal to such a node would influence the partitioning of carbon within the subtending leaf. Soybeans (Glycine max L. Merr. cv Hodgson 78) were grown to full bloom in a controlled environment chamber, and then deflowered at all nodes except the eighth. This treatment resulted in an 83% increase in the number of pods at the eighth node. At 24 days after flowering, one-half of the treated plants were girdled above the untreated node. Forty-two hours later, the eighth trifoliolate was pulsed with (14)CO(2) and sampled for radiolabeled starch and water-soluble compounds (WSC) at 0.5, 2, 4, 8, 12, and 24th after labeling.When no girdling was applied above the increased pod load at the eighth node more label was accumulated by the pod walls (+6.9%) and seeds (+6.3%) when compared to the controls. Starch accumulation was not altered in the labeled leaf of the nongirdled plants. When the stem was girdled above the eighth node, significantly less starch was retained in the labeled leaf. Girdling also resulted in an increase in label accumulation by the pod walls (+5.4%) and seeds (+6.6%). These data suggest that the plant will change the distribution patterns of assimilate to supply added sink demand before altering the partitioning of recently fixed carbon in the subtending leaf. PMID- 16663755 TI - In vitro characterization of tomato fruit softening : the use of enzymically active cell walls. AB - Cell wall isolated from pericarp of normal tomato (Lycopersicon esculentum Mill. cv ;Rutgers') fruit released pectic polymers in a reaction apparently mediated by wall-bound polygalacturonase that appears with the onset of ripening. Release was negligible in wall preparations from normal green and the ripening mutant rin fruit. Pectin solubilization was most extensive at pH 2.5 with a less significant peak at 5.5. Brief exposure to low (1.5) or high (7.5) pH resulted in reduction of autolytic activity, which was also inhibited by high temperature, Ca(2+), and treatments employed to dissociate protein from cell wall. Uronic acid solubilization was significantly enhanced by 150 millimolar NaCl and by increasing temperature within the physiological range. These data indicate that the release of polyuronide from isolated cell walls is enzymic and may provide a convenient and reliable system for the study of softening metabolism. PMID- 16663756 TI - Tissue distribution of acetyl-coenzyme a carboxylase in leaves. AB - Acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide ligase (ADP forming), EC 6.4.1.2] is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C(3)- and C(4)-plants. The mesophyll tissues of the C(3)-plants Pisum sativum and Allium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using (125)I-streptavidin as a probe, revealed biotinyl proteins of molecular weights 62,000, 51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.In the C(4)-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways. PMID- 16663757 TI - Effect of the defoliant thidiazuron on ethylene evolution from mung bean hypocotyl segments. AB - The effect of the defoliant thidiazuron (N-phenyl-N'1,2,3-thiadiazol-5-ylurea) on ethylene evolution from etiolated mung bean hypocotyl segments was examined. Treatment of hypocotyl segments with concentrations of thidiazuron equal to or greater than 30 nanomolar stimulated ethylene evolution. Increased rates of ethylene evolution from thidiazuron-treated tissues could be detected within 90 minutes of treatment and persisted up to 30 hours after treatment. Radioactive methionine was readily taken up by thidiazuron-treated tissues and was converted to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and an acidic conjugate of ACC. Aminoethoxyvinylglycine, aminooxyacetic acid, cobalt chloride, and alpha aminoisobutyric acid reduced ethylene evolution from treated tissues. An increase in the endogenous content of free ACC coincided with the increase in ethylene evolution following thidiazuron treatment. Uptake and conversion of exogenous ACC to ethylene were not affected by thidiazuron treatment. No increases in the extractable activities of ACC synthase were detected following thidiazuron treatment. PMID- 16663758 TI - Isopentenyl Pyrophosphate cis-1,4-Polyisoprenyl Transferase from Guayule (Parthenium argentatum Gray). AB - Electron micrographs of the mesophyll cells of guayule Parthenium argentatum Gray leaves show deposits of cis-polyisoprene (rubber) in the cytoplasm in the vicinity of mitochondria and chloroplasts and demonstrate that the rubber synthesizing enzymes are present in guayule leaves. The terminal step in the synthesis of cis-polyisoprene from isopentenyl pyrophosphate (IPP) catalyzed by isopentenyl pyrophosphate cis-1,4-polyisoprenyl transferase has been demonstrated in crude leaf extracts by the enzymic incorporation of [(14)C]isopentenyl pyrophosphate into the polymer and the recovery of [(14)C]levulinic acid following ozonolysis. The rubber transferase activity in the crude extracts of guayule leaves was 5.8 nanomoles isopentenyl pyrophosphate incorporated per milligram protein per hour. This is the first description of the rubber transferase from a nonlaticiferous plant.The specific activity (in units of nanomoles IPP converted per milligram protein per hour) of the partially purified enzyme following chromatography on diethylaminoethyl-cellulose columns was 41.7 units and contained 0.29 units of IPP isomerase activity and 0.08 units of farnesyl pyrophosphate synthetase activity. The rubber transferase requires reduced glutathione and Mg(2+) for maximal activity. There was no incorporation of IPP into cis-1,4-polyisoprene in the absence of rubber particles as primer, and Langmuir isotherm plots showed that the specific activity of the enzyme was proportional to the concentration of the enzyme on the surface of the rubber particles. For a given rubber particle distribution, enzyme activity was proportional to time, IPP concentration, and rubber concentration. The addition of 0.4 millimolar dimethylallyl pyrophosphate to the rubber transferase reaction resulted in a 2-fold increase in the incorporation of IPP into rubber. A comparison was made of the relative activities of rubber transferase in different species of Parthenium, Ficus, and Euphorbia. PMID- 16663759 TI - Selection, Isolation, and Characterization of Cadmium-Resistant Datura innoxia Suspension Cultures. AB - Datura innoxia cells from suspension cultures were selected for their ability to grow and divide rapidly in normally lethal concentrations of cadmium. Cells resistant to 12.5, 25, 50, 100, 160, 200, and 250 micromolar cadmium chloride were isolated and utilized to initiate cell suspension cultures resistant to this toxic metal ion. Variant cell lines retained their ability to grow in cadmium after being grown in its absence for more than 400 generations. Resistance to cadmium was correlated with the synthesis of low molecular weight, cysteine-rich, cadium-binding proteins. Synthesis of these proteins was induced rapidly in cadmium-resistant cells in response to a challenge of cadmium. Induction was detectable within one hour after exposure of the cells to the metal ion. Accumulation of protein bound cadmium reached a maximum eight to twelve hours following exposure. Metal-binding proteins were not detectable in the cadmium sensitive D. innoxia cells from which resistant cells were derived. PMID- 16663760 TI - Oxygen Uptake and Photosynthesis of the Red Macroalga, Chondrus crispus, in Seawater: Effects of Light and CO(2) Concentration. AB - With an experimental system using mass spectrometry techniques and infra-red gas analysis of CO(2) developed for aquatic plants, we studied the responses to various light intensities and CO(2) concentrations of photosynthesis and O(2) uptake of the red macroalga Chondrus crispus S. The CO(2) exchange resistance at air-water interface which could limit the photosynthesis was experimentally measured. It allowed the calculation of the free dissolved CO(2) concentration. The response to light showed a small O(2) uptake (37% of net photosynthesis in standard conditions) compared to C(3) plants; it was always higher than dark respiration and probably included a photoindependent part. The response to CO(2) showed: (a) an O(2) uptake relatively insensitive to CO(2) concentration and not completely inhibited with high CO(2), (b) a general inhibition of gas exchanges below 130 microliters CO(2) per liter (gas phase), (c) an absence of an inverse relationship between O(2) and CO(2) uptakes, and (d) a low apparent K(m) of photosynthesis for free CO(2) (1 micromolar). These results suggest that O(2) uptake in the light is the sum of different oxidation processes such as the glycolate pathway, the Mehler reaction, and mitochondrial respiration. The high affinity for CO(2) is discussed in relation to the use of HCO(3) (-) and/or the internal CO(2) accumulation. PMID- 16663761 TI - Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots. AB - Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10(4)Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27 degrees C) and substantially attenuated at both 4 and 37 degrees C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots. PMID- 16663762 TI - Protein Synthesis in Embryos of Dormant and Germinating Agrostemma githago L. seeds. AB - The time course of protein synthesis in embryos of dormant and afterripened Agrostemma githago seeds was studied. In embryos of afterripened geminating seeds, protein synthesis increased in three successive stages: (a) concurrent with swelling; (b) during the lag phase between the completion of water uptake and the onset of growth; and (c) immediately after protrusion through the seed coat. Embryos of dormant seeds showed the first increase but not the second unless dormancy was broken by imbibition at 4 degrees C. This indicates that dormancy affects processes prior to the onset of growth. The third increase was largely due to higher oxygen availability after the rupture of the seed coat and not to actual growth. It could also be elicited in dormant embryos by isolating them from the seeds.Electrophoretic analysis of the newly synthesized proteins demonstrated that the patterns of dormant and afterripened embryos became significantly different in both axes and cotyledons only just prior to the onset of axis elongation. Thereafter, the differences became larger.When afterripened or dormant seeds were transferred from a low, germination-permitting to a high, germination-inhibiting temperature, the seeds germinated at the high temperature if they had completed the lag phase to a sufficient extent at the low temperature. This shows that the processes during the lag phase were inhibited by the high temperature while the onset of growth was not affected. PMID- 16663763 TI - Sugar transport into protoplasts isolated from developing soybean cotyledons : I. Protoplast isolation and general characteristics of sugar transport. AB - A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Merr. cv Wye) cotyledons. Studies of sucrose and hexose uptake into these protoplasts show that the plasmalemma of cotyledons during the stage of rapid seed growth contains a sucrose-specific carrier which is energetically and kinetically distinct from the system(s) involved in hexose transport. For example, sucrose, but not hexose uptake: (a) is inhibited by alkaline pH and the nonpermeant SH modifier, p-chloromercuribenzene sulfonic acid; (b) is stimulated by fusicoccin; (c) shows both a saturable and a linear component of uptake in response to substrate concentration; and (d) displays a sharp temperature response (high Q(10) value and high activation energies). PMID- 16663764 TI - Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons : II. Sucrose Transport Kinetics, Selectivity, and Modeling Studies. AB - The effects of metabolic inhibitors, pH, and temperature on the kinetics of sucrose uptake protoplasts isolated from developing soybean Glycine max L. cv Wye cotyledons were studied. Structural requirements for substrate recognition by the sucrose carrier were examined by observing the effects of potential alternate substrates for the saturable component on sucrose uptake.Uptake by the three components (saturable, sulfhydryl reagent-sensitive nonsaturable, and diffusive) was calculated over a range of sucrose concentrations. The saturable component dominated uptake at external sucrose concentrations below 12 millimolar and was approximately equal to the nonsaturable and diffusive components at 44 and 22 millimolar external sucrose, respectively. The three uptake components showed different temperature sensitivities.Increasing external pH decreased both the linear component and the V(max) calculated for the saturable component. Conversely, increasing pH increased the calculated K(m) (sucrose) for the saturable component.Sucrose uptake by the saturable component was insensitive to several mono- and divalent cations. Competition for uptake of 0.5 millimolar sucrose by several sugars suggested that the beta-d-fructofuranoside bond and molecular size of sucrose were particularly important in sugar recognition by the saturable component carrier. PMID- 16663765 TI - Salt stimulation of phosphate uptake in maize root tips studied by p nuclear magnetic resonance. AB - The effects of external salt and inorganic phosphate (Pi) on the concentrations of vacuolar Pi, and cytoplasmic Pi, ATP, glucose-6-phosphate and UDP-glucose in maize root tips were examined using (31)P nuclear magnetic resonance spectroscopy. We observed a more than two-fold stimulation of Pi uptake from 10 millimolar KH(2)PO(4) solutions when root tips were exposed to 100 millimolar NaCl + CaCl(2). This stimulation of Pi uptake was associated with an increase in the concentration of cytoplasmic Pi in root tip cells. Thus, the molar ratio of cytoplasmic Pi to Pi + ATP + glucose-6-phosphate + UDP-glucose increased greatly in root tips exposed to salt and Pi. We speculate that it is this disturbance in relative concentrations of cytoplasmic phosphates (which we show are normally tightly regulated) that is responsible for both the greater rate of uptake of Pi by vacuoles of excised maize root tips, and the previously documented stimulation of Pi translocation from root to shoot in whole maize plants exposed to salt and Pi. PMID- 16663766 TI - Energetics of proline transport in corn mitochondria. AB - The mechanism of proline entry into the matrix region of isolated corn mitochondria (Zea mays L. Mo17 x B73) was investigated by measuring osmotically induced changes of mitochondrial size (changes in A(520)) in combination with oxygen uptake measurements. Using NADH oxidation to generate the electrochemical gradient, we have determined that proline transport is stereospecific and that it can be inhibited by the proline analog l-thiazolidine-4-carboxylic acid.The energetics of proline transport was investigated by measuring the effects of FCCP (p-trifluoromethoxycarbonyl cyanide phenylhydrazone) and valinomycin on mitochondrial swelling and substrate oxidation. Proline transport and resulting oxidation were found to be partially dependent upon the energy of the electrochemical gradient. At low proline concentrations, entry was found to be primarily independent of the gradient (based on insensitivity to FCCP), whereas at higher proline concentrations a gradient-dependent mechanism became involved. Results with valinomycin indicated that proline transport and oxidation are dependent upon the pH potential across the membrane rather than the electrical (membrane) potential. PMID- 16663767 TI - Characterization of peroxidase in plant cells. AB - Two peroxidases, one anionic and one cationic, have been purified from the proteins secreted by peanut (Arachis hypogaea L. var Virginia 56R) cells in suspension culture. These two peroxidases apparently have identical catalytic properties. PMID- 16663768 TI - Concentration Gradients of trans-Zeatin Riboside and trans-Zeatin in the Maize Stem: Measurement by a Specific Enzyme Immunoassay. AB - A sensitive, specific enzyme immunoassay (EIA) for trans-zeatin riboside (ZR) and trans-zeatin (Z) in the 0.3 to 30 picomole range has been described. The reliability of the method for measuring ZR + Z in partially purified extracts of Zea mays L. tissues was verified by highperformance liquid chromatography. EIA measurements showed that there was a concentration gradient of ZR + Z along the length of the Zea stem. The topmost internodes, internodes 7 and 8 counting from the coleoptilar node, had the highest concentration ( approximately 130 picomoles per gram fresh weight). Moving basipetally, the concentration dropped approximately 10-fold to a minimum at internode 4, and then increased slightly in internodes 2 and 3. There were also gradients within each internode. The five lowest internodes contained the highest concentrations toward their apical end, the region which included the node; this asymmetry was less pronounced near the top of the plant. PMID- 16663769 TI - A New Type of Hexose Monophosphate Shunt in Chlorella sorokiniana: CO(2) Release from Differentially Labeled Glucose. AB - Using differentially labeled glucose as a substrate to probe the operation of the hexose monophosphate shunt (pentose cycle) in Chlorella sorokiniana, we found that the labeling patterns for the release of (14)CO(2) over the first 5 minutes are compatible with the operation of the recently described L-type pentose shunt. Experimentally, this L-type differs from the F-type or ;textbook' variety in that no radioactivity is obtained from C-2 labeled glucose, and the small amount derived from C-6 labeled glucose is due to a second pass of the glucose molecule (derived from the pentose cycle) through the pentose cycle. PMID- 16663770 TI - Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts. AB - Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H(+)/e(-) stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria. PMID- 16663771 TI - Proline and glycine betaine influence protein solvation. AB - Glutamine synthetase from barley (Hordeum distichum L.) is precipitated by polyethylene glycol (PEG). Proline, in a concentration-dependent manner, reduces the amount of enzyme precipitated by PEG, although the effect of the imino acid can be counteracted by raising the level of PEG. The effect of PEG is a function of mer number and concentration and the influence of both elements can be ameliorated by proline. PEG-induced enzyme precipitation is a function of pH, as is its interaction with both proline and betaine in the reaction. The lack of effect of amount of enzyme on the proline and PEG effects supports the conclusion that, in this system, proline and PEG do not function through interaction with the protein. Other compounds, such as glycine, glucose, and sucrose, can decrease the PEG-induced precipitation of the enzyme, although glycerol was not active under the conditions employed.The results are consistent with the proposition that a protein-containing system in which high concentrations of proline and/or betaine are present, is better ;protected' against the biologically unfavorable consequences of dehydration-induced thermodynamic perturbation. PMID- 16663772 TI - Tissue Distribution of beta-Cyanoalanine Synthase in Leaves. AB - beta-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form beta-cyanoalanine and H(2)S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor x Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of beta-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.THE TISSUE DISTRIBUTIONS FOR THIS ENZYME WERE DETERMINED FOR PEA, LEEK, AND SORGHUM: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of beta cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism. PMID- 16663773 TI - On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds. AB - Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme. PMID- 16663774 TI - Ultrasonic acoustic emissions from the sapwood of cedar and hemlock : an examination of three hypotheses regarding cavitations. AB - Measurements are reported of ultrasonic acoustic emissions (AEs) measured from sapwood samples of Thuja occidentalis L. and Tsuga canadensis (L.) Carr. during air dehydration. The measurements were undertaken to test the following three hypotheses: (a) Each cavitation event produces one ultrasonic AE. (b) Large tracheids are more likely to cavitate than small tracheids. (c) When stem water potentials are >-0.4 MPa, a significant fraction of the water content of sapwood is held by ;capillary forces.' The last two hypotheses were recently discussed at length by M. H. Zimmermann. Experimental evidence consistent with all three hypotheses was obtained. The evidence for each hypothesis respectively is: (a) the cumulative number of AEs nearly equals the number of tracheids in small samples; (b) more water is lost per AE event at the beginning of the dehydration process than at the end, and (c) sapwood samples dehydrated from an initial water potential of 0 MPa lost significantly more water before AEs started than lost by samples dehydrated from an initial water potential of about -0.4 MPa. The extra water held by fully hydrated sapwood samples may have been capillary water as defined by Zimmerman.We also report an improved method for the measurement of the ;intensity' of ultrasonic AEs. Intensity is defined here as the area under the positive spikes of the AE signal (plotted as voltage versus time). This method was applied to produce a frequency histogram of the number of AEs versus intensity. A large fraction of the total number of AEs were of low intensity even in small samples (4 mm diameter by 10 mm length). This suggests that the effective ;listening distance' for most AEs was less than 5 to 10 mm. PMID- 16663775 TI - Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : III. Reduction of Photorespiration by a Limited C(4) Pathway of Photosynthesis in Flaveria ramosissima. AB - The initial products of photosynthesis by the C(3) species Flaveria cronquistii, the C(4) species F. trinervia, and the C(3)-C(4) intermediate species F. ramosissima were determined using a pulse-chase technique with (14)CO(2) (12)CO(2). The intermediate species F. ramosissima incorporated at least 42% of the total soluble (14)C fixed into malate and aspartate after 10 seconds of photosynthesis in (14)CO(2), as compared with 90% for the C(4) species F. trinervia and 5% for the C(3) species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in (12)CO(2), although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C(4) pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C(3) and C(4) plants. PMID- 16663776 TI - UV-B Inhibition of Phytochrome-Mediated Anthocyanin Formation in Sinapis alba L. Cotyledons : Action Spectrum and the Role of Photoreactivation. AB - An action spectrum was measured for ultraviolet (UV) radiation-induced damage to (inhibition of) phytochrome-induced anthocyanin formation in cotyledons of 40 hour-old Sinapis alba L. seedlings. The action spectrum showed maximum effectiveness in the 260 to 280 nanometer waveband with little effect above 295 nanometers. The damaging effect of UV could be photorepaired by subsequent exposure to sunlight or to long wavelength (360 nanometers) UV radiation. Because this form of damage is subject to photorepair (photoreactivation), it is probably due to the formation of pyrimidine dimers, and the results suggest that it would not be ecologically relevant even if there was an increase in solar UV due to a decrease in stratospheric ozone levels of about 30%. If a dark period of more than 1 hour is interspersed between the phytochrome induction and the UV irradiation, the inhibition of the phytochrome induction gradually decreases with increasing dark period. PMID- 16663777 TI - Light and Genetic Determinants in the Control of Specific Chloroplast Transcripts in Chlamydomonas reinhardtii. AB - We have looked for chloroplast genes whose expression is controlled by light by comparing the abundance of specific chloroplast transcripts in light-grown and dark-grown cells of Chlamydomonas reinhardtii. In addition, we have investigated whether genetic components influence expression of such genes.Northern blot analyses of specific separated transcripts showed that in certain strains (e.g. CC-278), several chloroplast transcripts from the Hpa II 5 region of the chloroplast chromosome were more abundant in dark-grown cells than in light-grown cells. The increased abundance of these Hpa II 5-specific transcripts was dependent on the genetic background: we have isolated mutants and we have found distantly related wild type strains which do not overaccumulate these transcripts in the dark.The strains which overaccumulated the Hpa II 5-specific transcripts, and only these strains, died after several cell divisions following transfer to the dark. Overaccumulation appears to be a necessary but not sufficient prerequisite for commitment to cell death in the dark.By analogy to bacterial systems, we speculate that a critical event associated with accumulation of one of these specific transcripts involves control at the level of transcription initiation or termination-antitermination. PMID- 16663778 TI - A simple plant nutrient solution purification method for effective removal of trace metals using controlled pore glass-8-hydroxyquinoline chelation column chromatography. AB - Column chelation chromatography on controlled pore glass-8-hydroxyquinoline was demonstrated to be a very efficient method for removing trace metal contaminants from concentrated macronutrient salt solutions used to prepare nutrient media. By using (63)Ni and (65)Zn radio-isotopes as tracers, controlled pore glass-8 hydroxyquinoline column packings were found to retain 99.9% of the radiotracer and quantitative recovery of the radioisotopes from these columns was obtained by eluting with 1.2 n HCl. This method has several advantages over liquid-liquid extraction methods of purification which previously have been used in plant micronutrient research. PMID- 16663779 TI - Effect of submergence on the cell wall composition of deep-water rice internodes. AB - The cell wall composition of internodes of deep-water rice plants (Oryza sativa L. cv Habiganj Aman II) which were induced to grow rapidly by submergence in water was compared to that of nonsubmerged plants which grew slowly. No differences could be detected in cellulose, uronic acid, and lignin content expressed on a dry weight basis. Cell wall preparations of rapidly growing, submerged internodes contained more hydroxyproline and had a higher hydration capacity than those of control internodes. The silicon content of submerged rice internodes was considerably lower than that of air-grown plants. The role of silicon as a structural component of the cell wall of grasses is discussed in relation to lodging of deep-water rice plants after the flood waters have receded. PMID- 16663780 TI - Interaction of water supply and N in wheat. AB - The purpose of this study was to investigate effects of N nutrition and water stress on stomatal behavior and CO(2) exchange rate in wheat (Triticum aestivum L. cv Olaf). Wheat plants were grown hydroponically with high (100 milligrams per liter) and low (10 milligrams per liter) N. When plants were 38 days old, a 24 day water stress cycle was begun. A gradual increase in nutrient solution osmotic pressure from 0.03 to 1.95 mega Pascals was achieved by incremental additions of PEG-6,000. Plants in both N treatments adjusted osmotically, although leaf water potential was consistently lower and relative water content greater for low N plants in the first half of the stress cycle. Leaf conductance of high N plants appeared greater than that of low N plants at high water potentials, but showed greater sensitivity to reductions in water potential as indicated by earlier stomatal closure during the stress cycle. The apparent greater stomatal sensitivity of high N plants was associated with a curvilinear relationship between leaf conductance and leaf water potential; low N plants exhibited more of a threshold response. Trends in [CO(2)](INT) throughout the stress cycle indicated nonstomatal effects of water stress on CO(2) exchange rate were greater in high N plants. Although estimates of [CO(2)](INT) were generally lower in high N plants, they were relatively insensitive to leaf water potential-induced changes in leaf conductance. In contrast, [CO(2)](INT) of low N plants dropped concomitantly with leaf conductance at low leaf water potentials. Oxygen response of CO(2) exchange rate for both treatments was affected less by reductions in water potential than was CO(2) exchange rate at 2.5% O(2), suggesting that CO(2) assimilation capacity of the leaves was affected more by reductions in leaf water potential than were processes related to photorespiration. PMID- 16663781 TI - Acetaldehyde Oxime, A Product Formed during the In Vivo Nitrate Reductase Assay of Soybean Leaves. AB - Evolution of nitrogen oxides (NO((x)), primarily as nitric oxide) from soybean (Glycine max [L.] Merr.) leaves during purged in vivo nitrate reductase assays had been reported; however, these reports were based on a method that had been used for determination of NO((x)) in air. This method also detects other N compounds. Preliminary work led us to doubt that the evolved N was nitric oxide. Studies were undertaken to identify the N compound evolved from the in vivo assay that had been reported as NO((x)). Material for identification was obtained by cryogenic trapping and fractional distillation, and by chemical trapping procedures. Mass spectrometry, ultraviolet spectroscopy, and (15)N-labeled nitrate were used to identify the compounds evolved and to determine whether these compounds were derived from nitrate. Acetaldehyde oxime was identified as the predominant N compound evolved and this compound is readily detected by the method for NO((x)) determination. Substantial quantities of acetaldehyde oxime (16.2 micromoles per gram fresh weight per hour) were evolved during the in vivo assay. Small amounts of nitrous oxide (0.63 micrograms N per gram fresh weight per hour) were evolved, but this compound is not detected as NO((x)). Acetaldehyde oxime and nitrous oxide were both produced as a result of nitrate ((15)NO(3) (-)) reduction during the assay. PMID- 16663782 TI - Benzyl viologen-mediated counteraction of diquat and paraquat phytotoxicities. AB - There was reason from bacterial and algal systems to expect that pretreatments with a paraquat analog might confer tolerance against a subsequent paraquat treatment. Thus, a series of compounds were tested for protective activity against bipyridinium herbicides. These included other bipyridinium compounds and derivatives, as well as compounds having similar or more positive redox potentials than paraquat and compounds known to increase or maintain high superoxide dismutase activity levels in plants.Only treatments with benzyl viologen, a benzyl analog of paraquat, protected Spirodela oligorrhiza (Kurz) Hegelm. colonies from otherwise damaging levels of diquat.NADP photoreduction by isolated thylakoids was inhibited by the same concentrations of paraquat, diquat, and benzyl viologen given separately. Thus, the benzyl viologen-mediated tolerance against the bipyridinium herbicides is probably not due to a direct interaction at the thylakoid level.Superoxide dismutase activity was about 50% higher in broken plastids of benzyl viologen-treated plants compared to controls, which may partly explain the observed tolerance. PMID- 16663783 TI - Respiration during Postharvest Development of Soursop Fruit, Annona muricata L. AB - Fruit of soursop, Annona muricata L., showed increased CO(2) production 2 days after harvest, preceding the respiratory increase that coincided with autocatalytic ethylene evolution and other ripening phenomena. Experiments to alter gas exchange patterns of postharvest fruit parts and tissue cylinders had little success.The respiratory quotient of tissue discs was near unity throughout development. 2,4-Dinitrophenol uncoupled respiration more effectively than carbonylcyanide m-chlorophenylhydrazone; 0.4 millimolar KCN stimulated, 4 millimolar salicylhydroxamic acid slightly inhibited, and their combination strongly inhibited respiration, as did 10 millimolar NaN(3). Tricarboxylic acid cycle members and ascorbate were more effective substrates than sugars, but acetate and glutarate strongly inhibited.Disc respiration showed the same early peak as whole fruit respiration; this peak is thus an inherent characteristic of postharvest development and cannot be ascribed to differences between ovaries of the aggregatetype fruit. The capacity of the respiratory apparatus did not change during this preclimacteric peak, but the contents of rate-limiting malate and citrate increased after harvest.It is concluded that the preclimacteric rise in CO(2) evolution reflects increased mitochondrial respiration because of enhanced supply of carboxylates as a substrate, probably induced by detachment from the tree. The second rise corresponds with the respiration during ripening of other climacteric fruits. PMID- 16663784 TI - Low Temperature-Induced GA(3) Sensitivity of Wheat : I. Characterization of the Low Temperature Effect on Isolated Aleurone OF KITE. AB - Gibberellic acid (GA(3)) sensitivity (measured as alpha-amylase production) of the isolated aleurone tissue/deembryonated seed of two wheat (Triticum aestivum L. var Kite and var Aroona) varieties each containing either one of the dwarfing genes, Rht1 and Rht2, was increased significantly as a result of low temperature treatment. The magnitude of the low temperature-induced increase occurred without any change in the lag time of alpha-amylase production. This low temperature induction of GA(3) sensitivity was found to be operative in aleurone tissue of only those varieties having at least one of the three Rht alleles. It is likely, therefore, that the low temperature treatment effect which ;cures' or circumvents the genetic lesions manifest in the Rht1 and Rht2 genotypes is the same as that effective in the Rht3-containing genotype and probably involves an increase in hormone (GA(3)) receptor sites. Furthermore, this increase appears to be a quantitative temporal one. PMID- 16663785 TI - Low Temperature-Induced GA(3) Sensitivity of Wheat : II. Changes in Lipids Associated with the Low Temperature-Induced GA(3) Sensisivity of Isolated Aleurone of Kite. AB - Exposure of isolated aleurone tissue from the wheat (Triticum aestivum) variety Kite which contains the Rht2 allele, to low temperature (5 degrees C) for 20 h prior to addition of exogenous GA(3), resulted in significant changes in the content of lipids, especially phospholipids. Significant low temperature-induced changes in both the head group and acyl contents of two phospholipids, phosphatidylcholine and phosphatidylethanolamine, were detected. More importantly, these changes displayed a very close temporal relationship with the low temperature-induced increase in GA(3) sensitivity. Further, this relationship was paralleled by a highly significant correlation between the changes in the phospholipids and the changes in alpha-amylase production. These results underline the possibility that the GA(3) receptor sites are membrane-based lipids. PMID- 16663786 TI - Development and Essential Oil Content of Secretory Glands of Sage (Salvia officinalis). AB - Scanning electron microscopy of sage (Salvia officinalis L.) leaves confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head. Gas liquid chromatographic analysis of the oil content of the most numerous gland species (capitate stalked, capitate sessile with one and with eight secretory cells) indicated only minor quantitative differences in essential oil composition. Thus, each gland type is capable of producing the four major monoterpene families (p-menthanes, pinanes, bornanes and thujanes) characteristic of sage. PMID- 16663787 TI - Critical oxygen pressure for growth and respiration of excised and intact roots. AB - A method based on the measurement of ATP/ADP ratios is described. It permits the determination of the critical respiratory oxygen pressure of any organ, or part of any organ, of an intact plant. The data obtained by this method with intact maize (Zea mays L. INRA 508) root tips are compared with polarographic determinations on similar excised tissues.When internal O(2) transport from the aerial part was prevented, the critical oxygen pressure found for the respiration of intact tips was similar to that found with excised tips. It was close to 10 kilopascals in a humid atmosphere and about 30 kilopascals in a liquid medium. Flooding of the gas spaces by vacuum infiltration did not modify these results. When internal O(2) transport from the aerial parts of the plant occurred, significantly lower values were obtained in liquid medium for the critical oxygen pressure, which shifted from more than 21 to 6 kilopascals. The higher values observed with excised root tips, compared to those obtained with intact tissues, can be explained by the lack of internal O(2) transport, rather than by the flooding of gas spaces.Data are presented which show that root growth started to be limited at a significantly higher pressure than the respiration. These results are attributed to nonrespiratory oxidative processes with a low affinity for O(2) involved in root elongation. PMID- 16663788 TI - Control of Seed Germination by Abscisic Acid : II. Effect on Embryo Water Uptake in Brassica napus L. AB - Germination of rape (Brassica napus L.) seeds proceeds in two phases, an initial imbibition phase and a subsequent growth phase. The time courses of water uptake, O(2) uptake, and ATP accumulation demonstrate that exogenous abscisic acid (ABA, 0.1 millimoles per liter) specifically prevents the embryo from entering the growth phase. The inhibition of water uptake by ABA is a rapid (lag-phase about 1 hour) and fully reversible process which appears to be the cause rather than the result of changes of the energy metabolism. In untreated seeds, an osmotic pressure (polyethylene glycol 6000) of 11 bars is required for a simulation of the ABA effect on water uptake. However, in ABA-treated seeds an osmotic pressure of only 3 bars is sufficient to suppress water uptake. Thus, ABA lowers the ability of the embryo to absorb water under osmotic stress. In a two-factor analysis of the simultaneous action of ABA and osmoticum on germination, a complete synergistic interaction between these factors was found while ABA and cycloheximide exhibit independent (multiplicative) coaction. These results are interpreted in terms of a common controlling point of ABA and osmotic stress in the water relations of germinating seeds. PMID- 16663789 TI - Intracellular transport and posttranslational cleavage of oat globulin precursors. AB - The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The alpha and beta globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The alpha and beta subunits appeared during the same chase period and assembled into a 12S oligomer.The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the alpha and beta subunits forming a 12S oligomer. PMID- 16663790 TI - Inhibition by auxins of 4-methyleneglutamic Acid synthesis in tissue cultures of peanut seeds. AB - Callus cultures of peanut (Arachis hypogaea L. cv Valencia Tennessee Red) cotyledons grown on Linsmaier and Skoog medium containing normal levels of auxin and cytokinin do not synthesize either 4-methyl-eneglutamic acid or 4 methyleneglutamine, which nonprotein amino acids are normally found in significant amounts in peanut plants. If mature peanut embryos (with cotyledons removed) are germinated and grown on a similar medium containing no added phytohormone, normal levels of these two amino acids accumulate. The addition of an auxin, however, prevents formation of 4-methyleneglutamic acid and 4 methyleneglutamine; typical levels of other free amino acids are seen and excised embryos so cultured develop into apparently otherwise normal plants. Kinetin addition to embryo cultures has little or no effect. 4-Methyleneglutamine is formed when 4-methyleneglutamic acid is added to embryo cultures maintained on auxin-containing medium, indicating that the phytohormone does not block amidation but rather the biosynthesis of 4-methyleneglutamic acid. PMID- 16663791 TI - Diurnal carbohydrate metabolism of barley primary leaves. AB - The carbohydrate content of barley (Hordeum vulgare L.) leaves was measured over a 24-hour cycle. Nonstructural carbohydrate accumulation was linear after the 1st hour of light, whereas utilization in the dark was fast initially and slowed as stored reserves were depleted. Sucrose was the most abundant storage form of carbohydrate in the primary leaf. Lesser amounts of starch, fructans, and hexoses were also present. Leaf reserves were almost completely remobilized by the end of the dark period. There was a lag in starch degradation following a light to dark transition. Lower rates of starch accumulation were observed at the beginning and at the end of the day. Fructan synthesis occurred primarily towards the end of the light period as rates of sucrose and starch synthesis decreased. The above results suggested that carbohydrate metabolism in primary barley leaves was controlled by light and by endogenous factors such as foliar sucrose levels. Measurements of specific [(14)C]sucrose activity in steady state labeled 7-day old barley primary leaves suggested the presence of at least two kinetically separate pools. Sucrose levels were higher and apparent turnover rates were lower in barley leaves in comparison to previous studies with other species. PMID- 16663792 TI - Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes. AB - 5-Keto-l-idionic acid ( identical with5-keto-d-gluconic acid, d-xylo-5 hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv ;Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.Label from dehydro-l-[1-(14)C]ascorbic acid, 2-keto-l [1-(14)C]idonic acid (l-xylo-2-hexulosonic acid), l-[1-(14)C]idonic acid, or 5 keto-l-[1-(14)C] idonic acid was incorporated into l-(+)-tartaric acid in high yields as it was in the l-[1-(14)C]ascorbic acid experiment. In a double label experiment involving a mixture of l-[1-(14)C]idonic acid and l-[2-(3)H]idonic acid, the (3)H/(14)C ratios of 5-keto-l-idonic acid and l-(+)-tartaric acid synthesized in young grape leaves were almost the same as the value of the l idonic acid fed. Label from 5-keto-l-[6-(14)C]idonic acid was incorporated into sugars and insoluble residue in the same way as l-[6-(14)C]ascorbic acid was metabolized in grapes.These results provide strong evidence that in grapes l-(+) tartaric acid is synthesized from the C(4) fragment that corresponds to the C1 to C4 group of the 5-keto-l-idonic acid derived from l-ascorbic acid via 2-keto-l idonic acid and l-idonic acid. PMID- 16663793 TI - Biochemical and x-ray diffraction analysis of concanavalin B crystals from jack bean. AB - Parallel biochemical and crystallographic studies have been carried out on the protein concanavalin B from Jack Bean (Canavalis ensiformis). The studies show the protein to be a monomer of 33,000 daltons with unexceptional amino acid composition and no covalently bound carbohydrate. The molecule contains a single, firmly bound zinc ion and we present evidence that the protein specifically binds nucleotide coenzymes including at least NADPH and flavin mononucleotide. An electron density map of the concanavalin B crystals (space group P6(1), a = b = 80.9 A, c = 102.2 A) has been calculated from phases based on six isomorphous heavy atom derivatives. A description of the structure of the protein based on the 5.0 A resolution x-ray diffraction study is provided. The structure appears to contain a substantial amount of alpha-helix as well as an extensive beta-sheet and these are organized into two domains of unequal size. The position of the bound zinc is indicated by anomalous difference Fouriers and the nucleotide binding site by conventional difference Fourier maps. PMID- 16663794 TI - Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4 cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir. AB - Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols. PMID- 16663795 TI - Characterization and Complexity of Wheat Developing Endosperm mRNAs. AB - Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA(+) RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)(+) RNAs, analyzed on isokinetic sucrose gradient with [(3)H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly (2/3) and (1/3) of the MB poly(A)(+) RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins. PMID- 16663796 TI - Nonosmotic inhibition by sugars of the ethylene-forming activity associated with microsomal membranes from carnation petals. AB - The activity of the membrane-bound ethylene-forming enzyme, previously reported in carnation (Dianthus caryophyllus L. cv White Sim) petals (Mayak, Legge, Thompson 1981 Planta 153: 49-55), is inhibited by sugars. Of the various sugars tested, sorbitol was the most effective and glucose the least. The effect of sugars was also evaluated on solubilized ethylene-forming enzyme activity, obtained by the use of 0.6% Nonidet NP-40 detergent. Similar to the membrane bound activity, the solubilized activity was also inhibited. Kinetic studies revealed that the inhibition by sugars is reversible, and that inhibition by sucrose is uncompetitive while that by sorbitol is competitive. During senescence of petals, a decline in sugar content and climacteric like increase in ethylene occurs. Hence, the physiological relevance of sugar inhibition and its possible involvement in the regulation of ethylene biosynthesis is suggested. PMID- 16663797 TI - Dependence of Seed Vigor during Germination on Carbohydrate Source in Endosperm Mutants of Maize. AB - Differences in seed vigor of four genotypes of maize (Zea mays L.), brittle-1 (bt1), shrunken-2 (sh2), sugary (su), and normal, in an isogenic background, were investigated. Excised whole embryos and axes were grown on Murashige and Skoog (MS) media containing various carbohydrate sources. Of the four genotypes examined, sh2 seeds had the lowest vigor, especially under germination stress conditions. Embryo dry weights of sh2 were less than su and normal but equal to bt1 and made up nearly 25% of the whole seed weight. The sh2 seeds and whole embryos had low starch levels compared with the other three genotypes. Sugar levels were comparable in the three endosperm mutants, which were two times higher than normal. Optimum growth of excised embryos of all genotypes was obtained on MS medium containing 5% sucrose. However, this concentration did not totally overcome poor germination and growth of sh2 embryos and axes. Axes of su and normal had greater growth rates than sh2 and bt1 on sucrose-free medium, although the difference between genotypes decreased when whole embryos were used. When ground endosperm was employed as the carbohydrate source, sh2 embryos germinated and grew poorly, particularly on normal endosperm. With a commercial corn starch as the carbohydrate source, sh2 germlings were shorter in length and displayed a greater loss in dry weight than the other genotypes. The poor growth of sh2 embryos on ground endosperm and starch media may indicate a dysfunction of the scutellum or axis in relation to carbohydrate metabolism and utilization. PMID- 16663798 TI - Freezing behavior of water in small pores and the possible role in the freezing of plant tissues. AB - Two model systems were used to study the freezing of water in small diameter pores. Water in pores having a diameter of less than 100 nanometers froze at lower temperatures than bulk water. Data obtained with a range of pore sizes were consistent with predicted values based on equations developed by Mazur (1965 Ann NY Acad Sci 125: 658-676), and Homshaw (1980 J Soil Sci 31: 399-414). The addition of solutes lowered the freezing point of water in small pores. We propose that the freezing behavior of water in small pores may account for some of the freezing patterns observed in plant tissues. In tissues where cells are tightly packed, share common walls, and lack intercellular spaces, the presence of water in cell wall microcapillaries would alter the freezing temperature of tissue water, impede the spread of ice, and facilitate supercooling. PMID- 16663799 TI - Cell wall development in maize coleoptiles. AB - The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 x Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5 arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages. PMID- 16663800 TI - Adenine nucleotide regulation of malate oxidation in isolated mung bean hypocotyl mitochondria. AB - We have studied the effects of ATP and ADP on the oxidation of malate by coupled and uncoupled mitochondria prepared from etiolated hypocotyls of mung bean (Vigna radiata L.).In coupled mitochondria, ATP (1 millimolar) increased pyruvate production and decreased oxaloacetate formation without altering the rate of oxygen consumption. ATP also significantly decreased oxaloacetate production and increased pyruvate production in mitochondria that were uncoupled by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone plus oligomycin.In coupled mitochondria, ADP (1 millimolar) increased the production of both pyruvate and oxaloacetate concomitantly with the acceleration of oxygen uptake to the state 3 rate. The effects of ADP were largely eliminated in uncoupled mitochondria. These results indicate that, whereas the ADP stimulation of oxaloacetate and pyruvate production in the coupled mitochondria is brought about primarily as the result of the accelerated rates of electron transport and NADH oxidation by the respiratory chain in state 3, ATP has significant regulatory effects independent of those that might be exerted by control of electron transport. PMID- 16663801 TI - Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions. AB - The effects of gibberellic acid (GA(3)) and calcium ions on the production of alpha-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA(3) or Ca(2+) show qualitative and quantitative changes in hydrolase production following incubation in either GA(3) or Ca(2+) or both. Incubation in H(2)O or Ca(2+) results in the production of low levels of alpha-amylase or acid phosphatase. The addition of GA(3) to the incubation medium causes a 10- to 20 fold increase in the amounts of these enzymes released from the tissue, and addition of Ca(2+) at 10 millimolar causes a further 8- to 9-fold increase in alpha-amylase release and a 75% increase in phosphatase release. Production of alpha-amylase isoenzymes is also modified by the levels of GA(3) and Ca(2+) in the incubation medium. alpha-Amylase 2 is produced under all conditions of incubation, while alpha-amylase 1 appears only when layers are incubated in GA(3) or GA(3) plus Ca(2+). The synthesis of alpha-amylases 3 and 4 requires the presence of both GA(3) and Ca(2+) in the incubation medium. Laurell rocket immuno electrophoresis shows that two distinct groups of alpha-amylase antigens are present in incubation media of aleurone layers incubated with both GA(3) and Ca(2+), while only one group of antigens is found in media of layers incubated in GA(3) alone. Strontium ions can be substituted for Ca(2+) in increasing hydrolase production, although higher concentrations of Sr(2+) are required for maximal response. We conclude that GA(3) is required for the production of alpha-amylase 1 and that both GA(3) and either Ca(2+) or Sr(2+) are required for the production of isoenzymes 3 and 4 of barley aleurone alpha-amylase. PMID- 16663802 TI - Subcellular localization of spermidine synthase in the protoplasts of chinese cabbage leaves. AB - Previous studies on the presence of spermidine synthase (EC 2.5.1.16) in the protoplasts of Chinese cabbage (Brassica pekinensis var Pak Choy) leaves had detected a small but significant fraction of the enzyme in a crude chloroplast fraction (Cohen, Balint, Sindhu 1981 Plant Physiol 68: 1150-1155). To establish whether this enzyme is truly a chloroplast component, we have isolated purified intact chloroplasts from protoplasts by density gradient centrifugation in silica sols (Ludox AM). Such chloroplasts contained all of the diaminopimelate decarboxylase (EC 4.1.1.20) of the protoplasts, but were essentially devoid of spermidine synthase. Control experiments showed that the latter had not been inactivated under conditions of isolation, purification, and assay of the intact chloroplasts. Isolation and assay of protoplast vacuoles in a further examination of the supernatant fluid containing the enzyme revealed a significant fraction of the enzyme in the vacuole fraction. However this fraction was found to contain similar proportions of a soluble enzyme, glucose 6-phosphate dehydrogenase. It has been concluded that vacuolar fractions are difficultly separable from soluble cytoplasmic material, which is probably the only compartment containing spermidine synthase. PMID- 16663803 TI - Transport of Benzyladenine and Gibberellin A(1) from Roots in Relation to the Dominance between the Axillary Buds of Pea (Pisum sativum L.) Cotyledons. AB - In etiolated, 5-day-old pea (Pisum sativum L.) seedlings a significantly more intensive growth of buds situated in the axil of the excised cotyledons was observed as early as 4 hours after decapitation and excision of one cotyledon of each pair. If [8-(14)C]benzyladenine ([(14)C]BA) was applied to roots of intact plants 10 hours prior to such decapitation and excision, significantly higher both total and specific (14)C activities were observed in buds situated on the side of the excised cotyledons as early as 4 hours after decapitation and excision. Although the removal of a substantial part of the root system carried out simultaneously with decapitation and excision of one cotyledon resulted in a decrease in total (14)C activity of buds, nevertheless a higher accumulation of (14)C activity was maintained in buds situated on the side of excised cotyledon. If [(14)C]BA was applied to roots of seedlings after they were decapitated and deprived of one cotyledon, both total and specific (14)C activities of buds situated on the side of excised cotyledons were significantly higher as early as the end of uptake of [(14)C]BA by roots, i.e. after 10 hours. On the other hand, [1,2-(3)H]gibberellin A(1) applied to roots of intact and/or decapitated and one cotyledon-deprived seedlings in the same way as [(14)C]BA did not appear in the buds until very much later and only in negligible amounts (i.e.(3)H activity). This indicates that the release of buds from apical dominance represents an active and selective process which can result from the ability of buds to utilize and/or synthesize only certain growth substances within a certain time interval. PMID- 16663804 TI - Protein bodies and vacuoles as lysosomes : investigations into the role of mannose-6-phosphate in intracellular transport of glycosidases in pea cotyledons. AB - We have failed to detect the presence of mannose-6-phosphate in the oligosaccharide moiety of glycoproteins from pea (Pisum sativum L. cv Burpeeana) cotyledons using an assay system sensitive to 10 picomoles of mannose-6 phosphate. We were also unable to demonstrate any retention of glycosidase activity from pea seedlings and pea cotyledons on Sepharose-coupled phosphomannosyl receptor proteins isolated from bovine liver which were, however, able to retain phosphomannosylated hexosaminidase purified from Dictyostelium discoideum secretions. Furthermore, although Sepharose-coupled phosphomannosylated hexosaminidase from Dictyostelium was able to bind phosphomannosyl receptors from bovine liver we were unable to detect the retention of any protein from acetone powder extracts of pea seedlings or from endoplasmic reticulum-associated proteins of pea cotyledons.Based on this collective evidence we conclude that mannose-6-phosphate does not appear to play a role in the targeting of hydrolytic enzymes from the endoplasmic reticulum to the protein bodies in pea cotyledons. PMID- 16663805 TI - Partial purification and characterization of arginine decarboxylase from avocado fruit, a thermostable enzyme. AB - A partially purified preparation of arginine decarboxylase (EC 4.1.1.19), a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained from the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60 degrees C, in the presence of 1.2 millimolar MnCl(2), 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The K(m), of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fully developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of V(app) (max) of these enzymes was 1613 and 68 nanomoles of CO(2) produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at 1 millimolar under similar conditions. The effect of different inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed. PMID- 16663806 TI - Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6 Bisphosphatase in Relation to Photosynthetic Rate in Maize. AB - The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6 bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30 degrees C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C(4) plants.Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells. PMID- 16663807 TI - Effects of Water Stress on Photosynthesis and Carbon Partitioning in Soybean (Glycine max [L.] Merr.) Plants Grown in the Field at Different CO(2) Levels. AB - The effects of water stress and CO(2) enrichment on photosynthesis, assimilate export, and sucrose-P synthase activity were examined in field grown soybean plants. In general, leaves of plants grown in CO(2)-enriched atmospheres (300 microliters per liter above unenriched control, which was 349 +/- 12 microliters per liter between 0500 and 1900 hours EST over the entire season) had higher carbon exchange rates (CER) compared to plants grown at ambient CO(2), but similar rates of export and similar activities of sucrose-P synthase. On most sample dates, essentially all of the extra carbon fixed as a result of CO(2) enrichment was partitioned into starch. CO(2)-enriched plants had lower transpiration rates and therefore had a higher water use efficiency (milligrams CO(2) fixed per gram H(2)O transpired) per unit leaf area compared to nonenriched plants. Water stress reduced CER in nonenriched plants to a greater extent than in CO(2)-enriched plants. As CER declined, stomatal resistance increased, but this was not the primary cause of the decrease in assimilation because internal CO(2) concentration remained relatively constant. Export of assimilates was less affected by water stress than was CER. When CERs were low as a result of the imposed stress, export was supported by mobilization of reserves (mainly starch). Export rate and leaf sucrose concentration were related in a curvilinear manner. When sucrose concentration was above about 12 milligrams per square decimeter, obtained with nonstressed plants at high CO(2), there was no significant increase in export rate. Assimilate export rate was also correlated positively with SPS activity and the quantitative relationship varied with CER. Thus, export rate was a function of both CER and carbon partitioning. PMID- 16663808 TI - Phosphate inhibition of spinach leaf sucrose phosphate synthase as affected by glucose-6-phosphate and phosphoglucoisomerase. AB - The inhibition patterns of inorganic phosphate (Pi) on sucrose phosphate synthase activity in the presence and absence of the allosteric activator glucose-6-P was studied, as well as the effects of phosphoglucoisomerase on fructose-6-P saturation kinetics with and without Pi. In the presence of 5 millimolar glucose 6-P, Pi was a partial competitive inhibitor with respect to both substrates, fructose-6-P and uridine diphosphate glucose. In the absence of glucose-6-P, the inhibition patterns were more complex, apparently because of the interaction of Pi at the activation site as well as the catalytic site. In addition, substrate activation by uridine diphosphate glucose was observed in the absence of effectors. The results suggested that Pi antagonizes glucose-6-P activation of sucrose phosphate synthase by competing with the activator for binding to the modifier site.The fructose-6-P saturation kinetics were hyperbolic in the absence of phosphoglucoisomerase activity, but became sigmoidal by the addition of excess phosphoglucoisomerase. The sigmoidicity persisted in the presence of Pi, but sucrose phosphate synthase activity was decreased. The apparent sigmoidal response may represent the physiological response of sucrose phosphate synthase to a change in hexose-P concentration because sucrose phosphate synthase operates in the cytosol in the presence of high activities of phosphoglucoisomerase. Thus, the enzymic production of an activator from a substrate represents a unique mechanism for generating sigmoidal enzyme kinetics. PMID- 16663809 TI - A Simple Technique of Studying Water Deficit Effects on Nitrogen Fixation in Nodules without Influencing the Whole Plant. AB - Cowpea (Vigna unguiculata L. Walp cv C-152) plants were grown in a system in which watering was withheld from the soil zone containing nodules, while the plants were able to maintain normal water status. The system was developed in a pot by making two soil zones, an upper and a lower separated by a gravel column between these two zones. Plants extended their roots into the lower layer of soil and were able to absorb water. The dry matter accumulation, photosynthesis rate, and leaf area development of the plant were not affected when the upper soil zone was dried, but the water potential of the nodules was lower than in the nodules in fully irrigated pots. Nitrogenase activity in the nodules obtained from plants stressed in the upper zone only was lower than in nodules obtained from fully irrigated plants. The present technique is helpful in distinguishing the direct water stress effects on nitrogen fixation compared to those mediated via photosynthate availability. PMID- 16663810 TI - Chemical and Biophysical Changes in the Plasma Membrane during Cold Acclimation of Mulberry Bark Cells (Morus bombycis Koidz. cv Goroji). AB - The lipid and protein composition of the plasma membrane isolated from mulberry (Morus bombycis Koidz.) bark cells was analyzed throughout the cold acclimation period under natural and controlled environment conditions. There was a significant increase in phospholipids and unsaturation of their fatty acids during cold acclimation. The ratio of sterols to phospholipids decreased with hardiness, primarily due to the large increase in phospholipids. The fluidity of the plasma membrane, as determined by fluorescent polarization technique, increased with hardiness. Electrophoresis of plasma membrane proteins including glycoproteins revealed change in banding pattern during the early fall to winter period. Some of the protein changes could be related to growth cessation and defoliation. However, minor changes in proteins also occurred during the most active period of hardening. Changes in glycoproteins were coincident both with changes in growth stages and with the development of cold hardiness. PMID- 16663811 TI - Characterization of the solubilized plasma membrane ATPase of red beet. AB - The plasma membrane ATP-phosphohydrolase (ATPase) from red beet (Beta vulgaris L.) storage tissue was solubilized with the zwitterionic detergent Zwittergent 3 14 from a plasma membrane-enriched fraction which was extracted with the anionic detergent, sodium deoxycholate. For both the extraction of extraneous proteins by deoxycholate and the solubilization of active plasma membrane ATPase by Zwittergent 3-14, the optimal concentration of detergent was 0.1% (weight per volume) with a detergent to protein ratio of 1.0 (milligram per milligram). The properties of the solubilized ATPase were found to be similar to the membrane bound enzyme with respect to pH optimum, substrate specificity, inhibitor sensitivity, and kinetics of K(+) stimulation. The solubilized ATPase preparation formed a rapidly turning over phosphoenzyme, the breakdown velocity of which was increased in the presence of 50 millimolar KCl. Solubilization with 0.1% Zwittergent 3-14 following extraction with 0.1% deoxycholate resulted in an increase in both ATPase activity and steady state phosphoenzyme level; however, a direct correspondence between the increase in ATPase activity and phosphorylation level did not exist. It is proposed that this discrepancy may be the result of a detergent-mediated modification of kinetic rate constants in the mechanism of the enzyme. PMID- 16663812 TI - Dynamic analysis of water stress of sunflower leaves by means of a thermal image processing system. AB - This paper describes a method for measuring the temperatures of all regions of an intact leaf by using an infrared scanning thermometer at wavelengths between 8 and 14 micrometers combined with a digital image processing system. Pictures obtained every 2 minutes from leaves of sunflower (Helianthus annuus L. cv Large Russian) plants subjected to increasing water stress showed that water deficit develops first at the margins of leaves, accompanied by stomatal closure and increase in temperature. Finally, the temperature of the entire leaf rises 3 to 5 degrees C above that of nonstressed leaves. When transpiration resumed, it did so first at the leaf margins and these proceeded nonuniformly inward.The results of these experiments indicate that there are significant differences in the temperature and water status of different parts of a leaf. This makes it important to determine causes of such behavior and decide in what part of a leaf the temperature and water status should be measured. The thermo-imaging method can be useful in monitoring short term temperature changes occurring in leaves undergoing water, chilling, and other stresses. PMID- 16663813 TI - Induction of heat shock protein messenger RNA in maize mesocotyls by water stress, abscisic Acid, and wounding. AB - Exposure of the excised growing region of the mesocotyl of young corn seedlings to heat shock stimulated the production of specific heat shock proteins and the intensification of synthesis of two proteins with a molecular weight of approximately 70,000. Water stress and abscisic acid also stimulated synthesis of these 70,000-dalton proteins, and other unique proteins distinct from those induced by heat shock. Growing tissues of intact corn mesocotyls exposed to heat shock, water stress, or abscisic acid accumulated mRNA species homologous to a cloned genomic probe of the 5' end of the 70,000-dalton Drosophila heat shock protein gene. Since cut segments of the mesocotyl under unstressed conditions produced a similar mRNA, we suggest that the hsp 70 gene is activated in corn by a variety of diverse stresses. Production of the mRNA is rapid, but transient, being induced within 3 hours of the imposition of the stress, but declining after reaching a maximum at 9 hours. PMID- 16663814 TI - Phycomyces: an increase in mechanical extensibility after a humidified wind stimulus. AB - The mechanical extensibilities of stage IVb Phycomyces were measured before and after a humidified wind stimulus. We find that when the humidity of the wind is greater than that of the ambient air, there is an increase in the mechanical extensibility of the cell wall. We also find that a step decrease in wind humidity results in a decrease in the mechanical extensibility of the cell wall. PMID- 16663815 TI - Pyruvate orthophosphate dikinase mRNA organ specificity in wheat and maize. AB - Polyadenylated RNA was isolated from leaves and seeds of a C(3) plant (Triticum aestivum L. cv Cheyenne, CI 8885) and from a C(4) plant (Zea mays L. cv Golden bantam). Each polyadenylated RNA preparation was translated in vitro with micrococcal nuclease-treated reticulocyte lysate. When the in vitro translation products were probed with antibodies to pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1), two sizes of polypeptide were identified. A 110 kilodalton polypeptide was found in the in vitro translation products of mRNA isolated exclusively from leaves of both wheat and maize. A 94 kilodalton polypeptide, similar to the PPDK polypeptide which can be extracted after in vivo synthesis in maize and wheat leaves and seeds, was found in the in vitro translation products obtained from wheat seeds and maize kernels.These results indicate that the mRNAs for PPDK polypeptides are organ-specific in both a C(4) and a C(3) plant. Hague et al. (1983 Nucleic Acids Res 11: 4853-4865) proposed that the larger size polypeptide of the in vitro translation polypeptide from maize leaf RNA contains a ;transit sequence' which permits entry into the chloroplasts of a polypeptide synthesized in vivo in maize leaf cell cytoplasm. It appears that in wheat leaves also the transit of synthesized PPDK polypeptide through an intracellular membrane may be required, while such a transit sequence seems not to be required within cells of wheat and maize seeds. PMID- 16663816 TI - Red-far red reversibility of anthocyanin synthesis in dark-grown and light pretreated seedlings. AB - The rate of escape from red-far red reversibility for anthocyanin synthesis is lower in dark-grown than in light-pretreated seedlings of Brassica oleracea L. Red Acre, Secale cereale L. Cougar, and Lycopersicon esculentum Mill. Beefsteak. This observation is consistent with the suggestion that there might be two pools of phytochrome, a labile one, characterized by fast disappearance of phytochrome far-red absorbing form, and a stable one, characterized by slow disappearance of phytochrome-far-red absorbing form, and that the proportion between the two pools might be different in dark-grown and light-pretreated seedlings. PMID- 16663817 TI - Metabolic response of river birch and European birch and European birch roots to hypoxia. AB - Flood tolerance of woody plants has been attributed to internal oxygen diffusion from shoot to root, metabolic adaptation within the root, or both. The purpose of this study was to compare several biochemical and physiological responses of birch roots to hypoxia in order to determine the nature of root metabolic adaptation to low oxygen tension. One-year-old seedlings of flood-tolerant river birch (Betula nigra L.) and flood-intolerant European birch (Betula pendula Roth) were transferred to solution culture, and the solutions were bubbled with air or nitrogen. After 18 days of hypoxia, total adenosine phosphate and ATP contents of river birch roots were 35% and 23% of controls, respectively, whereas those of European birch roots were 13% and 8%. Adenylate energy charge of river birch roots decreased between 6 and 12 days of hypoxia. In contrast, energy charge of European birch roots decreased after only 1 day of hypoxia. In vitro activity of cytochrome c oxidase and oxygen consumption capacity of excised roots from both birch species decreased under hypoxia. In vitro activity of alcohol dehydrogenase from roots of both species increased after 1 day of hypoxia. However, alcohol dehydrogenase activity from river birch roots increased 25-fold after 6 days of hypoxia, whereas that from European birch decreased back to control levels. Hypoxia decreased malate content of roots from both species. Metabolic adaptation within the root, rather than internal oxygen diffusion, appears to be responsible for the relative tolerance of river birch to hypoxia. PMID- 16663818 TI - Effects of a hydroxamate siderophore on iron absorption by sunflower and sorghum. AB - When Fe was supplied at 100 micromolar in nutrient solution of pH 7.5, 10 and 1 micromolar levels of the siderophore desferrioxamine B (DFOB), a microbial iron transport compound, significantly (alpha = 0.05) enhanced growth and reduced chlorosis of an Fe-inefficient variety of sorghum (Sorghum bicolor L.). Although significantly adverse effects resulted when both Fe and desferrioxamine B (DFOB) were added at 100 micromolar as FeDFOB, the plants were relatively healthy when grown with 100 micromolar DFOB plus 200 micromolar Fe. It was concluded that sorghum absorbed Fe from the pool of nonchelated, solubilized Fe, and utilized DFOB as a shuttle agent, in equilibrium with this pool, to transport Fe from finely suspended solid phase Fe particles to the membrane of absorbing root cells.In contrast to sorghum, absorption of Fe by the Fe-efficient species sunflower (Helianthus annuus L.) was related to the level of FeDFOB and independent of the level of solubilized, nonchelated Fe. The latter was decreased whenever the concentration of DFOB was equal to or greater than the concentration of total Fe. For an Fe concentration of 10 micromolar, significantly larger and greener plants were obtained when DFOB was present at 1, 10, or 100 micromolar than in the absence of DFOB. When grown with 100 micromolar FeDFOB, sunflower plants appeared larger and less chlorotic than those supplied with 100 micromolar Fe and no DFOB. Sunflower apparently was able to utilize FeDFOB more directly than was sorghum. It is suggested that sunflower acquires Fe after binding FeDFOB at membrane sites and/or by producing sufficient reductants in the rhizosphere to reduce biologically significant levels of Fe(III)DFOB to the less stable Fe(II)DFOB. PMID- 16663819 TI - Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb. AB - myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N ethylmaleimide inhibition by substrate and Mg(2+) together suggests sulfhydryl involvement at the active site. PMID- 16663820 TI - Termination of nutrient import and development of vein loading capacity in albino tobacco leaves. AB - The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with (14)CO(2) and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [(14)C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves. PMID- 16663821 TI - Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm. AB - The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60 fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6 bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6 bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6 bisphosphate and the other effectors in regulating FBPase are discussed. PMID- 16663822 TI - A development-dependent hemagglutinin from cucumber surfaces: I. Hemagglutinin activity of cotyledon surfaces. AB - During studies on the chemistry of plant surfaces, we observed that concomitant with the development of cucumber (Cucumis sativus L. cv ;ELEM') cotyledons an agglutinin that agglutinates human erythrocytes appeared on epicuticular surfaces. The agglutinin was released from cotyledon surfaces into distilled water by a 1-minute immersion (or even less). Homogenization of the washed cotyledons released residual agglutinating activity. The surface-located hemagglutinating activity was age-dependent and occurred in dark- and light-grown cotyledons. Agglutinating activities were present in light-grown cotyledons of 2- to 14-day-old seedlings and in dark-grown cotyledons of 3- to 17-day-old seedlings. Agglutinating activities were maximal in cotyledons of 3- to 4-day-old seedlings. No activity could be detected in dry seeds or in seedlings up to 2 days and after 17 days of germination. The hemagglutinating activity was specifically inhibited by N,N',N''-triacetylchitotriose, chitin, and chitosan. PMID- 16663823 TI - Effects of n(2) deficiency on transport and partitioning of C and N in a nodulated legume. AB - Nodulated root systems of white lupin (Lupinus albus L. cv Ultra: Rhizobium strain WU425) were exposed to Ar:O(2) (80:20, v/v) or Ar:N(2):O(2) (70:10:20, v/v/v) and C and N partitioning were examined over a 9- or 10-day period in comparison with control plants with nodulated roots retained in air. Accumulation of N ceased in plants exposed to Ar:O(2) or was much reduced in plants exposed to Ar:N(2):O(2), but net C assimilation rates and profiles of C utilization remained similar to those of control N(2)-fixing plants. There was, however, a proportional reduction in CO(2) evolution from nodulated roots of the Ar:O(2) treatment. Xylem N levels fell rapidly after application of Ar:O(2). C:N ratios of phloem sap of petioles and of stem base rose during the first day of Ar:O(2) treatment and then fell progressively back to levels close to that of control plants as leaf reserves of N became available for loading of phloem. Stem top phloem sap increased progressively in C:N ratio throughout Ar:O(2) treatment, presumably due to increasing shortage of xylem derived N for xylem to phloem exchange. Reexposure of Ar:O(2)-treated nodulated root systems to air prompted a rapid recovery of N(2) fixation and restoration of plant N status. Rates of N(2) fixation in plants whose roots were exposed to a range of N(2) concentrations indicated an apparent K(m) of 10% N(2) for the attached intact white lupin nodule. PMID- 16663824 TI - Hydrogen, oxygen, and carbon isotope ratios of cellulose from submerged aquatic crassulacean Acid metabolism and non-crassulacean Acid metabolism plants. AB - Isotope ratios of cellulose and cellulose nitrate from aquatic Crassulacean acid metabolism (CAM) and non-CAM plants were determined. Cellulose oxygen istope ratios for all plants that grew together were virtually identical, whereas large differences were observed for hydrogen isotope ratios of cellulose nitrate between CAM and non-CAM plants. Carbon isotope ratios of cellulose nitrate did not differentiate CAM from non-CAM plants. PMID- 16663825 TI - Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium. AB - Cell-free extracts of the unicellular red alga Cyanidium caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low molecular weight components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation, and mesobiliverdin cannot serve as a substrate.Incubation of cell extracts with biliverdin yields two products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same two products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The two products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliverdin transformation is therefore the Z ethylidine isomer. The time course for formation of the two products during incubation suggests that the early-eluting product is the precursor of the late eluting one. These results suggest that Z-ethylidine phycocyanobilin is the precursor of the E-ethylidine isomer, and that the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore. PMID- 16663826 TI - Inactivation of Glutamine Synthetase by Tabtoxinine-beta-lactam : Effects of Substrates and pH. AB - The inactivation of glutamine synthetase by tabtoxinine-beta-lactam, a phytotoxin produced by Pseudomonas syringae pv. tabaci, was shown to be irreversible. The chloroplast and cytosolic forms of the enzyme from pea leaves (Pisum sativum L.) were separated, purified, and found to be kinetically similar with K(m) values for glutamate of 6.7 and 4.3 millimolar and for ATP of 2.0 and 1.3 millimolar, respectively. Both forms were irreversibly inactivated by the toxin at equal rates. Using the chloroplast form, it was found that inactivation by tabtoxinine beta-lactam required ATP. Glutamate and low levels of ammonia (<2 millimolar) slowed the rate of inactivation, whereas high levels of ammonia (5, 20, and 50 millimolar) accelerated it. The inactivation proceeded at a faster rate as the pH was increased from pH 6.5 to 7.5. The role which cellular compartmentalization could play in the inactivation is discussed. PMID- 16663827 TI - Amine Transport in Riccia fluitans: Cytoplasmic and Vacuolar pH Recorded by a pH Sensitive Microelectrode. AB - The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH(4)Cl) and methylamine (CH(3)NH(3)Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode.On addition of 1 micromolar NH(4)Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH(4)Cl or 500 micromolar CH(3)NH(3)Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH(4) (+)/CH(3)NH(3) (+) uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH(4) (+); and second, alkalinization through protonation of NH(3) which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems. PMID- 16663828 TI - Effect of RNase on Zein Synthesis in Endosperms of brittle-2opaque-2 Maize Double Mutant. AB - Posttranscriptional degradation of zein mRNAs in Zea mays, presumably caused by high RNase activity, may be involved in reducing zein synthesis in the endosperms of zein-deficient mutants. Zein proteins prepared from mature endosperms of zein deficient double mutants such as brittle-2;opaque-2 showed an almost complete blockage of the synthesis of two major zein components, Z1 and Z2. However, these two components were present prior to 12 days postpollination. The changes in quantity and quality of zein synthesis in these double mutants correlated well with the rising RNase levels in the developing endosperm. In addition to its effect on zein synthesis, high RNase activity also influenced the stability of rRNAs in the brittle-2;opaque-2 double mutant at the later stages of endosperm development. These results suggest that RNase played a role in regulating zein synthesis in developing zein-deficient mutant endosperms. PMID- 16663829 TI - Levels of Endogenous Indole-3-acetic Acid in Achenes of Rosa rugosa during Dormancy Release and Germination. AB - Indole-3-acetic acid (IAA) in highly purified extracts of rose achenes (Rosa rugosa var rubra) was quantified by means of ion-pair reversephase high performance liquid chromatography with spectrofluorimetric detection. Changes in IAA content were determined during a 14-week 4 degrees C stratification, which leads to dormancy breakage, and during subsequent germination at 17 degrees C. IAA was also determined in achenes stratified in parallel at 17 degrees C, which does not induce release from dormancy. IAA decreased during the first 2 weeks of stratification both at 4 degrees C and at 17 degrees C. IAA remained low during the remaining 12 weeks of stratification at 4 degrees C, whereas it continued to decrease in achenes kept at 17 degrees C. An immediate increase in IAA during germination was followed by transients in the IAA level. The results suggest that IAA is without a regulating role in dormancy release although it seems to be involved in the germination process. PMID- 16663830 TI - Effect of a Brief CO(2) Exposure on Ethylene Production. AB - Ethylene production and respiration by Granny Smith apples were inhibited by treatment with 20% CO(2) for 2 hours. A similar effect was observed in tissue slices when treated at either 0 or 25 degrees C.The inhibition continued even after an extended aeration period. There is also an inhibition of ethylene emission in tissue slices incubated with exogenous 1-aminocyclopropane-1 carboxylic acid (ACC).In general, CO(2) treatment increased the ACC content of the tissue. These observations are consistent with the idea the action of CO(2) is directed toward the enzyme system responsible for the conversion of ACC into ethylene. PMID- 16663831 TI - Characteristics of a beta-Galactosidase Associated with the Stroma of Chloroplasts Prepared from Mesophyll Protoplasts of the Primary Leaf of Wheat. AB - Chloroplasts prepared from mesophyll protoplasts of the primary leaf of wheat (Triticum aestivum L. cv Egret) contain about 50% of the cellular beta galactosidase (EC 3.2.1.23) activity. More than 80% of this activity is associated with the stroma and most of the remainder, although tightly bound to the thylakoids, can be washed free with sodium pyrophosphate. The vacuole contained about 20% and the remaining enzyme was presumed to be cytoplasmic or associated with one of the other organelles. Both the vacuolar and chloroplast enzymes were capable of releasing galactose from the galactolipid monogalactosyldiacylglycerol. Apart from their distinct locations within the cells, we conclude that the enzymes are different because they differed with respect to assay pH-optimum, comparative activity against the synthetic substrates phenyl-beta-d-galactoside, 4-methylumbelliferyl-beta-d-galactoside, 6 bromo-2-naphthyl-beta-d-galactoside, the disaccharide lactose, and the inhibitors d-galactose and d-galactono-1,4-lactone. PMID- 16663832 TI - Selective Toxins and Analogs Produced by Helminthosporium sacchari: Production, Characterization, and Biological Activity. AB - Helminthosporium sacchari toxin and several lower molecular weight, nontoxic analogs were isolated from culture filtrates. Three isomers of the toxin (A, B, and C), each with four galactose units, were separated by high performance liquid chromatography. Isomer C had the highest and isomer A had the lowest toxicity to H. sacchari-susceptible sugarcane; resistant clones were not affected. Each toxin isomer was partially digested with a beta-galactofuranosidase and the resulting analogs (seven from each toxin isomer) were separated by reverse phase high performance liquid chromatography and identified. Each isomer of the analogs with 3 galactose units per mole also was partially digested and the arrangement of galactose units was determined. The compound with one galactose attached to position 2 of the bicyclic sesquiterpene and with 2 galactose units attached to position 13 (analog A(1,2)) was highly toxic to some but not to all clones of H. sacchari-susceptible sugarcane. Toxin analogs protected sensitive tissue against active toxin; protective effects of the analogs differed, but at least a 10-fold excess of analog was required. Analog C(2,1) was more effective at preventing toxin C-induced electrolyte losses than was any other analog. Each of the 3 galactose analog isomers protected better than did any of the 2-galactose compounds. The 1,1 analogs did not protect as well as did the 2,0 or 0,2 analogs. Thus, the sesquiterpene isomer, the number of galactose units, and the galactose arrangement pattern determine the effectiveness of the compound in induction of electrolyte loss and in prevention of toxininduced loss from sugarcane tissues. PMID- 16663833 TI - Threonine Synthase of Lemna paucicostata Hegelm. 6746. AB - Threonine synthase (TS) was purified approximately 40-fold from Lemna paucicostata, and some of its properties determined by use of a sensitive and specific assay. During the course of its purification, TS was separated from cystathionine gamma-synthase, establishing the separate identity of these enzymes. Compared to cystathionine gamma-synthase, TS is relatively insensitive to irreversible inhibition by propargylglycine (both in vitro and in vivo) and to gabaculine, vinylglycine, or cysteine in vitro. TS is highly specific for O phospho-l-homoserine (OPH) and water (hydroxyl ion). Nucleophilic attack by hydroxyl ion is restricted to carbon-3 of OPH and proceeds sterospecifically to form threonine rather than allo-threonine. The K(m) for OPH, determined at saturating S-adenosylmethionine (AdoMet), is 2.2 to 6.9 micromolar, two orders of magnitude less than values reported for TS from other plant tissues. AdoMet markedly stimulates the enzyme in a reversible and cooperative manner, consistent with its proposed role in regulation of methionine biosynthesis. Cysteine (1 millimolar) caused a slight (26%) reversible inhibition of the enzyme. Activities of TS isolated from Lemna were inversely related to the methionine nutrition of the plants. Down-regulation of TS by methionine may help to limit the overproduction of threonine that could result from allosteric stimulation of the enzyme by AdoMet.No evidence was obtained for feedback inhibition, repression, or covalent modification of TS by threonine and/or isoleucine. PMID- 16663834 TI - Deferral of senescence and abscission by chemical inhibition of ethylene synthesis and action in bean explants. AB - Three compounds known to inhibit ethylene synthesis and/or action were compared for their ability to delay senescence and abscission of bean explants (Phaseolus vulgaris L. cv Contender). Aminoethoxyvinyl-glycine (AVG), AgNO(3), and sodium benzoate were infiltrated into the petiole explants. Their effect on abscission was monitored by measuring the force required to break the abscission zone, and their effect on senescence was followed by measuring chlorophyll and soluble protein in the distal (pulvinus) sections. AVG at concentrations between 1 and 100 micromolar inhibited ethylene synthesis by about 80 to 90% compared to the control during sampling periods of 24 and 48 hours after treatment. This compound also delayed the development of abscission and senescence. Treatment with AgNO(3) at concentrations between 1 and 100 micromolar progressively reduced ethylene production, but to a lesser extent than AVG. The effects of AgNO(3) on senescence and abscission were quite similar to those of AVG. Sodium benzoate at 50 micromolar to 5 millimolar did not inhibit ethylene synthesis during the first 24 hours, but appreciably inhibited ethylene synthesis 48 hours after treatment. It also delayed the development of abscission and senescence. The effects of AVG, Ag(+), and sodium benzoate suggest that ethylene could play a major role in both the senescence induction phase and the separation phase in bean explants. PMID- 16663835 TI - Rhythmical changes in the sensitivity of cotton seedlings to herbicides. AB - Cotton (Gossypium hirsutum L.) seedlings that were grown under a photoperiod of 12 hours darkness and 12 hours light showed oscillations in their sensitivity to the herbicides sodium 5-(2-chloro-4-trifluoromethyl)-phenoxy)-2-nitrobenzoate (acifluorfen), butyl 2-(4-((5-(trifluoromethyl)-2 pyridinyl)oxy)phenoxy)propanoate (fluazifop) and 3-isopropyl-1H-2,1,3 benzothiadiazin-4(3H)-one 2,2-dioxide (bentazon). Sensitivity was expressed in appearance of necrotic areas on the cotyledons and in decreased growth of the shoot. The seedlings were least sensitive in the beginning and middle of the light period, then the sensitivity increased and reached its maximum during the beginning and middle of the dark period and then declined. Seedlings grown from germination under continuous light exhibited very small or no oscillations in sensitivity. The oscillations in sensitivity were entrained by one cycle of darkness and light. A cycle of 12 hours darkness and 12 hours light triggered the greatest oscillations while either increasing or decreasing the duration of the dark period resulted in smaller oscillations. Apparently, these oscillations in sensitivity to herbicides were endogenously controlled since after entrainment they continued irrespective of the light conditions. PMID- 16663836 TI - Abscisic Acid and its relationship to seed filling in soybeans. AB - The effect of exogenous abscisic acid (ABA) on the rate of sucrose uptake by soybean (Glycine max L. Merr.) embryos was evaluated in an in vitro system. In addition, the concentrations of endogenous ABA in seeds of three soybean Plant Introduction (PI) lines, differing in seed size, were commpared to their seed growth rates. ABA (10(-7) molar) stimulated in vitro sucrose uptake in soybean (cv ;Clay') embryos removed from plants grown in a controlled environment chamber, but not in embryos removed from field-grown plants of the three PI lines. However, the concentration of ABA in seeds of the three field-grown PI lines correlated well with their in situ seed growth rates and in vitro [(14)C] sucrose uptake rates.Across genotypes, the concentration of ABA in seeds peaked at 8.5 micrograms per gram fresh weight, corresponding to the time of most rapid seed growth rate, and declined to 1.2 micrograms per gram at physiological maturity. Seeds of the large-seeded genotype maintained an ABA concentration at least 50% greater than that of the small-seeded genotype throughout the latter half of seed filling. A higher concentration of ABA was found in seed coats and cotyledons than in embryonic axes. Seed coats of the large-seeded genotype always had a higher concentration of ABA than seed coats of the small-seeded line. It is suggested that this higher concentration of ABA in seed coats of the large-seeded genotype stimulates sucrose unloading into the seed coat apoplast and that ABA in cotyledons may enhance sucrose uptake by the cotyledons. PMID- 16663837 TI - Sugar regulation of plastid interconversions in epicarp of citrus fruit. AB - Seasonal transformations between chloroplasts and chromoplasts, as measured by changes in chlorophyll content, in the epicarp of degreening and regreening Citrus sinensis (L.) Osbeck cv Valencia fruit closely parallelled the accumulation and later loss of soluble sugars. At any stage of development, reversing the relative soluble sugar content in the epicarp by culturing pericarp segments on agar media with low (15 millimolar) or high (150 millimolar) sucrose concentrations reversed the direction of change in chlorophyll content. Fruit of C. madurensis Lour., which mature year around and do not regreen, also accumulated soluble sugars in the pericarp as degreening was initiated.The epicarp of C. sinensis fruit accumulated nitrogen, but total nitrogen concentrations and amino acid concentrations changed little, during degreening and regreening of C. sinensis fruit. Cessation of nitrogen fertilization reduced the tendency of pericarp segments to regreen in vitro during subsequent years, but regreening tendency was restored by inclusion of KNO(3) in the media.It is concluded that chloroplasts become chromoplasts and citrus fruit degreen partially in response to the accumulation of sugars in the epicarp and that the reverse transformation accompanying regreening of certain citrus species occurs when accumulated sugars disappear. Change in nitrogen flux to the fruit is probably not a factor in regulating seasonal transformations, but an abundance of nitrogen in the epicarp diminishes the effects of high sugar concentrations in inducing transformation of chloroplasts to chromoplasts, thereby retarding degreening and promoting regreening. PMID- 16663838 TI - Preferential Leaching of Pinitol from Soybeans during Imbibition. AB - Sugars and cyclitols leached from soybeans (Glycine max var Sparks) during imbibition were assayed as a function of time. Pinitol leached many times faster than carbohydrates. During the initial 20 minutes of imibition, the pinitol/carbohydrate ratio was 3.4, declining to 0.29 for fully imbibed seeds. The value for dry soybeans was 0.14. Hypochlorite treatment of seeds more than doubled the rate at which carbohydrates leached out, but had little effect on pinitol. A role in development of soil microorganisms is postulated for pinitol. PMID- 16663839 TI - Pyrophosphate and fructose 2,6-bisphosphate effects on glycolysis in pea seed extracts. AB - The participation of pyrophosphate-dependent phosphofructokinase (PPi-PFK) in plant glycolysis was examined using extracts from pea seeds (Pisum sativum L. cv Alaska). Glycolysis starting with fructose 6-phosphate was measured under aerobic conditions as the accumulation of pyruvate. Pyruvate accumulated in a medium containing PPi and adenosine diphosphate at about two-thirds of the rate in a medium containing adenosine diphosphate and adenosine triphosphate (ATP). The PPi dependent pyruvate accumulation had the same reactant requirements and sensitivity to glycolysis inhibitors, sodium fluoride, and iodoacetamide, as the well-established ATP-dependent glycolysis. Added fructose 2,6-bisphosphate stimulated both the PPi-dependent pyruvate accumulation and PPi-PFK activity whereas this modulator had no effect on ATP-dependent glycolysis or ATP-PFK. Collectively these results demonstrate a PPi-dependent glycolytic pathway in plants which is responsive to fructose 2,6-bisphosphate. PMID- 16663840 TI - Early effects of salinity on nitrate assimilation in barley seedlings. AB - The effect of NaCl and Na(2)SO(4) salinity on NO(3) (-) assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO(3) (-) transporter was affected very little; the major effect of the salts was on its activity. Both Cl(-) and SO(4) (2-) salts severely inhibited uptake of NO(3) (-). When compared on the basis of osmolality of the uptake solutions, Cl( ) salts were more inhibitory (15-30%) than SO(4) (2-) salts. At equal concentrations, SO(4) (2-) salts inhibited NO(3) (-) uptake 30 to 40% more than did Cl(-) salts. The absolute concentrations of each ion seemed more important as inhibitors of NO(3) (-) uptake than did the osmolality of the uptake solutions. Both K(+) and Na(+) salts inhibited NO(3) (-) uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.Unlike NO(3) (-) uptake, NO(3) (-) reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO(3) (-) in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the ;anaerobic' in vivo assay, was always greater than the actual in situ rate of NO(3) (-) reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO(3) (-) transporter, rather than that of the NO(3) (-) reduction system, may be a critical factor to plant survival during salt stress. PMID- 16663841 TI - Gibberellin A(4/7) and the Promotion of Flowering in Pinus radiata: Effects on Partitioning of Photoassimilate within the Bud during Primordia Diferentiation. AB - Gibberellin A(4/7) mixture (GA(4/7)), a highly effective promoter of early and enhanced flowering in the Pinaceae, caused a significant reallocation of dry matter and (14)C-photosynthate within terminal buds of Pinus radiata D. Don within 8 days of hormone treatment. Treatment with GA(4/7) to terminal shoots of vigorous, potentially flowering mature grafted propagules reduced the flow of photoassimilated (14)C and dry matter into the terminal bud as a whole, but significantly increased the dry matter and (14)C allocated within the bud to developing long-shoot primordia (potential seed-cone buds). This was accomplished at the expense of the structural tissues, the apical dome region, and the vegetative branch buds. Although GA(3) caused a similar reallocation of dry matter within the terminal bud, it was significantly less effective than GA(4/7) thus appears to have, in addition to any nutrient diversion abilities, a distinct morphogenic function in sexual differentiation. PMID- 16663842 TI - Physiological control of arginine decarboxylase activity in k-deficient oat shoots. AB - The effect of K-deficiency on the putrescine biosynthetic enzyme, arginine decarboxylase (ADC), was investigated by growing oat (Avena sativa L. var Victory) plants on a low-K, but otherwise complete nutrient medium in washed quartz sand for up to 18 days. Enzyme activity rose as the concentration of KCl was dropped to 0.6 millimolar or below. However, growth was not inhibited significantly at 0.6 millimolar KCl. ADC activity increased in the whole shoot of K-deficient oats throughout the period of 6 to 18 days, but remained constant in normal plants. At 18 days, ADC activity in entire K-deficient shoots was 6 times greater than in normal shoots, while in the first (oldest) leaf, ADC specific activity increased to more than 30 times the specific activity in the first leaf of normal plants. This effect was due to a moderate rise in total ADC activity in the first leaf between 6 and 18 days, accompanied by a significant decline in protein content. Replacing K(+) with Na(+) or Li(+) significantly inhibited the increase in ADC activity in K-deficient oats, while Rb(+) depressed the specific activity to a level below that in normal plants. An alternative putrescine biosynthetic enzyme, ornithine decarboxylase, was also examined. The specific activity of a pelletable form of the enzyme was increased 2-fold in the shoots of K-deficient oats. PMID- 16663843 TI - Influx and efflux of p in roots of intact maize plants : double-labeling with p and p. AB - Rates of P influx and efflux were determined in whole plants at ambient P concentrations comparable to those found in soil solutions. Maize (Zea mays L. var NC+59) seedlings were trimmed (endosperm and adventitious roots removed) and grown in a greenhouse in solution cultures at P concentrations of approximately 0.4 and 1.8 micromolar. Roots of intact plants previously exposed to (32)P labeled solutions at 0.2 and 2.0 micromolar P for 48 hours were rinsed 10 minutes in P-free solution and exposed to (33)P solutions at 0.2 and 2.0 micromolar for 10 minutes. Net depletion of (33)P from and appearance of (32)P in the ambient solution were used to measure influx and efflux. The ration of (32)P efflux to (33)P influx was about 0.68 at 0.2 micromolar and 0.08 at 2.0 micromolar. When plants were allowed to deplete P from solutions, the P concentration in the medium dropped to about 0.15 micromolar within 24 hours and 0.05 micromolar within 60 hours. Results indicate that P efflux is a substantial component of net P accumulation at P concentrations normally found in soil solutions. PMID- 16663844 TI - Role of calcium in the polar secretion of indoleacetic Acid. AB - The rate of auxin transport in sunflower hypocotyls (Helianthus annuus L. cv ;Russian mammoth') or corn coleoptiles (Zea mays L. cv ;WF9 x 38') was less in seedlings grown in Ca-deficient medium than in controls. The rate of IAA transport depended on the concentration of Ca in the root medium up to 1 millimolar. Further increases in auxin transport were observed when the isolated segments were incubated in medium containing up to 30 millimolar Ca. We suggest that the rate of auxin transport in plant tissue is dependent on the pool of ionic Ca in the extracellular space.Segments from Ca-deficient seedlings exhibited a high specific requirement for Ca(2+) in auxin transport. Magnesium, strontium, and several other divalent cations tested for their ability to replace Ca(2+) in restoring auxin transport showed no effect; partial replacement by lanthanum was observed.Auxin transport, or auxin flux through the segment, which is the result of IAA secretion by individual cells, was reduced in the low Ca(2+) segments due both to lowered velocity and to reduced capacity of transport. The requirement for Ca(2+) in the secretion of auxin is believed to be equivalent to the phenomenon observed in animal cell secretion, where the influx of Ca(2+) serves as a link between an external stimulus and the secretion response. PMID- 16663845 TI - Polar calcium flux in sunflower hypocotyl segments : I. The effect of auxin. AB - The flux of Ca(2+) at the apical or basal ends of short sunflower (Helianthus annuus L.) hypocotyl segments was monitored using a Ca(2+)-specific electrode. A higher Ca(2+) efflux was observed at the apical end relative to the basal end, indicating a net polar flux of Ca(2+). The extreme low mobility of Ca(2+) in the isolated segment makes it likely that the observed Ca(2+) fluxes are of localized origin, that is, from the parenchyma cells close to the exposed cut ends and may represent acropetal transport of Ca(2+) at the cellular level. The rate of Ca(2+) efflux depended on the concentration of Ca in the seedling medium. Incubation of hypocotyl segments in 10 mm CaCl(2) for 24 h did not eliminate the net acropetal flux of Ca(2+) at the apical end.IAA, as well as the synthetic auxin alpha naphthaleneacetic acid, significantly enhanced Ca(2+) efflux; the non-auxin analog, beta-naphthaleneacetic acid, was ineffective. The transport of auxin, not merely its presence in the medium, was found to be a requisite for the enhancement of Ca(2+) efflux since the presence of the auxin transport inhibitor 2,3,5-triiodobenzoic acid eliminated the auxin-promoted Ca(2+) efflux. A model for how auxin promotion of Ca(2+) efflux could play a role in promoting subsequent auxin secretion is proposed. Calcium probably serves as a ;second messenger', as it does in the secretion of various substances by animal cells. PMID- 16663846 TI - Calcium- and calmodulin-regulated phosphorylation of soluble and membrane proteins from corn coleoptiles. AB - In vitro phosphorylation of several membrane polypeptides and soluble polypeptides from corn (Zea mays var. Patriot) coleoptiles was promoted by adding Ca(2+). Ca(2+)-promoted phosphorylation of the membrane polypeptides was further increased in the presence of calmodulin. Both Ca(2+)-stimulated and Ca(2+)- and calmodulin-stimulated phosphorylations of membrane polypeptides were inhibited by chlorpromazine, a calmodulin antagonist. Ca(2+)-stimulated phosphorylation of soluble polypeptides increased with increasing Ca(2+) concentration. The calmodulin antagonists chlorpromazine and trifluoperazine inhibited the Ca(2+) promoted phosphorylation of soluble polypeptides. Added calmodulin promoted the Ca(2+)-dependent phosphorylation of a 98 kilodaltons polypeptide. Both Ca(2+) dependent and Ca(2+)-independent phosphorylations required Mg(2+) at an optimal concentration of 5 to 10 millimolar. Cyclic AMP was found to have no stimulatory effect on protein phosphorylation. Sodium molybdate, an inhibitor of protein phosphatase, increased the net phosphorylation of several polypeptides. Rapid loss of radioactivity from the phosphorylated polypeptides following incubation in unlabeled ATP indicated the presence of phosphoprotein phosphatase activity. PMID- 16663847 TI - Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation. AB - Active polyamine metabolism occurs in Glycine max (L.) seeds during development. Most (>/=97%) of putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad) are present as free forms in the growing embryo. In the cotyledon or embryonic axis, Put decreases to a nearly undetectable level, while Spd level sharply increases as seed dry weight accumulation progresses. Spm level in the axis also increases along with the Spd level. There is little change in Spm level in the cotyledons. Maturation and dehydration results in a slight reduction of Spd level in the cotyledons. Cad is present in relatively large quantities (5.5-12 micromoles per gram dry weight) in the axes of mature soybean seeds. Only traces of Cad, as expressed on a dry weight basis, are found in the developing or mature cotyledons. The synthesis and accumulation of Cad in the axis begins at the time when the axis or the seed accumulates 30 to 50% of its maximum dry weight. The Cad accumulation (0.8 nanomole per axis per day) proceeds until the later stages of dehydration. When soybean plants are subjected to complete defoliation and shade during the midpoint of seed maturation, Cad accumulation in the axis and seed dry weight accumulation ceased almost immediately. The treatment, however, does not affect the viability of soybean seeds. PMID- 16663848 TI - Polyamine Anabolism in Germinating Glycine max (L.) Seeds : Dynamics of Cadaverine and Putrescine Formation in the Embryonic Axis. AB - Active polyamine biosynthesis occurs in the embryonic axis, but not in the cotyledons, during germination of Glycine max (L.) cv Williams seeds and subsequent growth of the young seedlings. The hypocotyl and radicle synthesize and accumulate considerable amounts of cadaverine (Cad) and putrescine (Put) during the early stages of growth. Most of the amino acid precursors for the diamines are supplied from breakdown of the cotyledonary protein.In the axis, Cad synthesis which is catalyzed by l-lysine decarboxylase precedes the onset of growth (dry weight accumulation) of the axis and its accumulation continues as the growth progresses. After 2 days of imbibition, Cad is synthesized and accumulated at 37.4 nanomoles per axis per hour for at least 6 days. The rate then gradually decreases as the supply of free l-lysine from the cotyledons diminishes. Approximately 40 to 50% of the lysine resulting from breakdown of the cotyledonary protein ends up in Cad in the hypocotyl and radicle. Cad represents about 3.5% of the axis nitrogen derived from the cotyledons, which is equivalent to about 50% of the lysine content ( approximately 7%) of the seed protein.The synthesis and accumulation of Put in the axis also precedes the onset of growth of the axis. Both l-arginine decarboxylase and l-ornithine decarboxylase are involved in catalyzing the Put formation. The increased content of spermidine (Spd), but not spermine (Spm), parallels growth of the axis. Spm is maintained at a nearly undetectable level. After 2-day imbibition, Put and Spd are synthesized and accumulated at 0.94 and 0.17 nanomoles per axis per hour, respectively. The rate of Put accumulation, like that of Cad, decreases about 8 days after imbibition. The hypocotyl and radicle contain millimolar concentrations of Cad and Put which are primarily associated with the elongated zones. In contrast, Spd level or the molar ratio of Spd:Put appears to decrease as cell differentiation or elongation progresses. PMID- 16663849 TI - Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye. AB - Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20 degrees C (nonhardened rye, RNH) or 5 degrees C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20 degrees C indicated that RNH and RH thylakoids contained the same proportions of fast (alpha) and slow (beta) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for alpha components increased and the rate constant for beta components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5 degrees C. PMID- 16663850 TI - Redox activity at the surface of oat root cells. AB - Electron transport activity at the cell surface of intact oat seedlings (Avena sativa L. cv Garry) was examined by measuring the oxidation and/or reduction of agents in the medium bathing the roots. Oxidation of NADH with or without added electron acceptors and reduction of ferricyanide by an endogenous electron donor were detected. The activities appear to be due to electron transfer at, or across, the plasma membrane and not due to reagent uptake or leakage of oxidants or reductants. NADH-ferricyanide oxidoreductase activity was also detected in plasma membrane-enriched preparations from Avena roots. Based on redox responses to pH, various ions, and to a variety of electron donors and acceptors, the results indicate that more than one electron transport system is present at the plasma membrane. PMID- 16663851 TI - Stomatal responses to water stress and to abscisic Acid in phosphorus-deficient cotton plants. AB - Cotton (Gossypium hirsutum L.) plants were grown in sand culture on nutrient solution containing adequate or growth-limiting levels of P. When water was withheld from the pots, stomata of the most recently expanded leaf closed at leaf water potentials of approximately -16 and -12 bars in the normal and P-deficient plants, respectively. Pressure-volume curves showed that the stomata of P deficient plants closed when there was still significant turgor in the leaf mesophyll. Leaves of P-deficient plants accumulated more abscisic acid (ABA) in response to water stress, but the difference was evident only at low water potentials, after initiation of stomatal closure. In leaves excised from unstressed plants, P deficiency greatly increased stomatal response to ABA applied through the transpiration stream. Kinetin blocked most of this increase in apparent sensitivity to ABA. The effect of P nutrition on stomatal behavior may be related to alterations of the balance between ABA and cytokinins. PMID- 16663852 TI - Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme. AB - Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH(4) (+) indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH(4) (+) feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH(4) (+)-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.Using (15)N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, (15)N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of (15)N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the (15)N labeling pattern and effects of MSX, NH(4) (+) assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization. PMID- 16663853 TI - Fusicoccin and Air Pollutant Injury to Plants : Evidence for Enhancement of SO(2) but Not O(3) Injury. AB - Garden peas (Pisum sativum L. cv Alsweet) and a tomato mutant (Lycopersicon esculentum Mill. var flacca) were sprayed with fusicoccin, a fungal toxin affecting membrane transport properties, before exposure to SO(2) or O(3). Tomatoes treated with 10 micromolar fusicoccin and exposed to SO(2) (0.6 microliter per liter for 2 hours) exhibited twice as much foliar necrosis as untreated plants exposed to SO(2). Peas treated with fusicoccin and exposed to SO(2) (0.7 to 1.0 microliter per liter for 2 hours) exhibited 2 to 6 times more injury than untreated plants exposed to SO(2). Peas treated with fusicoccin and exposed to O(3) had less injury than untreated plants exposed to O(3) (0.1 to 0.3 microliter per liter for 2 hours). Several lines of evidence suggested that the fusicoccin enhancement of SO(2) injury is not the result of increased gas exchange, i.e. the tomato mutant has permanently open stomata under all conditions, and in peas fusicoccin had no effect on SO(2) or H(2)O flux in plants exposed to 0.12 microliter per liter SO(2). However, a 21% greater leaf conductance in fusicoccin treated versus untreated plants indicated the possibility of some differences in gas exchange for peas exposed to 1.0 microliter per liter SO(2). PMID- 16663854 TI - Reactions of corn root tissue to calcium. AB - Washing corn (Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca(2+) over the first 10 to 15 minutes. Upon transfer to K(+) containing solutions, the tissue shows a short period of rapid K(+) influx which subsequently declines. Addition of 0.1 millimolar Ca(2+) decreases the initial rapid K(+) influx, but increases the sustained rate of K(+) and Cl(-) uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca(2+) is more effective than higher concentrations for the initial inhibition, and that Mg(2+) will substitute.The inhibition arises from a mild shock affect of restoring Ca(2+). With 0.1 millimolar Ca(2+) net H(+) efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca(2+) rapidly produces increased K(+) influx and blocks net H(+) efflux for only a few minutes; blockage is preceded by a brief net H(+) influx which may restore and increase ion transport by reactivating the plasmalemma H(+)-ATPase.Stimulation of electrogenic H(+)-pumping with fusicoccin eliminates the shock responses and minimizes Ca(2+) effects on K(+) influx. Fusicoccin also strongly decreases Ca(2+) influx, but has no effect on Ca(2+) efflux. Ice temperatures and high pH decreased Ca(2+) efflux, but uncoupler and chlorpromazine did not.It is suggested that the inhibitory and promotive actions of Ca(2+) are manifested through decreases or increases in the protonmotive force. PMID- 16663855 TI - Characterization of the transport of oxaloacetate by pea leaf mitochondria. AB - Mitochondria isolated from pea (Pisum sativum L.) leaves are able to transport the keto acid, oxaloacetate, from the reaction medium into he mitochondrial matrix at high rates. The rate of uptake by the mitochondria was measured as the rate of disappearance of oxaloacetate from the reaction medium as it was reduced by matrix malate dehydrogenase using NADH provided by glycine oxidation. The oxaloacetate transporter was identifed as being distinct from the dicarboxylate and the alpha-ketoglutarate transporters because of its inhibitor sensitivities and its inability to interact with other potential substrates. Phthalonate and phthalate were competitive inhibitors of oxaloacetate transport with K(i) values of 60 micromolar and 2 millimolar, respectively. Butylmalonate, an inhibitor of the dicarboxylate and alpha-ketoglutarate transporters, did not alter the rate of oxaloacetate transport. In addition, a 1000-fold excess of malate, malonate, succinate, alpha-ketoglutarate, or phosphate had little effect on the rate of oxaloacetate transport. The K(m) for the oxaloacetate transporter was about 15 micromolar with a maximum velocity of over 500 nanomoles per milligram mitochondrial protein/min at 25 degrees C. No requirement for a counter ion to move against oxaloacetate was detected and the highest rates of uptake occurred at alkaline pH values. An equivalent transporter has not been reported in animal mitochondria. PMID- 16663856 TI - Cross-linking of soluble extensin in isolated cell walls. AB - The extensin component of primary cell walls has generally been considered to be an intrinsically insoluble cell wall glycoprotein. Recent data have established that cell wall extensin is in fact secreted in a soluble monomeric form which slowly becomes insolubilized in the cell wall probably through the oxidative formation of isodityrosine cross-links. We now show that isolated cell walls from aerated root slices of Daucus carota have the capacity to insolubilize extensin through the formation of isodityrosine. This in vitro cross-linking is specific for the extensin glycoprotein, as other wall proteins are not cross-linked by the isolated wall system. Although extensin can be cross-linked in solution by peroxidase and H(2)O(2), dityrosine and not isodityrosine is the phenolic cross link formed. Wall-catalyzed cross-linking of soluble extensin is inhibited by l ascorbate, and both the initial rate and total extent of cross-linking are inhibited by acidic pH in the physiological range (pH 4 to 6). We suggest several mechanisms by which acid might inhibit cross-linking and propose that cytoplasmic factors (ascorbate and/or hydrogen ions) may regulate the solubility of extensin in vivo. PMID- 16663857 TI - Effects of nitrogen dioxide and nitrate nutrition on growth and nitrate assimilation in bean leaves. AB - The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO(2) (0-0.5 parts per million) on growth, K, photosynthetic pigment, N contents, and the activities of enzymes of N assimilation was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves. Exposing 7-day old bean seedlings for 5 days continuously to 0.02 to 0.5 parts per million NO(2) increased plant height, fresh weight, chlorophyll, carotenoid, organic N and nitrate contents, and nitrate reductase and glutamate synthase activities in the leaves of seedlings supplied with no external N. At 20 millimolar nitrate, most of the parameters examined were inhibited except for organic N and nitrate contents and glutamate synthase activity which increased in most cases. Generally, with an increase in NO(2) concentration, the stimulatory effect declined and/or the inhibitory effect increased. A 3-hour exposure of 12-day-old bean seedlings to 0.1 to 2.0 parts per million NO(2) increased nitrate content and nitrate reductase activity at each nutrient nitrate level except for a slight inhibition of enzyme activity during exposure to 2.0 parts per million NO(2) at 20 millimolar nitrate. The experiments demonstrated that the effect of NO(2) is strongly influenced by nutrient N level and that NO(2) is assimilated into organic nitrogenous compounds to serve as a source of N, only to a limited extent. PMID- 16663858 TI - Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves. AB - The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO(2) concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves. PMID- 16663859 TI - CO(2) and O(2) Exchange in Two Mosses, Hypnum cupressiforme and Dicranum scoparium. AB - Photosynthetic CO(2) and O(2) exchange was studied in two moss species, Hypnum cupressiforme Hedw. and Dicranum scoparium Hedw. Most experiments were made during steady state of photosynthesis, using (18)O(2) to trace O(2) uptake. In standard experimental conditions (photoperiod 12 h, 135 micromoles photons per square meter per second, 18 degrees C, 330 microliters per liter CO(2), 21% O(2)) the net photosynthetic rate was around 40 micromoles CO(2) per gram dry weight per hour in H. cupressiforme and 50 micromoles CO(2) per gram dry weight per hour in D. scoparium. The CO(2) compensation point lay between 45 and 55 microliters per liter CO(2) and the enhancement of net photosynthesis by 3% O(2)versus 21% O(2) was 40 to 45%. The ratio of O(2) uptake to net photosynthesis was 0.8 to 0.9 irrespective of the light intensity. The response of net photosynthesis to CO(2) showed a high apparent K(m) (CO(2)) even in nonsaturating light. On the other hand, O(2) uptake in standard conditions was not far from saturation. It could be enhanced by only 25% by increasing the O(2) concentration (saturating level as low as 30% O(2)), and by 65% by decreasing the CO(2) concentration to the compensation point. Although O(2) is a competitive inhibitor of CO(2) uptake it could not replace CO(2) completely as an electron acceptor, and electron flow, expressed as gross O(2) production, was inhibited by both high O(2) and low CO(2) levels. At high CO(2), O(2) uptake was 70% lower than the maximum at the CO(2) compensation point. The remaining activity (30%) can be attributed to dark respiration and the Mehler reaction. PMID- 16663860 TI - Separation Procedure and Partial Characterization of Two NAD(P)H Dehydrogenases from Cauliflower Mitochondria. AB - A procedure was developed to separate and partially purify two NAD(P)H dehydrogenases from the inner membrane of cauliflower (Brassica oleracea L.) mitochondria. The procedure used Triton X-100 extraction followed by (NH(4))(2)SO(4) precipitation and gel filtration (Sepharose G-200 column) chromatography. The first dehydrogenase fraction (which eluted in the column void volume) was specific for NADH, was stimulated by KCl addition, and was inhibited by acidic pH, sulfhydryl reagents, and elevated temperature. This fraction contained two major polypeptides with molecular weights of about 57,600 and 32,600 daltons. The fraction exhibited electron paramagnetic resonance (EPR) signals associated with a reduced (ferredoxin-type) iron-sulfur center.A second dehydrogenase fraction was eluted from the column after removal of the first dehydrogenase. This fraction oxidized NADH and NADPH, was stable at high temperatures, and had a broad pH optima that ranged from 6.0 to 7.8. Although it was relatively insensitive to additions of monovalent and divalent cations, its activity was sensitive to incubation with sulfhydryl reagents. The second dehydrogenase fraction contained five major polypeptides and lacked the iron sulfur protein EPR signals shown by the first dehydrogenase fraction.The dehydrogenase fractions represent three potential sites of entry to mitochondrial electron transport; two sites for NADH and a third site for NADPH. PMID- 16663861 TI - Regulation of Aspartate Kinase Isoenzymes in Barley Mutants Resistant to Lysine plus Threonine : Construction and Analysis of Combinations of the Lt1a, Lt1b, and Lt2 Mutant Genes. AB - Two homozygous mutant lines of barley (Hordeum vulgare L.) R3202 (Lt1b/Lt1b) and R3004 (Lt2/Lt2), are resistant to lysine plus threonine. They contain aspartate kinase isoenzymes with lost or decreased feedback sensitivity to lysine in either isoenzyme AKII (R3202) or isoenzyme AKIII (R3004). A homozygous double mutant line (Lt1b/Lt1b, Lt2/Lt2) has now been constructed that grows vigorously on 8 millimolar lysine, 8 millimolar threonine, and 1 millimolar arginine. Both AKII and AKIII from the double mutant have altered lysine sensitivities, identical to those previously observed in R3202 and R3004, respectively. Aspartate kinase activity in extracts of leaves, roots, and the maturing endosperm of the double mutant was much less sensitive to lysine inhibition than the enzyme in comparable extracts of the parent cv Bomi, suggesting that aspartate kinase is expressed in a similar manner in different tissues of barley.A further mutant, R2501, resistant to lysine plus threonine has now given rise to a homozygous line (Lt1a/Lt1a), which had previously not been possible. AKII isolated from the homozygous line was completely insensitive to 10 millimolar lysine; however, the combined action of 10 millimolar lysine and 0.8 millimolar S-adenosylmethionine inhibited it by 60%, demonstrating the retention of some of the regulatory characteristics of the wild type enzyme. PMID- 16663862 TI - Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition. AB - Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O(2) uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 x 10(-14) counts per O(2) molecule) was used to estimate the O(2) uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O(2) uptake (1.2 microliters O(2) per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues. PMID- 16663863 TI - Effect of nodulation on assimilate remobilization in soybean. AB - The objectives of this work were to determine the effect of nodulation on dry matter, reduced-N, and phosphorus accumulation and partitioning in above-ground vegetative parts and pods of field-grown soybean (Glycine max [L.] Merr. cv Harosoy).From comparison of nodulated and nonnodulated isolines, it was estimated that nodulated plants attained 81 and 71% of total-plant (above ground) N from uptake of soil N in 1981 and 1982, respectively. These data, along with visibly greener leaves of nodulated plants, led us to assume that nonnodulated plants were under a moderate N stress relative to nodulated plants. Nonnodulated plants accumulated less total-plant N and partitioned less dry matter and N to the pods, compared with nodulated plants. This occurred even though net photosynthesis, as estimated by rate and amount of dry matter accumulation, was the same for both nonnodulated and nodulated plants. Rate of dry matter and reduced-N accumulation in pods was less for nonnodulated than for nodulated plants while duration of podfill was similar for both isolines. From these data we concluded that moderate N stress affected partitioning of photosynthate rather than net photosynthesis, and that N played a role in translocation of photosynthate to the pods. Total plants (above-ground portion) and pods of both nodulated and nonnodulated plants accumulated similar amounts of phosphorus, which indicated that phosphorus and N accumulation were independent.Remobilization of nitrogen and phosphorus from vegetation to pods preceded dry matter remobilization. It appeared that either more nitrogen accumulation prior to podfill, or continued nitrogen assimilation during podfill would increase nitrogen and dry matter partitioning to pods, but that increasing photosynthesis without concomitantly increasing nitrogen input may not necessarily result in enhanced seed production. PMID- 16663864 TI - Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts. AB - Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559(HP), cytochrome b-559(LP), and cytochrome aa(3) by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa(3) in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559(HP) but contain cytochrome b-559(LP) (E(m7.5) = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559(LP) in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559(LP) indicating that this cytochrome is not directly involved in this pathway. PMID- 16663865 TI - Increased mitochondrial DNA and RNA polymerase activity in ethylene-treated potato tubers. AB - A purified mitochondrial fraction was isolated from potato (Solanum tuberosum L.) tubers respiring normally at 23 degrees C or at an accelerated rate in response to treatment with ethylene (10 microliters per liter).A pronounced increase in various mitochondrial enzymic activities was observed in response to exposure of the whole tubers to ethylene. Cytochrome c oxidase activity increased more than 50%, DNA polymerase activity increased about 2-fold, and RNA polymerase activity increased 2.5-fold. Moreover, DNA or RNA polymerase activities of mitochondria isolated from tubers not treated with ethylene were not affected by ethylene treatment in vitro. Respiratory control ratios decreased from 2.84 to 1.50 with increasing periods of ethylene treatment from 0 to 15 hours. None of these changes were observed in untreated tubers. It is concluded that the stimulation of respiration by ethylene in potato tubers is accompanied in vivo by an enhancement of mitochondrial enzymic activity of both membrane-associated enzymes which participate in the mitochondrial oxidative electron transport as well as soluble enzymes which are not directly involved in respiration. PMID- 16663866 TI - Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism. AB - A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K(m) for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate. PMID- 16663867 TI - Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii. AB - We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO(2), but its synthesis was induced under low concentrations of CO(2) (air levels of CO(2)). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO(2) to growth on air levels of CO(2), (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species. PMID- 16663868 TI - Acid phosphatases and seed shriveling in triticale. AB - Seed shriveling in the man-made intergeneric hybrid, triticale (x Triticosecale Wittmack) appears to be related to increased activity of endosperm acid phosphatases including para-nitrophenyl phosphatase, ATPase, ADPase, phosphatidic phosphatase, and glucose-1-phosphatase that occur specifically at later stages of seed development. These hydrolases may reduce endogenous substrates for starch synthesis, deplete energy supply for maintenance and biosynthesis of tissue growth, and deassemble membrane structures resulting in a partially filled endosperm and localized necrosis. Electrophoretic isozyme patterns of endosperm acid phosphatase exhibited distinctive differences between lines producing plump and shriveled seeds indicating a divergent role of the isozymes in these two different seed conformations. PMID- 16663869 TI - A diatom light-harvesting pigment-protein complex : purification and characterization. AB - A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3 cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species. PMID- 16663870 TI - Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene. AB - To understand the mechanism and molecular properties of the tonoplast-type H(+) translocating ATPase, we have studied the effect of Cl(-), NO(3) (-), and 4,4' diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H(+)-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H(+)-pumping ATPase generates a membrane potential (Deltapsi) and a pH gradient (DeltapH) that make up two interconvertible components of the proton electrochemical gradient (Deltamuh(+)). A permeant anion (e.g. Cl(-)), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([(14)C]thiocyanate distribution) and stimulated formation of a pH gradient ([(14)C]methylamine distribution). However, Cl(-)-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl(-) independent of the proton electrochemical gradient. Unlike the plasma membrane H(+)-ATPase, the Cl(-)-stimulated ATPase was inhibited by NO(3) (-) (a permeant anion) and by DIDS. In the absence of Cl(-), NO(3) (-) decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO(3) (-) of Cl(-)-stimulated pH gradient formation and Cl(-)-stimulated ATPase activity was noncompetitive. In the absence of Cl(-), DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl(-)-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H(+)-ATPase by NO(3) (-) or DIDS suggest that the vanadate-insensitive H(+)-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion sensitive H(+)-ATPase has channels that conduct anions is yet to be established. PMID- 16663871 TI - Evidence for a KCl-Stimulated, Mg-ATPase on the Golgi of Corn Coleoptiles. AB - Membranes of corn (Zea mays, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient centrifugation and the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase was selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelles. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCl, Mg(2+) ATPase of the PM-rich fraction from a step gradient was 6.0 to 6.5, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To reduce PM contamination, Golgi-rich fractions obtained from step or rate-zonal gradients were recentrifuged isopycnically on linear sucrose gradients. The distribution of KCl, Mg(2+)-ATPase activity was measured at pH 6.5 and 7.5. The pH 6.5 ATPase was coincident with UDPG-sterol glucosyl transferase, a PM marker, while the pH 7.5 ATPase overlapped with latent UDPase, a Golgi marker. These results provide strong evidence for a KCl, Mg(2+)-ATPase, active at pH 7.5, associated with the Golgi membranes of corn coleoptiles. PMID- 16663872 TI - Effects of Methomyl and Helminthosporium maydis Toxin on Matrix Volume, Proton Motive Force, and NAD Accumulation in Maize (Zea mays L.) Mitochondria. AB - Methomyl and Helminthosporium maydis race T toxin block oxidative phosphorylation in mitochondria isolated from maize plants with Texas male sterile cytoplasm (T) but not in mitochondria isolated from those with Normal cytoplasm (N) (Bednarski, Izawa, Scheffer 1977 Plant Physiol 59: 540-545). Moreover, they have been reported to cause specific swelling in T mitochondria (Miller, Koeppe 1971 Science 173: 67-69; Koeppe, Cox, Malone 1978 Science 201: 1227-1229). We could not detect, by direct volume measurements, any change induced by these compounds in the mitochondrial matrix space. We show here that the proton motive force, which in maize mitochondria is composed of a large transmembrane potential and of a low transmembrane pH difference, is absent in T mitochondria incubated in the presence of methomyl or of Helminthosporium maydis race T toxin, while it is unchanged in N mitochondria. Methomyl and Helminthosporium maydis race T toxin induce, independently of the collapse of the proton motive force, a release of the cofactors NAD and coenzyme A from the mitochondrial matrix space. In particular, we show that NAD is transported in maize mitochondria, and that this transport, which is not dependent on the proton motive force, is inhibited by methomyl or Helminthosporium maydis race T toxin. PMID- 16663873 TI - Photoadaptation in marine phytoplankton : changes in spectral absorption and excitation of chlorophyll a fluorescence. AB - The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations. PMID- 16663874 TI - Carbon Assimilation Characteristics of the Aquatic CAM Plant, Isoetes howellii. AB - The relationship between malic acid production and carbon assimilation was examined in the submerged aquatic Crassulacean acid metabolism (CAM) plant, Isoetes howellii Engelmann. Under natural conditions free-CO(2) level in the water was highest at 0600 hours and (14)CO(2) assimilation rates in I. howellii were also highest at this time. After 0900 hours there was a similar pattern in (a) rate of free-CO(2) depletion from the water, (b) reduction of carbon assimilation rates, and (c) rate of deacidification in leaves. Rates of daytime deacidification increased under CO(2)-free conditions and as irradiance intensity increased. Nighttime CO(2) uptake was estimated to contribute one-third to one half of the total daily gross carbon assimilation. CO(2) uptake, however, accounted for only one-third to one-half of the overnight malic acid accumulation. Internal respiratory CO(2) may be a substrate for a large portion of overnight acid accumulation as leaves incubated overnight without CO(2) accumulated substantial levels of malic acid. Loss of CAM occurred in emergent leaf tips even though submerged bases continued CAM. Associated with loss of CAM in aerial leaves was an increase in total chlorophyll, a/b ratio, and carotenoids, and a decrease in leaf succulence. delta(13)C values of I. howellii were not clearly distinguishable from those for associated non-CAM submerged macrophytes. PMID- 16663875 TI - Properties of Thylakoid Membranes of the Mangroves, Avicennia germinans and Avicennia marina, and the Sugar Beet, Beta vulgaris, Grown under Different Salinity Conditions. AB - Properties of thylakoids isolated from leaves of three salt tolerant species, Avicennia germinans L., Avicennia marina var resinifera, and Beta vulgaris L., were not affected by the salinity in which the plants were grown. With increase in the growth salinity from 50 to 500 millimolar NaCl, there were no major effects on the per chlorophyll concentrations of lipids or proteins, or on the rates of uncoupled electron transport per chlorophyll mediated by either the whole chain or the partial reactions of photosystems I and II. Responses of the partial and whole chain reactions to variation in the sorbitol and NaCl concentrations in the assay media were independent of the salinity experienced during leaf growth and not substantially different from those of a salt-sensitive species, Cucurbita sativus L. Uncoupled rates of electron flow from water to p benzoquinone mediated by photosystem II were insensitive to the NaCl concentration unless thylakoids were rendered Cl(-) deficient by treatment with uncoupler under alkaline conditions. Loss of 65% to 85% of the photosystem II activity in these Cl(-)-deficient thylakoids was restored by addition of 10 to 20 millimolar Cl(-). PMID- 16663876 TI - Differential role of glutamate dehydrogenase in nitrogen metabolism of maize tissues. AB - Both calli and plantlets of maize (Zea mays L. var Tuxpeno 1) were exposed to specific nitrogen sources, and the aminative (NADH) and deaminative (NAD(+)) glutamate dehydrogenase activities were measured at various periods of time in homogenates of calli, roots, and leaves. A differential effect of the nitrogen sources on the tissues tested was observed. In callus tissue, glutamate, ammonium, and urea inhibited glutamate dehydrogenase (GDH) activity. The amination and deamination reactions also showed different ratios of activity under different nitrogen sources. In roots, ammonium and glutamine produced an increase in GDH-NADH activity whereas the same metabolites were inhibitory of this activity in leaves. These data suggest the presence of isoenzymes or conformers of GDH, specific for each tissue, whose activities vary depending on the nutritional requirements of the tissue and the state of differentiation. PMID- 16663877 TI - l-Canavanine Transport and Utilization in Developing Jack Bean, Canavalia ensiformis (L.) DC. [Leguminosae]. AB - l-Canavanine, the guanidinooxy structural analog of l-arginine, is an important nonprotein amino acid of many leguminous plants with nitrogen storage a major proported role. l-[Guanidinooxy-(14)C]canavanine, [(14)C] urea, and [(15)N]urea were injected separately into the fleshy, green cotyledons of 9-day old jack bean plants, Canavalia ensiformis (L.) DC. [Leguminosae]. There was significant transport of canavanine from the cotyledons to the aboveground portions of the plant, but not to the roots. Within 1.5 hours of isotope administration, the remaining labeled canavanine was divided equally between the cotyledons and the aboveground portions of the plant. During the 48-hour postinjection period, the contribution of l-[guanidinooxy-(14)C]canavanine to the total (14)carbon of the cotyledons decreased rapidly while it increased in the aboveground portions of the plant.[(14)C]Urea is degraded very rapidly; only 4.4% of the initial dose remained after 1.5 hours. Urea is catabolized so effectively within the cotyledons that not even 2% of the administered urea can be detected in tissues outside of these storage organs. [(15)N]Urea supplied to the developing coytledons leads to rapid (15)N incorporation into the amino nitrogen of glutamic acid and/or glutamine (28% (15)N abundance after 3 hours). Other amino acids are labeled but less heavily. The data are consistent with the proported role for l canavanine of nitrogen storage within the developing cotyledons and cotyledonary canavanine is transported very effectively to the aboveground portions of the plant. It is not yet clear how efficiently this transported canavanine supports the nitrogen metabolism of the developing plant. PMID- 16663878 TI - Imidazolinones: potent inhibitors of acetohydroxyacid synthase. AB - The imidazolinones, a new chemical class of herbicides, were shown to be uncompetitive inhibitors of acetohydroxyacid synthase from corn. This is the first common enzyme in the biosynthetic pathway for valine, leucine, and isoleucine. The K(i) for the imidazolinones tested ranged from 2 to 12 micromolar. These results may explain the mechanism of action of these new herbicides. PMID- 16663879 TI - Role of Pectinesterase in pH-Dependent Interactions between Pea Cell Wall Polymers. AB - Extracts of etiolated pea (Pisum sativum L.) shoots converted soluble pectin from the seedlings to a trichloroacetic acid-insoluble form. This activity coincided with pectinesterase peaks separated from the extracts by gel filtration and ion exchange. The conversion of pectin to the trichloroacetic acid-insoluble form and pectinesterase exhibited identical responses to pH, with activity only above pH 6. The formation of trichloroacetic acid-insoluble pectin in pea cell walls and their extracts is due to de-esterification of the pectin by pectinesterase and not to binding between pectin and a protein, as reported in the literature. PMID- 16663880 TI - Induction of aluminum tolerance in wheat seedlings by low doses of aluminum in the nutrient solution. AB - Preincubation of wheat (Triticum aestivum L. Thell.) seedlings in a nutrient solution containing low doses of aluminum (0.5 microgram per milliliter for tolerant cultivar Atlas 66 and 0.1 microgram per milliliter for the sensitive cultivar Grana) enabled substantial root regrowth of varieties grown in a lethal aluminum concentration, despite an increased accumulation of aluminum in root tissue of the pretreated seedlings. The distribution of aluminum in the subcellular fractions remained unchanged. The increase in tolerance was completely abolished by the addition of cycloheximide. Aluminum ions at sublethal concentrations significantly increased the incorporation of [(14)C]valine and [(3)H]thymidine in roots. The possible role of the synthesis of the inducible aluminum binding protein in the mechanism of aluminum tolerance is discussed. PMID- 16663881 TI - Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale. AB - Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(-) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(-) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(-) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(-) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO(2) to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(-) nodules. M. gale spore(-) nodules showed variable effectivity, though all had low CO(2) to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation. PMID- 16663883 TI - Steady state proline levels in salt-shocked barley leaves. AB - Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na(+), and K(+) were measured at intervals. Na(+) and K(+) concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na(+) concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na(+) concentration. For a given Na(+) concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress. PMID- 16663882 TI - Regulation of excitation energy in a wheat mutant deficient in light-harvesting pigment protein complex. AB - Chloroplasts of the CD3 wheat mutant were deficient primarily in chlorophyll of light harvesting pigment proteins (LHPP) 1 and 2 and CP1a. The reduced level of protein associated with chlorophyll of LHPP1 and LHPP2 and the reduced level of low molecular weight polypeptides between 23 and 29 kilodaltons confirmed that the CD3 mutant was deficient in the LHPP complex. The high fluorescence emission ratio at 740 (F740) to 686 nanometers (F686) observed from chloroplasts of normal wheat following light induced phosphorylation of the LHPP complex was not noted from mutant chloroplasts. The long wavelength peak fluorescence emission (F740) was shifted to a shorter wavelength peak (F725) and was reduced in intensity compared to that of normal wheat thylakoids. The ratio of variable fluorescence to maximum fluorescence, a measure of PSII photochemical efficiency, was the same for the normal wheat and mutant leaves. The ratios of uncoupled photosystem I/photosystem II electron transport rates for mutant and normal wheat chloroplasts were similar at saturating light suggesting that absorbed excitation energy was distributed to the two photosystem reaction centers of the mutant in a similar manner as in the normal wheat. Proteins of the LHPP complex were differentially phosphorylated by action of a membrane protein kinase when both normal wheat and CD3 mutant thylakoids were irradiated without an electron transport chain acceptor. Even though the F740/F686 ratio was low in mutant thylakoids, the phosphorylation of the 27-kilodalton LHPP polypeptide was consistent with the mutant being in a state II condition. The data gave rise to the suggestion that the F740/F686 ratio might not indicate excitation energy distribution to the two photosystems in the mutant. PMID- 16663884 TI - Selection and characterization of a carrot cell line tolerant to glyphosate. AB - Cultured carrot (Daucus carota L.) cells were adapted to growing in 25 millimolar glyphosate by transfer into progressively higher concentrations of the herbicide. Tolerance was increased 52-fold, and the adaptation was stable in the absence of glyphosate. The uptake of glyphosate was similar for adapted and nonadapted cells. Activity of the enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase was 12-fold higher in the adapted line compared to nonadapted cells, while activities of shikimate dehydrogenase and anthranilate synthase were similar in the two cell types. The adapted cells had higher levels of free amino acids-especially threonine, methionine, tyrosine, phenylalanine, tryptophan, histidine, and arginine-than did nonadapted cells. Glyphosate treatment caused decreases of 50 to 65% in the levels of serine, glycine, methionine, tyrosine, phenylalanine, and tryptophan in nonadapted cells, but caused little change in free amino acid levels in adapted cells.The adaptation reported here supports the growing body of evidence linking tolerance to glyphosate with increased levels of the enzyme 5 enolpyruvylshikimic acid-3-phosphate synthase. The elevated levels of aromatic amino acids, which may confer resistance in adapted cells, suggest that control of the shikimate pathway may be altered in these cells. PMID- 16663885 TI - Benzyladenine-induced increase in DNA content per chloroplast in intact bean leaves. AB - Benzyladenine (BA) treatment was found to induce chloroplast DNA (ctDNA) synthesis after it had stopped in primary leaves of light-grown intact bean plants (Phaseolus vulgaris L.). The leaves were treated with BA from 7 days after sowing. Chloroplasts were isolated and the ctDNA content per chloroplast was determined. Chloroplast division occurred until 13 days after sowing in untreated leaves. BA stimulated the division keeping the level of ctDNA content per chloroplast the same as that in the untreated controls. After the division period, the ctDNA content per chloroplast increased in BA-treated leaves, but not in controls. Consequently, ctDNA per leaf (or per cell) increased immediately after the beginning of BA treatment, but remained constant in the control leaves. PMID- 16663886 TI - Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L. AB - The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5' phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH(4) (+) added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH(4) (+), the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH(4) (+) from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO(3) (-) as a nitrogen source. Using [(35)S]SO(4) (2-) as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH(4) (+) was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH(4) (+) may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis. PMID- 16663887 TI - Characterization and localization of the ATPase associated with pea chloroplast envelope membranes. AB - Chloroplast envelope membranes isolated from Pisum sativum seedlings have been found to contain a Mg-ATPase activity (specific activity 50-175 nanomoles per minute per milligram protein). The ATPase had a broad pH optimum between 7.0 and 9.5. The activity was not inhibited by oligomycin, N,N'-dicyclohexylcarbodiimide, ouabain, or antibodies directed against chloroplast coupling factor 1; nor was the activity stimulated by monovalent cations. However, the ATPase was inhibited by vanadate, molybdate, and adenylyl imidodiphosphate.The ATPase hydrolyzed a broad range of nucleoside triphosphates, but did not hydrolyze ADP, AMP, or pyrophosphate. The K(m) for Mg-ATP was determined to be 0.2 millimolar. The ATPase was found to be distinct from ADPase and pyrophosphatase activities also present in pea envelope membranes.The ATPase was determined to be located on the inner membrane of the envelope after resolution of inner and outer membranes by sucrose density gradient centrifugation. PMID- 16663888 TI - Image Analysis of Geo-Induced Inhibition, Compression, and Promotion of Growth in an Inverted Helianthus annuus L. Seedling. AB - The growth responses of a sunflower seedling (Helianthus annuus L.), subjected to repeated inversion, were characterized by time-lapse recording in conjunction with video image analysis. The investigation revealed a characteristic response pattern and established that the directional movement of the seedling is achieved by both inhibition and stimulation of growth in the normal growing regions. The complex growth changes in contiguous regions of the hypocotyl are such as seem to be inexplicable in terms of an environmentally imposed gradient of a single growth substance. PMID- 16663889 TI - Efficiency of cold hardiness induction by desiccation stress in four winter cereals. AB - A number of defined desiccation treatments without low temperature exposure were able to induce freezing tolerance in 20 cultivars of winter cereals. A maximal degree of freezing tolerance was induced in epicotyls at 24 degrees C in 24 hours at 40% relative humidity in rye and wheat, 7 days at 54% RH in barley, and 4 days at 70% RH in oats. Freezing tolerance was not correlated to water content of the plants after desiccation treatment but was related to the genetic capacity of the cultivars to frost harden. Levels of freezing tolerance induced by desiccation were similar to those induced by cold acclimation in rye and wheat, but considerably less in barley and oats. This is associated with a more rapid desiccation injury in barley and oats, precluding the completion of the hardening process. PMID- 16663890 TI - Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis. AB - The nature of the inorganic carbon (C(i)) species actively taken up by cyanobacteria CO(2) or HCO(3) (-) has been investigated. The kinetics of CO(2) uptake, as well as that of HCO(3) (-) uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO(2) was higher than that for HCO(3) (-). Though the calculated V(max) was the same in both cases, the maximum rate of uptake actually observed was higher when HCO(3) ( ) was supplied. C(i) uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO(2) was the species supplied. Observations of photosynthetic rate as a function of intracellular C(i) level (following supply of CO(2) or HCO(3) (-) for 5 seconds) led to the inference that HCO(3) (-) is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C(i) transport apparatus binds CO(2) and releases HCO(3) (-) at or near a membrane porter. The latter transports HCO(3) (-) ions to the cell interior. PMID- 16663891 TI - Localization and capacity of proton pumps in roots of intact sunflower plants. AB - Proton extrusion by roots of intact sunflower plants (Helianthus annuus L.) was studied in nutrient solutions or in agar media with a pH indicator. Proton extrusion was enhanced by either iron deficiency, addition of fusicoccin, or single salt solutions of ammonium or potassium salts. The three types of proton extrusion differ in both localization along the roots and capacity. From their sensitivity to ATPase inhibitors it seems justified to characterize them as proton pumps driven by plasma membrane APTases.Enhanced proton extrusion induced by preferential cation uptake from (NH(4))(2)SO(4) or K(2)SO(4) was uniformly distributed over the whole root system. In contrast, the enhancement effect of fusicoccin was confined to the basal root zones and that of iron deficiency to the apical root zones. Also the rates of proton extrusion per unit of root fresh weight differed remarkably and increased in the order: Fusicoccin << K(2)SO(4) < (NH(4))(2)SO(4) < iron deficiency.Under iron deficiency the average values of proton extrusion for the whole root system are 5.6 micromoles H(+) per gram fresh weight per hour; however, for the apical root zones values of about 28 micromoles H(+) can be calculated. This high capacity is most probably related to the iron deficiency-induced formation of rhizodermal transfer cells in the apical root zones. It can be assumed that the various types of root-induced acidification of the rhizosphere are of considerable ecological importance for the plant-soil relationships in general and for mobilization of mineral nutrients from sparingly soluble sources in particular. PMID- 16663892 TI - Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3 Glucanase in Pea Pods by Pathogens and Elicitors. AB - Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and beta-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and beta-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and beta-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and beta-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and beta-1,3-glucanase. PMID- 16663893 TI - Effects of Respiratory Inhibitors on Respiration, ATP Contents, and the Circadian Conidiation Rhythm of Neurospora crassa. AB - Effects of respiratory inhibitors on the circadian clock, respiratory activity, and ATP content were examined in Neurospora crassa. All inhibitors, potassium cyanide, sodium azide, antimycin A, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), shifted the phase of the conidiation rhythm. All the phase response curves were similar and resembled that for cycloheximide, but were different from the phase response curve for light. Phase shifting by azide and CCCP was proportional to the lowering of respiratory activity and ATP content, but such a correlation was not observed for cyanide and antimycin A. In particular, cyanide at a concentration of 0.5 millimolar completely depleted ATP of the cultures but did not significantly shift their phase. Their results suggest that large shifts caused by these inhibitors are not due to a decrease in energy from respiratory activity. PMID- 16663894 TI - Effect of water stress on the chloroplast antioxidant system: I. Alterations in glutathione reductase activity. AB - The effect of water stress on glutathione reductase and catalase activities was evaluated in leaf blades of field-grown winter wheat (Triticum aestivum L.). Wheat was sown at two seeding rates under both irrigated and dryland conditions. Flag leaves from dryland plants sown at 60 kilograms/hectare showed no change in either glutathione reductase or catalase activities per unit leaf area, while leaves from the basal portion of the canopy exhibited a 273% increase in glutathione reductase activity and a 60% increase in catalase activity. Glutathione reductase activity in dryland plants sown at 120 kilograms/hectare increased 25% in flag leaves and 225% in basal leaves. No change in catalase activity was observed in either flag or basal leaves from these same plants. The increase in glutathione reductase activity in response to water stress was observed when activity was expressed on either a per unit leaf area, protein, or chlorophyll basis. No change in catalase activity was detected when enzyme activity was expressed on a protein basis. PMID- 16663895 TI - Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Avena. AB - Forty-six monoclonal antibodies have been prepared against 124 kilodalton phytochrome from Avena sativa cv Garry. Clones grown in mice have yielded ascites fluids with antibodies which bind to three distinct regions of the molecule, as visualized by immunoblot analysis of proteolytically produced peptides of the protein. One antibody group (type 1) recognizes an antigenic domain(s) that lies within 6 kilodaltons of the amino terminus of the molecule, a region critical to correct protein-chromophore interaction. The second group (type 2) binds to an antigenic site(s) present within the chromophore-containing half of the molecule that is adjacent to the domain recognized by the type 1 antibodies. The third group (type 3) recognizes an antigenic site(s) that resides in the nonchromophoric, carboxy terminal end of the molecule between 88 and 97 kilodaltons from the amino terminus. One of the type 1 antibodies cross-reacts with apparently undegraded 120 kilodalton phytochrome from zucchini, and therefore may be useful for identifying conserved domains which are essential to the regulatory role of the photoreceptor. PMID- 16663896 TI - Methylation Pattern of Radish (Raphanus sativus) Nuclear Ribosomal RNA Genes. AB - The methylation pattern of radish Raphanus sativus nuclear rDNA has been investigated using the Hpa II, Msp I, and Hha I restriction enzymes. The presence of numerous target sites for these enzymes has been shown using cloned rDNA fragments. A large fraction of the numerous rDNA units are heavily methylated, being completely resistant to Hpa II and Hpa I. However, specific sites are constantly available in another fraction of the units and are therefore unmethylated. The use of different probes allowed us to demonstrate that hypomethylated sites are present in different regions. Major hypomethylated Hha I sites have been mapped in the 5' portion of 25S rRNA coding sequence. Among the hypomethylated fraction, different methylation patterns coexist. It has been possible to demonstrate that methylation patterns are specific for particular units. The Hha I pattern of rDNA in tissues of different developmental stages was analyzed. Evidence for possible tissue specific differences in the methylation pattern is reported. PMID- 16663897 TI - Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation. AB - Pectic polysaccharides from the roots of cucumber (Cucumis sativus L.) grown in liquid culture medium with or without calcium (1 mm CaCl(2)) were studied after extraction successively by hot water and Na hexametaphosphate solution. The Ca(2+) starvation-treatment caused a striking reduction in content of extracted pectic polysaccharide; from an equivalent weight of cell walls, only 33.1% of the control level was extracted from root cell walls of plants cultured under Ca(2+) deficiency. The extracted pectic polysaccharides were fractionated into neutral and acidic polymers by a DEAE-Sephadex column. The acidic polymers, which represented more than 76% of the yield, appeared to be a major fraction of extracted pectic polysaccharides. The changes of molecular size and glycosyl residue composition of this fraction were compared for the control and Ca(2+) deprived samples. The results indicate that Ca(2+) deficiency caused structural changes which could involve both branching pattern and extent of contiguous galacturonosyl units in the water-solubilized pectic polysaccharides. Ca(2+) starvation also led to a notable decrease in molecular size of the hexametaphosphate-solubilized polysaccharides and, to a lesser extent, of the water-solubilized fraction as well. In addition, polygalacturonase activity in tissue homogenates increased remarkably with the Ca(2+) deficiency, whereas beta galactosidase activity did not undergo a change. Thus, it appears that one major effect of Ca(2+) deprivation was to stimulate polygalacturonase activity, an effect which could be involved in the control of the breakdown of pectic polysaccharides in the cell walls. PMID- 16663898 TI - Regulatory effect of cytokinin on secondary xylem fiber formation in an in vivo system. AB - The regulatory effect of cytokinin on the formation of secondary xylem fibers was studied in the hypocotyl of young Helianthus annuus L. plants. Positive correlation was found between the kinetin supplied (0.25-0.5 micrograms/gram) to the growth medium and the rate of fiber formation within and between the vascular bundles. Reducing the root originated cytokinin supply, either by root removal or by lowering the transpiration rate, diminished the number of newly formed secondary xylem fibers. This decrease was considerably reversed in the presence of 0.5 microgram/gram kinetin. Early pulse exposure of kinetin had a temporary promoting effect on fiber differentiation at low concentrations and a permanent inhibitory effect at high concentration. PMID- 16663899 TI - Seasonal Shifts of Photosynthesis in Portulacaria afra (L.) Jacq. AB - Portulacaria afra (L.) Jacq., a perennial facultative Crassulacean acid metabolism (CAM) species, was studied under natural photoperiods and temperatures in San Diego, California. The plants were irrigated every fourth day throughout the study period. Measurements of (14)CO(2) uptake, stomatal resistance, and titratable acidity were made periodically from July 1981 through May 1982. P. afra maintained C(3) photosynthesis during the winter and the spring. Diurnal acid fluctuations were low and maximal (14)CO(2) uptake occurred during the day. The day/night ratio of carbon uptake varied from 5 to 10 and indicated little nocturnal CO(2) uptake. CAM photosynthesis occurred during the summer and a mixture of both C(3) and CAM during the fall. Large acid fluctuations of 100 to 200 microequivalents per gram fresh weight were observed and maximal (14)CO(2) uptake shifted to the late night and early morning hours. Daytime stomatal closure was evident. A reduction in the day/night ratio of carbon uptake to 2 indicated a significant contribution of nocturnal CO(2) uptake to the overall carbon gain of the plant. The seasonal shift from C(3) to CAM was facilitated by increasing daytime temperature and accompanied by reduced daytime CO(2) uptake despite irrigation. PMID- 16663900 TI - Metabolism of Monoterpenes : Early Steps in the Metabolism of d-Neomenthyl-beta-d Glucoside in Peppermint (Mentha piperita) Rhizomes. AB - Previous studies have shown that the monoterpene ketone l-[G-(3)H] menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaves of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-beta-d-glucoside which is then transported to the rhizome (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). Analysis of the disposition of l-[G (3)H]menthone applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis and transport of the monoterpenyl glucoside to be determined, and gave strong indication that the glucoside was subsequently metabolized in the rhizome. Studies with d-[G-(3)H]neomenthyl-beta-d-glucoside as substrate, using excised rhizomes or rhizome segments, confirmed the hydrolysis of the glucoside as an early step in metabolism at this site, and revealed that the terpenoid moiety was further converted to a series of ether-soluble, methanol soluble, and water-soluble products. Studies with d-[G-(3)H]neomenthol as the substrate, using excised rhizomes, showed the subsequent metabolic steps to involve oxidation of the alcohol back to menthone, followed by an unusual lactonization reaction in which oxygen is inserted between the carbonyl carbon and the carbon bearing the isopropyl group, to afford 3,4-menthone lactone. The conversion of menthone to the lactone, and of the lactone to more polar products, were confirmed in vivo using l-[G-(3)H]menthone and l-[G-(3)H]-3,4-menthone lactone as substrates. Additional oxidation products were formed in vivo via the desaturation of labeled neomenthol and/or menthone, but none of these transformations appeared to lead to ring opening of the p-menthane skeleton. Each step in the main reaction sequence, from hydrolysis of neomenthyl glucoside to lactonization of menthone, was demonstrated in cell-free extracts from the rhizomes of flowering mint plants. The lactonization step is of particular significance in providing a means of cleaving the p-menthane ring to afford an acyclic carbon skeleton that can be further degraded by modifications of the well known beta-oxidation sequence. PMID- 16663901 TI - gamma-Guanidinobutyraldehyde Dehydrogenase of Vicia faba Leaves. AB - gamma-Guanidinobutyraldehyde dehydrogenase was purified 27-fold in 40% yield from extracts of Vicia faba leaves. High specificity exist only for gamma guanidinobutyraldehyde and gamma-aminobutyraldehyde; the K(m) value was 3.4 micromolar for gamma-guanidinobutyraldehyde, 25 micromolar for gamma aminobutyraldehyde, and 84 micromolar (case of gamma-guanidinobutyraldehyde) for NAD, respectively. The enzyme had a molecular weight of approximately 83,000. Optimal pH and temperature for activity were 9.5 and 45 degrees C, respectively. The enzyme was inhibited strongly by p-chloromercuribenzoate, N-ethylmaleimide, and zincon (2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene). PMID- 16663902 TI - Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica. AB - The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO(3) (-) grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH(4) (+) have high NADPH-GDH activity, the levels of which increase with increasing NH(4) (+) supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH(4) (+) grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH(4) (+). PMID- 16663903 TI - Developmental regulation of the synthesis of proteins encoded by stored mRNA in radish embryos. AB - Major radish (Raphanus sativus L. cv National) proteins synthesized at the beginning of germination have been characterized by their migration in two dimensional electrophoresis.The use of 15-minute labelings shows that these proteins are encoded by stored mRNA. They undergo little or no posttranslational modification. Other proteins become detectable only after 1 hour of imbibition, and are probably encoded by newly synthesized mRNA. Comparison with proteins synthesized during embryogenesis, late germination, or with those present in dry embryos allows the classification of the proteins encoded by stored mRNA into two sets:The first set is synthesized also during late embryogenesis and is present in dry embryos. Some of these polypeptides are no longer synthesized later in germination while the others continue to be synthesized. The corresponding stored mRNAs can be considered as remnants of mRNA actively translated during embryogenesis.The second set is synthesized only during early germination. Their messengers appear during late embryogenesis although they are apparently not translated at this stage, but translation can be induced by a desiccation treatment. These polypeptides may play a particular role during early germination. PMID- 16663904 TI - Role of Silicon in Diatom Metabolism : Cyclic Nucleotide Levels, Nucleotide Cyclase, and Phosphodiesterase Activities during Synchronized Growth of Cylindrotheca fusiformis. AB - Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn(2+) for activity, though Mg(2+) gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes.Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5'-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium.Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear. PMID- 16663905 TI - Extensor and flexor protoplasts from samanea pulvini : I. Isolation and initial characterization. AB - Protoplasts were isolated from extensor and flexor regions of open pulvini of the nyctinastic tree Samanea saman. Both types of protoplasts undergo many changes during isolation. Extensor protoplasts are univacuolate in vivo, but some become multivacuolate. All flexor protoplasts are univacuolate. In an open pulvinus, extensor cells have a higher osmotic pressure than flexor cells. However, both types of protoplasts can be isolated with optimal yield using the same osmoticum (0.5 molar sorbitol) in the digestion medium. This suggests that some leakage of osmoticum occurs during harvest or digestion, especially from extensor tissue. Despite these changes, both types of protoplasts extrude protons in response to 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/10(6) protoplasts/minute), demonstrating that the protoplasts are metabolically active and that proton transport mechanisms must be at least partially functional. The changes in vacuolar structure and osmotic pressure are what one might expect if the protoplasts, which are isolated from open pulvini, take on characteristics of cells in a closed pulvinus. PMID- 16663906 TI - Extensor and Flexor Protoplasts from Samanea Pulvini : II. X-Ray Analysis of Potassium, Chlorine, Sulfur, Phosphorus, and Calcium. AB - Concentrations of K, Cl, P, S, and Ca in extensor and flexor protoplasts from open pulvini of the nyctinastic tree Samanea saman were estimated using x-ray microanalysis. This technique is particularly suitable when absolute numbers of protoplasts are low, because less than 100 protoplasts are required to obtain statistically significant data. Flexor protoplasts contain similar concentrations of P and S but almost twice as much K and Cl as extensor protoplasts. Low levels of total measurable osmoticum suggest that extensive leakage has occurred during protoplast isolation. Both extensor and flexor protoplasts appear to contain some unidentified osmoticum not detectable by x-ray analysis. Extensor protoplasts must have more unidentified osmoticum to compensate for their lower levels of K and Cl. PMID- 16663907 TI - Nickel in higher plants: further evidence for an essential role. AB - Soybeans (Glycine max [L.] Merr.) grown in Ni-deficient nutrient solutions accumulated toxic urea concentrations which resulted in necrosis of their leaflet tips, a characteristic of Ni deficiency. Estimates of the Ni requirement of a plant were made by using seeds produced with different initial Ni contents. When compared to soybeans grown from seeds containing 2.5 nanograms Ni, plants grown from seeds containing 13 nanograms Ni had a significantly reduced incidence of leaflet tip necrosis. Plants grown from seeds containing 160 nanograms Ni produced leaves with almost no leaflet tip necrosis symptoms. Neither Al, Cd, Sn, nor V were able to substitute for Ni.In other experiments, a small excess of EDTA was included in the nutrient solution in addition to that needed to chelate micronutrient metals. Under these conditions, nodulated nitrogen-fixing soybeans had a high incidence of leaflet tip necrosis, even when 1 micromolar NiEDTA was supplied. However, in nutrient solutions containing inorganic sources of N, 1 micromolar NiEDTA almost completely prevented leaflet tip necrosis, although no significant increase in leaf urease activity was observed. Cowpeas (Vigna unguiculata [L.] Walp) grown in Ni-deficient nutrient solutions containing NO(3) and NH(4) also developed leaflet tip necrosis, which was analogous to that produced in soybeans, and 1 micromolar NiEDTA additions prevented these symptoms.These findings further support our contention that Ni is an essential element for higher plants. PMID- 16663908 TI - Carbohydrate level and growth of tomato plants: I. The effect of carbon dioxide enrichment and diurnally fluctuating temperatures. AB - To what extent can the influence of environment on greenhouse tomato plants (Lycopersicon esculentum L.) be explained by a linear response of the growth rate to carbohydrate level? To answer this question, young tomato plants were transplanted in January and March and grown for one-month periods under natural light either with or without CO(2) enrichment to 1000 microliters per liter and either under a constant minimum temperature of 15 degrees C or a minimum temperature that fluctuated between 20 degrees C for 12 hours including the photoperiod and 10 degrees C for the remaining 12 hours. The carbohydrate levels were measured for plants under all growth conditions at several times in the diurnal cycle.At the average irradiance in January, 2.3 megajoules per square meter per day, plants under CO(2) enrichment grew 15% faster and had, on average, total nonstructural carbohydrate levels 27 milligrams per gram greater and soluble carbohydrates 3 milligrams per gram greater at all hours of the day than did plants grown under ambient CO(2). Plants grown under fluctuating diurnal temperature grew slightly faster and had total nonstructural carbohydrate levels on average 8 milligrams per gram greater than plants grown under a more constant temperature. At the average irradiance in March-April, 4.3 megajoules per square meter per day, growth and carbohydrate level increased with CO(2) enrichment under the control temperature regime but not under fluctuating temperatures. Plants in all growth regimens grew faster than in January.Over all plantings and growth regimens, relative growth rates were more highly correlated to total nonstructural carbohydrate levels than they were to irradiance, CO(2), or temperature. PMID- 16663909 TI - Decline in Energy Reserves of Chlorella sorokiniana upon Exposure to Ozone. AB - Ozone exposure has been shown to increase the loss of K from Chlorella cells due to an increase in passive permeability and a depolarization of membrane potential. One factor which likely influences or can be influenced by these changes is the energy state of the cell. To study this relationship, various indicators of cell energy status were examined in the presence and absence of O(3).The active uptake of chloride and deoxyglucose is nearly completely inhibited by O(3) at a dose at which cellular death, measured by plating efficiency, is minimal. Glucose-stimulated respiration, dependent upon ATP/ADP balance, is depressed to a greater degree than endogenous respiration in ozonated cells. Total ATP and glucose-6-phosphate levels also decrease but not as rapidly, and labeled intermediates of glucose metabolism are lost.Thus, exposure to O(3) results in a depletion of the cell's energy reserves as substantiated by changes observed in processes which both utilize and generate ATP. This loss in energy reserves occurs at the same exposure level of O(3) as do the changes in passive transport properties. Thus, we cannot tell which occurs first; and the processes seem to be linked with respect to O(3) injury. PMID- 16663910 TI - Effects of short-term n(2) deficiency on N metabolism in legume nodules. AB - The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O(2) treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O(2) treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N(2) supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species. PMID- 16663911 TI - Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop. AB - The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3 phosphoglycerate (PGA)-dependent O(2) evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C(4) mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O(2) evolution in the range of 90 to 135 micromoles O(2) per milligram chlorophyll per hour, and up to 180 micromoles O(2) per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O(2) evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K(m) (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O(2) evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O(2) evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O(2) evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C(4) mesophyll chloroplast. C(3) spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K(i) values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively. PMID- 16663912 TI - Purification and initial characterization of lipase from the scutella of corn seedlings. AB - The lipase from the scutella of corn (Zea mays) MO-17 seedlings was purified 272 fold to apparent homogeneity as evidenced by sodium dodecyl sulfate polyacrylamide gel electrophoresis and double immunodiffusion. The procedure involved isolation of the lipid bodies, extraction with diethyl ether, DE-52 ion exchange chromatography, and sucrose density gradient centrifugation. The enzyme had an approximate molecular weight of 270,000 daltons after sucrose density gradient centrifugation, and 65,000 daltons after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The lipase contained no cysteine and its molecular weight in sodium dodecyl sulfate was not reduced by beta mercaptoethanol. The amino acid composition as well as a biphasic partition using Triton X-114 revealed the enzyme to be a hydrophobic protein. Rabbit gamma globulin containing antibodies raised against the purified lipase formed one precipitin line with the lipase in a double diffusion test, and precipitated all the lipase activity from a solution. PMID- 16663913 TI - Assimilatory Power (Postillumination CO(2) Uptake) in Leaves: Measurement, Environmental Dependencies, and Kinetic Properties. AB - Assimilatory power was measured in ten C(3) species by means of a rapid-response gas exchange device as the total amount of CO(2) fixed in N(2)-CO(2) atmosphere after switching the light off. Different steady-state levels of the assimilatory power were obtained by varying light intensity and O(2) and CO(2) concentrations during the preexposition periods in the leaf chamber.Within the limits of the linear part of the CO(2) curve of photosynthesis in N(2), the assimilatory power is constant, being sufficient for the assimilation of about 20 nanomoles CO(2) per square centimeter leaf. The pool starts to decrease with the onset of the CO(2) saturation of photosynthesis. Increase in O(2) concentration from 0 to 100% at 350 microliters CO(2) per liter produces a considerable decrease in the assimilatory power.THE MESOPHYLL CONDUCTANCE (M) WAS FOUND TO BE PROPORTIONAL TO THE ASSIMILATORY POWER (A): M = mA. The most frequently occurring values of the proportionality constant (m) (called the specific efficiency of carboxylation) were concentrated between 0.03 and 0.04 centimeter per second per nanomole A per square centimeter but the measured extreme values were 0.01 and 0.06 centimeter per second per nanomole A per square centimeter. The specific rate of carboxylation (the rate per unit A) showed a hyperbolic dependence on CO(2) conentration with the most frequent values of K(m) (CO(2)) ranging from 25 to 35 micromolar in the liquid phase of mesophyll cells (extremes 23 and 100 micromolar).It is concluded that the CO(2(-) ) and light-saturated rate of photosynthesis is limited by the reactions of the formation of the assimilatory power and not by ribulose-1,5-bisphosphate carboxylase. O(2) is a competitive consumer of the assimilatory power, and the inhibitory effect of O(2) on photosynthesis is caused mainly by a decrease in the pool of the assimilatory power at high O(2) concentrations. In intact leaves, the kinetic properties of ribulose-1,5-bisphosphate carboxylase seem to be variable. PMID- 16663914 TI - Gel electrophoretic profiles of proteinases in dark-germinated flax seeds. AB - The proteinases present in dark-germinated flax seeds (Linum usitatissimum) were studied as a function of germination at 25 degrees C. A majority of activity was present in basic proteinases with an acidic pH optimum and a temperature optimum of 45 degrees C in the digestion of hemoglobin. Electrophoresis in a sodium dodecyl sulfate-polyacrylamide mixture which had been polymerized with gelatin was used to separate proteins in extracts of seedlings. Subsequent activation of proteinases with Triton X-100 and resultant digestion of gelatin proved to be very reproducible and afforded detection and good quantification of various proteinase zones. An ethylenediaminetetraacetate-sensitive proteinase zone, P4 (about 60,000 daltons), appeared at day 3 after imbibition and attained maximum activity at day 4. This correlates with a rapid loss in vivo of the glyoxysomal enzyme, isocitrate lyase (EC 4.1.3.1). Ethylenediaminetetraacetate also slowed the loss of isocitrate lyase activity in extracts of 4-day seedlings in a dose dependent manner. The addition of leupeptin, alpha-tolylsulfonyl fluoride, Pepstatin A, p-chloromercuribenzoate, or 1,10-phenanthroline prior to, during, or after exchange of Triton X-100 for sodium dodecyl sulfate had almost no inhibitory effect upon proteinases in 4-day seedlings. PMID- 16663915 TI - Phenol metabolism, phytoalexins, and respiration in potato tuber tissue treated with Fatty Acid. AB - Potato (solanum tuberosum L. cv Katahdin) tuber discs treated with arachidonic acid become necrotic and accumulate sesquiterpenoid phytoalexins. The arachidonic acid also causes increases in both phenylalanine ammonia lyase and lignin, but no change in total alcohol-soluble phenols. Linoleic acid does not alter any of these parameters. A high concentration of nonanoic acid promotes both necrosis and accumulation of low levels of phytoalexins, but decreased levels of phenols, phenylalanine ammonia lyase, and lignin. The respiration of the control discs and those treated with linoleic acid declines by 24 hours after treatment, but the respiration of arachidonic acid-treated discs remains constant for at least 48 hours. PMID- 16663916 TI - Pea Xyloglucan and Cellulose : III. Metabolism during Lateral Expansion of Pea Epicotyl Cells. AB - Lateral expansion of the third internodes of pea epicotyls was evoked by treatment with either 2,4-dichlorophenoxyacetic acid (2,4-D) or ethylene gas. During growth, 2,4-D enhanced and ethylene inhibited the deposition of xyloglucan and cellulose in the cell wall, with the result that the wall framework (ghost) from ethylene-treated swollen tissue was much thinner than that from 2,4-D treated. The level of activity of xyloglucan synthase, alkali-insoluble beta glucan synthases, and endo-1,4-beta-glucanases were all enhanced by 2,4-D treatment but not by ethylene. Both 2,4-D and ethylene treatments led to increased osmotic potential in the swelling tissues. Accordingly, swelling after 2,4-D treatment was accompanied by xyloglucan degradation, concomitant with substantial net synthesis, but swollen tissue as a result of ethylene treatment was characterized by walls whose integrity was weakened by relatively low levels of newly deposited polysaccharides rather than by the degradation. PMID- 16663917 TI - Increase of natural N enrichment of soybean nodules with mean nodule mass. AB - The (15)N abundance of soybean (Glycine max L. Merrill var Harosoy) nodules is usually greater than it is for other tissues or for atmospheric N(2). Results of experiments in which nodules were separated by size show that the magnitude of the (15)N enrichment is correlated with nodule mass. The results support the hypothesis that (15)N enrichment of nodules results from differential N isotopic fractionation for synthesis of nodule tissue versus synthesis of compounds for export from the nodule. The physiological significance of this hypothesis is that it requires that a substantial fraction of the N for nodule tissue synthesis in (15)N-enriched nodules be N recently fixed within the same nodule. PMID- 16663918 TI - De novo arginine biosynthesis in leaves of phosphorus-deficient citrus and poncirus species. AB - Young, fully expanded leaves from 7-month-old P-deficient citrus rootstock seedlings had levels of nonprotein arginine that were 10- to 50-fold greater than those from P-sufficient control plants. Arginine content of the protein fraction increased 2- to 4-fold in P-deficient leaves. Total arginine content, which averaged 72 +/- 6 micromoles per gram dry weight of P-sufficient leaf tissue (mean +/- se, n = the four rootstocks) was 207, 308, 241, and 178 micromoles in P deficient leaves from Citrus limon cv rough lemon, Poncirus trifoliata x C. sinensis cv Carrizo citrange and cv Troyer citrange, and P. trifoliata cv Australian trifoliate orange, respectively. For each rootstock, the accumulation of arginine paralleled an increase in the activity of the pathway for the de novo biosynthesis of arginine. The ratio of the nanomoles NaH(14)CO(3) incorporated into the combined pool of arginine plus urea per gram fresh weight intact leaf tissue during a 3-hour labeling period for P-deficient to P-sufficient plants was 91:34, 49:11, 35:11, and 52:41, respectively. When P-deficient plants were supplied with P, incorporation of NaH(14)CO(3) into arginine plus urea was reduced to the level observed for the P-sufficient control plants of the same age and arginine ceased to accumulate. Arginase and arginine decarboxylase activity were either unaffected or slightly increased during phosphorus deficiency. Taken together, these results provide strong evidence that arginine accumulation during phosphorus deficiency is due to increased activity of the de novo arginine biosynthetic pathway. PMID- 16663919 TI - Evidence for energy-dependent C-photoassimilate retention in isolated tobacco mesophyll cells. AB - (14)C-photoassimilate release from isolated tobacco (Nicotiana tabacum L. cv Xanthi nc.) mesophyll cells was gradual and much of the photoassimilate was retained for several hours. (14)C-product retention was enhanced by Ca(2+) and impaired by EDTA and ATP. Mg(2+) reduced the ATP-enhanced (14)C-efflux suggesting that ATP impairs product retention by modifying cell membrane permeability, possibly as a result of its chelating properties.Various metabolic inhibitors increased (14)C-efflux indicating an energy requirement for (14)C-product retention. Profiles of (14)C-product distribution in the cells and suspending medium indicated that (14)C-efflux from cells treated with carbonylcyanide p trifluoromethoxyphenylhydrazone occurred by passive diffusion. Coupled with the observation that exogenous sucrose increased (14)C-efflux and the demonstration of (14)C-sucrose uptake, the results suggest that product retention is partially due to the activity of an energy-dependent uptake process which recovers much of the photoassimilate lost from the cells.Under certain conditions, carbonylcyanide p-trifluoromethoxyphenylhydrazone reduced (14)C-efflux. The apparently contradictory effects of the uncoupler appeared to be related to the intracellular photoassimilate concentration and may be explained by the partitioning of photoassimilate within the cells. PMID- 16663920 TI - Association between Membrane Phase Properties and Dehydration Injury in Soybean Axes. AB - Axes of soybean seeds are tolerant to dehydration at 6 hours of imbibition, but susceptible to dehydration injury if dried at 36 hours of imbibition. Smooth microsomal membranes were isolated from axes imbibed for 6 hours (dehydration tolerant state) and 36 hours (dehydration susceptible state) before and after dehydration treatment. The phase properties and the lipid composition of the membrane fraction were investigated. Wide angle x-ray diffraction patterns of microsomal membranes from axes imbibed for 6 or 36 hours indicated a liquid crystalline to gel phase transition at approximately 7 degrees C. Membranes from axes dehydrated at 6 or 36 hours of imbibition and rehydrated for 2 hours exhibited a phase transition at 7 degrees C and 47 degrees C, respectively. Changes in fatty acid saturation did not account for the changes in phase properties. However, the increased phase transition temperature of the membranes from dehydration injured axes was associated with an increase in free fatty acid:phospholipid molar ratio and a decrease in phospholipid:sterol ratio. These results suggests that dehydration prompted a deesterification of the linkage between glycerol and fatty acid side chains of the phospholipid molecules in the membrane. The resultant increase in free fatty acid content in the membrane is thought to alter the fluidity and phase properties of the membrane and contribute to dehydration injury. PMID- 16663921 TI - Effects of decreased net carbon exchange on carbohydrate metabolism in sugar beet source leaves. AB - The relationship between CO(2) concentration and starch synthesis and degradation was studied by measuring leaf starch content and disappearance of (14)C-starch. At a concentration of 340 microliters CO(2) per liter, starch accumulated without degradation of previously synthesized starch. Degradation of starch began when CO(2) concentration was lowered, but its synthesis continued. At 120 microliters CO(2) per liter rates of synthesis and degradation were equal. Even at the CO(2) compensation point, synthesis of starch continued. Concomitant starch synthesis and mobilization supported export from the leaf. Changes in starch metabolism that occur when photosynthesis is CO(2)-limited provide a means to study regulation of starch metabolism and carbon allocation in translocating leaves. PMID- 16663922 TI - Assimilation of NO(3) Taken Up by Plants in the Light and in the Dark. AB - An experiment was conducted to determine the extent that NO(3) (-) taken up in the dark was assimilated and utilized differently by plants than NO(3) (-) taken up in the light. Vegetative, nonnodulated soybean plants (Glycine max L. Merrill, ;Ransom') were exposed to (15)NO(3) (-) throughout light (9 hours) or dark (15 hours) phases of the photoperiod and then returned to solutions containing (14)NO(3) (-), with plants sampled subsequently at each light/dark transition over 3 days. The rates of (15)NO(3) (-) absorption were nearly equal in the light and dark (8.42 and 7.93 micromoles per hour, respectively); however, the whole plant rate of (15)NO(3) (-) reduction during the dark uptake period (2.58 micromoles per hour) was 46% of that in the light (5.63 micromoles per hour). The lower rate of reduction in the dark was associated with both substantial retention of absorbed (15)NO(3) (-) in roots and decreased efficiency of reduction of (15)NO(3) (-) in the shoot. The rate of incorporation of (15)N into the insoluble reduced-N fraction of roots in darkness (1.10 micromoles per hour) was somewhat greater than that in the light (0.92 micromoles per hour), despite the lower rate of whole-plant (15)NO(3) (-) reduction in darkness.A large portion of the (15)NO(3) (-) retained in the root in darkness was translocated and incorporated into insoluble reduced-N in the shoot in the following light period, at a rate which was similar to the rate of whole-plant reduction of (15)NO(3) (-) acquired during the light period. Taking into account reduction of NO(3) (-) from all endogenous pools, it was apparent that plant reduction in a given light period ( approximately 13.21 micromoles per hour) exceeded considerably the rate of acquisition of exogenous NO(3) (-) (8.42 micromoles per hour) during that period. The primary source of substrate for NO(3) (-) reduction in the dark was exogenous NO(3) (-) being concurrently absorbed. In general, these data support the view that a relatively small portion (<20%) of the whole-plant reduction of NO(3) (-) in the light occurred in the root system. PMID- 16663923 TI - Inorganic Carbon Source for Photosynthesis in the Seagrass Thalassia hemprichii (Ehrenb.) Aschers. AB - Photosynthetic carbon uptake of the tropical seagrass Thalassia hemprichii (Ehrenb.) Aschers was studied by several methods. Photosynthesis in buffered seawater in media in the range of pH 6 to pH 9 showed an exponentially increasing rate with decreasing pH, thus indicating that free CO(2) was a photosynthetic substrate. However, these experiments were unable to determine whether photosynthesis at alkaline pH also contained some component due to HCO(3) (-) uptake. This aspect was further investigated by studying photosynthetic rates in a number of media of varying pH (7.8-8.61) and total inorganic carbon (0.75-13.17 millimolar). In these media, photosynthetic rate was correlated with free CO(2) concentration and was independent of the HCO(3) (-) concentration in the medium. Short time-course experiments were conducted during equilibration of free CO(2) and HCO(3) (-) after injection of (14)C labeled solution at acid or alkaline pH. High initial photosynthetic rates were observed when acidic solutions (largely free CO(2)) were used but not with alkaline solutions. The concentration of free CO(2) was found to be a limiting factor for photosynthesis in this plant. PMID- 16663924 TI - Effect of Oxygen Concentration on C-Photoassimilate Transport from Leaves of Salvia splendens L. AB - Partitioning and transport of recently fixed photosynthate was examined following (14)CO(2) pulse-labeling of intact, attached leaves of Salvia splendens L. maintained in an atmosphere of 300 microliters per liter CO(2) and 20, 210, or 500 milliliters per liter O(2). Under conditions of increasing O(2) (210, 500 milliliters per liter), a smaller percentage of the recently fixed (14)C in the leaf was allocated to starch, whereas a greater percentage of the fixed (14)C appeared in amino acids, particularly serine. The increase in (14)C in amino acids was reflected in material exported from source leaves. A higher percentage of (14)C in serine, glycine, and glutamate was recovered in petiole extracts when source leaves were maintained under elevated O(2) levels. Although pool sizes of these amino acids were increased in both the leaves and petioles with increasing photorespiratory activity, no significant changes in either (14)C distribution or concentration of transport sugars (i.e. stachyose, sucrose, verbascose) were observed. The data indicate that, in addition to being recycled intracellularly into Calvin cycle intermediates, amino acids produced during photorespiration may also serve as transport metabolites, allowing the mobilization of both carbon and nitrogen from the leaf under conditions of limited photosynthesis. PMID- 16663925 TI - Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin. AB - Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses. PMID- 16663926 TI - Gene Expression and Synthesis of Phytohemagglutinin in the Embryonic Axes of Developing Phaseolus vulgaris Seeds. AB - Phytohemagglutinin (PHA), the major seed lectin of the common bean (Phaseolus vulgaris), is found largely in the cotyledons, but is also present in the embryonic axis. At mid-maturation, the percentage of total protein synthesis which is directed towards making PHA is 5 to 10 times greater in the cotyledons than in the axes. This lower rate of synthesis in the axes is correlated with a lower abundance of mRNA for PHA, as determined by dot blot hybridization using a cDNA clone for PHA. Manen and Pusztai (Planta 1982 155: 328-334) have claimed on the basis of immunocytochemical evidence that, in the axis, PHA is found in the cytosol although it is present in protein bodies in the cotyledons. In the cotyledons, PHA is synthesized on rough endoplasmic reticulum, and its transport to the protein bodies via the Golgi complex is associated with specific posttranslational processing steps (Vitale and Chrispeels, J Cell Biol 1984 In press). A cytosolic localization of axis PHA would be an indication of a different site of synthesis and transport pathway. The results presented here indicate that the site of synthesis of PHA and the posttranslational modifications of PHA are the same in the axes as in the cotyledons. Since in the cotyledons these modifications take place in the endoplasmic reticulum, the Golgi, and the protein bodies, it appears that the transport pathway and the site of accumulation of PHA in the axes is similar to that in the cotyledons. On the basis of our evidence, we suggest that the subcellular localization of PHA in the axes should be reexamined. PMID- 16663927 TI - Characterization of a cadmium-binding complex of cabbage leaves. AB - The chemical nature of a principal, inducible cadmium-binding complex which accumulates in cabbage leaves (Wagner and Trotter 1982 Plant Physiol 69: 804-809) was studied and compared with that of animal metallothionein and copper-binding proteins isolated from various organisms. The apparent molecular weight of native cabbage complex and carboxymethylated ligand of the complex under native conditions as determined by gel filtration was about 10,000 daltons. Under denaturing conditions their apparent molecular weights were about 2000 daltons. Ligand of native complex contained 37, 28, and 9 residue per cent of glutamic acid-glutamine, cysteine, and glycine, respectively, and low aromatic residue, serine and lysine content. The high acidic and low hydrophobic residue content explain the behavior of complex on electrophoresis in the presence and absence of sodium dodecyl sulfate. Its isoelectric point was below 4.0 and it bound 4 to 6 moles cadmium per mole ligand in what appear to be cadmium-mercaptide chromophores. The complex was found to be heat stable, relatively protease insensitive, and lacking in disulfide bonds. Attempts to determine the primary sequence of reduced native complex and carboxymethylated, cleaved ligand using the Edman degradation procedure were unsuccessful. An electrophoretic procedure is described for preparative isolation of purified complex and a method is described for monitoring ligand of complex as its fluorescent dibromobimane adduct. PMID- 16663928 TI - A Quantitative Simulation Model for H-Amino Acid Cotransport To Interpret the Effects of Amino Acids on Membrane Potential and Extracellular pH. AB - The H(+) cotransport of neutral and acidic amino acids induces transient depolarizations of oat coleoptile (Avena sativa L., var Victory) plasma membranes. The depolarizations, which are completed within 1 or 2 minutes, are followed by repolarizations that are nearly completed within another 2 or 3 minutes. Cysteine induced a two-phased alkalinization of the tissue free space during the electrical changes. The first phase was a rapid, linear increase in pH that coincided with the depolarization; the second phase was a slower, also linear, increase in pH that coincided with the repolarization. Reacidification did not occur until cysteine was withdrawn. Five other acidic, basic, and neutral amino acids also induced persistent alkalinization of the free space.The notable features of these measurements are that free-space pH was measured more directly than previously, that pH changes corresponded in time to the electrical potential changes, and that reacidification of the free space did not occur. The latter observation indicates that net H(+) efflux did not occur during repolarization and that the repolarizing current was carried by some other ion. We propose that repolarization could have depended upon depolarization-induced changes in passive K(+) fluxes combined with an enhanced H(+) extrusion that increased until it equaled, but did not exceed, the enhanced influx of H(+).In support of the feasibility of our hypothesis, we present a quantitative simulation model for cotransport. The simulation model also provides an interpretation of the unique electrical effects of histidine and the basic amino acids. In addition, the model focuses attention upon the difficulties of interpreting H(+)-anion cotransport. PMID- 16663929 TI - Conversion and Distribution of Cobalamin in Euglena gracilis z, with Special Reference to Its Location and Probable Function within Chloroplasts. AB - Cobalamin is essentially required for growth by Euglena gracilis and shown to be converted to coenzyme forms promptly after feeding cyanocobalamin. Concentrations of coenzymes, methylcobalamin, and 5'-deoxyadenosylcobalamin, reached about 1 femtomole/10(6) cells 2 hours after feeding cyanocobalamin to cobalamin-limited cells. Cobalamins all were bound to proteins in Euglena cells and located in subcellular fractions of chloroplasts, mitochondria, microsomes, and cytosol. Incorporated cobalamin into chloroplasts was localized in thylakoids. Methylcobalamin existed in chloroplasts, mitochondria, and cytosol, while 5' deoxyadenosylcobalamin was in mitochondria and the cytosol, 2 h after feeding cyanocobalamin to Euglena cells. Quantitative alterations of methylcobalamin and 5'-deoxyadenosylcobalamin in chloroplasts suggest their important functions as coenzymes in this organelle. The occurrence of functional cobalamins in chloroplasts has not been reported in other photosynthetic eukaryotes. PMID- 16663930 TI - Inhibition by fusicoccin of germination of pea seeds. AB - Fusicoccin inhibits the germination of pea (Pisum sativum L. cv Progress 9) seeds by decreasing the growth of the embryonal axis and by stimulating the fresh weight increase of the cotyledons. The growth of isolated embryonal axes in the presence of sucrose and KCl is stimulated by fusicoccin. The effect of fusicoccin on the seeds is not counteracted by sucrose and KCl. Fusicoccin promotes preferentially in the cotyledons a hyperpolarization of the transmembrane electric potential and an increase in the uptake capacity, suggesting the reinforcement of the sink strength of the cotyledons in comparison with the one of the embryonal axis and therefore the inhibition of translocation from the cotyledons of some substance necessary for the growth of the embryonal axis. PMID- 16663931 TI - Injury, Stomatal Conductance, and Abscisic Acid Levels of Pea Plants following Ozone plus Sulfur Dioxide Exposures at Different Times of the Day. AB - Pea (Pisum sativum L. cv Alsweet) plants were exposed to mixtures of ozone plus sulfur dioxide at different times of the day. Injury, evaluated either as necrosis or chlorophyll, was greatest at midday when stomatal conductance was greatest. Abscisic acid levels were similar over the day, and showed no relation to stomatal conductance. PMID- 16663932 TI - Relationship between Mineral Nitrogen Influx and Transpiration in Radish and Tomato. AB - Net ammonium and nitrate influx were independent of transpiration rate for intact seedlings of both a wild species of radish (Raphanus raphanistrum) and a wilty tomato mutant (Lycopersicon esculentum Mill. cv RR flacca). PMID- 16663933 TI - Presence in Photosystem II Core Complexes of a 34-Kilodalton Polypeptide Required for Water Photolysis. AB - Photosystem II (PSII) reaction center core complexes have been isolated and characterized from wild type (WT) Scenedesmus obliquus and from its LF-1 mutant. LF-1 thylakoids are blocked on the oxidizing side of PSII and have a reduced Mn content. Visible absorption and low temperature fluorescence spectra of both core complexes are identical and resemble those reported for spinach (Satoh, Butler 1978 Plant Physiol 61: 373-379). Lithium dodecyl sulfate-polycrylamide gel electrophoresis reveals that a protein alteration, originally observed in thylakoid membranes (Metz, Wong, Bishop 1980 FEBS Lett 114: 61-66), is retained in the PSII core particles. That is, a 34-kilodalton (kD) polypeptide, present in the WT core complex, is missing in the mutant, and the core complex of the mutant contains a 36-kD protein not present in the WT. The 34-kD intrinsic protein is also observed in O(2)-evolving PSII preparations and PSII core complexes from spinach. It is distinct from the 33-kD extrinsic protein first reported by T. Kuwabara and N. Murata (1979 Biochim Biophys Acta 581: 228-236). We suggest that the 34-kD protein is a site of Mn binding in the PSII membrane. PMID- 16663934 TI - Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans. AB - Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks. PMID- 16663935 TI - Lipase activities in scutella of maize lines having diverse kernel lipid content. AB - The maize lines, Illinois High Oil, Illinois Low Oil, and their F(1) generation contained about 18%, 0.5%, and 10%, respectively, of kernel lipids, which were present mostly in the scutella. We explored to see if the activities of gluconeogenic enzymes which appeared in postgerminative growth were proportional to the lipid content in each maize line. This proportionality was found to be valid in lipase, but the two glyoxysomal enzymes, catalase and isocitrate lyase, were the same in the three maize lines irrespective of the lipid content. The results suggest a difference in the genetic control of the gluconeogenic enzymes and a co-selection for high lipid content and high lipase activity through breeding. PMID- 16663936 TI - Urea-Elicited Changes in Relative Electrophoretic Mobility of Certain Glycinin and beta-Conglycinin Subunits. AB - Six molar urea in sodium dodecyl sulfate-polyacrylamide gels altered the relative electrophoretic mobility of several soybean protein subunits. Glycinin acidic polypeptide components A(3) and A(4) could be resolved from the other acidic polypeptides. A variant of the delta' subunit of beta-conglycinin was identified. PMID- 16663937 TI - Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories. AB - Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO(3) (-) and Mg(2+) concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C(3)); P. maximum (C(4) phosphoenolpyruvate carboxykinase); P. milioides (C(3)/C(4)); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C(3)); P. miliaceum (C(4) NAD malic enzyme); Zea mays and Sorghum bicolor (C(4) NADP malic enzyme); Moricandia arvensis (C(3)/C(4)); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C(3) species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO(2) and Mg(2+) activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID- 16663938 TI - Disulfiram metabolism in isolated mesophyll cells and inhibition of photosynthesis and cyanide-resistant respiration. AB - Tetraethylthiuram disulfide (disulfiram) stimulated medium acidification when added at a concentration of 0.4 millimolar to illuminated or nonilluminated suspensions of Asparagus sprengeri Regel mesophyll cells. Similar concentrations inhibited photosynthesis and cyanide-resistant respiration. The reduction product of disulfiram, diethyldithiocarbamic acid, accumulated in concentrations sufficient to account for the observed acidification. PMID- 16663939 TI - Gravitropism in Higher Plant Shoots : III. Cell Dimensions during Gravitropic Bending; Perception of Gravity. AB - Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130 degrees . Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing.Settling of amyloplasts was observed in cells of the starch sheath.Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending. PMID- 16663940 TI - Characteristics of glutamate dehydrogenase in mitochondria prepared from corn shoots. AB - The amination of alpha-ketoglutarate (alpha-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca(2+). The NADH-GDH purified 161 fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca(2+) in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca(2+) had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca(2+).About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca(2+) had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca(2+). In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar alpha-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca(2+), 10 and 50 millimolar NH(4) (+), and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation. PMID- 16663941 TI - An inhibitor of catalase induced by cold in chilling-sensitive plants. AB - An inhibitor of catalase accumulated when leaves of chilling-sensitive species were stored in the dark at 0 degrees C. The inhibitor could be removed from crude extracts by passing them through a column of Sephadex G-25. After this treatment, the catalase activity of extracts of chilled tissues was found to be equal to that of extracts from unchilled leaves. When chilled tissues were incubated at 20 degrees C, the inhibitor of catalase was lost, unless the tissues had been irreversibly damaged. It specifically inhibited plant catalase, and had no effect on mammalian catalase, plant malic dehydrogenase, or plant superoxide dismutase.Despite the presence of catalase inhibitor in extracts of chilled plants, no increase in the level of H(2)O(2) in chilled tissues was found, suggesting either that the inhibitor is compartmentalized and not in contact with catalase in vivo, or that the level of H(2)O(2) is controlled by means other than through catalase activity. Plant tissues normally contain H(2)O(2) which is destroyed by catalase when they are damaged. After chilling, H(2)O(2) leaking from already injured cells would not be so readily removed by the inhibited catalase, and could contribute to further injury by acting as a source of free radical oxidants. PMID- 16663942 TI - Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L. AB - Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N(2)-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides. PMID- 16663943 TI - Precocious Germination during In Vitro Growth of Soybean Seeds. AB - Immature Glycine max (L.) Merrill seeds were grown and matured in liquid medium at 25 degrees C under fluorescent light. In standard medium containing minerals, 146 millimolar sucrose and 62.5 millimolar glutamine (osmolality 0.24), precocious germination seldom occurred with a starting seed size of less than 300 milligrams fresh weight. Frequency of precocious germination increased with increased starting seed size. Sucrose concentration strongly affected precocious germination while glutamine concentration had no effect. Starting with 300 to 350 milligrams fresh weight seeds, treatments which reduced the sucrose concentration or lowered the osmolality of the culture medium stimulated precocious germination, and increased the fresh weight growth but not the dry weight growth of seeds. Increasing the osmolality to 0.38 with sucrose or mannitol prevented precocious germination without reducing dry weight accumulation in seeds. In medium with initially low osmolality, precocious germination was inhibited by addition of 1 to 100 micromolar abscisic acid to the medium without a reduction in seed growth. During growth and maturation of large soybean seeds in vitro, precocious germination and other abnormal tissue growth can be prevented by high sucrose or mannitol concentrations in the medium or by addition of abscisic acid. PMID- 16663944 TI - Abscisic Acid Metabolism in Relation to Water Stress and Leaf Age in Xanthium strumarium. AB - Intact plants of Xanthium strumarium L. were subjected to a water stress-recovery cycle. As the stress took effect, leaf growth ceased and stomatal resistance increased. The mature leaves then wilted, followed by the half expanded ones. Water, solute, and pressure potentials fell steadily in all leaves during the rest of the stress period. After 3 days, the young leaves lost turgor and the plants were rewatered. All the leaves rapidly regained turgor and the younger ones recommenced elongation. Stomatal resistance declined, but several days elapsed before pre-stress values were attained.Abscisic acid (ABA) and phaseic acid (PA) levels rose in all the leaves after the mature ones wilted. ABA-glucose ester (ABA-GE) levels increased to a lesser extent, and the young leaves contained little of this conjugate. PA leveled off in the older leaves during the last 24 hours of stress, and ABA levels declined slightly. The young leaves accumulated ABA and PA throughout the stress period and during the 14-hour period immediately following rewatering. The ABA and PA contents, expressed per unit dry weight, were highest in the young leaves. Upon rewatering, large quantities of PA appeared in the mature leaves as ABA levels fell to the pre-stress level within 14 hours. In the half expanded and young leaves, it took several days to reach pre-stress ABA values. ABA-GE synthesis ceased in the mature leaves, once the stress was relieved, but continued in the half expanded and young leaves for 2 days.Mature leaves, when detached and stressed, accumulated an amount of ABA similar to that in leaves on the intact plant. In contrast, detached and stressed young leaves produced little ABA. Detached mature leaves, and to a lesser extent the half expanded ones, rapidly catabolized ABA to PA and ABA-GE, but the young leaves did not. Studies with radioactive (+/-)-ABA indicated that in young leaves the conversion of ABA to PA took place at a much lower rate than in mature ones. Leaves of all ages rapidly conjugated PA to PA-glucose ester. Furthermore, when half expanded leaves were stressed on the intact plant, their rate of ABA catabolism was enhanced, an effect not observed in the young leaves.In conclusion, young leaves on intact Xanthium plants produce little stress-induced ABA themselves, but due to import and a low rate of catabolism accumulate more ABA and PA than mature leaves. PMID- 16663945 TI - A Comparison between Prolamellar Bodies and Prothylakoid Membranes of Etioplasts of Dark-Grown Wheat Concerning Lipid and Polypeptide Composition. AB - The aim of the present investigation was to find factors critical for the co existence of prolamellar bodies and prothylakoids in etioplasts of wheat (Triticum aestivum L. cv Starke II). The lipid composition of the prolamellar body and prothylakoid fractions was qualitatively similar. However, the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.6 +/- 0.1), as was the lipid content on a protein basis. Protochlorophyllide was present in both fractions. The dominating protein of the prolamellar body fraction was protochlorophyllide oxidoreductase. This protein was present also in prothylakoid fractions. The other major protein of the prothylakoid fraction was the coupling factor 1, subunit of the chloroplast ATPase. From the lipid and protein data, we conclude that prolamellar bodies are formed when monogalactosyl diacylglycerol is present in larger amounts than can be stabilized into planar bilayer prothylakoid membranes by lamellar lipids or proteins. PMID- 16663946 TI - Localization of Galactolipid Biosynthesis in Etioplasts Isolated from Dark-Grown Wheat (Triticum aestivum L.). AB - Etioplasts were isolated from leaves of dark-grown wheat (Triticum aestivum L. var Starke II). Galactolipid biosynthesis was assayed in an envelope-rich fraction and in the fraction containing the rest of the etioplast membranes by measuring incorporation of (14)C from uridine-diphospho[(14)C]galactose into monogalactosyl diacylglycerol and digalactosyl diacylglycerol. More than half of the galactolipid biosynthetic capability was found in the fraction of inner etioplast membranes. This fraction was subfractioned into fractions enriched in prolamellar bodies and membrane vesicles (prothylakoids), respectively. All membrane fractions obtained from etioplasts were able to carry out galactolipid biosynthesis, although the activity was very low in prolamellar body-enriched fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed markedly different polypeptide patterns between the different fractions. It is concluded that the capability of galactolipid biosynthesis of etioplasts probably is not restricted to the envelope, but is also present in the inner membranes of this plastid. PMID- 16663947 TI - Starch Degradation Metabolism towards Sucrose Synthesis in Germinating Araucaria araucana Seeds. AB - As starch is the main seed reserve material in both species of Araucaria of South America, A. araucana and A. angustifolia, it is important to understand starch breakdown in both embryo and megagametophyte tissues of Araucaria seeds. Sugar analysis by thin layer chromatography indicates that sucrose is the main sugar produced in both tissues. Enzyme reactions coupled to benzidine oxidation indicate that sucrose is the main sugar moved from the megagametophyte to the growing regions of the embryo via the cotyledons.Phosphorylase was detected in both embryo and megagametophyte tissues by the formation of [(32)P]glucose-1-P and by formation of [(14)C] amylopectin from [(14)C]glucose-1-P. The enzyme activity increases 5-fold in both embryo and gametophyte to a peak 18 hours after the start of imbibition. Debranching enzyme, alpha-glucosidase, and hexokinase are also present in both embryonic and megagametophytic tissues.Branched glucan oligosaccharides accumulate during this time, reaching a maximum 40 hours after imbibition starts, and decline after germination occurs.The pattern of activity of the enzymes studied in this work suggests that starch degradation is initiated by alpha-amylase and phosphorylase in the embryo and by phosphorylase mainly in the megagametophyte. Sucrose-P synthase seems to be the enzyme responsible for sucrose synthesis in both tissues. PMID- 16663948 TI - Effects of Chemical Treatments upon Photosynthetic Parameters in Soybean Seedlings. AB - The effects of various chemical treatments upon photosynthesis, soluble leaf protein, CO(2) compensation point, and leaf light transmission in soybean, Glycine max (L.) Merr., seedlings were examined following varying response periods after application at 14 to 17 days postemergence. The compounds N(6) benzyladenine (BA), 2-(4-chlorophenoxy)-2-methylpropanoic acid (CPMP), (4 chlorophenoxy)acetic acid (CPA), rhodanine-N-acetic acid (RAA), and 2,3,5 triiodobenzoic acid (TIBA) significantly increased soluble protein and decreased senescence, measured by leaf light transmission, at CO(2) concentrations below the compensation point in a survival chamber. All compounds except BA significantly decreased transmission values under ambient atmospheric conditions. In statistically significant experiments, applications of 3.49 millimolar CPMP increased net photosynthesis on a leaf area basis by an average of 14.4% at all trifoliolate positions with increases generally requiring response periods of 12 days or longer. RAA at 1.31 and 2.61 millimolar increased net photosynthesis by 19 to 36% following 13-day response periods. CPMP and other compounds tested had no effect upon the CO(2) compensation point after 4- to 8-day response periods. The effects of CPMP and RAA upon net photosynthesis and soluble protein appeared to involve a combined stimulation of protein synthesis and an antisenescent effect. There were no indications that any of the photosynthetic changes observed resulted from direct differential effects upon ribulose bisphosphate carboxylase oxygenase. The assays for soluble protein and light transmission responded more consistently to the chemicals than did photosynthesis. PMID- 16663949 TI - Detection of chemicals inhibiting photorespiratory senescence in a large scale survival chamber. AB - A large scale survival chamber was developed as a screen for detecting chemical treatments that extend the survival time of illuminated soybean seedlings at CO(2) concentrations below the compensation point. In theory, extended survival should indicate potential for improved crop performance via decreased photorespiration and increased photosynthetic efficiency. An automated control system regulated CO(2) concentrations, temperature and plant watering during a continuous CO(2)-removal photoperiod of 72 hours. An endogenously controlled circadian rhythm of net photosynthesis occurred throughout the continuous light treatment.Spray applications of 3.49 millimolar 2-(4-chlorophenoxy)-2 methylpropanoic acid (CPMP) significantly decreased leaf chlorophyll loss, compared with the control, after 72 hours of subcompensation-point stress. Treatment with CPMP also consistently increased leaf chlorophyll per unit area under nonstress greenhouse conditions. These effects may be due to increases in specific leaf weight produced by CPMP although the compound did not consistently act as a height retardant. The compound, 3-butyl-2-hydroxy-4H-pyrido[1,2 a]pyrimidin-4-one (BHPP), inhibited senescence under low CO(2) conditions but did not decrease leaf light transmission at ambient CO(2) levels. The cytokinin N(6) benzyladenine (BA) retarded low CO(2) stress senescence although greening effects were not observed. Neither 2-hydroxy-3-butynoic acid (HBA) nor its butyl ester, inhibitors of glycolate oxidase, influenced low CO(2) survival. Cyclohexanecarboxylic acid (CHCA) and sodium naphthenate had no effect upon subcompensation-point senescence.Antisenescence effects of CPMP, BHPP, and BA do not appear to be directly attributable to effects upon the competing carbon paths of photosynthesis and photorespiration. Protection against low CO(2) stress and increased chlorophyll synthesis under nonstress conditions may represent separate effects upon plastids by some of the compounds. This screen will identify compounds which inhibit photorespiratory senescence without decreasing the CO(2) compensation point. PMID- 16663950 TI - Purification and Properties of the Constitutive Arginase of Evernia prunastri. AB - Constitutive arginase (molecular weight 330,000) 920-fold purified from Evernia prunastri thallus, is activated by putrescine, l-ornithine, and agmatine with K(a) values of 2.7, 1.1, and 5.8 millimolar, respectively. Constitutive arginase is also activated by endogenous l-arginine, reaching its maximum activity at 16 hours of incubation on Tris-HCl (pH 9.15) with a subsequent decrease. Urea behaves as a mixed inhibitor of the enzyme with a K(i) value of 2.6 millimolar. Atranorin and evernic acid behave as in vitro activators of the enzyme; usnic acid does not have any significant effect as activator. PMID- 16663951 TI - Intracellular localization of lipoxygenases-1 and -2 in germinating soybean seeds by indirect labeling with protein a-colloidal gold complexes. AB - Soybean lipoxygenases-1 and -2 were localized intracellularly in seeds at various stages of germination by indirect labeling of cryosections with protein A colloidal gold complexes. Two sizes of gold particles (Au(5) and Au(16)) were used in single- and double-labeling experiments. In primary leaves, lipoxygenases are demonstrated to occur in vacuolating parenchyma cells but not in massive, nondifferentiated cells. In cotyledons, both isoenzymes are localized in the cytoplasm of storage parenchyma cells and in an aberrant type of protein bodies, occurring in hypodermis and vascular bundle sheath cells. No association has been found with either protein bodies in storage parenchyma cells or lipid bodies, mitochondria, and other organelles in any type of cell. The possible significance of lipoxygenase in the metabolism of storage lipids and its possible function as a regulatory enzyme are discussed on the basis of the random distribution throughout the cytoplasm of storage parenchyma cells and the course of biochemical processes during seed germination. PMID- 16663952 TI - Identification of two methionine transfer RNA genes in the maize mitochondrial genome. AB - Two methionine transfer RNA (tRNA) genes were identified in the maize mitochondrial genome by nucleotide sequence analysis. One tRNA gene was similar in nucleotide sequence and secondary structure to the initiator methionine tRNA genes of eubacteria and higher plant chloroplast genomes. This tRNA gene also had extensive nucleotide homology (99%) with an initiator methionine tRNA gene described for the wheat mitochondrial genome. The other methionine tRNA gene sequence was distinct and more closely resembled an elongator methionine tRNA. PMID- 16663953 TI - Use of Carbon Isotopes to Estimate Incorporation of Added CO(2) by Greenhouse Grown Tomato Plants. AB - A method is presented which uses the (13)C and (14)C isotope abundance in CO(2) enriched greenhouse crops to determine the percentage of plant organic carbon derived from artificially added CO(2). In a greenhouse experiment with CO(2) concentrations elevated to 1100 +/- 100 microliters per liter during part of the daylight hours and maintained at normal atmospheric concentrations (340 microliters per liter) during the rest of the time, it was shown by (14)C analysis that between 41% and 42% of the carbon in tomato plants (Lycopersicon esculentum var 4884) came from the artificially added CO(2). Similar results were obtained from (13)C analyses when the CO(2) pressure-dependent isotope separation was taken into account. PMID- 16663954 TI - Activation and de novo synthesis of hydrogenase in chlamydomonas. AB - Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.The adaptation process is very sensitive to the availability of iron. Iron deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase. PMID- 16663955 TI - Is HCO(3) Transport in Anabaena a Na Symport? AB - Na(+) strongly promoted HCO(3) (-) transport in Anabaena variabilis. The effect was highly specific to this cation. Kinetic analysis indicated a progressive decrease in the K(m) (HCO(3) (-)) of the transport system with increasing Na(+) concentration. V(max) was also affected. We raise the possibility that the transport is a Na(+)-HCO(3) (-) symport; alternatively, that a Na(+)-H(+) antiport (or Na(+)-OH(+) symport) system mediates the efflux of the OH(-) ions derived from the entering HCO(3) (-) ions, and that this antiport can rate-limit HCO(3) (-) influx. PMID- 16663956 TI - In Vitro Inhibition of the Plastid and Cytosolic Isozymess of 6-Phosphogluconate Dehydrogenase from Developing Endosperm of Ricinus communis by Fructose 2,6 bisphosphate. AB - Activities of the cytosolic and plastid isozymes of 6-phosphogluconate dehydrogenase from developing endosperm of Ricinus communis L. seeds were inhibited in vitro by hexosebisphosphates. Inhibition constants for glucose 1,6 bisphosphate were 221 and 209 micromolar for the cytosolic and plastid isozymes, respectively, and corresponding values for fructose 2,6-bisphosphate were 10.5 and 8.6 micromolar. In each case inhibition was of a mixed noncompetitive nature relative to 6-phosphogluconate. While the levels and distribution of fructose 2,6 bisphosphate in castor oil seed endosperm cells are not yet known, the levels reported to occur in leaf cytosol would be high enough to significantly inhibit carbon flux through the pentosephosphate pathway due to inhibition of 6 phosphogluconate dehydrogenase activity. PMID- 16663957 TI - Effects of Salicylic and Acetylsalicylic Acids on the Scotonastic and Photonastic Leaflet Movements of Cassia fasciculata. AB - Salicylic and acetylsalicylic acids applied on excised leaves of Cassia fasciculata modify the dark-induced (scotonastic) and light-induced (photonastic) leaflet movements. They inhibit the scotonastic movements in a dose-dependent manner from 1 x 10(-4) to 1 x 10(-3) molar and they promote the photonastic movements at an optimum concentration of 5 x 10(-4) molar. These results suggest that these phenolic compounds do not act specifically on the K(+) uptake, which was shown to be inhibited by their action on other materials. PMID- 16663958 TI - Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules. AB - I have measured acetylene diffusion through plant tissues including nodules from several species of legume-vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O(2) where there is no N(2) reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations. PMID- 16663959 TI - Chronobiology of Aging in Albizzia julibrissin: I. An Automated, Computerized System for Monitoring Leaflet Movement; The Rhythm in Constant Darkness. AB - We are using leaflet movements in Albizzia julibrissin as a model system for investigating the chronobiology of aging. To monitor leaflet movements during long dark periods with adequate temporal and spatial resolution, we designed an automated, computerized photoelectric monitoring system. Each of 12 leaflet pairs was positioned in an individual light-proof container, with one leaflet immobilized. The angle of the mobile leaflet was monitored by a photosensor array using a low intensity infrared beam. Leaflet position was determined by custom developed software, using information on the shading patterns of the sensors. Data on leaflet angle as a function of time were collected and stored on a floppy disc and then printed in numerical and graphical form.Oscillations of young, middle-aged, and old leaflets persist during 7 d of darkness with a periodicity close to 24 h. Period length appears to be age-independent, but rhythmic wave form is age-dependent. The older the leaflet, the earlier and more completely it opens and the less completely it closes. PMID- 16663960 TI - beta-Glucosidase Activity in Corn Roots: Problems in Subcellular Fractionation. AB - Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that beta-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the beta glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed. PMID- 16663961 TI - Mechanism of amino Acid uptake by sugarcane suspension cells. AB - The amino acid carriers in sugarcane suspension cells were characterized for amino acid specificity and the stoichiometry of proton and potassium flux during amino acid transport.Amino acid transport by sugarcane cells is dependent upon three distinct transport systems. One system is specific for neutral amino acids and transports all neutral amino acids including glutamine, asparagine, and histidine. The uptake of neutral amino acids is coupled to the uptake of one proton per amino acid; one potassium ion leaves the cells for charge compensation. Histidine is only taken up in the neutral form so that deprotonation of the charged imidazole nitrogen has to occur prior to uptake. The basic amino acids are transported by another system as uniport with charge compensating efflux of protons and potassium. The acidic amino acids are transported by a third system. Acidic amino acids bind to the transport site only if the distal carboxyl group is in the dissociated form (i.e. if the acidic amino acid is anionic). Two protons are withdrawn from the medium and one potassium leaves the cell for charge compensation during the uptake of acid amino acids. Common to all three uptake systems is a monovalent positively charged amino acidproton carrier complex at the transport site. PMID- 16663962 TI - Subcellular Localizations of Two Dolichos biflorus Lectins. AB - The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRM]) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCl, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied [(125)I]CRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions. PMID- 16663963 TI - Development and Distribution of a Lectin from the Stems and Leaves of Dolichos biflorus. AB - The stems and leaves of the Dolichos biflorus plant contain a lectin that cross reacts with antiserum against the seed lectin. This cross-reactive material (CRM) was followed during early seedling growth, stem elongation, and seed development using a specific radioimmunoassay.No CRM was detected in developing seeds, but very low levels were found in dormant and imbibed seeds. As germination proceeds, the CRM accumulates at the apex of both etiolated and green seedlings in the epicotyl and leaves. Lower amounts of CRM are found in the cotyledons and hypocotyl, but no CRM was detected in the roots.The amount of CRM in the first and second stem internodes increases during elongation and gradually declines after the completion of elongation. Approximately 80% of the CRM in the stems of 19-day-old Dolichos biflorus plants is associated with the elongating tissues. These results are discussed with respect to the possible roles of lectins in plants. PMID- 16663964 TI - Localization and Substrate Specificity of Glycosidases in Vacuoles of Nicotiana rustica. AB - Vacuoles isolated from Nicotiana rustica var brasilia have been shown to contain significant levels of glycosidase activity when assayed using p-nitrophenyl glycosides as substrates. The substrate specificity for the glycosidases in the vacuolar fraction closely paralleled that found in the protoplasts, and the leaf tissue from which the vacuoles were isolated. The substrate specificity of the vacuolar enzyme(s) was different from glycosidic activity found in the commercial digestive enzyme preparations used to isolate the protoplasts from leaf tissue. It was demonstrated that 70 to 90% of the glycosidases that were found in the protoplasts appeared to be localized within the vacuole, when the p-nitrophenyl substrates alpha- and beta-;d-galactose, beta-d-glucose, and alpha-d-mannose were used. Neither the vacuolar nor the protoplast enzymes were active towards the naturally occurring phenolic glycoside, rutin. alpha-Mannosidase appears to be a valuable marker enzyme for vacuoles isolated from mesophyll leaf cells of tobacco. PMID- 16663965 TI - Formation of Tryptophol Galactoside and an Unknown Tryptophol Ester in Euglena gracilis. AB - The unicellular alga Euglena gracilis Klebs ;Z' converted exogenous indole-3 ethanol (trytophol) to two major metabolites: tryptophol galactoside and an unknown compound, and to minor amounts of indole-3-acetic acid, tryptophol acetate, and tryptophol glucoside. The unknown was hydrolyzed to tryptophol by methanolic ammonia and should therefore be a tryptophol ester. The galactoside was identified as 2-(indol-3-yl)ethyl-beta-d-galactopyranoside. This structure was established by comparison with an authentic standard involving chromatographic methods, ultraviolet and mass spectroscopy, enzymic and acid hydrolysis, and identification of the galactose in the hydrolysate. By forming tryptophol galactoside, Euglena differs from the higher plants examined so far, for which the corresponding glucoside is the only sugar conjugate of tryptophol detected. PMID- 16663966 TI - Sugar transport in isolated corn root protoplasts. AB - Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K(m) of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42 degrees C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50 degrees C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed. PMID- 16663967 TI - Carbon Balance of Sorghum Plants during Osmotic Adjustment to Water Stress. AB - The daily (24-hour) carbon balances of whole sorghum plants (Sorghum bicolor L. Moench cv BTX616) were continuously measured throughout 15 days of water stress, followed by rewatering and 4 more days of measurements. The plants were grown under controlled environment conditions typical of warm, humid, sunny days. During the first 12 days, osmotic potentials decreased in parallel with decreased water potentials to maintain pressure potentials near 0.5 kilojoules per kilogram (5 bars). Immediately before rewatering on day 15, the water potential was -3.0 kilojoules per kilogram. Osmotic adjustment at this point was 1.0 kilojoules per kilogram, as measured by the decrease in the water potential at zero turgor from its initial value of -1.4 kilojoules per kilogram.Gross input of carbon was less but the fraction retained was greater because a smaller fraction was lost through respiration in stressed plants than in unstressed plants. This was attributed to a lower rate of biomass synthesis, and conversely a higher rate of storage of photosynthate, due to inhibition of leaf expansion. The reduction in the cost associated with biomass synthesis more than balanced any metabolic cost of osmotic adjustment. The net daily gain of carbon was always positive in the stressed plants.There was a large burst of respiration on rewatering, due to renewed synthesis of biomass from stored photosynthate. Over the next 3 days, osmotic adjustment was lost and the daily carbon balance returned to that typical of nonstressed plants. Thus, osmotic adjustment allowed the stressed plants to accumulate biomass carbon throughout the cycle, with little additional metabolic cost. Carbon stored during stress was immediately available for biomass synthesis on rewatering. PMID- 16663968 TI - Photosynthesis in Tall Fescue : V. Analysis of High PSI Activity in a Decaploid Genotype. AB - Previous work in our laboratory (Krueger, Miles 1981 Plant Physiol 68: 1110-1114) indicated that a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) which exhibits unusually high net photosynthesis rates also had high potential rates of photosynthetic electron transport through photosystem I (PSI) compared to the typical hexaploid genotype (V6-802). Analysis of electron transport activity revealed that the oxidizing side of PSI as the major site of difference. Examination of the whole thylakoids and subchloroplast particle protein components of the common hexaploid and the decaploid genotypes had major polypeptide differences at 30, 21, and 12.5 kilodaltons. These differences could not be assigned to a specific physiological function in PSI. The decaploid had increased P(700) and plastocyanin content on a chlorophyll basis. Antibodies raised against fescue plastocyanin were used to quantitate plastocyanin in crude (Triton X-100) solubilized extracts of plant material. Results showed that the decaploid had 16% and 40% more plastocyanin on a weight and area basis, respectively. The antibodies did not inhibit electron transport (diaminodiurene to methyl viologen) in isolated thylakoids strengthening the hypothesis of plastocyanin as an internal mobile electron shuttle. The trend of inhibition of plastocyanin by KCN was similar in the two genotypes but the decaploid had 15 to 20% higher rates of electron flow under nearly all inhibiting conditions. PMID- 16663969 TI - Changes of Sucrose-Phosphate Synthase Activity in Barley Primary Leaves during Light/Dark Transitions. AB - The activity of sucrose-phosphate synthase (SPS) in 9-day-old barley (Hordeum vulgare L.) primary leaves was measured over a 24-hour period. Extractable enzyme activity was constant in the light, decreased 50 to 60% during the first one-half hour of darkness, and then returned to full activity before the start of the normal light period. Decreases of SPS activity in the dark were fully reversed by less than 10 minutes of illumination. In contrast to results with barley, the measurable activity of SPS in soybean, spinach, and pea leaves was unchanged during the first hour of darkness. Changes of SPS activity in barley primary leaves were stable upon gel filtration. The exact biochemical mechanism responsible for the enzyme activity changes in barley leaf extracts is unknown. The above findings support the suggestion by de Fekete (1973 Eur J Biochem, 10: 73-80) that SPS is controlled by posttranslational protein modification. These results are discussed in relation to the regulation of photosynthetic sucrose metabolism. PMID- 16663970 TI - A comparison of methods for determining compartmental analysis parameters. AB - The traditional method for determining compartmental analysis parameters relies on a visual selection of data points to be used for regression of data from each cellular compartment. This method is appropriate when the compartments are kinetically discrete and are easily discernible. However, where treatment effects on compartment parameters are being evaluated, a more objective method for determining initial parameters is desirable.Three methods were examined for determining initial isotopic contents and half-times of (86)Rb elution from cellular compartments using theoretical data with known parameters. Experimental data from roots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) and barley (Hordeum vulgare L.) intact seedlings were also used. The three methods were a visually assisted, linear regression on data of semilog plot of isotope elution versus time, a microcomputer-assisted, linear regression on semilog plot where maximization of the square of the correlation coefficient (r(2)) was the criterion to determine data points needed for each regression and a mainframe computer-assisted, direct nonlinear regression on elution data using a model of the sum of three exponential decay functions. The visual method resulted in the least accurate estimates of compartmental analysis parameters. The microcomputer assisted and nonlinear regression methods calculated the parameters equally well. PMID- 16663971 TI - Mycorrhizal effects on potassium fluxes by northwest coniferous seedlings. AB - In ectomycorrhizae, the relative abilities of mycobiont and host plant to take up and store inorganic nutrients are not easily determined due to the intimate physical relationship of the two components forming the association. Since compartmental analysis of solute elution can estimate cellular compartment pool sizes and unidirectional fluxes across membranes, we have used this method to study ectomycorrhizal coniferous roots. Rubidium-86, used as a tracer for potassium, was loaded into and eluted from intact roots of nonmycorrhizal and mycorrhizal (with the fungus Hebeloma crustuliniformme [Bull.: St. Amans Quel] Douglas fir (Pseudotsuga menziesii [Mirb.] Franco), western hemlock (Tsuga heterophylla [Raf.] Sarg.) and Sitka spruce (Picea sitchensis [Bong.] Carr.) seedlings.Mycorrhizas significantly increased (86)Rb uptake rates while decreasing the amount of (86)Rb released to the external solution. Using compartmental analysis, the flux data suggest that the primary mycorrhizal effects were to increase inward potassium fluxes across the fungal tonoplast and to decrease potassium efflux across the fungal tonoplast, as compared with nonmycorrhizal seedling roots. The result was greater potassium storage, presumably in the fungal vacuole. The three coniferous species responded differently to fungal infection with respect to potassium fluxes. Both cytoplasmic and vacuolar fluxes for mycorrhizal hemlock were 2-fold greater than for spruce and 3-fold greater than for Douglas fir. These results demonstrate the usefulness of compartmental analysis for study of ion fluxes in intact mycorrhizal root systems and suggest that the fungal tonoplast may be the site for regulation of potassium fluxes in these coniferous roots. PMID- 16663972 TI - Surface galactolipids of wheat protoplasts as receptors for soybean agglutinin and their possible relevance to host-parasite interaction. AB - Soybean agglutinin, a lectin specific for N-acetyl-d-galactosamine and d galactose, was previously shown to agglutinate wheat leaf protoplasts (Larkin 1978 Plant Physiol 61: 626-629). We investigated the receptors for soybean agglutinin on the plasma membrane of these protoplasts. After treatment of the protoplasts with galactose oxidase, they were no longer agglutinated by the lectin, whereas upon reduction of the galactose oxidase-treated protoplasts with sodium borohydride the susceptibility to agglutination was restored. Analysis of the glycolipids of protoplasts surface labeled by the galactose oxidase borotritide method, revealed that the radioactivity was mainly present in monogalactosyldiglyceride and digalactosyldiglyceride. The same galactolipids were identified as the only receptors for soybean agglutinin by direct binding of the (125)I-labeled lectin to a thin layer chromatogram of the glycolipids of wheat leaf protoplasts. PMID- 16663973 TI - Inhibitors of lipase activities in soybean and other oil seeds. AB - In the cotyledon extracts of seedlings of many oil seeds, including soybean, sunflower, cucumber, and peanut, the in vitro lipase activity was too low to account for the observed in vivo lipolysis. The low in vitro lipase activity was due to the presence of lipase inhibitors in the extracts. The inhibitors from soybean were characterized based on their effects on the hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases. The inhibitors were not dialyzable and unaltered by RNase and beta-galactosidase treatment. However, they were sensitive to heating and protease digestion. The inhibitory effect of the inhibitors was expressed irrespective of the sequence of the addition of lipase, substrate, and inhibitors to the assay medium. The inhibitory effect was equally expressed when the inhibitors were added either before or after the lipase reaction had been in progress. The inhibitory effect of the inhibitors was independent of the amount of lipase present in the assay, but was dependent on the amount of substrate added. High substrate concentration eliminated totally the inhibitory effect of the inhibitors. Most of the inhibitors were recovered in the soluble fraction in subcellular fractionation. They were present in the 2-4S and not in the 7S, and 11S (storage proteins) protein fraction. There was a gradual decrease of the inhibitors in the cotyledons in the postgerminative growth. We suggest that the inhibitors are proteins which bind to the surface of the substrate micelles. The binding prevents the normal functioning of lipase which acts on the interfacial area between the aqueous medium and the micelle surface. PMID- 16663974 TI - Photo- and Metabolite Regulation of the Synthesis of Ribulose Bisphosphate Carboxylase/Oxygenase and the Phycobiliproteins in the Alga Cyanidium caldarium. AB - In the eukaryotic and unicellular alga Cyanidium caldarium the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and the light gathering proteins phycocyanin (PC) and allophycocyanin (APC) is under the control of light and glucose, which is a metabolizable carbon source for this organism. Light promotes the synthesis of these proteins while glucose has a strong inhibitory effect on this process. All subunits of the proteins mentioned above are in vitro translation products of poly (A)(-)-RNA (Steinmuller, Kaling, Zetsche 1983 Planta 159: 308-313). Both factors-light and glucose-exert their effects mainly by modulation of the level of translatable messenger RNA for these proteins. Under autotrophic growth conditions the level of translatable RuBPCase , PC-, and APC-messenger RNA is high, whereas in the presence of glucose the level of these mRNAs is low or not detectable at all. PMID- 16663975 TI - Loss of recovery capacity of plasmalemma k influx after cutting in chlorsulfuron pretreated maize roots. AB - Active K(+) influx was studied in apical segments from maize (Zea mays L., hybrid lines XL 342) and pea (Pisum sativum L. var Laxton superbo) seedlings pretreated with the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2 yl) aminocarbonyl]benzenesulfonamide).Even though both plants were sensitive to chlorsulfuron, a strong inhibition of K(+) uptake only was evident in maize root segments after 12 hours pretreatment with 10 micromolar chlorsulfuron. The inhibition was revealed only when maize root segments were washed for 2 hours before uptake measurements. This was done in order to recover K(+) influx inhibited by cutting injury. Consequently, we demonstrated that roots from chlorsulfuron pretreated maize seedlings lost the capacity to recover from cutting injury by washing. By contrast, K(+) influx in pea roots was not inhibited by chlorsulfuron because pea roots notoriously do not exhibit the ;washing' effect. PMID- 16663976 TI - Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L. AB - Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe(III)EDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H(+) (1H(+)/2 electrons) and K(+) ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts. PMID- 16663977 TI - Role of gibberellin in the growth response of submerged deep water rice. AB - We have shown previously that ethylene, which accumulates in the air spaces of submerged stem sections of rice (Oryza sativa L. cv "Habiganj Aman II"), is involved in regulating the growth response caused by submergence. The role of gibberellins in the submergence response was studied using tetcyclacis (TCY), a new plant growth retardant, which inhibits gibberellin biosynthesis. Stem sections excised from plants that had been watered with a solution of 1 micromolar TCY for 7 to 10 days did not elongate when submerged in the same solution or when exposed to 1 microliter per liter ethylene in air. Gibberellic acid (GA(3)) at 0.3 micromolar overcame the effect of TCY and restored the rapid internodal elongation in submerged and ethylene-treated sections to the levels observed in control sections that had not been treated with TCY. The effect of 0.01 to 0.2 micromolar GA(3) on internodal elongation was enhanced two- to eight fold when 1 microliter per liter ethylene was added to the air passing through the chamber in which the sections were incubated. GA(3) and ethylene caused a similar increase in cell division and cell elongation in rice internodes. Thus, ethylene may cause internodal elongation in rice by increasing the activity of endogenous GAs. In internodes from which the leaf sheath had been peeled off, growth in response to submergence, ethylene and GA(3) was severely inhibited by light. PMID- 16663978 TI - Concentrations of Abscisic Acid and Indole-3-Acetic Acid in Soybean Seeds during Development. AB - Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in seed parts were determined during reproductive development of soybean plants (Glycine max [L.] Merr. cv ;Chippewa 64'). The concentration of ABA and IAA changed independently in individual seed parts with time. Measurement of the level of ABA and IAA in whole seeds masked the changes which occurred in individual seed tissues. The concentration of ABA was generally highest and that of IAA was generally lowest in the embryonic axis of soybean seeds. In the testa, the IAA concentration was generally highest while the ABA concentration was generally the lowest compared to other parts of the seed. PMID- 16663979 TI - Effects of pod removal on the transport and accumulation of abscisic Acid and indole-3-acetic Acid in soybean leaves. AB - Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the second most recently expanded trifoliolate leaf were determined during reproductive development of soybean (Glycine max [L.] Merr cv ;Chippewa 64'). The concentration of ABA in leaves was constant during most of the seed filling period until the seeds began to dry. The concentration of IAA in the leaves decreased throughout development. Removal of pods 36 hours prior to sampling resulted in increased concentrations of ABA in leaves during the period of rapid pod filling but had little effect on the concentration of IAA in leaves. ABA appears to accumulate in leaves after fruit removal only when fruits represent the major sink for photosynthate.ABA and IAA moving acropetally and basipetally in petioles of soybean were estimated using a phloem exudation technique. ABA was found to move mostly in the basipetal direction in petioles (away from laminae). IAA, primarily in the form of ester conjugate(s), was found to be moving acropetally (toward laminae) in petioles. The highest amount of IAA ester(s) was found in petiole exudate during the mid and late stages of seed filling. Removal of fruits 36 hours prior to exudation reduced the amount of IAA ester recovered in exudate, suggesting that fruits were a source of the IAA conjugate in petiole exudate. PMID- 16663980 TI - Cell wall cementing materials of grass leaves. AB - Treatment of grass leaves with either a purified pectin lyase of Aspergillus japonicus or a purified xylanase of Trichoderma viride could lead to the isolation of some single leaf cells. However, a mixture of pectin lyase and xylanase brought about more rapid isolation of single cells than did either of the two enzymes alone, indicating a synergistic effect. Analysis of the components released from oat cell walls by the enzymes indicated that both homogalacturonans with a high degree of esterification and a kind of glucuronoarabinoxylan with ferulic acid ester may play a role in cell wall cementing in grass leaves. PMID- 16663981 TI - Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots. AB - The characteristics of Ca(2+) transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca(2+) and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0.The Ca(2+)-dependent modulation protein, calmodulin, was tested for its effect on Ca(2+) transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca(2+) transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca(2+) into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca(2+) transport activity. The inhibition of Ca(2+) transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca(2+) uptake into root endoplasmic reticulum. PMID- 16663982 TI - Photosynthesis and Ribulose 1,5-Bisphosphate Concentrations in Intact Leaves of Xanthium strumarium L. AB - The interacting effects of the rate of ribulose 1,5-bisphosphate (RuBP) regeneration and the rate of RuBP utilization as influenced by the amount and activation of RuBP carboxylase on photosynthesis and RuBP concentrations were resolved in experiments which examined the kinetics of the response of photosynthesis and RuBP concentrations after step changes from a rate-saturating to a rate-limiting light intensity in Xanthium strumarium. Because RuBP carboxylase requires several minutes to deactivate in vivo, it was possible to observe the effect of reducing the rate of RuBP regeneration on the RuBP concentration at constant enzyme activation state by sampling very soon after reducing the light intensity. Samples taken over longer time periods showed the effect of changes in enzyme activation at constant RuBP regeneration rate on RuBP concentration and photosynthetic rate. Within 15 s of lowering the light intensity from 1500 to 600 microEinsteins per square meter per second the RuBP concentration in the leaves dropped below the enzyme active site concentration, indicating that RuBP regeneration rate was limiting for photosynthesis. After longer intervals of time, the RuBP concentration in the leaf increased as the RuBP carboxylase assumed a new steady state activation level. No change in the rate of photosynthesis was observed during the interval that RuBP concentration increased. It is concluded that the rate of photosynthesis at the lower light intensity was limited by the rate of RuBP regeneration and that parallel changes in the activation of RuBP carboxylase occurred such that concentrations of RuBP at steady state were not altered by changes in light intensity. PMID- 16663983 TI - Isolation and Characterization of Swainsonine from Texas Locoweed (Astragalus emoryanus). AB - Swainsonine (1,2,8-trihydroxyoctahydroindolizine) was isolated from locoweed (Astragalus emoryanus) that grows in Texas. Using a biological assay as a measure of activity and purity, a relatively straightforward purification of the compound is described. The purified material was a potent inhibitor of jack bean alpha mannosidase and also of glycoprotein processing. The positive ion electron impact mass spectra of this compound was identical to that of authentic swainsonine. Nuclear magnetic resonance analysis also confirmed that the material was swainsonine. PMID- 16663984 TI - A Numerical Approach to Measurement of CO(2) Exchange Transients by Infrared Gas Analysis. AB - Open flow-through systems coupled to infrared gas analyzers have been frequently employed in the study of CO(2) exchange transients such as the postillumination burst observed in leaves of C(3) plants. A major limitation associated with use of such systems is their non-steady state response to rapid changes in leaf CO(2) exchange rate. A previous publication outlined a numerical approach to model the analyzer response as a function of CO(2) exchange rate and thus permit estimation of the postillumination burst (Peterson 1983 Plant Physiol 73: 978-982). The model is critically analyzed within the framework of the physics of solute dispersion as previously described for linear flow systems. Thus, the numerical simulation is validated on the basis of physical principle. Additional improvements to the previous model are described which enhance the accuracy and efficiency of use of this technique for estimation of photorespiration. PMID- 16663985 TI - Tissue cultures derived from ineffective root nodules of alfalfa : callus initiation and enzymic comparisons. AB - Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti.Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions. PMID- 16663986 TI - Comparative Investigations on the Metabolism of 2-(2,4-Dichlorophenoxy)Isobutyric Acid in Plants and Cell Suspension Cultures of Lycopersicon esculentum. AB - The metabolism of [(14)CH(3)]2-(2,4-dichlorophenoxy)isobutyric acid (DIB) was studied in plants and cell suspension cultures of Lycopersicon esculentum Mill. sp. ;Lukullus'. Both plants and cells in suspension culture showed a rapid uptake of DIB from nutrient media. The metabolites, isolated by extraction with methanol and separated by chromatographic methods, were identified by enzymic, chemical, and spectrometric methods. Two conjugates of the carboxyl with 2 and 3 moles glucose per mole DIB and, to a smaller extent, its beta-d-glucopyranosyl ester, were formed in both intact plants and cell suspension cultures, but there were quantitative differences. PMID- 16663987 TI - Gravitropism in Higher Plant Shoots : II. Dimensional and Pressure Changes during Stem Bending. AB - Dimensional changes during gravitropic bending of cocklebur (Xanthium strumarium L.) dicot stems were measured using techniques of stereo photogrammetry. The differential growth is from an increased growth rate on the bottom of the stem and a stopping or contraction of the top.Contraction of the top was especially evident upon release and immediate bending of horizontal stems that had been restrained between stiff wires for 36 hours. The energy for this could have been stored in both the top and bottom, since the bottom elongated, and the top contracted.Forces developed during bending were measured by fastening a stem tip to the end of a bar with attached strain gauges and recording electrical output from the strain gauges. Restrained mature cocklebur stems continued to accumulate potential energy for bending for about 120 hours, after which the recorded force reached a maximum.Pressures within castor bean (Ricinus communis L.) stems were also measured with 3.5-millimeter diameter pressure transducers. As expected, the pressure on the bottom of the restrained plants increased with time; pressures decreased in vertical controls, tops of restrained stems, and bottoms of free bending stems. Pressures increased in tops of free-bending stems. When restrained plants were released, pressure on the bottom decreased and pressure on the top increased. Results suggest a possible role for cell contraction in the top of stems bending upward in response to gravity. PMID- 16663988 TI - Metabolic Regulation during Glyceollin Biosynthesis in Green Soybean Hypocotyls. AB - The accumulation of the isoflavonoid phytoalexin, glyceollin, occurs in hypocotyls of green soybean seedlings (Glycine max L. Merr. cv Harosoy 63) in response to the injection of a glucan elicitor isolated from the mycelial walls of the fungus, Phytophthora megasperma f. sp. glycinea. This accumulation, which levels off after 24 hours, is preceded by a dramatic, transient rise in extractable activities of two early enzymes in the biosynthetic pathway, phenylalanine ammonia-lyase (PAL) and p-coumaryl CoA ligase (pCL). The maximum amount of extractable activity occurs 12 to 16 hours after elicitor treatment and is coincident with the most rapid period of glyceollin accumulation. These results suggest a regulatory role for these early enzymes in the biosynthesis of this secondary metabolite. High performance liquid chromatography analysis of the early intermediates in the pathway further corroborates this hypothesis. The relative pool size and rate of turnover of p-coumaric acid, an early intermediate in glyceollin production, increase during the period of rapid increases in enzyme activities. Removal of cotyledons from elicitor-treated seedlings reduces glyceollin accumulation approximately 70%. This limitation of phytoalexin accumulation by cotyledon removal is correlated with a similar cotyledon effect on reduction of extractable activities of both PAL and pCL as well as a decrease in the flux of carbon through the p-coumaric acid pool. This research further supports the hypothesis that early enzymic steps in a biosynthetic pathway diverting carbon from primary to secondary metabolites function as regulatory control points. PMID- 16663989 TI - Leaf Carbohydrate Status and Enzymes of Translocate Synthesis in Fruiting and Vegetative Plants of Cucumis sativus L. AB - Carbon partitioning in the leaves of Cucumis sativus L., a stachyose translocating plant, was influenced by the presence or absence of a single growing fruit on the plant. Fruit growth was very rapid with rates of fresh weight gain as high as 3.3 grams per hour. Fruit growth was highly competitive with vegetative growth as indicated by lower fresh weights of leaf blades, petioles, stem internodes and root systems on plants bearing a single growing fruit compared to plants not bearing a fruit. Carbon exchange rates, starch accumulation rates and carbon export rates were higher in leaves of plants bearing a fruit. Dry weight loss from leaves was higher at night from fruiting plants, and morning starch levels were consistently lower in leaves of fruiting than in leaves of vegetative plants indicating rapid starch mobilization at night from the leaves of fruiting plants. Galactinol, the galactosyl donor for stachyose biosynthesis, was present in the leaves of fruit-bearing plants at consistently lower concentration than in leaves of vegetative plants. Galactinol synthase, and sucrose phosphate synthase activities were not different on a per gram fresh weight basis in leaves from the two plant types; however, stachyose synthase activity was twice as high in leaves from fruiting plants. Thus, the lower galactinol pools may be associated with an activation of the terminal step in stachyose biosynthesis in leaves in response to the high sink demand of a growing cucumber fruit. PMID- 16663990 TI - Purification and Characterization of Extracellular Pectinesterases from Phytophthora infestans. AB - Constitutively produced extracellular pectinesterases from culture filtrates of the potato late blight fungus Phytophthora infestans were purified and characterized. One enzyme (PE II) was purified to homogeneity. Sodium dodecyl sulfate electrophoresis of the second enzyme (PE I) revealed two protein bands; there are indications that both proteins are pectinesterases, which were not separable by a number of different techniques. Thus, P. infestans might produce three pectinesterases in vitro. Enzyme activities were optimal in the neutral pH range and were largely dependent on the presence of NaCl or CaCl(2) in the reaction medium. The molecular weight of the PE I-complex was between 45 and 48 kilodaltons, and the one of PE II was between 35 and 40 kilodaltons. Further investigations will help us to clarify the role of these enzymes during pathogenesis. PMID- 16663991 TI - Comparative Water Relations of Phaseolus vulgaris L. and Phaseolus acutifolius Gray. AB - Leaf area expansion, dry weight, and water relations of Phaseolus vulgaris L. and P. acutifolius Gray were compared during a drying cycle in the greenhouse to understand the characteristics which contribute to the superior drought tolerance of P. acutifolius. Stomates of P. acutifolius closed at a much higher water potential than those of P. vulgaris, delaying dehydration of leaf tissue. P. acutifolius had a more deeply penetrating root system, which also contributes to its drought tolerance. Root-shoot ratios did not differ between the two species either under well watered or water stressed conditions. Leaf osmotic potential was also similar in the two species, with no apparent osmotic adjustment during water stress. These results indicate that P. acutifolius postpones dehydration and suggest that sensitive stomates and a deeply penetrating root system are characteristics which, if incorporated into cultivated beans, might increase their drought tolerance. PMID- 16663992 TI - Stoichiometry of proton translocation coupled to substrate oxidation in plant mitochondria. AB - The proton translocation coupled to the electron flux from succinate, exogenous NADH, and NAD(+)-linked substrates (malate and isocitrate) to cytochrome c and to oxygen was studied in purified potato (Solanum tuberosum) mitochondria using oxygen and ferricyanide pulse techniques. In the presence of valinomycin plus K(+) (used as a charge compensating cation), optimum values of H(+)/2 e(-) were obtained when low amounts of electron acceptors (oxygen or ferricyanide) were added to the mitochondria (1-2 nanogram [2 e(-)] equivalents per milligram protein). The stoichiometry of proton translocation to electron flux was unaffected in the presence of N-ethylmaleimide, an inhibitor of the Pi/H(+) symport. With succinate as substrate, H(+)/2 e(-) ratios were 4.0 +/- 0.2 and 3.7 +/- 0.3 with oxygen and ferricyanide as electron acceptors, respectively. With exogenous NADH, H(+)/2e(-) ratios were 4.1 +/- 0.9 and 3.4 +/- 0.2, respectively. The proton translocation coupled to the oxidation of NAD(+)-linked substrates (malate, isocitrate) was dependent upon the presence of adenylates (ADP, AMP, or ATP). For malate (+ glutamate) oxidation the observed H(+)/2 e(-) ratios were increased from 3.6 +/- 2.2 to 6.5 +/- 0.5 in the presence of 20 micromolar ADP. PMID- 16663993 TI - Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm. AB - Cytidine 5'-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn(2+) being preferred and giving maximum activity at 2.5 millimolar. The estimated K(m) for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K(m) for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K(m) estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold. PMID- 16663994 TI - Spinach nitrate reductase: purification, molecular weight, and subunit composition. AB - Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 +/- 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000. PMID- 16663995 TI - N and C NMR determination of allantoin metabolism in developing soybean cotyledons. AB - The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state (13)C and (15)N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO(2). About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO(2). The (13)C-(15)N bonds of [5-(13)C;1-(15)N] and [2 (13)C;1,3-(15)N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH(4) (+) in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH(4) (+) produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin. PMID- 16663996 TI - Effect of pH on Orthophosphate Uptake by Corn Roots. AB - Orthophosphate (Pi) influx in washed corn roots was studied with experimental conditions allowing a distinction of pH effects on Pi ionization in the medium and on the transport system itself. There appeared to be no relationship between the pH dependencies of membrane potential, H(+) secretion, and (32)Pi influx. The Pi uptake versus pH curves were compared to the calculated ones describing the concentrations of the different ionized Pi forms in the medium and in the cell walls; the latter were obtained using the theoretical model described by Sentenac and Grignon (1981) Plant Physiol 68: 415-419). The conclusion was that the transported form is H(2)PO(4) (-) and the concentration sensed by the transport system is the local one. The ionic compositions of experimental media were manipulated to ensure constant pH and various H(2)PO(4) (-) concentrations, or constant H(2)PO(4) (-) concentration and various pH values in the walls. The kinetic analysis of the results in the micromolar range showed that the transport system has an intrinsic sensitivity to pH, and is switched from a low activity state at pH > 6 to a high activity one at pH < 4 (pH in the walls). This change could be triggered by the protonation of a group with pK 5.5. PMID- 16663997 TI - Economy of water, carbon, and nitrogen in the developing cowpea fruit. AB - The nutritional economy of the fruit of cowpea (Vigna unguiculata (L.) Walp cv Vita 3) was assessed quantitatively from intake and utilization of carbon, nitrogen, and water. Fruits failed to make net gains of CO(2) from the atmosphere during daytime, although pod photosynthesis did play a role in the fruit's carbon economy by refixing a proportion of the fruit's respired CO(2). Of every 100 units by weight of carbon entering the fruit, 70.4 were finally incorporated into seeds, 10.3 remained as nonmobilizable material in pod walls, and the remaining 19.3 were lost in fruit respiration. Phloem supplied 97% of the fruit's carbon and 72% of its nitrogen. The xylem contribution of nitrogen occurred mainly in early growth. Ninety-six% of the fruit's nitrogen was incorporated into seeds, approximately 10% of this mobilized from the senescing pod. The mean transpiration ratio of the fruit was very low-8 milliliters water transpired per gram dry matter accumulated. Models of carbon, nitrogen, and water flow were constructed for the two consecutive 11 day periods of fruit development, and indicated a considerably greater entry of water through xylem and phloem than could be accounted for in changes in fruit tissue water and transpiration loss. This discrepancy was greater in the second half of fruit growth and was interpreted as evidence that a significant fraction of the water entering the fruit through phloem cycled back to the parent plant via the xylem. PMID- 16663998 TI - Diurnal water balance of the cowpea fruit. AB - The vascular network of the cowpea (Vigna unguiculata [L.] Walp.) fruit exhibits the anatomical potential for reversible xylem flow between seeds, pod, and parent plant. Feeding of cut shoots with the apoplast marker acid fuchsin showed that fruits imported regularly via xylem at night, less frequently in early morning, and only rarely in the afternoon. The dye never entered seeds or inner dorsal pod strands connecting directly to seeds. Root feeding (early morning) of intact plants with (32)PO(4) or (3)H(2)O rapidly (20 min) labeled pod walls but not seeds, consistent with uptake through xylem. Weak subsequent (4 hours) labeling of seeds suggested slow secondary exchange of label with the phloem stream to the fruit. Vein flap feeding of subtending leaves with [(14)C]sucrose, (3)H(2)O, and (32)PO(4) labeled pod and seed intensely, indicating mass flow in phloem to the fruit. Over 90% of the (14)C and (3)H of fruit cryopuncture phloem sap was as sucrose and water, respectively. Specific (3)H activities of transpired water collected from fruits and peduncles were assayed over 4 days after feeding (3)H(2)O to roots, via leaf flaps, or directly to fruits. The data indicated that fruits transpired relatively less xylem-derived (apoplastic) water than did peduncles, that fruit and peduncle relied more heavily on phloem-derived (symplastic) water for transpiration in the day than at night, and that water diffusing back from the fruit was utilized in peduncle transpiration, especially during the day. The data collectively support the hypothesis of a diurnally reversing xylem flow between developing fruit and plant. PMID- 16663999 TI - Permeability of the suberized mestome sheath in winter rye. AB - Mestome sheath cells of winter rye (Secale cereale L. cv Puma) deposit suberized lamellae in their secondary cell walls. Histochemical tests including acid digestion and staining with Sudan IV and Chelidonium majus root extract were used to detect the presence of suberin in the primary cell wall. There was no evidence of a Casparian band between adjacent mestome sheath cells. Fluorescent dye techniques were used to trace solute movement through the rye leaf apoplast. Calcofluor white M2R, a fluorescent dye which binds to cell walls as it moves apoplastically, proved to be too limited in its mobility in leaves to test mestome sheath permeability. Trisodium 3-hydroxy-5,8,10 pyrene trisulfonate, a fluorescent dye which is mobile in the apoplast, moved easily up the vascular bundles in the transpiration stream, and diffused outward from the veins to the epidermal cell walls within minutes of reaching a particular level in the leaf. We conclude that the suberized mestome sheath of rye leaves is freely permeable to solutes moving apoplastically through radial primary cell walls. PMID- 16664000 TI - Abscisic Acid and cutout in cotton. AB - A decline in growth, flowering, and boll (fruit) retention is referred to as cutout in cotton (Gossypium hirsutum L.). Fruit load affects cutout, possibly through hormonal effects. Experiments were conducted to test the hypothesis that fruits are a source of abscisic acid (ABA) that moves into fruiting branches and growing points where it inhibits growth, flowering, and boll retention. Removal of the flower or young boll at the first node of fruiting branches did not decrease the ABA content of fruiting branches or the abscission zone at the second node. Effects on ABA content of the boll at the second node varied. In one field test, ABA content of bolls at the second node decreased with successive harvests as bolls were removed from first node positions of several fruiting branches. Thus, the effect was cumulative and was not limited to individual branches. Removal of the flower or boll at the first node increased boll retention at the second node. Removal of all flowers during the first 3 weeks of flowering delayed the decreases in growth, flowering, and boll retention that occurred as fruit load increased. But, the ABA content of fruiting branches and mainstem apices was not decreased by early defruiting and did not increase with increasing fruit load. The results do not support the hypothesis that fruits are a source of ABA that moves into fruiting branches and growing points where it then inhibits growth, flowering, and boll retention. PMID- 16664001 TI - Studies of Root Function in Zea mays: III. Xylem Sap Composition at Maximum Root Pressure Provides Evidence of Active Transport into the Xylem and a Measurement of the Reflection Coefficient of the Root. AB - The cut ends of excised Zea mays roots were sealed to a pressure transducer and their root pressures recorded. These rose approximately hyperbolically to a maximum value of 4.21 +/- 0.34 bar after 30 to 40 minutes. Xylem exudate could not be collected at this pressure since the flow rate was zero. Samples of exudate were collected at lower applied pressures (DeltaP), however, and Deltapi, the osmotic pressure difference between them and the solution bathing the root, was measured by freezing point depression. A plot of DeltaP/Deltapi against J(v)/Deltapi, where J(v) is the volume flux, proved to be a straight line whose intercept, equal to sigma, the reflection coefficient, was 0.853 +/- 0.016. The maximum xylem concentrations of various chemical species were found by a similar extrapolative method and compared with those in the cell sap. This indicated that (a) Ca(2+), Mg(2+), NO(3) (2-), SO(4) (2-), and most amino acids move from the cells to the xylem down an electrochemical potential gradient; (b) relative to these ions H(+), NH(4) (+), glutamine and asparagine are actively transported into the xylem; and (c) H(2)PO(4) (-), and K(+) are actively retained in the symplasm. PMID- 16664002 TI - Studies of Root Function in Zea mays: IV. Effects of Applied Pressure on the Hydraulic Conductivity and Volume Flow through the Excised Root. AB - The volume flux, J(v), and the osmotic driving force, sigma big up tri, openpi, across excised root systems of Zea mays were measued as a function of big up tri, openP, the hydrostatic pressure difference applied across the root, using the pressure jump method previously described (Miller DM 1980 Can J Bot 58: 351-360). J(v) varied from 5.3% to 142% of its value in intact transpiring plants as a result of the application of pressure differences from -2.4 to 2.4 bar. The calculated hydraulic conductivity was 5.9 x 10(-4) cubic centimeters per second per bar per gram root and was independent of pressure. A model of root function similar to those appearing in the literature failed to provide quantitative accord with the data. A proposed model, which includes the effect of volume flux on the distribution of solutes in the symplasm, predicts accurately J(v) big up tri, openpi, and the xylem solute concentration as a function of big up tri, openP. PMID- 16664003 TI - Transient Nature of a (1 --> 3), (1 --> 4)-beta-d-Glucan in Zea mays Coleoptile Cell Walls. AB - Excised Zea mays L. embryos were cultured on Linsmaier and Skoog medium. Coleoptiles were sampled at regular intervals and the length, fresh weight, cell wall weight, and cell wall neutral sugar composition were determined. A specific beta-d-glucanase from Bacillus subtilis was used to determine the content of a (1 --> 3),(1 --> 4)-beta-d-glucan.Coleoptiles elongated through the 5th day following imbibition with the most rapid elongation occurring between days 3 and 4. The greatest net rate of incorporation of cell wall per coleoptile occurred between the 2nd and 3rd days when deposition of approximately one-third of the maximum net glucan level was observed. By day 5, the amount of glucan present had increased 34-fold from the 6 micrograms per coleoptile on day 1 and accounted for about 14% of the cell wall (w/w). Thereafter, the glucan content declined until only 3.3% (w/w) remained by day 10. In this 10-day interval, xylose increased 32% and cellulose content doubled, while proportions of other neutral sugars changed less dramatically.These results are consistent with a possible role for the beta d-glucan in elongation of the Zea coleoptile. Moreover, changes in the quantity of this wall component clearly reflect the dynamic nature of plant cell wall polysaccharides. An evaluation of glucan dynamics in vivo suggests that in vitro autolysis studies employing Zea coleoptile walls may overestimate the actual rate of glucan turnover in the intact tissue. PMID- 16664004 TI - Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site. AB - In maize chloroplasts, the ratio of HCO(3) (-) (anion) binding sites to high affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO(3) (-) to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO(3) (-) and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO(3) (-) binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO(3) (-) with a dissociation constant near 80 micromolar. The location of the HCO(3) (-) binding site, and the low-affinity atrazine binding site, is not known. PMID- 16664005 TI - Maintenance carbon cycle in crassulacean Acid metabolism plant leaves : source and compartmentation of carbon for nocturnal malate synthesis. AB - The reciprocal relationship between diurnal changes in organic acid and storage carbohydrate was examined in the leaves of three Crassulacean acid metabolism plants. It was found that depletion of leaf hexoses at night was sufficient to account quantitatively for increase in malate in Ananas comosus but not in Sedum telephium or Kalanchoe daigremontiana. Fructose and to a lesser extent glucose underwent the largest changes. Glucose levels in S. telephium leaves oscillated diurnally but were not reciprocally related to malate fluctuations.Analysis of isolated protoplasts and vacuoles from leaves of A. comosus and S. telephium revealed that vacuoles contain a large percentage (>50%) of the protoplast glucose, fructose and malate, citrate, isocitrate, ascorbate and succinate. Sucrose, a major constituent of intact leaves, was not detectable or was at extremely low levels in protoplasts and vacuoles from both plants.In isolated vacuoles from both A. comosus and S. telephium, hexose levels decreased at night at the same time malate increased. Only in A. comosus, however, could hexose metabolism account for a significant amount of the nocturnal increase in malate. We conclude that, in A. comosus, soluble sugars are part of the daily maintenance carbon cycle and that the vacuole plays a dynamic role in the diurnal carbon assimilation cycle of this Crassulacean acid metabolism plant. PMID- 16664006 TI - Inhibitory effects of water deficit on maize leaf elongation. AB - The growth rate of maize leaves has been investigated for plants grown in pots in controlled conditions and subjected to slow drying over a period of days. The elongation of leaves experiencing water deficit was inhibited primaily during the afternoon. No decrease in the turgor of the growing cells could be detected at that time. Solute concentration in the growing cells increased in tissue experiencing water deficit, but this was shown to occur after the growth rate had fallen. Calculations of the rate of solute accumulation necessary to maintain these concentrations indicated that the rate was less in slowly growing than in rapidly growing cells. The growing tissue of well-watered leaves excreted protons into the apoplastic space, but this acidification decreased in tissue exposed to water deficit. The pH of the apoplastic space correlated with the growth rate of the tissue. In vitro acidification of isolated, frozen-thawed tissue, maintained under constant tension, increased wall extensibility. The results suggest that one role of proton excretion may be to promote wall-loosening events necessary for cell enlargement, and that inhibition of this process may have reduced growth rate in leaves exposed to water deficit. PMID- 16664007 TI - Strongly acidic auxin indole-3-methanesulfonic Acid: synthesis of [C]indole-3 methanesulfonic Acid and studies of its chromatographic, spectral, and biological properties. AB - A radiochemical synthesis is described for [(14)C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [(14)C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles. PMID- 16664008 TI - Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization. AB - Mg:ATP-dependent H(+) pumping has been studied in microsomal vesicles from 24 hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Delta psi). DeltapH was measured as the decrease of absorbance of Acridine orange and Delta psi as the shift of absorbance of bis(3-propyl-5 oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Delta pH and Delta psi generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Delta pH generation is not inhibited by NO(3) (-). These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H(+)-ATPases. Both intravesicular acidification and Delta psi generation are influenced by anions: Delta pH increases and Delta psi decreases following the sequence SO(4) (2-), Cl(-), Br(-), NO(3) (-). ATP dependent H(+) pumping strictly requires Mg(2+). It is very specific for ATP (apparent K(m) 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Delta pH generation is inhibited by CuSO(4) and diethylstilbestrol as well as vanadate. Delta pH generation is specificially stimulated by K(+) (+ 80%) and to a lesser extent by Na(+) and choline (+28% and +14%, respectively). The characteristics of H(+) pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials. PMID- 16664009 TI - Inositol and sugars in adaptation of tomato to salt. AB - Tomato (Lycopersicon esculentum Mill. cv New Yorker) plants subjected to 100 millimolar NaCl plus Hoagland nutrients exhibited a pattern of wilting, recovery of turgor, and finally recovery of growth at a reduced level, which required 3 days. During the nongrowing, adaptation phase there were immediate increases in free hexoses and sucrose which declined to near control levels as growth resumed. There was a steady increase in myo-inositol content which reached its maximal level at the time of growth resumption. The myo-inositol level then remained elevated for the remainder of the experiment. Myo-inositol constituted two-thirds of the soluble carbohydrate in leaves and three-fourths of the soluble carbohydrate in roots of salt-adapted plants. Plants which were alternated daily between salt and control solutions accumulated less myo-inositol and exhibited less growth than the continuously salt-treated plants. In L. pennellii and in salt-tolerant and salt-sensitive breeding lines selected from L. esculentum x L. pennellii BC(1) and F(8), myo-inositol content was highest in the most tolerant genotypes, intermediate in the normal cultivar, and lowest in the sensitive genotype after treatment with salt. PMID- 16664010 TI - Quantifying Apoplastic Flux through Red Pine Root Systems Using Trisodium, 3 hydroxy-5,8,10-pyrenetrisulfonate. AB - The fluorescent compound trisodium, 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) was used to quantify the apoplastic flux through red pine (Pinus resinosa Ait.) root systems-that portion of the total water flux reaching the xylem without ever crossing a semipermeable membrane. Flow was induced by pressure through detopped root systems, and by transpiration through intact seedlings. Apoplastic flux was determined by multiplying total flux by the ratio of PTS concentration in the xylem exudate to PTS concentration in the bathing medium.Under aeration, apoplastic flux was less than 1% of total flux. Under anaerobic conditions, up to 50% of total flux was apoplastic suggesting that anaerobic conditions change the pathway of water flow into root xylem. The change under anaerobic conditions was reversible. Detopped root systems under pressure and intact seedlings under transpiration gave similar results. In detopped root systems, the magnitude of the pressure gradient may alter the apoplastic contribution to total flux. PMID- 16664011 TI - Proteins and peroxidase in callus and suspension cultures of apple : a study using ultrathin-layer isoelectric focusing, sensitive silver staining of proteins, and peroxidase isozyme visualization. AB - Different methods for the isolation of soluble proteins were applied to cell cultures of three apple cultivars (Malus sylvestris Mill.), best results being obtained with a rapid technique based on freezing and thawing. Ultrathin-layer isoelectric focusing followed by an improved silver staining method has shown that proteins from apple callus cultures consist of some 60 to 80 zones, with isoelectric points mainly between pH 4 to 7. Depending on protein content, adequate silver staining is achieved with 50 to 500 cells. Protein patterns of callus cultures allowed clear discrimination of cultivars. Protein and peroxidase isozyme patterns in cell saps of suspension cultures show striking differences during the growth cycle, whereas the protein patterns from the nutrient media were constant over the entire cultivation period and closely resembled the patterns of stationary phase and callus cells. PMID- 16664012 TI - Virulence properties of strains of agrobacterium on the apical and Basal surfaces of carrot root discs. AB - Most pathogenic strains of Agrobacterium are able to induce crown gall or hairy root on both the apical surface (facing the root tip) and the basal surface (facing the shoot) of carrot (Daucus carota L.) root discs. Tumorigenic strains carrying mutations in the shoot inhibition region of the T-DNA (TL-DNA genes 1 and 2) are markedly attenuated on the basal surface but remain virulent on the apical surface. Coinoculation of two attenuated tumorigenic strains, with mutations in gene 1 and gene 2, respectively, resulted in restoration of virulence on the basal surface. Wild type hairy root-inducing strains can be divided into two groups: those that are virulent on both apical and basal surfaces and those that are virulent only on the apical surface. alpha Naphthalene acetic acid stimulated virulence of hairy root strain TR7, belonging to the latter group, on the basal surface. Attenuated virulence on the basal surface can be explained in terms of an auxin deficiency in the basal tissues and unidirectional auxin transport to the apical surface. PMID- 16664013 TI - Onset of Alcohol Dehydrogenase Synthesis during Microsporogenesis in Maize. AB - During male gametophyte development the synthesis of several proteins occurs from transcripts of the haploid genome. Alcohol dehydrogenase (ADH1), a developmentally regulated protein, was chosen for study to determine the stage at which its synthesis was initiated and the pattern of its synthesis during microsporogenesis. The ability of ADH to reduce p-nitro blue tetrazolium chloride in situ was used as an indicator of enzyme activity. Maize strains heterozygous for adh1 were utilized to provide an internal control, 50% of the grains being adh1(+) and 50% being adh1(-). No ADH activity was detectable when tetrads were first formed after meiosis. Activity was initially detected soon after the tetrads began to break apart but before the microspores in the tetrads had completely separated. The transcription of the adh1 gene from the haploid genome must thus occur very soon after meiosis is completed. ADH activity increases at a constant rate thereafter until microspore mitosis when an increase in the rate takes place which lasts until generative cell division. Thereafter, there is a marked decrease in the rate of accumulation of ADH activity. PMID- 16664014 TI - Initiation of Postmeiotic beta-Galactosidase Synthesis during Microsporogenesis in Oilseed Rape. AB - The synthesis of beta-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-beta-d-galactoside is used as substrate giving a blue-green final reaction product. We have recently detected oilseed rape plants which are heterozygous for the beta-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The present study has identified the developmental phase at which synthesis of the enzyme commenced. Activity is absent in microsporocytes, tetrads, and at microspore release. Enzyme activity is first detected in the young microspores and, by early vacuolate period, there is an increase in the rate of enzyme activity. A second period of increased enzyme synthesis occurred prior to generative cell division, although the rate is reduced in mature pollen. PMID- 16664015 TI - Effect of Ferredoxin on the Diaphorase Activity of Cyanobacterial Ferredoxin-NADP Reductase. AB - The interaction of ferredoxin-NADP reductase (FNR) and ferredoxin (Fd) results in an enhanced rate of reaction and a shift of the pH optimum for the FNR-mediated diaphorase reaction. Low concentrations of NaCl (<100 millimolar), favorable for formation of the FNR:Fd complex, further magnify the alteration of the diaphorase reaction; the activity is enhanced 3-fold and pH optimum is shifted from 9.5 to 7.8. The Fd-stimulated diaphorase activity of FNR may result either from a conformational change of the enzyme and/or from a transition from a two electron to a one electron reaction. PMID- 16664016 TI - Immunochemical Characterization of Nitrate Reductase Forms from Wild-Type (cv Williams) and nr(1) Mutant Soybean. AB - Soybean (Glycine max L. Merr.) leaves contain two forms of nitrate reductase (NR) NAD(P)H:NR and NADH:NR. Wild-type (cv Williams), nr(1) mutant and an unrelated cultivar (Prize) were grown with either no N source or with nitrate. Crude extracts were assayed for NR activities and the enzyme forms were purified on blue Sepharose. Analyses were done by polyacrylamide gel electrophoresis and ;Western blotting' using antibodies specific for NR. NAD(P)H:NR was identified as the constitutive NR present in wild-type and Prize, but was absent from the mutant. All three soybean lines contained nitrate-inducible NADH:NR with highest activity at pH 7.5. The results showed that NAD(P)H:NR and constitutive NR were one in the same and confirmed the presence of NADH:NR with pH 7.5 optimum. PMID- 16664017 TI - Photosynthesis and Inorganic Carbon Accumulation in the Acidophilic Alga Cyanidioschyzon merolae. AB - The intracellular pH and membrane potential were determined in the acidophilic algae Cyanidoschyzon merolae as a function of extracellular pH. The alga appear to be capable of maintaining the intracellular pH at the range of 6.35 to 7.1 over the extracellular pH range of 1.5 to 7.5. The membrane potential increase from -12 millivolts (negative inside) to -71 millivolts and thus DeltamuH(+) decreased from -300 to -47 millivolts over the same range of extracellular pH. It is suggested that the DeltamuH(+) may set the upper and lower limits of pH for growth. Photosynthetic performance was also determined as a function of pH. The cells appeared to utilize CO(2) from the medium as the apparent K(m(co(2))) was 2 to 3 micromolar CO(2) over the pH range of 1.5 to 7.5 C. merolae appear to possess a ;CO(2) concentrating' mechanism. PMID- 16664018 TI - Conditions for strict autotrophic culture of tobacco callus. AB - Organic gelling agents such as agar and agarose provide a heterotrophic substrate for growth of illuminated tobacco callus. When green cells are incubated in CO(2) free air on a medium lacking sucrose but solidified with 1% agar, an increase in relative dry weight is sustained through two passages. Similar results with different inoculum sources, and with three brands of agar and two forms of agarose, suggest this is a general phenomenon. A fully autotrophic culture system was developed employing polyurethane pads to support cells in a liquid medium lacking sucrose. Growth was negligible in two passages in CO(2)-free air, and increased with each added increment in CO(2) concentration. PMID- 16664019 TI - Laboratory-Produced CO(2) Calibration Gases. AB - A simple, inexpensive apparatus for making mixtures of accurately known amounts of CO(2) and CO(2)-free atmospheric air is described. Calibration gases with CO(2) contents of 200 to 1500 microliters per liter produced with the apparatus had concentrations which were within 10 microliters per liter of the target concentration. PMID- 16664020 TI - Foliar sterols in soybeans exposed to chronic levels of ozone. AB - Soybeans (Glycine max) exposed to chronic levels of ozone showed a linear decrease in biomass with increasing concentration. The foliar free sterols increased while the steryl ester, and the steryl glycosides, a minor component, decreased with increasing pollutant concentration. Of the free sterols, stigmasterol showed the largest increase, followed by sitosterol; campesterol, however, decreased. All steryl esters decreased; sitosterol showed the largest decrease and campesterol the least. PMID- 16664021 TI - Blue and Green Light-Induced Phototropism in Arabidopsis thaliana and Lactuca sativa L. Seedlings. AB - Exposure time-response curves for blue and green light-induced phototropic bending in hypocotyls of Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. seedlings are presented. These seedlings show significant phototropic sensitivity up to 540 to 550 nanometers. Since wave-lengths longer than 560 nanometers do not induce phototropic bending, it is suggested that the response to 510 to 550 nanometers light is mediated by the specific blue light photoreceptor of phototropism. We advise care in the use of green ;safelights' for studies of phototropism. PMID- 16664022 TI - Abscisic Acid accumulation in spinach leaf slices in the presence of penetrating and nonpenetrating solutes. AB - Abscisic acid (ABA) accumulated in detached, wilted leaves of spinach (Spinacia oleracea L. cv Savoy Hybrid 612) and reached a maximum level within 3 to 4 hours. The increase in ABA over that found in detached turgid leaves was approximately 10-fold. The effects of water stress could be mimicked by the use of thin slices of spinach leaves incubated in the presence of 0.6 molar mannitol, a compound which causes plasmolysis (loss of turgor). About equal amounts of ABA were found both in the leaf slices and in detached leaves, whereas 2 to 4 times more ABA accumulated in the medium than in the slices. When spinach leaf slices were incubated with ethylene glycol, a compound which rapidly penetrates the cell membrane causing a decrease in the osmotic potential of the tissue and only transient loss of turgor, no ABA accumulated. Ethylene glycol was not inhibitory with respect to ABA accumulation. Spinach leaf slices incubated in both ethylene glycol and mannitol had ABA levels similar to those found when slices were incubated with mannitol alone. Increases similar to those found with mannitol also occurred when Aquacide III, a highly purified form of polyethylene glycol, was used. Aquacide III causes cytorrhysis, a situation similar to that found in wilted leaves. Thus, it appears that loss of turgor is essential for ABA accumulation.When spinach leaf slices were incubated with solutes which are supposed to disturb membrane integrity (KHSO(3), 2-propanol, or KCl) no increase in ABA was observed. These data indicate that, with respect to the accumulation of ABA, mannitol caused a physical stress (loss of turgor) rather than a chemical stress (membrane damage). PMID- 16664023 TI - Light quality effects on corn chloroplast development. AB - Corn was grown under greenhouse and controlled light quality conditions incluing full spectrum, red (R), and far-red (FR) sources. Young leaf samples were analyzed for pigments, pigment-proteins, membrane polypeptides, and ultrastructure. Chloroplast development in full spectrum white light was similar to that found in R but different from that found in FR plus low R. Compared to greenhouse and R, FR plus low R (670-760) repressed the formation of photosystem I reaction center protein (CP1 + CP1a) and enhanced those of photosystem II (CPa) in both bundle sheath and mesophyll cells. Photosystem II polypeptides were present in both cell types, with the 46 and 34 kilodalton proteins predominant in mesophyll cells. Bundle sheath cells contained relatively more of the 51 kilodalton and less of the 46 kilodalton proteins. However, they also contained measurable amounts of ribulose bisphosphate carboxylase which may interfere with estimates of the 51 kilodalton protein. PMID- 16664024 TI - Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells. AB - Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis. PMID- 16664025 TI - Inhibition of the k-stimulated ATPase of the plasmalemma of pinto bean leaves by ozone. AB - Three varieties of Phaseolus vulgaris which differ in their sensitivity to ozone were examined for changes in some physiological and structural plasma membrane characteristics. Plasma membrane vesicles were prepared from control and ozone treated (0.2 to 0.5 microliters per liter ozone for 5 hours) leaf tissue, and the (K(+) + Mg(2+))-ATPase activity determined and compared. No major changes were observed in the resistant varieties. The sensitive variety showed a severe inhibition of ATPase activity which was largely due to a decrease in the K(+) stimulated component. This inhibition was completely reversed by the addition of sulfhydryl compounds.Ozone-induced plasma membrane permeability changes may be effected by damage to membrane proteins, perhaps by oxidation of amino acid sulfhydryl groups to disulfide and sulfenic moieties. PMID- 16664026 TI - Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles. AB - The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl(-) and inhibited by NO(3) (-). NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K(2)SO(4) supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K(+). KCl and K(2)SO(4) support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet. PMID- 16664027 TI - Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr). AB - The relationship between ureide N and N(2) fixation was evaluated in greenhouse grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N(2) fixation in soybean and lima bean correlated with ureide N. In soybean, N(2) fixation, ureide N, acetylene reduction, and nodule mass were correlated when N(2) fixation was inhibited by applying KNO(3) solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N(2) fixation. Ureide N determinations in field studies with soybean correlated with N(2) fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N(2) fixation was estimated by (15)N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N(2) fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N(2) fixation and ureide N. N(2) fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N(2) fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N(2) fixation. PMID- 16664028 TI - Crassulacean Acid Metabolism and Crassulacean Acid Metabolism Modifications in Peperomia camptotricha. AB - Peperomia camptotricha, a tropical epiphyte from Mexico, shows variable forms of Crassulacean acid metabolism (CAM). Young leaves exhibit CAM-cycling, while mature leaves show an intermediate type of metabolism, between CAM and CAM cycling, having approximately the same amount of nighttime gas exchange as daytime. Metabolism of young leaves appears independent of daylength, but mature leaves have a tendency toward more CAM-like metabolism under short days (8 hours). Large differences in the physical appearance of plants were found between those grown under short daylengths and those grown under long daylengths (14 hours). Some anatomical differences were also detected in the leaves. Water stress caused a switch to CAM in young and mature leaves, and as water stress increased, they shifted to CAM-idling. PMID- 16664029 TI - Effect of Chilling Temperatures upon Cell Cultures of Tomato. AB - The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv ;VF36,' and cv ;VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28 degrees C, and 3 to 8 days at 12 degrees C. No growth was observed at 8 degrees C, indicating an abrupt limit to growth between 8 and 12 degrees C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8 degrees C for up to 2 weeks. When cultures kept at 8 degrees C for up to 30 days were transferred to 28 degrees C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0 degrees C, and for cells of ;VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9 degrees C but additional growth at 8 degrees C did not occur, nor could growth be maintained by subculture at 8 or 9 degrees C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10 degrees C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them. PMID- 16664030 TI - Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells. AB - Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a K(m) of 5 to 10 millimolar, stimulation being due to the anion, Cl(-), and not the cation, K(+), and was not inhibited by vanadate, but was inhibited by NO(3) (-). The activity is tentatively identified as the tonoplast ATPase. PMID- 16664031 TI - Acclimation to low temperature by microsomal membranes from tomato cell cultures. AB - Sealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon esculentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strongly temperature dependent with a Q(10) of 2 and a maximum rate near 35 degrees C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30 degrees C. A comparison was made between membranes from cell cultures grown at 28 degrees C and 9 degrees C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9 degrees C. PMID- 16664032 TI - Effect of Water Stress on the Carbohydrate Metabolism of Citrullus lanatus Seeds during Germination. AB - Gluconeogenesis in Citrullus lanatus seeds is a post germinative event. Increases in isocitrate lyase activity and incorporation of radioactivity from [2 (14)C]acetate into sugars occur only after radicle emergence. During germination, the seeds appear to rely on carbohydrate as the respiratory substrate. At this time, glycolysis, the pentose phosphate pathway, and the tricarbocyclic acid cycle seem to be functional. Utilization of raffinose during germination appears to be important.Water stress, which completely inhibits germination, has a marked effect on carbohydrate metabolism. The rate of (14)CO(2) release from [2 (14)C]acetate, [1-(14)C]glucose, and [6-(14)C]glucose is lower in the stressed seeds than the control seeds during the respiratory lag phase. However, in the stressed seeds neither glycolysis, the pentose phosphate pathway, nor the tricarboxylic acid cycle is completely inhibited. In contrast to the control seeds in which raffinose content sharply declines after 12 h of incubation, raffinose content in the stressed seeds remains fairly constant.The respiratory lag phase of the control seeds coincides with a lower reducing substance content, glucose content, and fructose content than in the stressed seeds during the corresponding incubation period. PMID- 16664033 TI - Red Light Stimulates an Increase in Intracellular Calcium in the Spores of Onoclea sensibilis. AB - Red light (R) stimulates an increase in the total concentration of intracellular calcium in the spores of Onoclea sensibilis L. as determined by atomic absorption spectroscopy. Subsequent exposure to far-red light inhibits the R-induced increase in intracellular calcium. The majority of the increase occurs 5 minutes after the onset of irradiation. The calcium antagonist, La(3+), inhibits both germination and the R-induced increase in intracellular calcium. The R-induced increase in calcium is sufficient to account for an increase in the concentration of intracellular calcium ions from 0.1 micromolar to 1 to 10 micromolar. Large detectable changes in other elements tested are not required for germination. PMID- 16664034 TI - Photosynthetic Carbon Assimilation in a Shootless Orchid, Chiloschista usneoides (DON) LDL: A Variant on Crassulacean Acid Metabolism. AB - Photosynthetic carbon assimilation in the roots of a shootless orchid Chiloschista usneoides (DON) LDL involves the synthesis and accumulation of malic acid from CO(2) in darkness. Malic acid is consumed in the light.The roots do not possess stomata or any means of diurnally regulating the diffusive conductance of the pathway between the internal gas phase of the plant and the atmosphere. Regulation of internal CO(2) concentration near to atmospheric levels avoids a large net loss of CO(2) to the atmosphere during malic acid consumption in the light.The water-absorbing function of the velamen conflicts with the photosynthetic function of the roots. Plants with water-saturated velamina do not acquire CO(2) from the atmosphere at night. PMID- 16664035 TI - Genotypic variability of soybean response to agrobacterium strains harboring the ti or ri plasmids. AB - Twenty four diverse cultivars of soybean (Glycine max [L.] Merrill) and three lines of its annual wild progenitor Glycine soja Sieb and Zucc. were tested for their response to Agrobacterium strains harboring either the Ti (tumor-inducing) plasmid (pTi) from Agrobacterium tumefaciens or the Ri (root-inducing) plasmid (pRi) from Agrobacterium rhizogenes following uniform wounding and inoculation. Based upon gall weight at 8 weeks postinfection, three G. max cultivars (Biloxi, Jupiter, and Peking) and one G. soja line, Plant Introduction (PI) 398.693B, were judged highly susceptible to A. tumefaciens strain A348 (pTiA6), ten genotypes moderately susceptible, 11 weakly susceptible, and two nonsusceptible. Of 26 genotypes inoculated with strain R1000 (pRiA4b), only seven responded in a clearly susceptible fashion by forming small, fleshy roots at internodal infection sites. Cotyledons excised from 1- or 3-day old seedlings of Peking and Biloxi cultivars also formed galls when infected in vitro with agrobacteria carrying either the Ti or Ri plasmid. Tumor lines established from cotyledon and stem galls induced by A. tumefaciens A348 (pTiA6) exhibited the T-DNA borne traits of phytohormone-independent growth and octopine synthesis. Additionally, DNA isolated from cultured tumors hybridized with labeled T-DNA probe. PMID- 16664036 TI - Spectral Dependence of Photoregulation of Inorganic Nitrogen Metabolism in Chlamydomonas reinhardii. AB - The utilization of NO(3) (-) by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of NO(2) (-) and NH(4) (+) to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The stimulation of NO(3) (-) utilization and of its associated NO(2) (-) and NH(4) (+) excretions saturated at very low quantum fluxes of blue light (15 microequivalents per square meter per second) in Chlamydomonas reinhardii cells sparged with CO(2)-free air and irradiated with 50 microequivalents per square meter per second background red light. The wavelength dependence data of this stimulation correlated closely with the in situ photoactivation of nitrate reductase and also with the light induced increase in its biosynthesis and/or assembly.These results indicate that the photoregulation of inorganic N metabolism in C. reinhardii is mainly due to the blue light modulation of nitrate reductase. Although flavins are the most suitable candidates to act as physiological photoreceptors, the wavelength dependence data only show a major peak in the blue region between 400 and 500 nanometers. PMID- 16664037 TI - Cytokinin-modulated gene expression in excised pumpkin cotyledons. AB - Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A)(+) mRNA isolated from cotyledons incubated with 10(-4) molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10(-9) to 10(-4) molar) of benzyladenine. Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxypyruvate reductase activity; thus, the enzyme synthesis does not appear to be controlled by organelle genes. Hydroxypyruvate reductase synthesis is inhibited by cycloheximide, cordycepin, and to a certain degree by actinomycin D. These data support the view of a close association between cytokinin action and gene expression. PMID- 16664038 TI - Inorganic Carbon Uptake by Chlamydomonas reinhardtii. AB - The rates of CO(2)-dependent O(2) evolution by Chlamydomonas reinhardtii, grown with either air levels of CO(2) or air with 5% CO(2), were measured at varying external pH. Over a pH range of 4.5 to 8.5, the external concentration of CO(2) required for half-maximal rates of photosynthesis was constant, averaging 25 micromolar for cells grown with 5% CO(2). This is consistent with the hypothesis that these cells take up CO(2) but not HCO(3) (-) from the medium and that their CO(2) requirement for photosynthesis reflects the K(m)(CO(2)) of ribulose bisphosphate carboxylase. Over a pH range of 4.5 to 9.5, cells grown with air required an external CO(2) concentration of only 0.4 to 3 micromolar for half maximal rates of photosynthesis, consistent with a mechanism to accumulate external inorganic carbon in these cells. Air-grown cells can utilize external inorganic carbon efficiently even at pH 4.5 where the HCO(3) (-) concentration is very low (40 nanomolar). However, at high external pH, where HCO(3) (-) predominates, these cells cannot accumulate inorganic carbon as efficiently and require higher concentrations of NaHCO(3) to maintain their photosynthetic activity. These results imply that, at the plasma membrane, CO(2) is the permeant inorganic carbon species in air-grown cells as well as in cells grown on 5% CO(2). If active HCO(3) (-) accumulation is a step in CO(2) concentration by air grown Chlamydomonas, it probably takes place in internal compartments of the cell and not at the plasmalemma. PMID- 16664039 TI - Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury. AB - Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately -5 degrees C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log(10) mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of -2.5 degrees C and -3.0 degrees C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at -5 degrees C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants. PMID- 16664040 TI - Allocation and Turnover of Photosynthetically Assimilated CO(2) in Leaves of Glycine max L. Clark. AB - The allocation and turnover of photosynthetically assimilated (14)CO(2) in lipid and protein fractions of soybean (Glycine max L. Clark) leaves and stem materials was measured. In whole plant labeling experiments, allocation of photosynthate from a pulse of (14)CO(2) into polymeric compounds was: 25% to proteins in 4 days, 20% to metabolically inert cell wall products in 1 to 2 days, 10% to lipids in 4 days, and 4% to starch in 1 day. The amount of (14)C labeled photosynthate that an actively growing leaf (leaf 4) used for its own lipid synthesis immediately following pulse labeling was about 25%. The (14)C of labeled proteins turned over with half-lives of 3.8, 3.3, and 4.1 days in leaves 1, 2, and 3, respectively; and turnover of (14)C in total shoot protein proceeded with a half life of 5.2 days. Three kinetic (14)C turnover patterns were observed in lipids: a rapid turnover fraction (within a day), an intermediate fraction (half-life about 5 days), and a slow turnover fraction. These results are discussed in terms of previously published accounts of translocation, carbon budgets, carbon use, and turnover in starch, lipid, protein, and cell wall materials of various plants including soybeans. PMID- 16664041 TI - Endogenous Rhythms in Photosynthesis, Sucrose Phosphate Synthase Activity, and Stomatal Resistance in Leaves of Soybean (Glycine max [L.] Merr.). AB - Experiments were conducted with soybean (Glycine max [L.] Merr. cv ;Ransom') plants to determine if diurnal rhythms in net carbon dioxide exchange rate (CER), stomatal resistance, and sucrose-phosphate synthase (SPS) activity persisted in constant environmental conditions (constant light, LL; constant dark DD) and to assess the importance of these rhythms to the production of nonstructural carbohydrates (starch, sucrose, and hexose). Rhythms in CER, stomatal resistance, and SPS activity were observed in constant environmental conditions but the rhythms differed in period length, amplitude, and phase. The results indicated that these photosynthetic parameters are not controlled in a coordinated manner. The activity of UDPG pyrophosphorylase, another enzyme involved in sucrose formation, did not fluctuate rhythmically in constant conditions but increased with time in plants in LL. In LL, the rhythm in CER was correlated positively with fluctuations in total chlorophyll (r = 0.810) and chlorophyll a (r = 0.791) concentrations which suggested that changes in pigment concentration were associated with, but not necessarily the underlying mechanism of, the rhythm in photosynthetic rate. Assimilate export rate, net starch accumulation rate, and leaf sucrose concentration also fluctuated in constant light. No single photosynthetic parameter was closely correlated with fluctuations in assimilate export during LL; thus, assimilate export may have been controlled by interactions among the endogenous rhythms in CER, SPS activity, or other metabolic factors which were not measured in the present study. PMID- 16664042 TI - Light-Dependent Oxygen Uptake, Glycolate, and Ammonia Release in l-Methionine Sulfoximine-Treated Chlamydomonas. AB - Glycolate and ammonia excretion plus oxygen exchanges were measured in the light in l-methionine-dl-sulfoximine treated air-grown Chlamydomonas reinhardii. At saturating CO(2) (between 600 and 700 microliters per liter CO(2)) neither glycolate nor ammonia were excreted, whereas at the CO(2) compensation concentration (<10 microliters per liter CO(2)) treated algae excreted both glycolate and ammonia at rates of 37 and 59 nanomoles per minute per milligram chlorophyll, respectively. From the excretion values we calculate the amount of O(2) consumed through the glycolate pathway. The calculated value was not significantly different from the component of O(2) uptake sensitive to CO(2) obtained from the difference between O(2) uptake of the CO(2) compensation point and at saturating CO(2). This component was about 40% of stationary O(2) uptake measured at the CO(2) compensation point. From these data we conclude that glyoxylate decarboxylation in air-grown Chlamydomonas represents a minor pathway of metabolism even in conditions where amino donors are deficient and that processes other than glycolate pathway are responsible for the O(2) uptake insensitive to CO(2). PMID- 16664043 TI - Effect of path or sink anoxia on sugar translocation in roots of maize seedlings. AB - After feeding the scutellum of young maize seedlings with a labeled analog of glucose, 2-deoxy-d-glucose, the progress of radioactivity along the root was followed when either 70% of the path or the whole root were in strict anoxic conditions, and was compared with the translocation pattern of aerobic seedlings. Special care was taken to suppress the internal O(2) transport and to control its occurrence.In air, the radioactive compounds accumulated from 30 minutes in the root tip mainly as an analog of sucrose. When the whole root was anoxic, the progress of the radioactivity was very slow and never reached the tip which did not survive more than 8 hours. When 70% of the path was in strict anoxia and the sink (root tip) in air, the translocation was not impaired and the radioactivity accumulated in the tips as fast as in aerobic controls. The addition of 3 millimolar NaF, which inhibits the fermentative energy production, did not modify these results. It is concluded that long distance transport in maize sieve tubes has no special energy requirements and is controlled by source-sink relationships. The inhibition of sugar supply in anoxic root tips is attributed to an effect on unloading processes rather than on sink metabolism. PMID- 16664044 TI - Transport and metabolism of 1'-fluorosucrose, a sucrose analog not subject to invertase hydrolysis. AB - The novel sucrose derivative 1'-fluorosucrose (alpha-d-glucopyranosyl-beta- d-1 deoxy-1-fluorofructofuranoside) was synthesized in order to help define mechanisms of sucrose entry into plant cells. Replacement of the 1'-hydroxyl by fluorine very greatly reduces invertase hydrolysis of the derivative (hydrolysis at 10 millimolar 1'-fluorosucrose is less than 2% that of sucrose) but does not reduce recognition, binding, or transport of 1'-fluorosucrose by a sucrose carrier. Transport characteristics of 1'-fluorosucrose were studied in three different tissues. The derivative is transported by the sucrose carrier in the plasmalemma of developing soybean cotyledon protoplasts with a higher affinity than sucrose (K(m) 1'-fluorosucrose 0.9 millimolar, K(m) sucrose 2.0 millimolar). 1'-Fluorosucrose is a competitive inhibitor of sucrose uptake with an apparent K(i) also of 0.9 millimolar, while the K(i) of sucrose competition of 1' fluorosucrose uptake was 2.0 millimolar. Thus, both sugars are recognized at the same binding site in the plasmalemma. Both sucrose and 1'-fluorosucrose show very similar patterns of phloem translocation from an abraded leaf surface through the petiole indicating that recognition of 1'-fluorosucrose by sucrose carriers involved in phloem loading is likely as well.1'-Fluorosucrose is a very poor substrate for invertase and as such is absorbed only slowly by corn root segments, a tissue in which sucrose hydrolysis by a cell wall invertase is required prior to active hexose uptake.The kinetics of 1'-fluorosucrose uptake by soybean cotyledon protoplasts indicate that membrane passage and substrate release to the protoplast interior are rate limiting to transport. Recognition of sucrose at the inner membrane surface of the carrier protein is apparently different than recognition and binding at the external surface. PMID- 16664046 TI - Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C(4) Plants : An Effect of pH. AB - Photoactivation of phosphoenolpyruvate carboxylase in C(4) plants is detected more efficiently when activity is assayed at suboptimum pH (e.g. 7.2); the magnitude of the light effect is often larger at low phosphoenolpyruvate concentration.Darkness and low assay pH induce an allosteric behavior (positive cooperativity with phosphoenolpyruvate) which is relieved in light or by higher pH; thus, normal Michaelis-Menten kinetics are exhibited only when the enzyme is extracted during the day and assayed at pH 8.2.Light activation, pH, and substrate level appear to be components of a regulatory device suppressing the activity in darkness and enhancing it under light. PMID- 16664045 TI - Characterization of Peroxisomes from the Alga Bumilleriopsis filiformis. AB - The Xanthophycean alga Bumilleriopsis filiformis possesses peroxisomes which on electron micrographs show a mostly spherical or ovoid shape with a diameter in the range of 0.3 micrometer. Their granular matrix is usually of moderate electron density and in a very few cases contains amorphous inclusions. No associations with other organelles could be observed.During separation in a sucrose gradient, the peroxisomes from Bumilleriopsis equilibrate at a density of 1.22 grams per cubic centimeter. Glycolate oxidase and glyoxylate-glutamate aminotransferase were found in the isolated organelles along with catalase and uricase. However, no further leaf peroxisomal enzymes were detected. This is the first time that an alga of the group of Xanthophyceae has been demonstrated to possess a glycolate oxidase.The organelles from Bumilleriopsis differ from leaf peroxisomes also by the absence of enzymes of the beta-oxidation pathway. All enzymes for the degradation of fatty acids which were tested are located solely in the mitochondria. PMID- 16664047 TI - Role of calmodulin inhibition in the mode of action of ophiobolin a. AB - Calmodulin has been isolated from the root of Zea mays. It activates the bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase and has electrophoretic mobility very similar to that of bovine brain calmodulin. Ophiobolin A, a fungal toxin, interacts with the maize calmodulin. The interaction is not reversed by dilution or denaturation in SDS and results in the loss of ability of the calmodulin to activate the phosphodiesterase. The inhibition is much faster in the presence than in the absence of Ca(2+). The electrophoretic mobility of ophiobolin A-treated calmodulin is less than that of untreated calmodulin. Several similarities are found between the inhibition of maize calmodulin by ophiobolin A in vitro and the effects of ophiobolin A on excised roots. Both are irreversible and time-dependent. The concentration of ophiobolin A for half-maximal inhibition of calmodulin in the phosphodiesterase assay is similar to that for phytotoxicity. In both cases ophiobolin A derivatives behave similarly, i.e. 18-bromo-19-methoxyophiobolin A is as potent as ophiobolin A, while 3-anhydro-ophiobolin A and 6-epi-ophiobolin A are less potent. A smaller amount of active calmodulin was measured in the extract from ophiobolin A-treated roots than in those from untreated roots. The present study suggests that calmodulin is a target molecule in the root for the toxicity of ophiobolin A. PMID- 16664048 TI - Osmoregulation in Cotton in Response to Water Stress : III. Effects of Phosphorus Fertility. AB - Cotton (Gossypium hirsutum) (L.) was grown in a sand and nutrient solution system at two levels of phosphorus (0.5 and 5.0 millimolar). Within each phosphorus treatment, plants were either watered daily or acclimated to water stress by subjection to several water stress cycles.Stress acclimation increased leaf starch at the low phosphorus level, but not at the high phosphorus level. High phosphorus increased leaf sucrose and glucose concentration in both acclimated and nonacclimated plants, but had little effect on osmotic adjustment or the relationship between turgor and water potential.In nonacclimated plants, high phosphorus increased both leaf conductance and photosynthesis at high water potentials. In acclimated plants, high phosphorus increased photosynthesis but decreased conductance, thus increasing water use efficiency at the single leaf level. PMID- 16664049 TI - Photosynthate supply and utilization in alfalfa : a developmental shift from a source to a sink limitation of photosynthesis. AB - Long-term carbon dioxide enrichment, (14)CO(2) feeding, and partial defoliation were employed as probes to investigate source/sink limitations of photosynthesis during the development of symbiotically grown alfalfa. In the mature crop, long term CO(2) enrichment does not affect the rates of net photosynthesis, relative growth, (14)C export to nonphotosynthetic organs, or the rates of (14)C label incorporation into leaf sucrose, starch, or malate. The rate of glycolate labeling is, however, substantially reduced under these conditions. When the mature crop was partially defoliated, a considerable increase in net photosynthesis occurred in the remaining leaves. In the seedling crop, long-term CO(2) enrichment increased dry matter accumulation, primarily as a result of increases in leaf starch content. Although the higher rates of starch synthesis are not maintained, the growth enhancement of the enriched plants persisted throughout the experimental period. These results imply a source limitation of seedling photosynthesis and a sink limitation of photosynthesis in more mature plants. Consequently, both the supply and the utilization of photosynthate may limit seasonal photosynthesis in alfalfa. PMID- 16664050 TI - Metabolic Interactions between Spinach Leaf Nitrite Reductase and Ferredoxin-NADP Reductase: Competition for Reduced Ferredoxin. AB - Steady state rates of NADP reduction decline upon commencement of nitrite reduction in reconstituted chloroplast preparations. Similarly, steady state rates of nitrite reduction are lower, but not zero, during concurrent NADP reduction. These results imply that competition for substrate occurs and suggest that nitrite reduction can successfully compete for reduced ferredoxin, even at high rates of NADP reduction. PMID- 16664051 TI - Nitrate inhibition of legume nodule growth and activity : I. Long term studies with a continuous supply of nitrate. AB - The synthesis and accumulation of nitrite has been suggested as a causative factor in the inhibition of legume nodules supplied with nitrate. Plants were grown in sand culture with a moderate level of nitrate (2.1 to 6.4 millimolar) supplied continuously from seed germination to 30 to 50 days after planting. In a comparison of nitrate treatments, a highly significant negative correlation between nitrite concentration in soybean (Glycine max [L.] Merr.) nodules and nodule fresh weight per shoot dry weight was found even when bacteroids lacked nitrate reductase (NR). However, in a comparison of two Rhizobium japonicum strains, there was only 12% as much nitrite in nodules formed by NR(-)R. japonicum as in nodules formed by NR(+)R. japonicum, and growth and acetylene reduction activity of both types of nodules was about equally inhibited. In a comparison of eight other NR(+) and NR(-)R. japonicum strains, and a comparison of G. max, Phaseolus vulgaris, and Pisum sativum, the concentration of nitrite in nodules was unrelated to nodule weight per plant or to specific acetylene reduction activity. The very small concentration of nitrite found in P. vulgaris nodules (0.05 micrograms NO(2) (-)-N per gram fresh weight) was probably below that required for the inhibition of nitrogenase based on published in vitro experiments, and yet the specific acetylene reduction activity was inhibited 83% by nitrate. The overall results do not support the idea that nitrite plays a role in the inhibition of nodule growth and nitrogenase activity by nitrate. PMID- 16664052 TI - Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply. AB - Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR(+) rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR(-) rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N(2) fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR(+) and one NR(-). The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%). PMID- 16664053 TI - Electrogenic proton translocation by the ATPase of sugarcane vacuoles. AB - Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation. PMID- 16664054 TI - Effect of temperature on h(2) evolution and acetylene reduction in pea nodules and in isolated bacteroids. AB - Nitrogenase (EC 1.7.99.2) activity in pea (Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H(2) evolution in air and H(2) evolution in Ar:O(2). With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H(2) evolution in Ar:O(2) was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H(2) evolution in Ar:O(2) with increased temperature, indicating that either acetylene reduction or H(2) evolution in Ar:O(2) are inadequate measures of nitrogenase activity at higher temperature. PMID- 16664055 TI - Isolation and Antigenic Characterization of Corn Mitochondrial F(1)-ATPase. AB - Corn mitochondrial F(1)-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 4 degrees C was substantially activated by ATP, while enzyme stored at -70 degrees C in 25% glycerol was not. Enzyme in glycerol remained fully active (8-9 micromoles P(i) released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60 degrees C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F(1)-ATPase were determined by SDS-polyacrylamide gel electrophoresis to be 58,000 (alpha), 55,000 (beta), 35,000 (gamma), 22,000 (delta), and 12,000 (epsilon). Monoclonal and polyclonal antibodies used in competitive binding assays demonstrated that corn mitochondrial F(1)-ATPase was antigenically distinct from the chloroplastic CF(1) ATPases of corn and spinach. Monoclonal antibodies against antigenic sites on spinach CF(1)-ATPase beta and gamma subunits were used to demonstrate that those sites were either changed substantially or totally absent from the mitochondrial F(1)-ATPase. PMID- 16664056 TI - Kinetic Analysis of Corn Mitochondrial F(1)-ATPase. AB - The activation and catalytic mechanism of corn mitochondrial F(1) were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg(2+) and Ca(2+) were found to be the most effective at promoting ATP hydrolysis. The substrates were hydrolyzed in the order GTP > ITP > ATP regardless of which nucleotide was used for activation. While ATP and GTP hydrolysis exhibited kinetics typical of other ATPases, ITP showed a transition from negative to positive cooperativity at low substrate concentrations. Bicarbonate was found to affect primarily the kinetics of ATP hydrolysis. AMP-PNP proved to be a potent inhibitor with respect to ATP hydrolysis. The results are discussed in terms of possible catalytic mechanisms and the similarities of the corn mitochondrial F(1) to other ATPases. PMID- 16664057 TI - Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes. AB - The ATP-dependent proton-pumping activity of soybean (Glycine max L.) root microsomes is predominantly nitrate sensitive and presumably derived from the tonoplast. We used microsomes to characterize anion effects on proton pumping of the tonoplast vesicles using two distinctly different techniques.Preincubation of the vesicles with nitrate caused inhibition of proton pumping and ATPase activity, with similar concentration dependence. Fluoride, which preferentially inhibits the plasma membrane ATPase, inhibited ATPase activity strongly at concentrations which did not affect proton pumping activity.Addition of potassium salts, after a steady-state pH gradient is established in the absence of such salts, caused an increased pH gradient which was due to alleviation of Delta Psi and subsequent increased influx of H(+) into these vesicles. This anion-induced increase in the pH gradient could be used as a measure of the relative anion permeabilities, which were of the order Br(-) = NO(3) (-) > Cl(-) >> SO(4) (2-). Phosphate and fluoride caused no increase in the pH gradient. Since the concentration dependence of KCl- and KNO(3)-induced quenching exhibited a saturable component, and since H(+) uptake was increased by only certain anions, the data suggest that there may be a relatively specific anion channel associated with tonoplast-derived vesicles. PMID- 16664058 TI - Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings. AB - The aim of this work was to examine the possibility that fructose 2,6 bisphosphate (Fru-2,6-P(2)) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P(2) is known to inhibit cytoplasmic fructose 1,6 bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P(2) was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P(2) in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P(2), respectively) were sufficient to account for the highest observed rates of Fru 2,6-P(2) metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3 phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6 P(2) level. Variation in the level of Fru-2,6-P(2) in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination. PMID- 16664059 TI - Uptake and reduction of [N]nitrate by intact soybean plants in the dark. AB - Experiments were conducted to determine if nitrate ((15)N-labeled) was taken up and assimilated by intact soybean (Glycine max [L.] Merr. cv Williams) plants during extended periods of dark. Nitrate was taken up by soybean roots throughout a 12-hour dark period. The (15)N-labeled nitrogen was also translocated to the plant shoots, but at a slower rate than the rate of accumulation in the roots. Much of the nitrogen ((15)N-labeled) was present in a nonreduced form, although considerable soluble-reduced nitrogen ((15)N-labeled) accumulated throughout the dark period. The (15)N-labeled, soluble-reduced nitrogen fraction accounted for nearly 30% of the total (15)N found in plant roots and more than 63% of the total (15)N found in plant tops after 12 hours of dark. This provided evidence that intact soybean plants take up and metabolize significant quantities of nitrate to reduced N forms in the dark.In addition to nitrate influx during the dark, it was shown that there was a concomitant loss of (15)N-labeled nitrogen compounds from previously (15)N-labeled plants to a natural abundance (15)N nutrient solution. Thus, evidence was obtained which indicated that light was not directly essential for flux and reduction of nitrate by intact soybean plants. PMID- 16664060 TI - Phosphorylation of wheat germ initiation factors and ribosomal proteins. AB - The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M(r) = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro. PMID- 16664061 TI - Glucose and glycine metabolism in regenerating tobacco protoplasts: followed nondestructively by nuclear magnetic resonance spectroscopy. AB - The metabolic states and the uptake and metabolism of [1-(13)C]glucose, [2 (13)C]glycine, and [(15)N]glycine in intact Nicotiana tabacum L. (cv Xanthi) mesophyll protoplasts were measured by (13)C and (15)N nuclear magnetic resonance spectroscopy. Changes in the concentration of metabolites during the first two days of culture in darkness were followed. Protoplasts isolated in 0.55 molar mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 hours of culture. Uptake of glucose and synthesis of glucose-derived metabolites were observed, indicating activity of glycolysis and the tricarboxylic acid cycle. Addition of glycine caused the accumulation of serine in dark cultured protoplasts, via the photorespiratory pathway. Glutamate dehydrogenase and glutamine synthetase activities in photorespiratory NH(4) (+) assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 millimolar glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system. PMID- 16664062 TI - Regulation of NO(3) Influx in Barley : Studies Using NO(3). AB - Short-term (10 minutes) measurements of plasmalemma NO(3) (-) influx (phi(oc)) into roots of intact barley plants were obtained using (13)NO(3) (-). In plants grown for 4 days at various NO(3) (-) levels (0.1, 0.2, 0.5 millimolar), phi(oc) was found to be independent of the level of NO(3) (-) pretreatment. Similarly, pretreatment with Cl(-) had no effect upon plasmalemma (13)NO(3) (-) influx. Plants grown in the complete absence of (13)NO(3) (-) (in CaSO(4) solutions) subsequently revealed influx values which were more than 50% lower than for plants grown in NO(3) (-). Based upon the documented effects of NO(3) (-) or Cl( ) pretreatments on net uptake of NO(3) (-), these observations suggest that negative feedback from vacuolar NO(3) (-) and/or Cl(-) acts at the tonoplast but not at the plasmalemma. When included in the influx medium, 0.5 millimolar Cl(-) was without effect upon (13)NO(3) (-) influx, but NH(4) (+) caused approximately 50% reduction of influx at this concentration. PMID- 16664063 TI - Nitrogen nutrition and metabolic interconversions of nitrogenous solutes in developing cowpea fruits. AB - Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticeably deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, serine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development. PMID- 16664064 TI - Topography of the protein complexes of the chloroplast thylakoid membrane : studies of photosystem I using a chemical probe and proteolytic digestion. AB - The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6 trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of approximately 62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light harvesting chlorophyll protein (LHCPI) complex are not labeled by [(14)C]TNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [(14)C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronase-treated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed. PMID- 16664065 TI - Putrescine uptake in saintpaulia petals. AB - Putrescine uptake and the kinetics of this uptake were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments of [(3)H] or [(14)C] putrescine were done on single petals at room temperature at various pH values. The results show that putrescine uptake occurs against a concentration gradient at low external putrescine concentration (0.5-100 micromolar) and follows a concentration gradient at higher external putrescine concentrations (100 micromolar to 100 millimolar). 2,4-Dinitrophenol and carbonylcyanide-m chlorophenylhydrazone, two uncouplers, had no effect on putrescine uptake. Uptake rates were constant for 2 hours, reaching a maximum after 3 to 4 hours. Putrescine uptake depended markedly on the external pH and two maxima were observed: at low external concentrations of putrescine, the optimum was at pH 5 to 5.5; at higher concentrations the optimum was at pH 8. PMID- 16664066 TI - Peptide alcohols as promoters of nitrate and ammonium ion uptake in plants. AB - Several aryl-carbamoyl dipeptide alcohols increased the uptake of nitrate and ammonium ions into corn root segments by up to 50% and 90%, respectively. The most effective one was N-carbobenzoxy-l-prolyl-l-valinol. Foliar application of this compound to underfertilized corn plants caused an increase in the rate of plant growth in the greenhouse and provided modest (6-10%) corn yield increases in field tests in Delaware. PMID- 16664067 TI - Promotion by Ethylene of the Capability to Convert 1-Aminocyclopropane-1 carboxylic Acid to Ethylene in Preclimacteric Tomato and Cantaloupe Fruits. AB - The intact fruits of preclimacteric tomato (Lycopersicon esculentum Mill) or cantaloupe (Cucumis melo L.) produced very little ethylene and had low capability of converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. When these unripe tomato or cantaloupe fruits were treated with ethylene for 16 hours there was no increase in ACC content or in ethylene production rate, but the tissue's capability to convert ACC to ethylene increased markedly. Such an effect was also observed in fruits of tomato mutants rin and nor, which do not undergo ripening and the climacteric increase in ethylene production during the senescence. The development of this ethylene-forming capability induced by ethylene increased with increasing ethylene concentration (from 0.1 to 100 microliters per liter) and duration (1 to 24 hours); when ethylene was removed this capability remained high for sometime (more than 24 hours). Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene in tomato fruit. These data indicate that the development of the capability to convert ACC to ethylene in preclimacteric tomato and cantaloupe fruits are sensitive to ethylene treatment and that when these fruits are exposed to exogenous ethylene, the increase in ethylene-forming enzyme precedes the increase in ACC synthase. PMID- 16664068 TI - Glutamine Transfer from Xylem to Phloem and Translocation to Developing Leaves of Populus deltoides. AB - The distribution of (14)C from xylem-borne [(14)C]glutamine, the major nitrogen compound moving in xylem sap of cottonwood (Populus deltoides Bartr. ex Marsh), was followed in rapidly growing shoots with a combination of autoradiographic, microautoradiographic, and radioassay techniques. Autoradiography and (14)C analyses of tissues showed that xylem-borne glutamine did not move with the transpiration stream into mature leaves. Instead, most of it was transferred from xylem to phloem in the upper stem and then translocated to young developing tissues. Microautoradiography showed that metaxylem parenchyma, secondary xylem parenchyma, and rays were the major areas of uptake from xylem vessels in the stem. Accumulation in phloem (high (14)C concentrations in sieve tubes) took place in internodes subtending recently mature leaves. Little (14)C from xylem borne glutamine was found in phloem of mature leaves, which indicates restricted retransport of glutamine that did enter the leaf. In the primary tissues of the upper stem, most (14)C was found in the phloem. Cottonwood stems have an efficient uptake and transfer system that enhances glutamine movement to developing tissues of the upper stem. PMID- 16664069 TI - Comparative Distribution and Metabolism of Xylem-Borne Amino Compounds and Sucrose in Shoots of Populus deltoides. AB - The transport and metabolism of xylem-borne amino compounds and sucrose were investigated in rapidly growing shoots of cottonwood (Populus deltoides Bartr. ex Marsh.). (14)C-labeled glutamine, threonine, alanine, glutamic acid, aspartic acid, and sucrose were applied to the base of severed stems for transport in xylem. Distribution and metabolism of the compounds were followed with autoradiography, microautoradiography, and radioassay. Three utilization patterns were observed: (a) little alanine and sucrose was transported to the laminae of either mature leaves or developing leaves. These compounds were taken up from xylem free-space and utilized in adjacent tissue; (b) threonine also did not move into mature leaves but was translocated to developing leaves or utilized in the stem; (c) glutamic acid and aspartic acid were transported directly into the laminae of mature leaves via the xylem. Relatively less (14)C was retained in stems compared to the other compounds.Metabolism of the test compounds also differed considerably. (14)C from amino acids moved primarily into organic acids and protein. The (14)C from sucrose was widely distributed among the chemical fractions, with a high percentage found in structural carbohydrates. Clearly, cottonwood stems contain efficient uptake and transfer systems that differentiate among various compounds moving from root to shoot in xylem. PMID- 16664070 TI - Potassium Transport in Corn Roots : III. Perturbation by Exogenous NADH and Ferricyanide. AB - It has recently been reported that plasmalemma electron transport may be involved in the generation of H(+) gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K(+) ((86)Rb(+)) influx, K(+) ((86)Rb(+)) efflux, net apparent H(+) efflux, and O(2) consumption in 2-centimeter corn (Zea mays [A632 x Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O(2) consumption and K(+) uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O(2) consumption but caused a dramatic inhibition of K(+) influx. Moreover, net apparent H(+) efflux was significantly inhibited following NADH exposure in 4 hour washed root segments.Exogenous ferricyanide inhibited K(+) influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H(+) efflux. Additionally, both reagents substantially altered K(+) efflux at both the plasmalemma and tonoplast.These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the ;wound response' exhibited by corn roots. PMID- 16664071 TI - Characterization of a Selenium-Independent Glutathione Peroxidase From Euglena gracilis. AB - Light or dark grown Euglena gracilis strains contain similar levels of glutathione (GSH) peroxidase. Cells in midstationary phase of growth contained the highest level of the enzyme. The enzyme was purified 280-fold to homogeneity from the permanently bleached strain, E. gracilis var bacillaris W(3)BUL. The native enzyme has a molecular weight of 130,000 as measured by gel permeation chromatography, and contains four subunits (mol wt 31,500) as measured by sodium dodecyl sulfate gel electrophoresis. A variable amount of a higher molecular weight form of the enzyme (approximate mol wt 250,000) was detected but not further characterized. The enzyme has an isoelectric point of 4.7. No selenium could be detected in the purified enzyme. The enzyme is active with H(2)O(2) and a variety of organic hydroperoxides, including 13-hydroperoxylinoleic acid, and is specific for GSH as the thiol substrate. Apparent K(m) values for H(2)O(2), t butyl hydroperoxide, and GSH were 0.03, 1.5, and 0.7 millimolar, respectively. A comparison of selenium-dependent and selenium-independent GSH peroxidases from various eukaryotic sources is presented. PMID- 16664072 TI - Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins : Evidence for the Inactivation of 6'-Hydroxyamatoxins. AB - Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, alpha-amanitin, 6'-deoxy alpha-amanitin, and 6'-O-methyl-alpha-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and alpha-amanitin was much less effective than the other two amatoxins, even though K(i) values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of alpha-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the alpha-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role. PMID- 16664073 TI - In vivo regulation of de novo methionine biosynthesis in a higher plant (lemna). AB - Administration of methionine to growing Lemna had essentially no effect on accumulation of sulfate sulfur in protein cysteine, but decreased accumulation into cystathionine and its products (homocysteine, methionine, S methylmethioninesulfonium salt, S-adenosylmethionine, and S-adenosylhomocysteine) to as low as 21% that of control plants, suggesting that methionine regulates its own de novo synthesis at cystathionine synthesis. Methionine caused only a slight reduction (to 80% that of control plants) in the accumulation of sucrose carbon into the 4-carbon moieties of cystathionine and products. This observation was puzzling since cystathionine synthesis proceeds by incorporation of equivalent amounts of sulfur (from cysteine) and 4-carbon moieties (from O phosphohomoserine). The apparent inconsistency was resolved by the demonstration in Lemna (Giovanelli, Datko, Mudd, Thompson 1983 Plant Physiol 71: 319-326) that de novo synthesis of the methionine 4-carbon moiety occurs not only via the established transsulfuration route from O-phosphohomoserine, but also via the ribose moiety of 5'-methylthioadenosine. It is now clear that the more accurate assessment of the flux of sulfur (and 4-carbon moieties) through transsulfuration is provided by the amount of (35)S from (35)SO(4) (2-) that accumulates in cystathionine and its products, rather than by the corresponding measurements with (14)C. These studies therefore unequivocally demonstrate in higher plants that methionine does indeed feedback regulate it own de novo synthesis in vivo, and that cystathionine synthesis is a locus for this regulation. PMID- 16664074 TI - Stomatal Limitation to Carbon Gain in Paphiopedilum sp. (Orchidaceae) and Its Reversal by Blue Light. AB - Leaves from Paphiopedilum sp. (Orchidaceae) having achlorophyllous stomata, show reduced levels of stomatal conductance when irradiated with red light, as compared with either the related, chlorophyllous genus Phragmipedium or with their response to blue light. These reduced levels of stomatal conductance, and the failure of isolated Paphiopedilum stomata to open under red irradiation indicates that the small stomatal response measured in the intact leaf under red light is indirect.The overall low levels of stomatal conductance observed in Paphiopedilum leaves under most growing conditions and their capacity to increase stomatal conductance in response to blue light suggested that growth and carbon gain in Paphiopedilum could be enhanced in a blue light-enriched environment. To test that hypothesis, plants of Paphiopedilum acmodontum were grown in controlled growth chambers under daylight fluorescent light, with or without blue light supplementation. Total photosynthetic photon flux density was kept constant in both conditions. Blue light enrichment resulted in significantly higher growth rates-of up to 77%-over a 3 to 4 week growing period, with all evidence indicating that the blue light effect was a stomatal response. Manipulations of stomatal properties aimed at long-term carbon gains could have agronomic applications. PMID- 16664075 TI - Stomatal Responses to CO(2) in Paphiopedilum and Phragmipedium: Role of the Guard Cell Chloroplast. AB - A role of the guard cell chloroplasts in the CO(2) response of stomata was investigated through a comparison of the leaf gas exchange characteristics of two closely related orchids: Paphiopedilum harrisianum, which lacks guard cell chloroplasts and Phragmipedium longifolium, which has chlorophyllous guard cells. Leaves of both species had an apparent quantum yield for assimilation of about 0.05, with photosynthesis saturating at 0.300 to 0.400 millimoles per square meter per second. CO(2) curves were obtained by measuring steady-state assimilation and stomatal conductance under 0.180 or 0.053 millimoles per square meter per second white light, or darkness, at 0 to 400 microliters per liter ambient CO(2). The response of assimilation to changes in CO(2) was similar in the two species, but the response of conductance was consistently weaker in Paphiopedilum than in Phragmipedium. The data suggest involvement of guard cell chloroplasts in the stomatal response to CO(2) and in the coupling of assimilation and conductance in the intact leaf. PMID- 16664076 TI - A Model for HCO(3) Accumulation and Photosynthesis in the Cyanobacterium Synechococcus sp: Theoretical Predictions and Experimental Observations. AB - A simple model based on HCO(3) (-) transport has been developed to relate photosynthesis and inorganic carbon fluxes for the marine cyanobacterium, Synechococcus sp. Nageli (strain RRIMP N1). Predicted relationships between inorganic carbon transport, CO(2) fixation, internal carbonic anhydrase activity, and leakage of CO(2) out of the cell, allow comparisons to be made with experimentally obtained data. Measurements of inorganic carbon fluxes and internal inorganic carbon pool sizes in these cells were made by monitoring time courses of CO(2) changes (using a mass spectrometer) during light/dark transients. At just saturating CO(2) conditions, total inorganic carbon transport did not exceed net CO(2) fixation by more than 30%. This indicates CO(2) leakage similar to that estimated for C(4) plants.For this leakage rate, the model predicts the cell would need a conductance to CO(2) of around 10(-5) centimeters per second. This is similar to estimates made for the same cells using inorganic carbon pool sizes and CO(2) efflux measurements. The model predicts that carbonic anhydrase is necessary internally to allow a sufficiently fast rate of CO(2) production to prevent a large accumulation of HCO(3) (-). Intact cells show light stimulated carbonic anhydrase activity when assayed using (18)O-labeled CO(2) techniques. This is also supported by low but detectable levels of carbonic anhydrase activity in cell extracts, sufficient to meet the requirements of the model. PMID- 16664077 TI - Simulation of dehydration injury to membranes from soybean axes by free radicals. AB - Smooth microsomal membranes were isolated from axes of soybean (Glycine max L. Merr.) seeds at the dehydration-tolerant (6 hours of imbibition) and dehydration susceptible (36 hours of imbibition) stages of development and were exposed to free radicals in vitro using xanthine-xanthine oxidase as a free radical source. Wide angle x-ray diffraction studies indicated that the lipid phase transition temperature of the microsomal membranes from the dehydration-tolerant axes increased from 7 to 14 degrees C after exposure to free radicals, whereas those from the dehydration-susceptible axes increased from 9 to 40 degrees C by the same free radical dose. The increased phase transition temperature was associated with a decrease in the phospholipid:sterol ratio, and an increase in the free fatty acid:phospholipid ratio. There was no significant change in total fatty acid saturation, which indicated that free radical treatment induced deesterification of membrane phospholipid, and not a change in fatty acid saturation. Similar compositional and structural changes have been previously observed in dehydration-injured soybean axes suggesting that dehydration may induce free radical injury to cellular membranes. Further, these membranes differ in their susceptibility to free radical injury, presumably reflecting compositional differences in the membrane since these membranes were exposed to free radicals in the absence of cytosol. PMID- 16664078 TI - Gravitropic Responses of Partially Decapitated Corn Coleoptiles with and without Applied [C]Indoleacetic Acid. AB - The curvature of corn seedling (Zea mays L. Mo17 x B73) coleoptiles which had been half-decapitated and supplied with [(14)C]indoleacetic acid (IAA) (3.2 micromolar, 51 milliCuries per millimole) was determined during a 3-hour period of gravitational stimulation. Curvature of such half-decapitated coleoptiles was found to be similar in rate and extent to that of intact coleoptiles responding to gravity. Gravitational stimulation was accomplished by reorienting seedlings to a horizontal position, either up or down with respect to the removed half of the coleoptile tips.The first set of experiments involved placing aluminum foil barriers along one of the two cut surfaces to restrict the movement of IAA into tissues. The initiation and extent of curvature of these half-decapitated coleoptiles was dependent upon the orientation of the removed half-tip and the accompanying barrier. The distribution of radioactivity from [(14)C] IAA after 3 hours indicated that the specific lateral movement of label was also dependent upon orientation of the removed half-tip of the coleoptile. A specific movement to the lower side of approximately 14% of the total recovered radioactivity was found in coleoptiles in which the [(14)C]IAA was supplied across a transverse cut surface. In contrast, specific movement of only 4% was found for application across a longitudinal cut surface.A second series of experiments was conducted using 1.0 and 3.2 micromolar [(14)C]IAA (51 milliCuries per millimole) supplied to half-decapitated coleoptiles without inserted barriers. The 3.2 micromolar concentration adequately replaced the removed coleoptile half-tips in terms of straight growth, but it did not result in as much curvature as shown by coleoptiles of intact seedlings. The 1 micromolar concentration was not adequate to replace the removed half-tip in straight growth, but resulted in gravitropic curvature nearly as great as that produced by the higher concentration.The data presented here suggest that strong auxin gradients are not produced in response to gravity stimulation based on the recovered radioactivity from [(14)C]IAA. However, it is evident that auxin is required for the development of normal gravitropic responses. It is possible, therefore, that an important early role of this movement is not to cause a large stimulation of growth on the lower side but to decrease growth on the upper side of a gravitropically responding coleoptile. PMID- 16664079 TI - Reversal of chlorsulfuron-induced inhibition of mitotic entry by isoleucine and valine. AB - Pea (Pisum sativum L. cv Alaska) root tips were excised and cultured aseptically in White's medium. Cultures were treated immediately or after a 24 hour equilibration time with 28 nanomolar chlorsulfuron plus isoleucine and valine (each 0.1 millimolar), isoleucine and valine, or untreated. The percentage of mitotic figures in untreated control roots sampled immediately after excision showed a transitory drop and recovery within 24 hours (an excision effect). In chlorsulfuron-treated roots, the percentage of mitotic figures did not recover. In roots treated with chlorsulfuron plus isoleucine and valine, a complete recovery did occur. If roots were treated with chlorsulfuron 24 hours after excision, the percentage of mitotic figures was reduced to near 0 by 8 hours. In roots treated with chlorsulfuron plus isoleucine and valine, no reduction in mitotic figures occurred. The complete reversal of chlorsulfuron-inhibited mitotic entry by isoleucine and valine implicates these amino acids, in some manner, with the control of cell cycles progression. PMID- 16664080 TI - Extinction coefficients of chlorophyll a and B in n,n-dimethylformamide and 80% acetone. AB - We found inconsistencies in the commonly used data for chlorophyll analysis in 80% acetone. Recently developed extinction coefficients for chlorophyll b in N,N dimethylformamide (DMF) based on values from 80% acetone are low as a result of these inconsistencies. We determined extinction coefficients of chlorophyll a (Chl a) and chlorophyll b (Chl b) in DMF for wavelengths of 618 to 665 nanometers. The simultaneous equations necessary for quantifying Chl a, Chl b, or total Chl in DMF in the absence of other chlorophyllous pigments are: Chl a = 12.70A(664.5) - 2.79A(647); Chl b = 20.70 A(647) - 4.62A(664.5); total Chl = 17.90A(647) + 8.08A(664.5), where A = absorbance in 1.00 centimeter cuvettes and Chl = milligrams per liter.N,N-Dimethylformamide is a very convenient solvent for Chl extraction since it is effective on intact plant parts and Chl is quite stable in DMF. There was no difference in the amount of Chl extracted when plant tissue was stored for 1 or 3 days at three temperatures, with or without solvent added. PMID- 16664081 TI - Viviparous-1 mutation in maize conditions pleiotropic enzyme deficiencies in the aleurone. AB - The viviparous-1 (vp1) mutation in maize (Zea mays L.) conditions a unique pleiotropic phenotype: premature germination of the embryo and failure to synthesize anthocyanin (flavonoid) pigments in the aleurone. By using a B-A translocation, it is possible to analyze the basis for the anthocyaninless phenotype of vp1 in the absence of vivipary. Anthocyaninless vp1 aleurones were found to be deficient in at least three enzymes of flavonoid biosynthesis (phenylalanine ammonia lyase, chalcone synthase, and UDPG-flavonoid glucosyltransferase) as well as in several other metabolically unrelated enzymes that show pronounced increases in late stages of aleurone development. The set of structural genes encoding such enzymes is postulated to be under the regulation of the vpl gene. PMID- 16664082 TI - Light and Temperature Regulation of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson. AB - During the early morning period, light and temperature exert distinctively different influences on the gas exchange patterns of the Crassulacean acid metabolism plant Opuntia erinacea through their effects on acid metabolism. An initial decrease in CO(2) uptake was triggered by illumination and was apparently due to a decreased CO(2) diffusion gradient through light-mediated decarboxylation of malate. In contrast, the morning burst of CO(2) uptake occurred at high temperature presumably in response to increases in both stomatal conductance and the CO(2) diffusion gradient, resulting from the temperature regulated fixation of endogenous CO(2), primarily into malate. Subsequent stomatal closure, apparently due to elevated levels of internal CO(2) through rapid decarboxylation of malate at high temperature, was primarily responsible for the final termination of early morning Crassulacean acid metabolism. PMID- 16664083 TI - Further Evidence that Cytoplasmic Acidosis Is a Determinant of Flooding Intolerance in Plants. AB - We present two pieces of evidence that regulation of cytoplasmic pH near neutrality is a prerequisite for survival of root tips during hypoxia. First, blackeye peas and navy beans show earlier cytoplasmic acidosis under hypoxia than soybeans or pumpkin or maize, and die earlier. Second, when cytoplasmic acidosis in maize root tips is greatly retarded by treatment with 25 millimolar Ca(NO(3))(2), they remain viable under hypoxia for a much longer period of time than untreated hypoxic root tips. We also show that viability of maize root tips is unaffected by the supply of exogenous sugar (and so on the rate of ethanolic fermentation) for at least 16 hours of hypoxia. PMID- 16664084 TI - Posttranslational processing of proteins in vacuoles and protein bodies is inhibited by monensin. AB - A number of proteins that accumulate in vacuoles and protein bodies undergo posttranslational processing at these accumulation sites. These processing steps include proteolytic cleavage (e.g. pea lectin, soybean glycinin, and rice lectin) and the removal of some sugar residues from oligosaccharide side-chains (e.g. bean phytohemagglutinin). Treatment of immature rice embryos with the sodium ionophore monensin slows down the proteolytic processing of the rice lectin precursor (M(r) 23,000) to mature rice lectin (M(r) 10,000 and 8,000). Treatment of developing bean cotyledons with monensin slows down the removal of peripheral N-acetylglucosamine residues from the oligosaccharide side-chains of phytohemagglutinin. The results are consistent with the interpretation that these processing steps, which occur in vacuoles or protein bodies, are carried out by enzymes with an acidic pH optimum, and that monensin slows down processing by alkalinization of the vacuoles or protein bodies. PMID- 16664085 TI - C Nuclear Magnetic Resonance Study of Mannitol Cycle and Trehalose Synthesis during Glucose Utilization by the Ectomycorrhizal Ascomycete Cenococcum graniforme. AB - (13)C nuclear magnetic resonance spectroscopy has been used to follow the utilization of glucose for the synthesis of carbohydrates in the ectomycorrhizal ascomycete Cenococcum graniforme. The fate of (13)C label was analyzed in vivo and in mycelial extracts. The major carbohydrates produced from [1-(13)C]glucose and [6-(13)C]glucose were mannitol and trehalose. Mannitol was mainly synthesized via a direct route from glucose. Scrambling of the (13)C label was observed to occur in trehalose during glycolysis. From the analysis of the scrambling patterns, it is concluded that the mannitol cycle was operative and that a large part of the carbon of glucose was used to form trehalose after cycling through the mannitol pool. The activities of NAD-mannitol-l-P dehydrogenase (EC 1.1.1.17) and NADP-mannitol dehydrogenase (EC 1.1.1.138), which participate in the mannitol cycle relative to the activity of glycolytic enzymes, provide evidence that the cycle is important for NADPH production. PMID- 16664086 TI - Action Spectrum of the Activity of Acifluorfen-methyl, a Diphenyl Ether Herbicide, in Chlamydomonas eugametos. AB - Light is required for the herbicide activity of diphenyl ether herbicides. An action spectrum of acifluorfen-methyl activity with Chlamydomonas eugametos (Moewus) determined that cell death occurred at two peaks of light; 450 and 670 nanometers. These data indicate both chlorophyll and carotenoids, but not riboflavin, are involved in herbicide toxicity. PMID- 16664087 TI - Diurnal variation of asparaginase in developing pea leaves. AB - Levels of asparaginase activity from developing pea leaves (Pisum sativum) were found to change on a daily basis, increasing during the light period and decreasing in the dark. During extended periods of light, high levels of activity were maintained, while prolonged dark reduced activity to a low value. Half expanded leaves exhibited the greatest change in activity over the photoperiod. Very young or mature leaves displayed little or no diurnal variation in asparaginase activity. PMID- 16664088 TI - Fermentative Metabolism of Chlamydomonas reinhardtii: II. Role of Plastoquinone. AB - Evidence is presented to substantiate a chloroplastic respiratory pathway in the green alga, Chlamydomonas reinhardtii, whereby reducing equivalents generated during the degradation of starch enter the thylakoidal chain at the plastoquinone site catalyzed by NADH-plastoquinone reductase. In this formulation, the reduced plastoquinone is oxidized either by the photoevolution (photosystem I) of H(2) under anaerobic conditions or by O(2) during dark respiration. PMID- 16664089 TI - Isolation of amyloplasts from developing maize endosperm. AB - Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4 degrees C or 5 hours at 30 degrees C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl(2). After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes. PMID- 16664090 TI - Glutamine metabolism in corn kernels cultured in vitro. AB - The fate of glutamine, the major source of nitrogen supplied to the developing maize endosperm, has been examined in endosperm tissues of corn caryopsis grown under sterile conditions. In the culture system, [U-(14)C]glutamine was included in the medium or was injected directly into the endosperm. Samples were harvested at intervals up to 168 hours. Protein and starch fractions were then separated and analyzed for their (14)C content. At 168 hours, 31% of the total label incorporated in the endosperm was in zein, 15% in glutelin, and 24% in starch. When individual amino acids and sugars in the endosperm powders were analyzed, the (14)C still remaining in the glutamine accounted for only 12 to 14% of the total radioactivity. PMID- 16664091 TI - Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue. AB - Sugar release from the pedicel tissue of maize (Zea mays L.) kernels was studied by removing the distal portion of the kernel and the lower endosperm, followed by replacement of the endosperm with an agar solute trap. Sugars were unloaded into the apoplast of the pedicel and accumulated in the agar trap while the ear remained attached to the maize plant. The kinetics of (14)C-assimilate movement into treated versus intact kernels were comparable. The rate of unloading declined with time, but sugar efflux from the pedicel continued for at least 6 hours and in most experiments the unloading rates approximated those necessary to support normal kernel growth rates. The unloading process was challenged with a variety of buffers, inhibitors, and solutes in order to characterize sugar unloading from this tissue.Unloading was not affected by apoplastic pH or a variety of metabolic inhibitors. Although p-chloromercuribenzene sulfonic acid (PCMBS), a nonpenetrating sulfhydryl group reagent, did not affect sugar unloading, it effectively inhibited extracellular acid invertase. When the pedicel cups were pretreated with PCMBS, at least 60% of sugars unloaded from the pedicel could be identified as sucrose. Unloading was inhibited up to 70% by 10 millimolar CaCl(2). Unloading was stimulated by 15 millimolar ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid which partially reversed the inhibitory effects of Ca(2+). Based on these results, we suggest that passive efflux of sucrose occurs from the maize pedicel symplast followed by extracellular hydrolysis to hexoses. PMID- 16664092 TI - A developmentally regulated hydroxyproline-rich glycoprotein from the cell walls of soybean seed coats. AB - In soybean seeds the level of hydroxyproline is regulated in a developmental and tissue-specific manner. The seed coat contains approximately 77% of the total hydroxyproline in the seed at all stages of development. We determined the ratio of hydroxyproline to dry weight in a number of tissues within the seed; however, only the seed coat shows an increase in this ratio during development. Within the many cell layers of the seed coat, hydroxyproline is most abundant in the external layer. The hydroxyproline is present as an hydroxyproline-rich cell wall glycoprotein. The protein is rich in hydroxyproline (36%), lysine (11%), proline (10%), histidine (9%), tyrosine (9%), and serine (8%). The carbohydrate portion is 90 mole% arabinose and 10 mole% galactose. The arabinose residues are attached to hydroxyproline mostly in the form of trisaccharides. The apparent molecular weight of this glycoprotein is 100,000 daltons. PMID- 16664093 TI - Effects of Aminooxyacetate and Aminoacetonitrile on Glycolate and Ammonia Release by the Cyanobacterium Anabaena cylindrica. AB - Aminooxyacetate and aminoacetonitrile cause increased excretion of glycolate by the cyanobacterium Anabaena cylindrica. Both compounds also reduce NH(4)-N release induced by methionine sulfoximine in non-nitrogen-fixing cultures. Changes in amino acid pool sizes together with changes in activities of some enzymes related to glycolate metabolism show that glyoxylate to glycine conversion and glycine to serine conversion are inhibited by aminooxyacetate and aminoacetonitrile, respectively. The results also verify that photorespiratory glycolate metabolism via amination of glyoxylate is operative in A. cylindrica. PMID- 16664094 TI - Inhibition of phytochrome synthesis by gabaculine. AB - Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein. PMID- 16664095 TI - Chitosan-elicited callose synthesis in soybean cells as a ca-dependent process. AB - A new method for the rapid and quantitative fluorometric determination of callose is described. In suspension-cultured cells of Glycine max, synthesis of callose starts within 20 minutes of treatment with chitosan and parallels over hours the accumulation of 1,3-linked glucose in the wall. Poly-l-lysine also elicits callose synthesis. The effect of chitosan is enhanced by Polymyxin B at low concentrations; this antibiotic alone at higher concentrations can also induce callose synthesis. Callose synthesis is immediately stopped when external Ca(2+) is bound by ethylene glycolbis-(2-aminoethyl ether)-N,N'-tetraacetate or cation exchange beads, and partly recovers upon restoration of 15 micromolar Ca(2+).Callose synthesis is observed only when membrane perturbation causing electrolyte leakage from the cells is induced by one of the above treatments. It does not appear to be due to de novo synthesis or proteolytic activation of 1,3 beta-d-glucan synthase. It is concluded that this Ca(2+)-dependent enzyme is directly activated by the influx of Ca(2+) occurring concomitantly with the leakage of cell constituents. This suggestion is also discussed in conjunction with the chitosan-induced synthesis of phytoalexin in the same cells. PMID- 16664096 TI - Rapid suppression of extension growth in dark-grown wheat seedlings by red light. AB - Continuous recordings were made using a linear displacement transducer to investigate short-term growth responses of intact dark-grown wheat (Triticum aestivum L. cv Maris Huntsman) seedlings to red light. To eliminate any effect of light prior to the experimental treatments, the seedlings were grown and mounted on the transducer apparatus in total darkness. The growth kinetics after irradiation were complex and appeared to consist of three successive phases of growth deceleration. When the tip of the intact coleoptile was irradiated with red light from two opposite fiber bundles (fluence rate: 2 x 64 micromoles per square meter per second) for varying periods of time (10 seconds, 1 minute, 5 minutes, continuous), a decrease in extension rate was detectable after a latent period of 8 to 10 minutes. Up to 30 minutes after the start of the irradiation treatment, there was no difference in the kinetics of inhibition (about 20 to 25% inhibition) between the different lengths of irradiation. Extension rate reached a minimum (65% inhibition) at about 85 minutes, after which growth acceleration toward the dark control rate was observed. Far-red reversibility of the rapid effect of red light on growth was not observed, even when far-red light was given only 4 seconds after the end of 10 seconds red light. Short (15 seconds) far-red light did not induce a response. PMID- 16664097 TI - Sexual Interaction in Heterothallic Strains of Closterium peracerosum-strigosum littorale: Partial Characterization of a Male-Specific Sexual Pheromone, Protoplast-Releasing Substance. AB - A sexual pheromone, named the protoplast releasing substance (PRS), was formed by mating type minus cells. PRS activates mating type plus cells and results in the release and fusion of protoplasts within distended conjugation-papilla in paired cells and in the release and disruption of protoplasts in unpaired mating type plus cells. In an agar barrier system, the activation of mating type plus cells was markedly inhibited by treatment with pronase (5-10 micrograms per milliliter), proteinase (100 micrograms per milliliter), and alpha-mannosidase (10 micrograms per milliliter). Trypsin (10-100 micrograms per milliliter) had no effect on the activation in the agar barrier system. The results suggest that PRS is a glycoprotein with pronase-sensitive and trypsin-insensitive structure. PMID- 16664098 TI - Nitrate Uptake and Partitioning by Corn Root Systems : Differential Effects of Ammonium among Genotypes and Stages of Root Development. AB - The relative effects of ammonium on nitrate uptake and partitioning during induction were compared among decapitated seedlings of three corn (Zea mays L.) genotypes at two developmental stages. This study tested the hypothesis that root systems efficient at translocating products of ammonium assimilation away from sites of nitrate uptake or reduction would exhibit less inhibition of nitrate uptake by ammonium compared to root systems with inefficient N translocation efficiency. Inhibition of nitrate uptake by ammonium was relatively slight at day 5 ranging from 0% to 20% among the three genotypes, as compared to greater inhibition, from 20% to 37%, at day 8. Five-day-old roots exhibited negligible xylem translocation capacity in comparison with those grown for 8 days. Thus, although the capability to translocate ammonium assimilates out of the root increased between days 5 and 8, inhibitory effects of ammonium also increased. In the absence of ammonium, nitrate uptake per unit root mass increased between days 5 and 8. This increased activity of the uptake system was proportionally more sensitive to ammonium.Partitioning of entering nitrate into the reduction process was positively correlated with lateral root development of the inbred root systems at 5 and 8 days. This is supportive of a localization of a major portion of nitrate reduction occurring in root apical regions. Nitrate reduction was the partitioning process most severely inhibited by ammonium in all cases, ranging from 39% to 55% inhibition. In contrast, ammonium-inhibition of nitrate accumulation in the root tissue and translocation via xylem vessels varied with genotype and root age.Two mechanisms of ammonium-inhibition of nitrate are implicated, one which directly affects nitrate reduction and the uptake system associated with it, and another which may involve potassium as an intermediate regulator of nitrate accumulation in the root tissue and nitrate translocation out of the root tissue. PMID- 16664099 TI - Epicuticular Lipid Accumulation on the Leaves of Lycopersicon pennellii (Corr.) D'Arcy and Lycopersicon esculentum Mill. AB - A comparison was made of epicuticular lipid accumulation on leaves of Lycopersicon pennellii and Lycopersicon esculentum Mill. cv VF36 from 5 to 16 weeks of age. Epicuticular lipids were a small fraction of the leaf dry weight (0.16%) of 5-week-old ;VF36', and increased to only 0.96% of the leaf dry weight after an additional 12 weeks of growth. In contrast, leaves from 5-week-old and 17-week-old L. pennellii plants had, respectively, 0.94% and 19.9% of their total dry weight in epicuticular lipid. Lipid accumulation was not affected by drought stress. Leaf position appears to influence the amount of lipid on the leaf surface. A glycolipid appears to be exuded from the terminal cell of glandular trichomes found on the leaves, stems, peduncles, calyxes, and fruits of L. pennellii. PMID- 16664100 TI - Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex : A Source of Acetyl-CoA and NADH for Fatty Acid Biosynthesis. AB - The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 mumoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD(+), and coenzyme-A and the products as NADH, CO(2), and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis. PMID- 16664101 TI - Reduced Apparent Photorespiration by the C(3)-C(4) Intermediate Species, Moricandia arvensis and Panicum milioides. AB - The CO(2)/O(2) specificity factor of sucrose gradient purified ribulose 1,5 bisphosphate carboxylase/oxygenase from the C(3)-C(4) intermediate plants Moricandia arvensis (79 +/- 1) and Panicum milioides (89 +/- 2) was similar to the respective values of the enzyme from the closely related C(3) species, Moricandia foetida (80 +/- 5) and Panicum laxum (86 +/- 2). Thus, the kinetic properties of this bifunctional enzyme do not explain the reduced rates of photorespiration exhibited by either of these intermediate species.Dark/light ratios for aminoacetonitrile-sensitive (14)CO(2) evolution during decarboxylation of exogenous [1-(14)C]glycine by leaf discs had values of 9.0 with M. arvensis and 11.8 with P. milioides. Equivalent ratios with M. foetida and P. laxum were 2.5 and 3.2, respectively. Similar results were obtained using [1-(14)C]glycolate as the exogenous photorespiratory substrate, with dark/light (14)CO(2) evolution ratios for the C(3)-C(4) and C(3) leaf discs averaging 6.6 and 2.0, respectively. Stimulating photosynthetic CO(2) fixation by progressively increasing photon flux density from 0 to 1900 micromoles per square meter per second caused a concomitant reduction in (14)CO(2) evolution from leaf discs of M. arvensis and P. milioides supplied with [1-(14)C]glycine. Conversely, inhibition of photosynthesis by DCMU or the Calvin cycle inhibitor dl-glyceraldehyde increased (14)CO(2) evolution in the light to rates comparable to those in the dark. The data suggest that P. milioides and M. arvensis are capable of a more efficient internal recycling of photorespiratory CO(2) via ribulose bisphosphate carboxylase/oxygenase than closely related C(3) plants, and that this may partially account for the reduced rates of apparent photorespiration by these intermediate species. PMID- 16664102 TI - Reversal of the effects of aging in soybean seeds. AB - Accelerated aging predisposed seeds to imbibition injury. Slowing the rate of hydration prevented the loss of germinability due to imbibition injury. Germinability of accelerated aged seeds (50 hours) was increased from 10 to 90% by controlling the rate of imbibition. Slow hydration also prevented seed electrolyte leakage. This may indicate that cell membrane permeability or rupture was a major factor contributing to the loss of germinability after aging.Reversal of the effects of aging (repair) was accomplished by slowly inbibing and then redrying seeds (priming). This treatment lowered steep water conductivity by a factor of 2 to 5. Priming also increased the per cent germination of low vigor seeds. The mechanism of this reversal was probably metabolic because it depended on temperature, seed moisture, and treatment duration.Priming doubled the survival of seeds in the accelerated aging vigor test. The ;rejuvenation' was accepted as evidence for metabolic repair. Since the ;vigor' of seeds was increased by priming, metabolic repair probably included other subcellular components as well as the plasma membrane. PMID- 16664103 TI - Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss. AB - Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [(3)H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [(3)H]leucine or [(14)C]proline.Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss. PMID- 16664104 TI - Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea. AB - A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC(90)in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response. PMID- 16664105 TI - Evaluation of a system for the imposition of plant water stress. AB - A system which imposes a range of water stress levels was developed and evaluated. Water stress was controlled by employing a screen to suspend roots above a water column of known height. Levels of water stress were imposed by changing water column height and/or hydraulic conductivity of the medium in the column. The system was evaluated in a series of growth chamber experiments in which sunflowers (Helianthus annuus L. cv NK894) were given three levels of water availability for a period of 3 weeks. Third leaf midday water potentials at the end of the trials ranged from -0.73 +/- 0.04 to -2.35 +/- 0.17 megapascals in waterstressed plants compared to -0.40 +/- 0.02 megapascals for control plants. Repetition of experiments showed no statistical differences in leaf water potentials, plant leaf areas, or plastochron indices between trials. During the experiments, the severity and pattern of water stress developments was related to both water column height and conductivity of the medium. Control plants exhibited normal diurnal water relations and transpirational behavior. Use of this system avoids many problems associated with other techniques and provides a means for subjecting plants to reproducible water stress levels for extended periods of time. PMID- 16664106 TI - Stabilization of pyruvate, pi dikinase regulatory protein in maize leaf extracts. AB - The objective of this study was to determine the biochemical basis for genetic variability in pyruvate,Pi dikinase (PPDK) activity among inbred lines of maize (Zea mays L.). Although in vitro PPDK activity varied more than 5-fold among eight maize inbreds, immunochemical determinations of the proportion of leaf soluble protein as PPDK revealed no significant differences among the inbreds. Genetic differences in the stability of PPDK activity in crude homogenates over 5 hours were not evident, but PPDK from some inbreds could not be activated in vitro. In vitro PPDK activation in crude homogenates could be restored by addition of casein (1% w/v) to homogenization media, and to a lesser extent, by gentle homogenization in a mortar. The major effect of casein appeared to be on processes other than proteolysis, as casein exerted its effects during tissue homogenization, rather than later. During homogenization, PPDK did not lose its ability to undergo in vitro activation; instead, it was instability of the regulatory protein responsible for PPDK activation that was the cause of the lack of PPDK activation in homogenates prepared without casein. PMID- 16664107 TI - Relationships between CO(2) Exchange Rates and Activities of Pyruvate,Pi Dikinase and Ribulose Bisphosphate Carboxylase, Chlorophyll Concentration, and Cell Volume in Maize Leaves. AB - The relationships between CO(2)-exchange rate (CER), DNA and chlorophyll (Chl) concentrations, pyruvate,Pi dikinase (PPDK) and ribulose bisphosphate carboxylase (RuBPCase) activities in ten maize (Zea mays L.) genotypes were investigated. The in vivo degrees of activation of PPDK and RuBPCase were estimated to make meaningful comparisons with CER. In leaves at a photosynthetic photon flux density (PPFD) of 720 micromoles per square meter per second, in vivo PPDK degree of activation was 80% of that of PPDK fully activated in vitro, whereas RuBPCase could not be further activated in vitro, suggesting that RuBPCase was fully activated in vivo. CER varied about 50% among the genotypes tested. Significant genetic differences were observed for the average weight of a cell (estimated by gram fresh weight per milligram DNA), but this character was not correlated with CER expressed on a fresh weight basis. CER was correlated with Chl concentration, and with estimates of the in vivo degree of activation of PPDK and RuBPCase. We concluded that in maize, CER is controlled by the metabolic components of photosynthesis rather than by membrane resistances to CO(2). If the latter factor were controlling CER, then smaller cells with higher amounts of exposed cell surface area per unit cell volume would have lower resistance to CO(2) diffusion, and therefore higher CER. When data were expressed on a DNA basis (proportional to a per cell basis), results indicated that larger cells (i.e. those with higher fresh weight per milligram DNA) have a higher content of Chl, and higher PPDK and RuBPCase activities, resulting in higher CER than in smaller cells. PMID- 16664108 TI - Starch and Sucrose Synthesis in Phaseolus vulgaris as Affected by Light, CO(2), and Abscisic Acid. AB - Phaseolus vulgaris L. leaves were subjected to various light, CO(2), and O(2) levels and abscisic acid, then given a 10 minute pulse of (14)CO(2) followed by a 5 minute chase with unlabeled CO(2). After the chase period, very little label remained in the ionic fractions (presumed to be mostly carbon reduction and carbon oxidation cycle intermediates and amino acids) except at low CO(2) partial pressure. Most label was found in the neutral, alcohol soluble fraction (presumed sucrose) or in the insoluble fraction digestable by amyloglucosidase. Sucrose formation was linearly related to assimilation rate (slope = 0.35). Starch formation increased linearly with assimilation rate (slope = 0.56) but did not occur if the assimilation rate was below 4 micromoles per square meter per second. Neither abscisic acid, nor high CO(2) in combination with low O(2) (thought to disrupt control of carbon metabolism) caused significant perturbations of the sucrose/starch formation ratio. These studies indicate that the pathways for starch and sucrose synthesis both are controlled by the rate of net CO(2) assimilation, with sucrose the preferred product at very low assimilation rates. PMID- 16664109 TI - Host recognition in the Rhizobium-soybean symbiosis : evidence for the involvement of lectin in nodulation. AB - Rhizobium japonicum mutant strain HS111 was previously shown to be defective in the rate of initiation of infection leading to subsequent nodule formation (1984 Plant Physiol 74: 84-89). Mutant strain HS111's defect in nodulation can be phenotypically reversed to wild type levels by pretreatment with root exudates from all soybean varieties that have been tested. The data indicate that lectin Rhizobium interaction is necessary for the phenotypic reversal of the nodulation characteristics of mutant strain HS111. Pretreatment of strain HS111 with soybean seed lectin mimics the effect of root exudate pretreatment. In addition, the presence of 30 millimolar d-galactose, a hapten of soybean seed lectin, in the root exudate or soybean seed lectin pretreatment solution prevents enhancement of nodulation of strain HS111. Pretreatment of mutant strain HS111 in soybean root exudate which has had galactose-specific lectin(s) removed by affinity chromatography (affinity eluate) results in no enhancement of nodulation by strain HS111. Lectin(s) subsequently removed from the affinity column possesses 100% of the stimulatory activity originally found in the root exudate. Pretreatment of strain HS111 in root exudate from a soybean seed line (T102) known to lack seed lectin due to an insertion in the structural gene results in the reversal of the defective nodulation phenotype. This latter result indicates that the lectin found in soybean root exudate is genetically distinct from the seed lectin. It is apparently this root lectin that is involved in nodulation. PMID- 16664110 TI - Characterization of a Purified Photosystem II-Phycobilisome Particle Preparation from Porphyridium cruentum. AB - Detergent preparations isolated from thylakoids of the red alga Porphyridium cruentum, in a sucrose, phosphate, citrate, magnesium chloride medium consist of phycobilisomes and possess high rates of photosystem II activity. Characterization of these particles shows that the O(2)-evolving activity is stable for several hours and the pH optimum is about 6.5 to 7.2. Response of the system to light, electron donors and acceptors, and inhibitors verify that the observed activity, measured both as O(2) evolution and 2,6-dichlorophenol indophenol reduction, is due to photosystem II. Furthermore, photosystem II is functionally coupled to the phycobilisome in this preparation since green light, absorbed by phycobilisomes of P. cruentum, is effective in promoting both O(2) evolution and 2,6-dichlorophenol-indophenol reduction. Photosystem II activity declines when light with wavelengths shorter than 665 nm is removed. Both 3-(3,4 dichlorophenyl)-1,1-dimethylurea and atrazine inhibit photosystem II activity in this preparation, indicating that the herbicide binding site is a component of the photosystem II-phycobilisome particle. PMID- 16664111 TI - Production of a Lectin in Tissue Cultures of Dolichos biflorus. AB - Callus cultures have been produced from the epicotyl and leaves, hypocotyl, and roots of germinating Dolichos biflorus seeds. These cultures were initiated on media containing 2,4-dichlorophenoxyacetic acid and kinetin, transferred to media with increased amounts of these hormones, and then maintained on hormone-free media. Extracts of these cultures were examined by radioimmunoassays specific for the lectin from the seeds of this plant and for a lectin that is present only in the stems and leaves of the intact plant. Although the seed lectin was not detected in any cultures, the stem and leaf lectin was produced in those cultures grown on the hormone free media. Lectin isolated from these cultures had subunits identical in electrophoretic mobilities to the subunits from the lectin isolated from intact stems and leaves. Levels of this lectin decreased when the cells were transferred back to media containing hormones and increased again upon transfer to the hormone-free media. The absence of exogenous hormones and the production of lectin were also correlated with the rapid growth and greening of the cells. Immunofluorescence and immunocytochemical studies on sections of cultured cells indicated that the stem and leaf lectin is associated with the cytoplasm as well as the cell wall as has been found in previous studies on the subcellular localization of this lectin in the intact plant. PMID- 16664112 TI - Cytokinin metabolism in phaseolus embryos : genetic difference and the occurrence of novel zeatin metabolites. AB - The metabolism of trans-[8-(14)C]zeatin was examined in embryos of Phaseolus vulgaris cv Great Northern (GN) and P. lunatus cv Kingston (K) in an attempt to detect genetic variations in organized plant tissues. Metabolites were fractionated by HPLC, and identified by chemical and enzymic tests and GC-MS analyses. Five major metabolites were recovered from P. vulgaris embryo extracts: ribosylzeatin, ribosylzeatin 5'-monophosphate, an O-glucoside of ribosylzeatin, and two novel metabolites, designated as I and II. Based on results of degradation tests and GC-MS analyses, I and II were tentatively identified as O ribosyl derivatives of zeatin and ribosylzeatin. In embryos of P. lunatus, however, metabolites I and II were not present. The major metabolites were ribosylzeatin, ribosylzeatin 5'-monophosphate, and the O-glucosyl derivatives of zeatin and ribosylzeatin. The zeatin metabolites recovered were the same for embryos of different sizes but their quantities varied with embryo size and incubation time. The genetic differences appear to be embryo-specific and may be useful in the studies of the possible relationship between abnormal interspecific hybrid embryo growth and hormonal derangement in Phaseolus. In addition, analyses of both organized (intact) and unorganized (callus) tissues of the same genotype may provide an opportunity to address the problem of differential expression of genes regulating cytokinin metabolism during plant development. PMID- 16664113 TI - Isolation and Characterization of an Elicitor of Necrosis Isolated from Intercellular Fluids of Compatible Interactions of Cladosporium fulvum (Syn. Fulvia fulva) and Tomato. AB - Intercellular fluids of compatible race-cultivar interactions of Cladosporium fulvum and tomato contain specific elicitors of necrosis. These elicitors which are of fungal origin induce chlorosis and necrosis in resistant but not in susceptible plants. With the tomato cultivar Sonatine (carrying resistance gene Cf9, resistant to the fungal races 0, 4, 5, 2, 2.4, and 2.4.5 but susceptible to race 2.4.5.9) as the test plant for assaying necrosis-inducing activity, we isolated and partially characterized an elicitor of necrosis on this cultivar. The elicitor bound to CM-Sephadex but not to DEAE-Sephadex; it was stable to heat (10 minutes at 100 degrees C), HCl (0.01 normal), NaOH (0.01 normal), and NaIO(4) (0.02 molar), sensitive to pronase and protease (from Bacillus polymyxa) but not to other proteases such as alpha-chymotrypsin and trypsin. After electrophoresis of partially purified elicitor preparations under low pH conditions, the necrosis inducing activity was association with a peptide with an apparent molecular weight of 5500. Races 0, 4, 5, 2.4, and 2.4.5 but not race 2.4.5.9 produced this elicitor in high yields. The elicitor is probably a product of avirulence gene A9 which is present in all races except in race 2.4.5.9 and induces necrosis in cultivars carrying resistance gene Cf9. PMID- 16664114 TI - A Low Molecular Weight Polypeptide Which Accumulates upon Inhibition of Porphyrin Biosynthesis in Maize. AB - Levulinic acid, an inhibitor of porphyrin biosynthesis, causes marked accumulation of a low molecular weight polypeptide in greening maize (Zea mays L.) leaves. Additional compounds which interfere with porphyrin synthesis (e.g. aminooxyacetate, iron-chelators, 4,6-dioxoheptanoic acid) had a similar effect. The polypeptide accumulated in the cytosol and could not be detected in the plastid stroma. Its molecular weight was estimated as 4800 daltons by electrophoresis in sodium dodecyl sulfate-acrylamide gels containing urea and glycerol. The accumulation of the polypeptide did not result from inhibition of chlorophyll or protoheme syntheses. Compounds which caused its accumulation markedly reduced the activity of nitrite reductase. It is suggested that the accumulation is caused by inhibition of siroheme synthesis which interferes with the formation of nitrite or sulfite reductase. PMID- 16664115 TI - Photosynthesis, Morphology, Leaf Anatomy, and Cytogenetics of Hybrids between C(3) and C(3)/C(4)Panicum Species. AB - The Laxa group of the Panicum genus contains species which have CO(2) exchange and anatomical characteristics intermediate to C(3) and C(4) photosynthetic types (C(3)/C(4)), and also species characterized as C(3). Hybrids were made between two of the C(3)/C(4) species and two C(3) species. Carbon dioxide exchange and morphological, leaf anatomical, and cytogenetic characteristics of F(1) hybrids between Panicum milioides Nees. ex Trin (C(3)/C(4)) and P. laxum Mez. (C(3)), P. spathellosum Doell (C(3)/C(4)) and P. boliviense Hack. (C(3)), and P. spathellosum and P. laxum were studied. There were no consistent differences in apparent photosynthesis, although two of the three hybrids had higher net CO(2) uptake than the C(3) parent. Values of inhibition of apparent photosynthesis by 21% O(2), CO(2) loss in the light, and CO(2) compensation concentration for the hybrids were between those of the parents. All three hybrids showed leaf anatomical traits, especially organelle quantities in the bundle sheath cells, between those of their respective parents. Linear regression of CO(2) compensation concentration on the percentage of mitochondria and chloroplasts in vascular bundle sheaths of the parents and hybrids gave correlation coefficients of -0.94. This suggests that the reduction in CO(2) loss in the C(3)/C(4) species, and to a lesser degree in the F(1) hybrids, was due to development of organelles and perhaps a higher proportion of leaf photorespiration in bundle sheaths. The overall morphology of the hybrids was so different from the parents that they could be described as new taxonomic forms. The chromosomes in the hybrids were mainly unpaired or paired as bivalents indicating possible homology between some parental genomes. PMID- 16664116 TI - Histochemical technique : a general method for quantitative enzyme assays of single cell ;extracts' with a time resolution of seconds and a reading precision of femtomoles. AB - Biochemists who study single cells have been constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods we describe will remove this limitation. In brief, nanogram tissue samples are dissected from frozen-dried tissue. The samples are ;extracted' in microdroplets of assay cocktail. The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. In the development of this method, we evaluated several parameters required for optimization; the most important of these evaluations, including numerous empirically derived relationships, are reported here and in supplemental material provided with reprints.With these methods, assays of pyruvate orthophosphate dikinase on samples enriched in bundlesheath cells and mesophyll cells of Flaveria brownii yielded the predictable results. Assays of this enzyme in guard cells dissected from Vicia faba leaflets gave results like those recently reported by another laboratory for protoplasts derived from these cells. The results of assays by this method and by enzymic cycling for NAD(P)triose-P dehydrogenase were comparable. Phosphoenolpyruvate carboxylase, the most extensively studied enzyme activity, was present at high levels in guard cells, which has been demonstrated previously in other reports based on diverse assay approaches. PMID- 16664117 TI - Diurnal regulation of phosphoenolpyruvate carboxylase from crassula. AB - Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K(m) for phosphoenolpyruvate and reducing V(max). With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K(m) for phosphoenolpyruvate, but having little effect on V(max). The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K(m) for phosphoenolpyruvate and an increased V(max), but the night, or insensitive, form shows only an increase in V(max) in response to glucose-6-phosphate. PMID- 16664118 TI - Control of Seed Germination by Abscisic Acid : III. Effect on Embryo Growth Potential (Minimum Turgor Pressure) and Growth Coefficient (Cell Wall Extensibility) in Brassica napus L. AB - The physical mechanism of seed germination and its inhibition by abscisic acid (ABA) in Brassica napus L. was investigated, using volumetric growth (= water uptake) rate (dV/dt), water conductance (L), cell wall extensibility coefficient (m), osmotic pressure ( product operator(i)), water potential (Psi(i)), turgor pressure (P), and minimum turgor for cell expansion (Y) of the intact embryo as experimental parameters. dV/dt, product operator(i), and Psi(i) were measured directly, while m, P, and Y were derived by calculation. Based on the general equation of hydraulic cell growth [dV/dt = Lm/(L + m) (Delta product operator - Y), where Delta product operator = product operator(i) - product operator of the external medium], the terms (Lm/(L + m) and product operator(i) - Y were defined as growth coefficient (k(G)) and growth potential (GP), respectively. Both k(G) and GP were estimated from curves relating dV/dt (steady state) to product operator of osmotic test solutions (polyethylene glycol 6000).During the imbibition phase (0-12 hours after sowing), k(G) remains very small while GP approaches a stable level of about 10 bar. During the subsequent growth phase of the embryo, k(G) increases about 10-fold. ABA, added before the onset of the growth phase, prevents the rise of k(G) and lowers GP. These effects are rapidly abolished when germination is induced by removal of ABA. Neither L (as judged from the kinetics of osmotic water efflux) nor the amount of extractable solutes are affected by these changes. product operator(i) and Psi(i) remain at a high level in the ABA-treated seed but drop upon induction of germination, and this adds up to a large decrease of P, indicating that water uptake of the germinating embryo is controlled by cell wall loosening rather than by changes of product operator(i) or L. ABA inhibits water uptake by preventing cell wall loosening. By calculating Y and m from the growth equation, it is further shown that cell wall loosening during germination comprises both a decrease of Y from about 10 to 0 bar and an at least 10-fold increase of m. ABA-mediated embryo dormancy is caused by a reversible inhibition of both of these changes in cell wall stability. PMID- 16664119 TI - Fucosylation of membrane proteins in soybean cultured cells : effects of tunicamycin and swainsonine. AB - Cultures of soybean cells incorporate [5,6-(3)H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose(15-17)-N-acetylglucosamine(2) (GlcNAc(2)) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose(12-13)-GlcNAc(2) structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.The membrane glycoproteins were doubly-labeled by incubating soybean cells in [(3)H]fucose and [(14)C]mannose. By repeated separation on Biogel P-4, six glycopeptides were purified that ranged in size from Hexose(8)GlcNAc(2) to Hexose(15-17)-GlcNAc(2). The three larger glycopeptides (I, II, III) were highly labeled with [(3)H]fucose and also contained [(14)C] mannose. Evidence that both isotopes were in the same glycopeptide was obtained by the finding that the mannose-labeled glycopeptides were shifted to smaller-sized structures when the [(3)H]fucose was removed by mild acid hydrolysis. Glycopeptide IV also contained [(3)H]fucose and [(14)C] mannose but only part of the [(3)H]fucose was released by mild hydrolysis. Glycopeptides V and VI contained only small amounts of tritium, but were labeled with [(14)C]mannose. None of the six glycopeptides was susceptible to the action of endo-beta-N-acetylglucosaminidase H, and none of these glycopeptides was bound to columns of concanavalin A-sepharose. The smaller glycopeptides (IV, V, VI) were partially susceptible to alpha-mannosidase digestion, but this enzyme did not release any radioactive mannose from the larger-sized glycopeptides. These data indicate that the fucosylated glycopeptides are N-linked structures containing fucose, mannose, GlcNAc, and probably other sugars, and that the mannose units are blocked by other sugars. Thus, these results indicate that plant membrane glycoproteins contain a significant amount of modified oligosaccharide side chains. PMID- 16664120 TI - Properties of Ornithine Carbamoyltransferase from Pisum sativum L. AB - Some properties of ornithine carbamoyltransferase from chloroplasts isolated from leaves of Pisum sativum L. (cv Marzia) were compared with those of the enzyme partially purified (316-fold) from shoots of seedlings after 3 weeks of cultivation.Both preparations showed a pH optimum at pH 8.3 and had the same affinity to ornithine (K(m) = 1.2 millimolar) as well as to carbamoyl phosphate (K(m) = 0.2 millimolar). The approximate molecular weight determined by gel sieving was 77,600.A desalted ammonium sulfate precipitate from 14-day seedlings (inclusive roots and senescing cotyledons) was applied on a column of anion exchanger. The elution pattern showed one peak of ornithine carbamoyl-transferase activity. This elution pattern was the same as observed for the enzyme from chloroplasts.The results suggest the presence of one form of ornithine carbamoyl transferase in pea seedlings. PMID- 16664121 TI - Cell surfaces in plant-microorganism interactions : v. Elicitors of fungal and of plant origin trigger the synthesis of ethylene and of cell wall hydroxyproline rich glycoprotein in plants. AB - Treatment of melon hypocotyls or petioles with an elicitor from Colletotrichum lagenarium, a fungal pathogen of melons, causes an initial transitory inhibition of protein synthesis and, after 18 hours, induces the synthesis of a plant cell wall hydroxyproline-rich glycoprotein (HRGP). Microgram amounts of elicitor are sufficient for maximum elicitation of HRGP when the elicitor is injected into hypocotyls. High elicitor concentrations have a strong inhibitory effect on total protein synthesis. Ethylene is increased early in elicitor-treated plant material, and may be involved in HRGP elicitation. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, both elicitor induced ethylene and elicitor-induced HRGP are inhibited. On the other hand, 1 aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene, triggers the synthesis of HRGP to the same extent as the elicitor of C. lagenarium, and partly restores in elicitor-treated petioles the synthesis of ethylene and of HRGP after previous inhibition by aminoethoxyvinylglycine. Elicitation of HRGP occurs in other systems, such as soybeans when inoculated with an elicitor from Phytophtora megasperma f. sp. glycinea, and when melons are incubated with an elicitor isolated from their cell walls. PMID- 16664122 TI - Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development. AB - Changes in the amounts of thylakoid galactolipids and proteins were monitored for 96 hours following iron resupply to iron-deficient sugar beet (Beta vulgaris L. cv F58-554H1) plants. During this period of iron nutrition-mediated chloroplast development, the amount of galactolipid per leaf area increased linearly with time. Assuming galactolipids are an index for the amount of thylakoids, then there was a linear synthesis of thylakoid membranes during regreening. Total thylakoid protein synthesis, however, lagged behind galactolipid synthesis, suggesting that proteins are inserted secondarily into the galactolipid matrix of the thylakoid membrane during development.Iron deficiency caused an increase in the free chlorophyll band under the conditions of gel electrophoresis used. Of the chlorophyll proteins resolved, the chlorophyll protein associated with photosystem I was most diminished in iron-deficient tissue, and appeared to recover most rapidly. Changes in the light-harvesting chlorophyll proteins are also discussed.The number of polypeptides resolved by lithium dodecyl sulfate polyacrylamide gel electrophoresis was higher in iron-deficient thylakoids. During regreening, the number of resolved polypeptides decreased. PMID- 16664123 TI - Effect of translocation-hindering procedures on source leaf photosynthesis in cucumber. AB - THREE TREATMENTS WHICH ALTERED TRANSLOCATION RATE WERE APPLIED TO CUCUMBER PLANTS: Girdling of source leaf petiole; removal of all aerial sinks; removal of all source leaves except one. Two different effects were observed, one short-term (during the initial 6 hours), and one long-term (detected after several days).The short-term effect was observed exclusively in girdled leaves and involved a reduction in (14)CO(2) fixation rate paralleled by an increase in stomatal resistance. The effects were maximal after 3 hours with subsequent recovery. Stomatal closure apparently resulted from the 5 to 10% water deficit temporarily detected in girdled leaves which probably induced the observed temporary increases in abscisic acid content. Kinetin counteracted the effects of girdling.The long-term effect was detected 3 days after girdling and 3 to 5 days after sink manipulation. An increase or decrease in (14)CO(2) fixation rate was observed when the sink-source ratio was increased or decreased respectively, accompanied by a respective decrease or increase in starch content. Changes in the relative amount of (14)CO(2) incorporated into various photosynthetic products were also observed. Stomatal closure was not involved, and the decrease in CO(2) fixation was not counteracted by kinetin. PMID- 16664124 TI - p-Fluorophenylalanine-Induced Restriction of Ion Uptake and Assimilation by Maize Roots. AB - Roots of decapitated maize seedlings (Zea mays L.) were exposed for 12 hours to 1.0 millimolar KNO(3) (98.5 atom per cent (15)N) in the presence and absence (control) of 0.1 millimolar p-fluorophenylalanine (FPA), an analog of the amino acid phenylalanine. FPA decreased nitrate uptake but had little effect on potassium uptake. In contrast, accumulation of both ions in the xylem exudate was greatly restricted. The proportion of reduced (15)N-nitrogen that was translocated at each time was also restricted by FPA. These observations are interpreted as indicating that synthesis of functional protein(s) is required for nitrate uptake and for transport of potassium, nitrate, and reduced-(15)N from xylem parenchyma cells into xylem elements. The effect of FPA on nitrate reduction is less clear. Initially, FPA limited nitrate reduction more than nitrate uptake, but by 8 hours the cumulative reduction of entering nitrate was similar ( approximately 35%) in both control and FPA-treated roots. A relationship between nitrate uptake and nitrate reduction is implied. It is suggested that nitrate influx regulates the proportion of nitrate reductase in the active state, and thereby regulates concurrent nitrate reduction in decapitated maize seedlings. PMID- 16664125 TI - Analysis of the Effect of Light and Temperature on the Fluence Response Curves for Germination of Rumex obtusifolius. AB - Both red light (10 minutes) and 35 degrees C treatment (60 minutes) stimulate the germination of seeds of Rumex obtusifolius otherwise maintained in darkness at 25 degrees C. Fluence response curves were determined for the effect of red light to stimulate germination of seeds with and without 35 degrees C treatment. The endogenous far-red absorbing form (Pfr) level in the seeds was determined using short saturating fluences of wavelengths of light which maintain different proportions of phytochrome as Pfr at equilibrium. In the seed batches investigated, the endogenous Pfr level was found to be 4% or less of the total phytochrome. High dark germination after 35 degrees C treatment does not result from an increase in sensitivity of the whole population to Pfr. Calculated fluence response curves for germination which best fit the experimental data suggest that seeds germinate in darkness after 35 degrees C treatment because of a nonphytochrome-related process (overriding factor). PMID- 16664126 TI - Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae. AB - As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [(14)C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [(14)C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[(14)C]CABP complex with a combination of polyethylene glycol-4000 and MgCl(2). Plots of the relationship between concentrations of [(14)C] CPBP in the reaction mixture and the precipitated [(14)C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [(14)C]CPBP since the dissociation constant of CABP with Rubisco is 10(-11) molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C(4) plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one sixth that of C(3) plant leaves and was affected by the CO(2) concentration during growth. The content of Euglena and blue-green algae is also reported. PMID- 16664127 TI - Phosphatidylglycerol and chilling sensitivity in plants. AB - The hypothesis that molecular species of thylakoid phosphatidylglycerol containing two saturated fatty acids (disaturated phosphatidylglycerol) confer chilling sensitivity upon plants was tested by analyzing the fatty acid composition of phosphatidylglycerols isolated from leaves of a range of plants expected to have different sensitivities to chilling temperatures.;Saturated' fatty acids (palmitate plus stearate plus hexadeca-trans-3-enoate) as a proportion of total phosphatidylglycerol fatty acids varied from 51 to 80 mole per cent in the plants analyzed but appeared to be rigidly fixed for a given plant species, being unaffected by leaf maturity or by environment.Hexadeca-trans 3-enoate occurred only at the sn-2 position, whereas C-18 fatty acids occurred only at the sn-1 position of thylakoid phosphatidylglycerol. Therefore, the proportion of disaturated molecular species could be predicted accurately from the total fatty acids of phosphatidylglycerol.Disaturated molecular species accounted for <25% of the total phosphatidylglycerol from leaves of chilling resistant plants and for 50 to 60% of the phosphatidylglycerol in leaves from some of the most chilling-sensitive plants. However, not all chilling-sensitive plants contained high proportions of disaturated phosphatidylglycerol; solanaceous and other 16:3-plants and C(4) grasses may be important exceptions. Nonetheless, proportions of disaturated phosphatidylglycerol increased concomitantly with increasing chilling sensitivity of plants within a genus. PMID- 16664128 TI - Biosynthesis and Intracellular Transport of 11S Globulin in Developing Pumpkin Cotyledons. AB - In vitro studies to explore the biosynthesis of 11S globulin developing cotyledons of pumpkin (Cucurbita sp.) demonstrated that 11S globulin is synthesized on membrane-bound polysomes. M(r) of the translation products (preproglobulin) synthesized by the poly(A)(+)-RNA isolated from developing cotyledons were determined to be 64,000 and 59,000, which are larger than those of the mature globulin subunit (62,000 and 57,000). Preproglobulin is then cotranslationally processed by cleavage of the signal peptide to produce proglobulin. In vivo pulse-chase experiments showed the sequential transformation of the single-chain proglobulin to mature globulin subunit (disulfide-linked doublet polypeptides) indicating posttranslational modification of the proglobulin.Subcellular fractionation of the pulse-chased intact cotyledons showed that the [(35)S]methionine label is detectable in proglobulin in rough endoplasmic reticulum shortly after the pulse label. With time, the labeled proteins move into other cellular fractions: proglobulin in the density = 1.24 grams per cubic centimeter fractions after 30 minutes and mature globulin subunit associated with protein bodies after 1 to 2 hours. The distribution of proglobulin in sucrose density gradients did not correspond with those of catalase (microbody marker) or fumarase (mitochondria marker). An accumulation of proglobulin occurred in the density = 1.24 grams per cubic centimeter fractions, whereas the mature globulin was scarcely detectable in this fraction. In contrast, proglobulin was not detected by immunochemical blotting analysis in the protein bodies prepared under the mild conditions from cotyledon protoplasts. The results suggest that the d = 1.24 grams per cubic centimeter fractions are engaged in the translocation of proglobulin into the protein bodies. PMID- 16664129 TI - Differential control of ethylene-induced gene expression and respiration in carrot roots. AB - Ethylene treatment of carrot roots elicits a developmental program encompassing an increase in respiration rate and changes in gene expression. Both phenomena are potentiated when ethylene is administered in O(2). Our previous studies showed that both respiration and a number of ethylene specific mRNAs increase together in response to ethylene through some 21 hours, whereas thereafter respiration continues to rise, while the level of induced mRNAs drops. Herein we ask whether an experimentally effected drop in the respiration rate within the first 21 hours caused by the withdrawal of ethylene, or substitution of air for O(2) in the continued presence of ethylene, is linked to a drop in the level of ethylene-induced mRNA. Quantitative estimation of two ethylene evoked mRNAs by dot blot hybridization with appropriate cDNA clones has shown that under the specified treatment the induced mRNA levels remain constant while the respiration rate drops, suggesting that gene expression, as reflected in induced mRNA levels, and respiration rate are separately regulated facets of the ethylene response. PMID- 16664130 TI - Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus). AB - The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c(1)-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCl fractionation procedure, contained one Cyt of c type (formally c(1) with Em(7.0) of +240 millivolts), two b type Cyt with Em(7.0) values of +100 and -25 millivolts, ferredoxin-like centers presumably linked to succinic- and NADH-dehydrogenases, and a Rieske-type iron sulfur center (g(y) = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-1 showed sensitivity to antimycin A, myxothiazol, and n-2 hepthyl-1-hydroxyquinoline N-oxide with I(50) of 12 nanomolar, 30 nanomolar, and 0.1 micromolar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined. PMID- 16664131 TI - Isoelectric focusing of plant cell protoplasts: separation of different protoplast types. AB - The surface charge of plant protoplasts has been measured by a new technique, isoelectric focusing. The protoplasts were loaded in a dextran density gradient over which a pH gradient was superimposed. When voltage was applied, protoplasts moved to a point in the gradient corresponding to their isoelectric point (pI). The pI of the protoplasts varied with the compounds used for pH gradient generation. Using commercial ampholytes for pH gradient formation, the pI of all protoplasts tested was 4.4 +/- 0.2, and viability following electrophoresis was low. Using an acetate/acetic acid mixture to generate the pH gradient, the pI of protoplasts varied from 3.7 to 5.3 depending on the species and tissue type of the parental cells. Postelectrophoresis viability was high. Using isoelectric focusing techniques, it was possible to separate mixtures of protoplasts derived from different species of plants. PMID- 16664132 TI - Uptake of Amino Acids and Other Organic Compounds by Lemna paucicostata Hegelm. 6746. AB - A survey of the capacity of Lemna paucicostata to take up organic compounds such as might be present in the natural environment of this plant has identified eight discrete transport systems. Reciprocal inhibition studies defined the preferred substrates for these systems as follows: (a) neutral l-alpha-amino acids, (b) basic amino acids, (c) purine bases, (d) choline, (e) ethanolamine, (f) tyramine, (g) urea, and (h) aldohexoses. Each of these systems takes up its preferred substrates at high rates. At low concentrations, each Lemna frond during each minute takes up amounts which would be found in volumes ranging from 0.4 (tyramine) to 3.9 (urea) times its own volume. The two systems for amino acid transport both showed kinetics of the biphasic type, so that uptake by each can be described as the composite result of two Michaelis-Menten processes. The neutral amino acid system neither transports basic amino acids nor is inhibited by these compounds. The basic amino acid system does not transport neutral amino acids but is strongly inhibited by some, but not all, of these compounds. It is argued that the maintenance of these active, specific, and discrete systems in Lemna suggests they play important roles permitting this plant to utilize organic compounds occurring naturally in its environment. PMID- 16664133 TI - Ureide metabolism in leaves of nitrogen-fixing soybean plants. AB - In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [(14)C]urea-dependent NH(3) and (14)CO(2) production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH(3) per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH(3) and CO(2) production from the utilization of [2-(14)C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH(3) production. The almost complete sensitivity of NH(3) and CO(2) production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH(3) and two molecules of CO(2). PMID- 16664134 TI - Natural Occurrence of Indole-3-acetylaspartate and Indole-3-acetylglutamate in Cucumber Shoot Tissue. AB - Indole-3-acetylaspartate and indole-3-acetylglutamate were isolated from 1-week old, green cucumber shoots that had not been pretreated with auxin. Using isotope dilution techniques, we found 129 micrograms (0.42 micromoles) of indoleacetylglutamate and 33 micrograms (0.11 micromoles) of indoleacetylaspartate per kilogram of fresh tissue. PMID- 16664135 TI - Inhibition of glycine oxidation by carboxymethoxylamine, methoxylamine, and acethydrazide. AB - Carboxymethoxylamine (amino-oxyacetate), methoxylamine, and acethydrazide are shown to be effective, although not completely specific, inhibitors of glycine oxidation by the isolated glycine decarboxylase multienzyme complex, mitochondria, protoplasts, and leaf discs from peas. The inhibition probably results from a reaction between these compounds and the pyridoxal 5-phosphate cofactor of the enzyme. PMID- 16664136 TI - Intercellular nodule localization and nodule specificity of xanthine dehydrogenase in soybean. AB - The distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellular localization of xanthine dehydrogenase within soybean nodules was determined. Polyclonal antibodies against purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slightly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test.Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by allopurinol and was observed only in the infected nodule cells of these species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase. PMID- 16664137 TI - Isolation of mitochondria from soybean leaves on discontinuous percoll gradients. AB - A technique to isolate mitochondria from chamber-grown soybeans (Glycine max cv Williams) was developed. The mitochondria were isolated by centrifugation on discontinuous Percoll gradients which yielded a sharp band of mitochondria contaminated by only 4% of the total chlorophyll in the gradient. Contamination by peroxisomes was also slight. The isolated mitochondria oxidized malate plus glutamate, NADH, and malate with respiratory control. They also showed cyanide insensitive, alternative pathway activity which was inhibited by salicylhydroxamic acid. PMID- 16664138 TI - Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts: Characteristics and Inhibitor Specificities. AB - The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30 degrees C), protoplasts accumulated phosphate at the rate of 3.9 +/- 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be -37 +/- 3 millivolts. Initiation of photosynthesis lowered the membrane potential to -39 +/ 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of -33 +/- 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated. PMID- 16664139 TI - Characterization of glutamine synthetase isoforms from chlorella. AB - Ion-exchange chromatography of extracts derived from Chlorella sorokiniana mutant strain (oxygen resistant) yielded two separate activity peaks of glutamine synthetase (GS). GS(I) and GS(II) were purified 220- and 187-fold and have molecular weights of approximately 398,000 and 360,000, respectively. Both enzymes are composed of eight identical subunits with a subunit molecular weight of 47,000 for GS(I) and 43,000 for GS(II). The amino acid composition, catalytic, and immunological properties for both enzymes are similar. PMID- 16664140 TI - Nuclear, Virescent Mutants of Zea mays L. with High Levels of Chlorophyll (a/b) Light-Harvesting Complex during Thylakoid Assembly. AB - We have found nuclear, recessive mutants in Zea mays L. where assembly of the major chlorophyll (a/b) light-harvesting complex (LHC) was not delayed relative to most other thylakoid protein complexes during thylakoid biogenesis. This contrasts with the normal development of maize chloroplasts (NR Baker, R Leech 1977 Plant Physiol 60: 640-644). All four mutants examined were allelic and virescent, and displayed visibly higher yields of leaf Chl fluorescence during greening. Fully greened mutants had normal leaf Chl fluorescence yield and normal levels of LHC, and grew to maturity under field conditions. Therefore, delayed LHC assembly is not an obligate feature of thylakoid differentiation.Assigning the molecular basis for the mutation should provide information concerning reguation of LHC assembly. Several possibilities are discussed. The pleiotropic mutant phenotype is not attributable to defects in thylakoid glycerolipid synthesis. Thylakoids isolated from greening mutant leaf sections had elevated glycerolipid/Chl ratios. In addition, both the molar distribution and acyl composition of four major glycerolipids were normal for developing mutant thylakoids. PMID- 16664141 TI - Metabolism of Monoterpenes : Evidence for the Function of Monoterpene Catabolism in Peppermint (Mentha piperita) Rhizomes. AB - l-Menthone of peppermint leaves is reduced to d-neomenthol which is glucosylated and transported to the rhizome, whereupon the beta-d-glucoside is hydrolyzed, the aglycone oxidized back to l-menthone, and this ketone converted to l-3,4-menthone lactone. l-[G-(3)H]-3,4-Menthone lactone and its labeled progenitors, when incubated with excised mint rhizomes, gave rise to nonvolatile lipids as well as polar metabolites. The lipids thus generated consisted of labeled squalene and phytosterols in the nonsaponifiable fraction and C(14)-C(26) fatty acids in the saponifiable fraction. These results imply degradation of the terpenoid to acetylcoenzyme A and reduced pyridine nucleotide, and reincorporation of label via these products. Starch and soluble carbohydrates were also found to be labeled; however, chemical degradation of the [(3)H]glucose obtained on hydrolysis of starch indicated the presence of tritium only on interior carbons, suggesting that labeling had occurred via reduced pyridine nucleotides. Analysis of the labeled organic acids revealed the presence of several hydroxy methylacyl intermediates suggesting the operation of a modified beta-oxidation pathway in the degradation of the acyclic terpenoid skeleton. The results indicate that monoterpenes transported to the rhizome are oxidized to yield acetyl-coenzyme A and reduced pyridine nucleotides, and suggest that metabolic turnover of monoterpenes in mint represents a mechanism for recycling carbon and energy from foliar terpenes into other metabolites of the rhizome. PMID- 16664142 TI - Changing Kinetic Properties of Fructose-1,6-bisphosphatase from Pea Chloroplasts during Photosynthetic Induction. AB - After dark-light transitions, there is a delay in photosynthetic CO(2) fixation by isolated pea chloroplasts in the range of some minutes. In order to assess the physiological significance of light modulation of enzyme activity in the control of induction, we made estimates of the kinetic parameters of fructose-1,6 bisphosphatase immediately upon release from pea chloroplasts in the dark and after illumination for various time periods. The Michaelis constant for fructose 1,6-bisphosphate decreased and maximal velocities increased during induction. It seems likely that light activation of this enzyme is one of the factors contributing to the overcoming of the lag period in photosynthetic CO(2) fixation. PMID- 16664143 TI - Influence of photosynthesis and chlorophyll synthesis on polypeptide accumulation in greening euglena. AB - Two-dimensional gel electrophoresis resolves total cellular protein from Euglena gracilis klebs var bacillaris Cori into 640 polypeptides, 79 of which are induced by light exposure. The inhibition of chloroplast translation by streptomycin, the direct inhibition of photosynthesis as well as the indirect inhibition of chlorophyll synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the specific inhibition of photosynthesis but not chlorophyll synthesis by DCMU in the presence of 17 millimolar ethanol failed to inhibit the accumulation of 40 polypeptides. These polypeptides appear to be synthesized on cytoplasmic ribosomes and their accumulation is independent of the developmental status of the chloroplast. Streptomycin but not DCMU completely inhibited the accumulation of six polypeptides which are undetectable in mutants lacking chloroplast DNA suggesting that these polypeptides are translated on chloroplast ribosomes. The accumulation of seven polypeptides which are detectable in mutants lacking chloroplast DNA was also inhibited by streptomycin but not by DCMU suggesting that the accumulation of these polypeptides is dependent upon stabilization by a chloroplast translation product. The accumulation of 12 polypeptides was inhibited by streptomycin and by DCMU under conditions in which chlorophyll synthesis was inhibited, but not under conditions in which chlorophyll synthesis was unaffected by DCMU. The inhibition by DCMU of the accumulation of these polypeptides appears to be due to the inhibition of chlorophyll synthesis suggesting that they are components of pigment protein complexes. The accumulation of six polypeptides was inhibited under all conditions in which photosynthesis was inhibited suggesting that the accumulation of these polypeptides is dependent upon a product of photosynthesis. PMID- 16664144 TI - Dual action of respiratory inhibitors: inhibition of germination and prevention of dormancy induction in lettuce seeds. AB - ;Grand Rapids' lettuce Lactuca sativa L. seeds germinate readily at 15 degrees C but poorly at 25 degrees C in darkness. When held in dark at 25 degrees C for an extended period, the ungerminated seeds become dormant as shown by their inability to germinate or transfer to 15 degrees C in darkness. Induction of dormancy at 25 degrees C was prevented by exposure to CN(-), azide, salicylhydroxamic acid (SHAM), dinitrophenol, and pure N(2) as determined by subsequent germination at 15 degrees C on removal of inhibitors. The effectiveness of inhibitors to break dormancy declined as dormancy intensified. At relatively low levels, CN(-), SHAM, and azide promoted dark germination at 25 degrees C while at high levels they were inhibitory. Uptake of O(2) by seeds held at 25 degrees C for 4 days in 1.0 millimolar KCN was inhibited by 67% but was promoted 61% when KCN was removed. Correspondingly greater inhibition (79%) and promotion (148%) occurred when 1.0 millimolar SHAM was added to KCN solution. When applied alone, SHAM had little effect on O(2) uptake. These data indicate that Cyt pathway of respiration plays a dominant role in the control of both dormancy induction and germination of lettuce seeds, and ;alternative pathway' is effectively engaged in presence of CN(-). The channeling of respiratory energy use for processes governing germination or dormancy is subject to control by physical and chemical factors.A scheme is proposed that illustrates compensatory use of energy for processes controlling dormancy induction and germination. A block of germination, e.g. by low water potential polyethylene glycol solution or a supraoptimal temperature spares energy to be utilized for dormancy induction while a block of dormancy induction by low levels of CN(-) (similar to GA and light effects) drives germination. Blocking both processes by inhibitors (e.g. CN(-), CN(-) + SHAM) presumably leads to accumulation of ;reducing power' with consequent improvement in O(2) uptake and oxidation rates of processes controlling germination or dormancy induction upon removal of the inhibitors. PMID- 16664145 TI - Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet. AB - The light-dependent development of the photosynthetic apparatus in the first leaf of the C(4) plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5 bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times. PMID- 16664146 TI - Nodule-specific polypeptides from effective alfalfa root nodules and from ineffective nodules lacking nitrogenase. AB - In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development. PMID- 16664147 TI - Biosynthesis of mannose-containing lipid-linked oligosaccharides by solubilized enzyme preparation from cultured soybean cells. AB - Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at -10 degrees C for 1 month. The solubilized enzyme synthesized [(14)C]Man(3)GlcNAc(2)-pyrophosphoryl-polyprenol and [(14)C]Man(5)GlcNAc(2) pyrophosphoryl-polyprenol when incubated with GDP-[(14)C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man(3)GlcNAc(2)-lipid and Man(5)GlcNAc(2)-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man(6)GlcNAc(2)-lipid, Man(7)GlcNAc(2)-lipid, and Man(8)GlcNAc(2) lipid occurred when the above incubations were supplemented with dolichyl phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man(5)GlcNAc(2)-lipid come directly from GDP mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose. PMID- 16664148 TI - An Analysis of Growth Regulator Interactions and Gene Expression during Auxin Induced Cell Elongation Using Cloned Complementary DNAs to Auxin-Responsive Messenger RNAs. AB - We have examined the effects of cytokinin, fusicoccin, and ethylene on auxin induced changes in gene expression during auxin-promoted cell elongation in soybean (Glycine max L. Merr. cv Wayne) using cloned cDNAs to two auxin responsive mRNAs (Walker, Key 1982 Proc Natl Acad Sci USA 79: 7185-7989). RNA blot analyses demonstrate that under conditions of cytokinin inhibition of auxin promoted cell elongation the levels of these two auxin-responsive mRNAs is unaltered. Fusicoccin-promoted elongation is not associated with an enhanced expression of these two mRNAs, suggesting that the increased levels of these mRNAs observed during auxin-promoted cell elongation are not simply due to enhanced rates of cell elongation. We have also determined that ethylene plays no apparent role in the regulation of expression of these mRNAs. However, the auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and alpha-naphthalene acetic acid all enhance an accumulation of these mRNAs. We conclude that the regulation of these mRNAs is directly dependent on auxin. That auxin-promoted cell elongation is dependent upon the increased accumulation of these mRNAs remains to be determined. PMID- 16664149 TI - Photorespiration-induced reduction of ribulose bisphosphate carboxylase activation level. AB - Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C(3) crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O(2), 350 microliters per liter CO(2), balance N(2)), but not in nonphotorespiratory conditions (2% O(2), 350 microliters per liter CO(2), balance N(2)). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg(2+) for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg(2+) and pH but rather is associated with metabolite flux through the photorespiratory pathway. PMID- 16664150 TI - Separation of the Mg-ATPases from the Ca-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots. AB - Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm(3)). Activities of NADH cytochrome (Cyt) c reductase, Ca(2+)-ATPase, and Mg(2+)-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca(2+)-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm(3), a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm(3), a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm(3), and of vanadate-inhibited ATPase at 1.16 g/cm(3). The Ca(2+)-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate inhibited ATPase on a sucrose gradient; approximately 70% of the Ca(2+) phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg(2+), was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species. PMID- 16664151 TI - Sucrose Concentration at the Apoplastic Interface between Seed Coat and Cotyledons of Developing Soybean Seeds. AB - The apoplastic sucrose concentration at the interface between cotyledons and surrounding seed coats of developing soybeans (Glycine max L. Merr. cv Wye) was found by three indirect methods to be in the range of 150 to 200 millimolar. This is an order of magnitude higher than has been reported elsewhere for soybean. It was also higher than the overall sucrose concentrations in the cotyledons and seed coats, each of which was approximately 90 millimolar. By defoliating plants 24 hours before measurement, both the overall sucrose concentration in the cotyledons and the interfacial apoplastic sucrose concentration were reduced by three-fourths. However, there was no day/night difference in overall tissue sucrose concentration of cotyledons or seed coats from intact plants suggesting the existence of a homeostatic mechanism compensating for the diurnal photosynthetic cycle. About 7 hours were required for a tritiated polyethylene glycol-900 solution to fully permeate developing cotyledons (from approximately 220 milligram fresh weight embryos), implying high diffusion resistance through the tissue.These results indicate that a high interfacial sucrose concentration may exist in vivo. They suggest that the saturable carrier-mediated component of sucrose uptake may be of little physiological significance in the outermost cell layers of the cotyledons. PMID- 16664152 TI - Shoot Turgor Does Not Limit Shoot Growth of NaCl-Affected Wheat and Barley. AB - The aim of this work was to test the hypothesis that the reduced growth rate of wheat and barley that results when the roots are exposed to NaCl is due to inadequate turgor in the expanding cells of the leaves. The hypothesis was tested by exposing plants to 100 millimolar NaCl (which reduced their growth rates by about 20%), growing them for 7 to 10 days with their roots in pressure chambers, and applying sufficient pneumatic pressure in the chambers to offset the osmotic pressure of the NaCl, namely, 0.48 megapascals. The results showed that applying the pressure had no sustained effect (relative to unpressurized controls) on growth rates, transpiration rates, or osmotic pressures of the cell sap, in either the fully expanded or currently expanding leaf tissue, of both wheat and barley. The results indicate that the applied pressure correspondingly increased turgor in the shoot although this was not directly measured. We conclude that shoot turgor alone was not regulating the growth of these NaCl-affected plants, and, after discussing other possible influences, argue that a message arising in the roots may be regulating the growth of the shoot. PMID- 16664153 TI - Evidence for a plasmalemma redox system in sugarcane. AB - A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O(2) consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K(+), 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH. PMID- 16664154 TI - Effects of low temperature and respiratory inhibitors on calcium flux in plant mitochondria. AB - The effects of low temperature on uptake and release of (45)Ca(2+) were studied with sound, well-coupled mitochondria extracted at room temperature from avocado (Persea americana Mill, cv Fuerte) fruits. Low Ca(2+) concentrations (10 micromolar) were employed to simulate physiological conditions. At 25 degrees C, the rate of Ca(2+) uptake decreased with time, whereas at 5 degrees C the initial rate, though lower, remained linear. As a consequence total uptake at 5 degrees C was substantially greater than at 25 degrees C for periods greater than 5 min. Preincubation of mitochondria at 5 degrees C enhanced subsequent Ca(2+) uptake at 25 degrees C. Ca(2+) uptake was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and by ruthenium red, but neither KCN nor salicylhydroxamic acid separately or together had any major inhibitory effect. Preloaded mitochondria held for 60 min in a Ca-free medium lost little Ca(2+) at 25 degrees C and none at 5 degrees C, except in the presence of ruthenium red or CCCP. PMID- 16664155 TI - ADP Is a Competitive Inhibitor of ATP-Dependent H Transport in Microsomal Membranes from Zea mays L. Coleoptiles. AB - An anion-sensitive ATP-dependent H(+) transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H(+) transport observed: strict dependence on Cl(-); inhibition by SO(4) (2-) and NO(3) (-); insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl(2) (H(+)/Ca(2+) antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl(2) with a K(m) value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H(+) transport significantly. However, ADP was a potent competitive inhibitor with a K(i) value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure. PMID- 16664156 TI - Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons. AB - We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) alpha-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with alpha galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that alpha-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process. PMID- 16664157 TI - Ethylene Promotes the Capability To Malonylate 1-Aminocyclopropane-1-carboxylic Acid and d-Amino Acids in Preclimacteric Tomato Fruits. AB - When whole unripe green tomato fruits (Lycopersicon esculentum Mill, cv T(3)) were treated with ethylene (10 microliters per liter) for 18 hours, the fruit's ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to N-malonyl-ACC (MACC) increased markedly and such an effect was also observed in fruits of mutant nor, which cannot ripen normally. The promotion of the capability to malonylate ACC by ethylene increased with the increasing ethylene concentration from 0.1 to 100 microliters per liter and with increasing duration of ethylene treatment up to 8 hours; a longer duration of ethylene treatment did not further increase the malonylation capability. When ethylene was withdrawn, the promotion disappeared within 72 hours. Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene. Ethylene treatment also promoted the fruits' capability to conjugate d-amino acids and alpha-amino-isobutyric acid. Since the increase in the tissue's capability to malonylate ACC was accompanied by an increase in the extractable activity of ACC and d-amino acid malonyltransferase, ethylene is thought to promote the development of ACC/d-amino acid malonyltransferase in unripe tomato fruits. PMID- 16664158 TI - Naringin levels in citrus tissues : I. Comparison of different antibodies and tracers for the radioimmunossay of naringin. AB - The preparation of a tritiated radiotracer that was used in the radioimmunoassay of naringin (naringenin-7-O-alpha-rhamnosyl- (1-2)-beta-d-glucopyranoside) and which was synthesized by reduction of the carbonyl group of the flavanone is reported. The resulting assay has a detection limit of 0.5 picomole per 0.1 milliliter, is specific for the 7-neohesperidoside substitution on flavanones, and can measure naringin in crude extracts of plant tissues. This radioimmunoassay is compared with three other naringin immunoassays which use antibodies raised against two different haptens and different tracers labeled with (125)I or (3)H. The applicability of the methods to the quantification of naringin and other flavanone neohesperidosides in citrus tissue is discussed. PMID- 16664159 TI - Naringin Levels in Citrus Tissues : II. Quantitative Distribution of Naringin in Citrus paradisi MacFad. AB - The quantitative distribution of the flavanone-7-neohesperidoside, naringin, in seeds, seedlings, young plants, branches, flowers, and fruit of Citrus paradisi Macfad., cv ;Duncan' was analyzed by radioimmunoassay. High levels of naringin were associated with very young tissue and lower levels were found in older tissues. Seed coats of ungerminated seeds and young shoots had high naringin concentrations whereas cotyledons and roots had very low concentrations. Light grown seedlings contained nearly twice as much naringin as etiolated seedlings and, in young plants and branches, the naringin content was highest in developing leaves and stem tissue. In flowers, the ovary had the highest levels of naringin, accounting for nearly 11% of the fresh weight. There was a net increase in the total naringin content of fruits during growth. However, due to the large increase in fruit size, there was a concomitant decrease in the naringin concentration as the fruit matured. PMID- 16664160 TI - Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine. AB - The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K(+)/H(+) antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K(+)/H(+) exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K(+)/H(+) antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane. PMID- 16664161 TI - Mode of Methomyl and Bipolaris maydis (race T) Toxin in Uncoupling Texas Male Sterile Cytoplasm Corn Mitochondria. AB - Bipolaris maydis race T toxin (BmT), and its functional analog, methomyl, uncoupled Texas male-sterile (T) cytoplasm mitochondria by decreasing the resistance of the inner membrane to protons. However, unlike protonophoric or ionophoric agents, BmT toxin and methomyl induced irreversible swelling. Packed volume measurements showed that mitochondrial volume was irreversibly increased by methomyl and BmT toxin indicating that mitochondria no longer functioned as differentially permeable osmometers. The decreased resistance of inner mitochondrial membranes to protons and the loss of osmotic volume regulation suggests that methomyl and BmT toxin induced the formation of hydrophilic pores in T mitochondrial inner membranes. PMID- 16664162 TI - Effect of External pH on the Internal pH of Chlorella saccharophila. AB - The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2 [(14)C]oxazolidine-2,4-dione ([(14)C]DMO) or [(14)C]benzoic acid ([(14)C]BA) between the cells and the surrounding medium. [(14)C]DMO was used at external pH of 5.0 to 7.5 while [(14)C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH. PMID- 16664163 TI - Effects of Ethephon, 1-Aminocyclopropane-1-carboxylic Acid, and Inhibitors of Ethylene Synthesis on the Gravitropically Induced Movement of Mimosa pudica Pulvinus. AB - Primary pulvini of Mimosa pudica L. displaced from their position display gravitropic movements beginning about 15 minutes after their reorientation. Ethephon, an ethylene-releasing compound, and 1-aminocyclopropane-1-carboxylic acid, an intermediate in ethylene biosynthesis, enhance these movements at a concentration as low as 10 nanomolar. Inhibitors of ethylene synthesis (l-alpha (2-aminoethoxyvinyl)glycine, (aminooxy)acetic acid, and Co(2+)) reduce the amplitude of the movements. The promotive action of 1-aminocyclopropane-1 carboxylic acid is abolished by l-alpha-(2-aminoethoxyvinyl)glycine. These results permit one to conclude that ethylene may modify the curvature movement but not the initiation of the gravitropic reaction. With reference to the pulvinus functioning based on turgor variations and ion migrations inside the organ, namely K(+) acting as the osmoticum, the data suggest that ethylene may act by increasing the membrane permeability to water and/or by altering an ion pump. PMID- 16664164 TI - Determination of Endogenous Indole-3-Acetic Acid in Plagiochila arctica (Hepaticae). AB - Endogenous indole-3-acetic acid (IAA) was found in axenically cultured gametophytes of the leafy liverwort, Plagiochila arctica Bryhn and Kaal., by high performance liquid chromatography with electrochemical detection. Identification of the methylated auxin was confirmed by gas chromatography-mass spectrometry. Addition of 57 micromolar IAA to cultures increased relative production of ethylene. This is the first definitive (gas chromatography-mass spectrometry) demonstration of the natural occurrence of IAA in a bryophyte. PMID- 16664165 TI - Controls on na influx in corn roots. AB - We have investigated the effects of hyperpolarization and depolarization, and the presence of K(+) and/or Ca(2+), on (22)Na(+) influx into corn (Zea mays L.) root segments. In freshly excised root tissue which is injured, Na(+) influx is unaffected by hyperpolarization with fusicoccin, or depolarization with uncoupler (protonophore), or by addition of K(+). However, added Ca(2+) suppresses Na(+) influx by 60%. In washed tissue which has recovered, Na(+) influx is doubled over that of freshly excised tissue, and the influx is increased by fusicoccin and suppressed by uncoupler. This energy-linked component of Na(+) influx is completely eliminated by low concentrations of K(+), leaving the same level and kind of Na(+) influx seen in freshly excised roots. The K(+)-sensitive energy linkage appears to be by the carrier for active K(+) influx. Calcium is equally inhibitory to Na(+) influx in washed as in fresh tissue. Other divalent cations are only slightly less effective. Net Na(+) uptake was about 25% of (22)Na(+) influx, but proportionately the response to K(+) and Ca(2+) was about the same.The constancy of K(+)-insensitive Na(+) influx under conditions known to hyperpolarize and depolarize suggests that if Na(+) transport is by means of a voltage-sensitive channel, the rise or fall of channel resistance must be proportional to the rise or fall in potential difference. The alternative is a passive electroneutral exchange of (22)Na(+) for endogenous Na(+). The data suggest that an inwardly directed Na(+) current is largely offset by an efflux current, giving both a small net uptake and isotopic exchange. PMID- 16664166 TI - Metabolic Basis for Injury to Plants from Combinations of O(3) and SO(2): Studies with Modifiers of Pollutant Toxicity. AB - Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum Mill. flacca (mutant tomato) were chosen to evaluate the metabolic basis for plant injury from combinations of O(3) + SO(2). The plants were exposed under conditions reported to specifically alter O(3) or SO(2) toxicity; light versus dark exposures, and treatment with the fungal metabolite fusicoccin (FC), the O(3) injury inhibitor N [2-(2-oxo-1-imidazolidiny) ethyl]-N'-phenylurea (EDU), and the SO(2) injury stimulator diethyldithiocarbamate (DDTC). Plants were grown in controlled environment chambers and exposed to combinations of O(3) (0.05-0.2 microliters per liter) and SO(2) (0.1-0.3 microliters per liter) for 2 hours. Peas treated with FC had the same or greater injury (quantified by visual rating) with O(3) + SO(2) exposures compared to plants not treated with FC. For plants with open stomata in the dark as well as light, i.e. FC-treated peas and tomatoes, there was no change or an increase in foliar necrosis with O(3) + SO(2) exposures in the dark versus light. Peas treated with EDU had an almost complete absence of O(3) injury, no change in SO(2) injury, and moderate decreases in injury from combinations of O(3) + SO(2) compared to plants not treated with EDU. Tomatoes treated with DDTC showed the same or less injury compared to plants not treated with DDTC and exposed to O(3) or SO(2). The plant responses to the experimental treatments and O(3) + SO(2) resembled O(3) responses more than SO(2) responses. The evidence for O(3)-like responses are: no change or increase in injury in the light versus dark, and EDU-induced decreases in injury. Evidences for SO(2)-like responses are: incomplete protection from injury with EDU, and no change or increased injury to FC-treated versus untreated plants. Thus, a metabolic mechanism affected by both pollutants may be associated with the combination injury, e.g. effects the plasma membrane. PMID- 16664167 TI - Seed Germination in Chenopodium album L: Relationships between Nitrate and the Effects of Plant Hormones. AB - Effects of ethylene, gibberellins, and kinetin on the germination of two lots of Chenopodium album L. seeds, collected from the field in 1982 and 1983, were studied in relation to the availability of nitrate. The experiments were conducted in darkness and at temperatures ranging from 12 to 32 degrees C. Ethylene induced over 75% germination in the 1983 seed but had little effect on the 1982 seed. Nitrate was only slightly promotive in either of the two seed lots. A combination of ethylene and nitrate, however, acted synergistically on 1982 seed, resulting in as much germination as that induced in 1983 seed by ethylene alone. In 1983 seed, a combination of ethylene and nitrate was only marginally more effective than ethylene. A similar relationship was observed in the effects of gibberellic acid(4+7) (GA(4+7)) and nitrate on seeds from the two lots. The 1982 seed, which responded synergistically to combinations of nitrate with ethylene or GA(4+7) was found to contain an extremely low endogenous level of nitrate as compared to 1983 seed. Thus, high levels of either endogenous or applied nitrate appeared to enhance the germination response to ethylene or GA(4+7).Kinetin had no effect on 1982 seed and only a small promotive effect on 1983 seed. There was no synergism between kinetin and nitrate in either of the seed lots. PMID- 16664168 TI - Water Stress Reduces Ozone Injury via a Stomatal Mechanism. AB - Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at -35 or -80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes. PMID- 16664169 TI - Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b: Studies on Pigment Protein Complexes and Thylakoid Protein Phosphorylation. AB - Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A 2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [gamma-(32)P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants. PMID- 16664170 TI - Identification and Quantification of Indole-3-methanol in Etiolated Seedlings of Scots Pine (Pinus sylvestris L.). AB - Combined gas chromatography-mass spectrometry has been used to identify indole-3 methanol in a purified buffer extract from etiolated seedlings of Pinus sylvestris L. Quantitative estimates obtained by high performance liquid chromatography with fluorescence detection, corrected for losses occurring during purification, indicated that etiolated seedlings of P. sylvestris contained 19.7 +/- 1.4 nanograms (+/- standard deviation) indole-3-methanol per gram fresh weight. The stability of indole-3-methanol at different pH levels was investigated. The rate of conversion, to a less polar unidentified substance, was enhanced with increasing acidity. PMID- 16664171 TI - Chloroplast Function in Guard Cells of Vicia faba L. : Measurement of the Electrochromic Absorbance Change at 518 nm. AB - Stomatal conductance is coupled to leaf photosynthetic rate over a broad range of environmental conditions. We have investigated the extent to which chloroplasts in guard cells may contribute to this coupling through their photosynthetic activity. Guard cells were isolated by sonication of abaxial epidermal peels of Vicia faba. The electrochromic band shift of isolated guard cells was probed in vivo as a means of studying the electric field that is generated across the thylakoid membranes by photosynthetic electron transport and dissipated by photophosphorylation. Both guard cells and mesophyll cells exhibited fast and slow components in the formation of the flash-induced electrochromic change. The spectrum of electrochromic absorbance changes in guard cells was the same as in the leaf mesophyll and was typical of that observed in isolated chloroplasts. This observation indicates that electron transport and photophosphorylation occur in guard cell chloroplasts. Neither the fast nor the slow component of the absorbance change was observed in the presence of the uncoupler carbonylcyanide p trifluoromethoxy-phenylhydrazone which confirms that the absorbance change was caused by the electric field across the thylakoid membranes. The magnitude of the fast rise was reduced by half in the presence of 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Therefore, photosystem II is functional and roughly equal in concentration to photosystem I in guard cell chloroplasts. The slow rise was abolished by 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone indicating the involvement of the cytochrome b(6)/f complex in electron transport between the two photosystems. Relaxation of the absorbance change was irreversibly retarded in cells treated with the energy transfer inhibitor, N,N' dicyclohexylcarbodiimide. The slowing of the rapid decay kinetics by N,N' dicyclohexylcarbodiimide confirms that the electrical potential across the thyalkoid membrane is dissipated by photophosphorylation. These results show that guard cell chloroplasts conduct photosynthetic electron transport in a manner similar to that in mesophyll cells and provide the first evidence that photophosphorylation occurs in guard cells in vivo. PMID- 16664172 TI - Translocation of photosynthates into vacuoles in spinach leaf protoplasts. AB - A method was developed for the isolation of vacuoles from the mesophyll protoplasts of spinach leaf, employing the discontinuous Ficoll density gradient centrifugation technique. Isolated vacuole preparations were judged to be free from other organellar fractions based on the assays of marker enzyme activities of individual organelles.Using this isolation method, a time-dependent translocation of (14)C-labeled photosynthates into vacuoles was determined. In contrast to a significant transport of (14)C organic acids such as malate and citrate within 10 to 15 minutes (14)C neutral sugars and amino acids were barely transported into vacuoles during 40 minutes incubation, in spite of the fact that a relatively large amount of these compounds are found in the vacuoles. It was also found that a majority of [(14)C]sucrose remains in the cytosol, apparently not actively moving into the vacuoles. Overall results appear to suggest that vacuoles are not actively engaged in photosynthetic carbon metabolism in spinach leaf protoplasts. PMID- 16664173 TI - Stimulation of ethylene production in bean leaf discs by the pseudomonad phytotoxin coronatine. AB - Coronatine is a toxin produced by Pseudomonas syringae pv. glycinea which induces the same chlorotic response in bean leaves as does infection by the bacterial pathogen. Although the structure of coronatine is known, the biological mode of action is not. One possible clue to its activity is the ethyl-substituted cyclopropane side chain of the molecule. This part structure (1-amino-2 ethycyclopropane-1-carboxylic acid or AEC) is an analog of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC).When coronatine was applied to bean leaf discs in solution, or to intact leaves through prick application, a substantial stimulation of ethylene production was measured. This stimulation was concomitant with an increase in ACC content of the tissue, and occurred under the same conditions as did the chlorotic response to the toxin. The stimulation of ethylene production was inhibited by aminoethoxyvinylglycine, an inhibitor of ACC synthesis. These results, along with those of experiments using l-[U (14)C]methionine, indicated that the stimulation involved de novo production of ethylene via the methionine pathway.The whole, unhydrolyzed coronatine molecule is probably necessary to elicit both the ethylene and chlorosis responses since neither hydrolysis product (coronafacic acid and coronamic acid AEC]) is effective alone. A naturally occurring analog of coronatine, coronafacoylvaline, also stimulated ethylene production and caused chlorosis. However, the unrelated pseudomonad phytotoxin phaseolotoxin, which also causes chlorosis, did not stimulate ethylene production. Ethylene thus may have a specific role in the coronatine toxic syndrome. PMID- 16664174 TI - Bicarbonate-Reversible and Irreversible Inhibition of Photosystem II by Monovalent Anions. AB - We tested a number of inhibitory monovalent anions for their primary site of action on photosystem II(PSII) in chloroplasts. We find that the inhibitory effects of F(-), HCO(2) (-), NO(2) (-), NO(3) (-), and CH(3)CO(2) (-) are all reversed by addition of a high concentration of HCO(3) (-). This class of anions competitively inhibits H(14)CO(3) (-) binding to PSII. All of those anions tested reduced H(14)CO(3) (-) binding more in the light than in the dark. We conclude that the primary inhibitory site of action of a number of monovalent anions is at the HCO(3) (-) binding site(s) on the PSII complex. The carbonic anhydrase inhibitor gold cyanide, and also azide, inhibit PSII but at a site other than the HCO(3) (-) binding site. We suggest that the unique ability of HCO(3) (-) to reverse the effects of inhibitory anions reflects its singular ability to act as a proton donor/acceptor at the anion binding site. A similar role has been proposed for non-substrate-bound HCO(3) (-) on carbonic anhydrase by Yeagle et al. (1975 Proc Natl Acad Sci USA 72: 454-458). PMID- 16664175 TI - Anaerobic stress in germinating castor bean, ethanol metabolism, and effects on subcellular organelles. AB - Endosperms from castor beans (Ricinus communis) germinated for 0 to 6 days were exposed to anoxia for 0 to 15 hours. Ethanol, the only alcohol detected by gas chromatography in the tissue, accumulates to a concentration of 15 millimolar during the first 2 to 4 hours of anoxia and subsequently decreases. The absolute amount of ethanol varies from 10 micromoles per 5-day endosperm after 4 hours anoxia to less than 1 micromole in 2-day endosperm after 4 hours. Lactate content is 2 micromoles or less per endosperm. Alcohol dehydrogenase and pyruvate decarboxylase activities, which are localized in cytosolic fractions, are not greatly affected by anoxia. The recoveries of the marker enzymes and protein in endoplasmic reticulum (ER) and mitochondrial fractions decrease during anoxia. After 15 hours, the recovery of NADPH cytochrome c reductase is 15% of that in controls, fumarase is 50%, and catalase is 75%.Glyoxysomes and ER are capable of converting ethanol to acetaldehyde which was measured using the fluorogenic reagent, 5,5-dimethyl-1,3-cyclohexanedione. The glyoxysomal activity is dependent on a hydrogen peroxide-generating substrate and the ER is dependent on NADPH. However, these activities are less than 3% of the alcohol dehydrogenase activity. PMID- 16664176 TI - Paraquat resistance in conyza. AB - A biotype of Conyza bonariensis (L.) Cronq. (identical to Conyza linefolia in other publications) originating in Egypt is resistant to the herbicide 1,1' dimethyl-4,4'-bipyridinium ion (paraquat). Penetration of the cuticle by [(14)C]paraquat was greater in the resistant biotype than the susceptible (wild) biotype; therefore, resistance was not due to differences in uptake. The resistant and susceptible biotypes were indistinguishable by measuring in vitro photosystem I partial reactions using paraquat, 6,7-dihydrodipyrido [1,2 alpha:2',1'-c] pyrazinediium ion (diquat), or 7,8-dihydro-6H-dipyrido [1,2 alpha:2',1'-c] [1,4] diazepinediium ion (triquat) as electron acceptors. Therefore, alteration at the electron acceptor level of photosystem I is not the basis for resistance. Chlorophyll fluorescence measured in vivo was quenched in the susceptible biotype by leaf treatment with the bipyridinium herbicides. Resistance to quenching of in vivo chlorophyll fluorescence was observed in the resistant biotype, indicating that the herbicide was excluded from the chloroplasts. Movement of [(14)C] paraquat was restricted in the resistant biotype when excised leaves were supplied [(14)C]paraquat through the petiole. We propose that the mechanism of resistance to paraquat is exclusion of paraquat from its site of action in the chloroplast by a rapid sequestration mechanism. No differential binding of paraquat to cell walls isolated from susceptible and resistant biotypes could be detected. The exact site and mechanism of paraquat binding to sequester the herbicide remains to be determined. PMID- 16664177 TI - Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L. AB - A spectral, immunochemical, and proteolytic characterization of native 120 kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified ( approximately 200-fold) in the phytochrome-far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr lambda(max) at 730 nanometers, a spectral change ratio (DeltaA(r)/DeltaA(fr)) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH(2) terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH(2) terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr lambda(max) to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers. PMID- 16664178 TI - Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves. AB - Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6 phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P(2)ase) were identified in leaves of corn (Zea mays), a C(4) plant. Fru-6-P,2K and Fru-2,6-P(2)ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C(3) species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6 phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P(2)ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P(2)ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C(4) Fru-6-P,2K and Fru-2,6-P(2)ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites. PMID- 16664179 TI - Electron Spin Resonance Studies of Ionic Permeability Properties of Thylakoid Membranes of Beta vulgaris and Avicennia germinans. AB - Measurement of intrathylakoid aqueous volumes by electron spin resonance spectroscopy was used to study ionic permeability properties of thylakoid membranes isolated from Beta vulgaris L. and Avicennia germinans L. The thylakoids behaved as perfect osmometers in the presence of sorbitol and betaine. Thylakoids exposed to hypertonic solutions of NaCl and KCl shrank and subsequently swelled, requiring 10 minutes to regain their original volume. The initial influx rate calculated from the kinetics of changes in intrathylakoid volume in response to 450 millimolar gradients of NaCl and KCl was 2.3 x 10(-13) moles per square centimeter per second. These data show that the passive permeability to NaCl and KCl was low. PMID- 16664180 TI - Rapid osmotic adjustment by a succulent halophyte to saline shock. AB - The objective of this research was to measure the short term osmotic adjustment of Salicornia europaea L. ssp. rubra (A. Nels) Breitung when suddenly exposed to 100 millimolar NaCl. Plants were grown hydroponically, shocked with 100 millimolar NaCl added to the culture solution, and stem tips analyzed for free inorganic ions and small organic molecules at intervals up to 72 hours. In the first 2 hours, the calculated leaf osmoticum showed a net increase of 158.8 millimolar most of which was free Mg(2+) (+135.3 millimolar). Total sugars increased almost 5-fold by the 6th hour, enough to provide sufficient osmoticum for the cytoplasm if only partially confined there. By 24 hours, all measured osmotica had decreased except Na(+), Mg(2+), Cl(-), and proline, with the net increase being 208 millimolar. By 72 hours, there was a net gain of 356 millimolar in osmotica of the stem tips, due to Na(+) (+233.3 millimolar), Cl(-) (+306.7 millimolar), and a small increase in sugar and proline (+3.5 millimolar), with all other osmotica decreasing in concentration. Compatible osmotica did not change sufficiently to account for osmotic balance between vacuole and cytoplasm; consequently, there must have been a reapportionment of osmotica within the cell in the short time duration of this experiment. PMID- 16664181 TI - Proton transport in isolated vacuoles from corn coleoptiles. AB - Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of [(14)C]methylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and alpha mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl(-), and inhibited by NO(3) (-), carbonyl cyanide-m-chlorophenylhydrazone, N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, diethylstilbestrol, and 7 chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously characterized in corn coleoptiles (Mettler et al. 1982 Plant Physiol 70: 1738 1742) is derived from the tonoplast. PMID- 16664182 TI - Acclimation Processes in the Light-Harvesting System of the Cyanobacterium Anacystis nidulans following a Light Shift from White to Red Light. AB - Cyanobacteria acclimate to changes in light by adjusting the amounts of different cellular compounds, for example the light-harvesting macromolecular complex. Described are the acclimatization responses in the light-harvesting system of the cyanobacterium Anacystis nidulans following a shift from high intensity, white light to low intensity, red light.The phycocyanin and chlorophyll content and the relative amount of the two linker peptides (33 and 30 kilodaltons) in the phycobilisome were studied. Both the phycocyanin and chlorophyll content per cell increased after the shift, although the phycocyanin increased relatively more. The increase in phycocyanin was biphasic in nature, a fast initial phase and a slower second phase, while the chlorophyll increase was completed in one phase. The phycocyanin and chlorophyll responses to red light were immediate and were completed within 30 and 80 hours for chlorophyll and phycocyanin, respectively. An immediate response was also seen for the two phycobilisome linker peptides. The amount of both of them increased after the shift, although the 33 kilodalton linker peptide increased faster than the 30 kilodalton linker peptide. The increase of the content of the two linker peptides stopped when the phycocyanin increase shifted from the first to the second phase. We believe that the first phase of phycocyanin increase was due mainly to an increase in the phycobilisome size while the second phase was caused only by an increase in the amount of phycobilisomes. The termination of chlorophyll accumulation, which indicates that no further reaction center chlorophyll antennae were formed, occurred parallel to the onset of the second phase of phycocyanin accumulation. PMID- 16664183 TI - Iron Deficiency Decreases Suberization in Bean Roots through a Decrease in Suberin-Specific Peroxidase Activity. AB - The suberin content of young root parts of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prelude was determined. The aliphatic components that could be released from suberin-enriched fractions by LiAID(4) depolymerization were identified by gas chromatography-mass spectrometry. In the normal roots, the major aliphatic components were omega-hydroxy acids and dicarboxylic acids in which saturated C(16) and monounsaturated C(18) were the dominant homologues. Iron-deficient bean roots contained only 11% of the aliphatic components of suberin found in control roots although the relative composition of the constituents was not significantly affected by iron deficiency. Analysis of the aromatic components of the suberin polymer that could be released by alkaline nitrobenzene oxidation of bean root samples showed a 95% decrease in p hydroxybenzaldehyde, vanillin, and syringaldehyde under iron-deficient conditions. The inhibition of suberin synthesis in bean roots was not due to a decrease in Fe-dependent omega-hydroxylase activity since normal omega hydroxylation could be demonstrated, both in vitro with microsomal preparations and in situ by labeling of omega-hydroxy and dicarboxylic acids with [(14)C]acetate. The level of the isozyme of peroxidase that is specifically associated with suberization was suppressed by iron deficiency to 25% of that found in control roots. None of the other extracted isozymes of peroxidase was affected by the iron nutritional status. The activity of the suberin-associated peroxidase was restored within 3 to 4 days after application of iron to the growth medium. The results suggest that, in bean roots, iron deficiency causes inhibition of suberization by causing a decrease in the level of isoperoxidase activity which is required for polymerization of the aromatic domains of suberin, while the ability to synthesize the aliphatic components of the suberin polymer is not impaired. PMID- 16664184 TI - Proteolysis of endogenous substrates in senescing oat leaves : I. Specific degradation of ribulose bisphosphate carboxylase. AB - Proteolysis of ribulose bisphosphate carboxylase (RuBPCase) during senescence was monitored using oat leaf segments (Avena sativa cv Victory), kept in the dark. We here report the development of a novel approach for measuring protein degradation of endogenous substrates both in situ and in vitro in crude extracts using specific antibodies against highly purified polypeptides. The proteolytic products were separated on sodium dodecyl sulfate-gels. They were then electrotransferred onto nitrocellulose paper and identified with specific antibodies to both the large and small subunits of RuBPCase. We could show differences in pH optima between two proteases degrading the subunits of RuBPCase. While both subunits were best hydrolyzed in acid and basic pH, they degraded differently at neutral pH. Furthermore, the large subunit displayed a different pattern of degradative products at the different pH levels. Older leaf segments, which were incubated in darkness, underwent enhanced proteolysis, as compared with young ones. These results show the advantages of the assay in demonstrating: (a) in situ proteolysis of specific substrates in crude extracts without further purification; (b) in vitro differential proteolysis of endogenous substrates during senescence. PMID- 16664185 TI - Carbohydrates stimulate ethylene production in tobacco leaf discs : I. Interaction with auxin and the relation to auxin metabolism. AB - Various naturally occurring carbohydrates, applied at a concentration range of 1 to 100 mm, stimulated ethylene production for several days in indoleacetic acid (IAA)-treated or untreated tobacco (Nicotiana tabacum L. cv ;Xanthi') leaf discs. The lag period for this sugar-stimulated ethylene production was 8 to 12 hours after excision in the untreated leaf discs, but less than 2 hours in the IAA treated ones. Among the tested carbohydrates, 12 were found to increase synergistically ethylene production, with d-galactose, sucrose, and lactose being the most active; mannitol and l-glucose had no effect. The extent and duration of the increased ethylene production was dependent upon the type of sugar applied, the tissue's age, and the existence of both exogenous IAA and sugar in the medium. Sucrose appeared to elicit a continuous IAA effect for 48 hours, as expressed by increased ethylene production, even when IAA was removed from the medium after a 4-hour pulse. Sucrose stimulated both the uptake and decarboxylation of [1-(14)C]IAA, as well as the hydrolysis of the esteric and amide IAA conjugates formed in the tissue after application of free IAA. This gradual hydrolysis was accompanied by a further accumulation of a third IAA metabolite. Moreover, synthetic indole-3-acetyl-l-alanine increased ethylene production mainly with sucrose, and this effect was accompanied by its increased decarboxylation and turnover pattern suggesting that release of free IAA was involved. An esteric IAA conjugate, tentatively identified by GC retention time was found to be the major component (84%) of the naturally occurring IAA conjugates in tobacco leaves. Accordingly the sucrose-stimulated ethylene production in tobacco leaves can be ascribed mainly to the sucrose-stimulated hydrolysis of the esteric IAA conjugate. PMID- 16664186 TI - Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : II. Sites of Stimulation in the Ethylene Biosynthesis Pathway. AB - Galactose, sucrose, and glucose (50 millimolar) applied to tobacco leaf discs (Nicotiana tabacum L. cv ;Xanthi') during a prolonged incubation (5-6 d) markedly stimulated ethylene production which, in turn, could be inhibited by aminoethoxyvinylglycine (2-amino-4-(2'-aminoethoxy)-trans-3-butenoic acid) (AVG) or Co(2+) ions. These three tested sugars also stimulated the conversion of l [3,4-(14)C]methionine to [(14)C]1-amino-cyclopropane-1-carboxylic acid (ACC) and to [(14)C]ethylene, thus indicating that the carbohydrates-stimulated ethylene production proceeds from methionine via the ACC pathway. Sucrose concentrations above 25 mm considerably enhanced ACC-dependent ethylene production, and this enhancement was related to the increased respiratory carbon dioxide. However, sucrose by itself could directly promote the step of ACC conversion to ethylene, since low sucrose concentrations (1-25 mm) enhanced ACC-dependent ethylene production also in the presence of 15% CO(2).The data suggest that the stimulation of ethylene production by sugars in tobacco leaf discs results from enhancement of ACC formation as well as from the conversion of ACC to ethylene, when both steps could be involved in regulation of ethylene biosynthesis. PMID- 16664187 TI - Acifluorfen-induced isoflavonoids and enzymes of their biosynthesis in mature soybean leaves : whole leaf and mesophyll responses. AB - Mature soybean (Glycine max L. cv Harosoy 63) leaves normally contain kaempferol 3-glycosides but they accumulate no other flavonoids. Whole leaves sprayed with the diphenyl ether herbicide Acifluorfen and maintained in the light developed small necrotic lesions and accumulated isoflavone aglycones, isoflavone glucosides, and pterocarpans. Isoflavonoid accumulation was preceded by induced activity for chalcone synthase (CHS) and by increased activity for phenylalanine ammonia-lyase (PAL) and UDP-glucose:isoflavone 7-O-glucosyl transferase (IGT). PAL and CHS activity was highest between 24 and 30 hours after treatment, isoflavone aglycones and pterocarpans at 48 hours, IGT at 72 hours, and isoflavone glucosides at 96 hours.Mesophyll cells isolated from control leaves contained no activity for PAL, CHS, or IGT and no flavonoids of any class. Cells isolated from treated leaves at the stage of maximal enzyme activity or isoflavonoid content contained PAL (12% of the whole leaf activity), CHS (24%), IGT (20%), and 25% of the whole leaf isoflavone glucosides, but only traces, presumably as contaminants, of the other flavonoids. We suggest that the isoflavone glucosides were synthesized and accumulated in intact mesophyll cells as soluble detoxification products, while the isoflavone aglycones and pterocarpans accumulated in the epidermis or extracellularly within the mesophyll. To our knowledge this is the first report of tissue-specific induction of isoflavonoid glucosides and key enzymes of their biosynthesis in any plant. PMID- 16664188 TI - Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt Tolerant Cell Lines of Citrus aurantium. AB - Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na(+), K(+), and Cl(-) when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na(+) and Cl( ) uptake, and all these cell lines took up similar or even larger amounts of Na(+) and Cl(-) than the NaCl-sensitive cell line. Exposure of cells of NaCl sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K(+) from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K(+) are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na(2)SO(4), but rather poor tolerance to K(+) introduced as either K(2)SO(4) or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na(+) by K(+). Analyses of internal Na(+), K(+), and Cl(-) concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance. PMID- 16664189 TI - Sucrose synthase of soybean nodules. AB - SUCROSE SYNTHASE (UDPGLUCOSE: d-fructose 2-alpha-d-glucosyl transferase, EC 2.4.1.13) has been purified from the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules. The native enzyme had a molecular weight of 400,000. The subunit molecular weight was 90,000 and a tetrameric structure is proposed for soybean nodule sucrose synthase. Optimum activity in the sucrose cleavage and synthesis directions was at pH 6 and pH 9.5 respectively, and the enzyme displayed typical Michaelis-Menten kinetics. Soybean nodule sucrose synthase had a high affinity for UDP (K(m), 5 micromolar) and a relatively low affinity for ADP (apparent K(m), 0.13 millimolar) and CDP (apparent K(m), 1.1 millimolar). The K(m) for sucrose was 31 millimolar. In the synthesis direction, UDPglucose (K(m), 0.012 millimolar) was a more effective glucosyl donor than ADPglucose (K(m), 1.6 millimolar) and the K(m) for fructose was 3.7 millimolar. Divalent cations stimulated activity in both the cleavage and synthesis directions and the enzyme was very sensitive to inhibition by heavy metals. PMID- 16664190 TI - Ribulose 1,5-Bisphosphate Carboxylase Synthesis during Heat Shock. AB - Ribulose 1,5-bisphosphate carboxylase (RuBPCase) was chosen as a model protein to study how heat shock (HS) affects both chloroplast protein synthesis and the nuclear-chloroplast interaction in production of chloroplast proteins. Experiments were performed using highly chlorophyllous, soybean (Glycine max L. Merr. var Corsoy) cell suspension cultures active in chloroplast protein synthesis. Synthesis of RuBPCase large (L) and small (S) subunits was followed by in vivo labeling, and corresponding mRNA levels were examined by Northern and dot hybridization analyses. Results demonstrate that L and S synthesis declines with increasing HS temperatures (33-40 degrees C) and reaches minimum levels (20-30% of control) at temperatures of maximum HS protein synthesis (39-40 degrees C). Recovery of L and S synthesis following a 2-hour HS at 38 or 40 degrees C was also studied. The changes in S synthesis during HS and recovery correlate with the steady state levels of S mRNA. In contrast, changes in L synthesis show little relationship to the corresponding mRNA levels; levels of L mRNA remain relatively unchanged by HS. We conclude that chloroplast protein synthesis shows no greater sensitivity to HS than is observed for cytoplasmic protein synthesis and that transport of proteins into the chloroplast (e.g.S subunit) continues during HS. Furthermore, there is no apparent coordination of L and S subunit mRNA levels under the conditions examined. PMID- 16664191 TI - Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris. AB - The pH-dependent fluorescence quenching of acridine orange was used to study the Na(+)- and K(+)-dependent H(+) fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na(+)-dependent H(+) flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na(+) concentration and was competitively inhibited by amiloride (K(i) = 0.1 millimolar). The potential-dependent component of H(+) flux showed an approximately linear dependence on Na(+) concentration. In contrast, the K(+) dependent H(+) flux apparently consisted of a single component which showed an approximately linear dependence on K(+) concentration, and was insensitive to amiloride. Based on the Na(+)- and K(+)-dependent H(+) fluxes, the passive permeability of the vesicle preparation to Na(+) was about half of that to K(+).The apparent K(m) for Na(+) of the electroneutral Na(+)/H(+) exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na(+)/H(+) antiport which can account for the estimated in vivo accumulation ratio for Na(+) into the vacuole. PMID- 16664192 TI - Antioxidant levels in germinating soybean seed axes in relation to free radical and dehydration tolerance. AB - The axis of soybean seeds suffer dehydration injury if they are dried to 10% moisture at 36 hours of imbibition, but tolerate this stress if dried at 6 hours of imbibition. Deesterification of membrane phospholipids has been correlated with the increased permeability and increased lipid phase transition temperatures of membranes from dehydration injured tissues. Deesterification, measured as increased free fatty acid:phospholipid and decreased phospholipid:sterol ratios, occurred primarily when the tissue was in the dry state and did not change significantly (P 3)-beta-d-Glucan Isolated From Zea Shoot Cell Wall Preparations. AB - A small quantity of (1-->3)-beta-d-glucan was extracted with a (1-->3),(1-->4) beta-d-glucan by hot water after treatment of the insoluble fraction of a buffer homogenate of Zea shoots with 3 molar LiCl. An ammonium sulfate precipitation procedure effected a separation of the (1-->3)-beta-d-glucan from the more prevalent (1-->3),(1-->4)-beta-d-glucan. The minor component polysaccharide precipitated at a concentration of 20% ammonium sulfate (w/v) and was, as a consequence of precipitation, rendered insoluble in water. The insoluble products were dissolved in 1 normal NaOH followed by neutralization with CH(3)COOH. The purified polysaccharide accounted for approximately 0.3% of total hot water extract. It consisted mostly of glucose and its average mol wt was estimated to be about 7.0 x 10(4), based on elution from a calibrated Sepharose CL-4B column. Methylation analysis and enzymic hydrolysis or partial acid-hydrolysis of the polysaccharide followed by analysis of the hydrolysate showed that the polysaccharide consisted of (1-->3)-beta-linked glucose residues. PMID- 16664199 TI - Effectiveness of intermittent light treatments on anthocyanin synthesis in dark grown and light-pretreated seedlings. AB - Differences in the extent of anthocyanin production between intermittent light treatments with short and long dark intervals between successive irradiations are more pronounced in dark-grown than in light-pretreated cabbage seedlings. This observation is consistent with the hypothesis, based on destruction kinetics data, that there might be two pools of phytochrome, a labile one and a stable one, present in different proportions in dark-grown and light-pretreated seedlings, and suggests that light-dependent changes of the stable to labile phytochrome ratio might be physiologically significant in the photoregulation of photomorphogenic responses. PMID- 16664200 TI - Physiological and metabolic responses of winter wheat to prolonged freezing stress. AB - Survival and cold hardiness declined gradually when cold-hardened Fredrick winter wheat (Triticum aestivum L.) was maintained at -6 degrees C for several weeks. Moisture content of crown and root tissue did not change significantly during this period. Uptake of O(2) and accumulation of (86)Rb by root tissue declined abruptly upon exposure to -6 degrees C, whereas a concomitant negative effect of freezing on these metabolic processes was not observed in crown tissue. Electron spin resonance spectroscopic analysis of microsomal membrane preparations from crown tissue revealed no evidence of gross changes in the physical properties of the bulk lipids even when seedlings were killed. The results provide biochemical evidence that seedling damage due to prolonged exposure to a mild freezing stress is due to disruption of key metabolic process in the root while cells within the crown remain viable. PMID- 16664201 TI - Influence of culture age and spermidine treatment on the accumulation of phenolic compounds in suspension cultures. AB - The influence of cell age on phenol accumulation was examined by determining the quantity of individual phenols which accumulated in Paul's scarlet rose cultures of increasing age. During log-phase growth (days 7 and 11), only gallic acid and epicatechin-catechin were detected; whereas, during early and late stationary phase (days 14 and 35), several other phenols were present in addition to gallic acid and epicatechincatechin. When stationary-phase cultures were provided with a supplement of sucrose and spermidine, a treatment previously shown to arrest the senescence of rose cultures (Muhitch, Edwards, Fletcher 1983 Plant Cell Rep 2: 82 84), the cells then accumulated a higher level and a wider assortment of phenols. These results suggest that extending the lifespan of mature nondividing cell cultures offers a means of increasing the yield of secondary products by cultured cells. PMID- 16664202 TI - Interaction between Senescence and Wounding in Oat Leaves. AB - A study was made of the influence of wounding on the senescence of standard oat leaf segments in the dark. Wounding was by either subdividing the 3 centimeter long segments into 5 millimeter subsegments, gently scraping the adaxial surface of the segments with a sharp blade, making transverse linear cuts, or by making many small holes with a needle. Wounding considerably delayed the loss of both chlorophyll and protein in the dark and the amount of inhibition was roughly proportional to the intensity of wounding. With surface wounding, the inhibition of senescence was detectable from the first day of dark incubation; other methods caused moderate promotion of senescence for the first 2 days but decreased the loss of chlorophyll and protein thereafter. A number of senescence-modifying substances acted similarly on both unwounded and wounded segments, but the amount of chlorophyll and protein in the wounded segments was always more than in the respective controls. Cytokinins, however, provided an exception, since their effect was actually decreased by wounding. The proteases operating at pH 4.1 and 6.6 were both clearly less active in the wounded leaves than in controls. The possible mechanism of this inhibitory effect of wounding on senescence is discussed. PMID- 16664203 TI - A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant. AB - The nodulation characteristics of soybean (Glycine max) mutant nts382 are described. The mutant nodulated significantly more than the parent cultivar Bragg in the presence and absence of several combined nitrogen sources (KNO(3), urea, NH(4)Cl, and NH(4)NO(3)). The number of nodules on the tap root and on lateral roots was increased in the mutant line. In the presence of KNO(3) and urea, nitrogenase activity was considerably higher in nts382 than in Bragg. Mutant plants were generally smaller than wild-type plants. Although nts382 is a supernodulator, inoculation with Rhizobium japonicum was necessary to induce nodule formation and both trial strains CB1809 (= USDA136) and USDA110 elicited the mutant phenotype. Segregation of M(3) progeny derived from a M(2) wild-type plant indicated that the mutant character is inherited as a Mendelian recessive. The mutant is discussed in the context of regulation of nodulation and of hypotheses that have been proposed to explain nitrate inhibition of nodulation. PMID- 16664204 TI - Development of three copper metalloenzymes in clover leaves. AB - Subterranean clover (Trifolium subterraneum L. cv Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without Cu. After Cu deficiency had developed, Cu(2+) was added to some deficient plants and Cu content, protein content, and activities of three Cu metalloenzymes (diamine oxidase [EC1.4.3.6], ascorbate oxidase [EC1.10.3.3] and o-diphenol oxidase [EC1.10.3.1]) were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.Copper deficiency had little effect on protein concentrations, but markedly depressed enzyme activities and Cu concentration in all leaf blades assayed. Within 4 d of adding Cu(2+) to Cu deficient plants, Cu concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu(2+) was added.The results suggest that active holoenzymes of diamine oxidase, ascorbate oxidase, and o-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development. PMID- 16664205 TI - Energetics of sucrose transport into protoplasts from developing soybean cotyledons. AB - The accumulation of tetraphenylphosphonium (TPP(+)), 5,5'-dimethyl-oxazolidine 2,4-dione (DMO), and a micro pH electrode were used to measure membrane potential, intracellular and extracellular pH, respectively, upon the addition of exogenous sucrose to soybean cotyledon protoplasts. Addition of sucrose caused a specific and transient (a) depolarization of the membrane potential (measured by TPP(+) accumulation), (b) acidification of the intracellular pH (measured by DMO accumulation), and (c) alkalization of the external medium (measured by a micro pH electrode). The time course for all these changes was similar (i.e. 5 to 10 minutes). Based on the rate of sucrose uptake and alkalization of the external medium, a stoichiometry of 1.02 to 1.10 for proton to sucrose was estimated. These data strongly support a proton/sucrose cotransporting mechanism in soybean cotyledon cells. PMID- 16664206 TI - Photosynthesis involvement in the mechanism of action of diphenyl ether herbicides. AB - Photosynthesis is not required for the toxicity of diphenyl ether herbicides, nor are chloroplast thylakoids the primary site of diphenyl ether herbicide activity. Isolated spinach (Spinacia oleracea L.) chloroplast fragments produced malonyl dialdehyde, indicating lipid peroxidation, when paraquat (1,1'-dimethyl-4,4' bipyridinium ion) or diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] were added to the medium, but no malonyl dialdehyde was produced when chloroplast fragments were treated with the methyl ester of acifluorfen (methyl 5-[2-chloro-4 (trifluoromethyl)phenoxy]-2-nitrobenzoic acid), oxyfluorfen [2-chloro-1-(3-ethoxy 4-nitrophenoxy)-4-(trifluoromethyl)benzene], or MC15608 (methyl 5-[2-chloro-4 (trifluoromethyl)phenoxy]-2-chlorobenzoate). In most cases the toxicity of acifluorfen-methyl, oxyfluorfen, or MC15608 to the unicellular green alga Chlamydomonas eugametos (Moewus) did not decrease after simultaneous treatment with diuron. However, diuron significantly reduced cell death after paraquat treatment at all but the highest paraquat concentration tested (0.1 millimolar). These data indicate electron transport of photosynthesis is not serving the same function for diphenyl ether herbicides as for paraquat. Additional evidence for differential action of paraquat was obtained from the superoxide scavenger copper penicillamine (copper complex of 2-amino-3-mercapto-3-methylbutanoic acid). Copper penicillamine eliminated paraquat toxicity in cucumber (Cucumis sativus L.) cotyledons but did not reduce diphenyl ether herbicide toxicity. PMID- 16664207 TI - Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L. AB - Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-(3)H]+/-ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species. PMID- 16664208 TI - Effect of multiple factor source-sink manipulation on nitrogen and carbon assimilation by soybean. AB - The objectives of this study were to determine the effect of light enhancement and hastened reproductive development on nitrogen and dry matter accumulation by field-grown soybean (Glycine max [L.] Merr.). The impacts of photosynthate supply and reproductive development on change in the season-long profiles of in vivo leaf nitrate reductase (NR) activity and root nodule acetylene reduction (AR) activity were evaluated.Light enhancement resulted in significant increases in dry matter accumulation, root nodule fresh weight and AR activity. Seed yield was increased in both light enhanced treatments in 1978 and in one in 1979.Hastened flowering and seed development was accomplished through photoperiod manipulation within a single genotype. Seasonal decline in leaf NR activity was most rapid in plants entering reproductive development early. An early increase in root nodule fresh weight and AR activity was also observed in response to this treatment and was followed similarly by early decline.The addition of high levels of soil applied nitrogen increased leaf NR activity and delayed late season decline in NR activity for both control and early reproductive plants. Nitrate supply was therefore implicated as limiting to leaf NR activity during the decline associated with flowering and early seed development. A limited additional increase in leaf NR activity was observed in response to light enhancement plus soil-applied nitrogen. As no significant increase in leaf NR activity was observed in response to light enhancement alone, leaf nitrate supply was further implicated as more limiting to leaf NR activity than was photosynthate supply during flowering and early seed development. PMID- 16664209 TI - Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines. AB - The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells x Beeson and Wells x Corsoy) were compared in 1979.The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering. PMID- 16664210 TI - In Situ Measurement of Epidermal Cell Turgor, Leaf Water Potential, and Gas Exchange in Tradescantia virginiana L. AB - A combined system has been developed in which epidermal cell turgor, leaf water potential, and gas exchange were determined for transpiring leaves of Tradescantia virginiana L. Uniform and stable values of turgor were observed in epidermal cells (stomatal complex cells were not studied) under stable environmental conditions for both upper and lower epidermises. The changes in epidermal cell turgor that were associated with changes in leaf transpiration were larger than the changes in leaf water potential, indicating the presence of transpirationally induced within-leaf water potential gradients. Estimates of 3 to 5 millimoles per square meter per second per megapascal were obtained for the value of within-leaf hydraulic conductivity. Step changes in atmospheric humidity caused rapid changes in epidermal cell turgor with little or no initial change in stomatal conductance, indicating little direct relation between stomatal humidity response and epidermal water status. The significance of within-leaf water potential gradients to measurements of plant water potential and to current hypotheses regarding stomatal response to humidity is discussed. PMID- 16664211 TI - O(2)-insensitive photosynthesis in c(3) plants : its occurrence and a possible explanation. AB - Leaves of C(3) plants which exhibit a normal O(2) inhibition of CO(2) fixation at less than saturating light intensity were found to exhibit O(2)-insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O(2) insensitive photosynthesis has been reported in nine other C(3) species and usually occurred when the intercellular CO(2) pressure was about double the normal pressure. A lack of O(2) inhibition of photosynthesis was always accompanied by a failure of increased CO(2) pressure to stimulate photosynthesis to the expected degree. O(2)-insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O(2) and CO(2) insensitive at physiological CO(2) pressures. Postillumination CO(2) exchange kinetics showed that O(2) and CO(2) insensitivity was not the result of elimination of photorespiration.It is proposed that O(2) and CO(2) insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO(2) assimilation. PMID- 16664212 TI - Vacuolar localization of wound-induced carboxypeptidase inhibitor in potato leaves. AB - The wound-induced carboxypeptidase inhibitor in potato leaves was shown to be localized in the central vacuoles of the cells. The inhibitor was quantified by immunological assays (ELISA) in protoplasts and vacuoles isolated from upper unwounded leaves of 5- to 6-week old potato plants that had been wounded on their lower leaves 48 hours earlier to induce the accumulation of the carboxypeptidase inhibitor. The regulation of the synthesis and compartmentation of the inhibitor is similar to that of wound-induced serine proteinase Inhibitors I and II in potato and tomato leaves and appears to be part of an induced defense response against attacking pests. PMID- 16664213 TI - Role of Gibberellins in the Environmental Control of Stem Growth in Thlaspi arvense L. AB - Field pennycress (Thlaspi arvense L.) is a winter annual that requires a cold treatment for the induction of stem elongation. An inbred line was selected in which no stem elongation was observed in plants grown for 6 months at 21 degrees C regardless of the prevailing photoperiod. Increased exposure time of plants grown initially at 21 degrees C to cold (2 degrees C) induced a greater rate of stem elongation when the plants were returned to 21 degrees C. Moreover, longer cold treatments resulted in a greater maximum stem height and reduced the lag period for the onset of measurable internode elongation. The optimal temperature range for thermoinduced stem growth was broad: rates of stem growth in plants maintained for 4 weeks at either 2 degrees or 10 degrees C were virtually identical. However, a 4-week thermoinductive treatment at 15 degrees C resulted in a greater lag period for the initiation of stem elongation and a decreased growth rate. The rate of cold-induced stem elongation was greater in plants subjected to long days than for plants subjected to short days following the cold treatment. Thus, photoperiod does not control the induction of stem elongation, but does regulate stem elongation in progress. Exogenous gibberellin A(3) (GA(3)) was able to substitute for the cold requirement, but elicited a greater response in plants maintained under long days than short days. This indicates that photoperiod influences the plant's sensitivity to GAs. The GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride, inhibited low temperature induced stem elongation, and this inhibition was completely reversed by exogenous GA(3). These results suggest that cold-induced stem elongation in field pennycress is mediated by some change in the endogenous GA status. PMID- 16664214 TI - Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties. AB - NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr(1)-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c(1)NR, and the form eluted with NADH was designated c(2)NR. Nitrate-grown nr(1) mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c(1)NR and c(2)NR had similar pH optima of 6.5, sedimentation behavior (s(20,w) of 5.5-6.0), and electrophoretic mobility. However, c(1)NR was more active with NADPH than with NADH, while c(2)NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c(1)NR) and 0.19 millimolar (c(2)NR). The iNR from the mutant had a pH optimum of 7.5, s(20,w) of 7.6, and was less mobile on polyacrylamide gels than c(1)NR and c(2)NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants. PMID- 16664215 TI - Photosynthetic and stomatal responses of spinach leaves to salt stress. AB - The gas exchange of spinach plants, salt-stressed by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar, was studied 3 weeks after starting NaCl treatment. Photosynthesis became light saturated at 1100 to 1400 micromoles per square meter per second in salt-treated plants and at approximately 2000 micromoles per square meter per second in control plants. Photosynthetic capacity of the mesophyll measured as a function of intercellular partial pressure of CO(2) at the light intensity prevailing during growth and at light saturation were both decreased in the salttreated plants. The CO(2) compensation points and relative enhancements of photosynthesis at low O(2) were not affected by salinity. The lower photosynthetic rates in salt-treated leaves at 450 micromoles per square meter per second were associated with a 70% reduction in stomatal conductance and low intercellular CO(2) (219 microbars; cf. 285 microbars for controls). Increasing photon flux density to light saturation extended the linear portions of the CO(2) response curves, increased stomatal conductances, increased intercellular CO(2) in the salt-treated plants, but lowered it in controls, and accentuated differences in photosynthetic rate (area basis) between the treatments.Leaves from salt-treated plants were thicker but contained about 73% of the chlorophyll per unit area of control plants. When photosynthetic rates were expressed on a chlorophyll basis there was no difference in initial slope of assimilation versus intercellular CO(2) between treatments. Photosynthetic rates (chlorophyll basis) at light saturation differed only by 20% which was also observed earlier with isolated, intact chloroplasts (Robinson et al. 1983 Plant Physiol 73: 238 242).Measurement of carbon isotope ratio revealed less discrimination against (13)C with salt treatment and confirmed the persistence of low intercellular partial pressures of CO(2) during plant growth. The development of a thicker leaf with less chlorophyll per unit area during salt treatment permitted stomatal conductance and intercellular partial pressure of CO(2) to decline without restricting photosynthesis and had the benefit of greatly increasing water use efficiency. PMID- 16664216 TI - Separation and quantitation of polyamines in plant tissue by high performance liquid chromatography of their dansyl derivatives. AB - High performance liquid chromatography in combination with fluorescence spectrophotometry can be used to separate and quantitate polyamines (putrescine, cadaverine, spermidine, spermine), prepared as their dansyl derivatives, from plant tissue. The procedure gives sensitive and consistent results for polyamine determinations in plant tissue. In a standard mixture, the minimal detection level was less than 1 picomole of polyamines. PMID- 16664217 TI - Role of polyamines in gibberellin-induced internode growth in peas. AB - To determine the requirement for polyamines in gibberellin (GA) induced internode growth polyamine content was measured in internodes of peas of various internode phenotypes (slender, tall, dwarf, nana) with and without applied gibberellin (GA(3)) and polyamine synthesis inhibitors. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. The amounts of polyamines in the different genetic lines of peas, which differed in internode lengths and extractable GA content, correlated with the extent of internode elongation. High polyamine concentrations were associated with young internodes and decreased with internode expansion. Extremely short internodes of nana plants without GA exhibited equal or higher amine concentrations relative to internodes of other lines of peas and GA-stimulated nana seedlings. The polyamine synthesis inhibitors, alpha-difluoromethylornithine and alpha-difluoromethylarginine, independently or in combination, inhibited polyamine accumulation and internode elongation of tall peas and GA-stimulated nana plants. Agmatine and putrescine restored growth and endogenous polyamine content to variable degrees. However, exogenous polyamines were not effective in promoting growth unless intracellular amines were partially depleted.These results suggest that polyamines do not have a role in cell elongation, but may be required to support cell proliferation. Polyamines do not mediate the entire action of GA in internode growth of peas since GA induction of growth involves both cell division and cell elongation, whereas polyamines appear to affect cell division only. PMID- 16664218 TI - Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L. AB - High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of (32)P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively. PMID- 16664219 TI - Chloroplast-Diphenyl Ether Interactions II. AB - Acifluorfen, a p-nitrodiphenyl ether herbicide, is inhibitory to those photosynthetic functions that require a functioning chloroplast envelope. Functions involving the stroma are also affected. Acifluorfen does not lyse intact spinach chloroplasts, yet does increase the sensitivity of CO(2)-dependent O(2) evolution to exogenous inorganic phosphate without directly affecting the function of the phosphate translocator. Acifluorfen penetrates into the chloroplast stroma in a light-independent fashion. Once inside, it causes the inactivation of light and dithiothreitol-activated fructose 1,6-bisphosphatase. Light-activated glyceraldehyde-3-phosphate dehydrogenase (NADP) is also inactivated by acifluorfen.These data suggest that acifluorfen stimulates a pathway for inactivation of fructose 1,6-bisphosphatase and glyceraldehyde 3 phosphate dehydrogenase (NADP) which uses oxygen as a terminal oxidant and which involves thioredoxin and ferredoxin-thioredoxin reductase. PMID- 16664220 TI - l-Aspartate Transport into Pea Chloroplasts : Kinetic and Inhibitor Evidence for Multiple Transport Systems. AB - The kinetics of l-aspartate transport into pea chloroplasts was studied in the presence and absence of transport inhibitors to determine whether multiple aspartate carriers exist. Transport was measured by the silicone oil centrifugation technique. Reciprocal plots of concentration-dependent transport rates were biphasic, indicating the presence of two transport components, distinguishable on the basis of their affinity for aspartate. These transport components, called high affinity and low affinity transport could also be distinguished on the basis of their apparent substrate saturability and their sensitivity to media pH. The apparent K(m) for high affinity transport was 30 micromolar. The K(m) for low affinity transport was not determined. To test whether these transport components could also be distinguished on the basis of inhibitor sensitivity and to assess the value of inhibitors for distinguishing multiple aspartate translocators, a survey of several classes of potential inhibitors was conducted. High affinity aspartate transport was inhibited by p chloromercuribenzenesulfonate and mersalyl, both sulfhydryl-reactive reagents; diethyl pyrocarbonate, a histidine-reactive reagent; and nigericin and carbonyl cyanide m-chlorophenylhydrazone, both ionophores. Low affinity aspartate transport was not inhibited by p-chloromercuribenzenesulfonate or nigericin, but preliminary results suggest it was sensitive to diethyl pyrocarbonate. Because the high and low affinity transport components could be distinguished not only by their sensitivity to media pH and substrate saturability, but also by their sensitivity to various inhibitors, we concluded that they may represent different transport systems or carriers. PMID- 16664221 TI - Enhanced Phytochrome Sensitivity and Its Reversal in Amaranthus albus Seeds. AB - Seed of Amaranthus alus L. develop an enhanced sensitivity to the farred absorbing form of phytochrome after prolonged imbibition at temperatures >32 degrees C. The enhanced sensitivity developed at 40 degrees C could be reversed by subsequent treatment at 20 degrees C and similarly reestablished by repeating a 40 degrees C treatment. It is concluded that relative sensitivity to the far red absorbing form of phytochrome may be readily manipulated in seeds of A. albus. PMID- 16664222 TI - Evidence for an ATP-Dependent Proton Pump on the Golgi of Corn Coleoptiles. AB - Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on sucrose density gradients, and ATP-dependent proton pumping activity was localized by the techniques of [(14)C]methylamine uptake and quinacrine fluorescence quenching. Two peaks of proton pumping activity were detected: a light peak (1.07 grams/cubic centimeter) corresponding to the previously characterized tonoplast-type H(+)-ATPase, and a second peak (1.13 grams/cubic centimeter) which coincided with the Golgi markers, latent UDPase, and glucan synthase I. The second peak was lighter than that of the plasma membrane marker, uridine diphosphoglucose-sterol glucosyltransferase (1.16 grams/cubic centimeter) and was not inhibited by vanadate, an inhibitor of the plasma membrane ATPase. The activity was also better correlated with the Golgi cisternae marker, glucan synthase I, than with latent UDPase, a secretory vesicle marker, but a secretory vesicle location cannot be ruled out. The tonoplast-type and Golgi proton pumps were similar in several respects, including a pH optimum at 7.2, stimulation by chloride, inhibition by diethylstilbestrol and N,N'-dicyclohexylcarbodiimide (DCCD), insensitivity to oligomycin and azide, and nucleotide specificity for Mg(2+)-ATP. However, the Golgi H(+) pump was much less sensitive to nitrate and iodide, and more sensitive to the anion channel blockers, 4-acetamido-4' isothiocyano-2,2'-stilbene sulfonic acid (SITS) and 4,4'-diisothiocyano-2,2' stilbene disulfonic acid (DIDS) than the tonoplast-type H(+)-pump. The Golgi pump, but not the tonoplast-type pump, was stimulated by valinomycin in the presence of KCl. It is concluded that the Golgi of corn coleoptiles contains a KCl-stimulated H(+)-ATPase which can acidify the interior of Golgi cisternae and associated vesicles. PMID- 16664223 TI - Wheat invertases : characterization of cell wall-bound and soluble forms. AB - Wheat coleoptiles have two distinct invertases, a soluble and a cell wall-bound form as indicated by results from cytochemical and biochemical studies. These enzyme activities differ in their pH optima, chromatographic behavior on diethylaminoethyl cellulose, kinetic properties, thermal stability, and response to light treatment. The soluble invertase was purified to near homogeneity by diethylaminoethyl-cellulose, concanavalin-A Sepharose, and Sephacryl S-300 chromatography. The overall purification was 175-fold with a recovery of about 26%. The holoenzyme has an apparent molecular weight of 158,000 and subunit molecular weight of 53,000 as estimated by polyacrylamide gel electrophoresis under denaturing conditions. Illumination of wheat seedlings caused an increase in the cell wall, but not the soluble, invertase activity. PMID- 16664224 TI - Physiological and isotopic aspects of photosynthesis in peperomia. AB - Physiological and isotopic aspects of several Peperomia species were investigated. All but one species had C(3)-like stomatal behavior, in that stomata were open during the day and closed during the night. In these species, most atmospheric CO(2) uptake occurred during the day. Concurrent with this stomatal behavior, there were Crassulacean acid metabolism-like acid fluctuations in most species. Carbon and hydrogen isotope ratios of cellulose nitrate from Peperomia reflect their physiological behavior. The delta(13)C values of cellulose nitrate from Peperomia species were similar to values observed in C(3) plants and consistent with the daytime uptake of exogeneous CO(2) via the C(3) photosynthetic pathway. The deltaD values of cellulose nitrate from Peperomia species approach those of Crassulacean acid metabolism plants. These elevated deltaD values are caused by fractionations occurring during biochemical reactions and not as a consequence of water relations. PMID- 16664225 TI - Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat. AB - Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation. PMID- 16664226 TI - Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase. AB - Polyphenol oxidase (PPO) was extensively purified to homogeneity from d'Anjou pear (Pyrus communis L.) by extraction in the presence of the phenolic binder AG 2-X8 andTriton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Purification of pear PPO was achieved after chromatography on Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns. Only after the columns were run at room temperature rather than at 4 degrees C were sharp peaks and good resolution obtained. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to successful purification. Three separate fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide slab gel electrophoresis and corresponded to relative mobilities of 0.41, 0.43, and 0.73. The effect of dimethylsulfoxide on enzyme activity was investigated and found to increase significantly the activity of purified pear PPO over the control. PMID- 16664227 TI - Effects of morphactin and other auxin transport inhibitors on soybean senescence and pod development. AB - Because triiodobenzoic acid increases pod number, albeit variably, in soybean (Glycine max), we tested other auxin-transport inhibitors. Morphactins, especially methylchlorflurenol (MCF), were found to be very active (optimal concentration 10 micromolar) when sprayed onto the foliage. Applications at 1 week after the start of flowering were most effective, producing a 40% increase in pod number with little inhibition (12%) of stem elongation. MCF increased the number of pods initiated (reaching 1 cm length) at least partially by prolonging the initiation period, while pod abortion (failure of pods > 1 cm long) remained low. Generally, MCF did not increase seed yield (dry weight/plant); more, but smaller seeds, were formed by the treated plants. The promotive effect of MCF on pod initiation seems to be independent of its inhibition of stem elongation, which is insignificant at 10 micromolar. MCF delayed pod maturation by 3 to 4 days, while foliar yellowing, blade abscission, and petiole abscission were retarded by 2, 4, and 2 days, respectively. MCF has only a small effect on senescence and that could be indirect, due to a delay in pod development. Other auxin-transport inhibitors tested, including N-1-naphthylphthalamic acid, produced little or no increase in pod number; however, 0.1 millimolar 5-[2' carboxyphenyl]-3-phenylpyrazole caused a 27% increase. These results implicate auxin as a potential regulator of pod development, and they show that soybean seed yield is not simply sink limited. PMID- 16664228 TI - A membrane-bound protease in microsomes of spinach callus. AB - A di-isopropyl phosphorofluoridate-sensitive endopeptidase activity against some minor components of heat-denatured alpha-casein was detected in the endoplasmic reticulum and Golgi body-rich fraction of spinach callus. The activity was not solubilized with 0.05% sodium deoxycholate, but with 0.5% sodium cholate. The activity was strongly inhibited by deoxycholate (0.2-0.5%), di-isopropyl phosphorofluoridate, p-chloro-mercuric benzoate, o-phenanthroline, NiCl(2), CuCl(2), and ZnSO(4), and moderately by phenylmethylsulfonyl fluoride, l-1 tosylamide-2-phenylethyl chloromethyl ketone, iodoacetic acid, ethylenediaminetetraacetate, and FeSO(4), and slightly by chymostatin. The inhibitory effect of o-phenanthroline was partially recovered with the addition of FeSO(4) and ZnSO(4). PMID- 16664229 TI - Involvement of ethylene in the action of the cotton defoliant thidiazuron. AB - The effect of the defoliant thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-ylurea) on endogenous ethylene evolution and the role of endogenous ethylene in thidiazuron-mediated leaf abscission were examined in cotton (Gossypium hirsutum L. cv Stoneville 519) seedlings. Treatment of 20- to 30-day-old seedlings with thidiazuron at concentrations equal to or greater than 10 micromolar resulted in leaf abscission. At a treatment concentration of 100 micromolar, nearly total abscission of the youngest leaves was observed. Following treatment, abscission of the younger leaves commenced within 48 hours and was complete by 120 hours. A large increase in ethylene evolution from leaf blades and abscission zone explants was readily detectable within 24 hours of treatment and persisted until leaf fall. Ethylene evolution from treated leaf blades was greatest 1 day posttreatment and reached levels in excess of 600 nanoliters per gram fresh weight per hour (26.7 nanomoles per gram fresh weight per hour). The increase in ethylene evolution occurred in the absence of increased ethane evolution, altered leaf water potential, or decreased chlorophyll levels. Treatment of seedlings with inhibitors of ethylene action (silver thiosulfate, hypobaric pressure) or ethylene synthesis (aminoethoxyvinylglycine) resulted in an inhibition of thidiazuron-induced defoliation. Application of exogenous ethylene or 1 aminocyclopropane-1-carboxylic acid largely restored the thidiazuron response. The results indicate that thidiazuron-induced leaf abscission is mediated, at least in part, by an increase in endogenous ethylene evolution. However, alterations of other phytohormone systems thought to be involved in regulating leaf abscission are not excluded by these studies. PMID- 16664230 TI - Anaerobic Formation of d-Lactate and Partial Purification and Characterization of a Pyruvate Reductase from Chlamydomonas reinhardtii. AB - d-Lactate accumulation in Chlamydomonas reinhardtii was dependent on anaerobic conditions. As much as 50% of the (14)C after 2 minutes of photosynthetic (14)CO(2) fixation moved into d-lactate from sugar phosphates if the cells became anaerobic for short time periods. No lactate accumulated in the dark until the O(2) concentration decreased to less than 0.1%. Lactate was determined to be of the d-configuration using stereospecific lactate dehydrogenases. d-Lactate produced anaerobically by algae grown on 5% CO(2) was only slowly metabolized aerobically in the light or dark, and in the dark, only a trace of the lactate was excreted.A pyruvate reductase (d-lactate: diphosphopyridine nucleotide oxidoreductase, EC 1.1.1.28) was partially purified 47-fold from Chlamydomonas. Because this enzyme catalyzes an essentially irreversible reaction in the direction of pyruvate reduction, it is considered to be a pyruvate reductase. The reductase activity in extracts of Chlamydomonas was 30 micromoles per hour per milligram chlorophyll. For the partially purified enzyme, the apparent K(m) (pyruvate) was 0.5 millimolar, and the pH optimum was 7.0. Studies with cycloheximide and chloramphenicol indicated that the enzyme was constitutive in aerobic cells. Potassium phosphate stimulated the reductase, and high salt and dithiothreitol were required for stability. The enzyme demonstrated substrate inhibition and was inhibited by ATP. Pyruvate reductase was separated from a hydroxypyruvate reductase by gel filtration chromatography, indicating the presence of separate reductases for these two substrates in Chlamydomonas. PMID- 16664231 TI - Subcellular and Developmental Distribution of beta-Cyanoalanine Synthase in Barley Leaves. AB - The subcellular and developmental distribution of beta-cyanoalanine synthase (EC 4.4.1.9), which catalyzes the reaction between cysteine and HCN to form beta cyanoalanine and H(2)S, were investigated in barley (Hordeum vulgare) leaves. Total leaf activity was 1.1 micromoles per minute per gram fresh weight. Sucrose density gradients of lysed mesophyll protoplasts of barley revealed the exclusive or predominant localization of beta-cyanoalanine synthase in the mitochondria. The enzyme was absent from both vacuole and chloroplast fractions.beta Cyanoalanine synthase activity was distributed over the entire length of the barley leaf. Activity was dependent on the developmental stage, with a 3.5-fold higher activity in the oldest (apical) compared to the youngest (basal) parts of the leaf. The corresponding difference in activity for mesophyll protoplasts isolated from these parts was 7.5-fold. In younger leaf seagments, the nonchlorophyllous tissues accounted for up to 70% of the total beta-cyanoalanine synthase activity. These results are discussed with reference to the formation of HCN as a substrate in barley leaves. PMID- 16664232 TI - Stereospecificity of the glucose carrier in sugar beet suspension cells. AB - The stereospecificity of the binding site on the glucose carrier system in sugar beet suspension culture cells was determined using a series of aldo and keto hexose sugars and sugar alcohols. Specificity was determined as competition with [(14)C]glucose transport and glucose/proton symport.The binding site of the glucose carrier system was specific for the stereo orientation of the three equatorial OH groups on the three carbons opposite the oxygen and for the CH(2)OH group. Hexopyranose isomers with the same orientation at the three OH groups (carbons 2, 3, and 4 of C-1 d-glucose), but not with the CH(2)OH group, have only little (1-C d-glucose) or no effect (1-C d-idose and myoinositol) on d-glucose uptake. The C-1 l-sorbose molecule matches the C-1 d-glucose at many points including the stereo configuration of the CH(2)OH group, but it had no effect on d-glucose uptake perhaps because of an interference of the OH group adjacent to the CH(2)OH substituent. The d-glucose analogs, 3-O-methylglucose and glucosamine, were the most effective in binding to the glucose carrier. The isomers d-fructose, d-galactose, and d-mannose have separate distinctive proton cotransport systems. However, in starved cells they compete with d-glucose uptake, but the competition is for the available energy and not the carrier binding site. PMID- 16664233 TI - Chlorophyll-Proteins and Electron Transport during Iron Nutrition-Mediated Chloroplast Development. AB - Chlorophyll-protein complexes and electron transport activities were measured during iron nutrition-mediated chloroplast development in sugar beet (Beta vulgaris L. cv F58-554H1). Results showed that the chlorophyll-protein complexes associated with the reaction centers of photosystem I (CP1) and photosystem II (CPa) and the electron transport activities of these two photosystems per leaf area increased rapidly during the first 24 to 48 hours of iron resupply to iron deficient sugar beet plants. Bulk chlorophyll and the amounts of light-harvesting chlorophyll-proteins increased after a lag period of 24 hours. The changes in chlorophyll-proteins with time were apparently the cause of an initial increase, then decrease, in the chlorophyll a/b ratio during iron resupply. There was evidence that iron deficiency diminished photosystem I more than photosystem II. We propose that there are two distinct phases in iron nutrition-mediated chloroplast development: (a) the commencement of the synthesis of the lipid matrix of the thylakoid membrane, including a fully functioning electron transport (and photosynthetic) system, during the first 24 hours of iron resupply; and (b) after 24 to 48 hours, the formation of the bulk of the thylakoid proteins, including the light-harvesting chlorophyll-proteins with which the large increase in total chlorophyll is associated. PMID- 16664234 TI - Brassinosteroid-induced epinasty in tomato plants. AB - The effects of root treatments of brassinosteroid (BR) on the growth and development of hydroponically grown tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were evaluated. There was a dramatic increase in petiole bending when the plants were treated with 0.5 to 1.0 micromolar BR. The leaf angle of the treated plants was almost three times that of untreated controls. BR-induced epinasty appeared to be due to stimulation of ethylene production. Excised petioles from BR-treated plants produced more than twice as much ethylene as did untreated controls. As ethylene production increased, the degree of petiole bending also increased, and inhibition of ethylene production by AOA or CoCl(2) also inhibited epinasty. BR-treated plants had increased levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaf tissue. ACC appeared to accumulate primarily in the petioles with the greatest amount of ACC accumulating in the youngest petioles. Time course evaluations revealed that BR treatment stimulated ACC production. As ACC accumulated, ethylene increased, resulting in epinasty. Little or no ACC was found in the xylem sap, indicating that there was a signal transported from the roots which stimulated ACC synthesis in the leaf tissue. PMID- 16664235 TI - Studies on N-glycosylation by elongating tissues and membranes from pea stems. AB - Glucosamine and mannose were incorporated into oligosaccharides linked to either polar membrane-lipids or to asparagine residues of endogenous proteins in apical growing tissues of the etiolated pea stem. The glycolipids were subject to turnover in pulse-chase tests and protein-linked oligosaccharides accumulated with time, as expected for a precursor-product relationship. The newly formed glycoproteins were hydrolyzed by endo-beta-N-acetylglucosaminidase H to oligosaccharides in the same size range as those released by dilute acid from the lipid-linked oligosaccharides formed during the pulse. The glycoproteins were also partly degraded to free N-acetylglucosamine by beta-N-acetylhexosaminidase. Affinity of the carbohydrate moiety of the protein for concanavalin A increased between the beginning and the end of the chase, indicating processing following core glycosylation.The addition of UDP-N-acetyl-[(14)C]glucosamine plus external peptide acceptors (derived from carboxymethylated alpha-lactalbumin) to membrane preparations from the pea stem resulted in peptide glycosylation at the expense of lipid-linked oligosaccharide. Glycosylation of endogenous protein acceptors did not take place via lipid intermediates but directly from the sugar nucleotide substrate. Tunicamycin inhibited glycosyltransfer to both glycolipids and added peptides, but not to endogenous protein. It is concluded that limiting factors for N-glycosylation by pea membranes in vitro could include the unavailability of endogenous acceptors or the inability to fully elongate and internalize lipid precursors, but is not due to any limitation in capacity for N-glycosylation. PMID- 16664236 TI - Differential expression of kunitz and bowman-birk soybean proteinase inhibitors in plant and callus tissue. AB - Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred. PMID- 16664237 TI - Metabolism of Oat Leaves during Senescence : VIII. The Role of L-Serine in Modifying Senescence. AB - The mechanism whereby l-serine specifically promotes the dark senescence of detached oat (Avena) leaves has been examined. The fact that this promotion is strong in darkness but very weak in white light has been explained, at least in part, by the finding that added serine is partly converted to reducing sugars in light. Labeled serine gives rise to (14)C-sugars and (14)CO(2). In the absence of CO(2), serine does cause chlorophyll loss in light and undergoes a decreased conversion to sugar.As to the large promotion of protease activity which accompanies senescence in the dark, reported earlier, careful purification of the proteases shows that the l-[(14)C]serine is not incorporated into these enzymes, although it is incorporated into the total protein. Cycloheximide decreases the overall synthesis both of protease and of total protein, but again [(14)C]serine does not impart radioactivity to the purified acid proteases. Even when serine is simply added to the protease assay the proteolysis is significantly increased. It is concluded that serine promotes the protease activity by synergizing with the enzyme, or by activating an apoenzyme. PMID- 16664238 TI - Cyclic variations in nitrogen uptake rate in soybean plants. AB - Uptake of NO(3) (-) by nonnodulated soybean plants (Glycine max L. Merr. cv Ransom) growing in flowing hydroponic culture at 22 and 14 degrees C root temperatures was measured daily during a 31-day growth period. Ion chromatography was used to determine removal of NO(3) (-) from solution during each 24-hour period. At both root-zone temperatures, rate of NO(3) (-) uptake per plant oscillated with a periodicity of 3 to 5 days. The rate of NO(3) (-) uptake per plant was consistently lower at 14 degrees C than 22 degrees C. The lower rate of NO(3) (-) uptake at 14 degrees C during the initial 5 to 10 days was caused by reduced uptake rates per gram root dry weight, but with time uptake rates per gram root became equal at 14 and 22 degrees C. Thereafter, the continued reduction in rate of NO(3) (-) uptake per plant at 14 degrees C was attributable to slower root growth. PMID- 16664239 TI - Partial purification of a tonoplast ATPase from corn coleoptiles. AB - The tonoplast ATPase from corn coleoptile membranes was solubilized using a two step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO(3) with an estimated K(i) of 10 millimolar. The specific activity of the KNO(3)-sensitive ATPase was increased 29 fold during purification. N,N'-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl(-) and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000 dalton proteins. PMID- 16664240 TI - Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves. AB - In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of (15)N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of alpha-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process. PMID- 16664241 TI - Effects of fungal elicitor on lignin biosynthesis in cell suspension cultures of soybean. AB - Soybean (Glycine max L.) cells cultured in B5 medium produce extremely low amounts of lignin. However, modification in the growth medium, by lowering the concentration of NO(-) (3) and PO(2-) (4), results in the lignification of these cells without affecting levels of cell wall-esterified 4-coumaric and ferulic acid. The production of an extracellular, macromolecular complex by the cultured soybean cells (Moore TS Jr 1973 Plant Physiol 51: 529-536) allows a rapid, nondestructive solubilization of the lignin which can be estimated by reaction with phloroglucinol in free solution. This system has been used to study the effects of fungal elicitor on the synthesis of lignin in soybean cells. The inclusion of very low levels of an elicitor fraction from the cell walls of Phytophthora megasperma in the medium in which lignification of the soybean cells occurs suppressed both the accumulation of extracellular lignin and phloroglucinol staining of the cell walls without affecting the levels of bound hydroxycinnamic acids. The activity profiles of phenylalanine ammonia-lyase (EC 4.3.1.5) and isoenzymes of 4-coumarate:CoA ligase (EC 6.2.1.12) were compared in lignifying and elicitor-treated cell cultures as was the activity of chalcone synthase, an enzyme of flavonoid biosynthesis. The measured activities of these enzymes in cell cultures treated with elicitor were considerably lower than in untreated cells. PMID- 16664242 TI - A simple method for estimating intactness of spinach leaf protoplasts by glycolate oxidase assay. AB - A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O(2) consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle. PMID- 16664243 TI - Cell wall yield properties of growing tissue : evaluation by in vivo stress relaxation. AB - Growing pea stem tissue, when isolated from an external supply of water, undegoes stress relaxation because of continued loosening of the cell wall. A theoretical analysis is presented to show that such stress relaxation should result in an exponential decrease in turgor pressure down to the yield threshold (Y), with a rate constant given by phiepsilon where phi is the metabolically maintained irreversible extensibility of the cell wall and epsilon is the volumetric elastic modulus of the cell. This theory represents a new method to determine phi in growing tissues.Stress relaxation was measured in pea (Pisum sativus L.) stem segments using the pressure microprobe technique. From the rate of stress relaxation, phi of segments pretreated with water was calculated to be 0.08 per megapascal per hour while that of auxin-pretreated tissue was 0.24 per megapascal per hour. These values agreed closely with estimates of phi made by a steady state technique. The yield threshold (0.29 megapascal) was not affected by auxin. Technical difficulties with measuring phi by stress relaxation may arise due to an internal water reserve or due to changes in phi subsequent to excision. These difficulties are discussed and evaluated.A theoretical analysis is also presented to show that the tissue hydraulic conductance may be estimated from the T((1/2)) of tissue swelling. Experimentally, pea stems had a swelling T((1/2)) of 2.0 minutes, corresponding to a relative hydraulic conductance of about 2.0 per megapascal per hour. This value is at least 8 times larger than phi. From these data and from computer modeling, it appears that the radial gradient in water potential which sustains water uptake in growing pea segments is small (0.04 megapascal). This means that hydraulic conductance does not substantially restrict growth. The results also demonstrate that the stimulation of growth by auxin can be entirely accounted for by the change in phi. PMID- 16664244 TI - Superoxide Dismutase from Lens esculenta: Purification and Properties. AB - Superoxide dismutase has been purified to homogeneity from Lens esculenta cotyledons and shoots. The two forms appeared to be identical. The purified enzyme contained two electrophoretically distinct bands. It contained two ions of Cu and two ions of Zn. Gel filtration experiments indicate a molecular weight of about 33,000. The spectrum of ultraviolet and visible regions and electron paramagnetic resonance were similar to those of Cu-Zn mammalian superoxide dismutase. PMID- 16664245 TI - Carbon Transport and Root Respiration of Split Root Systems of Phaseolus vulgaris Subjected to Short Term Localized Anoxia. AB - The influence of anoxia on carbon transport and root respiration was evaluated by applying [U-(14)C]sucrose to the foliage. Translocation patterns to the root systems of two dry edible bean genotypes (Phaseolus vulgaris L.) were examined after a 3-day exposure to aerated and nonaerated environments. Localized anoxia of root systems was simulated by growing roots in split configurations and exposing half of the system to anoxic conditions. Anoxia of the root system for 72 hours reduced the movement of (14)C label into the roots with concurrent accumulations in the hypocotyl region. The translocation of (14)C label to anoxic roots was less than 50% of the aerated controls of both genotypes. Most of the (14)C label translocated to anoxic root systems was excluded from respiratory metabolism during the 3-hour pulse/chase period and was an order of magnitude less than the aerated controls. These observations suggest that the bulk of (14)C label which entered the root during the anoxic period was unavailable for metabolism by the enzymes of glycolysis and/or was diluted by a relatively large metabolite pool. A higher percentage of (14)C label was translocated to the aerated half of the localized anoxia treatment relative to the half of the aerated controls. The proportion of (14)C label translocated to the root system in the aerated control was 20 and 16% compared to 28 and 25% in the aerated localized anoxia treatment for the genotypes Seafarer and line 31908, respectively. Line 31908 partitioned a greater percentage of (14)C-labeled compounds to the actively growing fraction of the root system in the localized anoxia treatment than did Seafarer. This suggests a greater reliance on previously stored carbohydrate for immediate root growth in Seafarer than in line 31908. PMID- 16664246 TI - Content of adenine nucleotides and orthophosphate in exporting and importing mature maize leaves. AB - Events of reactivation by re-illumination were studied in predarkened detached mature maize leaves, which were arranged as distal sources and proximal sinks; the latter were kept in CO(2)-free atmosphere and were either illuminated or darkened. Adenine nucleotide (AdN) content and orthophosphate (Pi) concentrations were measured 10 minutes, 30 minutes, and 2, 7, and 14 hours after the onset of re-illumination. For comparison, mature leaves attached to the plant were analyzed.The sum of AdN increased up to 7 hours of re-illumination, then dark sinks and their sources showed decreasing amounts of AdN, while the increase continued up to 14 hours in sources and illuminated sinks. In leaves attached to the plant, no further increase in AdN level followed the 7-hour mark. The amount of individual AdN (ATP, ADP, AMP) differed considerably in sources and sinks of the detached leaves. Although both the source supplying the illuminated sink and the source supplying the dark sink were treated the same, they showed striking differences in AdN contents. Such relations were also observed, when ATP/ADP ratios and Pi concentrations were compared. The influence a sink can exert on its source suggests a participation of the physiological events in the sink on the regulation of AdN and Pi metabolism in the source. PMID- 16664247 TI - Photocontrol of Peanut (Arachis hypogaea L.) Embryo and Ovule Development in Vitro. AB - The purpose of the present study was to determine the nature of the photoreceptor that controls the light-dependent development of the peanut ovule and embryo, and to determine whether this photoreceptor activity is located in the ovular or the embryonic tissue. Peanut ovules were aseptically excised from surface sterilized gynophores (pegs) and cultured in darkness, continuous far red (FR), or given 15 minutes daily irradiations with white (W), red (R), blue (B); FR, R/FR, FR/R light for 21 days. Increases in the volume of ovules cultured in dark, continuous FR, FR, or R/FR were significantly greater than for ovules cultured in W, R, FR/R, or B radiation. Halving the ovule or removing the micropyle did not affect the relative change in ovule volume in response to different light treatments. When the light sources were altered after the first 10 days of culture, the ovules exposed first to darkness or FR radiation began to increase in ovule volume which then ceased after subsequent exposure to W, R, or B radiation. Likewise, if W, R, or B radiation was given during the first 10 days, ovule development was inhibited, and could be stimulated by exposure to darkness or FR light. Growth of embryos, when removed from the influence exerted by ovular tissue, was unaffected by the light. However, when embryos remained attached to the ovular tissue, their growth was stimulated in darkness or by FR light and inhibited by W, R, or B light. The R/FR reversibility indicates that the photoreceptor that regulates ovular growth is phytochrome and that the maternal ovular tissue appears to be the site of photoreception, which may then transmit some developmental signal to the embryo. PMID- 16664248 TI - An electron transport system in maize roots for reactions of glutamate synthase and nitrite reductase : physiological and immunochemical properties of the electron carrier and pyridine nucleotide reductase. AB - A non-heme iron containing protein which bears an antigenic similarity to ferredoxin from spinach leaves (Spinacia oleracea L.) has been identified in extracts prepared from young roots of maize (Zea mays L., hybrid W64A x W182E). The ferredoxin-like root electron carrier could substitute for ferredoxin in a cytochrome c reduction system in which pyridine nucleotide (NADPH) reduces the root electron carrier in a reaction catalyzed by ferredoxin-NADP(+) reductase (EC 1.6.7.1) from spinach leaves. However, the root electron carrier did not mediate the photoreduction of NADP(+) in an illuminated reconstituted chloroplast system.A pyridine nucleotide reductase which shares identical immunological determinants with the ferredoxin-NADP(+) reductase from spinach leaves has also been characterized from maize roots. Root pyridine nucleotide reductase mediated the transfer of electrons from either NADPH or NADH to cytochrome c via ferredoxin or the root electron carrier. Under chemical reducing conditions with sodium dithionite and bicarbonate, the ferredoxin-like root electron carrier served as an electron carrier for the ferredoxin-requiring glutamate synthase (EC 1.4.7.1) and nitrite reductase (EC 1.7.7.1) obtained from maize roots or leaves. In the presence of root pyridine nucleotide reductase and root electron carrier, either NADPH or NADH served as the primary electron donor for glutamate synthesis in extracts from maize roots or leaves. The electron transport system originating with NADH or NADPH, was, however, not able to mediate the reduction of NO(2) (-) to NH(3). PMID- 16664249 TI - Isolation and Partial Characterization of a New Lectin from Seeds of the Greater Celandine (Chelidonium majus). AB - Seeds of the greater celandine (Chelidonium majus L.) contain a lectin which could be isolated using a combination of affinity chromatography on chitin and ion exchange chromatography on sulphopropyl-Sephadex. The purified lectin was partially characterized with respect to its biochemical and physicochemical properties. It is a small dimeric protein composed of two different subunits of M(r) 9,500 and 11,500, respectively. Its amino acid composition is typified by high contents of glycine and cysteine. No covalently bound carbohydrate could be detected. Hapten inhibition experiments indicated that the lectin exhibits specificity towards oligomers of N-acetylglucosamine, the potency of inhibition increasing with chain length up to four residues. The greater celandine lectin is the first lectin to be isolated from a species belonging to the plant family Papaveraceae (poppy family). Although it represents a new type of plant lectin, resemblances to phytohemaglutinins from diverse taxonomic origin are obvious. PMID- 16664250 TI - Lectin in Vegetative Tissues of Adult Barley Plants Grown under Field Conditions. AB - A study of the distribution of lectins over different vegetative tissues of barley (Hordeum vulgare L.) plants, which were grown under normal crop conditions, indicated that lectin occurs in roots, leaves, and developing ears. Isolation and characterization of both root and leaf lectins led to the conclusions (a) that they are indistinguishable from the embryo lectin and (b) that the total lectin content of these vegetative organs is many times higher than that of the embryo. Finally, in vivo labeling experiments demonstrated that the lectin is synthesized de novo in roots and leaves. PMID- 16664251 TI - Phytochrome control of specific mRNA levels in developing pea buds : the presence of both very low fluence and low fluence responses. AB - We have examined phytochrome regulated changes in transcript abundance for 11 different light regulated mRNAs in developing pea buds. Fluence-response curves were measured for changes in transcript abundance in response to red light pulses in both the low and very low fluence ranges. Most transcripts show only low fluence responses, with a threshold of approximately 10 micromoles per square meter. All of the low fluence responses are reversible by far red light. One transcript shows a very low fluence response, with a threshold of approximately 10(-4) micromoles per square meter. As expected, the very low fluence response is not far red reversible and in fact can be induced by far red light.Various fluences of red light were also used as pretreatments before transferring seedlings to continuous white light. One transcript responds to pretreatments in the very low fluence range, several respond to pretreatments in the low fluence range (including chlorophyll a/b binding protein RNA and ribulose-1,5 bisphosphate carboxylase RNA), and several show no response to the red light under these conditions. The threshold of these low fluence responses is approximately 10(2) micromoles per square meter, one order of magnitude greater than the threshold of the low fluence responses to red light alone.The transcripts may also be grouped by their responses to white light treatment alone. Three of the clones correspond to transcripts whose abundance decreases after a 24 hour white light treatment. The remainder of the mRNAs increase between 2- and 10-fold in response to the 24 hour white light. PMID- 16664252 TI - Accumulation of heat shock proteins in field-grown cotton. AB - Cotton (Gossypium hirsutum L.) plants grown under field water deficits exhibited an 80 to 85% reduction in leaf area index, plant height, and dry matter accumulation compared with irrigated controls. Midday photosynthetic rates of dryland plants decreased 2-fold, and canopy temperatures increased to 40 degrees C at 80 days after planting compared with canopy temperatures of 30 degrees C for irrigated plants. Leaves from dryland plants which had exhibited canopy temperatures of 40 degrees C for several weeks accumulated stainable levels of polypeptides with apparent molecular weights of 100, 94, 89, 75, 60, 58, 37, and 21 kilodaltons. These polypeptides did not accumulate in leaves from irrigated plants.Addition of [(35)S]methionine to leaves of growth chamber-grown cotton plants and subsequent incubation at 40 degrees C for 3 hours radiolabeled polypeptides with molecular weights similar to those that accumulate in dryland cotton leaves. These data suggest that the proteins which accumulate in water stressed cotton leaves at elevated temperatures (40 degrees C) are heat shock proteins and that these proteins can accumulate to substantial levels in field stressed plants. PMID- 16664253 TI - Photosynthetic gene expression and cellular differentiation in developing maize leaves. AB - We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits of the two CO(2) fixing enzymes and the major chlorophyll-binding protein. These three proteins are differentially compartmentalized in the two photosynthetically active cell types of the leaf. Previous studies have shown that accumulation of the two carboxylases commences 2 to 4 cm from the base of the leaf (Mayfield SP, WC Taylor Planta 161: 481-486) at a position where bundle sheath and mesophyll cells show morphological evidence of maturation. The light-harvesting chlorophyll a/b protein accumulates progressively from the leaf base, as does its mRNA, in spite of its localization in mesophyll cells after cellular differentiation occurs. While small quantities of phosphoenolpyruvate carboxylase mRNA are detectable in the basal region of the leaf, significant mRNA accumulation is coincident with that of the polypeptide at 4 to 6 cm from the leaf base, the region where bundle sheath and mesophyll cells exhibit fully differentiated morphologies. mRNAs encoding the small and large subunits of ribulose 1,5-bisphosphate carboxylase accumulate to significant levels before bundle sheath cells are fully differentiated and before their polypeptides are detectable. Cytological examination indicates that this is the position at which the maturation of intermediate vascular bundles is first evident. Cytosolically localized small subunit mRNA and chloroplast-localized large subunit mRNA are complexed with polyribosomes at all positions of the leaf. PMID- 16664254 TI - Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD. AB - A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD(+) at 25 degrees C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl(2), and 5 millimolar alpha-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD(+) efflux was observed instead. Furthermore, the rate of NAD(+) accumulation in the matrix space was strongly inhibited by the analog N-4 azido-2-nitrophenyl-4-aminobutyryl-3'-NAD(+). When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD(+) content. This led to a slow decrease of NAD(+)-linked substrates oxidation by isolated mitochondria The rate of NAD(+) efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD(+) transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size. PMID- 16664255 TI - Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants. AB - From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 x 10( 8). Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance. PMID- 16664256 TI - Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.). AB - Gibberellins (GAs) A(1), A(5), and A(29) were identified, and also GA(32) was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25 0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning --> paper chromatography --> reverse phase C(18) high performance liquid chromatography (HPLC) --> bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C(18) HPLC at Rts where GA(4/7) and GA(8) (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA(32) (112 ng of GA(3) equivalents per gram fresh weight), and minor amounts (0.5 ng of GA(3) equivalents) for each of GA(1) and GA(5), respectively, based on the microdrop assay. PMID- 16664257 TI - Photosynthetic Activity in the Flower Buds of ;Valencia' Orange (Citrus sinensis [L.] Osbeck). AB - Flower buds of ;Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix (14)CO(2) into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total (14)C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the (14)C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO(2) per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO(2) into different compounds suggests that this CO(2) assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting. PMID- 16664258 TI - Recognition of Superior Photosynthetic Efficiency in the Field Using the CO(2) Depletion Technique. AB - The relationship between CO(2) exchange rate (CER) and growth of crops in the field was investigated in Connecticut Broadleaf tobacco (Nicotiana tabacum) using the CO(2)-depletion technique. A particular objective was to determine if modest (i.e. <10%) varietal differences could be distinguished in mean CER. Statistical analysis of numerous CER values obtained over a wide range of irradiances during the course of the season indicated that differences of as little as 7% in the mean CER between varieties would be significant (n approximately 400). The usefulness of the CO(2)-depletion technique in detecting modest differences in photosynthetic efficiency has thus been demonstrated. These results are discussed in relation to the prospects for introducing and detecting genetic traits which would diminish photorespiration and increase CER and growth. PMID- 16664259 TI - Stable-carbon isotopic composition of maple sap and foliage. AB - The (13)C/(12)C ratios of Acer grandidentatum sap sugar collected during the dormant period are compared to those of buds, leaves, and wood developed over the following growing season. As the primary carbon source for cellulose manufacture at initiation of annual growth in deciduous trees, sap sucrose would be expected to have an isotopic composition similar to first-formed cellulose. Although constancy in concentration and (13)C/(12)C ratios of the maple sap sugar suggests any gains or losses (e.g. to maintenance metabolism) do not appreciably alter composition, the (13)C/(12)C ratios of cellulose of the enlarging buds in the spring are quite distinct from those of the sap sugar, seemingly precluding a simple direct biochemical pathway of sap sucrose-->glucose-->cellulose in favor of a more complex pathway with greater likelihood of isotopic fractionation. The (13)C/(12)C ratios of the leaves and in the growth ring were initially similar to the sap sugar but decreased steadily over the growing season. PMID- 16664260 TI - Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells. AB - Suspension cultures of carrot (Daucus carota, line C1), tobacco (Nicotiana tabacum, line TX1), and Nicotiana plumbaginifolia (line NP) were frozen under controlled conditions with trehalose as the sole cryoprotectant. Maximal post thaw viability (71-74%), measured by phenosafranin dye exclusion, was obtained with the C1 cells following a 24hour pretreatment with 5 or 10% trehalose and with 40% trehalose as the cryoprotectant during freezing. TX1 cells pretreated for 24 hours with 10% trehalose and cryoprotected with 40% trehalose during freezing showed 47% viability following thawing as determined by phenosafranin dye exclusion. The NP cells required a 3 to 6 day pretreatment with 10% trehalose and 40% trehalose as a cryoprotectant at the time of freezing for the recovery of viable cells. Growing cells were recovered when the C1 and NP cells treated as described were plated on agar-solidified medium following thawing. PMID- 16664261 TI - Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa. AB - We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b(5) or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum. PMID- 16664262 TI - Tyrosine and Phenylalanine Ammonia Lyase Activities during Shoot Initiation in Tobacco Callus Cultures. AB - Both phenylalanine ammonia lyase and tyrosine ammonia lyase were detected in tobacco (Nicotiana tabacum L. Wisconsin 38) callus. The enzymes were separated from each other by Sephadex G-200 column chromatography. Increased activity of tyrosine ammonia lyase was observed during culture of tobacco callus under shoot forming conditions, while activity of phenylalanine ammonia lyase increased during culture under non-organ-forming conditions. Confirmation of these findings was obtained by examining the incorporation of [(14)C]tyrosine and [(14)C]phenylalanine into p-coumarate and trans-cinnamate, respectively. PMID- 16664263 TI - Differential Induction of Endoproteinases during Senescence of Attached and Detached Barley Leaves. AB - Endoproteinase activities and species were compared during dark-induced senescence of attached and detached primary barley leaves by isoelectric focusing and polyacrylamide gel electrophoresis of cell-free extracts. Neither of the two major endoproteinases (EP1 and EP2) changed in amounts during senescence of attached leaves, nor did new endoproteinases appear. In contrast, during senescence of detached leaves, both EP1 and EP2 activities increased and four new species of endoproteinases appeared. Proteolytic activity was evenly distributed throughout attached leaves, but activity in the detached leaf increased sharply from the tip to the base with the four new higher molecular weight species of proteinases present only in the bottom half of the leaf nearest the cut end. Thus a wound response may be superimposed on the processes of senescence in detached leaves. Cycloheximide and kinetin both inhibited the increase of EP1, EP2, and the induction of the four new endoproteinases; chloramphenicol had no effect. Indications are that both the increases in activity and the induction of new species of proteinases were the result of activity of cytoplasmic ribosomes.Hydrolysis of total protein and ribulose-1,5-bisphosphate carboxylase protein in vivo was somewhat faster in detached than attached leaves. The difference, however, was much less than would be expected from the great increase in proteolytic activity in detached leaves. PMID- 16664264 TI - Metabolism of tryptophol in higher and lower plants. AB - Bacteria, thallophytes, and seed plants (107 species), supplied with exogenous indole-3-ethanol (tryptophol), formed one or more of the following metabolites: O acetyl tryptophol, an unknown tryptophol ester (or a set of structurally closely related esters), tryptophol glucoside, tryptophol galactoside, indole-3-acetic acid (IAA), and indole-3-carboxylic acid. The unknown ester was formed by all species examined; O-acetyl tryptophol appeared sporadically in representatives of most major taxonomic groups. Tryptophol galactoside was found in the algae Chlorella, Euglena, and Ochromonas. The glucoside was formed by many eucaryotic plants, but not by bacteria; it was a significant tryptophol metabolite in vascular plants. IAA, if detectable at all, was usually a minor metabolite, as should be expected, if tryptophol oxidase responds to feedback inhibition by IAA. Indole-3-carboxylic acid, formed by a few fungi and mosses, was the only tryptophol metabolite detected which is likely to be formed via IAA. PMID- 16664265 TI - Effect of Nitrate and Ammonium Nutrition of Nonnodulated Phaseolus vulgaris L. on Phosphoenolpyruvate Carboxylase and Pyruvate Kinase Activity. AB - Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with 3.5 or 10 millimolar N in either the form of NO(3) (-) or NH(4) (+), after being grown on N free nutrient solution for 8 days. The pH of the nutrient solutions was either 6 or 4. The cell sap pH and the extractable activities of phosphoenolpyruvate carboxylase and of pyruvate kinase from roots and primary leaves were measured over several days.The extractable activity of phosphoenolpyruvate carboxylase (based on soluble protein) from primary leaves increased with NO(3) (-) nutrition, whereas with NH(4) (+) nutrition and on N-free nutrient solution the activity remained at a low level. Phosphoenopyruvate carboxylase activity from the roots of NH(4) (+)-fed plants at pH 4 was finally somewhat higher than from the roots of plants grown on NO(3) (-) at the same pH. There was no difference in activity from the root between the N treatments when pH in the nutrient solutions was 6. The extractable activity of pyruvate kinase from roots and primary leaves seemed not to be influenced by the N nutrition of the plants.The results are discussed in relation to the physiological function of both enzymes with special regard to the postulated functions of phosphoenolpyruvate carboxylase in C3 plants as an anaplerotic enzyme and as part of a cellular pH stat. PMID- 16664266 TI - Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane. AB - The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1 acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts. PMID- 16664267 TI - Auxin and Fusicoccin Enhancement of beta-Glucan Synthase in Peas : An Intracellular Enzyme Activity Apparently Modulated by Proton Extrusion. AB - Fusicoccin (FC), like indoleacetic acid (IAA), causes Golgi-localized beta-1,4 glucan synthase (GS) activity to increase when applied to pea third internode segments whose GS activity has declined after isolation from the plant. This suggests that GS activity is modulated by H(+) extrusion; in agreement, vanadate and nigericin inhibit the GS response. The GS response is not due to acidification of the cell wall. Treatment of tissue with heavy water, which in effect raises intracellular pH, mimics the IAA/FC GS response. However, various treatments that tend to raise cytoplasmic pH directly, other than IAA- or FC induced H(+) extrusion, failed to increase GS activity, suggesting that cytoplasmic pH is not the link between H(+) extrusion and increased GS activity. Although FC stimulates H(+) extrusion more strongly than IAA does, FC enhances GS activity at most only as much as, and often somewhat less than, IAA does. This and other observations indicate that GS enhancement is probably not due to membrane hyperpolarization, stimulated sugar uptake, or changes in ATP level, but leave open the possibility that GS is controlled by H(+) transport-driven changes in intracellular concentrations of ions other than H(+). PMID- 16664268 TI - Identification of Endogenous Gibberellins from Salix pentandra. AB - Gibberellins A(1), A(19), A(20), and A(29) have been identified by sequential high-performance liquid chromatography retention time (Rt) and combined gas chromatography-mass spectrometry (Rt and characteristic mass spectra) from elongating shoots of Salix pentandra L. Gibberellins A(1) and A(19) were also detected in purified extracts from male and female flowers (catkins) of S. pentandra. PMID- 16664269 TI - Amino Acid sequence of a novel calmodulin from the unicellular alga chlamydomonas. AB - An amino acid sequence for a Chlamydomonas calmodulin has been elucidated with emphasis on the characterization of differences that are unique to Chlamydomonas and Dictyostelium calmodulin. While the concentration of calmodulin required for half-maximal activation of plant NAD kinase varies among vertebrate, higher plant, algal, and slime mold calmodulins, only calmodulins from the unicellular alga Chlamydomonas and the slime mold Dictyostelium show increased maximal activation of NAD kinase (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796 798; Marshak, Clarke, Roberts, Watterson 1984 Biochemistry 23: 2891-2899). The same preparations of calmodulin do not show major differences in phosphodiesterase or myosin light chain kinase activator activity.We report here that a Chlamydomonas calmodulin has four primary structural features similar to Dictyostelium that are not found in other calmodulins characterized to date: an altered carboxy terminus including a novel 11-residue extension for Chlamydomonas calmodulin, unique residues at positions 81 and 118, and an unmethylated lysine at position 115. The only amino acid sequence identity unique to Chlamydomonas and Dictyostelium calmodulin is the presence of a lysine at position 115 instead of a trimethyllysine. These studies indicate that the methylation state of lysine 115 may be important in the maximal NAD kinase activator activity of calmodulin and support the concept that calmodulin has multiple functional domains in addition to multiple structural domains. PMID- 16664270 TI - Effect of low temperature and calcium on survival and membrane properties of isolated winter wheat cells. AB - Isolated cells obtained by enzymic digestion of young primary leaves of cold hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of (86)Rb was generally decreased by increasing concentrations of Ca(2+), but after longer periods of incubation, the inhibiting effect of high Ca(2+) levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at -1 degrees C, while in the presence of 10 millimolar Ca(2+) viability declined only gradually over a 5-week period. Ice encasement markedly reduced (86)Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from -1 to 2 and -3 degrees C, but the presence of Ca(2+) in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from 10 to -30 degrees C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at -1 degrees C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane. PMID- 16664271 TI - Translucent Tissue Defects in Solanum tuberosum L: I. Alterations in Amyloplast Membrane Integrity, Enzyme Activities, Sugars, and Starch Content. AB - Kennebec (cv) potatoes randomly developed translucent areas in their centrally located pith-parenchymal cells during storage. These defective areas were characterized as having reduced starch concentration and increased levels of free sugars (i. e. sucrose and glucose) and inorganic phosphate. Electron micrographs of potato tubers stored at 10 degrees +/- 1 degrees C for 8 months indicated that the amyloplast membrane was still intact and continuous around starch granules in both normal and prematurely sweetened tissue. The total activities of phosphorylase and sucrose-6-P synthase were elevated 5.4- and 3.8-fold, respectively, in the defective tissue compared to healthy nonsweetened tubers while there were no significant differences in the levels of sucrose synthase, UDPglucose pyrophosphorylase, invertase, or alpha-amylase. Total and specific activities of acid phosphatase were only slightly elevated in translucent tissue but their increase was significant (P < 0.05, t test) over that seen in healthy tubers. The premature sweetening in storage may have been indirectly triggered by moisture and heat stress experienced during development. Translucency eventually led to physical deterioration of the tissue. PMID- 16664272 TI - H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products. AB - Two distinct membrane fractions containing H(+)-ATPase activity were prepared from red beet. One fraction contained a H(+)-ATPase activity that was inhibited by NO(3) (-) while the other contained a H(+)-ATPase inhibited by vanadate. We have previously proposed that these H(+)-ATPases are associated with tonoplast (NO(3) (-)-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP(2-), and Mg(2+) concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg(2+)) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP(2-) in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP(2-) complex.The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H(+)-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg(2+) could modulate ATPase activity at both the tonoplast and plasma membrane. PMID- 16664273 TI - Sodium and potassium fluxes and compartmentation in roots of atriplex and oat. AB - K(+) and Na(+) fluxes and ion content have been studied in roots of Atriplex nummularia Lindl. and Avena sativa L. cv Goodfield grown in 3 millimolar K(+) with or without 3 or 50 millimolar NaCl. Compartmental analysis was carried out with entire root systems under steady-state conditions.Increasing ambient Na(+) concentrations from 0 to 50 millimolar altered K(+), in Atriplex, as follows: slightly decreased the cytoplasmic content (Q(c)), the vacuolar content (Q(v)), and the plasma membrane influx and efflux. Xylem transport for K(+) decreased by 63% in Atriplex. For oat roots, similar increases in Na(+) altered K(+) parameters as follows: plasma membrane influx and efflux decreased by about 80%. Q(c) decreased by 65%, and xylem transport decreased by 91%. No change, however, was observed in Q(v) for K(+). Increasing ambient Na(+) resulted in higher (3 to 5-fold) Na(+) fluxes across the plasma membrane and in Q(c) of both species. In Atriplex, Na(+) fluxes across the tonoplast and Q(v) increased as external Na(+) was increased. In oat, however, no significant change was observed in Na(+) flux across the tonoplast or in Q(v) as external Na(+) was increased. In oat roots, Na(+) reduced K(+) uptake markedly; in Atriplex, this was not as pronounced. However, even at high Na(+) levels, the influx transport system at the plasma membrane of both species preferred K(+) over Na(+).Based upon the Ussing-Teorell equation, it was concluded that active inward transport of K(+) occurred across the plasma membrane, and passive movement of K(+) occurred across the tonoplast in both species. Na(+), in oat roots, was actively pumped out of the cytoplasm to the exterior, whereas, in Atriplex, Na(+) was passively distributed between the free space, cytoplasm, and vacuole. PMID- 16664274 TI - Localization of cytokinin biosynthetic sites in pea plants and carrot roots. AB - The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8 (14)C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-(14)C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N(6)-(Delta(2)-isopentyl_adenine-5'-monophosphate, 6-(4-hydroxy-3-methyl-2 butenyl-amino)-9-beta-ribofuranosylpurine-5'- monophosphate, N(6)-(Delta(2) isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta ribofuranosylpurine, N(6)-(Delta(2)-isopentenyl)adenine, and 6-(4-hydroxy-3 methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones. PMID- 16664275 TI - Photosynthetic Development of Anaerobically Grown Rice (Oryza sativa) after Exposure to Air. AB - During anaerobic germination, rice produces a coleoptile devoid of carotenoid and chlorophyll. Further development and greening of the shoot occur upon exposure of the seedlings to air. In this study, a comparison was made between anaerobically (N(2)) germinated rice, greened upon exposure to air, and air/dark (A/D) germinated seedlings, greened upon exposure to light. After exposure to air, N(2) grown seedlings had a 76-hour lag before net oxygen evolution occurred compared to a 6-hour lag for A/D-grown seedlings. After 98 h of greening, N(2)-grown seedlings reached a rate of oxygen evolution equivalent to that of A/D-grown seedlings after 24 hours. Chlorophyll and carotenoid content showed a similar lag, but did not reach the level found in A/D-grown seedlings even after 124 hours of exposure to air. RuBPcase activity also lagged in N(2)-grown seedlings, ultimately reaching greater values than in the ;greened' A/D-grown seedlings. Phosphoenolpyruvate carboxylase activity was constant and low in all treatments except for a transient increase after 24 hours of greening of the N(2)-grown seedlings. PMID- 16664276 TI - Seasonal Changes in the Photosynthetic Rate in Apple Trees : A Comparison between Fruiting and Nonfruiting Trees. AB - Seasonal changes in photosynthesis of apple trees (Malus domestica Borkh.) were monitored to examine the effect of source-sink interactions on photosynthesis and photorespiration. Elevated photosynthetic rates were observed during two periods of the growing season and correlated with the fruiting process. The first period of increased photosynthetic rates was during the bloom period, when spur leaves on flowering shoots exhibited up to 25% higher photosynthetic rates than vegetative spur leaves on a leaf area basis. CO(2) assimilation rates were also higher in fruiting trees than nonfruiting trees during the period of rapid fruit growth from July to September. Photorespiration, dark respiration, leaf resistance, and transpiration exhibited no seasonal changes which correlated to the presence or absence of fruit. These data represent the first comprehensive examination of the effects of flowering/fruit formation on photosynthesis and photorespiration in perennial plants. PMID- 16664277 TI - Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybeans. AB - The effects of drought stress on soybean nodule conductance and the maximum rate of acetylene reduction were studied with in situ experiments performed during two seasons and under differing field conditions. In both years drought resulted in decreased nodule conductances which could be detected as early as three days after water was withheld. The maximum rate of acetylene reduction was also decreased by drought and was highly correlated with nodule conductance (r = 0.95). Since nodule conductance is equal to the nodule surface area times the permeability, the relationship of these variables to both whole-plant and unit nodule nitrogenase activity was explored. Drought stress resulted in a decrease in nodule gas permeability followed by decreases in nodule surface area when drought was prolonged. Under all conditions studied acetylene reduction on a unit nodule surface area basis was highly correlated with nodule gas permeability (r = 0.92). A short-term oxygen enrichment study demonstrated nodule gas permeability may limit oxygen flux into both drought-stressed and well-watered nodules of these field-grown soybeans. PMID- 16664278 TI - Chloramphenicol stimulation of light harvesting chlorophyll protein complex accumulation in a chlorophyll B deficient wheat mutant. AB - As compared with normal wheat leaves, the chlorina wheat mutant, designated CD3, has a high chlorophyll a/b ratio and a deficiency in the light harvesting chlorophyll protein (LHCP) complex. Applications of 200 micrograms per milliliter of d-threo-chloramphenicol to etiolated seedlings decreased the chlorophyll a/b ratio and increased the accumulation of the 27 kilodalton LHCP polypeptide and the LHCP complex in thylakoids of the mutant during greening. These data led to the suggestion that a protein encoded in chloroplast genes impaired either transcriptional, translational, or posttranslational events in CD3 wheat limiting the accumulation of the LHCP complex. The LHCP complex which accumulated in chloramphenicol treated wheat appeared functional even though chlorophyll protein complex accumulations were altered greatly in the wheat thylakoids. LHCP polypeptides were phosphorylated by action of a membrane protein kinase but yet photosystem II electron transport was impaired. The chloramphenicol treatment increased the photosystem I/photosystem II ratio of electron transport and the fluorescence emission ratio at 740 to 686 nanometers relative to those of untreated wheat. Chloramphenicol prevented development of normal granal thylakoids in normal wheat chloroplasts but not in those of the CD3 mutant. Elongated stacked thylakoids were observed in normal wheat. Net-like membranes and vesicles were noted in the stroma of chloroplasts from treated mutant seedlings. PMID- 16664279 TI - Characterization of 4,4'-Diisothiocyano-2,2'-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution in Isolated Chloroplasts : Evidence for a Common Binding Site on the C(4) Phosphate Translocator for 3 Phosphoglycerate, Phosphoenolpyruvate, and Inorganic Phosphate. AB - 3-Phosphoglycerate (PGA)-dependent O(2) evolution by mesophyll chloroplasts of the C(4) plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of O(2) evolution. The extent of inhibition of O(2) evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophyll, concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA dependent O(2) evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorganic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent O(2) evolution by isolated chloroplasts of the C(3) plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C(4) chloroplasts.Oxalacetate-dependent O(2) evolution and its stimulation by the uncoupler, NH(4)Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C(4) crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA. PMID- 16664280 TI - Oxygen Uptake and Photosynthesis of the Red Macroalga, Chondrus crispus, in Seawater: Effects of Oxygen Concentration. AB - With an experimental system developed for aquatic plants using the mass spectrometry technique and infrared gas analysis of CO(2), we studied the responses to various O(2) concentrations of gas exchanges with the red macroalga Chondrus crispus S. The results were as follows. (a) Irrespective of the CO(2) concentration, net photosynthesis was O(2) sensitive with a 45 to 70% stimulation at 2% O(2). Even with high CO(2), a significant Warburg effect was detected. (b) Although photosynthesis was CO(2) sensitive, O(2) photoconsumption was only weakly affected by CO(2) even at high CO(2) where it was still photodependent. (c) O(2) photoconsumption was always sensitive to O(2) concentration whatever the CO(2) concentration, but with O(2) exceeding 20% the kinetics disagreed with the Michaelis-Menten model, with saturation being reached more rapidly. With various CO(2) concentrations, the apparent K(m) (O(2)) ranged from 4 to 16% O(2) with a relatively constant V(max) (O(2)) of about one-third the V(max) (CO(2)). (d) Dark respiration seemed to be O(2) insensitive. These results are discussed in relation to the nature of the processes able to consume O(2) in the light, and seem to be consistent with a significant involvement of a Mehlertype reaction. PMID- 16664281 TI - Continuous Measurements of the Free Dissolved CO(2) Concentration during Photosynthesis of Marine Plants: Evidence for HCO(3) Use in Chondrus crispus. AB - An experimental system consisting of a gas exchange column linked to an assimilation chamber has been developed to record continuously the free dissolved CO(2) concentration in seawater containing marine plants. From experiments performed on the red macroalga Chondrus crispus (Rhodophyta, Gigartinales), this measurement is in agreement with the free CO(2) concentration calculated from the resistance to CO(2) exchanges in a biphasic system (gas and liquid) as earlier reported. The response time of this apparatus is short enough to detect, in conditions of constant pH, a photosynthesis-caused gradient between free CO(2) and HCO(3) (-) pools which half-equilibrates in 25 seconds. Abolished by carbonic anhydrase, the magnitude of this gradient increases with decreasing time of seawater transit from the chamber to the column apparatus. But its maximum magnitude (0.35 micromolar CO(2)) is negligible compared to the difference between air and free CO(2) (11.4 micromolar CO(2)). This illustrates the extent of the physical limiting-step occurring at the air-water interface when inorganic carbon consumption in seawater is balanced by dissolving gaseous CO(2). The direction of this small free CO(2)/HCO(3) (-) gradient indicates that HCO(3) (-) is consumed during photosynthesis. PMID- 16664282 TI - Quantitative analysis of pathways of methionine metabolism and their regulation in lemna. AB - Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 growing under standard conditions, and used to quantitate pathways of methionine metabolism. Synthesis of S-adenosylmethionine (AdoMet) is the major pathway for methionine metabolism, with over 4 times as much methionine metabolized by this route as accumulates in protein. More than 90% of AdoMet is used for transmethylation. Methyl groups of choline, phosphatidylcholine, and phosphorylcholine are major end products of this pathway. Flux through methylthio recycling is about one third the amount of methionine accumulating in protein. Spermidine synthesis accounts for at least 60% of the flux through methylthio recycling. The results obtained here, together with those reported for methionine-supplemented plants (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839), indicate that methionine supplementation reduced methylneogenesis by no more than the small amount expected from the reduced entry of sulfate sulfur into methionine (Giovanelli, Mudd, Datko, 1985 Plant Physiol 77: 450-455). Methionine supplementation had no significant effect on transmethylation or methylthio recycling. The combined data provide the first comprehensive estimates of the quantitative relationships of major pathways for methionine metabolism and their control in plants. PMID- 16664283 TI - Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook. AB - The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO(2) assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species. PMID- 16664284 TI - Dicyclohexylamine-induced shift of biosynthesis from spermidine to spermine in plant protoplasts. AB - An improved analytical method, based on high pressure liquid chromatography, has been developed for the simultaneous determination of the polyamines and S adenosyl-containing compounds in extracts of plant protoplasts. The method involves simple procedures for sample preparation and permits quantification of 1 picomole or less for all the compounds. This method has been used to study the effects of dicyclohexylamine, an inhibitor of plant spermidine synthase (Sindhu, R. K., S. S. Cohen 1984 Plant Physiol 74: 645-649), on biosynthesis of polyamines and 1-aminocyclopropane-1-carboxylate in protoplasts derived from Chinese cabbage leaves. Dicyclohexylamine effectively inhibits spermidine synthase in vivo. Inhibition of the synthesis of spermidine by dicyclohexylamine resulted in a stimulation of spermine synthesis, without significant effect on the synthesis of 1-aminocyclopropane-1-carboxylate. Decarboxylated S-adenosylmethionine is present in control Chinese cabbage protoplasts at approximately 10(-18) moles per cell, and dicyclohexylamine caused an increase of this metabolite of up to 10-fold in a 4-hour period. The increase in decarboxylated S-adenosylmethionine permitted an increased synthesis of spermine. These findings suggest that the availability of decarboxylated S-adenosylmethionine may be rate-limiting for the synthesis of spermine in plant protoplasts. PMID- 16664285 TI - Changes in Nonstructural Carbohydrates in Different Parts of Soybean (Glycine max [L.] Merr.) Plants during a Light/Dark Cycle and in Extended Darkness. AB - Diurnal patterns of nonstructural carbohydrate (starch, sucrose, and hexose sugars) concentration were characterized in different parts (leaves, petioles, stems, and roots) of vegetative soybean (Glycine max [L.] Merr.) plants. Pronounced changes in all carbohydrate pools were observed in all plant parts during the normal photosynthetic period; however, starch accumulation within leaves accounted for more than 80% of the nonstructural carbohydrate accumulated by the plant during the light period. Efficiency of utilization of starch and sucrose during the normal dark period differed among organs, with leaves being most efficient in mobilizing starch reserves and roots being most efficient in utilizing sucrose reserves. The vast majority (about 85%) of the whole plant carbohydrate reserves present at the end of the photosynthetic period were utilized during the normal dark period. Sink leaf expansion ceased in plants transferred to extended darkness and the cessation in leaf expansion corresponded with carbohydrate depletion in the subtending source leaf and the remainder of the plant. Collectively, the results indicated that under the conditions employed, leaves are the whole plant's primary source of carbon at night as well as during the day. PMID- 16664286 TI - Effects of nutritional stress on the storage proteins of soybeans. AB - The effects of sulfur deficiency on the complement of proteins laid down in developing seeds of soybean (Glycine max L. Merr) have been examined. Sulfur deficiency caused a 40% decrease in the level of glycinins and a contrasting elevation in the level of beta-conglycinins. The subunit composition of these proteins was also affected. There was in particular a 3-fold increase in the beta subunit of beta-conglycinins in the sulfur-deficient seeds, and this accumulated largely as the B(0)-isomer of beta-conglycinins, a protein which while virtually devoid of methionine and cysteine retains the physical properties of a normal 7S storage protein. These data demonstrate that a high degree of selectivity can be exerted by environmental stress over the accumulation of proteins in developing seeds. PMID- 16664287 TI - Immunological evidence for the presence of ribulose bisphosphate carboxylase in guard cell chloroplasts. AB - Ribulose bisphosphate carboxylase (Rubisco) has been found in Vicia faba L. guard cell chloroplasts by two immunological methods, using antibodies raised against highly purified subunits of ribulose bisphosphate carboxylase. Indirect cytoimmunofluorescence revealed binding of antibodies against both the small and the large subunits of ribulose bisphosphate carboxylase. Binding was observed only after partial digestion of guard cell walls by 4% Cellulysin to facilitate antibody penetration. After electrophoresis of a homogenate of guard cell protoplasts, the presence of both subunits was also revealed by immunolabeling technique. Positive response required the inhibition of proteolysis which appeared to be active upon homogenization. PMID- 16664288 TI - Partial purification and properties of phleinase induced in stem base of orchardgrass after defoliation. AB - Phleinase induced in stem base of orchardgrass (Dactylis glomerata L.) after defoliation was partially purified with ammonium sulfate precipitation, DEAE Sephadex chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The molecular weight of phleinase was 57,000 as determined by gel chromatography. The enzyme showed normal Michaelis-Menten kinetics and its K(m) value was 91 millimolar for phlein of mean degree of polymerization 60 as substrate. Reaction velocity of the enzyme was proportional to molarity of phlein irrespective of its chain length (mean degree of polymerization, 30 to 314). Phleinase attacked terminal fructosyl linkage of phlein by multi-chain mechanism. Phleinase cleaved beta-2,6 linkage, beta-2,6 linkage branched with beta-2,1 linkage, and beta-2,1 linkage of fructan in order of affinity, but not sucrose. Phleinase exhibited an optimum activity at pH 5.5 at 40 degrees C. Its complete inactivation occurred at 60 and 70 degrees C without and with phlein, respectively. Heat inactivation of the enzyme was enhanced by p chloromercuribenzoate and protected partially by l-cysteine. The enzyme was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and Hg(2+). The modes of action of phleinase were compared with those of the related enzymes. PMID- 16664289 TI - Free space iron pools in roots: generation and mobilization. AB - A rapid and simple method for the determination of a ferric iron pool in the free space of roots is described. Formation of this pool depended on the source of iron in the nutrient solution. During growth in water culture at pH 5 to 6 with Fe-ethylenediaminetetraacetate, a free space pool of 500 to 1000 nanomoles Fe per gram fresh weight was formed in the roots of bean (Phaseolus vulgaris L. var. Prelude), maize (Zea mays L. var. Capella), and chlorophytum (Chlorophytum comosum [Thunb.] Jacques). No significant pool (less than 100 nanomoles per gram fresh weight) was formed with ferrioxamine. Upon impending Fe deficiency, bean and chlorophytum were able to mobilize this pool. Fe-deficient bean plants mobilized iron from the free space iron pool of another plant in the same vessel. PMID- 16664290 TI - Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris. AB - ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V(max). Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V(max) value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V(max) value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn(2+) and Mg(2+) were highly sigmoidal. The activator concentration at half V(max) values for Mn(2+) and Mg(2+) were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively. PMID- 16664291 TI - Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids : characterization and reconstitution into liposomes. AB - We have used the nonionic detergent octyl-beta-d-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P(700), and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI(*), is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.In addition, the PSI reaction center complex CPI isolated from preparative gels and PSI-B were reconstituted into lecithin liposomes for structural analysis using freeze-fracture electron microscopy. The results suggest that the native PSI complex produces 12- to 13 nanometer particles, while the PSI reaction center, depleted of LHCI and peripheral proteins, produces particles with an average diameter of 10 nanometers. PMID- 16664292 TI - Nitrate Reductase from Monoraphidium braunii: Immunocytochemical Localization and Immunological Characterization. AB - Homogeneous nitrate reductase (EC 1.6.6.2) from Monoraphidium braunii was obtained by means of affinity chromatography in blue-Sepharose and gel filtration. After electrophoresis in polyacrylamide, gel slices containing pure nitrate reductase were disrupted and injected into previously unimmunized rabbits. The antiserum produced after several weeks was found to inhibit the different activities of nitrate reductase to a similar degree. Monospecificity of the antiserum was demonstrated by Ouchterlony double diffusion and crossed immunoelectrophoresis. The antibodies were purified by immunoabsorption to Sepharose-bound nitrate reductase.The intracellular location of nitrate reductase in green algae was examined by applying an immunocytochemical method to M. braunii cells. Ultrathin frozen sections were first treated with immunopurified anti-nitrate reductase monospecific antibodies, followed by incubation with colloidal gold-labeled goat antirabbit immunoglobulin G as a marker. The enzyme was specifically located in the pyrenoid region of the chloroplast. PMID- 16664293 TI - Reversibility of Photosynthetic Inhibition in Cotton after Long-Term Exposure to Elevated CO(2) Concentrations. AB - Cotton (Gossypium hirsutum L. cv Stoneville 213) was grown at 350 and 1000 microliters per liter CO(2). The plants grown at elevated CO(2) concentrations contained large starch pools and showed initial symptoms of visible physical damage. Photosynthetic rates were lower than expected based on instantaneous exposure to high CO(2).A group of plants grown at 1000 microliters per liter CO(2) was switched to 350 microliters per liter CO(2). Starch pools and photosynthetic rates were monitored in the switched plants and in the two unswitched control groups. Photosynthetic rates per unit leaf area recovered to the level of the 350 microliters per liter CO(2) grown control group within four to five days. To assess only nonstomatal limitations to photosynthesis, a measure of photosynthetic efficiencies was calculated (moles CO(2) fixed per square meter per second per mole intercellular CO(2)). Photosynthetic efficiency also recovered to the levels of the 350 microliters per liter CO(2) grown controls within three to four days.Recovery was correlated to a rapid depletion of the starch pool, indicating that the inhibition of photosynthesis is primarily a result of feedback inhibition. However, complete recovery may involve the repair of damage to the chloroplasts caused by excessive starch accumulation. The rapid and complete reversal of photosynthetic inhibition suggests that the appearance of large, strong sinks at certain developmental stages could result in reduction of the large starch accumulations and that photosynthetic rates could recover to near the theoretical capacity during periods of high photosynthate demand. PMID- 16664294 TI - Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L. AB - The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the ;apoplastic' ABA, increased before ;bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period. PMID- 16664295 TI - Effects of polyethylene-glycol-induced osmotic stress on transpiration and photosynthesis in pinto bean leaf discs. AB - A new leaf disc chamber allows measurements of chlorophyll fluorescence and CO(2) and H(2)O vapor exchanges during infusion of solution into the cut edge of the disc. Polyethylene glycol (molecular weight, 6000) was used to apply a mild external osmotic stress to the leaf disc within this chamber. This stress rapidly caused a temporary increase in transpiration. This increase was reversed (5-6 minutes later) and after 20 to 25 min, the stomates nearly completely closed. Internal CO(2) (calculated) and leaf temperature followed the transpiration measurements. However, chlorophyll fluorescence (small rise) followed internal CO(2) (small rise). This complete sequence of events resembles those caused by exposure of leaves to certain air pollutants which have been seen to cause such a transient increase followed by a decrease in stomatal closure. PMID- 16664296 TI - Role of ABA in Maturation of Rapeseed Embryos. AB - Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investigate the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development. PMID- 16664297 TI - Effects of Quantum Flux Density on Photosynthesis and Chloroplast Ultrastructure in Tissue-Cultured Plantlets and Seedlings of Liquidambar styraciflua L. towards Improved Acclimatization and Field Survival. AB - Liquidambar styraciflua L. seedlings and tissue-cultured plantlets were grown under high, medium, or low (315, 155, or 50 microeinsteins per square meter per second photosynthetically active radiation) quantum flux densities. Net photosynthesis, chlorophyll content, and chloroplast ultrastructure of leaves differentiated from these conditions were investigated. Seedling photosynthetic rates at light saturation were positively related to light pretreatments, being 6.44, 4.73, and 2.75 milligrams CO(2) per square decimeter per hour for high, medium, and low light, respectively. Cultured plantlets under all light conditions had appreciably higher photosynthetic rates than noncultured seedlings; corresponding rates were 12.14, 13.55, and 11.36 milligrams CO(2) per square decimeter per hour. Chlorophyll in seedlings and plantlets was significantly higher in low light-treated plants. Seedling leaves had chloroplasts with abundant starch regardless of light pretreatment. In high light, starch granules were predominant and associated with disrupted granal structure. Low light seedling chloroplasts had smaller starch grains and well formed grana. In contrast, tissue culture-differentiated leaves were devoid of starch; grana were well organized in higher quantum flux density treatments, but disorganized at low flux densities. PMID- 16664299 TI - Reassessment of an apparent hyperactive form of phosphofructokinase from plants. AB - In the assay of phosphofructokinase (PFK) from endosperm of germinating castor bean (Ricinus communis L.) there is a transient stimulation of initial activity by fructose 2,6-bisphosphate. This activation is due to metabolism of a limited amount of pyrophosphate (a contaminant of commercial ATP) by PPi:fructose 6 phosphate phosphotransferase (PFP), which is present in the extract. Both this activity and the amount of pyrophosphate contamination are sufficient to account for the initial increase in apparent PFK activity. The transient burst of activity is dependent on both of the above factors. Based on studies of a similar hyperactive PFK, others have proposed that PFK and PFP may be interconverted (Balogh et al. 1984 FEBS Lett 169: 287-292). The evidence for such conversions is reinterpreted in the context of the current results. PMID- 16664298 TI - Target molecular size of the red beet plasma membrane ATPase. AB - Radiation inactivation of the red beet (Beta vulgaris L.) plasma membrane ATPase was carried out using gamma-ray radiation from a (137)Cs source. Inactivation of vanadate-sensitive ATPase activity by gamma-ray radiation followed an exponential decline with increasing total dose, indicating a single target size calculated to have a molecular weight of about 228,000. Since the catalytic subunit of the red beet plasma membrane ATPase has been demonstrated to have a molecular weight of about 100,000 by dodecyl-sulfate gel electrophoresis following (32)P phosphorylation, it is suggested that the native enzyme may exist, at least, as a dimer of catalytic subunits. PMID- 16664300 TI - Linear sucrose transport in protoplasts from developing soybean cotyledons. AB - Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells. PMID- 16664301 TI - Pollen sporoplasts: dissolution of pollen walls. AB - 4-Methylmorpholine N-oxide monohydrate (MMNO.H(2)O), a potent solvent for polysaccharides, is an effective vehicle for release of membrane-enclosed male gametophytes (sporoplasts) from spore walls. This release occurs in minutes when pollen (Lilium longiflorum Thunb.) is suspended in a melt of MMNO.H(2)O at 75 degrees C. Continued heating at 75 degrees C leads to distintegration of the exine ;shell' which coalesces into immiscible globules in the MMNO melt. These observations provide a general procedure for preparation of pollen sporoplasts and sporoplast outer membranes, and offer a new method for dissolving the sporopollenin component of the spore wall. PMID- 16664302 TI - Sites of gibberellin biosynthesis in pea seedlings. AB - Potential sites of gibberellin biosynthesis in 10-day-old ;Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [(14)C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation. PMID- 16664303 TI - Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase. AB - Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO(3) (-) medium were characterized. The in vivo NO(3) (-) reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO(2) (-) reductase as compared with the wild type. This coincided with higher accumulation of NO(2) (-) by the variant than by the wild type cells after transfer from alanine medium to NO(3) ( ) medium. NO(2) (-) accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions. PMID- 16664304 TI - Pyruvate, pi dikinase in bundle sheath strands as well as in mesophyll cells in maize leaves. AB - Mesophyll protoplasts and bundle sheath strands were isolated from maize leaves. Light microscopic observation showed the preparations were pure and without cross contamination. Protein blot analysis of mesophyll and bundle sheath cell soluble protein showed that the concentration of pyruvate orthophosphate dikinase (EC 2.7.9.1) is about one-tenth as much in the bundle sheath cells as in mesophyll cells, but about eight times greater than that found in wheat leaves, on the basis of soluble protein. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was barely detectable in the bundle sheath cells, while ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and NADP-dependent malic enzyme (EC 1.3.1.37) were exclusively present in the bundle sheath cells and were absent in the mesophyll cells. Whereas pyruvate, Pi dikinase was previously considered localized only in mesophyll cells of C(4) plants, these results clearly demonstrate the presence of appreciable quantities of the enzyme in the bundle sheath cells of the C(4) species maize. PMID- 16664305 TI - Presence of Host-Plasma Membrane Type H-ATPase in the Membrane Envelope Enclosing the Bacteroids in Soybean Root Nodules. AB - An improved method is described for the isolation of membrane envelope enclosing the bacteroids (peribacteroid membrane) from soybean (Glycine max L.) root nodules. The ATPase activity of the peribacteroid membrane from infected roots is compared with that of the plasma membrane from uninfected roots. The two ATPases are similar in terms of their vanadate sensitivities, pH optima, and mineral cation requirements, and show antigenic cross-reactivity. However, the ATPase of peribacteroid membrane is more sensitive to stimulation by NH(4) (+). ATP dependent proton translocation across the peribacteroid membrane was demonstrated in broken protoplasts of infected cells, by the use of fluorescence microscopy with acridine orange. It is suggested that acidification of the peribacteroid space by the peribacteroid membrane ATPase results in the conversion of NH(3) to NH(4) (+) in this space and thereby facilitates the removal of fixed-nitrogen from the bacteroid. PMID- 16664306 TI - Metabolic Conversion of Amino Acids Loaded in the Vacuole of Chara australis Internodal Cells. AB - Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, gamma-aminobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by l-methionine-d,l-sulfoximine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism. PMID- 16664307 TI - Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields. AB - Measurements of O(2) uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO(2) evolution and yields of dry matter. O(2) uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O(2) uptake in the dark. In both populations, adding malate and glycine stimulated O(2) uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O(2) uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.Transverse and cross sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume. PMID- 16664308 TI - Uptake of glutamine by the scutellum of germinating barley grain. AB - Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up [(14)C]glutamine at an initial rate of about 10 micromoles.gram(-1).hour(-1) in the standard assay conditions (pH 5, 30 degrees C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent K(m) of about 2 millimolar and a V(max) of about 25 micromoles.gram(-1).hour(-1).The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine.Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella. PMID- 16664309 TI - Behavior of Lipoxygenase during Establishment, Senescence, and Rejuvenation of Soybean Cotyledons. AB - Lipoxygenase protein and activity were examined during establishment, senescence, and rejuvenation of soybean cotyledons. Lipoxygenase protein, as determined on ;Western' immunoblots, and lipoxygenase-1 and -2/3 activities decreased during mobilization of seed reserves 3 to 9 days following planting. Lipoxygenase-1 activity decreased more rapidly than lipoxygenase-2/3 and was not detectable by 11 days after planting. Lipoxygenase protein increased after day 11 while lipoxygenase-2/3 activity continued to decline. During the later stages of cotyledon senescence, both lipoxygenase protein and lipoxygenase-2/3 activity decreased. Upon rejuvenation, lipoxygenase-2/3 activity, but not that of lipoxygenase-1, increased. These results demonstrate that elevated lipoxygenase activity does not represent a universal characteristic of senescent plant tissue. PMID- 16664310 TI - Protein and RNA Synthesizing Activities during Sexual Process of Heterothallic Closterium. AB - Protein and RNA synthesizing activities increased markedly during the mating process and decreased during the maturation stage of zygotes in heterothallic strains of Closterium peracerosum-strigosum-littoale, KAS-4-29 (mating-type minus) and KAS -4-30 (mating-type plus) and a homothallic Closterium acerosum. Different proteins were synthesized at the different stages of the mating process, suggesting that a sequential expression and repression of mating genes occur for the mating-specific protein synthesis during the sexual reproduction. PMID- 16664311 TI - Regulation of potato tuber protein accumulation by gibberellic Acid. AB - Many studies have shown that gibberellic acid (GA(3)) inhibits tuberization in potato (Solanum tuberosum L.). In this study, we have utilized the 40 kilodalton glycoprotein, patatin, as a marker for biochemical events associated with the process of tuberization. To determine the effects of exogenous applications of GA(3) on the induction of the accumulation of this major tuber protein, we measured patatin levels in tubers from treated whole plants, petioles from a single-node cutting system with GA(3) applied in a lanolin paste, and stolon tips cultured in vitro on an inductive medium supplemented with GA(3). In all three systems, GA(3) inhibited the accumulation of patatin and the major 15 and 22 kilodalton tuber proteins. This effect appeared to be selective since most of the other proteins were not affected and, in tubers, at least one protein was stimulated by GA(3). These results suggest that GA(3) can reverse biochemical events of tuberization in tubers as well as prevent the accumulation of the major tuber proteins in other inducible tissues. PMID- 16664313 TI - Photoperiodic Regulation of Photosynthate Partitioning in Leaves of Digitaria decumbens Stent. AB - In leaves of pangolagrass (Digitaria decumbens Stent.), the proportion of photosynthate partitioned into starch adjusts to a change in daylength within 24 hours. After a single 14-hour long day, the relative starch accumulation rate is approximately 50% of that under 7-hour short days. This rapid response was exploited to study the light requirement for the perception of changes in daylength. It was found for short day-grown plants that: (a) 7-hour daylength extensions with dim white light (below the light compensation point for photosynthesis); (b) 7-hour daylength extensions with dim far red light (wavelengths greater than 690 nanomoles); or (c) 0.5-hour night-break irradiations with bright white light were all capable of producing about one-half of the effect of a 7-hour daylength extension with bright light. However, long periods of bright light were not required for a complete effect, since a 7-hour shifted short day (i.e. beginning 7 hours later than usual) was as effective as a 14-hour-long day itself. There was also a critical daylength between 11 and 12 hours for the transition between short-day and long-day partitioning patterns. Photoperiod determination depends, at least in part, on a nonphotosynthetic photoreceptor sensitive to both visible and far red irradiation. The duration of the photosynthetic period, as shown in experiments with low-pressure sodium lamps, does not by itself determine the response to daylength. PMID- 16664312 TI - Abnormal processing of the modified oligosaccharide side chains of phytohemagglutinin in the presence of swainsonine and deoxynojirimycin. AB - Phytohemagglutinin, the glycoprotein lectin of the common bean, Phaseolus vulgaris, has both high-mannose (Man(8-9)GlcNAc(2)) and modified oligosaccharide side chains. The modified side chains have glucosamine, mannose, fucose, and xylose in the molar ratios 2:3.8:0.6:0.5, and are resistant to hydrolysis by endoglycosidase H. Synthesis and processing of side chains in the presence of 1 deoxynojirimycin, an inhibitor of alpha-glucosidase, results in the formation of chains which are all alike. They are sensitive to endoglycosidase H, do not contain fucose, and are largely resistant to alpha-mannosidase. This indicates that they are probably high-mannose chains blocked by terminal glucose residues. Synthesis and processing of side chains in the presence of swainsonine, an inhibitor of alpha-mannosidase II, results in the formation of normal high mannose chains, and of modified chains which contain fucose residues, are resistant to endoglycosidase H, and can be distinguished from normal modified chains only by the presence of extra mannose residues.Processing of the phytohemagglutinin modified chains of PHA under normal conditions involves the attachment of peripheral N-acetylglucosamine residues in the Golgi complex and their subsequent removal in the protein bodies. The attachment of the N acetylglucosamine residues is largely inhibited by deoxynojirimycin but still occurs in the presence of swainsonine. The results presented in this work show that processing of the asparagine-linked oligosaccharides is under the control of several glycosidases and glycosyltransferases and involves the formation of intermediate products. PMID- 16664314 TI - Proteins in intercellular washing fluid from noninoculated and rust-affected leaves of wheat and barley. AB - Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19 degrees C, four infection-related CBB-stainable proteins were detected in IWF.To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25 degrees C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley.IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19 degrees C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium. PMID- 16664315 TI - Photoinhibition of Photosynthesis in Broken Chloroplasts as a Function of Electron Transfer Rates during Light Treatment. AB - Photoinhibition was studied in osmotically broken chloroplasts isolated from spinach leaves (Spinacia oleracea L.). Both whole chain electron transport (measured as ferricyanide-dependent O(2) evolution in the presence of NH(4)Cl) and photosystem II activity (measured as O(2) evolution in the presence of either silicomolybdate plus 3-(3,4-diphenyl)-1,1 dimethylurea or parabenzoquinone) showed similar decreases in activity in response to a photoinhibitory treatment (8 minutes of high light given in the absence of an electron acceptor other than O(2)). Photosystem I activity was less affected. Photoinhibition of silicomolybdate reduction was largely reversible by an 8 minute dark incubation following the light treatment. Decreasing the O(2) concentration during photoinhibition below 2% increased photoinhibition of whole chain electron transport. Addition of superoxide dismutase to the reaction medium did not affect photoinhibition. Photoinhibition of both photosystem I and photosystem II activity increased as the rate of electron transfer during the treatment increased, and was largely prevented when 3-(3,4-diphenyl)-1,1-dimethylurea was present during the photoinhibition period. Noncyclic photophosphorylation was decreased as a consequence of whole chain electron transfer photoinhibition. Since diphenyl carbazide added after light treatment did not relieve photoinhibition of dichlorophenol indophenol reduction, we conclude that the site of inhibition is located within or near the photosystem II reaction center. PMID- 16664316 TI - Isolation and Partial Purification of Prophenoloxidase from Daucus carota L. Cell Cultures. AB - The enzyme, phenoloxidase, was isolated and partially purified as an inactive enzyme, a proenzyme, from plant cell cultures of Daucus carota, Nicotiana tabacum, and Haplopappus gracilis. The prophenoloxidase was found to be specifically activated by Ca(2+) or Mn(2+) ions in concentrations above 1 millimolar. Calmodulin was not involved in this activation. Concentrations of Ca(2+) or Mn(2+) below 1 millimolar could not induce activation of the prophenoloxidase, but if trypsin was added simultaneously with Ca(2+) or Mn(2+) at a concentration of 1 millimolar or below, the proenzyme was converted to its active form. The inactive form of phenoloxidase was found to be a soluble enzyme, whereas after activation the enzyme aggregated, and a significant amount of the enzyme activity could become pelleted. PMID- 16664317 TI - Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae. AB - Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH(3), NO(3) (-), or N(2). Expressed as a function of relative growth rate, steady state cellular P contents and PO(4) assimilation rates did not vary with N-source. However, N source did alter the maximal PO(4)-limited growth rate achieved by the cultures: the NO(3) (-) and N(2) cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH(3) grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO(3) (-)-grown cells were the smallest (627 +/- 34 micromoles C . 10(-9) cells), while NH(3)-grown cells were largest (900 +/- 44 micromoles C . 10(-9) cells) and N(2)-fixing cells were intermediate (726 +/- 48 micromoles C . 10(-9) cells) in size. In the NO(3) (-)-and N(2)-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH(3)-grown cells. Heterocysts were absent in NH(3) grown cultures but were present in both the N(2) and NO(3) (-) cultures. In the NO(3) (-)-grown cultures C(2)H(2) reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N(2)-fixing cultures the acetylene:N(2) ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.Compared with NH(3), the assimilation of NO(3) (-) and N(2) resulted either in a decrease in cellular C (NO(3) (-) and N(2) cultures) or in a lower maximal growth rate (N(2) culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO(3) (-) or N(2) compared with NH(3). PMID- 16664318 TI - Modeling the C Economy of Anabaena flos-aquae: Estimates of Establishment, Maintenance, and Active Costs Associated with Growth on NH(3), NO(3), and N(2). AB - Steady state cultures of Anabaena flos-aquae were established over a wide range of phosphate-limited growth rates while N was supplied as either NH(3), NO(3) ( ), or N(2) gas. At growth rates greater than 0.03 per hour, rates of gross and net carbon fixation were similar on all N sources. However, at lower growth rates (<0.03 per hour) in the NO(3) (-) and N(2) cultures, gross photosynthesis greatly exceeded net photosynthesis. The increase in photosynthetic O(2) evolution with growth rate was greatest when N requirements were met by NO(3) (-) and least when met by NH(3). These results were combined with previously reported measurements of cellular chemical composition, N assimilation, and acetylene reduction (Layzell, Turpin, Elrifi 1985 Plant Physiol 78: 739-745) to construct empirical models of carbon and energy flow for cultures grown at 30, 60, and 100% of their maximal growth rate on all N sources. The models suggested that over this growth range, 89 to 100% of photodriven electrons were allocated to biomass production in the NH(3) cells, whereas only 49 to 74% and 54 to 90% were partitioned to biomass in the NO(3) (-)-and N(2)-grown cells, respectively. The models were used to estimate the relative contribution of active, maintenance, and establishment costs associated with NO(3) (-) and N(2) assimilation over the entire range of growth rates. The models showed that the relative contribution of the component costs of N assimilation were growth rate dependent. At higher growth rates, the major costs for NO(3) (-) assimilation were the active costs, while in N(2) fixing cultures the major energetic requirements were those associated with heterocyst establishment and maintenance. It was concluded that compared with NO(3) (-) assimilation, N(2) fixation was energetically unfavorable due to the costs of heterocyst establishment and maintenance, rather than the active costs of N(2) assimilation. PMID- 16664319 TI - Diurnal changes in allocation of newly fixed carbon in exporting sugar beet leaves. AB - Storage of newly fixed carbon as starch and sucrose follows a regular daily pattern in exporting sugar beet leaves under constant day length and level of illumination. Up to the final two hours of the light period, when starch storage declines, a nearly constant proportion of newly fixed carbon was allocated to carbohydrate storage, principally starch. Sucrose is stored only early in the light period, when there is little accumulation of starch. Pulse labeling with (14)CO(2) revealed that considerable starch synthesis was taking place at this time. Starch made the previous day was not mobilized during this period but breakdown of newly synthesized starch may occur when carbon flow into sucrose synthesis increases early in the day. At the end of the day, starch storage declined from the constant level observed during most of the day, but no diversion of label into export of specific alternative compounds could be detected. Lowered storage of starch persisted when the 14-hour light period was lengthened. Changed allocation of recently fixed carbon to sucrose and starch at the beginning and end of the light period was not the result of outright inactivation of pathways but of regulation of carbon flow. PMID- 16664320 TI - Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves. AB - This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H(+) --> d-ribitol-5-P + NADP(+). The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. PMID- 16664321 TI - Studies on Genetic Male-Sterile Soybeans : IV. Effect of Male Sterility and Source of Nitrogen Nutrition on Accumulation, Partitioning, and Transport of Nitrogen. AB - Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male sterility (ms(1)) and nodulation (rj(1)), was used to investigate the effects of reproductive tissue development and source of nitrogen nutrition on accumulation, transport, and partitioning of nitrogen in a greenhouse experiment. Nodulated plants were supplied nitrogen-free nutrient solution, and nonnodulated plants were supplied nutrient solution containing 20 millimolar KNO(3). Plants were sampled from flowering until maturity (77 to 147 days after transplanting).Accumulation rates of nitrogen in whole plants during reproductive growth were not significantly different among the four plant types. Nitrogen accumulation in the sterile, nonnodulated plants, however, ceased 2 weeks earlier than in fertile, nonnodulated or fertile and sterile, nodulated plants. This early cessation in nitrogen accumulation resulted in sterile, nonnodulated plants accumulating significantly less whole plant nitrogen by 133 days after transplanting (DAT) than fertile, nonnodulated plants. Thus, changing the site of nitrogen assimilation from nodules (N(2)-fixing plants) to roots and leaves (NO(3)-fed plants) resulted in similar whole-plant nitrogen accumulation rates in fertile and sterile plants, despite the absence of seed in the latter.Leaflet and stem plus petiole tissues of both types of sterile plants had significantly higher nitrogen concentrations after 119 DAT than both types of fertile plants. Significantly higher concentrations and exudation rates of nonureide, reduced nitrogen in xylem sap of sterile than of fertile plants after 105 DAT were observed. These latter results indicated possible cycling of nonureide, reduced nitrogen from the downward phloem translocation stream to the upward xylem translocation stream in roots of sterile plants. Collectively, these results suggest a lack of sinks for nitrogen utilization in the shoots of sterile plants. Hence, comparison of nitrogen accumulation rates for sterile and fertile plants does not provide a definitive test of the hypothesis that reproductive tissue development limits photosynthate availability for support of N(2) fixation and nitrate assimilation in determinate soybeans.Nitrogen assimilation during reproductive growth met a larger proportion of the reproductive-tissue nitrogen requirement of nitrate-dependent plants (73%) than of N(2)-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N(2)-fixing than in nitrate-dependent plants. N(2)-fixing plants partitioned nitrogen to reproductive tissue more efficiently than nitrate dependent plants as the reproductive tissues of the former and latter contained 65 and 55%, respectively, of the whole-plant nitrogen at the time that nitrogen accumulation in reproductive parts had ceased (133 DAT). PMID- 16664322 TI - Intracellular pH Regulation during NO(3) Assimilation in Shoot and Roots of Ricinus communis. AB - Ricinus communis L. was used to test the Dijkshoorn-Ben Zioni hypothesis that NO(3) (-) uptake by roots is regulated by NO(3) (-) assimilation in the shoot. The fate of the electronegative charge arising from total assimilated NO(3) (-) (and SO(4) (2-)) was followed in its distribution between organic anion accumulation and HCO(3) (-) excretion into the nutrient solution. In plants adequately supplied with NO(3) (-), HCO(3) (-) excretion accounted for about 47% of the anion charge, reflecting an excess nutrient anion over cation uptake. In vivo nitrate reductase assays revealed that the roots represented the site of about 44% of the total NO(3) (-) reduction in the plants. To trace vascular transport of ionic and nitrogenous constituents within the plant, the composition of both xylem and phloem saps was thoroughly investigated. Detailed dry tissue and sap analyses revealed that only between 19 and 24% of the HCO(3) (-) excretion could be accounted for from oxidative decarboxylation of shoot-borne organic anions produced in the NO(3) (-) reduction process. The results obtained in this investigation may be interpreted as providing direct evidence for a minor importance of phloem transport of cation-organate for the regulation of intracellular pH and electroneutrality, thus practically eliminating the necessity for the Dijkshoorn-Ben Zioni recycling process. PMID- 16664323 TI - Transport and Partitioning of CO(2) Fixed by Root Nodules of Ureide and Amide Producing Legumes. AB - Nodulated and denodulated roots of adzuki bean (Vigna angularis), soybean (Glycine max), and alfalfa (Medicago sativa) were exposed to (14)CO(2) to investigate the contribution of nodule CO(2) fixation to assimilation and transport of fixed nitrogen. The distribution of radioactivity in xylem sap and partitioning of carbon fixed by nodules to the whole plant were measured. Radioactivity in the xylem sap of nodulated soybean and adzuki bean was located primarily (70 to 87%) in the acid fraction while the basic (amino acid) fraction contained 10 to 22%. In contrast, radioactivity in the xylem sap of nodulated alfalfa was primarily in amino acids with about 20% in organic acids. Total ureide concentration was 8.1, 4.7, and 0.0 micromoles per milliliter xylem sap for soybean, adzuki bean, and alfalfa, respectively. While the major nitrogen transport products in soybeans and adzuki beans are ureides, this class of metabolites contained less than 20% of the total radioactivity. When nodules of plants were removed, radioactivity in xylem sap decreased by 90% or more. Pulse chase experiments indicated that CO(2) fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO(2) fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO(2) fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen. PMID- 16664324 TI - Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light. AB - Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO(2) assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in (15)N-labeled NH(3), glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25 degrees C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The (15)N-enrichment of various amino acids derived from these (15)N-substrates were examined. The amido-N of glutamine was the first (15)N-labeled product in leaves incubated with (15)NH(4)Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly (15)N-labeled products during incubation with [(15)N]glycine. In contrast, aspartate and alanine were the first (15)N-labeled products when [(15)N] glutamate was used. These results indicate that NH(3) was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues. PMID- 16664325 TI - S-adenosylmethionine decarboxylase and spermidine synthase from chinese cabbage. AB - The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virus-infected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO(2) and of decarboxylated S-adenosylmethionine (dSAM). The K(m) for SAM is 38 micromolar. The reaction is not stimulated by Mg(++) or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5.1.16), present in leaf or protoplast extracts in many fold excess over SAM decarboxylase, has been purified approximately 1900-fold in steps involving ammonium sulfate precipitation, affinity chromatography, and gel filtration on Sephacryl S-300. Standardization of the Sephacryl column by proteins of known molecular weight yielded values of 35,000 and 81,000 for the decarboxylase and synthase, respectively. PMID- 16664326 TI - Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydromyricetin to Its Flavan 3,4-Diol (Leucodelphinidin) and to (+)-Gallocatechin by Reductases Extracted from Tissue Cultures of Ginkgo biloba and Pseudotsuga menziesii. AB - Extracts of callus or cell suspension cultures from petioles of Ginkgo biloba catalyzed the production of (+)-gallocatechin (2,3-trans-3,5,7,3',4',5' hexahydroxy-flavan) from (+)-dihydromyricetin (5'-hydroxy-dihydroquercetin) along with the expected 3,4-cis-diol intermediate, leucodelphinidin, in a NADPH dependent double-step reductase reaction at pH 7.4. The latter diol, isolated from the above incubation mixture, produced (+)-gallocatechin in a NADPH dependent reaction. Extracts from tissue cultures derived from needles of Pseudotsuga menziesii (Douglas fir) also produced significant amounts of the 3,4 diol from dihydromyricetin. (+)-Dihydromyricetin, purified via paper chromatography from leaves of Leptarrhena pyrolifolia, was reduced by NaBH(4) to the presumed 3,4-trans-diol and acid epimerized to the 3,4-cis-diol. PMID- 16664327 TI - Use of N-Bromoacetyl-l-ornithine to Study l-Ornithine and l-Arginine Biosynthesis in Soybean (Glycine max L.) Cell Cultures. AB - The effect of the inhibitor N(2)-bromoacetyl-l-ornithine (NBAO) on the biosynthesis of ornithine in higher plants, was investigated using soybean cells (Glycine max L. var Mandarin), grown in suspension culture. The NBAO was found to reduce the specific activity of the enzyme N(2)-acetyl-l-ornithine: l-glutamate N acetyltransferase (EC 2.3.1.35). In contrast, the specific activity of the enzyme acetyl coenzyme A:L-glutamate N-acetyltransferase (EC 2.3.1.1), which is also involved in N-acetylglutamate biosynthesis, was not significantly changed. Estimation of the concentrations of free amino acids in the soluble fraction of the cells showed that while ornithine levels were decreased, glutamic acid levels were increased in the presence of NBAO. While arginine levels initially increased in the presence of NBAO, they finally decreased near the end of the growth period. Evidence was obtained that the initial increase in arginine levels was due to the inhibition of arginase (EC 3.5.3.1) by N(2)-bromoacetyl l-ornithine. We conclude that the reaction catalyzed by N(2)-acetyl-l-ornithine:l-glutamate N acetyl transferase is a rate limiting reaction in vivo. PMID- 16664328 TI - Photosynthetic and respiratory rates of two psychrophilic diatoms. AB - The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3 10 and Nitzschia sp. K3-3 for cells grown at 0 degrees C were 8 to 10 microliters O(2) evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10 degrees C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0 degrees C, and 6 to 10 for cells grown at 10 degrees C. When cells grown at 0 degrees C were immediately shifted to 10 degrees C or cells grown at 10 degrees C were shifted to 0 degrees C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O(2) evolution showed much less immediate adaptation, especially on the up shift, 0 degrees to 10 degrees C. The chlorophyll a content of 0 degrees C grown cells was about 0.5% of dry weight, in 10 degrees C grown cells 1.3% (strain K3-10) and 2.2% (strain K3 3). In addition to a diminished chlorophyll a content in 0 degrees C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10 degrees C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0 degrees C grown cells of Chaetoceros sp. strain K3-10 as compared to 10 degrees C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0 degrees C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins. PMID- 16664329 TI - Effects of Light Intensity and Oxidized Nitrogen Sources on Hydrogen Production by Chlamydomonas reinhardii. AB - Chlamydomonas reinhardii cells, after a period of dark anaerobic adaptation, evolve H(2) not only in the dark but also in the light. Our results show that high irradiances impair prolonged H(2) evolution, while under low irradiances or darkness H(2) evolution proceeds for more than 50 hours. NO(3) (-) and NO(2) (-) suppress H(2) evolution both in the dark or under low irradiance. Apparently the cells prefer these oxidized nitrogen sources to protons as electron acceptors, since both NO(3) (-) and NO(2) (-) become reduced to NH(4) (+), which is excreted to the culture medium in high amounts. H(2) evolution started once these oxidized anions were largely depleted from the medium. Moreover, H(2) evolution was consistently associated with NH(4) (+) excretion even if NH(4) (+) was already present in high amounts in the medium. This observation indicates that the cells utilize not only their carbohydrate but also their protein reserves as sources of reducing power for H(2) evolution. This conclusion was supported by the observation that when nitrogen-starved cells were made anaerobic in a nitrogen free medium, they not only evolved H(2) at very high rates but excreted concomitantly NH(4) (+) up to concentrations in the millimolar range. PMID- 16664330 TI - Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts. AB - Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16 kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.Location of PPDK in C(3) plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C(3) plant leaves as it is in the case of C(4) plants. PMID- 16664331 TI - Homology among 3S and 7S Globulins from Cereals and Pea. AB - The globulins from wheat caryopses were found to consist primarily of protein sedimenting at approximately 3S and 7S. These proteins displayed a molecular weight distribution similar to that of the purified vicilin-like fractions from oat and pea, with variations occurring in the isoelectric points and relative quantities of their major subunits. concanavalin A Sepharose chromatography suggested that the major polypeptides of the wheat (3S + 7S) fraction are glycosylated. Western blot analysis using antioat (3S + 7S) globulin immunoglobulin G revealed the vicilins from pea and the globulin fractions of oat, wheat, barley, rye, corn, and rice to contain immunologically homologous polypeptides. Major groups of polypeptides were shared by all the cereals and pea, including subunits of approximately 75, 50, 40 kilodaltons and 20 to 25 kilodaltons. These results indicate that legume-like 3S and 7S globulins have been conserved and are being expressed in cereals. PMID- 16664332 TI - Determination of intracellular osmotic volume and sodium concentration in dunaliella. AB - A new method to measure intracellular volume in Dunaliella was developed, where lithium ions are used as monitors of the extracellular volume. Li(+) is shown to be impenetrable to the intracellular volume, insignificantly absorbed to the algae, and is rapidly and evenly distributed within the extracellular volume. The method is suggested to be free of several limitations and consistent errors present in several previously employed techniques.Using the new technique it is shown that both Dunaliella salina and Dunaliella bardawil adjust to a constant cellular volume when grown in a medium containing salt concentrations ranging from 0.5 molar to 4 molar NaCl. That volume is 90 femtoliter per cell for D. salina and 600 femtoliter per cell for D. bardawil. Nonosmotic volume accounts for about 10% of the total cell volume.The intracellular sodium concentration, as determined with the new technique, was under all experimental conditions tested below 100 millimolar. This was true both for cells grown on 0.5 to 4 molar NaCl, and during the osmoregulatory process. It is thus concluded that intracellular NaCl is a minor contributor to the overall intracellular osmotic pressure in Dunaliella. PMID- 16664333 TI - Leaf Conductance in Relation to Rate of CO(2) Assimilation: I. Influence of Nitrogen Nutrition, Phosphorus Nutrition, Photon Flux Density, and Ambient Partial Pressure of CO(2) during Ontogeny. AB - Plants of Zea mays were grown with different concentrations of nitrate (0.6, 4, 12, and 24 millimolar) and phosphate (0.04, 0.13, 0.53, and 1.33 millimolar) supplied to the roots, photon flux densities (0.12, 0.5, and 2 millimoles per square meter per second), and ambient partial pressures of CO(2) (305 and 610 microbars). Differences in mineral nutrition and irradiance led to a large variation in rate of CO(2) assimilation per unit leaf area (A, 11 to 58 micromoles per square meter per second) when measured under standard conditions. The variation was shown, with the plants that had received different amounts of nitrate, to be related to variations in the nitrogen and chlorophyll contents, and phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylase activities per unit leaf area. Irrespective of growth treatment, A and leaf conductance to CO(2) transfer (g), measured under standard conditions were in almost constant proportion, implying that intercellular partial pressure of CO(2) (p(i)), was almost constant at 95 microbars. The same proportionality was maintained as A and g increased in an initially nitrogen-deficient plant that had been supplied with abundant nitrate. It was shown that p(i) measured at a given ambient partial pressure was not affected by the ambient partial pressure at which the plants had been grown, although it was different when measured at different ambient partial pressures. This suggests that the close coupling between A and g in these experiments is not associated with sensitivity of stomata to change in p(i).Similar, though less comprehensive, experiments were done with Gossypium hirsutum, and yielded similar conclusions, except that the proportionality between A and g at normal ambient partial pressure of CO(2) implied P(i) approximately 200 microbars. PMID- 16664334 TI - Leaf Conductance in Relation to Rate of CO(2) Assimilation: II. Effects of Short Term Exposures to Different Photon Flux Densities. AB - When photon flux density incident on attached leaves of Zea mays L. was varied from the equivalent of 0.12 of full sunlight to full sunlight, leaf conductance to CO(2) transfer, g, changed in proportion to the change in rate of CO(2), assimilation, A, with the result that intercellular partial pressure of CO(2) remained almost constant. The proportionality was the same as that previously found in g and A measured at one photon flux density in plants of Zea mays L. grown at different levels of mineral nutrition, light intensities, and ambient partial pressures of CO(2). In shade-grown Phaseolus vulgaris L. plants, A as photon flux density was increased from about 0.12 up to about 0.5 full sunlight, the proportionality being almost the same in plants grown at low and at high light intensity.When photon flux density incident on the adaxial and abaxial surfaces of the isolateral leaves of Eucalyptus pauciflora Sieb. ex Spreng was varied, g and A also varied proportionally. The leaf conductance in a particular surface was affected by the photon flux density at the opposite surface to a greater extent than was expected on the basis of transmittance. The results indicated that stomata may, in some way, be sensitive to the photon flux absorbed within the leaf as a whole. PMID- 16664335 TI - Leaf Conductance in Relation to Rate of CO(2) Assimilation: III. Influences of Water Stress and Photoinhibition. AB - Rates of CO(2) assimilation and leaf conductances to CO(2) transfer were measured in plants of Zea mays during a period of 14 days in which the plants were not rewatered, and leaf water potential decreased from -0.5 to -8.0 bar. At any given ambient partial pressure of CO(2), water stress reduced rate of assimilation and leaf conductance similarly, so that intercellular partial pressure of CO(2) remained almost constant. At normal ambient partial pressure of CO(2), the intercellular partial pressure of CO(2) was estimated to be 95 microbars. This is the same as had been estimated in plants of Zea mays grown with various levels of nitrogen supply, phosphate supply and irradiance, and in plants of Zea mays examined at different irradiances.After leaves of Phaseolus vulgaris L. and Eucalyptus pauciflora Sieb. ex Spreng had been exposed to high irradiance in an atmosphere of CO(2)-free N(2) with 10 millibars O(2), rates of assimilation and leaf conductances measured in standard conditions had decreased in similar proportions, so that intercellular partial pressure of CO(2) remained almost unchanged. As the conductance of each epidermis that had not been directly irradiated had declined as much as that in the opposite, irradiated surface it was hypothesized that conductance may have been influenced by photoinhibition within the mesophyll tissue. PMID- 16664336 TI - UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons. AB - The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase beta-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by beta-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons. PMID- 16664337 TI - Binding of a Phosphorylated Inhibitor to Ribulose Bisphosphate Carboxylase/Oxygenase during the Night. AB - The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured at various times during the purification of the enzyme from leaves of Nicotiana tabacum which were collected either 1 hour before the start of the photoperiod (predawn) or in the middle of the photoperiod (midday). The activity of the enzyme in extracts of the predawn leaves (0.8 units/mg enzyme) was consistently about 2-fold lower than that measured in extracts of midday leaves (1.7 units/mg enzyme). The activity of the predawn enzyme was increased to that of the midday enzyme following removal of CO(2) and Mg(2+) (deactivation), (NH(4))(2)SO(4) precipitation, or incubation in SO(4) (2-) (18 millimolar required for one-half maximal increase). Following purification to >95% homogeneity, the predawn enzyme was found to have approximately 0.5 moles of bound organic phosphate per mole of enzyme active sites, while the midday enzyme had only approximately 0.08 moles of bound organic phosphate per mole of enzyme active sites. Deactivation of the predawn enzyme or treatment with 0.2 molar SO(4) (2-) resulted in the removal of most of the bound organic phosphate. These findings support the hypothesis that following the night period about 50% of the enzyme is catalytically inactive because of the tight-binding of a small molecular weight, phosphorylated inhibitor at the active site. PMID- 16664338 TI - Nature and Amount of Auxin in Algae : IAA from Extracts of Caulerpa paspaloides (Siphonales). AB - Evidence for the occurrence of indole 3-acetic acid in Caulerpa paspaloides extracts was obtained by bioassay, by high-performance liquid chromatography with an electrochemical detector, and by capillary gas chromatography combined with mass spectrometry. The amount of indole 3-acetic acid present was estimated to be about 1 milligram per kilogram fresh weight, with an error limit of one order of magnitude. This is in the range reported from angiosperms. PMID- 16664339 TI - Partial purification and characterization of granule-bound starch synthases from normal and waxy maize. AB - The granule-bound starch-synthases from normal and waxy maize kernels have been solubilized, partially purified, and characterized. Two broad categories of starch synthases were revealed with representatives in the soluble phase and also on granules from both normal and waxy maize though the activity associated with granules from waxy was small. Data for native molecular weights, kinetic parameters, and immunological relatedness are used to demonstrate that the granule-bound isozymes from normal maize are different from the soluble enzymes. These distinct granule-bound enzymes are missing in waxy maize granules and a further novel form of starch synthase is revealed. These findings are discussed in relation to the type of starch produced in tissue affected by the waxy mutation. PMID- 16664340 TI - Analysis of Photosynthetic Antenna Function in a Mutant of Arabidopsis thaliana (L.) Lacking trans-Hexadecenoic Acid. AB - Several lines of evidence support the proposal that the unusual chloroplast specific lipid acyl group Delta3,trans-hexadecenoic acid (trans-C(16:1)) stimulates the formation or maintenance of the oligomeric form of the light harvesting chlorophyll a/b complex (LHCP). To assess the functional significance of this apparent association we have analyzed LHCP structure and function in a mutant of Arabidopsis thaliana (L.) which lacks trans-C(16:1) by electrophoretic analysis of the protein-chlorophyll complexes and by measurements of chlorophyll fluorescence under a variety of conditions. By these criteria the putative oligomeric form of LHCP appears to be slightly more labile to detergent-mediated dissociation in the mutant. The oligomeric PSI chlorophyll-protein complex, associated with PSI, was also more labile to detergent-mediated dissociation in the mutant, suggesting a previously unsuspected association of trans-C(16:1) with the PSI complex. However, no significant effect of the mutation on the efficiency of energy transfer from LHCP to the photochemical reaction centers was observed under any of the various conditions imposed. Also, the stability of the chlorophyll-protein complexes to temperature-induced dissociation was unaffected in the mutant. The role of trans-C(16:1) is very subtle or is only conditionally expressed. PMID- 16664341 TI - Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway during Induction of Photosynthesis in Maize Leaves. AB - Changes in the level of metabolites of the C(4) cycle and reductive pentose phosphate (RPP) pathway were measured simultaneously with induction of photosynthesis in maize (Zea mays L.) to evaluate what may limit carbon assimilation during induction in a C(4) plant.After 20 minutes in the dark, there was an immediate rise in photosynthesis during the first 30 seconds of illumination, followed by a gradual rise approaching steady-state rate after 20 minutes of illumination. Among metabolites of the C(4) cycle, there was a net increase in the level of C(3) compounds (the sum of pyruvate, alanine, and phosphoenolpyruvate) during the first 30 seconds of illumination, while there was a net decrease in the level of C(4) acids (malate plus aspartate). The total level of metabolites of the C(4) cycle underwent a sharp increase during this period. At the same time, there was a sharp rise in the level of intermediates of the RPP pathway (ribulose-1,5-bis-phosphate, 3-phosphoglycerate, dihydroxyacetonephosphate, and fructose-1,6-bisphosphate) during the first minute of illumination. The net increase of carbon among intermediates of the C(4) cycle and RPP pathway was far above that of carbon input from CO(2) fixation, and the increase in intermediates of the RPP pathway could not be accounted for by decarboxylation of C(4) acids, suggesting that an endogenous source of carbon supplies the cycles. After 3 minutes of illumination there was a gradual rise in the levels of intermediates of the C(4) cycle and in the total level of metabolites measured in the RPP pathway. This rise in metabolite levels occurs as photosynthesis gradually increases and may be required for carbon assimilation to reach maximum rates in C(4) plants. This latter stage of inductive autocatalysis through the RPP pathway may contribute to the final buildup of these intermediates. PMID- 16664342 TI - Membrane transport in isolated vesicles from sugarbeet taproot : I. Isolation and characterization of energy-dependent, h-transporting vesicles. AB - Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H(+) transport, and this is consistent with the observation that H(+)-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N' dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I(-) > Br(-) > Cl(-) while F(-) was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl(-), alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H(+)/alkali cation exchange. Based upon the properties of the H(+)-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary. PMID- 16664343 TI - Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport. AB - The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [(14)C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [(14)C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H(+)/sucrose antiport may be operating on the vesicle membrane. PMID- 16664344 TI - Reprogramming of Protein Synthesis from a Developmental to a Germinative Mode Induced by Desiccation of the Axes of Phaseolus vulgaris. AB - Immature seeds of Phaseolus vulgaris cv Taylor's Horticultural removed from the pod at 32 days of development do not germinate unless first subjected to desiccation. Our results show that premature drying not only redirects metabolism from a developmental to a germination program but it does so permanently, thus effecting an irreversible switch. This is shown by in vitro protein synthesis, and analysis of poly(A)(+) mRNA with a cDNA probe specific for phaseolin message. For example, the pattern of proteins synthesized in vitro by the mRNA fraction from fresh and prematurely dried axes show strong similarities; on the other hand, the mRNA population from rehydrated axes code for a different set of proteins. Also, the message for phaseolin is preserved following the normal maturation process and premature desiccation of seeds. Following rehydration of immature seeds at the desiccation-tolerant stage, this message is no longer detectable in the axes. PMID- 16664345 TI - Effect of Leaf Detachment on Chlorophyll Fluorescence during Chilling Experiments. AB - The effect of leaf detachment on chlorophyll fluorescence was analyzed for Zea mays, Cucumis sativus, Phaseolus vulgaris, and Echinochloa crus-galli. Results clearly indicate that detachment hastens the decrease in chlorophyll fluorescence during the course of chilling experiments. For maize and bean, the activity of photosystem II of chloroplasts isolated from detached leaves is lower than that of chloroplasts isolated from attached leaves. There are also large differences in ionic loss between detached and attached leaves of barnyard grass which could correlate with changes in leaf water status. The detached leaves lost some 50% of their total ionic content. Finally, detachment alters the ranking of the species with regard to their chilling tolerance. PMID- 16664346 TI - High temperature-induced thermotolerance in pollen tubes of tradescantia and heat shock proteins. AB - Growing pollen tubes of Tradescantia paludosa are protected from inhibition of growth at 41 degrees C by a prior exposure to gradually increasing temperatures. Heat shock proteins (hsps) are not synthesized by pollen tubes as determined by labeling with [(35)S]methionine and two-dimensional gel electrophoresis, during either a heat shock at 41 degrees C or a gradual temperature increase to 41 degrees C. A comparison after two-dimensional electrophoresis of silver-stained spots and radioactive spots after autoradiography of an extract of ungerminated pollen mixed with a trace amount of [(35)S]methionine-labeled vegetative tissue heat shocked at 41 degrees C to act as a hsps marker, indicates that the majority, if not all, of the major hsps are not present in the pollen grain at anthesis. The type of thermotolerance seen with pollen tubes can thus be achieved without the presence or the new synthesis of the hsps. PMID- 16664347 TI - Decrease in Three Messenger RNA Species Coding for Chloroplast Proteins in Leaves of Barley Infected with Erysiphe graminis f. sp. hordei. AB - Cloned hybridization probes have been used to investigate the effect of infection of susceptible Hordeum vulgare cv Prior by Erysiphe graminis f. sp. hordei on the abundance of host mRNAs coding for the large (LSU) and precursor to the small (SSU) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase and the rapidly metabolized 32kD thylakoid protein (32kDP). In leaf RNA preparations from control (noninoculated) plants the amount of mRNA for the LSU and SSU declined from 7 to 11 days after sowing, whereas that for the 32kDP increased over this period. Following inoculation at 6 days after sowing, the abundance of each of the mRNA species was significantly reduced below that of controls at 1, 3, and 5 days later. Results indicate that infection causes a rapid and extensive reduction in host mRNA species coding for proteins with important photosynthetic functions. PMID- 16664348 TI - Degradation of beta-Conglycinin in Early Stages of Soybean Embryogenesis. AB - This study focuses on the role of protein turnover in the accumulation of the storage protein beta-conglycinin (7S protein) during soybean embryogenesis. The results of pulse:chase experiments using (3)H-leucine indicate that the turnover of the subunits of beta-conglycinin by proteolysis is more rapid in early stages of cotyledon maturation than in later stages. PMID- 16664349 TI - Identification of Ten Gibberellins from Sporophytes of the Tree fern, Cyathea australis. AB - Ten gibberellins (GAs) have been identified by Kovats retention indices and full mass spectra from GC-MS analysis of purified extracts of sporophytes of the tree fern, Cyathea australis. These include the known GA(1), GA(4), GA(9), GA(15), GA(24), GA(35), and GA(58) and three new GAs, 12beta-hydroxyGA(9) (GA(69)), 12alpha-hydroxyGA(9) (GA(70)) and 12beta-hydroxyGA(4) (GA(71)). The structure of GA(71) was established by the preparation and characterization of its methyl ester (as a metabolite of GA(4) methyl ester in a culture of prothallia of Lygodium japonicum). PMID- 16664350 TI - Phytoluminographic detection of dynamic variations in leaf gaseous conductivity. AB - Gas exchange and plant luminescence (delayed light emission) of a single red kidney bean leaf undergoing synchronous oscillations in gas exchange were recorded and analyzed. Introduction of 1.1 microliter per liter SO(2) during these oscillations produced increases in plant luminescence that, when averaged over a portion of the leaf, oscillated in phase with the gas exchange oscillations. However, examination of a video record of the plant luminescence showed not only that luminescence intensities tended to be localized within discrete areas of the leaf, but that the time-dependence of luminescence intensities within these regions varied considerably from the period, amplitude, and often phase of the overall gas exchange oscillations. The video recording also showed that changes in luminescence intensities appeared to migrate across the leaf in wave-like patterns. These data are interpreted in terms of localized fluctuations in gaseous conductances of the leaf. PMID- 16664351 TI - Chloroplast Biogenesis 49 : Differences among Angiosperms in the Biosynthesis and Accumulation of Monovinyl and Divinyl Protochlorophyllide during Photoperiodic Greening. AB - Various angiosperms differed in their monovinyl and divinyl protochlorophyllide biosynthetic capabilities during the dark and light phases of photoperiodic growth. Some plant species such as Cucumis sativus L., Brassica juncea (L.) Coss., Brassica kaber (DC.) Wheeler, and Portulaca oleracea L. accumulated mainly divinyl protochlorophyllide at night. Monocotyledonous species such as Avena sativa L., Hordeum vulgare L., Triticum secale L., Zea mays L., and some dicotyledonous species such as Phaseolus vulgaris L., Glycine max (L.) Merr., Chenopodium album L., and Lycopersicon esculentum L. accumulated mainly monovinyl protochlorophyllide at night.Under low light intensities meant to simulate the first 60 to 80 minutes following daybreak divinyl protochlorophyllide appeared to contribute much more to chlorophyll formation than monovinyl protochlorophyllide in species such as Cucumis sativus L. Under the same light conditions, species which accumulated mainly monovinyl protochlorophyllide at night appeared to form chlorophyll preferably via monovinyl protochlorophyllide.THESE RESULTS WERE INTERPRETED IN TERMS OF: (a) a differential contribution of monovinyl and divinyl protochlorophyllide to chlorophyll formation at daybreak in various plant species; and (b) a differential regulation of the monovinyl and divinyl protochlorophyllide biosynthetic routes by light and darkness. PMID- 16664352 TI - Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots. AB - Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared. PMID- 16664353 TI - In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1. AB - Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M(r)) precursor was converted to the M(r) 29,500 thylakoid membrane polypeptide whereas the M(r) 30,000 precursor was converted to the M(r) 26,000 product. Furthermore, the M(r) 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M(r) 29,500, although the presence of antibodies did not prevent the precursor of M(r) 30,000 from being converted to the mature M(r) 26,000 polypeptide. The mature fraction of M(r) 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes. PMID- 16664354 TI - Alterations in Growth, Photosynthesis, and Respiration in a Starchless Mutant of Arabidopsis thaliana (L.) Deficient in Chloroplast Phosphoglucomutase Activity. AB - A mutant of Arabidopsis thaliana (L.) Heynh. which lacks leaf starch was isolated by screening for plants which did not stain with iodine. The starchless phenotype, confirmed by quantitative enzymic analysis, is caused by a single recessive nuclear mutation which results in a deficiency of the chloroplast isozyme of phosphoglucomutase. When grown in a 12-h photoperiod, leaves of the wild-type accumulated substantial amounts of starch but lower levels of soluble sugars. Under these conditions, the mutant accumulated relatively high levels of soluble sugars. Rates of growth and net photosynthesis of the mutant and wild type were indistinguishable when the plants were grown in constant illumination. However, in a short photoperiod, the growth of the mutant was severely impaired, the rate of photosynthesis was depressed relative to the wild-type, and the rate of dark respiration, which was high following the onset of darkness, exhibited an uncharacteristic decay throughout the dark period. The altered control of respiration by the mutant, which may be related to the relatively high levels of soluble carbohydrate that accumulate in the leaf and stem tissue, is believed to be partially responsible for the low growth rate of the mutant in short days. The depressed photosynthetic capacity of the mutant may also reflect a metabolic adaptation to the accumulation of high levels of soluble carbohydrate which mimics the effects of alterations in source/sink ratio. The activities of sucrose phosphate synthase and acid invertase are significantly higher in the mutant than in the wild-type whereas ADP-glucose pyrophosphorylase activity is lower. This suggests that the activities of these enzymes may be modulated in response to metabolite concentrations or flux through the pathways. PMID- 16664355 TI - Hexose kinases from the plant cytosolic fraction of soybean nodules. AB - The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K(m) 0.075 millimolar) than fructose (K(m) 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K(+) ions for maximum activity. The enzyme was specific for fructose (apparent K(m) 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 +/- 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed. PMID- 16664356 TI - Adaptation of Tobacco Cells to NaCl. AB - Cell lines of tobacco (Nicotiana tabacum L. var Wisconsin 38) were obtained which are adapted to grow in media with varying concentrations of NaCl, up to 35 grams per liter (599 millimolar). Salt-adapted cells exhibited enhanced abilities to gain both fresh and dry weight in the presence of NaCl compared to cells which were growing in medium without NaCl (unadapted cells). Tolerance of unadapted cells and cells adapted to 10 grams per liter NaCl was influenced by the stage of growth, with the highest degree of tolerance exhibited by cells in the exponential phase. Cell osmotic potential and turgor varied through the growth cycle of unadapted cells and cells at all levels of adaptation, with maximum turgor occurring at approximately the onset of exponential fresh weight accumulation.Adaptation to NaCl led to reduced cell expansion and fresh weight gain, while dry weight gain remained unaffected. This reduction in cell expansion was not due to failure of the cells to maintain turgor since cells adapted to NaCl underwent osmotic adjustment in excess of the change in water potential caused by the addition of NaCl to the medium. Tolerance of the adapted cells, as indicated by fresh or dry weight gain, did not increase proportionately with the increase in turgor. Adaptation of these glycophytic cells to NaCl appears to involve mechanisms which result in an altered relationship between turgor and cell expansion. PMID- 16664357 TI - Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl. AB - Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate (35)S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26 kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress. PMID- 16664358 TI - Abscisic Acid accelerates adaptation of cultured tobacco cells to salt. AB - Adaptation of tobacco (Nicotiana tabacum L. var Wisconsin 38) cells to NaCl was accelerated by (+/-) abscisic acid (ABA). In medium with 10 grams per liter NaCl, ABA stimulated the growth of cells not grown in medium with NaCl (unadapted, S-0) with an increasing response from 10(-8) to 10(-4) molar. ABA (10(-5) molar) enhanced the growth of unadapted cells in medium with 6 to 22 grams per liter NaCl but did not increase the growth of cells previously adapted to either 10 (S 10) or 25 (S-25) grams per liter NaCl unless the cells were inoculated into medium with a level of NaCl higher than the level to which the cells were adapted. The growth of unadapted cells in medium with Na(2)SO(4) (85.5 millimolar), KCl (85.5 or 171 millimolar), K(2)SO(4) (85.5 millimolar) was also stimulated by ABA. ABA (10(-8)-10(-4) molar) did not accelerate the growth of unadapted cells exposed to water deficits induced by polyethylene glycol (molecular weight 8000) (5-20 grams per 100 milliliters), sorbitol (342 millimolar), mannitol (342 millimolar) or sucrose (342 millimolar). These results suggest that ABA is involved in adaptation of cells to salts, and is not effective in promoting adaptation to water deficits elicited by nonionic osmotic solutes. PMID- 16664359 TI - Importance of Environmental pH during Root Development on Phosphate Absorption. AB - Wheat seedlings (Triticum aestivum L. cv Gamenya) were grown for 4 days in culture solutions of differing pH prior to studying their subsequent short-term absorption of (32)Pi from solutions of the same or different pH.Increasing pH of the absorption solution from 5.5 to 7.0 or 8.0 depressed (32)Pi absorption from 1 and 10 micromolar Pi but had little effect at 100 and 1000 micromolar Pi. Increasing the pH of the culture solution from 4.5 to 6.5 doubled or trebled subsequent (32)Pi absorption from nearly all absorption solutions over a wide range of Pi concentrations, pH, and nutrient compositions.When seedlings were transferred between culture pH treatments 4.5 and 6.5, their capacity for (32)Pi absorption remained unchanged for at least 5 hours and adjusted by 60 to 80% after 24 hours and completely after 48 hours. This suggests that the root's capacity to absorb Pi responds to pH through slow structural changes in its mechanism of Pi absorption. P content and concentration of wheat seedlings reflected the response of (32)Pi absorption to culture pH.It is suggested that absorption pH affects an activity component of the process for Pi absorption and culture pH affects a capacity component. Failure to recognize the capacity component of the pH response explains why previously published results for short term (32)Pi absorption conflict with those for long-term P accumulation in plants. PMID- 16664360 TI - Interactions between Mild NaCl Stress and Red Light during Lettuce (Lactuca sativa L. cv Grand Rapids) Seed Germination. AB - The sensitivity of lettuce (Lactuca sativa L. cv Grand Rapids) seeds to red light was reduced by NaCl concentrations which had no effect upon the germination of continuously illuminated seeds. The germination capacity of the seeds was fully restored by increased red light exposures. Indirect evidence indicates that NaCl does not affect the photoconversion of red-absorbing form of phytochrome to the far-red absorbing form of phytochrome. Instead, the increased red light requirements are attributable to increases in the threshold levels of the far-red absorbing form of phytochrome necessary to induce germination and to changes in the slopes of the fluence-response curves. Results also show that the sensitivity of the seeds to NaCl decreased as the time between red light irradiation and the imposition of NaCl stress increased. PMID- 16664361 TI - Direct Observation of Reversible and Irreversible Stomatal Responses of Attached Sunflower Leaves to SO(2). AB - The effects of SO(2) on stomatal aperture of attached sunflower leaves were observed with a remote-control light microscope system that permitted continuous observation of stomatal responses over periods of several hours. The relationship between actual stomatal aperture and stomatal conductance, measured with a porometer, also was examined on leaves before and after exposure to SO(2).A distinction between uninjured and injured regions was clearly visible on leaves after exposure to 1.5 microliters per liter SO(2) for less than an hour. During the exposure, the mean value of apertures for many stomata, which indicates stomatal conductance and transpiration rate, tended to decrease simultaneously in the uninjured and injured regions. However, the rate of decrease in the injured region was slower than that in the uninjured region because of a transient opening induced by water-soaking in the injured region. The transient opening was less common in stomata near veins and veinlets.There was a good correlation between pore width and stomatal conductance measured with a porometer before exposure to SO(2). This correlation continued in leaves exposed to SO(2) until visible, irreversible injury occurred, but then it disappeared.The results of these experiments indicate the necessity of continuous observation of individual stomata under the microscope to understand the effects of air pollutants such as SO(2) on stomatal behavior. PMID- 16664362 TI - Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles. AB - Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H(+)-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H(+)-transport. At saturating (3 millimolar) concentrations of Mg(2+):ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H(+)-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO(3), unlike the ATP-dependent H(+)-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H(+)-translocating pyrophosphatases. Both potassium and a permanent anion (NO(3) (-) > Cl(-)), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N' dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1 phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction. PMID- 16664363 TI - CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays. AB - Conditions for optimal CO(2) fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO(2) fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO(2) assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V(max) for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C(3) plants. PMID- 16664364 TI - Oxygen-Dependent Exclusion of Sodium Ions from Shoots by Roots of Zea mays (cv Pioneer 3906) in Relation to Salinity Damage. AB - Using radio-tracers, we measured Na(+) and K(+) accumulation in roots and transport to shoots in Zea mays (cv Pioneer 3906) as a function of NaCl concentration and O(2) partial pressure in the nutrient solution. Under fully aerobic conditions, roots partially excluded Na(+) from the shoots over a wide range of NaCl concentration (0.2-200 millimolar). With root anoxia, the exclusion mechanism broke down so that much greater amounts of Na(+) reached the shoots, with simultaneous inhibition of K(+) transport. The ratio Na(+)/K(+) entering the shoot consequently increased 90 to 200 times. Increases in Na(+) transport were first detected when the O(2) partial pressure was reduced from ambient (21% v/v) to 15%, whereas K(+) transport was not inhibited until O(2) concentrations were <5%. Since soil O(2) deficiency can often accompany high salinity in irrigation agriculture, failure of the Na(+) exclusion mechanism may be a contributory factor in salinity damage of salt-sensitive glycophytes. PMID- 16664365 TI - Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii. AB - Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO(2) is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO(2) required for the half-maximal rate of photosynthetic O(2) evolution (K(0.5)[CO(2)]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO(2) fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K(0.5)(CO(2)) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO(2) is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO(2) from HCO(3) (-) at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO(3) (-) transport for internal accumulation might occur at the level of the chloroplast envelope. PMID- 16664366 TI - An evaluation of the role of ethylene in herbicidal injury induced by picloram or clopyralid in rapeseed and sunflower plants. AB - The role of ethylene in herbicidal injury induced by 4-amino-3,5,6 trichloropicolinic acid (picloram) or 3,6-dichloropicolinic acid (clopyralid) was investigated in sunflower (Helianthus annuus L.) and rapeseed (Brassica napus L. cv Altex). Picloram induces herbicide injury in both species, whereas clopyralid induces injury only in sunflower. Picloram applied to the third leaf of a rapeseed plant increased ethylene evolution several-fold. Clopyralid had no effect on ethylene production in rapeseed. In sunflower, both picloram and clopyralid elevated ethylene production. Ethylene biosynthesis induced by the herbicide treatment was not restricted to treated areas. When clopyralid was applied only to the lower stem and cotyledons of sunflower, the herbicide treatment resulted in an increase in the rate of ethylene production from the true leaves. Increased ethylene production preceded or coincided with the onset of morphological responses induced by a herbicide application to a susceptible species. The contrast in ethylene production by these two plant species cannot be accounted for by differences in absorption and translocation of clopyralid and picloram.Treatment with aminoethoxyvinylglycine (AVG) before picloram or clopyralid application prevented an increase in ethylene production. Pretreatment with AVG also delayed the development of morphological changes induced by picloram or clopyralid. It appears that enhanced ethylene biosynthesis after application of picloram or clopyralid to the susceptible plant species was a factor involved in resulting morphological changes. PMID- 16664367 TI - Errors in measuring water potentials of small samples resulting from water adsorption by thermocouple psychrometer chambers. AB - The adsorption of water by thermocouple psychrometer assemblies is known to cause errors in the determination of water potential. Experiments were conducted to evaluate the effect of sample size and psychrometer chamber volume on measured water potentials of leaf discs, leaf segments, and sodium chloride solutions. Reasonable agreement was found between soybean (Glycine max L. Merr.) leaf water potentials measured on 5-millimeter radius leaf discs and large leaf segments. Results indicated that while errors due to adsorption may be significant when using small volumes of tissue, if sufficient tissue is used the errors are negligible. Because of the relationship between water potential and volume in plant tissue, the errors due to adsorption were larger with turgid tissue. Large psychrometers which were sealed into the sample chamber with latex tubing appeared to adsorb more water than those sealed with flexible plastic tubing. Estimates are provided of the amounts of water adsorbed by two different psychrometer assemblies and the amount of tissue sufficient for accurate measurements of leaf water potential with these assemblies. It is also demonstrated that water adsorption problems may have generated low water potential values which in prior studies have been attributed to large cut surface area to volume ratios. PMID- 16664368 TI - Leaf k interaction with water stress inhibition of nonstomatal-controlled photosynthesis. AB - The relationship between leaf K(+) concentration, in vitro dehydration, and nonstomatal-controlled photosynthesis was investigated using leaf slices that were vacuum infiltrated with media containing varying sorbitol concentrations. The leaf slices were from plants either supplied with complete or K(+)-deficient medium throughout a 35-day growth period. During this time, leaf K(+) concentration, water potential, osmotic potential, and turgor pressure were monitored. Leaf K(+) concentration averaged 239 micomoles per gram (fresh weight) in control plants, and dropped to 74.3 micromoles per gram (fresh weight) in K(+) deficient plants. Less negative osmotic potentials and resultant turgor loss in K(+)-deficient plants indicated that the osmotically active pool of cellular K(+) was lower in those plants.The decrease in leaf K(+) concentration enhanced the dehydration inhibition of photosynthesis. For example, increasing sorbitol from 0.33 to 0.5 molar during incubation inhibited photosynthesis in the controls by 14% or less. This same protocol resulted in an inhibition of photosynthesis by as much as 41% in K(+)-deficient tissue. In contrast to the data obtained with leaf slices, dehydration inhibition of isolated chloroplast photosynthesis was not affected by K(+) status of parent plant material. These data are consistent with the hypothesis that one effect of leaf K(+) deficiencies on photosynthetic response to dehydration may be mediated by extra-choloroplastic factors.Ammonium ions, which facilitate stromal alkalinization, reversed the increased sensitivity of K(+)-deficient leaf slice photosynthesis to cell dehydration. However, NH(4) (+) had no effect on photosynthesis of K(+)-deficient leaf slices under nonhypertonic conditions. These data suggest that endogenous extra-chloroplastic K(+) may modulate dehydration inhibition of photosynthesis, possibly by facilitating stromal alkalinization. PMID- 16664369 TI - Ethylene and the growth of rice seedlings. AB - Etiolated whole rice seedlings enclosed in sealed vials produced ethylene at a rate of 0.9 picomole per hour per seedling. When 2-centimeter-long shoots were subdivided into 5-millimeter-long sections, the sections containing the tip of the shoot evolved 37% of the total ethylene with the remaining 63% being produced along a gradient decreasing to the base of the shoot. The tip of the coleoptile also had the highest level of the ethylene precursor 1-aminocyclopropane-1 carboxylic acid and of the ethylene-forming enzyme activity. Ethylene is one of the factors controlling coleoptile elongation. Decapitation of the seedling reduced ethylene evolution to one-third its original level and inhibited coleoptile growth. In short-term experiments, the growth rate of decapitated seedlings was restored to almost that of intact seedlings by application of ethylene at a concentration of 10 microliters per liter. Apart from ethylene, O(2) also participates in the control of coleoptile growth. When rice seedlings were grown in a gas mixture of N(2) and O(2), the length of the coleoptiles reached a maximum at a concentration of 2.5% O(2). Lower and higher concentrations of O(2) reduced coleoptile growth. The effect of exogenous ethylene on coleoptile growth was also O(2) dependent. PMID- 16664370 TI - Effects of CO(2) and O(2) on the Photosynthetic O(2) Evolution of Spirodela polyrrhiza Turions. AB - Net photosynthetic rates of Spirodela polyrrhiza turions, at low O(2) levels, were 6.2 and 38.8 micromoles O(2) per gram fresh weight per hour at 1 millimolar HCO(3) (-) and CO(2) saturation, respectively, and much lower in a regular low-pH growth solution. Air equilibration O(2) concentrations decreased rates considerably, except at CO(2) saturation. The surfacing rate of turions in various inorganic carbon surroundings correlated positively with their photosynthetic rates, but were the same at high and low O(2) levels. The relevance of these findings in relation to environmental conditions conductive to germination of autotrophically growing turions is discussed. PMID- 16664371 TI - Temperature Dependence of Carbon Isotope Fractionation in CAM Plants. AB - The carbon isotope fractionation associated with nocturnal malic acid synthesis in Kalanchoe daigremontiana and Bryophyllum tubiflorum was calculated from the isotopic composition of carbon-4 of malic acid, after appropriate corrections. In the lowest temperature treatment (17 degrees C nights, 23 degrees C days), the isotope fractionation for both plants is -4 per thousand (that is, malate is enriched in (13)C relative to the atmosphere). For K. daigremontiana, the isotope fractionation decreases with increasing temperature, becoming approximately 0 per thousand at 27 degrees C/33 degrees C. Detailed analysis of temperature effects on the isotope fractionation indicates that stomatal aperture decreases with increasing temperature and carboxylation capacity increases. For B. tubiflorum, the temperature dependence of the isotope fractionation is smaller and is principally attributed to the normal temperature dependences of the rates of diffusion and carboxylation steps. The small change in the isotopic composition of remaining malic acid in both species which is observed during deacidification indicates that malate release, rather than decarboxylation, is rate limiting in the deacidification process. PMID- 16664372 TI - Displacement of ca by na from the plasmalemma of root cells : a primary response to salt stress? AB - A microfluorometric assay using chlorotetracycline (CTC) as a probe for membrane associated Ca(2+) in intact cotton (Gossypium hirsutum L. cv Acala SJ-2) root hairs indicated displacement of Ca(2+) by Na(+) from membrane sites with increasing levels of NaCl (0 to 250 millimolar). K(+)((86)Rb) efflux increased dramatically at high salinity. An increase in external Ca(2+) concentration (10 millimolar) mitigated both responses. Other cations and mannitol, which did not affect Ca(2+)-CTC chelation properties, were found to have no effect on Ca(2+) CTC fluorescence, indicating a Na(+)-specific effect. Reduction of Ca(2+)-CTC fluorescence by ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid, which does not cross membranes, provided an indication that reduction by Na(+) of Ca(2+)-CTC fluorescence may be occurring primarily at the plasmalemma. The findings support prior proposals that Ca(2+) protects membranes from adverse effects of Na(+) thereby maintaining membrane integrity and minimizing leakage of cytosolic K(+). PMID- 16664373 TI - Localization, purification, and characterization of shikimate oxidoreductase dehydroquinate hydrolyase from stroma of spinach chloroplasts. AB - The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The chromatographic behavior of the bifunctional protein SORase/DHQase on several separation materials with extracts from stroma compared with leaf extracts showed only one peak of enzymic activity originating from the stroma. (b) Polyacrylamide gel electrophoresis (PAGE) of these extracts followed by specific staining resulted in the same pattern without a band of extraplastidic enzyme. (c) In protoplast fractionation experiments it was shown that SORase/DHQase was present only in the soluble chloroplast protein fraction.An improved purification procedure for SORase/DHQase from stroma of chloroplasts, yield 40%, 1600 times as pure, gave essentially one protein band on sodium dodecyl sulfate-PAGE. Our results demonstrate that both enzyme functions are carried out by a single polypeptide. Nondenaturing PAGE exhibited a pattern of four bands with SORase/DHQase showing that they differ in charge but not in their molecular weight. Molecular weight was determined to be 67 kilodaltons (gel filtration) and 59 kilodaltons (PAGE) for all four forms. It was proven they were not due to artifacts. The four forms show similar kinetic properties, their K(m) and pH optima differing only very slightly. Response to some metabolites is reported. PMID- 16664374 TI - Physiological potential for survival of propagules of crassulacean Acid metabolism species. AB - Terminal stem joints from three opuntias were detached and maintained for 160 days under natural climatic conditions in the winter and summer. Neither Crassulacean acid metabolism (CAM) nor CAM-idling, as evidenced by a diurnal malate flux, was maintained throughout the two periods; ceasing earlier in the summer period. A 13 to 20% fresh weight loss occurred over the winter period, as opposed to a 30 to 40% loss over the summer period, although tissue water potentials remained above -1.5 megapascals. Chlorophyll and protein contents remained essentially constant in the winter but decreased in the summer. Starch content decreased slightly over the winter but more significantly over the summer. Mucilage content increased slightly in winter and declined slightly in summer. The initiation of rooting was found to be inversely related to spine density and dependent upon orientation and season. Comparison of these data suggest rooting coincided with the cessation of CAM-idling in both climatic periods and was uncoupled from the occurrence of precipitation. The physiological limit for survival of these propagules after detachment was lower than anticipated being of only a few months' duration. PMID- 16664375 TI - O(2) uptake in the light in chlamydomonas: evidence for persistent mitochondrial respiration. AB - The nature of the process responsible for the stationary O(2) uptake occurring in the light under saturating CO(2) concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O(2) uptake and evolution in the algal suspension. First, we observed that the O(2) uptake rate was constant (about 0.5 micromoles of O(2) per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O(2) uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O(2) uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O(2) uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO(2) concentration or at the CO(2) compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O(2) uptake processes might be present in these two algal species. PMID- 16664376 TI - Chloroplast Protein Synthesis in the Chromophytic Alga Olisthodiscus luteus: Cell Cycle Analysis. AB - This study represents the first report on chloroplast protein synthesis during the synchronous cell growth of a chromophytic (chlorophyll a,c) plant. When the unicellular alga Olisthodiscus luteus is maintained on a 12-hour light:12-hour dark cycle, cell and chloroplast number double every 24 hours. A temporal separation between these two events occurs. Measurements of chloroplast and total cellular protein values suggest that polypeptide synthesis occurs mainly in the light portion of the cell cycle, and pulse chase studies demonstrate that chloroplast proteins made in the light are not degraded in the dark. Data support the following conclusions: (a) a similar complement of chloroplast DNA coded proteins is made at all phases of the light portion of the cell cycle, and (b) chloroplast protein synthesis is a light rather than a cell cycle mediated response. PMID- 16664377 TI - Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast. AB - Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (;Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [(14)C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of (14)C-label into sink leaves during steady state labeling of a source leaf with (14)CO(2). Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l [(14)C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[(14)C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll. PMID- 16664378 TI - Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves. AB - When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation. PMID- 16664379 TI - Pyruvate Decarboxylase from Zea mays L. : I. Purification and Partial Characterization from Mature Kernels and Anaerobically Treated Roots. AB - Pyruvate decarboxylase (PDC) was purified from mature, dry maize kernels and from roots of anaerobically treated maize seedlings and partially characterized. PDC was purified to a specific activity of 96 units per milligram protein from kernels and to 41 units per milligram protein from root. The subunit molecular masses were estimated to be 61,000 and 60,000 for kernel PDC and 59,000 and 58,000 for root PDC. The pH optimum for each enzyme was 5.8. Since the pH optimum is nearly one pH unit below the value reported for the cytoplasm of anaerobically metabolizing maize roots (pH 6.7 +/- 0.2), we investigated the effects of pH 5.8 and 6.6 on the cooperative kinetics observed for PDC from each source. The maximum Hill coefficients (n(H)) were much greater at each pH for the kernel PDC (pH 5.8, n(H) = 2.5 and pH 6.6, n(H) = 3.2) than for the root PDC (pH 5.8, n(H) = 1.4 and pH 6.6, n(H) = 1.8). The cooperative kinetics observed with respect to pyruvate were asymmetric. Potassium inhibited maize PDC and was competitive with pyruvate (root PDC K(i) = 16 millimolar and kernel PDC K(i) = 10 millimolar). PMID- 16664380 TI - Chlorophyll fluorescence characteristics associated with hydration level in pea cotyledons. AB - In order to study the effects of desiccation on a photosynthetic system, light harvesting and light-induced electron transport processes were examined in pea cotyledons at various moisture levels, using in vivo fluorescence excitation spectra and fluorescence induction kinetics. Water sorption isotherms yielded thermodynamic data that suggested very strong water binding between 4 to 11% water, intermediate sorption between water contents of 13 to 22%, and very weak binding at moisture contents between 24 to 32%. The fluorescence properties of the tissue changed with the moisture contents, and these changes correlated generally with the three regions of water binding. Peak fluorescence and fluorescence yield remained at low levels when water content was limited to the tightly bound regions, below 12%. Several new peaks appeared in the chlorophyll a excitation spectrum and both peak fluorescence and fluorescence yield increased at intermediate water-binding levels (12-22%). At moisture contents where water is weakly bound (>24%), peak fluorescence and fluorescence yield were maximum and the fluorescence excitation spectrum was unchanging with further increases in water content.The state of water is an important component in the energy transfer and electron transport system. At hydration levels where water is most tightly bound, energy transfer from pigments is limited and electron transport is blocked. At intermediate water binding levels, energy transfer and electron transport increase and, in the region of weak water binding, energy transfer and electron transport are maximized. PMID- 16664381 TI - Intracellular calcium and calmodulin involvement in protoplast fusion. AB - (45)Ca(2+) uptake was compared between fusogenic and nonfusogenic Daucus carota L. protoplasts. Fusogenic protoplasts took 10 minutes to reach calcium equilibrium compared to 5 minutes in the nonfusogenic protoplasts. Intracellular stores of calcium were manipulated by isolating protoplasts in different calcium regimes. Lowering of intracellular calcium lowered fusion potential, while raising intracellular stores of calcium enhanced fusion potential. Regardless of the amount of calcium sequestered in a store, mobilization with A23187 increased fusion levels within 10 minutes. Calmodulin antagonists were potent inhibitors of protoplast fusion. This inhibition was obtained by treating cells with the calmodulin antagonists during protoplast isolation. A23187, however, only allowed a partial recovery from this inhibition, indicating that calcium flux alone was not sufficient for maximum fusion potential. On the basis of the evidence presented, we propose that calcium fluxes during protoplast isolation activate a calmodulin-mediated biochemical process that is necessary for the formation or maintenance of a fusion permissive state. PMID- 16664382 TI - Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization. AB - Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [(14)C]acetate or [(14)C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3 phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [(14)C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid --> CDP-diacylglycerol -->- > phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane. PMID- 16664383 TI - Physiological changes in cultured sorghum cells in response to induced water stress : I. Free proline. AB - Ten varieties of Sorghum bicolor (L.) Moench were grown as callus cultures under conditions of water stress, which was induced by addition of polyethylene glycol (molecular weight 8000) in the medium. Growth and free proline were estimated in the control and water-stressed cultures. In all varieties, proline levels were low in the absence of water stress and the levels increased in response to water stress. However, the magnitude of these increases were not correlated with stress tolerance of the individual varieties in culture. Thus increase in proline seems to be an incidental consequence of stress in vitro and not an adaptive response to combat water stress in sorghum. PMID- 16664384 TI - In situ measurement of plant water potentials by equilibration with microdroplets of polyethylene glycol 8000. AB - Microdroplets (3-5 nanoliters) of polyethylene glycol 8000 solution were allowed to equilibrate with plant water potential by placing the microdroplet on an abraded surface and covering it with mineral oil to prevent evaporation. Osmolality was followed by cryoscopic measurements, accurate to about +/-0.1 bar, on subnanoliter samples.Under constant environmental conditions, apparent equilibrium between microdroplet and plant water potentials was attained in about 1 to 2 hours. Microdroplet osmolality responded promptly to treatments (illumination, excision, osmotica) which changed plant water status. The values obtained for plant water potentials appeared to be physiologically reasonable. However, comparison with values obtained by other means (dewpoint hygrometry, treatment of tissue with polyethylene glycol solutions, calculation from turgor and osmotic pressures) suggest that they might be somewhat more negative than the actual tissue water potential.Aside from the advantage of providing in situ measurements of plant water status, the method is not temperature sensitive and requires only about 10 square millimeters of surface area, which allows its use on even small structures with little interference by shading or with gas exchange. PMID- 16664385 TI - Light and thiol activation of maize leaf glycerate kinase : the stimulating effect of reduced thioredoxins and ATP. AB - Glycerate kinase (EC 2.7.1.31) from maize (Zea mays) leaves was shown to be regulated by light/dark transition. The enzyme more than doubled in activity after either the leaves or isolated mesophyll chloroplasts were illuminated with white light for 10 minutes. Rate of inactivation in the dark was faster in leaves than in the isolated chloroplast fraction. The stimulating effect of light could be mimicked in crude preparations by addition of 10 or 50 millimolar dithiothreitol or 100 millimolar 2-mercaptoethanol. The thiol treatment resulted in 8- to 10-fold activation of glycerate kinase, with the highest rates in the range of 27 to 30 micromoles per mg chlorophyll per hour. Activation was not accompanied by any changes in the apparent M(r) value of glycerate kinase as determined by gel filtration (M(r) = 47,000). In contrast to maize glycerate kinase, the enzyme from spinach was not affected by either light or thiol exposure.Partially purified maize glycerate kinase was activated up to 3-fold upon incubation with a mixture of spinach thioredoxins m and f and 5 millimolar dithiothreitol. The thioredoxin and dithiothreitol-treated glycerate kinase could be further stimulated by addition of 2.5 millimolar ATP. The results suggest that glycerate kinase from maize leaves is capable of photoactivation by the ferredoxin/thioredoxin system. The synergistic effect of ATP and thioredoxins in activation of the enzyme supports the earlier expressed view that the ferredoxin/thioredoxin system functions jointly with effector metabolites in light-mediated regulation during photosynthesis. PMID- 16664386 TI - Transmembrane Proton Electrochemical Gradients in Dark Aerobic and Anaerobic Cells of the Cyanobacterium (Blue-Green Alga) Anacystis nidulans: Evidence for Respiratory Energy Transduction in the Plasma Membrane. AB - The transmembrane proton electrochemical potential gradient Deltamu(H+) in whole cells of Anacystis nidulans was measured in aerobic and anaerobic dark conditions using the distribution, between external medium and cell interior, of radioactively labeled weak acids (acetylsalicyclic acid, 5,5-dimethyloxazolidine 2,4-dione) or bases (imidazole, methylamine), and permeant ions (tetraphenylphosphonium cation, thiocyanate anion), as determined by flow dialysis. Alternatively, the movements across the plasma membrane of DeltapH indicating atebrin or 9-aminoacridine, and of DeltaPsi-indicating 8-anilino-l naphthalenesulfonate were qualitatively followed by fluorescence measurements. Attempts were made to discriminate between the individual chemiosmotic gradients across the cytoplasmic (plasmalemma) and the intracytoplasmic (thylakoid) membranes. By use of the ionophores nigericin, monensin, and valinomycin, the components of the proton motive force, namely the proton concentration gradient DeltapH and the electric membrane potential DeltaPsi were shown to be mutually exchangeable within the range of external pH values tested (3.2-11.0). Both components were depressed by the uncoupler carbonylcyanide m chlorophenylhydrazone, though inhibition of DeltapH was much more pronounced than that of DeltaPsi, notably in the alkaline pH(0) range. The total proton electrochemical gradient across the plasma membrane was significantly higher in aerobic than in anaerobic cells and increased markedly (i.e. became more negative) towards lower pH(0) values. This increase was paralleled by a similar increase in the rate of endogenous respiration of the cells. At the same time the ATPase inhibitor dicyclohexylcarbodiimide only slightly affected the proton motive force across the plasma membrane of aerobic cells. The results will be discussed in terms of a respiratorily competent plasma membrane in Anacystis nidulans. PMID- 16664387 TI - Effect of calmodulin antagonists on auxin-induced elongation. AB - Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of alpha-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6 aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of (3)H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation. PMID- 16664388 TI - Membrane Development in the Cyanobacterium, Anacystis nidulans, during Recovery from Iron Starvation. AB - Deprivation of iron from the growth medium results in physiological as well as structural changes in the unicellular cyanobacterium Anacystis nidulans R2. Important among these changes are alterations in the composition and function of the photosynthetic membranes. Room-temperature absorption spectra of iron-starved cyanobacterial cells show a chlorophyll absorption peak at 672 nanometers, 7 nanometers blue-shifted from its normal position at 679 nanometers. Iron-starved cells have decreased amounts of chlorophyll and phycobilins. Their fluorescence spectra (77K) have one prominent chlorophyll emission peak at 684 nanometers as compared to three peaks at 687, 696, and 717 nanometers from normal cells. Chlorophyll-protein analysis of iron-deprived cells indicated the absence of high molecular weight bands. Addition of iron to iron-starved cells induced a restoration process in which new components were initially synthesized and integrated into preexisting membranes; at later times, new membranes were assembled and cell division commenced. Synthesis of chlorophyll and phycocyanins started almost immediately after the addition of iron. The absorption peak slowly returned to its normal wavelength within 24 to 28 hours. The fluorescence emission spectrum at 77K changed over a period of 14 to 24 hours during which the 696- and 717-nanometer peaks grew to their normal levels, and the 684 nanometer peak moved to 687 nanometers and its relative intensity decreased to its normal level. Analysis of chlorophyll-protein complexes on polyacrylamide gels showed that high molecular weight chlorophyll-protein bands were formed during this time, and that low molecular weight bands (related to photosystem II) disappeared. The origin of the fluorescence emission at 687 and 696 nanometers is discussed in relation to the specific chlorophyll-protein complexes formed during iron reconstitution. PMID- 16664389 TI - Identification of Cytokinins from Xylem Exudate of Phaseolus vulgaris L. AB - The principal biologically active cytokinins in xylem exudate of young Phaseolus vulgaris L. plants were identified by bioassay, high-performance liquid chromatography, enzymic degradation and combined gas chromatography-mass spectrometry (selected ion monitoring) a zeatin riboside, zeatin nucleotide, dihydrozeatin riboside, dihydrozeatin nucleotide, O-glucosyl zeatin, O-glycosyl dihydrozeatin, O-glucosyl dihydrozeatin riboside, and O-glucosyl dihydrozeatin nucleotide. Trace amounts of O-glucosyl zeatin riboside and O-glucosyl zeatin nucleotide were also detected. PMID- 16664390 TI - Biphasic fluence response curves for induction of seed germination. AB - Fluence-response curves for the induction of seed germination after 24 hours pretreatment at 35 degrees C of Rumex obtusifolius and Arabidopsis thaliana show two phases of response: (a) a very low fluence-response (10(-4) - 10(-1) micromoles per square meter) and (b) a low fluence-response (1 - 10(3) micromoles per square meter). PMID- 16664391 TI - Proteolytic activity in anther extracts of fertile and cytoplasmic male sterile petunia. AB - Proteolytic activity is compared in anther extracts from Petunia parodii fertile and cytoplasmic male sterile lines. It is characterized relative to developmental stage of the anthers, effect of variable incubation times, pH of isolation buffers, and degradation of marker proteins. In fertile anthers, proteolytic activity increases at the end of microsporogenesis and peaks early in microgametogenesis. Degradation is most severe in extracts of fertile anthers and in high molecular weight proteins and reaches its maximum within 20 minutes. Degradation of marker proteins is greatest at pH 5.6 to 8.0 in fertile anther extracts and is eliminated under strong acid conditions (pH 2.8 to 4.0) in both fertile and cytoplasmic male sterile anther extracts. Marker proteins degrade more severely in extracts of fertile anthers; however, the order of substrate sensitivity-myosin > phosphorylase b > bovine serum albumin and ovalbumin > beta galactosidase-is the same in extracts from fertile and cytoplasmic male sterile anthers. PMID- 16664392 TI - Promotion of ethylene evolution and ripening of tomato fruit by galactose. AB - Mature green tomato fruit (Lycopersicon esculentum Mill. cv ;Rutgers') were infiltrated with 0.5 milliliters of sterilized, distilled water alone, or containing enough glucose or galactose to allow for a concentration of 400 micrograms per gram fresh weight after infiltration. All fruit showed a transient peak of ethylene production within 6 hours due to the slight wounding by vacuum infiltration. In addition, galactose-treated fruit underwent a temporal increase in ethylene production after 25 hours, whereas, the glucose and H(2)O-control fruit did not. Furit infiltrated with galactose subsequently produced ethylene and ripened substantially earlier than glucose or H(2)O-control fruit. PMID- 16664393 TI - The Biosynthesis of the Pyrenocines in Cultures of Pyrenochaeta terrestris. AB - [(14)C]Acetate, [(14)C]formate, and methyl[(14)C]methionine all serve as precursors of pyrenocines A, B, and C when added to cultures of Pyrenochaeta terrestris (Hansen) Gorenz, Walker, and Larson, the pathogen responsible for disease known as pink root of onion (Allium cepa L.). This information supports the hypothesis that these metabolites are methyl-substituted polyketides in origin. Pyrenocine A arises from acetate via uncharacterized intermediates and is subsequently converted to pyrenocine B. The biosynthetic role of pyrenocine C remains uncertain. PMID- 16664394 TI - Protein phosphorylation in plant mitochondria. AB - Protein kinase activity was detected in osmotically lysed mitochondria isolated from etiolated seedlings of corn, pea, soybean, and wheat, as well as from potato tubers. Ther kinase(s) phosphorylated both endogenous polypeptides and exogenous, nonmitochondrial proteins when supplied with ATP and Mg(2+). Eight to fifteen endogenous mitochondrial polypeptides were phosphorylated. The major mitochondrial polypeptide labeled in all species migrated during denaturing electrophoresis with an apparent monomeric molecular weight of 47,000. Incorporation of phosphate into endogenous proteins appeared to be biphasic, being most rapid during the first 1 to 2 minutes but slower thereafter. The kinase activity was greatest at neutral and alkaline pH values and utilized ATP with a K(m) of approximately 200 micromolar. The kinase was markedly inhibited by CaCl(2) but was essentially unaffected by NaF, calmodulin, oligomycin, or cAMP. These data suggest that plant mitochondrial protein phosphorylation may be similar to protein phosphorylation in animal mitochondria. PMID- 16664395 TI - Changes in carbohydrate composition in wheat and pea seedlings induced by calcium deficiency. AB - Wheat (Triticum aestivum L. cv Jubilar) seedlings were grown for 10 days in hydroponics with or without calcium. In the leaves, Ca deficiency caused the level of ethanol soluble carbohydrate to increase between 2-and 10-fold, enhanced dark respiration and decreased CO(2) fixation capacity. Sucrose was the major carbohydrate to accumulate in wheat roots. PMID- 16664396 TI - Augmented growth equation for cell wall expansion. AB - The Growth Equation representing the relative rate of irreversible wall expansion is augmented with an elastic expansion component. Some of the utility of this augmented Growth Equation is demonstrated through selected applications. PMID- 16664397 TI - Zeatin Metabolism in Fruits of Phaseolus: Comparison between Embryos, Seedcoat, and Pod Tissues. AB - Zeatin metabolites were isolated from seedcoats and pod tissues of Phaseolus vulgaris and P. lunatus. The differences observed previously between P. vulgaris and P. lunatus embryos, i.e. the formation of O-ribosyl derivatives in the former and O-glucosyl derivatives in the latter, could also be detected in seedcoats, although the levels of these metabolites were much lower and there was a concomitant increase of breakdown products (adenine, adenosine and AMP). Inner pod wall tissues of both genotypes metabolized zeatin at a slow rate and the major metabolite was the mononucleotide of zeatin. The array of metabolites recovered was not influenced by the extraction method (cold ethanol or modified Bieleski solution). PMID- 16664398 TI - Reduced phytic Acid content does not have an adverse effect on germination of soybean seeds. AB - Altering the level of phytic acid phosphorus by nutritional means had no effect on the ability of soybean (Glycine max L. [Merr.], cv ;Williams 79') seeds to germinate under laboratory or greenhouse conditions. Dry matter moved out of the cotyledons at similar rates whether the germinating seeds initially contained low (0.19), medium (0.59), or high (1.00 milligram per seed) phytic acid phosphorus. Growth of roots and shoots from 3 to 9 days after planting was similar for seeds containing low and medium levels of phytic acid phosphorus. The medium level of phytic acid resembles that found in field-grown seed, so it is clear that soybean seeds normally contain a phosphorus reserve far above that needed for germination and early seedling growth. PMID- 16664399 TI - Starch Branching Enzymes from Maize : Immunological Characterization using Polyclonal and Monoclonal Antibodies. AB - Spleen cells from mice immunized with starch branching enzymes were fused with cells from the mouse myeloma Sp2/0-AG14 cell line to form hybridomas. Those hybridomas producing antibodies against the branching enzyme were screened by the enzyme-linked immunosorbent assay using purified branching enzyme as the antigen. Three monoclonal cell lines (1A1D7, 1A1C3 and 4D2A9D8) were found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with maize branching enzyme I in addition to branching enzymes IIa and IIb. Three other monoclonal cell lines (4D2D10, 4D2F9, and 2A6C12) were also selected which were found to produce antibodies showing positive enzyme-linked immunosorbent assay reactions with branching enzymes IIa and IIb only.Amino acid composition and peptide maps obtained after trypsin or chymotrypsin digestion show that there is no difference between branching enzyme IIa and IIb but they are significantly different from branching enzyme I which, along with immunological data, suggests that only two forms of starch branching enzyme may be present in maize kernels.Immunological cross-reaction was also found between the starch branching enzyme from maize kernels and the glycogen branching enzyme from Escherichia coli using polyclonal antibodies against starch branching enzyme I or IIa and IIb or E. coli glycogen branching enzyme, suggesting some immunological similarities between maize starch branching enzymes and E. coli glycogen branching enzyme. PMID- 16664400 TI - A novel method of natural cryoprotection : intracellular glass formation in deeply frozen populus. AB - Correlating measurements from differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and survival of twigs after two-step cooling experiments, we provide strong evidence that winter-hardened Populus balsamifera v. virginiana (Sarg.) resists the stresses of freezing below -28 degrees C by amorphous solidification (glass formation) of most of its intracellular contents during slow cooling (/=30 degrees C). This second phase depends on O(2): loss of responsiveness is accelerated at lower O(2) concentration. Phase II is only slightly affected by gibberellic acid. The results are discussed in terms of variation of phytochrome and of a reaction along the transduction chain initiated by phototransformation of this pigment, which is finally expressed in germination. PMID- 16664424 TI - Plant morphological and biochemical responses to field water deficits: I. Responses of glutathione reductase activity and paraquat sensitivity. AB - The effects of water deficits on plant morphology and biochemistry were analyzed in two photoperiodic strains of field-grown cotton (Gossypium hirsutum L.). Plants grown under dryland conditions exhibited a 40 to 85% decrease in leaf number, leaf area index, leaf size, plant height, and total weight per plant. Gross photosynthesis decreased from 0.81 to 0.47 milligram CO(2) fixed per meter per second and the average midday water, osmotic, and turgor potentials decreased to -2.1, -2.4, and 0.3 megapascals, respectively.There was a progressive increase in glutathione reductase activity and in the cellular antioxidant system in the leaves of stressed plants compared to the irrigated controls. The stress-induced increases in enzyme activity occurred at all canopy positions analyzed.Irrigation of the dryland plots following severe water stress resulted in a 50% increase in leaf area per gram fresh weight in newly expanded leaves of both strains over the leaves which had expanded under the dryland conditions. Paraquat resistance (a relative measure of the cellular antioxidant system) decreased in the strain T25 following irrigation. Glutathione reductase activities remained elevated in the T25 and T185 leaves which were expanded fully prior to irrigation and in the leaves which expanded following the irrigation treatment. PMID- 16664425 TI - Elevation in the Sucrose Content of the Shoot Apical Meristem of Sinapis alba at Floral Evocation. AB - Nanogram tissue samples from apical meristems of Sinapis alba were assayed for sucrose, total soluble hexosyl equivalents ( identical with glucose and fructose plus hexoses from sucrose hydrolysis), and total soluble glucosyl equivalents ( identical with glucose plus glucose from sucrose hydrolysis). On dry weight basis, sucrose concentration increased by more than 50% within 10 hours after the start of either a long photoperiod or a short photoperiod displaced by 10 hours in the 24-hour cycle (;displaced short day'). (These treatments induce flower initiation) Glucose and fructose concentrations were close to zero in vegetative meristems and remained low compared to sucrose in meristems of induced plants. Within a single meristem, the peripheral and the central zones had similar concentrations of sucrose. Our results indicate that an early physiological event in floral transition is the accumulation of sucrose in the meristem. PMID- 16664426 TI - Reorganization of Thylakoid Components during Chloroplast Development in Higher Plants after Transfer to Darkness : Changes in Photosystem I Unit Components, and in Cytochromes. AB - It was shown earlier that in etiolated bean (Phaseolus vulgaris, var. red kidney) leaves exposed to continuous light for a short time and then transferred to darkness a reorganization of their photosystem II (PSII) unit components occurs. This reorganization involves disorganization of the light-harvesting complex of PSII (LHC-II), destruction of its chlorophyll b and the 25 kilodalton polypeptide, and reuse of its chlorophyll a for the formation of additional, small in size, PSII units (Argyroudi-Akoyunoglou, Akoyunoglou, Kalosakas, Akoyunoglou 1982 Plant Physiol 70: 1242-1248). The present study further shows that parallel to the PSII unit reorganization a reorganization of the PSI unit components also occurs: upon transfer to darkness the 24, 23, and 21 kilodalton polypeptides, components of the light-harvesting complex of PSI (LHC-I), are decreased, the 69 kilodalton polypeptide, component of the chlorophyll a-rich P700-protein complex (CPI), is increased and new smallsized PSI units are formed. Concomitantly, the cytochrome f/chlorophyll and the cytochrome b/chlorophyll ratios are gradually increased. This suggests that the concentration of the electron transport components is also modulated in darkness to allow for adequate electron flow to occur between the newly synthesized PSII and PSI units. PMID- 16664427 TI - Fe reduction in cell walls of soybean roots. AB - Reduction of Fe(III)EDTA by excised roots of soybean seedlings (Glycine max L.) is stimulated by l-malate in the bathing solution. Reduction occurs much more rapidly with roots of seedlings grown in the absence of iron than with roots of seedlings grown with iron. Cell-wall preparations from these roots catalyze reduction of Fe(III)EDTA by NADH. They also contain NAD(+)-dependent l-malate dehydrogenase. Enzymic activity of the cell-wall preparations is not affected by previous iron nutrition of the plants, but the amount of l-malate in the roots is increased when seedlings have been deprived of iron. We propose that reduction of iron before absorption by soybean roots occurs in the cell-wall space, with l malate secreted from the roots serving as the source of electrons. Part of the iron reductase activity of the cell walls can be solubilized by extraction with 1 molar NaCl. The enzyme has been partially purified. PMID- 16664428 TI - Pyruvate Decarboxylase from Zea mays L. : 2. Examination of Hysteretic Kinetics. AB - A significant lag phase was observed in the accumulation of product for the reaction catalyzed by pyruvate decarboxylase (PDC) purified from mature maize kernels. The effects of pH, pyruvate, potassium chloride, PDC concentration, and Mg(2+)-thiamine pyrophosphate upon this lag and upon the observed cooperativity were investigated. PDC preincubated with Mg(2+)-thiamine pyrophosphate for six days had Michaelis-Menten kinetics, a Hill number of 1, and no apparent lag phase. The degree of saturation of PDC with Mg(2+)-thiamine pyrophosphate appears to have a central role in controlling the lag phase and the degree of cooperativity. PMID- 16664429 TI - The effect of low osmotic potential on nitrite reduction in intact spinach chloroplasts. AB - The effect of water stress (reduced osmotic potential) on photosynthetic nitrite reduction was investigated using intact, isolated spinach (Spinacia oleracea) chloroplasts. Nitrite-dependent O(2) evolution was inhibited 39% at -29.5 bars osmotic potential, relative to a control at -11 bars. In the presence of an uncoupler of photophosphorylation this inhibition was not seen. Reduced osmotic potential did not inhibit either methyl viologen reduction or photosynthetic O(2) reduction. These results indicate that an inhibition of electron transport to ferredoxin cannot account for the observed inhibition of nitrite-dependent O(2) evolution. In vitro assay of nitrite reductase activity showed that the interaction of the enzyme with nitrite was not affected by changes in the concentrations of ions or molecules that might be caused by water stress conditions. These results indicate that the most likely site for the effect of water stress on chloroplastic nitrite reduction is the interaction of ferredoxin with nitrite reductase. PMID- 16664430 TI - Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum. AB - Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv ;Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10(10) cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10(10) bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10(10) bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.To test the hypothesis that this reduced synthesis of exopolysaccharide by bacteroids is related to the low free O(2) concentration within nodules, strain RT2 was grown on l-arabinose/succinate/glutamate/nitrate medium both aerobically and anaerobically. Anaerobiosis caused a 92% reduction in total exopolysaccharide synthesis, with amounts averaging only 123 micrograms per 10(10) cells. Anaerobically cultured cells also failed to bind soybean lectin. These results suggest that the low free O(2) content of the nodules may be responsible for the reduced exopolysaccharide synthesis by the bacteroids. PMID- 16664431 TI - Induction of a Specific N-Methyltransferase Enzyme by Long-Term Heat Stress during Barley Leaf Growth. AB - Previous work showed that the indole alkaloid gramine accumulates in the upper leaves (e.g. the fifth) of barley as a response to high growth temperatures. The biosynthesis of gramine proceeds from tryptophan to 3-aminomethylindole (AMI); sequential N-methylations of AMI then yield N-methyl-3-aminomethylindole (MAMI) and gramine.To determine whether high-temperature stress increases the activity of gramine pathway enzymes, leaf tissue from plants grown at various temperatures was assayed for N-methyltransferase (NMT) activity using AMI and MAMI as substrates in both in vivo and in vitro assays. NMT activity in expanding fifth leaves was increased 8- to 20-fold by growth at high temperatures (35 degrees C day/30 degrees C night) compared to cool temperatures (15 degrees C/10 degrees C). Several days of high temperature were required for full induction of NMT activity. No induction of NMT activity occurred in leaves which had completed expansion in cool conditions before exposure to high temperature.To investigate NMT induction at the protein level, NMT activity was purified to homogeneity and used to produce polyclonal antibodies. Throughout enzyme purification, relative NMT activities towards AMI and MAMI remained constant, consistent with a single NMT enzyme. Immunoblot analysis showed that a large increase in NMT polypeptide coincided with induction of NMT activity by heat stress. Our results point to a type of high-temperature regulation of gene expression that is quite distinct from heat shock. PMID- 16664432 TI - Enzymic capacities of purified cauliflower bud plastids for lipid synthesis and carbohydrate metabolism. AB - Isolated cauliflower (Brassica oleracea) bud plastids, purified by isopycnic centrifugation in density gradients of Percoll, were found to be highly intact, to be practically devoid of extraplastidial contaminations, and to retain all the enzymes involved in fatty acid, phosphatidic acid, and monogalactosyldiacylglycerol synthesis. Purified plastids possess all the enzymes needed to convert triose phosphate to starch and vice versa, and are capable of conversion of glycerate 3-phosphate to pyruvate for fatty acid synthesis. They are also capable of oxidation of hexose phosphate and conversion to triose phosphate via the oxidative pentosephosphate pathway. Cauliflower bud plastids prove to be, therefore, biochemically very flexible organelles. PMID- 16664433 TI - Association between Symptom Development and Inhibition of Ornithine Carbamoyltransferase in Bean Leaves Treated with Phaseolotoxin. AB - The chlorosis symptom that characterizes the halo blight disease of Phaseolus vulgaris L. is caused by phaseolotoxin produced by the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola. Phaseolotoxin is hydrolyzed by plant peptidases to N(delta)(N'-sulpho-diaminophosphinyl) -l-ornithine which also causes chlorosis and is reported to be an irreversible inhibitor of ornithine carbamoyltransferase (OCTase). We have examined the hypothesis that inhibition of OCTase is the primary action of phaseolotoxin that leads to chlorosis.Chlorotic spots appeared on the primary leaves of P. vulgaris seedlings during the 2 days following leaf prick application of a minimum of 30 picomole phaseolotoxin. OCTase in extracts of the lesions was reduced to 20%, or less, of the activity in controls. Four hours after the application of phaseolotoxin the concentration of free ornithine increased more than 2-fold. Other amino acids, especially glutamine and asparagine-but not arginine-increased later. Chlorophyll remained at a constant level in the phaseolotoxin-treated tissue and the appearance of chlorosis was due to the increase in chlorophyll in the surrounding unaffected tissue.Clear halo symptoms developed only on primary leaves of the youngest seedlings (treated 6-7 days after germination). Lesions did not develop on primary leaves of seedlings more than 14 days old, in which the chlorophyll concentration had reached a maximum. OCTase also was inhibited in the symptomless tissue from older leaves treated with phaseolotoxin, but there was no accumulation of amino acids, including ornithine. A single appliction of 200 nanomoles arginine resulted in the complete regreening of the chlorosis caused by phaseolotoxin. Soluble protein was lower in the chlorotic tissue than in the controls, but increased to greater than the control value following the appliction of arginine. These results suggest that phaseolotoxin-induced chlorosis results from reduced chlorophyll synthesis that is associated with arginine-starvation in the tissue where OCTase is inhibited. PMID- 16664434 TI - Effects of Ni Deficiency on Some Nitrogen Metabolites in Cowpeas (Vigna unguiculata L. Walp). AB - Cowpeas grown in nutrient solutions, from which Ni had been removed by a ligand exchange technique, accumulated urea in most tissues. Urea levels were highest (up to 3.1 percent dry weight) in necrotic leaf tips. Urea accumulation in Ni deficient cowpea tissues amounted to about 1 percent of the total N. The accumulation of urea was presumably associated with the catabolism of N compounds in older tissues and the redistribution of N catabolites within the plant during the reproductive growth. The exclusion of N salts from the nutrient media at a late stage of growth, either with or without added Ni, led to a general amelioration of urea accumulation and a lower level of the related amino acid, arginine, in root and stem tissue. Plant leaves that contained toxic levels of urea and displayed necrotic symptoms had tissue Ni levels ranging from less than 0.01 to 0.15 mug Ni per gram dry weight. Nickel concentrations in tissue from plants not treated with Ni, were initially very low, but increased as the cowpeas matured. Apparently, there was a source of Ni contamination in the Ni-deficient growth media which provided a source of Ni for uptake by the plants during growth. Ureide levels were low and unaffected by Ni deprivation. No evidence for free purines or uric acid accumulation in plant tissues could be found. It is hypothesized that Ni (and urease) participates in the normal N metabolism of these plants during the reproductive phase of growth. PMID- 16664435 TI - Hemicelluloses of cell walls of a proso millet cell suspension culture. AB - Cell wall composition of a stable suspension of proso millet (Panicum miliaceum L. cv Abarr) cells is similar to those of tissues and cell suspensions of other graminaceous species. Extraction of hemicelluloses with step-wise increasing concentrations of alkali yields materials that, like those of embryonal cells of maize coleoptiles, comprise mostly glucuronoarabinoxylan, xyloglucan, and small amounts of (1-3),(1-4)-beta-d-glucan. As in the walls of embryonal cells of the maize coleoptile, 5-arabinosyl and 3-arabinosyl comprise much higher proportions of the total hemicellulosic sugars than in walls of developed or elongated cells. Unlike cells of many dicotyledonous species, millet cells do not elongate or undergo observable differentiation during the stationary phase of culture, and consequently, their wall composition is remarkably consistent throughout the culture cycle. The proso millet cell suspension culture constitutes a reasonable model for study of cell wall biogenesis in embryonal cells of a graminaceous species, but because of marked changes in the composition of hemicelluloses in these species during cell enlargement, additional model systems should be sought. PMID- 16664436 TI - Tensile strength of cell walls of living cells. AB - A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered. PMID- 16664437 TI - Purification and properties of glyoxysomal lipase from castor bean. AB - The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes. PMID- 16664438 TI - Phospholipid composition of a plasma membrane-enriched fraction from developing soybean roots. AB - Phospholipid polar head group and fatty acid composition were determined for plasma membrane enriched fractions from developing soybean root (Glycine max [L.] Merr. cult. Wells II). Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots at pH 7.8 and in the presence of 5 millimolar ethylenediaminetetraacetate, 5 millimolar ethyleneglycol bis (beta-aminoethyl ether)N,N tetraacetic acid, and 10 millimolar NaF. Lipid extracts analyzed for phospholipid composition revealed two major phospholipid components: phosphatidylcholine and phosphatidylethanolmine. Minor phospholipid components identified were phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol. Lipid degradation by endogenous phospholipase D during membrane isolation at pH 6.5 and in the absence of chelating agents and NaF resulted in the recovery of large amounts of phosphatidic acid. Phosphatidylcholine was the principal substrate for phospholipase D.Fatty acid composition was determined for plasma membrane phosphatidylcholine and phosphatidylethanolamine from meristematic and mature root tissue. The fatty acids identified were 16:0, 18:0, 18:1, 18:2, and 18:3. Fatty acid composition varied with both phospholipid class and the developmental stage of the root. Results suggest that differences in the composition of the major phospholipids of plasma membrane from meristemaic and mature root sections occur in the fatty acids and not in the polar head groups. These differences and those found in the composition of the polar head groups of the minor phospholipid components, e.g. phosphatidylglycerol, may be significant for structure-function relationships within the membrane. PMID- 16664439 TI - Identification and characterization of latex-specific proteins in opium poppy. AB - Latex from the opium poppy, Papaver somniferum L., was analyzed by polyacrylamide gel electrophoresis (PAGE). Two latex-specific bands were identified in protein samples of poppy latex using one-dimensional native PAGE. Second dimension analysis with SDS-PAGE indicates that these proteins have a relative molecular weight of approximately 20 kilodaltons. We have termed these polypeptides the major latex proteins (MLPs). Polyclonal antibodies prepared against the MLPs were used to probe protein gel blots of latex and poppy tissues known to lack laticifers. Laticifer-free tissues showed no reaction with anti-MLP immunoglobulin G indicating that MLPs are found only in poppy latex. MLP distribution was also examined in mature opium poppy tissues by immunocytochemistry. Laticifers were differentially labeled by fluorescein isothiocyanate secondary labeling of anti-MLP immunoglobulin G and could easily be identified in both transverse and longitudinal section. Fractionation studies of isolated latex showed that MLPs are concentrated in the latex cytosol and not in alkaloidal vesicles. Analysis of latex proteins by conventional two dimensional electrophoresis indicates that the two MLP bands are composed of several distinct polypeptides with similar relative molecular weights. The pIs of these molecules range from 6.0 to 3.5. The role(s) of MLPs in laticifer metabolism has not been determined. PMID- 16664440 TI - Selection of Nicotiana tabacum Haploids of High Photosynthetic Efficiency. AB - Anthers of Nicotiana tabacum cultivar Wisconsin 38 were treated with the mutagen ethyl methane sulfonate and cultured to obtain populations of haploid plants of high genetic variability. The selection of these populations by their photosynthetic efficiency was carried out in a hydroponic culture chamber with a CO(2) atmosphere concentration close to the compensation point. Plants that survived 45 days of treatment were grown in pots in a greenhouse and their performance was compared to a population of unselected haploids. The growth characteristics, net photosynthesis, and chlorophyll content were measured and the haploid character verified. Selected plants were propagated by in vitro culture of buds and then diploidized to obtain seeds. Growth and other characteristics of the plants obtained were compared with those of the parental variety (Wisconsin 38) in a field assay. Growth, dry weight, leaf area, and net photosynthesis of selected plants were higher than in control plants. PMID- 16664441 TI - Assaying ornithine and arginine decarboxylases in some plant species. AB - A release of (14)CO(2) not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-(14)C]- or [U-(14)C]ornithine. The total (14)CO(2) produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH(4))(2)SO(4) did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant (14)CO(2) release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris.HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard (14)CO(2)-based method by assessing labeled Put and/or Agm-formed in the presence of added aminoguanidine and/or ornithine when needed-is recommended especially when crude or semicrude plant extracts are assayed.When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids. PMID- 16664442 TI - Activities of arginine and ornithine decarboxylases in various plant species. AB - In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to V(max), ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. alpha-Difluoromethylornithine and alpha-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species.No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed.In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum. PMID- 16664443 TI - Light Scattering as an Indicator of the Energy State in Leaves of the Crassulacean Acid Metabolism Plant Kalanchoe pinnata. AB - Both transmittance changes in a weak beam of green light (light scattering) and the slow decay of chlorophyll a fluorescence were used as indicators of the energy state of leaves of a Crassulacean acid metabolism plant, Kalanchoe pinnata, at frequent intervals during 12-hour light/12-hour dark cycles. To induce light scattering and fluorescence changes, leaves were exposed to red light for 6 minutes. When measurements were made during the light period, the leaves were kept in darkness for 6 minutes before illumination. In the middle of the light period, when malic acid decarboxylation was very active and stomatal conductance was low, light scattering changes were small and indicated that the energy state of leaves was low. This result was supported by determination of adenylate levels. Light scattering and ATP/ADP ratios increased during the late light period when the tissue was deacidified. Illumination produced maximum light scattering changes between the 2nd and 5th hour of the dark period, when rates of dark CO(2) fixation were highest. Light scattering and fluorescence measurements taken from leaves, which were illuminated with red or far-red light in the presence or absence of O(2) showed that, in addition to linear electron transport, K. pinnata has the potential for both cyclic and pseudocyclic electron transport. The results are relevant with regard to the high ATP demand during Crassulacean acid metabolism. PMID- 16664444 TI - 5'-Methylthioadenosine Nucleosidase and 5-Methylthioribose Kinase Activities and Ethylene Production during Tomato Fruit Development and Ripening. AB - 5'-Methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase activities were measured in crude extracts of tomato fruits (Lycopersicon esculentum Mill cv Rutgers) during fruit development and ripening. The highest activity of MTA nucleosidase (1.2 nanomoles per milligram protein per minute) was observed in small green fruits. The activity decreased during ripening; at the overripe stage only 6.5% of the peak activity remained. MTR kinase activity was low at the small green stage and increased thereafter until it reached peak activity at the breaker stage (0.7 nanomoles per milligram protein per minute) followed by a sharp decline at the later stages of fruit ripening. 1-Amino cyclopropane-1-carboxylic acid (ACC) levels peaked at the red stage, while ethylene reached its highest level at the light-red stage. Several analogs of MTA and MTR were tested as both enzyme and ethylene inhibitors. Of the MTA analogs examined for their ability to inhibit MTA nucleosidase, 5'-chloroformycin reduced enzyme activity 89%, whereas 5'-chloroadenosine, 5'-isobutylthioadenosine, 5' isopropylthioadenosine, and 5'-ethylthioadenosine inhibited the reaction with MTA by about 40%. 5'-Chloroformycin and 5'-chloroadenosine inhibited ethylene production over a period of 24 hours by about 64 and 42%, respectively. Other analogs of MTA were not effective inhibitors of ethylene production, whereas aminoethoxyvinylglycine showed a 34% inhibition over the same period of time. Of the MTR analogs tested, 5-isobutylthioribose was the most effective inhibitor of both MTR-kinase (41%) and ethylene production (35%). PMID- 16664445 TI - A Comparison of Two Sucrose Synthetase Isozymes from Normal and shrunken-1 Maize. AB - The maize sucrose synthetase isozyme (SS2) present in sh1 endosperm, sh1 seedlings, and in suspension culture cells was purified to homogeneity from each of these tissues by sequential ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gel filtration chromatography, and affinity elution with UTP from a carboxymethyl-cellulose column. Cyanogen bromide digests were used to demonstrate that the SS2 isozymes in these different tissues are structurally identical and are therefore the product of the same gene. The sucrose synthetase produced by the Sh1 gene (SS1) was purified by modification of the SS2 procedure and was used in comparative analyses of the two isozymes. Ouchterlony assays demonstrated that SS1 and SS2 have partial antigenic identity. The two isozymes have similar enzyme kinetics in the sucrose cleavage reaction but differ in their relative activities with ADP and TDP. The amino acid compositions of SS1 and SS2 are similar, and proteolytic digests revealed that they share limited structural homologies. PMID- 16664447 TI - Control of seed coat thickness and permeability in soybean : a possible adaptation to stress. AB - Although the seed coat, through its thickness and permeability, often regulates seed germination, very little is known about the control of its development. Using soybean (Glycine max [L.] Merrill) explants, podbearing cuttings in which defined solutions can be substituted for the roots, we have demonstrated that cytokinin and mineral nutrients moving through the xylem can control soybean seed coat development. Lack of cytokinin and minerals in the culture solution, causes a thicker, less permeable seed coat to develop. The seeds with thickened coats will imbibe water rapidly if scarified; furthermore, these scratched seeds also germinate and produce normal plants. Inasmuch as stress (e.g. drought) decreases mineral assimilation and cytokinin production by the roots, the resulting delay in germination could be an adaptive response to stress. PMID- 16664446 TI - The Effect of Sugars on the Binding of [Hg]-p-Chloromercuribenzenesulfonic Acid to Leaf Tissues. AB - Replacement of mannitol with sucrose decreases the binding of [(203)Hg]-p chloromercuribenzenesulphonic acid (PCMBS) to Vicia faba leaf discs without epidermis. This decrease is optimal for 20 minutes on incubation, is concentration-dependent, and is also found with maltose and raffinose. In parallel experiments, the addition of sucrose, maltose, and raffinose during PCMBS pretreatment was shown to increase subsequent uptake of [U-(14)C]sucrose. In contrast, d- or l-glucose, 3-O-methylglucose, galactose, fructose, palatinose, turanose, or melibiose had no effect either on PCMBS binding or on [(14)C]sucrose uptake. The sucrose-induced decrease of PCMBS binding is retained after a cold and ionic shock. Measurements of specific activities of membrane fractions prepared from tissues incubated in labeled PCMBS show that the decrease concerns the 120,000 gravity pellet, but that very mild procedures must be chosen to prevent redistribution of label in the supernatant. Altogether, the data provide new support to the hypothesis that the active site of the sucrose carrier contains a group sensitive to PCMBS. PMID- 16664448 TI - Voltage Noise in Acer pseudoplatanus Cells : Basic Cellular Noise and Gramicidin a Induced Noise. AB - The present contribution is devoted to studying the electrical noise of Acer pseudoplatanus cells in culture suspensions. Spontaneous voltage noise of the cells was recorded by means of a microelectrode inserted in the vacuole. The small signal impedance of the cell was measured so that it was possible to study the intensity spectra of the noise. We recorded intensity spectra with cells incubated in 10(-3) molar gramicidin A. Difference spectra showed characteristics of a channel noise. By using the calculated conductance of gramicidin A in an artificial membrane, and by simplifying assumptions for the ionic transports through plasmalemma and tonoplast, we were able to estimate the electrochemical potential difference for K(+) ions across the plasmalemma (3.2 +/- 1 millivolt). PMID- 16664449 TI - Anacystis nidulans Demonstrates a Photosystem II Cation Requirement Satisfied Only by Ca or Na. AB - Anacystis nidulans exhibits a total loss of photosystem II (PSII) activity upon incubation in a nutrient medium deficient in Ca(2+) and Na(+) and containing a divalent cation chelator. This loss of activity is light-dependent, which corresponds to an energy requirement. Likewise, Ca(2+) efflux takes place only in cells incubated in light. The loss of PSII activity is reversible by addition of submillimolar amounts of either Ca(2+) or Na(+) to the external medium but not by the addition of any other cation. Restoration of lost PSII activity also requires light. Light saturation curves for partially depleted cells demonstrate both lower maximum O(2) evolution rates and decreased relative quantum yields when compared to control cells. Partial electron transport reactions isolate the site of the Ca(2+)/Na(+) effect to the reaction center itself or immediately on its oxidizing side and exclude the water-splitting complex. O(2) flash yields decline during cation depletion, indicating a decrease in the number of functional PSII reaction centers, but the maximum turnover rate for still functional reaction centers does not decline. Thus, PSII of A. nidulans exhibits an all-or-none cation requirement, satisfied only by Ca(2+) or Na(+). PMID- 16664450 TI - A simple procedure to overcome polyethelene glycol toxicity on whole plants. AB - A procedure is described that can be used to minimize toxic effects of polyethylene glycol (PEG) to plants. The procedure is based on recycling nutrient solutions containing PEG-6000 through two plant cultures. Tomato plants grown in 0.3 megapascals PEG solutions used after two growth cycles exhibited minimal toxic effects. Long-term responses like dry matter production and chlorophyll content as well as short-term responses like CO(2) fixation rates and leaf conductance were severely inhibited by fresh PEG-6000 and only slightly reduced by recycled PEG-6000. Complete osmotic adjustment was obtained with tomatoes grown in recycled but not in fresh PEG solutions. PMID- 16664451 TI - Enhanced N-Transfer from a Soybean to Maize by Vesicular Arbuscular Mycorrhizal (VAM) Fungi. AB - Using a split-root technique, roots of soybean plants were divided between two pots. In one of the two pots, two maize plants were grown and half of those pots were inoculated with the vesicular arbuscular mycorrhizal (VAM) fungus, Glomus fasciculatus. Fifty-two days after planting, (15)N-labeled ammonium sulfate was applied to the pots which contained only soybean roots. Forty-eight hours after application, significantly higher values for atom per cent (15)N excess were found in roots and leaves of VAM-infected maize plants as compared with the non VAM-infected maize plants. Results indicated that VAM fungi did enhance N transfer from one plant to another. PMID- 16664452 TI - In Vitro Biosynthesis of Vicia faba Polyphenoloxidase. AB - Poly A(+) mRNA was isolated from Vicia faba leaves and translated in vitro using a rabbit reticulocyte translation system. From analysis of the total translation products, the major proteins synthesized in vitro were 32 kilodaltons and 20 kilodaltons. When antibodies to Vicia faba polyphenoloxidase were added, a specific immunoprecipitable protein was observed. This protein's molecular weight was shown to be similar to that of the isolated enzyme (45 kilodaltons). The isolated enzyme successfully competed with the in vitro synthesized product for antipolyphenoloxidase. In addition, the in vitro synthesized product was not immunoprecipitated with antitomato peroxidase and comigrated with isolated and/or iodinated enzyme in sodium dodecylsulfate-polyacrylamide gel electrophoresis. Using in vitro translation and specific immunoprecipitation, a primary translation product corresponding to Vicia faba polyphenoloxidase was identified as a 45 kilodaltons protein. PMID- 16664453 TI - High efficiency transformation of cultured tobacco cells. AB - Tobacco calli were transformed at levels up to 50% by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harboring the binary transfer-DNA vector, pGA472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and, less so, on the expression of the vir genes of the tumor-inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essental factor for efficient transformation of higher plants. PMID- 16664454 TI - Nucleotide Sequence of the F(1)-ATPase alpha Subunit Gene from Maize Mitochondria. AB - The alpha subunit of the F(1)-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpalpha, that appears to code for the F(1)-ATPase alpha subunit of Zea mays. The atpalpha gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize alpha polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial alpha subunit and the tobacco chloroplast CF(1) and Escherichia coli alpha subunits are 54 and 51%, respectively. The origin of the atpalpha gene is discussed. PMID- 16664455 TI - Regeneration in alfalfa tissue culture: stimulation of somatic embryo production by amino acids and N-15 NMR determination of nitrogen utilization. AB - The production of somatic embryos in alfalfa (Medicago sativa L., cv Regen S) is increased 5- to 10-fold by alanine and proline. However, utilization of nitrogen for synthesis of protein from alanine, proline, glutamate, and glycine is not qualitatively different, even though the latter two amino acids do not increase somatic embryo formation. These determinations were made by (15)N labeling with detection by nuclear magnetic resonance. Overall metabolism of the nitrogen of proline, alanine, glutamate, and glycine is also similar in two regenerating and nonregenerating genotypes with similar germplasm, except that the levels of free amino acids are consistently higher in the nonregenerating line. In addition, when regeneration is suppressed in either of the two regenerating lines, the level of intracellular free amino acids increases. This increased level of metabolites is the only direct evidence provided by analysis of nitrogen metabolism of differences between the regenerating and nonregenerating states in alfalfa. PMID- 16664456 TI - Regeneration in Alfalfa Tissue Culture: Characterization of Intracellular pH During Somatic Embryo Production by Solid-State P-31 NMR. AB - The composition of phosphorus-containing compounds of intact lyophilized alfalfa tissue has been determined, in part, by solid-state (31)P nuclear magnetic resonance. The tissue (Medicago sativa L., cv Regen S; and some of its crosses) was grown in culture under both nonregenerating and regenerating conditions, the latter enhanced by the addition of specific amino acids. Analysis of the (31)P nuclear magnetic resonance spectra shows that regeneration is favored when metabolism occurs without the production of a low average intracellular pH. PMID- 16664457 TI - Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : V. Modulation of the Spinach Leaf Cytosolic Fructose 1,6-Bisphosphatase Activity in Vitro by Substrate, Products, pH, Magnesium, Fructose 2,6-Bisphosphate, Adenosine Monophosphate, and Dihydroxyacetone Phosphate. AB - How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg(2+) is required as an activator. There is a wide pH optimum, especially at high Mg(2+). The substrate dependence is not markedly pH dependent. High concentrations of Mg(2+) and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg(2+) dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg(2+) and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg(2+) dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg(2+), but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S(0.5) for fructose 1,6 bisphosphate. A model is proposed to account for the interaction between these effectors. PMID- 16664458 TI - Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : VI. Regulation of the Cytosolic Fructose 1,6-Bisphosphatase in Spinach Leaves by an Interaction between Metabolic Intermediates and Fructose 2,6-Bisphosphate. AB - In this article, we propose a model describing how the spinach leaf cytosolic fructose 1,6-bisphosphatase is regulated in vivo by an interaction between fructose 2,6-bisphosphate and metabolic intermediates during photosynthesis. Previously published results are reanalyzed to provide a description of the way in which fluxes and metabolites vary in spinach leaves, depending on the relation between the supply of photosynthate and the demand for sucrose. The activity of the spinach leaf cytosolic fructose 1,6-bisphosphatase has been assayed in conditions simulating those expected in leaves as the rate of sucrose synthesis increases in response to rising rates of photosynthesis, and as sucrose synthesis declines in response to accumulation of sucrose in the leaf so that more starch is synthesized. The results show that regulation of the cytosolic fructose 1,6 bisphosphatase by alterations of fructose 2,6-bisphosphate, dihydroxyacetone phosphate, adenosine monophosphate, and phosphate can account for the alterations of flux found in vivo. The properties of a regulatory network, which allows the distribution of triose P between the Calvin cycle, sucrose synthesis, and starch synthesis to be balanced and adjusted, are described. PMID- 16664459 TI - Effects of Nitrate Application on Amaranthus powellii Wats. : I. Changes in Photosynthesis, Growth Rates, and Leaf Area. AB - Physiological effects of different nitrate applications were studied using the C(4) plant, Amaranthus powellii Wats. Plants were grown in a controlled environment chamber and watered daily with nutrient solutions containing 45, 10, 5, or 1 millimolar nitrate. Chloride and sulfate were used to keep the cation and phosphate concentrations constant. Total leaf nitrogen concentration, chlorophyll concentration, specific leaf mass, leaf area, relative growth rate, relative leaf growth rate, unit leaf rate (increase of dry mass per unit leaf area per day), net photosynthetic rate, and incident quantum yield decreased with decreasing nitrate concentration. The per cent decrease of unit leaf rate was similar to the decrease of light-saturated net photosynthetic rate; however, the decrease in relative growth rate was less than that of unit leaf rate because leaf area ratio (leaf area per unit dry mass) increased with decreasing nitrate concentration. Essential mineral concentrations per unit leaf area were about equal among all treatments. Leaf expansion, determined by stomatal density, decreased except for the 1 millimolar treatment which showed relatively more cell expansion but less cell division. Decreased nitrate application was correlated with higher osmotic potentials and lower pressure potentials (determined by pressure-volume curves), whereas leaf water potentials were equal among treatments. Even though total leaf area and shoot mass decreased with decreasing applied nitrate, the increase of the leaf area ratio may be related to selection for the highest possible growth rate. PMID- 16664460 TI - Effects of Nitrate Application on Amaranthus powellii Wats : II. Stomatal Response to Vapor Pressure Difference is Consistent with Optimization of Stomatal Conductance. AB - The hypothesis of optimal stomatal conductance predicts conductance should vary with changes of the vapor pressure difference between leaf and air (VPD) to keep the partial derivative of transpiration rate (E) with respect to assimilation rate (A) constant ( partial differentialE/ partial differentialA = lambda). Stomatal conductance of Amaranthus powellii Wats. decreased strongly with increasing VPD for leaves with high total leaf nitrogen concentrations; whereas, it decreased slightly with increasing VPD for leaves with low leaf nitrogen concentrations. The calculated value of partial differentialE/ partial differentialA was constant for leaves with high leaf nitrogen concentrations but was not constant for leaves with low leaf nitrogen concentrations. However, the predicted values of stomatal conductance, transpiration rate, and assimilation rate for a constant lambda = 220 moles H(2)O/mole CO(2) and the measured values fit the y = x line for all nitrate treatments. These data extend the experimental support for the optimal water use efficiency hypothesis for a C(4) plant grown under different nitrate concentrations. PMID- 16664461 TI - Effects of Nitrate Application on Amaranthus powellii Wats. : III. Optimal Allocation of Leaf Nitrogen for Photosynthesis and Stomatal Conductance. AB - Optimal allocation of leaf nitrogen maximizes daily CO(2) assimilation for a given leaf nitrogen concentration. According to the hypothesis of optimization, this condition occurs when the partial derivative of assimilation rate with respect to leaf nitrogen concentration is constant. This hypothesis predicts a linear increase of assimilation rate with leaf nitrogen concentration under constant conditions. Plants of Amaranthus powellii Wats. were grown at 1, 5, 10, or 45 millimolar nitrate to obtain leaves with different nitrogen concentrations. Assimilation rate at 340 microbar CO(2)/bar, stomatal conductance, CO(2)- and light-saturated net photosynthetic rate, the initial slope of the CO(2) response of photosynthesis, ribulose-1,5'-bisphosphate carboxylase activity, and phosphoenolpyruvate carboxylase activity were linearly related to estimated or actual leaf nitrogen concentration. The data are consistent with the optimal use of leaf nitrogen. This hypothesis and the hypothesis of optimal stomatal conductance were combined to determine the relationship between conductance and leaf nitrogen concentration. The slope of conductance versus leaf nitrogen concentration was not significantly different than the slope predicted by the combination of the two hypotheses. Stomatal conductance was linearly related to leaf nitrogen in the field and the slope decreased with lower xylem pressure potentials in a manner consistent with the hypotheses. Finally, apparent maximum stomatal aperture of isolated abaxial epidermal strips was linearly related to leaf nitrogen suggesting stomatal conductance and assimilation rate are controlled in parallel by leaf nitrogen concentration or some factor correlated with leaf nitrogen. PMID- 16664462 TI - Stem Extension Rate in Light-Grown Plants : Effects of Photo- and Thermoperiodic Treatments on the Endogenous Circadian Rhythm in Chenopodium rubrum. AB - Low temperature pulses have two effects on the circadian rhythm exhibited by stem extension rate of green Chenopodium rubrum plants. First, low temperature pulses have the same effect on the phasing of the rhythm as a dark period interrupting continuous light. Second, low temperature pulses stimulate stem extension rate during the 10 hours immediately following the end of the pulse. A difference in temperature between soil and air increases this effect. In any case, it is the change in temperature which is essential and not a specific temperature. Effects of light and temperature on phasing and amplitude of the rhythm explain why the maximal stem growth is observed under normal photo-thermoperiodic conditions, i.e. a high temperature during the photoperiod and a low temperature during the dark period. PMID- 16664463 TI - Isoflavonoid Formation as an Indicator of UV Stress in Bean (Phaseolus vulgaris L.) Leaves : The Significance of Photorepair in Assessing Potential Damage by Increased Solar UV-B Radiation. AB - Induction of the isoflavonoid pigment, coumestrol (3,9-dihydroxy-6H-benzofuro [3,2-c][1] benzopyran-6-one), in primary leaves of beans (Phaseolus vulgaris L. var Saxa) by ultraviolet (UV) radiation was used as a quantifiable marker for UV damage to a plant system. Coumestrol was induced only by wavelengths below 300 nanometers and its formation could be reversed by treatment with white, but not red light after the UV irradiation period. Formation of coumestrol by UV could also be prevented over a period of 14 hours by simultaneous irradiation with blue light provided that the blue fluence rate was high enough. The results suggest that coumestrol formation is mediated via UV-induced pyrimidine dimer formation in the plant DNA and the photorepair properties of blue light are discussed with respect to possible increases in solar UV due to stratospheric ozone depletion. PMID- 16664464 TI - Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth. AB - Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect-90% inhibition-after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K(m) (app) of arginine decarboxylase for its substrate and reduced the V(max) (app) by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed. PMID- 16664465 TI - Aminopeptidase activity from germinated jojoba cotyledons. AB - One major and two minor aminopeptidase activities from germinated jojoba (Simmondsia chinensis) cotyledon extracts were separated by ammonium sulfate precipitation and chromatofocusing. None of the activities were inhibited by 1,10 phenanthroline.The major aminopeptidase, purified 260-fold, showed a pH optimum of 6.9 with leucine-p-nitroanilide as substrate, a molecular weight estimated at 14,200 by electrophoretic analysis, and an isoelectric point of 4.5 according to the chromatofocusing pattern. Activity was inhibited by p-chloromercuribenzoate, slightly stimulated by 1,10 phenanthroline and 2-mercaptoethanol, and not influenced by Mg(2+) or diethyl pyrocarbonate. Inhibition by p chloromercuribenzoate was prevented by the presence of cysteine in the assay. Leucine-p-nitroanilide and leucine-beta-naphthylamide were the most rapidly hydrolyzed of 11 carboxy-terminal end blocked synthetic substrates tested. No activity on endopeptidase or carboxypeptidase specific substrates was detected. The major aminopeptidase showed activity on a saline soluble, jojoba seed protein preparation and we suggest a possible physiological role for the enzyme in the concerted degradation of globulin reserve proteins during cotyledon senescence. PMID- 16664466 TI - Control of plastidic glycolipid synthesis and its relation to chlorophyll formation. AB - Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv "Halo") and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [(14)C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [(14)C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [(14)C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [(14)C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [(14)C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity. PMID- 16664467 TI - Abscisic Acid Accumulation by Roots of Xanthium strumarium L. and Lycopersicon esculentum Mill. in Relation to Water Stress. AB - Plants of Xanthium strumarium L. and Lycopersicon esculentum Mill. cv ;Rheinlands Ruhm' were grown in solution culture, and control and steam-girdled intact plants were stressed. Detached roots of both species were stressed to different extents in two ways: (a) either in warm air or, (b) in the osmoticum Aquacide III. The roots of both species produced and accumulated progressively more abscisic acid (ABA), the greater the stress inflicted by either method. ABA-glucose ester levels in Xanthium roots were not affected by water stress and were too low to be the source of the stress-induced ABA. The fact that ABA accumulated in detached roots and in roots of girdled plants proves that ABA was synthesized in the roots and not merely transported from the shoots.Maximum ABA accumulation in detached roots occurred after 60 to 70% loss of fresh weight. In Xanthium roots, ABA levels continued to increase for at least 11 hours, and no catabolism was apparent when stressed roots were immersed in water, although the roots did stop accumulating ABA. When osmotically stressed, Xanthium roots reached a maximum ABA level after 2 hours, but ABA continued to rise in the medium.Under optimal stress conditions, endogenous ABA levels increased 100 times over their prestress values in detached roots of Xanthium, and 15 times in Lycopersicon under nonoptimal stress, when endogenous ABA was expressed as concentrations based on tissue water content. These are much greater relative increases than observed in the leaves (15 times in Xanthium, 3 times in Lycopersicon), although the roots contain substantially less ABA than the leaves in all circumstances. The results suggest that the endogenous level of ABA in roots could rise appreciably prior to leaf wilt, and could modify the plant's water economy before the leaves become stressed. PMID- 16664468 TI - Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant. AB - Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division. PMID- 16664469 TI - Seed growth rate and carbohydrate pool sizes of the soybean fruit. AB - The relationships between various carbohydrate pools of the soybean (Glycine max [L.] Merrill) fruit and growth rate of seeds were evaluated. Plants during midpod fill were subjected to various CO(2) concentrations or light intensities for 7 days to generate different rates of seed growth. Dry matter accumulation rates of seeds and pod wall, along with glucose, sucrose, and starch concentrations in the pod wall, seed coat, and embryo were measured in three-seeded fruits located from nodes six through ten. Seed growth rates ranged from 4 to 37 milligrams.day( 1).fruit(-1). When seed growth rates were greater than 12 milligrams.day( 1).fruit(-1), sucrose concentration remained relatively constant in the pod wall (1.5 milligrams.100 milligrams dry weight(-1)), seed coat (8.5 milligrams.100 milligrams dry weight(-1)), and embryo (5.0 milligrams.100 milligrams dry weight( 1)). However, sucrose concentrations decreased in all three parts of the fruit as growth rate of the seeds fell below 12 milligrams.day(-1).fruit(-1). This relationship suggests that at high seed growth rates, flux of sucrose through the sucrose pools of the fruit was more important than pool size for growth. Starch concentration in the pod wall remained relatively constant (2 milligrams.100 milligrams dry weight(-1)) at higher rates of seed growth but decreased as seed growth rates fell below 12 milligrams.day(-1).fruit(-1). This suggests that pod wall starch may buffer seed growth under conditions of limiting assimilate availability. There was no indication that carbohydrate pools of the fruit were a limitation to transport or growth processes of the soybean fruit. PMID- 16664470 TI - Identification of 3-(O-beta-Glucosyl)-2-Indolone-3-Acetylaspartic Acid as a New Indole-3-Acetic Acid Metabolite in Vicia Seedlings. AB - A new indole-3-acetic acid metabolite was isolated from broad bean (Vicia faba L. cv Chukyo) seedlings. It was a conjugate of dioxindole-3-acetic acid, aspartic acid, and glucose and was identified as 3-(O-beta-glucosyl)-2-indolone-3 acetylaspartic acid (molecular weight 484) from ultraviolet, infrared, nuclear magnetic resonance, and mass spectra. Its natural content in 4-day-old Vicia seedlings was estimated to be 8.6 nanomoles per gram fresh weight. It was suggested that oxidation of indole-3-acetic acid not accompanied by decarboxylation might regulate endogenous level of the hormone. PMID- 16664471 TI - Presence of Heat Shock mRNAs in Field Crown Soybeans. AB - Our laboratory has extensively defined many parameters of the heat shock (HS) response in etiolated soybean (Glycine max [L.] Merr.) hypocotyls, including the identification of cDNA clones for mRNAs encoding several low molecular weight HS proteins. We have now investigated the response of mature plants to a HS in a growth chamber and to high temperature stress under field conditions. Soybean plants show induction of HS mRNAs when the temperature of the chamber is rapidly shifted from 28 degrees C to 45 degrees C. This temperature of induction is significantly higher than the optimal induction temperature for etiolated hypocotyls, probably reflecting the ability of mature plants to lower their leaf temperatures below the ambient air temperature through transpirational cooling. Samples of soybean leaves were taken from an irrigated and a nonirrigated field during a 24-h period when midday temperatures reached 40 degrees C. Several HS mRNAs were present in samples from both fields, although the levels of these mRNAs were much higher in nonirrigated leaves. This differential response of HS mRNA steady state levels was not a response to water stress, since water-stressed plants at 28 degrees C did not induce HS mRNAS. Rather, these quantitative differences are probably due to differences in actual leaf temperatures between irrigated and nonirrigated leaves. The presence of these HS mRNAS in field-grown plants suggests that HS proteins are produced as part of the normal plant response to high temperature. PMID- 16664472 TI - Role of ethylene in the senescence of isolated hibiscus petals. AB - Senescence of petals isolated from flowers of Hibiscus rosa-sinensis L. (cv Pink Versicolor) was associated with increased ethylene production. Exposure to ethylene (10 microliters per liter) accelerated the onset of senescence, as indicated by petal in-rolling, and stimulated ethylene production. Senescence was also hastened by basal application of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminooxyacetic acid, an inhibitor of ethylene biosynthesis, effectively inhibited ethylene production by petals and delayed petal in-rolling. In marked contrast to these results with mature petals, immature petals isolated from flowers the day before flower opening did not respond to ethylene in terms of an increase in ethylene production or petal in-rolling. Furthermore, treatment with silver thiosulfate the day before flower opening effectively prevented petal senescence, while silver thiosulfate treatment on the morning of flower opening was ineffective. Application of ACC to both immature and mature petals greatly stimulated ethylene production indicating the presence of an active ethylene forming enzyme in both tissues. Immature petals contained less free ACC than mature, presenescent petals and appeared to possess a more active system for converting ACC into its conjugated form. Thus, while the nature of the lack of responsiveness of immature petals to ethylene is unknown, ethylene production in hibiscus petals appears to be regulated by the control over ACC availability. PMID- 16664473 TI - Effects of CO(2) Enrichment and Carbohydrate Content on the Dark Respiration of Soybeans. AB - During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO(2) per liter air, was 1.45 milligrams CO(2) evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO(2) per liter air). There was a higher foliar nonstructural carbohydrate level (e.g. sucrose and starch) in the CO(2) enriched compared with CO(2) normal plants. For example, leaves of enriched plants displayed levels of nonstructural carbohydrate equivalent to 174 milligrams glucose per gram dry weight compared to the 84 milligrams glucose per gram dry weight found in control plant leaves. As the leaves of CO(2) enriched plants approached full expansion, both the foliar respiration rate and carbohydrate content of the CO(2) enriched leaves decreased until they were equivalent with those same parameters in the leaves of control plants. A strong positive correlation between respiration rate and carbohydrate content was seen in high CO(2) adapted plants, but not in the control plants.Mitochondria, isolated simultaneously from the leaves of CO(2) enriched and control plants, showed no difference in NADH or malate-glutamate dependent O(2) uptake, and there were no observed differences in the specific activities of NAD(+) linked isocitrate dehydrogenase and cytochrome c oxidase. Since the mitochondrial O(2) uptake and total enzyme activities were not greater in young enriched leaves, the increase in leaf respiration rate was not caused by metabolic adaptations in the leaf mitochondria as a response to long term CO(2) enrichment. It was concluded, that the higher respiration rate in the enriched plant's foliage was attributable, in part, to a higher carbohydrate status. PMID- 16664474 TI - Triosephosphates modulate leaf mitochondrial phosphorylation by inhibition and uncoupling of electron transport. AB - The effect of TP (triosephosphates:glyceraldehyde-3 phosphate, GAP, +dihydroxyacetone phosphate, DHAP) on respiration, phosphorylation and matrix ATP/ADP ratios of isolated oat mesophyll mitochondria was investigated. With both malate and NADH, a 50% inhibition of state 3-phosphorylation was induced by about 15 to 20 millimolar GAP and 30 to 40 millimolar DHAP. However, the nature of the inhibition appeared to be different with the two respiratory substrates. In the presence of NADH, TP did not inhibit the rate of state 3 (addition of ADP) O(2) consumption. In fact, depending on concentration, TP gradually increased the rates measured without ADP towards those seen under state 3, acting as uncouplers. When malate was the substrate for respiration, state 3 rates were decreased. The effect was comparable to that of rotenone and could be abolished by the addition of NADH. These observations indicate a dual action of TP: inhibition of electron transport around site I and uncoupling. In any case, the intramitochondrial ATP/ADP ratio decreased upon addition of TP. The effective TP concentrations as well as the changes in mitochondrial ATP/ADP ratios were comparable to results on changes of compartmental pool sizes of adenylates and other metabolites during dark/light transition of oat mesophyll protoplasts (R. Hampp, M. Goller, H. Fullgraf, and I. Eberle 1985 Plant Cell Physiol 24: 99). The possible role of TP in the regulation of mitochondrial respiration in the light, as well as modes of interference, are discussed. PMID- 16664475 TI - Possible control of maize leaf sucrose-phosphate synthase activity by light modulation. AB - Sucrose phosphate synthase (SPS) activity was measured in extracts of maize (Zea mays L.) and soybean (Glycine max L. [Merr.]) leaves over a single day/night cycle. There was a 2- to 3-fold postillumination increase in extractable enzyme activity in maize leaves, whereas the activity of soybean SPS was only about 30% higher in extracts prepared from light- compared to dark-adapted leaves. Alterations in extractable maize leaf SPS activity correlated with light/dark transitions suggesting that the enzyme may be light modulated. Diurnal variations of extractable maize leaf SPS activity were also observed in a greenhouse experiment. A transition from high (light) to low (dark) extractable SPS activity occurred near the light compensation point for photosynthesis (about 20 micromole photons per square meter per second). Further increases in irradiance did not increase extractable SPS activity. Substrate affinities for uridine 5' diphosphoglucose (Michaelis constant = 3.5 and 5.1 millimolar) and fructose-6 phosphate (half maximal concentration = 1.0 and 2.5 millimolar) were lower for partially purified SPS obtained from light compared to dark acclimated maize leaves. Light-induced changes in extractable SPS activity were stable for at least one column chromatography step. The above results indicate that light induced changes in SPS activity may be important in controlling the photosynthetic production of sucrose. PMID- 16664476 TI - Altered Leaf Structure and Function in Triazine-Resistant Common Groundsel (Senecio vulgaris). AB - Anatomical and physiological characteristics of leaves of triazinesusceptible and -resistant biotypes of common groundsel (Senecio vulgaris L.) were studied in order to explain the differences in light-saturated photosynthetic rates previously reported. Leaves were of uniform leaf plastochron index from greenhouse-grown plants. Susceptible plants had greater leaf fresh and dry weights and leaf areas, while resistant plants had greater specific leaf mass (mg fresh weight/cm(2)). Susceptible plants had greater amounts of total chlorophyll per unit leaf weight and a higher chlorophyll a/b ratio. Soluble protein in leaves was higher in susceptible chloroplasts on a weight and area basis, but similar to resistant chloroplasts on a unit chlorophyll basis. Activity of ribulose 1,5-bisphosphate carboxylase was higher in resistant plants on a fresh weight, leaf area, and milligram chlorophyll basis. Stomatal frequency, length, and arrangement were similar between biotypes, as were transpiration and conductance. Resistant leaves had less air space (v/v), more cells in palisade and spongy mesophyll, and a greater volume of palisade tissue than spongy, when compared to susceptible leaves. Differences in leaf structure and function between biotypes are probably due to a complex of developmental adaptations which may be only indirectly related to modified photosystem II in resistant plants. These results indicate that the consistently lower rates of net photosynthesis and yield in resistant plants cannot be explained solely on the basis of these leaf characteristics. Several possible mechanisms to account for reduced productivity are suggested. PMID- 16664477 TI - Biosynthesis and Degradation of a Wheat Embryo Cytokinin-Binding Protein during Embryogenesis and Germination. AB - The accumulation and degradation of a wheat (Triticum durum) embryo cytokinin binding protein (CBF-1) was followed during embryo development and germination by its N(6)-benzyladenine (BA) binding activity and immunological reactivity (rocket immunoelectrophoresis and Western blotting). Both BA binding activity and CBF-1 appeared at 2 weeks post-anthesis and rose sharply between 2 to 4 weeks before leveling off to approximately 47 micrograms per embryo (9% of the soluble embryo protein at maturity). In vitro translation of polyadenylated RNA from 20-day-old embryos yielded a polypeptide which was immunoprecipitable with anti-CBF-1 IgG and migrated closely to the 54-kilodalton CBF-1 polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon germination, both the amount of CBF-1 and BA binding activity dropped to low levels within 3 days. The data are discussed in relation to the possible role of CBF-1 as a regulator of cytokinin availability, and comparisons are drawn between the structural and biosynthetic similarities found between CBF-1 and the vicilin storage proteins of legumes. An improved method for isolating undegraded CBF-1 from whole seeds is also presented. PMID- 16664478 TI - Differentially Regulated Isozymes of 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Seedlings of Vigna radiata [L.] Wilczek. AB - Two isozymes of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) designated DS-Mn and DS-Co were separated from seedlings of Vigna radiata [L.] Wilczek by DEAE-cellulose column chromatography. DS-Mn was activated 2.6-fold by 0.4 millimolar manganese, had an activity optimum of 7.0, and was substrate inhibited by erythrose-4-phosphate (E4P) concentrations in excess of 0.5 millimolar. In contrast, DS-Co had an activity optimum at pH 8.8, required E4P concentrations of at least 4 millimolar to approach saturation, and exhibited an absolute requirement for divalent cation (cobalt being the best). Regulatory properties of the two isozymes differed dramatically. The activity of DS-Mn was activated by chorismate (noncompetitively against E4P and competitively against phosphoenolpyruvate), and was inhibited by prephenate and l-arogenate (competitively against E4P and noncompetitively against phosphoenolpyruvate in both cases). Under standard assay conditions, l-arogenate inhibited the activity of DS-Mn 50% at a concentration of 155 micromolar and was at least 3 times more potent than prephenate on a molar basis. Weak inhibition of DS-Mn by l-tryptophan was also observed, the magnitude of inhibition increasing with decreasing pH. The pattern of allosteric control found for DS-Mn is consistent with the operation of a control mechanism of sequential feedback inhibition governing overall pathway flux. DS-Co was not subject to allosteric control by any of the molecules affecting DS-Mn. However, DS-Co was inhibited by caffeate (3,4 dihydroxycinnamate), noncompetitively with respect to either substrate. The striking parallels between the isozyme pairs of 3-deoxy-d-arabino-heptulosonate-7 phosphate synthase and chorismate mutase are noted-one isozyme in each case being tightly regulated, the other being essentially unregulated. PMID- 16664480 TI - Extremely low frequency resistance fluctuations of chara membrane. AB - The membrane resistivity of resting characean cells was measured and found to fluctuate unpredictably about its resting level. The amplitude of these fluctuations relative to the mean resistivity was normally somewhat larger than the same ratio for the vacuolar voltage; further the resistivity and voltage fluctuations seemed not to be strongly correlated. PMID- 16664479 TI - The Compartmentation of Abscisic Acid and beta-d-Glucopyranosyl Abscisate in Mesophyll Cells. AB - beta-d-Glucopyranosyl abscisate (ABA-GE) is synthesized in Xanthium strumarium L. leaves during water stress. Following recovery from stress, the amount of ABA-GE does not decline. These observations led to the hypothesis that ABA-GE is sequestered in the vacuole where it is metabolically inert. The localization of abscisic acid (ABA) and ABA-GE was investigated by a dimethyl sulfoxide (DMSO) compartmentation method and by direct isolation of vacuoles.With the DMSO compartmentation method it was shown that in Xanthium mesophyll cells ABA was in a compartment not accessible to DMSO, presumably the chloroplast, whereas ABA-GE was in a compartment accessible to DMSO, presumably the vacuole. Neutral red, which accumulates in the vacuoles, showed a similar DMSO concentration dependence for its release from the cells as ABA-GE.Vacuoles isolated from Vicia faba L. leaf protoplasts contained 22% of the total ABA and 91% of the ABA-GE. Some of the ABA in the vacuole preparations was probably due to cytoplasmic contamination. These findings indicate that ABA-GE is sequestered in the vacuoles of mesophyll cells where the conjugated form of ABA is removed from the active ABA pool. PMID- 16664481 TI - Intermediates in the formation of the chlorophyll isocyclic ring. AB - Cell-free, organelle-free synthesis of Mg-2,4-divinylpheoporphyrin a(5) (MgDVP) from Mg-protoporphyrin IX monomethyl ester (Mg-Proto Me) has been described (Wong and Castelfranco 1984 Plant Physiol 75: 658-661). This system consists of plastid membrane and stromal fractions and requires O(2), NAD(P)H and S adenosylmethionine (SAM). The synthetic 6-methyl-beta-ketopropionate analog of Mg Proto Me was converted to MgDVP by the same catalytic system in the presence of O(2) and NADPH. SAM was not required. A compound (X) displaying the kinetic behavior of an intermediate was isolated from reaction mixtures with Mg-Proto Me as the substrate, but not with the 6-methyl-beta-ketopropionate analog as the substrate. X was identified as the 6-methyl-beta-hydroxypropionate analog of Mg Proto Me by conversion to the dimethyl ester with CH(2)N(2) and comparison with authentic 6-beta-hydroxydimethyl ester. X was converted to MgDVP by the same catalytic system in the presence of O(2) and NADPH. We conclude that the conversion of Mg-Proto Me to MgDVP proceeds through the 6-beta-hydroxy and the 6 beta-ketopropionate esters in agreement with earlier suggestions. PMID- 16664482 TI - Properties of the Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase System. AB - Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, the enzyme system responsible for the formation of the chlorophyll isocyclic ring, exhibits requirements for both essential sulfhydryls and essential disulfides. It is inhibited by N-ethylmaleimide, dithiothreitol, and beta-mercaptoethanol, but not by sodium arsenite. This enzyme system shows some substrate specificity: (a) the 6-side-chain of the macrocycle can either be a methyl propionate ester, or its beta-hydroxy or beta-keto derivatives; (b) the 7-side-chain can either be a propionic acid or a methyl propionate ester; (c) both the 4-vinyl and the 4-ethyl series can serve as substrates, at least at the beta-keto ester level; (d) the activity appears to be lost if the side-chain in the 2-position is reduced from a vinyl to an ethyl. PMID- 16664483 TI - H extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments treated with permeant weak acids. AB - The rapid uptake of weak acids permeant in the uncharged form is accompanied in maize and wheat root segments by a hyperpolarization of the transmembrane electrical potential and an increase in K(+) uptake, suggesting a stimulation of the plasmalemma H(+) pump. The evaluation of weak acid-induced H(+) extrusion must take into account the alkalinization of the medium due to the rapid uptake of the uncharged form of the acid, partially masking the proton pump-mediated extrusion of H(+). The data corrected for this interference show that the lipophilic butyric acid and trimethyl acetic acid induce in maize and in wheat root segments a significant increase in ;real' H(+) extrusion, roughly matching the increase in net K(+) uptake. The presence of K(+) significantly increases the rate of uptake of the weak acid, possibly as a consequence of an alkalinization of the cytosol associated with K(+) absorption. In maize root segments, the effects of fusicoccin and those of butyric acid on both K(+) uptake and H(+) extrusion are clearly synergistic, thus suggesting distinct modes of action. These results support the view that the activity of the plasmalemma H(+) pump is regulated by the value of cytosolic pH. PMID- 16664484 TI - Sexual Agglutination in the Unicellular Green Alga Chlamydomonas eugametos: Identification and Properties of the Mating Type plus Agglutination Factor. AB - Gametes of the unicellular green alga Chlamydomonas eugametos agglutinate via their flagella. The mating type plus agglutination factor was solubilized by relatively mild treatments such as a short pH shock or an osmotic shock indicating that it is an extrinsic membrane component. It was also extracted in the nonionic detergent Triton X-100. A simple two-step procedure consisting of gel filtration over Sepharose 4B-cross-linked followed by anion exchange chromatography of the void volume yielded an electrophoretically pure preparation of a single high molecular weight glycoprotein. The agglutination factor sedimented as a 9.3 S particle (assuming a density of 1.50) in sucrose gradients. This low value, compared with the high apparent molecular weight seen during gel filtration and electrophoresis, suggests that the agglutination factor is a rod like molecule. This was confirmed by viewing rotary-shadowed preparations in the electron microscope. A population of long slender molecules was revealed (328 +/- 20 nanometers), many of which had a knob at one end and a flexible region about one fourth of the length from the other end. PMID- 16664485 TI - Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons. AB - The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the beta-subunit of beta-conglycinin, one of the major storage proteins of soybean seeds. The amount of the alpha'-and alpha-subunits of beta conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the beta-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and beta-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against beta conglycinin, there was an increased amount of pre-beta-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-beta-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of beta-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the beta-subunit of beta-conglycinin in developing cultured soybean cotyledons. PMID- 16664486 TI - Intracellular Localization of Lunularic Acid and Prelunularic Acid in Suspension Cultured Cells of Marchantia polymorpha. AB - Intracellular localization of lunularic acid and prelunularic acid in suspension cultured cells of Marchantia polymorpha L. was studied. The sum of both compounds was determined as lunularic acid group (LNAs) because of the instability of prelunularic acid to convert into lunularic acid.Mechanical disruption of the cells followed by differential centrifugation showed that LNAs was associated with the supernatant of 100,000g centrifugation. Protoplasts isolated from the cells were osmotically ruptured and the distribution of LNAs among the organelles was examined by discontinuous density gradient centrifugation of the protoplast contents. Successful isolation of intact chloroplasts, mitochondria and peroxisomes free from cytoplasm indicated that LNAs was not accumulated in these organelles. Flotation techniques resulted in an efficient isolation of pure vacuoles and revealed that LNAs was distributed almost equally in the vacuoles and cytoplasm. PMID- 16664487 TI - Effect of frost hardening on lipid and Fatty Acid composition of chloroplast thylakoid membranes in two wheat varieties of contrasting hardiness. AB - Lipid and fatty acid composition of chloroplast thylakoid membranes was determined in two varieties of wheat (Triticum aestivum L.), the hardy Miranovskaja and the sensitive Penjamo. Plants were grown at room temperature or under frost hardening conditions (1.5 degrees C). Changes in lipid and fatty acid composition of the isolated thylakoids could be related to the temperature dependence of light-stimulated proton uptake. Changes in the thylakoid phospholipids upon hardening of the two varieties did not show any direct relation with low temperature tolerance of light-dependent H(+) uptake; neither did changes in phospholipid fatty acid chain lengthening to 20 and 22 C-atoms in combination with increased desaturation up to 6 double bonds. Increased low temperature tolerance of light-induced H(+) uptake by hardening was correlated with the following glycolipid changes: maintained glycolipid level, a proportionally increased digalactosyl diglyceride fraction, a decrease in thylakoid monogalactosyl diglyceride, increased sulfolipid fatty acid chain lengthening (20 and 22 C-atoms), and increased sulfolipid desaturation (4-6 double bonds). We suggest that the above mentioned changes in glycolipids have adaptive value for low temperature tolerance of light-dependent proton uptake. PMID- 16664488 TI - Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems. AB - Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed. PMID- 16664489 TI - 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Carrot Root (Daucus carota) Is a Hysteretic Enzyme. AB - Roots of carrots (Daucus carota) contain three activities of 3-deoxy-d-arabino heptulosonate 7-phosphate (DAHP) synthase, the enzyme that catalyzes the first step of the shikimate pathway. The three activities, enzymes I, II, and III, are separated by chromatography on phosphocellulose. Enzyme III, purified to electrophoretic homogeneity, has a native molecular weight of 103,000 and consists of two identical subunits of 53,000 daltons each. Double reciprocal plots of reaction velocity versus substrate concentration yield K(m) values of 0.03 and 0.07 millimolar for P-enolpyruvate and erythrose-4-P, respectively. Both products, DAHP and orthophosphate, inhibit the enzyme. Enzyme III is a hysteretic enzyme that is activated by physiological concentrations of l-tryptophan and Mn(2+), both of which also partially eliminate the hysteretic lag. Feedback activation of carrot DAHP synthase by tryptophan is interpreted to be an early regulatory signal for polyphenol biosynthesis. The three carrot DAHP synthase isoenzymes share antigenic determinants. PMID- 16664490 TI - Potassium Transport in Corn Roots : IV. Characterization of the Linear Component. AB - A detailed examination was conducted on the linear, or first-order kinetic component for K(+)((86)Rb(+)) influx into root segments of both low- and high salt grown corn seedlings (Zea mays [A632 x Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl(-) in the uptake solution by either SO(4) (2-), H(2)PO(4) (-), or NO(3) (-) caused a significant (50-60%) and specific inhibition of the linear component of K(+) influx. The anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid, was found to abolish saturable Cl(-) influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K(+) uptake system; this inhibition was identical to that observed when Cl(-) was replaced by other anions in the K(+) uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K(+) channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K(+) influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high salt roots to absorb tetraethylammonium.Our present results indicate that the linear component of K(+) influx may occur by a passive process involving transmembrane K(+) channels. Fluxes through these K(+) channels may be partly coupled to a saturating Cl(-) influx mechanism. PMID- 16664491 TI - Studies on the photoactivation of the water-oxidizing enzyme : I. Processes limiting photoactivation in hydroxylamine-extracted leaf segments. AB - In weak yet optimal light intensity, complete photoactivation of the water oxidizing enzyme in NH(2)OH-extracted wheat (Triticum aestivum, var Oasis) leaf segments could be obtained only after long dark preincubation. Photoactivation was not affected by ethylenediaminetetraacetate or inhibitors of photophosphorylation and protein synthesis, but was partially inhibited by a divalent cation ionophore. Complete photoactivation required ligation of approximately 4 Mn by the water oxidizing enzyme.WITHOUT DARK PREINCUBATION, PHOTOSYSTEM II (PSII) WAS SUSCEPTIBLE TO WEAK LIGHT PHOTOINHIBITION RESULTING IN: (a) 50% maximum decrease in photooxidation of artificial electron donors by PSII: (b) increased times for the variable fluorescence rise (with 3-(3,4 dichlorophenyl)-1,1-dimethyl urea): (c) abolishment of photoactivation: and (d) the imposition of sensitivity to inhibitors of photophosphorylation and 70S but not 80S protein synthesis on subsequent light-dependent recovery from photoinhibition and recovery of O(2) evolution. Decrease in susceptibility to photoinhibition and increase in rates of photoactivation resulting from dark preincubations proved closely correlated. Neither protein synthesis nor increases in abundances of thylakoid Mn(2+) and Ca(2+) were required for escape from photoinhibition. However, photoactivation of the wateroxidizing enzyme in NH(2)OH extracted Chlamydomonas occurred in absence of dark preincubation and protein synthesis. Results are discussed in the context of disassembly/reassembly/resynthesis of specific PSII polypeptides. PMID- 16664492 TI - Enzymic degradation of allantoate in developing soybeans. AB - A Mn(2+)-dependent enzymic breakdown of allantoate has been detected in crude and partially purified extracts of developing soybeans. The products detected were CO(2), NH(3), glyoxylate, labile glyoxylate derivatives, and low levels of urea. Urea is initially produced at less than 10% the rate of urease-independent CO(2) release indicating that the activity is not allantoate amidinohydrolase (i.e. urea is not directly cleaved off allantoate). The urease-independent CO(2) releasing activity has an apparent K(m) of 1.0 millimolar for allantoate. Ethylenediaminetetraacetate, borate, and acetohydroxamate (all at 10 millimolar) inhibit the enzymic production of NH(3), CO(2), and labile glyoxylate derivatives from allantoate. However, the potent urease inhibitor, phenyl phosphordiamidate does not inhibit CO(2) and NH(3) release indicating that the action of acetohydroxamate is not due to its inhibition of urease. That the allantoatedegrading activity was more than 5-fold greater in seed coats than in embryos is consistent with the data of Rainbird et al. (Plant Physiol 1984 74: 329-334) which indicate that available ureides are metabolized before reaching the embryo. 2-Ethanolthio, 2'ureido, acetic acid (NH(2)COHNCHCO(2)HSCH(2)CH(2)OH), the first allantoate-derived product detected by HPLC analysis, is an addition produced of mercaptoethanol with an unidentified enzymically produced ureido intermediate that is not derived from ureidoglycolate or oxalurate. PMID- 16664493 TI - Structure and possible ureide degrading function of the ubiquitous urease of soybean. AB - Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800 804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection ("Western Blot"). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid. PMID- 16664494 TI - Pyrimidine nucleoside uptake by petunia pollen: specificity and inhibitor studies on the carrier-mediated transport. AB - Transport of pyrimidine nucleosides into germinating Petunia hybrida pollen is carrier-mediated, and, except for thymidine, is inhibited by the energy poisons N,N'-dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 2,4 dinitrophenol, and carbonylcyanide-m-chlorophenylhydrazone. Kinetic studies with analogs deoxyuridine and 5-bromodeoxyuridine show that they too are taken up faster than thymidine and inhibited by the energy poisons. These and other analogs inhibit uridine and cytidine transport more than thymidine, as do the inhibitors parachloromercuribenzoic acid, N-ethylmaleimide, phenylarsine oxide, o phenanthroline, ethylene diamenetetraacetate, and ethylene glycol-bis (beta aminoethyl ether) N,N,N'N'-tetraacetic acid. Citrate, phosphate, succinate, and tartrate inhibited uptake of all pyrimidine nucleosides. The specific inhibitor of nucleoside transport in animal cells, nitrobenzylthioinosine, has little effect on pollen transport. Uridine and deoxyuridine accumulate against a concentration gradient, suggesting active transport. Except for thymidine, however, transported nucleosides were found to be extensively phosphorylated. Until mutant plants are found which do not phosphorylate uridine, it is not possible to decide unequivocally between active and nonactive transport for uridine. However, consistent with a low level of DNA synthesis in germinating Petunia pollen, it is clear that thymidine transport is nonactive and relatively slow. It is apparent from these experiments that a more sensitive way to study DNA repair in this pollen would be to use 5-bromodeoxyuridine or deoxyuridine instead of thymidine to label repaired DNA. The results show that pollen has the transport systems necessary to take up pyrimidine nucleosides from Petunia styles, where it is known that the concentration of free nucleosides increase after pollination. PMID- 16664495 TI - Purification and Partial Characterization of Ribulose Bisphosphate Carboxylase Holoenzyme and Its Subunits from Chlorella sorokiniana and Use of Its Antigen Affinity-Purified Antibodies in Specific Immunoprecipitation and Immunoadsorption Procedures. AB - Chlorella sorokiniana ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified to homogeneity with yields of 35 to 40%. Molecular weights of the holoenzyme and its large subunit (LS) and small subunit (SS) were estimated to be 562,000, 55,000, and 15,800, respectively. Amino acid compositions of LS from C. sorokiniana and spinach were similar, whereas the compositions of their SS were very different. Antisera prepared against holoenzyme, LS, and SS were purified by antigen-affinity column chromatography. Purified anti-holoenzyme immunoglobulin G (IgG) and anti-LS IgG cross-reacted with holoenzyme and LS but not with SS. Anti SS IgG reacted neither with holoenzyme nor with LS. Because purified anti holoenzyme IgG or the anti-LS IgG inhibited RuBPCase activity, antibody preparations were titered by the amount of (35)S-labeled RuBPCase immunoprecipitated. Approximately 40% of the total RuBPCase activity in cell homogenates was tightly particulate-bound and was solubilized with 0.5% Nonidet P 40 without inhibition of enzyme activity. Direct-immunoprecipitation and indirect immunoadsorption procedures, with affinity-purified anti-holoenzyme IgG, gave specific and quantitative recovery of (35)S-labeled RuBPCase from cell extracts containing Nonidet P-40. Affinity-purified anti-LS IgG and anti-SS IgG were used to immunoprecipitate either the LS or SS antigens synthesized in vitro in a mRNA dependent in vitro translation assay system. Rocket immunoelectrophoresis was used to quantify as little as 50 nanograms of RuBPCase antigen in cell extracts. PMID- 16664496 TI - Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana. AB - By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady state cultural conditions. PMID- 16664497 TI - Peroxidases and glycosidases in intercellular fluids from noninoculated and rust affected wheat leaves : isozyme assay on nitrocellulose blots from two dimensional gels. AB - Proteins in intercellular washing fluid (IWF) from noninoculated and stem rust affected wheat leaves were separated by isoelectric focusing and polyacrylamide gel electrophoresis under nondenaturing conditions, transferred to nitrocellulose membranes, and assayed in situ for peroxidase and glycosidase activity.Two infection-related peroxidase isozymes were detected in addition to more than ten that were present only in noninoculated plants. One other peroxidase isozyme was present in much higher concentration in IWF from infected leaves than in IWF from noninoculated leaves.IWF contained many polymorphic glycosidases. A new method is described to localize the glycosidase isozymes accurately on nitrocellulose blots for evaluation of their substrate specificities: each blot was developed with the appropriate p-nitrophenyl glycoside to reveal glycosidase activity, then reprobed for concanavalin A-binding glycoproteins to serve as an internal reference frame for blot-to-blot comparisons. This procedure also provided information on glycosylation of the isozymes.The locations of at least 15 (out of 37) isozymes were coincident with concanavalin A binding, including those of all 10 alpha-d mannosidases, and of 6 beta-d-xylosidases. On five areas of the blots there was coincidence of beta-d-xylosidase and alpha-l-arabinosidase activity. Three of these areas corresponded to three of the most prominent Coomassie brilliant blue stainable IWF proteins in gels. Isozymes that could convert p-nitrophenyl-beta-d glucoside, -beta-d-galactoside, and/or -beta-d-fucoside revealed a complex pattern of partially overlapping substrate specificities: two isozymes utilized both glucose and fucose derivatives, one utilized all three derivatives, and several others converted only one of the three substrates. No enzymes were detected with activity on p-nitrophenyl-beta-d-galactosaminide, -beta-l-fucoside, or -alpha-d-galactoside. No additional glycosidases were detected in IWF from stem rust-affected leaves. PMID- 16664498 TI - Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L. AB - The light-dependent pH changes in the suspending medium of guard cell protoplasts (GCP) from Vicia faba were studied. Upon illumination, the medium was initially slightly alkalinized and then acidified. The extent of alkalinization was lower in CO(2)-free air than in normal air. This initial alkalinization was inhibited by DCMU. Acidification in CO(2)-free air became observable in shorter duration of light exposure than that in normal air. The rate of acidification was higher in CO(2)-free air than in normal air. The CO(2) level of the medium decreased in the light, and increased in the dark. (14)CO(2) uptake was enhanced 2- to 3-fold by light, but not in the presence of DCMU. These results indicate that photosynthetic CO(2) fixation does take place in GCP and that the initial alkalinization is due to this photosynthetic CO(2) uptake. Diethylstilbestrol, a nonmitochondrial membrane-bound ATPase inhibitor, inhibited the acidification, suggesting that the acidification resulted from H(+) extrusion by GCP. The acidification in light was also prevented by KCN, and partly by DCMU. Possible mechanisms of alkalinization and acidification are discussed in relation to guard cell metabolism. PMID- 16664499 TI - Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L. AB - Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K(+) and malate. DCMU inhibited the increase of K(+) and malate, and consequently swelling.Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement. PMID- 16664500 TI - Varying Photoperiod, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and CO(2) Uptake in Thalassiosira fluviatilis (Bacillariophyceae). AB - The purpose of this research was to test the hypothesis that acclimation of the unicellular marine alga, Thalassiosira fluviatilis Hustedt, to short photoperiods results in decreased cellular concentrations of ribulose 1,5-bisphosphate carboxylase/oxygenase and decreased rates of light-saturated CO(2) uptake. Cells were acclimated to photoperiods of 6:18, 12:12, and 18:6 h:h light:dark, and concentrations of the large subunit of the enzyme and responses of CO(2) uptake to varying irradiance were measured. Concentrations of the large subunit, which weighed approximately 50 kilodaltons, were conserved while rates of CO(2) uptake under light saturation and limitation, and cellular contents of chlorophyll a increased as photoperiod decreased. Apparently, these cells acclimate to short photoperiods by increasing rates of CO(2) uptake under saturating irradiances by increasing in vivo activation of ribulose 1,5-bisphosphate carboxylase/oxygenase. Also, chlorophyll-specific concentrations and specific activities of the enzyme appear to be lower and higher, respectively, in diatomaceous algae than in higher plants. PMID- 16664501 TI - Sucrose and Starch Synthesis in Spinach Plants Grown under Long and Short Photosynthetic Periods. AB - The flow of carbon into sucrose and starch was investigated in fully expanded primary leaves of spinach using the long to short day transition and partial defoliation as tools to manipulate sucrose/starch synthesis. Transfer from 12 hour to 7 hour photosynthetic periods resulted in a 4-fold increase in the initial rate of starch synthesis, a 50% increase in the initial rate of sucrose synthesis, a 30% increase in leaf sucrose, and a 40% decrease in fructose, 2,6 biphosphate. In addition, sucrose synthesis rates in cells isolated from shortened daylength plants are 80% higher than in cells isolated from control plants. These results show that, in spinach, an increase in the rates of both sucrose and starch synthesis can occur under short day conditions. In contrast, when short day plants are partially defoliated, starch levels remain high, fructose 2,6-biphosphate levels remain low, but the level of leaf sucrose drops by 50%. Thus, when demand exceeds supply, starch synthesis has priority over filling of leaf sucrose pools in the short day plant. PMID- 16664502 TI - The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism. AB - Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K(+) which was accompanied by an equimolar net influx of H(+). These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K(+) during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K(+) and H(+) fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K(+) concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K(+) efflux and H(+) influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K(+)/H(+) exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death. PMID- 16664503 TI - Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels. AB - Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development. PMID- 16664504 TI - Effect of increased temperature in apical regions of maize ears on starch synthesis enzymes and accumulation of sugars and starch. AB - Apical florets of maize (Zea mays L.) ears differentiate later than basal florets and form kernels which have lower dry matter accumulation rates. The purpose of this study was to determine whether increasing the temperature of apical kernels during the dry matter accumulation period would alter the difference in growth rate between apical and basal kernels. Apical regions of field-grown maize (cultivar Cornell 175) ears were heated to 25 +/- 3 degrees C from 7 days after pollination to maturity (tip-heated ears) and compared with unheated ears (control). In controls, apical-kernel endosperm had 24% smaller dry weight at maturity, lower concentration of sucrose, and lower activity of ADP-Glc starch synthase than basal-kernel endosperm, whereas ADP-Glc-pyrophosphorylase (ADPG PPase) activities were similar. In tip-heated ears apical-kernel endosperm had the same growth rate and final weight as basal-kernel endosperm and apical kernels had higher sucrose concentrations, higher ADP-Glc starch synthase activity, and similar ADPG-PPase activity. Total grain weight per ear was not increased by tip-heating because the increase in size of apical kernels was partially offset by a slight decrease in size of the basal- and middle-position kernels. Tip-heating hastened some of the developmental events in apical kernels. ADPG-PPase and ADP-Glc starch synthase activities reached peak levels and starch concentration began rising earlier in apical kernels. However, tip-heating did not shorten the period of starch accumulation in apical kernels. The results indicate that the lower growth rate and smaller size of apical kernels are not solely determined by differences in prepollination floret development. PMID- 16664505 TI - Subcellular localization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea leaves. AB - The subcellular distribution of the enzymes of de novo pyrimidine nucleotide biosynthesis was investigated in pea (Pisum sativum L. cv Progress No. 9) leaves. Aspartate carbamoyltransferase, the committed step of the pathway, was found to be strictly confined to the chloroplasts. Dihydro-orotase, orotate phosphoribosyl transferase, and orotidine decarboxylase activities were also found only in the plastids. The remaining enzyme of the pathway, dihydroorotate dehydrogenase, was shown to be mitochondrial. PMID- 16664506 TI - Effects of Acifluorfen on Endogenous Antioxidants and Protective Enzymes in Cucumber (Cucumis sativus L.) Cotyledons. AB - The herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate) causes strong photooxidative destruction of pigments and lipids in sensitive plant species. Antioxidants and oxygen radical scavengers slow the bleaching action of the herbicide. The effect of acifluorfen on glutathione and ascorbate levels in cucumber (Cucumis sativus L.) cotyledon discs was investigated to assess the relationship between herbicide activity and endogenous antioxidants. Acifluorfen decreased the levels of glutathione and ascorbate over 50% in discs exposed to less than 1.5 hours of white light (450 microeinsteins per square meter per second). Coincident increases in dehydroascorbate and glutathione disulfide were not observed. Acifluorfen also caused the rapid depletion of ascorbate in far-red light grown plants which were photosynthetically incompetent.Glutathione reductase, dehydroascorbate reductase, superoxide dismutase, ascorbate oxidase, ascorbate free radical reductase, peroxidase, and catalase activities rapidly decreased in acifluorfen-treated tissue exposed to white light. None of the enzymes were inhibited in vitro by the herbicide. Acifluorfen causes irreversible photooxidative destruction of plant tissue, in part, by depleting endogenous antioxidants and inhibiting the activities of protective enzymes. PMID- 16664507 TI - Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1- >3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase. AB - Polyclonal antibodies raised against barley (1-->3,1-->4)-beta-d-glucanase, alpha amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley. In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding alpha amylase or (1-->3,1-->4)-beta-d-glucanase, while in the aleurone alpha-amylase and (1-->3,1-->4)-beta-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1-->3,1-->4)-beta-d-glucanase, alpha-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation products were identical for each enzyme. PMID- 16664508 TI - Photochemical Apparatus Organization in the Chloroplasts of Two Beta vulgaris Genotypes. AB - The composition and structural organization of thylakoid membranes of a low chlorophyll mutant of Beta vulgaris was investigated using spectroscopic, kinetic and electrophoretic techniques. The data obtained were compared with those of a standard F1 hybrid of the same species. The mutant was depleted in chlorophyll b relative to the hybrid and it had a higher photosystem II/photosystem I reaction center (Q/P(700)) ratio and a smaller functional chlorophyll antenna size. Analysis of thylakoid membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the mutant lacked a portion of the chlorophyll a/b light-harvesting complex but was enriched in the photosystem II reaction center chlorophyll protein complex. Comparison of functional antenna sizes and of photosystem stoichiometries determined electrophoretically were in good agreement with those determined spectroscopically. Both approaches indicated that about 30% of the total chlorophyll was associated with photosystem I and about 70% with photosystem II. A greater proportion of photosystem II(beta) was detected in the mutant. The results suggest that a higher photosystem II to photosystem I ratio in the sugar beet mutant has apparently compensated for the smaller photosystem II chlorophyll light-harvesting antenna in its chloroplasts. Moreover, a lack of chlorophyll a/b light-harvesting complex correlates with the abundance of photosystem II(beta). It is proposed that a developmental relationship exists between the two types of photosystem II where photosystem II(beta) is a precursor form of photosystem II(alpha) occurring prior to the addition of the chlorophyll a/b light-harvesting complex and grana formation. PMID- 16664509 TI - ATP Production by Respiration and Fermentation, and Energy Charge during Aerobiosis and Anaerobiosis in Twelve Fatty and Starchy Germinating Seeds. AB - The respiration and fermentation rates were compared in germinating seeds of 12 different cultivated species from five families. In air, fermentation contributes significantly to the energy metabolism only in some species (pea, maize), but is generally negligible when compared to respiration. The fermentation rate under anoxia was related either to the metabolic activity under air or to the adenine nucleotide content of the seeds: it was generally higher in seeds which contain starchy reserves (rice, maize, sorghum, pea), than in seeds which do not contain starch (lettuce, sunflower, radish, turnip, cabbage, flax); however, it was similar in wheat, sorghum (starchy seeds), and soya (nonstarchy seeds). The value of the energy charge of all the seeds was lower under anoxia than in air: after 24 hours under anoxia, it was higher than 0.5 in the starchy seeds and in soya and it was around 0.25 in the other fatty seeds. PMID- 16664510 TI - Germination, respiration, and adenylate energy charge of seeds at various oxygen partial pressures. AB - The effect of O(2) partial pressure on the germination and the respiration of 12 cultivated species was studied. The reciprocal of the time necessary to observe rootlet emergence in 50% of the seeds was used to approach the germination rate. The maximum germination and respiration rates were reached in most seeds at O(2) pressures close to that of air. Decreasing the O(2) pressure produced a gradual decrease of the germination rate. The seeds could be classed in two groups according to their response to low O(2) pressures. Group I includes lettuce, sunflower, radish, turnip, cabbage, flax, and soybean: at O(2) pressures close to 2 kilopascals, the germination in this group was stopped and the adenylate energy charge was lower than 0.6. Group II includes rice, wheat, maize, sorghum, and pea. The germination rate of these seeds was also gradually decreased by lowering the O(2) partial pressure but germination still occured, very slowly, at 0.1 kilopascal; the adenylate energy charge remained higher than 0.6. These differences in the germination rates and adenylate energy charge values could not be explained by differences in the sensitivity of respiration to O(2). PMID- 16664511 TI - Measurement of the water potential of stored potato tubers. AB - A method of measuring the water potential of stored potato tubers (Solanum tuberosum L.) was needed to investigate the relationship of bacterial soft rot in tubers to water potential. Pressure chamber measurements, while useful for tubers with functional stolons, cannot be made on stored tubers. Measurements could be made on excised tissue pieces in a hygrometer chamber and with hygrometers implanted into tubers. We report here our evaluation of these hygrometric methods using a comparison with the pressure chamber on tubers harvested with stolons intact.In tubers of high water potential, measurements on excised tissue were as much as 0.5 megapascals lower than the pressure chamber, probably due to turgor driven expansion of the sample when freed from constraints imposed by surrounding tissue. Good agreement (+/-0.05 megapascals) was found between the implanted hygrometer and the pressure chamber at potentials higher than -0.5 megapascals. At lower water potentials, both hygrometer measurements were higher than the pressure chamber. Respirational heating of the tissue contributed to the increase in the excised tissue samples, but not with the implanted hygrometers because of the hygrometer design. The osmotic pressure balanced the pressure chamber measurement of potential at -0.7 megapascals, but was too small to do so at lower potentials. At most, 25% of this discrepancy can be accounted for by dilution by apoplastic water. We believe that the pressure chamber measurement is too low at low water potentials and that the error is associated with air bubbles in the xylem. At low potentials air emerged from xylem vessels along with sap, and fewer xylem emitted sap as potentials decreased. PMID- 16664513 TI - ;Dark-Lethality' of Certain Chlamydomonas reinhardtii Strains Is Prevented by Dim Blue Light. AB - Cells of certain Chlamydomonas reinhardtii strains dramatically swell and ultimately disintegrate when grown in the dark but not in the light. This effect of darkness is delayed: cells undergo about two divisions before swelling. Measurements on O(2) uptake and ATP content indicate that the cells grown in the dark are not deficient in energy supply. Dim blue light not effective in photosynthesis prevents ;dark-lethality'. Inhibition of protein synthesis during the latent period with cycloheximide also prevents dark-lethality. We propose that blue light stimulates synthesis of one or more proteins which are part of or are important in assembly of an osmoregulatory complex. PMID- 16664512 TI - Photosynthetic Responses to Dynamic Light Environments by Hawaiian Trees : Time Course of CO(2) Uptake and Carbon Gain during Sunflecks. AB - Gas exchange responses to rapid changes in light were studied in a C(3) tree, Claoxylon sandwicense Muell-Arg and a C(4) tree, Euphorbia forbesii Sherff that are native to the understory of a mesic Hawaiian forest. When light was increased to 500 micromoles per meter per second following a 2 hour preexposure at 22 micromoles per meter per second, net CO(2) uptake rates and stomatal conductance gradually increased for over 1 hour in C. sandwicense but reached maximum values within 30 minutes in E. forbesii. Calculation of the intercellular CO(2) pressures indicated that the primary limitation to CO(2) uptake during this induction was nonstomatal in both species. The photosynthetic response to simulated sunflecks (lightflecks) was strongly dependent on the induction state of the leaf. Total CO(2) uptake during a lightfleck was greater and the response was faster after exposure of the leaf to high light than when the leaf had been exposed only to low light for the previous 2 hours. During a series of lightflecks, induction resulted in increased CO(2) uptake in successive lightflecks. Significant postillumination CO(2) fixation was evident and contributed substantially to the total carbon gain, especially for lightflecks of 5 to 20 seconds' duration. PMID- 16664514 TI - Possible Regulatory Roles of Cytokinins : NADH Oxidation by Peroxidase and a Copper Interaction. AB - Apparently free-base cytokinins can interact with cupric ions in a specific manner. Oxidation of NADH by a horseradish peroxidase system was strongly promoted by such cytokinins provided cupric ions were present. Oxidation was promoted by 5 micromolar kinetin, zeatin, 6-benzylaminopurine (BA), or 6 (Delta(2)-isopentenylamino)purine (2iP) but not by adenine, 6-methylaminopurine or 6,6-dimethylaminopurine. The 6-methylaminopurine promoted oxidation at 500 micromolar but adenine and 6,6-dimethylaminopurine did not. Activity of the free base purines correlated well with their activity in cell-division assays. However, addition of methoxymethyl-, cyclohexyl-, or tetrahydropyranyl- at N-9 of BA or of ribosyl- at N-9 of BA, 2iP, kinetin, or zeatin eliminated activity in the peroxidase system. In a nonenzymic system containing cupric ions, all of the bases, including adenine, inhibited the Cu(2+) -stimulated oxidation of ascorbic acid. As in the peroxidase system, the N-9 derivatives were inactive. The cytokinin promotion of NADH oxidation by peroxidase may result from an interaction of the hormones with copper, with peroxidase conferring a specificity similar to the cytokinin specificity observed in growth and development. PMID- 16664515 TI - A Rapid Increase in Spinach Leaf Fructose 2,6-Bisphosphate Occurs during a Light to Dark Transition. AB - Spinach leaf fructose 2,6-bisphosphate levels increase rapidly during the first 15 minutes of a normal dark period followed by a gradual decline during the next 5 hours. The regulatory mechanism responsible for the dark-induced rise in fructose 2,6-bisphosphate levels can be counteracted by a brief exposure to light intensities greater than 1 microeinstein per square meter per second. PMID- 16664516 TI - Nucleotide sequence of ATPase subunit 6 gene of maize mitochondria. AB - The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and characterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homology with the 5' end of the cytochrome oxidase subunit II gene of maize mitochondria, suggesting recombination between the two genes. PMID- 16664517 TI - In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate. AB - The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K(a)) values were obtained for each bisphosphate were: for glucose 1-6-P(2), 0.5 to 1.8; fructose 2,6-P(2), 6 to 11.7; and fructose 1,6-P(2), 7 micromolar, respectively. Fructose 2,6-P(2) is known to occur in both plant and animal tissues at changing levels encompassing the K(a) values found in this study; hence, these results implicate fructose 2,6-P(2) as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P(2) has not been found in plants, and the method for measuring glucose 1,6-P(2) by monitoring the activation of phosphoglucomutase is not specific. PMID- 16664518 TI - Role of Ca and EGTA on Stomatal Movements in Commelina communis L. AB - Ca(2+) (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'tetraacetic acid (EGTA) (2 millimolar), a Ca(2+) chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl(2). The results demonstrate the importance of Ca(2+) in the regulation of stomatal closure and point to a role of Ca(2+) in the regulation of K(+) efflux from stomatal guard cells. PMID- 16664519 TI - Nitrate reductase of green algae is located in the pyrenoid. AB - Antibodies against nitrate reductase from Monoraphidium braunii have been used to determine the antigenic relationships of nitrate reductases from different green algae. Nitrate reductases from Chlamydomonas reinhardii, Chlorella fusca, Dunaliella salina, and Scenedesmus obliquus, were inhibited by, and cross-reacted with, antibodies raised against the enzyme from Monoraphidium braunii.These antibodies were also used to determine, by immunoelectron microscopy, the intracellular location of nitrate reductase in the aforementioned green algae. In all cases, the enzyme was specifically located in the pyrenoid. PMID- 16664520 TI - Photocontrol of Hypocotyl Elongation in Light-Grown Cucumis sativus L. : Responses to Phytochrome Photostationary State and Fluence Rate. AB - The effects of the calculated photostationary state of phytochrome (phi(c)) and the photon fluence rate on the elongation growth of the hypocotyl of light-grown seedlings of Cucumis sativus L. are examined. Two threshold responses to phi(c) are found at values of 0.06 and 0.43. At phi(c) = 0.06, there is no response at any fluence rate. In the phi(c) range 0.1 to 0.43, elongation growth does not respond to changes in phi(c). Above the second threshold (phi(c) = 0.43), there is a strong response to changes in phi(c). At all values of phi(c) at and above 0.1, there is a response to fluence rate. A linear relationship can be demonstrated between a factor comprised of the logarithm of phytochrome cycling rate (a fluence-rate-dependent process) and phi(c), and the growth response. PMID- 16664521 TI - Carbon balance and water relations of sorghum exposed to salt and water stress. AB - The daily (24 hour) changes in carbon balance, water loss, and leaf area of whole sorghum plants (Sorghum bicolor L. Moench, cv BTX616) were measured under controlled environment conditions typical of warm, humid, sunny days. Plants were either (a) irrigated frequently with nutrient solution (osmotic potential -0.08 kilojoules per kilogram = -0.8 bar), (b) not irrigated for 15 days, (c) irrigated frequently with moderately saline nutrient (80 millimoles NaCl + 20 millimoles CaCl(2).2H(2)O per kilogram water, osmotic potential -0.56 kilojoules per kilogram), or (d) preirrigated with saline nutrient and then not irrigated for 22 days.Under frequent irrigation, salt reduced leaf expansion and carbon gain, but water use efficiency was increased since the water loss rate was reduced more than the carbon gain. Water stress developed more slowly in the salinized plants and they were able to adjust osmotically by a greater amount. Leaf expansion and carbon gain continued down to lower leaf water potentials.Some additional metabolic cost associated with salt stress was detected, but under water stress this was balanced by the reduced cost of storing photosynthate rather than converting it to new biomass. Reirrigation produced a burst of respiration associated with renewed synthesis of biomass from stored photosynthate.It is concluded that although irrigation of sorghum with moderately saline water inhibits plant growth in comparison with irrigation with nonsaline water, it also inhibits water loss and allows a greater degree of osmotic adjustment, so that the plants are able to continue growing longer and reach lower leaf water potentials between irrigations. PMID- 16664522 TI - Membrane Electrical Noise in Chara corallina: I. A Low Frequency Spectral Component. AB - Some aspects of the membrane physiology of the giant-celled alga Chara corallina have been studied using the techniques of membrane noise analysis. Cellular voltage noise was measured by means of a microelectrode inserted in the vacuole of the cell. The significant feature of spectra estimated from voltage noise signals in these cells was a low-frequency component possibly associated with the active, electrogenic proton pumping which occurs in these cells. The effect of high external pH is described. PMID- 16664523 TI - Direct demonstration of duvatrienediol biosynthesis in glandular heads of tobacco trichomes. AB - Biosynthesis of the diterpenes, alpha and beta 4,8,13-duvatriene-1,3-diol, has been observed in detached, intact glandular heads from trichomes of Nicotiana tabacum, Tobacco Introduction 1068. This result shows directly that the glandular head portion of the trichome is capable of duvatrienediol biosynthesis. In additional experiments, all of the [(14)C] duvatrienediol formed from sodium [2 (14)C]acetate by leaf midrib sections was recovered with trichome exudate and surface washes. None was found in trichome stalk, epidermal or subepidermal tissue extracts. Also, removal of glandular heads and exudate from midrib sections reduced or eliminated duvatrienediol biosynthetic capacity. Together these results strongly suggest that glandular heads are the primary, and perhaps the only, site of duvatrienediol biosynthesis in this plant.Incubation of detached, intact glandular heads with sodium [(14)C]acetate in the dark or incubation in the light in the presence of DCMU reduced incorporation into duvatrienediols by 97%. These results suggest that chloroplasts which are abundant in glandular heads are involved in the biogenesis of these compounds. PMID- 16664524 TI - Factors affecting ice nucleation in plant tissues. AB - Factors affecting the ice nucleation temperature of plants and plant tissues were examined. The mass of a sample had a marked effect on ice nucleation temperature. Small tissue samples supercooled to -10 degrees C and were not accurate predictors of the nucleation temperature of intact plants in either laboratory or field experiments. This effect was not unique to plant tissues and was observed in autoclaved and control soil samples. Ice nucleation temperatures of bean, corn, cotton, and soybean seedlings were influenced by the length of subzero exposure, presence of ice nucleation active bacteria, and leaf surface wetness. The number of factors influencing ice nucleation temperature suggested that predicting the freezing behavior of plants in the field will be complex. PMID- 16664525 TI - Influence of shoot structure on light interception and photosynthesis in conifers. AB - The influence of shoot structure on net photosynthesis was evaluated under field conditions for the central Rocky Mountain (United States) conifers Picea engelmannii (Parry ex Engelm.), Abies lasiocarpa ([Hook] Nutt.), and Pinus contorta (Engelm.). In all species, the greater number of needles per unit stem length on sun shoots correlated with a smaller silhouette leaf area to total leaf area ratio (STAR). Decreased STAR was due primarily to greater needle inclination toward the vertical, plus some needle mutual shading. However, photosynthesis expressed on a total leaf area basis did not decrease in sun shoots (lower STAR) but remained nearly constant at approximately 3 micromoles per square meter per second over a wide range of STAR (0.1 to 0.3). Relatively low light saturation levels of 200 to 1400 microeinsteins per square meter per second and diffuse light to 350 microeinsteins per meter per second maintained photosynthetic flux densities in inclined and/or shaded needles at levels comparable to those in unshaded needles oriented perpendicular to the solar beam. As a result, net CO(2) uptake per unit stem length increased as much as 2-fold in sun shoots (low STAR) in direct proportion to increasing needle density. PMID- 16664526 TI - Stimulation of glutathione synthesis in photorespiring plants by catalase inhibitors. AB - The effect of various herbicides on glutathione levels in barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), soybean (Glycine max [L.] Merr.), and corn (Zea mays L.) was examined. Illumination of excised barley, tobacco, and soybean plants for 8 hours in solution containing 2 millimolar aminotriazole (a catalase inhibitor) resulted in an increase in leaf glutathione from 250 to 400 nanomoles per gram fresh weight to 600 to 1800 nanomoles per gram fresh weight, depending on the species tested. All of this increase could be accounted for as oxidized glutathione. Between 25 and 50% of this oxidized glutathione was reduced when plants were darkened for 16 hours, but there was no significant decline in total glutathione. Another catalase inhibitor, thiosemicarbazide, was as effective as aminotriazole in elevating glutathione in soybean but was less effective in barley and tobacco. Glyphosate, an inhibitor of aromatic amino acid biosynthesis, had no significant effect on glutathione levels in any of the plants examined. Whereas methyl viologen (paraquat), which is a sink for photosystem I electrons, caused oxidation of leaf glutathione in all of the plants but did not increase the total amount of glutathione present. PMID- 16664527 TI - Resistance of citrus fruit to mass transport of water vapor and other gases. AB - The resistance of oranges (Citrus sinensis L. Osbeck) and grapefruit (Citrus paradisi Macf.) to ethylene, O(2), CO(2), and H(2)O mass transport was investigated anatomically with scanning electron microscope and physiologically by gas exchange measurements at steady state. The resistance of untreated fruit to water vapor is far less than to ethylene, CO(2) and O(2). Waxing partially or completely plugs stomatal pores and forms an intermittent cracked layer over the surface of fruit, restricting transport of ethylene, O(2), and CO(2), but not of water; whereas individual sealing of fruit with high density polyethylene films reduces water transport by 90% without substantially inhibiting gas exchange.Stomata of harvested citrus fruits are essentially closed. However, ethylene, O(2) and CO(2) still diffuse mainly through the residual stomatal opening where the relative transport resistance (approximately 6,000 seconds per centimeter) depends on the relative diffusivity of each gas in air. Water moves preferentially by a different pathway, probably through a liquid aqueous phase in the cuticle where water conductance is 60-fold greater. Other gases are constrained from using this pathway because their diffusivity in liquid water is 10(4)-fold less than in air. PMID- 16664528 TI - Glycine-Glomus-Rhizobium Symbiosis: II. Antagonistic Effects between Mycorrhizal Colonization and Nodulation. AB - Soybean (Glycine max [L.] Merr.) plants grown in pot cultures were inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe and Rhizobium japonicum strain 61A118 at planting (G(1)R(1)) or at 20 days (G(20)R(20)), or with one of the endophytes after the other has colonized the host root (G(1)R(20), G(20)R(1)). Nodulated (PR(1)) and VAM (G(1)N) dipartite associations, or nonsymbiotic plants (PN) using nutrient solutions with N, P, or N + P concentrations providing endophyte-equivalent nutrient inputs were used as controls. The delayed tripartite associations received the appropriate N, P, or N + P amendment while one or both endophytes were absent during the first 20 days of growth. Prior inoculation with one endophyte significantly inhibited development of the other. Root hexose sugar concentrations were negatively correlated with VAM colonization (r = -0.89), nodule activity (r = -0.91), and root P content (r = -0.93). Nodule (r = 0.97) and root (r = 0.96) P content correlated positively with VAM colonization. Nodule weight or VAM-fungal biomass were significantly greater in associations grown with only one endophyte. Dry weights of the PN, G(1)N, PR(1), and G(20)R(20) plants were significantly greater than those of tripartite plants inoculated at planting with either or both endophytes. Interendophyte inhibition is attributed to competition for root carbohydrates, and this effect apparently also affects overall plant productivity. The objective of the study was to determine if the timing of endophyte introduction and establishment affected the development of the other symbiotic partners. PMID- 16664529 TI - Chloroplast biogenesis 51 : modulation of monovinyl and divinyl protochlorophyllide biosynthesis by light and darkness in vitro. AB - It is shown that the monovinyl and divinyl protochlorophyllide biosynthetic patterns of etiolated maize (Zea mays L.), and cucumber (Cucumis sativus L.) seedlings and of their isolated etiochloroplasts can be modulated by light and darkness as was shown for green photoperiodically grown plants (E. E. Carey, C. A. Rebeiz 1985 Plant Physiol. 79: 1-6). In etiolated corn and cucumber seedlings and isolated etiochloroplasts poised in the divinyl protochlorophyllide biosynthetic mode by a 2 hour light pretreatment, darkness induced predominantly the biosynthesis of monovinyl protochlorophyllide in maize and of divinyl protochlorophyllide in cucumber. When etiolated seedlings and their isolated etiochloroplasts were poised in the monovinyl protochlorophyllide biosynthetic mode by a prolonged dark-pretreatment, light induced mainly the biosynthesis of divinyl protochlorophyllide in both maize and cucumber. PMID- 16664530 TI - Potassium and sodium absorption kinetics in roots of two tomato species : lycopersicon esculentum and lycopersicon cheesmanii. AB - Excised roots of the tomato species, Lycopersicon esculentum Mill. cv Walter (the commercial species) and of Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull. (a wild species from the Galapagos Islands), were used in comparative studies of their absorption of K(+) and Na(+). Uptake of (86)Rb-labeled K(+) and (22)Na labeled Na(+) by excised roots of ;Walter' and L. cheesmanii varied as a function of genotype and tissue pretreatment with or without K(+). Excised roots of ;Walter' consistently absorbed more (86)Rb-labeled K(+) than those of L. cheesmanii. Absorption of K(+) from solutions ranging from 0.01 to 0.2 millimolar KCl showed saturation kinetics in both K(+)-pretreated and K(+)-depleted roots of ;Walter,' and for K(+)-depleted roots of L. cheesmanii. K(+)-pretreated roots of L. cheesmanii had exceedingly low rates of K(+) uptake with strikingly different, linear kinetics. Pretreatment with K(+) caused a decrease in rates of K(+) uptake in both genotypes. Potassium depleted roots of L. cheesmanii absorbed Na(+) at a greater rate than those of ;Walter,' whereas K(+)-pretreated roots of ;Walter' absorbed Na(+) at a greater rate than those of L. cheesmanii. The results confirm and extend previous conclusions to the effect that closely related genotypes may exhibit widely different responses to the two alkali cations, K(+) and Na(+). PMID- 16664531 TI - Potassium transport in two tomato species : lycopersicon esculentum and lycopersicon cheesmanii. AB - The commercial tomato, lycopersicon esculentum Mill. cv Walter, and its wild relative, Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull., were grown for 30 days under controlled conditions and in solution culture varying in its content of Na(+) and K(+). Subsequently, (86)Rb-labeled K(+) uptake and distribution were studied. From all solutions, ;Walter' consistently absorbed (86)Rb-labeled K(+) at a higher rate in micromoles per gram fresh weight per 30 minutes than L. cheesmanii. L. cheesmanii distributed a greater proportion of the absorbed K(+) from its root to its shoot. When 0.6 millimolar NaNO(3) replaced 0.6 millimolar KNO(3) in the pretreatment solution, intact plants of both genotypes followed a similar pattern as when they were pretreated with K(+) only, but a greater percentage of the absorbed K(+) remained in the roots. Leaf slices of L. cheesmanii plants deprived of K(+) for 6 days showed a greater rate of K(+) uptake than did slices from ;Walter' plants pretreated the same way. Stem slices of L. cheesmanii, however, had a lower uptake rate than did those of ;Walter'. Both leaf and stem slices of ;Walter' plants, pretreated 6 days with 0.6 millimolar NaNO(3) substituting for 0.6 millimolar KNO(3) in their growth medium, had greater rates of (86)Rb-labeled K(+) uptake from 0.5 and 20 millimolar KCl solutions than did slices of L. cheesmanii. These marked differences in patterns of ion uptake and translocation indicate that these genotypes of tomato have evolved different mechanisms to deal with K(+) and Na(+) in their environments. PMID- 16664532 TI - Differential expression of the ribulose bisphosphate carboxylase large subunit gene in bundle sheath and mesophyll cells of developing maize leaves is influenced by light. AB - We have investigated the influence of light on the mRNA of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in mesophyll and bundle sheath cells of the C(4) plant Zea mays. The gene is transcribed in both cell types in leaves of seedlings grown in the dark. However, upon illumination, the level of mRNA declines and becomes undetectable in mesophyll cells after 72 hours. On the contrary, in bundle sheath cells the two transcripts of the same gene increase to peak after 24 hours of greening and then decrease to a steady state level. This study was made possible by the development of improved methods for the isolation of two maize leaf cell types suitable for the extraction of high quality RNAs at different developmental states, i.e. etiolated, greening and green. PMID- 16664533 TI - Effect of foliar applications of urea on accelerated senescence of maize induced by ear removal. AB - Field grown maize (Zea mays L. cv B73 x Mo17) plants, with and without ears, were sprayed with urea solutions to determine whether foliar application of N could prevent or delay the accelerated loss of reduced N from the leaf and leaf senescence induced by ear removal. Urea sprays were applied at 7, 14, and 21 days after anthesis in three separate and equal applications that provided a total of 67 kilograms N per hectare or 1 gram N per plant. Treatments were arranged in a 2 x 2 factorial in a randomized complete block with five replicates. Appropriate plant and leaf samplings and assays were made.In response to spray treatments, net increases of reduced N were detected in the whole shoot and plant parts, especially the stalk of the earless plants and grain of the eared plants. There was no effect of urea spray treatment on the normal loss of N from the leaves or rate of senescence of the eared plants or on the accelerated loss of N from the leaves or rate of senescence induced by ear removal. Grain and stover yields were unaffected by the spray treatment.Apparently the plants were unable to utilize the urea N applied to the vegetation (primarily leaves) after anthesis to enhance or extend the accumulation of dry weight by either eared or earless plants. PMID- 16664534 TI - Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles. AB - Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K(m) of about 0.23 millimolar for MgATP, is only slightly affected by K(+) or Cl(-) and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO(3) (-) > Cl(-)) and has a K(m) of about 0.7 millimolar. Auxins decrease the K(m) to about 0.35 millimolar, with no significant effect on the V(max), while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport. PMID- 16664535 TI - Drought-induced increases in abscisic Acid levels in the root apex of sunflower. AB - Abscisic acid (ABA) levels in 3-mm apical root segments of slowly droughted sunflower plants (Helianthus annuus L. cv Russian Giant) were analyzed as the methyl ester by selected ion monitoring gas chromatography-mass spectrometry using characteristic ions. An internal standard, hexadeuterated ABA (d6ABA) was used for quantitative analysis. Sunflower seedlings, grown in aeroponic chambers, were slowly droughted over a 7-day period. Drought stress increased ABA levels in the root tips at 24, 72, and 168 hour sample times. Control plants had 57 to 106 nanograms per gram ABA dry weight in the root tips (leaf water potential, -0.35 to -0.42 megapascals). The greatest increase in ABA, about 20-fold, was found after 72 hours of drought (leaf water potential, -1.34 to -1.47 megapascals). Levels of ABA also increased (about 7- to 54-fold) in 3-mm apical root segments which were excised and then allowed to dessicate for 1 hour at room temperature. PMID- 16664536 TI - Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells. AB - Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [(3)H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [(3)H]leucine labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20 degrees C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins. PMID- 16664537 TI - Voltage-dependent channels found in the membrane fraction of corn mitochondria. AB - Transmembrane channels have been found in the membrane fraction of corn (Zea mays W64AN) mitochondria that exhibit a remarkable resemblance to the voltage dependent anion-selective channels (VDAC) located in the outer membrane of animal (Rattus norvegicus), protist (Paramecium aurelia), and fungal (Neurospora crassa) mitochondria. The channels in corn were demonstrated to be essentially identical to VDAC channels in three characteristic properties: (a) single channel conductance magnitude, (b) weak anion selectivity, and (c) nature of voltage dependence. These findings led us to conclude that the channels present in corn mitochondria are VDAC channels. This discovery may have repercussions concerning the regulation and function of higher plant mitochondria, and the causation of higher plant excitability. PMID- 16664538 TI - Stereochemical aspects of the biosynthesis of the side chain of 9beta, 19 cyclopropyl sterols in maize seedlings treated with tridemorph. AB - 9beta, 19-Cyclopropyl sterols such as 24-methyl pollinastanol accumulate dramatically in maize (Zea mays L. var LG 11) seedlings treated with Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide (M. Bladocha, P. Benveniste, Plant Physiol 1983 41: 756-762). In contrast to the situation in control plants where 24-ethyl sterols predominate largely, 24-methyl sterols were more than 98% of total cyclopropyl sterols. In addition, 24-methyl cyclopropyl sterols were a mixture of (24-R)- and (24-S)-24-methyl epimers and are similar in that respect to the 24-methyl cholesterol of control plants. The presence of two epimers at C-24 has been previously explained by the operation of two routes (M. Zakelj, L. J. Goad, Phytochemistry 1983 22: 1931-1936). One may proceed via Delta(24(28))- and Delta(24(25))-sterols to produce the (24-R)-24-methyl epimer. The other route may involve reduction of either a Delta(24(28))-, a Delta(23)-, or a Delta(25)-sterol intermediate to give the (24-S)-24-methyl epimer. Such intermediates have been searched for in excised Zea mays axes grown aseptically in the presence of Tridemorph and either [5-(14)C]mevalonic acid, or [Me-(14)C]-l methionine. Whereas Delta(24(28))- and Delta(24(25))-cyclopropyl sterols were found in relatively large amounts, only traces of radioactivity were associated with Delta(25)-sterols. Gas chromatography/mass spectrometry analysis of the sterols from axes grown in the presence of [Me-(2)H(3)]-l-methionine showed that Delta(24(28))-cyclopropyl sterols contained only two (2)H atoms at C-28 as expected and that the 24-methyl pollinastanol fraction contained species with two (2)H atoms and no species with three (2)H atoms. These results indicate that both (24-R)- and (24-S)-epimers originate from a common Delta(24(28)) precursor. After incubation of the axis with [5-(14)C,(4-R)-4-(3)H(1)]mevalonic acid, the 24 methyl pollinastanol had a (3)H:(14)C atomic ratio of 4:6 which is consistent with the intermediacy of a Delta(24(25))-sterol. All these data are in accordance with a pathway where Delta(24(28))-cyclopropyl sterols are isomerized to give Delta(24(25))-cyclopropyl sterols which in turn would be reduced nonregiospecifically to yield both (24-R)- and (24-S)-24-methyl pollinastanols. A plausible mechanism for the reduction step is discussed. PMID- 16664539 TI - The senescence of detached leaves of tropaeolum. AB - The senescence of detached Tropaeolum majus leaves was compared with that described earlier for Avena. Tropaeolum was chosen as being not only a dicot but also as having a nearly circular leaf, thus needing only the smallest minimum of wounding, since wounding delays the loss of chlorophyll and protein in darkness. Tropaeolum resembles Avena in that closing the stomata osmotically or with ABA causes rapid senescence in light. As in Avena also, n-hexanol and alpha,alpha' dipyridyl delay senescence in darkness but cause ;bleaching' of chlorophyll in light. Unlike Avena, however, kinetin and gibberellic acid, which delay senescence in the dark in both species, do so in Tropaeolum without causing any significant stomatal opening. The senescence of Tropaeolum leaves is actually promoted by fusicoccin, which powerfully delays senescence in Avena, although fusicoccin does cause stomatal opening in darkness in both species. Thus, many of the phenomena of senescence are alike in the monocot and dicot, but there are several significantly different responses to the senescence-modifying reagents. It is concluded that while stomatal closure accelerates senescence in both species, stomatal opening is not directly linked to the prevention of leaf senescence. PMID- 16664540 TI - A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots. AB - Two types of ATP-dependent calcium (Ca(2+)) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca(2+) uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO(3) (-). The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg(2+).Like the tonoplast H(+)-ATPase activity, vanadate-insensitive Ca(2+) accumulation was stimulated by 20 millimolar Cl(-) and inhibited by 10 micromolar 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid or 50 micromolar N,N'-dicyclohexylcarbodiimide. This Ca(2+) transport system had an apparent K(m) for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca(2+) transport was abolished by compounds that eliminated a pH gradient and Ca(2+) dissipated a pH gradient (acid inside) generated by the tonoplast-type H(+)-ATPase. These results provide compelling evidence that a pH gradient generated by the H(+)-ATPase drives Ca(2+) accumulation into right-side-out tonoplast vesicles via a Ca(2+)/H(+) antiport. This transport system was saturable with respect to Ca(2+) (K(m) apparent = 14 micromolar). The Ca(2+)/H(+) antiport operated independently of the H(+)-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca(2+) accumulation. Ca(2+) transport by this system may be one major way in which vacuoles function in Ca(2+) homeostasis in the cytoplasm of plant cells. PMID- 16664541 TI - Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling. AB - The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O(2) evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids. PMID- 16664542 TI - Transport and Assimilation of Nitrogen by Stichococcus bacillaris Grown in the Presence of Methionine Sulfoximine. AB - Stichococcus bacillaris Naeg., a green soil alga, can grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining a high level of NADPH-glutamate dehydrogenase activity. MSX-grown cells can utilize both NH(4) (+) and NO(3) (-) as nitrogen source for growth. [(14)C]Methylammonium is not metabolized by S. bacillaris, and is transported by a carrier system that obeys Michaelis Menten kinetics, and is insensitive to MSX. PMID- 16664543 TI - The equilibrium of the reaction catalyzed by sucrose phosphate synthase. AB - The equilibrium constant for the reaction catalyzed by sucrose phosphate synthase was reported 25 years ago to be 3250 at pH 7.5. It has been redetermined and found to be about 2 in the direction of synthesis and 6 to 10 when measured in the opposite direction. PMID- 16664545 TI - Rapid oscillations in fructose 2,6-bisphosphate levels in plant tissues. AB - The fructose 2,6-bisphosphate (Fru 2,6-P(2)) content of pea, Pisum sativum, roots and leaves were measured following flooding with water and found to change in times of minutes and to exhibit oscillatory-type changes. Each organ changes its Fru 2,6-P(2) content in a unique pattern in response to environmental disturbances such as flooding or light. For example, when roots of intact illuminated pea plants are flooded, roots decrease their Fru 2,6-P(2) content while simultaneously leaves increase their Fru 2,6-P(2) content; but both organs exhibit oscillatory-type patterns within flooding time of about 30 minutes. Half change times can be as rapid as 2 to 3 minutes. The endogenous extractable activity of the root pyrophosphate-dependent phosphofructokinase also exhibits an oscillatory pattern upon root immersion slightly after Fru 2,6-P(2) changes occur. We postulate from these results that Fru 2,6-P(2) is a primary signal molecule which enables plants to regulate their metabolism to cope with changing environments. PMID- 16664544 TI - Corn mitochondrial protein synthesis in response to heat shock. AB - Corn (Zea mays L., W23(N), OH43(N), and reciprocal single cross hybrid) seedling mitochondria respond to a 10 degrees C temperature shift (27-37 degrees C) by incorporating a greater amount of [(35)S]methionine into acid-insoluble material than mitochondria incubated at the original growing temperature (27 degrees C). This increase is in part manifested in the enhanced synthesis of a 52 kilodaltons protein. At both temperatures mitochondria of two inbreds and their reciprocal hybrids synthesize normal (N) cytoplasm proteins sensitive to chloramphenicol and insensitive to cyclohexamide treatment. The 52 kilodaltons protein is found in the supernatants of pelleted (15,000g, 5 min) mitochondria after heat shock. The role of this protein in the heat shock response is discussed in light of the implication of mitochondria as the primary cellular target to temperature stress. PMID- 16664546 TI - Purification and properties of glutamine synthetase from spinach leaves. AB - The chloroplastic glutamine synthetase of spinach leaves has been purified to homogeneity using affinity chromatography. This involves a tandem ;reactive blue A-agarose' and ;reactive red-A-agarose' as the final step in the procedure. This procedure results in a yield of 18 milligrams of pure glutamine synthetase per kilogram of starting material. The purity of our enzyme has been demonstrated on both one- and two-dimensional polyacrylamide gels.Purified glutamine synthetase has a molecular weight of 360,000 daltons and consists of eight 44,000 dalton subunits. The K(m) is 6.7 millimolar for glutamate, 1.8 millimolar for ATP (synthetase assay), and 37.6 millimolar for glutamine (transferase assay). The isoelectric point is 6.5 and the pH optima are 7.3 in the synthetase assay and 6.4 in the transferase assay. The irreversible, competitive inhibitors methionine sulfoxamine and phosphinothricin have K(i) values of 0.1 millimolar and 6.1 micromolar, respectively. Amino acid analysis has been carried out and the results compared with published analyses for other isoforms of glutamine synthetase. PMID- 16664547 TI - An investigation into the role of photosynthesis in regulating ATP levels and rates of h efflux in isolated meosphyll cells. AB - Aerated and stirred 10-ml suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used for simultaneous measurements of net H(+) efflux and steady-state ATP levels.Initial rates of medium acidification indicated values for H(+) efflux in the light and dark of 0.66 and 0.77 nanomoles H(+)/10(6) cells per minute, respectively. When the medium pH was maintained at 6.5, with a pH-stat apparatus, rates of H(+) efflux remained constant. Darkness or DCMU, however, stimulated H(+) efflux by 100% or more. Darkness increased ATP levels by 33% and a switch from dark to light reduced ATP levels by 31%. In the absence of aeration, illumination prevented the accumulation of respiratory CO(2) and the buffering capacity of the medium was about 50% less than that found in the nonilluminated nonaerated medium. As a result, rates of pH decline were similar even though the dark rate of H(+) efflux was approximately 50% greater.Proposals that photosynthesis stimulates H(+) efflux are based on changes in the rate of pH decline. The present data indicate that photosynthesis inhibits H(+) efflux and that changes in rates of pH decline should not be equated with changes in the rate of H(+) efflux. PMID- 16664548 TI - Imunohistochemical Localization of alpha-Amylase in Cotyledons of Vigna mungo Seedlings. AB - We studied the localization of alpha-amylase with indirect fluorescence microscopy in transversely sectioned cotyledons of Vigna mungo seedlings. Tissue sections were fixed in periodate-lysine-paraformaldehyde and treated with anti alpha-amylase immunoglobulin G followed by fluorescein isothiocyanate labeled goat anti-rabbit immunoglobulin G. alpha-Amylase appeared in the cells farthest from vascular bundles on the second day of growth and appeared gradually closer to the vascular bundles as growth progressed. The pattern of alpha-amylase appearance was similar in detached cotyledons, indicating that attachment of the embryonic axis has no effect on this pattern. However, in attached cotyledons, alpha-amylase disappeared from the regions where starch grains had been digested, but in detached cotyledons there was no disappearance of alpha-amylase, and digestion was slower than in intact cotyledons. PMID- 16664549 TI - Biosynthesis of alpha-Amylase in Vigna mungo Cotyledon. AB - In vitro translation of RNA extracted from Vigna mungo cotyledons showed that alpha-amylase is synthesized as a polypeptide with a molecular mass of 45,000, while cotyledons contain a form of alpha-amylase with a molecular mass of 43,000. To find out whether the 45,000 molecular mass polypeptide is a precursor to the 43,000 found in vivo, the cell free translation systems were supplemented with canine microsomal membrane; when mRNA was translated in the wheat germ system supplemented with canine microsomes, the 45,000 molecular mass form was not processed to a smaller form but the precursor form was partly processed in the membrane-supplemented reticulocyte lysate system. When V. mungo RNA was translated in Xenopus oocyte system, only the smaller form (molecular mass 43,000) was detected. Involvement of contranslational glycosylation in the maturating process of the alpha-amylase was ruled out because there was no effect of tunicamycin, and the polypeptide was resistant to endo-beta-H or endo-beta-D digestion. We interpret these results to mean that the 45,000 molecular mass form is a precursor with a signal peptide or transit sequence, and that the 43,000 molecular mass is the mature form of the protein. PMID- 16664550 TI - Constant Phycobilisome Size in Chromatically Adapted Cells of the Cyanobacterium Tolypothrix tenuis, and Variation in Nostoc sp. AB - Phycobilisomes of Tolypothrix tenuis, a cyanobacterium capable of complete chromatic adaptation, were studied from cells grown in red and green light, and in darkness. The phycobilisome size remained constant irrespective of the light quality. The hemidiscoidal phycobilisomes had an average diameter of about 52 nanometers and height of about 33 nanometers, by negative staining. The thickness was equivalent to a phycocyanin molecule (about 10 nanometers). The molar ratio of allophycocyanin, relative to other phycobiliproteins always remained at about 1:3. Phycobilisomes from red light grown cells and cells grown heterotrophically in darkness were indistinguishable in their pigment composition, polypeptide pattern, and size. Eight polypeptides were resolved in the phycobilin region (17.5 to 23.5 kilodaltons) by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Half of these were invariable, while others were variable in green and red light. It is inferred that phycoerythrin synthesis in green light resulted in a one for one substitution of phycocyanin, thus retaining a constant phycobilisome size. Tolypothrix appears to be one of the best examples of phycobiliprotein regulation with wavelength. By contrast, in Nostoc sp., the decrease in phycoerythrin in red light cells was accompanied by a decrease in phycobilisome size but not a regulated substitution. PMID- 16664551 TI - Interspecific Variation in SO(2) Flux : Leaf Surface versus Internal Flux, and Components of Leaf Conductance. AB - The objective of this study was to clarify the relationships among stomatal, residual, and epidermal conductances in determining the flux of SO(2) air pollution to leaves. Variations in leaf SO(2) and H(2)O vapor fluxes were determined using four plant species: Pisum sativum L. (garden pea), Lycopersicon esculentum Mill. flacca (mutant of tomato), Geranium carolinianum L. (wild geranium), and Diplacus aurantiacus (Curtis) Jeps. (a native California shrub). Fluxes were measured using the mass-balance approach during exposure to 4.56 micromoles per cubic meter (0.11 microliters per liter) SO(2) for 2 hours in a controlled environmental chamber. Flux through adaxial and abaxial leaf surfaces with closed stomata ranged from 1.9 to 9.4 nanomoles per square meter per second for SO(2), and 0.3 to 1.3 millimoles per square meter per second for H(2)O vapor. Flux of SO(2) into leaves through stomata ranged from approximately 0 to 8.5 (dark) and 3.8 to 16.0 (light) millimoles per square meter per second. Flux of H(2)O vapor from leaves through stomata ranged from approximately 0 to 0.6 (dark) to 0.4 to 0.9 (light) millimole per square meter per second. Lycopersicon had internal flux rates for both SO(2) and H(2)O vapor over twice as high as for the other species. Stomatal conductance based on H(2)O vapor flux averaged from 0.07 to 0.13 mole per square meter per second among the four species. Internal conductance of SO(2) as calculated from SO(2) flux was from 0.04 mole per square meter per second lower to 0.06 mole per square meter per second higher than stomatal conductance. For Pisum, Geranium, and Diplacus stomatal conductance was the same or slightly higher than internal conductance, indicating that, in general, SO(2) flux could be predicted from stomatal conductance for H(2)O vapor. However, for the Lycopersicon mutant, internal leaf conductance was much higher than stomatal conductance, indicating that factors inside leaves can play a significant role in determining SO(2) flux. PMID- 16664552 TI - Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria. AB - The properties of the soluble moiety (F(1)) of the mitochondrial H(+)-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F(1) preparation contained major polypeptides corresponding to alpha, beta, gamma, delta, and epsilon of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F(1)-ATPase, like the native enzyme, was inhibited by azide (I(50) = 10 micromolar), nitrate (I(50) = 7-10 millimolar), 4,4' diisothiocyano-2,2'-stilbene disulfonic acid (I(50) = 1-3 micromolar), and 7 chloro-4-nitrobenzo-2-oxa-1,3-diazole (I(50) = 3 micromolar). F(1)-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F(1)-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F(1). In contrast to the membrane bound enzyme, the F(1)-ATPase was not inhibited by oligomycin or by N,N' dicyclohexylcarbodiimide. The mitochondrial F(1)-ATPase from oat roots is analogous to other known F(1)F(0)-ATPases. PMID- 16664553 TI - Competition for in vitro [h]gibberellin a(4) binding in cucumber by gibberellins and their derivatives. AB - The gibberellin (GA) binding properties of a cytosol fraction from hypocotyls of cucumber (Cucumis sativus L. cv National Pickling) were examined using a DEAE filter paper assay, [(3)H]GA(4), and over 20 GAs, GA derivatives and other growth regulators. The results demonstrate structural specificity of the binding protein for gamma-lactonic C-19 GAs with a 3 beta-hydroxyl and a C-6 carboxyl group. Additional hydroxylations of the A, C, or D ring of the ent-gibberellane skeleton and methylation of the C-6 carboxyl impede or abolish binding affinity. Bioassay data are generally supported by the in vitro results but significantly GA(9) and GA(36), both considered to be precursors of GA(4) in cucumber, show no affinity for the binding protein. The results are discussed in relation to the active site of the putative GA(4) receptor in cucumber. PMID- 16664554 TI - Interaction of wheat germ ca-dependent protein kinases with calmodulin antagonists and polyamines. AB - The two soluble Ca(2+)-dependent protein kinases resolved from wheat (Triticum aestivum) embryo (protein kinases I and II) are inhibited by the phenothiazine derived calmodulin antagonists trifluoperazine fluphenazine, and chlorpromazine. Protein kinases I and II are also inhibited by a variety of other calmodulin antagonists (including calmidazolium, amitriptyline, and iprindole), phosphodiesterase inhibitors (including flufenamic acid and papavarine) and by lanthanides. A number of compounds that inhibit mammalian Ca(2+) - and phospholipid-activated protein kinase (protein kinase C) including quercetin, polymixin B sulfate, and polyamines (as well as phenothiazine derivatives) also inhibit protein kinases I and II. Poly-l-lysine and poly-l-ornithine activate both plant Ca(2+)-dependent protein kinases. PMID- 16664555 TI - Ethylene production by suspension-cultured pear fruit cells as related to their senescence. AB - Suspension-cultured pear fruit cells produce low levels of ethylene during growth and division in auxin containing medium. When deprived of auxin, division gradually ceases and ethylene production falls to barely discernible levels. However, notable ethylene production can now be induced by indoleacetic acid, CuCl(2), or 1-aminocyclopropane-1-carboxylic acid. If the auxin-deprived cells are transferred to ;aging' medium that lacks auxin but contains 0.4 molar mannitol, inducible ethylene production increases several-fold reaching levels of 40 to 60 nanoliters/10(6) cells per hour. Maximum inducible ethylene productivity is attained at varying times (1-6 days) after transfer to aging medium and appears to be temporally related to cell survival, i.e. the time of subsequent cell death. It is argued that auxin depletion initiates senescence which, in turn, leads to a transient increase in inducible ethylene production and eventual death. The limitations and potentials of the suspension-cultured pear cells as a system for the study of cellular senescence are discussed. PMID- 16664556 TI - A role for fructose 2,6-bisphosphate in regulating carbohydrate metabolism in guard cells. AB - Fructose 2,6-bisphosphate (Fru2,6P(2)) appears to function as a regulator metabolite in glycolysis and gluconeogenesis in animal tissues, yeast, and the photosynthetic cells of leaves. We have investigated the role of Fru2,6P(2) in guard-cell protoplasts from Vicia faba L. and Pisum sativum L. (Argenteum mutant), and in epidermal strips purified by sonication from all cells except for the guard cells. Guard-cell protoplasts were separated into fractions enriched in cytosol and in chloroplasts by passing them through a nylon net, followed by silicone oil centrifugation. The cytosol contained a pyrophosphate: fructose 6 phosphate phosphotransferase (involved in glycolysis) which was strongly stimulated by Fru2,6P(2). A cytosolic fructose 1,6-bisphosphatase (a catalyst of gluconeogenesis) was inhibited by Fru2,6P(2). There was virtually no fructose 1,6 bisphosphatase activity in guard-cell chloroplasts of V. faba. It is therefore unlikely that the starch formed in these chloroplasts originates from imported triose phosphates or phosphoglycerate.The level of Fru2,6P(2) in guard-cell protoplasts and epidermal strips was about 0.1 to 1 attomole per guard cell in the dark (corresponding to 0.05 to 0.5 nanomole per milligram chlorophyll) and increased three- to tenfold within 15 minutes in the light. Within the same time span, hexose phosphate levels in guard-cell protoplasts declined to approximately one-half, indicating that acceleration of glycolysis involved stimulation of reactions using hexose phosphates. The level of Fru2,6P(2) in guard cells appears to determine the direction in which carbohydrate metabolism proceeds. PMID- 16664558 TI - Identification and Isolation of Single Cells that Produce Somatic Embryos at a High Frequency in a Carrot Suspension Culture. AB - A system was established in which single cells differentiated to embryos at a high frequency. Small spherical single cells from a carrot (Daucus carota L. cv "Kurodagosun") cell suspension culture were obtained by fractionation through sieving, using nylon screens and then density gradient centrifugation in Percoll solutions. Eighty-five to 90% of these small single cells differentiated to embryos when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (5 x 10(-8) molar), zeatin (10(-6) molar), and mannitol (0.2 molar) for 7 days, followed by transfer to a medium containing zeatin (10(-7) molar) but no auxin. This indicates that there are at least two phases in the differentiation of embryos from single cells. The progression of the first phase required exogenous auxin, whereas that of the second phase was inhibited by the same growth regulator. The relationship between the morphology and potency for embryogenesis from single cells was discussed. The system established here is a useful one for investigation of differentiation process from a single cell to a whole plant via embryogenesis, especially in its early stage. PMID- 16664557 TI - Light Intensity Adaptation and Phycobilisome Composition of Microcystis aeruginosa. AB - Phycobilisomes isolated from Microcystis aeruginosa grown to midlog at high light (270 microeinsteins per square meter per second) or at low light intensities (40 microeinsteins per square meter per second) were found to be identical. Electron micrographs established that they have a triangular central core apparently consisting of three allophycocyanin trimers surrounded by six rods, each composed of two hexameric phycocyanin molecules. The apparent mass of a phycobilisome obtained by gel filtration is 2.96 x 10(6) daltons. The molar ratio of the phycobiliproteins per phycobilisome is 12 phycocyanin hexamers:9 allophycocyanin trimers. The electron microscopic observations combined with the phycobilisome apparent mass and the phycobiliprotein stoichiometry data indicate that M. aeruginosa phycobilisomes are composed of a triangular central core of three stacks of three allophycocyanin trimers and six rods each containing two phycocyanin hexamers. Adaptation of M. aeruginosa to high light intensity results in a decrease in the number of phycobilisomes per cell with no alteration in phycobilisome composition or structure. PMID- 16664559 TI - Photoinhibition and Reactivation of Photosynthesis in the Cyanobacterium Anacystis nidulans. AB - The susceptibility of photosynthesis to photoinhibition and its recovery were studied on cultures of the cyanobacterium Anacystis nidulans. Oxygen evolution and low temperature fluorescence kinetics were measured. Upon exposure to high light A. nidulans showed a rapid decrease in oxygen evolution followed by a quasi steady state rate of photosynthesis. This quasi steady state rate decreased with increasing photon flux density of the photoinhibitory light. Reactivation of photosynthesis in dim light after the photoinhibitory treatment was rapid: 85 to 95% recovery occurred within 2 hours. In the presence of the translation inhibitor, streptomycin (250 micrograms per milliliter), no reactivation occurred. We also found that the damage increased dramatically if the high light treatment was done with streptomycin added. A transcription inhibitor, rifampicin, did not inhibit the reactivation process. Based on these data we conclude that the photoinhibitory damage observed is the net result of a balance between the photoinhibitory process and the operation of the repairing mechanism(s). PMID- 16664560 TI - Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume. AB - Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, 1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at -1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, -2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to -1.8 megapascals and the chloroplasts had higher rates of CO(2)-dependent O(2) evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20 degrees C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20 degrees C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20 degrees C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [(14)C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [(14)C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology. PMID- 16664561 TI - Lectins in castor bean seedlings. AB - The amounts of the two lectins (ricin and Ricinus communis agglutinin) in tissues of castor bean seedlings were followed during germination and early growth. For measurement, lectins in extracts were separately eluted from Sepharose columns; an antibody to the agglutinin was also used to detect the lectins by immunodiffusion. The endosperm of the dry seed contains 3.5 mg total lectin (5.6% of the total seed protein), which declines by 50% by day 4 and more rapidly thereafter as the tissue is completely consumed. The cotyledons of the dry seed also contain lectins but the amounts are less than 1% of those in the endosperm, and, as in the endosperm, they are constituents of the albumin fraction of the isolated protein bodies. No lectins were detected in the green cotyledons of 10 day seedlings that had been exposed to light from day 5. The embryonic axes of 2 day seedlings contained very small amounts of lectins but they were not detectable in the aerial parts of seedlings grown for 3 weeks or in cells from endosperm grown in tissue culture.The ability of proteinases and glycosidases (isolated from endosperm of 4-day seedlings) to hydrolyze the lectins was examined. No hydrolysis of the two lectins was observed, but the subunits, separated by reduction with 2-mercaptoethanol, were hydrolyzed slowly by a proteinase and some release of mannose was observed in the presence of the glycosidases. Ricin was converted to its subunits by cysteine and an enzyme in an endosperm extract accelerated chain separation by glutathione. PMID- 16664562 TI - Compartmentation and equilibration of abscisic Acid in isolated xanthium cells. AB - The compartmentation of endogenous abscisic acid (ABA), applied (+/-)-[(3)H]ABA, and (+/-)-trans-ABA was measured in isolated mesophyll cells of the Chicago strain of Xanthium strumarium L. The release of ABA to the medium in the presence or absence of DMSO was used to determine the equilibration of ABA in the cells. It was found that a greater percentage of the (+/-)-[(3)H]ABA and the (+/-)-trans ABA was released into the medium than of the endogenous ABA, indicating that applied ABA did not equilibrate with the endogenous material.Therefore, in further investigations only the compartmentation of endogenous ABA was studied. Endogenous ABA was released from Xanthium cells according to the pH gradients among the various cellular compartments. Thus, darkness, high external pH, KNO(2), and droughtstress all increased the efflux of ABA from the cells. Efflux of ABA from the cells in the presence of 0.6 m mannitol occurred within 30 seconds, but only 8% of the endogenous material was released during the 20 minute treatment. PMID- 16664563 TI - Relationship of Xylem Embolism to Xylem Pressure Potential, Stomatal Closure, and Shoot Morphology in the Palm Rhapis excelsa. AB - Xylem failure via gas embolism (cavitation) induced by water stress was investigated in the palm Rhapis excelsa (Thumb.) Henry. Xylem embolism in excised stems and petioles was detected using measurements of xylem flow resistance: a decrease in resistance after the removal of flow-impeding embolisms by a pressure treatment indicated their previous presence in the axis. Results supported the validity of the method because increased resistance in an axis corresponded with: (a) induction of embolism by dehydration, (b) increased numbers of cavitations as detected by acoustic means, (c) presence of bubbles in xylem vessels. The method was used to determine how Rhapis accommodates embolism; results suggested four ways. (a) Embolism was relatively rare because pressure potentials reach the embolism-inducing value of about -2.90 megapascals only during prolonged drought. (b) When embolism did occur in nature, it was confined to the relatively expendable leaf xylem; the stem xylem, which is critical for shoot survival, remained fully functional. (c) Even during prolonged drought, the extent of embolism is limited by complete stomatal closure, which occurred at the xylem pressure potential of -3.20 +/- 0.18 megapascals. (d) Embolism is potentially reversible during prolonged rains, since embolisms dissolved within 5 h at a pressure potential of 0.00 megapascals (atmospheric), and xylem sap can approach this pressure during rain. PMID- 16664564 TI - Behavior and viability of tobacco protoplasts in response to electrofusion parameters. AB - This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field. PMID- 16664565 TI - Paclobutrazol Inhibits Abscisic Acid Biosynthesis in Cercospora rosicola. AB - Three plant growth regulators, paclobutrazol, ancymidol, and decylimidazole, which are putative inhibitors of gibberellin (GA) biosynthesis, were studied to determine their effect on abscisic acid (ABA) biosynthesis in the fungus Cercospora rosicola. All three compounds inhibited ABA biosynthesis, and paclobutrazol was the most effective, inhibiting ABA 33% at 0.1 micromolar concentrations. In studies using (E,E,)-[1-(14)C] farnesyl pyrophosphate, it was shown that ancymidol blocked biosynthesis prior to farnesyl pyrophosphate (FPP), whereas paclobutrazol and decylimidazole acted after FPP. The three inhibitors did not prevent 4'-oxidation of (2Z,4E)-alpha-ionylideneacetic acid. C. rosiciola metabolized ancymidol by demethylation to alpha-cyclopropyl-alpha-(p hydroxyphenyl)-5-pyrimidine methyl alcohol. Paclobutrazol was not metabolized by the fungus. Information that these plant growth regulators inhibit ABA as well as GA biosynthesis should prove useful in determining the full range of action of these compounds. PMID- 16664566 TI - Localization of carbamoylphosphate synthetase and aspartate carbamoyltransferase in chloroplasts. AB - The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed. PMID- 16664567 TI - Oxidase reactions of tomato anionic peroxidase. AB - Tomato (Lycopersicon esculentum Mill) anionic peroxidase was found to catalyze oxidase reactions with NADH, glutathione, dithiothreitol, oxaloacetate, and hydroquinone as substrates with a mean activity 30% that of horseradish peroxidase; this is in contrast to the negligible activity of the tomato enzyme as compared to the horseradish enzyme in catalyzing an indoleacetic acid-oxidase reaction with only Mn(2+) and a phenol as cofactors. Substitution of Ce(3+) for Mn(2+) produced an 18-fold larger response with the tomato enzyme than with the horseradish enzyme, suggesting a significant difference in the autocatalytic indoleacetic acid-oxidase reactions with these two enzymes. In attempting to compare enzyme activities with 2,4-dichlorophenol as a cofactor, it was found that reaction rates increased exponentially with both increasing cofactor concentration and increasing enzyme concentration. While the former response may be analogous to allosteric control of enzyme activity, the latter response is contrary to the principle that reaction rate is proportional to enzyme concentration, and additionally makes any comparison of enzyme activity difficult. PMID- 16664568 TI - A Structural Comparison of the Acidic Extracellular Polysaccharides from Rhizobium trifolii Mutants Affected in Root Hair Infection. AB - The structures of the acidic extracellular polysaccharides (EPSs) from several R. trifolii mutants were compared by examining their compositions and their sugar linkages as determined by methylation analysis. These mutant strains were derived from the wild-type R. trifolii ANU843 and were unable to induce normal root hair curling (Hac- phenotype) or nodulation response (Nod- phenotype) in clover plants. These strains included several transposon Tn5-induced Nod-mutants, strain ANU871, which possesses a 40 to 50 kilobase deletion of the resident Sym plasmid, and strain ANU845 which is missing the Sym plasmid (pSym-). Strains ANU845(pSym-) containing either plasmid pRt150 or pBR1AN were also used. The recombinant plasmid pRt150 restores only root hair curling capacity to ANU845 while plasmid pBR1AN (an R. trifolii pSym) restores both root hair curling and nodulation capacity to this strain. Our composition and methylation results show that the EPSs from all these strains have the same glycosyl and pyruvyl linkages. Thus we suggest that neither the nod genes involved in root hair curling nor the entire pSym encodes for the arrangement of glycosyl or pyruvyl residues in these EPSs. Whether or not the nod genes dictate the location of acetyl or beta hydroxybutyrate substituent groups remains to be determined. PMID- 16664569 TI - Localization of Cytochrome b-559 in the Chloroplast Thylakoid Membranes in Spinach. AB - Cytochrome b-559 was purified from spinach leaves and antibodies were made against it in rabbit. Using affinity-purified, monospecific antibodies, we have found that cytochrome b-559, which is closely associated with the primary photochemical activity of photosystem II, is localized exclusively in the grana thylakoids. PMID- 16664570 TI - C(6)-[benzene ring]-indole-3-acetic Acid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic Acid in plants. AB - Indole-3-acetic acid (IAA) labeled with (13)C in the six carbons of the benzene ring is described for use as an internal standard for quantitative mass spectral analysis of IAA by gas chromatography/selected ion monitoring. [(13)C(6)]IAA was compared to the available deuterium labeled compounds and shown to offer the advantages of nonexchangeability of the isotope label, high isotopic enrichment, and chromatographic properties identical to that of the unlabeled compound. The utility of [(13)C(6)]IAA for measurement of endogenous IAA levels was demonstrated by analysis of IAA in Lemna gibba G-3. PMID- 16664571 TI - Purification to homogeneity of pyrroline-5-carboxylate reductase of barley. AB - An enzyme has been purified to homogeneity from barley seedlings which has ;proline dehydrogenase' and the pyrroline-5-carboxylic acid reductase activities. The purification achieved is 39,000-fold as calculated from the proline dehydrogenase activity. The subunit molecular weight of the protein is 30 kilodaltons. The native enzyme has molecular weights up to 480 kilodaltons, depending on the buffer environment. From the pH profiles, the specific activities and thermodynamic considerations, it is concluded that the plant proline dehydrogenase functions in vivo as a pyrroline-5-carboxylate reductase. PMID- 16664572 TI - The Initiation of Auxin Autonomy in Tissue from Tobacco Plants Carrying the Auxin Biosynthesizing Genes from the T-DNA of Agrobacterium tumefaciens. AB - Tobacco (Nicotiana tabacum cv Havana 425) plants containing the indole-3-acetic acid biosynthesizing genes (1 and 2) from the T-DNA of Agrobacterium tumefaciens strain T37-ADH(2) (mutated at the cytokinin biosynthesis gene 4) were used to study the physiological basis of the suppression and reinitiation of the auxin autonomous phenotype. The plants, though normal in appearance and cross-fertile with nontransformed, wild type tobacco, are shown to contain multiple copies of genes 1 and 2. Plants carrying these genes respond to inoculation by Agrobacterium strains mutated at genes 1 and 2 in a virulent fashion. Despite the presence and potential in planta activity of these genes, pith explants from such plants require auxin or tryptophan for growth in vitro, as does wild type tobacco. In both cases the indole-3-acetic acid levels increase rapidly in pith explants cultured on tryptophan-containing medium. However, only the tissues containing genes 1 and 2 grow subsequently on auxin-free medium and accumulate indole-3-acetic acid to levels that support growth. The capacity of such tissues to utilize naphthalene acetamide as an auxin suggests that gene 2 is rapidly activated during the reinitiation process. PMID- 16664573 TI - Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L. AB - Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate sensitive and KNO(3)-insensitive ATPase. The ATPase has high specificities both for substrate and Mg(2+) ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl(-). Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9 degrees C) and lower (6.7 degrees C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5 degrees C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury. PMID- 16664574 TI - Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L. AB - Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO(3) (-)-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg(2+), a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes. PMID- 16664575 TI - Abscisic Acid Control of Lectin Accumulation in Wheat Seedlings and Callus Cultures : Effects of Exogenous ABA and Fluridone. AB - Wheat germ agglutinin is found in wheat embryos and a similar lectin is present in the roots of older plants. We report here that 10 micromolar abscisic acid (ABA) produces an average two to three-fold enhancement in the amount of lectin in the shoot base and the terminal portion of the root system of hydroponically grown wheat seedlings. Although ABA stunts seedling growth, a similar growth inhibition produced by ancymidol is not accompanied by elevated lectin levels. To further clarify the role of ABA, wheat callus cultures were employed. Callus derived from immature embryos was grown on growth medium containing various combinations of ABA and 2,4-dichlorophenoxyacetic acid. Those grown in the presence of 10 micromolar ABA exhibit the largest increases in lectin compared to material grown on other regimes. The involvement of ABA in lectin accumulation was further probed with fluridone, an inhibitor of carotenoid synthesis which has also been linked to depressed levels of endogenous ABA. Wheat seedlings grown in the presence of 1 or 10 milligrams per liter fluridone have few or no carotenoids, and wheat germ agglutinin levels in the shoot base and roots are lower compared to controls. The greatest effect (a 39% reduction in the shoot base) is produced at an herbicide concentration of 10 milligrams per liter. Exogenous 10 micromolar ABA greatly stimulates lectin accumulation in the presence of fluridone, but the levels are not as high as those produced by ABA alone. These results indicate that lectin synthesis is under ABA control in both wheat embryos and adult plants. PMID- 16664576 TI - Demonstration and solubilization of lycopene cyclase from capsicum chromoplast membranes. AB - The conversion of phytoene into beta-carotene was demonstrated previously in chromoplast membranes prepared from Capsicum fruits (B. Camara et al. 1982 Eur J Biochem 127: 255-258). The direct cyclization of lycopene into beta-carotene and the successful solubilization of the enzymic activity involved in this reaction is reported. PMID- 16664577 TI - Evidence for a specific uptake system for iron phytosiderophores in roots of grasses. AB - Roots of grasses in response to iron deficiency markedly increase the release of chelating substances (;phytosiderophores') which are highly effective in solubilization of sparingly soluble inorganic Fe(III) compounds by formation of Fe(III)phytosiderophores. In barley (Hordeum vulgare L.), the rate of iron uptake from Fe(III)phytosiderophores is 100 to 1000 times faster than the rate from synthetic Fe chelates (e.g. Fe ethylenediaminetetraacetate) or microbial Fe siderophores (e.g. ferrichrome). Reduction of Fe(III) is not involved in the preferential iron uptake from Fe(III)phytosiderophores by barley. This is indicated by experiments with varied pH, addition of bicarbonate or of a strong chelator for Fe(II) (e.g. batho-phenanthrolinedisulfonate). The results indicate the existence of a specific uptake system for Fe(III)phytosiderophores in roots of barley and all other graminaceous species. In contrast to grasses, cucumber plants (Cucumis sativus L.) take up iron from Fe(III)phytosiderophores at rates similar to those from synthetic Fe chelates. Furthermore, under Fe deficiency in cucumber, increased rates of uptake of Fe(III)phytosiderophores are based on the same mechanism as for synthetic Fe chelates, namely enhanced Fe(III) reduction and chelate splitting. Two strategies are evident from the experiments for the acquisition of iron by plants under iron deficiency. Strategy I (in most nongraminaceous species) is characterized by an inducible plasma membrane-bound reductase and enhancement of H(+) release. Strategy II (in grasses) is characterized by enhanced release of phytosiderophores and by a highly specific uptake system for Fe(III)phytosiderophores. Strategy II seems to have several ecological advantages over Strategy I such as solubilization of sparingly soluble inorganic Fe(III) compounds in the rhizosphere, and less inhibition by high pH. The principal differences in the two strategies have to be taken into account in screening methods for resistance to ;lime chlorosis'. PMID- 16664578 TI - Temperature characteristics and adaptive potential of wheat ribosomes. AB - The translational efficiency of wheat ribosomes was studied as a function of an in vivo temperature pretreatment of wheat seedlings (Triticum aestivum L.). Ribosomes were isolated from heat-pretreated (36 degrees C) and reference (4 degrees C, 20 degrees C) wheat seedlings. The efficiency of the ribosomes in translating polyuridylic acid was assayed. Ribosomes from heat-pretreated seedlings exhibit a threefold enhanced incorporation rate of phenylalanine as compared to ribosomes from wheat seedlings adapted to 20 or 4 degrees C. This difference develops within 24 hours after onset of the heat treatment of seedlings following a 3 hour lag phase. The temperature induced changes can be traced back to the cytoplasmic ribosomes, since cycloheximide inhibits translation almost completely. Thermal inactivation of ribosomes occurs at 45 degrees C, irrespective of the temperature pretreatment of the wheat seedlings. Specific differences in the yield of ribosomes, in the polyribosomal profiles, and in the apparent Arrhenius' activation energy of protein synthesis were observed depending on the age and the temperature pretreatments. The results presented here are considered an important molecular correlation to phenotypical temperature adaptation of in vivo protein synthesis in wheat (M Weidner, C Mathee, FK Schmitz 1982 Plant Physiol 69: 1281-1288). PMID- 16664579 TI - Studies on Freezing Injury in Plant Cells : II. Protein and Lipid Changes in the Plasma Membranes of Jerusalem Artichoke Tubers during a Lethal Freezing in Vivo. AB - Plasma membranes were isolated from both unfrozen and frozen tissues of Jerusalem artichoke tubers (Helianthus tuberosus L.) in high purity utilizing an aqueous two-polymer phase partition system. Although the recovery of the plasma membranes was decreased significantly by freezing of tissues even at the nonlethal temperature (-5 degrees C), the isolated plasma membrane samples were considered to be representative of the plasma membranes in situ. Freezing of the tissues at sublethal temperatures resulted in marked changes in the chemical composition of the plasma membrane. Those are losses of sterols and phosphatidylethanolamine from the plasma membranes, and a change of specific proteins with relatively high molecular weights into low molecular weight peptides. These specific proteins were designated as frost susceptible proteins. The properties of the plasma membrane ATPase seem to be not affected so much by the in vivo freezing of cells. However, inhibition of the plasma membrane ATPase by N,N' dicyclohexylcarbodiimide (DCCD) was relatively low before and after freezing in vivo at the nonlethal temperature at -5 degrees C, but was markedly enhanced by freezing in vivo at sublethal temperatures below -10 degrees C. From the results, it is assumed either that the enzyme molecule was partially modified, especially at the presumed DCCD binding sites or that the DCCD had become more accessible to the enzyme as a result of increased permeability of the plasma membranes. These observed changes are discussed in connection with the mechanism of cell injury. PMID- 16664580 TI - Binding of Butyl Gallate to Plant Mitochondria : II. Relationship to the Presence or Absence of the Alternative Pathway. AB - [(14)C]butyl gallate was used in binding studies to investigate the cyanide resistant respiratory pathway in mitochondria isolated from a variety of sources displaying varying levels of cyanide resistance. Highly cyanide-resistant mitochondria were isolated from aroid spadices, while moderately cyanide resistant mitochondria were isolated from either mung bean (Vigna radiata L.) hypocotyls or carbon dioxide/oxygen/ethylene-treated tubers. Totally cyanide sensitive mitochondria were isolated from untreated tubers and rat liver. With one exception, all the plant mitochondria showed a reversible butyl gallate binding site which saturated at a level of 1.0 to 2.0 nanomoles per milligram protein. The exception, freshly harvested white potato tubers (<1 month from harvest), showed little specific butyl gallate binding, and also showed no appreciable induction of the cyanide-resistant pathway following carbon dioxide/oxygen/ethylene treatment. Only a low level, linear binding, well below that seen with plant mitochondria, was observed with rat liver mitochondria. Taken together, these results suggest a model for the interaction of the alternative pathway with the cytochrome pathway. In this model, the butyl gallate binding site (alternative oxidase) is a constitutive component in those mitochondria that are capable of developing the alternative pathway, and the binding sites associated with a second, inducible component that functions to couple the oxidase to the cytochrome pathway. PMID- 16664581 TI - A Circadian Rhythm in the Number of Daughter Cells in Synchronous Chlorella fusca var vacuolata. AB - Chlorella fusca var vacuolata cells were transferred to continuous darkness or weak light (0.07 watts per square meter) (both were called waiting time, WT) after a 12-hour light and 12-hour dark schedule. A daily dilution is performed at the end of the light/dark schedule, resulting in always the same average production of 18 autospores per mother cell. After 12 and 24 hours of WT in darkness, the production of autospores in a subsequent light/dark schedule was 50 and 100%, respectively. If the WT was performed in weak light (0.07 watts per square meter) the lowest production was obtained, independently of the length of WT. However, an interruption of this weak light by dark pulses (3 hours) increased the autospore production by an amount that depends upon the phase of the circadian rhythm, and varied up to 70% of the control (WT in permanent darkness). If the WT (total darkness) was interrupted by light pulses of 0.5 hour (white, same as used for growth), a phase response curve of productivity resulted. Pulses between the 12th and 18th hour of WT in darkness gave a 3-hour delay of maximum; later on pulses shifted the maximum autospore production 3 hours ahead. PMID- 16664582 TI - Characterization of 12-oxo-phytodienoic Acid reductase in corn: the jasmonic Acid pathway. AB - 12-Oxo-phytodienoic acid reductase, an enzyme of the biosynthetic pathway that converts linolenic acid to jasmonic acid, has been characterized from the kernel and seedlings of corn (Zea mays L.). The molecular weight of the enzyme, estimated by gel filtration, was 54,000. Optimum enzyme activity was observed over a broad pH range, from pH 6.8 to 9.0. The enzyme had a K(m) of 190 micromolar for its substrate, 12-oxo-phytodienoic acid. The preferred reductant was NADPH, for which the enzyme exhibited a K(m) of 13 micromolar, compared with 4.2 millimolar for NADH. Reductase activity was low in the corn kernel but increased five-fold by the fifth day after germination and then gradually declined. PMID- 16664583 TI - Phenotypical temperature adaptation of protein turnover in desert annuals. AB - Protein synthesis and protein degradation rates were measured in three desert annual species at four different experimental temperatures. The taxa chosen for this study were the C(3) winter annuals, Bowlesia incana Ruiz & Pavon and Plantago insularis Eastw., and a C(4) summer annual, Atriplex elegans (Moq.) D. Dietr. Peak rates of protein synthesis correlated well with the preferred habitat temperatures of B. incana and A. elegans; optima occurred at 25 and 35 degrees C, respectively. Plants of P. insularis showed an optimum protein synthesis rate at 35 degrees C; however, this optimum rate was considerably lower than for the other two species. Higher activation energies for protein synthesis tended to parallel adaptation to higher temperature habitats. Responses of protein degradation to temperature in A. elegans and B. incana were consistent with their natural thermal regimes, when evaluated for the transition from 25 to 35 degrees C. Again, protein degradation in P. insularis shows an intermediate response to temperature during the 25 to 35 degrees C transition. PMID- 16664584 TI - Relationship between Photosynthesis and Protein Synthesis in Maize: I. Kinetics of Translocation of the Photoassimilated Carbon from the Ear Leaf to the Seed. AB - To gain a better understanding of the biochemical basis for partitioning of photosynthetically fixed carbon between leaf and grain, a (14)CO(2) labeling study was conducted with field-grown maize plants 4 weeks after flowering. The carbon flow was monitored by separation and identification of (14)C assimilates and (14)C storage components within each tissue during the chase period (from 4 to 96 hours) following a 5 minute (14)CO(2) pulse. In the labeled ear leaf, the radioactivity strongly decreased to reach, at the end of the experiment, about 12% of the total incorporated radioactivity, mostly associated with sucrose and proteins. Nevertheless, an unexpected reincorporation of radioactivity was observed either in leaf starch or proteins, the day following the pulse. Conversely, the radioactivity in the grain increased to attain 66% of the total incorporated (14)C after a 96 hour chase. The photosynthates, mostly sucrose, organic and free amino acids, rapidly translocated towards the developing seeds, served as precursors for the synthesis of seed storage compounds, starch, and proteins. They accumulate in free form for 24 hours before being incorporated within polymerized storage components. This delay is interpreted as a necessary prerequisite for interconversions prior to the polycondensations. In the grain, the labeling of the storage molecules, either in starch or in storage protein groups (salt-soluble proteins, zein, and glutelin subgroups), was independent of their chemical nature but dependent on their pool size. PMID- 16664585 TI - Relationship between Photosynthesis and Protein Synthesis in Maize: II. Interconversions of the Photoassimilated Carbon in the Ear Leaf and in the Intermediary Organs to Synthesize the Seed Storage Proteins and Starch. AB - The mechanisms priming the production, the movement, and the transient and final storage of the photoassimilated carbon in the maize plant were examined at the metabolic level during the formation of the seed, with the ultimate aim to identify metabolic steps restricting grain yield and explaining the delay of formation of the reserve molecules. Under normal field conditions, we show that maize directly supplies the developing seed with the photoassimilated carbon which undergoes numerous interconversions from the ear leaf to the grain. The proteins, either in the leaf or in the seed, are primarily synthesized from incoming amino acids. Nevertheless, a secondary in situ synthesis of amino acids provides the proteins with new amino acids. The amino acids of this second set, slowly synthesized in the seed from the photosynthetic carbon skeletons, are not detected in their free form but immediately and regularly incorporated into the seed proteins, in such a way that, after 4 days of chase, the proportion of the radioactive labeling of the amino acids of the different storage protein groups corresponds to their amino acid composition. In the leaf, the labeling of proteins also arises from different metabolisms, but mainly from the photosynthetic metabolism. Contrary to the seed proteins, the time course of the labeled leaf proteins implies a rapid turnover. The second labeling of starch and proteins in the ear leaf involves a reassimilation of CO(2), a process optimizing the carbon uptake in maize. PMID- 16664586 TI - Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: I. Pigment Composition. AB - The use of n-octyl-beta-d-glucopyranoside along with sodium dodecyl sulfate improves the retention of chlorophyll (Chl) by chlorophyll-protein complexes (CPs) prepared from thylakoids of Euglena gracilis Klebs var bacillaris Cori and yields several additional complexes. Thylakoids from wild-type (WT) cells, solubilized in these detergents and subjected to polyacrylamide gel electrophoresis at 0 degrees C, yield the following CPs, in order of relative molecular weight, containing the pigments shown in parentheses with their respective molar ratios where determined: CP Ia (Chl a, diadinoxanthin and beta carotene; 100:12:5); CP I (Chl a and beta-carotene; 100:6-12); CPx (Chl and carotenoids); LHCP(2) (light-harvesting CP oligomer) (Chl a, Chl b, diadinoxanthin and neoxanthin; 12:4:3:1); CPy (Chl a, diadinoxanthin and beta carotene; 100:14:8); CPa (Chl a and beta-carotene; 100:18-25) and LHCP (monomer) (Chl a, Chl b, diadinoxanthin and neoxanthin; 12:6:4:1). The LHCP complexes retain up to 40% of the total Chl and 80% of the Chl b in the thylakoids. CP Ia contains only a trace of Chl b (Chl a/b [mol/mol] = 62). The lower amount of Chl b in Euglena (about 10% of Chl a + b) compared to higher plants (about 30% of Chl a + b) is probably a consequence of the lower Chl b (relative to Chl a) in the LHCPs of Euglena rather than of fewer LHCPs being present. G(1)BU, Gr(1)BSL, and O(4)BSL, mutants of bacillaris low in Chl b (1-2% of Chl a + b), lack the CP Ia, LHCP, and LHCP(2) found in wildtype (WT); G(1) and O(4) also lack CPy. The mutants contain reduced amounts of Chl a (two-thirds of WT in Gr(1) and one-third in G(1) and O(4)) and neoxanthin (20-40% of WT) but retain levels of beta carotene and diadinoxanthin close to those in cells of WT. The CPs remaining in the mutants have pigment compositions very similar to their counterparts from WT. PMID- 16664587 TI - Effects of Irradiance on Crassulacean Acid Metabolism in the Epiphyte Tillandsia usneoides L. (Bromeliaceae). AB - Spanish moss (Tillandsia usneoides L.) was collected in South Carolina, maintained in a greenhouse, then exposed to five levels of photosynthetic photon flux density (PPFD) for 3 weeks. Following this treatment, plants were sampled for chlorophyll concentrations, nocturnal acid accumulations, and photosynthetic responses to subsequent exposure at a range of PPFD. No acclimation to PPFD was observed; all plants exhibited similar patterns of nocturnal CO(2) uptake and acid accumulation regardless of initial PPFD treatment. These patterns revealed that at a PPFD level of approximately 200 micromoles per square meter per second (daytime integrated PPFD of 10 moles per square meter per day), CAM saturated or, in low-PPFD plants, was optimal. The results of this study indicate that adaptation to high PPFD is not necessarily a requirement of CAM. PMID- 16664588 TI - Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: II. Polypeptide Composition. AB - Chlorophyll-protein complexes (CPs) obtained from thylakoids of Euglena gracilis Klebs var bacillaris Cori contain the following polypeptides (listed in parentheses in order of prominence after Coomassie R-250 staining of polyacrylamide gels): CP Ia (66, 18, 22, 22.5, 27.5, 21, 28, 24, 25.5, and 26 kilodaltons [kD]); CP I (66 kD); CPx (41 kD); LHCP(2) (an oligomer of LHCP) (26.5, 28, and 26 kD); CPy (27 and 19 kD); CPa (54 kD); and LHCP (26.5, 28, and 26 kD). Mutants of bacillaris low in chlorophyll b (Gr(1)BSL, G(1)BU, and O(4)BSL; Chl a/b [mol/mol] = 50-100) which lack CP Ia, LHCP(2), and LHCP also lack or are deficient in polypeptides associated with these complexes in wild type cells. Mutants G(1) and O(4), which also lack CPy, lack the CPy-associated polypeptides found in wild-type and Gr(1). Using an antiserum which was elicited by and reacts strongly and selectively with the SDS-treated major polypeptide (26.5 kD) of the LHCP complexes of wild-type, this polypeptide is undetectable in the mutants (<<0.25% of the level in wild-type on a cell basis); the antiserum does not react with the SDS-treated 28 kD polypeptide of the Euglena LHCP complexes and cross-reacts only very weakly with components in SDS-treated cells of Chlamydomonas reinhardtii Dangeard and chloroplasts of Spinacia oleracea L. cv Winter Bloomsdale. Rates of photosynthesis of the wild-type and mutant cells of Euglena are approximately equal on a cell basis when measured at light saturation, consistent with the selective loss of major antenna components but not CP I or CPa from the mutants. PMID- 16664589 TI - Osmotic Response of Sugar Beet Source Leaves at CO(2) Compensation Point. AB - As sugar beet source leaves lowered the CO(2) concentration to compensation point in a closed atmosphere, leaf thickness and relative water content decreased. Leaf water potential declined rapidly from -0.5 to -1.4 megapascals. At 340 microliters CO(2) per liter, water potential and sucrose, glucose, and fructose contents were steady in photosynthesizing source leaves. Within 90 minutes after leaves were exposed to a CO(2) concentration at the compensation point, leaf sucrose content declined to 60% of the preteatment level, rapidly in the first 30 minutes and then more slowly. During the subsequent 200 minutes, sucrose content increased to 180% of pretreatment level. Glucose and fructose remained unchanged during the treatment. Degradation of starch was sufficient to account for the additional sucrose that accumulated. Labeled carbon lost from starch appeared in sucrose and several other compounds that likely contributed to the recovery in leaf water content. PMID- 16664590 TI - Compensation point and isotopic characteristics of c(3)/c(4) intermediates and hybrids in panicum. AB - Leaf CO(2) compensation points and stable hydrogen, oxygen and carbon isotope ratios were determined for Panicum species including C(3)/C(4) intermediate photosynthesis plants, hybrids between C(3)/C(4) intermediates and C(3) plants, C(3) and C(4) plants in the Panicum genus as well as several other C(3) and C(4) plants. C(3) plants had the highest compensation points, followed by hybrids, C(3)/C(4) intermediates, and C(4) plants. delta(13)C values of cellulose nitrate and saponifiable lipids from C(4) plants were about 10 per thousand higher than those observed for cellulose nitrate and saponifiable lipids of C(3)/C(4) intermediates, hybrids, and C(3) plants. Oxygen isotope ratios of cellulose as well as those of leaf water were similar for all plants. There was substantial variability in the deltaD values of cellulose nitrate among the plants studied. In contrast, such variability was not observed in deltaD values of water distilled from the leaves, nor in the deltaD values of the saponifiable lipids. Variability in deltaD values of cellulose nitrate from C(3)/C(4) intermediates, hybrids, C(3), and C(4) plants is due to fractionations occurring during biochemical reactions specific to leaf carbohydrate metabolism. PMID- 16664591 TI - Ethylene-induced increase in fructose-2,6-bisphosphate in plant storage tissues. AB - TREATMENT OF CARROT ROOTS WITH ETHYLENE LED TO: (a) a doubling of the fructose 2,6-bisphosphate content; (b) a general increase in the concentration of glycolytic intermediates; and (c) an increase in the extractable activity of fructose-6-phosphate,2-kinase, the enzyme synthesizing fructose-2,6-bisphosphate from fructose-6-phosphate and adenosine triphosphate. PMID- 16664592 TI - Carbon and nitrogen assimilation and partitioning in soybeans exposed to low root temperatures. AB - Low root temperature effects on vegetative growth of soybean (Harosoy 63 x Rhizobium japonicum USDA 16) were examined in 35 day old plants exposed to temperatures of 15 degrees C (shoots at 25 degrees C) for an 11 day period. Duing this period various aspects of C and N assimilation and partitioning were monitored including shoot night and nodulated root respiration, C and N partitioning to six plant parts, C(2)H(2) reduction, H(2) evolution, leaf area, transpiration, net photosynthesis, and N(2) fixation. The low temperature treatment resulted in a decrease in the net rate of N(2) fixation but nitrogenase relative efficiency increased. In response, the plant retained N in the tissues of the nodulated root and decreased N partitioning to young shoot tissues, thereby inducing the remobilization of N from older leaves, and reducing leaf area development. The leaf area specific rate of net photosynthesis was not affected over the study period; however, shoot and nodulated root respiration declined. Consequently, C accumulated in mature leaves and stems, partly in the form of increased starch reserves. Three possibilities were considered for increasing low temperature tolerance in nodulated soybeans: (a) decrease in temperature optima for nitrogenase, (b) increased development of nodules and N(2) fixation capacity at low temperature, and (c) alterations in the pattern of C and N partitioning in response to low temperature conditions. PMID- 16664593 TI - Identification of Indole-3-Acetylglutamate from Seeds of Glycine max L. AB - Indole-3-acetylglutamate was isolated from seeds of Glycine max L. cv Hark and identified by gas chromatography/mass spectrometry of the bis-methyl ester. Quantitative evaluation indicated that Hark soybeans contain 7.4 micromoles per kilogram of the glutamate conjugate. Conjugates with aspartate and glutamate have now been shown to account for essentially all of the bound indole-3-acetic acid present in seeds of soybean. PMID- 16664594 TI - Isolation of Indole-3-Acetyl Amino Acids using Polyvinylpolypyrrolidone Chromatography. AB - Amino acid conjugates of IAA can be chromatographed on polyvinylpolypyrrolidone (PVPP) using either acidic methanol or aqueous buffers as eluents. In the aqueous system, elution of the compounds is pH-dependent, and the pattern obtained suggests that hydrophobic interactions contribute substantially to the chromatographic behavior of IAA peptides on PVPP. Purification of soybean seed extracts by PVPP chromatography produced fractions containing indole-3 acetylaspartate and indole-3-acetylglutamate, based on chromatographic and mass spectral analysis, as well as three other indolic compounds, tentatively identified as N-acyl tryptophan derivatives. PVPP chromatography provides an effective preliminary purification of IAA peptides from plant extracts prior to their separation by other techniques such as high performance liquid chromatography. PMID- 16664595 TI - Temperature Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of BASF 13.338 (4-Chloro-5-Dimethylamino-2-Phenyl 3(2H)Pyridazinone). AB - When Triticum vulgare cv HD 2189 seedlings were grown in the presence of 125 micromolar BASF 13.338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), the rate of electron transport (H(2)O --> methyl viologen) in chloroplast thylakoids isolated from the treated seedlings was higher (by 50%) as compared to the control at assay temperatures above 30 degrees C. Below 30 degrees C, however, the rate with the treated seedlings was lower than the control rate. The temperature dependence of the rate of photosystem I electron transport (2-6 dichlorophenol indophenol-reduced --> methyl viologen) in the treated system was similar to that in the control. At high temperatures (>30 degrees C), with diphenyl carabazide as electron donor, the rates of electron transfer (diphenyl carbazide --> methyl viologen) were similar in the treated and in the control thylakoids. Direct addition of BASF 13.338 to the assay mixture for the measurement of rate of electron transport (H(2)O --> methyl viologen) in the thylakoids isolated from the control plants did not cause any change in the temperature dependence of photosynthetic electron transport. These results suggested that the donor side of photosystem II became tolerant to heat in the treated plants. Chlorophyll a fluorescence emission was monitored continuously in the leaves of control and BASF 13.338 treated wheat seedlings during continuous increase in temperature (1 degrees C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55 degrees C; this decrease was biphasic in the control and monophasic in the treated plants. PMID- 16664596 TI - Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein. AB - The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.The previously reported dissociation of the binding protein large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP. PMID- 16664597 TI - Cell Wall and Cytoplasmic Isozymes of Radish beta-Fructosidase Have Different N Linked Oligosaccharides. AB - When 36-hour-old dark grown radish seedlings are transferred to far-red light, there is a decrease in cytoplasmic beta-fructosidase (betaF) and an increase in cell wall betaF compared to the dark controls. Cytoplasmic and cell wall-bound beta-fructosidase are both glycoproteins and exhibit high antigenic similarities, but differ according to charge heterogeneity and carbohydrate microheterogeneity. Growth of radish seedlings in the presence of tunicamycin results in a partial inhibition of betaF glycosylation but nonglycosylated betaF still accumulates in the cell wall under far-red light. Thus, glycosylation is not necessary for intracellular transport, for correct targetting, or for wall association of an active betaF. The nonglycosylated cytoplasmic and cell wall betaF forms have the same relative molecular mass but glycosylated forms have different oligosaccharide side-chains, with respect to size and susceptibility to alpha mannosidase and endoglycosidase D digestion. The oligosaccharides of both forms are partly removed by endoglycosidase H when betaF is denatured. Isoelectric focusing analysis of betaF shows that the cell wall-associated isozymes are more basic than the cytoplasmic isozymes, and that the charge heterogeneity also exists within a single plant. A time course of changes in betaF zymograms shows a far red light stimulation of the appearance of the basic forms of the enzyme. However, the more basic cell wall specific betaF forms are not present when N glycosylation is prevented with tunicamycin. These results indicate that cytoplasmic and cell wall betaF probably have common precursor polypeptides and basic cell wall forms arise via processing events which are tunicamycin sensitive. PMID- 16664598 TI - Synthesis of the phytoalexin pisatin by a methyltransferase from pea. AB - Previous labeling studies in vivo suggest that the terminal step of (+)pisatin biosynthesis in Pisum sativum L. is methylation of the phenol (+)6a hydroxymaackiain (HMK). We have found that extracts from pea seedlings perform this reaction, using S-adenosylmethionine as the methyl donor. The enzyme activity was induced by microbial infection or treatment with CuCl(2), which elicit pisatin synthesis, though some activity was also present in healthy tissues. It has been reported that CuCl(2)-treated pea tissue provided with ( )HMK or (-)maackiain can synthesize (-)pisatin. Our extract showed no methyltransferase activity dependent on either of these substrates. Methylation of (+)maackiain was detectable, but much slower than that of (+)HMK. PMID- 16664599 TI - Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)? AB - Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei. PMID- 16664600 TI - Variations in leaf soluble amino acids and ammonium content in subtropical seagrasses related to salinity stress. AB - A survey of leaf soluble amino acids was conducted for four subtropical seagrasses grown at several salinities. Proline functioned as an organic osmoticum in Halodule wrightii Aschers., Thallasia testudinum Banks ex Koenig, and Ruppia maritima L., while alanine functioned in an osmoregulatory capacity in Halophila engelmanni Aschers. When light-and salinity-induced variations in leaf NH(4) and amide-N levels were compared in Halodule, Thalassia, and Halophila, ability to regulate leaf NH(4) levels was correlated with osmoregulatory capacity and maintenance of selected amino acid contents. PMID- 16664601 TI - Quantitative determination of indole-3-acetic Acid in sugarbeet leaves using a double standard isotope dilution gas chromatographic assay. AB - Quantitative levels of indole-3-acetic acid (IAA) were determined in leaf blades of two sugarbeet cultivars by a double standard isotope dilution assay using column chromatography followed by reverse phase C(18) high performance liquid chromatography and gas-liquid chromatography with nitrogen thermionic detection. The double standard method was validated as a quantitative tool by gas chromatography/selected ion monitoring mass spectrometry using 2,',4',5',6',7' d(5)-IAA as the internal standard. Progenies of one breeding line that had been selected for a high taproot to leaf weight ratio were used to correlate IAA levels with varying leaf and plant size at day 31 from germination. In spite of size differences, no significant difference in IAA levels per unit leaf weight could be found. The possible relationship between day 31 leaves and IAA content at an earlier stage of development is discussed in the text. A second analysis used four developmental leaf stages, classified as expanding, recently mature, aging, and senescing leaves. Expanding leaves contained the most IAA, senescing leaves contained the least IAA, with recently mature leaves and aging leaves containing intermediate amounts. The DNA content of each of the four developmental leaf stages was determined and DNA levels per gram fresh weight were found to be constant at all developmental stages. PMID- 16664602 TI - Manganese toxicity to chlorophyll synthesis in tobacco callus. AB - Tobacco (Nicotiana tabacum) pith explants were grown on manganese containing medium. At moderate concentration (10 millimolar), manganese selectively inhibited chlorophyll synthesis, resulting initially in growth of white callus. Several weeks later the white callus turned brown due to the accumulation of a pigment identified as protoporphyrin IX by its elution profile using high performance liquid chromatography, by its absorption spectrum, and by its fluorescence properties. At a concentration of 100 millimolar manganese the pigment accumulated without growth of the explant. PMID- 16664603 TI - A group translocator for sucrose assimilation in tonoplast vesicles of sugarcane cells. AB - Existence of a group translocator for sucrose transfer into vacuoles of sugarcane (Saccharum sp.) cells has been further confirmed by the use of tonoplast vesicles isolated from intact vacuoles. The group translocator depends on external UDP Glucose (Glc) and, via a series of enzymic reactions within the tonoplast, sucrose phosphate and sucrose are deposited inside the vesicles. Fructose-6 phosphate was not required for UDP-Glc uptake, nor was it taken up. None of the other sugar phosphates tested were taken up nor were the nucleotide sugars, UDP Galactose and ADP-Glc. The uptake of UDP-Glc was concentration-dependent with a K(m) of 1.2 millimolar and a V(max) of 83.3 nanomoles per minute per milligram protein. The optimum pH for UDP-Glc uptake was 7.0. Uptake of UDP-Glc was inhibited by para-chloromercuribenzene-sulfonic acid, UDP, and GDP; carbonyl cyanide m-chlorophenylhydrazone inhibited to a lesser extent. PMID- 16664604 TI - Immunochemistry on cryptomonad biliproteins. AB - A SURVEY IS MADE OF THE IMMUNOCHEMICAL BEHAVIOR OF FOUR OF THE SIX KNOWN TYPES OF CRYPTOMONAD BILIPROTEINS: phycocyanins 612 and 645 and phycoerythrins 545 and 566. They were compared both among themselves and to selected biliproteins isolated from blue-green and red algae. All the cryptomonad biliproteins were shown to be closely related to each other by Ouchterlony double diffusion technics. An antigenic relationship among all the cryptomonad biliproteins and B phycoerythrin (red alga) and C-phycoerythrin (blue-green alga) was established. Only a very marginal cross-reactivity was found between C-phycocyanin (blue-green algae) and the cryptomonad biliproteins. These results suggest a common ancestor for the photosynthetic units of all three biliprotein-containing phyla. PMID- 16664605 TI - Biogenesis and light regulation of the major light harvesting chlorophyll-protein of diatoms. AB - The apoprotein of the major light harvesting pigment-protein complex from the diatom Phaeodactylum tricornutum (UTEX 646) is composed of two similar polypeptides of 17.5 and 18.0 kilodaltons (kD). The in vivo synthesis of these polypeptides is inhibited by the 80s protein synthesis inhibitor cycloheximide, but not by the 70s ribosome inhibitor chloramphenicol. When total poly(A)(+) RNA was used in in vitro protein synthesis, a number of polypeptides were synthesized with a dominant product at 22 kD. When the polypeptides were immunoprecipitated with monospecific antibodies to the 17.5 and 18.0 polypeptides, a single protein zone of 22 kD was detected. Immunoprecipitation with preimmune serum failed to precipitate detectable levels of protein at any relative molecular weight (M(r)). These findings indicate that the two apoprotein polypeptides of the diatom light harvesting pigment-protein are translated from polyadenylated message on cytoplasmic ribosomes as either a single or two (or more) similar M(r) precursor proteins. These findings also suggest that this protein is encoded in the nucleus.Photosynthetic light adaptation features of P. tricornutum UTEX 646 indicate that it responds to low light by increasing cell size and numbers of photosystem I and II reaction centers per cell, but does not change photosynthetic rate per cell or photosynthetic unit sizes significantly. When low light cells are exposed to higher photon flux densities, the in vivo incorporation of label into the apoprotein of the light harvesting complex decreases. In contrast, high light grown cells show rapid (<3 hour) increases in apoprotein synthesis when exposed to low light levels. This is the first demonstration of a specific role of photon flux density in regulating the synthesis of a major light harvesting pigment-protein during photosynthetic light adaptation. PMID- 16664606 TI - The Effects of N Nutrition on the Water Relations and Gas Exchange Characteristics of Wheat (Triticum aestivum L.). AB - The purpose of this study was to characterize leaf photosynthetic and stomatal responses of wheat (Triticum aestivum L.) plants grown under two N-nutritional regimes. High- and low-N regimes were imposed on growth-chamber-grown plants by fertilizing with nutrient solutions containing 12 or 1 millimolar nitrogen, respectively. Gas-exchange measurements indicated not only greater photosynthetic capacity of high-N plants under well-watered conditions, but also a greater sensitivity of CO(2) exchange rate and leaf conductance to CO(2) and leaf water potential compared to low-N plants. Increased sensitivity of high-N plants was associated with greater tissue elasticity, lower values of leaf osmotic pressure and greater aboveground biomass. These N-nutritional-related changes resulted in greater desiccation (lowered relative water content) of high-N plants as leaf water potential fell, and were implicated as being important in causing greater sensitivity of high-N leaf gas exchange to reductions in water potential. Water use efficiency of leaves, calculated as CO(2) exchange rate/transpiration, increased from 9.1 to 13 millimoles per mole and 7.9 to 9.1 millimoles per mole for high- and low-N plants as water became limiting. Stomatal oscillations were commonly observed in the low-N treatment at low leaf water potentials and ambient CO(2) concentrations, but disappeared as CO(2) was lowered and stomata opened. PMID- 16664607 TI - Acclimation to High CO(2) in Monoecious Cucumbers : I. Vegetative and Reproductive Growth. AB - CO(2) concentrations of 1000 compared to 350 microliters per liter in controlled environment chambers did not increase total fruit weight or number in a monoecious cucumber (Cucumis sativus L. cv Chipper) nor did it increase biomass, leaf area, or relative growth rates beyond the first 16 days after seeding. Average fruit weight was slightly, but not significantly greater in the 1000 microliters per liter CO(2) treatment because fruit numbers were changed more than total weight. Plants grown at 1000 and 350 microliters per liter CO(2) were similar in distribution of dry matter and leaf area between mainstem, axillary, and subaxillary branches. Early flower production was greater in 1000 microliters per liter plants. Subsequent flower numbers were either lower in enriched plants or similar in the two treatments, except for the harvest at fruiting when enriched plants produced many more male flowers than the 350 microliters per liter treatments. PMID- 16664608 TI - Acclimation to High CO(2) in Monoecious Cucumbers : II. Carbon Exchange Rates, Enzyme Activities, and Starch and Nutrient Concentrations. AB - Carbon exchange capacity of cucumber (Cucumis sativus L.) germinated and grown in controlled environment chambers at 1000 microliters per liter CO(2) decreased from the vegetative growth stage to the fruiting stage, during which time capacity of plants grown at 350 microliters per liter increased. Carbon exchange rates (CERs) measured under growth conditions during the fruiting period were, in fact, lower in plants grown at 1000 microliters per liter CO(2) than those grown at 350. Progressive decreases in CERs in 1000 microliters per liter plants were associated with decreasing stomatal conductances and activities of ribulose bisphosphate carboxylase and carbonic anhydrase. Leaf starch concentrations were higher in 1000 microliters per liter CO(2) grown-plants than in 350 microliters per liter grown plants but calcium and nitrogen concentrations were lower, the greatest difference occurring at flowering. Sucrose synthase and sucrose-P synthase activities were similar in 1000 microliters per liter compared to 350 microliters per liter plants during vegetative growth and flowering but higher in 350 microliters per liter plants at fruiting. The decreased carbon exchange rates observed in this cultivar at 1000 microliters per liter CO(2) could explain the lack of any yield increase (MM Peet 1986 Plant Physiol 80: 59-62) when compared with plants grown at 350 microliters per liter. PMID- 16664609 TI - Proteinase inhibitor I accumulation in tomato suspension cultures : induction by plant and fungal cell wall fragments and an extracellular polysaccharide secreted into the medium. AB - Suspension-cultured cells of tomato accumulate proteinase Inhibitor I as the sucrose is depleted from 1% to less than 0.1% in the culture medium. Inhibitor I can be prematurely induced to accumulate in the cells by the addition to the medium of the proteinase inhibitor inducing factor, trigalacturonic acid, ethylene glycol chitin, or chitosan. In cultures grown in 0.6% initial sucrose with no inducers added, a uronic acid-rich extracellular polysaccharide appears in the medium during growth of the cells. This extracellular polysaccharide apparently contains an ;endogenous inducer' of Inhibitor I synthesis. When the partially purified polysaccharide is added to the culture medium, Inhibitor I accumulation is induced. Proteinase inhibitors also accumulate in tobacco and alfalfa suspension-cultured cells as the cell cultures age. As with the tomato cultures, a uronic acid-rich component(s) appears in the media prior to inhibitor accumulation. These data suggest that an endogenous inducer may be activating proteinase inhibitor genes through a similar mechanism in all three types of cells. PMID- 16664610 TI - Influence of Free Fatty Acids, Lysophosphatidylcholine, Platelet-Activating Factor, Acylcarnitine, and Echinocandin B on 1,3-beta-d-Glucan Synthase and Callose Synthesis. AB - The activity of 1,3-beta-d-glucan synthase assayed in the presence of digitonin in a microsomal preparation from suspension-cultured cells of Glycine max can be fully inhibited by unsaturated fatty acids, trienoic acids being most effective. Lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B can also fully inhibit the enzyme. Inhibition is observed both when the enzyme is activated by Ca(2+) or by trypsinization. At low amounts some of the substances can also cause stimulation. These effects all may result from a displacement of certain endogenous phospholipids necessary for optimal activity of the 1,3-beta-d-glucan synthase.In the absence of digitonin the enzyme activity is greatly stimulated by lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B within a certain concentration range, presumably by rendering the microsomal vesicles permeable to the substrate and Ca(2+). Dibucaine does not cause such an effect.Acylcarnitine and Echinocandin B at low concentrations can induce callose synthesis in vivo; this effect is enhanced by chitosan. At higher concentrations the two substances and polyunsaturated fatty acids cause severe electrolyte leakage. The effects are discussed in regard to the induction of callose synthesis by enforced Ca(2+) influx, and its modulation by membrane lipids. PMID- 16664611 TI - Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : II. Partial Activity, Inhibitor, and Complementation Analyses. AB - Soybean (Glycine max [L.] Merr.) leaves have been shown to contain three forms of nitrate reductase (NR). Two of the forms, which are present in leaves of wild type (cv. Williams) plants grown in the absence of NO(3) (-), are termed constitutive and designated c(1)NR and c(2)NR. The third form, which is present in NO(3) (-)-grown mutant (nr(1)) plants lacking the constitutive forms, is termed inducible and designated iNR. Samples of c(1)NR, c(2)NR, and iNR obtained from appropriately treated plants were analyzed for the presence of partial activities, response to inhibitors, and ability to complement a barley NR which lacks the molybdenum cofactor (MoCo) but is otherwise active.The three forms were similar to most assimilatory NR enzymes in that they (a) exhibited NADH cytochrome c reductase, reduced flavin mononucleotide-NR, and reduced methyl viologen-NR partial activities; (b) were inhibited by p-hydroxymercuribenzoate at the site of initial electron transport through each enzyme; (c) were more inhibited by CN(-) in their reduced enzyme state as compared with their oxidized state; and (d) complemented a MoCo-defective NR (e.g. contained cofactors with characteristics similar to the MoCo found in barley NR and commercial xanthine oxidase). However, among themselves, they showed dissimilarities in their response to treatment with HCO(3) (-) and CN(-), and in their absolute ability to complement the barley NR. The site of effect for these treatments was the terminal cofactor-containing portion of each enzyme. This indicated that, although a terminal cofactor (presumably a MoCo) was present in each form, structural or conformational differences existed in the terminal cofactor-protein complex of each form. PMID- 16664612 TI - In VivoP NMR Studies of Corn Root Tissue and Its Uptake of Toxic Metals. AB - Excised corn root tissue has been evaluated for its viability, integrity of compartmentation, intracellular pH gradients, total mobile phosphorus content and nucleotide concentrations under different levels of acidity, and mineral stresses using in vivo(31)P nuclear magnetic resonance spectroscopy at 21 to 23 degrees C. Perfusion with Al(3+) ion at low pH (4.0) for 20 hours caused the overall concentration of nucleotides in the cytoplasm to decrease significantly relative to the control. Respiratory activity as measured by O(2) uptake decreased by a comparable amount over this time period. The addition of glucose to the Al containing perfusate negated the inhibitory effects on the respiratory system. Treatment of the tissue with paramagnetic manganese ion while perfusing in the presence of O(2) allowed for the observation of the sequence of events leading to the irreversible trapping of Mn(2+) in the vacuole. Pretreatment of the roots with Mg(2+) prevented Mn(2+) migration to the vacuole over the time period of this experiment. Hypoxia prevented all but a limited uptake of Mn(2+) into the cytoplasm of the root tips. No evidence of Mn(2+) complexation of either cytoplasmic or vacuole Pi suggests that the energy derived from O(2) consuming processes is necessary for the facilitated movement of this divalent cation. PMID- 16664613 TI - Changes in Protein Composition and Mn Abundance in Photosystem II Particles on Photoactivation of the Latent O(2)-Evolving System in Flash-Grown Wheat Leaves. AB - Protein composition and Mn abundance were compared between the two photosystem II (PSII) particle preparations obtained before and after photoactivation of the latent O(2)-evolving system in intermittently flashed wheat leaves. The following results have been obtained: (a) nonphotoactivated PSII particles were devoid of two extrinsic proteins which corresponded to the 24 and 16 kilodalton proteins in spinach particles, although the particles contained all the intrinsic proteins and the 33 kilodalton extrinsic protein. (b) The two extrinsic proteins absent in nonphotoactivated PSII particles were present in nonphotoactivated thylakoids, but were easily removed by a hypotonic shock followed by brief sonication. Such removal of the proteins did not occur in photoactivated thylakoids. (c) Nonphotoactivated PSII particles contained 1.5 Mn/400 chlorophyll, while photoactivated particles contained 8 Mn/400 chlorophyll. (d) Nonphotoactivated thylakoids contained 6 Mn/400 chlorophyll, but most of them were removed from thylakoids by a hypotonic shock in the presence of ethylenediaminetetraacetate. Such removal of Mn did not occur in photoactivated thylakoids. PMID- 16664614 TI - Structural requirements for the binding of phenylglycosides to the surface of protoplasts. AB - A variety of phenylglycosides have been synthesized and tested for binding to the surface of protoplasts from suspension-cultured cells of "Paul's Scarlet" rose (Rosa sp.). Multivalent phenylglycosides in the form of Yariv antigens (1,3,5, tri-[p-glycosyloxyphenylazo]-2,4,6,-trihydroxybenzene) agglutinated the protoplasts. Fluorescence-labeled derivatives of other monovalent and polyvalent phenyl-beta-glycosides did not bind to the protoplast surface. Agglutination was induced by Yariv antigens only if these probes contained beta-anomeric, O glycosidic linkages. Yariv antigens containing alpha-anomeric or thio-glycosidic linkages did not agglutinate protoplasts. These same structural features of Yariv antigens were also required for the precipitation of gum arabic-Yariv antigen complexes. The results suggest that plasma membranes of "Paul's Scarlet" rose protoplasts contain arabinogalactan-proteins that interact with phenyl-beta glycosides. The results further show that binding at these plasma membrane sites is not solely dependent upon the carbohydrate portion of single phenylglycosides, but may also require specific spatial orientations of adjacent phenylglycosides. PMID- 16664615 TI - Isolation and Partial Characterization of the Major Amide-Linked Conjugate of Indole-3-Acetic Acid from Phaseolus vulgaris L. AB - A major indole-3-acetic acid conjugate from Phaseolus vulgaris seed has been isolated and partially characterized. It is a 3 kilodalton peptide with apparently 2 indole-3-acetyl moieties in amide linkage per peptide. The indole-3 acetic acid component was identified by gas chromatography-mass spectrometry and the peptide characterized by polyacrylamide gel electrophoresis, by amino acid analysis using dabsyl derivatives and by its Fourier transform-infrared spectrum. This is the first higher molecular weight amide-linked indole-3-acetic acid conjugate to be characterized from higher plants. PMID- 16664616 TI - Alternative Path Mediated ATP Synthesis in Roots of Pisum sativum upon Nitrogen Supply. AB - Changes in the efficiency of root respiration were examined on intact plants of Pisum sativum L. cv Rondo after addition of nitrate or ammonium to the culture solutions. Nitrate was absorbed immediately after addition and elicited a respiratory rise (O(2)-uptake as well as CO(2)-production) to 160% at most. This occurred both in roots of plants fixing N(2) and in those of non-nodulated plants pregrown for 1 or 2 weeks on a nitrogen-free culture solution. In older plants, used after 2 weeks of N-free growth, the full capacity of the cytochrome path was engaged in root respiration. This was demonstrated by the absence of an effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone in the presence of 25 millimolar salicylhydroxamate, an inhibitor of the alternative path. In these plants more than 90% of the nitrate-induced stimulation of root respiration was salicylhydroxamate-sensitive. In young plants, used after 1 week of N-free growth, the cytochrome path was not saturated. Its activity increased instantaneously at the expense of alternative path activity, which initially dropped to zero and subsequently increased to 160% of the control 7 hours after nitrate supply. The rate of photosynthesis rose to 120% of the control, but not before 1 hour after nitrate supply, suggesting that the stimulation of root respiration was not due to a higher rate of photosynthesis. Experiments with plants grown with a split-root system showed that respiration rate and alternative path activity only increased in the root halves exposed to nitrogen. Ammonium was equally effective as nitrate in stimulating root respiration. These results lead to the conclusion that alternative-path mediated root respiration contributes to synthesis of ATP during at least the first 24 hours following nitrogen supply. PMID- 16664617 TI - Distinction between Cytosol and Chloroplast Fructose-Bisphosphate Aldolases from Pea, Wheat, and Corn Leaves. AB - A reinvestigation of cytosol and chloroplast fructose-1,6-bisphosphate (FBP) aldolases from pea (Pisum sativum L.), wheat (Triticum aestivum L.) and corn leaves (Zea mays L.) revealed that the two isoenzymes can be separated by chromatography on diethylaminoethyl (DEAE)-cellulose although the separation was often less clear-cut than for the two aldolases from spinach leaves. Definite distinction was achieved by immunoprecipitation of the two isoenzymes with antisera raised against the respective isoenzymes from spinach leaves. The proportion of cytosol aldolase as part of total aldolase activity was 8, 9, 14, and 4.5% in spinach (Spinacia oleracea L.), pea, wheat, and corn leaves, respectively. For corn leaves we also obtained values of up to 15%. The K(m) (FBP) values were about 5-fold lower for the cytosol (1.1-2.3 micromolar concentration) than for the chloroplast enzymes (8.0-10.5 micromolar concentration). The respective K(m) (fructose-1-phosphate, F1P) values were about equal for the cytosol (1.0-2.3 millimolar concentration) and for the chloroplast aldolase (0.6-1.7 millimolar concentration). The ratio V (FIP)/V (FBP) was 0.20 to 0.27 for the cytosol and 0.07 to 0.145 for the chloroplast aldolase. Thus, cytosol and chloroplast aldolases from spinach, pea, wheat, and corn leaves differ quite considerably in the elution pattern from DEAE-cellulose, in immunoprecipitability with antisera against the respective isoenzymes from spinach leaves, and in the affinity to FBP. PMID- 16664618 TI - Effects of ethephon on aging and photosynthetic activity in isolated chloroplasts. AB - Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 25 degrees C with 2-chloroethylphosphonic acid (ethephon) under light (0.16 milliwatts per square centimeter) and dark conditions. Ethephon at 1 micromolar (0.1445 ppm), 0.1 and 1 millimolar, or 5 microliters ethylene promoted the deterioration of chloroplasts, increased proteolysis, and reduced the chlorophyll content and PSI and PSII during 72 hours under both light and dark conditions. The decline in PSI and PSII occurred prior to a measurable loss of chlorophyll. The loss of photosynthetic activity affected by ethephon was initiated prior to 12 hours of incubation. After 24 hours in light, 0.1 millimolar (1.445 ppm) epthephon significantly reduced PSI and PSII and promoted the total free amino acid liberation in isolated chloroplasts. In darkness the rate of loss of PSI activity was about 50% of that in light. After 24 hours, in light at 1 millimolar epthephon, PSII activity was 55% of the control, yet nearly 90% of the chlorophyll remained, which indicates that the loss of thylakoid integrity was promoted by ethephon. Ethylene injected in the chloroplast medium at 5 microliters (0.22 micromolar per milliliter) reduced PSI by nearly 50% of the initial in 12 hours. In leaf sections floated in 5 microliters per milliliter suspension medium, a 36% loss of chlorophyll of the control in 36 hours was observed. Cycloheximide at 0.5 millimolar masked the effect of 1 millimolar ethephon and maintained the initial chlorophyll content during the 72 hour period. PMID- 16664619 TI - Development of (1-->3,1-->4)-beta-d-Glucan Endohydrolase Isoenzymes in Isolated Scutella and Aleurone Layers of Barley (Hordeum vulgare). AB - An immunological assay has been used to investigate the synthesis of (1-->3,1- >4)-beta-glucanase (EC 3.2.1.73) isoenzymes from isolated barley aleurone layers and scutella. Enzyme release from both tissues is enhanced by 1 micromolar gibberellic acid and 10 millimolar Ca(2+), although increases induced by gibberellic acid are observed only in the presence of Ca(2+). Isoenzyme I is synthesized predominantly in the scutellum, while isoenzyme II is synthesized exclusively in the aleurone. A third, putative isoenzyme III has been detected in significant proportions in scutellar secretions and may also be secreted from aleurone layers. Both gibberellic acid and Ca(2+) appear to preferentially enhance isoenzyme II secretion from the aleurone and isoenzyme III secretion from scutella. The patterns of isoenzyme secretion are suggestive of tissue-specific differences in expression of the genes which code for (1-->3,1-->4)-beta glucanase isoenzymes. Qualitatively similar results were obtained with barley cultivars harvested in Australia and North America. PMID- 16664620 TI - Bound auxin metabolism in cultured crown-gall tissues of tobacco. AB - Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [(14)C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [(14)C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66 transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [(14)C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [(14)C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation. PMID- 16664621 TI - Appearance and accumulation of c(4) carbon pathway enzymes in developing maize leaves and differentiating maize a188 callus. AB - Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C(4) photosynthesis, for cell morphology, and for (14)C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later.Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C(3) leaves and from etiolated C(4) leaf tissues, but differing from the corresponding enzymes in the mature leaf.Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology.Analysis of the labeled products of (14)CO(2) fixation by nonchlorophyllous calli indicated beta-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in beta-carboxylation products was even greater. Analysis of enzyme levels in young leaf sections by protein blot and of (14)C labeling patterns in the present study are in general agreement with enzyme activity determinations of previous studies, providing additional information about PPDK levels, and supporting the model proposed for developing young leaves.These results suggest that maize leaves begin to express C(4) enzymes during ontogeny through several stages from greening and cell differentiation as seen in the callus and then shoot formation, and finally acquire capacity for full C(4) photosynthesis during leaf development concomitant with the development of Kranz anatomy and accumulation of large amounts of enzymes involved in carbon metabolism. PMID- 16664622 TI - Appearance and accumulation of c(4) carbon pathway enzymes in developing wheat leaves. AB - Soluble protein has been extracted from sections of wheat leaves, from base to tip, and the content of several key enzymes of photosynthetic carbon assimilation in each section has been determined by the protein blot method. In the first leaf, ribulose 1,5-bisphosphate carboxylase (RuBPC) (EC 4.1.1.39) in the basal 0 to 1 centimeter section is about 12% the level in the tip section, whereas phosphoenolpyruvate carboxylase (EC 4.1.1.31) is present in small amounts in the basal section and does not change much in the tip. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) first appears in the 4 to 6 centimeter section and increases gradually with development to 10-fold in the tip. Malic enzyme, NADP dependent (EC 1.3.1.37) also appears in the 4 to 6 centimeter section but remains low to the tip.Fixation of (14)CO(2) by wheat leaf base sections resulted in 42% of total incorporation into malate and aspartate, indicating beta-carboxylation, whereas in the tip section these labeled compounds were only 8% of the total. Although the amount of PPDK in wheat leaves is only 1 to 3% of that in maize leaves, this C(3) PPDK may have a limited role in photosynthesis leading to formation of C(4) compounds. The possibility of a further role, similar to that in C(4) plants, but for intracellular carbon transport in wheat leaves is discussed. The presence of malic dehydrogenase, NADP-specific (EC 1.1.1.82) in wheat leaf chloroplasts was shown, a necessary though not sufficient condition for such a proposed role. Assuming each of the four enzymes associated with C(4) carbon transport were fully active in vivo during photosynthesis, PPDK would still be rate limiting, even in the leaf tip where its activity is maximal. Possible evolutionary and breeding implications are discussed. PMID- 16664623 TI - Changes of Ribulose Bisphosphate Carboxylase/Oxygenase Content, Ribulose Bisphosphate Concentration, and Photosynthetic Activity during Adaptation of High CO(2) Grown Cells to Low-CO(2) Conditions in Chlorella pyrenoidosa. AB - Changes of some photosynthetic properties of high-CO(2) grown cells of Chlorella pyrenoidosa during adaptation to low-CO(2) conditions have been investigated. The K(m) value of photosynthesis of the high-CO(2) grown cells for dissolved inorganic carbon was 3.3 millimolar and decreased to 25 to 30 micromolar within 4 hours after transferring to air. In the presence of saturating CO(2) concentrations the photosynthetic activity of the high-CO(2) grown cells was 1.5 times as high as that of the low-CO(2) grown cells. There was a significant rise of the photosynthetic activity during adaptation of the high-CO(2) grown cells to air, followed by a steady decrease. The activity of ribulose 1,5-bisphosphate carboxylase/oxygenase in both the high- and low-CO(2) grown cells was close to the photosynthetic activity of the cells. The concentration of ribulose 1,5 bisphosphate (RuBP) was higher in the low-CO(2) adapting and low-CO(2) grown cells than in the high-CO(2) grown cells regardless of the photosynthetic rate. This seems to be due to an increased RuBP regeneration activity during adaptation followed by maintenance of the new higher concentration. The RuBP level always exceeded the concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP binding sites in both the high- and low-CO(2) grown cells at any dissolved inorganic carbon concentration. PMID- 16664624 TI - Oxygen exchange in ulva using a bare platinum electrode with 4 microsecond saturating light flashes. AB - Illuminating algae (Ulva species) on a bare platinum electrode with 4 microseconds of saturating light flashes leads to the familiar four-step oscillatory pattern of O(2) exchange. However, it appears that this O(2) exchange is a composite of O(2) evolution and endogenous O(2) uptake in the algae. We have added the O(2) uptake component to the O(2) exchange curve in order to obtain the corrected O(2) evolution flash sequence which shows a similar four-step oscillation as reported previously, but with negligible damping over the first three oscillations. PMID- 16664625 TI - Water stress enhances expression of an alpha-amylase gene in barley leaves. AB - The amylases of the second leaves of barley seedlings (Hordeum vulgare L. cv Betzes) were resolved into eight isozymes by isoelectric focusing, seven of which were beta-amylase and the other, alpha-amylase. The alpha-amylase had the same isoelectric point as one of the gibberellin-induced alpha-amylase isozymes in the aleurone layer. This and other enzyme characteristics indicated that the leaf isozyme corresponded to the type A aleurone alpha-amylase (low pI group). Crossing experiments indicated that leaf and type A aleurone isozymes resulted from expression of the same genes.In unwatered seedlings, leaf alpha-amylase increased as leaf water potential decreased and ABA increased. Water stress had no effect on beta-amylase. alpha-Amylase occurred uniformly along the length of the leaf but beta-amylase was concentrated in the basal half of the leaf. Cell fractionation studies indicated that none of the leaf alpha-amylase occurred inside chloroplasts.Leaf radiolabeling experiments followed by extraction of alpha-amylase by affinity chromatography and immunoprecipitation showed that increase of alpha-amylase activity involved synthesis of the enzyme. However, water stress caused no major change in total protein synthesis. Hybridization of a radiolabeled alpha-amylase-related cDNA clone to size fractionated RNA showed that water-stressed leaves contained much more alpha-amylase mRNA than unstressed plants. The results of these and other studies indicate that regulation of gene expression may be a component in water-stress induced metabolic changes. PMID- 16664626 TI - Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii. AB - Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO(2) in the presence of 3-(3,4-dichlorophenyl)-1,1 dimethylurea when adapted under an atmosphere of H(2) demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H(2) while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H(2). Both reactions occurred at rates approximately one third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO(2) was not detected. PMID- 16664627 TI - Association of the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardii with Photoreduction and the Oxyhydrogen Reaction. AB - The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO(2) assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chlamydomonas reinhardii 11-32/6. Photoreduction but not photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO(2) fixation was inhibited more than 95% by 10 micromolar 2,5 - dibromo - 3 - methyl - 6 - isopropyl - p - benzoquinone (DBMIB), a concentration which did not affect respiratory activity. In cells, both photoreduction and the oxyhydrogen reaction exhibited a similar sensitivity to salicylhydroxamic acid (SHAM) showing approximately 90% inhibition by 7 millimolar concentration. Photosynthesis was inhibited only 30% by the same concentration of SHAM. Antimycin A (18 micromolar, 10 micrograms per milliliter) inhibited both photoreduction (80%) and the oxyhydrogen reaction (92%) in cells with the oxyhydrogen reaction being approximately 10 times more sensitive to lower concentrations of the inhibitor. Antimycin A at 18 micromolar concentration did not inhibit photosynthetic CO(2) fixation unless the cells were adapted to an atmosphere of N(2) and the reaction conducted anaerobically. Photosynthesis, photoreduction, and the oxyhydrogen reaction coupled to CO(2) fixation were all inhibited greater than 90% by 10 micromolar carbonylcyanide-p trifluoromethoxyphenylhydrazone. ATP added to chloroplasts adapted to an atmosphere of H(2) could support CO(2) uptake in the dark. These results are interpreted as evidence that photoreduction and the oxyhydrogen reaction involve some common components of thylakoidal electron transport pathways in Chlamydomonas including NAD(P)H-plastoquinone oxidoreductase and the plastoquinone pool. An O(2)-consuming thylakoidal or mitochondrial reaction is an additional component of the oxyhydrogen reaction. PMID- 16664628 TI - Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.). AB - Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg(2+) ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K(m) of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO(3) (-) with S(0.5) value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, alpha-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca(2+) and Mn(2+). Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate. PMID- 16664629 TI - Alternative Respiratory Path Capacity in Plant Mitochondria: Effect of Growth Temperature, the Electrochemical Gradient, and Assay pH. AB - Influence of growth temperature on the capacity of the mitochondrial alternative pathway of electron transport was investigated using etiolated corn (Zea mays L.) seedlings. These seedlings were grown to comparable size in either a warm (30 degrees C) or a cold (13 degrees C) temperature regime, and then their respiration rates were measured as O(2) uptake at 25 degrees C. The capacity of the alternative pathway (KCN-insensitive O(2) uptake) was found essentially to double in shoots of cold-grown seedlings. This increased capacity slowly developed over several days growth in the cold, but was lost within 1 day when the seedlings were exposed to a warm regime. When mitochondria were isolated from the shoots of these seedlings, a greater potential for flow through the alternative path was observed in mitochondria from the cold-grown seedlings with all substrates used (an average increase of 84%). Using exogenous NADH as the substrate, the effect of the electrochemical gradient on measurable capacities of the cytochrome and alternative pathways was investigated in mitochondria from both etiolated seedlings and thermogenic spadices. The uncoupler FCCP (p trifluoromethoxycarbonylcyanide phenylhydrazone) was used to diminish the electrochemical gradient when desired. In corn (Zea mays L.) shoot and mung bean (Vigna radiata L.) hypocotyl mitochondria, which have relatively low capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the electrochemical gradient was found not to influence the potential for flow through the alternative path. However, in mitochondria from skunk cabbage (Symplocarpus foetidus L.) and voodoo lily (Sauromatum guttatum Schott) spadices, which have high capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the gradient occurred at the expense of flow through the alternative pathway. These results suggest that in mitochondria of thermogenic spadices, the combined capacities of the cytochrome and alternative paths exceed the capacity of the exogenous NADH dehydrogenase. The effect of assay pH on measurable capacities of the cytochrome and alternative paths was determined over a pH range of 5.6 to 8.8 using exogenous NADH as the mitochondrial substrate. When the electrochemical gradient was present, it limited the electron transport rate and little effect of assay pH was observed. However, when formation of the gradient was prevented through inclusion of FCCP, measurable capacities of the cytochrome and alternative paths were found to be greatly influenced by pH. This experiment also revealed that the potential for respiratory control is largely dependent upon the assay pH. PMID- 16664630 TI - Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes. AB - Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K(m) of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting K(m) of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S(0.5) of about 30 micromolar and production of H(2)O and H(2)O(2), without superoxide involvement. The ratio NAD(P)H:O(2) averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to O(2) without release of intermediates. PMID- 16664631 TI - d-Glycerate Transport by the Pea Chloroplast Glycolate Carrier : Studies on [1 C]d-Glycerate Uptake and d-Glycerate Dependent O(2) Evolution. AB - The transport of glycolate and d-glycerate, the substrate and end product of the photorespiratory carbon pathway, respectively, by isolated intact pea (Pisum sativum) chloroplasts has been compared. d-Glycerate uptake was inhibited by the 2-hydroxymonocarboxylates, glycolate, glyoxylate, and d-lactate. Phosphate and phosphoglycerate and triose phosphates were without effect when the assays were carried out for two seconds. Glycolate was found to be a competitive inhibitor of d-glycerate uptake and the presence of glycolate in the chloroplast stroma strongly enhanced d-glycerate uptake from the medium. For optimal rates of d glycerate-dependent O(2) evolution by pea chloroplasts, phosphate, ADP or ATP, and a mediator of cyclic electron flow had to be added. The inhibition of d glycerate-dependent O(2) evolution by triose phosphates and 2 hydroxymonocarboxylates was tested. The inhibition of d-glycerate-dependent O(2) evolution by these metabolites did not correlate with their effects on glycerate transport. Thus, metabolism of d-glycerate, rather than its transport, limits the rate of glycerate-dependent oxygen evolution. The ramifications of d-glycerate metabolism on the interpretation of d-glycerate uptake data obtained with prolonged incubations are discussed. We conclude that d-glycerate and glycolate transport are mediated by the same transport system. PMID- 16664632 TI - Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L. AB - Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA(1,9,12,15,19,20,24,29,44,51,53). Traces of GA(8) and GA(25) were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA(19) and GA(44) were most abundant, while GA(12,15,20,24,29,53) occurred in lesser amounts. Only small amounts of GA(1,9,51) were present. The levels of GA(8) and GA(25) were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA(1,8,19,20,29,44,53)) and another whose members are either nonhydroxylated (GA(9,12,15,24,25)) or lack a C-13 hydroxyl group (GA(51)). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA(12): an early C-13 hydroxylation pathway, leading ultimately to GA(1) and GA(8) and a C-13 deoxy pathway culminating in the formation of GA(9) and GA(51). PMID- 16664633 TI - Lack of Control by Early Pistillate Ethylene of the Accelerated Wilting of Petunia hybrida Flowers. AB - Well before pollen tube penetration, ethylene has begun to disseminate from pollinated styles of Petunia hybrida flowers. Previous stigmatic application of aminoethoxyvinylglycine (AVG) completely prevented this ethylene synthesis, indicating that the endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) in pollen is not readily converted on the stigma. Compared to other flower parts, the capacity of the ethylene forming enzyme was largest in the stigma. When applied to the stigma, ACC caused ethylene synthesis, but did not accelerate wilting, unless high concentrations (20 nanomols) were used. Upon pollination or stigma wounding, the early ethylene evolved exclusively from the gynoecium, much later followed by the synthesis of corolla ethylene. Employing wideneck Erlenmeyer flasks, the competitive inhibitor of ethylene action, norbornadiene, was applied to entire flowers in situ, with delaying effects on wound-induced wilting. In contrast, norbornadiene treatment of styles alone, using capillaries, could not postpone wilting. Pollination with foreign pollen species did not lead to accelerated corolla wilting, notwithstanding considerable synthesis of ethylene during the first 5 hours. In situ treatment of the stigma with AVG considerably delayed wound- and pollination-induced wilting. Removal of the entire AVG-treated style 6 hours after stigma wounding still allowed for the postponement of the accelerated wilting, even at very low concentrations of AVG. It is concluded that early stylar ethylene does not play a role in the acceleration of wilting but that, much later, corolla ethylene does, induced by a mobile wilting factor from the stigma, which is ACC. PMID- 16664634 TI - C(3)-C(4) Intermediate Species in Alternanthera (Amaranthaceae) : Leaf Anatomy, CO(2) Compensation Point, Net CO(2) Exchange and Activities of Photosynthetic Enzymes. AB - Two naturally occurring species of the genus Alternanthera, namely A. ficoides and A. tenella, were identified as C(3)-C(4) intermediates based on leaf anatomy, photosynthetic CO(2) compensation point (Gamma), O(2) response of small ghe, Cyrillic, light intensity response of small ghe, Cyrillic, and the activities of key enzymes of photosynthesis. A. ficoides and A. tenella exhibited a less distinct Kranz-like leaf anatomy with substantial accumulation of starch both in mesophyll and bundle sheath cells. Photosynthetic CO(2) compensation points of these two intermediate species at 29 degrees C were much lower than in C(3) plants and ranged from 18 to 22 microliters per liter. Although A. ficoides and A. tenella exhibited similar intermediacy in small ghe, Cyrillic, the apparent photorespiratory component of O(2) inhibition in A. ficoides is lower than in A. tenella. The small ghe, Cyrillic progressively decreases from 35 microliters per liter at lowest light intensity to 18 microliters per liter at highest light intensity in A. tenella. It was, however, constant in A. ficoides at 20 to 25 microliters per liter between light intensities measured. The rates of net photosynthesis at 21% O(2) and 29 degrees C by A. ficoides and A. tenella were 25 to 28 milligrams CO(2) per square decimeter per hour which are intermediate between values obtained for Tridax procumbens and A. pungens, C(3) and C(4) species, respectively. The activities of key enzymes of C(4) photosynthesis, phosphoenolpyruvate carboxylase, pyruvate Pi dikinase, NAD malic enzyme, NADP malic enzyme and phosphoenolpyruvate carboxykinase in the two intermediates, A. ficoides and A. tenella are very low or insignificant. Results indicated that the relatively low apparent photorespiratory component in these two species is presumably the basis for the C(3)-C(4) intermediate photosynthesis. PMID- 16664635 TI - Primary Role of the Cytoplasmic Membrane in Thermal Acclimation Evidenced in Nitrate-Starved Cells of the Blue-Green Alga, Anacystis nidulans. AB - The lipid phase transition of the cytoplasmic membrane and the chilling susceptibility were studied in nitrate-starved Anacystis nidulans cells. Nitrate starvation resulted in the disappearance of the thylakoid membrane system, without any effect on chilling susceptibility. The chilling susceptibility of the algal cells depended on the growth temperature. Temperatures of lipid phase transitions of the cytoplasmic membranes were detected by chilling-induced spectral changes in the carotenoid region, in vivo. These values were identical to those of cultures containing intact thylakoid systems. Our results suggest that cytoplasmic membrane plays a determinative role in the thermal acclimation of the alga cells. PMID- 16664636 TI - Chilling Sensitivity in Oryza sativa: The Role of Protein Phosphorylation in Protection against Photoinhibition. AB - The effects of exposure to low temperature on photosynthesis and protein phosphorylation in chilling-sensitive and cold-tolerant plant species were compared. Chilling temperatures resulted in light-dependent loss of photosynthetic electron transport in chilling-sensitive rice (Oryza sativa L.) but not in cold-tolerant barley (Hordeum vulgare L.). Brief exposure to chilling temperatures (0-15 degrees C, 10 min) did not cause a significant difference in photosynthetic O(2) evolution capacity in vivo between rice and barley. Analysis of in vivo chlorophyll fluorescence in chilling-sensitive rice suggests that low temperatures cause an increased reduction of the plastoquinone pool that could result in photoinhibitory damage to the photosystem II reaction centers. Analysis of (32)P incorporation into thylakoid proteins both in vivo and in vitro demonstrated that chilling temperature inhibited protein phosphorylation in rice, but not in barley. Low temperature (77 K) fluorescence analysis of isolated thylakoid membranes indicated that state I to state II transitions occurred in barley, but not in rice subjected to chilling temperatures. These observations suggest that protein phosphorylation may play an important role in protection against photoinhibition caused by exposure to chilling temperatures. PMID- 16664637 TI - Translucent Tissue Defects in Solanum tuberosum L. : II. Alterations in Lipolytic Acyl Hydrolase, Lipoxygenase, and Morphology of Mitochondria and Amyloplasts. AB - We analyzed a physiological defect that involved translucent-like tissue which occurred randomly in potato tubers (Solanum tuberosum L., cv Kennebec) after 8 months of storage. The translucent areas had reduced lipoxygenase (0.73-fold) and lipolytic acyl hydrolase (0.27-fold) activities. The effect(s) of these reduced enzyme activities in vivo is uncertain, but they may have influenced composition, turnover and permeability of membranes because potato lipid is primarily membranous in nature. Electron micrographs of the translucent tissue revealed a discernible decrease in the number of starch granules compared to normal/healthy tissue. A few remaining amyloplasts, which still contained starch granules, possessed large electrondense areas (stroma) within the organelle. Mitochondria in translucent tissue appeared to be present in increased numbers, were aggregated, had fewer but swollen cristae, and, morphologically, were of irregular size and shape suggestive of division. The result of this tuber defect appeared, in part, to be an exaggerated or accelerated form of senescence. PMID- 16664638 TI - Isolation and Characterization of a New Minor Chlorophyll a/b-Protein Complex (CP24) from Spinach. AB - We have identified a new minor chlorophyll a/b-protein complex in the thylakoid membranes of spinach (Spinacia oleracea L.), which migrates as a green band below CPII on mildly denaturing polyacrylamide gels. This complex, designated CP24, was isolated from octyl glucoside/sodium dodecyl sulfate solubilized spinach grana membrane fractions by preparative gel electrophoresis and has been characterized as to its spectral properties and polypeptide composition. CP24 has a room temperature absorption maximum at 668 nanometers, a chlorophyll a/b ratio between 0.8 and 1.2, and contains three or four polypeptides between 20 and 23 kilodaltons. CP24 was also identified in grana membrane preparations from peas (Pisum sativum) and barley (Hordeum vulgare). We postulate that CP24 functions as a linker component in photosystem II, acting to orient the photosystem II light harvesting components to ensure efficient energy transfer to the reaction center. PMID- 16664639 TI - Regulation of Corn Leaf Nitrate Reductase : I. Immunochemical Methods for Analysis of the Enzyme's Protein Component. AB - NADH:nitrate reductase was extracted from corn leaves (Zea mays L. W64A x W182E) and purified on blue Sepharose. After the nitrate reductase was further purified by polyacrylamide gel electrophoresis, it was used to immunize mice and a rabbit. Western blots of crude leaf extracts were used to demonstrate monospecificity of the mouse ascitic fluids and the rabbit antiserum. The electrophoretic properties of purified corn and squash NADH:nitrate reductases in both native and denatured states were shown to be similar using western blotting with mouse ascitic fluid. The corn leaf enzyme has a 115,000 polypeptide subunit like that of squash. Western blots could detect 3 to 10 nanograms of nitrate reductase protein. But the detection of proteolytic degradation products using western blotting was inconsistent and remains to be established. An enzyme-linked immunosorbent assay (ELISA) was developed for quantifying nitrate reductase protein in the crude extracts of corn leaves. Using a standard curve based on nitrate reductase activity, the ELISA for corn nitrate reductase could detect 0.5 to 10 nanograms of nitrate reductase protein and was adequately sensitive for quantitative analysis of nitrate reductase in crude extracts of leaves even when activity levels were very low. When the ELISA was used to compare the nitrate reductase protein content of corn roots and leaves, these tissues were estimated to contain 0.24 to 0.5 and 4 to 5 micrograms nitrate reductase protein/gram root and leaf, respectively. PMID- 16664640 TI - Regulation of Corn Leaf Nitrate Reductase : II. Synthesis and Turnover of the Enzyme's Activity and Protein. AB - The appearance and disappearance of NADH:nitrate reductase (NR) in the leaves of corn (Zea mays L. W64A x W182E) were studied using activity assays, an enzyme linked immunosorbent assay (ELISA) and western blotting. N-starved, etiolated corn plants were treated with nutrients containing either 35 millimolar NH(4) nitrate or K-nitrate and immediately thereafter given light. The curve for enhancement of NR activity had three phases: 1 hour lag, 5 hour rapid increase, and steady state. The pattern for NR protein, as measured with the ELISA, also had three phases, but the increase was more rapid and the steady state was established earlier. To differentiate the effects of N nutrition from those of light, N-starved etiolated plants were given N nutrients 4 hours before light. During the dark pretreatment, NR activity and protein increased. When the light was turned on the NR activity and protein increased very rapidly without a lag. Western blots of polyacrylamide gels of native and denatured crude extracts showed that NADH:NR polypeptide was absent prior to treatment with N nutrients, but appeared after nitrate was given in dark or light. A low level of NR activity was found in N-starved, etiolated plants and it was shown by western blotting to be an NR form with a different electrophoretic mobility in nondenaturing gels. Since this minor NR form was not influenced by either nitrate or light, it was designated a constitutive NR. Dark decay of NR activity and protein was also studied. After the plants which had been in light with N nutrients for 24 hours were transferred to dark, the NR activity dropped by 30% within 1 hour, but the NR protein did not decrease. This inactivation of NR was further supported by returning the plants to the light after 1.5 hours of dark and finding the activity restored without change in NR protein. After the initial activity drop, a parallel decrease in NR activity and protein was observed, which was likely due to irreversible degradation by proteolysis. PMID- 16664641 TI - Photocontrol of gibberellin metabolism in situ in maize. AB - Mature maize seeds were labeled with 10 to 100 pg per seed of [(3)H] gibberellins (GA) and [(3)H]GA glucosyl conjugate-like substances by feeding [(3)H]GA(20) of high specific activity (2.3 Curies per millimole) during seed maturation. The dry seeds, which contained 14% [(3)H]GA(20), 7% putative [(3)H]GA(1) and 78% [(3)H]GA glucosyl conjugate-like metabolites, were imbibed and germinated in the dark and under incandescent light. In both light and dark the proportion of [(3)H]GA conjugate-like metabolities declined (relative to that in the mature dry seeds) during imbibition and up to germination at hour 36. This decline was accompanied by increases in the proportions of [(3)H]GA(20) and putative [(3)H]GA(1) thereby indicating hydrolysis, which was greater in the dark than in the light. The proportions of [(3)H]GA conjugate-like substances in light-grown germinants were higher (121 and 141% of dark-grown) at 24 and 48 hour harvests and this statistically significant pattern was sustained up to 120 hours after imbibition. Conversely, the proportions of [(3)H]GA(20) and putative [(3)H]GA(1) were lower in the light-grown seedlings. Thus, during imbibition, hydrolysis (de conjugation) of [(3)H]GA glucosyl conjugate-like substances apparently occurred, and occurred more rapidly in the dark than in the light. Subsequently, during germination the reformation of [(3)H]GA conjugate-like substances was less rapid in the dark than in the light. The observation that dark-imbibed seeds and dark grown seedlings have higher proportions of putative free [(3)H]GAs, relative to [(3)H]GA conjugate-like substances, is consistent with the increased shoot elongation (etiolation) that occurs in dark-grown maize seedlings, and may indicate a homeostatic role for GAs and their conjugates in shoot elongation of maize germinants. PMID- 16664642 TI - Limitations on Leaf Nitrate Reductase Activity during Flowering and Podfill in Soybean. AB - The objective of this study was to identify factors which limit leaf nitrate reductase (NR) activity as decline occurs during flowering and beginning seed development in soybean (Glycine max [L.] Merr. cv Clark). Level of NR enzyme activity, level of reductant, and availability of NO(3) (-) as substrate were evaluated for field-grown soybean from flowering through leaf senescence. Timing of reproductive development was altered within one genotype by (a) exposure of Clark to an artificially short photoperiod to hasten flowering and podfill, and (b) the use of an early flowering isoline. Nitrogen (N) was soil-applied to selected plots at 500 kilograms per hectare as an additional variable. Stem NO(3) (-) concentration and in vivo leaf NR activity were significantly correlated (R(2) = 0.69 with nitrate in the assay medium and 0.74 without nitrate in the medium at P = 0.001) across six combinations of reproductive and soil N treatment. The supply of NO(3) (-) from the root to the leaf tissue was the primary limitation to leaf NR activity during flowering and podfill. Levels of NR enzyme and reductant were not limiting to leaf NR activity during this period. PMID- 16664643 TI - Hormonal Regulation of alpha-Amylase Gene Transcription in Wild Oat (Avena fatua L.) Aleurone Protoplasts. AB - The time of appearance and relative amounts of alpha-amylase mRNA in wild oat (Avena fatua L.) aleurone protoplasts incubated with 1 micromolar gibberellin A(4) (GA(4)) were closely correlated with the amounts of alpha-amylase enzyme secreted by the protoplasts. In the absence of GA(4), or when protoplasts were incubated with 25 micromolar abscisic acid (ABA) together with 1 micromolar GA(4) no alpha-amylase mRNA was detected and only very low levels of alpha-amylase were secreted. Nuclei were isolated in high yields (65-71%) from aleurone protoplasts and in an in vitro transcription system displayed characteristics of a faithful DNA-dependent RNA synthesizing system. The time course of incorporation of [(3)H] UTP suggested that the RNA synthesized was mainly ;run off' transcription and therefore that the transcripts produced in vitro were those being synthesized in the protoplasts at the times when the nuclei were isolated. By hybridizing in vitro synthesized [(32)P]RNA to barley alpha-amylase cDNA and control filters we have estimated that 90 +/- 10 ppm of the transcripts synthesized by nuclei isolated from GA(4) treated protoplasts can be attributed to alpha-amylase sequences and that statistically insignificant amounts of these transcripts are obtained from control and GA(4) plus ABA treatments. The results suggest that GA(4) and ABA influence the transcription of alpha-amylase genes in aleurone protoplasts of wild oat. PMID- 16664644 TI - Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'. AB - The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the ;empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl(2), or by pretreatments up to 20 minutes with p chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H(2)O(2), by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The ;steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates. PMID- 16664645 TI - Temperature response of plasma membranes in tuber-bearing solanum species. AB - Permeability coefficients (Kp) of nonelectroytes were determined at several temperatures for nonacclimated and acclimated plasma membranes of the frost sensitive Solanum tuberosum and the frost resistant Solanum commersonii. In nonacclimated membranes, Kp were equal at 25 degrees C for the two species. Kp decreased with decreased temperature in both species; however, the decrease was much greater in the frost sensitive S. tuberosum than in frost resistant S. commersonii.Kp increased with cold acclimation. After acclimation the temperature sensitivity of Kp or activation energy (Ea) for S. tuberosum was similar to that of S. commersonii; however, Kp for S. tuberosum were lower than those for S. commersonii at all temperatures.The differences in Kp and activation energy indicate plasma membrane differences between the species before acclimation. After acclimation there was still a difference between the plasma membranes of the two species as well as a difference between the nonacclimated and acclimated membranes of the same species. PMID- 16664646 TI - Inactivation of serine:glyoxylate and glutamate:glyoxylate aminotransferases from tobacco leaves by glyoxylate in the presence of ammonium ion. AB - Serine:glyoxylate and glutamate:glyoxylate aminotransferases (SGAT and GGAT), which catalyze the formation of glycine from glyoxylate during photorespiration, have been purified >300-fold from tobacco leaf extracts. Incubation with glyoxylate in the absence of amino acid substrate resulted in the time- and concentration-dependent inhibition of the partially purified enzymes. The second order rate constants for glyoxylate inhibition were 1.25 and 0.175 per millimolar per minute for SGAT and GGAT, respectively. The enzymes of highest specific activity were not inhibited by 5 millimolar glyoxylate alone but when 1 millimolar NH(4) (+) was added to 1 millimolar glyoxylate for 10 min in the absence of amino donor, SGAT was irreversibly inhibited more than 50%. GGAT was inhibited 50% in 10 minutes by 15 millimolar NH(4) (+) and 1 millimolar glyoxylate. By itself, NH(4) (+) was a reversible inhibitor; SGAT and GGAT were inhibited 50% at 6 millimolar and 50 millimolar, respectively. The irreversible inhibition occurred only with glyoxylate and NH(4) (+) added together; oxalate, formate, acetaldehyde, pyruvate, hydroxypyruvate, and alpha-ketoglutarate did not inhibit either in the presence or absence of NH(4) (+). Glyoxylate and NH(4) (+) could form a carbinolamine which is a serine analog and might bind irreversibly to the enzymes. Under conditions in vivo in which reassimilation of NH(4) (+) is reduced or blocked, the activity of SGAT may be inhibited and if glyoxylate is available, as in leaf peroxisomes, irreversible inhibition may occur. PMID- 16664647 TI - A comparison of the submergence response of deepwater and non-deepwater rice. AB - Twelve cultivars of rice (Oryza sativa L.), representing deepwater, short statured, and semidwarf types, were tested for their response to submergence. The magnitude of the response varied between cultivars; however, all cultivars responded to submergence by rapid growth once internodal elongation had started. Three of these cultivars were tested for elongation capacity at four ages. The deepwater rice was capable of rapid internodal elongation in response to submergence at 4 weeks of age. Growth of the short-statured and semidwarf cultivars was not stimulated by submergence until about 10 weeks of age. In air, the internodes of deepwater rice grew slower than did those of the short-statured and semidwarf cultivars. We also investigated the elongation response of stem sections of all 12 cultivars to an atmosphere containing 3% O(2), 6% CO(2), 91% N(2) (all by volume), and 1 microliter per liter ethylene. We found that the response of each of the non-deepwater cultivars was qualitatively and quantitatively similar to that of the deepwater rice. PMID- 16664648 TI - Microsomal flavonoid 3'-monooxygenase from maize seedlings. AB - Identification of flavonoid 3'-monooxygenase establishes another reaction in the biosynthesis of flavonoid compounds in maize (Zea mays L.). The flavonoid 3' hydroxylase was obtained as a microsomal enzyme preparation by buffer extraction of 5 day old maize seedlings and ultracentrifugation. Seedlings were exposed to light 24 hours prior to enzyme extraction. The extraction buffer required the addition of sucrose or glycerin and dithiothreitol to obtain an active hydroxylase that retained its activity on storage at -70 degrees C. Enzymic activity required O(2) and NADPH, was optimum at pH 8.5 and 30 degrees C, and could be inhibited 79% by carbon monoxide. Carbon monoxide inhibition could be reduced to 21% by irradiation of the samples with 450 nanometer light during incubation. Kaempferol, a flavonol; naringenin, a flavanone; and apigenin, a flavone, all served as substrates for the hydroxylase. Treatment of the microsomal enzyme preparation, previously reduced with sodium dithionite, with carbon monoxide gave a 455 nanometer absorption peak which disappeared on oxidation of the preparation with the formation of a 420 nanometer peak. These results suggest a cytochrome P-450 type monooxygenase enzyme. The concentration of cytochrome P-450 was 0.21 nanomoles per milligram protein. Identification of the monooxygenase provides further biochemical information about a biosynthetic sequence for which the genetics have been studied intensely. PMID- 16664649 TI - Photosynthesis, Leaf Anatomy, and Morphology of Progeny from Hybrids between C(3) and C(3)/C(4)Panicum Species. AB - Species in the Laxa group of Panicum have C(3) or C(3)/C(4) photosynthesis based on leaf anatomical and CO(2) exchange characteristics. Hybrids were previously made between C(3)/C(4) and C(3) species in this group (RH Brown et al. 1985 Plant Physiol 77: 653-658). In this paper, CO(2) exchange, morphological, and leaf anatomical characteristics of F(2) or F(5) progeny from colchicine-induced amphiploids of C(3)/C(4) x C(3) hybrids (Panicum milioides Nees ex Trin. [C(3)/C(4)] x Panicum laxum Mez [C(3)] and Panicum spathellosum Doell [C(3)/C(4)] x Panicum boliviense Hack. [C(3)]) were studied.There were no differences found in morphology or physiology between the amphiploids and the F(1) hybrids from which they were produced. In the segregating progeny, CO(2) compensation concentration and photorespiration values typical of C(3), but not of C(3)/C(4) plants, were recovered. Progeny were found from both crosses which possessed O(2) inhibition of apparent photosynthesis typical of the parents, and in the case of the P. milioides x P. laxum cross, leaf anatomy and overall plant morphology typical of the parents were observed in some progeny. The progeny were found to possess recombinations of various traits associated with reduced photorespiration, so that no correlation existed among O(2) inhibition of apparent photosynthesis, CO(2) compensation concentration, and leaf anatomical traits. One plant was especially noteworthy in possessing leaf anatomy typical of C(3)/C(4) plants, but with CO(2) exchange characteristics of C(3) plants. PMID- 16664650 TI - Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern. AB - The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N(6)-(Delta(2)-isopentenyl)adenine-8-(14)C (i(6) Ade-8-(14)C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i(6) Ade-8 (14)C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded. PMID- 16664651 TI - Platanetin: A Potent Natural Uncoupler and Inhibitor of the Exogenous NADH Dehydrogenase in Intact Plant Mitochondria. AB - Platanetin is a 3,5,7,8-tetrahydroxy, 6-isoprenyl flavone isolated from the bud scales of the plane tree (Platanus acerifolia Willd.). Its effects on the oxidative activities of isolated potato and mung bean mitochondria have been studied. The most noticeable effect is the selective inhibitory effect of this compound on the activity of the external NADH dehydrogenase of the inner membrane. A 50% inhibition of the NADH oxidation rate is obtained at a 2 micromolar concentration. This activity is probably due to the flavonoid structure and the high lipophilicity of platanetin associated with the presence of the isoprenyl chain. Another important effect of platanetin is its uncoupling activity on oxidative phosphorylation. The presence of easily dissociable hydroxyl groups and the high lipophilicity of platanetin allow a potent H(+) transfer through the mitochondrial inner membrane. This uncoupling activity is comparable to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Platanetin is therefore the most active natural uncoupler known at the present time (full uncoupling at 2 micromolar with succinate as substrate). At higher concentrations (10 micromolar and more), platanetin can transfer electrons from the mitochondrial inner membrane to O(2); the branching point of this KCN salicylhydroxamic acid insensitive platanetin dependent oxidative pathway is located at the level of flavoproteins, no transfer occurring when succinate is the substrate. The redox properties of platanetin are in accord with such an activity. PMID- 16664652 TI - Induction of pathogenesis-related proteins in tobacco leaves. AB - The synthesis of pathogenesis-related proteins (PR proteins), induced in tobacco leaves in response to infection with tobacco mosaic virus or treatment with salicylic acid, was studied with in vivo pulse-labeling experiments. PR proteins synthesis began after a lag phase of about 8 hours in leaf discs treated with salicylic acid and after more than 18 hours in those infected with tobacco mosaic virus. In both cases, the synthesis declined rapidly after 50 hours. The results show that the accumulation of PR proteins results from de novo synthesis and not from degradation of preexisting precursors and that the induced synthesis is transient like other stress-inducible proteins. The proteins have a half-life of at least 50 hours. The induction of these PR proteins was not inhibited by either 25 micrograms per milliliter of actinomycin D or 200 micrograms per milliliter of alpha-amanitin, which completely inhibited the increase of peroxidase activity in tobacco mosaic virus-infected leaf and the induction of heat shock proteins in tobacco leaf discs. These findings indicate that the induction of PR proteins is not regulated by a transcriptional step but by a translational step. PMID- 16664653 TI - Stability of chloroplastic triazine resistance in rutabaga backcross generations. AB - Triazine resistance originally observed in a weed biotype of birdsrape (Brassica campestris L.) has been transferred through cytoplasmic substitution into rutabaga (Brassica napus ssp. Rapifera [Metzg.] Minsk.) by conventional backcrossing. Photosynthetic function and resistance to triazines were examined in six backcross generations of rutabaga as well as in the original parents. Chloroplast thylakoid membranes were isolated and their sensitivity to atrazine, metribuzin, and diuron assayed by measuring the inhibition of photoreduction of 1,6-dichlorophenol indophenol as well as the alteration of in vitro chlorophyll fluorescence rise characteristics. Both assay methods indicated that triazine resistance persisted in all rutabaga backcross generations, and that it involved triazine binding sites in chloroplasts. There was little resistance to diuron. In vivo chlorophyll fluorescence was also monitored, in the absence of herbicides, as an indicator of the electron transfer properties of the chloroplast photosystem II complex. The results indicated that electron transport from Q(A) to Q(B) was slower (as indicated by a larger intermediate level fluorescence during the transient rise) in the triazine resistant parents as well as in all the rutabaga backcross generations. PMID- 16664654 TI - Effect of thidiazuron, a cytokinin-active urea derivative, in cytokinin-dependent ethylene production systems. AB - Cytokinins are known to stimulate ethylene production in mungbean hypocotyls synergistically with indoleacetic acid (IAA), in mungbean hypocotyls synergistically with Ca(2+), and in wilted wheat leaves. Thidiazuron, a substituted urea compound, mimicked the effect of benzyladenine (BA) in all three systems. In the Ca(2+) + cytokinin system and the IAA + cytokinin systems of mungbean hypocotyls, thiadiazuron was slightly more active than BA at equimolar concentration. In mungbean hypocotyls exogenously applied IAA was rapidly conjugated into IAA asparate, and this conjugation process was effectively inhibited by thidiazuron, as by cytokinins. In the wilted wheat leaves system, 10 micromolar thidiazuron exerted stress ethylene production equal to that exerted by 1 millimolar BA, indicating that thidiazuron is more active than BA by two orders. The structure-activity relationship of thidiazuron and its thiadiazolylurea analogs in stimulating Ca(2+)-dependent ethylene production in mungbean hypocotyls was found to agree well with the structure-activity relationship of these derivatives in promoting the growth of callus tissues. These results indicate that thidiazuron and its derivatives are highly active to mimic the adenine-type cytokinin responses in promoting ethylene production and that the structure-activity relationship in promoting the growth of callus and in promoting ethylene production is similar. PMID- 16664655 TI - Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration. AB - When cells of Anacystis nidulans strain R2 grown under high CO(2) conditions (3%) were transferred to low CO(2) conditions (0.05%), their ability to accumulate inorganic carbon (C(i)) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO(2) adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C(i)-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C(i)-accumulating capability. The incorporation of [(35)S]sulfate into membrane proteins was greatly reduced during low CO(2) adaptation. Radioautograms of the (35)S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C(i) transport by A. nidulans strain R2 and its synthesis under low CO(2) conditions leads to high C(i)-transporting activity. PMID- 16664656 TI - Seed dormancy in red rice : v. Response to azide, hydroxylamine, and cyanide. AB - The activity of NaN(3) (0.5 millimolar), hydroxylamine-HCl (10-18 millimolar), and potassium cyanide (1 millimolar) as dormancy-breaking agents of dehulled red rice (Oryza sativa) is pH-dependent such that medium pH values favoring formation of the uncharged chemical species resulted in the highest germination percentages. There was no promotive effect of pH itself in the range of 3 to 10. The minimum contact times for maximum response (>/=90% germination) to NaN(3), KCN, and NH(2)OH-HCl are 8 hours at pH 4, 24 hours at pH 8, and 72 hours at pH 6 or 7, respectively, for exposure commencing at the start of imbibition. Dehulled seeds, imbibed first in water, show only slightly reduced germination when subsequently transferred to solutions of dormancy-breaking chemicals.Intact seeds remain dormant in the presence of NaN(3), KCN, or NH(2)OH-HCl unless partially dry-afterripened. The pH dependence of these chemicals is reduced in intact, afterripening seeds. PMID- 16664657 TI - Starch phosphorylase inhibitor from sweet potato. AB - A protein, starch phosphorylase inhibitor, was purified from the root of sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had a molecular weight of 250,000 and could be composed of five identical subunits. The isoelectric point of the inhibitor was 4.63. It was a noncompetitive inhibitor toward the sweet potato enzyme with a K(i) value of 1.3 x 10(-6) molar when glucose-1-P was the variable substrate. Because cross-reacting materials of rabbit antiphosphorylase inhibitor of sweet potato were found in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain, the occurrence of this protein seemed universal in higher plants. By an immunofluorescence technique, the inhibitor was located in the amyloplast and cell wall where phosphorylase was also found. This implies that they may interact in vivo, and the inhibitor may play an unknown regulatory role against the plant enzyme. PMID- 16664658 TI - Ethylene-promoted conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in peel of apple at various stages of fruit development. AB - Internal ethylene concentration, ability to convert 1-amino-cyclopropane-1 carboxylic acid (ACC) to ethylene (ethylene-forming enzyme [EFE] activity) and ACC content in the peel of apples (Malus domestica Borkh., cv Golden Delicious) increased only slightly during fruit maturation on the tree. Treatment of immature apples with 100 microliters ethylene per liter for 24 hours increased EFE activity in the peel tissue, but did not induce an increase in ethylene production. This ability of apple peel tissue to respond to ethylene with elevated EFE activity increased exponentially during maturation on the tree. After harvest of mature preclimacteric apples previously treated with aminoethoxyvinyl-glycine, 0.05 microliter per liter ethylene did not immediately cause a rapid increase of development in EFE activity in peel tissue. However, 0.5 microliter per liter ethylene and higher concentrations did. The ethylene concentration for half-maximal promotion of EFE development was estimated to be approximately 0.9 microliter per liter. CO(2) partially inhibited the rapid increase of ethylene-promoted development of EFE activity. It is suggested that ethylene-promoted CO(2) production is involved in the regulation of autocatalytic ethylene production in apples. PMID- 16664659 TI - ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings. AB - Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16 d-old tall and dwarf pea (Pisum sativum L.) seedlings. [(14)C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (;Alaska') and dwarf (;Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue. PMID- 16664660 TI - Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells. AB - Two active calcium (Ca(2+)) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca(2+) transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl(-)-stimulated, NO(3) (-)-inhibited ATPase activity on sucrose density gradients. Ca(2+) transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The K(m) for MgATP and Ca(2+) were 0.1 mm and 21 micromolar, respectively. The predominant Ca(2+) transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca(2+) transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I(50) = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca(2+) dependent kinetics were complex with an apparent K(m) ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca(2+)/H(+) antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca(2+) transport systems are proposed to restore and maintain cytoplasmic Ca(2+) homeostasis under changing cellular and environmental conditions. PMID- 16664661 TI - Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations. AB - Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using (14)C-labeled polyamines were carried out on single petals, at room temperaure (20 degrees C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K(m) values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca(2+), Mg(2+), and K(+) at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca(2+) inhibited and K(+) enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0 degrees C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20 degrees C as well as a 68% inhibition with 20 millimolar NaSCN. PMID- 16664662 TI - Studies on the Mechanism of Regulation of the mRNA Level for a Soybean Storage Protein Subunit by Exogenous l-Methionine. AB - In previous studies (GL Creason et al. 1983 Biochem Biophys Res Commun 117: 658 662; LP Holowach et al. 1984 Plant Physiol 74: 576-583), we have shown that when soybean (Glycine max L. Merrill cv Provar) cotyledons are cultured in medium supplemented with l-methionine, the beta-subunit of 7S protein and beta-mRNA are absent. We have carried out further studies on the mechanism of the methionine action. In one experiment, cotyledons were cultured for 16 days with or without methionine. After 4 days, some cotyledons were transferred from methionine supplemented to basal (no methionine) medium and vice versa. In basal medium, beta-subunit was detected at 4 days whereas in methionine-supplemented medium, no beta-subunit was present. When cotyledons were transferred from basal to methionine-supplemented medium, the beta-subunit increased within a 4 day period and then remained constant (on a per cotyledon basis). This result indicated that methionine was not acting by accelerating the degradation of the beta-subunit. Four days after transfer from supplemented to basal medium cotyledons contained beta-subunit, thus demonstrating that the inhibition was reversible. During this time, the uncombined methionine declined from 7 to 1.5 mumoles methionine per gram fresh weight. When beta-mRNA was measured by in vitro translation, functional beta-mRNA was absent in tissue that was not accumulating beta-subunit. The messenger RNA for the beta-subunit had a half-life of about 1 day in the presence of methionine. Hybridization of cotyledon mRNA with cDNA complementary to beta-mRNA revealed that the 1700 nucleotide beta-mRNA was not present in supplemented cotyledons. Thus, expression of the beta-subunit gene is controlled at the level of transcription, RNA processing, or RNA turnover, rather than at the level of translation. PMID- 16664663 TI - Host-Pathogen Interactions : XXIX. Oligogalacturonides Released from Sodium Polypectate by Endopolygalacturonic Acid Lyase Are Elicitors of Phytoalexins in Soybean. AB - Recent studies have demonstrated that an apparently homogeneous preparation of an alpha-1,4-d-endopolygalacturonic acid lyase (EC 4.2.2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max [L.] Merr. cv Wayne) and that this pectin degrading enzyme released heat-stable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate (KR Davis et al. 1984 Plant Physiol 74: 52-60). The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P-6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of alpha-1,4-d-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-beta-l-5-threohexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) galactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor active fraction contained mostly undeca-alpha-1,4-d-galactosyluronic acid that contained 4,5-unsaturated galactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively. These results confirm and extend previous observations that oligogalacturonides derived from the pectic polysaccharides of plant cell walls can serve as regulatory molecules that induce phytoalexin accumulation in soybean. These results are consistent with the hypothesis that oligogalacturonides play a role in disease resistance in plants. PMID- 16664664 TI - Short-term measurement of carbon isotope fractionation in plants. AB - Combustion-based studies of the carbon-13 content of plants give only an integrated, long-term value for the isotope fractionation associated with photosynthesis. A method is described here which permits determination of this isotope fractionation in 2 to 3 hours. To accomplish this, the plant is enclosed in a glass chamber, and the quantity and isotopic content of the CO(2) remaining in the atmosphere are monitored during photosynthesis. Isotope fractionation studies by this method give results consistent with what is expected from combustion studies of C(3), C(4), and Crassulacean acid metabolism plants. This method will make possible a variety of new studies of environmental and species effects in carbon isotope fractionation. PMID- 16664665 TI - Antheraxanthin, a light harvesting carotenoid found in a chromophyte alga. AB - The pigments of the chromophyte freshwater alga, Chrysophaera magna Belcher were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) to reveal the presence of chlorophylls a and c, beta carotene, fucoxanthin, and antheraxanthin. The presence of antheraxanthin was verified by comparison of TLC R(F) values, HPLC retention times, and absorption features to those of authentic, synthetic antheraxanthin. Antheraxanthin accounted for about 15% of the total carotenoid content of C. magna. The molar ratio of the major carotenoids was antheraxanthin:fucoxanthin:beta-carotene, 1:2.3:3.3. The whole-cell absorption spectrum revealed a broad band between 470 and 520 nanometers which was attributed to fucoxanthin and antheraxanthin in vivo. Upon extraction in hydrocarbon, this broad absorption region was lost. The in vivo fluorescence excitation spectrum for 680 nm emission revealed the energy transfer activities and light harvesting roles of chlorophylls a and c, and fucoxanthin. In addition, an excitation band was resolved at 487 nanometers which could be attributed only to antheraxanthin. Comparison of whole-cell fluorescence excitation spectra of C. magna with the diatom Phaeodactylum tricornutum, which possesses fucoxanthin but not antheraxanthin, supports the assignment of the 487 nm band to antheraxanthin. This is the first report of a photosynthetic light harvesting function of the xanthophyll, antheraxanthin. This carotenoid broadens the absorption cross-section for photosynthesis in C. magna and extends light harvesting into the green portion of the spectrum. PMID- 16664666 TI - Increased Abscisic Acid Biosynthesis during Plant Dehydration Requires Transcription. AB - Excised pea plants were rapidly dehydrated to turgor pressures of 1.5 to 2.0 bars. After a 30 minute lag, abscisic acid (ABA) levels increased approximately 100-fold in the dehydrated plants. Pretreatment of plants with the transcription inhibitors actinomycin D or cordycepin or with an inhibitor of cytoplasmic protein synthesis prior to plant dehydration inhibited the synthesis of ABA. These results suggest that dehydration induced synthesis of ABA requires nuclear gene transcription. PMID- 16664667 TI - Concomitant changes in high temperature tolerance and heat-shock proteins in desert succulents. AB - Raising the day/night air temperatures from 30 degrees C/20 degrees C to 50 degrees C/40 degrees C increases the high temperature tolerated by Agave deserti, Carnegiea gigantea, and Ferocactus acanthodes by 6 degrees C to 8 degrees C; the increase is about half completed in 3 days and fully completed in 10 days. A 25 to 27 kilodalton protein concomitantly accumulates for all three desert succulents upon transfer to 50 degrees C/40 degrees C, while accumulation of other heat "heat-shock" proteins is species specific. Some of the induced proteins are more abundant at 3 days, while others (including the 25-27 kilodalton protein) remain after completion of high temperature acclimation. PMID- 16664668 TI - Measurement of the Enzyme-CO(2)-Mg Form of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase. AB - When the amount of activation of ribulose 1,5-bisphosphate carboxylase has been measured, two forms of the enzyme, not one, are actually determined experimentally. Only the enzyme-activator CO(2)-Mg(2+) form can bind ribulose bisphosphate for reaction with substrate CO(2) or O(2). A method is presented which measures only this catalytically active form by stabilizing it with ribulose bisphosphate just before dilution and assay in Mg(2+)-free reaction medium. PMID- 16664669 TI - Plastid DNA content in a cultured soybean line capable of photoautotrophic growth. AB - The levels of chloroplast DNA in a cultured photoautotrophic soybean (Glycine max [L.] Merr. v Corsoy) cell line were determined by molecular hybridization. The cells were also grown photomixotrophically and heterotrophically as suspension cultures and the level of plastid DNA was found to be constant at approximately 26% of the total cellular DNA in all three growth modes. By comparison, total cellular DNA extracted from plants of the same variety used as the explant source for the cultured cells contained 12.3 to 18.9% (leaves and seeds) and 6.1 to 8.9% (roots) plastid DNA. PMID- 16664670 TI - Adaptation to CO(2) Level and Changes in the Phosphorylation of Thylakoid Proteins during the Cell Cycle of Chlamydomonas reinhardtii. AB - The photosynthetic performance of synchronously grown Chlamydomonas reinhardtii alternated rhythmically during the cell cycle. The activity of the "CO(2) concentrating mechanism" including the ability to accumulate CO(2) internally and the activity of carbonic anhydrase peaked after 6 to 9 hours of light and reached minimum after 6 to 9 hours of dark. Consequently, the apparent photosynthetic affinity to extracellular CO(2) alternated rhythmically. At the end of the dark period the cells behaved as if they were adapted to high CO(2) even though they were continuously aerated with air. Results from experiments in which the light or dark periods were extended bear on the interaction between the internal (cell cycle or biological clock) and the external (light) signal. The observed rhythmical alterations in photosynthetic V(max) may result from changes in PSII activity. The latter may be partly explained by the capacity for phosphorylation of thylakoid proteins, which reached maximum after 9 hours of light and decreased toward the dark period. PMID- 16664671 TI - Sugar utilization by developing wild type and shrunken-2 maize kernels. AB - To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose. PMID- 16664672 TI - Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture. AB - The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2',4'-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4 dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site. PMID- 16664673 TI - Photocontrol of chloroplast and mitochondrial polypeptide levels in euglena. AB - Two dimensional polyacrylamide gel electrophoresis resolved protein from intact chloroplasts of wild type Euglena gracilis Klebs var. bacillaris Cori into 185 polypeptides of which 55 were localized on the whole cell polypeptide map. Of these chloroplast polypeptides, the relative amounts of 49 increased, the relative amounts of two decreased, and the relative amounts of four polypeptides were unaltered by exposure of dark grown resting cells to light for 72 hours. Proteins from intact purified mitochondria obtained from a bleached mutant (W(10)BSmL) lacking plastids were resolved into 193 polypeptides of which 44 were localized on the whole cell polypeptide map from wild type cells. Of these mitochondrial polypeptides, the relative amount of one increased, the relative amounts of 12 were unaltered, and the relative amounts of 31 decreased after exposure of the dark grown resting cells to light. Since it is known that the development of the chloroplast in Euglena occurs without a net increase in total cellular protein and without a change in the size of the cellular amino acid pools, the degradation of mitochondrial polypeptides represents a major source of amino acids for the synthesis of chloroplast polypeptides. PMID- 16664674 TI - Separation of alcohol-soluble proteins (zeins) from maize into three fractions by differential solubility. AB - The prolamin of maize (Zea mays L.), zein, was extracted from endosperm meal with 60% (v/v) 2-propanol/1% (v/v) 2-mercaptoethanol either directly or subsequent to extraction with 90% (v/v) 2-propanol. The zein extracted with 90% 2-propanol was essentially made up of 20 to 24 kilodalton polypeptides (alpha-zein) while that extractable with 60% 2-propanol/1% 2-mercaptoethanol contained, in addition to alpha-zein, 17 to 18 kilodalton methionine-rich polypeptides and a 27 kilodalton proline-rich polypeptide. While zein was separated into three fractions by differential solubility in 90% 2-propanol and 30% 2-propanol/30 millimolar sodium acetate (pH 6) using two different fractionation protocols. Each of the three solubility fractions (SF1, SF2, and SF3) had a unique polypeptide composition. Based on results obtained from two inbreds, K55 and W64A, the SF1 constituted 75 to 80% of the total zein and included as major components 20 to 24 kilodalton polypeptides and a minor 10 kilodalton polypeptide. The SF2 made up 10 to 15% of the total zein and included exclusively 17 to 18 kD methionine-rich polypeptides. A 27 kilodalton proline-rich component constituted the SF3 and contributed 5 to 10% to total zein. PMID- 16664675 TI - Separation and Characterization of Two Endopeptidases from Cotyledons of Germinating Vigna mungo Seeds. AB - Two major endopeptidases were present in cotyledons of germinating Vigna mungo seeds, as detected by the zymogram after polyacrylamide gel electrophoresis. They were not detectable in cotyledons of dry seeds, but their intensities on the zymogram increased during germination. During incubation of detached cotyledons, however, the activities showed only a slight increase for 5 days. These two endopeptidases could be separated by Sephacryl S-200 column chromatography. One of them was found to be a serine-endopeptidase as judged by phenylmethylsulfonylfluoride and diisopropyl fluorophosphate inhibition. The other was a sulfhydryl-endopeptidase because of its dependency on 2 mercaptoethanol and inhibition by leupeptin, chymostatin, and antipain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicatd that the two endopeptidases digested the Vigna mungo seed globulin subunits at different rates. The serine enzyme digested the 56 kilodalton subunit at first, but the sulfhydryl enzyme digested the 54 kilodalton peptide more efficiently than the 56 kilodalton peptide. The pattern of digestion of globulin by the combination of the serine- and sulfhydryl-endopeptidases was similar to that using crude enzyme extracts. PMID- 16664676 TI - Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L. AB - Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6 benzylaminopurine and abscisic acid affected (14)C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6 benzylaminopurine and for abscisic acid within 10 to 12 minutes of application. PMID- 16664677 TI - Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods. AB - The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (+/-1 degrees C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45 degrees C, the transition was at 7 degrees C while for plants from the same clone, grown at 20 degrees C, it was at -2 degrees C. For mung bean and tomato the transition was between 9 and 12 degrees C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids. PMID- 16664678 TI - Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate. AB - The effect of nitrate on N(2) fixation and the assimilation of fixed N(2) in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, (15)N(2) fixation and relative abundance of ureides ([ureides/(ureides + nitrate + alpha-amino nitrogen)] x 100) in xylem exudate, gave similar results for the effect of nitrate on N(2) fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N(2) fixation precedes the loss of the potential of bacteroids to fix N(2). This in turn precedes the loss of the potential of nodules to assimilate fixed N(2). PMID- 16664679 TI - Salt sensitivity in wheat : a case for specific ion toxicity. AB - Two selected lines of bread wheat, Triticum aestivum L., differing in their relative salt resistance, were grown in isosmotic solutions of different ionic compositions to investigate sensitivity to specific ions. Growth rates and ion accumulation were determined. The salt composition of the various solutions had little effect on the growth of the salt-resistant line, but significantly affected that of the salt-sensitive line. Specifically, solutions containing high Na(+) concentrations were more toxic than those containing high Cl(-) concentrations or high concentrations of nutrient ions. There were few differences in ion accumulation between lines in a given treatment, although the sensitive line tended to accumulate more Na(+) than the tolerant line in the salt treatments with high Na(+) concentrations. The overall results provide evidence that there is a definite specific ion effect which is related to salt sensitivity in wheat. It is suggested that superior compartmentation of toxic ions, principally Na(+), may be a mechanism of salt resistance in this case. PMID- 16664680 TI - Light and CO(2) Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves. AB - The requirements for activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) were investigated in leaves of Arabidopsis wild type and a mutant incapable of light activating rubisco in vivo. Upon illumination with saturating light intensities, the activation state of rubisco increased 2-fold in the wild-type and decreased in the mutant. Activation of fructose 1,6-bisphosphate phosphatase was unaffected by the mutation. Under low light, rubisco deactivated in both the wild-type and the mutant. Deactivation of rubisco in the mutant under high and low light led to the accumulation of high concentrations of ribulose 1,5-bisphosphate. Inhibiting photosynthesis with methyl viologen prevented ribulose 1,5-bisphosphate accumulation but was ineffective in restoring rubisco activation to the mutant. Net photosynthesis and the rubisco activation level were closely correlated and saturated at a lower light intensity in the mutant than in wild-type. At CO(2) concentrations between 100 and 2000 microliters per liter, the activation state was a function of the CO(2) concentration in the dark but was independent of CO(2) concentration in the light. High CO(2) concentration (1%) suppressed activation in the wild-type and deactivation in the mutant. These results support the concept that rubisco activation in vivo is not a spontaneous process but is catalyzed by a specific protein. The absence of this protein, rubisco activase, is responsible for the altered characteristics of rubisco activation in the mutant. PMID- 16664681 TI - Incorporation of proline and aromatic amino acids into cell walls of maize coleoptiles. AB - Sections excised from maize coleoptiles incorporated radioactivity from proline, tyrosine, and phenylalanine into structural components of the cell wall. Only about 2% of radioactivity from proline taken up by sections was incorporated into cell wall; about 24% of that incorporated was in hydroxyproline and the rest remained in proline. In contrast, as much as 40% of the radioactivity from phenylalanine and 30% from tyrosine was incorporated into cell wall material. Most of this radioactivity was in saponifiable ferulic acid. Small amounts of p coumaric and diferulic acid were found, but only a small fraction of the hemicellulose can possibly be immobilized directly through cross-linking of diferulic esters. Substantial amounts of radioactivity from aromatic amino acids remained insoluble after strong alkali extractions of wall material, and a large fraction of polysaccharide was solubilized by dilute alkali following oxidation of phenolics by acidic NaClO(2). Hence, hemicellulosic material in the cell walls of maize coleoptiles may be organized and cross-linked primarily through alkali resistant etherified aromatics. PMID- 16664682 TI - Blooms of cyanobacteria on the potomac river. AB - Blooms of cyanobacteria have appeared on the Potomac River near Washington, DC in years of drought and low river volume. The location of the bloom may be related to tidal activity. In 1983, the bloom of Microcystis aeruginosa used ammonia as its nitrogen source and contained low levels of toxic peptides. Cells collected from this bloom proved to be homogeneous and were an excellent source material for the isolation of proteins involved in photosynthesis. PMID- 16664683 TI - Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone. AB - The effect of gibberellic acid and Ca(2+) on the accumulation of alpha-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) alpha-amylases. There is no significant hybridization between the two alpha-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI alpha-amylases. It is shown that although the synthesis of the high pI alpha-amylase proteins depends on the presence of Ca(2+) in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca(2+). The accumulation of low pI alpha-amylase mRNA is also not affected by the presence or absence of Ca(2+) in the incubation medium. These results establish gibberellic acid, not Ca(2+), as the principal regulator of alpha-amylase mRNA accumulation in barley aleurone, while Ca(2+) controls high pI alpha-amylase synthesis at a later step in the biosynthetic pathway. PMID- 16664684 TI - Carbon dioxide and nitrite photoassimilatory processes do not intercompete for reducing equivalents in spinach and soybean leaf chloroplasts. AB - Previously, C Baysdorfer and JM Robinson (1985 Plant Physiol 77: 318-320) demonstrated that, in a reconstituted spinach chloroplast system, NADP photoreduction functioning at most maximal rate and reductant demand, was the successful competitor with NO(2) (-) photoreduction for reduced ferredoxin. This resulted in a repression of NO(2) (-) reduction until all NADP available had been almost totally reduced. Further experiments, employing isolated, intact spinach leaf plastids and soybean leaf mesophyll cells, were conducted to examine competition for reductant between CO(2) and NO(2) (-) photoassimilation, in situ. In isolated, intact plastid preparations, regardless of whether the demand for reductant by CO(2) photoassimilation was high (5 millimolar ;CO(2)') with rates of CO(2) fixation in the range 40 to 90 micromoles CO(2) fixed per hour per milligram chlorophyll, low (0.5 millimolar ;CO(2)') with rates in the range 5 to 8 micromoles CO(2) per hour per milligram chlorophyll, or zero (no ;CO(2)'), NO(2) (-) photoreduction displayed equal rates in the range of 8 to 22 micromoles per hour per milligram chlorophyll. In the absence of ;CO(2)', but in the presence of saturating white light, 3-phosphoglycerate photoreduction at rates of 82 to 127 micromoles per hour per milligram chlorophyll did not repress, and occasionally stimulated concomitant rates of NO(2) (-) reduction which ranged from 23.4 to 38.5. Conversely, in plastid preparations, NO(2) (-) at levels of 50 to 100 micromolar, stimulated plastid CO(2) fixation when ;CO(2)' was saturating with respect to carboxylation. Further, levels of NO(2) (-) in the range 250 to 2500 micromolar, stimulated soybean leaf mesophyll cell net CO(2) fixation as much as 1.5-fold if ;CO(2)' was saturating with respect to CO(2) fixation. It appeared likely that, in high light in vivo, CO(2) and NO(2) (-) photoassimilatory processes are not forced to intercompete for reduced ferredoxin in the intact chloroplast. PMID- 16664685 TI - Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae : Physical and Immunological Characterizations. AB - Distinctive properties are identified in the molecular structure of ribulose, 1,5 bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common. PMID- 16664686 TI - Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers. AB - Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA(3)) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA(3). In contrast, GA(3) is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA(3) is similar to that observed for alpha-amylase. Treating aleurone layers with both GA(3) and abscisic acid prevents all the GA(3) effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA(3) response, it is concluded that the proteases represent the GA(3)-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins. PMID- 16664687 TI - Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica. AB - Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH(4) (+) was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N(2) fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO(2) fixation, and NH(3) release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate. PMID- 16664688 TI - The C-S Lyases of Higher Plants : Isolation and Properties of Homogeneous Cystine Lyase from Broccoli (Brassica oleracea var botrytis) Buds. AB - Cystine lyase degrades l-cystine by a beta-elimination to form cysteine persulfide, pyruvate, and ammonia. This enzyme is common in Brassica sp. and has been purified to homogeneity from extracts of broccoli (Brassica oleracea var botrytis) buds. Two isozymes were separated on DEAE-Fractogel columns and the first peak, cystine lyase I further purified to homogeneity. The purified enzyme had a narrow range of substrate specificity with l-cystine and S-alkyl-l-cysteine sulfoxides being the primary substrates. The K(m) for l-cystine was 1.9 millimolar and for S-ethyl-l-cysteine sulfoxide was 15.6 millimolar, suggesting that l-cystine would be preferred in vivo. Using gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the holoenzyme was estimated as 152,000 composed of subunits of approximately 49,000. This strongly suggests the native enzyme is a trimer. The presence of carbohydrate in the native enzyme was detected at the level of 5.8% on a weight basis. Except for the ability to utilize l-cystine as a substrate there are many similarities between cystine lyase I and the alliin lyase of onion (Allium cepa). PMID- 16664689 TI - Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase. AB - The exchange properties of the activator CO(2) of spinach ribulose-1,5 bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5 bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO(2) on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate trapped CO(2). The exchange kinetics of the activator CO(2) were monitored by activation of the enzyme to steady state in the presence of (12)CO(2), followed by addition of (14)CO(2) and determination of the amount of labeled CO(2) trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K(obs)) for exchange with both the purified enzyme (0.45 min(-1)) and in illuminated chloroplasts (0.18 min(-1)) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO(2) was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation. PMID- 16664690 TI - On the Relationships of Net CO(2) Assimilation and Leaf Expansion to Vegetative Growth in Lycospersicum esculentum, var Jubilee. AB - Relationships between net plant CO(2) exchange rate (CER) and canopy development were examined in ;jubilee' tomato over the initial 4 weeks of vegetative growth. A comparison was made between two plant groups that were alternatively exposed to 200 or 800 microeinsteins per square meter per second midday irradiation to establish a differential in net CER. Plants exposed to higher irradiation demonstrated a 2- to 4-fold greater net photosynthetic rate per leaf area and 100% average higher net CO(2) assimilation rate/plant. day. However, leaf-stem growth differed by <50% suggesting a poor relationship to CER. Leaf area growth rate (LAGR) of individual leaves appeared closely related to CER during initial leaf expansion but a greater function of order of emergence in successive leaf growth. LAGR on a per plant basis increased linearly with leaf dry weight but appeared more limited by factors determining maximum leaf enlargement and rate of new leaf development. Net CO(2) assimilation/leaf area and leaf starch consistently declined with time while net CO(2) assimilation plant/day approached a constant rate following 2 to 3 weeks growth. Composite results suggested a simple relationship for sucessive growth where accumulated leaf carbohydrate in excess of 200 milligrams/plant.day could be expected to be partitioned to other plant segments. PMID- 16664691 TI - Fructose 1,6-Bisphosphatase Form B from Synechococcus leopoliensis Hydrolyzes both Fructose and Sedoheptulose Bisphosphate. AB - The substrate specificity of purified fructose bisphosphatase form B from Synechococcus leopoliensis (EC 3.1.3.11; cf. K-P Gerbling, M Steup, E Latzko 1985 Eur J Biochem 147: 207-215) has been investigated. Of the phosphate esters tested only fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were hydrolyzed by the enzyme. Both sugar bisphosphates were cleaved at the carbon 1-ester. Fructose- and sedoheptulose bisphosphate stabilized the activated (i.e. tetrameric) state of the enzyme and prevented a slow inactivation that is observed in the absence of sugar bisphosphates. With the activated enzyme, kinetic constants (half-saturating substrate concentrations, maximal reaction velocity, and the catalytical constant) were similar for both fructose- and sedoheptulose bisphosphate. The data suggest that fructose bisphosphatase form B from Synechococcus leopoliensis can catalyze both bisphosphatase reactions within the reductive pentose phosphate cycle. PMID- 16664692 TI - Phycomyces: fine structure analysis of the growing zone. AB - Fine structure analysis of the stage IVb Phycomyces sporangiophore growing zone (GZ) was performed during steady-state growth using a computer-video digitizer and recorder. By simultaneously measuring the trajectory of two independent particles above and within the GZ, we have confirmed the previous findings of R. Cohen and M. Delbruck (1958 J Cell Comp Physiol 52: 361-388) that the GZ is not uniform. We have been unable to confirm their findings that counterclockwise rotation exists in a mature sporangiophore. The rates of rotation and elongation change independently as a function of position in the GZ. This change is not linear as would be expected if the GZ were uniform. The importance of this finding is discussed in terms of the fibril reorientation model. PMID- 16664693 TI - Kinetics of Ca/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L. AB - Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were used to study the kinetics of a Ca(2+)/H(+) antiport across this membrane. Ca(2+)-dependent H(+) fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. DeltapH-dependent Ca(2+) influx was measured radiometrically. Both H(+) efflux and Ca(2+) influx displayed saturation kinetics and an identical dependence on external calcium with apparent K(m) values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg(2+) but was inhibited by La(3+) > Mn(2+) > Cd(2+). The apparent K(m) for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested. PMID- 16664694 TI - Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: I. Isolation and Characterization of Pigment-Protein Complexes. AB - Three pigment-protein complexes were isolated from the marine diatom Phaeodactylum tricornutum (Bohlin) by treatment of thylakoid membrane fragments with 1% Triton X-100 at 4 degrees C followed by centrifugation on sucrose density gradients. The major complex contains chlorophyll a, c(1), c(2), and the carotenoid fucoxanthin (chlorophyll a: c(1): c(2): fucoxanthin = 1.0: 0.09: 0.28: 2.22) bound to an apoprotein doublet of 16.4 and 16.9 kilodaltons. This complex accounts for >70% of the total pigment and 20 to 40% of the protein in the thylakoid membranes. Efficient coupling of chlorophyll c and fucoxanthin absorption to chlorophyll a fluorescence supports a light-harvesting function for the complex. A minor light-harvesting complex containing chlorophyll a, c(1), and c(2) but no fucoxanthin (chlorophyll a: c(1): c(2) = 1.0: 0.23: 0.26) was also isolated at Triton: chlorophyll a ratios between 20 and 40. These pigments are bound to a similar molecular weight apoprotein doublet. The third complex isolated was the P700-chlorophyll a protein, the reaction center of photosystem I, which showed characteristics similar to those isolated from other plant sources. The yield of the chlorophyll a/c-fucoxanthin complex was shown to respond strongly to changes in light intensity during growth, accounting for most of the changes in cellular pigmentation. PMID- 16664695 TI - Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: II. Distribution of Excitation Energy between the Photosystems. AB - The distribution of excitation energy between photosystems I and II (PSI and PSII) was investigated in the marine diatom Phaeodactylum tricornutum (Bohlin) using light-induced changes in fluorescence yield and rate of modulated O(2) evolution. The intensity dependence of the fast fluorescence rise in dark adapted cells (+/-DCMU) suggests that light absorbed by the major antenna complex was not delivered preferentially to PSII but is more equally distributed between the photosystems. Reversible, slow fluorescence yield changes measured in the absence of DCMU were correlated with decreased initial fluorescence and rate constants for PSII photochemistry, increased variable fluorescence, alteration of the fluorescence excitation and emission spectra, and could be effected by either 510 nm (PSII) or 704 nm (PSI) light. Slow, reversible fluorescence yield changes were also observed in the presence of DCMU, but were characterized by a loss of both initial and variable fluorescence and could not be induced by PSI light. The absence of slow changes in the yield of fluorescence and rate of modulated O(2) evolution, following addition or removal of PSI background light to modulated PSII excitation, does not support regulation of excitation energy density in PSI at the expense of PSII. The results suggest that adjustments are made at the level of excitation energy transfer to the PSII reaction center which prevent prolonged loss of photosynthetic capacity. Energy distribution is regulated by ionic distributions independently of the plastoquinone pool redox state. These differences in light-harvesting function are probably a response to the aquatic light field and may account for the success of diatoms in low and variable light environments. PMID- 16664696 TI - Seasonal Fluctuations of Lectins in Barks of Elderberry (Sambucus nigra) and Black Locust (Robinia pseudoacacia). AB - Elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia) agglutinins, which are abundantly present in the bark of both species, display seasonal fluctuations with regard to their content in this tissue. These seasonal changes result apparently from a circa-annual rhythm of lectin accumulation and depletion during autumn and spring, respectively. Because the bark of trees can be considered as a type of vegetative storage tissue, the results suggest that bark lectins behave as typical storage proteins. PMID- 16664697 TI - Diurnal changes in volume and solute transport coefficients of phaseolus roots. AB - Volume (J(v)) and solute (J(s)) fluxes through Phaseolus root systems were observed over a 24-hour period. The volume flux was varied in a pressure chamber by altering the hydrostatic pressure in 10 steps, from 0 to 0.41 megapascals. All root systems showed strong diurnal peaks in volume flux. The five transport coefficients (sigma, omega, J(s) (*), L(p), and pi(*)) were estimated from a nonlinear least squares algorithm. Analysis of the data revealed that all the coefficients exhibited a diurnal rhythm. When the total differential of the volume flux was considered it was possible to show that the diurnal changes in volume flux were due to a complex interaction between the diurnally shifting coefficients with the role of each highly dependent on the level of volume flux. At low volume fluxes, omega, J(s) (*), and pi(*) accounted for nearly all the diurnal change in volume flux. At high volume fluxes, however, the major influence shifted to L(p) and pi(*), while omega and J(s) (*) became relatively unimportant. Thus, pi(*) was the only coefficient of interest across the entire range of J(v) and appeared to be the single most important one in determining the diurnal rhythm of J(v) under conditions of a constant applied pressure. PMID- 16664698 TI - Effect of phaseolotoxin on the synthesis of arginine and protein. AB - Mesophyll cells in discs cut from primary leaves of Phaseolus vulgaris L. were exposed to a concentration of phaseolotoxin that inhibited ornithine carbamoyltransferase (OCTase) measured in an extract of the tissue. This treatment also blocked incorporation of exogenous [(14)C] ornithine into protein arginine of the mesophyll cells. By contrast more than 80% of the [(14)C]ornithine supplied to untreated tissue was incorporated into protein arginine in 565 minutes. Protein synthesis in mesophyll cells was unaffected by phaseolotoxin because treated tissue continued to incorporate [(14)C]leucine into protein at the same rate as the untreated control. The phaseolotoxin-treated tissue should therefore remain metabolically competent and this prediction was reinforced by the finding that the rate of photosynthetic O(2) evolution per unit chlorophyll was similar for tissue from the phaseolotoxin-induced chlorosis and from green healthy tissue. Phaseolotoxin also blocked OCTase but not protein synthesis in exponentially growing cell suspension cultures. Phaseolotoxin rapidly inhibited growth of Escherichia coli and this effect was rapidly reversed by arginine. Thus, the toxic effects of phaseolotoxin may be attributed to the inhibition of OCTase which, in turn, blocks arginine synthesis. Protein accumulation is blocked as a consequence, but protein synthesis is unaffected. Chlorosis is due to reduced chlorophyll synthesis and this is presumably a consequence of the lower protein level in affected tissue. PMID- 16664699 TI - The Effect of Abscisic Acid on the Freezing Tolerance of Callus Cultures of Lotus corniculatus L. AB - The effects of growth temperature (2 degrees C and 24 degrees C), abscisic acid (ABA) concentration, duration of exposure to ABA, and light were assessed for their ability to induce acclimation to freezing temperatures in callus cultures of Lotus corniculatus L. cv Leo, a perennial forage legume. The maximal expression of freezing tolerance was achieved on B(5) media containing 10(-5) molar ABA, at 24 degrees C for 7 or 14 days. Under these culture conditions, the freezing tolerance of the callus approximated that observed in field grown plants. In contrast, low temperatures (2 degrees C) induced only a limited degree of freezing tolerance in these cultures. Viability was assessed by tetrazolium reduction and by regrowth of the callus. The two assays often differed in their estimates of absolute freezing tolerance. Regression analysis of the temperature profile suggested that there may be two or more distinct populations of cells differing in freezing tolerance, which may have contributed to the variability between viability assays. PMID- 16664700 TI - Structural Analysis of Secreted Root Slime from Maize (Zea mays L.). AB - Secreted slime isolated from the incubation medium of Zea mays roots maintained axenically contains fucose, arabinose, xylose, galactose, and glucose as the major monosaccharides. The slime preparation contains low levels (3% weight/weight [w/w]) of uronic acids. Methylation analysis reveals an extraordinarily diverse range of glycosyl residues. The fucosyl residues are primarily terminal (60%) and 3-linked (33%) with a relatively small proportion being 2-linked (6%). The methylation data are consistent with, but not proof of, the presence of a range of polymers including arabinogalactan-proteins (AGPs), xyloglucans, arabinoxylans, and glucans in the slime. The specific binding of the beta-glucosyl Yariv reagent, a dye which binds and precipitates AGPs, to the slime preparation and to the outer periclinal epidermal cell wall surface in root sections, is further evidence for the presence of AGPs. Low levels of phenolic acids (approximately 0.17% w/w), in particular trans-ferulic acid, and protein (approximately 6% w/w) were also detected. PMID- 16664701 TI - Diurnal Phototropism in Solar Tracking Leaves of Lavatera cretica. AB - On a clear day, leaf laminas of Lavatera cretica tracked the solar position throughout the day. The laminar azimuth did not diverge from the solar azimuth by more than 12 degrees from sunrise to sunset. Tracking of the solar elevation started 1 to 2 hours after sunrise and ceased 1 to 2 hours before sunset. On an overcast day, the laminas reoriented horizontally. After sunset, following a clear day, the laminas performed a nocturnal reorientation, with three well defined phases. During the initial phase the laminas relaxed their strained sunset-facing orientation to one perpendicular to their petioles. This equilibrium configuration was maintained throughout the following phase, which was apparently concerned with time-measuring. During the final phase, the laminas reoriented, before sunrise, to a position facing the direction of the anticipated sunrise. This directional information is phototropic and was retained for 3 to 4 diurnal cycles, probably in the pulvinus itself, which is the site of the response. Laminas of plants transferred from sunlight either to darkness, or to a simulated natural photoperiod under overhead illumination, were facing the originally anticipated direction of sunrise at the time of each of the three to four subsequent sunrises (after which they reverted to the dark orientation in darkness, or to the horizontal one with overhead illumination). Cotyledonary laminas required directional information for the nocturnal reorientation during 3 or 4 cycles of simulated sunrise to sunset transitions. PMID- 16664702 TI - Inhibition of Adenosine Triphosphatase Activity from a Plasma Membrane Fraction of Acer pseudoplatanus Cells by 2,2,2-Trichloroethyl 3,4-Dichlorocarbanilate. AB - 2,2,2-Trichloroethyl 3,4-dichlorocarbanilate (SW26) is toxic for Acer pseudoplatanus cell cultures. It inhibited the cellular proton extrusion and depolarized the plasmalemma. In vitro, it inhibited the plasma membrane ATPase. SW 26 was also inhibitory to membrane ATPases of other origins-plant (maize shoot), fungus (Schizosaccharomyces pombe), and animal (dog kidney)-with about the same efficiency (7.5 micromolar < I(50) < 22 micromolar). It did not inhibit the oligomycin-sensitive ATPase from purified plant mitochondria, nor molybdate sensitive soluble phosphatases. SW26 was more specific for plasma membrane ATPases than diethylstilbestrol or vanadate. A Lineweaver-Burk plot analysis showed that inhibition kinetics were purely noncompetitive (K(i) = 14.7 micromolar) below 20 micromolar. Above this concentration, the inhibition pattern was not consistent with Michaelis-Menten kinetics, and a Hill plot representation revealed a positive cooperativity. PMID- 16664703 TI - Organ-Specific Arabinogalactan-Proteins of Lycopersicon peruvianum (Mill) Demonstrated by Crossed Electrophoresis. AB - Extracts of style, petal, leaf, petiole, stem, and callus derived from stems of wild tomato (Lycopersicon peruvianum) contain characteristic sets of arabinogalactan-proteins. This is demonstrated by crossed electrophoresis in which Yariv reagent, which specifically binds to and precipitates arabinogalactan proteins, is incorporated into the second gel. PMID- 16664704 TI - Effects of Dimethyl Sulfoxide and pH on Indoleacetic Acid-Induced Lateral Root Formation in the Radish Seedling Root. AB - Segments (2.5 cm) cut from 3-day-old seedling roots of radish (Raphanus sativus L. ;Scarlet Globe') were cultured in medium with or without indoleacetic acid (IAA). Lateral root primordia frequency, determined for the central centimeter of segments, was dependent on IAA concentration and on conditions affecting IAA uptake. Dimethyl sulfoxide treatment, or a relatively low medium pH, greatly enhanced the response to exogenous IAA. It was concluded that a permeation barrier exists between the external medium and the hormone responsive sites within the radish seedling root. PMID- 16664705 TI - Regulation of the NAD Malic Enzyme from Crassula. AB - Using size exclusion chromatography, the nicotinamide adenine dinucleotide malic enzyme purified to near homogeneity from leaves of Crassula argentea was found to exist in at least three aggregational states (dimer, tetramer, and octamer). These forms differ in their apparent kinetic characteristics in initial rate assays, but all display similar characteristics at the steady state. The presence of 50 millimolar malate during chromatography causes a shift in favor of the smaller forms with the tetramer predominating. The native enzyme, when diluted 1/1000 and incubated 18 hours in buffer of high ionic strength, changes its steady state kinetic parameters to ones which indicate a low activity and low affinity for malate. When 50 millimolar malate or 50 micromolar coenzyme A are present the loss of activity and increase in K(m) is reduced. When both malate and coenzyme A are present the effects in minimizing the change in kinetic characteristics are additive. PMID- 16664706 TI - Growth Response of a Succulent Plant, Agave vilmoriniana, to Elevated CO(2). AB - Large (about 200 grams dry weight) and small (about 5 grams dry weight) specimens of the leaf succulent Agave vilmoriniana Berger were grown outdoors at Phoenix, Arizona. Potted plants were maintained in open-top chambers constructed with clear, plastic wall material. Four CO(2) concentrations of 350, 560, 675, and 885 microliters per liter were used during two growth periods and two water treatments. Small and large plants were grown for 6 months, while a few large plants were grown for 1 year. Wet-treatment plants received water twice weekly, whereas dry-treatment plants received slightly more water than they would under natural conditions. Plant growth rates in all treatments were significantly different between small and large specimens, but not between 6 month and 1 year large plants. Only the dry-treatment plants exhibited statistically different growth rates between the CO(2) treatments. This productivity response was equivalent to a 28% and 3-fold increase when mathematically interpolated between CO(2) concentrations of 300 and 600 microliters per liter for large and small plants, respectively. PMID- 16664707 TI - Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis. AB - alpha-dl-Difluoromethylornithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaCl(2). alpha-dl-Difluoromethylarginine, a specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus. PMID- 16664708 TI - Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.). AB - Exposure of intact alfalfa nodules to (15)N(2) showed that in bacteroids the greatest flow of (15)N was to NH(3). Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more (15)N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to (15)N(2) or vacuum infiltration with ((15)NH(4))(2)SO(4) in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH(3) produced by N(2) fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the (15)N-labeled amide group of glutamine. Data from (15)NH(4) (+) feeding support some direct amidation of Asp to form Asn. PMID- 16664709 TI - Compartmental efflux analysis: an evaluation of the technique and its limitations. AB - Efflux analysis is an established tool for characterizing the exchange properties of multicomponent systems. In this report, we have simulated several three- and four-compartment systems with error-free and imperfect data, the errors being designed to mimic actual, nonbiological variability in isotope efflux studies. The data sets were analyzed using computerized nonlinear regression techniques to identify the important aspects of actual experimental design (uptake times, efflux collection schedules, and total efflux times), and to consider the possibility that a properly designed and executed experiment might fail to resolve compartmentation correctly. The results showed that for any of the systems simulated, including those with error-free four-component data, a reasonable three-component fit was obtainable. Resolution of the additional compartment was not always possible. In correctly resolved systems, failure to estimate the correct decay constants was common, especially when the half-times were separated by less than an order of magnitude. We conclude that efflux analysis, by itself, lacks the power to provide reliable information about multicompartment systems. PMID- 16664710 TI - Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells. AB - The water-soluble polysaccharides (SEPS) secreted into the medium by suspension cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified alpha-1,4 endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS. PMID- 16664711 TI - A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: I. Phasing in Relation to the Light-Off Signal. AB - Evidence is presented of an endogenous rhythm in flowering response to far-red (FR) irradiation, with a period of about 12 h (hence semidian rhythm), which persists through at least three cycles in constant conditions of continuous light at 27 degrees C and has a marked influence on the flowering response in Pharbitis nil to a subsequent inductive dark period. The phase of the rhythm is not influenced by real time nor by the time from imbibition or from the beginning of the light period. Rather, it is fed forward from the beginning of the FR interruption to the beginning of the inductive dark period. The period of the rhythm is not affected by irradiance but is longer at cooler temperature. When there are two FR interruptions during the preceding light period, it is primarily the later one which determines the phase of the rhythm, although some interactions are evident. There appears to be an abrupt rephasing of the rhythm at the beginning of the inductive dark period. No overt rhythms which could be used as "clock hands" for the semidian rhythm were detected in photosynthesis, stomatal opening, or translocation. PMID- 16664712 TI - A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: II. The Involvement of Phytochrome. AB - The semidian ( approximately 12 h) periodicity in the effect of far-red (FR) interruptions of the light period preceding inductive darkness on flowering in Pharbitis nil appears to be mediated by phytochrome: (a) promotion by interruptions 2 hours before inductive darkness (-2 hours) and inhibition at -8 hours are greater the higher the proportion of FR/R+FR during the interruption; (b) brief FR exposures followed by darkness are even more effective than FR throughout; (c) the effect of brief FR is reversed by subsequent R; (d) R interruptions of an FR background are most promotive at -8 hours, when FR is most inhibitory. Promotive FR interruptions at -2 or -14 hours shorten the critical dark period whereas inhibitory FR interruptions at -8 hours lengthen it. We conclude that the semidian rhythm is controlled by a ;timing pool' of phytochrome FR absorbing form (Pfr) which disappears rapidly in darkness: four different estimates from our experiments indicate that Pfr was reduced to the level set by FR within 20 to 45 minutes in darkness. However, flowering may also be influenced by a ;metabolic pool' of Pfr with a delayed loss in darkness, the time of which can be advanced or retarded by shifting the semidian rhythm. PMID- 16664713 TI - Effects of light and spermine on senescence of hydrilla and spinach leaves. AB - Light treatment markedly accelerated chlorophyll loss in Hydrilla (Hydrilla verticillata [L.f.] Royle) over dark treatment whereas such acceleration could not be observed in spinach (Spinacia oleracea L.) leaf segments. Spermine, a polyamine, retarded the loss of chlorophyll in the dark but markedly accelerated this loss in the light during senescence of Hydrilla leaves. However, such effect of spermine in the dark was not so pronounced in spinach. The loss of protein was slower in the light than in the dark in both the species. Spermine arrested the loss of protein (as in spinach) or even raised the protein level over initial (as in Hydrilla). Loss of both soluble and insoluble protein was slower in light than in darkness. Spermine treatment, either in light or darkness, markedly accelerated the loss of soluble protein but raised the level of insoluble protein over initial in both the species. The pattern of change in alpha-amino nitrogen in either species could be correlated well with that of protein level. In Hydrilla, light increased the soluble protein fraction over initial and this rise was prevented by cycloheximide and not by chloramphenicol. Also, spermine augmented the protease activity (both acid and neutral) while light retarded the rise in protease activity during senescence of either species. Although spermine treatment reduced the leaching of alpha-amino nitrogen and electrolytes in Hydrilla, it augmented the same in spinach. PMID- 16664714 TI - Interactions between Ethylene, CO(2), and ABA on GA(3)-Induced Amylase Synthesis in Barley Aleurone Tissue. AB - Gibberellic acid-induced synthesis and release of alpha-amylase in barley aleurone tissue was inhibited by abscisic acid. This inhibition was relieved by simultaneous application of ethylene ranging in concentration from 0.1 to 100 microliters per liter. When CO(2) was applied, it eliminated the effect of 0.1 microliter per liter ethylene and reimposed the abscisic acid inhibition. All concentrations of CO(2) tested from 400 to 10(5) microliters per liter counteracted the effect of 0.1 microliter per liter ethylene, but had no observable effect on any higher concentration of ethylene. The results indicate that some processes necessary for embryo growth may be subject to regulation by ethylene and carbon dioxide at naturally occurring concentrations of the gases. PMID- 16664715 TI - Photosynthetic Adaptation by Synechococcus leopoliensis in Response to Exogenous Dissolved Inorganic Carbon. AB - Synechococcus leopoliensis was grown over a wide range of dissolved inorganic carbon (DIC) concentrations (4-25,000 micromolar) which were obtained by varying culture pH (6.2-9.6) and the CO(2) concentration of the gas stream (36-50,000 microliters per liter). The [DIC] required to half-saturate photosynthesis (K((1/2)) (DIC)) was found to vary depending upon the ambient DIC concentration at which the cells were grown. Low [DIC] grown cells exhibited low values of K((1/2)) (DIC) (4.7 micromolar) whereas cells grown at high [DIC] exhibited high values of K((1/2)) (DIC) (1-2.5 millimolar). Intermediate concentrations of DIC produced intermediate values. Changes in K((1/2)) (DIC) appeared to be solely a function of [DIC] and were independent of both culture pH and CO(2) concentration. As changes in K((1/2)) (DIC) occur in response to DIC concentrations commonly found in natural systems we suggest this adaptation may be of ecological significance. PMID- 16664716 TI - Protein Phosphorylation in Amyloplasts Isolated from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.). AB - Highly purified amyloplasts were isolated from cultured cells of sycamore (Acer pseudoplatanus L.). Incubation of amyloplasts with [gamma-(32)P]-ATP resulted in the labeling of more than ten polypeptides. Pulsechase experiments showed the reversibility of the process with some but not all of the polypeptides. The phosphorylation reaction of one polypeptide, M(r) 100, was shown to be calcium dependent. Although exogenously added pig brain calmodulin had no effect, the calmodulin antagonist W-7 strongly inhibited phosphorylation of the 100 kilodaltons polypeptide. The presence of endogenous calmodulin, about 1 to 3 micrograms per milligram protein, in the amyloplast preparation was estimated by activation of phosphodiesterase in vitro. PMID- 16664717 TI - Age-Dependent Discrimination between Stereoisomers of 1-Amino-2-Ethylcyclopropane 1-Carboxylic Acid in Carnation Petals. AB - The ability of carnation petals (Dianthus caryophyllus L. cv White Sim) of different ages to convert the cis and trans isomers of 1-amino-2 ethylcyclopropane-1-carboxylic acid (AEC) to 1-butene was studied. Young petals, which produce ethylene at a low rate, convert both cis- and trans-AEC to 1-butene with low efficiency and at equal rates. In senescing petals, the rate of conversion of cis-AEC remains low, but there is a marked increase in the rate of trans-AEC conversion. Thus there is a clear evidence of stereodiscrimination between the isomers. Stimulating the rate of senescence by treatment with either 1-aminocyclopropane-1-carboxylic acid or ethylene further increases the rate of trans-AEC conversion. Delaying of petal senescence by silver thiosulphate or aminooxyacetic acid inhibits the rise in trans-AEC conversion. PMID- 16664718 TI - Labeling of fructans in winter wheat stems. AB - Fructans synthesized from newly formed assimilates accumulate in wheat stems as nonstructural carbohydrates. Experiments performed tested the hypothesis that the fructose moiety from translocated sucrose is used preferentially in biosynthesis of these fructans. Results indicated: (a) a large percentage of labeled sucrose was translocated and unloaded in an unaltered state; and (b) sucrose contributed its fructose moiety to fructan synthesis in stems. PMID- 16664719 TI - Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L. AB - Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (alpha and beta) during Phaseolus vulgaris root development shows that the alpha polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the beta polypeptide becomes the root predominant GS monomer. The alpha/beta ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the alpha/beta ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris. PMID- 16664720 TI - Anomalous nonstoichiometry detected by dual label analysis of ribulose 1,5 bisphosphate carboxylase. AB - When ribulose-1,5-bisphosphate carboxylase is assayed under N(2) using [(3)H]ribulose 1,5-bisphosphate and (14)CO(2), [(3)H]3-phosphoglycerate and [(14)C]3-phosphoglycerate are produced in nonstoichiometric amounts in a ratio which approaches 7 at low concentrations of CO(2) (2 micromolar) assuming a 1:1 ratio at V(max) (280 micromolar). The log of the molar ratio varies as a linear function of log[CO(2)]. Nonstoichiometry could be explained by CO(2) contaminatio of the reactants or tritium contamination of the products. However, the magnitude of CO(2) contamination required (18 +/- 4 micromolar) is far in excess of controlled CO(2) (<0.1 micromolar), and the required tritium contaminant would have to vary from 30 to 85% of the purified 3-phosphoglycerate at the 58 and 2 micromolar CO(2) assay levels, respectively. This contrasts with detectable tritium contamination which is only 1 to 4% and correctable. Nonstoichiometry is evident using either 1 or 5 labeled [(3)H]ribulose 1,5-bisphosphate. When 3 phosphoglycerate is reisolated as glycerate the (3)H/(14)C ratio remains unchanged. PMID- 16664721 TI - Preferential Photosynthetic Uptake of Exogenous HCO(3) in the Marine Macroalga Chondrus crispus. AB - The rate of HCO(3) (-) uptake by the red macroalga Chondrus crispus has been investigated. Unbalanced concentrations of free CO(2) and HCO(3) (-), generated by the photosynthetic activity, were detected in steady state conditions by using an exchange column apparatus linked to an assimilation chamber. Observing the variations of this gradient as influenced by the time of seawater transit from the assimilation chamber towards the column allowed an experimental determination of: (a) the actual gradient created by the photosynthetic activity, (b) the rate constant of the chemical conversion of free CO(2) to HCO(3) (-). With a value of 0.115 per second at pH 8.92, this rate constant was in good agreement with a previous estimation. By using a simple model, we show that the photosynthetic rate of HCO(3) (-) consumption can be estimated by the product of the actual gradient and the rate constant. In the conditions of the experiments reported here, this rate represented more than 90% of the whole photosynthetic flux. PMID- 16664722 TI - Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays. AB - Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H(+) excretion and inhibits the uptake of K(+) and alpha-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv "Seneca Chief"). H(+) excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H(+) excretion is retarded by the ATPase inhibitors N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H(+) ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H(+) transport, but that the activity may be an important regulator of H(+) excretion. The 90% inhibition of K(+) ((86)Rb(+)) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of alpha-aminoisobutyric acid. The possibility exists that a portion of the K(+) and most of the alpha aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K(+) uptake system may also regulate K(+) fluxes. PMID- 16664723 TI - Transport of gibberellin a(1) in cowpea membrane vesicles. AB - The permeability properties of gibberellin A(1) (GA(1)) were examined in membrane vesicles isolated from cowpea hypocotyls. The rate of GA(1) uptake was progressively greater as pH decreased, indicating that the neutral molecule is more permeable than anionic GA(1). Membrane vesicles used in this study possessed a tonoplast-type H(+)-translocating ATPase as assayed by MgATP-dependent quenching of acridine orange fluorescence and methylamine uptake. However, GA(1) uptake was not stimulated by MgATP. At concentrations in excess of 1 micromolar, GA(1), GA(5), and GA, collapsed both MgATP-generated and artifically imposed pH gradients, apparently by shuttling H(+) across the membrane as neutral GA. The relatively high permeability of neutral GA and the potentially detrimental effects of GA in uncoupling pH gradients across intracellular membranes supports the view that GA(1) accumulation and compartmentation must occur by conversion of GA(1) to more polar metabolites. PMID- 16664724 TI - Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots. AB - The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45 70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M(r) = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation. PMID- 16664725 TI - Changes in Amines and Biosynthetic Enzyme Activities in p-Fluorophenylalanine Resistant and Wild Type Tobacco Cell Cultures. AB - The levels of free amines and the activities of their biosynthetic enzymes were measured in a p-fluorophenylalanine resistant Nicotiana tabacum L. cv Xanthi cell line (TX4) which accumulates high levels of cinnamoylamides, and a wild type cell line (TX1). Putrescine in TX1 and spermidine in TX1 and TX4 increased 4-fold by day 4 but declined by day 8 of the culture period. Spermine levels were consistently low, while tyramine was not found in TX1 until day 9 when a gradual rise was noted. Ornithine decarboxylase activity in TX1 and TX4 increased slightly through day 2 but declined gradually thereafter. S-Adenosylmethionine decarboxylase activity remained low throughout the culture period, and tyrosine and arginine decarboxylases in TX1 were very low in activity. In contrast, the activities of tyrosine and arginine decarboxylases were elevated in TX4, but a 3 fold increase in tyramine after a subculture was not accompanied by a rise in tyrosine decarboxylase. However, tyrosine decarboxylase activity did increase during a second rise in tyramine levels in aging cells, late in the culture period. Although significant differences exist in amine levels, between TX4 and TX1, it is unclear how altered amine metabolism relates to p-fluorophenylalanine resistance. PMID- 16664726 TI - The immunologically conserved phycobilisome-thylakoid linker polypeptide. AB - We have isolated phycobilisomes from two classes of red algae, several subdivisions of the cyanobacteria, and the cyanelles of Cyanophora paradoxa. In addition to the major light harvesting biliproteins, these phycobilisomes also contain several other polypeptides, the largest of which ranges from 75 to 120 kilodaltons in the different species surveyed. This protein, previously isolated and characterized from three species, was shown to be the final emitter of excitation energy in phycobilisomes and is also thought to be involved in the attachment of the phycobilisomes to the thylakoid membrane. We have obtained polyclonal antibodies to the 95 kilodalton polypeptide isolated from phycobilisomes of the cyanobacterium, Nostoc sp. This protein shares no common antigenic determinants with either the alpha or beta subunits of allophycocyanin, or any of the other biliproteins, as determined by the sensitive Western immunoblotting technique. However, this antiserum cross-reacts with the highest molecular weight polypeptide of all the rhodophytan and cyanobacterial phycobilisomes tested. That these proteins are immunologically related, but are unrelated to other biliproteins, is reminiscent of previous immunological studies of biliproteins which showed that while the three major spectroscopically distinct classes of biliproteins (phycoerythrin, phycocyanin, and allophycocyanin) shared no common antigenic determinants, there was a strong antigenic determinant to specific biliprotein classes which crossed taxonomic divisions. PMID- 16664727 TI - Activity of the natural algicide, cyanobacterin, on angiosperms. AB - Cyanobacterin is a secondary metabolite produced by the cyanobacterium (blue green alga) Scytonema hofmanni. The compound had previously been isolated and chemically characterized. It was shown to inhibit the growth of algae at a concentration of approximately 5 micromolar. Cyanobacterin also inhibited the growth of angiosperms, including the aquatic, Lemna, and terrestrial species such as corn and peas. In isolated pea chloroplasts, cyanobacterin inhibited the Hill reaction when p-benzoquinone, K(3)Fe(CN)(6), dichlorophenolindophenol, or silicomolybdate were used as electron acceptors. The concentration needed to inhibit the Hill reaction in photosystem II was generally lower than the concentration of the known photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1 dimethyl urea. Cyanobacterin had no effect on electron transport in photosystem I. The data indicate that cyanobacterin inhibits O(2) evolving photosynthetic electron transport in all plants and that the most probable site of action is in photosystem II. PMID- 16664728 TI - Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria. AB - The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase. PMID- 16664729 TI - The effects of osmotic tissue dehydration and air drying on morphology and energy transfer in two species of porphyra. AB - Studies were conducted to document the effects on morphology and energy transfer in photosynthesis of severe tissue dehydration induced either by air-drying or by immersing the tissues of two Porphyra species in hyperosmotic solutions. These studies showed that the dehydration-tolerant intertidal alga, Porphyra perforata J.Ag., was almost unaffected by either of these treatments, while the dehydration sensitive Porphyra nereocystis Anders. was damaged similary by both treatments. Damage to that sensitive species was characterized by ruptured organelles as seen by interference microscopy as well as by increased fluorescence emission at 682 nanometers emanating from allophycocyanin. These results suggest that a disruption of energy transfer between allophycocyanin and chlorophyll a occurs because of the damage to membranes following tissue dehydration, and that the increase in the yield of phycobilin fluorescence is a good indicator of these phenomena. Thus, air-drying and osmotic-dehydration appear to have similar physiological consequences in a dehydration-sensitive alga but almost no effect in a tolerant species. PMID- 16664730 TI - Catalytic activity of maize leaf phosphoenolpyruvate carboxylase in relation to oligomerization. AB - The relationship between the state of oligomerization and activity of purified maize leaf phosphoenolpyruvate carboxylase using size exclusion high performance liquid chromatography was examined. Maximum activities of 35 to 38 micromoles per minute per milligram protein were found when 100% of the enzyme was in its tetrameric form. The effects of the sulfhydryl group modifiers CuCl(2) and p chloromercuribenzoate on enzyme inhibition and the state of aggregation of the protein complex were examined. Aggregation of the enzyme is temperature and pH sensitive with low temperature and high pH favoring depolymerization. Stability of the tetrameric form is largely dependent upon histidyl residues, and to some extent this explains the biphasic response of enzyme activity to changes in MgCl(2) concentrations. Modification of the tetramer's histidyl residues by the inhibitor diethylpyrocarbonate (0.125 millimolar) results in its dissociation to the dimeric form and loss of activity. Subsequent treatment with 0.4 molar hydroxylamine results in reassociation to the tetramer and restoration of enzymic activity. PMID- 16664731 TI - Effect of Calcium on Tuberization in Potato (Solanum tuberosum L.). AB - Tuberization response of single-node leaf cuttings from induced potato plants (Solanum tuberosum L.) was reversed when pretreated with 5 millimolar ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) + 50 micromolar calcium ionophore (A23187) and resumed when transferred to a CaCl(2) containing medium. Tuberization was inhibited by LaCl(3), chlorpromazine, and trifluoperazine at 5 to 10 micromolar. These results suggest a role for calcium in the tuberization process. PMID- 16664732 TI - Detection of the messenger RNA encoding for the ferredoxin-dependent glutamate synthase in maize leaf. AB - Ferredoxin-dependent glutamate synthase (EC 1.4.7.1), glutamate oxoglutarate aminotransferase (glutamate synthase) (GOGAT) messenger RNA was extracted from maize (Zea mays L.) leaves and partially purified through oligo(dT)-cellulose chromatography and ultracentrifugation in a sucrose gradient. mRNA were translated in vitro using a reticulocyte system. The glutamate synthase subunit was characterized by immunoprecipitation with antibodies raised against the rice (Oryza sativa L.) ferredoxin-glutamate synthase. The in vitro synthesized protein and the 145 kilodaltons genuine maize leaf subunit of GOGAT were found to comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. PMID- 16664733 TI - Inorganic Carbon Uptake during Photosynthesis : I. A Theoretical Analysis Using the Isotopic Disequilibrium Technique. AB - Equations have been developed which quantitatively predict the theoretical time course of photosynthetic (14)C incorporation when CO(2) or HCO(3) (-) serves as the sole source of exogenous inorganic carbon taken up for fixation by cells during steady state photosynthesis. Comparison between the shape of theoretical (CO(2) or HCO(3) (-)) and experimentally derived time-courses of (14)C incorporation permits the identification of the major species of inorganic carbon which crosses the plasmalemma of photosynthetic cells and facilitates the detection of any combined contribution of CO(2) and HCO(3) (-) transport to the supply of intracellular inorganic carbon. The ability to discriminate between CO(2) or HCO(3) (-) uptake relies upon monitoring changes in the intracellular specific activity (by (14)C fixation) which occur when the inorganic carbon, present in the suspending medium, is in a state of isotopic disequilibrium (JT Lehman 1978 J Phycol 14: 33-42). The presence of intracellular carbonic anhydrase or some other catalyst of the CO(2)-HCO(3) (-) interconversion reaction is required for quantitatively accurate predictions. Analysis of equations describing the rate of (14)C incorporation provides two methods by which any contribution of HCO(3) (-) ions to net photosynthetic carbon uptake can be estimated. PMID- 16664734 TI - Inorganic Carbon Uptake during Photosynthesis : II. Uptake by Isolated Asparagus Mesophyll Cells during Isotopic Disequilibrium. AB - The species of inorganic carbon (CO(2) or HCO(3) (-)) taken up a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO(2) or HCO(3) (-) transport, during steady state photosynthesis, is achieved by monitoring the changes (by (14)C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO(2) or HCO(3) (-) transport) and experimental time-courses of (14)C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO(2), rather than HCO(3) (-), is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO(2) is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO(3) (-) transport, as the incorporation of (14)C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO(2) uptake alone. The contribution of HCO(3) (-) to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO(3) (-) concentration. The evidence for direct HCO(3) (-) transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of (14)C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO(2), which is partially alleviated by a high extracellular concentration of HCO(3) (-). PMID- 16664735 TI - Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants. AB - Rates of CO(2) assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO(2) in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25 degrees C. Electron transport activity for CO(2) assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O(2), and between fluorescence and rates of CO(2) assimilation at 19 millibars but not 200 millibars O(2). The values of fluorescence measured at about 220 microbars intercellular CO(2) were not greatly affected by increasing O(2) from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO(2) partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO(2) partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO(2) partial pressures which reversed to a positive correlation at higher values of CO(2) partial pressures. Similar results were observed in the C(3) species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C(4) species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO(2) assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q(E)) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed. PMID- 16664736 TI - The Azolla-Anabaena azollae Relationship : XII. Nitrogenase Activity and Phycobiliproteins of the Endophyte as a Function of Leaf Age and Cell Type. AB - Nitrogenase activity was measured in leaves along the main stem axes of Azolla pinnata R. Br. The activity was negligible in leaves of the apical region, rapidly increased to a maximum as leaves matured, and declined in aging leaves. In situ absorption and fluorescence emission spectra were obtained for individual vegetative cells and heterocysts in filaments of the A. pinnata and Azolla caroliniana endophytes removed from the cavities of progressively older leaves. These spectra unequivocally demonstrate the occurrence of phycobiliproteins in the two cell types of both endophytes at the onset of heterocyst differentiation in filaments from young leaves, during the period of maximal nitrogenase activity in filaments from mature leaves, and in filaments from leaves entering senescence. Phycobiliproteins of the A. caroliniana endophyte were purified and extinction coefficients determined for the phycoerythrocyanin, phycocyanin, and allophycocyanin. The phycobiliprotein content and complement of sequential leaf segments from main stem axes and of vegetative cell and heterocyst preparations were measured in crude extracts. There was no obvious alteration of the phycobiliprotein complement associated with increasing heterocyst frequency of the endophyte in sequential leaf segments and the phycobiliprotein complement of heterocysts was not appreciably different from that of vegetative cells. These findings indicate that the phycobiliprotein complement of the vegetative cell precursor is retained in the heterocysts of the endophyte. PMID- 16664737 TI - Potassium-Dependent Increase in RNA and Protein Synthesis in the Early Phase of Incubation of the Thermodormant Phacelia tanacetifolia Seeds. AB - The seeds of Phacelia tanacetifolia Benth. cv Bleu Clair incubated at 30 degrees C in the dark did not germinate and did not activate K(+) uptake capacity. The administration of 1 millimolar K(+) in the early phase of incubation stimulated RNA and protein synthesis. The possible role of K(+) in promoting the marcromolecular syntheses during the early phase of germination is discussed. PMID- 16664738 TI - Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system. AB - The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO(3) (-) was faster than that of Cl(-). Phosphate and sulfate crossed the tonoplast only slowly. 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p chloromercuribenzene sulfonate and HgCl(2) stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K(m) for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP. PMID- 16664739 TI - Uptake of proline by the scutellum of germinating barley grain. AB - Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up 1 millimolar l-[(14)C]proline at an initial rate of about 6.5 micromoles gram(-1) fresh weight hour(-1) (pH 5, 30 degrees C). The uptake had a pH optimum at 5. The bulk of the uptake (93%) was via carrier-mediated active transport. All of the 19 l-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. d-Proline inhibited this system as strongly as l-proline. Nine of the 16 l-amino acids tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain. PMID- 16664740 TI - Properties of a Partially Purified Nucleoside Triphosphatase (NTPase) from the Chloroplast Envelope of Pea. AB - The Mg-nucleoside triphosphatase activity associated with the inner envelope membrane of the pea chloroplast is comprised of at least two components, a major activity that is sensitive to vanadate and sodium fluoride and a minor insensitive activity. The vanadate/fluoride sensitive activity has been partially purified (about 35-fold) from Triton X-100 solubilized membranes by DEAE-Sephadex chromatography and sucrose density gradient centrifugation. The partially purified enzyme resembles the membrane-bound activity in requiring either Mg(2+) or Mn(2+), having a broad specificity for nucleoside triphosphates, having a K(m) for ATP of 0.18 millimolar, and being inhibited by N-ethylmaleimide, but insensitive to sodium azide and dicyclohexylcarbodiimide. The partially purified enzyme obtained after sucrose gradient centrifugation has a markedly increased sensitivity to inhibition by inorganic pyrophosphate compared with the less pure enzyme. Pyrophosphate is not a substrate of either the membrane-bound or partially purified enzyme. PMID- 16664741 TI - Leaf and Stem CO(2) Uptake in the Three Subfamilies of the Cactaceae. AB - Net CO(2) uptake over 24-hour periods was examined for the leaves and for the stems of 11 species of cacti representing all three subfamilies. For Pereskia aculeata, Pereskia grandifolia, and Maihuenia poeppigii (subfamily Pereskioideae), all the net shoot CO(2) uptake was by the leaves and during the daytime. In contrast, for the leafless species Carnegiea gigantea, Ferocactus acanthodes, Coryphantha vivipara, and Mammillaria dioica (subfamily Cactoideae), all the shoot net CO(2) uptake was by the stems and at night. Similarly, for leafless Opuntia ficus-indica (subfamily Opuntioideae), all net CO(2) uptake occurred at night. For leafy members of the Opuntioideae (Pereskiopsis porteri, Quiabentia chacoensis, Austrocylindropuntia subulata), at least 88% of the shoot CO(2) uptake over 24 hours was by the leaves and some CO(2) uptake occurred at night. Leaves responded to the instantaneous level of photosynthetically active radiation (PAR) during the daytime, as occurs for C(3) plants, whereas nocturnal CO(2) uptake by stems of O. ficus-indica and F. acanthodes responded to the total daily PAR, as occurs for Crassulacean acid metabolism (CAM) plants. Thus, under the well-watered conditions employed, the Pereskioideae behaved as C(3) plants, the Cactoideae behaved as CAM plants, and the Opuntioideae exhibited characteristics of both pathways. PMID- 16664742 TI - Effects of environmental stresses on the cell cycle of two marine phytoplankton species. AB - Cell cycle phase durations of cultures of Hymenomonas carterae Braarud and Fagerl, a coccolithophore, and Thalassiosira weissflogii Grun., a centric diatom, in temperature-, light- or nitrogen-limited balanced growth were determined using flow cytometry. Suboptimal temperature caused increases in the duration of all phases of the cell cycle (though not equally) in both species, and the increased generation time of nitrogen-limited cells of both species was due almost wholly to expansion of G(1) phase. In H. carterae light limitation caused only G(1) phase to expand, but in T. weissflogii both G(2) + M and G(1) were affected. These results are discussed in relation to cell division phasing patterns of these two species and to models of phytoplankton growth. Simultaneous measurements of protein and DNA on individual cells indicated that under all conditions, the protein content of cells in G(1) was a constant proportion of that of G(2) + M cells. Simultaneous measurements of RNA and protein on each cell indicated that the amounts of these two cell constituents were always tightly correlated. Under conditions of nitrogen limitation both protein and RNA per cell decreased to less than one-third of the levels found in nonlimited cells. This indicates, at least for nitrogen-replete cells, that neither protein nor RNA levels are likely to act as the trigger for cell cycle progression. Strict control by cell size is also unlikely since mean cell volume decreased as growth rates were limited by light and nitrogen supply, but increased with decreasing temperature. PMID- 16664743 TI - Temperature and leaf osmotic potential as factors in the acclimation of photosynthesis to high temperature in desert plants. AB - Seasonal changes in the high temperature limit for photosynthesis of desert winter annuals growing under natural conditions in Death Valley, California were studied using an assay based upon chlorophyll fluorescence. All species of this group were 6 to 9 degrees C more tolerant of high temperature at the end of the growing season (May) than at its beginning (February). Over this same time period, the mean daily maximum air temperatures increased by 12 degrees C. Laboratory studies have demonstrated that increases in thermal tolerance could be induced by increasing growth temperature alone. For plants growing under field conditions there was also a good correlation between the thermal tolerance of leaves and the osmotic potential of leaf water, indicating that increases in the concentrations of some small molecules might also confer increased thermal tolerance. Isolated chloroplast thylakoids subjected to increasing concentrations of sorbitol could be demonstrated to have increased thermal tolerance. PMID- 16664744 TI - Reconstitution of the Light Harvesting Chlorophyll a/b Pigment-Protein Complex into Developing Chloroplast Membranes Using a Dialyzable Detergent. AB - Conditions were developed to isolate the light-harvesting chlorophyll-protein complex serving photosystem II (LHC-II) using a dialyzable detergent, octylpolyoxyethylene. This LHC-II was successfully reconstituted into partially developed chloroplast thylakoids of Hordeum vulgare var Morex (barley) seedlings which were deficient in LHC-II. Functional association of LHC-II with the photosystem II (PSII) core complex was measured by two independent functional assays of PSII sensitization by LHC-II. A 3-fold excess of reconstituted LHC-II was required to equal the activity of LHC developing in vivo. We suggest that a linker component may be absent in the partially developed membranes which is required for specific association of the PSII core complex and LHC-II. PMID- 16664745 TI - Proline accumulation and the adaptation of cultured plant cells to water stress. AB - The transfer of cultured tomato cells (Lycopersicon esculentum cv VFNT-Cherry) to a low water potential environment resulted in an increased dry weight to fresh weight ratio accompanied by a rapid accumulation of proline. Proline content continued to increase as osmotic adjustment and growth occurred. The initial increase in proline concentration was accompanied by a drop in turgor. However, proline levels continued to increase with a gain in turgor during osmotic adjustment. Thus, the accumulation of proline depended not only on cell water potential, or on the initial loss of turgor but more closely on cell osmotic potential. The ultimate level of proline depended on the level of adaptation. Proline levels remained high after more than 100 cell generations in low water potential media, but declined rapidly after transfer to media with a less negative water potential. Addition of exogenous proline to the medium during water stress and during osmotic downshock alleviated the normally resulting inhibition of growth. The results suggest a positive role for proline accumulation in adaptation of cells to changing external water potentials. PMID- 16664746 TI - New artificial electron donors for in vitro assay of nitrate reductase isolated from cultured tobacco cells and other organisms. AB - The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techne SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < Momega < 94 kilodaltons. PMID- 16664747 TI - Glycoproteins in the matrix of glyoxysomes in endosperm of castor bean seedlings. AB - The matrix of glyoxysomes from endosperm of castor bean (Ricinus communis cv Hale) seedlings has been analyzed for the presence of glycosylated proteins. Glyoxysome preparations were monitored for organelle homogeneity by electron microscopy and enzyme marker activities. Glyoxysomes were essentially free of endoplasmic reticulum, mitochondria, and protein bodies. At least eight glyoxysomal matrix glycopeptides ranging in size from 39 to 160 kilodaltons were identified by their affinity for concanavalin A. The glyoxysomal glycoproteins were shown to be radioactively labeled when endosperm was allowed to incorporate glucosamine. Incorporation of glucosamine was inhibited by tunicamycin under conditions which did not inhibit protein synthesis. Hydrolysis of glyoxysomal extracts and subsequent analysis by paper chromatography showed that the labeled precursor was incorporated into the glycoprotein without prior dispersion of the label into amino acids. The present data demonstrate the occurrence of N-linked, high mannose oligosaccharides on polypeptides of the glyoxysomal matrix. This finding is discussed in relation to pathways of protein maturation and transport during glyoxysomal biogenesis. PMID- 16664748 TI - The Effects of Streptomycin, Desiccation, and UV Radiation on Ice Nucleation by Pseudomonas viridiflava. AB - Streptomycin (100 micrograms per milliliter), desiccation (over CaSO(4)), and ultraviolet radiation (4500 microwatts per square centimeter at 254 nanometers for 15 minutes) reduced ice nucleation activity by Pseudomonas viridiflava strain W-1 as determined by freezing drops of the bacterial suspensions. Highest residual ice nucleation activity by dead cells was obtained by desiccation, although no freezing above -3.5 degrees C was detected. The rate and extent of loss of ice nucleation activity following streptomycin and ultraviolet treatment was affected by preconditioning temperature. At 21 degrees C and above, loss of activity by dead cells was rapid and irreversible. PMID- 16664749 TI - Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase. AB - Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme. PMID- 16664750 TI - Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves. AB - The enzyme aldose (aldehyde) reductase was partially purified (142-fold) and characterized from Euonymus japonica leaves. The reductase, a dimer, had an average molecular weight of 67,000 as determined by gel filtration on Sephadex G 100. The enzyme was NADPH specific and reduced a broad range of substrates including aldoses, aliphatic aldehydes, and aromatic aldehydes. Maximum activity was observed at pH 8 in phosphate and Tris-HCl buffers and at pH 8.6 to 9.0 in glycine-NaOH buffer using dl-glyceraldehyde or 3-pyridinecarboxaldehyde as substrate. NADP was a competitive inhibitor with respect to NADPH with a K(i) of 60 micromolar. Glycerol was an uncompetitive inhibitor to dl-glyceraldehyde (K'(i) = 460 millimolar). The Euonymus enzyme was inhibited by sulfhydryl inhibitor, phenobarbital, and high concentrations of Li(2)SO(4). Pyrazol and metal chelating agents inhibited the enzyme slightly. Enzyme activity was detected in the leaves and berries of Celastrus orbiculatus and several species of Euonymus. Probable function of this enzyme is to reduce d-galactose to galactitol, a characteristic metabolite in phloem sap of members of the Celastraceae family. PMID- 16664751 TI - The Loss of the Circadian Rhythm in Photosynthesis in an Old Strain of Gonyaulax polyedra. AB - Cultures of Gonyaulax polyedra Stein maintained in the laboratory for 15 to 20 years, including an axenic strain isolated in 1960, have gradually lost the ability to survive in darkness. G. polyedra (70A), isolated in 1970 and maintained in a 12:12 light:dark cycle, now tolerates continuous darkness for a much shorter time than a strain isolated in 1981. I have compared the properties of strain 70A with those of this newer strain (81N), to investigate changes in Gonyaulax with length of time in culture, which may account for poor survival in darkness. When grown in continuous light (13, 12, or 4.5 watts per square meter), strains 70A and 81N have similar growth rates, yields, cell diameters, protein contents, C/N ratios, respiration rates, pigment complements, and photosynthetic rates. When entrained by a light:dark cycle (12L:12D), 70A showed no photosynthesis rhythm, although such a rhythm was formerly present. However, the circadian rhythms in bioluminescence and cell division were normal in both strains. Thus, the circadian clock is apparently still intact in 70A as in 81N. The rate of photosynthesis in strain 70A was constant at a low level, the consequent smaller accumulation of photosynthetic products probably accounting for the limited survival in darkness. The defect in strain 70A may be the loss of a component either directly affecting P(max) or necessary for transduction from the circadian clock to photosynthesis. PMID- 16664752 TI - Identification of a highly conserved domain on phytochrome from angiosperms to algae. AB - A monoclonal antibody (Pea-25) directed to phytochrome from etiolated peas (Pisum sativum L., cv Alaska) binds to an antigenic domain that has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sample buffer extracts prepared from lyophilized tissue samples or freshly harvested algae. Pea-25 immunostained an approximately 120-kilodalton polypeptide from a variety of etiolated and green plant tissues, including both monocotyledons and dicotyledons. Moreover, Pea-25 immunostained a similarly sized polypeptide from the moss Physcomitrella, and from the algae Mougeotia, Mesotaenium, and Chlamydomonas. Because Pea-25 is directed to phytochrome, and because it stains a polypeptide about the size of oat phytochrome, it is likely that Pea-25 is detecting phytochrome in each case. The conserved domain that is recognized by Pea-25 is on the nonchromophore bearing, carboxyl half of phytochrome from etiolated oats. Identification of this highly conserved antigenic domain creates the potential to expand investigations of phytochrome at a cellular and molecular level to organisms, such as Chlamydomonas, that offer unique experimental advantages. PMID- 16664753 TI - Flash-Induced Delayed Light Emission Patterns of Red Kidney Bean Leaves: Evidence of a New Component Dependent upon Tissue Integrity. AB - Studies of flash-induced delayed light emission profiles of dark-adapted intact plant tissues revealed a previously unreported component of plant luminescence. Only partially evident in intact chloroplasts and totally absent in broken chloroplasts, this peak may reflect the interaction of one or more light activated enzyme systems with photosynthetic electron transport. PMID- 16664754 TI - Biochemistry of fern spore germination: protease activity in ostrich fern spores. AB - Protease activities were detected in quiescent and germinating spores of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Peak endopeptidase, aminopeptidase, and carboxypeptidase activities were detected 12 to 24 hours after spores began imbibing under light. There was a correlation between activities of proteases, the onset of a decline in levels of soluble protein, and an increase in levels of free amino acids. The earliest visible event of spore germination, breakage of the spore coat and protrusion of a rhizoid cell, was observed after peak protease activity, 48 to 72 hours after the start of imbibition. Results of this study demonstrate similarities in the pattern of protease activities during germination of ostrich fern spores to those of some seeds. PMID- 16664755 TI - Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263). AB - We have measured the exchange of (18)O between CO(2) and H(2)O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of (18)O from CO(2) in the fluid outside the cells provides a method to study CO(2) and HCO(3) (-) kinetics in suspensions of algae that contain carbonic anhydrase since (18)O loss to H(2)O is catalyzed inside the cells but not in the external fluid. Low-CO(2) cells of Chlorella vulgaris (grown with air) were added to a solution containing (18)O enriched CO(2) and HCO(3) (-) with 2 to 15 millimolar total inorganic carbon. The observed depletion of (18)O from CO(2) was biphasic and the resulting (18)C content of CO(2) was much less than the (18)O content of HCO(3) (-) in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO(3) (-) into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 x 10(-4) s(-1) due to our experimental errors. The Fick's law rate constant for entry of CO(2) to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO(2) flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO(2) entry into the cell or both. The CO(2) hydration activity inside the cells was 160 times the uncatalyzed CO(2) hydration rate. PMID- 16664756 TI - A Model for the Regulation of K Influx, and Tissue Potassium Concentrations by Negative Feedback Effects upon Plasmalemma Influx. AB - By means of a modified Michaelis-Menten equation for K(+) influx, which includes terms for root and external K(+) concentrations (root [K(+)] and [K(+)](0), respectively) it is possible to predict the manner in which short-term (perturbation) fluxes of K(+) into roots of barley plants (Hordeum vulgare cv Fergus) vary with root [K(+)] and [K(+)](0). Influx values derived from this equation were used to predict changes of root and shoot [K(+)] and K(+) absorption rates (as functions of time and [K(+)](0)) from a knowledge of K(+) efflux, relative growth rates of roots and shoots, and the partitioning of absorbed K(+) between these organs. A microcomputer program was employed to model these changes in low-salt plants following transfer to solutions in which [K(+)](0) was maintained at values ranging from 5 to 1000 millimoles per cubic meter. The model was operated on the basis of 10 minute absorption periods which provided data for continuous ;updating' of tissue [K(+)]. The simulations were undertaken for periods corresponding to 30 days. During this time the model accurately predicted the manner in which K(+) influx and root and shoot [K(+)] gradually approach values which are essentially independent of [K(+)](0). The computer program was also used to predict the outcome of changing various external and internal parameters of the proposed regulatory system. The results of these simulations are discussed in the context of current models for negative feedback control of ion fluxes. PMID- 16664757 TI - Methionine methyl group metabolism in lemna. AB - To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed. PMID- 16664758 TI - The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays. AB - Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K(m) (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K(i) of about 6 millimolar. For comparison the K(i) for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K(a) values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l glutamate, l-aspartate, and beta-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts. PMID- 16664759 TI - Distribution of pyrophosphate:fructose 6-phosphate phosphotransferase in maize leaves. AB - In leaves of maize (Zea mays) the activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) is much less than that of ATP:fructose 6-phosphate 1 phosphotransferase. A sequential extraction technique was used to study the location of PFP in this tissue. When compared with enzymes known to be restricted to specific locations in maize, the distribution of PFP activity in the sequential extracts indicated that PFP is located predominantly, if not exclusively, in the mesophyll cytoplasm. Although confined to the same site as sucrose synthesis, the level of PFP activity is inadequate to contribute significantly to the gluconeogenic flux from fructose 1,6-bisphosphate to fructose 6-phosphate. The absence of PFP activity from the bundle-sheath demonstrates that this activity is not essential for glycolysis in higher plants. PMID- 16664760 TI - Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate. AB - The H(+)-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl(-) and NO(3) (-). N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K(m) of the enzyme for ATP but caused relatively little alteration of the V(max). Nitrate increased K(m) only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl(-). Other monovalent anions that specifically increased the K(m) of the ATPase for ATP were, in order of increasing K(i), SCN( ), ClO(4) (-), and ClO(3) (-). Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO(3) (-), was a competitive inhibitor of the ATPase but ADP and NO(3) (-) did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO(3) (-) does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg.ADP and Mg.ATP protected the enzyme from inactivation by NEM but Mg.ADP was the more potent of the two. Chloride and NO(3) (-) exerted little or no effect on the protective actions of Mg.ADP and Mg.ATP suggesting that neither Cl(-) nor NO(3) (-) are involved in substrate binding. PMID- 16664761 TI - Chromatographic resolution of h-translocating pyrophosphatase from h translocating ATPase of higher plant tonoplast. AB - Membrane vesicles derived from the tonoplast of Beta vulgaris L. possess two predominant phosphohydrolase activities: a Cl(-)-stimulated, NO(3) (-)-inhibited ATPase, and a K(+)-stimulated, Na(+)-inhibited inorganic pyrophosphatase (PPase). The solubilization of tonoplast vesicles with 2% (w/v) Triton X-100 containing 4 millimolar MgCl(2) and 1 millimolar ethylenediamine tetracetic acid, as protectants, gives high yields of both the ATPase and PPase in soluble form. Chromatography of the solubilized membranes on Sephacryl-400 results in the separation of the two enzymes. The PPase and ATPase are purified 4- and 17-fold, respectively, with quantitative recovery. The separated enzymes show negligible activity towards the other's substrate and the separated PPase only hydrolyzes pyrophosphate. The separated enzymes show mineral ion requirements identical to those of the corresponding pump and hydrolytic activities in native tonoplast and both solubilized enzymes are subject to phospholipid activation. PMID- 16664762 TI - Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening. AB - Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity. PMID- 16664763 TI - Remobilization patterns of C and N in soybeans with different sink-source ratios induced by various night temperatures. AB - The effects of increased sink-source ratios, induced by elevating night temperatures, on remobilization of (14)C-assimilates and N within field-grown soybeans (Glycine max [L.] Merr.) was investigated from preflowering to maturity. Raising the mean minimum night temperature for the entire growing season from 10 (check, uncontrolled) to 16 degrees C increased seed growth without appreciable effect on final leaf area. Increasing this temperature to 24 degrees C increased seed growth and reduced final leaf area. Leaves, stems, petioles, and pods acted as intermediate storage sites for (14)C assimilates. Only plants with higher night temperatures remobilized some of the stored assimilates during the period of rapid seed growth. Even the seeds in the 24 degrees C plants with the largest sink-source ratios did not utilize all the C-assimilates potentially available for remobilization. Nitrogen was readily remobilized from petioles, stems, and pods of all treatments as early as the beginning of seed development, but from the leaves only during late seed-filling. However, only plants with elevated night temperatures tended to remobilize all of the available N from vegetative tissues and pods. We concluded that a larger portion of stored assimilates may be remobilized to the seed if a strong seed sink can be sustained. It also appeared that with increasing sink-source ratios, N shortage might limit seed yield before a lack of C-assimilates would. A proposed model for soybean assimilate demand, distribution, partitioning, and remobilization is presented. PMID- 16664764 TI - Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules. AB - The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS(n1)) and a band (GS(n2)) similar to the single band (GS(r)) present in root extracts. In nodule extracts of P. lunatus, the GS(n1) band was detected, but the GS(n2) band was barely detectable. In contrast to P. vulgaris, the GS(n2) band and the GS(r) band of P. lunatus appeared to be different. The electrophoretic mobility of the GS(n1) band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS(n1) activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS(n1) of increasing electrophoretic mobility during the course of nodule development. PMID- 16664766 TI - Interferences and specificity of the 1-aminocyclopropane-1-carboxylic Acid assay with the hypochlorite reagent. AB - 1-Aminocyclopropane-1-carboxylic Acid (ACC), the immediate precursor of ethylene is routinely assayed by converting it into ethylene with NaOCl, and the ethylene liberated is then determined by gas chromatography (MCC Lizada, SF Yang 1979 Anal Biochem 100: 140-145). However, certain materials which may be present in crude plant extracts or in enzyme reaction mixtures interfere with this assay procedure. Mono, and di-alkyl amines cause poor yields of ethylene from ACC. Ethanol in the presence of NH(3) or amines but in the absence of ACC can produce ethylene under the assay procedure. The characteristics of these interfering reactions were studied and precautions to avoid these problems are suggested. Recovery of ACC during its extraction and purification from plant extracts were tested and are discussed. PMID- 16664765 TI - Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors. AB - A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides. PMID- 16664767 TI - Subcellular Localization of Alkaloids and Dopamine in Different Vacuolar Compartments of Papaver bracteatum. AB - Fractionation of Papaver bracteatum Arya II Lindl. latex on Renografin step gradients revealed that 43% of the dopamine was compartmentalized along with alpha-mannosidase (40%) in vacuoles sedimenting in the 2% fraction. Twenty-two percent of the dopamine was in the supernatant, but a corresponding amount (18%) of alpha-mannosidase was also present suggesting vacuole breakage during isolation. By subcellular fractionation of protoplasts from cultured P. bracteatum cells, the 1,000g sedimenting organelles have been identified as the major site of accumulation of the morphinan alkaloid, thebaine (99+/-0.8%), and the benzophenanthridine alkaloid, sanguinarine (96+/-3%). Although the 1,000g pellet also contained 33+/-4% of the total alkaloid precursor, dopamine, and half of the total vacuolar marker enzyme, alpha-mannosidase, 62+/-10% of the amine was localized in the 100,000g supernatant. A differential distribution of the alkaloids was discovered upon resolution of the lysed protoplasts on Renografin step gradients. Over 40% of the dopamine was in the supernatant with 15% in a 2% Renografin band. The remainder was evenly distributed in denser fractions of the gradient. The 4 to 8% interface, previously found to contain the largest amount of thebaine and small amounts of sanguinarine and dopamine, has been shown to be enriched in vacuoles by electron microscopy. Using a histofluorescence method, dopamine compartmentation in vacuoles of intact cultured cells was corroborated. In summary, dopamine, sanguinarine, and thebaine occur in vacuoles of different densities. A large fraction of the total dopamine in cultured cells was found in the 100,000g supernatant along with 37% of the alpha-mannosidase suggesting that the amine may be sequestered in more fragile vacuoles than the alkaloids. The possibility that some dopamine may be cytosolic cannot be ruled out. PMID- 16664768 TI - Ultrastructural Changes in Potato Tuber Pith Cells during Brown Center Development. AB - Electron microscopy revealed that subjecting ;Russet Burbank' potato (Solanum tuberosum L.) plants to 2 days of cool temperature growing conditions (18 degrees C days/10 degrees C nights) did not produce visible damage or changes in tuber pith tissue when compared to warm-grown tubers (23 degrees C days/18 degrees C nights). However, damage to some tuber pith cells was observed after 5 days of cool treatment. Eight days of cool treatment produced extensive alterations in cell structure. The cytoplasm of the cool-treated tuber pith cells had become highly vesiculated and there was evidence of complete destruction of amyloplast membranes and tonoplasts. In many cases the starch grains appeared to be undergoing hydrolysis suggesting total disruption of normal cell function. Sixteen days of cool treatment were sufficient to produce visible brown center development in all cool-grown tubers examined. Electron microscopy of these tissues revealed that, although some organelles were still present, the cytoplasm had become extremely vesiculated and lacked any resemblance to that of tissue from warm-grown tubers. Gross, irregular thickening of cell walls was also detected. PMID- 16664769 TI - Postanthesis nitrate assimilation in winter wheat : in situ flag leaf reduction. AB - When adequate levels of soil NO(3) (-) are available, concurrent NO(3) (-) absorption and assimilation, and mobilization of vegetative N reserves accumulated prior to anthesis, may be used to supply N to developing wheat (Triticum aestivum L.) kernels. Vegetative wheat components (stems, leaves, spike) are known to possess NO(3) (-) reductase activity, but the in situ utilization of NO(3) (-) translocated to the shoot has not been studied. Assimilation and partitioning of (15)N was determined in winter wheat ;Doublecrop.' At 7 days after anthesis, the stem immediately above the peduncle node was heat girdled to block phloem export from the flag leaf. Control plants were not girdled. One day later, 50 micromoles of (15)NO(3) (-) (98 atom percent (15)N) was injected into the penultimate internodal lacuna, after which (15)NO(3) (-) utilization was determined sequentially over a 5 day period. Based on differences in spike accumulation of reduced (15)N excess between treatments and the amount of reduced (15)N excess remaining in the flag leaf, it was estimated that the flag leaf contributed 37% of the total reduced (15)N excess in the injected shoot. The lower shoot contribution was 18% and that of the peduncle plus spike was 45%. PMID- 16664770 TI - (1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization. AB - A (1-->3)-beta-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1-->3)-beta-glucan by methylation analysis and by glucanase digestion. (1-->3)-beta-Glucan synthase activity is markedly stimulated by Ca(2+); activation is half-maximal at about 50 micromolar Ca(2+) and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg(2+), Mn(2+), and Sr(2+), also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by beta-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein. PMID- 16664771 TI - Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis. AB - A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1 naphthylphthalamic acid (NPA), glucan synthetase-II, and K(+)-stimulated, vanadate-inhibited Mg(2+) ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%. PMID- 16664772 TI - Quantitative Measurements of Hexokinase Activity in the Shoot Apical Meristem, Leaf Primordia, and Leaf Tissues of Dianthus chinensis L. AB - Hexokinase was measured by quantitative histochemical techniques in the apical meristem, primordia, and leaves of Dianthus chinensis L. The structural stages of development in the leaves sampled were determined by light and scanning electron microscopy. The results showed that activity decreased from the youngest primordia (1500 millimoles per kilogram dry weight per hour) to the mature leaves (200 millimoles per kilogram dry weight per hour) and that an intermediate leaf, the fourth youngest, showed the same declining pattern from its base to its tip. Surface views and measurements of these leaves revealed their basipetal maturation as seen by cell size, stomatal development, trichome differentiation, cuticular appearance, and leaf thickness. The intermediate leaf showed features representative of several stages in structural differentiation. It was concluded that the changes in hexokinase activity among the leaves of a shoot and within an individual leaf are similar and correlate with the degree of structural differentiation of the leaves. PMID- 16664773 TI - Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress. AB - Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress. PMID- 16664774 TI - The role of dark carbon dioxide fixation in root nodules of soybean. AB - The magnitude and role of dark CO(2) fixation were examined in nodules of intact soybean plants (Harosoy 63 x Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO(2) fixation, based on a 2 minute pulse-feed with (14)CO(2) under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO(2) fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO(2) within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO(2) fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O(2) (80:20) did not affect dark CO(2) fixation, while exposure to O(2):CO(2) (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO(2) fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity. PMID- 16664775 TI - Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to Glycosidases. AB - Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with alpha-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean alpha-mannosidase. (d) Treatment of the native proteins with alpha-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate denatured or pronase-digested glycoproteins removes all alpha-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized alpha-mannosidase. PMID- 16664776 TI - Root Respiration in White Spruce (Picea glauca [Moench] Voss) Seedlings in Relation to Morphology and Environment. AB - Roots of white spruce seedlings (Picea glauca [Moench] Voss) undergo respiratory changes during the year that are related to changing metabolic requirements. An alternative pathway is always present, functions during most of the year, and operates maximally during periods of root and shoot growth. Although some differences in respiration and the apportioning of respiration can be correlated to root morphology, the environment and the stage of shoot development are also important controls. Differences in respiration related to root morphology are not manifest in mitochondrial structure, but overall rates were found to correlate with the number of mitochondria present. Root respiration in seedlings grown under root growth capacity conditions reflects root and shoot growth at that time rather than root growth capacity. PMID- 16664777 TI - Oxygen Stimulation of Apparent Photosynthesis in Flaveria linearis. AB - A plant was found in the C(3)-C(4) intermediate species, Flaveria linearis, in which apparent photosynthesis is stimulated by atmospheric O(2) concentrations. A survey of 44 selfed progeny of the plant showed that the O(2) stimulation of apparent photosynthesis was passed on to the progeny. When leaves equilibrated at 210 milliliters per liter O(2) were transferred to 20 milliliters per liter O(2) apparent photosynthesis was initially stimulated, but gradually declined so that at 30 to 40 minutes the rate was only about 80 to 85% of that at 210 milliliters per liter O(2). Switching from 20 to 210 milliliters per liter caused the opposite transition in apparent photosynthesis. All other plants of F. linearis reached steady rates within 5 minutes after switching O(2) that were 20 to 24% lower in 210 than in 20 milliliters per liter O(2). At low intercellular CO(2) concentrations and low irradiances, O(2) inhibition of apparent photosynthesis of the aberrant plant was similar to that in normal plants, but at an irradiance of 2 millimoles quanta per square meter per second and near 300 microliters per liter CO(2) apparent photosynthesis was consistently higher at 210 than at 20 milliliters per liter O(2). In morphology and leaf anatomy, the aberrant plant is like the normal plants in F. linearis. The stimulation of apparent photosynthesis at air levels of O(2) in the aberrant plant is similar to other literature reports on observations with C(3) plants at high CO(2) concentrations, high irradiance and/or low temperatures, and may be related to limitation of photosynthesis by triose phosphate utilization. PMID- 16664778 TI - Responses of cultured parsley cells to elicitors from phytopathogenic fungi : timing and dose dependency of elicitor-induced reactions. AB - Cultured parsley cells (Petroselinum crispum) responded to treatment with heat released soluble cell-wall fragments (elicitors) from several different phytopathogenic fungi by forming coumarin derivatives (phytoalexins). This response was preceded in all cases by large but transient increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). The activities of two hydrolytic enzymes, chitinase and 1,3-beta-glucanase, also increased strongly in elicitor-treated cells, whereas the activities of three enzymes participating in primary metabolism were affected differently by the elicitor treatment. Glucose-6 phosphate dehydrogenase increased, phosphofructokinase remained almost constant, and pyrophosphate:fructose-6-phosphate phosphotransferase declined sharply in activity. Different amounts of cell-wall preparations from various phytopathogenic fungi were required for maximum elicitor activity. While three oomycetes (Phytophthora spp.) yielded the most active elicitors studied (maximum coumarin accumulation at concentrations of about 10 microgram per milliliter), cell-wall preparations from an ascomycete and three deuteromycetes gave comparable results only at 10 to 100 times higher concentrations. Optimal induction of PAL, 4CL, and chitinase with Phytophthora elicitor required only about 1 microgram per milliliter, whereas 1,3-beta-glucanase induction showed a dose dependence similar to that observed for coumarins. The elicitor concentration had pronounced effects not only on the extent, but also on the timing of all induced reactions. PMID- 16664779 TI - Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.). AB - The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F(0) to F(1) ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N'-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme. PMID- 16664780 TI - Cell Surfaces in Plant-Microorganism Interactions : VI. Elicitors of Ethylene from Colletotrichum lagenarium Trigger Chitinase Activity in Melon Plants. AB - Treatment of melon leaves or seedlings with elicitors of Colletotrichum lagenarium, a fungal pathogen of melon, increases chitinase activity. In treated leaves, chitinase is enhanced within the first 6 hours and becomes 2 to 10 times higher than in control leaves after 24 hours. Ethylene is increased simultaneously and is correlated with chitinase elicitation. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, both elicitor induced ethylene and elicitor-induced chitinase are inhibited. This inhibition is overcome by added exogenous ethylene. On the other hand, 1-aminocyclopropane-1 carboxylic acid the direct precursor of ethylene, triggers chitinase activity. Chitinase elicitation is thought to be a protein synthesis dependent process, as it does not occur in the presence of cycloheximide. PMID- 16664781 TI - Cross-linking patterns in salt-extractable extensin from carrot cell walls. AB - Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as "extensin-1" to distinguish it from "extensin-2," a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated ( congruent with84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix. PMID- 16664782 TI - The role of carbohydrate in maintaining extensin in an extended conformation. AB - Monomers of the plant cell wall glycoprotein extensin are secreted into the wall where they become cross-linked to each other to form a rigid matrix. Expression of the extensin matrix is correlated with the inhibition of further cell elongation during normal development, with increased resistance to virulent pathogens and with other physiological responses characterized by wall strengthening. Carbohydrates make up about two-thirds of the mass of extensin. Arabinose oligomers linked to hydroxyproline residues represent 95% of the total carbohydrate with the remainder occurring as single residues of galactose linked to some serine residues. Electron microscopy of shadowed extensin shows the glycosylated form to be an easily visualized and highly elongated molecule. In contrast, extensin that has been deglycosylated with anhydrous hydrogen fluoride is difficult to resolve in the EM. Glycosylated extensin elutes from a gel filtration column much more rapidly than does the deglycosylated form, and from this analysis we have calculated respective Stokes' radii of 89 and 11 Angstroms for these molecules. Others have shown that inhibition of extensin glycosylation has no effect on its secretion or insolubilization in the cell wall, but that this extensin cannot inhibit cell elongation. It is likely that carbohydrate moieties keep extensin in an extended conformation and that extensin must be in this conformation to form a cross-linked matrix that can function properly in vivo. PMID- 16664783 TI - Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria. AB - Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features. PMID- 16664784 TI - Reversal of Abscisic Acid-Induced Stomatal Closure by trans-Cinnamic and p Coumaric Acid. AB - Abscisic acid (ABA)-induced increase in stomatal diffusive resistance (SDR) in excised leaves of bean (Phaseolus vulgaris L. cv Pencil Pod) and maize (Zea mays L. cv Golden Bantam) is inhibited by low concentrations of trans-cinnamic acid (TCA) (1 micromolar) and p-coumaric acid (PCA) (10 micromolar) when given together with ABA (10 micromolar) in the transpiration stream through the cut end of the petiole or leaf blade. A concentration effect is observed both in the ABA action and its reversal by phenolic acids. Leaves having attained a high diffusive resistance in ABA solution recover rapidly when transferred to water. ABA (10 micromolar) induced closure of the stomata in onion, Allium cepa L. and Vicia faba epidermal peels. This is associated with loss of K(+) from guard cells. In the presence of TCA (10 micromolar) and PCA (10 micromolar) K(+) is retained in the guard cells with open stomata. The dark closure of stomata is also inhibited by TCA and PCA. It is suggested that these phenolic acids may inhibit the ABA effect by competing with or acting on some ABA-specific site, probably located on the plasma membrane, regulating flux of K(+) ions. A weak association of ABA with the plasma membrane is envisaged because of the rapid recovery obtained upon transferral of the leaves to water. PMID- 16664785 TI - The effect of dormancy on the heat shock response in gladiolus cormels. AB - Cormels of Gladiolus X gandavensis Van Houtte respond to heat shock by an induced synthesis of heat shock proteins. Synthesis of some of the non-heat shock proteins is concomitantly reduced. The ability of dormant cormels to synthesize heat shock proteins (hsps) and to repress the synthesis of non-hsps is greater than that of nondormant ones. A hsp of apparent molecular weight 68 kilodaltons is synthesized only in dormant cormels or in cormels that lost their dormancy after long storage at 25 degrees C. The synthesis of hsps at 40 degrees C, but not at 25 degrees C, is promoted by abscisic acid in nondormant cormels. Methionine incorporation into hsps declines after a 4-hour incubation period at 40 degrees C. Induction of hsps is stronger if exposure to extreme temperature is done gradually. PMID- 16664786 TI - Characterization of water stress and low temperature effects on flower induction in citrus. AB - Experiments were conducted with containerized ;Tahiti' lime (Citrus latifolia Tan.) trees in order to define conditions needed to induce flowering. Cyclical or continuous water stress for 4 to 5 weeks induced flowering. Moderate (-2.25 megapascals, midday) or severe (-3.5 megapascals, midday) water stress as measured by leaf xylem pressure potential, for as little as 2 weeks induced flowering, but the response was more significant in severely stressed trees. Low temperature (18 degrees C day/10 degrees C night) induced a time dependent flowering response much like that of moderate water stress. Significantly negative leaf xylem pressure potentials as compared to controls were found only under water stress treatment, suggesting that a common stress-linked event, separate from low plant water potential is involved in floral induction. Leafless, immature cuttings from mature, field-grown trees were induced to flower by water stress treatment, suggesting that leaves are not essential for a flower inductive response. PMID- 16664787 TI - The effect of dormancy on glucose uptake in gladiolus cormels. AB - The uptake of glucose and of 3-O-methyl-d-glucose by cormel slices of Gladiolus X gandavensis Van Houtte was studied in relation to cormel dormancy. Uptake was higher in nondormant cormels. Incubation of nondormant cormels with abscisic acid (ABA) reduced their uptake capacity. Treatment of dormant cormels with 6 benzyladenine (BA) did not affect their uptake rate. ABA and BA promoted O(2) uptake, indicating that differences in uptake are not related to differences in energy supply. PMID- 16664788 TI - Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum. AB - Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO(3) (-) or NH(4) (+) induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH(4) (+) addition resulted in O(2) consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO(3) (-) or NH(4) (+) resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O(2) evolution, dark respiration, and dark C-fixation are shown to be consistent with this model. PMID- 16664789 TI - Carbohydrate partitioning and the capacity of apparent nitrogen fixation of soybean plants grown outdoors. AB - Patterns of leaf carbohydrate partitioning and nodule activity in soybean plants grown under natural conditions and the irradiance level required to produce sufficient carbohydrate to obtain maximum rates of apparent N(2)-fixation (acetylene reduction) were measured. Soybean plants, grown outdoors, maintained constant levels of leaf soluble sugars while leaf starch pools varied diurnally. When root temperature was kept at 25 degrees C and shoot temperature was allowed to vary with ambient temperature, the plants maintained constant rates of apparent N(2)-fixation and root+nodule respiration. Results from a second experiment, in which the entire plant was kept at 25 degrees C, were similar to those of the first experiment. Shoot carbon exchange rate of plants from the second experiment was light saturated at photosynthetic photon flux densities between 400 and 600 micromoles per square meter per second. When plants were subjected to an extended 40-hour dark period to deplete carbohydrate reserves, apparent N(2)-fixation was unaffected during the first 10 hours of darkness, decreased rapidly between 10 and 16 hours, and plateaued at one-third the initial level thereafter. After the extended dark period, plants were exposed to photosynthetic photon flux density from 200 to 1000 micromoles per square meter per second for 10 hours. Photosynthetic photon flux densities of 200 micromoles per square meter per second and greater resulted in maximum leaf soluble sugar content and nodule activity. Leaf starch content increased with irradiance levels up to 600 micromoles per square meter per second with no further increase at higher irradiance levels. Results presented here indicate that maximum nodule activity occurs at irradiance levels that do not saturate the plant's photosynthetic apparatus. This response would allow for maximum N(2)-fixation to occur in a nodulated legume during periods of inclement weather. PMID- 16664790 TI - Uptake of Choline and Ethanolamine by Lemna paucicostata Hegelm. 6746. AB - Lemna paucicostata Hegelm. 6746 possesses specific systems for uptake of choline and ethanolamine. Each is distinct from the six other systems for uptake of organic compounds so far identified in this plant. Both systems show biphasic kinetics, so that uptake by them can be described as the composite result of two Michaelis-Menten processes. Inhibitor studies are reported which indicate the very strict structural specificity of each system. The kinetic constants of choline uptake are such that, at an external concentration of 0.65 micromolar, the total requirement of the plant for this compound would be met, 41% via the high affinity system and 59% via the lower. At an external concentration of 2.4 micromolar ethanolamine, an amount of this compound sufficient to form the total choline of the plant would be supplied, 59% via the high affinity system and 41% via the lower. These, and other observations, strongly support the physiological importance of these systems under natural conditions. PMID- 16664791 TI - Short-Term Flooding Effects on Gas Exchange and Quantum Yield of Rabbiteye Blueberry (Vaccinium ashei Reade). AB - Roots of 1.5-year-old ;Woodard' rabbiteye blueberry plants (Vaccinium ashei Reade) were flooded in containers or maintained at container capacity over a 5 day period. Carbon assimilation, and stomatal and residual conductances were monitored on one fully expanded shoot/plant using an open flow gas analysis system. Quantum yield was calculated from light response curves. Carbon assimilation and quantum yield of flooded plants decreased to 64 and 41% of control values, respectively, after 1 day of flooding and continued decreasing to 38 and 27% after 4 days. Stomatal and residual conductances to CO(2) also decreased after 1 day of flooding compared with those of unflooded plants with residual conductance severely limiting carbon assimilation after 4 days of flooding. Stomatal opening occurred in 75 to 90 minutes and rate of opening was unaffected by flooding. PMID- 16664792 TI - Phosphoserine aminotransferase in soybean root nodules : demonstration and localization. AB - Phosphoserine aminotransferase activity was detected in the plant and bacteroid fractions from soybean (Glycine max) root nodules. Both total and specific activities increased in the plant fraction during nodule development. Serine pyruvate aminotransferase activity was not detectable in the plant or bacteroid fractions of these nodules. Sucrose density gradient fractionation indicated a proplastid localization for phosphoserine aminotransferase. The data presented support a role for this enzyme in carbon supply to purine biosynthesis in the pathway of ureide biogenesis in soybean nodules. PMID- 16664793 TI - Quantum Yields of CAM Plants Measured by Photosynthetic O(2) Exchange. AB - The quantum yield of photosynthetic O(2) exchange was measured in eight species of leaf succulents representative of both malic enzyme type and phosphoenolpyruvate carboxykinase type CAM plants. Measurements were made at 25 degrees C and CO(2) saturation using a leaf disc O(2) electrode system, either during or after deacidification. The mean quantum yield was 0.095 +/- 0.012 (sd) moles O(2) per mole quanta, which compared with 0.094 +/- 0.006 (sd) moles O(2) per mole quanta for spinach leaf discs measured under the same conditions. There were no consistent differences in quantum yield between decarboxylation types or during different phases of CAM metabolism. On the basis of current notions of compartmentation of CAM biochemistry, our observations are interpreted to indicate that CO(2) refixation is energetically independent of gluconeogenesis during deacidification. PMID- 16664794 TI - Accumulation of Chlorophyll, Chloroplastic Proteins, and Thylakoid Membranes during Reversion of Chromoplasts to Chloroplasts in Citrus sinensis Epicarp. AB - In vitro culture of pericarp segments from fruit of Citrus sinensis (L.) Osbeck cv Valencia was used to determine the temporal sequence in development of chloroplasts from chromoplasts during regreening of the epicarp. Regreening of chromoplasts closely resembled greening of etioplasts, except that regreening proceeded much more slowly. Chlorophyll, the light-harvesting chlorophyll a/b binding protein of photosystem II, the chlorophyll a binding protein of reaction center P-700 of photosystem I, thylakoid membranes, and adenosine triphosphate synthetase were all detected at very low levels in degreened epicarp. All of these increased in parallel during regreening of the epicarp. Ribulose 1,5 bisphosphate carboxylase (RuBPCase) levels were high in degreened epicarp and declined for the first 10 days of culture before reaccumulating in the regreening segments. Light was necessary for the accumulation of all of the chloroplastic components. A lack of exogenous nitrogen did not prevent the accumulation of any chloroplastic component except Ru-BPCase, although accumulation of the other components was reduced. Sucrose at 150 millimolar in media lacking nitrogen markedly inhibited the accumulation of chlorophyll and light-harvesting chlorophyll a/b-protein. PMID- 16664795 TI - Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System. AB - Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were transformed with a kanamycin resistance marker (neomycin phosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant. PMID- 16664796 TI - Can lateral redistribution of auxin account for phototropism of maize coleoptiles? AB - Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip. PMID- 16664797 TI - Evidence for Cyclic Electron Flow around Photosystem II in Chlorella pyrenoidosa. AB - Electron flow around photosystem II was investigated in Chlorella pyrenoidosa. Using a bare platinum O(2) electrode, simultaneous measuremnts were made of steady-state photosynthesis in continuous light, the yield of oxygen (Y(o(2) )) produced by a superimposed saturating xenon flash, and the change in fluorescence yield of a weak flash triggered before and 70 microseconds after the saturating flash. Throughout most of the continuous photosynthesis-irradiance curve, normalized O(2) flash yields (Y(o(2) )/Y(o(2)max)) and normalized variable fluorescence yields (Deltaphi/Deltaphi') were linearly correlated with a slope of 1.0. As photosynthetic rates reached light saturation, however, the variable fluorescence yields remained relatively constant while O(2) flash yields decreased. These results strongly suggest that there is a cyclic electron flow around photosystem II in unpoisoned intact cells at light saturation and supraoptimal light intensities. PMID- 16664798 TI - Immunocytochemical Analysis Shows that Glyoxysomes Are Directly Transformed to Leaf Peroxisomes during Greening of Pumpkin Cotyledons. AB - The functional transition of glyoxysomes to leaf peroxisomes occurs during greening of germinating pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). The immunocytochemical protein A-gold method was employed in the analysis of the transition using glyoxysomal specific citrate synthase immunoglobulin G and leaf peroxisomal specific glycolate oxidase immunoglobulin G. The labeling density of citrate synthase was decreased in the microbodies during the greening, whereas that of glycolate oxidase was dramatically increased. Double labeling experiments using different sizes of protein A-gold particles show that both the glyoxysomal and the leaf peroxisomal enzymes coexist in the microbody of the transitional stage indicating that glyoxysomes are directly transformed to leaf peroxisomes during greening. PMID- 16664799 TI - Isolation of Sperm Cells from the Pollen of Plumbago zeylanica. AB - Intact sperm cells of Plumbago zeylanica were released from mature pollen grains near anthesis using osmotic shock with 20% sucrose. Sperm cell yields of up to 75% can be attained by differential centrifugation using a clinical centrifuge with concentrations of up to 8.8 x 10(6) cells/milliliter. Such ;gametoplasts' remain intact for up to 24 hours according to Evan's blue exclusion and can be used for characterization or physiological manipulation. PMID- 16664800 TI - The high mannose oligosaccharide of phytohemagglutinin is attached to asparagine 12 and the modified oligosaccharide to asparagine 60. AB - Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, has a high mannose and a modified (fucosylated) oligosaccharide on each polypeptide. Fractionation by high performance liquid chromatography of tryptic digests of [(3)H]fucose or [(3)H]glucosamine labeled phytohemagglutinin, followed by amino acid sequencing of the isolated glycopeptides, shows that the high mannose oligosaccharide is attached to Asn(12) and the modified oligosaccharide to Asn(60) of the protein. In animal glycoproteins, high mannose chains are rarely found at the N-terminal side of complex chains. PMID- 16664801 TI - A rapid, simple synthesis and purification of abscisic Acid glucose ester. AB - A simple and rapid technique was developed to synthesize abscisic acid glucose ester. The free acid of abscisic acid (ABA) was combined with CsHCO(3) to form the Cs salt of ABA. The Cs salt of ABA was then combined with acetobromo-alpha-d glucose tetraacetate, and the tetraacetate derivative of abscisic acid glucose ester was formed. Acetate groups were selectively removed from the glucose moiety with a crude enzyme preparation derived from Helianthus annuus seeds. Abscisic acid glucose ester was purified via silica gel column chromatography and identified by micro NMR. PMID- 16664802 TI - Stimulation of Nodulation in the Clover-Rhizobium trifolii 0403 System by Penicillin and Mecillinam. AB - The number of nodules produced per clover seedling inoculated with Rhizobium trifolii 0403 can be increased almost 2-fold by the addition of penicillin or mecillinam. Two-day-old dutch white clover seedlings grown in 250 milliliter boston round jars containing agar-solidified plant growth medium were inoculated with exponentially growing Rhizobium trifolii 0403 cells. Penicillin or mecillinam (100 micrograms per milliliter) were added immediately or after 24 hours. Following 42 days growth, 10 replicate sets of 5 plants for each treatment were assayed for nodule number, plant dry weight, and Kjeldahl nitrogen. Both antibiotics increased nodule number, plant dry weight, and Kjeldahl nitrogen. Increases in nodule number and dry weight were statistically significant. The range of values in Kjeldahl nitrogen was so extensive as to make the data insignificant at the P < 0.05 level, however nodule number, plant dry weight, and Kjeldahl nitrogen displayed a significant correlation with each other. There were no significant differences in treatment with either antibiotic or with time of treatment. Nodule number increased by about 85%, and plant dry weight and nitrogen increased by about 30%. PMID- 16664803 TI - Amino Acid Transport in Suspension-Cultured Plant Cells : VI. Influence of pH Buffers, Calcium, and Preincubation Media on l-Leucine Uptake. AB - The rate at which l-leucine was transported into suspension-cultured Nicotiana tabacum cv Wisconsin 38 cells increased more than 2-fold over a period of hours when the cells were preincubated in a 1% sucrose solution. This increase in uptake rate was eliminated if certain tris buffers were included in the preincubation solution while other buffers had little effect. Calcium could reverse the effect of the inhibitory buffers only if the buffer and calcium were present together from the beginning of the preincubation period. It was the amine group of the inhibitory buffers which was responsible for the inhibition. Preincubation in a complete culture medium (EM Linsmaier, F Skoog 1965 Physiol Plant 18: 100-127) led to minimal changes in l-leucine uptake rate over a 10 hour preincubation period indicating that the uptake rate was stabilized by this medium. The complete medium stabilized the l-leucine uptake rate as a result of its ionic composition and not because of its osmolarity. Most of the increased uptake rate observed after preincubation in a 1% sucrose solution could be inhibited by 2,4-dinitrophenol or carbonyl cyanide m-chlorophenyl hydrazone, or high concentrations of l-phenyl-alanine or l-leucine. Therefore much of the increase could be accounted for by an increase in active transport of l-leucine. PMID- 16664804 TI - A Comparison of Oleic Acid Metabolism in the Soybean (Glycine max [L.] Merr.) Genotypes Williams and A5, a Mutant with Decreased Linoleic Acid in the Seed. AB - The metabolism of oleoyl coenzyme A (CoA) was examined in developing seed from two soybean (Glycine max [L.] Merr.) genotypes: Williams, a standard cultivar and A5, a mutant containing nearly twice the oleic acid (18:1) content of Williams. The in vitro rates of esterification of oleoyl-CoA to lysophosphatides by acyl CoA: lysophosphatidylcholine acyltransferase was similar in both genotypes and lysophosphatidyl-ethanolamine was a poor substrate. Crude extracts desaturated exogenous [1-(14)C]dioleoyl phosphatidylcholine at 14% of the rate achieved with [1-(14)C]oleoyl-CoA, and 50 micromolar lysophosphatidylcholine. The desaturase enzyme also required NADH for full activity. Extracts from Williams contained 1.5 fold more oleoyl phosphatidylcholine desaturase activity, on a fresh weight basis, than did A5 and appeared to have a similar affinity for oleoyl-CoA. There was 1.2- to 1.9-fold more linoleic acid (18:2) in phosphatidylcholine from Williams than from A5, measured at two stages of development, but both genotypes had a similar distribution of fatty acids in the one and two positions. Phosphatidylethanolamine in A5 contained relatively more linoleic acid (18:2) in the one position than did Williams. The increased oleic acid (18:1) content in A5 appeared to be a result of decreased rates of 18:1 desaturation of oleoyl phosphatidylcholine in this genotype. PMID- 16664805 TI - Continuous monitoring of plant water potential. AB - Plant water potential was monitored continuously with a Wescor HR-33T dewpoint hygrometer in conjunction with a L51 chamber. This commercial instrument was modified by replacing the AC-DC mains power converter with one stabilized by zener diode controlled transistors. The thermocouple sensor and electrical lead needed to be thermally insulated to prevent spurious signals. For rapid response and faithful tracking a low resistance for water vapor movement between leaf and sensor had to be provided. This could be effected by removing the epidermis either by peeling or abrasion with fine carborundum cloth. A variety of rapid plant water potential responses to external stimuli could be followed in a range of crop plants (sunflower (Helianthus annuus L., var. Hysun 30); safflower (Carthamus tinctorious L., var. Gila); soybean (Glycine max L., var. Clark); wheat (Triticum aestivum L., var. Egret). These included light dark changes, leaf excision, applied pressure to or anaerobiosis of the root system. Water uptake by the plant (safflower, soybean) mirrored that for water potential changes including times when plant water status (soybean) was undergoing cyclical changes. PMID- 16664806 TI - Transcriptional and Posttranscriptional Control of Phaseolin and Phytohemagglutinin Gene Expression in Developing Cotyledons of Phaseolus vulgaris. AB - The expression of phaseolin and phytohemagglutinin (PHA) in the developing cotyledons of a normal (Greensleeves) and a PHA-deficient (Pinto 111) cultivar of Phaseolus vulgaris was investigated. Phaseolin mRNA translational activity and abundance were present at similar levels in both cultivars. In contrast, PHA mRNA translational activity and abundance in Pinto 111 were less than 1% of the levels measured in Greensleeves. Using nuclear runoff assays, the transcription rate of phaseolin gene sequences was similar in both cultivars. The transcription rate of PHA gene sequences in Pinto 111 was only 20% of that measured in Greensleeves. Comparison of the transcription rates with the relative mRNA amounts measured in RNA blot hybridizations indicated that the normally expressed storage protein gene mRNAs were very stable with half-lives greater than several days. Because a low level of PHA gene transcription in Pinto 111 was measurable but no PHA mRNA accumulated, these results suggest that the PHA deficiency in Pinto 111 is due to a reduced transcription rate and possibly an instability of the mRNA. PMID- 16664807 TI - Inhibitors of proton pumping: effect on passive proton transport. AB - Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H(+) conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H(+) conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H(+). However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H(+) conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically. PMID- 16664808 TI - Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex. AB - Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b(6)/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700 chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b(6), and Rieske Fe-S center protein in cytochrome b(6)/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b(6)/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b(6), and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems. PMID- 16664809 TI - The influence of ammonium and chloride on potassium and nitrate absorption by barley roots depends on time of exposure and cultivar. AB - Net uptakes of K(+) and NO(3) (-) were monitored simultaneously and continuously for two barley (Hordeum vulgare) cultivars, Prato and Olli. The cultivars had similar rates of net K(+) and NO(3) (-) uptake in the absence of NH(4) (+) or Cl( ). Long-term exposure (over 6 hours) to media which contained equimolar mixtures of NH(4) (+), K(+), Cl(-), or NO(3) (-) affected the cultivars very differently: (a) the presence of NH(4) (+) as NH(4)Cl stimulated net NO(3) (-) uptake in Prato barley but inhibited net NO(3) (-) uptake in Olli barley; (b) Cl(-) inhibited net NO(3) (-) uptake in Prato but had little effect in Olli; and (c) NH(4) (+) as (NH(4))(2)SO(4) inhibited net K(+) uptake in Prato but had little effect in Olli. Moreover, the immediate response to the addition of an ion often varied significantly from the long-term response; for example, the addition of Cl(-) initially inhibited net K(+) uptake in Olli barley but, after a 4 hour exposure, it was stimulatory. For both cultivars, net NH(4) (+) and Cl(-) uptake did not change significantly with time after these ions were added to the nutrient medium. These data indicate that, even within one species, there is a high degree of genotypic variation in the control of nutrient absorption. PMID- 16664810 TI - Primary structure of a proline-rich zein and its cDNA. AB - Eighty-five cDNA clones for gamma-zein (proline-rich zein) from a cDNA expression library were isolated using specific antibody and cDNA probes. Nucleotide sequences of seven independent clones were determined and found to be identical in regions where they overlapped. The primary structure of the mature protein, determined from the sequence of one near full-length clone, consists of 204 amino acids. It has a molecular weight of 21,824 daltons, about 5 kilodaltons less than that estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal one-half of the sequence contained eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln Pro-His-Pro-Cys-Pro-Cys-Gln. The codon specifying the third proline in the hexapeptide repeating units is identical (CCG) in all of the eight repeats. The coding region has a very high G-C content (69.8%). The multiple charge components of gamma-zein detected by isoelectric focusing do not seem to be encoded by members of a multigene family. Moreover, it was found that the codon preference in gamma-zein is, in fact, the base preference in the wobble position. A codon usage value was devised to express this phenomenon. PMID- 16664811 TI - Amino Acid transport in protoplasts isolated from soybean leaves. AB - We isolated large quantities of mesophyll protoplasts from source and sink leaves of soybean plants and examined them for amino acid uptake. Accumulation of amino acids in isolated protoplasts was linear for at least 40 minutes. Uptake kinetics revealed the presence of both saturable and linear components. Increasing external pH decreases the uptake. The uncoupler, carbonyl cyanide p trifluoromethoxyphenylhydrazone at 15 micromolar inhibited and fusicoccin at 10 micromolar stimulated amino acid uptake. Our data are consistent with a proton cotransport mechanism for the uptake of l-glutamine and alpha-amino isobutyric acid into soybean mesophyll cells. PMID- 16664812 TI - Causes for the Disappearance of Photosynthetic CO(2) Fixation with Isolated Spinach Chloroplasts. AB - When isolated spinach chloroplasts are illuminated, photosynthesis and CO(2) fixation die off within 30 to 90 minutes. Even when air levels of CO(2) are used which maintain high and rate-saturating amounts of ribulose 1,5-bisphosphate inside the plastids, CO(2) fixation declines. The decline begins with a drop in activity of the ribulose 1,5-bishosphate carboxylase/oxygenase, specifically loss of the enzyme-activator CO(2)-Mg(2+) form. Next, the light reactions cause gradual leakage of the carboxylase and other stromal proteins to the suspending medium. The chloroplast outer envelope appears to reseal and protect the thylakoids since there is little change in the ferricyanide-dependent Hill reaction. In the dark, under otherwise identical conditions, leakage of carboxylase does not occur. PMID- 16664813 TI - Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. AB - I have developed promoter expression binary vectors based on the tumor-inducing plasmid of Agrobacterium tumefaciens to facilitate elucidation of plant gene regulation. Promoter activity can be determined by inserting DNA fragments into the multiple cloning sites of the vectors forming transcriptional and/or translational fusions between the cat structural gene and an inserted promoter region. The activity of the nopaline synthase (nos) promoter was demonstrated with the vector. However, three animal promoters tested with this system showed no measurable activity in plant cells. Examination of 40 independently derived transformed tissues revealed a 200-fold difference in the nos promoter activity. Furthermore, there is no apparent correlation between the neomycin phosphotransferase and chloramphenicol acetyltransferase activities, although both genes are closely linked and under control of identical nos promoters. These results indicate that vast differences in promoter activity of transferred genes can occur within the same cell, as well as in independently derived cell lines. PMID- 16664814 TI - Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina. AB - A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na(+), K(+), and Ca(2+) concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K(+), and 0.3 millimolar Ca(2+) are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K(+) concentration is maintained constant over a wide range of media K(+) concentrations (0.5-10 millimolar), leading to a ratio of K(+) in the cells to K(+) in the medium of 10 to 1,000. Severe limitation of external K(+), induces loss of K(+) and increase in Na(+) inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na(+) and accumulate K(+) and that intracellular Na(+) and K(+) concentrations are carefully regulated. The contribution of the intracellular Na(+) and K(+) salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%. PMID- 16664815 TI - Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves. AB - A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4 degrees C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl(2), 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58 degrees C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25 degrees C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide. PMID- 16664816 TI - A Temperature-Sensitive Chlorophyll b-Deficient Mutant of Sweetclover (Melilotus alba). AB - The ch4 mutant of sweetclover (Melilotus alba) has previously been demonstrated to be partially deficient in chlorophyll and to have a higher ratio of chlorophyll a to b than normal plants. We were able to substantiate these findings when plants were grown at 23 degrees C and lower (permissive temperatures). However, when grown at 26 degrees C (nonpermissive temperature) the plants produced small yellow leaves which exhibited one-twentieth the chlorophyll content of normal plants. Affected leaves did not increase their chlorophyll content when plants were incubated at permissive temperatures, but leaves which developed at the lower temperature contained increased amounts of chlorophyll. Similarly, only new leaves, not previously grown leaves, exhibited the yellow phenotype when the mutant plant was shifted from the permissive temperature to the nonpermissive temperature. Ribulose 1,5-bisphosphate carboxylase activity was decreased by half, relative to normal plants, in the mutant plants grown at the nonpermissive temperature, indicating that general protein synthesis was not greatly impaired and that the effect of the mutation was perhaps specific for chlorophyll content. HPLC analysis indicated that carotenoid content was not diminished to the same extent as chlorophyll and we have determined that the thylakoid protein kinase is not altered, as is the case for other chlorophyll b-deficient mutants. Experiments suggest that changes in photoperiod may be able to modulate the effect of temperature. PMID- 16664817 TI - Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway. AB - Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids. PMID- 16664818 TI - Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings. AB - FARNESYL TRANSFERASE (FARNESYL PYROPHOSPHATE: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400 fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 +/- 3,000 from its behavior on a calibrated G 100 Sephadex molecular sieving column. Mg(2+) ion at 4 millimolar gave the greatest stimulation of activity; Mn(2+) ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K(m) = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K(m) = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K(m) = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes-a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates. PMID- 16664819 TI - Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana. AB - Chlorella sorokiniana cells, cultured for 12 hours in 30 millimolar ammonium medium, contained an ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzyme with subunits having a molecular weight of 53,000. In vitro translation of total cellular poly(A)(+) RNA, isolated from fully induced cells, resulted in synthesis of an NADP-GDH antigen with a molecular weight of 58,500. The 58,500 dalton antigen was processed in vitro, with a 100,000g supernatant prepared from broken fully induced Chlorella cells, to a protein with a molecular weight of 53,000. These data support the inference that the NADP-GDH subunit (M(r) = 53,000) is initially synthesized as a larger precursor protein (M(r) = 58,500). By use of a cytochemical staining procedure, dependent upon NADP-GDH catalytic activity, the holoenzyme was shown to be chloroplast-localized. An immunoelectron microscopy procedure, employing anti-NADP-GDH immunoglobulin G and Protein A-gold complex, showed that NADP-GDH antigen was absent from the nucleus but present in both the chloroplast and cytosol. Since synthesis of the enzyme can be inhibited by cycloheximide, the detection of NADP-GDH antigen in the cytosol was probably due to binding of the NADP-GDH antibody to nascent polypeptide chains of the precursor-protein being synthesized on cytosolic 80S ribosomes. PMID- 16664820 TI - Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L. AB - Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO(2) and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, (15)NH(4)Cl was injected into the central pith of stem sections. The kinetics of (15)N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of (15)N derived from (15)NH(4)Cl was almost completely inhibited. Injections of amido-(15)N glutamine demonstrated a potential for (15)N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism. PMID- 16664821 TI - Changes in adenine nucleotides and energy charge in isolated winter wheat cells during low temperature stress. AB - Adenylate energy charge (AEC) and adenine nucleotide levels of isolated winter wheat (Triticum aestivum L. cv Kharkov 22 MC) cells exposed to various low temperature stresses were determined. During ice encasement at -1 degrees C, nucleotide levels decreased gradually in approximate relation to a decline in cell viability. AEC values remained high even after 5 weeks of icing when cell viability was severely reduced. When isolated cell suspensions were exposed to various cooling and freezing regimes ranging from -10 to -30 degrees C, cell damage was dependent on the minimum temperature imposed and the duration of exposure to the freezing stress. The levels of all three adenine nucleotides declined with increasing severity of the imposed stress, but AEC values remained high even at -30 degrees C when nearly all of the cells were killed. The addition of 10 millimolar Ca(2+) to cell suspensions enhanced survival during low temperature stresses, but did not influence nucleotide levels other than through its effect on cell viability. These results indicate that impairment of the ion transport system during the early stages of ice encasement prior to a detectable decline in cell viability cannot be attributed to changes in the adenylate energy charge system of the cell. PMID- 16664822 TI - Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase. AB - Ethyl methane sulfonate treatment was used to induce a mutation in the nuclear gene encoding the chloroplast isozyme of phosphoglucose isomerase in Clarkia xantiana. The mutation, which proved allelic to wild type activity, was backcrossed to wild type for five generations so that the two could be compared in a near isogenic background. An immunological analysis showed that the mutant, when homozygous, reduced the activity of the isozyme by about 50%. In contrast to wild type, the mutant showed little change in leaf starch level over a diurnal period or following a 72-hour continuous light treatment. By the end of the diurnal light period, the mutant accumulated only about 60% as much starch as wild type. However, mutant leaves had an increased sucrose level presumably because photosynthate was directly exported from the chloroplasts. The mutant also exhibited reduced leaf weight. These changes in metabolism and growth suggest that the wild type level of plastid phosphoglucose isomerase activity is necessary to achieve wild type carbohydrate status. PMID- 16664823 TI - Effect of Photon Fluence Rate on Oxygen Evolution and Uptake by Chlamydomonas reinhardtii Suspensions Grown in Ambient and CO(2)-Enriched Air. AB - A closed system consisting of an assimilation chamber furnished with a membrane inlet from the liquid phase connected to a mass spectrometer was used to measure O(2) evolution and uptake by Chlamydomonas reinhardtii cells grown in ambient (0.034% CO(2)) or CO(2)-enriched (5% CO(2)) air. At pH = 6.9, 28 degrees C and concentrations of dissolved inorganic carbon (DIC) saturating for photosynthesis, O(2) uptake in the light (U(o)) equaled O(2) production (E(o)) at the light compensation point (15 micromoles photons per square meter per second). E(o) and U(o) increased with increasing photon fluence rate (PFR) but were not rate saturated at 600 micromoles photons per square meter per second, while net O(2) exchange reached a saturation level near 500 micromoles photons per square meter per second which was nearly the same for both, CO(2)-grown and air-grown cells. Comparison of the U(o)/E(o) ratios between air-grown and CO(2)-grown C. reinhardtii showed higher values for air-grown cells at light intensities higher than light compensation. For both, air-grown and CO(2)-grown algae the rates of mitochondrial O(2) uptake in the dark measured immediately before and 5 minutes after illumination were much lower than U(o) at PFR saturating for net photosynthesis. We conclude that noncyclic electron flow from water to NADP(+) and pseudocyclic electron flow via photosystem I to O(2) both significantly contribute to O(2) exchange in the light. In contrast, mitochondrial respiration and photosynthetic carbon oxidation cycle are regarded as minor O(2) consuming reactions in the light in both, air-grown and CO(2)-grown cells. It is suggested that the "extra" O(2) uptake by air-grown algae provides ATP required for the energy dependent CO(2)/HCO(3) (-) concentrating mechanism known to be present in these cells. PMID- 16664824 TI - Immunological comparisons of nitrate reductase of different plant species using monoclonal antibodies. AB - Six monoclonal antibodies against different epitopes of maize leaf nitrate reductase were used to compare plant nitrate reductases in enzyme linked immunosorbent assay and enzyme activity inhibition tests. The number of cross reacting antibodies was shown to vary with species according to phylogenetic classification, ranging from five (sugarcane) to one (dicotyledonous species). Cross-reactions were restricted to higher plant nitrate reductases. PMID- 16664825 TI - Effect of Oxygen on the Contribution of Respiration to the CO(2) Compensation Point in Wheat and Bean Leaves. AB - The CO(2) compensation point at 21% O(2) (Gamma(21)) and at 2% O(2) (Gamma(2)), and the rate of dark CO(2) efflux at 21% O(2) (R(n)) were measured in adult wheat (Triticum aestivum L, cv Gabo) leaves at the end of the night and after a period of photosynthesis of 5 h at 800 mubar CO(2). The values of Gamma(21) and R(n) significantly increased after the light period, due to the stimulation of respiration by carbohydrates. In contrast, Gamma(2) did not increase after the same period of photosynthesis, suggesting that the respiratory component of Gamma(2) was not stimulated by carbohydrates. In a different experiment, Gamma(21), Gamma(2), and R(n) were studied during the growth period of bean (Phaseolus vulgaris L, cv Hawkesbury Wonder) leaves. The values of Gamma(21) and R(n) were high in young leaves, and decreased rapidly in parallel during maturation. However, Gamma(2) presented relatively low values in growing bean leaves, and a model predicted that the observed values of Gamma(2) should have been considerably higher if their respiratory component was considered to be as large as that of Gamma(21). The results suggest that the rate of respiration in the light contributing to the CO(2) compensation point in wheat and bean leaves is smaller at low O(2) levels than at ambient levels. PMID- 16664826 TI - Purification and Properties of a Glycoprotein Processing alpha-Mannosidase from Mung Bean Seedlings. AB - The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [(3)H]mannose from the [(3)H]Man(9)GlcNAc as well as for releasing mannose from p-nitrophenyl-alpha-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca(2+) was the most effective cation for reversing this inhibition. Mn(2+) was considerably less effective than Ca(2+) and Mg(2+) was without effect. The processing mannosidase was inhibited by alpha1,2- and alpha1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and alpha1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [(3)H]Man(9)GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man(5)GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific alpha1,2-mannosidase. PMID- 16664827 TI - Respiratory CO(2) as Carbon Source for Nocturnal Acid Synthesis at High Temperatures in Three Species Exhibiting Crassulacean Acid Metabolism. AB - TEMPERATURE EFFECTS ON NOCTURNAL CARBON GAIN AND NOCTURNAL ACID ACCUMULATION WERE STUDIED IN THREE SPECIES OF PLANTS EXHIBITING CRASSULACEAN ACID METABOLISM: Mamillaria woodsii, Opuntia vulgaris, and Kalanchoe daigremontiana. Under conditions of high soil moisture, nocturnal CO(2) gain and acid accumulation had temperature optima at 15 to 20 degrees C. Between 5 and 15 degrees C, uptake of atmospheric CO(2) largely accounted for acid accumulation. At higher tissue temperatures, acid accumulation exceeded net carbon gain indicating that acid synthesis was partly due to recycling of respiratory CO(2). When plants were kept in CO(2)-free air, acid accumulation based on respiratory CO(2) was highest at 25 to 35 degrees C. Net acid synthesis occurred up to 45 degrees C, although the nocturnal carbon balance became largely negative above 25 to 35 degrees C. Under conditions of water stress, net CO(2) exchange and nocturnal acid accumulation were reduced. Acid accumulation was proportionally more decreased at low than at high temperatures. Acid accumulation was either similar over the whole temperature range (5-45 degrees C) or showed an optimum at high temperatures, although net carbon balance became very negative with increasing tissue temperatures. Conservation of carbon by recycling respiratory CO(2) was temperature dependent. At 30 degrees C, about 80% of the dark respiratory CO(2) was conserved by dark CO(2) fixation, in both well irrigated and water stressed plants. PMID- 16664828 TI - Changes in Gene Expression during Tomato Fruit Ripening. AB - Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)(+) and poly(A)(-) RNAs. Peak levels of total RNA, poly(A)(+) RNA, and poly(A)(+) RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)(+) RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)(+) RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products. PMID- 16664829 TI - Protein-synthesizing activity of maize-shoot chromatin : I. Conditions and component requirements. AB - The evidence presented in this paper suggests that purified plant chromatin, similar to mammalian (SR Umansky et al., Eur J Biochem 1980 105: 117-129), has the ability to incorporate amino acids into acid precipitable material. The polypeptide-synthesizing system of chromatin seems to differ substantially from the classical polyribosomal translation mechanism in cytoplasm. When chromatin purified from 5-day-old etiolated maize (Zea mays) shoots was incubated with (14)C-labeled amino acids, label was incorporated into the trichloroacetic acid precipitable product. Chloramphenicol, pactamycin, and actinomycin D inhibited the incorporation almost completely, whereas treatment with cycloheximide, puromycin, or aurintricarboxylic acid did not affect the labeling. Preincubation with pancreatic RNase was also without effect, but treatment of chromatin with DNase I caused about 25% depression of label incorporation. A wheat germ translation system or its single components have no effect on the chromatin polypeptide-synthesizing activity beyond that expected for a simple addition. The protein-synthesizing system is tightly bound to chromatin and could not be removed by dissociation in 1 molar NaCl. The mean molecular weight of the major protein fraction synthesized in the presence of chromatin was 21 to 24 kilodaltons. PMID- 16664830 TI - Polar Calcium Flux in Sunflower Hypocotyl Segments : II. The Effect of Segment Orientation, Growth, and Respiration. AB - Calcium flux in sunflower (Helianthus annuus L. cv Russian mammoth) hypocotyl was measured with a Ca(2+) electrode as the increase or decrease in Ca(2+) in an aqueous solution (10 micromolar CaCl(2)) in contact with either the basal or apical end of 20 millimeter segments. Ca(2+) efflux was significantly higher at the apical end compared with the basal end; this apparent polarity was maintained even when the segments were inverted. No significant difference was observed in the cation exchange capacity of apical and basal cell walls that could explain the difference in Ca(2+) efflux at opposite ends of the hypocotyl segment. The presence of exogenous indoleacetic acid (IAA) in the segment medium resulted in the promotion of both Ca(2+) efflux and segment elongation. However, osmotic inhibition of the IAA-induced elongation did not result in inhibiting the IAA induced Ca(2+) efflux. Ca(2+) efflux was inhibited by cyanide. Lowering the temperature from 25 degrees C also caused the gradual reduction of Ca(2+) efflux; at 5 degrees C the hypocotyl segments showed a net absorption of Ca(2+) from the segment medium. These findings support the suggestion that: (a) the observed Ca(2+) efflux in hypocotyl segments is probably the manifestation of the system which maintains the transmembrane Ca(2+) gradient at the cellular level. (b) The acropetal polarity of Ca(2+) efflux may be the result of the involvement of Ca(2+) in the basipetal transport of IAA. PMID- 16664831 TI - Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark. AB - The ammonium induction of the chloroplast-localized NADP-specific glutamate dehydrogenase (NADP-GDH) was shown not to be a light-dependent process per se in Chlorella sorokiniana. In the dark without exogenous organic substrates, the cells synthesized low levels of fully active NADP-GDH, provided endogenous starch reserves had not been depleted. When cells were supplied with exogenous acetate, the rate of induction of NADP-GDH activity per milliliter of culture in the dark was equal to or slightly greater than the rate observed under photosynthetic conditions without an organic carbon source. Glucose supported only a low rate of induction of NADP-GDH activity in the dark. Both acetate and glucose inhibited induction of enzyme activity in the light. The NADP-GDH holoenzyme had at least 7 different electrophoretic forms. These forms differed in net charge and/or molecular weight. Their difference in molecular weight was due to the presence of 2 subunits with similar antigenic properties but different molecular weights (M(r) = 55,500 and 53,000; alpha-and beta-subunits, respectively). Depending upon the cultural conditions and length of the induction period, a wide variation was observed in the alpha:beta subunit ratio and in the numbers and sizes of the NADP GDH holoenzymes. PMID- 16664832 TI - Chlorophyll a Fluorescence and Photosynthetic and Growth Responses of Pinus radiata to Phosphorus Deficiency, Drought Stress, and High CO(2). AB - Needles from phosphorus deficient seedlings of Pinus radiata D. Don grown for 8 weeks at either 330 or 660 microliters CO(2) per liter displayed chlorophyll a fluorescence induction kinetics characteristic of structural changes within the thylakoid chloroplast membrane, i.e. constant yield fluorescence (F(O)) was increased and induced fluorescence ([F(P)-F(I)]/F(O)) was reduced. The effect was greatest in the undroughted plants grown at 660 mul CO(2) L(-1). By week 22 at 330 mul CO(2) L(-1) acclimation to P deficiency had occurred as shown by the similarity in the fluorescence characteristics and maximum rates of photosynthesis of the needles from the two P treatments. However, acclimation did not occur in the plants grown at 660 mul CO(2) L(-1). The light saturated rate of photosynthesis of needles with adequate P was higher at 660 mul CO(2) L(-1) than at 330 mul CO(2) L(-1), whereas photosynthesis of P deficient plants showed no increase when grown at the higher CO(2) concentration. The average growth increase due to CO(2) enrichment was 14% in P deficient plants and 32% when P was adequate. In drought stressed plants grown at 330 mul CO(2) L(-1), there was a reduction in the maximal rate of quenching of fluorescence (R(Q)) after the major peak. Constant yield fluorescence was unaffected but induced fluorescence was lower. These results indicate that electron flow subsequent to photosystem II was affected by drought stress. At 660 mul CO(2) L(-1) this response was eliminated showing that CO(2) enrichment improved the ability of the seedlings to acclimate to drought stress. The average growth increase with CO(2) enrichment was 37% in drought stressed plants and 19% in unstressed plants. PMID- 16664833 TI - Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119. AB - The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N(2) fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O(2) evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron deficient cells. These results strongly suggest an involvement of boron in N(2) fixation in cyanobacteria. PMID- 16664834 TI - The physiological significance of phenylacetic Acid in abscising cotton cotyledons. AB - The physiological role of phenylacetic acid (PAA) as an endogenous regulator of cotyledon abscission was examined using cotton (Gossypium hirsutum L. cv LG 102) seedlings. Application of 100 micromolar or more PAA to leafless cotyledon abscission-zone explants resulted in the retardation of petiole abscission and a decrease in the rise of ethylene evolution that normally accompanies aging of these explants in vitro. The partial inhibition of ethylene evolution in these explants by PAA was indirect since application of this compound stimulated short term (<24 hours) ethylene production. PAA treatment partially suppressed the stimulation of petiole abscission elicited by either ethylene or abscisic acid. Both free and an acid-labile, bound form of PAA were identified in extracts prepared from cotyledons. No discernible pattern of changes in free or bound PAA was found during the course of ethylene-induced cotyledon abscission. Unlike indole-3-acetic acid, transport of PAA in isolated petiole segments was limited and exhibited little polarity. On the whole, these results are not consistent with the direct participation of PAA in the endogenous regulation of cotyledon abscission. PMID- 16664835 TI - Regulation of Fructan Metabolism in Leaves of Barley (Hordeum vulgare L. cv Gerbel). AB - Excised primary leaf blades of barley (Hordeum vulgare L. cv Gerbel) rapidly synthesized large quantities of fructan in the light and, upon transfer to the dark, they rapidly degraded it again. In the course of such a light/dark cycle the activities of sucrose-sucrose-fructosyltransferase (SST), fructan hydrolase, and invertase were measured in cell-free extracts of the blades. SST activity increased 20-fold within 24 hours in the light and disappeared again upon transfer to the dark during a similar period of time. Cycloheximide inhibited the increase of SST activity in the light indicating de novo synthesis. The loss of SST activity in the dark, however, was unaffected by cycloheximide. No SST activity appeared in the light if photosynthesis was inhibited by lowering the CO(2) concentration in the atmosphere. However, SST activity and fructan synthesis were induced even in the dark and at a low CO(2) concentration when the leaf blades were immersed in a solution of sucrose. Several other sugars, maltose and fructose in particular, had the same effect. Trehalose induced SST activity but no fructan synthesis occurred. The activities of fructan hydrolase and invertase changed little during the light/dark cycle. It is suggested that the control of SST activity in conjunction with the supply of photosynthates plays a key role in the regulation of fructan metabolism. PMID- 16664836 TI - Heat inactivation of starch synthase in wheat endosperm tissue. AB - The effect of temperature on accumulation of starch was studied in grain slices of wheat (Triticum aestivum cv SUN9E), taken 15 days after anthesis. As compared with pretreatment of such slices at 25 degrees C, pretreatment at 30 or 35 degrees C reduced the subsequent conversion of sucrose to starch. In contrast to rice (Oryza sativa cv Calrose), pretreatment of wheat soluble starch synthase in vitro at 30 degrees C or higher temperatures reduced its activity. In zymograms using nondenaturing polyacrylamide gel electrophoresis followed by activity staining, the slowest migrating band represented the most temperature sensitive isozyme. Although preincubation of a soluble enzyme sample in vitro at 25 degrees C did not result in loss of starch synthase activity, it did result in a gradual shift of zymogram banding pattern toward faster migrating species. Pretreatment of isolated starch granules at 40 degrees C increased their bound starch synthase activity. Both soluble and bound enzymes in the grains of whole wheat plants lost activity when the plants were held above 30 degrees C for 30 minutes or longer. Both activities lost from the grains after a 1 hour treatment at 37 degrees C were restored in 1 to 2 days by a return to 21 degrees C. In slices, inactivation of the soluble starch synthase was increased by incubation with 2,4 dinitrophenol. It is tentatively suggested that in vivo heat inactivation of soluble starch synthase may be a direct effect of heat on the enzyme protein and that of bound enzyme an indirect effect involving metabolic factors. PMID- 16664837 TI - Proton Fluxes as a Response to External Salinity in Wild Type and NaCl-Adapted Nicotiana Cell Lines. AB - Addition of 100 millimolar KCl, NaCl, or Na(2)SO(4) strongly promoted acidification of the medium by cells of Nicotiana tabacum/gossii in suspension culture. Acidification was greater in the case of NaCl-adapted than in that of wild type cells, and strikingly so in KCl medium when fusicoccin (FC) was present. Back-titration indicated that net proton secretion in KCl medium was increased 4-fold by FC treatment in the case of adapted cells; but was not even doubled in wild type cells. Membrane potential was higher in NaCl-adapted cells. FC treatment hyperpolarized wild, but not NaCl-adapted cells, suggesting a higher degree of coupling between H(+) efflux and K(+) influx in adapted cells; FC enhanced net K(+) uptake in adapted but not in wild cells. Acidification by cells suspended in 10 millimolar KCl was highly sensitive to vanadate, but that after addition of 100 millimolar KCl or NaCl was much less sensitive. Addition of 100 millimolar NaCl to wild type cells already provided with 10 millimolar KCl briefly accelerated, then slowed down the rate of acidification. If the addition was made after acidification had already ceased, alkalization was observed, particularly in the presence of FC. The results are consistent with the operation of a Na(+)-H(+) antiporter. PMID- 16664838 TI - Rapid Degradation of Abnormal Proteins in Vacuoles from Acer pseudoplatanus L. Cells. AB - In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([(14)C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([(14)C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole. PMID- 16664839 TI - Incorporation of UDPGlucose into Cell Wall Glucans and Lipids by Intact Cotton Fibers. AB - The [(14)C] moiety from [(3)H]UDP[(14)C]glucose was incorporated by intact cotton fibers into hot water soluble, acetic-nitric reagent soluble and insoluble components, and chloroform-methanol soluble lipids; the [(3)H] UDP moiety was not incorporated. The (3)H-label can be exchanged rapidly with unlabeled substrate in a chase experiment. The cell wall apparent free space of cotton fibers was in the order of 30 picomoles per milligram of dry fibers; 25 picomoles per milligram easily exchanged and about 5 picomoles per milligram more tightly adsorbed. At 50 micromolar UDPglucose, 70% of the [(14)C]glucose was found in the lipid fraction after both a short labeling period and chase. The percent of [(14)C]glucose incorporated into total glucan increased slightly with chase, but the fraction of total glucans incorporated into insoluble acetic-nitric reagent (cellulose) did increase within a 30-minute chase period. The data supports the concept that glucan synthesis, including cellulose, as well as the synthesis of steryl glucosides, acetylated steryl glucosides, and glucosyl-phosphoryl-polyprenol from externally supplied UDPglucose occurs at the plasma membrane-cell wall interface. The synthase enzymes for such synthesis must be part of this interfacial membrane system. PMID- 16664840 TI - Low temperature development of winter rye leaves alters the detergent solubilization of thylakoid membranes. AB - Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5 degrees C were extracted with the nonionic detergents Triton X 100 and octyl glucoside. Less total chlorophyll was extracted from 5 degrees C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5 degrees C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20 degrees C thylakoids than from 5 degrees C thylakoids. When 20 and 5 degrees C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the (32)Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5 degrees C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20 degrees C membranes. Since 20 and 5 degrees C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X 100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature. PMID- 16664841 TI - Turgor regulation of sucrose transport in sugar beet taproot tissue. AB - Sink tissues that store osmotically active compounds must osmoregulate to prevent excessively high turgor. The ability to regulate turgor may be related to membrane transport of solutes and thus sink strength. To study this possibility, the kinetics of sugar uptake were determined in sugar beet (Beta vulgaris L.) taproot tissue discs over a range of cell turgors. Sucrose uptake followed biphasic kinetics with a high affinity saturating component below 20 millimolar and a low affinity linear component at higher concentrations. Glucose uptake exhibited only simple saturation type kinetics. The high affinity saturating component of sucrose and glucose uptake was inhibited by increasing cell turgor (decreasing external mannitol concentrations). The inhibition was evident as a decrease in V(max) but no effect on K(m). Sucrose uptake by tissue equilibrated in dilute buffer exhibited no saturating component. Ethylene glycol, a permeant osmoticum, had no effect on uptake kinetics, suggesting that the effect was due to changes in cell turgor and not due to decreased water potential per se. p (Chloromercuri)benzene sulfonic acid (PCMBS) inhibited sucrose uptake at low but not high cell turgor. High cell turgor caused the tissue to become generally leaky to potassium, sucrose, amino acids, and reducing sugars. PCMBS had no effect on sucrose leakage, an indication that the turgor-induced leakage of sucrose was not via back flow through the carrier. The ability of the tissue to acidify the external media was turgor dependent with an optimum at 300 kilopascals. Acidification was sharply reduced at cell turgors above or below the optimum. The results suggest that the secondary transport of sucrose is reduced at high turgor as a result of inhibition of the plasma membrane ATPase. This inhibition of ATPase activity would explain the reduced V(max) and leakiness to low molecular weight solutes. Cell turgor is an important regulator of sucrose uptake in this tissue and thus may be an important determinant of sink strength in tissues that store sucrose. PMID- 16664842 TI - Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation. AB - Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with (14)CO(2), stem sections were cut and the efflux characteristics of (14)C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2 hour chase. Therefore, sucrose leakage from stem segments prepared following a 75 minute chase period was assumed to be characteristic of phloem unloading. The efflux of (14)C assimilates from the phloem was enhanced by 1 millimolar p chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [(14)C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [(14)C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds. PMID- 16664843 TI - Immunocytochemical localization and time course of appearance of an anionic peroxidase associated with suberization in wound-healing potato tuber tissue. AB - Thin sections of wound-healing potato tuber tissue were stained with rabbit antibody prepared against a suberization-associated anionic peroxidase and then stained with a goat anti-rabbit antibody-fluorescein conjugate. When these sections were examined with an epiilluminating fluorescence microscope, bright green fluorescent linear deposits were observed on the inner side of cell walls in the periderm layer. Initial deposits which were often not contiguous throughout the wall were first observed in some cells after 3 days of wound healing and subsequently these layers became more pronounced so that all 6 day old periderm cells had green fluorescent layers on their inner walls. This fluorescence was not present in the walls of parenchyma cells or in the walls of periderm cells treated with preimmune serum and anti-rabbit IgG-FITC conjugate. Thin sections of wound-healing potato tissue which were stained with anti peroxidase antibody and a goat anti-rabbit antibody-rhodamine conjugate exhibited a similar time course of development with a bright reddish-orange fluorescent layer observed on the inside wall of periderm cells. The production of this suberization-associated anionic peroxidase in wound-healing tissue was also demonstrated by an immunobinding dot blot assay which showed that the largest increase in the enzyme level occurred between 4 and 6 days of wound-healing. The present results support the hypothesis that this anionic peroxidase is involved in the deposition of the aromatic polymeric domain of suberin. PMID- 16664844 TI - myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination. AB - Radiolabeled d-[1-(3)H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the (3)H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained (3)H, showing that de novo synthesis of myo-inositol from [1-(3)H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The (3)H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, (3)H percentages in hexose residues were higher than their mole percentages. The results suggest that myo inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo inositol oxidation pathway. PMID- 16664845 TI - Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots. AB - A H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H(+)-PPase catalyzed the electrogenic transport of H(+) into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb(+) = K(+) > Cs(+); but it was inhibited by Na(+) or Li(+). Maximum rates of transport required at least 20 millimolar K(+) and the K(m) for this ion was 4 millimolar. Fluoride inhibited both DeltapH formation and K(+)-dependent PPase activity with an I(50) of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H(+)-ATPase (e.g. a disulfonic stilbene or NO(3) (-)) had no effect on the PPase activity. Vanadate and azide were also ineffective. H(+)-pumping PPase was inhibited by 7-chloro-4 nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N' dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H(+)-PPase and the H(+)-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H(+) pumping activity was strongly affected by Mg(2+) and pyrophosphate (PPi) concentrations. At 5 millimolar Mg(2+), H(+) pumping showed a K(m(aPP) ) for PPi of 15 micromolar. The rate of H(+) pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H(+) PPase. PMID- 16664846 TI - Kernel abortion in maize : I. Carbohydrate concentration patterns and Acid invertase activity of maize kernels induced to abort in vitro. AB - Kernels cultured in vitro were induced to abort by high temperature (35 degrees C) and by culturing six kernels/cob piece. Aborting kernels failed to enter a linear phase of dry mass accumulation and had a final mass that was less than 6% of nonaborting field-grown kernels. Kernels induced to abort by high temperature failed to synthesize starch in the endosperm and had elevated sucrose concentrations and low fructose and glucose concentrations in the pedicel during early growth compared to nonaborting kernels. Kernels induced to abort by high temperature also had much lower pedicel soluble acid invertase activities than did nonaborting kernels. These results suggest that high temperature during the lag phase of kernel growth may impair the process of sucrose unloading in the pedicel by indirectly inhibiting soluble acid invertase activity and prevent starch synthesis in the endosperm. Kernels induced to abort by culturing six kernels/cob piece had reduced pedicel fructose, glucose, and sucrose concentrations compared to kernels from field-grown ears. These aborting kernels also had a lower pedicel soluble acid invertase activity compared to nonaborting kernels from the same cob piece and from field-grown ears. The low invertase activity in pedicel tissue of the aborting kernels was probably caused by a lack of substrate (sucrose) for the invertase to cleave due to the intense competition for available assimilates. In contrast to kernels cultured at 35 degrees C, aborting kernels from cob pieces containing all six kernels accumulated starch in a linear fashion. These results indicate that kernels cultured six/cob piece abort because of an inadequate supply of sugar and are similar to apical kernels from field-grown ears that often abort prior to the onset of linear growth. PMID- 16664847 TI - Kernel Abortion in Maize : II. Distribution of C among Kernel Carbohydrates. AB - This study was designed to compare the uptake and distribution of (14)C among fructose, glucose, sucrose, and starch in the cob, pedicel, and endosperm tissues of maize (Zea mays L.) kernels induced to abort by high temperature with those that develop normally. Kernels cultured in vitro at 30 and 35 degrees C were transferred to [(14)C]sucrose media 10 days after pollination. Kernels cultured at 35 degrees C aborted prior to the onset of linear dry matter accumulation. Significant uptake into the cob, pedicel, and endosperm of radioactivity associated with the soluble and starch fractions of the tissues was detected after 24 hours in culture on labeled media. After 8 days in culture on [(14)C]sucrose media, 48 and 40% of the radioactivity associated with the cob carbohydrates was found in the reducing sugars at 30 and 35 degrees C, respectively. This indicates that some of the sucrose taken up by the cob tissue was cleaved to fructose and glucose in the cob. Of the total carbohydrates, a higher percentage of label was associated with sucrose and a lower percentage with fructose and glucose in pedicel tissue of kernels cultured at 35 degrees C compared to kernels cultured at 30 degrees C. These results indicate that sucrose was not cleaved to fructose and glucose as rapidly during the unloading process in the pedicel of kernels induced to abort by high temperature. Kernels cultured at 35 degrees C had a much lower proportion of label associated with endosperm starch (29%) than did kernels cultured at 30 degrees C (89%). Kernels cultured at 35 degrees C had a correspondingly higher proportion of (14)C in endosperm fructose, glucose, and sucrose. These results indicate that starch synthesis in the endosperm is strongly inhibited in kernels induced to abort by high temperature even though there is an adequate supply of sugar. PMID- 16664848 TI - Monoclonal Antibodies to Plant Growth Regulators: III. Zeatinriboside and Dihydrozeatinriboside. AB - Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme linked immunosorbent assay is also discussed. PMID- 16664849 TI - Cellular Interactions during the Mating Process in Chlamydomonas eugametos. AB - A method to determine the mating competence of Chlamydomonas eugametos was developed. The contribution of each mating type in the pair formation was investigated using asymmetric gamete mixtures. It was established that pair formation is not mediated by a pheromonal attraction mechanism between partner gametes, but depends on collision chances. On the other hand, it was demonstrated that during transient contacts between partner gametes the flagellar agglutinability of both partners is stimulated, evidently to prepare a successful mating. The plus mating type was generally less agglutinable than the minus mating type and was a rate-limiting factor in the mating process. PMID- 16664850 TI - Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double Stranded cDNA from the Purified mRNA. AB - A specific polysome immunoadsorption procedure, employing soluble rabbit anti NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)(+)RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)(+)RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single stranded cDNA and double-stranded cDNA molecules. PMID- 16664851 TI - Photobiology of phytochrome-mediated growth responses in sections of stem tissue from etiolated oats and corn. AB - The far-red reversibility of the phytochrome-controlled stimulation of elongation of coleoptile sections by low fluence red light has been characterized in subapical coleoptile sections from dark-grown Avena sativa L., cv Lodi seedlings. The fluence dependence of the far-red reversal was the same whether or not the very low fluence response is also expressed. The capacity of far-red light to reverse the red light-induced response began to decline if the far-red light was given more than 90 minutes after the red irradiation. Escape was complete if the far red irradiation was given more than 240 minutes after the red irradiation. Sections consisting of both mesocotyl and coleoptile tissue from dark-grown Avena seedlings were found to have physiological regulation of the very low fluence response by indole 3-acetic acid and low external pH similar to that seen for sections consisting entirely of coleoptile tissue. The fluence-dependence of the red light-induced inhibition of mesocotyl elongation was studied in mesocotyl sections from dark grown Zea mays L. hybrid T-929 seedlings. Ten micromolar indole 3-acetic acid stimulates the control elongation of the sections, while at the same time increasing the sensitivity of the tissue for the light-induced inhibition of growth by a factor of 100. PMID- 16664852 TI - Enzymes of alpha,alpha-Trehalose Metabolism in Soybean Nodules. AB - Metabolism of trehalose, alpha,d-glucopyranosyl-alpha,d-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max [L.] Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6 phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6 phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and beta-glucose-1-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6 phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase. PMID- 16664853 TI - H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots. AB - The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H(+)-pumping in membrane vesicles from corn (Zea mays L. cv WF9 x MO17) roots and obtain a preparation in which vanadate-sensitive H(+)-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H(+)-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO(3) (-) and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO(3) (-) and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H(+)-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H(+)-pumping by vanadate in this membrane preparation indicated that most of the H(+)-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K(m) for ATP (0.7 millimolar) of the H(+)-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials. PMID- 16664854 TI - Stage-Specific State I-State II Transitions during the Cell Cycle of Euglena gracilis. AB - In synchronized Euglena gracilis (light-dark regime of 14:10 hours) the successive formation of the photosynthetic apparatus during cell ontogeny is correlated with large changes in photosynthetic efficiency (P Brandt, B von Kessel 1983 Plant Physiol 72: 616-619; B Kohnke, P Brandt 1984 Biochim Biophys Acta 766: 156-160). This observation led us to investigate the functional association of the chlorophyll a/b light-harvesting protein complex (LHCP) with photosystem I or II, because changes in energy flow to photosystem I or II and in energy transfer between the two photosystems can be a reason for these alterations. As criterion for the association of the LHCP with photosystem I or II, state transitions were determined after 15 minutes preillumination using wave lengths of 725 or 620 nanometers. The state transitions were determined from measurements of fluorescence induction at room temperature, and fluorescence kinetics at 77 K. According to the obtained data (a) mobile LHCP is present only between the 6th and the 10th hour of the light-time of the cell cycle and (b) this functional relation of the LHCP to photosystem I only at this stage of Euglena chloroplast development is not accompanied by a decrease in stacking. A model for the organization of the newly inserted LHCP within the photosynthetic apparatus of E. gracilis is discussed. PMID- 16664855 TI - Serine hydroxymethyltransferase from soybean root nodules : purification and kinetic properties. AB - Serine hydroxymethyltransferase has been purified 1,550-fold from the plant fraction of soybean (Glycine max [L]. Merr. cv Williams) nodules. The pH optimum for the enzyme was at 8.5. The native molecular weight was 230,000 with a subunit molecular weight of 55,000 which suggested a tetramer of identical subunits. The enzyme kinetics for the enzyme were Michaelis-Menten; there was no evidence for cooperativity in the binding of either substrates or product inhibitors. There were two K(m) values for serine at 1.5 and 40 millimolar. The K(m) for l tetrahydrofolate was 0.25 millimolar. l-Methyl-, l-methenyl-, and l-methylene tetrahydrofolates were all noncompetitive inhibitors with l-tetrahydrofolate with K(i) values of 1.8, 3.0, and 2.9 millimolar, respectively. Glycine was a competitive inhibitor with serine with a K(i) value of 3.0 millimolar. The intersecting nature of the double reciprocal plots together with the product inhibition data suggested an ordered mechanism with serine the first substrate to bind and glycine the last product released. The enzyme was insensitive to a wide range of metabolites which have previously been reported to affect its activity. These results are discussed with respect to the roles of serine hydroxymethyltransferase and the one-carbon metabolite pool in control of the carbon flow to the purine biosynthetic pathway in ureide biogenesis. PMID- 16664856 TI - Soybean lectin and related proteins in seeds and roots of le and le soybean varieties. AB - The localizations of soybean lectin (SBL) and antigenically related proteins in cotyledons and roots of lectin positive (Le(+)) and lectin negative (Le(-)) soybean cultivars were compared by light level immunocytochemistry using antibodies produced against the 120 kilodalton (kD) native seed lectin tetramer or its subunits. Lectin is present in the protein bodies of cotyledons cells as are two other seed proteins, the Kunitz trypsin inhibitor and the storage protein glycinin. Analysis of single seed extracts by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels using the same antibodies, reveals up to 4 milligrams of the 30 kD seed lectin protein is present per seed in the Le(+) varieties. There is no detectable lectin in the protein bodies of Le(-) cotyledons as determined by immunocytochemistry and immunoblotting. Enzyme-linked immunosorbent assay confirmed this result to a sensitivity of less than 20 nanograms per seed. In contrast, the roots of both Le(+) and Le(-) plants bind the seed lectin antibody during immunocytochemistry, with fluorescence mainly localized in vacuole-like bodies in the epidermis. Root extracts contain a 33 kD polypeptide that binds anti-SBL antibody at an estimated minimal level of 20 nanograms per 4 day seedling, or 2.0 nanograms per primary root tip. This polypeptide is also present in the embryo axis and in leaves. The latter also contain a 26 kD species that binds seed lectin antibody. The 30 kD seed lectin subunit, however, is not detectable in roots or leaves. PMID- 16664857 TI - Two indirect methods for detecting ureide synthesis by nodulated legumes. AB - Two methods were developed for the detection of altered ureide metabolism in legume nodules. Both techniques are based on the positive correlation between the presence of high xanthine dehydrogenase (EC 1.2.1.37) specific activity in nodules and the ability of those nodules to produce the ureides, allantoin and allantoic acid. In the first method, nodulated legumes are treated for 2 weeks with a soil drench of allopurinol. After allopurinol treatment, leaves of N(2) fed, ureide-producing legumes, soybean, cowpea, and lima bean, became very chlorotic. Leaves of KNO(3) (-) or NH(4)Cl-fed ureide-producing legumes were unaffected by the allopurinol treatment. Leaves of the amide-producing legumes, alfalfa, clover, peak, and lupin, were unaffected by the allopurinol treatment with N(2), KNO(3), or NH(4)Cl as nitrogen source. These experiments showed that long-term allopurinol treatments are useful in differentiating between ureide- and amide-producing legumes when effectively nodulated. A second method was developed for the rapid, qualitative estimation of xanthine dehydrogenase activity in legume nodules. This method utilizes pterin, an alternate substrate for xanthine dehydrogenase. Xanthine dehydrogenase hydroxylates pterin in the presence of NAD(+) to produce isoxanthopterin. When exposed to long wave ultraviolet light (365 nanometers), isoxanthopterin emits blue fluorescence. When nodules of ureide-producing legumes were sliced in half and placed in microtiter plate wells containing NAD(+) and pterin, isoxanthopterin was observed after 6 hours of incubation at room temperature. Allopurinol prevented isoxanthopterin production. When slices of amide-producing legume nodules were placed in wells with pterin and NAD(+), no blue fluorescence was observed. The production of NADH by xanthine dehydrogenase does not interfere with the fluorescence of isoxanthopterin. These observations agree with the high specific activity of xanthine dehydrogenase in nodules of ureide-producing legumes and the low activity measured in amide-producing nodules. The wild soybean, Glycine soja Sieb. and Zucc., was examined for ureide synthesis. Stems of wild soybean plants had a high ureide abundance with N(2) as sole nitrogen source when nodulated with either Rhizobium fredii or Bradyrhizobium japonicum. Ureide abundance declined when nitrate or ammonium was added to the nutrient solution. Nodule slices of these plants produced isoxanthopterin when incubated with pterin. Nodule crude extracts of G. soja had high levels of xanthine dehydrogenase activity. Both Glycine max and G. soja plants were found to produce ureides when plants were inoculated with fast-growing R. fredii. The two methods described here can be used to discriminate ureide producers from amide producers as well as detect nitrogen-fixing legumes which have altered ureide metabolism. A nodulated legume that lacks xanthine dehydrogenase activity as demonstrated by the pterin assay cannot produce ureides since ureide synthesis has been shown to require xanthine dehydrogenase activity both in vivo and in vitro. A nodulated legume that remains green during allopurinol treatment also lacks ureide synthesis since the leaves of ureide-producing legumes are very chlorotic following allopurinol treatment. PMID- 16664858 TI - Isolation and Initial Characterization of Constitutive Nitrate Reductase Deficient Mutants NR328 and NR345 of Soybean (Glycine max). AB - Two nitrate reductase deficient mutants of soybean (Glycine max [L.] Merr. cv Bragg) were isolated from approximately 10,000 M(2) seedlings, using a direct enzymic assay in microtiter plates. Stable inheritance of NR345 and NR328 phenotypes has been demonstrated through to the M(5) generation. Both mutants were affected in constitutive nitrate reductase activity. Assayable activities of cNR in nitrate-free grown seedlings was about 3 to 4% of the control for NR345 and 14 to 16% of the control for NR328. Both mutants expressed inducible NR during early plant development and were sensitive to nitrate and urea inhibition of nodulation. These new mutants will allow an extension of the characterization of nitrate reductases and their function in soybean. Preliminary evidence indicates that NR345 is similar to the previously isolated mutant nr(1), while NR328 is different. PMID- 16664859 TI - Effects of Orthophosphate and Adenosine 5'-Phosphate on Threonine Synthase and Cystathionine gamma-Synthate of Lemna paucicostata Hegelm. 6746. AB - Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine gamma-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine gamma synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts. PMID- 16664860 TI - Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes. AB - In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark. PMID- 16664861 TI - The responses of isolated plant mitochondria to external nicotinamide adenine dinucleotide. AB - The effects of added NAD on substrate oxidation by turnip (Brassica rapa L.) and beetroot (Beta vulgaris L.) mitochondria were investigated. State 3 malate and 2 oxoglutarate oxidation rates with turnip mitochondria were stimulated 25 to 40% by external NAD. Following NAD-depletion this stimulation by NAD was increased to 70 to 80%. With purified beetroot mitochondria, state 3 malate and 2-oxoglutarate oxidation rates were only marginally increased (10-15%) by the addition of NAD but after NAD-depletion treatments this stimulation increased to 55%. The effect of added NAD on oxidation rates could be reduced by preloading mitochondria with NAD in the presence of succinate. Oxidation rates were found to be most sensitive to the addition of external NAD when rotenone was present. The uptake of external NAD into beetroot mitochondria appeared to be composed of both an active and a diffusive component. The active component displayed saturation kinetics with an approximate K(m) of 0.105 +/- 0.046 millimolar. These results provide further evidence, reported previously with potato mitochondria, that NAD can move across the inner membrane of plant mitochondria. They are particularly significant with respect to beetroot mitochondria which in contrast to other plant mitochondria, have not demonstrated any response to added NAD. PMID- 16664862 TI - Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase. AB - Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M(2) seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH(2)-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c(1)NR), constitutive NADH:NR (c(2)NR), and nitrate-inducible NR (iNR). PMID- 16664863 TI - Buffer capacities of leaves, leaf cells, and leaf cell organelles in relation to fluxes of potentially acidic gases. AB - Since environmental pollution by potentially acidic gases such as SO(2) causes proton release inside leaf tissues, homogenates of needles of spruce (Picea abies) and fir (Abies alba) and of leaves of spinach (Spinacia oleracea) and barley (Hordeum vulgare) were titrated and buffer capacities were determined as a function of pH. Titration curves of barley leaves were compared with titration curves of barley mesophyll protoplasts. From the protoplasts, chloroplasts and vacuoles were isolated and subjected to titration experiments. From the titration curves, the intracellular distribution of buffering capacities could be deduced. Buffering was strongly pH-dependent. It was high at the extremes of pH but still significant close to neutrality. Owing to its large size, the vacuole was mainly responsible for cellular buffering. However, on a unit volume basis, the cytoplasm was much more strongly buffered than the vacuole. Potentially acidic gases are trapped in the anionic form. They release protons when trapped. The magnitude of diffusion gradients from the atmosphere into the cells, which determines flux, depends on intracellular pH. In the light, the chloroplast stroma, as the most alkaline leaf compartment, has the highest trapping potential. Acidification of the chloroplast stroma inhibits photosynthesis. The trapping potential of the chloroplast is followed by that of the cytosol. Compared with the cytoplasm, the vacuole possesses little trapping potential in spite of its large size. It is particularly small in the acidic vacuoles of conifer needles. In the physiological pH range (slightly above neutrality), chloroplast buffering was about 1 microequivalents H(+) per milligram chlorophyll per pH unit or 35 microequivalents H(+) per milliliter per pH unit in barley or spinach chloroplasts. This compares with SO(2)-generated H(+) production of somewhat more than 1 microequivalent H(+) per milligram chlorophyll per hour, which results from observed SO(2) uptake of leaves when stomata were open and the atmospheric SO(2) concentration was 0.4 microliters per liter (GE Taylor Jr, DT Tingey 1983 Plant Physiol 72: 237-244). At lower SO(2) concentrations, similar H(+) generation inside the cells requires correspondingly longer exposure times. PMID- 16664864 TI - Neutral peptidases in the stroma of pea chloroplasts. AB - One endopeptidase (EP1) and at least three aminopeptidases (AP1, AP2, and AP3) were discovered in the stroma of chloroplasts isolated from pea seedlings (Pisum sativum L.), and purified over 100-fold. EP1 requires added Mg(2+) or Ca(2+) for activity, may have an additional tightly bound metal atom, and is inhibited by sulfhydryl reagents but not by serine residue-directed inhibitors. It is reversibly inhibited by dithiothreitol. Its specificity is for the bond between two adjacent Ala or Gly residues. Its molecular mass is 93 kilodaltons, estimated on a gel filtration column. Aminopeptidase activities were detected with the aid of different amino acyl-beta-naphthylamides as substrates. They were resolved into at least three individual proteins by gel filtration and DEAE-cellulose chromatography, having apparent molecular masses of 269,000 (AP1), 84,000 (AP2), and 42,000 (AP3) daltons, respectively. Each has a unique specificity for substrates, with AP1 hydrolyzing only the Prolyl-beta-naphthylamide. None of the APs require added divalent cations for activity, but the possibility of a tightly bound metal function was suggested in AP2 and AP3 (not AP1) from effects of inhibitors. A probable sulfhydryl residue function was indicated for all three, from inhibition by p-hydroxymercuribenzoate and Zn(2+). All these peptidases had pH optima at 7.7. PMID- 16664865 TI - Polyphenoloxidase in higher plants: immunological detection and analysis of in vitro translation products. AB - Antibodies to broad bean polyphenoloxidase (PPO) were used to detect and demonstrate that the PPOs found in several different plants have many similarities in common. Crude extracts from leaves of broad bean, bush bean, lettuce, mung bean, pea, soybean, spinach, tobacco, and tomato contained enzyme which cross-reacted with polyclonal anti-PPO in Ouchterlony double diffusion analysis. The results suggested that plant polyphenoloxidase from a wide range of species may contain similar antigen determinants. Poly A(+) mRNA was isolated from leaves of each plant species and translated in vitro using a rabbit reticulocyte translation system. An in vitro synthesized product corresponding to PPO from each species was identified after specific immunoprecipitation with anti PPO. The molecular weight of this in vitro product was similar in all plants examined and found to be approximately 45 kilodaltons. Peptide maps of the in vitro synthesized product from all plant species were similar and showed at least three peptides in common. Plant PPOs may have more structural similarities than commonly though in spite of the great variety in observed isoenzyme forms. PMID- 16664866 TI - Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures. AB - Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6beta hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6beta-hydroxylase was purified 3.3 fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe(2+) ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-(14)C]-2 oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO(2) from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6beta-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures. PMID- 16664867 TI - Physiological Changes in Cultured Sorghum Cells in Response to Induced Water Stress : II. Soluble Carbohydrates and Organic Acids. AB - Eight cultivars Sorghum bicolor (L.) Moench were grown as callus cultures under induced, prolonged water stress (8 weeks), with polyethylene glycol in the medium. Concentrations of soluble carbohydrates and organic acids in callus were measured at the end of the growth period to determine differences in response to prolonged water stress. Sucrose, glucose, fructose, and malate were the predominant solutes detected in all callus at all water potentials. All cultivars had high levels of solutes in the absence of water stress and low levels in the presence of prolonged water stress. However, at low water potentials, low levels of solutes were observed in drought-tolerant cultivar callus and high solute levels were observed in drought-susceptible cultivar callus. Estimated sucrose concentrations were significantly higher in water-stressed, susceptible cultivar callus. Large solute concentrations in susceptible cultivar callus were attributed to osmotic adjustment and/or reduced growth during water stress. PMID- 16664868 TI - Differential synthesis in vitro of barley aleurone and starchy endosperm proteins. AB - To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the alpha-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, beta-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of alpha-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for alpha-amylase, these hormones have the opposite effect on ASI mRNA levels. PMID- 16664869 TI - The Conversion of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid to 1 Aminocyclopropane-1-Carboxylic Acid in Plant Tissues. AB - Since 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the major conjugate of 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues, is a poor ethylene producer, it is generally thought that MACC is a biologically inactive end product of ACC. In the present study we have shown that the capability of watercress (Nasturtium officinale R. Br) stem sections and tobacco (Nicotiana tabacum L.) leaf discs to convert exogenously applied MACC to ACC increased with increasing MACC concentrations (0.2-5 millimolar) and duration (4-48 hours) of the treatment. The MACC-induced ethylene production was inhibited by CoCl(2) but not by aminoethoxyvinylglycin, suggesting that the ACC formed is derived from the MACC applied, and not from the methionine pathway. This was further confirmed by the observation that radioactive MACC released radioactive ACC and ethylene. A cell-free extract, which catalyzes the conversion of MACC to ACC, was prepared from watercress stems which were preincubated with 1 millimolar MACC for 24 hours. Neither fresh tissues nor aged tissues incubated without external MACC exhibited enzymic activity, confirming the view that the enzyme is induced by MACC. The enzyme had a K(m) of 0.45 millimolar for MACC and showed maximal activity at pH 8.0 in the presence of 1 millimolar MnSO(4). The present study indicates that high MACC levels in the plant tissue can induce to some extent the capability to convert MACC to ACC. PMID- 16664870 TI - ADPglucose Pyrophosphorylase Is Encoded by Different mRNA Transcripts in Leaf and Endosperm of Cereals. AB - Western blots of soluble protein from wheat, rice, and corn showed that ADPglucose pyrophosphorylase subunits have a size of 50 kilodaltons from endosperm tissue and 43 and 46 kilodaltons from leaf. Antisera to ADPglucose pyrophosphorylase precipitated in vitro translation products of 73 and 76 kilodaltons when leaf poly(A)(+) RNA was used, whereas endosperm mRNA directed the synthesis of 50 and 56 kilodalton polypeptides. To further study the nature of these mRNA species, an ADPglucose pyrophosphorylase cDNA clone from rice endosperm polyadenylated RNA was obtained and used as a hybridization probe. Northern blots showed that ADPglucose pyrophosphorylase mRNA was slightly larger in leaf (2100 bases) than in endosperm tissue (1900 bases). These studies indicated that in cereals there are at least two tissue specific forms of ADPglucose pyrophosphorylase that are encoded by distinct mRNA transcripts. Analysis of genomic DNA by Southern blotting suggested that ADPglucose pyrophosphorylase is encoded by a small gene family. PMID- 16664871 TI - The transport of indole-3-acetic Acid in boron- and calcium-deficient sunflower hypocotyl segments. AB - Transfer of sunflower (Helianthus annuus L. cv Russian Mammoth) seedlings from complete nutrient solution to solutions deficient in either boron or calcium resulted in a steady decline in the rate of auxin transport, compared to seedlings that remained in the complete solution. In seedlings transferred to solutions deficient in both B and Ca, the decline in auxin transport was greater than seedlings deficient in only one element. The transfer of B- or Ca-deficient seedlings back to the complete solution prevented further decline in auxin transport, but auxin transport did not increase to the same level as seedlings maintained in complete solution. The significant reduction in auxin transport during the early stages of B or Ca deficiency was not related to (a) reduced growth rate of the hypocotyl, (b) increased acropetal movement of auxin, or (c) lack of respiratory substrates in the hypocotyl. In addition, no difference was found in the water-extractable total and ionic Ca in B-deficient and control nondeficient hypocotyls, indicating a direct effect of B on auxin transport, rather than indirectly by affecting Ca absorption. The rate of auxin transport in hypocotyls deficient in either B or Ca, was inversely correlated with K(+) leakage and rate of respiration. The data presented strongly support the view that there are separate sites for B and Ca in the basipetal transport of the plant hormone indoleacetic acid. PMID- 16664872 TI - Boron and calcium sites involved in indole-3-acetic Acid transport in sunflower hypocotyl segments. AB - Sunflower (Helianthus annuus L. cv Russian Mammoth) hypocotyl segments deficient in either B or Ca exhibited a higher rate of potassium leakage, compared to nondeficient segments. Potassium leakage, used here as an indication of membrane integrity, was completely reversed by the addition of H(3)BO(3) or Ca(NO(3))(2) to the incubation medium of the B-deficient or Ca-deficient hypocotyl segments, respectively. This role of B and Ca in membrane integrity, which may be important in the entry and exit of auxin in cells, is identified as the first site of action for each of these two essential elements in the basipetal secretion of auxin. A second site for B is postulated because auxin transport was not restored, even when K(+) leakage has been completely reversed to the nondeficient level, when B-deficient hypocotyls were incubated in B solution. This lack of reversibility of auxin transport implied that the incubation for 2 h in B solution was not enough to restore the auxin transport process. However, since the transfer of B-deficient seedlings to B solutions prevented further deterioration of auxin transport, these observations suggest that: (a) either an intact seedling, or a longer period of incubation of the hypocotyl in B solution, is required for the synthesis or maintenance of the functional second site for B; (b) B is probably essential in the synthesis of a ligand, which may or may not be needed to bind B, but which is essential in the basipetal transport of auxin. The second site for Ca in auxin transport, is indicated by the complete reversal of its inhibition in Ca-deficient hypocotyl, when incubated in Ca solution. The second site for Ca is thought to be directly involved in the secretion of auxin, in which Ca probably plays the role of a second messenger, as in stimulus response coupling. The two sites for Ca can be distinguished from each other by their cation specificity. The requirement for Ca in the first site can be substituted by other divalent cations, while the second site is highly specific for Ca. PMID- 16664873 TI - Thiol-dependent regulation of glycerate metabolism in leaf extracts : the role of glycerate kinase in c(4) plants. AB - We have recently reported that the activity of maize leaf glycerate kinase [EC 2.7.1.31] is regulated in vivo by the light/dark transition, possibly involving the ferredoxin/thioredoxin mechanism, and that the stimulating effect of light can be mimicked in vitro by incubation of crude leaf extract with reducing compounds (LA Kleczkowski, DD Randall 1985 Plant Physiol 79: 274-277). In the present study it was found that the time course of thiol activation of the enzyme was substantially dependent on the presence of some low molecular weight inhibitor(s) of activation found both in leaf extracts and mesophyll chloroplasts. Activity of glycerate kinase from maize as well as wheat leaves increased upon greening of etiolated plants and was correlated with the development of photosynthetic apparatus in these species. The maize enzyme was strongly activated by thiols at all stages of development from etiolated to green seedlings. Thiol activation of glycerate kinase was observed for a number of C(4) plants, notably of the nicotinamide adenine dinucleotide phosphate-malic enzyme type, with the strongest effect found for the enzyme from leaf extracts of maize and sorghum (10- and 8-fold activation, respectively). Among the C(3) species tested, only the enzyme from soybean leaves was affected under the same conditions (1.6-fold activation). This finding was reflected by an apparent lack of cross-reactivity between the enzyme from maize leaves and antibodies raised against purified spinach leaf glycerate kinase. We suggest that, in addition to its role as a final step of photorespiration in leaves, glycerate kinase from C(4) species may serve as a part of the facilitative diffusion system for the intercellular transport of 3-phosphoglycerate. Simultaneous operation of both the passive and the facilitative diffusion mechanisms of 3-phosphoglycerate transport in C(4) plants is postulated. PMID- 16664874 TI - Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: I. Initial Products of CO(2) Assimilation and Levels of Whole Leaf C(4) Metabolites. AB - Labeling patterns from (14)CO(2) pulses to leaves and whole leaf metabolite contents were examined during photosynthetic induction in Flaveria trinervia, a C(4) dicot of the NADP-malic enzyme subgroup. During the first one to two minutes of illumination, malate was the primary initial product of (14)CO(2) assimiltion (about 77% of total (14)C incorporated). After about 5 minutes of illumination, the proportion of initial label to aspartate increased from 16 to 66%, and then gradually declined during the following 7 to 10 minutes of illumination. Nutrition experiments showed that the increase in (14)CO(2) partitioning to aspartate was delayed about 2.5 minutes in plants grown with limiting N, and was highly dampened in plants previously treated 10 to 12 days with ammonia as the sole N source. Measurements of C(4) leaf metabolites revealed several transients in metabolite pools during the first few minutes of illumination, and subsequently, more gradual adjustments in pool sizes. These include a large initial decrease in malate (about 1.6 micromoles per milligram chlorophyll) and a small initial decrease in pyruvate. There was a transient increase in alanine levels after 1 minute of illumination, which was followed by a gradual, prolonged decrease during the remainder of the induction period. Total leaf aspartate decreased initially, but temporarily doubled in amount between 5 and 10 minutes of illumination (after its surge as a primary product). These results are discussed in terms of a plausible sequence of metabolic events which lead to the formation of the intercellular metabolite gradients required in C(4) photosynthesis. PMID- 16664875 TI - Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times. AB - The metabolism of fixed (14)CO(2) and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C(4) dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C(4) cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of (14)C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1-->3 of each C(4) acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1-->3 of both C(4) acids after 20 minutes illumination. Thus, while the turnover rate of the (14)C-4 label in both C(4) acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C(4) acids. The implied source of (14)C for labeling of the 1-->3 positions of the C(4) acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C(4) cycle. PMID- 16664876 TI - Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions. AB - Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved. PMID- 16664877 TI - The Response of Leaf Water Potential and Crassulacean Acid Metabolism to Prolonged Drought in Sedum rubrotinctum. AB - Plants of Sedum rubrotinctum R. T. Clausen were studied in a green-house over a 2 year period without watering. Only the apical leaves survived and were turgid at the end of the experiment. The midday leaf water potential of these apical leaves was -1.20 megapascals, while the leaf water potential of comparable leaves on well-watered control plants was -0.20 megapascals. The unwatered plants appear to have maintained turgor by means of an osmotic adjustment. After 2 years without water the plants no longer exhibited a nocturnal accumulation of titratable acidity. However, the daytime levels of titratable acidity of the unwatered plants were more than 2-fold greater than the levels in well-watered control plants. Well-watered plants of S. rubrotinctum exhibited seasonal shifts in biomass stble carbon isotope ratios, indicating a greater proportion of day versus night CO(2) uptake in the winter than in the summer. The imposition of water stress prevented the expression of this seasonal rhythm and restricted the plants to dark CO(2) uptake. PMID- 16664878 TI - Cytokinin activity induced by thidiazuron. AB - The diphenylurea derivative thidiazuron induces a variety of cytokinin responses. Levels above 5 x 10(-9) molar and 4 x 10(-7) molar stimulate maximum soybean callus growth and radish cotyledon expansion, respectively. A wider range of dose response related effects follows thidiazuron induced tobacco plant regeneration. Analysis of soybean callus extracts strongly suggests that thidiazuron treatment creates an accumulation and/or synthesis of purine cytokinins, able to induce the growth, expansion and regeneration, mentioned above. PMID- 16664879 TI - A rapid leaf-disc sampler for psychrometric water potential measurements. AB - An instrument was designed which facilitates faster and more accurate sampling of leaf discs for psychrometric water potential measurements. The instrument consists of an aluminum housing, a spring-loaded plunger, and a modified brass plated cork borer. The leaf-disc sampler was compared with the conventional method of sampling discs for measurement of leaf water potential with thermocouple psychrometers on a range of plant material including Gossypium hirsutum L., Zea mays L., and Begonia rex-cultorum L. The new sampler permitted a leaf disc to be excised and inserted into the psychrometer sample chamber in less than 7 seconds, which was more than twice as fast as the conventional method. This resulted in more accurate determinations of leaf water potential due to reduced evaporative water losses. The leaf-disc sampler also significantly reduced sample variability between individual measurements. This instrument can be used for many other laboratory and field measurements that necessitate leaf disc sampling. PMID- 16664880 TI - Distinctive Light and CO(2)-Fixation Requirements of Nitrate and Ammonium Utilization by the Cyanobacterium Anacystis nidulans. AB - The effect of light intensity on the rates of ammonium and nitrate uptake and of CO(2) fixation has been determined in intact Anacystis nidulans cells. Ammonium uptake became saturated at photon flux values of about 60 microeinsteins per square meter per second, whereas both nitrate uptake and CO(2) fixation reached saturation at about 250 microeinsteins per square meter per second, the rates of the two latter processes being tightly correlated at any light intensity assayed. Inhibition of ammonium assimilation resulted in the loss of correlation between CO(2) fixation and nitrate uptake, the latter process exhibiting then a reduced light requirement. The results establish a clear distinction between ammonium utilization and nitrate utilization with regard to their light requirement and to the nature of their dependence upon CO(2) fixation. PMID- 16664881 TI - Comparative Phloem mobility of nickel in nonsenescent plants. AB - (63)Ni was applied to nonsenescent source leaves and found to be transported to sink tissues in pea (Pisum sativum L.) and geranium plants (Pelargonium zonale L.). The comparative mobilities (percent tracer transported out of source leaf /% (86)Rb transported) for (63)Ni in peas was 2.12 and in geranium 0.25. The value for the phloem mobile (86)Rb was 1.00. By contrast, the comparative mobility of (45)Ca, which is relatively immobile in the phloem, was low (0.05 in peas, 0.00 in geranium). Interruption of the phloem pathway between source and sink leaves by steam girdling almost completely inhibited (63)Ni accumulation in the sink leaves of both species. We conclude that Ni is transported from nonsenescent source leaves to sink tissues via the phloem of leguminous and nonleguminous plants. PMID- 16664882 TI - Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L. AB - During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, gamma-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa. PMID- 16664883 TI - No Latex Starch Utilization in Euphorbia esula L. AB - Utilization of leaf, stem, root, and latex starch was monitored in Euphorbia esula L. plants. Leaf, stem, and root starch decreased rapidly during a 52 day light starvation period while latex starch did not. Scanning electron and light microscope studies provided additional evidence that no changes in latex starch granules had occurred. Amylase activity (6.6 units per milligram protein) could be isolated from latex. However, latex starch granules were extremely resistant to enzymic hydrolysis by latex amylases, Bacillus subtilis alpha-amylase, and by amyloglucosidase from Aspergillus niger. Results indicate that latex starch grains do not function as utilizable carbohydrate in this species under these conditions. PMID- 16664884 TI - Relationship between Mefluidide Treatment and Abscisic Acid Metabolism in Chilled Corn Leaves. AB - Mefluidide, N-(2,4-dimethyl-5[([trifluoromethyl]sulfonyl) amino] phenyl)acetamide, a synthetic plant growth regulator, was capable of triggering an increase in endogenous free abscisic acid content when corn (Zea mays L.) plants were grown in a nonstress, day/night, temperature regime (26 degrees C) with sufficient moisture supply. The relevance of such an abscisic acid increase prior to chilling exposure and the water relations during chilling are discussed in reference to the mefluidide protection of the chilled corn plants. PMID- 16664885 TI - A Secondary Processing Site in the Precursor of the Small Subunit of Ribulose Bisphosphate Carboxylase of Chlamydomonas reinhardtii y-1. AB - When the precursor of ribulose bisphosphate carboxylase of Chlamydomonas reinhardtii y-1 is bound to antibodies and treated with the soluble cell fraction, it is cleaved to the mature form (M(r) 16,500) via an intermediate of M(r) 18,500. Although this intermediate has only been observed in vitro, it may be produced during processing of the precursor in vivo. PMID- 16664886 TI - Synthesis of a photosystem I polypeptide of 15 kilodaltons in isolated etiochloroplasts of wheat. AB - Etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings synthesized a membrane polypeptide of 15 kilodaltons. One-dimensional peptide mapping with Staphylococcus aureus V8 protease revealed that the 15 kilodaltons polypeptide is the subunit 5 of photosystem I reaction center complex. PMID- 16664887 TI - Microspectrofluorometric Measurement of Chloroplast DNA in Dividing and Expanding Leaf Cells of Spinacia oleracea. AB - Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide. PMID- 16664888 TI - Tissue Distribution and Subcellular Localization of Prephenate Aminotransferase in Leaves of Sorghum bicolor. AB - The tissue and subcellular distribution of prephenate aminotransferase, an enzyme of the shikimate pathway, was investigated in protoplasts from leaves of Sorghum bicolor. Activity was detected in purified epidermal and mesophyll protoplasts, and in bundle sheath strands. After fractionation of mesophyll and epidermal protoplasts by differential centrifugation, 92% of the total prephenate aminotransferase activity was detected in the plastid fraction. PMID- 16664889 TI - Aphidicolin Inhibition of DNA Synthesis and Germination in Spores of Anemia phyllitidis L. Sw. AB - Aphidicolin inhibits DNA synthesis and nuclear division in spores of Anemia phyllitidis. In spite of blocked DNA replication, spores germinate under continuous dark conditions, if induced by addition of 5 x 10(-5) grams per milliliter gibberellic acid. Differentiation of aphidicolin-treated prothallia indicate the existence of a prepattern in the dry spore which is realized independent of cell division during early events of spore germination. PMID- 16664890 TI - Chorismate Mutase Isoenzymes from Selected Plants and Their Immunological Comparison with the Isoenzymes from Sorghum bicolor. AB - The isoenzyme pattern of chorismate mutase (EC 5.4.99.5) was examined by diethylaminoethyl-cellulose chromatography in a wide variety of plants. All plants contained a regulated form of chorismate mutase (CM-1), and most contained an additional, unregulated form (CM-2). The regulatory properties of CM-1 differed significantly between plants. Antisera prepared against CM-1 and CM-2 from Sorghum bicolor were used to test immunological cross reaction of chorismate mutases from other plants. There was a high degree of similarity between chorismate mutase isoenzymes from Sorghum bicolor and Zea mays and some with Hordeum vulgare, but all other species studied were antigenically distinct from sorghum. No homology between the structure of CM-1 and CM-2 was detected within any species. PMID- 16664891 TI - Nitrate reductase of primary roots of red spruce seedlings : effects of acidity and metal ions. AB - Nitrate reductase activity (NRA) was found in primary roots, but not in foliage of red spruce (Picea rubens Sarg.) seedlings. Nitrate induced NRA:NH(4) (+) did not induce and slightly depressed NRA in older seedlings. Induction required 8 hours and, once induced, NRA decreased slowly in the absence of exogenous NO(3) ( ). Seedlings were grown in perlite with a complete nutrient solution containing NH(4) (+) to limit NR induction. Established seedlings were stressed with nutrient solutions at pH 3, 4, or 5 supplemented with Cl(-) salts of Al, Cd, Pb, or Zn each at two concentrations. NRA in primary root tips was measured at 2, 14, 28, and 42 days. NRA induction was greatest at pH 3, and remained high during the period of study. NRA induction at pH 4 was lower. Metal ions suppressed NRA at pH 3 and 5, but enhanced NRA at pH 4. It is concluded that acidity and soluble metals in the root environment of red spruce are unlikely to be important factors in nitrogen transformations in red spruce roots. PMID- 16664892 TI - Modified Light-Induced Absorbance Changes in dim Y Photoresponse Mutants of Trichoderma. AB - A brief pulse of blue light induces the common soil fungus Trichoderma harzianum to sporulate. Photoresponse mutants with higher light requirements than the wild type are available, including one class, dim Y, with modified absorption spectra. We found blue-light-induced absorbance changes in the blue region of the spectrum, in wild-type and dim Y mutant strains. The light-minus-dark difference spectra of the wild type and of several other strains indicate photoreduction of flavins and cytochromes, as reported for other fungi and plants. The difference spectra in strains with normal photoinduced sporulation have a prominent peak at 440 nm. After actinic irradiation, this 440 nanometer difference peak decays rapidly in the dark. In two dim Y photoresponse mutants, the difference spectra were modified; in one of these, LS44, the 440 nanometer peak was undetectable in difference spectra. Detailed study of the dark-decay kinetics in LS44 and the corresponding control indicated that the 440 nanometer difference peak escaped detection in LS44 because it decays faster than in the control. The action spectrum of the 440 nm difference peak is quite different from that of photoinduced sporulation. The light-induced absorbance changes are thus unlikely to be identical to the primary photochemical reaction triggering sporulation. Nevertheless, these results constitute genetic evidence that physiologically relevant pigments participate in these light-induced absorbance changes in Trichoderma. PMID- 16664893 TI - Metabolism of Unsaturated Monogalactosyldiacylglycerol Molecular Species in Arabidopsis thaliana Reveals Different Sites and Substrates for Linolenic Acid Synthesis. AB - Synthesis of unsaturated monogalactosyldiacylglycerol (MGDG) was examined in a mutant of Arabidopsis thaliana (L.) Heynh. containing reduced levels of hexadecatrienoic (16:3) and linolenic (18:3) acids in leaf lipids. Molecular species composition and labeling kinetics following the incorporation of exogenous [(14)C]fatty acids suggest that at least two pathways and multiple substrates are involved in desaturation of linoleic acid (18:2) to 18:3 for production of unsaturated galactolipids. A reduction in 18:3/16:3 MGDG and an increase in 18:2/16:2 MGDG, together with labeling kinetics of these molecular species following the incorporation of exogenous [(14)C]12:0 fatty acids, suggests that a chloroplastic pathway for production of 18:3 at the sn-1 position of MGDG utilizes 18:2/16:2 MGDG as a substrate. This chloroplastic (prokaryotic) pathway is deficient in the mutant. When exogenous [(14)C]18:1 was supplied, a eukaryotic (cytoplasmic) pathway involving the desaturation of 18:2 to 18:3 on phosphatidylcholine serves as the source of 18:3 for the sn-2 position of MGDG. This eucaryotic pathway predominates in the mutant. PMID- 16664894 TI - Effects of nitrogen dioxide and nitrate nutrition on nodulation, nitrogenase activity, growth, and nitrogen content of bean. AB - The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO(2) (0-0.5 parts per million) on nodulation and in vivo acetylene reduction activity of the roots and on growth and nitrate and Kjeldahl N concentration in shoots was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) plants. Exposing 8-day old seedlings for 6 hours each day, for 15 days, to 0.02 to 0.5 parts per million NO(2) decreased total nodule weight at 0 and 1 millimolar nitrate, and nitrogenase (acetylene reduction) activity at all concentrations of nitrate. The pollutant had little effect on root fresh or dry weights. Shoot growth was inhibited by NO(2). The NO(2) exposure increased nitrate concentration in roots only at 20 millimolar nutrient nitrate. Exposure to NO(2) markedly increased Kjeldahl N concentration in roots but generally decreased that in shoots. The experiments demonstrated that nutrient N level and NO(2) concentration act jointly in affecting nodulation and N fixing capability, plant growth and composition, and root/shoot relationships of bean plants. PMID- 16664895 TI - Sodium Transport and Compartmentation in Spergularia marina: Partial Characterization of a Functional Symplasm. AB - In this paper, a combination of tracer uptake, efflux, and pulse-chase techniques is applied to the problem of compartmentation of Na(+) ((24)Na(+)) in the roots of intact, midvegetative Spergularia marina (L.) Griseb. plants. An approach is presented for conducting useful compartmental analysis when it is known that the assumptions required for straightforward interpretations of influx and efflux studies are invalid. Linear rates of (24)Na(+) accumulation in both roots and shoots were attained within at most a few minutes following the start of labeling. Shoot (24)Na(+) contents equaled root contents within about 20 minutes. Analysis of root accumulation rates, and compartmental and pulse-chase efflux studies indicated that the unidirectional flux rates involved were at least an order of magnitude greater than linear rates of root and shoot accumulation. These rapid fluxes involved only a small portion of the total root Na(+) (about 1%). The results suggest the existence of a small symplastic compartment, distinct from the ;bulk cytoplasm,' rapidly exchanging with the medium, and responsible for delivery of Na(+) to the xylem. The physical identity of this compartment and its physiological significance are discussed with respect to precedents in the literature. PMID- 16664896 TI - Structural Relationship among the Rice Glutelin Polypeptides. AB - When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the alpha and beta subunits are proteolytic products of the 51 kD precursor protein. The poly(A)(+) RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form. PMID- 16664897 TI - Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots. AB - Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [(15)N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [(15)N] H(4) (+) or [(15)N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [(15)N]H(4) (+) was provided, mitochondria isolated from light-grown pea shoots synthesized [(15)N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [(15)N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [(15)N]glycine was used, pea mitochondria synthesized [(15)N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [(15)N]glycine and synthesis of [(15)N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [(15)N]H(4) (+) by the [(14)N]H(4) (+) pool in the mitochondria. The majority of the [(15)N]H(4) (+) released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [(15)N]glycine and little synthesis of [(15)N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [(15)N]glutatmate was synthesized at a rate of 7% of that of [(15)N]serine synthesis by corn mitochondria. PMID- 16664898 TI - Membrane Electrical Noise in Chara corallina: II. Effects of Inhibitors on the Low Frequency Spectral Component. AB - Certain inhibitors have been found to affect the low frequency spectral component of the electrical noise power spectrum in Chara corallina. Application of the ATPase inhibitor N,N'-dicyclohexylcarbodiimide removed the low frequency spectral component, strengthening the case that the component is produced by active proton pumping. Cytocholasin B, which inhibits cyclosis in internodes of C. corallina, removed the low frequency spectral component in a time-dependent fashion which was correlated with the cessation of streaming. The protonophore carbonyl cyanide m-chlorophenylhydrazone did not produce consistent effects on the low frequency spectral component in these cells. PMID- 16664899 TI - Glyoxysomal Malate Synthase of Cucumber: Molecular Cloning of a cDNA and Regulation of Enzyme Synthesis during Germination. AB - A cDNA clone for the glyoxysomal enzyme malate synthase was isolated from a cDNA library made with polyadenylated RNA from the cotyledons of germinating Cucumis sativus L. This cloned DNA sequence was used as a probe to characterize changes in the amounts of malate synthase gene transcripts in cotyledons of cucumber seeds grown both in the light and in the dark. Malate synthase gene transcripts increase in amount to a peak at day 3 or day 4, and thereafter decline. In the light, this rate of decline is significantly greater than in the dark. Measurement of the changes in the amounts of malate synthase by assaying enzyme activity directly, and by immunological reaction with a specific antiserum indicate that the developmentally regulated synthesis of malate synthase in germinating cucumber is brought about primarily by changes in the amount of malate synthase gene transcripts, rather than through a control of translation. Similarly, the effect of light on the amount of malate synthase correlates precisely with its effect on the abundance of malate synthase gene transcripts. PMID- 16664900 TI - Stomatal Responses to Light and Drought Stress in Variegated Leaves of Hedera helix. AB - Direct and indirect mechanisms underlying the light response of stomata were studied in variegated leaves of the juvenile phase of Hedera helix L. Dose response curves of leaf conductance were measured with blue and red light in leaves kept in normal or in an inverted position. In the green portions of the leaves, the sensitivity to blue light was nearly 100 times higher than that to red light. No response to red light was observed in the white portions of the leaves up to 90 micromoles per square meter per second. Red light indirectly affected leaf conductance while blue light had a direct effect. Leaf conductance was found to be more sensitive to drought stress and showed a more persistent aftereffect in the white portions of the leaves. A differential effect of drought stress on the responses to blue and red light was also observed. PMID- 16664901 TI - Increased Arginine Biosynthesis during Phosphorus Deficiency : A Response to the Increased Ammonia Content of Leaves. AB - The accumulation of arginine in leaves of four citrus rootstock cultivars during P deficiency has been demonstrated to be due to increased de novo synthesis rather than decreased catabolism or increased protein degradation (E Rabe, CJ Lovatt, 1984, Plant Physiol 76: 747-752). In this report, we provide evidence (a) that the increased activity of the arginine biosynthetic pathway observed for citrus rootstocks grown under P-deficient conditions for 7 months is due to an increase in the concentration of ammonia in leaves of P-deficient plants and (b) that ammonia accumulation and removal through arginine systhesis are early responses to phosphorus deficiency for both a woody perennial, rough lemon (Citrus limon), and an herbaceous annual, summer squash (Cucurbita pepo). Transferring 5-day-old squash plants to a phosphorus-deficient nutrient solution for only 10 days resulted in a 2-fold increase in the concentration of nitrate in the youngest fully expanded leaves (YFE). Concomitantly, the specific activity of nitrate reductase doubled and the ammonia content of P-deficient YFE leaves increased to a concentration significantly greater that of leaves from healthy control plants (P < 0.05). Consistent with increased availability of ammonia, the incorporation of NaH(14)CO(3) into arginine plus urea doubled during phosphorus deficiency and arginine accumulated. Despite the accumulation of nitrate and ammonia in YFE leaves during phosphorus deficiency, the total nitrogen content of these leaves was less than that of the healthy control plants. Similar results were obtained for rough lemon. Nitrate content of the YFE leaves increased 1.5- and 3.0-fold in plants deprived of phosphorus for 6 and 12 weeks, respectively. Ammonia content of the leaves increased as P deficiency progressed to 1.4 +/- 0.08 mg (+/- se, n = 4) per gram dry weight, a level 1.8-fold greater than that of the P-sufficient control plants. During P deficiency de novo arginine biosynthesis in rough lemon increased 10-fold. Immersing the petiole of YFE leaves from P-sufficient squash and rough lemon plants in 50 millimolar NH(4) (+) for 3 hours resulted in the accumulation of ammonia in the leaves, and a 4-fold increase in the incorporation of NaH(14)CO(3) into arginine plus urea. Taken together, these results provide strong evidence that the accumulation of nitrate and ammonia in leaves is an early response of both woody and herbaceous plants to P deprivation. The data are consistent with the hypothesis that increased de novo arginine biosynthesis in leaves during P deficiency is in response to ammonia content of the leaves. PMID- 16664902 TI - Role of Ethylene in Lactuca sativa cv ;Grand Rapids' Seed Germination. AB - Promotion of thermoinhibited (30 degrees C) lettuce (Lactuca sativa cv ;Grand Rapids') seed germination by ethylene is similar to the action of the gas in other hormonal systems. Ethylene was more active than propylene and ethane was inactive. An inhibitor of ethylene production, aminoethoxy-vinylglycine, reduced ethylene evolution and germination. Inhibitors of ethylene action such as, 5 methyl-7-chloro-4-ethoxycarbonylmethoxy-2,1,3-benzothiadiazole, 2,5 norbornadiene, and silver thiosulfate inhibited germination and the effect was reversed by the addition of ethylene to the gas phase. The action of ethylene appears to be due to the promotion of radial cell expansion in the embryonic hypocotyl. The action of N6-benzyladenine and fusiccocin, which also overcome thermoinhibition, appears to be due to a promotion of hypocotyl elongation. None of the germination promoters studied appeared to function by lowering the mechanical resistance of the endosperm to embryonic growth. Data presented here are consistent with the view that ethylene plays a role in lettuce seed germination under thermoinhibited and normal conditions. PMID- 16664903 TI - Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Changing Partial Pressure of O(2) and Light in Phaseolus vulgaris. AB - The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (rubisco) activity in Phaseolus vulgaris was studied under moderate CO(2) and high light, conditions in which photosynthesis in C(3) plants can be insensitive to changes in O(2) partial pressure. Steady state RuBP concentrations were higher, the calculated rate of RuBP use was lower and the activation state of rubisco was lower in low O(2) relative to values observed in normal O(2). It is suggested that the reduced activity of rubisco observed here is related to feedback effects which occur when the rate of net CO(2) assimilation approaches the maximum capacity for starch and sucrose synthesis (triose phosphate utilization). The activation state of rubisco was independent of O(2) partial pressure when light or CO(2) was limiting for photosynthesis. Reduced activity of rubisco was also observed at limiting light. However, in this species light dependent changes in the concentration of an inhibitor of rubisco controlled the apparent V(max) of rubisco in low light while changes in the CO(2)-Mg(2+) dependent activation of rubisco controlled the apparent V(max) in high light. PMID- 16664904 TI - Effects of NaCl and CaCl(2) on Ion Activities in Complex Nutrient Solutions and Root Growth of Cotton. AB - Sodium displaces Ca(2+) from membranes (GR Cramer, A Lauchli, VS Polito Plant Physiol 1985 79: 207-211) and this can be related to the (Ca(2+))/(Na(+))(2) activity ratio in the external solution (GR Cramer, A Lauchli 1986 J Exp Bot 37: 321-330). Supplemental Ca(2+) is known to mitigate the adverse effects of salinity on plant growth. In this report we investigated the effects of NaCl (0 250 millimolar) and Ca(2+) (0.4 and 10 millimolar) on the ion activities in solution and on root growth of cotton (Gossypium hirsutum L.). Ion activities were analyzed using the computer program, GEOCHEM. Most ion activities in a 0.1 modified Hoagland solution were significantly reduced by both NaCl and supplemental Ca(2+). Ion-pair formation and precipitation were significant for some ions, especially phosphate. Root growth of 6-day-old seedlings was stimulated by low NaCl concentrations (25 millimolar). At higher NaCl concentrations, root growth was inhibited; the concentration at which this occurred depended on the Ca(2+) concentration and the growth index used. Supplemental Ca(2+) mitigated the inhibition of root growth caused by NaCl. There was a curvilinear relationship between root growth and the (Ca(2+))/(Na(+))(2) ratio in the nutrient solution. The mechanisms by which Na(+) and Ca(2+) may affect root growth are discussed. PMID- 16664905 TI - Changes in Soybean (Glycine max [L.] Merr.) Glycerolipids in Response to Water Stress. AB - Soybean (Glycine max [L.] Merr.) plants with the first trifoliate leaf fully expanded were exposed to 4 and 8 days of water stress. Leaf water potentials dropped from -0.6 megapascal to -1.7 megapascals after 4 days of stress; then to 3.1 megapascals after 8 days without water. All of the plants recovered when rewatered. The effects of short-term drought stress on triacylglycerol, diacylglycerol, phospholipid, and galactolipid metabolism in the first trifoliate leaves was determined. Leaf triacylglycerol and diacylglycerol content increased 2-fold during the first 4 days of stress and returned to control levels 3 days after rewatering. The polar lipid fraction, which contained phospholipids and galactolipids, changed little during this time. The linolenic acid (18:3) content of the triacylglycerol and diacylglycerol increased 25% during stress and the polar lipid 18:3 content decreased 15%. The pattern of glycerolipid labeling, after applying [2-(14)C]acetate to intact leaves was altered by water stress. After 4 days of water stress the radioactivity of phosphatidic acid + phosphatidylinositol, phosphatidylcholine, triacylglycerol, and diacylglycerol increased between 4 and 9% (compared to control plans) while radioactivity of phosphatidylethanolamine, monogalactosyldiglyceride, and digalactosyldiglyceride decreased 2 to 11%. These data indicated that increased levels of triacylglycerol and diacylglycerol observed during water stress were attributed to de novo synthesis rather than breakdown or reutilization of existing glycerolipids and fatty acids. PMID- 16664906 TI - Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. AB - A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins. PMID- 16664907 TI - Phase transitions in liposomes formed from the polar lipids of mitochondria from chilling-sensitive plants. AB - The thermal response of mitochondrial polar lipids from a variety of chilling sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15 degrees C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16 degrees C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15 degrees C for cucumber (Cucumus sativus L.) fruit, at 14 degrees C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15 degrees C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature induced alteration in the structure of cell membranes. PMID- 16664908 TI - Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves. AB - The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 +/- 3,000 daltons, an isoelectric point of 4.8 +/- 0.1, and a relative electrophoretic mobility of 0.58 +/- 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N alpha-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 +/- 0.5 degrees C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK(a) values of 6.8 +/- 0.1 and 8.2 +/- 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-alpha-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 x 10(-9) molar. In agreement with the enzyme specificity, only N-alpha tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators. PMID- 16664909 TI - Uptake and processing of the precursor to the small subunit of ribulose 1,5 bisphosphate carboxylase by leucoplasts from the endosperm of developing castor oil seeds. AB - Intact leucoplasts from the endosperm of developing castor oil seed were isolated by Percoll density gradient centrifugation. The precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase from pea was synthesized in vitro from hybrid-selected mRNA. Leucoplasts imported this precursor by an ATP-requiring mechanism similar to that described in chloroplasts (AR Grossman et al. 1980 Nature 285: 625-628). The small subunit precursor was processed to a molecular weight that was identical with that of the mature pea small subunit. These results show that leucoplasts, though specialized for fatty acid biosynthesis and not photosynthesis, have a mechanism of protein import similar to that of chloroplasts. PMID- 16664910 TI - Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L. AB - Extracts of resting and germinating (3 days at 20 degrees C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent. PMID- 16664911 TI - Synthesis and processing of cellulase from ripening avocado fruit. AB - The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum. PMID- 16664912 TI - Rhizosphere acidification as a response to iron deficiency in bean plants. AB - Iron deficiency in higher plants causes accumulation of salts of organic acids in the roots, the most characteristic being citrate. We show that citrate and malate accumulate in beans (Phaseolus vulgaris L. var Prelude), not because of a lack of the iron-containing enzyme aconitase (EC 4.2.1.3), but in close coupling to the extrusion of protons during rhizosphere acidification, one of the ;Fe-efficiency' reactions of dicotyledonous plants. When proton excretion is induced in roots of control bean plants by addition of fusicoccin, only malate, not citrate, is accumulated. We propose that iron deficiency induces production of organic acids in the roots, which in beans leads to both proton excretion and an increased capacity to reduce ferric chelates via the induced electron transfer system in the root epidermis cells. PMID- 16664913 TI - H and k electrogenic exchanges in corn roots. AB - The membrane potential difference, the net H(+) exchange rate, the K(+) net flux, and the K(+) ((86)Rb(+)) influx were measured in excised corn roots as functions of the K(+) concentration in the medium at various pH values, in the presence of poorly permeant anions. The roots behaved as a K(+)/H(+) exchange system. By comparing the results in normal or hypoxic conditions, or in the presence of vanadate, it was possible to distinguish the active components of membrane potential and transports from the passive ones. The magnitude of the electrogenic potential was not related to the active H(+) extrusion rate. At pH 6, the variations of the electrogenic potential resulted from variations of the stoichiometry of the active H(+)/K(+) exchange. The same relationship between this stoichiometry and the K(+) concentration was observed in conditions ensuring different membrane polarizations (pH 6, pH 4, or pH 6 with fusicoccin). Both metabolic and Mg-ATPase specific inhibitors stopped the active H(+) transport and the net K(+) influx. Nevertheless, the tracer influx in the presence of vanadate remained higher than the passive influx calculated from the permeability coefficient determined in hypoxia. It is proposed that vanadate uncouples the K(+) moiety of the H(+)/K(+) antiport and allows it to mediate isotopic exchanges. PMID- 16664914 TI - Assay of photosynthetic oxygen evolution from single protoplasts. AB - A semiquantitative assay for light-dependent O(2) evolution by a single mesophyll protoplast is described. The assay indicator is the density of aerotactic bacteria (Pseudomonas aeruginosa, ATCC 10145; ;Engelmann experiment') attracted to the protoplast. Quantification is by dark field microphotometry. The sensitivity is about 50 femtomoles O(2) per protoplast per minute. The results demonstrate the biphasic nature of O(2) evolution of a single protoplast during photosynthetic induction. Computerized data acquisition yields traces which, until a steady state of photosynthetic O(2) evolution is reached, are identical to ordinary O(2) electrode traces. PMID- 16664915 TI - A mutant of Arabidopsis deficient in c(18:3) and c(16:3) leaf lipids. AB - Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 16:3 and 18:3 fatty acids and has correspondingly increased levels of the 16:2 and 18:2 precursors due to a single recessive nuclear mutation. The kinetics of in vivo labeling of lipids with [(14)C]acetate and quantitative analysis of the fatty acid compositions of individual lipids suggests that reduced activity of a glycerolipid n-3 desaturase is responsible for the altered lipid composition of the mutant. The effects of the mutation are most pronounced when plants are grown at temperatures above 26 degrees C but are relatively minor below 18 degrees C, suggesting a temperature-sensitive enzyme. Since the desaturation of both 16- and 18-carbon fatty acids is altered, it appears that the affected enzyme lacks specificity with respect to acyl group chain length and that it is located in the chloroplast where 16:3-monogalactosyldiglyceride is synthesized. Because the degree of unsaturation of all the major glycerolipids was similarly affected by the mutation, it is inferred that either the affected desaturase does not exhibit head group specificity or there is substantial transfer of trienoic acyl groups between different lipid classes. Both chloroplast and extrachloroplast lipids are equally affected by the mutation. Thus, either the desaturase is located both outside and inside the chloroplast, or 18:3 formed inside the chloroplast is reexported to other cellular sites. PMID- 16664916 TI - Stomatal responses to light and leaf-air water vapor pressure difference show similar kinetics in sugarcane and soybean. AB - Stomatal responses to light and humidity (vapor pressure difference, VPD) are important determinants of stomatal conductance. Stomatal movements induced by light are the result of a transduction of the light stimulus into modulated ion fluxes in guard cells and concomitant osmotic adjustments and turgor changes. It is generally assumed that this transduction process is a general stomatal property, with different environmental stimuli integrated into guard cell metabolism through their modulation of ion fluxes. In contrast with this notion, the VPD response, which is unique because both its triggering signal and the turgor changes required for aperture modulations involve water molecules, has been considered to be hydropassive and thus independent of guard cell metabolism. We used a kinetic approach to compare the light and VPD responses in order to test the hypothesis that hydropassive changes in guard cell turgor could be faster than the metabolism-dependent light responses. Changes in stomatal conductance in intact leaves of sugarcane and soybean were measured after application of step changes in VPD and in light. In spite of a 5-fold difference in overall rates between the two species, the response rates following light or VPD steps were similar. Although a coincidental kinetic similarity between two mechanistically different responses cannot be ruled out, the data suggest a common mechanism controlling stomatal movements, with the VPD stimulus inducing metabolic modulations of ion fluxes analogous to other stomatal responses. PMID- 16664917 TI - A sensitive diffusion plate assay for screening inhibitors of protease activity in plant cell fractions. AB - Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins. PMID- 16664918 TI - Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina. AB - The uptake of K(+) and Ca(2+) in Dunaliella salina is mediated by two distinct carriers: a K(+) carrier with a high selectivity against Na(+), Li(+), and choline(+) but not towards Rb(+), K(+), Cs(+), or NH(4) (+), and a Ca(2+) carrier with a high selectivity against Mg(2+). The latter is specifically blocked by La(3+) and by Cd(2+). Apparent K(m) values for K(+) and Ca(2+) uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K(+) and Ca(2+) uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H(+)-ATPases and protonionophores inhibit K(+) and Ca(2+) uptake and accelerate K(+) efflux. The results suggest that an H(+)-ATPase in the cell membrane provides the driving force for K(+) and Ca(2+) uptake. Efflux measurements from (86)Rb(+) and (45)Ca(2+) loaded cells suggest that part of the intracellular K(+) and most of the intracellular Ca(2+) is nonexchangeable with the extracellular pool. Correlations between phosphate and K(+) contents and the effect of phosphate on K(+) efflux suggest intracellular associations between K(+) and polyphosphates. On the basis of these results, it is suggested that: (a) K(+) and Ca(2+) uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H(+) ATPase of the plasma membrane. (b) Part of the intracellular K(+) is associated with polyphosphate bodies, while most of the intracellular Ca(2+) is accumulated in intracellular organelles in the algal cells. PMID- 16664919 TI - Photosynthesis Is Not Involved in the Mechanism of Action of Acifluorfen in Cucumber (Cucumis sativus L.). AB - The possible role of photosynthesis in the mechanism of action of the herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate; AF) was examined. The sensitivity to AF of cotyledons of cucumber (Cucumis sativus L.) which had been grown under far red light (FR) and white light were compared. FR grown tissues which were photosynthetically imcompetent were hypersensitive to AF under white light and had approximately the same relative response to AF under blue and red light as green, white-light-grown tissues. Ultrastructural damage was apparent in FR-grown, AF-treated tissues within an hour after exposure to white light, with cytoplasmic and plastidic disorganization occurring simultaneously. In cucumber cotyledon tissue which had been greening for various time periods, there was no correlation between photosynthetic capacity and herbicidal efficacy of AF. PSII inhibitors (atrazine and DCMU) and the photophosphorylation inhibitor, tentoxin, had no effect on AF activity. Atrazine did not reduce AF activity at any concentration or light intensity tested, indicating that there is no second, photosynthetic-dependent mechanism of action operating at low AF concentrations or low fluence rates. Carbon dioxide-dependent O(2) evolution of intact chloroplasts of spinach (Spinacia oleracea L.) had an AF I(50) of 125 micromolar compared to 1000 micromolar for cucumber, whereas AF was much more herbicidally active in tissues of cucumber than of spinach. Differences in activity could not be accounted for by differences in uptake of AF. Our results indicate that there is no photosynthetic involvement in the mechanism of action of AF in cucumber. PMID- 16664920 TI - Saturable uptake of indol-3yl-acetic Acid by maize roots. AB - The uptake of 5-[(3)H]indol-3yl-acetic acid (IAA(*)) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA degrees ) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K(m) of 0.285 micromolar and apparent V(max) 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA(*) took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport. PMID- 16664921 TI - Partial characterization of cadmium-binding protein from roots of tomato. AB - Cd-binding protein was extracted from tomato roots and purified on QAE-Sephadex A 25 and on Sephadex G-75 in 1 molar KCl buffer. The protein preparation was light brown and contained predominantly Cd and small amounts of Zn and Cu. Polyacrylamide gel electrophoresis at pH 6.9 removed the brown material from protein which now bound mostly Cd and some Cu. The apparent molecular weight was 3,100 daltons in high ionic strength medium (1 molar KCl buffer) and 21,500 daltons at low ionic strength. Ionic strength also affected the apparent molecular weight of the Cd-binding protein in crude root extracts. The protein contained 26% cysteine, 53% glutamic acid/glutamine, and 2.8 gram atoms (Cd+Zn+Cu)/mole. The (Cd+Zn+Cu):cysteine ratio was 1:2.3. Circular dichroism measurements indicated Cd-thiolate coordination. The tomato Cd-binding protein was more similar to phytochelatins than to animal metallothioneins. PMID- 16664922 TI - Hydrolysis of storage proteins in barley endosperms : analysis of soluble products. AB - Soluble products, released by the hydrolysis of hordeins into the media of barley (Hordeum vulgare cv. Perth) half-seeds were analyzed. Large polypeptide fragments (methanol-insoluble) were identified using the Western immunoblot technique with the antibodies prepared against B and C polypeptides of hordein. A number of hordein IgG-reacting bands were noted in the samples from dry kernels. In samples incubated in the absence of gibberellic acid, polypeptide fragments in the size range of 25 to 30 kilodaltons appeared within 24 hours, and those in the size range of 40 kilodaltons became more prominent. In samples incubated in the presence of gibberellic acid, polypeptide fragments in the size range of 45 to 67 kilodaltons were less apparent and those in the size range of less than 15 kilodaltons were more pronounced. The hordein-related polypeptide fragments were present in low amounts after 72 hours in the presence of gibberellic acid. Methanol-soluble peptides were fractionated, on the basis of size, into two broad peaks. In the absence of gibberellic acid, there was no significant change in their profile over a 72 hour incubation period. In the presence of this growth substance, however, there was a decrease in the proportion of large size peptides (50-70 amino acid residues in length), and an increase in the levels of small peptides (15-35 amino acid residues in length) and amino acids. Our interpretation of the results is that the release of the initial large polypeptide fragments from hordein proteins is mediated by a protease(s) whose appearance is not dependent on the exogenously added gibberellic acid. Further hydrolysis is, however, mediated by proteases induced in the presence of this growth substance. PMID- 16664923 TI - Rapeseed embryo development in culture on high osmoticum is similar to that in seeds. AB - The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. The osmotic conditions used block germination of predesiccation stage embryos but were not sufficient to prevent desiccation stage embryos from taking up water and germinating. At all stages tested, the osmotically treated embryos had approximately normal levels of cruciferin mRNA. Measurements of endogenous abscisic acid (ABA) levels by radioimmunoassay indicated that the osmotic effects on germination and gene expression were not mediated by elevated embryonic ABA. Comparison of the kinetics of osmotic and ABA effects on gene expression showed that the osmotic effect is more rapid. These results are consistent with the hypothesis that ABA acts by inhibiting water uptake, which mechanically prevents germination and affects gene expression in some unknown manner. PMID- 16664924 TI - Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases. AB - We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:beta-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1-->3)-beta-glucan synthase on both Ca(2+) and a beta-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay. PMID- 16664925 TI - Membrane Operational Impedance Spectra in Chara corallina Estimated by Laplace Transforms Analysis. AB - The membrane operational impedance spectrum of Chara corallina Klein ex Willd. (R. Brown) cells is investigated using Laplace transform analysis. The spectrum changes with both amplitude and sign of the electrical stimulation when time- and voltage-dependent K(+) channels contribute to the membrane conductance. We compare the advantages and disadvantage of this technique for studying membrane impedance with those of the alternating current method and the white noise method. PMID- 16664926 TI - Light, temperature, and anthocyanin production. AB - Temperature affects the total amount, the time course, and the red/far-red effectiveness ratio of light-dependent anthocyanin production in Brassica oleracea L. seedlings. Some of the effects of temperature on anthocyanin production in cabbage are in agreement with the predictions of a model proposed by JK Wall and CB Johnson (1983 Planta 159: 387-397) for the effects of temperature on the state of phytochrome and on the expression of phytochrome mediated high irradiance responses, but others are not. The lack of a complete agreement between experimental results and model predictions might be due to factors related to the experimental system used or to limitations of the model or both. PMID- 16664927 TI - Energy Facilitated Na Uptake in Excised Corn Roots via P and Na NMR. AB - The uptake of sodium ions by excised corn root tips (Zea mays L. cv FRB-73) was monitored by (23)Na nuclear magnetic resonance (NMR) methods in the presence of a membrane impermeable shift reagent under different metabolic conditions. In addition, for the first time, the energy status as well as the intracellular pH associated with this influx was concurrently evaluated by (31)P NMR. The rate of sodium ion uptake decreased (in order) as the normal metabolic state was changed by the addition of cyanide, anaerobic condition, and carbonyl cyanide trifluoromethoxy phenyl-hydrazone treatment. The results suggest that the proton electrochemical potential of the plasma membrane may facilitate the influx of Na(+). PMID- 16664928 TI - The induction of ethylene production from pear cell culture by cell wall fragments. AB - Macerase, a pectinase-containing enzyme mixture, was used to digest cell walls isolated from cultured pear cells. Following digestion, the reaction mixture was boiled to inactivate enzymes. Addition of soluble aliquots of the mixture to suspension cultures of pear cells led to a rapid and transient production of ethylene by the cells. PMID- 16664929 TI - Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells. AB - Glycoproteins released from carrot cells into culture media were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by staining with Coomassie brilliant blue or with the periodic-acid Schiff procedure. The appearance or disappearance of two glycoproteins of M(r) 65,000 (GP65) and M(r) 57,000 (GP57) was closely related to the formation of somatic embroys. GP65 was released specifically from embryogenic cells cultured in a medium without 2,4-dichlorophenoxyacetic acid, in which they can form somatic embryos. GP57 was released from the same embryogenic cells, if they were cultured in a medium with 2,4-dichlorophenoxyacetic acid, in which they cannot form somatic embryos. Nonembryogenic cells which cannot form somatic embryos, released only GP57. PMID- 16664930 TI - The effect of salt concentration on auxin stability in culture media. AB - The concentrations of indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were followed for 35 days in cell-free liquid medium containing 100, 50, or 0% Murashige-Skoog (MS) salt base. Although the concentrations of NAA or 2,4-D remained constant the level of IAA decreased to only 11% of the original concentration after 35 days in the presence of 100% MS salt base. The observed rate of IAA degradation was accelerated by the presence of MS salts. PMID- 16664931 TI - Photosynthesis of Ulva sp: III. O(2) Effects, Carboxylase Activities, and the CO(2) Incorporation Pattern. AB - Ulva, a common green seaweed, performs at the biochemical level as a typical C(3) plant. Over 90% of label was found in glycerate 3-phosphate following a 3 second (14)C pulse in the light, and the label was subsequently transferred to sugars. Also, the level of ribulose-1,5-bisphosphate carboxylase activity in crude extracts was about 10 times higher than that of phosphoenolpyruvate carboxylase. Concerning gas exchange, photosynthetic rates of Ulva showed no O(2) sensitivity, indicating that photorespiratory CO(2) losses are repressed as in C(4) plants. This apparent anomaly could be explained by the efficient HCO(3) (-) uptake system of Ulva which might concentrate CO(2) to the chloroplasts, thus suppressing the oxygenase activity of ribulose-1,5-bisphosphate carboxylase. PMID- 16664932 TI - Effect of Monochromatic Light on Proton Efflux of the Blue-Green Alga Anabaena variabilis. AB - Light-induced proton efflux of Anabaena variabilis was found to be biphasic, the second phase being inhibited by the ATPase inhibitor nitrofen (2,4-dichloro-1-[4 nitrophenoxy]benzene). The first, fast phase was triggered by monochromatic light of 707 nanometers, whereas the second, slower phase was not. With 707 nanometers, light, respiratory O(2) uptake was inhibited. Using light composed of two wavelengths (616 and 707 nanometers) a marked enhancement of both O(2) evolution as well as the second phase of proton efflux was observed. The first phase was not enhanced. Thus, phase II is driven by both photosystems. As concluded from the action spectrum phase I is markedly determined by photosystem-I activity. Altogether the data show that two different mechanisms of light-induced proton efflux exist on the cytoplasmic membrane of Anabaena, the slower one being dependent on ATP and linear photosynthetic electron flow. PMID- 16664933 TI - Light Dependent Reduction of Pertechnetate (TcO(4)) by Broken Chloroplasts. AB - Arguments are given for a ferredoxin-mediated reduction of TcO(4) (-), preponderantly into extractable Tc(V) complexes, by illuminated, broken chloroplasts. Photosynthetic O(2)- and NADP-reduction competitively inhibit Tc incorporation. As for O(2), the reaction can be stimulated by the auto-oxidizable electron acceptor methyl viologen. Furthermore TcO(4) (-) can function as terminal acceptor in the diaphorase reaction, with NADPH as electron donor. PMID- 16664934 TI - A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. AB - Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K(m) for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective ;PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed. PMID- 16664935 TI - Synthesis of phytochrome apoprotein and chromophore are not coupled obligatorily. AB - G Gardner and HL Gorton (1985 Plant Physiol 77: 540) demonstrated that gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid) inhibits the initial synthesis and resynthesis of spectrophotometrically detectable phytochrome in pea, maize, and oat. We show that the level of immuno-detectable phytochrome in pea is unaffected by the presence of gabaculine at a concentration that reduces spectrophotometrically detectable phytochrome up to 10-fold. This result indicates that gabaculine inhibits chromophore synthesis without affecting phytochrome apoprotein synthesis and that chromophore-less phytochrome is stable in the cell. PMID- 16664936 TI - Abscisic Acid Accumulation by in Situ and Isolated Guard Cells of Pisum sativum L. and Vicia faba L. in Relation to Water Stress. AB - Isolated guard cells, prepared by sonication of epidermal peels, were used to investigate the endogenous level of abscisic acid (ABA) in the guard cells of turgid and stressed leaves of Vicia faba L. and the argenteum (arg) mutant of Pisum sativum L. The guard cells of V. faba and arg were found to contain 18 and 8 times more ABA, respectively, when isolated from stressed leaves than from turgid leaves. Isolated guard cells of V. faba were also directly stressed with the osmoticum Aquacide III. These guard cells were capable of producing stress induced ABA to at least 3 times their ABA level when non-stressed. PMID- 16664937 TI - Potassium transport in suspension culture cells and protoplasts of carrot. AB - The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15: 473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0.1 millimolar KCl, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues. PMID- 16664938 TI - Rubber production in guayule: determination of rubber producing potential. AB - Optimum conditions for the rapid, efficient, nondestructive determination of rubber producing potential in guayule (Parthenium argentatum) were established. The rubber producing potential may be defined as the ability of the plant material to synthesize rubber from a precursor under specified conditions. To achieve this, stem slices taken from the first 5 centimeters of branches were incubated with [(14)C]acetate as precursor in 0.1 molar phosphate buffer (pH 6.5) at 26 degrees C for 16 hours in the light. The (14)C from labeled acetate and acetyl coenzymeA were efficiently incorporated into rubber whereas the (14)C from both mevalonic acid (MVA) and isopentenylpyrophosphate (IPP) were poorly incorporated. Incorporation of 68.6% of the (14)C from labeled IPP into the acetone extractable material suggests that most of the IPP was channeled down the lower terpenoid branch of the polyisoprene biosynthetic pathway. The incorporation of (14)C from labeled acetate into rubber was most efficient at temperatures between 20 and 25 degrees C. The rubber producing potential was also found to be dependent on light intensity. The roots which represent about one third of the plant biomass not only had the highest rubber producing potential but also contained the highest amount of rubber (7.6%), indicating that the root system could be a major source of rubber. The mature stem bark also had a high rubber content and rubber producing potential, whereas the young stem had a low rubber content and a lower potential for producing rubber. The leaves showed little potential to incorporate labeled acetate into rubber and no more than 0.5% rubber was found in guayule leaves. PMID- 16664939 TI - Phytochrome Control of Specific mRNA levels in Developing Pea Buds : Kinetics of Accumulation, Reciprocity, and Escape Kinetics of the Low Fluence Response. AB - We have examined the time course for accumulation of each of 12 different nuclear gene transcripts in pea buds after irradiating dark grown seedlings with a single pulse low fluence red light (10(3) micromoles per square meter delivered in 100 seconds). The 12 time courses can be grouped into four general classes. Six transcripts (including RNAs coding for the chlorophyll a/b binding protein and ribulose-1,5-bisphosphate carboxylase) accumulate at a linear rate during 24 hours in darkness following the light pulse. Two transcripts increase rapidly at first but then reach a plateau after 3 hours and remain at that level for the next 21 hours. Another two transcripts exhibit a prolonged lag period before beginning to accumulate, and do not reach significant accumulation rates until 12 to 16 hours after the red light pulse. One transcript appears to undergo a transient increase in abundance in response to red light, but this is superimposed on a background of slowly increasing abundance of this RNA in control plants. This response, unlike all the others, exhibits reciprocity failure in experiments in which the same fluence of light is given over periods ranging between 50 and 4000 seconds.We have also examined the kinetics with which each of these 12 responses escapes from phytochrome-far-red absorbing form control by attempting to reverse the induction with far-red light given at various times after the red light pulse. Again, several different patterns are apparent for the different transcripts. The time at which far red reversibility first begins to be lost, the rate at which it is lost, and the final extent of reversibility remaining after 7 hours in the dark all differ for different transcripts. In addition, we have observed that some responses retain virtually complete photoreversibility for at least 7 hours. In some cases, a comparison of the time course and escape kinetic data indicates that relatively rapid turnover of the RNA must occur. It is not clear whether or not the rate of turnover is influenced by phytochrome. PMID- 16664940 TI - Malate Metabolism in Leaf Mitochondria from the Crassulacean Acid Metabolism Plant Kalanchoe blossfeldiana Poelln. AB - The mechanisms and the controlling factors of malate oxidation by mitochondria from leaves of Kalanchoe blossfeldiana Poelln. plants performing Crassulacean acid metabolism were investigated using Percollpurified mitochondria. The effects of pH and of various cofactors (ATP, NAD(+), coenzyme A) on malate dehydrogenase (EC 1.1.1.37) and malic enzyme (EC 1.1.1.39) solubilized from these mitochondria were examined. The crucial role of cofactor concentrations in the mitochondrial matrix on the pathways of malate oxidation is shown. The distribution of the electrons originating from malate between the different electron transport pathways and its consequence on the phosphorylation yield was studied. It was found that, depending on the electron transport pathway used, malate oxidation could yield from 3 to 0 ATP. Assayed under conditions of high reducing power and high energy charge, the ability of malic enzyme to feed electrons to the cyanide resistant nonphosphorylating alternative pathway was found to be higher than that of other dehydrogenases linked to the functioning of the Krebs cycle (pyruvate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase). The physiological significance of such a functional relationship between malic enzyme activity and the nonphosphorylating alternative pathway is discussed in relation to Crassulacean acid metabolism. PMID- 16664941 TI - Effect of Betaine on Enzyme Activity and Subunit Interaction of Ribulose-1,5 Bisphosphate Carboxylase/Oxygenase from Aphanothece halophytica. AB - The presence of betaine, a quaternary ammonium compound, at a concentration (0.5 molar) reported to accumulate inside Aphanothece halophytica in response to increasing external salinity, slightly promoted ribulose-1,5-bisphosphate (RuBP) carboxylase activity. KCl at 0.25 molar inhibited RuBP carboxylase about 55%. Betaine relieved the inhibition by 0.25 m KCl and the original uninhibited activity was restored at 1 m betaine. Other osmoregulatory solutes such as sucrose and glycerol also reduced KCl inhibition, though to a lesser extent than betaine. Proline had no effect. The protective effect of betaine against KCl inhibition of RuBP carboxylase activity was also observed in other cyanobacteria, i.e. Synechococcus ACMM 323, Plectonema boryanum, and Anabaena variabilis, and in the photosynthetic bacterium Rhodospirillum rubrum but not in Chromatium vinosum. Apart from betaine, other quaternary ammonium compounds, i.e. sarcosine and trimethylamine-N-oxide (TMAO), but not glycine, also protected the enzyme against KCl inhibition and the effectiveness of such compounds appeared to correlate with the extent of N-methylation. Heat and cold inactivation of the enzyme could be protected by either betaine or KCl. However, best protection occurred when both betaine and KCl were present together. The K(m) (CO(2)) was not altered by either betaine or KCl, nor when they were present together. However, the K(m) (RuBP) was increased about 5-fold by KCl, but was unaffected by betaine. The presence of betaine together with KCl lowered the KCl-raised K(m) (RuBP) by about half. The extent of the dissociation of the enzyme molecule under the condition of low ionic strength was reduced by either betaine or KCl alone and more so when they were present together. Glycine, sarcosine, and TMAO were more effective than betaine or KCl in lowering the extent of the dissociation of the enzyme molecule. PMID- 16664942 TI - Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : I. Salinity during Growth Modulates the Proton Pump in the Halophyte Atriplex nummularia. AB - Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H(+)-translocating Mg(2+)-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN(-) accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H(+)-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN(-) accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP dependent SCN(-) accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO(3) (-) inhibition was observed. PMID- 16664943 TI - Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : II. K Strongly Promotes Development of Membrane Potential in Vesicles from Cotton Roots. AB - Mg(2+)-ATP-dependent H(+)-translocation has been studied in membrane vesicles derived from the roots of Gossypium hirsutum L. var. Acala San Jose 2. Establishment of a positive membrane potential was followed by measuring SCN(-) accumulation; establishment of DeltapH across the vesicle membranes by measuring quinacrine fluorescence quenching. High specificity for ATP was shown, and H(+) translocation was oligomycin stable. The pH profile for H(+)-translocation showed an optimum at 5.5. The relationship between SCN(-) accumulation and ATP concentration was approximately Michaelian; the apparent K(m) was 0.7 millimolar. K-2-(N-morpholino)ethanesulfonic acid strongly promoted ATP-dependent SCN(-) uptake (up to 180% stimulation). The effect was not given by Na-Mes. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone totally inhibited SCN(-) accumulation, both in the presence and absence of K-2(N-morpholino)ethanesulfonic acid. Vanadate at 200 micromolar inhibited SCN(-) uptake by about 10 to 40% in the absence of K(+), but more strongly in its presence (about 60%). NO(3) (-) at 100 millimolar inhibited initial rate of quinacrine quenching by about 25%. The NO(3) (-) insensitive fraction was activated by K(+); and inhibited by 200 micromolar vanadate to about 40%, provided K(+) was present. Saline conditions during the growth of the plants had no appreciable effect on the observed characteristics of H(+)-translocation. PMID- 16664944 TI - The Multiple Forms of alpha-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz : A Comparative Study. AB - alpha-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of alpha-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific alpha-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five alpha amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of alpha-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins. PMID- 16664945 TI - Biochemical characterization of soybean ovary growth from anthesis to abscission of aborting ovaries. AB - Soybean (Glycine max [L.] Merr.) ovary growth was measured from anthesis to 6 days after anthesis (DAA) to establish a timetable of biochemical events that might be useful in identifying processes that initiate abscission. Two procedures were developed to provide samples with either high or low percent pod set for ;IX93-100,' a semideterminate line having long racemes. Characteristics measured were fresh and dry weight, soluble and insoluble protein, soluble carbohydrate, starch, RNA, and DNA. Setting ovaries grew more rapidly than abscising ovaries. Since there was a daily increase in ovary weight in both groups, all measured characteristics showed daily increases when expressed on perovary basis. Statistically significant differences between groups were detected between 2 and 5 DAA for most characteristics. When chemical composition was expressed on concentration basis, starch level was significantly higher in setting ovaries at 5 and 6 DAA. Regression analysis showed that these deviations between setting and abscising samples started between anthesis and 1 DAA. We conclude that processes leading to eventual shedding of fertilized ovaries (called flower abortion in soybeans) commence soon after anthesis of the shed flower, and that setting and abscising ovaries do not differ in protein, soluble carbohydrate, starch, or nucleic acid content when abscission processes begin. PMID- 16664946 TI - Carbohydrate Level and Growth of Tomato Plants: II. The Effect of Irradiance and Temperature. AB - The growth response of tomato (Lycopersicon esculentum L.) to temperature and irradiance may be related to carbohydrate concentration. Plants in the exponential phase of vegetative growth were grown under temperatures ranging from 9 to 36 degrees C and under low or high irradiances of approximately 110 or 370 microeinsteins per square meter per second photosynthetically active radiation for a 12 hour photoperiod. The relative growth rate, leaf area ratio, net assimilation rate and whole plant carbohydrate levels were measured. At high irradiance, relative growth rate was 43% faster and total nonstructural carbohydrate concentration was 41% greater than at low irradiance. The change in carbohydrate with irradiance could explain the growth response. Plant growth was fastest at 25 degrees C and decreased parabolically at lower and higher temperatures with a half-maximal rate at 13 and 36 degrees C. Total nonstructural carbohydrate decreased between 13 and 23 degrees C and remained constant at higher temperatures. Soluble sugar concentrations varied little with temperature above 13 degrees C except for sucrose, whose level rose above 30 degrees C. The change in carbohydrate with temperature could not explain the growth response. Above 23 degrees C tomato plants appeared to regulate growth rate to maintain a relatively constant nonstructural carbohydrate concentration. PMID- 16664947 TI - Purification and properties of the h-translocating ATPase from the plasma membrane of tomato roots. AB - The proton-translocating, plasma membrane ATPase was purified from tomato roots. At the final stage of purification approximately 80% of the protein was found in a single band with an apparent molecular weight of 90 kilodaltons. Cross-linking studies indicated that the ATPase normally exists as a trimer of catalytic subunits. No evidence was found for any additional subunits. The pH optimum for ATP hydrolysis by the purified protein was 6.5. Activity was stimulated by K(+), especially at low pH, and inhibited by vanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol; nitrate was weakly inhibitory. Activity was stimulated by lysolecithin but inhibited by sonicated phospholipids. The inhibition by lipids could be prevented if octylglucoside was added with the lipids; the combination of octylglucoside and lipids actually stimulated activity. The purified protein could be reconstituted into liposomes and catalyzed ATP-dependent, vanadate sensitive proton translocation. PMID- 16664948 TI - Purification and Properties of UDP-GlcNAc:Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc Transferase from Mung Bean Seedling. AB - The N-acetylglucosamine (GlcNAc) transferase that catalyzes the formation of dolichyl-pyrophosphoryl-GlcNAc-GlcNAc from UDP-GlcNAc and dolichyl-pyrophosphoryl GlcNAc was solubilized from the microsomal enzyme fraction of mung beans with 1.5% Triton X-100, and was purified 140-fold on columns of DE-52 and hydroxylapatite. The partially purified enzyme preparation was quite stable when stored in 20% glycerol and 0.5 millimolar dithiothreitol, and was free of GlcNAc 1-P transferase and mannosyl transferases. The GlcNAc transferase had a sharp pH optimum of 7.4 to 7.6 and the K(m) for dolichyl-pyrophosphoryl-GlcNAc was 2.2 micromolar and that for UDP-GlcNAc, 0.25 micromolar. The enzyme showed a strong requirement for the detergent Triton X-100 and was stimulated somewhat by the divalent cation Mg(2+). Uridine nucleotides, especially UDP and UDP-glucose inhibited the enzyme as did the antibiotic, diumycin. However, a variety of other antibiotics including tunicamycin were without effect. The product of the reaction was characterized as dolichyl-pyrophosphoryl-GlcNAc-GlcNAc. PMID- 16664949 TI - Preparation and properties of mitochondria from cowpea nodules. AB - Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O(2) uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD(+)-malic enzyme which was 12-fold lower in the mitochondria from nodules. PMID- 16664950 TI - Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules. AB - A method is described for isolating mitochondria from nodules of cowpea (Vigna unguiculata [L.] Walp.) under completely anaerobic conditions. The mitochondria were immediately active when incubated aerobically with substrates, and their respiration rates were higher than mitochondria prepared in air. The mitochondria lacked fumarate reductase and were not inhibited by 5% CO(2). When incubated under microaerobic conditions, their respiration could be measured by leghemoglobin spectroscopy. Microaerobic respiration was inhibited approximately 50% by 1 millimolar malonate, and was completely inhibited by cyanide. O(2) uptake and the ATP/O ratio declined under microaerobic conditions, and therefore ATP production may be low in the environment of infected nodule cells. PMID- 16664951 TI - Changes in Cytokinin Concentrations in Xylem Extrudate following Infection of Eucalyptus marginata Donn ex Sm with Phytophthora cinnamomi Rands. AB - The concentrations of zeatin-type and isopentenyladenine-type cytokinins were reduced in the xylem extrudate collected from seedlings of Eucalyptus species following infection by Phytophthora cinnamomi Rands. The use of an enzyme-linked immunosorbent assay (ELISA) allowed the detection of these cytokinins over the range of 0.3 to 7 picomoles for the isopentenyladenine-type and 1 to 1000 picomoles for the zeatin-type. Isopentenyladenine-type cytokinins occurred in concentrations less than 10% of the zeatin-type, but they could be readily detected and measured. This is the first report of their presence in xylem. The sensitivity of the assay allowed a short collection period (30 minutes) reducing any confusion with trauma-induced changes. Infection of the susceptible species Eucalyptus marginata Donn. ex Sm. resulted in significant reduction of zeatin type cytokinins within 3 days of infection, and at 14 days postinfection the concentration of both cytokinin types was reduced to 26% of uninoculated controls. No reduction in cytokinins occurred with the field resistant Eucalyptus calophylla R. Br. It is suggested that failure of cytokinin transport from the root system may be responsible for the failure in water transport and symptoms of P. cinnamomi infection observed in infected susceptible eucalypts. PMID- 16664952 TI - Maize leaf adenylate kinase : purification and partial characterization. AB - Adenylate kinase (EC 2.7.4.3) from leaves of maize (Zea mays) was purified to homogeneity using (NH(4))(2)SO(4) fractionation, followed by chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75SF, and Green A dye-ligand columns. The purified enzyme had specific activity of about 1,550 micromoles ADP produced per minute per milligram protein, and the ratio of velocities of the reverse (utilization of ATP) to forward (formation of ATP) reaction was about 1.5. The M(r) value of adenylate kinase, determined by electrophoresis in dissociating conditions and by gel filtration, was 29,000 and 31,000 respectively, suggesting monomeric nature of the enzyme. Purified preparations were stable for at least 1 month at 0 to 4 degrees C. Magnesium ions were essential for activity of adenylate kinase in both directions of the reaction. Optimal rates in the forward direction were observed at the magnesium to ADP ratio of about 0.6 to 0.8. For the reverse reaction, ATP served as a substrate only when complexed with magnesium, while AMP reacted as a free species. The enzyme preferentially utilized adenine ribonucleotides in both directions of the reaction. The nucleoside triphosphate-binding site of adenylate kinase was fairly nonspecific with regard to nucleotide species. On the other hand, the primary amino group of either adenine and cytosine moieties was essential for effective binding to the nucleoside monophosphate site of the enzyme. PMID- 16664953 TI - Limitation of Photosynthesis by Carbon Metabolism : I. Evidence for Excess Electron Transport Capacity in Leaves Carrying Out Photosynthesis in Saturating Light and CO(2). AB - It has been investigated how far electron transport or carbon metabolism limit the maximal rates of photosynthesis achieved by spinach leaves in saturating light and CO(2). Leaf discs were illuminated with high light until a steady state rate of O(2) evolution was attained, and then subjected to a 30 second interruption in low light, to generate an increased demand for the products of electron transport. Upon returning to high light there is a temporary enhancement of photosynthesis which lasts 15 to 30 seconds, and can be up to 50% above the steady state rate of O(2) evolution. This temporary enhancement is only found when saturating light intensities are used for the steady state illumination, is increased when low light rather than darkness is used during the interruption, and is maximal following a 30 to 60 seconds interruption in low light. Decreasing the temperature over the 10 to 30 degrees C range led to the transient enhancement becoming larger. The temporary enhancement is associated with an increased ATP/ADP ratio, a decreased level of 3-phosphoglycerate, and increased levels of triose phosphate and ribulose 1,5-bisphosphate. Since electron transport can occur at higher rates than in steady state conditions, and generate a higher energy status, it is concluded that leaves have a surplus electron transport capacity in saturating light and CO(2). From the alterations of metabolites, it can be calculated that the enhanced O(2) evolution must be accompanied by an increased rate of ribulose 1,5-bisphosphate regeneration and carboxylation. It is suggested that the capacity for sucrose synthesis ultimately limits the maximal rates of photosynthesis, by restricting the rate at which inorganic phosphate can be recycled to support electron transport and carbon fixation in the chloroplast. PMID- 16664954 TI - Limitation of Photosynthesis by Carbon Metabolism : II. O(2)-Insensitive CO(2) Uptake Results from Limitation Of Triose Phosphate Utilization. AB - The occurrence of O(2)-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves. In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions. Upon switching to low O(2) in O(2)-insensitive conditions the ATP/ADP ratio fell dramatically within one minute. The P-glycerate pool increased over the same time. Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air. The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of O(2) of 21 millibars than at 210 millibars. These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O(2)-insensitive conditions. PMID- 16664955 TI - Use of P NMR to Assess Effects of DNP on ATP Levels in Vivo in Barley Roots. AB - Previous work has shown that undissociated 2,4-dinitrophenol (DNP) both increases the permeability of roots to ions and alters the membrane lipids of barley roots. Anionic DNP is the main entrant form but has no effect on permeability or on the membrane lipids. The amount of anionic DNP taken up by the roots is sufficient, that were it in free solution in the cytoplasm, the DNP would uncouple oxidative phosphorylation, and thereby inhibit ATP synthesis. The present work was undertaken to assess whether DNP alters ATP levels when it is taken up by barley roots. (31)P nuclear magnetic resonance spectra were used to monitor, in vivo, levels of ATP, cytoplasmic phosphate, vacuolar phosphate, and other phosphate compounds in barley roots in the presence of 10 micromolar DNP at pH 5 and pH 7. The spectra indicate that no change in the level of ATP or the cytoplasmic pH occurred in the roots in the presence of DNP for as long as 20 hours. Thus, the effects of undissociated DNP are effects directly on the root membranes and do not involve inhibition of ATP synthesis. Furthermore, the results explain why anionic DNP has no effect on ion uptake and accumulation. PMID- 16664956 TI - Heterogeneity of catalase in maturing and germinated cotton seeds. AB - To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000. PMID- 16664957 TI - Changes in the Activity of Catalase (EC 1.11.1.6) in Relation to the Dormancy of Grapevine (Vitis vinifera L.) Buds. AB - Catalase activity in grapevine (Vitis vinifera L.) buds cv. ;Perlette.' increased to a maximum in October and thereafter decreased within 3 months to less than half its maximal rate. The decrease in catalase activity coincided with the decline in temperature during winter. The rate of sprouting of buds forced at 23 degrees C was negatively related to the activity of catalase. Artificial chilling of grapevine canes at 5 degrees C resulted in a 25% decrease of catalase activity in the buds after 3 days and 31% after 17 days. The activity of catalase increased to the control level only 96 hours after removing canes from 5 degrees C to room temperature. Efficient buddormancy breaking agents, such as thiourea and cyanamide decreased catalase activity to 64 and 50% of the controls respectively, while the activity of peroxidase remained the same under those conditions. A less efficient dormancy breaking agent dinitro-ortho-cresol, did not decrease catalase activity. PMID- 16664958 TI - Effect of 2,4-dichlorophenoxyacetic Acid on the expression of embryogenic program in carrot. AB - Embryogenesis in a wild carrot cell line, W001C, can start and progress up to the first morphogenetic stage (the globular-stage embryo) in 2,4-dichloropenoxyacetic acid (2,4-D). To clarify the quantitative effect of 2,4-D on this cell line, morphological and biochemical criteria have been used to monitor embryogenesis in the presence of increasing concentrations of 2,4-D. The biochemical criteria are the ability to inactivate cycloheximide and the expression of an embryogenic polypeptide E(1). The results show that 2,4-D can affect embryogenesis in a quantitative manner but never fully suppresses embryogenesis unless it is coupled with high cell density. PMID- 16664959 TI - Electrical noise measurements on red beet vacuoles : another way to detect the ATPase activity. AB - The vacuolar potential (V(vac)) and its fluctuations were recorded in red beet vacuoles (Beta vulgaris L.). Measurements with vacuoles in their suspension medium gave V(vac) = 10 +/- 2 millivolts (referred to the external medium) when 3 molar KCl microelectrodes were used. Buffering the microelectrode filling solution at pH 7.7 reversed the sign of the potential: V(vac) = -7 +/- 2 millivolts. The magnitude of the potential fluctuations was lowered by dilution (5-1000 times) with the suspension medium containing components released by the cells during the mechanical preparation. Fluctuations were decreased by 50 millimolar KNO(3) while they were enhanced by 5 millimolar ATP-Mg. No noticeable change in membrane resistance was detected. The presence of an ATPase bound to the tonoplast may explain the recorded noise spectra. These spectra imply a close connection between the rate of ATPase functioning and the magnitude of ionic fluxes across the tonoplast. It is suggested that noise analysis could be used to detect ATPase (or related enzyme) activity in vacuoles. Possible use of H(+) diffusion through a buffered microelectrode, to modify intravacuolar pH, is also suggested. PMID- 16664960 TI - Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts. AB - Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W 7. Ethyleneglycol-bis(beta- aminoethylether)-N,N'-tetraacetic acid a Ca(2+) chelator, La(3+), a surface Ca(2+) antagonist, and verapamil, a Ca(2+) channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La(3+). A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion. PMID- 16664961 TI - Determinants of substrate specificity and the role of metal in the reactions of ribulosebisphosphate carboxylase/oxygenase. AB - Recent studies have provided a fairly detailed view of the various intermediates involved in the reactions of ribulosebisphosphate carboxylase and the manner in which the catalytically essential metal atom might catalyze their interconversions. A better understanding of how the enzyme distinguishes between its alternate substrates, CO(2) and O(2), has also emerged. The results of these studies should prove useful in anticipating possible ways in which the enzyme's substrate specificity might be manipulated. Together, the techniques that are described constitute a powerful methodology for more refined experimentation aimed at understanding the curious reactivities of ribulosebisphosphate carboxylase. PMID- 16664962 TI - The Fatty Acid composition of phosphatidylglycerol and sulfoquinovosyldiacylglycerol of higher plants in relation to chilling sensitivity. AB - The fatty acid composition of phosphatidylglycerol and sulfoquinovosyldiacylglycerol has been measured in the leaves of 27 species of higher plants from six families whose members differed in their degrees of chilling sensitivity. The content of high melting point fatty acids (represented by the sum of hexadecanoic, trans-3-hexadecenoic and octadecanoic acids) in phosphatidylglycerols varied little between members of the same plant family and was not obviously related to the relative chilling sensitivity of members of that family. The saturated fatty acid content (hexadecanoic + octadecanoic acids) of sulfoquinovosyldiacylglycerols also appeared to be characteristic of a plant family, although some exceptions were found. In one case, (Carica papaya) the content of saturated fatty acids in sulfoquinovosyldiacylglycerol was sufficiently high to suggest that this lipid could undergo phase separations above 0 degrees C. It is concluded that the content of high melting point fatty acids in leaf phosphatidylglycerol is not a direct indication of the chilling sensitivity of a plant, but rather may be a reflection of the genetic origin of that plant. PMID- 16664963 TI - Requirement for Ethylene Synthesis and Action during Relief of Thermoinhibition of Lettuce Seed Germination by Combinations of Gibberellic Acid, Kinetin, and Carbon Dioxide. AB - Application of exogenous ethylene in combination with gibberellic acid (GA(3)), kinetin (KIN), and/or CO(2) has been reported to induce germination of lettuce seeds at supraoptimal temperatures. However, it is not clear whether endogenous ethylene also plays a mediatory role when germination under these conditions is induced by treatment regimes that do not include ethylene. Therefore, possible involvement of endogenous ethylene during the relief of thermoinhibition of lettuce (Lactuca sativa L. cv Grand Rapids) seed germination at 32 degrees C was investigated. Combinations of GA(3) (0.5 millimolar), KIN (0.05 millimolar), and CO(2) (10%) were used to induce germination. Little germination occurred in controls or upon treatment with ethylene, KIN, or CO(2). Neither KIN nor CO(2) affected the rate of ethylene production by seeds. Both germination and ethylene production were slightly promoted by GA(3). Treatments with GA(3)+CO(2), GA(3)+KIN, or GA(3)+CO(2)+KIN resulted in approximately 10-to 40-fold increases in ethylene production and 50 to 100% promotion of germination as compared to controls. Initial ethylene evolution from the treated seeds was greater than from the controls and a major surge in ethylene evolution occurred at the time of visible germination. Application of 1 millimolar 2-aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene synthesis, in combination with any of above three treatments inhibited the ethylene production to below control levels. This was accompanied by a marked decline in germination percentage. Germination was also inhibited by 2,5-norbornadiene (0.25-2 milliliters per liter), a competitive inhibitor of ethylene action. Application of exogenous ethylene (1-100 microliters per liter) overcame the inhibitory effects of AVG and 2,5 norbornadiene on germination. The results demonstrate that endogenous ethylene synthesis and action are essential for the alleviation of thermoinhibition of lettuce seeds by combinations of GA(3), KIN, and CO(2). It also appears that these treatment combinations do not act exclusively via promotion of ethylene evolution as the application of exogenous ethylene alone did not promote germination. PMID- 16664964 TI - Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit. AB - Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r(ss)) of 1,6-diphenyl-1,3,5 hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r(ss) for plastid membranes labeled with DPH, 1-[4 trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r(oo)) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues. PMID- 16664965 TI - Synthesis of a Putative c-Type Cytochrome by Intact, Isolated Pea Chloroplast. AB - In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea (Pisum sativum L.) chloroplasts were incubated with [(14)C]-5-aminolevulinic acid. The major labeled band (M(r) = 43 kilodaltons by lithium dodecyl sulfate-polyacrylamide gel electrophoresis) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H(2)O(2) stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [(14)C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome; however, the possibility that it might be a protein containing a covalently linked linear tetrapyrrole was not ruled out. PMID- 16664966 TI - Biosynthesis of protoheme and heme a from glutamate in maize. AB - The heme and chlorophyll precursor delta-aminolevulinic acid (ALA) can be formed by two biosynthetic routes: from the intact carbon skeleton of glutamate via a five-carbon pathway, which occurs in chloroplasts and bluegreen algae, and by ALA synthase-catalyzed condensation of succinyl-CoA and glycine, which occurs in bacteria and animal mitochondria. The biosynthetic route of plant mitochondrial heme a was determined by incubating terminal epicotyl sections of 8-day-old etiolated Zea mays seedlings in the dark with l-1-[(14)C]glutamate (which can be incorporated into ALA only via the five-carbon route) or 2-[(14)C]glycine (which would be incorporated via ALA synthase). Label incorporation was measured in highly purified protoheme and heme a. In 12-hour incubations, label uptake was greater than 70%. Total cellular protoheme was labeled 29.7 times more effectively by glutamate than glycine. Heme a was labeled 4.1 times more effectively by glutamate than by glycine. To assess the relative ability of the two amino acids to contribute label to the farnesyl moiety of heme a, label incorporation into total cellular nonsaponifiable lipids was measured. Glycine labeled this fraction 11.3 times more effectively than glutamate. Thus, a contribution by glycine to the farnesyl moiety may account for the small amount of label appearing in heme a. Our results indicate that in etiolated maize, noncovalently bound hemes, including mitochondrial heme a, are made mostly, and possibly entirely, from ALA synthesized via the five-carbon pathway. There is little or no contribution from ALA formed via ALA synthase, and no evidence was found for the operation of this enzyme in maize. PMID- 16664967 TI - Root respiration associated with nitrate assimilation by cowpea. AB - Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using (15)NO(3) (-), and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO(2) per plant per hour for growth and maintenance. Adding 10 millimolar NO(3) (-) to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO(3) (-) from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N(2) fixation. PMID- 16664968 TI - Phototropism in Hypocotyls of Radish : I. Isolation and Identification of Growth Inhibitors, cis- and trans-Raphanusanins and Raphanusamide, Involved in Phototropism of Radish Hypocotyls. AB - Three growth inhibitors which might be involved in phototropism of Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls, were isolated as crystalline forms from light-exposed radish seedlings and identified as cis- and trans-raphanusanins and 6-methoxy-2,3,4,5-tetrahydro-1,3-oxazepin-2 one (designated raphanusamide). The cis- and trans-raphanusanins inhibited growth of etiolated radish hypocotyls at concentrations higher than 1.5 micromolar, raphanusamide at concentrations higher than 20 micromolar. PMID- 16664969 TI - Phototropism in Hypocotyls of Radish : II. Role of cis- and trans-Raphanusanins, and Raphanusamide in Phototropism of Radish Hypocotyls. AB - When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light (0.1 watt per square meter), the growth rate at the lighted side was strongly inhibited for the first 2 hours, while that at the shaded side showed no change. After 2.5 hours growth on the lighted side recovered gradually, while that on the shaded side was slightly inhibited. The neutral growth inhibitors, cis- and trans-raphanusanins and raphanusamide, were determined in the lighted and shaded sides from 1 hour before until 2 hours after the start of unilateral illumination. In the lighted side, cis- and trans-raphanusanins increased by 0.5 hour after the start of illumination, reached 3 to 3.5-fold greater concentrations than in the shaded side after 1 hour, and then decreased gradually. Raphanusamide increased in the lighted side to a 3-fold greater concentration than that in the shaded one 2 hours after the start of the illumination. Unilateral applications of cis- and trans-raphanusanins and raphanusamide suppressed the growth of the hypocotyl on the applied side more than that on the opposite one, causing the hypocotyls to bend towards the site of application. The data suggest that phototropic curvature in radish is caused by the light-induced synthesis of growth-inhibiting cis- and trans-raphanusanins, and raphanusamide at the site of illumination. PMID- 16664970 TI - Identification of Pea Gibberellins by Studying [C]GA(12)-Aldehyde Metabolism. AB - Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with [(14)C]GA(12)-7-aldehyde ([(14)C]GA(12)ald) would serve as appropriate [(14)C]markers for the recovery of naturally-occurring gibberellins (GAs). The [(14)C]GA(12)ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using [4,5 (14)C]mevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high performance liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as [(14)C]markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co eluting with each [(14)C]marker. These [(14)C] and UV-absorbing peaks were shown to contain GA(53), GA(44), GA(20), GA(19), and GA(17) by GC-MS. The finding of GA(53) is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using [(14)C]GA(12)ald metabolites as [(14)C]markers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species. PMID- 16664971 TI - Photoperiod modification of [C]gibberellin a(12) aldehyde metabolism in shoots of pea, line g2. AB - In G2 peas (Pisum sativum L.) apical senescence occurs only in long days (LD), and indeterminate growth is associated with elevated gibberellin (GA) levels in the shoot in short days (SD). Metabolism of GA(12) aldehyde was investigated by feeding shoots grown in SD or LD with [(14)C]GA(12) aldehyde through the cut end of the stem for 0.5 to 6 hours in the light and analyzing the tissue extract by high performance liquid chromatography. More radioactive products were detected than can be accounted for by the two GA metabolic pathways previously known to be present in peas. Three of the major products appear to be GA conjugates, but an additional pathway(s) of GA metabolism may be present. The levels of putative C(20) GAs, [(14)C]GA(53), [(14)C]GA(44), [(14)C]GA(19), and/or [(14)C] GA(17), were all elevated in SD as compared to LD. Putative [(14)C]GA, was slightly higher in LD than in SD. Putative [(14)C]GA(53) was a major metabolite after 30 minutes of treatment in SD but had declined after longer treatment times to be replaced by elevated levels of putative [(14)C] GA(44) and [(14)C]GA(19/17). Metabolism of GA(20) was slow in both photoperiods. Although GA(20) and GA(19) are the major endogenous GAs as determined by gas chromatography-mass spectrometry, putative [(14)C]GA(20) and [(14)C]GA(19) were never major products of [(14)C]GA(12) aldehyde metabolism. Thus, photoperiod acts in G2 peas to change the rate of GA(53) production from GA(12) aldehyde, with the levels of the subsequent GAs on the 13-OH pathway being determined by the amount of GA(53) being produced. PMID- 16664972 TI - Purification and measurement of abscisic Acid and indoleacetic Acid by high performance liquid chromatography. AB - A procedure was selected for the simultaneous extraction and purification of abscisic acid (ABA) and indoleacetic acid (IAA). Unnecessary steps were eliminated and an accumulation of aqueous phase was avoided. The superior performance of diethyl ether (compared to ethyl acetate) for bulk purification and the superior resolution provided by 250 millimeter columns packed with 5 micrometer spherical particles of strong anion exchanger and octadecylsilane (C18) greatly facilitated the purification of samples. A fixed-wavelength (254 nanometer) ultraviolet detector and a fluorescence detector connected in series on a high performance liquid chromatograph permitted nondestructive monitoring and measurement of ABA and IAA. Derivatization was not necessary for chromatography or for detection. Isocratic elution with simple mobile phases gave sharp peaks. A few simple precautions minimized losses. Recoveries through the entire procedure averaged about 75% for ABA and about 50% for IAA. Purified ABA and IAA fractions were usually free of interfering contaminants. Identities were confirmed by gas chromatography-mass spectrometry. PMID- 16664973 TI - Characterization and Solubilization of the Alternative Oxidase of Sauromatum guttatum Mitochondria. AB - The alternative oxidase activity of Sauromatum guttatum spadix mitochondria has been investigated as to its developmental expression and tissue localization. Mitochondria rich in alternative oxidase activity were found in a yellow cortex tissue present to varying degrees within the appendix, male floral, and sterile floral regions of the spadix. During a 5-day period just prior to anthesis, the alternative oxidase activity present in the appendix region was found to increase over 10-fold. On the following day when the appendix region becomes thermogenic, cytochrome oxidase activity was found to decrease by 92%, effectively forcing electron flow through the alternative oxidase. A procedure for efficient solubilization of the alternative oxidase from appendix region mitochondria was developed. The alternative oxidase thus solubilized was sensitive to heat inactivation and trypsin digestion. The activity showed inhibition characteristics expected of the alternative oxidase in that it was sensitive to salicylhydroxamic acid and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, but relatively insensitive to KCN and antimycin A. Essential sulfhydryl group(s) were indicated by reversible inhibition by p-chloromercuribenzoic acid. The solubilized alternative oxidase was most active in the detergent N,N-bis-(3-d glucoamidopropyl)-deoxycholamide and had a pH optimum of 6.8. PMID- 16664974 TI - Growth, partitioning, and harvest index of tuber-bearing solanum genotypes grown in two contrasting peruvian environments. AB - Ten Solanum potato genotypes, including four primitive species and six hybrids, were grown to maturity near 230 and 3273 meters in elevation at two sites, Coast and Sierra, in Peru. Growth data, with emphasis upon tubers and leaves, were collected periodically to analyze the plant components which differed in these contrasting environments. Nine of the Solanum species/cultivars effectively partitioned dry matter into tubers with values reaching 73 to 85% of the total plant at mature harvest in the Sierra but dropping to 33 to 75% on the Coast. These harvest index differences were, however, accompanied by no consistent changes in total leaf area, specific leaf area, nor number of tuber initiated. Consistent differences did occur in having shorter plants in the Sierra and an increased tuber dry matter percentage, 20 to 28%, in the Sierra compared to 14 to 21% on the Coast. Linear relationships exist between plant tuber harvest index versus tuber yield and versus total plant dry matter on both the Coast and in the Sierra. PMID- 16664975 TI - Identification with Monoclonal Antibodies of a Second Antigenic Domain on Avena Phytochrome that Changes upon Its Photoconversion. AB - An enzyme-linked immunosorbent assay that revealed an antigenic difference between the red-absorbing and far-red-absorbing forms of phytochrome (Pr and Pfr, respectively) near its amino terminus (Cordonnier M-M, H Greppin, LH Pratt 1985 Biochemistry 24: 3246-3253) was used to screen eight additional monoclonal antibodies directed to phytochrome from etiolated oats. While six of these antibodies detected Pr and Pfr with equal affinity, two of them, designated Oat-9 and Oat-16, bound to Pfr 1.6 to 2.3 times better than to Pr. Competitive enzyme linked immunosorbent assays indicate (a) that Oat-9 and Oat-16 probably bind to the same domain on phytochrome and (b) that this domain is at least 3.5 nanometers away from the epitope near its amino terminus that was shown earlier to change upon phototransformation. Neither the absorbance spectra of Pr and Pfr, nor the rate of dark reversion of Pfr to Pr, was influenced by the presence of Oat-9. Immunoblotting of sodium dodecyl sulfate polyacrylamide gels after electrophoretic separation of phytochrome fragments obtained by endogenous proteolytic digestion indicates that Oat-16 binds to an epitope located on the chromophore half of this chromoprotein. The observation that the epitope recognized by Oat-9 and Oat-16 is also present on at least some of the immunochemically distinct phytochrome that is obtained from green oat shoots (Shimazaki Y, LH Pratt 1985 Planta 164: 333-344), together with the evidence that this epitope undergoes a change upon photoransformation, indicates that it may play an important role in phytochrome function. PMID- 16664976 TI - Translocation of paclobutrazol, a gibberellin biosynthesis inhibitor, in apple seedlings. AB - The [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1- yl)-pentan-3 ol] (paclobutrazol, PP333) measured in apple seedlings (;York Imperial' Malus domestica Borkh) was confirmed by gas chromatography-mass spectrometry. Data showed that paclobutrazol was taken up through roots and transported primarily in the xylem through the stems and accumulated in leaves. No detectable basipetal movement of paclobutrazol in apple seedlings was found. PMID- 16664977 TI - The Water Oxidation Complex of Chlamydomonas: Accumulation and Maturation of the Largest Subunit in Photosystem II Mutants. AB - Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl(2) washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O(2) evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex. PMID- 16664978 TI - H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin. AB - The effect of fusicoccin on Mg:ATP-dependent H(+)-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the DeltapH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H(+)-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H(+) pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H(+)-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H(+) pumping as much as fusicoccin, while the physiologically inactive derivative 8 oxo-9-epideacetylfusicoccin did not. Stimulation of H(+)-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H(+)-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V(max) of the plasma membrane H(+) ATPase rather than a decrease in its apparent K(m) for Mg:ATP. PMID- 16664979 TI - Phosphoethanolamine bases as intermediates in phosphatidylcholine synthesis by lemna. AB - The pathway for synthesis of phosphatidylcholine, the dominant methyl-containing end product formed by Lemna paucicostata, has been investigated. Methyl groups originating in methionine are rapidly utilized by intact plants to methylate phosphoethanolamine successively to the mono-, di-, and tri-methyl (i.e. phosphocholine) phosphoethanolamine derivatives. With continued labeling, radioactivity initially builds up in these compounds, then passes on, accumulating chiefly in phosphatidylcholine (34% of the total radioactivity taken up by plants labeled to isotopic equilibrium with l-[(14)CH(3)]methionine), and in lesser amounts in soluble choline (6%). Radioactivity was detected in mono- and dimethyl derivatives of free ethanolamine or phosphatidylethanolamine only in trace amounts. Pulse-chase experiments with [(14)CH(3)]choline and [(3)H] ethanolamine confirmed that phosphoethanolamine is rapidly methylated and that phosphocholine is converted to phosphatidylcholine. Initial rates indicate that methylation of phosphoethanolamine predominates over methylation of either phosphatidylethanolamine or free ethanolamine at least 99:1. Although more studies are needed, it is suggested this pathway may well turn out to account for most phosphatidylcholine synthesis in higher plants. Phosphomethylethanolamine and phosphodimethylethanolamine are present in low quantities during steady-state growth (18% and 6%, respectively, of the amount of phosphocholine). Radioactivity was not detected in CDP-choline, probably due to the low steady-state concentration of this nucleotide. PMID- 16664980 TI - Regulation of Carbon Flow by Nitrogen and Light in the Red Alga, Gelidium coulteri. AB - The red alga Gelidium coulteri Harv. photosynthetically fixed [(14)C] bicarbonate at high rates under defined conditions in unialgal laboratory culture. The fixation rate and flow of photosynthate into various end products were dependent on the nitrogen status of the tissue. Plants fed luxury levels of nitrogen (approximately 340 micromolar) showed fixation rates several-fold higher than those seen for plants starved for nitrogen. The addition of NO(3) (-) or NH(4) (+) to such starved plants further inhibited fixation over at least the first several hours after addition. The majority of (14)C after incubations of 30 minutes to 8 hours was found in the compounds floridoside, agar and floridean starch. In addition, amino acids and intermediate compounds of the reductive pentose phosphate pathway, glycolytic pathway and tricarboxylic acid cycle were detected. Nitrogen affected the partitioning of labeled carbon into these compounds. Plants under luxury nitrogen conditions had higher floridoside levels and markedly lower amounts of agar and starch than found in plants limited for nitrogen. Amino acid, phycobiliprotein and chlorophyll levels were also significantly higher in nitrogen-enriched plants. Addition of NO(3) (-) to starved plants led to an increase in floridoside, tricarboxylic acid cycle intermediates and amino acids within 1 hour and inhibited carbon flow into agar and starch. Carbon fixation in the dark was only 1 to 7% of that seen in the light. Dark fixation of [(14)C]bicarbonate yielded label primarily in tricarboxylic acid cycle intermediates, amino acids and polysaccharides. Nitrogen stimulated amino acid synthesis at the expense of agar and starch. Floridoside was only a minor component in the dark. Pulse-chase experiments, designed to show carbon turnover, indicated a 2-fold increase in labeling of agar over 96 hours of chase in the light. No increases were seen in the dark. Low molecular weight pools, including floridoside, decreased 2- to 5-fold over this period under both light and dark conditions. Nitrogen status did not influence turnover. There was little or no organic carbon released into the culture medium over this period. The results are consistent with biosynthetic pathways to floridoside and agar that share the common intermediate UDP-d-galactose. It is hypothesized that synthesis of floridoside is regulated by nitrogen and light at the enzymic level. PMID- 16664981 TI - Transient Activation of Plasmalemma K Efflux and H Influx in Tobacco by a Pectate Lyase Isozyme from Erwinia chrysanthemi. AB - A purified pectate lyase isozyme derived from Erwinia chrysanthemi induced rapid net K(+) efflux and H(+) influx in suspension-cultured tobacco cells. Comparable fluxes of other ions (Na(+), Cl(-)) were not observed. The K(+) efflux/H(+) influx response began within 15 minutes after addition of enzyme to cell suspensions and continued for approximately 1 hour after which cells resumed the net H(+) efflux exhibited prior to enzyme treatment. The response was not prolonged by a second enzyme dose 1 hour after the first. The K(+)/H(+) response was characterized by saturation at low enzymic activity (2 x 10(-3) units per milliliter), and inhibition by the protonophore, carbonyl cyanide m chlorophenylhydrazone, and was not associated with membrane leakiness caused by structural cell wall damage. The total K(+) loss and H(+) uptake induced by enzyme was one-fourth to one-third that induced by Pseudomonas syringae pv. pisi and did not reduce cell viability. These results indicate that pectate lyase induces a K(+) efflux/H(+) influx response in tobacco similar to but of shorter duration than that induced by P. syringae pv. pisi during the hypersensitive response. Pectate lyase or other cell wall degrading enzymes may therefore influence the induction of hypersensitivity. PMID- 16664982 TI - Recovery of turgor by wilted, excised cabbage leaves in the absence of water uptake : a new factor in drought acclimation. AB - Cabbage leaves excised from a fully turgid plant wilt within 20 minutes to 2 hours (depending on plant age) with a loss of about 10% relative water content (RWC). If droughted for 2 to 4 days in a high relative humidity leaf chamber, they may acclimate, recovering their turgor without the absorption of water, in fact at a loss of 15 to 25% RWC. This turgor recovery in the absence of water uptake occurs only if (a) the rate of water loss is slow enough (about 1-5% RWC per day after the first 24 hours drought loss of about 15% RWC), (b) if the leaves are no longer growing actively. Osmotic adjustment accompanies the turgor adjustment, but cannot be the cause in the absence of water uptake. The recovery of turgor by wilted cabbage leaves in the absence of water uptake cannot be explained by (a) transfer of reserve water from apoplast to symplast either from the cell walls or from the vessel lumens by cavitation or (b) metabolic loss of dry matter and gain of water. It can be explained by a contraction of the cell walls around the partially dehydrated protoplasts, until they regain their elastic extensibility. These proposed cell wall changes during drought acclimation are therefore the opposite of those occurring during growth. This hypothesis therefore explains the long recognized inverse relation between growth and acclimation. Two predictions of this hypothesis were tested and substantiated. PMID- 16664983 TI - Control of photosynthetic sucrose synthesis in barley primary leaves: role of fructose 2,6-bisphosphate. AB - Levels of fructose 2,6-bisphosphate (F2,6BP) and related metabolites were measured in 8- or 9-day-old barley (Hordeum vulgare L.) primary leaves throughout a 24 hour cycle. Young barley leaves contained about 0.4 nanomole F2,6BP per milligram chlorophyll at the end of a 12 hour dark period. F2,6BP levels increased rapidly following a dark-to-light transition and then decreased to about 0.1 nanomole per milligram chlorophyll after 5 or 10 minutes of light. Low levels of F2,6BP were detected in barley primary leaves throughout the day. A 10 fold increase in F2,6BP was observed during the first hour of the dark period and then levels of this metabolite decreased slowly for the next several hours. Only small diurnal fluctuations were noted in barley leaf glucose 6-phosphate and uridine 5'-diphosphoglucose levels. There were rapid changes in whole leaf F2,6BP levels when the light intensity was altered. High F2,6BP levels in the dark were not observed after short photosynthetic periods. Results obtained with barley primary leaves support the suggestion that F2,6BP is involved in regulating the flow of photosynthate from the chloroplast to sucrose. Extractable sucrose phosphate synthase activity was inversely related to barley primary leaf F2,6BP levels. This finding may indicate that the activities of sucrose-phosphate synthase and cytosolic fructose 1,6-bisphosphatase in barley primary leaves are metabolically coordinated. PMID- 16664984 TI - The Relationship between H(2) Evolution and Acetylene Reduction in Pisum sativum Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity. AB - Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H(2) evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H(2) or (3)H(2) in the presence of acetylene. In all cases, the rate of H(2) evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H(2) evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup(-) strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H(2) evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup(-) strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H(2) evolution by Hup(+) strains were similar in magnitude to their H(2)-saturated rates of Hup activity. The curvilinear relationship between H(2) evolution and acetylene reduction for a representative Hup(+) strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity. PMID- 16664985 TI - Fermentative Metabolism of Chlamydomonas reinhardii: III. Photoassimilation of Acetate. AB - The anaerobic photodissimilation of acetate by Chlamydomonas reinhardii F-60 adapted to a hydrogen metabolism was studied utilizing manometric and isotopic techniques. The rate of photoanaerobic (N(2)) acetate uptake was approximately 20 mumoles per milligram chlorophyll per hour or one-half that of the photoaerobic (air) rate. Under N(2), cells produced 1.7 moles H(2) and 0.8 mole CO(2) per mole of acetate consumed. Gas production and acetate uptake were inhibited by monofluoroacetic acid (MFA), 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and by H(2). Acetate uptake was inhibited about 50% by 5% H(2) (95% N(2)). H(2) in the presence of MFA or DCMU stimulated acetate uptake and the result was interpreted to indicate a transition from oxidative to reductive metabolism. Carbon-14 from both [1-(14)C]- and [2-(14)C]acetate was incorporated under N(2) or H(2) into CO(2), lipids, and carbohydrates. The methyl carbon of acetate accumulated principally (75-80%) in the lipid and carbohydrate fractions, whereas the carboxyl carbon contributed isotope primarily to CO(2) (56%) in N(2). The presence of H(2) caused a decrease in carbon lost from the cell as CO(2) and a greater proportion of the acetate was incorporated into lipid. The results support the occurrence of anaerobic and light-dependent citric acid and glyoxylate cycles which affect the conversion of acetate to CO(2) and H(2) prior to its conversion to cellular material. PMID- 16664986 TI - The metabolism of sunflower phytoalexins ayapin and scopoletin: plant-fungus interactions. AB - The coumarin phytoalexins ayapin and scopoletin accumulate in longitudinal stem sections of sunflower (Helianthus annuus L., Compositae) following inoculation with fungi both pathogenic (Alternaria helianthi) and nonpathogenic (Helminthosporium carbonum) to this plant. Both compounds were induced more rapidly, and they attained higher levels in tissue inoculated with the heterologous pathogen H. carbonum as compared with the sunflower pathogen A. helianthi. Similarly, scopoletin and ayapin accumulated to comparatively low concentrations following inoculation with a second sunflower pathogen, Phoma macdonaldii. Scopoletin was biosynthesized de novo following inoculation, although levels of its glucoside scopolin exceeded those of the aglucone in both infected and control tissues. Both scopoletin and scopolin were routinely detected in trace amounts in uninoculated tissue. In contrast, ayapin was not detected as a component of uninfected plants. When [(14)C]scopoletin was supplied to induced sunflower stem sections about 36% of the recovered radioactivity was in the form of ayapin. In vitro studies demonstrated that A. helianthi possessed the ability to rapidly degrade both scopoletin and ayapin, whereas H. carbonum was much less efficient in these traits. The differential degradation of these compounds by phytopathogenic fungi which do not attack sunflower is also discussed. PMID- 16664987 TI - Analysis of Stomatal and Nonstomatal Components in the Environmental Control of CO(2) Exchange in Leaves of Welwitschia mirabilis. AB - In well-watered plants of Welwitschia mirabilis, grown in the glass-house under high irradiance conditions, net CO(2) assimilation was almost exclusively observed during the daytime. The plants exhibited a very low potential for Crassulacean acid metabolism, which usually resulted in reduced rates of net CO(2) loss for several hours during the night. In leaves exposed to the diurnal changes in temperature and humidity typical of the natural habitats, CO(2) assimilation rates in the light were markedly depressed under conditions resembling those occurring during midday, when leaf temperatures and the leaf-air vapor pressure differences were high (36 degrees C and 50 millibars bar(-1), respectively). Studies on the relationship between CO(2) assimilation rate and intercellular CO(2) partial pressure at various temperatures and humidities showed that this decrease in CO(2) assimilation was largely due to stomatal closure. The increase in the limitation of photosynthesis by CO(2) diffusion, which is associated with the strong decline in stomatal conductance in Welwitschia exposed to midday conditions, may significantly contribute to the higher (13)C content of Welwitschia compared to the majority of C(3) species. PMID- 16664988 TI - Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes. AB - We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 x 10(5) protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts. PMID- 16664989 TI - Cyanobacterial Acclimation to Photosystem I or Photosystem II Light. AB - The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O(2) evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions. PMID- 16664990 TI - Developmental Loss of Photosystem II Activity and Structure in a Chloroplast Encoded Tobacco Mutant, Lutescens-1. AB - Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF(s) particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light dependent phosphorylation of PSII polypeptides and flash-induced O(2) evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes. PMID- 16664991 TI - Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod. AB - Cell-free extracts capable of converting [(14)C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [(14)C]-labeled GAs, prepared enzymically from [(14)C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA(12) --> GA(53) --> GA(44) --> GA(19) --> GA(20). The pH optima for the enzymic conversions of [(14)C]GA(53), [(14)C]GA(44) and [(14)C]GA(19) were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe(2+), alpha-ketoglutarate and O(2) for activity, and ascorbate stimulated the conversion of [(14)C]GA(53) and [(14)C]GA(19). Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA(53) and GA(19) are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA(44) remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA(20) content of spinach plants in LD than in SD. PMID- 16664992 TI - Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. AB - Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories. PMID- 16664993 TI - The Two-Isozyme System of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Nicotiana silvestris and Other Higher Plants. AB - Two isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, denoted DS-Mn and DS-Co, were identified following DEAE-cellulose chromatography of crude extracts prepared from suspension-cultured cells of Nicotiana silvestris. The strikingly different properties of the isozymes allowed the development of assays for the selective detection of either isozyme in samples containing a mixture of the two. The DS-Mn isozyme required the sulfhydryl reductant, dithiothreitol, for activity and was stimulated by manganese. Activation by dithiothreitol was slow relative to catalysis, accounting for a hysteretic progress curve that was observed when reactions were started with inactive enzyme. The DS-Co isozyme was inhibited by dithiothreitol and required a divalent cation for activity. At optimal cation concentrations of 10 millimolar (magnesium), 0.5 millimolar (cobalt), and 0.5 millimolar (manganese), relative activities obtained were 100, 85, and 20, respectively. The substrate saturation curves with respect to erythrose 4-phosphate differed markedly when the two isozymes were compared. As little as 0.5 millimolar erythrose 4-phosphate saturated DS-Mn, whereas a 10-fold higher concentration was needed for saturation of DS-Co. The pH optimum of DS-Mn was 8.0, while that of the DS-Co isozyme was 8.6. Leaves of both N. silvestris and spinach also exhibited the DS-Mn/DS-Co isozyme arrangement, and the subcellular location of DS-Mn was shown to be the chloroplast compartment. By application of the differential assays for DAHP synthase isozymes, various monocotyledonous and dicotyledonous plants yielded data indicating the general presence of the DS-Mn/DS-Co isozyme pair in higher plants. PMID- 16664994 TI - Photosynthesis of F(1) Hybrids between C(4) and C(3)-C(4) Species of Flaveria. AB - Photosynthetic characteristics were studied in several F(1) hybrids between C(4) and C(3)-C(4) species of Flaveria. Stable carbon isotope ratios, O(2) inhibition of apparent photosynthesis, and phosphoenolpyruvate carboxylase activities in the hybrids were similar to the means for the parents. Values of CO(2) compensation concentrations were nearer to those of the C(4) parent and apparent photosynthesis was below that of both parents, being only 60 and 74% of that of the lowest (C(3)-C(4)) parent in two experiments. Reductions of CO(2) compensation concentration and O(2) inhibition of apparent photosynthesis as well as increases in carbon isotope ratios and phosphoenolpyruvate carboxylase activities compared to values in C(3)-C(4) species suggest transfer of a limited degree of C(4) photosynthesis to the F(1) hybrids. However, the lower apparent photosynthesis of the hybrids suggests that transfer of C(4) characteristics to non-C(4) species is detrimental unless characteristics associated with C(4) photosynthesis are fully developed. There was a highly significant negative correlation (r = -0.90) between CO(2) compensation concentration and the logarithm of phosphoenolpyruvate carboxylase activity in the parents and hybrids, suggesting involvement of this enzyme in controlling the CO(2) compensation concentration. Although bundle-sheath cells were more developed in leaves of hybrids than in C(3)-C(4) parents, they appeared to contain lower quantities of organelles than those of the C(4) parent. Reduced quantities of organelles in bundle-sheath cells could indicate incomplete compartmentation of partial pathways of the C(4) cycle in the hybrids. This may mean that the reduction of CO(2) compensation and O(2) inhibition of apparent photosynthesis relative to the C(3)-C(4) parents is less dependent on fully developed Kranz anatomy than is increased apparent photosynthesis. PMID- 16664995 TI - Opine synthesis in wild-type plant tissue. AB - Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid. PMID- 16664996 TI - Cessation of assimilate uptake in maturing soybean seeds. AB - In vitro assimilate uptake and metabolism were evaluated in embryos of known age isolated from seeds at mid-podfilling through physiological maturity. The capacity of isolated Wye soybean embryos to take up exogenous [(14)C]sucrose dropped nearly 4-fold in less than 1 week at incipient cotyledon yellowing. This drop in rate of sucrose uptake coincided with cessation of seed growth as well as rapid decline in leaf photosynthetic rate that preceded leaf yellowing. Conversely, the rate of [(3)H]glutamine uptake by cotyledons increased as they yellowed. Yellow cotyledons also rapidly converted exogenous [(3)H]glutamine to ethanolinsoluble components, but converted little exogenous [(14)C]sucrose to ethanol-insoluble components, primarily because of greatly reduced sucrose uptake. Sustained import and metabolism of amino acids remobilized from senescing leaves may prolong seed growth beyond loss of photosynthetic competency and sucrose availability. PMID- 16664997 TI - A possible second role for calmodulin in biological clock-controlled processes of euglena. AB - The response of the Euglena gracilis (Klebs strain Z) photosynthesis circadian rhythm to three calmodulin antagonists was examined. In the presence of an antagonist, the photosynthetic reactions were uncoupled from the biological clock. Instead of the highly predictable rhythmic pattern characteristic of a biological clock-controlled circadian rhythm, the photosynthetic rate appears to be influenced by the light/dark cycle. The rate of O(2) evolution increases throughout the light portion of the cycle and does not decrease until the cells are exposed to darkness. Shortterm exposure to a calmodulin antagonist (2 hour pulses) failed to cause phase shifts in the timing of the rhythm. This suggests that calmodulin is not part of the clock controlling photosynthesis and that it has a clock-related role different from that reported for the cell division rhythm in Euglena. PMID- 16664998 TI - Factors Influencing Spore Germination and Early Gametophyte Development in Anemia mexicana and Anemia phyllitidis. AB - Spores of Anemia mexicana Klotzsch and Anemia phyllitidis (L.) Swartz were tested comparatively to investigate the effects of various treatments on spore germination and early gametophyte development in light and darkness. The optimum pH for induction of spore germination is approximately 6. Both species have a minimum 8 hour light insensitive preinduction phase for spore germination. An additional 8 to 12 hours of light are needed to induce 50% germination in A. phyllitidis while at least 24 hours of light are needed for A. mexicana spores. A. phyllitidis has greater sensitivity to the four gibberellic acids tested (GA(3), GA(4), GA(7), and GA(13)) than A. mexicana for induction of spore germination in darkness. In both species the greatest response was observed with GA(4) and GA(7). GA(13) was clearly the least effective. Gametophytes of each species are 100 times more sensitive to their own antheridiogen than to the antheridiogen of the other species. AMO-1618 (1 millimolar), fenarimol (1 mm), and ancymidol (0.1 mm) had essentially no effect on light-induced germination. The latter two did, however, inhibit gametophyte development. PMID- 16664999 TI - Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements. AB - Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O(2) uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate stimulated O(2) uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O(2) uptake is due to the activation of a peroxidase catalyzing reduction of O(2). This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O(2) uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots. PMID- 16665000 TI - Metabolism of gibberellin a(12)-7-aldehyde by soybean cotyledons and its use in identifying gibberellin a(7) as an endogenous gibberellin. AB - The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill-about 10 nanograms GA(3) equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. The metabolism of [(14)C]-GA(12)-7-aldehyde (GA(12)ald)-the universal GA precursor-by intact, mid-pod-fill, soybean cotyledons and their cell-free extracts was investigated. In 4 hours, extracts converted GA(12)ald to two products [(14)C]GA(12) (42% yield) and [(14)C]GA(15) (7%). Within 5 minutes, intact embryos converted GA(12)ald to [(14)C]GA(12) and [(14)C]GA(15) in 15% yield; 4 hour incubations afforded at least 22 products (96% total yield). The putative [(14)C]GA(12) was identified as a product of [(14)C]GA(12)ald metabolism on the basis of co-chromatography with authentic GA(12) on a series of reversed and normal phase high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) systems, and by a dual feed of the putative [(14)C]GA(12) and authentic [(14)C]GA(12) to cotyledons of both peas and soybeans. The [(14)C]GA(15) was identified as a metabolite of [(14)C]GA(12)ald by capillary gas chromatography (GC)-mass-spectrometry-selected ion monitoring, GC-radiocounting, HPLC, and TLC. By adding the [(14)C] metabolites of [(14)C]GA(12)ald to a different and larger extract (about 0.2 kg fresh weight of soybean reproductive tissue) and purifying endogenous substances co-chromatographing with these metabolites, at least two GA-like substances were obtained and one identified as GA(7) by GC-mass spectrometry. Since [(14)C]GA(9) was not found as a [(14)C]metabolite of [(14)C]GA(12)ald, soybean embryos might have a pathway for biosynthesis of active, C-19 gibberellins like that of the cucurbits; GA(12)ald - > GA(12) --> GA(15) --> GA(24) --> GA(36) --> GA(4) --> GA(7). PMID- 16665001 TI - Utilization of Previously Accumulated and Concurrently Absorbed Nitrogen during Reproductive Growth in Maize : Influence of Prolificacy and Nitrogen Source. AB - A prolific maize (Zea mays L.) genotype was grown to physiological maturity under greenhouse conditions to examine the effects of reproductive sink demand on (a) the remobilization of N accumulated during vegetative growth, and (b) the partitioning of N accumulated concurrent with ear development. One- and two-eared plants were treated with either a NO(3) (-) or NH(4) (+) source of (15)N-labeled N during reproductive growth. Plants with two ears enhanced grain production, N remobilization from the stalk and roots, and N translocation to the grain from concurrently assimilated N. But, remobilization of leaf-N was unaffected by ear number. In addition, N uptake and total dry matter accumulation during the reproductive period were also unaffected, although P uptake was greater in the two-eared plants. Less than 15% of the total K(+) uptake was accumulated after silking while during this time more than 40% of the total N and more than 50% of the total P were absorbed. The data also indicate that with NO(3) (-) nutrition, internal recirculation of K(+) between shoots and roots may play a prominent role in the transport of nitrogenous solutes during grain development. N source had no effect on dry matter production and N uptake of both one- and two-eared plants. However, slightly greater partitioning of labeled-N from the NH(4) (+) source to the grain was observed in the two-eared plants. PMID- 16665002 TI - Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) and Corn (Zea mays L.) Seedlings: I. N Study. AB - Nitrate reduction in roots and shoots of 7-day-old barley seedlings, and 9-day old corn seedlings was investigated. The N-depleted seedlings were transferred for 24 h or 48 h of continuous light to a mixed nitrogen medium containing both nitrate and ammonium. Total nitrate reduction was determined by (15)N incorporation from (15)NO(3) (-), translocation of reduced (15)N from the roots to the shoots was estimated with reduced (15)N from (15)NH(4) (+) assimilation as tracer, and the translocation from the shoots to the roots was measured on plants grown with a split root system. A model was proposed to calculate the nitrate reduction by roots from these data. For both species, the induction phase was characterized by a high contribution of the roots which accounted for 65% of the whole plant nitrate reduction in barley, and for 70% in corn. However, during the second period of the experiment, once this induction process was finished, roots only accounted for 20% of the whole plant nitrate reduction in barley seedlings, and for 27% in corn. This reversal in nitrate reduction localization was due to both increased shoot reduction and decreased root reduction. The pattern of N exchanges between the organs showed that the cycling of reduced N through the plant was important for both species. In particular, the downward transport of reduced N increased while nitrate assimilation in roots decreased. As a result, when induction was achieved, the N feeding of the roots appeared to be highly dependent on translocation from the leaves. PMID- 16665003 TI - Studies on the Photoactivation of the Water-Oxidizing Enzyme: II. Characterization of Weak Light Photoinhibition of PSII and Its Light-Induced Recovery. AB - Inactivation of the water splitting enzyme complex in leaves or isolated chloroplasts results in increased susceptibility of photosystem II (PSII) to damage by light. Photoinhibition under this condition occurs in very weak light. The site of damage is exclusive of the water splitting complex yet still on the oxidizing side of PSII, as the Q(B) locus is unaffected while photoreduction of silicomolybdate is inhibited. The kinetics of loss in PSII activity are more complex than apparent first-order, and the quantum efficiency is low. We observe no evidence of deletion from thylakoid membranes of any PSII polypeptide as a consequence of photoinhibition, although recovery from the photoinhibition is dependent upon both light and 70S protein synthesis. Enhanced synthesis of two proteins occurs during recovery, only one of which (D2) appears to be causally related to the recovery. We present a model which describes the relationship of weak light photoinhibition and its recovery to photoactivation of the S-state water oxidizing complex. PMID- 16665004 TI - Effects of Respiration Inhibitors and Uncouplers on Dark- and Light-Induced Leaflet Movements of Cassia fasciculata. AB - Respiration inhibitors, in particular KCN and NaN(3), inhibited slightly the dark induced (scotonasty) as well as the light-induced (photonasty) leaflet movements of Cassia fasciculata: they act only at concentrations higher than 1 millimolar and 0.1 millimolar, respectively. Amytal induced a stronger inhibitory effect on scotonasty. Salicylhydroxamic acid, which inhibits the cyanide-insensitive respiration pathway, was also poorly effective when applied alone. KCN and salicylhydroxamic acid applied together increased the inhibition. Uncouplers of oxidative phosphorylation were very effective: 2,4-dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone inhibited the scotonastic movements at concentrations higher than 10 mum and 1 mum, respectively. Although uncouplers reduced the photonastic movements at higher concentrations, they promoted leaflet opening at other concentrations in an unexpected way. PMID- 16665005 TI - Variation in cadmium accumulation potential and tissue distribution of cadmium in tobacco. AB - Variation in Cd accumulation between Nicotiana species but not varieties has been observed in seedlings grown in solution culture with moderate-to-low levels of Cd. Nicotiana tabacum has been characterized as a leaf and root accumulator while Nicotiana rustica is shown to be primarily a root accumulator, having about half the leaf Cd per gram dry weight of N. tabacum. This phenotype is retained in the mature N. rustica plant. To characterize these two species which differ in their modes of Cd accumulation, tissue Cd distribution, partitioning of metal in soluble and insoluble fractions and the contribution of soluble Cd-binding proteins (peptides) to total plant Cd was assessed using mature solution cultured plants. Metal accumulation was highest in the most mature leaves and in young roots. The preponderance of young roots in N. rustica may, in part, account for low leaf/high root Cd accumulation in this species. While Cd-binding peptides appear to be a principal form of Cd in leaves and roots of seedlings and these also occur in mature leaves, Cd is equally distributed between soluble (about 80% as Cd-binding peptide) and uncharacterized insoluble forms in mature plant roots. PMID- 16665006 TI - Photocontrol of Dark Circadian Rhythms in Stomata of Phaseolus vulgaris L. AB - Stomatal diffusion resistance in primary leaves of Phaseolus vulgaris L. which had been grown in light:dark cycles followed a marked circadian rhythm when the plants were transferred to continuous darkness. Reentrainment of the rhythm required more than one inductive change in photoperiod. The phasing of the rhythm of dark stomatal opening was contolled primarily by the light-on (dawn) signal, whereas the rhythm of dark closure was related to the light-off (dusk) signal. The evidence points to a dual control of the circadian clock in which a product of photosynthesis plays a major role. No evidence for phytochrome involvement in the phasing of the rhythm was found. An influence of phytochrome on the amplitude of the stomatal rhythm was observed in which removal of phytochrome-far-red absorbing form caused rapid damping. PMID- 16665007 TI - Effects of Gabaculine on Pigment Biosynthesis in Normal and Nutrient Deficient Cells of Anacystis nidulans. AB - Pigment biosynthesis in the cyanobacterium, Anacystis nidulans, was examined in the presence of gabaculine (5-amino-1,3-cyclohexadienyl-carboxylic acid). At 20 micromolar, this inhibitor blocked the biosynthesis of both chlorophyll and phycocyanin. Analogs of gabaculine were not effective as inhibitors of chlorophyll or phycocyanin biosynthesis. Iron- and phosphate-deficient cultures were 2- to 4-fold more sensitive to the inhibitor than were normal or nitrate deficient cultures. Inhibition resulted in the excretion of a mixture of organic acids by the cells. delta-Aminolevulinic acid was a principle component of the mixture, identified by thin layer chromatography. Excretion of delta aminolevulinic acid occurred following a brief lag after gabaculine addition. It remained linear for nearly 24 hours and was dependent upon illumination. However, high light inhibited excretion. Apparently, gabaculine blocks chlorophyll biosynthesis after the formation of delta-aminolevulinic acid in cyanobacteria. PMID- 16665008 TI - Regeneration of Magnesium-2,4-Divinylpheoporphyrin a(5) (Divinyl Protochlorophyllide) in Isolated Developing Chloroplasts. AB - A preparation of developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons was found capable of synthesizing divinyl protochlorophyllide (magnesium-2,4-divinylpheoporphyrin a(5)) in the presence of glutamate, adenosine triphosphate, reducing power, S-adenosyl-l-methionine, and molecular oxygen. Both adenosine triphosphate and molecular oxygen were absolutely required while each of the other three was strongly promotive. Organelle intactness was essential. The divinyl protochlorophyllide (Pchlide) formed in vitro could be completely phototransformed. Regeneration of Pchlide was not inhibited by 0.3 millimolar chloramphenicol. The initial in vitro rate of Pchlide regeneration was considerably higher than the rate of Pchlide synthesis observed when greened cucumber seedlings were returned to darkness. However, Pchlide synthesis in vitro fell off exponentially with a half-life of approximately 21 minutes, whereas Pchlide synthesis in vivo was linear for at least 100 minutes. It is likely that the leveling off of the in vitro rate is due to the loss of chloroplast integrity during the incubation, because neither adding more cofactors, nor phototransforming the accumulated Pchlide in the middle of the incubation period, restored the high initial rate of Pchlide synthesis. PMID- 16665009 TI - Mode of action of abscisic Acid in barley aleurone layers : induction of new proteins by abscisic Acid. AB - As part of a continuing effort to elucidate the mode of action of abscisic acid (ABA) in barley (Hordeum vulgare L. cv Himalaya) aleurone layers, we have investigated the induction of several polypeptides by ABA in this tissue. There were nine ABA-induced polypeptides as observed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and considerably more (at least 16 spots) on a two-dimensional gel. These proteins started to show enhanced synthesis 2 to 4 hours after ABA treatment, and their synthesis continued for at least 48 hours. In vitro translation using total RNA isolated from ABA-treated aleurone layers indicated that translatable mRNA levels of these proteins essentially paralleled the levels of in vivo synthesized proteins. The most abundant of the ABA-induced proteins was a 29 kilodalton polypeptide which was also synthesized in tissue incubated without ABA. In vivo synthesis of this protein declined as ABA concentration was decreased, with 1 nanomolar ABA approaching control level. Cell fractionation experiments located the 29 kilodalton major ABA-induced protein in 1,000g and 13,000g pellets; most other induced proteins were in the 80,000g supernatant. The 29 kilodalton protein appeared to be sensitive to degradation by sulfhydryl type proteases. As expected, the induction of these proteins by ABA was suppressed by gibberellic acid. Phaseic acid, the first stable metabolite of ABA, suppressed the gibberellic acid-enhanced alpha-amylase synthesis but was unable to induce the ABA-induced proteins. None of the ABA-induced proteins were secreted into the incubation medium. A 36 kilodalton ABA-induced protein showed cross-reactivity with antibody against a barley lectin specific for glucosamine, galactosamine, and mannosamine. PMID- 16665010 TI - Gibberellins in Suspensors of Phaseolus coccineus L. Seeds. AB - Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C(19)-gibberellins, GA(1), GA(4), GA(5), GA(6), GA(8), and one C(20)-GA, GA(44). The major GAs present were GA(1) and GA(8). Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system. PMID- 16665011 TI - Pea Xyloglucan and Cellulose : IV. Assembly of beta-Glucans by Pea Protoplasts. AB - The synthesis and assembly of xyloglucan were examined during early stages of wall regeneration by protoplasts isolated from growing regions of etiolated peas. During early stages of cultivation, fluorescence microscopy showed that the protoplast surface bound Calcofluor and ammonium salt of 8-anilino-1-naphthalene sulfonic acid and, in time, it also bound fluorescent fucose-binding lectin. Based on chemical analysis, 1,3-beta-glucan was the main polysaccharide formed by protoplasts and xyloglucan and cellulose were minor wall components. Binding between cellulose and xyloglucan was not as strong as that in tissues of intact pea plants, i.e. mild alkali could dissolve most xyloglucan from the protoplast. However, the addition of exogenous pea xyloglucan into the culture medium stimulated the deposition of new polysaccharides into the protoplast wall and enhanced the close association of newly formed xyloglucan with cellulose. PMID- 16665012 TI - Developmental Changes in Photosynthetic Gas Exchange in the Polyol-Synthesizing Species, Apium graveolens L. (Celery). AB - Developmental changes in photosynthetic gas exchange were investigated in the mannitol synthesizing plant celery (Apium graveolens L. ;Giant Pascal'). Greenhouse-grown plants had unusually high photosynthetic rates for a C(3) plant, but consistent with field productivity data reported elsewhere for this plant. In most respects, celery exhibited typical C(3) photosynthetic characteristics; light saturation occurred at 600 micromoles photons per square meter per second, with a broad temperature optimum, peaking at 26 degrees C. At 2% O(2), photosynthesis was enhanced 15 to 25% compared to rates at 21% O(2). However, celery had low CO(2) compensation points, averaging 7 to 20 microliters per liter throughout the canopy. Conventional mechanisms for concentrating CO(2) were not detectable. PMID- 16665013 TI - Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize : Effect of DCMU, Antimycin A, CCCP, and Phlorizin. AB - Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK. PMID- 16665014 TI - Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K-Induced Cytosol Alkalinization. AB - In maize root segments fusicoccin induced a consistent increase in cell sap pH (taken as representative of vacuolar pH). This effect was markedly enhanced by the presence of K(+) in the medium, whereas in the absence of fusicoccin K(+) did not significantly influence cell sap pH. Treatment with a weak acid at 2 mm concentration inhibited the uptake of a different ((14)C-labeled) weak acid fed at a lower concentration, thus suggesting that acidification of the cytoplasm inhibits weak acid uptake. Fusicoccin and K(+) increased the rate of uptake of 5,5-dimethyloxazolidine-2,4-dione, butyric acid, or isobutyric acid slightly when fed separately, strongly when fed in combination. The synergism between fusicoccin and K(+) in stimulating weak acid uptake was parallel to that observed for the stimulation of H(+) extrusion. Application of the weak acid distribution method to a condition of ;quasi-equilibrium' indicated that fusicoccin induces a cytosolic pH increase of about 0.14 unit. These results are interpreted as providing circumstantial evidence that fusicoccin- and K(+)- induced stimulation of H(+) extrusion led to an alkalinization of the cytosol, and that other early metabolic responses, such as an increase in malate level, are a consequence of the increase in cytosolic pH. PMID- 16665015 TI - Accumulation of Putrescine during Chilling Injury of Fruits. AB - Putrescine (Put) increased 68% in lemon (Citrus limon (L.) Burm. f. cv Bearss) flavedo, 39% in grapefruit (C. paradisi Macf. cv Marsh) flavedo, 49% in grapefruit juice, and 149% in pepper (Capsicum annuum L. cv Early Calwonder) pericarp when fruits were stored at chilling temperatures. In lemon flavedo, the coefficient of correlation (r(2)) between Put concentration with severity of chilling was 0.90 and Put levels almost doubled; the injury index going from 1 to 2 units. Pepper pericarp, which was the most chilling-sensitive tissue tested (injury index going from 1 to 3.8 units), showed the greatest difference in Put accumulation (166 to 413 nanomoles per gram fresh weight) between storage temperatures of 7.2 and 1 degrees C. The least difference (338 to 470 nanomoles per gram fresh weight) was found in grapefruit flavedo between storage temperatures of 15.5 and 4.4 degrees C; the injury index going from 1 to 1.3 units. PMID- 16665016 TI - Isoelectric focusing of oat root membranes. AB - The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad range (pH 3-10) or narrow-range (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient. Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures. PMID- 16665017 TI - Identification of endogenous gibberellins from sorghum. AB - Gibberellins (GA) A(1), A(19), and A(20) were identified in shoot cylinders containing the apical meristems from sorghum (Sorghum bicolor L.). Extracts were purified by sequential SiO(2) partition chromatography and reversed-phase C(18) high performance liquid chromatography and biologically active (dwarf rice cv Tan ginbozu microdrop assay) fractions were subjected to gas chromatography-selected ion monitoring. Based on the use of [(3)H]GA and [(2)H](d(2))GA internal standards, amounts of GA(1), GA(19), and GA(20) were estimated to be 0.7, 8.8, and 1.5 namograms per gram dry weight of tissue, respectively. PMID- 16665018 TI - A Calcium-Activated Phytase from Pollen of Lilium longiflorum. AB - A phytase was isolated and partially purified from the pollen of Lilium longiflorum Thumb. Optimum activity was at pH 8.0. The phytase was activated by Ca(2+) and Sr(2+) but not by the other divalent cations tested. Activity was inhibited by ethylenediaminetetraacetate. The phytase had a temperature optimum of 55 to 60 degrees C and an activation energy of about 12,700 calories/mole. Extraction of L. longiflorum pollen with 0.1% Triton X-100 increased recovery of the phytase by nearly 4-fold. The phytase had a molecular weight of about 88,000 as determined by gel filtration chromatography and a K(m) value of 7.2 micromolar for phytic acid in the presence of Ca(2+). PMID- 16665019 TI - Effect of SO(2) and O(3) on Production of Antioxidants in Conifers. AB - Production of antioxidants was investigated in needles of fir (Abies alba Mill.) and spruce (Picea abies (L.) Karst) after exposure to low concentrations of SO(2), O(3), and a combination of both pollutants. Glutathione reacted most sensitively to pollutants followed by vitamin E and vitamin C. In spruce needles, the overall increase of antioxidants after exposure to air pollutants was lower than in needles of fir. SO(2) was more potent than O(3). Maximum increase of antioxidants was found in needles after exposure of trees to SO(2) + O(3). PMID- 16665020 TI - Gene Expression in Developing Wheat Endosperm : Accumulation of Gliadin and ADPglucose Pyrophosphorylase Messenger RNAs and Polypeptides. AB - The developmental accumulation pattern of messenger RNA transcripts and polypeptides for wheat gliadins and ADPglucose pyrophosphorylase was determined using cDNA and antibody probes. Gliadin mRNA was detected on Northern and RNA dot blots at 3 days after flowering, it increased 100-fold by 10 days and decreased subsequent to 14 days. The abundant mRNAs encoding alpha/beta- and gamma-type gliadins and mRNA for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, accumulated coordinately. Despite the coordinate accumulation of their mRNA transcripts, the accumulation of gliadin and ADPglucose pyrophosphorylase polypeptides, as determined by Western blot, differed significantly. The time at which gliadin and ADPglucose pyrophosphorylase mRNAs began accumulating was also the time when the overall pattern of gene expression, as seen by two-dimensional gel electrophoresis of in vitro translation products, changed most significantly. However, the accumulation of a number of other mRNAs or polypeptides having unknown function occurred at other times during endosperm development. The pattern of expression in the earliest stages of development was strikingly similar to that of coleoptile, another rapidly growing, nonphotosynthetic tissue. Thus, the pattern of gene expression reflects the program of development observed cytologically. PMID- 16665021 TI - Effects of Ca on Amino Acid Transport and Accumulation in Roots of Phaseolus vulgaris. AB - Ca(2+) stimulates the uptake of alpha-aminoisobutyric acid (AIB) into excised or intact Phaseolus vulgaris L. roots by a factor of two. In roots depleted of Ca(2+) by preincubation with ethylenediaminetetraacetate, ethyleneglycol-bis(beta aminoethyl ether)-N,N'-tetraacetic acid, or streptomycin, the stimulatory effect is 7- to 10-fold. In the presence of Ca(2+), roots accumulate AIB more than 100 fold; Ca(2+)-depleted roots only equilibrate with AIB. Radioautography shows [(14)C]AIB to be present in all cells after 90 min. Although Ca(2+)-depleted roots lose accumulated [(14)C]AIB about 10 times faster than roots supplied with Ca(2+), this increased efflux is not the main cause for the decrease in net uptake observed. The latter is rather due to a less negative membrane potential Deltapsi in Ca(2+) depleted roots (-120 mV --> -50 mV). The basic feature explaining all the results of Ca(2+) deficiency is an increase in general membrane permeability. No indication of a specific regulatory function of Ca(2+) in membrane transport of roots has been obtained. PMID- 16665022 TI - Ca uptake by endoplasmic reticulum from zucchini hypocotyls : the use of chlorotetracycline as a probe for ca uptake. AB - Ca(2+) uptake into microsomal vesicles was measured using the fluorescent probe chlorotetracycline. The Ca(2+) uptake was ATP-dependent and did not occur in the presence of the calcium ionophore A23187. There was a linear relationship between the rate of ATP-dependent fluorescence increase using chlorotetracycline and ATP dependent (45)Ca(2+) uptake, indicating that chlorotetracycline can be used as a quantitative probe for Ca(2+) uptake. The fluorescent probe allows measurements to be made in real time, and avoids the use of radioisotopes. Ca(2+) transport was associated with endoplasmic reticulum on linear gradients when the endoplasmic reticulum was in either rough or smooth form. The Ca(2+) uptake had a pH optimum of 7.5, a K(m) for ATP of 0.1 millimolar, a K(m) for Ca(2+) of about 70 nanomolar, and was stimulated 2-fold by calmodulin. Vanadate inhibited uptake completely at a concentration of 50 micromolar, half-maximally at 5 micromolar. Carbonyl cyanide 4-(trifluoromethoxy)-phenyl-hydrazone, oligomycin, azide, and nitrate caused only slight inhibition. Dicyclohexylcarbodiimide (DCCD) stimulated slightly at concentrations as high as 400 micromolar. The hormones gibberellic acid, indoleacetic acid, and abscisic acid at 10 micromolar had no significant effect. Myo-inositol 1,4,5-trisphosphate did not cause release of Ca(2+) after uptake. The properties of the enzyme suggest that it has a functional role in regulating cytosolic Ca(2+) levels. Based on the lack of an effect by hormones, it may not act as a mediator of second messenger roles of Ca(2+). The inhibition by vanadate and slight stimulation by DCCD may be useful as a ;signature' for this endoplasmic reticulum Ca(2+) uptake system. PMID- 16665023 TI - Enzyme Dynamics of the Resurrection Plant Selaginella lepidophylla (Hook. & Grev.) Spring during Rehydration. AB - The activities of 10 enzymes involved in carbohydrate metabolism were measured in both desiccated and rehydrated fronds of the desiccation-tolerant pteridophyte Selaginella lepidophylla (Hook. & Grev.) Spring. Enzyme conservation was defined as the ratio of desiccated to hydrated frond enzyme activity. The mean level of conservation was 74%, with nine of the 10 enzymes showing significant activity increases (P<0.05) during hydration. The mean of photosynthetic enzyme conservation was significantly lower (P=0.05) than the mean for glycolytic and respiratory enzymes combined. Chloramphenicol inhibited the normal activity increase in ribulose bisphosphate carboxylase and (NADPH)triose-P dehydrogenase but not pyruvate kinase upon rehydration. Cycloheximide did not affect the normal activity increase for these three enzymes. It is concluded that substantial enzyme conservation is beneficial for rapid resumption of metabolic activity in resurrection plants. PMID- 16665024 TI - Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine. AB - The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH(2)OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH(2)OH. To demonstrate that the terminal activity was readily inactivted by NH(2)OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH(2)OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH(2)OH appears to be competitive versus NO(3) (-). Since NO(3) (-), as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO(3) (-)), protect the enzyme from NH(2)OH inactivation, it is suggested that NH(2)OH binds to the nitrate active site. The NH(2)OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN(-). NH(2)OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance. PMID- 16665025 TI - Sucrose synthase activity in developing wheat endosperms differing in maximum weight. AB - Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the "rate limiting" enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels. PMID- 16665026 TI - Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth. AB - The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and beta-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and beta-conglycinin specific antibodies. The three subunits of beta-conglycinin were preferentially metabolized. Of the three subunits of beta-conglycinin, the larger alpha and alpha' subunits are rapidly degraded, generating new beta-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the beta-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of beta conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth. PMID- 16665027 TI - Effects of pH on Activity and Activation of Ribulose 1,5-Bisphosphate Carboxylase at Air Level CO(2). AB - The effects of pH on catalysis and activation characteristics of spinach ribulose 1,5-bisphosphate (RuBP) carboxylase were examined at air level of CO(2). Catalysis at limiting CO(2) was independent of pH over the range of pH 8.2 to 8.8 However, the kinetics of activation and the apparent equilibrium between the activated and inactivated forms of the enzyme were strongly dependent upon the pH and the presence or absence of the substrate RuBP. When incubated at air level of CO(2) at pH 8.2 in the absence of RuBP, the enzyme activation state was approximately 75% of that achieved with saturating CO(2) at that pH. The extent of activation increased with pH reaching 100% at pH values of 8.6 or higher. Adding RuBP to the activation medium after equilibrium activation state had been established decreased the apparent equilibrium activation level at pH values below 8.6. This effect was reversed at pH values above 8.6. Activation of inactive enzyme by CO(2) and Mg(2+) was inhibited dramatically at pH values below 8.6 and less so at pH values above 8.6. Studies showed that binding of RuBP to the inactive form of the enzyme was pH dependent with tighter binding occurring at lower pH values. It is suggested that the tight binding of RuBP to the inactive enzyme tends to decrease the equilibrium concentration of the activated form at pH values less than 8.6. These studies indicate that stromal pH could have a strong effect on the activation state of this enzyme in vivo, and possible feedback interactions which might adjust the apparent V(max) to match the rate of RuBP regeneration are discussed. PMID- 16665028 TI - Effects of Atmospheric CO(2) Enrichment on the Growth and Mineral Nutrition of Quercus alba Seedlings in Nutrient-Poor Soil. AB - One-year-old dormant white oak (Quercus alba L.) seedlings were planted in a nutrient-deficient forest soil and grown for 40 weeks in growth chambers at ambient (362 microliters per liter) or elevated (690 microliters per liter) levels of CO(2). Although all of the seedlings became severely N deficient, CO(2) enrichment enhanced growth by 85%, with the greatest enhancement in root systems. The growth enhancement did not increase the total water use per plant, so water use efficiency was significantly greater in elevated CO(2). Total uptake of N, S, and B was not affected by CO(2), therefore, tissue concentrations of these nutrients were significantly lower in elevated CO(2). An increase in nutrient-use efficiency with respect to N was apparent in that a greater proportion of the limited N pool in the CO(2)-enriched plants was in fine roots and leaves. The uptake of other nutrients increased with CO(2) concentration, and P and K uptake increased in proportion to growth. Increased uptake of P by plants in elevated CO(2) may have been a result of greater proliferation of fine roots and associated mycorrhizae and rhizosphere bacteria stimulating P mineralization. The results demonstrate that a growth response to CO(2) enrichment is possible in nutrient-limited systems, and that the mechanisms of response may include either increased nutrient supply or decreased physiological demand. PMID- 16665029 TI - Photosynthesis at low water potentials in sunflower: lack of photoinhibitory effects. AB - The losses in chloroplast capacity to fix CO(2) when photosynthesis is reduced at low leaf water potential (psi(1)) have been proposed to result from photoinhibition. We investigated this possibility in soil-grown sunflower (Helianthus annuus L. cv IS894) using gas exchange techniques to measure directly the influence of light during dehydration on the in situ chloroplast capacity to fix CO(2). The quantum yield for CO(2) fixation as well as the rate of light- and CO(2)-saturated photosynthesis were strongly inhibited at low psi(1). The extent of inhibition was the same whether the leaves were exposed to high or to low light during dehydration. When intercellular partial pressures of CO(2) were decreased to the compensation point, which was lower than the partial pressures resulting from stomatal closure, the inhibition of the quantum yield was also unaffected. Photoinhibition could be observed only after high light exposures were imposed under nonphysiological low CO(2) and O(2) where both photosynthesis and photorespiration were suppressed. The experiments are the first to test whether gas exchange at low psi(1) is affected by potentially photoinhibitory conditions and show that the loss in chloroplast capacity to fix CO(2) was entirely the result of a direct effect of water availability on chloroplast function and not photoinhibition. PMID- 16665030 TI - Variation in amounts of pyruvate, orthophosphate dikinase, and some other enzymes of the c(4) pathway in some wheat species. AB - First leaves and flag leaves of the wheat species Triticum aestivum cv Anza (6x), T. boeoticum Boiss (2x) L. were examined for content of pyruvate, orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and ribulose 1,5 bisphosphate carboxylase (RuBPC) by protein blot analyses using antibodies to maize leaf enzymes and by activity assays. In agreement with previous reports, the amount of RuBPC per mesophyll cell was about 3 times more in the hexaploid species, T. aestivum, than in the diploid species, T. boeoticum, both in first leaves and in flag leaves. In contrast, the level of PPDK polypeptide was nearly 3-fold higher per unit leaf area in the first leaf and 63% higher in the flag leaf of this diploid species compared to this hexaploid species. There was no significant difference in the levels of polypeptide and enzyme activity of PEPC between diploid and hexaploid wheat. Despite this significantly greater level of PPDK in the diploid species, the actual amount of PPDK could still supply only a limited amount of the enzyme activity necessary to provide phosphoenolpyruvate (PEP) for any putative intracellular C(4) carbon shuttle providing carbon to RuBPC. Thus, this difference in enzyme amount could not by itself account for the reported high rates of net photosynthesis at high light intensity in T. boeoticum. Together with reported anatomical differences between the diploid and hexaploid species, however, this biochemical difference may be of physiological importance. PMID- 16665031 TI - The role of transamination in the synthesis of homoserine in peas. AB - Incubation of intact pea plants (Pisum sativum), or detached shoots, in continuous light caused a substantial increase (up to 4-fold in 2 days) in levels of homoserine. Amino acids supplied to leaves in the transpiration stream enhanced the accumulation, with glutamate, aspartate, and asparagine causing similar enhancement. Aminooxyacetate (AOA), a transamination inhibitor, at 1 millimolar prevented the accumulation. (14)C-labeling experiments showed that succinate was a good source of carbon for homoserine synthesis; carbon from aspartate or asparagine was also incorporated into homoserine. For each precursor, the transfer of label was prevented by AOA. The keto acid analog of homoserine was rapidly transaminated in leaves to give homoserine. The results suggest that accumulating homoserine is synthesised by transamination rather than being derived from aspartate via the aspartate kinase/homoserine dehydrogenase pathway. The latter pathway was shown to be operating in the chloroplasts, and was sensitive to threonine (but was not inhibited by AOA), suggesting that this path has a role in synthesis of aspartate-derived amino acids but is not involved in the accumulation of excess homoserine in the pea. PMID- 16665032 TI - The Occurrence of Abscisic Acid and Abscisyl-beta-d-Glucopyranoside in Developing and Mature Citrus Fruit as Determined by Enzyme Immunoassay. AB - The contents of (+)-cis-abscisic acid (ABA) and alkaline-hydrolyzable ABA conjugate(s) were analyzed by means of enzyme immunoassay in partially purified extracts of developing and mature sweet orange fruit (Citrus sinensis [L.] Osbeck cv Washington navel). A relatively small increase in ABA was observed in the fruit exocarp during the natural color transition from green to orange. At the same time, the ABA-conjugate level increased approximately 12-fold in this tissue. The contents of ABA and ABA-conjugate equaled 15.0 +/- 0.7 and 107.8 +/- 2.1 nanomoles per gram fresh weight, respectively, in the exocarp at harvest. Other tissues also contained considerable quantities of these compounds. Whereas the highest ABA content was observed in the exocarp, the highest ABA-conjugate content was observed in the central vascular axis of the fruit and equaled 187.0 +/- 10.3 nanomoles per gram fresh weight. The only immunoreactive conjugate found in significant quantity in mature fruit was identified as abscisyl-beta-d glucopyranoside (ABA-GE) based on (a) immunological cross-reactivity, (b) thin layer chromatography co-chromatography with authentic standards in two solvent systems, (c) susceptibility to both chemical and enzymic degradation, and (d) mass spectroscopy. PMID- 16665033 TI - Bacteroids Are Stable during Dark-Induced Senescence of Soybean Root Nodules. AB - Physiological and biochemical markers of metabolic competence were assayed in bacteroids isolated from root nodules of control, dark-stressed, and recovered plants of Glycine max Merr. cv ;Woodworth.' Nitrogenase-dependent acetylene reduction by the whole plant decreased to 8% of control rates after 4 days of dark stress and could not be detected in plants dark stressed for 8 days. However, in bacteroids isolated anaerobically, almost 50% of initial acetylene reduction activity remained after 4 days of dark stress but was totally lost after 8 days of dark stress. Bacteroid acetylene reduction activity recovered faster than whole plant acetylene reduction activity when plants were dark stressed for 8 days and returned to a normal light regimen. Significant changes were not measured in bacteroid respiration, protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, or in bacteroid proteolytic activity throughout the experiment. Immunoblots of bacteroid extracts revealed the presence of nitrogenase component II in control, 4-day dark stressed, and 8-day dark-stressed plants that were allowed to recover under a normal light regimen, but not in 8-day dark-stressed plants. Our data indicate that dark stress does not greatly affect bacteroid metabolism or induce bacteroid senescence. PMID- 16665034 TI - Pea chloroplast glutathione reductase: purification and characterization. AB - Glutathione reductase (EC 1.6.4.2) was purified from intact pea (Pisum sativum) chloroplasts by a method which includes affinity chromatography on ADP-agarose. Fractions from the affinity column which had glutathione reductase activity consisted of polypeptides of 60 and 32 kilodaltons. Separation of the proteins by electrophoresis on native gels showed that glutathione reductase activity was associated with 60 kilodalton polypeptides and not with the 32 kilodalton polypeptides. Antibodies to spinach whole leaf glutathione reductase (60 kilodaltons) cross-react with the chloroplast 60 kilodalton glutathione reductase but not the 32 kilodalton polypeptides. In the absence of dithiothreitol the 60 kilodalton polypeptides showed a shift in apparent molecular weight on sodium dodecyl sulfate gels to 72 kilodaltons. Dithiothreitol did not alter the activity of the chloroplast enzyme. Chloroplast glutathione reductase is relatively insensitive to NADPH. PMID- 16665035 TI - Spinach Leaves Desaturate Exogenous [C]Palmitate to Hexadecatrienoate : Evidence that de Novo Glycerolipid Synthesis in Chloroplasts Can Utilize Free Fatty Acids Imported from Other Cellular Compartments. AB - Long-chain (14)C-fatty acids applied to the surface of expanding spinach leaves were incorporated into all major lipid classes. When applied in diethyleneglycol monomethyl ether solution, as done by previous workers, [(14)C]palmitic acid uptake was much lower than that of [(14)C] oleic acid. However, when applied in a thin film of liquid paraffin the rate of [(14)C] palmitic acid metabolism was rapid and virtually complete. Considerable radioactivity from [(14)C]palmitate incorporated into lipids following either application method gradually appeared in polyunsaturated C(16) fatty acids esterified to those molecular species of galactolipids previously thought to be made using only fatty acids synthesized and retained within the chloroplast. Evidence for the incorporation of radioactivity from exogenous [(14)C]oleate into those same molecular species of galactolipids was less compelling. The unexpected availability of fatty acids bound to extrachloroplastidal lipids for incorporation into galactolipids characteristically assembled entirely within the chloroplast emphasizes the need to reassess interrelations between the "prokaryotic" and "eukaryotic" pathways of galactolipid formation. PMID- 16665036 TI - Carbon assimilation in carrot cells in liquid culture. AB - Assimilation of carbohydrates by carrot (Daucus carota L. cv Danvers) cells in liquid culture was studied to delineate the major metabolic pathways used in transformation of external carbohydrates to UDP-glucose. The cells grown on either sucrose or glucose for several years proved equally capable of utilizing each of these sugars. Sucrose was rapidly hydrolyzed extracellularly to glucose and fructose, and glucose was preferentially taken up. Uptake of fructose was slower and delayed until glucose was nearly depleted from the medium. Concentrations of cellular sugars, mainly glucose and sucrose, increased during late logarithmic phase of growth and decreased during the plateau phase. Continuous labeling of the cells with d-[(14)C]glucose resulted in rapid accumulation of radioactivity in glucose-6-phosphate and UDP-glucose. Because there was virtually no uptake of sucrose, UDP-glucose was likely derived from glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase and not directly from sucrose. Concentrations of major nucleotides and nucleotide sugars were maximal during the early logarithmic phase of growth and decreased several-fold in the stationary phase. A modified ;energy charge' for adenylates calculated with the omission of AMP decreased steadily from 0.9 to 0.8 during the course of culture cycle. An analogous uracil nucleotide ratio was considerably lower (0.85) during early culture, decreased to about 0.7 for the entire logarithmic phase, and returned to initial values as cells entered stationary phase. The uracil nucleotide ratio may provide a useful index to assess the coupling between the energy available in phosphoanhydride bond in adenine nucleotides and the demand for sugar for polysaccharide synthesis through uridine diphosphate-sugar pools. PMID- 16665037 TI - Biosynthesis of the fucose-containing xyloglucan nonasaccharide by pea microsomal membranes. AB - Pea microsomal membranes catalyze the transfer of [(14)C]fucose (Fuc) from GDP-[U (14)C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [(14)C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc(4) Xyl(3) Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, alpha-linked in a terminal nonreducing position. By comparison, in incubations where GDP [(14)C] fucose is absent and replaced by UDP-[(3)H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[(14)C]fucose and UDP-[(3)H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP fucose. PMID- 16665038 TI - Responses of Adaxial and Abaxial Stomata of Normally Oriented and Inverted Leaves of Vicia faba L. to Light. AB - Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This ;coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO(2). PMID- 16665039 TI - Asparagine Biosynthesis in Alfalfa (Medicago sativa L.) Root Nodules. AB - Rapid direct conversion of exogenously supplied [(14)C]aspartate to [(14)C] asparagine and to tricarboxylic cycle acids was observed in alfalfa (Medicago sativa L.) nodules. Aspartate aminotransferase activity readily converted carbon from exogenously applied [(14)C]aspartate into the tricarboxylic acid cycle with subsequent conversion to the organic acids malate, succinate, and fumarate. Aminooxyacetate, an inhibitor of aminotransferase activity, reduced the flow of carbon from [(14)C]aspartate into tricarboxylic cycle acids and decreased (14)CO(2) evolution by 99%. Concurrently, maximum conversion of aspartate to asparagine was observed in aminooxyacetate treated nodules (30 nanomoles asparagine per gram fresh weight per hour. Metabolism of [(14)C]aspartate and distribution of nodulefixed (14)CO(2) suggest that two pools of aspartate occur in alfalfa nodules: (a) one involved in asparagine biosynthesis, and (b) another supplying a malate/aspartate shuttle. Conversion of [(14)C]aspartate to [(14)C]asparagine was not inhibited by methionine sulfoximine, a glutamine synthetase inhibitor, or azaserine, a glutmate synthetase, inhibitor. The data did not indicate that asparagine biosynthesis in alfalfa nodules has an absolute requirement for glutamine. Radioactivity in the xylem sap, derived from nodule (14)CO(2) fixation, was markedly decreased by treating nodulated roots with aminooxyacetate, methionine sulfoximine, and azaserine. Inhibitors decreased the [(14)C]aspartate and [(14)]asparagine content of xylem sap by greater than 80% and reduced the total amino nitrogen content of xylem sap (including nonradiolabeled amino acids) by 50 to 80%. Asparagine biosynthesis in alfalfa nodules and transport in xylem sap are dependent upon continued aminotransferase activity and an uninterrupted assimilation of ammonia via the glutamine synthetase/glutamate synthase pathway. Continued assimilation of ammonia apparently appears crucial to continued root nodule CO(2) fixation in alfalfa. PMID- 16665040 TI - Regulation of Plasma Membrane beta-Glucan Synthase from Red Beet Root by Phospholipids : Reactivation of Triton X-100 Extracted Glucan Synthase by Phospholipids. AB - Extraction of red beet root plasma membranes with the detergent Triton X-100 at a level of 2.0% (weight/volume) resulted in the depletion of over 90% of total membrane phospholipid and the reduction of glucan synthase activity by 80 to 90%. Reconstitution of the delipidated Triton X-100, 100,000g fraction in the presence of phospholipids restored glucan synthase activity. The most effective phospholipid was phosphatidyl-ethanolamine, which restored 110 to 144% of the original activity at 0.5% (weight/volume). Glucan synthase in the phospholipid reactivated Triton X-100-treated fraction was enriched 9-fold in specific activity relative to microsomal membranes but was unstable in digitonin. These results support the hypothesis that glucan synthase activity is regulated by its phospholipid environment. PMID- 16665041 TI - Joint Action of O(3) and SO(2) in Modifying Plant Gas Exchange. AB - The joint action of O(3) and SO(2) stress on plants was investigated by determining the quantitative relationship between air pollutant fluxes and effects on stomatal conductance. Gas exchange measurements of O(3), SO(2), and H(2)O vapor were made for Pisum sativum L. (garden pea). Plants were grown under controlled environments, and O(3), SO(2), and H(2)O vapor fluxes were evaluated with a whole-plant gas exchange chamber using the mass-balance approach. Maximum O(3) and SO(2) fluxes per unit area (2 sided) into leaves averaged 8 nanomoles per square meter per second with exposure to either O(3) or SO(2) at 0.1 microliters per liter. Internal fluxes of either O(3) or SO(2) were reduced by up to 50% during exposure to combined versus individual pollutants; the greatest reduction occurred with simultaneous versus sequential combinations of the pollutants. Stomatal conductance to H(2)O was substantially altered by the pollutant exposures, with O(3) molecules twice as effective as SO(2) molecules in inducing stomatal closure. Stomatal conductance was related to the integrated dose of pollutants. The regression equations relating integrated dose to stomatal conductance were similar with O(3) alone, O(3) plus added SO(2), and O(3) plus SO(2) simultaneously; i.e. a dose of 100 micromoles per square meter produced a 39 to 45% reduction in conductance over nonexposed plants. With SO(2) alone, or SO(2) plus added O(3), a dose of 100 micromoles per square meter produced a 20 to 25% reduction in conductance. When O(3) was present at the start of the exposure, then stomatal response resembled that for O(3) more than the response for SO(2). This study indicated that stomatal responses with combinations of O(3) and SO(2) are not dependent solely on the integrated dose of pollutants, but suggests that a metabolic synergistic effect exists. PMID- 16665042 TI - The Role of Glycerol and Inorganic Ions in Osmoregulatory Responses of the Euryhaline Flagellate Chlamydomonas pulsatilla Wollenweber. AB - The green euryhaline flagellate Chlamydomonas pulsatilla Wollenweber, isolated from a coastal marine environment, was grown exponentially over the salinity range of 10 to 200% artificial seawater (ASW). The cellular volume and aqueous space of the alga, measured by [(14)C] mannitol and (3)H(2)O tracer analyses of centrifuged cell pellets, ranged between 2.3 and 3.1 picoliters and between 1.5 and 2.1 picoliters, respectively. The nonaqueous space determined in those analyses (28-35%) was consistent with the cell composition of the alga. The glycerol content of the alga increased almost linearly with increasing salinity; its contribution to intracellular osmolality at 200% ASW was about 57%. The contribution of amino acids and soluble carbohydrates to the cell osmotic balance was small. Intracellular ion concentrations determined by analyzing centrifuged cell pellets of known [(14)C]mannitol space by atomic absorption spectrophotometry, and by neutron activation analyses of washed cells were similar. At 10% ASW, potassium and magnesium were the major cations, and chloride and phosphate were the major anions. The sodium and chloride content of the alga increased with increasing salinity; at 200% ASW the intracellular concentration of both sodium and chloride was about 400 millimolar. The intracellular osmolality (pi(int)) matched closely the external osmolality (pi(ext)) over the entire salinity range except at 10% ASW where pi(int) exceeded pi(ext) by 120 to 270 milliosmoles per kilogram H(2)O. PMID- 16665043 TI - Identification of granule-bound starch synthase in potato tubers. AB - Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase. PMID- 16665044 TI - Simultaneous Measurements of Cytoplasmic K Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina. AB - The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K(+)-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (K(o) (+)), the resting conductance (membrane conductance G(m), 0.85 +/- 0.25 Siemens per square meter (+/-standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K(+) activities (a(K+)), judged most reliable, gave values of 117 +/- 67 millimolar; stable a(K+) values were 77 +/- 31 millimolar. Equilibrium potentials for K(+) (Nernst equilibrium potential) (E(K)) calculated, using either of these data sets, were near the mean membrane potential (V(m)). On a cell-to-cell basis, however, E(K) was generally negative of the V(m), despite an electrogenic contribution from the Chara proton pump. When K(o) (+) was increased to 1.0 millimolar or above, G(m) rose (by 8- to 10 fold in 10 millimolar K(o) (+)), the steady state I-V characteristics showed a region of negative slope conductance, and V(m) followed E(K). These results confirm previous studies which implicated a K(o) (+)-induced and voltage dependent permeability to K(+) at the Chara plasma membrane. They provide an explanation for transitions between apparent K(o) (+)-insensitive and K(o) (+) sensitive (;K(+) electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells. PMID- 16665045 TI - Oxygen Isotope Exchange between Metabolites and Water during Biochemical Reactions Leading to Cellulose Synthesis. AB - Cellulose was produced heterotrophically from different carbon substrates by carrot tissue cultures and Acetobacter xylinum (a cellulose-producing bacterium) and by castor bean seeds germinated in the dark, in each case in the presence of water having known concentration of oxygen-18 ((18)O). We used the relationship between the amount of (18)O in the water and in the cellulose that was synthesized to determine the number and (18)O content of the substrate oxygens that exchanged with water during the reactions leading to cellulose synthesis. Our observations support the hypothesis that oxygen isotope ratios of plant cellulose are determined by isotopic exchange occurring during hydration of carbonyl groups of the intermediates of cellulose synthesis. PMID- 16665046 TI - Oxygen and Hydrogen Isotope Ratios of Water from Photosynthetic Tissues of CAM and C(3) Plants. AB - Water samples from photosynthetic tissues of C(3) and Crassulacean acid metabolism (CAM) plants that grew together in the field were extracted and the stable oxygen and hydrogen isotope ratios determined. During the day, (18)O/(16)O and deuterium/hydrogen (D/H) ratios of water from CAM plants were lower than those observed in water from C(3) plants. The patterns of diurnal variation (or lack thereof) in isotope ratios of plant water are consistent with the gross anatomical and physiological characteristics of the plants studied here. Our observations support the previously advanced hypothesis that high D/H ratios in cellulose nitrate prepared from CAM plants relative to those for C(3) plants are not caused by greater deuterium enrichment in the water in CAM plants, but rather by isotopic fractionations associated with different biochemical reactions in the two types of plants. PMID- 16665047 TI - Symplastic Transport in Ipomea tricolor Source Leaves : Demonstration of Functional Symplastic Connections from Mesophyll to Minor Veins by a Novel Dye Tracer Method. AB - A novel method for the delivery of the fluorescent dye Lucifer Yellow CH to the cytosol of a source leaf mesophyll cell was devised which utilized a preencapsulation of the dye in phospholipid vesicles (liposomes). The liposomes were easily injected into the vacuoles of leaf cells of Beta vulgaris or Ipomea tricolor, where fusion with the tonoplast resulted in the release of the dye into the cytosol. Subsequent cell-to-cell movement of the dye was readily followed by fluorescence microscopy. Using this liposome technique symplastic continuity from the the mesophyll to the minor veins of the source leaf of Ipomea tricolor was demonstrated. This agreed with ultrastructural studies which demonstrated the presence of plasmodesmata between all cells from the mesophyll to the minor veins. The symplastic movement of dye from the injected mesophyll cell to the minor veins was unaffected by pretreatment of the leaf tissues with 2 millimolar p-chloromercuribenzenesulfonic acid. Pretreatment of the leaf tissues at alkaline pH (3-[N-morpholino] propanesulfonic acid-KOH, pH 8.0) had no apparent effect on dye movement between adjacent mesophyll cells but inhibited the movement of dye into and along the minor veins. Thus, although there were no apparent barriers to symplastic solute movement in this leaf, symplastic barriers could be imposed by the experimental conditions used. PMID- 16665048 TI - Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages. AB - Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv ;Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O(2) uptake by mature leaves measured at 25 degrees C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O(2) uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O(2) uptake declined consistently with leaf age. The stimulation of O(2) uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O(2) uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O(2) uptake by KCN and NaN(3) in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O(2) uptake by SHAM was not expected, and the reason for it is not clear. PMID- 16665049 TI - Activity of acyl carrier protein isoforms in reactions of plant Fatty Acid metabolism. AB - Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K(m) of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K(m) of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described. PMID- 16665050 TI - Accumulation of C-radiolabel in leaves and fruits after injection of [C]tryptophan into seeds of soybean. AB - Injection of [(14)C]tryptophan into one seed in a soybean fruit resulted in recovery of radiolabel in a fraction that cochromatographed with indoleacetic acid (IAA) through three successive high performance liquid chromatography separations. Label was found in the putative IAA fraction in all of the fruit tissues sampled and in the blade of the leaf subtending the pod into which the radiolabeled tryptophan had been injected. This suggested that IAA or an IAA precursor was transported from seeds to other parts of the fruit and to subtending leaves. PMID- 16665051 TI - Seasonal Changes in the Kinetic Parameters of a Photosynthetic Fructose-1,6 Bisphosphatase Isolated from Peltigera rufescens. AB - The kinetic parameters of the photosynthetic fructose-1,6-bisphosphatase isolated from Peltigera rufescens (Weis) Mudd. were measured on a seasonal basis and during a laboratory-induced temperature acclimation. Both the substrate affinity and Ea changed on a seasonal basis. During the summer, the Ea decreased from 91.8 to 62.3 kilojoules per mole. The K(m) fructose-1,6-bisphosphate measured at temperatures above 25 degrees C was also found to decrease by 50%. This seasonal change in K(m) can be induced by growing the lichen under appropriate conditions for 2 weeks, and is correlated to a change in the net photosynthetic rates. It is hypothesized that this change in fructose-1,6-bisphosphatase is related to the seasonal temperature acclimation process that has been previously reported in this species. PMID- 16665052 TI - Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6 Bisphosphatase from the Lichen Peltigera rufescens. AB - Two enzymes capable of hydrolyzing fructose-1,6-bisphosphate (FBP) have been isolated from the foliose lichen Peltigera rufescens (Weis) Mudd. These enzymes can be separated using Sephadex G-100 and DEAE Sephacel chromatography. One enzyme has a pH optimum of 6.5, and a substrate affinity of 228 micromolar FBP. This enzyme does not require MgCl(2) for activity, and is inhibited by AMP. The second enzyme has a pH optimum of 9.0, with no activity below pH 7.5. This enzyme responds sigmoidally to Mg(2+), with half-saturation concentration of 2.0 millimolar MgCl(2), and demonstrates hyperbolic kinetics for FBP (K(m) = 39 micromolar). This enzyme is activated by 20 millimolar dithiothreitol, is inhibited by AMP, but is not affected by fructose-2-6-bisphosphate. It is hypothesized that the latter enzyme is involved in the photosynthetic process, while the former enzyme is a nonspecific acid phosphatase. PMID- 16665053 TI - Glyphosate inhibits photosynthesis and allocation of carbon to starch in sugar beet leaves. AB - Application of glyphosate (N-[phosphonomethyl] glycine) to exporting leaves of sugar beet (Beta vulgaris, L.) during the day lowered stomatal conductance and carbon fixation. Allocation of newly fixed carbon to foliar starch accumulation was nearly completely inhibited, being decreased by the same amount as net carbon fixation. In contrast, decreasing net carbon fixation in untreated leaves by lowering CO(2) concentration caused starch accumulation to decrease, but only in the same proportion as net carbon fixation. Shikimate level increased 50-fold in treated leaves but the elevated rate of carbon accumulation in shikimate was only 4% of the decrease in the rate of starch accumulation. Application of steady state labeling with (14)CO(2) to exporting leaves confirmed the above changes in carbon metabolism, but revealed no other major daytime differences in the (14)C content of amino acids or other compounds between treated and control leaves. Less (14)C accumulated in treated leaves because of decreased fixation, not increased export. The proportion of newly fixed carbon allocated to sucrose increased, maintaining export at the level in control leaves. Returning net carbon exchange to the rate before treatment restored starch accumulation fully and prevented a decrease in export during the subsequent dark period. PMID- 16665054 TI - NADH Nitrate Reductase and NAD(P)H Nitrate Reductase in Genetic Variants and Regenerating Callus of Maize. AB - Different organs of maize seedlings are known to contain different complements of NADH and NAD(P)H nitrate reductase (NR) activity. The study of the genetic programming that gives rise to such differences can be initiated by looking for genetic variants exhibiting different patterns of distribution of the above enzymes. We demonstrate in this work that scutella of very young maize seedlings contain NADH NR almost exclusively and that this activity is gradually replaced, as the seedling ages, with NAD(P)H NR. Leaves in the seedlings contain exclusively the NADH NR activity. A genetic variant is described that contains much reduced levels of NAD(P)H NR activity but not of NADH NR activity in the scutellum. This same variant exhibits a relatively low level of NAD(P)H NR but normal NADH NR activity in seedling root tips. These observations suggest that the genetic program used to specify the scutellar complement of NR activity shares some common components with the genetic program used to determine the young root tip complement of NR activities. Parts of regenerating callus at different stages of differentiation were examined to determine when the differences in NR complement begin to appear. The same pattern of NADH NR and NAD(P)H NR activities was found in unorganized as well as in organized callus, in recognizable root-like and even in green shoot-like material, both activities being present in all these tissues. An examination of the NR complement in different organs of a number of siblings originating from a cross involving transposon Mu-containing parents and having different levels of leaf NADH NR activity shows that the leaf NADH NR activity content and the scutellum NAD(P)H NR activity content are relatively independent of each other, indicating that the genetic programs specifying the NR content of these organs are not tightly coupled, if at all. PMID- 16665055 TI - The Biosynthesis of a Novel Nicotine Alkaloid in the Trichomes of Nicotiana stocktonii. AB - N-Hydroxyacylnornicotine, newly discovered from fresh plant tissue, was found entirely in the trichome exudate produced at the epidermis of the aerial part of Nicotiana stocktonii. Nicotine and nornicotine, but not N-hydroxyacylnornicotine, were present inside of the trichomes as well as other internal parts of the plant. Only nicotine was found in bleeding sap squeezed from cut roots or stems. Feeding of leaves with 2'-(14)C-labeled nicotine primarily yielded labeled nicotine, nornicotine, and N-hydroxyacylnornicotine. When similarly labeled nornicotine was fed to leaves as a precursor, a labeled N-hydroxyacylnornicotine was obtained, with a higher specific activity than with the [2'-(14)C]nicotine feeding. Based on these results, a synthesis route is suggested where nicotine is converted in the leaf to nornicotine, followed by trichome conversion of nornicotine to N-hydroxyacylnornicotine, and rapid secretion of this product. PMID- 16665056 TI - Effects of Arsenite, Sulfite, and Sulfate on Photosynthetic Carbon Metabolism in Isolated Pea (Pisum sativum L., cv Little Marvel) Chloroplasts. AB - Photosynthetic CO(2)-fixation in isolated pea (Pisum sativum L., cv Little Marvel) chloroplasts during induction is markedly inhibited by 0.4 millimolar sulfite. Sulfate at the same concentration has almost no effect. The (14)CO(2) fixation pattern indicates that the primary effect of sulfite is inhibition of the reaction catalyzed by ribulose bisphosphate carboxylase and a stimulation of export of intermediates out of the chloroplasts. Inhibition of light modulation of stromal enzyme activity does not appear to account for the toxicity of SO(2) in this Pisum variety. Arsenite at 0.2 millimolar concentrations inhibits light activation and inhibits photosynthetic CO(2) fixation. The (14)CO(2)-fixation pattern indicates that the primary effect of arsenite is inhibition of light activation of reductive pentose phosphate pathway enzyme activity. PMID- 16665057 TI - Sodium stimulation of uptake hydrogenase activity in symbiotic Rhizobium. AB - Initial observations showed a 100% increase in H(2)-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H(2) evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na(+) (29 versus 9 milligrams per kilogram) and less K(+) (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na(+) concentration of clay-grown plants with a much smaller reduction in K(+) concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na(+) strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na(+) concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na(+) increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.). PMID- 16665058 TI - Localization and Quantitative Determination of Ferredoxin-NADP Oxidoreductase, a Thylakoid-Bound Enzyme in the Cyanobacterium Anabaena sp. Strain 7119. AB - Thylakoid membrane preparations obtained from mechanically disrupted (sonicated) cells of the cyanobacterium Anabaena sp. strain 7119 show a membrane-bound ferredoxin-NADP(+) oxidoreductase (EC 1.18.1.2) as determined either by specific antibodies or by using the ferredoxin-dependent NADPH-cytochrome c reductase activity, which is a specific test for this enzyme. However, in contrast with higher plant thylakoids, a low yield of the cyanobacterial reductase-only about 20% of the total amount of this protein estimated in whole cell homogenates-was obtained as a membrane-bound form when Mg(2+) was present during the disruption treatment. It is noteworthy that the addition of water-soluble nonionic polymers, namely polyethylene glycol and polyyinylpyrrolidone, dramatically increased the yield of the thylakoid-bound reductase, reaching values up to 80 to 85% of the total enzyme. Using these thylakoid membrane preparations, a quantitative determination of the reductase has been performed for the first time for cyanobacterial thylakoids. The value determined by immunoelectrophoresis-from 8 to 10 nanomoles per micromole of chlorophyll-is clearly higher than those reported for chloroplast thylakoids. PMID- 16665059 TI - Enzymes of Glucose Oxidation in Leaf Tissues : The Distribution of the Enzymes of Glycolysis and the Oxidative Pentose Phosphate Pathway between Epidermal and Mesophyll Tissues of C(3)-Plants and Epidermal, Mesophyll, and Bundle Sheath Tissues of C(4)-Plants. AB - The distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C(3)-plants, pea and leek, and two C(4)-plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue. PMID- 16665060 TI - Function of Rhizodermal Transfer Cells in the Fe Stress Response Mechanism of Capsicum annuum L. AB - A variety of red pepper (Capsicum annuum L., cv Yaglik) responds to Fe deficiency stress with simultaneously enhanced H(+) extrusion, reduction of ferric ions and synthesis of malic and citric acid in a swollen subapical root zone densely covered with root hairs. It is demonstrated that these stress responses temporally coincide with the development of rhizodermal and hypodermal transfer cells in this root zone. During stress response the transfer cells show a marked autofluorescence which could arise from endogenous iron chelators of the phenolic acid type. The presence of organelle-rich cytoplasm which often exhibits rotational cytoplasmic streaming points to high physiological activity and makes these cells, with their increased plasmalemma surface, particularly well suited for the entire stress response mechanism. Since Fe stress-induced acidification is diminished by vanadate and erythrosin B, both specific inhibitors of plasmalemma ATPases, it seems reasonable to suppose that H(+) pumping from transfer cells is activated by an ATPase located in their plasmamembrane. H(+) extrusion is also shown to be inhibited by abscisic acid. Raised phosphoenolpyruvate carboxylase activity and simultaneous accumulation of malate in the swollen root zone point to the action of a pH stat preventing a detrimental rise in cytoplasmic pH of transfer cells during enhanced H(+) extrusion. The simultaneous increase in citric acid concentration favors chelation of iron at the site of its uptake and thus ensures long distance transport to the areas of metabolic demand. A direct link between citrate accumulation and ferric ion reduction as proposed in recent literature further supports the crucial role of transfer cells in the response to Fe deficiency stress. PMID- 16665061 TI - Cooperative Effects of Light and Temperature on the Activity of Phosphoenolpyruvate Carboxylase from Amaranthus paniculatus L. AB - The phosphoenolpyruvate carboxylase of Amaranthus paniculatus shows in vitro optimum affinity (S(0.5)) to phosphoenolpyruvate at a relatively high temperature (about 35 degrees C); even in the presence of activators, it functions efficiently only above 25 to 27 degrees C. At lower temperatures, a steep increase of activity with temperature is observed, due to the high activation energy for the catalyzed reaction. The same behavior in vivo could amplify the photoactivation of the enzyme to a large extent, since the night/day transition is soon followed by a considerable rise in leaf temperature. PMID- 16665062 TI - 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Ochromonas malhamensis: A System to Study the Relationship between Enzyme Activity and Rate of Steroid Biosynthesis. AB - 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme of the isoprenoid pathway, was found to be predominantly microsomal in Ochromonas malhamensis, a chrysophytic alga. Detection of HMG-CoA reductase requires the presence of 1% bovine serum albumin during cell homogenization, and the activity is stimulated by the presence of Triton X-100. The enzyme has a pH optimum of 8.0 and an absolute requirement for NADPH. When grown in 10 micromolar mevinolin, a competitive inhibitor of HMG-CoA reductase, O. malhamensis shows a 10- to 15-fold increase in HMG-CoA reductase activity (after washing) with little or no effect on cell growth rate. Cultures can be maintained in 10 micromolar mevinolin for months. O. malhamensis produces a large amount (1% dry weight) of poriferasterol, a product of the isoprenoid pathway. The addition of 10 micromolar mevinolin initially blocked poriferasterol biosynthesis by >90%; within 2 days the rate of synthesis returned to normal levels. Immediately after mevinolin was washed from the 2-day culture, there was a transient 2.5-fold increase in the rate of poriferasterol biosynthesis. The rate of poriferasterol biosynthesis and the level of HMG-CoA reductase activity both fell to control levels within hours. PMID- 16665063 TI - Photosynthesis and Growth of Water Hyacinth under CO(2) Enrichment. AB - Water hyacinth (Eichhornia crassipes [Mart.] Solms) plants were grown in environmental chambers at ambient and enriched CO(2) levels (330 and 600 microliters CO(2) per liter). Daughter plants (ramets) produced in the enriched CO(2) gained 39% greater dry weight than those at ambient CO(2), but the original mother plants did not. The CO(2) enrichment increased the number of leaves per ramet and leaf area index, but did not significantly increase leaf size or the number of ramets formed. Flower production was increased 147%. The elevated CO(2) increased the net photosynthetic rate of the mother plants by 40%, but this was not maintained as the plants acclimated to the higher CO(2) level. After 14 days at the elevated CO(2), leaf resistance increased and transpiration decreased, especially from the adaxial leaf surface. After 4 weeks in elevated as compared to ambient CO(2), ribulose bisphosphate carboxylase activity was 40% less, soluble protein content 49% less, and chlorophyll content 26% less; whereas starch content was 40% greater. Although at a given CO(2) level the enriched CO(2) plants had only half the net photosynthetic rate of their counterparts grown at ambient CO(2), they showed similar internal CO(2) concentrations. This suggested that the decreased supply of CO(2) to the mesophyll, as a result of the increased stomatal resistance, was counterbalanced by a decreased utilization of CO(2). Photorespiration and dark respiration were lower, such that the CO(2) compensation point was not altered. The photosynthetic light and CO(2) saturation points were not greatly changed, nor was the O(2) inhibition of photosynthesis (measured at 330 microliters CO(2) per liter). It appears that with CO(2) enrichment the temporary increase in net photosynthesis produced larger ramets. After acclimation, the greater total ramet leaf area more than compensated for the lower net photosynthetic rate on a unit leaf area basis, and resulted in a sustained improvement in dry weight gain. PMID- 16665064 TI - Regulation of Gene Expression in Developing Zea mays Embryos: Protein Synthesis during Embryogenesis and Early Germination of Maize. AB - Polypeptides synthesized in dissected embryos of Zea mays at different stages of embryogenesis and early germination have been characterized by their migration in two-dimensional gel electrophoresis. This analysis has been carried out with in vivo labeled polypeptides from excised embryos and with proteins synthesized in vitro in the rabbit reticulocyte system directed by poly(A(+)) RNA isolated from the different developmental stages. We have identified three main sets of expressed polypeptides: (a) embryonic set: this group of polypeptides is synthesized in young and mature embryos but not in early germination; (b) maturation set: this group of polypeptides is not present in young embryos and appears during the maturation period. Some of these polypeptides are still present in early germination while others disappear from stored mRNAs in dry embryos. One particular group from this set can be induced prematurely in young embryos by incubation with abscisic acid; and (c) germination set: this group of polypeptides is not expressed in the maturation period and appears after brief imbibition of the dry embryos. PMID- 16665065 TI - Light modulation of maize leaf phosphoenolpyruvate carboxylase. AB - Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH(4))(2)SO(4) fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition. PMID- 16665066 TI - Salinity and Nitrogen Effects on Photosynthesis, Ribulose-1,5-Bisphosphate Carboxylase and Metabolite Pool Sizes in Phaseolus vulgaris L. AB - Salinity (100 millimolar NaCl) was found to reduce photosynthetic capacity independent of stomatal closure in Phaseolus vulgaris. This reduction was shown to be a consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) rather than a reduction in the leaf content of photosynthetic machinery. In control plants, photosynthesis became RuBP-limited at approximately 1.75 moles RuBP per mole 2-carboxyarabinitol bisphosphate binding sites. Salinization caused the RuBP pool size to reach this limiting value for CO(2) fixation at much lower values of intercellular CO(2). Plants grown at low nitrogen and +/- NaCl became RuBP limited at similar RuBP pool sizes as the high nitrogen-grown plants. At limiting RuBP pool sizes and equal values of intercellular CO(2) photosynthetic capacity of salt-stressed plants was less than control plants. This effect of salinity on RuBPCase activity could not be explained by deactivation of the enzyme or inhibitor synthesis. Thus, salinity reduced photosynthetic capacity by reducing both the RuBP pool size by an effect on RuBP regeneration capacity and RuBPCase activity by an unknown mechanism when RuBP was limiting. PMID- 16665067 TI - K-Nutrition, Growth Bud Formation, and Amine and Hydroxycinnamic Acid Amide Contents in Leaf Explants of Nicotiana tabacum Variety Xanthi n.c. Cultivated in Vitro. AB - The effects of K-nutrition on growth (increase of fresh weight), bud formation (time of emergence, number of buds), and amine and hydroxycinnamic acid amide contents in foliar explants of Nicotiana tabacum cv Xanthi n.c. cultivated in vitro were examined. In K-deficient medium and in high K medium growth and bud formation were markedly inhibited. Marked changes of amine content (a diamine, putrescine; a phenolic amine, phenethylamine) were observed after a few days of culture. No apparent relationship was found between these amines and growth or bud differentiation. In contrast, changes in hydroxycinnamic acid levels were shown to correlate well with growth and bud formation. The greatest stimulation of budding and growth was correlated with the greatest accumulation of these amides. The highest contents of hydroxycinnamic acid amides were found during the first 15 days in culture when intensive cell division took place. Then they declined sharply after 26 days in culture as the rate of cell division decreased and differentiation occurred. PMID- 16665068 TI - Ca-stimulated secretion of alpha-amylase during development in barley aleurone protoplasts. AB - The effects of gibberellic acid (GA(3)) and Ca(2+) on the synthesis and secretion of alpha-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA(3), CaCl(2), or both. Incubation of protoplasts in medium containing both GA(3) and Ca(2+), however, causes an increase in the alpha-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA(3) or Ca(2+) alone. Isoelectric focusing shows that adding Ca(2+) to incubation media containing GA(3) increases the levels of alpha-amylase isozymes having high isoelectric points (pI). In the presence of GA(3) alone, only isozymes with low pIs accumulate. The increase in alpha-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca(2+) to maintain elevated levels of alpha-amylase release. Immunoelectrophoresis shows that Ca(2+) stimulates the release of low-pI alpha-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA(3) alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl(2). The response is not specific for Ca(2+) since Sr(2+) can substitute, although less effectively than Ca(2+). Pulse-labeling experiments show that alpha-amylase isozymes produced by aleurone protoplasts in response to GA(3) and Ca(2+) are newly synthesized. The effects of Ca(2+) on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on alpha-amylase stability or on protoplast viability. We conclude that Ca(2+) directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca(2+)-stimulated enzyme release. PMID- 16665069 TI - Genetic Regulation of Development in Sorghum bicolar: I. Role of the Maturity Genes. AB - The photoperiodic behavior and other developmental and morphological differences of 11 maturity genotypes (as identified by JR Quinby 1967, Adv Agron 19: 267-305) of the milo group of Sorghum bicolor (L.) Moench were studied under 8, 10, 12, and 14 hour photoperiods. Sorghum is a quantitative short day plant. The genotypes studied differ in genes which modify photoperiodic behavior and thus maturity; the alleles are designated as Ma(1), ma(1), Ma(2), ma(2), Ma(3), ma(3), and ma(3) (R) (single symbols indicate homozygosity at the indicated gene loci). Based on floral initiation (differentiation) under 10, 12, and 14 hour photoperiods the 11 genotypes were assigned to three clases: (I) flower initiation delayed by 12 hour photoperiods (all genotypes with Ma(1)Ma(2) but not ma(3) (R)), (II) flower initiation delayed by 14 hour photoperiods (all genotypes with Ma(1)ma(2), ma(1)Ma(2), or ma(1)ma(2) but not ma(3) (R)), (III) flower initiation not drastically delayed by 14 hour photoperiods (all genotypes with ma(3) (R)). All of the class III genotypes were taller, had longer leaf sheaths, narrower and longer leaf blades, and less leaf area, than the other genotypes. In addition, the class III genotypes initiated rapid culm and thus internode elongation sooner after floral initiation than any of the class I or II genotypes. Dry weight did not differ between the class III genotypes and the others. The rate of leaf emergence in the class III genotypes and all others was indistinguishable until after floral initiation in the former. The allelic combination unique to class I, Ma(1)Ma(2), makes plants very photoperiod sensitive without causing observable changes in morphology or other development events. The allelic combination unique to class III, ma(3) (R), makes plants relatively photoperiod insensitive and results in several differences in morphology and development. PMID- 16665070 TI - Genetic Regulation of Development in Sorghum bicolor: II. Effect of the ma(3) Allele Mimicked by GA(3). AB - The class III, ma(3) (R)-containing maturity genotypes of Sorghum bicolor (L.) Moench differ from all of the other maturity genotypes in that they flower earlier, exhibit rapid shoot elongation sooner after floral differentiation, and exhibit several morphological differences. In the present study, a class III, ma(3) (R)-containing genotype (44M) was compared to two other genotypes (60M, 80M) that differed only at the third maturity locus. GA(3) treatment caused 60M and 80M to undergo floral differentiation earlier, to exhibit an increase in shoot elongation rate sooner after floral initiation, and to exhibit morphological changes including: increased leaf blade and leaf sheath length, decreased leaf width and leaf area, as well as increased dry weight, especially in the culm weight. Since every major morphological character which is different in class III genotypes was changed by GA(3) treatment of the class II genotypes (60M, 80M), thereby causing them to be more like the class III genotypes, it is clear that application of GA(3) makes maturity genotypes with Ma(3) or ma(3) behave as if they contained ma(3) (R). All of these results support the hypothesis that the ma(3) (R) allele increases gibberellin activity in the maturity genotypes with that allele. PMID- 16665071 TI - Immunochemical comparison of glutamine synthetases from some solanaceae plants. AB - The presence of different glutamine synthetase isoenzymes in different Solanaceae plants and their relative antigenicities against antiglutamine synthetase from tomato leaf serum were studied. All the plants tested showed one glutamine synthetase isoenzyme except for Mandragora autumnalis, which showed two, after discontinuous polyacrylamide gel electrophoresis and specific in situ assay. Antigenicities were compared by the double immunodiffusion technique. The Nicotiana glauca enzyme showed equal reactivity to that of Lycopersicon esculentum, but its antigenicity was higher than Withania frutescens, Datura stramonium, and Hyoscyamus niger. The study of relative antigenicities permitted differentiation of the glutamine synthetase enzymes from uncultivated species of Solanaceae. PMID- 16665072 TI - Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors. AB - The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype. PMID- 16665073 TI - Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre Steady-State Study. AB - Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-alpha-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 +/- 0.5 degrees C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k(cat) and k(cat)/K(m) reflect the ionization of two groups with pK(a) values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K(m) are pH independent. Among the substrates examined, N-alpha-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 x 10(-10) molar at the optimum pH value (approximately 7.5). PMID- 16665074 TI - Effect of Osmotic Stress on Carbon Metabolism in Chlamydomonas reinhardtii: Accumulation of Glycerol as an Osmoregulatory Solute. AB - NaCl, KCl, and sucrose at equiosmolar concentrations had similar inhibitory effects on photosynthetic carbon metabolism by the freshwater green alga, Chlamydomonas reinhardtii. Inhibitory concentrations of these solutes altered the products of photosynthetic (14)CO(2) incorporation, resulting in reduced incorporation into starch, sugar phosphates, lactate, and glycolate, but caused an accumulation of glycerol both intracellularly and in the medium. PMID- 16665075 TI - Detection of Xylem Cavitation in Corn under Field Conditions. AB - We report the detection of cavitation events in corn (Zea mays) plants growing under field conditions in Greeley, CO. To our knowledge this study reports the first successful attempt to monitor continuously for long periods the cavitation events of a crop plant using acoustic detection techniques. Cavitation events occur in corn plants using acoustic detection techniques. Cavitation events occur in corn plants irrigated daily when the xylem pressure potentials fall below about -1.0 megapascals. In unirrigated corn we estimate that approximately half of all vessels cavitate on any one day when xylem pressure potentials fall below about -1.8 megapascals. We postulate that root pressure developed every night in irrigated and unirrigated corn is adequate to rejoin cavitated water columns. PMID- 16665076 TI - A highly sensitive method for quantitative determination of abscisic Acid. AB - An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry. PMID- 16665077 TI - Short-Term and Long-Term Responses of Crassulacean Acid Metabolism Plants to Elevated CO(2). AB - For the leaf succulent Agave deserti and the stem succulent Ferocactus acanthodes, increasing the ambient CO(2) level from 350 microliters per liter to 650 microliters per liter immediately increased daytime net CO(2) uptake about 30% while leaving nighttime net CO(2) uptake of these Crassulacean acid metabolism (CAM) plants approximately unchanged. A similar enhancement of about 30% was found in dry weight gain over 1 year when the plants were grown at 650 microliters CO(2) per liter compared with 350 microliters per liter. Based on these results plus those at 500 microliters per liter, net CO(2) uptake over 24 hour periods and dry weight productivity of these two CAM succulents is predicted to increase an average of about 1% for each 10 microliters per liter rise in ambient CO(2) level up to 650 microliters per liter. PMID- 16665078 TI - Inhibition by ethylene of polyamine biosynthetic enzymes enhanced lysine decarboxylase activity and cadaverine accumulation in pea seedlings. AB - Exposing etiolated pea seedlings to ethylene which inhibited the activity of arginine decarboxylase and S-adenosylmethionine decarboxylase caused an increase in the level of cadaverine. The elevated level of cadaverine resulted from an increase in lysine decarboxylase activity in the tissue exposed to ethylene. The hormone did not affect the apparent K(m) of the enzyme, but the apparent V(max) was increased by 96%. While lysine decarboxylase activity in the ethylene-treated plants increased in both the meristematic and the elongation zone tissue, cadaverine accumulation was observed in the latter only. The enhancement by ethylene of the enzyme activity was reversed completely 24 hours after transferring the plants to an ethylene-free atmosphere. It is postulated that the increase in lysine decarboxylase activity, and the consequent accumulation of cadaverine in ethylene-treated plants, is of a compensatory nature as a response to the inhibition of arginine and S-adenosylmethionine decarboxylase activity provoked by ethylene. PMID- 16665079 TI - High CO(2) Requiring Mutant of Anacystis nidulans R(2). AB - Some physiological characteristics of a mutant (E(1)) of Anacystis nidulans R(2), incapable of growing at air level of CO(2), are described. E(1) is capable of accumulating inorganic carbon (C(i)) internally as efficiently as the wild type (R(2)). The apparent photosynthetic affinity for C(i) in E(1), however, is some 1000 times lower than that of R(2). The kinetic parameters of ribulose 1,5 bisphosphate carboxylase/oxygenase from E(1) are similar to those observed in R(2). The mutant appears to be defective in its ability to utilize the intracellular C(i) pool for photosynthesis and depends on extracellular supply of Ci in the form of CO(2). The very high apparent photosynthetic K(m) (CO(2)) of the mutant indicate a large diffusion resistance for CO(2). Data obtained here are used to calculate the permeability coefficient for CO(2) between the bulk medium and the carboxylation site of cyanobacteria. PMID- 16665080 TI - Cell-specific photosynthetic gene expression in maize determined using cell separation techniques and hybridization in situ. AB - Bundle sheath strands and mesophyll cell extracts have been isolated from maize (Zea mays L.) leaves using a mechanical disruption-filtration technique. Northern blot analysis showed that phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) mRNA accumulates only in mesophyll cells. The mechanisms regulating the cell specific expression of this gene must, therefore, be at either the level of RNA transcription or that of mRNA turnover. The first successful application of hybridization to mRNA molecules in photosynthetic plant tissue sections is described. Results obtained from this in situ study corroborate our finding that PEPCase mRNA accumulates only in mesophyll cells as well as the previously reported (Link, G, DM Coen, L Bogorad 1978 Cell 15: 725-731) finding that the accumulation of ribulose 1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) large subunit mRNA is restricted to bundle sheath cells. Demonstrating the differential accumulation of PEPCase mRNA and RuBPCase mRNA by utilizing the in situ hybridization technique paves the way for its use as a powerful tool in relating cellular differentiation to gene expression during plant development. PMID- 16665081 TI - Effects of fructose 2,6-bisphosphate on phosphoglucomutase from plants. AB - Fructose 2,6-bisphosphate affects phosphoglucomutase from plant and animal sources in a similar way. As previously found with rabbit muscle phosphoglucomutase, fructose 2,6-bisphosphate cannot substitute for glucose 1,6 bisphosphate as a cofactor in the reaction catalyzed by phosphoglucomutase from potato tubers, pea seeds, and string-beans. In the presence of glucose 1,6 bisphosphate, fructose 2,6-bisphosphate inhibits phosphoglucomutase from potato tubers. Activation of phosphoglucomutase from plant sources by fructose 2,6 bisphosphate reported by others was probably due to contamination of the commercial preparation of fructose 2,6-bisphosphate by glucose 1,6-bisphosphate. PMID- 16665082 TI - Response of various cucurbits to infection by plasmid-harboring strains of agrobacterium. AB - Tumor formation in cucurbit cultivars resulting from infection by various strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes is environmentally affected. In all instances, tumors could be induced on excised cotyledons while inoculating attached cotyledons or stems resulted in no tumor formation. In addition, buttercup squash (Cucurbita maxima Duch. buttercup) was most susceptible to tumor formation, while butterbush squash (Cucurbita maxima Duch. butterbush) failed to form tumors when inoculated with any of the strains of Agrobacterium. Other tested cucurbit cultivars showed intermediate susceptibility to tumor induction by the various Agrobacterium strains. PMID- 16665083 TI - Protoplasmic Swelling as a Symptom of Freezing Injury in Onion Bulb Cells : Its Simulation in Extracellular KCl and Prevention by Calcium. AB - Freezing injury, in onion bulb tissue, is known to cause enhanced K(+) efflux accompanied by a small but significant loss of Ca(2+) following incipient freezing injury and swelling of protoplasm during the postthaw secondary injury. The protoplasmic swelling of the cell is thought to be caused by the passive influx of extracellular K(+) into the cell followed by water uptake. Using outer epidermal layer of unfrozen onion bulb scales (Allium cepa L. cv Big Red), we were able to stimulate the irreversible freezing injury symptoms, by bathing epidermal cells in 50 millimolar KCl. These symptoms were prevented by adding 20 millimolar CaCl(2) to the extracellular KCl solution. Our results provide evidence that loss of cellular Ca(2+) plays an important role in the initiation and the progression of freezing injury. PMID- 16665084 TI - Organelle Movements along Actin Filaments and Microtubules. AB - Organelle movements involving microtubules and actin filaments are a conspicuous and important feature of many plant cells. Movements have recently been supported in preparations of demembranated cytoplasm and reconstituted from purified proteins. The favored mechanism involves organelles carrying a force-generating ATPase moving along a track provided by either actin filaments or microtubules. Cytoplasmic free Ca(2+) concentration regulates at least some organelle movements. PMID- 16665085 TI - Canopy photosynthesis and its relationship to plant productivity in near-isogenic cotton lines differing in leaf morphology. AB - A 2-year study was conducted to determine the relationships between plant canopy photosynthesis, canopy light interception, and plant productivity of cotton (Gossypium hirsutum L.) exhibiting differing leaf morphologies. The near-isogenic lines were from a single background (MD 65-11) and represented the leaf shapes Normal (small leaf lobing), Sub-Okra (intermediate leaf lobing), Okra (large leaf lobing), and Super Okra (severe leaf lobing). The F(1) of a cross Normal x Okra (intermediate leaf lobing) and the F(2) (segregating 1:2:1 for Normal Sub-Okra, and Okra, respectively) were also grown. Reduced plant canopies were produced by Okra and Super Okra lines, which translated into increased light penetration to the ground, and hence, in reduced canopy photosynthesis. Integrated canopy photosynthesis (ICAP) was significantly associated with light interception by the plant canopy. Part of the remaining variability in ICAP was associated with confounding factors associated with plant maturity and other unmeasured genotypic factors. Intermediate (F(1) and Sub-Okra) and normal leaf types displayed the largest ICAP values in both years. Lint production was positively related to ICAP (R(2) = 0.53). The combination of high ICAP values and competitive lint yields indicate that intermediate lobed leaf morphologies offer promise as productive sources of physiological variation for cotton germplasm development. PMID- 16665086 TI - The response of foliar gas exchange to exogenously applied ethylene. AB - The responsiveness to ethylene of net photosynthesis and stomatal conductance to water vapor in intact plants was investigated in 13 herbaceous species representing seven plant families. Exposures were conducted in an open, whole plant exposure system providing controlled levels of irradiance, air temperature, CO(2), relative humidity, and ethylene concentration. Net photosynthesis and stomatal conductance to water vapor in units of moles per square meter per second were measured on recently expanded leaves in control and ethylene-treated plants using a remotely operated single-leaf cuvette. The ethylene concentration was either 0 or 210 micromoles per cubic meter and was maintained for 4 hours. Species varied substantially in the response of their foliar gas exchange to ethylene. In 7 of the 13 species, net photosynthesis was inhibited statistically by 4 hours of ethylene exposure. As a function of the rate in control plants, the responses were most pronounced and statistically significant in Arachis hypogaea (-51.1%), Gossypium hirsutum (-31.7%), Glycine max (-24.8%), Cucurbita pepo ( 20.4%), Phaseolus vulgaris (-18.4%), Setaria viridis (-17.5%), and Raphanus sativus (-4.4%). Whereas the responsiveness of net photosynthesis to ethylene among the 13 species showed no specific taxonomic associations, the responsiveness was positively correlated with the intrinsic rate of net photosynthesis. Stomatal conductance to water vapor after 4 hours of ethylene exposure declined statistically in 6 of the 13 species. As a function of control rates, the most marked and statistically significant responses of stomatal conductance were in Glycine max (-53.6%), Gossypium hirsutum (-51.2%), Arachis hypogaea (-42.7%), Phaseolus vulgaris (-38.6%), Raphanus sativus (-26.8%), and Solanum tuberosum (-23.4%). Although ethylene-induced changes in net photosynthesis and stomatal conductance were positively correlated, there were species-specific exceptions in which net photosynthesis declined after 4 hours of exposure without a concurrent change in stomatal conductance, stomatal conductance declined without a change in net photosynthesis, and the decline in stomatal conductance substantially exceeded the corresponding decline in net photosynthesis. Thus, the responsiveness to ethylene of net photosynthesis and stomatal conductance to water vapor were not consistently synchronous or equivalent among the 13 species. It is concluded that foliar gas exchange is responsive to exogenously applied ethylene in many plant species. The sensitivity of foliar gas exchange to ethylene may play a role in general plant response to environmental stress in which one of the physiological sites of action for endogenously produced stress ethylene in the leaf is the plant's photosynthetic capacity and/or stomatal conductance to water vapor. PMID- 16665087 TI - Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue. AB - Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O(2) and greatest at 0.06%, the lowest O(2) concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O(2) level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-(14)C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [(14)C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis. PMID- 16665088 TI - Purification and properties of hypoxically induced lactate dehydrogenase from barley roots. AB - Using Affigel Blue and oxamate-agarose affinity chromatography, lactate dehydrogenase (LDH) was purified 2000-fold from hypoxically induced barley roots. Molecular weights of the native and sodium dodecyl sulfate-denatured LDH protein were 157 and 40 kilodaltons, respectively, indicating a tetramer. Purified barley LDH was very similar in size and kinetic properties to potato LDH. However, their amino acid compositions differed substantially and antibodies raised against barley LDH did not cross-react with potato LDH on immunoblots, implying that the barley and potato LDHs are not closely related proteins. In vivo [(35)S] methionine labeling and immunoprecipitation experiments indicated that hypoxia increased the rate of LDH protein synthesis, and immunoblot analysis showed that LDH protein levels rose during hypoxia. We conclude that increased enzyme synthesis plays a major part in the induction of LDH enzyme activity by low O(2) levels in barley roots. PMID- 16665089 TI - ATPase in lipid body membranes of castor bean endosperm. AB - Lipid body membranes purified from castor seed endosperm of dry seeds and 4 d old seedlings were found to have an ATPase activity associated with them. This was confirmed by equilibrium density centrifugation of the membranes using acid lipase as a marker enzyme. The specific activity ranged from 45 to 200 nanomoles per milligram protein per minute. The pH optimum was 9.0 but at pH 7.5 nearly 40% of the maximum activity was retained. The apparent K(m) for Mg-ATP was 0.5 millimolar. A divalent cation was required for activity and Mg(2+) was the most effective. Other nucleoside triphosphates were also hydrolyzed but there was no hydrolysis of pyrophosphate or p-nitrophenylphosphate. The ATPase was not inhibited by oligomycin, vanadate, dicyclohexylcarbodiimide, or molybdate but was inhibited by sodium azide. Washing the membranes with increasing concentrations of NaCl removed up to 60% of the ATPase activity but none was removed by 3 millimolar ethylene-diaminetetraacetate. PMID- 16665090 TI - Intercellular localization of nitrate reductase in roots. AB - Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO(3) (-) for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO(3) (-) likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO(3) (-), the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO(3) (-) occurs during uptake and reduction of exogenous NO(3) (-). Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO(3) ( ). It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO(3) ( ), (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO(3) (-) is taken up by cells of the cortex and stele, and/or (c) NO(3) (-) is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO(3) (-) reduction. PMID- 16665091 TI - Polyamine stimulation of protein phosphorylation in isolated pea nuclei. AB - The phosphorylation of several proteins in isolated nuclei from Pisum sativum L. was stimulated by spermine. Although spermine increased the general protein phosphorylation by 10 to 20%, it increased the phosphorylation of a 47 kilodalton polypeptide by 150%. By comparison other polyamines, spermidine, putrescine, and cadavarine had far less effect on the phosphorylation of the 47 kilodalton or any other polypeptide. Sodium fluoride was able to inhibit the phosphorylation of the 47 kilodalton polypeptide in the control, implying the participation of protein phosphatase(s) in the phosphorylation of nuclear proteins. Spermine stimulated the phosphorylation of the 47 kilodalton polypeptide over the controls, even in the presence of NaF. This result indicates that spermine probably activates a nuclear kinase, a conclusion supported also by thiophosphorylation data. The inability of ethyleneglycol-bis (beta-amino-ethyl ether)-N, N'-tetraacetic acid and Compound 48/80, a calmodulin antagonist, to inhibit this spermine stimulated phosphorylation renders improbable any role of calcium and calmodulin in mediating this response. PMID- 16665092 TI - Comparison of IAA-Induced and Low Temperature-Induced GA(3) Responsiveness and alpha-Amylase Production by GA(3) Insensitive Dwarf Wheat Aleurone. AB - Rht3-containing gibberellic acid (GA(3)) insensitive deembryonated wheat (Triticum aestivum L. var Cappelle Desprez x Minister Dwarf) aleurone, that can be made responsive to GA(3) by low temperature, can also be rendered GA(3) sensitive by preincubation with indoleacetic acid (IAA). The IAA-induced response of the dwarf selection is concentration-dependent, relatively sensitive, and similar in magnitude to that induced by low temperature. Other auxins also induce GA(3) responsiveness to a greater or lesser degree. IAA has no apparent effect on the wild type (rht, tall) selection. PMID- 16665093 TI - A Possible Role for Indoleacetic Acid, Low Temperature, and Phospholipid Metabolism in the Induction of GA(3) Responsiveness in GA(3) Insensitive (Rht3 Containing) Dwarf Wheat Aleurone. AB - Preincubation of dwarf, Rht3-containing deembryonated seed for 4 hours in 342 nanomolar indoleacetic acid (IAA) induced maximum sensitivity to GA(3). In addition, the 4-hour IAA pretreatment caused a 2-fold increase in total phospholipids which coincided identically on a temporal basis with the induced GA(3) sensitivity. Changes in absolute levels of individual phospholipids and their acyl groups were recorded and compared with the changes observed in several Rht-containing aleurone tissues which were induced to develop GA(3) sensitivity by exposure to low temperature (5 degrees C). Several distinct similarities between all tissues were recorded as they develop GA(3) sensitivity. One parameter, the percentage phospholipid composition, was quite similar in all tissues after they had become maximally sensitive to GA(3), suggesting that there is at least one membrane phospholipid composition which is particularly responsive to GA(3). The results indicate that (a) the basis of the GA(3) insensitivity of the Rht mutation resides in an aberrant phospholipid/fatty acid composition and/or metabolism; (b) exposure to low temperature (5 degrees C) for 20 hours or longer, or 342 nanomolar IAA for 4 hours or longer reverses or corrects the genetic lesion, enabling the tissue to adopt a GA(3) responsive membrane composition. Finally, an hypothesis is discussed which indicates that IAA may play a controlling role in the mobilization of endospermal reserves, at least in Rht3-containing wheat aleurone. PMID- 16665094 TI - Kinetic properties of phosphoenolpyruvate carboxylase from c(3), c(4), and c(3) c(4) intermediate species of flaveria (asteraceae). AB - Flaveria cronquistii (C(3)), F. chloraefolia (C(3)-C(4)), F. floridana (C(3) C(4)), F. pubescens (C(3)-C(4)), F. anomala (C(3)-C(4)), F. linearis (C(3)-C(4)), F. brownii (C(4)), F. palmeri (C(4)), F. trinervia (C(4)) and F. australasica (C(4)), comprising 10 out of the 21 known species of the genus Flaveria (Asteraceae), were included in a comparative study of the kinetic and regulatory properties of green leaf phosphoenolpyruvate (PEP) carboxylase. At least three kinetically distinct enzyme-forms were identified on the basis of their affinities for PEP and the degree of allosterism with respect to this substrate. The kinetic properties of PEP carboxylase of most of the species seemingly were modified in vivo depending on the growth conditions of the plants. K(m)(PEP(free))-values of the enzyme from the five C(3)-C(4) intermediate species ranged from 6 micromolar (F. chloraefolia, low light-grown) to 38 micromolar (F. pubescens, high light-grown). In contrast, the K(m) for PEP of PEP carboxylase from the C(3) species F. cronquistii (13 micromolar) apparently was not influenced by growth conditions. The response of the enzyme from the C(3) and C(3)-C(4) species was hyperbolic in all cases. A second isoform with a lower affinity for PEP (88-100 micromolar), but also hyperbolic kinetics was found in the C(4) species F. brownii, whereas in the three other C(4) species examined a PEP carboxylase with a still lower affinity for PEP (187-221 micromolar) and sigmoidal kinetics was present. These isozyme-related kinetic data were supported by analyses of the elution behavior of the enzyme during anion-exchange chromatography on DEAE-Trisacryl M. The results are discussed with respect to the evolution of C(4) photosynthesis in the Flaveria genus. PMID- 16665095 TI - Lipid peroxidation in higher plants : the role of glutathione reductase. AB - To study the role of glutathione reductase in lipid peroxidation, bean leaves (Phaseolus vulgaris) cv Fori were treated with the herbicide acifluorfen-sodium (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Acifluorfen is a potent inducer of lipid peroxidation. In beans, decrease of acid-soluble SH compounds and lipid peroxidation, measured as ethane evolution, were the toxic events after treatment of leaves with acifluorfen. As a primary response to peroxidation, increased production of antioxidants, such as vitamin C and glutathione, was found. This was followed by elevation of glutathione reductase activity. Enhanced activity of the enzyme prevented both further decline of acid soluble SH-compounds and lipid peroxidation. Increased production of antioxidants and elevated activity of antioxidative enzymes, like glutathione reductase, seem to be a general strategy to limit toxic peroxidation in plants. PMID- 16665096 TI - The effects of benzyladenine, cycloheximide, and cordycepin on wilting-induced abscisic Acid and proline accumulations and abscisic Acid- and salt-induced proline accumulation in barley leaves. AB - Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA) treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt shocked leaves was observed both when benzyladenine was added at the beginning of or after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level. PMID- 16665097 TI - Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum: II. Effects of NO(3) and NH(4) Addition to CO(2) Efflux in the Light. AB - The effects of nitrate and ammonium addition on net and gross photosynthesis, CO(2) efflux and the dissolved inorganic carbon compensation point of nitrogen limited Selenastrum minutum Naeg. Collins (Chlorophyta) were studied. Cultures pulsed with nitrate or ammonium exhibited a marked decrease in both net and gross photosynthetic carbon fixation. During this period of suppression the specific activity of exogenous dissolved inorganic carbon decreased rapidly in comparison to control cells indicating an increase in the rate of CO(2) efflux in the light. The nitrate and ammmonium induced rates of CO(2) efflux were 31.0 and 33.8 micromoles CO(2) per milligram chlorophyll per hour, respectively, and represented 49 and 48% of the rate of gross photosynthesis. Nitrate addition to cells at dissolved inorganic carbon compensation point caused an increase in compensation point while ammonium had no effect. In the presence of the tricarboxylic acid cycle inhibitor fluoroacetate, the nitrate-induced change in compensation point was greatly reduced suggesting the source of this CO(2) was the tricarboxylic acid cycle. These results are consistent with the mechanism of N-induced photosynthetic suppression outlined by Elrifi and Turpin (1986 Plant Physiol 81: 273-279). PMID- 16665098 TI - Insensitivity of the diageotropica tomato mutant to auxin. AB - The sensitivity of excised hypocotyl segments to indoleacetic acid (IAA) in two assays, ethylene production and elongation, was determined in the ethylene requiring tomato (Lycopersicon esculentum Mill.) mutant, diageotropica (dgt), and its isogenic parent, cv VFN8. Endogenous (uninduced) ethylene synthesis rates were slightly lower in dgt hypocotyls than in VFN8 hypocotyls. Ethylene production was essentially unaffected by IAA in dgt, but was stimulated up to 10 fold by 10 micromolar IAA in VFN8. Elongation of dgt hypocotyls was also insensitive to concentrations of IAA as high as 100 micromolar, as compared to significant elongation of VFN8 hypocotyls in response to 0.1 micromolar IAA. A range of IAA analogs active in VFN8 was also ineffective in stimulating elongation of dgt hypocotyls, suggesting that the differences were not due to rapid metabolism of IAA by dgt tissues. Auxin-induced elongation of VFN8 hypocotyls was unaffected by 2,3,5-triiodobenzoic acid and naphthylphthalamic acid, indicating that polar auxin transport was not a factor in these experiments. Exogenous and auxin-induced ethylene had no effect on the elongation respone of either genotype, nor did exogenous ethylene restore the sensitivity of dgt hypocotyls to IAA. Despite their apparent insensitivity to auxin, dgt hypocotyls elongated dramatically and synthesized ethylene rapidly in response to 1.2 micromolar fusicoccin. These results suggest that the primary effect of the dgt mutation is to reduce the sensitivity of the tissue to auxin. As altered regulation of ethylene synthesis is only one symptom of this fundamental deficiency, dgt should more properly be considered to be the auxin-insensitive tomato mutant. PMID- 16665099 TI - Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay. AB - This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N(2)O) were identified by GC and GC/MS as the main N products formed. NO and N(2)O produced from soybean leaflets were both labeled with (15)N when (15)N nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N(2)O were identified as air (N(2), O(2)), CO(2), methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N(2)O (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N(2)O were not evolved from leaves of the nr(1) soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO((x)) (NO and NO(2)). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Merr., and Pueraria thunbergiana Benth. evolved lower levels of NO((x)). PMID- 16665100 TI - Effects of water stress on the organic Acid and carbohydrate compositions of cotton plants. AB - Two photoperiodic cotton (Gossypium hirsutum L.) strains (T185 and T466) which had been empirically selected because of poor performance and two strains (T25 and T256) selected because of enhanced performance under field water stress were evaluated for stress-induced changes in their organic acids and carbohydrates. Profiles and quantitation of organic acids and carbohydrates from aqueous extractions of cotton leaf tissue were determined by high performance liquid chromatography. In all cases, the water-stressed plants showed two to five times greater amounts of organic acids and carbohydrates over the values determined for the irrigated samples. Under stress, sucrose accumulation was observed in wilting strains (poor performers) probably related to rate of translocation out of the leaf. The most dramatic response to water stress was the accumulation of citric acid in strains T25 and T256 as compared to T185 and T466. Citric/malic acid ratios for both the irrigated and water-stressed samples of T25 and T256 were twice those of T185 and T466. PMID- 16665101 TI - Properties of Bromphenol Blue as an Electron Donor for Higher Plant NADH: Nitrate Reductase. AB - Bromphenol blue, which was reduced with dithionite, was found to support nitrate reduction catalyzed by squash NADH:nitrate reductase at a rate about 5 times greater than NADH with freshly prepared enzyme and 10 times or more with enzyme having been frozen and thawed. Kinetic analysis of bromphenol blue as a substrate for squash nitrate reductase yielded apparent K(m) values of 60 micromolar for bromphenol blue at 10 millimolar nitrate and 500 micromolar for nitrate at 0.2 millimolar bromphenol blue. With the same preparation of enzyme the apparent K(m) values were 9 micromolar for NADH at 10 millimolar nitrate and 50 micromolar nitrate at 0.1 millimolar NADH. Bromphenol blue was found to be a noncompetitive inhibitor versus NADH with a K(i) of 0.3 millimolar. When squash NADH:nitrate reductase activity was inactivated with p-hydroxymercuribenzoate or denatured by heating at 40 degrees C, the bromphenol blue nitrate reductase activity was not lost. These results were taken to indicate that bromphenol blue and NADH donated electrons to nitrate reductase at different sites. When monoclonal antibodies prepared against corn and squash nitrate reductases were used to inhibit the nitrate reductase activities supported by NADH, bromphenol blue, and methyl viologen, differential inhibition was found which tended to indicate that the three electron donors were interacting with the enzyme at different sites. One monoclonal antibody prepared against squash nitrate reductase inhibited all three activities of both corn and squash nitrate reductase. It appears this antibody may bind to a highly conserved antigenic site in the nitrate binding region of the enzyme. PMID- 16665102 TI - Changes in Protein Synthesis in Rapeseed (Brassica napus) Seedlings during a Low Temperature Treatment. AB - Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18 degrees C for 48 hours, one half of the seedlings was transferred to 0 degrees C for another 48 hour period, the other half being kept at 18 degrees C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [(35)S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0 degrees C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6 bisphosphate carboxylase. These changes are also detectable after shorter treatments. PMID- 16665103 TI - Regulation of nitrate reductase activity in cultured spinach cells as studied by an enzyme-linked immunosorbent assay. AB - An enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. Immunoblotting analysis of the subunit of NR also supported this result. It is concluded that the regulation of NR in spinach cells as a function of culture age is mediated by changes in the amount of the enzyme protein rather than by activation and inactivation of the preexisting proteins. PMID- 16665104 TI - Secretion of Zeatin, Ribosylzeatin, and Ribosyl-1'' -Methylzeatin by Pseudomonas savastanoi: Plasmid-Coded Cytokinin Biosynthesis. AB - Cytokinin production by strains of the phytopathogenic bacterium Pseudomonas syringae pv savastanoi was measured by immunoaffinity chromatography of the culture medium on immobilized anti-cytokinin antibodies, followed by high performance liquid chromatography, radioimmunoassay and mass spectrometry. P. savastanoi strain PB213-2 secretes zeatin (80 nanograms per milliliter) and ribosylzeatin (80 nanograms per milliliter). Even higher levels of zeatin (400 nanograms per milliliter) are produced by the olive-specific strain EW1006, which also produces 180 nanograms per milliliter of the recently identified cytokinin, ribosyl-1'' -methylzeatin. The amounts secreted were approximately 1000 times greater than those secreted by Agrobacterium tumefaciens (DA Regier, RO Morris 1982 Biochem Biophys Res Commun 104: 1560-1566). Examination of cytokinin production by plasmid deletion mutants of PB213-2 and EW1006 indicated that cytokinin biosynthesis was specified, at least in part, by plasmid-borne genes. A fragment of the 105 kilobase pair plasmid from EW1006 was cloned into Escherichia coli where its expression resulted in dimethylallyl transferase activity and the secretion of zeatin. PMID- 16665105 TI - Properties of Solubilized UDP-GlcNAc: Dolichyl Phosphate-GlcNAc-1-P-Transferase from Soybean Cultured Cells. AB - The GlcNAc-1-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP GlcNAc and dolichyl phosphate. The GlcNAc-1-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The K(m) value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GlcNAc 1-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg(2+). The addition of unlabeled GDP-mannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity. This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-[(3)H]-GlcNAc from pyrophosphatase degradation. The GlcNAc-1-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations. PMID- 16665106 TI - Betaine aldehyde oxidation by spinach chloroplasts. AB - Chenopods synthesize betaine by a two-step oxidation of choline: choline --> betaine aldehyde --> betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H(2)O(2)-mediated reaction cannot be ruled out. PMID- 16665107 TI - The Fate of Chloroplast Proteins during Photooxidation in Carotenoid-Deficient Maize Leaves. AB - Maize seedlings, treated with the herbicide norflurazon to produce a deficiency in carotenoid pigments, were grown in low-fluence-rate light. Under these conditions, which induced chlorophyll biosynthesis while minimizing photooxidation, carotenoid-deficient seedlings showed identical patterns of chloroplast protein accumulation compared with normal seedlings. Carotenoid pigments thus play no direct role in regulating the accumulation of chloroplast proteins. When shifted to high-fluence-rate light, chlorophyll was rapidly photooxidized in carotenoid-deficient seedlings. Chloroplast proteins showed varying degrees of sensitivity to photooxidation. The P-700 apoprotein of photosystem I was rapidly degraded. Most stromal and thylakoid proteins either decreased progressively in photooxidative conditions or appeared to be unaffected. The relative quantity of the light-harvesting chlorophyll a/b-binding protein of photosystem II increased significantly in the first few hours of high fluence-rate light. It then appeared to be only minimally affected 18 hours after complete photooxidation of chlorophyll. PMID- 16665108 TI - Osmotic Adjustment in Leaves of VA Mycorrhizal and Nonmycorrhizal Rose Plants in Response to Drought Stress. AB - Osmotic adjustment in Rosa hybrida L. cv Samantha was characterized by the pressure-volume approach in drought-acclimated and unacclimated plants brought to the same level of drought strain, as assayed by stomatal closure. Plants were colonized by either of the vesicular-arbuscular mycorrhizal fungi Glomus deserticola Trappe, Bloss and Menge or G. intraradices Schenck and Smith, or were nonmycorrhizal. Both the acclimation and the mycorrhizal treatments decreased the osmotic potential (Psi(pi)) of leaves at full turgor and at the turgor loss point, with a corresponding increase in pressure potential at full turgor. Mycorrhizae enabled plants to maintain leaf turgor and conductance at greater tissue water deficits, and lower leaf and soil water potentials, when compared with nonmycorrhizal plants. As indicated by the Psi(pi) at the turgor loss point, the active Psi(pi) depression which attended mycorrhizal colonization alone was 0.4 to 0.6 megapascals, and mycorrhizal colonization and acclimation in concert 0.6 to 0.9 megapascals, relative to unacclimated controls without mycorrhizae. Colonization levels and sporulation were higher in plants subjected to acclimation. In unacclimated hosts, leaf water potential, water saturation deficit, and soil water potential at a particular level of drought strain were affected most by G. intraradices. G. deserticola had the greater effect after drought preconditioning. PMID- 16665109 TI - A system for manipulating the membrane Fatty Acid composition of soybean cell cultures by adding tween-Fatty Acid esters to their growth medium : basic parameters and effects on cell growth. AB - The development of a system for modifying the membrane fatty acid composition of cultured soybean cells (Glycine max [L.] Merr.) is described. Tween-fatty acid esters carrying specific fatty acids were synthesized and added to the medium of suspension cultures. Cells transferred large quantities of exogenous fatty acids from Tweens to all acylated membrane lipids; up to 50% of membrane fatty acids were exogenously derived. C15 to C20 saturated fatty acids and C16, C18, and C20 unsaturated fatty acids with either cis or trans double bonds were incorporated into lipids. Cells elongated saturated fatty acids of C16 or less, and unsaturated fatty acids with cis double bonds were further desaturated. No other types of modifications were observed. Growth ceased in cells treated with excessive concentrations of Tween-fatty acid esters, but frequently not for several days. Cessation of cell growth was correlated with changes in membrane fatty acid composition resulting from incorporation of large amounts of exogenous fatty acids into membrane lipids, although cells tolerated large variations in fatty acid composition. Maximum tolerable Tween concentrations varied widely according to the fatty acid supplied. Potential uses of this system and implications of the observed modifications on the pathway of incorporation are discussed. PMID- 16665110 TI - Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis. AB - Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-(14)C]-fatty acids and in cells treated with [1-(14)C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-(14)C]acetate or Tween-[1-(14)C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-(14)C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1 (14)C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism. PMID- 16665111 TI - Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells. AB - PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged. PMID- 16665112 TI - Distribution and Metabolism of sym-Homospermidine and Canavalmine in the Sword Bean Canavalia gladiata cv Shironata. AB - The unusual polyamines, sym-homospermidine (homoSPD) and canavalmine (CAN), were found in the seed of Canavalia species such as C. gladiata, C. ensiformis, and C. brasilensis, but not in those of other leguminous crops. To examine the distribution and metabolism of homoSPD and CAN in sword bean, C. gladiata cv Shironata, polyamine analysis was carried out throughout the life cycle of this plant. During seed germination, putrescine (PUT), spermidine (SPD), and spermine (SPM) were accumulated in the radicle and hypocotyl. HomoSPD and CAN were, however, maintained at very low levels over a 6-day period of germination. In nodulated sword bean plants, a large quantity of homoSPD was found in the root nodule. CAN was detected exclusively in the senescent nodule at very low concentrations. These polyamines were not detected in any other organs including root, stem, leaf, vine, flower, and pod, while PUT, SPD, and SPM were always found in those organs. As plants reached the reproductive stage, homoSPD and CAN appeared in the immature seed and their concentrations increased as seed formation progressed. By contrast, the level of SPM continuously decreased during seed development. In developing seeds, considerable accumulation of canavanine, an analog of arginine, which is a precursor in polyamine biosynthesis, was also observed. PMID- 16665113 TI - Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities. AB - Incubation of barley (Hordeum vulgare L. cv Himalaya) half-seeds with gibberellic acid enhances the secretion of ribonuclease and deoxyribonuclease from aleurone tissue (MJ Chrispeels, JE Varner 1967 Plant Physiol 42: 398-406; L Taiz, JE Starks 1977 Plant Physiol 60: 182-189). These activities were over 50-fold greater in medium of half-seeds incubated with gibberellic acid than in control medium. Ribonuclease and deoxyribonuclease activities initially appeared in the medium 24 to 48 hours after hormone induction and increased for up to 96 hours. Both activities had a pH optimum of 6.0 and a temperature optimum of 55 degrees C. When the medium from gibberellic acid-treated half-seeds was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the major ribonuclease and deoxyribonuclease activity bands comigrated. The two enzyme activities remained associated throughout a 2,700-fold purification employing ammonium sulfate fractionation, Heparin-Agarose affinity chromatography, and Reactive Blue 2-Agarose affinity chromatography. Also accompanying the ribonuclease and deoxyribonuclease activities throughout purification was the ability to hydrolyze the 3'-phosphoester linkage of 3'-AMP. The purified protein was composed of a single polypeptide with an apparent molecular weight of 36 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is concluded that in response to gibberellic acid, barley aleurone tissue secretes a nuclease having ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities. PMID- 16665114 TI - Lipid Composition of Plasma Membranes and Tonoplasts Isolated from Etiolated Seedlings of Mung Bean (Vigna radiata L.). AB - The lipid composition of plasma membranes and tonoplasts from etiolated mung bean hypocotyls was examined in detail. Phospholipids, sterols, and ceramide monohexoside(s) were the major lipid classes in both membranes. The content of phospholipids on a protein basis was higher in the tonoplast, but the content of total sterols was similar in both membranes. Accordingly, the sterol to phospholipid molar ratio in the plasma membrane was higher than that of the tonoplast. Phosphatidylethanolamine and phosphatidylcholine comprised the major phospholipids in both membranes. Phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were identified as minor phospholipid components. The content of phosphatidylinositol and phosphatidylglycerol was relatively high in the tonoplast, comprising 11 and 5% of the total phospholipids, respectively. Although special care was taken against the degradative action of phospholipase D and phosphatidic acid phosphatase during the isolation of these membranes, by adding EDTA, EGTA, KF, choline, and ethanolamine to the homogenizing medium, significant amounts of phosphatidic acid, about 15% of the total phospholipids, were detected in the plasma membrane. On the other hand, the content of phosphatidic acid in tonoplasts and other membrane fractions was very low. This fact may indicate that high levels of phosphatidic acid occur naturally in plasma membranes. Phosphatidylglycerol in both membranes and phosphatidylinositol in the tonoplast contained high levels of palmitic acid, which comprised more than 50% of the total fatty acids. Significant differences were observed in the sterol compositions of plasma membranes and tonoplasts. More than 90% of the sterols in the plasma membrane were unesterified, while the tonoplast was enriched in glycosylated sterols, especially acylated sterylglycosides. Ceramide monohexoside was found to be specifically located in these membranes, in particular, in the tonoplast, in which it comprised nearly 17% of the total lipids. PMID- 16665115 TI - Diacylglycerol acyltransferase in maturing oil seeds of maize and other species. AB - Diacylglycerol acyltransferase (EC 2.3.1.20) activity was detected in the microsomal fractions of maturing maize scutellum, soybean cotyledon, peanut cotyledon, and castor bean endosperm. The activity detected was high enough to account for the in vivo rate of triacylglycerol synthesis. The activity of the maize enzyme was characterized using diolein micelles prepared by sonication in Tween 20 as the substrate. The activity was highest at pH values of 6 to 7. The activity was proportional to the amount of enzyme added, and the reaction rate was linear for about 2 minutes. The enzyme was not inactivated by Tween 20, Zwitterion 3-08, Triton-X 100, and cholate, but was inactivated completely by sodium dodecyl sulfate. The enzyme was active on linoleoyl coenzyme A (CoA), palmitoyl CoA, and oleoyl CoA, although the activity was highest on linoleoyl CoA. Endogenous diacylglycerol was present in the microsomes, and the enzyme activity was only partially dependent on the addition of external diolein. Subcellular fractionation of the total scutellum extract in sucrose density gradients was performed. By comparing the migration of the enzyme between rate and equilibrium centrifugation, and between equilibrium centrifugation in the presence and absence of magnesium ions in the preparative media, the enzyme was shown to be associated with the rough endoplasmic reticulum. Some of the above findings on the maize enzyme were extended to the enzymes from castor bean, soybean, and peanuts. PMID- 16665116 TI - Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii. AB - The flux of glycolate through the C(2) pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO(2) (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air grown cells reflects the presence of a CO(2) concentrating mechanism which raises the internal CO(2) level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO(2) concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C(2)-cycle inhibitors during CO(2) fixation. PMID- 16665117 TI - Photosynthetic responses of leaves to water stress, expressed by photoacoustics and related methods : I. Probing the photoacoustic method as an indicator for water stress in vivo. AB - The effect of leaf desiccation on the photosynthetic activities in vivo was probed by the photoacoustic method. The aim of this research was: (a) To study the photoacoustic signal per se in varied conditions in order to develop this tool as a probe for stress conditions in vivo. (b) To obtain results pertaining to electron transport activities in vivo, and confirm conclusions based on work with isolated chloroplasts, which could otherwise be the result of nonspecific damage occurring during their isolation. Leaf discs from tobacco (Nicotiana tabacum L.) were routinely used, with other species tested also for comparison. Rapid leaf desiccation caused changes in the low frequency photoacoustic signal, attributed both to the mechanism of signal transduction, influenced by changes in the structural parameters of the leaf, and to the direct (nonstomatal) inhibition of gross photosynthesis. The dependence of the photothermal part of the signal on the frequency indicated the presence of two photothermal components, one of which persisted only at low modulation frequencies (below about 100 Hz) and which largely increased with the desiccation treatment. This component was ascribed to a thermal wave which reaches the leaf surface. The other nonvariable photothermal component was ascribed to a thermal wave propagating from the chloroplasts to the surface of the mesophyll cell. Only this component is considered in the ratio of the O(2) signal to the photothermal signal, which is used to estimate the quantum yield of photosynthesis. The specific dependence of the latter ratio on the frequency yielded a comparative quantum yield parameter from its extrapolation to zero frequency, and also indicated stress induced changes in the diffusion of O(2) through the mesophyll cell, reflected by changes in its characteristic slope. The (zero frequency extrapolated) quantum yield was markedly reduced with the progression of the water stress, indicating the inhibition of (gross) phototosynthetic electron transport in vivo. This result was expressed even more emphatically by the stronger inhibition of the photochemical energy storage, obtained by photoacoustic measurements at a high modulation frequency. PMID- 16665118 TI - Photosynthetic Responses of Leaves to Water Stress, Expressed by Photoacoustics and Related Methods : II. The Effect of Rapid Drought on the Electron Transport and the Relative Activities of the Two Photosystems. AB - The effect of rapid dehydration of detached tobacco leaves (Nicotiana tabacum L.) on the photochemical apparatus of photosynthesis was studied in vivo by a combination of methods: photoacoustics, chlorophyll a fluorescence, and cytochrome f difference spectroscopy. It was shown that the inhibition of gross O(2) evolution was mainly caused by inactivation of PSII: (a) The saturation curve of cytochrome-f photooxidation by farred (>710 nanometers) light was resistant to the stress, leading to the conclusion that photosystem I (PSI) was largely unaffected by the stress. (b) The extent of the chlorophyll a variable fluorescence arising from photosystem II (PSII) decreased with the progression of the stress, but was largely unaffected when the leaf was preincubated with electron donors to PSII, such as hydroxylamine. It is concluded that the drought damage to PSII occurred on the photooxidative side. Despite the extensive inhibition of PSII and the relative preservation of PSI, the apparent PSII/PSI activity balance was somewhat larger in stressed leaves than in the control, as indicated by photoacoustic measurements of Emerson enhancement. These measurements were performed continuously under conditions which favor transitions to either state 1 or 2, showing that the transition to state 2 was considerably inhibited. Simultaneous measurements of chlorophyll fluorescence induction at 680 and 730 mm at room temperature were also used to probe changes in energy distribution between PSII and PSI and indicated that the transition from a dark adapted state to state 2 was also affected in water-stressed leaves. The saturation curve of the far-red light effect in Emerson enhancement was not changed by the stress, giving another independent evidence for the drought resistance of PSI activity. This apparent preservation of the imbalance in photochemical activities in favor of PSII, despite the fact that PSII is strongly inhibited, and PSI is not, supports a previous suggestion that the electron transfer between the two photosystems is not random but that a large extent of PSII and PSI units are specifically linked. PMID- 16665119 TI - Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: I. A P NMR Description of Acid-Load Effects. AB - Modifications of cytoplasmic pH in Acer pseudoplatanus L. cells cultivated in suspension have been induced by acid-loads and studied by using (31)P nuclear magnetic resonance spectroscopy. The initial drop of cytoplasmic pH, observed in the first minutes of exposure to weak lipophilic acids, was followed by a slow recovery to reach a plateau phase with a pH value lower than the initial one. Conversely, removal of the acid led to a sharp increase of cytoplasmic pH with in most cases an overshoot toward more alkaline values than the initial one and a subsequent decrease to more acidic values. This shows that A. pseudoplatanus cells powerfully regulate their cytoplasmic pH both on the acid side of their normal pH, during the acid-load, and on the alkaline side, after removal of acid. Similar results were obtained with different types of acid-loads, i.e. treatments with propionic or benzoic acid or bubbling with CO(2)-enriched air. This indicates that the occurrence of pH regulation does not depend upon the method used to acid-load the cells. The time courses of cytoplasmic pH observed for A. pseudoplatanus and also Catharanthus roseus cells are similar to those recorded for animal cells but different from those described for other plant materials for which no recovery phase was observed. This can be explained by different balances between the initial rate of proton influx brought in by the acids, and the capacity of proton consumption by the regulatory mechanisms. The existence of the recovery phase offers a unique possibility to study the regulation of the cytoplasmic pH of plant cells, as it has been done in animal systems. PMID- 16665120 TI - Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: II. Possible Mechanisms Involved in pH Regulation during Acid-Load. AB - Qualitative and quantitative aspects of the mechanisms involved in the regulation of cytoplasmic pH during an acid-load have been studied in Acer pseudoplatanus cells. Two main processes, with about the same relative importance, account for the removal of H(+) from the cytoplasm, namely a ;metabolic consumption' of protons and the excretion of protons or proton-equivalents out of the cells. The metabolic component corresponds to a change in the equilibrium between malate synthesis and degradation leading to a 30% decrease of the malate content of the cells during the period of cytoplasmic pH regulation. Various conditions which severely inhibit the activity of the plasmalemma proton pump ATPase reduce, at most by 50%, the excretion of H(+). This suggests that, besides the plasmalemma proton-pump, other systems are involved in the excretion of proton-equivalents. Indirect information on qualitative and quantitative features of these systems is described, which suggests the involvement of Na(+) and HCO(3) (-) exchanges in the regulation of cytoplasmic pH of acid-loaded cells. PMID- 16665121 TI - Spatial distributions of potassium, solutes, and their deposition rates in the growth zone of the primary corn root. AB - Densities of osmoticum and potassium were measured as a function of distance from the tip of the primary root of Zea mays L. (cv WF9 x mo17). Millimeter segments were excised and analyzed for osmotic potential by a miniaturized freezing point depression technique, and for potassium by flame spectrophotometry. Local deposition rates were estimated from the continuity equation with values for density and growth velocity. Osmotic potential was uniform, -0.73 +/- 0.05 megapascals, throughout the growth zone of well-watered roots. Osmoticum deposition rate was 260 muosmoles per gram fresh weight per hour. Potassium density fell from 117 micromoles per gram in the first mm region to 48 micromoles per gram at the base of the growth zone. Potassium deposition rates had a maximum of 29 micromoles per gram per hour at 3.5 millimeters from the tip and were positive (i.e. potassium was being added to the tissue) until 8 millimeters from the tip. The results are discussed in terms of ion relations of the growing zone and growth physics. PMID- 16665122 TI - Characterization of Triazine-Resistant and -Susceptible Isolines of Canola (Brassica napus L.). AB - Morphometric, electrophoretic, and immunological procedures were used to probe the structural and physiological differences between triazine-resistant (R) and susceptible (S) isolines of canola (Brassica napus L.). The R biotype exhibited increased grana stacking and decreased amounts of starch compared to the S biotype. Likewise, characters associated with an increase in grana stacking (lower chlorophyll a/b ratio, increased chlorophyll a/b light-harvesting complex, and relatively lower amounts of the P700 chlorophyll a protein and chloroplast coupling factor) were all observed in the R isoline of canola. Proteins which occur with approximately equal frequency in grana and stroma lamellae (plastocyanin, cutochrome f) or present only in the stroma (ribulose 1,5 bisphosphate carboxylase/oxygenase) were not quantitatively different in the two biotypes. Gross anatomical parameters (volume of epidermis, palisade mesophyll, spongy mesophyll, and air space) were similar in the two isolines. Thus, the triazine-resistance mutation does not confer a shade-type anatomy despite the chloroplast changes that are characteristic of shade biotypes or shade adaptions. These data indicate that the differences in chloroplast structure noted previously in comparisons of nonisonuclear R and S weed biotypes reflect differences in the triazineresistance factor rather than characters unrelated to triazine resistance. PMID- 16665123 TI - Rooting cofactor activity of plant phytoalexins. AB - The pterocarpinoid phytoalexins, glyceollin, pisatin, and phaseollin, stimulated adventitious root formation in a mung bean rooting bioassay only in the presence of indoleacetic acid (rooting cofactor activity). Relatively low (5 to 50 micrograms per milliliter) concentrations of the phytoalexins were effective. The phytoalexins also increased the numbers of root primordia formed, therefore suggesting that they interacted with an early process in root formation. PMID- 16665124 TI - Carbon Accumulation during Photosynthesis in Leaves of Nitrogen- and Phosphorus Stressed Cotton. AB - Leaves of cotton (Gossypium hirsutum L.) accumulate considerable dry mass per unit area during photosynthesis. The percentage of C in that accumulated dry mass was estimated as the regression coefficient (slope) of a linear regression relating C per unit area to total dry mass per unit area. Plants were grown on full nutrients or on N- or P-deficient nutrient solutions. In the fully nourished controls, the mass that accumulated over a 9-hour interval beginning at dawn contained 38.6% C. N and P stress increased the C concentration of accumulated mass to 49.7% and 45.1%, respectively. Nutrient stress also increased the starch concentration of accumulated mass, but starch alone could not account for the differences in C concentration. P stress decreased the estimated rate of C export from source leaves, calculated as the difference between C assimilation and C accumulation. The effect of P stress on apparent export was very sensitive to the C concentration used in the calculation, and would not have been revealed with an assumption of unchanged C concentration in the accumulated mass. PMID- 16665125 TI - Apoplastic and symplastic pathways of atrazine and glyphosate transport in shoots of seedling sunflower. AB - [(14)C]Atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) and [(14)C]glyphosate (N-[phosphonomethyl]glycine) were xylem fed to sunflower shoots at 100 micromolar for 1 hour in the light, then placed in the dark at 100% relative humidity for 1, 4, 7, or 10 hours. The distribution of atrazine and glyphosate between shoot parts, in the leaves, and between the aoplast and symplast of the leaf was determined. The apoplastic concentrations and distribution patterns of atrazine and glyphosate in the leaves were evaluated using a pressure dehydration technique, our results were compared to the previously reported distribution patterns of the naturally occurring apoplastic leaf solutes, and the apoplastic dye PTS (trisodium 3-hydroxy-5,8,10 pyrenetrisulfonate). The pattern of atrazine and glyphosate distribution in the shoot, and between the leaf apoplast and symplast, was found to reflect the potential of these herbicides to enter the shoot symplast. The results of this study are discussed with respect to current theories of xenobiotic transport in plants, and have been found to be consistent with the intermediate permeability hypothesis for xenobiotic transport. PMID- 16665126 TI - P Nutrition during Seed Development : Leaf Senescence, Pod Retention, and Seed Weight of Soybean. AB - Soybean (Glycine max [L.] Merrill) leaf senescence, which may partially result from mineral redistribution, appears to limit grain yield. Two experiments were designed to test the effects of supplemental inorganic phosphate (Pi), K, malate, and methionine (Met) infusions on senescence and yield. A novel stem infusion technique using pediatric intravenous kits was developed to supply these nutrients throughout seed growth. An average of 48.4 milliliters per plant was successfully infused into lower stem internodes during a 4 to 6 week period. Senescence was unaffected by K or malate infusions, but was delayed by Pi infusions (up to 8 days) and by increased nutrient solution Pi levels (up to 21 days) in separate experiments. Treatments which delayed senescence also improved yield as much as 3-fold, due primarily to increased pod retention and secondarily to increased seed size. Met infusions further increased pod retention at the lower, infused nodes, and thus increased total plant yield also. The influence of higher Pi levels during reproductive growth on soybean pod retention and yield may have been the result of sustained sucrose export due to altered C partitioning in leaves. The role of Met in improving yield was not clear. However, these results clearly demonstrate the importance of adequate Pi for delaying senescence and improving pod retention and yield. PMID- 16665127 TI - Stem infusions enhanced methionine content of soybean storage protein. AB - The quality of soybean (Glycine max [L.] Merrill) seed storage protein is limited by its low methionine (Met) content. Met supplementation of an in vitro soybean cotyledon culture has been shown to increase Met content by 21.9% due to an inhibition of the synthesis of the Met-devoid beta subunit of 7S storage protein (JF Thompson et al. 1981, Phytochemistry 20: 941-945). The objective of this research was to determine if Met supplementation of intact plants would result in a similar improvement in soybean protein quality. A solution including 10 millimolar d,l malic acid plus 10 millimolar K(2)HPO(4) with or without 20 millimolar d,l Met or 20 millimolar Na(2)SO(4) was infused throughout seed development into lower stem internodes of soybeans (cv ;Williams 79' or ;Williams 82') grown under both greenhouse and field conditions. Pediatric intravenous kits were used to infuse an average of 51.2 milliliters per plant. Met content of whole soybean seeds from intact plants receiving Met infusions increased by as much as 22.7%. Even greater (up to 31.0%) increases in cysteine (Cys) content were noted, indicating that soybean plants are able to metabolize Met to Cys, or that supplemental Met allows Cys accumulation by some other mechanism. Electrophoretic patterns showed a dramatic decrease in the synthesis of the beta subunit of 7S storage protein when Met was supplemented, and this effect was not confined to seeds at the lower nodes. In addition, seeds from upper compared to lower plant nodes (regardless of infusion treatment) had greater protein content (45.0 versus 41.6 w/w%), and different protein composition, as indicated by significantly different amino acid profiles. Methionine supplementation of intact soybean plants improved protein quality through an alteration in storage protein composition. PMID- 16665128 TI - Amino Acid Composition Along the Transport Pathway during Grain Filling in Wheat. AB - The amino acid composition of endosperm cavity sap and of sieve tube saps from the flag leaf, peduncle, rachis, grain pedicel, and grain were determined for wheat plants just past the mid-half of grain filling. On a mole percent basis, glutamine accounted for almost half of the amino acids in sieve tube sap from the peduncle and ear. Other protein amino acids, plug gamma-aminobutyrate, were present in varying, but mostly low (a few mole percent) proportions. The amino acid composition of phloem exudate resembled that of the mature wheat grain. The proportions of amino acids in the endosperm cavity were generally similar to those of the sieve tube sap supplying the grain. Cysteine, however, while virtually absent from sieve tube sap, comprised 1 to 2 mole percent of amino acids in the endosperm cavity, suggesting it is transported in a different form. Also, alanine and, to a lesser extent, glutamate were relatively more prominent in endosperm cavity sap than in the sieve tube sap. Thus, while most amino acids were more concentrated in the sieve tube sap than in the endosperm cavity sap, alanine and glutamate appeared to be moving from the sieve tube to the endosperm cavity in the absence of, or perhaps even against, their concentration gradients. PMID- 16665129 TI - Accumulation and Conversion of Sugars by Developing Wheat Grains : VI. Gradients Along the Transport Pathway from the Peduncle to the Endosperm Cavity during Grain Filling. AB - Gradients along the transport pathway from the peduncle to the endosperm cavity were examined during grain filling in wheat. Sieve tube exudate was collected from severed aphid stylets established on the peduncle and rachis and on the vascular bundles in the creases of grains. Phloem exudate could also be collected from broken grain pedicels, and by puncturing the vascular bundle in the grain crease with a needle. Stylets on excised grains persisted exuding, indicating that grain sieve tubes are capable of loading solutes. There was little, if any, discernible gradient in osmolality or solute composition (sucrose, total amino acids) of sieve tube contents along the phloem pathway from the peduncle to the rachis or along the rachis itself. Neither was a gradient detected in osmolality along the sieve tube pathway from the rachis through the rachilla and grain stalk to the crease. Demonstrable solute gradients occurred only across those tissues of the grain crease between the crease sieve tubes and the endosperm cavity, a distance of just 1 millimeter. However, while the sucrose concentration in the sieve tubes was almost tenfold that in the endosperm cavity sap, total amino acids were only threefold higher, and the potassium concentrations of the two were equal. Our observations strongly implicate the movement of assimilates from the sieve tubes and across the crease tissues as important control points in grain filling. PMID- 16665130 TI - Effect of heat shock on the metabolism of glutathione in maize roots. AB - High performance liquid chromatography analyses revealed that glutathione (GSH) and cysteine are two of the major low molecular weight thiol compounds in maize root extracts. Treatment of maize roots to heat shock temperatures of 40 degrees C resulted in a decrease of cysteine levels and an increase of GSH levels. Pulse labeling of maize roots with [(35)S]cysteine showed that the rate of incorporation of (35)S into GSH or glutathione disulfide (GSSG) in heat shocked tissues was twice that in nonheat shocked tissues. In addition, extracts from heat shocked maize, barley, and soybean tissues contained an unidentified low molecular weight compound that increased from 1.2- to 8-fold within 2 hours of heat shock treatment depending on the tissue and plant involved. Our results indicate that during heat shock there is an increase in the activity of the GSH synthetizing capacity in maize root cells. The elevated synthesis of GSH may be related to the cells capacity to cope with heat stress conditions. PMID- 16665131 TI - A New Lectin from Meadow Saffron (Colchicum automnale). AB - A lectin has been isolated from tubers of the meadow saffron (Colchicum autumnale). It is an octameric protein (M(r) 100,000) composed of 4A- and 4B subunits of M(r) 15,000 and 10,000, respectively. It is a glycoprotein with 4.4% carbohydrate, the main sugars are (N-acetyl-) glucosamine, mannose, fucose, and xylose. Although the Colchicum autumnale agglutinin (CAA) agglutinates human red blood cells, it has a much higher activity with rabbit erythrocytes. With respect to its carbohydrate-binding specificity CAA behaves rather unusually as it is inhibited by lactose, galactose, N-acetylgalactosamine and related sugars when assayed with human red blood cells but not in assays with rabbit erythrocytes. PMID- 16665132 TI - 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Potato Tuber (Solanum tuberosum L.). AB - 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was purified to electrophoretic homogeneity from tubers of Solanum tuberosum L. cv Superior. The enzyme is a dimer with a native molecular weight of 110,000. The enzyme appears to be hysteretic. The enzyme activity is stimulated by Mn(2+) and l-tryptophan. Chromatofocusing resolved two forms of the enzyme with isoelectric points of 7.8 and 8.4, respectively. The enzyme closely resembles an analogous activity previously isolated from roots of Daucus carota (JA Suzich, JFD Dean, KM Herrmann 1985 Plant Physiol 79: 765-770). PMID- 16665133 TI - Effects of Tabtoxinine-beta-Lactam on Nitrogen Metabolism in Avena sativa L. Roots. AB - The effects of tabtoxinine-beta-lactam (T-beta-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO(3) and 0.5 mm T-beta-L solution for 24 hours took up T-beta-L and lost approximately 90% of their root GS activity. [(3)H]-T-beta-L taken up by roots of seven-day-old oat seedlings was associated with GS immunoprecipitated from the extract of these roots. Total nitrate uptake and in vivo NR activity were decreased approximately 50% in the T-beta-L treated roots. However, T-beta-L uptake did not affect the induction phases of nitrate uptake or reduction, nor did it inhibit in vitro NR activity. Thus, the decrease in nitrate uptake and reduction is a secondary effect of T-beta-L action. Roots of seven-day-old oat seedlings were inoculated with Pseudomonas syringae pv tabaci (Tox+) and the pathogen population in the rhizosphere was estimated by dilution plate count; 6 x 10(13) bacteria were recovered after 3 days, as compared to the original inoculation with 7 x 10(9) bacteria, indicating a significant growth of the pathogen in the rhizosphere. The bacteria recovered from the rhizosphere caused chlorosis in tobacco leaves and produced T-beta-L in culture; 1 x 10(14) bacteria were recovered from roots of seedlings inoculated with P. syringae pv tabaci (Tox-) using the same inoculation and assay procedure as for the pv tabaci (Tox+). Extracts of surface-sterilized roots previously inoculated with P. syringae pv tabaci (Tox+) did not produce viable bacterial cultures when plated out on a complete medium. Oat seedlings growing in sand culture and inoculated with P. syringae pv tabaci (Tox+) had developed chlorosis, and root GS activity had declined to less than 10% of controls after 3 days. Conversely, seedlings inoculated with P. syringae pv tabaci (Tox-) never developed chlorosis and maintained normal levels of GS activity. All oat plants inoculated with P. syringae pv tabaci (Tox+) died within 7 days after inoculation as compared to the plants inoculated with P. syringae pv tabaci (Tox-) which grew to maturity. PMID- 16665134 TI - Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine. AB - When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [(15)N]H(4) (+) prior to supplying the inhibitor. Analyses of the (15)N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [(15)N]H(4) (+), are (14)N enriched and are not apparently derived from (15)N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes. PMID- 16665135 TI - Contribution of Metabolites of Photosynthesis to Postillumination CO(2) Assimilation in Response to Lightflects. AB - In the shade plant Alocasia macrorrhiza grown in low light, photosynthetic CO(2) assimilation during a 5 second lightfleck plus postillumination CO(2) assimilation can allow up to 60% more photosynthesis than that which occurs during 5 seconds of steady state light of the same intensity (RL Chazdon, RW Pearcy 1986 Oecologia. 69: 524-531). Metabolites of photosynthesis were measured to determine if the pool of ribulose 1,5-bisphosphate (RuBP) could account for all of the postillumination CO(2) assimilation following a lightfleck in Alocasia. It was found that the pool of triose-P was much larger than that of RuBP and could account for five times more postillumination CO(2) assimilation than could RuBP. The same trend was seen in the sun plant Phaseolus vulgaris when it was grown in the shade. In contrast, sun-grown Alocasia and Phasiolus did not have a large pool of triose-P relative to RuBP following a lightfleck. In sun plants, carbon may rapidly be converted to RuBP in the light whereas in shade plants there may be a restriction in the path between the triose-P and RuBP pools. It is hypothesized that in shade plants the buildup of triose-P rather than RuBP during the lightfleck prevents inhibition of electron transport which may otherwise occur because of competition for ATP between the two kinases of the photosynthetic carbon reduction cycle. Utilization of the triose-P for postillumination CO(2) fixation would require the capacity for significant postillumination ATP synthesis. The extensive grana stacking and large intrathylakoid space which accompanies the high level of chlorophyll in low-light grown Alocasia could be an important contributing factor to postillumination ATP formation. PMID- 16665136 TI - Synthesis of globulins in maize embryos. AB - The two major components of the globulin fraction in Zea mays embryos are specified by the Prot gene. Pulse-chase analysis of protein synthesis in cultured, immature embryos indicates that the smaller Prot-specific polypeptide, PROT, is derived from the larger polypeptide, PROT'. These experiments also demonstrate that PROT' is derived from a short-lived precursor polypeptide, prePROT'. The primary Prot-specific translation product, as detected by in vitro translation of immature embryo RNA, is of a lower apparent molecular weight than pre-PROT', suggesting the involvement of co- and/or post-translational modification in the production of prePROT'. PMID- 16665137 TI - The complete amino Acid sequence for the anaerobically induced aldolase from maize derived from cDNA clones. AB - A cDNA library was synthesized from maize anaerobic root mRNA and screened with cDNA specific to the anaerobically induced Zea mays cytoplasmic aldolase. At least 1% of the cDNA of the library corresponded to maize cytoplasmic aldolase. The sequence of four overlapping cDNA clones encoded a protein of molecular weight 38,611 homologous to aldolase. These cDNAs were polymorphic at three bases and one of these cDNAs had a different, shorter 3'-untranslated region. No known eukaryotic poly(A) addition site was detected. The derived amino acid sequences of maize was compared to the sequence of aldolase of trypanosome, Drosophila, and two mammalian isozymes, A and B. Of these, maize cytoplasmic aldolase was found to have the highest homology (55%) with rabbit aldolase A. PMID- 16665138 TI - Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence. AB - When intact corn leaves were provided millimolar concentrations of d-mannose through the transpiration stream photosynthesis was inhibited; 5.7 millimolar resulted in a 50% inhibition of the carbon exchange rate. This inhibition was partially reversible by the addition of orthophosphate to the feeding solution. Mannose metabolism by corn leaves was limited in that it did not act as a resource for sucrose or starch synthesis. Mannose 6-phosphate accumulated in the leaf tissues and was slowly metabolized by a pathway involving mannose 1 phosphate. Correlated with the mannose-6-phosphate accumulation were decreases in ATP, orthophosphate, sucrose, and phosphoenolpyruvate and increases in starch and maltose. When provided in the transpiration stream mannose had access to both mesophyll and bundle sheath cells. Mannose feeding led to oscillations in steady state chlorophyll fluorescence emission (680 nanometers) and an elimination of the Kautsky effect during fluorescence induction. Pyridoxal 5-phosphate and 2,4 dinitrophenol were found to be inhibitors of CO(2) exchange when provided in the transpiration stream of intact corn leaves. However, Pyridoxal 5-phosphate induced a quenching of steady state fluorescence while 2,4-dinitrophenol led to an increase in fluorescence emission. PMID- 16665139 TI - The influence of dwarfing interstocks on the distribution and metabolism of xylem applied [h]gibberellin a(4) in apple. AB - The influence of an interstock of the dwarfing cultivar M9 and the nondwarfing cultivar MM115 on the distribution and metabolism of labeled gibberellic acid A(4) ([(3)H]GA(4)) of high specific radioactivity (5.18 x 10(10) becquerel per millimole) applied to the xylem of the rootstock in grafted apple (Malus x domestica Borkh.) trees was compared. Free [(3)H] GA-like metabolites of [(3)H]GA(4), including putative GA(1), GA(2), GA(3), and GA(34), as well as various (3)H-putative GA glucosyl conjugates were detected in stem segments from both cultivars. M9 interstocks reduced the total uptake of [(3)H]GA(4) and decreased the proportion of (3)H metabolites transported to the shoots and leaves of scions. The M9 interstock tissue and adjacent rootstock and scion tissue retained a much greater amount and a higher proportion of the label than did comparable tissue of the nondwarfing MM115 interstock. In addition, the amount and proportion of free [(3)H]GAs was higher, and the proportion of putative [(3)H]GA glucosyl conjugates lower, in M9 interstocks compared to MM115. These effects of the dwarfing interstock on GA distribution and metabolism indicate a significant role for GAs in any satisfactory explanation of the dwarfing mechanism in apple. PMID- 16665140 TI - Stimulation of delta-Aminolevulinic Acid Formation in Algal Extracts by Heterologous RNA. AB - Formation of the chlorophyll and heme precursor delta-aminolevulinic acid (ALA) from glutamate in soluble extracts of Chlorella vulgaris, Euglena gracilis, and Cyanidium caldarium was stimulated by addition of low molecular weight RNA derived from greening algae or plant tissue. Enzyme extracts were prepared for the ALA formation assay by high-speed centrifugation, partial RNA depletion, and gel filtration through Sephadex G-25. RNA was extracted from greening barley epicotyls, greening cucumber cotyledon chloroplasts, and growing cells of Chlorella, Euglena, Chlamydomonas reinhardtii, and Anacystis nidulans, freed of protein, and fractionated on DEAE-cellulose to yield an active component corresponding to the tRNA-containing fraction. RNA from homologous and heterologous species stimulated ALA formation when added to enzyme extracts, and the degree of stimulation was proportional to the amount of RNA added. Algal enzyme extracts were stimulated by algal RNAs interchangeably, with the exception of RNA prepared from aplastidic Euglena, which did not stimulate ALA production. RNA from greening cucumber cotyledon chloroplasts and greening barley epicotyls stimulated ALA formation in algal enzyme incubations. In contrast, tRNA from Escherichia coli, both nonspecific and glutamate-specific, as well as wheat germ, bovine liver, and yeast tRNA, failed to reconstitute ALA formation. Moreover, E. coli tRNA inhibited ALA formation by algal extracts, both in the presence and absence of added algal RNA. Chlorella extracts were capable of catalyzing aminoacyl bond formation between glutamate and both the activity reconstituting and nonreconstituting RNAs, indicating that the inability of some RNAs to stimulate ALA formation was not due to their inability to serve as glutamyl acceptors. The first step in the ALA-forming reaction sequence has been proposed to be activation of glutamate via aminoacyl bond formation with a specific tRNA, analogous to the first step in peptide bond formation. Our results suggest that the RNA that is required for ALA formation may be functionally distinct from the glutamyl-tRNA species involved in protein synthesis. PMID- 16665141 TI - Effects of NaCl and CaCl(2) on Cell Enlargement and Cell Production in Cotton Roots. AB - In many crop species, supplemental Ca(2+) alleviates the inhibition of growth typical of exposure to salt stress. In hydroponically grown cotton seedlings (Gossypium hirsutum L. cv Acala SJ-2), both length and weight of the primary root were enhanced by moderate salinities (25 to 100 millimolar NaCl) in the presence of 10 millimolar Ca(2+), but the roots became thinner. Anatomical analysis showed that the cortical cells of these roots were longer and narrower than those of the control plants, while cortical cells of roots grown at the same salinities but in the presence of only 0.4 millimolar Ca(2+) became shorter and more nearly isodiametrical. Cell volume, however, was not affected by salinities up to 200 millimolar NaCl at either 0.4 or 10 millimolar Ca(2+). Our observations suggest Ca(2+)-dependent effects of salinity on the cytoskeleton. The rate of cell production declined with increasing salinity at 0.4 millimolar Ca(2+) but at 10 millimolar Ca(2+) was not affected by salinities up to 150 millimolar NaCl. PMID- 16665142 TI - In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase. AB - Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with (32)P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [gamma-(32)P]ATP or [beta-(32)P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation ( approximately 25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6 phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants. PMID- 16665143 TI - Compatible and incompatible rhizobia alter membrane potentials of soybean root cells. AB - Inoculation with Rhizobium japonicum or R. meliloti reduced the electrical transmembrane potential (E(m)) of soybean (Glycine max [L.] Merr.) root cells within 1 day. The response could be attributed to altered diffusion potential (E(D)). E(m) values return to control levels by the second day after inoculation, but again were reduced in R. meliloti-inoculated tissue on the seventh day. Increased concentrations of sodium phosphate in the perfusion solution magnified the effects of inoculation on E(m). Neither heat-killed rhizobia nor living cells of Pseudomonas fluorescens elicited the response. The E(m) and E(D) of nodule cells were nearly 20% lower than corresponding values from adjacent cortical cells of the root. PMID- 16665144 TI - Exoamylase activity in vacuoles isolated from pea and wheat leaf protoplasts. AB - Vacuoles isolated from pea (Pisum sativum), and wheat (Triticum aestivum) leaf protoplasts contained considerable activities of electrophoretically highly mobile exoamylases. Vacuoles from spinach (Spinacia oleracea) leaf and photoautotrophic Chenopodium rubrum suspension culture cell protoplasts were devoid of amylolytic activity. Endoamylase activity was in all cases associated primarily with the chloroplast. PMID- 16665145 TI - Endogenous Auxin and Ethylene in the Lichen Ramalina duriaei. AB - Indole-3-acetic acid (IAA) levels and ethylene evolution rates were measured in a fruticose lichen Ramalina duriaei collected from carob trees growing in northeast Israel. IAA levels were estimated by gas liquid chromatography with electron capture detection of the pentafluorobenzyl ester and also by enzyme-linked immunosorbent assay following methylation. The identity of the isolated IAA was confirmed by gas chromatography-mass spectrometry of both the methyl and the pentafluorobenzyl ester. IAA levels in lichens 1 year after transplanting to an air-polluted urban site were found to be lower than in the control thalli left at a nonpolluted, rural site. The material from the latter contained about 2.5 micrograms per gram fresh weight free IAA and 0.5 microgram per gram fresh weight conjugated IAA, while the urban material contained 0.3 microgram per gram each of free and conjugated IAA. Ethylene production rate was 1.0 nanoliter per gram fresh weight per hour in the material from the rural site and 1.5 nanoliters per gram fresh weight per hour in material from the urban site. PMID- 16665147 TI - Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh. AB - Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C(18) columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3'-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid. PMID- 16665146 TI - The effects of copper nutrition and developmental state on the biosynthesis of diamine oxidase in clover leaves. AB - The biosynthesis of diamine oxidase (DAO; EC 1.4.3.6) in leaf blades of subterranean clover (Trifolium subterraneum L. cv Seaton Park) was followed by labeling whole plants with (14)CO(2). A pulse-chase experiment where DAO was immunoprecipitated with anti-DAO antibodies showed that only leaf primordia and the youngest emerged leaves were able to synthesize the enzyme. The amount of DAO in young leaves of clover grown with a range of Cu treatments was determined by its enzymic activity and by single radial immunodiffusion against anti-DAO antibodies; both parameters were highly correlated with the Cu concentration of the leaf. Further, anti-DAO antibodies reacted against apo-DAO prepared in vitro indicating that apo-DAO was absent from Cu-deficient leaves. These results suggest that the biosynthesis of DAO in young clover leaves is controlled by the Cu concentrations of the leaves. Poly(A) mRNA purified from leaf primordia and young emerging leaves of plants with either a high Cu or low Cu supply was translated in wheat germ and rabbit reticulocyte cell-free systems. No differences between the two Cu treatments could be seen in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the translation products after fluorography. However, anti-DAO antibodies did not detect any DAO synthesized in vitro from either treatment. PMID- 16665148 TI - The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro. AB - A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase). PMID- 16665149 TI - Restriction enzyme analysis of tomato chloroplast and chromoplast DNA. AB - Plastid DNA was isolated from the chloroplasts of tomato (Lycopersicon esculentum var Traveler 76) leaves and the chromoplasts of ripe tomato fruit. Comparisons of the two DNAs were made by restriction endonuclease analysis using PvuII, HpaI, and Bg1I. No differences in the electrophoretic banding patterns of the restricted plastid DNAs were detected, indicating that no major rearrangements, losses, or gains of plastid DNA accompany the transition from chloroplast to chromoplast. PMID- 16665150 TI - Electrical evidence for turgor inhibition of proton extrusion in sugar beet taproot. AB - Sections of sugar beet (Beta vulgaris L.) taproot were incubated in various concentrations of mannitol. At 0.4, 0.6, and 0.8 molar, the membrane electrical potential difference (E(m)) averaged about -130 millivolts; at 0.2 molar, about 90 millivolts; and at 0 molar, between -60 and -80 millivolts. Additions of 10 millivolts acetate to the incubation solutions (all at pH 5) enhanced the membrane polarity to about -200 millivolts. We conclude from these and previous findings that high turgor inhibits proton extrusion in the sugar beet, but that proton extrusion can be activated in fully turgid tissue by acidification of the cytoplasm. A possible function of this turgor effect may be the control of turgor itself. PMID- 16665151 TI - Effects of wounding on cytokinin activity in cucumber cotyledons. AB - Three known physiological responses to exogenous cytokinins were measured in wounded and nonwounded cotyledons from cucumber (Cucumis sativus L. cv Marketer) seedlings grown in darkness. Enhanced cell division, chlorophyll formation, and cotyledon expansion were detected in wounded cotyledons. The data suggest that wounding enhances endogenous cytokinin activity. PMID- 16665152 TI - Symplastic growth and symplasmic transport. AB - IN CURRENT USAGE, THE ADJECTIVE SYMPLASTIC HAS TWO DIFFERENT MEANINGS: in the term, symplastic growth, as defined by Priestley; and in discussions of transport through the symplast, as defined by Munch. To avoid confusion, it is recommended that symplastic be reserved to characterize growth deformation, that symplasmic be used to refer to transport through the symplasm, and apoplasmic, to refer to the apoplasm. PMID- 16665153 TI - Presence and identification of polyamines in xylem and Phloem exudates of plants. AB - Polyamines were identified by high performance liquid chromatography (benzoylation) and by thin layer chromatography (dansylation) in xylem exudates from stems of sunflowers (Helianthus annuus [L.]), mung bean (Vigna radiata [L.] Wilczek), grapevine (Vitis vinifera [L.] cv Grenache), and orange (Citrus sinensis [L.] Osbeck, cv Valencia), as well as in phloem sap (using elution into EDTA) of sunflower and mung bean plants. Putrescine was the major polyamine detected, ranging in concentrations of 150 to 9200 picomoles per milliliter exudate, whereas only trace amounts of spermine were detected. High amounts of putrescine and spermidine were found in EDTA eluates (possibly phloem sap) as compared with elution into water. Concentrations of putrescine and spermidine in xylem exudates were related to the physiological conditions of the plants prior to exudate collection. More putrescine was found in exudates of older than in younger sunflower plants, and salt stress applied to sunflower plants resulted in a higher concentration of putrescine and spermidine in the exudate. The presence and abundance of putrescine and spermidine in xylem and phloem exudates indicate that polyamines may be translocated in plants. This long-distance translocation further supports the hypothesis that polyamines have a regulatory role in plant growth and response to stress. PMID- 16665154 TI - In Vivo Magnetic Resonance Microscopy of Changing Water Content in Pelargonium hortorum Roots. AB - Magnetic resonance imaging (MRI) was used to nondestructively observe changes in water content in roots of Pelargonium hortorum x Bailey during a period of relatively rapid transpiration. Anatomical regions of the root could be differentiated with a spatial resolution of 0.1 x 0.1 mm. MRI shows great potential for study of plant-water relations. PMID- 16665155 TI - Species variation in the predawn inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase. AB - The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (==40 degrees C), the complex-type alpha-amylase isoform, resistant to hydrolytic digestion by endo-beta-N-acetylglucosaminidase H (Endo-beta-H) was predominantly secreted, whereas at lower temperatures (<==15 degrees C), the isoform susceptible to Endo-beta-H attack was the major molecular form secreted. PMID- 16665162 TI - Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata: I. Changes during Flower Development and Pollination. AB - Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination. PMID- 16665163 TI - Metabolic changes associated with adaptation of plant cells to water stress. AB - Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from (15)N labeling kinetics after substituting [(15)N]ammonium and [(15)N]nitrate for the (14)N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and gamma aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and gamma-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and gamma-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low (15)N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation. PMID- 16665164 TI - Kinetic Characterization of Nitrite Uptake and Reduction by Chlamydomonas reinhardtii. AB - Kinetics of nitrite uptake and reduction by Chlamydomonas reinhardtii cells growing phototrophically has been studied by means of progress curves and the Michaelis-Menten integrated equation. Both uptake and reduction processes exhibited hyperbolic saturation kinetics, the nitrite uptake system lacking a diffusion component. Nitrite uptake and reduction showed significant differences in K(s) for nitrite at pH 7.5 (1.6 versus 20 micromolar, respectively), optimal pH, activation energy values, and sensitivity toward reagents of sulfhydryl groups. K(s) values for nitrite uptake were halved in cells subjected to darkness or to nitrogen-starvation. Nitrate inhibited nitrite uptake by a partially competitive mechanism. The same inhibition pattern was found for nitrite uptake by C. reinhardtii mutant 305 cells incapable of nitrate assimilation. The results demonstrate that C. reinhardtii cells take up nitrite via a highly specific carrier, probably energy-dependent, kinetically responsive to environmental changes, distinguishable from the enzymic nitrite reduction and endowed with an active site for nitrite not usable for nitrate transport. PMID- 16665165 TI - Evidence for Phytochrome Regulation of Gibberellin A(20) 3beta-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L. AB - The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA(20)), the product (GA(1)), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA(1) under red irradiation and in darkness. The lele plants grew in response to GA(20) and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA(20) and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3beta-hydroxylation of GA(20) to GA(1) in genotype lele is due to a Pfr-induced blockage in the expression of that activity. PMID- 16665166 TI - Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants. AB - Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant. PMID- 16665167 TI - Carbon dioxide enhances the development of the ethylene forming enzyme in tobacco leaf discs. AB - Since CO(2) is known to stimulate ethylene production by promoting the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, the effect of CO(2) on the activity and the development of the ethylene forming enzyme (EFE) was studied in tobacco (Nicotiana tabacum L. cv Havana 425 and Xanthi) leaf discs. In addition to previous observations that EFE activity is dependent on CO(2) concentration and is saturable with 2% CO(2), present data show two saturation curves at 2% and 10% CO(2). Promotion of EFE development was dependent also on CO(2) concentration (saturated at 2% CO(2)) and duration (maximum at 24 in the dark), and was abolished by 20 micromolar cycloheximide. Application of exogenous ethylene (20 microliters per liter) or light treatment further increased the CO(2)-enhanced development of EFE, implying that these two factors can also affect EFE development via interaction with CO(2). The results suggest that CO(2) exerts its stimulatory effect on the conversion of ACC to ethylene by enhancing not only the activity but also the synthesis of EFE in leaf discs. PMID- 16665168 TI - Cytokinin-induced ethylene biosynthesis in nonsenescing cotton leaves. AB - The influence of cytokinins on ethylene production was examined using cotton leaf tissues. Treatment of intact cotton (Gossypium hirsutum L. cv LG 102) seedlings with both natural and synthetic cytokinins resulted in an increase in ethylene production by excised leaves. The effectiveness of the cytokinins tested was as follows: thidiazuron >> BA >> isopentyladenine >/= zeatin >> kinetin. Using 100 micromolar thidiazuron (TDZ), an initial increase in ethylene production was observed 7 to 8 hours post-treatment, reached a maximum by 24 hours and then declined. Inhibitors of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis and its oxidation to ethylene reduced ethylene production 24 hours post-treatment; however, by 48 hours only inhibitors of ACC oxidation were effective. The increase in ethylene production was accompanied by a massive accumulation of ACC and its acid-labile conjugate. TDZ treatment resulted in a significant increase in the capacity of tissues to oxidize ACC to ethylene. Endogenous levels of methionine remained constant following TDZ treatment. It was concluded that the stimulation of ethylene production in cotton leaves following cytokinin treatment was the result of an increase in both the formation and oxidation of ACC. PMID- 16665169 TI - Effect of Osmotic Stress on Ion Transport Processes and Phospholipid Composition of Wheat (Triticum aestivum L.) Mitochondria. AB - The effect of osmotic stress on wheat (Triticum aestivum L.) mitochondrial activity and phospholipid composition was investigated. Preliminary growth measurements showed that osmotic stress (-0.25 or -0.5 megapascal external water potential) inhibited the rate of shoot dry matter accumulation while root dry matter accumulation was less sensitive. We have determined that differences in sensitivity to osmotic stress existed between tissues at the mitochondrial level. Mitochondria isolated from roots or shoots of stressed seedlings showed respiratory control and ADP/O ratios similar to control seedlings which indicates that stressed mitochondria were well coupled. However, under passive swelling conditions in a KCl reaction mixture, the rate and extent of valinomycin-induced swelling of shoot mitochondria were increased by osmotic stress while root mitochondria were largely unaffected. Active ion transport studies showed efflux transport by stressed-shoot mitochondria to be partially inhibited since mitochondrial contraction required the addition of N-ethylmaleimide or nigericin. Efflux ion transport by root mitochondria was not inhibited by osmotic stress which indicates that stress-induced changes in ion transport were largely limited to shoot mitochondria. Characterization of mitochondrial fatty acid and phospholipid composition showed an increase in the percentage of phosphatidylcholine in stressed shoot mitochondria compared to the control. Mitochondrial fatty acid composition was not markedly altered by stress. No significant changes in either the phospholipid or fatty acid composition of stressed root mitochondria were observed. Hence, these results suggest that a tissue-specific response to osmotic stress exists at the mitochondrial level. PMID- 16665170 TI - Mineral Nutrient Requirements of a Loblolly Pine (Pinus taeda) Cell Suspension Culture : Evaluation of a Medium Formulated from Seed Composition Data. AB - The mineral nutrient requirements of Pinus taeda cells were explored using quantitative cell culture growth measurements. An appraisal was thereby made of the critical features of a novel and successful medium which was developed specifically for this gymnosperm using chemical composition data for developing seeds, and characterized by generally high concentration of all micronutrients, high magnesium, and low calcium. The high magnesium concentration was found not to be detrimental and possibly beneficial whereas the calcium level bordered on a deficiency threshold. Within the microelements high iodide was found to be essential, as was a higher borate level than is present in media developed for angiosperms. High zinc concentrations were also beneficial, with normal levels permitting slower but nevertheless healthy growth. An improved medium was thereby formulated which was stress-free and exhibited broader genotype specificity. This new formulation has proved very successful in maintaining long-term growth of highly uniform and apparently meristematic suspension cultures of Pinus radiata. PMID- 16665171 TI - Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch). AB - The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4 methyleneglutamine. Pulse-feeding nodulated roots with (15)N(2) gas showed asparagine to be the principal nitrogen product exported from N(2)-fixing nodules. Maintaining root systems in an N(2)-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4 Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N(2) fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of (15)N-nitrate, and a (15)N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N(2) or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N(2). Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N(2) and nitrate are being used in peanuts. PMID- 16665172 TI - Cryptochrome, Phytochrome, and the Photoregulation of Anthocyanin Production under Blue Light. AB - The principle of equivalent light action predicts that two light treatments (wavelengths ;lambda(1) and lambda(2)) producing the same Pfr/P ratio (phi(lambda1) = phi(lambda2)) and the same rate of phytochrome photoconversion (k(lambda1) = k(lambda2)) are perceived by phytochrome as being the same and should produce the same effect. The results of experiments based on the principle of equivalent light action indicate that cryptochrome is involved in the photoregulation of anthocyanin production elicited by blue light in tomato seedlings. This was also the case for one strain of cabbage seedlings. For another strain of cabbage seedlings, the results suggest that cryptochrome is either not involved or that the state of phytochrome is the principal limiting factor. PMID- 16665173 TI - Comparison of spectral properties of phytochromes from different preparations. AB - The spectral parameters of phytochrome in vitro described in several recent studies have been determined in purer and less degraded phytochrome samples than those used by Butler, Hendricks and Siegelman more than 20 years ago. There are considerable differences between the old and the new data. It seems logical that the researchers interested in phytochrome-mediated photomorphogenesis should use the new data for ;native' phytochrome instead of the old ones, still in common use. A comparison of the spectral properties of phytochrome described in old and recent studies show that the differences among the ;new' data are as large or larger, depending on the particular parameter and wavelength region considered, than the differences between ;old' and ;new' data. Therefore, if one should decide to use the new data instead of the old ones, one must also decide which set of new data should be used. The latter is a difficult choice. No matter what the choice, it should be open to revision in the future, the limitations associated with the choice should be noted, and the use of any set of spectral parameters of phytochrome should be made with extreme caution. PMID- 16665174 TI - Enhancement of [C]Sucrose Export from Source Leaves of Vicia faba by Gibberellic Acid. AB - The effect of gibberellic acid (GA(3)) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA(3) (10 micromolar) applied to the source leaf, enhanced export of [(14)C]sucrose (generated by (14)CO(2) fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA(3) was applied 24 hours prior to the (14)CO(2) pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA(3) increased the proportion of sucrose exported. After a 10-min exposure to [(14)C]GA(3), radioactivity was found only in the source leaf. Following a 2 hour exposure to [(14)C]GA(3), radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [(14)C]GA(3) in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA(3) effect in the lower stem with time after treatment. We conclude that in the short term, GA(3) enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA(3) is outside the source leaf. GA(3) accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA(3) is rapidly metabolized in the lower stem thus attenuating any GA effect. PMID- 16665175 TI - Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase. AB - The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl(2), carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg(2+) at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K(act)(CO(2)) of rubisco activation in the reconstituted system was 4 micromolar CO(2), compared to a K(act)(CO(2)) of 25 to 30 micromolar CO(2) for the previously reported spontaneous CO(2)/Mg(2+) activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO(2) and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro. PMID- 16665176 TI - Synthesis and Turnover of Proteins in Proplastids and Chloroplasts of Euglena gracilis. AB - Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg(2+). ATP and Mg(2+) also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg(2+) have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose 1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids. PMID- 16665177 TI - An NADH-Dependent Acetoacetyl-CoA Reductase from Euglena gracilis: Purification and Characterization, Including Inhibition by Acyl Carrier Protein. AB - An NADH-dependent acetoacetyl-CoA reductase from Euglena gracilis variety bacillaris was extensively purified and characterized. Two different isoelectric forms of the reductase with identical characteristics otherwise were found. The reductase was noncompetitively inhibited by acyl carrier protein, K(i) 5.6 micromolar at pH 5.4; this inhibition decreased with increasing pH or ionic strength. Coenzyme A was a competitive inhibitor, K(i) 230 micromolar. Kinetic parameters with respect to acetoacetyl-CoA and NADH were sensitive to changes in pH and ionic strength. PMID- 16665178 TI - Malate inhibition of phosphoenolpyruvate carboxylase from crassula. AB - Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate(2-) concentration. Of the reaction components only HCO(3) (-) increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K(m), but no effect on V(max). Enzyme preincubated with malate shows the same effect of malate on K(m), but in addition V(max) is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate. PMID- 16665179 TI - Abscission of mango fruitlets as influenced by enhanced ethylene biosynthesis. AB - Experiments were conducted on developing fruitlet explants of two mango (Mangifera indica L.) cultivars to establish the source and dynamics of ethylene production prior to and during fruitlet abscission. Abscission of all fruits in the samples occurred at approximately 86 and 74 hours postharvest in ;Keitt' and ;Tommy Atkins,' respectively. Increased abscission began 26 hours from harvest and was preceded by enhanced ethylene synthesis. Enhanced ethylene production initiated approximately 48 hours prior to abscission and increased to a maximum near the time of fruitlet abscission. The seed produced the highest amount of ethylene on a per gram fresh weight basis. The pericarp, however, was the main source of ethylene on an absolute basis, since it represented more than 85% of total fruitlet weight. Pedicels containing the abscission zone produced no detectable ethylene prior to or at the moment of abscission. Fumigation of ;Tommy Atkins' fruitlets with 1, 15, or 100 microliters per liter ethylene accelerated abscission by 24 to 36 hours in comparison with unfumigated controls. Diffusion of ethylene from distal fruitlet tissues to the abscission zone triggers the events leading to separation of the fruit from the tree. PMID- 16665180 TI - Use of the pressure vessel to measure concentrations of solutes in apoplastic and membrane-filtered symplastic sap in sunflower leaves. AB - A simple, repeatable, and accurate method is described for the collection of apoplastic and membrane-filtered symplastic sap fractions, and for the determination of the origin of these fractions within the leaf. The apoplastic distribution patterns of the naturally occurring apoplastic leaf solutes, and the apoplastic dye PTS (trisodium 3-hydroxy-5, 8, 10-pyrenetrisulfonate) were compared. Aliquots of sap were expressed from detached sunflower leaves in a pressure chamber over intervals of 0.02 to 0.04 megapascal. Three distinct fractions were detected in the expressed sap volume. These were successively released and identified as a petiole-midrib fraction, a minor vein-cell wall fraction, and a mixed fraction consisting of a contribution from the minor vein cell wall with an increasing proportion of membrane-filtered cell sap. PMID- 16665181 TI - Detection of glycosylated and deglycosylated extensin precursors by indirect competitive ELISA. AB - A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the rapid quantitation of the glycosylated and deglycosylated forms of the monomeric soluble extensin precursor subunits P1 and P2. A log-linear response range for each kind of precursor in the competition curve was between 0.01 and 100 nanograms per milliliter. PMID- 16665182 TI - Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm. AB - ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles alpha glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30 degrees C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate. PMID- 16665183 TI - The Effect of pH, O(2), and Temperature on the CO(2) Compensation Point of Isolated Asparagus Mesophyll Cells. AB - The effect of pH, O(2) concentration, and temperature on the CO(2) compensation point (capital GHE, Cyrillic[CO(2)]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO(2) depending upon experimental conditions. The capital GHE, Cyrillic(CO(2)) increases with increasing temperature. The rate of increase is dependent upon the O(2) concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O(2) concentrations. The differential effect of temperature on capital GHE, Cyrillic(CO(2)) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O(2)-sensitive component of capital GHE, Cyrillic(CO(2)) remains constant over this range. The capital GHE, Cyrillic(CO(2)) of Asparagus cells at 25 degrees C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O(2) concentration. It is suggested that the pH-dependence of capital GHE, Cyrillic(CO(2)) is related to the ability of the cell to take up CO(2) from the aqueous environment. The correlation between high HCO(3) (-) concentrations and low capital GHE, Cyrillic(CO(2)) at alkaline pH indicates that extracellular HCO(3) (-) facilitates the uptake of CO(2), possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O(2)- and temperature-dependence of capital GHE, Cyrillic(CO(2)) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO(2) to a level sufficient to reduce or suppress photorespiration. PMID- 16665184 TI - The Effects of Light and Gravity on the Horizontal Curvature of Roots of Gravitropic and Agravitropic Arabidopsis thaliana L. AB - In an attempt to study and distinguish the effects of light and gravity on the direction of horizontal root growth, wild-type and an agravitropic mutant of Arabidopsis thaliana L., aux-1 were examined. The mutant aux-1 seedling roots are agravitropic but do respond to light, thus allowing the effects of light and gravity on roots to be studied separately. It is shown that in addition to the recognized negative phototropic and positive gravitropic responses of the root, there are also horizontal curvatures (clockwise or counterclockwise) induced by both unilateral light and gravity. The effects of light and gravity in inducing the horizontal curvature of roots are synergistic when both act in the same direction, and are antagonistic when acting in opposite directions. The results indicate that light and gravity interact to determine the direction and magnitude of the horizontal curvature of roots. PMID- 16665186 TI - Plant factors controlling seed set in maize : the influence of silk, pollen, and ear-leaf water status and tassel heat treatment at pollination. AB - In maize (Zea mays L.) large decreases in kernel number result when low water potentials (Psi(w)) and high temperatures occur during pollination. To gain insight into the basis for the decreased seed set, silk, pollen, and ear-leaf Psi(w), the capability for silk osmotic adjustment, and pollen appearance were measured to determine their relationship to seed set. A multiple-eared or prolific (high carbohydrate availability to the pistillate inflorescence) hybrid (B73 x FR25), a heat sensitive hybrid (WF9 x A632), and a commercial hybrid (B73 x Mo17) were studied. A cross-pollination experiment, with pollination limited by pollen amount, was conducted to determine the impact on seed set of water and heat stressing the tassel and water stressing the ear. At low Psi(w), silk Psi(w) and seed set were decreased whereas pollen Psi(w), appearance, and viability were unaffected. High temperature resulted in a 2 megapascal decrease in pollen Psi(w), visually damaged pollen being shed, decreased pollen viability, and, in two of the hybrids, substantially decreased pollen shed. Prolificacy did not result in increased silk solute accumulation but did result in superior seed production by the pistillate inflorescence at low Psi(w). The magnitude of the decrease in silk solute potential was small (0.2 megapascal) and similar for all genotypes. One hybrid maintained a relatively high silk turgor but this hybrid also decreased the most in seed production when the pistillate inflorescence was water deficient. These results indicated an adverse effect of high temperature on pollen development, a positive relationship between seed production and silk water status, and no advantage to high silk turgor after silk emergence in maintaining seed production. Additionally, there was no evidence of variation in silk solute regulation capability among hybrids which varied in prolificacy, a trait important in drought tolerance, but the seed production of the pistillate inflorescence of the prolific hybrid was least affected by water deficit. PMID- 16665185 TI - Action of Heavy Metals on Hill Activity and O(2) Evolution in Anacystis nidulans. AB - Addition of 5 micromolar Cu(2+), Cd(2+), and Zn(2+) was inhibitory to 10 micromolar H(2)O(2)-supported Hill activity (dichlorophenolindophenol reduction) and O(2) evolution in membrane preparation from Anacystis nidulans. The reversal of Cd(2+) and Zn(2+) inhibition, in contrast to Cu(2+), by exogenously added catalase (EC 1.11.1.6) suggested that the former cations were inhibitory to H(2)O(2) degradation. Ascorbic acid (20 micromolar) supported 27% of the Hill activity which was insensitive to DCMU (10 micromolar) and the remaining activity, attributable to the DCMU sensitive process, was sensitive to inhibition by Cu(2+) only. It is suggestive that the action site of Cd(2+) and Zn(2+) is located between the electron donation sites of H(2)O(2) and ascorbic acid, while that of Cu(2+) is located beyond it. Electron donation by reduced glutathione was insensitive to DCMU and Cu(2+), indicating that the action site of Cu(2+) is prior to its electron donation site. Further, the phenanthroline (10 micromolar) reversal of Cu(2+) inhibition of Hill activity suggested a tentative action site of Cu(2+) at the level of cytochrome. PMID- 16665187 TI - Biphasic fluence-response curves for phytochrome-mediated kalanchoe seed germination : sensitization by gibberellic Acid. AB - The fluence-response curves for the effect of two red pulses separated by 24 hours on the germination of Kalanchoe blossfeldiana Poelln. cv Vesuv seeds, incubated on gibberellic acid (GA(3)) are biphasic for suboptimal concentrations. The response in the low fluence range corresponds with a classical red/far-red reversible phytochrome mediated reaction. GA(3) induces an additional response in the very low fluence range, which is also phytochrome mediated. The sensitivity to phytochrome-far-red absorbing form (Pfr), however, is increased about 20,000 fold, so that even far-red fluences become saturating. Both in the very low and low fluence response range, the maximal responses induced by saturating fluences are modulated by the GA(3) concentration. GA(3) having no direct influence on the phytochrome phototransformations, alters the Pfr requirement and determines the responding seed population fraction in the very low and low fluence range. The effet of GA(3) appears to be on the transduction chain of the phytochrome signal. PMID- 16665188 TI - Assimilate Unloading from Maize (Zea mays L.) Pedicel Tissues : I. Evidence for Regulation of Unloading by Cell Turgor. AB - Sugar and (14)C-assimilate release from the pedicel tissue of attached maize (Zea mays L.) kernels was studied following treatment with solute concentrations of up to 800 millimolal. Exposure and collection times ranged from 3 to 6 hours. Sugar and (14)C-assimilate unloading and collection in agar traps was reduced by 25 and 43%, respectively, following exposure to 800 millimolal mannitol. Inhibition of unloading was not specific to mannitol, since similar concentrations of glucose, fructose, or equimolar glucose plus fructose resulted in comparable inhibition. Ethylene glycol, a rapidly permeating solute which should not greatly influence cell turgor, did not inhibit (14)C-assimilate unloading. Based on these results, we suggest that inhibition of unloading by high concentrations of sugar or mannitol was due to reduced pedicel cell turgor. Changes in pedicel cell turgor may play a role in the regulation of assimilate transfer within the maize kernel. PMID- 16665189 TI - Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.). AB - Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A(5) (GA(5)) as 1- and 1,2-[(3)H]GA(5) (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [(3)H]GA-like metabolites were separated from the highly H(2)O-soluble [(3)H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted --> isocratic eluted reversed-phase C(18) high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [(3)H] GA(5) were identified as GA(1), GA(3), and GA(6) by GC-SIM. The major highly water soluble metabolite of [(3)H]GA(5) at all levels of substrate GA(5) had chromatographic characteristics similar to authentic GA(1)-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [(3)H]GA(5) feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA(1), GA(3), GA(5) methyl ester, GA(6), GA(22), GA(29) (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA(1), GA(3), GA(5), and GA(8) (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA(6) and GA(29) would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA(5). Increasing substrate GA(5) levels increased the proportion of metabolites with HPLC Rts similar to GA(1), GA(6), and especially GA(1) glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA(3), GA(3)-glucosyl ester, and a postulated conjugate of GA(6). There was evidence that high doses of substrate GA(5) induced new metabolites which often, but not always, differed from GA(1), GA(3), and GA(6) in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M(+) and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities. PMID- 16665190 TI - Regulation of sucrose efflux from soybean leaf discs. AB - Net sucrose efflux from discs of fully expanded leaves of soybean (Glycine max [L.] Merr.) plants was studied to characterize sucrose efflux into the apoplast. Net sucrose efflux had a Q(10) of 2.3, was linear for at least 3.5 hours, and was selective for sucrose over glucose. Sulfhydryl group inhibitors reduced sucrose efflux by up to 80%. There was a biphasic promotion of sucrose efflux by KCl with an apparent saturable component up to about 20 millimolar, above which the effect was linear. Sucrose efflux was promoted by NaCl as a linear function of concentration. Monovalent cation ionophores did not affect sucrose efflux, regardless of external KCl concentration. Light in the absence of added HCO(3) increased sucrose efflux by about 20%. Sucrose efflux was promoted by increasing pH from 4 to about 8, above which no additional effect was observed. When leaf discs were bathed at pH 6.0, the protonophore carbonyl cyanide m chlorophenylhydrazone (CCCP) increased sucrose efflux by about 25%. CCCP in the presence of valinomycin had the same effect as CCCP alone. Inhibition of plasmalemma ATPase activity with N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, or orthovanadate increased sucrose efflux. These data indicate that sucrose efflux from soybean leaf discs is not a result of simple leakage but is a regulated process. PMID- 16665191 TI - The Partial Purification and Characterization of Nuclear and Mitochondrial Uracil DNA Glycosylase Activities from Zea mays Seedlings. AB - Uracil-DNA glycosylase activities from etiolated Zea mays seedling nuclei and mitochondria were partially purified and characterized. Nuclei and mitochondria were separated using sucrose differential and step gradient centrifugation. Experiments with osmotically shocked organelles indicated that enzyme activity from mitochondria was soluble, whereas nuclear enzyme activity was only partially soluble under the conditions tested. Purification using DEAE-cellulose and Affigel Blue column chromatography yielded distinct elution profiles from both columns for each of the organellar enzyme activities. Final purification was 490- and 850- fold for the nuclear and mitochondrial uracil-DNA glycosylase, respectively. Characterization studies demonstrated significant differences between the nuclear and mitochondrial uracil-DNA glycosylase with respect to K(m), temperature, and pH activity optimum, the effect of salts, and substrate preference. Molecular weight as determined by gel filtration was 18,000 for enzymes from both sources. Both were also sensitive to the sulfhydryl group blocking agent N-ethylmaleimide. A number of uracil analogs were tested for their ability to inhibit nuclear and mitochondrial uracil-DNA glycosylase activities. 5 Azauracil, uracil, 6-aminouracil, 6-azauracil, 5-aminouracil, and 5-fluorouracil all inhibited both activities to variable degrees. PMID- 16665192 TI - Gibberellin-Induced Changes in the Populations of Translatable mRNAs and Accumulated Polypeptides in Dwarfs of Maize and Pea. AB - Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize (d-5) and pea (Progress No. 9) lack endogenous gibberellin (GA(1)) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [(35)S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs. PMID- 16665193 TI - Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis. AB - Eight mutants of barley (Hordeum vulgare cv Maris Mink) lacking the chloroplast isozyme of glutamine synthetase (EC 6.3.1.2.) were isolated by their inability to grow under photorespiratory conditions. The cytoplasmic isozyme of glutamine synthetase was present in the leaves of all the mutants, with activities comparable to the wild-type (10-12 nanokatals per gram fresh weight). The mutant plants developed normally and were fully fertile under conditions that minimize photorespiration. In 1% O(2) the rate of CO(2) fixation in leaves of one of the mutants, RPr 83/32, was the same as the wild-type, but in air this rate declined to 60% of the wild-type after 30 minutes. During this time the ammonia concentration in leaves of the mutant rose from 1 to 50 micromoles per gram fresh weight. Such ammonia accumulation in air was found in all the mutant lines. In back-crosses with the parent line, F(1) plants were viable in air. In the F(2) generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic; we designate them gln 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling. PMID- 16665194 TI - Al and Ca Alteration of Membrane Permeability of Quercus rubra Root Cortex Cells. AB - This study was undertaken to quantify the effect of aluminum and calcium on membrane permeability. The influence of Ca(2+) (0.2-3.7 millimolar) and Al(3+) (0 3.7 millimolar) on the permeability of root cortical cells of Quercus rubra was measured using three nonelectrolytes (urea, methyl urea, and ethyl urea) as permeators of progressively increasing lipid solubility. Water permeability was also measured. Al(3+) (a) increased membrane permeability to the nonelectrolytes, (b) decreased the membrane's partiality for lipid permeators, and (c) decreased membrane permeability to water. Ca(2+) had effects on permeability that were opposite to those of Al(3+). When Al(3+) and Ca(2+) were tested in combination, these opposite effects counteracted each other. The results suggest that Al(3+) altered the architecture of membrane lipids. PMID- 16665195 TI - Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis. AB - An O(2) electrode system with a specially designed chamber for ;whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O(2) concentrations on photosynthetic O(2) evolution. At pH = 5.5 and 20% O(2), cells grown in HCO(3) (-) medium (low CO(2), pH >/= 9.0) exhibited a higher affinity for external CO(2) (K((1/2))(CO(2)) = 40 +/- 6 micromolar) than the cells grown for at least 24 hours in high-CO(2) medium (pH = 6.5), (K((1/2))(CO(2)) = 94 +/- 16 micromolar). With O(2) 3),(1-->4)-beta-d-glucosyl sequences. Substrates with (1-->4)-beta-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1-->3)-beta-glucans such as laminarin were not hydrolyzed. When (1-->3),(1-->4)-beta-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 x 10(4)). Activity against a water soluble (1-->3),(1-->4)-beta-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1-->3),(1-->4)-beta-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1-->3),(1-->4)-beta-d-glucan that contained blocks of (1-->4) linkages and/or more than a single contiguous (1-->3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1- >3),(1-->4)-beta-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase. PMID- 16665204 TI - Photosynthetic Assimilation of NO(3) by Intact Cells of the Cyanobacterium Anacystis nidulans: Influence of NO(3) and NH(4) Assimilation on CO(2) Fixation. AB - Illuminated suspensions of Anacystis nidulans, supplied with saturating concentrations of CO(2) evolved O(2) at a greater rate when nitrate was simultaneously present. The extent of the stimulation of noncyclic electron flow induced by nitrate was dependent on light intensity, being maximal under light saturating conditions. Accordingly, nitrate depressed the rate of CO(2) fixation at limiting but not at saturating light, this depression reflecting the competition between both processes for assimilatory power. In contrast, ammonium stimulated CO(2) fixation at any light intensity assayed, the stimulation being dependent on the incorporation of ammonium to carbon skeletons. The positive effect of ammonium on CO(2) fixation also appeared to occur when nitrate was the nitrogen source, since with either nitrogen source an increase in the incorporation of newly fixed carbon into acid-soluble metabolites took place. From these results, the in vivo partitioning of assimilatory power between photosynthetic nitrogen and carbon assimilation and the quantitative and qualitative effects of inorganic nitrogen assimilation on CO(2) fixation are discussed. PMID- 16665205 TI - Responses of Two CAM Species to Different Irradiances during Growth and Susceptibility to Photoinhibition by High Light. AB - Two CAM species, Kalanchoe daigremontiana Hamet et Perrier and Hoya carnosa (L.) R. Br., were grown under a range of five photon flux area densitites (PFD) and then characterized. Significant acclimation to shade was indicated by progressive decreases in leaf thickness, rates of respiratory O(2) uptake, light compensation point, maximum rates of photosynthetic O(2) evolution, nocturnal acid accumulation, and delta(13)C values, and increases in chlorophyll concentration and absolute levels of room temperature (25 degrees C) and 77K fluorescence. Quantum yields (as measured by O(2) exchange) and the ratio of variable 77K fluorescence over the maximum yield (F(v)/F(m)) were relatively constant across the treatments. The only significant deviation from the above characteristics was in H. carnosa grown under full glasshouse PFD, where it apparently experienced photoinhibition. Following a photoinhibitory treatment, K. daigremontiana exhibited increases in the light compensation point and progressively greater reductions in the quantum yield, maximum photosynthetic rate, F(v)/F(m), and the variable component of room temperature fluorescence with increasing shade during growth. Thus although Crassulacean acid metabolism plants can adjust to shaded conditions, they are susceptible to photoinhibition when exposed to higher PFD than that experienced during growth. PMID- 16665206 TI - Changes in k, rb, and na transport to shoots after anoxia. AB - The effect of anoxia on subsequent uptake and transport of K, Rb, and Na was examined with seedlings of barley (Hordeum vulgare L.), corn (Zea mays L.), and tall fescue (Lolium x Festuca hybrid derivative) to further our understanding of xylem loading. Roots were incubated in solutions depleted of O(2) by flushing with N(2) gas. After 1 hour exposure, plants were returned to aerated solutions for 16 hours prior to measuring uptake and transport. For each species, anoxia pretreatment significantly enhanced Na transport to the shoot. The rate of Na accumulation into roots, however, was not affected. There was no enhancement of either K or Rb accumulation in shoots, indicating specificity for Na transport. A minimum exposure to anoxia of 30 minutes and a minimum of 12 hours elapsed time was necessary to achieve the maximum rate of Na transport to the shoot in barley seedlings. Accumulation of Na in the shoot of both the control and anoxia pretreated barley plants was inhibited by anoxia and by addition of the proline analog, l-azetidine-2-carboxylic acid, during the uptake period. Enhancement of Na transport was associated with a proportional increase in the rate of synthesis of a membrane bound protein with a molecular weight of 78,000 daltons. PMID- 16665207 TI - Acifluorfen Enhancement of Cryptochrome-Modulated Sporulation following an Inductive Light Pulse. AB - Acifluorfen enhancement of blue-light mediated phototropism suggested that this diphenyl-ether herbicide augments the light reaction (TY Leong, WR Briggs 1983 Plant Physiol 70: 875-881). The separation of the possible direct interaction of acifluorfen with light reactions from interactions with dark pathways has been elucidated in this paper with Trichoderma harzianum. Acifluorfen at 30 micromolar, given for 5 hours in the growth medium, stimulated the conidiation of Trichoderma in response to blue light without apparently affecting growth. Enhanced conidiation could be elicited by dipping cultures into medium with acifluorfen both before as well as 0.5 hour after inductive blue light. This postphotoinduction stimulation indicates that acifluorfen does not directly augment the effect of light by interacting with cryptochrome(s) in Trichoderma. Instead, acifluorfen most probably interacted with the dark reactions following photoinduction. PMID- 16665208 TI - Permeabilizing soybean protoplasts to macromolecules using electroporation and hypotonic shock. AB - The percentage of soybean cell culture protoplasts permeabilized by electroporation was dependent on the voltage and the number of successive pulses that were applied. Best results were obtained with two 50 milliseconds, 400 volts per centimeter pulses after which 78% of the surviving protoplasts had been permeabilized to the fluorescent dye calcein. Quantitation of the volume of extracellular fluid taken up was performed using radioactive inulin (molecular weight 5000-5500). Typically between 20 and 40 nanoliters of fluid was taken up by 10(6) protoplasts. Electroporation and hypotonic shock treatments (M Saleem, AJ Cutler 1986 J Plant Physiol 124: 11-21) were compared with respect to the volume of fluid taken up under optimum conditions. Electroporation produced 10 times more uptake than hypotonic shock treatment. In all experiments there was a direct relationship between the number of protoplasts lysed and both the amount of fluid taken up and the percentage of surviving protoplasts that were permeabilized. PMID- 16665209 TI - Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Concentrations of CO(2) in Maize Leaves. AB - The rate of CO(2) assimilation and levels of metabolites of the C(4) cycle and reductive pentose phosphate pathway in an attached leaf of maize (Zea mays L) were measured over a range of intercellular CO(2) concentration (Ci) of 10 to 190 microliters per liter. The CO(2) assimilation rate was saturated at a Ci of around 175 microliters per liter. The levels of ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate decreased substantially with increasing Ci. The levels of 3-phosphoglycerate, phosphoenolpyruvate (PEP), and pyruvate increased with increasing Ci. The level of dihydroxyacetone phosphate increased moderately from Ci of 10 microliters per liter to 20 to 50 microliters per liter and stayed almost constant over the rest of the range of Ci investigated. The levels of fructose 6-phosphate did not show any significant changes over the range of Ci. The levels of glucose 6-phosphate decreased slightly with increasing Ci. Although photosynthetically inactive pools of malate, asparate, and alanine could mask real changes in levels of the photosynthetically active pools of these compounds, the apparent levels of these compounds and the total amount of intermediates in the C(4) cycle (malate, aspartate, pyruvate, PEP, and alanine) increased with increasing Ci. The results suggest that there is carbon input into the C(4) cycle from the reductive pentose phosphate pathway which increases the level of total intermediates of the C(4) cycle with increasing Ci. PMID- 16665210 TI - Effect on Root Growth of Endogenous and Applied IAA and ABA: A Critical Reexamination. AB - Applications of indole-3yl-acetic acid (IAA) and abscisic acid (ABA) were done on two-day-old intact maize (cv LG 11) roots. The effect of the treatment on the root growth depends on their initial elongation rate. The slow growing roots were all inhibited by exogenous IAA and ABA at any concentrations used whereas for the fast growing roots their elongation was promoted by these two hormones at low concentrations. Quantitative analyses of endogenous IAA and ABA were performed using the gas chromatography-mass spectrometry technique. Detection and quantification of endogenous IAA and ABA were done on the zone of the root implicated in elongation. These techniques were achieved by electron impact on the IAA-Me-heptafluorobutyryl derivative and by negative ion chemical ionization with NH(3) on the ABA-Me ester derivative. A negative correlation between the growth and the endogenous content of these two hormones was obtained. ABA presented a larger range of endogenous level than IAA on the whole population of roots tested. When using applied IAA and ABA at different concentrations the same differentiating effect on the growth was observed. This allowed us to conclude that for identical concentrations, IAA has a more powerful effect on root elongation than ABA. Present results are discussed in relation to previous data related to the role of IAA and ABA in the growth and gravireaction of maize roots. PMID- 16665211 TI - Mechanism of Action of Pseudomonas syringae Phytotoxin, Syringomycin : Stimulation of Red Beet Plasma Membrane ATPase Activity. AB - Syringomycin, a peptide toxin produced by the phytopathogen Pseudomonas syringae pv syringae preferentially stimulated (2-fold) the vanadate-sensitive ATPase activity associated with the plasma membrane of red beet storage tissue. The toxin had a very slight effect on the tonoplast ATPase and had no detectable effect on the mitochondrial ATPase. Optimal stimulation was achieved with 10 to 50 micrograms of syringomycin per 25 micrograms of membrane protein. Treatment of membranes with 0.1% (weight/volume) deoxycholate eliminated the activation effect, and enzyme solubilized with Zwittergent 3-14 was not affected by syringomycin. ATPase activity was activated to the same extent at KCl concentrations ranging from 0 to 50 millimolar. Valinomycin, nigericin, carbonylcyanide p-trifluoromethoxyphenylhydrazone, and gramicidin did not increase the plasma membrane ATPase activity. However, these ionophores did not hinder the ability of syringomycin to stimulate the activity. We suggest that syringomycin does not increase ATPase activity by altering membrane ion gradients nor directly interacting with the enzyme, but possibly through regulatory effectors or covalent modification of the enzyme. PMID- 16665212 TI - Fructan Content and Fructosyltransferase Activity during Wheat Seed Growth. AB - The objective of this research was to determine the changes in fructan content and the activity of fructosyltransferases during the growth of wheat seeds (Triticum aestivum L. Thell, cv Caldwell). The total fructan content of the seeds decreased significantly during seed growth. The trisaccharide and tetrasaccharide content increased from 6 to 28 days post anthesis (DPA) and then declined, but these changes are not statistically significant. The content or concentration of longer chain polymers did decline significantly (64.55-6.52 milligrams per gram dry weight). Free fructose also decreased significantly during seed growth indicating that the fructose liberated from the decrease in fructan content was utilized by the seed. Sucrose increased significantly from 6 to 12 DPA, then declined significantly from 12 to 28 DPA. Sucrose:sucrose fructosyltransferase activity was greatest from 6 to 12 DPA (averaging 0.16 micromole of fructose transferred per seed per hour), then declined rapidly (0.04 micromole of fructose transferred per seed per hour). The estimated activity of fructan:fructan fructosyltransferase followed a similar pattern. The increase in sucrose concentration and high enzyme activity suggests that fructans were synthesized during the lag phase of seed growth. PMID- 16665213 TI - Effect of silver ions on ethylene biosynthesis by tomato fruit tissue. AB - Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC +/- silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC. PMID- 16665214 TI - Phloem Unloading in Developing Leaves of Sugar Beet : II. Termination of Phloem Unloading. AB - Phloem unloading in developing leaves of Beta vulgaris L. (;Klein E' multigerm) occurred from successively higher order branches of veins as leaves matured. Phloem unloading was studied in autoradiographs of leaf samples taken at various times during the arrival of a pulse of (14)C-labeled photoassimilate. Extension of mass flow of sieve element contents into leaf vein branches was determined from the high level of radiolabel in veins soon after first arrival of the pulse. Rapid entry, indicative of mass flow through open sieve pores, occurred down to the fourth division of veins in young, importing leaves and to the fifth or terminal branch in importing regions near the zone of transition from sink to source. The rate of unloading decreased with leaf age, as evidenced by the increased time required for the vein-mesophyll demarcation to become obscured. The rate of import per unit leaf area, measured by steady state labeling with (14)CO(2) also decreased as a leaf matured. The decline in import appeared to result from progressive changes that increased resistance to unloading of sieve elements and eventually terminated phloem unloading. PMID- 16665215 TI - Voltage-Dependent K-Channel in Protoplasmic Droplets of Chara corallina: A Single Channel Patch Clamp Study. AB - Passive transport of potassium through the plasma membrane of a protoplasmic droplet isolated from large internodal cells of Chara corallina Klein ex Willd., em, R.D.W. has been investigated using the patchclamp technique. When the membrane is hyperpolarized the conductance of a single K(+)-channel is of the order of magnitude of 100 picoSiemens and is reduced by tetraethylammonium chloride. Its open time is voltage dependent. This voltage-dependent K(+)-channel displays rectifying properties. The channel density is about 0.1 channel per square micrometer of membrane. When the membrane is depolarized the conductance of a single channel is of the order of magnitude of 30 picoSiemens and is insensitive to tetraethylammonium chloride. These results suggest that K(+) channels are incorporated in the plasma membrane during membranogenesis of a protoplasmic droplet. They constitute further evidence for the existence of voltage-dependent K(+)-channels in plant cells. PMID- 16665216 TI - Isoenzymes of Glutamine Synthetase in Roots of Pea (Pisum sativum L. cv Little Marvel) and Alfalfa (Medicago media Pers. cv Saranac). AB - Cell organelles have been isolated from protoplast lysates and total homogenates obtained from root tips of Pisum sativum L. (cv Little Marvel) and Medicago media Pers. (cv Saranac) grown in hydroponics with nitrate nutrient solutions. Density gradient and differential centrifugation procedures have been used to prepare mitochondria-and plastid-enriched fractions in which glutamine synthetase (GS) activity was estimated. Even when purified protoplasts were gently ruptured, significant breakage of plastids occurred during preparation as shown by the high proportion of nitrite reductase recovered in the soluble fraction. Of the total GS activity recovered, up to 20% was associated with the plastid fraction, depending on the source of plant material and the GS assay utilized; when corrected for recovery of the plastid marker nitrite reductase, it was calculated that 15 to 57% of alfalfa and 14 to 64% of pea root GS was located in the plastids. A true biosynthetic assay in which glutamine production was monitored by high performance liquid chromatography was devised to estimate the physiological significance of the transferase and the semibiosynthetic assays currently used for activity measurements. When compared with the true and semibiosynthetic assays, the transferase assay for GS appeared to underestimate the root plastid enzyme. Root plastid GS was partially purified by ion-exchange chromatography, and results show that the isoenzyme found in root plastids is different from chloroplastic or cytosolic GS. PMID- 16665217 TI - Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons. AB - Evidence for the involvement of Ca(2+) and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U (14)C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-(14)C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca(2+), whereas lipolytic acyl hydrolase proved to be insensitive to Ca(2+). Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca(2+). Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC(50) values ranging from 10 to 15 micromolar. Thus the Ca(2+) induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca(2+) on phospholipase D is independent of calmodulin. The role of Ca(2+) as a second messenger in the initiation of membrane lipid degradation is discussed. PMID- 16665218 TI - Influences of leaf temperature on photosynthetic carbon metabolism in wheat. AB - Net photosynthetic assimilation rate (A), extractable activities of three photosynthetic enzymes, and the concentrations of six metabolites were determined for wheat (Tricum aestivum L.) leaves as leaf temperature was varied under photorespiring (350 microliters per liter CO(2) and 21% O(2)) and under nonphotorespiring conditions (800 microliters per liter CO(2) and 2% O(2)). The extractable activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) and fructose-1,6-bisphosphatase declined with increasing leaf temperature from 15 to 45 degrees C. Leaf concentrations of ribulose-1,5-bisphosphate (RuBP) declined slightly between 15 and 25 degrees C but increased to a level which is 4 to 5 times the binding site concentration of Rubisco at leaf temperatures of 35 and 45 degrees C. Leaf concentrations of 3-phosphoglycerate, fructose-6-phosphate, and glucose-6-phosphate all declined with increasing leaf temperature. Outside of the limitations imposed by photorespiration, it is proposed that under high light and at suboptimal temperatures, A is limited by rate of utilization of triose phosphate; at optimal temperatures, by the availability of substrate (CO(2) and RuBP) under photorespiring conditions or utilization of triose phosphate under nonphotorespiring conditions; and at supraoptimal temperatures, by the activation state of Rubisco. PMID- 16665219 TI - Purification and Partial Kinetic and Physical Characterization of Two Chloroplast Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels. AB - Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either alpha- or beta-subunits with molecular weights of 55,500 or 53,000, respectively. The alpha-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the beta-isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar). Purification of isoenzymes was achieved by (NH(4))(2)SO(4) fractionation, gel-filtration, anion exchange fast protein liquid chromatography, and affinity chromatography. The alpha- and beta-isoenzymes were separated by their differential binding to Type 4 nicotinamide adenine dinucleotide phosphate-Sepharose. Both isoenzymes bound to an antibody affinity column to which purified antibody (prepared against beta isoenzyme) was covalently attached. Peptide mapping of the subunits showed them to have a high degree of sequence homology. Both subunits were synthesized in vitro from precursor protein(s) with a molecular weight of 58,500. Although the subunits have similar chemical, physical, and antigenic properties, their holoenzymes have strikingly different ammonium K(m) values. The ammonium K(m) of the beta-isoenzyme remained constant at approximately 75 millimolar, whereas this K(m) of the alpha-isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration. PMID- 16665220 TI - Galactose inhibits the conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in aged tobacco leaf discs. AB - d-Galactose has been shown to have toxic and growth inhibitory effects in plants. When applied at levels of 50 millimolar to tobacco (Nicotiana tabacum L. cv Xanthi) leaf discs galactose caused a rapid increase in ethylene production during the first 2 days of incubation, followed by a rapid return to the basal level on the third day. This pattern of galactose-stimulated ethylene production was accompanied by increased formation of 1-aminocyclopropane-1-carboxylic acid (ACC), which accumulated without being metabolized to ethylene or to the ACC conjugate. The inhibitory effect of galactose (50 millimolar) on the conversion of ACC of ethylene was relieved partially by d-glucose or sucrose (50 millimolar), and completely by CO(2) (10%), which were shown to enhance this conversion by themselves. Consequently, application of galactose plus any one of these compounds increased ethylene production and decreased free ACC levels. The data suggest that galactose toxicity may result in both an increased ethylene production as well as in accumulation of free ACC in aged discs. The increased ethylene production rates and ACC levels may, in turn, play a role in the development of symptoms associated with galactose toxicity. PMID- 16665221 TI - Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells. AB - The kinetics of accumulation (per milliliter of culture) of the alpha- and beta- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The beta-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the alpha-subunit holoenzyme(s). After 120 minutes, the alpha-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using (35)SO(4) and immunochemical procedures, the rate of synthesis of the alpha-subunit was shown to be greater than the beta-subunit during the first 80 minutes of induction. The alpha- and beta-subunits had different rates of degradation during the induction period (t((1/2)) = 50 versus 150 minutes, respectively) and during the deinduction period (t((1/2)) = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed. PMID- 16665222 TI - The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH. AB - The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO(2) supplied from the spontaneous dehydration of HCO(3) (-) thereby showing that C. reinhardii has the ability to utilize exogenous HCO(3) (-). Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K((1/2)) (DIC) from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO(3) addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO(2) transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO(2) and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga. PMID- 16665223 TI - The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N Limited Selenastrum minutum. AB - Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In (14)CO(2) pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 mumoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 mumoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279). PMID- 16665224 TI - 5-enolpyruvylshikimate 3-phosphate synthase, the target enzyme of the herbicide glyphosate, is synthesized as a precursor in a higher plant. AB - Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5 enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30- to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M(r)) 53900 +/- 900 as compared to M(r) 45500 +/- 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures. PMID- 16665225 TI - High speed HPLC analysis of polyamines in plant tissues. AB - A high speed high performance liquid chromatography (HPLC) method for the quantification of putrescine, spermidine, and spermine in biological samples is described. The dansylation is followed by a sample cleanup. The isocratic HPLC analysis with acetonitrile:H(2)O (72:28 volume/volume) on 10 centimeter long, 3 micrometer octadecyl silica columns (3 millimeter inner diameter) takes only 4.5 minutes. By our method about 100 analyses can be done in 1 day. PMID- 16665226 TI - UDP-Glucose-Dependent Sucrose Translocation in Tonoplast Vesicles from Stalk Tissue of Sugarcane. AB - Tonoplast vesicles isolated from stalk parenchyma tissue of sugarcane plants transport sucrose via a uridine diphosphate glucose (UDPGlc)-dependent group translocator. No sucrose transport via an ATP-dependent system could be detected. The products of UDPGlc uptake in the vesicles were sucrose and sucrose phosphate which, upon hydrolysis with alkaline phosphatase and invertase, showed that both hexose moieties are derived from UDPGlc. PMID- 16665227 TI - The effect of sodium chloride on solute potential and proline accumulation in soybean leaves. AB - Two cultivars of soybean (Glycine max [L.] Merr.) were grown in solution with up to 100 millimolar NaCl. Leaf solute potential was -1.1 to -1.2 megapascals in both cultivars without NaCl. At 100 millimolar NaCl leaf solute potential was 3.1 to -3.5 megapascals in Bragg and -1.7 megapascals in Ransom. The decrease in solute potential was essentially proportional to the concentration of NaCl. In both salt susceptible Bragg and salt semitolerant Ransom, leaf proline was no more than 0.4 micromole per gram fresh weight at or below 20 millimolar NaCl. At 40 and 60 millimolar NaCl, Bragg leaf proline levels were near 1.2 and 1.9 micromoles per gram fresh weight, respectively. Proline did not exceed 0.5 micromole per gram fresh weight in Ransom even at 100 millimolar NaCl. Proline accumulated in Bragg only after stress was severe enough to induce injury; therefore proline accumulation is not a sensitive indicator of salt stress in soybean plants. PMID- 16665228 TI - Control of the development of iron-efficiency reactions in potato as a response to iron deficiency is located in the roots. AB - Roots of potato plants (Solanum tuberosum cv Bintje) growing on low Fe nutrient solution developed the characteristic Fe efficiency reactions, such as high ferric reductase activity, proton extrusion and increased root hair formation. Roots from a tuber with sprout removed, when grown on Fe-free nutrient solution, also expressed these reactions; transfer to iron-containing medium resulted in their complete disappearance within 10 days. Roots growing on 2% sucrose in sterile Murashige-Skoog medium increased their ferric reductase activity upon withholding Fe and formed transfer cells. It is concluded that potato roots themselves control the development of Fe-efficiency reactions, and that the shoot may exert a modulating influence on their expression. PMID- 16665229 TI - A rapid method to test for chloroplast DNA involvement in atrazine resistance. AB - A point mutation in the chloroplast psbA gene at codon 264 resulting in an animo acid substitution (ser-gly) manifests itself as atrazine resistance in all recognized weed species studied to date. The single base substitution overlaps a highly conserved Mae1 restriction site which is present in susceptible but not in resistant plants. This restriction enzyme, recently commercialized, has been used to show that it is now possible to discriminate rapidly between the two biotypes without the need for DNA sequencing. PMID- 16665230 TI - Membrane rupture is the common cause of damage to chloroplast membranes in leaves injured by freezing or excessive wilting. AB - The effects of freezing and desiccation of spinach leaves (Spinacia oleracea L. cv Yates) on the thylakoid membranes were assessed using antibodies specific for thylakoid membrane proteins. The peripheral part of the chloroplast coupling factor ATPase (CF1) was used as a molecular marker for chemical membrane damage by chaotropic solutes. Plastocyanin, a soluble protein localized inside the closed thylakoid membrane system, was a marker for damage by mechanical membrane rupture. After freezing and wilting of leaves which resulted in damage, very little CF1 was detached from the membranes, whereas almost all plastocyanin was released from the thylakoids. It is suggested that in vivo dehydration both by freezing and desiccation results in membrane rupture rather than in the dissociation of peripheral thylakoid membrane proteins. PMID- 16665231 TI - Metabolic Requirement of Cucurbita pepo for Boron. AB - Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [(3)H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [(14)C]leucine into protein and respiration as measured by O(2) consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O(2) consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems. PMID- 16665232 TI - Glutamine Synthetase Isoenzymes in the Green Soil Alga Stichococcus bacillaris Naeg. AB - Two forms of glutamine synthetase (GS(1) and GS(2)) have been separated from cells of Stichococcus bacillaris by fast protein liquid chromatography. The activities of the two isoenzymes were influenced by the composition of the media employed; thiol reagents were essential for stabilizing GS(2) but they suppressed GS(1) activity. The activity of each isoenzyme was, therefore, determined following separate purification procedures. Growth conditions influenced both isoenzymes; GS(2) showed maximum activity under photoautotrophic conditions, whereas GS(1) showed maximum activity under heterotrophic conditions. PMID- 16665233 TI - Effect of pH on IAA Uptake by Maize Root Segments. AB - The uptake of [5-(3)H]indoleacetic acid (IAA) by Zea mays L. root segments involves nonsaturable and saturable processes. The pH optimum of the saturable component was found to be 5.0. The proton ionophore carbonylcyanide p trifluoromethoxyphenylhydrazone inhibited at 100 micromolar the saturable component of IAA uptake but had no effect on non-saturable uptake. This indicates that the saturable component of IAA uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongation zone of the root will stimulate nonsaturable and saturable uptake of IAA in that zone. PMID- 16665234 TI - Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots. AB - Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato. PMID- 16665235 TI - Chilling-Enhanced Photooxidation : The Peroxidative Destruction of Lipids during Chilling Injury to Photosynthesis and Ultrastructure. AB - Chilling-induced photooxidation was studied in detached leaves of chilling sensitive (CS) cucumber (Cucumis sativus L.) and chilling resistant (CR) pea (Pisum sativum L.). The rates of photosynthesis and respiration, measured as O(2) exchange, were found to be comparable in the two species over a temperature range of 5 to 35 degrees C. Chilling at 5 degrees C for 12 hours in high light (1000 microeinsteins per square meter per second) decreased CO(2) uptake 75% in detached pea leaves whereas CO(2) uptake by cucumber was reduced to zero within 2 hours. Respiration was unaffected in either species by the chilling and light treatment. Although ultrastructural alterations were apparent in chloroplasts of both species, cucumber's were affected sooner and more severely. The mechanism of photooxidative lipid peroxidation was investigated by following the production of ethane gas under a variety of conditions. Maximum ethane production occurred in the CS cucumber at low temperature (5 degrees C) and high light (1000 microeinsteins per square meter per second). Atrazine, an inhibitor of photosynthetic electron transport, almost completely halted this chilling- and light-induced ethane production. These data, taken with those reported in an accompanying article (RR Wise, AW Naylor 1986 Plant Physiol 83: 278-282) suggest that the superoxide anion radical is generated in cucumber chloroplasts (probably via a Mehler-type reaction) during chilling-enhanced photooxidation. Parallel experiments were conducted on pea, a CR species. Detached pea leaves could only be made to generate ethane in the cold and light if they were pretreated with the herbicide parquat, a known effector of O(2) (-) production. Even so, pea showed no lipid peroxidation for 6 hours, at which time ethane production began and was at a rate equal to that for the chilled and irradiated cucumber leaves. The results indicate that pea has an endogenous mechanism(s) for the removal of toxic oxygen species prior to lipid peroxidation. This mechanism breaks down in pea after 6 hours in the cold, light, and the presence of paraquat. PMID- 16665236 TI - Chilling-enhanced photooxidation : evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. AB - Chilling temperatures (5 degrees C) and high irradiance (1000 microeinsteins per square meter per second) were used to induce photooxidation in detached leaves of cucumber (Cucumis sativus L.), a chilling-sensitive plant. Chlorophyll a, chlorophyll b, beta carotene, and three xanthophylls were degraded in a light dependent fashion at essentially the same rate. Lipid peroxidation (measured as ethane evolution) showed an O(2) dependency. The levels of three endogenous antioxidants, ascorbate, reduced glutathione, and alpha tocopherol, all showed an irradiance-dependent decline. alpha-Tocopherol was the first antioxidant affected and appeared to be the only antioxidant that could be implicated in long-term protection of the photosynthetic pigments. Results from the application of antioxidants having relative selectivity for (1)O(2), O(2) (-), or OH indicated that both (1)O(2) and O(2) (-) were involved in the chilling- and light-induced lipid peroxidation which accompanied photooxidation. Application of D(2)O (which enhances the lifetime of (1)O(2)) corroborated these results. Chilling under high light produced no evidence of photooxidative damage in detached leaves of chilling-resistant pea (Pisum sativum L.). Our results suggest a fundamental difference in the ability of pea to reduce the destructive effects of free radical and (1)O(2) production in chloroplasts during chilling in high light. PMID- 16665237 TI - Diurnal changes in maize leaf photosynthesis : I. Carbon exchange rate, assimilate export rate, and enzyme activities. AB - Diurnal changes in photosynthetic parameters and enzyme activities were characterized in greenhouse grown maize plants (Zea mays L. cv Pioneer 3184). Rates of net photosynthesis and assimilate export were highest at midday, coincident with maximum irradiance. During the day, assimilate export accounted for about 80% of net carbon fixation, and the maximum export rate (35 milligrams CH(2)O per square decimeter per hour) was substantially higher than the relatively constant rate maintained through the night (5 milligrams CH(2)O per square decimeter per hour). Activities of sucrose phosphate synthase and NADP malate dehydrogenase showed pronounced diurnal fluctuations; maximum enzyme activities were generally coincident with highest light intensity. Reciprocal light/dark transfers of plants throughout the diurnal cycle revealed that both enzymes were deactivated by 30 minutes of darkness during the day, and they could both be substantially activated by 30 minutes of illumination at night. During 24 hours of extended darkness, sucrose phosphate synthase activity declined progressively to an almost undetectable level, but was activated after 1.5 hours of illumination. Thus, the diurnal fluctuation in maize sucrose phosphate synthase can be explained by some form of light modulation of enzyme activity and is not due to an endogenous rhythm in activity. No diurnal fluctuations were observed in the activities of NADP-malic enzyme or fructose 6-phosphate-2-kinase. Phosphoenolpyruvate carboxylase was activated by light to some extent (about 50%) when activity was measured under suboptimal conditions in vitro. The results suggested that the rates of sucrose formation and assimilate export were closely aligned with the rate of carbon fixation and the activation state of sucrose phosphate synthase. PMID- 16665238 TI - Diurnal Changes in Maize Leaf Photosynthesis : II. Levels of Metabolic Intermediates of Sucrose Synthesis and the Regulatory Metabolite Fructose 2,6 Bisphosphate. AB - Diurnal changes in the regulatory metabolite, fructose-2,6-bisphosphate (F26BP), and key metabolic intermediates of sucrose biosynthesis were studied in maize (Zea mays L. cv Pioneer 3184) during a day-night cycle. Whole leaf concentrations of dihydroxyacetonephosphate (DHAP) and fructose 1,6-bisphosphate changed markedly during the photoperiod. DHAP concentration was correlated positively with the rate of sucrose formation in vivo (assimilate export plus sucrose accumulation) and extractable activity of sucrose phosphate synthase (SPS). The changes closely followed net photosynthetic rate, which tracked irradiance. The other metabolic intermediates measured (glucose 6-phosphate, fructose 6 phosphate, and UDP-glucose) were either relatively constant over the 24 hour period or changed in a different pattern. Diurnal changes in leaf F26BP concentrations were pronounced, and fundamentally different than the pattern reported with other species. F26BP concentration decreased at the beginning of the day and remained low and constant; a 3- to 4-fold increase occurred with darkness, and slowly declined thereafter. In general, leaf F26BP concentration was negatively correlated with net photosynthetic rate, and also leaf DHAP concentration. Consequently, co-ordination of the regulation of cytosolic fructose 1,6-bisphosphatase and SPS was apparent. The results support the postulate that in maize leaves the activation state of SPS may be dependent on availability of DHAP and possibly other metabolites. PMID- 16665239 TI - Diurnal Changes in Maize Leaf Photosynthesis : III. Leaf Elongation Rate in Relation to Carbohydrates and Activities of Sucrose Metabolizing Enzymes in Elongating Leaf Tissue. AB - Maize (Zea mays L. cv. Pioneer 3184) leaf elongation rate was measured diurnally and was related to diurnal changes in the activities of sucrose metabolizing enzymes and carbohydrate content in the elongating portion of the leaf. The rate of leaf elongation was greatest at midday (1300 hours) and was coincident with the maximum assimilate export rate from the distal portion of the leaf. Leaf elongation during the light period accounted for 70% of the total observed increase in leaf length per 24 hour period. Pronounced diurnal fluctuations were observed in the activities of acid and neutral invertase and sucrose phosphate synthase. Maximum activities of sucrose phosphate synthase and acid invertase were observed at 0900 hours, after which activity declined rapidly. The activity of sucrose phosphate synthase was substantially lower than that observed in maize leaf source tissue. Neutral invertase activity was greatest at midday (1200 hours) and was correlated positively with diurnal changes in leaf elongation rate. There was no significant change in the activity of sucrose synthase over the light/dark cycle. Sucrose accumulation rate increased during a period when leaf elongation rate was maximal and beginning to decline. Maximum sucrose concentration was observed at 1500 hours, when the activities of sucrose metabolizing enzymes were low. At no time was there a significant accumulation of hexose sugars. The rate of starch accumulation increased after the maximum sucrose concentration was observed, continuing until the end of the light period. There was no delay in the onset of starch mobilization at the beginning of the dark period, and essentially all of the starch was depleted by the end of the night. Mobilization of starch in the elongating tissue at night could account for a significant proportion of the calculated increase in the tissue dry weight due to growth. Collectively, the results suggested that leaf growth may be controlled by the activities of certain sucrose metabolizing enzymes and may be coordinated with assimilate export from the distal, source portion of the leaf. Results are discussed with reference to diurnal photoassimilation and export in the distal, source portion of the leaf. PMID- 16665240 TI - The occurrence of phytoferritin and its relationship to effectiveness of soybean nodules. AB - Polyacrylamide gel electrophoresis and electron microscopy revealed that accumulation of iron-protein in soybean nodules is influenced by nodule age, mutation in bradyrhizobia, and rhizobial/bradyrhizobial strain-soybean cultivar interactions. Iron-protein concentrations (micrograms per milligram protein) were inversely related to heme concentrations (nanomoles per milligram protein), with correlation coefficients (r values) ranging from -0.98 in young nodules to -0.83 in mature ones. Bradyrhizobium japonicum symbiotic mutants HS 129 and HS 145 (Nod(+) Fix(-)) produced nodules high in iron-protein. Electrophoresis of homogenate prepared from nodules on Lee 68 produced by B. japonicum HS 129 yielded two different forms of the iron-proteins, 570 and 600 kilodaltons. The 570 kilodalton iron-protein isolated by preparative polyacrylamide gel electrophoresis behaved like horse-spleen-ferritin in responses to iron-stains, heat stability, ultraviolet absorption spectrum, iron unloading and reloading, and characteristic appearance in electron micrographs. These properties led to the conclusion that the 570 kilodalton iron-protein is phytoferritin. The nodule phytoferritin differed from horse-spleen-ferritin in electrophoretic mobility, serological properties, and molecular size and was distinct from most other known phytoferritins in that it was composed of different subunit types. PMID- 16665241 TI - Some kinetic and regulatory properties of the pea mitochondrial pyruvate dehydrogenase complex. AB - The pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea (Pisum sativum L., cv Little Marvel) leaf mitochondria. The pH optimum for the overall reaction was 7.6. The divalent cation requirement was best satisfied by Mg(2+). Reaction velocity was maximal at 40 degrees C. Pyruvate was a better substrate than 2-oxo-butyrate; other 2-oxo acids were not substrates. Michaelis constants for substrates were; pyruvate, 57 micromolar; NAD, 122 micromolar; Coenzyme-A, 5 micromolar; Mg(2+), 0.36 millimolar; Mg-thiamine pyrophosphate, 80 nanomolar. The products, NADH and acetyl-Coenzyme-A, were linear competitive inhibitors with respect to NAD and Coenzyme A. Inhibition constants were 18 and 10 micromolar, respectively. Glyoxylate inhibited complex activity only in the absence of thiol reagents. Glyoxylate inhibition was competitive with respect to pyruvate with an inhibition constant of 51 micromolar. Among mitochondrial metabolites examined as potential effectors, only ADP with an inhibition constant of 0.57 millimolar could be of physiological significance. PMID- 16665242 TI - Some properties of pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase. AB - Reactivation of the pea mitochondrial pyruvate dehydrogenase complex was the result of dephosphorylation catalyzed by phospho-pyruvate dehydrogenase phosphatase, an intrinsic component of the complex. Phosphatase activity was dependent upon divalent metal ions, with Mg(2+) more effective than Mn(2+) or Co(2+). The Michaelis constants for Mg(2+), Mn(2+), and Co(2+) were 3.8, 1.7, and 1.4 millimolar, respectively. Neither the rate nor the extent of activation of the phosphatase by Mg(2+) or Mn(2+) was effected by up to 100 units per assay of megamodulin. Calcium ions did not activate pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase, and low concentrations of Ca(2+) antagonized activation by other divalent cations. Phosphatase activity was inhibited by fluoride and ortho-phosphate but not by molybdate or vanadate. Krebs cycle intermediates, adenylates, polyamines, amino acids, and phosphoamino acids were without effect upon pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase activity in vitro. PMID- 16665243 TI - Photosynthesis Decrease and Stomatal Control of Gas Exchange in Abies alba Mill. in Response to Vapor Pressure Difference. AB - The responses of steady state CO(2) assimilation rate (A), transpiration rate (E), and stomatal conductance (g(s)) to changes in leaf-to-air vapor pressure difference (DeltaW) were examined on different dates in shoots from Abies alba trees growing outside. In Ecouves, a provenance representative of wet oceanic conditions in Northern France, both A and g(s) decreased when DeltaW was increased from 4.6 to 14.5 Pa KPa(-1). In Nebias, which represented the dry end of the natural range of A. alba in southern France, A and g(s) decreased only after reaching peak levels at 9.0 and 7.0 Pa KPa(-1), respectively. The representation of the data in assimilation rate (A) versus intercellular CO(2) partial pressure (C(i)) graphs allowed us to determine how stomata and mesophyll photosynthesis interacted when DeltaW was increased. Changes in A were primarily due to alterations in mesophyll photosynthesis. At high DeltaW, and especially in Ecouves when soil water deficit prevailed, A declined, while C(i) remained approximately constant, which may be interpreted as an adjustment of g(s) to changes in mesophyll photosynthesis. Such a stomatal control of gas exchange appeared as an alternative to the classical feedforward interpretation of E versus DeltaW responses with a peak rate of E. The gas exchange response to DeltaW was also characterized by considerable deviations from the optimization theory of IR Cowan and GD Farquhar (1977 Symp Soc Exp Biol 31: 471-505). PMID- 16665244 TI - The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls. AB - Root and hypocotyl plasma membrane H(+)-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K(m) for ATP.Mg(2+) and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences. PMID- 16665245 TI - Triazine Resistance without Reduced Vigor in Phalaris paradoxa. AB - A triazine-resistant (R) biotype of Phalaris paradoxa L. (hood canarygrass) was superior to a triazine-susceptible (S) biotype in seed-germinability and seedling emergence. It was equal or superior to the S-biotype in growth under noncompetitive conditions. Rates of CO(2) uptake by R-plants were similar to those of S-plants, except at very low photon flux densities, where S-plants exhibited higher rates of CO(2) uptake. Fluorescence induction curves of chloroplasts isolated from R-plants indicated an alteration in photosystem II. Analysis of the light dependence of electron transport shows a reduction in quantum yield (Q(y)) in R- compared to S-chloroplasts. The same analysis, however, shows for R-chloroplasts an increase in the light-saturated electron transport rate (V(max)). The increase in V(max) compensates for the reduction of Q(y) over a wide range of photon flux densities, which may explain the similarity between R- and S-biotypes in photosynthetic potential and growth. PMID- 16665246 TI - Cytokinin Biochemistry in Relation to Leaf Senescence : II. The Metabolism of 6 Benzylaminopurine in Soybean Leaves and the Inhibition of Its Conjugation. AB - The metabolism of [(3)H]6-benzylamino purine was studied in presenescent and early senescent soybean (Glycine max [L.] Merr.) leaves. In both types of leaves, the metabolism was essentially the same. The principal metabolite was identified as beta-(6-benzylaminopurin-9-yl)alanine by mass spectral studies, which included discharge ionization-secondary ion mass spectrometry and pulsed positive ion negative ion-chemical ionization mass spectrometry. Conversion to this alanine conjugate was found to be inhibited 2,4-dichlorophenoxyacetic acid and 5,7 dichloroindoleacetic acid. PMID- 16665247 TI - Regulation of Assimilate Partitioning in Soybean : Initial Effects following Change in Nitrate Supply. AB - Increased concentrations of nitrate in a nutrient solution (2, 5, and 10 millimolar KNO(3)) were correlated with increased shoot:root ratios of non nodulated soybeans (Glycine max [L.] Merr.) grown in sand culture. While altering the pattern of C and N partitioning, the N treatments did not affect whole plant photosynthesis over the study period. To determine the mechanism responsible for the observed changes in assimilate partitioning, detailed C and N budgets were worked out with plants from each N treatment over three consecutive 4-day periods of midvegetative growth. The information for the C and N budgets from the 2 and 10 millimolar NO(3) (-) treatments was combined with data on the composition of xylem and phloem exudates to construct a series of models of C and N transport and partitioning. These models were used to outine a ;chain-reaction' of cause and-effect relationships that may account for the observed changes in assimilate partitioning in these plants. The proposed mechanism identifies two features which may be important in regulating the partitioning of N and other nutrients within the whole plant. (a) The concentration of N in the phloem is highly correlated with the N concentration in the xylem. (b) The amount of N which cycles through the root-from phloem imported from the shoot to xylem exported by the root-is regulated by the root's requirement for N: only that N in excess of the root's N requirements is returned to the shoot in the xylem. Therefore, roots seem to have the highest priority for N in times of N stress. PMID- 16665248 TI - The simultaneous determination of carbon dioxide release and oxygen uptake in suspensions of plant leaf mitochondria oxidizing glycine. AB - The construction and operation of a device for continuous measurement of CO(2) release by suspensions of respiring mitochondria is described. A combination of this device with a Clark-type O(2) electrode was used for simultaneous measurement of respiration and of CO(2) release by spinach and pea leaf mitochondria with glycine as substrate. Both mitochondrial preparations showed high rates of respiration and high respiratory control ratios. The addition of oxaloacetate not only inhibited O(2) uptake substantially, but also greatly stimulated glycine oxidation as monitored by CO(2) release. In spinach leaf mitochondria, the maximal rates of glycine oxidation thus obtained, were two times higher than the rate of glycine oxidation required at average rates of photorespiration. It is concluded from these results that under saturating conditions the capacity of glycine oxidation by intact mitochondria exceeds the capacity of glycine-dependent respiration. PMID- 16665249 TI - Changes in the levels of major sulfur metabolites and free amino acids in pea cotyledons recovering from sulfur deficiency. AB - Changes in levels of sulfur metabolites and free amino acids were followed in cotyledons of sulfur-deficient, developing pea seeds (Pisum sativum L.) for 24 hours after resupply of sulfate, during which time the legumin mRNA levels returned almost to normal. Two recovery situations were studied: cultured seeds, with sulfate added to the medium, and seeds attached to the intact plant, with sulfate added to the roots. In both situations the levels of cysteine, glutathione, and methionine rose rapidly, glutathione exhibiting an initial lag. In attached but not cultured seeds methionine markedly overshot the level normally found in sulfur-sufficient seeds. In the cultured seed S adenosylmethionine (AdoMet), but not S-methylmethionine, showed a sustained rise; in the attached seed the changes were slight. The composition of the free amino acid pool did not change substantially in either recovery situation. In the cultured seed the large rise in AdoMet level occurred equally in nonrecovering seeds. It was accompanied by 6-fold and 10-fold increases in gamma-aminobutyrate and alanine, respectively. These effects are attributed to wounding resulting from excision of the seed. (35)S-labeling experiments showed that there was no significant accumulation of label in unidentified sulfur-containing amino compounds in either recovery situation. It was concluded from these results and those of other workers that, at the present level of knowledge, the most probable candidate for a ;signal' compound, eliciting recovery of legumin mRNA level in response to sulfur-feeding, is cysteine. PMID- 16665250 TI - Effects of drought on primary photosynthetic processes of cotton leaves. AB - The effects of drought on Photosystem II (PSII) fluorescence and photosynthetic electron transport activities were analyzed in cotton. Water stress did not modify the amplitude of leaf variable fluorescence at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) nor at 77 K. It is therefore concluded that photon collection, their distribution between the two photosystems, and PSII photochemistry are unaffected by the stress. In droughted leaves at room temperature under low exciting light, the transitory maximum (F(p)) and steady state (F(t)) fluorescence levels are increased; under high exciting light, F(p) level and the rise time from the initial level (F(o)) to F(p) are unchanged, whereas F(p) to F(t) decay time is increased. These results infer that the drought slows the rate of plastoquinone reoxidation. This conclusion agrees with a larger proportion of reduced primary PSII electron acceptor Q(A) measured at the steady state under low light. In thylakoids isolated from droughted leaves, PSII mediated electron flow was the same as in thylakoids from control leaves, whereas PSI mediated electron transport was inhibited. It is shown that water stress does not induce sensitization to photoinhibition in cotton. PMID- 16665251 TI - Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain. AB - Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (-)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used (18)O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under (18)O(2), the product was labeled with 99% efficiency; no label was incorporated by metabolism in H(2) (18)O. Pisatin synthesized by pea pods in the presence of (18)O(2) or H(2) (18)O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H(2)O and not from O(2). Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown. PMID- 16665252 TI - Isolation and in vitro translation of polysomes from mature rye leaves. AB - Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status. PMID- 16665253 TI - Intracellular and Extracellular Cyclic Nucleotides in Wild-Type and White Collar Mutant Strains of Neurospora crassa: Temperature Dependent Efflux of Cyclic AMP from Mycelia. AB - Cyclic AMP and cyclic GMP were released into the growth medium of mycelia of Neurospora crassa wild-type strains St.L.74A and Em5297a and by white collar-1 and white collar-2 mutant strains. After growth for 6 days at 18 degrees C, there were 2.19 (St.L.74A), 5.83 (Em5297a), 1.38 (white collar-1), and 1.10 (white collar-2) nanomoles of cyclic AMP per gram dry weight of mycelia in the growth medium. These values corresponded to concentrations of cyclic AMP of between approximately 10 and 50 nanomolar. The corresponding values for extracellular cyclic GMP were typically less than 6% of the values for cyclic AMP. Following transfer to fresh medium, cyclic AMP efflux was demonstrated for each of the strains, and the amount of cyclic AMP exported into the fresh medium was greater at 25 degrees C than 6 degrees C. Intracellular cyclic AMP and cyclic GMP were also measured in each of the strains. The values for cyclic AMP were in the same range as those in the literature (approximately 0.5 to 1.5 nanomoles per gram dry weight of mycelia). However, the corresponding intracellular cyclic GMP values were less than 1% of the cyclic AMP values, i.e. more than 50 times lower than the value previously reported for the St.L.74A wild-type. Transfer of mycelia after 6 days at 18 degrees C to fresh media and incubation for 2 hours at 25 degrees C or 6 degrees C did not consistently affect the intracellular level of cyclic AMP or cyclic GMP in the strains examined. We could detect no change in intracellular cyclic AMP when mycelia of the St.L.74A wild-type strain were irradiated with blue light for periods of up to 3.0 hours at 18 degrees C, or in cyclic AMP and cyclic GMP for irradiation times of up to 1 minute at 6 degrees C. We propose that the plasma membrane of Neurospora crassa is permeable to cyclic nucleotides, and the export of cyclic nucleotides into the growth medium may be a means of regulating intracellular levels. We conclude that three factors that affect carotenogenesis in Neurospora crassa (blue light, temperature, and the white collar mutations) have no appreciable effect on the total measurable intracellular cyclic nucleotides in this organism. There was no extracellular or intracellular cyclic AMP or cyclic GMP in the crisp-1 mutant strain, which suggested either that adenylate cyclase (which is absent in crisp-1) catalyzes the synthesis of both cyclic AMP and cyclic GMP or that the crisp-1 mutation somehow results in a deficiency of two enzymes (adenylate and guanylate cyclase). PMID- 16665255 TI - Salinity reduces membrane-associated calcium in corn root protoplasts. AB - Calcium is an important factor in the ability of plants to resist salt stress, possibly because of its role in maintaining membrane integrity. We studied the effects of NaCl stress on membrane-associated Ca in corn root protoplasts (Zea mays L. cv Pioneer 3377) using the fluorescent Ca probe chlorotetracycline (CTC). Protoplasts were isolated from the cortex of primary roots of corn seedlings (Gronwald and Leonard, Plant Physiol 1982 70: 1391-1395). After a 30 minute incubation in 50 micromolar CTC, the protoplasts were exposed to isosmotic treatment solutions containing various concentrations of NaCl just before fluorimetric analysis. Increasing NaCl concentrations caused a progressive reduction in net CTC fluorescence, to 50% of control values at 150 mm NaCl. NaCl did not displace CTC from the cells, nor did it directly interfere with Ca-CTC binding. Tests with CsCl, RbCl, KCl, LiCl, Na(2)SO(4), NaNO(3), and NaBr indicated that the reduction in CTC fluorescence was not specific to either Na or Cl, but may have been due to increased ionic strength of the treatment solutions. Like CTC fluorescence, root growth of intact corn seedlings was not specifically sensitive to Na, but was inhibited by several monovalent cations in the order Li > Cs >> Rb > Na > K. CTC fluorescence at 100 mm NaCl was restored to unstressed levels by increasing Ca concentrations. Since our salt treatments were isosmotic, we conclude that the ionic component of salt stress displaces Ca from membranes of corn root cells. PMID- 16665254 TI - Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions in Vitro and in Vivo. AB - Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40 degrees C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1- >2)-beta-glucan, (1-->3)-beta-glucan, (1-->6)-beta-glucan, (1-->3, 1-->4)-beta glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period. PMID- 16665256 TI - myo-Inositol Trisphosphate Mobilizes Calcium from Fusogenic Carrot (Daucus carota L.) Protoplasts. AB - To determine whether or not inositol trisphosphate (IP(3)) mobilizes calcium in higher plant cells, we investigated the effect of IP(3) on Ca(2+) fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in (45)Ca(2+)-containing medium and the (45)Ca(2+) associated with the protoplasts was monitored with time. Addition of IP(3) (20 micromolar) caused a 17% net loss of the accumulated (45)Ca(2+) within 4 minutes. There was a reuptake of (45)Ca(2+) and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP(6)), also stimulated (45)Ca(2+) efflux from the protoplasts. Both the IP(3(-) ) and the IP(6(-) )induced (45)Ca(2+) efflux were inhibited by the calmodulin antagonist, trifluoperazine. PMID- 16665257 TI - Subcellular Metabolite Levels in Spinach Leaves : Regulation of Sucrose Synthesis during Diurnal Alterations in Photosynthetic Partitioning. AB - The alterations of subcellular metabolite levels during the day in spinach leaves have been investigated using nonaqueous density gradient centrifugation to separate chloroplasts, cytosol, and vacuole. The results provide direct evidence for the role of sucrose phosphate synthase and cytosolic fructose 1,6 bisphosphatase in regulating sucrose synthesis in leaves and also show that the phosphate translocator is kinetically limiting in vivo. PMID- 16665258 TI - Phytic Acid Metabolism in Lily (Lilium longiflorum Thunb.) Pollen. AB - The accumulation of phytic acid during development of lily (Lilium longiflorum Thunb.) pollen and its degradation during germination have been studied. A substantial amount of phytic acid accumulates in lily pollen by 5 days before anthesis, and little change occurs during subsequent maturation. Mature lily pollen contains 7 to 8 micrograms phytic acid per milligram pollen. Considerable degradation of phytic acid occurs by 15 minutes of incubation in glucose culture medium, and very little is left by 3 hours. No partially phosphorylated myo inositol accumulates during germination. The breakdown of phytic acid proceeds at a constant rate during this time period. The rate is calculated to be 0.037 microgram phytic acid/milligram pollen/minute. Two phytases are detected in germinated lily pollen extract using high performance liquid chromatography with an anion exchange column (diethylaminoethyl-5PW). The results suggest that one of the phytases is already present in mature ungerminated lily pollen and the other one is newly synthesized during germination from a long-lived, pre-existing mRNA. PMID- 16665259 TI - Spring filling of xylem vessels in wild grapevine. AB - Xylem vessels in grapevines Vitis labrusca L. and Vitis riparia Michx. growing in New England contained air over winter and yet filled with xylem sap and recovered their maximum hydraulic conductance during the month before leaf expansion in late May. During this period root pressures between 10 and 100 kilopascals were measured. Although some air in vessels apparently dissolved in ascending xylem sap, results indicated that some is pushed out of vessels and then out of the vine. Air in the vessel network distal to advancing xylem sap was compressed at about 3 kilopascals; independent measurements indicated this was sufficient to push air across vessel ends, and from vessels to the exterior through dead vine tips, inflorescence scars, and points on the bark. Once wetted, vessel ends previously air-permeable at 3 kilopascals remained sealed against air at pressures up to 2 and 3 megapascals. Permeability at 3 kilopascals was restored by dehydrating vines below -2.4 megapascals. We suggest that the decrease in permeability with hydration is due to formation of water films across pores in intervascular pit membranes; this water seal can maintain a pressure difference of roughly 2 megapascals, and prevents cavitation by aspirated air at xylem pressures less negative than -2.4 megapascals. PMID- 16665260 TI - Alteration of Cell-Wall Water Content and Elasticity in Douglas-Fir during Periods of Water Deficit. AB - Three populations of seedlings of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) were subjected to recurrent drought cycles. Pressure-volume curves were developed to derive the osmotic and pressure potentials as functions of the symplastic water over a wide range of relative water content. The drought cycles induced large reversible changes in the relative water content of the apoplasm and in the bulk modulus of elasticity. These responses have not been reported previously in any species of higher plant. Our data suggest that turgor regulation in Douglas-fir is mediated by structural and biochemical factors associated with the cell wall, rather than by osmotic adjustment. PMID- 16665261 TI - Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L. AB - A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 x 10(-5) molar abscisic acid (ABA) for 7 days at 25 degrees C survived slow cooling to -60 degrees C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N(2) after slow cooling to -40 or -60 degrees C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of -28 degrees C after 5 days of ABA treatment. Ethanol (2 x 10(-2) molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25 degrees C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10(-4) molar) treated alfalfa cells (Medicago sativa L.) grown at 25 degrees C hardened from an initial LT(50) of -5 degrees C to an LT(50) of -23 degrees C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days. PMID- 16665262 TI - Phosphorylation of Thylakoid Proteins of Oryza sativa: In Vitro Characterization and Effects of Chilling Temperatures. AB - The phosphorylation of thylakoid proteins of rice (Oryza sativa L.) was studied in vitro using [gamma-(32)P]ATP. Several thylakoid proteins are labeled, including the light-harvesting complex of photosystem II. Protein phosphorylation is sensitive to temperature, pH, and ADP, ATP, and divalent cation concentrations. In the range pH 7 to 8.2, phosphorylation of the light-harvesting polypeptides declines above pH 7.5, whereas labeling of several other thylakoid polypeptides increases. Increasing divalent cation concentration from 3 to 20 millimolar results in a decrease in phosphorylation of the 26 kilodalton light harvesting complex polypeptide and increased phosphorylation of several other polypeptides. ADP has an inhibitory effect on the phosphorylation of the light harvesting complex polypeptides. Phosphorylation of the 26 kilodalton light harvesting polypeptide requires 0.45 millimolar ATP for half-maximal phosphorylation, compared to 0.3 millimolar for the 32 kilodalton phosphoprotein. Low temperature inhibits the phosphorylation of thylakoid proteins in chilling sensitive rice. However, phosphorylation of histones by thylakoid-bound kinase(s) is independent of temperature in the range of 25 to 5 degrees C, suggesting that the effect of low temperature is on accessibility of the substrate, rather than on the activity of the kinase. PMID- 16665263 TI - Functional Organization of Chlorophyll a and Carotenoids in the Alga, Nannochloropsis salina. AB - Chlorophyll-protein complexes were isolated from a yellow-green alga, Nannochloropsis salina after mild detergent treatment and gel electrophoresis. Three different complexes were obtained which correspond to the three major kinds of chlorophyll-proteins isolated from spinach chloroplasts by the same procedure and previously identified as reaction center complexes for photosystems I and II and a light-harvesting complex. The analogy between the algal complexes and those from spinach was drawn from their absorption and fluorescence spectra and relative pigment content. The identities and amounts of the major carotenoids associated with each isolated complex were determined by HPLC. Although the reaction center complexes accounted for only 14% of the total chlorophyll, they were highly enriched in beta-carotene, whereas the light-harvesting complex contained a high proportion of xanthophylls (mainly violaxanthin and vaucheriaxanthin-ester). Fluorescence excitation spectra of the algal membranes showed that one or both of the major xanthophylls may act as antenna pigment for photosynthesis. PMID- 16665264 TI - The Susceptibility of Photosynthesis to Photoinhibition and the Capacity of Recovery in High and Low Light Grown Cyanobacteria, Anacystis nidulans. AB - The susceptibility of photosynthesis to photoinhibition and the rate of its recovery were studied in the cyanobacterium Anacystis nidulans grown at a low (10 micromoles per square meter per second) and a high (120 micromoles per square meter per second) photosynthetically active radiation. The rate of light limited photosynthetic O(2) evolution was measured to determine levels of photoinhibition and rates of recovery. Studies of photoinhibition and recovery with and without the translation inhibitor streptomycin demonstrated the importance of a recovery process for the susceptibility of photosynthesis to photoinhibition. We concluded that the approximately 3 times lower susceptibility to photoinhibition of high light than of low light grown cells, significantly depended on high light grown cells having an approximately 3 times higher recovery capacity than low light grown cells. It is suggested that these differences in susceptibility to photoinhibition and recovery depends on high light grown cells having a higher turnover rate of photosystem II protein(s) that is(are) the primary site(s) of photodamage, than have low light grown cells. Furthermore, we demonstrated that photoinhibition of A. nidulans may occur under physiological light conditions without visible harm to the growth of the cell culture. The results give support for the hypotheses that the net photoinhibitory damage of photosystem II results from the balance between the photoinhibitory process and the operation of a recovery process; the capacity of the latter determining significant differences in the susceptibility of photosynthesis to photoinhibition of high and low light grown A. nidulans. PMID- 16665265 TI - Genes expressed in the male gametophyte of flowering plants and their isolation. AB - Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period. PMID- 16665266 TI - Genetic Regulation of Development in Sorghum bicolor: III. Asynchrony of Thermoperiods with Photoperiods Promotes Floral Initiation. AB - Asynchronous thermo- and photoperiods were observed to hasten floral differentiation (initiation) in the short day plant Sorghum bicolor (L.) Moench. Shifting the 12 hour thermoperiod forward 0.5 or 2.5 hours hastened floral initiation compared to controls with synchronous thermo-and photoperiods. Delaying the day-night temperature change until 2.5 hours after either the dark light or light-dark transition also hastened floral initiation. Inasmuch as sorghum milo-type maturity genotypes containing the genes designated Ma(1)Ma(2) (class I) were most responsive to asynchrony of the thermo- and photoperiods while those containing ma(3) (R) (class III) were not responsive, the maturity genes appear to control the plant's response to both photoperiod and temperature. Gibberellic acid promoted the effect of thermo- and photoperiod asynchrony on floral initiation. The results suggest that in sorghum both temperature and photoperiod may act as phase setting signals in what has previously been termed photoperiodism. PMID- 16665267 TI - Imidazolinones and acetohydroxyacid synthase from higher plants: properties of the enzyme from maize suspension culture cells and evidence for the binding of imazapyr to acetohydroxyacid synthase in vivo. AB - Acetohydroxyacid synthase has been purified from maize (Zea mays, var Black Mexican Sweet) suspension culture cells 49-fold by a combination of ion exchange chromatography, gel filtration, and hydroxyapatite chromatography. Use of the nondenaturing, zwitterionic detergent 3-([3-cholamidopropyl]dimethyl-ammonio)-1 propanesulfonate was necessary to dissociate the enzyme from the heterogeneous, high molecular weight aggregates in which it appears to reside in vitro. The solubilized maize acetohydroxyacid synthase had a relative molecular mass of 440,000. The purified enzyme was highly unstable. Acetohydroxyacid synthase activities in crude extracts of excised maize leaves and suspension cultured cells were reduced 85 and 58%, respectively, by incubation of the tissue with 100 micromolar (excised leaves) and 5 micromolar (suspension cultures) of the imidazolinone imazapyr prior to enzyme extraction, suggesting that the inhibitor binds tightly to the enzyme in vivo. Binding of imazapyr to maize acetohydroxyacid synthase could also be demonstrated in vitro. Evidence is presented which suggests that the interaction between imazapyr and the enzyme is reversible. Imazapyr also exhibited slow-binding properties when incubated with maize cell acetohydroxyacid synthase in extended time course experiments. Initial and final K(i) values for the inhibition were 15 and 0.9 micromolar, respectively. The results suggest that imazapyr is a slow, tight-binding inhibitor of acetohydroxyacid synthase. PMID- 16665268 TI - Net CO(2) Assimilation Rate of Grapevine Leaves in Response to Trunk Girdling and Gibberellic Acid Application. AB - Net CO(2) assimilation rate (A), stomatal conductance (g(s)), and weight per unit leaf area (W) were determined on Thompson Seedless grapevines grown in the field. Treatments included fruit set applications of gibberellic acid (40 milligrams gibberellic acid (GA(3)) per liter) to vines, shoots and clusters, alone and in combination with trunk girdling. Leaf A and g(s) were measured prior to and 3, 6, and 13 days after fruit set. Weight per unit leaf area was determined on leaves collected subsequent to gas exchange measurements. Leaf A of girdled vines was reduced approximately 30% when compared to the control 13 days after treatment. The reduction in A due to girdling was not as great when vines were sprayed with GA(3). GA(3) sprays alone had no significant effect on A. Stomatal conductance was reduced by girdling 13 days after treatment. Weight per unit leaf area was 17% greater for trunk girdled vines when compared to the controls. Results indicate GA(3) affected net CO(2) assimilation rate only on girdled vines, a treatment which increased weight per unit leaf area. PMID- 16665269 TI - Evidence for Inorganic Carbon Transport by Intact Chloroplasts of Chlamydomonas reinhardtii. AB - Isolated intact chloroplasts from wall-less mutants of Chlamydomonas reinhardtii accumulate inorganic carbon (C(i)) from the medium provided the cells had been adapted to low CO(2) photoautotrophic growth conditions. Chloroplasts from cultures grown on high (5%) CO(2) or photoheterotrophically with acetate did not accumulate inorganic carbon. Chloroplast C(i) accumulation from low CO(2) grown cells was light dependent and was inhibited by uncouplers and inhibitors of electron transport. In a model for C(i) accumulation by Chlamydomonas, it is proposed that CO(2) diffuses into the cell and C(i) accumulation occurs in the chloroplast. PMID- 16665270 TI - Male Germ Unit Isolation from Three Tricellular Pollen Species: Brassica oleracea, Zea mays, and Triticum aestivum. AB - A technical procedure is described to follow the in vitro release of the ;male germ unit' and the sperm cells from three tricellular pollen species (Brassica, Zea, and Triticum). The condition of the sperm cell was controlled using light microscopy. In addition, for the first time, the sperm cells viability has been checked by the fluorochromatic reaction test. These preliminary results indicate that this procedure appears to be a prerequisite for the successful preparation of purified viable sperm cells. PMID- 16665271 TI - Soybean seed growth in response to long-term exposures to differing oxygen partial pressures. AB - Short-term studies have indicated that alterations in the oxygen partial pressure (pO(2)) around developing soybean (Glycine max [L.] Merr.) seeds may alter seed growth characteristics. A 2-year field study was undertaken to determine the effects on seed development of long-term exposures of individual pods to either sub-ambient or supra-ambient pO(2). Pod chambers were used through which fixed pO(2) were continuously flowed throughout seed development. No effects on maturity date were observed from exposures to either sub-ambient or supra-ambient pO(2). On the other hand, seed weight was reduced by 0.10 pO(2) in both years of the study implicating an O(2) limitation on seed growth rate at this fairly high pO(2). In 1 of the 2 years, supra-ambient pO(2) resulted in increased seed weight. PMID- 16665272 TI - Inhibition of Adenosine Triphosphatase Activity from a Plasma Membrane Fraction of Acer pseudoplatanus Cells by Carbanilate Derivatives. AB - Thirty-one carbanilate derivatives were assayed for their capabilities to inhibit the ATPase activity of a plasma membrane fraction from Acer pseudoplatanus cells. At a concentration of 100 micromolar, nine compounds strongly inhibited the ATPase activity, with I(50) ranging from 14.5 micromolar to 35 micromolar. These molecules were also inhibitory to plasma membrane ATPases of other origins: plant (maize shoot), yeast (Schizosaccharomyces pombe), and animal (dog kidney). The most efficient molecule appeared to be 2,2,2-trichloroethyl 3,4 dichlorocarbanilate. PMID- 16665273 TI - Effect of Inorganic Orthophosphate on in Vitro Activity of NADH-Nitrate Reductase Isolated from 2-Row Barley Leaves. AB - Inorganic orthophosphate (25 millimolar in assay media; Pi) was found to increase in vitro activity of NADH-nitrate reductase (NR) isolated from 2-row barley (Hordeum vulgare L.) leaves with a saturating concentration of nitrate (2 millimolar) but to decrease it with low nitrate levels (<0.1 millimolar). The response to nitrate concentrations was Pi specific. The Lineweaver-Burk plot showed that Pi increases the apparent K(m) for nitrate as well as V(max), whereas it does not alter the K(m) for NADH significantly. These results suggest that the interaction between a molybdenum site of the enzyme and Pi results in alteration of the properties of NR molecule. PMID- 16665274 TI - Enzymic synthesis of caffeoylglucaric Acid from chlorogenic Acid and glucaric Acid by a protein preparation from tomato cotyledons. AB - The phenylpropane metabolism of tomato (Lycopersicon esculentum Mill) cotyledons was investigated. The HPLC analysis revealed two hydroxycinnamic-acid conjugates as major components, identified as chlorogenic acid (5-O-caffeoylquinic acid) and caffeoylglucaric acid (2-O- or 5-O-caffeoyl-glucaric acid). Quantitative analyses indicated a precursor-product relationship between the chlorogenic and caffeoylglucaric acids. Protein preparations from tomato cotyledons were found to catalyze the formation of caffeoylglucaric acid with chlorogenic acid as acyl donor and free glucaric acid as acceptor molecule. This enzyme activity, possibly to be classified as hydroxycinnamoylquinic acid:glucaric acid hydroxycinnamoyltransferase, acts together with hydroxycinnamoyl-CoA: quinic acid hydroxycinnamoyltransferase. PMID- 16665275 TI - Response of Cytosolic-Isozyme and Plastid-Isozyme Levels of 3-Deoxy-d-arabino Heptulosonate 7-Phosphate Synthase to Physiological State of Nicotiana silvestris in Suspension Culture. AB - Two isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase are partitioned into plastid (DS-Mn) and cytosolic (DS-Co) compartments of at least several higher plants (RA Jensen 1986 Rec Adv Phytochem 20: 257-258). Differential variation of isozyme levels and in the timing of their expression was observed during growth of Nicotiana silvestris in suspension culture. The ratio of DS-Co to DS-Mn varied about fivefold in comparison of the different physiological stages of growth. Cultures maintained in exponential phase for >10 generations (EE cells) possessed balanced-growth properties and did not exhibit the considerable variation of isozyme levels found during the initial 2 to 3 generations of exponential growth (E cells) that followed subculture of stationary-phase cultures. The plastid isozyme level declined substantially in stationary phase, responded immediately to subculture, and reached a peak in early exponential growth similar to the steady-state level of DS-Mn in EE cells. In contrast, the cytosolic isozyme level peaked in late exponential growth. A recent history of stationary-phase physiology appeared to foster elevated synthesis of DS-Co since the steady-state level of DS-Co in EE cells was much lower than in E cells. PMID- 16665276 TI - Potential-dependent anion transport in tonoplast vesicles from oat roots. AB - Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Deltapsi) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H(+)-pumping pyrophosphatase, which is K(+) stimulated and anion insensitive. The relative rate of DeltaPsi dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN(-) (100) > NO(3) (-) (72) = Cl(-) (70) > Br(-) (62) > SO(4) (2-) (5) = H(2)PO(4) (-) (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Deltapsi dissipation by Cl(-) and NO(3) (-), but not by SCN(-), was saturable. The K(m) values for Cl( ) and NO(3) (-) uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H(+)-coupled Cl(-) transporter on the same vesicles, the potential-dependent Cl(-) transport was insensitive to 4,4'-diisothiocyano-2,2'-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl( ) transport in these vesicles. The potentials generated by the H(+)-translocating ATPase and H(+)-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl(-) conductance on the anion-sensitive H(+)-ATPase was found. PMID- 16665277 TI - Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H Coupled Anion Transport. AB - Chloride or nitrate decreased a pH gradient (measured as [(14)C]methylamine accumulation) in tonoplast-enriched vesicles. The DeltapH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H(+)-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K(+) gradient and nigericin. 4,4' Diisothiocyano-2,2'-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl(-). Two possible models to account for this anion dependent decrease of DeltapH are: (a) H(+) loss is accompanied by Cl(-) or NO(3) (-) efflux from the vesicles via H(+)/anion symport systems on the tonoplast and (b) H(+) loss is accompanied by Cl(-) or NO(3) (-) uptake into the vesicles via H(+)/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl(-) and NO(3) (-) stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H(+)-coupled anion transport systems. PMID- 16665278 TI - Quantification of the kinetin effect on protein synthesis and degradation in senescing wheat leaves. AB - Wheat leaves (Triticum aestivum L. cv San Agustin INTA) were detached when they reached maximum expansion, put individually in tubes containing water and left in darkness. After 3 days the protein content had decreased to 46% of the initial value. When the leaves were placed in 1 micromolar kinetin, they retained 60% of the initial protein content for the same period. This effect was observed only when leaves were treated with kinetin within the first 24 hours after detachment. The action of kinetin on both protein synthesis and degradation was quantitatively measured. Synthesis was estimated by the incorporation of l [(3)H]leucine into proteins. It was higher in kinetin treated than in non treated leaves. It contributed to about 14 micrograms of protein retention per leaf in 3 days. Measurement of protein degradation, evaluated by the decay of radioactivity in leaf proteins previously labeled with l-[(3)H] leucine or as the difference between rates of protein synthesis and protein content, showed that kinetin decreased protein breakdown rates. It accounted for about 186 micrograms of protein retention per leaf in 3 days. Hence, kinetin action on protein breakdown was 13-fold average higher than its action on synthesis for the conservation of leaf protein. This difference is higher in early stages of the process. PMID- 16665279 TI - Effects of prostaglandins e(2) and f(2alpha) on the electrofusion of pea mesophyll protoplasts. AB - The effects of prostaglandins E(2) and F(2alpha) on the electrofusion of pea (Pisum sativum cv Ran 1) mesophyll protoplasts were examined. Prostaglandins E(2) and F(2alpha) influenced electrofusion by lowering the threshold voltage necessary for fusion of dielectrophoretically arranged pairs of protoplasts. The direct current voltage threshold decreased with increasing Ca(2+) concentration up to 0.1 millimolar CaCl(2) and the effects of prostaglandins E(2) and F(2alpha) were more pronounced when CaCl(2) was present in the medium. Treatment with calcium channel blocker methoxy verapamil did not change the prostaglandin effects, while the addition of ethyleneglycol-bis (beta-aminoethyl either) N,N,N',N'-tetraacetic acid, which binds free Ca(2+), increased the threshold voltage. Influence of prostaglandins E(2) and F(2alpha) and Ca(2+) on the membrane fluidity was investigated by analysis of pyrene fluorescence spectra. The values of the ratio between the maximum fluorescence emission intensities of the excimer and the monomer forms (I(ex)/I(mon)) indicated that prostaglandins and Ca(2+) decrease the membrane fluidity. It is proposed that electrically evoked displacement of plasmalemma components takes part in the fusion process (U Zimmermann 1982 Biochim Biophys Acta 694: 227-277). We suggest that prostaglandins E(2) and F(2alpha) facilitate the electrofusion of pea mesophyll protoplasts by changing the fluidity of plasmalemma. PMID- 16665280 TI - Influx of na, k, and ca into roots of salt-stressed cotton seedlings : effects of supplemental ca. AB - High Na(+) concentrations may disrupt K(+) and Ca(2+) transport and interfere with growth of many plant species, cotton (Gossypium hirsutum L.) included. Elevated Ca(2+) levels often counteract these consequences of salinity. The effect of supplemental Ca(2+) on influx of Ca(2+), K(+), and Na(+) in roots of intact, salt-stressed cotton seedlings was therefore investigated. Eight-day-old seedlings were exposed to treatments ranging from 0 to 250 millimolar NaCl in the presence of nutrient solutions containing 0.4 or 10 millimolar Ca(2+). Sodium influx increased proportionally to increasing salinity. At high external Ca(2+), Na(+) influx was less than at low Ca(2+). Calcium influx was complex and exhibited two different responses to salinity. At low salt concentrations, influx decreased curvilinearly with increasing salt concentration. At 150 to 250 millimolar NaCl, (45)Ca(2+) influx increased in proportion to salt concentrations, especially with high Ca(2+). Potassium influx declined significantly with increasing salinity, but was unaffected by external Ca(2+). The rate of K(+) uptake was dependent upon root weight, although influx was normalized for root weight. We conclude that the protection of root growth from salt stress by supplemental Ca(2+) is related to improved Ca-status and maintenance of K(+)/Na(+) selectivity. PMID- 16665281 TI - The effects of salt on the pattern of protein synthesis in barley roots. AB - The effect of salt stress on the incorporation of [(35)S]methionine into protein was examined in roots of barley (Hordeum vulgare L. cv California Mariout 72). Plants were grown in nutrient solution with or without 200 millimolar NaCl. Roots of intact plants were labeled in vivo and proteins were extracted and analyzed by fluorography of two-dimensional gels. Although the protein patterns for control and salt-stressed plants were qualitatively similar, the net synthesis of a number of proteins was quantitatively changed. The most striking change was a significant increase of label in two protein pairs that had pIs of approximately 6.3 and 6.5. Each pair consisted of proteins of approximately 26 and 27 kilodaltons (kD). In roots of control plants, the 27-kD proteins were more heavily labeled in the microsomal fraction relative to the 26-kD proteins, whereas the 26-kD proteins were enriched in the post 178,000 g supernatant fraction; in roots of salt treated plants, the 26- and 27-kD proteins were more intensely labeled in both fractions. Labeling of the 26- and 27-kD proteins returned to control levels when salt-stressed plants were transferred to nutrient solution without NaCl. No cross-reaction was detected between the antibody to the 26-kD protein from salt-adapted tobacco cells and the 26- and 27-kD proteins of barley. PMID- 16665282 TI - Inhibitory Effects of a Pectin-Enriched Tomato Cell Wall Fraction on Agrobacterium tumefaciens Binding and Tumor Formation. AB - A pectin-enriched soluble cell wall fraction (CWF) prepared from suspension cultured tomato cells inhibits binding of Agrobacterium tumefaciens to these cells. It was hypothesized that the CWF contains the plant surface binding site for A. tumefaciens (NT Neff, AN Binns 1985 Plant Physiol 77: 35-42). Experiments described here demonstrate that tomato CWF inhibited tumor formation on potato slices and Agrobacterium binding to intact tomato cells in a dose-dependent fashion. Boiling the fraction reduced both its binding and tumor inhibitory activities. Tumor inhibitory activity was titrated out by increased concentrations of bacterial inocula with no inhibition apparent at 1 x 10(8) bacteria per milliliter. These results indicate that a tomato CWF is enriched for a putative A. tumefaciens binding site which may also be involved in tumor formation in potato. PMID- 16665283 TI - Virulence of Agrobacterium tumefaciens Strain A281 on Legumes. AB - This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains. PMID- 16665284 TI - Uptake and Metabolism of Carbohydrates by Bradyrhizobium japonicum Bacteroids. AB - Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with (14)C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O(2) (2% in the gas phase). Uptake and conversion of (14)C to CO(2) were measured at intervals up to 90 minutes. Of the five compounds studied, UDP glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO(2), and fructose was respired at a rate at least double that of glucose. Sucrose and glucose were converted to CO(2) at a very low but measurable rate (<0.1 nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO(2) at a rate 30 times greater than the conversion of carbon Number 6 to CO(2), indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase. PMID- 16665285 TI - Selective Solubilization of Membrane Proteins Differentially Labeled by p Chloromercuribenzenesulfonic Acid in the Presence of Sucrose. AB - Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [(203)Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethyleneglycol-bis-(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3 cholamidopropyl)-dimethylammonio] 1-propane-sulfonate) (CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to include the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS labeled proteins (;background' proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mm EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions. PMID- 16665286 TI - Cation amelioration of aluminum toxicity in wheat. AB - Aluminum is a major constituent of most soils and limits crop productivity in many regions. Amelioration is of theoretical as well as practical interest because understanding amelioration may contribute to an understanding of the mechanisms of toxicity. In the experiments reported here 2-day-old wheat (Triticum aestivum L. cv Tyler) seedlings with 15-millimeter roots were transferred to solutions containing 0.4 millimolar CaCl(2) at pH 4.3 variously supplemented with AlCl(3) and additional amounts of a chloride salt. Root lengths, measured after 2 days in the test solutions, were a function of both Al activity and the cation activity of the added salt. Percent inhibition = 100 {Al(3+)}/({Al(3+)} + K(m) + alpha{C}(beta)) where {Al(3+)} is the activity of Al(3+) expressed in micromolar, {C} is the activity of the added cation expressed in millimolar, and K(m) (= 1.2 micromolar) is the {Al(3+)} required for 50% inhibition in the absence of added salt. For Ca(2+), Mg(2+), and Na(+) the values of alpha were 2.4, 1.6, and 0.011, respectively, and the values for beta were 1.5, 1.5, and 1.8, respectively. With regard to relative ameliorative effectiveness, Ca(2+) > Mg(2+) approximately Sr(2+) >> K(+) approximately Na(+). Other cations were tested, but La(3+), Sc(3+), Li(+), Rb(+), and Cs(+) were toxic at potentially ameliorative levels. The salt amelioration is not solely attributable to reductions in {Al(3+)} caused by increases in ionic strength. Competition between the cation and Al for external binding sites may account for most of the amelioration. PMID- 16665287 TI - Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules. AB - Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O(2) (Ar:O(2)) and air (Air) atmospheres affected N assimilation (NH(4)NO(3) and N(2) fixation) and the partitioning of photosynthate to roots and nodules. Application of NH(4)NO(3) to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O(2) or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O(2) to the other (Air/Ar:O(2)), and (c) Ar:O(2) to both sides (Ar:O(2)/Ar:O(2)). Results indicated that dry matter and current photosynthate ((14)C) were selectively partitioned to nodules and roots where N(2) was available. Both root and nodule growth on the Air side of Air/Ar:O(2) plants was significantly greater than the Ar:O(2) side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N(2) availability. The Ar:O(2) sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N(2)-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O(2) atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half root systems in Air while specific nitrogenase activity of nodules in Ar:O(2) was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N(2) fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N(2)-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots. PMID- 16665288 TI - Hevea Linamarase-A Nonspecific beta-Glycosidase. AB - In the leaf tissue of the cyanogenic plant Hevea brasiliensis, which contains large amounts of linamarin, there is no specific linamarase. In Hevea leaves only one beta-glucosidase is detectable. It is responsible for the cleavage of all beta-glucosides and beta-galactosides occurring in Hevea leaf tissue, including the cyanogenic glucoside linamarin. Therefore, the enzyme is referred to as a beta-glycosidase instead of the term beta-glucosidase. This beta-glycosidase has a broad substrate spectrum and occurs in multiple forms. These homo-oligomeric forms are interconvertible by dissociation-association processes. The monomer is a single protein of 64 kilodaltons. PMID- 16665289 TI - Involvement of Carrot Cell Surface Proteins in Attachment of Agrobacterium tumefaciens. AB - The initial step in tumor formation by Agrobacterium tumefaciens is the site specific attachment of the bacteria to plant cells. A similar attachment to plant tissue culture cells has been observed. Binding to carrot suspension culture cells was not dependent on the presence of divalent cations and was not inhibited by the addition of mannose, alpha-methyl mannoside, galactose, arabinose, glucosamine, 2-deoxyglucose, or 0.25 molar NaCl to the culture medium. The ability of the carrot cells to bind A. tumefaciens was markedly reduced by elution of the cells with dilute detergent or CaCl(2) or by incubation of the cells with proteolytic enzymes. The carrot cells were not killed by these treatments and recovered the ability to bind A. tumefaciens within 3 to 6 hours. A. tumefaciens did not bind to carrot cells which had been induced to form embryos (AG Matthysse, RHG Gurlitz 1982 Physiol Plant Pathol 21: 381-387). A comparison of the peptides eluted from embryos and from uninduced cells using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there were several changes in extractable polypeptides after embryo induction. One or more of the polypeptides present before embryo induction and absent from embryos may be involved in the binding of A. tumefaciens to the carrot cell surface. PMID- 16665290 TI - H-ATPase Activity from Storage Tissue of Beta vulgaris: IV. N,N' Dicyclohexylcarbodiimide Binding and Inhibition of the Plasma Membrane H-ATPase. AB - The molecular weight and isoelectric point of the plasma membrane H(+)-ATPase from red beet storage tissue were determined using N,N'-dicyclohexylcarbodiimide (DCCD) and a H(+)-ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [(14)C]-DCCD at 0 degrees C, a single 97,000 dalton protein was visualized on a fluorograph of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [(14)C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H(+)-ATPase. An antibody raised against the plasma membrane H(+)-ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H(+)-ATPase to be 6.5. PMID- 16665291 TI - Drought Stress and Elevated CO(2) Effects on Soybean Ribulose Bisphosphate Carboxylase Activity and Canopy Photosynthetic Rates. AB - Soybean (Glycine max [L.] cv Bragg) was grown at 330 or 660 microliters CO(2) per liter in outdoor, controlled-environment chambers. When the plants were 50 days old, drought stress was imposed by gradually reducing irrigation each evening so that plants wilted earlier each succeeding day. On the ninth day, as the pots ran out of water CO(2) exchange rate (CER) decreased rapidly to near zero for the remainder of the day. Both CO(2)-enrichment and drought stress reduced the total (HCO(3) (-)/Mg(2+)-activated) extractable ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity, as expressed on a chlorophyll basis. In addition, drought stress when canopy CER values and leaf water potentials were lowest, reduced the initial (nonactivated) RuBPCase activity by 50% compared to the corresponding unstressed treatments. This suggests that moderate to severe drought stress reduces the in vivo activation state of RuBPCase, as well as lowers the total activity. It is hypothesized that stromal acidification under drought stress causes the lowered initial RuBPCase activities. The K(m)(CO(2)) values of activated RuBPCase from stressed and unstressed plants were similar; 15.0 and 12.6 micromolar, respectively. RuBP levels were 10 to 30% lower in drought stressed as compared to unstressed treatments. However, RuBP levels increased from near zero at night to around 150 to 200 nanomoles per milligram chlorophyll during the day, even as water potentials and canopy CERs decreased. This suggests that the rapid decline in canopy CER cannot be attributed to drought stress induced limitations in the RuBP regeneration capability. Thus, in soybean leaves, a nonstomatal limitation of leaf photosynthesis under drought stress conditions appears due, in part, to a reduction of the in vivo activity of RuBPCase. Because initial RuBPCase activities were not reduced as much as canopy CER values, this enzymic effect does not explain entirely the response of soybean photosynthesis to drought stress. PMID- 16665292 TI - Ureide Catabolism of Soybeans : II. Pathway of Catabolism in Intact Leaf Tissue. AB - Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit (14)CO(2) release from [2,7-(14)C]allantoin (urea labeled), but inhibits urea dependent CO(2) release >/=99.9% under similar conditions. Furthermore, (14)CO(2) and [(14)C] allantoate are the only detectable products of [2,7-(14)C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-(14)C]allantoin), its catabolism to [(14)C]allantoate, (14)CO(2), [(14)C]glyoxylate, [(14)C]glycine, and [(14)C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH(4) (+) and CO(2) and that carbons 4 and 5 enter the photorespiratory pathway. PMID- 16665293 TI - Pressure probe and isopiestic psychrometer measure similar turgor. AB - Turgor measured with a miniature pressure probe was compared to that measured with an isopiestic thermocouple psychrometer in mature regions of soybean (Glycine max [L.] Merr.) stems. The probe measured turgor directly in cells of intact stems whereas the psychrometer measured the water potential and osmotic potential of excised stem segments and turgor was calculated by difference. When care was taken to prevent dehydration when working with the pressure probe, and diffusive resistance and dilution errors with the psychrometer, both methods gave similar values of turgor whether the plants were dehydrating or rehydrating. This finding, together with the previously demonstrated similarity in turgor measured with the isopiestic psychrometer and a pressure chamber, indicates that the pressure probe provides accurate measurements of turgor despite the need to penetrate the cell. On the other hand, it suggests that as long as precautions are taken to obtain accurate values for the water potential and osmotic potential, turgor can be determined by isopiestic psychrometry in tissues not accessible to the pressure probe for physical reasons. PMID- 16665294 TI - Origin of growth-induced water potential : solute concentration is low in apoplast of enlarging tissues. AB - We developed a new method to measure the solute concentration in the apoplast of stem tissue involving pressurizing the roots of intact seedlings (Glycine max [L.] Merr. or Pisum sativum L.), collecting a small amount of exudate from the surface of the stem under saturating humidities, and determining the osmotic potential of the solution with a micro-osmometer capable of measuring small volumes (0.5 microliter). In the elongating region, the apoplast concentrations were very low (equivalent to osmotic potentials of -0.03 to -0.04 megapascal) and negligible compared to the water potential of the apoplast (-0.15 to -0.30 megapascal) measured directly by isopiestic psychrometry in intact plants. Most of the apoplast water potential consisted of a negative pressure that could be measured with a pressure chamber (-0.15 to -0.28 megapascal). Tests showed that earlier methods involving infiltration of intercellular spaces or pressurizing cut segments caused solute to be released to the apoplast and resulted in spuriously high concentrations. These results indicate that, although a small amount of solute is present in the apoplast, the major component is a tension that is part of a growth-induced gradient in water potential in the enlarging tissue. The gradient originates from the extension of the cell walls, which prevents turgor from reaching its maximum and creates a growth-induced water potential that causes water to move from the xylem at a rate that satisfies the rate of enlargement. The magnitude of the gradient implies that growing tissue contains a large resistance to water movement. PMID- 16665295 TI - Immunocytochemical Localization of Ribulose-1,5-Bisphosphate Carboxylase in the Pyrenoid and Thylakoid Region of the Chloroplast of Chlamydomonas reinhardtii. AB - The distribution of the large and small subunits of ribulose-1,5-bisphosphate carboxylase in the chloroplast of Chlamydomonas reinhardtii was studied by immunoelectron microscopy by labeling Lowicryl-embedded sections with antibody to each subunit followed by protein A-gold. In light-harvested synchronously dividing cells, antibodies to each subunit heavily labeled the pyrenoid, whereas the thylakoid region of the plastid was lightly labeled. By estimating the volume of each chloroplast compartment, it was determined that approximately 40% of the total small subunit in the plastid and 30% of the large subunit are localized in the thylakoid region, presumably in the stroma. In synchronously dividing cells exposed to an extended dark period, the amount of labeling of the pyrenoid region by antibody to the small subunit stayed constant, but the labeling of the thylakoid region decreased. In stationary phase cells, the proportion of the label over the pyrenoid is higher than in synchronously dividing cells suggesting that the pyrenoid may be a storage organelle. PMID- 16665296 TI - Influence of water relations and temperature on leaf movements of rhododendron species. AB - Rhododendron maximum L. and R. Catawbiense L. are subcanopy evergreen shrubs of the eastern United States deciduous forest. Field measurements of climate factors and leaf movements of these species indicated a high correlation between leaf temperature and leaf curling; and between leaf water potential and leaf angle. Laboratory experiments were performed to isolate the influence of temperature and cellular water relations on leaf movements. Significant differences were found between the patterns of temperature induction of leaf curling in the two species. Leaves of the species which curled at higher temperatures (R. catawbiense) also froze at higher leaf temperatures. However, in both cases leaf curling occurred at leaf temperatures two to three degrees above the leaf freezing point. Pressure volume curves indicated that cellular turgor loss was associated with a maximum of 45% curling while 100% or more curling occurred in field leaves which still had positive cell turgor. Moisture release curves indicated that 70% curling requires a loss of greater than 60% of symplastic water which corresponds to leaf water potentials far below those experienced in field situations. Conversely, most laboratory induced changes in leaf angle could be related to leaf cell turgor loss. PMID- 16665297 TI - Mode of action studies on nitrodiphenyl ether herbicides: I. Use of barley mutants to probe the role of photosynthetic electron transport. AB - 5-[2-Chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI), is a potent nitrodiphenyl ether herbicide which causes rapid leaf wilting, membrane lipid peroxidation, and chlorophyll destruction in a process which is both light- and O(2)-dependent. These effects resemble those of other nitrodiphenyl ether herbicides. Unlike paraquat, the herbicidal effects of DPEI are only slightly reduced by pretreatment with the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. DPEI is a weak inhibitor of photosynthetic electron transport (I(50) 15 micromolar for water to paraquat) in vitro, with at least one site of action at the cytochrome b(6)f complex. Ultrastructural studies and measurements of ethane formation resulting from lipid peroxidation indicate that mutants of barley lacking photosystem I (PSI) (viridis-zb(63)) or photosystem II (viridis-zd(69)) are resistant to paraquat but susceptible to DPEI. The results indicate that electron transfer through both photosystems is not essential for the toxic effects of nitrodiphenyl ether herbicides. Furthermore, the results show that neither cyclic electron transport around PSI, nor the diversion of electrons from PSI to O(2) when NADPH consumption is blocked are essential for the phytotoxicity of nitrodiphenyl ether herbicides. PMID- 16665298 TI - Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases. AB - The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg(2+), ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3 phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll. PMID- 16665299 TI - Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. AB - Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro. PMID- 16665300 TI - Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton. AB - Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves. PMID- 16665301 TI - Phosphatidylinositol Cycle Metabolites in Samanea saman Pulvini. AB - The major metabolites of the phosphatidylinositol cycle from extracts of [(32)PO(4)]- and [(3)H]-inositol-labeled Samanea saman pulvini were separated. The membrane localized phosphoinositides were separated by thin layer chromatography, identified by comparison with purified lipid standards, and quantitated based on incorporation of radiolabel. The ratio of radioactivity in phosphatidylinositol:phosphatidylinositol 4-phosphate:phosphatidylinositol 4,5 bisphosphate is about 32:8:1. The aqueous inositol phosphates were separated by anion exchange chromatography using conventional liquid chromatography and by high performance liquid chromatography (HPLC) and were identified by comparison with standards. Analysis by HPLC reveals that (32)P-labeled pulvini have inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate that co migrate with red blood cell inositol phosphates, but (3)H-inositol-labeled pulvini appear to have a variant profile. PMID- 16665302 TI - Effect of Acid treatment of plant cuticles on sorption of selected auxins. AB - Sorption characteristics of 2-(1-naphthyl)acetic acid (NAA), 2-(1 naphthyl)acetamide (NAAm), and 2,4-dichlorophenoxyacetic acid (2,4-D) were determined for cuticles enzymically isolated from mature tomato (Lycopersicon esculentum Mill. cv Sprinter) and pepper (Capsicum annuum L.) fruit. Sorption equilibrium for NAA and 2,4-D by tomato cuticular membranes (CM) and dewaxed cuticular membranes (DCM) was achieved within 24 hours at 25 degrees C. The average K (partition coefficient) values for NAA in tomato CM and DCM were 166 and 204, respectively, whereas the corresponding K values for 2,4-D were 292 and 383, respectively. Sorption equilibrium for 2,4-D and NAA in pepper cuticles was not achieved after 18 and 63 days, respectively. Sorption equilibrium for NAAm in tomato and pepper CM and DCM was attained within 48 hours. Acid pretreatment (2.0 n HCl, 10 minutes) had no effect on NAA, 2,4-D, or NAAm sorption by tomato CM and DCM, or on NAAm sorption by pepper CM and DCM. Acid pretreatment of pepper CM and DCM led to slightly lower K(pH) (apparent partition coefficient) values for both NAA and 2,4-D. More significantly, sorption equilibrium for NAA and 2,4-D in pepper CM and DCM was achieved within 24 hours after acid treatment. PMID- 16665303 TI - Site of Synthesis of the Enzymes of the Pyrimidine Biosynthetic Pathway in Oat (Avena sativa L.) Leaves. AB - Heat-bleached oat (Avena sativa L. cv Porter) leaves lacking 70S chloroplast ribosomes have been used to demonstrate that four chloroplast-localized enzymes of pyrimidine nucleotide biosynthesis: aspartate carbamoyl-transferase, dihydroorotase, orotidine phosphoribosyl-transferase, and orotidine-5'-phosphate decarboxylase, are synthesized on cytoplasmic ribosomes. Two other chloroplast enzymes, carbamoyl phosphate synthetase, involved in both pyrimidine and arginine biosynthesis, and ornithine carbamoyltransferase, an enzyme of arginine biosynthesis, were also shown to be made on 80S ribosomes. PMID- 16665304 TI - Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells. AB - The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-alpha-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-alpha-1,4-polygalacturonase-treated walls by treatment with an endo-beta-1,4 glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-beta-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides. PMID- 16665305 TI - Phototropism in Hypocotyls of Radish : III. Influence of Unilateral or Bilateral Illumination of Various Light Intensities on Phototropism and Distribution of cis and trans-Raphanusanins and Raphanusamide. AB - When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light at 0.05, 0.1, or 1 watt per square meter, the suppression of the growth rate on the lighted side depended on the light intensity. The growth rate at the shaded side was only a little affected by the illumination at 0.05 and 0.1 watt per square meter but considerably suppressed by that at 1 watt per square meter. Upon a continuous unequal bilateral illumination, the growth rate was more strongly suppressed on the side of the higher intensity than on the side of the lower one, resulting in phototropic curvature toward the light source of the higher intensity. It was calculated from correlation analysis of light intensity and growth rate that, on an average, 6.9% of the irradiation applied to one side reached the opposite side. The amounts of cis- and trans-raphanusanins and raphanusamide in hypocotyls subjected to unilateral or unequal bilateral illumination increased much more at the side of the lighted or the higher intensity than at the opposite side. The present study demonstrates that phototropism in radish hypocotyl is correlated with and we conclude caused by a gradient of growth inhibition in the hypocotyl, depending on irradiation-induced amounts of cis- and trans-raphanusanins and raphanusamide. PMID- 16665306 TI - Fatty Acid Specificity and Selectivity of the Chloroplast sn-Glycerol 3-Phosphate Acyltransferase of the Chilling Sensitive Plant, Amaranthus lividus. AB - Chilling sensitivity of plants is strongly correlated with the presence of high levels of a species of chloroplast phosphatidylglycerol that contains two saturated fatty acids. The most straightforward synthetic pathway for this lipid would require the primary acylation of sn-glycerol 3-phosphate (G3P) with a saturated fatty acid (palmitic acid) rather than with oleic acid, an unsaturated acid. This selective incorporation would differ markedly from the reported properties of the chloroplast G3P acyltransferases of pea and spinach, two chilling resistant plants and thus we have studied the chloroplast G3P acyltransferase of Amaranthus lividus, a chilling sensitive plant. In contrast to our results and those of others (M. Frentzen et al. 1983 Eur J Biochem 129: 629 636 and previous work) with the pea and spinach enzymes, the amaranthus chloroplast G3P acyltranferase did not select oleic acid donors from a mixture of oleic and palmitic acid donors (either coenzyme A or acyl carrier protein thioesters). Instead the fatty acid composition of the synthesized 1-acyl G3P faithfully reflected the composition of the acyl donor mixture. However, the amaranthus enzyme did strongly select against incorporation of stearic acid. The properties of the amaranthus G3P acyltransferase are consistent with this enzyme having the major role in synthesis of the disaturated phosphatidylglycerol species. PMID- 16665307 TI - Active Uptake of Amino Acids by Leaves of an Epiphytic Vascular Plant, Tillandsia paucifolia (Bromeliaceae). AB - Specialized epidermal trichomes on the leaves of the epiphyte, Tillandsia paucifolia (Bromeliaceae) accumulate amino acids from solution. Simultaneous net uptake of 17 amino acids was determined using high performance liquid chromatography. Uptake occurs against concentration gradients at least as high as 10(4). PMID- 16665308 TI - An empirical function for the description of root growth. AB - Vertical growth of young cress roots (Lepidium sativum L.) which have not yet attained their steady state is analyzed in terms of the relative elemental rate of elongation (RELEL). To this end, an empirical model function is suggested which describes, by means of six parameters, the movement of marker particles on the root surface. These parameters are interpreted as morphological and physiological quantities. Their determination by independent measurements is in good agreement with that obtained by fitting the model function to the experimental data. The RELEL is then evaluated analytically, so that numerical smoothing and differentiating algorithms with all their problems are avoided. The result demonstrates that-apart from the root cap-nearly the whole root is elongating, including the root hair region and part of the basal portion beyond it. This explains the basal curvature component that is observed during the graviresponse of cress roots. PMID- 16665309 TI - Evidence for a specific glutamate/h cotransport in isolated mesophyll cells. AB - Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO(4). Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H(+)/10(6) cells.minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-(14)C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K(+) efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells. PMID- 16665310 TI - N and C NMR determination of methionine metabolism in developing soybean cotyledons. AB - The metabolism of d- and l-methionine by immature cotyledons of soybean (Glycine max, L. cv Elf) grown in culture has been investigated using solid-state (13)C and (15)N nuclear magnetic resonance. d-Methionine is taken up by the cotyledons and converted to an amide, most likely by N-malonylation. About 16% of the l methionine taken up is incorporated intact into protein, and 25% remains as soluble methionine. Almost two-thirds of the l-methionine that enters the cotyledons is degraded. The largest percentage of this is used in transmethylation of the carboxyl groups of pectin. Methionine is not extensively converted to polyamines. We attribute the stimulation of growth of the cotyledons by exogenous methionine to the bypassing of a rate-limiting methyl-transfer step in the synthesis of methionine itself, and subsequently of pectins and proteins. PMID- 16665311 TI - Proline accumulation and its implication in cold tolerance of regenerable maize callus. AB - Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H99(3)H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19 degrees C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the medium to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to the culture media also increased the tolerance of callus to 4 degrees C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4 degrees C for 90 days which is more than double the typical survival time of callus. These studies show that proline and conditions which induce proline accumulation increase the cold tolerance of regenerable maize callus. PMID- 16665312 TI - Membrane Transport in Isolated Vesicles from Sugarbeet Taproot: III. Effects of Fluoride on ATPase Activity and Transport. AB - The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride. PMID- 16665313 TI - Auxin reduces the synthesis of major vacuolar proteins in tobacco mesophyl protoplast. AB - We have established that polypeptides whose synthesis is reduced by 2,4 dichlorophenoxyacetic acid during in vitro culture of tobacco mesophyll protoplasts are secreted into the vacuole where they constitute the bulk of labeled proteins. In addition, these proteins continue to be synthesized in protoplast-derived cultured cells and their synthesis is strictly correlated with the size of the cell, i.e. with vacuolar size. PMID- 16665314 TI - The Mechanics of Injury to Isolated Protoplasts following Osmotic Contraction and Expansion. AB - Micro-osmotic manipulation was used to determine the influence of osmotic contraction on the expansion potential of individual protoplasts isolated from rye (Secale cereale L. cv Puma) leaves. For protoplasts isolated from leaves of nonacclimated plants (NA protoplasts), osmotic contraction in sufficiently hypertonic solutions (>1.53 osmolal) predisposed the protoplasts to lysis during osmotic expansion when they were returned to isotonic conditions (0.53 osmolal). In contrast, for protoplasts isolated from leaves of cold acclimated plants (ACC protoplasts), osmotic contraction in either 2.6 or 4.0 osmolal solutions was readily reversible. Following osmotic contraction, the resting tension (gamma(r)) of NA protoplasts was similar to that determined for protoplasts in isotonic solutions (i.e. 110 +/- 22 micronewtons per meter). In contrast, gamma(r) of ACC protoplasts decreased from 164 +/- 27 micronewtons per meter in isotonic solutions to values close to zero in hypertonic solutions. Following expansion in hypotonic solutions, gamma(r)'s of both NA and ACC protoplasts were similar for area expansions over the range of 1.3 to 1.6. Following osmotic contraction and reexpansion of NA protoplasts, hysteresis was observed in the relationship between gamma(r) and surface area-with higher values of gamma(r) at a given surface area. In contrast, no hysteresis was observed in this relationship for ACC protoplasts. Direct measurements of plasma membrane tension (gamma) during osmotic expansion of NA protoplasts from hypertonic solutions (1.53 osmolal) revealed that gamma increased rapidly after small increments in surface area, and lysis occurred over a range of 1.2 to 8 millinewtons per meter. During osmotic expansion of ACC protoplasts from hypertonic solutions (2.6 osmolal), there was little increase in gamma until after the isotonic surface area was exceeded. These results are discussed in relation to the differences in the behavior of the plasma membrane of NA and ACC protoplasts during osmotic contraction (i.e. endocytotic vesiculation versus exocytotic extrusion) and provide a mechanistic interpretation to account for the differential sensitivity of NA and ACC protoplasts to osmotic expansion from hypertonic solutions. PMID- 16665315 TI - Leaf photosynthesis and conductance of selected triticum species at different water potentials. AB - Leaf gas exchange characteristics of a desert annual (Triticum kotschyi [Boiss.] Bowden) and the wheat cultivar TAM W-101 (Triticum aestivum L. em Thell) were compared over a range of leaf water potentials from -0.50 to -2.9 megapascals. At an ambient [CO(2)] of 330 microliters per liter, T. kotschyi had higher conductance and CO(2) assimilation (A) at a given water potential than T. aestivum. Under well watered conditions, A versus internal CO(2) concentration (C(i)) response curves for both species were similar in shape and magnitude, and the higher A of T. kotschyi at an ambient [CO(2)] of 330 microliters per liter was mostly related to the higher stomatal conductance of T. kotschyi. The higher conductance of T. kotschyi than T. aestivum under well watered conditions was associated with higher C(i) and lower water use efficiency. Under water deficits, however, C(i) at 330 microliters per liter ambient [CO(2)] did not differ significantly between species. T. kotschyi had higher A under water deficits than T. aestivum primarily because its A versus C(i) response curves had higher A at C(i) values above about 150 microliters per liter. The results show that conductance played an important role in the high A of T. kotschyi under well watered conditions, but under water deficits the high A of T. kotschyi was related more to the maintenance of a higher capacity for mesophyll photosynthesis. PMID- 16665316 TI - Localization of Phosphorus and Cation Reserves in Lilium longiflorum Pollen. AB - Transmission electron microscopy of pollen from Lilium longiflorum Thunb. reveals electron-dense inclusions in storage body organelles ubiquitous in the cytosol. In ungerminated pollen, these inclusions are rounded in appearance and appressed to the inner surface of the smooth membrane of the storage body. During pollen germination, these inclusions become less rounded, smaller, and enclosed in storage bodies that have developed crenated membranes. Energy dispersive x-ray analysis reveals high levels of P, Mg, K, and Ca in the inclusions relative to other regions of the cytosol in which elemental signals can be obtained. The elemental composition and the degradation of inclusions during germination are offered as evidence for storage of phytin in these structures which are thus analogous to phytin storage globoids of seed tissues. PMID- 16665317 TI - Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation. AB - Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chlamydomonas MGA 161, was unusual in having high H(2) but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH(4)Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations; starch degradation, and formation of ethanol, H(2), and CO(2) increased with the addition of NH(4)Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO(2) as its main products, with H(2), formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H(2) evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells. PMID- 16665318 TI - Interaction of Herbicides and Quinone with the Q(B)-Protein of the Diuron Resistant Chlamydomonas reinhardtii Mutant Dr2. AB - We have used the diuron-resistant Dr2 mutant of Chlamydomonas reinhardtii which is altered in the 32 kilodalton Q(B)-protein at amino acid 219 (valine to isoleucine), to investigate the interactions of herbicides and plastoquinone with the 32 kilodalton Q(B)-protein. The data contained in this report demonstrate that the effects of this mutation are different from those of the more completely characterized mutant which confers extreme resistance to triazines in higher plants. The mutation in C. reinhardtii Dr2 confers only slight resistance to a number of inhibitors of photosynthetic electron transport. Extreme triazine resistance results from an increase in the binding constant of the herbicide with the 32 kilodalton Q(B)-protein, in contrast the diuron binding constant for chloroplasts isolated from wild-type (sensitive) Chlamydomonas and the resistant Dr2 are indistinguishable. We conclude that the altered structure in the 32 kilodalton Q(B)-protein of Dr2 does not directly affect the diuron binding site. This mutation appears to alter the steric properties of the binding protein in such a way that diuron and plastoquinone do not directly compete for binding. This steric perturbation confers mild resistance to other herbicidal inhibitors of photosynthesis and alters the kinetics of Q(A) to Q(B) electron transfer. PMID- 16665319 TI - Investigation of the CO(2) Dependence of Quantum Yield and Respiration in Eucalyptus pauciflora. AB - In leaves of C(3) plants, the rate of nonphotorespiratory respiration appears to be higher in darkness than in the light. This change from a high to a low rate of carbon loss with increasing photon flux density leads to an increase in the apparent quantum yield of photosynthetic CO(2) assimilation at low photon flux densities (Kok effect). The mechanism of this suppression of nonphotorespiratory respiration is not understood, but biochemical evidence and the observation that a Kok effect is often not observed under low O(2), has led to the suggestion that photorespiration might be involved in some way. This hypothesis was tested with snowgum (Eucalyptus pauciflora Sieb. ex Spreng.) using gas exchange methods. The test was based on the assumption that if photorespiration were involved, then it would be expected that the intercellular partial pressure of CO(2) would also have an influence on the Kok effect. Under normal atmospheric levels of CO(2) and O(2), a Kok effect was found. Changing the intercellular partial pressure of CO(2), however, did not affect the estimate of nonphotorespiratory respiraton, and it was concluded that its decrease with increasing photon flux density did not involve photorespiration. Concurrent measurements showed that the quantum yield of net assimilation of CO(2) increased with increasing intercellular partial pressure of CO(2), and this increase agreed closely with predictions based on recent models of photosynthesis. PMID- 16665320 TI - Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals. AB - Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22 degrees C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22 degrees C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3 degrees C for 10 to 17 days and then transferrd to 22 degrees C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3 degrees C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3 degrees C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death. PMID- 16665321 TI - Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L. AB - Activities of key enzymes of the Calvin cycle and C(4) metabolism, rates of CO(2) fixation, and the initial products of photosynthetic (14)CO(2) fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv ;Toria.' Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5 phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C(4) metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of (14)CO(2) assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO(2) during light. However, respiratory losses were very high during the dark period. PMID- 16665322 TI - Internode Length in Pisum: Gene na May Block Gibberellin Synthesis between ent 7alpha-Hydroxykaurenoic Acid and Gibberellin A(12)-Aldehyde. AB - The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA(12)-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde in the na mutant. Feeds of ent-[(3)H]kaurenoic acid and [(2)H]GA(12)-aldehyde to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize [(2)H]GA(12)-aldehyde to a number of[(2)H]C(19)-GAs including GA(1). However, there was no indication in na genotypes for the metabolism of ent-[(3)H]kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolised ent [(3)H]kaurenoic acid to radioactive compounds that co-chromatographed with GA(1), GA(8), GA(20), and GA(29). However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-[(3)H]kaurenoic acid were similar for both Na and na plants and contained ent-16alpha,17-dihydroxykaurenoic acid and ent-6alpha,7alpha,16beta,17-tetrahydroxykaurenoic acid (both characterized by GC MS), suggesting that the metabolites arose from side branches of the main GA biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7alpha hydroxylation. PMID- 16665323 TI - UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product. AB - A re-examination of the kinetic properties of UDP-glucose: (1-->3)-beta-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca(2+) and millimolar concentrations of beta-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V(max) of the enzyme, and by lowering the apparent K(m) for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg(2+) markedly enhances the affinity of the mung bean enzyme for Ca(2+) but not for beta-glucoside; with saturating Ca(2+), Mg(2+) only slightly stimulates further production of glucan. However, the presence of Mg(2+) during synthesis, or NaBH(4) treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca(2+), beta-glucoside-activated cotton fiber enzyme, with or without Mg(2+), is very similar in size to callose isolated from cotton fibers, but is a linear (1-->3)-beta-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg(2+)in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg(2+) on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme. PMID- 16665324 TI - Direct measurement of turgor and osmotic potential in individual epidermal cells : independent confirmation of leaf water potential as determined by in situ psychrometry. AB - The pressure probe, which is routinely used to measure the turgor potential (Psi(p)) of individual epidermal cells in Tradescantia virginiana (L.), has also been used to sample small volumes of vacuolar fluid from these same cells (as low as 0.02 nl) for measurement of cellular solute (osmotic) potential (Psi(s)) in a micro freezing point osmometer. The water potential components Psi(p) and Psi(o) have been used to calculate the total water potential of individual epidermal cells (Psi(cell)) which has then been directly compared to the total leaf water potential (Psi(leaf)) measured psychrometrically. The relation of Psi(leaf) and Psi(cell) to leaf transpiration indicates that in T. virginiana, a relatively straightforward relation exists between the level of water flow through the leaf tissue, and the DeltaPsi within the leaf, between two points along the water flow pathway. Substantial agreement was found between the two independent, in situ methods of measuring Psi when extrapolated to zero transpiration conditions. These results are discussed with respect to the thermodynamics of water transport in plant tissues. PMID- 16665325 TI - (2-Chloroethyl)phosphonic Acid Promotes Germination of Immature Spores of Ceratopteris richardii Brongn. AB - Freshly collected spores of strain Hn-n of Ceratopteris richardii Brongn. require storage for several months before attaining maximum germination rate. Treatments using (2-chloroethyl)phosphonic acid increased germination rate in freshly collected spores and decreased germination rate in older spores. PMID- 16665326 TI - Cytokinin Utilization by Adenine-Requiring Mutants of the Yeast Saccharomyces cerevisiae. AB - By monitoring the growth of several adenine auxotrophs of the yeast Saccharomyces cerevisiae on cytokinin-supplemented media, we have demonstrated that this organism can utilize some of these derivatives as a source of adenine. Growth of a mutant lacking adenylosuccinate synthetase suggests that the conversion of cytokinins to adenine does not involve a hypoxanthine intermediate and may be catalyzed by an enzyme analogous to cytokinin oxidase. PMID- 16665327 TI - Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase. AB - When Frankia HFPCcI3 was grown in culture at oxygen O(2) levels ranging from 2 to 70 kilopascals O(2), under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO(2) and showed a marked optimum close to growth pO(2). Vesicles were thin walled at low pO(2) and very thick walled at high pO(2). Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO(2). PMID- 16665328 TI - Action of proline on stomata differs from that of abscisic Acid, g-substances, or methyl jasmonate. AB - Methyl jasmonate (MJ) and a mixture of G(1), G(2), and G(3) (G-substances) inhibited stomatal opening in abaxial epidermis of Commelina benghalensis and complete closure occurred at 10(-6) molar MJ, or 10(-3) molar G-substances compared to 10(-5) molar abscisic acid (ABA). Proline, even at 10(-3) molar caused only a partial stomatal closure. Apart from ABA, other endogenous plant growth regulators do regulate stomata. Reduction in the stimulation by fusicoccin and complete stomatal closure, at 30 millimolar KCl or less, were affected by ABA, MJ, or G-substances, but not by proline. The action of MJ or G-substances was similar to ABA in decreasing proton efflux and the levels of potassium, malate, or reducing sugars. Proline, however, interfered with starch-sugar interconversion but had no effect on proton efflux or potassium content of epidermis. PMID- 16665329 TI - Carbonic Anhydrase-Dependent Inorganic Carbon Uptake by the Red Macroalga, Chondrus crispus. AB - The rate of photosynthetic carbon uptake of Chondrus crispus Stack-house plants, at various CO(2) concentrations and pretreated with carbonic anhydrase (CA) inhibitors, was determined using an air-suspension, differential infra-red gas analyzer technique. It was found that the CA inhibitors, acetazolamide, dextran bound acetazolamide (DBI, which does not permeate cell membranes), and subtilisin (a protease that attacks the cell surface) inhibit photosynthetic carbon uptake in C. crispus. Inhibition was greatest at low CO(2) concentrations, and decreased at CO(2) saturation. Acetazolamide inhibited carbon uptake to a greater extent than DBI. The data support the conclusion that C. crispus plants utilize HCO(3) ( ) for photosynthesis, and that both cell-surface and internal CA are involved in the photosynthetic uptake of inorganic carbon. PMID- 16665330 TI - Correction of Freezing Artifacts in the Application of Electron Paramagnetic Resonance for Quantification of the Reduction of Ferric Hydroxamate by Chlorella vulgaris. AB - Electron paramagnetic resonance has been used to quantitate reduction of ferric ion bound to hydroxamate siderophores by iron-stressed fungi (DJ Ecker et al. 1982 J Biol Chem 257:8623-8626) and by iron-stressed Chlorella vulgaris (ATCC 11468) (FCT Allnutt, WD Bonner 1984 J Plant Nutr 7: 427-435). Application of this technique to quantitation of bound ferric ion has been questioned due to the possibility of freezing artifacts. Dipole-dipole interaction which resulted in broadening of the g = 4.3 signal was observed. Quantitation was affected when peak to trough amplitude was applied to quantitate the ferric ion but could be corrected by the application of internal standards and maintenance of constant cell content in the samples. PMID- 16665331 TI - Expression from heterologous promoters in electroporated carrot protoplasts. AB - Plasmids were constructed that contained promoters of ;plant' genes fused to the bacterial gene for chloramphenicol acetyl transferase. The promoters were isolated from a developmentally regulated Zea mays seed storage protein gene and from the mannopine synthase gene of the octopine type Ti plasmid of Agrobacterium tumefaciens which is constitutively expressed in crown gall tumors. These plasmids were introduced into carrot protoplasts made permeable by electroporation. Expression of chloramphenicol acetyl transferase activity directed by both promoters was positively correlated with DNA concentration. The efficiency of gene transfer was increased by inclusion of polyethylene glycol and by optimization of the voltage used in electroporation. PMID- 16665332 TI - Abscisic Acid accumulation is not required for proline accumulation in wilted leaves. AB - Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt. PMID- 16665333 TI - Regulation of nitrite reductase : cell-free translation and processing. AB - A protein with molecular mass of 67 kilodaltons is immunoprecipitated from in vitro translated products obtained from rabbit reticulocyte lysate primed with polyadenylated RNA from nitrate treated illuminated pea seedlings. This protein resembles the native nitrite reductase because of its competitive elimination when immunoprecipitation of in vitro translated products was carried out in the presence of cold unlabeled nitrite reductase or in vivo labeled pea leaf extract. This protein is of slightly higher molecular weight than that of the native nitrite reductase. Proteinaceous extracts from chloroplasts convert the in vitro product to the same molecular weight as the native peptide. The conversion appears to occur in two steps. Polyadenylated RNA from nitrate deficient plants or from nitrate-treated plants transferred to darkness do not support the synthesis of nitrite reductase. It is concluded that nitrate and light modulate the synthesis of the enzyme nitrite reductase by regulating the availability of mRNA for the enzyme. PMID- 16665334 TI - Endogenous cytokinins in the ribosomal RNA of higher plants. AB - Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2 methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A(260) unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests. PMID- 16665335 TI - Plasma Membrane Lipid Alterations Associated with Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma). AB - Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free beta-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing beta-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane. PMID- 16665336 TI - A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species. AB - Starch, sucrose, and fructose 2,6-bisphosphate (F2, 6BP) levels were measured in pea (Pisum sativum L.), maize (Zea mays L.), onion (Allium cepa L.) and soybean (Glycine max L.) leaves throughout a light/dark cycle. Leaf starch accumulated in pea, maize, and soybean but not in onion. Sucrose was a major leaf storage reserve in pea, maize, and onion but was only found at low levels in soybean. In all species examined, the most dramatic changes in F2,6BP concentration coincided with light/dark transitions. During the light period F2,6BP levels were about 0.1 nanomole/milligram chlorophyll in soybean source leaves and there was a small increase in effector concentration in the dark. Levels of F2,6BP were also low in pea and maize leaves during the light period but then increased 10- or 20-fold in the dark. Dark onion leaf F2,6BP levels were about 1.1 to 1.3 nanomole/milligram chlorophyll and these values decreased by 20 to 30% in the light. Thus, three different patterns were identified that describe diurnal F2,6BP levels in source leaves. These results support the suggestion that F2,6BP is involved in the regulation of sucrose biosynthesis. However, it was not possible to demonstrate that high levels of F2,6BP are essential for starch synthesis in the chloroplast. PMID- 16665337 TI - Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds. AB - THE DISTRIBUTION OF PYROPHOSPHATE: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles.min.milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6 P and ATP with K(m) values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P(2). The activation is caused by lowering the K(m) for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P(2) and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K(m) of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K(m) for fructose 6-P. The ATP content appears to be at saturating levels for PFK. PMID- 16665338 TI - Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum. AB - In this study we examined the effect of heat stress (up to 6 hours at 43 degrees C) on the biosynthesis and transport of phytohemagglutinin (PHA) in cotyledons of developing seeds of the common bean, Phaseolus vulgaris. Heat stress resulted in a decrease of total protein synthesis and an enhancement of the synthesis of heat shock proteins and PHA. Pulse chase experiments showed that a considerable proportion of the newly synthesized PHA was present in the endoplasmic reticulum (ER)/Golgi fraction and did not readily chase-out. Analysis with endoglycosidase H showed that the oligosaccharide sidechains of PHA were almost entirely in the high mannose configuration, indicating that most of the newly synthesized PHA was in the ER. However, some of the PHA became fucosylated at 43 degrees C, indicating fucosyltransferase activity. That the biosynthesis and secretion of fucosyl-containing cell wall polymers proceeded normally at 43 degrees C provided evidence that certain Golgi functions (i.e. transport to the cell wall) remained unaffected by heat stress. The ER obtained from these heat stress cotyledons had a greater density (1.16 g. cm(-3) at 43 degrees C instead of 1.14 g.cm(-3) at 22 degrees C) in sucrose gradients. Ultrastructural observations showed that the width of the lumen of the ER cisternae had increased from 20 nanometers at 22 degrees C to 60 to 80 nanometers at 43 degrees C; the lumen was filled with electrondense material presumed to be protein. The experiments are interpreted as evidence that heat stress imposes a block in the transport of PHA out of the ER. Whether heat stress affects the ER itself or alters the conformation of PHA, thereby preventing its transport, is not clear. PMID- 16665339 TI - Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum. AB - Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K(m) values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg(2+) and Mn(2+). PMID- 16665340 TI - Purification, properties, and distribution of ascorbate peroxidase in legume root nodules. AB - All aerobic biological systems, including N(2)-fixing root nodules, are subject to O(2) toxicity that results from the formation of reactive intermediates such as H(2)O(2) and free radicals of O(2). H(2)O(2) may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN(3), CO, and C(2)H(2) were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of (1/2) V(max) was 70 micromolar for ascorbate and 3 micromolar for H(2)O(2). The high affinity of ascorbate peroxidase for H(2)O(2) indicates that this enzyme, rather than catalase, is responsible for most H(2)O(2) removal outside of peroxisomes in root nodules. PMID- 16665341 TI - Comparison of the growth promoting activities and toxicities of various auxin analogs on cells derived from wild type and a nonrooting mutant of tobacco. AB - A naphthaleneacetic acid tolerant mutant isolated from a mutagenized culture of tobacco mesophyll protoplasts and impaired in root morphogenesis has been previously characterized by genetic analysis. To understand the biochemical basis for naphthaleneacetic acid resistance, cells derived from this mutant and from wild-type tobacco were compared for their ability to respond to various growth regulators. The growth promoting abilities and cytotoxicities of auxin analogs were different for mutant and wild-type cells. These different activities were not correlated with increased rate of conjugation or breakdown of the auxins by mutant cells. These observations, as well as previous studies on the interaction of the mutant with Agrobacterium, suggest that mutant resistance to auxins is not a result of a specific modification of the process by which auxins induce cell killing, but to a more general alteration of the cellular response to auxin. A screening of auxin-related molecules which induce cell death in wild-type cells but not mutant cells without promoting growth in either was performed. p Bromophenyleacetic acid was found to display these characteristics. PMID- 16665342 TI - Auxin effect on the transmembrane potential difference of wild-type and mutant tobacco protoplasts exhibiting a differential sensitiity to auxin. AB - The effects of 1-naphthaleneacetic acid (NAA) and other auxin analogs on the transmembrane potential difference (Em) were compared on tobacco protoplasts isolated from two genotypes differing in their sensitivity to auxins. For both types, NAA modifies Em by inducing at low doses a hyperpolarization, the amplitude of which increased with auxin concentration. Above an optimal concentration this hyperpolarization was reduced and even nullified. However, for the mutant type, this electrical response was shifted toward higher NAA concentrations, as its growth response. In the presence of structural analogs of auxin which have been showed to modify the dose-response curve for growth, the Em was altered: the growth-stimulatory molecule (picloram) initiated hyperpolarization, whereas the growth-inhibitory substance (4-bromophenylacetic acid) caused depolarization. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity. PMID- 16665343 TI - Chlorophyll-Protein Complexes from the Red-Tide Dinoflagellate, Gonyaulax polyedra Stein : Isolation, Characterization, and the Effect of Growth Irradiance on Chlorophyll Distribution. AB - A comparision of high (330 microeinsteins per meter squared per second) and low (80 microeinsteins per meter squared per second) light grown Gonyaulax polyedra indicated a change in the distribution of chlorophyll a, chlorophyll c(2), and peridinin among detergent-soluble chlorophyll-protein complexes. Thylakoid fractions were prepared by sonication and centrifugation. Chlorophyll-protein complexes were solubilized from the membranes with sodium dodecyl sulfate and resolved by Deriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I' to IV') were evident in preparations of high light cells. Both high molecular weight complexes I and I' were dominated by chlorophyll a absorption and associated with minor amounts of chlorophyll c. Both complexes II and II' were chlorophyll a-chlorophyll c(2) protein complexes devoid of peridinin and unique to dinoflagellates. The chlorophyll a:c(2) molar ratio of both complexes was 1:3, indicating significant chlorophyll c enrichment over thylakoid membrane chlorophyll a:c ratios of 1.8 to 2:1. Low light complex III differed from all other high or low light complexes in that it possessed peridinin and had a chlorophyll a:c(2) ratio of 1:1. Low light complexes IV and V and high light complexes III' and IV' were spectrally similar, had high chlorophyll a:c(2) ratios (4:1), and were associated with peridinin. The effects of growth irradiance on the composition of chlorophyll-protein complexes in Gonyaulax polyedra differed from those described for other chlorophyll c containing plant species. PMID- 16665344 TI - Losses of Polyol through Leaching in Subarctic Lichens. AB - Upon rewetting, lichens lose polyols through leaching. We quantified leaching losses for 21 species under simulated rainfall. Polyol concentrations in these lichens range from 1.0 to 8.8%, with a mean of 2.8%. Leaching losses range up to about 7.5 mg (polyol)/g (lichen dry weight) in a typical rain event. The rate of polyol leaching declines exponentially, becoming negligible within 1 hour of continuous rain. The response of polyol leaching rate to rainfall intensity and amount varies between species-six species showed no response, one had increased leaching with increased rainfall intensity, four had increased leaching with increased amount of rainfall, and one had decreased leaching with increased total amount of rainfall. Polyol leaching rates are positively correlated with polyol concentration for 20 species. Literature values of average daily growth rates for subarctic lichens are of the same order of magnitude as leaching rates, suggesting that polyol leaching is an important part of the carbon budget of lichens. PMID- 16665345 TI - Glutathione Status and Protein Synthesis during Drought and Subsequent Rehydration in Tortula ruralis. AB - Glutathione status and its relationship to protein synthesis during water deficit and subsequent rehydration have been examined in the drought-tolerant moss, Tortula ruralis. During slow drying there is a small decrease in total glutathione but the percentage of oxidized glutathione (GSSG) increases. During rapid drying there is little change in total glutathione but a small increase in GSSG. On rehydration of slowly dried moss, GSSG rapidly declines to normal level. But when rapidly dried moss is rehydrated, there is an immediate, sharp increase in GSSG as a percentage of total glutathione. After 2 hours of rehydration GSSG starts declining and reaches a normal level in about 6 hours. When an increasing degree of steady state water deficit is imposed on the moss tissue with polyethylene glycol 6000, there is a progressive decrease in protein synthesis but an increase in oxidized glutathione. When 5 millimolar GSSG is supplied exogenously during rehydration of rapidly dried or slowly dried moss, protein synthesis is strongly inhibited. In vitro protein synthesis supported by moss mRNA is also inhibited by more than 85% by 150 micromolar GSSG. The role of glutathione status in water deficit-induced inhibition of protein synthesis is discussed. PMID- 16665346 TI - A possible role for abscisic Acid in regulation of photosynthetic and photorespiratory carbon metabolism in barley leaves. AB - The influence of abscisic acid (ABA) on carbon metabolism, rate of photorespiration, and the activity of the photorespiratory enzymes ribulose bisphosphate oxygenase and glycolate oxidase in 7-day-old barley seedlings (Hordeum vulgare L. var. Alfa) was investigated. Plants treated with ABA had enhanced incorporation of labeled carbon from (14)CO(2) into glycolic acid, glycine, and serine, while (14)C incorporation into 3-phosphoglyceric acid and sugarphosphate esters was depressed. Parallel with this effect, treated plants showed a rise in activity of RuBP oxygenase and glycolic acid oxidase. The rate of photorespiration was increased twofold by ABA treatment at IO(-6) molar while the CO(2)-compensation point increased 46% and stomatal resistance increased more than twofold over control plants. PMID- 16665347 TI - The Lectins of Sophora japonica: I. Purification Properties and N-Terminal Amino Acid Sequences of Two Lectins from Leaves. AB - Two lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like the Sophora seed lectin, LLI and LLII are tetrameric glycoproteins containing a single subunit with respect to size. The subunits of LLI (32 kilodaltons) and LLII (34 kilodaltons) are slightly larger than those of the seed lectin (29.5 kilodaltons). The three Sophora lectins display indistinguishable specificities, amino acid compositions, specific hemagglutinin activities, and extinction coefficients. Although very closely related to the seed lectin, the leaf and seed lectins are not immunologically identical and they differ in subunit molecular weights, carbohydrate content, and in the pH sensitivity of their hemagglutinin activities. N-terminal amino acid sequence analysis shows that although they are homologous proteins, the three Sophora lectins are products of distinct genes. PMID- 16665348 TI - A calcium-dependent but calmodulin-independent protein kinase from soybean. AB - A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar ( approximately 2 micromolar). The protein kinase activity was stimulated 100-fold by >/=10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium dependent and -independent activities were weakly stimulated (5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass. PMID- 16665404 TI - Nitrate-induced changes in protein synthesis and translation of RNA in maize roots. AB - Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [(35)S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [(35)S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots. PMID- 16665405 TI - Anapleurotic CO(2) Fixation by Phosphoenolpyruvate Carboxylase in C(3) Plants. AB - The role of phosphoenolpyruvate carboxylase in photosynthesis in the C(3) plant Nicotiana tabacum has been probed by measurement of the (13)C content of various materials. Whole leaf and purified ribulose bisphosphate carboxylase are within the range expected for C(3) plants. Aspartic acid purified following acid hydrolysis of this ribulose bisphosphate carboxylase is enriched in (13)C compared to whole protein. Carbons 1-3 of this aspartic acid are in the normal C(3) range, but carbon-4 (obtained by treatment of the aspartic acid with aspartate beta-decarboxylase) has an isotopic composition in the range expected for products of C(4) photosynthesis (-5 per thousand), and it appears that more than half of the aspartic acid is synthesized by phosphoenolpyruvate carboxylase using atmospheric CO(2)/HCO(3) (-). Thus, a primary role of phosphoenolpyruvate carboxylase in C(3) plants appears to be the anapleurotic synthesis of four carbon acids. PMID- 16665406 TI - ABA Levels and Sensitivity in Developing Wheat Embryos of Sprouting Resistant and Susceptible Cultivars. AB - A sprouting-resistant and a sprouting-susceptible wheat cultivar were utilized to examine the role of ABA levels and sensitivity responses in wheat embryonic germination. Endogenous embryonic ABA levels were measured in both cultivars throughout grain maturation utilizing a new and sensitive ABA immunoassay. Embryonic ABA levels of each cultivar were similar with the sprouting-susceptible cultivar having about a 25% lower ABA level than that of the sprouting-resistant cultivar. Larger differences between the cultivars were noted in sensitivity to ABA, as measured by capability of ABA to block embryonic germination. ABA inhibited embryonic germination much more effectively in the sprouting-resistant cultivar. PMID- 16665407 TI - Consequence of Absence of Nitrate Reductase Activity on Photosynthesis in Nicotiana plumbaginifolia Plants. AB - Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR(-)nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen and nitrate but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second (14)CO(2) pulse, the total (14)C incorporation of the mutant leaves was approximately 20% of that of the control. The (14)C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second (14)CO(2) pulse followed by a 60 second chase with normal CO(2), (14)C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus. PMID- 16665408 TI - Red Light-Dependent CO(2) Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO(2) Fixation. AB - Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O(2) concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality. PMID- 16665409 TI - Effects of Cold Hardening on the Regulation of Polyamine Levels in Wheat (Triticum aestivum L.) and Alfalfa (Medicago sativa L.). AB - When leaves of wheat (Triticum aestivum L.) are exposed to a cold hardening temperature, a major accumulation of putrescine (6-9 times) takes place. Spermidine accumulates to a lesser extent and, conversely, spermine decreases slightly. These variations are completely reversible when plants are returned to initial growing conditions. A similar response is obtained with crowns. During cold hardening, arginine decarboxylase activity remains near its initial level while a considerable loss of activity is observed in control plants. Ornithine decarboxylase and diamine oxidase activity levels are not substantially modified by the treatment. Alfalfa (Medicago sativa L.) also accumulates putrescine under low temperature stress, indicating that this phenomenon is not typical of cereals. The physiological significance of this accumulation of putrescine is still unexplained but the results obtained suggest the involvement of polyamines in the biochemical processes of cold hardening. PMID- 16665410 TI - Phosphatidylcholine Synthesis in Castor Bean Endosperm : I. Metabolism of l Serine. AB - Endosperm halves from 3-day-old castor bean (Ricinus communis var Hale) were incubated for 30 minutes with l-[(14)C]serine, after which label was observed in ethanolamine, choline, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, ethanolaminephosphate, and CDPethanolamine, but not in cholinephosphate or CDPcholine. Only later did significant amounts of isotope become incorporated into cholinephosphate and CDPcholine. The choline kinase inhibitor hemicholinium-3 prevented the incorporation of label from serine into cholinephosphate and CDPcholine, reduced the incorporation of [(14)C]choline into phosphatidylcholine by 65%, but inhibited the incorporation of label into phosphatidylcholine from serine by only 15%. The inhibitor did not prevent the incorporation of labeled methyl groups from S-adenosyl-l-methionine into phosphatidyldimethylethanolamine plus phosphatidylcholine. The amount of incorporation of label from the methyl donor was only 8% of that from choline into phosphatidylcholine. The implications of these results for the pathway and regulation of phosphatidylcholine synthesis from the water-soluble precursors are discussed. PMID- 16665411 TI - Regulation of phosphoenolpyruvate carboxylase activity in maize leaves. AB - The aim of this work was to investigate how light regulates the activity of phosphoenolpyruvate carboxylase in vivo in C(4) plants. The properties of phosphoenolpyruvate carboxylase were investigated in extracts which were rapidly prepared (in less than 30 seconds) from darkened and illuminated leaves of Zea mays. Illumination resulted in a significant decrease in the S(0.5)(phosphoenolpyruvate) but there was no change in V(max). The form of the enzyme from illuminated leaves was less sensitive to malate inhibition than was the form from darkened leaves. At low concentrations of phosphoenolpyruvate, the activity of the enzyme was strongly stimulated by glucose-6-phosphate, fructose-6 phosphate, triose-phosphate, alanine, serine, and glycine and was inhibited by organic acids. The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light and in the dark. It displayed low activity in a simulated ;dark' cytosol and high activity in a simulated ;light' cytosol, but activities were different for the enzyme from darkened compared to illuminated leaves. PMID- 16665412 TI - Compositional and Thermal Properties of Thylakoid Polar Lipids of Nerium oleander L. in Relation to Chilling Sensitivity. AB - The polar lipid classes from thylakoids of Nerium oleander L. were studied with the aim of relating changes in their composition and thermal behavior with reported changes in the transition temperature of their polar lipids and chilling sensitivity of their leaves. With an increase in growth temperature, the transition temperature of phosphatidylglycerol increased from 16 degrees C to 26 degrees C, and for sulfoquinovosyldiacylglycerol from 19 degrees C to 24 degrees C. Transitions in the other lipid classes were below -10 degrees C for plants grown at both growth temperature. The major changes in the molecular species of phosphatidylglycerol, with increasing growth temperature, were an increase in 1 oleoyl-2-palmitoyl phosphatidylglycerol from 21 to 39% and a decrease in 1-oleoyl 2-trans-3-hexadecanoic phosphatidylglycerol from 51 to 25%. Although the disaturated species increased from 8 to 23%, the maximum was less than that reported for chilling-sensitive plants. There was no change in the sum of the palmitic, hexadeca-trans-3-enoic and stearic acids. Dipalmitoyl sulfoquinovosyldiacylglycerol increased from 12 to 20% and 1-linolenoyl-2 palmitoyl sulfoquinovosyldiacylglycerol decreased from 40 to 30%. It is concluded that the increase in the transition temperature of the polar lipids and the sensitivity of acclimated oleander plants to chilling could not be predicted by the absolute sum of the saturated fatty acids or disaturated molecular species in phosphatidylglycerol. The polar lipid transition appears to be a product of mixing of both high and low melting-point lipids. PMID- 16665413 TI - Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata). AB - The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p chlorophenylsulfonate, but not by iodoacetate and CuCl(2). It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4 degrees C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies. PMID- 16665414 TI - Specific peroxidase isoenzymes are correlated with organogenesis. AB - We have examined isoperoxidase patterns obtained from buffer-, salt-, and enzyme extractable fractions and correlated them with histological changes in tobacco (Nicotiana tabacum L., cv Wisc. 38) ;epidermal' explants induced to produce either callus, vegetative buds, or floral buds. By utilizing a combination of extraction and electrophoretic procedures different from any hitherto used for this kind of investigation, we were able to resolve 47 isoperoxidases distributed between the three types of fractions. The majority of these isoperoxidases were common to all explants regardless of their developmental fate. Correspondingly, a number of histological changes were observed in all explants (e.g. the initiation of cell division by day 2, lignin deposition by day 4, and the formation of clustered tracheary elements by day 8). We have made correlations between 25 isoperoxidases and specific developmental events based on the time when certain isoperoxidases were detected relative to observed histological changes: 3 were correlated with desuppressed/sustained cell division, 3 to 6 with lignification/tracheary element maturation, 7 with callus formation, 1 with localized suppression of growth, 3 with determinate axial organization, 4 with leaf development, and 1 with stamen development. These results suggest that a continued investigation using this system could lead to a better understanding of the role of specific isoperoxidases in different developmental processes. PMID- 16665415 TI - Fructose 2,6-bisphosphate and plant carbohydrate metabolism. AB - The control of the fructose 2,6-bisphosphate (Fru2,6P(2)) concentration and its possible role in controlling carbohydrate synthesis and degradation are discussed. This regulator metabolite is involved in the fine tuning of photosynthetic metabolism, and in controlling photosynthetic partitioning, and may also be involved in the response to hormones, wounding, and changing water relations. Study of the mechanisms controlling Fru2,6P(2) concentrations could reveal insights into how these responses are mediated. However, the detailed action of Fru2,6P(2) requires more attention, especially in respiratory metabolism where the background information about the compartmentation of metabolism between the plastid and cytosol is still inadequate, and the potential role of pyrophosphate has to be clarified. PMID- 16665416 TI - Cytosolic ATP-Dependent Phosphofructokinase from Spinach. AB - ATP-dependent 6-phosphofructokinase (PFK) activity is present in both chloroplastic and in nonchloroplastic fractions isolated from spinach protoplasts. The activity in the extra-chloroplastic fraction was stimulated 2- to 3.5-fold by 25 mm inorganic phosphate (Pi), the chloroplast-associated activity was inhibited 2- to 5-fold. The Pi stimulated activity was ATP-dependent and was not an artifact due to the presence of fructose 6-P, Pi, pyrophosphatase, and pyrophosphate fructose 6-P 1-phosphotransferase (PFP). PFK activities, which expressed characteristics similar to those separated from protoplasts, could be separated following ammonium sulfate fractionation of crude extracts; the ammonium sulfate treatment also separated both PFK activities from PFP. It is concluded that spinach leaves contain a cytosolic PFK. This activity is relatively stable, is stimulated by Pi over a wide pH range, is not a result of the transformation of another enzyme activity, and has an activity that is similar to, or slightly less than, that of the cytosolic PFP. PMID- 16665417 TI - Hydroxylation of N-(Delta-Isopentenyl)adenine to Zeatin: Relative Activities of Organ Systems from Actinidia arguta. AB - By incubating explants from Actinidia arguta seedlings on a nutrient medium supplemented with 20 to 30 micromolar N(6)-(Delta(2)-isopentenyl)adenine (i(6)Ade) and then measuring zeatin (io(6)Ade) accumulation in tissues, the distribution of i(6)Ade hydroxylase activities in whole plants could be determined. Based on analyses with three entire plants, it is estimated that, as an organ system, roots contain approximately 68% of the plant's hydroxylase, while stems and leaves account for about 26% and 6%, respectively, of the total activity. Depending on the part of the root examined, hydroxylase activities ranged from 20 to 148 nanomoles io(6)Ade accumulated per gram fresh weight per 24 hours of incubation. Stem activities ranged from 17 to 165 nanomoles per gram fresh weight per 24 hours with the lowest activities being found at the tip. Leaf activities were substantially lower (1-10 nanomoles per leaf depending on position) than either root or stem. PMID- 16665418 TI - Energization and activation of inorganic carbon uptake by light in cyanobacteria. AB - The requirement of the inorganic carbon (C(i)) transport system for light in cyanobacteria was investigated in Anabaena variabilis by the filtering centrifugation technique and in a mutant (E(1)) isolated from Anacystis nidulans using a gas exchange system. C(i) transport capability increased with time of preillumination and decreased following darkening. Full activity could not be obtained by operating either photosystem II (PSII) or photosystem I alone. 3(3,4 Dichlorophenyl)-1,1 dimethylurea strongly inhibited C(i) uptake. Very low activity of PSII was sufficient to activate C(i) uptake. However, in the presence of dithiothreitol PSII activity was not required. We conclude that light may be required to activate as well as to energize C(i) uptake in cyanobacteria. PMID- 16665419 TI - Effects of Extracellular Extracts from Leaves on the Tuberization of Cuttings of Potato (Solanum tuberosum L.). AB - Extracellular extracts from leaves were applied to moderately induced cuttings of potato. The more inductive cycles the plants from which the leaf extracts were obtained had experienced, the lower the percentages of tuberization in thcuttings and the lower the tuber yields on the central buds. PMID- 16665420 TI - Photoinhibition and zeaxanthin formation in intact leaves : a possible role of the xanthophyll cycle in the dissipation of excess light energy. AB - Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O(2), 0% CO(2)) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F(v), or maximum fluorescence, F(m), and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of beta-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O(2) and 0% CO(2) resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F(v)/F(m), determined with the modulated fluorescence technique at room temperature, and the photon yield of O(2) evolution, similar to previous findings (O Bjorkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis. PMID- 16665421 TI - Catalase Degradation in Sunflower Cotyledons during Peroxisome Transition from Glyoxysomal to Leaf Peroxisomal Function. AB - First order rate constants for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing (14)C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly (P = 0.05) slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day(-1) in contrast to the constants ranging from 0.304 day(-1) to 0.515 day(-1) during the other developmental stages. Density labeling experiments comprising labeling of catalase with (2)H(2)O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of (14)C labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes. PMID- 16665422 TI - The Physiological Basis for Cytokinin Induced Increases in Pod Set in IX93-100 Soybeans. AB - Previous investigations have shown the feasibility of increasing pod number on legumes by the application of 6-benzylaminopurine (BA) directly to the raceme. These investigations were designed to determine what reproductive parameter was affected by cytokinin application, and if these applications were overcoming a deficiency in root-produced cytokinins during late flowering. Five individual main stem racemes on greenhouse grown soybeans (Glycine max L. Merr.) were treated with 2 millimolar BA. A single application of BA when pods appeared at 25 to 50% of the proximal floral positions resulted in a 58% increase in pod set due primarily to a 33% reduction in floral abscission. Applications of BA at later intervals also resulted in significant reductions in total abscission. When three applications of BA were imposed on the upper five nodes of field grown soybeans, total pod number and seed weight were significantly increased in this section of the canopy by 27 and 18%, respectively. Throughout the flowering period, root pressure exudate was sampled for the subsequent separation and quantification of zeatin, dihydrozeatin, zeatin riboside, dihydrozeatin riboside, and isopentenyladenine. Total cytokinin flux peaked from 0 to 9 days after flowering began, and then dropped to one-half of this level by 15 days postanthesis. The probability that a flower would initiate a pod was directly related to the concentration of total cytokinins present in the exudate when the flower opened. PMID- 16665423 TI - Soybean pod set enhancement with synthetic cytokinin analogs. AB - The previously reported activity of benzyladenine and selected other cytokinin analogs to increase pod set in soybean (Glycine max [L.] Merr.) was further investigated to define the structure-activity relationship and evaluate the effects of the cytokinins on yield parameters. Enhancement of pod set was found to be greatest with N-6 saturated alkyl substituted analogs, and was only weakly associated with activity in a callus growth bioassay. The response of yield parameters to increasing pod load was evaluated by applying various cytokinin analogs having a range of pod set enhancement activity. The increased pod load at the treated nodes was not compensated by a reduction in pod number on the remainder of the plant. However, there was a compensatory decrease in seed size. Overall, a significant trend to greater total seed weight per plant was associated with the increased pod number. Initial evaluations indicated that foliar applications of select cytokinins could temporarily increase pod number. However, the increases in pod number obtained with foliar treatments were too small to be of practical utility and were not maintained to maturity. PMID- 16665424 TI - Formation of delta-Aminolevulinic Acid from Glutamic Acid in Algal Extracts : Separation into an RNA and Three Required Enzyme Components by Serial Affinity Chromatography. AB - Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to delta-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg(2+), and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH(4))(2)SO(4) at 70% saturation. The proteins recovered from the (NH(4))(2)SO(4) precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH(4))(2)SO(4) precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography. PMID- 16665425 TI - Plant Morphological and Biochemical Responses to Field Water Deficits : II. Responses of Leaf Glycerolipid Composition in Cotton. AB - The effects of water deficits on leaf glycerolipid composition were analyzed in two photoperiodic strains of field grown cotton (Gossypium kirsutum L.) that differ in sensitivity to drought. Leaves from plants grown under dryland conditions exhibited increased dry weight and specific leaf weight. The average midday leaf water potential in the dryland treatment decreased to -1.9 and -2.4 megapascals, respectively, for the T25 and T185 genotypes. Total leaf lipid content of plants exposed to dryland conditions was 5.9 and 7.5% of leaf dry weight for strain T25 and T185, respectively. The difference in leaf lipid content between these genotypes was caused by water deficits and was attributed to loss of both phospholipids and glycolipids in strain T25. There was no apparent loss of phospholipids due to water deficits in the T185 genotype; however, a significant loss of glycolipids was partially compensated by a 2-fold increase in triacylglycerol. No change in triacylglycerol was found between treatments in T25 leaves. Water deficit caused a significant decline in the relative degree of acylunsaturation in phospholipids and glycolipids from both genotypes; however, the double bond index for triacylglycerol increased in both genotypes. It is believed that the observed responses of leaf lipid composition to dryland conditions may be an additional criterion for characterization and selection of new drought-tolerant cotton genotypes. PMID- 16665426 TI - Carbon dioxide exchange rates, ribulose bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase activities, and kernel growth characteristics of maize. AB - Four high-yield-potential maize hybrids (FS854, CB596 x LH38, B73 x LH38, and B73 x Mo17) and four inbred lines (LH38, CB59G, Mo17, and B73) were grown in the field to study traits associated with leaf area duration (LAD) and the relationship between LAD and kernel growth characters. Based on decline in chlorophyll, leaf N concentration, CO(2) exchange rate, and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase) activities, the hybrid B73 x Mo17 had a significantly shorter LAD than the other three hybrids. The shorter LAD was not due to maturity because B73 x Mo17 is in a maturity class similar to the other hybrids except CB59G x LH38, which is approximately 1 week earlier. At the time of grain maturity, leaves of B73 x Mo17 had lost all chlorophyll and CO(2) exchange and carboxylase activities. The other three hybrids, however, retained green leaves which still had 20% of the maximum CO(2) exchange rate. In addition, B73 x Mo17 remobilized leaf N more extensively. For all hybrids, declines in CO(2) exchange were closely correlated with declines in PEPCase activity, whereas the relationship between CO(2) exchange and Rubisco activity was weak. Responses of the inbred lines predicted, to some extent, physiological characteristics of the hybrids. CB59G and LH38 both had a longer LAD than either B73 or Mo17 as judged by decline in chlorophyll, leaf N, CO(2) exchange rate, and Rubisco and PEPCase activities. With the exception of B73 x LH38, kernel growth characteristics of the hybrids were related to LAD. Effective filling period (EFP) measured in days was 32.9, 31.5, 30.8, and 30.4 for FS854, CB59G x LH38, B73 x LH38, and B73 x Mo17, respectively. For FS854 and CB59G x LH38, the longer EFP was associated with a larger kernel weight. These data suggested that late season photoassimilate resulting from longer LAD could be utilized by the kernels of these two hybrids. For B73 x Mo17, the shorter LAD and EFP was associated with a kernel dry matter accumulation rate (10.1 milligrams per kernel per day) which was significantly higher than for the other three hybrids. Thus, the more rapid leaf senescence of B73 x Mo17 appeared to be coordinated with efficient leaf N remobilization and a relatively short grain-filling period characterized by rapid kernel dry matter accumulation. PMID- 16665427 TI - Effect of Ear Removal on CO(2) Exchange and Activities of Ribulose Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase of Maize Hybrids and Inbred Lines. AB - The effects of ear removal on gas exchange traits, chlorophyll, and leaf N profiles, and activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase were examined using four maize hybrids (B73 x Mo17, B73 x LH38, FS854, and CB59G x LH38) and four inbred lines (B73, Mo17, LH38, and CB59G) as experimental material. A diverse genotypic response to ear removal was observed which was generally typified by (a) greatly accelerated loss of chlorophyll, leaf N, enzyme activities, and CO(2) exchange relative to controls for B73, B73 x Mo17, and B73 x LH38, (b) intermediate rate of decline for leaf constituents for FS854, LH38, and Mo17, or (c) loss of leaf constituents at similar or slower rates than for control plants for CB59G and CB59G x LH38. For all genotypes which had accelerated senescence relative to controls, loss of CO(2) exchange activity was correlated with increased internal CO(2) concentrations. Thus, it was concluded that metabolic factors and not stomatal effects were responsible for loss of CO(2) exchange activity. Loss of chlorophyll, leaf N, and enzyme activities correlated well with loss of CO(2) exchange activity only for some of the genotypes. Accelerated leaf senescence in response to ear removal for the inbred line B73 and the hybrids B73 x Mo17 and B73 x LH38, as well as the apparent delayed leaf senescence for the inbred line CB59G and the hybrid CB59G x LH38 show that the contrasting responses to ear removal, rapid versus delayed senescence, can be transmitted as dominant traits to F(1) hybrids. The intermediate response by some genotypes, and the dominance of contrasting senescence traits, suggested a relatively complex inheritance for expression of the ear removal response. PMID- 16665428 TI - Nitrate induced regulation of nodule formation in soybean. AB - Nodule formation was inhibited by exposing soybean plants to nitrate in plastic growth pouches. Exposure to 15 millimolar nitrate resulted in a 2.5-fold decrease in the number of nodules formed in the region of the primary root above the mark made at the time of inoculation to indicate the position of the root tip. Serial section analysis of Bradyrhizobium infections in this region revealed that infection initiation was inhibited approximately 3-fold by exposure to nitrate. Both initial cortical cell divisions and infection thread formation were inhibited. If exposure to nitrate was delayed for 18 hours after the time of inoculation, inhibition was much reduced. This indicates that most of the nitrate sensitive events of infection were functionally complete within less than 18 hours. Exposure to nitrate for periods of 4 to 24 hours after inoculation, followed by transfer to no-nitrate conditions for the remainder of the time, did not result in substantial inhibition of nodule number. This indicates that the effects of nitrate on infection initiation can be almost entirely reversible. Split towel pouches were used to physically separate portions of the primary root exposed to nitrate and portions of the root exposed to rhizobia. In experiments where nitrate was applied either below or above the inoculated region of the primary root, the degree of inhibition of nodulation was not correlated with either the external concentration of nitrate in contact with root cells undergoing infection or with the internal concentration of nitrate in the infectible region of the root. These results indicate that nitrate itself may not directly inhibit infection initiation or induce host regulatory responses. PMID- 16665429 TI - Ca stimulated neutral lipase activity in castor bean lipid bodies. AB - The membranes of lipid bodies from the endosperm of seeds of Ricinus communis have long been known to contain an acid lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3). The means by which fat hydrolysis is initiated and controlled in the endosperm of the young seedling are not yet understood, although it is generally assumed that the acid lipase is the enzyme responsible for the conversion of stored triacylglycerols to fatty acids and glycerol. However, the enzyme from seeds is not an effective catalyst at cytoplasmic pH since it has a pH optimum at 4.5 and is virtually inactive above pH 6.0. The results described in this paper show that during early growth of castor seeds the lipid bodies acquire a lipase which is active at neutral pH values. The lipase is absent from dry seeds, appears at day 3, and increases rapidly in activity until day 5. The pattern of appearance of the lipase mirrors that of other enzymes involved in the conversion of fat to sugar. The lipase is stimulated 40-fold by 30 micromolar free Ca(2+) and the activity at pH 7.0 to 7.5 adequately accounts for the known rate of triacylglycerol hydrolysis in vivo. PMID- 16665430 TI - Metabolism of 5-methylthioribose to methionine. AB - During ethylene biosynthesis, the H(3)CS- group of S-adenosylmethionine is released as 5'-methylthioadenosine, which is recycled to methionine via 5 methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, [(14)C]MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of [ribose-U-(14)C]MTR with avocado extract resulted in the production of [(14)C]formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, l-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O(2) in the conversion of MTR to methionine is discussed. PMID- 16665431 TI - In vivo 1-aminocyclopropane-1-carboxylate synthase activity in internodes of deepwater rice : enhancement by submergence and low oxygen levels. AB - Inasmuch as the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase cannot be measured in homogenates of deepwater rice internodes (Oryza sativa L.), we have employed an in vivo assay to determine the activity of this enzyme. This assay is based on the accumulation of ACC in tissue kept under N(2). Submergence of whole plants or stem sections containing the uppermost, developing internode enhances the in vivo activity of ACC synthase in the stem. This stimulation of in vivo ACC-synthase activity is especially pronounced in the region of the internode containing the intercalary meristem and the elongation zone above it. Enhancement of in vivo ACC-synthase activity is evident after 2 hours of submergence and shows a peak after 4 hours. Reduced levels of atmospheric O(2), which promote ethylene synthesis and growth in internodes of deepwater rice, also enhance the in vivo activity of ACC synthase. Our results are consistent with the hypothesis that induction of ACC-synthase activity at low partial O(2) pressures is among the first biochemical events leading to internodal growth in deepwater rice. PMID- 16665432 TI - Euphorbia escula L. Root and Root Bud Indole-3-Acetic Acid Levels at Three Phenologic Stages. AB - Endogenous indoleacetic acid (IAA) levels of Euphorbia esula L. primary root and root buds were examined at three phenologic stages. High performance liquid chromatography coupled with fluorescence detection and gas chromatography-mass spectrometry, using (13)C(6)[benzene ring]-indole-3-acetic acid as internal standard, were used to measure root bud free and bound IAA levels in vegetative, full flower, and post-flower plants. Highest levels of free IAA (103 nanograms per gram fresh weight) were found in root buds during full flower. Esterified and amide IAA increased significantly in root buds of full flower and post-flower plants, but were not detectable in root buds of vegetative plants. Primary rootfree IAA was highest in vegetative and full flower plants (34.5 nanograms per gram fresh weight) and decreased by 50% in post-flower plants. PMID- 16665433 TI - Characterization of Polypeptides Corresponding to Clones of Maize Zein mRNAS. AB - Zeins, the storage proteins of maize (Zea mays) are a complex group of polypeptides encoded by a large multigene family. The alpha-zein proteins, which account for about 70% of the total, show both size and charge heterogeneity. Although clones corresponding to several different alpha zeins have been characterized, it has not been possible to correlate these sequences with individual zein polypeptides. By translating in Xenopus oocytes RNAs transcribed in vitro from cloned zein mRNAs, we were able to identify the encoded proteins among native zeins or zeins synthesized in oocytes with total zein mRNA. There was no correlation between the isoelectric points of these proteins and the homology of their coding DNA sequences, as the proteins encoded by two closely homologous cDNAs migrated with greater charge heterogeneity than those encoded by less homologous clones. In addition, the size of the proteins as determined by SDS polyacrylamide gel electrophoresis did not always correlate with the length of the protein deduced from the DNA sequence. The ability to match cloned zein sequences to individual native proteins will enable the genetic mapping of cloned genes as well as the analysis of their translational regulation. PMID- 16665434 TI - Comparison of in situ and in vitro regulation of soybean seed growth and development. AB - The growth characteristics of soybean (Glycine max [L.] Merr.) embryos in culture and seeds in situ were found to be similar, but developmental differences were observed. Embryos placed in culture when very small (<2 milligrams dry weight) failed to attain the maximal growth rates attained by embryos which were more mature when placed in culture. When nutrient levels were maintained in the culture medium, embryos continued to grow indefinitely, reaching dry weights far in excess of seeds matured in situ. Apparently, maternal factors were important in early and late development during the determination of maximum growth rate and the cessation of growth. Embryo growth rate was not affected by substituting glucose plus fructose for sucrose in the medium, nor by hormone treatments, including abscisic acid. Glutamine was found to give substantially better growth than glutamate, however. Contrary to prior reports, the response of soybean embryo growth rate to irradiance was found to be primarily an artifact of the effect of irradiance on media temperature. Across seven genotypes the correlation coefficient between seed growth rate in situ and embryo growth rate in vitro was 0.94, indicating essentially all of the variability of in situ seed growth rate between cultivars could be attributed to inherent growth rate differences associated with the embryos. The response to temperature was very similar for both embryos in culture and seeds in situ at temperatures below 30 degrees C. Beyond that temperature, embryo growth rate continued to increase, while seed growth rate did not. The implication is that in situ seed growth rate is determined by the inherent growth potential of the embryo at low to moderate temperatures; however, at higher temperatures, the maternal plant is unable to support the rapid growth rates that the embryo is capable of attaining under conditions of unlimited assimilate supply. PMID- 16665435 TI - Phytochrome Chromophore Biosynthesis : Both 5-Aminolevulinic Acid and Biliverdin Overcome Inhibition by Gabaculine in Etiolated Avena sativa L. Seedlings. AB - Etiolated Avena sativa L. seedlings grown in the presence of gabaculine (5-amino 1,3-cyclohexadienylcarboxylic acid) contained reduced levels of phytochrome as shown by spectrophotometric and immunochemical assays. Photochromic phytochrome levels in gabaculine-grown plants were estimated to be 20% of control plants, while immunoblot analysis showed that the phytochrome protein moiety was present at approximately 50% of control levels. Gabaculine-grown seedlings administered either 5-aminolevulinic acid or biliverdin exhibited a rapid increase of spectrophotometrically detectable phytochrome. Phytochrome concentrations estimated immunochemically did not similarly increase throughout treatment with either compound. Similar experiments with 5-amino[4-(14)C] levulinic acid showed radiolabeling of phytochrome with kinetics that paralleled the spectrally detected increase. These results are consistent with (a) the intermediacy of both 5-aminolevulinic acid and biliverdin in the biosynthetic pathway of the phytochrome chromophore and (b) the lack of coordinate regulation of chromophore and apoprotein synthesis in Avena seedlings. PMID- 16665436 TI - Transport and Compartmentation of 1-Aminocyclopropane-1-Carboxylic Acid and Its Structural Analog, alpha-Aminoisobutyric Acid, in Tomato Pericarp Slices. AB - The uptakes of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene, and its structural analog, alpha-aminoisobutyric acid (alphaAIB) by tomato pericarp slices were investigated. Both uptakes show a biphasic (saturable-linear) dependence on external concentration of the transported amino acid. At low concentrations, ACC uptake is competitively inhibited by alphaAIB and vice versa. Both uptakes also are inhibited by other neutral amino acids but not by acidic or basic amino acids. ACC and alphaAIB uptakes are metabolically dependent and are increased with time of tissue incubation. alphaAIB efflux patterns from pericarp slices indicated three distinct alphaAIB compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole. The bulk of the alphaAIB taken up by pericarp tissue is sequestered into the vacuole. The ability of pericarp tissue to accumulate alphaAIB in the vacuole declines with fruit development. PMID- 16665437 TI - Photonastic Control of Leaflet Orientation in Melilotus indicus (Fabaceae). AB - Leaflet orientation in Melilotus indicus (L.) All. Is under photonastic control during the day and nyctinastic control during the night, but also exhibits a diaphototropic (solar-tracking) response. Detached leaves with the two lateral leaflets excised were used to study the solar-tracking capability of the terminal leaflet. Perception of the photonastic excitation is located in the pulvinule. The lower (abaxial) and upper (adaxial) surfaces perceive photonastic excitation, which results in concomitant contraction of the side exposed to light and/or expansion of the opposite side. Steady state laminar elevation is determined by the fluence rates of the light incident simultaneously on the opposite sides. Light sensitivity of the lower side exceeds that of the upper. Response to photonastic excitation of either side is affected by angle of incidence of the light, but angular dependence is restricted to a limited range of angle of incidence, which differs for the two sides. This may be accounted for by the different topography of the two pulvinar surfaces and the localization in them of the light-sensitive tissues. PMID- 16665438 TI - Salinity stress induced tissue-specific proteins in barley seedlings. AB - Protein changes induced by salinity stress were investigated in two barley cultivars, California Mariout, a salt-tolerant variety and Prato, a salt sensitive variety. Rapidly growing young barley seedlings were exposed to NaCl and the newly synthesized proteins were resolved on two dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. Salinity induces distinct protein changes in root and shoot tissues. In roots, the salinity effects are identical in both cultivars. First, salinity modulates the synthesis of two different sets of proteins, one of which is elevated, and the other, depressed. Second, six new proteins are induced all of which are low in molecular weight, 24 to 27 kilodaltons, with an isoelectric point range of 6.1 to 7.6. In contrast to roots, salinity induces cultivar-specific shoot proteins. Five new shoot proteins are induced whose molecular weights and isoelectric points fall within the range of 20 to 24 kilodaltons and 6.3 to 7.2, respectively. Three of the newly induced proteins are unique to Prato. In addition, salinity inhibits the synthesis of a majority of shoot proteins. The new proteins produced in roots and shoots are unique to each tissue and their induction is apparently regulated coordinately during salinity stress. PMID- 16665439 TI - Demonstration of ATP-Dependent, Ubiquitin-Conjugating Activities in Higher Plants. AB - Ubiquitin is a highly conserved, 76-amino acid polypeptide with several important regulatory functions in both plants and animals that all arise from its covalent ligation to other cellular proteins. Here, we demonstrate that higher plants have the capacity to conjugate ubiquitin to other plant proteins in vitro. Using (125)I-labeled human ubiquitin as a substrate, conjugating activities were observed in crude etiolated tissue extracts from all species tested, including oats, rye, barley, corn, zucchini squash, pea, soybean, and sunflower. The reaction has a soluble distribution, is specific for ATP, and requires the protease inhibitor, leupeptin, to protect ubiquitin from inactivation during the assay. Conjugation is inhibited by N-ethylmaleimide and high concentrations of 2 mercaptoethanol suggesting that the mechanism of ubiquitin ligation in plants involves a similar thiolester intermediate to that found in the mammalian pathway. The conjugating activity in etiolated oat extracts is extremely labile with a half-life of about 20 minutes at 30 degrees C. Detectable but low ATP stimulated, conjugating activities were also observed in extracts from dry seeds and green leaves of oats. In addition to this conjugating activity, crude plant extracts have the capacity to degrade ubiquitin-protein conjugates formed in vitro. These results demonstrate that higher plants contain several of the enzymic activities necessary for ubiquitin's functions and provide a method for assaying ubiquitin conjugation in vitro. PMID- 16665440 TI - Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development. AB - Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [(35)S]sulfate into protein. Incorporation of [(35)S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the alpha and beta globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis. PMID- 16665441 TI - Accumulation and Conversion of Sugars by Developing Wheat Grains : VII. Effect of Changes in Sieve Tube and Endosperm Cavity Sap Concentrations on the Grain Filling Rate. AB - The extent to which wheat grain growth is dependent on transport pool solute concentration was investigated by the use of illumination and partial grain removal to vary solute concentrations in the sieve tube and endosperm cavity saps of the wheat ear (Triticum aestivum L.). Short-term grain growth rates were estimated indirectly from the product of phloem area, sieve tube sap concentration, and (32)P translocation velocity. On a per grain basis, calculated rates of mass transport through the peduncle were fairly constant over a substantial range in other transport parameters (i.e. velocity, concentration, phloem area, and grain number). The rates were about 40% higher than expected; this probably reflects some unavoidable bias on faster-moving tracer in the velocity estimates. Sieve tube sap concentration increased in all experiments (by 20 to 64%), with a concomitant decline in velocity (to as low as 8% of the initial value). Endosperm cavity sucrose concentration also increased in all experiments, but cavity sap osmolality and total amino acid concentration remained nearly constant. No evidence was found for an increase in the rate of mass transport per grain through the peduncle in response to the treatments. This apparent unresponsiveness of grain growth rate to increased cavity sap sucrose concentration conflicts with earlier in vitro endosperm studies showing that sucrose uptake increased with increasing external sucrose concentration up to 150 to 200 millimolar. PMID- 16665442 TI - Degradation of the 32 kD Herbicide Binding Protein in Far Red Light. AB - White light (400-700 nanometers) supports the activity of photosystem I (PSI) and photosystem II while far red light (>/=700 nanometers) supports PSI almost exclusively. In intact fronds of Spirodela oligorrhiza, turnover of the 32 kilodaltons herbicide binding protein is stimulated under both these light conditions, although not in the dark or at wavelengths >730 nanometers. As is the case in white light, the far red light induced degradation of the protein is inhibited by DCMU. The means by which far red light operates is unclear. Hypotheses considered include: PSI activated proteolysis, PSI-induced formation of semiquinone anions, and PSI-generated free radicals. PMID- 16665443 TI - The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis. AB - A mutant of Arabidopsis thaliana with reduced content of C(18:3) and C(16:3) fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO(2)-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes. PMID- 16665444 TI - Purification and properties of starch hydrolyzing enzymes in mature roots of sugar beets. AB - Mature roots of sugar beets, which accumulate large amounts of sucrose but not starch, nevertheless contained acid and neutral amylases, judging from their pH optima, as well as pullulanase. Acid and neutral amylases were partially purified by procedures including fractionation with ammonium sulfate, ion exchange column chromatography, and gel filtration. Acid amylase was classified as an exoamylase, since it produced only glucose from soluble starch, amylopectin. beta-limit dextrin, and rabbit liver glycogen. Neutral amylase was classified as an endoamylase, since it liberated maltose as the main product plus a small amount of glucose and oligosaccharides, and was capable of hydrolyzing beta-limit dextrin. Pullulanase was purified to apparent homogeneity by procedures including fractionation with ammonium sulfate, Diethylaminoethyl-cellulose column chromatography and affinity chromatography. Pullulanase was capable of hydrolyzing soluble starch, amylopectin, beta-limit-dextrin, and pullulan. Debranching of amylopectin was further evident by an increase in extinction coefficient, and by a shift of lambda(max) from 530 to 560 nm when the debranched amylopectin formed a complex with I(2)-KI. PMID- 16665445 TI - Glutamine Synthetase of Nicotiana plumbaginifolia: Cloning and in Vivo Expression. AB - We have characterized the distinct forms of glutamine synthetase (GS) which are present in leaves and roots of Nicotiana plumbaginifolia. Mature leaves contain a single GS polypeptide (44 kilodaltons in size) which is localized to the stroma of intact chloroplasts. In contrast, the GS polypeptide in roots is distinct in size (38 kilodaltons) and charge. A lectin stain of leaf soluble protein indicates that the size difference of these mature GS polypeptides is not the result of posttranslational glycosylation. cDNA clones encoding a GS mRNA of N. plumbaginifolia were characterized and used as molecular probes to examine GS transcripts in leaves and roots. GS mRNA hybrid-selected from leaves or roots translated in vitro into distinct GS primary translation products (49 or 38 kilodaltons). The 49 kilodalton GS primary translation product, specific to leaf poly(A)RNA is proposed to be a precursor to the mature 44 kilodalton chloroplast stromal GS polypeptide. The 38 kilodalton GS primary translation product encoded by root GS mRNA, corresponds in size to the polypeptide encoded by the GS cDNA clones characterized. Southern blot analysis of nuclear DNA indicates that there are several different genomic fragments encoding GS in N. plumbaginifolia. PMID- 16665446 TI - Polyamine uptake in carrot cell cultures. AB - Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca(2+). The effects of Ca(2+) were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca(2+) and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca(2+) concentration range between 10 micromolar and 1 millimolar. La(3+) nullified the stimulatory effect of 10 micromolar Ca(2+) on putrescine uptake and that of 1 millimolar Ca(2+) on spermidine uptake. La(3+) at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule. PMID- 16665447 TI - Effects of Chilling and ABA on [H]Gibberellin A(4) Metabolism in Somatic Embryos of Grape (Vitis vinifera L. x V. rupestris Scheele). AB - Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. x V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [(3)H]GA(4) (of high specific activity, 4.81 x 10(10) becquerel per millimole) for 48 hours at 26 degrees C. Chilling had little effect on the total amount of free [(3)H]GA-like metabolites formed during incubation at 26 degrees C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [(3)H] metabolites formed at 26 degrees C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA(12) aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [(3)H]GA(4) treatment at 26 degrees C, reduced the uptake of [(3)H] GA(4) but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26 degrees C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs). PMID- 16665448 TI - Sucrose Metabolism in Netted Muskmelon Fruit during Development. AB - Sugar content and composition are major criteria used in judging the quality of netted muskmelon (Cucumis melo L. var reticulatus) fruit. Sugar composition and four enzymes of sucrose metabolism were determined in ;Magnum 45' muskmelon fruit at 10-day intervals beginning 10 days after pollination (DAP) until full-slip (35 DAP). Sugar content increased in both outer (green) mesocarp and inner (orange) mesocarp between 20 and 30 DAP. The major proportion of total increase in sugar was attributed to sucrose accumulation. The large increase in sucrose relative to glucose and fructose was accompanied by a dramatic decrease in acid invertase activity, which was highest in both tissues at 10 and 20 DAP, and increases in sucrose phosphate synthase (SPS) and sucrose synthase activities. The green tissue had a lower proportion of total sugar as sucrose, greater invertase activity, and less SPS activity than the orange tissue. Changes in relative sucrose content were highly correlated with changes in enzyme activity. The results strongly suggest that increases in the proportion of sucrose found in melon fruit were associated with a decline in acid invertase activity and an increase in SPS activity approximately 10 days before full-slip. Therefore, these enzymes apparently play a key role in determining sugar composition and the quality of muskmelon fruit. PMID- 16665449 TI - A Kinetic Analysis of Phytochrome Controlled Mesocotyl Growth in Zea mays Seedlings. AB - Mesocotyl elongation in 4 day old etiolated seedlings immediately following 3 hours of white light (3 h W) is reversibly controlled by phytochrome. Time-lapse video measurements were made of the 5 millimeter zone just below the coleoptile which is the main growth region of the mesocotyl. The growth kinetics were determined for five contiguous 1 millimeter zones subtending the coleoptile node for nonirradiated seedlings, for seedlings given 3 h W, and 3 h W followed by terminal far-red (FR) or red subsequent to the far-red (FR/R) irradiation. Each zone in nonirradiated seedlings exhibits exponential elongation kinetics during the early stages of elongation. This finding suggests that during elongation, a growth limiting factor is also exponentially increasing. Following 3 h W differences in the kinetic responses were found for each zone. In all zones, the inhibitory effect following the 3 h W is totally FR reversible. The effect of FR is reversed by R. The upper zone exhibits the fastest response and is the most plastic in its growth response. The three upper zones all exhibit spontaneous and sharp recoveries with time. It is suggested that the control by phytochrome is not inductive but rather continuous, the controlling factor being either the level of the far red-absorbing form of phytochrome (Pfr) or the ratio Pfr to total phytochrome. PMID- 16665450 TI - An evaluation of 2,5-norbornadiene as a reversible inhibitor of ethylene action in deepwater rice. AB - Partial submergence of deepwater rice (Oryza sativa L. cv Habiganj Aman II) elicits three responses: enhancement of internodal elongation, inhibition of leaf growth, and promotion of adventitious root formation. All three responses can be induced in isolated stem sections by treatment with ethylene. Dose-response curves indicate that the responses are linearly related to the logarithm of the ethylene concentration over two orders of magnitude. Application of the cyclic olefin 2,5-norbornadiene (NBD) to ethylene-treated sections results in a parallel shift in dose-response curves to higher ethylene concentrations, indicating that NBD behaves as a competitive inhibitor of ethylene action. Internodal elongation of stem sections is promoted by gibberellic acid (GA(3)) in the absence of exogenous ethylene. Endogenous ethylene levels do not increase in GA(3)-treated sections, and application of NBD does not prevent GA(3)-promoted elongation. To the contrary, NBD treatment results in increased growth at intermediate GA(3) concentrations. These results support the idea that ethylene acts through endogenous GA in promoting growth in deepwater rice. NBD applied to GA(3)-treated stem sections results in increased ethylene production. This enhancement of ethylene formation is reversed by application of either ethylene or propylene, indicating that ethylene biosynthesis in rice internodes is under negative feedback control. PMID- 16665451 TI - Purification and properties of Acid phosphatase-1 from a nematode resistant tomato cultivar. AB - In tomato the acid phosphatase-1 isozyme (Apase-1) is inherited as a single locus linked to the nematode resistance gene (Mi). The Apase-1(1) electrophoretic variant has been purified from a tomato cell suspension culture using ion exchange and concanavalin A sepharose affinity chromatography. A cellulose acetate electrophoresis method was used to distinguish Apase-1(1) rapidly from other Apase isozymes in tomato. The subunit molecular weight of the purified enzyme was estimated to be 31,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native size of the enzyme, which is reported to be a dimer, was determined to be approximately 51,000 by high performance liquid chromatography gel filtration. Apase-1(1) has a lower pH optimum and a distinct substrate specificity as compared to Apases extracted from tomato fruit or from other plant species. The amino acid composition of Apase-1(1) is similar to that of a potato Apase. PMID- 16665452 TI - Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato. AB - The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato. PMID- 16665453 TI - Specific mRNA and rRNA Levels in Greening Pea Leaves during Recovery from Iron Stress. AB - Hydroponically grown pea seedlings (Pisum sativum L., cv Alaska) were subjected to Fe stress for 10 to 16 days to produce mature chlorotic leaves. Greening was initiated by adding Fe to the nutrient solution. The levels of chlorophylls, chloroplast, and cytoplasmic rRNAs, and specific chloroplast- and nucleus-encoded mRNAs were all significantly lower in leaves developing during iron stress than in nonstressed leaves. In plants greening after addition of Fe, nuclear transcripts encoding chlorophyll a/b-binding protein and the small subunit of ribulose bisphosphate carboxylase/oxygenase increased about 5-fold in abundance following an 18 to 24 hour lag, as did the chloroplast-encoded transcript for the large subunit of the carboxylase/oxygenase. Chloroplast rRNA showed an increase over that in continually stressed control leaves only after a 40 hour lag. The chloroplast-encoded transcript encoding the Q(B)-binding 32 kilodalton polypeptide of Photosystem II showed little change during greening. Chlorophyll itself increased gradually after a lag period of 24 hours, with an increase in chlorophyll a slightly preceding that of chlorophyll b. Kinetic considerations suggest that the changes observed represent a coordinate series of events initiated by readdition of Fe and occurring in parallel. Though accumulation of mRNA for light-harvesting, chlorophyll-a/b-binding protein might limit chlorophyll accumulation at the onset, subsequent changes in the mRNA do not parallel chlorophyll changes. All three of the mRNAs showing recovery on addition of Fe to Fe-stressed plants undergo sharp diurnal fluctuations in abundance. Such fluctuations are comparable to those in nonstressed controls (mRNA for light harvesting protein) or considerably more pronounced (mRNAs for carboxylase large and small subunits). The carboxylase small subunit mRNA and that for light harvesting chlorophyll-binding protein were measured under constant conditions of light and temperature. Though a rhythm in greening leaves was hard to detect, it was prominent in the Fe-sufficient controls, persisting undamped through three full cycles for both mRNAs, and hence is probably circadian. PMID- 16665454 TI - Identification of a receptor protein in cotton fibers for the herbicide 2,6 dichlorobenzonitrile. AB - The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and apparently specific inhibitor of cellulose synthesis in higher plants. We have synthesized a photoreactive analog of DCB (2,6-dichlorophenylazide [DCPA]) for use as an affinity-labeling probe to identify the DCB receptor in plants. This analog retains herbicide activity and inhibits cellulose synthesis in cotton fibers and tobacco cells in a manner similar to DCB. When cotton fiber extracts are incubated with [(3)H]DCPA and exposed to ultraviolet light, an 18 kilodalton polypeptide is specifically labeled. About 90% of this polypeptide is found in the 100,000g supernatant, the remainder being membrane-associated. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of this polypeptide indicate that it is an acidic protein which has a similar size in its native or denatured state. The amount of 18 kilodalton polypeptide detectable by [(3)H]DCPA-labeling increases substantially at the onset of secondary wall cellulose synthesis in the fibers. A similar polypeptide, but of lower molecular weight (12,000), has been detected upon labeling of extracts from tomato or from the cellulosic alga Chara corallina. The specificity of labeling of the 18 kilodalton cotton fiber polypeptide, coupled with its pattern of developmental regulation, implicate a role for this protein in cellulose biosynthesis. Being, at most, only loosely associated with membranes, it is unlikely to be the catalytic polypeptide of the cellulose synthase, and we suggest instead that the DCB receptor may function as a regulatory protein for beta-glucan synthesis in plants. PMID- 16665455 TI - The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection. AB - The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nod-phenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6 dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PS1 and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs. PMID- 16665456 TI - The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl. AB - Extracellular components released from mycelia of the alpha and beta races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible alpha race and compatible beta race. Elicitor activity, however, was greater in the alpha race fractions. Elicitor purified from alpha race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function. PMID- 16665457 TI - A Tight-Seal Whole Cell Study of the Voltage-Dependent Gating Mechanism of K Channels of Protoplasmic Droplets of Chara corallina. AB - The biophysical properties of voltage-dependent K(+)-channels of protoplasmic droplets of Chara corallina Klein ex Willd., em, R.D.W. were investigated using the tight-seal whole cell method. Two potassium currents were observed in voltage clamp mode and they can be used to explain the transient membrane potential time course observed in current-clamp mode. The K(+)-channels are identified by the effect of tetraethylammonium chloride which blocks both currents. A two-state, constant dipole moment model is used to fit the voltage-conductance curve. From this model the minimum equivalent gating charge involved in the gating mechanism of K(+)-channels of Chara can be estimated. PMID- 16665458 TI - Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection. AB - Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus. PMID- 16665459 TI - Rapid Effects of IAA on Cell Surface Proteins from Intact Carrot Suspension Culture Cells. AB - Suspension cultures of carrot (Daucus carrota L.) which had an absolute requirement for exogenously supplied auxin were grown in medium containing indoleacetic acid (IAA) as the sole auxin source. Putative cell surface proteins were extracted from the intact cells. Resupply of IAA to cultures partially depleted of auxin resulted in rapidly increased activities of three enzyme activities subsequently extracted. Two of the enzyme activities which increased, peroxidase and pectinesterase, have been implicated in the literature as important to cell wall development, structure, and growth. The other enzyme activity which was increased, IAA oxidase, may be involved in the degradation of IAA In vivo. Polypeptides in the extracts were found to increase equally as rapidly as the enzymes in response to IAA as determined with sodium dodecyl sulfate-polyacrylamide electrophoretic gels stained with silver. It is not known whether the changes in enzyme and polypeptide levels in the protein extracts were due to auxin effects on protein synthesis, transport, or extractability. PMID- 16665460 TI - Effect of Night Temperature on the Activity of Sucrose Phosphate Synthase, Acid Invertase, and Sucrose Synthase in Source and Sink Tissues of Rosa hybrida cv Golden Times. AB - Sucrose phosphate synthase and acid invertase activities in the mature leaves of roses (Rosa hybrida cv Golden Times) were greater in plants grown under a higher night temperature than under a lower temperature regime. In young shoots, the activity of acid invertase was promoted by the lower temperature while that of sucrose synthase was increased at the higher temperature. At both temperatures benzyladenine when applied to the axillary bud stimulated sucrose phosphate synthase activity and advancement of its peak of activity in the leaf subtending to the bud, and also stimulated sucrose synthase activity in the young shoot. At the lower temperature, application of benzyladenine to the axillary bud stimulated acid invertase activity in the young shoot but not in the leaves. PMID- 16665461 TI - Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. AB - Leaf extracts of both Nicotiana tabacum and Nicotiana sylvestris contain multiple forms of catalase (H(2)O(2):H(2)O(2) oxidoreductase, EC 1.11.1.6) which are separable at different pH values by chromatofocusing columns. Marked changes in distribution of these catalases occur during seedling development and leaf maturation. The form of catalase eluting first (peak 1) was predominant during early seedling growth and present at all stages of development. Two more acidic forms (peaks 2 and 3) appeared later and comprised 29% of the total activity by 11 days postgermination. Mature leaves of N. tabacum contained peak 1 catalase, but peaks 2 and 3 represented 62% of the total activity. No interconversion of peaks 1, 2, and 3 was detected. The three forms of catalase differed in thermal stability with peak 1 > peak 2 >> peak 3. For N. sylvestris, t((1/2)) at 55 degrees C was 31.5 and 3.0 min for peaks 1 and 3, respectively, and for N. tabacum, t((1/2)) was 41.5 and 3.2 min, respectively. All forms of catalase in tobacco show peroxidatic (measured as ethanol to acetaldehyde conversion) as well as catalatic activities. However, for both Nicotiana species the ratio peroxidatic/catalatic activity is at least 30-fold higher in peak 3 than in peaks 1 and 2. Chromatofocusing of extracts from spinach leaves separated at least four peaks of catalase activity, one of which had a 10-fold higher ratio of peroxidatic/catalatic activity than the others. Short-term growth (5 days) of tobacco seedlings under atmospheric conditions suppressing photorespiration (1% CO(2)/21% O(2)) reduced total catalase activity and caused a decline in peak 1 catalase and a substantial increase in the activity of peaks 2 and 3 relative to air-grown seedlings at the same stage. PMID- 16665462 TI - Photosynthesis, Leaf Anatomy, and Cellular Constituents in the Polyploid C(4) Grass Panicum virgatum. AB - Photosynthetic gas exchange, activities of six key C(4) cycle enzymes, amounts of soluble protein, chlorophyll, and DNA, and various leaf anatomical and structural features were measured in naturally occurring tetraploid and octaploid plants of the NAD-malic enzyme type C(4) grass Panicum virgatum L. On a leaf area basis, the photosynthetic rate and concentrations of DNA, soluble protein, and chlorophyll were 40 to 50% higher, and enzyme activities 20 to 70% higher in the octaploid than in the tetraploid. Photosynthetic cells in the octaploid were only 17 to 19% larger in volume, yet contained 33 to 38% more chloroplasts than cells in the tetraploid. On a per cell basis the contents of DNA, soluble protein, and chlorophyll, activities of carboxylating photosynthetic enzymes, and carbon assimilation rate were all doubled in octaploid compared with tetraploid cells. Since cellular volume did not double with genome doubling, cellular constituents were more concentrated in the cells of the octaploid. The influences of polyploidy were balanced between mesophyll and bundle sheath cells since the changes in physical and biochemical parameters with ploidy level were similar in both cell types. We conclude that photosynthetic activity in these two polyploid genotypes of P. virgatum is determined by enzyme activities and concentrations of biochemical constituents, and that selection for smaller cell volume has led to higher photosynthetic rates per unit leaf area in the octaploid. The ratio of DNA content to cellular volume is a major factor determining the concentrations of gene products in cells. The number of chloroplasts, however, is controlled more by cellular volume than by the number of nuclear chromosomes. PMID- 16665463 TI - In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm. AB - Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net (14)C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of (14)C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q(10) suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis). PMID- 16665464 TI - In Vitro Sugar Transport in Zea mays L. Kernels : II. Characteristics of Sugar Absorption and Metabolism by Isolated Developing Embryos. AB - In vitro sugar transport into developing isolated maize embryos was studied. Embryo fresh and dry weight increased concomitantly with endogenous sucrose concentration and glucose uptake throughout development. However, endogenous glucose and fructose concentration and sucrose uptake remained constant. The uptake kinetics of radiolabeled sucrose, glucose, and fructose showed a biphasic dependence on exogenous substrate concentration. Hexose uptake was four to six times greater than sucrose uptake throughout development. Carbonylcyanide-m chlorophenylhydrazone and dinitrophenol inhibited sucrose and glucose uptake significantly, but 3-O-methyl glucose uptake was less affected. The uptake of 1 millimolar sucrose was strongly pH dependent while glucose was not. Glucose and fructose were readily converted to sucrose and insoluble products soon after absorption into the embryo. Thus, sucrose accumulated, while glucose pools remained low. Based on the findings of this and other studies a model for sugar transport in the developing maize kernel is presented. PMID- 16665465 TI - Comparisons of Photosynthetic Responses of Xanthium strumarium and Helianthus annuus to Chronic and Acute Water Stress in Sun and Shade. AB - We have examined the effects of mild, chronic water stress and acute water stress on two water stress sensitive plants, Xanthium strumarium and Helianthus annuus. Using a combination of the leaf disc O(2) electrode to measure the light responses of photosynthesis and 77 K fluorescence to monitor damage to the primary photochemistry, we have found the following: (a) The CO(2) saturated rate of photosynthesis at high light is the most water stress sensitive parameter measured. (b) The apparent quantum yield (moles O(2) per mole photons) was slightly, if at all, affected by mild water stress (>-1.5 megapascals). (c) Severe water stress (<-1.5 megapascals) reduced the quantum yield of photosynthesis regardless of whether the stress was applied in sun or shade. The light independent reduction of quantum yield was not associated with a reduction in 77 K fluorescence (F(v)/F(m)) indicating that the quantum yield reduction was not the result of damage to primary photochemistry. (d) The diel fluctuation in 77 K fluorescence seen in sun-exposed control leaves was greatly exaggerated in water stressed leaves because of enhanced decline in 77 K fluorescence in the morning. The rate of recovery was similar in both control and water stressed leaves. Shaded leaves showed no change in 77 K fluorescence regardless of whether water stress was imposed or not. (e) The water stress sensitive plants used in these experiments did not recover from acute water stress severe enough to reduce the quantum yield or chronic water stress which lasted long enough that light dependent damage to primary photochemistry occurred. PMID- 16665466 TI - Structural, Functional, and Evolutionary Analysis of Ribulose-1,5-Bisphosphate Carboxylase from the Chromophytic Alga Olisthodiscus luteus. AB - Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg(2+) have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO(3) and Mg(2+) for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO(2) and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase catalyzed CO(2) fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site. PMID- 16665467 TI - Measurement of Indole-3-Acetic Acid in Peach Fruits (Prunus persica L. Batsch cv Redhaven) during Development. AB - The amount of indole-3-acetic acid (IAA) was measured in peach fruits by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution assay with [(13)C(6)]IAA as an internal standard throughout the growing season. Ethylene evolution of the fruit was also measured. IAA levels were 25 nanograms per gram fresh weight, 18 days after anthesis. Both IAA levels and rates of ethylene evolution declined to their lowest levels (7 nanograms IAA per gram fresh weight and 0.01 nanoliter ethylene per gram per hour) in the second stage of fruit growth. Endogenous levels of free-IAA and ethylene evolution increased in the last stage of peach fruit development to 32 nanograms per gram fresh weight and 0.27 nanoliter per gram per hour, respectively. IAA amounts peaked in the ovules 67 days after anthesis. PMID- 16665468 TI - A requirement for sucrose in xylem sap flow from dormant maple trees. AB - The response of excised stem segments of several tree species to freezing and thawing cycles was studied. All species studied (Thuja occidentalis, Fagus grandifolia, and Betula papyrifera) except maple (Acer spp.) exuded sap while freezing and absorbed on thawing. Maple stems absorbed sap while freezing and exuded sap during the thaw only when sucrose was present in the vessel solution. Increased concentration of sucrose in the vessel sap led to increased exudation. In the absence of sucrose, maple stems absorbed sap on thawing. The presence of sucrose enhanced sap absorption during freezing cycles in maples. In general, large sugars, disaccharides and larger, could substitute for sucrose in the maple exudation response while sugar hexoses could not. The results are discussed in relation to the O'Malley-Milburn model (1983 Can J Bot 61: 3100-3106) of sap flow in maples. PMID- 16665469 TI - Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase. AB - Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C(4) PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C(4) plant species. Neither antiserum reacted strongly with any C(3) leaf proteins. The molecular weight of the PEPC polypeptide from C(4) leaves and legume nodules appears to be similar. PMID- 16665470 TI - Increased Lysine and Seed Storage Protein in Rice Plants Recovered from Calli Selected with Inhibitory Levels of Lysine plus Threonine and S-(2 Aminoethyl)cysteine. AB - Experiments were designed to test whether variation in percent lysine in seed proteins could be recovered in plants regenerated from callus subjected to inhibitory levels of lysine plus threonine. Anther-derived callus was subjected to 1 millimolar lysine plus threonine for three successive passages and then once to the same concentration of S-(2-aminoethyl)cysteine. Plants were regenerated from the resistant callus. Plants recovered directly from tissue culture were normal in color, size and were 50% or less fertile. Second and third generation plants produced a wide range of variants including albinos, deep green plants both short and tall, and totally fertile as well as partially fertile plants. All regenerated plants produced chalky or opaque seed. One unique second generation line had 14% more lysine in seed storage proteins than the controls. This characteristic was transmitted to the next generation. The high lysine plants had reduced seed size with significantly higher levels of seed storage protein than the controls. The phenotypes recovered provide experimental materials for basic studies in protein synthesis and lysine metabolism and may become a source of material for rice breeding. PMID- 16665471 TI - Identification of Gibberellins in Norway Spruce (Picea abies [L.] Karst.) by Combined Gas Chromatography-Mass Spectrometry. AB - Gibberellins A(1) (GA(1)), A(3) and A(9) were identified from extracts of shoots of 6-month old Norway spruce (Picea abies) seedlings by the use of sequential reverse and normal phase high performance liquid chromatography (HPLC), bioassay, radioimmunoassay (RIA) and combined gas chromatography-mass spectrometry (GC-MS). The bioassay and RIA were used after fractionation by HPLC to detect the GA containing fractions, which were then examined by GC-MS. The GAs identified are considered to be endogenous. PMID- 16665472 TI - A role for ca in the elicitation of rishitin and lubimin accumulation in potato tuber tissue. AB - Calcium and strontium ions enhanced rishitin but not lubimin accumulation in tuber tissue of potato (Solanum tuberosum cv Kennebec) treated with arachidonic acid (AA). The same cations in the presence of poly-l-lysine (PL) enhanced the accumulation of lubimin more than rishitin. In contrast, Mg(2+) did not affect AA elicited rishitin and lubimin accumulation and inhibited the accumulation of these compounds following application of PL. AA-elicited potato tuber tissue remained sensitive to the stimulatory effects of Ca(2+) and Sr(2+) up to 24 h after application of AA, but PL-elicited tuber tissue was sensitive to Ca(2+) and Sr(2+) for only 6 hours after PL application. Ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid and La(3+) both inhibited rishitin and lubimin accumulation elicited by AA. The inhibition by either agent was overcome by the addition of Ca(2+). Calcium was more effective in overcoming lanthanum inhibition when applied simultaneously than when applied 12 hours later. Lanthanum was only effective in inhibiting rishitin and lubimin accumulation when applied within 3 hours of the application of AA. Inhibition of phytoalexin accumulation was greater when La(3+) was applied simultaneously with AA compared to La(3+) application after AA application to discs. These observations suggest that the mobilization of calcium may play a central regulatory role in the expression of phytoalexin accumulation following elicitation in potato tissue. PMID- 16665473 TI - Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes. AB - The effects of NO(3) (-) and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H(+)-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO(3) when assayed at 24 degrees C or above and was tentatively identified as the tonoplast H(+)-ATPase. A smaller peak of H(+) ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO(3) and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H(+)-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO(3) inhibited ATP hydrolysis by the tonoplast ATPase at 12 degrees C and the initial rate of proton transport was stimulated by 50 millimolar KNO(3). The time course for fluorescence quench indicated that addition of ATP in the presence of KNO(3) caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO(3) was explained by the temperature-dependent delay of the inhibition by KNO(3). The plasma membrane H(+)-ATPase maintained a pH gradient in the presence of KNO(3) for up to 30 minutes at 24 degrees C. PMID- 16665474 TI - Synthesis and Assembly of the Polypeptides of Photosystem I and II in Isolated Etiochloroplasts of Wheat. AB - Synthesis and assembly of photosystems (PS) I and II polypeptides in etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings were studied. The isolated etiochloroplasts synthesized PSI polypeptides of 66 and 15 kilodaltons, PSII polypeptides of 46 and 42 kilodaltons, and atrazine-binding 34 to 32 kilodalton polypeptide. Their assembly processes in the thylakoid membrane were studied by pulse-chase labeling with [(35)S]methionine, mild solubilization of the thylakoid membrane with Triton X 100, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The newly synthesized polypeptides of 66, 46, 42, 34, and 32 kilodaltons were first integrated into the complexes of 7.5, 5.9, 7.5, 6.3, and 7.5 Svedberg units, respectively, in 20 minutes. After the chase with excess amount of methionine for 100 min, they were found in complexes of 9.5, 9.1, 9.1, 9.1, and 9.1 Svedberg units, respectively. In this condition, stained polypeptides of PSI and PSII were found in the complexes of 11.1 and 10.3 Svedberg units, respectively. These results indicated that newly synthesized PSI or PSII polypeptides are integrated into intermediate complexes, but not complete complexes in the isolated etiochloroplasts. The relationship between the processing of the atrazine-binding 32 kilodalton polypeptide and its assembly into the PSII complex is also discussed. PMID- 16665475 TI - Indole-3-Ethanol Oxidase in Phycomyces blakesleeanus Bgff: Characterization of the Enzyme. AB - Indole-3-ethanol oxidase (IEt oxidase) from Phycomyces blakesleeanus Bgff.(P.b.) is a 56 kD polypeptide as determined by gel filtration. The reaction products are indole-3-acetaldehyde (IAAld) and, possibly, H(2)O(2). Enzyme activity (33-45% ammonium sulfate fraction) shows a broad pH optimum and simple Michaelis-Menten kinetics (K(m) 7 micromolar, Hill coefficient 0.95). Flavin adenine dinucleotide increases enzyme activity particularly under anaerobic conditions. Iodoacetate and HgCl(2) drastically inhibit the enzyme. With IAAld, product inhibition is observed at micromolar concentrations. IAA and some other acidic substituted indoles reduce enzyme activity but only at higher concentrations. PMID- 16665476 TI - Changes in Cotyledon mRNA during Ethylene Inhibition of Floral Induction in Pharbitis nil Strain Violet. AB - Floral induction in seedlings of Pharbitis nil strain Violet, with one cotyledon removed, was manipulated by applying various ethylene treatments to the remaining cotyledon during a 16 hour inductive dark period. Exposure of cotyledons to ethylene (100 microliters per liter) for 4 hours at different times during the dark period inhibited flowering to some extent, with inhibition being greater towards the end of the dark period. RNA from cotyledons given a 16 hour dark period (induced) or exposed to 100 microliters per liter ethylene throughout the dark period, which completely inhibited flowering, was examined. The poly(A)(+)RNA was translated in vitro using a wheat germ system, and the resulting translation products were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were substantial qualitative and quantitative differences between the poly(A)(+)RNA extracted from induced cotyledons and that from those exposed to ethylene throughout the dark period. Some of these changes are similar to those observed when flowering was inhibited by photoperiodic treatments (M Lay-Yee, RM Sachs, MS Reid 1987 Planta. In press). The significance of these findings to our understanding of the molecular control of flower induction is discussed. PMID- 16665477 TI - Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves. AB - The rate of CO(2) assimilation and levels of metabolites of the C(4) cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO(2) concentration of 350 microliters per liter and a limiting CO(2) concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO(2) with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of approximately 2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO(2). These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO(2) to Rubisco through the C(4) cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C(4) cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C(4) cycle can be raised to mediate higher photosynthesis rate. PMID- 16665478 TI - Fatty acids of rice coleoptiles in air and anoxia. AB - The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [(14)C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O(2), as it does in other plants. PMID- 16665479 TI - (1-->3)-beta-d-Glucan Synthase from Sugar Beet : II. Product Inhibition by UDP. AB - The mode of inhibition of UDP, one of the products of the reaction catalyzed by (1-->3)-beta-d-glucan synthase in sugar beet (Beta vulgaris L.) was investigated. In the absence of added UDP, the enzyme, in the presence of Ca(2+), Mg(2+), and cellobiose, exhibited Michaelis-Menten kinetics and had an apparent K(m) of 260 micromolar for UDP-glucose. Complex effects on the kinetics of the (1-->3)-beta-d glucan synthase were observed in the presence of UDP. At high UDP-glucose concentrations, i.e. greater than the apparent K(m), UDP behaved as a competitive inhibitor with an apparent K(i) of 80 micromolar. However, at low UDP-glucose concentrations, reciprocal plots of enzyme activity versus substrate concentration deviated sharply from linearity. This unusual effect of UDP is similar to that reported for fungal (1-->3)-beta-d-glucan synthase. However, papulacandin B, a potent inhibitor of this fungal enzyme, had no effect on the plant (1-->3)-beta-d-glucan synthase isolated from sugar beet petioles. The inhibitory effect of UDP was also compared with other known inhibitors of glucan synthases. PMID- 16665480 TI - New light on the cholodny-went theory. AB - The existence of different auxin sensitivities in epidermal and subepidermal tissues (KV Thimann, CL Schneider 1938 Plant Physiol 25: 627-641) suggests a refinement to the Cholodny-Went theory which overcomes some of the difficulties associated with it. A model is presented to account for the inverse tropic responses of shoots and roots through differences in the respective locations of the auxin-sensitive tissues. PMID- 16665481 TI - An avidin-biotin solid phase ELISA for femtomole isopentenyladenine and isopentenyladenosine measurements in HPLC purified plant extracts. AB - A solid phase enzyme immunoassay was developed for isopentenyladenine (iP) and isopentenyladenosine (iPA) quantitation in HPLC purified plant extracts. It was performed on antigen-coated microtitration plates on which bound antibodies were indirectly labeled by the means of a biotinylated goat anti-rabbit antibody and an avidin-alkaline phosphatase conjugate. Less than 3 femtomoles of iP or iPA were easily detected and the measuring range extended from 3 femtomole to 1 picomole. The reproducibility has been tested and intra- and interassay variations did not exceed 5.0%. The specificity of iPA antibodies was good, as determined by cross-reactivity measurements with other adenylic compounds. The specificity of the measurements for iP and iPA was demonstrated by analysis of the immunoreactivity of fractions obtained by HPLC separation of a methanolic tobacco leaf extract. PMID- 16665482 TI - Effects of buthionine sulfoximine on cd-binding Peptide levels in suspension cultured tobacco cells treated with cd, zn, or cu. AB - Inhibition of cell growth and accumulation of Cd-binding peptide were measured in cultured tobacco cells exposed to buthionine sulfoximine. This inhibitor of glutathione metabolism caused little or no reduction of growth (at 0.1 millimolar) in the absence of Cd, but growth was greatly reduced in cultures exposed to buthionine sulfoximine and >/=22 micromolar Cd. Decreased cell growth was directly correlated with decreased levels of Cd-binding peptide and increased levels of what is thought to be free Cd. Zinc inhibited growth of tobacco cells only at the highest levels examined (900-1800 micromolar Zn), but buthionine sulfoximine had no additional significant effect. Similar results were observed for Cu (45-90 micromolar). Results suggest that synthesis of plant Cd-peptide involves gamma-glutamylcysteine synthetase or a related enzyme and that Zn accumulation in tobacco cells does not cause formation of significant Cd-peptide ligand. PMID- 16665483 TI - Influence of light on the ferredoxin-dependent glutamate synthase in maize leaves. AB - The ferredoxin (Fd)-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) activities are carried out by two immunochemically distinct enzyme proteins in maize leaves (Zea mays W64A and W182E). Continuous irradiation of etiolated tissue at 75 micro einsteins per square meter per second for 24 hours resulted in a 3-fold increase on a fresh weight basis in the activity of the Fd-dependent glutamate synthase and a slight decrease in the activity of the NADH-dependent enzyme. There was also a significant increase of the Fd-glutamate synthase protein during greening of etiolated tissue. PMID- 16665484 TI - Water Transfer in an Alfalfa/Maize Association : Survival of Maize during Drought. AB - We investigated the possibility of interspecific water transfer in an alfalfa (Medicago sativa L.) and maize (Zea mays L.) association. An alfalfa plant was grown through two vertically stacked plastic tubes. A 5 centimeter air gap between tubes was bridged by alfalfa roots. Five-week old maize plants with roots confined to the top tube were not watered, while associated alfalfa roots had free access to water in the bottom tube (the -/+ treatment). Additional treatments included: top and bottom tubes watered (+/+), top and bottom tubes droughted (-/-), and top tube droughted after removal of alfalfa root bridges and routine removal of alfalfa tillers (-(*)). Predawn leaf water potential of maize in the -/+ treatment fell to -1.5 megapascals 13 days after the start of drought; thereafter, predawn and midday potentials were maintained near -1.9 megapascals. Leaf water potentials of maize in the -/- and -(*) treatments declined steadily; all plants in these treatments were completely desiccated before day 50. High levels of tritium activity were detected in water extracted from both alfalfa and maize leaves after (3)H(2)O was injected into the bottom -/+ tube at day 70 or later. Maize in the -/+ treatment was able to survive an otherwise lethal period of drought by utilizing water lost by alfalfa roots. PMID- 16665485 TI - Studies on 17,24 kD Depleted Photosystem II Membranes : I. Evidences for High and Low Affinity Calcium Sites in 17,24 kD Depleted PSII Membranes from Wheat versus Spinach. AB - Analyses were made of the effects of extraction of the 17,24 kilodalton extrinsic proteins from spinach versus wheat photosystem II (PSII) membranes on Ca abundance and O(2) evolution capacity determined in the absence and presence of either Cl(-) or Ca(2+). Extraction of these proteins from spinach PSII routinely diminished steady state O(2) evolution by about 70% when assayed in the presence of sufficient Cl(-). Additionally, O(2) evolution of 17,24 kilodalton-less spinach PSII membranes showed about 2-fold more enhancement by Ca(2+) than by Cl( ) during assay. When the same extraction and assay procedures were applied to wheat PSII membranes, we observed, in contrast to 17,24 kilodalton-less spinach PSII, only about 50% inhibition of O(2) evolution and about 2-fold greater enhancement by Cl(-) than by Ca(2+). Irrespective of differences in the magnitude of enhancement of O(2) evolution by Ca(2+)versus Cl(-) in spinach versus wheat, the K(m) values for Cl(-) (about 1.7 millimolar) and Ca(2+) (about 1.5 millimolar) were similar for both type preparations. The abundance of Ca specifically associated with fully functional PSII (about 2 and about 3 Ca/200 chlorophyll for spinach and wheat, respectively) was diminished to about 1 per 200 chlorophyll upon 17.24 kilodalton protein depletion. Further treatment of wheat 17,24 kilodalton-less PSII in darkness with 2 molar NaCl/1 millimolar ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid/20 micromolar A23187(2) made O(2) evolution highly dependent on Ca(2+) addition, much like the 17,24 kilodalton-less spinach PSII. Analyses of this Ca(2+) effect on O(2) evolution revealed both high (K(m) about 65 micromolar) and low (K(m) about 1.5 millimolar) affinity Ca(2+) sites in wheat 17,24 kilodalton-less PSII. The results suggest that during 17,24 kilodalton extraction by NaCl, spinach PSII is more susceptible than wheat PSII to loss of high affinity Ca and irreversible inhibition of O(2) evolution. PMID- 16665486 TI - Metabolism of C-zeatin in phaseolus embryos : occurrence of o xylosyldihydrozeatin and its ribonucleoside. AB - The metabolism of trans-[8-(14)C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5'-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus. PMID- 16665487 TI - Carbon Flow and Metabolic Specialization in the Tissue Layers of the Crassulacean Acid Metabolism Plant, Peperomia camptotricha. AB - Leaves of Peperomia camptotricha contain three distinct upper tissue layers and a one-cell thick lower epidermis. Light and dark CO(2) fixation rates and the activity of ribulose bisphosphate carboxylase/oxygenase and several C(4) enzymes were determined in the three distinct tissue layers. The majority of the C(4) enzyme activity and dark CO(2) fixation was associated with the spongy mesophyll, including the lower epidermis; and the least activity was found in the median palisade mesophyll. In contrast, the majority of the C(3) activity, that is ribulose bisphosphate carboxylase/oxygenase and light CO(2) fixation, was located in the palisade mesophyll. In addition, the diurnal flux in titratable acidity was greatest in the spongy mesophyll and lowest in the palisade mesophyll. The spatial separation of the C(3) and C(4) phases of carbon fixation in P. camptotricha suggests that this Crassulacean acid metabolism plant may have low photorespiratory rates when it exhibits daytime gas exchange (that is, when it is well watered). The results also indicate that this plant may be on an evolutionary path between a true Crassulacean acid metabolism plant and a true C(4) plant. PMID- 16665488 TI - The effect of vanadate on proton-sucrose cotransport in ricinus cotyledons. AB - The effects of orthovanadate on the uptake of sucrose by Ricinus cotyledons and on sucrose-coupled proton influx were measured in order to gain insight into the relationship to the plasma membrane proton pump. Vanadate had no effect on short term sucrose uptake. In longterm experiments (>30 min) sucrose uptake was progressively inhibited, but only at high external sucrose concentrations. Vanadate did not affect proton efflux pumping in the absence of sucrose and neither did it change the initial rate of sucrose-coupled proton influx. However, it enhanced the maximal level of sucrose-induced alkalinization of the medium at all sucrose concentrations tested. This is interpreted as an inhibiting effect of vanadate on the proton pump that recycles protons during sucrose-proton cotransport. The sensitivity towards vanadate indicates that this proton pump is an ATPase. A second proton-translocating system, that is insensitive to vanadate, is postulated to function in the absence of sucrose. PMID- 16665489 TI - Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions. AB - Cobalamin is essential for growth of Euglena gracilis and photosynthesis. Methylcobalamin in Euglena chloroplasts (Y Isegawa, Y Nakano, S Kitaoka, 1984 Plant Physiol 76: 814-818) functions as a coenzyme of methionine synthetase. The requirement of cobalamin for photosynthesis appeared remarkably high in Euglena grown under the dark-precultured condition. The required amount of cobalamin for normal photosynthetic activity was 7.4 x 10(-11) molar, while 7.4 x 10(-10) molar cobalamin was required for normal growth. The lowered photosynthetic activity in cobalamin-limited cells was restored 20 hours after feeding cyanocobalamin or methionine to cobalamin-limited cells. Lowering of photosynthetic activity was due to loss of photosystem I activity. This photosynthetic activity was recovered after supplementation by methionine or cobalamin. The results suggest that methionine serves for the stabilization of photosystem I. This paper is the first report of the physiological function of cobalamin in chloroplasts of photosynthetic eukaryotes. PMID- 16665490 TI - Proton Transport in Plasma Membrane and Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : A Comparative Study of Ion Effects on DeltapH and DeltaPsi. AB - The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon DeltapH and DeltaPsi revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce DeltaPsi. For tonoplast vesicles, the relative order for this effect was KI > KBr >/= KCl > KClO(3) > K(2)SO(4) while for plasma membrane vesicles, a different order KI > KNO(3) >/= KBr >/= KClO(3) > KCl > K(2)SO(4) was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K(+)/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized. PMID- 16665491 TI - Inhibition of Spinach Leaf NADPH(NADH)-Glyoxylate Reductase by Acetohydroxamate, Aminooxyacetate, and Glycidate. AB - Acetohydroxamate (AHA) and aminooxyacetate (AOA) were found to be potent inhibitors of purified NADPH(NADH)-dependent glyoxylate reductase from spinach (Spinacia oleracea) leaves. AHA was a noncompetitive (ro mixed) inhibitor of the NADPH-dependent activity of the reductase with a K(i) of 0.33 millimolar. With NADH serving as a cofactor, AHA preferentially bound to the same form of the enzyme as glyoxylate, exhibiting a K(i) of 0.31 millimolar. Glycine hydroxamate and l-glutamic acid-gamma-hydroxamate were also inhibitory, but to a lesser extent than AHA. Inhibition by AOA (K(i) of 1.8 millimolar) was enhanced by increased concentrations of glyoxylate, indicating that the inhibitor preferentially reacted with the glyoxylate-bound form of the enzyme. Glycidate, an effector of glycolate metabolism in leaves, was found to be a much weaker inhibitor of the enzyme with a K(i) of 21 millimolar. While the inhibition by both AHA and AOA was fully reversible, glycidate acted as a tight-binding inhibitor. These findings are discussed with respect to the use of AHA, AOA, and glycidate as inhibitors of photorespiratory carbon metabolism in leaves. Caution is recommended in the use of these inhibitors with intact tissue experiments due to their lack of specificity. PMID- 16665492 TI - A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light. AB - This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH(4)Cl preincubation in the light. Treatment with NH(4)Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH(4)Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH(4)Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH(3), 2-OG)-dependent O(2) evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2 OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH(3) assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively. PMID- 16665493 TI - Isolation and Characterization of a Carotenoid-Associated Thylakoid Protein from the Cyanobacterium Anacystis nidulans R2. AB - A carotenoid-associated membrane protein was isolated from Anacystis nidulans R2 thylakoids. Sodium pyrophosphate and sodium bromide washed thylakoids were solubilized with the nonionic detergents dodecyl-beta-D-maltoside and octyl-beta D-glucopyranoside, and these detergent extracts were fractionated on a sucrose density gradient. A yellow fraction from the sucrose gradient was further purified by anion-exchange and organomercuric-affinity column chromatography to yield a fraction virtually free of chlorophyll and highly enriched in both carotenoids and a 42 kilodalton polypeptide. Evidence presented in this paper suggests that the carotenoid-containing 42 kilodalton protein is thylakoid associated rather than cytoplasmic membrane associated. The purified 42 kilodalton polypeptide was used to raise polyclonal antibodies in rabbits. Immuno chemical detection of the 42 kilodalton polypeptide on Western blots demonstrated an increased accumulation of this polypeptide in cells grown under high-light conditions relative to cells grown under low light. PMID- 16665494 TI - Relationship between Respiration and CAM-Cycling in Peperomia camptotricha. AB - Mature leaves of well-watered Peperomia camptotricha show Crassulacean acid metabolism (CAM). Young leaves show CAM-cycling in which CO(2) uptake occurs during the day concomitant with a marked diurnal fluctuation of organic acids as in CAM. Evidence is presented suggesting that respiration is the source of CO(2) for nocturnal acid synthesis in leaves exhibiting CAM-cycling. Respiratory quotients for these leaves were consistently much less than unity despite the fact that the leaves metabolize starch. The conservation of CO(2) by refixation into acids at night represents about 17% of the total photosynthetically fixed CO(2) and about 50% of the total respiratory CO(2). PMID- 16665495 TI - Metabolism of Monoterpenes : Metabolic Fate of (+)-Camphor in Sage (Salvia officinalis). AB - The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2 campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the beta-d-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1 carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-[G-(3)H]camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess beta-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2[U-(14)C]campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C(14) to C(20)) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid and isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots. PMID- 16665496 TI - Inhibition of the photosynthetic activities of isolated spinach chloroplasts by phosphonate compounds. AB - The effects of three closely related phosphonate compounds on several photosynthetic activities of isolated chloroplasts were investigated. Phosphonoformic and phosphonopropionic acid were found to inhibit both CO(2) fixation and the reduction of 3-phosphoglyceric acid, with CO(2) fixation being more sensitive. In contrast, phosphonoacetic acid was only slightly inhibitory. The lack of inhibition appeared to be due to its inability to enter the stroma via the phosphate translocator. Measurements of changes in stromal metabolite levels following the inhibition of CO(2) fixation by either phosphonoformic or phosphonopropionic acid indicated that the activity of ribulose bisphosphate carboxylase/oxygenase was reduced. Studies with the isolated enzyme confirmed that both of these compounds were effective competitive inhibitors of the carboxylase activity of the enzyme. PMID- 16665497 TI - A method for the separation and partial purification of the three forms of nitrate reductase present in wild-type soybean leaves. AB - A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC 1.6.6.2; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC 1.6.6.1) was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b(556) type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported. PMID- 16665498 TI - The Effect of Temperature on the Occurrence of O(2) and CO(2) Insensitive Photosynthesis in Field Grown Plants. AB - The sensitivity of photosynthesis to O(2) and CO(2) was measured in leaves from field grown plants of six species (Phaseolus vulgaris, Capsicum annuum, Lycopersicon esculentum, Scrophularia desertorum, Cardaria draba, and Populus fremontii) from 5 degrees C to 35 degrees C using gas-exchange techniques. In all species but Phaseolus, photosynthesis was insensitive to O(2) in normal air below a species dependent temperature. CO(2) insensitivity occurred under the same conditions that resulted in O(2) insensitivity. A complete loss of O(2) sensitivity occurred up to 22 degrees C in Lycopersicon but only up to 6 degrees C in Scrophularia. In Lycopersicon and Populus, O(2) and CO(2) insensitivity occurred under conditions regularly encountered during the cooler portions of the day. Because O(2) insensitivity is an indicator of feedback limited photosynthesis, these results indicate that feedback limitations can play a role in determining the diurnal carbon gain in the field. At higher partial pressures of CO(2) the temperature at which O(2) insensitivity occurred was higher, indicating that feedback limitations in the field will become more important as the CO(2) concentration in the atmosphere increases. PMID- 16665499 TI - Involvement of Reversible Inactivation in the Regulation of Nitrate Reductase Enzyme Levels in Chlamydomonas reinhardtii. AB - All nitrate reductase-related activities of Chlamydomonas reinhardtii wild-type and mutant 305 cells were degraded in vivo under conditions in which the reversible inactivation could take place. When the enzyme was in the inactive form, half-lives of all nitrate reductase-related activities in wild and mutant 305 strains decreased significantly. The only nitrate reductase-related activity present in mutant 104, nitrate reductase-diaphorase, was incapable of undergoing reversible inactivation and was not degraded under any of the conditions tested. Addition of nitrate to inactive nitrate reductase of mutant 305 caused the in vivo reactivation of the enzyme and halted its degradation. Our results indicate that reversibly inactivated nitrate reductase from C. reinhardtii is the main target for a degradation system, and that nitrate reductase related diaphorase must be integrated in a reversibly inactive nitrate reductase complex to undergo degradation. A physiological role for the interconversion process of nitrate reductase can be understood on the basis of these facts. PMID- 16665500 TI - Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs. AB - We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Deltapsi) could also be detected indirectly as (45)Ca(2+) uptake. Hmt toxin added to T mitochondria dissipated the steady state Deltapsi similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C(41)H(68)O(12) and Cpd IV: C(25)H(44)O(6)), reduced native toxin (RT2C: C(41)H(84)O(13)) and Pm toxin (band A: C(33)H(60)O(8), produced by the fungus, Phyllosticta maydis) were effective in dissipating Deltapsi and decreasing Ca(2+) uptake with the following order: Pm (100) >> HmT (23-30) > Cpd XIII (11-25) >> RT2C (0-4-1.8) > Cpd IV (0.2-1.0). In contrast, the toxins and analogs had no effect on Deltapsi formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C(41)H(68)O(13)) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Deltapsi in energized mitochondria is caused mainly by the electrogenic extrusion of H(+), the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H(+). The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H(+) leakage. PMID- 16665501 TI - Chilling sensitivity of cucumber cotyledon protoplasts and seedlings. AB - Cucumber (Cucumis sativus L.) seedlings are more sensitive to chilling stress when transferred to low temperature from the night cycle than from the day cycle. However, greater damage occurs when chilling is carried out in light than in dark. Freshly isolated protoplasts are extremely sensitive to damage when chilled at 4 degrees C in light, but suffer significantly less injury when chilled in dark. If freshly isolated protoplasts are pre-chill conditioned at 27 degrees C in either light or dark for a few hours prior to exposure to various chilling stresses, subsequent chilling damage is markedly reduced. Damage to chilled protoplasts also is reduced if cultures are placed in dark instead of light immediately following removal from low temperature. Experiments utilizing the cell wall synthesis inhibitor, dichlorobenzonitrile, showed that cell wall regeneration during the pre-chill conditioning period at 27 degrees C does not appear to be associated with the enhanced chilling tolerance observed in these cultures. The results obtained in this investigation suggest that the physiological properties of cucumber cotyledon protoplasts accurately reflect those of intact seedlings, and hence provide a good system for studies into the mechanism of chilling damage in plants. PMID- 16665502 TI - Biosynthesis of Sulfoquinovosyldiacylglycerol in Higher Plants: The Incorporation of SO(4) by Intact Chloroplasts in Darkness. AB - Intact spinach chloroplasts incorporated (35)SO(4) (2-) into sulfoquinovosyldiacylglycerol in the dark at rates equivalent to those previously reported for illuminated chloroplasts provided that either ATP itself or an ATP generating system was added. No additional reductant was necessary for SQDG synthesis by chloroplasts. The optimal concentration of ATP was between 2 and 3 millimolar. Rates of synthesis up to 2.6 nanomoles per milligram chlorophyll per hour were observed. UTP, GTP, and CTP could not substitute for ATP. Incubation of UTP with ATP (1:1) stimulated synthesis of sulfoquinovosyldiacylglycerol. No additional stimulation of the reaction was observed upon addition of other nucleoside triphosphates with ATP. For the generation of ATP in the chloroplast, addition of dihydroxyacetone phosphate alone did not promote synthesis of sulfoquinovosyldiacylglycerol, but in combination with inorganic phosphate and oxaloacetate, rates of synthesis up to 3.2 nanomoles per milligram chlorophyll per hour were observed. Dark synthesis was optimal in the presence of 2 millimolar dihydroxyacetone phosphate, 2 millimolar oxaloacetate, and 1 millimolar KH(2)PO(4). PMID- 16665503 TI - Purine Nucleoside Transport in Petunia Pollen Is an Active, Carrier-Mediated System Not Sensitive to Nitrobenzylthioinosine and Not Renewed during Pollen Tube Growth. AB - Adenosine and guanosine are transported into Petunia hybrida pollen by a saturable, carrier-mediated mechanism. The energy poisons carbonylcyanide-m chlorophenylhydrazone, 2,4-dinitrophenol, 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole, and N,N'-dicyclohexylcarbodiimide all inhibit uptake, suggesting an energy coupled (active) transport process. Transport takes place against a concentration gradient, strongly favoring an active transport mechanism. The purine nucleoside transport in Petunia pollen differs from that already reported for pyrimidine nucleosides in that it exhibits a significantly higher K(m) for nucleoside and is not so severely inhibited by the polyamine, spermine. Like that for the pyrimidine nucleosides uridine and cytosine, however, the system exhibits a broad pH optimum, is inhibited by sulfydryl-binding reagents, while the potent inhibitors of nucleoside transport in animal cells, nitrobenzylthioinosine and dipyridamole, have no effect. Transport of both purine and pyrimidine nucleosides in germinating pollen decreases steadily with time, a finding consistent with reports that RNA synthesis and DNA repair are early events of pollen germination and tube elongation. However, since these precursors are often used to demonstrate nucleic acid synthesis, it cannot be ruled out that the lack of precursor transport itself leads to scoring nucleic acid synthesis as negative. The results indicate that the newly synthesized pollen tube membranes contain little or no nucleoside transporters. PMID- 16665504 TI - Evaluation of Polyamine and Proline Levels during Low Temperature Acclimation of Citrus. AB - The polyamines (PA) putrescine (Put), spermidine (Spd), and spermine (Spm) were measured during 3 weeks exposure to cold hardening (15.6 degrees C day and 4.4 degrees C night) and nonhardening (32.2 degrees C day and 21.1 degrees C night) temperature regimes in three citrus cultivars: sour orange (SO) (Citrus aurantium L.), ;valencia' (VAL) (Citrus sinensis L. Osbeck), and rough lemon (RL) (Citrus jambhiri Lush). The changes in PA were compared to the amount of free proline, percent wood kill and percent leaf kill. A 2- to 3-fold increase in Spd concentrations were observed in hardened RL, SO, and VAL leaves compared to nonhardened leaves. Spermidine reached its highest level of approximately 200 nanomoles per gram fresh weight after 1 week of acclimation in both SO and VAL leaves, while RL spermidine content continued to increase up to the third week of acclimation. Spm levels in acclimated VAL and RL leaves increased 1- to 4-fold. However, SO leaves Spm content decreased with acclimation. Putrescine levels in SO and VAL increased 20 to 60% during the first 2 weeks of acclimation then declined after 3 weeks. RL putrescine content was not affected by cold acclimation. The data presented here provided direct relationship between increased Spd concentration and citrus cold hardiness. Free proline was 3- to 6 fold higher in acclimated than in nonacclimated trees. Results also demonstrate that in acclimated versus nonacclimated citrus trees the absolute amount rather than the ratio of increase in free proline is more important in predicting their ability to survive freezing stress. PMID- 16665505 TI - The Effect of Abscisic Acid and Other Inhibitors on Photosynthetic Capacity and the Biochemistry of CO(2) Assimilation. AB - Abscisic acid (ABA) was shown to reduce the photosynthetic capacity of a leaf through an apparent inhibition of ribulose 1,5-bisphosphate (RuBP) carboxylase (RuBPCase) activity, in addition to promoting stomatal closure. By comparison with the effects of other inhibitors of photosynthesis (cyanazine, methyl viologen, sodium azide, nigericin, sodium cyanide) on whole leaf photosynthesis, RuBPCase activity and metabolite pool sizes, it was demonstrated that the biochemical basis for the apparent effect of ABA on RuBPCase activity was not the result of reduced substrate availability, decarbamylation of the enzyme, or synthesis of carboxyarabinitol 1-phosphate, the naturally occurring tight-binding inhibitor of the enzyme. An inhibition of photosynthetic capacity showing the same biochemical characteristics as ABA-fed leaves was observed in plants grown under saline conditions. We suggest that the common link between environmental stress and reductions in photosynthetic capacity may be ABA. We hypothesize that ABA may affect plasma membrane function and thus indirectly RuBPCase activity through altered ion fluxes. The results of feeding cyanazine, methyl viologen, and nigericin provide additional evidence that regulation of RuBPCase activity by carbamylation/decarbamylation is related to the extent to which the capacity for ATP formation limits photosynthesis. PMID- 16665506 TI - Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants. AB - THE RATE AND SENSITIVITY TO INHIBITORS (KCN AND SALICYLHYDROXAMIC ACID[SHAM]) OF RESPIRATORY OXYGEN UPTAKE HAS BEEN INVESTIGATED IN PHOTOSYNTHETIC ORGANS OF SEVERAL FRESHWATER AQUATIC PLANT SPECIES: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O(2) uptake was also increased by the uncoupler. PMID- 16665507 TI - Expression of circaseptan and circannual rhythmicity in the imbibition of dry stored bean seeds. AB - Water uptake of seeds of Phaseolus vulgaris L. cv Limburg was monitored in a controlled atmosphere every day at solar noon for a period of 2 years. The imbibition rate, determined as the mean of five replicas, was related to the lunar cycle, maximal water uptake occurring between the new phases of the cycle. The monthly mean value of imbibition rate, calculated from the daily measurements, displayed a circannual rhythm reaching maximal values in August September and minimal values in February-March. These monthly values were positively correlated with the mean of the maximal temperatures recorded daily outside. PMID- 16665508 TI - A Mutant of Synechococcus PCC7942 Incapable of Adapting to Low CO(2) Concentration. AB - Some properties of a mutant (RK1) of Synechococcus PCC7942, which requires high CO(2) for growth, are described. The photosynthetic affinity for inorganic carbon (C(i)) in RK1 was about 40 times lower than that in the wild type (WT) when grown at 3% CO(2) (H-cells) and did not change during 10 hours of exposure to low CO(2) (air containing 0.04% CO(2)). The gas exchange of WT and RK1 cells was measured using an open gas-analysis system. All the measurements were performed at a CO(2) concentration of 400 microliters per liter under the conditions where photosynthetic CO(2) fixation is inhibited. When the suspension of H-cells of WT or RK1 was illuminated, the rate of CO(2) influx from the gas phase into the suspension was low and addition of carbonic anhydrase during illumination released only a small amount of CO(2) from the medium into the gas phase. The rate of CO(2) influx and the amount of CO(2) released by carbonic anhydrase were increased in WT during low CO(2) adaptation. These changes did not occur in RK1 during exposure to low CO(2). Cytoplasmic membrane from H-cells of WT or RK1 contained small amount of 42-kilodalton polypeptide. Exposure of RK1 to low CO(2) did not have significant effect on the amount of 42-kilodalton polypeptide, while the same treatment on WT resulted in a large increase of this polypeptide. The RK1 mutant appears to be defective in its ability to utilize the intracellular C(i) pool for photosynthesis and also to transmit a low CO(2) signal for inducing the functional and compositional changes observed in WT during low CO(2) adaptation. PMID- 16665509 TI - Seed Dormancy in Red Rice : VI. Monocarboxylic Acids: A New Class of pH-Dependent Germination Stimulants. AB - The weak acid character of many previously identified, but otherwise chemically dissimilar, dormancy-breaking compounds may contribute to their physiological activity. To test this idea, short chain monocarboxylic acids of one to six carbons, for which no previous reports of such activity exist, were incubated with dormant, dehulled red rice (Oryza sativa) seeds. Greater than 90% germination was observed after 24 hours of imbibition with 19 millimolar formic, 53 millimolar acetic, 20 millimolar propionic, 28 millimolar butyric, 20 millimolar valeric, or 16 millimolar caproic acid followed by 7 to 14 days incubation on water at 30 degrees C. Dormancy-breaking activity was pH-dependent. Incubation medium pH values that favored formation of the protonated species resulted in the highest germination percentages. There was no promotive effect of medium pH itself in the range of 3 to 7. In contrast, germination of intact seeds was less than 40% in the presence of 55 millimolar monocarboxylic acids at pH 3, unless seeds were partially dry-afterripened. The pH-dependent activity of these acids was maintained during afterripening of intact seeds. The results are consistent with the idea that the dissociable proton of weak acids is responsible for their dormancy-breaking activity. Many other weak acids may break seed dormancy but have been over-looked due to the rigid pH dependence necessary for activity. PMID- 16665510 TI - Vacuolar/Extravacuolar Distribution of Aminopeptidases in Giant Alga Chara australis and Partial Purification of One Such Enzyme. AB - The presence of two major aminopeptidases (aminopeptidases I and II) in the giant alga Chara australis was shown using polyacrylamide gel electrophoresis. Partially purified aminopeptidase I had a molecular weight of about 120,000, hydrolyzed both leucine-beta-naphthylamide (pH optimum 6.0) and alanine-beta naphthylamide (pH optimum 7.5), and was located both inside and outside the vacuole. Aminopeptidase I was inhibited by p-chloromercuribenzoic acid, iodoacetic acid, 1,10-phenanthroline, and N-tosyl-l-phenylalanine chloromethyl ketone. Aminopeptidase II hydrolyzed alanine-beta-naphthylamide but not leucine beta-naphthylamide and was located only outside the vacuole. PMID- 16665511 TI - Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues : Enhanced in Vitro Activity of the Enzyme in the Presence of Copper-Imidazole Complexes. AB - The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N(6)-(Delta(2)-isopentenyl)-adenine-2,8-(3)H (i(6) Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N(6)-side chain of i(6)Ade. PMID- 16665512 TI - Purification and characterization of ethylene inducing proteins from cellulysin. AB - Ethylene inducing proteins were partially purified and characterized from the cell wall digesting enzyme mixture, Cellulysin. Purification included binding to Sephacryl S-200, isoelectric focusing, molecular sieving on Sephadex G-75, agarose electrophoresis, and sizing using a Superose 12 column. At least three active proteins were obtained from the Sephadex G-75 fraction that move towards the cathode during nondenaturing agarose electrophoresis. These three protein fractions separated by preparative agarose electrophoresis contain polypeptide patterns that are very similar on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions contain three main Coomassie blue stained bands of about 10, 14, and 18 kilodaltons. Gel filtration of the major fraction on a Superose 12 column yields an active peak with an apparent molecular weight of 27,000. Proteolytic enzymes, in the presence of urea, destroy the ethylene inducing activity. We conclude that the ethylene inducing factor (EIF) that we have isolated from Cellulysin is protein. Similar ethylene inducing factors are present in Cellulase RS. Ethylene inducing components from pectinase, Pectolyase, and Rhozyme do not bind to Sephacryl like EIF from Cellulysin. Thus, the components responsible for the ethylene inducing activity in these latter enzyme preparations differ from that of EIF. PMID- 16665513 TI - Kinetics of l-Valine Uptake in Suspension-Cultured Cells and Protoplast-Derived Cells of Tobacco: Comparison of Wild-Type and the Val-2 Mutant. AB - A kinetic analysis was made of l-valine uptake in protoplast-derived cells (mesophyll protoplasts cultured for 6 days) and in suspension-cultured cells of tobacco (Nicotiana tabacum L., cv Xanthi). Cells from wild-type and Val(r)-2 mutant plants were compared. A low-K(m) component was found in protoplast-derived cells (K(m) = 45 +/- 5 micromolar) as well as in suspension-cultured cells (K(m) = 84 +/- 21 micromolar). In the mutant cells the V(max) of this component was 12- to 14-fold less than in wild-type cells. A second component (K(m) = 2.4 +/- 0.7 millimolar) was found in suspension-cultured cells but not in protoplast-derived cells; its V(max) was the same in wild-type and mutant cells. A third component was apparently unsaturable (linear component). It was present in protoplast derived cells but not in suspension-cultured cells, and had the same magnitude in wild-type and mutant cells. The results are discussed with reference to the uptake of l-valine in leaf tissue, in which the three kinetic components have been found simultaneously. The reduced V(max) of the low-K(m) component in the Val(r)-2 mutant, and the differential expression of the other two components in suspension-cultured cells and protoplast-derived cells indicate that the kinetically distinguishable components represent physically distinct transport systems. PMID- 16665514 TI - Developmental Control of CAM in Peperomia scandens. AB - Experiments were conducted to examine the development of photosynthetic carbon metabolism in Peperomia scandens, a tropical epiphyte. Leaves were sampled during a 10-day period when they were between 30 to 165 days old. P. scandens exhibits a C(3) to CAM-cycling to CAM shift during maturation with the magnitude of CAM increasing with age. Initially, during both day and night, no significant CO(2) uptake or diurnal acid flux was evident. C(3) gas exchange was detected at 41 days of age with a gradual shift towards CAM gas exchange maximized thereafter. An acidity flux of 130 to 150 microequivalents per gram fresh weight was evident by 41 days. Between 40 and 90 days, the leaves shifted their CO(2) uptake pattern from a daytime to a nighttime peak. After 90 days, the leaves remained in CAM. The delta(13)C values became progressively less negative as the leaves matured. In the 30-day-old leaves, the delta(13)C value was -21.1% while in the 165-day old leaves the delta(13)C value was -18.3%. The time-dependent shift from C(3) to CAM-cycling to CAM in P. scandens does not appear to result from changes in water, light, or temperature regimes since these variables were constant for all leaves sampled. PMID- 16665515 TI - Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves. AB - A new image instrumentation system for quantitative analysis of the rapid change in intensity of chlorophyll fluorescence during dark-light transition (CFI, chlorophyll fluorescence induction), which is a sensitive indicator of the various reactions of photosynthesis, was developed and its performance was evaluated. This system made it possible to resolve CFI at any small leaf area (about 1 square millimeter) of a whole leaf when the plant was illuminated by blue-green light at more than 50 micromoles photons per square meter per second. In order to test the usefulness of this system, we applied it to analyze the effect of SO(2) on photosynthetic apparatus in attached sunflower leaves. Dynamic CFI imaging over the whole single leaf, where there was no visible injury, indicated not only the local changes in photosynthetic activity but also the site of inhibition in photosynthetic electron transport system in chloroplasts. The new instrumentation system will be useful for the analytical diagnosis of various stress-actions on plants in situ. PMID- 16665516 TI - Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. AB - Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-[(14)C] fucose and UDP-[(14)C]xylose to xyloglucan occurred mainly in dictyosomeenriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with [(3)H]fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles. PMID- 16665517 TI - Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air Grown Chlamydomonas. AB - Inorganic carbon (C(i)) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO(2). Both air-grown cells, that have a CO(2) concentrating system, and 5% CO(2)-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C(i)) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO(2)-grown cells also accumulated some C(i), although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO(2) fixation by high CO(2)-grown, wild type cells at both pH 5.1 and 7.3. These observations imply that 5% CO(2)-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase. PMID- 16665518 TI - Acyl coenzyme a preference of diacylglycerol acyltransferase from the maturing seeds of cuphea, maize, rapeseed, and canola. AB - In their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations from maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of triacylglycerols from the acyl groups at 24, 32, and 40 degrees C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24 degrees C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40 degrees C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology. PMID- 16665519 TI - Relationship between Fructose 2,6-Bisphosphate and Carbohydrate Metabolism in Darkened Barley Primary Leaves. AB - Initial dark fructose 2,6-bisphosphate levels in 10-day-old barley (Hordeum vulgare L.) leaves increased when the photosynthetic period was lengthened, when the temperature during the prior photosynthetic period was reduced, and following leaf excision. These treatments also increased the leaf sucrose concentration. Conversely, a decrease in dark fructose 2,6,-bisphosphate occurred during extended darkness, with increasing leaf age and when photosynthate in the leaf was reduced by earlier low light treatments. These variations in fructose 2,6 bisphosphate content correlate with known changes in dark respiration. These findings suggest, but do not conclusively prove, a causal relationship between dark fructose 2,6-bisphosphate levels and dark respiration rates. PMID- 16665520 TI - Purification and Characterization of Two Benzoyl-l-Tyrosine p-Nitroanilide Hydrolases from Etiolated Leaves of Zea mays L. AB - Two benzoyl-l-tyrosine p-nitroanilide hydrolases (BTPAases I and II) were purified from the etiolated leaves of Zea mays L. and characterized. BTPAase I was electrophoretically homogeneous and consisted of two identical subunits having a molecular weight of 53,000. The molecular weight of BTPAase II was 65,000. The Michaelis constants for substrate, BTPA, were 4 millimolar and 1.3 millimolar for BTPAases I and II, respectively. Based on the action of various inhibitors on both enzyme activities, these enzymes were classified as serine proteases. BTPAase I showed caseinolytic activity at neutral pH and the activity was strongly inhibited by the serine protease inhibitors. PMID- 16665521 TI - Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron. AB - Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10(-8) molar and above, but had no inhibitory effects on growth at 10(-9) molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10(-8) molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as alpha-amino-n butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of alpha-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10(-4) molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10(4) times greater than that required to inhibit growth. Tracer studies with (15)N demonstrate that chlorsulfuron inhibits the incorporation of (15)N into valine, leucine, and isoleucine. The alpha-amino-n butyrate accumulated in the presence of chlorsulfuron and [(15)N]H(4) (+) was heavily labeled with (15)N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to alpha-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as alpha-amino-N donors. PMID- 16665522 TI - Determination of Betaines by Fast Atom Bombardment Mass Spectrometry : Identification of Glycine Betaine Deficient Genotypes of Zea mays. AB - A rapid, sensitive, and selective method for the determination of betaines is described and discussed. The method entails derivatizing the quaternary ammonium compounds to increase their sensitivity to detection by fast atom bombardment mass spectrometry. Sensitivity of detection increases markedly as the length of the carbon chain of the alcohol used to esterify the betaine carboxylic acid group is increased (C4 > C3 > C2 > C1 > C0). The lower limit of detection of glycine betaine as the n-propyl ester is 0.05 nanomole per microliter of glycerol. Betaine aldehyde can be readily derivatized to the di-n-butyl or di-n propyl acetal derivatives which exhibit lower limits of detection of about 5 picomoles and 10 picomoles per microliter of glycerol, respectively. Accurate quantification of these compounds is accomplished by the use of deuterium labeled internal standards or quaternary ammonium compound homologs of distinct mass. Methods for the synthesis of these internal standards are reported. Some applications of these methods are illustrated with stable isotope tracer studies on the kinetics of metabolism of choline to betaine aldehyde and glycine betaine in spinach leaf discs, and the identification of several Zea mays genotypes which appear deficient in glycine betaine. Tracer studies with deuterium labeled betaine aldehyde suggest that the deficiency of glycine betaine in one sweet corn hybrid is probably not due to a deficiency in the capacity to oxidize betaine aldehyde. PMID- 16665523 TI - Purification and properties of Acid phosphatase from plump and shriveled seeds of triticale. AB - A major triticale (X Triticosecale Wittmack) endosperm acid phosphatase (EC 3.1.2.2) (APase) from sib-lines producing plump and shriveled seed was purified 140- and 230-fold to a specific activity of 94 and 153 micromoles per minute per milligram protein respectively, by ammonium sulfate fractionation, ion-exchange chromatography, chromatofocusing, affinity column chromatography, and gel filtration. The purified enzyme from both materials is a monomeric glycoprotein with an apparent molecular weight of 45,700 +/- 500 containing 12% carbohydrate and an apparent isoelectric point of pH 5.9. It hydrolyzes tri- and di-phosphate of nucleosides as well as phosphate esters and exhibits characteristics of ATP hydrolase and phosphatase. About 2-fold more of the APase was isolated from shriveled seeds, and the purified enzyme exhibited 3- and 5-fold higher V(max) for p-nitrophenyl phosphate and ATP, respectively, than that of plump seed. The I(50) for Pi concentration was 5.5-fold higher in APase of shriveled seed than the plump one. These varied quantitative and kinetic properties substantiate the role of APase in lines with shriveled seeds being reduction of starch accumulation by depleting substrates and energy supply in the cytosol. PMID- 16665524 TI - Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. AB - Effects of varying light intensity and nitrogen nutrition on photosynthetic physiology and biochemistry were examined in the sun plant Phaseolus vulgaris (common bean) and in the shade plant Alocasia macrorrhiza (Australian rainforest floor species). In both Phaseolus and Alocasia, the differing growth regimes produced large changes in photosynthetic capacity and composition of the photosynthetic apparatus. CO(2)-saturated rates of photosynthesis were linearly related to leaf nitrogen (N) content in both species but photosynthesis per unit leaf N was markedly higher for Phaseolus than for Alocasia. Photosynthetic capacity was also higher in Phaseolus per unit ribulose 1,5-bisphosphate (RuBP) carboxylase (RuBPCase) protein. The leaf content of RuBPCase was linearly dependent on leaf N content in the two species. However, the proportion of leaf N which was RuBPCase was greater in Phaseolus than in Alocasia and was more sensitive to growth conditions, ranging from 6% of leaf N at low light to 20% at high light. In Alocasia, this range was much less, 6 to 11%. However, chlorophyll content was much more sensitive to light intensity in Alocasia. Thus, the RuBPCase/chlorophyll ratio was quite responsive to N availability and light intensity in both species (but for different reasons), ranging from 6 grams per gram for Phaseolus and 2 grams per gram for Alocasia at high leaf N and 1.5 gram per gram for Phaseolus and 0.5 gram per gram for Alocasia at low leaf N. These large changes in the proportions of components of the photosynthetic apparatus had marked effects on the sensitivity of these species to photoinhibition. These environmental effects also caused changes in the absolute levels of metabolites of the photosynthetic carbon reduction cycle. Concentrations of RuBP and P glycerate were approximately 2-fold higher in high light-grown than low light grown Phaseolus and Alocasia when expressed on a leaf area basis. However, if metabolite pool sizes are expressed on the basis of the RuBPCase catalytic site concentration, then they were little affected by the marked changes in leaf makeup. There appears to be fundamental differences between these species in the mechanism of sun-shade adaptation and N partitioning in the photosynthetic apparatus that result in significant differences in the N-use efficiency of photosynthesis between Phaseolus and Alocasia but similar RuBPCase:substrate:product ratios despite these differences. PMID- 16665525 TI - Equivalence between Pfr and Cyclic AMP in the Induction of d-Usnic Acid Dehydrogenase in the Lichen Evernia prunastri. AB - d-Usnic acid dehydrogenase is induced in Evernia prunastri thalli by a supply of exogenous d-usnic acid in light. This effect is enhanced by red light pulses through a two step way: a very rapid increase of activity after the first 10 minutes of red light, which is not reversed by far-red light, and a slow enhancement following successive red light pulses at the beginning of each hour of incubation. The last response is completely reversed by far-red following red light. Although induction of the enzyme is not achieved in the dark, 0.1 and 0.5 millimolar cyclic AMP, or 0.1 millimolar dibutyryl cyclic AMP substitutes light action and, then, the enzyme is produced. In addition, phytochrome-far red absorbing form-increases the amount of endogenously produced cyclic AMP and this effect is shown to be photoreversible when ethylenediaminetetraacetic acid is inhibiting adenylate cyclase. PMID- 16665526 TI - Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves. AB - Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q(B) polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves. PMID- 16665527 TI - Gas exchange characteristics of the sorghum-striga host-parasite association. AB - Gas exchange characteristics are reported for both members of the sorghum-Striga host-parasite association. Both Striga hermonthica (Del.) Benth and Striga asiatica (L.) Kuntze had transpiration rates considerably in excess of those of sorghum (Sorghum bicolor (L.) Moench, cv CSH1). Stomatal conductance in both Striga spp. showed little response to periods of darkness and moderate water stress. Low rates of net CO(2) fixation and high rates of dark respiration led to no net daily (24 hours) C gain, and Striga would appear to be reliant on its host for photosynthate. Infection of sorghum plants with either S. hermonthica or S. asiatica reduced host photosynthetic capacity. Infected sorghum plants were also more prone to water stress, but reduced rates of CO(2) fixation could not be accounted for in terms of lower stomatal conductance. Lower stomatal conductances were associated with an increase in water use efficiency (WUE) in uninfected sorghum; however, Striga-infected sorghum plants had lower WUE than those of uninfected plants. We suggest that Striga exerts a specific effect on processes affecting C acquisition in sorghum leaves. The water relations of S. hermonthica and S. asiatica are not characteristic of plants growing in semiarid environments and are more likely to reflect the nature of the parasitic life-style. Despite transfer of water and solutes from host to parasite, the reduction in C fixation observed in infected sorghum plants appears to be the major determinant of growth reductions observed in sorghum supporting Striga. PMID- 16665528 TI - A second extensin-like hydroxyproline-rich glycoprotein from carrot cell walls. AB - The insoluble extensin matrix of dicot cell walls has been studied most fruitfully by examining the salt-extractable precursors to this matrix. Multiple extensin-like hydroxyproline-rich glycoproteins (HRGPs) have been isolated, or their existence inferred, from tomato, potato, bean, soybean, melon, carrot, and other plants. We and others previously have studied a carrot extensin which we call extensin-1. Here we report on the properties of extensin-2, a second salt extractable hydroxyproline-rich glycoprotein from carrot. Like extensin-1, extensin-2 contains large amounts of hydroxyproline, serine, histidine, and lysine. In contrast, its tyrosine content is only about one-third that of extensin-1. Arabinose and galactose are the most abundant neutral sugars in both proteins, and nearly identical buoyant densities in CsCl suggest a similar proportion of carbohydrate in each. The size of extensin-2 is about half the size of extensin-1 based on: (a) the measured lengths of shadowed molecules (about 40 versus 84 nanometers); (b) the migration of extensin-2 in acid-urea gels relative to monomers, dimers, and trimers of extensin-1; and (c) the Stokes' radii of these molecules as determined by gel filtration chromatography. Electron microscopy of shadowed extensin-2 molecules indicates that they contain kinks, which may indicate the presence of intramolecular isodityrosine cross-links, but intermolecular cross-links, either with other extensin-2 molecules or extensin-1 molecules, are observed rarely if ever. PMID- 16665529 TI - Oxidation of indole-3-acetic Acid-amino Acid conjugates by horseradish peroxidase. AB - The stability of 21 amino acid conjugates of indole-3-acetic acid (IAA) toward horseradish peroxidase (HRP) was studied. The IAA conjugates of Arg, Ile, Leu, Tyr, and Val were oxidized readily by peroxidase. Those of Ala, beta-Ala, Asp, Cys, Gln, Glu, Gly, and Lys were not degraded and their recovery was above 92% after 1 hour incubation with HRP. A correlation between the stability of IAA conjugates toward peroxidase-catalyzed oxidation and the hydrophobicity of the amino acid moiety conjugated to IAA was demonstrated. Polar amino acid conjugates of IAA are more resistant to HRP-catalyzed oxidation. PMID- 16665530 TI - Ketose reductase activity in developing maize endosperm. AB - Ketose reductase (NAD-dependent polyol dehydrogenase EC 1.1.1.14) activity, which catalyzes the NADH-dependent reduction of fructose to sorbitol (d-glucitol), was detected in developing maize (Zea mays L.) endosperm, purified 104-fold from this tissue, and partially characterized. Product analysis by high performance liquid chromatography confirmed that the enzyme-catalyzed reaction was freely reversible. In maize endosperm, 15 days after pollination, ketose reductase activity was of the same order of magnitude as sucrose synthase activity, which produces fructose during sucrose degradation. Other enzymes of hexose metabolism detected in maize endosperm were present in activities of only 1 to 3% of the sucrose synthase activity. CaCl(2), MgCl(2), and MnCl(2) stimulated ketose reductase activity 7-, 6-, and 2-fold, respectively, but had little effect on NAD dependent polyol dehydrogenation (the reverse reaction). The pH optimums for ketose reductase and polyol dehydrogenase reactions were 6.0 and 9.0, respectively. K(m) values were 136 millimolar fructose and 8.4 millimolar sorbitol. The molecular mass of ketose reductase was estimated to be 78 kilodaltons by gel filtration. It is postulated that ketose reductase may function to metabolize some of the fructose produced during sucrose degradation in maize endosperm, but the metabolic fate of sorbitol produced by this reaction is not known. PMID- 16665531 TI - Investigation of the role of phosphorus in symbiotic dinitrogen fixation. AB - The interactive effects of phosphorus supply and combined nitrogen (nitrate) on dry matter and nitrogen accumulation by nodulated soybean (Glycine max L. Merr.) plants, and the relative effects of phosphorus supply on nodule number, mass, and function in comparison to host plant growth were used to investigate the role of phosphorus in symbiotic dinitrogen fixation. Mixed positive and negative phosphorus by nitrogen source interactions indicated that severe phosphorus deficiency markedly impaired both host plant growth and symbiotic dinitrogen fixation and that symbiotic dinitrogen fixation has a higher phosphorus requirement for optimal functioning than either host plant growth or nitrate assimilation. In the whole plant phosphorus concentration range of 0.8 to 1.5 grams per kilogram dry weight, plants supplied with 20 millimolar nitrate accumulated significantly more dry matter and nitrogen than symbiotic plants without nitrate. This suggested that the higher phosphorus requirement for symbiotic dinitrogen fixation is internal rather than being associated with differences in the ability of roots in the two nitrogen regimes to absorb phosphorus from the external solution. Increasing the phosphorus concentration in plants solely dependent on dinitrogen fixation resulted in highly significant (P = 0.0001) increases in whole plant nitrogen concentration as well as highly significant increases (P = 0.0001) in whole plant dry matter and nitrogen accumulation. This indicated a greater responsiveness of symbiotic dinitrogen fixation than of host plant growth to improvement in phosphorus nutrition. The large increases in whole plant nitrogen concentration were associated with about 3.5-fold increases in the ratio of nodule mass to whole plant mass and about 2 fold increases in specific acetylene reduction (nitrogenase) activity of the nodules. The large increase in nodule mass (>30-fold) between the 0 and 2.0 millimolar phosphorus levels resulted from 11- and 3-fold increases in nodule number per plant and average mass of individual nodules, respectively. Root mass per plant over the same concentration range increased 3.5-fold. These results indicate that phosphorus has specific roles in nodule initiation, growth, and functioning in addition to its involvement in host plant growth processes. PMID- 16665532 TI - An enzyme-linked immunosorbent assay (ELISA) for in vitro pollen growth based on binding of a monoclonal antibody to the pollen tube surface. AB - An indirect method of quantifying in vitro pollen growth has been developed. This is based on the finding that a monoclonal antibody (PCBC3), which has a primary specificity for alpha-l-arabinofuranosyl residues, binds to the surface of in vitro grown pollen tubes of the ornamental tobacco, Nicotiana alata. This binding was quantified using an enzyme linked immunosorbent assay (ELISA). The method was used to determine the effects of 2-deoxy-d-glucose and nonanoic acid on in vitro pollen growth. PMID- 16665533 TI - Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts. AB - A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3' chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system. PMID- 16665534 TI - Quantitation of the O(2)-Dependent, CO(2)-Reversible Component of the Postillumination CO(2) Exchange Transient in Tobacco and Maize Leaves. AB - The postillumination transient of CO(2) exchange and its relation to photorespiration has been examined in leaf discs from tobacco (Nicotiana tabacum) and maize (Zea mays). Studies of the transients observed by infrared gas analysis at 1, 21, and 43% O(2) in an open system were extended using the nonsteady state model described previously (Peterson and Ferrandino 1984 Plant Physiol 76: 976 978). Cumulative CO(2) exchange equivalents (i.e. nanomoles CO(2)) versus time were derived from the analyzer responses of individual transients. In tobacco (C(3)), subtraction of the time course of cumulative CO(2) exchange under photorespiratory conditions (21 or 43% O(2)) from that obtained under nonphotorespiratory conditions (1% O(2)) revealed the presence of an O(2) dependent and CO(2)-reversible component within the first 60 seconds following darkening. This component was absent in maize (C(4)) and at low external O(2):CO(2) ratios (i.e. <100) in tobacco. The size of the component in tobacco increased with net photosynthesis as irradiance was increased and was positively associated with inhibition of net photosynthesis by O(2). This relatively simple and rapid method of analysis of the transient is introduced to eliminate some uncertainties associated with estimation of photorespiration based on the maximal rate of postillumination CO(2) evolution. This method also provides a useful and complementary tool for detecting variation in photorespiration. PMID- 16665535 TI - Induction of Freezing Tolerance in Spinach during Cold Acclimation. AB - Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5 degrees C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10(5) counts per minute per microgram at 25 degrees C or 5 x 10(4) counts per minute per microgram at 5 degrees C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation. PMID- 16665536 TI - Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins. AB - Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5 degrees C. During cold acclimation at 5 degrees C and deacclimation at 25 degrees C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25 degrees C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25 degrees C were in vivo radiolabeled, without wounding or injury, to high specific activities with [(35)S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5 degrees C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M(r) of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M(r) 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5 degrees C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25 degrees C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M(r) 79,000, pI 4.8) similar to that of a HSP (M(r) 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights. PMID- 16665537 TI - Use of a light-induced respiratory transient to measure the optical cross section of photosystem I in chlorella. AB - A method has been developed whereby the magnitude of a transient in O(2) uptake attributable to photosystem (PS) I activity, following single-turnover laser flashes of varying energy, can be used to measure the optical cross section of PSI. As measurements are made under the identical physiological conditions for which the cross section of PSII has previously been determined (AC Ley, DC Mauzerall 1982 Biochim Biophys Acta 680: 96-105), it is now possible to simultaneously measure the cross section of both photosystems in intact, photosynthetically competent cells, without the use of inhibitors or artificial mediators of electron transport. Plots of light-saturation behavior of the respiratory oscillation following pulses at 596 nanometers indicate a mean optical cross section similar to that of PSII at this wavelength, but suggest significant heterogeneity in the cross section of PSI. If this method measures only PSI activity, this result implies that there exist units with different numbers of identical chromophores, or units having populations of chromophores with different absorption spectra. PMID- 16665538 TI - Azolla-Anabaena Relationship : XIII. Fixation of [N]N(2). AB - The major radioactive products of the fixation of [(13)N]N(2) by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [(13)N]N(2)-derived (13)NH(4) (+) after longer incubation periods was attributed to the spatial separation between the site of N(2)-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [(13)N]N(2), but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [(13)N]N(2)-derived (13)NH(4) (+), indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena. PMID- 16665539 TI - Inhibition by Salicylhydroxamic Acid, BW755C, Eicosatetraynoic Acid, and Disulfiram of Hypersensitive Resistance Elicited by Arachidonic Acid or Poly-l Lysine in Potato Tuber. AB - The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) was more persistent. These results are consistent with previous reports that SHAM and BW755C are reversible inhibitors of lipoxygenase and easily oxidized by potato while ETYA acts as an irreversible inhibitor. Disulfiram (tetraethylthiuram disulfide) also inhibited AA elicitor activity. SHAM was most effective if applied at the time of AA treatment, having no effect if applied 6 hours afterward. SHAM was effective in the presence of MES or MOPS buffers but not in acetate-buffered or unbuffered solutions; neither BW755C nor ETYA exhibited this restriction. However, SHAM, BW755C, and ETYA also were inhibitors of browning and sesquiterpene accumulation elicited in potato by poly-l-lysine, which, unlike AA, is not a lipoxygenase substrate. SHAM effectiveness also was restricted to 6 hours after treatment with poly-l-lysine. While the results with AA support a role for lipoxygenase, those with poly-l-lysine may be evidence that these compounds are having other effects in potato tissue. PMID- 16665540 TI - Osmotic dependence of the transmembrane potential difference of broadbean mesocarp cells. AB - Pod walls of broadbean (Vicia faba L. cv Aguadulce) were harvested at the import (S(1)), at the transition (S(2)) or at the export (S(3)) phase for assimilate transport. Measurements of the transmembrane potential difference (PD) of mesocarp cells were made under various osmotic conditions. Internal osmotic potentials and cell turgor were calculated from osmolality measurements of cell saps recovered by freeze-thawing, after correction for the contribution of the free-space solution. Changes in the mannitol concentration of the medium altered the PD within a few minutes, and new stable values of PD were reached within 20 minutes after the osmotic change. With mannitol as the osmoticum, the most negative PD was measured at an external osmotic potential of -0.70 megapascals (MPa) for S(1) and S(2), while the most negative was at -0.40 MPa for S(3). Ethylene glycol, a permeant osmoticum, had little effect on PD, showing that the PD was sensitive to turgor, not to solute potential per se. For S(1) and S(2), the PD was less negative for turgor potentials lower than 0.1 MPa or greater than 0.3 MPa. S(3) samples exhibited a different turgor dependence, with a sharp optimum of the negativity of the PD at 0.3 MPa. The data are consistent with the proposal that the proton pump acts as a transducer of the osmotic conditions. They show that the osmotic sensitivity of the PD of mesocarp cells of broadbean changes with the stage of development of the pod. PMID- 16665541 TI - Regulation of soybean nitrogen fixation in response to rhizosphere oxygen: I. Role of nodule respiration. AB - Nitrogen fixation (acetylene reduction) rates of nodules on intact field-grown soybean (Glycine max) subjected to altered oxygen concentration (0.06-0.4 cubic millimeter per cubic millimeter) returned to initial rates during an 8-hour transitory period. Hydroponically grown soybean plants also displayed a transitory (1-4 hours) response to changes in the rhizosphere oxygen concentration after which the fixation rates returned to those observed under ambient oxygen concentrations. It was hypothesized that soybean nodules contain a regulatory mechanism which maintains a stable oxygen concentration inside nodules at a sufficiently low concentration to allow nitrogenase to function. A possible physiological mechanism which could account for this regulation is adjustment in nodule respiration activity such that nodule oxygen concentration and nitrogen fixation are maintained at stable levels. Experiments designed to characterize the non-steady-state oxygen response and to test for the presence of nodule respiratory control are presented. Non-steady-state acetylene reduction and nodule respiration (oxygen uptake) rates measured after alterations in the external oxygen concentration indicated that the regulatory mechanism required 1 to 4 hours to completely adjust to changes in the external oxygen concentration. Steady-state nodule respiration, however, did not respond to alterations in the rhizosphere oxygen concentration. It was concluded that soybean nodules can adjust to a wide range of rhizosphere oxygen concentrations, but the mechanism which controls nitrogen fixation rates does not involve changes in the nodule respiration rate. PMID- 16665542 TI - Regulation of Soybean Nitrogen Fixation in Response to Rhizosphere Oxygen: II. Quantification of Nodule Gas Permeability. AB - Nodule nitrogen fixation rates are regulated by a mechanism which is responsive to the rhizosphere oxygen concentration. In some legumes, this oxygen-sensitive mechanism appears to involve changes in the gas permeability of a diffusion barrier in the nodule cortex. In soybean evidence for such a mechanism has not been found. The purpose of this research was to make quantitative measurements of soybean nodule gas permeability to test the hypothesis that soybean nodule gas permeability is under physiological control and responsive to the rhizosphere oxygen concentration. Intact hydroponically grown soybean plants were exposed to altered rhizosphere oxygen concentrations, and the nodule gas permeability, acetylene reduction and nodule respiration rates were repeatedly assayed. After a change in the external oxygen concentration, nitrogenase activity and nodule respiration rates displayed a short-term transient response after which the values returned to rates similar to those observed under ambient oxygen conditions. In contrast to steady-state nitrogenase activity and nodule respiration, nodule gas permeability was dramatically affected by the change in oxygen concentration. Decreasing the external oxygen concentration to 0.1 cubic millimeter per cubic millimeter resulted in a mean increase in nodule gas permeability of 63%. Increasing the rhizosphere oxygen concentration resulted in decreased nodule gas permeability. These data are consistent with the hypothesis that soybean nodules are capable of regulating nitrogen fixation and nodule respiration rates in response to changes in the rhizosphere oxygen concentration and indicate that the regulatory mechanism involves physiological control of the nodule gas permeability. PMID- 16665543 TI - Changes in Photosynthetic Capacity and Photosynthetic Protein Pattern during Tomato Fruit Ripening. AB - Levels of polypeptides participating in the photosynthetic light and dark reactions have been measured during fruit ripening in tomato. Photosynthetic proteins were identified by Western blot analysis with heterologous antibodies. The concentrations of proteins of photosystem (PS) I (14 kilodaltons), of PSII (47-kilodalton reaction center protein, 32-kilodalton ;Q(B) binding' protein and light harvesting complex proteins), of the photosynthetic electron transport chain (ferredoxin-NADP-oxidoreductase and plastocyanin), and of the stroma (ribulose-1,5-bisphosphate carboxylase) decrease during the ripening process. The 32-kilodalton protein and plastocyanin were detectable in pericarp protein preparations of ripe tomato fruits. Absorbance difference spectrophotometry provided information on the relative concentrations of PSII and PSI reaction centers in leaf and green fruit tissue of tomato. These results indicate that green fruit pericarp of tomato is photosynthetically active. Photosynthetic activity decreases during chloroplast/chromoplast differentiation. This is consistent with changes that occur at the transcript level of photosynthesis specific proteins during the differentiation process. PMID- 16665544 TI - Temperature-induced protein conformational changes in barley root plasma membrane enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility. AB - A protein spin label and lipid spin probes were used to study the temperature dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20 degrees C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12 degrees C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14 degrees C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes. PMID- 16665545 TI - Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane Enriched Microsomes: II. Intrinsic Protein Fluorescence. AB - The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase. PMID- 16665546 TI - Purification and species distribution of rubisco activase. AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms. PMID- 16665547 TI - Calmodulin mRNA in Barley (Hordeum vulgare L.) : Apparent Regulation by Cell Proliferation and Light. AB - Calmodulin is encoded by a 650-nucleotide mRNA in higher plants. This messenger was identified in barley and pea by a combination of in vitro translation and blot hybridization experiments using anti-sense RNA produced from an eel calmodulin cDNA probe. In all plant tissues tested, calmodulin mRNA represents between 0.01 and 0.1% of the total translatable mRNA population. Calmodulin mRNA levels are three- to fourfold higher in the meristematic zone of the first leaf of barley. At all other stages of leaf cell differentiation, calmodulin mRNA levels are nearly identical. During light-induced development in barley leaves, the relative proportion of translatable calmodulin mRNA declines about twofold. Cytoplasmic mRNAs that may encode calmodulin-like proteins were also detected. The levels of several of these putative Ca(2+)-binding protein mRNAs are modulated during the course of light-induced barley leaf cell development. PMID- 16665548 TI - C nuclear magnetic resonance study of acetate incorporation into malate during ca uptake by isolated leaf tissues. AB - (13)C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from (13)C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-(13)C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-(13)C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a (13)C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin. PMID- 16665549 TI - Nitrogenase Activity in Trifolium subterraneum L. in Relation to the Uptake of Nitrate Ions. AB - An experiment was conducted to test the hypothesis that, when nitrogenase and nitrate reductase both contribute to the nitrogen nutrition of a nodulated legume, nitrogenase activity is inversely proportional to the rate of accumulation of organic nitrogen derived from the reduction of nitrate. Trifolium subterraneum L. plants, inoculated with Rhizobium trifolii and sown as small swards, were allowed to establish a closed canopy and steady rates of growth, dinitrogen fixation, and nitrogen accumulation. Swards were then supplied with nutrient solutions of 0, 0.5, 1.0, or 2.5 mm NO(3) (-) with a 29.69% enrichment of (15)N and allowed to grow for a further 33 days. Harvests were made to measure dry weight, nitrogen accumulation, (15)N accumulation, NO(3) (-) content and nitrogenase activity by acetylene reduction assay. Since the (15)N of the plant organic matter could have been derived only from the NO(3) (-) of the nutrient solution, its rate of accumulation provided a measure of the rate of NO(3) (-) reduction. It was found that as this rate increased in response to external NO(3) (-) concentration the rate of nitrogenase activity decreased proportionately. It is concluded that the reduction of nitrate and the reduction of dinitrogen act in a complementary manner to supply a plant with organic nitrogen for growth. PMID- 16665550 TI - The Nitrogen Use Efficiency of C(3) and C(4) Plants: I. Leaf Nitrogen, Growth, and Biomass Partitioning in Chenopodium album (L.) and Amaranthus retroflexus (L.). AB - The effect of applied nitrogen (N) on the growth, leaf expansion rate, biomass partitioning and leaf N levels of Chenopodium album (C(3)) and Amaranthus retroflexus (C(4)) were investigated. At a given applied N level, C. album had 50% greater leaf N per unit area (N(a)) than A. retroflexus. Nitrate accumulated at lower N(a) in A. retroflexus than C. album. A. retroflexus was more productive than C. album at high N, but C. album was more productive at low N. At high applied N, nitrogen use efficiency (NUE), expressed either as net assimilation rate (NAR) per unit N or relative growth rate per unit N, was greater in A. retroflexus than C. album. However, at low applied N, C. album had a greater NUE on both an NAR and growth basis than A. retroflexus. The leaf area partitioning coefficient was similar in the species at high N, but was greater in A. retroflexus than C. album at low N. At low N, greater leaf area partitioning apparently lowered leaf N in A. retroflexus to levels at which necrosis occurred. In C. album by contrast, leaf area partitioning declined to a greater degree with declining N than it did in A. retroflexus, so that leaf N did not decline as much. Consequently, low N C. album plants did not lose leaf area to necrosis and had a greater NAR and NUE at low applied N than A. retroflexus. PMID- 16665551 TI - The Nitrogen Use Efficiency of C(3) and C(4) Plants: II. Leaf Nitrogen Effects on the Gas Exchange Characteristics of Chenopodium album (L.) and Amaranthus retroflexus (L.). AB - The effect of leaf nitrogen (N) on the photosynthetic capacity and the light and temperature response of photosynthesis was studied in the ecologically similar annuals Chenopodium album (C(3)) and Amaranthus retroflexus (C(4)). Photosynthesis was linearly dependent on leaf N per unit area (N(a)) in both species. A. retroflexus exhibited a greater dependence of photosynthesis on N(a) than C. album and at any given N(a), it had a greater light saturated photosynthesis rate than C. album. The difference between the species became larger as N(a) increased. These results demonstrate a greater photosynthetic N use efficiency in A. retroflexus than C. album. However, at a given applied N level, C. album allocated more N to a unit of leaf area so that photosynthetic rates were similar in the two species. Leaf conductance to water vapor increased linearly with N(a) in both species, but at a given photosynthetic rate, leaf conductance was higher in C. album. Thus, A. retroflexus had a greater water use efficiency than C. album. Water use efficiency was independent of leaf N in C. album, but declined with decreasing N in A. retroflexus. PMID- 16665552 TI - Isolation and Characterization of ABA-Insensitive Cell Lines of Carrot. AB - While abscisic acid (ABA) exerts multiple effects on somatic embryogenesis, the most pronounced of these effects is the arrestment of torpedo-stage embryos, preventing them from developing into plantlets. In order to understand the mechanism of ABA inhibition of plantlet formation, we have isolated seven ABA insensitive cell lines capable of developing into plantlets in the presence of ABA. These ABA-insensitive cell lines, whose frequency of appearance is 7 x 10( 6), have been isolated from a haploid cell line of Daucus carota L. var Juwarot. Surprisingly, all seven cell lines exhibit auxin insensitivity as evidenced by their ability to produce heart-stage embryos in various auxins including 2,4 dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid, and indolacetic acid. Three of the cell lines, ABA 1, ABA 15, and ABA 17, have been further characterized. We found that all three showed lower levels of ABA uptake which may be the cause of ABA insensitivity. However, the uptake of 2,4-D is higher in the three cell lines than in the wild type. The basis of the interaction between ABA and 2,4-D responses is discussed. PMID- 16665553 TI - Synthesis of the low molecular weight heat shock proteins in plants. AB - Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots. PMID- 16665554 TI - Regulation of plant morphology by growth retardants: effects on phytohormone levels in soybean seedlings determined by immunoassay. AB - The effects of the growth retardants tetcyclacis, a norbornenodiazetine, and LAB 150 978, a dioxanylalkenyl triazole, on seedling growth and endogenous levels of phytohormone-like substances in Glycine max L. cv Maple Arrow were studied. The levels of phytohormone-like substances in the root and in the various shoot tissues were analyzed by immunoassay. After seed treatment with both compounds, shoot growth was reduced more intensively than root growth. Both compounds decreased, on a fresh weight basis, the amount of various immunoreactive gibberellins when compared with the levels in control plants, especially in the shoot tip. Likewise, the growth retardants lowered the levels of abscisic acid like material, particularly in the primary leaf, the epicotyl and the root. In contrast, the levels of trans-zeatin-riboside and dihydrozeatin-riboside-type cytokinins were considerably elevated by the growth retardants, mainly in the primary leaf, epicotyl, and hypocotyl. On the other hand the level of isopentenyladenosine-like material was less influenced. In general, the immunoreactive 3-indoleacetic acid content in the different plant parts was changed only slightly. It is assumed that besides their effect on gibberellin content both compounds interfere directly or indirectly with the regulation of the endogenous levels of abscisic acid and cytokinins. This might be seen as an additional mode of action of growth retardants explaining some side effects on developmental processes of treated plants, e.g. delayed senescence and enhanced chlorophyll concentration in the leaves. PMID- 16665555 TI - Characteristics of a phosphatidylinositol exchange activity of soybean microsomes. AB - Microsome fractions from hypocotyls of dark-grown soybean (Glycine max [L.] Merrill) seedlings incorporated myo-inositol into phosphatidylinositol by an exchange reaction stimulated by Mn(2+) (optimum at 10 mm) and cytidine nucleotides (CMP = CDP approximately CTP) but not by Mg(2+) or nucleotides other than cytidine nucleotides. The activity was membrane associated, with an optimum pH of 8, stimulated by auxin, and inhibited by certain thiol reagents or by heating above 40 degrees C. With radioactive inositol, phosphatidylinositol was the only radioactive product. That turnover was by myo-inositol exchange was verified from experiments where unlabeled inositol replaced already incorporated inositol with approximately the same kinetics as for the incorporation of label. Both the incorporation and the displacement reactions were stimulated by Mn(2+) and CMP and both were responsive to auxin with comparable dose dependency. Corresponding exchange activities with choline or ethanolamine were not observed. The phosphatidylinositol-myo-inositol exchange activity was low or absent from plasma membrane, tonoplast, and mitochondria enriched fractions. The activity co localized on free-flow electrophoresis and aqueous two-phase partition with NADPH cytochrome c reductase and latent IDPase, markers for endoplasmic reticulum and Golgi apparatus, respectively. With microsomes incubated with both ATP and inositol, polyphosphoinositides were unlabeled demonstrating separate locations for the inositol exchange and phosphatidylinositol kinase reactions. Thus, the auxin-responsive inositol turnover activity of soybean membranes is distinct from the usual de novo biosynthetic pathway. It is not the result of a traditional D type phospholipase and appears not to involve plasma membrane-associated polyphosphoinositide metabolism. It most closely resembles previously described phosphatidylinositol-myo-inositol exchange activities of plant and animal endoplasmic reticulum. PMID- 16665556 TI - Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum. AB - The distribution of phosphoribulose kinase (PRK) in the cyanelles of Cyanophora paradoxa Korschikoff and Glaucocystis nostochinearum Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum against ribulose 1,5 bisphosphate carboxylase/oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extracts of C. paradoxa showed that 93% of the enzyme was in the soluble fraction. Quantitative immunoelectron microscopy showed that more than 99% of the PRK in the cyanelle of C. paradoxa was localized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algal chloroplasts do not contain phosphoribulose kinase. PMID- 16665557 TI - Interaction of cell turgor and hormones on sucrose uptake in isolated Phloem of celery. AB - Phloem tissue isolated from celery (Apium graveolens L.) was used to investigate the regulation of sucrose uptake by turgor (manipulated by 50-400 milliosomolal solutions of polyethylene glycol) and hormones indoleacetic acid (IAA) and gibberillic acid (GA(3)). Sucrose uptake was enhanced under low cellular turgor (increase in the V(max)). Furthermore, enhancement of sucrose uptake was due to a net increase in influx rates since sucrose efflux was not affected by cell turgor. Manipulations of cell turgor had no effect on 3-O-methyl glucose uptake. When 20 millimolar buffer was present in uptake solutions, low turgor-induced effects were observed only at low pH range (4.5-5.5). However, the effect was extended to higher external pH (up to 7.5) when buffer was omitted from uptake solutions. A novel interaction between cellular turgor and hormone treatments was observed, in that GA(3) (10 micromolar) and IAA (0.1-100 micromolar) enhanced sucrose uptake only at moderate turgor levels. The hormones elicited little or no response on sucrose uptake under conditions of low or high cell turgor. Low cell turgor, IAA, GA(3), and fusicoccin caused acidification by isolated phloem segments in a buffer-free solution. It is suggested that enhanced sucrose uptake in response to low turgor and/or hormones was mediated through the plasmalemma H(+)-ATPase and most likely occurred at the site of loading. PMID- 16665558 TI - Identification of polysaccharide hydrolases involved in autolytic degradation of zea cell walls. AB - Cell walls of Zea mays (cv L.G.11) seedlings labeled with (14)C were treated with alpha-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 m LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previously found to occur in cell walls during auxin induced extension. PMID- 16665559 TI - Effect of paclobutrazol on water stress-induced abscisic Acid in apple seedling leaves. AB - Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) in water-stressed leaves from control apple seedlings, and also from apple seedlings treated for 28 days with paclobutrazol ([2RS, 3RS]-1-[4-chlorophenyl] 4,4-dimethyl-2-[1,2,4-triazol-1-yl] pentan-3-ol). The ELISA quantitative estimates were also validated by gas chromatography-electron capture detector and lettuce seed germination inhibition bioassay. Paclobutrazol treatment reduced endogenous ABA levels by about one-third, and prevented the marked accumulation of water-stress-induced ABA that occurred in untreated seedlings. The presence of ABA in the apple leaf extracts was confirmed by gas chromatography-mass spectrometry. PMID- 16665560 TI - Effects of CO(2) and O(2) on Photosynthesis and Growth of Autotrophic Tobacco Callus. AB - Mean inhibition of net photosynthesis in autotrophic tobacco callus by 21 and 40% O(2) was 30 and 47%, respectively, similar to intact leaves. Increasing CO(2) concentrations (500-2000 microliters per liter) produced a steady decline in percent inhibition at both O(2) concentrations, indicating that O(2) inhibition resulted primarily from photorespiration. Net photosynthetic rate was plotted as a function of CO(2) concentration at 1, 21, and 40% O(2) for calculation of kinetic constants. Values for V(max) were similar at all O(2) concentrations (x = 5.27 mumol CO(2) per gram fresh weight per hour), indicating that O(2) inhibition of net photosynthesis was fully reversible by CO(2). To determine whether CO(2) and O(2) produced similar effects on growth, autotrophic callus was incubated for three weeks in atmospheres of normal air, high CO(2), high O(2) and high CO(2)/high O(2). Growth in high CO(2) was nearly double that in normal air. High O(2) decreased growth significantly relative to air, but growth in high CO(2)/high O(2) was similar to that in air. Lack of CO(2) reversal of growth inhibition by O(2) indicates that prolonged exposure to high O(2) results in toxicity arising from a nonphotorespiratory source. Growth of heterotrophic callus (2% sucrose), however, was not inhibited by 40 or 60% O(2), suggesting that O(2) toxicity in autotrophic callus results primarily from disruption of photosynthetic functions. PMID- 16665561 TI - Rapid phytochrome regulation of wheat seedling extension: light pretreatment extends coupling time, increases response lag, and decreases light sensitivity. AB - Rapid effects of light on wheat seedling extension growth were monitored by sensitive transducer techniques. Seedlings grown in complete darkness responded to light by a marked deceleration of extension growth after a mean latent period of 10.4 minutes. Pulses (5 minutes) of red (660 nanometers), green (530 nanometers), and far-red (730 nanometers) light caused marked extension rate depression, and the red effect could not be reversed by 730 nanometers far-red. Pulses of 1 second red (72 micromoles per square meter) were effective, and were reversible by immediate long wavelength (759 nanometers) far-red. Seedlings pretreated with 2 minute broadband green light (0.6 micromole per square meter), 28 hours prior to the experimental light treatments, displayed similar extension rate decelerations in response to red light, but after a longer mean lag of 23.75 minutes. No response was observed with red light treatments of less than 1 minute, and the effects of 5 minutes of red light were fully reversible by 5 minutes of 730 nanometers far-red. Fluence-response curves showed that nonpretreated seedlings were approximately 100 times more sensitive to far-red absorbing form of phytochrome than were those given prior light treatment. Although the fluence-response relationship for nonpretreated seedlings matched the photoconversion kinetics of phytochrome reasonably well, that for the pretreated seedlings indicated a requirement for repeated photoconversions for maximum action. The results are discussed in relation to the possibility that phytochrome may regulate the availability, or the activity, of a component of its own transduction chain. PMID- 16665562 TI - High-temperature sensitivity and its acclimation for photosynthetic electron transport reactions of desert succulents. AB - Photosynthetic electron transport reactions of succulent plants from hot deserts are able to tolerate extremely high temperatures and to acclimate to seasonal increases in temperature. In this study, we report the influence of relatively long, in vivo, high-temperature treatments on electron transport reactions for two desert succulents, Agave deserti and Opuntia ficus-indica, species which can tolerate 60 degrees C. Whole chain electron transport averaged 3 degrees C more sensitive to a 1-hour high-temperature treatment than did PSII (Photosystem II) which in turn averaged 3 degrees C more sensitive than did PSI. For plants maintained at day/night air temperatures of 30 degrees C/20 degrees C, treatment at 50 degrees C caused these reactions to be inhibited an average of 39% during the first hour, an additional 31% during the next 4 hours, and 100% by 12 hours. Upon shifting the plants from 30 degrees C/20 degrees C to 45 degrees C/35 degrees C, the high temperatures where activity was inhibited 50% increased 3 degrees C to 8 degrees C for the three electron transport reactions, the half times for acclimation averaging 5 days for A. deserti and 4 days for O. ficus indica. For the 45 degrees C/35 degrees C plants treated at 60 degrees C for 1 hour, PSI activity was reduced by 54% for A. deserti and 36% for O. ficus-indica. Acclimation leads to a toleration of very high temperatures without substantial disruption of electron transport for these desert succulents, facilitating their survival in hot deserts. Indeed, the electron transport reactions of these species tolerate longer periods at higher temperatures than any other vascular plant so far reported. PMID- 16665563 TI - Physiological Studies of a Synthetic Gibberellin-Like Bioregulator: II. Effect of Site of Application on Biological Activity. AB - The biological activity of the synthetic gibberellin agonist AC-94,377 (1-[3 chlorophthalimido]-cyclohexanecarboxamide) in certain plants is strictly dependent on the site of application. Root application of AC-94,377 at concentrations greater than or equal to 1 micromolar to seedlings of dwarf corn (Zea mays L. var d(5)), dwarf rice (Oryza sativa L. cv Tan-ginbozu), and sunflower (Helianthus annuus L. cv NK265) seedlings resulted in readily measurable gibberellin-like biological activity. Application of up to 10 micrograms of AC-94,377 to the shoots of these same species had no effect. AC 94,377 was metabolized to more polar products in both dwarf corn and sunflower seedlings. After 4 days of continuous root treatment with [(14)C]AC-94,377, greater than 70% of the recovered (14)C was found in the form of unmetabolized AC 94,377. In contrast, only 30 to 40% of the recovered (14)C was unmetabolized 4 days after shoot treatment. Translocation studies demonstrated that the movement of [(14)C]AC-94,377 was limited and occurred almost exclusively in an apoplastic fashion. Four days after leaf treatment, less than 1.5% (corn) or 4% (sunflower) of the recovered radioactivity had moved away from the treated area. It was concluded that the lack of biological activity of AC-94,377 following shoot treatment resulted principally from limited phloem mobility and to a lesser extent from accelerated metabolic breakdown. PMID- 16665564 TI - Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron. AB - Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map. PMID- 16665565 TI - Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea: Further Evidence on the Dimer-Tetramer Interconversion. AB - Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at -70 degrees C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage-which is composed of an equilibrium mixture of dimer and tetramer-with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. Thus the question of whether some covalent modification of the enzyme, such as phosphorylation, is required to permit complete changes in equilibrium remains open. PMID- 16665566 TI - pH Effects on the Activity and Regulation of the NAD Malic Enzyme. AB - The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg(2+) and NAD(+) but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate(-2). This is characteristic of an induced conformational change. The slope of the V(max) or V(max)/K(m) profiles is steeper on the alkaline side of the pH optimum. The K(m) for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K(m pH6.7) = 9.2 millimolar, K(m pH7.7) = 28.3 millimolar). Similar changes in K(m) have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V(max)/K(m) profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme. PMID- 16665567 TI - Influence of internal sugar levels on apoplasmic retrieval of exogenous sucrose in source leaf tissue. AB - Sugar levels in Beta vulgaris leaves were increased by heat-girdling the petiole and returning the plant to the controlled-environment chamber for 10 and 34 hours. After 10 hours, sucrose influx into the treated leaves was similar to the controls, although sucrose levels increased from 2.1 to 5.3 micromoles per milligram chlorophyll. However, after a 34-hour treatment, sucrose levels increased from 2.1 to 11.5 micromoles per milligram chlorophyll. In this instance, sucrose influx decreased relative to the untreated controls. Decreasing sugar levels by DCMU treatment resulted in a small stimulation of sucrose influx. A similar DCMU treatment applied to leaves of Allium cepa also resulted in an increase in sucrose influx. However, in A. cepa we could not attribute this increase to a lowering of sugar levels, as the kinetic profiles obtained from control leaves did not vary from each other throughout the day, despite considerable changes in sugar levels. Additionally, it appeared that sucrose uptake in onion may be set at some point and remains invariant throughout the day. Similar studies were also conducted on discs cut from mature leaves of Spinacia oleracea var America. Between 1 and 8 hours after the onset of the photoperiod, the sucrose content of the spinach leaves increased from 2.6 to 9.3 micromoles per milligram chlorophyll. A comparison of the kinetic profiles obtained from leaf discs, taken at these times, indicated that sucrose uptake was not influenced by these changes in internal sugar levels. The relationship between the above findings and ;trans' inhibition of exogenous sucrose uptake is discussed. Although intermediate changes in sugar levels in sugar beet leaves did not appear to affect sucrose influx, autoradiographic studies revealed that these changes dramatically affected the partitioning of exogenously supplied [(14)C]sucrose. Our results indicate that while intermediate changes in internal sugar levels have little effect on sucrose influx across the plasmalemma, they may dramatically affect partitioning between the phloem and the mesophyll vacuole. PMID- 16665568 TI - Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species. AB - Experiments were conducted with several Panicum species (representing the different C(4) subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C(4) > C(3)-C(4) intermediate > C(3). In illuminated leaves, SPS activities were similar among the three C(4) subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C(3) species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C(4) subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C(4) subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation. PMID- 16665569 TI - Partial Purification and Characterization of Uracil-DNA Glycosylase Activity from Chloroplasts of Zea mays Seedlings. AB - A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K(m) was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl(2), or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA containing organelles of eucaryotic cells. PMID- 16665570 TI - Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl. AB - The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers. PMID- 16665571 TI - Gibberellin-mediated synergism of xylogenesis in lettuce pith cultures. AB - Major gibberellins (GAs) in lettuce (Lactuca sativa L. cv Romaine) pith explants have been identified by gas chromatography-mass spectrometry (GC-MS) or GC selected ion monitoring (GC-SIM) as GA(1), 3-epi-GA(1), GA(8), GA(19), and GA(20). Treatment of pith explants with indole-3-acetic acid (IAA) (57 micromolar) plus kinetic (0.5 micromolar) induced xylogenesis. In this xylogenic treatment, the concentration of a biologically active, polar GA-like substance(s) increased during the first 2 days of culture, although all of the above GAs decreased (as measured by GC-SIM). In non-xylogenic treatments, where explants were cultured without exogenous hormones, or with IAA or kinetin alone, the concentration of the biologically active, polar GA-like substance(s) decreased during the first two days of culture, as did all of the above GAs (as measured by GC-SIM). Treatment of pith explants with exogenous GA(1) alone did not induce xylogenesis, but GA(1) at very low concentrations (0.0014 and 0.003 micromolar) synergized xylogenesis in the IAA plus kinetin-treated cultures. These results suggest that changes in the concentration of certain endogenous GAs may be involved in xylogenesis mediated by IAA plus kinetin in lettuce pith cultures. PMID- 16665572 TI - Gibberellins in Relation to Growth and Flowering in Pharbitis nil Chois. AB - Flowering can be modified by gibberellins (GAs) in Pharbitis nil Chois. in a complex fashion depending on GA type, dosage, and the timing of treatment relative to a single inductive dark period. Promotion of flowering occurs when GAs are applied 11 to 17 hours before a single inductive dark period. When applied 24 hours later the same GA dosage is inhibitory. Thus, depending on their activity and the timing of application there is an optimum dose for promotion of flowering by any GA, with an excessive dose resulting in inhibition. Those GAs highly promotory for flowering at low doses are also most effective for stem elongation (2,2-dimethyl GA(4) >> GA(32) > GA(3) > GA(5) > GA(7) > GA(4)). However, the effect of GAs on stem elongation contrasts markedly with that on flowering. A 10- to 50-fold greater dose is required for maximum promotion of stem elongation, and the response is not influenced by time of application relative to the inductive dark period. These differing responses of flowering and stem elongation raise questions about the use of relatively stable, highly bioactive GAs such as GA(3) to probe the flowering response. It is proposed that the ;ideal' GAs for promoting flowering may be highly bioactive but with only a short lifetime in the plant and, hence, will have little or no effect on stem elongation. PMID- 16665573 TI - Gibberellins, Endogenous and Applied, in Relation to Flower Induction in the Long Day Plant Lolium temulentum. AB - Changes in endogenous gibberellin-like substances (GAs) and related compounds in the shoot apices of Lolium temulentum during and after flower induction by one long day was examined for plants grown in three consecutive years. The total GA level in the shoot apical tissue was high (up to 42 micrograms per gram dry weight, or 3 x 10(-5) molar GA(3) equivalents), increasing several-fold on the day after the long day and then declining. Of the many GA-like substances present, the putative polyhydroxylated components-with HPLC retention times between those of GA(8) (three hydroxyls) and GA(32) (four hydroxyls), and accounting for about a quarter of the total GA activity-were most consistent and striking in their changes. Their level in the apices increased 3- to 5-fold on the day after the long day and then subsided. When various GAs were applied to plants in noninductive short days, flower initiation was induced by several, most notably by GA(32), GA(5), 2,2-dimethyl GA(4), GA(3), and GA(7). GA(32) was most like one long day in eliciting a strong flowering response while having little effect on stem growth, whereas GA(1) had the opposite effect. It is suggested that highly hydroxylated C-19 GAs may play a central role in the induction of flowering in this long-day plant. PMID- 16665574 TI - Interaction of indoleacetic Acid with its inositol and glucoside conjugates in Avena coleoptile curvature. AB - Avena coleoptile curvature is promoted by indoleacetic acid (IAA) IAA-glucoside, and IAA-inositol when these substances are applied in agar to the decapitated apical end of deseeded plantlets. Absorption of [(3)H]IAA-inositol over a wide range of concentrations during the 20 hour period of incubation is only 20 to 50% of the applied amount, compared with 85 to 92% of uptake of the applied [(3)H]IAA at equimolar concentrations. The absorption of IAA-glucoside could not be readily measured. The stimulation by both IAA-conjugates is very similar to that of free IAA at low concentrations (0.2 and 0.4 micromolar), but much less at higher concentrations. The interaction of free IAA with IAA-glucoside is additive or synergistic (depending on concentration). The interaction of free IAA with IAA inositol is an inhibition (i.e. less than additive). The simultaneous application of equimolar concentrations of free IAA does not change the chromatographic pattern of the metabolic products of [(3)H] IAA-inositol. One of the more polar metabolites of [(3)H]IAA-inositol has chromatographic characteristics similar to the major polar metabolite of free [(3)H]IAA on an isocratically eluted reversed phase C(18) high performance liquid chromatography system that separates a number of IAA sugar and amino acid conjugates from each other, and from free IAA. PMID- 16665575 TI - Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. AB - Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. PMID- 16665576 TI - Altered Gene Expression during Auxin-Induced Root Development from Excised Mung Bean Seedlings. AB - Changes in the pattern of protein synthesis and in the translatable mRNA population have been examined during auxin-induced root development from excised mung bean seedlings. Several proteins, predominantly of low molecular weight and high pI, as shown by two-dimensional polyacrylamide gel electrophoresis, are synthesized specifically by auxin-treated tissue. These auxin-induced proteins appear between 6 and 12 hours of auxin treatment, reach a maximum at 24 hours, and decline at 48 hours. Untreated seedlings (placed in Hoagland solution), known to produce small number of roots at the cut end probably due to endogeneous auxin accumulated at the cut end through basipetal transport, show low level synthesis of auxin-specific proteins. Antiauxin treatment that completely inhibits auxin induced rooting also prevents the appearance of auxin-induced proteins. The induction of a group of three to four proteins appears to be specific to antiauxin treatment. In vitro translation of mRNA from auxin-treated tissue, but not of mRNA from antiauxin-treated tissue, yields several polypeptides of low molecular weight and high pI. Since the auxin-induced proteins precede root development and are synthesized transitorily, it is likely that they play some regulatory role during the initiation of root development. The result show that auxin-induced root formation involves altered gene expression. PMID- 16665577 TI - Osmotic Adjustment in Cotton (Gossypium hirsutum L.) Leaves and Roots in Response to Water Stress. AB - The relative magnitude of adjustment in osmotic potential (psi(s)) of water stressed cotton (Gossypium hirsutum L.) leaves and roots was studied using plants raised in pots of sand and grown in a growth chamber. One and three water-stress preconditioning cycles were imposed by withholding water, and the subsequent adjustment in solute potential upon relief of the stress and complete rehydration was monitored with thermocouple psychrometers. Both leaves and roots exhibited a substantial adjustment in psi(s) in response to water stress with the former exhibiting the larger absolute adjustment. The osmotic adjustment of leaves was 0.41 megapascal compared to 0.19 megapascal in the roots. The roots, however, exhibited much larger percentage osmotic adjustments of 46 and 63% in the one and three stress cycles, respectively, compared to 22 and 40% in the leaves in similar stress cycles. The osmotically adjusted condition of leaves and roots decreased after relief of the single cycle stress to about half the initial value within 3 days, and to the well-watered control level within 6 days. In contrast, increasing the number of water-stress preconditioning cycles resulted in significant percentage osmotic adjustment still being present after 6 days in roots but not in the leaves. The decrease in psi(s) of leaves persisted longer in field-grown cotton plants compared to plants of the same age grown in the growth chamber. The advantage of decreased psi(s) in leaves and roots of water-stressed cotton plants was associated with the maintenance of turgor during periods of decreasing water potentials. PMID- 16665578 TI - Determination by Gas Chromatography-Mass Spectrometry of [N(5)]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue. AB - In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [(15)N(5)]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium ((2)H)-labeled internal standards. Incorporation levels of the [(15)N(5)]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5'-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-(14)C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-(14)C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of (14)C incorporation into cytokinins declined. PMID- 16665579 TI - Dynamic Relation between Expansion and Cellular Turgor in Growing Grape (Vitis vinifera L.) Leaves. AB - Measurements of the growth and water relations of expanding grape (Vitis vinifera L.) leaves have been used to determine the relationship between leaf expansion rate and leaf cell turgor. Direct measurement of turgor on the small (approximately 15 micrometer diameter) epidermal cells over the midvein of expanding grape leaves was made possible by improvements in the pressure probe technique. Leaf expansion rate and leaf water status were perturbed by environmentally induced changes in plant transpiration. After establishing a steady state growth rate, a step decrease in plant transpiration resulted in a rapid and large increase in leaf cell turgor (0.25 megapascal in 5 minutes), and leaf expansion rate. Subsequently, leaf expansion rate returned to the original steady state rate with no change in cell turgor. These results indicate that the expansion rate of leaves may not be strongly related to the turgor of the leaf cells, and that substantial control of leaf expansion rate, despite changes in turgor, may be part of normal plant function. It is suggested that a strictly physical interpretation of the parameters most commonly used to describe the relationship between turgor and growth in plant cells (cell wall extensibility and yield threshold) may be inappropriate when considering the process of plant cell expansion. PMID- 16665580 TI - Changes in Protein Patterns and Translatable Messenger RNA Populations during Cold Acclimation of Alfalfa. AB - Changes in the rate and pattern of protein synthesis and in translatable mRNA population during cold acclimation of alfalfa (Medicago falcata cv Anik) seedlings have been examined. There appears to be a positive correlation between the increase in ability to synthesize proteins at 4 degrees C and the increase in freezing resistance (survival at -10 degrees C). Results obtained with three different approaches using sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern visualized by (a) staining, (b) immunoblotting and autoradiography, and (c) fluorography of in vivo labeled proteins, show that at least eight polypeptides are newly synthesized during cold acclimation. Results of analysis of in vitro translation products of mRNA from nonacclimated and acclimated seedlings show the appearance of new translatable mRNAs. It is concluded that changes in gene expression occur during cold acclimation, most probably at the transcriptional level. PMID- 16665581 TI - Fluxes of h and k in corn roots : characterization and stoichiometries using ion selective microelectrodes. AB - We report here on an experimental system that utilizes ion-selective microelectrodes to measure the electrochemical potential gradients for H(+) and K(+) ions within the unstirred layer near the root surface of both intact 4-day old corn seedlings and corn root segments. Analysis of the steady state H(+) and K(+) electrochemical potential gradients provided a simultaneous measure of the fluxes crossing a localized region of the root surface. Net K(+) influx values obtained by this method were compared with unidirectional K(+) ((86)Rb(+)) influx kinetic data; at any particular K(+) concentration, similar values were obtained by either technique. The ionspecific microelectrode system was then used to investigate the association between net H(+) efflux and net K(+) influx. Although the computed H(+):K(+) stoichiometry is dependent upon the choice of diffusion coefficients, the values obtained were extremely variable, and net K(+) influx rarely appeared to be charge-balanced by H(+) efflux. In contrast to earlier studies, we found the cortical membrane potential to be highly K(+) sensitive within the micromolar K(+) concentration range. Simultaneous measurements of membrane potential and K(+) influx, as a function of K(+) concentration, revealed similar K(m) values for the depolarization of the potential (K(m) 6-9 micromolar K(+)) and net K(+) influx (K(m) 4-7 micromolar K(+)). These data suggest that K(+) may enter corn roots via a K(+)-H(+) cotransport system rather than a K(+)/H(+) antiporter. PMID- 16665582 TI - Factors affecting the level of kanamycin resistance in transformed sunflower cells. AB - A 230 base pair DNA segment containing the sequences 5' to the 700 to 750 nucleotide (nt) transcript 7' (ORF 3; RF Barker, KB Idler, DV Thompson, JD Kemp 1983 Plant Mol Biol 2: 335-350) of the octopine tumor inducing plasmid pTiA6 has been isolated. This region has (a) 180 base pairs of DNA upstream of the TATA box, (b) the start of RNA synthesis, and (c) the entire 5' untranslated region of the gene. We have fused this presumed promoter fragment to the neomycin phosphotransferase II (NPTII) gene from Tn5 in a plant expression cassette. After recombination into a tumor inducing plasmid delivery plasmid, this cassette confers selectable kanamycin resistance to transformed sunflower cells. Removal of the out-of-frame ATG in the 5' leader sequence of the NPTII gene by two different modifications increased both the levels of NPTII enzyme activity and the ID(50) for kanamycin in the tumor cells. The promoter region of the transcript 7 gene gives levels of kanamycin resistance equivalent to the nopaline synthase promoter and octopine synthase promoter when used in the same constructions and assayed in the same tissues. PMID- 16665583 TI - Flavone limitations to root nodulation and symbiotic nitrogen fixation in alfalfa. AB - Transcription of the nodABC genes in Rhizobium meliloti is required for root nodule formation in alfalfa (Medicago sativa L.) and occurs when specific compounds, such as the flavone luteolin, are supplied by the host plant. Results reported here indicate how luteolin in the root and rhizosphere can affect subsequent N(2) fixation and plant growth. Previous experiments with ;Hairy Peruvian 32' (HP32), an alfalfa population produced from ;Hairy Peruvian' (HP) by two generations of selection for increased N(2) fixation and growth, found that HP32 had more root nodules and fixed more N(2) than the parental HP population. In the present study, flavonoid extracts of HP32 seedling roots are shown to contain a 60% higher concentration of compounds that induce transcription of a nodABC-lacZ fusion in R. meliloti than comparable extracts of HP roots. Chromatographic data indicated that HP32 roots had a 77% higher concentration of luteolin than HP roots. Adding 10 micromolar luteolin to the rhizosphere of HP seedlings increased nodulation, N(2) fixation, total N, and total dry weight but had no effect on nitrate assimilation. These data show that normal levels of flavone nodulation signals in the rhizosphere of HP alfalfa can limit root nodulation, symbiotic N(2) fixation, and seedling growth and suggest that one mechanism for increasing N(2) fixation can be the genetic enhancement of specific biochemical signals which induce nodulation genes in Rhizobium. PMID- 16665584 TI - Intracellular localization of heat shock proteins in maize. AB - The intracellular distribution of the maize root heat shock proteins (hsp) was studied as a step toward understanding their physiological function. Linear sucrose density centrifugation was employed to separate organelles so the relative quantities of hsp in different subcellular compartments could be analyzed in a single preparation. Gradient fractions were assayed for the presence of hsp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and for marker enzyme activities. Analyses of 15 to 60% gradients showed five hsp to be organelle associated. Hsp 25 and 72 were in fractions containing closely equilibrating Golgi and endoplasmic reticulum marker activities, while hsp 18, 29, and 72 were in fractions containing overlapping plasma membrane, mitochondria, and glyoxysomal marker activities. Hsp larger than 72 kilodaltons were not present in gradient fractions. A second fractionation scheme achieved better separation of the two sets of closely equilibrating organelles. When a 13,000g centrifugation step to remove mitochondria was employed prior to gradient centrifugation, hsp 29 was absent from the gradient fractions. If the buoyant density of the endoplasmic reticulum was shifted by either maintaining the ribosomes on the membrane or removing them, a corresponding shift in the equilibrium positions of hsp 25 and 72 occurred. Hsp 18 and 70 remained in plasma membrane-containing fractions irrespective of these treatments. PMID- 16665585 TI - Acetaldehyde and ethanol biosynthesis in leaves of plants. AB - Leaves of terrestrial plants are aerobic organs, and are not usually considered to possess the enzymes necessary for biosynthesis of ethanol, a product of anaerobic fermentation. We examined the ability of leaves of a number of plant species to produce acetaldehyde and ethanol anaerobically, by incubating detached leaves in N(2) and measuring headspace acetaldehyde and ethanol vapors. Greenhouse-grown maize and soybean leaves produced little or no acetaldehyde or ethanol, while leaves of several species of greenhouse-grown woody plants produced up to 241 nanograms per milliliter headspace ethanol in 24 hours, corresponding to a liquid-phase concentration of up to 3 milligrams per gram dry weight. When leaves of 50 plant species were collected in the field and incubated in N(2), all higher plants produced acetaldehyde and ethanol, with woody plants generally producing greater amounts (up to 1 microgram per milliliter headspace ethanol concentration). Maize and soybean leaves from the field produced both acetaldehyde and ethanol. Production of fermentation products was not due to phylloplane microbial activity: surface sterilized leaves produced as much acetaldehyde and ethanol as did unsterilized controls. There was no relationship between site flooding and foliar ethanol biosynthesis: silver maple and cottonwood from upland sites produced as much acetaldehyde and ethanol anaerobically as did plants from flooded bottomland sites. There was no relationship between flood tolerance of a species and ethanol biosynthesis rates: for example, the flood intolerant species Quercus rubra and the flood tolerant species Quercus palustris produced similar amounts of ethanol. Cottonwood leaves produced more ethanol than did roots, in both headspace and enzymatic assays. These results suggest a paradox: that the plant organ least likely to be exposed to anoxia or hypoxia is rich in the enzymes necessary for fermentation. PMID- 16665586 TI - Alcohol Dehydrogenase and Pyruvate Decarboxylase Activity in Leaves and Roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.). AB - Pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) are responsible for the anaerobic production of acetaldehyde and ethanol in higher plants. In developing soybean embryos, ADH activity increased upon imbibition and then declined exponentially with development, and was undetectable in leaves by 30 days after imbibition. PDC was not detectable in soybean leaves. In contrast, ADH activity remained high in developing cottonwood seedlings, with no decline in activity during development. ADH activity in the first fully expanded leaf of cottonwood was 230 micromoles NADH oxidized per minute per gram dry weight, and increased with leaf age. Maximal PDC activity of cottonwood leaves was 10 micromoles NADH oxidized per minute per gram dry weight. ADH activity in cottonwood roots was induced by anaerobic stress, increasing from 58 to 205 micromoles NADH oxidized per minute per gram dry weight in intact plants in 48 hours, and from 38 to 246 micromoles NADH oxidized per minute per gram dry weight in detached roots in 48 hours. Leaf ADH activity increased by 10 to 20% on exposure to anaerobic conditions. Crude leaf enzyme extracts with high ADH activity reduced little or no NADH when other aldehydes, such as trans-2-hexenal, were provided as substrate. ADH and PDC are constitutive enzyme in cottonwood leaves, but their metabolic role is not known. PMID- 16665587 TI - Leaf magnesium alters photosynthetic response to low water potentials in sunflower. AB - We grew sunflower (Helianthus annuus L.) plants in nutrient solutions having nutritionally adequate but low or high Mg(2+) concentrations and determined whether photosynthesis was effected as leaf water potentials (psi(w)) decreased. Leaf Mg contents were 3- to 4-fold higher in the plants grown in high Mg(2+) concentrations (10 millimolar) than in those grown in low concentrations (0.25 millimolar). These contents were sufficient to support maximum growth, plant dry weight, and photosynthesis, and the plants appeared normal. As low psi(w) developed, photosynthesis was inhibited but moreso in high Mg leaves than in low Mg leaves. The effect was particularly apparent under conditions of light- and CO(2)-saturation, indicating that the chloroplast capacity to fix CO(2) was altered. The differential inhibition observed in leaves of differing Mg contents was not observed in leaves having differing K contents, suggesting that the effect may have been specific for Mg. Because Mg(2+) inhibits photophosphorylation and coupling factor activities at concentrations likely to occur as leaves dehydrate, Mg may play a role in the inhibition of chloroplast reactions at low psi(w), especially in leaves such as sunflower that markedly decrease in water content as psi(w) decreases. PMID- 16665588 TI - Water transport in maize roots : measurement of hydraulic conductivity, solute permeability, and of reflection coefficients of excised roots using the root pressure probe. AB - A root pressure probe has been used to measure the root pressure (P(r)) exerted by excised main roots of young maize plants (Zea Mays L.). Defined gradients of hydrostatic and osmotic pressure could be set up between root xylem and medium to induce radial water flows across the root cylinder in both directions. The hydraulic conductivity of the root (Lp(r)) was evaluated from root pressure relaxations. When permeating solutes were added to the medium, biphasic root pressure relaxations were observed with water and solute phases and root pressure minima (maxima) which allowed the estimation of permeability (P(Sr)) and reflection coefficients (sigma(sr)) of roots. Reflection coefficients were: ethanol, 0.27; mannitol, 0.74; sucrose, 0.54; PEG 1000, 0.82; NaCl, 0.64; KNO(3), 0.67, and permeability coefficients (in 10(-8) meters per second): ethanol, 4.7; sucrose, 1.6; and NaCl, 5.7. Lp(r) was very different for osmotic and hydrostatic gradients. For hydrostatic gradients Lp(r) was 1.10(-7) meters per second per megapascal, whereas in osmotic experiments the hydraulic conductivity was found to be an order of magnitude lower. For hydrostatic gradients, the exosmotic Lp(r) was about 15% larger than the endosmotic, whereas in osmotic experiments the polarity in the water movement was reversed. These results either suggest effects of unstirred layers at the osmotic barrier in the root, an asymmetrical barrier, and/or mechanical effects. Measurements of the hydraulic conductivity of individual root cortex cells revealed an Lp similar to Lp(r) (hydrostatic). It is concluded that, in the presence of external hydrostatic gradients, water moves primarily in the apoplast, whereas in the presence of osmotic gradients this component is much smaller in relation to the cell-to-cell component (symplasmic plus transcellular transport). PMID- 16665589 TI - beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism. AB - Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form beta-carotene from NaH(14)CO(3) or [U-(14)C]-3 phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-(14)C]-3-PGA or 3 to 6 millimolar NaH(14)CO(3) was fully sufficient to supply beta-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles (14)C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the beta-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO(2) to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute beta-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis. PMID- 16665590 TI - Acclimation of Ribulose Bisphosphate Carboxylase and mRNAs to Changing Irradiance in Adult Tobacco Leaves: Differential Expression in LSU And SSU mRNA. AB - The transfer of Nicotiana tabacum plants grown in low light (60 micromoles quanta per square meter per second) to higher light (360 micromoles quanta per square meter per second) was previously shown to induce adaptive stimulation of photosynthetic capacities. The variations of ribulose bisphosphate carboxylase/oxygenase (RubisCo) expression in mature leaves was examined as a result of this acclimation. Maximum or initial activities increased markedly after low- to high-light transfer with a maximum effect after 2 to 3 days. The higher activity is mainly explained by RubisCo protein synthesis as shown by immunorocket technique. Small subunits of RubisCo (SSU) mRNA relative content determined by hybridization of total RNA with DNA probe by Dot-blot method, followed the same pattern as RubisCo quantity. The magnitude of this response was amplified when more contrasting light conditions (25 versus 360 micromoles per square meter per second) were established on the same leaf: RubisCo activity, RubisCo protein, and SSU mRNA contents decreased in the shaded zone and increased in the high-light zone within 1 day. After 2 days the shade/light ratio was 1 to 3 for RubisCo protein and 1 to 4 for SSU-RNA, whereas the ratios remained equal to one in controls. Hybridization of the same RNA extracts with large subunits of RubisCo (LSU) probe showed no variation in LSU-RNA content. So in green adult leaves, the expression of SSU and LSU genes is regulated differently. The observed white light quantitative effect on RubisCo expression was not dependent on the photosynthetic rate or assimilate content since low CO(2) concentration around the leaf after the light shift did not modify the response. PMID- 16665591 TI - Influence of leaf age on photosynthesis, enzyme activity, and metabolite levels in wheat. AB - The rate of photosynthesis under high light (1000 micromole quanta per square meter per second) and at 25 degrees C was measured during development of the third leaf on wheat plants and compared with the activity of several photosynthetic enzymes and the level of metabolites. The rate of photosynthesis reached a maximum the 7th day after leaf emergence and declined thereafter. There was a high and significant correlation between the rate of photosynthesis per leaf area and the activities of the enzymes ribulose 5-phosphate kinase (r = 0.91), ribulose 1,5-bisphosphate (RuBP) carboxylase (r = 0.94), 3 phosphoglycerate (PGA) kinase (r = 0.82), and fructose 1,6-bisphosphatase (r = 0.80) per leaf area. There was not a significant correlation of photosynthesis rate with chlorophyll content. The rate of photosynthesis was strongly correlated with the level of PGA (r = 0.85) and inversely correlated with the level of triose phosphate (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) (r = 0.92). RuBP levels did not change much during leaf development; therefore photosynthesis rate was not correlated with the level of RuBP. The rate of photosynthesis was at a maximum when the ratio of PGA/triose phosphate was high, and when the ratio of RuBP/PGA was low. Although several enzymes change in parallel with leaf development, the metabolite changes suggest the greatest degree of control may be through RuBP carboxylase. The sucrose content of the leaf was highest under high rates of photosynthesis. There was no evidence that later in leaf development, photosynthesis (measured under high light and at 25 degrees C) was limited by utilization of photosynthate. PMID- 16665592 TI - Photosynthesis by intact isolated chloroplasts on solid support. AB - A new approach to measurements of photosynthesis by isolated chloroplasts has been devised. Intact isolated chloroplasts were trapped in the cavities of membrane filters. The thin layers of chloroplasts so obtained were assayed for O(2) evolution and CO(2) assimilation in leaf-chambers. Photosynthetic gas exchange could be demonstrated to take place either in a closed or a flow-through system. The chloroplasts were morphologically intact as shown by light or scanning electron microscopy and displayed stable rates of photosynthesis in the presence of phosphate and alkaline phosphatase. The methods described open the way to in vitro measurement of photosynthesis, by chloroplasts under conditions more closely resembling those in leaves. PMID- 16665593 TI - Control of the Photosynthetic Apparatus of Acetabularia mediterranea by Blue Light : Analysis by Light-Saturation Curves. AB - During growth, Acetabularia mediterranea requires the action of blue light to maintain high rates of photosynthesis. In the present study, blue light-dependent alterations of the photosynthetic apparatus, which can be detected by analysis of light-saturation curves and by measurements of partial reactions of the photosynthetic electron transport chain, are described. Light-saturation curves of photosynthesis in vivo were measured with a new closed oxygen electrode system after culture of Acetabularia in continuous red or blue light. These curves were compared to those of 2,6-dichlorophenol-indophenol reduction by isolated chloroplast membranes. The analysis lead to the following statements: (a) only one reaction limits electron transport rates in vitro (dichlorophenol-indophenol reduction) at all light intensities irrespective of the light quality during growth, and (b) the limiting step is light driven and located in the reaction center of photosystem II. Presumably, this same reaction determines the flow of electrons under low light intensities in vivo in cells from white, blue, and red light. In addition to photosynthesis, the rates of dark respiration changed due to the action of blue light. Concomitantly, the light compensation point of apparent photosynthesis was shifted during monochromatic irradiations. PMID- 16665594 TI - Iron Specifically Protects Corn Protoplasts from T-Toxin of Cochliobolus heterostrophus. AB - Ferric ion reduced the damaging effects of T-toxin, a series of linear beta polyketols produced by the pathogenic fungus Cochliobolus heterostrophus, on leaf mesophyll protoplasts from susceptible T-cytoplasm corn. Of nine metals tested, only ferric and ferrous ions had this effect. Despite the presence of 12 available oxygen atoms in each T-toxin molecule, there was no evidence for the formation of an aqueous Fe(2+)- or Fe(3+)-T-toxin complex. The protective effect of iron was eliminated by a molar excess of EDTA. Iron had no effect on the sensitivity of T-cytoplasm mitochondria to T-toxin, even at a 1000-fold molar excess, nor did it protect roots of T-cytoplasm corn seedlings from inhibition by T-toxin. The mechanism by which iron specifically protects protoplasts from T toxin is not understood, but time lapse experiments suggest that iron acts on some intracellular site to modify T-toxin sensitivity and not on a transport system at the cell surface. PMID- 16665595 TI - Maize root growth and localized indol-3yl-acetic Acid treatment: a new methodological approach. AB - Resin beads loaded with indol-3yl-acetic acid (IAA) were used as asymmetrical donors along the elongation zone of intact primary Zea mays L. roots. A strong curvature, towards and above the bead, occurred when IAA was applied at a mean distance of 2.20 mm from the tip. No curvature was detected after applications at 3.89 and 5.71 mm from the tip. Correspondence analysis, a new methodological approach in plant hormone studies, permitted the evaluation of the relative influence of several factors on the curvature observed for each root. The parameters considered were the initial growth rate, the exact location of the bead (1.64-2.73 millimeters from tip) and the quantity of IAA absorbed. Roots which grew rapidly bent earlier than slowly growing ones and the more basal the treatment was, the less curvature occurred. Surprisingly, the amount of IAA taken up (between 1.2 and 2.2 times the endogenous IAA content) was found to have no influence on either the time-course or the magnitude of this growth inhibition (curvature). The usefulness of this multivariate analysis is also discussed. PMID- 16665596 TI - Salt Stress Increases the Level of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Mesembryanthemum crystallinum. AB - Mesembryanthemum crystallinum responds to salt stress by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM). During this transition the activity of phosphoenolpyruvate carboxylase (PEPCase) increases in soluble protein extracts from leaf tissue. We monitored CAM induction in plants irrigated with 0.5 molar NaCl for 5 days during the fourth, fifth, and sixth week after germination. Our results indicate that the age of the plant influenced the response to salt stress. There was no increase in PEPCase protein or PEPCase enzyme activity when plants were irrigated with 0.5 molar NaCl during the fourth and fifth week after germination. However, PEPCase activity increased within 2 to 3 days when plants were salt stressed during the sixth week after germination. Immunoblot analysis with anti-PEPCase antibodies showed that PEPCase synthesis was induced in both expanded leaves and in newly developing axillary shoot tissue. The increase in PEPCase protein was paralleled by an increase in PEPCase mRNA as assayed by immunoprecipitation of PEPCase from the in vitro translation products of RNA from salt-stressed plants. These results demonstrate that salinity increased the level of PEPCase in leaf and shoot tissue via a stress induced increase in the steady-state level of translatable mRNA for this enzyme. PMID- 16665597 TI - Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate. AB - Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID(50) concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes. PMID- 16665598 TI - Sucrose phosphatase associated with vacuole preparations from red beet, sugar beet, and immature sugarcane stem. AB - The specific phosphatase, sucrose phosphate phosphohydrolase (sucrose phosphatase, EC 3.1.3.24) was present in vacuole preparations from storage tissue of red beet (Beta vulgaris L.), sugar beet (Beta vulgaris L. cultivar Kawemono), and immature sugarcane (Saccharum spp. hybrid, cultivar NCO 310). In red beet vacuole preparations the specific activity of sucrose phosphatase, using the naturally occurring vacuole marker, betanin, as reference, was higher than the specific activity of cytoplasmic markers, phosphoenolpyruvate carboxylase and glucose 6-phosphate dehydrogenase, suggesting that sucrose phosphatase is associated with the vacuoles. High speed centrifugation of lysed vacuoles did not result in precipitation of the enzyme indicating that the enzyme is not tightly bound to the tonoplast. Sucrose phosphatase was more sensitive to inhibition by sodium vanadate and less sensitive to ammonium molybdate than was the nonspecific phosphatase which was also present in the extracts. Sucrose phosphatase might be part of the group translocator proposed recently to operate in the tonoplast of sugarcane and red beet. PMID- 16665599 TI - Induction of defense responses in cultured parsley cells by plant cell wall fragments. AB - Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased beta-1,3-glucanase activity when treated with either a purified alpha-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense. PMID- 16665600 TI - Responses of Rj(1) and rj(1) Soybean Isolines to Inoculation with Bradyrhizobium japonicum. AB - We evaluated the symbiotic phenotypes of nodulation-restrictive and normal soybean isolines by inoculating Clark (genotypically Rj(1)Rj(1)) and mutant Clark rj(1) (genotypically rj(1)rj(1)) seedlings in plastic growth pouches. Nodules first appeared on Clark seedlings inoculated with Bradyrhizobium japonicum USDA 94 after 6 days. The mean number of nodules per plant was 13.9 +/- 0.8 after 24 days. In contrast, Clark-rj(1) seedlings first nodulated at 12 days, and the mean number of nodules per plant was only 1.7 +/- 0.3 at 24 days. Segments from infectible zones of primary roots, i.e. near the position occupied by the root tip at the time of inoculation, were sectioned serially. Clark roots contained cortical cell divisions and a few infection threads in question mark-shaped root hairs by 2 days after inoculation. Typical nodules developed soon thereafter. Analogous serially sectioned segments from Clark-rj(1) roots lacked these responses. This prompted us to section nodules and adjacent tissues from other parts of Clark and Clark-rj(1) roots. Clark roots contained cortical cell divisions, many associated with infected root hairs. Cortical cell divisions occasionally were present in Clark-rj(1), and a few infection threads were visible in surface cells. The presence of infection threads within Clark-rj(1) nodules was confirmed by transmission electron microscopy. Thus, although B. japonicum USDA 94 fails to elicit the wild-type spectrum of responses in the infectible zones of primary roots, it can infect Clark-rj(1) via infection threads. PMID- 16665601 TI - The Linamarin beta-Glucosidase in Costa Rican Wild Lima Beans (Phaseolus lunatus L.) Is Apoplastic. AB - Analysis of mesophyll protoplasts and cell wall extracts of leaf discs of Costa Rican wild lima bean (Phaseolus lunatus L.) shows that the linamarase activity is confined to the apoplast. Its substrate linamarin, together with the related enzyme hydroxynitrile lyase, is found inside the cells. This compartmentation prevents cyanogenesis from occurring in intact tissue, and suggests that linamarin has to be protected during any translocation across the linamarase rich apoplast. PMID- 16665602 TI - Substrate Specificities of N-Acetylglucosaminyl-, Fucosyl-, and Xylosyltransferases that Modify Glycoproteins in the Golgi Apparatus of Bean Cotyledons. AB - As part of their posttranslational maturation process, newly synthesized glycoproteins that contain N-linked oligosaccharide side chains pass through the Golgi apparatus, where some of their oligosaccharides become modified by carbohydrate processing reactions. In this paper, we report the presence of Golgi localized enzymes in plant cells (Phaseolus vulgaris cotyledons) that transfer GlcNAc, fucosyl, and xylosyl residues to the oligosaccharide side chains of glycoproteins. All three enzyme activities are involved in the transformation of high mannose side chains into complex glycans. As judged by acceptor specificity studies, at least two GlcNAc residues can be added to the nonreducing side of high mannose oligosaccharides, which have been trimmed by alpha-mannosidase(s). A Man(5)(GlcNAc)(2)-peptide serves as the acceptor for the first GlcNAc added. The second GlcNAc can be added only after the prior removal of two additional mannose residues, ultimately yielding (GlcNAc)(2)Man(3)(GlcNAc)(2)-peptide. Fucosyltransferase can transfer fucose to GlcNAcMan(5)(GlcNAc)(2)Asn, GlcNAcMan(3)(GlcNAc)(2)Asn, and (GlcNAc)(2)Man(3)(GlcNAc)(2)Asn; xylosyltransferase exhibits significant activity toward the latter two substrates only. These results suggest an overlapping sequence of oligosaccharide modification in the Golgi apparatus that, in regard to GlcNAc and fucose additions, is analogous to pathways of oligosaccharide processing reported for animal cells. To our knowledge, this is the first report characterizing a xylosyltransferase involved in N-linked oligosaccharide modification, an activity that is apparently absent in most animal cells. PMID- 16665603 TI - Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L. AB - Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO(2) exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine. PMID- 16665604 TI - Nitrogen Enhancement of Phosphate Transport in Roots of Zea mays L. : I. Effects of Ammonium and Nitrate Pretreatment. AB - The effect of nitrogen status on phosphorous uptake and translocation was examined in 6-day-old dark-grown decapitated maize seedlings exposed to 25 micromolar phosphorous. Transfer to complete solutions containing 1 millimolar ammonium resulted in an increase in phosphorous uptake rate after 6 to 8 hours. The stimulus remained effective for at least 5.5 hours upon subsequent transfer to nitrogen-free solutions. Pretreatments for 16 hours with either nitrate or ammonium resulted in enhanced rates of subsequent phosphorous uptake and in enhanced translocation to the xylem of the exogenously supplied phosphorous. Both processes reached a plateau following pretreatment with 0.1 to 1.0 millimolar concentrations of either nitrogen ion. Further enhancement occurred with 10 millimolar nitrate, but not with 10 millimolar ammonium pretreatment. Although nitrogen pretreatments slightly increased the quantity of exogenous phosphorous retained in the root tissue, most of the extra phosphorous taken up by the nitrogen-pretreated seedlings was translocated to the xylem. The enhanced translocation, however, did not totally account for the increase in uptake implying a specific stimulation of the uptake process. PMID- 16665605 TI - Nitrogen Enhancement of Phosphate Transport in Roots of Zea mays L: II. Kinetic and Inhibitor Studies. AB - Exposure to 1 millimolar ammonium pretreatments increased V(max) for phosphorous uptake in dark-grown decapitated maize seedlings without a statistically measurable change in K(m). Sulfate uptake also was stimulated. The stimulation in phosphorous uptake due to ammonium pretreatment was greater in seedlings grown without phosphorous than in those grown with 25 micromolar phosphorous. The stimulus was not expressed unless the entire root system was pretreated with ammonium, and pretreatment of a part of the root system inhibited phosphorous uptake by the remaining part unless it also had been pretreated. Pretreatment with the amino acid analogs p-fluoro-dl-phenylalanine and l-azetidine-2 carboxylic acid (AZ) restricted phosphorous uptake in seedlings that were pretreated with ammonium and in those that were not, but the effect of ammonium pretreatment was not completely eliminated by the analogs. In general, translocation of the entering phosphorous was affected similarly to uptake by experimental treatments. Enhanced translocation, however, was not sufficient to account quantitatively for the increase in uptake, and an increased uptake was still evident when translocation was completely prevented by 50 micromolar AZ pretreatment. PMID- 16665606 TI - Photosystem Stoichiometry and Excitation Distribution in Chloroplasts from Surface and Minus 20 Meter Blades of Macrocystis pyrifera, the Giant Kelp. AB - The photochemical apparatus organization in the thylakoid membrane of Macrocystis pyrifera, the giant kelp, was investigated. Chloroplasts were isolated from surface and minus 20 meter blades. Photosynthetic electron-transport complex quantitation revealed ratios of photosystem (PS) II/cytochrome b(6)-f/PSI = 1.8:3.3:1.0 in surface and 2.2:2.3:1.0 in minus 20 meter blades. The apparent photosynthetic unit size of chloroplasts from minus 20 meter blades (chlorophyll/P700 = 1485:1) was about 45% larger than that of surface blades (chlorophyll/P700 = 1025:1). The larger photosynthetic unit size of minus 20 meter blades is attributed to the substantially lower intensity of sunlight reaching the minus 20 meter habitat. In different chloroplast preparations, the effective absorption cross section of PSI and PSII to 670 nanometer light (chlorophyll a) and 481 nanometer light (chlorophyll c and fucoxanthin) was investigated. The results showed larger functional antenna size for PSII (about 90%) and for PSI (about 50%) in minus 20 meter than in surface blades. Moreover, the efficiency of utilization of 481 nanometer light by Macrocystis chloroplasts was equal to that of 670 nanometer light. It is concluded that the chlorophyll c fucoxanthin complex in brown algae enables the highly efficient utilization of blue-green wavelengths of the nearshore marine environment and contributes to the dominance of M. pyrifera in this habitat. PMID- 16665607 TI - Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature. AB - Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3 degrees C developed more freezing resistance than cells cultured at 3 degrees C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [(14)C]leucine incorporation. Protein synthesis continued at 3 degrees C, but net cell growth was stopped. Most of the major proteins detected at 23 degrees C were synthesized at 3 degrees C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3 degrees C. Comparative electrophoretic analysis of [(14)C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3 degrees C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23 degrees C. Quantitative analysis of [(14)C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth. PMID- 16665608 TI - Cottonseed malate synthase : purification and immunochemical characterization. AB - Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds. PMID- 16665609 TI - Cottonseed malate synthase : biogenesis in maturing and germinated seeds. AB - The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and imbibition, then increases again following germination. The research reported herein is a comparative study of the synthesis and acquisition of MS into glyoxysomes as they occur in maturing and germinated seeds. Rate-zonal centrifugation of cotyledon extracts revealed that the 5 Svedberg unit (S) cytosolic form of MS was the only form present at 42 days postanthesis (DPA) when activity was first detectable. At later stages (48 DPA, 0 day, 26 hours, and 48 hours), both the 5S and glyoxysomal 20S forms were present, with the 20S form becoming much more prevalent. Western blot analyses revealed that no other form(s) of MS were present in the phosphate-buffered gradients, and that 5S and 20S forms had the same subunit molecular weight in maturing and germinated seeds. Comparisons of radiospecific activity of MS immunoprecipitates following in vivo labeling with [(35)S]methionine for varying time intervals provided strong evidence for a 5S-precursor to 20S-product relationship during both seed maturation and seedling growth. Comparisons of MS labeled in vivo and in vitro in wheat germ and rabbit reticulocyte lysates programmed with poly(A)(+)RNA (from maturing and germinated seeds) revealed no detectable differences in subunit molecular weights. These results reinforced our other data indicating that MS was synthesized in the cytosol and acquired by glyoxysomes in both maturing and germinated cotton seeds without involvement of an intervening aggregate pool in the endoplasmic reticulum, or via processing of a cleavable precursor molecule. MS was translated from poly(A)(+)RNA extracted from 28 DPA cotton seeds. This was nearly 2 weeks before MS activity or protein was detected in vivo. This finding invites further study on the regulation of RNA transcripts during maturation. PMID- 16665610 TI - Studies on genetic male-sterile soybeans : v. Effects of male-sterility on the function and glycerolipid composition of chloroplast thylakoids. AB - Soybean (Glycine max L. Merr.) germplasm, isogenic except for loci controlling male sterility (ms(1)), was utilized to study the effects of reproductive development on certain aspects of photosynthesis. Plants were sampled at various times between flowering (77 days after transplanting) and maturity (147 days after transplanting). During that period photosynthetic rates declined more rapidly in the male-sterile genotypes than male-fertile genotypes; and after 105 days, the sterile genotypes maintained low but relatively constant carbon exchange rates. The decline of leaf photosynthesis in the male-sterile genotype occurred concomitantly with an inhibition of the photosynthetic electron transport chain associated with photosystem II. Changes in photosystem I activities, cytochrome f levels, and chlorophyll a/b ratios per se were not responsible for the decline in whole leaf photosynthesis. These conditions were independent of the source of nitrogen nutrition. Lipid analyses of the thylakoids revealed that a loss of phosphatidylglycerol was highly correlated with the inhibition of photosystem II activity. These results suggested a relation between the decline in leaf carbon exchange and the decline in photosynthetic electron transport activity. PMID- 16665611 TI - Differential effect of auxin on in vivo extensibility of cortical cylinder and epidermis in pea internodes. AB - The effect of auxin indole-3-acetic acid (IAA) on growth and in vivo extensibility of third internode sections from red light grown pea seedlings (Pisum sativum L. cv Alaska) and the isolated tissues (cortex plus vascular tissue = cortical cylinder, and epidermis) was investigated. Living tissue was stretched at constant force (creep test) in a custom-built extensiometer. In the intact section, IAA-induced increase in total (E(tot)), elastic (E(el)), and plastic (E(pl)) extensibility is closely related to the growth rate. The extensibility of the cortical cylinder, measured immediately after peeling of intact sections incubated for 4 hours in IAA, is not increased by IAA. Epidermal strips, peeled from growing sections incubated in IAA, show a E(pl) increase, which is correlated to the growth rate of the intact segments. The isolated cortical cylinder expands in water; IAA has only a small growth-promoting effect. The extensibility of the cortical cylinder is not increased by IAA. Epidermal strips contract about 10% on isolation. When incubated in IAA, they do not elongate, but respond with an E(pl) increase. The amount of expansion of the cortical cylinder and contraction of the epidermis (tissue tension), measured immediately following excision and peeling, stays constant during IAA-induced growth of intact sections. The results support the hypothesis that IAA induces growth of the intact section by causing an E(pl) increase of the outer epidermal wall. The driving force comes from the expansion of the cortical cylinder which is under constant compression in the intact section. PMID- 16665612 TI - Effect of H Excretion on the Surface pH of Corn Root Cells Evaluated by Using Weak Acid Influx as a pH Probe. AB - The initial rate of (2-(14)C)acetic acid (AA) uptake by corn roots was used for probing the dependency of the root cell surface pH on H(+) excretion. AA influx was linearly related to AA concentration, dependent on the concentration of the undissociated form (AH), unaffected by variations of the membrane potential, and was thus assumed to result mainly from the free diffusion of AH across the membrane. Various treatments (vanadate, dicyclohexylcarbodiimide, hypoxia, nitrate, root ageing, fusicoccin) were used to vary H(+) flux while the medium pH was maintained constant. There was a positive relation between AA influx and the net H(+) efflux. This relation disappeared when the proton buffering strength of the absorption medium was increased. These results indicate that the pH at the membrane surface was lowered by H(+) excretion, even in situations where the bulk (pH) was unaffected. The depressive effect of vanadate on AA influx was counteracted by acidifying the medium in order to estimate this pH shift: -1.2 pH units in 12.5 millimolar K(2)SO(4) (pH 6.8). Substituting AA by butyric acid showed that this estimation was not dependent of the probe used. PMID- 16665613 TI - Photosystem I-Mediated Regulation of Water Splitting in the Red Alga, Porphyra sanjuanensis. AB - The marine red alga, Porphyra sanjuanensis is found mainly in the high intertidal zone and at low tide subject to frequent and extreme water stress, often accompanied by high temperatures and light intensities. Such exposures can lead to severe desiccation which is accompanied by the progressive loss of photosynthetic activity. Even following the loss of more than 90% of the thallus water content the alga recovers rapidly when returned to seawater. This stress induced, reversible inactivation of photosynthesis is believed to be a protective adaptation which prevents photodamage to the exposed alga. Effects of light, inhibitors of water splitting, and electron donors to PSI on variable fluorescence and water splitting suggest that activity of the oxygen evolving complex is regulated by the PSI-driven reduction of a component of intersystem electron transport. PMID- 16665614 TI - Early and late heat shock proteins in wheats and other cereal species. AB - Coleoptiles and roots of 3-day-old seedlings from five cereal species (Triticum aestivum L., T. durum Desf., Hordeum vulgare L., Secale cereale L., and Triticale) respond to heat shock at 40 degrees C by synthesizing a new set of 13 strong bands (as revealed by one-dimensional sodium dodecyl sulfate gel electrophoresis) as well as some 20 degrees C proteins. Heat shock proteins (HSPs) belong to three different size groups: high molecular mass HSPs in the 103 to 70 kilodalton range, intermediate molecular mass HSPs in the 62 to 32 kilodalton range, and low molecular mass HSPs about 17 to 16 kilodalton in size. At the beginning of the heat shock coleoptiles show a reduced ability to synthesize intermediate molecular mass HSPs but after 4 hours at 40 degrees C they exhibit fully developed HSP patterns identical to that found in roots. Synthesis of early HSPs declines after 7 hours of treatment followed by the appearance of a new set of 12 protein bands (late HSPs) in the ranges 99 to 83, 69 to 35, and 15 to 14 kilodaltons. After 12 hours at 40 degrees C, three other late HSPs of 89, 45, and 38 kilodalton are induced. The induction of late HSPs after 7 hours at 40 degrees C appears to be associated with an enhancement of radioactive methionine incorporation into proteins. PMID- 16665615 TI - Pontentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells : kinetin enhances mitochondrial fluorescence. AB - Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3' diheptyloxacarbocyanine iodide [DiOC(7)(3)] accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhanced within 5 to 10 minutes after addition of 0.1 millimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane. PMID- 16665616 TI - Regulation of nodulation in the soybean-Rhizobium symbiosis : strain and cultivar variability. AB - Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod(+) on McCall] and USDA257 [Nod(-) on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall x 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions. PMID- 16665617 TI - Characterization of a cytosolic aconitase in higher plant cells. AB - Protoplasts obtained from sycamore (Acer pseudoplatanus) cell suspensions were found to be highly intact. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured leaving the fragile amyloplasts largely intact. Aconitase hydratase (citrate [isocitrate] hydro lyase, EC 4.2.1.3) activity of sycamore cells was associated with two protein fractions, one present in the cytosol while the second is of mitochondrial origin. Chromatography on DEAE-trisacryl did not separate the aconitase hydratase isoenzymes. EPR studies established that both isoenzymes exhibited an EPR signal at g = 2.03 once oxidized. PMID- 16665618 TI - Solute Accumulation in Tobacco Cells Adapted to NaCl. AB - Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na(+) and Cl(-) as principal solutes for this adjustment. Although the intracellular concentrations of Na(+) and Cl(-) correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na(+) and Cl(-) did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na(+) and Cl(-). The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na(+) and Cl(-) are principal components of osmotic adjustment, organic solutes also may make significant contributions. PMID- 16665619 TI - Potassium influx into maize root systems : influence of root potassium concentration and ambient ammonium. AB - Potassium influx into roots of dark-grown decapitated maize seedling (Zea mays L., cv Pioneer 3369A) was examined in presence and absence of ambient ammonium and at various root potassium concentrations. Six-day old seedlings which were dependent on the endosperm reserves for their energy source were exposed to KCl (labeled with (86)Rb) ranging from 5 to 200 micromolar. At both low (13 micromoles per gram fresh weight) and high (100 micromoles per gram fresh weight) root potassium concentration, isotherms indicated two potassium influx systems, one approaching saturation at 50 to 100 micromolar potassium and an additional one tentatively considered to be linear. A mixed-type inhibition by ammonium for the low-concentration saturable system was indicated by a concomitant decrease in V(max) and increase in K(m). High root potassium concentration decreased V(max) but had little effect on K(m) of this system. The rate constant for the second quasilinear system was decreased by ambient ammonium and by high root potassium status. Transfer of high potassium roots to potassium-free solutions resulted in an increase in influx within 2 hours; by 24 hours influx significantly exceeded that of roots not previously exposed to potassium. In roots of both low and high root potassium concentrations, potassium influx was restricted progressively as ambient ammonium increased to about 100 micromolar, but there was little further inhibition as ammonium concentrations increased beyond that to 500 micromolar. The data imply that potassium influx has two components, one subject to inhibition by ambient ammonium and one relatively resistant. PMID- 16665620 TI - Endogenous NO(3) in the Root as a Source of Substrate for Reduction in the Light. AB - An experiment was conducted to investigate the reduction of endogenous NO(3) (-), which had been taken up by plants in darkness, during the course of the subsequent light period. Vegetative, nonnodulated soybean plants (Glycine max [L]. Merrill, ;Ransom') were exposed to 1.0 millimolar (15)NO(3) (-) for 12 hours in darkness and then returned to a solution containing 1.0 millimolar (14)NO(3) ( ) for the 12 hours ;chase' period in the light. Another set of plants was exposed to (15)NO(3) (-) during the light period to allow a direct comparison of contributions of substrate from the endogenous and exogenous sources. At the end of the (15)NO(3) (-) exposure in the dark, 70% of the absorbed (15)NO(3) (-) remained unreduced, and 83% of this unreduced NO(3) (-) was retained in roots. The pool of endogenous (15)NO(3) (-) in roots was depleted at a steady rate during the initial 9 hours of light and was utilized almost exclusively in the formation of insoluble reduced-N in leaves. Unlabeled endogenous NO(3) (-), which had accumulated in the root prior to the previous dark period, also was depleted in the light. When exogenous (15)NO(3) (-) was supplied during the light period, the rate of assimilation progressively increased, reflecting an increased rate of uptake and decreased accumulation of NO(3) (-) in the root tissue. The dark absorbed endogenous NO(3) (-) in the root was the primary source of substrate for whole-plant NO(3) (-) reduction in the first 6 hours of the light period, and exogenous NO(3) (-) was the primary source of substrate thereafter. It is concluded that retention of NO(3) (-) in roots in darkness and its release in the following light period is an important whole-plant regulatory mechanism which serves to coordinate delivery of substrate with the maximal potential for NO(3) ( ) assimilation in photosynthetic tissues. PMID- 16665621 TI - Activity of Glyceraldehyde-3-Phosphate Dehydrogenase-NADP in Developing Leaves of Light-Grown Dianthus chinensis L. AB - Quantitative histochemical methods were used to measure glyceraldehyde-3 phosphate dehydrogenase-NADP activity in developing and mature leaves of Dianthus chinensis L. The activity values were compared on the basis of illumination of developing leaves, the maturity of stomata, and the activity of enzymes concerned with carbohydrate oxidation. Activity on a kilogram dry weight per hour basis increased from the youngest primordia (50 mmol) to fully expanded leaves (1900 mmol) and then declined in older, senescing leaves. Enzymic activity increased from leaf base (400 mmol) to tip (1000 mmol) in leaf pair four, the tip of which was exposed to light, while activity of enzymes of carbohydrate oxidation decline. We propose that this leaf is beginning the transition to autotrophy, but does not fix significant amounts of atmospheric carbon because the stomata are not functional until leaf pair six. PMID- 16665622 TI - Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes. AB - A maize (Zea mays L. cv LG 11) root homogenate was prepared and centrifuged to sediment the mitochondria. The pellet (6 KP) and the supernatant (6 KS) were collected and fractionated on linear sucrose density gradients. Marker enzymes were used to study the distribution of the different cell membranes in the gradients. The distribution of the ATP- and pyrophosphate-dependent proton pumping activities was similar after 3 hours of centrifugation of the 6 KS or the 6 KP fraction. The pumps were clearly separated from the mitochondrial marker cytochrome c oxidase and the plasmalemma marker UDP-glucose-sterolglucosyl transferase. The pyrophosphate-dependent proton pump might be associated with the tonoplast, as the ATP-dependent pump, despite the lack of a specific marker for this membrane. However, under all the conditions tested, the two pumps overlapped the Golgi markers latent UDPase and glucan synthase I and the ER marker NADH cytochrome c reductase. It is therefore not possible to exclude the presence of proton pumping activities on the Golgi or the ER of maize root cells. The two pumps (but especially the pyrophosphate-dependent one) were more active (or more abundant) in the tip than in the basal part of maize roots, indicating that these activities might be important in growth processes. PMID- 16665623 TI - Study of Energy Storage Processes in Bundle Sheath Cells of Zea mays. AB - Photochemical energy storage in isolated bundle sheath cells from Zea mays was examined. Photoacoustic spectroscopy was used in this study to monitor energy storage processes. The presence of methyl viologen or addition of substrates which activated carbon fixation, prevented energy storage processes through the electron transport system. The energy storage was inhibited completely by dibromothymoquinone (DBMIB) and DCMU, inhibitors of noncyclic electron flow. However, the reductants such as dithiothreitol and ascorbate increased the energy storage. It was concluded that photosystem (PS) I may be reduced by some electron donor(s) other than water and that PSII only partially participates in PSI reduction. It is postulated that the role of PSII is to regulate PSI electron transport and prevent its overoxidation. In the presence of high level of malate, photoacoustic spectroscopy indicated a low energy storage which may be due to induction of energy utilization in carbon assimilation. PMID- 16665624 TI - Atrazine Resistance in Chenopodium album: Low and High Levels of Resistance to the Herbicide Are Related to the Same Chloroplast PSBA Gene Mutation. AB - In Chenopodium album two different levels of atrazine resistance have been found according to following criteria: lethal dose and leaf fluorescence curve. The intermediate (I) phenotype is represented by a low level of resistance and a typical I fluorescence curve. It arose at high frequency, within one generation, after self-pollination of particular plants displaying a susceptible (S) phenotype. The resistance phenotype (Ri) has a high level of resistance and presents a typical resistant fluorescence curve. It appeared after self pollination of chemically treated I plants. The I, Ri, and also R (resistant plants found in atrazine treated fields) phenotypes contain a serine to glycine mutation at amino acid position 264 in the chloroplast psbA gene product. The steady state level of the psbA gene transcript is not modified between S, I, Ri, and R phenotypes. PMID- 16665625 TI - Impairment of Photorespiratory Carbon Flow into Rubber by the Inhibition of the Glycolate Pathway in Guayule (Parthenium argentatum Gray). AB - Cut shoots of guayule (Parthenium argentatum Gray) were treated with four inhibitors of the glycolate pathway (alpha-hydroxypyridinemethanesulfonic acid; isonicotinic acid hydrazide, glycine hydroxamate, and amino-oxyacetate, AOA) in order to evaluate the role of photorespiratory intermediates in providing precursors for the biosynthesis of rubber. Photorespiratory CO(2) evolution in guayule leaves was severely inhibited by AOA. Application of each of the four inhibitors has resulted in a significantly decreased incorporation of (14)C into rubber fractions suggesting that the glycolate pathway is involved in the biosynthesis of rubber in guayule. However, the application of each of the glycolate pathway inhibitors showed no significant effect on photosynthetic CO(2) fixation in the leaves. The inhibitors individually also reduced the incorporation of labeled glycolate, glyoxylate, and glycine into rubber, while the incorporation of serine and pyruvate was not affected. The effective inhibition of incorporation of glycolate pathway intermediates in the presence of AOA was due to an inhibition of glycine decarboxylase and serine hydroxymethyltransferase. It is concluded that serine is a putative photorespiratory intermediate in the biosynthesis of rubber via pyruvate and acetyl coenzyme A. PMID- 16665626 TI - Photosynthetic characteristics of a photoautotrophic cell suspension culture of soybean. AB - A soybean suspension culture (SB-P) which can grow photoautotrophically in 5% CO(2) will not grow in ambient CO(2) levels. This elevated CO(2) requirement seems to be due to the additive effects of a number of factors. The in vivo activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) is much lower in the SB-P cells, compared to soybean plants. This may be due to the low light intensity used to culture the cells, which has been shown to decrease both the amount and activity in whole plants, resulting in a low rate of net photosynthesis. The RuBPcase activation level is also lowered in air CO(2) levels. The presence of the liquid medium raises the cells CO(2) compensation concentration (the CO(2) concentration reached when the rates of CO(2) fixed by photosynthesis and the CO(2) respired by the cells are equal). These factors, coupled with the high respiratory loss of CO(2) all contribute to reduced net photosynthesis in air, resulting in a photosynthetic capacity that is inadequate for cell survival. Active cell division, low photosynthetic capacity, elevated respiration, and a low ratio of RuBPcase(initial)/phosphoenolpyruvate carboxylase are traits that SB-P cells share with young leaf cells, indicating SB-P cell physiology may be comparable to that of young expanding leaves rather than to that of mature leaves. PMID- 16665627 TI - Protein trafficking in plant cells. AB - The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation. PMID- 16665628 TI - Boron stem infusions stimulate soybean yield by increasing pods on lateral branches. AB - Studies were carried out to determine if supplemental B (H(3)BO(3)) and Ca (CaCl(2)) injected via a stem infusion technique into soybeans could stimulate yield by increasing pods on lateral branches, seed number, and overall seed yield. Boron treatments caused a significant 84.8% increase in the number of lateral pods/plant and a 17.6% increase in total seed weight/plant. This corresponded to a seed yield of 4170 kilograms per hectare in the B-treated plants compared to 3540 kilograms per hectare in the injected control plants, indicating that B deficiency may have been a factor in limiting yield of control plants. Ca treatments tended to accentuate the negative yield effects of apparent B deficiency. PMID- 16665629 TI - Evidence for transglutaminase activity in plant tissue. AB - An extract prepared from the apical meristematic region of etiolated pea seedlings was able to catalyze the incorporation of putrescine into trichloroacetic acid precipitable material. The enzyme was found to be soluble and followed a typical Michaelis-Menten kinetics when N-N-dimethyl casein was used as a substrate. Its activity was promoted by Ca(2+) and inhibited by Cu(2+) and dl-dithiothreitol. Other polyamines competed with putrescine as substrates and cadaverine was the most potent inhibitor of putrescine incorporation. Plant transglutaminase is capable of recognizing specific sites in substrates described for animal transglutaminase, like insulin, fibrinogen, pepsin, and thrombin. However, it can also use as substrates cellulase and creatine kinase which have not been described for transglutaminase from other sources. PMID- 16665630 TI - Pathotoxin effects in sorghum are also produced by mercuric chloride treatment. AB - Pathogenic isolates of Periconia circinata produce a host-specific toxin (PC toxin) and cause a root and crown rot in susceptible genotypes of sorghum. Treatment with PC-toxin leads to selective development of disease symptoms and an increase in synthesis of a group of acidic, low molecular weight proteins only in susceptible genotypes. Treatment of sorghum seedlings or excised root tips with HgCl(2) resulted in responses indistinguishable from those produced by treatment with PC-toxin, but the effects were not genotype specific. PMID- 16665631 TI - Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases. AB - Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C(4) enzyme to inhibition by malate is also unaffected by preincubation with these ligands. PMID- 16665632 TI - Comparison of a commercial ELISA assay for indole-3-acetic Acid at several stages of purification and analysis by gas chromatography-selected ion monitoring-mass spectrometry using a c(6)-labeled internal standard. AB - Quantitative analysis of indole-3-acetic acid (IAA) using selected ion monitoring gas chromatography-mass spectrometry (GC-MS) with (13)C(6)[benzene ring]-IAA as the internal standard was used to compare the quantitative accuracy of commercial enzyme-linked immunoabsorbent assay (ELISA) kits. Plant materials differed in the amount of purification required prior to use of ELISA for reliable estimates to be made. Purification similar to that obtained by at least one high performance liquid chromatographic (HPLC) step was generally necessary prior to ELISA analysis of plant materials. Additional levels of purification appeared to be required for some plant materials prior to HPLC in order to obtain an accurate estimate by ELISA techniques. In no case was it possible to obtain reasonable estimates of IAA from crude extracts or even from acidic fractions of extracts of plant tissues. GC-MS techniques provide a rapid and simple method for checking the validity of ELISA techniques. Quantitative GC-MS, or a similar technique that provides an independent quantitative validation, should, whenever possible, be applied to each new plant material under study if use of the ELISA is planned. PMID- 16665633 TI - Comparison of tissue preparation methods for assay of nicotinamide coenzymes. AB - To prepare tissues for analysis of NAD(+), NADH, NADP(+), and NADPH, common practice is to freeze samples in liquid nitrogen, often followed by freeze drying, before extraction in HCl or NaOH. With cucumber (Cucumis sativus L.) cotyledons, prefreezing in liquid nitrogen or slower freezing to -20 degrees C yielded substantially lower values for NADH and NADPH than obtained from samples homogenized immediately in acid or base. Freeze-drying after freezing in liquid nitrogen generally caused even lower values of those coenzymes. We suggest that direct extraction is more likely to yield accurate results with cotyledons and other plant parts. PMID- 16665634 TI - Drought-Induced Changes in Protein Patterns of Brassica napus var. oleifera Roots. AB - Drought-induced changes in two-dimensional silver stained protein patterns of Brassica napus L. var. oleifera M. root system were detected both at quantitative and qualitative levels. Particularly, 13 new polypeptides of low molecular weight were evidenced in the drought-stressed tap root, 12 of which were also present in the short tuberized roots, a specific drought-induced root type. The reversibility of these modifications, observed after 3 days rehydration, suggests that they might be involved in drought tolerance. PMID- 16665635 TI - Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase. AB - An anion-sensitive H(+)-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H(+) ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO(3) (-), N,N'-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H(+)-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K(m) for the H(+)-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H(+)-ATPase are very similar to those described for a number of plant cell tonoplast H(+)-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation. PMID- 16665636 TI - Transport Properties of the Tomato Fruit Tonoplast : II. Citrate Transport. AB - Citrate transport across the membrane of tomato fruit tonoplast vesicles was investigated. In the tonoplast vesicles, [(14)C]methylamine uptake was stimulated 10-fold by MgATP and strongly inhibited by NO(3) (-). Under identical experimental conditions, [(14)C]citrate uptake was inhibited by 5 millimolar free Mg(2+), and this inhibition was reversed in the presence of ATP, presumably by ATP chelation of free Mg(2+). No evidence was obtained in support of energy linked ATP stimulation of citrate uptake. Citrate uptake showed saturation kinetics, and was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid and by other organic acids. The pH-dependence of uptake suggested that citrate(3 ) was the transported species. Our results indicate that citrate transport across the tomato fruit tonoplast occurs by facilitated diffusion of citrate(3-). The carrier shares some features in common with anion channels in that it is relatively nonspecific for organic acids and is inhibitable by 4,4' diisothyocyano-2,2'-stilbenedisulfonic acid. PMID- 16665637 TI - The use of cytotoxic plant lectins in cancer therapy. AB - As part of their defense mechanism against herbivores or phytophagous insects, many plant tissues contain lectins. Some of these lectins are potent toxins which kill animal cells by arresting protein synthesis. An attractive strategy for developing specifically cytotoxic chemotherapeutic agents is to link cell type specific monoclonal antibodies to potent toxins. The plant protein ricin has emerged as the toxin of choice for such constructs. PMID- 16665638 TI - Role of Catecholamines in Promotion of Flowering in a Short-Day Duckweed, Lemna paucicostata 6746. AB - l-Epinephrine, l-norepinephrine, and l-isoproterenol substantially promote flowering under a photoperiodic regime of 8 hours light and 16 hours darkness in Lemna paucicostata 6746 when grown on the modified Bonner-Devirian medium devoid of ethylenediaminetetraacetic acid. If catecholamines are provided to plants at 10(-4) molar level prior to transferring them to the short-day regime, they not only induce more floral primordia but also significantly improve flower development and sustain the flowers for a longer period. Propranolol (10(-4) molar), a beta-adrenergic blocking agent, partially suppresses flowering and the inhibition of flowering is relieved by catecholamines. PMID- 16665639 TI - Plant Morphological and Biochemical Responses to Field Water Deficits: III. Effect of Foliage Temperature on the Potential Activity of Glutathione Reductase. AB - Activity of glutathione reductase has been related to stress tolerance; however, these enzyme assays are generally conducted at 25 degrees C. Foliage temperature varies greatly in the field in response to soil water availability and ambient conditions and this may affect enzyme response. This study was conducted to determine the effect of changing foliage temperature on glutathione reductase activity of wheat under field conditions. Wheat leaf glutathione reductase was purified and the temperature response of the enzyme was determined at 2.5 degrees C intervals between 12.5 and 45 degrees C. These data, in conjunction with continuous measurements of field-grown wheat foliage temperatures, were used to compare the temperature-related changes in potential glutathione reductase activities in water stressed and control plants. Assuming saturating substrate levels, the results indicate that early in the season the daily potential enzyme activity of the irrigated and stressed plants could never have reached the daily activity predicted from the 25 degrees C (room temperature) measurements. Later in the season, the daily potential activity of the irrigated plants was lower, and the daily potential activity of the stressed plants was higher, than the activities predicted from the 25 degrees C determinations. These results suggest that a better understanding of the regulation of plant metabolism will be obtained by linking continuous temperature measurements of plant foliage with enzyme responses to temperature. PMID- 16665640 TI - Effect of photosynthetic photon flux density on carboxylation efficiency. AB - The effect of photosynthetic photon flux density (PPFD) on photosynthetic response (A) to CO(2) partial pressures between 35 pascals and CO(2) compensation point (Gamma) was investigated, especially below PPFD saturation. Spinacia oleracea cv ;Atlanta,' Glycine max cv ;Clark,' and Arbutus unedo were studied in detail. The initial slope of the photosynthetic response to CO(2) ( partial differentialA/ partial differentialC[Gamma]) was constant above a PPFD of about 500 to 600 micromoles per square meter per second for all three species; but declined rapidly with PPFD below this critical level. For Gamma there was also a critical PPFD (approximately 200 micromoles per square meter per second for S. oleracea and G. max; 100 for A. unedo) above which Gamma was essentially constant, but below which Gamma increased with decreasing PPFD. All three species showed a dependence of partial differentialA/ partial differentialC(Gamma) on PPFD at low PPFD. Simulated photosynthetic responses obtained with a biochemically based model of whole-leaf photosynthesis were similar to measured responses. PMID- 16665641 TI - Glycine-Glomus-Rhizobium Symbiosis: V. Effects of Mycorrhiza on Nodule Activity and Transpiration in Soybeans under Drought Stress. AB - Soybean (Glycine max [L.] Merr.) plants were nodulated (Bradyrhizobium japonicum) and either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or left uncolonized. All plants were grown unstressed for 21 days initially. After this period, some VAM and non VAM plants were exposed to four 8-day drought cycles while others were kept well watered. Drought cycles were terminated by rewatering when soil moisture potentials reached -1.2 megapascal. Nodule development and activity, transpiration, leaf conductance, leaf and root parameters including fresh and dry weight, and N and P nutrition of VAM plants and of non-VAM, P-fed plants grown under the same controlled conditions were compared. All parameters, except N content, were greater in VAM plants than in P-fed, non-VAM plants when under stress. The opposite was generally true in the unstressed comparisons. Transpiration and leaf conductance were significantly greater in stressed VAM than in non-VAM plants during the first half of the final stress cycle. Values for both VAM and non-VAM plants decreased linearly with time during the cycle and converged at a high level of stress (-1.2 megapascal). Effects of VAM fungi on the consequences of drought stress relative to P nutrition and leaf gas exchange are discussed in the light of these findings and those reported in the literature. PMID- 16665642 TI - Glycine-Glomus-Rhizobium Symbiosis : VI. Photosynthesis in Nodulated, Mycorrhizal, or N- and P-Fertilized Soybean Plants. AB - Soybean (Glycine max [L.] Merr. cv Hobbit) plants were grown in a growth chamber for 56 days in a phosphorus- and nitrogen-deficient soil and were colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd) Gerd. and Trappe and Rhizobium japonicum strain USDA 136, or by either organism alone, or by neither. Non-VAM plants received supplemental phosphorus and nonnodulated plants supplemental nitrogen to achieve the same rate of growth in all treatments. Plants of all four treatments had the same (P > 0.05) dry weights at harvest, but VAM plants had higher rates of CO(2) exchange (CER, P < 0.05) and lower leaf P concentrations (P < 0.01). Leaf nitrogen concentrations were lower in nodulated than in nitrogen-supplemented plants (P < 0.01) while starch concentrations were higher (P < 0.01). There was a significant negative relationship between nitrogen and starch (r = -0.989). Statistical evaluation of the data showed that some parameters (CER, leaf area and phosphorus content) were associated with phosphorus nutrition (or the presence of the VAM fungus), others (leaf fresh weight and root dry weight) with nitrogen nutrition (or the presence of Rhizobium), and some (leaf nitrogen and starch content) by both factors. The development of microsymbiont structures and nodule activity were significantly lower in the tripartite association than in plants colonized by one endophyte only. The findings suggest that endophyte effects go beyond those of simple nutrition and associated source-sink relationships. PMID- 16665643 TI - Light and acetate regulate a mitochondrial malate dehydrogenase. AB - A malate dehydrogenase was purified from the unicellular green alga Chlorogonium elongatum Dangeard. The enzyme was localized in the mitochondria by immunogold electron microscopy and was found to be present on the cristae. The concentration of the enzyme is regulated by acetate and light. In cells cultured heterotrophically with acetate as carbon source the activity and the concentration of the enzyme is 5- to 6-fold higher than in autotrophic cells. In mixotrophically cultured cells (light and acetate) the enzyme level attains only half of the value of that in heterotrophic cells. Acetate induces an increase of the enzyme concentration while light has an inhibitory effect on this process. PMID- 16665644 TI - Modulation of Pea Membrane beta-Glucan Synthase Activity by Calcium, Polycation, Endogenous Protease, and Protease Inhibitor. AB - beta-Glucan synthase activity in plant membranes can be markedly altered by a multiplicity of apparently unrelated factors. In pea epicotyl membranes it is enhanced by low and inhibited by high concentrations of added Ca(2+), trypsin or soluble pea protease. Ca(2+) stimulates preexisting synthase activity, particularly in the presence of polycations (spermidine), but protease treatments activate and, with time, inactivate synthase zymogen. Endogenous pea protease activity is also associated with washed pea membrane and appears to be responsible for the decay observed with time in the beta-glucan synthase activity. Endogenous pea protease activity is inhibited by thiol inhibitors, e.g. iodoacetamide and Hg(2+), and by a heat-stable peptide, molecular weight approximately 10,000, that is found in supernatants of pea extracts. These protease inhibitors have the capacity to protect beta-glucan synthase activity from denaturation or its zymogen from activation due to endogenous or added protease activity. Evidence is described which supports the proposal that 1,4 beta-glucan synthase is destroyed and possibly converted to 1,3-beta-glucan synthase activity by protease action, and that the latter may then be greatly enhanced by Ca(2+) and polycations. PMID- 16665645 TI - Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling. AB - When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO(2) and H(2) evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO(2) and H(2) evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function. PMID- 16665646 TI - Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures. AB - Dihydrodipicolinate synthase, the first enzyme unique to lysine biosynthesis in higher plants, was purified about 5100-fold from suspension-cultured cells of wheat (Triticum aestivum var Chinese Spring). The synthase has an average molecular weight of 123,000 as determined by gel filtration and exhibited maximum activity at pH 8.0. The kinetics of the condensation reaction are compatible with a "Ping Pong" mechanism in which pyruvate reacts first with the enzyme to form a Schiff base. Pyruvate and l-aspartic-beta-semialdehyde (ASA) have respective K(m) values of 11.76 and 0.80 millimolar. Allosteric inhibition was observed with increasing concentrations of l-lysine and its structural analogs, including threo 4-hydroxy-l-lysine and S-(2-aminoethyl)-l-cysteine, with respective I(0.5) values of 51, 141, and 288 micromolar. These amino acids were competitive inhibitors with respect to ASA and noncompetitive inhibitors with respect to pyruvate. We propose that the binding site for lysine overlaps with the ASA binding site, possibly by an attachment of the common alanyl moiety. The wheat enzyme was inhibited by Zn(2+), Cd(2+), and Hg(2+) and also by sulfhydryl inhibitors, p (hydroxymercuri)benzoic acid and p-chloromercuribenzenesulfonic acid. PMID- 16665647 TI - Rubisco Activase Mediates ATP-Dependent Activation of Ribulose Bisphosphate Carboxylase. AB - The activation level of ribulosebisphosphate carboxylase following preincubation with ribulose 1,5-bisphosphate was increased by ATP and ribulosebisphosphate carboxylase activase in the absence of thylakoids or illumination. Maximal activation was obtained with 0.5 millimolar ATP in the presence of an ATP regenerating system (phosphoenolpyruvate and pyruvate kinase). Without the ATP regenerating system, activation was lower, linearly dependent on ATP concentration up to 1.0 millimolar, and was strongly inhibited by ADP. PMID- 16665648 TI - Uptake and Fate of Ethephon ([2-Chloroethyl]phosphonic Acid) in Dormant Weed Seeds. AB - Although ethephon ([2-chloroethyl]phosphonic acid) is often used as a form of liquid ethylene in studies of seed germination, it is not known if ethylene evolved from ethephon in the seed is sufficient to elicit the desired response and/or if ethephon has a regulatory action that alone accounts for the response. For these reasons we studied the uptake and fate of [1,2-(14)C]ethephon in dormant seeds of Avena fatua, Sinapis arvensis, Thlaspi arvense, and Chenopodium album. The radioactivity within the seeds was separated into a labile carbon labeled ethephon/ethylene fraction (64-87%) and, following extraction in methanol chloroform-water (12:5:3), into fractions associated with insoluble (12-29%) and soluble (3-8%) seed constituents. The radioactivity associated with seed constituents was reduced 5 to 75% by hot alkaline hydrolysis (2.5 n KOH, 70 degrees C for 1 hour). Although a small portion of the ethephon (or metabolite of ethephon/ethylene) taken up by the seeds is tightly bound to the tissues, our results indicate that, at the appropriate external concentrations of ethephon, the amount of ethylene evolved from ethephon within the seeds is sufficient to produce the desired ethylene mediated responses. However, factors affecting the decomposition of ethephon must be considered in the decision as to whether to use ethephon as a liquid supply of ethylene. PMID- 16665649 TI - Functional size of photosynthetic electron transport chain determined by radiation inactivation. AB - Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H(2)O to methylviologen) was 623 +/- 37 kilodaltons; for photosystem II (H(2)O to dimethylquinone/ferricyanide), 174 +/- 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 +/- 11 kilodaltons. The difference between 364 +/- 22 (the sum of 174 +/- 11 and 190 +/- 11) kilodaltons and 623 +/- 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b(6)/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed. PMID- 16665650 TI - Errors in the Measurement of Root Pressure and Exudation Volume Flow Rate Caused by Damage during the Transfer of Unsupported Roots between Solutions. AB - Plants of Zea mays were grown with their roots confined to growing tubes, consisting of cylindrical or spherical glass tubes fitted at the bottom with a stopcock. Nutrient solution was circulated past the roots, and when a plant was 21 to 25 days old, the stopcock was closed, the root excised from the plant and connected to an apparatus which measured root pressure and exudation volume flow rate. The stopcock was opened and solution was again circulated through the growing tube without dropping the level of the liquid bathing the root in the process. Measurements of pressure and flow rate were made continuously during a period in which the solution was replaced, first by draining and refilling the tube in situ, and second by replacing the growing tube with a beaker of solution. Both these manipulations caused at least temporary and frequently permanent drops in root pressure and flow rate. Plants were also grown in cylindrical tubes with a support medium of either glass beads or Raschig rings which filled the growing tubes. It is shown that the solution bathing these roots could be repeatedly replaced by draining and refilling with no visible effect on the measurements. It is recommended, therefore, that in future, support be provided for the roots of all experimental plants grown by solution culture. PMID- 16665651 TI - Coordinate, Organ-Specific and Developmental Regulation of Ribulose 1,5 Bisphosphate Carboxylase Gene Expression in Amaranthus hypochondriacus. AB - The expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in roots, stems, cotyledons, and leaves of amaranth during the development of these tissues. The highest accumulation of LSU and SSU polypeptides occurred in cotyledons and leaves. Their steady state levels were approximately 20-fold lower in stems, while in roots neither LSU and SSU polypeptides nor their respective mRNAs could be detected. In cotyledons and leaves accumulation of these two polypeptides reached peak levels during the expansion stage of each tissue and then declined, reflecting changes in the synthesis, not turnover, of these proteins. In cotyledons and stems, the rates of synthesis of LSU and SSU polypeptides correlated with the levels of their respective mRNA, suggesting regulation primarily at the transcriptional level. In contrast, the dramatic and specific decrease in the synthesis of these two proteins during the last stages of development of the leaves could only partially be accounted for by the modest reduction in their mRNAs. Neither the translatability of these mRNAs, as assayed in cell-free systems, nor the stability of LSU and SSU polypeptides were altered, thus implying that control was being exerted at the translational level. During the development of these different organs, the expression of the LSU and SSU genes were generally coordinately regulated both at the levels of protein synthesis and mRNA accumulation. PMID- 16665652 TI - Changes in the Cationic Regulation of Structure and Function in Thylakoids Isolated from Wheat Seedlings Treated with BASF 13.338. AB - When wheat seedlings (Triticum vulgare cf HD 2189) were grown in the presence of BASF 13.338 (4-chloro-5-[dimethylamino]-2-phenyl-3[2H]-pyridazinone), there was a decrease in the ratio of linolenic acid to linoleic acid in the thylakoid membrane lipids (JB St John 1976 Plant Physiol 57: 38) and an increase in the ratio of photosystem II to photosystem I (RM Mannan, S Bose 1984 Photochem Photobiol 41: 63). Accompanying these gross structural changes were alterations in the cationic regulation of structure and functioning of the thylakoid membranes: (a) Mg(2+)-induced increase in the room temperature fluorescence was totally absent; (b) Mg(2+)-induced increase in absorbance at 560 nm, indicative of granal stacking, was slightly higher in thylakoids isolated from the BASF 13.338 treated plants suggesting an increased degree of stacking; and (c) absorption changes in the red and Soret regions of the absorption spectrum, normally resulting from the addition of divalent cation or alkyl anion, or from osmotic shrinkage were almost totally absent in thylakoid membranes isolated from BASF 13.338 treated plants. These observations have been interpreted in terms of: (a) significant alterations in the lipid matrix of the thylakoids from treated plants, (b) absence of cation-induced reorganization of the pigment-protein complexes in the horizontal plane of the treated thylakoid membranes suspended in low salt medium, and (c) absence of dynamic changes even within the individual pigment-protein complexes of treated thylakoids. PMID- 16665653 TI - Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl. AB - Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (+/-) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K(2)SO(4), Na(2)SO(4), sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Psipi) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Psipi differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Psipi difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na(+), K(+), Cl(-), free amino acids, or organic acids could not account for the Psipi differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na(+), K(+), and Cl(-) was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Psipi. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment. PMID- 16665654 TI - Subunit Structure of Spinach Leaf ADPglucose Pyrophosphorylase. AB - To initiate structural studies of the ADPglucose pyrophosphorylase from spinach an improved purification procedure was devised. The modified purification scheme allowed the isolation of 20 to 30 milligrams pure enzyme from 10 kilogram of spinach leaves. Electrophoresis of the purified enzyme confirmed an earlier study which showed that the enzyme was putatively composed of two subunits (Copeland L, J Preiss 1981 Plant Physiol 68: 996-1001). The two subunits migrate as 51 and 54 kilodalton proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both proteins can be detected on Western blots of leaf homogenates prepared under denaturing conditions suggesting that both subunits exist in vivo. Anion-exchange chromatography in the presence of urea allowed resolution of the 51 and 54 kilodalton proteins. They possess different N terminal amino acid sequences and tryptic peptide maps. Western blot analysis reveals that the 51 and 54 kilodalton proteins are antigenically dissimilar. The 51 but not the 54 kilodalton protein is immunologically related to the ADPglucose pyrophosphorylase from maize endosperm and potato tuber. PMID- 16665655 TI - Effects of diclofop and diclofop-methyl on the membrane potentials of wheat and oat coleoptiles. AB - Electrophysiological measurements were made on the mesophyll cells of wheat (Triticum aestivum L. cv Waldron) and oat (Avena sativa L. cv Garry) coleoptiles treated either with the herbicide diclofop-methyl (methyl 2-(4-(2',4' dichlorophenoxy)phenoxy)propanoate), or it's primary metabolite diclofop, (2-(4 (2',4'-dichlorophenoxy)phenoxy)-propanoic acid). Application of a 100 micromolar solution of diclofop-methyl to wheat coleoptiles had little or no effect on the membrane potential (E(M)), however in oat, E(M) slowly depolarized to the diffusion potential (E(D)). At pH 5.7, 100 micromolar diclofop rapidly abolished the electrogenic component of the membrane potential in both oat and wheat coleoptiles with half-times of 5 to 10 minutes and 15 to 20 minutes, respectively. The concentrations giving half-maximal depolarizations in wheat were 20 to 30 micromolar compared to 10 to 20 micromolar in oat. The depolarizing response was not due to a general increase in membrane permeability as judged from the E(M)'s response to changes in K(+), Na(+), Cl(-), and SO(4) (2-), before and after treatment with diclofop and from its response to KCN treatment. In both plants, diclofop increased the membrane permeability to protons, making the E(M) strongly dependent upon the external pH in the range of pH 5.5 to pH 8.5. The effects of diclofop can best be explained by its action as a specific proton ionophore that shuttles protons across the plasmalemma. The rapidity of the cell's response to both diclofop-methyl (15-20 minutes) and diclofop (2-5 minutes) makes the ionophoric activity a likely candidate for the earliest herbicidal event exhibited by these compounds. PMID- 16665656 TI - Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina. AB - A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using (32)P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions. PMID- 16665657 TI - Knudsen-transitional flow and gas pressurization in leaves of nelumbo. AB - Pressures in gas spaces of leaves of the lotus Nelumbo are higher than ambient pressure. The pressurization capacity of leaves was studied as a function of leaf temperature, and the composition of air entering evacuated leaves was used to calibrate the pore sizes which determine flow in these leaves. The adaxial side of the leaf of Nelumbo has two distinct regions in terms of gas exchange characteristics. There is a region of relatively high mean pore diameter in the center of the leaf opposite the point of petiole insertion. Gas exchange between the remainder of the leaf (>99% by area) and the atmosphere is restricted by "pores" with an effective mean diameter less than 0.03 micrometer. As a result, a flowthrough ventilation operates within each leaf. Air enters the leaf across the expanse of the lamina, and escapes back to the atmosphere through the highly porous region at the center of the lamina. PMID- 16665658 TI - Leaf traits associated with flavonol glycoside genes in soybean. AB - In the soybean (Glycine max (L.) Merr.), the gene combination Fg1 Fg3 is responsible for the glycosylation in the biosynthesis of kaempferol triglucoside (K9) in leaves. The presence of K9 is associated with reduction in chlorophyll content, specific leaf mass, photosynthetic rate and stomatal frequency. Blocking the action of Fg1 Fg3 with the magenta flower gene wm prevents formation of K9 and restores leaf traits to normal. A direct effect of K9 on leaf development is postulated. PMID- 16665659 TI - Membrane deterioration in senescing carnation flowers : coordinated effects of phospholipid degradation and the action of membranous lipoxygenase. AB - The lipid fluidity of microsomal membranes from the petals of cut carnation flowers decreases as the flowers senesce. A comparable change in fluidity was induced by in vitro aging of microsomal membranes from young flowers under conditions in which membranous lipoxygenase-like activity was active. There was no change in fluidity when the membranes were aged in the presence of inhibitors of lipoxygenase or were heat-denatured prior to aging. Membranes from naturally senesced flowers and membranes that had been aged in vitro both sustained an increase in saturated:unsaturated fatty acid ratio that accounted for the decrease in lipid fluidity, and in both instances there was evidence for depletion of the unsaturated fatty acids, linoleic acid, and linolenic acid, which are substrates for lipoxygenase. Loss of lipid phosphate reflecting breakdown of membrane phospholipids preceded the depletion of unsaturated fatty acids attributable to the lipoxygenase-like activity. The data have been interpreted as indicating that fatty acid substrates for membrane-associated lipoxygenase-like activity are made available by the initiation of phospholipid degradation, and that the utilization of these substrates results in a selective depletion of unsaturated fatty acids from the membrane and an ensuing decrease in bulk lipid fluidity. PMID- 16665660 TI - Phytochrome Regulation of the Response to Exogenous Gibberellins by Epicotyls of Vigna sinensis. AB - The elongation rate of cowpea epicotyls from whole cowpea (Vigna sinensis) seedlings and derooted and debladed plants (explants) increased after the main light period (8-hour duration) was extended with either continuous low intensity tungsten light or brief (5 minutes) far-red (FR) irradiation. This end-of-day FR effect was reversed by red (R) irradiation suggesting the involvement of phytochrome. These results confirm and extend those obtained previously with other species. Localization studies indicate the epicotyl to be the site of the photoreceptor. Treatment of cowpea seedlings with paclobutrazol, a gibberellin (GA) biosynthetic inhibitor, abolished the FR promoted epicotyl elongation, indicating a role for GAs in this process. There was no significant difference in epicotyl elongation rates of R plus FR irradiated explants treated with GA(1) or GA(20) and R irradiated explants treated with GA(1). However, R irradiation inhibited subsequent epicotyl elongation of GA(20) treated explants. Moreover, the observation, using GC-MS, that GA(1) and GA(20) are native GAs in cowpea lends support to the concept that phytochrome may control the conversion of endogenous GA(20) to GA(1) in cowpea. PMID- 16665661 TI - Pressure-Driven Extrusion of Intracellular Substances from Bean and Pea Cotyledons during Imbibition. AB - Intracellular substances leak from imbibing cotyledons of grain legumes during imbibition. This work reports the discovery of a biophysical process by which intracellular substances are driven from cotyledons during imbibition. Light and scanning electron microscopy were used to examine bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.) cotyledons and the material released from them into imbibition water. A large fraction of the visible materials released from excised bean and pea cotyledons during the first 30 minutes of imbibition consisted of convoluted or helical streams of material which rapidly emerged from the cotyledons surfaces. Large streams of material from bean cotyledons contained starch grains and protein bodies, and smaller streams from bean and pea cotyledons probably contained protein bodies. The forms of streams were characteristic of a viscous fluid which had been forced by pressure through irregular orifices. The sites of extrusion from bean cotyledons were multicellular blisters which formed on the surfaces of imbibing cotyledons. In 6 hours, pea and bean cotyledons leaked from 1 to 11 micrograms protein per milligram of seed dry weight. The quantities of protein leaked primarily depended on cultivar. PMID- 16665662 TI - 5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1 Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L. AB - A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5'-azido-N-1 naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [(3)H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K(0.5) = 2.8 x 10(-7) molar. The K(0.5) for NPA under these conditions is 2 x 10(-8) molar, indicating that the affinity of Az NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [(3)H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [(3)H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment. PMID- 16665663 TI - Light Induced Polarity of Redox Reactions in Leaves of Elodea canadensis Michx. AB - This paper reports that extracellular reductase activity in leaves of Elodea canadensis, hitherto never associated with polar processes thought to be involved in bicarbonate utilization, also shows a very marked polarity in light. The effect of ferricyanide, applied to the lower side of illuminated leaves, was a depolarization of the membrane electrical potential of up to 110 millivolts, while no depolarization was induced when ferricyanide was applied to the upper side. In the dark ferricyanide induced a depolarization when applied to either the upper or to the lower side of the leaf. Staining with tetrazolium salts, specific indicators for reductase activity, resulted in the formation of a precipitate on the lower side of the leaf when illuminated and on both sides in the dark. The precipitate was only located along the plasmalemma. PMID- 16665664 TI - Photocontrol of sorghum leaf phosphoenolpyruvate carboxylase : characterization of messenger RNA and of photoreceptor. AB - The mechanism underlying the light effect on phosphoenolpyruvate carboxylase (PEPC) from the C(4) plant sorghum (Sorghum vulgare Pers., var Tamaran) leaves was investigated. Following exposure to light a new isozyme of PEPC, specific for the green leaf and responsible for primary CO(2) fixation in photosynthesis, was established. Northern blot experiments revealed the presence of PEPC mRNA showing a molecular weight of 3.4 kilobases. During the greening process, concomitant to enzyme activity, PEPC protein and PEPC messenger RNA amounts increased considerably. This photoresponse was shown to be under phytochrome control. PMID- 16665665 TI - Mass-Action Expressions of Ion Exchange Applied to Ca, H, K, and Mg Sorption on Isolated Cells Walls of Leaves from Brassica oleracea. AB - The cation exchange properties of cell walls isolated from collard (Bassica oleracea var acephala D.C.) leaves were investigated. Cation sorption on cell walls was described by mass-action expressions of ion exchange, rather than by the traditional Donnan equilibrium. The mass-action expressions enable the selectivity of the wall for one cation over another to be determined unambiguously from ion exchange isotherms. We found that: (a) the cation composition of the wall varied as a function of the solution cation concentration, solution cation composition, and pH in a way predicted by mass action; (b) the affinity of the wall for divalent cations increased as the equivalent fraction of divalent cation on the wall increased, and as the concentration of divalent cations in solution increased; (c) the selectivity of the wall for any metal cation pair was not altered by the concentration of H(+) in solution or on the wall; (d) H(+) sorption on the wall may be treated as a cation exchange reaction making it possible to calculate the relative affinity of the wall for metal cation pairs from H(+)-metal (Me) titration curves; and (e) the relative affinity of the wall for the cations we studied was: H(+) >> (K(+) >/= Ca(2+)) > Mg(2+). A cation-exchange model including surface complexes is consistent with observed cation selectivity. We conclude that metal cations interact with the wall to minimize or eliminate long-range electrostatic interactions and suggest that this may be due to the formation of site-specific cation-wall surface complexes. PMID- 16665666 TI - Contiguous organization of nitrogenase genes in a heterocystous cyanobacterium. AB - The organization of the three structural nitrogen fixation (nif) genes that encode nitrogenase (nif K and nif D) and nitrogenase reductase (nif H) have been examined in a number of cyanobacteria. Hybridization of Anabaena 7120 nif gene probes to restriction endonuclease-digested genomic DNA has shown (a) that cyanobacteria incapable of N(2) fixation have no regions of DNA with significant homology to the three nif probes, (b) that Pseudanabaena sp., a nonheterocystous cyanobacterium, has a contiguous nif KDH gene cluster, and (c) that in contrast with other heterocystous cyanobacteria, Fischerella sp. has a contiguous nif KDH gene cluster. PMID- 16665667 TI - Cell wall proteins at low water potentials. AB - We investigated the proteins extractable from cell walls of stem tissues when plants were subjected to low water potentials (low psi(w)). Dark-grown soybean seedlings (Glycine max [L.] Merr.) showed decreased stem growth when the roots were exposed to vermiculite having low water content (psi(w) = -3 bar). After a time, growth resumed but at a reduced rate relative to the controls. The extractable protein increased in the cell walls as psi(w) decreased, especially a 28-kilodalton protein in the young tissue. In contrast, a 70 kilodalton protein, mainly extractable from mature cell walls, appeared to decrease slightly at low psi(w). No hydroxyproline was present in either protein, which shows that neither protein is related to extensin. The level of the 28 kilodalton protein increased in the cell wall of the dividing region soon after the initial growth inhibition, and it appeared in the elongating tissue at about the time growth resumed. The correlation between growth and these protein changes suggests that the two events could be related. PMID- 16665668 TI - Actin of chara giant internodal cells: a single isoform in the subcortical filament bundles and a larger, immunologically related protein in the chloroplasts. AB - Internodal cells of Chara corallina Klein ex. Wild have been studied to determine the number of actin isoforms they contain and whether actin occurs at locations in the cortical cytoplasm outside the filament bundles. A monoclonal antibody to chicken actin is specific for actin in numerous animal cells but binds to two Chara proteins after their separation by two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-M(r)) and isoelectric point (5.5) while the other is distinctly different (58,000-M(r), isoelectric point = 4.8). Because it is indetectable in cells whose actin bundles have been extracted, the 43,000-M(r) protein is assigned to the bundles and concluded to be rare or absent in the remaining cortical cytoplasm. The 58,000-M(r) protein, in contrast, does not extract with the actin bundles. It was localized within the chloroplasts by immunofluorescence and by the dependence of proteolysis on the permeabilization of the chloroplast envelope. PMID- 16665669 TI - Control of wild carrot somatic embryo development by antioxidants : a probable mode of action of 2,4-dichlorophenoxyacetic Acid. AB - As we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass. Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) reductase was found to be considerably more active in proliferating than in developing cultures, whereas no difference was evident in the case of dehydroascorbate (DHA) reductase. Both GSH and AA concentrations in these cells are governed by 2,4-D. These results show that redox status is a strong determinant of proliferative versus developmental growth and indicate that the mode of action of 2,4-D in this system may be explained at least in part by its influence on endogenous antioxidant levels. PMID- 16665670 TI - Banana Ripening: Implications of Changes in Internal Ethylene and CO(2) Concentrations, Pulp Fructose 2,6-Bisphosphate Concentration, and Activity of Some Glycolytic Enzymes. AB - In ripening banana (Musa acuminata L. [AAA group, Cavandish subgroup] cv. Valery) fruit, the steady state concentration of the glycolytic regulator fructose 2,6 bisphosphate (Fru 2,6-P(2)) underwent a transient increase 2 to 3 hours before the respiratory rise, but coincident with the increase in ethylene synthesis. Fru 2,6-P(2) concentration subsequently decreased, but increased again approximately one day after initiation of the respiratory climacteric. This second rise in Fru 2,6-P(2) continued as ripening proceeded, reaching approximately five times preclimacteric concentration. Pyrophosphate-dependent phosphofructokinase glycolytic activity exhibited a transitory rise during the early stages of the respiratory climacteric, then declined slightly with further ripening. Cytosolic fructose 1,6-bisphosphatase activity did not change appreciably during ripening. The activity of ATP-dependent phosphofructokinase increased approximately 1.6 fold concurrent with the respiratory rise. A balance in the simultaneous glycolytic and gluconeogenic carbon flow in ripening banana fruit appears to be maintained through changes in substrate levels, relative activities of glycolytic enzymes and steady state levels of Fru 2,6-P(2). PMID- 16665671 TI - Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules. AB - The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO(2) fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O(2). Phloemgirdling was effected 20 hours and exposure to Ar:O(2) was effected 2 to 3 hours before initiation of experiments. Nodule and root CO(2) fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O(2) decreased nodule CO(2) fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO(2) fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to (14)CO(2). In contrast to nodules, roots exported very little radioactivity, and most of the (14)C was exported as organic acids. The nonphotosynthetic CO(2) fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO(2) assimilation. Nodules fixed CO(2) at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO(2) fixation. The results of this study showed that exposure of nodules to Ar:O(2) reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO(2) fixation, nitrogenase activity, nodule-specific respiration, and transport of (14)C photoassimilate to nodules. These results indicate that nodule CO(2) fixation in alfalfa is associated with N assimilation. PMID- 16665672 TI - Assessment of spatial distribution of growth in the elongation zone of grass leaf blades. AB - Knowledge about the spatial distribution of growth is essential for understanding the leaf growth process. In grasses the elongation zone is located at the base of the leaf blade and is enclosed by sheaths of older leaves. Assessment of spatial growth distribution, therefore, necessitates use of a destructive method. We used a fine needle to make holes through bases of tillers at the location of the leaf elongation zone of tall fescue (Festuca arundinacea Schreb.), then measured the displacement of the holes after a 6 or 24 h interval. Needle holes caused a 22 to 41% decrease in daily leaf elongation so experiments were conducted to investigate if the spatial distribution of growth in the elongation zone was altered. Leaf elongation rate was reduced similarly when needle holes were made within or above the zone where cell elongation occurs. Distribution of elongation within the zone was the same when estimated by displacement of needle holes or ink marks placed on the epidermis of the elongation zone after surrounding tissue had been removed. Making holes at different locations within the elongation zone did not differentially affect the relative contribution of the damaged or undamaged parts to leaf elongation. These findings demonstrate that needle holes or ink marks in paired leaves can be used to estimate the relative distribution of growth in the elongation zone of undamaged tall fescue leaf blades. PMID- 16665673 TI - Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species. AB - Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C(4) plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C(4) subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O(2) evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O(2) evolution (29 to 58 micromoles O(2) evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide. PMID- 16665674 TI - Production and Preliminary Characterization of Monoclonal Antibodies against Cationic Peanut Peroxidase. AB - Ten monoclonal antibodies (McAbs) have been produced against the cationic peroxidase from peanut suspension cell culture. Eight of these antibodies were found to be of the immunoglobulin (Ig)G(1) subclass and two were of IgA subclass. A combination of competitive enzyme-linked immunosorbent assay, Western blotting analysis, and direct antigen-binding assay revealed that the antibodies are directed against four different epitopes on the cationic peroxidase and the McAbs can be subdivided into four groups. Only group A inhibits peroxidase activity. Group B and D bind equally well to the native and the denatured form of cationic peroxidase, whereas the remaining McAbs react with more or less reduced affinity to the denatured antigen. Group C probably recognizes a conformation-dependent epitope. All the McAbs cross react weakly with the anionic peanut peroxidase, suggesting a structural nonidentity as well as some similarity between these two peroxidase isozymes. Cross reactivities of these McAbs with peroxidases of various plant species were also demonstrated. PMID- 16665675 TI - Inhibition of na/h antiport activity in sugar beet tonoplast by analogs of amiloride. AB - The effects of amiloride and a series of amiloride analogs have been tested on the Na(+)/H(+) antiport activity in intact vacuoles and tonoplast vesicles isolated from sugar beet cell suspension cultures. There is a competitive interaction between amiloride analogs and sodium. Substitution of one or both H atoms of the 5-amino group of amiloride (apparent K(i) about 150 micromolar) resulted in a 3- to 200-fold increase in inhibitory potency of the antiport activity. PMID- 16665676 TI - In organello transcription in maize mitochondria and its sensitivity to inhibitors of RNA synthesis. AB - Purified mitochondrial preparations from etiolated maize shoots support the incorporation of radioactivity from labeled UTP into RNA. The incorporation is linear with time for up to 2 hours, shows Michaelis-Menton kinetics with respect to the concentration of the labeled substrate, UTP, and has salt and pH optima which are different than those previously reported for RNA synthesis by isolated chloroplasts. When a crude mitochondrial preparation is subjected to isopycnic sucrose gradient centrifugation, the bulk of the RNA synthetic activity co sediments with mitochondrial marker enzymes and with the mitochondrial 26S and 18S rRNAs. Maize mitochondrial RNA synthesis is prevented by actinomycin D and ethidium bromide but unaffected by alpha-amanitin. It is strongly inhibited by rifampicin at concentrations which have no effect on nuclear and chloroplast RNA synthesis, but only moderately inhibited by rifampicin at concentrations which completely inhibit bacterial RNA synthesis. The optimization, cell fractionation, and inhibitor data all suggest that contaminating organelles and bacteria do not contribute appreciably to the RNA synthesis in purified mitochondrial preparations. PMID- 16665677 TI - Involvement of superoxide radical in extracellular ferric reduction by iron deficient bean roots. AB - The recent proposal of Tipton and Thowsen (Plant Physiol 79: 432-435) that iron deficient plants reduce ferric chelates in cell walls by a system dependent on the leakage of malate from root cells was tested. Results are presented showing that this mechanism could not be responsible for the high rates of ferric reduction shown by roots of iron-deficient bean (Phaseolus vulgaris L. var Prelude) plants. The role of O(2) in the reduction of ferric chelates by roots of iron-deficient bean plants was also tested. The rate of Fe(III) reduction was the same in the presence and in the absence of O(2). However, in the presence of O(2) the reaction was partially inhibited by superoxide dismutase (SOD), which indicates a role for the superoxide radical, O(2) ([unk]), as a facultative intermediate electron carrier. The inhibition by SOD increased with substrate pH and with decrease in concentration of the ferrous scavenger bathophenanthroline disulfonate. The results are consistent with a mechanism for transmembrane electron transport in which a flavin or quinone is the final electron carrier in the plasma membrane. The results are discussed in relation to the ecological importance that O(2) ([unk]) may have in the acquisition of ferric iron by dicotyledonous plants. PMID- 16665678 TI - Transient Induction of Phenylalanine Ammonia-Lyase and 4-Coumarate: CoA Ligase mRNAs in Potato Leaves Infected with Virulent or Avirulent Races of Phytophthora infestans. AB - Infection of potato leaves with the fungal pathogen Phytophthora infestans (Pi) resulted in the rapid stimulation of phenylpropanoid metabolism. Increases in the activities of several mRNAs, including those encoding phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), were detectable within a few hours postinoculation, as demonstrated by two-dimensional gel electrophoresis of proteins synthesized in vitro. This effect was closely mimicked by application of Pi culture filtrate through cut leaf stems. PAL and 4CL mRNA activities were also rapidly and transiently induced in potato cell suspension cultures by treatments with Pi culture filtrate or arachidonic acid. This induction was exploited to generate cDNA probes complementary to PAL and 4CL mRNAs. Blot hybridizations using these probes revealed almost immediate, transient and coordinate increases in the transcription rates and subsequent changes in the amounts of PAL and 4CL mRNAs in leaves treated with Pi culture filtrate. Similar changes in the mRNA amounts were found in infected leaves of potato cultivars carrying resistance genes R1 (cv Datura) or R4 (cv Isola), independent of whether a virulent or an avirulent Pi pathotype was used for inoculation. These results are discussed in relation to recent cytological observations with the same potato cultivars and Pi pathotypes. PMID- 16665679 TI - Synthesis of only two heat shock proteins is required for thermoadaptation in cultured cowpea cells. AB - Cell cultures of a heat sensitive genotype of cowpea (Vigna unguiculata) were adapted to tolerate moderate levels of heat by maintaining cells at 32, 36, and 38 degrees C over many cell generations. Cells adapted to 32 and to 36 degrees C did not produce the typical heat shock proteins (HSP). Cells adapted to 38 degrees C synthesized two new proteins, which appear to be a subset of the HSP. In many temperature sensitive organisms it is thought that HSP confer thermotolerance. However, we hypothesize that specific proteins are associated with heat tolerance in cowpea, other heat tolerant plants (species such as sorghum and millet), and adapted cells which provide them with enhanced heat tolerance. From present data we suggest two proteins (70 and 80 kilodaltons) are strongly associated with heat tolerance and heat adaptation. PMID- 16665680 TI - Changes in the level of free and ester indol-3yl-acetic Acid in growing maize roots. AB - The levels of free and ester-linked indole-3-acetic acid (IAA) in different parts of the maize root were measured using gas chromatography-mass spectrometry (selected ion monitoring). In roots of 2-day-old plants, the distribution of free and ester IAA differed both along the root and between stele and cortex. The levels of IAA and IAA esters were then measured in whole roots and in the elongation zone using roots of different ages. The level of ester IAA decreased steadily with time. In contrast, the level of free IAA in the elongation zone was found to increase after a few days of culture at which time the rate of root growth was decreasing. PMID- 16665681 TI - The development of an indirect enzyme linked immunoassay for abscisic Acid. AB - An indirect method of enzyme-linked-immunosorbent-assay (ELISA) is reported for abscisic acid (ABA), utilising a thyroglobulin-ABA conjugate for coating wells. The assay can use commercially available monoclonal antibodies, is sensitive to as little as 20 picograms ABA per well, and is much more conservative of antibody than direct methods. The most dilute ABA standards did not retain their antigenicity during storage, so ABA standard sets were diluted immediately prior to use. The indirect ELISA was used successfully to estimate ABA concentrations in developing cotyledons of Pisum sativum L., after only little preliminary purification. It was validated for this tissue through the use of gas chromatography-electron capture detection (GC-EC), and capillary GC-selected ion monitoring (GC-MS-SIM) using [(2)H(6)]ABA as an internal standard. Full spectrum GC-mass spectrometry was also used to verify that ABA was present in a sample assayed quantitatively by both ELISA and GC-MS-SIM. PMID- 16665682 TI - Regulation by Phospholipids and Kinetic Studies of Plant Membrane-Bound UDP Glucose Sterol beta-d-Glucosyl Transferase. AB - Solubilization and partial purification of the microsomal UDP-glucose sterol glucosyl transferase activity from maize coleoptiles by chromatography on DEAE cellulose resulted in a highly delipidated (>95%) and inactive enzymic preparation. Addition of sterols revealed part of the activity and subsequent addition of phospholipids further increased the activity. Negatively charged phospholipids were shown to be by far the best activators. The purification step also produced the elimination of two interfering microsomal enzymic activities: UDPase and steryl glucoside acyl transferase. The removal of these two enzymic activities was a prerequisite for kinetic studies including product-inhibition studies, since the substrates of these two latter enzymes are the products of UDPG-SGTase activity. The results of the kinetic studies strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. Finally the effect of different phospholipids on the kinetic parameters of the reaction was studied. Both phosphatidylcholine and phosphatidylglycerol significantly decrease K(m sterol) (and not K(m-UDPglucose)) and increase the reaction V(max). The decrease of K(m-sterol) is similar with both phospholipids whereas the increase of V(max) is much greater with phosphatidylglycerol than with phosphatidylcholine. PMID- 16665683 TI - Inhibition of pear fruit ripening by mannose. AB - Softening of the flesh and the rise in ethylene evolution and respiration associated with ripening in pear (Pyrus communis L.) fruit was delayed when mannose was vacuum infiltrated into intact fruit. The extent of delay could be modified by altering the concentration or the volume of mannose applied to the fruit. Inhibition of ripening was associated with phosphorylation of mannose to mannose 6-phosphate (M6P), and accumulation of M6P was associated with lowered levels of inorganic phosphate (Pi), glucose 6-phosphate (G6P), and ATP in the fruit tissue. Subsequently, however, as the M6P was metabolized, the levels of Pi, G6P, and ATP increased and ripening processes were concomitantly released from inhibition. Hence, the degree of inhibition by mannose or the release from inhibition was related to the level of M6P in the fruit and its rate of metabolism. The data provide correlative evidence to support a view that one inhibitory effect of mannose is depletion of Pi in the cell as a result of phosphorylation of mannose to M6P. Inhibition of ripening by mannose was not alleviated by co-application of glucose as a competitive substrate for the hexokinase(s), or by Pi, presumably the depleted metabolite. Also, incubation of tissue disks with M6P resulted in inhibition of ethylene production and respiration. The structural analogs of mannose, glucosamine, and 2-deoxyglucose, which have been shown to mimic mannose action in several plant tissues, did not cause inhibition of ripening of pear fruit comparable with that associated with mannose. Both analogs stimulated respiration, and glucosamine caused only a small inhibition of softening and ethylene evolution. Another mannose analog, alpha methylmannoside, did inhibit fruit ripening though to a lesser extent than mannose. Its influence was also associated with accumulation of M6P and a decrease of Pi levels. We conclude that the mannose effect may, in part, be due to M6P toxicity, as well as by depletion of Pi. PMID- 16665684 TI - Compartmental efflux analysis and removal of extracellular cadmium from roots. AB - Profiles of (109)Cd efflux from roots into three solutions were determined for young intact plants of Agrostis gigantea and maize. The solutions were (a) nutrient culture medium containing 3 micromolar Cd at room temperature, (b) ice cold 5 millimolar CaCl(2), and (c) ice-cold 5 millimolar PbCl(2). Efflux profiles were clearly resolved into three easily discernible components having fast, medium, and slow exchange rates. These results were unexpected for the situation where some intracellular Cd was present both as extractable Cd-binding peptide and in electron-dense granules within the cytoplasm and the vacuoles. Adding a fourth compartment to the curve-fitting model produced a splitting of the fast exchanging component. Use of these efflux kinetics to estimate Cd fluxes through membranes was inappropriate. However, they were useful in determining optimal washing times for the removal of extracellular Cd. A 10 minute wash in ice-cold 5 millimolar CaCl(2) is recommended for this purpose for Agrostis and maize roots. PMID- 16665685 TI - Factors affecting the activation state and the level of total activity of ribulose bisphosphate carboxylase in tobacco protoplasts. AB - The relationship between the activation state and the level of total activatable activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) was examined in tobacco protoplasts. When darkened protoplasts were illuminated, both activation and total activity increased, but at different rates; the t((1/2)) were 2.3 and 6.7 minutes, respectively. The light response of rubisco activation state and total activity, measured after 15 minutes of illumination, were similar but their responses to light transitions and photosynthetic inhibitors were different. When irradiance was reduced from saturating to subsaturating, deactivation of rubisco in protoplasts was immediate, whereas there was little change in total activity during the first 20 minutes following the transition. The light-induced increases in activation state and total activity were inhibited by nigericin, but activation was more sensitive exhibiting a response similar to that of photosynthesis. Treatment of tobacco protoplasts and leaves with methyl viologen at limiting irradiance increased rubisco activation, but inhibited the light-induced increase in total activity. These results indicate that light activation of rubisco is mechanistically distinct from the light-dependent changes in total activity in tobacco, a species containing carboxyarabinitol 1 phosphate, an endogenous inhibitor of total rubisco activity. PMID- 16665686 TI - Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX 2380). AB - We report the changes in the concentrations and (18)O contents of extracellular CO(2) and HCO(3) (-) in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring (18)O exchange between CO(2) and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO(2) hydration rate in different samples of cells that were grown on bubbled air (low-CO(2) conditions). This activity was only weakly inhibited by ethoxzolamide with an I(50) near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO(2)-starved cells there is considerable generation of CO(2) from intracellular stores, a factor that can cause errors in measurement of net CO(2) uptake unless accounted for. It was demonstrated that use of (13)C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO(2) readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO(3) ( ) was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO(3) (-) from Synechococcus. PMID- 16665687 TI - Subcellular Localization of Amines and Activities of Their Biosynthetic Enzymes in p-Fluorophenylalanine Resistant and Wild-Type Tobacco Cell Cultures. AB - Three levels of free amines and the activities of their biosynthetic enzymes were measured in subcellular fractions of two cell lines of Nicotiana tabacum L. cv Xanthi. The TX4 cell line, a p-fluorophenylalanine resistant culture which accumulates high levels of cinnamoylamides, was compared to the wild-type culture TX1. In cells harvested on day 6 of the growth cycle, nearly all free putrescine, spermidine, and tyramine was found in the supernatant fraction of both cell lines. Although a consistent portion of ornithine decarboxylase activity was detected in the nuclear-enriched fractions of TX1 and TX4, the largest levels of activity were in the supernatants of both lines. In TX1, arginine decarboxylase activity was low relative to that of ornithine decarboxylase, but, in the TX4 line arginine decarboxylase levels in the cytosol were substantially elevated. Tyrosine decarboxylase was not detected in 6-day-old TX1 cells, but significant amounts of activity were measured in the 1000g and supernatant fractions of TX4. S-Adenosylmethionine decarboxylase activity was low in both cell lines and was located predominantly in the supernatant. PMID- 16665688 TI - Quantification of abscisic Acid in a single maize root. AB - Quantitative analyses of abscisic acid in the elongating zone of a single maize root (Zea mays L. cv LG 11) were performed by gas chromatography-mass spectrometry using negative chemical ion ionization. Data showed that the more abscisic acid, the slower the growth, but a large dispersion of individual values was observed. We assume that abscisic acid is perhaps not correlated only to the growth rate. PMID- 16665689 TI - Uptake and accumulation of the herbicides chlorsulfuron and clopyralid in excised pea root tissue. AB - The herbicides chlorsulfuron and clopyralid were taken up rapidly by excised pea root tissue and accumulated in the tissue to concentrations ten and four times those in the external medium, respectively. Uptake was related linearly to external herbicide concentration over a wide concentration range, implying that transport across the membrane is by nonfacilitated diffusion. Uptake of both compounds was influenced by pH, with greatest uptake at low pH. The pH dependence of uptake suggests that the herbicides (both of which are weak acids) are transported across the plasma membrane in the undissociated form, and accumulate in the cytoplasm by an ion trap mechanism. Most of the absorbed herbicide effluxed from the tissue when it was transferred to herbicide-free buffer, indicating that the accumulation was not due to irreversible binding. Consequently, both herbicides remain available for transfer to the phloem. These results can explain the high reported phloem mobility of clopyralid in intact plants. The low phloem mobility of chlorsulfuron must be accounted for by factors that override its ability to accumulate in the symplast. PMID- 16665690 TI - Partial purification of gibberellin oxidases from spinach leaves. AB - Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA(12) --> GA(53) --> GA(44) - > GA(19) --> GA(20). Two of these, GA(53) oxidase and GA(19) oxidase, were separable from the other two, GA(44) oxidase and GA(12) 13-hydroxylase, by anion exchange high performance liquid chromatography (HPLC). Apparent molecular weights of the four enzymes as determined by gel filtration HPLC are: GA(12) 13 hydroxylase, 28,400; GA(53) oxidase, 42,500; GA(44) oxidase, 38,100; GA(19) oxidase, 39,500. GA(44) oxidase was purified approximately 100-fold in 0.3% yield by a combination of ammonium sulfate fractionation, anion exchange HPLC, phenyl Sepharose chromatography and gel filtration HPLC. PMID- 16665691 TI - Free amino Acid content and metabolic activities of setting and aborting soybean ovaries. AB - Fruits of soybean (Glycine max [L.] Merr.) that are destined to abscise shortly after anthesis grow more slowly than fruits that will be retained. In this work, amino acid composition, protein metabolism, and nucleic acid metabolism were studied in setting and abscising soybean ovaries from anthesis to 6 days after anthesis to provide additional evidence of chemical processes associated with abscission. Principal free amino acids were asparagine, aspartic acid, glutamic acid, serine, and glutamine. Percent aspartate and glutamate declined as the ovaries grew, with aspartate declining more in abscising and glutamate more in setting ovaries. Percent glutamate was positively correlated to percent abscission throughout the period. Proline, serine, and leucine were positively correlated to abscission from 0 to 2 days after anthesis, whereas significant negative correlations were observed at these ages for ethanolamine and arginine. (75)Se fed as selenate and (14)C fed as sucrose, glycine, and alanine were readily incorporated into soluble and insoluble proteins in a 24-hour in vitro incubation. Radioactivity of total proteins, expressed on a perovary basis, was negatively correlated with percent abscission and positively correlated with ovary weight. [(14)C]Glutamine and serine followed the opposite pattern, with greater protein labeling in abscising than in setting ovaries. When data were expressed as disintegrations per minute per milligram ovary fresh weight, protein labeling from alanine was seen to be significantly greater in abscising ovaries at anthesis and throughout the sampling period. Nucleic acid labeling from uridine was highly correlated to ovary weight; labeling from thymidine was greater in setting than abscising ovaries at anthesis and in abscising ovaries at later stages of development. It is concluded that abscising ovaries can continue amino acid metabolism almost up to the date of separation from the raceme, and that the involvement of alanine, glutamine, aspartate, glutamate, and other amino acids in soybean flower abortion deserves further study. PMID- 16665692 TI - Nitric Oxide Emissions from Soybean Leaves during in Vivo Nitrate Reductase Assays. AB - Recent work identified acetaldehyde oxime as the predominant product purged by inert gases from anaerobic in vivo nitrate reductase (NR) assays of soybean (Glycine max [L.] Merr.) leaves. Another recent study supported earlier research findings which identified the primary product evolved from soybean leaves as nitric oxide (NO). This paper provides evidence that eliminates acetaldehyde oxime and confirms that NO is the primary nitrogenous product purged from the in vivo NR assay system. A portion of the evidence is based on the high water solubility of acetaldehyde oxime. Other evidence presented is the failure by chemical and spectrophotometric means to detect oximes in gases emitted in the purging of the reaction medium or in the leaf tissues. The gaseous product from the in vivo NR assay system reacted identically to NO standards and did not resemble acetaldehyde oxime standards. It was concluded that the predominant N product within the leaves was nitrite and that the predominant gaseous N product evolved from the assay was NO. PMID- 16665693 TI - Citrate, Malate, and Succinate Concentration in Exudates from P-Sufficient and P Stressed Medicago sativa L. Seedlings. AB - Under certain stress conditions roots exude organic molecules, which may facilitate the uptake of nutrients. The objective of this research was to identify and measure the effect of low P upon the exudation of organic acids by roots of alfalfa (Medicago sativa L.) seedlings. Surface-sterilized alfalfa seeds were grown aseptically in sterile sand using an apparatus specially designed for the addition of +/-P nutrient solutions and for the collection of root exudates. Citric, malic, and succinic acids were detected in the root exudates of 24-day old alfalfa seedlings. Citrate exudation from the roots of P-stressed alfalfa was 182% that of plants receiving a complete nutrient solution. The increased release of citrate may provide a mechanism by which P-stressed plants enhance the availability of P in the rhizosphere. PMID- 16665694 TI - Promotion of Stomatal Opening by Indoleacetic Acid and Ethrel in Epidermal Strips of Vicia faba L. AB - Indole-3-acetic acid (IAA), at concentrations of 0.01 to 1.0 millimolar, and ethephon (0.3% v/v Ethrel) promote stomatal opening when applied to epidermal peels of Vicia faba L. in light or dark. The effect of ethylene is seen by 30 minutes and maximal opening (over two times that of untreated controls) occurs after only 60 to 90 minutes in the light. Stomatal opening by IAA and Ethrel in both light and dark is prevented by 0.14 millimolar AgCl. It is suggested that the effect of added IAA, but not that of light, is linked to ethylene production. The possible role of ethylene in stomatal opening during fungal infection is discussed. The stomates of Vicia faba provide a new system to study the effects of ethylene on certain membrane-regulated processes. PMID- 16665695 TI - Immunological conservation of phycobilisome rod linker polypeptides. AB - Monospecific polyclonal antibodies raised against the phycobilisome rod linker polypeptides of Nostoc sp. were used to assess structural similarities among phycobilisome linker proteins from diverse, phycobilisome-containing organisms. While a remarkable conservation of antigenic determinants was demonstrated for two of these rod linker proteins, the third linker polypeptide appeared to be conserved only among closely related species. PMID- 16665696 TI - Construction and Homologous Expression of a Maize Adh1 Based NcoI Cassette Vector. AB - The alcohol dehydrogenase I (Adh1) gene of maize (Zea mays L.) was employed as a source of transcriptional, posttranscriptional, and translational regulatory sequences in the construction of an expression vector. By transforming the translation-initiating ATG and an ATG three triplets upstream from the translational termination triplet into NcoI sites (5'-CCATGG-3'), the maize Adh1 gene was converted into a cassette vector allowing one-step placement of any structural gene under Adh1 regulatory control. We inserted the structural gene for chloramphenicol acetyl transferase (CAT) into this cassette vector and found that this construct expressed the cat gene when transfected into maize protoplasts. Significant expression was observed with a construct that contained only 146 base pairs of Adh1 sequence upstream of the transcription-initiation site. Derivatives with a further 266 or 955 base pairs of contiguous Adh1 upstream sequences increased CAT expression approximately 5-fold or 8-fold, respectively. PMID- 16665697 TI - Quantitative Estimates of Phosphorus Concentrations within Lupinus luteus Leaflets by Means of Electron Probe X-ray Microanalysis. AB - Phosphorus contents of epidermal vacuoles and mesophyll cells of Lupinus lutens leaflets were measured by electron probe x-ray microanalysis of fully hydrated, bulk frozen samples. Quantitation was achieved using standard solutions containing colloidal graphite to simulate cell contents and the derived nonlinear relationship between peak over background ratio and concentration improved the accuracy of the analytical procedure. Inorganic phosphate contents of mesophyll cells were shown to be highly dependent on phosphate nutrition. Comparison with data obtained from conventional analysis leads to the suggestion that in heterogeneous tissues the inorganic phosphate concentrations of the cytoplasm may show greater variation than observed in the cytoplasm of simple plant systems such as cell suspension and root tips. PMID- 16665698 TI - Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover. AB - Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24. PMID- 16665699 TI - Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L. AB - Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M(r). The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds. PMID- 16665700 TI - Far-Red Light Reflection from Green Leaves and Effects on Phytochrome-Mediated Assimilate Partitioning under Field Conditions. AB - The influence of plant spacing and row orientation on spectral distribution of light received by growing soybean (Gylcine max [L.] Merr.) plants was measured under field conditions. Light absorption, reflection and transmission of individual leaves showed that most of the blue and red was absorbed while most of the far-red was either reflected or transmitted. Plants growing in the field received different ratios of far-red relative to red, depending on nearness and/or orientation of other vegetation. Plants grown in close-spaced rows, or high population densities, received higher far-red/red ratios than did those grown in wide rows, or sparse populations. Heliotropic movements of the leaves also contributed to the far-red reflection patterns associated with row orientation. Under field conditions, differences in far-red/red ratios associated with nearness of competing vegetation became more pronounced with low solar angle near the end of the day. Plants exposed to far-red for 5 minutes at the end of each day in controlled environments, and those grown in close-spaced rows in the field, developed longer internodes and fewer branches. Red, far-red photoreversibility in the controlled environment study indicated involvement of phytochrome. Dry matter partitioning among plant components in the field was related to far-red/red light ratio received during growth and development. PMID- 16665701 TI - The Nitrogen Use Efficiency of C(3) and C(4) Plants : III. Leaf Nitrogen Effects on the Activity of Carboxylating Enzymes in Chenopodium album (L.) and Amaranthus retroflexus (L.). AB - The relationships between leaf nitrogen content per unit area (N(a)) and (a) the initial slope of the photosynthetic CO(2) response curve, (b) activity and amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC), and (c) chlorophyll content were studied in the ecologically similar weeds Chenopodium album (C(3)) and Amaranthus retroflexus (C(4)). In both species, all parameters were linearly dependent upon leaf N(a). The dependence of the initial slope of the CO(2) response of photosynthesis on N(a) was four times greater in A. retroflexus than in C. album. At equivalent leaf N(a) contents, C. album had 1.5 to 2.6 times more CO(2) saturated Rubisco activity than A. retroflexus. At equal assimilation capacities, C. album had four times the Rubisco activity as A. retroflexus. In A. retroflexus, a one to one ratio between Rubisco activity and photosynthesis was observed, whereas in C. album, the CO(2) saturated Rubisco activity was three to four times the corresponding photosynthetic rate. The ratio of PEPC to Rubisco activity in A. retroflexus ranged from four at low N(a) to seven at high N(a). The fraction of organic N invested in carboxylation enzymes increased with increased N(a) in both species. The fraction of N invested in Rubisco ranged from 10 to 27% in C. album. In A. retroflexus, the fraction of N(a) invested in Rubisco ranged from 5 to 9% and the fraction invested in PEPC ranged from 2 to 5%. PMID- 16665702 TI - Localization of Carbohydrate Metabolizing Enzymes in Guard Cells of Commelina communis. AB - The localization of enzymes involved in the flow of carbon into and out of starch was determined in guard cells of Commelina communis. The guard cell chloroplasts were separated from the rest of the cellular components by a modification of published microfuge methods. The enzymes of interest were then assayed in the supernatant and chloroplast fractions. The chloroplast yield averaged 75% with 10% cytoplasmic contamination. The enzymes involved in starch biosynthesis, ADPglucose pyrophosphorylase, starch synthase, and branching enzyme, are located exclusively in the chloroplast fraction. The enzymes involved in starch degradation show a more complex distribution. Phosphorylase is located in both the supernatant and chloroplast fraction, 50% in each fraction. Most of the amylase and debranching enzyme activity is present in the supernatant (70%) fraction. The majority of the rest of the enzymes involved in the degradation of starch to malate and synthesis of starch from a hexose precursor were also investigated. All of the enzymes were present in the chloroplast except for hexokinase and phosphofructokinase. The inability to assay these enzymes could possibly have been due to the lack of or low activity of the enzymes or to nonoptimal assay conditions. PMID- 16665703 TI - Glyphosate effects on carbon assimilation and gas exchange in sugar beet leaves. AB - The mechanism responsible for the inhibition of net carbon exchange (NCE) which was reported previously (DR Geiger et al. 1986 Plant Physiol 82: 468-472) was investigated by applying glyphosate [N-(phosphonomethyl)glycine] to exporting leaves of sugar beet (Beta vulgaris L.). Leaf internal CO(2) concentration (C(i)) remained constant despite decreases in stomatal conductance and NCE following glyphosate treatment, indicating that the cause of the inhibition was a slowing of carbon assimilation rather than decreased conductance of CO(2). Throughout a range of CO(2) concentrations, NCE rate at a given C(i) declined gradually, with the time-series of response curves remaining parallel. Gas exchange measurements revealed that disruption of chloroplast carbon metabolism was an early and important factor in mediating these glyphosate effects, perhaps by slowing the rate of ribulose bisphosphate regeneration. An increase in the CO(2) compensation point accompanied the decrease in NCE and this increase was hastened by stepwise lowering of the ambient CO(2) concentration. Eventually the CO(2) compensation point approached the CO(2) level of air and the difference between internal and external CO(2) concentrations decreased. In control and in glyphosate-treated plants, both carbon assimilation and photorespiration at atmospheric CO(2) level were inhibited to a similar extent of air level of O(2). Maintaining leaves in low O(2) concentration did not prevent the decline in NCE rate. PMID- 16665704 TI - Glyphosate effects on carbon assimilation, ribulose bisphosphate carboxylase activity, and metabolite levels in sugar beet leaves. AB - Application of a 17-millimolar solution of glyphosate (GLP) to sugarbeet (Beta vulgaris L.) leaves resulted in an immediate and rapid decline in the level of ribulose bisphosphate (RuBP). Phosphoglyceric acid level began to decrease about 2 hours following the decline in RuBP level. Photosynthesis rate declined linearly with RuBP level, but only when the RuBP level had decreased to about twice the RuBP carboxylase active site concentration. This occurred about 4 hours following GLP-application. At this time starch synthesis also declined abruptly. The activation state of RuBP carboxylase did not change for 8 hours following GLP application and then decreased slightly from 70 to 50% when the RuBP level fell below the RuBP carboxylase active-site concentration. Triose-phosphate, hexose phosphate, and adenylate energy charge did not change for 8 hours following GLP application. These data indicate that GLP induced a depletion of carbon or phosphate or both from the photosynthetic carbon reduction cycle, reducing the rate of regeneration of RuBP, photosynthesis, and starch synthesis, while having little effect upon the rate of sucrose synthesis and transport. PMID- 16665705 TI - Characterization of Nitrate Reductases from Corn Leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris: Sensitivity to a Proteinase Extracted from Corn Roots. AB - The sensitivity of the two forms of nitrate reductase, NR(I) and NR(II), obtained from the primary leaf of corn, to a limited action corn root proteinase has been examined. The corn inactivating protein (CIP) inhibited the overall reaction (NADH-NR) and the two partial reactions, cytochrome c reductase and reduced methyl viologen NR (MV-NR) of both forms of NR. NADH-cytochrome c reductase was more sensitive to the protease than MV-NR. NR(II) was less sensitive to inactivation than NR(I). When NR(I) and NR(II) were inactivated and then subjected to native gel electrophoresis the protein bands associated with MV-NR activity shifted from an R(m) value of 0.32 to 0.61 for NR(I) and from an R(m) of 0.28 to 0.60 for NR(II). For Chlorella NR these values are 0.32 and 0.70. The initial cleavage of the 116 kilodalton subunit of NR(I) yielded fragments of 84 and 80 kilodaltons after a 5 minute incubation with CIP. With longer incubation times smaller fragments were also identified. For the Chlorella NR the initial cleavage products are approximately 68 and 25 kilodaltons. Longer incubation times also led to smaller fragments. The products of hydrolysis by this limited action protease are quite different for the corn and Chlorella NRs. PMID- 16665706 TI - Phototropism in Hypocotyls of Radish: IV. Flank Growth and Lateral Distribution of cis- and trans-Raphanusanins in the First Positive Phototropic Curvature. AB - The first positive phototropic curvature induced by a pulse of unilateral white irradiation (0.1 watt per square meter, 30 seconds) of etiolated and de-etiolated Sakurajima radish (Raphanus sativus var hortensis f. gigantissimus Makino) hypocotyls was analyzed in terms of differential growth and growth inhibitor contents of the hypocotyls. In both etiolated and de-etiolated hypocotyls, the growth rates at the lighted sides were suppressed whereas those at the shaded ones showed no change. De-etiolation treatment induced a larger difference between the growth rates at the lighted and shaded sides of the hypocotyls, resulting in a larger curvature of de-etiolated seedlings than of etiolated ones. The contents of growth inhibitors, cis- and trans-raphanusanins, increased in the lighted but not in the shaded halves of the hypocotyls of etiolated seedlings. In de-etiolated seedlings, the two inhibitors increased due to the de-etiolation treatment. When de-etiolated seedlings were exposed to a pulse of unilateral irradiation the level of the two inhibitors remained high along the lighted side for 1 h following the light pulse, whereas at the shaded side the contents of the inhibitors abruptly decreased upon transfer to the dark, the difference between their amounts in the lighted and shaded sides being larger than in etiolated seedlings. Another growth inhibitor, raphanusamide, did not respond to the phototropic stimulus, although its amounts increased by the de-etiolation treatment. These data suggest that cis- and trans-raphanusanins are involved in the first positive phototropic response of radish hypocotyls, and that de etiolation magnifies the phototropic response through induction of a larger lateral gradient of the raphanusanins in the hypocotyls by the phototropic stimulus. PMID- 16665707 TI - Osmotically induced proton extrusion from carrot cells in suspension culture. AB - Addition of 200 mm of a polyol to anthocyanin containing carrot (Daucus carota L.) cells in suspension culture decreased turgor pressure to zero and induced hyperpolarization of the membrane potential and acidification of the medium due to H(+) extrusion. These changes were shown to be slightly affected by vanadate. In parallel, a decrease in intracellular ATP and total adenylate concentrations were observed. However, when the osmoticum was NaCl acidification of the medium occurred in the absence of considerable changes in intracellular ATP concentration. These results are interpreted as indicating that a drop of turgor, by addition of a polyol, triggers a proton extrusion activity which is only slightly inhibited by vanadate but apparently ATP utilizing. The observed decrease in ATP level occurs without a change in respiration rate and is accompanied by a drop in total adenylate pool. However when NaCl is the osmoticum it is assumed that Delta(muH+) is enhanced through a Na(+)/H(+) antiporter. The difference between the two types of osmotica as related to their ability to penetrate through the cellular membrane is discussed. PMID- 16665708 TI - Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells. AB - Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo [2-(3)H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [(3)H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)). An additional [(3)H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP(2) on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction. PMID- 16665709 TI - Master Regulatory Genes, Auxin Levels, and Sexual Organogeneses in the Dioecious Plant Mercurialis annua. AB - In Mercurialis annua L. (2n = 16) genes for sex determination are considered as major regulator genes controlling stamen and ovary development and sexual phenotypes. After stamen induction, sterility determinants control sporogenous tissue and pollen formation. Moreover, exogenous auxins are able to induce male flowers on female plants. In order to verify if sex and sterility genes have an effect on indole-3-acetic acid (IAA) contents of these plants, various wild or genetically constructed strains were assayed. The IAA levels of their apices were determined by HPLC followed by gas chromatography, selected ion monitoring, mass spectrometry. Results show that high auxin levels are linked to male phenotypes. The genes inducing maleness and the determinants of restored male fertility appear to control and modulate the IAA content. Close correspondence between the number of these dominant genes and IAA levels was established. A final hypothesis about the control of sexual specialization by phytohormones induced by the presence of these genes is discussed. PMID- 16665710 TI - Protein synthesis associated with quiescence and senescence in auxin-starved pear cells. AB - Pear fruit cells (Pyrus communis L. cv Passe Crassane) stopped dividing when subcultured in a bioreactor under auxin starvation in the presence of 0.37 molar mannitol. The cessation of cell division was preceded by the accumulation of a specific basic polypeptide of 24 kilodalton. Readdition of 2.3 micromolar 2,4 dichlorophenoxyacetic acid (2,4-D) neither caused a resumption of cell division nor depressed the accumulation of this polypeptide. Under complete auxin starvation, cells began to die at day 18. In vivo radioactive labeling of proteins followed by two-dimensional electrophoresis showed that during auxin starvation the synthesis of some polypeptides including the 24 kilodalton one (referred to as homeostasis-related proteins, HRPs) was decreased while the synthesis of some others (referred as senescence-related proteins, SRPs) was increased. Readdition of 2.3 micromolar 2,4-D postponed the onset of cell death by 10 to 15 days while supplementation with 7.6 micromolar abscisic acid advanced cell death by 8 days. Two-dimensional analysis of protein synthesis indicated that both hormones interact on the synthesis of these two groups of polypeptides. The levels of most HRPs were maintained or increased in the presence of auxin, while the levels of the SRPs were decreased by auxin and increased by abscisic acid. Short and long-term effects of 2,4-D and abscisic acid on the synthesis of specific polypeptides were observed, allowing a discrimination between the direct and indirect effect of both hormones on the development of cell senescence. PMID- 16665711 TI - Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves : estimation by alternate substrate utilization. AB - The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1'-deoxy-1'-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [(14)C]Sucrose and [(14)C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [(14)C]sucrose was supplied than when [(14)C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%. PMID- 16665712 TI - Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts. AB - Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K(+) and Cl(-), respectively. Whole cell K(+) currents activated exponentially during step depolarizations, while voltage-dependent Cl(-) channels were activated by hyperpolarizations. Single K(+) channel conductance was 40 +/- 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs(+) and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 +/- 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn(2+) and ethacrynic acid. Whole-cell Cl(-) currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development. PMID- 16665713 TI - Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring. AB - Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [(2)H(4)]IAA (m/z 194, 134) or [(2)H(5)]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [(2)H(3)]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue. PMID- 16665714 TI - Influence of mannose on the apoplasmic retrieval systems of source leaves. AB - Experiments were conducted in which d-mannose was supplied to mature Beta vulgaris L. (sugar beet) leaves, via the transpiration stream, to perturb photosynthetic carbon allocation by sequestering cytosolic Pi. Biochemical and enzymic analyses conducted on this tissue indicated that mannose 6-P was present, that it was only slowly metabolized, and that after a 24-hour pretreatment sugar metabolism was slightly perturbed. However, sucrose retrieval by the mesophyll tissue was greatly impaired in 24-hour mannose-pretreated tissue, a response which was due in part to mannose acting as an osmoticum. Inhibition of glucose, fructose, and arginine uptake into mannose-treated sugar beet leaf discs indicated that mannose may elicit a general perturbation of all membrane transport processes. This conclusion was supported by our finding that sucrose efflux was increased from mannose-treated tissue. Analysis of adenine nucleotide levels showed that whereas these levels declined over the first 3 to 6 hours of the mannose treatment, by 24 hours they had recovered to near control values. Similar experiments conducted on Nicotiana rustica indicated that whereas mannose 6-P was present in mature leaves, it remained at a much lower level than that found in sugar beet. Sucrose uptake into N. rustica was insensitive to mannose pretreatment. However, glucosamine treatment, which is also thought to sequester cytosolic Pi, inhibited sucrose uptake in both N. rustica and B. vulgaris. Further, experiments conducted on N. tabacum L. var Xanthii showed that mannose caused an inhibition of sucrose uptake, indicating that a range of sensitivity to mannose exists between closely related species. These results are discussed in terms of possible mechanisms of inhibition. PMID- 16665715 TI - Relationship between Salt Tolerance and Resistance to Polyethylene Glycol-Induced Water Stress in Cultured Citrus Cells. AB - Salt-tolerant selected cells of Shamouti orange (Citrus sinensis) and Sour orange (Citrus aurantium) grew considerably better than nonselected cells at any NaCl concentration tested up to 200 millimolar. Also, the growth response of each treatment was identical in the two species. However, the performance of cells of the two species under osmotic stress induced by polyethylene glycol (PEG), which is presumably a nonabsorbed osmoticum, was significantly different. The nonselected Shamouti cell lines were significantly more sensitive to osmotic stress than the selected cells. The salt adapted Shamouti cells were apparently also adapted to osmotic stress induced by PEG. In Sour orange, however, the selected lines had no advantage over the nonselected line in response to osmotic stress induced by PEG. This response was also similar quantitatively to the response of the selected salt-tolerant Shamouti cell line. It seems that the tolerance to salt in Shamouti, a partial salt excluder, involves an osmotic adaptation, whereas in Sour orange, a salt accumulator, such an adaptation apparently does not occur. PEG-induced osmotic stress causes an increase in the percent dry weight of salt-sensitive and salt-tolerant cells of both species. No such increase was found under salt stress. The size of control and stressed cells is not significantly different. PMID- 16665716 TI - Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations. AB - Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl(2). On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl(2) the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K(+) concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl(2) and probably played a key role in determining growth capacity. (b) Internal concentrations of Na(+), Ca(2+), or Cl(-) could not be directly correlated with growth. (C) Internal Mg(2+) concentration could be significant only in the presence of high external Ca(2+) concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca(2+))/(Na(+))(2) concentration is crucial for growth. Ratios above 0.5 x 10( 4) per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca(2+))/(Na(+))(2) ratio and the absolute external concentration of NaCl. However, a correlation between internal K(+) concentration and growth capacity seemed independent of external NaCl concentration. PMID- 16665717 TI - Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons. AB - The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulselabeled with [(35)S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, gamma and delta. The pH optimum for the vacuole mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg(2+), and Cu(2+), but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles. PMID- 16665718 TI - Reduction of purothionin by the wheat seed thioredoxin system. AB - Thioredoxin h, the thioredoxin characteristic of heterotrophic plant tissues, was purified to homogeneity from wheat endosperm (flour) and found to resemble its counterpart from carrot cell cultures. In the presence of NADPH, homogeneous thioredoxin h and partially purified wheat endosperm thioredoxin reductase (NADPH), (EC 1.6.4.5), purothionin promoted the activation of chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11). Under these conditions, NADPH provided the reducing equivalents for a series of thiol reactions in which (a) thioredoxin reductase reduced thioredoxin h thereby converting it from disulfide (S-S) to sulfhydryl (SH) form; (b) the sulfhydryl form of thioredoxin h reduced the disulfide form of purothionin-a 5 kilodalton seed storage protein with 4 S-S bridges; and (c) the sulfhydryl form of purothionin reductively activated fructose-1,6-bisphosphatase. The results show that, since thioredoxin h does not react effectively with fructose-1,6-bisphosphatase, the thioredoxin system can activate an enzyme through purothionin by secondary thiol redox control. In a related type reaction, purothionin, inhibited the activity of either Escherichia coli or calf thymus ribonucleotide reductase with reduced thioredoxin as hydrogen donor. The results suggest that purothionin competes with ribonucleotide reductase for reducing equivalents from thioredoxin. Thus, inhibition of deoxyribonucleotide synthesis should be considered a possible mechanism when examining the toxic effects of purothionin on mammalian cells in S-phase. PMID- 16665719 TI - Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue. AB - The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5'-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5' nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25 fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5'-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O(2) concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O(2). 5'-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O(2) concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O(2) concentration. PMID- 16665720 TI - Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum. AB - In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b(5) and NADH-cytochrome b(5) reductase from the microsomal fraction of Pisum sativum. The cytochrome b(5) component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b(5) component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer. PMID- 16665721 TI - Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy. AB - Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-(13)C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-(13)C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the (13)C NMR spectra due to loss of the label as (13)CO(2). Uptake of [3-(13)C]pyruvate by the cells was considerably slower than with [2-(13)C]acetate; however, the labelling patterns were similar with the exception of increased [3-(13)C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-(13)C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine. PMID- 16665722 TI - Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor. AB - Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [(14)C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [(14)C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [(14)C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [(3)H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures. PMID- 16665723 TI - Activities of isolated mitochondria and mitochondrial enzymes from aerobically and anaerobically germinated barnyard grass (echinochloa) seedlings. AB - Activity of mitochondria isolated from whole seedlings of Echinochloa crus-galli (L.) Beauv. var oryzicola germinated under aerobic and anaerobic conditions for 5 to 7 days was investigated. Mitochondria from both treatments exhibited good respiratory control and ADP/O ratios. Although O(2) uptake was low in anaerobic mitochondria, activity rapidly increased when the seedlings were transferred to air. Mitochondria from both aerobically and anaerobically grown seedlings of E. crus-galli var oryzicola maintained up to 66% of their initial respiration rate in the presence of both cyanide and salicylhydroxamic acid, and the inhibitory effects of cyanide and azide were additive. In addition, antimycin A was not an effective inhibitor of respiration. Reduced-minus-oxidized absorption spectra revealed that cytochromes a, a(3), and b were reduced to a greater extent and cytochrome c was reduced to a lesser extent in anaerobically germinated seedlings relative to that in aerobically germinated seedlings. An absorption maximum in the cytochrome d region of the spectrum was reduced to the same extent under both germination conditions and an absorption maximum at 577 nm was present only in anaerobically germinated seedlings. Anaerobically germinated seedlings contained 70% of the cytochrome c oxidase activity found in air grown seedlings. Upon exposure to air, the developmental pattern of this enzyme in anaerobically germinated seedlings was similar to air controls. Succinate dehydrogenase activity in anaerobic seedlings was only 45% of the activity found in aerobically germinated seeds, but within 1 hour of exposure to air, the activity had increased to control levels. The results suggest that mitochondria isolated from E. crus-galli var oryzicola differ from other plants studied and that the potential for mitochondrial function during anaerobiosis exists. PMID- 16665724 TI - Physiological Changes in Portulacaria afra (L.) Jacq. during a Summer Drought and Rewatering. AB - The changes of titratable acidity, enzyme activity, water status, and pigment composition were studied in Portulacaria afra (L.) Jacq. during a normal summer drought and rewatering. Two groups of plants were grown outside under a clear plastic canopy with water stress initiated at 2-week intervals in May 1986. Drought resulted in a linear decrease of fresh weight for 80 days and there was no further fresh weight change for the next 65 days. Nocturnal CO(2) uptake remained measurable for 83 days. Cessation of exogenous CO(2) uptake corresponded to the point where the pressure potential (Psi(p)) became zero. Ribulose-1, 5 bisphosphate (RuBP) and phosphoenolpyruvate carboxylase were reduced to 50% of this activity by the end of the drought period. Phosphoenolpyruvate carboxykinase activity was undetectable after 120 to 140 days of drought. Chlorophyll (Chl) levels decreased with a preferential loss of Chl a over Chl b. Carotenoid content was relatively constant over the course of the drought period. After 145 days of drought, plants responded to rewatering within 24 hours; Psi(p) became positive and daytime CO(2) uptake resumed after 24 hours. After 3 days, RuBP carboxylase activity reached control levels. Activity of the CAM pathway recovered after 5 days, as noted by increased diurnal acid fluctuations. Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase activity fully recovered within 6 days. Chl levels were greater than control levels within 5 days. Chl a/b ratios took 27 days to return to control levels. The results indicated that P. afra can withstand a normal summer drought by utilizing the CAM and CAM-idling pathway for 130 to 140 days. The plants respond rapidly to rewatering because of the conservation of enzyme activity and the quick recovery of Psi(p). PMID- 16665725 TI - Does gibberellic Acid induce the transfer of lipase from protein bodies to lipid bodies in barley aleurone cells? AB - We have examined the effect of gibberellic acid (GA(3)) on the distribution of the enzyme responsible for mobilizing storage triacylglycerol in aleurone cells of Hordeum vulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA(3) for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism. PMID- 16665726 TI - Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study. AB - The effect of temperature in the range from 10 to 35 degrees C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C(4) plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V(max) for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C(4) enzyme has an inflection at 27 degrees C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K(m) for the two enzymes reveals the startling fact that at temperatures above 20 degrees C an increasing temperature causes an increase in K(mPEP) for the CAM enzyme while the C(4) enzyme displays a decreased K(m) as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15 degrees C to 70% at 35 degrees C. For the C(4) enzyme the percent inhibition drops from about 60% at 20 degrees C to 2% at 30 degrees C. Similar opposite behavior of the two enzymes is found with the K(i) for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25 degrees C. PMID- 16665727 TI - Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits. AB - Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant. PMID- 16665728 TI - Monoclonal Antibodies to Glycoprotein Antigens of a Fungal Plant Pathogen, Phytophthora megasperma f. sp. glycinea. AB - Genetic studies of plants and their pathogens indicate that dominant alleles for resistance in hosts are complemented by corresponding dominant alleles for avirulence in pathogens. Products of these genes have not yet been identified. We have produced murine monoclonal antibodies (mAbs) to extracellular antigens of the fungal soybean pathogen Phytophthora megasperma f. sp. glycinea (Pmg, race 1) as part of a larger effort to identify antigenic determinants associated with particular avirulence genes. Thirty-six independent mAbs have been characterized by binding to Western blots of Pmg extracellular glycoproteins and by enzyme linked immunosorbent assay with glycoproteins modified by treatment with periodate, alpha-mannosidase, and endo-beta-N-acetylglucosaminidase H. The mAbs are predominantly carbohydrate-specific and can be placed in six groups based on interactions with Pmg glycoproteins. Binding patterns of various mAbs to Western blots indicate that Pmg proteins may have single or multiple types of attached carbohydrate antigens. Races of Pmg with differing avirulence genes exhibit more characteristic differences by Western blot analysis than by protein staining of glycoprotein profiles. Several of the mAbs show much higher reaction levels to glycoproteins from race 1 than from two other races. All of the glycoprotein specific mAbs cross-react with purified mycelial walls. PMID- 16665729 TI - CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue. AB - A method for the solubilization and reconstitution of red beet (1,3)-beta-d glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1 propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg(2+) prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment. PMID- 16665730 TI - Involvement of Macromolecule Biosynthesis in Auxin and Fusicoccin Enhancement of beta-Glucan Synthase Activity in Pea. AB - In pea stem segments whose cuticle has been made permeable by abrading it, actinomycin D (ActD) and 80S ribosomal protein synthesis inhibitors such as cycloheximide (CHI) inhibit enhancement by indole 3-acetic acid (IAA) of the activity of the cell wall biosynthetic enzyme, glucan synthase I (GS). This supersedes earlier, negative results with inhibitors, obtained with segments having an intact cuticle, which prevents adequate inhibitor uptake. Since these inhibitors also block IAA-stimulated H(+) extrusion, which according to earlier results is involved in the GS response, the significance of these inhibitions would be ambiguous without additional evidence. ActD does not inhibit fusicoccin (FC) enhancement of GS activity, which indicates existence of a post transcriptional control mechanism for GS, but does not preclude involvement of transcription in the GS response to IAA. Although protein synthesis inhibitors such as CHI do not block FC-stimulated H(+) extrusion, they do inhibit FC enhancement of GS activity, indicating an involvement of protein synthesis in the GS response to FC, and presumably also to IAA. However, protein synthesis inhibitors (but not ActD) by themselves paradoxically elevate GS activity, less strongly than IAA does but resembling the IAA enhancement in several characteristics. These results suggest that IAA may enhance GS activity at least in part by inhibiting the synthesis or action of a labile repressor of the transcription of, or a labile destabilizer of, mRNA for GS or some polypeptide that enhances GS activity. However, resemblances between the IAA and FC effects on GS suggest that IAA also has a posttranscriptional GS-enhancing action like that of FC. Lipid biosynthesis may be involved in this aspect of the response since both IAA and FC enhancements of GS activity are inhibited by cerulenin. PMID- 16665731 TI - Characterization of osmotin : a thaumatin-like protein associated with osmotic adaptation in plant cells. AB - Cultured tobacco (Nicotiana tabacum var Wisconsin 38) cells adapted to grow under osmotic stress synthesize and accumulate a 26 kilodalton protein (osmotin) which can constitute as much as 12% of total cellular protein. In cells adapted to NaCl, osmotin occurs in two forms: an aqueous soluble form (osmotin-I) and a detergent soluble form (osmotin II) in the approximate ratio of 2:3. Osmotin-I has been purified to electrophoretic homogeneity, and osmotin-II has been purified to 90% electrophoretic homogeneity. The N-terminal amino acid sequences of osmotins I and II are identical through position 22. Osmotin-II appears to be much more resistant to proteolysis than osmotin-I. However, it cross-reacts with polyclonal antibodies raised in rabbits against osmotin-I. Osmotin strongly resembles the sweet protein thaumatin in its molecular weight, amino acid composition, N-terminal sequence, and the presence of a signal peptide on the precursor protein. Thaumatin does not cross-react with antiosmotin. An osmotin solution could not be detected as sweet at a concentration at least 100 times that of thaumatin which could be detected as sweet. Immunocytochemical detection of osmotin revealed that osmotin is concentrated in dense inclusion bodies within the vacuole. Although antiosmotin did not label organelles, cell walls, or membranes, osmotin appeared sparsely distributed in the cytoplasm. PMID- 16665733 TI - Growth Rates and Carbohydrate Fluxes within the Elongation Zone of Tall Fescue Leaf Blades. AB - Investigations were performed to better understand the carbon economy in the elongation zone of tall fescue leaf blades. Plants were grown at constant 21 degrees C and continuous 300 micromoles per square meter per second photosynthetic photon flux density where leaf elongation was steady for several days. Elongation occurred in the basal 20 mm of the blade (0-20 millimeters above the ligule) and was maximum at 9 to 12 millimeters. Eight 3-millimeter long segments were sampled along the length of the elongation zone and analyzed for water-soluble carbohydrates. Sucrose concentration was high in the zone of cell division (0-6 millimeters) whereas monosaccharide concentration was high at and distal to the location where cell elongation terminated (20 millimeters). Fructan concentration increased in the basal part, then remained constant at about 85% of the total mass of water-soluble carbohydrates through the remainder of the elongation zone. Data on spatial distribution of growth velocities and substance contents (e.g. microgram fructan per millimeter leaf length) were used to calculate local net rates of substance deposition (i.e. excess rates of substance synthesis and/or import over substance degradation and/or export) and local rates of sucrose import. Rates of sucrose import and net deposition of fructan were positively associated with local elongation rate, whereas net rates of sucrose deposition were high in the zone of cell division and those of monosaccharide were high near the termination of elongation. At the location of most active elongation imported sucrose (29.5 milligrams per square decimeter per hour) was used largely for synthesis of structural components (52%) and fructan (41%). PMID- 16665732 TI - The Effect of Pseudomonas putida Colonization on Root Surface Peroxidase. AB - Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions. PMID- 16665734 TI - Acid-Induced Stomatal Opening in Vicia faba L. and the Role of Guard Cell Wall Elasticity. AB - When epidermal peels of Vicia faba L. were treated with solutions of varying pH, stomatal aperture was significantly increased at pH 4.0, 3.0, and 2.7 in darkness, but not in light. This effect was independent of the presence of KCl in the medium. Using a short-term plasmolytic method, estimates were made of the osmotic pressure (II(i)) and the volumetric elastic modulus of guard cells, the aperture of which varied due to pretreatments at different pH, in darkness or light. In darkness, the lower pH pretreatments induced an increase in II(i) and a decrease in volumetric elastic modulus. In comparison to the response in unbuffered solutions, 10 and 25 millimolar Mes buffer at pH 6.5 significantly reduced the degree of stomatal opening induced by light or by fusicoccin. These results indicate that acid-induced stomatal opening is, at least partially, due to an increase in guard cell wall elasticity which occurs in association with changes in II(i). It is suggested that the observed inhibitory effect of Mes buffer on stomatal opening may be due to a reduction in the degree of acidification of the guard cell wall and a consequent decrease of cell wall elasticity. PMID- 16665735 TI - Assimilate Unloading from Maize (Zea mays L.) Pedicel Tissues : II. Effects of Chemical Agents on Sugar, Amino Acid, and C-Assimilate Unloading. AB - Sugar, amino acid, and (14)C-assimilate release from attached maize (Zea mays L.) pedicels was studied following treatment with several chemical inhibitors. In the absence of these agents, sugar release was nearly linear over a 7-hour period. At least 13 amino acids were released with glutamine comprising over 30% of the total. Release was not affected by potassium concentration, 10-minute pretreatments with p-chloromercuribenzene sulfonic acid (PCMBS) or dithiothreitol, and low concentrations of CaCl(2). Three hours or more exposure to PCMBS, dinitrophenol, N-ethylmaleimide, or 2,4,6-trinitrobenzene sulfonic acid strongly inhibited (14)C-assimilate, sugar, and amino acid release from the pedicel. These treatments also reduced (14)C-assimilate movement into the kernel bases. It is, therefore, likely that reduced unloading, caused by these relatively long-term exposures to chemical inhibitors, was related to reduced translocation of assimilates into treated kernels. Whether this effect is due to disruption of kernel metabolism and sieve element function or reduced assimilate unloading and subsequent accumulation of unlabeled assimilates within the pedicel tissues cannot be determined at this time. PMID- 16665736 TI - Leaves of the Orchid Twayblade (Listera ovata) Contain a Mannose-Specific Lectin. AB - A new lectin was isolated from leaves of the twayblade (Listera ovata). It is a dimeric protein built up of two subunits of M(r) 12,500. This lectin, which is the first to be isolated from a species of the family Orchidaceae, exhibits exclusive specificity towards mannose. PMID- 16665737 TI - Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants. AB - The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O(2) (-), OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L. PMID- 16665738 TI - Establishment of thermotolerance in maize by exposure to stresses other than a heat shock does not require heat shock protein synthesis. AB - Maize (Zea mays) seedlings were pretreated prior to heat shock with either a progressive water stress of -0.25 megapascal PEG/hour from 0 to -1.25 megapascal over a 6-hour time period, or various concentrations of copper, cadmium, or zinc for 4 days. When the subsequent heat shock of 40 or 45 degrees C was administered for 3 hours, the seedlings showed an induced thermotolerance to these temperatures, which were otherwise lethal to control (water grown) seedlings. Thermotolerance was exhibited by both the root and the shoot of pretreated seedlings, even though the water and heavy metal stresses were applied only to the roots. Neither of these pretreatments had induced the synthesis of detectable levels of heat shock proteins (Hsps) at the time of heat shock. Pretreatment of seedlings with a progressive heat shock of 2 degrees C/hour from 26 to 36 degrees C, which did induce Hsps 18, 70, and 84, resulted in tolerance of a severe water stress of -1.5, -1.75, or -2.0 megapascal for 24 hours. But these seedlings producing Hsps were no better protected against water stress than those pretreated with a progressive water stress which did not produce Hsps. Hsps appear not to act as general stress proteins and their presence is not always required for the establishment of thermotolerance. PMID- 16665739 TI - Interaction between Green and Far-Red Light on the Low Fluence Rate Chloroplast Orientation in Mougeotia. AB - Continuous irradiation of Mougeotia with linearly polarized green light (550 nanometers, 0.2 watt per square meter) induces a change in the orientation of its chloroplast from profile to face position, if the electrical vector of the green light is vibrating normal to the cell axis. This change is complete within 25 minutes of the onset of irradiation. In contrast, if the electrical vector of the green light is parallel to the cell axis, no chloroplast reorientation is induced, even with a fluence rate as high as 3 watts per square meter. Furthermore, unpolarized far-red light (727 nanometers, 2 watts per square meter) given alone has no effect on chloroplast reorientation. Simultaneous and continuous irradiation with polarized green light, regardless of its plane of polarization, together with unpolarized far-red light, however, does lead to chloroplast reorientation. These data indicate that, in addition to the red absorbing form of phytochrome, there exists in Mougeotia another sensory pigment absorbing green light. PMID- 16665740 TI - Sodium Requirement for Photosynthesis and Its Relationship with Dinitrogen Fixation and the External CO(2) Concentration in Cyanobacteria. AB - Cells of Anabaena PCC 7119 and of a mutant strain of Nostoc muscorum unable to fix dinitrogen, grown at pH 8 and under low CO(2) tension (air), showed a reduced capacity for photosynthesis when cultured in the absence of sodium, this inhibition being followed by symptoms of photooxidation, such as chlorosis, oxygen consumption in the light, and decrease of superoxide dismutase activity. The impairment of photosynthesis preceded that of nitrogenase activity, indicating that the requirement for sodium in photosynthesis was independent of its effects on nitrogen metabolism. However, when cyanobacteria were grown at pH 6.3 or under high CO(2) tensions, sodium was not required for photosynthesis and no symptoms of photooxidation were observed. PMID- 16665741 TI - Intracellular pH of Cotton Embryos and Seed Coats during Fruit Development Determined by P Nuclear Magnetic Resonance Spectroscopy. AB - The pH of the phosphate-containing compartments of developing cotton seed coat and embryo tissues was determined by means of (31)P nuclear magnetic resonance spectroscopy. The pH values of these tissues varied as a function of developmental age. From 27 to approximately 38 days postanthesis, a strong pH differential existed between the two tissues; the seed coat was up to 1.4 pH units more acid than developing cotton embryos. The pattern of pH values found with this technique agrees with pH values of tissue homogenates in distilled water. The results confirm an earlier suggestion that seed coat cells are more acidic than embryo cells during key developmental stages of the seed. The pH differential between these two tissues causes abscisic acid to diffuse from seed coats to embryos against its apparent concentration gradient to prevent viviparous germination, despite a higher abscisic acid concentration in the embryo. PMID- 16665742 TI - Regulation of photosynthetic carbon metabolism in cucumber by light intensity and photosynthetic period. AB - The effects of photosynthetic periods and light intensity on cucumber (Cucumis sativus L.) carbon exchange rates and photoassimilate partitioning were determined in relation to the activities of galactinol synthase and sucrose phosphate synthase. Carbon assimilation and partitioning appeared to be controlled by different mechanisms. Carbon exchange rates were influenced by total photon flux density, but were nearly constant over the entire photoperiod for given photoperiod lengths. Length of the photosynthetic periods did influence photoassimilate partitioning. Assimilate export rate was decreased by more than 60% during the latter part of the short photoperiod treatment. This decrease in export rate was associated with a sharp increase in leaf starch acccumulation rate. Results were consistent with the hypothesis that starch accumulation occurs at the expense of export under short photoperiods. Galactinol synthase activities did not appear to influence the partitioning of photoassimilates between starch and transport carbohydrates. Sucrose phosphate synthase activities correlated highly with sugar formation rates (sucrose, raffinose, stachyose + assimilate export rate, r = 0.93, alpha = 0.007). Cucumber leaf sucrose phosphate synthase fluctuated diurnally in a similar pattern to that observed in vegetative soybean plants. PMID- 16665743 TI - Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction. AB - Light inactivation of glucose 6-phosphate dehydrogenase is rapid and occurs before photosynthetic O(2) evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced change in the kinetic parameters of glucose 6-phosphate dehydrogenase is in maximal velocity. PMID- 16665744 TI - Breakdown of Phosphatidylinositol during the Elicitation of Phytoalexin Production in Cultured Carrot Cells. AB - Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. An inhibitor of Ca(2+)-phospholipid dependent protein kinase (protein kinase C), 1-(5 isoquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor. Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca(2+)-mediated processes including calmodulin. PMID- 16665745 TI - Identification of endogenous gibberellins from oilseed rape. AB - Oilseed rape (Brassica napus, canola variety ;Westar') plants were grown in greenhouse conditions and shoots were harvested during the final stages of shoot elongation. Leaves and immature pods were removed and the remaining stem tissue was extracted and purified. The extract was chromatographed on sequential, step eluted silica gel partition and reverse-phase C(18) HPLC columns, and gibberellin (GA)-like substances were detected using the ;Tan-ginbozu' dwarf rice microdrop assay. Purified fractions showing GA-like activity were analyzed by capillary gas chromatography-mass spectrometry (GC-MS) and GC-selected ion monitoring (GC-SIM). Gibberellins A(1), A(3), and iso-A(3) were identified by full spectrum GC-MS with GA(1) being the most abundant GA in the stem tissue. Gibberellins A(19) and A(20) were identified by GC-SIM and are logical precursors of the GA(1). PMID- 16665746 TI - Microelectrode Measurements on Red Beet Vacuole : Biological Effect of Na OR NO(3) Ions, Diffusing from the Microelectrode. AB - Glass microelectrodes filled with 3 molar KCl are widely used to measure intracellular potentials and it is usual to try to minimize their electrolyte loss. In these experiments we have used the ionic leak of our microelectrodes, filled with various salt solutions, to introduce a given ion into the red beet vacuole. This allowed us to show that NO(3) (-) ions reduce the magnitude of the current spectral density while they do not change the resistance of the tonoplast. This is true when NO(3) (-) is either added to the external medium or used as the microelectrode filling solution. This can be interpreted by a NO(3) ( ) effect on the vacuolar side of the tonoplast, resulting in an inhibition of the ion transporting ATPase. Replacing K(+) by Na(+) ions in the medium has no effect on tonoplast resistance (R(s)). On the contrary, when ions leaking from the microelectrode are H(+), Li(+) or K(+), R(s) is close to 4 kilohm square centimeter, whereas R(s) is of the order of 30KOmega square centimeter when Na(+) are the leaking ions. We also found a possible correlation between the presence of a Lorentzian in the current spectral density (cut-off frequency = 2 hertz) and a Cl(-) efflux from the vacuole. This could be explained by the existence of Cl( ) channels on the tonoplast. PMID- 16665747 TI - Chloroplast as a Locale of L-myo-Inositol-1-Phosphate Synthase. AB - Chloroplasts from 5 to 7 day old Vigna radiata seedling, grown under alternate light/dark conditions or from green Euglena gracilis Z. cells have been found to harbor L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) activity. In contrast, dark-grown V. radiata seedlings, or streptomycin-bleached Euglena cells exhibit either reduced or no enzyme activity. An apparent enhancement of the chloroplastic inositol synthase by growth in presence of light is observed. PMID- 16665748 TI - Genetic Regulation of Development in Sorghum bicolor: IV. GA(3) Hastens Floral Differentiation but Not Floral Development under Nonfavorable Photoperiods. AB - Sorghum bicolor (L.) Moench lines with genetic differences in photoperiod requirement were planted in the field near Plainview, Texas (about 34 degrees northern latitude) around June 1 and treated with gibberellic acid (GA(3)) solutions applied in the apical leaf whorl. GA(3) hastened the date of floral differentiation (initiation). The greatest responses to GA(3) were by 90M and 100M, the latest of the genotypes, for which floral initiation dates were hastened an average of 19.5 and 21.7 days, respectively, for the 4 years beginning in 1980. There were very small differences in dates of anthesis between control and GA(3)-treated plants. Microscopic examination of apical meristems collected between the date of floral initiation of GA(3)-treated plants and the later date of initiation of control plants revealed: (a) several morphological characteristics of floral differentiation in the apical meristem of treated plants, (b) consistent occurrence of vegetative morphology in control plants, (c) a few meristems from GA(3)-treated plants that appeared to be regressing in floral development and thus possibly exhibiting dedifferentiation. Dedifferentiation of prepanicle primordia into leaves would explain the observed equal or greater number of leaves in GA(3)-treated plants rather than the expected smaller number. It is apparent that the presence of a morphological differentiated floral meristem in sorghum does not drive subsequent floral development in the absence of inductive photoperiods. This further suggests that initial floral differentiation and subsequent floral development may be controlled separately in sorghum. PMID- 16665749 TI - N-ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light. AB - The direct incorporation of (15)NH(4)Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during (15)NH(4) assimilation in the presence of malate. In 4 minutes the (15)N label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of (15)NH(4)Cl and Na(15)NO(2) greatly increased the (15)N-label into glutamine but did not qualitatively change the order of the incorporation of (15)N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in [(14)C]2-oxoglutarate transport into the chloroplast as well as an increase in the export of [(14)C]glutamate out of the chloroplast. Little [(14)C]glutamine was detected in the medium of the chloroplast preparations. The stimulation of (15)N-incorporation and [(14)C]glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2 oxoglutarate translocator) observed in the presence of exogenous malate. PMID- 16665750 TI - Elicitation of Necrosis in Vigna unguiculata Walp. by Homogeneous Aspergillus niger Endo-Polygalacturonase and by alpha-d-Galacturonate Oligomers. AB - Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi. Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di- and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di- and monogalacturonate did not. PMID- 16665751 TI - Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Phaseolus vulgaris L. AB - Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 m Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colletotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed. PMID- 16665752 TI - Phosphorylation of Photosystem II Components, CP43 Apoprotein, D1, D2, and 10 to 11 Kilodalton Protein in Chloroplast Thylakoids of Higher Plants. AB - Phosphorylated thylakoid proteins of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, D1, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, D1 and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the O(2)-evolving PSII reaction center complex. PMID- 16665753 TI - The effect of light and phytochrome on 1-aminocyclopropane-1-carboxylic Acid metabolism in etiolated wheat seedling leaves. AB - While light-grown wheat leaves produced ethylene at a low rate of <0.1 nanomoles per gram per hour and contained 1-aminocyclopropane-1-carboxylic acid (ACC) at low levels of <2.5 nanomoles per gram, etiolated wheat leaves produced ethylene at a rate of 2 nanomoles per gram per hour and accumulated concentrations of ACC at levels of 40 nanomoles per gram. Upon illumination of 8-day-old etiolated wheat seedlings with white light, the ethylene production rate increased initially, due to the activation of ethylene-forming activity, but subsequently declined to a low level (0.1 nanomoles per gram per hour) at the end of the 6 hour illumination. This light-induced decline in ethylene production rate resulted from a decline (more than 35 nanomoles per gram) in ACC level, which was accompanied by a corresponding increase in 1-(malonylamino)cyclopropane-1 carboxylic acid content. These data indicate that illumination promoted ACC malonylation, resulting in reduced ACC level and consequently reduced ethylene production. However, light did not cause any significant increase in the extractable ACC-malonyltransferase activity. The effect of continuous white light on promotion of ACC malonylation was also observed in intermittent white light or red light. A far-red light treatment following red light partially reversed the red light effect, indicating that phytochrome participates in the promotion of ACC malonylation. PMID- 16665754 TI - Polysomes from winter rye seedlings grown at low temperature : I. Size class distribution, composition, and stability. AB - We have studied the influence of growth at low temperature on size class distribution, stability and composition of leaf cytoplasmic polysomes from rye seedlings (Secale cereale, cv Puma) grown at 5 degrees C and at 20 degrees C. Leaves of seedlings grown at 5 degrees C contain 2.7 times more cytoplasmic polysomes (expressed on a DNA basis) and the polysome size class distribution is skewed toward larger polysomes. These changes were more pronounced in the free polysome fraction than in the membrane-bound fraction. The melting point of the total ribosome fraction from cold-grown leaves was decreased by 3.7 degrees C. Electrophoresis did not reveal any difference in the rRNA or in core-ribosomal proteins (KCl nondissociable) following growth at low temperature. Some differences were noted in peripheral ribosomal proteins. This study is the first to examine the effect of growth at low and high temperatures on polysome metabolism using plants of similar developmental stage. Polysome quantity, polymerization, melting point and peripheral ribosomal proteins in rye seedlings are modified during growth at low temperature. PMID- 16665755 TI - Modulation of water stress effects on photosynthesis by altered leaf k. AB - Wheat irrigated with nutrient solutions containing 0, 0.2, 0.5, 1, 2, or 6 millimolar K(+) had maximum photosynthetic rates at 1 to 2 millimolar K(+) concentrations. Rates in the 6 millimolar K(+)-grown plants were not higher than the 2 millimolar K(+)-grown wheat, and rates were inhibited below 0.5 millimolar K(+). Photosynthesis was measured by both attached whole leaf CO(2) uptake and by (14)CO(2) fixation of leaf slices in solution. Exposure of leaf slices from 0.2, 2, and 6 millimolar K(+)-grown wheat to various assay media water potentials showed that photosynthesis of the 0.2 millimolar K(+)-grown wheat decreased from control (high water potential) rates by 35%, that of the 2 millimolar K(+)-grown wheat by 20.4%, and that of the 6 millimolar K(+)-grown wheat by only 8.3% at 3.11 megapascals. Also, photosynthesis of the 6 millimolar K(+)-grown wheat was enhanced by 28% over that of the 2 millimolar K(+) wheat at the most severe water stress (-3.11 megapascals), indicating that the excess leaf K(+) in the 6 millimolar K(+)-grown wheat partially reversed dehydration effects on photosynthesis. Oligomycin eliminated the protective effects of high K(+) on photosynthesis in dehydrated leaf slices. These results suggest that the protective effect of high K(+) under water stress may involve the exchange of K(+) in the cytoplasm for stroma H(+), thus altering stromal pH and restoring photosynthesis. The protective effect of high K(+) was also observed in attached whole leaf photosynthesis of in situ water-stressed wheat grown on 0.2, 2, and 6 millimolar K(+). Under water stress, rates of the 6 millimolar K(+)-grown wheat were enhanced by 66.2% and 113.9% over that of 2 millimolar K(+)-grown wheat in two separate experiments. Internal CO(2) concentration of the 6 millimolar K(+) grown wheat was lower than that of the 0.2 and 2 millimolar K(+)-grown wheat. These results suggest that the high K(+) effects on chloroplast photosynthesis seen in leaf slices also occur at the whole plant level. PMID- 16665756 TI - Sink removal and leaf senescence in soybean : cultivar effects. AB - Three cultivars of soybean (Glycine max [L.] Merr. cvs Harper, McCall, and Maple Amber) were grown in the field and kept continuously deflowered throughout the normal seedfill period. For all cultivars, deflowering led to delayed leaf abscission and a slower rate of chlorophyll loss. Compared to control plants, photosynthesis and ribulose 1,5-bis-phosphate carboxylase/oxygenase (Rubisco) level declined slightly faster for deflowered Harper, but for both McCall and Maple Amber, leaves of deflowered plants maintained approximately 20% of maximum photosynthesis and Rubisco level 1 month after control plants had senesced. Deflowering led to decreased leaf N remobilization and increased starch accumulation for all cultivars, but cultivars differed in that for McCall and Maple Amber, N and starch concentrations slowly but steadily declined over time whereas for Harper, N and starch concentrations remained essentially constant over time. SDS-PAGE of leaf proteins indicated that for all cultivars, deflowering led to accumulation of four polypeptides (80, 54, 29, and 27 kilodaltons). Western analysis using antisera prepared against the 29 and 27 kilodalton polypeptides verified that these polypeptides were the glycoproteins previously reported to accumulate in vacuoles of paraveinal mesophyll cells of depodded soybean plants. The results indicated that depending on the cultivar, sink removal can lead to either slightly faster or markedly slower loss of photosynthesis and Rubisco. This difference, however, was not associated with the ability to synthesize leaf storage proteins. For any particular cultivar, declines in chlorophyll, photosynthesis, and Rubisco were initiated at approximately the same time for control and deflowered plants. Thus, even though cultivars differed in rate of decay of photosynthetic rate and Rubisco level in response to sink removal, the initiation of leaf senescence was not influenced by presence or absence of developing fruits. PMID- 16665757 TI - Photosynthetic Gas Exchange Characteristics of Wheat Flag Leaf Blades and Sheaths during Grain Filling: The Case of a Spring Crop Grown under Mediterranean Climate Conditions. AB - The rate of net CO(2) assimilation (A), the stomatal (g(s)) and residual (g(r)) conductances to CO(2), the intercellular CO(2) concentration, the CO(2) compensation points at 21% O(2) (Gamma(21)) and at 2% O(2) (Gamma(2)), and the amounts of dry matter, nitrogen, and carbohydrates were determined, from anthesis through grain filling, in the flag leaf blade and sheath of spring wheat (Triticum aestivum L. cv Kolibri). The nitrogen content and the rate of net CO(2) assimilation declined slowly until the onset of senescence in both organs, about 3 weeks after anthesis. During senescence the reduction of A in both organs was not primarily caused by a decrease in g(s); the main factor is the decrease in g(r). From values of Gamma(21) and Gamma(2) it is suggested that the rate of respiration in the light contributing to the CO(2) compensation point is higher in sheaths than in blades irrespective of the O(2) level considered. The role of sheaths storing and later transporting assimilates to the developing grains seems to be more important for shoot yield than that of sheaths functioning as photosynthetic organs after the onset of senescence occurs. It is suggested that accumulation of carbohydrates in leaves might somehow trigger senescence in the flag leaf blade and sheath simultaneously. PMID- 16665758 TI - Membrane Potential and Proton Cotransport of Alanine and Phosphate as Affected by Permeant Weak Acids in Lemna gibba. AB - The treatment of Lemna gibba plants with the weak acids (trimethylacetic acid and butyric acid), used as tools to decrease intracellular pH, induced a hyperpolarization of membrane potential, dependent on the concentration of the undissociated permeant form of the weak acid and on the value of the resting potential. Measurements were carried out both with ;high potential' and ;low potential' plants and the maximum values af acid induced hyperpolarizations were about 35 and 71 millivolts, respectively. Weak acids influenced also the transient light-dark membrane potential changes, typical for photosynthesizing material, suggesting a dependence of these changes on an acidification of cytoplasm. In the presence of the weak acids, the membrane depolarization induced by the cotransport of alanine and phosphate with protons was reduced; the maximum reduction (about 90%) was obtained with alanine during 2 millimolar trimethylacetic acid perfusion at pH 5. A strong inhibition of the uptake rates (up to 48% for [(14)C]alanine and 68% for (32)P-phosphate) was obtained in the presence of the weak acids, both by decreasing the pH of the medium and by increasing the concentration of the acid. In these experimental conditions, the ATP level and O(2) uptake rates did not change significantly. These results constitute good evidence that H(+)/solute cotransport in Lemna, already known to be dependent on the electrochemical potential difference for protons, is also strongly regulated by the cytoplasmic pH value. PMID- 16665759 TI - Molecular species specificity of phospholipid breakdown in microsomal membranes of senescing carnation flowers. AB - During senescence of cut carnation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine substrates. 16:0(*)/16:0(*), 16:0/18:2(*), and 18:1(*)/18:1(*) phosphatidylcholine were degraded only minimally over a 3 hour period by microsomes isolated from senescing flowers. By contrast, [U (14)C]phosphatidylcholine, which comprises various molecular species including those containing polyunsaturated acyl chains, and 18:0/20:4(*) phosphatidylcholine were extensively degraded. Under identical conditions, but in the absence of added radiolabeled substrate, endogenous 18:2/18:2, 18:1/18:3, and 18:2/18:3 phosphatidylcholine were selectively depleted from the membranes. During natural senescence of the flowers, there was a sharp decline in microsomal 16:0/18:1 and 18:1/18:2 phosphatidylcholine, whereas molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain remained unchanged or decreased only slightly. The data have been interpreted as indicating that provision of particular molecular species susceptible to lipase attack is a prerequisite to phospholipid catabolism in senescing membranes. PMID- 16665760 TI - Differential Effects of a Substituted Pyridazinone, BASF 13-338, on Pathways of Monogalactosyldiacylglycerol Synthesis in Arabidopsis. AB - We have examined the effects of the substituted pyridazinone herbicide, 4-chloro 5-(dimethylamino)-2-phenyl-3(2H)pyridazinone (BASF 13-338, Sandoz 9785), on the desaturation of linoleic acid (18:2) on different molecular species of monogalactosyldiacylglycerol (MGDG) and phosphatidylcholine (PC) in leaf tissue of Arabidopsis thaliana (L.) Heynh. Specific changes in lipid composition allowed identification of different substrates for desaturation of 18:2 to linolenic acid (18:3). 18:2/16:2 MGDG was desaturated in the chloroplast to form 18:3/16:3 MGDG. Levels of 18:3/16:3 MGDG were reduced by treatment with BASF 13-338, suggesting that both the formation of 18:3 at the sn-1 position, and the formation of 16:3 at the sn-2 position of 18:2/16:2 MGDG were inhibited by this compound. Kinetic studies using exogenously incorporated [(14)C] 18:1 indicated that 18:2/18:3 MGDG originated from an 18:2/18:3 diglyceride precursor derived from PC. The formation of 18:3 at the sn-1 position of 18:2/18:3 MGDG was also inhibited by BASF 13-338. In contrast the desaturation of 18:2 proposed to occur at the sn-2 position of PC outside the chloroplast, was not affected. PMID- 16665761 TI - Comparison of fluence-response relationships of phototropism in light- and dark grown buckwheat. AB - Fluence-response relationships of phototropism in light- and dark-grown buckwheat (Fagopyrum esculentum Moench.) were compared using systematically varied fluence rates and irradiation times of unilateral monochromatic blue light. Etiolated seedlings respond to most fluence rates in a tri-phasic manner. Phase one differs from classic first positive in that reciprocity is not observed and the peak occurs at a wide variety of fluences, often orders of magnitude less than those characteristic of first positive. Light-grown plants display this pattern only when stimulated by low fluence rates. Phase three is an ascending arm directly related to irradiance time and is comparable to classic second positive. Phase two is a nearly indifferent zone separating phases one and three. At the lowest fluence rates, the maximal observed curvature is greater for dark-grown than for light-grown plants and the former curve more in response to short (2-second) exposures than do the latter. At the highest fluence rates, the maximal observed curvature is much greater for light-grown than for dark-grown seedlings, particularly at irradiation times of 2 to 3 minutes or more. Tropic curvatures correlate positively with increasing fluence rate up to some inflection range, above which the relationship becomes negative. This inflection range is approximately two orders of magnitude higher for light-grown plants. PMID- 16665762 TI - Latency of Plasma Membrane H-ATPase in Vesicles Isolated by Aqueous Phase Partitioning : Increased substrate Accessibility or Enzyme Activation. AB - The properties of the plasma membrane H(+)-ATPase and the cause of its latency have been studied using a highly purified plasma membrane fraction from oat (Avena sativa L., cv Victory) roots, prepared by aqueous two-phase partitioning. The ATPase has a maximum specific activity (at 37 degrees C) in excess of 4 micromoles inorganic phosphate per milligram protein per minute in the presence of nondenaturing surfactants. It is inhibited by more than 90% by vanadate, is specific for ATP, has a pH optimum of 6.5, and is stimulated more than 4-fold by 50 millimolar K(+) in the presence of low levels of the nondenaturing surfactants Triton X-100 and lysolecithin. This ;latent' activity is usually explained as being a result of the inability of ATP to reach the ATPase in right-side out, sealed vesicles, until they are disrupted by surfactants. Consistent with this idea, trypsin digestion significantly inhibited the ATPase only in the presence of the surfactants. Electron spin resonance spectroscopy volume measurements confirmed that surfactant-free vesicles were mostly sealed to molecules similar to ATP. However, the Triton to protein ratio required to disrupt vesicle integrity completely is 10-fold less than that needed to promote maximum ATPase activity. We propose that plasma membrane ATPase activation is due not solely to vesicle disruption and accessibility of ATP to the ATPase but to the surfactants activating the ATPase by altering the lipid environment in its vicinity or by removing an inhibitory subunit. PMID- 16665763 TI - Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures. AB - Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of -20 degrees C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [(35)S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants. PMID- 16665764 TI - Enzymology of Fructan Synthesis in Grasses: Properties of Sucrose-Sucrose Fructosyltransferase in Barley Leaves (Hordeum vulgare L. cv Gerbel). AB - Fructan synthesis was induced in excised primary leaf blades of Hordeum vulgare L. cv Gerbel by illumination in 30 millimolar fructose. This treatment induced a 26-fold increase of sucrose-sucrose-fructosyltransferase (SST, EC 2.4.1.99) activity within 24 hours. Acid invertase (EC 3.2.1.26) activity remained about constant. By preparing protoplasts from induced leaves, approximately 80% of the invertase activity was removed with the cell walls while SST was retained. The protoplast homogenate was used to partially purify and characterize SST. Acid precipitation (pH 4.75) and anion exchange chromatography (fast protein liquid chromatography on Mono ;Q') resulted in a recovery of about 80% of total SST activity. The principal activity (SST 1), accounting for 85% of the activity recovered, was purified about 200-fold. It was essentially free of invertase activity and catalyzed the synthesis of a trisaccharide which co-chromatographed with isokestose (1F-beta-fructosylsucrose). The remaining 15% of SST activity (SST 2) was purified about 35-fold. It retained substantial invertase activity and catalyzed the synthesis of only one trisaccharide which co-chromatographed with kestose (6F-beta-fructosylsucrose). It is concluded that barley leaves which store mainly fructan of the phlein type (beta-2-6 polyfructosylsucrose), nevertheless contain sucrose-sucrose 1F-beta-d-fructosyltransferase as the key enzyme of fructan synthesis. PMID- 16665765 TI - Facilitated transport of glucose in isolated Phloem segments of celery. AB - In isolated phloem segments of celery (Apium graveolens L.), a tissue highly specific for sucrose and mannitol uptake, glucose uptake occurs at very low rates and exhibits biphasic kinetics. Nonpenetrating inhibitors such as parachloromercuribenzene sulfonic acid did not inhibit glucose uptake. However, uptake was greatly inhibited by penetrating inhibitors such as N-ethylmaleimide and carbonylcyanide-m-chlorophenyl hydrazone. Carbonylcyanide-m-chlorophenyl hydrazone inhibition of uptake was reversed by washing and addition of thiol reagents to uptake solutions. Phlorizin, a competitive inhibitor of glucose caused moderate inhibition of uptake only after 3 hours of tissue exposure. Low pH, fusicoccin, and low turgor which enhance H(+)-sugar cotransport did not alter uptake rates. Furthermore, glucose did not induce alkalinization of the uptake media. Efflux analysis indicated that the presence of 50 millimolar unlabeled glucose in the wash media enhanced exchange of the labeled glucose across the tonoplast. Results indicate that the glucose carrier is not located at the plasmalemma but appears to be present at the membrane of an intracellular compartment, most likely the tonoplast. Carrier-mediated glucose transport in this tissue is proposed to be a facilitated diffusion. PMID- 16665766 TI - P NMR Study of Elicitor Treated Phaseolus vulgaris Cell Suspension Cultures. AB - The addition of an elicitor (glucan) to Phaseolus vulgaris cell suspension cultures increased the formation of the phytoalexin phaseollin. Intracellular pH and phosphate concentrations were studied with (31)P nuclear magnetic resonance spectroscopy on elicitor-treated cells which were aerated during the nuclear magnetic resonance measurement. The pH of the vacuole and to a lesser extent the pH of the cytoplasm were affected at 10 minutes after elicitor addition; a decrease in pH from 5.3 to 4.8 was noted in the vacuole and from 7.46 to 7.28 in the cytoplasm. The ratio between the amount of Pi in the vacuole to that in the cytoplasm also changed within 10 minutes after elicitor addition. The signal for ATP (beta-ATP) was low after elicitor addition and was high again 23 hours after elicitation. Forty-eight hours after elicitor addition, vacuolar and cytoplasmic pH had almost returned to their initial values. The rapid change in vacuolar and cytoplasmic pH may cause the change of metabolism that occurs in elicitor-treated P. vulgaris cells. PMID- 16665767 TI - Genotype-dependent leaf senescence in maize : inheritance and effects of pollination-prevention. AB - Objective of research was (a) to evaluate the influence of pollination-prevention on various metabolic parameters of the two maize inbreds B73 and B14A and their F1, and (b) to gain information on the inheritance of leaf senescence, in response to pollination-prevention. The results show that the visual pattern of leaf senescence, in response to prevention of ear pollination, contrasts markedly between the two inbred lines. Relative to control plants, prevention of ear pollination, causes a premature senescence in B73 and B73 x B14A plants, while leaves of unpollinated B14A remain green and similar in appearance to pollinated controls. Furthermore, prevention of ear pollination induces a sizable reduction of dry matter accumulation of all above-ground material and changes in various metabolic parameters. An accumulation of sucrose in the leaves of unpollinated B73 and B73 x B14A plants is correlated with the development of premature senescence. Finally, the genetic analysis supports suggestions that a single dominant gene is responsible for the differences observed, in the visual pattern of leaf senescence, in response to prevention of ear pollination. PMID- 16665768 TI - Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium. AB - RESEARCH ON THE BIOSYNTHESIS OF ABSCISIC ACID (ABA) HAS FOCUSED PRIMARILY ON TWO PATHWAYS: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in (18)O(2). It was found that in stressed leaves three atoms of (18)O from (18)O(2) are incorporated into the ABA molecule, and that the amount of (18)O incorporated increases with time. One (18)O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in (18)O(2) shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more (18)O into the tertiary hydroxyl group at C-1' after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, (18)O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied (14)C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional (18)O incorporated during 8'-hydroxylation of ABA to phaseic acid. PMID- 16665769 TI - An Extracellular Protein from Phytophthora parasitica var nicotianae Is Associated with Stress Metabolite Accumulation in Tobacco Callus. AB - The most abundant extracellular protein produced by Phytophthora parasitica var nicotianae at early stages of rapid growth in culture has a molecular weight of 46 kilodaltons and has been designated Ppn 46e. Culture conditions for the production of this protein have been optimized and the protein has been purified by gel filtration and ion-exchange chromatography. Ppn 46e is a soluble, acidic protein (pI 4.67). The amino acids Asx (aspartic acid or asparagine), alanine, glycine, Glx (glutamic acid or glutamine), and serine are the most abundant at 13.4%, 12.3%, 12.1%, 9.3%, and 9.3% of the residues, respectively. The purified protein is, by weight, 1.8% glucose, 1.6% mannose, and 0.5% galactose. A bioassay for Ppn 46e based on tobacco callus has been developed. In this assay as little as 20 nanograms (4.3 x 10(-13) mole) Ppn 46e causes the accumulation of the sesquiterpenoid phytoalexin, capsidiol, as estimated by gas chromatography. Levels of capsidiol of 25 micrograms per gram fresh weight were elicited by 80 nanograms Ppn 46e per callus piece. Pretreatment of the protein with either pronase or by boiling resulted in a loss of elicitor activity. Periodate treatment, which inactivates glucan elicitors, did not affect the ability of Ppn 46e to cause capsidiol accumulation. Monospecific antibodies to Ppn 46e were raised in mice. Western blotting experiments employing these antibodies showed that Ppn 46e was present in infected tobacco plants. Dot blotting experiments revealed the presence of the Ppn 46e epitope(s) in Phytophthora megasperma, P. cactorum, P. cinnamomi, and P. infestans but not in Fusarium. PMID- 16665770 TI - Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides. AB - Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc(3)Man(9)(GlcNAc)(2), was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man(6-9)[GlcNAc](2)), mannosidase II (removes mannose residues from GlcNAcMan(5)[GlcNAc](2)), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities. PMID- 16665771 TI - Characterization of Iron Uptake from Ferrioxamine B by Chlorella vulgaris. AB - Iron uptake from two Fe(3+)-hydroxamate siderophores, ferrioxamine B and Fe(3+) rhodotorulate, by iron-stressed Chlorella vulgaris (ATCC strain 11468) was evaluated with some comparison to iron uptake from synthetic and organic acid ferric chelates. Iron-stress induced iron uptake from ferrioxamine B. Dissipation of the electrochemical gradient, via uncouplers, inhibited iron uptake. Respiratory inhibitors gave variable results, an indication that a direct link to respiration was not apparent. Vanadate inhibition of iron uptake indicated that an ATPase or phosphate intermediate could be involved in the uptake mechanism. Divalent cations manifested variable effects dependent on the cation and chelator used. These data confirm that C. vulgaris has an inducible iron-uptake system for Fe(3+)-hydroxamic acid siderophores which may involve a different mechanism than that observed for other chelates. PMID- 16665772 TI - Evaluation of Reductive Release as a Mechanism for Iron Uptake from Ferrioxamine B by Chlorella vulgaris. AB - The involvement of ferric reduction in the iron uptake mechanism of iron-stressed Chlorella vulgaris from ferrioxamine B was investigated. Some comparative data for ferric-citrate was also obtained. EPR and a spectrophotometric assay were used to measure Fe(3+) reduction. These two methods differed in the absolute quantity but not in effectors of ferric reduction. The mechanism governing ferric reduction was investigated by use of respiratory inhibitors, uncouplers, alternative electron acceptors, and ATPase inhibitors. Reduction appears to play a role in iron uptake from both Fe(3+)-deferrioxamine B and Fe(3+)-citrate; however, the involvement of photoreduction in Fe(3+)-citrate uptake implies multiple reductive mechanisms could be involved. PMID- 16665773 TI - Nuclear gene-regulated expression of chloroplast genes for coupling factor one in maize. AB - In order to gain a better understanding of the interaction between the chloroplast and nuclear genomes in controlling the expression of plastid genes and the biosynthesis of chloroplast proteins, maize (Zea mays) nuclear gene mutant hcf(*)-38, in which alpha and beta subunits of coupling factor one (CF(1)) are almost completely missing was studied. The mutant possesses all the other subunits of CF(1) but several peptides of photosystem II are present in reduced amounts. A competitive hybridization experiment showed the presence of the same plastid mRNA species in mutant and wild-type plants except for slightly lower levels of some transcripts in the mutant. Northern hybridization and dot blot hybridization experiments showed the features of transcripts for alpha and beta subunits of CF(1) in the mutant to be similar to those in the wild-type maize although their levels are somewhat lower in the mutant. In vivo and in organello protein labeling experiments with L-[(35)S]Met have shown that alpha and beta subunits of CF(1) are synthesized, assembled into CF(1), and probably associated with thylakoid membranes in mutant plants. It is concluded that they are subsequently degraded. PMID- 16665774 TI - Carbohydrate, organic Acid, and amino Acid composition of bacteroids and cytosol from soybean nodules. AB - Metabolites in Bradyrhizobium japonicum bacteroids and in Glycine max (L.) Merr. cytosol from root nodules were analyzed using an isolation technique which makes it possible to estimate and correct for changes in concentration which may occur during bacteroid isolation. Bacteroid and cytosol extracts were fractionated on ion-exchange columns and were analyzed for carbohydrate composition using gas liquid chromatography and for organic acid and amino acid composition using high performance liquid chromatography. Analysis of organic acids in plant tissues as the phenacyl derivatives is reported for the first time and this approach revealed the presence of several unknown organic acids in nodules. The time required for separation of bacteroids and cytosol was varied, and significant change in concentration of individual compounds during the separation of the two fractions was estimated by calculating the regression of concentration on time. When a statistically significant slope was found, the true concentration was estimated by extrapolating the regression line to time zero. Of 78 concentration estimates made, there was a statistically significant (5% level) change in concentration during sample preparation for only five metabolites: glucose, sucrose, and succinate in the cytosol and d-pinitol and serine in bacteroids. On a mass basis, the major compounds in bacteroids were (descending order of concentration): myo-inositol, d-chiro-inositol, alpha,alpha-trehalose, sucrose, aspartate, glutamate, d-pinitol, arginine, malonate, and glucose. On a proportional basis (concentration in bacteroid as percent of concentration in bacteroid + cytosol fractions), the major compounds were: alpha-aminoadipate (94), trehalose (66), lysine (58), and arginine (46). The results indicate that metabolite concentrations in bacteroids can be reliably determined. PMID- 16665775 TI - Effect of nitrate on the organic Acid and amino Acid composition of legume nodules. AB - Nitrate supplied to legume plants inhibits the activity of nitrogenase in Rhizobium bacteroids in root nodules. The accumulation of amino N which is known to occur in Glycine max (L.) Merr. nodules as nitrogenase activity declines was studied in more detail by analysis of changes in free amino acid composition in response to high nitrate supply. A 6-fold increase in asparagine concentration in Bradyrhizobium japonicum bacteroids was found about the time of maximum nitrogenase inhibition. However, the accumulation of amino acids in soybean nodules lagged behind the inhibition of nitrogenase. Furthermore, in studies of a second legume, Phaseolus vulgaris (L.) inoculated with two different strains of Rhizobium phaseoli, a high nitrate treatment inhibited nitrogenase but had no significant effect on amino acid composition of nodules. The possibility that nitrate may interfere with the supply of carbon substrates to bacteroids was examined by the analysis of organic acids in legume nodules supplied with nitrate. Nitrate had a small (10-20%) negative effect on the concentration of tricarboxylic acid cycle acids in P. vulgaris nodules. However, in G. max nodules, high nitrate treatment resulted in significant increases in the concentration of malate, succinate, fumarate, and citrate. Thus, carbon deprivation of bacteroids also seems unlikely as a cause of the inhibition of nitrogenase by nitrate. There was a transient increase in ammonium concentration in P. vulgaris nodules in response to high nitrate treatment. This effect was rapid relative to other effects of nitrate on nodule composition and was roughly coincident with the rapid decline in acetylene reduction activity. PMID- 16665776 TI - Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase. AB - Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranslationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor. PMID- 16665777 TI - Effect of altered sink: source ratio on photosynthetic metabolism of source leaves. AB - When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of (14)C detected in sugar phosphates and UDPglucose following (14)CO(2) supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO(2) fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans (14)C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO(2) fixation rate was constant for several days. PMID- 16665778 TI - beta-Oxidation and Glyoxylate Cycle Coupled to NADH: Cytochrome c and Ferricyanide Reductases in Glyoxysomes. AB - Glyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contamination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate of 340 nanomole electrons per minute per milligram glyoxysomal protein. Palmitoyl-CoA oxidation could be coupled to ferricyanide or cytochrome c reduction. Complete oxidation of palmitoyl-CoA, yielding 14 nanomole electrons/per nanomole palmitoyl-CoA, was demonstrated with the acceptors, NAL, cytochrome c, and ferricyanide. Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by beta-oxidation and the glyoxylate cycle. PMID- 16665779 TI - Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm. AB - Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca(2+) and Mg(2+) stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane. PMID- 16665780 TI - Nickel: a micronutrient essential for higher plants. AB - Nickel was established as an essential micronutrient for the growth of temperate cereal crops. Grain from barley (Hordeum vulgare L. cv ;Onda'; containing 40 to 80 nanograms of Ni per gram dry weight) grown in solution culture with negligible Ni concentrations (< 30 nanograms of Ni per liter) exhibited greatly reduced germination rates (i.e. 50% less than grain from Ni-adequate plants) and seedling vigor of the viable grain was greatly depressed. Grain containing less than 30 nanograms per gram dry weight was inviable. Under Ni-deficient conditions, barley plants fail to produce viable grain because of a disruption of the maternal plant's normal grain-filling and maturation processes that occur following formation of the grain embryo. The observations that (a) barley plants fail to complete their life cycle in the absence of Ni and (b) addition of Ni to the growth medium completely alleviates deficiency symptoms in the maternal plants satisfies the essentiality criteria; thus, Ni should be considered a micronutrient for cereals. Because Ni is required by legumes, and is now established as essential for cereals, we conclude that Ni should be added to the list of micronutrients essential for all higher plant growth. PMID- 16665781 TI - Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit. AB - We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green ;Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO(2) evolution by 400 micrograms per gram fresh weight galactose. 1 Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol. PMID- 16665782 TI - Plastid Development in Pisum sativum Leaves during Greening : I. A Comparison of Plastid Polypeptide Composition and in Organello Translation Characteristics. AB - Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A(+)) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis. PMID- 16665783 TI - Plastid Development in Pisum sativum Leaves during Greening : II. Post Translational Uptake by Plastids as an Indicator System to Monitor Changes in Translatable mRNA for Nuclear-Encoded Plastid Polypeptides. AB - When isolated pea plastids are incubated with translation products of poly(A(+)) mRNA they specifically import precursor molecules of plastid polypeptides. Etioplasts and chloroplasts import the same polypeptides from identical translation products, and, the imported polypeptides can be well resolved by two dimensional gel electrophoresis. Therefore, the posttranslational uptake system using isolated chloroplasts can monitor changes in the abundance of translatable plastid-targeted messages. Poly(A(+)) mRNA was isolated from peas at various times during greening and analyzed by this technique. (a) After 48 hours of illumination of dark-grown plants, the relative portion of nuclear encoded messages for plastid targeted proteins had increased by a factor of 2. The percentage of polypeptides recovered in the stroma fraction increased from about 50 to 65%. (b) More than 140 imported polypeptide species could be detected in fluorograms of two-dimensional gels, most of which could be identified throughout the time course of greening. At least 37 imported polypeptides decreased and 36 increased in relative abundance during greening of darkgreen plants. (c) In most cases, where differences in translatable messages were seen between dark- and light-grown plants, they were accompanied by parallel changes in polypeptide abundance. PMID- 16665784 TI - Purification and Partial Characterization of a Hydroxyproline-Rich Glycoprotein in a Graminaceous Monocot, Zea mays. AB - Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). As HRGPs are often at the cell surface, we used the intact cell elution technique (100 millimolar AlCl(3)) to isolate soluble surface proteins from Zea mays cell suspension cultures. Further fractionation of the trichloroacetic acid-soluble eluate on the cation exchangers phospho-cellulose and BioRex-70 gave several retarded, hence presumably basic fractions, which also contained hydroxyproline (Hyp). One of these fractions yielded a pure HRGP after a final purification step involving Superose-6 gel filtration. As this HRGP was unusually rich in threonine, (25 mole%) we designated it as a threonine hydroxyproline-rich glycoprotein (THRGP); it contained about 27% carbohydrate occurring exclusively as arabinosylated Hyp, predominantly as the monosaccharide (15%), and trisaccharide (25%) with 48% Hyp nonglycosylated-a characteristically graminaceous monocot profile. Amino acid analysis confirmed the basic character, and gave a low alanine content. Reaction with Yariv artificial antigen was negative. These characteristics show that the THRGP is not an arabinogalactan protein. On the other hand, antibodies raised against tomato extensin P1 cross reacted significantly with the THRGP; this cross-reactivity and the above analytical data provide the best evidence to date for the presence of extensin in a graminaceous monocot. PMID- 16665785 TI - Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes. AB - An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a(2)b(2)) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n(H) = 0.55 +/- 0.05 for NADPH and 0.22 +/- 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes. PMID- 16665786 TI - Monoclonal antibodies against fusicoccin with binding characteristics similar to the putative fusicoccin receptor of higher plants. AB - Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [(3)H]-nor-fusicoccin-alcohol of high specific activity (1.5 x 10(14)Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 x 10(-9) molar and 1.85 x 10(-9) molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 x 10(-9) molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [(3)H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. PMID- 16665787 TI - The timing and rate of phytic Acid accumulation in developing soybean seeds. AB - The time-course of phosphorus (P) accumulation in the phytic acid fraction of developing soybean (Glycine max [L.] Merr. cv ;Williams 79') seeds as well as the relation of phytic acid P to total P content were determined. Phytic acid was detected early in embryogenesis in field-grown soybeans and accumulated in a linear fashion throughout most of seed development. Although the observed rates of accumulation ranged from 18.7 micrograms phytic acid P per seed per day in pods positioned low on the plant to 33.6 micrograms in pods positioned high on the plant, the final concentrations were the same in all cases. Nearly all of the P translocated to developing seeds was incorporated into phytic acid from the third week after flowering until physiological maturity, with the sum of nonphytic acid P compounds remaining constant. Phytic acid accumulation was also linear throughout development when soybean plants were grown in solutions having nutrient P levels that ranged from severely limiting (2.0 milligrams P per liter) to excess (50 milligrams P per liter). However, there was a pronounced effect on rate of accumulation, which ranged from 7.2 micrograms phytic acid per seed per day with limiting nutrient P to 44.7 micrograms with excess P. The change in level of phytic acid accounted for most of the alteration in total seed P that was caused by altering the P status of the plants. These results support the view that phytic acid synthesis is involved in P homeostasis of the developing soybean seed. PMID- 16665788 TI - Tabtoxinine-beta-Lactam Transport into Cultured Corn Cells : Uptake via an Amino Acid Transport System. AB - Tabtoxinine-beta-lactam (T-beta-L), a unique amino acid, is a toxin produced by several closely related pathovars of Pseudomonas syringae. These chlorosis inducing pathogens establish themselves in the apoplastic space of their hosts where they release the toxin. We have examined the transport of T-beta-L into cultured corn (Zea mays cv Black Mexican) cells using [(14)C]T-beta-L. The pH optimum of the uptake of the toxin was between 4.0 and 5.5 pH units. Toxin uptake was inhibited by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone, and by the sulfhydryl re-agent, N-ethylmaleimide. Tabtoxinine-beta-lactam transport exhibited saturation kinetics that were described by the Michaelis-Menton equation for toxin concentrations of 1 millimolar and less. However, the transport of toxin in concentrations greater than 1 millimolar was not described by Michaelis-Menten kinetics. Glutamate and alanine exhibited similar transport kinetics with a transition to non-Michaelis-Menten kinetics when the amino acid concentration exceeded 1 millimolar. Hill numbers for glutamate, alanine, and T beta-L ranged from 0.6 to 0.8. Methionine, alanine, tyrosine, glutamine, glutamate, and arginine were inhibitors of toxin transport. Alanine was a competitive inhibitor of the transport of T-beta-L and of glutamate. The data are consistent with T-beta-L being transported into the plant cell through an amino acid transport system. PMID- 16665789 TI - Effects of Temperature on H Secretion and Uptake by Excised Flexor Cells during Dark-Induced Closure of Samanea Leaflets. AB - Previous studies reveal that dark-induced closure of Samanea leaflets is accompanied by H(+) secretion from flexor motor cells. We now report that flexor tissue excised in the light, incubated in a weakly buffered bathing solution, and then darkened at different temperatures (18 degrees C-30 degrees C) acidified the medium (indicating net H(+) efflux) at all temperatures tested, but most rapidly at the highest temperature. However, pH changes reversed direction after 20 to 70 minutes; the lower the temperature, the later pH reversal occurred, and the lower the pH at reversal and after 45 minutes. These data provide a basis for the previously reported promotive effect of low temperature on dark-induced leaflet closure, assuming net H(+) and K(+) fluxes are opposite in direction. Net H(+) efflux at all temperatures tested was greater when the impermeant molecule iminodiacetate replaced small permeant anions in the bathing solution, suggesting that H(+) uptake is coupled to anion uptake, probably via a H(+)/anion symport system. When permeant anions were deficient, the amount of malate in the tissue increased, presumably by new synthesis. Malate synthesis would substitute for H(+)/anion uptake in charge balance and in providing H(+) for cytoplasmic pH regulation. PMID- 16665790 TI - Nitrogen Utilization in Lemna: I. Relations between Net Nitrate Flux, Nitrate Reduction, and in Vitro Activity and Stability of Nitrate Reductase. AB - Cultures of Lemna gibba L. G3 were maintained at a constant, N-limited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day(-1). Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemna is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity. PMID- 16665791 TI - Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using NO(3). AB - (13)N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs. PMID- 16665792 TI - Nitrogen Utilization in Lemna: III. Short-Term Effects of Ammonium on Nitrate Uptake and Nitrate Reduction. AB - The effects of ammonium application on nitrate utilization were studied in N limited cultures of Lemna gibba L. G3. Addition of ammonium instantaneously inhibited net nitrate uptake by at least 60%, followed by a slight recovery. The inhibition was equally clear after near-complete inactivation of glutamine synthetase by application of l-methionine-d,l-sulfoximine. Experiments where (13)N-labeled nitrate was used as an influx tracer revealed that ammonium specifically inhibited influx, but did not promote nitrate efflux. Nitrate accumulation was relatively more inhibited than nitrate reduction and net uptake. Nitrate reductase, extracted and assayed in vitro in the presence of the thiol proteinase inhibitor leupeptin, was unaffected by short-term treatment of the plants with either nitrate, ammonium, or ammonium nitrate. Nitrate reductase activity recovered in the absence of leupeptin was considerably lower; however, it was enhanced by all the nitrogen sources, with ammonium as the most potent. It is argued that the effect of ammonium on nitrate utilization in Lemna is due to inhibition of nitrate influx, and that the effect should be attributed to ammonium itself, not to a newly formed nitrogen derivative. The decreased nitrate flux caused a decrease in nitrate reduction, whereas the activity of nitrate reductase per se rather is stabilized by presence of ammonium. PMID- 16665793 TI - Reduction State of Q and Nonradiative Energy Dissipation during Photosynthesis in Leaves of a Crassulacean Acid Metabolism Plant, Kalanchoe daigremontiana Hamet et Perr. AB - Fluorescence was measured in leaves of the CAM plant Kalanchoe daigremontiana using a pulse modulation technique at room temperature. During a 12-h light period at 500 micromole photons per square meter per second (400-700 nanometers) in air containing 350 microbar CO(2), the component of fluorescence quenching related to the reduction state of Q, the primary electron transport acceptor of PSII, remained fairly constant and showed that only 20% of Q were in the reduced form. The reduction state was slightly increased at the onset and at the end of the light period. By contrast, the nonphotochemical component of fluorescence quenching which is a measure of the fraction of nonradiative deexcitation underwent marked diurnal changes. Nonradiative energy conversion was low during the phase of most active malic acid decarboxylation in the middle of the light period when uptake of atmospheric CO(2) was negligible, and when internal CO(2) partial pressures were higher than in air; this allowed for high rates of CO(2) reduction in the chloroplasts. Nonradiative energy conversion was high during the early and the late light period when atmospheric CO(2) was taken up and internal CO(2) partial pressures were below air level. Manipulation of the internal CO(2) partial pressure during the late light period by increasing or decreasing the external CO(2) partial pressure to 1710 and 105 microbar, respectively, led to changes in the magnitude of energy dependent fluorescence quenching which were consistent with the relationship between nonradiative energy dissipation and internal CO(2) partial pressure observed during the diurnal cycle. Again, the reduction state of Q was hardly affected by these treatments. Thus, changes in electron transport rate during the diurnal CAM cycle at a given photon flux density lead primarily to alterations in the rate of nonradiative energy dissipation, with the reduction state of Q being maintained at a relatively low and constant level. Conditions are described under which nonphotochemical dissipation of excitation energy reaches a maximum value and the reduction state of Q is increased. PMID- 16665794 TI - Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP Pyridoxal: Evidence for an Active-Site Amino Group. AB - UDP-pyridoxal competitively inhibits the Ca(2+)-, cellobiose-activated (1-->3) beta-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K(i) of 3.8 +/- 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V(max) of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 +/- 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (1-->3)-beta-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca(2+) and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1 ->3)-beta-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[(3)H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca(2+) and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP [(3)H]pyridoxal in the presence of Mg(2+) and copurifies with (1-->3)-beta-glucan synthase activity. PMID- 16665795 TI - Superoxide Dismutase as an Anaerobic Polypeptide : A Key Factor in Recovery from Oxygen Deprivation in Iris pseudacorus? AB - The perennating organ, the rhizome, was chosen for examination of response to anoxia in the species Iris pseudacorus L., Iris germanica L. var Quechei, and Glyceria maxima (Hartm.) Holmberg. These monocots are known to differ in their tolerance of anoxia. Intact rhizomes were subjected to periods of prolonged anoxia of up to 28 days and superoxide dismutase (SOD) activity was determined in a 48 hour postanoxic recovery phase. Tests were performed to ensure the accuracy of the measured enzyme activities. In the most anoxia tolerant species, I. pseudacorus, SOD activity rose continuously during the period of imposed anoxia, and levels were maintained in the postanoxic recovery phases: 28 days brought about a 13-fold increase to 1576 U SOD per milligram protein. Small increases were found in the less anoxia tolerant I. germanica during anoxic/postanoxic phases, while a drop in activity was recorded in the least anoxia tolerant G. maxima. However, initial levels in G. maxima were more than twice as high as in the other two species. Experiments applying cycloheximide to anoxic rhizome slices of I. pseudacorus inhibited the increase in SOD activity. This indicates that SOD is, paradoxically, induced under anoxia and we suggest that in this species SOD is one of the enzymes identified as anaerobic polypeptides. The significance of the induction of an ;oxygen-protecting' enzyme during complete oxygen deprivation is discussed with regard to a possible critical role during recovery from anoxic stress. PMID- 16665796 TI - Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803 : Inactivation of the psbA Gene Family. AB - psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center. PMID- 16665797 TI - Metabolization of elemental sulfur in wheat leaves consecutive to its foliar application. AB - The qualitative and quantitative aspects of elemental sulfur metabolization in wheat leaves and its effect upon photosynthetic metabolism were studied through the application of micronized sulfur upon the third leaf. Energy-dispersive x-ray analysis combined with scanning electron microscopy emphasized the existence of a sulfur peak associated with a strong potassium peak in the spectra of different tissue regions for treated leaves only, supplying an original evidence of sulfur uptake. Experiments with(35)S-labeled micronized sulfur showed that about 2% of the labeled S was absorbed and metabolized into cystine, methionine, glutathione, and sulfate. The close correlation between the excess of oxygen uptake and oxygen needs for sulfur oxidation in conjunction with the absence of hydrogen sulfide released by treated leaves support direct and fast oxidation of sulfur into sulfate according to a pathway still unclear but independent of photosynthetic CO(2) metabolism in treated leaf. The mechanisms involved in the primary metabolism of element sulfur in wheat therefore appear to be different from those in fungi. PMID- 16665798 TI - Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells. AB - Cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, produce phytochelatins (poly[gamma-glutamylcysteinyl]glycines) when exposed to cadmium. The synthesis of these peptides is accompanied by a decline in cellular levels of glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, inhibits the sustained production of phytochelatins. However, phytochelatin synthesis can occur in the presence of buthionine sulfoximine provided that sufficient glutathione is available. These results indicate that glutathione is a substrate for phytochelatin synthesis. The protein synthesis inhibitor cycloheximide does not affect the initial production of phytochelatin. PMID- 16665799 TI - Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum. AB - The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht fruhstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22 degrees C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H linked activity, with a K(a) value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K(i) of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K(i) value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell. PMID- 16665800 TI - Osmotic adjustment, symplast volume, and nonstomatally mediated water stress inhibition of photosynthesis in wheat. AB - At low water potential (psi(w)), dehydration reduces the symplast volume of leaf tissue. The effect of this reduction on photosynthetic capacity was investigated. The influence of osmotic adjustment on this relationship was also examined. To examine these relationships, comparative studies were undertaken on two wheat cultivars, one that osmotically adjusts in response to water deficits (;Condor'), and one that lacks this capacity (;Capelle Desprez'). During a 9-day stress cycle, when water was withheld from plants grown in a growth chamber, the relative water content of leaves declined by 30% in both cultivars. Leaf osmotic potential (psi(s)) declined to a greater degree in Condor plants. Measuring psi(s) at full turgor indicated that osmotic adjustment occurred in stressed Condor, but not in Capelle plants. Two methods were used to examine the degree of symplast (i.e. protoplast) volume reduction in tissue rapidly equilibrated to increasingly low psi(w). Both techniques gave similar results. With well-watered plants, symplast volume reduction from the maximum (found at high psi(w) for each cultivar) was the same for Condor and Capelle. After a stress cycle, volume was maintained to a greater degree at low psi(w) in Condor leaf tissue than in Capelle. Nonstomatally controlled photosynthesis was inhibited to the same degree at low psi(w) in leaf tissue prepared from well-watered Condor and Capelle plants. However, photosynthetic capacity was maintained to a greater degree at low psi(w) in tissue prepared from stressed Condor plants than in tissue from stressed Capelle plants. Net CO(2) uptake in attached leaves was monitored using an infrared gas analyzer. These studies indicated that in water stressed plants, photosynthesis was 106.5% higher in Condor than Capelle at ambient [CO(2)] and 21.8% higher at elevated external [CO(2)]. The results presented in this report were interpreted as consistent with the hypothesis that there is a causal association between protoplast (and presumably chloroplast) volume reduction at low psi(w) and low psi(w) inhibition of photosynthesis. Also, the data indicate that osmotic adjustment allows for maintenance of relatively greater volume at low psi(w), thus reducing low psi(w) inhibition of chloroplast photosynthetic potential. PMID- 16665801 TI - Plant plasma membrane proteins : immunological characterization of a major 75 kilodalton protein group. AB - A major 75 kD protein group from the tomato plasma membrane was semipurified on polyacrylamide gels and used to raise a rabbit antiserum. The resulting antiserum recognized a single 75 kilodalton band from phase partitioned tomato plasma membrane (from both suspension cells and mature, green fruit) after resolution on one-dimensional polyacrylamide gels. Two-dimensional polyacrylamide gel analysis of proteins from tomato plasma membrane showed that the 75 kilodalton antiserum recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons (mean = 75.6 kilodaltons) and with isoelectric point values ranging from 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This antiserum was shown to bind protoplast surface epitopes by indirect immunofluorescence. The presence of this protein group in both monocotyledonous and dicotyledonous plants was established by immunoblotting the tomato 75 kilodalton antiserum against proteins obtained from plasma membrane-enriched fractions from corn roots and soybean roots. The data suggest that this 75 kilodalton protein group is a major proteinaceous component of the plant plasma membrane. PMID- 16665802 TI - Effect of salinity on leaf gas exchange in two populations of a c(4) nonhalophyte. AB - Gas exchange measurements were made on plants from two natural populations differing in salt tolerance of Andropogon glomeratus, a C(4) nonhalophyte, to examine the effect of salinity on components responsible for differences in photosynthetic capacity. Net CO(2) uptake and stomatal conductance decreased with increasing salinity in both populations, but to a greater extent in the inland (nontolerant) population. The intercellular CO(2) concentrations increased with increasing salinity in the inland population, but decreased in the marsh (tolerant) population. Water use efficiency decreased as salinity increased in the inland population, and remained unchanged in the marsh population. Carboxylation efficiency decreased and CO(2) compensation points increased with increasing salinity in both populations, but to a lesser extent in the marsh population. Carboxylation efficiencies were higher with 2% relative to 21% atmospheric O(2) in salt stressed plants, suggesting that a decrease in the carboxylation:oxygenation ratio of ribulose 1,5-bisphosphate carboxylase/oxygenase was partly responsible for the decrease in photosynthetic capacity. Populational differences in photosynthetic capacity were the result of greater salinity-induced changes in carboxylation efficiency in the inland population, and not due to differences in the stomatal limitation to CO(2) diffusion. PMID- 16665803 TI - Histochemical technique: densitometry of nanogram quantities of proteins separated in one-dimensional microslab gels. AB - We describe methods for densitometry of electrophoretically separated proteins in 25-millimeter microslab gels. The methods are sufficiently sensitive to use with several individually excised plant cells, for which we describe an extraction procedure. In brief, submicrogram samples are excised from freeze-dried plant tissue. Extraction takes place under oil in microliter droplets of detergent. Proteins are separated by one-dimensional microelectrophoresis (HM Poehling, V Neuhoff 1980 Electrophoresis 1: 90-102) and then stained by a sensitive Coomassie procedure (V Neuhoff, R Stamm, E Hansjorg 1985 Electrophoresis 6: 427-448). The resulting profile is scanned by a computerized densitometer based on the Leitz Diavert MPV Microphotometer. Evaluations and typical data demonstrate the high performance of this system. PMID- 16665804 TI - Photosynthesis in Flaveria brownii A.M. Powell : A C(4)-Like C(3)-C(4) Intermediate. AB - Leaves of Flaveria brownii exhibited slightly higher amounts of oxygen inhibition of photosynthesis than the C(4) species, Flaveria trinervia, but considerably less than the C(3) species, Flaveria cronquistii. The photosynthetic responses to intercellular CO(2), light and leaf temperature were much more C(4)-like than C(3)-like, although 21% oxygen inhibited the photosynthetic rate, depending on conditions, up to 17% of the photosynthesis rate observed in 2% O(2). The quantum yield for CO(2) uptake in F. brownii was slightly higher than that for the C(4) species F. trinervia in 2% O(2), but not significantly different in 21% O(2). The quantum yield was inhibited 10% in the presence of 21% O(2) in F. brownii, yet no significant inhibition was observed in F. trinervia. An inhibition of 27% was observed for the quantum yield of F. cronquistii in the presence of 21% O(2). The photosynthetic response to very low intercellular CO(2) partial pressures exhibited a unique pattern in F. brownii, with a break in the linear slope observed at intercellular CO(2) partial pressure values between 15 and 20 mubar when analyzed in 21% O(2). No significant break was observed when analyzed in 2% O(2). When taken collectively, the gas-exchange results reported here are consistent with previous biochemical studies that report incomplete intercellular compartmentation of the C(3) and C(4) enzymes in this species, and suggest that F. brownii is an advanced, C(4)-like C(3)-C(4) intermediate. PMID- 16665805 TI - Ice Encasement Injury to Microsomal Membranes from Winter Wheat Crowns : I. Comparison of Membrane Properties after Lethal Ice Encasement and during a Post Thaw Period. AB - The functional and physical properties of cellular membranes isolated from Triticum aestivum, cvs Norstar and Fredrick, were altered coincident with changes in composition after a lethal ice-encasement stress and further during a 6 hour post-thaw period. Crowns encased in ice for a duration which inhibited regrowth, exhibited enhanced rates of electrolyte leakage. Furthermore, the recovery of total microsomal protein and phospholipid declined, suggesting that some membrane degradation had been induced during the anoxic stress. The microviscosity of microsomes and liposomes prepared from such membranes increased during stress, and this was correlated with a 2- to 4-fold increase in the free fatty acid levels in the microsomal fraction. There was, however, only a relatively minor change in fatty acid unsaturation during the ice-encasement stress. The process continued during a 6 hour aerobic post-thaw treatment, but the pattern was somewhat different. During this phase, the leakage of electrolytes was further increased and the recovery of microsomal protein and phospholipid continued to decline, indicating general degradation; but, in contrast to the anoxic phase, the degree of fatty acid unsaturation declined markedly, indicating lipid peroxidation. PMID- 16665806 TI - Pathways of Fatty Acid hydroperoxide metabolism in spinach leaf chloroplasts. AB - The metabolism of 13-hydroperoxylinolenic acid was examined in protoplasts and homogenates prepared from mature leaves of spinach (Spinacia oleracea L.). Chloroplast membranes were the principal site for metabolism of the compound by at least two highly hydrophobic enzyme systems, hydroperoxide lyase and hydroperoxide dehydrase, the new name for an enzyme system formerly known as hydroperoxide isomerase and hydroperoxide cyclase. Hydroperoxide lyase was most active above pH 7 and could be separated from hydroperoxide dehydrase by anion exchange chromatography. Hydroperoxide dehydrase, measured by the formation of both alpha-ketol product and 12-oxo-phytodienoic acid, had its optimum activity in the range of pH 5 to 7. Lyase was more active than dehydrase activity when the enzymes were extracted by homogenization. The reverse was true when the enzyme activities were measured in protoplasts, which are isolated by gentle extraction methods. The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response. In the absence of injury, the normal pathway of fatty acid hydroperoxide metabolism is probably by hydroperoxide dehydrase activity. The molecular weights of both the lyase and dehydrase were approximately 220,000, as estimated by gel filtration. PMID- 16665807 TI - The C-S Lyases of Higher Plants : Direct Comparison of the Physical Properties of Homogeneous Alliin Lyase of Garlic (Allium sativum) and Onion (Allium cepa). AB - Garlic and onion alliin lyases, although from closely related species, have many differences. The two enzymes differ in their K(m) values, pH optima, and isoelectric points. There is a major difference in their molecular weight and subunit structure. The garlic holoenzyme has a molecular weight of 85,000 and consists of two subunits of molecular weight 42,000. The onion enzyme has a holoenzyme molecular weight of 200,000 composed of four subunits of molecular weight 50,000. The onion enzyme is much more difficult to dissociate into its subunits which suggests differences in subunit interaction between the two enzymes. The dimeric stucture of the garlic and the tetrameric structure of the onion enzyme is consistent with a coenzyme content (pyridoxal-5'-phosphate) equivalent to one mole per subunit. The two enzymes vary vastly in their spectra, the onion enzyme having a lower pyridoxal-5'-phosphate absorbance at 430 nanomoles and an inability to react with l-cysteine. Both enzymes are glycoproteins and bind to concanavalin A-Sepharose columns. The onion alliin lyase binds more tightly than the garlic enzyme. The amino acid content of both enzymes is similar as is the carbohydrate content. However, upon hydrolysis the onion lyase does yield more mannose units than the garlic enzyme which is consistent with the former's stronger affinity for concanavalin A. PMID- 16665808 TI - An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants. AB - An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others. PMID- 16665809 TI - Development of the alt Mutant of Pisum sativum L. AB - The alt (albina-terminalis) mutant of Pisum sativum L. germinates normally, produces several nodes, and then above a sharp transition produces 2 to 3 bleached nodes, ceases growth, and eventually dies. Green nodes have normal chlorophyll content, absorption spectra, photosynthetic rates, and ultrastructure. In bleaching tissues, the chloroplasts degenerate rapidly, followed by extensive disruption and loss of the remaining cytoplasm and organelles. Application of tissue extracts of normal genotypes of pea, corn, and bean stimulates apical development of alt. The resulting tissues have essentially normal structure and function. Application of thiamine, thiamine monophosphate, and thiamine pyrophosphate also stimulate normal apical development at concentrations of 1 micromolar and above. Partial characterization of the stimulus from pea seed extracts is consistent with thiamine as the active factor. PMID- 16665810 TI - Protein Synthesis during Rehydration of Rapidly Dried Tortula ruralis: Evidence for Oxidation Injury. AB - Rapidly dried Tortula ruralis, a drought-tolerant moss, is known to synthesize proteins on rehydration at a much lower rate than the slowly dried moss. The reasons for this low rate of protein synthesis are unclear. We have found that during rehydration of rapidly dried moss, there is a negative correlation between the rate of protein synthesis and the tissue levels of oxidized glutathione (GSSG) and lipid peroxidation. When rapidly dried moss, which is known to show extensive solute leakage, is rehydrated in the presence of 100 millimolar K(+), 5 millimolar Mg(2+), 1 millimolar ATP, and 1 millimolar GTP, either separately or together, there is no stimulation of protein synthesis. When it is hydrated in the presence of either 5 millimolar glucose-6-phosphate or 0.1 millimolar NADPH, protein synthesis is stimulated but the stimulation is transitory. A second addition of either of these two chemicals causes a second transient stimulation of protein synthesis. A transitory decrease in the rate of GSSG accumulation is observed during rehydration in the presence of glucose-6-phosphate or NADPH. Both glucose-6-phosphate and NADPH are known to reverse GSSG-induced inhibition of protein synthesis in rabbit reticulocyte lysate. Results of the present study suggest that the rate of protein synthesis during rehydration of rapidly dried moss is not limited by the availability of ions or energy sources. Since exogenously applied GSSG has been shown to inhibit in vivo and in vitro protein synthesis and since it is known to accumulate during rehydration of rapidly dried, but not slowly dried, moss, it is suggested that the low rate of protein synthesis during rehydration of the rapidly dried moss is, at least in part, due to endogenous GSSG. PMID- 16665811 TI - Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus. AB - The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline. PMID- 16665812 TI - Bacillus thuringiensis section sign-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects. AB - The crystal proteins, or section sign-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests. PMID- 16665813 TI - Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides. AB - Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways to valine, isoleucine, and leucine. It is the target of two structurally unrelated classes of herbicides, the sulfonylureas and the imidazolinones. Genomic clones encoding ALS have been isolated from the higher plants Arabidopsis thaliana and Nicotiana tabacum, using a yeast ALS gene as a heterologous hybridization probe. Clones were positively identified by the homology of their deduced amino acid sequences with those of yeast and bacterial ALS isozymes. The tobacco and Arabidopsis ALS genes have approximately 70% nucleotide homology, and encode mature proteins which are approximately 85% homologous. Little homology is seen between the amino acid sequences of the presumptive N-terminal chloroplast transit peptides. Both plant genes lack introns. The tobacco ALS gene was isolated from a line of tobacco which is resistant to the sulfonylurea herbicides due to an alteration in ALS. The tobacco gene which was isolated codes for an ALS that is sensitive to the herbicides, as assayed by transformation of the gene into sensitive tobacco cells. PMID- 16665814 TI - Isozymes of beta-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.). AB - Four isozymes of beta-N-acetylhexosaminidase (beta-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated beta-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p aminophenyl-N-acetyl-beta-d-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn(2+) charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. beta-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p nitrophenol from both p-nitrophenyl-N-acetyl-beta-d-glucosaminide and p nitrophenyl-N-acetyl-beta-d-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K(m) value for the two substrates and pH optima of the isozymes are comparable to beta-NAHAs from other plant sources. PMID- 16665815 TI - Biochemistry of Oleoresinosis : Monoterpene and Diterpene Biosynthesis in Lodgepole Pine Saplings Infected with Ceratocystis clavigera or Treated with Carbohydrate Elicitors. AB - Elevated levels of monoterpenes and diterpene resin acids are produced in the stems of lodgepole pine (Pinus contorta var latifolia) saplings when wounded and inoculated with the blue-stain fungus Ceratocystis clavigera or when wounded and treated with a pectic fragment from tomato leaves (PIIF) or a fungal cell wall fragment (chitosan). This induced defensive response (hyperoleoresinosis) is the result of a transient rise in the ability to biosynthesize cyclic monoterpenes and diterpene resin acids as measured by the in vivo incorporation of label from [U-(14)C]sucrose relative to untreated controls, and is accompanied by a corresponding rise in the levels or activities of the relevant terpene cyclases as determined by in vitro assay using labeled acyclic precursors. The results indicate that juvenile P. contorta responds to infection and biotic elicitors much like the mature tree, and they suggest that the Pinaceae possess a mechanism for elicitor recognition and induced defense similar to that of other higher plants. PMID- 16665816 TI - Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum. AB - Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization. PMID- 16665817 TI - Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles. AB - Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell. PMID- 16665818 TI - Carbon isotope ratios demonstrate carbon flux from c(4) host to c(3) parasite. AB - Carbon isotope ratios of mature leaves from the C(3) angiosperm root hemiparasites Striga hermonthica (Del.) Benth (-26.7 per thousand) and S. asiatica (L.) Kuntze (-25.6 per thousand) were more negative than their C(4) host, sorghum (Sorghum bicolor [L.] Moench cv CSH1), (-13.5 per thousand). However, in young photosynthetically incompetent plants of S. hermonthica this difference was reduced to less than 1 per thousand. Differences between the carbon isotope ratios of two C(3)-C(3) associations, S. gesnerioides (Willd.) Vatke-Vigna unguiculata (L.) Walp. and Oryza sativa L.-Rhamphicarpa fistulosa (Hochst.) Benth differed by less than 1 per thousand. Theoretical carbon isotope ratios for mature leaves of S. hermonthica and S. asiatica, calculated from foliar gas exchange measurements, were -31.8 and -32.0 per thousand, respectively. This difference between the measured and theoretical delta(13)C values of 5 to 6 per thousand suggests that even in mature, photosynthetically active plants, there is substantial input of carbon from the C(4) host. We estimate this to be approximately 28% of the total carbon in S. hermonthica and 35% in S. asiatica. This level of carbon transfer contributes to the host's growth reductions observed in Striga-infected sorghum. PMID- 16665819 TI - Wavelength Effect on the Action of a N-Phenylimide S-23142 and a Diphenylether Acifluorfen-Ethyl in Cotyledons of Cucumber (Cucumis sativus L.) Seedlings. AB - Specific wavelengths of light required for expression of phytotoxic activity of S 23142 (N-[4-chloro-2-fluoro-5-propargyloxy]phenyl-3,4,5,6-tetra- hydrophthalimide) and acifluorfen-ethyl (ethyl-5-[2-chloro-4 (trifluoromethyl)phenoxy]-2-nitro benzoic acid) were determined in cotyledons of cucumber seedlings using the Okazaki Large Spectrograph. Leakage of amino acids from the cotyledons was measured as an indication of the phytotoxic activity. The wavelength effects showed common major peaks of activity at 550 and 650 nanometers and a minor peak at 450 nanometers for both herbicides, indicating a common primary photoreaction. Concomitant application of DCMU (3-[3,4 dichlorophenyl]-1,1-dimethylurea) with S-23142 had little influence on the effective wavelengths for S-23142 activity. Light of 450 and 650 nanometers was relatively less effective in achlorophyllous tissue grown in far red light than in green tissue. These results strongly suggest that the phytotoxic action of S 23142 and diphenylethers involves multiple photoreactions and that one of the photoreceptor pigments may be chlorophyll or its related pigment, although photosynthesis is not involved. PMID- 16665820 TI - Phosphatidylinositol specific phospholipase C of plant stems : membrane associated activity concentrated in plasma membranes. AB - A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1.4.10) assayed at pH 6.6 and at 30 degrees C cleaved phosphatidylinositol such that more than 85% of the product was inositol-1-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all major cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by two-phase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium. PMID- 16665821 TI - Kinetic Analysis of Resistance to Paraquat in Conyza: Evidence that Paraquat Transiently Inhibits Leaf Chloroplast Reactions in Resistant Plants. AB - Paraquat resistance has been claimed to be due to a sequestration of the herbicide before it reaches chloroplasts. This is based on the sensitivity of photosystem I in isolated thylakoids to paraquat, and autoradiographic analyses showing label from paraquat near veins 4 hours after treatment of a resistant biotype. Conversely, the enzymes of the superoxide detoxification pathway were found to be at constitutively elevated levels in intact class A chloroplasts of the resistant biotype of Conyza bonariensis (L.) Cronq. Evidence is presented here that physiologically active levels of paraquat rapidly inhibit chloroplast function in both the resistant and sensitive biotype, before the first sequestration was visualized. This inhibition is transient (completed in 2 hours) in the resistant biotype and irreversible in the sensitive type. Intact class A chloroplasts of the resistant biotype with or without paraquat are less susceptible to photoinduced membrane damage than the sensitive biotype without paraquat, as measured by ethane evolution. These data support a hypothesis that the ability to prevent superoxide damage keeps the resistant biotype viable while paraquat or its metabolites are being sequestered. PMID- 16665822 TI - Generation of a membrane potential by electron transport in plasmalemma-enriched vesicles of cotton and radish. AB - Plasmalemma-enriched vesicles were isolated from cotton roots (Gossypium hirsutum L. cv Acala San Jose 2) and from germinating radish seeds (Raphanus sativa L. cv Tondo Rosso Quarantino). When 100 millimolar ascorbate was added to the grinding medium, the addition of ferricyanide to either preparation led to an inside positive membrane potential as measured by the accumulation of thiocyanate. It is suggested that electrons from ascorbate were being transported electrogenically across the membrane to ferricyanide, resulting in an accumulation of protons within the vesicle. The redox activity of the vesicles has some similarities to that occurring in intact cells, thus providing a simpler system to study the components and effects of transmembrane electron transport. PMID- 16665823 TI - Procedure to Isolate Viable Sperm Cells from Corn (Zea mays L.) Pollen Grains. AB - Sperm cells were isolated from corn (Zea mays L.) tricellular pollen grains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 x 10(6) per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts. PMID- 16665824 TI - Photoinduced Seed Germination of Oenothera biennis L: I. General Characteristics. AB - General characteristics of light-induced germination of Oenothera biennis L. seeds were investigated at 24 degrees C. During dark imbibition, seeds reached maximal respiration in 7 hours and maximal water content and photosensitivity in 24 hours. After dark imbibition of 24 hours, seeds required a long exposure (>36 hours) to red or white light for maximal germination. Two photoperiods (12 and 2 hours) separated by a period of darkness of 10 to 16 hours gave near maximal germination. For the two photoperiod regime, the first light potentiates a reversible phytochrome response by the second light. A 35 degrees C treatment for 2 to 3 hours in the dark immediately prior or subsequent to 8 hours of light caused a higher percentage of germination. A 2 hour treatment at 35 degrees C also potentiates a reversible phytochrome response. Halved seeds germinated at 100% in light or darkness indicating that the light requirement of the seeds is lost in the halving procedure. After-ripened seeds required less light and germinated more rapidly and at higher percentages than seeds tested shortly after maturation. PMID- 16665825 TI - Photoinduced Seed Germination of Oenothera biennis L: II. Analysis of the Photoinduction Period. AB - The photoinduction period of Oenothera biennis L. seed germination was analyzed by varying the photoinduction temperature and by substituting red light pulses for continuous red light. At 24 degrees C, seeds require 36 hours of continuous red light for maximal percent germination. The optimal photoinduction temperature is 32 degrees C, with higher and lower temperatures being strongly inhibitory. A 30 minute exposure to far-red light, given immediately after a red light period of 1 to 36 hours, reduces germination by about 25%. Seeds escape from far-red inhibition with a half-time of 5 to 10 hours, depending on the length of the red exposure that precedes the far-red light. Periodic 15 minute pulses of red light can substitute for continuous red light in stimulating germination. Ted red light pulses, with 6 hours of darkness between successive pulses, cause maximal germination. The response to periodic red light is fully reversible by far-red light. Probit analysis of the periodic light response shows that as the length of the dark periods between successive pulses increases, less incident light is needed to induce germination but the population variance in light sensitivity remains constant. Probit analysis of the temperature response shows that as the photoinduction temperature increases from 16 to 32 degrees C, less incident light is needed to induce germination and the population variance in light sensitivity also increases. PMID- 16665826 TI - Intracellular Carbon Partitioning in Chlamydomonas reinhardtii. AB - Using enzymic and isotope techniques the intracellular partitioning of newly fixed carbon was studied in synchronized cells of Chlamydomonas reinhardtii. Starch and growth metabolism, i.e. the use of carbon in biosynthesis, were found to be the major sinks for photosynthetically fixed carbon in the alga. Sucrose does not accumulate in significant quantities. The amount of carbon partitioned either into starch or growth varies during the 12 hour light/12 hour dark cell cycle. Starch is accumulated at the beginning and at the end of the light period while a net breakdown is observed in the middle of the light period and in the dark. In contrast, nonsynchronized cells accumulate starch all the time in the light which suggests that carbon partitioning is controlled by the cell cycle. Labeled bicarbonate is incorporated into starch even at times when the total intracellular level of starch is decreasing. This indicates a turnover of the starch pool in the light with synthesis and degradation occurring simultaneously and at different rates. PMID- 16665827 TI - Mutations in Chlamydomonas reinhardtii Conferring Resistance to the Herbicide Sulfometuron Methyl. AB - Chlamydomonas reinhardtii mutants resistant to the herbicide sulfometuron methyl (SM) were isolated and characterized. Growth of C. reinhardtii is sensitive to inhibition by SM at a concentration of 1 micromolar. Four mutants resistant to 10 to 100-fold higher concentrations were isolated. All possess a form of acetolactate synthase (ALS) whose specific activity in cell extracts is 100- to 1000-fold more resistant to SM than is the specific activity of wild-type enzyme. Only one mutant had abnormally low ALS specific activity in the absence of SM. All mutations were inherited as single lesions in the nuclear genome and were expressed in heterozygous diploids. The mutations in two strains mapped to linkage group IX about 30 centimorgans from streptomycin resistance and on the same side of the centromere, and in genetic crosses between mutants no segregation was observed. Accordingly, all mutations are tentatively assigned to gene smr-1. Herbicide resistance appears to be suitable as a selectable marker for molecular transformation in this organism. PMID- 16665828 TI - Transport and Metabolism of a Sucrose Analog (1'-Fluorosucrose) into Zea mays L. Endosperm without Invertase Hydrolysis. AB - 1'-Fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L., Pioneer Hybrid 3320) kernels with the same magnitude and kinetics as sucrose. (14)C-Label from [(14)C]FS and [(14)C]sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was absorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of [(14)C] sucrose from supplied [(14)C]glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggests that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded from the phloem, hexoses are not specifically needed for uptake into maize endosperm. PMID- 16665829 TI - Polyamine oxidase from water hyacinth: purification and properties. AB - Polyamine oxidase was purified to homogeneity from leaves of water hyacinth by the criterion of sodium dodecyl sulfate gel electrophoresis (SDS disc PAGE). The enzyme showed a high specificity for spermidine and spermine (K(m) values 28 micromolar and 20 micromolar, respectively). The optimal pH of the enzyme for both spermidine and spermine was 6.5. The molecular weight of the enzyme estimated by Sephadex G-200 gel filtration was 87,000, while SDS disc PAGE gave a single band at the molecular weight of 60,000. Octamethylenediamine and quinacrine were strong inhibitors of the enzyme, but p-chloromercuribenzoate was without effect. A prosthetic group in the enzyme was identified as flavin adenine dinucleotide. PMID- 16665830 TI - Xanthophylls and abscisic Acid biosynthesis in water-stressed bean leaves. AB - Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to (14)CO(2) and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in (12)CO(2) for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from (14)CO(2) were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of (14)C from (14)CO(2) into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of (18)O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with (18)O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves. PMID- 16665831 TI - Conversion of xanthoxin to abscisic Acid by cell-free preparations from bean leaves. AB - Cell-free extracts from the leaves of Phaseolus vulgaris L. convert xanthoxin to abscisic acid. The enzyme activity in dialyzed or acetone-precipitated extracts shows a strong dependence on either NAD or NADP. The enzyme activity appears to be cytosolic with no significant activity observed in chloroplasts. The activity was observed in extracts from roots of Phaseolus vulgaris, and also in extracts prepared from the leaves of Pisum sativum L., Zea mays L., Cucurbita maxima Duchesne, and Vigna radiata L. Neither water stress nor cycloheximide appear to significantly affect the level of enzyme activity in leaves. No intermediates between xanthoxin and abscisic acid were detected. PMID- 16665832 TI - Mesophyll Resistances to SO(2) Fluxes into Leaves. AB - Uptake of label from solutions containing (35)SO(2), H(35)SO(3) (-) and (35)SO(3) (2-) into mesophyll protoplasts, vacuoles, and chloroplasts isolated from young barley leaves was measured at different pH values. Uptake was fast at low pH, when the concentration of SO(2) was high, and low at high pH, when the concentration of SO(2) was low. When the resistance (R) of plasmalemma, tonoplast, and chloroplast envelope to the penetration of SO(2) was calculated from rates of uptake of label, comparable values were obtained for the different biomembranes at low pH values. R was close to 8000 seconds per meter and permeability coefficients were close to 1.25 x 10(-4) meters per second. Under these conditions R may describe resistance to SO(2) diffusion across a lipid bilayer. At higher pH values, R decreased. As R was calculated on the assumption that SO(2) is the only penetrating molecular species, the data suggest that carrier-mediated anion transport contributes to the uptake of sulfur at physiological pH values thereby decreasing apparent R(SO(2) ). The contribution of anion transport appeared to be smaller for transfer across the plasmalemma than for transfer across the tonoplast. It was large for transfer across the chloroplast envelope. The phosphate translocator of the chloroplast envelope catalyzed uptake of SO(3) (2-) into chloroplasts at neutral pH. Uptake was decreased in the presence of high levels of phosphate or sulfate and by pyridoxal phosphate. SO(2) transfer into cells leads to the intracellular liberation of one or two protons, depending on pH and oxidizing conditions. When the divalent sulfite anion is exchanged across the chloroplast envelope, bisulfite formation results in proton uptake in the chloroplast stroma, whereas SO(2) uptake into chloroplasts lowers the stroma pH. PMID- 16665833 TI - Flux of SO(2) into Leaf Cells and Cellular Acidification by SO(2). AB - A comparison of fluxes of SO(2) from the atmosphere into leaves with fluxes across biomembranes revealed that, apart from the cuticle, the main barrier to SO(2) entry into leaves are the stomates. SO(2) fluxes into leaves can be calculated with an accuracy sufficient for many purposes on the assumption that the intracellular SO(2) concentration is zero. SO(2) entering green leaf cells is trapped in the cytoplasm. In the light, the products formed in its reaction with water are processed particularly in the chloroplasts. Flux of SO(2) to the acidic central vacuole of leaf cells is insignificant. Intracellular acidification of barley mesophyll protoplasts by SO(2) was measured by the uptake of (14)C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. The measured acidification was similar to the acidification calculated from known buffer capacities and the rate of SO(2) influx when the H(+)/SO(2) ratio was assumed to be 2. A comparison of photosynthesis inhibition by SO(2) with calculated acidification revealed different mechanisms of inhibition at low and at high concentrations of SO(2). At very low concentrations, inhibition by SO(2) was even smaller than expected from calculated acidification. The data suggest that, if acidification cannot be compensated by pH-stabilizing cellular mechanisms, it is a main factor of SO(2) toxicity at low SO(2) levels. At high levels of SO(2), anion toxicity and/or radical formation during oxidation of SO(2) to sulfate may play a large role in inhibition. PMID- 16665834 TI - In Vitro Gibberellin A(1) Binding in Zea mays L. AB - The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]gibberellin A(1) (GA(1)) to a soluble macromolecular component present in the cytosol was demonstrated at 4 degrees C by Sephadex G 200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the (3)H-activity bound to this protein was largely [(3)H]GA(1) and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA(1). Both biologically active and inactive GAs and non-GAs were able to inhibit GA(1) binding. [(3)H]GA(1) binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography. PMID- 16665835 TI - Classification and Structural Comparison of Full-Length cDNAs for Pathogenesis Related Proteins. AB - Fourteen cDNA clones of pathogenesis-related (PR) proteins, PR1a and PR1b of tobacco were obtained and classified into six groups based on restriction enzyme maps. To assign the groups to different classes of PR1 proteins, all the clones were partially sequenced and compared with amino acid sequences of PR1a and PR1b. Two groups of these corresponded to PR1a and four to PR1b. The results indicate that there are at least two kinds of PR1a mRNAs and four kinds of PR1b mRNAs. In fact, one cDNA insert hybridized to at least six to seven DNA fragments in restriction enzyme fragments of Samsun NN genomic DNA, indicating that the PR1 protein genes exist as a multigene family in the tobacco genome. Two sequences of essentially full-length cDNAs for PR1a and PR1b were determined and compared. The coding sequences of two cDNAs share 93% homology and the deduced amino acid sequences of PR1a and PR1b precursors, which are synthesized as larger precursors containing signal peptides, are 91% homologous. The homology of mature PR1a and PR1b regions is higher than that of larger precursors, 94% in the nucleotide sequence and 93% in the amino acid sequence, whereas that of the signal peptide regions is 80 and 90%, respectively. The hydropathy patterns and the secondary structures predicted by Chou-Fasman rules are similar to tomato PR protein in the half-side of the C terminus, which suggests that the half-C terminus side is important for the function of PR1 proteins. PMID- 16665836 TI - Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants. AB - Phosphoenolpyruvate carboxylase (PEPC) from several C(3) plants was compared to maize PEPC by immunoblotting using an antibody against maize PEPC and by peptide mapping. In C(3) gramineous plants, PEPCs of slightly different monomeric sizes were detected as two bands for wheat and barley leaves, as three bands for etiolated maize leaves and as four bands for rice leaves by SDS-polyacrylamide gel electrophoresis and immunoblotting, whereas only one PEPC band was detected for maize leaves, a C(4) plant, or tobacco leaves, a dicotyledonous C(3) plant. The peptide fragment patterns of the lower molecular weight PEPC (major band in immunoblotting) in wheat leaves was similar to that of maize PEPC in peptide mapping by protein staining or by immunological detection, but the upper one (minor band) had a different pattern from the lower one in peptide mapping by immunological detection and few peptide fragments from this were recognized by the anti-(maize) PEPC antibody. These results suggest that there are multiple forms of PEPC subunits in the gramineous plants tested, and the major PEPC has a primary structure similar to that of maize PEPC. To obtain information about the expression of PEPCs in C(3) plants, changes in the amount of PEPC protein were investigated during the greening of rice and wheat seedlings. Judging from the regulation by light, there were two types of PEPCs in greening rice seedlings, one induced by light and the other reduced by it. Greening wheat seedlings also show a PEPC band induced by light. These findings indicate that some PEPCs in C(3) gramineous plants not only have structures similar to that of maize PEPC, but also are regulated by light in a similar manner. PMID- 16665837 TI - Form of inorganic carbon involved as a product and as an inhibitor of c(4) Acid decarboxylases operating in c(4) photosynthesis. AB - These studies demonstrated that CO(2) rather than HCO(3) (-) is the inorganic carbon metabolite produced by the C(4) acid decarboxylases involved in C(4) photosynthesis (chloroplast located NADP malic enzyme, mitochondrial NAD malic enzyme, and cytosolic phosphoenolpyruvate [PEP] carboxykinase). The effect of varying CO(2) or HCO(3) (-) as a substrate for the carboxylation reaction catalyzed by these enzymes or as inhibitors of the decarboxylation reaction was also determined. The K(m)CO(2) was 1.1 millimolar for NADP malic enzyme and 2.5 millimolar for PEP carboxykinase. For these two enzymes the velocity in the carboxylating direction was substantially less than for the decarboxylating direction even with CO(2) concentrations at the upper end of the range of expected cellular levels. Activity of NAD malic enzyme in the carboxylating direction was undetectable. The decarboxylation reaction of all three enzymes was inhibited by added HCO(3) (-). For NADP malic enzyme CO(2) was shown to be the inhibitory species but PEP carboxykinase and NAD malic enzyme were apparently inhibited about equally by CO(2) and HCO(3) (-). PMID- 16665838 TI - Mechanism of c(4) photosynthesis: the size and composition of the inorganic carbon pool in bundle sheath cells. AB - We sought to characterize the inorganic carbon pool (CO(2) plus HCO(3) (-)) formed in the leaves of C(4) plants when C(4) acids derived from CO(2) assimilation in mesophyll cells are decarboxylated in bundle sheath cells. The size and kinetics of labeling of this pool was determined in six species representative of the three metabolic subgroups of C(4) plants. The kinetics of labeling of the inorganic carbon pool of leaves photosynthesizing under steady state conditions in (14)CO(2) closely paralleled those for the C-4 carboxyl of C(4) acids for all species tested. The inorganic carbon pool size, determined from its (14)C content at radioactivity saturation, ranged between 15 and 97 nanomoles per milligram of leaf chlorophyll, giving estimated concentrations in bundle sheath cells of between 160 and 990 micromolar. The size of the pool decreased, together with photosynthesis, as light was reduced from 900 to 95 microeinsteins per square meter per second or as external CO(2) was reduced from 400 to 98 microliters per liter. A model is developed which suggests that the inorganic carbon pool existing in the bundle sheath cells of C(4) plants during steady state photosynthesis will comprise largely of CO(2); that is, CO(2) will only partially equlibrate with bicarbonate. This predominance of CO(2) is believed to be vital for the proper functioning of the C(4) pathway. PMID- 16665839 TI - Studies on Octylphenoxy Surfactants : III. Sorption of Triton X-100 by Isolated Tomato Fruit Cuticles. AB - Sorption characteristics of a polyethoxy (EO) derivative of octylphenol (OP) were determined for enzymically isolated mature tomato (Lycopersicon esculentum Mill. cv Sprinter) fruit cuticles at 25 degrees C. Sorption was followed using (14)C labeled OP + 9.5EO (Triton X-100). Solution pH (2.2-6.2) did not affect surfactant sorption by tomato fruit cuticular membranes (CM). Surfactant concentration (0.001-1.0%, w/v) had a marked impact on sorption. Sorption equilibrium was reached in 24 hours for OP + 9.5EO concentrations below the critical micelle concentration (CMC), whereas 72 to 120 hours were required to reach equilibrium with concentrations greater than the CMC. Regardless of when equilibrium was attained, initial sorption of OP + 9.5EO occurred rapidly. Partition coefficients (K) of approximately 300 were obtained at pre-CMC concentrations, whereas at the highest concentration (1.0%), K values were approximately 15- to 20-fold lower. Sorption was higher for dewaxed CM (DCM) than for CM. At OP + 9.5EO concentrations below the CMC, the amount (millimoles per kilogram) sorbed by CM and DCM increased sharply as the CMC was reached. After an apparent plateau in the amount sorbed at concentrations immediately below and above the CMC, sorption by CM and DCM increased dramatically with OP + 9.5EO concentrations greater than the CMC (0.5 and 1.0%). In contrast, sorption of OP + 5EO (Triton X-45) by CM and DCM differed from one another at relatively high (0.5 and 1.0%) concentrations, where sorption by DCM increased with increasing concentration, but plateaued for the CM. Sorption of OP + 9.5EO was also related to CM concentration, with an inverse relationship existing between sorption and CM at concentrations less than 3.33 milligrams per milliliter. PMID- 16665840 TI - Regulation of light-harvesting chlorophyll protein biosynthesis in greening seedlings : a species comparison. AB - The biosynthesis of the chlorophyll a/b binding protein associated with photosystem II (LHC-II) was characterized during light-induced greening of etiolated barley (Hordeum vulgare [L.] cv Boone), maize (Zea mays [L.] Pioneer 3148), pea (Pisum sativum [L.] cv Progress 9), and soybean (Glycine max [L.] Merr. cv Ransom 2). Northern blot analysis revealed that pea LHC-II mRNA was present in dark-grown seedlings and accumulated rapidly within 1 hour following illumination with white light. In contrast, the accumulation of LHC-II mRNA was delayed in barley and soybean until 2 to 4 hours after illumination began. Single radial immunodiffusion analysis revealed that LHC-II polypeptides began to accumulate in all species between 4 and 8 hours although the protein was present in detectable levels at earlier times in certain species. In a pattern similar to the LHC-II protein accumulation, chlorophyll accumulated at increased rates between 4 and 8 hours of greening in all species following an initial delay. The absence of coordination between LHC-II mRNA and LHC-II protein accumulation that was clearly observed in pea suggested that transcription is not the factor that limits LHC-II complex formation during chloroplast development. The accumulation of chlorophyll and LHC-II protein appeared to coincide suggesting that chlorophyll biosynthesis may be a factor that limits LHC-II complex formation. PMID- 16665841 TI - Photosynthetic Characteristics of the C(3)-C(4) Intermediate Parthenium hysterophorus. AB - The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO(2) compensation concentration (20-25 microliters per liter at 30 degrees C and 21% O(2)) and by a reduced sensitivity of net photosynthesis to 21% O(2). In contrast, the related C(3) species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO(2) compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O(2). Furthermore, in P. hysterophorus the CO(2) compensation concentration is relatively less sensitive to changes in O(2) concentrations and shows a biphasic response to changing O(2), with a transition point at about 11% O(2). Based on these results, P. hysterophorus is classified as a C(3)-C(4) intermediate. The activities of diagnostic enzymes of C(4) photosynthesis in P. hysterophorus were very low, comparable to those observed in the C(3) species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10 29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to (14)CO(2) (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial (14)C products (77-84%), with 3)-linked beta-d galactosyl backbone chains having side chains of (1-->6)-linked beta-d-galactosyl residues, to which alpha-l-arabinofuranosyl residues were attached in the outer regions. One prominent feature of the primary root AGPs was that they contained appreciable amounts of l-fucose, which was presumably responsible for expression of the serological activity. In their immunological reactions with rabbit anti radish leaf AGP antibody, the root AGPs were shown to share common antigenic determinant(s) with those of seed and leaf AGPs. PMID- 16665860 TI - Water is allocated differently to chloroplasts in sun and shade leaves. AB - Hydrogen-1 nuclear magnetic resonance spectroscopy was used to study water allocation in cell compartments of sun and shade leaves. NMR spectra of Acer platanoides were resolved into two peaks that were assigned to chloroplast and nonchloroplast water. Sun leaves contained 1.7 times more water per unit area of surface than shade leaves, and the water was allocated differently. Chloroplasts in sun leaves contained 17% of the total leaf water versus 47% in shade leaves. Comparing equal leaf surface areas, the chloroplasts in shade leaves contained 60% more water than those in sun leaves. PMID- 16665861 TI - Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum. AB - Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens. PMID- 16665862 TI - Elicitation of diacetylenic compounds in suspension cultured cells of eggplant. AB - Induction of stress metabolites in the suspension cultured cells of eggplant (Solanum melongena L.) was examined. When autoclaved RNase A or nigeran, both of which are nonspecific phytoalexin elicitors in bean cells, were added to the cell culture of eggplant, greatly enhanced levels of three compounds were observed. One of them was cis-pentadeca-6-ene-1,3-diyne-5,15-diol, a novel diacetylenic compound. This compound has considerable fungitoxic activity. Also identified was falcarindiol, another fungitoxic diacetylenic compound previously reported as one of the phytoalexins in infected tomato fruits and leaves. Elicited compounds preferentially accumulated in the culture medium rather than in the cells and decreased to original levels during prolonged culturing. The elicitation of these compounds was closely correlated with cellular damage in terms of the decrease of growth rate and was inhibited by 10 micromolar cycloheximide. PMID- 16665863 TI - Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection. AB - Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome. PMID- 16665864 TI - Isolation and Biological Activities of Four Selective Toxins from Helminthosporium carbonum. AB - A new and simpler purification procedure was developed for host selective toxins from Helminthosporium carbonum race 1. Four analogs or forms of toxin with the same selectivity as the fungus were isolated from culture fluids; two forms (HC toxins III and IV) have not been reported by other workers. Crystals of the major form of toxin (HC toxin I) were recovered in high yields (>80 milligrams per liter of culture fluid) without the use of high performance or preparative thin layer liquid chromatography. ED(50) values, based on inhibition of root growth of susceptible seedlings, for HC toxins I, II, III, and IV were 0.2, 0.4, 2.0, and 20 micrograms per milliliter, respectively. The specific activity of crystalline HC toxin I matched the most active preparation reported previously; the preparation of HC toxin II was more active than that reported previously. Resistant seedlings tolerated 100-fold higher concentrations of each form of toxin than did susceptible seedlings. Hydrolysis of the epoxide group of HC toxin I to a diol destroyed toxicity to susceptible and resistant seedlings. The data suggest that the same mechanisms are affected in resistant and susceptible plants. PMID- 16665865 TI - Specific Selenium-Containing Macromolecules in the Marine Diatom Thalassiosira pseudonana. AB - Thalassiosira pseudonana Husedt (Hasle and Heimdal) clone 3H was grown in axenic culture in artificial seawater medium containing 10(-8) molar Na(2) (75)SeO(3). Biochemical distribution of radiolabeled Se was determined by solvent extraction techniques, gel filtration, and polyacrylamide gel electrophoresis. Of the total cellular Se, 51% was protein bound. Two soluble macromolecules of 21 and 29 kilodaltons contained (75)Se. These results are the first to provide evidence of specific Se-containing compounds in a photosynthetic organism. Glutathione peroxidase (GSH-Px) activity was measured in cell-free extracts and on nondenaturing polyacrylamide gels by a glutathione-reductase coupled assay. Two enzymes showing GSH-Px activity were present. One enzyme was active with H(2)O(2) and tert-butyl hydroperoxide (tBOOH); consistent with known Sedependent GSH-Pxs, but the other enzyme was only active with tBOOH. Co-migration of the H(2)O(2) active GSH-Px and (75)Se on nondenaturing polyacrylamide gels provides evidence that T. pseudonana contains a Sedependent GSH-Px. The molecular weight of one of the (75)Se-labeled macromolecules is identical with the weight of previously characterized GSH-PX subunits. We conclude that the obligate requirement for Se in Thalassiosira pseudonana is due in part to the presence of the selenoenzyme glutathione peroxidase. PMID- 16665866 TI - Do Stomata Respond to CO(2) Concentrations Other than Intercellular? AB - Most studies on stomatal responses to CO(2) assume that guard cells respond only to intercellular CO(2) concentration and are insensitive to the CO(2) concentrations in the pore and outside the leaf. If stomata are sensitive to the CO(2) concentration at the surface of the leaf or in the stomatal pore, the stomatal response to intercellular CO(2) concentration will be incorrect for a ;normally' operating leaf (where ambient CO(2) concentration is a constant). In this study asymmetric CO(2) concentrations for the two surfaces of amphistomatous leaves were used to vary intercellular and leaf surface CO(2) concentrations independently in Xanthium strumarium L. and Helianthus annuus L. The response of stomata to intercellular CO(2) concentration when the concentration at the leaf surface was held constant was found to be the same as the response when the surface concentration was varied. In addition, stomata did not respond to changes in leaf surface CO(2) concentration when the intercellular concentration for that surface was held constant. It is concluded that stomata respond to intercellular CO(2) concentration and are insensitive to the CO(2) concentration at the surface of the leaf and in the stomatal pore. PMID- 16665867 TI - Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol. AB - In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4 d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [(15)N]xanthine from (15)N(2) at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible (15)N-labeling of asparagine from (15)N(2) was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery. PMID- 16665868 TI - ABA-Regulation of Two Classes of Embryo-Specific Sequences in Mature Wheat Embryos. AB - We have previously described the isolation and characterization of ABA-enhanced sequences from developing wheat embryos. Here we use in vivo RNA labeling and the inhibitors alpha-amanitin and cycloheximide to determine the level at which ABA acts to modulate these sequences in cultured mature embryos. Sequences fell into two classes: one, represented by the 7S globulin clone, p511, appears to be regulated at the level of transcription, while the other, represented by the early methionine-labeled polypeptide (E(m))-protein clone, p1015, has an additional posttranscriptional component. In mature embryos cultured in the absence of ABA, mRNA levels of p511 and p1015 declined rapidly until neither was detected at 3 days postimbibition. Levels of p511 increased in mature embryos cultured in the presence of ABA, but remained low in the presence of ABA + alpha amanitin, suggesting p511 RNA is regulated at the level of transcription. Levels of p1015, in contrast, remained high not only in the presence of ABA, but also in the presence of ABA + alpha-amanitin or alpha-amanitin alone. This suggests p1015 regulation might be at the level of selective RNA stability. Cycloheximide had no detectable effect on ABA-mediated stabilization of p1015, suggesting that newly synthesized proteins are not involved. E(m)-protein synthesis rates closely paralleled E(m) RNA levels, suggesting E(m) expression is not controlled at the level of translation. PMID- 16665869 TI - Light Affects Flagellar Agglutinability in Chlamydomonas eugametos by Modification of the Agglutinin Molecules. AB - The effect of light on the sexual competence of a light-sensitive mating type minus strain (mt(-)) of Chlamydomonas eugametos obtained by crossing a light sensitive mating type plus strain (mt(+)) with a light-insensitive mt(-) strain is described. As previously demonstrated for the mt(+) parent, this study of one of the mt(-) offspring shows that (a) a light-sensitive mechanism affects flagellar agglutinability in a rapid process that does not require protein synthesis; (b) only the activity of the flagellar agglutinins (glycoproteins responsible for agglutination) is susceptible to light while agglutinins on the cell body surface are not affected by light. We further demonstrate that (a) membrane vesicles naturally released from nonagglutinable dark gametes remain inactive. Extracts of these vesicles also remain inactive even though they contain agglutinin-like components; (b) inactive mt(-) agglutinin is present in extracts of flagella from nonagglutinable dark gametes by comparison of its chromatographic, electrophoretic, and immunogenic properties with those of active agglutinin. When purified of all other flagellar proteins, it remains inactive; (c) a monoclonal antibody directed against the sexual agglutination site of the mt(-) agglutintin discriminates between active and inactive agglutinins when present in a native state on the flagellar surface, but is unable to discriminate between them when they are denatured in sodium dodecyl sulfate-electrophoresis gels and blotted onto nitrocellulose. Taken collectively these observations suggest that light activation involves the chemical modification of the agglutinins in situ on the flagellar surface. PMID- 16665870 TI - Ornithine decarboxylase, polyamines, and pyrrolizidine alkaloids in senecio and crotalaria. AB - When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here-using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors-endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S. riddellii and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence-with relatively very high levels of these compounds-in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ- or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence. PMID- 16665871 TI - Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels. AB - Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis. PMID- 16665872 TI - NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots. AB - Evidence was found for a Na(+)/H(+) antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg(2+), and the pH gradient gradually dissipated. When 50 millimolar K(+) or Na(+) was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K(+) to vesicles from salt-grown roots. However, when 50 millimolar Na(+) was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K(+) and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na(+)/H(+) exchange. The Na(+)/H(+) exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na(+) concentration, with an apparent K(m) for Na(+) of 9 millimolar. The rate of Na(+)/H(+) exchange with 10 millimolar Na(+) was inhibited 97% by 0.1 millimolar dodecyltriethylammonium. PMID- 16665873 TI - Gibberellins and Light Regulated Petiole Growth in Thlaspi arvense L. AB - Petiole growth in Thlaspi arvense L. was stimulated when a basic 8 hour photoperiod (4.20 milliwatts per square centimeter) was extended with low intensity light (0.16 milliwatt per square centimeter) from incandescent lamps. The day length extension was effective only when the light contained high proportions of far red light. Exogenous gibberellin A(3) (GA(3)) could partially substitute for the promotive effect of the extended photoperiod. Moreover, the GA biosynthesis inhibitor 2-chlorocholine chloride inhibited the increase in petiole growth induced by the extended photoperiod. However, evidence was obtained indicating that gibberellins do not mediate the effect of the extended photoperiod. First, petiole growth was greater in plants receiving both exogenous GA(3) and a day length extension than the sum of the effects of the two treatments alone. Second, petioles were sensitive to exogenous GA(3) only during the early stages of leaf development, whereas mature (but not senescent) leaves continued to respond to an extension of the photoperiod. Third, the cellular basis for growth induced by extending the photoperiod was different from that observed with GA(3). It was concluded that light and gibberellins are both important in the overall regulation of petiole growth, but act through independent mechanisms. PMID- 16665874 TI - Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron. AB - The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of (14)C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5 norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of (14)C-2,4 dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment. PMID- 16665875 TI - Fluorescence Quenching and Gas Exchange in a Water Stressed C(3) Plant, Digitalis lanata. AB - A leaf cuvette has been adapted for use with a pulse-modulation fluorometer and an open gas exchange system. Leaf water potential (psi) was decreased by withholding watering from Digitalis lanata EHRH. plants. At different stages of water deficiency the photochemical (q(Q)) and nonphotochemical (q(E)) fluorescence quenching was determined during the transition between darkness and light-induced steady state photosynthesis of the attached leaves. In addition, the steady state CO(2) and H(2)O gas exchange was recorded. Following a decrease of leaf water potential with increasing water deficiency, the transition of photochemical quenching was almost unaffected, whereas nonphotochemical quenching increased. This is indicative of an enhanced thylakoid membrane energization during the transition and is interpreted as a partial inhibition of either the ATP generating or the ATP consuming reaction sequences. Complete reversion of the stress induced changes was achieved within 6 hours after rewatering. In contrast to the variations during transition, the final steady state values of q(Q) and q(E) remained unchanged over the entire stress range from -0.7 to -2.5 megapascals. From these results we conclude that, once established, electron transport via photosystem II and the transmembrane proton gradient remain unaffected by water stress. These data are indicative of a protective mechanism against photoinhibition during stress, when net CO(2) uptake is limited. PMID- 16665876 TI - Subcellular localization and characterization of amylases in Arabidopsis leaf. AB - Amylolytic enzymes of Arabidopsis leaf tissue were partially purified and characterized. Endoamylase, starch phosphorylase, d-enzyme (transglycosylase), and possibly exoamylase were found in the chloroplasts. Endoamylase, fraction A2, found only in the chloroplast, was resolved from the exoamylases by chromatography on a Mono Q column and migrated with an R(F) of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, A1, has an R(F) of 0.23 on the polyacrylamide gel. Viscometric analysis showed that A1 has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. A1, however, can be distinguished from A3 by having much higher amylolytic activity in succinate buffer than acetate buffer, and having much less reactivity with amylose. A1 probably is also localized in the chloroplast, and contributes to the 30 to 40% higher amylolytic activity of the chloroplast preparation in succinate than acetate buffer at pH 6.0. The high activity of d-enzyme compared to the amylolytic activity in the chloroplast suggests that transglycosylation probably has an important role during starch degradation in Arabidopsis leaf. Extrachloroplastic amylase, A3, has an R(F) of 0.55 on 7% electrophoretic gel and constitutes 80% of the total leaf amylolytic activity. The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic. PMID- 16665877 TI - Characterization of d-Enzyme (4-alpha-Glucanotransferase) in Arabidopsis Leaf. AB - Two major forms of d-enzyme (4-alpha-glucanotransferase, EC 2.4.1.25) were successfully separated from most of the amylase activity using FPLC-Mono Q column chromatography. Transfer of a maltosyl group was observed upon the incubation of d-enzyme with maltotriose and d-[U-(14) C]glucose. About 4.5% of the radioactivity was transferred to maltotriose in 2 hours. End product analysis showed the accumulation of glucose and maltopentaose from maltotriose within the first 10 minutes of the reaction. Several other maltodextrins were also observed with longer incubation times, although maltose was never produced. A quantitative measurement of maltodextrin production from the reaction of [(14) C]maltotriose with d-enzyme showed that the quantity of maltotriose decreased from 100% to 31% after 3 hours incubation, while glucose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, and higher maltodextrins increased in amount. Glucose is the major product throughout the course of the reaction of d enzyme with maltotriose. Maltotriose, in addition to glucose, are the major products in the reaction of d-enzyme with maltodextrins with a chain length greater than maltotriose. This study confirms the existence of a transglycosylase that disproportionates maltotriose and higher maltodextrins by transferring maltosyl or maltodextrinyl groups between maltodextrins resulting in the production of glucose and different maltodextrins, but not maltose, in leaf tissue with enzymic properties very similar to the previously reported d-enzyme in potato. PMID- 16665878 TI - Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells. AB - One cell strain with stable tolerance to allyl alcohol (AA(r)) was selected from 6 x 10(8) suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA(r) cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA(r) cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells. PMID- 16665879 TI - Immunogold Localization of the L3 Protein of Maize Lipid Bodies during Germination and Seedling Growth. AB - We have used antibodies directed against the 16.5 kilodalton protein L3, the most abundant integral protein of maize (Zea mays L. cv Mo 17) lipid bodies, to follow the fate of this protein in scutellar parenchyma cells of maize during germination and subsequent seedling growth. Using gel electrophoresis and immunoblotting as well as immunocytochemical electron microscopy, we found that the amount of L3 decreases gradually during the first 3 to 4 days of seedling growth and more rapidly over the course of the next several days. Immunogold localization of the protein on thin sections indicated that L3 is found exclusively in the surface phospholipid monolayer of lipid bodies. The density of L3 in the surface layer of individual lipid bodies does not change during seedling growth; therefore, the decrease in the amount of L3 can be attributed to a decrease in the number of lipid bodies rather than to selective removal of protein components from the surface of all lipid bodies. Thus, L3 is apparently degraded at the same time as the matrix lipid of each lipid body. Unlike lipase, L3 does not appear to be transferred to other cellular compartments such as vacuoles during late stages of seedling growth. PMID- 16665880 TI - Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures. AB - The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to -8 degrees C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to -8 degrees C and thawing, but nonacclimated stems were injured by freezing to temperatures between -2.2 and -5.6 degrees C and thawing. Injury to nonacclimated stems at freezing temperatures below -2.2 degrees C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz. PMID- 16665881 TI - Changes in Free and Conjugated Indole 3-Acetic Acid and Abscisic Acid in Young Cotton Fruits and Their Abscission Zones in Relation to Fruit Retention during and after Moisture Stress. AB - Experiments were conducted with field-grown cotton (Gossypium hirsutum L.) in 1985 and 1986 to determine effects of water deficit on levels of conjugated indole 3-acetic acid (IAA) and abscisic acid (ABA) in young fruits (bolls) and their abscission zones in relation to boll retention. Tissues were harvested three times during an irrigation cycle in 1985. They were harvested twice during an irrigation cycle and once after irrigation in 1986 to determine extent of recoveries of measured parameters. As reported earlier, the free IAA content of abscission zones decreased with moisture stress. Irrigation caused a partial recovery in free IAA content of abscission zones and caused a partial recovery in rate of boll retention. In contrast to free IAA, conjugated IAA increased with water deficit, both in 3-day-old bolls and in their abscission zones. Bolls contained much more ester IAA than their abscission zones. Some, but not all, of the increase in ester IAA in bolls during moisture stress could have come from a conversion of amide-linked IAA. Amide IAA decreased slightly during stress and increased after irrigation, but the concentration was low relative to ester IAA. Free and conjugated ABA both increased during stress and decreased after irrigation. However, the concentration of conjugated ABA remained relatively high in abscission zones. Ester IAA, being more resistant than free IAA to enzymic destruction during stress, may hasten recovery of fruit retention after relief of stress by providing a source of free IAA in abscission zones to inhibit continued abscission. PMID- 16665882 TI - Effect of Cold Acclimation on Bulk Tissue Electrical Impedance: II. Measurements with Alfalfa and Birdsfoot Trefoil at Nonfreezing Temperatures. AB - Stem and electrode electric impedance at 14 frequencies were monitored during cold acclimation of alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.). Cold acclimation significantly increased high frequency (e.g. 1.11 megahertz) resistance and reactance but not low frequency (49 hertz) resistance and reactance of both species. High frequency resistance of living stems was equivalent to the average resistance at all frequencies of dead stems and the resistance of dead stems (y) was related to stem water content (x):y = 9.28 - 4.11x + 0.47x(2), R = 0.92. The low-high frequency (49 hertz/1.11 megahertz) resistance ratio decreased during cold acclimation. A time constant believed to be a function of membrane resistance and capacitance was not affected by cold acclimation. PMID- 16665883 TI - Elimination of Differences in the Mobility of Flax Isoperoxidases on PAGE by Digestion with alpha-Mannosidase. AB - In the flax (Linum usitatissimum) genotype Stormont cirrus, anodic peroxidases from the genotroph S migrate more slowly on PAGE and SDS-PAGE than the corresponding peroxidases from the genotroph L. When purified isoperoxidases S2 and L2 were digested with alpha-mannosidase, the difference in mobility was eliminated. Treatment with alpha-fucosidase and beta-xylosidase also altered the mobility of S2 and L2, but affected the sensitivity to the action of endo-beta-N acetylglucosaminidase H of only S2. Our results suggest differences in posttranslational processing of the carbohydrate moiety between S and L isoperoxidases. These differences were also found in other S and L glycoenzymes (anodic acid phosphatases) as well as in the peroxidases of other flax genotypes. PMID- 16665884 TI - Involvement of stromal ATP in the light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact isolated chloroplasts. AB - Light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and stromal ATP content were measured in intact isolated spinach chloroplasts. Treatments which decreased stromal ATP, such as incubation with the ATP analog beta,gamma-methylene adenosine triphosphate or with the energy transfer inhibitor phloridzin inhibited the light activation of rubisco. In the absence of added inorganic phosphate (Pi), light activation of rubisco was inhibited, coincident with low stromal ATP. Addition of methyl viologen restored both stromal ATP and rubisco activity to levels observed in the presence of Pi. Activation of rubisco was inhibited in the presence of 2 millimolar dihydroxyacetone phosphate or 3 phosphoglycerate and stromal ATP was also decreased under these conditions. Both were partially restored by increasing the Pi concentration. The strong correlation between activation state of rubisco and stromal ATP concentration in intact chloroplasts under a wide variety of experimental conditions indicates that light activation of rubisco is dependent on ATP and proportional to the ATP concentration. These observations can be explained in terms of the rubisco activase protein, which mediates activation of rubisco at physiological concentrations of CO(2) and ribulose-1,5-bisphosphate and is dependent upon ATP. PMID- 16665885 TI - Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins. AB - A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation. PMID- 16665886 TI - Growth, in vivo extensibility, and tissue tension in developing pea internodes. AB - The relationship between growth, in vivo extensibility, and tissue tension in the first 3 internodes of 5, 6, and 7 day-old pea plants (Pisum sativum L. cv Alaska), grown under continuous red light was investigated. The upper 15 millimeters of each internode was marked with ink and its elongation growth measured over the next subsequent 8 hours. In vivo extensibility was measured by stretching living tissue at constant force (creep test) in a custom-built extensiometer. Tissue tension was determined by (a) measuring the rate of expansion of the isolated cortical cylinder after adding water and the amount of contraction of the epidermis after peeling, and (b) by use of the ;split section test.' A good correlation between rate of elongation growth, in vivo extensibility, and tissue tension was established. The epidermis peeled from the growing third internode of 7 day-old plants and measured immediately showed a plastic extensibility (E(pl) twice that of peels from nongrowing excised sections. This high E(pl)-value was lost on incubation of the sections in distilled water, and was subsequently restored by incubating the sections in auxin (indole-3-acetic acid). We conclude that the in situ growth of the internodes is a function of tissue-tension, which provides the driving force of organ growth, and the extensibility (E(pl) of the outer epidermal wall, which is in the growing plant in a ;loosened' state. We furthermore suggest that in the intact plant auxin is causally involved in the wall loosening process in the epidermis. PMID- 16665887 TI - Acid and alkaline invertases in suspension cultures of sugar beet cells. AB - Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K(m) values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K(m) values for sucrose. PMID- 16665888 TI - Effects of soil strength on the relation of water-use efficiency and growth to carbon isotope discrimination in wheat seedlings. AB - The ratio of carbon accumulation to transpiration, W, of wheat (Triticum aestivum L.) seedlings increased with increasing soil strength, measured as soil penetrometer resistance, and this was already apparent at the two leaf stage. The ratio was negatively correlated with carbon isotope discrimination, in accord with theory. This means that decrease in intercellular partial pressure of CO(2) accounted for an important part of the increase in W with increasing soil strength. Despite a lower CO(2) concentration in the leaves at high soil strength, assimilation rate per unit leaf area was enhanced. Greater ribulose 1,5 bisphosphate carboxylase activity confirmed that photosynthetic capacity was actually increased. This pattern of opposite variation of assimilation rate and of stomatal conductance is unusual. The ratio of plant carbon mass to leaf area increased markedly with increasing soil strength, mainly because of a greater investment of carbon into roots than into shoots. A strong negative correlation was found between this ratio and carbon isotope discrimination. For a given increase in discrimination, decrease in carbon mass per leaf area was proportionally larger than decrease in assimilation rate, so that relative growth rate was positively correlated to carbon isotope discrimination. PMID- 16665889 TI - Interrelations between Carbon Dioxide and Ethylene on the Stimulation of Cocklebur Seed Germination. AB - Interrelations between CO(2) and C(2)H(4) on promotion of seed germination were examined in more detail at 23 degrees C with presoaked upper seeds of Xanthium pennsylvanicum Wallr. The germination-promoting effect of C(2)H(4) decreased gradually as its application time was delayed during a soaking period, whereas CO(2) was most promotive in application at 5 days of soaking, then its effect declined. CO(2) and C(2)H(4) were additive in earlier soaking periods and synergistic in later periods. Such changes in germination behavior in response to CO(2) and/or C(2)H(4) during a soaking period were closely associated with growth responsiveness of the axial tissues, but not of the cotyledonary ones. Growth responsiveness of axial tissues to CO(2) or C(2)H(4) disappeared finally during a soaking period, but their extinct responsiveness to any one of these gases was almost fully restored in the simultaneous presence of the other. The extinct responsiveness to CO(2) was partially recovered by a preexposure to C(2)H(4). This suggests that in the later period of soaking, unlike the case in a very early period of soaking, the C(2)H(4)-sensitive phase for seed germination precedes the CO(2)-sensitive phase in which CO(2) potentiated axial growth. The restoration of CO(2) responsiveness in axial growth occurred not only after C(2)H(4) treatment but also after exposure to 8 or 33 degrees C or after KCN treatment. Thus, secondarily dormant Xanthium seeds could germinate in response to CO(2) alone, when they were previously exposed for shortterms not only to C(2)H(4) but also 8 degrees C, 33 degrees C, or KCN. PMID- 16665890 TI - Effects of Previous Pollination and Stylar Ethylene on Pollen Tube Growth in Petunia hybrida Styles. AB - The effect of ethylene on the growth rate of pollen tubes in styles of Petunia hybrida was examined. Apart from its strong inhibition of pollination-induced ethylene synthesis, aminoethoxyvinylglycine, placed on the stigma, did not impede tube growth. The inhibitors of the action of ethylene, silver thiosulfate and 2,5 norbornadiene, were similarly ineffective. Application of the ethylene precursor, 1-amino-cyclopropane-1-carboxylic acid, onto the stigma at different intervals prior to pollination evoked synthesis of ethylene, but was without effect on tube growth. However, prepollination (by 24 hours) with Nicotiana tabacum pollen, significantly enhanced tube growth of Petunia pollen. This enhancement was not counteracted by the pretreatment of stigmas with aminoethoxy-vinylglycine. It is concluded that the ethylene associated with pollination is without effect on pollen tube growth in the style, but that other pollination-induced factors may lead to an acceleration of growth. PMID- 16665891 TI - Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis. AB - The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than ;mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits. PMID- 16665892 TI - Stability and Dissociation of the Large Subunit RuBisCO Binding Protein Complex in Vitro and in Organello. AB - We are studying the stability of the binding protein which associates with newly synthesized large subunits of ribulose bisphosphate carboxylase. In chloroplast extracts, it has been shown that a dodecameric complex of the large subunit binding protein dissociates extensively into binding protein monomers and 7S (117 kilodaltons) large subunit-containing complexes in the presence of ATP. The concentrations of ATP which bring this about are quite low, prompting some investigators to suggest that the dodecameric complex might not exist in vivo. We have found, however, that in concentrated chloroplast extracts, at protein concentrations which are closer to those which occur in organello, the dissociation of the binding protein complex by ATP is much less extensive. For this reason, we have tested the stability of the binding protein in organello, by illuminating chloroplasts followed by lysis and polyacrylamide gel electrophoresis of the extracts. Radioactive large subunits associated with the dodecameric binding protein dissociated extensively in the light. The results are consistent with the idea that the high molecular weight form of the binding protein can function as a reservoir of large subunits which can be tapped in vivo, in a reaction dependent on light and ATP. PMID- 16665893 TI - Choline oxidation by intact spinach chloroplasts. AB - Plants synthesize betaine by a two-step oxidation of choline (choline --> betaine aldehyde --> betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness (AD Hanson et al. 1985 Proc Natl Acad Sci USA 82: 3678-3682). We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers nor the Calvin cycle inhibitor glyceraldehyde greatly affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO(2) fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed. PMID- 16665894 TI - Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays. AB - Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1(-) mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO(3) (-). Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO(3) (-) to NO(2) (-). These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl(-) mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours. PMID- 16665895 TI - The Lectins of Sophora japonica: II. Purification, Properties, and N-Terminal Amino Acid Sequences of Five Lectins from Bark. AB - Five N-acetyl-galactosamine-specific lectins were isolated from the bark of the legume tree Sophora japonica. These lectins are immunologically and structurally very similar, but not identical, to the Sophora seed and leaf lectins. The carbohydrate specificities and hemagglutinin activities of these lectins are indistinguishable at pH 8.5 but their activities differ markedly at pH values below 8. All five lectins are tetrameric glycoproteins made up of different combinations of subunits of about 30,000, 30,100, 33,000 M(r) containing 3% to 5% covalently attached sugar. These lectins are the overwhelmingly dominant proteins in bark, but they do not appear to be present in other tissues. Amino terminal sequence analysis indicates that at least two distinct lectin genes are expressed in bark. PMID- 16665896 TI - Transcription of Two Photosynthesis-Associated Nuclear Gene Families Correlates with the Presence of Chloroplasts in Leaves of the Variegated Tomato ghost Mutant. AB - Leaves of the tomato ghost mutant show a variegated green/white phenotype due to a somatically unstable genetic block in carotenoid biosynthesis. Colored carotenoids are not synthesized in white leaves; consequently, chlorophyll is destroyed by photooxidation and the plastids formed show little development of internal membrane structures. Carotenoid biosynthesis proceeds to wild type levels in green tissue, thus chlorophyll accumulates and chloroplasts develop normally. The presence of green sectors allows for the production through tissue culture of variegated green/white plants, in which growth is supported by the photosynthetic green tissue. Thus, ghost is the first plant carotenoid mutant that can be grown to maturity. We determined the steady state mRNA levels for two nuclear gene families that code for chloroplast proteins: rbcS, which codes for the small subunit of ribulose-1-5-bisphosphate carboxylase; and cab, which codes for chlorophyll a/b binding protein. In ghost plants grown in light, the steady state mRNA levels for both gene families were low in white leaves but were similar to wild type in green leaves. Light regulation of the transcripts studied was observed in both ghost green and white leaves. Transcription experiments conducted on nuclei isolated from green and white leaves indicate that the low levels of cytoplasmic mRNAs observed in the absence of colored carotenoids and/or light are due to reduced rates of transcription. We conclude that maximum transcription of rbcS and cab genes in leaves of mature tomato plants requires both light and mature chloroplasts. PMID- 16665897 TI - Effect of shading individual soybean reproductive structures on their abscisic Acid content, metabolism, and partitioning. AB - Pod set in soybean is related to carbon partitioning and may be, at least partially, regulated by abscisic acid (ABA) concentrations. The studies reported here examine the relationship between carbon and ABA partitioning, reproductive abscission and ABA metabolism. The partitioning of radiolabeled ABA and photoassimilates from leaves to flowers and endogenous ABA concentrations were determined in shaded and unshaded reproductive structures. Aluminum foil was gently placed over individual soybean reproductive structures for 48 hours at 0, 4, 12, 17, and 22 days after anthesis (DAA). Shading of flowers at 12, 17, and 22 DAA resulted in significantly reduced concentration of ABA. However, shading had no effect on the catabolism of exogenously supplied [(3)H] ABA. The shading treatment on the first four of the five dates reduced partitioning of photoassimilates and ABA from the subtending leaf to the flower. Shading of reproductive structures also caused a significant reduction in the amount of assimilate exported from the subtending leaf, at 17 DAA. We conclude that shade induced premature reproductive abscission in soybean is not stimulated by high levels of ABA within reproductive structures, but that ABA may inhibit abscission of reproductive structures by playing a role in preferential assimilate partitioning. PMID- 16665898 TI - Separation of Chlorophylls c(1) and c(2) from Pigment Extracts of Pavlova gyrans by Reversed-Phase High Performance Liquid Chromatography. AB - Chlorophylls c(1) and c(2) have been separated from total pigment extracts of the alga Pavlova gyrans Butcher using a reversed-phase high-performance liquid chromatography system. Pigments were separated on a 5 micrometer C(18) column (25 centimeters x 4.6 millimeters) using a gradient of methanol-acetonitrile-water. Other photosynthetic pigments were also well resolved by the system used. The separation system described may replace current thin layer chromatography methods for qualitative and quantitative determination of chlorophyll c species. PMID- 16665899 TI - Physiological Site of Ethylene Effects on Carbon Dioxide Assimilation in Glycine max L. Merr. AB - The physiological site of ethylene action on CO(2) assimilation was investigated in intact plants of Glycine max L., using a whole-plant, open exposure system equipped witha remotely operated single-leaf cuvette. The objective of the study was met by investigating in control and ethylene-treated plants the (a) synchrony in response of CO(2) assimilation, stomatal conductance to water vapor, and substomatal CO(2) partial pressure; (b) response of CO(2) assimilation as a function of a range of substomatal CO(2) partial pressures; and (c) response of CO(2) assimilation as a function of a range of photon flux densities. After exposure to 410 micromoles per cubic meter of ethylene for 2.0 hours, CO(2) assimilation and stomatal conductance declined in synchrony, while substomatal CO(2) partial pressure remained unchanged until exposure times equaled and exceeded 3.0 hours. Because incipient changes in CO(2) assimilation occurred without a change in the CO(2) partial pressure in the leaf interior, it is concluded that both stomatal physiology and the chloroplast's CO(2) assimilatory capacity were initial sites of ethylene action. After 3.5 hours the effect of ethylene on stomatal conductance and CO(2) assimilation exhibited saturation kinetics, and the effect was substantially more pronounced for stomatal conductance than for CO(2) assimilation. Based on the response of CO(2) assimilation to a range of substomatal CO(2) partial pressures, ethylene did not affect either the CO(2) compensation point or carboxylation efficiency at subsaturating CO(2) partial pressures. Above-ambient supplies of CO(2) did not alleviate the diminished rates of CO(2) assimilation. In partitioning the limitations imposed on CO(2) assimilation in control and ethylene-treated plants, the stomatal component accounted for only 16 and 4%, respectively. The response of CO(2) assimilation to a range of photon flux densities suggests that ethylene reduced apparent quantum yield by nearly 50%. Thus, the pronounced decline in net photosynthetic CO(2) assimilation in the presence of ethylene was due more to a loss in the mesophyll tissue's intrinsic capacity to assimilate CO(2) than to a reduction in stomatal conductance. PMID- 16665900 TI - An investigation of the mineral content of barley grains and seedlings. AB - The Ca, Mg, K, and P content of dry barley (Hordeum vulgare) grains and seedlings was investigated using energy dispersive x-ray analysis and neutron activation analysis. Energy dispersive x-ray analysis of protein bodies in aleurone cells showed that these bodies contained very little Ca in relation to P, Mg, and K. Neutron activation analysis also showed that the endosperm contained very little Ca in relation to the other three elements. Surface sterilization and soaking treatments brought about slight loss of Ca but substantial loss of K from embryos. Over 6 days of growth the seedling plant gained minerals from the endosperm. PMID- 16665901 TI - Dihydroxyacetone phosphate reductase in plants. AB - Two forms of dihydroxyacetone phosphate reductase are present in spinach, soybean, pea, and mesophyll cells of corn leaves. An improved homogenizing medium was developed to measure this activity. The enzyme was detectable only after dialysis of the 35 to 70% saturated (NH(4))(2)SO(4) fraction and the two forms were separated by chromatography on either DEAE cellulose or Sephacryl S-200. About 80% of the reductase was one form in the chloroplast and the rest was a second form in the cytosol as determined by chromatography and by fractionation of subcellular organelles. The amount of activity detectable in the chloroplast fraction was 10.7 micromoles of dihydroxyacetone phosphate reductase per hour per milligram chlorophyll from spinach leaves and 4.9 from pea leaves. The chloroplast form eluted first from DEAE cellulose and, being smaller, it eluted second from Sephacryl S-200. Activity of the chloroplast form was stimulated 3- to 5-fold by the addition of 1 millimolar dithiothreitol or 50 microgram reduced Escherichia coli thioredoxin or 4 micrograms spinach thioredoxin to the assay mixture. This stimulation was not observed with monothiols. Activity of the cytosolic form was not affected by either reduced thioredoxin or dithiothreitol. PMID- 16665902 TI - Glucosinolate Biosynthesis: Sulfation of Desulfobenzylglucosinolate by Cell-Free Extracts of Cress (Lepidium sativum L.) Seedlings. AB - After removal of myrosinase activity by concanavalin A-Sepharose 4B chromatography, cell-free extracts of light-grown cress (Lepidium sativum L.) seedlings, catalyzed the sulfation of desulfobenzylglucosinolate (K(m), 0.23 millimolar) to benzylglucosinolate using PAPS (K(m), 1 millimolar) as sulfur donor. Sulfotransferase activity, which was optimal at pH 9.0, was stimulated by MgCl(2), MnCl(2), beta-mercaptoethanol, and dithiothreitol and was inhibited by ZnSO(4) and SH-reagents. The enzyme also sulfated desulfoallyglucosinolate to allylglucosinolate (sinigrin) but was inactive towards all phenylpropanoids and flavonoids tested. PMID- 16665903 TI - Catabolism of Cyanogenic Glycosides by Purified Vicianin Hydrolase from Squirrel's Foot Fern (Davallia Trichomanoides Blume). AB - Vicianin hydrolase, which catalyzes the hydrolysis of vicianin (K(m), 4.9 millimolar) to (R)-mandelonitrile and vicianose at an optimum pH of 5.5, was extensively purified from the young fronds and fiddleheads of the squirrel's foot fern (Davallia trichomanoides Blume) using DEAE-cellulose and Ultrogel HA chromatography. The native molecular weight of the enzyme was 340,000, and the isoelectric point was 4.6 to 4.7. SDS-PAGE analysis yielded three polypeptides with molecular weights of 56,000, 49,000, and 32,500. The enzyme hydrolyzed only a narrow range of glycosides and, among cyanogenic glycosides, exhibited a strict requirement for (R)-epimers and a preference for disaccharides over monosaccharides. (R)-Amygdalin, (R)-prunasin and p-nitrophenyl-beta-d-glucoside were hydrolyzed at 27, 14, and 3%, respectively, of the rate of vicianin hydrolysis. Mixed substrate studies showed that (R)-vicianin, (R)-prunasin, and p nitrophenyl-beta-d-glucoside competed for the same active site. The enzyme was significantly inhibited by castanospermine, delta-gluconolactone, and p chloromercuriphenylsulfonate. Failure to recognize concanavalin A-Sepharose 4B and to stain with periodic acid-Schiff reagent indicated that the enzyme was not a glycoprotein. PMID- 16665904 TI - A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity. AB - The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V(max) with no apparent effect on K(m). The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation. PMID- 16665905 TI - Antitranspirant associated abscisic Acid effects on the water relations and yield of transplanted bell peppers. AB - Greenhouse and field experiments were performed to determine if increased leaf resistance induced by exogenous application of abscisic acid (ABA) could enhance the water status of transplanted bell pepper seedlings. Seedling survival and yield were also monitored in the field experiment. When seedlings were transplanted into either wet or dry potting mix in the greenhouse, ABA increased leaf resistance and leaf water potential. In the field, plots were irrigated either immediately after, or 1 day after transplanting. Under both treatments, ABA application resulted in increased leaf resistance and water potential, but seedling survival and yield were enhanced due to ABA only in plots which were irrigated 1 day after transplanting. It is concluded that antitranspirant application can reduce transplant shock and increase yield of bell pepper. PMID- 16665906 TI - Lipid Saturation Induced Microviscosity Increase Has No Effect on the Reducibility of Flash-Oxidized Cytochrome f in Pea Thylakoids. AB - Homogeneous catalytic hydrogenation was used to modify the level of fatty acid unsaturation of thylakoid membranes in the pea chloroplast. Fluidity alteration has been monitored simultaneously using the spin-label probe, 16-doxyl stearate. Even in the case of 30% hydrogenation, no change in the reduction rate of flash oxidized cytochrome f was observed, in contrast to the fact that the same decrease in the double-bond content of the thylakoid membrane resulted in a pronounced inhibition in the full-chain electron transport. We conclude that the rate of lateral diffusion of reduced plastoquinone is unaffected by the lowering of the fluidity of the thylakoid lipid matrix. PMID- 16665907 TI - Polyamine content of long-keeping alcobaca tomato fruit. AB - Fruit of tomato landrace Alcobaca, containing the recessive allele alc, ripen more slowly, with a reduced level of ethylene production, and have prolonged keeping qualities. The levels of polyamines in pericarp tissues of alc and ;wild type' Alc (cv Rutgers and Alcobaca-red) fruit were measured by HPLC in relation to ripening. Putrescine was the predominant polyamine with a lower content of spermidine, while spermine was just detectable. The level of putrescine was high at the immature green stage and declined in the mature green stage. In Alc fruit the decline persisted but in alc fruit the putrescine level increased during ripening to a level similar to that present at the immature green stage. There was no pronounced change or difference in spermidine levels. The enhanced polyamine level in alc fruit may account for their ripening and storage characteristics. PMID- 16665908 TI - The dependence of stomatal closure on protein synthesis. AB - Seven different inhibitors of the synthesis of protein and RNA, all of which are found to delay the senescence of detached oat leaves in darkness, also cause the opening of the stomata in the dark. The concentration ranges for activity on the two processes agree closely. Four other compounds of similar effects on RNA and protein synthesis, but which are inactive on senescence, correspondingly fail to open the stomata. This not only strengthens the relationship between stomatal closure and senescence, but-more important-provides strong evidence that continued protein synthesis is necessary to keep foliar stomata closed. PMID- 16665909 TI - Identification of Proteins Correlated with Increased Freezing Tolerance in Bromegrass (Bromus inermis Leyss. cv Manchar) Cell Cultures. AB - Cellular and extracellular protein profiles from Bromus inermis Leyss. cv Manchar cell suspension cultures cold hardened by low temperature and abscisic acid (ABA) treatment were analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellular proteins (25, 165, 190, and 200 kilodaltons) increased by low temperature growth and cellular proteins (20, 25, 28, 30, 32, 37, 40, 45, 200 kilodaltons) increased by exogenous ABA treatment were identified. Low temperature treatment inhibited the synthesis of a 22 kilodalton protein and ABA treatment resulted in the synthesis of two extracellular proteins (17 and 21 kilodaltons). Low temperature and ABA-induced hardening conditions increased or induced a 25 and a 200 kilodalton protein. The 25 and a 30 kilodalton protein previously shown to be enriched by ABA-induced hardening conditions at both 3 and 23 degrees C temperatures co-fractionated with the crude membrane fraction (30,000g sediment). The 200 kilodalton protein was detected in the 30,000g supernatant. Two-dimensional analysis of the crude membrane fraction resolved the 30 kilodalton protein band into a major polypeptide with an apparent isoelectric point of 6.85. PMID- 16665910 TI - Binding of glycolytic enzymes to a particulate fraction in carrot and sugar beet storage roots : dependence on metabolic state. AB - Numerous studies have demonstrated a rapid increase in the respiration rate during aging of slices of tuber and storage roots. To determine the molecular mechanisms of this phenomenon, the role of enzyme binding to the subcellular particulate fraction has been assessed in carrot (Daucus carota L.) and sugar beet (Beta vulgaris L.). Soluble versus particulate fractions were separated by centrifugation at 16,000g and both fractions assayed for the activities of six glycolytic enzymes. Preparations from sliced and aged tissues showed elevated percentages of five enzymes associated with the particulate fraction as compared with controls. The stimulation of respiration which occurs during aging of underground storage organ slices may result, in part, from an association of enzymes with the particulate fraction of the cell promoting an elevated glycolytic rate. PMID- 16665911 TI - Effects of temperature on h uptake and release during circadian rhythmic movements of excised samanea motor organs. AB - A previous study revealed that Samanea saman leaflets open more completely and close less completely as temperature is increased. We now demonstrate that, as temperature is increased, extensor cells release more H(+) during their swelling phase (opening), but flexor motor cells release less H(+) during their swelling phase (closure). PMID- 16665912 TI - Whole Plant CO(2) Exchange Measurements for Nondestructive Estimation of Growth. AB - A computer controlled semiclosed net CO(2) exchange measurement system, employing an infrared gas analyzer and mass flow controllers to inject pure CO(2) at preset rates, has been developed for measuring whole plant net CO(2) exchange and net C gain in a controlled environment (i.e. CO(2), light, and temperature). Data for tomato (Lycoperscicon esculentum cv Campbell 19 VF) and rose (Rosa hybrida cv Samantha) plants grown for 4 and 17 day periods, respectively, clearly show that net C gain measured and computed using nondestructive CO(2) analysis equaled the increase in C content determined by chemical analysis following destruction of the test plants. The analysis of C gain based on CO(2) exchange allows estimation of biomass production and growth of a single population of plants under varying light and CO(2) conditions without physically handling the test plants. PMID- 16665913 TI - Different rates of metabolism of two chloroacetanilide herbicides in pioneer 3320 corn. AB - The in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two- to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application. Since the initial step in detoxification is conjugation of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjugation, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K(m)app = 1.69 millimolar) than for metolachlor (K(m)app = 8.9 millimolar). PMID- 16665914 TI - Cell-specific expression of pyruvate, pi dikinase : in situ mRNA hybridization and immunolocalization labeling of protein in wheat seed. AB - Pyruvate, Pi dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway. However, its metabolic role in C3 plants remains uncertain. Northern blot analyses of PPDK mRNAs from wheat leaves and seeds probed with maize PPDK cDNA indicates the presence of organ-specific mRNAs. Immunofluorescent labeling of protein in wheat seed demonstrate that the PPDK polypeptide and the ribulose-1, 5 bisphosphate carboxylase small subunit polypeptide are localized predominantly in the aleurone layer and the chlorophyllous pericarp tissue, respectively. This differential distribution of the two polypeptides in wheat seed is paralleled by the differential localization of the their mRNAs as revealed by in situ hybridization. These results suggest a distinct role of cytoplasmic PPDK in seeds, which is different from the well established role in C4 photosynthesis. PMID- 16665915 TI - In Vivo Regulation of Threonine and Isoleucine Biosynthesis in Lemna paucicostata Hegelm. 6746. AB - Little, if any, regulation of threonine synthesis was observed in Lemna paucicostata Hegelm. 6746 supplemented with concentrations of threonine and/or isoleucine that allow for uptake of these amino acids in amounts sufficient for total plant requirements, and that increase tissue concentrations of soluble threonine manyfold. High tissue concentrations of soluble threonine generated endogenously in isoleucine-supplemented plants were no more effective in regulation than a similar concentration of threonine accumulated from the medium. These studies exclude also major regulation of threonine biosynthesis by bivalent repression by threonine plus isoleucine. Isoleucine biosynthesis was severely inhibited by supplementation with isoleucine, but not with threonine or methionine. The fivefold increase in soluble threonine in isoleucine-supplemented plants suggests that threonine dehydratase is a major locus for feedback regulation of isoleucine synthesis. It is concluded that regulation of threonine biosynthesis differs from that of the other amino acids of the aspartate family (isoleucine, methionine, and lysine), each of which strongly feedback regulates its own synthesis. Methionine supplementation had a negligible effect on the tissue concentration of soluble threonine, indicating that threonine is not important in balancing changes of flux into methionine by equivalent changes of flux through the step catalyzed by aspartokinase. PMID- 16665916 TI - Nitrite uptake into intact pea chloroplasts : I. Kinetics and relationship with nitrite assimilation. AB - The uptake of nitrite into intact pea chloroplasts was observed and its relationship with internal nitrite reduction was assessed. Net nitrite uptake exhibited saturation kinetics and an alkaline pH preference. This evidence questioned the accepted major role for neutral HNO(2) permeation and its reported influence on photosynthesis. The possible involvement of a nitrite permeation channel or transport protein is discussed. Net nitrite uptake curves were closely comparable with those for nitrite reduction within the chloroplast. Net nitrite uptake into chloroplasts was profoundly influenced by darkness, incubation temperature, and plant nitrate nutrition. Of several inorganic salts tested, sulfite was the only anion to exhibit a distinct inhibition of nitrite uptake. In contrast, nitrite uptake could be stimulated by the presence of certain cations, particularly at acidic pH values. It was concluded that nitrite uptake was closely related to stromal pH, internal nitrite accumulation, and nitrite reduction. The possible dependence of nitrite reduction on nitrite uptake rather than electron transport is discussed. External ATP and NAD(P)H did not significantly affect net nitrite uptake. This suggested that cytoplasmic ATP or reductant could not directly support nitrite uptake and, possibly, nitrite assimilation. PMID- 16665917 TI - Nitrite Uptake into Intact Pea Chloroplasts : II. Influence of Electron Transport Regulators, Uncouplers, ATPase and Anion Uptake Inhibitors and Protein Binding Reagents. AB - The relationship between net nitrite uptake and its reduction in intact pea chloroplasts was investigated employing electron transport regulators, uncouplers, and photophosphorylation inhibitors. Observations confirmed the dependence of nitrite uptake on stromal pH and nitrite reduction but also suggested a partial dependance upon PSI phosphorylation. It was also suggested that ammonia stimulates nitrogen assimilation in the dark by association with stromal protons. Inhibition of nitrite uptake by N-ethylmaleimide and dinitrofluorobenzene could not be completely attributed to their inhibition of carbon dioxide fixation. Other protein binding reagents which inhibited photosynthesis showed no effect on nitrite uptake, except for p chlormercuribenzoate which stimulated nitrite uptake. The results with N ethylmaleimide and dinitrofluorobenzene tended to support the proposed presence of a protein permeation channel for nitrite uptake in addition to HNO(2) penetration. On the basis of a lack of effect by known anion uptake inhibitors, it was concluded that the nitrite uptake mechanism was distinct from that of phosphate and chloride/sulfate transport. PMID- 16665918 TI - Polyamine levels in petunia genotypes with normal and abnormal floral morphologies. AB - We characterized the polyamine pathway in Petunia hybrida genotypes that were either wild type or that had been identified as having altered floral morphology. Analysis of four normal morphology lines revealed two patterns of endogenous levels of putrescine and arginine decarboxylase: two with higher levels of putrescine, two with lower levels of putrescine. Analysis of F1 and backcross progeny between high putrescine and low putrescine strains is consistent with their differences being due to a dominant allele for low putrescine content and arginine decarboxylase activity. Four Petunia mutants with floral morphology changes were also screened. One of these mutants, alf, showed high levels of putrescine and high levels of arginine decarboxylase late in development; these high levels were found whether the alf line was present in either of the two types of normal morphology genetic backgrounds that had been characterized. PMID- 16665919 TI - Temperature-induced reversals of rotation in phycomyces. AB - The steady state extension and rotation rates of the Phycomyces sporangiophore were measured as a function of temperature. Maximum growth occurred at 27 degrees C; maximum rotation at 28 degrees C. The rotation to extension ratio, a qualitative parameter of cell wall structure, is affected differently by high and low temperatures. Steady state counterclockwise rotation, as opposed to the normal clockwise rotation, was found at both high and low temperatures. The extensional and rotational responses to step changes in temperature were also measured. The conclusions are drawn that a relative decrease in the lysis rate of wall polymer is responsible for the decrease in growth rate at low temperatures, and that a relative increase in the rate of wall synthesis and cross-linking is responsible for the decrease in growth rate at high temperatures. It is suggested that reversals in rotation result from changes in the handedness of the wall's helical structure. PMID- 16665920 TI - Purification and characterization of arcelin seed protein from common bean. AB - Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common. Both were glycoproteins having similar subunit M(r), deglycosylated M(r), and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity of arcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin affinity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein. PMID- 16665921 TI - Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : I. Water Relations of Seed and Fruit Development. AB - Total water potential (psi), solute potential, and turgor potential of field grown muskmelon (Cucumis melo L.) fruit tissue (pericarp) and seeds were determined by thermocouple psychrometry at 5-day intervals from 10 to 65 days after anthesis (DAA). Fruit maturity occurred between 44 and 49 DAA, and seed germination ability developed between 35 and 45 DAA. Pericarp psi was essentially constant at approximately -0.75 megapascal (MPa) from 10 to 25 DAA, then decreased to a minimum value of -1.89 MPa at 50 DAA before increasing to -1.58 MPa at 65 DAA. Seed psi remained relatively constant at approximately -0.5 MPa from 10 to 30 DAA then decreased to -2.26 MPa at 50 to 60 DAA before increasing to -2.01 MPa at 65 DAA. After a rapid increase to 20 DAA, seed fresh weight declined until 30 DAA due to net water loss, despite continuing dry weight gain. As fruit and seed growth rates decreased, turgor potential initially increased, then declined to small values when growth ceased. A disequilibrium in psi was measured between seeds and pericarp both early and late in development. From 20 to 40 DAA, the psi gradient was from the seed to the tissue, coinciding with water loss from the seeds. From 50 to 65 DAA, seed psi decreased, causing a reversal of the psi gradient and a slight increase in seed water content. The partitioning of solutes between symplast and apoplast may create and maintain psi gradients between the pericarp and seed. The low solute potential within the pericarp due to solute accumulation and loss of cellular compartmentation during ripening and sensecence may be involved in prevention of precocious germination of mature seeds. PMID- 16665922 TI - Dissociation and reassembly of soybean clathrin. AB - Coated vesicles (CVs), approximately 85 nanomolar in diameter, were obtained from etiolated soybean hypocotyls using discontinuous sucrose gradients. The CVs were treated with 4 molar urea and the vesicle membranes were then removed by centrifugation. When the supernatant subsequently was dialyzed against isolation buffer, sedimentable complexes were obtained. Electron microscopic examination of the pelleted complexes shows many spherical, 65 nanometer baskets consisting of a polygonal lattice free of internal membrane. LDS-PAGE reveals that a 185 kilodalton clathrin heavy chain is enriched in both the basket and CV pellets. However, the overall protein pattern is more complex than that of comparable brain fractions. The results are discussed in terms of the similarities between soybean and brain clathrins and the function of CVs in vivo. PMID- 16665923 TI - Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach. AB - Two major alpha-glucan phosphorylases (I and II) from leaves of the C(4) plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C(3) plant spinach. The two alpha-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K(m) = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C(3) plant spinach with mesophyll cell phosphorylase of the C(4) plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn. PMID- 16665924 TI - Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase. AB - Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10(-5) molar external free Ca(2+). The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca(2+) influx, inhibits this Ca(2+) stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca(2+) concentrations in the presence of physiological concentrations of Mg(2+) and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca(2+). The Ca(2+) mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca(2+). In dark-kept chloroplasts the steady-state concentration of free stromal Ca(2+) is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca(2+) influx into chloroplasts does not only influence the cytosolic concentration of free Ca(2+) but also regulates enzymatic processes inside the chloroplast. PMID- 16665925 TI - Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum. AB - The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St. Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both fast and slow fluorescence transients from S. secundatum guard cells showed a rapid rise and then remained at a steady level. Neither variable fluorescence increase (induction) nor decrease (quenching), properties normally associated with photosystem II, was observed from these chloroplasts. These fluorescence kinetics did not change either with alterations of the specimen preparation procedure or with alterations of the excitation light intensities and wavelengths. These results indicate that guard cell chloroplasts in this variety of S. secundatum do not conduct normal photosystem II electron transport. Light regulation of stomatal conductance in intact leaves of this plant did occur, however, and was similar to light regulation observed in other species. The conductance of the green portion of the leaves was much greater in the light than in the dark, and was much greater than the conductance of the albino portion of the leaves. Stomata in the green portion of the leaves also showed greater opening in blue light than in red light. These results provide evidence that stomatal regulation in this variety of S. secundatum does not rely on photosystem II electron transport in guard cell chloroplasts. PMID- 16665926 TI - Phytochrome regulation of greening in barley-effects on chlorophyll accumulation. AB - Red light treatment of dark-grown 6-day-old barley seedlings (Hordeum vulgare L.) strongly reduces the lag in chlorophyll accumulation in subsequent white light over that found in dark control seedlings placed under white light. Fluence response studies show that the effect has both very low fluence and low fluence components. Kinetic studies indicate that the reduction in lag begins immediately following either a low fluence or a very low fluence red irradiation, with the initial rate of change significantly lower after the very low fluence treatment and showing sharp far red-absorbing form of phytochrome dependence. In both cases, the effect is maximal after roughly 4 hours, either remaining fairly constant (very low fluence) or declining somewhat (low fluence) thereafter. Saturating far red light alone yields a response equivalent to very low fluence red, and will reverse only the low fluence component of the red response. Escape from far red reversibility occurs gradually over about a 3 hour period. Since the kinetics described here differ from those in the literature related to phytochrome effects on transcription of the mRNA for the light-harvesting chlorophyll a/b-binding protein, we conclude that the phytochrome-regulated component of chlorophyll accumulation is not limited by transcription of the mRNA for its major apoprotein. Leaf segments vacuum-infiltrated with water retain the capacity to green in white light. If they are infiltrated with mannitol solutions of various concentrations, their capacity to green declines sharply at concentrations above 0.2 molar. These results bear on interpretation of run-on transcription experiments with isolated nuclei: preparation of the nuclei involves enzymic digestion of the tissue in the presence of 0.7 molar mannitol for 2.5 hours, to obtain protoplasts prior to breaking the cells. The results here make it unlikely that normal transcriptional regulation is occurring during this procedure. PMID- 16665927 TI - Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis. AB - Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium. PMID- 16665928 TI - Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus. AB - Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings. PMID- 16665929 TI - Comparison of Starch and ADP-Glucose Pyrophosphorylase Levels in Nonembryogenic Cells and Developing Embryos from Induced Carrot Cultures. AB - Cultures of carrot (Daucus carota L.) in a medium without added 2,4 dichlorophenoxyacetic acid were separated into fractions of embryos at different stages of development (large globular and heart, torpedo, and germinating) and nonembryogenic cells. The average starch content per cell in these fractions was similar. However, due to the smaller sizes of the cells of the embryos relative to the nonembryogenic cells, starch content per weight of tissue was higher in the embryos. The ADP-glucose pyrophosphorylase activity per cell in the nonembryogenic cells was double that of the embryo cells. Furthermore, the ratio of ADP-glucose pyrophosphorylase to starch was over 2-fold higher in the nonembryogenic cells, indicating that starch content is not simply determined by ADP-glucose pyrophosphorylase levels. ADP-glucose pyrophosphorylase activity of all culture fractions was directly proportional to the level of a single 50 kilodalton polypeptide detected by immunoblot analysis, using antiserum raised to the purified spinach leaf enzyme. In the same immunoblot analysis, novel polypeptides of 63 and 100 kilodalton were detected in embryos but were absent from nonembryogenic cells. This is one of the few reported examples of specific proteins which differentially accumulate in embryos and nonembryogenic cells. PMID- 16665930 TI - Comparison of Phosphoenolpyruvate-Carboxykinase from Autotrophically and Heterotrophically Grown Euglena and Its Role during Dark Anaerobiosis. AB - Euglena gracilis (1224-5/9) contains phosphoenolpyruvate carboxykinase when grown autotrophic with CO(2) in the light. Its yield is higher when an additional carbon source like glucose has been added. The enzyme is lacking in cells provided with CO(2) alone and kept in the dark, whereas highest yields result if both glucose and CO(2) are provided together in the dark. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephacryl S-300 and affinity chromatography on GMP-Sepharose. The latter step was most effective to protect the enzyme from inactivation. Its homogeneity was tested electrophoretically and immunologically. Enzymes from autotrophic and heterotrophically grown cells have identical pH optima and similar isoelectric points. The molecular weight was different: 761,000 for the enzyme from autotrophic and 550,000 for that from heterotrophic cells as determined by gel filtration. The subunit molecular weight of both enzymes is nearly the same. The kinetic data of the enzymes are slightly different. Glycolytic and tricarboxylic acid cycle intermediates are of limited influence on enzyme activity and inhibitory in unphysiological high concentrations. From Ouchterlony double immunodiffusion and enzyme-linked immunosorbent assay, it is evident that the enzyme is localized in the cytosol. With the latter quantification test the phosphoenolpyruvate carboxykinase protein content was found 10 times higher in heterotrophically grown cells than when cultivated under autotrophic conditions. PMID- 16665931 TI - Synthesis of Glycolate from Pyruvate via Isocitrate Lyase by Tobacco Leaves in Light. AB - Tobacco (Nicotiana tabacum var Havana Seed) leaf discs were supplied tracer quantities of [2-(14)C]- and [3-(14)C]pyruvate for 60 minutes in steady state photosynthesis with 21% or 1% O(2), and the glycolate oxidase inhibitor alpha hydroxy-2-pyridinemethanesulfonic acid was then added for 5 or 10 minutes to cause glycolate to accumulate. The [3-(14)C]pyruvate was converted directly to glycolate as shown by a 50% greater than equallabeled (14)C in C-2 of glycolate, and the fraction of (14)C in C-2 increased in 1% O(2) to 80% greater than equal labeled. This suggests the pathway using pyruvate is less O(2)-dependent than the oxygenase reaction producing glycolate from the Calvin cycle. The formation of glycolate from pyruvate in the leaf discs was time-dependent and with [2-(14)C]- and [3-(14)C]pyruvate supplied leaf discs the C-2 of glyoxylate derived from C-2 of isocitrate was labeled asymmetrically in a manner similar to the asymmetrical labeling of C-2 of glycolate under a number of conditions. Thus glycolate was probably formed by the reduction of glyoxylate. Isocitric lyase activity of tobacco leaves was associated with leaf mitochondria, though most of the activity was in the supernatant fraction after differential centrifugation of leaf homogenates. The total enzyme activity was at least 35 micromoles per gram fresh weight per hour. The relative contribution of the pathway to the glycolate pool is unknown, but the results support the existence of a sequence of reactions leading to glycolate synthesis during photosynthesis with pyruvate, isocitrate, and glyoxylate as intermediates. PMID- 16665932 TI - Photoinduced Seed Germination of Oenothera biennis L: III. Analysis of the Postinduction Period by Means of Temperature. AB - The postinduction period of Oenothera biennis L. seed germination was examined by temperature treatments. For all experiments, seeds received a standard 24 hour/24 degrees C preinduction period and 12 hour/32 degrees C photoinduction period. Germination is inhibited by postinduction temperatures above 32 degrees C. When seeds are briefly incubated at 44 degrees C and then transferred to 28 degrees C, they germinate at a much lower percentage than 28 degrees C controls. When thermally inhibited seeds are placed in the dark at 28 degrees C for 20 hours, they can be promoted to germinate by a single pulse of red light. Seeds incubated at 12 degrees C or below immediately after photoinduction enter a lag period in which they germinate slowly or not at all for a long time and then resume germination. The length of the lag period is exponentially related to the postinduction temperature. When seeds are incubated at a low temperature and then transferred to a warm temperature, they germinate much more rapidly than seeds not incubated at a low temperature. A model is proposed which is consistent with these and additional results. In the model, a germination promoter is irreversibly formed from a precursor and the synthesis of the precursor is favored at low temperatures and its degradation is favored at high temperatures. PMID- 16665934 TI - Carbon Cost of the Fungal Symbiont Relative to Net Leaf P Accumulation in a Split Root VA Mycorrhizal Symbiosis. AB - Translocation of (14)C-photosynthates to mycorrhizal (+ +), half mycorrhizal (0+), and nonmycorrhizal (00) split-root systems was compared to P accumulation in leaves of the host plant. Carrizo citrange seedlings (Poncirus trifoliata [L.] Raf. x Citrus sinensis [L.] Osbeck) were inoculated with the vesicular-arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith. Plants were exposed to (14) CO(2) for 10 minutes and ambient air for 2 hours. Three to 4% of recently labeled photosynthate was allocated to metabolism of the mycorrhiza in each inoculated root half independent of shoot P concentration, growth response, and whether one or both root halves were colonized. Nonmycorrhizal roots respired more of the label translocated to them than did mycorrhizal roots. Label recovered in the potting medium due to exudation or transport into extraradical hyphae was 5 to 6 times greater for (+ +) versus (00) plants. In low nutrient media, roots of (0+) and (+ +) plants transported more P to leaves per root weight than roots of (00) plants. However, when C translocated to roots utilized for respiration, exudation, etc., as well as growth is considered, (00) plant roots were at least as efficient at P uptake (benefit) per C utilized (cost) as (0+) and (+ +) plants. Root systems of (+ +) plants did not supply more P to leaves than (0+) plants in higher nutrient media, yet they still allocated twice the (14)C-photosynthate to the mycorrhiza as did (0+) root systems. This indicates there is an optimal level of mycorrhizal colonization above which the plant receives no enhanced P uptake yet continues to partition photosynthates to metabolism of the mycorrhiza. PMID- 16665933 TI - Comparative analysis of polyadenylated RNA complexity in soybean hypocotyl tissue and cultured suspension cells. AB - Growth parameters of suspension culture cells of soybean (Glycine max L.) were compared between cells grown in medium with (+) auxin and without (-) auxin. Growth rates were greater for (+) auxin cells. Cells transferred to (-) auxin medium primarily expanded in size while (+) auxin cells initially divided and then expanded. Two methods were used to estimate polyadenylated RNA sequence complexity. Kinetic analysis gave a sum of component complexity values of 36,000 and 64,000 diverse poly(A) RNA sequences of about 1,400 nucleotides in (+) and ( ) auxin grown cells, respectively. The most striking difference between these cell populations was the increase in the poly(A) RNA sequence complexity in cells grown in medium without auxin. RNA complexities were also determined by the saturation of ;single' copy DNA by poly(A) RNAs from (+) and (-) auxin suspension cells. These saturation studies estimated the total complexity of (+) and (-) auxin suspension cells as 41,000 and 57,000 diverse sequences, respectively. Suspension cells in auxin-depleted medium produced about 20,000 more diverse sequences than (+) auxin cells. Comparisons of poly(A) complexities were also made among auxin-treated and untreated hypocotyl cells from the intact plant relative to suspension culture cells. Mixed populations of poly(A) RNA from these tissues and cells allowed the determination of shared sequences among them. When all combinations of poly(A) RNA were mixed, the percentage of ;single' copy DNA that saturated was equivalent to diverse sequence complexity estimates of about 60,000. When mixed poly(A) RNA from suspension cells from (+) and (-) auxin medium were compared, they shared about 40,000 sequences and (-) auxin cells contained an additional 20,000. Both (+) and (-) tissue culture cells shared a subset of about 20,000 sequences with cells from (+) and (-) auxin treated hypocotyl. A third subset of about 20,000 sequences was shared by (-) auxin suspension cells and hypocotyl treated with or without auxin, a subset most of which were not shared by (+) auxin suspension cells. Kinetic and saturation data estimates of poly(A) RNA complexity compared favorably and indicated that exogenous auxin treatment can dramatically alter the complexity of all classes of poly(A) RNAs in cultured cells. PMID- 16665935 TI - Characterization of the RNA Required for Biosynthesis of delta-Aminolevulinic Acid from Glutamate : Purification by Anticodon-Based Affinity Chromatography and Determination That the UUC Glutamate Anticodon Is a General Requirement for Function in ALA Biosynthesis. AB - The heme and chlorophyll precursor delta-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3' terminus of yeast tRNA(Phe(GAA)) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA(Glu(UUC)) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A(260) unit added was as much as 50 times greater with the affinity purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in plants and algae. PMID- 16665936 TI - Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion. AB - Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [gamma-(32)P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M(r) 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M(r) 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M(r) 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic (32)P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses. PMID- 16665937 TI - Hypochlorite Disinfection Influences the GA(3)-Induced Synthesis of alpha-Amylase Isozymes by Isolated Barley Aleurone Layers. AB - Aleurone layers isolated from half-seeds of Himalaya barley (Hordeum vulgare cv Himalaya) disinfected in hypochlorite solutions containing 1.0% available chlorine synthesized significantly less alpha-amylase in response to gibberellic acid than layers derived from half-seeds disinfected in 0.1% hypochlorite. This effect of hypochlorite involved neither a differential decrease in the synthesis of group A or B alpha-amylase isozymes nor a general decrease in alpha-amylase synthesis attributable to fewer viable aleurone cells in layers from half-seeds disinfected with 1% hypochlorite. Our results emphasize the need to evaluate the potential effects of routine disinfection procedures used in physiological and biochemical studies. PMID- 16665938 TI - Isolation and sequence of tryptic peptides from the proton-pumping ATPase of the oat plasma membrane. AB - In crude extracts of plant tissue, the M(r) = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4 degrees C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases. PMID- 16665939 TI - Trends in carbohydrate depletion, respiratory carbon loss, and assimilate export from soybean leaves at night. AB - To evaluate assimilate export from soybean (Glycine max [L.] Merrill) leaves at night, rates of respiratory CO(2) loss, specific leaf weight loss, starch mobilization, and changes in sucrose concentration were measured during a 10-hour dark period in leaves of pod-bearing ;Amsoy 71' and ;Wells II' plants in a controlled environment. Lateral leaflets were removed at various times between 2200 hours (beginning dark period) and 0800 hours (ending dark period) for dry weight determination and carbohydrate analyses. Respiratory CO(2) loss was measured throughout the 10-hour dark period. Rate of export was estimated from the rate of loss in specific leaf weight and rate of CO(2) efflux. Rate of assimilate export was not constant. Rate of export was relatively low during the beginning of the dark period, peaked during the middle of the dark period, and then decreased to near zero by the end of darkness. Rate of assimilate export was associated with rate of starch mobilization and amount of starch reserves available for export. Leaves of Amsoy 71 had a higher maximum export rate in conjunction with a greater total change in starch concentration than did leaves of Wells II. Sucrose concentration rapidly declined during the first hour of darkness and then remained constant throughout the rest of the night in leaves of both cultivars. Rate of assimilate export was not associated with leaf sucrose concentration. PMID- 16665940 TI - Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture. AB - Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with (13)C(6)-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR(1)) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2 phenylindole stained cells showed that jsR(1) has the same amount of DNA per nucleus as the parent line (PL). All jsR(1) cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR(1) at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR(1) had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR(1) and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR(1) had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity. PMID- 16665941 TI - Regulation of isocitrate lyase gene expression in sunflower. AB - A cDNA sequence that encodes a portion of sunflower (Helianthus annuus L.) seedling isocitrate lyase was selected from a lambda gt11 cDNA library derived from sunflower seedling cotyledon poly(A)(+) messenger RNA. The library was screened for bacteriophage recombinants that expressed antigens which reacted with antisera directed against cotton seed isocitrate lyase. The isolated cDNA hybridized with a 2 kilobase RNA species that was first detectable in maturing sunflower embryos 19 days after flowering and remained at a constant low level through seed desiccation. The prevalence of this transcript in sunflower cotyledons increased by about 10-fold within 2 days after inhibition in darkness, and transcript levels began to decrease by 5 days after imbibtion. During the first 2 days of germination and growth of sunflower seedlings in light, the rate of isocitrate lyase mRNA accumulation was greater than the rate observed during this period in dark-grown seedlings, giving peak levels about 2-fold higher than corresponding levels in dark-grown seedlings. Illumination of seedlings also promoted an earlier, and more rapid decline of isocitrate lyase transcripts. Peak levels of isocitrate lyase mRNA preceded a corresponding peak in immunologically detectable isocitrate lyase polypeptides by about 24 hours. Isocitrate lyase expression in sunflower cotyledons is developmentally regulated and is modulated, in seedlings, by exposure to light. Mechanisms that control these processes appear to function primarily at the level of mRNA accumulation and are likely to involve changes in transcription rates and/or mRNA stability. PMID- 16665942 TI - CO(2) Assimilation and Activities of Photosynthetic Enzymes in High Chlorophyll Fluorescence Mutants of Maize Having Low Levels of Ribulose 1,5-Bisphosphate Carboxylase. AB - Photosynthetic properties were examined in several hcf (high chlorophyll fluorescence 11, 21, 42 and 45) nuclear recessive mutants of maize which were previously found to have normal photochemistry and low CO(2) fixation. Mutants usually either died after depletion of seed reserves (about 18 days after planting), or survived with slow growth up to 7 or 8 weeks. Both the activity and quantity of ribulose 1,5-bisphosphate carboxylase (Rubisco) were low in the mutants (5-25% of the normal siblings on a leaf area basis) and the loss of Rubisco tended to parallel the reduction in photosynthetic capacity. The Rubisco content in the mutants was often marginal for photosynthetic carbon gain, with some leaves and positions along a leaf having no net photosynthesis, while other leaves had a low carbon gain. Conversely, the activities of C(4) cycle enzymes, phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malate dehydrogenase, and NADP-malic enzyme, were the same or only slightly reduced compared to the normal siblings. The mutants had about half as much chlorophyll content per leaf area as the normal green plants. However, the Rubisco activity in the mutants was low on both a leaf area and chlorophyll basis. Low Rubisco activity and lower chlorophyll content may both contribute to the low rates of photosynthesis in the mutants on a leaf area basis. PMID- 16665943 TI - Two different families of hydroxyproline-rich glycoproteins in melon callus: biochemical and immunochemical studies. AB - Two different families of hydroxyproline-rich glycoproteins, HRGP(1) and HRGP(2), have been isolated from melon callus and separated by ion exchange chromatography on CM-sepharose. HRGP(1) corresponds to an arabinogalactan protein. The sugar portion of HRGP(1) accounts for 94% of the molecule and contains galactose (66%) and arabinose (34%); these residues are present as polysaccharide side chains attached to hydroxyproline. Hydroxyproline is the main amino acid residue (46%) of the protein moiety. The arabinogalactan protein nature of HRGP(1) has been checked by its ability to positively react with the beta-glucosyl Yariv antigen; the (3)H-labeled deglycosylated HRGP(1) also called HRP(1) migrates upon electrophoresis as a single band of molecular weight 76,000. HRGP(2) was fractionated by affinity chromatography on heparin-Ultrogel into three different glycoproteins, HRGP(2a,2b) and (2c). Two of these glycoproteins behave as polycations (HRGP(2b) and (2c)) and are chemically distinct from HRGP(2a). HRGP(2b) is the most abundant component and contains 41% protein and 50% sugar. Hydroxyproline, lysine, tyrosine, and arabinose are the most prominent residues of their respective moiety. The glycosylation pattern of hydroxyproline indicates that HRGP(2b) is related to and possibly a precursor of the wall HRGP; as in melon cell wall HRGP, Hyp-Ara(3) predominates, and small amounts of a putative Hyp-Ara(5) a hitherto unreported hyp-arabinoside, are recorded. The molecular weight of HRP(2b), the protein portion of HRGP(2b) is 55,000 +/- 5,000, as estimated after deglycosylation of the molecule with trifluoromethane sulfonic acid. Antibodies have been raised against HRGP(2b) and HRP(2b). Immunodiffusion shows that each antigen (HRGP(2b) or HRP(2b)) reacts with its own IgG, and cross reacts with the heterologous IgG, thereby indicating the presence of common (unglycosylated) and specific (glycosylated and deglycosylated) epitopes. The arabinogalactan protein HRGP(1) is not recognized by either antibody and HRGP(2b) does not react with the Yariv antigen. Immunoprecipitation of (3)H-labeled HRP(1) and HRP(2b) in the presence of goat antirabbit IgG, followed by gel electrophoresis, allows to recover HRP(2b) only. Again, HRP(2b) is immunoprecipitated by the two antisera. PMID- 16665944 TI - Lipid peroxidation is a consequence of elicitor activity. AB - Elicitor-active preparations from the fungal pathogen of bean Colletotrichum lindemuthianum stimulated the accumulation of products characteristic of lipid peroxidation in treated bean tissues. Bean suspension cells treated with crude and purified elicitors accumulated ;lipofuscin-like pigment' (LEP) and malondialdehyde. The accumulation of LFP after about 6 h of treatment coincided with the onset of visible browning and production of the bean phytoalexins kievitone, phaseollin, and phaseollinisoflavan. The induction of phytoalexins and accumulation of LFP were also triggered by treatments with generators of activated oxygen species, xanthine:xanthine oxidase and Fe:ethylenediaminedi-o hydroxyphenylacetic acid. These data suggest that generation of active oxygen species may be involved in lipid peroxidation triggered by elicitors. PMID- 16665945 TI - Induced Changes in Chloroplast Protein Accumulation during Heat Bleaching in Euglena gracilis. AB - When growing cultures of light-grown Euglena gracilis Z are exposed to slightly elevated temperatures (33 degrees C) there is a time-dependent decrease in chlorophyll (bleaching) and a gradual transformation of chloroplasts into rudimentary plastids. A study was undertaken whose primary objective was to document major changes in polypeptide composition in the stroma and in thylakoids of cells that have been exposed to the bleaching temperature for up to 57 hours. A novel polypeptide of about 60,000 to 63,000 M(r) whose function is presently unknown, accumulates in the stroma and in thylakoids in response to growth at the bleaching temperature. The levels of the large and small subunit of ribuolosebisphosphate carboxylase, on the other hand, decrease to very low levels at about 33 hours and remain very low for the duration of the temperature treatment. Of two polypeptides associated with the light-harvesting chlorophyll protein complex of photosystem II (28,000 and 24,500 M(r)) only the level of the smaller polypeptide decreases at the elevated temperature. The levels of 28,000 M(r) species remain virtually unchanged throughout the temperature treatment period. Changes in chloroplast polypeptide composition were also studied in cells that were allowed to recover at room temperature from an initial treatment at 33 degrees C. Bleaching Euglena could provide a useful tool for studying the interaction between the nucleus and chloroplast genetic system that govern the development and maintenance of this vital organelle to plants. PMID- 16665946 TI - Ecophysiological Significance of CO(2)-Recycling via Crassulacean Acid Metabolism in Talinum calycinum Engelm. (Portulacaceae). AB - High levels of variability in gas exchange characteristics and degree of CAM cycling were found in the same and different individuals of Talinum calycinum Engelm. collected from rock outcrops in Missouri. Differences in CO(2) assimilation were mostly correlated with differences in shoot conductance to CO(2) not shoot internal CO(2) concentration. As found previously, CAM acid fluctuations were evident in well-watered plants exhibiting C(3) gas exchange patterns (CAM-cycling) and also in drought-stressed plants with stomata closed, or nearly so, day and night (CAM-idling). Drought stress also resulted in rapid stomatal closure, conserving water during droughts. Maximal CO(2) uptake rates occurred below 35 degrees C; higher temperatures induced decreases in CO(2) assimilation and conductance while shoot internal CO(2) concentrations remained similar. Plant water-use-efficiency was severely curtailed at temperatures above 30 degrees C. Tissue acid fluctuations were the result of changes in malic acid concentrations. Calculations of the amount of water potentially conserved by CAM cycling yielded values of approximately 5 to 44% of daytime water loss. Thus, CAM cycling may be an important adaptation minimizing water loss by perennial succulents growing in shallow soil on rock outcrops. PMID- 16665947 TI - Datura stramonium Agglutinin : Location in the Seed and Release upon Imbibition. AB - The distribution of Datura stramonium agglutinin over different tissues of D. stramonium L. seeds was visualized by immunocytochemical techniques and quantified by agglutination assays. The lectin occurs predominantly in the outer seed tissues (seed coat and seed epidermis), where it is associated, at least in part, with the cell walls. Developing D. stramonium seeds secrete newly synthesized lectin polypeptides into the incubation medium, which confirms the extracellular location of the lectin. Imbibition of mature decoated seeds results in a rapid and highly specific release of lectin. Indeed, imbibition solutions contain almost exclusively the lectin together with a few other carbohydrate binding proteins; this is indicative of the predominance of these proteins in the seed surface layer. The presence of important amounts of lectin in the outer tissues of the seed is consistent with a possible role in the mediation of cell cell interactions. PMID- 16665948 TI - Evidence for the presence of a sucrose carrier in immature sugar beet tap roots. AB - The objectives of this work were to determine the path of phloem unloading and if a sucrose carrier was present in young sugar beet (Beta vulgaris L.) taproots. The approach was to exploit the characteristics of the sucrose analog, 1' fluorosucrose (F-sucrose) which is a poor substrate for acid invertase but is a substrate for sucrose synthase. Ten millimolar each of [(3)H]sucrose and [(14)C]F sucrose were applied in a 1:1 ratio to an abraded region of an attached leaf for 6 hours. [(14)C]F-sucrose was translocated and accumulated in the roots at a higher rate than [(3)H]sucrose. This was due to [(3)H]sucrose hydrolysis along the translocation path. Presence of [(3)H]hexose and [(14)C]F-sucrose in the root apoplast suggested apoplastic sucrose unloading with its subsequent hydrolysis. Labeled F-sucrose uptake by root tissue discs exhibited biphasic kinetics and was inhibited by unlabeled sucrose, indicating that immature roots have the ability for carrier-mediated sucrose transport from the apoplast. Collectively, in vivo and in vitro data indicate that despite sucrose hydrolysis by the wall-bound invertase, sucrose hydrolysis is not entirely essential for sugar accumulation in this tissue. PMID- 16665949 TI - Time course of acetylene reduction in nodules of five actinorhizal genera. AB - The rate of acetylene reduction was measured as a function of time after addition of 10% acetylene in Alnus, Casuarina, Ceanothus, Datisca, and Myrica. The maximum rate occurred after 45 to 60 seconds and was maintained for an additional 0.5 to 4 minutes before a decline in rate to 30 to 90% of the maximum. The rate then recovered to a value of 63 to 98% of the maximum. Removal of the shoot and lower roots did not affect nodule activity. PMID- 16665950 TI - Reversion of Aberrant Plants Transformed with Agrobacterium rhizogenes Is Associated with the Transcriptional Inactivation of the T(L)-DNA Genes. AB - Transgenic plants harboring the left transfer DNA (T(L)-DNA) of the root inducing plasmid of Agrobacterium rhizogenes show many developmental abnormalities. We observed frequent appearance of normal looking lateral (revertant) shoots from such aberrant plants. Unlike aberrant shoots of the plant, revertant shoots exhibited a very high growth rate and set viable seeds. Sexual and vegetative reproduction studies showed inheritance of the revertant phenotype. Southern hybridization experiments demonstrated that the T-DNA pattern was identical in aberrant and revertant shoots, indicating that the revertant phenotype was not due to deletion or rearrangement of the T-DNA genes. Specific T-DNA transcripts were not expressed in revertant shoots. Thus, the revertant phenotype appears to result from the transcriptional inactivation of T-DNA genes. We propose that similar events in the past may have mediated horizontal acquisition of T(L)-DNA genes by ancestors of the genus Nicotiana, which are still found as silent endogenous T-DNA in present day untransformed Nicotiana species. PMID- 16665951 TI - Dual Effect of Light on the Gibberellin- and Nitrate-Stimulated Seed Germination of Sisymbrium officinale and Arabidopsis thaliana. AB - Red light (R) has a dual effect on the seed germination of the two related species Arabidopsis thaliana and Sisymbrium officinale. The two species provide different means to separate the light-effects. In S. officinale, stimulation of germination by R depends on the stimultaneous presence of nitrate (light-effect I). The effect of both factors is completely blocked by tetcyclacis, an inhibitor of gibberellin (GA)-biosynthesis. Addition of a mixture of gibberellins A(4) and A(7) (GA(4+7)) antagonizes the inhibition. In the absence of nitrate, R shifts germination to lower GA-requirement (light-effect II). In A. thaliana a similar second light-effect is seen on the GA-requirement of GA-deficient ga-1 mutant seeds. R stimulates germination of wild type seeds in water (light-effect I). For both species, light-effect I shows a fluence threshold value of approximately 10( 5) moles per square meter, which is independent of the nitrate concentration. Increasing nitrate concentrations narrow the fluence-range required for maximal germination whereby the product of nitrate concentration and fluence value determines the germination level, indicating a multiplicative interaction between R and nitrate. Fluence-response curves for light-effect II are similar for both species. Germination occurs in the range of 10(-6) to 10(-2) moles per square meter fluence. The maximal level of germination is determined by the level of dark-germination and light-effect II. Increasing GA(4+7) concentrations induce a shift to lower fluence values. It is shown that in the second effect the co action of R and exogenous GA(4+7) is clearly additive. It is concluded that light effect I induces a chain of events leading to GA biosynthesis. Light-effect II seems to enhance the sensitivity of the seeds to GAs. PMID- 16665952 TI - The Urtica dioica Agglutinin Is a Complex Mixture of Isolectins. AB - Rhizomes of stinging nettle (Urtica dioica) contain a complex mixture of isolectins. Ion exchange chromatography with a high resolution fast protein liquid chromatography system revealed six isoforms which exhibit identical agglutination properties and carbohydrate-binding specificity and in addition have the same molecular structure and virtually identical biochemical properties. However, since the U. dioica agglutinin isolectins differ definitely with respect to their amino acid composition, it is likely that at least some of them are different polypeptides coded for by different genes. PMID- 16665953 TI - Evaluation of selectable markers for obtaining stable transformants in the gramineae. AB - Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 x 10(-5) to 2 x 10(-6)) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome. PMID- 16665954 TI - Intracellular compartmentation of ions in salt adapted tobacco cells. AB - Na(+) and Cl(-) are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na(+) and Cl(-) were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of (22)Na(+) and (36)Cl(-) efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells. PMID- 16665955 TI - HPLC separation and indirect ultraviolet detection of phosphorylated sugars. AB - An HPLC method for the separation and analysis of phosphorylated sugars is presented. Ion-exchange chromatography coupled to indirect ultraviolet detection has produced good resolution and sensitivity. Fructose 6-P, glucose 6-P, ribose 5 P, 3-phosphoglyceric acid, ribulose 1,5-P(2), fructose 1,6-P(2), and sedoheptulose 1,7-P(2) can be separated at a sensitivity down to 10 nanomoles. The system resolves 2-carboxy-D-arabinitol 1,5-P(2) from 2-carboxy-D-ribitol 1,5 P(2). The natural inhibitor of ribulose bisphosphate carboxylase, 2-carboxy-D arabinitol 1-P, has been separated from its 5-P isomer and most other phosphorylated compounds. This method is applied to identification of the products obtained upon ion-exchange purification of synthetic 2-carboxyarabinitol 1-P. PMID- 16665956 TI - Studies on the mode of action of acifluorfen-methyl in nonchlorophyllous soybean cells : accumulation of tetrapyrroles. AB - Phytotoxic effects of the herbicide acifluorfen-methyl on nonchlorophyllous soybean cells were estimated by (86)Rb leakage. An action spectrum study showed maximum injury at 350 to 450 nanometers, with lesser activity between 450 and 700 nanometers. Cells treated in the dark with acifluorfen-methyl accumulated fluorescent pigments with the spectral characteristics of protoporphyrin IX. The action spectrum of acifluorfen-methyl matched the absorption spectrum of this tetrapyrrole, and the extent of cellular damage in the light was related to the degree of fluorescent pigment accumulation. We propose that the phytotoxicity of diphenyl ether herbicides could be explained by their ability to cause abnormal accumulations of tetrapyrroles, which in turn induce lethal photooxidative reactions. PMID- 16665957 TI - limitation of Alternative Respiratory Pathway Activity in Grapefruit Flavedo Tissue by Oxygen Availability. AB - The capacity of the alternative respiratory pathway increased in the flavedo tissue of ;Marsh' grapefruit (Citrus paradisi Macf.) stored at 5 degrees C for 2 weeks or longer. Elevated O(2) levels during respiratory assays enhanced respiration by the tissue at 20 degrees C but not at 5 degrees C. At 20 degrees C, salicylhydroxamic acid alone was inhibitory to O(2) uptake only in elevated O(2). In conventional Warburg studies, alternative pathway respiration may be limited by the low solubility and/or slow rate of O(2) diffusion into plant tissues, such as grapefruit flavedo, and may be responsible for the apparent low utilization of the alternative pathway potential observed in other studies. PMID- 16665958 TI - Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination. AB - Hydrogen peroxide steady state levels of 5 micromolar were determined in soybean (Glycine max) embryonic axes incubated for 2 hours and in axes pretreated with aminotriazole or cyanide, where these levels were 50 and 1 micromolar, respectively. The activities of catalase (105 picomoles H(2)O(2) per minute per axis), peroxidase (10-44 picomoles H(2)O(2) per minute per axis), glutathione peroxidase (3 picomoles H(2)O(2) per minute per axis) and superoxide dismutase (3.5 units per axis), were also determined. Catalase seems to be the most important H(2)O(2) consuming enzyme at the physiological concentration of H(2)O(2). A short treatment with aminotriazole, while substantially increasing H(2)O(2) level, did not affect the growth of the axes. The production of superoxide anion by the mitochondria isolated from soybean axes was measured from the superoxide dismutase-sensitive rate of adrenochrome formation in the presence of NADH or succinate as substrate and amounted to 1.3 and 0.8 nanomole O(2) (-) per minute per milligram protein, respectively. According to the stoichiometry of O(2) (-) and H(2)O(2) dismutation reactions, it is apparent that about 0.9 to 1.5% of the total oxygen uptake proceeds through the formation of the free intermediates of the partial reduction of oxygen. PMID- 16665959 TI - Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis. AB - Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction ;E,' was pure plasma membrane. The other, more electronegative and designated fraction ;C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two phase partitioning. Purified plasma membrane obtained from microsomes by two phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations. PMID- 16665960 TI - Characterization of root hair cell walls as potential barriers to the infection of plants by rhizobia : the carbohydrate component. AB - The sugar compositions of root hairs of a variety of plant species were determined. Root hairs of legumes had very similar compositions, whereas those of different families varied widely. Only approximately 50% of the weight of the root hairs of legumes could be accounted for by sugar. Up to 80% of the weight of root hairs from other sources could be accounted for by sugars. Protein made up 5 to 8% of the weight of root hairs of dicots but only 1.3% in corn. Comparison between cell walls from various root cell types within legumes showed that the polysaccharide compositions of root epidermal, root hair, and root cortical cells were very similar. Cotton root hairs were markedly different from walls of mesophyll and epidermal cells of cotyledons from cotton. PMID- 16665961 TI - Kinetics of the Acidification Capacity of Aleurone Layer and Its Effect upon Solubilization of Reserve Substances from Starchy Endosperm of Wheat. AB - The capacity of the isolated barley aleurone layer for endosperm acidification has been demonstrated (J Mikola, M Virtanen 1980 Plant Physiol 66: S-142). The kinetics of this acidification by isolated wheat aleurone layer and its effect on starchy endosperm solubilization are reported. PMID- 16665962 TI - Molecular Comparison of Pyrophosphate- and ATP-Dependent Fructose 6-Phosphate 1 Phosphotransferases from Potato Tuber. AB - The aim of this work was to compare the molecular properties of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) and ATP:fructose 6 phosphate 1-phosphotransferase (PFK). Both enzymes were purified to apparent homogeneity from potato tubers (Solanum tuberosum cv Record). Neither PFP nor PFK preparations contained detectable activity of the other enzyme. PFP was composed of two polypeptides of apparent molecular weight 58,000 and 55,700 whereas PFK contained four polypeptides of apparent molecular weight between 46,300 and 53,300. Chemical cleavage of individual PFP and PFK polypeptides gave a different set of fragments for each polypeptide. On Western blots antisera against PFP failed to cross-react with any of the four PFK polypeptides, and antibodies against PFK failed to bind to either of the PFP polypeptides. Antibodies that immunoprecipitate PFP activity had no effect on PFK activity. Conversely, antibodies against the four PFK polypeptides precipitated the activity of PFK, but not that of PFP. This work shows that potato tuber PFP and PFK are composed of distinct, unrelated polypeptides and indicate that interconversion between PFP and PFK is unlikely. PMID- 16665963 TI - Selenium as Inducer of Glutathione Peroxidase in low-CO(2)-Grown Chlamydomonas reinhardtii. AB - Culture of the green alga Chlamydomonas reinhardtii in the medium containing sodium selenite caused the activity of ascorbate peroxidase to disappear and the appearance of glutathione peroxidase. The induced maximum activity of glutathione peroxidase reached 350 micromole (milligram chlorophyll hour)(-1) under assay conditions used. The enzymic properties of the selenite-induced glutathione peroxidase closely resembled those of animal glutathione peroxidase that contains selenium. PMID- 16665964 TI - Anthocyanin production in chl-rich and chl-poor seedlings. AB - Screening by chlorophyll (Chl) affects photoconversion rates and photoequilibrium ratios of phytochrome in vivo and may cause distortion of the action spectra of photomorphogenesis (N Kazarinova-Fukshansky, M Seyfried, E Schafer 1985 Photochem Photobiol 41: 689-702). Inhibitors that reduce the Chl content of seedlings are sometimes used in photomorphogenesis research to decrease the effects of Chl screening on the state of phytochrome in vivo. Streptomycin is one of the inhibitors that can be used for this purpose. The effects of streptomycin on phytochrome-mediated anthocyanin accumulation in young seedlings are significantly different in closely related systems. The use of ;Chl-bleachers' in photomorphogenesis studies may produce undesirable side effects. At the level of the expression of a photoregulated response, the effects of differences in the state of phytochrome between water-grown Chl-rich and inhibitor-treated Chl-poor seedlings may be difficult to evaluate because they may be masked by the effects of the inhibitor on the response. PMID- 16665965 TI - Chlorophyll a Fluorescence Yield as a Monitor of Both Active CO(2) and HCO(3) Transport by the Cyanobacterium Synechococcus UTEX 625. AB - Simultaneous measurements have been made of inorganic carbon accumulation (by mass spectrometry) and chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625. The accumulation of inorganic carbon by the cells was accompanied by a substantial quenching of chlorophyll a fluorescence. The quenching occurred even when CO(2) fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO(2) or HCO(3) (-) transport. Measurement of chlorophyll a fluorescence yield of cyanobacteria may prove to be a rapid and convenient means of screening for mutants of inorganic carbon accumulation. PMID- 16665966 TI - Partial purification and characterization of the major endoamylase of mature pea leaves. AB - An endoamylase from leaves of pea (Pisum sativum) was purified to near homogeneity by affinity chromatography and ultrafiltration with a yield of about 20%. The purified protein had a specific activity of 686 to 1300 units per milligram protein. Molecular weights of 45 and 41 kilodalton were determined by SDS-PAGE and molecular sieve chromatography, respectively. The purified protein exhibited an action pattern commensurate with that of an endoamylase and exhibited properties indicating it to be very similar to cereal grain alpha amylases (calcium requirement, stability to heat, lability to low pH-values, insensitivity to sulfhydryl reagents). Leaf frationation studies indicated that the enzyme was not primarily located in assimilatory mesophyll cells. Chloroplasts isolated from the leaves were found to contain endoamylases, but their activities represented only a small proportion of the total amylolytic potential of the leaf and reflected for the most part properties quite different from those exhibited by the purified enzyme. PMID- 16665967 TI - Reduced Cytosolic Fructose-1,6-Bisphosphatase Activity Leads to Loss of O(2) Sensitivity in a Flaveria linearis Mutant. AB - The mutant plant of Flaveria linearis characterized by Brown et al. (Plant Physiol. 81: 212-215) was studied to determine the cause of the reduced sensitivity to O(2). Analysis of CO(2) assimilation metabolites of freeze clamped leaves revealed that both 3-phosphoglycerate and ribulose 1,5-bisphosphate were high in the mutant plant relative to F. linearis with normal O(2) sensitivity. The k(cat) of ribulose-1,5-bisphosphate carboxylase (RuBPCase) was equal in all plant material tested (range 18-22 s(-1)) indicating that no tight binding inhibitor was present. The degree of RuBPCase carbamylation was reduced in the mutant plant relative to the wild-type plant. Since 3-phosphoglycerate was high in the mutant plant and photosynthesis did not exhibit properties associated with RuBPCase limitations, we believe that the decarbamylation of RuBPCase was a consequence of another lesion in photosynthesis. Fructose 1,6-bisphosphate and its precursors, such as the triose phosphates, were in high concentration in the mutant plant relative to the wild type. The concentrations of the product of the fructose 1,6-bisphosphatase reaction, fructose 6-phosphate, and its isomer, glucose 6-phosphate, were the same in both plants. We found that the mutant plant had up to 75% less cytosolic fructose 1,6-bisphosphatase activity than the wild type but comparable levels of stromal fructose 1,6-bisphosphatase. We conclude that the reduced fructose-1,6-bisphosphatase activity restricts the mutant plant's capacity for sucrose synthesis and this leads to reduced or reversed O(2) sensitivity. PMID- 16665968 TI - The generation of singlet oxygen (o(2)) by the nitrodiphenyl ether herbicide oxyfluorfen is independent of photosynthesis. AB - The mechanism of action of the p-nitrodiphenyl ether herbicides has remained ambiguous because of conflicting reports in the literature. The diphenyl ether herbicide oxyfluorfen causes a light induced consumption of oxygen which resembles the electron acceptor reaction of paraquat. However, this reaction is not linked to the transport of electrons through photosystem I. This conclusion is based on the observation that the rate of oxygen consumption, in the presence of oxyfluorfen, does not demonstrate a first order rate dependence on light intensity. Using the bleaching of N,N-dimethyl p-nitrosoaniline as a specific detector of singlet oxygen, we demonstrate that oxyfluorfen is a potent generator of this toxic radical. The production of singlet oxygen occurs in the presence of inhibitors of photosynthetic electron transport (oxyfluorfen at 10(-4) molar and paraquat) and also under temperature conditions (3 degrees C) which prevent electron transport. This light induced reaction results in oxygen consumption and is the primary cause of lethality for oxyfluorfen. The production of singlet oxygen occurs rapidly and at low herbicide concentrations (10(-9) molar). The reaction occurs without photosynthetic electron transport but does require an intact thylakoid membrane. PMID- 16665969 TI - Active Transport of CO(2) by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry. AB - Mass spectrometry has been used to confirm the presence of an active transport system for CO(2) in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO(3) in the absence of Na(+) (to prevent HCO(3) (-) transport). Upon illumination the cells rapidly removed almost all the free CO(2) from the medium. Addition of carbonic anhydrase revealed that the CO(2) depletion resulted from a selective uptake of CO(2), rather than a total uptake of all inorganic carbon species. CO(2) transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO(2) fixation but had little effect on CO(2) transport. In iodoacetamide poisoned cells, transport of CO(2) occurred against a concentration gradient of about 18,000 to 1. Transport of CO(2) was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO(2) transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na(+) (<100 microequivalents per liter), but not of K(+), stimulated CO(2) transport as much as 2.4-fold. Unlike Na(+)-dependent HCO(3) (-) transport, the transport of CO(2) was not inhibited by high concentrations (30 milliequivalents per liter) of Li(+). During illumination, the CO(2) concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO(2) transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO(3) (-) to CO(2) would rapidly raise the CO(2) concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO(2) transport was balanced by HCO(3) (-) efflux. PMID- 16665970 TI - The effect of 1,4-dihydropyridines on the initiation and development of gametophore buds in the moss funaria. AB - The plant hormone cytokinin stimulates target caulonemata of Funaria to form buds that develop into the leafy gametophyte. Previous reports have shown that increases in intracellular Ca(2+) occur during hormone-activated budding concomitant with an alteration in the polarity of the organelles in the bud site. In order to ascertain the involvement of voltage-dependent Ca(2+) channels in this phenomenon, we have employed dihydropyridines (DHP), compounds noted for their ability to alter Ca(2+) flux through potential-sensitive channels. Addition of the DHP agonists (+)202-791 and CGP 28392 (100 micromolar) induces bud initials on every target cell including the tip cell. Application of the DHP antagonist (-)202-791, in the presence of cytokinin (1 micromolar benzyladenine), inhibits budding 96%. Similarly, nifedipine blocks cytokinin-induced budding 87% and its effect on budding can be inactivated with a pulse of ultraviolet light. These results are consistent with the idea that cytokinin induces the budding response by increasing Ca(2+) entry through voltage-operated channels. We suggest that cytokinin activation of Ca(2+) channels is the first action of the hormone and that subsequent cytokinin-induced mechanisms are operating to maintain budding, since DHP-induced initials rarely develop into complete buds. PMID- 16665971 TI - Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum. AB - Mass spectrometric analysis of O(2) and CO(2) exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH(4)Cl resulted in nearly a 250% increase in the rate of TCA cycle CO(2) efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O(2) consumption in both light and dark. Anaerobiosis inhibited the CO(2) release caused by NH(4)Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O(2) consumption in the light. This implied that the Mehler reaction did not play a significant role in O(2) consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions. PMID- 16665972 TI - Uniformity of the microsymbiont population from soybean nodules with respect to buoyant density. AB - The microsymbiont population in soybean root nodules (Glycine max L. cv Williams 82 inoculated with Bradyrhizobium japonicum 2143) was characterized during symbiotic development to determine the extent of heterogeneity in this population. The microsymbiont population was isolated by centrifugation through a continuous sucrose gradient (44 to 57% weight to weight ratio) and appeared homogeneous at each age examined up to 26 days after planting based on the symmetrical distribution of the population, enzyme activities, poly-beta hydroxybutyrate contents, protein contents, and viabilities. Some differences in viability, protein content, and acetylene reduction activity were observed at later ages. The population migrated to progressively lighter buoyant densities with increasing age until a density equivalent to 48% sucrose was reached. The changing density correlated directly with the increasing poly-beta hydroxybutyrate to protein ratio. The acetylene reduction activity, based on microsymbiont concentration, followed the same developmental pattern as whole nodules. On a protein basis, the decline of acetylene reduction activity was later and reflected the decrease in protein content per cell. These results suggested that the microsymbiont population, which resulted from inoculation of B. japonicum 2143 onto Williams 82 cultivar of soybeans, developed as a homogeneous population. PMID- 16665973 TI - Photosynthetic Carbon Fixation in Guard Cell Protoplasts of Vicia faba L. : Evidence from Radiolabel Experiments. AB - Photosynthetic carbon fixation in guard cells was reexamined in experiments with highly purified guard cell protoplasts from Vicia faba L. irradiated with red light. The fate of (14)CO(2) (4.8 microcuries of NaHCO(3); final concentration: 100 micromolar) supplied to these preparations was investigated with two dimensional paper, and thin layer chromatography. Rates of CO(2) fixation were 5- to 8-fold higher in the light than in darkness. Separation of acid-stable products into water-insoluble, neutral, and anionic fractions showed that more radioactivity was incorporated into the neutral fraction in the light than in the dark. In the dark, malate and aspartate comprised 90% of the radiolabel found in the anionic fraction, whereas in the light, radioactivity was also found in 3 phosphoglyceric acid (PGA), sugar monophosphates, sugar diphosphates, and triose phosphates. Phosphorylated compounds contained up to 60% of the label in the light-treated anionic fraction. Phosphatase treatment and rechromatography of labeled sugar diphosphate showed the presence of ribulose, a specific metabolite of the photosynthetic carbon reduction pathway (PCRP). In time-course experiments, labeled PGA was detected within 5 seconds. With time, the percentage of label in PGA decreased and that in sugar monophosphate increased. We conclude that PGA is a primary carboxylation product of the PCRP in guard cells and that the activity of the PCRP, and phosphoenolpyruvate-carboxylase is metabolically regulated. PMID- 16665974 TI - Phytochrome regulation of greening in barley : effects on mRNA abundance and on transcriptional activity of isolated nuclei. AB - Red light treatment of etiolated barley (Hordeum vulgare L.) seedlings causes an increase in the relative abundance of the mRNA for light-harvesting, chlorophyll a/b-binding polypeptides, and a decrease in the relative abundance of the mRNA for the NADPH:protochlorophyllide oxidoreductase. It also increases transcriptional activity of subsequently isolated nuclei for the mRNA for the chlorophyll-binding polypeptides and reduces it for the reductase. These results confirm those published previously. Fluence-response and kinetic studies support the hypothesis that the abundance of mRNA for the chlorophyll-binding polypeptides may be transcriptionally limited. They do not support the same hypothesis for the reductase. Red light treatments in the very low fluence range significantly decrease transcriptional activity in isolated nuclei for RNAs hybridizable by the reductase probe, but have little effect on mRNA abundance. By contrast, red light treatments in the low fluence range bring about a sharp decrease in reductase mRNA abundance with little further effect on transcription, suggesting light regulation at the level of mRNA stability rather than transcription. The fluence-response relationships for increase in abundance of mRNA for the chlorophyll-binding polypeptides is similar to that published elsewhere for elimination of the lag period of chlorophyll accumulation in barley. However, kinetic studies published elsewhere show that for elimination of the lag in chlorophyll accumulation, the dependence on the far red-absorbing form of phytochrome concentration is significantly different from the dependence of the transcriptional changes, suggesting that although transcription might be what limits mRNA abundance for the chlorophyll-binding proteins, the mRNA abundance can not be what limits chlorophyll accumulation per se. PMID- 16665975 TI - Mobilization and utilization of cyanogenic glycosides: the linustatin pathway. AB - In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside linamarin (2 beta-d-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the endosperm. After onset of germination, the cyanogenic diglucoside linustatin (2-[6-beta-d glucosyl-beta-d-glucopyranosyloxy]-2- methylpropionitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The linustatin-splitting diglucosidase and the beta cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at this time. Based on these observations the following pathway for the in vivo mobilization and metabolism of cyanogenic glucosides is proposed: storage of monoglucosides (in the endosperm)-glucosylation-transport of the diglucoside (out of the endosperm into the seedling)-cleavage by diglucosidase-reassimilation of HCN to noncyanogenic compounds. The presence of this pathway demonstrates that cyanogenic glucosides, typical secondary plant products serve in the metabolism of developing plants as N-storage compounds and do not exclusively exhibit protective functions due to their repellent effect. PMID- 16665976 TI - separation and Immunological Characterization of Membrane Fractions from Barley Roots. AB - Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) and PM fractions were separated on sodium dodecyl sulfate gels, and immunoblots were used to test for cross-contamination between the fractions. Proteins that cross-reacted with antibodies to the PM ATPase from corn roots and Neurospora were greatly enriched in the PM fraction, as were proteins that cross reacted with monoclonal antibodies to an arabinogalactan protein from the PM of tobacco cells. Proteins that cross-reacted with antibodies to the 58- and 72 kilodalton subunits of the tonoplast ATPase of red beet storage tissue were greatly enriched in the TG fraction. The results with immunoblots and enzyme assays indicated that there was little cross-contamination between the tonoplast and PM vesicles. The molecular weights and isoelectric points of the PM ATPase and the tonoplast ATPase subunits were also determined using immunoblots of two dimensional gels of the PM and TG proteins. PMID- 16665977 TI - Polysomes, Messenger RNA, and Growth in Soybean Stems during Development and Water Deficit. AB - The polysome status and populations of polysomal mRNA were examined in different regions of dark-grown soybean (Glycine max [L.] Merr.) stems that contained either dividing, elongating, or mature (nongrowing) cells. There was a developmental gradient of polysome content in which the dividing tissue had the highest levels and the mature tissue the lowest. A few hours after transplanting the seedlings to vermiculite having low water content (water potential Psi(w) = 0.29 megapascals), stem growth rate decreased to 30% of well-watered controls and the polysome content decreased most in the dividing and elongating tissues. After 24 to 36 hours, stem growth and polysome content recovered gradually. In vitro translation products of polysomal mRNA from dividing, elongating or mature tissue were examined on two-dimensional gels. In well-watered controls, each of the stem regions was enriched in a small subset of the polysomal mRNA population, probably because of developmentally regulated gene expression. Exposing plants to low Psi(w) for 24 hours induced a change in the relative abundance of a small number of polysomal mRNAs in the elongating and mature tissues, but not in the dividing tissue. After 24 to 72 hours at low Psi(w), the changes in polysomal mRNA population were reversed in the elongating tissue. The data indicate that changes in stem growth at low water potential are associated with changes in polysome status and polysomal mRNA in the elongating tissue. PMID- 16665978 TI - Organic Constituents and Complexation of Nickel(II), Iron(III), Cadmium(II), and plutonium(IV) in Soybean Xylem Exudates. AB - The xylem exudates of soybean (Glycine max cv Williams), provided with fixed N, were characterized as to their organic constituents and in vivo and in vitro complexation of plutonium, iron, cadmium, and nickel. Ion exchange fractionation of whole exudates into their compound classes (organic acid, neutral, amino acid, and polyphosphate), followed by thinlayer electrophoresis, permitted evaluation of the types of ligands which stabilize each element. The polyvalent elements plutonium(IV) and iron(III) are found primarily as organic acid complexes, while the divalent elements nickel(II) and cadmium(II) are associated primarily with components of the amino acid/peptide fraction. For plutonium and cadmium, it was not possible to fully duplicate complexes formed in vivo by back reaction with whole exudates or individual class fractions, indicating the possible importance of plant induction processes, reaction kinetics, and/or the formation of mixed ligand complexes. The number and distribution of specific iron- and nickel containing complexes varies with plant age and appears to be related to the relative concentration of organic acids and amino acids/peptides being produced and transported in the xylem as the plant matures. PMID- 16665979 TI - Ice-Encasement Injury to Microsomal Membranes Isolated from Winter Wheat Crowns : II. Changes in Membrane Lipids during Ice Encasement. AB - The physical properties and chemical composition of microsomal membranes were examined during a 7 day period of ice encasement in crown tissue of winter wheat (Triticum aestivum L. cv Norstar). Membrane damage, detected as an increase in microviscosity and electrolyte leakage, began between 1 and 3 days of icing, and was associated with a reduction in the recovery of microsomal membranes from stressed tissue, an increase in the microsomal free fatty acid:total fatty acid ratio, and a decrease in the phospholipid:total fatty acid ratio. These trends were amplified between 3 and 7 days of ice encasement. Examination of the free and total fatty acid fractions showed there was a slight, but not statistically significant (P = 0.05) reduction in the degree of unsaturation of the total fatty acid fraction. The composition of the free and total fatty acid fractions were very similar during ice encasement. Furthermore, analysis of phospholipid classes revealed no significant change in the relative amounts of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, or lysophospholipids in microsomal membranes during icing. Membrane injury during ice encasement apparently involves hydrolysis of the ester bond between glycerol and the acyl groups of the phospholipid resulting in loss of the phosphate-containing polar head group and a concomitant accumulation of free fatty acids in the bilayer. PMID- 16665980 TI - Variable Chlorophyll a Fluorescence and CO(2) Uptake in Water-Stressed White Spruce Seedlings. AB - Regulation of photosynthetic activity can contribute to the prevention of photodamage in stress resistant plants during exposure to drought or low temperatures. Responses to increasing levels of water stress were examined in seedlings of the stress resistant forest conifer, white spruce (Picea glauca [Moench.] Voss). Some seedlings were grown under aseptic in vitro conditions and others in pots. In relatively resistant in vivo seedlings, photosynthetic activities changed slowly in response to increasing water stress. Highly sensitive in vitro seedlings responded to water deficits similarly to in vivo seedlings but over a much shorter time scale. Fluorescence, CO(2) exchange, and stomatal conductance data reported here suggest possible mechanisms for the regulation of photochemical activity in these plants. PMID- 16665981 TI - Phytochrome Photoconversion in Vivo: Comparison between Measured and Predicted Rates. AB - The measured rates of phytochrome photoconversion in vivo, in etiolated cabbage (Brassica oleracea L.) seedlings and cucumber (Cucumis sativus L.) cotyledons, under blue, red, and far red irradiation, are significantly different from those predicted on the basis of the spectral photon flux distributions of the light sources and optical parameters of purified phytochrome. The geometrical relationships between the light source and the irradiated sample affect the rate of phytochrome photoconversion, which is significantly faster in cabbage seedling laying flat on white, wet filter paper than in seedlings in a vertical position. Light reflected from the white filter paper on the bottom of the dish contributes significantly to phytochrome photoconversion. Substituting the white filter paper with a less reflective black one results in a significant decrease of the rate of phytochrome photoconversion in cucumber cotyledons. PMID- 16665982 TI - Lipid-sugar interactions : relevance to anhydrous biology. AB - The ability of seeds and other anhydrous plant forms to survive the withdrawal of water must involve a mechanism for protecting the integrity of cellular membranes. Evidence from animal systems implicates sugars as protective components, and we have tested the changes in mesomorphic phase state of phospholipid model membranes upon hydration and dehydration in the presence of sucrose and/or sucrose plus raffinose. X-ray diffraction studies of dry dimyristoylphosphatidylcholine (DMPC) indicate that the presence of sucrose lowers the chain order/disorder transition temperature to that of hydrated lipid; likewise, the lamellar repeat spacings showed the dry DMPC/sucrose mixture to be similar to that of the hydrated lipid. These results support the proposed potential of sugars to substitute for water in biomembranes. If sucrose is to serve as a protectant during desiccation of seeds, its tendency to crystallize would lessen its effectiveness. Raffinose is known to serve as an inhibitor of sucrose crystallization, and is abundant in seeds. The addition of raffinose to make DMPC/sucrose/raffinose mixtures (1/1/0.3 mass ratio) prevented sucrose crystallization, suggesting this as a possible in vivo role for raffinose. PMID- 16665983 TI - Nitrate absorption by corn roots : inhibition by phenylglyoxal. AB - Nitrate transport in excised corn (Zea mays L.) roots was inhibited by phenylglyoxal, but not by 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) or fluorescein isothiocyanate (FITC). Inhibition of nitrate uptake by a 1 hour treatment with 1 millimolar phenylglyoxal was reversed after 3 hours, which was similar to the time needed for induction of nitrate uptake. If induction of nitrate uptake occurs by de novo synthesis of a nitrate carrier, then the resumption of nitrate uptake in the inhibitor-treated roots may occur because of turnover of phenylglyoxal-inactivated nitrate carrier proteins. All three chemicals inhibited chloride uptake to varying degrees, with FITC being the strongest inhibitor. While inhibition due to DIDS was reversible within 30 minutes, both FITC and phenylglyoxal showed continued inhibition of chloride uptake for up to 3 hours after removal from the uptake solution. Assuming that the anion transporter polypeptide(s) carries a positive charge density at or near the transport site, the results indicate that the nitrate carrier does not carry any lysyl residues that are accessible to DIDS or FITC, whereas the chloride carrier does. Both chloride and nitrate carriers, however, seem to possess arginyl residues that are accessible to phenylglyoxal. PMID- 16665984 TI - Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings. AB - Tomato seedlings (Lycopersicon esculentum Mill.) chilled starting at different times during the light/dark cycle were most chilling-sensitive at the end of the dark period (AI King, MS Reid, BD Patterson 1982 Plant Physiol 70: 211-214). Low temperature tolerance was regained with as little as 10 minutes of light exposure. Low light intensities were less effective than high light intensities in reducing sensitivity, and the length of exposure to light directly influenced sensitivity. Seedlings kept at low night temperatures prior to chilling were also less injured following chilling. Light also restored chilling tolerance to seedlings whose roots were removed. Supplying cut shoots with sucrose, glucose, or fructose reduced chilling sensitivity and largely eliminated the diurnal difference in sensitivity. Endogenous carbohydrate content was correlated with changes in chilling sensitivity; starch and sugar content fell markedly during the dark period. Increased concentrations of sugars were detected 15 minutes after the start of the light period. This evidence all suggests that changes in chilling sensitivity over the diurnal period are regulated by the light cycle. It also suggests that increased sensitivity at the end of the dark period could be due to carbohydrate depletion, and that chilling tolerance following light exposure is likely due to carbohydrate accumulation or closely related events. PMID- 16665985 TI - Light-Induced Proton Release by the Cyanobacterium Anabaena variabilis: Dependence on CO(2) and Na. AB - Light-induced acidification by the cyanobacterium Anabaena variabilis is biphasic (a fast phase I and slow phase II) and shown to be sodium-dependent with an optimum concentration of 40 to 60 millimolar Na(+). Cells grown under low CO(2) concentrations at pH 9 (i.e. mainly HCO(3) (-) present in the medium) exhibited the slow phase II of proton efflux only, while cells grown under low CO(2) concentrations at pH 6.3 (i.e. CO(2) and HCO(3) (-) present) exhibited both phases. Light-induced proton release of phase I was dependent on inorganic carbon available in the bathing medium with an apparent K(m) for CO(2) of 20 to 70 micromolar. As was concluded from the CO(2) dependence of acidification measured at different pH of the bathing medium, bicarbonate inhibited phase-I acidification noncompetetively. Acidification was inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Apparently, acidification of phase I is due to a light-dependent uptake of CO(2) being converted to HCO(3) (-) by a carbonic anhydrase-like function of the HCO(3) (-)-transport system (M Volokita, D Zenvirth, A Kaplan, L Reinhold 1984 Plant Physiol 76: 599-602) before or during entering the cell, thus releasing one proton per CO(2) converted to HCO(3) (-). PMID- 16665986 TI - Temperature-Sensitive, Photosynthesis-Deficient Mutants of Chlamydomonas reinhardtii. AB - Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35 degrees C. Whereas nonconditional mutants required acetate for growth at both 25 and 35 degrees C, the conditional mutants have normal photoautotrophic growth at 25 degrees C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35 degrees C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35 degrees C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis deficient phenotypes was observed. PMID- 16665988 TI - Gas Exchange Analysis of the Relative Importance of Stomatal and Biochemical Factors in Photosynthetic Induction in Alocasia macrorrhiza. AB - When leaves of Alocasia macrorrhiza adapted to 10 micromole quanta per square meter per second were transferred to 500 micromole quanta per square meter per second, the rate of photosynthetic CO(2) assimilation increased for over 45 minutes. For the first 10 to 15 minutes, increases in both stomatal conductance and the leaf's photosynthetic capacity were responsible for the increase in assimilation rate. Thereafter, continuing increases in stomatal conductance were almost entirely responsible for further increases in assimilation rate. When conductances were initially high, assimilation rates 1 minute after the increase in photon flux density could be more than six times as high as for similar leaves with initially low conductance. Further increases in assimilation rate in these leaves with high conductance were predominantly due to increases in the induction state at the biochemical level and followed an exponential time course. When stomatal conductances were initially low, then increases in conductance were predominantly responsible for the increases in assimilation rate, with both following a sigmoidal time course. In these leaves, it was important to also consider the effect of cuticular water loss on the calculation of the intracellular partial pressure of CO(2), and an assessment of the relative importance of stomatal conductance differed considerably from one that did not include cuticular water loss. PMID- 16665987 TI - Exogenous NO(3) Influx and Endogenous NO(3) Efflux by Two Maize (Zea mays L.) Inbreds during Nitrogen Deprivation. AB - The influence of nitrogen stress on net nitrate uptake resulting from concomitant (15)NO(3) (-) influx and (14)NO(3) (-) efflux was examined in two 12-day-old inbred lines of maize. Plants grown on (14)NO(3) (-) were deprived of nitrogen for up to 72 hours prior to the 12th day and then exposed for 0.5 hour to 0.15 millimolar nitrate containing 98.7 atom% (15)N. The nitrate concentration of the roots declined from approximately 100 to 5 micromolar per gram fresh weight during deprivation, and (14)NO(3) (-) efflux was linearly related to root nitrate concentration. Influx of (15)NO(3) (-) was suppressed in nitrogen-replete plants and increased with nitrogen deprivation up to 24 hours, indicating a dissipation of factors suppressing influx. Longer periods of nitrogen-deprivation resulted in a decline in (15)NO(3) (-) influx from its maximal rate. The two inbreds differed significantly in the onset and extent of this decline, although their patterns during initial release from influx suppression were similar. Except for plants of high endogenous nitrogen status, net nitrate uptake was largely attributable to influx, and genetic variation in the regulation of this process is implied. PMID- 16665990 TI - Biosynthesis of delta-Aminolevulinic Acid in Chlamydomonas reinhardtii: Study of the Transamination Mechanism Using Specifically Labeled Glutamate. AB - The first committed intermediate of chlorophyll biosynthesis, delta aminolevulinic acid (ALA), is synthesized from glutamate in the plant cell. The last step of ALA synthesis is a transamination reaction which converts glutamate 1-semialdehyde (GSA) to ALA. The mechanism of the transamination was examined by using glutamate, specifically labeled with either 1-(13)C or (15)N, as substrate for ALA synthesis. After incubating with crude enzymes extracted from Chlamydomonas reinhardtii, the distribution of labels in purified ALA molecules was examined by nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry. We found that both isotopes were present in the same ALA molecule. We interpret the results to mean that intermolecular transamination occurs during the conversion of GSA to ALA. PMID- 16665989 TI - Enzyme activities associated with maize kernel amyloplasts. AB - Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6 phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development. PMID- 16665991 TI - Translocation of Sulfate in Soybean (Glycine max L. Merr). AB - Sulfate translocation in soybean (Glycine max L. Merr) was investigated. More than 90% of the sulfate entering the shoot system was recoverable in one or two developing trifoliate leaves. In young plants, the first trifoliate leaf contained between 10 to 20 times as much sulfate as the primary leaves, even though both types of leaf had similar rates of transpiration and photosynthesis. We conclude that most of the sulfate entering mature leaves is rapidly loaded into the phloem and translocated to sinks elsewhere in the plant. This loading was inhibited by carbonylcyanide m-chlorophenylhydrazone and selenate. At sulfate concentrations below 0.1 millimolar, more than 95% of the sulfate entering primary leaves was exported. At higher concentrations the rate of export increased but so did the amount of sulfate remaining in the leaves. Removal of the first trifoliate leaf increased two-fold the transport of sulfate to the apex, indicating that these are competing sinks for sulfate translocated from the primary leaves. The small amount of sulfate transported into the mesophyll cells of primary leaves is a result of feedback regulation by the intracellular sulfate pool, not a consequence of their metabolic inactivity. For example, treatment of plants with 2 millimolar aminotriazole caused a 700 nanomoles per gram fresh weight increase in the glutathione content of primary leaves, but had no effect on sulfate aquisition. PMID- 16665992 TI - Phloem mobility of xenobiotics: I. Mathematical model unifying the weak Acid and intermediate permeability theories. AB - A passive diffusion model has been developed which simultaneously accounts for the dependence of phloem mobility on permeability and acid dissociation. The model is consistent with the observation that the addition of an acid moiety to an otherwise phloem immobile compound may enhance that compound's ability to move in the phloem. However, acid trapping in the basic phloem is not the only enhancement factor. Acid functionalization also lowers the effective permeability usually towards its optimum value. The unified theory predicts that for a given acid dissociation constant there is an optimum permeability and conversely for a given permeability there is an optimum dissociation constant. PMID- 16665993 TI - Phloem Mobility of Xenobiotics: II. Bioassay Testing of the Unified Mathematical Model. AB - TWO BIOASSAYS WERE USED TO TEST PHLOEM MOBILITY OF SELECTED XENOBIOTIC COMPOUNDS: (a) excised bean leaf assay; (b) rooted bean leaf assay. Compounds assayed were N alkylpyridiniums with systematic variation in octanol-water partition coefficients (log K(ow)), substituted benzoic acids with about the same log K(ow) value but variable acidities. Results of the assays strongly conform, quantitatively, to the predictions of the unified mathematical model. Results also indicate that the membrane permeability value of a compound, which depends directly on log K(ow) value, is the overriding factor in determining phloem mobility. When the weak acid functionality of a compound confers increased phloem mobility, it does so principally by making the log K(ow) value, and consequently the membrane permeability of the compound more optimal. PMID- 16665994 TI - The Kinetics of Chlorate Uptake by XD Tobacco Cells. AB - The uptake of [(36)Cl]chlorate by the 14U variant of the XD cell line of Nicotiana tobaccum L. cv Xanthi was investigated to examine the use of chlorate as a nitrate analog in transport studies. The kinetics of chlorate uptake against concentration was complex. Evidence was obtained, e.g., by means of nitrate competition, that these kinetics could be resolved into two components indicating the existence of two influx mechanisms: a saturable high affinity transport system (HATS) and a low affinity transport system (LATS) that showed first order kinetics. HATS has an apparent K(m) for chlorate of 0.3 millimolar, and a marked pH dependence. The V(max) dropped about fivefold as the pH was changed from the optimum pH (5.5-6.5), while the K(m) remained virtually unchanged. The activity of HATS was completely inhibited by 15 millimolar nitrate and was less sensitive to chloride. LATS was inhibited by chloride and showed some inhibition by nitrate. It was concluded that [(36)Cl]chlorate can be used as an analog for nitrate uptake studies only in a limited low concentration range where HATS is the main route for chlorate influx. PMID- 16665995 TI - A Rapid and Simple Procedure for Purification of Indole-3-Acetic Acid Prior to GC SIM-MS Analysis. AB - A simple and rapid procedure for the purification of indole-3-acetic acid prior to gas chromatography-selected ion monitoring-mass spectrographic analysis was developed using an amino anion exchange minicolumn and a short high resolution C18 column. Since multiple samples can be prepared at one time, the procedure is more rapid and the sample preparation time is reduced to one-third that normally required. In addition, the final recovery was improved by 40 to 50% over that of a solvent partitioning procedure. PMID- 16665996 TI - Induced k efflux from cultured rose cells : effects of protein-synthesis inhibitors. AB - Inhibitors of translation, cycloheximide, emetine, and puromycin, and inhibitors of transcription, actinomycin D and cordycepin, stimulate a net efflux of K(+) from cultured cells of Rosa damascena. In the case of cycloheximide and emetine, this efflux bears many similarities to the efflux induced by ultraviolet radiation, including a lag period of 0.25 to 2.5 hours and a limited total loss of K(+). The efflux is transient, and continued incubation of cells with cycloheximide and emetine allows the cells to recover the K(+); after this, the cells no longer release K(+) in response to UV or to cycloheximide treatment. This suggests that the efflux process depends on continued protein synthesis. Other treatments such as cerulenin, low temperature (0 degrees C), and high temperature (40 degrees C) also inhibit the UV- and cycloheximide-induced K(+) efflux, suggesting that the induction of efflux may involve the synthesis of new plasma membrane. PMID- 16665997 TI - Role of ethylene and cytokinins in the initiation of lateral shoot growth in bromeliads. AB - Aechmea victoriana var discolor L. B. Foster and Aechmea dactylina Bal. are commercially propagated in vitro through lateral shoot growth. A modified Murashige and Skoog medium is used which contains both BA and IAA. These growth substances were shown in the present study to synergistically stimulate the production of ethylene by the cultured plants. The stimulation of ethylene production is correlated with the outgrowth of the lateral buds. The rise in ethylene production was concluded to induce lateral shoot growth, because: (a) outgrowth of the shoots was blocked by preventing an increase in ethylene production, (b) 1-aminocyclopropane-1-carboxylic acid (ACC), the natural precursor of ethylene biosynthesis, substituted for IAA in the promotion of ethylene production and lateral bud outgrowth. Although ACC could substitute for IAA, it could not substitute for BA; therefore, cytokinins are concluded to be essential for lateral bud outgrowth in vitro in Aechmea. These results suggest that cytokinins and ethylene both play roles in natural lateral bud initiation and that the cytokinin function involves two stages of the process. PMID- 16665998 TI - Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump. AB - Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H(+)-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H(+)-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump. PMID- 16665999 TI - Characterization of native and modified extensin monomers and oligomers by electron microscopy and gel filtration. AB - We isolated hydroxyproline-rich extensin precursors from suspension-cultured tomato, cucumber, and sycamore-maple by salt-elution of intact cells and cell wall preparations. Cation exchange chromatography and HPLC gel filtration resolved these precursors into monomeric and oligomeric fractions, confirmed by amino acid analysis, immunological cross-reactivity, and TEM visualization. After rotary shadowing monomers appeared as flexuous rods with a contour length of 70 to 100 nanometers and a ;persistence length' (maximum linear displacement) of 44 to 51 nanometers. Oligomers were larger branched assemblies with occasional pores. Native extensin monomers gave uniform gel filtration retention times (Rts), but the Rts of HF-deglycosylated monomers varied depending on concentration, implying ionic interaction between the highly basic deglycosylated monomers and a weakly cationic gel matrix. Succinylation of the deglycosylated monomers reversed the net charge, and restored the retention time to that of glycosylated monomers, confirming the ionic interaction. Succinylation enhanced visualization of the deglycosylated monomers, which previously were barely discernible flexuous rods. The persistence length:contour length ratios of succinylated deglycosylated monomers (tomato sdP2) and glycosylated monomers (sP2) were the same, implying a similar molecular flexibility for both glycosylated and deglycosylated monomers at room temperature. These molecular properties are consistent with suggestions that extensin monomers reptate into the wall as a transmural protein ;weft' which becomes progressively cross-linked forming a network penetrated by the cellulose ;warp.' PMID- 16666000 TI - Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica. AB - Gibberellin A(1) (GA(1)), 3-epi-GA(1), GA(4), GA(9), 11alpha-hydroxyGA(12), 12alpha-hydroxyGA(12), GA(15), GA(17), GA(19), GA(20), GA(25), GA(37), GA(40), GA(58), GA(69), GA(70), and GA(71) have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4'-dihydrophaseic acid, and the epimeric ent 6alpha, 7alpha, 16alpha, 17-(OH)(4) and ent-6alpha, 7alpha, 16beta, 17-(OH)(4) derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16alpha, 17- and 16beta, 17-dihydroxy-16, 17-dihydro derivatives of GA(12), were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA(4), GA(9), 12alpha- and 12beta-hydroxyGA(12), GA(15), GA(25), and GA(37) were identified by the same criteria, as well as abscisic acid, phaseic acid, 8'-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA(40) has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11alpha- and 12beta-hydroxyGA(12). The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum. PMID- 16666001 TI - Herbicide Resistance in Datura innoxia: Cross-Resistance of Sulfonylurea Resistant Cell Lines to Imidazolinones. AB - Cells resistant to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl were isolated from a predominantly haploid cell suspension culture of Datura innoxia P. Mill. Exponentially growing cell colonies (aggregates of about 40 cells) were mutagenized with ethyl methane sulfonate, subcultured for 10 days to allow growth recovery and plated on a medium containing either chlorsulfuron or sulfometuron methyl at a concentration (10(-8) molar) which killed wild type cells. Surviving clones were picked up after 3 to 4 weeks, further proliferated as callus or cell suspension cultures, and tested for their resistance to both the sulfonylureas and imidazolinones, a chemically different class of herbicides. The variants were stable and showed high (100- to 1000-fold) resistance to the sulfonylureas. While some also exhibited cross resistance to imidazolinones, others showed no cross-resistance at all or, as in one case, greater sensitivity than wild type cells to the imidazolinones. Both classes of herbicides tested inhibited acetolactate synthase activity isolated from wild type cells. The acetolactate synthase of the resistant variants, however, was found to be resistant to the sulfonylureas and also to the imidazolinone(s) in those cells showing cross-resistance to the latter. The lack of cross-resistance observed in some cases provides evidence that the two groups of herbicides have slightly different sites on the acetolactate synthase molecule. PMID- 16666002 TI - Effect of apoplastic solutes on water potential in elongating sugarcane leaves. AB - Solute concentration in the apoplast of growing sugarcane (Saccharum spp. hybrid) leaves was measured using one direct and several indirect methods. The osmotic potential of apoplast solution collected directly by centrifugation of noninfiltrated tissue segments ranged from -0.25 megapascal in mature tissue to 0.35 megapascal in tissue just outside the elongation zone. The presence of these solutes in the apoplast manifested itself as a tissue water potential equal to the apoplast osmotic potential. Since the tissue was not elongating, the measurements were not influenced by growth-induced water uptake and no significant tension was detected with the pressure chamber. Further evidence for a significant apoplast solute concentration was obtained from pressure exudation experiments and comparison of methods for estimating tissue apoplast water fraction. For elongating leaf tissue the centrifugation method could not be used to obtain direct measurements of apoplast solute concentration. However, several other observations suggested that the apoplast water potential of -0.35 to -0.45 megapascal in elongating tissue had a significant osmotic component and small, but significant tension component. Results of experiments in which exudate was collected from pressurized tissue segments of different ages suggested that a tissue age-dependent dynamic equilibrium existed between intra- and extracellular solutes. PMID- 16666003 TI - Daytime and nighttime carbon balance and assimilate export in soybean leaves at different photon flux densities. AB - To evaluate daytime and nighttime carbon balance and assimilate export in soybean (Glycine max [L.] Merrill) leaves at different photon flux densities, rates of CO(2) exchange, specific leaf weights, and concentrations of sucrose and starch were measured at intervals in leaves of pod-bearing ;Amsoy 71' and ;Wells II' plants grown in a controlled environment room. Assimilate export was estimated from CO(2) exchange and change in specific leaf weight. Total diurnal assimilate export was similar for both cultivars. Large cultivar differences existed, however, in the partitioning of carbon into starch reserves and the relative amounts of assimilate exported during the day and the night. Total amounts of both daytime and nighttime export increased with increasing photon flux density, as did sucrose and starch concentrations, specific leaf weight, and rate of respiratory carbohydrate loss at night. Cultivar differences in nighttime rate of export were more closely related to the differences in amount of assimilate available at the end of the day than to differences in daytime rate of net CO(2) assimilation. Daytime rates of export, however, were closely related to daytime rates of net CO(2) assimilation within each cultivar. The total amount of starch depleted during the 10-hour night increased as starch concentration at the beginning of the night increased. PMID- 16666004 TI - Increased Chorismate Mutase Levels as a Response to Wounding in Solanum tuberosum L. Tubers. AB - Discs excised from Solanum tuberosum L. cv White Rose tubers demonstrated a 4.5 fold increase in chorismate mutase activity 48 hours after excision. Incubation in the presence of cycloheximide (25 micromolar) or actinomycin D (100 micromolar) completely inhibited the wound response suggesting de novo synthesis of chorismate mutase. Ratios of activity in the presence of the activator tryptophan to that in the absence of tryptophan remained relatively constant during the induction period. This indicated either a constant ratio of tryptophan sensitive to tryptophan insensitive isozymes, or that only one form of chorismate mutase was present. Chromatography of crude extracts on three different columns yielded only one peak of chorismate mutase activity, activated by tryptophan in each case. Incubation under white light had no effect on chorismate mutase activity when compared to dark controls. PMID- 16666005 TI - Early Activation by Ethylene of the Tonoplast H-Pumping ATPase in the Latex from Hevea brasiliensis. AB - The treatment of Hevea brasiliensis (rubber tree) bark by chloro-2-ethyl phosphonic acid (ethrel), an ethylene-producing compound, induces a significant increase in the tonoplast H(+)-translocating ATPase activity in the latex during the first 24 hours after the application of the stimulating agent. Moreover, the tonoplast-bound ATPase is highly activated when vacuoles (lutoids) are resuspended in ultrafiltrated cytosol. This effect is amplified during ethrel stimulation. Preliminary assays to characterize the endogenous effector(s) suggest that the activator(s) could be a heat-resistant compound with a low molecular weight, most likely an anion. The activation of the tonoplast-bound ATPase and the associated activation of the protons translocation across the lutoid membrane, could explain the cytosolic alkalinization observed in latex following the ethrel treatment of Hevea bark, which results in an enhanced rubber production. PMID- 16666006 TI - Floral Induction in a Photoperiodically Insensitive Duckweed, Lemna paucicostata LP6 : Role of Glutamate, Aspartate, and Other Amino Acids and Amides. AB - The effects of 20 amino acids and two amides were studied on the flowering of a photoperiodically insensitive duckweed, Lemna paucicostata LP6. Alanine, asparagine, aspartate, cystine, glutamate, glutamine, glycine, lysine, methionine, proline, serine, and threonine induced flowering under a photoperiodic regime of 16 hours light and 8 hours darkness. Among these, glutamate and aspartate were found to be the most effective for flower induction. These acids could initiate flowering even at 5 x 10(-7) molar level, though maximal flowering (about 80%) was obtained at 10(-5) molar. Change in the photoperiodic schedule or the pH of the nutrient medium did not influence glutamate- or aspartate-induced flowering. The low concentrations at which glutamate and aspartate are effective suggests that they may have a regulatory role rather than simply acting as metabolites. PMID- 16666007 TI - Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH. AB - Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K(+) concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5 7.5), allowing extremely small changes in H(+) fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid. PMID- 16666008 TI - Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis. AB - Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO(3) (-) or NH(4) (+), the solutions being maintained at pH 5.5. In NO(3) (-)-fed plants excess nutrient anion over cation uptake was equivalent to net OH(-) efflux, and the total charge from NO(3) (-) and SO(4) (2-) reduction equated to the sum of organic anion accumulation plus net OH(-) efflux. In NH(4) (+)-fed plants a large H(+) efflux was recorded in close agreement with excess cation over anion uptake. This H(+) efflux equated to the sum of net cation (NH(4) (+) minus SO(4) (2-)) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO(3) (-) that is taken up and reduced in NO(3) (-)-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH(4) (+)-fed plants absorbed NH(4) (+) was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO(3) (-)-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients. PMID- 16666009 TI - Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries. AB - The biosynthesis and processing of the Galanthus nivalis agglutinin were studied in vivo in ripening snowdrop ovaries. Using labeling and pulse chase labeling experiments it could be demonstrated that the snowdrop lectin is synthesized as a precursor of relative molecular weight (M(r)) 15,000 which is posttranslationally converted into the authentic lectin polypeptide of M(r) 13,000 with a half-life of about 6 hours. Gel filtration of an extract of [(3)H]leucine labeled ovaries on Sepharose 4B showed that a significant portion of the newly synthesized lectin is associated with the particulate fraction. When the organellar fraction was fractionated on isopycnic sucrose gradients this lectin banded in the same density region as the endoplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase. Both radioactivity in lectin and in enzyme activity shifted towards a higher density in the presence of 2 millimolar Mg-acetate indicating that the labeled lectin was associated with the rough ER. Labeled lectin could be chased from the ER with a half-life of 4 hours and then accumulated in the soluble fraction. Whereas the ER-associated lectin contains exclusively polypeptides of M(r) 15,000 the soluble fraction contains both precursor molecules and mature lectin polypeptides. The snowdrop lectin in the ER is fully capable of binding immobilized mannose. It is associated into tetramers with an appropriate molecular weight of 60,000. These results indicate that newly synthesized snowdrop lectin is transiently associated with the ER before transport and processing. PMID- 16666010 TI - Ion fluxes and abscisic Acid-induced proline accumulation in barley leaf segments. AB - The increase in proline induced by ABA, a process stimulated by NaCl or KCl in barley leaves, did not occur when Na(+) (or K(+)) was present in the external medium as the gluconate salt, namely with an anion unable to permeate the plasma membrane. However, proline increase was restored, to different extents, by the addition of various chloride salts but not by ammonium chloride. Moreover, it was shown that the stimulation of the process by NaCl (or KCl) was variously affected by the presence of different salts; all the ammonium salts (10 millimolar NH(4) (+) concentration) inhibited this stimulation almost completely. Inhibition by NH(4) (+) was accompanied by a decreased Na(+) influx (-40%). Also, in the case of Na-gluconate, Na(+) uptake was reduced and the addition of Cl(-) as the calcium or magnesium salt (but not as ammonium salt) restored both the ion influxes and the increase in proline typical of NaCl treatments. Both 4,4' diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), an anion transport inhibitor, and tetraethylammonium chloride (TEA), a K(+) channels-blocking agent, caused, as well as with a reduction of ion influxes, an inhibition of the proline accumulation. The inhibition was practically total with 1 millimolar DIDS and about 80% with 20 millimolar TEA. A possible role of ion influxes in the process leading to the increase in proline induced by ABA is proposed. PMID- 16666011 TI - Dual Role of Methionine in the Biosynthesis of Diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii. AB - Biosynthesis of the polar group of diacylglyceryl-O-4'-(N,N,N trimethyl)homoserine (DGTS) was studied in intact cells of Chlamydomonas reinhardtii Dangeard. Among the three C(4) amino acids tested, only l-methionine could specifically inhibit the photosynthetic incorporation of [(14)C]NaHCO(3) into the polar group of DGTS. The radioactivity in l-[(14)C]methionine, which was labeled at either the C3 + C4, the C1, or the methyl carbon, was efficiently incorporated into the polar group of DGTS. These results suggest that the C(4) backbone and the S-methyl group of l-methionine are precursors to the C(4) backbone and the N-methyl groups of DGTS, respectively. PMID- 16666012 TI - Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization : A C NMR Study. AB - Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-(13)C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the (13)C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the (13)C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-(13)C]trehalose and [1,6-(13)C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble (13)C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-(13)C]alanine and [2 (13)C]-, [3-(13)C]-, and [4-(13)C]glutamate and glutamine. From the analysis of the intramolecular (13)C enrichment of these amino acids, it is concluded that [3 (13)C]pyruvate, arising from [1-(13)C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular (13)C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO(2) fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH(4) (+) assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia. PMID- 16666013 TI - Induction of barley leaf urease. AB - Foliar urea application on barley plants increased leaf urease activity for 5 hours with a peak of 20-fold at 2 hours. To discern the mode of urease induction, urea with or without inhibitors and [(35)S]methionine were incubated with leaf sections for different lengths of time. Urease was extracted, partially purified, electrophoresed, and then quantified by fluorogram. Five urease (U) isozymes were separated by PAGE. U(a) and U(b) might be polymers or complexes that occurred only at the peak of induced activity. U(1) and U(2) appeared at 0.5 and 0.75 hour, respectively, after urea induction, peaked at 2 hours, and persisted only in treated leaves for several additional hours indicating that they are transient inducible forms. U(3) was the constitutive form present in control and treated leaves. Induction with cordycepin or cycloheximide completely prevented urea stimulated activity and nullified the existence of isozymes U(a), U(b), U(1), and U(2). (35)S-U(1), which was labeled in the last hour of induction, appeared on fluorogram 1 hour after induction, peaked at 2 hours, and declined at 3 hours. Results indicated that de novo synthesis of urease is activated by the influx of urea. PMID- 16666014 TI - Fluorescence characteristics of photoinhibition and recovery in a sun and a shade species of the red algal genus porphyra. AB - The effects of light treatment (2000 micromole photons per square meter per second) for varying periods (up to 60 minutes) on chlorophyll fluorescence characteristics and light-limited rates of O(2) evolution were examined in two Porphyra species. Brief light exposures (5-60 seconds) produced a large decrease in variable fluorescence which was not accompained by photoinhibition of light limited O(2) evolution rates. This rapid decrease in variable fluorescence was suppressed by carbonylcyanide m-chlorophenylhydrazone, indicating that it was related to formation of a proton gradient across the thylakiod membranes. A second phase of fluorescence quenching started after 5 minutes of illumination in the case of the shade species, Porphyra nereocystis Anderson, and after 30 minutes of illumination in the case of the sun species, Porphyra perforata J. Agardh. The rate of fluorescence quenching in the second phase was similar to the rate of photoinhibition of light-limited O(2) evolution in both cases. The dark recovery of variable fluorescence in light-treated plants was also biphasic consisting of a rapid first phase and a slower second phase in both the Porphyra species. Recovery of P. perforata was more complete than that of P. nereocystis over the same recovery period. This greater capacity for recovery could represent a mechanism by which P. perforata is more resistant to photoinhibition than P. nereocystis. PMID- 16666015 TI - Export of organic materials from developing fruits of pea and its possible relation to apical senescence. AB - In the G2 line of peas (Pisum sativum L.) senescence and death of the apical bud occurs only in long days (LD) in the presence of fruits. Removal of the fruits prevents apical senescence. One possible reason for the senescence-inducing effect of fruit is that the fruits produce a senescence-inducing factor which moves to the apical bud and is responsible for the effect. For this to be possible there must be a transport mechanism by which material may move from the pods to the apex. To examine the extent of fruit export, pods were labeled via photoassimilation of (14)CO(2) beginning 12 days after anthesis. Under LD conditions, 1.14% of label fixed was transported from the pods with only 10.5% of this found in the apical bud and youngest leaves after 48 hours, the remainder being found principally in other developing fruits and mature leaves. During the onset of apical senescence, less total label was actually exported to the apical bud than at other times. In addition, more total export occurred from pods in short days than in LD, with the apical bud receiving a greater percentage than in LD. Thus the amount and distribution of export would not seem to support the idea of specific export of targeted senescence-promoting compounds. Girdling of the fruit peduncle did not change the characteristics of export suggesting movement via an apoplastic xylem pathway. PMID- 16666016 TI - Mechanism of export of organic material from the developing fruits of pea. AB - Pisum sativum L. fruits export a small quantity of radiolabeled substances to other plant parts after the fruits are allowed to photosynthesize in the presence of (14)CO(2). Export was uninhibited by peduncle girdling suggesting an apoplastic route for transport of material, presumably by ;reverse' flow in the peduncle xylem. To determine if any diurnal water potential gradient formed between pea leaves and fruit might be responsible for the observed export, the water potentials of the various organs were monitored over 24 hours. Water potential differences of up to 7.5 bars existed between leaves and fruit in long photoperiods, and up to 2.5 bars in short photoperiods. Pulses of (14)CO(2) labeling indicated that initial delivery of exported label was to ;transpirational sinks,' with subsequent redistribution of label to metabolic sinks. Export to the apical bud appeared to be direct via the xylem. Application of membrane-impermeable inulin to a surgically opened seed coat ;cup' resulted in export mainly to the subtending leaf with little redistribution. Simultaneous application of sucrose to the seed coat resulted in more extensive distribution of the sucrose, consistent with reloading of the sucrose into mature leaf phloem. Thus, export of material from fruits appears to occur via a xylem pathway in response to transpirationally derived water potential gradients. PMID- 16666017 TI - Inhibition of stem elongation in cucumis seedlings by blue light requires calcium. AB - The effects of blue light and calcium on elongation of hypocotyl segments of Cucumber (Cucumis sativa L. cv Burpee's Pickler) were studied. Cucumber seedlings grown in dim red light showed a rapid decline in the rate of hypocotyl elongation when irradiated with high intensity (100 micromoles per square meter per second) blue light. In intact, 4-day-old seedlings the inhibition began within 2 minutes after the onset of blue-light irradiation and reached a maximum of approximately 55% within 4 minutes. Hypocotyl segments cut from 4-day-old seedlings also showed an inhibition of elongation in response to blue light when segments were floated on aqueous buffer and exposed to blue light for 3 hours. In the presence of 2 micromolar indole-3-acetic acid, blue light caused a 50% inhibition of elongation. Buffering free calcium in the incubation medium with 0.1 millimolar ethylene glycol bis(-aminoethyl ether)- N,N,N',N'-tetraacetic acid eliminated the blue-light inhibition of segment elongation. Several experiments confirmed a specific requirement for calcium for the blue-light-induced inhibition of segment elongation. Treating segments with 0.2 micromolar fusicoccin abolished the inhibition of elongation by blue light as did buffering the medium at pH 4. Adding 1 millimolar ascorbate to incubation medium also eliminated the inhibition of segment elongation caused by blue light. Several compounds implicated in cell wall redox reactions alter the magnitude of the blue-light-induced inhibition. The activity of peroxidase isolated from the cell-wall free space of cucumber hypocotyls was inhibited by ascorbate and low pH. The results are consistent with the hypothesis that blue light inhibits elongation by inducing an increase in cell-wall peroxidase activity and implicate calcium ions in the response to blue light. PMID- 16666018 TI - Variation of Transhexadecenoic Acid Content in Two Triazine Resistant Mutants of Chenopodium album and Their Susceptible Progenitor. AB - Two atrazine resistant nutants of Chenopodium album L. and their susceptible progenitor were analyzed for lipid composition. In the phosphatidyldiacylglycerol the Delta3-trans-hexadecenoic acid (C16:1 trans) percentage was higher in the two resistant phenotypes. However, this difference appears later in the development of the leaves and is not clearly observed in young leaves and seedlings. Thus, the increase of the C16:1 trans during the leaf development of the resistant phenotypes is probably a secondary effect of the psbA mutation that arises in compensation for some photosynthesis deficiency. The significance of the lipid differences shown between the two resistant mutants is discussed in terms of whether they are responsible of the two different levels of herbicide resistance observed in the field. PMID- 16666019 TI - Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts. AB - Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U (14)C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-(14)C]acetate or sn-[U-(14)C]glycerol-3-P in the presence of Mg(2+) and unlabeled UDP-Gal resulted in high (14)C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase. PMID- 16666020 TI - Photoperiodic and genetic control of carbon partitioning in peas and its relationship to apical senescence. AB - Apical senescence but not flower initiation is delayed by short days (SD) compared to long days (LD) in pea plants (Pisum sativum L.) of genotype E Sn Hr. We recently reported that delay of senescence correlated with slower reproductive development, suggesting that fruits are weaker sinks for assimilates under delayed senescence conditions. Thus, we have examined assimilate partitioning in peas to determine if genotype and photoperiod regulate relative sink strength. Assimilate diversion by developing fruit has been implicated in senescence induction. A greater percentage of leaf-exported (14)C was transported to fruits and a smaller percentage to the apical bud of G2 peas (genotype E Sn Hr) in LD than in SD. Relatively more of the (14)C delivered to the apical bud of G2 peas was transported to flower buds than to young leaves in LD as compared to SD. There was no striking photoperiodic difference in carbon partitioning in genetic lines without the Sn Hr allele combination. The Sn Hr allele combination and photoperiod may regulate the relative strength of reproductive and vegetative sinks. Photoperiodic differences in sink strength early in reproduction suggest that these genes regulate sink strength by affecting the physiology of the whole plant. High vegetative sink strength in SD may maintain assimilate supply to the apical bud, delaying senescence. PMID- 16666021 TI - PEPCase Transcript Levels in Mesembryanthemum crystallinum Decline Rapidly upon Relief from Salt Stress. AB - Mesembryanthemum crystallinum plants respond to water stress by changing their pathway of carbon assimilation from C(3) to Crassulacean acid metabolism (CAM). Stressed plants are characterized by elevated levels of phosphoenolpyruvate carboxylase (PEPCase) mRNA, protein, and enzyme activity. We wanted to determine whether CAM is a reversible response to environmental conditions or a developmentally programmed adaptation that is irreversibly expressed once induced. Plants were osmotically stressed by irrigation with 500 millimolar NaCl for 12 days to elicit CAM. Salt was then thoroughly flushed from the soil and PEPCase protein and transcript levels were monitored. PEPCase mRNA levels dropped by 77% within 2.5 hours after salt removal. PEPCase activity and polypeptide levels declined more slowly, with a half-life of 2 to 3 days. These results show that PEPCase expression in M. crystallinum is a reversible response to stress that is regulated at the level of transcription or stability of the PEPCase mRNA. PMID- 16666022 TI - Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa. AB - Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [(35)S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS PAGE and fluorography of membrane proteins labeled in vivo. PMID- 16666023 TI - Respiratory Properties of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation. AB - Respiratory activities were compared among rice seedlings germinated in air for 6 days (aerobic seedlings), those germinated under water for 5 days (submerged seedlings), and those grown in air for 1 day after 5 days' submerged germination (air-adapted seedlings). The respiratory activity of the submerged seedlings increased rapidly on transfer to air and reached a plateau at 16 hours in air. Respiration of the submerged seedlings was as sensitive to cyanide as those of aerobic and air-adapted seedlings. 2,4-Dinitrophenol had no effect on the respiration of the submerged seedlings, but stimulated those of the other two types of seedlings. Mitochondria from three types of seedlings did not differ in the ADP/O ratio and the respiratory control ratio (RCR) when succinate was oxidized. However, mitochondria from submerged seedlings (submerged mitochondria) showed poor RCR of about unity when malate was oxidized. Both the rate of succinate oxidation and succinate dehydrogenase activity were low in submerged mitochondria, but increased during air adaptation. Although submerged mitochondria oxidized malate very slowly, this activity increased after exposure to air without any increase in malate dehydrogenase activity. When NAD(+) was added to submerged mitochondria, oxidation of malate was restored to the level of the aerobic controls. Addition of NAD(+) enhanced the state 3 rate in submerged mitochondria, and RCR recovered to nearly the same value as that of the aerobic controls. Similar effects of NAD(+) on 2-oxoglutarate oxidation were observed. All these defects in submerged mitochondria were repaired during air adaptation. These results suggest that NAD(+)-linked substrate oxidation was low in submerged mitochondria because of NAD(+) deficiency, and that the oxidation increased with an increasing level of NAD(+) during air adaptation. PMID- 16666024 TI - Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize. AB - Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed. PMID- 16666025 TI - Differential Expression in Bundle Sheath and Mesophyll Cells of Maize of Genes for Photosystem II Components Encoded by the Plastid Genome. AB - We have explored the cell-specific expression of plastid genes encoding five photosystem II proteins (PSII-A, -B, -C, -D and cytochrome (Cyt) b-559 encoded by plastid genes psbA, B, C, D and E plus F, respectively) and one encoding the photosystem I protein PSI-A1 in bundle sheath and mesophyll cells (BSC and MC) at different stages of photoregulated development in leaves of dark-grown maize seedlings. Two types of cell-specific mRNA ccumulation patterns have been discerned. First is the PSII-A type: the mRNA is present in small but about equal amounts in both cell types in etiolated leaves; then, upon illumination, the level of mRNA increases progressively and remains high in MC but not BSC of green leaves. Second is the Cyt b-559 type: MC of etiolated leaves have about twice as much of this mRNA as do BSC; about a twofold to threefold transient increase is induced by light in MC but not in BSC. The genes for the PSII-C and -D proteins are complementary to a sizable family of transcripts over a large range of sizes; the patterns of changes in pools of some of these transcripts are of the PSII-A type and others are of the Cyt b-559 type. The DNA region encoding the PSII-B protein also hybridizes to multiple transcripts; among them is a prominent 2.2 kilobase transcript that follows the Cyt b-559 accumulation pattern. In contrast, the pattern of PSI-A1 mRNA accumulation is the same in both cell types. Accumulation of the PSII-B and -D proteins is light-dependent and coordinate. Both are much more abundant in MC than in BSC. PSII-A is detectable in MC of etiolated leaves and increases during photoregulated chloroplast maturation in both cell types, however, to a much greater extent in MC. We conclude that the expression of each of these plastidencoded photosystem II genes in BSC and MC is photoregulated differently; both transcript abundance and translation are regulated. PMID- 16666026 TI - Bark and Leaf Lectins of Sophora japonica Are Sequestered in Protein-Storage Vacuoles. AB - The leguminous tree Sophora japonica contains a family of closely related, but distinct, lectins. Different members of this family are independently expressed in seeds, leaves, and bark (CN Hankins, J Kindinger, LM Shannon 1987 Plant Physiol 83: 825-829; 1988 Plant Physiol 86: 67-10). The inter-, and intracellular distribution of the bark and leaf lectins was studied by indirect postembedding immunogold electron microscopy. Aldehyde fixed bark and leaves postifixed with OsO(4) and embedded in LR White resin permitted sensitive and specific immunogold labeling while maintaining cellular ultrastructure. The leaf and bark tissue cells contain protein-filled storage vacuoles which occupy most the cell's interior volume. The leaf and bark vacuoles closely resemble the protein bodies, or protein storage vacuoles, of seed cotyledons. The leaf and bark lectins were found to be exclusively sequestered in the protein-storage vacuoles of these tissues. PMID- 16666027 TI - Gradients of Intercellular CO(2) Levels Across the Leaf Mesophyll. AB - Most current photosynthesis models, and interpretations of many wholeleaf CO(2) gas exchange measurements, are based on the often unstated assumption that the partial pressure of CO(2) is nearly uniform throughout the airspaces of the leaf mesophyll. Here we present measurements of CO(2) gradients across amphistomatous leaves allowed to assimilate CO(2) through only one surface, thus simulating hypostomatous leaves. We studied five species: Eucalyptus pauciflora Sieb. ex Spreng., Brassica chinensis L., Gossypium hirsutum L., Phaseolus vulgaris L., and Spinacia oleracea L. For Eucalyptus, maximum CO(2) pressure differences across the leaf mesophyll were 73 and 160 microbar when the pressures outside the lower leaf surface were 310 and 590 microbar, respectively. Using an approximate theoretical calculation, we infer that if the CO(2) had been supplied equally at both surfaces then the respective mean intercellular CO(2) pressures would have been roughly 12 and 27 microbar less than the pressures in the substomatal cavities in these cases. For ambient CO(2) pressures near 320 microbar, the average and minimum pressure differences across the mesophyll were 45 and 13 microbar. The corresponding mean intercellular CO(2) pressures would then be roughly 8 and 2 microbar less than those in the substomatal cavities. Pressure differences were generally smaller for the four agricultural species than for Eucalyptus, but they were nevertheless larger than previously reported values. PMID- 16666028 TI - Hormone action on transmembrane electron and h transport. AB - A possible involvement of two different systems in proton translocation was investigated by simultaneous measurement of transmembrane electron flow and proton secretion in a pH-stat combined with a redoxstat. The pH gradient between cytoplasm and apoplast is probably maintained by an H(+) -pumping ATPase and by a second proton extrusion system, which seems to be linked to a redox chain with NAD(P)H as electron donor. Indole acetic acid inhibits both e(-) and H(+) efflux, but only if the ;electron draw' from the outside is not too high. The electron draw depends on the hexacyanoferrate level at the plasmalemma surface and on the Ca(2+) concentration. The inhibiting effect of auxin on e(-) and H(+) efflux in the presence of hexacyanoferrate can be only detected at low levels of bivalent cations and of the artificial electron acceptor. The inhibition of e(-) and H(+) efflux by auxin requires high oxygen levels. The influence of auxin on both e(-) and H(+) transfer disappears below 2 kilopascals O(2), a level which does not influence respiration. Ethanol and fusicoccin do not increase the e(-) flux, probably because the electron transfer from the plasma membrane to HCF III is the limiting step. If electron transfer is reduced by IAA pretreatment, ethanol increases e(-) flux. Fusicoccin decreases e(-) and increases H(+) efflux if the rates have been lowered previously by indole acetic acid pretreatment. This effect depends on high oxygen levels and is reversible by lowering oxygen pressure. Auxin and Ca(2+) change e(-) flow and H(+) ejection in a 1:1 ratio. PMID- 16666029 TI - Hexachloroiridate IV as an Electron Acceptor for a Plasmalemma Redox System in Maize Roots. AB - Hexachloroiridate IV, a new artificial electron acceptor for the constitutive plant plasma membrane redox system has been investigated. It appeared not to permeate through biological membranes. Due to its higher redox potential, it is a more powerful electron acceptor than hexacyanoferrate III (ferricyanide) and even micromolar concentrations are rapidly reduced. Hexachloroiridate IV increased H(+) efflux over a concentration range of 0.05 to 0.1 millimolar. Lower concentrations slightly inhibited proton extrusion. Calcium stimulated both proton and electron transfer rates. Like hexacyanoferrate III-reduction, irridate reduction was inhibited by auxin. PMID- 16666030 TI - Induction and Accumulation of Heat Shock-Specific Poly(A) RNAs and Proteins in Soybean Seedlings during Arsenite and Cadmium Treatments. AB - Northern blot hybridization analyzes revealed that poly(A(+)) RNAs homologous to eight heat shock (HS)-specific cDNA clones were induced by arsenite (As) or Cd treatments. The mRNAs accumulated slower, and maximum accumulations were consistently lower than HS-induced levels. Prolonged treatment with low concentrations (50-100 micromolar) of As for 6 hours, or Cd for 12 hours, resulted in decreased accumulations of HS-specific mRNAs. This response resembled the ;autoregulation' observed during continuous 40 degrees C HS. However, no autoregulation was evident when soybean seedlings were exposed to high concentrations of As (250 micromolar) or Cd (1 millimolar) for 12 hours. The cDNA probe pCE54 detected a second higher molecular weight poly(A(+)) RNA following As or Cd treatments which accumulated concomitantly with the lower molecular weight HS-specific poly(A(+)) RNA. The patterns of low molecular weight HS polypeptides from in vitro translations induced by HS, As, and Cd, and analyzed by one dimensional and two-dimensional SDS-PAGE, were similar but temporal differences were apparent. In addition to HS proteins, many control proteins were also detected in both in vitro and in vivo labeling patterns from As and, to a lesser extent, Cd treatments. The chemical agents used in this study apparently induced the accumulation and translation of HS messages in vivo but not in the selective manner as observed during HS treatment. PMID- 16666031 TI - In vitro synthesis and processing of tomato fruit polygalacturonase. AB - The in vitro processing of tomato fruit polygalacturonase (PG) (poly[1,4-alpha-d galacturonide]glucanohydrolase, EC 3.2.1.15) was studied. Complete chemical deglycosylation of a mixture of mature, purified PG 2A and PG 2B isozymes (45 and 46 kilodaltons; respectively) with trifluoromethane sulfonic acid yielded a single polypeptide of 42 kilodaltons. Similarly, N-terminal amino acid sequencing of the PG 2A/2B isozyme mixture yielded a single 21 amino acid N-terminal sequence, suggesting that the two isozymes result from differential post translational processing of a single polypeptide. Translation of PG mRNA in vitro results in the synthesis of a single polypeptide with an apparent molecular weight of 54 kilodaltons. Nucleotide sequence analysis of a full-length PG cDNA clone indicates that the large size difference between the PG in vitro translation product and the mature isozymes is due to the presence of a 71 amino acid (8.2 kilodaltons) domain at the N-terminus of in vitro translated PG, consisting of a hydrophobic signal sequence followed by a highly charged prosequence. To determine the precise cleavage site of the signal sequence, PG mRNA was translated in vitro in the presence of canine pancreas microsomal membranes. This resulted in the production of two glycosylated PG processing intermediates with apparent molecular weights of 58 and 61 kilodaltons. The PG processing intermediates were shown to be sequestered within the lumen of the microsomal membranes by protease protection and centrifugational analysis. Deglycosylation of the PG processing intermediates with endoglycosidase H yielded a single polypeptide with an apparent molecular weight of 54 kilodaltons. The production of two distinct, glycosylated processing intermediates from the single in vitro translated PG polypeptide suggests a mechanism by which the differential glycosylation observed for the mature PG 2A and PG 2B isozymes may occur. Edman degradation of (3)H-labeled 58 and 61 kilodalton PG processing intermediates indicates that the site of signal sequence cleavage is after amino acid 24 (serine). These results suggest that the proteolytic processing of PG occurs in at least two steps, the first being the co-translational removal of the 24 amino acid signal sequence and the second being the presumed post-translational removal of the remaining highly charged 47 amino acid prosequence. PMID- 16666032 TI - Purification of pyruvate kinase from germinating castor bean endosperm. AB - Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme. PMID- 16666033 TI - Diurnal growth of tall fescue leaf blades : I. Spatial distribution of growth, deposition of water, and assimilate import in the elongation zone. AB - Tall fescue leaf blades elongate at near constant rates during most of the light and dark periods of the diurnal cycle, with the dark rate being higher by 60 to 65%. Our objective was to determine relationships among diurnal rates of leaf elongation, deposition of water and deposition of dry matter (DM) into the elongation zone. Two separate experiments were conducted, both with a 15-hour photoperiod and constant 21 degrees C at the growth zone. Increased rates of leaf elongation in darkness were due to proportionally increased rates of elongation of 4-millimeter segments of the elongation zone. Length of the total elongation zone was 30 millimeters in both light and darkness. The spatial distribution of water contents in the elongation zone varied little during the diurnal cycle. Thus, dark stimulation of leaf elongation rate (+65%) and of water deposition (+77%) into elongation zones were similar. Water content per unit leaf length increased by 50% between the basal and distal limits of the elongation zone, indicating that tissue also grew in the lateral and vertical dimensions. Longitudinal growth of tissue, however, allowed 5 to 7 times more water deposition into the elongation zone than growth in cross-sectional area. This relationship was similar in light and darkness. In both light and darkness net rates of DM deposition (microgram per millimeter leaf length per hour) increased from the zone of cell division towards the region of most active elongation, 10 to 15 millimeters from the ligule, then decreased towards the distal end of the elongation zone. Net DM deposition rates (microgram per hour) integrated over the 30-millimeter elongation zone were similar during light and darkness. Thus, DM in the elongation zone was diluted during darkness as a result of increased water deposition. Net DM deposition rates at and above the distal end of the elongation zone were clearly positive during the light, but were close to zero or negative in darkness. Thus, DM deposition into the elongation zone and the adjacent recently expanded tissue was differentially affected in the diurnal cycle, DM deposition occurred in both tissues in light, but was restricted to the elongation zone in darkness. PMID- 16666034 TI - Diurnal Growth of Tall Fescue Leaf Blades : II. Dry Matter Partitioning and Carbohydrate Metabolism in the Elongation Zone and Adjacent Expanded Tissue. AB - The spatial distributions of net deposition rates of water soluble carbohydrate free dry matter (WSC-free DM) and WSC were evaluated within and above the elongation zone of tall fescue (Festuca arundinacea Schreb.) leaf blades during light and darkness. Imported DM used for WSC-free DM synthesis during darkness (67% of the total in experiment I and 59% in experiment II) was greater than during light (47% in both experiments), suggesting that the 65% higher leaf elongation rate during darkness was accompanied by higher rates of synthesis of cellular structural components. Deposition rates of WSC in the basal and central part of the elongation zone (0-20 mm from the ligule) were similar during light and darkness, but above 20 millimeters WSC deposition occurred during light and WSC loss occurred during darkness. WSC deposition and loss throughout the elongation zone and the recently expanded tissue were mostly due to net synthesis and degradation of fructan. Fructan was predominantly low molecular weight and contributed about 50% of the total osmotic partial pressure of WSC. In the most actively growing region, where fructan synthesis was most rapid, no diurnal change occurred in molecular weight distribution of fructan. WSC solute concentrations were diluted in the most actively growing tissue during darkness because net monosaccharide and fructan deposition were unaltered and sucrose deposition was decreased, but growth-associated water deposition was increased by 77%. Net rates of fructan synthesis and degradation were not related to tissue sucrose concentration, but appeared to respond to the balance between assimilate import and assimilate use in synthesis of cellular structural components (i.e. WSC-free DM) and deposition of monosaccharides. Fructan synthesized in tissue during most active elongation was degraded when the respective tissue reached the distal limit of the elongation zone where assimilate import in darkness was insufficient to maintain synthetic processes associated with further differentiation of cells. PMID- 16666035 TI - Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex. AB - We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released (14)CO(2) from [ureido-(14)C]ureidoglycolate under conditions in which urease is not detectable. CO(2) and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K(m) of 85 micromolar for ureidoglycolate. Glyoxylate and CO(2) are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO(2) and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2'-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [(14)C]urea production from [2,7-(14)C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585 591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of (14)CO(2) release from [2,7-(14)C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of (14)CO(2) is not dependent on the accumulation of free intermediates. That [2,7 (14)C]allantoate dependent (14)CO(2) release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido carbons of allantoate can be completely degraded to CO(2) without a urea intermediate. PMID- 16666036 TI - The regulation of gelation of Phloem exudate from cucurbita fruit by dilution, glutathione, and glutathione reductase. AB - The average glutathione equivalent concentration in phloem exudate collected from squash fruit (Cucurbita moschata [Duchesne] Poir. var Butternut) and pumpkin fruit (Cucurbita pepo [L.] var Jack-o-lattern) was 1.02 and 0.60 millimolar, respectively. Glutathione reductase (EC 1.6.4.2) activity in phloem exudate from squash and pumpkin fruit averaged 0.48 and 1.74 micromole NADPH oxidized per minute per milliliter, respectively. Protein concentrations in fruit phloem exudates averaged 67 milligrams per milliliter for squash and 57 milligrams per milliliter for pumpkin. The phloem-specific P-proteins account for most of the protein content of exudate. Pure exudate from fruit does not gel for hours or days, but when diluted with neutral or alkaline aqueous solutions, exudate gels rapidly. Exudate solutions undergo biphasic pH changes with dilution. We suggest that P-protein undergoes conformational change upon dilution, exposing titratable groups and sulfhydryl residues. Oxidation of the latter forms the intermolecular disulfide bridges of the gel. The gelation of diluted exudate is regulated by factors (oxygen, pH, glutathione, NADPH) which affect the maintenance of reduced sulfhydryl residues and the activity of glutathione reductase. While these factors may also act in vivo to regulate redox conditions in phloem, their relationship to hypothetical sol/gel transitions or motile and nonmotile phases in the transport conduit is unknown. PMID- 16666037 TI - Cytokinin Fluxes during Floral Induction in the Long Day Plant Sinapis alba L. AB - Sinapis alba is a long-day (LD) plant that can be induced to flower by a single LD. A number of changes normally occurring in the meristem of plants subjected to the LD can be produced in short day by a single application of a cytokinin to the apical bud. However, flower buds are not produced indicating that evocation by the cytokinin is only partial. In this work, the cytokinin content of root exudate, obtained under vacuum, and of leaf exudate, obtained by the EDTA-method, has been analyzed comparatively in vegetative and induced plants, using reversed phase HPLC coupled to the Amaranthus bioassay. The results show that, as early as 16 hours after the start of the LD, there is an increase of cytokinin activity in both the root and leaf exudates of induced plants. These observations fit nicely with previous results obtained on Sinapis, and they indicate that cytokinins are part of the floral stimulus in this species. PMID- 16666038 TI - beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : I. Identification of an Endogenous beta-Linked Glucolipid Activator. AB - Heat-stable activators of membranous beta-glucan synthase have been isolated from the supernatant fraction of crude mung bean (Vigna radiata) extracts by DEAE cellulose and silica-gel chromatography. One of the activators has been partially purified and characterized on the basis of susceptibility to various enzymes and by analysis of the products formed upon total acid hydrolysis, alkaline methanolysis, and beta-glucosidase digestion. This activator has the characteristics of a 1,2-dioleoyl diglyceride containing beta-linked glucose residue(s) at the C-3 position. When expressed per mole of glucosyl residues, the maximal K(a) value of the activator is estimated to be 25 micromolar. Both the intact glucosyl and fatty acid moiety are essential to the stimulatory effect of the activator. PMID- 16666039 TI - beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : II. Comparison of Effects of an Endogenous beta-Linked Glucolipid with Synthetic n Alkyl beta-d-Monoglucopyranosides. AB - n-Alkyl (C(6)-C(12)) beta-d-monoglucopyranosides have been found to be highly potent activators of mung bean beta-glucan synthase in vitro, increasing the V(max) of the enzyme as much as 60-fold and with K(a) values as low as 10 micromolar. Activation is highly specific for the beta-linked terminal glucose residue; other alkyl glycosides such as, octyl-alpha-glucoside, dodecyl beta maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on beta-glucan synthesis under a variety of conditions, it is proposed that the alkyl beta-glucosides are structural analogs of the native glucolipid activator of beta-glucan synthase isolated from mung bean extracts. PMID- 16666040 TI - Influence of Drought Acclimation and CO(2) Enrichment on Osmotic Adjustment and Chlorophyll a Fluorescence of Sunflower during Drought. AB - Osmotic adjustment occurred during drought in expanded leaves of sunflowers (Helianthus annuus var Hysun 30) which had been continuously exposed to 660 microliters CO(2) per liter or had been previously acclimated to drought. The effect was greatest when the treatments were combined and was negligible in nonacclimated plants grown at 340 microliters CO(2) per liter. The concentrations of ethanol soluble sugars and potassium increased during drought but they did not account for the osmotic adjustment. The delay in the decline in conductance and relative water content and in the loss of structural integrity with increasing drought was dependent on the degree of osmotic adjustment. Where it was greatest, conductance fell from 5.8 millimeters per second on the first day of drought to 1.3 millimeters per second on the fourth day and was at approximately the same level on the eighth day. The relative water content remained constant at 85% for three days and fell to 36% on the sixth day. There was no evidence of leaf desiccation even on the eighth day. In contrast, the conductance of leaves showing minimal adjustment fell rapidly after the first day of drought and was negligible after the fourth, at which time the relative water content was 36%. By the sixth day of drought, areas near the margins of the leaves were desiccating and the plants did not recover upon rewatering. Despite the differences in the rate of change of conductance and relative water content during drought, photosynthetic electron transport activity, inferred from measurements of chlorophyll a fluorescence in vivo and PSII activity of isolated thylakoids, remained functional until desiccation occurred. PMID- 16666041 TI - Action spectra for nitrate and nitrite assimilation in blue-green algae. AB - Action spectra for the assimilation of nitrate and nitrite have been obtained for several blue-green algae (cyanobacteria) with different accessory pigment composition. The action spectra for both nitrate and nitrite utilization by nitrate-grown Anacystis nidulans L-1402-1 cells exhibited a clear peak at about 620 nanometers, corresponding to photosystem II (PSII) C-phycocyanin absorption, the contribution of chlorophyll a (Chl a) being barely detectable. The action spectrum for nitrate reduction by a nitrite reductase mutant of A. nidulans R2 was very similar. All these action spectra resemble the fluorescence excitation spectrum of cell suspensions of the microalgae monitored at 685 nanometers-the fluorescence band of Chl a in PSII. In contrast, the action spectrum for nitrite utilization by nitrogen-starved A. nidulans cells, which are depleted of C phycocyanin, showed a maximum near 680 nanometers, attributable to Chl a absorption. The action spectrum for nitrite utilization by Calothrix sp. PCC 7601 cells, which contain both C-phycoerythrin and C-phycocyanin as PSII accessory pigments, presented a plateau in the region from 550 to 630 nanometers. In this case, there was also a clear parallelism between the action spectrum and the fluorescence excitation spectrum, which showed two overlapped peaks with maxima at 562 and 633 nanometers. The correlation observed between the action spectra for both nitrate and nitrite assimilation and the light-harvesting pigment content of the blue-green algae studied strongly suggests that phycobiliproteins perform a direct and active role in these photosynthetic processes. PMID- 16666042 TI - Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates. AB - The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process. PMID- 16666043 TI - P-NMR Spectroscopy of Roots of Intact Corn Seedlings. AB - The application of (31)P nuclear magnetic resonance spectroscopy to the study of metabolism in roots of intact corn seedlings is described. (31)P-NMR spectra of developmentally distinct parts of primary roots of whole seedlings are presented. The spectra are of quality comparable to those of excised pieces of plant tissue. PMID- 16666044 TI - Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity. AB - A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F(1) progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus. PMID- 16666045 TI - Early Inhibition of Photosynthesis during Development of Mn Toxicity in Tobacco. AB - Early physiological effects of developing Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) were examined in glass-house/water cultured plants grown at high (summer) and low (winter) photon flux. Following transfer of plants to solutions containing 1 millimolar Mn(2+), sequential samplings were made at various times for the following 9 days, during which Mn accumulation by leaves increased rapidly from approximately 70 on day 0 to approximately 1700 and approximately 5000 microgram per gram dry matter after 1 and 9 days, respectively. In plants grown at high photon flux, net photosynthesis declined by approximately 20 and approximately 60% after 1 and 9 days, respectively, and the onset of this decline preceded appearance (after 3 to 4 days) of visible foliar symptoms of Mn toxicity. Intercellular CO(2) concentrations and rates of transpiration were not significantly affected; moreover, the activity of the Hill and photosystem I and II partial reactions of chloroplasts remained constant despite ultimate development of severe necrosis. Though the activity of latent or activated polyphenol oxidase increased in parallel with Mn accumulation, neither leaf respiration nor the activity of catalase [EC 1.11.1.6] and peroxidase [EC 1.10.1.7] were greatly affected. These effects from Mn toxicity could not be explained by any changes in protein or chlorophyll abundance. Additionally, they were not a consequence of Mn induced Fe deficiency. Therefore, inhibition of net photosynthesis and enhancement of polyphenol oxidase activity are early indicators of excess Mn accumulation in tobacco leaves. These changes, as well as leaf visual symptoms of Mn toxicity, were less severe in plants cultured and treated at low photon flux even though the rates of leaf Mn accumulation at high and low photon flux were essentially equivalent. PMID- 16666047 TI - Positive selection for male-sterile mutants of Arabidopsis lacking adenine phosphoribosyl transferase activity. AB - Three mutants of Arabidopsis thaliana deficient in adenine phosphoribosyl transferase activity were isolated by selecting for germination of seeds on a medium containing 0.1 millimolar 2,6-diaminopurine. In each of the mutants, diaminopurine resistance was due to a recessive nuclear mutation at a locus designated apt. The mutants grow more slowly than wild type, and are male sterile due to abortion of pollen development after the meiotic divisions of the pollen mother cells. The reliability and ease with which the mutants can be selected should afford novel opportunities to investigate purine metabolism, pollen development, and genetic problems which require the ability to select for loss-of function mutations. PMID- 16666046 TI - Evidence for Effects on the in Vivo Activity of Ribulose-Bisphosphate Carboxylase/Oxygenase during Development of Mn Toxicity in Tobacco. AB - The progressive decrease in net photosynthesis accompanying development of Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) is a result of effects on in vivo activity of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco, EC 4.1.1.39). This conclusion is supported by: (a) decrease in rates of CO(2) depletion during measurements of CO(2) compensation, (b) increase in leaf RuBP concentrations, (c) progressive decreases in rate-constants of RuBP loss (light to dark transition analyses) with progressive increases of leaf Mn concentrations, and (d) restoration of diminished rates of net photosynthesis to control rates by elevated CO(2) (5%). Moreover, elevated CO(2) (1100 microliters per liter) during culture of Mn treated plants decreased elevated RuBP concentrations to control levels and alleviated foliar symptoms of Mn toxicity. These effects of Mn toxicity on in vivo activity of rubisco were not expressed by in vitro kinetic analyses of rubisco prepared under conditions to sequester Mn or to adsorb polyphenols or their oxidation products. Similarly, the in vitro activity of fructose bisphosphatase (EC 3.1.3.11) was unaffected by Mn toxicity. PMID- 16666048 TI - Rapid wall relaxation in elongating tissues. AB - Reported differences in the relaxation of cell walls in enlarging stem tissues of soybean (Glycine max [L.] Merr.) and pea (Pisum sativum L.) cause measurements of the yield threshold turgor, an important growth parameter, to be in doubt. Using the pressure probe and guillotine psychrometer, we investigated wall relaxation in these species by excising the elongating tissue in air to remove the water supply. We found that the rapid kinetics usually exhibited by soybean could be delayed and made similar to the slow kinetics previously reported for pea if slowly growing or mature tissue was left attached to the rapidly growing tissue when relaxation was initiated. The greater the amount of attached tissue, the slower the relaxation, suggesting that slowly growing tissue acted as a water source. Consistent with this concept was a lower water potential in the rapidly elongating tissue than in the slowly growing tissue. Previous reports of wall relaxation in pea included slowly growing tissue. If this tissue was removed from pea, relaxation became as rapid as usually exhibited by soybean. It is concluded that the true relaxation of cell walls to the yield threshold requires only a few minutes and that the yield threshold should be constant during so short a time, thus reflecting the yield threshold in the intact plant before excision. Under these conditions, the yield threshold was close to the turgor in the intact plant regardless of the species. The presence of slowly growing or mature tissue delays wall relaxation and should be avoided during such measurements. However, this delay can be used to advantage when turgor of intact growing tissues is being measured using excised tissues because turgor does not change for a considerable time after excision. PMID- 16666049 TI - Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina. AB - Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H(+) pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H(+) pumping activities. N,N'-dicyclohexylcarbodiimide inhibited the ATPase strongly (I(50) = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4' Stilbenedisulfonic acid affected both enzyme activities and their associated H(+) pumping activities. This is in contrast to the H(+)-PPase of higher plants which is 4,4'-stilbenedisulfonic acid insensitive. PMID- 16666050 TI - Regulation of Acyl Carrier Protein Messenger RNA Levels during Seed and Leaf Development. AB - We have examined the expression of acyl carrier protein (ACP) mRNA levels and ACP activity in leaves, where fatty acids function primarily in membrane synthesis, and in developing soybean seeds, where fatty acids are primarily used for oil storage. An RNA probe transcribed from a synthetic spinach ACP-I gene hybridized on Northern blots to ACP mRNA from both seed and leaf tissue from soybean, spinach, and rapeseed. In each species, the ACP transcript from leaf was slightly larger than that from seed. Both the amounts of ACP protein and the levels of ACP mRNA were substantially higher in young leaf tissue of spinach and soybean when compared to mature leaf tissue. Light-grown spinach leaves also contained higher ACP activity and accumulated more ACP mRNA than dark-grown leaves. ACP mRNA levels measured in developing soybean seeds peaked at 20 days after flowering then decreased 10-fold by 70 days after flowering. In each tissue, the developmental changes in ACP protein levels can be accounted for by changes in ACP mRNA abundance. Comparison of the relative prevalence of mRNA and protein for ACP and lectin in soybean seeds suggests a major difference in mRNA translational efficiency and/or protein stability for these two proteins. PMID- 16666051 TI - Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2. AB - We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M(r) 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M(r) 160,000 inhibit phosphatase activity by approximately 70%. PMID- 16666052 TI - Role of Photosynthetic Reactions in the Activity of Carbonic Anhydrase in Synechococcus sp. (UTEX 2380) in the Light : Inhibitor Studies Using the O Exchange in C/O-Labeled Bicarbonate. AB - The role of the photosystems in the exchange of (18)O between species of inorganic carbon and water was studied in suspensions of the cyanobacterium Synechococcus sp. (UTEX 2380) using membrane-inlet mass spectrometry. This (18)O exchange is caused by the hydration-dehydration cycle of CO(2) and is catalyzed by carbonic anhydrase. We observed the complex (18)O exchange kinetics including dark-light-dark transients in suspensions of whole cells and found these to be identical to the (18)O exchange kinetics of physiologically fully active spheroplast preparations. There was no enhancement effect of inorganic nitrogen on inorganic carbon accumulation. Membrane preparations exhibited no uptake of inorganic carbon and very little carbonic anhydrase activity, although these membranes were photosynthetically fully competent. DCMU, the inhibitor of photosystem II, eliminated almost entirely the (18)O exchange activity of whole cells in the light. But this effect of DCMU could be reversed by addition of the electron donor couple 3,6-diaminodurene/ascorbate, suggesting the involvement of photosystem I in the events leading to (18)O exchange. Iodoacetamide, an inhibitor of CO(2) fixation, enhanced the (18)O exchange in whole cell suspensions and inhibited neither the uptake of inorganic carbon nor the dehydration of bicarbonate in the light. The proton carrier carbonylcyanide m chlorophenylhydrazone and the inhibitors diethylstilbestrol and N,N' dicyclohexyl carbodiimide affecting the membrane potential, totally abolished (18)O exchange in the light. From (18) O-labeled inorganic carbon experiments we conclude that one of the roles of photosystem I is to provide the active uptake of inorganic carbon into the cells, where carbonic anhydrase catalyzes the interconversion between CO(2) and HCO(3) (-) resulting in the (18)O exchange from inorganic carbon to water. PMID- 16666053 TI - Specific Labeling of the Phosphate Translocator in C(3) and C(4) Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2' -Disulfo-4,4' Diisothiocyano] Diphenylethane). AB - The phosphate translocator protein of C(3) and C(4) mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2 ditritio-1,2-(2,2' -disulfo-4,4' -diisothiocyano) diphenylethane ([(3)H](2) DIDS). Intact mesophyll chloroplasts were isolated from the C(3) plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C(4) plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 mum [(3)H](2)-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [(3)H](2)-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C(3) phosphate translocator) for both C(3) and C(4) chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29 kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C(3) and C(4) phosphate translocator protein(s). PMID- 16666054 TI - Regulation of nonphosphorylating electron transport pathways in soybean cotyledon mitochondria and its implications for fat metabolism. AB - The respiration of mitochondria isolated from germinating soybean cotyledons was strongly resistant to antimycin and KCN. This oxygen uptake was not related to lipoxygenase which was not detectable in purified mitochondria. The antimycin resistant rate of O(2) uptake was greatest with succinate as substrate and least with exogenous NADH. Succinate was the only single substrate whose oxidation was inhibited by salicyl hydroxamic acid alone, indicating engagement of the alternative oxidase. Concurrent oxidation of two or three substrates led to greater involvement of the alternative oxidase. Despite substantial rotenone resistant O(2) uptake with NAD-linked substrates, respiratory control was observed in the presence of antimycin, indicating restriction of electron flow through complex I. Addition of succinate to mitochondria oxidizing NAD-linked substrates in state four stimulated O(2) uptake substantially, largely by engaging the alternative oxidase. We suggest that these properties of soybean cotyledon mitochondria would enable succinate received from the glyoxysome during lipid metabolism to be rapidly oxidized, even under a high cytosolic energy charge. PMID- 16666055 TI - Effects of Elevated CO(2) Concentrations on Glycolysis in Intact ;Bartlett' Pear Fruit. AB - Mature intact ;Bartlett' pear fruit (Pyrus communis L.) were stored under a continuous flow of air or air + 10% CO(2) for 4 days at 20 degrees C. Fruit kept under elevated CO(2) concentrations exhibited reduced respiration (O(2) consumption) and ethylene evolution rates, and remained firmer and greener than fruit stored in air. Protein content, fructose 1,6-bisphosphate levels, and ATP:phosphofructokinase and PPi:phosphofructokinase activities declined, while levels of fructose 6-phosphate and fructose 2,6-bisphosphate increased in fruit exposed to air + 10% CO(2). These results are discussed in light of a possible inhibitory effect of CO(2) at the site of action of both phosphofructokinases in the glycolytic pathway, which could account, at least in part, for the observed reduction in respiration. PMID- 16666056 TI - Efficiency of nodule initiation in cowpea and soybean. AB - When serial dilutions of a suspension of Bradyrhizobium japonicum strain 138 were inoculated onto both soybean and cowpea roots, the formation of nodules in the initially susceptible region of the roots of both hosts was found to be linearly dependent on the log of the inoculum dosage until an optimum dosage was reached. Approximately 30- to 100-fold higher dosages were required to elicit half-maximal nodulation on cowpea than on soybean in the initially susceptible zone of the root. However, at optimal dosages, about six times as many nodules formed in this region on cowpea roots than on soybean roots. There was no appreciable difference in the apparent rate of nodule initiation on these two hosts nor in the number of inoculum bacteria in contact with the root. These results are consistent with the possibility that cowpea roots have a substantially higher threshold of response to symbiotic signals from the bacteria than do soybean roots. Storage of B. japonicum cells in distilled water for several weeks did not affect their viability or efficiency of nodule initiation on soybean. However, the nodulation efficiency of these same cells on cowpea diminished markedly over a 2 week period. These differential effects of water storage indicate that at least some aspects of signal production by the bacteria during nodule initiation are different on the two hosts. Mutants of B. japonicum 138 defective in synthesis of soybean lectin binding polysaccharide were defective in their efficiency of nodule initiation on soybean but not on cowpea. These results also suggest that B. japonicum may produce different substances to initiate nodules on these two hosts. PMID- 16666057 TI - Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases. AB - The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3' nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture. PMID- 16666058 TI - Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants. AB - Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1 phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose 1-phosphate, which interferes with UDP-glucose formation as an analog of glucose 1-phosphate. PMID- 16666059 TI - Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti. AB - Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10(5) to 10(6) bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development. PMID- 16666060 TI - Changes in the Level of [C]Indole-3-Acetic Acid and [C]Indoleacetylaspartic Acid during Root Formation in Mung Bean Cuttings. AB - Changes in the levels of [(14)C]indole-3-acetic acid (IAA) and [(14)C]indole acetylaspartic acid (IAAsp) were examined during adventitious root formation in mung bean (Vigna radiata [L.] R. Wilcz. ;Berken') stem cuttings. IAAsp was identified by GC-MS as the primary conjugate in IAA-treated cuttings. During root formation in IAA-treated cuttings, the level of [(14)C]IAAsp increased rapidly the first day and then declined; [(14)C]IAA was rapidly metabolized and not detected after 12 hours. PMID- 16666061 TI - Cytoplasmic distribution of heat shock proteins in soybean. AB - Previous analyses of the distribution of heat shock (hs) proteins in soybean (Glycine max L. Merr., var Wayne) have demonstrated that a fraction of the low molecular weight hs protein associates with ribosomes during hs. To more specifically characterize the nature of this association, isokinetic centrifugation of ribosomes through sucrose gradients was used to separate monosomes from polysomes. The present analysis demonstrated that hs proteins were bound to polysomes but not monosomes. Treatment of polysomes with puromycin, K(+), and Mg(2+), which caused dissociation of ribosomes into 40S and 60S subunits, also caused dissociation of the hs proteins. Using the procedure of Nover et al. (1983, Mol. Cell Biol, 3: 1628-1655), a hs granule fraction was also isolated. As in tomato cells, hs granules from soybean seedlings contained the low molecular weight hs proteins as a primary component and a number of other non hs proteins of relative molecular mass 30 to 40 kilodaltons and 70 to 90 kilodaltons. On metrizamide gradients they exhibited a buoyant density of 1.20 to 1.21 grams per cubic centimeter, typical of ribonucleoprotein particles. Heat shock granules were characterized as unique cytoplasmic particles based on protein composition and buoyant density. Isopycnic centrifugation of ribosome preparations demonstrated that they contained hs granules, but the hs proteins bound to polysomes were not released by KCI/EDTA treatment. Thus, the polysome bound hs proteins and the granule-bound hs proteins appear to represent two distinct populations of hs proteins in the cytoplasm. Heat shock granules were not distinguishable from ribosomes at the level of resolution used in transmission electron microscopy. PMID- 16666062 TI - Fructan Content and Synthesis in Leaf Tissues of Festuca arundinacea. AB - The concentration of fructan in tall fescue (Festuca arundinacea Schreb.) changes during growth and in response to environment. The objective of this research was to compare the fructan concentration and fructosyl-transferase activity of tall fescue leaf tissues. Expanding leaves, inner and outer sheaths, and expanded blades of greenhouse-grown tall fescue plants were assayed for fructan concentration and fructosyl-transferase activity. Leaf sheaths contained significantly more nonstructural carbohydrate than did the expanded blade. Sheaths also contained a greater percentage of fructan with more than six sugar residues (long chain fructan), than either the expanded blade or expanding leaf. Expanding leaves contained a greater concentration of fructose and oligosaccharides than did sheath or blade tissues. Expanding leaves also had the greatest fructosyl-transferase activity measured either as radiolabel incorporated into fructans in tissue pieces or protein extracts. Activity of fructosyl-transferase was greater in expanding leaf tissue than in sheath tissues. PMID- 16666063 TI - Low bundle sheath carbonic anhydrase is apparently essential for effective c(4) pathway operation. AB - Bundle sheath cells from leaves of a variety of C(4) species contained little or no carbonic anhydrase activity. The proportion of total leaf carbonic anhydrase in extracts of bundle sheath cells closely reflected the apparent mesophyll cell contamination of bundle sheath cell extracts as measured by the proportion of the mesophyll cell marker enzymes phosphoenolpyruvate carboxylase and pyruvate,Pi dikinase. Values of about 1% or less of the total leaf activity were obtained for all three enzymes. The recorded bundle sheath carbonic anhydrase activity was compared with a calculated upper limit of carbonic anhydrase activity that would still permit efficient functioning of the C(4) pathway; that is, a carbonic anhydrase level allowing a sufficiently high steady state [CO(2)] to suppress photorespiration. Even before correcting for mesophyll cell contamination the activity in bundle sheath cell extracts was substantially less than the calculated upper limit of carbonic anhydrase activity consistent with effective C(4) function. The results accord with the notion that a deficiency of carbonic anhydrase in bundle sheath cells is vital for the efficient operation of the C(4) pathway. PMID- 16666064 TI - Quantitative and rapid estimation of h fluxes in membrane vesicles : software for analysis of fluorescence quenching and relaxation. AB - Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na(+)/H(+) antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na(+) -dependent fluorescence changes are derived and can be used for K(m) determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations. PMID- 16666065 TI - Auxin-Stimulated NADH Oxidase Purified from Plasma Membrane of Soybean. AB - NADH oxidation by plasma membrane vesicles purified from hypocotyls of etiolated soybean seedlings by two-phase partition was stimulated 2- to 3-fold by auxins, indole-3-acetic acid, 2,4-dichlorophenoxy acetic acid (2,4-D), and alpha naphthaleneacetic acid. The stimulation was concentration dependent in the presence or absence of detergent with a maximum for 2,4-D at 1 micromolar. The NADH oxidation activity was solubilized with the zwitterionic detergent CHAPS and purified by ion exchange chromatography and gel filtration approximately 2000 fold over the total homogenate. Both the partially purified fraction and an active band from nondenaturing gel electrophoresis revealed the same three bands when analyzed by denaturing gel electrophoresis. When obtained from plasma membrane vesicles from the region of rapid cell elongation, the NADH oxidase complex retained auxin responsiveness throughout purification (3- to 5-fold stimulation by 1 micromolar 2,4-D). PMID- 16666066 TI - Changes in the C-Labeled Cell Wall Components with Chase Time after Incorporation of UDP[C]Glucose by Intact Cotton Fibers. AB - Intact, in vitro-grown cotton fibers will incorporate [(14)C]glucose from externally supplied UDP[(14)C]glucose into a variety of cell wall components including cellulose; this labeled fraction will continue to increase up to 4 hours chase time. In the fraction soluble in hot water there was no significant change in total label; however, the largest fraction after the 30 minute pulse with UDP[(14)C]glucose was chloroform-methanol soluble (70%) and showed a significant decrease with chase. The lipids that make up about 85% of this fraction were identified by TLC as steryl glucosides, acylated steryl glucosides, and glucosyl-phosphoryl-polyprenol. Following the pulse, the loss of label from acylated steryl glucosides and glucosylphophoryl-polyprenol was almost complete within 2 hours of chase; steryl glucosides made up about 85% of the fraction at that chase time. The total loss in the lipid fraction (about 100 picomoles per milligram dry weight of fiber) with chase times of 4 hours approximates the total gain in the total glucans. PMID- 16666067 TI - Expression of a maize storage protein gene in petunia plants is not restricted to seeds. AB - Genes encoding maize seed storage proteins, zeins, are expressed in developing endosperm tissue. To determine whether the DNA sequences controlling the developmental expression of these genes are recognized in dicots, we introduced a gene encoding a M(r) 19,000 zein protein into petunia by Agrobacterium tumefaciens mediated transformation. Southern blot analysis of DNA from regenerated transgenic plants showed that between 1 and 12 copies of the zein gene were integrated at various locations in the petunia genome. S1 nuclease mapping with 5' and 3' probes for zein mRNA showed that transcription of the gene was correctly initiated and terminated in seeds of the transgenic plants. The mRNA was first detected in petunia seeds 10 days after pollination and disappeared 17 days after pollination. However, only small amounts of zein transcripts were synthesized and protein could not be detected at any stage of development. We also found low levels of zein mRNA in leaves, stems, and flowers of the transgenic plants, suggesting that DNA sequences responsible for developmental regulation are not readily recognized in petunia plants. PMID- 16666068 TI - Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil. AB - Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of beta-carotene when grown outdoors under defined conditions. The beta-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all trans beta-carotene and 40% 9-cis beta-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of beta-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9 cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9 cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of beta-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer. PMID- 16666069 TI - The Glycine-Glomus-Rhizobium Symbiosis : VII. Photosynthetic Nutrient-Use Efficiency in Nodulated, Mycorrhizal Soybeans. AB - Four consecutive trifoliate leaves of 56-day-old symbiotic or nonsymbiotic soybean plants were evaluated individually for CO(2) exchange rates (CER), leaf area and dry weight, and leaf N, P, and starch concentrations. Plants had been inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae and Rhizobium japonicum, with either of the endophytes alone, or with neither at time of planting. Plants lacking one or both endophytes received N and/or P fertilizers to produce plants of equal total leaf dry weight in all four treatments. Photosynthetic P-use efficiency (CER per unit leaf P) was higher in the leaves of VAM plants than in P-fertilized plants regardless of the N source (N(2) fixation or combined N). Photosynthetic N-use efficiency was also higher in VAM than in non-VAM plants, but it was affected by the N source, with higher CER in the nodulated plants. The greatest differences in CER, starch accumulation and leaf area were found between the nonsymbiotic plants and those with both endophytes. Statistical evaluations of leaf parameters for treatment or nutrient concentration (N and P) effects between the tri-partite and the nonsymbiotic treatments showed significant changes in concentration of P, but not N, with decreasing leaf age. Both endophytes apparently enhance CO(2) fixation at N and/or P concentrations lower than those of the nonsymbiotic plants. The effects of the endophytes on CO(2) fixation were additive. PMID- 16666070 TI - Root Exudates of Various Host Plants of Rhizobium leguminosarum Contain Different Sets of Inducers of Rhizobium Nodulation Genes. AB - Rhizobium promoters involved in the formation of root nodules on leguminous plants are activated by flavonoids in plant root exudate. A series of Rhizobium strains which all contain the inducible Rhizobium leguminosarum nodA promoter fused to the Escherichia coli lacZ gene, and which differ only in the source of the regulatory nodD gene, were recently used to show that the regulatory nodD gene determines which flavonoids are able to activate the nodA promoter (HP Spaink, CA Wijffelman, E Pees, RJH Okker, BJJ Lugtenberg 1987 Nature 328: 337 340). Since these strains therefore are able to discriminate between various flavonoids, they were used to determine whether or not plants that are nodulated by R. leguminosarum produce different inducers. After chromatographic separation of root exudate constituents from Vicia sativa L. subsp. nigra (L.), V. hirsuta (L.) S.F. Gray, Pisum sativum L. cv Rondo, and Trifolium subterraneum L., the fractions were tested with a set of strains containing a nodD gene of R. leguminosarum, R. trifolii, or Rhizobium meliloti, respectively. It appeared that the source of nodD determined whether, and to what extent, the R. leguminosarum nodA promoter was induced. Lack of induction could not be attributed to the presence of inhibitors. Most of the inducers were able to activate the nodA promoter in the presence of one particular nodD gene only. The inducers that were active in the presence of the R. leguminosarum nodD gene were different in each root exudate. PMID- 16666071 TI - Differential Two-Dimensional Protein Patterns as Related to Tissue Specificity and Water Conditions in Brassica napus var oleifera Root System. AB - Differential two-dimensional protein patterns as related to tissue specificity and water conditions were investigated within Brassica napus var oleifera root system. The different parts of the root system (tap root, lateral roots, and drought-induced short roots) were analyzed under various moisture regimes (regular watering at field capacity, progressive drought stress, and rewatering). Tissue specificity was evident from 25 differences in protein patterns (qualitative and quantitative) between well-watered lateral and tap roots. Twice as many polypeptides (52) were drought-affected and the response to the water stress was shown to be similar in both root types. In addition, more than half of the polypeptides detected as organ-specific were affected by drought. Based upon the trend of variation observed under drought and rehydration, three categories of polypeptides could be defined that might be differently involved in drought susceptibility or tolerance. A highly differentiated protein pattern characterized the drought-induced short roots. This pattern appeared as far from the watered as from the water-stressed normal roots. In particular, 13 unique polypeptides were detected which could be relevant to their adaptive morphogenesis and/or their specific drought tolerance induction. Upon rehydration, their polypeptide pattern and their specific morphology returned to a normal well-watered lateral root type. PMID- 16666072 TI - Indole-3-acetic Acid oxidation and crocin bleaching by horseradish peroxidase. AB - During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory. PMID- 16666073 TI - Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe. AB - Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I(-), ClO(3) (-), NO(3) (-), Br(-), or SCN(-), acridine orange reported lower pH gradients than either quinacrine or [(14)C]methylamine. Acridine orange, but not quinacrine, reduced [(14)C]methylamine accumulation when NO(3) (-) was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO(3) (-) increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24 degrees C than at 12 degrees C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H(+), possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO(3) (-)/H(+) symport involved in the export of NO(3) (-) from the vacuole is probably an artefact caused by acridine orange. PMID- 16666074 TI - Translational alterations in maize leaves responding to pathogen infection, paraquat treatment, or heat shock : polysome dissociation and accumulation of a 57 kilodalton protein. AB - Translational alterations occur in maize (Zea mays L.) leaves stressed by pathogen infection or herbicide paraquat treatment. These translational changes include: (a) dissociation of large polysomes to small polysomes, monosomes, and subunits; (b) a decreased rate of total protein synthesis; and (c) a reduced synthesis of several proteins by polysomes in vitro. The polysome dissociation was neither due to an extraction artifact nor to degradation of RNA by RNase. The protein patterns of polysomes isolated from leaves inoculated with Bipolaris maydis at 6 to 48 hours showed an increase in the intensity of a 57 kilodalton protein. When inoculated with less virulent pathogens, such as B. zeicola, Exserohilum turcicum, or Colletotrichum graminicola, the protein was accumulated in polysomes of leaves at 24 to 48 hours after inoculation. The 57 kilodalton protein was also accumulated in polysomes of maize leaves responding to heat shock or herbicide paraquat treatments. The purified 57 kilodalton protein reassociated with polysomes isolated from healthy leaves and inhibited polysomal translation in vitro. Since the 57 kilodalton protein is rapidly accumulated in maize polysomes in response to various biological and environmental stresses and may affect protein synthesis, it may be involved in translational regulation of maize leaves during stress response. PMID- 16666075 TI - Cyclohexanedione Herbicides Are Selective and Potent Inhibitors of Acetyl-CoA Carboxylase from Grasses. AB - Biochemical studies of plant species susceptible to the cyclohexanedione herbicides, alloxydim, sethoxydim, and clethodim, have demonstrated that these selective grass herbicides inhibit acetyl-coenzyme A carboxylase, the second enzyme common to both fatty acid and flavonoid biosynthetic pathways. The K(i)s for the cyclohexanediones tested ranged from 0.02 to 1.95 micromolar, depending on the species. The enzyme isolated from broadleaf plants was much less sensitive to inhibition by these herbicides (K(i)s from 53 micromolar to 2.2 millimolar). These results may explain the mechanism of action of these herbicides and their selectivity for monocotyledonous species. PMID- 16666076 TI - Missense Mutation in the Chlamydomonas Chloroplast Gene that Encodes the Rubisco Large Subunit. AB - The 69-12Q mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase activity, but retains holoenzyme protein. It results from a mutation in the chloroplast large-subunit gene that causes an isoleucine-for-threonine substitution at amino-acid residue 173. Considering that lysine-175 is involved in catalysis, it appears that mutations cluster at the active site. PMID- 16666077 TI - Localization of mRNAs for the Small and Large Subunits of Rubisco Using Electron Microscopic in Situ Hybridization. AB - In situ hybridization coupled with electron microscopy has been used to locate mRNAs for the small and large subunits of ribulose 1,5-bisphosphate carboxlase in young leaf tissue of tobacco (Nicotiana tabacum L.) plants. The endogeneous mRNAs were hybridized with either a biotinylated DNA probe for the small subunit or large subunit and subsequently visualized using avidin-ferritin conjugates at the electron microscope level. In the tissue incubated with the small subunit cDNA probe, the cytoplasm was uniformly labeled with ferritin indicating the presence of the target mRNA; this was particularly visible in cells which had under-gone some structural damage. In the case of the LSU probe, the ferritin marker was shown to be exclusively associated with the plastid stroma in intact leaf cells. The compartmentation of cytoplasmic small subunit mRNA versus plastid large subunit mRNA has been confirmed by direct visualization of in situ hybridization. PMID- 16666078 TI - Receptor-Mediated Endocytosis in Plants is Energetically Possible. AB - A detailed examination of the arguments of Cram (1980 New Phytol 84: 1-17) against the uptake of major nutrients in plants by endocytosis shows that the arguments do not exclude the possibility of receptor-mediated endocytosis. PMID- 16666079 TI - N-terminus conservation in the terminal pigment of phycobilisomes from a prokaryotic and eukaryotic alga. AB - High molecular weight polypeptides from phycobilisomes, believed to be involved in facilitating the energy flow from phycobilisomes to thylakoids, are conserved in the prokaryote Nostoc sp. and the eukaryote Porphyridium cruentum. Partial N terminal sequence analysis of the phycobilisome-polypeptides of Nostoc (94 kilodalton) and Porphyridium (92 kilodalton) revealed 55% identity in the first 20 residues, but no significant homology with sequences of other phycobiliproteins or phycobilisome-linkers. Polypeptides (94 and 92 kilodalton) from Nostoc thylakoids free of phycobilisomes, previously presumed to be involved in the phycobilisome-thylakoid linkage (M Mimuro, CA Lipschultz, E Gantt 1986 Biochim Biophys Acta 852: 126) exhibit the same immunocrossreactivity but are different from the 94 kilodalton-phycobilisome polypeptide by having blocked N termini and a different amino acid composition. PMID- 16666080 TI - Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles. AB - A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H(+) co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies. PMID- 16666081 TI - Superoxide free radicals are produced in glyoxysomes. AB - The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O(2) (-) radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O(2) (-) radicals in glyoxysomes by endogenous metabolites suggests new active oxygen related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism. PMID- 16666082 TI - Evidence for a Na/H Antiporter in Membrane Vesicles Isolated from Roots of the Halophyte Atriplex nummularia. AB - The ATP-dependent establishment of a positive membrane potential (measured as S(14)CN(-)-accumulation) in membrane vesicles isolated from the roots of Atriplex nummularia Lindl. was not inhibited by NaMes and KMes at concentrations up to 140 millimolar. On the other hand, the formation of DeltapH (measured as (14)C methylamine accumulation or quenching of quinacrine fluorescence), was depressed by NaMes concentrations as low as 30 millimolar. Supply of NaMes after the DeltapH had been established brought about partial dissipation within 30 seconds. Extent of dissipation of DeltapH increased with NaMes concentration over the range tested (up to 180 millimolar). The H(+)/Na(+) exchange indicated by these results was not due to the creation of a Na(+) diffusion potential. Formation of DeltapH in these vesicles was stable to NO(3) (-) up to 100 millimolar; further, the dissipating effect of Na(+) supply was apparent on a DeltapH formed in the presence of 30 millimolar NO(3) (-). Additional evidence that the origin of the membrane vesicles observed in this investigation was not the tonoplast and was probably the plasmalemma included the vanadate sensitivity of the establishment of the membrane potential. PMID- 16666083 TI - Oxaloacetate and malate transport by plant mitochondria. AB - The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinlanderin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10( 3). From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport. PMID- 16666084 TI - Pressure probe technique to study transpiration in phycomyces sporangiophores. AB - The growth equation for the rate of water uptake is augmented with a transpiration term. The obtained augmented growth equations are used to develop methodology which employs the pressure probe to measure transpiration rates from single plant cells. Experiments are conducted on the sporangiophores of Phycomyces blakesleeanus to demonstrate this technique. PMID- 16666085 TI - Toxic Effects of Copper on Photosystem II of Spinach Chloroplasts. AB - The room temperature fluorescence induction of chloroplasts was utilized as a probe to locate the site of inhibition on PSII by copper. It was found that, while the initial fluorescence yield was hardly affected, the variable fluorescence yield was lowered without significant change in its kinetics. Addition of DCMU, or abolishing oxygen evolution capability by Tris treatment, did not alter this basic inhibition pattern. Copper was also found to lower the fluorescence yield of chloroplasts treated with linolenic acid which inhibited the secondary electron transport on both oxidizing and reducing sides of PSII. The data indicate that copper adversely affects the primary charge separation at the PSII reaction center. We suggest that the inhibition is due to creation of a lesion close to the reaction center, leading to increased dissipation of incoming excitation energy to heat. PMID- 16666086 TI - Correlated induction of nitrate uptake and membrane polypeptides in corn roots. AB - Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the (35)S methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane. PMID- 16666087 TI - Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots. AB - Proton-translocating ATPase (H(+)-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37 degrees C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H(+) ATPase, probably at the catalytic site. PMID- 16666088 TI - A preliminary analysis of Fatty Acid synthesis in pea roots. AB - Subcellular fractions from pea (Pisum sativum L.) roots have been prepared by differential centrifugation techniques. Greater than 50% of the recovered plastids can be isolated by centrifugation at 500g for 5 minutes. Plastids of this fraction are largely free from mitochondrial and microsomal contamination as judged by marker enzyme analysis. De novo fatty acid biosynthesis in pea roots occurs in the plastids. Isolated pea root plastids are capable of fatty acid synthesis from acetate at rates up to 4.3 nanomoles per hour per milligram protein. ATP, bicarbonate, and either Mg(2+) or Mn(2+) are all absolutely required for activity. Coenzyme A at 0.5 millimolar improved activity by 60%. Reduced nucleotides were not essential but activity was greatest in the presence of 0.5 millimolar of both NADH and NADPH. The addition of 0.5 millimolar glycerol 3-phosphate increased activity by 25%. The in vitro and in vivo products of fatty acid synthesis from acetate were primarily palmitate, stearate, and oleate, the proportions of which were dependent on experimental treatments. Fatty acids synthesized by pea root plastids were recovered in primarily phosphatidic acid and diacylglycerol or as water soluble derivatives and the free acids. Lesser amounts were found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and monogalactosyldiacylglycerol. PMID- 16666089 TI - A developmentally regulated hydroxyproline-rich glycoprotein in maize pericarp cell walls. AB - We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell walls of developing kernels were fractionated on a CsCl density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed. PMID- 16666090 TI - Plasmalemma redox activity in the diatom thalassiosira: a possible role for nitrate reductase. AB - Plasmalemma redox activity in the diatom Thalassiosira is competitively inhibited by antiserum prepared against algal nitrate reductase (NR), and fluorescent labeling experiments reveal the binding of NR antiserum to the cell surface. Furthermore, the external electron acceptor Cu bathophenanthroline disulfonate causes immediate inhibition of intracellular primary amine production. A model is proposed in which plasmalemma-bound nitrate reductase reduces extracellular electron acceptors and intracellular nitrate and also acts as a trans-plasmalemma proton pump. PMID- 16666091 TI - Ammonia Production and Assimilation in Glutamate Synthase Mutants of Arabidopsis thaliana. AB - Ammonia production and assimilation(1) were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH(4) (+) continued to accumulate. The accumulation of NH(4) (+) was also seen after an initial lag of 30 minutes in 2% O(2), 350 microliters per liter of CO(2) and after 90 minutes in 2% O(2), 900 microliters per liter of CO(2). The accumulation of NH(4) (+) in normal air and low O(2) was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O(2), 1% CO(2) in the light, the NH(4) (+) which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH(4) (+) in glutamate synthase mutants and prevented the reassimilation of NH(4) (+) upon return to the dark. The addition of MSO also resulted in the accumulation of NH(4) (+) in glutamate synthase mutants in the light and in 21% O(2), 1% CO(2). These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH(4) (+) and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH(4) (+) by a GS/NADH-GluS cycle. PMID- 16666092 TI - Cytokinin Secretion by Frankia sp. HFP ArI3 in Defined Medium. AB - Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N(6)- (Delta(2)-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography mass spectrometry, at levels of approximately 1 ng/ml culture medium. PMID- 16666093 TI - Dark Respiration during Photosynthesis in Wheat Leaf Slices. AB - The metabolism of [(14)C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-(14)C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO(2). An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after (14)CO(2) production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of (14)CO(2) over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO(2) as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO(2) was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the (14)CO(2) release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark. PMID- 16666094 TI - Development of accelerated net nitrate uptake : effects of nitrate concentration and exposure time. AB - Upon initial nitrate exposure, net nitrate uptake rates in roots of a wide variety of plants accelerate within 6 to 8 hours to substantially greater rates. Effects of solution nitrate concentrations and short pulses of nitrate (3, 1-->4)-beta-Glucan 4 Glucanohydrolases. AB - Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1-->3, 1-->4)-beta-glucanase isoenzyme II. It is shown that one of the barley (1-->3, 1-->4)-beta-glucanase genes is located on chromosome 1. PMID- 16666138 TI - Spectral, Photophysical, and Stability Properties of Isolated Photosystem II Reaction Center. AB - Photosystem II reaction center (RC) preparations isolated from spinach (Spinacea oleracea) by the Nanba-Satoh procedure (O Nanba, K Satoh 1987 Proc Natl Acad Sci USA 84: 109-112) are quite labile, even at 4 degrees C in the dark. Simple spectroscopic criteria were developed to characterize the native state of the material. Degradation of the RC results in (a) blue-shifting of the red-most absorption maximum, (b) a shift of the 77 K fluorescence maximum from approximately 682 nm to approximately 670 nm, and (c) a shift of fluorescence lifetime components from 1.3-4 nanoseconds and >25 nanoseconds to approximately 6 7 nanoseconds. Fluorescence properties at 77 K seem to be a more sensitive spectral indicator of the integrity of the material. The >25 nanosecond lifetime component is assigned to P680(+) Pheophytin(-) recombination luminescence, which suggests a correlation between the observed spectral shifts and the photochemical competence of the preparation. Substitution of lauryl maltoside for Triton X-100 immediately after RC isolation stabilizes the RCs and suggests that Triton may be responsible for the instability. PMID- 16666139 TI - DNA and RNA Levels in Bundle Sheath and Mesophyll Cells of Pearl Millett (Pennisetum americanum). AB - The DNA content of bundle sheath cells and mesophyll protoplasts from the C(4) plant pearl millet (Pennisetum americanum, Tift 23DB) was determined by microspectrophotometry to be 1.8 to 2.3 and 3.2 to 4.0 picograms/nucleus, respectively. Measurement of RNA by ultraviolet spectroscopy indicated that bundle sheath cells contain twice as much RNA as mesophyll cells. PMID- 16666140 TI - Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway. AB - We have used (13)C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of (13)C between carbons atoms 1 and 6 of [1-(13)C] or [6 (13)C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the (13)C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the (13)C sugars were supplied in vivo to the intact plant. The (13)C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of (13)C in all glucosyl carbons was quantified by (13)C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background (13)C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6 phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues. PMID- 16666141 TI - P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina. AB - The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy. The (13)C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T(1) relaxation rates of the (13)C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The (31)P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in (31)P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites. PMID- 16666142 TI - Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection. AB - Chitinase and beta-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv "Dot") pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were beta-1,3 glucanases. The different molecular forms of chitinase and beta-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and beta-1,3 glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and beta-1,3 glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both beta-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [(3)H]chitin, as well as in their relative lysozyme activity. Similarly, the two beta-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin. PMID- 16666143 TI - Haloxyfop Inhibition of the Pyruvate and the alpha-Ketoglutarate Dehydrogenase Complexes of Corn (Zea mays L.) and Soybean (Glycine max [L.] Merr.). AB - The grass-specific herbicide haloxyfop, ((+/-)-2-[4-((3-chloro-5 (trifluoromethyl)-2-pyridinyl)oxy)-phenoxy] propionic acid) has been shown to inhibit lipid synthesis and respiration, to cause the accumulation of amino acids, and not to affect cellular sugar or ATP levels. Thus studies were carried out with enzyme activities from corn (Zea mays L.) (haloxyfop sensitive) and soybean (Glycine max [L.] Merr.) (haloxyfop tolerant) to locate the possible inhibition sites among the glycolytic and tricarboxylic acid (TCA) cycle enzymes. Following along the oxidative metabolism pathway of sugars, the pyruvate dehydrogenase complex (PDC) was the first enzyme among the glycolytic enzymes that demonstrated noticeable inhibition by 1 millimolar haloxyfop. Kinetic studies with corn and soybean PDC from both purified etioplasts and mitochondria gave K(i) values of from 1 to 10 millimolar. Haloxyfop also inhibited the activity of the TCA cycle enzyme, the alpha-ketoglutarate dehydrogenase complex (alpha-KGDC) which carries out the same reaction as PDC except for the substitution of alpha-ketoglutarate for pyruvate as one of the substrates. The K(i) values were somewhat lower in this case (near 1 millimolar). The relatively high K(i) values for both enzyme complexes would indicate that these may not be the herbicidal sites of inhibition, but it is possible that the herbicide could be concentrated in compartments and/or the substrate concentrations may be well below optimal. Likewise little difference was seen in the haloxyfop inhibition of the enzyme activities from the sensitive species, corn, and from the tolerant species, soybean, so the selectivity of the herbicide is not evident from these results. The inhibition of the PDC and alpha-KGDC as the mode of action of haloxyfop is, however, consistent with the observed physiological effects of the herbicide, and these are the only enzymic activities so far found to be sensitive to haloxyfop. PMID- 16666144 TI - Formation of UDP-Xylose and Xyloglucan in Soybean Golgi Membranes. AB - Soybean (Glycine max) membranes co-equilibrating with Golgi vesicles in linear sucrose gradients contained UDP-glucuronate carboxy-lyase and xyloglucan synthase activities. Digitonin solubilized and increased the activity of the membrane bound UDP-glucuronate carboxy-lyase. UDP-xylose did not inhibit the transport of UDP-glucuronate into the lumen of Golgi vesicles but repressed the decarboxylation of the translocated UDP-glucuronate. The results suggest that UDP glucuronate is transported into the vesicles by a specific carrier and decarboxylated to UDP-xylose within the lumen. On incubation of UDP [(14)C]glucuronate with Golgi membranes in the presence of UDP-glucose, [(14)C]xylose-labeled xyloglucan was formed. Although the K(m) value of UDP glucuronate for the decarboxylation was 240 micromolar, the affinity of UDP glucuronate for xyloglucan formation (31 micromolar) was similar to that of UDP xylose (28 micromolar), suggesting a high turnover of UDP-xylose. The biosynthesis of UDP-xylose from UDP-glucuronate probably occurs in Golgi membranes, where xyloglucan subsequently forms from UDP-xylose and UDP-glucose. PMID- 16666145 TI - Response of Two Wheat Cultivars to CO(2) Enrichment under Subambient Oxygen Conditions. AB - Two cultivars of wheat (Triticum aestivum L. cvs Sonoita and Yecora Rojo) were grown to maturity in a growth chamber within four sub-chambers under two CO(2) levels (350 or 1000 microliters per liter) at either ambient (21%) or low O(2) (5%). Growth analysis was used to characterize changes in plant carbon budgets imposed by the gas regimes. Large increases in leaf areas were seen in the low O(2) treatments, due primarily to a stimulation of tillering. Roots developed normally at 5% O(2). Seed development was inhibited by the subambient O(2) treatment, but this effect was overcome by CO(2) enrichment at 1000 microliters per liter. Dry matter accumulation and seed number responded differently to the gas treatments. The greatest dry matter production occurred in the low O(2), high CO(2) treatment, while the greatest seed production occurred in the ambient O(2), high CO(2) treatment. Growth and assimilation were stimulated more by either CO(2) enrichment or low O(2) in cv Yecora Rojo than in Sonoita. These experiments are the first to explore the effect of whole plant low O(2) treatments on growth and reproduction. The finding that CO(2) enrichment overcomes low O(2)-induced sterility may help elucidate the nature of this effect. PMID- 16666146 TI - Salinity affects intracellular calcium in corn root protoplasts. AB - Previous work with the fluorescent Ca probe chlorotetracycline (CTC) showed that salinity displaces Ca from membranes of root cells. Using a variety of indirect approaches, we studied whether salinity displaces Ca from the cell surface or from internal membranes of corn (Zea mays L. cv Pioneer 3377) root protoplasts. Preloading the cells with supplemental Ca counteracted subsequent NaCl effects on CTC fluorescence. CTC quenching by exogenous EGTA was not competitive with CTC quenching by NaCl. The Ca channel reagent (+)-202-791 had significant interactions with the effect of NaCl on CTC fluorescence. The effect of NaCl on CTC fluorescence was attenuated by pretreatment with Li, but was restored by inositol. Salinity increased Na influx, decreased Ca influx, and increased Ca efflux from the cells. Fluorescence anisotropy indicated that NaCl decreased the fluidity of the external face of the plasmalemma but increased the fluidity of cell membranes in general. Our results suggest that salinity displaces Ca associated with intracellular membranes through activation of the phosphoinositide system and depletion of intracellular Ca pools. PMID- 16666147 TI - Hierarchical Response of Light Harvesting Chlorophyll-Proteins in a Light Sensitive Chlorophyll b-Deficient Mutant of Maize. AB - The light-sensitive chlorophyll b (Chl b)-deficient oil yellow-yellow green (OY YG) mutant of maize (Zea mays) grown under conditions of high light exhibits differential reductions in the accumulation of the three major Chl b-containing antenna complexes and characteristic changes in thylakoid architecture. When observed by freeze-fracture electron microscopy, the most notable changes in the OY-YG thylakoid structure are: (a) a major reduction in the number of 8 nanometer particles of the protoplasmic fracture face of stacked membrane regions (PFs) paralleled by a 60% reduction in the chlorophyll-proteins (CP) associated with the peripheral light harvesting complex (LHCII) for photosystem II (PSII) and which give rise to the LHCII oligomer/monomer (CPII(*)/CPII) bands on mildly dissociated green gels; (b) a sizable decrease in the proportion of 11 to 13 nanometer particles of the protoplasmic fracture face of unstacked membrane regions (PFu) that parallels the loss of light harvesting complex I (LHCI) antennae from photosystem I (PSI) centers and a 40% reduction of the band containing CP1 and LHCI (CPI(*)) on mildly dissociating green gels; (c) an unchanged or slightly increased average size of particles of the exoplasmic fracture face of stacked (or appressed) membrane regions (EFs) along with a relative increase in CP29, the postulated bound LHC of PSII, and of CP47 and CP43, PSII core antenna complexes. This latter result sets the OY-YG mutant apart from all other Chl b-deficient mutants studied to date, all of which possess EFs particles that are substantially reduced in size. Based on these findings, we postulate that the bound LHCII associated with EFs particles consists mostly of CP29 chlorophyll proteins and very little, if any, CPII(*)/CPII chlorophyll proteins. Indeed, the CPII(*)/CPII chlorophyll proteins may be exclusively associated with the ;peripheral' LHCII units that give rise to 8 nanometer PF particles. The differential effect of the Chl b deficiency on the accumulation of the three main antenna complexes (CPII(*)/CPII>CPI(*)>CP29) suggests, furthermore, that there is a hierarchy among Chl b-binding proteins, and that this hierarchy might be an integral part of long-term photoregulation mediating Chl b partitioning in the chloroplast. PMID- 16666148 TI - Compensatory Alterations in the Photochemical Apparatus of a Photoregulatory, Chlorophyll b-Deficient Mutant of Maize. AB - Characterization of the functional organization of the photochemical apparatus in the light sensitive chlorophyll b-deficient oil yellow-yellow green (OY-YG) mutant of maize (Zea mays) is presented. Spectrophotometric and kinetic analysis revealed substantially lower amounts of the light harvesting complex of photosystem II (LHCII-peripheral) in high light-grown OY-YG thylakoids. However, accumulation of a tightly bound LHCII appears unaffected by the lesion. Changes in photosystem (PS) stoichiometry include lower amounts of PSII with characteristic fast kinetics (PSII(alpha)) and a substantial accumulation of PSII centers with characteristic slow kinetics (PSII(beta)) in the thylakoid membrane of the OY-YG mutant. Thus, PSII(beta) is the dominant photosystem in the mutant chloroplasts. In contrast to wild type, roughly 80% of the mutant PSII(beta) centers are functionally coupled to the plastoquinone pool and are probably localized in the appressed regions of the thylakoid membrane. These centers, designated PSII(beta)-Q(B)-reducing (Q(B) being the secondary electron quinone acceptor of PSII), are clearly distinct from the typical PSII(beta)-Q(B) nonreducing centers found in the stroma lamellae of wild-type chloroplasts. It is concluded that the observed changes in the stoichiometry of electron-transport complexes reflect the existence of a regulatory mechanism for the adjustment of photosystem stoichiometry in chloroplasts designed to correct any imbalance in light absorption by the two photosystems. PMID- 16666149 TI - Isolation and Characterization of the Amyloplast Envelope-Membrane from Cultured White-Wild Cells of Sycamore (Acer pseudoplatanus L.). AB - To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi translocator, the amount of this carrier-protein (31-kilodalton) in the white wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes. PMID- 16666150 TI - Differential inhibition and activation of two leaf dihydroxyacetone phosphate reductases : role of fructose 2,6-bisphosphate. AB - The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH(4))(2)SO(4). Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C(3) photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis. PMID- 16666151 TI - Effect of the electrochemical proton gradient and anions on the ATPase activity of soybean submitochondrial particles. AB - Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38 degrees C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg ATP plus phosphate. Under the latter conditions, collapse of DeltamuH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by DeltamuH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles. PMID- 16666152 TI - Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts. AB - When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La(3+)) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl(2)), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La(3+) -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La(3+) -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La(3+) after washing without EDTA and released most of the bound La(3+) by washing with 1 millimolar EDTA. This tightly bound La(3+) seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La(3+) -treated protoplasts after washing. zeta-potentials of protoplasts were -39.0+/-3.2 millivolts, -16.7 +/- 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La(3+) (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 +/- 2.2 millivolts) in the presence of 1 millimolar La(3+). These effects of La(3+) on zeta-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La(3+) exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La(3+) were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently. PMID- 16666153 TI - RuBP Limitation of Photosynthetic Carbon Fixation during NH(3) Assimilation : Interactions between Photosynthesis, Respiration, and Ammonium Assimilation in N Limited Green Algae. AB - The effects of ammonium assimilation on photosynthetic carbon fixation and O(2) exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O(2) evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light. PMID- 16666154 TI - Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts. AB - The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN(3) or 0.5 millimolar Cu(2+). In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu(2+), was insensitive to inhibition by NaN(3). The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide insensitive component, which may be due to complexation with organic counterions and phenolics. PMID- 16666155 TI - Effect of Restricted Root Growth on Carbohydrate Metabolism and Whole Plant Growth of Cucumis sativus L. AB - The effects of varied rooting volumes on root growth and source leaf carbohydrate metabolism were studied in greenhouse-grown cucumber (Cucumis sativus L cv Calypso) plants. Plants were grown for 7 weeks in container volumes that ranged from 0.4 to 5.9 liters. Plants grown in the smaller containers exhibited less leaf expansion, lower root and shoot weight, and fewer lateral stems than plants grown in the 5.9 liter containers. Shoot/root ratio was not altered by the container volume, suggesting coordination of root and shoot growth due to rooting volume. Source leaf carbon exchange rates, assimilate export rates, and starch accumulation rates for plants grown in 0.4 liter containers were approximately one-half or less in comparison to those for plants grown in 5.9 liter containers. Starch concentrations per unit leaf area were maintained at high levels in source leaves of plants grown in 0.4 liter containers over the entire day/night cycle. Lower extractable galactinol synthase activities and higher galactinol concentrations occurred in leaves of plants grown in 0.4 liter container volumes. The reduced sink demand, induced by restricted root growth, may have led to increased starch concentrations and to a reduction in stachyose biosynthesis in cucumber source leaves. PMID- 16666156 TI - Experimental Studies on Lateral Root Formation in Radish Seedling Roots: II. Analysis of the Dose-Response to Exogenous Auxin. AB - Application of indoleacetic acid (IAA) and other auxins causes cultured radish (Raphanus sativus L. ;Scarlet Globe') seedling root segments to produce an increased frequency (FR, no. cm(-1)) of lateral roots (LR); in the absence of auxin, segments spontaneously form about 6 LR cm(-1). A dose-response study has revealed that the increase in FR follows a biphasic Michaelis-Menten relationship with the medium concentration of the undissociated form of IAA ([IAAH](m)). The fitted curve for phase I has a maximum response level (R(max)) of 5.2 LR per centimeter above the spontaneous FR; the [IAAH](m) giving half-maximal response (C(1/2)) is 21 nanomolar. For phase II, the values for R(max) and C(1/2) are 56 LR per centimeter and 11 micromolar, respectively. The response is variable in the transition concentration region between the two phases; in that region (but not, or much less commonly, at higher or lower [IAAH](m)), LR initiation may resume or continue after the first day. At and above 100 micromolar [IAAH](m), the roots are hyperstimulated and generally fail to respond. The developmental stage of LR formed in medium with very low [IAAH](m) (10 nanomolar) is enhanced compared to LR formed in medium lacking auxin; the stage is diminished at higher auxin levels, in inverse correlation with FR. Trends in the responses to NAA and IBA were similar, but NAA required only 0.03 times the dose of IAA, while IBA required 6 times the dose of IAA. These findings may be of use in a search for possible auxin receptors involved with LR initiation. PMID- 16666157 TI - Glycoprotein synthesis in maize endosperm cells: the nucleoside diphosphate sugar: dolichol-phosphate glycosyltransferases. AB - Microsomal membrane preparations from maize (Zea mays L., inbred A636) endosperm cultures contained enzymes that transferred sugar moieties from uridine diphosphate-N-acetylglucosamine, guanosine diphosphate-mannose, and uridine diphosphate-glucose to dolichol-phosphate. These enzyme activities were characterized with respect to detergent and pH optima, substrate kinetic constants, and product and antibiotic inhibition constants. It was demonstrated by mild acid hydrolysis and high performance liquid chromatography that the products of the N-acetylglucosamine transferases were N-acetylglucosamine pyrophosphoryl-dolichol and N,N'-diacetyl-chitobiosyl-pyrophosphoryl-dolichol and that the product of the mannose transferase was mannosyl-phosphoryl-dolichol. A large proportion of the products of the glucose transferase activity was stable to mild acid hydrolysis. However, the proportion that was labile was identified as glucosyl-phosphoryl-dolichol. Rate zonal sedimentation and isopycnic banding in linear sucrose density gradients in the presence of 1 millimolar ethylenediaminetetraacetic acid indicated that the glycosyltransferase activities were located in the endoplasmic reticulum. The glycosyltransferases were not solubilized by 500 millimolar KCl or by sequential washes with tris (hydroxymethyl)aminomethane and water, treatments that release peripheral membrane proteins. Solubilization was achieved with low concentrations of Triton X-100. When sealed microsomal vesicles were incubated with trypsin for 30 minutes in absence of detergent, the activity of N-acetylglucosaminyl-transferase was substantially reduced, while the activity of the glucosyl-transferase was somewhat reduced. Activity of the mannosyl-transferase was resistant to inactivation by incubation with trypsin unless Triton was present. PMID- 16666158 TI - Nonaqueous purification of maize mesophyll chloroplasts. AB - A nonaqueous fractionation method to obtain highly purified mesophyll chloroplasts from lyophilized leaves of Zea mays L. is described. The levels of several metabolites including pyruvate were determined in the purified mesophyll chloroplast fractions which were prepared from leaves exposed to different light intensities. The role of pyruvate in the regulation of pyruvate,Pi dikinase in these chloroplasts under different light intensities is discussed. PMID- 16666159 TI - Analysis of mRNAs that Accumulate in Response to Low Temperature Identifies a Thiol Protease Gene in Tomato. AB - We have studied the induction of gene expression at low temperature by cloning mRNAs that accumulate when unripe tomato (Lycopersicon esculentum) fruit are incubated at 4 degrees C. Two cloned mRNAs, C14 and C17, accumulate relatively rapidly in response to cold treatment, while a third, C19, displays a delayed response. Significant levels of these mRNAs were not detected during fruit ripening at normal temperature. We have analyzed gene expression at different temperatures and detect half-maximal accumulation of the C14 and C17 mRNAs at 16 degrees C and 11 degrees C, respectively, and have observed that sustained gene expression requires continuous cold treatment. Furthermore, the level of C14 and C17 gene expression in cold-tolerant (hybrid L. esculentum/Lycopersicon pimpinellifolium) fruit is different from that in cold-sensitive (L. esculentum) fruit. DNA sequence analysis indicates that the C14 mRNA encodes a polypeptide with a region that is homologous to the plant thiol proteases actinidin and papain and to the animal thiol protease cathepsin H. We conclude from these experiments that low temperature selectively induces the expression of specific genes and that one such gene encodes a thiol protease. PMID- 16666160 TI - Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii. AB - The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism. PMID- 16666161 TI - Role of Magnesium in the Binding of Substrate and Effectors to Phosphoenolpyruvate Carboxylase from a CAM Plant. AB - The binding of phosphoenolpyruvate, malate, and glucose 6-phosphate to phosphoenolpyruvate carboxylase purified from Crassula argentea Thunb. was measured using both the intrinsic tryptophan fluorescence of the enzyme and the extrinsic fluorescence of the complex of 8-anilino-1-napthalenesulfonate with the enzyme. It was found that the substrate phosphoenolpyruvate can bind in the absence of magnesium but is bound in greater quantities and more tightly when magnesium is present. Malate reduces the binding of phosphoenolpyruvate, while glucose 6-phosphate increases the binding of the substrate. Glucose 6-phosphate requires magnesium to bind to the enzyme, while malate does not. The general trends from the binding experiments using fluorescence methods were confirmed by activity determinations using assays performed in the absence of magnesium. PMID- 16666162 TI - Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate. AB - Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of alpha-amino-n butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate. PMID- 16666163 TI - Stimulation of CAM Photosynthesis in Kalanchoe blossfeldiana by Transferring to Nitrogen-Deficient Conditions. AB - Kalanchoe blossfeldiana Poelln. cv Hikan plants were grown hydroponically with nutrient solution containing 5 millimolar NO(3) (-) (or NH(4) (+)) for 1 to 2 months and then transferred to nutrient solution containing no nitrogen. CO(2) uptake at night, nocturnal increase in titratable acidity, and activity of phosphoenolpyruvate carboxylase increased after the transfer. Thus, transfer to nitrogen-deficient conditions stimulates Crassulacean acid metabolism (CAM photosynthesis) in K. blossfeldiana. The importance of the plant nitrogen status (nitrogen-withdrawal status) for induction and stimulation of CAM photosynthesis is discussed. PMID- 16666164 TI - Characteristics of Photosynthate Partitioning during Chloroplast Development in Avena Leaves. AB - The first leaves (40 millimeters long) of 4-day-old light-grown Avena sativa L. cv Victory I seedlings contained a complete age sequence of cells from the base to the tip, and within these tissues all stages of chloroplast development could be observed. Although chloroplasts underwent progressive development, a marked increase in number of thylakoids per granum, in chloroplast volume, and in chlorophyll content occurred in the region between 20 and 30 millimeters from the base. Photosynthetic CO(2) fixation (per unit chlorophyll) increased markedly during chloroplast development and closely followed structural changes in chloroplasts. It was also found that the partitioning of photosynthates differed greatly in the segment from 30 to 40 millimeters (at the tip of the leaf) compared with the segment nearer to the leaf base, although both total (14)CO(2) fixation and chlorophyll content per segment did not change significantly along the length of the leaves. As the thylakoid system reached full maturation, partitioning of photosynthates into sucrose increased but partitioning decreased into starch, lipids, and phosphorylated intermediates. PMID- 16666165 TI - Interrelationship of Polyamine and Ethylene Biosynthesis during Avocado Fruit Development and Ripening. AB - Concentrations of polyamines (PA) and the activities of the PA-synthesizing enzymes ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) extracted from the mesocarp tissue of avocado (Persea americana Mill, cv ;Simmonds') fruits at different stages of development were compared with DNA content and the activities of 5'-methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase. Putrescine, spermidine, and spermine were at their peak concentrations during the early stages of fruit development (362, 201, and 165 nanomoles per gram fresh weight, respectively, at 15 days from full bloom), then declined to 30% or less at full maturity. Agmatine showed only a slight change in concentration throughout the fruit development. The activity of ODC, which was low during flowering (8 nmoles per milligram protein per hour), increased more than threefold during the first 2 months then declined at the later stages of fruit development, while ADC activity showed only a slight increase. DNA content followed a similar pattern of change as that of PA and ODC. The decline in DNA and ODC activity suggest a lack of correlation between cell proliferation and PA at the later stages of the avocado fruit development. It is also possible that any cell division which may take place during the latter stages of the fruit development is not sufficient to alter the pattern of PA biosynthesis. MTA nucleosidase and MTR kinase activities increased during the first 15 days of fruit development followed by a slight decline at 60 and 90 days from full bloom. At 120 days (1 month before full maturity) both MTA nucleosidase and MTR kinase activities increased significantly. During maximum ethylene synthesis, MTA nucleosidase and MTR kinase activities were approximately fivefold and eightfold, respectively, higher than during maximum PA synthesis. The data indicate that the MTA molecules produced during PA and ethylene synthesis are actively metabolized to MTR and MTR-1-P, the two intermediates involved in the regeneration of S adenosylmethionine from MTA. The data also suggest that the PA and ethylene biosynthetic pathways are not actively competing for the same substrates at any given stage of the avocado fruit development and ripening. PMID- 16666166 TI - Abscisic Acid-regulated gene expression in relation to freezing tolerance in alfalfa. AB - A comparison of abscisic acid (ABA)-induced and cold-acclimation-induced freezing tolerance in two alfalfa cultivars (Medicago falcata cv Anik and Medicago sativa v Trek) indicates that ABA alone can increase freezing tolerance to some extent, but for the development of maximum tolerance, cold acclimation is essential. Analysis of in vivo-labeled proteins of ABA-treated seedlings reveals that ABA causes several changes in the pattern of protein synthesis. While some of these changes appear to be similar to those induced by cold acclimation, others seem to be specific to ABA treatment. From a cDNA library constructed against poly(A(+)) RNA of a freezing-tolerant alfalfa cultivar, Anik, a cDNA clone, pSM1409, has been isolated. Expression of the gene corresponding to this clone, as determined by northern hybridization, is regulated most likely at the transcriptional level by cold acclimation and exogenously supplied ABA. However, the increase in the transcript level is much greater in the freezing-tolerant cultivar Anik than in the relatively freezing-sensitive cultivar, Trek. The role of ABA in the acquisition of freezing tolerance is discussed. PMID- 16666167 TI - Protein Synthesis during Natural and Precocious Soybean Seed (Glycine max [L.] Merr.) Maturation. AB - Protein synthesis was studied during precocious and natural soybean seed (Glycine max [L.] Merr.) maturation. Developing seeds harvested 35 days after flowering were precociously matured through controlled dehydration. Total soluble proteins and proteins labeled with [(35)S]methionine were extracted from control, developing seeds and from precociously and naturally matured seeds and were analyzed by one-dimensional PAGE and fluorography. The results demonstrated that several polypeptides which were designated "mature polypeptides," were synthesized de novo during precocious and natural seed maturation. Two of these polypeptides, 31 and 128 kilodalton in mass, also stained intensely with Coomassie blue, suggesting their abundant accumulation during seed maturation. Results from in vitro translation experiments showed that the mRNAs corresponding to these "maturation polypeptides" accumulated during precocious maturation and in naturally matured seeds, but not in seeds freshly harvested 35 days after flowering (control). The role of the "maturation polypeptides" is currently unknown; however, their presence and that of their corresponding mRNAs was coincident with the ability of matured seeds to establish seedling growth. This study has demonstrated that precocious seed maturation treatments may be extremely useful for investigations of metabolic events and molecular control mechanisms affecting soybean seed maturation. PMID- 16666168 TI - Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA. AB - Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin. PMID- 16666169 TI - Enzymic and Photosynthetic Characteristics of Reciprocal F(1) Hybrids of Flaveria pringlei (C(3)) and Flaveria brownii (C(4)-Like Species). AB - The activities of key C(4) enzymes in gel-filtered, whole-leaf extracts and the photosynthetic characteristics for reciprocal F(1) hybrids of Flaveria pringlei (C(3)) and F. brownii (C(4)-like species) were measured to determine whether any inherited C(4)-photosynthetic traits are responsible for their reduced CO(2) compensation concentration values (AS Holaday, S Talkmitt, ME Doohan Plant Sci 41: 31-39). The activities of phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and NADP-malic enzyme (ME) for the reciprocal hybrids are only about 7 to 17% of those for F. brownii, but are three- to fivefold greater than the activities for F. pringlei. The low activities of these enzymes in the hybrids appear to be the result of a partial dominance of F. pringlei genes over certain F. brownii genes. However, no such dominance occurs with respect to the expression of genes for NADP-malate dehydrogenase, which is as active in the hybrids as in F. brownii. In contrast to the situation with the enzymes above, cytoplasmic factors appear to determine the inheritance of NAD-ME. The NAD-ME activity in each hybrid is comparable to that in the respective maternal parent. Pulse-chase (14)CO(2) incorporation analyses at ambient CO(2) levels indicate that the hybrids initially assimilate 7 to 9% of the total assimilated CO(2) into C(4) acids as compared to 3.5% for F. pringlei. In the hybrids, the percentage of (14)C in malate decreases from an average of 6.5 to 2.1% after a 60-second chase in (12)CO(2)/air. However, this apparent C(4)-cycle activity is too limited or inefficient to substantially alter CO(2) exchange from that in F. pringlei, since the values of net photosynthesis and O(2) inhibition of photosynthesis are similar for the hybrids and F. pringlei. Also, the ratio of the internal to the external CO(2) concentration and the initial slopes of the plot of CO(2) concentration versus net photosynthesis are essentially the same for the hybrids and F. pringlei. At 45 micromoles CO(2) per mole and 0.21 mole O(2) per mole, the hybrids assimilate nearly fivefold more CO(2) into C(4) acids than does F. pringlei. Some turnover of the malate pool occurs in the hybrids, but the labelling of the photorespiratory metabolites, glycine and serine, is the same in these plants as it is in F. pringlei. Thus, although limited C(4)-acid metabolism may operate in the hybrids, we conclude that it is not effective in altering O(2) inhibition of CO(2) assimilation. The ability of the hybrids to assimilate more CO(2) via phosphoenolpyruvate carboxylase at low levels of CO(2) than does F. pringlei may result in an increased rate of reassimilation of photorespiratory CO(2) and CO(2) compensation concentrations below that of their C(3) parent. If the hybrids do possess a limited C(4) cycle, it must operate intracellularly. They are not likely to have inherited an intercellular compartmentation of C(4) enzymes, since F. brownii has incomplete compartmentation of key C(3) and C(4) enzymes. PMID- 16666171 TI - Ethylene-induced gene expression in carnation petals : relationship to autocatalytic ethylene production and senescence. AB - Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression. PMID- 16666170 TI - Preparation of Corn Root Plasmalemma with Low Mg-ATPase Latency and High Electrogenic H Pumping Activity after Phase Partitioning. AB - Crude plasma membranes of corn (Zea mays L.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H(+) transport, was purified by phase partitioning. The percentage of inside-out vesicles (10%) was determined from the Mg-ATPase latency, revealed with lysophosphatidylcholine. A Triton X-100 treatment described previously (JP Grouzis, R Gibrat, J Rigaud, C Grignon 1987 Biochim Biophys Acta 903: 449-464) was applied to phase-partitioned plasma membranes. The percentage of catalytic sites freely accessible to Mg-ATP increased to 50% after Triton X-100 treatment. Treated vesicles remained capable of electrogenic H(+)-pumping, as demonstrated by Mg:ATP-dependent quinacrine fluorescence quenching and oxonol absorbance shift. As expected from the large increase of the catalytic sites accessibility, increases of the dye responses were observed. Concanavalin A binding was estimated from microelectrophoretic measurements of individual vesicles. Statistical analysis of concanavalin A binding and Mg-ATPase latency suggest that treated membranes have lost their asymmetric structure. PMID- 16666172 TI - Carbon dioxide fixation by detached cereal caryopses. AB - Immature detached cereal caryopses from barley (Hordeum vulgare L. var distichum cv Midas) and wheat (Triticum aestivum L. cv Sicco) were shown to be capable of fixing externally supplied (14)CO(2) in the light or dark. Green cross cells and the testa contained the majority of the (14)C-labeled material. Some (14)C labeled material was also found in the outer, or transparent, layer and in the endosperm/embryo fraction. More (14)C was recovered from caryopses when they were incubated in (14)CO(2) without the transparent layer, thus suggesting that this layer is a barrier to the uptake of CO(2). In all cases, significant amounts of (14)C-labeled material were found in caryopses after dark incubation with (14)CO(2). Interestingly, CO(2) fixation in the chlorophyll-less mutant Albino lemma was significantly greater in the light than in the dark. The results indicate that intact caryopses have the ability to translocate (14)C-labeled assimilate derived from external CO(2) to the endosperm/embryo. Carboxylating activity in the transparent layer appears to be confined to phosphoenolpyruvate carboxylase activity but that in the chloroplast-containing cross-cells may be accounted for by both ribulose-1,5-bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase activity. Depending on a number of assumptions, the amount of CO(2) fixed is sufficient to account for about 2% of the weight of starch found in the mature caryopsis. PMID- 16666173 TI - Ethylene production by sunflower cell suspensions : effects of plant growth retardants. AB - From a variety of undifferentiated plant cell suspensions, 2,4 dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole x gram fresh weight(-1) x hour(-1) during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene forming enzyme was influenced. PMID- 16666174 TI - Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles. AB - Putrescine and spermidine uptake in carrot (Daucus carota L., cv "Tip top") protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K(m) = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca(2+) affected only surface binding but not transport into the cells. Nonpermeant polycations such as La(3+) and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast. PMID- 16666175 TI - High performance liquid chromatography of the dansyl derivatives of putrescine, spermidine, and spermine. AB - A high performance liquid chromatographic (HPLC) method, based on dansylation and fluorescence detection, is described for the estimation of putrescine, spermidine, and spermine in lichen (Evernia prunastri [L.]) samples. Because of the high concentrations of phenols and salts, dansylation was followed by a pre HPLC purification step. Both flow rate and mobile phase (methanol:water) followed a gradient for optimum resolution on a reverse-phase column. Amounts as small as 0.3 picomole of standard polyamines could be detected. In applying the method to lichens, it was found that 5.45% (w/w) of the exogenous putrescine taken up by the thallus was unbound in the algal partner and that 60% (w/w) was conjugated in the thallus, perhaps to lichen phenolics. PMID- 16666176 TI - Effect of nitrogen nutrition on endosperm protein synthesis in wild and cultivated barley grown in spike culture. AB - In normal growth conditions, total protein percent (salt soluble plus hordein fractions) in the endosperm at maturity in barley cultivar Hordeum vulgare L. cv ;Ruth' was about 14%, whereas in an accession of wild barley, Hordeum spontaneum Koch line 297, it was about 28%. Spike culture experiments were conducted to ascertain whether there were basic differences between the two genotypes under conditions of widely different nitrogen supply. Spikes of each genotype were grown from 8 to 25 days after flowering in in vitro culture in a growth medium containing 0 to 4 grams per liter nitrogen supplied as NH(4)NO(3). Spikes were pulse-labeled at intervals from 12 to 24 days after flowering with 3.7 megabecquerel of [(3)H]leucine to determine relative rates of synthesis of hordein-1 and hordein-2 polypeptides. At low nitrogen levels ;Ruth' had a lower protein content than 297, but at increasing nitrogen levels its protein content increased rapidly and reached a maximum (35%) higher than 297 (30%). The relative contribution of the hordein fraction to total protein increased mainly with time, and hordein-1 to total hordein increased mainly with nitrogen level, in both genotypes. There appeared to be no fundamental limitations in the capacity of ;Ruth' to accumulate protein; 297 appears to have a greater basal level of nitrogen availability under normal conditions. PMID- 16666177 TI - Effect of temperature on spatial and temporal aspects of growth in the primary maize root. AB - In the range 16 to 29 degrees C, increases in temperature caused large (two-to threefold) increases in growth velocity, growth strain rate, and biomass deposition rate in primary roots of maize, Zea mays L. Temperature had small effects on root diameter, fresh weight density, and dry weight density, and negligible effects on length of the growth zone and growth strain at particular positions. PMID- 16666178 TI - Stress and activity of molybdenum-containing complex (molybdenum cofactor) in winter wheat seeds. AB - Molybdenum, applied in vivo, restored the damage from low temperature with winter wheat (Triticum aestivum, var "Sadovo 1") grown on acid soil and, in addition, sharply increased productivity (G Salcheva, D Georgieva, 1982; G Salcheva et al., 1977, 1979). Two fractions with molybdenum-cofactor activity in seeds were detected. One of them has a molecular weight of about 230 kilodaltons corresponding to xanthine oxidase activity and leaf nitrate reductase activity. The other has a molecular weight of about 60 kilodaltons. The ratio between the molybdenum-cofactor activity of these fractions was different in ;mother' seeds used in the experiment, in seeds obtained from the damaged plants, and in seeds obtained from the damaged plants restored by in vivo molybdenum addition. Every one of these fractions consisted of several components in which molybdenum cofactor activity and stability in vitro was different. We suggest that plants store molybdenum as molybdenum carriers in these low molecular weight fractions. PMID- 16666179 TI - Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.). AB - The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm. PMID- 16666180 TI - Anaerobiosis induces transcription but not translation of sucrose synthase in maize. AB - This report examines the effect of anaerobic stress on the expression of sucrose synthase in maize (Zea mays L.). Following 24 hours of anaerobic treatment, alcohol dehydrogenase displayed the classical characteristics of induction: increased mRNA and protein levels. However, there was no detectable increase in sucrose synthase specific proteins by either native or denaturing Western blot analysis nor was there an increase in sucrose synthase activity. Anaerobic treatment did induce significantly higher steady state levels of sucrose synthase mRNA. Even though previous work has implicated sucrose synthase as an anaerobically induced protein, the data in this report suggest that sucrose synthase is not inducible at the protein level by anaerobic treatment. PMID- 16666181 TI - Mechanisms of salinity tolerance in plants. AB - The mechanisms of salt stress response and tolerance have eluded definition despite reasonable success in defining their physiological manifestations. In this review, we consider the integrated salt metabolism of plants, essentially as a problem in meganutrient physiology. Two critical aspects of cellular and organismal metabolism are given particular attention-those involved in the control and integration of Na(+) acquisition and allocation in plants and those involved in readjustment of other aspects of metabolism, especially those involving carbon as a resource. PMID- 16666182 TI - Simultaneous Transport of CO(2) and HCO(3) by the Cyanobacterium Synechococcus UTEX 625. AB - A mass spectrometer was used to simultaneously follow the time course of photosynthetic O(2) evolution and CO(2) depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO(2) and HCO(3) (-) were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO(3) (-) transport by Na(+) addition had no effect on ongoing CO(2) transport. This result is interpreted to indicate that the CO(2) and HCO(3) (-) transport systems are separate and distinctly different transport systems. Measurement of CO(2)-dependent photosynthesis indicated that CO(2) uptake involved active transport and that diffusion played only a minor role in CO(2) acquisition in cyanobacteria. PMID- 16666183 TI - Growth Responses of Plantago major L. ssp. pleiosperma (Pilger) to Changes in Mineral Supply : Evidence for Regulation by Cytokinins. AB - Plants of an inbred line of Plantago major ssp. pleiosperma were subjected to an alteration in mineral supply. Observed responses of growth rate and shoot to root ratio are thought to be induced by changes in endogenous cytokinin concentration and not by mineral concentration in plant tissue. PMID- 16666184 TI - Regulation of ribulose-1,5-bisphosphate carboxylase activity by the activase system in lysed spinach chloroplasts. AB - Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO(2) and Mg(2+) by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of DeltapH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between DeltapH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO(2) and Mg(2+). PMID- 16666185 TI - Adaptive Potential of Wheat Ribosomes toward Heat Depends on the Large Ribosomal Subunit and Ribosomal Protein Phosphorylation. AB - In a study of the translational efficiency of ribosomal subunits as a function of an in vivo temperature pretreatment, ribosomes were isolated from heat-pretreated (36 degrees C) and reference (20 degrees C) wheat seedlings (Triticum aestivum L.). The efficiency of recombined subunits in translating polyuridylic acid was assessed. A threefold increase in the rate of incorporation of phenylalanine by ribosomes from heat-pretreated plants was due to the large ribosomal subunit. This adaptive temperature effect was not correlated with a higher thermal stability of ribosomes or subunits from heat-pretreated seedlings, and two dimensional gel electrophoresis failed to detect structural alterations of ribosomal proteins. Phosphorylation of ribosomal proteins in vitro showed no differences between ribosomes or subunits from heat-pretreated and reference plants. Incubation with [(32)P]orthophosphate in vivo led to twice the amount of phosphate in ribosomal proteins from heat-pretreated wheat seedlings. This result is important with respect to the evaluation of the molecular basis of enhanced translational efficiency of ribosomes isolated from heat-pretreated wheat seedlings. PMID- 16666186 TI - Carbohydrate Responsive Proteins in the Roots of Pennisetum americanum. AB - The effect of changes in carbohydrate status on the synthesis of specific proteins was investigated in millet (Pennisetum americanum L., Leeke, Tift 23B(1)E(1)) seedlings grown in sterile solution culture. Carbohydrate status was altered by extended darkness and sucrose feeding. Root proteins from intact seedlings were labeled with [(35)S]methionine, phenol-extracted, separated by two dimensional gel electrophoresis, and visualized by autoradiography. In four separate experiments, two proteins showed a consistent change in labeling when root carbohydrate levels were varied between 200 and 1000 micromole hexose per gram residual dry weight. Labeling of the first protein (P(47), M(r) 47 kD) increased as the carbohydrate levels rose above 500 micromole hexose per gram residual dry weight. Labeling of the second protein (P(34), M(r) 34 kD) increased as carbohydrate levels declined from 500 to 200 micromole hexose per gram residual dry weight. Under extreme conditions, when carbohydrate levels fell below 100 micromole hexose per gram residual dry weight, the labeling pattern of most proteins was drastically altered. It is suggested that P(47) and P(34) are ;carbohydrate responsive proteins,' i.e. proteins whose concentrations are controlled either directly or indirectly by tissue carbohydrate status. In contrast, the changes in protein labeling that occur once carbohydrate pools are depleted may be involved in adaptation to periods of prolonged starvation. PMID- 16666187 TI - Respiration and protein synthesis in nongrowing cultured pear fruit cells in response to ethylene and modified atmospheres: a model system for fruits postharvest. AB - The respiration of pear fruit (Pyrus communis L. Passe Crassane) cells was monitored after subculture into an auxin-free, mannitol-enriched medium in which the cells remained viable but did not grow. Respiration rates were affected by the presence or absence of sucrose in the medium even though the cells retained reserves of sucrose and starch. Provided the medium contained respirable carbohydrate, exposure to ethylene (1-10 microliters per liter) increased the respiration rate with some acceleration of cell death. In the range from 10 to 2% oxygen by volume, the respiration rate of the cells decreased with oxygen concentration resulting in some prolongation of cell life. Thus, in their responses to ethylene and modified atmospheres, the cells reflected the behavior of harvested fruits. Having defined conditions under which respiration rate could be varied without apparent influence on the quiescent state of the cells, we sought a connection between maintenance respiration and protein turnover. Relative rates of protein synthesis were assessed by measuring ribosome distribution between monosomes and polysomes. In general, the higher the respiration rate the higher the proportion of polysomes supporting the thesis that protein turnover is a variable component of maintenance metabolism. Protein turnover in cells incubated in the presence or absence of sucrose was measured as retained alpha-amino-(3)H following a pulse of (3)H(2)O. Turnover was shown to be a quantitatively important component of the maintenance budget and to be more rapid in cells in media supplemented with sucrose through the chase period. The experiments illustrate that cultured cells may be used to explore aspects of the maintenance metabolism of resting or senescent cells that are not amenable to study in bulky fruit tissues. PMID- 16666188 TI - A test of the air-seeding hypothesis using sphagnum hyalocysts. AB - "Air-seeding" is a proposed mechanism for the initiation of water stress embolism in dead plant cells. During air-seeding, external air is drawn into the lumen of a dead plant cell through a pore or crack in the cell wall. The resulting bubble may expand to fill the lumen, thus embolizing the cell. The data presented confirm that Sphagnum hyalocysts can embolize by air-seeding when the pressure difference across the air-water meniscus is given by DeltaP = 0.3/D (derived from the capillary equation), where DeltaP is the pressure difference across the meniscus (megapascal), and D is the diameter (micrometer) of the pore through which the air bubble enters. PMID- 16666189 TI - Calcium Effects on Stomatal Movement in Commelina communis L. : Use of EGTA to Modulate Stomatal Response to Light, KCl and CO(2). AB - Stomatal movements depend on both ion influx and efflux; attainment of steady state apertures reflects modulation of either or both processes. The role of Ca(2+) in those two processes was investigated in isolated epidermal strips of Commelina communis, using the Ca(2+) chelator EGTA to reduce apoplastic [Ca(2+)]. The results suggest that a certain concentration of Ca(2+) is an absolute requirement for salt efflux and stomatal closure. EGTA (2 millimolar) increased KCl-dependent stomatal opening in darkness and completely inhibited the dark induced closure of initially open stomata. Closure was inhibited even in a KCl free medium. Thus, maintenance of stomata in the open state does not necessarily depend on continued K(+) influx but on the inhibition of salt efflux. Opening in the dark was stimulated by IAA in a concentration-dependent manner, up to 15.4 micrometer without reaching saturation, while the response to EGTA leveled off at 9.2 micrometer. IAA did not inhibit stomatal closure to the extent it stimulated opening. The response to IAA is thus consistent with a primary stimulation of opening, while EGTA can be considered a specific inhibitor of stomatal closing since it inhibits closure to a much larger degree than it stimulates opening. CO(2) causes concentration-dependent reduction in the steady state stomatal aperture. EGTA completely reversed CO(2)-induced closing of open stomata but only partially prevented the inhibition of opening. PMID- 16666190 TI - Stachyose Synthesis in Source Leaf Tissues of the CAM Plant Xerosicyos danguyi H. Humb. AB - Leaf tissues from Xerosicyos danguyi H. Humb., a succulent member of the Cucurbitaceae, were found to possess both galactinol synthase activity and the capacity for photosynthetic production of stachyose, the phloem transport oligosaccharide common to other nonsucculent cucurbits. The amounts of stachyose isolated from leaf tissues, and the extractable activity of galactinol synthase, were somewhat higher in leaf tissues obtained from plants operating in the Crassulacean acid metabolism (CAM) mode (well watered plants) compared to leaf tissues from plants operating in the CAM-idling mode (water-stressed plants). In contrast, in leaf discs, the photosynthetic incorporation of label into stachyose following pulse labeling with (14)CO(2) was similar for stressed and for nonstressed tissues. Stachyose could be extracted from, and was synthesized photosynthetically by, leaf discs which contained no vascular tissues, indicating that synthesis of stachyose can occur in photosynthetic mesophyll cells of Xerosicyos. PMID- 16666191 TI - Uronic Acid products release from enzymically active cell wall from tomato fruit and its dependency on enzyme quantity and distribution. AB - Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed. PMID- 16666192 TI - Factors associated with the instability of nitrate-insensitive proton transport by maize root microsomes. AB - Proton transport catalyzed by the nitrate-insensitive, vanadate-sensitive H(+) ATPase in microsomes from maize (Zea mays L.) roots washed with 0.25 molar KI decreased as a function of time at 0 to 4 degrees C. The rate of proton transport was approximately one-half of that by freshly isolated microsomes after 6 to 18 hours of cold storage. The decrease in proton transport coincided with losses in membrane phosphatidylcholine and was not associated with a change in vanadate sensitive ATP hydrolysis. A technique based on a protocol developed for the reconstitution of Neurospora crassa plasma membrane H(+)-ATPase (DS Perlin, K Kasamo, RJ Brooker, CW Slayman 1984 J Biol Chem 259: 7884-7892) was employed to restore proton transport activity to maize microsomes. These results indicated that the decline in proton transport by maize root membranes during cold storage was not due to degradation of the protein moiety of the H(+)-ATPase, but was due to the loss of phospholipids. PMID- 16666193 TI - Establishment of control parameters for in situ, automated screening of sustained hydrogen photoproduction by individual algal colonies. AB - An apparatus was constructed which allowed automated screening of individual microalgal colonies for sustained ability to photoevolve H(2) during anaerobic photosynthesis. The main components of this apparatus were a microcomputer, a He Ne laser mounted on a computer-controlled X-Y translation stage, a flow-through chamber which contained an agar plate of colonies, and a H(2) detector which interfaced with the microcomputer for data collection. The system was capable of detecting a minimum production rate of 1 nanomole of H(2) per hour per colony and provided an efficient means of screening relatively large numbers of algal colonies. Examination of the effect of the spacing of colonies on the agar plate, light intensity, stability of colonies within a screening period, colony age, chlorophyll content, and colony size on H(2) yield indicated that, under optimum conditions, yields from genetically uniform colonies varied by no more than a factor of 2 in their H(2)-producing ability. Therefore, colonies of algae whose H(2) yields lie outside this intrinsic twofold variability can be identified and selected as natural variants or mutants. A description of the construction and of the apparatus is presented, and the experimental results used to establish the control parameters for Chlamydomonas reinhardtii colonies are discussed. PMID- 16666194 TI - Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons. AB - Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv ;Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [(35)S]Na(2)SO(4) was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues. PMID- 16666195 TI - Enzymic cross-linkage of monomeric extensin precursors in vitro. AB - Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase. PMID- 16666196 TI - In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium. AB - Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K(+), along with small but significant loss of cellular Ca(2+). During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, ;protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca(2+) from membrane by extracellular K(+) and subsequent perturbation of K(+) transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either -8.5 degrees C or -11.5 degrees C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (-11.5 degrees C; irreversible) caused a specific and substantial loss of membrane-associated Ca(2+) compared to control. Loss of membrane-associated Ca(2+) caused by moderate stress (-8.5 degrees C; reversible) was much less relative to -11.5 degrees C treatment. Ion efflux and Ca(2+)-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl(2). Our results suggest that the loss of membrane-associated Ca(2+), in part, plays a role in initiation and progression of freezing injury. PMID- 16666197 TI - Changes in Abscisic Acid and Indoleacetic Acid before and after Anthesis Relative to Changes in Abscission Rates of Cotton Fruiting Forms. AB - Cotton (Gossypium hirsutum L.) fruiting forms exhibit pronounced changes, with age, in their probability of abscission. Large floral buds rarely abscise, but after anthesis the young fruits (bolls) have a high probability of abscising. Abscission rate reaches a peak about 5 to 6 days after anthesis and then gradually decreases. An experiment was conducted to try to determine the reason for the rapid and pronounced increase in probability of abscission just after anthesis. Cotton was grown in the field and fruiting forms of various ages from 9 days before to 9 days after anthesis were all harvested the same day and subsequently analyzed for ABA and IAA. The concentration of ABA decreased slightly at anthesis and increased gradually thereafter. In contrast, the concentration of IAA was high before anthesis and then decreased at anthesis to about one-fifth the previous concentration. IAA remained low for at least 4 days after anthesis and then increased rapidly between 7 and 9 days after anthesis. The high concentration of IAA in floral buds before anthesis is probably a major factor in their resistance to abscission. Likewise, the low concentration of IAA at anthesis and for about 4 days thereafter may promote fruit abscission during the young boll stage. PMID- 16666198 TI - Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl. AB - Treatment with acifluorfen-methyl (AFM), methyl 5-(2-chloro-4-[tri-fluoromethyl] phenoxy)-2-nitrobenzoate, inhibited protochlorophyllide synthesis in dark-held, delta-amino levulinic acid-fed, excised cotyledons of cucumber (Cucumis sativus L.). Protochlorophyllide and protoporphyrin IX levels in AFM-treated cotyledons were inversely related and dependent on AFM concentration; as the herbicide dose increased, protoporphyrin IX levels also increased with a concomitant loss of protochlorophyllide. Significant protoporphyrin IX accumulation was induced by concentrations of AFM from the linear region of the membrane disruption dose response curve. The pattern of precursor accumulation seen in HPLC chromatograms from extracts of AFM-treated tissue indicate that Mg insertion into the tetrapyrrole ring is inhibited, suggesting interference with Mg-chelatase. An inhibitor of delta-amino levulinic acid synthesis, gabaculine (3-amino-2,3 dihydrobenzoic acid), completely blocked the membrane disruption activity of AFM in illuminated cotyledons. Protoporphyrin IX accumulating in AFM-treated tissues may serve as the primary photosensitizer for initiating lipid peroxidation. PMID- 16666199 TI - Effects of ozone and peroxyacetyl nitrate on polar lipids and Fatty acids in leaves of morning glory and kidney bean. AB - To compare the effects of ozone and peroxyacetyl nitrate (PAN) on leaf lipids, fatty acids and malondialdehyde (MDA), morning glory (Pharbitis nil Choisy cv Scarlet O'Hara) and kidney bean (Phaseolus vulgaris L. cv Gintebo) plants were exposed to either ozone (0.15 microliter per liter for 8 hours) or PAN (0.10 microliter per liter for up to 8 hours). Ozone increased phospholipids in morning glory and decreased in kidney bean at the initial stage (2-4 hours) of exposure, while it scarcely changed glycolipids, the unsaturated fatty acids, and MDA in both plants. A large reduction of glycolipids occurred 1 day after ozone exposure in both plants. PAN caused marked drops in phospholipids and glycolipids in kidney bean at relatively late stage (6-8 hours) of exposure, while it increased phosphatidic acid and decreased the unsaturated fatty acids, an increase which was accompanied by a large increase in MDA. These results suggest that ozone may not directly oxidize unsaturated fatty acids at the initial stage of exposure, but may alter polar lipid metabolism, particularly phospholipids. On the other hand, PAN may abruptly and considerably degrade phospholipids and glycolipids by peroxidation or hydrolysis at the late stage of exposure. The present study shows that ozone and PAN affect polar lipids in different manners. PMID- 16666200 TI - Isolation of Sperms from the Pollen Tubes of Flowering Plants during Fertilization. AB - Sperm cells have been isolated from pollen tubes growing in style segments of the dicotlyledon Rhododendron macgregoriae and the monocotyledon Gladiolus gandavensis by the in vivo/in vitro method at various stages of fertilization. Pollen tubes emerged from the cut end of the style into agar medium, and more than 95% contained sperm cells. Sperm cells were released from the pollen tubes by osmotic shock or by placing styles in wall-degrading enzymes: 0.5% macerozyme and 1% cellulase. The isolated sperms were ellipsoidal protoplasts of diameter about 2 x 3 micrometers in Gladiolus and about 3 x 4 micrometers in Rhododendron. After isolation, a proportion of the sperm cells occurred in pairs linked at one end by finger-like connections. The pairs of isolated sperms were dimorphic in terms of surface area and volume. By cutting the styles at various positions and times after pollination, the potential exists to detect changes in sperm gene expression associated with fertilization. PMID- 16666201 TI - Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii. AB - Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake. PMID- 16666202 TI - Cyanide-resistant respiration in light- and dark-grown soybean cotyledons. AB - Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv ;Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O(2) uptake rates were determined. O(2) uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O(2) uptake in tissues more than 9 days old. Mitochondria in which O(2) uptake was coupled to ATP synthesis were isolated from all tissues. O(2) uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria. PMID- 16666203 TI - Abscisic Acid Is an Endogenous Inhibitor in the Regulation of Mannanase Production by Isolated Lettuce (Lactuca sativa cv Grand Rapids) Endosperms. AB - The production of mannanase, a cell-wall-degrading carbohydrase, can be manipulated in isolated lettuce (Lactuca sativa cv Grand Rapids) endosperms by changes in the volume of buffer in which they are incubated. The enzyme is produced when endosperms are incubated in a large volume, but not when incubated in a small volume, which is suggestive that an endogenous, diffusible inhibitor of mannanase production is being lost from the endosperms in a large volume (JD Bewley, P Halmer 1980/1981 Israel J Bot 29: 118-132). We have investigated the possibility that the phytohormone abscisic acid (ABA) is involved in this regulation of mannanase production in isolated lettuce endosperms. We find several correlations between the presence of the endogenous inhibitor and of ABA, i.e. (a) a ;leachate' prepared from isolated lettuce endosperms induces synthesis of ABA-specific proteins in barley aleurone layers, indicating that incubation of endosperms in a large volume results in the diffusion of ABA therefrom into the surrounding medium; (b) fractionation of the components of a leachate by either polyvinylpyrrolidone-chromatography of C(18) reversed-phase high performance liquid chromatography fails to separate the endogenous inhibitor from authentic ABA; and (c) changes in the incubation volume of endosperms result in changes in the amount of extractable ABA in the endosperms, as detected by ELISA. These results are consistent with a role for endogenous ABA in the regulation of mannanase production in isolated lettuce endosperms. PMID- 16666204 TI - Photosynthetic activities in the petunia corolla. AB - Pink Petuniahybrida (cv Hit Parade Rosa) corollas were found to contain photosynthetically active chloroplasts. The corolla chloroplasts were similar to those of green leaves in size and structure. The chlorophyll (Chl) content of Petunia corollas increased during early stages of flower development, reaching a maximum just before anthesis. Chloroplasts isolated from corollas at this stage, carried out photosystem I-dependent electron transport at rates which were two thirds of those measured in chloroplasts from green leaves, but full chain electron transport at only one-quarter of the rate carried out by chloroplasts from green leaves. Both the light saturated rate and the quantum yield for electron transport were lower in corolla chloroplasts, which also required lower intensities for light saturation. Reduced efficiency of photosystem II photoreactions in the corolla was also indicated by the ratio between variable and constant components of Chl fluorescence, which was lower in corollas compared to green leaves. The induction time of Chl fluorescence was at least three times shorter in corollas compared to green leaves, indicating a smaller number of functional photosystem II centers (per Chl) in the corolla. It is suggested that corolla chloroplasts of Petunia might have a role in flower developmental processes. PMID- 16666205 TI - Stable Transformation of Soybean Callus by DNA-Coated Gold Particles. AB - Immature soybean (Glycine max L.) embryos from commercially important cultivars were the targets of rapidly accelerated, DNA-coated, gold particles. Protoplasts were prepared from these tissues and propagated in culture under selection conditions for the introduced neomycin phosphotransferase II gene. Kanamycin resistant calli were obtained at a rate of approximately 10(-5). Enzyme assays and Southern blot hybridization confirmed the expression of the foreign gene and its stable integration into the soybean genome. Our results show that particle acceleration can be used for the introduction of foreign DNA into the soybean genome and indicate the technique may be useful in the recovery of engineered plants by transformation of regenerable tissues. PMID- 16666206 TI - Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons. AB - The activities of ribulose bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), as indicators of autotrophic and nonautotrophic CO(2) fixation, were measured in excised cotyledons of Pinus radiata D. Don cultured for 21 days under shoot-forming (SF) and nonshoot-forming (NSF) conditions. The activity of RuBPC was found to increase in both SF and NSF cultures during the initial 5 days of culture. However, it leveled off from day 5 to day 10 and subsequently began to decrease until the end of the culture period under the SF conditions. In contrast, in the NSF cultures, RuBPC activity increased until day 15, when it was twofold higher than the maximum activity found in the SF cultures. An increase in PEPC activity of about 2.5 times the level of activity in freshly excised cotyledons was observed during the initial 5 days of culture under the SF conditions. PEPC activity began to decline after day 5 until it reached the level of activity seen in NSF cotyledons by day 15. In contrast, the activity of PEPC did not show any significant increase during the initial 10 days of culture under the NSF conditions. The K(m) (phosphoenolpyruvate) of PEPC from SF cotyledons was about 35% higher than that of NSF cotyledons. Cotyledons from two culture periods (days 5 and 15) were incubated for 15 seconds with NaH(14)CO(3). The label in the malate and asparatate fractions as a percentage of total (14)C incorporation was 3 times higher in the SF cotyledons than in the NSF cotyledons. A higher incorporation of (14)C into products of photosynthesis under the NSF conditions was also observed. PMID- 16666207 TI - Utilization of microbial siderophores in iron acquisition by oat. AB - Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K(m) of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms. PMID- 16666208 TI - Alternative methods of photosynthetic carbon assimilation in marine macroalgae. AB - Two green macroalgae, Codium decorticatum and Udotea flabellum, differ photosynthetically. Codium had high O(2)-sensitive, and Udotea low O(2) insensitive, CO(2) compensation points; Codium showed a Warburg effect at seawater dissolved inorganic carbon levels and had photorespiratory CO(2) release, whereas Udotea did not. Seawater dissolved inorganic carbon levels did not saturate photosynthesis. For Codium, but not Udotea, the Warburg effect was increased by ethoxyzolamide, a carbonic anhydrase inhibitor, at high but not low pH. Isolated chloroplasts from both macroalgae showed a Warburg effect that was ethoxyzolamide-insensitive. In both macroalgae, chloroplastic and extrachloroplastic carbonic anhydrase activity was present. P-enolpyruvate carboxykinase (PEPCK) carboxylating activity in Udotea extracts was equivalent to that of ribulose bisphosphate carboxylase, and enzyme activities for C(4) acid metabolism and P-enolpyruvate regeneration were sufficient to operate a limited C(4)-like system. In Udotea, malate and aspartate were early-labeled photosynthetic products that turned over within 60 seconds. Photorespiratory compounds were much less labeled in Udotea. Low dark fixation rates ruled out Crassulacean acid metabolism. A limited C(4)-like system, based on PEPCK, is hypothesized to be the mechanism reducing photorespiration in Udotea. Codium showed no evidence of photosynthetic C(4) acid metabolism. Marine macroalgae, like terrestrial angiosperms, seem to have diverse photosynthetic modes. PMID- 16666209 TI - Alcohol dehydrogenase and ethanol in the stems of trees : evidence for anaerobic metabolism in the vascular cambium. AB - Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out. PMID- 16666210 TI - Molecular characterization of oat seed globulins. AB - We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a highly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin). PMID- 16666211 TI - Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa. AB - Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity. PMID- 16666212 TI - Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: I. Excretion of Acid Phosphatase by Tomato Plants and Suspension-Cultured Cells. AB - Both tomato (Lycopersicon esculentum cv VF 36) plants and suspension cultured cells show phosphate starvation inducible (psi) excretion of acid phosphatase (Apase). Apase excretion in vitro was proportional to the level of exogenous orthophosphate (Pi). Intracellular Apase activity remained the same in both Pi starved and sufficient cells, while Apase excreted by the starved cells increased by as much as six times over unstressed control cells on a dry weight basis. At peak induction, 50% of total Apase was excreted. Ten day old tomato seedlings grown without Pi showed slight growth reduction versus unstressed control plants. The Pi-depleted roots showed psi enhancement of Apase activity. Severely starved seedlings (17 days) reached only one-third of the biomass of unstressed control plants but, because of a combination of psi Apase excretion by roots and a shift in biomass to this organ, they excreted 5.5 times the Apase activity of the unstressed control. Observed psi Apase excretion may be part of a phosphate starvation rescue system in plants. The utility of the visible indicator dye 5 bromo-4-chloro-3-indolyl-phosphate-p-toluidine as a phenotypic marker for plant Apase excretion is demonstrated. PMID- 16666213 TI - Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase. AB - Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [(35)S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins. PMID- 16666214 TI - Overwintering Periwinkle (Vinca minor L.) Exhibits Increased Photosystem I Activity. AB - The effects of natural, overwintering conditions on photosystem I and photosystem II activity were examined in isolated thylakoids of periwinkle (Vinca minor L.), an endemic, cold-tolerant, herbaceous evergreen. DCMU-Insensitive photosystem I activity (ascorbate/dichlorophenolindophenol --> methylviologen) exhibited a twofold increase in light-saturated rates upon exposure to low temperature and freezing stress with no effect on the apparent quantum yield of this reaction. DCMU-Sensitive photosystem II activity (H(2)O --> dichlorlophenolindophenol) exhibited only minor fluctuations in light-saturated rates but a 50% decrease in the apparent quantum yield of this reaction upon exposure to overwintering conditions. This was correlated with a decrease in the 77 degrees K fluorescence emission at 694 nanometers. These functional changes occurred with no detectable changes in the relative chlorophyll contents of the chlorophyll-protein complexes or the chlorophyll-thylakoid protein. The chlorophyll a/b varied less than 10% during any single growth year. Analyses of total leaf extracts indicated that all lipid classes exhibited increased levels of linoleic and linolenic acid. Neither the trans-Delta(3)-hexadecenoic acid level nor the ratio of oligomeric:monomeric light harvesting of photosystem II was affected by exposure to winter stress. The content of the major chloroplast lipids monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidyl-diacyl-glycerol, and sulfoquinovosyldiacylglycerol exhibited minor fluctuations, whereas phosphatidylcholine and phosphatidylethanolamine content doubled on a mole percent or chlorophyll basis. We conclude that the previously reported increase in photosystem I activity during controlled, low temperature growth is observed during exposure to natural overwintering conditions. This appears to occur with minimal changes in the structure and composition of the photosynthetic apparatus of periwinkle. PMID- 16666215 TI - Sink metabolism in tomato fruit : I. Developmental changes in carbohydrate metabolizing enzymes. AB - In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145 7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined. PMID- 16666216 TI - Sink Metabolism in Tomato Fruit : II. Phloem Unloading and Sugar Uptake. AB - Analysis of [(3)H]-(fructosyl)-sucrose translocation in tomato (Lycopersicon esculentum Mill.) indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast followed by extracellular hydrolysis. Apoplastic sucrose, glucose, and fructose concentrations were estimated as 1 to 7, 12 to 49, and 8 to 63 millimolar, respectively in the tomato fruit pericarp tissue. Hexose concentrations were at least four-fold greater than sucrose at all developmental stages. Short-term uptake of [(14)C]sucrose, -glucose, and -fructose in tomato pericarp disks showed first order kinetics over the physiologically relevant concentration range. The uptake rate of [(14)C]-(glucosyl)-1'-fluorosucrose was identical to the rate of [(14)C]sucrose uptake, suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m chlorophenylhydrazone. Together these results suggest that although sucrose is at least partially hydrolyzed in the apoplast, sucrose may enter the metabolic carbohydrate pool directly. In addition, sugar uptake across the plasma membrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato fruit is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast. PMID- 16666217 TI - Sink Metabolism in Tomato Fruit : III. Analysis of Carbohydrate Assimilation in a Wild Species. AB - Carbohydrate composition and key enzymes involved in carbohydrate metabolism were assayed throughout development of Lycopersicon esculentum and L. chmielewskii fruit. Translocation and assimilation of asymmetric sucrose and total soluble solids content was also determined in both species. The data showed that L. chmielewskii accumulated less starch than L. esculentum, and this was related to a lower level of ADPglucose pyrophosphorylase and a higher level of phosphorylase in L. chmielewskii. L. chmielewskii accumulated sucrose throughout fruit development rather than glucose and fructose which were accumulated by L. esculentum. A low level of invertase and nondetectable levels of sucrose synthase were associated with the high level of sucrose in L. chmielewskii. Translocation and assimilation of asymmetrically labeled sucrose indicated that sucrose accumulated in L. chmielewskii fruit was imported and stored directly in the fruit without intervening metabolism along the translocation path. In contrast, the relatively low level of radioactive sucrose found in L. esculentum fruit appeared to arise from hydrolysis and resynthesis of sucrose. The possible relationship between the level of soluble solids and differences in carbohydrate metabolism in sink tissue of the two species is discussed. PMID- 16666218 TI - Partial Purification and Characterization of NADP-Isocitrate Dehydrogenase from Immature Pod Walls of Chickpea (Cicer arietinum L.). AB - NADP(+)-isocitrate dehydrogenase (threo-DS-isocitrate: NADP(+) oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4 degrees C and had an absolute requirement for a divalent cation, either Mg(2+) or Mn(2+), for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP(+), dl-isocitrate, Mn(2+), and Mg(2+). Their K(m) values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP(+). Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP(+) at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP(+) and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP(+) IDH in situ is likely to be controlled by intracellular NADPH to NADP(+) ratio as well as by the concentration of various substrates and products. PMID- 16666219 TI - Cold Acclimation in Arabidopsis thaliana. AB - The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24 degrees C, increased in freezing tolerance from an initial 50% lethal temperature (LT(50)) of about -3 degrees C to an LT(50) of about -6 degrees C after 24 hours at 4 degrees C; LT(50) values of -8 to -10 degrees C were achieved after 8 to 9 days at 4 degrees C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A(+)) RNA isolated from control and cold treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4 degrees C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4 degrees C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides. PMID- 16666220 TI - First evidence for polyamine conjugation mediated by an enzymic activity in plants. AB - An enzyme activity, found for the first time in plants, mainly located in the 22,000g supernatant of the crude extract of sprout apices of Helianthus tuberosus L. cv OB1 tubers, is able in vitro to covalently bind polyamines to endogenous substrates of different molecular weights. The major assay parameters, such as pH, dithiothreitol, and extract concentrations were optimized. The time course of the reaction, the dependence on putrescine concentration, its competition with histamine, the capacity to bind spermidine and spermine better than putrescine, the stability of the reaction product and analysis of the latter by sodium dodecyl sulfate polyacrylamide gel electrophoresis and thin-layer chromatography suggest that putrescine is linked to endogenous substrates by means of an enzyme reaction that shows some similarities with transglutaminase activities detected in animals. However, the activities of the crude extract and of a fraction eluted from a Sephadex G25 column were not affected by CaCl(2) concentrations lower or equal to 5 millimolar; 4 or 10 millimolar EGTA caused a very small reduction; higher concentrations of CaCl(2) and 15 millimolar or more of EDTA were inhibitory. N,N'-dimethylcasein was not recognized as a substrate. These data indicate that this activity also displays some characteristics which are different from those of animal transglutaminases. PMID- 16666221 TI - Rearrangement of the chloroplast thylakoid at chilling temperature in the light. AB - The leaves of chilling-sensitive pumpkin (Cucurbita pepo L.) showed symptoms reminiscent of photoinhibition when kept for 4 days at 5 degrees C in moderate light. A decrease was observed in the variable part of chlorophyll alpha fluorescence, apparent quantum yield, and maximum rate of O(2) evolution. Chloroplast whole-chain electron transport activity measured from chloroplast thylakoids had decreased to 51% of the control value. Photosystem II (PSII) activity decreased by only 9%, suggesting that photoinhibition was not responsible for the loss of electron transport activity. An increase in the proportion of PSII(beta) (measured as a beta(max) value) was observed after the chilling treatment. Fractionation of thylakoid membranes showed a 42% increase in PSII activity in the nonappressed region while that in the appressed region decreased slightly. This was accompanied by a decrease in the ratio of the length of appressed to nonappressed thylakoid membranes. Leaf photosynthesis largely recovered within 24 hours of returning to the original growth conditions. We suggest that the increase in the proportion of PSII(beta) during chilling in light plays a role in protecting PSII from photoinhibitory damage. PMID- 16666222 TI - A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves. AB - Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of (14)C accumulated in the leaf supplied with (14)CO(2) throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO(2) concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant. PMID- 16666223 TI - Endogenous Gibberellins in Elongating Shoots of Clones of Salix dasyclados and Salix viminalis. AB - Elongating shoots of rapidly growing clones of Salix viminalis L. (clone 683-4) and Salix dasyclados Wimm. (clone 908) harvested in early August were analyzed for endogenous gibberellins (GA). Distribution of GA-like activity, determined by Tan-ginbozu dwarf rice microdrop bioassay after reverse phase C(18) high performance chromatography, was similar for both species. For S. dasyclados, combined gas chromatography-selected ion monotoring (GC-SIM) yielded identifications of GA(1), GA(8), GA(19), GA(20), and GA(29). Identifications of GA(4) and GA(9) were also made using co-injections of known amounts of [17, 17 (2)H(2)]GAs. By bioassay, the main activity was GA(19)-like in both species. Gibberellin A(1), GA(19), and GA(20) concentrations were approximated by GC-SIM using co-injections of known amounts of [17,17-(2)H(2)]GAs. Both bioassay and GC SIM results indicated very high concentrations of GA(19) and GA(20) (about 6000 nanograms per kilogram fresh weight shoot tissue using GC-SIM: 800 ng using bioassay), compared to the concentration of GA(1) (about 130 nanograms per kilogram fresh weight using either GC-SIM or bioassay). PMID- 16666224 TI - Boron Protection for O(2) Diffusion in Heterocysts of Anabaena sp. PCC 7119. AB - The effect of boron on nitrogenase activity has been studied. When cells were dependent on N(2) fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization. PMID- 16666225 TI - Intercellular communication-filling in the gaps. AB - Coordination and synchrony of a variety of cellular activities in tissues of plants and animals occur as a consequence of the transfer of low molecular weight biosynthetic and signaling molecules through specialized structures (plasmodesmata in plant cells and gap junctions in mammalian cells) that form aqueous channels between contacting cells. Investigations with rat liver demonstrated that cell-cell communication is mediated by a 32 kilodalton polypeptide that forms a hexameric pore structure in the plasma membrane. Following association with the same structure in a contiguous cell, a trans double membrane channel is created that has been termed a gap junction. In plant tissue, long tubelike structures called plasmodesmata are suggested to serve a similar cell-cell linking function between cytoplasmic compartments. Although morphologically distinct, dynamic observations suggest similarities in transport properties between gap junctions and plasmodesmata. Recent work now provides evidence that these functional similarities may reflect a more profound identity between the paradigm animal gap junction polypeptide (32 kilodalton rat liver polypeptide) and an immunologically homologous protein localized to plant plasma membrane/cell wall fractions that may be a component of plasmodesmata. PMID- 16666226 TI - Root excision decreases nutrient absorption and gas fluxes. AB - The roots of barley plants (Hordeum vulgare L. cv Steptoe) were monitored before and after excision for net uptake of carbon dioxide, oxygen, ammonium, potassium, nitrate, and chloride and for their content of sucrose, glucose, fructose, and malic acid. All fluxes began to attenuate within 2 hours after excision. Net potassium uptake returned to control levels 6 hours after excision, but carbon dioxide, oxygen, ammonium, and nitrate fluxes continued to diminish for the remainder of the observation period. The addition of 0.1 molar glucose or 0.1 molar sucrose to excision medium had no significant effect on these changes in ion and gas fluxes. Net chloride uptake was negligible for all treatments. Sugar and malic acid content of the root declined after excision. Sucrose and glucose levels remained depressed for the entire observation period, whereas fructose and malic acid returned to control levels after 9 hours. These results indicate that excision has profound, adverse effects on root respiration and the absorption of mineral nitrogen. PMID- 16666227 TI - Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems. AB - Most of the activity of an alpha-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic alpha-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the alpha-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this alpha-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the beta-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction. PMID- 16666228 TI - A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels. AB - The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO(2) but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-(14)C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-(14)C]glycolate, showing there is a large excess of this enzyme in wild-type plants. PMID- 16666229 TI - Habituation in in vitro soybean cultures. AB - The habituation of soybean (Glycine max) callus can be induced rapidly, by exposing the tissue to small amounts (10(-9)molar) of compounds including 2.4 dinitrophenol and phenoxyisobutyric acid for brief periods of time. Such compounds reportedly exhibit antiauxin activity. Various soybean callus phenotypes have been isolated which continue to exhibit hormone habituation 14 months following the initiation of the experiment. Protein changes in habituated tissue under selected hormonal regimes were detected indicating changes at the level of gene expression. Habituated tissue exhibits hormonal autonomy in a manner similar to crown gall tissue, suggesting that such studies may help elucidate the mechanism of induction of crown gall disease and genetic transformation by Agrobacterium. PMID- 16666230 TI - Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina. AB - The Intracellular Na(+) concentration in the halotolerant alga Dunaliella salina was measured in intact cells by (23)Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 +/- 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the (23)Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na(+) ions and glycerol are codistribbuted within the cell volume. PMID- 16666231 TI - Gas Exchange Analysis of the Fast Phase of Photosynthetic Induction in Alocasia macrorrhiza. AB - When leaves of Alocasia macrorrhiza that had been preconditioned in 10 micromoles photons per square meter per second for at least 2 hours were suddenly exposed to 500 micromoles photons per square meter per second, there was an almost instantaneous increase in assimilation rate. After this initial increase, there was a secondary increase over the next minute. This secondary increase was more pronounced in high CO(2) (1400 microbars), where assimilation rate was assumed to be limited by the rate of regeneration of ribulose 1,5-bisphosphate (RuBP). It was absent in low CO(2) (75 microbars), where RuBP carboxylase/oxygenase (Rubisco) was assumed to be limiting. It was therefore concluded that it represented an increase in the capacity to regenerate RuBP. This fast-inducing component not only gained full induction rapidly, but also lost it rapidly in low photon flux density (PFD) with a half time of 150 to 200 seconds. It was concluded that in environments with fluctuating PFD, this fast-inducing component is an important factor in determining a leaf's potential for photosynthetic carbon gain. It is especially important during brief periods (<30 seconds) of high PFD that follow moderately long periods (1 to 10 minutes) of low PFD. PMID- 16666232 TI - Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency : I. Solute Concentrations in Leaves and Chloroplasts from Spinach Plants under NaCl or NaNO(3) Salinity. AB - Spinach (Spinacia oleracea var "Yates") plants in hydroponic culture were exposed to stepwise increased concentrations of NaCl or NaNO(3) up to a final concentration of 300 millimoles per liter, at constant Ca(2+)-concentration. Leaf cell sap and extracts from aqueously isolated spinach chloroplasts were analyzed for mineral cations, anions, amino acids, sugars, and quarternary ammonium compounds. Total osmolality of leaf sap and photosynthetic capacity of leaves were also measured. For comparison, leaf sap from salt-treated pea plants was also analyzed. Spinach plants under NaCl or NaNO(3) salinity took up large amounts of sodium (up to 400 millimoles per liter); nitrate as the accompanying anion was taken up less (up to 90 millimoles per liter) than chloride (up to 450 millimoles per liter). Under chloride salinity, nitrate content in leaves decreased drastically, but total amino acid concentrations remained constant. This response was much more pronounced (and occurred at lower salt concentrations) in leaves from the glycophyte (pea, Pisum sativum var "Kleine Rheinlanderin") than from moderately salt-tolerant spinach. In spinach, sodium chloride or nitrate taken up into leaves was largely sequestered in the vacuoles; both salts induced synthesis of quarternary ammonium compounds, which were accumulated mainly in chloroplasts (and cytosol). This prevented impairment of metabolism, as indicated by an unchanged photosynthetic capacity of leaves. PMID- 16666233 TI - Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency: II. Solute Distribution between Chloroplasts and Extrachloroplastic Space under Excess or Deficiency of Sulfate, Phosphate, or Magnesium. AB - Spinach (Spinacia oleracea var "Yates") plants grown hydroponically were exposed to an excess or deficiency of various mineral ions. Solutes were measured in leaf extracts and in isolated intact chloroplasts. Under phosphate (120 millimoles per liter NaH(2) PO(4)), sulfate (200 millimolar per liter (Na(2) SO(4)), or magnesium excess (150 millimolar per liter MgCl(2)), concentrations of these ions in leaf extracts increased, but in chloroplasts, concentrations of all ions remained constant. Concentrations of quarternary ammonium compounds in chloroplasts increased. Under mild phosphate or magnesium deficiency, concentrations of these ions decreased in chloroplasts less than in whole leaf extracts. Under severe sulfate deficiency causing chlorosis in younger leaves, sulfate concentrations in chloroplasts remained even unchanged, despite a drastic decrease of sulfate concentrations both in green and in chlorotic leaves. Together with results from a companion study (G Schroppel-Meier, WM Kaiser 1988 Plant Physiol 87: 822-827) our data demonstrate that leaf cells are able to keep the concentrations of several mineral ions rather constant in metabolically active compartments even at extremely large variations of ion concentrations in the culture solution and in the leaves. PMID- 16666234 TI - Effect of CO(2) Concentration on Protein Biosynthesis and Carbonic Anhydrase Expression in Chlamydomonas reinhardtii. AB - The effect of external inorganic carbon (C(i)) concentrations on protein biosynthesis and carbonic anhydrase (CA) mRNA abundance were examined in the eukaryotic alga Chlamydomonas reinhardtii. Transfer of high CO(2) (5%) grown algae to air levels of CO(2) resulted in the transitory synthesis of two polypeptides of approximately 49,000 and 52,000 daltons as well as prolonged synthesis and accumulation of the 37,000 dalton CA monomer and an unidentified 20,000 dalton polypeptide. The gene coding for carbonic anhydrase was isolated from a genomic expression library and subjected to restriction endonuclease analysis. Southern blot analysis of chromosomal DNA indicates that only a single copy of the gene is present. The 2.5 kilobase DNA fragment hybridizes specifically to a 1.4 kilobase transcript in RNA isolated from air-grown cells and from cells grown on 5% CO(2) that have been exposed to air levels of CO(2). Maximum mRNA abundance was observed after 1 to 3 hours of exposure to air. Transfer of air-grown cells to a high CO(2) environment resulted in the elimination of the CA transcript after 60 minutes of exposure. Changes in CA transcript abundance in response to external C(i) concentrations occurred in the presence or absence of light. PMID- 16666235 TI - Influence of manganese deficiency and toxicity on isoprenoid syntheses. AB - Twenty-eight day old wheat (Triticum aestivum L. cv Stacy) response to varying Mn concentration (10.1-10,000 micromolar) in nutrient solution was measured. Manganese concentrations in the most recently matured leaves (blade 1) were 0.21 to 19.03 mmol Mn per kilogram dry weight, respectively. Fresh and dry weights increased to a maximum at the 5 micromolar Mn nutritional level (0.37 millimole Mn per kilogram dry weight) and were decreased at Mn above and below this concentration. Blade 1 chloroplast pigment concentrations increased up to the 20 micromolar Mn nutritional level (1.98 millimole Mn per kilogram dry weight) and decreased at higher Mn concentrations. Thylakoid Mn content was above 1 mole Mn/100 mole chloroplast at Mn nutrition levels which resulted in greatly decreased plant growth. Total phytoene biosynthesis was decreased by Mn deficiency and toxicity. In vitro ent- kaurene synthesis was greatly influenced by Mn concentration with a maximal biosynthesis at 1 micromolar Mn and decreases at Mn levels above and below this concentration. In vivo blade 1 gibberellic acid equivalent concentrations were maximal at 20 parts per million Mn nutrition solution levels (1.98 millimole Mn per kilogram dry weight) and decreased at Mn tissue concentrations above and below this value; additionally, gibberellic acid concentrations were reciprocal to extracted C(20) alcohol concentrations. Mn influence on gibberellin and chloroplast pigment biosyntheses exactly matched the measured changes in growth. PMID- 16666236 TI - Synergistic enhancement of ethylene production and germination with kinetin and 1 aminocyclopropane-1-carboxylic Acid in lettuce seeds exposed to salinity stress. AB - Relief of salt (0.1 molar NaCl) stress on germination of lettuce (Lactuca sativa L., cv Mesa 659) seeds occurred with applications of 0.05 millimolar kinetin (KIN) and 1 to 10 millimolar 1-aminocyclopropane 1-carboxylic acid (ACC). Treatment with KIN enhanced the pregermination ethylene production under saline condition. A synergistic or an additive enhancement of pregermination ethylene production and germination occurred under saline condition in the presence of KIN and a saturating dose (10 millimolar) of ACC. No KIN-ACC synergism was noted in ethylene production or germination under nonsaline condition. Addition of 1 millimolar aminoethoxyvinylglycine (AVG) inhibited the KIN-enhanced pregermination ethylene production (85 to 89%) and germination (58%) under saline condition but not the synergistic effect of KIN + ACC on ethylene production. Under nonsaline condition, AVG had no effect on germination even though ethylene production was strongly inhibited. Alleviation of salt stress by KIN was inhibited in a competitive manner by 2,5-norbornadiene (NBD) (0.02-0.2 milliliter per liter), and the addition of ACC and/or ethylene reduced this inhibition. An increase in the pregermination ethylene production and germination occurred also by cotylenin E (CN) under saline condition. However, neither AVG (1 millimolar) nor NBD (0.02 to 0.2 milliliter per liter) prevented the relief of salt stress by CN. Thus, KIN may alleviate salt stress on germination by promoting both ACC production and its conversion to ethylene. Rapid utilization of ACC may be the basis for the synergistic or the additive effect of KIN plus ACC. The need for ethylene production and action for the relief of salt stress is circumvented by a treatment with CN. PMID- 16666237 TI - The relation of anatomy to water movement and cellular response in young barley leaves. AB - Young barley (Hordeum vulgare L. cv Arivat) leaves were examined anatomically and physiologically to infer the pathway of transpirational water movement and to explain why the growing region is more responsive to osmotic stress than the expanded blade. Vessels with open lumens extend from the intercalary meristem to the expanded blade, and all vessels are clustered in five vascular bundles that are separated by 20 closely packed mesophyll cells. Heat pulse transport data confirmed the anatomical suggestion that water moves through the growing region in vessels and not intercellularly, and also showed that stress reduces xylem water transport within 1 minute while transpiration remained unaffected. Water equal in volume to twice that expected in the xylem, and which exchanges more readily with water in the nutrient solution than with most water in tissues, can be extracted easily from growing tissues. It is hypothesized that this water is xylem plus cell wall water, that osmotic stress will quickly reduce its in situ water potential, and that stress causes growth to stop because cells in the growing region can respond rapidly to changes in water potential around them. In the expanded blade, bundles containing vessels are three and eight cells away from the closest and next substomatal cavities. This allows xylem water loss to occur predominantly through the closest stomata, and the expanded blade is believed to be less responsive because effects of stress on xylem water potential are confined largely to cells immediately around the vessels. PMID- 16666238 TI - Determination of the Processing Sites of an Arabidopsis 2S Albumin and Characterization of the Complete Gene Family. AB - The most abundant isoform of the 2S albumin present in seeds of Arabidopsis thaliana has been sequenced and the corresponding gene isolated. Examination of the protein and DNA sequences allows the determination of the exact proteolytic cleavage sites during posttranslational processing. Like other 2S albumins, that of Arabidopsis is made as a prepropeptide. After removal of the signal peptide, the propeptide is cleaved at four other points, giving two subunits linked by a disulfide bridge(s). Comparison of these cleavage sites with those of 2S albumins of Brassica napus and Bertholletia excelsa suggests that while individual cleavage sites between species are conserved, the four processing sites within a species are not similar, suggesting that up to four different proteases may be involved in processing 2S albumins. The Arabidopsis 2S albumin gene was used to isolate the entire gene family. There are four genes, tightly linked in a tandem array. None of the genes contains an intron. Comparison of the predicted protein sequences shows that only one of the genes can encode the isoform determined by protein analysis to be the most abundant, and therefore this gene is certain to be expressed. It is possible that some or all of the other three genes are also active. PMID- 16666239 TI - Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2). AB - Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO(2) compensation points at different O(2) levels, which is typical of C(4) plants, yet it does show about 4% inhibition of net photosynthesis by 21% O(2) at 30 degrees C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C(4) pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C(3) cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial (14)C products in whole leaves and extrapolation of pulse labeling curves to zero time indicated that about 80% of the CO(2) is fixed into C(4) acids (malate and aspartate), whereas about 20% of the CO(2) directly enters the C(3) cycle. This is consistent with the high activity of enzymes for CO(2) fixation by the C(4) pathway and the substantial activity of enzymes of the C(3) cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C(3) photosynthesis in the mesophyll cells and should be considered a C(4) like species. PMID- 16666240 TI - Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls I : Purification of Novel Enzymes Capable of Dissociating Feraxan Fragments from Zea mays Coleoptile Cell Wall. AB - Three novel beta-xylan xylanohydrolases capable of dissociating ferulated arabinoxylan (Feraxan) from maize (Zea mays L. hybrid B73 x Mo17) coleoptile sections and two conventional beta-xylan xylanohydrolases (xylanases) were purified from a Bacillus subtilis industrial enzyme preparation (Novo Ban L-120). The Feraxan-dissociating enzymes (designated as feraxanases) exhibit optimum activities between pH 6.5 and 7.0 and have common molecular weights of 45 kilodaltons as studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two xylanases exhibit their optimum activities between pH 4.5 and 6.0 and have common molecular weights of 27 kilodaltons. Feraxanases liberate oligomeric fragments, which accounted for the following percentages of walls of Zea mays coleoptile sections that had been pretreated by boiling in 80% ethanol: 76% of the ferulic acid, 96% of the arabinose, 71% of the xylose, 27% of the galactose, 50% of the uronic acid, and 4% of the glucose. Monomers, dimers, trimers, or tetramers were not found among enzyme digestion products. The enzymes hydrolyzed both Feraxan in intact cell wall and maize arabinoxylans extracted from walls by alkaline solutions but did not degrade other substrates including larch arabinoxylan and Rhodymenia xylan. Structural analyses of the fragments released by the enzymes from the maize cell wall indicated the presence of 2,4/3,4-linked-xylopyranosyl, terminal-arabinofuranosyl, 5-linked arabinofuranosyl, 4-linked-xylopyranosyl, terminal-glucuronopyranosyl, and ferulic acid as major components. This result is consistent with the idea that most of the fragments were derived from Feraxan. Because of high enzyme specificity and substantial recovery of digestion products from maize cell walls, these new enzymes offer opportunities not only for enhanced structural analyses of cell walls but also for assistance in protoplast preparation from cereals. PMID- 16666241 TI - Glyphosate Induces 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Potato (Solanum tuberosum L.) Cells Grown in Suspension Culture. AB - The activity of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino heptulosonate 7-phosphate synthase, varies during the growth cycle of Solanum tuberosum L. cv superior cells in suspension culture. Maximum specific enzyme activity was observed midway through the linear phase of growth. When mid-log phase cells are exposed to glyphosate, the specific activity of the enzyme increases severalfold within 24 hours. The glyphosate-induced increase in enzyme activity is due to an increase in the amount of enzyme as determined by immunoblotting. Glyphosate (up to 2 millimolar) has no effect on the enzyme activity in vitro. Dehydroquinate synthase, the second enzyme of the shikimate pathway, is not induced by glyphosate. PMID- 16666242 TI - Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP Dependent Protein Kinase. AB - An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca(2+), calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a K(M) of 450 micromolar for ATP and a V(max) of 25 picomoles of (32)P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (K(i) 150 micromolar). PMID- 16666243 TI - The Effect of Temperature on the Level and Biosynthesis of Unsaturated Fatty Acids in Diacylglycerols of Brassica napus Leaves. AB - Experiments on the effects of temperature on the levels of unsaturated fatty acids and their rates of desaturation in Brassica napus leaf lipids have shown that significant differences occur in the composition of all diacylglycerols in the leaf between plants grown at high and low temperatures. In the major thylakoid diacylglycerols, monogalactosyl-diacylglycerol and digalactosyldiacylglycerol, not only is there an increase in the level of unsaturation at low temperatures, but there is a change in the balance between molecular species of chloroplastic origin (16/18C) and cytosolic origin (18/18C). Radioactivity tracer data indicate that at low temperatures there are two distinct phases of desaturation in the fatty acids of the major diacylglycerols of these leaves. A rapid phase, which appears in plants grown at low temperatures and results in the desaturation of palmitic acid to hexadecadienoic acid and oleic acid to linoleic acid may explain the high levels of unsaturated fatty acids found in the leaf diacylglycerols from plants grown at low temperatures. The appearance of this rapid phase is controlled by the temperature at which the plant is grown and is not subject to rapid variations in environmental temperature. PMID- 16666244 TI - Isoosmotic regulation of cotton and peanut at saline concentrations of k and na. AB - Peanut (Arachis hypogaea L.) and cotton (Gossypium hirsutum) plants were grown for 4 weeks in saline, isoosmotic rooting substrates with different proportions of K and Na. Isoosmotic media did not affect growth (except at the highest external K concentrations) or estimates of intracellular osmotic pressure in expanding leaves (i.e. osmotic pressure of leaf sap and intracellular osmotic pressure as calculated from pressure-volume curves). In expanded leaves, an increase in the proportion of external K increased sap osmotic pressure. The sum of [K+Na+Cl] in the sap of expanding and expanded leaves accounted for the effect of isoosmotic media on the concentration of osmolytes with high electrical conductance, so the difference between sap osmotic pressure and [K+Na+Cl] accounted for the concetration of osmolytes with low conductance. In expanding leaves, an increase in the proportion of external K increased [K+Na+Cl] and decreased the concentration of osmolytes with low conductance. In expanded leaves, an increase in the proportion of external K increased [K+Na+Cl] to approximately the same extent as sap osmotic pressure. Isoosmotic regulation was apparent in expanding leaves but not evident in expanded leaves. This suggests a turgor homeostat which can influence the concentration of organic solutes in expanding leaves but cannot control the import of inorganic solutes from a rooting medium nor the total production of organic solutes in plants with a low sink:source ratio. PMID- 16666245 TI - Catalysis of Ribulosebisphosphate Carboxylase/Oxygenase Activation by the Product of a Rubisco Activase cDNA Clone Expressed in Escherichia coli. AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase activity was obtained from a partially purified extract of Escherichia coli transformed with a 1.6-kilobase spinach (Spinacia oleracea L.) cDNA clone. This activity was ATP dependent. Catalysis of rubisco activation by spinach and cloned rubisco activase was accompanied by the same extent of carboxyarabinitol bisphosphate-trapped (14)CO(2) as occurred in spontaneous activation, indicating that rubisco carbamylation is one facet of the rubisco activase reaction. The CO(2) concentration required for one-half maximal rubisco activase activity was about 8 micromolar CO(2). These observations are consistent with the postulated role of rubisco activase in regulating rubisco activity in vivo. PMID- 16666246 TI - Explorations in the "inner sanctum of the photosynthetic process," the water oxidizing system. AB - This article summarizes the present state of knowledge about the organization of the water oxidizing polypeptide complex of photosynthesis and the mechanism of its operation. Spectacular advances have been made in recent years, but many questions remain to be answered. Among them are those concerned with the organization and binding of the functional Mn, its specific role in the oxidant storage that precedes the formation of one oxygen from two water molecules, and its cooperation with other redox-active constituents. Much is still to be learned also about the function of the individual polypeptides and about the role of the cofactors Ca(2+) and Cl(-). Differences between the organization of the water oxidizing complexes in green plants and cyanobacteria suggest, furthermore, that in vivo their water oxidizing mechanisms must operate under different, still unrecognized constraints. PMID- 16666247 TI - Preliminary Genetic Studies of the Phenotype of Betaine Deficiency in Zea mays L. AB - Glycinebetaine-deficient inbreds of Zea mays do not exhibit a general deficiency of nitrogenous solutes; the total free amino acid levels of betaine-deficient lines are not significantly less than those of inbreds which exhibit >100-fold higher betaine levels. Betaine-deficient inbreds are characterized by extremely low betaine: total free amino acid ratios (<0.0015). Highly significant correlations are demonstrated between the expected mid-parent and observed betaine:amino acid ratios of 30 hybrids of known pedigree. In 12 hybrids constructed from a betaine-deficient male parent (inbred 1506), the observed betaine:amino acid ratios of the hybrids are proportional to the betaine:amino acid ratios of the female parents (r = 0.83). Two hybrids, 1146 x 1074 and 1146 x 1506, were chosen for further genetic analysis. The common female parent (1146) and inbred 1074 both exhibit betaine:amino acid ratios of 0.090, a value which is approximately 90-fold greater than the betaine:amino acid ratio of inbred 1506. Hybrid 1146 x 1074 exhibits almost exactly twice the betaine:amino acid ratio of hybrid 1146 x 1506. If inbred 1506 is homozygous recessive for a single nuclear gene responsible for the phenotype of betaine deficiency, and if inbreds 1146 and 1074 are homozygous dominant for this allele, then this twofold difference in betaine:amino acid ratio must be associated with the homozygous dominant and heterozygous conditions, respectively, for 1146 x 1074 and 1146 x 1506. Evidence is presented from both greenhouse and field evaluations of F(2) populations of these hybrids that a single nuclear recessive gene is most likely responsible for the phenotype of betaine-deficiency in inbred 1506. Approximately 25% of the F(2) segregants from 1146 x 1506 exhibited extremely low betaine:amino acid ratios (<0.0015), whereas 0% of the F(2) segregants from 1146 x 1074 exhibited this phenotype. The segregation patterns with respect to betaine:amino acid ratio suggest a 1:2:1 segregation ratio for homozygous recessive:heterozygous:homozygous dominant individuals within the 1146 x 1506 F(2) population. PMID- 16666248 TI - S-adenosylmethionine-dependent inactivation and radiolabeling of 1 aminocyclopropane-1-carboxylate synthase isolated from tomato fruits. AB - 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase was partially purified from the homogenate of wounded tomato (Lycoperiscon esculentum Mill.) pericarp tissue by (NH(4))(2)SO(4) fractionation followed by conventional column chromatography with diethylaminoethyl-Sepharose, Sephadex G-150, Affi-Gel blue and hydroxylapatite. The partially purified ACC synthase preparation attained a specific activity of about 12,000 nmoles per hour per milligram protein. Employing this enzyme preparation, we confirmed that the ACC synthase was inactivated by its substrate, S-adenosyl-l-methionine (SAM), during its catalytic action. When the partially purified enzyme preparation was incubated with [3,4 (14)C]SAM and the resulting proteins were analyzed by sodium dodecyl sulfate-gel electrophoresis, only one radioactive protein band was observed. This protein was thought to be ACC synthase based on its molecular mass of 50 kD and on the fact that it was specifically bound to a monoclonal antibody against ACC synthase (AB Bleecker et al. 1986 Proc Natl Acad Sci USA 83, 7755-7759). These results suggest that the substrate SAM acts as an enzyme-activated inactivator of ACC synthase by covalently linking a fragment of SAM molecule to the active site of ACC synthase, resulting in the inactivation of the enzyme. PMID- 16666249 TI - Endogenous ethylene production is a potential problem in the measurement of nitrogenase activity associated with excised corn and sorghum roots. AB - Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of field-grown corn, Zea mays L. cv Funks G-4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots. PMID- 16666250 TI - Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.). AB - Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg(2+)-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg(2+), Mn(2+) and Ca(2+), whereas it was not affected by Sr(2+), K(+), or Na(+). The K(m) for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N'-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg(2+)-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F(1) ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692). PMID- 16666251 TI - The Influence of Leaf Age on C(4) Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C(4) Dicot. AB - Characteristics of C(4) photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C(4) dicot). The turnover of [4-(14)C] (malate plus aspartate) following a pulse with (14)CO(2) was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of (14)CO(2) incorporation in mid-age leaves was about 1.5-fold higher than in young leaves, and about 2.5-fold higher than in mature leaves. The rate of (14)CO(2) fixation was proportional to the total active pool of malate plus aspartate but was not correlated with the total photosynthetically derived inorganic carbon pool. The leaf's ability to concentrate inorganic carbon photosynthetically declined during leaf expansion, from 29 down to 7 nanomoles per milligram chlorophyll. Similarly, the active aspartate pool also declined during leaf expansion, from about 123 down to 20 nanomoles per milligram chlorophyll. Enhanced metabolism of aspartate to CO(2) and pyruvate in young leaves is suggested to facilitate the maintenance of high CO(2) levels in bundle sheath cells which are thought to have a higher conductance to CO(2). PMID- 16666252 TI - Oxidative decarboxylation of branched-chain 2-oxo Fatty acids by higher plant peroxisomes. AB - Peroxisomes from mung bean (Vigna radiata L.) hypocotyls catalyze, in the presence of branched-chain 2-oxo fatty acid, CoASH and NAD, the release of CO(2), and the formation of NADH and acyl-CoA. The acyl-CoA contains one carbon atom less than the branched-chain 2-oxo fatty acid and serves as substrate for the peroxisomal acyl-CoA oxidase. CO(2) release, NADH and acyl-CoA formation occur in 1:1:1 stoichiometry. For the first time the data demonstrate directly the oxidative decarboxylation of branched-chain 2-oxo fatty acids in higher plants and a location of this activity in the peroxisomes. PMID- 16666253 TI - Regulation of electrogenic proton pumping by auxin and fusicoccin as related to the growth of Avena coleoptiles. AB - The temporal relations between early responses to indoleacetic acid (IAA), proton secretion, hyperpolarization of the membrane potential, and growth change during the incubation of segments of oat (Avena sativa L.) coleoptiles in a low salt medium. When IAA is added after pretreatment of several hours, proton secretion increases after a latency of 7 minutes and reaches its maximum 10 to 15 minutes later. This timing coincides with both the increase in growth of the segments and the hyperpolarization of the membrane potential of parenchyma cells, consistent with the hypothesis that the change in membrane voltage reflects the activity of an electrogenic proton pump. The extent of IAA-induced hyperpolarization is substantially reduced by elevating [KCl](0), most likely because this increases the passive conductance of the membrane. Neither growth nor proton secretion is affected by high [KCl](0) (30 millimolar), indicating that neither process is limited by the magnitude of the membrane potential. These results are consistent with the acid growth hypothesis. Following short incubation times, however, IAA induced hyperpolarization and growth are detected within 10 minutes, while acidification of the medium is delayed for more than 40 minutes. This result is seemingly in conflict with the acid growth hypothesis, but in freshly cut tissue, the pH of the external medium may not reflect the pH of the epidermal cell walls. The temporal coincidence of auxin-induced growth and hyperpolarization suggests that in freshly isolated segments the hyperpolarization is a more sensitive indication of proton secretion than is acidification of the external aqueous environment. PMID- 16666254 TI - Effects of Analogs of the Fungal Sexual Pheromone Sirenin on Male Gamete Motility in Allomyces macrogynus. AB - The ability of various structural analogs of the sexual pheromone sirenin to attract male gametes of the aquatic fungus Allomyces macrogynus was determined. Previous studies had shown that several structural analogs and stereoisomers of natural l-sirenin were devoid of activity at physiological concentrations. We now report the discovery of a structural analog that exhibits biological activity indistinguishable from the natural pheromone. The bioassay system used to determine chemotaxis was calibrated using synthetic, racemic sirenin, which exhibited a threshold concentration for gamete attraction at an applied concentration of 10 picomolar. The new synthetic monohydroxy analog of sirenin also had a threshold concentration of 10 picomolar. In the process of developing a new total synthesis of sirenin, a variety of other analogs were prepared and tested. All of these analogs exhibited threshold concentrations at 1 micromolar or higher, although attraction at these higher concentrations still varied according to their structural resemblance to sirenin. Thus, the results of these studies demonstrate that the hydroxymethyl group attached to the six-membered ring of sirenin is not essential for biological activity at physiological concentrations. The studies with other analogs demonstrate that biological activity at any concentration involves a balance between hydrophilic hydroxyl groups and hydrophobic hydrocarbon groups in the structure. PMID- 16666255 TI - Effect of endogenously synthesized and exogenously applied ethanol on tomato fruit ripening. AB - Tomato (Lycopersicon esculentum Mill. var Castlemart) fruit ripening was inhibited by tissue concentrations of ethanol that were produced by either exposure to exogenous ethanol vapors or synthesis under anaerobic atmospheres. Ethanol was not detected in aerobically ripened tomato fruit. Ripening was not inhibited by exposure to methanol at an equivalent molar concentration to inhibitory concentrations of ethanol, while ripening was slightly more inhibited by n-propanol than by equivalent molar concentrations of ethanol. The mottled appearance of a few ripened ethanol-treated fruit was not observed in n-propanol treated fruit. PMID- 16666256 TI - Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Alocasia macrorrhiza in Response to Step Changes in Irradiance. AB - The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (Rubisco) activity and pool sizes of RuBP and P-glycerate were examined in the tropical understory species Alocasia macrorrhiza following step changes in photon flux density (PFD). Previous gas exchange analysis of this species following a step increase in PFD from 10 to 500 micromoles quanta per square meter per second suggested that the increase in photosynthetic rate was limited by the rate of increase of Rubisco activity for the first 5 to 10 minutes. We demonstrate here that the increase in photosynthetic rate was correlated with an increase in both the activation state of Rubisco and the total k(cat) (fully activated specific activity) of the enzyme. Evidence presented here suggests that a change in the pool size of the naturally occurring tight binding inhibitor of Rubisco activity, 2 carboxyarabinitol 1-phosphate, was responsible for the PFD-dependent change in the total k(cat) of the enzyme. RuBP pool size transiently increased after the increase in PFD, indicating that photosynthesis was limited by the capacity for carboxylation. After 5 to 10 minutes, RuBP pool size was again similar to the pool size at low PFD, presumably because of the increased activity of Rubisco. Following a step decrease in PFD from 500 to 10 micromoles quanta per square meter per second, Rubisco activity declined but at a much slower rate than it had increased in response to a step increase in PFD. This slower rate of activity decline than increase was apparently due to the slower rate of 2 carboxyarabinitol 1-phosphate synthesis than degradation and, to a lesser degree, to slower deactivation than activation. RuBP pool size initially declined following the decrease in PFD, indicating that RuBP regeneration was limiting photosynthesis. As Rubisco activity decreased, RuBP slowly increased to its original level at high PFD. The slow rate of activity loss by Rubisco in this species suggests a biochemical basis for the increased efficiency for CO(2) assimilation of successive lightfleck use by species such as A. macrorrhiza. PMID- 16666257 TI - The Effect of Adenine Nucleotides on Purified Phosphoenolpyruvate Carboxylase from the CAM Plant Crassula argentea. AB - The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the K(m) for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the K(m) for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the K(a) for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg(2+) was held constant and saturating, these effects cannot be ascribed to Mg(2+) chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the "R" type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day. PMID- 16666258 TI - Relationship between Steady-State Fluorescence Yield and Photosynthetic Efficiency in Spinach Leaf Tissue. AB - The relationship between steady-state photosynthetic efficiency, as moles CO(2) per mole of incident visible photons under 2% O(2), and chlorophyll fluorescence quenching has been investigated in intact leaf tissue of Spinacia oleracia. Fluorescence yield was measured using a pulse amplitude modulation technique that permitted rapid and sensitive resolution and quantitation of photochemical and nonphotochemical quenching coefficients. A highly linear relationship was observed between photosynthetic efficiency and the ratio of photochemical:nonphotochemical quenching coefficients for values of the latter less than 1.6. This relationship applied whether irradiance or CO(2) concentration was varied. The observed relationships between photochemical yield and fluorescence yield were compatible with the photosystem II model proposed by Butler and Kitajima (1975 Biochim Biophys Acta 376: 116-125). The results are discussed with respect to the proposed role of nonphotochemical quenching in regulating radiant energy utilization and also the applicability of fluorescence measurements as a means of estimation of the rate of photosynthetic electron transport. PMID- 16666259 TI - DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development. AB - We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated. PMID- 16666260 TI - Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases. AB - Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca(2+) -dependent protein kinase. The Ca(2+) dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as beta-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid binding. The Ca(2+) -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca(2+) -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca(2+) -dependent binding to phenyl Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca(2+) -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K(m) values for ATP, and in Ca(2+) independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca(2+) -dependent protein kinases present in the soluble phase. PMID- 16666261 TI - Purification and properties of glucoamylase from sugar beet cells in suspension culture. AB - Glucoamylase and alpha-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with alpha-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the alpha-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of alpha-mannosidase and alpha-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained alpha-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M(r) (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K(m) value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some alpha-amylases and the debranching enzyme. PMID- 16666262 TI - Xanthoxin Metabolism in Cell-free Preparations from Wild Type and Wilty Mutants of Tomato. AB - Extracts prepared from the turgid and water-stressed leaves of wild-type tomato (Lycopersicon esculentum Mill cv Ailsa Craig) and the wilty mutants sitiens, notabilis, and flacca were tested for their ability to metabolize xanthoxin to ABA. Extracts from wild type and notabilis converted xanthoxin at similar rates, while extracts from sitiens and flacca showed little or no activity. We also observed no activity when extracts of sitiens and flacca were mixed. Similar results were obtained when ABA aldehyde was used as a substrate, in that extracts from wild type and notabilis were equally active, but extracts from flacca and sitiens showed little activity. None of the tomato extracts showed significant activity with xanthoxin acid, xanthoxin alcohol, or ABA-1',4-'Trans-diol as substrates. Extracts from bean leaves (Phaseolus vulgaris L. cv Blue Lake) were similar to the wild-type tomato extracts in their ability to convert the various substrates to ABA, although excised bean leaves did convert ABA-1',4'-trans-diol and xanthoxin alcohol to ABA when these substances were taken up through the petiole. These results are consistent with a role for xanthoxin as a normal intermediate on the ABA biosynthetic pathway, and they suggest that ABA aldehyde is the final ABA precursor. PMID- 16666263 TI - Effects of Various Levels of CO(2) on the Induction of Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq. AB - In response to water stress, Portulacaria afra (L.) Jacq. (Portulacaceae) shifts its photosynthetic carbon metabolism from the Calvin-Benson cycle for CO(2) fixation (C(3)) photosynthesis or Crassulacean acid metabolism (CAM)-cycling, during which organic acids fluctuate with a C(3)-type of gas exchange, to CAM. During the CAM induction, various attributes of CAM appear, such as stomatal closure during the day, increase in diurnal fluctuation of organic acids, and an increase in phosphoenolpyruvate carboxylase activity. It was hypothesized that stomatal closure due to water stress may induce changes in internal CO(2) concentration and that these changes in CO(2) could be a factor in CAM induction. Experiments were conducted to test this hypothesis. Well-watered plants and plants from which water was withheld starting at the beginning of the experiment were subjected to low (40 ppm), normal (ca. 330 ppm), and high (950 ppm) CO(2) during the day with normal concentrations of CO(2) during the night for 16 days. In water-stressed and in well-watered plants, CAM induction as ascertained by fluctuation of total titratable acidity, fluctuation of malic acid, stomatal conductance, CO(2) uptake, and phosphoenolpyruvate carboxylase activity, remained unaffected by low, normal, or high CO(2) treatments. In well-watered plants, however, both low and high ambient concentrations of CO(2) tended to reduce organic acid concentrations, low concentrations of CO(2) reducing the organic acids more than high CO(2). It was concluded that exposing the plants to the CO(2) concentrations mentioned had no effect on inducing or reducing the induction of CAM and that the effect of water stress on CAM induction is probably mediated by its effects on biochemical components of leaf metabolism. PMID- 16666264 TI - Action Spectrum for Interaction between Visible and Far-Red Light on Face Chloroplast Orientation in Mougeotia. AB - The orientation of chloroplasts from profile to face position in Mougeotia can be controlled in two ways: by a typical phytochrome-mediated system or by continuous, simultaneous irradiation with far-red and visible light. In experiments with dichromatic irradiation of Mougeotia, the light conditions applied prevented the formation of a far-red-absorbing form of phytochrome gradient in the cell. An unpolarized background of far-red light and linearly polarized monochromatic light of different wavelengths and vibrating parallel to the cell axis, if given by themselves, were completely ineffective in producing any changes in chloroplast orientation. Given together, however, changes in chloroplast orientation were induced. The action spectrum for this interaction between constant far-red and variable visible light was maximal at 620 nanometers. The chloroplast response in these dichromatic light conditions required a prolonged duration of exposure to simultaneous continuous irradiation of high fluence energy. The vibrating plane of linearly polarized 620 nanometer light had no significant influence on interaction with far-red light in chloroplast movement. The results obtained are different from the typical low energy phytochrome-mediated chloroplast orientation. This new type of chloroplast photoresponse might be mediated by an unknown sensory pigment. PMID- 16666265 TI - Structural Effects of Cl and Other Anions on the Water Oxidizing Complex of Chloroplast Photosystem II. AB - To further our understanding of the role of Cl(-) and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl(-) free media of pH approximately 7 was enhanced by millimolar concentrations of the divalent anion SO(4) (2-) and also by divalent cations like Mg(2+) and Ca(2+); (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl(-) = Br(-) > NO(3) (-) > F(-) > ClO(4) (-); (c) at molar concentrations, SO(4) (2-) stabilized the binding of the 23 kilodalton polypeptide, while Cl(-) and Br(-) became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0 degrees C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl(-), or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by "lyotropic" actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains. PMID- 16666266 TI - Chloroplast osmotic adjustment and water stress effects on photosynthesis. AB - Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var ;Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Psi(w)) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Psi(w) reached -1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Psi(w) may involve stromal volume maintenance. PMID- 16666267 TI - Effects of temperature pretreatment in the dark on photosynthesis of the intact spinach chloroplast. AB - Isolated, intact spinach (Spinacia oleracea L. var. "Long Standing Bloomsdale") chloroplasts were heated in the dark and the effect of this treatment on photosynthetic activities was determined at 25 degrees C. Dark incubation of the chloroplasts for 10 minutes at 35 degrees C and pH 8.1 resulted in a 50% decline in CO(2) photoassimilation. This decline in photosynthetic performance was dependent upon time, temperature, and medium pH with the optimum effect at acidic pH values. Photosynthetic decline was not observed if MgATP, MgADP, or a mixture of fructose 1,6-bisphosphate, aldolase, and oxaloacetate or ribose 5-phosphate and oxaloacetate was added prior to but not after the temperature pretreatment. A chloroplast preparation reconstituted with thylakoids and stroma from pretreated (35 degrees C, 10 minutes, pH 8.1) intact chloroplasts and supplemented with ferredoxin, ADP, and NADP was photosynthetically competent, indicating that ATP coupled electron flow and the enzymes comprising the Benson-Calvin cycle remained stable during the dark treatment. In contrast, exposure of isolated thylakoids to 35 degrees C for 10 minutes uncoupled photophosphorylation from NADP and ferricyanide reduction. We propose that the decline of intact chloroplast photosynthesis is the result of a decrease in the content of or a change in the ratios of the adenine nucleotides. Maintenance of an adequate supply of adenine nucleotide is the effect of the externally added MgATP or of chloroplastic respiration of a sugar phosphate. PMID- 16666268 TI - Circadian Control of the Accumulation of mRNAs for Light- and Heat-Inducible Chloroplast Proteins in Pea (Pisum sativum L.). AB - The levels of the mRNAs for light-inducible, nuclear-coded chloroplast proteins vary rhythmically in pea (Pisum sativum L.) plants either grown in a dark-light cycle or under constant light conditions. This has been observed for the early light-inducible protein, the light-harvesting chlorophyll a/b protein, and the small subunit of the ribulose-1,5-bisphosphate carboxylase. The mRNA levels are high in the morning, exhibit a minimum in the first half of the night, and increase again during the second half of the night. The amplitude of fluctuation is between 5- and 10-fold. A similar change in the mRNA abundance was found for four nuclear encoded heat-shock proteins of 18, 24, 26, and 30 kilodaltons. The ability of plants to transcribe heat-shock genes upon heat-shock for 2 hours varies through the day. The maxima for induction are found in the second half of the night and the morning. The minima are reached during the afternoon. The degree of fluctuation is between 3- and 5-fold. The levels of mRNAs for cytosolic as well as for plastid heat-shock proteins oscillate in parallel. PMID- 16666269 TI - Stable Carbon Isotope Composition (deltaC), Water Use Efficiency, and Biomass Productivity of Lycopersicon esculentum, Lycopersicon pennellii, and the F(1) Hybrid. AB - Three tomatoes, Lycopersicon esculentum Mill. cv UC82B, a droughttolerant wild related species, Lycopersicon pennellii (Cor.) D'Arcy, and their F(1) hybrid, were grown in containers maintained at three levels of soil moisture. Season-long water use was obtained by summing over the season daily weight losses of each container corrected for soil evaporation. Plant biomass was determined by harvesting and weighing entire dried plants. Season-long water use efficiency (gram dry weight/kilogram H(2)O) was calculated by dividing the dry biomass by the season-long water use. The season-long water use efficiency was greatest in the wild parent, poorest in the domestic parent, and intermediate (but closer to the wild parent) in the F(1) hybrid. Instantaneous water-use efficiency (micromole CO(2)/millimole H(2)O) determined by gas exchange measurements on individual leaves was poorly correlated with season-long water use efficiency. However, the relative abundance of stable carbon isotopes of leaf tissue samples was strongly correlated with the season-long water use efficiency. Also, the isotopic composition and the season-long water use efficiency of each genotype alone were strongly negatively correlated with plant dry weight when the dry weight varied as a function of soil moisture. PMID- 16666270 TI - Effects of Air Pollutants on the Composition of Stable Carbon Isotopes, deltaC, of Leaves and Wood, and on Leaf Injury. AB - Air pollutants are known to cause visible leaf injury as well as impairment of photosynthetic CO(2) fixation. Here we evaluate whether the effects on photosynthesis are large enough to cause changes in the relative composition of stable carbon isotopes, delta(13)C, of plant tissue samples, and, if so, how the changes relate to visual leaf injury. For that purpose, several woody and herbaceous plant species were exposed to SO(2) + O(3) and SO(2) + O(3) + NO(2) for one month (8 hours per day, 5 days per week). At the end of the fumigations, the plants were evaluated for visual leaf lesions, and delta(13)C of leaf tissue was determined. Woody plants generally showed less visual leaf injury and smaller effects on delta(13)C of pollutant exposure than did herbaceous plants. If delta(13)C was affected by pollutants, it became, with few exceptions, less negative. The data from the fumigation experiments were consistent with delta(13)C analyses of whole wood of annual growth rings from two conifer tree species, Pseudotsuga menziesii and Pinus strobus. These trees had been exposed until 1977 to exhaust gases from a gas plant at Lacq, France. Wood of both conifer species formed in the polluted air of 1972 to 1976 had less negative delta(13)C values than had wood formed in the much cleaner air in 1982 to 1986. No similar, time-dependent differences in delta(13)C of wood were observed in trees which had been continuously growing in clean air. Our delta(13)C data from both relatively short-term artificial exposures and long-term natural exposure are consistent with greater stomatal limitation of photosynthesis in polluted air than in clean air. PMID- 16666271 TI - Polyamines in relation to growth in carrot cell cultures. AB - Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed. PMID- 16666272 TI - Light-affected ca fluxes in protoplasts from vallisneria mesophyll cells. AB - In Vallisneria gigantea Graebner mesophyll cells, red light irradiation induces cytoplasmic streaming by decreasing the Ca(2+) concentration in the cytoplasm, while far-red light irradiation inhibits it by increasing the concentration (S Takagi, R Nagai 1985 Plant Cell Physiol 26: 941-951). To examine the effects of light irradiation on Ca(2+) fluxes across the cell membrane, protoplasts are isolated from the mesophyll cells. Changes in Ca(2+) concentration in a solution bathing the protoplasts are monitored by spectrophotometry, using the Ca(2+) sensitive dye murexide. Red light irradiation induces an increase in Ca(2+) concentration, which means an efflux of Ca(2+) from the protoplasts. Subsequent far-red light irradiation produces a rapid decrease in Ca(2+) concentration down to the dark control level; however, this is not observed in the presence of the Ca(2+) -channel blocker nifedipine. Vanadate inhibits both the streaming and the Ca(2+) efflux induced by red light irradiation. The results suggest that red light and far-red light control Ca(2+) movements across the cell membrane, which in turn regulate the streaming. PMID- 16666273 TI - Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process. AB - Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C(1), xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO(3), an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O(2)-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A(2), which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity. PMID- 16666274 TI - Localization and toxic effects of cadmium, copper, and uranium in azolla. AB - The storage and distribution of copper, cadmium, and uranium and their effects on ionic contents in roots and shoots of Azolla filiculoides has been studied by x ray microanalysis. The relative content of copper was eightfold higher in the root than in the shoot, suggesting low mobility of this metal in Azolla plant. Cadmium relative content in the shoot was similar to its content in the root, hence its mobility was relatively high. The absence of significant uranium quantities in the shoot and its relative high content in the root suggest the immobility of this metal from Azolla root. Cadmium formed precipitates with phosphate and calcium in xylem cells of the shoot bundle and caused a two- to threefold increase in the content of phosphate in the root. Uranium in roots and cadmium in shoots were associated with calcium. All three treatments caused losses of potassium, chloride, and magnesium from Azolla roots. Accumulation of heavy metals in Azolla and their mobility from the root to the shoot can be correlated with damage caused by the loss of essential nutrients. PMID- 16666275 TI - Molecular Features Affecting the Biological Activity of the Host-Selective Toxins from Cochliobolus victoriae. AB - The structures of the toxins produced by Cochliobolus victoriae, victorin B, C, D, E, and victoricine, have recently been established. These toxins and modified forms of victorin C were tested for their effect on dark CO(2) fixation in susceptible oat (Avena sativa) leaf slices. Half-maximal inhibition of dark CO(2) fixation occurred with the native toxins in the range of 0.004 to 0.546 micromolar. An essential component for the inhibitory activity of victorin is the glyoxylic acid residue, particularly its hydrated aldehyde group. Removal of glyoxylic acid completely abolished the inhibitory activity of victorin, and the reduction of the aldehydo group transformed the toxin into a protectant. Conversion of victorin to its methyl ester resulted in diminution of inhibitory activity to 10% of the original activity of the toxin, whereas derivatization of the epsilon-amino group of the beta-hydroxylysine moiety resulted in a decrease of inhibitory activity to 1% of that of victorin C. However, the derivatized toxin retained its host selectivity. In addition, the opening of the macrocyclic ring of the toxin drastically reduced the inhibitory activity. PMID- 16666276 TI - Chromoplast-Specific Proteins in Capsicum annuum. AB - Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M(r) of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species. PMID- 16666277 TI - Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : I. Induction and Purification. AB - l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO(2) produced per minute per milligram protein at pH 8.4 and 30 degrees C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 +/- 300 daltons. PMID- 16666278 TI - Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization. AB - Properties of purified l-tyrosine decarboxylase (EC 4.1.1.25) from elicitor induced cell suspension cultures of Eschscholtzia californica Cham. and Thalictrum rugosum Ait. are described. l-Tyrosine decarboxylase is a dimeric enzyme with a molecular weight of 112,600 +/- 600 daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4 for the enzyme from E. californica and T. rugosum, respectively. The purified enzymes were stabilized in the presence of pyridoxal-5-phosphate. Optimum pH for the enzyme from both plants was found to be 8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5 phosphate. The enzyme decarboxylated l-tyrosine and l-beta-3,4 dihydroxyphenylalanine but was inactive toward l-phenylalanine and l-tryptophan. Apparent K(m) values of Eschscholtzia- and Thalictrum-decarboxylase for l tyrosine were 0.25 +/- 0.03 and 0.27 +/- 0.04 millimolar, respectively. Similar affinities were found for l-3,4-dihydroxyphenylalanine. Eschscholtzial-tyrosine decarboxylase was strongly inhibited by the phenylalanine analogue l-alpha aminooxy-beta-phenylpropionate and largely unaffected by d,l-alpha monofluoromethyl-3,4-dihydroxyphenylalanine and alpha-difluoromethyltyrosine. PMID- 16666279 TI - Acyl coenzyme a preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed. AB - The acyl coenzyme A (CoA) preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm (Butia capitata Becc.), maize (Zea mays L.), and rapeseed (Brassica napus L.) was tested. Each microsomal preparation was incubated with [(14)C-U]-glycerol-3-phosphate and either lauroyl CoA, oleoyl CoA, or erucoyl CoA, and the (14)C-lipid products were separated and quantitated. In the presence of oleoyl CoA, the microsomes from each of the three species produced lysophosphatidic acid, phosphatidic acid, diacylglycerol, and triacylglycerol with kinetics consistent with the operation of the glycerol phosphate pathway. In the presence of erucoyl CoA, the microsomes from all the three species did not produce di- or tri-acyl lipids. In the presence of lauroyl CoA, only the microsomes from palm, but not those from maize or rapeseed, synthesized di- and tri-acyl lipids. This lack of reactivity of lauroyl CoA was also observed in the microsomes from maturing castor bean, peanut, and soybean. In maize seed and rapeseed, but not palm seed, the kinetics of labeling suggest that lauroyl and erucoyl moieties of the acyl CoAs were incorporated into lysophosphatidic acid but failed to enter into phosphatidic acid and thus the subsequent lipid products. We propose that the high degree of acyl specificity of lysophosphatidyl acyltransferase is the blocking step in the synthesis of triacylglycerols using lauroyl CoA or erucoyl CoA. The significance of the findings in seed oil biotechnology is discussed. PMID- 16666280 TI - Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625. AB - Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO(2) fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q(a), the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O(2) photoreduction. Hence, O(2) could serve as an electron acceptor to allow oxidation of Q(a) even in the absence of CO(2) fixation. PMID- 16666281 TI - Effects of multivalent cations on cell wall-associated Acid phosphatase activity. AB - Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu(2+), Mg(2+), Zn(2+), and Mn(2+); unaffected by Ba(2+), Cd(2+), and Pb(2+); and inhibited by Al(3+). The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg(2+). On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al(3+) and Hg(2+) was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg(2+). In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca(2+) significantly reduced the effects of Hg(2+) or Al(3+), but not Mg(2+), to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg(2+) or Al(3+) which caused a Ca(2+)-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure. PMID- 16666282 TI - Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts. AB - Using the principle described by R McC Lilley, M Stitt, G Mader, HW Heldt (1982 Plant Physiol 70: 965-970), an apparatus for rapid fractionation of barley leaf (Hordeum vulgare) protoplasts by membrane filtration was built. From studies of ATP/ADP ratios, it is concluded that the quenching of metabolic reactions is very fast, making it possible to use the method for studies on metabolic interactions between different compartments in plant cells. The fractionation method was used to study the influence of photorespiration on ATP/ADP ratios in the chloroplasts, mitochondria, and cytosol of barley leaf protoplasts. The cytosolic ATP/ADP ratio was higher under photorespiratory conditions than under nonphotorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, had a very small effect on the ATP/ADP ratios in the different subcellular compartments during photosynthesis in nonphotorespiratory conditions (saturating CO(2)). In photorespiratory conditions (limiting CO(2)), on the other hand, aminoacetonitrile increased the ATP/ADP ratio in the chloroplasts and decreased the ATP/ADP ratios in the mitochondria and the cytosol. These results are consistent with the hypothesis, that during photorespiration glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol. PMID- 16666283 TI - Chemical Equivalence of Phosphoenzyme Reaction States in the Catalytic Mechanism of the Red Beet (Beta vulgaris L.) Plasma Membrane ATPase. AB - A comparison of two phosphoryl enzyme reaction states associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue was carried out to determine if their differences in reactivity toward ADP and K(+) was related to a structural difference in the site of phosphorylation. Using a pulse labeling method it was possible to produce preparations where either the ADP-sensitive and -insensitive phosphoenzyme forms or the ADP-insensitive phosphoenzyme form alone were trapped as trichloroacetic acid denatured protein. Following complete digestion with Pronase, both preparations yielded radioactive tripeptides with similar properties with respect to pH stability of the covalent bond linking the phosphate to the peptide, isoelectric point, and migration on cellulose thin layer plates. Since the preparation containing both intermediate reaction states behaved in a uniform manner during analysis and displayed properties similar to the preparation containing only the ADP-insensitive phosphoenzyme form, it was proposed that both phosphoenzyme forms were chemically equivalent and derived from the same region of the catalytic active site. The observation that ethyleneimine treatment of both preparations followed by trypsin digestion resulted in the production of tripeptides similar to the Pronase fragments would support this proposal since it suggests that the tripeptides from both phosphoenyzme states contain a lysine residue on the C terminal end and are adjacent to a cysteine residue on the N-terminal end. The chemical equivalence of these two phosphoenzyme reaction states suggests that their differences in reactivity toward ligands may be related to conformational changes associated with the catalytic and transport mechanism of this enzyme. PMID- 16666284 TI - Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state. AB - The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual labeled substrate ([gamma-(32)P]ATP and [5',8-(3)H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg(2+)-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg(2+) increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated. PMID- 16666285 TI - Steady-State Chlorophyll a Fluorescence Transients during Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum. AB - The assimilation of ammonium by the N-limited green alga Selenastrum minutum results in the suppression of photosynthetic electron flow from H(2)O to CO(2) (6, 7, 18). In this study, results are presented which describe the correponding change in steady-state chlorophyll a fluorescence. The addition of ammonium resulted in a transient decline in fluorescence followed by a marked increase. Fluorescence did not return to control levels until the added ammonium had been assimilated. Analysis of the fluorescence transients showed that ammonium assimilation resulted in a rapid increase in nonphotochemical quenching (Q(e)) peaking 10 to 15 seconds after ammonium addition. Q(e) then decreased dramatically reaching a minimum value approximately 45 seconds following ammonium addition and returned to the control level only after the added ammonium had been assimilated. There were no effects of ammonium addition on photochemical quenching (Q(q)) for approximately 10 to 15 seconds at which time both gross O(2) evolution (as measured by mass spectrometry) and Q(q) declined. In the presence of d,l-glyceraldehyde or when cells were held at the CO(2) compensation point, the addition of ammonium resulted in a decline in Qe 10 to 15 seconds after addition. The Q(e) peak and the Q(q) decline were absent. These results imply that the transient increase in Q(e) and the subsequent decline in Q(q) may be attributed to the decline in Calvin cycle activity during ammonium assimilation. The decline in Q(e) is apparently a direct result of ammonium assimilation. The observation that the Q(e) peak precedes the Q(q) decline would be consistent with the decreases in Calvin cycle carbon flow occurring at the kinase reactions prior to glyceraldehyde-3-phosphate dehydrogenase. PMID- 16666286 TI - Sucrose Synthase, a Cytosolic Enzyme in Protoplasts of Jerusalem Artichoke Tubers (Helianthus tuberosus L.). AB - The exact subcellular location of sucrose synthase (UDP-d-glucose: d-fructose 2 alpha-d-glucosyltransferase, EC 2.4.1.13) in Helianthus tuberosus tubers was studied by comparison of its activity in protoplasts with that of vacuoles isolated from them. Assuming 100% of the beta-N-acetylglucosaminidase activity to be of vacuolar origin, less than 5% of both the sucrose synthase activity and the extravacuolar marker NAD-malate dehydrogenase was detected in the vacuole preparations. Sucrose synthase is therefore an extravacuolar enzyme. Its role in the inulin metabolism of H. tuberosus is discussed. PMID- 16666287 TI - Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons. AB - In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome. PMID- 16666288 TI - Cytokinin Oxidase from Phaseolus vulgaris Callus Cultures : Affinity for Concanavalin. AB - Cytokinin oxidase activity from Phaseolus vulgaris cv Great Northern callus cultures exhibited affinity for the lectin concanavalin A. Over 80% of the activity extracted from the callus tissue bound to a concanavalin A-Sepharose 4B column. The bound activity was eluted from the column by the addition of methylmannose to the eluting buffer. On the basis of this result, it appears that most of the cyokinin oxidase activity present in Great Northern callus cultures exists in the form of a glycoprotein. The apparent pI of this enzyme, as estimated by chromatofocusing, is approximately 5.0. PMID- 16666289 TI - A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase. AB - We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 x 10(-7) molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl 4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild type plant were examined. The optimum temperature for the reaction is 45 degrees C, and the K(m) values for the substrates are 2.7 x 10(-6) molar for 4-methyl-5 hydroxyethylthiazole phosphate and 1.8 x 10(-6) molar for 2-methyl-4-amino-5 hydroxymethylpyrimidine pyrophosphate. PMID- 16666290 TI - Tonoplast stability and survival of isolated vacuoles in different buffers. AB - The mechanism of sucrose transport into vacuoles isolated from leaf tissue has been studied only in barley (Hordeum vulgare) mesophyll cells. In this tissue, sucrose transport was reported to be a facilitated diffusion. We have observed a facilitated diffusion of sucrose into vacuoles isolated from this tissue. However, no pH dependence was observed. Evidence is presented indicating that the pH dependence of sucrose uptake into vacuoles may be an artifact, reflecting tonoplast instability and survival of isolated vacuoles in different buffers. Apparently vacuoles do not withstand exposure to some commonly used buffers. PMID- 16666291 TI - Photooxidation of Plastids Inhibits Transcription of Nuclear Encoded Genes in Rye (Secale cereale). AB - Rye (Secale cereale cv Halo) seedlings treated with the herbicide Norflurazon SAN 9789 showed a reduced concentration of mRNA for the small subunit of ribulose-1,5 bisphosphate carboxylase and for the light-harvesting chlorophyll a/b protein. The inhibition of mRNA accumulation by Norflurazon occurred only in the presence of high light intensities and only after a period of days. The primary effect was an inhibition of the transcription rate that occurred within 1 day after exposure of the seedlings to light. PMID- 16666292 TI - Analysis of the reaction products from incubation of sugarcane vacuoles with uridine-diphosphate-glucose: no evidence for the group translocator. AB - Isolated sugarcane (Saccharum spp. hybrid H50-7209) vacuoles incorporate radioactivity during incubation with labeled UDP-glucose by a mechanism which was postulated to be responsible for sucrose storage in the vacuoles (UDP-glucose group translocator). Analysis of the reaction products in the medium revealed that several enzymic processes are going on during incubation with UDP-glucose such as production of hexose phosphates, UMP, and sugars, all of which seem unrelated to the incorporation of radioactivity into vacuoles. The incorporated radioactivity was identified mainly as (1-->3)-beta-glucan (callose) of polymerization grades up to more than 20. Callose occurs as a contaminant at the surface of isolated vacuoles coming from the plasmalemma. The properties of UDP glucose incorporation into the vacuolar preparation compared favorably with known properties of callose synthase. The low mol wt glucans that are found are probably degradation products of labeled callose due to hydrolases, which are liberated by centrifugation of vacuoles. The labeled disaccharide, which chromatographically had been formerly identified as sucrose, is laminaribiose. No sucrose (or sucrose phosphate) could be identified in the vacuole preparation after incubation with UDP-glucose. Thus, the mechanism of sucrose storage in sugarcane vacuoles is still open. PMID- 16666293 TI - High Performance Liquid Chromatography-Based Reevaluation of Disaccharides Produced upon Incubation of Sugarcane Vacuoles with UDP-Glucose. AB - A reanalysis of products formed after short-term incubation of sugarcane (Saccharum spp. hybrid cv H50-7209) vacuole preparations with uridine diphosphate [(14)C]glucose was performed. The results indicated that the ethanol-soluble substance previously identified as sucrose did not elute with sucrose when subjected to high performance liquid chromatography but had the same retention time as a disaccharide tentatively identified as laminaribiose. PMID- 16666294 TI - Chitinases and beta-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.). AB - To isolate chitinases and beta-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the beta-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and beta-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (M(r) 29.8 kD) both in IWF and homogenate. beta-1,3-Glucanase, however, showed two peaks in the IWF (M(r) 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five beta-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular beta-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection. PMID- 16666295 TI - Fluence-response curves and action spectra for the very low fluence and the low fluence response for the induction of kalanchoe seed germination. AB - Germination of Kalanchoe blossfeldiana Poelln. seeds is absolutely light requiring. Germination of one seed is the result of one out of three reactions, viz. the very low fluence response (VLFR), the low fluence response (LFR) and the high fluence response/high irradiance response. In order to demonstrate the involvement of phytochrome for both photoresponses, i.e. VLFR and LFR, action spectra for induction were determined. Fluence-response data are analyzed by means of probit analysis in order to calculate the seed population parameters, with special attention to mu, or the fluence for half-maximal induction, and B, the slope in the probit diagram. Laser light was used between 620 and 800 nanometers to analyze the VLFR. Phytochrome is responsible for both photoresponses: the VLFR action spectrum demonstrates an exponential decrease in apparent photoconversion cross-section (P(r) --> P(fr)) up to about 800 nanometers. Assuming that P(r):P(fr)-X and P(fr):P(fr)-X are the effectors for the VLFR and the LFR, respectively, we estimate an average induction threshold of about 0.003% P(r):P(fr) for the VLFR and about 1% P(fr):P(fr) for the LFR among individuals of the seed population. PMID- 16666296 TI - Is there a role for the 42 kilodalton polypeptide in inorganic carbon uptake by cyanobacteria? AB - Cyanobacterial cells accumulate substantial amounts of a membrane-associated 42 kilodalton polypeptide during adaptation to low CO(2) conditions. The role of this polypeptide in the process of adaptation and in particular in the large increase in the ability to accumulate inorganic carbon (C(i)), which accompanies this process, is not yet understood. We have isolated a mutant Synechococcus PCC7942 that does not accumulate the 42 kilodalton polypeptide. The mutant requires a high-CO(2) concentration for growth and exhibits a very low apparent photosynthetic affinity for extracellular C(i). The latter might be attributable to the observed defective ability of the mutant to utilize the intracellular C(i) pool for photosynthesis. The 42 kilodalton polypeptide does not appear to participate directly in the active transport of C(i), since the difference between the observed capabilities for CO(2) and HCO(3) (-) uptake of the mutant and the wild type is not sufficient to account for their different growth and photosynthetic performance. Furthermore, high CO(2)-grown wild-type cells, where we could not detect the 42 kilodalton polypeptide, transported CO(2) faster than the mutant. An analysis of the curves relating the rate of accumulation of C(i) to the concentration of CO(2) or HCO(3) (-) supplied, in the presence or absence of carbonic anhydrase, indicated that under the experimental conditions used here, CO(2) was the preferred C(i) species taken up by Synechococcus. PMID- 16666297 TI - Water deficit-induced changes in abscisic Acid, growth, polysomes, and translatable RNA in soybean hypocotyls. AB - Soybean seedlings (Glycine max L.) were germinated and dark-grown in water saturated vermiculite (water potential = -0.01 megapascal) for 48 hours, then transferred either to water-saturated vermiculite or to low water potential vermiculite (water potential = -0.30 megapascal). A decrease in growth rate was detectable within 0.8 hour post-transfer to low water potential vermiculite. A fourfold increase in the abscisic acid content of the elongating region was observed within 0.5 hour. At 24 hours post-transfer, hypocotyl elongation was severely arrested and abscisic acid reached its highest measured level: 3.7 nanograms per milligram dry weight (74-fold increase). A comparison of the polyA(+) RNA populations isolated at 24 hours post-transfer from the elongating region of water-saturated and low water potential vermiculite-grown seedlings was made by two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gel analysis of in vitro translation products. It revealed both increases and decreases in the relative amounts of a number of translation products. Rewatering seedlings grown in low water potential vermiculite at 24 hours post-transfer led to a total recovery in growth rate within 0.5 hour, while abscisic acid in the elongating hypocotyl region required 1 to 2 hours to return to uninduced levels. Application of 1.0 millimolar (+/-) abscisic acid to well watered seedlings resulted in a 48% reduction in hypocotyl growth rate during the first 2 hours after treatment. Plants treated with abscisic acid for 24 hours had a lower polysome content than control plants. However, hypocotyl growth inhibition in abscisic acid-treated seedlings preceded the decline in polysome content. PMID- 16666298 TI - Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals. AB - Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca(2+) in the presence of ATP. The specific activity of ATP-dependent uptake is approximately 20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca(2+) stimulation of ATP hydrolysis was not discernible above the high background of Ca(2+)-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca(2+) concentration, suggesting that Ca(2+) serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca(2+) at half maximal velocity of uptake (S(0.5)) was 12.5 micromolar and the Hill coefficient (n(H)) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca(2+), although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca(2+) into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca(2+) transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca(2+) into microsomal vesicles declined by approximately 70%. This occurred before the appearance of petal-inrolling and the climacteric like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S(0.5) or n(H), but V(max) for ATP-dependent Ca(2+) uptake decreased by approximately 40%. A similar decline in ATP-dependent uptake of Ca(2+) into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene. PMID- 16666299 TI - On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber. AB - Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD(+) or 3-acetylpyridine-NAD(+) by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute.milligram protein at pH 5 to 6. The K(m) values for 3-acetylpyridine-NAD(+) and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity. PMID- 16666300 TI - A survey of the pectic content of nonlignified monocot cell walls. AB - The primary cell walls of graminaceous monocots were known to have a low content of pectin compared to those of dicots, but it was uncertain how widespread this feature was within the monocots as a whole. Nonlignified cell walls were therefore prepared from 33 monocot species for determination of their pectin content. It was not possible to solubilize intact pectins quantitatively from the cell walls, and the pectin content was assessed from three criteria: the total uronic acid content; the content of alpha-(1,4')-D-galacturonan isolated by partial hydrolysis and characterized by electrophoresis and degradation by purified polygalacturonase; and the proportion of neutral residues in a representative pectic fraction solubilized by sequential beta-elimination and N,N,N'N'-cyclohexanediaminetetraacetic acid extraction. Low galacturonan contents were restricted to species from the Gramineae, Cyperaceae, Juncaceae, and Restionaceae. Other species related to these had intermediate galacturonan contents, and the remainder of the monocots examined had high galacturonan contents comparable with those of dicots. The other criteria of pectin content showed the same pattern. PMID- 16666301 TI - N,N-Dimethyltryptamine Production in Phalaris aquatica Seedlings: A Mathematical Model for its Synthesis. AB - The activities of three enzymes and the concentration of intermediates involved in the synthesis of N,N-dimethyltryptamine (DMT) from endogenous tryptophan (TRP) have been measured in vitro in seedlings of Phalaris aquatica L. cv Australian Commercial over 16 days after planting. The activities of tryptophan decarboxylase and the two N-methyl-transferases increased rapidly to maximal rates of substrate conversion at day 5 of 95, 1000, and 2200 micromoles per hour per milliliter, respectively. After these maximal rates, the activities decreased rapidly. The concentration of intermediates increased rapidly from zero in the seeds to maximal values of 25 and 53 micromolar at day 5 for tryptamine (T) and N methyltryptamine (MT), respectively, 1000 micromolar at day 6 for TRP, and 650 micromolar at day 8 for DMT. The concentration of DMT and of all the intermediates in its synthesis declined rapidly after the maximal value had been reached. A mathematical model of the pathway from TRP to DMT using these enzymes correctly predicts the concentrations of T and MT, intermediates whose concentration is determined only by the pathway, and confirms that these three enzymes are responsible for the in vivo synthesis of DMT. Kinetic studies are reported for these enzymes. Tryptophan decarboxylase uses pyridoxal phosphate (PALP) as a coenzyme and has the following kinetic constants: K(m) (PALP) = 2.5 micromolar, K(m) (TRP) = 200 micromolar, K(i) (MT) = 5 millimolar, and K(i) (DMT) = 4 millimolar. The N-methyltransferases use S-adenosylmethionine (SAM) as substrate; S-adenosylhomocysteine (SAH) is assumed to be the product. The mechanism of secondary indolethylamine-N-methyltransferase, determined by initial velocity studies, is rapid equilibrium random with formation of both dead end complexes. Secondary indolethylamine-N-methyltransferase methylates both MT and 5 methoxy-N-methyltryptamine (5MeOMT). The kinetic constants for the methylation of MT are: K(MT) = 40 +/- 6, K(SAM) = 55 +/- 15, K(DMT) = 60, K(SAH) = 4.3 +/- 0.4 micromolar with unity interaction factors. The kinetic constants for the conversion of 5MeOMT to 5-methoxy-N,N-dimethyltryptamine (5MeODMT) are K(5MeOMT) = 40 +/- 10, K(SAM) = 90 +/- 40, and K(SAH) = 2.9 +/- 0.3 micromolar with unity interaction factors, except for SAM-5MeODMT = 2.0 +/- 0.9 and SAH-5MeOMT = 0.45 +/- 0.25. The kinetic constants for primary indolethylamine N-methyltransferase are K(m) (T) = 20, K(m) (SAM) = 40, K(i) (DMT) = 450 micromolar with the substrates binding independently. PMID- 16666302 TI - Nodulin gene expression in effective alfalfa nodules and in nodules arrested at three different stages of development. AB - Nodulin gene expression was analyzed in effective and ineffective root nodules of alfalfa (Medicago sativa L. cv Iroquois) elicited by three different Rhizobium meliloti mutants: an exoB mutant having defective acidic exopolysaccharide that does not fluoresce on plates containing the fluorescent brightener Calcofluor; fix21, a spontaneous mutant that has defective lipopolysaccharide and is Calcofluor bright; and a Rhizobium mutant resulting from a Tn5 insertion in the nifH gene of the nif operon. The ineffective nodules elicited by these various mutant rhizobia are blocked at different stages of nodule development and have unique phenotypes. A distinctive pattern of nodulin gene expression as determined by in vitro translations of total nodule RNA characterizes each nodule phenotype. Seventeen nodulins are found in effective nodules including five leghemoglobins. Only one nodulin gene is expressed in the bacteria-free nodules elicited by the exoB mutant. Other nodulin genes (leghemoglobin and nine others) are expressed in fix21-induced nodules. The genes for nodule-enhanced glutamine synthetase as well as for all the other nodulins are expressed in nodules induced by the nifH mutant. The expression of genes for the nodulins, including leghemoglobin, is independent of the nitrogen-fixing ability of the nodule and appears to correlate with the differentiation of densely cytoplasmic host cells in the nodule and, to some extent, with bacterial release from infection threads. PMID- 16666303 TI - Ethylene Production by Attached Leaves or Intact Shoots of Tobacco Cultivars Differing in Their Speed of Yellowing during Curing. AB - Using an open air flow system, differences in the yellowing rate of leaves during curing were assessed in relation to ethylene production by shoots of intact seedlings or attached mature leaves of 60 day old tobacco (Nicotiana tabacum L.) plants. The rate of ethylene evolution from the leaves of the fast yellowing cultivars was significantly higher than in the slow yellowing ones. The same differences were obtained with shoots of intact seedlings. The findings suggest that it is possible to use ethylene production by seedlings as a selection criterion in screening for genotypic differences in the rate of yellowing. The ability of carbon dioxide (1%) to enhance ethylene production by attached leaves was significant in a slow, but not in a fast yellowing cultivar. However, similar amounts of ethylene were produced on administration of 1-aminocyclopropane-1 carboxylic acid to a slow and a fast yellowing cultivar. Exposure of attached leaves to exogenous ethylene (0.1 microliter per liter) accelerated the loss of chlorophyll and protein. This treatment was effective only for slightly yellow leaves and not for fully expanded green ones. The significance and possible use of ethylene in the flue-curing process are discussed. PMID- 16666304 TI - Oats Tolerant of Pseudomonas syringae pv. tabaci Contain Tabtoxinine-beta-Lactam Insensitive Leaf Glutamine Synthetases. AB - Pseudomonas syringae pv. tabaci, a commonly recognized leaf pathogen of tobacco, can infest the rhizosphere of many plants, including oats. Normal oat plants do not survive this infestation as a consequence of the complete and irreversible inactivation of all of their glutamine synthetases by tabtoxinine-beta-lactam (TbetaL), a toxin released by pv. tabaci. We have identified a population of oat (Avena sativa L. var Lodi) plants that are tolerant of pv. tabaci. The tolerant plants had no detectable TbetaL-detoxification mechanisms. Pathogen growth on these plant roots was not inhibited. These plants contain leaf glutamine synthetases (GS(1) and GS(2)) that were less sensitive to inactivation by TbetaL in vitro; these GSs have normal K(m) values for glutamate and ATP when compared with those of GS in control plants. Root glutamine synthetase of the tolerant plants was inactivated in vivo during infestation by the pathogen or by TbetaL in vitro. When growing without pv. tabaci, the tolerant plants contained normal levels of glutamine synthetase in their roots and leaves and normal levels of protein, ammonia, glutamate, and glutamine in their leaves. However, when the tolerant plants' rhizosphere was infested with pv. tabaci, the plant leaves contained elevated levels of glutamine synthetase activity, protein, ammonia, glutamate, and glutamine. No changes in glutamate dehydrogenase activity were detected in leaves and roots of pathogen-infested tolerant plants. PMID- 16666305 TI - Light effects on several chloroplast components in norflurazon-treated pea seedlings. AB - Changes occurring in several chloroplast components during Norflurazon-induced photobleaching of Pisum sativum seedlings were investigated. mRNA steady state levels of the chlorophyll a/b-binding protein of photosystem II, ferredoxin I, the small and large subunits of ribulose 1,5-bisphosphate carboxylase, and pEA214 and pEA207, two other light-responsive genes, were determined during chlorophyll photooxidation. Relative transcription rates were assayed in isolated nuclei. The results illustrate a complex set of interactions regulating expression of the nuclear and chloroplast genomes. Photobleaching was found to affect the expression of the various genes in different ways. While transcript levels of the chlorophyll a/b-binding protein decreased by more than 80% under photooxidative light conditions in carotenoid-deficient peas, levels of ferredoxin, the small and large subunits of ribulose 1,5-bisphosphate carboxylase, and pEA214 mRNAs were reduced by less than 50%. pEA207 mRNA levels, on the other hand, were resistant to the effects of photobleaching. Analyses of chlorophylls a and b and the chlorophyll a/b-binding protein suggest that accumulation of the protein and its mRNA are coordinated with chlorophyll abundance at several steps. In addition to post-transcriptional regulation at the level of mRNA and protein stability, there may exist coordination at the transcriptional stage. PMID- 16666306 TI - The Relationship between Inorganic Nitrogen Metabolism and Proline Accumulation in Osmoregulatory Responses of Two Euryhaline Microalgae. AB - Chlorella autotrophica, a euryhaline marine alga, and Stichococcus bacillaris, a salt-tolerant soil alga, grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining high levels of NADPH-glutamate dehydrogenase. Nitrate reductase showed no change in MSX-adapted cells. For both species, MSX-adapted cells retained their capacity to accumulate proline in response to salinity, and in S. bacillaris no major shift was observed in the presence of MSX toward the accumulation of sorbitol. Following transfer from 33 to 150% artificial seawater (ASW), both algae exhibited increases in organic solute levels without a lag. Within 6 h of this sudden increase in salinity, the levels of proline in C. autotrophica and of proline and sorbitol in S. bacillaris were similar to those found in steady state 150% ASW cultures. Following transfer from 33 to 150% ASW, S. bacillaris continued [(14)C] bicarbonate photoassimilation at a normal rate and maintained active enzymes of nitrogen assimilation. The incorporation of [(14)C]phenylalanine into proteins was inhibited for about 30 minutes in MSX-free cells and 90 minutes in MSX-adapted cells following transfer from 33 to 150% ASW; the recovery after these lag periods was almost complete. PMID- 16666307 TI - Survey of the Proteolytic Activities Degrading the Kunitz Trypsin Inhibitor and Glycinin in Germinating Soybeans (Glycine max). AB - The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti(a). Protease K1 cleaves Ti(a) to Ti(a) (m), the inhibitor form lacking the five carboxyl-terminal amino acid residues relative to Ti(a). Protease K1 is a cysteine protease that peaks in activity on day 4 after the beginning of imbibition, with maximal activity toward Ti(a) at pH 4. The characteristics of protease K1 are consistent with the involvement of this protease in the initial proteolysis of the Kunitz inhibitor during germination. Protease K2 also degrades Ti(a) at pH 4 but produces no electrophoretically recognizable products. It peaks later in seedling growth, at day 8. Protease K3 degrades Ti(a) to products other than Ti(a) (m). However, it is active at pH 8. Two proteolytic activities were identified that attack the major storage protein, glycinin. Protease G1 (which appears by 4 days after imbibition) specifically cleaves the acidic polypeptides of glycinin at pH 4, yielding a product approximately 1.5 kilodaltons smaller. Protease G1 is inhibited by metal chelators as well as by reagents reactive toward thiols. Protease G2 also degrades the glycinin acidic chains at pH 4, but without the appearance of electrophoretically recognizable products. Protease G2, while present at low levels in the dry seed, is found primarily in the cotyledons after 8 days of growth. PMID- 16666308 TI - The biophysical basis of elongation growth in internodes of deepwater rice. AB - Partial submergence induces rapid internodal elongation in deepwater rice (Oryza sativa L., cv Habiganj Aman II). We measured in vivo extensibility, tissue tension, hydraulic conductance and osmotic potential in the region of cell elongation in the uppermost internode. The in vivo extensibility of the internode, measured by stretching of living tissue with a custom-made constant stress extensiometer, rose rapidly following submergence of the plant. Both the elastic (E(el)) and plastic (E(pl)) extensibility increased when growth of the internode was induced. The submerged internode displayed tissue tension (elastic outward bending of longitudinally split internode sections); in air-grown control internodes, no such bending occurred. The hydraulic conductance, estimated from the kinetics of tissue shrinkage in 0.5 molar mannitol and subsequent swelling in distilled water, was not changed by submergence. The osmotic potential, measured with a dew-point hygrometer using frozen-thawed tissue, was only 18% less negative in the submerged internode than in the air-grown control. This indicates that osmoregulation takes place in rapidly elongating rice internodes. We suggest that the rapid expansion of the newly formed internodal cells of submerged plants is controlled by the yielding properties (E(pl)) of the cell walls. Experiments with excised stem sections indicate that gibberellin is involved in increasing the E(pl) of the elongating cell walls. PMID- 16666309 TI - Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid. AB - Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl(2), and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H(+)-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H(+)-translocating ATPase located at the plasma membrane of the mesophyll cells. PMID- 16666310 TI - Regulation of Gene Expression by Ethylene in Wild-Type and rin Tomato (Lycopersicon esculentum) Fruit. AB - Levels of ethylene biosynthesis and ethylene-inducible gene expression in wild type tomato (Lycopersicon esculentum) fruit and in nonripening fruit from the tomato mutant rin (ripening inhibitor) were compared in order to investigate the mechanism of ethylene action. Whereas wild-type tomato fruit dramatically increase the rate of ethylene biosynthesis at the onset of ripening, rin fruit constitutively produce ethylene at a low basal level. We have compared the mRNA levels and transcription rates of four cloned ethylene-inducible genes (JE Lincoln, S Cordes, E Read, RL Fischer 1987 Proc Natl Acad Sci USA 84: 2793-2797) during wild-type and rin fruit development. In wild-type fruit, both mRNA levels and transcription rates of these genes increase. The effect of the rin mutation on gene expression is different for each ethylene-inducible gene. In one case expression is completely suppressed, while in other instances it is either partially inhibited or relatively unaffected by the mutation. The mRNA levels of each of these genes in response to exogenous ethylene in rin fruit was also measured. The mRNAs for all four genes accumulate to similar levels in both ethylene treated rin and ethylene treated wild-type fruit. These results are discussed with regard to the response of plants to ethylene hormone at the level of gene expression. PMID- 16666311 TI - Water Deficit and Associated Changes in Some Photosynthetic Parameters in Leaves of ;Valencia' Orange (Citrus sinensis [L.] Osbeck). AB - Photosynthetic CO(2) assimilation, transpiration, ribulose-1,5-bisphosphate carboxylase (RuBPCase), and soluble protein were reduced in leaves of water deficit (stress) ;Valencia' orange (Citrus sinensis [L.] Osbeck). Maximum photosynthetic CO(2) assimilation and transpiration, which occurred before midday for both control and stressed plants, was 58 and 40%, respectively, for the stress (-2.0 megapascals leaf water potential) as compared to the control (-0.6 megapascals leaf water potential). As water deficit became more severe in the afternoon, with water potential of -3.1 megapascals for the stressed leaves vs. 1.1 megapascals for control leaves, stressed-leaf transpiration declined and photosynthetic CO(2) assimilation rapidly dropped to zero. Water deficit decreased both activation and total activity of RuBPCase. Activation of the enzyme was about 62% (of fully activated enzyme in vitro) for the stress, compared to 80% for the control. Water deficit reduced RuBPCase initial activity by 40% and HCO(3) (-)/Mg(2+)-saturated activity by 22%. However, RuBPCase for both stressed and control leaves were similar in K(cat) (25 moles CO(2) per mole enzyme per second) and K(m) for CO(2) (18.9 micromolar). Concentrations of RuBPCase and soluble protein of stressed leaves averaged 80 and 85%, respectively, of control leaves. Thus, reductions in activation and concentration of RuBPCase in Valencia orange leaves contributed to reductions in enzyme activities during water-deficit periods. Declines in leaf photosynthesis, soluble protein, and RuBPCase activation and concentration due to water deficit were, however, recoverable at 5 days after rewatering. PMID- 16666312 TI - Nutrient salts promote light-induced degradation of indole-3-acetic Acid in tissue culture media. AB - The disappearance of indole-3-acetic acid (IAA) from cell-free liquid culture medium was followed in response to nutrient salts found in Murashige-Skoog salt base, light, and pH range of 4 to 7. The loss of IAA was accelerated by light or Murashige-Skoog salts. However, the combination of both light and Murashige-Skoog salts acted synergistically to catalyze the destruction of over 80% of the original IAA within 7 days of continuous incubation. Under these same conditions, the loss of IAA was decreased to approximately 50% by adjusting the initial pH of the medium to 7. Iron was identified as the single major contributor to light catalyzed destruction of IAA. Removal of nitrates, which represented 87% of the molar salt composition, also reduced the light-catalyzed loss of IAA. Treatments that protected IAA from degradation, such as darkness or removal of iron from the medium, suppressed the growth of muskmelon (Cucumis melo. Naud., var. reticulatus) callus tissue cultured for 30 days. Treatments in the light that rapidly degraded IAA resulted in maximum growth. Consequently, the brief exposure to IAA prior to degradation was apparently sufficient to initiate physiological changes required for growth. Possible approaches to the preservation of IAA during incubation are discussed. PMID- 16666313 TI - Expression of leaf nitrate reductase genes from tomato and tobacco in relation to light-dark regimes and nitrate supply. AB - The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels. PMID- 16666314 TI - The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H Nitrate Reductase Enzyme from Soybean. AB - A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO((x))) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO((x)) formation. The Blue Sepharose purified soybean enzyme has a pH optimum of 6.75, an apparent K(m) for nitrite of 0.49 millimolar, and an apparent K(m) for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO((x)) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH(2)) and reduced methyl viologen (MV) can serve as electron donors for NO((x)) evolution activity. The NADPH-, FMNH(2) , and reduced MV-NO((x)) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH(2) and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO((x)). NADPH eluted both NR and NO((x)) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean. PMID- 16666315 TI - Alfalfa Root Exudates and Compounds which Promote or Inhibit Induction of Rhizobium meliloti Nodulation Genes. AB - Using a plate induction assay, we demonstrate that alfalfa exudes inducer of Rhizobium meliloti nodulation genes. The inducer is exuded from the infectible zone of the root, accumulates to at least 1 micromolar, and is not affected by 10 millimolar nitrate. No zones of inhibition are observed. A nodulation minus mutant line of alfalfa, MN-1008, exudes normal levels of inducer. R. meliloti grown in rich medium requires ten-fold higher concentrations of luteolin to achieve half-maximal induction as compared to cells grown in a minimal medium. Flavonoids other than luteolin are found to have activity in R. meliloti nodulation gene induction assays. The compounds apigenin and eriodictyol have activities two-fifths and one-seventh that of luteolin, respectively. Several of the flavonoids tested (morin = naringenin > kaempferol = chrysin > quercetin = fisetin = hesperitin) demonstrate antagonistic activity toward induction by luteolin. The most effective antagonist is the coumarin, umbelliferone. PMID- 16666316 TI - Reduction of turgor induces rapid changes in leaf translatable RNA. AB - The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23 degrees C air for 20 min. Poly(A)(+) RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)(+) RNAs increased in wilted plants. Most of the poly(A)(+) RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A lambdagt10 cDNA library was constructed using poly(A)(+) RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)(+) RNAs which are induced in wilted shoots. PMID- 16666317 TI - Long-term in vitro culture of wheat grains. AB - Wheat (Triticum aestivum, Triticum durum) grains were excised immediately following fertilization and cultured until maturity. A rachis fragment attached to the grain was required to ensure an increase in grain size for the first 10 days following fertilization. A (14)C-labeling study revealed that 8-day-old grains accumulated more dry matter into the ethanol-insoluble fraction when grown on agar rather than when immersed in liquid medium. Light enhanced the absorption of sucrose from the medium only in the latter case. In agar-based culture, when no contact was made between the grain surface and the medium, peeling off the outer pericarp layers increased sugar absorption, leading to a threefold increase in the amount of accumulated dry matter in the ethanol-insoluble fraction. Culturing of wheat grains with attached rachis fragment and peeled pericarp is recommended for maximum in vitro growth. PMID- 16666318 TI - Development of Oat Prothylakoids into Thylakoids during Greening Does Not Change Transmembrane Galactolipid Asymmetry but Preserves the Thylakoid Bilayer. AB - The lipase from Rhizopus arrhizus and the lipolytic acyl hydrolase from potato tubers have been used to determine the transmembrane distribution of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) in prothylakoids and thylakoids from oat (Avena sativa). Both galactolipids were found to be asymmetrically distributed. The molar outside/inside distribution was 70 +/- 8/30 +/- 8 for MGDG and 10 +/- 4/90 +/- 4 for DGDG in the prothylakoid membrane. Mature thylakoids presented a similar distribution, i.e. 63 +/- 4/37 +/ 4 for MGDG and 12 +/- 3/88 +/- 3 for DGDG. This distribution has been assessed under a variety of different conditions, namely (a) in media favoring thylakoid stacking or unstacking and inducing various membrane surface potentials, (b) in the presence of defatted bovine serum albumin which removed free fatty acids and partially lyso-galactolipids, (c) under various temperature conditions which resulted in different hydrolysis rates and degrees of fluidity of the membrane, and (d) in the presence of different enzyme concentrations which influenced the hydrolysis rate. The above distribution was found to be independent of the type of conditions used. Nonbilayer forming/bilayer forming lipid ratios suggest that both monolayers of the prothylakoid and the inner monolayer of oat thylakoid membranes should display lamellar structures (e.g. ratios <2.5). In contrast the outer monolayer of the thylakoid membrane should display non-lamellar configurations (e.g. ratio >2.5). Thus, it is concluded that the incorporation of chlorophyll-protein complexes into the nascent thylakoid membrane modifies neither the galactolipid nor the phospholipid transmembrane distribution. However, these complexes appear to be crucial to preserve a bilayer configuration to the greening membrane which, otherwise, would adopt nonlamellar structures. The possible origin of galactolipid transversal asymmetry which appears very early during the biogenesis of oat thylakoid membranes is discussed. PMID- 16666319 TI - Proton extrusion by wheat roots exhibiting severe aluminum toxicity symptoms. AB - The mechanisms of Al rhizotoxicity are not known, but disruption of membrane function has been a persistent hypothesis. The objective of this study was to establish whether cells of Al-cultured wheat roots (Triticum aestivum L. cv Tyler) exhibiting severe Al toxicity symptoms were capable of vigorous proton extrusion. The membrane electrical potential difference (E(m)) was measured in individual cells throughout the first centimeter of root tips during perfusion with Al solutions similar to or more concentrated than those of the culture medium. For both Al-cultured and control roots the resting E(m) was -100 millivolts, and 1 millimolar acetic acid induced cyanide-sensitive hyperpolarizations to -180 millivolts at a maximum rate of -30 millivolts per minute. Al, like Ca(2+), enhanced the negativity of the E(m) of cells already treated with acetic acid. Both acetic acid and fusicoccin stimulated net proton extrusion from Al-cultured and control roots, both of which also extruded protons in the absence of these stimulants. These results demonstrate that wheat roots exhibiting severe Al toxicity symptoms had an undiminished capacity to extrude protons, that the membranes were intact, and that ATP synthesis was sufficient to supply the proton-translocating ATPases. PMID- 16666320 TI - Localization of the Site of Perception of Thermoinductive Temperatures in Thlaspi arvense L. AB - This paper describes attempts to localize the site of perception of low temperatures (0-10 degrees C) during thermoinduction in Thlaspi arvense L. Reproductive development (stem elongation and flower formation) was observed when shoots were cooled to 4 degrees C for 4 weeks and then returned to 21 degrees C while maintaining the roots constant 21 degrees C. However, chilling the roots was ineffective for initiating reproductive development. The apparent site of perception of thermoinductive temperatures was further localized to the shoot tip (apex and immature leaves) by controlling the temperature of the shoot tip independently of the rest of the plant. Furthermore, excised apices regenerated flowering plants in organ culture only if they were subjected to a 4 week cold treatment. Grafting experiments also support the notion that the shoot tip or the apex is the site of perception of thermoinductive temperatures: noninduced shoot tips grafted onto bolting donors remained as vegetative rosettes. Paradoxically, it was found that the cells of the shoot tip are not the only ones capable of being thermoinduced. Shoots regenerated from leaf cuttings excised from thermoinduced plants exhibited all signs of reproductive development, while regenerated shoots from control leaves developed into vegetative rosettes. It is suggested that many cell types are capable of being thermoinduced and that the shoot tip may appear to be the site of perception of thermoinductive temperatures because structures associated with reproductive development originate from this tissue. PMID- 16666321 TI - Cercospora beticola Toxin Inhibits Vanadate-Sensitive H Transport in Corn Root Membrane Vesicles. AB - The effect of Cercospora beticola toxin on the transport of protons by vanadate sensitive ATPase was studied with corn (Zea mays) root microsomal vesicles prepared by differential centrifugation, sedimentation through a sucrose cushion, and washing with Triton X-100 plus KBr. In these preparations, addition of ATP induced intravesicular H(+)-accumulation as evidenced by a rapid quenching of the fluorescence of 9-amino-6-chloro-2-methoxy acridine. This quenching was relatively unaffected by inhibitors of mitochondrial and tonoplast-type ATPases, but was strongly reduced by inhibitors of plasma membrane H(+)-ATPase. C. beticola toxin markedly inhibited ATP dependent H(+)-transport, and this effect increased with the length of preincubation with the toxin. The same observations were made concerning ATPase activity. Inhibition of H(+)-transport was greater at pH 7.3 than at pH 5.7. Lineweaver-Burk plot analysis showed that inhibition kinetics were competitive with respect to ATP. These data suggest a direct effect of C. beticola toxin on vanadate-sensitive ATPase presumed to be associated with the plasma membrane. PMID- 16666322 TI - Identification of l-Tryptophan as an Endogenous Inhibitor of Embryo Germination in White Wheat. AB - An endogenous germination inhibitor(s) in wheat (Triticum aestivum L.) grain has been implicated in seed dormancy and germination, but its identity and mode of action have not been elucidated. We isolated and identified an endogenous germination inhibitor in white wheat grain and compared its activity with that of known compounds. A water extract of wheat bran chromatographed on Sephadex LH-20 yielded an inhibitory fraction that was detected by bioassay on embryos excised from dormant wheat seeds. The inhibitor was sequentially purified by DEAE cellulose, P-2 gel filtration, and C(18) reversed-phase HPLC and identified as l tryptophan by carbon and proton nuclear magnetic resonance spectroscopy, mass spectrometry, optical rotation, UV absorption spectrum, and chromatographic co elution with authentic tryptophan. Extractable tryptophan occurred in wheat grain and bran at about 1.3 to 1.5 and 2.7 to 5.9 milligrams per gram, respectively. Low millimolar concentrations of l-tryptophan similar to physiological levels and d-tryptophan were more inhibitory to embryos from dormant seeds than to embryos from nondormant seeds. Tryptophol and tryptamine, putative intermediates between tryptophan and IAA, were highly inhibitory and noninhibitory of embryo germination, respectively. IAA and 2,4-D also inhibited germination, but embryos from dormant and nondormant seeds responded similarly. l-Tryptophan may be an important germination inhibitor of excised embryos, but its role in situ remains to be determined. PMID- 16666323 TI - Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination. AB - The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling. PMID- 16666324 TI - Plant Plasma Membrane Proteins : II. Biotinylation of Daucus Carota Protoplasts and Detection of Plasma Membrane Polypeptides after Sds-Page. AB - The ability of two biotinylating reagents, sulfosuccinimidobiotin and sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate, to label plasma membrane proteins was examined. These compounds form covalent bonds with the free amino groups of proteins and label the proteins with biotin. Biotinylated proteins can be detected with avidin-peroxidase staining. Protoplasts isolated from embryogenic Daucus carota suspension cells were labeled with biotin and the membranes were separated on linear sucrose gradients. The conditions used for labeling the protoplasts did not cause protoplast rupture or loss of viability. The distribution of the biotin label in these linear sucrose gradients was analyzed and compared to the distribution of vanadate-sensitive ATPase activity, a marker for the plasma membrane. Both the biotin label and the vanadate sensitive ATPase activity were strongly localized in the gradient at peak density of 1.16 gram per cubic centimeter. When the protoplast surface was labeled, biotinylated polypeptides were detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis and polypeptides of 153, 94, 51, 30, 20, 17, and 14 kilodaltons were shown to be plasma membrane in origin. When a crude membrane pellet was labeled, numerous biotinylated polypeptides were distributed throughout the gradient. Because the position of the biotin label in the gradient is strongly correlated with the distribution of vanadate-sensitive ATPase, it is concluded that these biotinylating reagents are effective and reliable labels for proteins of the plant plasma membrane. Furthermore, these labels permit the positive identification of plasma membrane proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis and can serve as convenient markers for solubilization and purification of these proteins. PMID- 16666325 TI - Hordein gene expression in a low protein barley cultivar. AB - The low protein barley (Hordeum vulgare L.) cultivar ;Karl' is deficient in hordeins, the major storage protein fraction, but has normal levels of other endosperm protein fractions. We compared hordein gene expression in Karl with that in ;Traill,' a related cultivar that contains normal amounts of hordein. In plants grown in controlled environmental conditions, hordein accumulation in Karl was 55% of that in Traill. The amount of hordein synthesized at all developmental stages tested was lower in Karl, as shown by pulse labeling of excised spikes with [(3)H]leucine. To determine whether levels of hordein mRNAs were related to amounts of hordein synthesis, total RNA preparations from endosperms at three or four developmental stages were hybridized to cDNA probes corresponding to B and C hordein genes. Both B and C hordein mRNA levels were significantly lower in Karl than in Traill at intermediate developmental stages. It was concluded that the low-hordein character of Karl was regulated by the size of the hordein mRNA pool. Transcriptional and/or post-transcriptional controls may be involved in the regulation of hordein mRNA levels. PMID- 16666326 TI - Changes in protein synthesis induced in tomato by chilling. AB - Impaired chloroplast function is responsible for nearly two-thirds of the inhibition of net photosynthesis caused by dark chilling in tomato (Lycopersicon esculentum Mill.). Yet the plant can eventually recover full photosynthetic capacity if it is rewarmed in darkness at high relative humidity. As a means of identifying potential sites of chilling injury in tomato, we monitored leaf protein synthesis in chilled plants during this rewarming recovery phase, since changes in the synthesis of certain proteins might be indicative of damaged processes in need of repair. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins pulse labeled with [(35)S]methionine revealed discrete changes in the pattern of protein synthesis as a result of chilling. A protein of M(r) = 27 kilodaltons (kD), abundantly synthesized by unchilled plants, declined to undetectable levels in chilled plants. Reillumination restored the synthesis of this protein in plants rewarmed for 8 hours. Peptide mapping analysis showed the 27 kD protein to be the major chlorophyll a/b binding protein of the photosystem II light-harvesting complex (LHCP-II). The identity of this protein was confirmed by its immunoprecipitation from leaf extracts by a monoclonal antibody specific for the major LHCP-II species. While chilling abolished the synthesis of the major LHCP-II species, it also induced the synthesis of an entirely new protein of M(r) = 35 kD. The protein was synthesized on cytoplasmic ribosomes, and two-dimensional polyacrylamide gel electrophroesis showed it to exist as a single isoelectric species. This chilling-induced 35 kD protein is structurally distinct from the 27 kD LHCP-II and appears to be synthesized specifically in response to low temperature. While the 35 kD protein was found not to be associated with the chloroplast thylakoid membrane, chilling did cause selective changes in thylakoid membrane protein synthesis. The synthesis of two unidentified proteins, M(r) = 14 and 41 kD, and the beta-subunit of the chloroplast coupling factor were substantially reduced after chilling. These losses may provide clues as to the causes of the overall reduction in net photosynthesis caused by chilling. PMID- 16666327 TI - Bicarbonate inhibits ribulose-1,5-bisphosphate carboxylase. AB - Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) rapidly extracted from leaves of wheat (Triticum aestivum) and purified activated RuBPCO were incubated in the presence and absence of 20 millimolar HCO(3) (-) and changes in activation state were followed. Rapid inactivation occurred in the presence, but not in the absence, of HCO(3) (-). Effects of CO(2) concentration and pH during preincubation before assay on activation state of RuBPCO were investigated in equilibrium studies. Twenty percent inactivation occurred at high CO(2) concentration if pH was high, but not if it was low, suggesting that RuBPCO was inactivated by HCO(3) (-). The inactivation by HCO(3) (-) was more rapid than the dissociation of activating CO(2) in CO(2)-free buffer (both in the presence of 20 millimolar MgCl(2)), suggesting that HCO(3) (-) was bound to the active enzyme complex. The dissociation of inactivating HCO(3) (-) from the enzyme was slow enough that inhibition could be demonstrated in experiments with HCO(3) (-) treatments during preincubation and constant conditions during assay. Inorganic phosphate did not seem to interfere with the binding of HCO(3) (-). PMID- 16666328 TI - Sucrose and Malic Acid as the Compounds Exported to the Apical Bud of Pea following CO(2) Labeling of the Fruit : No Evidence for a Senescence Factor. AB - The G2 line of peas (Pisum sativum L.) displays senescence and death of the apical bud only in long days and in the presence of fruit. As the removal of fruit prevents senescence, one possible mechanism by which fruits induce senescence is that the fruits produce some ;senescence factor' under long day conditions, which is then transported to the apical bud. Allowing developing fruits to photosynthesize in the presence of (14)CO(2) results in the recovery of label in the apical bud. In order to determine the chemical nature of this radiolabeled material, fruits of G2 peas, growing under long days, were exposed to (14)CO(2) at the time when the first senescence symptoms start to appear. The radiolabeled material from apical buds was then extracted, purified, and identified. Using HPLC and GC-MS the major labeled compound found in the apical bud following exposure of pea fruits to (14)CO(2) was identified as sucrose, while malic acid was identified as the major ethyl acetate-soluble compound. These compounds accounted for about 73 and 16%, respectively, of the radioactivity in the apical bud. No other compounds were present in significant amounts. As neither of these chemicals is likely to have any kind of senescence effect, we report no evidence for a senescence factor. PMID- 16666329 TI - Cyanide metabolism in relation to ethylene production in plant tissues. AB - HCN is the putative product of C-1 and amino moieties of 1-aminocyclopropane-1 carboxylic acid (ACC) during its conversion to ethylene. In apple (Malus sylvestrus Mill.) slices or auxin-treated mungbean (Vigna radiata L.) hypocotyls, which produced ethylene at high rates, the steady state concentration of HCN was found to be no higher than 0.2 micromolar, which was too low to inhibit respiration (reported Ki for HCN to inhibit respiration was 10-20 micromolar). However, these tissues became cyanogenic when treated with ACC, the precursor of ethylene, and with 2-aminoxyacetic acid, which inhibits beta-cyanoalanine synthase, the main enzyme to detoxify HCN; the HCN levels in these tissues went up to 1.7 and 8.1 micromolar, respectively. Although ethylene production by avocado (Persea gratissima) and apple fruits increased several hundred-fold during ripening, beta-cyanoalanine synthase activity increased only one- to two fold. These findings support the notion that HCN is a co-product of ethylene biosynthesis and that the plant tissues possess ample capacity to detoxify HCN formed during ethylene biosynthesis so that the concentration of HCN in plant tissues is kept at a low level. PMID- 16666330 TI - Depression of Photosynthesis, Growth, and Yield in Field-Grown Green Pepper (Capsicum annuum L.) Exposed to Acidic Fog and Ambient Ozone. AB - The relationship among physiological, injury, growth, and yield responses was examined in field-grown green pepper (Capsicum annuum L. ;California Wonder') subjected to two airborne environmental stresses. The primary objectives were to determine if the stresses could cause alterations in the plant responses, and to determine if any stress induced alterations in physiological or injury responses were correlated with effects on growth or yield. Responses were monitored in green pepper exposed to simulated acidic fog alone, or in combination with ambient concentrations of ozone in open-top field chambers. Both highly acidic fog and ambient ozone depressed green pepper growth and yield responses via the inhibition of photosynthesis. Applications of highly acidic fog (i.e. two exposures of pH 1.68 fog per week for 11 weeks) caused a significant depression of net photosynthesis, reduction in leaf buffering capacity, and an extensive amount of leaf injury. These alterations closely paralleled decreases in growth and yield on a percentage basis. In contrast, ambient ozone had similar impacts on net photosynthesis, growth and yield, but enhanced leaf buffering capacity, and caused no visible injury. The pollutant-specific differences in plant response are discussed with respect to whole-plant carbon metabolism and physiological compensation. PMID- 16666331 TI - Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F. AB - A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S 200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. PMID- 16666332 TI - Amino Acid transport into membrane vesicles isolated from zucchini : evidence of a proton-amino Acid symport in the plasmalemma. AB - Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid transport was studied in membrane vesicles isolated from zucchini (Cucurbita pepo L. cv Black Beauty) hypocotyls. An imposed pH gradient (basic interior) was used to energize isolated membrane vesicles and drive amino acid transport. Proton-coupled amino acid accumulation was demonstrated for alanine, glutamate, glutamine, leucine, and tabtoxinine-beta lactam. Alanine transport into the isolated membrane vesicles was studied in detail. Alanine transport was protonophore sensitive and accumulation ratios exceeding 10 times that predicted by diffusion alone were observed. DeltapH Dependent alanine transport exhibited saturation kinetics, suggesting translocation was mediated via a carrier transport system. In support of that conclusion, 50 micromolar N,N'-dicyclohexylcarbodiimide, a hydrophobic modifier of protein carboxyls, completely inhibited proton-coupled alanine accumulation. Transport activity, equilibrated on a linear sucrose gradient, peaked at 1.16 grams per cubic centimeter and co-migrated with a plasmalemma marker (vanadate sensitive K(+)-Mg(2+)-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants. PMID- 16666333 TI - Inorganic Carbon Accumulation by Chlamydomonas reinhardtii: New Proteins are made During Adaptation to Low CO(2). AB - When the unicellular green alga Chlamydomonas reinhardtii is placed under low CO(2) conditions it adapts by making an inorganic carbon accumulating mechanism. Algal cells were labeled with (35)SO(4) (-2) during this adaptation period and labeled proteins specific for this low CO(2) adaptation were identified. Four major proteins were preferentially synthesized under low CO(2) conditions and had M(r) of 46, 44, 37, and 20 kilodaltons. The 37 kilodalton protein is most likely the periplasmic carbonic anhydrase previously identified as being part of the inorganic carbon accumulation mechanism of C. reinhardtii. The other three proteins have not been identified. The 46 and the 44 kilodalton proteins were not synthesized by a mutant algal strain, pmp-1, which cannot grow at low CO(2) concentrations. This strain does make the 37 and 20 kilodalton proteins, however. These data suggest that at least two or three proteins in addition to the periplasmic carbonic anhydrase are part of the inorganic carbon accumulation mechanism in C. reinhardtii. PMID- 16666334 TI - Immunological Characterization of Iron-Regulated Membrane Proteins in the Cyanobacterium Anacystis nidulans R2. AB - Antibodies cross-reactive with specific membrane proteins were used to investigate membrane development in Anacystis nidulans R2 during recovery from iron stress. Polyclonal antibodies prepared using the iron-regulated chlorophyll (Chl)-protein CPVI-4 (HB Pakrasi, HC Riethman, LA Sherman 1985 Proc Natl Acad Sci USA 82: 6903-6907) as antigen were characterized and used to identify three iron stress-induced polypeptides of 36, 35, and 34 kilodaltons on immunoblots of polyacrylamide gels. The 34 kilodalton protein was shown to be a component of the Chlbinding CPVI-4 complex. The 36 kilodalton protein is an unrelated, intrinsic membrane protein tightly regulated by iron (designated IrpA), whereas the 35 kilodalton immunoreactive component is an extremely abundant glycoprotein (GP35). An analysis of photosystem II (PSII)-associated Chl-proteins during recovery from iron stress demonstrates that CPVI-4 is associated with most of the Chl present in iron-starved cells, whereas the PSII core polypeptides are present in very low levels; upon recovery, CPVI-4 diminishes in abundance as the relative levels of the other PSII proteins increase. The abundance of CPVI-4 in iron-stressed cells and the distribution of Chl among individual Chl-proteins during recovery suggest a possible role for CPVI-4 in the direction of membrane assembly during recovery from iron stress. PMID- 16666335 TI - Use of a laser-driven photoacoustic detection system for measurement of ethylene production in cymbidium flowers. AB - A laser-based photoacoustic method was used for determination of ethylene (C(2)H(4)) production of emasculated orchid (Cymbidium) flowers in a flow-through system. The laser photoacoustic equipment consisted of a line-tuneable CO(2) laser in conjunction with a single-pass resonant acoustic cell. The minimum detection limit of the system for C(2)H(4) in air was 0.03 nanoliter per liter. C(2)H(4) production of intact Cymbidium (cv Mary Pinchess ;Del Rey') flowers was very low (0.015 nanoliter per gram per hour) and showed an increase within 3 hours following emasculation (removal of pollinia plus anthercap). Production peaked (0.14 nanoliter per gram per hour) 8 hours after emasculation and decreased thereafter. Production again increased 45 hours after emasculation. Coloration of the labellum appeared shortly after the first peak; wilting of the petals and sepals appeared during the second rise in ethylene production. The use of the laser photoacoustic technique in plant physiological studies is discussed. PMID- 16666336 TI - Incorporation of [C]Glucose into Cell Wall Polysaccharides of Cotton Roots: Effects of NaCl and CaCl(2). AB - Cotton (Gossypium hirsutum L. cv Acala SJ-2) seedlings were grown in nutrient solutions with four combinations of NaCl (0.1 and 150 millimolar) and CaCl(2) (1 and 10 millimolar) for 7 days, and then exposed to [(14)C]glucose for 5 hours. Uptake and incorporation of [(14)C]glucose into various cell wall fractions of the root tips were determined. At 1 millimolar Ca(2+), treatment with 150 millimolar NaCl slightly stimulated uptake but considerably inhibited glucose incorporation into noncellulosic and cellulosic polysaccharides. Supplemental Ca(2+) did not affect incorporation of glucose into the noncellulosic fraction (regardless of NaCl treatment) but completely alleviated the inhibitory effect of NaCl on glucose incorporation into cellulose. We suggest that high Na(+) concentrations reduce synthesis of cellulose in cotton roots via disturbance of plasma membrane integrity and that supplemental Ca(2+) counteracts this effect. The effects on cellulose biosynthesis are proposed to be related to Ca(2+) displacement from the plasma membrane. PMID- 16666337 TI - A refined technique to apply electrical currents to callus cultures. AB - A simple, inexpensive technique is described which can be used to pass small electrical currents of a few microamperes through callus cultures under sterile conditions without contaminating the tissue or the medium with electrode products. Application of 1 or 2 microamperes currents of either polarity stimulated growth as well as shoot regeneration in tobacco (Nicotiana tabacum var. virginica) callus cultures, indicating that these effects are due to the electrical fields set up by the exogenous currents, rather than by electrode products. PMID- 16666339 TI - Ethylene effect on extensin and peroxidase distribution in the subapical region of pea epicotyls. AB - In dark grown pea (Pisum sativum) seedlings ethylene causes the triple response in which elongation growth is inhibited, radial growth is promoted, and orientation of shoots to gravity is altered. The distribution of extensin and peroxidase activity in pea epicotyls upon ethylene treatment was studied by tissue printing on nitrocellulose paper. It was found that the localization of extensin and peroxidase activity changes after 72 and 96 hours of ethylene treatment. In untreated plants, peroxidase activity is detected only in the vascular bundles. Nonetheless, after 72 and 96 hours of ethylene treatment peroxidase activity is hardly detected in the vascular system but present in the epidermal and cortical cells. Extensin increases in the epidermal and cortical cells upon ethylene treatment but it also appears in the vascular system when peroxidase activity is no longer detected. PMID- 16666338 TI - Photosystem II in Guard Cells of Vicia faba: Immunological Detection. AB - Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts. PMID- 16666340 TI - Determination of Abscisic Acid in Pinus densiflora by Selected Ion Monitoring. AB - Abscisic acid (ABA) in the stem of akamatsu (Pinus densiflora) was identified and quantified by gas chromatography-mass spectrometry-selected ion monitoring using hexadeuterated ABA as an internal standard. tert-Butyldimethylsilyl ester was used as a derivative of ABA. This derivative had high sensitivity and selectivity for ABA determination. ABA concentrations in cambial region scrapings were independent of the cessation of cambial activity. PMID- 16666341 TI - Occurrence of alternative respiratory capacity in soybean and pea. AB - Capacity for the alternative respiratory pathway was assessed in leaf and root tissue of male-sterile and fertile soybean (Glycine max [L.] Merr.) plants and in leaf, embryonic axis, and epicotyl tissue as well as isolated mitochondria of pea (Pisum sativum L.) by measurement of oxygen uptake in the presence and absence of KCN and salicylhydroxamic acid. Male-sterile and fertile soybean tissues showed similar responses to the inhibitors, and both possessed a capacity for alternative respiration. We also found that tissue and isolated mitochondria from ;Progress No. 9' pea possessed alternative respiratory capacity similar to that of ;Alaska' pea. PMID- 16666342 TI - Inheritance of c(4) enzymes associated with carbon fixation in flaveria species. AB - Activities and subunit levels of three C(4) enzymes were determined for F(1) hybrids between C(4) and C(3)-C(4)Flaveria species. For phosphoenolpyruvate carboxylase and pyruvate orthophosphate, dikinase, enzyme amounts in the hybrids were close to the mid-parent means. However, activity and subunit levels of NADP malic enzyme were approximately one-half the mid-parent mean. PMID- 16666343 TI - When does the self-regulatory response elicited in soybean root after inoculation occur? AB - The inoculation of soybean (Glycine max L.) roots with Bradyrhizobium japonicum produces a regulatory response that inhibits nodulation in the younger regions of the roots. By exposing the soybean roots to live homologous bacteria for only a short period of time, the question of whether or not early interactions of rhizobia with root cells, prior to infection, elicit this regulatory response has been explored. B. japonicum cells mixed with infective bacteriophages were applied to the roots and then 6 or 24 hours later roots were again inoculated with phage-resistant rhizobia. Mixing of the rhizobia and bacteriophages caused bacterial lysis in 6 to 8 hours and allowed the bacteria to act as live symbionts on the root for only a few hours. However, the interaction of live homologous bacteria with the soybean roots for a few hours did not cause inhibition of nodulation in the younger regions of the roots. Results of these experiments indicate that the self-regulatory response in soybean is not rapidly produced by the early, pre-infection, interactions between rhizobia and the root cells. PMID- 16666344 TI - Phytochrome mediated regulation of sucrose phosphate synthase activity in maize. AB - The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30 minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet. PMID- 16666345 TI - Isolation of Intact Chloroplasts from Dunaliella tertiolecta. AB - Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO(2) was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O(2) evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O(2) and fixed 21 micromoles of CO(2) per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO(2) in air for 24 hours. On low CO(2) the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations. PMID- 16666346 TI - Organ-specific and developmental regulation of the nopaline synthase promoter in transgenic tobacco plants. AB - Control regions of the nopaline synthase (nos) gene have been widely used to express foreign genes in plants since the promoter is active in a wide variety of plant tissues. We report here the characteristics of the nos promoter activity in transgenic tobacco (Nicotiana tabacum) plants at various developmental stages. The promoter was highly active in the lower parts of a plant and gradually decreased in the upper parts. This vertical gradient was maintained throughout plant growth until the flowering stage when the overall promoter strength decreased significantly in the vegetative organs. However, in various flower organs, the nos promoter activities increased dramatically. Higher activity was observed in calyx, corolla, and stamens although the maximum promoter activity in each organ was found at different stages of flower development. The promoter activity in pistils was low and gradually increased in the ovaries after anthesis. In developing fruits, the nos promoter activity was strongly induced during the mid-stage of embryogenesis. These results indicate that the expression of the nos promoter is developmentally regulated and organ specific in transgenic tobacco plants. PMID- 16666347 TI - Dependence of in vivo ethylene production rate on 1-aminocyclopropane-1 carboxylic Acid content and oxygen concentrations. AB - 1-Aminocyclopropane-1-carboxylic acid (ACC) is aerobically oxidized in plant tissues to form ethylene by ethylene-forming enzyme (EFE). The effect of substrate (ACC and oxygen) concentrations on ethylene production rate by plant tissues was investigated. The K(m) value for O(2) in ethylene production varied greatly depending on the internal ACC content. When ACC levels in the tissue were low (below its K(m) value), the concentration of O(2) giving half-maximal ethylene production rate ([S](0.5)) ranged between 5 and 7%, and was similar among different tissues. As the concentration of ACC was increased (greater than its K(m) value), [S](0.5) for O(2) decreased markedly. In contrast, the K(m) value for ACC was not much dependent on O(2) concentration, but varied greatly among different plant tissues, ranging from 8 micromolar in apple (Malus sylvestris Mill.) tissue to 120 micromolar in etiolated wheat (Triticum aestivum) leaf. Such a great variation was thought to be due to the different compartmentation of ACC within the cells in different tissues. These kinetic data are consistent with the view that EFE follows an ordered binding mechanism in which EFE binds first to O(2) and then to ACC. PMID- 16666348 TI - Temporary disturbance of translocation of assimilates in douglas firs caused by low levels of ozone and sulfur dioxide. AB - Douglas firs (Pseudotsuga menziesii [Mirb.] Franco) are suffering strongly from air pollution in western Europe. We studied the effect of low concentrations of ozone (200 micrograms per cubic meter during 3 days) and sulfur dioxide (53 micrograms per cubic meter during 28 days) on translocation of assimilates in 2 year old Douglas firs. The trees were exposed to the pollutants and afterward transferred to a growth chamber adapted to the use of (14)CO(2). Root/soil respiration was measured daily. The results showed a significant decrease of the (14)CO(2) root/soil respiration during the first 1 to 2 weeks after exposure to either ozone or sulfur dioxide. The ultimate level of (14)CO(2) root/soil respiration did not differ significantly, which suggests a recovery of the exposed trees during the first weeks after exposure. PMID- 16666349 TI - Gene Expression in Developing Zea mays Embryos: Regulation by Abscisic Acid of a Highly Phosphorylated 23- to 25-kD Group of Proteins. AB - We have earlier identified a set of proteins of 23 to 25 kilodaltons (kD), covering an isoelectric point (pI) range of 6.2 to 8.2, which accumulate gradually during normal embryogenesis of Zea mays and disappear in early germination. These polypeptides can be induced prematurely in immature embryos by abscisic acid (ABA) treatment. We report here that the more acidic protein forms are due to post-translational phosphorylation of at least two polypeptides of 23 kD, pI 8.2 and 25 kD, pI 8.0. A polyclonal antiserum was obtained which recognizes all forms of both the 23-kD and 25-kD polypeptides. Recovery of cDNA clones corresponding to these proteins was accomplished by hybridization with cDNA made from size-selected mRNA enriched for these sequences. Hybrid selection experiments demonstrate that clone MA12 specifically hybridizes with mRNAs encoding the 23-kD and 25-kD protein set which are recognized by the antiserum. By Northern hybridization analysis, the RNA encoded by clone MA12 is shown to accumulate in mature embryos and to be induced in young embryos upon ABA incubation. PMID- 16666350 TI - Transformation of Soybean Cells Using Mixed Strains of Agrobacterium tumefaciens and Phenolic Compounds. AB - Cotyledon explants from germinated 1-day-old soybean seedling were inoculated with single or mixed strains of Agrobacterium tumefaciens. Mixed-strain infections with the supervirulent L,L-succinamopine type strain A281 (pTiBo542) and strain LBA4404 carrying an octopine type virulence (vir) region and a binary vector (pBin6) with a chimeric gene for kanamycin detoxification gave rise to tumors of which 25% were both kanamycin resistant and capable of hormone independent growth. Singlestrain inoculations with LBA4404 (pBin6) failed to give rise to kanamycin-resistant callus. Syringaldehyde, a compound which induces vir genes carried on the Ti plasmid, increased the number of galls incited on excised cotyledons by the weakly virulent octopine type strain A348 (pTiA6). Similar results were obtained with whole plants treated with this strain in the presence of the vir-inducing compound acetosyringone. Our results indicate that the recovery of transformed soybean cells can be enabled in some instances by coinfecting with a supervirulent strain or in other instances promoted by adding a phenolic compound to the inoculum. PMID- 16666351 TI - Do woody plants operate near the point of catastrophic xylem dysfunction caused by dynamic water stress? : answers from a model. AB - We discuss the relationship between the dynamically changing tension gradients required to move water rapidly through the xylem conduits of plants and the proportion of conduits lost through embolism as a result of water tension. We consider the implications of this relationship to the water relations of trees. We have compiled quantitative data on the water relations, hydraulic architecture and vulnerability of embolism of four widely different species: Rhizophora mangle, Cassipourea elliptica, Acer saccharum, and Thuja occidentalis. Using these data, we modeled the dynamics of water flow and xylem blockage for these species. The model is specifically focused on the conditions required to generate ;runaway embolism,' whereby the blockage of xylem conduits through embolism leads to reduced hydraulic conductance causing increased tension in the remaining vessels and generating more tension in a vicious circle. The model predicted that all species operate near the point of catastrophic xylem failure due to dynamic water stress. The model supports Zimmermann's plant segmentation hypothesis. Zimmermann suggested that plants are designed hydraulically to sacrifice highly vulnerable minor branches and thus improve the water balance of remaining parts. The model results are discussed in terms of the morphology, hydraulic architecture, eco-physiology, and evolution of woody plants. PMID- 16666352 TI - Mechanism of water stress-induced xylem embolism. AB - We investigated the hypothesis that water stress-induced xylem embolism is caused by air aspirated into functional vessels from neighboring embolized ones (e.g. embolized by physical damage) via pores in intervessel pit membranes. The following experiments with sugar maple (Acer saccharum Marsh.) support the hypothesis. (a) Most vessels in dehydrating stem segments embolized at xylem pressures < -3 megapascals; at this point the pressure difference across intervessel pits between air-filled vessels at the segment's ends and internal water-filled vessels was >3 megapascals. This same pressure difference was found to be sufficient to force air across intervessel pits from air injection experiments of hydrated stem segments. This suggests air entry at pits is causing embolism in dehydrating stems. (b) Treatments that increased the permeability of intervessel pits to air injection also caused xylem to embolize at less negative xylem pressures. Permeability was increased either by perfusing stems with solutions of surface tension below that of water or by perfusion with a solution of oxalic acid and calcium. The mechanism of oxalic-calcium action on permeability is unknown, but may relate to the ability of oxalate to chelate calcium from the pectate fraction of the pit membrane. (c) Diameter of pores in pit membranes measured with the scanning electron microscope were within the range predicted by hypothesis ( betaine aldehyde --> betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O(2)- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo(18)O- and (2)H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O(2) and reductant: a desaturase or an oxygenase. Simple syntheses for (2)H(3)-choline, (2)H(3), (18)O-choline, and (2)H(3), (18)O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with (2)H(2)O and by analyzing newly synthesized betaine. About 15% of the (2)H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% (2)H(2)O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of (2)H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of (18)O from (18)O(2) into newly synthesized betaine was compared with that from (18)O-labeled choline, in light and darkness. Incorporation of (18)O from (18)O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the (18)O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of (18)O from (18)O(2) approached that from (18)O choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic (16)O(2). These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase. PMID- 16666371 TI - Abscisic Acid is not the only stomatal inhibitor in the transpiration stream of wheat plants. AB - Xylem sap was collected from the transpiration stream of wheat (Triticum aestivum L.) plants and assayed for the presence of an inhibitor of transpiration using leaves detached from well-watered plants. Transpiration of detached leaves was reduced by nearly 60% by sap collected from plants in drying soil, and to a lesser extent (about 25%) by sap from plants in well-watered soil. As the soil dried the abscisic acid (ABA) concentration in the sap increased by about 50 times to 5 x 10(-8) molar. However, the ABA in the sap did not cause its inhibitory activity. Synthetic ABA of one hundred times this concentration was needed to reduce transpiration rates of detached leaves to the same extent. Furthermore, inhibitory activity of the sap was retained after its passage through an immunoaffinity column to remove ABA. Xylem sap was also collected by applying pressure to the roots of plants whose leaf water status was kept high as the soil dried. Sap collected from these plants reduced transpiration to a lesser extent than sap from nonpressurised plants. This suggests that the inhibitory activity was triggered partly by leaf water deficit and partly by root water deficit. PMID- 16666372 TI - Preparation and characterization of envelope membranes from nongreen plastids. AB - We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes. PMID- 16666373 TI - Resistance to Water Transport in Shoots of Vitis vinifera L. : Relation to Growth at Low Water Potential. AB - Apparent resistances to water transport in the liquid phase were determined from measurements of soil, root, basal shoot internode, shoot apex, and leaf water potentials and water flux in Vitis vinifera (cv White Riesling) during soil drying. Predawn water potential differences (DeltaPsi) in the shoots accounted for 20% of the total DeltaPsi between the soil and the shoot apex when plants were well-watered but increased to about 90% when shoot growth ceased. The DeltaPsi from soil to root was essentially constant during this period. At low water potential, the DeltaPsi in the shoot was persistent when transpiration was low (predawn) or completely prevented (plant bagging). The apparent hydraulic resistance between the basal shoot internode and most rapidly expanding leaf (or shoot apex) increased several-fold when water was withheld. Leaf and internode expansion both exhibited high sensitivity to increasing hydraulic resistance. Measurements of pneumatic resistance to air flow through frozen internode segments indicated progressive vapor-filling of vessels as soil drying progressed. From these observations and others in the literature, it was suggested that embolization may be a common occurrence and play an important role in the inhibition of shoot growth at moderate water deficits. PMID- 16666374 TI - Alterations in leaf carbohydrate metabolism in response to nitrogen stress. AB - A series of experiments was conducted to characterize alterations in carbohydrate utilization in leaves of nitrogen stressed plants. Two-week-old, nonnodulated soybean plants (Glycine max [L.] Merrill, ;Ransom'), grown previously on complete nutrient solutions with 1.0 millimolar NO(3) (-), were transferred to solutions without a nitrogen source at the beginning of a dark period. Daily changes in starch and sucrose levels of leaves were monitored over the following 5 to 8 days in three experiments. Starch accumulation increased relative to controls throughout the leaf canopy during the initial two light periods after plant exposure to N-free solutions, but not after that time as photosynthesis declined. The additional increments of carbon incorporated into starch appeared to be quantitatively similar to the amounts of carbon diverted from amino acid synthesis in the same tissues. Since additional accumulated starch was not degraded in darkness, starch levels at the beginning of light periods also were elevated. In contrast to the starch effects, leaf sucrose concentration was markedly higher than controls at the beginning of the first light period after the N-limitation was imposed. In the days which followed, diurnal turnover patterns were similar to controls. In source leaves, the activity of sucrose-P synthase did not decrease until after day 3 of the N-limitation treatment, whereas the concentration of fructose-2,6-bisphosphate was decreased on day 2. Restricted growth of sink leaves was evident with N-limited plants within 2 days, having been preceeded by a sharp decline in levels of fructose-2,6 bisphosphate on the first day of treatment. The results suggest that changes in photosynthate partitioning in source leaves of N-stressed plants resulted largely from a stable but limited capacity for sucrose formation, and that decreased sucrose utilization in sink leaves contributed to the whole-plant diversion of carbohydrate from the shoot to the root. PMID- 16666375 TI - Characterization of an HSP70 Cognate Gene Family in Arabidopsis. AB - Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5' regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species. PMID- 16666376 TI - Molecular cloning of complementary DNA encoding maize nitrite reductase: molecular analysis and nitrate induction. AB - Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20 26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. PMID- 16666377 TI - 4-amino-5-hexynoic Acid-a potent inhibitor of tetrapyrrole biosynthesis in plants. AB - 4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants. PMID- 16666378 TI - Polar transport of 1-naphthaleneacetic Acid determines the distribution of flower buds on explants of tobacco. AB - Upon addition of 1-naphthaleneacetic acid (1-NAA) and benzylaminopurine, flower buds developed on explants from flower stalks of Nicotiana tabacum L. cv Samsun cultured in vitro. At low concentrations of 1-NAA, buds emerged mainly at the basal edge, whereas at high concentrations they developed on the remaining surface. The optimum concentrations for the two groups of buds were 0.45 micromolar and 2.2 micromolar, respectively, and the shapes of the concentration versus response curves were similar. The level of benzylaminopurine in the medium affected neither the shape nor the optimum concentration of these curves. The distribution of the buds over the explants was shown to be caused by polar auxin transport, leading to accumulation at the basal side. First, in the presence of the inhibitors 2,3,5-triiodobenzoic acid and 1-naphthylphthalamic acid, both groups of buds had the same optimum concentration of 1 micromolar 1-NAA. Second, after 6 hours of culture applied 1-NAA had accumulated in the basal part of the explant. In the presence of 1-naphthylphthalamic acid, no transport or accumulation of applied 1-NAA occurred. PMID- 16666379 TI - Characterization of the na-requirement in cyanobacterial photosynthesis. AB - The Na(+) requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li(+) concentration was examined in air grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O(2) evolution) as a function of Na(+) concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na(+) (K((1/2))[Na(+)]) were calculated. The value of both the low and the high K((1/2))(Na(+)) was dependent upon extracellular DIC concentration. The low K((1/2))(Na(+)) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K((1/2))(Na(+)) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na(+) and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na(+) and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO(2) and HCO(3) (-) transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO(2) and HCO(3) (-) transport systems required Na(+), directly or indirectly, for full activity. However, micromolar levels of Na(+) were required for CO(2) transport while millimolar levels were required for HCO(3) (-) transport. These levels corresponded to those found for the low and high K((1/2))(Na(+)) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO(2) transport increased with increased DIC concentration relative to HCO(3) (-) transport. This change was reflected in a decrease in the Na(+) concentration required for maximum photosynthesis, in accord with the lower Na(+)-requirement for CO(2) transport. Lithium competitively inhibited Na(+)-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na(+)-dependent HCO(3) (-) transport. Lithium had little effect on CO(2) transport and only a small effect on the size of the pool it generated. Thus, CO(2) transport did not require a functional HCO(3) (-) transport system for full activity. Based on these observations and the differential requirement for Na(+) in the CO(2) and HCO(3) (-) transport system, it was proposed that CO(2) and HCO(3) (-) were transported across the membrane by different transport systems. PMID- 16666380 TI - Two-Dimensional Electrophoretic Studies of the Proteins and Polypeptides in Mature Pollen Grains and the Male Germ Unit of Plumbago zeylanica. AB - Three fractions (male germ unit [MGU]-rich, cytoplasmic-particulate and water soluble proteins) were isolated from pollen of Plumbago zeylanica L. Proteins were extracted using a phenol procedure and polypeptide patterns were compared on one- and two-dimensional polyacrylamide gels. The MGU-rich fraction contains the sperm and vegetative nucleus of the pollen grain and yielded 427 spots >33 kilodaltons. The cyto-plasmic-particulate fraction contained 515 spots >33 kilodaltons. The third fraction consisted of water-soluble proteins and polypeptides from the pollen cytoplasm, in which 285 spots (>33 kilodaltons) were identified. Of 133 polypeptide spots suitable for comparison, 18 were unique to the MGU-rich fraction, 3 to the cytoplasmic-particulate fraction, 14 to the water soluble fraction, 65 were common to two different fractions (and absent in one), and 33 were common to all three of the fractions examined. PMID- 16666381 TI - Properties of a small basic Peptide from pumpkin seeds. AB - A small basic peptide with an unusual amino acid composition has been isolated from the seeds of pumpkin, Cucurbita maxima. Amino acid analysis and sequence data show the protein to be about 36 residues in length, with an approximate composition Lys(1), Arg(14), Asp(3), (Glu + Gln)(15), Gly(1), Pro(1), Trp(1). On the basis of composition, the molecular weight is approximately 5000 daltons and the nitrogen content by weight is 20.4%. Twelve amino acids are entirely lacking. The peptide is slightly toxic to mouse B-16 melanoma cells, but its in vivo function is unknown. It does not appear to be derived from cucurbitin, the pumpkin storage globulin; however, it could be a storage peptide involved in nitrogen mobilization during the early stages of germination. PMID- 16666382 TI - Characterization of an Ethylene Overproducing Mutant of Tomato (Lycopersicon esculentum Mill. Cultivar VFN8). AB - Ethylene production rates and tissue ethylene concentrations were determined for the single-gene, Epinastic (Epi) tomato (Lycopersicon esculentum Mill.) mutant, and its parent, cv VFN8. The Epi phenotype was characterized by severe leaf epinasty, thickened stems and petioles, and a compact growth habit. In 4-day-old seedlings, ethylene production was significantly higher in Epi than in VFN8. Ethylene production rates also were higher for excised root, hypocotyl, cotyledon, and shoot tissue of 14-day-old Epi seedlings as compared with VFN8. The greatest difference in the ethylene production rate was observed in excised Epi shoot tissue, which was more than 2.5 times higher than in VFN8. Tissue ethylene concentrations of 19-, 25-, and 31-day-old Epi plants were 8, 172, and 307% higher than for VFN8, corresponding to increasing expression of the Epi phenotypic characteristics with age. The highest ethylene concentrations occurred in the shoot apex of both genotypes. Higher ethylene concentrations in Epi resulted from greater 1-aminocyclopropane-1-carboxylic acid content rather than increased ethylene-forming enzyme activity. The elevated ethylene levels in Epi did not result from increased auxin sensitivity. The sensitivity of root growth to inhibition by ethylene did not differ between VFN8 and Epi. Although elevated levels of ethylene in Epi plants apparently exacerbate its epinastic growth characteristics, other evidence indicates that this may not be the fundamental lesion. This mutant may provide a unique system for investigating the regulation of ethylene biosynthesis and the role of target cell types in plant development. PMID- 16666383 TI - Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.). AB - Endogenous indoleacetic acid (IAA) levels were examined in 7-day-old, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using (13)C(6)-[benzene ring]indoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene over-production by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3 ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms). PMID- 16666384 TI - Proteins under the Control of the Gene for Fe Efficiency in Tomato. AB - Fe-deficient dicotyledons develop Fe-efficiency reactions, such as proton extrusion and ferric chelate reduction activity, which are located in the plasma membranes of the root epidermal cells. The fer mutant of tomato (Lycopersicon esculentum Mill.) cannot develop these reactions. Membranes were isolated from roots of wild-type (FER) and mutant (fer) tomato plants grown on nutrient solution with high and low Fe concentrations. Two proteins were identified which are synthesized under the control of the FER gene. PMID- 16666385 TI - Cytokinin Biochemistry in Relation to Leaf Senescence: IV. Cytokinin Metabolism in Soybean Explants. AB - When [(3)H]dihydrozeatin riboside and [(3)H]zeatin riboside were supplied to soybean (Glycine max L.) explants (comprising one leaf, associated pods, and subtending stem) via the xylem at mid to late podfill, 0.1% of the supplied (3)H was extracted from the seeds. The distribution of (3)H in the explants was similar to that bound previously following uptake of [(3)H]zeatin riboside at earlier stages of pod development. Metabolites formed in the explants from (3)H labeled zeatin, zeatin riboside, and dihydrozeatin riboside were identified and related to the endogenous cytokinins shown to be present. When zeatin riboside and zeatin were supplied for 1 hour, zeatin nucleotide was the principal metabolite formed and this appeared to be the precursor of the other metabolites detected subsequently. Explants supplied with zeatin riboside or dihydrozeatin riboside for 1 hour, and then transferred to water for 20 to 24 hours, yielded leaf blades in which the main metabolites were O-glucosyldihydrozeatin, adenosine, and adenine. The metabolism of zeatin riboside in blades of explants at pre-podfill, early podfill, and mid to late podfill did not differ appreciably. The results are discussed in relation to leaf senescence and seed development. PMID- 16666386 TI - Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls. AB - The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [(3)H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [(3)H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K(d)) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [(3)H]NPA binding. PMID- 16666387 TI - Carbonyl sulfide: an inhibitor of inorganic carbon transport in cyanobacteria. AB - Cells of a high CO(2)-requiring mutant (E(1)) and wild type of Synechococcus PCC7942 were incubated with COS in the light, then suspended in COS-free medium and their CO(2) exchange was measured using an open gas-analysis system under the conditions where photosynthetic CO(2) fixation is inhibited. When the suspension of cells untreated with COS was illuminated, the rate of CO(2) uptake was high and addition of carbonic anhydrase during illumination released a large amount of CO(2) from the medium into the gas phase. The COS treatment in the light markedly reduced the rate of CO(2) uptake by the cells and the amount of CO(2) released by carbonic anhydrase. Incubation of cells with COS in the dark had no effect on the CO(2)-exchange profile. The COS concentration required for 50% inhibition of CO(2) uptake was about 25 micromolar when the concentration of inorganic carbon (C(i)) in the medium was 60 micromolar; higher C(i) concentrations reduced the inhibitory effect of COS. Measurement of C(i) uptake in E(1) cells by a silicone oil centrifugation method also indicated marked reduction of the activities of (14)CO(2) and H(14)CO(3) (-) uptake in the cells treated with COS in the light. The results demonstrated that COS is a potent inhibitor of C(i) transport. PMID- 16666388 TI - Hypoxic stress inhibits the appearance of wound-response proteins in potato tubers. AB - Potato (Solanum tuberosum L.) tubers respond to environmental stresses by alterations of macromolecular synthesis. In an aerobic environment tubers respond rapidly to wounding by synthesizing a set of proteins, the most prominent of which display apparent molecular weights of 78, 48, 38, and 31 kilodaltons. These proteins become intensely labeled by [(35)S]methionine within 2 hours of wounding. The 78 kilodalton polypeptide has been identified by immunoprecipitation as phenylalanine ammonia-lyase. By contrast, tubers incubated in hypoxic conditions for a period as short as 1.5 hours exhibit significantly reduced incorporation of amino acids such that newly synthesized polypeptides are not detected. However, a second set of proteins is synthesized by wounded tubers after prolonged incubation in a hypoxic environment. One peptide of this set is precipitated by an antibody directed against aldolase; several of these proteins may be enzymes of glycolysis necessary for anaerobic metabolism. The results indicate that there is a complex regulatory mechanism which allows mature potato tubers to respond to changes in the environment. PMID- 16666389 TI - Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I. AB - We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3 dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP(+) reductase. PMID- 16666390 TI - Metabolite diffusion into bundle sheath cells from c(4) plants: relation to c(4) photosynthesis and plasmodesmatal function. AB - The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function. PMID- 16666391 TI - Pulse-induced phototropisms in oat and maize coleoptiles. AB - Phototropisms induced by a pulse (1-30 seconds) of blue light in red-light-grown coleoptiles of oats (Avena sativa L.) and maize (Zea mays L.) were investigated in terms of fluence-response relationships and time courses. Phototropic stimulation was made by a laser beam (457.9 nanometers), allowing application of high-fluence pulses. The phototropic fluence-response curves for oats and maize revealed two peaks in the positive range, thus indicating the occurrence of two separable pulse-induced positive responses. The response at low fluences corresponded to the ;first positive curvature.' The response at high fluences was very small in oats, but was large in maize. Reciprocity was valid in this high fluence response (tested only for maize), indicating that it is distinct from the so-called ;second positive curvature.' In oats, the trough between the two positive responses fell into the negative range. This negative response, corresponding to the ;first negative curvature,' showed time courses distinct from those of ;first positive curvature:' the negative response was induced after a longer time lag and developed with a more gradual increase of the rate of bending. The maximal rate of the negative response was as high as one-half of that of first positive curvature. In maize, the trough between the two responses was in the positive range, and the time-course result revealed no apparent response counteracting the positive responses. Physiological and ecological implications of the pulse-induced phototropisms are discussed. PMID- 16666392 TI - Sugars and desiccation tolerance in seeds. AB - Soluble sugars have been shown to protect liposomes and lobster microsomes from desiccation damage, and a protective role has been proposed for them in several anhydrous systems. We have studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating soybean (Glycine max L. Merr. cv Williams), pea (Pisum sativum L. cv Alaska), and corn (Zea mays L. cv Merit) axes. The loss of desiccation tolerance during imbibition was monitored by following the ability of seeds to germinate after desiccation following various periods of preimbibition and by following the rates of electrolyte leakage from dried, then rehydrated axes. Finally, we analyzed the soluble sugar contents of the axes throughout the transition from desiccation tolerance to intolerance. These analyses show that sucrose and larger oligosaccharides were consistently present during the tolerant stage, and that desiccation tolerance disappeared as the oligosaccharides were lost. The results support the idea that sucrose may serve as the principal agent of desiccation tolerance in these seeds, with the larger oligosaccharides serving to keep the sucrose from crystallizing. PMID- 16666393 TI - Inhibition of ethylene biosynthesis by salicylic Acid. AB - Salicylic acid inhibited ethylene formation from ACC in self-buffered (pH 3.8) pear (Pyrus communis) cell suspension cultures with a K(1) (app) of about 10 micromolar after 1 to 3 hours incubation. Inhibition appeared noncompetitive. Among 22 related phenolic compounds tested, only acetylsalicylic acid showed similar levels of inhibition. Inhibition by salicylic acid was inversely dependent on the pH of the culture medium and did not require a continuous external supply of salicylate. When compared to known inhibitors of the ethylene forming enzyme, cobalt, n-propyl gallate, and dinitrophenol, inhibition by salicylic acid most closely resembled that by dinitrophenol but salicylic acid did not produce the same degree of respiratory stimulation. Results are discussed in terms of other known effects of salicylic acid on plants, pH-dependency, and the possible influence of salicylic acid on electron transport. PMID- 16666394 TI - A Starchless Mutant of Nicotiana sylvestris Containing a Modified Plastid Phosphoglucomutase. AB - A mutant (NS 458) of Nicotiana sylvestris (Spegazzini and Comes) unable to synthesize leaf starch was isolated in the M(2) generation following ethyl methanesulfonate mutagenesis by testing with iodine. Segregation ratios in reciprocal F(2) progenies showed that the starchless phenotype resulted from a recessive mutation in a single nuclear gene. DEAE-agarose chromatography showed that the mutant is grossly deficient in plastid phosphoglucomutase (EC 2.1.5.1) activity. The structure of the enzyme is changed, as evidenced by increased Michaelis constants and by the prolonged activation period (>40 minutes) observed when the enzyme is assayed in triethanolamine buffer rather than imidazole buffer. The activity of the wild-type enzyme with saturating glucose 6-P alone was 7% of the activity when saturating glucose 1,6-P(2) was also present. The results suggest that glucose 1,6-P(2) is both an effector and a dissociable reaction intermediate. The growth rate of mutant and wild-type plants were not significantly different in continuous light and on an 8-hour dark, 16-hour light cycle and the mutants grew normally under greenhouse conditions. The mutant supports growth during diurnal periods of darkness by vacuolar storage of sugars instead of chloroplast storage of starch. The simplification in metabolism achieved by blocking the diversion of plastid fructose-6-P to starch facilitates the induction of oscillations in CO(2) fixation. PMID- 16666395 TI - Correlation between Auxin Resistance and the Lack of a Membrane-Bound Auxin Binding Protein and a Root-Specific Peroxidase in Nicotiana tabacum. AB - The levels of a membrane-bound auxin binding protein (MABP) and a root-specific peroxidase (RSP) were studied in several tobacco (Nicotiana tabacum L.) cell lines including an auxin-resistant variant. Groups of cell lines were distinguished which behaved differentially with respect to MABP and RSP depending on the hormonal composition of the medium. In cell lines in which there existed a correlation between the presence or absence of MABP and that of RSP both phenotypes were expressed if kinetin (1-2 micromolar) was supplied. In contrast, neither MABP nor RSP could be detected under any hormonal conditions tested in the auxin-resistant variant which retains the ability to differentiate shoots but lacks the ability to differentiate roots. About an eightfold increase in the concentration of MABP and a dramatic increase in the activity of RSP occurred in a transformant by a mutant strain of Agrobacterium tumefaciens lacking an intact cytokinin gene when it was grown on medium containing 1 to 2 micromolar kinetin. A correlation between auxin resistance and the lack of MABP and RSP suggests that MABP might be involved in auxin-mediated root differentiation in tobacco. PMID- 16666396 TI - Effects of Irradiance and Methyl Viologen Treatment on ATP, ADP, and Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves. AB - Since activation of ribulose bisphosphate carboxylase (rubisco) by rubisco activase is sensitive to ATP and ADP in vitro, we aimed to test the correlation between ATP level and rubisco activation state in intact leaves of Spinacia oleracea L. in response to changes in irradiance and after feeding the electron acceptor methyl viologen. Leaves were exposed to various irradiances for 45 minutes at atmospheric partial pressures of CO(2) and O(2). After measuring the rate of CO(2) assimilation, leaves were freeze-clamped in situ and the punched discs assayed for rubisco activity, and amounts of ribulose bisphosphate (RuBP), ATP, and ADP. The photosynthetic rate and the activation state of rubisco increased with increasing irradiance but the levels of RuBP, ATP, and ADP were not greatly affected. Methyl viologen fed leaves under low irradiance had rubisco activation states of 93% compared to 51% in control leaves. The ATP content of the leaves was also significantly higher and the ratio of ATP to ADP was 4.1 in methyl viologen fed leaves compared to 2.2 in control leaves. From these results and other published results we conclude that a correlation between ATP level and rubisco activation can be observed in intact leaves, but that during changes in irradiance some additional factors are involved in regulating rubisco activation. PMID- 16666397 TI - Phosphatidylcholine synthesis: differing patterns in soybean and carrot. AB - The methylation steps in the biosynthesis of phosphatidylcholine by tissue culture preparations of carrot (Daucus carota L.) and soybean (Glycine max), and by soybean leaf discs, have been studied. Preparations were incubated with tracer concentrations of l-[(3)H(3)C]methionine and the kinetics of appearance of radioactivity in phosphomethylethanolamine, phosphodimethylethanolamine, phosphocholine, phosphatidylmethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, methylethanolamine, dimethylethanolamine, and choline followed at short incubation times. With soybean (tissue culture or leaves), an initial methylation utilizes phosphoethanolamine as substrate, forming phosphomethylethanolamine. The latter is converted to phosphatidylmethylethanolamine, which is successively methylated to phosphatidyldimethyethanolamine and to phosphatidylcholine. With carrot, again, an initial methylation is of phosphoethanolamine. Subsequent methylations occur at both the phospho-base and phosphatidyl-base levels. Both of these patterns differ qualitatively from that previously demonstrated in Lemna (SH Mudd, AH Datko 1986 Plant Physiol 82: 126-135) in which all three methylations occur at the phospho-base level. For soybean and carrot, some added contribution from initial methylation of phosphatidylethanolamine has not been excluded. These results, together with those from similar experiments carried out with water stressed barley leaves (WD Hitz, D Rhodes, AD Hanson 1981 Plant Physiol 68: 814 822) and salinized sugarbeet leaves (AD Hanson, D Rhodes 1983 Plant Physiol 71: 692-700) suggest that in higher plants some, perhaps all, phosphatidylcholine synthesis occurs via a common committing step (conversion of phosphoethanolamine to phosphomethylethanolamine) followed by a methylation pattern which differs from plant to plant. PMID- 16666398 TI - Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells. AB - Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K(a)) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate. PMID- 16666399 TI - Biosynthesis of Tetrapyrrole Pigment Precursors : Formation and Utilization of Glutamyl-tRNA for delta-Aminolevulinic Acid Synthesis by Isolated Enzyme Fractions from Chlorella Vulgaris. AB - The universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) is formed from glutamate (Glu) in algae and higher plants. In the postulated reaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase, and the product serves as a substrate for a reduction step catalyzed by a pyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reduced intermediate is then converted into ALA by a transaminase. An RNA and three enzyme fractions required for ALA formation from Glu have been isolated from soluble Chlorella extracts. The recombined fractions catalyzed ALA production from Glu or Glu-tRNA. The fraction containing the synthetase produced Glu-tRNA from Glu and tRNA in the presence of ATP and Mg(2+). The isolated product of this reaction served as substrate for ALA production by the partially reconstituted enzyme system lacking the synthetase fraction and incapable of producing ALA from Glu. The production of ALA from Glu-tRNA by this partially reconstituted system did not require free Glu or ATP, and was not affected by added ATP. These results show that (a) free Glu-tRNA is an intermediate in the formation of ALA from Glu, (b) ATP is required only in the first step of the reaction sequence, and NADPH only in a later step, (c) Glu-tRNA production is the essential reaction catalyzed by one of the enzyme fractions, (d) this enzyme fraction is active in the absence of the other enzymes and is not required for activity of the others. The specific Glu-tRNA synthetase required for ALA formation has an approximate molecular weight of 73,000 +/- 5,000 as determined by Sephadex G-100 gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffective in the the ALA-forming process. PMID- 16666400 TI - Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways. AB - Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K(+) content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of -0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K(+) content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K(+) content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K(+) content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K(+) gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K(+) gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K(+) uptake and starch loss. Comparison of K(+) content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K(+) content in the intact tissue than in isolated peels. These results are not consistent with K(+) (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism. PMID- 16666401 TI - Isolation of dihydroxyacetone phosphate reductase from dunaliella chloroplasts and comparison with isozymes from spinach leaves. AB - A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30 degrees C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg(2+) or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl(2) and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl(2), and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent K(m) (NADH) was 74 micromolar and K(m) (NADPH) was 81 micromolar. Apparent V(max) was 1100 mumoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide. PMID- 16666402 TI - Changes in Two Forms of Membrane-Associated Cellulase during Ethylene-Induced Abscission. AB - Only one form of membrane-associated cellulase was found previously in the lower petiolar pulvinus of Phaseolus vulgaris (cv Red Kidney). The cellulase has an isoelectric point (pI) of 4.5 (DE Koehler, LN Lewis 1979 Plant Physiol 63: 677 679). This enzyme was detected in abscission zones collected before the onset of abscission (control tissue), and was thought to represent a pre-secretory form of another cellulase, the abscission cellulase, which has a basic pI and is secreted during abscission. We now show that this acidic, membrane-associated cellulase is a glycoprotein, tightly bound to the membrane, with maximum activity at pH 5.1, and that it is not immunologically related to the abscission cellulase. Furthermore, when bean explants are induced to abscise with ethylene, the activity of the acidic cellulase declines rapidly to 50% of control levels in the first day. When abscission is fully developed, the membranes contain a basic form of cellulase with a pI of 8.0 to 9.0 and only trace levels of the acidic cellulase. The basic form is not a high mannose glycoprotein; it has maximum activity in a broad pH range (4.0-8.0) and is antigenically related to the abscission cellulase, which is induced during abscission and transported to the cell wall. Antibody raised against the abscission cellulase recognized two proteins in a crude membrane fraction from abscising tissue. One of those proteins comigrated with the abscission cellulase, and the other was 1 to 2 kilodaltons larger. Thus, during abscission, the acidic membrane-associated cellulase rapidly declines before the appearance of the abscission cellulase. We conclude that there is no conversion from the acidic cellulase to the basic cellulase and suggest that the acidic and basic cellulase isoenzymes are proteins derived from two different genes. PMID- 16666403 TI - Changes in the Levels of Calmodulin and of a Calmodulin Inhibitor in the Early Phases of Radish (Raphanus sativus L.) Seed Germination: Effects of Aba and Fusicoccin. AB - An inhibitor of Ca(2+)-calmodulin (Cam)-dependent brain phosphodiesterase was present in the soluble fraction of embryo axes from ungerminated radish (Raphanus sativus L.) seeds. This inhibitor is a Ca(2+)-dependent, Cam-binding protein; in fact: (a) its effect was strongly reduced by treatment with proteases; (b) the inhibition was counteracted by Cam but not by Ca(2+); (c) on gel filtration in the presence of Ca(2+), Cam co-chromatographed with the inhibitor. The inhibitor is heat stable and positively charged at pH 7.5. During early phases of germination, the fresh weight and the levels of DNA and RNA of embryo axes increased, the level of the inhibitor decreased, and the level of Cam increased. Abscisic acid (ABA) inhibited germination, the decrease of inhibitor, and the increase of Cam. Fusicoccin (FC) stimulated the increase in fresh weight but not the increase in the RNA and DNA levels; in this condition, the inhibitor level decreased and the increase in Cam level was higher than in the control. In the presence of both ABA and FC, there was an increase in fresh weight not accompanied by an increase in DNA and RNA levels; Cam increased and, on a fresh weight basis, reached the value of the control. These results indicate that the Ca(2+)-Cam system was activated in early germination of radish seeds by an increase in Cam and a decrease in the inhibitor levels, that FC, probably through the activation of membrane functions, increased Cam level, and that the ABA inhibition on germination was not mediated by the Ca(2+)-Cam system. PMID- 16666404 TI - Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in prunus wood. AB - Ice nuclei active at approximately -2 degrees C and intrinsic to woody tissues of Prunus spp. were shown to have properties distinct from bacterial ice nuclei. Soaking 5-centimeter peach stem sections in water for 4 hours lowered the mean ice nucleation temperature to below -4 degrees C, nearly 2 degrees C lower than stems inoculated with ice nucleation-active Pseudomonas syringae strain B301D. Ice nucleation activity in peach was fully restored by air-drying woody stem sections for a few hours. The ice nuclei in woody tissue were inactivated between 40 and 50 degrees C, but unaffected by treatment with bacterial ice nucleation inhibitors (i.e. NaOCl, tartaric acid, Triton XQS-20), sulfhydryl reagents (i.e. p-hydroxymercuribenzoate and iodine) and Pronase. Ice nuclei could not be dislodged from stems by sonication and were shown to be equally distributed in peach bud and internodal stem tissue on a per unit mass basis; outer and inner stem tissues were also indistinguishable in ice nucleation activity. Development of ice nuclei in immature peach and sweet cherry stems did not occur until midsummer and their formation was essentially complete by late August. Once formed the ice nuclei intrinsic to woody stems were stable and unaffected by seasonal changes in growth. The apparent physiological function of the ice nuclei is discussed in relation to supercooling and mechanisms of cold hardiness in Prunus spp. PMID- 16666405 TI - Effects of Growth Irradiance and Nitrogen Limitation on Photosynthetic Energy Conversion in Photosystem II. AB - Photosynthetic energy conversion was investigated in five species of marine unicellular algae, (Dunaliella tertiolecta, Thalassiosira pseudonana, T. weisflogii, Skeletorema costatum, Isochrysis galbana) representing three phylogenetic classes, which were grown under steady state conditions with either light or inorganic nitrogen as a limiting factor. Using a pump and probe fluorescence technique we measured the maximum change in variable fluorescence yields, the flash intensity saturation curves for the change in fluorescence yields and the kinetics of the decay in fluorescence yields. Under all growth irradiance levels nutrient replete cells exhibited approximately the same changes in fluorescence yields and similar fluorescence decay kinetics. The apparent relative absorption cross-section of photosystem II, calculated from the slope of the flash intensity saturation curves, generally increased as cells shade adapted. The decay kinetics of the fluorescence yield following a saturating pump flash can be expressed as the sum of three exponential components, with half times of 160 and 600 microseconds and 30 to 300 milliseconds. The relative contribution of each component did not change significantly with growth irradiance. As cells became more nitrogen limited, however, the maximum change in fluorescence yield decreased, and was accompanied by a decrease in the proportion of a 160 microsecond fluorescence decay component, which corresponds to the transfer of electrons from Q(a) (-) to Q(b). Changes in fluorescence yields were also accompanied by changes in the levels of D1, a protein which is integral in reaction center II, and CP47, a chlorophyll protein forming part of the core of photosystem II. These results are consistent with a loss of functional photosystem II reaction centers. Moreover, in spite of losses of total cellular chlorophyll, which invariably accompanied nitrogen limitation, the apparent absorption cross-sections of photosystem II increased. Our results suggest that nitrogen limitation leads to substantial decreases in photosynthetic energy conversion efficiency. PMID- 16666406 TI - A Nuclear Mutation in Nicotiana sylvestris Causing a Thiamine-Reversible Defect in Synthesis of Chloroplast Pigments. AB - We report the recovery of a nuclear recessive mutation in Nicotiana sylvestris (Spegazzini and Comes) producing a conditional disruption in the pathway for synthesis of chlorophyll a and b and carotenoids which is fully reversible by exogenous thiamine (0.3 micromolar). In the absence of supplemental thiamine, chlorophyll levels declined by 50% after 5 days, and fell to undetectable levels by 11 days. Mitochondrial (KCN sensitive) respiration rates remained normal in albino leaves (80% loss of chlorophyll), suggesting that chlorosis results primarily from a deficiency of thiamine in the chloroplasts. After thiamine removal, mutant plants produced at least 10 albino leaves with a substantial capacity for growth (0-15 centimeters; 70-fold increase in area), demonstrating sustained operation of many cellular functions in spite of chloroplast disruption. Activities of the plastid isozymes of phosphoglucomutase and phosphoglucoisomerase in albino leaves indicated that the decline in pigment synthesis does not result from a general loss of metabolic activity in chloroplast. Plastid pyruvate dehydrogenase from mutant and wild-type plants displayed a similar affinity for thiamine pyrophosphate, showing that chlorosis does not result from an alteration in this enzyme. Growth of albino leaves and ultrastructural evidence for thylakoid membranes in the chloroplasts suggest that a certain level of fatty acid synthesis is maintained after the interruption of pigment synthesis. Since thiamine deprivation is expected to block production of acetyl-coenzyme A from pyruvate by pyruvate dehydrogenase, acetyl-coenzyme A supporting fatty acid synthesis in albino leaves may be derived solely from mitochondrial acetate. PMID- 16666407 TI - Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase. AB - Chitinase and beta-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and beta-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and beta-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and beta-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by beta-1,3-glucanase alone. However, combinations of purified chitinase and beta-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and beta-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips. PMID- 16666408 TI - Immunocytolocalization of Glutamine Synthetase in Green Leaves and Cotyledons of Lycopersicon esculentum. AB - Glutamine synthetase was localized in leaves and cotyledons of young tomato (Lycopersicon esculentum Mill.) plants using immunogold techniques coupled to transmission electron microscopy. The enzyme occurs only in chloroplasts and is most probably a stroma constituent. PMID- 16666409 TI - Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii. AB - The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (-10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate). PMID- 16666410 TI - Phase shift of the circadian rhythm of lemna caused by pulses of a leucine analog, trifluoroleucine. AB - Pulses of a fluorinated analog of leucine, 5',5',5'-trifluoroleucine, reset the phase of the circadian rhythm of K(+) uptake in Lemna gibba G3 under continuous light conditions. The trifluoroleucine pulse caused the largest delay phase shifts during the early subjective phase but it caused only small phase advances. The action of trifluoroleucine was investigated and the following results were obtained. (a) The uptake of trifluoroleucine was essentially the same at all circadian phases, even though phase shifting was dramatically different at different phases. At effective phases, the magnitude of phase shifting was well correlated with the amount of trifluoroleucine taken up by the duckweed. (b) The trifluoroleucine pulse lowered the endogenous content of valine and leucine but these decreases did not correlate with phase shifting. (c) Protein synthesis was not affected by trifluoroleucine pulses which caused large phase shifts. (d) Pulses of 4-azaleucine, a different structural analog of leucine, also caused phase shifting. However, neither the direction nor the effective times of phase shifting were similar to those of trifluoroleucine. Taken together, these results negate the proposition that trifluoroleucine and azaleucine caused phase shift by disturbing amino acid metabolism and/or inhibiting protein synthesis, but they suggest instead that these analogs are incorporated into some protein(s) which are necessary for normal clock operation. PMID- 16666411 TI - The synthesis of a 19 kilodalton zein protein in transgenic petunia plants. AB - Transcriptional fusions composed of a 19 kilodalton zein cDNA, the 5' flanking region from a beta-phaseolin gene, and 3' flanking regions from either the phaseolin or a 15-kilodalton zein gene were introduced into Petunia by Agrobacterium-mediated transformation. The expression of both zein mRNA and protein in these transgenic plants was seed-specific and developmentally regulated. Both monocot (zein) and dicot (phaseolin) polyadenylation consensus sequences were recognized in Petunia. Analysis by immunoblotting showed that the M(r) of the zein protein corresponded to that of the mature protein, suggesting that recognition and cleavage of the signal sequence had occurred. While zein mRNA accumulated to approximately 1% of the total poly(A)(+) RNA in seeds of the transformed plants, zein protein was present at a much lower concentration than expected, at most being 0.005% of the total seed protein. These results suggest that the 19 kilodalton zein gene, in addition to lacking specific sequences required for efficient transcription in dicots, might also lack sequences required for the efficient synthesis, targeting, transport, or stabilization of the protein. PMID- 16666412 TI - Purification and assay of rubisco activase from leaves. AB - Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes. PMID- 16666413 TI - Characterization of the hexose transport system in maize root tips. AB - Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2 deoxy-d-glucose and 3-O-methyl-d-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments. PMID- 16666414 TI - Sites of inhibition by disulfiram in thylakoid membranes. AB - Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between Q(A), the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, Q(B). This site of inhibition is shown by a severalfold increase in the half-time of Q(-) (A) oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II; disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from H(2)O to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I. PMID- 16666415 TI - Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints. AB - The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex. PMID- 16666416 TI - Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium. AB - The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC. PMID- 16666417 TI - Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit. AB - Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO(2) and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit. PMID- 16666418 TI - l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism? AB - Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H(+) efflux from the cells by 111% and the uptake of l-[U (14)C]glutamate by 55%. Manometric experiments demonstrated that, unlike l methionine-d,l-sulfoximine, l-glutamate stimulated CO(2) evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and (14)CO(2) evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas (14)CO(2) evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l [U-(14)C]glutamate were higher in the light while rates of (14)CO(2) evolution were higher in the dark. The major labeled product of glutamate decarboxylation, gamma-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO(2) and gamma-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H(+)/l-glutamate uptake process. PMID- 16666419 TI - Glutamine transport and the role of the glutamine translocator in chloroplasts. AB - The transport of l-[(14)C]glutamine in oat (Avena sativa L.) and spinach (Spinacia oleracea L.) chloroplasts was studied by a conventional single-layer and a newly developed stable double-layer silicone oil filtering system. [(14)C]Glutamine was actively transported into oat chloroplasts against a concentration gradient. Metabolite uptake was greatly affected by the endogenous dicarboxylate pools, which could be easily changed by preloading the chloroplast with specific exogenous substrate. Glutamine uptake was decreased by 44 to 75% in oat chloroplasts preloaded with malate, 2-oxoglutarate (2-OG), and aspartate, but increased by 52% in chloroplasts preloaded with l-glutamate. On the other hand, the uptake of the other four dicarboxylates was decreased by 47 to 79% in chloroplasts preloaded with glutamine. In glutamine-preloaded chloroplasts the uptake of glutamine was inhibited only by l-glutamate. The observed inhibition by l-glutamate was competitive with an apparent K(i) value of 32.1 millimolar in oat and 6.7 millimolar in spinach chloroplasts. This study indicates that there are two components involved in glutamine transport in chloroplasts. The major component was mediated via a specific glutamine translocator. It was specific for glutamine and did not transport other dicarboxylates except l-glutamate. A K(0.5) value of 1.25 millimolar and V(max) of 45.5 micromoles per milligram of chlorophyll per hour were determined for the glutamine translocator in oat chloroplasts. The respective values were 1.0 millimolar and 16.7 micromoles per milligram of chlorophyll per hour in spinach chloroplasts. A three translocator model, involving the glutamine, dicarboxylate, and 2-OG translocators, is proposed for the reassimilation of photorespiratory NH(3) in chloroplasts of C(3) species. In this three-translocator model the additional transport of glutamine into the chloroplast is coupled to the export of glutamate via the glutamine translocator. This is an extension of the two-translocator model, involving the dicarboxylate and 2-OG translocators, proposed for spinach chloroplasts, (KC Woo, UI Flugge, HW Heldt 1987 Plant Physiol 84: 624-632). PMID- 16666420 TI - A New UV-A/B Protecting Pigment in the Terrestrial Cyanobacterium Nostoc commune. AB - A new ultraviolet (UV)-A/B absorbing pigment with maxima at 312 and 330 nanometers from the cosmopolitan terrestrial cyanobacterium Nostoc commune is described. The pigment is found in high amounts (up to 10% of dry weight) in colonies grown under solar UV radiation but only in low concentrations in laboratory cultures illuminated by artificial light without UV. Its experimental induction by UV as well as its capacity to efficiently protect Nostoc against UV radiation is reported. PMID- 16666421 TI - Correlation of Stomatal Conductance with Photosynthetic Capacity of Cotton Only in a CO(2)-Enriched Atmosphere: Mediation by Abscisic Acid? AB - Some evidence indicates that photosynthetic rate (A) and stomatal conductance (g) of leaves are correlated across diverse environments. The correlation between A and g has led to the postulation of a "messenger" from the mesophyll that directs stomatal behavior. Because A is a function of intercellular CO(2) concentration (c(i)), which is in turn a function of g, such a correlation may be partially mediated by c(i) if g is to some degree an independent variable. Among individual sunlit leaves in a cotton (Gossypium hirsutum L.) canopy in the field, A was significantly correlated with g (r(2) = 0.41, n = 63). The relative photosynthetic capacity of each leaf was calculated as a measure of mesophyll properties independent of c(i). This approach revealed that, in the absence of c(i) effects, mesophyll photosynthetic capacity was unrelated to g (r(2) = 0.06). When plants were grown in an atmosphere enriched to about 650 microliters per liter of CO(2), however, photosynthetic capacity remained strongly correlated with g even though the procedure discounted any effect of variable c(i). This "residual" correlation implies the existence of a messenger in CO(2)-enriched plants. Enriched CO(2) also greatly increased stomatal response to abscisic acid (ABA) injected into intact leaves. The data provide no evidence for a messenger to coordinate g with A at ambient levels of CO(2). In a CO(2)-enriched atmosphere, though, ABA may function as such a messenger because the sensitivity of the system to ABA is enhanced. PMID- 16666422 TI - Promoter strength comparisons of maize shrunken 1 and alcohol dehydrogenase 1 and 2 promoters in mono- and dicotyledonous species. AB - Promoter strengths of two maize alcohol dehydrogenase genes, Adh1 and Adh2, and the maize shrunken-1 gene, Sh1, were evaluated by transient expression in cultured protoplasts of Panicum maximum, Triticum monococcum, and Daucus carota. Promoter elements were ligated in correct and opposite orientations as transcriptional gene fusions to the chloramphenicol acetyl transferase gene containing the nopaline synthase 3' polyadenylation signal. The relative levels of gene expression were compared to the cauliflower mosaic virus 35S promoter. The full length Adh1 promoter (-1100 to +15) functioned in all species, but at a reduced level in D. carota. An Adh1 promoter deletion from -304 to -1100 did not express at detectable levels in any species nor did the Sh1 promoter construction. The Adh2 promoter (-860 to +90) only expressed in D. carota. The full length Adh1 promoter gave the highest level of CAT expression in the monocot cells but at levels which were approximately 30% compared to the CaMV 35S promoter. This was reduced further in D. carota to approximately 4%. These data suggest that at least some of the regulatory factors responsible for promoter function are somewhat species specific and that these differences should be considered in gene expression studies. PMID- 16666423 TI - The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves. AB - Corn seedlings (Zea mays cv W64A x W182E) were grown hydroponically, in the presence or absence of NO(3) (-), with or without light and with NH(4)Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO(3) (-). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO(3) (-) was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO(3) (-). Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO(3) (-) was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH(4)Cl(-) was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO(3) (-) or by some other unidentified factor, and that higher levels of NO(3) (-) are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO(3) (-) to NO(2) (-). PMID- 16666424 TI - Effects of Inorganic Phosphate on the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.). AB - The effects of inorganic phosphate on the plasma membrane H(+)-ATPase of red beet (Beta vulgaris L.) were studied. ATPase activity was inhibited weakly and noncompetitively by phosphate. This anion also relieved the inhibition caused by vanadate by displacing it from the enzyme. From this effect, a dissociation constant for phosphate of 25 millimolar and an extrapolated activity at infinite phosphate concentration of 84% of the activity without inhibitors were calculated. The partial inhibition by phosphate indicates the existence of a catalytically active enzyme-phosphate complex. In the presence of 24% dimethylsulfoxide, the inhibition of ATPase activity by phosphate is much greater than in its absence. This suggests that the active enzyme-phosphate complex could be converted into a covalent phosphoenzyme through a dehydration promoted by the low water activity of the medium. The inhibitory ability of phosphate in 24% dimethylsulfoxide was dependent on the presence of potassium. Potassium ions increased both the affinity for phosphate and the inhibition caused by an infinite phosphate concentration, suggesting that potassium stimulates both phosphate binding and phosphoenzyme formation. PMID- 16666425 TI - Changes in Accumulation and Synthesis of Transcripts Encoding Phycobilisome Components during Acclimation of Fremyella diplosiphon to Different Light Qualities. AB - We have used gene-specific DNA fragments as hybridization probes to quantitate the levels of transcripts encoding several phycobilisome polypeptides in the cyanobacterium Fremyella diplosiphon in response to changes in the light environment. While the levels of transcripts encoding allophycocyanin, the core linker polypeptide, and the constitutive phycocyanin subunits are similar in F. diplosiphon grown either in red or green light, the levels of other transcripts change dramatically. Transcripts encoding the inducible phycocyanin subunits are barely detected in green light-grown cells and very abundant in red light-grown cells, while the level of phycoerythrin mRNA is approximately 10-fold more in green than red light-grown cells. Quantitation of the phycoerythrin and inducible phycocyanin transcripts after transfer of cultures from green to red light and red to green light demonstrate that both increase rapidly upon exposure of cells to inductive illumination. The decrease in the phycoerythrin mRNA level in red light is much slower than the decline in the levels of the inducible phycocyanin transcripts in green light. Since the half-lives of the inducible phycocyanin and phycoerythrin transcripts do not change when F. diplosiphon is exposed to red or green illumination, the steady state levels of these mRNAs are primarily controlled by the rate of transcription. Therefore, the high level of phycoerythrin mRNA maintained for several hours after cultures are transferred from green to red illumination must result from continued transcription of the phycoerythrin gene set. Differences in expression from the phycoerythrin and inducible phycocyanin gene sets in response to light quality are discussed in terms of possible mechanisms involved in their regulation. PMID- 16666426 TI - Photoreversibility of the Effect of Red and Green Light Pulses on the Accumulation in Darkness of mRNAs Coding for Phycocyanin and Phycoerythrin in Fremyella diplosiphon. AB - DNA fragments encoding a red light-inducible phycocyanin gene and a green light inducible phycoerythrin gene have been used to investigate the effect of red and green pulses on the accumulation of phycocyanin and phycoerythrin mRNA in subsequent darkness. A red pulse promotes phycocyanin and suppresses phycoerythrin mRNA accumulation while a green pulse has an opposite effect on both transcript levels. The effect of a saturating light pulse is canceled by a subsequently given pulse of the other light quality. For a given mRNA, the positive and negative effects require the same fluence for saturation, whereas to saturate the phycoerythrin mRNA response requires at least twice as much light as to saturate the phycocyanin mRNA response. Calculations of the apparent extinction coefficients for the pigments mediating the light-regulated mRNA increase and decrease are of the order of 2 x 10(4) for phycocyanin mRNA and less than 10(4) for phycoerythrin mRNA. The data are consistent with the hypothesis that the light-induced increase and decrease of a particular phycobiliprotein mRNA is controlled by a single red/green photoreversible photosystem, but that phycoerythrin and phycocyanin mRNA levels are either controlled by two distinct photoreversible systems or that marked differences occur in the chain of events leading from photoperception to gene activation. These system(s) differ from most phytochrome systems in several ways: First, they remain fully on or off depending upon the light quality of the terminal irradiation. Second, they can be completely reversed by light of the appropriate wavelength after several hours of darkness without diminution of the effectiveness of the reversing light pulse. These two features argue against the existence of dark reversion or dark destruction of the biologically active moiety. Third, signal transduction is rapid-measurable mRNA changes occur even during a 10 minute irradiation. PMID- 16666427 TI - Degradation of Proteins Artificially Introduced into Vacuoles of Chara australis. AB - When an exogenous protein, bovine serum albumin, was introduced into the vacuole of a Chara australis internodal cell, it was degraded with time. This degradation proceeded only in the vacuole as far as could be observed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Degradation was inhibited by protease inhibitors such as antipain and leupeptin. Endogenous proteins introduced into the vacuole were also degraded there. Furthermore, intravacuolar cytoplasmic drops, which were often formed by cell ligation, seemed to be degraded in the vacuole. However, bovine serum albumin degradation did not proceed when mixed with isolated vacuolar sap. These results show that the vacuole in the Chara internodal cell has the capacity to degrade cellular proteins, but that cytoplasmic support is needed for this degrading activity to be maintained. PMID- 16666428 TI - Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport. AB - Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10 fold by MgATP. Two ATP-dependent Ca(2+) transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca(2+). A low affinity Ca(2+) uptake system (K(m) > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H(+)/Ca(2+) antiport. A high affinity Ca(2+) uptake system (K(m) = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca(2+) transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12 degrees C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca(2+)/H(+) antiport activity could only by partially ascribed to an effect of low temperature on H(+)-ATPase activity, ATP-dependent H(+) transport, passive H(+) fluxes, or passive Ca(2+) fluxes. These results suggest that low temperature directly affects Ca(2+)/H(+) exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca(2+)/H(+) exchange protein or by an indirect effect of temperature on lipid interactions with the Ca(2+)/H(+) exchange protein. PMID- 16666429 TI - Diurnal and Circadian Rhythms in the Accumulation and Synthesis of mRNA for the Light-Harvesting Chlorophyll a/b-Binding Protein in Tobacco. AB - The steady state level of mRNA for the light-harvesting chlorophyll a/b binding protein (LHCP) varies considerably during a day-night light cycle in expanding leaves of tobacco (Nicotiana tabacum, cv Wisconsin 38). The maximum (day) level is at least one hundred-fold higher than the minimum (night) level. The oscillation in the LHCP mRNA level continues for several days even under constant illumination. However, no such circadian rhythm can be detected in constant darkness. Plants that have been maintained in constant darkness for a prolonged period reinitiate the circadian rhythm upon illumination: the LHCP mRNA level resumes its rhythmic behavior with a period of roughly 24 hours. The same circadian fluctuation of LHCP mRNA is observed upon illuminating etiolated tobacco seedlings, demonstrating that the circadian rhythm is not dependent on the presence of mature chloroplasts. In vitro transcription experiments with nuclei isolated from green plants during various times of the day indicate that the diurnal as well as the circadian changes of the level of LHCP RNA are due, at least in part, to changes in the rate of LHCP mRNA synthesis. PMID- 16666430 TI - The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air. AB - Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O(3)), nonfiltered air (0.03 microliters per liter O(3)), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic (14)CO(2) uptake was measured in situ. Net photosynthesis, dark respiration, and CO(2) compensation concentration at 2 and 21% O(2) were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO(2) compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO(2) to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase. PMID- 16666431 TI - CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities. AB - The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO(2) fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level. PMID- 16666432 TI - Carbon Dioxide-Induced Oscillations in Fluorescence and Photosynthesis: Role of Thylakoid Membrane Energization in Regulation of Photosystem II Activity. AB - The response of CO(2) fixation to a sudden increase in ambient CO(2) concentration has been investigated in intact leaf tissue from spinach (Spinacia oleracea) using a dual channel infrared gas analyzer. Simultaneous with these measurements, changes in fluorescence emission associated with a weak, modulated measuring beam were recorded. Application of brief (2-3 seconds) dark intervals enabled estimation of the dark fluorescence level (F(o)) under both steady state and transient conditions. The degree of suppression of F(o) level fluorescence in the light was strongly correlated with nonphotochemical quenching under all conditions. During CO(2)-induced oscillations in photosynthesis under 2% O(2) the changes in nonphotochemical quenching anticipate changes in the rate of uptake of CO(2). At such low levels of O(2) and constant illumination, changes in the relative quantum efficiency of open photosystem II units were estimated as the ratio of the rate of CO(2) uptake and the photochemical quenching coefficient. Under the same conditions the relative quantum efficiency of photosystem II was found to vary inversely with the degree of nonphotochemical quenching. The relationship between changes in the rate of CO(2) uptake: photochemical quenching coefficient and nonphotochemical quenching was altered somewhat when the same experiment was conducted under 20% O(2). The results suggest that electron transport coupled to reduction of O(2) occurs to varying degrees with time during oscillations, especially when ambient O(2) concentrations are high. PMID- 16666433 TI - Heath Shock Enhances the Synthesis of a Lectin-Related Protein in Dolichos biflorus Cell Suspension Cultures. AB - Dolichos biflorus cell suspension cultures synthesize at least two lectins or lectin-like proteins that are related to the seed lectin from this plant. The synthesis of one of these proteins, DB57, is greatly enhanced in response to heat shock; this enhancement of synthesis is dependent upon the growth phase of the cells in culture. Early in the growth curve, when the cells are in lag phase, there is no detectable induction of DB57 synthesis above the levels found in control cells. Enhancement of DB57 synthesis becomes apparent during the last half of the exponential phase of growth at which time there is approximately a 10 fold increase in the amount of newly synthesized DB57 at 42 degrees C compared with the amount of DB57 synthesized at the control temperature of 27 degrees C. The implication of this finding is discussed with respect to role of lectins in plants. PMID- 16666434 TI - Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce. AB - Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 +/- 2 degrees C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (-)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development. PMID- 16666436 TI - Starch Phosphorylase Inhibitor Is beta-Amylase. AB - The proteinaceous noncompetitive inhibitor of starch phosphorylase isolated from the root of sweet potato (Ipomoea batatas [L.] Lam.) (TC Chang, JC Su 1986 Plant Physiol 80: 534-538) has been identified as a beta-amylase. The starch phosphorylase inhibitor and beta-amylase activities copurified to give a protein indistinguishable from commercial beta-amylase by electrophoretic and immunological methods, and the two activities showed parallel responses in pH, temperature, and inhibitor sensitivity tests. The amylolytic pattern of the inhibitor corresponded to that of beta-amylase and its inhibitory effect toward starch phosphorylase was due to neither deprivation of starch, the primer for the phosphorylase assay, nor the inhibitory effect of amylolytic products. PMID- 16666435 TI - A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP. AB - The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30 degrees C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M(r) of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems. PMID- 16666437 TI - Calcium Transport in Protoplasts Isolated from ml-o Barley Isolines Resistant and Susceptible to Powdery Mildew. AB - Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R S) barley isolines, Riso 5678/3(*) x Carlsberg II R and S, were used to test for differences in fluxes of Ca(2+) across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca(2+) postulated to exist in ml o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca(2+). Washout curves of (45)Ca(2+) from R and S protoplasts revealed three compartments-presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca(2+)). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca(2+) and our initial rates of (45)Ca-labeled Ca(2+) uptake, we show that measurement of the unidirectional influx of Ca(2+) across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly, measurement of the efflux of Ca(2+) across the plasmalemma is not possible since the activity of the pool of free cytoplasmic calcium is a factor of 350 smaller than the most rapid component of the washout experiment. This pool of cytoplasmic free Ca(2+) will wash out too rapidly and be too small to detect under the conditions of these experiments. PMID- 16666438 TI - Anion-Sensitive Mg ATP-Dependent Proton Pumping in Microsomal Membranes from Phycomyces blakesleeanus Bgff. AB - A light microsomal membrane fraction (collected on 28% weight/volume sucrose) from young mycelia of Phycomyces blakesleeanus Bgff. stationary cultures exhibited Mg(2+) ATP-dependent proton pumping as monitored with the dye neutral red or by accumulation of [(14)C]methylamine. The substrate kinetics for ATP (K(m(ATP)) 1.1 millimolar in the presence of 2 millimolar Mg(2+)), the competitive inhibition by ADP (K(i(ADP)) 0.8 millimolar), the anion sensitivity (stimulation by Cl(-), inhibition by NO(3) (-) and SO(4) (2-)), the reversal of acidification by Ca(2+), the inhibition by diethylstilbestrol and dicyclohexylcarbodiimide, as well as the absence of any inhibition by azide, molybdate, and vanadate strongly suggest a tonoplast-type ATPase driven proton pumping. The same membrane fraction showed no pyrophosphate driven H(+) translocation. Electron microscopic examination of the fungal mycelium demonstrated the presence of vacuoles of different sizes and other nonidentified vesicles but no typical Golgi stacks. PMID- 16666439 TI - Evidence for precursor forms of the low isoelectric point alpha-amylase isozymes secreted by barley aleurone cells. AB - Gibberellin-treated barley (Hordeum vulgare L.) aleurone cell protoplasts have been shown previously to contain two alpha-amylase isozymes which are not secreted (JV Jacobsen, JA Zwar, PM Chandler 1985 Planta 13: 430-438). This report shows that these intracellular forms are immunochemically related to the low isoelectric point but not the high isoelectric point group of alpha-amylase isozymes and that they arise by new synthesis like the secreted forms. Pulse chase studies show that the intracellular isozymes are precursors to the secreted isozymes. Conversion of the intra- to the extracellular forms involves decreases in isoelectric points with no change in size detectable by SDS-PAGE. The precursor isozymes were also detected in aleurone layer homogenates but they were unstable. They could be stabilized by various treatments including heating the homogenate to 70 degrees C for 10 minutes indicating that the instability was enzymically mediated. Using purified radioactive precursor isozymes, it was shown that instability did not involve inactivation but the conversion to secreted forms. The nature of the covalent modification associated with conversion was not determined but available data indicate that it does not involve glycosylation. PMID- 16666440 TI - A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme. AB - A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme. PMID- 16666441 TI - Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism. AB - Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate ;leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant. PMID- 16666442 TI - Determination of the Rate Limiting Step for Photosynthesis in a Nearly Isonuclear Rapeseed (Brassica napus L.) Biotype Resistant to Atrazine. AB - Plant biotypes that are resistant to S-triazines under most conditions often grow less vigorously and have lower quantum yields and lower maximum rates of photosynthesis. The photosynthetic reactions responsible for these effects were identified in whole leaves and thylakoids of nearly isonuclear lines of oilseed rape (Brassica napus L.). The lower quantum yield was a result of poor efficiency in the use of separated charge at the photosystem II reaction center. Charge separation occurred normally, but over 30% of the charges recombined instead of being used for oxygen evolution and for reduction capacity in photosystem I. The lower maximum rate of photosynthesis in the resistant biotype was set by the transfer of electrons between the primary, Q(A), and secondary, Q(B), acceptors of photosystem II. This charge transfer reaction became rate limiting in resistant biotypes. The decreased quantum yield and decreased maximum rate of photosynthesis are both believed to be consequences of changes in the 32 kilodalton herbicide binding protein. As such, it is likely that these traits will not be genetically separable. PMID- 16666443 TI - Structural Aspects of Photosystem I from Dunaliella salina. AB - A native PSI complex and a PSI core complex have been isolated from the halophilic green alga, Dunaliella salina. The composition and properties of these complexes are similar to previously described PSI complexes from spinach membranes. By growth on (14)C-NaHCO(3), it has been possible to isolate uniformly labeled (14)C-PSI complexes in order to determine PSI subunit stoichiometry. This analysis has shown a ratio of one copy of three low molecular weight subunits (22,000; 15,000; 8,000) per two copies of high molecular weight subunits (84,000). Using a (14)C-labeled cytochrome b(6)-f complex as an internal protein standard, it has been possible to estimate the molecular weight of a PSI core complex as about 330,000. This complex contains one P700, two 84,000 subunits, and one subunit of 22,000, 15,000, and 8,000. PMID- 16666444 TI - Phytochrome-Mediated Control of Diamine Oxidase Level in the Epicotyl of Etiolated Lentil (Lens culinaris Medicus) Seedlings. AB - Diamine oxidase (DAO; EC 1.4.3.6) levels are strongly reduced in epicotyls of 3 day-old etiolated lentil (Lens culinaris Medicus) seedlings upon exposure to continuous red and blue light, as compared to etiolated controls. Far-red light inhibits DAO activity to a lesser extent. A less marked effect can also be obtained by short (5-10 min) daily exposures. Phytochrome involvement in this light-mediated response has been demonstrated by red/far-red reversibility experiments. These findings provide the first evidence that mechanisms underlying the photoregulation of DAO level in the Leguminosae are related to photomorphogenesis and are essentially unrelated to the photosynthetic capacity of the seedling. PMID- 16666445 TI - Drought- and ABA-Induced Changes in Polypeptide and mRNA Accumulation in Tomato Leaves. AB - Drought stress triggers abscisic acid (ABA) biosynthesis resulting in ABA accumulation. The ABA-deficient tomato mutant, flacca (Lycopersicon esculentum Mill. cv Ailsa Craig), does not synthesize ABA in response to drought stress. This mutant has been used to distinguish polypeptides and in vitro translation products that are synthesized during drought stress in response to elevated ABA levels from those that are induced directly by altered water relations. A set of polypeptides and in vitro translation products was synthesized during drought stress in the wild type. These polypeptides and in vitro translation products were synthesized to a lesser extent in the drought-stressed ABA-deficient mutant. Treatment of flacca with ABA resulted in the synthesis of the drought-stress induced polypeptides and in vitro translation products. These results support the hypothesis that many of the polypeptides that are synthesized during drought are regulated by alterations in ABA concentration. Similarly, the mRNA population was altered by ABA during drought stress. PMID- 16666446 TI - Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxysomes and leaf peroxisomes). AB - The intraorganellar distribution of superoxide dismutase (SOD) (EC 1.15.1.1) in two types of plant peroxisomes (glyoxysomes and leaf peroxisomes) was studied by determinations of SOD latency in intact organelles and by solubilization assays with 0.2 molar KCl. Glyoxysomes were purified from watermelon (Citrullus vulgaris Schrad.) cotyledons, and their integrity, calculated on the basis of glyoxysomal marker enzymes, was about 60%. Under the same conditions, the latency of SOD activity determined in glyoxysomes was 40%. The difference between glyoxysomal intactness and SOD latency was very close to the percentage of isozyme Mn-SOD previously determined in glyoxysomes (LM Sandalio, LA Del Rio 1987 J Plant Physiol 127: 395-409). In matrix and membrane fractions of glyoxysomes, SOD exhibited a solubilization pattern very similar to catalase, a typical soluble enzyme of glyoxysomes. The analysis of the distribution of individual SOD isozymes in glyoxysomal fractions treated with KCl showed that Cu,Zn-SOD II, the major SOD isozyme in glyoxysomes, was present in the soluble fraction of these organelles, whereas Mn-SOD was bound to the glyoxysomal membrane. These data in conjunction with those of latency of SOD activity in intact glyoxysomes suggest that Mn-SOD is bound to the external side of the membrane of glyoxysomes. On the other hand, in intact leaf peroxisomes where only a Mn-containing SOD is present (LM Sandalio, JM Palma, LA Del Rio 1987 Plant Sci 51: 1-8), this isozyme was found in the peroxisomal matrix. The physiological meaning of SOD localization in matrix and membrane fractions of glyoxysomes and the possibility of new roles for plant peroxisomes in cellular metabolism related to activated oxygen species is discussed. PMID- 16666447 TI - Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm. AB - Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation. PMID- 16666448 TI - Effect of temperature on starch synthesis in potato tuber tissue and in amyloplasts. AB - A sharp temperature optimum is observed at 21.5 degrees C when the incorporation of [(14)C]sucrose into starch is measured with discs cut from developing tubers of potato (Solanum tuberosum L. cv Desiree). By contrast, increasing temperatures over the range 9 to 31 degrees C only enhance release of (14)C to respiratory CO(2) and incorporation of (14)C into the ethanolsoluble fraction. By comparison, starch synthesis in discs from developing corms of cocoyam (Colocasia esculenta L. Schott) is increased by raising the temperature from 15 to 35 degrees C. The significance of a relatively low temperature optimum for starch synthesis in potato is discussed in relation to the yield limitations imposed by continuously high soil temperatures. Amyloplasts isolated from protoplasts prepared from developing potato tubers contain activities of alkaline pyrophosphatase, NAD dependent glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, and phosphoglucomutase in addition to ADP-glucose-pyrophosphorylase, starch phosphorylase and starch synthase. Cell-free amyloplasts released by thinly slicing developing potato tubers synthesize starch from [(14)C]triose-phosphate generated from [(14)C]fructose-1,6-bisphosphate in the reaction medium. This starch synthesis is inhibited by addition of 10 millimolar inorganic phosphate and requires amyloplast integrity, suggesting the operation of a triose phosphate/inorganic phosphate exchange carrier at the amyloplast membrane. The temperature optimum at 21.5 degrees C observed with tissue discs is not observed with amyloplasts. PMID- 16666449 TI - Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol. AB - Allopurinol (1H-pyrazolo-[3,4-d]pyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (1H-pyrazolo-[3,4-d]pyrimidine-4,6 diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the O(2) concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure. PMID- 16666450 TI - Sugar uptake by maize endosperm suspension cultures. AB - Maize (Zea mays L.) endosperm suspension cultures are a useful model system for studying biochemical and physiological events in developing maize endosperm. In this report, sugar uptake by the cultures is characterized. Uptake of (14)C labeled fructose and l-glucose was linear with time, while the rate of uptake of radioactivity from sucrose increased over a 120 min period. Both saturable and linear components of uptake were observed for fructose, glucose, sucrose, 1' deoxy-1'-fluorosucrose, and maltose. Uptake of mannitol, sorbitol, and l-glucose took place at lower rates and was linear with concentration. Rates of incorporation of radioactivity from fructose and glucose exceeded that of sucrose at all concentrations tested. Kinetics of 1'-deoxy-1'-fluorosucrose uptake indicated that (14)C from sucrose can be taken up by a saturable carrier of intact sucrose as well as by invertase hydrolysis and subsequent uptake of hexoses. Cell wall invertase was demonstrated histochemically. Further study of fructose uptake at a concentration at which the saturable component predominated revealed sensitivity to metabolic inhibitors, respiratory uncouplers, the nonpermeant sulfhydryl reagent p-chloromercuribenzenesulfonic acid, and nigericin. Uptake was not affected by valinomycin plus K(+) and was stimulated by fusicoccin. Fructose and glucose uptake was not pH-sensitive below pH 7.0, whereas uptake of radioactivity from sucrose and 1'-deoxy-1'-fluorosucrose declined as the pH was increased above 5.0. Fructose uptake was not completely inhibited by glucose and vice versa, suggesting the presence of specific carriers. These results indicate that maize endosperm suspension cultures (a) absorb fructose via a typical, energy-requiring, carrier-mediated proton cotransport system; (b) possess saturable carriers for glucose and sucrose; and (c) also absorb sucrose via hexose uptake after sucrose hydrolysis by extracellular invertase. PMID- 16666451 TI - Light-Dependent Accumulation of Radiolabeled Plastid-Encoded Chlorophyll a Apoproteins Requires Chlorophyll a: I. Analysis of Chlorophyll-Deficient Mutants and Phytochrome Involvement. AB - The accumulation of radiolabeled plastid-encoded chlorophyll a-apoproteins is light dependent and is controlled at a posttranscriptional level. Illumination of dark-grown barley (Hordeum vulgare L.) with a brief pulse of red light induced the accumulation of radiolabeled chlorophyll a-apoproteins in subsequent protein synthesis assays. The induction of radiolabeled chlorophyll a-apoprotein accumulation was not affected by pretreatment of leaves with cycloheximide. Fluence response studies showed that a red light photoreceptor controls the accumulation of radiolabeled chlorophyll a-apoproteins with a threshold fluence of approximately 50 to 100 microeinsteins per square meter. While red light initiated chlorophyll a-apoprotein accumulation, this process was not reversed by a far red light treatment given immediately after the pulse of red light. The light pulse which initiated the accumulation of radiolabeled chlorophyll a apoproteins also induced the rapid conversion of protochlorophyllide to chlorophyll a. A chlorophyll-deficient mutant, xan-f(10), which is blocked in chlorophyll biosynthesis prior to protochlorophyllide formation, failed to accumulate radiolabeled chlorophyll a-apoproteins in the light even though transcripts for these apoproteins were present. A second mutant, xan-j(64), which accumulates chlorophyllide in the light but only low levels of chlorophyll a, also showed reduced accumulation of radiolabeled chlorophyll a-apoproteins upon illumination. These results suggest that the light-induced conversion of protochlorophyllide to chlorophyll a is necessary for accumulation of the plastid encoded chlorophyll a-apoproteins and one red light photoreceptor controlling this response is the protochlorophyllide holochrome. PMID- 16666452 TI - Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin. AB - The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)(+) RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity. PMID- 16666453 TI - The effects of salt stress on polypeptides in membrane fractions from barley roots. AB - Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [(35)S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium proton anti-porter in the tonoplast membrane. PMID- 16666454 TI - Sodium and Potassium Compartmentation and Transport across the Roots of Intact Spergularia marina. AB - The Na(+) and K(+) transport characteristics of Spergularia marina (L.) Griseb. were considered in order to compare the systems by which these two physiologically different cations are managed during initial acquisition and subsequent partitioning in midvegetative plants. Uptake of (22)Na(+) and (42)K(+) and redistribution of labels in pulse-chase studies were compared under steady state growth conditions or with the concentration of one of the ions elevated. At high external concentrations, the initial (42)K(+) accumulation and transport to the shoot was associated with a small, rapidly exchanging, cellular compartment similar to that previously indicated for Na(+) (D Lazof, JM Cheeseman 1986 Plant Physiol 81: 742-747). At 1 mol m(-3), K(+) was conducted to the shoot through a root compartment, the specific activity of which rose much more slowly than the rapidly exchanging compartment. After a lag of approximately 5 minutes, (42)K(+) translocation approached a constant rate with a half-time of 14 minutes compared to 5 minutes for (22)Na(+) or for (42)K(+) at higher external levels. At all external levels, prolonged translocation of (42)K(+) was measured when a 10 minute pulse was followed by an unlabeled chase, again suggesting a conducting compartment distinct from that for Na(+). It is suggested that the K(+) conducting compartment, possibly the ;bulk cytoplasm,' is associated with the active K(+) transport system generally found in higher plants. PMID- 16666455 TI - Sodium and potassium compartmentation and transport in the roots of intact lettuce plants. AB - In this report, we consider the accumulation in roots, and transport to the shoot, of Na(+) and K(+) in intact lettuce plants (Lactuca sativa cv Black-seeded Simpson). Plants were grown in modified Hoagland medium supplemented with 10 moles NaCl per cubic meter. At this salinity, significant levels of Na(+) were accumulated in roots and shoots, but there was no reduction in plant growth. Transport characteristics for both Na(+) and K(+) were qualitatively similar to those previously reported, for Spergularia marina, indicating that the results obtained with these experimental protocols are not limited to one unconventional experimental plant. The most pronounced difference in transport of the two ions was evident when transport was followed in a chase period after a 10 minute uptake pulse. For Na(+), there was an initially rapid, but small, loss of label to the medium, and very little movement to the shoot. For K(+), little label was lost from the plants, but translocation to the shoot proceeded for at least 60 minutes. The transport systems were further distinguished by treating the roots during labeling with 20 micrograms per milliliter cycloheximide. For K(+), both uptake and translocation were reduced by about 50%. For Na(+), root accumulation was stimulated more than five-fold, while transport to the shoot was reduced about 20%. Cycloheximide also modified the Na(+) transport characteristics such that continued translocation occurred during the chase period of pulse-chase studies. PMID- 16666456 TI - Paralytic Shellfish Toxins in Protogonyaulax tamarensis and Protogonyaulax catenella in Axenic Culture. AB - Paralytic shellfish toxin concentrations were measured and individual toxin profiles were monitored in axenic batch cultures of Protogonyaulax tamarensis and Protogonyaulax catenella. High pressure liquid chromatographic methods were used that allowed the separation of all 12 known paralytic shellfish poisons, including toxins C1, C2, and C3, from a single sample. In isolates of both Protogonyaulax species, total toxin levels were relatively low after inoculation, increased rapidly in early to mid-exponential growth to a value 100 to 300% of that at the initial time point, then decreased by 86 to 95% as the culture aged. Although the concentrations of individual toxins per cell followed the same general pattern as that seen for total moles of toxin per cell, variability in toxin profile with culture age was observed. In P. tamarensis, the mole percent of neosaxitoxin increased substantially from 8 to 44% as total toxin levels per cell decreased. A concomitant decrease in the mole percent of saxitoxin with culture age was noted. Although not as precipitous, changes in the mole percent of specific toxins from P. catenella were also observed. The mole percent of gonyautoxins I and IV increased, while that of gonyautoxins II and III decreased. These data suggest that the toxin profile in isolates of Protogonyaulax can change, sometimes significantly, with changing environmental variables. PMID- 16666457 TI - Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor. AB - Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse labeling experiments with [(14)C]acetate and [(3)H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids. PMID- 16666458 TI - Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels. AB - Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO(2) (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO(2) levels (5%). Photoautotrophy was indicated by the requirement for CO(2) and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C(4)Amaranthus species. The cells showed high dark respiration rates and had lower net CO(2) fixation under high O(2) conditions. Dark CO(2) fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO(2) conditions showed the unique properties of a high RuBPcase activation level in ambient CO(2) and a stable ability to show net CO(2) fixation in 21% O(2) conditions. PMID- 16666459 TI - Uptake and Assimilation of NO(3) and NH(4) by Nitrogen-Deficient Perennial Ryegrass Turf. AB - Assimilation of NO(3) (-) and NH(4) (+) by perennial ryegrass (Lolium perenne L.) turf, previously deprived of N for 7 days, was examined. Nitrogen uptake rate was increased up to four- to five-fold for both forms of N by N-deprivation as compared to N-sufficient controls, with the deficiency-enhanced N absorption persisting through a 48 hour uptake period. Nitrate, but not NH(4) (+), accumulated in the roots and to a lesser degree in shoots. By 48 hours, 53% of the absorbed NO(3) (-) had been reduced, whereas 97% of the NH(4) (+) had been assimilated. During the early stages (0 to 8 hours) of NO(3) (-) uptake by N deficient turf, reduction occurred primarily in the roots. Between 8 and 16 hours, however, the site of reduction shifted to the shoots. Nitrogen form did not affect partitioning of the absorbed N between roots (40%) and shoots (60%) but did affect growth. Compared to NO(3) (-), NH(4) (+) uptake inhibited root, but not shoot, growth. Total soluble carbohydrates decreased in both roots and shoots during the uptake period, principally the result of fructan metabolism. Ammonium uptake resulted in greater total depletion of soluble carbohydrates in the root compared to NO(3) (-) uptake. The data indicate that N assimilation by ryegrass turf utilizes stored sugars but is also dependent on current photosynthate. PMID- 16666460 TI - Effects of CO(2) Concentration on Rubisco Activity, Amount, and Photosynthesis in Soybean Leaves. AB - Growth at an elevated CO(2) concentration resulted in an enhanced capacity for soybean (Glycine max L. Merr. cv Bragg) leaflet photosynthesis. Plants were grown from seed in outdoor controlled-environment chambers under natural solar irradiance. Photosynthetic rates, measured during the seed filling stage, were up to 150% greater with leaflets grown at 660 compared to 330 microliters of CO(2) per liter when measured across a range of intercellular CO(2) concentrations and irradiance. Soybean plants grown at elevated CO(2) concentrations had heavier pod weights per plant, 44% heavier with 660 compared to 330 microliters of CO(2) per liter grown plants, and also greater specific leaf weights. Ribulose 1,5 bisphosphate carboxylase/oxygenase (rubisco) activity showed no response (mean activity of 96 micromoles of CO(2) per square meter per second expressed on a leaflet area basis) to short-term ( approximately 1 hour) exposures to a range of CO(2) concentrations (110-880 microliters per liter), nor was a response of activity (mean activity of 1.01 micromoles of CO(2) per minute per milligram of protein) to growth CO(2) concentration (160-990 microliters per liter) observed. The amount of rubisco protein was constant, as growth CO(2) concentration was varied, and averaged 55% of the total leaflet soluble protein. Although CO(2) is required for activation of rubisco, results indicated that within the range of CO(2) concentrations used (110-990 microliters per liter), rubisco activity in soybean leaflets, in the light, was not regulated by CO(2). PMID- 16666461 TI - Immunocytochemical localization of a wheat germ lysozyme in wheat embryo and coleoptile cells and cytochemical study of its interaction with the cell wall. AB - Among several wheat (Triticum aestivum L.) germ proteins able to lyse Micrococcus lysodeikticus, one lysozyme (W1A) was purified by ion-exchange chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. Polyclonal antibodies against this lysozyme were raised in rabbits. The in situ localization of W1A lysozyme was achieved by the indirect protein A-gold technique. Large amounts of W1A lysozyme were found in cell walls whereas intercellular spaces, cytoplasm, and organelles were nearly free of labeling. Specificity of labeling was assessed with several controls. In an attempt to detect the presence of binding sites, W1A lysozyme was complexed to colloidal gold. Particles were specifically distributed in large amounts over wheat embryo and coleoptile cell walls. The absence of labeling over isolated coleoptile cell walls treated with 0.1 and 0.4 molar potassium hydroxide for hemicellulose extraction indicated that W1A lysozyme binding sites were probably of hemicellulosic nature. PMID- 16666462 TI - Expression of Light-Harvesting Chlorophyll a/b-Protein Genes Is Phytochrome Regulated in Etiolated Arabidopsis thaliana Seedlings. AB - Phytochrome action results in a large and rapid increase in the light-harvesting chlorophyll a/b-protein (LHCP) mRNA level in etiolated seedlings of Arabidopsis thaliana: the RNA increase is detectable within 1 hour after 1 minute red illumination, reaches a maximum 30-fold higher than the dark level at ca. 2 hours, and decays back to dark levels by about 8 hours after the brief red illumination. S1 nuclease analysis distinguishes two kinds of mRNAs transcribed from the three members of the LHCP gene family previously characterized for Arabidopsis (LS Leutwiler, EM Meyerowitz, EM Tobin, 1986 Nucleic Acids Res 14: 4051-4064). One of these arises from the AB140 gene, while the other represents the product(s) of the AB165 and/or AB180 gene(s) (AB165/AB180 mRNA). In mature, white light-grown plants, the two kinds of mRNAs are present in nearly equal amounts. In contrast, in etiolated seedlings, 1 minute red light causes a sixfold greater increase in the level of AB140 mRNA than in the level of AB165/AB180 mRNA, although both levels are regulated by phytochrome action. The kinetics of the responses to 1 minute red light are similar for both kinds of transcripts. Additional evidence suggests that this differential expression is developmentally regulated. Because the AB140 gene offers an attractive target for further analysis of phytochrome-regulated gene expression in Arabidopsis, we have further characterized this gene by mapping its 5' and 3' transcript termini. PMID- 16666463 TI - Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by C NMR. AB - The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using (13)C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose. PMID- 16666464 TI - Enzymes of phosphatidylcholine synthesis in lemna, soybean, and carrot. AB - Cell-free extracts from Lemna and suspension cultured carrot (Daucus Carota L.) catalyze S-adenosylmethionine-dependent N-methylations of phosphoethanolamine, phosphomethylethanolamine, and phosphodimethylethanolamine; extracts of suspension cultured soybean (Glycine max), of phosphoethanolamine only. Material pelleted from each tissue between 15,000 and 100,000g catalyzes S adenosylmethionine-dependent N-methylations of phosphatidylmethylethanolamine and phosphatidyl-dimethylethanolamine, but not phosphatidylethanolamine. Extracts from each tissue catalyze CTP-dependent cytidylyltransfers to each of the three methylated phosphoethanolamine derivatives, forming the corresponding CDP derivatives. Some of the properties of the activities investigated are reported. On the basis of in vivo labeling experiments, we have proposed (AH Datko, SH Mudd 1988 Plant Physiol 88: 854-861) differing pathways for phosphatidylcholine synthesis in which, after a common committing step, N-methylation of phosphoethanolamine, subsequent methylations occur in Lemna almost exclusively at the phospho-base level; in soybean, at the phosphatidyl-base level; and in carrot, at both levels. Thus, among the activities investigated, at least those required for the operation of the proposed pathways have been positively demonstrated. The extent to which the present results explain the differences between these pathways is discussed, and a speculation offered as to how these differences may have arisen phylogenetically. PMID- 16666465 TI - Characterization of fructan from mature leaf blades and elongation zones of developing leaf blades of wheat, tall fescue, and timothy. AB - Water-soluble carbohydrate composition of mature (ceased expanding) leaf blades and the elongation zone of developing leaf blades was characterized in wheat (Triticum aestivum L.), tall fescue (Festuca arundinacea Schreb.), and timothy (Phleum pratense L.). These species were chosen because they differ in mean degree of polymerization (DP) of fructan in the mature leaf blade. Our objective was to compare the nature and DP of the fructan. Vegetative plants were grown with a 14-hour photoperiod and constant 21 degrees C at the leaf base. Gel permeation chromatography of leaf blade extracts showed that the apparent mean fructan DP increased in the order wheat < tall fescue < timothy. Apparent mean DP of elongation zone fructan was higher than that of leaf blade fructan in wheat and timothy, but the reverse occurred for tall fescue. Low DP (10) pools were found in both tissues of tall fescue and wheat, but concentration of low DP fructan was very low in either tissue of timothy. All three species have high DP fructan. Comigration with standards on thin-layer chromotography showed that wheat contained 1-kestose and a noninulin fructan oligomer series. Tall fescue contained neokestose, 1-kestose and higher oligosaccharides that comigrated with neokestose-based compounds and inulins. Thin-layer chromatography showed that small amounts of fructose-containing oligosaccharides were present in timothy. PMID- 16666466 TI - Immunogold localization of nitrate reductase in maize leaves. AB - Mature maize leaf tissue (Zea mays L.) was immunolabeled using a pre-embedding protocol with specific antibodies for nitrate reductase and protein A-colloidal gold. Immunogold label was found exclusively in the cytoplasm of mesophyll cells; no reaction was detected in bundle sheath cells. Chloroplasts, which were sliced open during cryosectioning, had no labeling. Thus, it appears nitrate reductase is localized exclusively in the cytoplasm of maize leaf mesophyll cells. No gold labeling was found on tissue sections embedded in L. R. White's or Lowicryl resin, indicating that previous chloroplast localization utilizing these protocols may be artifactual, resulting from shared epitopes or nonspecific antibody binding. PMID- 16666467 TI - The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System. AB - RNA is an essential component for the enzymic conversion of glutamate to delta aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA(Glu(UUC)), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA(Glu(UUC)) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA(Glu(UUC)) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [(3)H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA(Glu(UUC)) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA(Glu(UUC)) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA(Glu(UUC)) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [(3)H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract. PMID- 16666468 TI - Qualitative and Quantitative Analyses of Gibberellins in Vegetative Shoots of Normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 Seedlings of Zea mays L. AB - Gibberellins A(12) (GA(12)), GA(53), GA(44), GA(19), GA(17), GA(20), GA(29), GA(1), and GA(8) have been identified from extracts of vegetative shoots of normal (wild type) maize using full scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Seven of these gibberellins (GAs) have been quantified by capillary gas chromatography-selected ion monitoring using internal standards of [(14)C(4)]GA(53), [(14)C(4)]GA(44), [(2)H(2)] GA(19), [(13)C(1)]GA(20), [(13)C(1)]GA(29), [(13)C(1)]GA(1), and [(13)C(1)]GA(8). Quantitative data from extracts of normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 seedlings support the operation of the early 13-hydroxylation pathway in vegetative shoots of Zea mays. These data support the positions in the pathway blocked by the mutants, previously assigned by bioassay data and metabolic studies. The GA levels in dwarf-2, dwarf-3, and dwarf-5 were equal to, or less than, 2.0 nanograms per 100 grams fresh weight, showing that these mutants are blocked for steps early in the pathway. In dwarf-1, the level of GA(1) was very low (0.23 nanograms per 100 grams fresh weight) and less than 2% of that in normal shoots, while GA(20) and GA(29) accumulated to levels over 10 times those in normals; these results confirm that the dwarf-1 mutant blocks the conversion of GA(20) to GA(1). Since the level of GAs beyond the blocked step for each mutant is greater than zero, each mutated gene probably codes for an altered gene product, thus leading to impaired enzyme activities. PMID- 16666469 TI - Spinach Leaf Chloroplast CO(2) and NO(2) Photoassimilations Do Not Compete for Photogenerated Reductant: Manipulation of Reductant Levels by Quantum Flux Density Titrations. AB - Potential competition between CO(2) and NO(2) (-) photoassimilation for photogenerated reductant (e.g. reduced ferredoxin and NADPH) was examined employing isolates of mesophyll cells and intact chloroplasts derived from mature ;source' spinach leaves. Variations in the magnitude of incident light energy were used to manipulate the supply of reductant in situ within chloroplasts. Leaf cell and plastid isolates were fed with saturating CO(2) and/or NO(2) (-) to produce the highest demand for reductant by CO(2) and/or NO(2) (-) assimilatory processes (enzymes). Even in the presence of CO(2) fixation, NO(2) (-) reduction in intact leaf cell isolates as well as plastid isolates was maximal at light energies as low as 50 to 200 microeinsteins per second per square meter. Simultaneously, 500 to 800 microeinsteins per second per square meter were required to support maximal CO(2) assimilation. Regardless of the magnitude of the incident light energy, CO(2) assimilation did not repress NO(2) (-) reduction, nor were these two processes mutually repressed. These observations have been interpreted to mean that reduced ferredoxin levels in situ in the plastids of mature source leaf mesophyll cells were adequate to supply the concurrent maximal demands exerted by enzymes associated with CO(2) as well as with inorganic nitrogen photoassimilation. PMID- 16666470 TI - Mechanisms of starvation tolerance in pearl millet. AB - The response of pearl millet (Pennisetum glaucum [L.]) seedlings to prolonged starvation was investigated at the biochemical and ultrastructural level. After 2 days of darkness the bulk of the seedling carbohydrate reserves were depleted. After 8 days in the dark the respiratory rate had declined to less than 50% of its initial value and the plants had lost half of their total protein content. Unlike the situation with carbohydrate depletion, protein loss was restricted to specific organs. The secondary leaf and stem (including the apical meristem) showed little or no protein loss during this period. In the primary leaf, seed, and roots, protein loss was substantial. In spite of the high rate of protein degradation in the primary leaf and roots, these organs showed no ultrastructural changes suggestive of tissue, cellular, or subcellular degradation. In addition, ribulose bisphosphate carboxylase was not preferentially degraded during starvation and only a small decline in chlorophyll content was observed after 8 days in the dark. During the period from 8 to 14 days, cell death started at the tip of the primary leaf and gradually spread downward. Both shoot and root meristems remained alive up to 14 days. Consequently, the eventual death of the plant was due to the loss of the carbohydrate-producing regions rather than the meristems. We suggest that these results provide an explanation for the high degree of starvation tolerance exhibited by pearl millet. PMID- 16666471 TI - A comparative spin-label study of isolated plasma membranes and plasma membranes of whole cells and protoplasts from cold-hardened and nonhardened winter rye. AB - Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between -40 degrees C and 40 degrees C using the freely diffusing spin-label, 16 doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5 degrees C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used. PMID- 16666472 TI - Nuclear-Cytoplasmic Partitioning of Phytochrome-Regulated Transcripts in Pisum sativum. AB - Nuclear and cytoplasmic mRNAs for several phytochrome-regulated genes were examined in Pisum seedlings in order to investigate possible light effects on mRNA partitioning between the nucleus and cytoplasm. Transcripts from each of five light-regulated genes exhibited different responses to a variety of light treatments, but for each transcript we observed a characteristic linear relationship between nuclear and cytoplasmic levels over a wide range of total transcript abundance. Different mRNAs are characterized by different nuclear cytoplasmic ;partitioning coefficients', indicating that post-transcriptional events play a significant role in regulating the accumulation of these mRNAs during light induction. PMID- 16666473 TI - Effect of Light on Sterol Changes in Medicago sativa. AB - Most vascular plants contain Delta(5)-sterols as the predominant type; however, a few species such as Medicago sativa, have mainly Delta(7)-sterols. The Delta(7) sterols of alfalfa are isomers of the common Delta(5)-sterols and are generally assumed to be their immediate precursors. Light had a significant influence on the sterol status of M. sativa. High light intensity and a long day favored the accumulation of dihydrospinasterol; a short day and low light intensity, particularly darkness, favored spinasterol accumulation. These data for Delta(7) sterol plants agree with those reported for Delta(5)-sterol plants; light favors the accumulation of the monounsaturated 29 carbon sterols and darkness favors the accumulation of the diunsaturated sterols. Proposed is a mechanism to explain the effect of light on the accumulation of Delta(7)- and Delta(5)-sterols. PMID- 16666474 TI - Regulation of Assimilatory Sulfate Reduction by Cadmium in Zea mays L. AB - Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from (35)SO(4) (2-) into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5'-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5'-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves. PMID- 16666475 TI - Two Isoenzymes of NADH-dependent Glutamate Synthase in Root Nodules of Phaseolus vulgaris L: Purification, Properties and Activity Changes during Nodule Development. AB - The specific activity of plant NADH-dependent glutamate synthase (NADH-GOGAT) in root nodules of Phaseolus vulgaris L. is over threefold higher than the specific activity of ferredoxin-dependent GOGAT. The NADH-GOGAT is composed of two distinct isoenzymes (NADH-GOGAT I and NADH-GOGAT II) which can be separated from crude nodule extracts by ion-exchange chromatography. Both NADH-GOGAT isoenzymes have been purified to apparent homogeneity and shown to be monomeric proteins with similar M(r)s of about 200,000. They are both specific for NADH as reductant. An investigation of their kinetic characteristics show slight differences in their K(m)s for l-glutamine, 2-oxoglutarate, and NADH, and they have different pH optima, with NADH-GOGAT I exhibiting a broad pH optimum centering at pH 8.0 whereas NADH-GOGAT II has a much narrower pH optimum of 8.5. The specific activity of NADH-GOGAT in roots is about 27-fold lower than in nodules and consists almost entirely of NADH-GOGAT I. During nodulation both isoenzymes increase in activity but the major increase is due to NADH-GOGAT II which increases over a time course similar to the increase in nitrogenase activity. This isoenzyme is twice as active as NADH-GOGAT I in mature nodules. The roles and regulation of these two isoenzymes in the root nodule are discussed. PMID- 16666476 TI - Correlation between the Carbon Isotope Discrimination in Leaf Starch and Sugars of C(3) Plants and the Ratio of Intercellular and Atmospheric Partial Pressures of Carbon Dioxide. AB - Carbon isotope discrimination (Delta) was analyzed in leaf starch and soluble sugars, which represent most of the recently fixed carbon. Plants of three C(3) species (Populus nigra L. x P. deltoides Marsh., Gossypium hirsutum L. and Phaseolus vulgaris L.) were kept in the dark for 24 hours to decrease contents of starch and sugar in leaves. Then gas exchange measurements were made with constant conditions for 8 hours, and subsequently starch and soluble sugars were extracted for analysis of carbon isotope composition. The ratio of intercellular, p(i), and atmospheric, p(a), partial pressures of CO(2), was calculated from gas exchange measurements, integrated over time and weighted by assimilation rate, for comparison with the carbon isotope ratios in soluble sugars and starch. Carbon isotope discrimination in soluble sugars correlated strongly (r = 0.93) with p(i)/p(a) in all species, as did Delta in leaf starch (r = 0.84). Starch was found to contain significantly more (13)C than soluble sugar, and possible explanations are discussed. The strong correlation found between Delta and p(i)/p(a) suggests that carbon isotope analysis in leaf starch and soluble sugars may be used for monitoring, indirectly, the average of p(i)/p(a) weighted by CO(2) assimilation rate, over a day. Because p(i)/p(a) has a negative correlation with transpiration efficiency (mol CO(2)/mol H(2)O) of isolated plants, Delta in starch and sugars may be used to predict differences in this efficiency. This new method may be useful in ecophysiological studies and in selection for improved transpiration efficiency in breeding programs for C(3) species. PMID- 16666477 TI - Synthesis and biological activity of an azido derivative of paclobutrazol, an inhibitor of gibberellin biosynthesis. AB - A photolabile azido derivative of the kaurene oxidase inhibitor 1-(4 chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl) pentan-3-ol (paclobutrazol) has been synthesized for use as a photoaffinity labeling agent. The compound was tested as an inhibitor of the oxidation of ent-kaurene catalyzed by cell-free preparations from endosperm of Cucurbita maxima. The I(50) of the azido derivative was 9.5 nanomolar, which compares well with that of paclobutrazol (6.3 nanomolar in our measurements). The azido compound bound to Cytochrome P-450 in microsomes from Cucurbita maxima, and induced a Type II spectral change, with an apparent binding constant of 0.24+/-0.04 micromolar. PMID- 16666478 TI - Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons. AB - Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)(+) RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS(1)) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS(2)) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS(1) sevenfold after 72 hours, and decreased rapidly that for GS(2) and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS(1) mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS(1) genes were activated, while those for GS(2) were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves. PMID- 16666479 TI - Identification of membrane protein associated with sucrose transport into cells of developing soybean cotyledons. AB - The photolyzable sucrose derivative 6'-deoxy-6'-(4-azido-2-hydroxy) benzamidosucrose (6'-HABS), competitively inhibited the influx of [(14)C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of (125)I-labeled 6'-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl alpha-d-thioglucopyranoside. Glucose, raffinose, or phenyl alpha-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6' HABS was attached to 6-deoxy-6-aminoglucose and (125)I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter. PMID- 16666480 TI - A Proteinase from Germinating Barley : I. Purification and Some Physical Properties of a 30 kD Cysteine Endoproteinase from Green Malt. AB - A proteinase was purified from germinated barley (green malt from Hordeum vulgare L. cv Morex) by acidic extraction, ammonium sulfate fractionation and successive chromatographies on CM-cellulose, hemoglobin sepharose, Sephadex G-75 and organomercurial agarose columns. The overall purification and final recovery were 290-fold and 7.5%, respectively. The purified enzyme was homogeneous on analytical gel electrophoresis, yielding a single protein associated with protease activity. An apparent molecular weight of about 20 kilodaltons was estimated for the native enzyme from gel filtration. SDS-gel electrophoresis revealed a single polypeptide of about 30 kilodaltons. The optimum pH for the hydrolysis of hemoglobin was around 3.8. The enzyme was strongly inhibited by leupeptin but was insensitive to phenylmethylsulfonyl fluoride, indicating that it was a cysteine proteinase. It hydrolyzed several large proteins from various origins. The ability of the enzyme to digest barley storage proteins in vitro was examined using SDS-gel electrophoresis. The hydrolysis patterns obtained showed that the enzyme rapidly hydrolyzed the large hordein polypeptides into relatively small fragments. The results of this study suggest that this 30 kilodalton enzyme is one of the predominant cysteine proteinases secreted into the starchy endosperm during barley germination and that it plays a major role in the mobilization of storage proteins. PMID- 16666481 TI - Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities. AB - Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed. PMID- 16666482 TI - H Cotransports in Corn Roots as Related to the Surface pH Shift Induced by Active H Excretion. AB - The surface pH shift induced by active H(+) excretion in corn (Zea mays L.) roots was estimated using acetic acid influx as a pH probe (H Sentenac, C Grignon 1987 Plant Physiol 84: 1367-1372). At constant bulk pH, buffering the medium strongly reduced the magnitude of the surface pH shift. This was used to study the effect of surface pH shift on H(+) cotransports. In the absence of buffers, the surface pH shift increased with the bulk pH. Buffers decreased (32)Pi influx and this effect was stronger at pH 7.2 than at pH 5.8, and stronger in the absence than in the presence of an inhibitor of the proton pump (vanadate). Buffers exerted a similar depressive and pH-dependent effect on net NO(3) (-) uptake. They hyperpolarized the cell membrane, and stimulated (86)Rb(+) influx, K(+):H(+) net exchange, and malate accumulation. These results are consistent with the hypothesis that H(+) accumulation at the cell surface is effective in driving H(+) reentry. We concluded that the surface pH shift due to proton pump activity is involved in the energetic coupling of H(+) cotransports. PMID- 16666483 TI - Glycine supports in vivo reduction of nitrate in barley leaves. AB - Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO(2) (-) production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity. PMID- 16666484 TI - Monoclonal antibodies to 13-deoxy-gibberellins. AB - The production and characterization of two high affinity rat monoclonal antibodies to 13-deoxy-gibberellins is described. Hybrid myelomas were derived from rats immunized with an immunogenic keyhole limpet hemocyanin-gibberellin conjugate, linked at carbon-3 to gibberellin A(4) via a hemisuccinate bridge. The selected monoclonal antibodies were characterized by a competitive radioimmunoassay. PMID- 16666485 TI - Characterization of elemental sulfur in isolated intact spinach chloroplasts. AB - Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of (35)SO(4) (2-) resulted in the light-dependent formation of a chloroform soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S(8)) of elemental sulfur (S(0), valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S(0)) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S(8) under the experimental conditions used for analysis. PMID- 16666486 TI - Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts. AB - The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26 degrees C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with (35)S-methionine. Incubation at 40 degrees and 42 degrees C resulted in the synthesis of polypeptides not detectable at 26 degrees C. Introduction of a chimeric maize heat shock protein 70 promoter chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40 degrees C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress. PMID- 16666487 TI - Isolation of viable wheat male gametophytes of different stages of development and variations in their protein patterns. AB - Procedures have been designed to isolate viable immature male gametophytes from wheat (Triticum aestivum L.) anthers of different stages of development. Maceration of anthers by a micro-blender allowed for the release of numerous intact vacuolate microspores. Tris-buffered media prevented tricellular pollen grains from bursting during the isolation procedure. Proteins from the undamaged male gametophytes have been analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A set of new proteins was detected at the onset of the second pollen grain mitosis. The results demonstrate the opportunity for studies on haploid gene expression at the translational level. PMID- 16666488 TI - Interaction between Chloroplasts and Mitochondria in Microalgae: Role of Glycolysis. AB - In a mutant strain of Chlamydomonas reinhardtii devoid of active ribulose 1,5 bisphosphate carboxylase oxygenase, the addition of mitochondrial inhibitors in the dark resulted in a pronounced decrease in cellular ATP, a fall of the glucose 6-phosphate content, and a rise of the NADPH concentration. These biochemical changes were accompanied by an increase of the fluorescence level, showing changes in the redox state of the chloroplastic electron transport chain. Similar results were obtained in presence of an uncoupler. These data indicated that alterations in the mitochondrial electron transport chain in dark could affect the chloroplastic chain, probably through variations of the glycolysis activity. When mitochondrial oxidases were blocked, illumination of the algae reversed the effect of the inhibitors on the ATP and glucose 6-phosphate concentrations. This last result suggested that the chloroplastic photophosphorylations in these algae played a major role in the control of the glycolytic flux. PMID- 16666489 TI - Kinetic studies of the form of substrate bound by phosphoenolpyruvate carboxylase. AB - Phosphoenolpyruvate carboxylase isolated from maize (Zea mays L.) leaves was assayed with varying concentrations of free phosphoenolpyruvate at several fixed varying concentrations of free magnesium higher than required to saturate the enzyme reaction. These assays produced velocity data which were found to form a family of individual lines when plotted against free phosphoenolpyruvate or against total phosphoenolpyruvate, but not when plotted against the concentration of the complex of phosphoenolpyruvate with magnesium. In this latter case, the points from all the fixed-varying concentrations fell on the same line, which can be fitted to a modified Michaelis-Menten equation with a multiple correlation coefficient R(2) = 0.995. Similar results were obtained when the enzyme from the C(4) plant maize was assayed with manganese rather than magnesium and when phosphoenolpyruvate carboxylase from leaves of the C(3) plant wheat (Triticum vulgare Vill.) was assayed with magnesium. However, at pH 7.0 the enzyme from the Crassulacean acid metabolism plant Crassula argentea did not produce a satisfactory single line when plotted against the complex of metal ion and substrate, but did so when the assay pH was raised to 8.0. It is concluded that in general the preferred form of substrate for phosphoenolpyruvate carboxylase is the complex of phosphoenolpyruvate with the metal ion. PMID- 16666490 TI - Fructose 2,6-bisphosphate inhibition of phosphoglucomutase. AB - Fructose 2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate. Previous studies from our laboratory had shown that phosphoglucomutase was activated by fructose 2,6 bisphosphate in the absence of added glucose 1,6-bisphosphate. The fructose 2,6 bisphosphate activation previously reported was due to the presence of glucose 1,6-bisphosphate in the commercial preparation of fructose 2,6-bisphosphate. PMID- 16666491 TI - Root formation in deteriorated (aged) wheat embryos. AB - Inability of aged seeds to grow into successful plants in the field is primarily due to their age-associated loss of rooting ability. The present work describes an attempt to initiate roots in nonrooting aged embryos of wheat (Triticum aestivum L.). Data presented give a comparative study of root formation and seedling growth on different culture media. Such studies indicate that sucrose alone is enough to bring about root development in nonrooting aged embryos. PMID- 16666492 TI - Measurements of Proton Pumping in Arum maculatum Mitochondria. AB - Direct evidence is presented for the lack of a pH change in the medium associated with electron flow along the alternative, cyanide-resistant pathway in Arum maculatum mitochondria. When these mitochondria oxidize succinate via the cytochrome chain, the medium becomes acidified. However, when the main respiratory chain is blocked by cyanide or antimycin A, oxygen consumption continues, but no pH change occurs. This confirms the lack of a charge separation across the membrane, so that phosphorylation cannot be supported during operation of the alternative oxidase. PMID- 16666493 TI - Inhibition of somatic embryogenesis in orchardgrass by endogenous cytokinins. AB - Endogenous indoleacetic acid (IAA) and cytokinin concentrations were measured by high performance liquid chromatography in leaf sections of an orchardgrass (Dactylis glomerata L.) genotype which exhibited a high capacity for somatic embryogenesis in vitro and in two genotypes that did not exhibit this capacity. The nonembryogenic genotypes contained 3- to 4-fold higher concentrations of zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, and total cytokinins than the embryogenic genotype. There were no significant differences in IAA concentrations between genotypes. Cytokinin concentrations between basal and distal sections of embryogenic genotype were not different, but the IAA concentration was significantly greater in basal sections. Somatic embryogenesis was inhibited in the embryogenic genotype by 0.001 micromolar exogenously added zeatin. PMID- 16666494 TI - Evidence for the Nuclear Location of the Genes for Chloroplast IF-2 and IF-3 in Euglena. AB - The chloroplast translational initiation factors IF-2 and IF-3 from Euglena gracilis are present in low levels in dark-grown cells and can be induced by exposure of cells to light. Studies of the antibiotic sensitivity of the light induction of these factors indicates that both are encoded in the nuclear genome. PMID- 16666495 TI - Fusicoccin Activity and Binding in Arabidopsis thaliana. AB - I have examined the activity and specific binding of the phytotoxin fusicoccin using Arabidopsis thaliana L. Col-O. Fusicoccin (10 micromolar) stimulates both proton extrusion and enlargement in isolated Arabidopsis leaf discs. Radiolabeled fusicoccin specifically binds to membranes (13,000 to 100,000g subcellular fraction) from cultured cells of Arabidopsis. The specific binding of this phytotoxin to putative receptor sites in Arabidopsis membranes is both pH sensitive (pH optimum = 5.5 to 6.0) and heat-labile (10 min at 70 degrees C). The apparent dissociation constant for the specific binding at 20 degrees C is approximately 1.3 x 10(-8) molar. The results of this study are in general agreement with previous reports of fusicoccin binding and activity in other plant species. PMID- 16666496 TI - Rapid induction of na/h exchange activity in barley root tonoplast. AB - Na(+)/H(+) exchange activity in barley (Hordeum vulgare cv CM-72) root tonoplast was induced by Na(+) even in the presence of inhibitors of protein synthesis. Induction occurred with a half-time of only 15 minutes. When salt-treated roots were transferred to a nutrient solution containing no Na(+), the activity disappeared with a similar time course. The data suggest that Na(+)/H(+) exchange was due to activation of an existing protein rather than to de novo protein synthesis. PMID- 16666497 TI - Evidence from Solanum tuberosum in Support of the Dual-Pathway Hypothesis of Aromatic Biosynthesis. AB - Key branchpoint enzymes of aromatic amino acid biosynthesis, 3-deoxy-d-arabino heptulosonate 7-phosphate synthase (DS) and chorismate mutase (CM), have previously been shown to exist as separate compartmentalized isozymes in the chloroplasts and cytosol of tobacco, sorghum and spinach. Although additional examples of plants containing these isozyme pairs are accumulating, some studies in the literature report the presence of only the single plastidic DS or CM enzyme. Such apparent exceptions contradict the universality of pathway organization existing in higher plants that is implied by the dual-pathway hypothesis of aromatic biosynthesis. Since potato (Solanum tuberosum) exemplifies a case where only a single species of both DS and CM have been reported, we selected this system for further analysis. The DS-Mn and DS-Co isozyme pair, exhibiting all of the differential properties described in Nicotiana silvestris, have now been identified in S. tuberosum. Likewise, partial purification via DEAE cellulose chromatography revealed two isozymes of CM in disks excised from tubers of S. tuberosum. The differential regulatory properties of these isozymes were comparable to the CM-1 and CM-2 isozymes of N. silvestris. PMID- 16666498 TI - In vitro synthesis, assembly and function of a photosynthetic membrane protein. AB - Cell-free translation of Chlamydomonas reinhardtii RNA in the presence of photosynthetic membranes resulted in association of the herbicide binding (Qb) protein with membranes. Incubation of recovered membranes with high salt did not extract the polypeptide from membranes. Tryptic digestion of in vivo labeled membranes or membranes recovered from in vitro translation mixtures showed that Qb had similar orientation. In vitro translation in the presence of chloroplast membranes from cells exposed to high light intensity restored the membrane associated kinase activity lost by photoinhibition. Thus, in vitro synthesis resulted in functional integration of the Qb protein within the photosynthetic membrane. PMID- 16666499 TI - Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation. AB - We studied the effects of dl-alpha-difluoromethylarginine (DFMA) and dl-alpha difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines. PMID- 16666500 TI - Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus. AB - We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed. PMID- 16666501 TI - Temporal regulation of polygalacturonase gene expression in fruits of normal, mutant, and heterozygous tomato genotypes. AB - Molecular cloning of polygalacturonase (PG; EC 3.2. 1.15) from fruits of tomato (Lycopersicon esculentum Mill cv Rutgers) was accomplished by constructing a cDNA library from turning stage poly(A)(+) RNA in lambdagtll and immunoscreening with polyclonal antibodies raised against purified PG2. Both PG cDNA and antibody probes were used to quantify changes in PG gene expression in pericarp from normal, mutant, and heterozygous genotypes. Results show that PG mRNA, protein, and enzyme activity sequentially peak at the turning, ripe, and red ripe stages of Rutgers pericarp ripening, respectively. PG gene expression was attenuated greatly (0-15% of normal on a gram fresh weight basis) for PG mRNA, protein, and enzyme activity in five ripening-impaired mutants (rin, nor, Nr, Gr, and Long Keeper) tested. Maximum expression of the PG gene in heterozygotes of rin, nor, Nr, Gr, and Long Keeper (crosses with Rutgers) at the mRNA level was about 25, 13, 17, 5, and 62% of the Rutgers turning stage, at the protein level was about 166, 110, 15, 6, and 104% of the Rutgers ripe stage, and at the enzyme activity level was about 69, 37, 4, 1, and 50% of the Rutgers red ripe stage, respectively. No PG gene expression was found in preclimacteric fruits or vegetative tissues. PG mRNA was localized on both free and membrane-bound polyribosomes of ripening pericarp. In addition to transcriptional regulation, mechanisms contributing to mRNA stability, delayed protein accumulation, and posttranslational modifications may play important roles in the overall accumulation of PG activity during fruit ripening. PMID- 16666502 TI - Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells. AB - We have shown previously that inositol-1,4,5-trisphosphate (IP(3)) stimulates an efflux of (45)Ca(2+) from fusogenic carrot protoplasts (M Rincon, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP(3) might serve as a second messenger for the mobilization of intracellular Ca(2+) in higher plant cells. To determine whether or not IP(3) and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2 (3)H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [(3)H]inositol metabolites coeluted with inositol bisphosphate (IP(2)) and IP(3) when separated by anion exchange chromatography. However, we could not detect IP(2) or IP(3) when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP(2) and IP(3), were present in these cells. Thus, [(3)H] inositol metabolites other than IP(2) and IP(3) had coeluted on the anion exchange columns. The data indicate that either IP(3) is rapidly metabolized or that it is not present at a detectable level in the carrot cells. PMID- 16666503 TI - Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations. AB - Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO(2), little O(2) inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO(2) concentrating system. The CO(2)-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO(2) concentrations have inorganic carbon transport activity, but cells grown at 5% CO(2) do not. Four membrane-associated polypeptides (M(r) 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO(2) concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO(2)-concentrating system activity in response to CO(2) limitation. PMID- 16666504 TI - Ethylene biosynthesis-inducing protein from cellulysin is an endoxylanase. AB - The proteinaceous ethylene biosynthesis-inducing factor (EIF) that was purified from Cellulysin was also shown to contain a xylanase activity. In all nondenaturing protein separation methods employed (Sephacryl S-200 chromatography, and preparative isoelectric focusing and agarose electrophoresis), xylanase activity copurified with the ethylene biosynthesis inducing activity. Treatment with heat (60 degrees C) or proteases in 8 molar urea inhibited both ethylene-inducing and xylanase activities. Antibodies raised against purified EIF, which contains three polypeptides of 18, 14, and 10 kilodaltons, immunoprecipitated both ethylene biosynthesis-inducing and xylanase activities. The purified EIF contained no detectable cellulase, polygalacturonase, or protease activity. Other hydrolytic activities as estimated by using p-nitrophenyl derivatives of several sugars as substrates also were not detected. Different commercially available hydrolytic enzyme preparations were tested for both ethylene biosynthesis-inducing and xylanase activities. All enzymes tested contained xylanase activity, but only a few induced ethylene biosynthesis. Western blots of proteins separated by SDS-PAGE, using antibodies prepared against the non-denatured purified EIF, revealed two major bands of about 18 and 14 kilodaltons in EIF. These antibodies seem to be specific for these proteins from Trichoderma viride, because there was little cross-reactivity with the other proteins in Cellulysin and other commercial enzyme preparations. Based on these data, we suggest that EIF contains a specific xylanase activity which is involved in inducing ethylene biosynthesis. PMID- 16666505 TI - Low Temperature-Induced Decrease in trans-Delta-Hexadecenoic Acid Content Is Correlated with Freezing Tolerance in Cereals. AB - The effect of growth at 5 degrees C on the trans-Delta(3)-hexadecenoic acid content of phosphatidyl(d)glycerol was examined in a total of eight cultivars of rye (Secale cereale L.) and what (Triticum aestivum L.) of varying freezing tolerance. In these monocots, low temperature growth caused decreases in the trans-Delta(3)-hexadecenoic acid content of between 0 and 74% with concomitant increases in the palmitic acid content of phosphatidyl(d)glycerol. These trends were observed for whole leaf extracts as well as isolated thylakoids. The low growth temperature-induced decrease in the trans-Delta(3)-hexadecenoic acid content was shown to be a linear function (r(2) = 0.954) of freezing tolerance in these cultivars. Of the six cold tolerant dicotyledonous species examined, only Brassica and Arabidopsis thaliana L. cv Columbia exhibited a 42% and 65% decrease, respectively, in trans-Delta(3)-hexadecenoic acid content. Thus, the relationship between the change in trans-Delta(3)-hexadecenoic acid content of phosphatidyl(d)glycerol and freezing tolerance cannot be considered a general one for all cold tolerant plant species. However, species which exhibited a low growth temperature-induced decrease in trans-Delta(3)-hexadecenoic acid also exhibited a concomitant shift in the in vitro organization of the light harvesting complex II from a predominantly oligomeric form to the monomeric form. We conclude that the proposed role of phosphatidyl(d)glycerol in modulating the organization of light harvesting complex II as a function of growth temperature manifests itself to varying degrees in different plant species. A possible physiological role for this phenomenon with respect to low temperature acclimation and freezing tolerance in cereals is discussed. PMID- 16666506 TI - Preparation of chlamydomonas chloroplasts for the in vitro import of polypeptide precursors. AB - To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro. PMID- 16666507 TI - Protein kinase activities in tonoplast and plasmalemma membranes from corn roots. AB - Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H(+)-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K(m) value for MgATP(2-) of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg(2+) and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca(2+), but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H(+)-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination. PMID- 16666508 TI - Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide. AB - Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell. PMID- 16666509 TI - The Role of Ethylene in the Inhibition of Rooting under Low Oxygen Tensions. AB - A 60-fold increase in ethylene content was observed in stem cuttings of chrysanthemum (Chrysanthemum x morifolium Ramat.) held in aero-hydroponics under anoxic conditions during the 8 to 12 days necessary for adventitious root formation. Ethylene, 1-aminocyclopropane-1-carboxylic acid, and 10-(malonylamino) cyclopropane-1-carboxylic acid contents were highest in the immersed portion of the cuttings, but there was substantial ethylene produced by the anoxic, misted portions of the cutting above the liquid. Application of ethylene (10 microliters per liter) to chrysanthemum cuttings stimulated root development in cuttings held in high dissolved oxygen concentrations (8.0 milligrams per liter). Since the application of ethylene did not inhibit rooting in cuttings held at low dissolved oxygen concentrations (2.0 milligrams per liter), the inhibition of rooting under low oxygen concentrations is not mediated by the observed increase in endogenous ethylene content. PMID- 16666510 TI - Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean. AB - Since NO(3) (-) availability in the rooting medium seriously limits symbiotic N(2) fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO(3) (-) and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M(2) generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3 7) were more extensively nodulated (427 to 770 nodules plant(-1)) than the Williams parent (187 nodules plant(-1)) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO(3) (-); each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO(3) (-) than did Williams at 1.5 millimolar NO(3) (-). The NO(3) (-) tolerance did not appear to be related to an altered ability to take up or metabolize NO(3) (-), based on solution NO(3) (-) depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M(5) generation in all three mutants. PMID- 16666511 TI - Light-dependent kinetics of 2-carboxyarabinitol 1-phosphate metabolism and ribulose-1,5-bisphosphate carboxylase activity in vivo. AB - The light-dependent kinetics of the apparent in vivo synthesis and degradation of 2-carboxyarabinitol 1-phosphate (CA1P) were studied in three species of higher plants which differ in the extent to which this compound is involved in the light dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity. Detailed studies with Phaseolus vulgaris indicate that both the degradation and synthesis of this compound are light-stimulated, although light is absolutely required only for CA1P degradation. We hypothesize that the steady state level of CAIP at any particular photon flux density (PFD) represents a pseudo-steady state balance between ongoing synthesis and degradation of this compound. The rate of CA1P synthesis in P. vulgaris and the resultant reduction in the total catalytic constant of Rubisco were maximal at 200 micromoles quanta per square meter per second following a step decrease from a saturating PFD, and substantially faster than the rate of synthesis in the dark. Under these conditions an amount of CA1P equivalent to approximately 25% of the Rubisco catalytic site content was synthesized in less than 1 minute. The rate of synthesis was reduced at higher or lower PFDs. In Beta vulgaris, the rate of CA1P synthesis at 200 micromoles quanta per square meter per second was substantially slower than in P. vulgaris. In Spinacea oleracea, an apparent noncatalytic tight-binding of RuBP to deactivated sites on the enzyme was found to occur following a step decrease in PFD. When dark acclimated leaves of P. vulgaris were exposed to a step increase in PFD, the initial rate of CA1P degradation was also found to be dependent on PFD up to a maximum of approximately 300 to 400 micromoles quanta per square meter per second. The rate of degradation of this compound was similar in B. vulgaris. In S. oleracea, a step increase in PFD resulted in noncatalytic RuBP binding to Rubisco followed by an apparent release of RuBP and activation of the enzyme. The in vivo rate of change of Rubisco activity in response to an increase or decrease in PFD was similar between species despite the differences between species in the mechanisms used for the regulation of this enzyme's activity. PMID- 16666512 TI - Collapse of ATP-Induced pH Gradient by Sodium Ions in Microsomal Membrane Vesicles Prepared from Atriplex gmelini Leaves: Possibility of Na/H Antiport. AB - Sealed microsomal membrane vesicles were prepared from leaves of a 250 millimolar NaCl-grown halophyte (Atriplex gmelini C. A. Mey). The vesicles exhibited ATP dependent proton-transporting activity which was inhibited 60% by NO(3) (-) (50 millimolar) but not by vanadate (100 micromolar) and 23% by oligomycin (10 micrograms per milliliter), suggesting that tonoplast-derived vesicles were the major constituents of the preparation. The pH gradient established by the vesicles by ATP in the presence of oligomycin collapsed upon the addition of Na(+) salts. The vesicles took up Na(+) ions in the presence of ATP and this activity was canceled by gramicidin. These results suggest that Na(+) ions were taken up by the vesicles via a Na(+)-specific uptake system, possibly a Na(+)/H(+) antiport. PMID- 16666513 TI - Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies. AB - For the first time, the (31)P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 x 10(6) vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 +/- 0.06 and a mean trans-tonoplast DeltapH of 1.56 +/- 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H(+) exchanges at the tonoplast. The (31)P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 +/- 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content. PMID- 16666514 TI - Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves. AB - In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX. PMID- 16666515 TI - Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by C-NMR Spectroscopy. AB - The accumulation of malate by maize (Zea mays L.) root tips perfused with KH(13)CO(3) was followed by (13)C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH approximately 5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4 labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH(13)CO(3) treatment, (13)C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of approximately 6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment. PMID- 16666516 TI - Enhancement of cyanobacterial salt tolerance by combined nitrogen. AB - Presence of certain nitrogenous compounds in the growth medium significantly enhanced the salt tolerance of the fresh-water cyanobacterium Anabaena sp. strain L-31 as well as the brackish water cyanobacterium Anabaena torulosa. Among these, nitrate, ammonium, and glutamine were most effective followed by glutamate and aspartate. These nitrogenous compounds also inhibited Na(+) influx in both Anabaena spp. with the same order of effectiveness as that observed for protection against salt stress. The inhibition of Na(+) influx on addition of the nitrogenous substances was rapid; nitrate and ammonium inhibited Na(+) influx competitively. Proline and glycine did not affect Na(+) influx and also had no influence on the salt tolerance of either Anabaena sp. The observed protection was not consequent to a stimulatory effect of combined nitrogen on growth per se. Uptake of NO(3) (-) and NH(4) (+) increased during salt stress but was not correlated with growth. Intracellular levels of NO(3) (-) and NH(4) (+) were found to be inadequate to constitute a major component of the internal osmoticum. The results suggest that inhibition of Na(+) influx by combined nitrogen is a major mechanism for protection of cyanobacteria against salt stress. PMID- 16666517 TI - Fluorescence and Delayed Light Emission from Mesophyll and Bundle Sheath Cells in Leaves of Normal and Salt-Treated Panicum miliaceum. AB - A modified fluorescence microscope system was used to measure chlorophyll fluorescence and delayed light emission from mesophyll and bundle sheath cells in situ in fresh-cut sections from leaves of Panicum miliaceum L. The fluorescence rise in 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-treated leaves and the slow fluorescence kinetics in untreated leaves show that mesophyll chloroplasts have larger photosystem II unit sizes than do bundle sheath chloroplasts. The larger photosystem II units imply more efficient noncyclic electron transport in mesophyll chloroplasts. Quenching of slow fluorescence also differs between the cell types with mesophyll chloroplasts showing complex kinetics and bundle sheath chloroplasts showing a relatively simple decline. Properties of the photosynthetic system were also investigated in leaves from plants grown in soil containing elevated NaCl levels. As judged by changes in both fluorescence kinetics in DCMU-treated leaves and delayed light emission in leaves not exposed to DCMU, salinity altered photosystem II in bundle sheath cells but not in mesophyll cells. This result may indicate different ionic distributions in the two cell types or, alternatively, different responses of the two chloroplast types to environmental change. PMID- 16666518 TI - Influence of Phosphorus Nutrition on Growth and Carbon Partitioning in Glycine max. AB - Soybean plants (Glycine max [L.] Merr var Amsoy 71) were grown in growth chambers with high-phosphorus (high-P) and low-phosphorus (low-P) culture solutions. Low-P treatment reduced shoot growth significantly 7 days after treatment began. Root growth was much less affected by low-P, there being no significant reduction in root growth rate until 17 days had elapsed. The results suggest that low-P treatment decreased soybean growth primarily through an effect on the expansion of the leaf surface which was diminished by 85%, the main effect of low-P being on the rate of expansion of individual leaves. Low-P had a lesser effect on photosynthesis; light saturated photosynthetic rates at ambient and saturating CO(2) levels were lowered by 55 and 45%, respectively, after 19 days of low-P treatment. Low-P treatment increased starch concentrations in mature leaves, expanding leaves and fibrous roots; sucrose concentrations, however, were reduced by low-P in leaves and increased in roots. Foliar F-2,6-BP levels were not affected by P treatment in the light but in darkness they increased with high-P and decreased with low-P. The increase in the starch/sucrose ratio in low-P leaves was correlated primarily with changes in the total activities of enzymes of starch and sucrose metabolism. PMID- 16666519 TI - Multiphasic uptake of potassium by corn roots: no linear component. AB - Concentration-dependence data (LV Kochian, WJ Lucas Plant Physiol 1982 70: 1723 1731; [1983] 73: 208-215; [1985] 429-436; LV Kochian, J Xin-zhi, WJ Lucas [1985], 79: 771-776) for potassium uptake by corn roots have been claimed to be resolvable into a saturable and a linear component representing two separate uptake mechanisms. A comprehensive analysis shows, however, that the data are not compatible with this concept. They can, in contrast, be precisely represented by multiphasic isotherms, in agreement with many findings for uptake of potassium and other solutes. Solute uptake in plants is proposed to be mediated by multistate entities having carrier-like properties at low external solute concentrations and channel-like properties at high concentrations. PMID- 16666520 TI - Chloroplast thylakoid protein phosphatase is a membrane surface-associated activity. AB - Chloroplast thylakoid protein phosphatase activity was measured using (32)P labeled histone as an exogenous substrate and an assay of the (32)Pi released involving formation of a phosphomolybdate complex and organic extraction. The activity was liberated from wheat (Triticum aestivum) thylakoids by washing the membranes in NaCl-containing solutions followed by centrifugation. The liberated phosphatase activity had a pH optimum of approximately 6.75, was inhibited by addition of 10 millimolar EDTA or EGTA, and was stimulated by addition of millimolar amounts of dithiothreitol, magnesium, manganese, or calcium ions. The rate of thylakoid protein dephosphorylation was decreased following liberation of a portion of the protein phosphatase activity and was increased by addition of salt-liberated phosphatase fraction. These results suggest that at least a portion of wheat thylakoid protein phosphatase is a peripheral, rather than an integral, membrane protein. PMID- 16666521 TI - Release and Activity of Bound beta-Amylase in a Germinating Barley Grain. AB - In resting grains of Triumph barley (Hordeum vulgare L. cv Triumph) about 40% of the beta-amylase could be extracted with a saline solution, the remaining 60% being in a bound form. During seedling growth (20 degrees C), the bound form was released mainly between days 1 and 3. When a preparation containing bound beta amylase was incubated with an extract made of endosperms separated from germinating grains, release of bound beta-amylase took place and could be studied in vitro. The release was almost completely prevented by leupeptin and antipain, specific inhibitors of a group of SH-proteinases, but it was not inhibited by pepstatin A or EDTA, which inhibit some other barley proteinases. It is thus very likely that in a whole grain, at least the bulk of the bound beta-amylase is released by the proteolytic action of one or several SH-proteinases. When the bound beta-amylase was released by papain, its molecular weight was about 5000 daltons smaller than that of beta-amylase released by dithiothreitol. This indicates that the release is due to removal of a sequence of beta-amylase itself. A similar decrease in size took place during seedling growth. Bound beta amylase showed some activity against native starch and it hydrolyzed maltotetraose at a rate that was about 70% of the rate the same amount of bound beta-amylase gave after release. Bound beta-amylase is thus not inactive and it is likely that the slower rate of hydrolysis is due to steric hindrances which prevent substrates from reaching the active site. PMID- 16666522 TI - Glucosinolate uptake by developing rapeseed embryos. AB - Embryos excised from the seed of rapeseed (Brassica napus L.) accumulated glucosinolate from the culture medium. Uptake was saturable, subject to inhibition, varied with the developmental stage of the embryo but correlated with the time of accumulation of glucosinolates in situ. It is suggested that a carrier-mediated transport system is operating in the developing embryo. PMID- 16666523 TI - Water transport properties of cortical cells in roots of nitrogen- and phosphorus deficient cotton seedlings. AB - Growth-limiting deficiencies of N or P substantially decrease the hydraulic conductance of cotton (Gossypium hirsutum L.) roots. This shift could result from decreased hydraulic conductivity of cells in the radial flow pathway. A pressure microprobe was used to study water relations of cortical cells in roots of cotton seedlings stressed for N or P. During 10 days of seedling growth on a complete nutrient solution, root cell turgor was stable at 0.4 to 0.5 megapascal, the volumetric elastic modulus increased slowly from 6 to 10 megapascals, and the half-time for water exchange increased from 10 to 15 seconds. In seedlings transferred to N-free solution for 10 days, final values for each of those parameters were approximately doubled. Root cell hydraulic conductivity (cell Lp) was 1.4 x 10(-7) meters per second per megapascal at the time of transfer. In the well-nourished controls, cell Lp decreased over 10 days to 38% of the initial value, but in the N-stressed plants it decreased much more sharply, reaching 6% of the initial value after 10 days. Transfer to solutions without P or with an intermediate level of N also decreased cell Lp. The changes in root cell Lp were consistent with nutrient effects on intact-root water relations demonstrated earlier. However, cell Lp was about half that of the intact root, implying that substantial water flow may follow an apoplastic pathway, bypassing the cortical cells from which these values were derived. PMID- 16666524 TI - Endogenous Gibberellins and Shoot Growth and Development in Brassica napus. AB - Greenhouse-grown oilseed rape (Brassica napus, annual Canola variety ;Westar') plants were harvested at six dates from the vegetative phase until the early pod (silique)-fill/late flowering stage. Endogenous gibberellin (GA)-like substances were extracted from stems, purified, and chromatographed on silica gel partition columns prior to bioassay in serial dilution using the ;Tan-ginbozu' dwarf rice microdrop assay. The concentrations of total endogenous GA-like substances were low during vegetative stages (1 nanogram GA(3) equivalents/gram dry weight), and rose 300-fold by the time of floral initiation. After floral initiation the concentration of GA-like substances fell, then rose again during bolting to maximal levels during the early pod-fill stage (940 nanograms per gram dry weight). The qualitative profiles of GA-like substances varied across harvests, with higher proportions of a GA(1)-like substance at the early pod-fill stage. In a second study stems were similarly harvested at eight dates and the concentrations of endogenous GA(1), the principal bioactive native GA of oilseed rape, were determined by gas chromatography-selected ion monitoring using [17,17 (2)H]GA(1) as a quantitative internal standard. The concentration of GA(1) increased at about the time of floral initiation and then subsequently fell, thus confirming the pattern noted above for total GA-like substances. The exogenous application of paclobutrazol (PP333), a persistent triazole plant growth regulator (PGR) which blocks GA biosynthesis, or another triazole, triapenthenol (RSW0411), prevented flowering as well as bolting; plants remained at the vegetative rosette stage. These results imply a causal role for endogenous GA, in the control of bolting, which normally precedes anthesis. Further, the rise in the concentration of total endogenous GA-like substances, including GA(1), which was associated with floral initiation, and the prevention of visable floral development by the triazole PGRs, also indicates a role for endogenous GAs in the regulation of flowering in B. napus. PMID- 16666525 TI - Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges. AB - The vacuolar pH and the trans-tonoplast DeltapH modifications induced by the activity of the two proton pumps H(+)-ATPase and H(+)-PPase and by the proton exchanges catalyzed by the Na(+)/H(+) and Ca(2+)/H(+) antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the (31)P NMR technique. The H(+)-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast DeltapH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H(+)-ATPase. A ratio of H(+) translocated/ATP hydrolyzed of 1.97 +/- 0.06 (mean +/- standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H(+)-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H(+) ATPase, building a steady state DeltapH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na(+) were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using (23)Na NMR that sodium uptake was coupled to the H(+) efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca(2+)/H(+) antiport has been measured on isolated intact vacuoles. Ca(2+) uptake was strongly inhibited by NH(4)Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca(2+) were alkalinized by about 0.6 pH unit and this H(+) efflux was associated to a Ca(2+) uptake as demonstrated by measuring the external Ca(2+) concentration with a calcium specific electrode. Steady state accumulation ratios of Ca(2+) as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca(2+) uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca(2+)/H(+) antiport has a high potential capacity to regulate cytoplasmic concentration of calcium. PMID- 16666526 TI - Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds. AB - A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 +/- 1 degrees C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme. PMID- 16666527 TI - Protection of Pyruvate,Pi Dikinase from Maize against Cold Lability by Compatible Solutes. AB - Most C(4) species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C(4) pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0 degrees C) by the divalent cations Mn(2+), Mg(2+), and Ca(2+). There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl(2) (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg(2+) is required and Mn(2+) could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg(2+) had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl(2) was evaluated by incubation at 2 to 17 degrees C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11 degrees C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures. PMID- 16666528 TI - Increased thermal deactivation of excited pigments in pea leaves subjected to photoinhibitory treatments. AB - The photoacoustic technique was used to monitor thermal deexcitation of the photosynthetic pigments in intact pea leaves (Pisum sativum L.) submitted to photoinhibitory treatments. When the leaves were exposed to photon flux densities above 1000 micromoles per square meter per second, the amplitude of the photothermal component of the in vivo photoacoustic signal strongly increased. This high-light-induced stimulation of nonradiative energy dissipation (heat emission) was accompanied by an inverse change in the O(2) evolution activity and in the steady state emission of 685 nanometer chlorophyll fluorescence. The time course of these effects was shown to be very rapid, with a t(1/2) of around 15 minutes. When high-light-treated leaves were readapted to the dark, the heat emission changes were reversed, following somewhat slower kinetics. A reversible increase in the rate of light energy dissipation via radiationless transitions could be a photoprotective mechanism eliminating excess excitation energy from the photosynthetic reaction centers. Interestingly, this process does not operate at temperatures below about 12 degrees C. PMID- 16666529 TI - Regulation of pea epicotyl elongation by blue light : fluence-response relationships and growth distribution. AB - Irradiation with blue light causes a rapid decrease in stem elongation in Pisum sativum. Growing plants under continuous red light allowed us to study the fluence dependence and spatial distribution of blue-induced growth effects without interference from large changes in the ratio of the far-red absorbing form of phytochrome to total phytochrome. The magnitude of the inhibition generated by a 30-second pulse of blue light was linearly related to the log of the fluence applied over two orders of magnitude. Reciprocity held for irradiations with a pulse length shorter than the lag time for the response. The spatial distribution of inhibition was studied by marking the growing zone and photographing the stem at 10-minute intervals before, during, and after a 1-hour exposure to blue light. The region just below the hook does not undergo any perceptible change in growth rate while growth is nearly 100% inhibited in the base of the third internode. PMID- 16666530 TI - Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root. AB - Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [(3)H]glucosamine and [(35)S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties. PMID- 16666531 TI - Changes in the Activity of the Chloroplastic and Cytosolic Forms of Dihydroxyacetone Phosphate Reductase during Maturation of Leaves. AB - Young or mature rosette leaves from spinach (Spinacia oleracea L.) plants growing in the field, in the greenhouse, or in a growth chamber under a regimen of 8 hours light and 16 hours dark contained 15 to 50 nanomoles per minute per gram wet weight of NADH:dihydroxyacetone phosphate reductase activity. Of this activity, 75 to 87% was the chloroplastic isoform and 25 to 13% was the cytosolic form. When plants were induced to senesce, as measured by stem elongation and flowering, the percentage of the two reductase isoforms in rosette or stem leaves changed to about 12% as the chloroplastic and 88% as the cytosolic isoform. The change in enzyme activity of the rosette leaves occurred within 3 days, before phenotypic changes were observed. Likewise, when plants senesced in continuous darkness, the percentage of chloroplastic to cytosolic reductase changed from 80:20% to 25:75% after 62 hours before changes in total protein or chlorophyll occurred. The ratio of activities did not change in the first 16 hours of darkness or overnight. In each case the change in ratio resulted from about a 75% decrease in activity of the chloroplastic isoform and up to 14-fold increase in cytosolic isoform. In spinach leaves purchased at a local market primarily only the cytosolic isoform remained. When plants were returned to normal day-nights, after 62 hours in continuous darkness, the activity of the chloroplastic isoform increased, but not to control levels after 3 days, while the cytosolic enzyme decreased within 1 day to normal day-night values. Changes in activity were not due to changes during in vitro assays in activation by thioredoxin for the chloroplastic isoform or fructose 2,6-phosphate for the cytosolic isoform. PMID- 16666532 TI - Developmental regulation and the influence of plant sinks on vegetative storage protein gene expression in soybean leaves. AB - Soybeans (Glycine max L.) accumulate a storage glycoprotein which is abundant in vegetative tissues, but is only a minor component of seeds. Changes in vegetative storage protein gene expression in leaves of control and depodded plants were monitored throughout plant development. Western and Northern blot hybridization analysis of protein and mRNA levels, respectively, showed that expression of these genes was highly regulated during development. Expression correlated with periods when expected demand for mobilized leaf reserves by other plant sinks was low. Vegetative storage protein mRNA comprised about 0.5% of the total mRNA in immature leaves and declined at least 20-fold by flowering. Depodding or blockage of leaf petiole phloem transport increased these mRNAs to about 16% of the total mRNA. Transcript levels also increased dramatically after seed maturation, just before leaf senescence. Protein levels followed a similar pattern and were inversely related to the number of seed pods allowed to develop on the plants. The results support the role for these proteins as temporary storage molecules which can be rapidly synthesized or degraded according to the need for nutrients by other plant tissues. PMID- 16666533 TI - Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum. AB - NAD malic enzyme (EC 1.1.1.39), which is involved in C(4) photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C(4) species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M(r) of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C(4) species is an octamer of identical subunits. The purified NAD malic enzyme from each C(4) species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn(2+) and activation by fructose- 1,6 bisphosphate (FBP) or CoA. A cooperativity with respect to Mn(2+) was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K(0.5) (the concentration giving half-maximal activity) for Mn(2+). The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn(2+). In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn(2+). Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C(4) dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana. PMID- 16666534 TI - Thermal Acclimation of Photosynthetic Electron Transport Activity by Thylakoids of Saxifraga cernua. AB - Thermal acclimation by Saxifraga cernua to low temperatures results in a change in the optimum temperature for gross photosynthetic activity and may directly involve the photosynthetic apparatus. In order to test this hypothesis photosynthetic electron transport activity of S. cernua thylakoids acclimated to growth temperatures of 20 degrees C and 10 degrees C was measured in vitro. Both populations exhibited optimum temperatures for whole chain and PSII electron transport activity at temperatures close to those at which the plants were grown. Chlorophyll a fluorescence transients from 10 degrees C-acclimated leaves showed higher rates in the rise and subsequent quenching of variable fluorescence at low measuring temperatures; 20 degrees C-acclimated leaves showed higher rates of fluorescence rise at higher measuring temperatures. At these higher temperatures, fluorescence quenching rates were similar in both populations. The kinetics of State 1-State 2 transitions in 20 degrees C- and 10 degrees C-acclimated leaf discs were measured as changes in the magnitude of the fluorescence emission maxima measured at 77K. Leaves acclimated at 10 degrees C showed a larger F730/F695 ratio at low temperatures, while at higher temperatures, 20 degrees C acclimated leaves showed a higher F730/F695 ratio after the establishment of State 2. High incubation temperatures also resulted in a decrease in the F695/F685 ratio for 10 degrees C-acclimated leaves, suggesting a reduction in the excitation transfer from the light-harvesting complex of photosystem II to photosystem II reaction centers. The relative amounts of chlorophyll-protein complexes and thylakoid polypeptides separated electro-phoretically were similar for both 20 degrees C- and 10 degrees C-acclimated leaves. Thus, photosynthetic acclimation to low temperatures by S. cernua is correlated with an increase in photosynthetic electron transport activity but does not appear to be accompanied by major structural changes or different relative amounts in thylakoid protein composition. PMID- 16666535 TI - Seed Development in Phaseolus vulgaris L. cv Seminole: I. Developmental Independence of Seed Maturation. AB - Phaseolus vulgaris L. cv Seminole pods removed from the plant continued their development when incubated in suitable conditions. Seeds continued to grow and develop and pods and seeds passed through an apparently normal developmental sequence to dryness. Seed growth was at the expense of pod dry weight (DW) reserves. Losses of pod DW paralleled DW gains by seeds in detached pods and in pod cylinders containing a seed. The transfer activity was apparent only within the period 10 to 30 days after anthesis (DAA) with maximal activity between 15 to 20 DAA. This period corresponds to maximum pod growth and the attainment of maximal DW. Seeds are in only the early phase of seed growth at this time. No DW transfer was observed at developmental stages beyond 30 to 35 DAA when normal senescence DW losses in pods became evident and seeds were in the later phase of seed fill. Pods or pod cylinders remained green and succulent over the transfer period, later passing through yellowing and drying phases characteristic of normal development. DW transfer was dependent on funicle integrity and was readily detectable in pod cylinders after 7 days incubation. The DW transfer activity may contribute to continuing nutrition of seeds under conditions where the normal assimilate supply to seeds becomes limiting. Defoliation and water stress treatments applied to Phaseolus plants reduced seed yields but allowed persistence of seed maturation processes such that all seeds developing to dryness were capable of germination. PMID- 16666536 TI - Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen. AB - This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise. PMID- 16666537 TI - Light/Dark profiles of sucrose phosphate synthase, sucrose synthase, and Acid invertase in leaves of sugar beets. AB - The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves. PMID- 16666538 TI - Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum. AB - Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20 40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes. PMID- 16666539 TI - Effect on in Vitro Pollen Growth of an Isolated Style Glycoprotein Associated with Self-Incompatibility in Nicotiana alata. AB - The effect on in vitro pollen tube growth of an isolated style glycoprotein (S(2) glycoprotein) associated with self-incompatibility in Nicotiana alata was investigated. Tube growth of pollen bearing the S(2)-allele was inhibited, but tube growth of pollen bearing other alleles was not affected. Inhibition showed a dose response effect. The percentage of pollen grains that germinated was not significantly affected by the S(2)-glycoprotein. Growth of S(2)-pollen in the presence of the S(2)-glycoprotein resulted in increased binding to the pollen of monoclonal antibody (PCBC3) which has a primary specificity for alpha-l arabinofuranosyl residues. Growth of pollen bearing other alleles in the presence of the glycoprotein resulted in no increased binding of the antibody. PMID- 16666540 TI - The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase. AB - When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl(2) followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5 bisphosphate dependent rates. When sucrose gradients were used to purify ribulose 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay. PMID- 16666541 TI - Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity. AB - In high inorganic carbon grown (1% CO(2) [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO(2) uptake and to reduce the final internal inorganic carbon (C(i)) pool size reached. The relationship between CO(2) fixation rate and internal C(i) concentration in high C(i) grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High C(i) grown cells readily took up CO(2) but had little or no capacity for HCO(3) (-) uptake. These cells appear to possess a CO(2) utilizing C(i) pump that has a CA-like function associated with the transport step such that HCO(3) (-) is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low C(i) grown cells possess both CO(2) uptake and HCO(3) (-) uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO(2) and HCO(3) (-) uptake (such as the C(i) pump) may have been affected. The inhibitor had no apparent effect on internal CO(2)/HCO(3) (-) equilibria (internal CA function) in low C(i) grown cells. PMID- 16666542 TI - Molecular cloning and relationship to freezing tolerance of cold-acclimation specific genes of alfalfa. AB - Cold-acclimation-specific (CAS) gene expression has been examined by screening a cDNA library prepared from poly(A)(+) RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medicago falcata cv Anik). Three CAS cDNA clones, pSM784, pSM2201, and pSM2358, representing different sequence species, have been used to investigate the relative abundance and time-course of accumulation of corresponding transcripts. Results obtained show that the expression of these CAS genes is regulated in a coordinated manner most likely at the level of transcription. The expression of genes, as measured by mRNA abundance corresponding to the three CAS cDNA clones, is not stimulated or induced by heat shock, water stress, abscisic acid, or wounding. A positive correlation is observed between the expression of these cloned sequences and the degree of freezing-tolerance in four alfalfa cultivars. PMID- 16666543 TI - Immunogold localization of photosynthetic fructose-1,6-bisphosphatase in pea leaf tissue. AB - An enriched IgG serum fraction obtained from rabbits immunized against pea chloroplast fructose-1,6-bisphosphatase (FBPase) was used, coupled to colloidal gold (15 nanometer particles) goat anti-rabbit IgG, to analyze by electron microscopy the location of photosynthetic FBPase in pea (Pisum sativum L.) leaf ultrathin sections. In accordance with earlier biochemical studies on distribution of FBPase activity, the enzyme was visualized both in the stromal space and bound to the chloroplast membranes. Some gold particles also appear in the cytoplasm, which can be related to the presence in the cytosol of a high molecular weight precursor of this nuclear coded enzyme. PMID- 16666544 TI - Ethoxyzolamide Inhibition of CO(2)-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC7942. AB - Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (C(i)) conditions (1% CO(2); pH 8) were found to be photosynthetically dependent on exogenous CO(2). This was judged by the fact that they had a similar photosynthetic affinity for CO(2) (K(0.5)[CO(2)] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for C(i) measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO(2) in high C(i) cells. The addition of 200 micromolar EZ to high C(i) cells increased K(0.5)(CO(2)) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low C(i) cells (grown at 30 microliters CO(2) per liter of air) were less sensitive to EZ. EZ inhibition in high and low C(i) cells was largely relieved by increasing exogenous C(i) up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO(2) usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem II activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO(2) usage. High C(i) cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO(2) usage in high C(i) cells of Synechococcus PCC7942 may be due to inhibition of a ;CA-like' function associated with the CO(2) utilizing C(i) pump or due to inhibition of an internal CA activity, thus affecting CO(2) supply to ribulose bisphosphate carboxylase-oxygenase. PMID- 16666545 TI - Exopolysaccharides Produced by Phytopathogenic Pseudomonas syringae Pathovars in Infected Leaves of Susceptible Hosts. AB - Bacterial exopolysaccharide (EPS) was extracted from infected leaves of several host plants inoculated with phytopathogenic strains of Pseudomonas syringae pathovars. Extraction was by a facilitated diffusion procedure or by collection of intercellular fluid using a centrifugation method. The extracted EPS was purified and characterized. All bacterial pathogens which induced watersoaked lesions on their host leaves, a characteristic of most members of this bacterial group, were found to produce alginic acid (a polymer consisting of varying ratios of mannuronic and guluronic acids). Only trace amounts of bacterial EPS could be isolated from leaves inoculated with a pathovar (pv. syringae) which does not induce the formation of lesions with a watersoaked appearance. Guluronic acid was either present in very low amounts or absent in the alginic acid preparations. All bacterial alginates were acetylated (7-11%). Levan (a fructan) was apparently not produced as an EPS in vivo by any of the pathogens tested. PMID- 16666546 TI - Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942. AB - Intact cells and crude homogenates of high (1% CO(2)) and low dissolved inorganic carbon (C(i)) (30-50 microliters per liter of CO(2)) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of (18)O from CO(2) to H(2)O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low C(i) cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO(2). This activity is most apparent in the light, where (18)O-labeled CO(2) species are being taken up by the cells but the effluxing CO(2) has lost most of its label to water. In the dark, low C(i) cells catalyze the depletion of the (18)O enrichment of CO(2) and this activity is inhibited by both ethoxyzolamide and 2 (trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the C(i) pump and the C(i) pump is proposed as a potential site for the exchange of (18)O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low C(i) cells. This was inhibited by ethoxyzolamide with an I(50) of 50 to 100 micromolar in both low and high C(i) cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg(2+) in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction. PMID- 16666547 TI - CO(2) and O(2) Exchanges in the CAM Plant Ananas comosus (L.) Merr: Determination of Total and Malate-Decorboxylation-Dependent CO(2)-Assimilation Rates; Study of Light O(2)-Uptake. AB - Photosynthesis and light O(2)-uptake of the aerial portion of the CAM plant Ananas comosus (L.) merr. were studied by CO(2) and O(2) gas exchange measurements. The amount of CO(2) which was fixed during a complete day-night cycle was equal to the amount of total net O(2) evolved. This finding justifies the assumption that in each time interval of the light period, the difference between the rates of net O(2)-evolution and of net light atmospheric CO(2)-uptake give the rates of malate-decarboxylation-dependent CO(2) assimilation. Based upon this hypothesis, the following photosynthetic characteristics were observed: (a) From the onset of the light to midphase IV of CAM, the photosynthetic quotient (net O(2) evolved/net CO(2) fixed) was higher than 1. This indicates that malate decarboxylation supplied CO(2) for the photosynthetic carbon reduction cycle during this period. (b) In phase III and early phase IV, the rate of CO(2) assimilation deduced from net O(2)-evolution was 3 times higher than the maximum rate of atmospheric CO(2)-fixation during phase IV. A conceivable explanation for this stimulation of photosynthesis is that the intracellular CO(2)-concentration was high because of malate decarboxylation. (c) During the final hours of the light period, the photosynthetic quotient decreased below 1. This may be the result of CO(2)-fixation by phosphoenolpyruvate-carboxylase activity and malate accumulation. Based upon this hypothesis, the gas exchange data indicates that at least 50% of the CO(2) fixed during the last hour of the light period was stored as malate. Light O(2)-uptake determined with (18)O(2) showed two remarkable characteristics: from the onset of the light until midphase IV the rate of O(2) uptake increased progressively; during the following part of the light period, the rate of O(2)-uptake was 3.5 times higher than the maximum rate of CO(2) uptake. When malate decarboxylation was reduced or suppressed after a night in a CO(2)-free atmosphere or in continuous illumination, the rate of O(2)-uptake was higher than in the control. This supports the hypothesis that the low rate of O(2)-uptake in the first part of the light period is due to the inhibition of photorespiration by increased intracellular CO(2) concentration because of malate decarboxylation. In view of the law of gas diffusion and the kinetic properties of the ribulose-1,5-bisphosphate carboxylase/oxygenase, O(2) and CO(2) gas exchange suggest that at the end of the light period the intracellular CO(2) concentration was very low. We propose that the high ratio of O(2)-uptake/CO(2) fixation is principally caused by the stimulation of photorespiration during this period. PMID- 16666548 TI - Diamine Oxidase Activity in Different Physiological Stages of Helianthus tuberosus Tuber. AB - Diamine oxidase (DAO, EC 1.4.3.6) activity was examined in relation to polyamine content in Helianthus tuberosus L. during the first synchronous cell cycle induced in vitro by 2,4,-dichloro-phenoxyacetic acid in tuber slices and during the in vivo formation of the tuber. The optimal pH, buffer and dithiothreitol concentrations for the enzyme extraction and assay were determined. When added in the assay mixture, catalase enhanced DAO activity, while polyvinylpyrrolidone had no effect; both aminoguanidine and hydrazine inhibited enzyme activity. The time course of the reaction, based on the recovery of Delta(1)-pyrroline from labeled putrescine in lipophilic solvents, showed that it was linear up to 30 minutes; the K(m) of the enzyme for putrescine was of the order of 10(-4) molar. During the first cell cycle, DAO activity exhibited a peak at 15 hours of activation while putrescine content gave a peak at 12 hours. During tuber formation (from August till October) DAO activity was relatively high during the first phase of growth (cell division), decreased until flowering (end of September-early October), and then newly increased during the cell enlargement phase preceding the entry into dormancy (November). Maximum putrescine content was observed at the end of October. The increase in DAO activity paralleled the accumulation of putrescine. This indicates a direct correlation between the biosynthesis and oxidation of putrescine which, as already demonstrated in animal systems, occur simultaneously in physiological stages of intense metabolism such as cell division or organ formation. PMID- 16666549 TI - Quantification of cytokinin o-glucosides by negative ion mass spectrometry. AB - Pulsed positive ion-negative ion chemical ionization mass spectra of O-glucosyl zeatin and -dihydrozeatin, their ribosides, and their N-9 2-cyanoethyl and 2 chloro-2-cyanoethyl derivatives are reported. By methods based on these spectra, the levels of the glucosides were determined in soybean (Glycine max) leaves. PMID- 16666550 TI - Occurrence of H(2)-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus. AB - Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H(2) uptake by bacteroids and the relative efficiency of N(2) fixation (RE = 1 - [H(2) evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H(2)-uptake activity in symbiosis with O. compressus. The inability of these putative Hup(+) strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains. PMID- 16666551 TI - Hormonal characterization of a nonrooting naphthalene-acetic Acid tolerant tobacco mutant by an immunoenzymic method. AB - The comparative analysis of plant hormones was undertaken on a 1 naphthaleneacetic acid tolerant mutant and normal tobacco (Nicotiana tabacum cv Xanthi) plantlets. The mutant plantlet was scrubby and impaired in its root morphogenesis. Degeneration of the root meristem was studied on tissue sections; it appeared very fast (as early as the 3rd or 4th day after sowing), after which the root was further transformed into a callus. Indoleacetic acid (IAA), abscisic acid (ABA), and the isopentenyladenine (iP)- and trans-zeatin(Z)-type cytokinin levels were measured in terminal buds and root tips 13 days after sowing, by enzyme linked immunosorbent assay of high performance liquid chromatography fractions. Some differences appeared between the apical buds of the two genotypes, but the mutant tobacco differed from the wild type mainly by the presence of higher levels of IAA, ABA, and iP + isopentenyladenosine (iPA) in its small root. Thus, the IAA, ABA, and iP + iPA contents were increased by a factor of 15, 7, and 24 times, respectively, in mutant root compared to wild-type tobacco roots. Previous work has shown that the mutation impairs membrane polarization effects induced by auxin at the cell level. The present results would favor the hypothesis that the mutation has also affected the control of growth regulator accumulation in tissues. PMID- 16666552 TI - Mechanism of Photosynthetic Carbon Dioxide Uptake by the Red Macroalga, Chondrus crispus. AB - The aim of this study was to determine how Chondrus crispus, a marine red macroalga, acquires the inorganic carbon (C(i)) it utilizes for photosynthetic carbon fixation. Analyses of C(i) uptake were done using silicone oil centrifugation (using multicellular fragments of thallus), infrared gas analysis, and gas chromatography. Inhibitors of carbonic anhydrase (CA), the band 3 anion exchange protein and Na(+)/K(+) exchange were used in the study. It was found that: (a) C. crispus does not accumulate C(i) internally above the concentration attainable by diffusion; (b) the initial C(i) fixtion rate of C. crispus fragments saturates at approximately 3 to 4 millimolar C(i); (c) CA is involved in carbon uptake; its involvement is greatest at high HCO(3) (-) and low CO(2) concentration, suggesting its participation in the dehydration of HCO(3) (-) to CO(2); (d) C. crispus has an intermediate C(i) compensation point; and (e) no evidence of any active or facilitated mechanism for the transport of HCO(3) (-) was detected. These data support the view that photosynthetic C(i) uptake does not involve active transport. Rather, CO(2), derived from HCO(3) (-) catalyzed by external CA, passively diffuses across the plasma membrane of C. crispus. Intracellular CA also enhances the fixation of carbon in C. crispus. PMID- 16666553 TI - Repeated measurements of aperture for individual stomates. AB - Measurements of stomatal aperture in epidermal peels are used in a variety of physiological studies, but variability between stomates often hinders experimentation. We mount epidermal peels of Vicia faba over a microscopic grid that enables us to map the positions of individual stomates and thus measure the same individual stomates repeatedly, using a simple digitizing system. Repeated measurements of the same population of 15 stomates show much lower variability than measurements of different populations either on the same peel or on different peels. PMID- 16666554 TI - Immunocytochemical Localization of the Vacuolar H-ATPase in Maize Root Tip Cells. AB - The vacuolar H(+)-ATPase of maize (Zea mays L.) root tip cells has been localized at the EM level using rabbit polyclonal antibodies to the 69 kilodalton subunit and protein A-colloidal gold. Intracellular gold particles were detected mainly on the tonoplast and Golgi membranes. Only about 27% of the vacuoles were labeled above background. The absence of gold particles on the majority of vacuoles suggests either that the tonoplast H(+)-ATPase is degraded during tissue preparation or that the small vacuoles of root tip cells are specialized with respect to H(+)-ATP ase activity. The pattern of gold particles on the labeled vacuoles ranged from uniform to patchy. Virtually all of the Golgi bodies were labeled by the antibody, but the particle densities were too low to determine whether the H(+)-ATPase was associated with specific regions, such as the trans face. Cell wall-labeling was also observed which could be partially prevented by the inclusion of gelatin as a blocking agent. The immunocytochemical results confirm previous biochemical studies with isolated membrane fractions (A Chanson, L Taiz 1985 Plant Physiol 78: 232-240). PMID- 16666555 TI - Photosynthesis, Carbohydrate Metabolism, and Export in Beta vulgaris L. and Phaseolus vulgaris L. during Square and Sinusoidal Light Regimes. AB - Rates of photosynthesis, sucrose synthesis, starch accumulation and degradation were measured in sugar beet (Beta vulgaris L.) and bean (Phaseolus vulgaris L.) plants under a square-wave light regime and under a sinusoidal regime that simulated the natural daylight period. Photosynthesis rate increased in a measured manner in direct proportion to the increasing light level. In contrast to this close correspondence between photosynthesis and light, a lag in photosynthesis rate was seen during the initial hour under square-wave illumination. The leaf appeared to be responding to limits set by carbon metabolism rather than by gas exchange or light reactions. Under the sinusoidal regime starch degradation occurred during the first and last 2 hours of the photoperiod, likely in response to photosynthesis rate rather than directly to light level. Starch broke down when photosynthesis was below a threshold rate and accumulated above this rate. Under square-wave illumination, accumulation of starch did not begin until irradiance was at full level for an hour or more and photosynthesis was at or near its maximum. Under a sinusoidal light regime, sucrose synthesis rate comprised carbon that was newly fixed throughout the day plus that from starch degradation at the beginning and end of the day. Synthesis of sucrose from recently fixed carbon increased with increasing net carbon fixation rate while its formation from degradation of starch decreased correspondingly. The complementary sources of carbon maintained a relatively steady rate of sucrose synthesis under the changing daytime irradiance. PMID- 16666556 TI - Leaf Carbon Metabolism and Metabolite Levels during a Period of Sinusoidal Light. AB - Photosynthesis rate, internal CO(2) concentration, starch, sucrose, and metabolite levels were measured in leaves of sugar beet (Beta vulgaris L.) during a 14-h period of sinusoidal light, which simulated a natural light period. Photosynthesis rate closely followed increasing and decreasing light level. Chloroplast metabolite levels changed in a manner indicating differential activation of enzymes at different light levels. Starch levels declined during the first and last 2 hours of the photoperiod, but increased when photosynthesis rate was greater than 50% of maximal. Sucrose and sucrose phosphate synthase levels were constant during the photoperiod, which is consistent with a relatively steady rate of sucrose synthesis during the day as observed previously (BR Fondy et al. [1989] Plant Physiol 89: 396-402). When starch was being degraded, glucose 1-phosphate level was high and there was a large amount of glucose 6-phosphate above that in equilibrium with fructose 6-phosphate, while fructose 6-phosphate and triose-phosphate levels were very low. Likewise, the regulatory metabolite, fructose, 2,6-bisphosphate was high, indicating that little carbon could move to sucrose from starch by the triose-phosphate pathway. These data cast doubt upon the feasibility of significant carbon flow through the triose-phosphate pathway during starch degradation and support the need for an additional pathway for mobilizing starch carbon to sucrose. PMID- 16666557 TI - Mitochondrial Respiration Can Support NO(3) and NO(2) Reduction during Photosynthesis : Interactions between Photosynthesis, Respiration, and N Assimilation in the N-Limited Green Alga Selenastrum minutum. AB - Mass spectrometric analysis shows that assimilation of inorganic nitrogen (NH(4) (+), NO(2) (-), NO(3) (-)) by N-limited cells of Selenastrum minutum (Naeg.) Collins results in a stimulation of tricarboxylic acid cycle (TCA cycle) CO(2) release in both the light and dark. In a previous study we have shown that TCA cycle reductant generated during NH(4) (+) assimilation is oxidized via the cytochrome electron transport chain, resulting in an increase in respiratory O(2) consumption during photosynthesis (HG Weger, DG Birch, IR Elrifi, DH Turpin [1988] Plant Physiol 86: 688-692). NO(3) (-) and NO(2) (-) assimilation resulted in a larger stimulation of TCA cycle CO(2) release than did NH(4) (+), but a much smaller stimulation of mitochondrial O(2) consumption. NH(4) (+) assimilation was the same in the light and dark and insensitive to DCMU, but was 82% inhibited by anaerobiosis in both the light and dark. NO(3) (-) and NO(2) (-) assimilation rates were maximal in the light, but assimilation could proceed at substantial rates in the light in the presence of DCMU and in the dark. Unlike NH(4) (+), NO(3) (-) and NO(2) (-) assimilation were relatively insensitive to anaerobiosis. These results indicated that operation of the mitochondrial electron transport chain was not required to maintain TCA cycle activity during NO(3) (-) and NO(2) (-) assimilation, suggesting an alternative sink for TCA cycle generated reductant. Evaluation of changes in gross O(2) consumption during NO(3) (-) and NO(2) (-) assimilation suggest that TCA cycle reductant was exported to the chloroplast during photosynthesis and used to support NO(3) (-) and NO(2) (-) reduction. PMID- 16666558 TI - Genetic Variability in Carbon Fixation, Sucrose-P-Synthase and ADP Glucose Pyrophosphorylase in Maize Plants of Differing Growth Rate. AB - The net photosynthetic rate and the activities of ribulose 1,5 bisphosphate carboxylase (RubisCo), phosphoenolpyruvate carboxylase, sucrose-P-synthase, and ADP glucose-pyrophosphorylase, key enzymes of the leaf carbohydrate metabolism were compared in eight maize (Zea mays L.) genotypes presenting large differences in growth rate. The sucrose-P-synthase activity varied in the ratio 1 to 3 from the less active to the more active genotype and this variation was highly correlated with those in growth rate. ADP glucose pyrophosphorylase activity was not significantly different from one genotype to another whatever the basis for expression, leaf area, or soluble protein. The photosynthetic rate varied with similar amplitude (1:1) to the RubisCo activity or RubisCo quantity but the correlation with growth rate was highly significant for photosynthesis and nonsignificant for RubisCo or phosphoenolpyruvate carboxylase. So, in our series of genotypes the sucrose synthesis capacities as expressed by sucrose phosphate synthase activity seem to have a good predicting value for mean growth rate at a young stage. PMID- 16666559 TI - Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides. AB - Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells. PMID- 16666560 TI - Behavior of Free Aromatic Amino Acid Pools in Rosmarinic Acid-Producing Cell Cultures of Anchusa officinalis L. AB - The pool sizes of free l-phenylalanine and l-tyrosine, the precursors of rosmarinic acid in Anchusa officinalis L. cell suspension cultures, fluctuated during the culture cycle. The major increase in pool sizes was preceded by a peak of prephenate aminotransferase activity, while the subsequent decrease coincided with the presence of high activities of phenylalanine ammonia-lyase and tyrosine aminotransferase, the two entrypoint enzymes of the rosmarinic acid biosynthesis pathway. Timecourse feeding studies with linear growth stage cells revealed that the tyrosine pool turned over rapidly, consistent with direct participation in rosmarinic acid synthesis. Since externally applied l-tyrosine was rapidly incorporated into rosmarinic acid with little evidence of radioactively labeled intermediates, it is suggested that there exists a close coupling between the l tyrosine pool and the rosmarinic acid biosynthetic pathway, which may involve the channelling of intermediates both into and within the pathway. PMID- 16666561 TI - Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene. AB - The inhibitory effects of the cyclic olefin 2,5-norbornadiene (NBD) on ethylene action were tested in carnation (Dianthus caryophyllus L. cv White Sim) flowers. Treatment of flowers at anthesis with ethylene in the presence of 500 microliters per liter NBD increased the concentration of ethylene required to elicit a response (petal senescence), indicating that NBD behaves as a competitive inhibitor of ethylene action. Transfer of flowers producing autocatalytic ethylene and exhibiting evidence of senescence (petal in-rolling) to an atmosphere of NBD resulted in a rapid reduction in ethylene production, petal 1 aminocyclopropane-1-carboxylic acid synthase activity, 1-aminocyclopropane-1 carboxylic acid content, and ethylene forming enzyme activity. Removal of NBD resulted in recovery of ethylene biosynthesis. These results support the autocatalytic regulation of ethylene production during the climacteric stage of petal senescence and suggest that continued perception of ethylene is required for maintenance of ethylene biosynthesis. The inhibition of ethylene action by NBD after the flowers had reached the climacteric peak was associated with interruption of petal senescence as evidenced by reversal of senescence symptoms. This result is in contrast to the widely held belief that the rate of petal senescence is fixed and irreversible once petals enter into the ethylene climacteric. PMID- 16666562 TI - Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid. AB - In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using (13)[C(6)]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 +/- 6.5 versus 166.6 +/- 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 +/- 4.4 versus 263.7 +/- 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base. PMID- 16666563 TI - Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration. AB - Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT(50)) of -2.8 to -3.8 degrees C. In contrast, citrus leaves cold acclimated at a constant 10 degrees C in growth chambers were freezing tolerant to about -6 degrees C. The calculated freezing induced cellular dehydration at the LT(50) remained relatively constant for field grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10 degrees C in a controlled environment. Spinach leaves cold acclimated at 5 degrees C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about -1 degrees C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration. PMID- 16666564 TI - Stabilization of Isolated Photosystem II Reaction Center Complex in the Dark and in the Light Using Polyethylene Glycol and an Oxygen-Scrubbing System. AB - The photosystem II reaction center as isolated (O Nanba, K Satoh [1987] Proc Natl Acad Sci USA 84: 109-112) is quite dilute and very unstable. Precipitating the complex with polyethylene glycol and resuspending it in buffer without detergent concentrates the reaction center and greatly improves its stability at 4 degrees C in the dark as judged by light-induced electron transport activity. Furthermore, a procedure was developed to minimize photodestruction of polyethylene-glycol-concentrated material at room temperature in the light. The ability to stabilize the photosystem II reaction center should facilitate future photophysical, biochemical, and structural studies of the complex. PMID- 16666565 TI - Effects of Altered Carbohydrate Availability on Whole-Plant Assimilation of NO(3). AB - An experiment was conducted to investigate the relative changes in NO(3) (-) assimilatory processes which occurred in response to decreasing carbohydrate availability. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were exposed to 1.0 millimolar (15)NO(3) (-) for 6 hour intervals during a normal 12 hour light period and a subsequent period of darkness lasting 42 hours. Uptake of (15)NO(3) (-) decreased to 71 to 83% of the uptake rate in the light during the initial 18 hours of darkness; uptake then decreased sharply over the next 12 hours of darkness to 11 to 17% of the light rate, coincident with depletion of tissue carbohydrate reserves and a marked decline in root respiration. Changes also occurred in endogenous (15)NO(3) (-) assimilation processes, which were distinctly different than those in (15)NO(3) ( ) uptake. During the extended dark period, translocation of absorbed (15)N out of the root to the shoot varied rhythmically. The adjustments were independent of (15)NO(3) (-) uptake rate and carbohydrate status, but were reciprocally related to rhythmic adjustments in stomatal resistance and, presumably, water movement through the root system. Whole plant reduction of (15)NO(3) (-) always was limited more than uptake. The assimilation of (15)N into insoluble reduced-N in roots remained a constant proportion of uptake throughout, while assimilation in the shoot declined markedly in the first 18 hours of darkness before stabilizing at a low level. The plants clearly retained a capacity for (15)NO(3) (-) reduction and synthesis of insoluble reduced-(15)N even when (15)NO(3) (-) uptake was severely restricted and minimal carbohydrate reserves remained in the tissue. PMID- 16666566 TI - Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes. AB - Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO(3) or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used. PMID- 16666567 TI - Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L. AB - Chromatography on DEAE cellulose equilibrated with Pipes buffer resolved three forms of chorismate mutase (CM) from tubers and leaves of Solanum tuberosum: CM 1A and CM-1B were activated by tryptophan and inhibited by phenylalanine and tyrosine; CM-2 was unaffected by these aromatic amino acids. When compared to freshly excised discs, 3 day old tuber discs demonstrated a 4.5-fold increase in CM-1 activity following wounding. By contrast, CM-2 activity levels were not affected by this treatment. In aged tuber discs the CM-1:CM-2 activity ratio was 9:1. However, in green leaves the CM-1:CM-2 activity ratio was 1:4 suggesting organ specific regulation for the expression of these isozymes. The CM-1 isozymes isolated from both tubers and leaves shared similar native molecular weight values of 55,000, K(m) values of 40 to 56 micromolar, and inhibition by phenylalanine (110-145 micromolar concentrations required for 50% inhibition) and tyrosine (50-70 micromolar concentrations required for 50% inhibition). The resolution of CM-1 into two forms occurred only in the presence of Pipes buffer. When this buffer was replaced with Aces, Bes, imidazole or Tris, only a single peak of CM-1 activity was observed. In these buffers CM-2 eluted as a shoulder on the CM-1 peak. Analytical isoelectric focusing of the CM-1 fraction followed by assay of the gel yielded only one form of CM-1 with an isoelectric point of 5.0. Gel filtration studies with Pipes buffer yielded molecular weights of 60,000 for both CM-1A and CM-1B indicating these forms are not the result of aggregation. The two forms of CM-1 may be artifacts generated by Pipes buffer. PMID- 16666568 TI - Tomato Fruit Cell Wall Synthesis during Development and Senescence : In Vivo Radiolabeling of Wall Fractions Using [C]Sucrose. AB - The pedicel of tomato fruit (Lycopersicon esculentum Mill., cv ;Rutgers') of different developmental stages from immature-green (IG) to red was injected on the vine with 7 microcuries [(14)C(U)]sucrose and harvested after 18 hours. Cell walls were isolated from outer pericarp and further fractionated yielding ionically associated pectin, covalently bound pectin, hemicellulosic fraction I, hemicellulosic fraction II, and cellulosic fraction II. The dry weight of the total cell wall and of each cell wall fraction per gram fresh weight of pericarp tissue decreased after the mature-green (MG) stage of development. Incorporation of radiolabeled sugars into each fraction decreased from the IG to MG3 (locules jellied but still green) stage. Incorporation in all fractions increased from MG3 to breaker and turning (T) and then decreased from T to red. Data indicate that cell wall synthesis continues throughout ripening and increases transiently from MG4 (locules jellied and yellow to pink in color) to T, corresponding to the peak in respiration and ethylene synthesis during the climacteric. Synthesis continued at a time when total cell wall fraction dry weight decreased indicating the occurrence of cell wall turnover. Synthesis and insertion of a modified polymer with removal of other polymers may produce a less rigid cell wall and allow softening of the tissue integrity during ripening. PMID- 16666569 TI - A Gibberellin-Deficient Brassica Mutant-rosette. AB - A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and internode growth. The exogenous application of GA(3) by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances (;Tan-ginbozu' dwarf rice assay) of about one tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA(1) and GA(3) upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA(1) and GA(3) conjugates were present, but additionally, free GA(1) and GA(3) were abundant and identified by gas chromatography-mass spectrometry selected ion monitoring. The quantities of free GA(1) and GA(3) in the normal line and in rosette were quantified by GC-MS-SIM using [(2)H(2)]GA(1) as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA(1) per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA(1) and GA(3), while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA(1), or GA(3), than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA(1) and GA(3) in the regulation of these processes in Brassica. PMID- 16666570 TI - Hydroxycinnamoyl-CoA:Putrescine Hydroxycinnamoyltransferase in Tobacco Cell Cultures with High and Low Levels of Caffeoylputrescine. AB - A new hydroxycinnamoyl-CoA:putrescine hydroxycinnamoyltransferase (PHT) was detected in two variant lines of Nicotiana tabacum L. (TX1, TX4) accumulating markedly different levels of caffeoylputrescine. The enzyme accepted only the aliphatic diamines putrescine, cadaverine and 1,3-diaminopropane at a ratio of 100:33:8. Caffeoyl- and feruloyl-CoAs were the best acyl donors. The apparent K(m)-values for caffeoyl-CoA and putrescine were near 3 and 10 micromolar, respectively, at the pH-optimum of 10.0. PHT activity was quite similar in low producing TX1 and high producing TX4 cells, while some other biosynthetic enzymes (phenylalanine ammonia-lyase, ornithine decarboxylase) were greatly enhanced in TX4 cells, suggesting that PHT does not catalyze the rate-limiting step in hydroxycinnamoylputrescine formation. PMID- 16666571 TI - Evidence for a phytochrome-mediated phototropism in etiolated pea seedlings. AB - Entirely etiolated pea seedlings (Pisum sativum, L. cv Alaska) were tested for a phototropic response to short pulses of unilateral blue light. They responded with small curvatures resembling in fluence-dependence and kinetics of development a phytochrome-mediated phototropic response previously described in maize mesocotyls. Irradiations from above with saturating red or far-red light, either immediately before or after the unilateral phototropic stimulus, strongly reduced or eliminated subsequent positive phototropic curvature. Only blue light from above, however, entirely eliminated curvature at all fluences of stimulus. It is concluded that the phototropism is primarily a result of phytochrome action. PMID- 16666572 TI - Photorespiratory ammonia does not inhibit photosynthesis in glutamate synthase mutants of Arabidopsis. AB - Exposure of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mutants of Arabidopsis thaliana to photorespiratory conditions resulted in the accumulation of NH(4) (+) and the inhibition of photosynthesis. However, upon transfer from 2% O(2), 350 microliters per liter CO(2), to 21% O(2), 350 microliters per liter CO(2), net photosynthesis declined at a slower rate in methionine sulfoximine treated leaf discs relative to controls. The recovery of photosynthesis was also more rapid in MSO-treated leaf discs although ammonia levels were more than threefold higher. Photosynthesis in leaf discs treated with azaserine was inhibited more than controls when transferred to 21% O(2) and recovered less than controls when returned to 2% O(2) although NH(4) (+) levels were not significantly different. The results obtained are consistent with the view that the rapid inhibition of photosynthesis in the glutamate synthase mutants in photorespiratory conditions is not due to the accumulation of NH(4) (+) but rather to the depletion of amino donors for glyoxylate and the consequent effects of glyoxylate on the lack of return of carbon to the chloroplast. PMID- 16666573 TI - Purification and Characterization of Calmodulins from Papaver somniferum and Euphorbia lathyris. AB - Calmodulins (CaM) were isolated and characterized from two well-known latex producing plants, Papaver somniferum and Euphorbia lathyris. The molecular weights of both were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 17,000 comparable to that of bovine brain CaM. Amino acid compositions also compared similarly with those of known CaMs, with regard to the presence of trimethyllysine and the ratio of phenylalanine to tyrosine. The Cornish-Bowden equation (SDeltan) revealed strong statistical correlations of P. somniferum and E. lathyris CaM with those of other plants and animals, although their amino acid compositions were not identical. Both plant CaM stimulated CaM dependent cAMP phosphodiesterase: for Papaver somniferum the K(a) was found to be 1.09 nanomolar and for Euphorbia lathyris, 2.01 nanomolar. PMID- 16666574 TI - Stimulation of Isopentenyl Pyrophosphate Incorporation into Polyisoprene in Extracts from Guayule Plants (Parthenium argentatum Gray) by Low Temperature and 2-(3,4-Dichlorophenoxy) Triethylamine. AB - Rubber particles isolated from guayule (Parthenium argentatum Gray) stem homogenates contain a polyprenyl transferase which catalyzes the polymerization of isopentenyl pyrophosphate into polyisoprene. The polymerization reaction is stimulated with the addition of an allylic pyrophosphate initiator and forms a polymer of polyisoprene with a molecular weight distribution from 10(3) to 10(7). The polymerization reaction in crude stem homogenates is not affected by the addition of an initiator probably due to the high activity of isopentenyl pyrophosphate isomerase furnishing saturating levels of dimethylallyl pyrophosphate. Polyisoprene formation in stems of guayule plants exposed to cold winter temperatures increased from 15.4 milligrams per gram dry weight in October to 24.5 milligrams per gram dry weight in January and increased from 16.2 to 38.1 milligrams per gram dry weight in the same period by additionally treating the plants with 5000 ppm of 2-(3,4-dichlorophenoxy)triethylamine. The rate of polymerization of isopentenyl pyrophosphate into polyisoprene in stem homogenates of the cold treated plants increased from 12.1 nanomoles per hour per gram fresh weight in October to 144.3 nanomoles per hour per gram fresh weight in January and increased from 17.7 to 446.8 nanomoles per hour per gram fresh weight in the same period by additionally treating the plants with 5000 ppm of 2-(3,4 dichlorophenoxy)triethylamine. These results show that the increase in polyprenyl transferase activity partially accounts for the increase in polyisoprene synthesis in guayule plants exposed to low temperature and treated with 2-(3,4 dichlorophenoxy)triethylamine. PMID- 16666575 TI - Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies. AB - Monoclonal antibodies specific for sucrose phosphate synthase (SPS; EC 2.4.1.14) have been obtained for the first time. Three independent clones have been isolated which inhibited spinach (Spinacia oleracea L.) leaf SPS activity and facilitated the enzyme purification by immunoprecipitation. All three clones were specific for the spinach enzyme but neither inhibited nor precipitated the SPS present in tissue extracts of maize (Zea mays L.), barley (Hordeum vulgare L.), soybean (Glycine max L.), and sugar beet (Beta vulgaris L.). The inhibition of SPS activity by all three clones was reversible in the presence of UDPG, suggesting the presence of an epitope at the substrate-binding site. Immunoprecipitates of active enzyme preparations consistently revealed the presence of a 120 kilodalton polypeptide, indicating that the enzyme may be a homotetramer with a native molecular weight of about 480 kilodaltons. The occasional appearance of a 52 kilodalton polypeptide in the immunoprecipitates of some enzyme preparations was not the result of proteolysis, was not necessary for enzyme activity, and did not contain an antigenic site as revealed by Western blotting experiments. All three antibodies bind weakly to the SDS denatured 120 kilodalton subunit bound to nitrocellulose. The specific activity of the purified spinach enzyme was determined for the first time to be approximately 150 units per milligram SPS protein (pH 7.5 and 25 degrees C) based on quantitative immunoprecipitation of the enzyme. PMID- 16666576 TI - Detection of Norspermidine and Norspermine in Medicago sativa L. (Alfalfa). AB - Shoot meristem tissues of alfalfa, Medicago sativa L., were found by high performance liquid chromatography analyses to contain the uncommon polyamines, norspermidine and norspermine. The chemical structures of norspermidine and norspermine, purified from alfalfa, were confirmed by comparison of mass spectra with those from authentic standards. The discovery of norspermidine and norspermine in alfalfa implicates the presence of at least two biosynthetic enzymes, a polyamine oxidase and a previously uncharacterized aminopropyltransferase. PMID- 16666577 TI - Involvement of Calcium in the Photoperiodic Flower Induction Process of Pharbitis nil. AB - EGTA, a specific Ca(2+) chelator, inhibited the flowering response of Pharbitis nil when applied to the cotyledons immediately before the inductive dark period. Calcium sprayed 30 minutes after the EGTA blocked the effect of EGTA. The length of the critical dark period was increased both by EGTA and by LaCl(3). The calmodulin antagonists W-7 and chlorpromazine also reduced the flowering response. On the other hand, A23187, a calcium ionophore, increased the flowering response. Both EGTA and A23187 were effective at certain times of the photoperiod but had almost no effect when applied at other times. The results indicate that the level of endogenous Ca(2+) may be limiting for floral induction in Ph. nil. Ca(2+) seems to play a role during the early stages of the inductive dark period. PMID- 16666578 TI - Evidence for a gibberellin biosynthetic origin of ceratopteris antheridiogen. AB - The species-specific chemical messenger, antheridiogen A(Ce), mediates the differentiation of male gametophytes in the fern Ceratopteris. In order to investigate the biochemical origin of antheridiogen, the effect of the inhibitors, 2'-isopropyl-4'-(trimethylammoniumchloride)-5' methylphenylpiperidine-1-carboxylate (AMO-1618), 2-chloroethyl trimethylammonium chloride (CCC), and alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) on gametophytic sex expression was determined in C. richardii. Both AMO-1618 and ancymidol blocked the production of male gametophytes in three genetically defined strains of C. richardii that exhibit different sensitivities to antheridiogen. Antheridiogen supplementation overcame inhibition by AMO-1618 and ancymidol, except in one strain (HaC18) that is insensitive to antheridiogen supplementation. These data suggest that the synthesis of Ceratopteris antheridiogen, a taxon that is insensitive to exogenously supplied gibberellins, occurs via a pathway that may include steps in common with gibberellin biosynthesis or involves similar reactions. PMID- 16666579 TI - Effect of sodium nutrition on the ultrastructure of chloroplasts of c(4) plants. AB - Mesophyll chloroplasts from sodium-deficient compared to normal plants of the C(4) species Kochia childsii and Amaranthus tricolor were found to have significantly less stacking in their grana. On the other hand, no marked difference of thylakoid arrangement between bundle sheath chloroplasts from sodium-deficient and normal plants of A. tricolor were observed. PMID- 16666580 TI - Blue-Light Regulation of Epicotyl Elongation in Pisum sativum. AB - Blue light is known to induce suppression of stem elongation. To avoid the complication of blue-light-induced transformation of phytochrome we have adapted the procedure of measuring blue-light-induced suppression of stem elongation in Pisum sativum L. var Alaska grown under continuous red light. The resulting fluence-response curve for suppression of epicotyl elongation measured twenty four hours after a blue-light treatment is bell-shaped, with the peak of suppression between 10(0) and 10(1) micromoles per square meter, and no suppression at 10(4) micromoles per square meter. Suppression is first observed 5 and 11 hours after the blue-light treatment for the fourth and third internodes, respectively. No significant differences in elongation rates were noted for the 10(4) micromoles per square meter treated seedlings throughout the 24 hour period. Reciprocity holds for both third and fourth internodes in response to 10(1) and 10(4) micromoles per square meter of blue light over the range of irradiation times tested (10(0) to 10(4) seconds, 10(1) micromoles per square meter; 10(0) to 10(3) seconds, 10(4) micromoles per square meter). In contrast to the bell-shaped fluence-response obtained for epicotyl elongation, measurements of chlorophyll and carotenoid accumulation indicate increasing accumulation with increasing fluence. PMID- 16666581 TI - Effects of nitrogen on mesophyll cell division and epidermal cell elongation in tall fescue leaf blades. AB - Leaf elongation rate (LER) in grasses is dependent on epidermal cell supply (number) and on rate and duration of epidermal cell elongation. Nitrogen (N) fertilization increases LER. Longitudinal sections from two genotypes of tall fescue (Festuca arundinacea Schreb.), which differ by 50% in LER, were used to quantify the effects of N on the components of epidermal cell elongation and on mesophyll cell division. Rate and duration of epidermal cell elongation were determined by using a relationship between cell length and displacement velocity derived from the continuity equation. Rate of epidermal cell elongation was exponential. Relative rates of epidermal cell elongation increased by 9% with high N, even though high N increased LER by 89%. Duration of cell elongation was approximately 20 h longer in the high- than in the low-LER genotype regardless of N treatment. The percentage of mesophyll cells in division was greater in the high- than in the low-LER genotype. This increased with high N in both genotypes, indicating that LER increased with cell supply. Division of mesophyll cells adjacent to abaxial epidermal cells continued after epidermal cell division stopped, until epidermal cells had elongated to a mean length of 40 micrometers in the high-LER and a mean length of 50 micrometers in the low-LER genotype. The cell cycle length for mesophyll cells was calculated to be 12 to 13 hours. Nitrogen increased mesophyll cell number more than epidermal cell number: in both genotypes, the final number of mesophyll cells adjacent to each abaxial epidermal cell was 10 with low N and 14 with high N. A spatial model is used to describe three cell development processes relevant to leaf growth. It illustrates the overlap of mesophyll cell division and epidermal cell elongation, and the transition from epidermal cell elongation to secondary cell wall deposition. PMID- 16666582 TI - Evidence for a light dependent increase of phosphoglucomutase activity in isolated, intact spinach chloroplasts. AB - Phosphoglucomutase (PGM) activity was measured in spinach (Spinacia oleracea L.) chloroplasts. Initial enzyme activity in a chloroplast lysate was 5 to 10% of total activity measured with 1 micromolar glucose 1,6-bisphosphate (Glc 1,6-P(2)) in the assay. Initial PGM activity increased 2- to 3-fold when chloroplasts were illuminated for 10 minutes prior to enzyme measurement and then decreased slowly in the dark. Measurements of total enzyme activity were unchanged by prior light treatment. Initial PGM activity from light treated chloroplasts was sufficient to account for in vivo rates of starch synthesis. Changes in PGM activity were affected by stromal pH and orthophosphate concentration. Photosynthetic inhibitors, dl-glyceraldehyde, glycolaldehyde, and glyoxylate, decreased and 3 phosphoglyceric acid increased light induced changes of PGM activity. Dark preincubation of chloroplasts with 10 millimolar dithiothreitol had no effect upon initial PGM activity, suggesting that light effects did not involve a sulfhydryl mechanism. Hexose monophosphate levels increased in illuminated chloroplasts. Activation of PGM in a chloroplast lysate by Glc 1,6-P(2) was maximal between pH 7.5 and 8.5. Stromal concentrations of Glc 1,6-P(2) were between 20 and 30 micromolar for both light and dark incubated chloroplasts and these levels should saturate PGM activity. Light dependent alterations of enzyme activity may be due to changes of phosphorylated PGM levels in the stroma or are the result of changes in residual activity by the dephosphorylated form of the enzyme. The above results indicate that PGM activity in spinach chloroplasts may be regulated by light, stromal pH, and Glc 1,6-P(2) concentration. PMID- 16666583 TI - Cytokinin antagonist activity of substituted phenethylamines in plant cell culture. AB - A series of structurally related substituted phenethylamines shows extreme toxicity toward wild-type callus tissue cultures of tobacco (Nicotiana tabacum), soybean (Glycine max), corn (Zea mays), and sunflower (Helianthus annuus L.), but tobacco crown gall cultures are resistant to the compounds. The essential components that result in toxicity of the phenethylamines include one aromatic hydroxyl and one primary aliphatic amino group. A series of attenuated crown gall cultures, generated by transformation of tobacco with various modified Agrobacterium strains, has been used to demonstrate that the resistance of crown galls to the phenethylamines is due to the expression in these tissues of isopentenyl transferase, a first step in cytokinin biosynthesis. The toxicity of the compounds to tissue cultures is very rapid, but can be overcome by prior exposure of the calli to exogenous cytokinin. Because of the relationships we have observed between cytokinins and these compounds, we propose that the substituted phenethylamines may represent a class of chemicals that can be used as specific probes to further an understanding of cytokinin metabolism in plant tissues. PMID- 16666584 TI - Thermotolerance is developmentally dependent in germinating wheat seed. AB - During the initial 9 to 12 hours of imbibition, the imbibing wheat (Triticum aestivum L.) seed was found to exhibit substantial tolerance to high temperature relative to later times of imbibition. Tolerance was assessed by seed viability and seedling growth. This initial high temperature tolerance gradually declines with increasing time of seed imbibition. A range of 2 hour heat pretreatments (38 42 degrees C) prior to imposition of a 2 hour heat shock (51-53 degrees C) during this same 9 to 12 hour interval was unable to increase survival or seedling growth over that of seed that did not receive a pretreatment. However, after 9 to 12 hours of imbibition the pretreatment provided both increased survival and increased seedling growth, measured 120 hours later, i.e., classical thermotolerance could be acquired. This response is called a ;thermotolerance transition.' Isolated embryos responded in a similar manner using a 2,3,5 triphenyltetrazolium chloride assay for viability determination following heat treatments. The high temperature tolerance during early imbibition indicates that the thermotolerance transition involves the loss of an existing thermotolerance coincident with acquiring the ability to become thermotolerant following heat pretreatment. Despite the inability to acquire thermotolerance, heat shock protein synthesis was induced by heat shock immediately upon imbibition of wheat seed or isolated embryos. Developmentally regulated heat shock proteins of 58 to 60, 46, 40, and 14 kilodaltons were detected at 1.5 hours of imbibition following heat shock, but were absent or greatly reduced by 12 hours. Constitutive synthesis of 70 and 90 kilodalton hsp groups appeared to be greater at 1.5 hours of imbibition than at 12 hours of imbibition. PMID- 16666585 TI - Synthesis of Freezing Tolerance Proteins in Leaves, Crown, and Roots during Cold Acclimation of Wheat. AB - Protein synthesis was studied in leaves, crown, and roots during cold hardening of freezing tolerant winter wheat (Triticum aestivum L. cv Fredrick and cv Norstar) and freezing sensitive spring wheat (T. aestivum L. cv Glenlea). The steady state and newly synthesized proteins, labeled with [(35)S]methionine, were resolved by one- and two-dimensional polyacrylamide gels. The results showed that cold hardening induced important changes in the soluble protein patterns depending upon the tissue and cultivar freezing tolerance. At least eight new proteins were induced in hardened tissues. A 200 kilodalton (kD) (isoelectric point [pl] 6.85) protein was induced concomitantly in the leaves, crown, and roots. Two proteins were specifically induced in the leaves (both 36 kD, pl 5.55 and 5.70); three in the crown with M(r) 150 (pl 5.30), 45 (pl 5.75), and 44 kD (pl > 6.80); and two others in the roots with M(r) 64 (pl 6.20) and 52 kD (pl 5.55). In addition, 19 other proteins were synthesized at a modified rate (increased or decreased) in the leaves, 18 in the crown and 23 in the roots. Among the proteins induced or increased in hardened tissues, some were expressed at a higher level in the freezing tolerant cultivars than in the sensitive one, indicating a correlation between the synthesis and accumulation of these proteins and the degree of freezing tolerance. These proteins, suggested to be freezing tolerance proteins, may have an important role in the cellular adaptation to freezing. PMID- 16666586 TI - Actin and Myosin in pea tendrils. AB - We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K(+)-EDTA and Ca(2+) and is inhibited by Mg(2+). At low ionic strength, the Mg(2+)-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril. PMID- 16666587 TI - Acclimation of Photosynthesis to Elevated CO(2) in Five C(3) Species. AB - The effect of long-term (weeks to months) CO(2) enhancement on (a) the gas exchange characteristics, (b) the content and activation state of ribulose-1,5 bisphosphate carboxylase (rubisco), and (c) leaf nitrogen, chlorophyll, and dry weight per area were studied in five C(3) species (Chenopodium album, Phaseolus vulgaris, Solanum tuberosum, Solanum melongena, and Brassica oleracea) grown at CO(2) partial pressures of 300 or 900 to 1000 microbars. Long-term exposure to elevated CO(2) affected the CO(2) response of photosynthesis in one of three ways: (a) the initial slope of the CO(2) response was unaffected, but the photosynthetic rate at high CO(2) increased (S. tuberosum); (b) the initial slope decreased but the CO(2)-saturated rate of photosynthesis was little affected (C. album, P. vulgaris); (c) both the initial slope and the CO(2)-saturated rate of photosynthesis decreased (B. oleracea, S. melongena). In all five species, growth at high CO(2) increased the extent to which photosynthesis was stimulated following a decrease in the partial pressure of O(2) or an increase in measurement CO(2) above 600 microbars. This stimulation indicates that a limitation on photosynthesis by the capacity to regenerate orthophosphate was reduced or absent after acclimation to high CO(2). Leaf nitrogen per area either increased (S. tuberosum, S. melongena) or was little changed by CO(2) enhancement. The content of rubisco was lower in only two of the five species, yet its activation state was 19% to 48% lower in all five species following long term exposure to high CO(2). These results indicate that during growth in CO(2) enriched air, leaf rubisco content remains in excess of that required to support the observed photosynthetic rates. PMID- 16666588 TI - Main stem sink manipulation in wheat : effects on nitrogen allocation to tillers. AB - The role of main stem (MS) sink size on N use by field-grown soft red winter wheat (Triticum aestivum L. cv Hart) was determined. At Feeke's growth stage 8 (last leaf just visible), 100 micromoles of 99 atom% (15)N-ammonium was injected into the lower MS. At anthesis, MS sink size was adjusted by removal of 0, 33, 66, and 100% of the MS spikelets; tiller spikes were left intact. The MS and tiller average kernel size was unaffected by MS sink manipulations. The MS kernel N concentration increased when MS spikelets were removed. Tiller kernel N concentrations were unaffected except when the entire MS reproductive sink was removed, which caused an increase in tiller kernel N concentration. Net losses of MS vegetative N during grain fill were similar for all treatments except for plants lacking MS spikelets, which mobilized 30% less N from the MS. Labeled N was predominately (>90%) associated with the insoluble reduced N fraction of plant tissues at anthesis. Allocation of labeled N to tillers was not proportional to reduction in MS sink size. These results indicate that the reproductive sink on an individual culm has first priority for vegetative N mobilized during grain fill even when sink demand is reduced substantially. PMID- 16666590 TI - Soybean Seed Water Relations during in Situ and in Vitro Growth and Maturation. AB - Water, osmotic, and pressure potentials of soybean (Glycine max [L.] Merrill) embryos and related maternal tissues were measured during periods of seed growth and maturation to test the involvement of embryo water relations in seed maturation. Seeds were matured in situ or in an in vitro liquid culture medium in detached pods or as isolated seeds. Changes in water relations of embryo tissues were independent of maternal tissues. During seed maturation in situ, water and osmotic potentials in both embryo and maternal tissues declined sharply near the time of maximum dry weight. During in vitro seed culture with and without pods, water and osmotic potentials in axis and cotyledon tissues declined continuously during growth. Water and osmotic potentials of the seed coat, which was present only during in vitro seed culture with pods, changed little during the culture period. Positive turgor in the embryo was maintained beyond maximum dry weight and the loss of green color during in vitro culture but declined to zero at maturity in situ. The osmotic potential in embryo tissues declined from -1.1 megapascals at early pod fill to between -1.65 and -2.2 megapascals at maximum seed dry weight across all maturation environments. It is suggested that the decreasing osmotic potential in the growing soybean embryo reaches a threshold level that is associated with cessation of growth and onset of seed maturation. PMID- 16666589 TI - Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize. AB - Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts. PMID- 16666591 TI - Thermotolerance of isolated mitochondria associated with heat shock proteins. AB - Mitochondria isolated from 2-day-old etiolated soybean (Glycine max) seedlings which had been subjected to various heat shock treatments, i.e. (A) 28 degrees C (2 h), (B) 38 degrees C (2 h), (C) 38 degrees C (2 h)-42.5 degrees C (0.5 h), and (D) 38 degrees C (2 h)-42.5 degrees C (0.5 h)-28 degrees C (4 h), were monitored for O(2) uptake using an oxygen electrode. Mitochondria isolated after all four heat shock treatments were active in O(2) consumption at 28 degrees C in response to succinate and ADP (derived P/O ratios were 1.6, 1.7, 1.3, and 1.3, respectively.) The mitochondria from all four treatments were also active in O(2) uptake at 42.5 degrees C. However, only mitochondria isolated after treatment (C) were tightly coupling at 42.5 degrees C (derived ADP/O ratio was about 1.4). Combined with our earlier findings on the subcellular localization of heat shock proteins, our present data demonstrate that association of heat shock proteins with mitochondria by treatment (C) enables them to phosphorylate at 42.5 degrees C (i.e. they become thermotolerant). Isolated mitochondria from treatment (C) and treatment (A) were compared by electron microscopy. They appeared to be very similar and no significant ultrastructural differences were noted. PMID- 16666592 TI - Endogenous ABA in Growing Maize Roots: Light Effects. AB - The growth of intact maize (Zea mays L.) roots and the abscisic acid (ABA) content (measured by gas chromatography-mass spectrometry of the root tip were analyzed after a white-light treatment. The decrease of the elongation rate due to the illumination corresponded to a concomitant increase in the ABA found in the root. When selecting roots, on the basis of their growth rate, it was possible to show that the relation between growth and ABA content, previously reported in darkness was conserved after light treatments. Therefore, light decreased the root growth rate while it simultaneously increased the ABA content in the roots. This increase was higher than expected, demonstrating the complexity of the involvement of ABA on root growth. PMID- 16666593 TI - Product inhibition of potato tuber pyrophosphate:fructose-6-phosphate phosphotransferase by phosphate and pyrophosphate. AB - The product inhibition of potato (Solanum tuberosum) tuber pyrophosphate:fructose 6-phosphate phosphotransferase by inorganic pyrophosphate and inorganic phosphate has been studied. The binding of substrates for the forward (glycolytic) and the reverse (gluconeogenic) reaction is random order, and occurs with only weak competition between the substrate pair fructose-6-phosphate and pyrophosphate, and between the substrate pair fructose-1,6-bisphosphate and phosphate. Pyrophosphate is a powerful inhibitor of the reverse reaction, acting competitively to fructose-1,6-biphosphate and noncompetitively to phosphate. At the concentrations needed for catalysis of the reverse reaction, phosphate inhibits the forward reaction in a largely noncompetitive mode with respect to both fructose-6-phosphate and pyrophosphate. At higher concentrations, phosphate inhibits both the forward and the reverse reaction by decreasing the affinity for fructose-2,6-bisphosphate and thus, for the other three substrates. These results allow a model to be proposed, which describes the interactions between the substrates at the catalytic site. They also suggest the enzyme may be regulated in vivo by changes of the relation between metabolites and phosphate and could act as a means of controlling the cytosolic pyrophosphate concentration. PMID- 16666594 TI - Impairment of Tonoplast H-ATPase as an Initial Physiological Response of Cells to Chilling in Mung Bean (Vigna radiata [L.] Wilczek). AB - Biochemical alterations of cellular membranes in chilling-sensitive mung bean (Vigna radiata [L.] Wilczek) hypocotyls were investigated with reference to chilling injury. Reversible decreases in activities of tonoplast H(+)-ATPase and in vivo respiration became manifest within 24 hours of chilling when tissues suffered no permanent injury as assessed by electrolyte leakage and regrowth capacity. These changes were found to be the earliest cellular responses to chilling. A density-shift on a sucrose density gradient was observed in Golgi membranes early in the chilling treatment, suggesting that Golgi function and/or membrane biogenesis via the Golgi may have been altered upon chilling. After chilling more than 2 days, irreversible changes were generally produced in cellular membranes including the plasma membrane, endoplasmic reticulum, and mitochondria. Respiratory functions remained intact in mitochondria isolated from tissues prechilled for 24 hours, but were impaired after prechilling for 3 days. Given the important role of the tonoplast H(+)-ATPase in the active transport of ions and metabolites, the early decline in the tonoplast H(+)-ATPase activity may give rise to an alteration of the cytoplasmic environment and, consequently, trigger a series of degenerative reactions in the cells. PMID- 16666595 TI - Quantitative analysis of transpiration stream dynamics in an intact cucumber stem by a heat flux control method. AB - Water flux of transpiration stream in an intact stem of the 10 leaf stage cucumber plant (Cucumis sativus L. cv. Chojitsu-Ochiai) was measured by a novel system of heat flux control method with a resolution of 1 x 10(-3) grams per second and a time constant of 1 minute; two heat flux control sensors were attached to the seventh internode and the stem base. The transpiration stream responded clearly to leaf transpiration and root water absorption when the plant was exposed to light, and the water flux at the stem base corresponded to the transpiration rate per plant in steady state. Root water absorption lagged about 10 minutes behind leaf transpiration. Dynamics of water fluxes were affected by the lag of water absorption in roots, and temporary water loss caused by rapid increase in leaf transpiration was buffered by about 5% of the water content in the stem. PMID- 16666596 TI - Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock. AB - Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24 degrees C to 4 degrees C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24 degrees C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before. PMID- 16666597 TI - Localization of the Enzymes of Fructan Metabolism in Vacuoles Isolated by a Mechanical Method from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.). AB - Vacuoles isolated by a mechanical slicing method from developing tubers of Jerusalem artichoke (Helianthus tuberosus L.) contain activities of the two principal enzymes responsible for fructan synthesis: sucrose-sucrose fructosyl transferase and fructan-fructan fructosyl transferase. Both enzymes are associated with the vacuolar sap and not with the tonoplast. In vacuoles isolated from dormant tubers, the fructan-fructan fructosyl transferase activity remains in the vacuolar sap but the fructan exohydrolase activity is associated with the tonoplast. Fructan is hydrolysed by these vacuoles to fructose, which can be exported to the suspending medium. The localization of the enzymes of fructan metabolism in the vacuole has implications for the maintenance of fructan polymerisation. PMID- 16666598 TI - Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons. AB - Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33 CPO) in excised cucumber (Cucumis sativus L. cv ;Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33 CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue. PMID- 16666599 TI - Protein synthesis in isolated castor bean mitochondria is stimulated by cyanide. AB - Cyanide added to isolated castor bean (Ricinus communis L.) mitochondria supplemented with ATP and succinate (or NADH) significantly enhanced the rate and extent of organellar protein synthesis. Cyanide stimulated mitochondrial protein synthesis in a dose-dependent manner with an optimum stimulation of over twofold at 1 millimolar cyanide. At this concentration of cyanide, the mitochondrial respiratory activity, in the presence of succinate (or NADH) and ADP was inhibited by 90%. The stimulatory effect of cyanide on mitochondrial translation was reflected in the increased synthesis of all the proteins synthesized within the organelle. Preliminary evidence indicates a role for the alternative, salicylhydroxamic acid-sensitive, oxidase in the cyanide stimulation of protein synthesis. PMID- 16666600 TI - Mode of Action Studies on Nitrodiphenyl Ether Herbicides : II. The Role of Photosynthetic Electron Transport in Scenedesmus obliquus. AB - The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2 nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI) induces light- and O(2)-dependent lipid peroxidation and chlorophyll (Chl) bleaching in the green alga Scenedesmus obliquus. Under conditions of O(2)-limitation, these effects are diminished by prometyne and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), both inhibitors of photosynthetic electron transport. Mutants in which photosynthetic electron transport is blocked are also resistant to DPEI under conditions of O(2) limitation. Light- and O(2)-dependent lipid peroxidation and Chl bleaching are also induced by 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methoxyphthalide (DPEII), a diphenyl ether whose redox properties preclude reduction by photosystem I. However, these effects of DPEII are also inhibited by DCMU. Under conditions of high aeration, DCMU does not protect Scenedesmus cells from Chl bleaching induced by DPEI, but does protect against paraquat. DPEI, but not paraquat, induces tetrapyrrole formation in treated cells in the dark. This is also observed in a mutant lacking photosystem I but is suppressed under conditions likely to lead to O(2) limitation. Our results indicate that, in contrast to paraquat, the role of photosynthetic electron transport in diphenyl ether toxicity in Scenedesmus is not to reduce the herbicide to a radical species which initiates lipid peroxidation. Its role is probably to maintain a sufficiently high O(2) concentration, through water-splitting, in the algal suspension. PMID- 16666601 TI - Enhancement of Terpenoid Biosynthesis from Mevalonate in a Fraction of the Latex from Euphorbia lathyris. AB - A latex pellet fraction from Euphorbia lathyris incorporates mevalonate into triterpenols and their fatty acid esters. Conditions for improved incorporation were determined. CaCl(2) or CaCl(2) plus MnCl(2) stimulated biosynthesis, and the metal ion chelator, ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) enhanced stimulation. Ethylenediaminetetraacetic acid was almost as effective as EGTA, but phthalic acid and citric acid were relatively poor stimulators. The concentration of the Ca(2+)-EGTA complex was directly measured, and the incorporation data are best fitted by a curve that shows that the receptor for the complex is saturable. In the presence of the metal-chelate complex, the addition of fructose, 1,6-bisphosphate plus aldolase (triose-P) or malate provided additional stimulation. Incorporation was maximum at 40 micromolar R mevalonate, and inhibition occurred at higher concentrations. The apparent K(m) for R-mevalonate was 15 micromolar. Under improved reaction conditions, the rate of triterpenoid biosynthesis from mevalonate is 25 times faster than was previously observed (GJ Piazza, EJ Saggese, KM Spletzer [1987] Plant Physiol 83: 177-180). PMID- 16666602 TI - Partial Purification and Characterization of Aminopeptidase II from Chara australis. AB - Aminopeptidase II, one of the two major aminopeptidases in the giant alga Chara australis, was partially purified. Its molecular weight was estimated to be about 80,000 by gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that it is composed of a single polypeptide with a molecular weight of about 85,000. Aminopeptidase II hydrolyzed alanine-2 naphthylamide more efficiently than the naphthylamides of lysine and proline, and only weakly hydrolyzed the naphthylamides of arginine, phenylalanine, valine, and leucine. The optimal pH for the hydrolysis of alanine-2-naphthylamide was near 7.0. The activity of aminopeptidase II was inhibited by the SH-reagents p chloromercuribenzoic acid and N-ethylmaleimide and by the metal chelator 1,10 phenanthroline. PMID- 16666603 TI - Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. : Characterization, Solubilization, and Photoaffinity Labeling. AB - Using the novel radioligand, [(3)H]-9'-nor-fusicoccin-8'-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K(+), Mg(2+)-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [(3)H]-9'-nor-fusicoccin-8'-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (K(D)), K(D1) = 1.5 nanomolar and K(D2) = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID- 16666604 TI - Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance. AB - The effects of Cd on poly(gamma-glutamylcysteinyl)glycine [(gammaEC)(n)G] biosynthesis and formation of (gammaEC)(n)G:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl(2) was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd sensitive cells synthesized the same amount of (gammaEC)(n)G as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl(2). However, rates of (gammaEC)(n)G:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[(35)S]cysteine resulted in equivalent rates of incorporation into the (gammaEC)(n)G of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (gammaEC)(n)G synthesis in sensitive cells is operational and that relative overproduction of (gammaEC)(n)G is not the mechanism of Cd-tolerance in a Cd tolerant cell line of D. innoxia. Rapid formation of (gammaEC)(n)G:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells. PMID- 16666605 TI - Rapid in Vivo Acylation of Acyl Carrier Protein with Exogenous Fatty Acids in Spirodela oligorrhiza. AB - Posttranslational acylation of several chloroplast proteins with palmitic acid was recently demonstrated in Spirodela oligorrhiza (AK Mattoo, M Edelman [1987] Proc Natl Acad Sci USA 84: 1497-1501). We have now identified an in vivo acylated, soluble protein having an apparent M(r) of 10 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as an acylated form of acyl carrier protein (ACP). This 10-kilodalton protein is present in low abundance, and its acylation is light-stimulated. Turnover of the acyl moiety but not the apo-protein is rapid in the light. The acylated 10-kilodalton protein coelectrophoreses with in vitro synthesized palmitoyl-acyl carrier protein and is immunoprecipitated from soluble extracts with an antibody raised against spinach ACP. Cerulenin, an inhibitor of beta-ketoacyl-ACP synthetase, inhibited in vivo acylation of Spirodela ACP. Cell-free extracts of Spirodela plants were able to catalyze the transfer of palmitate from palmitoyl-CoA to ACP, suggesting the existence in higher plants of a pathway for acylation of ACP that involves transacylation from acyl-CoA. PMID- 16666606 TI - Cell wall proteins from sugar beet cells in suspension culture. AB - Several proteins were extracted from the purified cell walls of suspension cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall. PMID- 16666607 TI - Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L. AB - Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population. PMID- 16666608 TI - Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA. AB - The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [(14)C]trioleoylglycerol, releasing [(14)C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [(14)C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [(14)C]oleic acid produced from the lipase reaction to [(14)C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation. PMID- 16666609 TI - Plastid transcription activity and DNA copy number increase early in barley chloroplast development. AB - Plastid transcription activity and DNA copy number were quantified during chloroplast development in the first foliage leaf in dark-grown and illuminated barley (Hordeum vulgare L.) seedlings. Primary foliage leaves of seedlings given continuous illumination from 2 days post-imbibition reached a final mean length of 15 centimeters at 6.5 days, whereas primary leaves of dark-grown seedlings required 7 days to reach a similar length. Dividing cells were observed in the basal 0.5 to 1 centimeter of primary leaves until 5.5 days post-imbibition. Plastids isolated from cells located in the basal meristem of 4-day-old seedlings were small ( approximately 2 micrometers in diameter), exhibited low transcription activity and contained approximately 130 copies of plastid DNA per organelle. Cell size increased from 18 to 60 micrometers in a 1 to 3 centimeter region located adjacent to the leaf basal meristem. In this region, transcriptional activity per plastid increased 10-fold and DNA copy number increased from 130 to 210. Plastid transcriptional activity declined rapidly in illuminated plants with increasing leaf cell age and plastid DNA copy number also declined but with a slower time course. In dark-grown seedlings, plastid transcriptional activity declined more slowly than in illuminated plants while DNA copy number remained constant with increasing cell age. These data show that plastid transcriptional activity and DNA copy number increase early in chloroplast development and that transcriptional activity per DNA template varies up to 5-fold during barley leaf biogenesis. PMID- 16666610 TI - Purification and characterization of mitochondrial citrate synthase. AB - Mitochondrial citrate synthase was purified from leaves of Pisum sativum L. cv Progress 9. A three step purification was employed using ATP-Sepharose affinity chromatography which resulted in a 600-fold enrichment. Enzyme activity was assayed spectrophotometrically during greening of etiolated leaves under constant white light illumination. An increase (1.4 fold) in citrate synthase activity was observed in response to light. Immunoblot analysis of the same samples indicated a constant steady state level of citrate synthase on a per milligram protein basis. These investigations provide supportive evidence for the ability of this trichloroacetic acid cycle enzyme to be active in photosynthesizing tissue. PMID- 16666611 TI - Light-Stimulated Inositol Phospholipid Turnover in Samanea saman Pulvini : Increased Levels of Diacylglycerol. AB - Leaflet movement in Samanea saman is driven by an endogenous circadian clock and by light. We are investigating whether the effects of light on leaflet movement are mediated by increased inositol phospholipid turnover. We demonstrated previously that irradiation of excised pulvini with 15 to 30 seconds of white light decreases the levels of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and increases the levels of inositol phosphates. We now report that the diacylglycerol level increases after 30 seconds of white light but returns to below the control level after 10 minutes of white light. PMID- 16666612 TI - NADH Induces the Generation of Superoxide Radicals in Leaf Peroxisomes. AB - In peroxisomes isolated from pea leaves (Pisum sativum L.) the production of superoxide free radicals (O(2) (-)) by xanthine and NADH was investigated. In peroxisomal membranes, 100 micromolar NADH induced the production of O(2) (-) radicals. In the soluble fractions of peroxisomes, no generation of O(2) (-) radicals was observed by incubation with either NADH or xanthine, although xanthine oxidase was found located predominantly in the matrix of peroxisomes. The failure of xanthine to induce superoxide generation was probably due to the inability to fully suppress the endogenous Mn-superoxide dismutase activity by inhibitors which were inactive against xanthine oxidase. The generation of superoxide radicals in leaf peroxisomes together with the recently described production of these oxygen radicals in glyoxysomes (LM Sandalio, VM Fernandez, FL Ruperez, LA del Rio [1988] Plant Physiol 87: 1-4) suggests that O(2) (-) generation could be a common metabolic property of peroxisomes and further supports the existence of active oxygen-related roles for peroxisomes in cellular metabolism. PMID- 16666613 TI - Endogenous Abscisic Acid Content Correlates with Photon Fluence Rate and Induced Leaf Morphology in Hippuris vulgaris. AB - This research focused on studying how light and endogenous abscisic acid regulate leaf development in Hippuris vulgaris, a species of heterophyllic aquatic plant. Amounts of photosynthetically active radiation greater than 300 micromoles per square meter per second caused submerged H. vulgaris shoots to produce aerial type leaves. Abscisic acid was not detected in shoots grown under noninducing light quantities (100 micromoles per square meter per second), but was present at 13.4 nanograms per gram fresh weight in shoot tips after plants were exposed to 1 photoperiod of inducing light (500 micromoles per square meter per second). This supports a role for abscisic acid in the high light-induced heterophylly in H. vulgaris, and provides additional support for the general hypothesis that abscisic acid regulates leaf development in heterophyllic aquatic plants. No relationship was observed here between postphotoperiodic light treatments of various red/far red ratios and heterophylly in H. vulgaris. PMID- 16666614 TI - Activity ratios of ribulose-1,5-bisphosphate carboxylase accurately reflect carbamylation ratios. AB - Activity ratios and carbamylation ratios of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were determined for leaves of Phaseolus vulgaris and Spinacia oleracea exposed to a variety of partial pressures of CO(2) and O(2) and photon flux densities (PFD). It was found that activity ratios accurately predicted carbamylation ratios except in extracts from leaves held in low PFD. In particular, it was confirmed that the loss of RuBPCase activity in low partial pressure of O(2) and high PFD results from reduced carbamylation. Activity ratios of RuBPCase were lower than carbamylation ratios for Phaseolus leaves sampled in low PFD, presumably because of the presence of 2-carboxyarabinitol 1-phosphate. Spinacia leaves sampled in darkness also exhibited lower activity ratios than carbamylation ratios indicating that this species may also have an RuBPCase inhibitor even though carboxyarabinitol 1-phosphate has not been detected in this species in the past. PMID- 16666615 TI - Cold-induced sudden reversible lowering of in vivo chlorophyll fluorescence after saturating light pulses : a sensitive marker for chilling susceptibility. AB - In chilling-sensitive plants (Glycine max, Saintpaulia ionantha, Saccharum officinarum) a sudden reversible drop in chlorophyll fluorescence occurs during photosynthetic induction immediately following saturating light pulses at low temperatures in the range 4 to 8 degrees C. A comparison of two soybean cultivars of different chilling sensitivities revealed that this phenomenon, termed lowwave, indicates specific thresholds of low temperature stress. Its occurrence under controlled chilling can be regarded as a quantitative marker for screening chilling susceptibility in angiosperms. PMID- 16666616 TI - An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein. AB - We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division. PMID- 16666617 TI - Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg. AB - Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg(2+) bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg(2+) was altered by pretreatment with Mg(2+) and divalent cation chelating agents and by additions of Mg(2+) to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg(2+) can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with (28)Mg(2+) confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg(2+). It was concluded that changes in Mg(2+) associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg(2+) may be exclusively due to increased envelope-associated Mg(2+). The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg(2+) inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K(+) in the photosynthetic reaction media. PMID- 16666618 TI - Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia. AB - The photorespiratory activity of mesophyll protoplasts of Nicotiana plumbaginifolia has been clearly demonstrated by the presence of a Warburg effect, the occurrence of an important CO(2)-sensitive O(2) uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO(2) fixation was 60% lower at 50% O(2) compared to that measured at 2% O(2). Using (18)O(2) and mass spectrometry, we measured O(2) exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO(2) compensation point (47.4 micromoles O(2) per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO(2) concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the O(2) uptake to the same extent as CO(2) saturation. We conclude from these results that the major part of the CO(2)-sensitive O(2) uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO(2) saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and 1 millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO(2) compensation point at 20% O(2)), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% O(2), but was stronger at 50% O(2) (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar l-methionine-dl sulfoximine (l-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO(2) compensation point at 20% O(2) (30% for BHB and 20% for l-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate. PMID- 16666619 TI - Maturation of soybean somatic embryos and the transition to plantlet growth. AB - The maturation of soybean (Glycine max L. Merr.) somatic embryos was characterized. Maturation was assayed by evaluating the ability of somatic embryos to make the transition to a plantlet through a germination-like process. Somatic embryos were organized from cotyledons of immature soybean embryos. Maturation of somatic embryos occurred on a Murashige-Skoog basal medium supplemented with activated charcoal and 0.28 molar sucrose. After 8 weeks on this medium, somatic embryos exhibited vigorous, high frequency development to plantlets. The "germination" frequency (conversion) of somatic embryos, and plantlet recovery frequency varied concurrently with maturation period. Conversion and plant recovery required no exogenous growth regulators. Desiccation of immature somatic embryos under controlled humidity regimes resulted in increased frequency of conversion of immature somatic embryos. Morphological abnormalities appeared in the somatic embryos, but few were detrimental to conversion velocity. There was little effect of genotype on conversion velocity or frequency. PMID- 16666620 TI - Photosynthetic Genes of Petunia (Mitchell) Are Differentially Expressed during the Diurnal Cycle. AB - The petunia (Petunia [Mitchell]) chloroplast proteins, the chlorophyll a/b binding (Cab) proteins, and the small subunit of ribulose bisphosphate carboxylase (RbcS) are encoded by nuclear genes that are expressed in a light dependent manner. The steady-state concentrations of five cab mRNAs vary with a dramatic circadian rhythm in plants grown under a constant diurnal cycle (10 hours light, 14 hours dark). cab mRNA levels reach their maximum during the light period, but begin to drop prior to the dark period. These RNAs fall to their minimum concentration during the dark period and then begin to increase again in anticipation of the light. Within this general pattern, there are variations in expression among specific classes of cab genes. The light harvesting complex of photosystem II LHCII-type 1 cab mRNAs rise to a well-defined maximum at 2 hours prior to the dark period. All but one of these genes are expressed in anticipation of the light period. The LHCII type 2 cab mRNA and the LHC of photosystem I cab mRNA are expressed at more constant levels throughout the light period. The expression of these genes anticipates the light more than does the expression of the LHCII type 1 genes. The steady state mRNA levels for the petunia rbcS genes show no significant diurnal fluctuation. PMID- 16666621 TI - Occurrence and Localization of Phycoerythrin in Symbiotic Nostoc of Cycas revoluta and in the Free-Living Isolated Nostoc 7422. AB - The phycobiliprotein phycoerythrin was localized in symbiotic and free-living Nostoc of the cycad Cycas using immunocytochemistry. In symbiotic Nostoc, phycoerythrin was associated with the thylakoid membranes of vegetative cells and absent from heterocysts. Similar cellular/subcellular localization was observed between symbiotic Nostoc and the free-living Cycas isolate Nostoc 7422. PMID- 16666623 TI - Structure of a pectic polysaccharide fraction from zea shoots. AB - A pectic fraction, accounting for about 0.3% of the total cell wall polysaccharide, was derived from the hot water extract of an insoluble fraction of the buffer-homogenate of Zea shoots. The pectic polysaccharide fraction was characterized by fragmentation analysis after hydrolysis with acid and Erwinia carotovora pectate lyase. The results suggest that the fraction consists of mostly a linear homopolygalacturonan with neutral sugar components or a homogalacturonan and a rhamnogalacturonan with neutral sugar components. PMID- 16666622 TI - Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana. AB - After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO(3) (-) as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH(4) (+)-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein. PMID- 16666624 TI - Turgor and growth at low water potentials. AB - Turgor affects cell enlargement but has not been measured in enlarging tissue of intact plants when growth is inhibited by inadequate water. Mature or excised tissue can be problematic for these measurements because turgor may not be the same as in intact enlarging cells. Therefore, we measured the average turgor in the elongating region of intact stems of soybean (Glycine max [L.] Merr.) while the seedlings were exposed to low water potentials by transplanting to vermiculite of low water content. Stem growth was completely inhibited by the transplanting, and the average turgor decreased in the mature stem tissue. However, it did not decrease in the elongating region whether measured in intact or excised tissue (total of four methods). At the cellular level, turgor was uniform in the elongating tissue except at transplanting, when turgor decreased in a small number of cortical cells near the xylem. The reduced turgor in these cells, but constant turgor in most of the cells, confirmed that no general turgor loss had occurred but indicated that gradients in water potential extending from the xylem into the enlarging tissue were reduced, thus decreasing the movement of water into the tissue for cell enlargement. A modest growth recovery occurred after 2 days and was preceded by a recovery of the gradient. This suggests that under these conditions, growth initially was inhibited not by turgor loss but by a collapse of the water potential gradient necessary for the growth process. PMID- 16666625 TI - Dynamics of Imbibition in Phaseolus vulgaris L. in Relation to Initial Seed Moisture Content. AB - The seed moisture level marking the onset of imbibitional injury (breakpoint) was determined for two cultivars of Phaseolus vulgaris L. cvs ;Tendercrop' (TC) and ;Kinghorn Wax' (KW). At 20 degrees C the breakpoints were 0.15 gram H(2)O/gram dry weight (gram per gram) for TC and 0.11 gram per gram for KW. When seeds were imbibed at 5 degrees C, the breakpoints were 0.19 gram per gram (TC) and 0.16 gram per gram (KW). Below the breakpoint germination changed 4.6%/0.01 gram per gram for all treatments. Imbibition rates were maximal at 0.07 gram per gram and 0.33 gram per gram after 20 minutes imbibition. Rates of electrolyte leakage were correlated with the imbibition rate maximum at 0.07 gram per gram but were unaffected by the maximum at 0.33 gram per gram. The transition from tightly bound to semibound water occurred at 0.09 gram per gram and 0.11 gram per gram for KW and TC, respectively. T1 values increased exponentially as seed moisture decreased from 0.47 gram per gram to 0.05 gram per gram. (13)C-NMR sugar signals increased at moisture levels above 0.14 gram per gram and plateaued at approximately 0.33 gram per gram seed moisture. These results suggest that the breakpoint moisture level for imbibitional damage is a function of temperature while the injury process is similar at both 5 and 20 degrees C. Imbibition and leakage rate maxima reflect transitions in the states of seed water. NMR data support the application of the Water Replacement Hypothesis to seeds. Thus, imbibitional injury may be related to specific, temperature dependent moisture levels that are determined by water binding characteristics in the seed tissue. PMID- 16666626 TI - Time Course of mRNA Induction Elicited by Salt Stress in the Common Ice Plant (Mesembryanthemum crystallinum). AB - In the facultative halophyte Mesembryanthemum crystallinum (common ice plant), irrigation with solutions containing NaCl induces an alternate mode of carbon dioxide fixation, Crassulacean acid metabolism (CAM). The salt stress protocol which we have established facilitates the study of CAM induction and the correlation of changes in metabolism and gene expression. We have studied the time course of mRNA induction for phosphoenolpyruvate carboxylase (PEPCase) (gene: ppc) and several other enzymes of carbon metabolism during stress. While CAM is not fully established for at least 10 days after the start of stress, mRNA amounts for PEPCase and for other CAM enzymes, such as Pyruvate orthophosphate dikinase, increase between day 2 and 3 after stress induction. Increases continue for at least 5 days. Concomitant with the increase of CAM transcripts, fluctuations in the mRNA amounts for genes rbcS and cab were observed. Transcript levels for these proteins decreased several-fold during a 3 to 4 day period. PMID- 16666627 TI - Expression during Salt Stress and Nucleotide Sequence of cDNA for Ferredoxin-NADP Reductase from Mesembryanthemum crystallinum. AB - In the facultative halophyte Mesembryanthemum crystallinum (common ice plant) the enzyme ferredoxin-NADP(+)-reductase (FNR) is coded for by a small family of 2 to 3 genes. We have determined the expression characteristics as the plants adapt to high salt and the nucleotide sequence of a full-length cDNA coding for the precursor of this chloroplast-located enzyme. On a developmental scale amounts of FNR transcripts and protein are highest in young emerging leaves. The FNR cDNA is a member of a class of genes whose expression is only slightly affected by salt stress. Even less pronounced than mRNA fluctuations, the amount of FNR protein is unaffected by salt stress. The longest FNR cDNA found was 1,419 nucleotides in size. It consisted of 74 nucleotides 5'-leader sequence, 1,095 nucleotides of protein coding sequence encoding 365 amino acids, and 247 nucleotides 3-region excluding a short poly(A(+)) tail. As expected for a nucleus-coded chloroplast protein an amino terminal transit peptide (52 amino acids in length) was found. The mature FNR protein is predicted to contain 313 amino acids corresponding to a protein of M(r) 35,713. The deduced amino acid sequence of the mature FNR protein is 93.2 and 85.9% identical to those of spinach and pea. The transit peptide of pea and spinach have 55.8 and 69.2% identity with that from ice plant. PMID- 16666628 TI - Proton and copper adsorption to maize and soybean root cell walls. AB - A surface complexation model which has been used to describe inner-sphere complexation on metal oxide surfaces was applied to the adsorption of Cu by isolated cell walls of 4-day and 28-day-old maize (Zea mays L. cv WF9 x Mo17) and 21-day-old soybean (Glycine max [L.] Merr. cv Dare) roots. Concentration dependence of the titration data prevented the determination of unique pK and capacitance values for the 4-day maize cell walls, though mean values obtained for the intrinsic pK of the titratable carboxyl groups were 3.0 (4-day maize), 3.6 (28-day maize), and 3.0 (21-day soybean) as determined by potentiometric titration with either NaOH or HCl in 20 millimolar NaCl. The constant capacitance model was applied to Cu sorption data from rapid batch equilibrium experiments in an ionic medium of 20 millimolar NaClO(4). Speciation calculations indicated that the formation of a bidentate surface complex was sufficient to describe the experimental data for all three types of plant material, with only one value for the pK and capacitance density. The intrinsic constants of Cu complexation by a neutral site are: log K = -0.3 +/- 0.1, -0.2 +/- 0.3, and 0.9 +/- 0.1 for 4-day and 28-day maize, and 21-day soybean, respectively. The integral capacitance density parameter, which describes the relationship between surface charge density and electrical potential, is several times larger than for crystalline mineral surfaces. This result indicates that the surface electrical potential remains low even when the surface charge density is high. Such behavior is characteristic of gels and porous oxides. PMID- 16666629 TI - A developmentally regulated bud specific transcript in pea has sequence similarity to seed lectins. AB - We report a striking example of organ and stage specific gene expression in pea (Pisum sativum L.). We have identified a transcript to a previously isolated cDNA clone, pEA207 (WF Thompson et al. (1983) Planta 158: 487-500) which accumulates in the actively growing bud of the pea plant and is either absent or present at very low levels in the expanded leaves below the bud. The deduced amino acid sequence of pEA207 shows 49% similarity to the phytohemagglutinin lectin sequence of kidney bean (Phaseolus vulgaris) (LM Hoffman, DD Donaldson (1985) EMBO J 4: 883-889) and 37% similarity to that of the major pea seed lectin sequence (TJV Higgins et al. (1983) J Biol Chem 258: 9544-9549). It is also similar to seed lectins from five other legumes. All of the residues directly involved in metal binding by lectins are present in this sequence. We discuss the possibility that pEA207 encodes a sugar binding lectin-like polypeptide associated with the cell walls of actively growing plant cells. PMID- 16666630 TI - CO(2) Exchange, Cytogenetics, and Leaf Anatomy of Hybrids between Photosynthetically Distinct Flaveria Species. AB - Hybrids between the C(4)-like species, Flaveria brownii, A. M. Powell and the C(3)-C(4) intermediate species Flaveria linearis Lag., Flaveria floridana Johnston, and Flaveria oppositifolia (DC.) Rydb. exhibited bivalent chromosome pairing during meiosis and stainability of pollen was high, ranging from 51 to 95%. An F(2) population produced from an F. brownii x F. linearis F(1) hybrid, exhibited bivalent chromosome pairing and high pollen stainability indicating a high degree of fertility in the hybrid. Oxygen inhibition of apparent photosynthesis averaged 6.8% for F. brownii and 22.2% for the C(3)-C(4) species (in two experiments), and F(1) hybrids exhibited inhibitions which were intermediate to their parents. Values of carbon dioxide compensation concentration determined at low irradiance were 4.0, 34.0, and 6.5 microliters per liter for F. brownii, F. linearis and their F(1) hybrid, respectively. The mean value at low irradiance for 33 F(1) plants was 6.8 microliters per liter, and individual values ranged only from 3.7 to 11.7 microliters per liter. Anatomical characteristics for the F(1) hybrid leaves were intermediate to those of the parents, and there was considerable variation among F(2) plants derived from F. brownii x F. linearis. In the F(2) population delta(13)C values ranged from -27 per thousand to -20 per thousand. The expression of more C(4)-like characteristics by the F(1) hybrids in this study and their apparent high fertility make them promising specimens for producing segregating populations for use in C(4) inheritance studies. PMID- 16666631 TI - Apparent Inhibition of beta-Fructosidase Secretion by Tunicamycin May Be Explained by Breakdown of the Unglycosylated Protein during Secretion. AB - Suspension-cultured carrot (Daucus carota) cells synthesize and secrete beta fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of beta fructosidase as measured by the accumulation of the radioactive protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated beta-fructosidase is synthesized and is associated with an endoplasmic-reticulum rich microsomal fraction. Pulse-chase experiments show that the unglycosylated beta-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated beta-fructosidase; however, no radioactive, unglycosylated beta-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete beta-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated beta-fructosidase. In the presence of tunicamycin, there is no accumulation of beta-fructosidase activity or unglycosylated beta fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of beta-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall. PMID- 16666632 TI - Biosynthesis of Tetrapyrrole Pigment Precursors : Pyridoxal Requirement of the Aminotransferase Step in the Formation of delta-Aminolevulinate from Glutamate in Extracts of Chlorella vulgaris. AB - The aminotransferase that catalyzes the formation of delta-aminolevulinic acid from glutamate-1-semialdehyde or from glutamate in a reconstituted enzyme system was isolated and partially purified from Chlorella vulgaris. The apparent molecular weight of the aminotransferase was determined by Sephadex G-100 and Ultrogel AcA 54 gel filtration to be 60,000 +/- 5,000. Catalytic activity of the aminotransferase required pyrixodal phosphate (PALP). The cofactor could not be removed by gel filtration after exposure of the enzyme to PALP. Aminotransferase was inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The concentration of gabaculine required for half maximal inhibition was about 0.05 micromolar. Aminotransferase activity could be regained upon the removal of gabaculine by gel filtration and supplementing the assay medium with PALP. Neither the inhibitory action of gabaculine nor its reversibility was affected by preincubation of the enzyme with the keto acids levulinate and delta-aminolevulinic acid. PMID- 16666633 TI - beta-Amylase from Mustard (Sinapis alba L.) Cotyledons : Immunochemical Evidence for Synthesis de Novo during Photoregulated Seedling Development. AB - A polyclonal antiserum against mustard (Sinapis alba L.) beta-amylase was obtained by injecting a homogeneously purified enzyme preparation in rabbits. The formation of beta-amylase specific antibodies was confirmed by staining the precipitin line in double diffusion gel for beta-amylase activity. The monospecificity of antiserum against mustard beta-amylase was also ascertained by Western blotting. The antiserum efficiently recognised both the denatured and the native form of beta-amylase, but it did not cross-react with other higher plant beta-amylase. The mode of photoregulation of beta-amylase activity in mustard cotyledons was investigated by a variety of immunochemical techniques. Immunotitration experiments ruled out the possible contribution of enzyme activation/inactivation in photoregulation of beta-amylase activity. The use of single radial immunodiffusion, rocket immunoelectrophoresis, and immunotitration confirmed that the light mediated increase in beta-amylase activity quantitatively corresponds with the increase in beta-amylase protein level. The in vivo labeling with l-[(35)S] methionine and pulse chase studies of in vivo labeled beta-amylase protein revealed that the photoregulated increase in beta amylase activity in mustard cotyledon exclusively results from an increase in the rate of de novo synthesis of beta-amylase protein against a very low background rate of enzyme degradation. PMID- 16666634 TI - Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods. AB - The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids. PMID- 16666635 TI - Seed Dormancy in Red Rice : VII. Structure-Activity Studies of Germination Stimulants. AB - Many chemically dissimilar substances break dormancy of seeds, but the relationship between chemical structure and physiological activity is unknown. In this study, the concentrations of organic acids, esters, aldehydes, alcohols, and inorganic weak acids required to elicit 50% germination of initially dormant, dehulled red rice seeds (Oryza sativa) were determined. The activity of most substances was very highly and inversely correlated to lipophilicity as measured by octanol/water partition coefficients; chemicals with the highest partition coefficients required the lowest concentrations to elicit the germination response. Relative efficacy was also dependent upon the functional group; generally, monocarboxylic acids were more effective than aldehydes, esters, hydroxyacids, and alcohols. Relative hydrophobicity plots supported a modulating role of the functional group. Dormancy-breaking activity of methyl formate, formic acid, nitrite, azide, and cyanide was higher than predicted based on lipophilicity and apparently was related to molecular size; compounds with smaller molecular widths were required at lower concentrations to achieve the 50% germination response. PMID- 16666636 TI - Structure-activity relationships for xyloglucan oligosaccharides with antiauxin activity. AB - This work was designed to investigate the structural features required for a branched xyloglucan nonasaccharide (XG9; composition: glucose(4)xylose(3)galactose(1)fucose(1)) to exhibit anti-auxin activity in the pea (Pisum sativum L.) stem segment straight-growth bioassay. Oligosaccharides were prepared by cellulase-catalyzed hydrolysis of Rosa xyloglucan, and tested for auxin antagonism. The quantitatively major hepta-, octa-, and decasaccharides (XG7, XG8, and XG10) showed no antiauxin activity at the concentrations tested and did not interfere with the antiauxin effect of 10(-9) molar XG9 when coincubated at equimolar concentrations. The results indicate that the XG9 recognition system in pea stem segments is highly discriminating. A terminal alpha-l-fucose residue is essential for the antiauxin activity of XG9 and a neighboring terminal beta-d-galactose residue can abolish the activity; possible reasons for the effect of the galactose residue are discussed. A sample of XG9 extensively purified by gel-permeation chromatography followed by paper chromatography in two solvent systems still exhibited antiauxin activity with a concentration optimum around 10(-9) molar. This diminishes the likelihood that the antiauxin activity reported for previous nonsaccharide preparations was due to a compound other than XG9. PMID- 16666637 TI - Phosphatidylinositol(4,5)bisphosphate and Phosphatidylinositol(4)phosphate in Plant Tissues. AB - Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with [(3)H]myo-inositol or [(32)P]Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as d-myo inositol(1,4,5)trisphosphate and d-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected. PMID- 16666638 TI - Sugar content and activity of sucrose metabolism enzymes in milled rice grain. AB - Most rice (Oryza sativa L.) cultivars grown in the United States were selected for endosperm starch properties and not soluble sugar content. The minor pool of soluble sugar may affect the qualities of rice as a food. Some cultivar variation in soluble sugar content was detected in milled grain, essentially the starchy endosperm, of long grain varieties. Milled grain of cultivars Lemont and Texmati had a soluble sugar content of 0.21 and 0.35% (w/w), respectively, on a fresh weight basis. The dorsal portion of the milled grain contained the greatest amount of soluble sugar, approximately tenfold the amount found in the central core of the grain. Extracts of the milled grain contained sucrose-phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities, which were separated by anion exchange chromatography. The presence of sucrose-phosphate synthase in the rice endosperm suggested a mechanism for sucrose accumulation which might be involved in carbon partitioning during grain development. PMID- 16666639 TI - Isolation and Characterization of a Mutant of Chlamydomonas reinhardtii Deficient in the CO(2) Concentrating Mechanism. AB - A Chlamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO(2) concentrations but can grow on elevated CO(2). In a test cross, the high CO(2)-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO(2) accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO(2) by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO(2) levels to an environment low in CO(2). It is concluded that this mutant fails to induce the CO(2) concentrating system and is incapable of adapting to low CO(2) conditions. PMID- 16666640 TI - Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme. AB - A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system. PMID- 16666641 TI - Phase-Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells. AB - This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [(35)S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G(2) phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G(2) phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G(2) phase. In the G(1) and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)(+) RNA isolated at each phase. Three poly(A)(+) RNAs increased in amount from the G(1) to the S phase and one poly (A)(+) RNA increased preferentially from the G(2) phase to cytokinesis. PMID- 16666642 TI - Regulation of ribulose-1,5-bisphosphate carboxylase activity in response to diurnal changes in irradiance. AB - The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (Rubisco) activity and metabolite pool sizes in response to natural diurnal changes in photon flux density (PFD) was examined in three species (Phaseolus vulgaris, Beta vulgaris, and Spinacia oleracea) known to differ in the mechanisms used for this regulation. Diurnal regulation of Rubisco activity in P. vulgaris was primarily the result of metabolism of the naturally occurring tight-binding inhibitor of Rubisco, 2-carboxyarabinitol 1-phosphate (CA1P). In B. vulgaris, the regulation of Rubisco activity was the result of both changes in activation state and CA1P metabolism. In S. oleracea, Rubisco activity was regulated by a combination of changes in activation state and the binding/release of another tight binding inhibitor, probably RuBP. Despite these different mechanisms for the light regulation of Rubisco activity, the relationship between the in vivo activity of Rubisco and the PFD was the same for all three species. Rates of CA1P metabolism were thus sufficient to allow this mechanism to participate in the diurnal regulation of Rubisco activity as PFD changed at its normal rate. Furthermore, under natural conditions this regulatory mechanism was found to be important in controlling Rubisco activity over approximately the same range of PFD as did changes in activation state of the enzyme. Finally, this regulation of Rubisco activity resulted in relatively similar and saturating RuBP pool sizes for photosynthesis at all but the lowest PFD values in all three species. PMID- 16666643 TI - Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria. AB - Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD(+) in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon. PMID- 16666644 TI - Photosynthetic apparatus of pea thylakoid membranes : response to growth light intensity. AB - We investigated the effect of growth light intensity on the photosynthetic apparatus of pea (Pisum sativum) thylakoid membranes. Plants were grown either in a growth chamber at light intensities that ranged from 8 to 1050 microeinsteins per square meter per second, or outside under natural sunlight. In thylakoid membranes we determined: the amounts of active and inactive photosystem II, photosystem I, cytochrome b/f, and high potential cytochrome b(559), the rate of uncoupled electron transport, and the ratio of chlorophyll a to b. In leaves we determined: the amounts of the photosynthetic components per leaf area, the fresh weight per leaf area, the rate of electron transport, and the light compensation point. To minimize factors other than growth light intensity that may alter the photosynthetic apparatus, we focused on peas grown above the light compensation point (20-40 microeinsteins per square meter per second), and harvested only the unshaded leaves at the top of the plant. The maximum difference in the concentrations of the photosynthetic components was about 30% in thylakoids isolated from plants grown over a 10-fold range in light intensity, 100 to 1050 microeinsteins per square meter per second. Plants grown under natural sunlight were virtually indistinguishable from plants grown in growth chambers at the higher light intensities. On a leaf area basis, over the same growth light regime, the maximum difference in the concentration of the photosynthetic components was also about 30%. For peas grown at 1050 microeinsteins per square meter per second we found the concentrations of active photosystem II, photosystem I, and cytochrome b/f were about 2.1 millimoles per mol chlorophyll. There were an additional 20 to 33% of photosystem II complexes that were inactive. Over 90% of the heme-containing cytochrome f detected in the thylakoid membranes was active in linear electron transport. Based on these data, we do not find convincing evidence that the stoichiometries of the electron transport components in the thylakoid membrane, the size of the light-harvesting system serving the reaction centers, or the concentration of the photosynthetic components per leaf area, are regulated in response to different growth light intensities. The concept that emerges from this work is of a relatively fixed photosynthetic apparatus in thylakoid membranes of peas grown above the light compensation point. PMID- 16666645 TI - Changes in Amide-Linked and Ester Indole-3-Acetic Acid in Cotton Fruiting Forms during Their Development. AB - The concentration of free indoleacetic acid (IAA) is high in cotton (Gossypium hirsutum L.) fruiting forms before anthesis, but is low at and for a few days after anthesis. Amide-linked and ester IAA were measured in fruiting forms at 9, 6, and 3 days before anthesis; at anthesis; and at 2, 4, 7, and 9 days after anthesis to determine if free IAA decreased because it was converted to a conjugated form. That did not appear to be the case. While the major decrease in free IAA occurred during the 6 days before anthesis, ester IAA increased only a small amount and amide-linked IAA decreased even more than free IAA. During the 6 days before anthesis free IAA decreased from 0.62 to 0.12 micrograms per gram and amide-linked IAA decreased from 19.14 to 1.16 micrograms per gram dry weight. No evidence was found that a large amount of amide-linked IAA was converted to an insoluble form; flowers contained less than 1 microgram per gram of insoluble IAA. The free and amide-linked IAA must have been converted to other forms, perhaps by oxidation. Soluble amide-linked IAA remained low after anthesis. No ester IAA was detected 6 days before anthesis and only 0.08 microgram per gram dry weight was measured at anthesis. The concentration of ester IAA increased thereafter to 4.43 micrograms per gram at 9 days after anthesis. Therefore, amide linked IAA was the major form of IAA in flower buds and ester IAA was the major form in young fruits (bolls). Minimum concentrations of free and total IAA occurred during the 4 days after anthesis, a stage when cotton fruiting forms are most likely to abscise. The large decreases in free and amide-linked IAA during the 6 days before anthesis may indicate a rapid turnover of IAA in flower buds. But, the decrease in free IAA was not accompanied by a comparable increase in ester or amide-linked IAA. PMID- 16666646 TI - Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-beta-Glucanases and Chitinases. AB - Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [M(r)] 35 kilodaltons [kD], isoelectric point [pI] approximately 6.4) showed 1,3-beta-glucanase activity, while the other one (M(r)26 kD, pI approximately 6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-beta-glucanases isolated from other plant species, one additional 1,3-beta-glucanase (M(r)33 kD) and three additional chitinases (M(r) 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-beta-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense. PMID- 16666647 TI - Regulation of Catalase Activity in Leaves of Nicotiana sylvestris by High CO(2). AB - The effect of high CO(2) (1% CO(2)/21% O(2)) on the activity of specific forms of catalase (CAT-1, -2, and -3) (EA Havir, NA McHale [1987] Plant Physiol 84: 450 455) in seedling leaves of tobacco (Nicotiana sylvestris, Nicotlana tabacum) was examined. In high CO(2), total catalase activity decreased by 50% in the first 2 days, followed by a more gradual decline in the next 4 days. The loss of total activity resulted primarily from a decrease in CAT-1 catalase. In contrast, the activity of CAT-3 catalase, a form with enhanced peroxidatic activity, increased 3-fold in high CO(2) relative to air controls after 4 days. Short-term exposure to high CO(2) indicated that the 50% loss of total activity occurs in the first 12 hours. Catalase levels increased to normal within 12 hours after seedlings were returned to air. When seedlings were transferred to air after prolonged exposure to high CO(2) (13 days), the levels of CAT-1 catalase were partially restored while CAT-3 remained at its elevated level. Levels of superoxide dismutase activity and those of several peroxisomal enzymes were not affected by high CO(2). Total catalase levels did not decline when seedlings were exposed to atmospheres of 0.04% CO(2)/5% O(2) or 0.04% CO(2)/1% O(2), indicating that regulation of catalase in high CO(2) is not related directly to suppression of photorespiration. Antibodies prepared against CAT-1 catalase from N. tabacum reacted strongly against CAT-1 catalase from both N. sylvestris and N. tabacum but not against CAT-3 catalase from either species. This observation, along with the rapid changes in CAT-1 and the much slower changes in CAT-3 suggest that one form is not directly derived from the other. PMID- 16666648 TI - Variations in the Alternative Oxidase in Chlamydomonas Grown in Air or High CO(2). AB - Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O(2) uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O(2) uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O(2) uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO(2), the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO(2), which induced a CO(2) concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive. PMID- 16666649 TI - Compartmentation of Nucleotides in Corn Root Tips Studied by P-NMR and HPLC. AB - Corn (Zea mays L.) root tips were subjected to different conditions so that nucleotide levels varied over a wide range. Levels of nucleotides in corn root tips were measured using (31)P nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography. Results indicate: (a) Similar amounts of NTP and sugar nucleotides were observed by in vivo NMR and in extracts. In contrast, a significant amount of NDP observed in root tip extracts was not detected by in vivo NMR. Thus, for a given sample, [NTP]/[NDP] ratios determined in vivo by (31)P-NMR are always higher than ratios observed in extracts, deviating by approximately 4-fold at the highest ratios. The NMR-invisible pool of NDP appeared quite metabolically inert, barely changing in size as total cell NDP changed. We conclude that NDP in corn root tips is compartmented with respect to NMR visibility, and that it is the NMR-visible pool which responds dynamically to metabolic state. The NMR-invisible NDP could either be immobilized (and so have broad, undetectable NMR signals), or be complexed with species that cause the chemical shift of NDP to change (so it does not contribute to the NMR signal of free NDP), or both. (b) (31)P-NMR cannot distinguish between bases (A, U, C, and G) of nucleotides. HPLC analysis of root tip extracts showed that the relative amount of each base in the NTP and NDP pools was quite constant in the different samples. (c) In extracts, for each of the nonadenylate nucleotides, [NTP]/[NDP] was linearly proportional to [ATP]/[ADP], indicating near equilibrium in the nucleoside diphosphokinase (NDPK) reaction. However, the apparent equilibrium constants for the phosphorylation of GDP and UDP by ATP were significantly lower than 1, the true equilibrium constant for the NDPK reaction. Thus, for a given sample, [ATP]/[ADP] approximately [CTP]/[CDP] > [UTP]/[UDP] > [GTP]/[GDP]. This result suggests that the different NDPs in corn root tips do not have equal access to NDPK. PMID- 16666650 TI - Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity. AB - We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested. PMID- 16666651 TI - The glassy state in corn embryos. AB - The possibility is examined whether seeds may survive the desiccated state in part by vitrification, or the formation of a glassy state. Embryos excised from viable corn (Zea mays L.) seeds at low moisture contents show a series of low temperature first- and second-order phase transitions in the differential scanning calorimeter. These embryos produce normal seedlings if moistened. The thermal events can be duplicated almost entirely in both extracted lipids and purified commercial corn oil. They are therefore associated primarily with these bulk lipids, since membrane phospholipids are present in too small an amount to produce a detectable signal. When the bulk lipids have been extracted, a glass transition appears in the remaining material. At low water contents, it occurs above +40 degrees C and systematically falls to below -60 degrees C as the water content of the embryo rises to 20%. These data are consistent with our hypothesis that the desiccated state in seeds is a glassy state, and that imbibition of water reduces the glass transition temperature below ambient, allowing biochemical activity to resume. PMID- 16666652 TI - Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase. AB - The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays- a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate-resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity. PMID- 16666653 TI - The mechanism of herbicide resistance in tobacco cells with a new mutation in the q(b) protein. AB - A new mutant of the psbA gene conferring resistance to 2-chloro-4-(ethylamino)-6 (isopropylamino)-s-triazine (atrazine) was obtained by selection of photomixotrophic tobacco (Nicotiana tabacum cv Samsun NN) cells. The 264th codon AGT (serine) in the wild psbA gene was changed to ACT (threonine) in these mutant tobacco cells. All other higher plants resistant to atrazine exhibit a change to GGT (glycine) in this codon. Measurements of Hill reaction activity and chlorophyll fluorescence showed that the threonine 264-containing plastoquinone serving as secondary stable electron acceptor of PSII (Q(B) protein) had not only strong resistance to triazine-type herbicides but also moderate resistance to substituted urea-type herbicides. Threonine-type Q(B) protein showed especially strong resistance to methoxylamino derivatives of the substituted urea herbicides. The projected secondary structures of the mutant Q(B) proteins indicate that the cross-resistance of threonine 264 Q(B) protein to triazine and urea herbicides is mainly due to a conformational change of the binding site for the herbicides. However, the glycine 264 Q(B) protein is resistant to only triazine herbicides because of the absence of an hydroxyl group and not because of a conformational change. PMID- 16666654 TI - Chloroplast protein import : quantitative analysis of precursor binding. AB - The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose 1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0 degrees C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density. PMID- 16666655 TI - Deposition of Aliphatic Suberin Monomers and Associated Alkanes during Aging of Solanum tuberosum L. Tuber Tissue at Different Temperatures. AB - The effect of temperature on suberization of potato tuber tissue was measured by diffusive resistance and quantitative chemical procedures. The optimum temperature for formation of aliphatic suberin monomers and development of resistance to water vapor conduction was 26.4 degrees C whereas alkane synthesis was optimal at 18.6 degrees C. Low temperatures (<16.6 degrees C) reduced suberin monomer production more than alkane synthesis. PMID- 16666656 TI - Immunochemical Analysis Shows That an ATP/ADP-Translocator Is Associated with the Inner-Envelope Membranes of Amyloplasts from Acer pseudoplatanus L. AB - Pure preparations of intact amyloplasts and chloroplasts, free from mitochondrial contamination, were isolated from cultured cells of the white-wild and green mutant lines of sycamore (Acer pseudoplatanus L.), respectively. A specific rabbit antiserum against yeast mitochondrial cytochrome c(1) only cross-reacted with mitochondrial membranes from the white-wild sycamore cells. The outer and inner envelope-membranes of the two plastid-types were isolated and subsequently analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis to characterize polypeptide patterns in each fraction. Analysis by immunoblotting clearly showed that antiserum against the 29-kilodalton inorganic orthophosphate translocator isolated from pea chloroplasts cross-reacted with a 31-kilodalton polypeptide residing in the inner-envelope membranes from both sycamore chloroplasts and amyloplasts. In contrast, antiserum against the ADP/ATP translocator isolated from mitochondria of Neurospora crassa yielded a positive signal with a 32-kilodalton polypeptide in the inner-membranes isolated from amyloplasts, but not green-mutant chloroplasts. We propose that this 32 kilodalton polypeptide in the amyloplast envelope is a putative ATP/ADP translocator and its possible functional significance is discussed. PMID- 16666657 TI - Accumulation of alpha-Tocopherol in Senescing Organs as Related to Chlorophyll Degradation. AB - alpha-Tocopherol (alpha-T) has been identified, using gas chromatography-mass spectroscopy and (1)H- and (13)C-nuclear magnetic resonance, in senescing leaves of Melia azedarach L. The content of alpha-T increased concomitantly with the breakdown of chlorophyll in senescing Vinca and Melia leaves. An increase in alpha-T was found also in detached Melia leaves, senescing in either light or darkness and in senescing, ethylene-treated orange leaves and fruit. The possibility that phytol, which is released from chlorophyll by chlorophyllase is utilized for the biosynthesis of alpha-T is discussed. Senescing leaves of the low chlorophyllase plants, parsley and tobacco, did not contain alpha-T in measureable amounts. PMID- 16666658 TI - Supercooling characteristics of isolated peach flower bud primordia. AB - The amount of unfrozen water in dormant peach (Prunus persica [L.] Batsch, cv Redhaven) flower buds, isolated primordia, and bud axes was determined during freezing using pulse nuclear magnetic resonance methods. Differential thermal analysis studies were conducted on whole buds and isolated primordia in the presence of ice nucleation. The results showed that some of the water in isolated primordia remained supercooled in the presence of ice nucleation. Although most tissue water froze (57.5%) following ice nucleation at -2.5 degrees C, a considerable amount of water was found to supercool. In the presence of ice nucleation, increased hydration of isolated primordia resulted in the elimination of the supercooling characteristic. The structural integrity of isolated primordia appeared to be essential for supercooling. PMID- 16666659 TI - Physiological factors determining hydrogenase activity in nitrogen-fixing heterocystous cyanobacteria. AB - Four species of nitrogen-fixing heterocystous cyanobacteria were compared with respect to induction of hydrogenase activity. Two of the strains contained phycoerythrin and built up high levels of carbohydrate storage material when grown in batch culture under nitrogen-fixing conditions and continuous illumination. These strains did not exhibit hydrogenase activity. Lack of activity in the phycoerythrin-containing species was determined by cell-free assays measuring both hydrogen-evolving and hydrogen-uptake activities. Apparently, expression of hydrogenase is negatively correlated with the carbohydrate pool present and concurrent respiration. Furthermore, there is an apparent relationship between the presence of phycoerythrin, carbohydrate accumulation, and the absence of hydrogenase activity. PMID- 16666660 TI - Abscisic Acid Biosynthesis in Isolated Embryos of Zea mays L. AB - Previous labeling experiments with (18)O(2) have supported the hypothesis that stress-induced abscisic acid (ABA) is synthesized through an indirect pathway involving an oxygenated carotenoid (xanthophyll) as a precursor. To investigate ABA formation under nonstress conditions, an (18)O(2) labeling experiment was conducted with isolated embryos from in vitro grown maize (Zea mays L.) kernels. Of the ABA produced during the incubation in (18)O(2), three-fourths contained a single (18)O atom located in the carboxyl group. Approximately one-fourth of the ABA synthesized during the experiment contained two (18)O atoms. These results suggest that ABA synthesized in maize embryos under nonstress conditions also proceeds via the indirect pathway, requiring a xanthophyll precursor. It was also found that the newly synthesized ABA was preferentially released into the surrounding medium. PMID- 16666661 TI - Separation and characterization of four hexose kinases from developing maize kernels. AB - Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low K(m) values [K(m)(glucose) = 117 micromolar, K(m)(ATP) = 66 micromolar] whereas the other form had much higher K(m) values [K(m)(glucose) = 750 micromolar, K(m)(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The K(m)(fructose) was about 130 micromolar and the K(m)(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [K(i)(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ. PMID- 16666662 TI - Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina. AB - The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (beta(i)) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 +/- 0.029 (mean +/- standard error). At this pH, beta(i) was minimal and amounted to 0.933 +/- 0.11 millimoles H(+)/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 +/- 0.028. beta(i) was minimal in this pH region and amounted to 14.2 +/- 0.80 millimoles H(+)/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 +/- 0.026, where beta(i) was 20.35 +/- 2.66 millimoles H(+)/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X 100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the beta(i) values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H(+) pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells. PMID- 16666663 TI - Identification of a naturally occurring inhibitor of the conversion of 1 aminocyclopropane-1-carboxylic Acid to ethylene by carnation microsomes. AB - During cell-free experiments with membranes isolated from carnation petals (Dianthus caryophillus L. cv White Sim), the conversion of 1-aminocyclopropane-1 carboxylic acid into ethylene was blocked by a factor derived from the cytosol. Subsequent characterization of the inhibitor revealed that its effect was concentration dependent, that it was water soluble, and that it could be removed from solution by dialysis and addition of polyvinyl-polypyrrolidone. Activity profiles obtained after solvent partitioning over a range of pH values and after chromatography on silica gel, size exclusion gel, and ion exchange resins revealed that the inhibitor was a highly polar, low molecular weight species that was nonionic at low pH and anionic at pH values above 8. Use of selected solvent systems during paper and thin layer chromatography combined with specific spray reagents tentatively identified the compound as a hydroxycinnamic acid derivative. Base hydrolysis and subsequent comparison with known standards by high performance liquid chromatography, gas-liquid chromatography, and ultraviolet light spectroscopy established that the inhibitor was a conjugate with a ferulic acid moiety. Release of ferulic acid following treatment with beta glucosidase also indicated the presence of a glucose moiety, and unequivocal identification of the inhibitor as 1-O-feruloyl-beta-d-glucose was confirmed by gas chromatography-mass spectroscopy and by ultraviolet light, (1)H-, and (13)C- nuclear magnetic resonance spectroscopy. Feruloylglucose constituted about 0.1% of the dry weight of stage III (preclimacteric) carnation petals, but concentrations fell sharply during stage IV (climacteric), when ethylene production peaks and the flowers senesce. In a reaction mixture containing microsome-bound ethylene forming enzyme system, 98% of all ethylene production was abolished in the presence of 50 mum concentrations of the inhibitor. PMID- 16666664 TI - Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves. AB - We have examined the effect of mild water stress on photosynthetic chloroplast reactions of intact Phaseolus vulgaris leaves by measuring two parameters of ribulose bisphosphate (RuBP) carboxylase activity and the pool sizes of RuBP, 3 phosphoglycerate (PGA), triose phosphates, hexose monophosphates, and ATP. We also tested for patchy stomatal closure by feeding (14)CO(2). The k(cat) of RuBP carboxylase (moles CO(2) fixed per mole enzyme per second) which could be measured after incubating the enzyme with CO(2) and Mg(2+) was unchanged by water stress. The ratio of activity before and after incubation with CO(2) and Mg(2+) (the carbamylation state) was slightly reduced by severe stress but not by mild stress. Likewise, the concentration of RuBP was slightly reduced by severe stress but not by mild stress. The concentration of PGA was markedly reduced by both mild and severe water stress. The concentration of triose phosphates did not decline as much as PGA. We found that photosynthesis in water stressed leaves occurred in patches. The patchiness of photosynthesis during water stress may lead to an underestimation of the effect of stomatal closure. We conclude that reductions in whole leaf photosynthesis caused by mild water stress are primarily the result of stomatal closure and that there is no indication of damage to chloroplast reactions. PMID- 16666665 TI - Mild Water Stress of Phaseolus vulgaris Plants Leads to Reduced Starch Synthesis and Extractable Sucrose Phosphate Synthase Activity. AB - Mild water stress, on the order of -1.0 megapascals xylem water potential, can reduce the rate of photosynthesis and eliminate the inhibition of photosynthesis caused by O(2) in water-stress-sensitive plants such as Phaseolus vulgaris. To investigate the lack of O(2) inhibition of photosynthesis, we measured stromal and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase, and partitioning of newly fixed carbon between starch and sucrose before, during, and after mild water stress. The extractable activity of the fructose bisphosphatases was unaffected by mild water stress. The extractable activity of SPS was inhibited by more than 60% in plants stressed to water potentials of -0.9 megapascals. Water stress caused a decline in the starch/sucrose partitioning ratio indicating that starch synthesis was inhibited more than sucrose synthesis. We conclude that the reduced rate of photosynthesis during water stress is caused by stomatal closure, and that the restriction of CO(2) supply caused by stomatal closure leads to a reduction in the capacity for both starch and sucrose synthesis. This causes the reduced O(2) inhibition and abrupt CO(2) saturation of photosynthesis. PMID- 16666666 TI - Preparation and Properties of Antibodies against Indoleacetic Acid (IAA)-C5-BSA, a Novel Ring-Coupled IAA Antigen, as Compared to Two Other Types of IAA-Specific Antibodies. AB - Two types of indoleacetic acid (IAA) antigens have been described in the literature which show marked differences with respect to antibody specificity. In this communication an alternative antigen design is described. In the so-called IAA-C5-BSA conjugate, both the acetic acid group and the pyrrole moiety are presented free, as the covalent linkage between IAA and the carrier molecule is introduced in the benzene moiety. Antibodies were elicited in rabbits against this novel antigen. Using cross-reactivity data and the strategy of successive approximation for the measurements of auxin levels in the internodes of light grown broad bean (Vicia faba L. cv Superfine), the three types of antibodies are compared. PMID- 16666667 TI - Mannose analog 1-deoxymannojirimycin inhibits the Golgi-mediated processing of bean storage glycoproteins. AB - The asparagine-linked oligosaccharide chains of glycoproteins can be processed to form a wide variety of structures. The Golgi complex is the main compartment involved in this processing. In mammalian cells the first enzyme acting along the Golgi processing pathway is mannosidase I, whose action is a prerequisite for any further processing and which is inhibited by the mannose analog 1 deoxymannojirimycin (dMM). To have insights into the processing pathway in plant cells, we have studied the in vivo effect of dMM on the processing of the bean (Phaseolus vulgaris) storage proteins phaseolin and phytohemagglutinin, two well characterized plant glycoproteins. Cotyledons obtained from developing seeds were labeled with radioactive leucine, glucosamine, or fucose in the presence or absence of dMM. Treatment with dMM fully inhibited the acquisition of resistance to endo-beta-N-acetylglucosaminidase H by phaseolin and phytohemagglutinin and the incorporation of fucose into protein. Furthermore, the apparent molecular weight of the polypeptides of phaseolin and phytohemagglutinin synthesized in dMM treated cotyledons was consistent with the exclusive presence of oligommanose oligosaccharide chains which had not been processed in the Golgi complex. The inhibition of processing did not prevent exit from the Golgi complex, and most probably the storage proteins were correctly targeted to the protein bodies as indicated by the post-translational polypeptide cleavage of phaseolin. These results indicate that the action of a mannosidase is the first obligatory step of Golgi-mediated processing also in a plant cell and, together with data obtained in other laboratories on the in vitro specificity of glycosidases and glycosyltransferases present in the Golgi complex of plant cells, support the hypothesis that the key early reactions in Golgi-mediated processing are similar if not identical in plants and mammals. PMID- 16666668 TI - Growth Retardant-Induced Changes in Phototropic Reaction of Vigna radiata Seedlings. AB - The effect of growth retardants on phototropism has been studied in mung bean (Vigna radiata) seedlings. Ancymidol, tetcyclacis, and paclobutrazol inhibited phototropism while AMO 1618 and CCC were ineffective. The fluence-response relationships for phototropism of etiolated seedlings were similar to those previously described for monocots and other dicots. Ancymidol caused a shift in the maximum phototropic response to higher fluence of light. It is suggested that ancymidol may affect phototropism through an effect on the photoreceptor system. PMID- 16666669 TI - Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L. AB - The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba. PMID- 16666670 TI - Development of water conducting capacity in the root systems of young plants of corn and some other c4 grasses. AB - Development of the primary and early nodal roots was studied in Zea mays L., Zea mexicana (Schrad.) Reeves & Mangelsd., Sorghum bicolor (L.) Moench., and Sorghum sudanese (Piper) Stapf. in relation to shoot development. In all the types studied all roots reached lengths of about 30 centimeters before the late metaxylem (LMX) was open, and young plants with total root lengths of around 100 centimeters had almost no open LMX. On average, corn seedlings with up to 36 square centimeters of leaf had no open LMX. The name "immature apices" is suggested for such long but not fully functional roots. In plants up to 50 days old a fairly constant proportion of less than half the total root length had open LMX. A pilot study of stomatal resistance on days of high evaporative demand suggested that young seedlings may show higher resistance than older plants in the afternoon. Estimates of longitudinal permeability of corn roots with only early metaxylem vessels open indicate very steep gradients of water potential would develop under such conditions. PMID- 16666671 TI - Sodium Stimulates Growth of Amaranthus tricolor L. Plants through Enhanced Nitrate Assimilation. AB - Effects of Na application on the capacity of NO(3) (-) assimilation were studied in Na-deficient Amaranthus tricolor L. cv Tricolor plants. On day 30 after germination, Na-deficient A. tricolor plants received either 0.5 millimolar NaCl or KCl. The level of nitrate reductase activity doubled within 24 hours by the addition of Na and the enhanced level was maintained thereafter. When the plants were exposed to 2 millimolar (15)NO(3) (-), total (15)N taken up by the plants was greater in the Na-treated plants than in the K-treated plants within 24 hours of the Na treatment. Incorporation of (15)N into the 80% ethanol-insoluble nitrogen fraction of the Na-treated plants in the light period was about 260% of those of the K-treated plants indicating greater capacity of NO(3) (-) assimilation in the Na-treated plants. From these results, it was demonstrated that Na application to the Na-deficient A. tricolor plants promoted NO(3) (-) reduction and its subsequent assimilation into protein, resulting in growth enhancement. PMID- 16666672 TI - Sucrose metabolism in lima bean seeds. AB - Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase. PMID- 16666673 TI - Identification of actively filling sucrose sinks. AB - Certain actively filling plant sucrose sinks such as a seed, a tuber, or a root can be identified by measuring the uridine diphosphate and pyrophosphate dependent metabolism of sucrose. Sucrolysis in both active and quiescent sucrose sinks was tested and sucrose synthase was found to be the predominant sucrose breakdown activity. Sucrolysis via invertases was low and secondary in both types of sinks. Sucrose synthase activity dropped markedly, greater than fivefold, in quiescent sinks. The tests are consistent with the hypothesis that the sucrose filling activity, i.e. the sink strength, of these plant sinks can be measured by testing the uridine diphosphate and pyrophosphate-dependent breakdown of sucrose. Measuring the initial reactions of sucrolysis shows much promise for use in agriculture crop and tree improvement research as a biochemical test for sink strength. PMID- 16666674 TI - Specificity of aspartate aminotransferases from leguminous plants for 4 substituted glutamic acids. AB - Aspartate aminotransferase (glutamate-oxalacetate transaminase) was partially purified from extracts of germinating seeds of peanut (Arachis hypogaea), honey locust (Gleditsia triacanthos), soybean (Glycine max), and Sophora japonica. The ability of these enzyme preparations, as well as aspartate aminotransferase purified from pig heart cytosol, to use 4-substituted glutamic acids as amino group donors and their corresponding 2-oxo acids as amino group acceptors in the aminotransferase reaction was measured. All 4-substituted glutamic acid analogs tested were poorer substrates than was glutamate or 2-oxoglutarate. 2-Oxo-4 methyleneglutarate was least effective (lowest relative V(m)/K(m)) as a substrate for the enzyme from peanuts and honey locust, which are the two species studied that accumulate 4-methyleneglutamic acid and 4-methyleneglutamine. Of the different aminotransferases tested, the enzyme from honey locust was the least active with 2-oxo-4-hydroxy-4-methylglutarate, the corresponding amino acid of which also accumulates in that species. These results suggest that transamination of 2-oxo-4-substituted glutaric acids is not involved in the biosynthesis of the corresponding 4-substituted glutamic acids in these species. Rather, accumulation of certain 4-substituted glutamic acids in these instances may be, in part, the result of the inefficacy of their transamination by aspartate aminotransferase. PMID- 16666675 TI - Photosynthetic Plasticity in Flaveria brownii: Growth Irradiance and the Expression of C(4) Photosynthesis. AB - Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO(2) compensation point and the inhibition of photosynthesis by 21% O(2) were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C(4) cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO(2) compensation point and the degree of O(2) inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO(2) between Rubisco of the C(3) pathway and phosphoenolpyruvate carboxylase of the C(4) cycle was determined by exposing leaves to (14)CO(2) for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that approximately 94% of the CO(2) was fixed by the C(4) cycle in high light grown plants, versus approximately 78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C(4) photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C(4)-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth. PMID- 16666676 TI - Net CO(2) Assimilation and Carbohydrate Partitioning of Grapevine Leaves in Response to Trunk Girdling and Gibberellic Acid Application. AB - Leaf net CO(2) assimilation rate (A), stomatal conductance (g(s)), carboxylation efficiency, and foliar nonstructural carbohydrates were measured on mature, field grown Vitis vinifera L. (cv Thompson Seedless) vines that had been trunk girdled, sprayed with gibberellic acid, or both, shortly after anthesis. Girdling reduced A, g(s), and carboxylation efficiency when measured 2 weeks after imposition of the treatments. Diurnal measurements indicated that A of girdled vines was less than that of control vines between 1000 and 1800 hours. Gibberellic acid mitigated the depressing effect of girdling on g(s) during the same diurnal measurements. The concentrations of foliar carbohydrates were greatest for the girdled vines, followed by the combination treatment and were lowest for the control vines. Foliar carbohydrates were greater for girdled vines 4 weeks after the treatments were imposed, however, by this time there was no significant difference in A between the control and girdled vines. Two and 4 weeks after the experiment was initiated root carbohydrate concentrations were less for the girdled vines when compared to the control vines. The data indicate that the reduction in A of girdled grapevines is not associated with the accumulation of leaf nonstructural carbohydrates following the girdling treatment. PMID- 16666677 TI - Cytochrome P-450 from the Mesocarp of Avocado (Persea americana). AB - The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450. PMID- 16666678 TI - Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation: Carbon Isotope Discrimination in Photosynthesis and Respiration by the N-Limited Green Alga Selenastrum minutum. AB - The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO(2) fixation and respiratory CO(2) release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and alpha-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C(3) organism. PMID- 16666679 TI - Inhibition of chloroplastic respiration by osmotic dehydration. AB - The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of (14)CO(2) evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [(14)C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO(2) evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH(4)Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6 bisphosphatase/phosphofructokinase junction. PMID- 16666680 TI - Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine. AB - A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of (15)N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253 261) for derivatization of glutamine and asparagine with N-methyl-N-(tert butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [(14)N]asparagine and [(14)N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-(15)N- and double-(15)N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[(15)N] abundance of these amino acids was determined. We demonstrate that this approach to (15)N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-(15)N:double-(15)N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with (15)N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [(15)N]H(4) (+) are (14)N-enriched and must be derived primarily from protein turnover. PMID- 16666681 TI - Effect of light on the chloroplast division cycle and DNA synthesis in cultured leaf discs of spinach. AB - The effects of light on both the division cycle of chloroplasts and the synthesis of chloroplast DNA were investigated in cultured discs taken from the distal end of 2-centimeter spinach (Spinacia oleracea) leaves. Comparisons were made of discs cultured for a maximum of 4 days in a shaking liquid medium under continuous white light, darkness, and of discs cultured for 1 day in light following 3 days in darkness. In continuous white light the shortest generation time of chloroplasts observed in this study was 19.4 hours and the duration of spherical, ovoid, and dumbbell-shaped stages in the division cycle were 13.4, 2.8, and 3.1 hours, respectively. In darkness the generation times of chloroplasts extended to 51.5 hours. Under these conditions the duration of spherical, ovoid, and dumbbell-shaped stages were 22.8, 8.4, and 20.2 hours, respectively, suggesting that in darkness the separation of dumbbell-shaped chloroplasts may be the rate limiting step. When discs cultured in the dark were transferred to light, most dumbbell-shaped chloroplasts separated into daughter chloroplasts in less than an hour. Measurements of chloroplast DNA established that the cellular level of chloroplast DNA increased 10-fold over the 4 days of culture in continuous white light. Comparisons of the plastids of dark and light grown discs showed that the synthesis of chloroplast DNA was enhanced by light. Observations of DAPI stained dividing chloroplasts indicate that DNA partitioning can take place during the final stage of chloroplast division and that it does not precede plastid division. PMID- 16666682 TI - Characterization of potassium-dependent currents in protoplasts of corn suspension cells. AB - Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K(+) channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than -100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than -60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K(+) channels. These three currents, which are not generated by the plasmalemma H(+)-ATPase, are likely to assist in the regulation of the cellular K(+) fluxes and membrane potential. PMID- 16666683 TI - Localization of ferredoxin isoproteins in mesophyll and bundle sheath cells in maize leaf. AB - Four ferredoxin isoproteins were identified in the C(4) plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etiolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of etiolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C(4) plant. PMID- 16666684 TI - Correlation between Root-Generated Ionic Currents, pH, Fusicoccin, Indoleacetic Acid, and Growth of the Primary Root of Zea mays. AB - Correlations between root-generated ionic currents, extracellular pH, indoleacetic acid, fusicoccin, and growth were investigated. Current consistently entered the meristematic and elongating tissues of intact growing roots of Zea mays cv Golden Bantam. Mature root regions generated the outward limb of the current loop. Ion-substitution and pH-profile experiments suggested that the bulk of the ionic current was carried by H(+). Calcium ions did not carry current, but calcium may regulate the proton circulation since the proton current density was slightly larger in calcium-depleted media. Increased root elongation at low pH was associated with increased current density and an extended zone of inward current. Conversely decreased elongation at high pH was associated with a reduced current density and a more restricted zone of inward current. The effect of the fungal toxin fusicoccin was to increase the current density of the inward limb of the ion current and to increase root extension. Concentrations of indoleacetic acid that reduced root growth, also reduced the density of the inward current and shortened the inward current zone. The results emphasize the point that roots are electrically contiguous over many millimeters and that the electrophysiology of root growth is best studied in intact root systems. PMID- 16666685 TI - Regulation of pea mitochondrial pyruvate dehydrogenase complex : does photorespiratory ammonium influence mitochondrial carbon metabolism? AB - Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na(+). NH(4) (+), at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K(+) in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na(+) was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH(4) (+) is discussed in relation to a possible regulatory interaction between photorespiratory NH(4) (+) production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex. PMID- 16666686 TI - Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii. AB - Mass spectrometric measurements of dissolved free (13)CO(2) were used to monitor CO(2) uptake by air grown (low CO(2)) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the (13)CO(2) concentration in the medium to close to zero. Similar results were obtained with low CO(2) cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO(2) uptake revealed that the removal of CO(2) from the medium in the light was due to selective and active CO(2) transport rather than uptake of total dissolved inorganic carbon. In the light, low CO(2) cells and protoplasts incubated with carbonic anhydrase took up CO(2) at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO(2) uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO(2) cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO(2) compensation point. During the linear uptake of CO(2), low CO(2) cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O(2) evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O(2) evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO(2) uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO(2) had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O(2) evolution. These results show low CO(2) cells of Chlamydomonas are able to transport both CO(2) and HCO(3) (-) but CO(2) is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO(2) from the dehydration of HCO(3) (-). PMID- 16666688 TI - Involvement of Cl in the Increase in Proline Induced by ABA and Stimulated by Potassium Chloride in Barley Leaf Segments. AB - Stimulation by sodium or potassium chloride of the ABA-induced increase in proline was synergistically enhanced by CaCl(2) or MgCl(2) as well as by 1,3 bis[tris(hydroxymethyl)methylamino] propane chloride (BTP-Cl), N-methyl-d glucamine chloride (NMG-Cl), or 2-amino-2-hydroxymethyl-1,3-propandiol chloride (TRIS-Cl). This enhancing effect did not depend on the osmolarity and occurred when Cl(-) was higher than K(+) in the incubation medium, but not vice versa. When CaCl(2) or MgCl(2) or NMG-Cl were added, the higher the Cl(-):K(+) ratio in the external solution the higher was the increase in proline. When the excess of Cl(-) to K(+) was obtained with BTP-Cl the highest enhancing effect resulted with a Cl(-):K(+) ratio of 3:1 while, at a 5:1 ratio, the KCl stimulation was completely suppressed. The inhibiting effect of proline accumulation by NH(4) (+) and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene was reversed to varying degrees depending on the magnitude of the excess of Cl(-) on K(+) concentration in the medium. Also, the inhibition of proline accumulation obtained by tetraethylammonium chloride, monensin, and d-mannose was similarly reverted. These data suggest that Cl(-) elicits an increase in ABA-induced proline which needs the simultaneous presence of K(+) (or Na(+)) to take place. PMID- 16666687 TI - Nature of the light-induced h efflux and na uptake in cyanobacteria. AB - We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H(+) efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C(i)). Light and C(i) stimulated the uptake of sodium, but the stimulation by C(i) was observed only when C(i) was present at the time sodium was provided. Amiloride, a potent inhibitor of Na(+)/H(+) antiport and Na(+) channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na(+)/H(+) antiport and Na(+) uniport. PMID- 16666689 TI - Effects of the Phosphoenolpyruvate Carboxylase Inhibitor 3,3-Dichloro-2 (Dihydroxyphosphinoylmethyl)propenoate on Photosynthesis: C(4) Selectivity and Studies on C(4) Photosynthesis. AB - The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Panicum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of 1 millimolar PEP and 1 millimolar HCO(3) (-). When fed to leaf sections via the transpiration stream, DCDP at 1 millimolar strongly inhibited photosynthesis in C(4) species (79-98% inhibition for a range of seven C(4) species), but only moderately in C(3) species (12-46% for four C(3) species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C(4)) net photosynthesis to intercellular pCO(2) showed that carboxylation efficiency, as well as the CO(2) saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C(4) species is directly related to PEP carboxylase activity. A fivefold increase in intercellular pCO(2) over that occurring in P. miliaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO(2) by the bundle sheath cells makes any significant contribution to photosynthetic CO(2) assimilation in C(4) species. The results support the concept that C(4)-selective herbicides may be developed based on inhibitors of C(4) pathway reactions. PMID- 16666690 TI - In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters. AB - (31)P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6 P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from (31)P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in (31)P NMR studies. PMID- 16666691 TI - Cytokinin Production by Bradyrhizobium japonicum. AB - Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH 20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay. PMID- 16666692 TI - Evidence that Ribulose 1,5-Bisphosphate (RuBP) Binds to Inactive Sites of RuBP Carboxylase in Vivo and an Estimate of the Rate Constant for Dissociation. AB - The binding of ribulose 1,5-bisphosphate (RuBP) to inactive (noncarbamylated) sites of the enzyme RuBP carboxylase in vivo was investigated in Spinacia oleracea and Helianthus annuus. The concentrations of RuBP and inactive sites were determined in leaf tissue as a function of time after a change to darkness. RuBP concentrations fell rapidly after the change to darkness and were approximately equal to the concentration of inactive sites after 60 s. Variations in the concentration of inactive sites, which were induced by differences in the light intensity before the light-dark transition, correlated with the concentration of RuBP between 60 and 120 s after the change to darkness. These data are discussed as evidence that RuBP binds to inactive sites of RuBP carboxylase in vivo. After the concentration of RuBP fell below that of inactive sites (at times longer than 60 s of darkness), the decline in RuBP was logarithmic with time. This would be expected if the dissociation of RuBP from inactive sites controlled the decline in RuBP concentration. These data were used to estimate the rate constant for dissociation of RuBP from inactive sites in vivo. PMID- 16666693 TI - Involvement of the Plasma Membrane ATPase in the Osmoregulatory Mechanism of the Alga Dunaliella salina. AB - The unicellular halotolerant alga Dunaliella salina recovers normally from a hypertonic shock even when suspended in NaCl and buffer only. Furthermore, addition of Cu(2+), valinomycin and KCl, or permeable ions such as methyltriphenylphosphonium or thiocyanate, do not affect the recovery. However, treatment with two specific inhibitors of the plasma membrane adenosine triphosphatase (ATPase), diethylstilbestrol, or vanadate, fully inhibit the recovery. The inhibition is manifested by the inability of the cells to both synthesize glycerol and return to their original volume. The inhibitions are nonlethal, reversible and equally effective in the dark or the light. Since the plasma membrane ATPase is the only enzyme known to be inhibited by both diethylstilbestrol and vanadate, it is concluded that its activity is essential for the recovery of Dunaliella from a hypertonic shock. Mechanisms by which the plasma membrane ATPase may participate in the activation of glycerol production in the algae are discussed. PMID- 16666694 TI - Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella. AB - Neither Dunaliella cells grown with 5% CO(2) nor their isolated chloroplasts had a CO(2) concentrating mechanism. These cells primarily utilized CO(2) from the medium because the K((0.5)) (HCO(3) (-)) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K((0.5)) CO(2) was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO(2) concentrating mechanism was present that decreased the K(0.5) (CO(2)) to about 0.5 micromolar and K(0.5) (HCO(3) (-)) to 11 micromolar at pH 8. These K(0.5) values suggest that air-adapted cells preferentially concentrated CO(2) but could also use HCO(3) (-) from the medium. Chloroplasts isolated from air-adapted cells had a K((0.5)) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO(2). Chloroplasts from air-adapted cells, but not CO(2)-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO(2) concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO(2) concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO(2) concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport. PMID- 16666695 TI - Studies on the entry of fructose-2,6-bisphosphate into chloroplasts. AB - The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P(2)) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P(2) controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate, fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P(2) is present mainly in the cytosol. Here we present evidence that Fru-2,6-P(2) can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P(2) (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P(2) in the stroma. Elevated plastid Fru-2,6-P(2) levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P(2) enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6-P(2) on chloroplast enzymes. PMID- 16666696 TI - Dependence of Ethanolic Fermentation, Cytoplasmic pH Regulation, and Viability on the Activity of Alcohol Dehydrogenase in Hypoxic Maize Root Tips. AB - We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was like-wise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed. PMID- 16666697 TI - Evidence that the rb Locus Alters the Starch Content of Developing Pea Embryos through an Effect on ADP Glucose Pyrophosphorylase. AB - The aim of this work was to discover whether the rb locus of peas (Pisum sativum L.) affects seed starch content through action on an enzyme of starch synthesis in the developing embryo. The phenotypic effects of this locus are like those of the better characterised, unlinked r locus, which affects seed starch content through action on starch-branching enzyme. Embryos recessive at one or both of these loci (RRrbrb, rrRbRb, rrrbrb) have lower starch contents from an early stage of development than embryos dominant at these loci (RRRbRb). Maximum catalytic activities of enzymes of the pathway from sucrose to starch (sucrose synthase EC 2.4.1.13, UDP glucose pyrophosphorylase EC 2.7.7.9, ADP glucose pyrophosphorylase EC 2.7.7.27, ADP glucose-starch synthase EC 2.4.1.21, starch branching enzyme EC 2.4.1.18) were compared in developing embryos of three lines of rbrb peas and four lines of RbRb peas. The only consistent difference between the two sorts of embryo was in the activity of ADP glucose pyrophosphorylase, which was at least tenfold lower in rbrb than in RbRb embryos. The activity in rbrb embryos was in most cases less than the estimated rate of starch synthesis of RRRbRb embryos. We conclude that the effect of the rb locus on the starch content of pea seeds is mediated through an alteration in the activity of ADP glucose pyrophosphorylase in the developing embryo. PMID- 16666698 TI - Amino Acid and Peptide uptake in the scutella of germinating grains of barley, wheat, rice, and maize. AB - Scutella separated from germinating grains of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) took up the four amino acids and the three peptides tested from incubation media. The uptake of amino acids by wheat scutella was similar to that of barley scutella and was via at least four uptake systems: two nonspecific amino acid uptake systems, one system specific for proline, and another system specific for basic amino acids. The scutellum of rice apparently has two nonspecific systems and a system specific for the basic amino acids, but the proline-specific system is lacking. The scutellum of maize seems to have the same systems as the scutellum of rice, but one (or both) of the nonspecific systems differs from that of the other species studied in taking up arginine only slowly. No great differences were observed in the uptake of peptides in the four species studied. The rates of uptake of different amino acids and peptides were of the same order of magnitude in the four cereals. The fact that carboxypeptidase activities in the endosperms of wheat and barley are 20-to 100-fold higher than those in rice and maize, does thus not seem to be reflected in the uptake properties of the scutella. PMID- 16666699 TI - Biosynthesis of the Tonoplast H-ATPase from Oats. AB - To determine whether the tonoplast-type H(+)-ATPase was differentially synthesized in various parts of the oat seedling, sections of 4-day-old oat (Avena sativa L. var Lang) seedlings were labeled in vivo with [(35)S]methionine and ATPase subunits were precipitated with polyclonal antisera. ATPase subunits were detected in all portions of the seedling with the exception of the seed. Lesser amounts of the 60 and 72 kilodalton polypeptides of the ATPase were found in apical regions (0-5 millimeter) than in maturing regions (10-15, or 20-25 millimeter from the tip) of the roots or shoots. To initiate a study of the biosynthesis of the ATPase, the intracellular site of synthesis for two peripheral ATPase subunits was investigated. Poly(A) RNA from either free or membrane-bound polysomes was isolated and translated in vitro. Message encoding the 72 kilodalton (catalytic) subunit was found predominantly in mRNA isolated from membrane-bound polysomes. In contrast, the message for the 60 kilodalton (putative regulatory) subunit was found predominantly on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences (limit of resolution, approximately 1 kilodalton), suggesting the absence of cleaved precursors for the 72 or 60 kilodalton subunits. These data suggest a complex mechanism for the synthesis and assembly of the tonoplast ATPase. PMID- 16666700 TI - Characterization of a meta-Fluorotyrosine-Tolerant Cell Culture of Eschscholtzia californica Cham. AB - A cell line of Eschscholtzia californica selected for meta-fluorotyrosine (MFT) tolerance was found to have 10-fold increased levels of phenylalanine and tyrosine compared to the parent line, while most other amino acids were only increased 2-fold. Tracer experiments with shikimic acid in the presence of MFT showed that the biosynthesis of the aromatic amino acids was not impaired in the tolerant line. Feeding experiments with phenylalanine, tyrosine, or shikimic acid also revealed a reduced turnover of the pools of the aromatic amino acids in the variant. Thus undisturbed de novo biosynthesis of the aromatic amino acids and dilution of toxic effects of MFT by the enlarged pool sizes seemed to be the main reason for the acquired tolerance. Despite the enlarged availability of the precursor tyrosine, formation of the benzophenanthridine alkaloids was enhanced neither in the growth nor in the production medium. PMID- 16666701 TI - A role for ethylene in the metabolism of cyanide by higher plants. AB - The action of ethylene on the capacity of plant tissues to metabolize cyanide to beta-cyanoalanine was examined. Beta-cyanoalanine synthase (EC 4.4.1.9) catalyzes the reaction between cyanide and cysteine to form beta-cyanoalanine and hydrogen sulfide. Levels of beta-cyanoalanine synthase activity in tissues of 6 day old etiolated pea (Pisum sativum) seedlings were enhanced severalfold by 1 microliter per liter ethylene. The promotive effect of ethylene increased with increasing ethylene concentrations from 0.01 to 100 microliters per liter and with the period of exposure from 3 to 24 hours. Ethylene enhanced beta-cyanoalanine synthase activity in all regions of the seedling (shoots and roots, internodal regions, cotyledons). The promotive effect was eliminated by norbornadiene, a competitive inhibitor of ethylene action. Levels of beta-cyanoalanine synthase in seedlings of four other dicots (Phaseolus aureas, Glycine max, Lactuca sativa, Sinapis arvensis) and two monocots (Hordeum vulgares, Triticum aestivum) were also increased in response to ethylene. Our results suggest an important regulatory role for ethylene in the metabolism of cyanide by higher plants. PMID- 16666702 TI - Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. AB - The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M(r) of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states (as on protein blots). AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase. PMID- 16666703 TI - Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves. AB - Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. DeltapH-dependent sucrose transport exhibited saturation kinetics with an apparent K(m) of 1.20 +/- 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. DeltapH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because DeltapH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K(+), Mg(2+)-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue. PMID- 16666704 TI - Photomodulation of axis extension in sparse canopies : role of the stem in the perception of light-quality signals of stand density. AB - A fiber optic probe inserted into plant tissues was used to investigate the effects of canopy density on the light environment in different organs. The red:far-red ratio inside the stem of Datura ferox L. seedlings and the estimated phytochrome photoequilibrium were strongly reduced by the presence of neighbors forming canopies too sparse to cause any mutual shading at the level of the leaves. In such canopies, changes in plant density had little effects on the light regime inside the leaves of the succulent Aeonium haworthii (S.D.) Webb et Berth., particularly when the lamina was kept nearly normal to the direct rays of the sun. In field experiments using D. ferox and Sinapis alba L. seedlings, the elongation of the internodes responded to various types of localized light quality treatments that simulated different plant densities in sparse canopies. The responses were quantitatively similar to those elicited by changes in plant density. The evidence supports the hypothesis that, in stands formed by plants of similar size, the red:far-red ratio of the light that impinges laterally on the stems is among the earliest environmental cues that allow plants to detect local canopy density and adjust axis extension accordingly. PMID- 16666705 TI - P-Nuclear Magnetic Resonance Determination of Phosphate Compartmentation in Leaves of Reproductive Soybeans (Glycine max L.) as Affected by Phosphate Nutrition. AB - Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using (31)P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition ( betaine aldehyde --> betaine. Our previous experiments with intact chloroplasts, and in vivo(18)O(2) labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O(2) and reduced ferredoxin, the stromal fraction from spinach (Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of (18)O from (18)O(2) by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea (Pisum sativum L.), a species that does not accumulate betaine. The spinach choline oxidizing enzyme was stimulated by 10 millimolar Mg(2+), had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin. PMID- 16666759 TI - Role of Metabolites in the Reversible Light Activation of Pyruvate, Orthophosphate Dikinase in Zea mays Mesophyll Cells in Vivo. AB - Whole leaf and mesophyll cell concentrations of pyruvate, phosphoenolpyruvate (PEP), ATP, and ADP were determined in Zea mays during the reversible light activation of pyruvate, orthophosphate dikinase in vivo. Mesophyll cell levels of the four metabolites were estimated by extrapolation from values in freeze quenched leaf samples that were fractionated by differential filtration through nylon mesh nets (adapted from M Stitt, HW Heldt [1985] Planta 164: 179-188). During the 3 minutes required for complete light activation of dikinase, pyruvate levels in the mesophyll cell decreased (from 166 +/- 15 to 64 +/- 10 nanomoles per milligram of chlorophyll [nmol/mg Chl]) while PEP levels increased (from 31 +/- 4 to 68 +/- 4 nmol/mg Chl, with a transient burst of 133 +/- 16 nmol/mg Chl at 1 minute). Mesophyll cell levels of ATP increased (from 22 +/- 4 to 48 +/- 3 nmol/mg Chl) and ADP levels decreased (from 16 +/- 4 to 7 +/- 6 nmol/mg Chl) during the first minute of illumination. Upon darkening of the leaf and inactivation of dikinase, pyruvate levels initially increased in the mesophyll (from 160 +/- 30 to a maximum of 625 +/- 40 nmol/mg Chl), and then slowly decreased to about the initial value in the light over an hour. PEP levels dropped (from 176 +/- 5 to 47 +/- 3 nmol/mg Chl) in the first 3 minutes and remained low for the remainder of the dark period. Mesophyll levels of ATP and ADP rapidly decreased and increased, respectively, about twofold upon darkening. The trends observed for these metabolite levels in the mesophyll cell during the light/dark regulation of pyruvate, orthophosphate dikinase activity suggest that pyruvate and PEP do not play a major role in vivo in regulating the extent of light activation (dephosphorylation) or dark inactivation (ADP-dependent threonyl phosphorylation) of dikinase by its bifunctional regulatory protein. While the changes in ADP levels appear qualitatively consistent with a regulatory role for this metabolite in the light activation and dark inactivation of dikinase, they are not of a sufficient magnitude to account completely for the tenfold change in enzyme activity observed in vivo. PMID- 16666760 TI - Senescence-induced, thylakoid-bound diisopropylfluorophosphate-binding protein in spinach : induction pattern, localization, and some properties. AB - Changes in diisopropylfluorophosphate (DFP)-binding proteins during development and senescence of spinach (Spinacia oleracea) leaves were followed using [(3)H]DFP and sodium dodecylsulfate-polyacrylamide gel electrophoresis fluorography. Experiments using a series of aging stages of leaves attached to plants and ones with detached leaves stored in the dark both showed that a protein of 38 kilodaltons was the only major DFP-binding protein in the membrane fraction and that its DFP-binding increased markedly as senescence proceeded, corresponding with the degradation of leaf protein. DFP binding to the 38 kilodalton protein was not affected by membrane solubilization with Triton X-100, and gradually decreased upon preservation of the membranes. The DFP binding was inhibited completely by phenylmethane-sulfonyl fluoride and slightly by p chloromercuribenzoic acid, suggesting a serine protease-like character of the protein and a possible contribution of SH residues to the binding. Both differential and Percoll-gradient centrifugation indicated that the 38-kilodalton protein was localized in thylakoid membranes. The sedimentation behavior of the detergent-solubilized protein indicated that it belongs to a complex different from photosystem I, photosystem II, or coupling factor 1 of the ATP-synthesizing complex. PMID- 16666761 TI - Comparison of Photomorphogenic Responses to UV Light in Red and White Cabbage (Brassica oleracea L.). AB - Photoinhibition of hypocotyl growth in white cabbage (Brassica oleracea L., cv "Bianco Brunswick") is controlled by UV absorbing receptor(s) and the phytochrome system, while in red cabbage (cv "Rosso Olandese tardivo invernale") phytochrome can act without any requirement for the action of a specific UV receptor. Similar results have been obtained for the photoregulation of anthocyanin production. Twenty-four hour preirradiations with UV light or 692 nanometers light lead to the same increase in responsiveness of the system toward Pfr in a following dark period, suggesting a phytochrome promotion of subsequent light induction for both. PMID- 16666762 TI - Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules. AB - Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N(2)) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at -80 degrees C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 +/- 7 kilodaltons with a native molecular weight of 235 +/- 16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pl was estimated to be 6.6. The K(m) values for glutamine, alpha-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GOGAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Western blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs. PMID- 16666763 TI - Rhizosphere Acidification by Iron Deficient Bean Plants: The Role of Trace Amounts of Divalent Metal Ions: A Study on Roots of Intact Plants with the Use of C- and P-NMR. AB - Rhizosphere acidification by Fe-deficient bean (Phaseolus vulgaris L.) plants was induced by trace amounts of divalent metal ions (Zn, Mn). The induction of this Fe-efficiency reaction was studied by (14)CO(2) and (11)CO(2) fixation experiments, and with (31)P-NMR on roots of whole plants. The starting and ending of an acidification cycle was closely coupled to parallel changes in CO(2) fixation, within the maximal resolution capacity of 20 min. (31)P-NMR experiments on intact root systems showed one peak which was ascribed to vacuolar free phosphate. At the onset of proton extrusion this peak shifted, indicating increase of pH in the cells. Proton extrusion was inhibited, with a lag period of 2 hours, by the protein synthesis inhibitors cycloheximide and hygromycin. It is assumed that Zn and Mn induce proton extrusion in Fe-deficient bean roots by activating the synthesis of a short-living polypeptide; the NMR data suggest a role for this peptide in the functioning of a proton pumping ATPase in the plasma membrane. PMID- 16666764 TI - DNA Methylation and Embryogenic Competence in Leaves and Callus of Napiergrass (Pennisetum purpureum Schum.). AB - Quantitative and qualitative levels of DNA methylation were evaluated in leaves and callus of Pennisetum purpureum Schum. The level of methylation did not change during leaf differentiation or aging and similar levels of methylation were found in embryogenic and nonembryogenic callus. PMID- 16666765 TI - A Cerulenin Insensitive Short Chain 3-Ketoacyl-Acyl Carrier Protein Synthase in Spinacia oleracea Leaves. AB - A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryl- and hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated. PMID- 16666766 TI - Identification of Gibberellins A(1), A(3), and Iso-A(3) in Cultures of Azospirillum lipoferum. AB - Gibberellins A(1), A(3), and iso-A(3) were identified from aseptic cultures of Azospirillum lipoferum strain op 33 by capillary gas chromatography-mass spectrometry (GC-MS) and GC-MS-selected ion monitoring. There were 20 to 40 picograms (in GA(3) equivalents, estimated from bioassay) of gibberellins A(1) and A(3) per milliliter of cell culture (containing 10(9) cells). PMID- 16666767 TI - Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings. AB - Myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K(m), 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 +/- 5 kilodaltons and an isoelectric point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. Both subunits contained carbohydrate as shown by periodic acid Schiff staining. The purified enzyme hydrolyzed p-nitrophenyl-beta-d-glucoside (K(m), 2.0 millimolar) at an optimum pH of 6.5 in phosphate buffer. The indolizidine alkaloid castanospermine, a known inhibitor of O-glycosidases, competitively inhibited the hydrolyses of sinigrin (thioglucosidase activity) and p-nitrophenyl-beta-d-glucoside (O-glucosidase activity) with K(i) values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses. PMID- 16666768 TI - Tetrazolium Reduction by Guard Cells in Abaxial Epidermis of Vicia faba: Blue Light Stimulation of a Plasmalemma Redox System. AB - The stomata in the abaxial epidermis of Vicia faba were examined for the location of redox systems using tetrazolium salts. Three distinct redox systems could be demonstrated: chloroplast, mitochondrial, and plasmalemma. The chloroplast activity required light and NADP. Mitochondrial activity required added NADH and was suppressed by preincubation with KCN. The plasmalemma redox system in guard cells also required NADH, but was insensitive to KCN and was stimulated by blue light. The involvement of an NADH dehydrogenase in the blue light stimulated redox system in guard cells was suggested by the sensitivity to plantanetin, an inhibitor of NADH dehydrogenase. The redox system of mitochondria was the most active followed by that of plasmalemma. The activity of chloroplasts was the least among the three redox systems. The plasmalemma mediated tetrazolium reduction was stimulated by exogenous flavins and suppressed by Kl or phenylacetate, inhibitors of flavin excitation. We therefore conclude that an NADH-dependent, flavin mediated electron transport system, sensitive to blue light, operates in the plasmalemma of guard cells. PMID- 16666769 TI - Evidence for the uptake of sucrose intact into sugarcane internodes. AB - Application of [(14)C]fructosyl sucrose was used to determine whether sucrose cleavage was necessary for sucrose uptake by sugarcane (Saccharum spp.) internode tissue. Although approximately 25% of (14)C in the apoplast was present as fructose, indicating some sucrose cleavage, less than 15% of the label was randomized in the sucrose that remained in the tissue after a 30 minute osmoticum rinse. This is insufficient to support cleavage and resynthesis as the sole sucrose transport scheme. The lack of randomization of label between the glucose and fructose moieties of the sucrose molecule was taken as presumptive evidence that sucrose does not have to be cleaved prior to uptake by parenchyma cells in sugarcane internode tissue. PMID- 16666770 TI - Translational regulation of the synthesis of euglena fumarase by light and ethanol. AB - Ethanol addition or the exposure of dark-grown resting Euglena to light produced a transient increase in the rate of fumarase synthesis as measured by pulse labeling with [(35)S]sulfate and immunoprecipitation. The half-life of fumarase in the dark and in the light was similar. In the plastidless mutant W(3)BUL, fumarase synthesis was transiently induced by light indicating that fumarase induction is regulated by a nonchloroplast photoreceptor. In the plastidless mutant W(10)BSmL, light exposure did not increase the rate of fumarase synthesis while the addition of ethanol transiently induced fumarase synthesis. The nonchloroplast photoreceptor regulating fumarase synthesis appears to be absent or nonfunctional in W(10)BSmL. In vitro translation of Euglena RNA in a rabbit reticulocyte lysate showed that fumarase is synthesized as a precursor of 61.5 kilodaltons which is 1.5 kilodaltons larger than the mature protein. The levels of translatable RNA for fumarase were similar in the dark and after light exposure or ethanol addition. The transient increase in the in vivo rate of fumarase synthesis after light exposure or organic carbon addition was not accompanied by a concomitant increase in the level of translatable RNA for fumarase. The synthesis of fumarase in dark grown resting Euglena upon light exposure and ethanol addition is controlled at the translational rather than at the transcriptional level. PMID- 16666771 TI - Chloride inhibition of spinach nitrate reductase. AB - Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V(max) and K(m) for NO(3) (-) yielding V(max) values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K(m) values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K(m) of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed type inhibitor with a K(i) of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO(3) (-) complex. Binding of Cl(-) to the enzyme-NO(3) (-) complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase. PMID- 16666772 TI - Immunological comparison of the starch branching enzymes from potato tubers and maize kernels. AB - Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M(r) = 79,000) shares a high degree of similarity with maize branching enzyme I (M(r) = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species. PMID- 16666773 TI - Whole Plant and Leaf Steady State Gas Exchange during Ethylene Exposure in Xanthium strumarium L. AB - The effects of ethylene evolved from ethephon on leaf and whole plant photosynthesis in Xanthium strumarium L. were examined. Ethylene-induced epinasty reduced light interception by the leaves of ethephon treated plants by up to 60%. Gas exchange values of individual, attached leaves under identical assay conditions were not inhibited even after 36 hours of ethylene exposure, although treated leaves required a longer induction period to achieve steady state photosynthesis. The speed of translocation of recently fixed (11)C-assimilate movement was not seriously impaired following ethephon treatment; however, a greater proportion of the assimilate was partitioned downward toward the roots. Within 24 hours of ethephon treatment, the whole plant net carbon exchange rate expressed on a per plant basis or a leaf area basis had dropped by 35%. The apparent inhibition of net carbon exchange rate was reversed by physically repositioning the leaves with respect to the light source. Ethylene exposure also inhibited expansion of young leaves which was partially reversed when the leaves were repositioned. The data indicated that ethylene indirectly affected net C gain and plant growth through modification of light interception and altered sink demand without directly inhibiting leaf photosynthesis. PMID- 16666774 TI - Isolation of Fixed and Viable Eggs, Central Cells, and Embryo Sacs from Ovules of Plumbago zeylanica. AB - Three alternative protocols for light microscopy, electron microscopy, and biochemical characterization of isolated megagametophytic tissues are described employing enzymic maceration and microdissection of living and fixed ovular tissue of Plumbago zeylanica. Morphologically well preserved megagametophytes are obtained using fixed ovules in two different regimes (nearly 40 and 60% yield, respectively). Fluorescein diacetate-positive megagametophytic cells are recovered in nearly 20% of unfixed ovules using the third regime. PMID- 16666775 TI - Soluble Sugars as the Carbohydrate Reserve for CAM in Pineapple Leaves : Implications for the Role of Pyrophosphate:6-Phosphofructokinase in Glycolysis. AB - Neutral ethanol-soluble sugar pools serve as carbohydrate reserves for Crassulacean acid metabolism (CAM) in pineapple (Ananas comosus (L.) Merr.) leaves. Levels of neutral soluble sugars and glucans fluctuated reciprocally with concentrations of malic acid. Hexose loss from neutral soluble-sugar pools was sufficient to account for malic acid accumulation with about 95% of the required hexose accounted for by turnover of fructose and glucose pools. Hexose loss from starch or starch plus lower molecular weight glucan pools was insufficient to account for nocturnal accumulation of malic acid. The apparent maximum catalytic capacity of pyrophosphate:6-phosphofructokinase (PPi-PFK) at 15 degrees C was about 16 times higher than the mean maximum rate of glycolysis that occurred to support malic acid accumulation in pineapple leaves at night and 12 times higher than the mean maximum rate of hexose turnover from all carbohydrate pools. The apparent maximum catalytic capacity of ATP-PFK at 15 degrees C was about 70% of the activity required to account for the mean maximal rate of hexose turnover from all carbohydrate pools if turnover were completely via glycolysis, and marginally sufficient to account for mean maximal rates of acidification. Therefore, at low night temperatures conducive to CAM and under subsaturating substrate concentrations, PPi-PFK activity, but not ATP-PFK activity, would be sufficient to support the rate of glycolytic carbohydrate processing required for acid accumulation. These data for pineapple establish that there are at least two types of CAM plants with respect to the nature of the carbohydrate reserve utilized to support nighttime CO(2) accumulation. The data further indicate that the glycolytic carbohydrate processing that supports acidification proceeds in different subcellular compartments in plants utilizing different carbohydrate reserves. PMID- 16666776 TI - Malic enzymes of higher plants: characteristics, regulation, and physiological function. AB - The characteristics and distribution of the malic enzyme in plants is discussed as well as those features which appear to be limited to the plant NAD malic enzyme. Regulation of the malic enzyme as it relates to the physiological roles of this enzyme is also discussed. PMID- 16666777 TI - Immunodetection of the Ferredoxin-NADP Oxidoreductase-Binding Protein Complex in Thylakoids of Different Higher Plant Species. AB - Monospecific polyclonal antibodies against thylakoid ferredoxin-NADP(+) oxidoreductase and its binding protein from Spinacia oleracea were used to detect the presence of these proteins in different higher plants, including C(3), C(4), and Crassulacean acid metabolism species. A remarkable conservation of antigenic determinants in all the species analyzed was demonstrated for both the reductase and its binding protein. The association of these polypeptides in a complex was detected by immunoprecipitation. PMID- 16666778 TI - Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots. AB - Phytochrome is rapidly degraded in vivo after photoconversion from the stable red absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates. PMID- 16666779 TI - Low oxygen inhibition of photosynthesis is caused by inhibition of starch synthesis. AB - Photosynthesis of C(3) plants is occasionally inhibited upon switching from normal to low partial pressure of O(2). Leaves of Solanum tuberosum exhibited this effect reproducibly under saturating light and 700 microbars of CO(2). We determined the partitioning of recent photosynthate between starch and sucrose and measured the concentration of hexose monophosphates in the stroma and cytosol after nonaqueous fractionation. The reduction in the rate of photosynthesis upon switching to low partial pressure of O(2) was caused by reduced starch synthesis. The concentration of hexose monophosphates in the stroma fell and the glucose 6 phosphate to fructose 6-phosphate to fructose 6-phosphate ratio fell from 2.7 to 1.3, indicating an inhibition of phosphoglucoisomerase as described by K-J Dietz ([1985] Biochim Biophys Acta 839: 240-248). The concentration of hexose monophosphates in the cytosol increased, ruling out a sucrose synthesis limitation by reduced transport from the chloroplast as the explanation for low O(2) inhibition of photosynthesis. PMID- 16666780 TI - A fast and easy technique for the isolation of aleurone layers. AB - Although the aleurone layer of ceral grain seeds has many advantages for the study of gibberellin action, it has the disadvantage that the hand-isolation of the aleurone layers is time-consuming. To overcome this disadvantage, a commercially available pasta machine was modified and used to remove aleurone layers from imbibed barley (Hordeum vulgare) seeds. This equipment allows isolation of a thousand layers in 5 minutes compared to the 3 to 4 hours required to hand-isolate them. The machine-made aleurone layers are gibberellic-acid responsive and the response is similar both qualitatively and quantitatively to that of hand-isolated layers. PMID- 16666781 TI - An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants. AB - DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome. PMID- 16666782 TI - Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution. AB - The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of gamma-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity. PMID- 16666783 TI - Quantitation of indoleacetic Acid conjugates in bean seeds by direct tissue hydrolysis. AB - Gas chromatography-selected ion monitoring-mass spectral analysis using [(13)C(6)]indole-3-acetic acid (IAA) as an internal standard provides an effective means for quantitation of IAA liberated during direct strong basic hydrolysis of bean (Phaseolus vulgaris L.) seed powder, provided that extra precautions are undertaken to exclude oxygen from the reaction vial. Direct seed powder hydrolysis revealed that the major portion of amide IAA conjugates in bean seeds are not extractable by aqueous acetone, the solvent used commonly for IAA conjugate extraction from seeds and other plant tissues. Strong basic hydrolysis of plant tissue can be used to provide new information on IAA content. PMID- 16666784 TI - Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species. AB - We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C(4) species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C(4) enzyme has a group with a pK of about 6.0 (6.6 for the C(4) enzyme), as indicated from plots of the log V(max)/K(m) (V(max) = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C(4) enzyme-NADP-Mg(2+) complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K(m) increases, but V(max) remains constant. The log V(max)/K(m) plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K(m) and a decrease in V(max) for the C(4) enzyme. As in the case of the animal enzyme, the log V(max)/K(m) plot for the C(4) enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C(4) NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts. PMID- 16666785 TI - Maturation in larch : I. Effect of age on shoot growth, foliar characteristics, and DNA methylation. AB - The time course of maturation in eastern larch (Larix laricina [Du Roi] K. Koch) was examined by grafting scions from trees of different ages onto 2-year-old root stock and following scion development for several years. Height, diameter, foliar chlorophyll content, and rooting ability of scion-derived cuttings all varied linearly as a function of log(10) age. Chlorophyll content (milligrams per gram of dry weight) increased while height, diameter, and ability to root decreased with age (P < 0.01). The tendency toward orthotropic growth and branch formation per centimeter of main stem decreased abruptly between age 1 and 5 years (P < 0.01). Total chlorophyll content of both long and short shoot foliage increased by 30 to 50% with increasing age, but the chlorophyll a/b ratio did not change. Also, juvenile long shoot needles were significantly longer than mature (P < 0.01). Surprisingly, the juvenile scions produced more total strobili over two successive years, but the mature scions produced a significantly higher proportion of male strobili (P < 0.001 year 1; P < 0.02 year 2). The age-related changes in foliar traits were not associated with changes in DNA methylation between juvenile and mature scions. Using HPLC, we found that 20% of foliar DNA cytosine residues were methylated in both scion types. PMID- 16666786 TI - Microsomal phosphatidate phosphatase in maturing safflower seeds. AB - An assay system comprising sodium phosphatidate, phosphatidylcholine, and bovine serum albumin has been developed for the reproducible determination of phosphatidate phosphatase activity in maturing seeds of safflower (Carthamus tinctorius L.). The activity was detected in both membrane and soluble fractions, and the microsomal phosphatidate phosphatase was characterized. The optimum pH for Pi release was 6.7, and the activity depended on the concentration of Mg(2+). Phosphatidylcholine and bovine serum albumin stimulated the phosphatase reaction. This phosphatase was highly specific for phosphatidate; lysophosphatidate, and water-soluble phosphate esters did not serve as substrate. The specific activity was approximately 20 nanomoles per minute per milligram of protein, which was close to that of glycerol-phosphate acyltransferase and higher than that of diacylglycerol acyltransferase. Furthermore, the activity per seed was enough to account for the rate of triacylglycerol accumulation in vivo. The step of diacylglycerol formation by phosphatidate phosphatase does not appear to be rate limiting for triacylglycerol synthesis during seed maturation. PMID- 16666787 TI - Distribution of N within Pea, Lupin, and Soybean Nodules. AB - The (15)N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in (15)N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring (15)N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in (15)N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest (15)N enrichment. In soybean nodules, the (15)N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in (15)N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in (15)N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual (15)N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in (15)N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in (15)N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess (15)N of lupin and soybean bacteriods is offered. PMID- 16666788 TI - Maize leaf phosphoenolpyruvate carboxylase : oligomeric state and activity in the presence of glycerol. AB - Maize (Zea mays L.) leaf phosphoenopyruvate (PEP) carboxylase activity at subsaturating levels of PEP was increased by the inclusion of glycerol (20%, v/v) in the assay medium. The extent of activation was dependent on H(+) concentration, being more marked at pH 7 (with activities 100% higher than in aqueous medium) than at pH 8 (20% activation). The determination of the substrate concentration necessary to achieve half-maximal enzyme activity (S(0.5)) (PEP) and maximal velocity (V) between pH 6.9 and 8.2 showed a uniform decrease in S(0.5) in the presence of glycerol over the entire pH range tested, and only a slight decrease in V at pH values near 8. Including NaCl (100 millimolar) in the glycerol containing assay medium resulted in additional activation, mainly due to an increase in V over the entire range of pH. Glucose-6-phosphate (5 millimolar) activated both the native and the glycerol-treated enzyme almost to the same extent, at pH 7 and 1 millimolar PEP. Inhibition by 5 millimolar malate at pH 7 and subsaturating PEP was considerably lower in the presence of glycerol than in an aqueous medium (8% against 25%, respectively). Size-exclusion high performance liquid chromatography in aqueous buffer revealed the existence of an equilibrium between the tetrameric and dimeric enzyme forms, which is displaced to the tetramer as the pH was increased from 7 to 8. In the presence of glycerol, only the 400 kilodalton tetrameric form was observed at pH 7 or 8. However, dissociation into dimers by NaCl could not be prevented by the polyol. We conclude that the control of the aggregation state by the metabolic status of the cell could be one regulatory mechanism of PEP carboxylase. PMID- 16666789 TI - An Improved Method for Analysis of Polyamines in Plant Tissue by Precolumn Derivatization with o-Phthalaldehyde and Separation by High Performance Liquid Chromatography. AB - An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts. Polyamines were derivatized with o-phthalaldehyde and mercaptoethanol (OPT). The fluorescent derivatives were eluted from a C(18) column with the dimethylcyclohexylamine-phosphate buffer derived by T. Skaaden and T. Greibrokk ([1982] J Chromatogr 247: 111-122) after treatment to remove impurities in the buffer. The method had a sensitivity of 1-2 picomoles and completely resolved nine polyamines (agmatine, spermine, nor spermidine, spermidine, 3,5-homospermidine, 4,4-homospermidine, 1,3 diaminopropane, putrescine, and cadaverine) in 12 to 14 minutes. An optional ion exchange step was used to remove less basic amines (including amino acids) and to concentrate the crude extracts. This method was compared with benzoyl chloride derivatization. Use of the benzoyl chloride method vastly under-estimated the amount of polyamine in some plant extracts, a problem not encountered with the OPT procedure. Additionally, the OPT procedure resolved two isomers of homospermidine found in Azolla caroliniana. These two isomers were not resolved with the benzoylation method. Overall, the OPT method described here requires preparation and analysis time similar to other current methods but provides greater sensitivity and selectivity. PMID- 16666790 TI - Inhibition of Acid-Enhanced Elongation of Zea mays Root Segments by Galactose. AB - The effect of sugars and metabolic inhibitors on the elongation of Zea mays root segments was analyzed by a rhizometer which records the elongation of each of 32 root segments at the same time. Galactose suppressed the acid-enhanced rapid elongation after a lag period of 1.5 hours, but it did not inhibit the slow elongation at pH 7. Mannose was less inhibitory than galactose. Arabinose, xylose, glucose, sucrose, mannitol, and sorbitol caused no inhibition. When galactose was removed after a 1-hour treatment, the elongation was partially recovered. Cycloheximide and 2-deoxyglucose suppressed acid-enhanced elongation when these were applied at the same time as acid treatments, whereas cordycepin (3'-deoxyadenosine) inhibited elongation only if it was applied prior to acid treatment. Over the 9-hour period of elongation studied, the inhibition by galactose was comparable to that of cycloheximide. Since galactose has been reported to suppress the sugar metabolism necessary for the cell wall synthesis, the later phase of acid-enhanced elongation of root segments may at least partially depend on the synthesis or metabolism of cell wall components. The inhibition of root growth by galactose may be partially ascribed to a direct effect on the elongation process in roots, an effect that is enhanced by the acidification of the cell walls. PMID- 16666791 TI - Immunoaffinity techniques applied to the purification of gibberellins from plant extracts. AB - The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA(1), GA(3), GA(4), GA(5), GA(7), and GA(9) conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA(7) was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA(20) and GA(29) were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA(9) were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA(9) was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA(20) and GA(51) were also observed in testae. PMID- 16666792 TI - Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa). AB - Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate. PMID- 16666793 TI - Changes in Fructose 2,6-Bisphosphate Levels in Green Pepper (Capsicum annuum L.) Fruit in Response to Temperature. AB - The regulatory metabolite, fructose 2,6-bisphosphate (Fru 2,6-P(2)) was found in green pepper (Capsicum annuum L.). The Fru 2,6-P(2) level was found to: (a) rise rapidly in response to heat; (b) drop rapidly, followed by recovery, in response to cold storage of fruit and, (c) oscillate during cold storage of fruit. The possible existence of a relationship between chilling injury and Fru 2,6-P(2) is considered. PMID- 16666794 TI - In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana. AB - In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed. PMID- 16666795 TI - Correlation between Carbonic Anhydrase Activity and Inorganic Carbon Internal Pool in Strain Synechocystis PCC 6174. AB - Existence of an internal carbonic anhydrase was demonstrated in the cyanobacterium Synechocystis PCC 6714. The enzyme, present at a low specific activity, was inducible by limitation in inorganic carbon and inhibited both in vivo and in vitro by acetazolamide. The internal inorganic carbon pool as determined by mass spectrometry, was similarly modulated by the actual inorganic carbon growth regime; its building up was also sensitive to acetazolamide. A possible role of carbonic anhydrase in inorganic carbon metabolism regulation through the control of the dimension and nature of the inorganic carbon pool is discussed. PMID- 16666796 TI - Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings. AB - The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz [1985] Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg(2+)-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (alpha subunit) and 64,000 (beta subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for alpha and beta subunits. The amino acid composition of alpha and beta subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 +/- 24 and 405 +/- 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of alphabeta-heteromultimer. PMID- 16666797 TI - A Comparison between Quin-2 and Aequorin as Indicators of Cytoplasmic Calcium Levels in Higher Plant Cell Protoplasts. AB - Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca(2+) in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca(2+) indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (Quin-2) and the Ca(2+) indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca(2+) level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca(2+) levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca(2+) concentrations. Cytosolic Ca(2+) was observed to increase on treatment with azide and silver ions. PMID- 16666798 TI - Identification and Partial Characterization of the Denaturation Transition of the Light Harvesting Complex II of Spinach Chloroplast Membranes. AB - Differential scanning calorimetry was employed to investigate the structure of spinach (Spinacia oleracea) chloroplast membranes. In a low ionic strength Hepes buffered medium, major calorimetric transitions were resolved at 42.5 degrees C. (A), 60.6 degrees C (B), 64.9 degrees C (C(1)), 69.6 degrees C (C(2)), 75.8 degrees C (D), 84.3 degrees C (E), and 88.9 degrees C (F). A lipid melting transition was also commonly seen at 17 degrees C in scans starting at lower temperatures. The D transition was demonstrated by four independent methods to derive from denaturation of the light harvesting complex associated with photosystem II (LHC-II). Evidence for this conclusion was as follows: (a) the endotherm of the isolated LHC-II (74.0 degrees C) was very similar to that of D (75.8 degrees C); (b) the denaturation temperature of the 27 kilodalton LHC-II polypeptide determined in intact chloroplast membranes by thermal gel analysis was identical to the temperature of the D transition at pH 7.6 and after destabilization by shifting the pH to 6.6 or by addition of Mg(2+); (c) analysis of the stability of the LHC-II complex by electrophoresis in native gels demonstrated that the complex dissociates during the D transition, both at pH 7.6 and 6.6; and (d) the 77 Kelvin fluorescence maximum of LHC-II in chloroplasts was seen to shift to lower wavelengths (indicating gross denaturation of LHC-II), at the temperature of the D transition when examined at either of the above pHs. With this identification, five of the eight major endotherms of the chloroplast membrane have now been assigned. PMID- 16666799 TI - Photorespiratory rates in wheat and maize as determined by o-labeling. AB - A method was devised to quantify short-term photorespiratory rates in terrestrial plants using (18)O-intermediates of the glycolate pathway, specifically glycolate, glycine, and serine. The pathway intermediates were isolated and analyzed on a GC/MS to determine molecular percent (18)O-enrichment. Rates of glycolate synthesis were determined from (18)O-labeling kinetics of the intermediates, derived rate equations, and nonlinear regression techniques. Glycolate synthesis in wheat (Triticum aestivum L.), a C(3) plant, and maize (Zea mays L.), a C(4) plant, was stimulated by high O(2) concentrations and inhibited by high CO(2) concentrations. The synthesis rates were 7.3, 2.1, and 0.7 micromoles per square decimeter per minute under a 21% O(2) and 0.035% CO(2) atmosphere for leaf tissue of wheat, maize seedlings, and 3-month-old maize, respectively. Photorespiratory CO(2) evolution rates were estimated to be 27, 6, and 2%, respectively, of net photosynthesis for the three groups of plants under the above atmosphere. The results from maize tissue support the hypothesis that C(4) plants photorespire, albeit at a reduced rate in comparison to C(3) plants, and that the CO(2)/O(2) ratio in the bundle sheath of maize is higher in mature tissue than in seedling tissue. The pool size of the three photorespiratory intermediates remained constant and were unaffected by changes in either CO(2) or O(2) concentrations throughout the 10-minute labeling period. This suggests that photorespiratory metabolism is regulated by other mechanism besides phosphoglycolate synthesis by ribulose-1,5-bisphosphate carboxylase/oxygenase, at least under short-term conditions. Other mechanisms could be alternate modes of synthesis of the intermediates, regulation of some of the enzymes of the photorespiratory pathway, or regulation of carbon flow between organelles involved in photorespiration. The glycolate pool became nearly 100% (18)O-labeled under an atmosphere of 40% O(2). This pool failed to become 100% (18)O-enriched under lower O(2) concentrations. PMID- 16666800 TI - Procedures for the Generation of Mature Chlamydomonas reinhardtii Zygotes for Molecular and Biochemical Analyses. AB - Zygotes represent an important stage in the sexual cycle of the unicellular green alga Chlamydomonas reinhardtii. To study zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quantities and a method was developed for the extraction from zygotes of RNA that could be translated in vitro. PMID- 16666801 TI - Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development. AB - We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves. PMID- 16666803 TI - Vacuolar Acid hydrolysis as a physiological mechanism for sucrose breakdown. AB - Sucrose breakdown in mature acidic ;Persian' limes (Citrus aurantifolia [Christm.] Swing.) occurred at a rate of 30.6 picomoles per milliliter per day during 9 weeks storage at 15 degrees C. Neither enzyme of sucrose catabolism (sucrose synthase or acid/alkaline invertase) was present in extracts of mature storage tissue. The average vacuolar pH, estimated by direct measurement of sap from isolated vacuoles and by the methylamine method, was about 2.0 to 2.2. In vitro acid hydrolysis of sucrose at physiological concentrations in a buffered solution (pH 2.2) occurred at identical rates as in matured limes. The results indicate that sucrose breakdown in stored mature acidic limes occurs by acid hydrolysis. PMID- 16666802 TI - A mutant of Arabidopsis deficient in the chloroplast 16:1/18:1 desaturase. AB - Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 18-carbon and 16-carbon polyunsaturated fatty acids and increased levels of the 18:1 and cis-16:1 precursors due to a single nuclear mutation at a locus designated fadC. Analysis of the fatty acid compositions of individual lipids and the kinetics of lipid labeling with [(14)C]acetate in vivo indicate that the mutant lacks activity of the chloroplast glycerolipid omega-6 desaturase. As a result, lipids synthesized by the prokaryotic pathway are not desaturated further than 18:1 and 16:1. Lipids derived from the eukaryotic pathway are desaturated presumably by the endoplasmic reticulum 18:1 phosphatidylcholine desaturase. However, an increase in the level of 18:1 on all the phospholipids derived from the eukaryotic pathway in leaves of the mutant suggests that the mutation does exert an effect on the composition of extrachloroplast membranes. Synthesis of monogalactosyldiacylglycerol (MGD) by the prokaryotic pathway is reduced 30 to 35% in the mutant and there is a corresponding increase in MGD synthesis by the eukaryotic pathway. This shift in metabolism which results in a more unsaturated MGD pool, may reflect the existence of a regulatory mechanism which apportions lipid synthesis between the two pathways in response to alterations in the physical properties of the chloroplast membranes. PMID- 16666804 TI - Antigenic Crossreactivity between Bacterial and Plant Cytochrome P-450 Monoxygenases. AB - Although cytochrome P-450 monoxygenases mediate critical reactions in plant microsomes, characterization of their activities has been difficult due to their inherent instability and the lack of a crossreacting P-450 antibody. We have surveyed the effects of protein stabilizing agents on t-cinnamic acid hydroxylase (t-CAH), a prominent microsomal P-450, and on total P-450 monoxygenase content. Trans-cinnamic acid is the most effective protecting agent for t-CAH activity. Leupeptin, a broad spectrum protease inhibitor, stabilizes t-CAH activity and increases the apparent P-450 content more than serine protease inhibitors such as phenylmethylsulfonyl fluoride. The combination of t-cinnamic acid and protease inhibitors increase the level of detectable t-CAH activity 4- to 14-fold over the levels detected by previously published procedures. In order to estimate the molecular weights and diversity of the plant P-450 monoxygenases in wounded pea epicotyls, we have prepared two polyclonal antibodies against the Pseudomonas putida camphor hydroxylase (P-450(cam)). One of the heterologous antibodies cross reacts with constitutive microsomal polypeptides between 52 and 54 kilodaltons and several pea (Pisum sativum L.) mitochondrial proteins between 47 and 48 kilodaltons. The other polyclonal antibody cross-reacts strongly with two wound induced polypeptides (65 and 47 kilodaltons) and weakly with one constitutive polypeptide (58 kilodaltons). We conclude that at least two subclasses of plant P 450 monoxygenases share common epitopes with the bacterial P-450 enzyme. PMID- 16666805 TI - Host-Pathogen Interactions : XXXIII. A Plant Protein Converts a Fungal Pathogenesis Factor into an Elicitor of Plant Defense Responses. AB - This paper describes the effect of a plant-derived polygalacturonase-inhibiting protein (PGIP) on the activity of endopolygalacturonases isolated from fungi. PGIP's effect on endopolygalacturonases is to enhance the production of oligogalacturonides that are active as elicitors of phytoalexin (antibiotic) accumulation and other defense reactions in plants. Only oligogalacturonides with a degree of polymerization higher than nine are able to elicit phytoalexin synthesis in soybean cotyledons. In the absence of PGIP, a 1-minute exposure of polygalacturonic acid to endopolygalacturonase resulted in the production of elicitor-active oligogalacturonides. However, the enzyme depolymerized essentially all of the polygalacturonic acid substrate to elicitor-inactive oligogalacturonides within 15 minutes. When the digestion of polygalacturonic acid was carried out with the same amount of enzyme but in the presence of excess PGIP, the rate of production of elicitor-active oligogalacturonides was dramatically altered. The amount of elicitor-active oligogalacturonide steadily increased for 24 hours. It was only after about 48 hours that the enzyme converted the polygalacturonic acid into short, elicitor-inactive oligomers. PGIP is a specific, reversible, saturable, high-affinity receptor for endopolygalacturonase. Formation of the PGIP-endopolygalacturonase complex results in increased concentrations of oligogalacturonides that activate plant defense responses. The interaction of the plant-derived PGIP with fungal endopolygalacturonases may be a mechanism by which plants convert endopolygalacturonase, a factor important for the virulence of pathogens, into a factor that elicits plant defense mechanisms. PMID- 16666806 TI - Fatty Acid Acylated Proteins of the Halotolerant Alga Dunaliella salina. AB - The unicellular, wall-less alga Dunaliella salina has been shown to contain an array of proteins modified by the covalent attachment of fatty acids. Myristic acid (14:0) comprised approximately 80% by weight of the protein-linked acyl groups in samples derived from cells cultured in medium containing 1.7 molar NaCl and 93% in samples from cells grown in medium containing 3.0 molar NaCl. Palmitic and stearic acids accounted for most of the remaining protein-bound acyl chains. Approximately 0.2% of the incorporated radioactivity was estimated to be in linkage with protein. The bulk of acyl chains (about 99%) were resistant to cleavage by alkali, indicating a preponderance of amide bonding. The sodium dodecyl sulfate-polyacrylamide electrophoresis labeling pattern of proteins from [(3)H]myristic-labeled cells was significantly different from that of proteins from cells exposed to [(3)H]palmitate. The appearance of radioactivity in certain proteins was also influenced by the salinity of the culture medium. Thus growth in moderate (1.7 molar) salt favored the acylation of a 48-kilodalton polypeptide whereas in high (3.0 molar) salt, a 17-kilodalton polypeptide was more heavily labeled. PMID- 16666807 TI - Cytochrome mutants of bradyrhizobium induced by transposon tn5. AB - Transposon Tn5 was used to mutate Bradyrhizobium japonicum USDA 61N. From over 5000 clones containing Tn5, 12 were selected and purified using a chemical reaction to identify oxidase-deficient clones. Four classes of mutants were identified based on the alterations in cytochromes. Most of the mutants had alterations in more than one cytochrome. Southern hybridization analysis of restricted genomic DNA of a representative strain of each class demonstrated that each mutant had a single Tn5 insert. Thus a single Tn5 insert produced pleiotropic effects on cytochromes. One class, which was totally deficient in cytochromes aa(3) and c, produced ineffective nodules on soybeans. Most of the strains representing the other classes produced effective nodules but exceptions were observed in each class. Bacteroids of the wild-type strain contained cytochrome aa(3). Bacteroids from one class of mutants were totally devoid of cytochrome aa(3). Several of these strains produced effective symbioses indicating that cytochrome aa(3) is not required for an effective symbiosis in this DNA homology group II strain which normally has this terminal oxidase in bacteroids. PMID- 16666808 TI - Light intensity regulates the accumulation of the major light-harvesting chlorophyll-protein in greening seedlings. AB - Etiolated pea (Pisum sativum [L.] cv Progress 9) and barley (Hordeum vulgare [L.] cv Boone) seedlings greened under either low (40 microeinsteins per square meter per second) or high (550 microeinsteins per square meter per second) intensity light were analyzed for chlorophyll (Chl) content and the levels of mRNA and protein for the major light-harvesting chlorophyll (Chl)-protein of photosystem II (LHC-II). Low intensity plants accumulated Chl more rapidly than high intensity plants. Both single radial immunodiffusion analysis and mild sodium dodecyl sulfate-polyacrylamide gel electrophoresis green gels showed that low intensity plants also accumulated LHC-II protein more rapidly than high intensity plants, following a kinetic pattern similar to the total Chl data. In contrast, LHC-II mRNA levels appeared to be independent of LHC-II protein levels although pea and barley LHC-II mRNA exhibited different light intensity responses. The absence of coordination between LHC-II mRNA and protein levels suggested that the biosynthesis of LHC-II in greening seedlings is not limited by mRNA. A correlation (better than the 0.01 significance level) between LHC-II protein accumulation and Chl accumulation was found for both pea and barley. The accumulation of LHC-II protein was not linked to the development of photosynthetic electron transport. These results and the similar effect of light intensity on Chl content and LHC-II protein levels suggested that the availability of Chl may limit LHC-II protein accumulation in greening seedlings. PMID- 16666809 TI - A beta-Galactosidase from Radish (Raphanus sativus L.) Seeds. AB - A basic beta-galactosidase (beta-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl beta-d-galactoside and beta-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg(2+) and 4-chloromercuribenzoate. d-Galactono-(1-->4) lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed beta-Galase specifically split off beta-1,3- and beta-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the beta-galase alone but could be partially degraded by the enzyme after the treatment with a fungal alpha-l arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product. PMID- 16666810 TI - Identification and Partial Characterization of the Denaturation Transition of the Photosystem II Reaction Center of Spinach Chloroplast Membranes. AB - Sensitive differential scanning calorimetry was employed to investigate thylakoid membrane structure. Calorimetric scans of chloroplast membranes suspended in a low ionic strength Hepesbuffered medium revealed endothermic transitions centered at the following temperatures ( degrees C): A (42.5), B (60.6), C(1) (64.9), C(2) (69.6), D (75.8), E (84.3), and F (88.9). The B transition was demonstrated by several different methods to originate from denaturation of the photosystem II reaction center complex. Evidence for this conclusion is as follows: (a) the isolated reaction center complex denatures near the temperature of the B transition; (b) inorganic phosphate destablizes the isolated reaction center complex and the B endotherm to a similar extent; (c) heat inactivation of the photosystem II-mediated 1,5-diphenylcarbazide --> dichloroindophenol photoreaction occurs at the temperature of the B transition and is influenced in a manner similar to B by the presence of phosphate; (d) thermal gel analysis indicates that the 43 and 47 kilodalton polypeptides of the photosystem reaction center complex denature at the temperature of the B transition, both in the presence and absence of phosphate; (e) low temperature (77 Kelvin) fluorescence reveals that a change in photosystem II emission at 695 nanometers occurs during the B transition; and (f) ioxynil, a specific inhibitor of photosystem II, selectively stabilizes the B endotherm. With the identification of the B transition established, the origins of six of the eight major transitions of the chloroplast membrane have now been determined. PMID- 16666811 TI - deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels. AB - Values of delta(13)C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The delta(13)C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or delta(13)C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, delta(13)C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on delta(13)C should occur through its effect on the extent of leakage of CO(2) from the bundle sheath cells. In this case, delta(13)C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in delta(13)C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase. PMID- 16666812 TI - Iron induction of ferritin synthesis in soybean cell suspensions. AB - In animal cells specialized for iron storage, iron-induced accumulation of ferritin is known to result from a shift of stored mRNA from the ribonucleoprotein fraction to polysomes. Previous reports with bean leaves suggested that in plants iron induction of ferritin synthesis would result from a regulation at the transcriptional level (F van der Mark, F Bienfait, H van der Ende [1983] Biochem Biophys Res Commun 115:463-469). Soybean (Glycine max, cv Mandarin) cell suspension cultures have been used here to support these findings. Ferritin induction is obtained by addition of Fe-citrate to the culture medium. A good correlation is found between cellular iron content and the amount of ferritin accumulation. This protein accumulation corresponds to an increase of in vitro translatable ferritin mRNA. Addition of 4 micrograms actinomycin D per milliliter to the cultures inhibits completely in vivo RNA synthesis, whereas protein synthesis was poorly affected, at least for 24 hours. During the same time, this concentration of actinomycin D strongly inhibits the iron-induced synthesis of ferritin. These results show that in soybean cell cultures, the mechanism of regulation of ferritin synthesis in response to iron does not result from recruitment of preexisting mRNA. They confirm that in plant systems, ferritin synthesis results from increased transcription of the corresponding genes. PMID- 16666813 TI - Cyclopropyl sterol and phospholipid composition of membrane fractions from maize roots treated with fenpropimorph. AB - Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Delta(5)-sterols disappeared almost completely and were replaced by 9beta, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Delta(5) sterols. PMID- 16666814 TI - Heat shock response of germinating embryos of wheat : effects of imbibition time and seed vigor. AB - Seeds frequently face a hostile environment during early germination. In order to determine whether seeds have evolved unique mechanisms to deal with such environments, a survey of the heat shock response in isolated embryos of wheat (Triticum aestivum L.) was undertaken. Embryos simultaneously heat shocked and labeled following several different periods of prior imbibition up to 12 hours synthesized many groups of heat shock proteins (hsps) typical of other plant and animal systems. Also, five developmentally dependent hsps, present only in treatments imbibed less than 6 hours prior to heat shock, were detected. These proteins have relative molecular masses of 14, 40, 46, 58, and 60 kilodaltons. One of the developmentally dependent hsps is among the most highly labeled hsps found in early imbibed embryos. The possibility that this protein is the E(m) protein is discussed. The hypothesis that the capacity for hsp synthesis is affected by seed vigor was also tested. The heat shock responses of embryos from two high and two low vigor seed lots were compared using one- and two-dimensional electrophoresis of labelled protein extracts. The results indicate that both of the low vigor lots tested had weaker heat shock responses than their high vigor counterparts overall. Not all hsps were relatively less abundant in low vigor embryos. The developmentally dependent hsps showed little relationship to vigor. Some of the developmentally dependent hsps were actually made in greater amounts, relative to other proteins, in the low vigor seed lots. The results presented here demonstrate that imbibing embryos are capable of expressing an enhanced heat shock response, and that this response is related to seed vigor. PMID- 16666815 TI - Thermal damage to chloroplast envelope membranes. AB - Nuclear magnetic resonance was used to detect thermal injury to chloroplasts in vivo. A lesion occurs in the chloroplast envelope membrane at temperatures between 53 degrees C and 57 degrees C, depending on species, leaf condition, and heating rate. The injury is associated with a sudden loss of water from the chloroplast. PMID- 16666816 TI - Galactolipids of Thylakoid Pigment Protein Complexes Separated Electrophoretically from Thylakoids of Dunaliella salina Labeled with Radioactive Fatty Acids. AB - Experiments have been conducted to determine whether mono- and digalactosyldiacylglycerols (MGDG and DGDG) labeled by short term exposure of the green alga Dunaliella salina to exogenous radioactive lauric, myristic, or palmitic acids are preferentially recovered in one or more thylakoid protein complexes separated by electrophoresis on nondenaturing gels. A selective retention of certain lipid classes and molecular species by the major electrophoretically separable fractions, oligomeric form (LHCP-1) and monomeric form (LHCP-3) of hight-harvesting chlorophyll a/b protein complex, and major photosystem I protein complex (CPla), and nonprotein-bound lipids, was indicated. After 5 min of labeling with (3)H-palmitate the specific radioactivities of MGDG and DGDG molecular species recovered from LHCP-3 and CPla were slightly higher and lower, respectively, than specific radioactivities of equivalent molecular species from LHCP-1 and nonprotein-bound lipid. However, the differences were smaller than those which have been observed between whole thylakoids and the more highly labeled chloroplast envelopes from briefly labeled cells. PMID- 16666817 TI - Spectral Properties and Composition of Reaction Center and Ancillary Polypeptide Complexes of Photosystem II Deficient Mutants of Synechocystis 6803. AB - The polypeptide composition and spectral properties of three photosystem II (PSII) deficient mutants of the cyanobacterium Synechocystis 6803 have been determined. The levels of the 43 and 47 kilodalton chlorophyll-binding proteins and the reaction center component D2 are affected differently in each mutant; the 33 kD polypeptide of the oxygen-evolving complex is found at wild-type levels in all three. The 43 and 47 kilodalton proteins are implicated as important elements in the assembly and/or stability of the PSII reaction center, although the loss of one of these polypeptides does not lead to the loss of all PSII proteins. Low temperature fluorescence emission spectra of wild-type cells reveal chlorophyll attributable peaks at 687 (PSII), 696 (PSII), and 725 (photosystem I) nanometers. All three mutants retain the 725 nanometer fluorescence but lack the 696 nanometer peak. This suggests that the latter fluorescence arises from PSII reaction center chlorophyll or results from interactions among functional PSII components in vivo. Cells that contain the 43 kilodalton and lack the 47 kilodalton protein, retain the 687 fluorescence; furthermore, in as much as this fluorescence is absent from cells without the 43 kilodalton protein, the 687 nanometer peak is judged to emanate from the 43 kilodalton chlorophyll-protein. A new peak, probably previously obscured, is revealed at 691 nanometers in cells that retain the 47 kilodalton protein but lack the 43 kilodalton polypeptide, suggesting that emission near 691 nanometers can be attributed to the 47 kilodalton polypeptide. Membrane-bound phycobilisomes are retained in these cells as is coupled-energy transfer between phycocyanin and allophycocyanin. Energy transfer to photosystem I by way of phycocyanin excitation proceeds as in wild type cells despite the absence of certain PSII components. PMID- 16666818 TI - Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition. AB - The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1 (14)C]glutamate and [U-(14)C]glutamine were fed to excised cotyledons. After 4 hours, (14)C was incorporated into protein and released as (14)CO(2), but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ. PMID- 16666819 TI - Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis. AB - Tracer kinetic experiments were performed using [ureido-(14)C] citrulline, [1 (14)C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the (14)C-solution and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the (14)C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose (14)C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO(2) up to 4 hours after introduction of label. All of the (14)C in free and protein arginine could be accounted for in the C-6 position. Metabolism of (14)C-ornithine resulted in (14)C-incorporation into citrulline and free and protein arginine and the evolution of (14)CO(2). Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of (14)C. With the addition of 800 nanomoles unlabeled citrulline to the (14)C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of (14)C-citrulline increased 12-fold. The appearance of (14)C in free arginine and protein arginine was also delayed. In both (14)C-ornithine feedings all of the (14)C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine --> citrulline --> argininosuccinate --> arginine --> protein arginine. PMID- 16666820 TI - Characterization of Sucrolysis via the Uridine Diphosphate and Pyrophosphate Dependent Sucrose Synthase Pathway. AB - The breakdown of sucrose to feed both hexoses into glycolytic carbon flow can occur by the sucrose synthase pathway. This uridine diphosphate (UDP) and pyrophosphate (PPi)-dependent pathway was biochemically characterized using soluble extracts from several plants. The sucrolysis process required the simultaneous presence of sucrose, UDP, and PPi with their respective K(m) values being about 40 millimolar, 23 micromolar, and 29 micromolar. UDP was the only active nucleotide diphosphate. Slightly alkaline pH optima were observed for sucrose breakdown either to glucose 1-phosphate or to triose phosphate. Sucrolysis incrased with increasing temperature to near 50 degrees C and then a sharp drop occurred between 55 and 60 degrees C. The breakdown of sucrose to triose-P was activated by fructose 2,6-P(2) which had a K(m) value near 0.2 micromolar. The cytoplasmic phosphofructokinase and fructokinase in plants were fairly nonselective for nucleotide triphosphates (NTP) but glucokinase definitely favored ATP. A predicted stoichiometric relationship of unity for UDP and PPi was measured when one also measured competing UDPase and pyrophosphatase activity. The cycling of uridylates, UDP to UTP to UDP, was demonstrated both with phosphofructokinase and with fructokinase. Enzyme activity measurements indicated that the sucrose synthase pathway has a major role in plant sucrose sink tissues. In the cytoplasmic sucrose synthase breakdown pathway, a role for the PPi phosphofructokinase was to produce PPi while a role for the NTP phosphofructokinase and for the fructokinase was to produce UDP. PMID- 16666821 TI - Seed dormancy and responses of caryopses, embryos, and calli to abscisic Acid in wheat. AB - Preharvest sprouting of wheat (Triticum aestivum L.) is associated with inadequate seed dormancy. Although abscisic acid (ABA) has often been suggested to play a central role in developing seed, its involvement in dormancy of mature seed lacks firm experimental evidence and endogenous ABA levels are not well correlated with germinability. We examined genotypic and temporal variation in wheat seed and embryo germination responses to ABA and determined whether differential sensitivity of embryos to ABA extended to growth of embryo-derived calli. Germination of Parker 76 caryopses, which have little dormancy at maturity, was only slightly inhibited by ABA, whereas germination of Clark's Cream, a highly dormant genotype, was greatly inhibited. Responsiveness of caryopses to ABA and dormancy of seeds decreased concurrently during afterripening. Germination of embryos excised from dormant and nondormant seeds was more responsive to ABA but otherwise was similar to that of caryopses, indicating that differential response to ABA occurs in the embryo. Growth of calli derived from immature embryos of two sprouting-susceptible wheat genotypes exceeded growth of calli from Clark's Cream, but no distinct differences in response to ABA among the genotypes were apparent. We concluded that the action of ABA is similar in developing and mature seeds, that genotypic and temporal variation in embryo responsiveness to endogenous ABA may be involved in dormancy, and that ABA probably acts in concert with other endogenous constituents. PMID- 16666822 TI - Activation of higher plant phosphoenolpyruvate carboxylases by glucose-6 phosphate. AB - Studies of the response of phosphoenolpyruvate carboxylase from C(3) (wheat [Triticum aestivum L.]), C(4) (maize [Zea mays L.]), and Crassulacean acid metabolism (CAM) (Crassula) leaves to the activator glucose-6-phosphate as a function of pH showed that the binding of the activator and the response path to activation were essentially identical for all three enzymes. The level of affinity for the activator differed, with the CAM enzyme having the highest affinity and the maize enzyme the lowest. The observed pK values suggest that histidine and cysteine groups may be involved in activation by glucose-6 phosphate. The presence of glucose-6-phosphate protected the enzyme against inactivation of the activation response by p-chloromercuribenzoate. The maximal activation response to glucose-6-phosphate showed differences among the three enzymes including different pH optima and different pH profiles. Here the maize leaf enzyme showed a potential response about twice as great as that of the C(3) and CAM enzymes. PMID- 16666823 TI - Aerobic and anaerobic respiration in the intact spinach chloroplast. AB - Aerobic and anaerobic chloroplastic respiration was monitored by measuring (14)CO(2) evolution from [(14)C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6 bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O(2) or in N(2) with nitrite or oxaloacetate as electron acceptors. The pathway of (14)CO(2) evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N(2) were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O(2) was the best (2.4 nanomoles CO(2) per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N(2) with nitrite and oxaloacetate. PMID- 16666824 TI - Photophosphorylation after chilling in the light : effects on membrane energization and coupling factor activity. AB - The response of in situ photophosphorylation in attached cucumber (Cucumis sativus L. cv Ashley) leaves to chilling under strong illumination was investigated. A single-beam kinetic spectrophotometer fitted with a clamp-on, whole leaf cuvette was used to measure the flash-induced electrochromic absorbance change at 518 minus 540 nanometers (DeltaA(518-540)) in attached leaves. The relaxation kinetics of the electric field-indicating DeltaA(518-540) measures the rate of depolarization of the thylakoid membrane. Since this depolarization process is normally dominated by proton efflux through the coupling factor during ATP synthesis, this technique can be used, in conjuction with careful controls, as a monitor of in situ ATP formation competence. Whole, attached leaves were chilled at 5 degrees C and 1000 microeinsteins per square meter per second for up to 6 hours then rewarmed in the dark at room temperature for 30 minutes and 100% relative humidity. Leaf water potential, chlorophyll content, and the effective optical pathlength for the absorption measurements were not affected by the treatment. Light- and CO(2)-saturated leaf disc oxygen evolution and the quantum efficiency of photosynthesis were inhibited by approximately 50% after 3 hours of light chilling and by approximately 75% after 6 hours. Despite the large inhibition to net photosynthesis, the measurements of DeltaA(518-540) relaxation kinetics showed photophosphorylation to be largely unaffected by the chilling and light exposure. The amplitude of the DeltaA(518 540) measures the degree of energization of the photosynthetic membranes and was reduced significantly by chilling in the light. The cause of the decreased energization was traced to impaired turnover of photosystem II. Our measurements showed that the chilling of whole leaves in the light caused neither an uncoupling of photophosphorylation from photosynthetic electron transport nor any irreversible inhibition of the chloroplast coupling factor in situ. The sizeable inhibition in net photosynthesis observed after chilling in the light cannot, therefore, be attributed to any direct effect on photophosphorylation competence. PMID- 16666825 TI - Circadian Rhythm in the Expression of the mRNA Coding for the Apoprotein of the Light-Harvesting Complex of Photosystem II : Phytochrome Control and Persistent Far Red Reversibility. AB - The mRNA coding for light-harvesting complex of PSII (LHC-II) apoprotein is present in etiolated bean (Phaseolus vulgaris L.) leaves; its level is low in 5 day-old leaves, increases about 3 to 4 times in 9- to 13-day-old leaves, and decreases thereafter. A red light pulse induces an increase in LHC-II mRNA level, which is reversed by far red light, in all ages of the etiolated tissue tested. The phytochrome-controlled initial increase of LHC-II mRNA level is higher in 9- and 13-day-old than in 5- and 17-day-old bean leaves. The amount of LHC-II mRNA, accumulated in the dark after a red light pulse, oscillates rhythmically with a period of about 24 hours. This rhythm is also observed in continuous white light and in the dark following exposure to continuous white light, and persists for at least 70 hours. A second red light pulse, applied 36 hours after initiation of the rhythm, induces a phase-shift, which is prevented by far red light immediately following the second red light pulse. A persistent, but gradually reduced, far red reversibility of the red light-induced increase in LHC-II mRNA level is observed. In contrast, far red reversibility of the red light-induced clock setting is only observed when far red follows immediately the red light. It is concluded that (a) the light-induced LHC-II mRNA accumulation follows an endogenous, circadian rhythm, for the appearance of which a red light pulse is sufficient, (b) the circadian oscillator is under phytochrome control, and (c) a stable Pfr form, which exists for several hours, is responsible for sustaining LHC-II gene transcription. PMID- 16666826 TI - Degradation of 2-carboxyarabinitol 1-phosphate by a specific chloroplast phosphatase. AB - The catalytic degradation of 2-carboxyarabinitol 1-phosphate (CA 1-P), a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was investigated by chromatographic and spectroscopic analyses of the reaction products. Carboxy-labeled [(14)C]CA 1-P was incubated with a partially purified tobacco (Nicotiana rustica) chloroplast protein that has been shown previously to catalyze metabolism of CA 1-P to a form incapable of inhibiting Rubisco (ME Salvucci, GP Holbrook, JC Anderson, and G Bowes [1988] FEBS Lett 231: 197-201). In the presence and absence of NADPH, ion-exchange chromatography showed a progressive conversion of [2'-(14)C]CA 1-P to a labeled compound which coeluted with authentic carboxyarabinitol. Parallel assays with unlabeled CA 1-P showed a concomitant decrease in the ability of reaction samples to inhibit Rubisco activity. In separate experiments, a 1:1 stoichiometry was found between the release of inorganic phosphate from [2'-(14)C]CA 1-P and accumulation of the (14)C-labeled product. Liberation of inorganic phosphate was not observed when the tobacco enzyme was incubated with ribulose-1,5-bisphosphate, fructose-1,6 bisphosphate, glucose-1-phosphate, glucose-6-phosphate, or 6-phosphogluconate. Proton nuclear magnetic resonance spectroscopy of the labeled CA 1-P reaction product established its identity as carboxyarabinitol. We therefore propose that light-stimulated degradation of CA 1-P is catalyzed in vivo by a specific phosphatase, 2-carboxyarabinitol 1-phosphatase. Carboxyarabinitol 1-phosphatase activity was detected in the absence of NADPH, but increased threefold when 2 millimolar NADPH was present. Thus, while not required for the reaction, NADPH may play an important role in the regulation of CA 1-P degradation. PMID- 16666827 TI - Purification and Properties of 2-Carboxy-d-Arabinitol 1-Phosphatase. AB - Carboxyarabinitol 1-phosphatase (2-carboxy-d-arabinitol 1-phosphate phosphohydrolase), a chloroplast enzyme that metabolizes the naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, was isolated from tobacco (Nicotiana tabacum) leaves. The enzyme was purified more than 3500-fold using a protocol that included ammonium sulfate fractionation and gel filtration, ion-exchange, and hydrophobic interaction chromatography. Analysis of the final preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of a single polypeptide with a molecular mass of 53 kilodaltons. The enzyme exhibited an apparent K(m) (carboxyarabinitol 1-phosphate) of 33 micromolar and a pH optimum of 7.5. Enzyme activity did not require divalent cations and was unaffected by the metal chelators EDTA and cysteine. Carboxyarabinitol 1-phosphatase activity was inhibited by zinc, copper and molybdate and stimulated by sulfate. Chloroplast metabolites that affected activity included inorganic phosphate and ATP, which were inhibitory, and ribulose-1,5-bisphosphate, fructose-1,6-bisphosphate and NADPH which stimulated activity 2.5-fold. Activation of carboxyarabinitol 1-phosphatase activity by these positive effectors, together with the previously reported requirement for dithiothreitol, explain the light/dark modulation of carboxyarabinitol 1 phosphatase activity in vivo. PMID- 16666828 TI - Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans. AB - Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls. PMID- 16666829 TI - Molecular cloning and characterization of senescence-related genes from carnation flower petals. AB - The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased production of ethylene which plays an important role in regulating this developmental event. Three senescence-related cDNA clones were isolated from a cDNA library prepared from mRNA isolated from senescing petals. These cDNAs are representative of two classes of mRNAs which increase in abundance in senescing petal tissue. The mRNA for one class is present at low levels during the early stages of development and begins to accumulate in mature petals prior to the increase in ethylene production. The accumulation of this mRNA is reduced, but not eliminated, in petals treated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, or silver thiosulfate, an ethylene action inhibitor. In contrast, expression of the second class of mRNAs appears to be highly regulated by ethylene. These mRNAs are not detectable prior to the rise in ethylene production and increase in abundance in parallel with the ethylene climacteric. Furthermore, expression of these mRNAs is significantly inhibited by both aminooxyacetic acid and silver thiosulfate. Expression of these mRNAs in vegetative and floral organs was limited to floral tissue, and predominantly to senescing petals. PMID- 16666830 TI - Effects of Compounds Affecting Calcium Channels on Phytochrome- and Blue Pigment Mediated Pulvinar Movements of Cassia fasciculata. AB - Verapamil and nifedipine, known as calcium channel blockers, inhibited the phytochrome-mediated movements induced on Cassia fasciculata leaflets by a light off signal, whereas they had no effect on the ;blue' pigment-mediated movements induced by a light-on signal. LaCl(3) inhibited both types of reactions, but the inhibition of light-induced opening needed a 10 times higher concentration than that of dark-induced closure. Bay K 8644, an activator of calcium channels, increased the rate of dark-induced closure, whereas it had no effect on the light induced opening. These data suggest that calcium ions are not mobilized in the same way in the two types of movements: possibly from external stores in the phytochrome-mediated reaction and from internal stores in the ;blue' pigment mediated reaction. PMID- 16666831 TI - Developing Seeds of Ricinus communis L., When Detached and Maintained in an Atmosphere of High Relative Humidity, Switch to a Germinative Mode without the Requirement for Complete Desiccation. AB - Immature seeds of castor bean (Ricinus communis) removed from the capsule at 25 to 40 days after pollination (25-40 DAP) and placed in an atmosphere of high relative humidity undergo limited water loss, and germinate upon subsequent return to full hydration. This switch from a developmental to a germinative/growth mode at 40 DAP is reflected in a change in the types of proteins being synthesized in the endosperm; after partial drying, developmental protein synthesis ceases and germinative/growth-related proteins are produced. The nature and timing of these protein synthetic changes elicited upon imbibition are identical to those following premature desiccation/rehydration of 30 and 40 DAP seeds and upon imbibition of the mature dry seed. Enzymes involved in postgerminative reserve mobilization (l-leucyl-beta-naphthylamidase and isocitrate lyase) are induced upon imbibition, following partial drying at 40 DAP, to levels attained in the endosperms of germinated mature, and prematurely dried/rehydrated, seeds. The changes in protein synthesis resulting from partial drying are effected at the transcriptional and post-transcriptional level. Upon return to full hydration some new (i.e. germination and growth-related) mRNAs are synthesized, while others (associated with development) present in the partially dried endosperm decline. Thus developing seeds of castor bean do not have to experience substantial (whole seed) water loss to acquire the ability to germinate and grow upon subsequent imbibition. Seed detachment from the mother plant alone is not sufficient to elicit a switch to germination and growth processes. However, the length of time of detachment from the mother plant, in combination with some water loss may interact to elicit the "switch" from development to germination. PMID- 16666832 TI - Growth Patterns Inferred from Anatomical Records : Empirical Tests Using Longisections of Roots of Zea mays L. AB - Our objective was to test whether accurate growth analyses can be obtained from anatomical records and some mathematical formulas. Roots of Zea mays L. were grown at one of two temperatures (19 degrees C or 29 degrees C) and were prepared with standard techniques for light microscopy. Positions of cell walls were digitized from micrographs. The digitized data were averaged and smoothed and used in formulas to estimate growth trajectories, Z(t), velocities, v(z), and strain rates, r(z), where Z(t) is the location occupied by the cellular particle at time t; and v(z) and r(z) are, respectively, the fields of growth velocity and strain rate. The relationships tested are: for Z(t), t = n * c; v(z) = l(z) * f; and r(z) = f * ( partial differential/ partial differentialz (l(z))). In the formulas, n represents the number of cells between the origin and the position Z(t); l(z) is local cell length; the constant c, named the ;cellochron,' denotes the time for successive cells to pass a spatial point distal to the meristem; l(z) is local cell length, and f is cell flux. Growth trajectories and velocity fields from the anatomical method are in good agreement with earlier analyses based on marking experiments at the two different temperatures. Growth strain rate fields show an unexpected oscillation which may be due to numerical artifacts or to a real oscillation in cell production rate. PMID- 16666833 TI - Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.). AB - Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in lambdagt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca(2+)-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants. PMID- 16666834 TI - The Relationship between Ribulose Bisphosphate Concentration, Dissolved Inorganic Carbon (DIC) Transport and DIC-Limited Photosynthesis in the Cyanobacterium Synechococcus leopoliensis Grown at Different Concentrations of Inorganic Carbon. AB - To examine the factors which limit photosynthesis and their role in photosynthetic adaptation to growth at low dissolved inorganic carbon (DIC), Synechococcus leopoliensis was grown at three concentrations (as signified by brackets) of DIC, high (1000-1800 micromolar), intermediate (200-300 micromolar), and low (10-20 micromolar). In all cell types photosynthesis varied from being ribulose bisphosphate (RuBP)-saturated at low external [DIC] to RuBP-limited at high external [DIC]. The maximum rate of photosynthesis (P(max)) was achieved when the internal concentration of RuBP fell below the active site density of RuBP carboxylase/oxygenase (Rubisco). At rates of photosynthesis below P(max), photosynthetic capacity was limited by the ability of the cell to transport inorganic carbon and to supply CO(2) to Rubisco. Adaptation to low DIC was reflected by a decrease in the [DIC] required to half-saturate photosynthesis. Simultaneous mass-spectrometric measurement of rates of photosynthesis and DIC transport showed that the initial slope of the photosynthesis versus [DIC] curve is identical to the initial slope of the DIC transport versus [DIC] curve. This provided evidence that the enhanced capacity for DIC transport which occurs upon adaptation to low [DIC] was responsible for the increase in the initial slope of the photosynthesis versus [DIC] curve and therefore the decrease in the half saturation constant of photosynthesis with respect to DIC. Levels of RuBP and in vitro Rubisco activity varied only slightly between high and intermediate [DIC] grown cells but fell significantly (65-70%) in low [DIC] grown cells. Maximum rates of photosynthesis followed a similar pattern with P(max) only slightly lower in intermediate [DIC] grown cells than in high [DIC] grown cells, but much lower in low [DIC] grown cells. The changing response of photosynthesis to [DIC] during adaptation to low DIC, may be explained by the interaction between DIC transport limited and [RuBP]-limited photosynthesis. PMID- 16666835 TI - Reduced Accumulation of ABA during Water Stress in a Molybdenum Cofactor Mutant of Barley. AB - A barley (Hordeum vulgare L.) mutant (Az34) has been identified with low basal levels of abscisic acid (ABA) and with reduced capacity for producing ABA in response to water stress. The mutation is in a gene controlling the molybdenum cofactor resulting in a pleiotropic deficiency in at least three molybdoenzymes, nitrate reductase, xanthine dehydrogenase, and aldehyde oxidase. The mutant was found to lack aldehyde oxidase activity with several substrates including: (a) ABA aldehyde, a putative precursor of ABA; (b) an acetylenic analog of ABA aldehyde; and (c) heptaldehyde. Elevating the growth temperature from 18 to 26 degrees C caused mutant leaves to wilt and brown. Desiccation of mutant leaves was prevented by applying ABA. These results indicate that ABA biosynthesis at some developmental stages is dependent upon a molybdoenzyme which may be an aldehyde oxidase. PMID- 16666836 TI - Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells. AB - Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6 phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation. PMID- 16666837 TI - Assay and Electrophoresis of Superoxide Dismutase from Red Spruce (Picea rubens Sarg.), Loblolly Pine (Pinus taeda L.), and Scotch Pine (Pinus sylvestris L.) : A Method for Biomonitoring. AB - This paper reports a method for extracting the antioxidant enzyme superoxide dismutase (SOD) from the needles of red spruce (Picea rubens Sarg.), loblolly pine (Pinus taeda L.), and scotch pine (Pinus sylvestris L.) with high efficiency and free from interfering compounds. The extraction employs phosphate buffer with polyvinylpolypyrrolidone and Triton X-100 followed by dialysis overnight. The isozymes of SOD in each species were separated electrophoretically and tested for their sensitivity to KCN and H(2)O(2). An isozyme resistant to these inhibitors was found in the spruce but not the pine needles. The isozymes from the spruce needles were examined for individual responses to aging and H(2)O(2) inhibition. Four of the five CuZn isozymes in spruce were found to have increased significantly but equally by October of their first year and two of those four isozymes were found to be more sensitive to H(2)O(2). The response of the SOD isozymes in loblolly pine seedlings to O(3) was also examined and the isozymes were found to be induced equally. Because the SOD activity in the young pine needles was too low to electrophorese, the SOD activity from the pines in the O(3) experiment had to be partially purified using CHCl(3) and ethanol, then concentrated. PMID- 16666838 TI - Effects of Extracellular pH on UV-Induced K Efflux from Cultured Rose Cells. AB - Ultraviolet (UV) light causes a specific leakage of K(+) from cultured rose cells (Rosa damascena). During K(+) efflux, there is also an increase in extracellular HCO(3) (-) and acidification of the cell interior. We hypothesized that the HCO(3) (-) originated from intracellular hydration of respiratory CO(2) and served as a charge balancing mechanism during K(+) efflux, the K(+) and HCO(3) ( ) being cotransported out of the cell through specific channels. An alternative hypothesis which would yield similar results would be the countertransport of K(+) and H(+). To test these hypotheses, we studied the effect of a range of external pH values (pH 5-9), regulated by various methods (pH-stat, 100 millimolar Tris-Mes buffer, or CO(2) partial pressure), on the UV-induced K(+) efflux. Both UV-C (<290 nanometers) and UV-B (290-310 nanometers) induced K(+) efflux with a minimum at about pH 6 to 7, and greater efflux at pH values of 5, 8, and 9. Since pH values of 8 and 9 increased instead of reduced the efflux of K(+), these data are not consistent with the notion that the efflux of K(+) is dependent on an influx of H(+), a process that would be sensitive to external H(+) concentration. We suggest that the effect of pH on K(+) efflux may be mediated through the titration of specific K(+)-transporting proteins or channels in the plasma membrane. Since we could not detect the presence of carbonic anhydrase activity in cell extracts, we could not use the location of this enzyme to aid in our interpretation regarding the site of hydration of CO(2). PMID- 16666839 TI - Compartmentation of Peroxisomal Enzymes in Algae of the Group of Prasinophyceae : Occurrence of Possible Microbodies without Catalase. AB - Using the Prasinophycean algae Platymonas, Heteromastix, Pedinomonas, and Pyramimonas, subcellular distribution of the enzymes of glycolate metabolism and beta-oxidation pathway have been studied. Glycolate dehydrogenase, hydroxypyruvate reductase, and glutamate-glyoxylate aminotransferase are located in the mitochondria. In addition, the mitochondria of all four species contain acyl-coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and thiolase. In Platymonas, Heteromastix, and Pedinomonas, organelles with the characteristic structure of peroxisomes have been detected which also contain the enzymes acyl CoA oxidase, enoyl-CoA hydratase and thiolase. However, catalase could not be demonstrated in either the peroxisome-like organelles or in the whole cells. PMID- 16666840 TI - Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature. AB - Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35 degrees C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20 degrees C as those cultured at 25 degrees C. Both desaturases had negligible activity in seeds cultured at 35 degrees C compared to seeds cultured at 20 degrees C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35 degrees C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20 degrees C cultures. Activities were twofold lower at 25 degrees C and a further threefold lower in 35 degrees C cultures. Cultures from 25 and 35 degrees C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20 degrees C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not. PMID- 16666841 TI - Inactive Photosystem II Complexes in Leaves : Turnover Rate and Quantitation. AB - The flash-induced electrochromic shift, measured by the amplitude of the rapid absorbance increase at 518 nanometers (DeltaA518), was used to determine the amount of charge separation within photosystems II and I in spinach (Spinacia oleracea L.) leaves. The recovery time of the reaction centers was determined by comparing the amplitudes of DeltaA518 induced by two flashes separated by a variable time interval. The recovery of the DeltaA518 on the second flash revealed that 20% of the reaction centers exhibited a recovery half-time of 1.7 +/- 0.3 seconds, which is 1000 times slower than normally active reaction centers. Measurements using isolated thylakoid membranes showed that photosystem I constituted 38% of the total number of reaction centers, and that the photosystem I reaction centers were nearly fully active, indicating that the slowly turning over reaction centers were due solely to photosystem II. The results demonstrate that in spinach leaves approximately 32% of the photosystem II complexes are effectively inactive, in that their contribution to energy conversion is negligible. Additional evidence for inactive photosystem II complexes in spinach leaves was provided by fluorescence induction measurements, used to monitor the oxidation kinetics of the primary quinone acceptor of photosystem II, Q(A), after a short flash. The measurements showed that in a fraction of the photosystem II complexes the oxidation of Q(A) (-) was slow, displaying a half-time of 1.5 +/- 0.3 seconds. The kinetics of Q(A) (-) oxidation were virtually identical to the kinetics of the recovery of photosystem II determined from the electrochromic shift. The key difference between active and inactive photosystem II centers is that in the inactive centers the oxidation rate of Q(A) (-) is slow compared to active centers. Measurements of the electrochromic shift in detached leaves from several different species of plants revealed a significant fraction of slowly turning over reaction centers, raising the possibility that reaction centers that are inefficient in energy conversion may be a common feature in plants. PMID- 16666842 TI - Changes in photosynthesis and other chloroplast traits in lanceolate leaflet isoline of soybean. AB - Lanceolate leaflet soybean (Glycine max L. Merrill) has been known to photosynthesize more CO(2) per unit leaf area than normal leaflet soybean. The exact reason for this increase in photosynthetic rate is still unclear. The present study was undertaken to investigate the leaf photosynthetic rate and other physiological traits in relation to chloroplast of lanceolate leaflet soybean. Ontogenic changes in apparent photosynthesis (AP) were related primarily to variations in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit leaf area, and only secondarily to difference in specific activity of the said enzyme. Moreover, lanceolate leaflet consistently maintained a higher leaf AP, higher Rubisco activity, and more chloroplasts per unit leaf area basis than did normal leaflet soybean throughout leaf ontogeny. However, lanceolate soybean tended to have lower AP and Rubisco activity on a chloroplast basis. The superiority of leaf AP and other leaf physiological traits, expressed on a leaf area basis, in lanceolate leaflet soybean is associated with a corresponding increase in chloroplast number. PMID- 16666843 TI - Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : II. Regulation of ALA-Synthesizing Capacity by Phytochrome. AB - When dark-grown cucumber (Cucumis sativus L.) seedlings previously exposed to white light for 20 hours were returned to darkness, the ability of isolated chloroplasts to synthesize 5-aminolevulinic acid dropped by approximately 70% within 1 hour. The seedlings were then exposed to light, and the synthetic ability of the isolated chloroplasts was determined. Restoration of the synthetic capacity was promoted by continuous white or red light of moderate intensity. Intermittent red light was also effective. Blue and far-red light did not restore the synthetic capability. Blue light given after a red pulse did not enhance the effect of the red light. Far-red light given immediately after each red pulse prevented the stimulation due to intermittent red light. Restoration of the biosynthetic activity by in vivo light treatments was inhibited by cycloheximide indicating the requirement for translation on 80 S ribosomes for the in vivo light response. These findings suggest that the majority of the plastidic 5 aminolevulinic acid synthesis is under phytochrome regulation. PMID- 16666844 TI - Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma membrane vesicles. AB - Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H(+)-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H(+) was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H(+)-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, K(m) for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H(+)-ATPase activity in vivo. PMID- 16666845 TI - Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris. AB - The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse labeled with either [(3)H]glucosamine or [(3)H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M(r) 40,000 and most probably has four asparagine linked glycans. Pinto UI 111 LLP has M(r) 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [(3)H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin. PMID- 16666846 TI - Environmental Effects on Photorespiration of C(3)-C(4) Species : I. Influence of CO(2) and O(2) during Growth on Photorespiratory Characteristics and Leaf Anatomy. AB - The possibility of altering CO(2) exchange of C(3)-C(4) species by growing them under various CO(2) and O(2) concentrations was examined. Growth under CO(2) concentrations of 100, 350, and 750 micromoles per mole had no significant effect on CO(2) exchange characteristics or leaf anatomy of Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), or Flaveria trinervia (C(4)). Carboxylation efficiency and CO(2) compensation concentrations in leaves of F. floridana developed under the different CO(2) concentrations were intermediate to F. pringlei and F. trinervia. When grown for 12 days at an O(2) concentration of 20 millimoles per mole, apparent photosynthesis was strongly inhibited in Panicum milioides (C(3)-C(4)) and to a lesser degree in Panicum laxum (C(3)). In P. milioides, acute starch buildup was observed microscopically in both mesophyll and bundle sheath cells. Even after only 4 days exposure to 20 millimoles per mole O(2), the presence of starch was more pronounced in leaf cross-sections of P. milioides compared to those at 100 and 210 millimoles per mole. Even though this observation suggests that P. milioides has a different response to low O(2) with respect to translocation of photosynthate or sink activity than C(3) species, the concentration of total available carbohydrate increased in shoots of all species by 33% or more when grown at low O(2). This accumulation occurred even though relative growth rates of Festuca arundinacea (C(3)) and P. milioides grown for 4 days at 210 millimoles per mole O(2), were inhibited 83 and 37%, respectively, when compared to plants grown at 20 millimoles per mole O(2). PMID- 16666847 TI - Relationship between the Quantum Efficiencies of Photosystems I and II in Pea Leaves. AB - The irradiance dependence of the efficiencies of photosystems I and II were measured for two pea (Pisum sativum [L.]) varieties grown under cold conditions and one pea variety grown under warm conditions. The efficiencies of both photosystems declined with increasing irradiance for all plants, and the quantum efficiency of photosystem I electron transport was closely correlated with the quantum efficiency of photosystem II electron transport. In contrast to the consistent pattern shown by efficiency of the photosystems, the redox state of photosystem II (as estimated from the photochemical quenching coefficient of chlorophyll fluorescence) exhibited relationships with both irradiance and the reduction of P-700 that varied with growth environment and genotype. This variability is considered in the context of the modulation of photosystem II quantum efficiency by both photochemical and nonphotochemical quenching of excitation energy. PMID- 16666848 TI - C Fixation by Leaves and Leaf Cell Protoplasts of the Submerged Aquatic Angiosperm Potamogeton lucens: Carbon Dioxide or Bicarbonate? AB - Protoplasts were isolated from leaves of the aquatic angiosperm Potamogeton lucens L. The leaves utilize bicarbonate as a carbon source for photosynthesis, and show polarity; that is, acidification of the periplasmic space of the lower, and alkalinization of the space near the upper leaf side. At present there are two models under consideration for this photosynthetic bicarbonate utilization process: conversion of bicarbonate into free carbon dioxide as a result of acidification and, second, a bicarbonate-proton symport across the plasma membrane. Carbon fixation of protoplasts was studied at different pH values and compared with that in leaf strips. Using the isotopic disequilibrium technique, it was established that carbon dioxide and not bicarbonate was the form in which DIC actually crossed the plasma membrane. It is concluded that there is probably no true bicarbonate transport system at the plasma membrane of these cells and that bicarbonate utilization in this species apparently rests on the conversion of bicarbonate into carbon dioxide. Experiments with acetazolamide, an inhibitor of periplasmic carbonic anhydrase, and direct measurements of carbonic anhydrase activity in intact leaves indicate that in this species the role of this enzyme for periplasmic conversion of bicarbonate into carbon dioxide is insignificant. PMID- 16666849 TI - Enhancement of thermal injury to photosynthesis in wheat plants and thylakoids by high light intensity. AB - Thermal inhibition and photoinhibition of plants, which may occur simultaneously in nature, were investigated to determine whether the two causal stresses interact and to characterize any interactions that occurred. Photosynthetic rates of wheat (Triticum aestivum L. cv Len) seedlings declined gradually after temperature treatment increased from 22 to 42 degrees C or after photosynthetically active radiation (PAR) treatment increased from 450 to 2000 micromoles per square meter per second and fell rapidly after the stresses were simultaneously imposed. Stomatal conductance and internal CO(2) were affected little, indicating the interaction occurred in chloroplasts. Thylakoid whole chain electron transport, quantum yield, and saturating PAR intensity were decreased by high temperature and an additional amount by high PAR treatments. Photosystem reactions involving water oxidation were inhibited more than other reactions, and chlorophyll fluorescence transients indicated most inhibition was on the photooxidizing side of photosystem II. Injury was influenced little by the order in which the stresses were imposed and was always most severe when they were combined. Release of proteins from thylakoid membranes was not detected. Lability to the stresses was lowest in thylakoids from vegetative stage plants and increased as plants matured. We concluded that thermal injury is accentuated by high PAR, the two stresses may act at a common site near the water oxidizing complex, and their interaction may be involved in photosynthetic decline during adverse conditions. PMID- 16666850 TI - Chilling-Induced Lipid Degradation in Cucumber (Cucumis sativa L. cv Hybrid C) Fruit. AB - Chilling at 4 degrees C in the dark induced lipid degradation in cucumber (Cucumis sativa L.) fruit upon rewarming at 14 degrees C. Rates of ethane evolution by fruits rewarmed after 3 days of chilling were up to four-fold higher than those evolved by unchilled (14 degrees C) fruits (0.02-0.05 picomoles gram fresh weight(-1) hour(-1)). This potentiation of lipid peroxidation occurred prior to irreversible injury (requiring 3 to 7 days of chilling) as indicated by increases in ethylene evolution and visual observations. Decreases in unsaturation of peel tissue glycolipids were observed in fruits rewarmed after 3 days of chilling, indicating the plastids to be the site of the early phases of chilling-induced peroxidation. Losses in unsaturation of tissue phospholipids were first observed only after chilling for 7 days. Phospholipase D activity appeared to be potentiated in fruits rewarmed after 7 days of chilling as indicated by a decrease in phosphatidylcholine (and secondarily phosphatidylethanolamine) with a corresponding increase in phosphatidic acid. These results indicate that lipid peroxidation may have a role in conferring chilling injury. PMID- 16666851 TI - Calvin-Benson Cycle Enzymes in Guard-Cell Protoplasts from Vicia faba L: Implications for the Greater Utilization of Phosphoglycerate/Dihydroxyacetone Phosphate Shuttle between Chloroplasts and the Cytosol. AB - Activities of Calvin-Benson cycle enzymes were found in protoplasts of guard cells from Vicia faba L. The activities of NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD) and ribulose-1,5-bisphosphate carboxylase (RuBPC) were 2670 and 52 micromoles per milligrams chlorophyll per hour, respectively. Activities of NADP-GAPD and RuBPC in guard cells were increased by red light illumination, and the light activations were inhibited completely by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Enzymes related to the Calvin-Benson cycle such as 3-phosphoglycerate kinase (PGAK), triose phosphate (TP) isomerase, and fructose-1,6-bisphosphatase (FBPase) were shown to be present in guard-cell chloroplasts. From these results, we conclude that the photosynthetic carbon reduction pathway is present in guard-cell chloroplasts of Vicia faba. We compared these enzyme activities in guard cells with those in mesophyll cells. The activities of NADP-GAPD and PGAK were more than several-fold higher and that of TP isomerase was much higher in guard-cell chloroplasts than in mesophyll chloroplasts. In contrast, activities of RuBPC and FBPase were estimated to be roughly half of those in mesophyll chloroplasts. High activities of PGAK, NAD-GAPD, and TP isomerase were found in fractions enriched in cytosol of guard cells. Illumination of guard-cell protoplasts with red light increased the cellular ATP/ADP ratio from 5 to 14. These results support the interpretation that guard cells utilize a shuttle system (e.g. phosphoglycerate [PGA]/dihydroxyacetone phosphate [DHAP] shuttle) for an indirect transfer of ATP and reducing equivalents from chloroplasts to the cytosol. PMID- 16666852 TI - Induction of Proteinase Inhibitors in Tobacco Cell Suspension Culture by Elicitors of Phytophthora parasitica var. nicotianae. AB - An elicitor preparation obtained from Phytophthora parasitica var. nicotianae, a pathogen of tobacco, induced an accumulation of proteinase inhibitors and a stimulation of ethylene synthesis in a tobacco (Nicotiana tabacum) cell suspension culture. About 30 micrograms per milliliter of elicitor were necessary for maximal induction of proteinase inhibitor accumulation, and the response was detectable after 12 hours of incubation with elicitor. Accumulation of proteinase inhibitors required de novo protein synthesis, since cycloheximide completely inhibited its elicitation, and actinomycin D inhibited it partially. One of the inhibitors was purified by a procedure that included heating, (NH(4))(2)SO(4) precipitation, ion-exchange chromatography, and affinity chromatography. The purified inhibitor was shown to be a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 10,500. It inhibited trypsin but not chymotrypsin. PMID- 16666853 TI - Hydroxamic Acid glucosyltransferases from maize seedlings. AB - Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy 7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K(m) of 174 micromolar for DIMBOA and a K(m) of 638 micromolar for DIBOA; the enzyme in peak 1 has a K(m) of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K(m). The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize. PMID- 16666854 TI - b-Type Cytochromes in Higher Plant Plasma Membranes. AB - The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E(0)'). Cytochrome b-560.7 (E(0)' from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception. PMID- 16666855 TI - Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii. AB - A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K(m) for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H(2) adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166). PMID- 16666856 TI - Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury. AB - Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K(m) around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K(m) was decreased by about 3 fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K(m), while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze thaw stress. PMID- 16666857 TI - Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential. AB - In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize alpha-amylase/trypsin inhibitor and a tobacco mosaic virus induced pathogenesis-related protein. PMID- 16666858 TI - Salt Stress-Induced Cytoplasmic Acidification and Vacuolar Alkalization in Nitellopsis obtusa Cells : In VivoP-Nuclear Magnetic Resonance Study. AB - Time courses of cytoplasmic and vacuolar pH changes under salt stress were monitored by in vivo(31)P-nuclear magnetic resonance spectroscopy in intact cells of Nitellopsis obtusa. When cells were treated with 100 millimolar NaCl for 2 hours, the cytoplasmic pH deceased from 7.2 to 7.0, while the vacuolar pH increased from 4.9 to 5.2. This salt-induced breakdown of the pH gradient between the cytoplasm and the vacuole was also confirmed through direct measurements of change in vacuolar pH with a micro-pH electrode. We speculate that the intracellular pH changes induced by the salt stress mainly results from the inhibition of the H(+)-translocating pyrophosphatase in the vacuolar membrane, since this H(+)-translocating system is sensitive to salt-induced increase in the cytoplasmic [Na(+)] and a simultaneous decrease in the cytoplasmic [K(+)]. Since disturbance of the cytoplasmic pH value should have serious consequences on the homeostasis of living cells, we propose that the salt-induced intracellular pH changes are one of initial and important steps that lead to cell death. PMID- 16666859 TI - Role of Calcium in Phytochrome-Controlled Nyctinastic Movements of Albizzia lophantha Leaflets. AB - The involvement of Ca(2+) on phytochrome-controlled nyctinastic closure in Albizzia lophantha has been studied by testing the effect of the calcium ionophore 6S-[6alpha(2S(*),3S(*)),8beta(R(*)),9beta,11alpha]-5- methyl-amino)-2 [[3,9,11-trimethyl-8-[-1-methyl-2-oxo-2-(1H-pyrrol-2-yl) ethyl]-1,7 dioxaspiro[5.5]-undec-2yl] methyl]-4-benzoxazolecarboxylic acid (A23187) and the intracellular calcium antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). An external supply of Ca(2+) or calcium ionophore A23187 to the Albizzia leaflets emulates the effect of red light irradiation and counteracts the inhibitory effect of far red light. The intracellular calcium antagonist TMB-8 supplied to Albizzia leaflets inhibits the effect of red light, but had no effect on far red irradiated plants. This suggests a dependence between phytochrome action and intracellular free Ca(2+). We suggest that calcium acts as a phytochrome messenger on control of ion fluxes that drive turgor changes in pulvinular motor cells. PMID- 16666860 TI - Phosphatidylinositol 4,5-Bisphosphate Phospholipase C and Phosphomonoesterase in Dunaliella salina Membranes. AB - In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [(3)H]phosphatidylinositol 4,5-bisphosphate (PIP(2)). Based on release of [(3)H]inositol phosphates, the plasma membrane exhibited a PIP(2)-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast ;bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [(3)H]inositol phosphates released were recovered as [(3)H]inositol trisphosphate (IP(3)). These results suggest a plausible mechanism for the rapid breakdown of PIP(2) and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [(3)H]inositol phospholipids during these assays revealed that some of the [(3)H]-PIP(2) was converted to [(3)H]phosphatidylinositol 4-monophosphate (PIP) and to [(3)H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP(2)/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca(2+)]. We also report here that 100 micromolar GTPgammaS stimulates phosphospholipase C activity over a range of free [Ca(2+)]. Together, these results provide evidence that the plasma membrane PIP(2)-phospholipase C of D. salina may be subject to Ca(2+) and G-protein regulation. PMID- 16666861 TI - Relationship between Thermal Transitions and Freezing Injury in Pea and Soybean Seeds. AB - In an attempt to correlate freezable water with freezing injury, the thermal behavior of pea (Pisum sativum L.) and soybean (Glycine max L. Merr) seed parts at different moisture contents were compared with survival of the seeds when exposed to low temperatures. Thermal transitions between -150 and 10 degrees C were studied using differential scanning calorimetry. In pea, reduction of germinability, after exposure of seeds to temperatures between - 18 and - 180 degrees C, occurred at a constant moisture content (about 0.33 gram H(2)O/gram dry weight) regardless of the temperature; this moisture level was above that at which freezable water was first detectable by differential scanning calorimetry (0.26 gram H(2)O/gram dry weight). In contrast, damage to soybean seeds was observed at progressively lower moisture contents (from 0.33 to 0.20 gram H(2)O/gram dry weight) when the temperature was decreased from -18 degrees C to 50 degrees C. At -18 and -30 degrees C, moisture contents at which damage to soybean seeds was evident were above that at which freezable water was first detectable (0.23 gram H(2)O/gram dry weight). However, at -50, -80, and -180 degrees C, damage was evident even when freezable water was not detectable. The data suggest that, while the quantity of water is important in the expression of freezing injury, the presence of freezable water does not account for the damage. PMID- 16666862 TI - Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings. AB - The effect of nitrate application on glutamine synthetase activity in roots of pea (Pisum sativum L.) seedlings (2 weeks old) was studied. Separation of organelles from root fragments by sucrose density-gradient centrifugation revealed that both nitrite reductase and glutamine synthetase activities increased in root plastids as a response to nitrate application and that no such response was induced by ammonium application. Glutamine synthetase activity was also found to increase in plastids with distance from apex in nitrate-treated plants, the highest specific activity being located in the fourth 1-centimeter segment. Separation by SDS-PAGE and characterization by Western blotting showed that cytosolic glutamine synthetase contains one subunit polypeptide (28 kilodaltons) and that plastid glutamine synthetase contains both the 38 kilodalton subunit and a heavier subunit. When nitrate was present in the nutrient solution, the heavier subunit increased in abundance in protein fractions obtained from purified root plastids. PMID- 16666863 TI - Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation. AB - A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C(16:1) and C(18:1) lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes. PMID- 16666864 TI - Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole. AB - The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water. PMID- 16666865 TI - An immunological approach to gibberellin purification and quantification. AB - Gibberellin (GA) specific, high-affinity monoclonal antibodies have been used to assay the GA content of various plant tissues and to purify selected GAs by immunoaffinity chromatography. These immunological techniques may not stand alone as a general method of GA analysis. The results of this study indicate, however, that in conjunction with gas chromatography-mass spectrometry for positive GA identification, radioimmunoassay and immunoaffinity chromatography are extremely powerful tools for purifying and quantifying GAs from plant tissues. PMID- 16666866 TI - Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis. AB - Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38 degrees C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54 degrees C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36 degrees C. Temperatures below 36 degrees C or above 40 degrees C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34 degrees C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36 degrees C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38 degrees C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS. PMID- 16666867 TI - Ozone concentration in leaf intercellular air spaces is close to zero. AB - Transpiration and ozone uptake rates were measured simultaneously in sunflower leaves at different stomatal openings and various ozone concentrations. Ozone uptake rates were proportional to the ozone concentration up to 1500 nanoliters per liter. The leaf gas phase diffusion resistance (stomatal plus boundary layer) to water vapor was calculated and converted to the resistance to ozone multiplying it by the theoretical ratio of diffusion coefficients for water vapor and ozone in air (1.67). The ozone concentration in intercellular air spaces calculated from the ozone uptake rate and diffusion resistance to ozone scattered around zero. The ozone concentration in intercellular air spaces was measured directly by supplying ozone to the leaf from one side and measuring the equilibrium concentration above the other side, and it was found to be zero. The total leaf resistance to ozone was proportional to the gas phase resistance to water vapor with a coefficient of 1.68. It is concluded that ozone enters the leaf by diffusion through the stomata, and is rapidly decomposed in cell walls and plasmalemma. PMID- 16666868 TI - Sources of Carbon for Export from Spinach Leaves throughout the Day. AB - Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity. PMID- 16666869 TI - Protoporphyrin IX Content Correlates with Activity of Photobleaching Herbicides. AB - Several laboratories have demonstrated recently that photobleaching herbicides such as acifluorfen and oxadiazon cause accumulation of protoporphyrin IX (PPIX), a photodynamic pigment capable of herbicidal activity. We investigated, in acifluorfen-treated tissues, the in vivo stability of PPIX, the kinetics of accumulation, and the correlation between concentration of PPIX and herbicidal damage. During a 20 hour dark period, PPIX levels rose from barely detectable concentrations to 1 to 2 nanomoles per 50 cucumber (Cucumis sativus L.) cotyledon discs treated with 10 micromolar acifluorfen. When placed in 500 micromoles per square meter per second PAR, PPIX levels decayed logarithmically, with an initial half-life of about 2.5 hours. PPIX levels at each time after exposure to light correlated positively with the cellular damage that occurred during the following 1 hour in both green and yellow (tentoxin-treated) cucumber cotyledon tissues. PPIX levels in discs incubated for 20 hours in darkness correlated positively with the acifluorfen concentration in which they were incubated. In cucumber, the level of herbicidal damage caused by several p-nitrodiphenyl other herbicides, a p-chlorodiphenylether herbicide, and oxadiazon correlated positively with the amount of PPIX induced to accumulate by each of the herbicide treatments. Similar results were obtained with acifluorfen-treated pigweed and velvetleaf primary leaf tissues. In cucumber, PPIX levels increased within 15 and 30 minutes after exposure of discs to 10 micromolar acifluorfen in the dark and light, respectively. These data strengthen the view that PPIX is responsible for all or a major part of the photobleaching activity of acifluorfen and related herbicides. PMID- 16666870 TI - Characterization of a xylose-specific antiserum that reacts with the complex asparagine-linked glycans of extracellular and vacuolar glycoproteins. AB - Antibodies were raised against carrot (Daucus carota) cell wall beta-fructosidase that was either in a native configuration (this serum is called anti-betaF(1)) or chemically deglycosylated (anti-betaF(2)). The two antisera had completely different specificities when tested by immunoblotting. The anti-betaF(1) serum reacted with beta-fructosidase and many other carrot cell wall proteins as well as with many proteins in extracts of bean (Phaseolus vulgaris) cotyledons and tobacco (Nicotiana tabacum) seeds. It did not react with chemically deglycosylated beta-fructosidase. The anti-betaF(1) serum also reacted with the bean vacuolar protein, phytohemagglutinin, but not with deglycosylated phytohemagglutinin. The anti-betaF(2) serum reacted with both normal and deglycosylated beta-fructosidase but not with other proteins. These results indicate that the betaF(2) antibodies recognize the beta-fructosidase polypeptide, while the betaF(1) antibodies recognize glycan sidechains common to many glycoproteins. We used immunoadsorption on glycoprotein-Sepharose columns and hapten inhibition of immunoblot reactions to characterize the nature of the antigenic site. Antibody binding activity was found to be associated with Man(3)(Xyl)(GIcNAc)(2)Fuc, Man(3)(Xyl)(GIcNAc)(2), and Man(Xyl) (GIcNAc)(2) glycans, but not with Man(3)(GIcNAc)(2). Treatment of phytohemagglutinin, a glycoprotein with a Man(3)(Xyl)(GIcNAc)(2)Fuc glycan, with Charonia lampas beta xylosidase (after treatment with jack-bean alpha-mannosidase) greatly diminished the binding between the antibodies and phytohemagglutinin. We conclude, therefore, that the antibodies bind primarily to the xylosebeta, 1--> 2mannose structure commonly found in the complex glycans of plant glycoproteins. PMID- 16666871 TI - Composition and Structure of Starch from Taproots of Contrasting Genotypes of Medicago sativa L. AB - The objective of this study was to examine the composition and branch chain lengths of alfalfa (Medicago sativa L.) taproot starch during starch utilization and reaccumulation in response to defoliation. Genotypes were propagated vegetatively and well-established plants were sampled at defoliation and at weekly intervals thereafter. Starch granules from root tissues were dispersed in dimethyl sulfoxide and starch components separated using gel permeation chromatography. Root starches also were debranched enzymically, and branch chain lengths were examined. Results indicate that, irrespective of starch concentration, starch from taproots of the high starch genotype was composed of approximately 80% high molecular weight starch with I(2)-Kl absorbance characteristics similar to amylopectin. The remaining 20% of the starch was low molecular weight with I(2)-Kl absorbance characteristics similar to amylose. Starches of the low starch genotype contained approximately 85% high molecular weight polysaccharide at high root starch concentrations (>50 grams per kilogram). At low root starch concentrations (<10 grams per kilogram), starch from the low starch genotype had nearly equal proportions of low and high molecular weight polysaccharide. The I(2)-Kl absorbance properties of the low molecular weight starches from roots of the low starch genotype indicated that some branching may be present. The distribution of chain lengths from amylopectin did not change during starch degradation and reaccumulation for the high starch genotype. In the low starch genotype, the proportion of low molecular weight branches having a degree of polymerization between 1 and 30 was decreased at the very low starch concentrations observed on the 14th day of regrowth. Higher concentrations and/or quantities of starch in roots of the high starch genotype were not associated with greater rate of herbage regrowth, when compared to the low starch genotype. PMID- 16666872 TI - Adaptation of Chlamydomonas reinhardtii High-CO(2)-Requiring Mutants to Limiting CO(2). AB - Photosynthetic characteristics of four high-CO(2)-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO(2) concentrations. The four mutants represent two loci involved in the CO(2)-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO(2) concentration, indicating that the genetic lesion in each is expressed even at elevated CO(2) concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO(2) characteristic of the induction of the CO(2)-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO(2)-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO(2)-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon. PMID- 16666873 TI - Growth rates and assimilate partitioning in the elongation zone of tall fescue leaf blades at high and low irradiance. AB - Tall fescue (Festuca arundinacea Schreb.) leaf blades elongated 33% faster at continuous low than at continuous high irradiance (60 versus 300 micromoles per second per square meter photosynthetic photon flux density) when temperature of the leaf elongation zone was held constant at 21 degrees C. Increased rate of elongation was associated with a near proportional increase in length of the elongation zone (+38%). In contrast, growth in width and thickness was decreased at low irradiance, resulting in only a 12% increase in leaf area production and 5% less total growth-associated water deposition than at high irradiance. At low irradiance dry matter (DM) import into the elongation zone was 28% less, and 55% less DM was used per unit leaf area produced. DM use in synthesis of structural components (i.e. DM less water-soluble carbohydrates) was only 13% less at low irradiance, whereas water-soluble carbohydrates (WSC) deposition was 43% less. The lower rate of WSC deposition at low irradiance was associated with a higher net rate of monosaccharide deposition (+39%), whereas net deposition rates for sucrose (-27%) and fructan (-56%) were less than at high irradiance. Still, at low irradiance, net fructan accumulation accounted for 64% of WSC deposition, i.e. 25% of DM import, demonstrating the high sink strength of the leaf elongation zone. PMID- 16666874 TI - Transient Accumulation of Nitrite Reductase mRNA in Maize following the Addition of Nitrate. AB - Expression of the gene coding for nitrite reductase (NiR) is induced upon the addition of nitrate. We have analyzed this induction process in hydroponically grown maize (Zea mays L.) seedlings where the level of nitrate in the medium can be easily manipulated. There is a rapid induction of NiR mRNA upon addition of nitrate, increasing first in the roots and then in the leaves. The rapidity of the response depends on the nitrate concentration and the growth medium. However, the general pattern of expression is the same: the mRNA level increases, reaches a maximum, and then decreases, despite the fact that the nitrate concentration in the medium remains constant. This decline in mRNA level can be quite rapid, particularly in root tissue. If the nitrate is given as a pulse, the mRNA levels decrease even more rapidly. It is clear that the NiR mRNA is short-lived, with a half-life in the roots of less than 30 minutes. The NiR protein level, on the other hand, increases gradually somewhat after the increase in mRNA and remains at high levels at least for 24 hours after the addition of nitrate. PMID- 16666875 TI - Use of Carbon Oxysulfide, a Structural Analog of CO(2), to Study Active CO(2) Transport in the Cyanobacterium Synechococcus UTEX 625. AB - Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO(2). Although hydrolysis of COS (to CO(2) and H(2)S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO(2) transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO(2) and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H(2)S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H(2)S. The active transport of CO(2) was inhibited by COS in an apparently competitive manner. When Na(+)-dependent HCO(3) (-) transport was allowed in the presence of COS, the extracellular [CO(2)] rose considerably above the equilibrium level. This CO(2) appearing in the medium was derived from the dehydration of transported HCO(3) (-) and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO(2) was inhibited. These results showed that the Na(+)-dependent HCO(3) (-) transport was not inhibited by COS, whereas active CO(2) transport was inhibited. When COS was removed by gassing with N(2), a normal pattern of CO(2) uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO(3) (-) transport or CO(2) fixation. The steady state rate of CO(2) fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO(2) from the cells that occurred when CO(2) uptake was inhibited by COS. Neither CS(2) nor N(2)O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO(2) transport. PMID- 16666876 TI - Estimation of ammonium concentration in the cytosol of soybean nodules. AB - Analysis of ammonium concentration in the cytosol of soybean (Glycine max [L.] Merr.) root nodules gave high levels of error variation. When the separation of cytosol and bacteroids was deliberately delayed following nodule maceration, a large increase in ammonium concentration was found in the cytosol. When a series of samples was subjected to delay intervals of 0 to 60 minutes, extrapolation of the regression line to time zero indicated that the ammonium concentration in cytosol at the time of nodule maceration was essentially nil. The source of ammonium buildup following maceration was not found, but hydrolysis of free amino acids or ureides was ruled out. An extremely low concentration of ammonium in the cytosol is consistent with a model for movement of ammonia (or ammonium) from bacteroids to host cytoplasm by diffusion. PMID- 16666877 TI - C nuclear magnetic resonance study of suberized potato cell wall. AB - High-resolution, solid-state (13)C nuclear magnetic resonance (NMR) spectra are reported for suberized cell wall from potatoes (Solanum tuberosum L.). Through experiments combining the techniques of cross polarization and magic-angle spinning, we verified that suberin, like cutin, is a polyester and demonstrated that it also has phenylpropanoid groups characteristic of lignin. Roughly 50% of the suberized material consists of cell-wall polymers; aromatics and other unsaturated linkages outnumber methylene groups 2:1. In conjunction with traditional direct-polarization NMR results, these experiments provide support for prior suggestions that suberin and cell-wall components are chemically bonded via aromatic groups. PMID- 16666878 TI - Localization of the Enzymes Involved in the Photoevolution of H(2) from Acetate in Chlamydomonas reinhardtii. AB - The localization of a series of enzymes involved in the anaerobic photodissimilation of acetate in Chlamydomonas reinhardtii F-60 adapted to a hydrogen metabolism was determined through the enzymic analyses of the chloroplastic, cytoplasmic, and mitochondrial fractions obtained with a cellular fractionation procedure that incorporated cell wall removal by treatment with autolysine, digestion of the plasmalemma with the detergent digitonin, and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photoevolution of H(2) from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumarate by chloroplastic succinate dehydrogenase, and the oxidation of malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity for the enzymes assayed were sufficient to accommodate the observed rate of the photoanaerobic dissimilation of acetate and the photoevolution of H(2). PMID- 16666879 TI - cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark. AB - cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)(+):glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression. PMID- 16666880 TI - Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium. AB - The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed. PMID- 16666881 TI - Studies of the Regulation of Nitrate Influx by Barley Seedlings Using NO(3). AB - Using (13)NO(3) (-), effects of various NO(3) (-) pretreatments upon NO(3) (-) influx were studied in intact roots of barley (Hordeum vulgare L. cv Klondike). Prior exposure of roots to NO(3) (-) increased NO(3) (-) influx and net NO(3) (-) uptake. This ;induction' of NO(3) (-) uptake was dependent both on time and external NO(3) (-) concentration ([NO(3) (-)]). During induction influx was positively correlated with root [NO(3) (-)]. In the postinduction period, however, NO(3) (-) influx declined as root [NO(3) (-)] increased. It is suggested that induction and negative feedback regulation are independent processes: Induction appears to depend upon some critical cytoplasmic [NO(3) (-)]; removal of external NO(3) (-) caused a reduction of (13)NO(3) (-) influx even though mean root [NO(3) (-)] remained high. It is proposed that cytoplasmic [NO(3) (-)] is depleted rapidly under these conditions resulting in ;deinduction' of the NO(3) ( ) transport system. Beyond 50 micromoles per gram [NO(3) (-)], (13)NO(3) (-) influx was negatively correlated with root [NO(3) (-)]. However, it is unclear whether root [NO(3) (-)] per se or some product(s) of NO(3) (-) assimilation are responsible for the negative feedback effects. PMID- 16666882 TI - Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes. AB - Sugar beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically for 2 weeks in growth chambers with two levels of orthophosphate (Pi) supplied in half strength Hoagland solution. Low-P plants were supplied with 1/20th of the Pi supplied to control plants. With low-P treatment, the acid soluble leaf phosphate and total leaf P decreased by about 88%. Low-P treatment had a much greater effect on leaf area than on photosynthesis. Low-P decreased total leaf area by 76%, dry weight per plant by 60%, and the rate of photosynthesis per area at light saturation by 35%. Low-P treatment significantly decreased the total extractable activity of phosphoglycerate kinase (by 18%) and NADP-glyceraldehyde 3-phosphate dehydrogenase (by 16%), but did not decrease the total activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) and ribulose-5-phosphate kinase. Low-P treatment decreased the initial activities of three rate-limiting Calvin cycle enzymes, but had no effect on the initial activity of RuBPCase. Furthermore, low-P treatment significantly increased the total extractable activities of fructose-1,6-bisphosphatase (by 61%), fructose-1,6-bisphosphate aldolase (by 53%), and transketolase (by 46%). The results suggest that low-P treatment affected photosynthetic rate through an effect on RuBP regeneration rather than through RuBPCase activity and that the changes in Calvin cycle enzymes with low-P resulted in an increased flow of carbon to starch. PMID- 16666883 TI - Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides. AB - Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3 phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6 phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP(+) ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis. PMID- 16666884 TI - Fructose 2,6-bisphosphate hydrolyzing enzymes in higher plants. AB - The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases I, II, and III, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a K(m) value of 30 micromolar, to a mixture of fructose 2 phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the beta-anomeric form. Its apparent K(m) for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described by F. D. Macdonald, Q. Chou, and B. B. Buchanan ([1987] Plant Physiol 85: 13-16). Phosphatase III copurified with phosphofructokinase 2 and corresponded to the specific, low-K(m) (24 nanomolar) fructose-2,6-bisphosphatase purified and characterized by Y. Larondelle, E. Mertens, E. Van Schaftingen, and H. G. Hers ([1986] Eur J Biochem 161: 351-357). Three similar types of phosphatases were present in a crude extract of Jerusalem artichoke (Helianthus tuberosus) tuber. The concentration of fructose 2,6-bisphosphate decreased at a maximal rate of 30 picomoles per minute and per gram of fresh tissue in slices of Jerusalem artichoke tuber, upon incubation in 50 millimolar mannose. This rate could be accounted for by the maximal extractable activity of the low-K(m) fructose-2,6-bisphosphatase. A new enzymic method for the synthesis of beta glucose 1,6-bisphosphate from beta-glucose 1-phosphate and ATP is described. PMID- 16666885 TI - A comparison of the metabolic fate of Fatty acids of different chain lengths in developing oilseeds. AB - To determine if medium and long chain fatty acids can be appropriately metabolized by species that normally produce 16 and 18 carbon fatty acids, homogenates of developing Cuphea wrightii, Carthamus tinctorius, and Crambe abyssinica seeds were incubated with radiolabeled lauric, palmitic, oleic, and erucic acids. In all three species, acyl-CoA synthetase showed broad substrate specificity in synthesis of acyl-coenzyme A (CoA) from any of the fatty acids presented. In Carthamus, two- to fivefold less of the foreign FAs, lauric, and erucic acid was incorporated into acyl-CoAs than palmitic and oleic acid. Lauric and erucic acid also supported less glycerolipid synthesis in Carthamus than palmitic and oleic acid, but the rate of acyl-CoA synthesis did not control rate of glycerolipid synthesis. In all species examined, medium and long chain fatty acids were incorporated predominantly into triacylglycerols and were almost excluded from phospholipid synthesis, whereas palmitic and oleic acid were found predominantly in polar lipids. However, the rate of esterification of unusual fatty acids to triacylglycerol is slow in species that do not normally synthesize these acyl substrates. PMID- 16666886 TI - Biosynthesis of nonspecific lipid transfer proteins in germinating castor bean seeds. AB - The biosynthesis of nonspecific lipid transfer proteins (ns-LTPs) in germinating castor bean (Ricinus communis L.) seeds were investigated. Lipid transfer activities of ns-LTPs in the cotyledons, axis, and endosperm increased with growth after germination. The activity increases were accompanied by increased amounts of ns-LTPs in each tissue, as measured by immunoblot using anti-ns-LTP serum. These results suggest that the ns-LTPs are synthesized de novo in each tissue after germination and not activated from inactive proteins synthesized before germination. Comparison of the immunoblot products in each tissue from 4 day-old seedlings indicate the occurrence of tissue-specific isoforms of ns-LTPs; 9 kilodaltons (major) and 7 kilodaltons (minor) in the cotyledons, and 7 kilodaltons (major) and 9 kilodaltons (minor) in the axis, whereas only the 8 kilodalton ns-LTP is present in the endosperm. In vitro translation from poly(A)(+) RNAs from three tissues of castor bean seedlings and the detection of immunoprecipitated products indicate that translatable mRNAs for ns-LTPs exist in the three tissues a day before the synthesis of ns-LTPs; the translation products, which are 3.5 to 4.0 kilodaltons larger than ns-LTPs, were processed to the mature ns-LTPs. The production of mature ns-LTPs from translatable mRNAs without any delay suggests that gene expression of ns-LTPs in castor bean seedlings is controlled at a step before the formation of translatable mRNAs. PMID- 16666887 TI - Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity. AB - Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28 degrees C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport. PMID- 16666888 TI - Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA(1) Content of Tall and Dwarf Wheat Genotypes. AB - Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA(3). Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25 degrees C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA(1) levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25 degrees C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA(1) was higher at 25 degrees C than at 10 degrees C. At 10 degrees C the endogenous GA(1) was at a similar level in all the genotypes (except Rht2+3). At 25 degrees C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25 degrees C, but were only slight and often unsignificant at 11 degrees C. The response of leaf length to applied GA(3) in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA(1) and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles. PMID- 16666889 TI - Induction of alcohol dehydrogenase and lactate dehydrogenase in hypoxically induced barley. AB - In barley (Hordeum vulgare L.), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) are induced by anaerobiosis in both aleurone layers and roots. Under aerobic conditions, developing seeds of cv Himalaya accumulate ADH activity, which survives seed drying and rehydration. This activity consists almost entirely of the ADH1 homodimer. Activity of LDH also increases during seed development, but the level of activity in dry or rehydrated seeds is very low, indicating that this enzyme may not be involved in anaerobic glycolysis during the initial stages of germination. In contrast to ADH, the LDH isozymes present in developing seeds are similar to those found in uninduced and induced roots. Developmental expression of ADH and LDH was monitored from 0 to 24 days postgermination. Neither activity was induced to any extent in the germinating seeds; however, both enzymes were highly induced by anoxia in root tissue during development. Based on gel electrophoresis, this increase in activity results from the differential expression of different Adh and Ldh genes in root tissue. The changes in ADH and LDH activity levels were matched by changes in the amount of these particular proteins, indicating that the increase in activity results from de novo synthesis of these two proteins. The level of inducible LDH activity in an ADH1(-) mutant was not found to differ from cv Himalaya. We suggest that although the ADH(-) plants are more susceptible to flooding, they are not capable of responding to the lack of ADH1 activity by increasing the amount of LDH activity in root tissue. PMID- 16666890 TI - Electrolyte Leakage, Lipoxygenase, and Lipid Peroxidation Induced in Tomato Leaf Tissue by Specific and Nonspecific Elicitors from Cladosporium fulvum. AB - Glycoprotein nonspecific elicitor (NSE) and a specific elicitor preparation from intercellular fluids (SE) of tomato (Lycopersicon esculentum Mill. cv Bonny Best or Potentate) infected with race 2.4.5 of Cladosporium fulvum Cooke [syn. Fulvia fulva (Cooke) Ciferri] were injected into cv Sonatine (resistant to race 2.4.5) to compare electrolyte leakage, lipoxygenase activity, and lipid peroxidation induced in response to these elicitors. Increased electrolyte leakage was induced by NSE or SE; the leakage due to NSE but not to SE was inhibited by the nonsteroidal antiinflammatory drug (NSAID) piroxicam. Under normal photoperiod conditions, higher levels of lipoxygenase activity were detected 6 hours after injection with either elicitor. This activity peaked by 12 hours with both elicitors and declined to control levels by 24 hours when visible necrosis could be detected. Both NSE and SE-induced lipoxygenase was inhibited by piroxicam in vitro. Lipid peroxidation in elicitor-treated tissue was also assayed at 6, 12, and 24 hours after injection using the TBA test for malonaldehyde. Increased peroxidation was detected in response to NSE or SE at 12 hours with similar values obtained at 24 hours. With plants incubated in the dark, lipoxygenase, and lipid peroxidation were similarly induced in SE-injected tissue whereas necrosis induction by SE was light dependent. PMID- 16666891 TI - Hormones and Pod Development in Oilseed Rape (Brassica napus). AB - The endogenous levels of several plant growth substances (indole acetic acid, IAA; abscisic acid, ABA; zeatin, Z; zeatin riboside, [9R]Z; isopentenyladenine, iP; and isopentenyladenosine, [9R]iP were measured during pod development of field grown oilseed Rape (Brassica napus L. var oleifera cv Bienvenu) with high performance liquid chromatography and immunoenzymic (enzyme-linked immunosorbent assay, ELISA) techniques. Results show that pod development is characterized by high levels of Z and [9R]Z in 3 day old fruits and of IAA on the fourth day. During pod maturation, initially a significant increase of IAA and cytokinins was observed, followed by a progressive rise of ABA levels and a concomitant decline of IAA and cytokinin (except iP) levels. The relationship between hormone levels and development, especially pod number, seed number per pod, and seed weight determination, will be discussed. PMID- 16666892 TI - Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves. AB - Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO(2) uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de epoxidation), as well as nonphotochemical quenching (q(NP)), and the rate constant of radiationless energy dissipation (k(D)). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO(2) uptake differed widely and light saturation of CO(2) uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO(2) uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q(NP), increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO(2) uptake was approached, q(NP) showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k(D), which opens the possibility that zeaxanthin is related to the rapidly relaxing "high-energy-state quenching" in leaves. PMID- 16666893 TI - Zeaxanthin and the Induction and Relaxation Kinetics of the Dissipation of Excess Excitation Energy in Leaves in 2% O(2), 0% CO(2). AB - The relationship between the carotenoid zeaxanthin, formed by violaxanthin de epoxidation, and nonphotochemical fluorescence quenching (q(NP)) in the light was investigated in leaves of Glycine max during a transient from dark to light in 2% O(2), 0% CO(2) at 100 to 200 micromoles of photons per square meter per second. (a) Up to a q(NP) (which can vary between 0 and 1) of about 0.7, the zeaxanthin content of leaves was linearly correlated with q(NP) as well as with the rate constant for radiationless energy dissipation in the antenna chlorophyll (k(D)). Beyond this point, at very high degrees of fluorescence quenching, only k(D) was directly proportional to the zeaxanthin content. (b) The relationship between zeaxanthin and k(D) was quantitatively similar for the rapidly relaxing quenching induced in 2% O(2), 0% CO(2) at 200 micromoles of photons per square meter per second and for the sustained quenching induced by long-term exposure of Nerium oleander to drought in high light (B Demmig, K Winter, A Kruger, F-C Czygan [1988] Plant Physiol 87: 17-24). These findings suggest that the same dissipation process may be induced by very different treatments and that this particular dissipation process can have widely different relaxation kinetics. (c) A rapid induction of strong nonphotochemical fluorescence quenching within about 1 minute was observed exclusively in leaves which already contained a background level of zeaxanthin. PMID- 16666894 TI - Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures. AB - When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O(2), 0% CO(2) at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5 degrees C leaf temperature) in air and then held under control conditions in low light in air at 25 degrees C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5 degrees C did not recover at all. During a dark/light transient in 2% O(2), 0% CO(2) in low light at 5 degrees C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content. PMID- 16666895 TI - Cytokinin oxidase from wheat: partial purification and general properties. AB - As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N(6)-(Delta(2)-isopentenyl)adenosine to adenosine at a V(max) of 0.4 nanomol per milligram protein per minute at 30 degrees C and pH 7.5, the K(m) being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N(6)-(Delta(2)-isopentenyl)adenine and zeatin riboside are also degraded, but N(6)-(Delta(2)-isopentenyl)adenosine-5' monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N(6)-(Delta(2) isopentenyl)adenosine. The degradation of N(6)-(Delta(2)-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4 pyridyl)-N'-phenylurea. PMID- 16666896 TI - Hormone Levels and Apical Dominance in the Aquatic Fern Marsilea drummondii A. Br. AB - Terminal buds and successively subjacent lateral buds of the water fern, Marsilea drummondii, were examined to determine the pattern of hormone distribution in relation to apical dominance. Quantitative levels of indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin and zeatin riboside (Z and ZR), and isopentenyladenosine (iPA) were determined by a solid-phase immunoassay using polycional antihormone antibodies. Enzyme-linked immunosorbent assay was used following a one-step HPLC purification procedure to obtain the free hormones. Active shoot apices contained the most IAA and Z-type cytokinins and inhibited buds the least. No significant differences in ABA levels were found leading to the conclusion that ABA did not play any role in apical dominance. The normal precedence of the most rapid outgrowth of the youngest inhibited bud as observed previously in decapitated plants was well correlated with its very high level of iPA observed in this study. The same phenomenon was observed in the median buds but with a weaker amplitude. The presence of this storage form could indicate that a bud at its entry into quiescence eventually looses the ability to hydroxylate iPA to Z-type cytokinins when it is fully inhibited. IAA and Z + ZR are concluded to be essential for lateral bud growth. PMID- 16666897 TI - The Proton Electrochemical Transmembrane Gradients Generated by the Transfer Cells of the Haustorium of Polytrichum formosum and Their Use in the Uptake of Amino Acids. AB - The epidermal cells of the sporophyte haustorium of Polytrichum formosum are modified into transfer cells. These cells are located in a strategic place allowing them to control the exchanges between the two generations. Their plasmalemma creates proton gradients (Deltapsi and DeltapH) which increase during the development of the sporophyte. As the sporophyte grows from 2 to 4 cm long, the pH of the incubation medium of the haustoria decreases from 5.2 to 4.3, and the transmembrane potential difference (PD) hyperpolarizes form -140 to -210 millivolts. These gradients become rapidly larger than that generated by the plasmalemma of the basal cells of the sporophyte. They are used to energize the uptake of the solutes present in the apoplast of the gametophyte, particularly the amino acids. Below 20 micromolar alpha-aminoisobutyric acid uptake in the transfer cells is mediated by a saturable system and is optimal at acidic pH (4.0 and 4.5). It is strongly inhibited by compounds dissipating both Deltapsi and DeltapH (10 micromolar carbonylcyanide-m-chlorophenyl hydrazone) or only Deltapsi (0.1 molar KCl). The absorption of alpha-aminoisobutyric acid and of the other neutral amino acids tested induces an alkalinization of the medium and a depolarization of membrane potential difference which is concentration dependent. These data show that the uptake of amino acids by the transfer cells of the haustorium is a secondary translocation (proton-amino acid symport) energized by a primary translocation (proton efflux). More particularly, they show that transfer cells possess a membrane enzymic equipment particularly efficient to achieve the uptake of the solutes leaked in the apoplast from other cell types. PMID- 16666898 TI - Interruption of Somatic Embryogenesis in Daucus carota L. by 5-Bromodeoxyuridine. AB - Embryogenic Daucus carota L. cells grown in 9 micromolar 2.4 dichlorophenoxyacetic acid are resistant to greater than 5 micromolar 5 bromodeoxyuridine (BrdU). In contrast, 5 micromolar BrdU strongly inhibits somatic embryogenesis within 24 hours after transfer of cells to an auxin-free medium. DNA synthesis rates in control and BrdU-treated cultures are rapid and similar; however, the DNA content does not reach levels as great in the presence of BrdU as in control cultures. BrdU substitutes for thymidine in the DNA in 28% of the available sites 48 hours after auxin removal. Following DNA repair, somatic embryogenesis resumes. BrdU DNA incorporation leads to somatic embryogenesis inhibition and provides an alternative to auxin treatment for the interruption of carrot cell culture differentiation. PMID- 16666899 TI - An analysis of the control of phosphorylation-coupled respiration in isolated plant mitochondria. AB - The control of phosphorylation-coupled respiration in isolated turnip (Brassica rapa) mitochondria was investigated according to the principles of metabolic control analysis as developed by H. Kacser and J. A. Burns ([1973] Symp Soc Exp Biol 32: 65-104) and R. Heinrich and T. A. Rapoport ([1974] Eur J Biochem 42: 97 105). Inhibitor titration studies were used to determine quantitatively the amount of control exerted by four individual processes-cytochrome bc(1), cytochrome oxidase, H(+)-ATPase, and the adenine nucleotide carrier-on respiratory flux under ADP-excess (state 3) and ADP-limited (state 4) conditions with a range of respiratory substrates. Under state 3 conditions control strength was found to be distributed between cytochrome oxidase, cytochrome bc(1), and H(+)-ATPase in decreasing order of importance. The adenine nucleotide carrier exerted no control on respiratory flux under these conditions. Control strength at each step was found to vary with different substrates and with the respiratory flux as altered by ADP supply, i.e. virtually zero control strength at cytochrome oxidase and cytochrome bc(1) under state 4 conditions. PMID- 16666900 TI - Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast. AB - Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity. PMID- 16666901 TI - Two modes of rubidium uptake in sunflower plants. AB - The Rb(+)-uptake kinetics in K(+)-starved sunflower (Helianthus annuus) plants can be explained by the addition of two Michaelis-Menten equations. In contrast, Rb(+) uptake can be described by a single Michaelis-Menten equation in normal K(+) plants. Differences in the K(m)s and in the Arrhenius plots of Rb(+) uptake in the two types of plants suggest two uptake modes. PMID- 16666902 TI - A mutant of Arabidopsis deficient in desaturation of palmitic Acid in leaf lipids. AB - The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by elevated amounts of palmitic acid and a decreased amount of unsaturated 16-carbon fatty acids as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggested that the mutant is deficient in the activity of a chloroplast omega9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to monogalactosyldiacylglycerol (MGD). The mutant exhibited an increased ratio of 18- to 16-carbon fatty acids in MGD due to a change in the relative contribution of the prokaryotic and eukaryotic pathways of lipid biosynthesis. This appears to be a regulated response to the loss of chloroplast omega9 desaturase and presumably reflects a requirement for polyunsaturated fatty acids for the normal assembly of chloroplast membranes. The reduction in mass of prokaryotic MGD species involved both a reduction in synthesis of MGD by the prokaryotic pathway and increased turnover of MGD molecular species which contain 16:0. PMID- 16666903 TI - Crassulacean Acid Metabolism and Photochemical Efficiency of Photosystem II in the Adaxial and Abaxial Parts of the Succulent Leaves of Kalanchoe daigremontiana Grown at Four Photon Flux Densities. AB - Kalanchoe daigremontiana, a species possessing crassulacean acid metabolism, was grown at four photon flux densities (1300, 400, 60, and 25 micromole photons per square meter per second). In leaves which had developed at 1300 and 400 micromole photons per square meter per second, CO(2) was mainly incorporated through the lower, shaded leaf surfaces, and the chlorenchyma adjacent to the lower surfaces showed a higher degree of nocturnal acid synthesis than the chlorenchyma adjacent to the upper surfaces. In leaves acclimated to 60 and 25 micromole photons per square meter per second, the gradient in CAM activity was reversed, i.e. more CO(2) was taken up through the upper than through the lower surfaces and nocturnal acidification was higher in the tissue next to the upper surfaces. Total net carbon gain and total nocturnal acid synthesis were highest in leaves which had developed at 400 micromole photons per square meter per second. Chlorophyll content was markedly reduced in leaves which had developed at 1300 micromole photons per square meter per second, especially in the exposed adaxial parts. There was also a sustained reduction in photosystem II photochemical efficiency as indicated by measurements of the ratio of variable over maximum chlorophyll a fluorescence. These findings suggest that, at high growth photon flux densities, the reduced activity of the exposed portions of these succulent leaves is caused by (a) the adverse effects of excess light, (b) together with a genotypic component which favors CO(2) uptake and acid synthesis in the abaxial (lower) leaf parts even when light is not or only marginally excessive. This latter component is predominant at medium photon flux densities, e.g. at 400 micromole photons per square meter per second. It becomes overridden, however, under conditions of deep shade when strongly reduced light levels in the abaxial parts of the leaf chlorenchyma severely limit photosynthesis. PMID- 16666904 TI - Lipid Composition of Plasma Membranes and Endomembranes Prepared from Roots of Barley (Hordeum vulgare L.) : Effects of Salt. AB - Membrane fractions enriched in endoplasmic reticulum (ER), tonoplast and Golgi membranes (TG) and plasma membranes (PM) were prepared from barley (Hordeum vulgare L. cv CM 72) roots and the lipid compositions of the three fractions were analyzed and compared. Plants were grown in an aerated nutrient solution with or without 100 millimolar NaCl. Each membrane fraction had a characteristic lipid composition. The mole per cent of the individual phospholipids, glycolipids, and sterols in each fraction was not altered when roots were grown in 100 millimolar NaCl. The ER had the highest percentages of phosphatidylinositol and phosphatidylcholine of the three fractions (7 and 45 mole per cent, respectively, of the total lipid). The TG contained the highest percentage of glycosylceramide (13 mole per cent). The PM had the highest percentage of phosphatidylserine (3 mole per cent) and nearly equal percentages of phosphatidylethanolamine (15 mole per cent and phosphatidylcholine (18 mole per cent). The most abundant sterols in membranes prepared from barley roots were stigmasterol (10 mole per cent), sitosterol (50 mole per cent), and 24zeta-methylcholesterol (40 mole per cent of the total sterol). Salt-treated plants contained a slightly higher percentage of stigmasterol than controls. The percentage of stigmasterol increased with age and a simple cause and effect relationship between salt treatment and sterol composition was not observed. PMID- 16666905 TI - Nitrate effects on nitrate reductase activity and nitrite reductase mRNA levels in maize suspension cultures. AB - Nitrate reductase (NR) activity and nitrite reductase (NiR) mRNA levels were monitored in Black Mexican Sweet maize (Zea mays L.) suspension cultures after the addition of nitrate. Maximal induction occurred with 20 millimolar nitrate and within 2 hours. Both NR and NiR mRNA were transiently induced with levels decreasing after the 2 hours despite the continued presence of nitrate in the medium. Neither ammonia nor chlorate prevented the induction of NR. Furthermore, removal of nitrate, followed by its readdition 22 to 48 hours later, did not result in reinduction of activity or message. NR was synthesized de novo, since cycloheximide completely blocked its induction. Cycloheximide had no effect on the induction of NiR mRNA or on the transient nature of its induction. These results are similar to those reported previously for maize seedlings. PMID- 16666906 TI - Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. AB - The rate of CO(2) fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO(2) fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein. PMID- 16666907 TI - Developmental Control of Apiogalacturonan Biosynthesis and UDP-Apiose Production in a Duckweed. AB - Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, d-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed d [(3)H]glucuronic acid for 30 minutes, the accumulated UDP-[(3)H]apiose pool accounted for about 27% of the total phosphorylated [(3)H]pentose derivatives; in turions, the UDP-[(3)H]apiose pool accounted for only about 4% of the total phosphorylated [(3)H]pentose derivatives. We conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDP-apiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase. PMID- 16666908 TI - Partial Purification and Characterization of Three Flavonol-Specific Sulfotransferases from Flaveria chloraefolia. AB - Three distinct flavonol-specific sulfotransferases were partially purified from the shoot tips of Flaveria chloraefolia A. Gray by fractional precipitation with ammonium sulfate, followed by gel filtration on Sephacryl S-200, 3' phosphoadenosine 5-phosphate-Agarose affinity chromatography and chromatofocusing on Mono P. These enzymes exhibited expressed specificity for positions 3 of various flavonol acceptors and of 3' and 4' of flavonol 3-sulfate. The three sulfotransferases had similar molecular weights (35,000), exhibited no requirement for divalent cations and were not inhibited by SH group reagents. Their K(m) values for both the sulfate donor and the flavonol acceptors were of the same order of magnitude (ca. 0.2-0.4 micromolar). Except for the 3 sulfotransferase, which exhibited two optima at pH 6.5 and 8.5, the 3' and the 4' sulfotransferases had a pH optimum of 7.5. The three enzymes could be resolved only by chromatofocusing and were eluted at pH 5.4, pH 6.0, and pH 5.1 for the 3 , 3'- and 4'-sulfotransferases, respectively. The substrate specificity of these three enzymes is discussed in relation to the biosynthesis of polysulfated flavonols in F. chloraefolia. PMID- 16666909 TI - Electrogenic ATPase Activity on the Peribacteroid Membrane of Soybean (Glycine max L.) Root Nodules. AB - Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (DeltaPsi), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive DeltaPsi across the peribacteroid membrane was dependent upon ATP, inhibited by N,N'-dicyclohexyl-carbodiimide and vanadate, but insensitive to N ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO(3) (-) > NO(2) (-) > Cl(-) > acetate(-) > malate( ). The observation that 10 millimolar NO(3) (-) completely dissipated the membrane potential was of particular interest in view of the fact that NO(3) (-) inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed. PMID- 16666910 TI - Putrescine-induced wounding and its effects on membrane integrity and ion transport processes in roots of intact corn seedlings. AB - Interactions between putrescine and membrane function were examined with the use of a recently developed microelectrode system that enables us simultaneously to quantify membrane potentials and net K(+) fluxes associated with individual cells at the root surface of an intact corn (Zea mays L.) seedling. In contrast to the results of others, our analyses indicate that exogenous putrescine (0.5 millimolar), in the absence of calcium, does not maintain membrane stability. In addition, putrescine caused a wound response characterized by a gradual depolarization of the membrane potential and a considerable net efflux of K(+) from the root. In the presence of calcium, both short term (20 minutes) and long term (24 hours) exposure to a high concentration of exogenous putrescine (5 millimolar) also caused a reduction in the resting membrane potential and a significant K(+) efflux. However, preincubating corn roots in a solution containing the antioxidant ascorbate ameliorated the wounding effects of putrescine and slightly increased potassium uptake. A similar preincubation in the absence of calcium did not protect membranes against putrescine-induced damage. The ameliorating effect of ascorbate on putrescine-induced membrane damage suggests that the wounding response of high putrescine levels in corn roots involves the catabolism of the polyamine by a cell wall diamine oxidase, with the concomitant production of hydrogen peroxide and free radicals resulting in peroxidative damage of the plasmalemma. PMID- 16666911 TI - Regulation of 5-aminolevulinic Acid synthesis in developing chloroplasts : I. Effect of light/dark treatments in vivo and in organello. AB - Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were returned to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALA-synthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from dark-treated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feed-back inhibitor on ALA synthesis. Chloroplasts isolated from dark-treated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not turn over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment. PMID- 16666912 TI - Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence. AB - The distribution of photosynthetic activity over the area of a leaf and its change with time was determined (at low partial pressure of O(2)) by recording images of chlorophyll fluorescence during saturating light flashes. Simultaneously, the gas exchange was being measured. Reductions of local fluorescence intensity quantitatively displayed the extent of nonphotochemical quenching; quench coefficients, q(N), were computed pixel by pixel. Because rates of photosynthetic electron transport are positively correlated with (1 - q(N)), computed images of (1 - q(N)) represented topographies of photosynthetic activity. Following application of abscisic acid to the heterobaric leaves of Xanthium strumarium L., clearly delineated regions varying in nonphotochemical quenching appeared that coincided with areoles formed by minor veins and indicated stomatal closure in groups. PMID- 16666913 TI - Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen methyl. AB - The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from delta-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from delta-aminolevulinate was blocked, indicating a site of inhibition prior to the Mg-chelatase. The enzymic oxidation of protoporphyrinogen IX to protoporphyrin IX was highly sensitive to acifluorfen methyl, indicating that the site of action of the herbicide is the protoporphyrinogen oxidase. ((c) 1989 FMC Corporation. All rights reserved.). PMID- 16666914 TI - A Unique Phenotype in Heterozygotes of the Auxin-Insensitive Mutant of Tomato, diageotropica. AB - Tomato (Lycopersicon esculentum Mill.) plants heterozygous for the diageotropica (dgt) mutation exhibit a unique phenotype, termed ;mottled.' Unlike dgt, mottled individuals grow upright, exhibit normal root branching, and produce normal levels of ethylene in response to applied auxin. Leaves of mottled plants are deformed and reduced in size and are characterized by a mottled appearance on their surfaces with small dark-green islands clustered along the leaf veins. The lack of phenotypic overlap between dgt and mottled may represent interallelic interaction at a locus which influences auxin sensitivity or action in the tomato. PMID- 16666915 TI - Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : III. Stimulation of Enzymic Hydrolysis of Indole-3-Acetyl-l-Alanine. AB - The sucrose-stimulated in vivo hydrolysis of indole-3-acetyl-l-alanine (IAAIa) in tobacco (Nicotiana tabacum L.) leaf discs was confirmed by in vitro analysis of an IAAIa-hydrolyzing enzyme isolated from the same tissue. The enzymic activity could be stimulated by either aging of the tissue or by application of external IAA or sucrose. A combination of the above three treatments yielded maximal activity. PMID- 16666916 TI - Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration. AB - The rate of dark O(2) uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O(2) uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O(2) uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O(2) uptake were similar to those of cyanide, except that the percent stimulation of O(2) uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase. PMID- 16666917 TI - Preferential Loss of an Abundant Storage Protein from Soybean Pods during Seed Development. AB - A temporary vegetative storage protein, composed of similar 25 kilodalton and 27 kilodalton subunits, was found to be abundant in soybean (Glycine max (L.) Herr. var Hobbit) leaves, stems, pods, flower petals, germinated cotyledons, and less abundant in roots, nodules and seeds. Total pod protein was highest at 3 weeks after flowering and declined by 37% within 3 weeks during seed development. During this time the vegetative storage protein declined from 18% to 1.5% of the total pod protein and accounted for 45% of the protein lost from pods. This indicates that the vegetative storage protein makes a significant contribution to the pool of nutrients mobilized from pods for transport to developing seeds. PMID- 16666918 TI - Heat Shock Protein Synthesis Induced by Methomyl in Maize (Zea mays L.) Seedlings. AB - Exposure of plumules of intact maize seedlings (Zea mays L.) to S-methyl-N [(methylcarbamoyl)-oxy]thioacetimidate (methomyl) represses synthesis of several polypeptides normally made under control conditions and induces synthesis of polypeptides similar to maize heat shock polypeptides (HSPs). Three of the methomyl-induced polypeptides (18 kilodaltons) are recognized by antibodies raised against 18 kilodalton maize heat shock polypeptides. PMID- 16666919 TI - Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis. AB - Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [(35)S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation. PMID- 16666920 TI - Lethal hydroxyl radical production in paraquat-treated plants. AB - Bipyridinium herbicides, including paraquat and diquat, are believed to act by generating highly reactive, oxygen-centered free radicals within chloroplasts when treated plants are exposed to sunlight. This hypothesis has not yet been confirmed by direct chemical measurements of specific free radicals. We studied paraquat-treated plants using a new method able to detect and quantify formation of highly reactive and deleterious hydroxyl radicals (HO(*)), in which dimethyl sulfoxide (DMSO) is used as a molecular probe. DMSO is oxidized by HO(*) to form the stable, nonradical compound, methane sulfinic acid, which can be easily extracted from plant tissue and measured spectrophotometrically. Initial experiments revealed formation of extraordinary numbers of hydroxyl radicals in light-exposed, paraquat + DMSO-treated plants, equivalent at least to the cumulative number of HO(*) radicals per gram of fresh tissue that would be produced by 10,000 rads of gamma irradiation. This appears to be the greatest production of hydroxyl radicals yet observed in a biological system and is quite sufficient to explain the rapid death of top growth in paraquat-treated plants. PMID- 16666921 TI - Salinity stress increases cytoplasmic ca activity in maize root protoplasts. AB - High concentrations of NaCl immediately elevated cytoplasmic Ca activity in maize (Zea Mays L. cv Pioneer 3377) root protoplasts, as measured with the fluorescent probe Indo-1. The effect of salinity was inhibited by Li pretreatment but restored by inositol, suggesting that phosphoinositides mediate the stress response. PMID- 16666922 TI - Phosphate Starvation Inducible ;Bypasses' of Adenylate and Phosphate Dependent Glycolytic Enzymes in Brassica nigra Suspension Cells. AB - When Brassica nigra leaf petiole suspension cells were subjected to 7 days of inorganic phosphate (Pi) starvation the extractable activity of: (a) pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate phosphatase, and phosphoenolpyruvate carboxylase increased at least fivefold, (b) phosphorylating NAD-glyceraldehyde 3-phosphate dehydrogenase decreased about sixfold, and (c) ATP:fructose 6-phosphate 1-phosphotransferase, 3-phosphoglycerate kinase, pyruvate kinase, or NAD malic enzyme was not altered. Pi deprivation also resulted in significant reductions in extractable levels of Pi, ATP, ADP, fructose 2,6-bisphosphate, and soluble protein, but caused a sixfold elevation in free amino acid concentrations. No change in inorganic pyrophosphate concentration was observed following Pi starvation. It is hypothesized that pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP glyceraldehyde 3-phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion. PMID- 16666923 TI - Rapid cycling of autophosphorylation of a ca-calmodulin regulated plasma membrane located protein kinase from pea. AB - Plasma membrane vesicles from pea (Pisum sativum L.) seedlings contain an autophosphorylating calcium-activated protein kinase of relative molecular weight 18,000 (phosphoprotein 18, pp 18). Pulse chase analysis revealed that pp 18 autophosphorylation exhibited very rapid turnover. pp 18 was the only detectable plasma membrane protein to have this property. pp 18 has been highly purified by affinity chromatography and the final preparation contains peptides of relative molecular weight 67,000, 48,000, and 18,000. Highly purified pp 18 still showed rapid cycling of autophosphorylated phosphate attached to pp 18. Turnover of pp 18 autophosphorylation was accelerated by ADP, but this effect did not appear to be the back reaction of the kinase since inorganic phosphate accumulation is increased by ADP. A rapid cycling of phosphorylation is an ideal control point in signal transduction. PMID- 16666924 TI - Isoenzymes of Superoxide Dismutase in Nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp. AB - The activity and isozymic composition of superoxide dismutase (SOD; EC 1.15.1.1) were determined in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp. formed by Rhizobium phaseoll 3622, R. Ieguminosarum 3855, and Bradyrhizobium sp. BR7301, respectively. A Mn-SOD was present in Rhizobium and two in Bradyrhizobium and bacteroids. Nodule mitochondria from all three legume species had a single Mn-SOD with similar relative mobility, whereas the cytosol contained several CuZn-SODs: two in Phaseolus and Pisum, and four in Vigna. In the cytoplasm of V. unguiculata nodules, a Fe-containing SOD was also present, with an electrophoretic mobility between those of CuZn- and Mn-SODs, and an estimated molecular weight of 57,000. Total SOD activity of the soluble fraction of host cells, expressed on a nodule fresh weight basis, exceeded markedly that of bacteroids. Likewise, specific SOD activities of free-living bacteria were superior or equal to those of their symbiotic forms. Soluble extracts of bacteria and bacteroids did not show peroxidase activity (EC 1.11.1.7), but the nodule cell cytoplasm contained diverse peroxidase isozymes which were readily distinguishable from leghemoglobin components by electrophoresis. Data indicated that peroxidases and leghemoglobins did not significantly interfere with SOD localization on gels. Treatment with chloroform ethanol scarcely affected the isozymic pattern of SODs and peroxidases, and had limited success in the removal of leghemoglobin. PMID- 16666925 TI - Inhibitory effect of gabaculine on 5-aminolevulinate dehydratase activity in radish seedlings. AB - We have compared the activity of 5-aminolevulinate dehydratase (5-ALAD) with the amount of protein detected by specific antibodies in rocket immunoelectrophoresis. Parallel kinetic evolutions of enzymic activity and amount of antigen were observed in radish (Raphanus sativus L.) cotyledons, both in complete darkness or under standard far red light involving phytochrome. However, the treatment of seedlings with gabaculine leads to an important decrease in enzymic activity, while the specific protein content is maintained. This inhibition is not overcome by the addition of glutamic acid, but by 5 aminolevulinic acid which points to a specific control of 5-ALAD activity by its substrate. As there is no discrepancy between the enzymic activity and the amount of antigen during the time course development of seedlings, this could confirm a coordinate cellular control between 5-aminolevulinic acid formation and 5-ALAD protein synthesis, both being amplified by the action of phytochrome. PMID- 16666926 TI - Thermal regulation of phosphoenolpyruvate carboxylase and ribulose-1,5 bisphosphate carboxylase in c(3) and c(4) plants native to hot and temperate climates. AB - Exposure of leaf sections from 2-week-old seedlings of sorghum (Sorghum bicolor L.) (C(4) plant), corn (Zea mays L.) (C(4)), peanut (Arachis hypogaea L.) (C(3) plant), and soybean (Glycine max L.) (C(3)) to 40 or 45 degrees C for up to 4 hours resulted in significant increases in the levels of 102 kilodalton (C(4)), 52 kilodalton (C(3) and C(4)), and 15 kilodalton (C(3) and C(4)) polypeptides. These proteins comigrated, respectively, with authentic phosphoenolpyruvate carboxylase (PEPC) and the large (RLSU) and small (RSSU) subunits of ribulose-1,5 bisphosphate carboxylase (Rubisco) during both one- and two-dimensional SDS-PAGE and reacted with antisera raised against these enzymes. After 4 hours at 50 degrees C, levels of the polypeptides either remained relatively stable (PEPC, RLSU) or increased (RSSU) in sorghum and peanut (plants native to hot climates). In corn and soybean (plants native to temperate climates), levels of the proteins either fell sharply (corn) or showed strong evidence of incomplete processing and/or aggregation (soybean). In addition to changes in levels of the proteins, the activities of PEPC and Rubisco in extracts of leaves exposed to 50 degrees C fell by 84% and 11% of their respective control values in sorghum and by 54% each in peanut. In corn and soybean, the activities of both enzymes were depressed at 40 degrees C, with measured values at 50 degrees C not exceeding 5% of those from the nonstressed controls. PMID- 16666927 TI - Anaerobic induction of alanine aminotransferase in barley root tissue. AB - Alanine aminotransferase, otherwise called glutamate-pyruvate aminotransferase (GPT), activity increases up to fourfold during several days of anaerobic induction in barley (Hordeum vulgare L.) roots, reaching a maximum activity of 13 international units per gram fresh weight. This increase in activity paralleled the increase in alcohol dehydrogenase activity in the same root tissue. Upon return to aerobic conditions, the induced GPT activity declined with an apparent half-life of 2 days. The isozyme profile of GPT in barley root tissue comprised one band of activity; in maize there were three bands of activity, the bands with greater mobility had much lower activity. Native polyacrylamide gel electrophoresis indicated that the induction of GPT activity results from an increase in the level of activity of these bands; no other activities were detected. When root tissue was induced under different levels of hypoxia (0%, 2%, 5%, and 21% O(2)), changes in GPT activity were found to increase with lower levels of oxygen. Comparisons of GPT induction in barley, maize (Zea mays), rye, (Secale cereale) and wheat (Triticum aestivum) indicate that this enzyme is induced in the root tissue of all of these cereals; however, anaerobic root conditions do not result in the induction of GPT activity in leaf tissue. The dependence of GPT induction on high levels of nitrate in the media was tested by comparing activity levels in Hoagland solution and a nitrate-free nutrient solution. GPT activity was induced to similar levels under both conditions. These results indicate that alanine aminotransferase shows a very similar pattern of induction to alcohol dehydrogenase in barley root tissue and may be important in anaerobic glycolysis. PMID- 16666928 TI - Superoxide Dismutase Activity in Needles of Norwegian Spruce Trees (Picea abies L.). AB - The activity of superoxide dismutase was investigated in needles of spruce trees. To obtain maximum activity, needles were homogenized in the presence of Triton X 100 and polyvinylpyrrolidone. Superoxide dismutase activity was measured in dialyzed extracts with a modified epinephrine assay (HP Misra, I Fridovich [1972] J Biol Chem 247: 3170-3175) at pH 10.2. The extracts contained 70 to 120 units of superoxide dismutase per milligram protein. One unit of superoxide dismutase was completely inhibited in the presence of 20 micromolar NaCN. On native polyacrylamide gels three electromorphs were visualized after staining for activity. All three species were sensitive to CN(-) and H(2)O(2) and were therefore assumed to be Cu/Zn-superoxide dismutases. Superoxide dismutase activity was dependent on the age of the needles and declined by approximately 25% within 3 to 4 years. PMID- 16666929 TI - Zeatin Glycosylation Enzymes in Phaseolus: Isolation of O-Glucosyltransferase from P. lunatus and Comparison to O-Xylosyltransferase from P. vulgaris. AB - An enzyme catalyzing the formation of O-glucosylzeatin in immature embryos of Phaseolus lunatus was purified 2500-fold using ammonium sulfate precipitation followed by affinity and anion exchange chromatography. The enzyme uses trans zeatin as substrate (K(m) 28 micromolar) but not cis-zeatin, ribosylzeatin, or dihydrozeatin. Both UDP-glucose and UDP-xylose can serve as glycosyl donors, with K(m)s of 0.2 and 2.7 millimolar, respectively, for the formation of O glucosylzeatin and O-xylosylzeatin. In comparison, the UDPxylose-zeatin:O xylosyltransferase (JE Turner, DWS Mok, MC Mok, G Shaw [1987] Proc Natl Acad Sci USA 84: 3714-3717) isolated by the same procedures from P. vulgaris embryos uses only UDP-xylose as donor substrate and the K(m)s for both zeatin and UDP-xylose are much lower (2 and 3 micromolar, respectively). The chromatographic behavior on affinity columns and molecular weights (approximate M(r) 44,000 daltons) of the two enzymes are similar. Results from substrate competition experiments and enzyme separation by anion exchange HPLC indicate a single, distinct, zeatin O glycosylation enzyme occurs in embryos of each of these Phaseolus species. PMID- 16666930 TI - Partitioning of Noncyclic Photosynthetic Electron Transport to O(2)-Dependent Dissipative Processes as Probed by Fluorescence and CO(2) Exchange. AB - The partitioning of noncyclic photosynthetic electron transport between net fixation of CO(2) and collective O(2)-dependent, dissipative processes such as photorespiration has been examined in intact leaf tissue from Nicotiana tabacum. The method involves simultaneous application of CO(2) exchange and pulse modulated fluorescence measurements. As either irradiance or CO(2) concentration is varied at 1% O(2) (i.e. absence of significant O(2)-dependent electron flow), the quantum efficiency of PSII electron transport (phi(se)) with CO(2) as the terminal acceptor is a linear function of the ratio of photochemical:nonphotochemical fluorescence quenching coefficients (i.e. q(Q):q(NP)). When the ambient O(2) concentration is raised to 20.5% or 42% the q(Q):q(NP) is assumed to predict the quantum efficiency of total noncyclic electron transport (phi'(se)). A factor which represents the proportion of electron flow diverted to the aforementioned dissipative processes is calculated as (phi'(se) - phi(se))/phi'(se) where phi(se) is now the observed quantum efficiency of electron transport in support of net fixation of CO(2). Examination of changes in electron allocation with CO(2) and O(2) concentration and irradiance at 25 degrees C provides a test of the applicability of the Rubisco model to photosynthesis in vivo. PMID- 16666931 TI - Circadian Stomatal Rhythms in Epidermal Peels from Vicia faba. AB - Circadian rhythms in stomatal aperture and in stomatal conductance have been observed previously. Here we investigate circadian rhythms in apertures that persist in functionally isolated guard cells in epidermal peels of Vicia faba, and we compare these rhythms with rhythms in stomatal conductance in attached leaves. Functionally isolated guard cells kept in constant light display a rhythmic change in aperture superimposed on a continuous opening trend. The rhythm free-runs with a period of about 22 hours and is temperature compensated between 20 and 30 degrees C. Functionally isolated guard cell pairs are therefore capable of sustaining a true circadian rhythm without interaction with mesophyll cells. Stomatal conductance in whole leaves displays a more robust rhythm, also temperature-compensated, and with a period similar to that observed for the rhythm in stomatal aperture in epidermal peels. When analyzed individually, some stomata in epidermal peels showed a robust rhythm for several days while others showed little rhythmicity or damped out rapidly. Rhythmic periods may vary between individual stomata, and this may lead to desynchronization within the population. PMID- 16666932 TI - Gibberellic Acid stimulation of cucumber hypocotyl elongation : effects on growth, turgor, osmotic pressure, and cell wall properties. AB - Recently developed techniques have been used to reinvestigate the mechanism by which gibberellic acid (GA(3)) stimulates elongation of light-grown cucumber (Cucumis sativus L.) seedlings. Osmotic pressure and turgor pressure were slightly reduced in GA(3)-treated seedlings, which elongated 3.5 times faster than control seedlings. This indicated that GA(3) enhancement of growth was not controlled by changes in the osmotic properties of the tissues. Stress/strain (Instron) analysis revealed that plastic extension of the cell walls of GA(3) treated seedlings increased by up to 35% above the control values. Stress relaxation measurements on frozen-thawed tissue showed that T(0) the minimum relaxation time, was reduced following application of GA(3). In vivo wall relaxation (measured by the pressure block technique) showed that the wall yield coefficient was increased, and the yield threshold was slightly reduced. Thus GA(3) affected both the mechanical (viscoelastic) and biochemical (chemorheological) properties of the cell walls of light-grown cucumber. The previous hypothesis, that GA(3) stimulates cucumber hypocotyl growth by increasing osmotic pressure and cell turgor, is contradicted by our results. PMID- 16666933 TI - Auxin and Ethylene Regulation of Petiole Epinasty in Two Developmental Mutants of Tomato, diageotropica and Epinastic. AB - The epinastic growth responses of petioles to auxin and ethylene were quantified in two developmental mutants of tomato (Lycopersicon esculentum Mill.). In the wild type parent line, cultivar VFN8, the epinastic response of excised petiole sections was approximately log-linear between 0.1 and 100 micromolar indole-3 acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations, with a greater response to 2,4-D at any concentration. When ethylene synthesis was inhibited by aminoethoxyvinylglycine (AVG), epinasty was no longer induced by auxin, but could be restored by the addition of ethylene gas. In the auxin insensitive mutant, diageotropica (dgt), no epinastic response to IAA was observed at IAA concentrations that effectively induced epinasty in VFN8. In the absence of added IAA, epinastic growth of dgt petioles in 1.3 microliters per liter exogenous ethylene gas was more than double that of VFN8 petioles. IAA had little additional effect in dgt, but promoted epinasty in VFN8. These results confirm that tomato petiole cells respond directly to ethylene and make it unlikely that the differential growth responsible for epinasty results from lateral auxin redistribution. The second mutant, Epinastic (Epi), exhibits constitutively epinasty, cortical swelling, and root branching symptomatic of possible alternation in auxin or ethylene regulation of growth. Only minor quantitative differences were observed between the epinastic responses to auxin and ethylene of VFN8 and Epi. However, in contrast to VFN8, when ethylene synthesis or action was inhibited in Epi, auxin still induced 40 to 50% of the epinastic response observed in the absence of inhibitors. This indicates that the target cells for epinastic growth in Epi are qualitatively different from those of VFN8, having gained the ability to grow differentially in response to auxin alone. The dgt and Epi mutants provide useful systems in which to study the genetic determination of target cell specificity for hormone action. PMID- 16666934 TI - Lack of Systemic Suppression of Nodulation in Split Root Systems of Supernodulating Soybean (Glycine max [L.] Merr.) Mutants. AB - Wild-type soybean (Glycine max [L] Merr. cv Bragg) and a nitrate-tolerant supernodulating mutant (nts382) were grown in split root systems to investigate the involvement of the autoregulation response and the effect of timing of inoculation on nodule suppression. In Bragg, nodulation of the root portion receiving the delayed inoculation was suppressed nearly 100% by a 7-day prior inoculation of the other root portion with Bradyrhizobium japonicum strain USDA 110. Significant suppression was also observed after a 24-hour delay in inoculation. Mutant nts382 in the presence of a low nitrate level (0.5 millimolar) showed little, if any, systemic suppression. Root fresh weights of individual root portions were similar for both wild type and nts382 mutant. When nts382 was grown in the absence of nitrate, a 7-day delay in inoculation resulted in only 30% suppression of nodulation and a significant difference in root fresh weight between the two sides, with the delayed inoculated side always being smaller. Nodulation tests on split roots of nts382, nts1116, and wild-type cultivars Bragg, Williams 82, and Clark demonstrated a difference in their systemic suppression ability. These observations indicate that (a) autoregulation deficiencies in mutant nts382 result in a reduction of systemic suppression of nodulation, (b) some suppression is detectable after 24 hours with a delayed inoculation, (c) the presence of low nitrate affects the degree of suppression and the root growth, and (d) soybean genotypes differ in their ability to express this systemic suppression. PMID- 16666935 TI - beta-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments. AB - Antiserum was raised against the Avena sativa L. caryopsis beta-d-glucan fraction with an average molecular weight of 1.5 x 10(4). Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1-->3), (1-->4)-beta-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ beta-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis beta-d-glucans. These results support the idea that the degradation of (1-->3), (1-->4)-beta-d glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation. PMID- 16666936 TI - Post-transcriptional control of sucrose synthase expression in anaerobic seedlings of maize. AB - We have examined post-transcriptional control of expression of the anaerobically induced sucrose synthase 1 (SS1) isozyme mRNA encoded by the shrunken (Sh) gene of maize (Zea mays L.). The SS1 transcript level is increased in maize seedling roots during anaerobiosis without a concomitant increase in the SS1 protein level. We show that the anaerobic SS1 RNA was loaded onto polyribosomes and that SS1 proteins produced by in vitro translation of polyribosomal RNA from anaerobic roots and immature kernels were indistinguishable based on abundance and apparent molecular weight. [(35)S]Methionine uptake in control and anaerobically stressed seedling roots indicated a detectable, but only slight, increase in radiolabel in the SS1 polypeptide as compared to the sucrose synthase 2 isozyme, SS2. However, this slight increase in [(35)S]methionine uptake did not contribute to a detectable increase in the steady state level of SS1 protein relative to SS2 protein. Chase experiments with unlabeled methionine indicated that SS1 protein was relatively stable in the anaerobic environment. From these results we conclude that SS1 protein was not rapidly turned over in the anaerobic environment and that expression of anaerobically induced SS1 transcripts was blocked at some step beyond polyribosomal loading. PMID- 16666937 TI - Influence of cadmium on water relations, stomatal resistance, and abscisic Acid content in expanding bean leaves. AB - Ten day old bush bean plants (Phaseolus vulgaris L. cv Contender) were used to analyze the effects of 3 micromolar Cd on the time courses of expansion growth, dry weight, leaf water relations, stomatal resistance, and abscisic acid (ABA) levels in roots and leaves. Control and Cd-treated plants were grown for 144 hours in nutrient solution. Samples were taken at 24 hour intervals. At the 96 and 144 hour harvests, additional measurements were made on excised leaves which were allowed to dry for 2 hours. From the 48 hour harvest, Cd-treated plants showed lower leaf relative water contents and higher stomatal resistances than controls. At the same time, root and leaf expansion growth, but not dry weight, was significantly reduced. The turgor potentials of leaves from Cd-treated plants were nonsignificantly higher than those of control leaves. A significant increase (almost 400%) of the leaf ABA concentration was detected after 120 hours exposure to Cd. But Cd was found to inhibit ABA accumulation during drying of excised leaves. It is concluded that Cd-induced decrease of expansion growth is not due to turgor decrease. The possible mechanisms of Cd-induced stomatal closure are discussed. PMID- 16666938 TI - Transcriptional Analysis of Polygalacturonase and Other Ripening Associated Genes in Rutgers, rin, nor, and Nr Tomato Fruit. AB - We have studied the transcription of polygalacturonase (PG) and several other riponing-associated genes in wild-type tomato (Lycopersicon esculentum) fruit and three ripening-impaired mutants, rin, nor, and Nr. In wild-type fruit, the PG gene becomes transcriptionally active early in ripening and remains transcriptionally active during the ripening process. Fruit of the three ripening impaired mutants, which have reduced levels of PG mRNA, have correspondingly reduced PG transcription rates. Other ripening-associated genes showed diverse patterns of expression in the ripening-impaired mutant backgrounds. These results indicate that transcriptional activation of the PG gene is an important control point regulating the expression of PG during ripening in wild-type fruit and that PG expression in rin, nor, and Nr fruit is blocked at the level of transcription. A comparison of PG transcription rates and mRNA levels with those of other ripening-associated genes suggests that posttranscriptional processes may also contribute to the large accumulation of PG mRNA during ripening. PMID- 16666939 TI - Polyamine synthesis in maize cell lines. AB - Uptake of [(14)C]putrescine, [(14)C]arginine, and [(14)C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions. PMID- 16666940 TI - Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves. AB - A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl(2), and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl(2), PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl(2), PVP, pepstatin A, cellulase "Onozuka" RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained 0.25 gram H(2)O/gram dry weight), rapid cooling rates were optimal for maintaining seed vigor. If the seeds were cooled while at intermediate moisture levels (0.12 to 0.20 gram H(2)O per gram dry weight), there appeared to be no effect of cooling rate on seedling vigor. When seeds were very dry (< 0.08 gram H(2)O per gram dry weight), cooling rate had no effect on pea, but rapid cooling rates had a marked detrimental effect on soybean and sunflower germination. Glass transitions, detected by differential scanning calorimetry, were observed at all moisture contents in sunflower and soybean cotyledons that were cooled rapidly. In pea, glasses were detectable when cotyledons with high moisture levels were cooled rapidly. The nature of the glasses changed with moisture content. It is suggested that, at high moisture contents, glasses were formed in the aqueous phase, as well as the lipid phase if tissues had high oil contents, and this had beneficial effects on the survival of seeds at low temperatures. At low moisture contents, glasses were observed to form in the lipid phase, and this was associated with detrimental effects on seed viability. PMID- 16666955 TI - Role of Protein Synthesis in Regulation of Phycobiliprotein mRNA Abundance by Light Quality in Fremyella diplosiphon. AB - If green light-acclimated Fremyella diplosiphon cultures are transferred to red light, the transcription from the inducible phycocyanin gene set increases at least 30-fold within 60 minutes. This effect is inhibited completely by the protein synthesis inhibitors chloramphenicol and spectinomycin. Application of chloramphenicol 30 minutes after transfer of cultures to inductive red light prevents further phycocyanin mRNA accumulation within 10 minutes. If red light acclimated cells are transferred to green light, the phycocyanin transcript level declines by about 70% within 1 hour. Most of the green light-dependent decline results from the rapid cessation of transcription from the PC gene set. Chloramphenicol slows the decline to some extent by decreasing the rate of mRNA degradation in a light-independent manner. The accumulation of phycoerythrin mRNA after transfer of red light-acclimated cells to green light is also inhibited by chloramphenicol. However, there is no red light-dependent mechanism that rapidly halts phycoerythin mRNA synthesis after transfer of cultures from green to red light. Therefore, at least three light-dependent processes are involved in regulating phycobiliproteingene expression: chloramphenicol-sensitive processes required for the activation of phycocyanin and phycoerythrin gene sets and a chloramphenicol-insensitive process which blocks phycocyanin mRNA synthesis after transfer of cells from red to green light. PMID- 16666956 TI - Inhibition of Photosystem II Precedes Thylakoid Membrane Lipid Peroxidation in Bisulfite-Treated Leaves of Phaseolus vulgaris. AB - Exposure of leaves to SO(2) or bisulfite is known to induce peroxidation of thylakoid lipids and to inhibit photosynthetic electron transport. In the present study, we have examined the temporal relationship between bisulfite-induced thylakoid lipid peroxidation and inhibition of electron transport in an attempt to clarify the primary mechanism of SO(2) phytotoxicity. Primary leaves of bean (Phaseolus vulgaris L. cv Kinghorn) were floated on a solution of NaHSO(3), and the effects of this treatment on photosynthetic electron transport were determined in vivo by measurements of chlorophyll a fluorescence induction and in vitro by biochemical measurements of the light reactions using isolated thylakoids. Lipid peroxidation in treated leaves was followed by monitoring ethane emission from leaf segments and by measuring changes in fatty acid composition and lipid fluidity in isolated thylakoids. A 1 hour treatment with bisulfite inhibited photosystem II (PSII) activity by 70% without modifying Photosystem I, and this inhibitory effect was not light-dependent. By contrast, lipid peroxidation was not detectable until after the inhibition of PSII and was strongly light dependent. This temporal separation of events together with the differential effect of light suggests that bisulfite-induced inhibition of PSII is not a secondary effect of lipid peroxidation and that bisulfite acts directly on one or more components of PSII. PMID- 16666957 TI - Cytosolic phosphofructokinase from spinach leaves : I. Purification, characteristics, and regulation. AB - Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was enriched 2600-fold by (NH(4))(2)SO(4) fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6 phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi(2-), Cl(-), SO(4) (2-), NO(3) (-), HAsO(4) (2-), and HCO(3) (-) but was not affected by NH(4) (+). Pi and Mg(2+) changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg(2+) was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3 PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P(2) had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light. PMID- 16666958 TI - Cytosolic Phosphofructokinase from Spinach Leaves : II. Affinity for Mg and Nucleoside Phosphates. AB - Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg(2+). The free Mg(2+) concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg(2+) was independent of the MgATP(2-) concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg(2+). Free ATP (ATP(4-)) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP(2-) concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP(2-), respectively. It was proposed that free ATP competes for the MgATP(2-) binding site, whereas free Mg(2+) does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP(2-) requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP(2-) was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half maximum saturation was achieved at 9 and 14 micromolar MgATP(2-). PFK activity was inhibited by Ca(2+). The inhibition by Ca(2+) depends upon the total Mg(2+) concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca(2+) at 0.1 and 0.2 millimolar Mg(2+), respectively. At physiological concentrations of about 0.5 millimolar free Mg(2+), Ca(2+) would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5'-triphosphate substrate. Besides MgATP(2 ), MgUTP(2-), MgCTP(2-), and MgGTP(2-) could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg(2+) and Pi. PMID- 16666959 TI - Intact Chloroplasts Show Ca-Gated Switching between Localized and Delocalized Proton Gradient Energy Coupling (ATP Formation). AB - Intact chloroplasts were compared to isolated thylakoids as to whether storage of the organelle in high KCl medium caused the energy coupling reactions to show a delocalized or a localized proton gradient energy coupling response. With isolated thylakoids, the occurrence of one or the other energy coupling mode can be reversibly controlled by the concentration of mono- and divalent cations used for the thylakoid storage media. Calcium was shown to be the key ion and previous evidence suggested a Ca(2+)-controlled gating of H(+) fluxes in the thylakoid membrane system (G Chiang, RA Dilley [1987] Biochemistry 26: 4911-4916). Isolated, intact chloroplasts, which retained the outer envelope membranes during the 30 min or longer storage treatments in various concentrations of KCl and CaCl(2) (with sorbitol to maintain iso-osmotic conditions), were osmotically burst in a reaction cuvette and within 3 minutes were assayed for either a localized or a delocalized proton gradient energy coupling (ATP formation) mode. The intact chloroplast system was analogous to isolated thylakoids, with regard to the effects of KCl and CaCl(2) on the energy coupling mode. For example, adding 100 millimolar KCl to the intact organelle storage medium resulted in the subsequent ATP formation assay showing delocalized proton gradient coupling just as with isolated thylakoids. Adding 5 millimolar CaCl(2) to the 100 millimolar KCl storage medium resulted in a localized proton gradient coupling mode. Suspending thylakoids in stromal material previously isolated from intact chloroplast preparations and testing the energy coupling response showed that the stromal milieu has enough Ca(2+) to cause the localized coupling response even though there was about 80 millimolar K(+) in the intact chloroplasts used in this study (determined by atomic absorption spectrophotometry). Extrapolating the intact chloroplast data to the whole leaf level, we suggest that proton gradient energy coupling is normally of the localized mode, but under certain conditions it could be either localized or delocalized, depending on factors that affect the putative Ca(2+)-regulated proton flux gating function. PMID- 16666960 TI - Selective delipidation of the plasma membrane by surfactants : enrichment of sterols and activation of ATPase. AB - The influence of plasma membrane lipid components on the activity of the H(+) ATPase has been studied by determining the effect of surfactants on membrane lipids and ATPase activity of oat (Avena sativa L.) root plasma membrane vesicles purified by a two-phase partitioning procedure. Triton X-100, at 25 to 1 (weight/weight) Triton to plasma membrane protein, an amount that causes maximal activation of the ATPase in the ATPase assay, extracted 59% of the membrane protein but did not solubilize the bulk of the ATPase. The Triton-insoluble proteins had associated with them, on a micromole per milligram protein basis, only 14% as much phospholipid, but 38% of the glycolipids and sterols, as compared with the native membranes. The Triton insoluble ATPase could still be activated by Triton X-100. When solubilized by lysolecithin, there were still sterols associated with the ATPase fraction. Free sterols were found associated with the ATPase in the same relative proportions, whether treated with surfactants or not. We suggest that surfactants activate the ATPase by altering the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity. PMID- 16666961 TI - Uptake of Acidic and Basic Sugar Derivatives in Lemna gibba G1. AB - The uptake of acidic and basic sugar derivatives in Lemna gibba L. was studied. Uronic acids applied to the experimental solution (50 millimolar) induced a small decrease of the membrane potential (10 +/- 1 millivolt galacturonic acid, and 20 +/- 4 millivolt glucuronic acid). After incubation of the plants in a 0.1 millimolar solution of these substrates, no decrease in the concentration of reducing groups in the external solution was detected. Respiration increased by 31% with 50 millimolar galacturonic acid, whereas no effect was found with the same concentration of glucuronic acid. Glucosamine caused a considerable concentration-dependent membrane depolarization. ((14)C)glucosamine uptake followed Michaelis-Menten kinetics together with a linear component. Influx of this substrate was inhibited by glucose but the type of competition could not be clearly distinguished. Glucosamine, 50 millimolar, inhibited the respiration rate by 30%. The glucosamine uptake was pH-dependent, with maximum uptake at around pH 7. Lack of enhancement of uptake by low pH as well as the permanent membrane depolarization suggest a uniport mechanism for the charged species of the substrate and an electroneutral diffusion of the uncharged species. PMID- 16666962 TI - Transfer of C(4) Photosynthetic Characters through Hybridization of Flaveria Species. AB - Transfer of C(4) photosynthetic traits was studied through hybridization of Flaveria trinervia (Spreng.) Mohr (C(4)) and Flaveria brownii A.M. Powell (C(4) like) with Flaveria linearis Lag. (C(3)-C(4)) and the C(3) species Flaveria pringlei Gandoger (C(3)). Fertility was low, based on irregular chromosome pairing and low pollen stainability, except in F. brownii x F. linearis which had bivalent pairing and 76% stainable pollen. Hybrids had apparent photosynthesis values of 71 to 148% of the midparental means, while the CO(2) compensation concentration was similar to the C(4) or C(4)-like parent, except in hybrids having the C(3) species F. pringlei as a parent. Inhibition of apparent photosynthesis by O(2), and phosphoenolpyruvate carboxylase and NADP-malic enzyme activities and subunit levels in the hybrids were closer to the C(3) or C(3)-C(4) parent. The species F. brownii and F. trinervia were equal in their capacity to transfer reduced O(2) inhibition of AP and CO(2) compensation concentration values to hybrids with F. linearis (C(3)-C(4)), although hybrids with F. trinervia had higher PEPC activity. The O(2) inhibition of AP was correlated with the logarithm of activities of phosphoenolpyruvate carboxylase (r = -0.95) and NADP-malic enzyme (r = -0.87). These results confirm that C(4) traits can be transferred by hybridization of C(3)-C(4) and C(4) or C(4)-like species, with a higher degree of C(4) photosynthesis than exists in C(3)-C(4) species, and at least in F. brownii x F. linearis, fertile progeny are obtained. PMID- 16666963 TI - Diacylglycerol kinase from suspension cultured plant cells : purification and properties. AB - Diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from suspension-cultured Catharanthus roseus cells was extracted from a membrane fraction with 0.6% Triton X-100 and 150 millimolar NaCl and was purified about 900-fold by DEAE-cellulose, blue Sepharose, gel permeation, and phenyl-Sepharose chromatography. The enzyme is obviously membrane bound as activity in the cytosol could not be detected. In the presence of detergents such as Triton X-100 (3-[3 cholamidopropyl]dimethylamino)-1-propanesulfonate (Chaps), or deoxycholate, a molecular weight of about 250,000 was determined by gel filtration. In glycerol density gradients, the enzyme sedimented slightly more slowly than bovine serum albumin, indicating a molecular weight of less than 68,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzyme activity could be assigned to a protein of 51,000 daltons. As found previously for bacterial and animal diacylglycerol kinases, the purified enzyme was completely devoid of activity without the addition of phospholipids or deoxycholate. Cardiolipin was found to be most effective, whereas higher amounts of detergent were inhibitory. The enzyme needs divalent cations for activity, with Mg(2+) ions being the most effective. Apparent K(m) values for ATP and diacylglycerol were determined as 100 and 250 micromolar, respectively. PMID- 16666964 TI - Biosynthesis of the Cyanogenic Glucoside Dhurrin in Seedlings of Sorghum bicolor (L.) Moench and Partial Purification of the Enzyme System Involved. AB - The cyanogenic glucoside dhurrin is rapidly synthesized in etiolated seedlings of Sorghum bicolor (L.) Moench. The dhurrin content of the seedlings increases sigmoidally with the germination time. Shoots of 10 centimeters height contain 850 nanomoles of dhurrin per shoot corresponding to 6% of the dry weight. The biosynthetic activity sharply rises upon germination and reaches a maximum level of 10 nanomoles dhurrin/(hour x shoot) after 48 hours when the shoots are 3 centimeters high. This maximum level is followed by a sharp decline in activity when germination time exceeds 65 hours. Dhurrin and the dhurrin-synthesizing enzyme system are primarily located in the upper part of the etiolated shoot where both are evenly distributed between the coleoptile, the primary leaves and the upper 0.5 centimeter of the first internode including the shoot apex. Dhurrin constitutes 30% of the dry weight of the upper 1.2 centimeter of 10 centimeter high shoots. The seed and root contain neither dhurrin nor the dhurrin synthesizing enzyme system. The codistribution of dhurrin and the enzyme system throughout the seedling indicates that production and storage sites are located within the same cell. Purification of the dhurrin-synthesizing enzyme by gel filtration or by sucrose gradient centrifugations results in a tenfold increase in specific activity. Further purification is accompained by a decline in specific activity due to loss of essential components as demonstrated by reconstitution experiments. PMID- 16666965 TI - Changes in Level and Activity of Phospholipid Transfer Protein during Maturation and Germination of Maize Seeds. AB - The variations of the amounts of phospholipid transfer proteins (PLTP), determined by ELISA and immunoblotting methods, were followed during the maturation and germination of maize (Zea mays L.) seeds. Changes of the amounts of PLTP occur during seed maturation. The levels of PLTP, low in the first 3 weeks after fecondation, strongly raised 3 to 5 weeks after, then reached and maintained a high value (10% of total soluble proteins) during the last steps of maturation. These variations, determined by ELISA, are in accordance with the observations made by immunoblotting. Changes in phospholipid transfer activity were also found when protein extracts prepared from seeds at different stages of maturation were assayed for transfer activity. The levels of PLTP were also determined during the germination of maize seeds and the early growth of the plantlets, both in the endosperm and the aerial parts. While no major change was observed in the endosperm, a high increase in PLTP level was found in the aerial part of the plantlet, both by ELISA and immunoblotting. An enhancement of the phospholipid transfer activity was parallely observed in the protein extracts of plantlets at various stages of germination. These results are consistent with an in vivo correlation between the synthesis of phospholipid transfer protein, observed during the maturation and germination of maize seeds, and the biogenesis of membranes which involves intracellular movements of phospholipids. PMID- 16666966 TI - Carbamoyl phosphate synthetase, ornithine transcarbamylase, and aspartate transcarbamylase activities in the pea ovary : changes with senescence of the unpollinated ovary or with fruit set induced by gibberellic Acid. AB - Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), and aspartate transcarbamylase (ATC) were assayed in extracts from unpollinated ovaries of Pisum sativum L. CPS and OTC activities were, per milligram protein, the highest reported in a plant tissue, representing an estimated 0.1% of the protein in the ovary. The OTC/CPS and ATC/CPS ratios were about 100 and 0.5, respectively, indicating that most of the carbamoyl phosphate is used for arginine synthesis. The weight, protein content, and CPS, OTC, and ATC activities per ovary were determined during the senescence of the ovary and also during fruit set induced by treatment with gibberellic acid (GA(3)). In the nontreated ovary the weight and the protein first increased and then decreased dramatically, but the decrease in protein took place much earlier. In the GA(3)-treated ovaries the increase in weight was considerably greater than the increase in the protein. Whether or not the ovaries were treated with GA(3), CPS, OTC, and ATC activities closely followed the changes in protein, and thus their ratios and specific activities remained essentially constant. It appears that treatment with GA(3) increases the amount of protein and enzymic activities by preventing a large increase in the rate of protein degradation. In addition, the effects of acetylglutamate, ornithine, and UMP on CPS activity were studied. The pea enzyme exhibits regulatory properties intermediate between those of Escherichia coli and the ureotelic liver enzymes. PMID- 16666967 TI - Differential Accumulation of a Transcript Driven by the CaMV 35S Promoter in Transgenic Tobacco. AB - A transcriptional fusion composed of the CaMV 35S promoter, a 19 kD zein cDNA, and the 3' flanking regions from the 0' to 1' T(R) T-DNA genes was introduced into tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. The accumulation of RNA generated from this transcriptional fusion varied both temporally and spatially in all tissues examined in greenhouse-grown tobacco plants, suggesting that the CaMV 35S promoter is not constitutive. Younger, actively dividing leaf, stem, root, and flower tissues contained higher steady state levels of zein RNA than did older, more quiescent tissues. Zein RNA levels greatly decreased during seed development and were undetectable in the mature seed. In addition, the two RNA termination or processing signals present in our construct were differentially utilized during seed development. PMID- 16666968 TI - Aspartokinase of Lemna paucicostata Hegelm. 6746. AB - A sensitive and specific method was developed for assay of aspartokinase (EC 2.7.2.4) in crude extracts of Lemna paucicostata. Lysine inhibited approximately 93%, and threonine approximately 6%; together, these amino acids inhibited 99%. Inhibition by lysine was synergistically increased by S-adenosylmethionine, which by itself had no effect on activity. Essentially complete inhibition of threonine resistant activity was obtained with lysine, and of lysine-resistant activity with threonine. Inhibition by lysine and threonine was additive, with no indication of concerted inhibition. Aspartate concentration had no effect on the relative proportions of lysine- and threonine-sensitive activities. Aspartokinase activity was in large excess of that reported by other workers, the maximum capacity (V(max)) far exceeding the in vivo requirements. Estimations of rates of aspartokinase in vivo suggest that the step catalyzed by this enzyme may not be the overall ;rate-limiting' one for entry of 4-carbon units into the aspartate family of amino acids, and that feedback inhibition of this enzyme by lysine and threonine may not be a major factor in regulating flux through this step. PMID- 16666969 TI - Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase. AB - Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [(14)C]threonine and [(14)C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants. PMID- 16666970 TI - Comparison of Phase Shifts of the Circadian Rhythm of K Uptake in Lemna gibba G3 by Various Amino Acid Analogs. AB - Phase shifts of the circadian rhythm of K(+) uptake by Lemna gibba G3, caused by pulse administration of various amino acids analogs, were examined and compared. The various phase shifts were not due to any disturbance in the biosynthesis of amino acids, since the effective time of day and direction of the phase shift caused by analogs were not correlated with the standard amino acid which was modified. Effective analogs could be classified into three groups. The first group was effective during the middle subjective day and caused both advances and delays in phase. The second group was effective early in the subjective night, causing large delays and small phase advance. Analogs in the third group shifted the phase as did cycloheximide and were effective at the subjective dawn. Since the analogs of the third group were known to inhibit protein synthesis, it is likely that they shift the phase by lowering the level of some protein(s) important for the clock. By contrast, since the analogs in groups 1 and 2 are known to generate abnormal proteins, the different phase-shifting patterns caused by analogs in groups 1 and 2 suggest that at least two other proteins are important for the circadian timing loop. The amino acid analogs shift the phase as a result of their incorporation into these proteins instead of the standard amino acid. This probably alters the structure and/or activities of these proteins. PMID- 16666971 TI - Photoinhibition at low temperature in chilling-sensitive and -resistant plants. AB - Photoinhibition resulting from exposure at 7 degrees C to a moderate photon flux density (300 micromoles per square meter per second, 400-700 nanometers) for 20 hours was measured in leaves of annual crops differing widely in chilling tolerance. The incidence of photoinhibition, determined as the decrease in the ratio of induced to total chlorophyll fluorescence emission at 693 nanometers (F(v)/F(max)) measured at 77 Kelvin, was not confined to chilling-sensitive species. The extent of photoinhibition in leaves of all chilling-resistant plants tested (barley [Hordeum vulgare L.], broad bean [Vicia faba L.], pea [Pisum sativum L.], and wheat [Triticum aestivum L.]) was about half of that measured in chilling-sensitive plants (bean [Phaseolus vulgaris L.], cucumber [Cucumis sativus L.], lablab [Lablab purpureus L.], maize [Zea mays L.], pearl millet [Pennisetum typhoides (Burm. f.) Stapf & Hubbard], pigeon pea [Cajanus cajun (L.) Millsp.], sesame [Sesamum indicum L.], sorghum [Sorghum bicolor L. Moench], and tomato [Lycopersicon esculentum Mill.]). Rice (Oryza sativa L.) leaves of the indica type were more susceptible to photoinhibition at 7 degrees C than leaves of the japonica type. Photoinhibition was dependent both on temperature and light, increasing nonlinearly with decreasing temperature and linearly with increasing light intensity. In contrast to photoinhibition during chilling, large differences, up to 166-fold, were found in the relative susceptibility of the different species to chilling injury in the dark. It was concluded that chilling temperatures increased the likelihood of photoinhibition in leaves of both chilling-sensitive and -resistant plants. Further, while the photoinhibition during chilling generally occurred more rapidly in chilling-sensitive plants, this was not related directly to chilling sensitivity. PMID- 16666972 TI - Endopeptidases in the stroma and thylakoids of pea chloroplasts. AB - Three endopeptidases (EP2, EP3, and EP4) were identified after fractionation of pea (Pisum sativum, var Feltham First) chloroplast stromal extracts. All three were identified by their ability to cleave in vitro-synthesized preplastocyanin to lower molecular weight forms. EP2 is inhibited by phenylmethylsulfonylfluoride, and both EP2 and EP3 are inhibited by the heavy metal chelators 1,10-phenanthroline and EDTA. A further endopeptidase, EP5, was identified in Triton X-100 extracts of thylakoid membranes. Experiments involving contrifugation through a sucrose pad indicate that EP5 either has a high molecular weight or is associated with a thylakoid protein complex. EP5 is effectively inhibited by phenylmethylsulfonylfluoride, iodoacetate, and 1,10 phenanthroline, but not by EGTA. The implications of these results for the analysis of chloroplast protein maturation are discussed, and an improved protocol for the purification of the stromal processing peptidase is described which ensures the removal of EP2, the most active of the stromal peptidases analysed in this study. PMID- 16666973 TI - Aspartate aminotransferase in alfalfa root nodules : I. Purification and partial characterization. AB - Aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1 [AAT]), a key enzyme in the assimilation of C and N compounds, was purified from the cytosol of alfalfa (Medicago sativa L.) root nodules. Isoforms that increased during nodule development, AAT-2a, AAT-2b, and AAT-2c, were purified greater than 447-fold to apparent homogeneity, and high titer polyclonal antibodies were produced. The native molecular weight of the AAT-2 isoforms was approximately 80 kilodatons with a subunit molecular weight of 40 kilodatons, indicating that the holoenzymes are dimers. The AAT-2 isoforms comprised approximately 0.4% of the total soluble nodule protein. The AAT specific activity was measured in leaf, stem, root, and nodule organs, and zymograms of each were compared. Enzyme activity was 4- to 37-fold greater in effective (nitrogen fixing) nodules than in leaves, stems, and roots. Effective nodule AAT-specific activity was 3- to 8-fold greater than that of plant-controlled ineffective nodules. No differences in K(m) were observed between AAT-1 and AAT-2. Antibodies raised against AAT-2 were more selective against AAT-2 than AAT-1. Evidence obtained from zymograms suggests that the expression of alfalfa nodule AAT is controlled at two different gene loci, AAT-1 and AAT-2, resulting in different dimeric isoforms. PMID- 16666974 TI - An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos. AB - A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls zeatin, O-xylosylzeatin, N(6)-(Delta(2)-isopentenyl)adenine, or N(6)-(Delta(2) isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M(r) 55,000 +/- 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M(r) 25,000+/- 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes. PMID- 16666975 TI - Characterization of the Effects of Divalent Cations on the Coupled Activities of the H-ATPase in Tonoplast Vesicles. AB - The substrate requirement of the H(+)-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg(2+) or Mn(2+). The presence of Ca(2+) or Ba(2+) did not significantly affect the coupled activities. The addition of Cd(2+), Co(2+), Cu(2+), and Zn(2+) inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu(2+) and Co(2+) were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co(2+), Cu(2+), and Zn(2+) were effective inhibitors. However, this inhibition could be further modulated by free Co(2+), Cu(2+), and Zn(2+). While the equilibrium concentrations of Cd-ATP and free Cd(2+) were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H(+)-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5 hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in "indirect-link" mechanism. PMID- 16666976 TI - Nitrogen Nutrition of Nodules in Relation to ;N-Hunger' in Cowpea (Vigna unguiculata L. Walp). AB - Early growth, nodule development, and nitrogen fixation by two cultivars of cowpea (Vigna unguiculata L. Walp), one large-seeded (Vita 3; 146.0 +/- 0.9 milligrams seed dry weight, 4.1 +/- 0.2 milligrams seed N), the other small seeded (Caloona; 57.5 +/- 2.5 milligrams seed dry weight, 1.8 +/- 0.1 milligrams seed N), were compared under conditions of sand culture with nutrient solution free of combined N. The seed stocks used had been obtained from plants uniformly labeled with (15)N, thus enabling changes with time in distribution of cotyledon and fixed N among plant parts to be measured by isotope dilution. Caloona, but not Vita 3, showed physiological symptoms of ;N hunger,' i.e. transient loss of chlorophyll (visible yellowing) and N from the first-formed unifoliolate leaves at or around the onset of symbiotic functioning and N(2) fixation. The smaller seeded Caloona showed higher early nitrogenase activity than the larger-seeded Vita 3 and by 28 days had fixed 6.6 milligrams of N per milligram of seed N [mg N . (mg seed N)(-1)] versus only 3.5 mg N . (mg seed N)(-1) in Vita 3. Both cultivars lost around 30% of their initial seed N at germination, mostly as fallen cotyledons. Abscised cotyledons of Caloona contained 1.21 +/- 0.17% N; those of Vita 3 contained 2.61 +/- 0.37% N. When compared on the basis of cotyledon N available for seedling growth, Caloona was shown to have fixed 10.6 mg N . (mg seed N)(-1) and Vita 3 only 5.3 mg N . (mg seed N)(-1). Most of the cotyledon N withdrawn from the unifoliolate leaf pair of Caloona during ;N hunger' was committed to early nodule growth and, in total, 20 to 25% of the cotyledon N resource of this cultivar was ultimately invested in establishment of symbiosis compared with only 7% in Vita 3. PMID- 16666977 TI - Enzymes of ethylene biosynthesis. AB - The properties of enzymes involved in ethylene biosynthesis are reviewed and progress toward the purification of these enzymes is described. The enzyme whose activity usually limits ethylene biosynthesis is 1-aminocyclopropane-1 carboxylate (ACC) synthase. Even though its level in plants is extremely low, it has now been purified from several sources. The enzyme that converts ACC to ethylene does not survive homogenization, apparently because it is membrane-bound and because its activity requires membrane integrity. Properties of this enzyme have been elucidated in vivo and in vacuolar preparations which possess the capacity to convert ACC to ethylene. PMID- 16666978 TI - Regulation of Embryo Dormancy by Manipulation of Abscisic Acid in Kernels and Associated Cob Tissue of Zea mays L. Cultured in Vitro. AB - Sectors of Zea mays cobs, with and without kernels were cultured in vitro in the presence and absence of fluridone. Cultured kernels, cob tissue, and embryos developed similarly to those grown in the field. Abscisic acid (ABA) levels in the embryos were evaluated by enzyme-linked immunosorbant assay. ABA levels in intact embryos cultured in the presence of fluridone were extremely low and indicate an inhibition of ABA synthesis. ABA levels in isolated cob tissue indicate that ABA can be produced by cob tissue. Sections containing kernels cultured in the presence of fluridone were transferred to medium containing fluridone and ABA. Dormancy was induced in more than 50% of the kernels transferred from 13 to 15 days after pollination, but all of the kernels transferred at 16 days after pollination or later were viviparous. ABA recovered from kernels that were placed in medium containing fluridone and ABA suggest that ABA can be transported through the cob tissue into developing embryos and that ABA is required for induction of dormancy in intact embryos. PMID- 16666979 TI - Isolation and Partial Characterization of a Subtilisin Inhibitor from the Mung Bean (Vigna radiata). AB - The subtilisin inhibitor (MBSI-A) from the mung bean (Vigna radiata (L.) Wilczek) seed has been purified to homogeneity. MBSI-A consists of a single polypeptide chain of 119 residues, with a high content of glutamic acid/glutamine, aspartic acid/asparagine, valine, threonine, and proline (19, 12, 10, 9, and 8 residue percent, respectively). MBSI-A is a potent inhibitor of subtilisin Carlsberg, but is inactive toward bovine trypsin and alpha-chymotrypsin and the plant cysteinyl proteinase papain. The MBSI is located exclusively in the cytosol of the seed cotyledon cell, unlike the mung bean trypsin inhibitor (MBTI), which is located primarily in the protein bodies. Both MBSI and MBTI accumulate in the seed during the most active period of reserve protein accumulation, 12 to 18 days after flowering. During germination MBSI, like MBTI, is broken down beginning 2 to 3 days after seed imbibition. The disappearance of MBSI-A is accompanied by the transient appearance of a new inhibitor species, MBSI-D. The amino acid composition of MBSI-D suggests that it may be produced by the loss of approximately 20 amino acid residues from MBSI-A. PMID- 16666980 TI - Variation in Binding and Virulence of Agrobacterium tumefaciens Chromosomal Virulence (chv) Mutant Bacteria on Different Plant Species. AB - Chromosomal virulence (chv) mutants of Agrobacterium tumefaciens have been reported to be deficient in binding to cells of zinnia, tobacco, and bamboo. The mutants are nonpathogenic on stems of Kalanchoe, sunflower, tomato, Jerusalem artichoke, and tobacco, but they cause tumors on tubers of Solanum tuberosum. We used a root cap cell binding assay to test ability of cells from individual plants of 13 different plant species to bind parent or chv mutant bacteria. The same plants were then inoculated to test for disease response. Cells from nine of the plant species were grossly deficient in their abilities to bind mutant bacteria, and the plants inoculated with mutant bacteria failed to form tumors. In contrast, root cap cells as well as root hairs and root surfaces of S. tuberosum, S. okadae, and S. hougasii bound chv mutant bacteria as well as wild type. Nevertheless, S. tuberosum roots inoculated with mutant bacteria did not develop tumors. Although S. okadae plants inoculated with mutant bacteria formed a few tumors, and S. hougasii developed as many tumors in response to chv mutants as in response to the parent strain, the tumors induced by mutant bacteria were smaller. PMID- 16666981 TI - Immunogold localization of the citrus exocortis viroid-induced pathogenesis related proteinase p69 in tomato leaves. AB - Citrus exocortis viroid induces in tomato plants (Lycopersicon esculentum) synthesis and accumulation of a pathogenesis-related protein (P69) previously reported to be a proteinase (Vera P, Conejero V [1988] Plant Physiol 87: 58-63). By immunogold/transmission electron microscopy, we have studied the distribution of this protein in thin sections of parenchymatous leaf tissue. The enzyme was present intra- and extracellularly. The intracellular location was limited to the vacuole and was always associated with engulfed cell material. When extracellularly located, the enzyme was associated with a dispersed, electron dense material in the intercellular spaces. This latter location was confirmed after analysis of intercellular washing fluids obtained by vacuum infiltration of leaves. These observations provide new data for the understanding of viroid pathogenesis and the biological role of the pathogenesis-related proteinase P69. PMID- 16666982 TI - Cloning and characterization of root-specific barley lectin. AB - Cereal lectins are a class of biochemically and antigenically related proteins localized in a tissue-specific manner in embryos and adult plants. To study the specificity of lectin expression, a barley (Hordeum vulgare L.) embryo cDNA library was constructed and a clone (BLc3) for barley lectin was isolated. BLc3 is 972 nucleotides long and includes an open reading frame of 212 amino acids. The deduced amino acid sequence contains a putative signal peptide of 26 amino acid residues followed by a 186 amino acid polypeptide. This polypeptide has 95% sequence identity to the antigenically indistinguishable wheat germ agglutinin isolectin-B (WGA-B) suggesting that BLc3 encodes barley lectin. Further evidence that BLc3 encodes barley lectin was obtained by immunoprecipitation of the in vitro translation products of BLc3 RNA transcripts and barley embryo poly(A(+)) RNA. In situ hybridizations with BLc3 showed that barley lectin gene expression is confined to the outermost cell layers of both embryonic and adult root tips. On Northern blots, BLc3 hybridizes to a 1.0 kilobyte mRNA in poly(A(+)) RNA from both embryos and root tips. We suggest, on the basis of immunoblot experiments, that barley lectin is synthesized as a glycosylated precursor and processed by removal of a portion of the carboxyl terminus including the single N-linked glycosylation site. PMID- 16666983 TI - Protoplast volume:water potential relationship and bound water fraction in spinach leaves. AB - Methods used to estimate the (nonosmotic) bound water fraction (BWF) (i.e. apoplast water) of spinach (Spinacia oleracea L.) leaves were evaluated. Studies using three different methods of pressure/volume (P/V) curve construction all resulted in a similar calculation of BWF; approximately 40%. The theoretically derived BWF, and the water potential (Psi(w))/relative water content relationship established from P/V curves were used to establish the relationship between protoplast (i.e. symplast) volume and Psi(w). Another method of establishing the protoplast volume/Psi(w) relationship in spinach leaves was compared with the results from P/V curve experiments. This second technique involved the vacuum infiltration of solutions at a range of osmotic potentials into discs cut from spinach leaves. These solutions contained radioactively labeled H(2)O and sorbitol. This dual label infiltration technique allowed for simultaneous measurement of the total and apoplast volumes in leaf tissue; the difference yielded the protoplast volume. The dual label infiltration experiments and the P/V curve constructions both showed that below -1 megapascals, protoplast volume decreases sharply with decreasing water potential; with 50% reduction in protoplast volume occurring at -1.8 megapascals leaf water potential. PMID- 16666984 TI - Bacterial chitinase is modified and secreted in transgenic tobacco. AB - The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway. PMID- 16666985 TI - Characterization of Pea Chloroplast D-Enzyme (4-alpha-d-Glucanotransferase). AB - Pea (Pisum sativum L.) chloroplast D-enzyme (4-alpha-d-glucanotransferase, EC 2.4. 1.25) was purified greater than 750-fold and partially characterized. It is a dimer with a subunit M(r) of ca. 50,000. Optimal activity is between pH 7.5 and 8.0 with maltotriose as substrate and the enzyme's K(m) for maltotriose is 3.3 millimolar. Chloroplast D-enzyme converts maltotriose to maltopentaose and glucose via the exchange of alpha-1,4-glycosidic linkages. Maltotriose acts either as a donor or acceptor of a maltosyl group. The enzyme has highest activity with maltotriose as substrate. As initial substrate degree of polymerization is increased to maltoheptaose, D-enzyme activity drops to zero at 10 millimolar substrate concentrations and by 70% at 1 millimolar concentrations. The enzyme cannot use maltose as a substrate. Glucose was found to be a suitable acceptor substrate for this D-enzyme. Addition of glucose to incubation mixtures, or production of glucose by D-enzyme, prevents the synthesis of maltodextrins larger than maltopentaose. Removal of glucose produced by D-enzyme activity with maltotriose as substrate resulted in the synthesis of maltopentaose and maltodextrins with sufficient degrees of polymerization to be suitable substrates for pea chloroplast starch phosphorylase. The possible role of D-enzyme in pea chloroplast starch metabolism is discussed. PMID- 16666986 TI - Hybrid hybridoma cell lines which express anti-zeatin riboside, anti-abscisic Acid, and hybrid antibodies. AB - Hybrid hybridoma cell lines that secreted antibodies which reacted with two distinct plant hormones, abscisic acid (ABA) and zeatin riboside (ZR) were prepared and cloned. These cell lines were derived by polyethylene glycol mediated fusion of HAT-sensitive anti-ZR hybridoma cells with splenocytes harvested from a BALB/c mouse previously immunized with an ABA-bovine serum albumin conjugate. Chromatographic analyses indicated that these lines expressed two different isotypes, each associated with a specific immunologic reactivity, and that the populations of immunoglobulins secreted by these hybridomas included antibodies directed against each individual hapten as well as hybrid molecules which reacted simultaneously with both. Hybrid hybridomas such as these should provide antibody populations useful for simultaneous isolation of multiple plant hormones from individual plant samples. PMID- 16666987 TI - Kinetic comparison of cytokinin nucleosidase activity isolated from normally ripening and mutant tomato varieties. AB - Kinetic parameters for cytokinin nucleosidase activity which catalyzes the deribosylation of N(6)(Delta(2)-isopentenyl)adenosine (I(6)Ado) to produce the more "active" free base N(5)(Delta(2)-isopenetyl)adenine (I(6)Ade) were compared for a normally ripening tomato (Lycopersicon esculentum L.) cultivar Rutgers, and two mutant tomato varieties (Nor and Rin). K(m) for nucleosidase activity in Rutgers was lower (K(m) = 0.1 millimolar) than that in either Nor (K(m) = 0.14 millimolar) or Rin (K(m) = 0.13 millimolar). PMID- 16666988 TI - Evidence for the involvement of a specific cell wall layer in regulation of deep supercooling of xylem parenchyma. AB - Current theory indicates that the structure of the cell wall is integral to the ability of a tissue to exhibit deep supercooling. Our previous work has indicated that the structure of the pit membrane and/or amorphous layer (protective layer), rather than the entire cell wall, may play a major role in deep supercooling (21, 22). The present study indicates a shift in the low-temperature-exotherm of current year shoots of peach can be induced by soaking twigs in water over 3 to 10 days. Alternatively, these shifts can be inhibited by exposing tissues to 10( 4) molar cycloheximide. Ultrastructural observations indicated a marked alteration of the amorphous layer in xylem parenchyma of water-soaked tissue. Alterations consisted of an apparent loosening or partial dissolution of portions of the amorphous layer. Changes in the density or uniformity of the amorphous layer in cycloheximide-treated tissues were not as readily apparent. The appearance of the protoplast in tissue soaked in water for up to 10 days was characteristic of deacclimated cells. However, in tissue soaked in cycloheximide for the same period these changes were not evident. These observations further support our contention that the structure of the amorphous layer may play a key role in establishing and regulating the ability of a cell to exhibit deep supercooling. PMID- 16666989 TI - Effect of Ethylene Action Inhibitors upon Wound-Induced Gene Expression in Tomato Pericarp. AB - The contribution of wound-ethylene to wound-induced gene expression was investigated in unripe tomato pericarp using inhibitors of ethylene action. Wounded unripe tomato pericarp was treated with 2,5-norbornadiene or silver thiosulfate to inhibit specifically the induction of ethylene-dependent mRNA species. Poly(A)(+) RNAs isolated from these tissues after 12 hours of wounding were translated in vitro in a rabbit reticulocyte lysate system and [(35)S]methionine-labeled polypeptides were compared to unwounded controls after separation by one and two-dimensional polyacrylamide gel electrophoresis. Results show that mechanical wounding induces a dramatic shift in gene expression (over 50 mRNA species) but expression of less than 15% of these genes is affected by the treatment with ethylene action inhibitors. A selective decrease in mRNAs coding for a 37 kilodalton doublet and 75 kilodalton polypeptides is observed in 2,5-norbornadiene and silver thiosulfate treated wounded pericarp. Levels of hydroxyproline-rich glycoprotein mRNAs induced in wounded tissue were not influenced by inhibitors of ethylene action. PMID- 16666990 TI - Light Quality and Irradiance Level Interaction in the Control of Expression of Light-Harvesting Complex of Photosystem II: Pigments, Pigment-Proteins, and mRNA Accumulation. AB - Effects of red and blue light at irradiances from 1.6 to 28.3 micromolar per square meter per second on chloroplast pigments, light-harvesting pigment proteins associated with photosystem II, and the corresponding mRNA were evaluated in maize (Zea mays L.) plants (OP Golden Bantum) grown for 14 days under 14 hours light/10 hours dark cycles. Accumulation of pigments, pigment proteins, and mRNA was less in blue than in red light of equal irradiance. The difference between blue and red light, however, varied as a function of irradiance level, and the pattern of this variation suggests irradiance controlled activation/deactivation (switching) of blue-light receptor. The maximum reduction in blue light of mRNA and proteins associated with light harvesting complex occurs at lower irradiance levels than the maximum reduction of chlorophylls a and b. PMID- 16666991 TI - Arginine Metabolism in Developing Soybean Cotyledons: III. Utilization. AB - Tracerkinetic experiments were performed using l-[guanidino-(14)C]arginine, l-[U (14)C]arginine, l-[ureido-(14)C]citrulline, and l-[1-(14)C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free (14)C compounds and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine specific enzyme-linked assays. After 4 hours, 75% and 90% of the (14)C metabolized from [guanidino-(14)C]arginine and [U-(14)C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours (14)CO(2) was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra (14)CO(2) was evolved during the catabolism of ornithine-derived glutamate; (14)C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.The maximum specific radioactivity ratios between arginine in newly synthesized protein and total free arginine in the (14)C-citrulline and (14)C-ornithine experiments indicated that only 3% of the free arginine was in the protein precursor pool, and that argininosuccinate and citrulline were present in multiple pools. PMID- 16666992 TI - Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: III. Changes in Protein Secretion under Nutrient Stress. AB - Phosphate starvation increased the secretion of at least six proteins by suspension cultured tomato (Lycopersicon esculentum L. and L. pennellii) cells. Cells exhibited a biphasic response to phosphate (Pi) starvation. The early phase involved enhanced secretion of three proteins in response to transfer to a Pi depleted media, while biomass accumulation continued at the same rate as in the Pi-sufficient cells. Severe starvation, defined as inhibition of biomass accumulation, induced enhanced secretion of three additional proteins. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, media proteins were immunoblotted with antibodies reacting specifically to oligosaccharides processed by the Golgi apparatus. Binding patterns showed that the enhancement in secretion during both phases of starvation was Golgi-mediated. Cells undergoing severe starvation had a respiration rate approximately twice that of unstressed cells and secreted 4.4 times more protein into the media per unit biomass. These data suggest overlapping Pi starvation-specific and global stress responses in plant cells. Under these conditions, Golgi-mediated protein secretion is enhanced. We present evidence for phosphate starvation inducible enhancement of Pi uptake. Secreted proteins specific for N and Fe starvation are also identified. PMID- 16666993 TI - Polyamine metabolism of potato seed-tubers during long-term storage and early sprout development. AB - Growth potential of potato (Solanum tuberosum L.) plants is influenced by seed tuber age. After 24 days of growth, single-eye seedcores from 7-month-old seed tubers produced 64% more foliar dry matter than those from 19-month-old seed tubers, reflecting a higher growth rate. This study was initiated to determine if differences in polyamine (PA) metabolism are associated with aging and age reduced vigor of potato seed-tubers. As tubers aged in storage, putrescine (Put) increased 2.2-fold, while spermidine (Spd) and spermine (Spm) decreased 33% and 38%, respectively. Ethylene content of the tuber tissue also increased with advancing age, suggesting that during the aging process S-adenosylmethionine was directed toward ethylene biosynthesis at the expense of the PAs. Single-eye cores from 7- and 19-month-old tubers were sown and PA levels in core and shoot tissues were monitored during plant development. Put titer of younger cores increased 8.8 fold by 12 days. In contrast, the increase in Put over the initial titer in older cores was 2.9-fold. The reduced ability of older cores to synthesize Put during plant establishment is probably due to a 45% decline in ornithine decarboxylase activity between 12 and 16 days after planting. Lack of available Put substrate limited the biosynthesis of Spd and Spm, and thus their concentrations remained lower in older cores than in younger cores. Lower PA titer in older cores during plant establishment is thus coincident with reduced growth potential. Concentrations of Put and Spd were higher in shoots developing from older cores throughout the study, but there was no age-related difference in Spm content. In contrast, activities of arginine and S-adenosylmethionine decarboxylases were higher in shoots from younger cores during establishment. The results indicate that aging affects PA metabolism in both tuber and developing plant tissues, and this may relate to loss of growth potential with advancing seed-tuber age. PMID- 16666994 TI - Gibberellic Acid effects on greening in pea seedlings. AB - The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly. PMID- 16666995 TI - A New Reversed Phase-HPLC Method Resolving All Major Higher Plant Photosynthetic Pigments. AB - A new reversed phase-high performance liquid chromatography method has been developed to analyze the full complement of higher plant photosynthetic pigments (cis-neoxanthin, neoxanthin, violaxanthin, taraxanthin, anteraxanthin, lutein, zeaxanthin, cis-lutein, chlorophyll b, chlorophyll a, alpha- and beta-carotene). The separation is carried out on a C(18) column in about 10 minutes, using a single high-pressure pump and three different mobile phases in three isocratic steps. This method introduces a major improvement in higher plant photosynthetic pigment analysis, resolving in only 10 minutes all photosynthetic pigments while achieving good separation of lutein from its isomer zeaxanthin. Zeaxanthin is involved in the xanthophyll cycle, which recently has been proposed to play a significant role in the protection of the photosynthetic apparatus from photoinhibitory conditions (Demmig et al. [1987] Plant Physiol 84: 218-224). PMID- 16666996 TI - A N and N Nuclear Magnetic Resonance Study of Nitrogen Metabolism in Shoot Forming Cultures of White Spruce (Picea glauca) Buds. AB - Nitrogen-14 and nitrogen-15 nuclear magnetic resonance (NMR) spectra were recorded for freshly dissected buds of Picea glauca and for buds grown for 3, 6 and 9 weeks on shoot-forming medium. Resonances for Glu (and other alphaNH(2) groups), Pro, Ala, and the side chain groups in Gln, Arg, Orn, and gamma aminobutyric acid could be detected in in vivo(15)N NMR spectra. Peaks for alpha amino groups, Pro, NO(3) (-) and NH(4) (+) could also be identified in (14)N NMR spectra. Perfusion experiments performed for up to 20 hours in the NMR spectrometer showed that (15)N-labeled NH(4) (+) and NO(3) (-) are first incorporated into the amide group of Gln and then in the alphaNH(2) pool. Subsequently, it also emerges in Ala and Arg. These data suggest that the glutamine synthetase/ glutamate synthase pathway functions under these conditions. The assimilation of NH(4) (+) is much faster than that of NO(3) (-). Consequently after 10 days of growth more than 70% of the newly synthesized internal free amino acid pool derives its nitrogen from NH(4) (+) rather than NO(3) (-). If NH(4) (+) is omitted from the medium, no NO(3) (-) is taken up during 9 weeks and the buds support limited growth by utilizing their endogenous amino acid pools. It is concluded that NH(4) (+) and NO(3) (-) are both required for the induction of nitrate- and nitrite reductase. PMID- 16666997 TI - Manipulating membrane Fatty Acid compositions of whole plants with tween-Fatty Acid esters. AB - This paper describes a method for manipulating plant membrane fatty acid compositions without altering growth temperature or other conditions. Tween-fatty acid esters carrying specific fatty acids were synthesized and applied to various organs of plants growing axenically in glass jars. Treated plants incorporated large amounts of exogenous fatty acids into all acylated membrane lipids detected. Fatty acids were taken up by both roots and leaves. Fatty acids applied to roots were found in leaves, while fatty acids applied to leaves appeared in both leaves higher on the plant and in roots, indicating translocation (probably in the phloem). Foliar application was most effective; up to 20% of membrane fatty acids of leaves above the treated leaf and up to 40% of root membrane fatty acids were exogenously derived. Plants which took up exogenous fatty acids changed their patterns of fatty acid synthesis such that ratios of saturated to unsaturated fatty acids remained essentially unaltered. Fatty acid uptake was most extensively studied in soybean (Glycine max [L.] Merr.), but was also observed in other species, including maize (Zea mays L.), mung beans (Vigna radiata L.), peas (Pisum sativum L.), petunia (Petunia hybrida L.) and tomato (Lycopersicon esculentum Mill.). Potential applications of this system include studying internal transport of fatty acids, regulation of fatty acid and membrane synthesis, and influences of membrane fatty acid composition on plant physiology. PMID- 16666998 TI - Calcium activates an electrogenic proton pump in neurospora plasma membrane. AB - Calcium ionophoresis into coenocytic cells of Neurospora crassa activates the plasma membrane proton pump as measured by current-voltage analysis. This is direct evidence that intracellular calcium regulates the activity of a key transport enzyme found in higher plants and fungi. PMID- 16666999 TI - Immunocytochemical Localization of ADPglucose Pyrophosphorylase in Developing Potato Tuber Cells. AB - The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition. PMID- 16667000 TI - Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts. AB - The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [(14)C]5,5-dimethyl-2,4-oxazolidinedione and [(14)C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO(2) fixation by superoptimal medium Pi or by adding glyceraldehyde-an inhibitor of the Calvin cycle-leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific ;low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP(+) and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N'-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N' Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific ;low phosphate' proton leak in the thylakoids, and its origin is discussed. PMID- 16667001 TI - Root Respiration and Growth in Plantago major as Affected by Vesicular-Arbuscular Mycorrhizal Infection. AB - Effects of vesicular-arbuscular mycorrhizal (VAM) infection and P on root respiration and dry matter allocation were studied in Plantago major L. ssp. pleiosperma (Pilger). By applying P, the relative growth rate of non-VAM controls and plants colonized by Glomus fasciculatum (Thaxt. sensu Gerdemann) Gerdemann and Trappe was increased to a similar extent (55-67%). However, leaf area ratio was increased more and net assimilation rate per unit leaf area was increased less by VAM infection than by P addition. The lower net assimilation rate could be related to a 20 to 30% higher root respiration rate per unit leaf area of VAM plants. Root respiration per unit dry matter and specific net uptake rates of N and P were increased more by VAM infection than by P addition. Neither the contribution of the alternative respiratory path nor the relative growth rate could account for the differences in root respiration rate between VAM and non VAM plants. It was estimated that increased fungal respiration (87%) and ion uptake rate (13%) contributed to the higher respiratory activity of VAM roots of P. major. PMID- 16667002 TI - Abscisic Acid Suppression of Phenylalanine Ammonia-Lyase Activity and mRNA, and Resistance of Soybeans to Phytophthora megasperma f.sp. glycinea. AB - Etiolated hypocotyls of the resistant soybean (Glycine max [L.] Merr.) cultivar Harosoy 63 became susceptible to Phytophthora megasperma (Drechs.) f.sp. glycinea (Hildeb.) Kuan and Erwin race 1 after treatment with abscisic acid. Susceptibility was expressed by increases in lesion size and a major decrease in accumulation of the isoflavonoid phytoalexin, glyceollin. In untreated hypocotyls, activity of phenylalanine ammonia-lyase and accumulation of mRNA for this enzyme increased rapidly after infection, but these increases were suppressed in abscisic acid-treated hypocotyls. The results suggest the possibility that biosynthesis of glyceollin in the resistance response of soybeans may be controlled at the transcriptional level by changes in abscisic acid concentrations caused by infection. PMID- 16667003 TI - Analysis of aluminum and divalent cation binding to wheat root plasma membrane proteins using terbium phosphorescence. AB - A phosphorescent trivalent cation, terbium [Tb(III)], has been used to study the binding of different polyvalent cations to the proteins of wheat (Triticum aestivum L.) root plasma membranes. The phosphorescence emission intensity of Tb(III) was enhanced after Tb(III) binding to wheat root plasma membranes as a result of nonradiative resonance energy transfer from the membrane protein tyrosine and phenylalanine residues. Complex, saturable Tb(III) binding was observed, suggesting multiple binding sites. Bound Tb(III) could be displaced by divalent cations in the general order: Mn(II) > Ca(II) > Mg(II). Al(III) was very effective in reducing the protein-enhanced Tb(III) phosphorescence at pH values below 5. Al(III) also altered the Tb(III) phosphorescence lifetime, suggesting Al(III)-induced changes in membrane protein conformation. The more Al(III) sensitive wheat cultivar (Anza) bound Al(III) with higher affinity than the more tolerant cultivar (BH 1146). At pH 5.5 where Al(III) did not displace bound Tb(III), low levels of Al(III) reduced the ability of Mn(II) to decrease Tb(III) phosphorescence. The significance of these results is discussed with respect to the mechanisms of Al(III) tolerance in wheat and the potential beneficial effects of Al(III) in reducing Mn(II) phytotoxicity. PMID- 16667004 TI - Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : II. Fractionation and Partial Characterization of Feraxan Fragments Dissociated by a Bacillus subtilis Enzyme (Feraxanase). AB - Structural features of feruloylated arabinoxylan (feraxan) present in Zea mays L. (hybrid B 73 x Mo 17) coleoptile cell walls have been studied using a purified feraxan-dissociating enzyme (feraxanase) and an alpha-arabinofuranosidase. This experimental approach has demonstrated the following. (a) Feraxanase dissociated ca. 20% (dry weight basis) of the maize wall preparation. The predominant oligosaccharides enzymically liberated were allocated into seven major subfractions designated A-1 (0.8%), B-1 (1.6%), B-2 (2.4%), B-3 (4.6%), C-1 (1.0%), C-2 (4.2%), and C-3 (0.3%). Values in parentheses reflect the percentage of the wall associated with each subfraction. Subfractions represent samples enriched in different degrees of polymerization, sugar composition, linkage arrangements, and phenolic acid content. (b) B-1, B-2, and B-3 fractions are not feruloylated and have smaller molecular mass (less than 10(4) kilodaltons) and consist chiefly of t-arabinosyl-5-arabinosyl, 4-xylosyl, 2,4/3,4-xylosyl, and glucuronosyl residues, suggesting that these fragments constitute nonferuloylated regions of arabinoxylan. (c) C-2 and C-3 fractions contain ferulic acid (6.2% and 12.1%, respectively) and are similar to the B series in their sugar linkage arrangements but were derived from feruloylated regions. (d) Alkali treatment of the C-2 fraction decreases the molecular size of the fragment and liberates phenolic acids. The results suggest the presence of alkaline-labile links, probably diferulate bridges. (e) A-1 and C-1 fractions are larger (more than 5 x 10(5) kilodalton) and contain t-galactosyl-, 4-galactosyl, 2,4-rhamnosyl residues, galacturonic acid, and the sugar linkage arrangements common to other fractions. The A-1 fraction is not feruloylated, whereas C-1 fraction contains 0.5% ferulic acid. The presence of galactose, rhamnose, and galacturonic acid suggests that pectic polymers, probably homopolygalacturonans and rhamnogalacturonans, are linked to nonferuloylated and feruloylated segments of arabinoxylans. PMID- 16667005 TI - Direct measurement of k channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers. AB - Light-driven electron transfer reactions cause the active accumulation of protons inside thylakoids, yet at steady state the electrical potential difference across the thylakoid membrane is very small; therefore, there must be a flux of other ions to balance the charge that would otherwise be built up by the net movement of H(+). This paper presents direct measurements of ion movements through channels in the thylakoid membrane. These were made possible by fusing thylakoid vesicles from spinach (Spinacia oleracea L.) into planar lipid bilayers, using techniques developed originally to study sarcoplasmic reticulum. No Mg(2+) current was found, but voltage-dependent channels have been characterized, these being somewhat selective for K(+) over Cl(-). The data are consistent with a role for these channels in charge balance during light-driven H(+) movements. PMID- 16667006 TI - Control of alpha-Amylase Development in Cotyledons during and following Germination of Mung Bean Seeds. AB - Developmental patterns of alpha-amylase in Vigna radiata cotyledons during and following germination were quite different depending on the differences in the treatments of cotyledons during the imbibitional stage. When axis-detached cotyledons were imbibed in water with seed-coats attached, alpha-amylase activity did not increase and remained low. On the other hand, when the cotyledons were imbibed in water after seed-coat removal, the enzyme activity increased markedly. If the axis was attached to the cotyledons, alpha-amylase showed a marked development even under the former imbibition conditions. These changes in the enzyme activity were in parallel with those in the enzyme content, and the content, in turn, was dependent upon the availability of mRNA for alpha-amylase. We propose that the regulation of the development of alpha-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of alpha-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis. PMID- 16667007 TI - Sucrose Loading in Isolated Veins of Pisum sativum: Regulation by Abscisic Acid, Gibberellic Acid, and Cell Turgor. AB - Enzymatically isolated vein networks from mature pea (Pisum sativum L. cv Alaska) leaves were employed to investigate the properties of sucrose loading and the effect of phytohormones and cell turgor on this process. The sucrose uptake showed two components: a saturable and a first-order kinetics system. The high affinity system (K(m), 3.3 millimolar) was located at the plasmalemma (p chloromercuriphenylsulfonic acid and orthovanadate sensitivity). Further characterization of this system, including pH dependence and effects of energy metabolism inhibitors, supported the H(+)-sugar symport concept for sucrose loading. Within a physiological range (0.1-100 micromolar) and after 90 min, abscisic acid (ABA) inhibited and gibberellic acid (GA(3)) promoted 1 millimolar sucrose uptake. These responses were partially (ABA) or totally (GA(3)) turgor dependent. In experiments of combined hormonal treatments, ABA counteracted the GA(3) positive effects on sucrose uptake. The abolishment of these responses by p chloromercuriphenylsulfonic acid and experiments on proton flux suggest that both factors (cell turgor and hormones) are modulating the H(+) ATPase plasmalemma activity. The results are discussed in terms of their physiological relevance. PMID- 16667008 TI - Decreased Ethylene Biosynthesis, and Induction of Aerenchyma, by Nitrogen- or Phosphate-Starvation in Adventitious Roots of Zea mays L. AB - Plants of Zea mays L. cv TX5855 were grown in a complete, well oxygenated nutrient solution then subjected to nutrient starvation by omitting either nitrate and ammonium or phosphate from the solution. These treatments induced the formation of aerenchyma close to the apex of the adventitious roots that subsequently emerged from the base of the shoot, a response similar to that shown earlier to be induced by hypoxia. Compared with control plants supplied with all nutrients throughout, N- or P-starvation consistently depressed the rates of ethylene release by excised, 25 mm apical segments of adventitious roots. Some enzymes and substrates of the ethylene biosynthetic pathway were examined. The content of 1-amino cyclopropane-1-carboxylic acid (ACC) paralleled the differences in ethylene production rates, being depressed by N or P deficiency, while malonyl-ACC showed a similar trend. Activity of ACC synthase and of ethylene forming enzyme (g(-1) fresh weight) was also greater in control roots than in nutrient starved ones. These results indicate that much of the ethylene biosynthetic pathway is slowed under conditions of N- or P-starvation. Thus, by contrast to the effects of hypoxia, the induction of aerenchyma in roots of Zea mays by nutrient starvation is not related to an enhanced biosynthesis and/or accumulation of ethylene in the root tips. PMID- 16667010 TI - Identification of hydroxypyruvate and glyoxylate reductases in maize leaves. AB - At least two hydroxypyruvate reductases (HPRs), differing in specificity for NAD(P)H and (presumably) utilizing glyoxylate as a secondary substrate, were identified by fractionation of crude maize leaf extracts with ammonium sulfate. The NADH-preferring enzyme, which most probably represented peroxisomal HPR, was precipitated by 30 to 45% saturated ammonium sulfate, while most of the NADPH dependent activity was found in a 45 to 60% precipitate. The HPRs had similar low K(m)s for hydroxypyruvate (about 0.1 millimolar), regardless of cofactor, while affinities of glyoxylate reductase (GR) reactions for glyoxylate varied widely (K(m)s of 0.4-12 millimolar) depending on cofactor. At high hydroxypyruvate concentrations, the NADPH-HPR from the 30 to 45% precipitate showed negative cooperativity with respect to this reactant, having a second K(m) of 6 millimolar. In contrast, NADPH-HPR from the 45 to 60% precipitate was inhibited at high hydroxypyruvate concentrations (K(1) of 3 millimolar) and, together with NADPH-GR, had only few, if any, common antigenic determinants with NADH-HPR from the 30 to 45% fraction. Both NADPH-HPR and NADPH-GR activities from the 45 to 60% precipitate were probably carried out by the same enzyme(s), as found by kinetic studies. Following preincubation with NADPH, there was a marked increase (up to sixfold) in activity of NADPH-HPR from either crude or fractionated extracts. Most of this increase could be attributed to an artefact resulting from an interference by endogeneous NADPH-phosphatase, which hydrolyzed NADPH to NADH, the latter being utilized by the NADH-dependent HPR. However, in the presence of 15 millimolar fluoride (phosphatase inhibitor), preincubation with NADPH still resulted in over 60% activation of NADPH-HPR. The NADPH treatment stimulated the V(max) of the reductase but had no effect on its K(m) for hydroxypyruvate. Enzyme distribution studies revealed that both NADH and NADPH-dependent HPR and GR activities were predominantly localized in the bundle sheath compartment. Rates of NADPH-HPR and NADPH-GR in this tissue (over 100 micromoles per hour per milligram of chlorophyll each) are in the upper range of values reported for leaves of C(3) species. PMID- 16667009 TI - Isolation and partial characterization of the glutamate/aspartate transporter from pea leaf mitochondria using a specific monoclonal antibody. AB - A library of monoclonal antibodies directed against the proteins of the inner mitochondrial membrane was screened for antibodies that could bind to the glutamate/aspartate transporter of pea mitochondria and thereby inhibit its activity. One antibody, 2C7, had the property of inhibiting glutamate and aspartate-dependent oxaloacetate metabolism by pea mitochondria without affecting the metabolism of other substrates. The antibody specifically recognized a 21,000 dalton protein, which was tentatively identified as the glutamate/aspartate transporter. The antibody was used to follow the extraction of this protein by Triton X-114 and cardiolipin and the partial purification of the protein by centrifugation and chromatography on hydroxylapatite. The partially purified preparation was reconstituted into azolectin vesicles and shown to catalyze glutamate/glutamate and glutamate/aspartate exchange in an apparently nonelectrogenic manner. The antibody was shown to specifically bind to the glutamate/aspartate exchanger by its ability to inhibit this reconstituted exchange reaction. PMID- 16667011 TI - Changes in Net O(2) Exchange Induced by Inorganic Nitrogen in the Blue-Green Alga Anacystis nidulans. AB - The response of net O(2) exchange to light intensity by intact Anacystis nidulans cells in the presence of saturating NaHCO(3) concentrations followed a curve with an inflection near the light-compensation point. Addition of either KNO(3) or NH(4)Cl stimulated O(2) uptake in the dark and at light intensities below the light-compensation point. This resulted in steeper slopes of the curve calculated below and above the light-compensation point. At O(2) concentrations limiting dark respiration, addition of inorganic nitrogen had no effect on either dark respiration or O(2) exchange in the light. The apparent changes in photosynthetic yield observed under normal O(2) concentration disappeared when respiration was limited by O(2) availability, indicating that the effects of inorganic nitrogen on O(2) exchange at low light intensities are due to stimulation of respiration rather than to increases in photosynthetic yield. PMID- 16667012 TI - Absence of a Formate-Induced Release of Bicarbonate from Photosystem II. AB - Formate has been proposed to inhibit electron flow in photosystem II by replacing endogenous bound bicarbonate on the reaction center complex. A mass spectrometer was used to measure directly the CO(2)/HCO(3) (-) released when maize thylakoids, showing normal rates of electron flow, were treated with formate. Although the formate inhibited electron flow by 95%, no release (displacement) of CO(2)/HCO(3) (-) was detected. This is consistent with the concept that membrane-bound HCO(3) (-) is not a requirement for normal rates of electron flow through photosystem II. Moreover, formate and other monovalent anions do not inhibit electron flow by removing bound HCO(3) (-) but by binding to empty sites. The "bicarbonate effect" is a reversal, by high concentrations of exogenous bicarbonate, of anion inhibition of photosystem II. PMID- 16667013 TI - Carbon Partitioning among Leaves, Fruits, and Seeds during Development of Phaseolus vulgaris L. AB - Development of vegetative and floral buds was found to be a key factor in establishing the way carbon is distributed among growing leaves and fruits in Phaseolus vulgaris L. plants. Leaves emerged principally during a period 14 to 32 days after planting while flowers were produced during a 10- to 12-day period near the end of leaf emergence. Timing of anthesis established the sigmoidal time course for dry weight accumulated by the composite of all fruits on the plant. During the first 12 days following anthesis, fruit growth mainly consisted of elongation and dry weight accumulation by the pod wall. Thereafter, seed dry weight increased for about 1 week, decreased markedly for several days, and then increased again over the next 2 weeks. Accumulation of imported carbon in individual seeds, measured by steady-state labeling, confirmed the time course for dry weight accumulation observed during seed development. Seed respiration rate initially increased rapidly along with dry weight and then remained nearly steady until seed maturation. A number of developmental events described in the literature coincided with the different phases of diauxic growth. The results demonstrated the feasibility of relating current rates of carbon import in individual seeds measured with tracer (14)C to the rates of conversion of imported sucrose and use of the products for specific developmental processes. The resulting data are useful for evaluating the roles of conversion and utilization of imported sucrose in regulating import by developing seeds. PMID- 16667014 TI - Role of the Plasmalemma H-ATPase in Pseudomonas syringae-Induced K/H Exchange in Suspension-Cultured Tobacco Cells. AB - Activation of a host plasma membrane K(+) efflux/net H(+) uptake exchange by pathogenic pseudomonads plays an important role in the development of hypersensitivity in tobacco (Nicotiana tabacum). Involvement of the plasmalemma H(+)-pumping ATPase in this response was investigated. The exchange response of suspension-cultured tobacco cells to Pseudomonas syringae pv syringae was reduced 90% or more by ATPase inhibitors including vanadate, N-ethylmaleimide, and N,N' dicyclohexylcarbodiimide. The exchange was also strongly inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone and by slightly alkaline external pH. Respiratory inhibitors such as oligomycin and sodium azide reduced the exchange by 50% to 75%, while glycolysis inhibitors such as sodium arsenite and sodium iodoacetate decreased exchange by approximately 90%. These results suggest that plasmalemma H(+)-ATPase activity is required for the exchange response and that this may reflect a requirement for a plasmalemma pH and/or electrical potential gradient. PMID- 16667015 TI - Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene. AB - Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed. PMID- 16667016 TI - New ways to look at the architecture of plant cell walls : localization of polygalacturonate blocks in plant tissues. AB - We report the use of Ni(2+) and Co(2+) on free-hand sections of soybean (Glycine max L.) and Bidens sp to localize polygalacturonates. In soybean only the hourglass cells of the seedcoat stain intensely. In the pod the epidermis of the outer pod wall and a few layers of subepidermal cells stain lightly, while that part of the funiculus adjacent to the seedcoat palisade epidermal cells stains heavily and the neck of the funiculus close to the pod also stains. In Bidens stem sections, the walls of the collenchyma stain most intensely. PMID- 16667017 TI - Inhibitory action of auxin on root elongation not mediated by ethylene. AB - The inhibitory effects of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1 carboxylic acid (ACC) on elongation growth of pea (Pisum sativum L.) seedling roots were investigated in relation to the effects of these compounds on ethylene production by the root tips. When added to the growth solution both compounds caused a progressively increasing inhibition of growth within the concentration range of 0.01 to 1 micromolar. However, only ACC increased ethylene production in root tips excised from the treated seedlings after 24 hours. High auxin concentrations caused a transitory increase of ethylene production during a few hours in the beginning of the treatment period, but even in 1 micromolar IAA this increase was too low to have any appreciable effect on growth. ACC, but not IAA, caused growth curvatures, typical of ethylene treatment, in the root tips. IAA caused conspicuous swelling of the root tips while ACC did not. Cobalt and silver ions reversed the growth inhibitory effects induced by ACC but did not counteract the inhibition of elongation or swelling caused by IAA. The growth effects caused by the ACC treatments were obviously due to ethylene production. We found no evidence to indicate that the growth inhibition or swelling caused by IAA is mediated by ethylene. It is concluded that the inhibitory action of IAA on root growth is caused by this auxin per se. PMID- 16667018 TI - Effects of gradual increases in o(2) concentration on nodule activity in soybean. AB - The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO(2) without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO(2) under ambient conditions. A computer controlled gas blending apparatus was used to produce linear increases (ramps) in pO(2) around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H(2) production in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) were monitored continuously as pO(2) was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an O(2)-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions. PMID- 16667019 TI - Psychrometric pressure-volume analysis of osmoregulation in roots, shoots, and whole sporophytes of salinized ceratopteris. AB - Osmotic and turgor adjustment in roots, shoots, and whole sporophytes of a NaCl tolerant mutant and a NaCl-sensitive wild-type strain of the fern Ceratopteris richardii Brongn. were characterized following exposure to 60 millimolar NaCl, using a psychrometric approach to pressure-volume analysis. Water potential components of whole plants and shoots at full or ambient hydration were similar between strains. Roots of the mutant, however, had osmotic potentials at full turgor and water potentials at zero turgor which were lower (0.32 and 0.46 megapascal, respectively) than those in roots of the wild type after salinization. Although compromised in the absence of NaCl, sporophytes of the mutant strain were larger and much less necrotic in 60 millimolar NaCl after 17 days, compared to the wild type. Root growth in the mutant strain was unaffected by salinization and far exceeded root growth in the wild type. Correlation coefficients of the linear regression and the general consistency and precision of the pressure-volume data demonstrated the feasibility of using PV curves for estimating water potential components of roots. The technique should also provide a means of studying osmoregulation in a variety of other rapidly equilibrating plant tissues. PMID- 16667020 TI - Chlorophyll a Fluorescence Predicts Total Photosynthetic Electron Flow to CO(2) or NO(3)/NO(2) under Transient Conditions. AB - A model which predicts total photosynthetic electron flow from a linear regression of the relationship between corrected steady-state quantum yield and nonphotochemical quenching (E Weis, JA Berry [1987] Biochem Biophys Acta 894: 198 208) was formulated for N-limited cells of the green alga Selenastrum minutum. Unlike other models based on net CO(2) fixation, our model is based on total photosynthetic electron flow measured as gross O(2) evolution. This allowed for the prediction of total photosynthetic electron flow from water to both CO(2) fixation and NO(3) (-)/NO(2) (-) reduction. The linear regression equation predicting electron flow is of the form: J = I . Q(q)[0.4777-0.3282 Q(NP)] (where J = gross photosynthetic electron flow, I = incident PAR, Q(q) = photochemical quenching, Q(NP) = nonphotochemical quenching). During steady-state photosynthesis, over a range of irradiance, the model predicted a photosynthetic light saturation curve which was well correlated with that observed. Although developed under steady-state conditions, the model was tested during nonsteady state photosynthesis induced by transient nitrogen assimilation. The model predicted transient rates of gross O(2) evolution which were in excellent agreement with the rates observed under a variety of conditions regardless of whether CO(2) or NO(3) (-)/NO(2) (-) served as the physiological electron acceptor. The fluorescence transients resulting from ammonium and nitrate assimilation are discussed with respect to metabolic demands for reductant and ATP. PMID- 16667021 TI - Distribution of endogenous indole-3-acetic Acid and compression wood formation in reoriented branches of douglas-fir. AB - Five-year-old segments of intact 7-year-old branches of Douglas-fir (Pseudotsuga meziesii [Mirb.] Franco) were reoriented to determine the relation between indole 3-acetic acid (IAA) and the formation of compression wood. Eight branches per treatment were either left at their original angle (mean of 69 degrees , the control), or bent proximal to the segment to reorient it up or down 30 degrees . Differentiating xylem tissue from the upper and lower sides of each segment was collected and extracted separately for IAA analysis by in-line fluorescence detection of free IAA and IAA methyl ester after sequential C(16) reversed-phase high performance liquid chromatography. The IAA methyl ester was confirmed by gas chromatography-mass spectroscopy. Compression wood formed on the upper side of branches reoriented up and on the lower side of controls or branches reoriented down. IAA was present in all samples. The difference in IAA concentration between upper and lower sides was either not correlated, or negatively correlated in segments reoriented down, with both the occurrence of compression wood and the rate of new tracheid production. Mean concentrations for whole branch segments were not affected by the treatments, regardless of whether IAA concentrations were expressed on a surface area, weight, or cell basis. PMID- 16667022 TI - Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet. AB - An H(+)-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150 fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase. PMID- 16667023 TI - Two isozymes of dihydroxyacetone phosphate reductase in dunaliella. AB - Two isoforms of dihydroxyacetone phosphate reductase were present in Dunaliella tertiolecta. The major form was located in the chloroplast and the minor form in the cytosol. The chloroplastic reductase eluted first from a DEAE cellulose column followed immediately by the cytosolic form. Both forms were unstable and cold labile. Addition of 5 millimolar dithiothreitol helped to stabilize the enzymes. The cytosolic isoform of DHAP reductase was detected only if the cells were in an active log phase of growth. Then its activity was 20 to 30% of the total reductase activity. When cell cultures entered late log phase of growth the activity of the cytosolic form of the enzyme disappeared, but the chloroplastic form remained. The cytosolic DHAP reductase from Dunaliella has some properties similar to the cytosolic isoform from spinach leaves. Detergents inhibited both enzymes. However, neither form of the algal dihydroxyacetone phosphate reductase was stimulated by fructose 2,6-bisphosphate. In Dunaliella the properties of the chloroplastic form were those expected for glycerol production for osmoregulation, whereas the cytosolic form, like the reductases in leaves, is more likely involved in glycerol phosphate formation for lipid synthesis. PMID- 16667024 TI - Oxygen and carbon dioxide fluxes from barley shoots depend on nitrate assimilation. AB - A custom oxygen analyzer in conjunction with an infrared carbon dioxide analyzer and humidity sensors permitted simultaneous measurements of oxygen, carbon dioxide, and water vapor fluxes from the shoots of intact barley plants (Hordeum vulgare L. cv Steptoe). The oxygen analyzer is based on a calciazirconium sensor and can resolve concentration differences to within 2 microliters per liter against the normal background of 210,000 microliters per liter. In wild-type plants receiving ammonium as their sole nitrogen source or in nitrate reductase deficient mutants, photosynthetic and respiratory fluxes of oxygen equaled those of carbon dioxide. By contrast, wild-type plants exposed to nitrate had unequal oxygen and carbon dioxide fluxes: oxygen evolution at high light exceeded carbon dioxide consumption by 26% and carbon dioxide evolution in the dark exceeded oxygen consumption by 25%. These results indicate that a substantial portion of photosynthetic electron transport or respiration generates reductant for nitrate assimilation rather than for carbon fixation or mitochondrial electron transport. PMID- 16667025 TI - Enhanced ethylene emissions from red and norway spruce exposed to acidic mists. AB - Acidic cloudwater is believed to cause needle injury and to decrease winter hardiness in conifers. During simulations of these adverse conditions, rates of ethylene emissions from and levels of 1-aminocyclopropane-1-carboxylic acid (ACC) in both red and Norway spruce needles increased as a result of treatment with acidic mists but amounts of 1-malonyl(amino)cyclopropane-1-carboxylic acid remained unchanged. However, release of significant quantities of ethylene by another mechanism independent of ACC was also detected from brown needles. Application of exogenous plant growth regulators such as auxin, kinetin, abscisic acid and gibberellic acid (each 0.1 millimolar) had no obvious effects on the rates of basal or stress ethylene production from Norway spruce needles. The kinetics of ethylene formation by acidic mist-stressed needles suggest that there is no active inhibitive mechanism in spruce to prevent stress ethylene being released once ACC has been formed. PMID- 16667026 TI - Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development. AB - Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography mass spectrometry (GC-MS). From the suspensor four C(19)-GAs, GA(1), GA(4), GA(5), GA(6), and one C(20) GA, GA(44), were identified. From the embryo, five C(19)-GAs GA(1), GA(4), GA(5), GA(6), GA(60) and two C(20) GAs, GA(19) and GA(44) were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development. PMID- 16667027 TI - Properties of a Lipoxygenase in Green Algae (Oscillatoria sp.). AB - A lipoxygenase preparation was obtained from green algae Oscillatoria sp. and was shown to differ from previous described lipoxygenases in the positional specificity and pH characteristics of the dioxygenation reaction. The enzyme had a pH optimum at 8.8 and was inactive at pH 6. Oscillatoria lipoxygenase converted linoleic acid into two products: 13-hydroperoxylinoleic acid (52%) and 9 hydroperoxylinoleic acid (48%). The molecular weight of the enzyme was estimated at 124,000. Esculetin was found to be the best inhibitor of the enzyme activity. PMID- 16667028 TI - Incorporation of UDP-[C]Glucose into Xyloglucan by Pea Membranes. AB - The water-insoluble 1,4-beta-linked products formed from UDP-[(14)C]glucose by pea membranes were dissolved in hot dimethyl-sulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [(14)C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP xylose at concentrations equal to or less than UDP-[(14)C]glucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-beta-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc(4)Xyl(3), which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-alpha-d glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[(14)C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-beta-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan. PMID- 16667029 TI - Light adaptation/acclimation of photosynthesis and the regulation of ribulose-1,5 bisphosphate carboxylase activity in sun and shade plants. AB - The consequences of light adaptation and acclimation of photosynthesis on photosynthetic nitrogen use efficiency (NUE), particularly as it relates to the efficiency of ribulose-1,5-bisphosphate carboxylase (Rubisco) use in photosynthetic CO(2) assimilation, was studied in the sun species Glycine max and the shade species Alocasia macrorrhiza. Both G. max and A. macrorrhiza were found to possess the capacity for light acclimation of CO(2) assimilation, but over distinctly different ranges of photon flux density (PFD). For each species, light acclimation of photosynthesis had little effect on the rate of photosynthesis per unit Rubisco protein or the light response of Rubisco carbamylation and CA 1P metabolism. In contrast, photosynthesis per unit Rubisco protein was significantly higher in G. max than in A. macrorrhiza, due in part to a lower total (fully carbamylated) molar activity (activity per unit enzyme) of A. macrorrhiza Rubisco than that of G. max. Comparison of the light response of Rubisco regulatory mechanisms between G. max and A. macrorrhiza indicated some degree of adaptation, such that carbamylation was higher and CA 1P levels lower at lower PFDs in the shade species than the sun species. However, this adjustment was not sufficient for Rubisco in low light grown A. macrorrhiza to be fully active at the growth PFD. Photosynthesis in A. macrorrhiza appeared to become RuBP regeneration-limited at lower PFDs than G. max, and this was probably the determinant of the light saturated rate of photosynthesis in the shade species. The low efficiency of Rubisco use in A. macrorrhiza was a major contributing factor to its five- to sixfold lower photosynthetic NUE than G. max. Shade species such as A. macrorrhiza appear to make far from maximal use of Rubisco protein N. PMID- 16667030 TI - Selective and Reversible Inhibition of Active CO(2) Transport by Hydrogen Sulfide in a Cyanobacterium. AB - The active transport of CO(2) in the cyanobacterium Synechococcus UTEX 625 was inhibited by H(2)S. Treatment of the cells with up to 150 micromolar H(2)S + HS( ) at pH 8.0 had little effect on Na(+)-dependent HCO(3) (-) transport or photosynthetic O(2) evolution, but CO(2) transport was inhibited by more than 90%. CO(2) transport was restored when H(2)S was removed by flushing with N(2). At constant total H(2)S + HS(-) concentrations, inhibition of CO(2) transport increased as the ratio of H(2)S to HS(-) increased, suggesting a direct role for H(2)S in the inhibitory process. Hydrogen sulfide does not appear to serve as a substrate for transport. In the presence of H(2)S and Na(+) -dependent HCO(3) (-) transport, the extracellular CO(2) concentration rose considerably above its equilibrium level, but was maintained far below its equilibrium level in the absence of H(2)S. The inhibition of CO(2) transport, therefore, revealed an ongoing leakage from the cells of CO(2) which was derived from the intracellular dehydration of HCO(3) (-) which itself had been recently transported into the cells. Normally, leaked CO(2) is efficiently transported back into the cell by the CO(2) transport system, thus maintaining the extracellular CO(2) concentration near zero. It is suggested that CO(2) transport not only serves as a primary means of inorganic carbon acquisition for photosynthesis but also serves as a means of recovering CO(2) lost from the cell. A schematic model describing the relationship between the CO(2) and HCO(3) (-) transport systems is presented. PMID- 16667031 TI - Alteration of the physical and chemical structure of the primary cell wall of growth-limited plant cells adapted to osmotic stress. AB - Cells of tobacco (Nicotiana tabacum L.) adapted to grow in severe osmotic stress of 428 millimolar NaCl (-23 bar) or 30% polyethylene glycol 8000 (-28 bar) exhibit a drastically altered growth physiology that results in slower cell expansion and fully expanded cells with volumes only one-fifth to one-eighth those of unadapted cells. This reduced cell volume occurs despite maintenance of turgor pressures sometimes severalfold higher than those of unadapted cells. This report and others (NM Iraki et al [1989] Plant Physiol 90: 000-000 and 000-000) document physical and biochemical alterations of the cell walls which might explain how adapted cells decrease the ability of the wall to expand despite diversion of carbon used for osmotic adjustment away from synthesis of cell wall polysaccharides. Tensile strength measured by a gas decompression technique showed empirically that walls of NaCl-adapted cells are much weaker than those of unadapted cells. Correlated with this weakening was a substantial decrease in the proportion of crystalline cellulose in the primary cell wall. Even though the amount of insoluble protein associated with the wall was increased relative to other wall components, the amount of hydroxyproline in the insoluble protein of the wall was only about 10% that of unadapted cells. These results indicate that a cellulosic-extensin framework is a primary determinant of absolute wall tensile strength, but complete formation of this framework apparently is sacrificed to divert carbon to substances needed for osmotic adjustment. We propose that the absolute mass of this framework is not a principal determinant of the ability of the cell wall to extend. PMID- 16667032 TI - Metabolism of Hydroxypyruvate in a Mutant of Barley Lacking NADH-Dependent Hydroxypyruvate Reductase, an Important Photorespiratory Enzyme Activity. AB - A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO(2) fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O(2). There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO(2) efflux and (14)CO(2) release from l-[U-(14)C]serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either [(14)C]serine or (14)CO(2), the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied [(14)C]serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolise a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration. PMID- 16667033 TI - Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation. AB - A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast omega9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO(2) fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28 degrees C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes. PMID- 16667034 TI - Formation of the Photosynthetic Electron Transport System during the Early Phase of Greening in Barley Leaves. AB - The development of photochemical activity in isolated plastids during the early phase of greening of 5-day-old etiolated barley seedlings was studied and related to the appearance of chlorophyll-protein complexes. Photochemical activities of PSI (DCIPH(2) --> MV) and PSII (H(2)O --> DCIP, DPC --> DCIP) appeared at 1 and 1.5 hours after the onset of illumination, respectively. However, PSI + PSII activity (H(2)O --> MV, H(2)O --> NADP) appeared at 4 hours. The functional plastoquinone pool was noticed, at the latest, from 4 hours. Chloroplast preparations from seedlings of 1 h of greening showed O(2) uptake upon illumination in the absence of MV (-MV activity). This activity peaked at 2 hours of greening, then fell to zero by 6 hours. In contrast to the -MV activity, MV Hill activity began to increase at 2 hours. Although PSI activity appeared at 1 hour, it failed to reduce ferredoxin until 2 hours. NADP began to be photoreduced at 4 hours in accordance with the appearance of the ferredoxin:NADP reductase activity. After formation of PSI and PSII, electron transport systems between them and between PSI and NADP developed in coordination with each other. Thus, the whole electron transport from water to NADP began to operate at 4 hours. PMID- 16667035 TI - Induction of alpha-amylase inhibitor synthesis in barley embryos and young seedlings by abscisic Acid and dehydration stress. AB - An endogenous alpha-amylase inhibitor was found to be synthesized in embryos of developing barley grain (Hordeum vulgare cv Bonanza). Accumulation of this protein occurred late in development (stage IV), at the same time that endogenous abscisic acid (ABA) showed a large increase. The inhibitor could be induced up to 23-fold in isolated immature embryos (stage III) by culture in ABA. Precocious germination was also blocked in stage III embryos by ABA. Dehydration stress on the isolated immature embryos also induced higher levels of the inhibitor and ABA. An even greater response to dehydration stress was observed in young seedlings, where inhibitor content increased 20-fold and ABA increased 80-fold during water stress. The high degree of correlation between ABA and inhibitor contents in in situ embryos, dehydrated embryos and young seedlings, as well as the increase in inhibitor caused by exogenously applied ABA to isolated embryos, suggests that increased alpha-amylase inhibitor synthesis in response to dehydration stress is mediated by ABA. PMID- 16667036 TI - Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains. AB - Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg(2+) and exhibited hyperbolic kinetics with all substrates including Mg(2+). K(m) values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. Kinetic constants were determined in the presence of fructose-2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K(i) values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed. PMID- 16667037 TI - Decline of activity and quantity of ribulose bisphosphate carboxylase/oxygenase and net photosynthesis in ozone-treated potato foliage. AB - The effect of ozone (O(3)) on ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity and quantity and net photosynthesis in greenhouse-grown Solanum tuberosum L. cv ;Norland' foliage was studied in relation to oxidant induced premature senescence. Plants, 26 days old, were exposed to 0.06 to 0.08 microliters per liter O(3) from 1000 to 1600 hours for 4 days in a controlled environment chamber. On day 5, plants were exposed to a 6-hour simulated inversion in which O(3) peaked at 0.12 microliters per liter. Net photosynthesis declined in response to O(3) but recovered to near control levels 3 days after the exposure ended. Rubisco activity and quantity in control potato foliage increased and then decreased during the 12-day interval of the study. In some experiments foliage studied was physiologically mature and Rubisco activity had peaked when O(3) exposure commenced. In those cases, O(3) accelerated the decline in Rubisco activity. When less mature foliage was treated with O(3), the leaves never achieved the maximal level of Rubisco activity observed in control foliage and also exhibited more rapid decline in initial and total activity. Percent activation of Rubisco (initial/total activity) was not affected significantly by treatment. Quantity of Rubisco decreased in concert with activity. The decrease in activities is most likely due to a decrease in available protein rather than a decrease in the percentage of Rubisco activated in vivo. The reduction in the quantity of Rubisco, an important foliar storage protein, could contribute to premature senescence associated with toxicity of this air pollutant. PMID- 16667038 TI - Water relations of turgor recovery and restiffening of wilted cabbage leaves in the absence of water uptake. AB - A novel phenomenon in which wilted cabbage leaves appeared to regain positive turgor pressures without additional water uptake has been previously reported (J Levitt [1986] Plant Physiol 82: 147-153). These experiments were replicated and the biophysical nature of turgor recovery characterized. Leaf water potential and its components were assayed in hydrated, wilted, and desiccated leaves which appeared to regain turgor after wilting. The hypotheses that turgor recovery was due to an increased volumetric elastic modulus (epsilon), or alternatively the result of solute redistribution were tested. Quantitative evidence that turgor recovery occurs in excised leaves was found. Leaf turgor pressure in hydrated leaves ( approximately 0.6 megapascal) decreased to zero upon wilting. After continued desiccation, turgor pressure returned to approximately 0.3 megapascal even though leaf relative water content declined. The epsilon of hydrated leaves was large and there was no evidence of an increased epsilon in the turgor recovered leaves. Solute mobilization occurred during desiccation. The apoplastic osmotic potential decreased from -0.15 to -0.44 megapascal in hydrated and turgor recovered leaves, respectively, and solutes were transported from the lamina to the midrib tissue. Solute redistribution coupled with the high epsilon may have resulted in localized turgor recovery in specific cells in the desiccated leaves. PMID- 16667039 TI - Genetic transformation of maize cells by particle bombardment. AB - Intact maize cells were bombarded with microprojectiles bearing plasmid DNA coding for selectable (neomycin phosphotransferase [NPT II]) and screenable (beta glucuronidase [GUS]) marker genes. Kanamycin-resistant calli were selected from bombarded cells, and these calli carried copies of the NPT II and GUS genes as determined by Southern blot analysis. All such calli expressed GUS although the level of expression varied greatly between transformed cell lines. These results show that intact cells of important monocot species can be stably transformed by microprojectiles. PMID- 16667040 TI - Maize Photosystem I : Identification of the Subunit which Binds Plastocyanin. AB - Photosystem I (PSI) has been isolated from mesophyll chloroplasts of mature maize leaves. The isolated PSI (PSI-200) was used as starting material for preparing an antenna-depleted core (PSI-100). Both of these preparations appear to be quite analogous to PSI complexes isolated from other plant tissue sources, such as those from C3 plants, as judged from NADP photoreduction assays, immunoblotting, and the ability of the complexes to form a covalent crosslinked product with spinach plastocyanin. The study suggests that the PSI complex from a C4 plant is similar to that isolated from a C3 plant in that both contain the plastocyanin docking protein although the apparent molecular weight of these respective subunits differ slightly. PMID- 16667041 TI - Cell Walls of Tobacco Cells and Changes in Composition Associated with Reduced Growth upon Adaptation to Water and Saline Stress. AB - The relative mass of the cell walls of tobacco (Nicotiana tabacum L.) cells adapted to grow in medium containing 30% polyethylene glycol 8000 or 428 millimolar NaCl was reduced to about 50% of that of the walls of unadapted cells. Cellulose synthesis was inhibited substantially in adapted cells. The proportions of total pectin in walls of unadapted and adapted cells were about the same, but substantial amount of uronic acid-rich material from walls of cells adapted to either NaCl or polyethylene glycol was more easily extracted with cold sodium ethylenediamine tetraacetic acid solutions (NM Iraki et al. [1989] Plant Physiol. 91: 39-47). We examined the linkage composition of the pectic and hemicellulosic polysaccharides to ascertain chemical factors that may explain this difference in physical behavior. Adaptation to stress resulted in the formation of a loosely bound shell of polygalacturonic acid and rhamnogalacturonan. Pectins extracted from walls of adapted cells by either cold sodium ethylenediamine tetraacetic acid or hot ammonium oxalate were particularly enriched in rhamnose. Compared to pectins of unadapted cells, rhamnosyl units of the rhamnogalacturonans of adapted cells were more highly substituted with polymers containing arabinose and galactose, but the side groups were of greatly reduced molecular size. Possible functional roles of these modifications in cell wall metabolism related to adaptation to osmotic stress are discussed. PMID- 16667042 TI - Rapid effects of red light on the isopentenyladenosine content in scots pine seeds. AB - Red light (R) stimulates germination in Scots pine seed (Pinus sylvestris L.). The response is far red (FR) reversible. The dynamics of cytokinin changes following light treatment was investigated. Extracts were purified by immunoaffinity and high performance liquid chromatography. N(6)-(Delta(2) Isopentenyl) adenosine (iPA) and trans-zeatin riboside (ZR) were quantified by both UV-absorbance of high performance liquid chromatography peaks and by enzyme linked immunosorbent assay. Identification of iPA was accomplished by gas chromatography-mass spectrometry. Levels of cytokinins were low in seeds imbibed in the dark. Exposure of seeds imbibed in the dark for 5 hours to R for 15 minutes induced a strong, immediate but transitory increase in iPA content. This increase was not observed when the R treatment was followed by 10 minutes of FR or by storage in darkness before extraction. No ZR was detected during the first 8 hours of imbibition in any treatment. Addition of iPA via acetone enhanced seed germination in the dark. The results suggest that iPA may be involved in the R mediated release of dormancy of Scots pine seed. PMID- 16667043 TI - Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress. AB - The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (-28 bar) or 428 millimolar NaCl (-23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl(2) extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium. PMID- 16667044 TI - Light-Induced Polar pH Changes in Leaves of Elodea canadensis: I. Effects of Carbon Concentration and Light Intensity. AB - Leaves of the submerged aquatic Elodea canadensis Michx. exhibit a light induced polar pH reaction. In this study, the effects of light intensity and dissolved inorganic carbon concentration on this polar reaction were examined. At a light intensity of 100 watts per square meter the leaf showed a polar pH response when the dissolved inorganic carbon concentration was less than about 1 millimolar. The polar reaction was suppressed at a higher dissolved inorganic carbon concentration. This suppression was not due to the buffering capacity of bicarbonate. Because another weak acid, acetate, did not inhibit the polarity, but even had a small stimulatory effect, the effect of bicarbonate is also not due to acidification of the cytoplasm. The suppression of the polar reaction by CO(2)/HCO(3) (-) was relieved when the light intensity was increased. Apparently there is competition for product(s) of the photosynthetic light reactions between processes generating the polar reaction and the carbon fixation reactions. The possibility that the redox state of the cell regulates the generation of the polar reaction is discussed. PMID- 16667045 TI - Light-Induced Polar pH Changes in Leaves of Elodea canadensis: II. Effects of Ferricyanide: Evidence for Modulation by the Redox State of the Cytoplasm. AB - The effect of an extracellular electron acceptor, ferricyanide, on the light induced polar leaf pH changes of the submerged angiosperm Elodea canadensis in light and in darkness was determined. The rate of transmembrane ferricyanide reduction was stimulated by increased light intensity and was inhibited by inorganic carbon, indicating that changes in the redox state of the chloroplast were reflected at the plasma membrane. The addition of ferricyanide inhibited the light-induced polar leaf pH reaction. This effect could be balanced by increasing the light intensity. In the dark, the acidification induced by ferricyanide was not influenced by diethylstilbestrol at concentrations that completely inhibited the polar leaf pH changes. This indicates that the ferricyanide-induced H(+) extrusion and the H(+) transport during the polar reaction were mediated by different mechanisms. PMID- 16667046 TI - Auxin Levels Regulate the Expression of a Wound-Inducible Proteinase Inhibitor II Chloramphenicol Acetyl Transferase Gene Fusion in Vitro and in Vivo. AB - Proteinase inhibitor genes are expressed in solanaceous and leguminous plants following wounding of the foliage by mechanical methods. Previous studies have shown that a cloned proteinase inhibitor II-chloramphenicol acetyl transferase (pin2-CAT) chimeric gene is regulated in a wound-inducible manner in transgenic plants. In this study, we analyzed transgenic plant tissues for expression of the pin2-CAT gene in response to various plant hormones. We found that CAT activity was induced in tobacco (Nicotiana tabacum) callus incubated in the absence of any plant growth regulators. Addition of growth regulators to the medium thus permitted us to measure the effects of these substances on the activity of the pin2-CAT gene construction. Cytokinin (BAP) and ethylene (ethophon) even at low concentrations stimulated the expression of CAT activity by 25 to 50%. Abscisic acid at concentrations up to 4.4 x 10(-5) molar had no effect upon CAT activity, but increasing auxin (naphthalene acetic acid) levels completely inhibited the synthesis of CAT protein. Gibberellic acid had little effect except at very high concentration (2.9 x 10(5) molar). The kinetics of activation of the pin2-CAT gene were quite long (5 to 7 days) when unwounded calli were plated on media lacking auxin. This effect was documented for calli derived from several transformed plants, containing the full, chimeric pin2-CAT (pRT45) gene. In addition, calli from tissues transformed with wild-type vectors or from several plants transformed with pRT50 (a noninducible derivative of pRT45) were not induced by plating on media lacking auxin. Other naturally occurring and synthetic auxins had similar effects to naphthalene acetic acid in inhibiting the induction of the chimeric gene fusion. Finally, leaf discs from transformed plants were induced by incubation in MS liquid medium in the presence and absence of naphthalene acetic acid. NAA was also effective in down regulating the chimeric gene in whole plant tissues. PMID- 16667047 TI - Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings. AB - l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program. PMID- 16667048 TI - Whole plant senescence of sunflower following seedhead removal. AB - This study was undertaken to further clarify the relationship between seed development and monocarpic senescence of sunflower (Helianthus annuus L.). Field grown plants with and without seedheads were evaluated for rate and duration of accumulation of dry weight, reduced N, and P by whole shoots, and for partitioning of these constituents within the individual plant parts. Concurrent with seedhead removal, [(15)N]nitrate was applied to the plants in a selected area of the experimental plot. Whole plants (above ground portions) were harvested seven times during the seed-filling period and analyzed for dry weight, reduced N, and P. Although seedhead removal depressed the rates of dry weight, reduced N, and P accumulation by whole shoots, it extended the duration of accumulation of these constituents, relative to headed control plants. As a result, the final whole shoot dry weight and N and P contents at seed maturity were similar for deheaded and headed plants. Seedhead removal also affected the partitioning of dry matter, reduced N, and P but the relative proportions varied as a function of constituent and growth stage. Analysis of (15)N present in whole shoots at physiological maturity showed that similar amounts of nitrate were absorbed during the postflowering period by headed and deheaded plants. These data indicate that the absence of seeds does not affect the total accumulation of dry matter, reduced N, or P, by sunflower plants, but does alter the rates of accumulation and partitioning of these constituents. PMID- 16667049 TI - Hydrolysis of indole-3-acetic Acid esters exposed to mild alkaline conditions. AB - Ester conjugates of indole-3-acetic acid are hydrolyzed easily in basic solutions; however, quantitative data have not been available on the relationship between pH and rate of hydrolysis of the known ester conjugates. The use of basic conditions during extraction or purification of IAA by several laboratories suggested that a more systematic analysis of this process was needed. In this report we present data indicating: (a) that measurable hydrolysis of IAA-glucose (from standard solutions) and IAA-esters (from maize kernel extracts) occurs with only a few hours of treatment at pH 9 or above; (b) that the lability of some ester conjugates is even greater than that of IAA-glucose; and (c) that ester hydrolysis of standard compounds, IAA-glucose and IAA-p-nitrophenol, occurs in the ;three phase extraction system' proposed by Liu and Tillberg ([1983] Physiol Plant 57: 441-447). These data indicate that the potential for problems with inadvertent hydrolysis of ester conjugates of IAA exists even at moderate pH values and in the multiphase system where exposure to basic conditions was thought to be limited. PMID- 16667050 TI - Sequential Events in the Photoinhibition of Synechocystis under Sodium Stress. AB - Synechocystis (PCC 6714) is photoinhibited at low photon flux density when illuminated in medium devoid of Na(+). Photosynthetic activity and variable fluorescence yield were measured in intact cells during the course of inhibition and during recovery from inhibition in order to characterize sequential inhibitory events. Photosystem II antenna chlorophyll becomes functionally disconnected from the reaction center in the first stage of inhibition, although energy is still transferred from phycobilin pigments to the reaction center. This occurs simultaneously with, or subsequent to, the previously observed (J Zhao, JJ Brand [1988] Arch Biochem Biophys 264: 657-664) inactivation of O(2)-evolution units in Na(+)-stressed cells. Addition of Na(+) to the culture medium rapidly restores the O(2)-evolving system, but reassociation of photosystem II antenna chlorophyll is much slower and requires new protein synthesis. A site nearer to the reaction center of photosystem II becomes inactivated in a secondary inhibitory event. Recovery from secondary inhibition occurs slowly and requires protein synthesis. Cells recovering from secondary inhibition produce active photosystem II units with a fully functional complement of antenna chlorophyll. Synechocystis is photoinhibited in complete (Na(+)-containing) medium when illuminated at high photon flux density. These photoinhibited cells exhibit the same recovery behavior as cells at the second stage of inhibition in Na(+) deficient medium. PMID- 16667052 TI - Synthesis of Two Chromoplast-Specific Proteins During Fruit Development in Capsicum annuum. AB - The time-course of accumuiation of two membrane proteins during fruit ripening was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots in tissue extracts of Capsicum annuum L., vars Emerald Giant, Albino, and DNAP VS-12. The proteins, named ChrA and ChrB, were previously shown to occur specifically in chromoplasts. Fruit development was divided into five stages based on changes in color. ChrA was not detectable in the first three stages, but accumulated to a high level in the fully mature, red fruit. ChrB was not detectable in the first, mature-green stage of fruit maturation, but was found in the second stage, when carotenoid accumulation first appeared, and in all later stages. The patterns of accumulation in chromoplasts that develop from proplastids or leucoplasts are similar to those in chromoplasts that develop from chloroplasts. We conclude that ChrA and ChrB are probably synthesized de novo during chromoplast development. PMID- 16667051 TI - DNA Methylation is Reduced in DNasel-Sensitive Regions of Plant Chromatin. AB - Pea, barley, and corn chromatin was isolated and subjected to limited digestion with DNasel. The preferentially degraded, small molecular weight fraction, presumably consisting of sequences in an active state of expression, was isolated and its 5-methylcytosine content determined. The DNasel-sensitive chromatin fraction from all three plant species investigated contained a markedly reduced level of DNA methylation compared to total DNA. PMID- 16667053 TI - Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts. AB - Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O(2) evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O(2) evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O(2) was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO(2) photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO(2) fixation. PMID- 16667054 TI - Species and Environmental Variations in the Effect of Inorganic Phosphate on Sucrose-Phosphate Synthase Activity : Reliability of Assays Based Upon UDP Formation. AB - The effect of inorganic phosphate (Pi) on sucrose-phosphate synthase (SPS) activity was determined for the enzyme from five plant species (Nicotiana tabacum L., Spinacia oleracea L., Triticum aestivum L., Zea mays L., Glycine max L.) using two assay methods. The assay method based on determination of uridine diphosphate glucose- (UDPG) and fructose-6-phosphate-dependent sucrose formation was linear up to 15 minutes for all species tested. When assayed in this way, the effect of Pi at levels of 5 or 10 millimolar in the assay was variable, ranging from 0 to 35% inhibition of SPS activity. The assay method based on substrate dependent UDP formation was linear for some, but not for all of the species tested. Deviations from linearity were caused by loss of UDP from the assay medium. In some species, the extent of UDP loss was influenced by the level of Pi in the assay medium and, for at least one species (tobacco), it was influenced by the environment in which the plants were grown. The results indicated that (a) the role of Pi as an effector of SPS may vary depending on the species, and (b) the UDP assay method should be used with caution for assays of crude or desalted extracts, particularly when evaluating the effect of Pi on SPS activity. PMID- 16667055 TI - Production of an antibody specific for the propeptide of wheat germ agglutinin. AB - Wheat germ agglutinin (WGA) is synthesized as a proprotein with a glycosylated, 15 amino acid, carboxyl-terminal propeptide. This glycopeptide is cleaved from pro-WGA to produce the mature lectin during the transport of WGA to the protein bodies/vacuoles. To study the posttranslational modification of WGA, it would be useful to be able to differentiate between pro-WGA and mature WGA. Therefore, a peptide corresponding to the propeptide of WGA was synthesized (WGA-B 172-186), and an antiserum was raised in rabbits (anti-WGA-B 172-186). Anti-WGA-B 172-186 reacted with pure WGA-B 172-186 and pro-WGA in ELISA. Anti-WGA-B 172-186 was also specific for and readily differentiated between pro-WGA and mature WGA on Western blots. This provided an assay to monitor pro-WGA on Western blots before and after endo-beta-N-acetylglucosaminidase H digestion. Using this assay, direct evidence was obtained that the oligosaccharide of pro-WGA is of the high-mannose type. PMID- 16667056 TI - Inositol Trisphosphate Metabolism in Carrot (Daucus carota L.) Cells. AB - The metabolism of exogenously added d-myo-[1-(3)H]inositol 1,4,5-trisphosphate (IP(3)) has been examined in microsomal membrane and soluble fractions of carrot (Daucus carota L.) cells grown in suspension culture. When [(3)H]IP(3) was added to a microsomal membrane fraction, [(3)H]IP(2) was the primary metabolite consisting of approximately 83% of the total recovered [(3)H] by paper electrophoresis. [(3)H]IP was only 6% of the [(3)H] recovered, and 10% of the [(3)H]IP(3) was not further metabolized. In contrast, when [(3)H]IP(3) was added to the soluble fraction, approximately equal amounts of [(3)H]IP(2) and [(3)H]IP were recovered. Ca(2+) (100 micromolar) tended to enhance IP(3) dephosphorylation but inhibited the IP(2) dephosphorylation in the soluble fraction by about 20%. MoO(4) (2-) (1 millimolar) inhibited the dephosphorylation of IP(3) by the microsomal fraction and the dephosphorylation of IP(2) by the soluble fraction. MoO(4) (2-), however, did not inhibit the dephosphorylation of IP(3) by the soluble fraction. Li(+) (10 and 50 millimolar) had no effect on IP(3) metabolism in either the soluble or membrane fraction; however, Li(+) (50 millimolar) inhibited IP(2) dephosphorylation in the soluble fraction about 25%. PMID- 16667057 TI - Polypeptide composition and amino-terminal sequence of broad bean polyphenoloxidase. AB - Polyphenoloxidase was purified from broad bean (Vicia faba) leaves. The purified enzyme contained two immunocross-reactive proteins of approximately 60 to 65 and 43 to 45 kilodaltons. Further electrophoretic separation resolved these proteins into doublets with molecular mass of 61.5, 60, 44.5, and 43 kilodaltons, respectively. Each of the four polypeptides was transferred to Immobilon and subjected to microprotein sequencing. All the polypeptides showed the same amino acid sequence up to residue 9 but some variations occurred thereafter. The amino terminal sequence contained a large number of proline and serine residues. These results suggest that the four polypeptides were derived from a common parent form and that a posttranslational modification(s) must have occurred to account for the difference in their apparent size. PMID- 16667058 TI - Auxins induce alpha-amylase activity in pea cotyledons. AB - The report documents how the development of alpha-amylase activity in detached cotyledons of Pisum sativum cv Alaska is accelerated 2- to 12-fold during incubation with 1 micromolar to 10 micromolar 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, or with 4-chloroindoleacetic acid, an endogenous auxin from Pisum sativum. It seems probable that auxin from the embryonic axis induces alpha-amylase in the attached cotyledons during germination. PMID- 16667059 TI - Role of nonohmicity in the regulation of electron transport in plant mitochondria. AB - The relationship between the respiratory rate and the membrane ionic current on the protonmotive force has been investigated in percoll purified potato mitochondria. The dependence of the membrane ionic current on the membrane potential was monitored using a methyltriphenylphosphonium-sensitive electrode and determining the maximal net rate of depolarization following the addition of a respiratory inhibitor. We have confirmed that a nonohmic relationship exists between the ionic conductance and membrane potential. Addition of ATPase inhibitors markedly increased the initial rate of dissipation suggesting that in their absence the dissipation rate induced by respiratory inhibitors is partially offset by H(+)-efflux due to the hydrolysis of endogenous ATP. This was corroborated by direct measurement of endogenous ATP levels which decreased significantly following dissipation of the membrane potential. Results are discussed in terms of the regulation of electron transport in plant mitochondria in vivo. PMID- 16667060 TI - Salinity effects on photosynthesis in isolated mesophyll cells of cowpea leaves. AB - Mesophyll cells from leaves of cowpea (Vigna unquiculata [L.] Walp.) plants grown under saline conditions were isolated and used for the determination of photosynthetic CO(2) fixation. Maximal CO(2) fixation rate was obtained when the osmotic potential of both cell isolation and CO(2) fixation assay media were close to leaf osmotic potential, yielding a zero turgor pressure. Hypotonic and hypertonic media decreased the rate of photosynthesis regardless of the salinity level during plant growth. No decrease in photosynthesis was obtained for NaCl concentrations up to 87 moles per cubic meter in the plant growing media and only a 30% decrease was found at 130 moles per cubic meter when the osmotic potential of cell isolation and CO(2) fixation media were optimal. The inhibition was reversible when stress was relieved. At 173 moles per cubic meter NaCl, photosynthesis was severely and irreversibly inhibited. This inhibition was attributed to toxic effects caused by high Cl(-) and Na(+) accumulation in the leaves. Uptake of sorbitol by intact cells was insignificant, and therefore not associated with cell volume changes. The light response curve of cells from low salinity grown plants was similar to the controls. Cells from plants grown at 173 moles per cubic meter NaCl were light saturated at a lower radiant flux density than were cells from lower salinity levels. PMID- 16667061 TI - Polyamines as modulators of salt tolerance in rice cultivars. AB - The effect of NaCl on the endogenous levels of diamine, putrescine and polyamines, spermidine and spermine, was studied in the shoot system of salt tolerant and salt-sensitive lines of rice (Oryza sativa L.) cultivars during three growth stages. Salt stress increased the levels of diamine and polyamine in varying degrees among nine rice cultivars investigated. Salt tolerant AU1, Co43, and CSC1 were effective in maintaining high concentrations of spermidine and spermine, while the content of putrescine was not significantly altered in all the growth stages when plants were exposed to salinity. The salt sensitivity in rice was associated with excessive accumulation of putrescine and with low levels of spermidine and spermine in the shoot system of salt-sensitive cultivars Co36, CSC2, GR3, IR20, TKM4, and TKM9 under saline condition. One of the possible mechanisms of saline resistance was observed to be due to the highly increased polyamines against the low increase in diamines. Alternatively, the salt sensitivity could be due to high increase of diamines and an incapacity to maintain high levels of polyamines. PMID- 16667062 TI - Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO(2)-Requiring Phenotype : Evidence for a Central Role for Carboxysomes in the CO(2) Concentrating Mechanism. AB - Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (C(i)) and low C(i) grown cells leads the creation of a high C(i) requiring phenotype causing: (a) a dramatic increase in the K(0.5) (C(i)) for photosynthesis, (b) a loss of the ability to accumulate internal C(i), and (c) a decrease in the lag between the initial C(i) accumulation following illumination and the efflux of CO(2) from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO(2) concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and C(i) accumulation is developed in which the carboxysome plays a central role as both the site of CO(2) generation from HCO(3(-) ) and a resistance barrier to CO(2) efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells. PMID- 16667063 TI - Isolation and Characterization of High CO(2)-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942 : Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO(2) within the Carboxysome. AB - A total of 24 high CO(2)-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO(2), on agar media, but are incapable of growth at air levels of CO(2). All the mutants were able to accumulate inorganic carbon (C(i)) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO(2). On the basis of the rate of C(i) release following a light (5 minutes) --> dark transition two extreme phenotypes (fast and slow release mutants) and a number of ;intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast C(i) release, normal internal C(i) pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of (18)O from (18)O-labeled CO(2), 1% CO(2) requirement for growth in liquid media, normal affinity of carboxylase for CO(2), and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow C(i) release, increased internal C(i) pool, normal CA activity in crude extracts, normal internal (18)O exchange, a 3% CO(2) requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO(2), and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO(3) (-) to CO(2) inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H(+) import during photosynthesis. PMID- 16667064 TI - Reconstitution of Oxidative Phosphorylation and of Oligomycin-Sensitive ATPase by Five- and Six-Subunit Forms of Pea Mitochondrial F(1)-ATPase. AB - Five- and six-subunit forms of F(1)-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F(1)-ATPase were used to reconstitute oxidative phosphorylation in F(1)-depleted (ASU) as well as in F(1) and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six subunit F(1)-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP. PMID- 16667065 TI - Oxidation of Proline and Glutamate by Mitochondria of the Inflorescence of Voodoo Lily (Sauromatum guttatum). AB - In appendices of Sauromatum guttatum that are developing thermogenicity, mitochondria isolated from successive developmental stages of the inflorescence show an increase in the oxidation rates of proline and glutamate. A similar rise in the oxidation rates of these compounds is observed in mitochondria obtained from the spathe, a nonthermogenic organ of the inflorescence. Changes in oxidative metabolism were also observed in mitochondria isolated from sections of immature appendix treated with salicylic acid (SA) at 0.69 microgram per gram fresh weight indicating that they are induced by SA. At that concentration, however, SA has no effect on oxygen consumption by mitochondria in the presence of glutamate, proline, or malate. Furthermore, oxygen uptake by mitochondria in the presence of proline or glutamate is partially sensitive to salicylhydroxamic acid (SHAM) at concentrations greater than 2 millimolar when in the presence of 1 millimolar KCN. For NADH, succinate, and malate a high capacity of the alternative (cyanide-resistant) pathway is found that is completely sensitive to SHAM at 1.5 to 4 millimolar. The increase in the mitochondrial capacity to oxidize either amino acid is also found in four other Araceae species including both thermogenic and nonthermogenic ones. After anthesis, the rates of proline and glutamate oxidation decline. PMID- 16667066 TI - Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch. AB - We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, delta(18)O cellulose = 0.48 delta(18)O water + 24.1% per thousand. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of (18)O in the form of O(2) bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O(2) of normal (18)O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water. PMID- 16667067 TI - Light-induced spectral absorbance changes in relation to photosynthesis and the epoxidation state of xanthophyll cycle components in cotton leaves. AB - When cotton (Gossypium hirsutum L., cv Acaia SJC-1) leaves kept in weak light were suddenly exposed to strong red actinic light a spectral absorbance change took place having the following prominent characteristics. (a) It was irreversible within the first four minute period after darkening. (b) The difference in leaf absorbance between illuminated and predarkened leaves had a major peak at 505 nanometers, a minor peak at 465 nanometers, a shoulder around 515 nanometers, and minor troughs at 455 and 480 nanometers. (c) On the basis of its spectral and kinetic characteristics this absorbance change can be readily distinguished from the much faster electrochromic shift which has a peak at 515 nanometers, from the slow, so-called light-scattering change which has a broad peak centered around 535 nanometers and is reversed upon darkening, and from absorbance changes associated with light-induced chloroplast rearrangements. (d) The extent and time course of this absorbance change closely matched that of the deepoxidation of violaxanthin to zeaxanthin in the same leaves. (e) Both the absorbance change and the ability to form zeaxanthin were completely blocked in leaves to which dithiothreitol (DTT) had been provided through the cut petlole. DTT treatment also caused strong inhibition of that component of the 535 nanometer absorbance change which is reversed in less than 4 minutes upon darkening and considered to be caused by increased light scattering. Moreover, DTT inhibited a large part of nonphotochemical quenching of chlorophyll fluorescence in the presence of excessive light. However, DTT had no detectable effect on the photon yield of photosynthesis measured under strictly rate limiting photon flux densities or on the light-saturated photosynthetic capacity, at least in the short term. We conclude that it is possible to monitor light induced violaxanthin de-epoxidation in green intact leaves by measurement of the absorbance change at 505 nanometers. Determination of absorbance changes in conjunction with measurements of photosynthesis in the presence and absence of DTT provide a system well suited for future studies of meachanisms of dissipation of excessive excitation energy in intact leaves. PMID- 16667068 TI - The Heat Shock Response of Carrot : Protein Variations between Cultured Cell Lines. AB - We have defined several parameters surrounding the heat shock response of cultured cells of carrot (Daucus carota L.) and have found that these cells exhibit a typical "higher plant" heat shock response. In particular, the resolution of the heat shock proteins (hsps) by two-dimensional polyacrylamide gel electrophoresis (PAGE) has revealed a pattern of proteins very similar to the hsps from soybean; specifically, the low molecular weight class is composed of approximately 15 to 20 different polypeptides which likely represent different members of a small gene family. In addition, we have compared the (2-D) PAGE profiles of hsps isolated from several different cultured cell lines currently maintained in our laboratory and have found notable differences in the low molecular weight hsps between cell lines. Some of the differences appear to be quantitative, while others may be qualitative. Each of the cell lines was derived from a different seedling of the same seed stock of the same cultivar; thus, genetic differences should be minimized. In addition, two of the cell lines, which show clear differences, were initially derived from a single parental line, and thus arose from a single genetic stock. Possible explanations for the cell line differences observed here are either partial aneuploidy or modified gene regulation resulting from molecular changes during the time in culture (i.e. somaclonal variation). These observations serve to highlight the potential for variation that exists in cells in culture even for such a highly conserved response and gene set as the heat shock genes. PMID- 16667069 TI - Purification and Characterization of S-Adenosyl-l-methionine:6a-Hydroxymaackiain 3-O-Methyltransferase from Pisum sativum. AB - The isoflavonoid phytoalexin pisatin is synthesized by Pisum sativum in response to microbial infection and certain other forms of stress. An enzyme which synthesizes pisatin by methylating the 3-hydroxyl of (+)6a-hydroxymaackiain (HMK) was extracted from CuCl(2)-stressed pea seedlings. The enzyme was enriched 370 fold by (NH(4))(2)SO(4) precipitation, DEAE chromatography, chromatofocusing, and hydrophobic interaction chromatography (HIC), to a specific activity of 8.2 microkatals per gram protein. Enzyme activity profiles from chromatofocusing and HIC columns suggested the presence of two isozymes, of pl 5.2 and 4.9. Nondenaturing gel filtration of the HIC-purified enzyme gave a single peak of activity at the same elution volume as BSA (66 kilodaltons); the active fractions showed two proteins upon SDS-PAGE, of M(r) 66,000 and 43,000. The smaller protein was most abundant in chromatographic fractions containing peak enzyme activity throughout purification. In a partially purified preparation, this 43 kilodalton protein was the only one photoaffinity labelled by [(3)H]S-adenosyl-l-methionine. The purified enzyme preferred the (+) over the (-) stereoisomer of HMK and other pterocarpans; overall, (+)HMK was the best substrate. K(m) values were 2.3 micromolar for (+)HMK and 35 micromolar for S-adenosyl-l-methionine. The methyltransferase had a pH optimum of 7.9 and no apparent divalent cation requirement. PMID- 16667070 TI - Phospholipid-Dependence of Plant UDP-Glucose Sterol beta-d-Glucosyl Transferase : IV. Reconstitution into Small Unilamellar Vesicles. AB - The phospholipid dependence of the UDP-glucose sterol glucosyl transferase (UDPG SGTase) from maize coleoptiles was previously demonstrated using the partially purified and highly delipidated enzyme, in the presence of the detergent Triton X 100 (P Ullmann, P Bouvier-Nave, P Benveniste [1987] Plant Physiol 85: 51-55). We now report the reconstitution of the enzyme activity into unilamellar lipid vesicles. This was achieved by adding phospholipids, sterols and beta octylglucoside to the solubilized enzyme and passing the mixture through Sephadex G-50. The treatment led to almost complete removal of the detergents. The incorporation of UDPG-SGTase in the lipid vesicles was demonstrated by (a) coelution of the enzyme activity with the labeled lipid vesicles (average diameter: 260A) on a Sephacryl S-1000 column and (b) flotation experiments on metrizamide density gradients. Release of dithiobis-(2-nitro-benzoic acid) (DTNB) from DTNB-preloaded vesicles was very slow, indicating good membrane integrity of the vesicles. Treatment of the intact vesicles with the nonpermeant reagent p chloro-mercuribenzene sulfonate led to more than 95% inactivation of the total enzyme activity, i.e. the activity measured in the presence of Triton X-100 at permeabilizing concentration. This suggests an outward orientation for the active site of the enzyme. Finally, the enzyme was incorporated into vesicles of various phospholipid compositions and the kinetic parameters of the reactions were determined. Our results clearly show that the reconstituted UDPG-SGTase activity is stimulated to a large extent by negatively charged phospholipids. PMID- 16667071 TI - Cross-resistance to short residual sulfonylurea herbicides in transgenic tobacco plants. AB - Transgenic Nicotiana tabacum plants, produced by Agrobacterium tumefaciens mediated transformation with a mutant gene (csr1-1) coding for acetohydroxyacid synthase (AHAS) from a chlorsulfuron resistant Arabidopsis thaliana line GH50 (GW Haughn et al. [1988] Mol Gen Genet 211: 266-271; GW Haughn, C Somerville [1986] Mol Gen Genet 204: 430-434), were selected directly on 80 micrograms per liter (225 nanomolar) chlorsulfuron. The expression of csr-1 in two separate transgenic lines CHL-1 and CHL-2 was confirmed by biochemical and genetic analyses. The AHAS activity of GH50 and the equivalent component of AHAS activity in CHL-2 was resistant to three short residual sulfonylurea herbicides, DPX-M6316, DPX-A7881, and DPX-L5300, in addition to chlorsulfuron but not to the sulfonylurea CGA 131'036. Cross-resistance to the imidazolinones AC 263, 499, AC 252, 214, and AC 243,997 was not observed. Parallel observations were made on the inhibition of seedling growth in soil or on culture medium. The relevance of these findings for the application of transgenic plants in agriculture is discussed. PMID- 16667072 TI - Pyrroline-5-Carboxylate Reductase Is in Pea (Pisum sativum L.) Leaf Chloroplasts. AB - Proline accumulation is a well-known response to water deficits in leaves. The primary cause of accumulation is proline synthesis. Delta(1)-Pyrroline-5 carboxylate reductase (PCR) catalyzes the final reaction of proline synthesis. To determine the subcellular location of PCR, protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated by differential and Percoll density gradient centrifugation. PCR activity comigrated on the gradient with the activity of the chloroplast stromal marker NADPH-dependent triose phosphate dehydrogenase. We conclude that PCR is located in chloroplasts, and therefore that chloroplasts can synthesize proline. PCR activities from chloroplasts and etiolated shoots were compared. PCR activity from both extracts is stimulated at least twofold by 100 millimolar KCl or 10 millimolar MgCl(2). The pH profiles of PCR activity from both extracts reveal two separate optima at pH 6.5 and 7.5. Native isoelectric focusing gels of sampies from etiolated tissue reveal a single band of PCR activity with a pl of 7.8. PMID- 16667073 TI - Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata. AB - The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine --> ethanolamine - > phosphoethanolamine --> phosphatidylethanolamine. A possible alternative is: serine --> phosphatidylserine --> phosphatidylethanolamine --> ethanolamine --> phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO(2) from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives. PMID- 16667074 TI - Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy. AB - A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv "Sparkle") by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O(2) concentration can be calculated. With this method, we determined internal O(2) while measuring nitrogenase activity (C(2)H(2)) in detached pea nodules over a range of external O(2) concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O(2) concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C(2)H(2)) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors. PMID- 16667075 TI - Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules. AB - The interaction between carbon substrates and O(2) and their effects on nitrogenase activity (C(2)H(2)) were examined in detached nodules of pea (Pisum sativum L. cv "Sparkle"). The internal O(2) concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O(2) for 2 hours resulted in a 2- to 10-fold increase in internal O(2), and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O(2) was lowered. Nitrogenase activity was stimulated by succinate but only at high external O(2). Oxygen uptake increased linearly with external O(2) but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O(2) concentration within detached nodules. PMID- 16667076 TI - The occurrence of a ca-dependent period in the red light-induced late g1 phase of germinating adiantum spores. AB - The first cell cycle of Adiantum spores, which is induced by red light (R), was studied with regard to its temporal dependence on Pfr (the active form of phytochrome) and Ca(2+). Responses were quantified with increases of the spore content of DNA, thus limiting the investigation to the reactions taking place before the S phase of the cell cycle (i.e. during G0/G1/S transitions). Spores hydrated for more than 2 days in the standard medium (includes 1 millimolar free Ca(2+)) showed, after stimulation with a saturating R pulse, an increase of DNA beginning at about 25 hours and saturating at about 55 hours. Reversal by far-red light of the inductive effect of R was used to examine the temporal requirement for Pfr. Spores became dependent on the supply of external Ca(2+) when incubated in a low Ca medium (32 nanomolar free Ca(2+) with 10 millimolar EGTA); this culture condition was used, after observing that the DNA increase occurs similarly if Ca(2+) is supplied after the R pulse, to examine the temporal requirement for external Ca(2+). It was concluded that the G1 phase of the spore is separated into three subphases: (a) the Pfr-dependent period which immediately follows the R pulse and varies among individual spores from a few to about 25 hours, (b) the Ca(2+)-dependent period (about 10 hours) which occurs immediately before the S phase, and (c) a gap (15-20 hours) between the two periods. In the Ca(2+)-dependent period, spores require the presence of extracellular Ca(2+). This period occurs only during a determined time after the R pulse, and the competence of spores to enter the S phase is lost sharply if external Ca(2+) is not available continuously during this period. PMID- 16667077 TI - Seed Storage Protein Transcription and mRNA Levels in Brassica napus during Development and in Response to Exogenous Abscisic Acid. AB - Transcription rates and mRNA levels for Brassica napus seed storage protein families, cruciferin and napin, have been determined in embryos developing in the seed, as well as in embryos cultured with and without abscisic acid. Cruciferin and napin mRNAs are high during the cell expansion phase of embryo development, representing as much as 11 and 8%, respectively, of the total embryo mRNA. During the same time cruciferin and napin gene transcription rates, as measured in isolated nuclei, are also high. The data indicate that cruciferin mRNA is more stable than napin mRNA because while the napin transcription rate is higher than the cruciferin transcription rate, the cruciferin mRNA accumulates to higher levels. However, late in embryo development, both cruciferin and napin mRNAs seem to be less stable than earlier because comparable transcription rates result in lower mRNA levels. When embryos are cultured in the presence of abscisic acid, the levels of cruciferin and napin mRNAs are two- to threefold higher than in embryos cultured on basal medium. The transcription rates show a similar increase in the presence of abscisic acid, suggesting that abscisic acid is responsible for the increased mRNA level at least in part through an increase in the transcription rate of the two genes. PMID- 16667078 TI - Artefactual Origins of Cyclic AMP in Higher Plant Tissues. AB - A highly sensitive radioimmunoassay has been used to determine the levels of adenosine 3',5'-cyclic monophosphate (cAMP) in five higher plants (Lactuca sativa, Helianthus annuus, Oryza sativa, Pinus pinaster, Nicotiana tabacum). Particular attention was paid to the three main sources of errors in the characterization of cAMP in plants: presence of interfering substances in plant tissues; possible artefactual formation of cAMP from endogenous ATP during extraction, purification, and assay; and microbial origin of cAMP. In all the tested tissues, the cAMP level was below the detection limit of 0.5 picomole per gram fresh weight, a value much lower than those reported for similar materials of the same species in many previous studies. This result is not in favor of cAMP dependent regulations in higher plants. PMID- 16667079 TI - Distribution of Thylakoid Proteins between Stromal and Granal Lamellae in Spirodela : Dual Location of Photosystem II Components. AB - We have quantified the lateral distribution of 12 thylakoid proteins of Spirodela oligorrhiza by immunoblot analysis of detergent-derived granal and stromal lamellae. The immunological, ultrastructural, cytochemical, and biophysical measurements each indicated the expected overall separation of photosystem II (PSII) and photosystem I (PSI) components; however, certain proteins were not completely localized to one lamellar fraction. The apoproteins of the light harvesting chlorophyll a/b complex, subunit 1 of PSI and the components of the PSII reaction center (the 32 kilodalton, D2, and cytochrome b(559) proteins) were dually located between granal and stromal lamellae. Proteins associated exclusively with one of the membrane types were: in granal lamellae, the 43 and 51 kilodalton PSII proteins, and in stromal lamellae, the alpha and beta subunits of the proton ATPase. PMID- 16667080 TI - Molecular characterization of the major maize embryo globulin encoded by the glb1 gene. AB - One of the most abundant proteins in maize (Zea mays L.) embryos is the molecular weight 63,000 globulin encoded by the Glb1 gene. To obtain DNA clones corresponding to Glb1, a cDNA library corresponding to RNA from developing maize embryos was constructed in a lambda expression vector and screened with antibodies specific for Glb1-encoded proteins. Here we report the complete nucleotide sequence, as determined from two overlapping clones, of pcGlb 1S, a 2009 base pair clone containing the entire translated region of Glb1. The deduced amino acid sequence of pcGlb 1S shows similarities to 7S-type seed storage proteins of wheat and legumes. Southern blot analysis of maize DNA confirms previous genetic studies which had indicated the presence of a single copy of Glb1 per haploid genome. Northern blot analysis indicates that Glb1 transcripts are present throughout most of embryo development and that expression of this gene is limited to seed tissues. Embryos homozygous for a Glb1 null allele, in which Glb1-encoded proteins are not detectable, contain low levels of Glb1 transcripts which are a different size from those encoded by functional alleles. This suggests that the defect in the null allele is at the level of gene transcription or RNA processing. PMID- 16667081 TI - Increased 8-hydroxyguanine content of chloroplast DNA from ozone-treated plants. AB - The mechanism of ozone-mediated plant injury is not known but has been postulated to involve oxygen free radicals. Hydroxyl free radicals react with DNA causing formation of many products, one of which is 8-hydroxyguanine. By using high performance liquid chromatography with electrochemical detection, the 8-hydroxy 2'-deoxyguanosine (8-OHdG) content of a DNA enzymatic digest can be sensitively quantitated. Beans (Phaseolus vulgaris L.) and peas (Pisum sativum L.) were treated with an ozone regime that caused acute injury. Chloroplast DNA was obtained from plants harvested either immediately after ozone treatment or 24 hours later. Ozone-exposed plants in general had nearly two-fold higher levels of 8-OHdG as compared to control plants. In vitro treatment of DNA in buffer solution with ozone did not cause formation of 8-OHdG in DNA, even though ozone did react directly with the macromolecule per se. Exposure of isolated, illuminated chloroplasts to ozone caused nearly a seven-fold increase in the amount of 8-OHdG in the chloroplast DNA as compared to none-ozone-exposed chloroplasts. These results suggest that ozone exposure to plants causes formation of enhanced levels of oxygen free radicals, thus mediating formation of 8-OHdG in chloroplast DNA. The reaction of ozone with DNA per se did not cause formation of 8-OHdG. Therefore, it is the interaction of ozone with plant cells and isolated chloroplasts which mediates oxygen free radical formation. PMID- 16667082 TI - Relationship between Gene Dosage and Gene Expression in the Chloroplast of Chlamydomonas reinhardtii. AB - Expression of three chloroplast genes encoding proteins of different chloroplast complexes and the rRNA gene has been examined in cells having reduced numbers of chloroplast genomes as a result of growth in the presence of the thymidine analog 5-fluorodeoxyuridine. While accumulation of total mRNA for rpl2 (ribosomal protein L-1), rbcL (ribulose bisphosphate carboxylase large subunit) and atpA (alpha-subunit of ATP synthase) declined with gene copy number, the levels of translatable mRNA and rates of synthesis of these three proteins were largely unaffected. Accumulation of rRNA declined less precipitously than mRNA levels for the three proteins in response to the reduction in chloroplast genome number. Chlamydomonas appears to compensate for reductions in the number of chloroplast genomes at several different levels. Populations of cells with only one-fourth the wild-type amount of chloroplast DNA per cell on average have half the normal level of chloroplast ribosomes and nearly normal rates of CO(2) fixation and levels of specific chloroplast encoded proteins. These results suggest that normal cells accumulate a large excess of transcripts for chloroplast genes and that levels of expression of these genes are regulated by posttranscriptional mechanisms. PMID- 16667083 TI - Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis. AB - It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K(m) [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought. PMID- 16667084 TI - Hydrogen inhibition of nitrogen reduction by nitrogenase in isolated soybean nodule bacteroids. AB - Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N(2) reduction by nitrogenase. To evaluate the significance of H(2) inhibition in vivo, we have measured the apparent inhibition constant for H(2) inhibition of N(2) reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N(2) reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O(2) and various concentrations of N(2) and H(2). The apparent inhibition constant for H(2) under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H(2) inhibition of N(2) reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules. PMID- 16667085 TI - Ethylene-like activity of isocyanides. AB - Benzyl isocyanide, cyclohexyl isocyanide, benzyl isocyanate, methyl isocyanate, benzyl isothiocyanate, and methyl isothiocyanate were each tested for ethylene like activity in a pea (Pisum sativum) growth assay. Only the isocyanides gave an ethylene-like response; the concentration that gave a half-maximal response was approximately 10 to 15 microliters per liter for each isocyanide, and this activity was inhibited by norbornadiene, a competitive inhibitor of ethylene action. Since the isocyanides did not promote endogenous ethylene production, it was concluded that the isocyanides acted directly to give an ethylene-like response. The isocyanides were further shown to elicit ethylene-like activity in a potato (Solanum tuberosum) tuber respiration assay, in a carnation (Dianthus caryophyllus) senescence assay and in a carrot (Daucus carota L.) isocoumarin formation assay. The feasibility of employing azido derivatives of benzyl isocyanide to photoaffinity label ethylene receptors in vivo is discussed. PMID- 16667086 TI - Coated Vesicles Are Involved in the Transport of Storage Proteins during Seed Development in Pisum sativum L. AB - During seed development, various storage proteins and hydrolases accumulate in specialized storage vacuoles, the protein bodies, via an elaborate intracellular transport system involving the rough endoplasmic reticulum, the Golgi apparatus, and transit vesicles. Clathrin-coated vesicles, similar to those which transport lysosomal proteins to lysosomes, an organelle analogous to the vacuole, in animal cells, could be involved in this intracellular transport mechanism. Clathrin coated vesicles have been isolated from cotyledons of developing pea (Pisum sativum L.) seeds at the time of rapid protein accumulation and analyzed for the presence of protein body constitutents. A 23,000 M(r) polypeptide, corresponding to pea lectin precursor, was found associated with the vesicles, as determined by immunoblotting. The lectin precursor was apparently sequestered within the vesicles, as the polypeptide was only susceptible to proteolysis if detergents were included in the digestion buffer. A number of glycosidase activities, including alpha-mannosidase, alpha-galactosidase, and beta-N acetylhexosaminidase, were also associated with the vesicles. Thus, it appears that clathrin-coated vesicles are involved in the intracellular transport of storage proteins during seed development. PMID- 16667087 TI - Stromal Phosphate Concentration Is Low during Feedback Limited Photosynthesis. AB - It has been hypothesized that photosynthesis can be feedback limited when the phosphate concentration cannot be both low enough to allow starch and sucrose synthesis at the required rate and high enough for ATP synthesis at the required rate. We have measured the concentration of phosphate in the stroma and cytosol of leaves held under feedback conditions. We used non-aqueous fractionation techniques with freeze-clamped leaves of Phaseolus vulgaris plants grown on reduced phosphate nutrition. Feedback was induced by holding leaves in low O(2) or high CO(2) partial pressure. We found 7 millimolar phosphate in the stroma of leaves in normal oxygen but just 2.7 millimolar phosphate in leaves held in low oxygen. Because 1 to 2 millimolar phosphate in the stroma may be metabolically inactive, we estimate that in low oxygen, the metabolically active pool of phosphate is between negligible and 1.7 millimolar. We conclude that halfway between these extremes, 0.85 millimolar is a good estimate of the phosphate concentration in the stroma of feedback-limited leaves and that the true concentration could be even lower. The stromal phosphate concentration was also low when leaves were held in high CO(2), which also induces feedback-limited photosynthesis, indicating that the effect is related to feedback limitation, not to low oxygen per se. We conclude that the concentration of phosphate in the stroma is usually in excess and that it is sequestered to regulate photosynthesis, especially starch synthesis. The capacity for this regulation is limited by the coupling factor requirement for phosphate. PMID- 16667088 TI - Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine. AB - Previous researchers found that formation and function of nitrogen-fixing nodules on legume roots were severely inhibited by addition of exogenous ethylene. Nodule formation by Rhizobium meliloti on Medicago sativa was stimulated twofold when the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) was added with the inoculum. Stimulation of nodule formation by AVG showed a similar concentration dependence as inhibition of ethylene biosynthesis, suggesting that the primary action of AVG is the inhibition of ethylene biosynthesis. When AVG was added 2 to 3 days after inoculation, the number of nodules formed was still increased. On a per plant basis, however, the average nitrogen fixation was unchanged by AVG treatment and was independent of nodule number. PMID- 16667089 TI - Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants. AB - An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO(3) was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed. PMID- 16667090 TI - Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum. AB - In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or "twin" GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the "twin" GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD(-) strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development. PMID- 16667091 TI - Properties of a polycation-stimulated protein kinase associated with purified Avena phytochrome. AB - ATP-dependent polycation-stimulated phosphorylation of highly purified phytochrome preparations from etiolated Avena seedlings has been reported previously (Y-S Wong, H-C Cheng, DA Walsh, JC Lagarias [1986] J Biol Chem 261: 12089-12097). In this study, we present a more detailed description of the properties of this protein kinase based on the analysis of over 30 different Avena phytochrome preparations. ATP-dependent phosphorylation of phytochrome was strongly stimulated by a wide range of polycationic molecules, including synthetic and natural polypeptides as well as nonpeptide cationic polymers. Many of the compounds known to stimulate other known protein kinases (i.e., cyclic nucleotides, Ca(2+), calmodulin, diacylglycerol, phospholipids) were either inhibitory or nonstimulatory. Among the polycations, histone H1, polylysine, and polybrene were the most effective, giving average stimulations of four- to sevenfold. Polycation-stimulated protein phosphorylation was inhibited by elevated ionic strength; of the salts examined, magnesium pyrophosphate was a particularly potent inhibitor of the kinase activity. MgATP was preferred as the phosphoryl donor to either MgGTP or magnesium pyrophosphate. The K(m) for MgATP was estimated to be 30 micromolar when histone H1 was used as a protein substrate. The Pr form of phytochrome was always a better substrate than the Pfr form regardless of the polycation present. Polylysine-stimulated, phytochrome(preparation)-dependent phosphorylation of purified maize phosphoenolpyruvate carboxylase was observed, as well as phosphorylation of a number of polypeptides in crude soluble protein extracts from etiolated Avena seedlings. PMID- 16667092 TI - Axial and Radial Hydraulic Resistance to Roots of Maize (Zea mays L.). AB - A root pressure probe was employed to measure hydraulic properties of primary roots of maize (Zea mays L.). The hydraulic conductivity (Lp(r)) of intact root segments was determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. In hydrostatic experiments, Lp(r) was constant along the segment except for an apical zone of approximately 20 millimeters in length which was hydraulically isolated due to a high axial resistance. In osmotic experiments, Lp(r) decreased toward the base of the roots. Lp(r) (osmotic) was significantly smaller than Lp(r) (hydrostatic). At various distances from the root tip, the axial hydraulic resistance per unit root length (R(x)) was measured either by perfusing excised root segments or was estimated according to Poiseuille's law from cross-sections. The calculated R(x) was smaller than the measured R(x) by a factor of 2 to 5. Axial resistance varied with the distance from the apex due to the differentiation of early metaxylem vessels. Except for the apical 20 millimeters, radial water movement was limiting water uptake into the root. This is important for the evaluation of Lp(r) of roots from root pressure relaxations. Stationary water uptake into the roots was modeled using measured values of axial and radial hydraulic resistances in order to work out profiles of axial water flow and xylem water potentials. PMID- 16667093 TI - A new form of chlorophyll C involved in light-harvesting. AB - A new form of chlorophyll c has been isolated from the pyrmnesiophyte Pavlova gyrans Butcher. This pigment is spectrally similar to chlorophyll c(2), but all the absorption maxima (454, 583, and 630 nm in diethyl ether) are shifted 4 to 6 nanometers to longer wavelengths. The new pigment can be separated from other chlorophyll c-type pigments by reversed-phase high performance liquid chromatography and thin layer chromatography. Both chlorophylls c(1) and c(2) are found with the new chlorophyll c pigment in P. gyrans, and it has also been detected in the chrysophyte Synura petersenii Korsh. The light-harvesting function of the new chlorophyll c pigment is indicated by its presence along with chlorophyll c(1) and c(2) in a light-harvesting pigment-protein complex isolated from P. gyrans in which chlorophyll c pigments efficiently transfer absorbed light energy to chlorophyll a. PMID- 16667094 TI - Kinetin Enhanced 1-Aminocyclopropane-1-Carboxylic Acid Utilization during Alleviation of High Temperatures Stress in Lettuce Seeds. AB - The thermoinhibition at 35 and 32 degrees C of pregermination ethylene production and germination in lettuce (Lactuca sativa L. cv Mesa 659) seeds was synergistically or additively alleviated by 0.05 millimolar kinetin (KIN) and 10 millimolar 1-aminocyclopropane-1-carboxylic acid (ACC). The synergistic effect of KIN + ACC on ethylene production and germination at 35 degrees C was inhibited by Co(2+) (44-46%) but not by aminoethoxyvinyl glycine (AVG). The uptake of ACC by the seed was not influenced by KIN. Upon slitting of the seed coats (composed of pericarp, testa and endosperm), following the uptake of chemicals, ACC was readily converted into ethylene at all temperatures, and the synergistic effects of KIN + ACC at 35 degrees C were lost. At 35 degrees C, KIN acted synergistically with ACC or ethephon (ETH) in alleviating the osmotic restraint. At 25 degrees C, ETH was more active than KIN or KIN + ACC in overcoming the osmotic restraint. Thus, the integrity of the seed coats, the KIN-enhanced ACC utilization, and an interaction of KIN with the ethylene produced may be the basis for the synergistic or additive effects of KIN + ACC at high temperature. PMID- 16667095 TI - Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N Limited Green Alga Selenastrum minutum. AB - In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH(4) (+) addition we found that: (a) N accumulated in glutamine while glutamate and alpha-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH(4) (+) addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru 1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH(4) (+) addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme. PMID- 16667096 TI - Diclofop-methyl increases the proton permeability of isolated oat-root tonoplast. AB - Diclofop-methyl (methyl ester of 2-[4-(2',4'-dichlorophenoxy)phenoxy]propionate; 100 micromolar) and diclofop (100 micromolar) inhibited both ATP- and PPi dependent formation of H(+) gradients by tonoplast vesicles isolated from oat (Avena sativa L., cv Dal) roots. Diclofop-methyl (1 micromolar) significantly reduced the steady-state H(+) gradient generated in the presence of ATP. The ester (diclofop-methyl) was more inhibitory than the free acid (diclofop) at pH 7.4, but this relative activity was reversed at pH 5.7. Neither compound affected the rate of ATP or PPi hydrolysis by the proton-pumping enzymes. Diclofop-methyl (50, 100 micromolar), but not diclofop (100 micromolar), accelerated the decay of nonmetabolic H(+) gradients established across vesicle membranes. Diclofop-methyl (100 micromolar) did not collapse K(+) gradients across vesicle membranes. Both the (+)- and (-)-enantiomers of diclofop-methyl dissipated nonmetabolic H(+) gradients established across vesicle membranes. Diclofop-methyl, but not diclofop (each 100 micromolar), accelerated the decay of H(+) gradients imposed across liposomal membranes. These results show that diclofop-methyl causes a specific increase in the H(+) permeability of tonoplast. PMID- 16667097 TI - Osmotic Pressure of Aqueous Polyethylene Glycols : Relationship between Molecular Weight and Vapor Pressure Deficit. AB - Osmotic pressures (II) of aqueous solutions of polyethylene glycols (PEGs) of average relative molecular weight (M(r)) between 200 and 10,000 were measured using vapor pressure deficit osmometry. The relationships between molarity and II were described with high precision by second order polynomials for each of the PEGs studied. In contrast to previous reports, equivalent weights of different polymers in solution did not generate the same II; low M(r) PEGs generated a higher II than the higher M(r) PEGs. The effect of PEGs upon II represents an interaction between concentration and M(r). PMID- 16667098 TI - Functional Analysis of the Photosynthetic Apparatus of Prochlorothrix hollandica (Prochlorales), a Chlorophyll b Containing Procaryote. AB - Light-shade adaptation of the chlorophyll a/b containing procaryote Prochlorothrix hollandica was studied in semicontinuous cultures adapted to 8, 80 and 200 mumole quanta per square meter per second. Chlorophyll a contents based on dry weight differed by a factor of 6 and chlorophyll b by a factor of 2.5 between the two extreme light conditions. Light utilization efficiencies determined from photosynthesis response curves were found to decrease in low light grown cultures due to lower light harvesting efficiencies; quantum requirements were constant at limiting and saturating irradiances for growth. At saturating growth irradiances, changes in light saturated oxygen evolution rate originated from changes in chlorophyll a antenna relative to the number of reaction centers II. At light-limiting conditions both the number of reaction centers II and the antenna size changed. The amount of chlorophyll b relative to reaction center II remained constant. As in cyanobacteria, the ratio of reaction center I to reaction center II was modulated during light-shade adaptation. On the other hand, time constants for photosynthetic electron transport (4 milliseconds) were low as observed in green algae and diatoms. The occurrence of state one to two and state two to one transitions is reported here. Another feature linking photosynthetic electron transport in P. hollandica to that in the eucaryotic photosynthetic apparatus was blockage of the state one to two transition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Although chlorophyll b was reported in association with photosystem I, the 630 nanometer light effect does not exclude that chlorophyll b is the photoreceptor for the state one to two transition. PMID- 16667099 TI - Free and conjugated indole-3-acetic Acid in developing bean seeds. AB - The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed. PMID- 16667100 TI - Relationships between Cadmium, Zinc, Cd-Peptide, and Organic Acid in Tobacco Suspension Cells. AB - Responses of tobacco (Nicotiana tabacum) suspension cells to Cd and Zn were studied in the presence and absence of ligand of Cd-peptide in order to understand the role of this peptide versus other mechanisms in Cd and Zn accumulation and accommodation in plants. With 45 micromolar Cd and 300 micromolar Zn (non-growth-inhibiting levels), metals appeared rapidly within cells, and intracellular Cd and Zn reached medium concentrations after 6 to 10 hours. Cd-peptide was observed in response to Cd after 2 hours, but this form only accounted for approximately 30% of soluble Cd after 24 hours. Peptide was not observed in cells exposed to 300 micromolar Zn for up to 7 days. Organic acid to-metal stoichiometry indicated that endogenous organic acid content of cells was more than sufficient to complex absorbed metals and no evidence was found for stimulation of organic acid biosynthesis by Cd or Zn. Metal-complexing potential of organic acids for Cd and Zn versus endogenous cations is discussed as is vacuolar-extravacuolar distribution of metals. The absence of Cd-peptide does not limit Cd-accumulation in the system studied. Results suggest that tobacco suspension cells accommodate the presence of non-growth-inhibiting and growth inhibiting levels of Cd and Zn by sequestration in the vacuole as complexes with endogenous organic acids and that this may be a principal means for accommodation of Cd as well as Zn in the presence and absence of Cd-peptide. PMID- 16667101 TI - Selection and Characterization of Dunaliella salina Mutants Defective in Haloadaptation. AB - A technique for selection of Dunaliella mutants defective in their capacity to recover from osmotic shocks has been developed. The selection is based on physical separation of mutants on density gradients. This technique takes advantage of the fact that Dunaliella cells, when exposed to osmotic shocks, initially change volume and density due to water gain or loss and subsequently recover their volume and density by readjusting their intracellular glycerol. Eight mutants that do not recover their original density following hyperosmotic shocks have been isolated. The mutants grow similar to wild type cells in 1 molar NaCl, and recover like the wild type from hypotonic shocks but are defective in recovering from hypertonic shocks. A partial characterization of one of the mutants is described. PMID- 16667102 TI - An abundant, highly conserved tonoplast protein in seeds. AB - We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M(r) 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds. PMID- 16667103 TI - Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots. AB - Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity. PMID- 16667104 TI - Inheritance and expression of the mouse metallothionein gene in tobacco: impact on cd tolerance and tissue cd distribution in seedlings. AB - Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content. PMID- 16667105 TI - Effects of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea on the Cell Cycle in Euglena gracilis. AB - The cell cycle of the photosynthetic unicellular alga Euglena gracilis growing in phototrophic medium is regulated by light. To investigate the relationship of this cell cycle response to light stimulated photosynthesis, we have tested the effect of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on Euglena cell cycle transit. While DCMU does not block light stimulated cells from entering the S phase of the cell cycle, it does inhibit the transit through G(2)/M. The specificity of this response and its relationship to photosynthesis was studied by looking at the effect of DCMU on dark grown wild type cells, and on two bleached variants of Euglena (W(3)BUL and W(10)BSmL) that lack chloroplasts. The drug does block G(2)/M in these cells, but not entrance into the cell cycle. Our studies show that entrance of cells into the cell cycle from a quiescent state does not require active photosynthesis, and that DCMU has effects on G(2)/M transit that are independent of the photosynthetic capacity of the cells. PMID- 16667106 TI - Blue-Light Regulation of Specific Transcript Levels in Pisum sativum: Fluence Response, Time-Course, and Reciprocity Characteristics. AB - The expression of many nuclear genes in plants is light regulated. We have examined the fluence-response, time-course, and reciprocity characteristics of four nuclear, blue-light-regulated transcripts in Pisum sativum L. var Alaska: Cab RNA, pEA207 RNA, pEA215 RNA, and pEA25 RNA. To avoid complications due to blue-light-induced transformation of phytochrome, we have adapted the procedure of measuring blue-light-induced changes in steady-state-RNA levels in seedlings grown in continuous red light. The fluence-response curves for Cab and pEA215 RNA are bell-shaped, with peak accumulations at 10(2) and 10(1) micromoles per square meter, respectively. No response is observed at 10(4) micromoles per square meter. pEA25 RNA has threshold and saturation fluences below 10(-1) micromoles per square meter. pEA207 RNA has a threshold at 10(2) micromoles per square meter and saturates above 10(4) micromoles per square meter. pEA215 and Cab RNA start to increase within 1 hour after the 10(1) micromoles per square meter pulse, and finish accumulation by 5 hours. The decrease in pEA207 RNA in response to 10(4) micromoles per square meter and pEA25 RNA in response to 10(1) or 10(4) micromoles per square meter starts between 3 and 5 hours after the blue-light pulse. The Bunsen-Roscoe Law of Reciprocity is followed in the Cab, pEE215, pEA25, and pEA207 RNA responses to 10(1) and 10(4) micromoles per square meter. PMID- 16667107 TI - Inactivation of 1-Aminocyclopropane-1-Carboxylate Synthase by l-Vinylglycine as Related to the Mechanism-Based Inactivation of the Enzyme by S-Adenosyl-l Methionine. AB - The pyridoxal phosphate-dependent 1-aminocyclopropane-1-carboxylate (ACC) synthase catalyzes the conversion of S-adenosyl-l-methionine (AdoMet) to ACC, and is inactivated by AdoMet during the reaction. l-Vinylglycine was found to be a competitive inhibitor of the enzyme, and to cause a time-dependent inactivation of the enzyme. The inactivation required the presence of pyridoxal phosphate and followed pseudo-first-order kinetics at various concentrations of l-vinylglycine. The Michaelis constant for l-vinylglycine in the inactivation reaction (K(inact)) was 3.3 millimolar and the maximum rate constant (k(max)) was 0.1 per minute. These findings, coupled with the previous observations that the suicidal action of AdoMet involved a covalent linkage of the aminobutyrate portion of AdoMet to the enzyme, support the view that the mechanism-based inactivation of ACC synthase by the substrate AdoMet proceeds through the formation of a vinylglycine ACC synthase complex as an intermediate. PMID- 16667108 TI - Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation. AB - When grown under defined conditions Dunaliella bardawil accumulates a high concentration of beta-carotene, which is composed primarily of two isomers, all trans and 9-cis beta-carotene. The high beta-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low beta-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating beta-carotene. Protection against photoinhibition in the high beta-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated beta-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis beta carotene, photodestruction of all-trans beta-carotene, photodestruction of chlorophyll and cell death. PMID- 16667109 TI - Glycolaldehyde Inhibits CO(2) Fixation in the Cyanobacterium Synechococcus UTEX 625 without Inhibiting the Accumulation of Inorganic Carbon or the Associated Quenching of Chlorophyll a Fluorescence. AB - When studying active CO(2) and HCO(3) (-) transport by cyanobacteria, it is often useful to be able to inhibit concomitant CO(2) fixation. We have found that glycolaldehyde was an efficient inhibitor of photosynthetic CO(2) fixation in Synechococcus UTEX 625. Glycolaldehyde did not inhibit inorganic carbon accumulation due to either active CO(2) or HCO(3) (-) transport. When glycolaldehyde (10 millimolar) was added to rapidly photosynthesizing cells, CO(2) fixation was stopped within 15 seconds. The quenching of chlorophyll a fluorescence remained high ( EDTA > CaSO(4) = ScCl(3). In all cultivars, 30 minutes of desorption with citric acid depleted the rapidly exchanging, putative apoplasmic compartment, although some tightly bound Al remained in that compartment. The relationship between Al remaining after desorption and time in the uptake medium was nearly linear and no distinction was observed between Al-tolerant and Al-sensitive cultivars. However, uptake of Al by the Al-tolerant cultivars was increased by treatment with the protonophore 2,4-dinitrophenol (DNP), while uptake of Al by Al-sensitive cultivars was relatively unaffected. Such results suggest the possible involvement of an active exclusion mechanism in Al-tolerant cultivars of T. aestivum. PMID- 16667118 TI - Kinetics of Chlorophyll Accumulation and Formation of Chlorophyll-Protein Complexes during Greening of Chlamydomonas reinhardtii y-1 at 38 degrees C. AB - The initial kinetics of accumulation of chlorophylls (Chl) were analyzed during optimal greening of Chlamydomonas reinhardtii y-1 at 38 degrees C. Acetate was required for maximal synthesis of Chl, which occurred at a linear rate when degreened cells were exposed to light. During the first hour Chl a and b accumulated predominantly as geranylgeraniol esters, with lesser amounts of the species with more reduced alcohol side chains. When Chl synthesis was blocked either by treatment with gabaculine or by transfer to the dark, the distribution shifted to the more reduced forms. Similar kinetic patterns indicated that a common pool of chlorophyllides a and b provided substrate for the enzymatic system that performs esterification and reduction of the sldechain for each group of Chl. Chl b was essentially quantitatively integrated into light-harvesting complexes as indicated by energy transfer to Chl a. In the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis, Chl b did not accumulate and Chl a production was reduced about one-half. The results demonstrate that Chl a/b-protein complexes assemble rapidly during greening and that reduction of the alcohol side chain of the Chl is not required for assembly of these complexes. PMID- 16667119 TI - Characteristics of the Mg-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor. AB - The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg(2+)-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na(2)SO(3)-dependent Mg(2+)-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg(2+)-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg(2+)-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg(2+)-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t((1/2)) of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one. PMID- 16667120 TI - Development of two isogenic sweet corn hybrids differing for glycinebetaine content. AB - A hybrid of sweet corn, Zea mays L. (;1720'; Rogers Brothers Seed Co.), was found to be comprised of glycinebetaine-positive and glycinebetaine-deficient individuals in a 1:1 mixture. This phenomenon was traced to segregation for a single, nuclear, dominant gene determining leaf glycinebetaine content within the female inbred parent of this hybrid. Selection for homozygous recessive (glycinebetaine-deficient) and homozygous dominant (glycinebetaine-positive) genotypes of the female inbred parent enabled production of two isogenic versions of hybrid ;1720' differing with respect to a single copy of the dominant allele, by mating these female parent selections with the common homozygous recessive (glycinebetaine-deficient) male parent. These two isogenic hybrids are shown to differ by a factor of 300- to 400-fold in glycinebetaine titer of young expanding leaves of salinized plants, but exhibit no striking differences in the levels of free amino acids or the level of N-methylnicotinic acid (nicotinic acid betaine; trigonelline). The only significant difference between the two hybrids in terms of amino acid composition was found to be in the level of alanine under nonsalinized conditions. The betaine-deficient hybrid exhibited a 14% lower alanine level than the betaine-positive hybrid. Betaine deficiency was not associated with altered stress-induced accumulation of amino acids such as proline, serine, and asparagine plus aspartate, attesting to the high specificity of the genetic difference between these isogenic hybrids with respect to betaine accumulation. This germplasm offers unique opportunities to test whether a single dominant allele determining stress-induced betaine accumulation capacity influences stress resistance in maize. PMID- 16667121 TI - Genotypic Variation for Glycinebetaine among Public Inbreds of Maize. AB - Screening of a range of public maize (Zea mays L.) inbred lines for glycinebetaine (betaine) content over two growing seasons (1987 and 1988), using fast atom bombardment mass spectrometry, has identified 19 public inbred lines which all exhibit low betaine levels (<100 nanomoles per gram fresh weight). These include common inbreds such as A188, A619, B37, H95, N6, and Oh43. Several inbreds exhibit high betaine levels (3000 to 10000 nanomoles per gram fresh weight); in these strongly betaine-positive inbreds, betaine levels tended to be, on average, 1.38-fold greater in the 1988 growing season presumably in part due to field water deficits experienced during the drought of 1988. Where several different sources of the same inbred line were available (including cytoplasmic male sterile and restored lines of A632, B37, B73, Oh43, and WF9), betaine levels were found to be similar when the inbreds were tested in the same environment. Because W22-R/r-X1 was found to be strongly betaine-positive, it should be possible to map the putative recessive gene(s) determining betaine deficiency to specific chromosome(s) from monosomics resulting from crosses between W22-R/r-X1 and betaine-deficient lines. PMID- 16667122 TI - Activity of Ageing Carnation Flower Parts and the Effects of 1 (Malonylamino)cyclopropane-1-Carboxylic Acid-Induced Ethylene. AB - Peak levels of 1-aminocyclopropane-l-carboxylic acid (ACC) in flower parts of ageing carnations (Dianthus caryophyllus L. cv Scanea 3C) were detected 6 to 9 days after flower opening. The ethylene climacteric and the first visible sign of wilting was observed 7 days after opening. The concentration of conjugated ACC in these same tissues peaked at day three with reduction of 70% by day 4. From day 5 to day 9 all parts followed a diurnal pattern of increasing in conjugate levels 1 day and decreasing the next. Concentrations of conjugated ACC were significantly higher than those of ACC in all ageing parts. Preclimacteric petals treated with ACC or 1-(malonylamino)-cycloprane-1-carboxylic acid (MACC), started to senesce 30 to 36 hours after treatment. When petals were treated with MACC plus by 0.1 millimolar aminoethyoxyvinylglycine, premature senescence was induced, while ethylene production was suppressed relative to MACC-treated petals. Petals treated with MACC and silver complex produced ethylene, but did not senesce. The MACC-induced ethylene was inhibited by the addition of 1.0 millimolar CoC1(2). These results demonstrate MACC-induced senescence in preclimacteric petals. The patterns of ACC and MACC detected in the flower parts support the view that an individual part probably does not export an ethylene precursor to the remainder of the flower inducing senescence. PMID- 16667123 TI - Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves. AB - The freezing tolerance and incidence of two forms of freezing injury (expansion induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT(50) (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from -5 degrees C to -25 degrees C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT(50). However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10 degrees C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane. PMID- 16667124 TI - Interactions among Flavonoid nod Gene Inducers Released from Alfalfa Seeds and Roots. AB - Alfalfa (Medicago sativa L.) seeds and roots can create complex rhizosphere effects by releasing flavonoids that induce nodulation (nod) genes in Rhizobium meliloti. Previous reports identified luteolin and 4,4'-dihydroxy-2' methoxychalcone as strong inducers that are released from seeds and roots, respectively, and 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone as weaker inducers which are exuded by roots. As a first step toward identifying flavonoid interactions that may occur in the rhizosphere, combinations of these molecules were tested for transcriptional effects on a nodABC-lacZ fusion in R. meliloti. At low concentrations (e.g. 8.4 nanomolar), interactions of the three nod gene inducers from root exudate were additive. When the strong inducers 4,4'-dihydroxy 2'-methoxychalcone and luteolin were present separately at higher concentrations (e.g. 21 nanomolar), their effect could be decreased significantly by the weaker inducers 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone. In contrast, when low concentrations of luteolin from seed rinses and 4,4'-dihydroxy-2'-methoxychalcone from root exudate were present together, they produced synergistic increases in nod gene transcription. Tests with mixtures of the three nod gene inducers from root exudate indicated that alfalfa seedlings might easily decrease the strong inductive effect of the chalcone by releasing modest amounts of the weaker inducers. In addition, mixtures of luteolin and the nod gene inducers in root exudate suggested that interactions between nod gene inducers from seeds and roots may create a zone highly favorable to root nodule formation near the top of the primary root. PMID- 16667125 TI - Effects of Polyploidy on Photosynthetic Rates, Photosynthetic Enzymes, Contents of DNA, Chlorophyll, and Sizes and Numbers of Photosynthetic Cells in the C(4) Dicot Atriplex confertifolia. AB - Photosynthetic rates, chlorophyll content, and activities of several photosynthetic enzymes were determined per cell, per unit DNA, and per unit leaf area in five ploidal levels of the C(4) dicot Atriplex confertifolia. Volumes of bundle sheath and mesophyll protoplasts were measured in enzymatic digestions of leaf tissue. Photosynthetic rates per cell, contents of DNA per cell, and activities of the bundle sheath enzymes ribulose 1,5-bisphosphate carboxylase (RuBPC) and NAD-malic enzyme per cell were correlated with ploidal level at 99% or 95% confidence levels, and the results suggested a near proportional relationship between gene dosage and gene products. There was also a high correlation between volume of mesophyll and bundle sheath cells and the ploidal level. Contents of DNA per cell, activity of RuBPC per cell, and volumes of cells were correlated with photosynthetic rate per cell at the 95% confidence level. The mesophyll cells did not respond to changes in ploidy like the bundle sheath cells. In the mesophyll cells the chlorophyll content per cell was constant at different ploidal levels, there was less increase in cell volume than in bundle sheath cells with an increase in ploidy, and there was not a significant correlation (at 95% level) of phosphoenolpyruvate carboxylase activity or content and pyruvate,Pi dikinase activity with increase in ploidy. The number of photosynthetic cells per unit leaf area progressively decreased with increasing ploidy from diploid to hexaploid, but thereafter remained constant in octaploid and decaploid plants. Numbers of cells per leaf area were not correlated with cell volumes. The mean photosynthetic rates per unit leaf area were lowest in the diploid, similar in 4x, 6x, and 8x, and highest in the decaploid. The photosynthetic rate per leaf area was highly correlated with the DNA content per leaf area. PMID- 16667126 TI - Light Regulation of beta-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.). AB - The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of beta-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)(+)RNA with a probe derived from the coding region of a previously characterized soybean beta-tubulin gene. (MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of beta-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of beta-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of beta-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA. PMID- 16667127 TI - Heliotropic leaf movements in common beans controlled by air temperature. AB - Heliotropic leaf movements were examined in common beans (Phaseolus vulgaris cv Blue Lake Bush) under outdoor and laboratory conditions. Heliotropic leaf movements in well-watered plants were partly controlled by temperature, and appeared to be independent of atmospheric humidity and CO(2) concentration. When environmental conditions were held constant in the laboratory, increased air temperature caused bean leaves to orient more obliquely to a light source. Ambient CO(2), intercellular CO(2), and net photosynthesis were not correlated with the temperature-induced changes in heliotropic movements, nor did they significantly affect these movements directly. The effect of air temperature on leaf movements need not be mediated through a change in leaf water potential, transpiration, or leaf conductance. Air temperature modified laminar orientation in light through its effect on tissue temperature in the pulvinal region, not that of the lamina or petiole. However, under darkness the temperature effects on leaf movements were not expressed. Active heliotropic movements in response to air temperature allowed lamina temperature to remain close to the thermal optimum of photosynthesis. This temperature effect underlies a commonly observed pattern of leaf movements under well-watered conditions: a tendency for leaves to face the sun more obliquely on hot days than cool days. PMID- 16667128 TI - Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot. AB - Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion ( approximately 20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels. PMID- 16667129 TI - Increased Tolerance to Photoinhibitory Light in Paraquat-Resistant Conyza bonariensis Measured by Photoacoustic Spectroscopy and CO(2)-Fixation. AB - Tolerance to photoinhibition was compared between a paraquat-resistant and a sensitive biotype of Conyza bonariensis (L.). Cronq. Photoinhibitory damage was measured as a decrease in oxygen evolution or energy storage using photoacoustic spectroscopy, or as a decrease of (14)CO(2)-fixation. Prior to exposure to high fluence rates, both biotypes had similar quantum yields of oxygen evolution and energy storage. After exposure to high intensity light, the resistant biotype continued to evolve oxygen and to store energy with a high quantum yield while both energy storage and oxygen evolution were severely reduced in the sensitive biotype. CO(2)-fixation was less rapidly inhibited in the resistant biotype compared to the sensitive one. The data show that the paraquat resistant biotype with its high constitutive levels of the chloroplast localized enzymes of the oxygen detoxification pathway, is also partially protected from photoinhibition. This supports the theory that an enhanced radical scavenging system can give temporary protection against photooxidative damage from a variety of sources. PMID- 16667130 TI - Stoichiometry of Photosystem I, Photosystem II, and Phycobilisomes in the Red Alga Porphyridium cruentum as a Function of Growth Irradiance. AB - Cells of the red alga Porphyridium cruentum (ATCC 50161) exposed to increasing growth irradiance exhibited up to a three-fold reduction in photosystems I and II (PSI and PSII) and phycobilisomes but little change in the relative numbers of these components. Batch cultures of P. cruentum were grown under four photon flux densities of continuous white light; 6 (low light, LL), 35 (medium light, ML), 180 (high light, HL), and 280 (very high light, VHL) microeinsteins per square meter per second and sampled in the exponential phase of growth. Ratios of PSII to PSI ranged between 0.43 and 0.54. About three PSII centers per phycobilisome were found, regardless of growth irradiance. The phycoerythrin content of phycobilisomes decreased by about 25% for HL and VHL compared to LL and ML cultures. The unit sizes of PSI (chlorophyll/P(700)) and PSII (chlorophyll/Q(A)) decreased by about 20% with increase in photon flux density from 6 to 280 microeinsteins per square meter per second. A threefold reduction in cell content of chlorophyll at the higher photon flux densities was accompanied by a twofold reduction in beta-carotene, and a drastic reduction in thylakoid membrane area. Cell content of zeaxanthin, the major carotenoid in P. cruentum, did not vary with growth irradiance, suggesting a role other than light-harvesting. HL cultures had a growth rate twice that of ML, eight times that of LL, and slightly greater than that of VHL cultures. Cell volume increased threefold from LL to VHL, but volume of the single chloroplast did not change. From this study it is evident that a relatively fixed stoichiometry of PSI, PSII, and phycobilisomes is maintained in the photosynthetic apparatus of this red alga over a wide range of growth irradiance. PMID- 16667131 TI - Mechanisms of Aluminum Tolerance in Wheat : An Investigation of Genotypic Differences in Rhizosphere pH, K, and H Transport, and Root-Cell Membrane Potentials. AB - Control of rhizosphere pH and exclusion of Al by the plasma membrane have been hypothesized as possible mechanisms for Al tolerance. To test primarily the rhizosphere pH hypothesis, wheat cultivars (Triticum aestivum L. ;Atlas 66' and ;Scout'), which differ in Al tolerance, were grown in either complete nutrient solution, or 0.6 millimolar CaSO(4), with and without Al at pH 4.50. A microelectrode system was used to simultaneously measure rhizosphere pH, K(+), and H(+) fluxes, and membrane potentials (E(m)) along the root at various distances from the root apex. In complete nutrient solution, the rhizosphere pH associated with mature root cells (measured 10-40 millimeters from the root apex) of Al-tolerant ;Atlas 66' was slightly higher than that of the bulk solution, whereas roots of Al-sensitive ;Scout' caused a very small decrease in the rhizosphere pH. In CaSO(4) solution, no significant differences in rhizosphere pH were found between wheat cultivars, while differential Al tolerance was still observed, indicating that the rhizosphere pH associated with mature root tissue is not directly involved in the mechanism(s) of differential Al tolerance. In Al tolerant ;Atlas 66', growth in a CaSO(4) solution with 5 micromolar Al (pH 4.50) had little effect on net K(+) influx, H(+) efflux, and root-cell membrane potential measured in cells of mature root tissue (from 10-40 mm back from apex). However, in Al-sensitive ;Scout', Al treatment caused a dramatic inhibition of K(+) influx and both a moderate reduction of H(+) efflux and depolarization of the membrane potential. These results demonstrate that increased Al tolerance in wheat is associated with the increased ability of the tolerant plant to maintain normal ion fluxes and membrane potentials across the plasmalemma of root cells in the presence of Al. PMID- 16667132 TI - Polyamines in Rice Seedlings under Oxygen-Deficit Stress. AB - Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of (14)CO(2) from tissues fed with [(14)C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed. PMID- 16667133 TI - High affinity k uptake in maize roots: a lack of coupling with h efflux. AB - We report here on the putative coupling between a high affinity K(+) uptake system which operates at low external K(+) concentrations (K(m) = 10-20 micromolar), and H(+) efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K(+)((86)Rb(+)) influx, and the simultaneous measurement of net K(+) and H(+) fluxes associated with individual cells at the root surface with K(+)- and H(+)-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K(+) and H(+) ions within the unstirred layer at the root surface. No evidence for coupling between K(+) uptake and H(+) efflux could be found based on: (a) extremely variable K(+):H(+) flux stoichiometries, with K(+) uptake often well in excess of H(+) efflux; (b) dramatic time-dependent variability in H(+) extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K(+) uptake system (to changes in external pH) when net K(+) uptake, unidirectional K(+)((86)Rb(+)) influx, and K(+)-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K(+) absorption into maize root cells is not mediated by a K(+)/H(+) exchange mechanism. Instead, it is either due to the operation of a K(+)-H(+) cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K(+)-ATPase as reported for Escherichia coli and Saccharomyces. PMID- 16667134 TI - Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens. AB - The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (R(0)) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues. Putative transformed R(0) soybean plants were advanced to produce R(1) plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the R(0) plant, and that about 1/10 of these plants yielded transformed R(1) plants. The presence of the Nos-NPT II gene in DNAs isolated from both R(0) and R(1) plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R(1) soybean plants. PMID- 16667135 TI - Spatial and Temporal Influences on the Cell-Specific Distribution of Glycine Decarboxylase in Leaves of Wheat (Triticum aestivum L.) and Pea (Pisum sativum L.). AB - The distribution of glycine decarboxylase (GDC) in leaves of pea (Pisum sativum L.) and wheat (Triticum aestivum L.) has been investigated using immunogold labeling of the P-protein subunit of the GDC complex. Mitochondria in photosynthetic mesophyll cells were densely labeled, whereas those in nonphotosynthetic vascular parenchyma and epidermal cells were only weakly labeled. In pea leaves the density of immunogold labeling on mitochondria in the chloroplast-containing bundle sheath and stomatal guard cells was intermediate between that in mesophyll and epidermal cells. In both species the density of labeling on mitochondria in a cell appeared to reflect the photosynthetic capacity of the cell. This relationship was further examined in wheat where a natural developmental gradient exists along the lamina such that cell maturity increases with distance from the basal meristem. In this case the density of labeling on mesophyll cell mitochondria increased with photosynthetic development and with increasing maturity of the cell. Vascular cell mitochondria, however, became less densely labeled as the cells matured. The results indicate a close, positive correlation between the concentration of GDC in the mitochondria and the photosynthetic status of the host cell. This relationship is maintained effectively under the influence of both spatial (i.e. cellular differentiation across the lamina) and temporal (i.e. cellular development along the lamina) constraints. PMID- 16667136 TI - Primary Site of Action of Amitrole in Arabidopsis thaliana Involves Inhibition of Root Elongation but Not of Histidine or Pigment Biosynthesis. AB - Interference with histidine metabolism, inhibition of pigment biosynthesis, or both have been the principal candidates for the primary site of action of 3-amino 1,2,4-triazole (amitrole). Arabidopsis thaliana is sensitive to 1,2,4-triazole-3 alanine, a feedback inhibitor of histidine biosynthesis, and this effect is reversed by histidine. The combination of triazolealanine and histidine, however, does not reverse the herbicidal effect of amitrole. This indicates that amitrole toxicity is not caused by histidine starvation, nor is it caused by the accumulation of a toxic intermediate of the histidine pathway. Amitrole inhibits root elongation at lower concentrations than it causes pigment bleaching in the leaves. In contrast, fluridone, a known inhibitor of the carotenoid biosynthetic pathway does not block root elongation. Fluridone also inhibits carotenoid accumulation in etiolated seedlings in the dark, but amitrole does not. Last, gabaculine and acifluorfen, but not amitrole, prevent chlorophyll accumulation in greening etiolated seedlings of Arabidopsis. These experiments cast doubt on pigment biosynthesis as the primary site of action of amitrole. PMID- 16667137 TI - Increased Proteolysis of Senescing Rice Leaves in the Presence of NaCl and KCl. AB - NaCl and KCl enhanced the degradation of chlorophylls and proteins in detached rice (Oryza sativa) leaves in a concentration-dependent manner. Ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco) accounted for 73 to 80% of the protein lost by day 4 in the light. NaCl at 50 millimolar increased proteolysis by 21% over the control in 4 days, but the addition of cycloheximide reduced the increase to about one-half. Cycloheximide alone had no effect on proteolytic activity during this period. Leaf segments taken from 10-day-old seedlings contained the highest proteolytic activity. Both NaCl and KCl increased the activity of Rubisco-degrading endoproteinases (the amount of ninhydrin-positive compounds measured from HCl-hydrolyzates of trichloroacetic acid-soluble supernatant), but decreased the activity of hemoglobin- and Rubisco-degrading exoproteinases (the amount of ninhydrin-positive compounds measured directly from trichloroacetic acid-soluble supernatant). Efflux of amino acids from senescing leaf segments into the incubation media increased 7- and 12-fold in the presence of KCl and NaCl, respectively. The increased efflux resulted in a negative correlation between salt concentration and amino acid content of leaf segments at the later stage of senescence. It is concluded that, in addition to the induction of new proteinase synthesis, the increased efflux of protein hydrolyzates may play a significant role in increasing proteolysis of salt-treated leaves, especially at the later stages of senescence. PMID- 16667138 TI - Sensory transduction and electrical signaling in guard cells. AB - Guard cells are a valuable model system for the study of photoreception, ion transport, and osmoregulation in plant cells. Changes in stomatal apertures occur when sensing mechanisms within the guard cells transduce environmental stimull into the ion fluxes and biosynthesis of organic solutes that regulate turgor. The electrical events mediating sensory transduction in guard cells can be characterized with a variety of electrophysiological recording techniques. Recent experiments applying the patch clamp method to guard cell protoplasts have demonstrated activation of electrogenic pumps by blue and red light as well as the presence of potassium channels in guard cell plasmalemma. Light activation of electrogenic proton pumping and the ensuing gating of voltage-dependent ion channels appear to be components of sensory transduction of the stomatal response to light. Mechanisms underlying stomatal control by environmental signals can be understood by studying electrical events associated with ion transport. PMID- 16667139 TI - Reflected Far-Red Light Effects on Chlorophyll and Light-Harvesting Chlorophyll Protein (LHC-II) Contents under Field Conditions. AB - The influence of various colors of soil cover (mulch) on the farred/red (FR/R) ratio in upwardly reflected light and on concentrations of chlorophyll (Chl) and light-harvesting Chl protein (LHC-II) were measured under field conditions. The FR/R ratios above green surfaces were higher than over white surfaces. Even though plants (Gossyplum hirsutum L. cv PD-1) were grown in full sunlight, those that received higher FR/R ratios in upwardly reflected light were taller and had thinner leaves with higher concentrations of Chl and LHC-II. A controlled environment experiment showed FR/R control of Chl and LHC-II concentrations. The results illustrate the importance of spectral distribution of reflected light on plant growth and a potential means of altering the chemistry of leaf crops under field conditions. PMID- 16667140 TI - Cytokinin-to-Auxin Ratios and Morphology of Shoots and Tissues Transformed by a Chimeric Isopentenyl Transferase Gene. AB - Tissues transformed with the isopentenyl transferase (ipt) gene cloned from the T DNA region of the Ti plasmid or with the ipt gene placed under the control of the cauliflower mosaic virus 35S promoter (35S-ipt) were analyzed for auxin and cytokinin. Free and total indole-3-acetic acid (IAA) levels in 35S-ipt transformed Nicotiana tabacum and cucumber cells were reduced by 12 to 78% in comparison to untransformed tissues. In contrast, free IAA concentrations in 35S ipt-transformed Nicotiana plumbaginifolia were almost three times those of untransformed tissues, while total IAA levels were not significantly affected. Cytokinin levels in these antibodies were elevated an average of 300-fold resulting in a 24- to over 2000-fold increase in the cytokinin-to-auxin ratios. High cytokinin-to-auxin ratios correlated with the shooty phenotype of transformed tissues propagated in vitro in the absence of added growth hormones. We conclude that increased endogenous cytokinin-to-auxin ratios can induce cells to undergo morphogenesis and that elevated cytokinin levels can also induce auxin autonomous growth of transformed tissues without causing a corresponding increase in endogenous IAA levels. PMID- 16667141 TI - Enhanced-peroxidatic activity in specific catalase isozymes of tobacco, barley, and maize. AB - Separation of catalase isozymes from leaf extracts of three diverse plant species (Nicotiana sylvestris, Zea mays, Hordeum vulgare L.) revealed a distinct isozyme with enhanced peroxidatic activity (30-, 70-, 28-fold over typical catalase, respectively) which constituted 10 to 20% of the total catalase activity. In maize this isozyme is the product of the Cat3 gene, which is expressed only in mesophyll cells (AS Tsaftaris, AM Bosabalidis, JG Scandalios [1983] Proc Natl Acad Sci USA 80: 4455-4459). A mutation in barley reducing levels of peroxisomal catalase (AC Kendall et al. [1983] Planta 159: 505-511) does not reduce the amount of the isozyme with enhanced peroxidatic activity. Similarly, this isozyme is unaffected in dark-grown barley in spite of a 75% decrease in total catalase activity. These results suggest that this catalase isozyme is under separate genetic control in barley. This may also be the case in tobacco where the catalase isozyme with enhanced peroxidatic activity is an immunologically distinct protein (EA Havir, NA McHale [1989] Plant Physiol 89: 952-957). PMID- 16667142 TI - Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins. AB - Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces beta elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development. PMID- 16667143 TI - Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity. AB - Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [(3)H]GA(4) binding sites (K(d) approximately 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K(d) for [(3)H]GA(4) increased, whereas the maximum number of saturable [(3)H]GA(4) binding sites did not change significantly. The dissociation of [(3)H]GA(4) from its binding sites was complex and was best described by a bi exponential equation. AC-94,377 did not affect the rates of [(3)H]GA(4) dissociation from its binding sites. These results implied that AC-94,377 and [(3)H]GA(4) compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs. PMID- 16667144 TI - Characterization of a Radish Nuclear Gene Expressed during Late Seed Maturation. AB - To study long-lived mRNAs stored in radish (Raphanus sativus) seed, we have selected clones from a dry seed cDNA library by differential screening. One of these clones, p8B6, whose mRNAs are abundant in the dry seed, was characterized. This clone hybridizes to an RNA class of approximately 600 nucleotides whose accumulation begins during the desiccation phase, reaches its maximum level in the dry seed, and is no longer detectable in 12 hour old seedlings. mRNAs hybrid selected by p8B6 encode four polypeptides, but only two are compatible with the size class of RNAs detected by Northern analysis. Three of them have previously been identified as major ;early germination' polypeptides, and their synthesis has been shown to be induced prematurely in immature embryos by a desiccation treatment. The protein deduced from the p8B6 nucleotide sequence is 9 kilodaltons in size, highly hydrophilic, rich in Gly and Glu, and contains no Cys, Trp, and lie. The amino acid sequence shares good homology with that of two recently described seed proteins: a cotton late embryogenesis abundant protein and the wheat early methionine-labeled protein. Southern blot analysis suggests that the p8B6 sequence belongs to a very small gene family. The exact function of the product encoded by p8B6 remains to be determined. PMID- 16667145 TI - Hypoxic Induction of Anoxia Tolerance in Root Tips of Zea mays. AB - When root tips of fully aerobic, intact maize (Zea mays L.) seedlings are made anaerobic, viability normally is only 24 hours or less at 25 degrees C. We find that viability can be extended to at least 96 hours if seedlings are given a hypoxic pretreatment for 18 hours by sparging the solution with 4% O(2) in nitrogen (v/v) before anoxia. Fully aerobic root tips (sparged with 40% O(2)) had very low alcohol dehydrogenase (ADH) activity (per gram root fresh weight), and the level remained low under anoxia. In hypoxically pretreated roots, however, high levels of ADH activity were induced, and activity rose further during the initial 24 hours of anoxia, and then remained high at about 20 times that of controls in 40% O(2). ADH activity in roots in solution sparged with air (21% O(2)) was about three times that in 40% O(2). Improved viability of hypoxically pretreated root tips was associated with maintenance of a high energy metabolism (ATP concentration, total adenylates, and adenylate energy charge). Roots that were not pretreated lost 94% of the total adenylates and ATP at 24 hours of anoxia. The relation between induced ADH activity, energy metabolism, and improved anoxia-tolerance in acclimated maize root tips is discussed. PMID- 16667146 TI - A Chalcone and Two Related Flavonoids Released from Alfalfa Roots Induce nod Genes of Rhizobium meliloti. AB - Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium meliloti. Previous investigations identified the flavone luteolin as an active inducer in alfalfa seed extracts, but the nature of nod inducers released from roots has not been reported. Root exudate from 3-day-old alfalfa seedlings was purified and then assayed for biological activity with a nodABC-lacZ fusion in R. meliloti. Indentities of major nod inducers were established by spectroscopic analyses (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Major nod inducers, which were identified as 4',7-dihydroxyflavone, 4' 7-dihydroxyflavanone, and 4,4'-dihydroxy-2'-methoxychalcone, were released from seedling roots at 54, 22, and 20 picomole.plant(-1).day(-1), respectively. Luteolin was not found in these root exudates. The 4,4'-dihydroxy-2' methoxychalcone induced nod genes at a concentration one order of magnitude lower than luteolin and is the first naturally released chalcone reported to have this function. Moderate and weak nod-inducing activity was associated, respectively, with 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone. PMID- 16667147 TI - Whole Leaf Carbon Exchange Characteristics of Phosphate Deficient Soybeans (Glycine max L.). AB - Low phosphate nutrition results in increased chlorophyll fluorescence, reduced photosynthetic rate, accumulation of starch and sucrose in leaves, and low crop yields. This study investigated physiological responses of soybean (Glycine max [L.] Merr.) leaves to low inorganic phosphate (Pi) conditions. Responses of photosynthesis to light and CO(2) were examined for leaves of soybean grown at high (0.50 millimolar) or low (0.05 millimolar) Pi. Leaves of low Pi plants exhibited paraheliotropic orientation on bright sunny days rather than the normal diaheliotropic orientation exhibited by leaves of high Pi soybeans. Leaves of plants grown at high Pi had significantly higher light saturation points (1000 versus 630 micromole photons [400-700 nanometers] per square meter per second) and higher apparent quantum efficiency (0.062 versus 0.044 mole CO(2) per mole photons) at ambient (34 pascals) CO(2) than did low Pi leaves, yet stomatal conductances were similar. High Pi leaves also had significantly higher carboxylation efficiency (2.90 versus 0.49 micromole CO(2) per square meter per second per pascal), a lower CO(2) compensation point (6.9 versus 11.9 pascals), and a higher photosynthetic rate at 34 pascals CO(2) (19.5 versus 6.7 micromoles CO(2) per square meter per second) than did low Pi leaves. Soluble protein (0.94 versus 0.73 milligram per square centimeter), ribulose-1,5-bisphosphate carboxylase/oxygenase content (0.33 versus 0.25 milligram per square centimeter), and ribulose-1,5-bisphosphate carboxylase/oxygenase specific activity (25.0 versus 16.7 micromoles per square meter per second) were significantly greater in leaves of plants in the high Pi treatment. The data indicate that Pi stress alters the plant's CO(2) reduction characteristics, which may in turn affect the plant's capacity to accommodate normal radiation loads. PMID- 16667148 TI - Stable NaCl Tolerance of Tobacco Cells Is Associated with Enhanced Accumulation of Osmotin. AB - Osmotin is a major protein which accumulates in tobacco cells (Nicotiana tabacum L. var Wisconsin 38) adapted to low water potentials. Quantitation of osmotin levels by immunoblots indicated that cells adapted to 428 millimolar NaCl contained 4 to 30 times the level of osmotin found in unadapted cells, depending on the stage of growth. Unadapted cells accumulated low levels of osmotin with apparent isoelectric points, (pl) of 7.8 and >8.2. Upon transfer of NaCl-adapted cells to medium without NaCl and subsequent growth for many cell generations, the amount of osmotin declined gradually to a level intermediate between that found in adapted and unadapted cells. NaCl-adapted cells grown in the absence of NaCl accumulated both pl forms; however, the form accumulated by cells adapted to NaCl (pl > 8.2) was most abundant. Adapted cells grown in the absence of NaCl exhibited absolute growth rates and NaCl tolerance levels which were intermediate to those of NaCl-adapted and unadapted cells. The association between osmotin accumulation and stable NaCl tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCl. This stable alteration in gene expression probably affects salt tolerance. PMID- 16667149 TI - Water deficits and reproduction in maize : response of the reproductive tissue to water deficits at anthesis and mid-grain fill. AB - Reproductive development in maize (Zea mays L.) is vulnerable to plant water deficits during anthesis but becomes less sensitive as reproduction progresses. To determine whether changes in tissue water status correlated with the change in sensitivity, we examined the water potential (Psi(w)), osmotic potential (Psi(s)), and turgor of reproductive tissues during a short-term water deficit imposed at anthesis or mid-grain fill. Plants were grown in controlled environments in soil. At anthesis, leaf, husk, silk, and ovary Psi(w) of control plants was similar (-0.5 to -0.65 megapascal) at midday. When water was withheld, Psi(w) decreased to -1.75, -1.3, -1.2, and -1.0 megapascal in these tissues. Net water uptake by the ovaries was inhibited, but final dry weight, solute content, and total extractable carbohydrates were similar to the controls. At mid-grain fill, leaf, husk, grain, and embryo Psi(w) of control plants were -0.55, -0.35, 0.75, and -0.80 megapascal at midday. When water was withheld, leaf and husk Psi(w) decreased to -2.4 and -1.4 megapascal within 6 days. However, grain and embryo Psi(w) remained within 0.15 megapascal of control values. The grain continued to accumulate dry matter despite a net loss of water and a reduction in total solute content. These results indicate that the response of the reproductive tissues to plant water deficits varies with stage of grain development. The maintenance of a favorable water status only after grain filling is under way may explain, at least in part, the high sensitivity to plant water deficits early in reproductive development and the decrease in sensitivity as reproduction progresses. PMID- 16667150 TI - Purification and Characterization of Two Forms of Glutamine Synthetase from the Pedicel Region of Maize (Zea mays L.) Kernels. AB - Maize (Zea mays L.) kernel pedicels, including vascular tissues, pedicel parenchyma, placento-chalazal tissue, and the surrounding pericarp, contained two forms of glutamine synthetase (EC 6.3.1.2), separable by anion exchange chromatography under mildly acidic conditions. The earlier-eluting activity (GS(p1)), but not the later-eluting activity (GS(p2)), was chromatographically distinct from the maize leaf and root glutamine synthetases. The level of GS(p1) activity changed in a developmentally dependent manner while GS(p2) activity was constitutive. GS(p1) and GS(p2) exhibited distinct ratios of transferase to hydroxylamine-dependent synthetase activities (5 and 23, respectively), which did not change with kernel age. Purified pedicel glutamine synthetases had native relative molecular masses of 340,000, while the subunit relative molecular masses differed slightly at 38,900 and 40,500 for GS(p1) and GS(p2), respectively. Both GS forms required free Mg(2+) with apparent K(m)s = 2.0 and 0.19 millimolar for GS(p1) and GS(p2), respectively. GS(p1) had an apparent K(m) for glutamate of 35 millimolar and exhibited substrate inhibition at glutamate concentrations greater than 90 millimolar. In contrast, GS(p2) exhibited simple Michaelis-Menten kinetics for glutamate with a K(m) value of 3.4 millimolar. Both isozymes exhibited positive cooperativity for ammonia, with S(0.5) values of 100 and 45 micromolar, respectively. GS(p1) appears to be a unique, kernel-specific form of plant glutamine synthetase. Possible functions for the pedicel GS isozymes in kernel nitrogen metabolism are discussed. PMID- 16667151 TI - Culm strength of barley : correlation among maximum bending stress, cell wall dimensions, and cellulose content. AB - Grass culms are known to differ in breaking strength, but there is little physicochemical data to explain the response. The fourth internode of four brittle and two nonbrittle barley (Hordeum vulgare L.) strains were used for physical and chemical studies of culm strength. Inner and outer culm diameters of brittle strains (3.6 +/- 0.2 and 5.0 +/- 0.1 millimeters) were not significantly different from those of nonbrittle strains (3.9 +/- 0.2 and 5.2 +/- 0.2 millimeters). Maximum bending stress, at which the culm was broken, was 192 +/- 34 g/mm(2) for brittle and 490 +/- 38 g/mm(2) for nonbrittle strains. Wall thickness and cell dimensions of epidermal, sclerenchyma, and parenchyma cells were measured in culm cross sections. The area of cell wall per unit cell area for each tissue was significantly correlated with the maximum bending stress (r = 0.93 for epidermis, 0.90 for sclerenchyma, and 0.84 for parenchyma). Cell walls of brittle culms had 6 to 64% as much cellulose content as those of nonbrittle culms. Maximum bending stress correlated significantly with cellulose content of the cell walls (r = 0.93), but not with the contents of noncellulosic compounds. The lower cellulose content of the brittle culm was significantly correlated with brittleness. PMID- 16667152 TI - Oxygen Inhibition of Nitrate Reductase Biosynthesis in Detached Corn Leaves via Inhibition of Total Soluble Protein Synthesis. AB - Detached first leaves of 3-day-old corn seedlings (Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O(2) in the light. Nitrate accumulation in the leaves was not depressed by O(2). NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O(2) the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O(2)-treated leaves. Total soluble protein levels in leaves were slightly depressed by O(2) and shifting from O(2) to an air environment increased the protein level. Incorporation of [(35)S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O(2) environment relative to air, but both recovered when leaves were shifted from O(2) to air. Although O(2) accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O(2)-treated leaves. We concluded that O(2) inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation. PMID- 16667153 TI - Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean. AB - Suspension cultures of castor bean (Ricinus communis L.) which have been treated with pectic fragment elicitor rapidly accumulate lignin as measured by derivatization with thioglycolic acid. The responsiveness of cultured cells to elicitor is dependent on the stage of culture growth. In 6-day (maximally responsive) cultures, increases in lignin are first evident 3 hours after addition of pectic fragment elicitor with maximal rates of lignin synthesis between 4 and 10 hours. The abundance of lignin in cultures after 12 hours of elicitor treatment is 10- to 20-fold higher than in untreated control cultures and can thereby account for as much as 2% of the dry cell weight. Only intermediate sizes of pectic oligomer are active as elicitors of lignin. Half maximal accumulation of lignin occurs at 250 to 300 micrograms per milliliter of an optimal elicitor preparation with an average degree of polymerization of seven. We consider the synthesis of lignin in elicited cultures to be a mechanism of plant disease resistance which is induced by the elicitor. Plant peroxidases have been proposed to catalyze the last enzymatic steps in the biosynthesis of both lignin and hydrogen peroxide. Six extracellular isoenzymes of peroxidase (two anionic, designated A1 and A2, and four cationic, designated C2, C3, C4, and C7) are detectable in healthy suspension cultures of castor bean by native gel electrophoresis. Treatment of cultures with elicitor causes substantial changes in the activity of four of these species (A1, C2, C3, and C7). Elicitor treatment also results in the appearance of three new peroxidase isoenzymes that are not readily detectable in healthy cultures (C1, C5, and C6). Increases in the activities of these isoenzymes are concurrent with or slightly precede the accumulation of lignin in elicited 6-day cultures. By 12 hours after addition of elicitor, C1 becomes the most abundant extracellular isoperoxidase. The differential regulation of expression of peroxidase isoenzymes following elicitor treatment suggests that individual isoenzymes of peroxidase may have specific functional roles in the biosynthesis of disease-lignin. PMID- 16667154 TI - Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules. AB - Effective (N(2)-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N(2)-fixing) alfalfa recessive for the in(1) gene were compared to determine the effects of the in(1) gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in(1) effective nodules. PMID- 16667155 TI - Influence of low temperature on respiration and contents of phosphorylated intermediates in darkened barley leaves. AB - The aim of this work was to examine the effect of exposure of leaves to low temperatures (5 degrees C) upon the contents of phosphorylated intermediates and respiration in darkened barley (Hordeum vulgare L.) plants which differed in their carbohydrate status. In leaves that had previously been illuminated for 24 hours, there was a large increase in amounts of phosphorylated metabolites at 5 degrees C during the first 3 hours of darkness, compared with control plants kept at 30 degrees C. Hexose phosphates accounted for about two-thirds of this increase, which reached a peak after about 3 hours. At higher temperatures, there was a peak in the amount of fructose 2,6-bisphosphate and the rate of respiration which accompanied the transient increase in phosphorylated intermediates. At 5 degrees C the increase in phosphorylated intermediates was not accompanied by appreciable changes in fructose 2,6-bisphosphate, and there was a rapid decline in the rate of respiration. Leaves that had previously been darkened for 24 hours and that were low in carbohydrate failed to accumulate phosphorylated intermediates when exposed to low temperatures. The results are discussed with respect to the acclimation of carbohydrate metabolism to low temperatures. The results suggest that respiratory carbohydrate metabolism is strictly controlled even when the carbohydrate supply and glycolytic intermediates are abundant. The possibility that accumulation of hexose phosphates may be involved in acclimation of metabolism to low temperature is discussed. PMID- 16667156 TI - Increase of chalcone synthase mRNA in pathogen-inoculated soybeans with race specific resistance is different in leaves and roots. AB - Soybeans (Glycine max [L.] Merr.) respond to pathogens by producing isoflavonoid derived phytoalexins. Chalcone synthase (CHS) is the first enzyme of the flavonoid/isoflavonoid biosynthetic pathway. We investigated changes in the steady state levels of CHS mRNA and other specific mRNAs at increasing times after inoculation in two different race-specific interactions, one between leaves and the bacterium Pseudomonas syringae pv glycinea (Psg), and one between roots and the fungus, Phytophthora megasperma f. sp. glycinea (Pmg). The amount of CHS mRNA increases significantly in soybean leaves inoculated with an avirulent race of Psg but not with a virulent race or water. In contrast, the increase in CHS mRNA is similar in roots inoculated with zoospores of either an avirulent or virulent race of Pmg. CHS mRNA increases significantly in pathogen inoculated roots but not in uninoculated controls. Hydroxyproline-rich glycoprotein (HRGP) has been observed by others to increase in wounded or pathogen-inoculated plants. We report here that HRGP mRNA levels are greater in roots inoculated with an avirulent Pmg race than with a virulent race, but inoculation with either race causes a significant increase in HRGP mRNA with respect to controls. Calmodulin or ubiquitin mRNA do not increase in either uninoculated or inoculated roots and leaves. The possibility that race-specific resistance in soybeans is expressed differently in different organs of the plant is discussed. PMID- 16667157 TI - Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation. AB - Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent K(m) values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments. PMID- 16667158 TI - Effect of Growth Temperature on the Biosynthesis of Chloroplastic Galactosyldiacylglycerol Molecular Species in Brassica napus Leaves. AB - Brassica napus leaves developed at low temperature display rapid in situ desaturation of monogalactosyldiacylglycerol (MGDG) fatty acids leading to the production of hexadecatrienoic/linolenic acid. This was shown by radioactivity tracer experiments to occur via a sequence of desaturations proceeding from the initially synthesized palmitic/oleic acid molecular species to palmitic/linoleic acid, palmitoleic/linoleic acid, hexadecadienoic/linoleic acid, hexadecadienoic/linolenic acid, and finally to hexadecatrienoic/linolenic acid. The results suggest that there is increased activity in all five desaturation steps in leaves developed at low temperatures. Labeling data also indicate that there is another pool of MGDG which is more slowly desaturated before galactosylation to digalactosyldiacylglycerol (DGDG). Our data further suggest that relative rates of galactosylation of chloroplastic and cytosolic MGDG molecular species may regulate the final amounts of chloroplastic and cytosolic MGDG and DGDG in the leaf. We have proposed a model for chloroplastic biosynthesis and desaturation of galactosyldiacylglycerols in the leaves of Brassica napus, a 16:3 plant. PMID- 16667159 TI - Effects of cold-treatment on protein synthesis and mRNA levels in rice leaves. AB - The effects of cold on protein and RNA metabolism in leaves of rice (Oryza sativa L.) seedlings were investigated. Treatment with a diurnal cycle of 15/10 degrees C or 11/6 degrees C for up to 1 week resulted in progressive changes in the protein synthesis pattern after in vivo labeling of intact rice leaves with [(35)S]methionine. These changes were reversed when the seedlings were returned to normal growth temperatures. While de novo accumulation of several abundant proteins was suppressed, some polypeptides were consistently found to be cold induced. Synthesis of ribulose 1,5-bisphosphate carboxylase (Rubisco) was drastically reduced after 7 days of cold. Using immunoprecipitation of Rubisco, evidence was obtained that the suppression was greater for the small subunit (over 90%) than for the large subunit (80%), indicating a partial loss of coordination in their synthesis. Preformed Rubisco as well as other cold suppressed proteins were stable for up to 7 days at 11/6 degrees C. Cold sensitive rice cultivars responded with similar but more drastic changes in the protein synthesis pattern when compared to cold-tolerant varieties. The suppression of Rubisco synthesis by cold was shown to result from reduced levels of the mRNAs encoding both subunits; their decrease paralleled the lower protein synthesis of each. The levels of other chloroplast-encoded mRNAs, especially psaB, and of the nuclear encoded chlorophyll a/b binding protein, also strongly decreased in the cold, whereas the transcripts of the mitochondrial genes apt9, coxIII, and most nuclear genes analyzed were unaffected or only slightly reduced. These data indicate that some chloroplast functions are disturbed during cold stress. One nuclear gene known to be induced by water stress and ABA (Rab21) was also found to be induced by cold treatment. PMID- 16667160 TI - Analysis of Chromophytic and Rhodophytic Ribulose-1,5-Bisphosphate Carboxylase Indicates Extensive Structural and Functional Similarities among Evolutionarily Diverse Algae. AB - Ribulose-1,5-bisphosphate carboxylase (Rubisco) from the algae Olisthodiscus luteus (chromophyte) and Griffithsia pacifica (rhodophyte) are remarkably similar to each other. However, both enzymes differ significantly in the structure and function when compared to Rubisco from green algae and land plants. Analysis of purified Rubisco from O. luteus and G. pacifica indicates that the size of the holoenzyme and stoichiometry of the 55 and 15 kilodalton subunit polypeptides are approximately 550 kilodaltons and eight:eight for both algae. Antigenic determinants are highly conserved between the O. luteus and G. pacifica enzymes and differ from those of the spinach subunit polypeptides. Sequence similarity between the two algal large subunits has been further confirmed by one dimensional peptide mapping. Substrate ribulose bisphosphate has no effect on the rate of CO(2)/Mg(2+) activation of O. luteus and G. pacifica enzymes which contrasts to the extensive inhibition of spinach Rubisco activation at similar concentrations of this compound. In addition, the Michaelis constant for CO(2) and the inhibition constant for 6-phosphogluconate are similar for the O. luteus and G. pacifica catalyzed carboxylation reaction. Both values are intermediate to those observed for the tight binding spinach enzyme and weak binding prokaryotic (Rhodospirillum rubrum) enzyme. The biochemical similarities documented between O. luteus and G. pacifica may be due to a common evolutionary origin on the chromophytic and rhodophytic chloroplast but could also result from the fact that both subunit polypeptides are chloroplast DNA encoded in these algal taxa. PMID- 16667161 TI - Malate-Induced Hysteresis of Phosphoenolpyruvate Carboxylase from Crassula argentea. AB - The hysteretic behavior of phosphoenolpyruvate (PEP) carboxylase from Crassula argentea has been investigated. Incubation of the purified enzyme with the inhibitor malate prior to starting the reaction by the addition of PEP resulted in a kinetic lag of several minutes duration. The length of the lag was inversely proportional to the enzyme concentration, suggesting subunit association dissociation as the hysteretic mechanism, rather than a mechanism based on a slow conformational change in the enzyme. Dynamic laser light scattering measurements also support this conclusion, showing that the diffusion coefficient of malate incubated enzyme slowly decreased after the reaction was started by the addition of PEP. Lags were observed only at pH values of 7.5 or lower. Maximum lags were observed after 10 min of preincubation with malate. Fumarate and succinate, which like malate caused mixed inhibition, also caused lags. In contrast, no lag was induced by malate in the presence of PEP or by the competitive inhibitor phosphoglycolate. The activators glucose 6-phosphate and malonate decreased the malate-induced lag. PMID- 16667162 TI - Parallel effects of freezing and osmotic stress on the ATPase activity and protein composition of the plasma membrane of winter rye seedlings. AB - The objective of this study was to determine the influence of freezing versus hypertonic stress on the ATPase activity and polypeptide profile of the plasma membrane of nonacclimated winter rye leaves (Secale cereale L. cv Puma). Exposure of leaves to hypertonic sorbitol solutions resulted in a similar extent of injury as did freezing to subzero temperatures that resulted in equivalent osmotic stresses. When isolated with a two-phase partition system of aqueous polymers, the plasma membrane fractions of control, frozen, or hypertonically stressed leaves were of similar purity as judged by the distribution of marker enzyme activities. When assayed in the presence of Triton X-100 (0.05% w/w), ATPase activity was decreased only slightly in plasma membrane fractions isolated from either frozen or hypertonically stressed leaves. In contrast, the specific ATPase activity of the plasma membrane fractions assayed in the absence of Triton X-100 increased following freezing or hypertonic stress. As a result, the Triton X-100 stimulation of the ATPase activity decreased significantly from sixfold in control leaves to threefold in lethally stressed leaves and reflects an increase in the permeability of the plasma membrane vesicles. The increased permeability was also manifested as a decrease in H(+)-transport following exposure to freezing or hypertonic stress. Both freezing and hypertonic exposure at subzero temperatures altered the polypeptide profile of the plasma membrane, but with the exception of one polypeptide, there was no difference between the two treatments. PMID- 16667163 TI - Low CO(2) Prevents Nitrate Reduction in Leaves. AB - The correlation between CO(2) assimilation and nitrate reduction in detached spinach (Spinacia oleracea L.) leaves was examined by measuring light-dependent changes in leaf nitrate levels in response to mild water stress and to artificially imposed CO(2) deficiency. The level of extractable nitrate reductase (NR) activity was also measured. The results are: (a) In the light, detached turgid spinach leaves reduced nitrate stored in the vacuoles of mesophyll cells at rates between 3 and 10 micromoles per milligram of chlorophyll per hour. Nitrate fed through the petiole was reduced at similar rates as storage nitrate. Nitrate reduction was accompanied by malate accumulation. (b) Under mild water stress which caused stomatal closure, nitrate reduction was prevented. The inhibition of nitrate reduction observed in water stressed leaves was reversed by external CO(2) concentrations (10-15%) high enough to overcome stomatal resistance. (c) Nitrate reduction was also inhibited when turgid leaves were kept in CO(2)-free air or at the CO(2)-compensation point or in nitrogen. (d) When leaves were illuminated in CO(2)-free air, activity of NR decreased rapidly. It increased again, when CO(2) was added back to the system. The half-time for a 50% change in activity was about 30 min. It thus appears that there is a rapid inactivation/activation mechanism of NR in leaves which couples nitrate reductase to net photosynthesis. PMID- 16667164 TI - Effect of water deficits on seed development in soybean : I. Tissue water status. AB - Water deficits during seed filling often decrease seed size in soybean (Glycine max L.). The physiological basis for this response is not known but may result from direct effects of low seed water potential (Psi(w)) on the seed filling process. To determine whether low Psi(w) occurred in reproductive tissues of soybean, we monitored the water status (Psi(w), Psi(s), and Psi(p)) of leaf, pericarp, and seed (embryo and testa) tissue of greenhouse-grown plants subjected to a brief water deficit during the linear period of seed growth. Water deficits were imposed by withholding water and monitored in the reproductive tissues by thermocouple psychrometry. When water was abundant, leaf, pericarp, and seed Psi(w) were -0.5 to -0.7 megapascal at midday. When water was withheld, leaf Psi(w) decreased to -2.3 megapascals within 6 days. Pericarp Psi(w) also decreased to -1.9 megapascal during this time. Pericarp Psi(s) followed the decline in Psi(w), but osmotic adjustment was not evident as the pericarp lost turgor completely by day 6. However, seed Psi(w), Psi(s), and Psi(p) were not significantly different from the controls. These results indicate that the water status of the developing seeds of soybean is not altered by short-term water deficits severe enough to inhibit the metabolic activity of the maternal plant. Maintenance of a favorable water status may be important for the conservation of seed growth rate exhibited by soybean under dry conditions. PMID- 16667165 TI - Effect of Water Deficits on Seed Development in Soybean : II. Conservation of Seed Growth Rate. AB - Water deficits during seed filling decrease seed size in soybean (Glycine max L.). This may result from a reduction in the supply of assimilates from the maternal plant and/or an inhibition of seed metabolism. To determine whether maternal or zygotic factors limited seed growth, we examined the effects of a plant water deficit on the supply of sucrose to and its utilization by developing embryos. Plants were grown in the greenhouse, and water deficits were imposed by withholding water for a period of 6 days during linear seed fill. When water was withheld, leaf water potential decreased rapidly, inhibiting canopy photosynthesis completely within 3 days. However, seed dry weight (nodes 7-11) continued to increase at or near the control rate. The level of total extractable carbohydrates in leaf, stem, and pericarp tissue decreased by 70, 50, and 45%, respectively, indicating that reserves were mobilized to support seed growth. Cotyledon sucrose content decreased from about 60 milligrams per gram dry weight to 30 milligrams per gram dry weight. Similarly, the concentration of sucrose in the interfacial apoplast of the cotyledons decreased from approximately 100 millimolar to 50 millimolar. However, the rate of sucrose accumulation by excised embryos, measured in a short-term in vitro assay, increased in response to the water deficit. These results indicate that both source and sink activity in soybean are altered by water deficits to maintain the flux of assimilates to the developing embryos. This may explain why seed growth is maintained, albeit for a shorter duration, when soybean is exposed to water deficits during the seed filling period. PMID- 16667166 TI - Roles of carbohydrate supply and phytohormones in maize kernel abortion. AB - Kernels at the ear tip of field grown maize (Zea mays L.) often abort during flowering, resulting in significant yield loss. The objective of this study was to determine if abortion is initiated by an inadequate supply of carbohydrates for growth of ear tip kernels, and/or by a hormonal signal. Field grown maize plants were either unshaded or shaded during flowering to increase kernel abortion. Nonstructural carbohydrates, indoleacetic acid (IAA), abscisic acid (ABA), and cytokinins were measured in aborting and nonaborting kernels, before and after abortion occurred. Kernel abortion was initiated 8 days after anthesis (DAA) and was complete by 12 DAA, when kernels ceased dry weight accumulation. Concentrations of reducing sugars, sucrose, and starch in aborting kernels were not significantly different from those in nonaborting kernels up to 12 DAA. Also, total carbohydrate concentrations were higher in the cob of aborting than of nonaborting kernels from 8 to 26 DAA. These data suggest that kernel abortion is not initiated by an inadequate supply of carbohydrates. However, accumulation of reducing sugars in the cob of aborting kernels suggests that transfer of sugars from cob to kernels is impaired early in the abortion process. Differences in IAA, ABA, and cytokinin concentrations between aborting and nonaborting kernels were only observed after abortion was complete. Kernel abortion is apparently not initiated by a signal of these hormones. After completion of abortion, aborted kernels contained higher concentrations of ABA and lower concentrations of IAA than nonaborted kernels. There was also a trend toward higher concentrations of zeatin riboside in nonaborted kernels. Abscisic acid may promote kernel abortion after the process has been initiated. A mechanism for kernel abortion is discussed. PMID- 16667167 TI - Change in Photosynthetic Capacity over the Cell Cycle in Light/Dark-Synchronized Amphidinium carteri Is Due Solely to the Photocycle. AB - Cell cycle dependent photosynthesis in the marine dinoflagellate Amphidinium carteri was studied under constant illumination and light/dark (L/D) photocycles to distinguish intrinsic cell cycle control from environmental influences. Cells were grown in constant light and on a 14:10 L:D cycle at light intensities that would yield a population growth rate of 1 doubling per day. In the former case division was asynchronous, and cells were separated according to cell cycle stage using centrifugal elutriation. Cells grown on the L:D cycle were synchronized, with division restricted to the dark period. Cell cycle stage distributions were quantified by flow cytometry. Various cell age groups from the two populations were compared as to their photosynthetic response (photosynthetic rate versus irradiance) to determine whether or not the response was modulated primarily by cell cycle constraints or the periodic L/D cycle. Cell cycle variation in photosynthetic capacity was found to be determined solely by the L/D cycle; it was not present in cells grown in constant light. PMID- 16667168 TI - Ca-selective microelectrodes and their application to plant cells and tissues. AB - The use of Ca(2+)-selective microelectrodes is difficult because of some basic problems: (a) electrodes with submicron tips may display non-Nernstian slopes; (b) liquid membrane microelectrodes respond only slowly (within seconds) to changes in ion activity; (c) turgid plant cells with tough walls damage the sensitive tip. This article describes concisely recent advances in fabricating Ca(2+)-selective single and double-barreled microelectrodes and their intracellular applications to different plant cell materials. Beveling the electrodes, mixing the sensor components with polyvinylchloride, insulation of the hydrated glass, and stabilization of the tips with inert materials are considered the basic concepts to circumvent most difficulties. It is concluded that the Ca(2+)-electrode can be a useful tool in plant physiology, but in spite of recent progress this technique remains experimentally demanding. PMID- 16667169 TI - Xenobiotic Monitoring in Plants by F and H Nuclear Magnetic Resonance Imaging and Spectroscopy: Uptake of Trifluoroacetic Acid in Lycopersicon esculentum. AB - (19)F and (1)H nuclear magnetic resonance imaging and spectroscopy have been used to monitor the uptake of trifluoroacetic acid in stems and leaves of Lycopersicon esculentum. The movement and location of a xenobiotic have been demonstrated in vivo by a noninvasive technique. PMID- 16667170 TI - Role of Ethylene in the Geotropic Response of Bermudagrass (Cynodon dactylon L. Pers.) Stolons. AB - We studied the relationship between ethylene and gravity-induced upward bending of bermudagrass (Cynodon dactylon L. Pers.) stolons. Ethylene production begins within 3 hours of the onset of gravistimulation, and increases thereafter until the 15th hour, after which it declines. There is a close positive relationship between ethylene production and upward bending during the first 12 hours of gravistimulation. Incubation of stolons with AgNO(3) did not prevent ethylene evolution but delayed upward bending. In addition, ethylene production was 10 fold greater and peaked earlier in gravistimulated nodes incubated with 1 aminocyclopropane 1-carboxylic acid. The gravitational stimulation could be due to an increase in both 1-aminocyclopropane 1-carboxylic acid synthase and the ethylene forming enzyme. The results suggest that ethylene promotes the activity of indoleacetic acid. PMID- 16667171 TI - Further identification of endogenous gibberellins in the shoots of pea, line g2. AB - To interpret the metabolism of radiolabeled gibberellins A(12)-aldehyde and A(12) in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [(14)C]GA(12) and [(14)C]GA(12)-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). [(14)C]GA(12) was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [(14)C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA(53), GA(44), GA(19) and GA(20) was demonstrated and their HPLC peak identity ascertained. The (14)C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA(53) to 4% in GA(20). Calculated levels of GA(20), GA(19), GA(44), and GA(53) were 42, 8, 10, and 0.5 nanograms/gram, respectively. PMID- 16667172 TI - Antibodies against the Calcium-Binding Protein: Calsequestrin from Streptanthus tortuosus (Brassicaceae). AB - Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca(2+) within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind (45)Ca(2+) and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein. PMID- 16667173 TI - Effects of sodium chloride on the hydraulic conductivity of soybean root systems. AB - Root system hydraulic conductivity (L(P)) was measured on soybean (Glycine max [L.] Merr. var Harosoy) seedlings grown in solution culture and exposed to varying levels of osmotic stress. Hydroponic growth solutions were salinized by additions of NaCl, and the permeability of excised seedling root systems to water was measured. Conductance was estimated at high rates of water flux, where osmotic effects are negligible. L(P) was reduced as the salinity of the growth solution increased. Growth in NaCl for 14 days at -0.17 megapascals and -0.26 megapascals resulted in reductions in L(P) from that of controls by 27% and 72%, respectively. L(P) was correlated with the root/shoot biomass ratio (RS), with larger values of L(P) observed in seedlings with lower RS. PMID- 16667174 TI - Phosphoinositides in barley aleurone layers and gibberellic Acid-induced changes in metabolism. AB - Phospholipids of barley (Hordeum vulgare L. cv Himalaya) aleurone layers were labeled with myo-[2-(3)H]inositol or [(32)Pi], extracted, and analyzed by physical (chromatography) and chemical (deacylation) techniques. Three phospholipids were found to incorporate both myo-[2-(3)H]inositol and [(32)Pi] phosphatidylinositol, phosphatidylinositol-monophosphate, and phosphatidylinositol-bisphosphate. Stimulation of [(3)H]inositol prelabeled aleurone layers with GA(3) showed enhanced incorporation of label into phosphatidylinositol within 30 seconds and subsequent rapid breakdown. Stimulation of phosphatidylinositol labeling observed in these studies is the earliest response of aleurone cells to gibberellic acid reported. PMID- 16667175 TI - Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. AB - Chimeric genes containing a pollen-specific promoter from tomato (Lycopersicon esculentum) or the CaMV35S promoter were transiently expressed following their introduction into tobacco (Nicotiana tabacum) pollen using high velocity microprojectiles. Transient expression of the microprojectile-introduced genes in leaves and pollen was similar to that observed for these genes in stably transformed tobacco plants. PMID- 16667176 TI - Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa. AB - A phosphoinositide-specific phospholipase C activity was identified in oat root (Avena sativa, cv Victory) plasma membranes purified by separation in an aqueous two-phase polymer system. The enzyme is highly active toward inositol phospholipids but only minimally active toward phosphatidylethanolamine and phosphatidylcholine. Activity approaches maximal levels at 200 micromolar phosphatidylinositol 4-phosphate (PIP) and is highly dependent on calcium; it is inhibited by 1 millimolar EGTA and is activated by calcium with an apparent activation constant of 2 micromolar. At 10 micromolar calcium and 200 micromolar inositol phospholipid, the enzyme is specific for phosphatidylinositol 4,5 bisphosphate (PIP(2)) and PIP, which are hydrolyzed at 10 and 4 times, respectively, the rate of phosphatidylinositol (PI) hydrolysis. The principle water soluble products of hydrolysis, as determined by high performance liquid chromatography, are inositol 1,4,5-trisphosphate from PIP(2), inositol 1,4 bisphosphate from PIP, and inositol phosphate from PI. PMID- 16667177 TI - An Enzymatic Conversion of Lipoxygenase Products by a Hydroperoxide Lyase in Blue Green Algae (Oscillatoria sp.). AB - An enzyme has been isolated from blue-green algae Oscillatoria sp. which utilizes the product, 13-hydroperoxy-9, 11-octadecadienoic acid (13-HPOD), of lipoxygenase for its substrate. This enzyme, termed hydroperoxide lyase, converts the conjugated diene 13-hydroperoxide of linoleic acid to 13-oxotrideca-9, 11-dienoic acid. The structure of the latter has been determined by ultraviolet spectroscopy and mass spectrometry. 9-HPOD is not a substrate for this enzyme. The hydroperoxide lyase from Oscillatoria sp. has a maximum of activity at pH 6.4 and 30 degrees C. The molecular weight of the enzyme was estimated at 56,000. The enzyme was not inhibited by BW 755C, but was inhibited by molecules containing more than one hydroxyl group. Quercetin was found to be the best inhibitor of the enzyme activity. The purified hydroperoxide lyase from Oscillatoria sp. showed an apparent K(m) of 7.4 micromolar and a V(max) of 35 nanomoles per minute per milligram of protein for 13-HPOD. An enzymatic pathway for the biogenesis of oxodienoic acid from linoleic acid is proposed. This involves the sequential activity of lipoxygenase and hydroperoxide lyase enzymes. PMID- 16667178 TI - Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scutellum, and rapeseed cotyledon of maturing seeds. AB - Lysophosphatidate (LPA) acyltransferase (EC 2.3.1.51) in the microsomes from palm endosperm (Syagrus cocoides Martius), maize scutellum (Zea mays L.), and rapeseed cotyledon (Brassica napus L.) of maturing seeds were studied for their specificities toward the acyl moiety of the substrates lysophosphatidate and acyl coenzyme A (CoA). The LPA acceptor greatly influenced the acyl CoA specificity of the enzyme and vice versa. With 1-oleoyl-lysophosphatidate (LPA-18:1), the palm enzyme was equally active on oleoyl CoA and lauroyl CoA, whereas the maize and rapeseed enzymes were more active on oleoyl CoA than on lauroyl CoA. With 1 lauroyl-lysophosphatidate (LPA-12), which generated less activity than LPA-18:1, the palm enzyme was three times more active on lauroyl CoA than on oleoyl CoA. LPA-12 was an inactive substrate for the maize and rapeseed enzymes. The selectivity of the enzymes was also studied using a mixture of LPA-18:1 and LPA 12, as well as lauroyl CoA and oleoyl CoA. Under this selectivity condition and compared to the specificity condition, the enzymes from all the three seeds exerted stronger preference for oleoyl moiety in either the LPA or acyl CoA, and again, only the palm enzyme could act on LPA-12. Similar studies, although in lesser detail, showed that the enzymes from soybean and castor bean were similar to the maize and rapeseed enzymes in having little activity on substrates containing lauroyl moiety. The results demonstrate the importance of the acyl group in the sn-1 position of LPA in determining the acyl preference in the sn-2 position in phosphatidate synthesis. The palm enzyme appears to be the only one capable of synthesizing phosphatidates containing high amounts of lauric moieties. PMID- 16667179 TI - Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide. AB - The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca(2+) gradients, induced by the toxin in T corn was prevented by N,N'-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N'-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site. PMID- 16667180 TI - Properties of Solubilized Microsomal Lipase from Germinating Brassica napus. AB - Lipase (triacylglycerol acylhydrolase [EC 3.1.1.3.]) was extracted from the microsomal fraction of cotyledons of dark grown seedlings of Canola (Brassica napus L. cv Westar) by treatment with Triton X-100. The enzyme was partially purified by chromatography on Sephacryl S-300 and DEAE Bio-Gel and was stable when stored at -20 degrees C in 50% (v/v) glycerol. The lipase aggregated readily but the distribution of species present in solution could be controlled by nonionic detergents. A species with an apparent M(r) of about 250,000 was obtained by gel filtration chromatography in the presence of 1% (v/v) Triton X 100. Lipase activity was optimal near neutral pH, and the reaction approached maximum velocity at a concentration of 0.5 to 1 millimolar emulsified triolein. The reaction rate responded linearly to temperature up to about 40 degrees C and the hydrolytic process had an activation energy of 18 kilocalories per mole. Microsomal lipase lost about 20% and 80% activity when heat-treated for 1 hour at 40 degrees C and 60 degrees C, respectively. At appropriate concentrations, the detergents Triton X-100, n-octyl-beta-d-glucopyranoside, (3-[(3-cholamidopropyl-O dimethylammonio]-1-propanesulfonate, cetyl trimethylammonium bromide, and sodium dodecyl sulfate all inhibited lipase activity. n-Octyl-beta-d-glucopyranoside, however, was stimulatory in the 2 to 8 millimolar concentration range. The inhibitory effects of Triton X-100 were reversible. PMID- 16667181 TI - Low growth temperature-induced increase in light saturated photosystem I electron transport is cation dependent. AB - Thylakoid membranes isolated from cold tolerant, herbaceous monocots and dicots grown at 5 degrees C exhibit a 1.5-fold to 2.7-fold increase in light saturated rates of photosystem I (PSI) electron transport compared to thylakoids isolated from the same plant species grown at 20 degrees C. This was observed only when either water or reduced dichlorophenolindophenol was used as an electron donor. The apparent quantum yield for PSI electron transport was not affected by growth temperature. The higher light saturated rates of PSI electron transport in 5 degrees C thylakoids had an absolute requirement for the presence of Na(+) and Mg(+2). The accessibility of reduced dichlorophenolindophenol to the donor site was not affected by growth temperature since 5 degrees C and 20 degrees C thylakoids exhibited no significant difference in the concentration of this electron donor required for half-maximal PSI activity. The cation dependent higher rates of light saturated PSI activity were also observed when rye thylakoids were developed under intermittent light conditions at 5 degrees C. Thus, this cation effect on PSI activity appeared to be independent of light harvesting complex I and II. The extent of the in vitro reversibility of this cation effect appeared to be limited by an inherent decay process for PSI electron transport. The rate of decay for PSI activity was greatest when thylakoids were isolated in the absence of NaCl and MgCl(2). We conclude that exposure of plants to low growth temperatures induces a reorganization of thylakoid membranes which increases the light saturated rates of PSI electron transport with no change in the apparent quantum efficiency for this reaction. Cations are required to stabilize this reorganization. PMID- 16667182 TI - Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells. AB - The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K(m) of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30 degrees C. Several effectors known to inhibit ATPase (N,N'-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H(+) electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN(-) and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH-, OH-, and NH(2)- groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d amino acids generated under stress conditions. PMID- 16667183 TI - Flavanone Glycoside Biosynthesis in Citrus: Chalcone Synthase, UDP Glucose:Flavanone-7-O-Glucosyl-Transferase and -Rhamnosyl-Transferase Activities in Cell-Free Extracts. AB - Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species. PMID- 16667184 TI - Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane Enriched Microsomes: III. Effect of Temperature and Cations on Protein Sulfhydryl Reactivity. AB - Temperature and cations modified the reaction of barley (Hordeum vulgare L. cv Conguest) root plasma membrane protein sulfhydryl groups with N-4-(7-diethylamino 4-methylcoumarin-3-yl)-phenyl maleimide (CPM). The pseudo-first-order rate constants for the formation of fluorescent CPM-protein adducts increased as the temperature was raised above 30 degrees C, suggesting changes in protein conformation. Monovalent [K(I), Na(I), L(I)] and certain divalent cations [Ba(II), Mg(II)] increased the reaction rates. Other divalent cations [Ca(II), Mn(II), Ni(II), Co(II), Sr(II), Cd(II), Hg(II)] decreased the rate of fluorescent adduct formation. Na(I) promoted and Ca(II) delayed the onset of the temperature dependent increases in reaction rates. The results are discussed in terms of lipid-mediated, temperature-dependent changes in membrane protein conformation and ion-protein interactions. PMID- 16667185 TI - Abscisic Acid Levels during Early Seed Development in Sechium edule Sw. AB - The time-course growth of single tissues in pollinated and unpollinated ovules of Sechium edule Sw. is described in relation to the endogenous levels of abscisic acid. Quantitation of abscisic acid (ABA) in the minute amounts of material obtained after ovule dissection has been performed by using a highly specific and sensitive solid-phase radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA. While the absolute amount of ABA rises in both types of ovules, only in unpollinated ones does this leads to an increase in the hormone concentration. Infact in pollinated ovules the rapid growth following pollination prevents, through a dilution effect, the increase in ABA concentration. Growth patterns and endogenous ABA levels are similar for integuments and nucellus tissues either in pollinated or unpollinated ovules. It is suggested that the growth inhibition induced by the increase in ABA concentration after anthesis could be counteracted by the pollination triggered fast ovule growth. PMID- 16667186 TI - Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway. AB - Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO(2) assimilation or oxygen evolution, over a wide range of CO(2) concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO(2) near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO(2) concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO(3) (-), indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of (14)C-incorporation from (14)CO(2) into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO(2) occurs predominantly by direct CO(2) fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO(2) concentrations, permeability coefficients for CO(2) flux into bundle sheath cells were determined for a range of C(4) species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C(3) plants. These results support the concept that a CO(2) permeability barrier exists to allow the development of high CO(2) concentrations in bundle sheath cells during C(4) photosynthesis. PMID- 16667187 TI - CO(2) Concentrating Mechanism of C(4) Photosynthesis: Permeability of Isolated Bundle Sheath Cells to Inorganic Carbon. AB - Diffusion of inorganic carbon into isolated bundle sheath cells from a variety of C(4) species was characterized by coupling inward diffusion of CO(2) to photosynthetic carbon assimilation. The average permeability coefficient for CO(2) (P(CO(2) )) for five representatives from the three decarboxylation types was approximately 20 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. The average value for the NAD-ME species Panicum miliaceum (10 determinations) was 26 with a standard deviation of 6 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. A P(CO(2) ) of at least 500 micromoles per minute per milligram chlorophyll per millimolar was determined for cells isolated from the C(3) plant Xanthium strumarium. It is concluded that bundle sheath cells are one to two orders of magnitude less permeable to CO(2) than C(3) photosynthetic cells. These data also suggest that CO(2) diffusion in bundle sheath cells may be made up of two components, one involving an apoplastic path and the other a symplastic (plasmodesmatal) path, each contributing approximately equally. PMID- 16667188 TI - Mechanism of c(4) photosynthesis: a model describing the inorganic carbon pool in bundle sheath cells. AB - A theoretical model of the composition of the inorganic carbon pool generated in C(4) leaves during steady-state photosynthesis was derived. This model gives the concentrations of CO(2) and O(2) in the bundle sheath cells for any given net photosynthesis rate and inorganic carbon pool size. The model predicts a bundle sheath CO(2) concentration of 70 micromolar during steady state photosynthesis in a typical C(4) plant, and that about 13% of the inorganic carbon generated in bundle sheath cells would leak back to the mesophyll cells, predominantly as CO(2). Under these circumstances the flux of carbon through the C(4) acid cycle would have to exceed the net rate of CO(2) assimilation by 15.5%. With the calculated O(2) concentration of 0.44 millimolar, the potential photorespiratory CO(2) loss in bundle sheath cells would be about 3% of CO(2) assimilation. Among the factors having a critical influence on the above values are the permeability of bundle sheath chloroplasts to HCO(3) (-), the activity of carbonic anhydrase within these chloroplasts, the assumed stromal volume, and the permeability coefficients for CO(2) and O(2) diffusion across the interface between bundle sheath and mesophyll cells. The model suggests that as the net photosynthesis rate changes in C(4) plants, the level and distribution of the components of the inorganic carbon pool change in such a way that C(4) acid overcycling is maintained in an approximately constant ratio with respect to the net photosynthesis rate. PMID- 16667189 TI - In Vivo Biosynthetic Studies of the Dolichos biflorus Seed Lectin. AB - The in vivo biosynthesis of the Dolichos biflorus seed lectin was studied by pulse-chase labeling experiments using [(35)S] methionine and [(14)C]glucosamine. These studies demonstrate that each of the two mature lectin subunit types are derived by the processing of separate glycosylated precursors. The appearance of the precursor to subunit I before the precursor to subunit II supports the possibility raised by previous studies that both subunit types of this lectin may originate from a single gene product. PMID- 16667190 TI - Peroxisomal degradation of branched-chain 2-oxo acids. AB - Branched-chain 2-oxo acids which are formed by transamination of leucine, isoleucine, and valine are metabolized by the peroxisomes from mung bean (Vigna radiata L.) hypocotyls. Acylcoenzyme A (CoA) thio ester intermediates of the pathways were separated by reversed-phase high performance liquid chromatography. Retention time and cochromatography of individual acyl-CoA reference standards were used for identification of the acyl-CoA esters separated from the assay mixtures. Based on the results of identification and those of kinetic experiments, pathways of the peroxisomal degradation of 2-oxoisocaproate, 2 oxoisovalerate, and 2-oxo-3-methylvalerate are suggested. PMID- 16667191 TI - Structural Similarities between Spinach Chloroplast and Cytosolic Class I Fructose 1,6-Bisphosphate Aldolases : Immunochemical and Amino-Terminal Amino Acid Sequence Analysis. AB - Immunochemical studies using polyclonal antisera prepared individually against highly purified cytosolic and chloroplast spinach leaf (Spinacia oleracea) fructose bisphosphate aldolases showed significant cross reaction between both forms of spinach aldolase and their heterologous antisera. The individual cross reactions were estimated to be approximately 50% in both cases under conditions of antibody saturation using a highly sensitive enzyme-linked immunosorbent assay. In contrast, the class I procaryotic aldolase from Mycobacterium smegmatis and the class II aldolase from yeast (Saccharomyces cerevisiae) did not cross react with either type of antiserum. The 29 residue long amino-terminal amino acid sequences of the procaryotic M. smegmatis and the spinach chloroplast aldolases were determined. Comparisons of these sequences with those of other aldolases showed that the amino-terminal primary structure of the chloroplast aldolase is much more similar to the amino-terminal structures of class I cytosolic eucaryotic aldolases than it is to the corresponding region of the M. smegmatis enzyme, especially in that region which forms the first "beta sheet" in the secondary structure of the eucaryotic aldolases. Moreover, results of a systematic comparison of the amino acid compositions of a number of diverse eucaryotic and procaryotic fructose bisphosphate aldolases further suggest that the chloroplast aldolase belongs to the eucaryotic rather than the procaryotic "family" of class I aldolases. PMID- 16667192 TI - Properties of proteoliposomes containing fusicoccin receptors from maize. AB - We have recently described a fusicoccin (FC)-sensitive system reconstituted by inserting into liposomes FC-receptors and H(+)-ATPase-enriched preparations from maize tissues. While the proteoliposomes of maize H(+)-ATPase had been already investigated, those of FC-receptors required a careful characterization before use in the dual system. In particular, the influence of the phospholipid environment on time-course, reversibility, and pH-dependence of the FC-binding reaction has been studied by comparing these properties in microsome-bound, solubilized, and liposome-entrapped receptors. Similarities and differences between the results of this investigation and those previously obtained with FC receptors from spinach leaves suggest that functionally similar binding proteins from monocot and dicot plants have distinct structural features. PMID- 16667193 TI - Competitive Al Inhibition of Net Mg Uptake by Intact Lolium multiflorum Roots : I. Kinetics. AB - Aluminum impairs uptake of Mg(2+), but the mechanisms of this inhibition are not understood. The depletion technique was used to monitor net Mg(2+) uptake from nutrient solution by intact, 23-day-old plants of ryegrass (Lolium multiflorum Lam., cv Gulf and Wilo). Activities of Mg(2+) and monomeric Al species in nutrient solution were calculated and used as the basis for expressing the results. The kinetics of net Mg(2+) absorption was resolved into (a) a transpiration-dependent uptake component, (b) a metabolically mediated, discontinuous saturable component that is Al(3+) sensitive and p chloromercuribenzene sulfonic acid (PCMBS) resistant, and (c) a linear, carbonyl cyanide m-chlorophenylhydrazone resistant, Al(3+) sensitive component that might be a type of facilitated diffusion. Lowering the pH from 6.0 to 4.2 exerted a noncompetitive inhibition of net Mg(2+) uptake, while aluminum at 6.6 micromolar Al(3+) activity exerted competitive inhibition of net Mg(2+) uptake at pH 4.2. The Al(3+)-induced effect was obvious after 30 minutes. Cultivar-specific ability to retain a higher affinity for Mg(2+) by postulated transport proteins in the presence of Al(3+) might be one of the mechanisms of differential Al tolerance among ryegrass cultivars. PMID- 16667194 TI - Proteolytic Activity at Alkaline pH in Oat Leaves, Isolation of an Aminopeptidase. AB - Proteolytic activity in oat leaf extracts was measured with both azocasein and ribulose bisphosphate carboxylase (Rubisco) as substrates over a wide range of pH (3.0-9.2). With either azocasein or Rubisco activity peaks appeared at pH 4.8, 6.6, and 8.4. An aminopeptidase (AP) which hydrolyzes leucine-nitroanilide was partially purified. Purification consisted of a series of six steps which included ammonium sulfate precipitation, gel filtration, and two ionic exchange chromatographies. The enzyme was purified more than 100-fold. The apparent K(m) for leucine-nitroanilide is 0.08 millimolar at its pH optimum of 8.4. AP may be a cystein protease since it is inhibited by heavy metals and activated by 2 mercaptoethanol. Isolated chloroplasts were also able to hydrolyze leucine nitroanilide at a pH optimum of 8.4, indicating that AP could be localized inside the photosynthetic organelles. PMID- 16667195 TI - Monoclonal Antibodies against Pectin: Recognition of a Conformation Induced by Calcium. AB - Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the ;egg box' model. PMID- 16667196 TI - Isolation and Characterization of Protein Bodies in Lupinus angustifolius. AB - Using Nycodenz, a novel density gradient medium, we isolated intact protein bodies from developing seeds of Lupinus angustifolius L. (cultivar Unicrop) and achieved excellent separation from the endoplasmic reticulum, mitochondria, and other organelles. The distribution of the storage protein conglutin-beta was taken as evidence that up to 96% of the protein bodies remained intact on the gradients and banded at 1.25 grams per milliliter. The protein bodies also contained the three other abundant proteins present in L. angustifolius seeds: conglutins-alpha, -gamma, and -delta. Pulse labeling experiments were carried out to determine the site of proteolytic processing of conglutin-alpha, a legumin like 11Svedberg unit storage protein. Cotyledons aged either 33 or 40 days after flowering were pulsed with [(3)H]leucine. Protein bodies obtained from the cotyledons aged 33 days after flowering contained only the labeled precursors of conglutin-alpha with molecular weights 85,000, 72,000, and 64,000, even after a 4 hour chase of the radioactivity. Protein bodies obtained from the cotyledons aged 40 days after flowering contained the same radioactive precursors if the tissue had been pulsed for 2 hours, and the processing products of these precursors when the tissue had been chased for 4 hours. These studies confirm that the subcellular location of proteolytic cleavage of this legumin-like protein is the protein body, that this activity is detected only in protein bodies from lupin seeds aged between 33 and 40 days of seed development after flowering and that protein bodies from seeds younger than this contain only unprocessed conglutin alpha. PMID- 16667197 TI - Stress-induced accumulation of wheat germ agglutinin and abscisic Acid in roots of wheat seedlings. AB - Wheat germ agglutinin (WGA) levels in roots of 2-day-old wheat seedlings increased up to three-fold when stressed by air-drying. Similar results were obtained when seedling roots were incubated either in 0.5 molar mannitol or 180 grams per liter polyethylene glycol 6000, with a peak level of WGA after 5 hours of stress. Longer periods of osmotic treatment resulted in a gradual decline of WGA in the roots. Since excised wheat roots incorporate more [(35)S]cysteine into WGA under stress conditions, the observed increase of lectin levels is due to de novo synthesis. Measurement of abscisic acid (ABA) levels in roots of control and stressed seedlings indicated a 10-fold increase upon air-drying. Similarly, a five- and seven-fold increase of ABA content of seedling roots was found after 2 hours of osmotic stress by polyethylene glycol 6000 and mannitol, respectively. Finally, the stress-induced increase of WGA in wheat roots could be inhibited by growing seedlings in the presence of fluridone, an inhibitor of ABA synthesis. These results indicate that roots of water-stressed wheat seedlings (a) contain more WGA as a result of an increased de novo synthesis of this lectin, and (b) exhibit higher ABA levels. The stress-induced increase of lectin accumulation seems to be under control of ABA. PMID- 16667198 TI - Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6 bisphosphate concentration. AB - In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO(2). Glucose 6-phosphate (G-6-P), fructose 6 phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P(2)) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P(2) interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO(2) production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P(2)) concentration decreased to (1/2) that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P(2) concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P(2) concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P(2). PMID- 16667199 TI - Purification and Characterization of Pectinmethylesterase from Ficus awkeotsang Makino Achenes. AB - Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4 degrees C for at least 6 months, and at 60 degrees C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K(m) value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V(max) value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds. PMID- 16667200 TI - Negative pressures produced in an artificial osmotic cell by extracellular freezing. AB - A rigid artificial osmotic cell has been constructed using reverse osmosis membranes that were supported by metal grids from both sides to yield a high elastic modulus of the system. The cell could be subjected to changes of external water potential either by evaporation or by application of hypertonic solutions so that negative internal pressures or tensions (i.e. pressures smaller than atmospheric) could be built up. Negative pressures were also obtained by freeze induced dehydration when the cell was cooled to -1.5 degrees C and ice was formed on the outer surface. Tensions of up to -0.7 megapascals (-7 bars) could be established in the different types of experiments. Smaller tensions could be kept in the cell for several hours. Cavitations caused the pressure to increase instantaneously to values of about -0.1 megapascals (relative to atmospheric pressure) as theoretically expected. Cavitations could be reversed by pressurizing the system. The cell could be cooled to subzero temperatures while the cell solution was under tension. Intracellular freezing could be easily detected from an instantaneous increase in pressure. When the membrane was not supported by a grid from the inside (analogous to the situation in plant cells), no tensions could be built up in the system. The results support the idea of the incidence of negative pressures during freezing, if the wall is sufficiently rigid to prevent cell collapse and if the membrane does not separate from the cell wall. PMID- 16667201 TI - Increased Potassium Absorption Confers Resistance to Group IA Cations in Rubidium Selected Suspension Cells of Brassica napus. AB - Cell lines of suspension cultures of Brassica napus cv. Jet Neuf were identified for their ability to tolerate 100 millimolar Rb(+), a level which was double the normally lethal concentration. Ten spontaneous isolates were obtained from approximately 5 x 10(7) cells, one of which was reestablished as a cell suspension. This cell line, JL5, was also resistant to the other group IA cations Li(+), Na(+), K(+), and Cs(+)-and this trait was stable for at least 30 cell generations in the absence of Rb(+) selection pressure. The growth characteristics were similar to those of sensitive cells under nonselective conditions. The selected JL5 cells were shown by analysis to have effected more net accumulation of K(+) and Rb(+) and less of Na(+) than did the unselected cells. JL5 and unselected cells after 14 days of culture in basal medium contained 597.2 and 258.2 micromoles of K per gram dry weight, respectively. Michaelis-Menten kinetic analysis of K(+) influx showed that JL5 possessed an elevated phase 1 V(max), but there was no alteration in its K(m). This is the first time that a plant mutation has been shown to have both increased influx and net absorption of a major essential cation. PMID- 16667202 TI - Abscisic Acid ELISA: Organic Acid Interference. AB - Consideration must be exercised in determination of buffers and solutions used when carrying out enzyme-linked immunosorbent assays (ELISAs). A commercial monoclonal antibody kit for abscisic acid (Idetek, Inc.) gives significant false positives with tricarboxylic acid cycle intermediates. The organic acids or contaminants interfered with ELISA assays for ABA as indicated by deviations in the slopes of standard curves of ABA in the organic acids. The interference, in the case of alpha-ketoglutarate, was caused by a contaminant. Of the organic buffers tested-Tris, Tricine, and Hepes-only Hepes showed false-positive ABA. In addition, we present data indicating the presence of ABA in commercial mannitol and provide a simple procedure for removal of the ABA. PMID- 16667203 TI - Sensitivity of Altitudinal Ecotypes of the Wild Tomato Lycopersicon hirsutum to Chilling Injury. AB - The transition temperature of the leaf polar lipids and the critical temperature for chill-induced inhibition of photosynthesis was determined for three altitudinal ecotypes of the wild tomato Lycopersicon hirsutum. Photosynthesis was measured as CO(2)-dependent O(2) evolution at 25 degrees C after leaf slices were exposed to chilling temperatures for 2 hours at a moderate photon flux density of 450 micromoles per square meter per second. The transition temperature of the leaf polar lipids was detected from the change in the temperature coefficient of the fluorescence intensity of trans-parinaric acid. Chill-induced photoinhibition was evident in the three tomato ecotypes when they were chilled below a critical temperature of 10 degrees , 11 degrees , and 13 degrees C, respectively, for the high (LA1777), mid (LA1625), and low (LA1361) altitudinal ecotypes. The temperature differential, below the critical temperature, required to produce a 50% inhibition was also similar for the three ecotypes. A transition was detected in the leaf polar lipids of these plants at a temperature similar to that of the critical temperature for photoinhibition. The results show that the three tomato ecotypes are similar with respect to their critical temperature for chilling induced photoinhibition and the rate of their response to the chilling stress. They are, thus, similarly sensitive to chilling. PMID- 16667204 TI - Intracellular Localization of Enzymes of Fatty Acid-beta-Oxidation in the Alga Cyanidium caldarium. AB - The intracellular distribution of enzymes, participating in the beta-oxidation of fatty acids in the eucaryotic alga Cyanidium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and thiolase were present in the mitochondrial fraction (density: 1.19 gram per cubic centimeter). Activity of an acyl-CoA synthetase was found in the mitochondrial fraction as well as in a band where mitochondrial membrane apparently had accumulated (density: 1.17 gram per cubic centimeter). None of these enzymes were present in the peroxisomes (density: 1.23 gram per cubic centimeter). Results from cell fractionation as well as properties of beta-oxidation enzymes indicate a mitochondrial location of fatty acid degradation also in the algae Galdieria sulphuraria and Cyanidioschyzon merolae. PMID- 16667205 TI - Novel procedure for extraction of a latent grape polyphenoloxidase using temperature-induced phase separation in triton x-114. AB - Polyphenoloxidase from grape berries is extracted only by nonionic detergents with a hydrophilic-lipophilic balance between 12.4 and 13.5. The enzyme was partially purified in latent form, free of phenolics and chlorophylls, by using temperature phase partitioning in a solution of Triton X-114. This method permits the purification of the enzyme with the same fold purification as the commonly used method, but with a yield three times higher and a 90% reduction in time needed. The latent enzyme can be activated by different treatments, including trypsin and cationic and anionic detergents. Cetyltrimethylamonium bromide was found to be the most effective detergent activator, followed by sodium dodecyl sulfate. Polyphenoloxidase in grape berries, in spite of being an integral membrane protein, had an anomalous interaction with Triton X-114, remaining in the detergent-poor phase after phase separation. This could be explained by its having a short hydrophobic tail that anchors it to the membrane. PMID- 16667206 TI - Isolation of gibberellin precursors from heavily pigmented tissues. AB - The kauranoid precursors of gibberellins are difficult to isolate from heavily pigmented plant tissues. In this paper, we describe relatively simple and efficient procedures for the purification of these compounds from tissues containing chlorophyll and other high molecular weight pigments. Extracts of shoots from Thlaspi arvense L. were subjected first to size exclusion chromatography using ethyl acetate as the eluting solvent. This procedure resulted in the separation of kauranoids as a class of compounds from chlorophyll. Typically, a 90% reduction in mass of the kauranoid enriched fraction was observed. This fraction was subjected to reverse phase high performance liquid chromatography and individual fractions analyzed by combined gas chromatography-mass spectrometry. Five kauranoids were identified in shoot extracts of T. arvense: ent-kaur-16-ene, ent-kaur-16-en-19-ol, ent-kaur-16-en-19 oic acid, trachylobanoic acid, and 7beta, 13-dihydroxykaurenolide. The metabolic relationships of these compounds to the gibberellins previously identified in this species (JD Metzger, MC Mardaus [1986] Plant Physiol 80: 396-402) are discussed. In addition, the utility of size exclusion chromatography in preparative situations is demonstrated by the purification of ent-kaurenoic acid in milligram quantities from the florets of Helianthus annuus L. PMID- 16667207 TI - Low and High Temperature Limits to PSII : A Survey Using trans-Parinaric Acid, Delayed Light Emission, and F(o) Chlorophyll Fluorescence. AB - Many studies have shown that membrane lipids of chilling-sensitive plants begin lateral phase separation (i.e. a minor component begins freezing) at chilling temperatures and that chilling-sensitive plants are often of tropical origin. We tested the hypothesis that membranes of tropical plants begin lateral phase separation at chilling temperatures, and that plants lower the temperature of lateral phase separation as they invade cooler habitats. To do so we studied plant species in one family confined to the tropics (Piperaceae) and in three families with both tropical and temperate representatives (Fabaceae [Leguminosae], Malvaceae, and Solanaceae). We determined lateral phase separation temperatures by measuring the temperature dependence of fluorescence from trans parinaric acid inserted into liposomes prepared from isolated membrane phospholipids. In all families we detected lateral phase separations at significantly higher temperatures, on average, in species of tropical origin. To test for associated physiological effects we measured the temperature dependence of delayed light emission (DLE) by discs cut from the same leaves used for lipid analysis. We found that the temperature of maximum DLE upon chilling was strongly correlated with lateral phase separation temperatures, but was on average approximately 4 degrees C lower. We also tested the hypothesis that photosystem II (PSII) (the most thermolabile component of photosynthesis) of tropical plants tolerates higher temperatures than PSII of temperate plants, using DLE and F(o) chlorophyll fluorescence upon heating to measure the temperature at which PSII thermally denatured. We found little difference between the two groups in PSII denaturation temperature. We also found that the temperature of maximum DLA upon heating was not significantly different from the critical temperature for F(o) fluorescence. Our results indicate that plants lowered their membrane freezing temperatures as they radiated from their tropical origins. One interpretation is that the tendency for membranes to begin freezing at chilling temperatures is the primitive condition, which plants corrected as they invaded colder habitats. An alternative is that membranes which freeze at temperatures only slightly lower than the minimum growth temperature confer an advantage. PMID- 16667208 TI - Effects of Ambient and Acute Partial Pressures of Ozone on Leaf Net CO(2) Assimilation of Field-Grown Vitis vinifera L. AB - Mature, field-grown Vitis vinifera L. grapevines grown in open-top chambers were exposed to either charcoal-filtered air or ambient ozone partial pressures throughout the growing season. Individual leaves also were exposed to ozone partial pressures of 0.2, 0.4, or 0.6 micropascals per pascal for 5 hours. No visual ozone damage was found on leaves exposed to any of the treatments. Chronic exposure to ambient O(3) partial pressures reduced net CO(2) assimilation rate (A) between 5 and 13% at various times throughout the season when compared to the filtered treatment. Exposure of leaves to 0.2 micropascals per pascal O(3) for 5 hours had no significant effect on A; however, A was reduced 84% for leaves exposed to 0.6 micropascals per pascal O(3) when compared to the controls after 5 hours. Intercellular CO(2) partial pressure (c(i)) was lower for leaves exposed to 0.2 micropascals per pascal O(3) when compared to the controls, while c(i) of the leaves treated with 0.6 micropascals per pascal of 0(3) increased during the fumigation. The long-term effects of ambient O(3) and short-term exposure to acute levels of O(3) reduced grape leaf photosynthesis due to a reduction in both stomatal and mesophyll conductances. PMID- 16667209 TI - Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C(4) Plant, Panicum miliaceum L. : Identification and Kinetic Characterization. AB - The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-(3)H]1,2-(2,2' -disulfo-4,4' -diisothiocyano)diphenylethane ([(3)H]H(2)DIDS) and by using SDS-PAGE. Assay of (32)Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C(3) counterpart and can be regarded as C(4) types. PMID- 16667210 TI - Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO(2). AB - Photosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO(2) for growth, and a unique cotton cell line that grows at ambient CO(2) (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C(3) mesophyll leaf cells than the cell suspension cultures previously studied. The pattern of (14)C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO(2) fixation occurred primarily by the C(3) pathway. Photorespiration occurred at 330 microliters per liter CO(2), 21% O(2) as indicated by the synthesis of high levels of (14)C-labeled glycine and serine in a pulse-chase experiment and by oxygen inhibition of CO(2) fixation. Short-term CO(2) fixation in the presence and absence of carbonic anhydrase showed CO(2), not HCO(3) (-), to be the main source of inorganic carbon taken up by the low CO(2)-requiring cotton cells. The cells did not have a CO(2)-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low CO(2)-requiring cotton cells, present in the high CO(2)-requiring soybean cell lines, and absent in other high CO(2) cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO(2) concentrations. PMID- 16667211 TI - Heat-stable proteins and abscisic Acid action in barley aleurone cells. AB - [(35)S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100 degrees C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heatstable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered. PMID- 16667212 TI - Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits. AB - Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 +/- 0.3 micromole per hour per gram fresh weight and 2.2 +/- 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality. PMID- 16667213 TI - Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays. AB - Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c(1) subunits and by inference from comigration. PMID- 16667214 TI - Leaf anatomy of c(3)-c(4) species as related to evolution of c(4) photosynthesis. AB - This study was undertaken to examine the degree of Kranz anatomy development in the species intermediate to C(3) and C(4) types (C(3)-C(4)) in Panicum, Neurachne, Flaveria, and Moricandia. In each genus, C(3) and/or C(4) species were used for comparison. Leaf transections from each species were examined by light and transmission electron microscopy. The percentages of leaf photosynthetic cell profiles partitioned to bundle sheaths were higher in C(4) than in C(3) species, while C(3)-C(4) species tended to be in between. However, percentages for C(3) C(4) species in Moricandia and some C(3)-C(4)Flaveria species were not greater than C(3). When expressed on a cell profile area basis, C(3)-C(4) species partitioned more photosynthetic tissue to bundle sheaths than C(3) species in Moricandia, but not in Flaveria. Neurachne minor S. T. Blake (C(3)-C(4)) partitioned a very small portion of cell profile area to the inner bundle sheaths (5%) compared to Neurachne munroi F. Muell (C(4)) (21%). The percentage of organelles partitioned to bundle sheaths was much greater in C(3)-C(4) than in C(3) species. The average C(3) percentages for mitochondria plus peroxisomes were 19, 8, and 19.5% for Neurachne, Flaveria, and Moricandia, respectively, compared to 41, 29, and 46.5% for the C(3)-C(4) species. The CO(2) compensation concentration was negatively related to the partitioning of tissue to bundle sheaths and to the percentage of organelles in bundle sheaths. It is concluded that all of the C(3)-C(4) species examined have developed some degree of Kranz anatomy and that this altered anatomy is involved in their reduced apparent photorespiration. PMID- 16667215 TI - Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle : Evidence for Partial Oxidative and Reductive Pathways during Dark Ammonium Assimilation. AB - Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH(4) (+) in the dark under anaerobic conditions. Addition of NH(4) (+) to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO(2) efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H(14)CO(3) (-) to anaerobic cells assimilating NH(4) (+) results in the incorporation of radiolabel into the alpha-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH(4) (+) addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply alpha-ketoglutarate for glutamate production. During dark aerobic NH(4) (+) assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH(4) (+) assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH(4) (+) assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity. PMID- 16667216 TI - Growth and development temperature influences level of tolerance to high light stress. AB - The influence of growth and development temperature on the relative tolerance of photosynthetic tissue to high light stress at chilling temperatures was investigated. Two tuber-bearing potato species, Solanum tuberosum L. cv Red Pontiac and Solanum commersonii were grown for 4 weeks, at either 12 or 24 degrees C with 12 hours of about 375 micromoles per second per square meter of photosynthetically active radiation. Paired leaf discs were cut from directly across the midvein of leaflets of comparable developmental stage and light environment from each species at each growth temperature treatment. One disc of each pair was exposed to 1 degrees C and about 1000 micromoles per second per square meter photosynthetically active radiation for 4 hours, and the other disc was held at 1 degrees C in total darkness for the same duration. Photosynthetic tissue of S. tuberosum, developed at 12 degrees C, was much more tolerant to high light and low temperature stress than tissue developed under 24 degrees C conditions. Following the high light treatment, 24 degrees C-grown S. tuberosum tissue demonstrated light-limited and light-saturated rates that were approximately 50% of their paired dark controls. In contrast, the 12 degrees C grown tissue from S. tuberosum that was subjected to the light stress showed only a 18 and 6% reduction in light-limited and light-saturated rates of photosynthetic oxygen evolution, respectively. Tissue from 24 degrees C-grown S. commersonii was much less sensitive to the light stress than was tissue from S. tuberosum grown under the same conditions. The results presented here demonstrate that: (a) acclimation of S. tuberosum to lower temperature growth conditions with a constant light environment, results in the increased capacity of photosynthetic tissue to tolerate high light stress at chilling temperature and (b) following growth and development at relatively high temperatures S. commersonii, a frost- and heat-tolerant wild species, has a much greater tolerance to the high light stress at chilling temperature than does S. tuberosum cv Red Pontiac, a frost sensitive cultivated species. PMID- 16667217 TI - Limitation of CO(2) Assimilation and Regulation of Benson-Calvin Cycle Activity in Barley Leaves in Response to Changes in Irradiance, Photoinhibition, and Recovery. AB - The response of the Benson-Calvin cycle to changes in irradiance and photoinhibition was measured in low-light grown barley (Hordeum vulgare) leaves. Upon the transition from the growth irradiance (280 micromoles per square meter per second) to a high photoinhibitory irradiance (1400 micromoles per square meter per second), the CO(2) assimilation rate of the leaves doubled within minutes but high irradiance rapidly caused a reduction in quantum efficiency. Following exposure to high light the activities of NADP-malate dehydrogenase and fructose-1,6-bisphosphatase obtained near maximum values and the activation state of ribulose-1,5-bisphosphate carboxylase increased. The activity of the latter remained constant throughout the period of photoinhibitory irradiance, but the increase in the activities of fructose-1,6-bisphosphatase and NADP-malate dehydrogenase was transient decreasing once more to much lower values. This suggests that immediately following the transition to high light reduction and activation of redox-modulated enzymes occurred, but then the stroma became relatively oxidized as a result of photoinhibition. The leaf contents of glucose 6-phosphate and fructose 6-phosphate increased following exposure to high light but subsequently decreased, suggesting that following photoinhibition sucrose synthesis exceeded the rate of carbon assimilation. The ATP content attained a constant value much higher than that in low light. During photoinhibition the glycerate 3-phosphate content greatly increased while ribulose-1,5-bisphosphate decreased. The fructose-1,6-bisphosphate and triose phosphate contents increased initially and then remained constant. During photoinhibition CO(2) assimilation was not limited by ribulose-1,5-bisphosphate carboxylase activity but rather by the regeneration of the substrate, ribulose-1,5-bisphosphate, related to a restriction on the supply of reducing equivalents. PMID- 16667218 TI - Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures. AB - Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 +/- 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K(+). When the enzyme is dialyzed in the presence of K(+) and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K(+). The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K(+) concentrations increase. The change in threonine and K(+) concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark. PMID- 16667219 TI - Increased gene expression by the first intron of maize shrunken-1 locus in grass species. AB - The first intron of the shrunken-1 (Sh1) locus of maize was incorporated into constructs containing the chloramphenicol acetyltransferase gene (CAT) coupled with the nopaline synthase 3' polyadenylation signal. Transcription was driven with the 35S promoter of the cauliflower mosaic virus (CaMV) or the Sh1 promoter of maize. Transient gene expression was monitored following electroporation into protoplasts of Panicum maximum (guineagrass), Pennisetum purpureum (napiergrass), or Zea mays (maize). The 1028 base pair intron increased gene expression in cells of each species when transcription was driven with the 35S promoter. Eleven to 91 fold increases were observed. Expression levels observed in maize were two and eight times those observed in napiergrass and guineagrass, respectively. The 35S promoter gave CAT activity 10 to 100 times that observed with the Sh1 promoter. Whereas expression driven by the 35S promoter was reproducible, that observed with the Sh1 promoter proved quite variable. In similar constructs the first intron of the alcohol dehydrogenase-1 (Adh1) gene of maize led to increased gene expression of only 7 to 10% of that observed with the Sh1 first intron. The increased level of gene expression caused by the Sh1 first intron is approximately 10 times higher than that caused by any other plant introns that have been used. Thus, the Sh1 first intron may prove quite useful in increasing expression of foreign genes in monocots and possibly other plants. PMID- 16667220 TI - Combined low temperature-high light effects on gas exchange properties of jojoba leaves. AB - Jojoba (Simmondsia chinensis [Link] Schneider) is an important crop in desert climates. A relatively high frequency of periods of chilling and high photon flux density (PFD) in this environment makes photoinhibition likely, resulting in a reduction of assimilation capacity in overwintering leaves. This could explain the low net photosynthesis found in shoots from the field (4-6 micromoles per square meter per second) when compared to greenhouse grown plants (12-15 micromoles per square meter per second). The responses of photosynthesis and stomatal conductance to changes in absorbed PFD and in substomatal partial pressure of CO(2) were measured on jojoba leaves recovering from chilling temperature (4 degrees C) in high or low PFD. No measurable gas exchange was found immediately after chilling in either high or low PFD. For leaves chilled in low PFD, the original quantum yield was restored after 24 hours. The time course of recovery from chilling in high PFD was much longer. Quantum yield recovered to 60% of its original value in 72 hours but failed to recover fully after 1 week. Measurements of PSII chlorophyll fluorescence at 77 K showed that the reduced quantum yield was caused by photoinhibition. The ratio of variable to maximal fluorescence fell from a control level of 0.82 to 0.41 after the photoinhibitory treatment and recovery was slow. We also found a large increase in net assimilation rate and little closure of stomata as CO(2) was increased from ambient partial pressure of 35 to 85 pascals. For plants grown in full light, the increase in net assimilation rate was 100%. The photosynthetic response at high CO(2) concentration may constitute an ecological advantage of jojoba as a crop in the future. PMID- 16667221 TI - A Novel Effect in Phycomyces Phototropism : Positive Bending and Compensation Spectrum in Far UV. AB - A novel effect-positive phototropic bending under far UV irradiation (between 260 and 305 nanometers) at low intensities-is reported. Natural compensation points (intensities which cause no bending under unilateral irradiation) have been determined for different wavelengths. The curve connecting these points, the compensation spectrum, divides the intensity-wavelength plane into areas of negative and positive tropism. It is further shown that a highly asymmetrical pattern of light stimulus within the sporangiophore underlies the symmetrical growth response at each compensation point. This suggests that some unknown additional factor is involved in perceiving a UV stimulus at the level of the photoreceptor. It is also demonstrated here that positive tropism in the UV range is due to a lens effect. We conclude that the hypothesis of optical attenuation of the stimulus (considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism heretofore neglected by others. PMID- 16667222 TI - Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns. AB - Previous labeling studies of abscisic acid (ABA) with (18)O(2) have been mainly conducted with water-stressed leaves. In this study, (18)O incorporation into ABA of stressed leaves of various species was compared with (18)O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), (18)O was most abundant in the carboxyl group, whereas incorporation of a second and third (18)O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in (18)O(2). ABA from turgid bean leaves showed significant (18)O incorporation, again with highest (18)O enrichment in the carboxyl group. The (18)O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high (18)O enrichment in the carboxyl group, also much additional (18)O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more (18)O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1'-(18)OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of (18)O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange (18)O with water. On the basis of (18)O labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid. PMID- 16667223 TI - Purification and characterization of lysine-sensitive aspartate kinase from maize cell cultures. AB - Aspartate kinase is a feedback-regulated enzyme that controls the first step common to the biosynthesis of lysine, threonine, isoleucine, and methionine in plants. Aspartate kinase was purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures for physical and kinetic characterization studies. Partial purification and elution from an anion exchange column resolved two lysine-sensitive aspartate kinase isoforms. Both isoforms were purified >1,200 fold to a minimum specific activity of 18 units/milligram of protein. Both isoforms were sensitive to the lysine analogues S-2-aminoethyl-l-cysteine, l lysine ethyl ester, and delta-hydroxylysine. No threonine-sensitive form of aspartate kinase was detected at any stage during the purification. Additional purification steps were combined with preparative gel electrophoresis to obtain apparently homogeneous lysine-sensitive aspartate kinase. Aspartate kinase appeared to be a tetramer with a holoenzyme molecular weight of 254,000 and to be composed of 49,000 and 60,000 subunits. The tetramer appeared to disassociate during native gel electrophoresis to 113,000 dalton species that retained aspartate kinase activity. PMID- 16667224 TI - Hormonal regulation and distribution of peroxidase isoenzymes in the Cucurbitaceae. AB - Ethylene enhanced the levels of peroxidases in the roots, stems, leaves, and cotyledons of 2-week-old cucumber Cucumis sativus cv Poinsett 76 seedlings. Antibodies to the isoelectric point (pl) 9 and pl 4 isoenzymes were used in a radial immuno-diffusion assay to demonstrate that ethylene induced similar peroxidases in other cultivars of C. sativus, other species of Cucumis and other genera of Cucurbitaceae. Examination of ethylene-induced peroxidases, using isoelectric focusing gels, demonstrated the presence of a series of other peroxidases, mostly slightly acidic, whose isoelectric focusing pH was approximately 6. These pl 6 peroxidases were partially purified on a cation exchange column. Ouchterlony double diffusion gels indicated that these proteins cross-reacted with antibodies to both the pl 9 and pl 4 peroxidase. The data presented here suggest that the induction of peroxidase isoenzymes during ethylene-induced senescence is a common response in this family of plants. In addition, antibody and isoelectric focusing studies indicate that both acidic and basic peroxidase are highly conserved in members of this family. PMID- 16667225 TI - Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates. AB - By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulin dependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V(max)/K(m) ratio 200-fold lower than KM 14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity. PMID- 16667226 TI - Light/Dark modulation of enzyme activity in developing barley leaves. AB - Light/dark modulation of the ribulose-5-phosphate kinase, NADP(+)-glyceraldehyde 3-phosphate dehydrogenase, and fructose- 1,6-bisphosphatase activity was measured in the developing primary leaf of barley (Hordeum vulgare L.) seedlings. Ribulose 5-phosphate kinase and NADP(+) -glyceraldehyde-3-phosphate dehydrogenase were fully light activated even at the earliest developmental stage sampled. In contrast, light modulation of fructose- 1,6-bisphosphatase exhibited a complex response to leaf developmental status. Light stimulation of fructose- 1,6 bisphosphatase activity (measured at pH 8.0) increased progressively during leaf development. On the other hand, acid fructose- 1,6-bisphosphatase activity (measured at pH 6.0) was inhibited by light, and this light inhibition was greater in the base of the leaf than in the tip of the leaf. PMID- 16667227 TI - Water Transport in the Liana Bauhinia fassoglensis (Fabaceae). AB - To determine the efficiency of xylem conductance in the liana (woody vine) Bauhinia fassoglensis Kotschy ex Schweinf., we measured hydraulic conductance per unit stem length (measured K(h)), leaf-specific conductivity (LSC = K(h)/distal leaf area), transpiration rate (E), xylem water potential (epsilon), vessel number, and vessel diameter. The measured K(h) was 49% (se = 7%) of the predicted K(h) from Poiseuille's law. The mean LSC for unbranched stem segments was 1.10 x 10(-8) square meters per megapascal per second (se = 0.07). LSCs were much lower (about 0.2) at branch junctions. At midday, with E at 7 x 10(-8) meters per second, the measured drop in epsilon was about 0.08 megapascal per meter along the stems and branches and about 0.27 megapascal in going from stem to leaf. In addition, there was a drop of about 0.20 megapascal at branch junctions as predicted by E/LSC. In diurnal measurements leaf epsilon never dropped below about -1.2 megapascal. For long (e.g. 16 meters) stems, the predicted mid-day drop in epsilon through the xylem transport system might be great enough to have substantial physiological impact. PMID- 16667228 TI - Codon usage in higher plants, green algae, and cyanobacteria. AB - Codon usage is the selective and nonrandom use of synonymous codons by an organism to encode the amino acids in the genes for its proteins. During the last few years, a large number of plant genes have been cloned and sequenced, which now permits a meaningful comparison of codon usage in higher plants, algae, and cyanobacteria. For the nuclear and organellar genes of these organisms, a small set of preferred codons are used for encoding proteins. Codon usage is different for each genome type with the variation mainly occurring in choices between codons ending in cytidine (C) or guanosine (G) versus those ending in adenosine (A) or uridine (U). For organellar genomes, chloroplastic and mitochrondrial proteins are encoded mainly with codons ending in A or U. In most cyanobacteria and the nuclei of green algae, proteins are encoded preferentially with codons ending in C or G. Although only a few nuclear genes of higher plants have been sequenced, a clear distinction between Magnoliopsida (dicot) and Liliopsida (monocot) codon usage is evident. Dicot genes use a set of 44 preferred codons with a slight preference for codons ending in A or U. Monocot codon usage is more restricted with an average of 38 codons preferred, which are predominantly those ending in C or G. But two classes of genes can be recognized in monocots. One set of monocot genes uses codons similar to those in dicots, while the other genes are highly biased toward codons ending in C or G with a pattern similar to nuclear genes of green algae. Codon usage is discussed in relation to evolution of plants and prospects for intergenic transfer of particular genes. PMID- 16667229 TI - Leaf cuticles behave as asymmetric membranes : evidence from the measurement of diffusion potentials. AB - Cuticles were isolated enzymatically from the leaves of two maple species (Acer saccharum Marsh and A. platanoides L.) and from orange (Citrus aurantium L.). The cuticles were placed in a plastic cuvette and different concentrations of KCl were perfused over the physiological inner and outer surfaces while the electrical potential (E(10)) that developed across the cuticles and was caused by ion diffusion was measured. E(10) was always positive, indicating that the permeability of K(+) was always greater than that of Cl(-). Measured E(10) in cuticles did not fit the Goldman equation, whereas, E(10) measured during KCl diffusion across selected artificial membranes fit the equation. The magnitude of E(10) in cuticles and artificial membranes also was dependent on ionic strength, decreasing as ionic strength increased. These observations are explained by combining classical transport equations with equations that describe the equilibrium ion distribution between ionic double layers in the cuticle or membranes and the bathing solution. PMID- 16667230 TI - A novel role for light in the activation of ribulosebisphosphate carboxylase/oxygenase. AB - Light stimulated the activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) in a buffered lysed chloroplast system in the presence of saturating concentrations of ATP. This indicates a role for light in the rubisco activase activation system in addition to the previously identified requirement for the synthesis of ATP. Rubisco activation was nearly as great at low irradiance (10 micromoles of photons per square meter per second) as at high irradiance (1000 micromoles of photons per square meter per second). Light stimulation of activation occurred at both low bicarbonate (equivalent to air levels of CO(2)) and high bicarbonate (10 mm) concentrations. Light activation was inhibited by DCMU and glyoxylate. Methyl viologen did not inhibit light activation, and dithiothreitol did not stimulate activation in the dark, indicating that the ferredoxin/thioredoxin system was not involved. Following a transition of the lysed chloroplasts from light to dark, the light-dependent increase in activation ceased immediately. The experiments were conducted with chloroplasts from spinach (Spinacea oleracea L.), a species which was previously shown not to contain the endogenous inhibitor of rubisco, 2-carboxyarabinitol 1-phosphate. Assays of total rubisco activity in the light and dark confirmed the absence of such a tight binding inhibitor of activity. The observations reported here cannot be explained by current hypotheses of the role of light in rubisco activation and demonstrate that in addition to providing ATP needed for rubisco activase activity, at least one other light-dependent reaction is required for regulating the activation state of rubisco in vivo. PMID- 16667231 TI - Chrysoeriol and Luteolin Released from Alfalfa Seeds Induce nod Genes in Rhizobium meliloti. AB - Flavonoid signals from alfalfa (Medicago sativa L.) seed and root exudates induce transcription of nodulation (nod) genes in Rhizobium meliloti. The flavone luteolin previously was isolated from alfalfa seeds by other workers and identified as the first nod gene inducer for R. meliloti. Our recent study of ;Moapa 69' alfalfa root exudates found no luteolin but did identify three other nod gene inducers: 4,4'-dihydroxy-2'-methoxychalcone, 4',7-dihydroxyflavone, and 4',7-dihydroxyflavanone. The goal of the current study was to identify and quantify nod gene-inducing flavonoids that may influence Rhizobium populations around a germinating alfalfa seed. Aqueous rinses of Moapa 69 alfalfa seeds were collected and assayed for induction of a nodABC-lacZ fusion in R. meliloti. During the first 4 hours of imbibition, total nod gene-inducing activity was released from seeds at 100-fold higher rates than from roots of 72-hour-old seedlings. Five flavonoids were purified and identified by spectroscopic analyses (ultraviolet/visible absorbance, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Two very active nod gene inducing flavonoids, chrysoeriol (3'-methoxyluteolin) and luteolin, were identified in seed rinses. Luteolin required a higher concentration (18 nanomolar) than chrysoeriol (5 nanomolar) for half-maximum induction of nodABC lacZ in R. meliloti, and both were less active than 4,4'-dihydroxy-2' methoxychalcone (2 nanomolar) from root exudates. Seeds exuded three other luteolin derivatives: luteolin-7-O-glucoside, 5-methoxyluteolin, and 3',5 dimethoxyluteolin. Their combined quantities were 24-fold greater than that of luteolin plus chrysoeriol. Most nod gene-inducing activity of these luteolin derivatives apparently is associated with degradation to luteolin and chrysoeriol. However, their presence in large quantities suggests that they may contribute significantly to nod gene-inducing activity in the soil. These results indicate the importance of germinating seeds as a source of nod gene-inducing flavonoids and emphasize the quantitative and qualitative differences in those compounds around the seed and root. PMID- 16667232 TI - Comparison of lodging safety factor of untreated and succinic Acid 2,2 dimethylhydrazide-treated shoots of mulberry tree. AB - This study examined the lodging resistance of mulberry tree (Morus bombycis Koidz. cv Kenmochi) shoots treated or not treated with succinic acid 2,2 dimethylhydrazide (SADH). The lodging safety factor, an indicator of lodging resistance, was defined as the ratio of critical lodging load to the leaf fresh weight observed, provided that the distribution of the critical lodging load along the stem was similar to that of the leaf fresh weight observed. The critical lodging load was experimentally estimated by loading weights onto the stems. In the untreated trees, the lodging safety factor was maintained at about 3.2. In the SADH-treated trees, the stem elongation was inhibited to about 80% of that in the untreated trees, and the percentage of shoot dry matter partitioned into the leaves was always larger than that of the untreated trees. This dwarfing of the stem caused by SADH increased the critical lodging load supported by the unit stem dry weight, while this large investment of materials in leaves increased the leaf fresh weight supported by the unit stem dry weight. Since the increments canceled each other, the lodging safety factor of the SADH-treated shoots was similar to that of the untreated ones. These results suggest that the shoot formation of the mulberry tree is controlled to maintain the lodging safety factor at a constant level. PMID- 16667233 TI - Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine. AB - Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using (14)C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors. PMID- 16667234 TI - Carbon Isotope Discrimination in Coffee Genotypes Grown under Limited Water Supply. AB - Photosynthetic gas exchange, plant-water relations characteristics, and stable carbon isotope discrimination (Delta) were evaluated for five Coffea arabica L. genotypes growing under two soil moisture regimes in the field. The Delta of leaf tissue was strongly correlated (r = -0.95) with inherent water use efficiency (ratio of assimilation to stomatal conductance; A/g). The variation in inherent water use efficiency (WUE) among genotypes was 30% for plants irrigated weekly. The higher WUE exhibited by some of these plants resulted from reduced g rather than increased photosynthetic capacity at a given g. Withholding irrigation for 1 month caused Delta to decline substantially in expanding leaf tissue of all genotypes. A strong correlation (r = 0.92) was found between Delta and plant hydraulic efficiency estimated as the ratio of g to the diurnal range in leaf water potential (Psi(l)). The Delta values for plants irrigated weekly adequately predicted drought-induced changes in Delta (r = 0.99) and midday Psi(l) (r = 0.95). The results indicated that Delta might be used to evaluate several aspects of plant performance and response to specific environmental conditions, once suitable background physiological data have been gathered. PMID- 16667235 TI - Variations in the Levels of Chloroplast tRNAs and Aminoacyl-tRNA Synthetases in Senescing Leaves of Phaseolus vulgaris. AB - The relative amounts of chloroplast tRNAs(Leu), tRNA(Glu), tRNA(Phe), tRNAs(Thr), and tRNA(Tyr) and of chloroplastic and cytoplasmic aminoacyl-tRNA synthetases were compared in green leaves, yellowing senescing leaves, and N(6)-benzyladenine treated senescing leaves from bean (Phaseolus vulgaris, var Contender). Aminoacylation of the tRNAs using Escherichia coli aminoacyl-tRNA synthetases indicated that in senescing leaves the relative amount of chloroplast tRNA(Phe) was significantly lower than in green leaves. Senescing leaves treated with N(6) benzyladenine contained higher levels of this tRNA than untreated senescing leaves. No significant change in the relative amounts of chloroplast tRNAs(Leu), tRNAs(Thr), and tRNA(Tyr) was detected in green, yellow senescing, or N(6) benzyladine-treated senescing leaves. Relative levels of chloroplast tRNAs were also estimated by hybridization of tRNAs to DNA blots of gene specific probes. These experiments confirmed the results obtained by aminoacylation and revealed in addition that the relative level of chloroplast tRNA(Glu) is higher in senescing leaves than in green leaves. Transcription run-on assays indicated that these changes in tRNA levels are likely to be due to a differential rate of degradation rather than to a differential rate of transcription of the tRNA genes. Chloroplastic and cytoplasmic leucyl-, phenylalanyl-, and tyrosyl-tRNA synthetase activities were greatly reduced in senescing leaves as compared to green leaves, whereas N(6)-benzyladenine-treated senescing leaves contained higher enzyme activities than untreated senescing leaves. These results suggest that during senescence, as well as during senescence-retardation by cytokinins, changes in enzyme activities, such as aminoacyl-tRNA synthetases, rather than reduced levels of tRNAs, affect the translational capacity of chloroplasts. PMID- 16667236 TI - Field Study of the Interaction between Solar Ultraviolet-B Radiation and Drought on Photosynthesis and Growth in Soybean. AB - Soybean, Glycine max (L.) Merr. cv Essex, plants were grown in the field in a 2 x 2 factorial design, under ambient and supplemental levels of ultraviolet-B (UV-B) radiation (supplemental daily dose of 5.1 effective kilojoules per square meter) and were either well-watered or subjected to drought. Soil water potentials were reduced to -2.0 megapascals by the exclusion of natural precipitation in the drought plots and were maintained at approximately -0.5 megapascal by supplemental irrigation in well-watered plots. Plant growth and gas exchange characteristics were affected under both drought and supplemental UV-B radiation. Whole-leaf gas exchange analysis indicated that stomatal limitations on photosynthesis were only significantly affected by the combination of UV-B radiation and drought but substrate (ribulose bisphosphate) regeneration limitations were observed under either stress. The combined effect of both drought and UV-B radiation on photosynthetic gas exchange was a reduction in apparent quantum efficiency and the rapid appearance of biochemical limitations to photosynthesis concomitant with reduced diffusional limitations. However, the combination of stresses did not result in additive effects on total plant growth or seed yield compared to reductions under either stress independently. PMID- 16667237 TI - Activation of O(2) Uptake and NAD-Specific Isocitrate Dehydrogenase in Mitochondria Isolated from Cotyledons of Castor Bean by cis, trans-Abscisic Acid. AB - The rate of O(2) uptake and the activity of NAD-specific isocitrate dehydrogenase (NAD-ICDH) of mitochondria isolated from castor bean cotyledons were increased by added cis, trans-abscisic acid (ABA) in an in vitro system, while the NADP specific isocitrate dehydrogenase (NADP-ICDH) was not affected by cis, trans-ABA. Trans, trans-ABA showed only a slightly inhibitory effect on O(2) uptake. The V(max) value for the isotherm of isocitrate by the enzyme was also increased by cis, trans-ABA. The isocitrate K(m) value for the enzyme with cis, trans-ABA was calculated to be approximately 249.8 micromolar, while the S(0.5) for the enzyme without the ABA was 151.6 micromolar. The n value calculated from the slopes of Hill plots of the reaction velocity of NAD-ICDH against isocitrate concentration was 1.5 in the mitochondrial fraction in the absence of ABA, and cis, trans-ABA treatment decreased the value to 1.0. Cis, trans-ABA also partly overcame the inhibition of NAD-ICDH activity by ATP. PMID- 16667238 TI - Energy Requirement for the Import of Protein into Plastids from Developing Endosperm of Ricinus communis L. AB - Leucoplasts isolated from developing endosperm of Ricinus communis L. will import the precursor of the small subunit of ribulose bisphosphate carboxylase from pea shoots and process it to its mature molecular weight (SA Boyle, SM Hemmingsen, DT Dennis [1986] Plant Physiol 81: 817-822). This process requires energy in the form of ATP. GTP, CTP, and UTP are inactive. ADP will also satisfy the energy requirement, probably through the action of adenylate kinase in the envelope. Fatty acid biosynthesis which occurs within these leucoplasts also requires ATP for maximal activity. Phosphoenolpyruvate will stimulate fatty acid biosynthesis approximately three times as effectively as ATP through the generation of ATP within the organelle by the action of the plastid pyruvate kinase. However, phosphoenolpyruvate under similar conditions will not stimulate the uptake of the small subunit of ribulose bisphosphate carboxylase into leucoplasts. These results indicate that ATP is required outside the leucoplast for protein uptake and that internally generated ATP is not effective in this process. PMID- 16667239 TI - Characterization of ATP-Dependent Fructose 6-Phosphate 1-Phosphotransferase Isozymes from Leaf and Endosperm Tissues of Ricinus communis. AB - Plastid and cytosolic isozymes of ATP:fructose 6-phosphate 1-phosphotransferase (PFK(p) and PFK(c), respectively) have been isolated from leaves and developing endosperm tissues of the castor oil plant (Ricinus communis L). Endosperm PFK(p) has been purified to apparent homogeneity. Polyclonal antibodies raised against one of the four polypeptides associated with potato tuber PFK (molecular mass, 46 kilodaltons) immunoprecipitated developing endosperm and leaf PFK(p), but not PFK(c) isozymes. Western blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and analytical gel filtration show that PFK(p) from developing endosperm is a 220 kilodalton homotetramer composed of 57 kilodalton subunits. Kinetic studies of leaf PFK(p) and PFK(c) isozymes reveal both similarities and differences to the characteristics of the respective endosperm isozymes studied previously (WJ Garland, DT Dennis [1980] Arch Biochem Biophys 204: 302-317). The immunological and kinetic data suggest that leaf and developing endosperm PFK(p) are different but structurally related proteins. PMID- 16667240 TI - Nitrogen-induced changes in the growth and metabolism of developing maize kernels grown in vitro. AB - Cereal kernel growth and grain yield are functions of endosperm starch accumulation. The objective of this study was to examine how various metabolic factors in developing maize (Zea mays L.) endosperm influence starch deposition. Kernels were grown in vitro on medium with: (a) zero N (-N), (b) optimum N (+N), or (c) -N from 3 to 20 days after pollination followed by +N until maturity (+/ N) to produce different degrees of endosperm growth and to promote an enhancement of starch synthesis midway through development. At intervals, kernels were harvested and levels of enzyme activities and carbohydrate and N constituents examined. Endosperm starch and protein accumulation were decreased in -N compared to +N kernels, but relief of N starvation increased both constituents. With greater movement of N into +/-N kernels, endosperm sugar concentrations declined suggesting an inverse relationship between C and N transport. Unusually high concentrations of sugar in N stressed kernels did not appear to limit or enhance starch production. Rather, increased accumulation of starch in +/-N endosperm was correlated with significant increases in the enzymatic activities of sucrose synthase and PPi-linked phosphofructokinase, and to a lessor extent hexokinase. In addition, the occurrence of specific proteins of the albumin/globulin fraction either increased, decreased, or remained unchanged in relation to starch synthesis. These data suggest that lack of N limits starch deposition in maize endosperm primarily through an influence on synthesis of key proteins. PMID- 16667241 TI - Developmental Transition from Enzymatic to Acid Hydrolysis of Sucrose in Acid Limes (Citrus aurantifolia). AB - The sucrose breakdown mechanisms in juice sacs of acid lime (Citrus aurantifolia [Christm.] Swing.) were investigated throughout fruit development. All three enzymes of sucrose catabolism (sucrose synthase, acid, and alkaline invertase) are present during the initial stages. The activities of these enzymes declined rapidly and disappeared by stage 5 (80% development) but not before vacuolar pH had decreased to approximately 2.5. At this stage, sucrose breakdown occurs by acid hydrolysis. By attaining a vacuolar pH of 2.5 prior to enzyme disappearance, the cell maintains a continuous ability to break down sucrose throughout ontogeny. Thus, acid limes possess a unique and coordinated system for sucrose breakdown that involves both enzymatic and nonenzymatic pathways. PMID- 16667242 TI - Accumulation of apoplastic iron in plant roots : a factor in the resistance of soybeans to iron-deficiency induced chlorosis? AB - We hypothesized that the resistance of Hawkeye (HA) soybean (Glycine max L.) to iron-deficiency induced chlorosis (IDC) is correlated to an ability to accumulate a large pool of extracellular-root iron which can be mobilized to shoots as the plants become iron deficient. Iron in the root apoplast was assayed after efflux from the roots of intact plants in nutrient solution treated with sodium dithionite added under anaerobic conditions. Young seedlings of HA soybean accumulated a significantly larger amount of extracellular iron in their roots than did either IDC-susceptible PI-54619 (PI) soybean or IDC-resistant IS-8001 (IS) sunflower (Helianthus annus L.). Concurrently, HA soybean had much higher concentrations of iron in their shoots than either PI soybean or IS sunflower. The concentration of iron in the root apoplast and in shoots of HA soybean decreased sharply within days after the first measurements of extracellular root iron were made, in both +Fe and -Fe treatments. The accumulation of short-term iron reserves in the root apoplast and translocation of iron in large quantities to the shoot may be important characteristics of IDC resistance in soybeans. PMID- 16667243 TI - Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : III. Evidence for Functional Heterogeneity of the ALA Pool. AB - Gabaculine and 4-amino-5-hexynoic acid (AHA) up to 3.0 millimolar concentration strongly inhibited 5-aminolevulinic acid (ALA) synthesis in developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts, while they hardly affected protochlorophyllide (Pchlide) synthesis. Exogenous protoheme up to 1.0 micromolar had a similar effect. Exogenous glutathione also exhibited a strong inhibitory effect on ALA synthesis in organello but hardly inhibited Pchlide synthesis. Pchlide synthesis in organello was highly sensitive to inhibition by levulinic acid, both in the presence and in the absence of gabaculine, indicating that the Pchlide was indeed formed from precursor(s) before the ALA dehydratase step. The synthesis of Pchlide in the presence of saturating concentrations of glutamate was stimulated by exogenous ALA, confirming that Pchlide synthesis was limited at the formation of ALA. The gabaculine inhibition of ALA accumulation occurred whether levulinic acid or 4,6-dioxohepatonic acid was used in the ALA assay system. ALA overproduction was also observed in the absence of added glutamate and was noticeable after 10-minute incubation. These observations suggest that although Pchlide synthesis in organello is limited by ALA formation, it does not utilize all the ALA that is made in the in organello assay system. Gabaculine, AHA, and probably also protoheme, inhibit preferentially the formation of that portion of ALA that is not destined for Pchlide. A model proposing a heterogenous ALA pool is described. PMID- 16667244 TI - Characterization of a Rapid, Blue Light-Mediated Change in Detectable Phosphorylation of a Plasma Membrane Protein from Etiolated Pea (Pisum sativum L.) Seedlings. AB - When crude microsomal membranes from apical stem segments of etiolated Pisum sativum L. cv Alaska are mixed in vitro with gamma-[(32)P]ATP, a phosphorylated band of apparent molecular mass 120 kilodaltons can be detected on autoradiographs of sodium dodecyl sulfate electrophoresis gels. If the stem sections are exposed to blue light immediately prior to membrane isolation, this band is not evident. The response is observed most strongly in membranes from the growing region of the stem, but no 120 kilodalton radiolabeled band is detected in membranes from the developing buds. Fluence-response curves for the reaction show that the system responds to blue light above about 0.3 micromole per square meter, and the visible phosphorylation completely disappears above 200 micromoles per square meter. Reciprocity is valid for the system, because varying illumination time or fluence rate give similar results. If the stem segments are left in the dark following a saturating blue irradiation, the radio-labeled band begins to return after about 10 minutes and is as intense as that from the dark controls within 45 to 60 minutes. A protein that comigrates with the phosphorylated protein on polyacrylamide gels is also undetectable after saturating blue light irradiations. The fluence range in which the protein band disappears is the same as that for the disappearance of the phosphorylation band. Its dark recovery kinetics and tissue distribution also parallel those for the phosphorylation. In vitro irradiation of the isolated membranes also results in a phosphorylation change at that molecular mass, but in the opposite direction. Comparisons of the kinetics, tissue distribution, and dark recovery of the phosphorylation response with those published for blue light-mediated phototropism or rapid growth inhibition indicate that the phosphorylation could be linked to one or both of those reactions. However, the fluence-response relationships for the change in detectable phosphorylation match quite closely those reported for phototropism but not those for growth inhibition. Blue light has also been found to regulate the capacity for in vitro phosphorylation of a second protein. It has an apparent molecular mass of 84 kilodaltons and is localized primarily in basal stem sections. PMID- 16667245 TI - Inheritance of the Reversal of O(2) Response of Photosynthesis in a Flaveria linearis Mutant. AB - A mutant plant of Flaveria linearis Lag. expresses reversed O(2) response of photosynthesis (i.e. its apparent photosynthesis is stimulated at atmospheric O(2) levels). The objectives of this study were to determine the genetic inheritance of this trait and to investigate the biochemical mechanism for its expression. The mutant plant was crossed reciprocally with a plant of the closely related species Flaveria oppositifolia (DC.) Rydb. and also with another plant of F. linearis. Data on O(2) inhibition of apparent photosynthesis were analyzed on F(2) and F(3) progeny from these F(1) hybrids. In addition, test crosses (mutant x F(1) hybrid) and S(1) progeny from the mutant plant were also analyzed. All F(1) hybrids expressed inhibition of apparent photosynthesis and their progeny segregated in acceptable 3:1 and 13:3 (normal:reversed) ratios. There was little effect of environment on expression of the reversed O(2) response. Selected F(2) plants and the original mutant plant produced progeny in normal:reversed ratios which indicated the trait is controlled by two major genes which show dominant and recessive epistasis. Plants with greater than 20 nanomoles per gram fresh weight per minute of fructose-1, 6-bisphosphatase activity in the cytosol had normal O(2) response of photosynthesis. However, when plants had less than 20 nanomoles per gram fresh weight per minute of this enzyme activity in the cytosol, the O(2) was normal in some and reversed in others. It is proposed that low fructose bisphosphatase activity in the cytosol is controlled by a recessive gene (fbp). A second dominant gene is speculated to be hypostatic to the normal fructose bisphosphatase gene and controls the expression of an unknown factor that determines whether O(2) response of AP is reversed in the presence of fbp (i.e. when fructose bisphosphatase activity is low). PMID- 16667246 TI - New Methods for Extraction and Quantitation of Zeins Reveal a High Content of gamma-Zein in Modified opaque-2 Maize. AB - We have developed methods for quantitative extraction and analysis of zeins from maize (Zea mays L.) flour. Extraction involved solubilization of total endosperm proteins in an alkaline buffer containing SDS and 2-mercaptoethanol with subsequent precipitation of nonzein proteins by the addition of ethanol to 70%. Analysis of these proteins by SDS-PAGE with Coomassie blue staining and by Western blotting and ELISA assay with zein antibodies revealed that this extraction method is more quantitative than the traditional Landry-Moureaux procedure, especially for the beta- and gamma-zeins. This method was used to extract and analyze the zein content of several ;Quality Protein Maize' (QPM) varieties developed by the International Maize and Wheat Improvement Center. QPM varieties contain ;modifier genes' that confer a vitreous phenotype on opaque-2 genotypes, while maintaining the elevated levels of lysine and tryptophan characteristic of this mutant. This analysis revealed that the QPM types contain 2 to 4 times the amount of the gamma-zein than unmodified opaque-2 or normal maize varieties. Possible relationships between the high expression of the gamma zein and the modified opaque phenotype are discussed. PMID- 16667247 TI - Solubilization of covalently bound extensin from capsicum cell walls. AB - Acidified sodium chlorite cleaves isodityrosine and solubilizes covalently bound hydroxyproline-rich material from cell walls. This has been taken as evidence that isodityrosine acts as a cross-link holding the hydroxyproline-rich glycoprotein extensin in the cell wall. However, acidified chlorite was found to cleave peptide bonds in salt-soluble extensin and in bovine serum albumin (BSA). This invalidates the use of conventional acidified chlorite treatment to provide evidence for isodityrosine cross-links. The ratio of BSA:chlorite was important in determining peptidyl cleavage. At a ratio of 0.75:1.00 (mole amino acid residues/mole chlorite), or higher, peptidyl cleavage was not detected. Furthermore, in samples where a low concentration of radioactive extensin was present, BSA substantially protected the peptide bonds of the extensin against peptidyl cleavage during treatment with acidified chlorite, while not preventing the cleavage of isodityrosine. Therefore, acidified sodium chlorite plus BSA was a more specific reagent for the cleavage of isodityrosine than was acidified chlorite alone. This modified treatment solubilized in intact form the ;covalently bound' extensin from cell walls of Capsicum frutescens (chili pepper) suspension cultures, providing new evidence compatible with the view that extensin molecules are held in the cell wall by isodityrosine cross-links. PMID- 16667248 TI - Water Deficit and Abscisic Acid Cause Differential Inhibition of Shoot versus Root Growth in Soybean Seedlings : Analysis of Growth, Sugar Accumulation, and Gene Expression. AB - Roots often continue to elongate while shoot growth is inhibited in plants subjected to low-water potentials. The cause of this differential response to water deficit was investigated. We examined hypocotyl and root growth, polysome status and mRNA populations, and abscisic acid (ABA) content in etiolated soybean (Glycine max [L.] Merr. cv Williams) seedlings whose growth was inhibited by transfer to low-water potential vermiculite or exogenous ABA. Both treatments affected growth and dry weight in a similar fashion. Maximum inhibition of hypocotyl growth occurred when internal ABA levels (modulated by ABA application) reached the endogenous level found in the elongating zone of seedlings grown in water-deficient vermiculite. Conversely, root growth was affected to only a slight extent in low-water potential seedlings and by most ABA treatments (in some, growth was promoted). In every seedling section examined, transfer of seedlings into low-water potential vermiculite caused ABA levels to increase approximately 5- to 10-fold over that found in well-watered seedlings. Changes in soluble sugar content, polysome status, and polysome mRNA translation products seen in low-water potential seedlings did not occur with ABA treatments sufficient to cause significant inhibition of hypocotyl elongation. These data suggest that both variation in endogenous ABA levels, and differing sensitivity to ABA in hypocotyls and roots can modulate root/shoot growth ratios. However, exogenous ABA did not induce changes in sugar accumulation, polysome status, and mRNA populations seen after transfer into low-water potential vermiculite. PMID- 16667249 TI - Involvement of plasma membrane calcium influx in bacterial induction of the k/h and hypersensitive responses in tobacco. AB - An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K(+)/H(+) response characterized by specific plasma membrane K(+) efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca(2+) influx of 0.02 to 0.06 micromole per gram per hour as determined from (45)Ca(2+) uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K(+)/H(+) response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca(2+) influx was prevented by EGTA and calcium channel blockers such as La(3+), Co(2+), and Cd(2+) but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K(+)/H(+) response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca(2+) influx is required for the K(+)/H(+) and hypersensitive responses in tobacco. PMID- 16667250 TI - Free amino Acid composition of leaf exudates and Phloem sap : a comparative study in oats and barley. AB - Comparisons were made between the free amino acid composition in leaf exudates and that in pure phloem sap, using twin samples taken from a single leaf of two oat (Avena sativa L.) and three barley (Hordeum vulgare L.) varieties. Leaf exudate was collected in a 5 mm EDTA-solution (pH 7.0) from cut leaf blades and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Fluorescent derivatives of amino acids were obtained using 9-fluorenylmethyl chloroformate and were separated by means of high performance liquid chromatography. The total concentration of free amino acids varied considerably in the exudate samples. There was no correlation between the total amino acid content in the exudate samples and that of the corresponding phloem sap samples, but the amino acid composition of the corresponding samples was highly correlated (median R(2)-value 0.848). There was only limited between-plant variation in phloem sap amino acid composition. Nevertheless, in comparisons involving all samples, many of the amino acids showed significant correlations between their relative amounts in exudate and phloem sap. The results presented here indicate that the exudate technique holds great promise as an interesting alternative to the laborious and time-consuming stylet-cutting technique of obtaining samples for comparative studies of phloem sap. PMID- 16667251 TI - Effect of Ethephon on Protein Degradation and the Accumulation of ;Pathogenesis Related' (PR) Proteins in Tomato Leaf Discs. AB - The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5 bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69 (P Vera, V Conejero [1988] Plant Physiol 87: 58-63). In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other (35)S-labeled proteins as well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene. PMID- 16667252 TI - Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress. AB - Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na(+) was the same as for K(+), P(K) (+)/P(Na) (+) = 1, whereas the cation/anion selectivity (P(K) (+)/P(c1) (-)) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t((1/2)) = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a DeltapH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered. PMID- 16667253 TI - Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices. AB - Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30 degrees C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity. PMID- 16667254 TI - Gibberellic Acid Regulates Cell Wall Extensibility in Wheat (Triticum aestivum L.). AB - Mutations (Rht genes) blocking sensitivity to gibberellic acid (GA) were used to examine phytohormone mediated cell wall expansion in wheat (Triticum aestivum L.). Irreversible extensibility of immature leaf segments, as determined by stress/strain (instron) measurements, declined with Rht gene dose. Exogenous GA(3) significantly increased wall extensibility in the nonmutant controls but had no effect on the near-isogenic GA-insensitive genotypes. Furthermore, ancymidol, an inhibitor of gibberellin biosynthesis, diminished wall extensibility in the nonmutant control. Extensibility of immature segments was highly correlated with mature leaf sheath length (R = +0.95). The results indicate that wall yielding properties of expanding wheat leaves are associated with leaf cell expansion potential and that GA is involved in the determination of those properties. PMID- 16667256 TI - Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant. AB - Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly. PMID- 16667255 TI - Rubisco activity in guard cells compared with the solute requirement for stomatal opening. AB - We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5 bisphophate carboxylase/oxygenase (Rubisco) were determined by the [(14)C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to (14)CO(2) for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant. PMID- 16667257 TI - A squared michaelis-menten function of substrate concentration for plant mitochondrial respiration. AB - Dry and Wiskich ([1987] Arch Biochem Biophys 257: 92-99) have published data showing the response of plant mitochondrial respiration to increasing additions of oxaloacetate or malate when these substrates have been depleted by inhibition of succinate dehydrogenase by malonate, and coenzyme A (CoA) has been sequestered as acetyl-CoA by pyruvate dehydrogenase. In the presence of 2-oxoglutarate, it is shown that the response is given by a Michaelis-Menten curve, but in its absence, when malate has to supply substrate for dehydrogenation as well as to liberate CoA via malate dehydrogenase and citrate synthase, the response is presumably the product of two Michaelis-Menten functions, which can be approximated by the square of a single function. PMID- 16667258 TI - Nitrogenase Activity and Nodule Gas Permeability Response to Rhizospheric NH(3) in Soybean. AB - This study was conducted on soybean (Glycine max L. Merr.) nodules to determine if exogenous NH(3) exerts a controlling influence over nitrogenase activity through changes in nodule gas permeability (P), and if decreasing carbohydrate availability, as a result of low-light treatment, increases the sensitivity of root nodules to NH(3). Nodulated root systems of intact plants were exposed to one of several NH(3) concentrations ranging from 0 to 821 microliters per liter for an 8-hour period. Treatments were conducted under high-light (2300 micromoles per square meter per second) or low-light (800 micromoles per square meter per second) conditions. Increasing the NH(3) concentration and length of exposure of NH(3) caused a progressive decline in acetylene reduction activity (ARA). There was generally a greater reduction in ARA under the low-light treatment compared to the high-light treatment at a particular NH(3) concentration. The NH(3) concentration necessary to decrease P was greater than that needed to decrease ARA, and there was no evidence of a causal relationship between P and ARA in response to NH(3). PMID- 16667259 TI - Polymorphism of a photosystem I subunit caused by alloploidy in Nicotiana. AB - The photosystem I complex from Nicotiana tabacum, which has an alloploid genome, contains subunits of 17.5 and 18.5 kilodaltons whose N-terminal amino acid sequences are highly homologous. Comparative analysis of photosystem I subunits among N. tabacum and its ancestral plants, N. tomentosiformis and N. sylvestris, revealed that the 17.5 kilodalton subunit of N. tabacum derives from N. sylvestris, and the 18.5 kilodalton subunit from N. tomentosiformis. PMID- 16667260 TI - Differential Activation of Expression of a Suberization-Associated Anionic Peroxidase Gene in Near-Isogenic Resistant and Susceptible Tomato Lines by Elicitors of Verticillium albo-atratrum. AB - We tested whether the expression of the suberization-associated anionic peroxidase gene is involved in the timely appearance of the vascular suberized coating involved in the resistance of a tomato line to Verticillium albo-atrum. The mRNA for this peroxidase appeared at a higher level one day earlier in wound healing fruits of the resistant tomato line than in a near-isogenic susceptible line. Cell cultures from the resistant line, when treated with low levels (nanograms per milliliter) of fungal elicitor, generated the peroxidase mRNA and this apparent activation of the peroxidase gene expression could be detected in minutes, whereas the cells from the susceptible line hardly responded. PMID- 16667261 TI - Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet: III. Diurnal Changes in Carbon Partitioning and Carbon Export. AB - The effect of low phosphate supply (low P) was determined on the diurnal changes in the rate of carbon export, and on the contents of starch, sucrose, glucose, and fructose 2,6-bisphosphate (F2,6BP) in leaves. Low-P effects on the activities of a number of enzymes involved in starch and sucrose metabolism were also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers and the low-P treatment induced nutritionally. Low-P treatment decreased carbon export from the leaf much more than it decreased photosynthesis. At growth chamber photon flux density, low P decreased carbon export by 34% in light; in darkness, export rates fell but more so in the control so that the average rate in darkness was higher in low-P leaves. Low P increased starch, sucrose, and glucose contents per leaf area, and decreased F2, 6BP. The total extractable activities of enzymes involved in starch and sucrose synthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5 diphosphoglucose pyrophosphorylase, and sucrose-phosphate synthase. The activities of some enzymes involved in starch and sucrose breakdown were also increased by low P. We propose that plants adapt to low-P environments by increasing the total activities of several phosphatases and by increasing the concentrations of phosphate-free carbon compounds at the expense of sugar phosphates, thereby conserving Pi. The partitioning of carbon among the various carbon pools in low-P adapted leaves appears to be determined in part by the relative capacities of the enzymes for starch and sucrose metabolism. PMID- 16667262 TI - Spinach chloroplastic carbonic anhydrase: nucleotide sequence analysis of cDNA. AB - We have determined the nucleotide sequence of a cDNA encoding spinach (Spinacia oleracea) chloroplastic carbonic anhydrase (CA). The open reading frame encodes a protein consisting of a transit peptide and a mature CA protein with a predicted mass of 24, 116 daltons. This represents the first report of a nucleotide sequence of a plant CA. PMID- 16667263 TI - Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum). AB - A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400 fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V(max) per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M(r) of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38 degrees C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K(m) values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V(max)/K(m) values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1. ). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes. PMID- 16667264 TI - Characterization of abscisic Acid-induced ethylene production in citrus leaf and tomato fruit tissues. AB - Abscisic acid (ABA) significantly stimulated ethylene production in citrus (Citrus sinensis [L.] Osbeck, cv Shamouti orange) leaf discs. The extent of stimulation was dependent upon the concentration of ABA (0.1-1 milimolar) and the duration of treatment (15-300 minutes). Aging the discs before applying ABA increased ABA-induced ethylene production due to enhancement of both ethylene forming enzyme activity and the responsiveness of ABA. Discs excised from mature leaves were much more responsive to ABA than discs excised from young or senescing leaves. ABA stimulated ethylene production shortly after application, suggesting that ABA does not enhance ethylene production via the acceleration of senescence. The stimulating effect of ABA on ethylene production resulted mainly from the enhancement of 1-aminocylopropane-1-carboxylic acid synthesis. Stimulation of ethylene production by ABA in intact citrus leaves and tomato (Lycopersicon esculentum Mill., cv Castlemart) fruit was small but could be increased by various forms of wounding. PMID- 16667265 TI - Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues. AB - Exogenous [(14)C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole 3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [(14)C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels. PMID- 16667266 TI - Direct observation of cell wall structure in living plant tissues by solid-state C NMR spectroscopy. AB - Solid-state (13)C nuclear magnetic resonance (NMR) spectra of the following intact plant tissues were recorded by the crosspolarization magic-angle spinning technique: celery (Apium graveolens L.) collenchyma; carob bean (Ceratonia siliqua L.), fenugreek (Trigonella foenum-graecum L.), and nasturtium (Tropaeolum majus L.) endosperm; and lupin (Lupinus polyphyllus Lindl.) seed cotyledons. All these tissues had thickened cell walls which allowed them to withstand the centrifugal forces of magic angle spinning and which, except in the case of lupin seeds, dominated the NMR spectra. The celery collenchyma cell walls gave spectra typical of dicot primary cell walls. The carob bean and fenugreek seed spectra were dominated by resonances from galactomannans, which showed little sign of crystalline order. Resonances from beta(1,4')-d galactan were visible in the lupin seed spectrum, but there was much interference from protein. The nasturtium seed spectrum was largely derived from a xyloglucan, in which the conformation of the glucan core chain appeared to be intermediate between the solution form and solid forms of cellulose. PMID- 16667267 TI - Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves. AB - Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP(+), and Mg(2+) were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K(m) = 58 micromolar), but also inhibition by high substrate (K(i) = 4.95 millimolar) were observed at pH 7.0. l Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K(m) = 120 micromolar). At both pH conditions, activity response to NADP(+) exhibited Michaelis-Menten behavior with K(m) values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg(2+) suggested the presence of at least two Mg(2+) - binding sites with different affinity. The K(a) values for Mg(2+) obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg(2+) levels could be important for the physiological regulation of NADP-malic enzyme. PMID- 16667268 TI - Purification and Characterization of Acetyl-CoA Carboxylase from the Diatom Cyclotella cryptica. AB - Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a molecular weight of approximately 740 kilodaltons and appears to be composed of four identical biotin-containing subunits. The enzyme has maximal activity at pH 8.2, but enzyme stability is greater at pH 6.5. K(m) values for MgATP, acetyl-CoA, and HCO(3)- were determined to be 65, 233, and 750 micromolar, respectively. The purified enzyme is strongly inhibited by palmitoyl-CoA, and is inhibited to a lesser extent by malonyl-CoA, ADP, and phosphate. Pyruvate stimulates enzymatic activity to a slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxyphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar clethodim and 100 micromolar haloxyfop, respectively. PMID- 16667269 TI - Estimation of Carbon and Nitrogen Allocation during Stalk Elongation by C and N Tracing in Zea mays L. AB - Zea mays L. (cv Dea) plants grown to the stage of stalk elongation, were allowed to assimilate (13)CO(2) and (15)N-nitrates from 45 to 53 days after sowing. Isotopic abundances in labeled nutrients were slightly enriched compared to natural abundances. The new C in plant was acropetally distributed and the new N was preferentially accumulated in the sheath and stalk in the medium region. C input was 25-fold higher than N input. The new C in total plant C was 20%, whereas it was 10% for N. The stalk acted as a major sink because it accumulated, respectively, 27.5 and 47.5% of the C and N inputs. The new C in soluble carbohydrates was 76% in growing organs (upper stalk) and only 39% in source leaves, whereas it was 43% and 13% in starch, respectively. New N in nitrates+amino-acids spanned in the range from 20% (leaf) to 50% (stalk). New C and N in soluble proteins were, respectively, 13.4 and 3.8% in leaves, 8.8 and 9.6% in stalk, and 8.7 and 14.3% in roots. In the middle stalk and leaves, the proteins and carbohydrates represent an equivalent C and N source for remobilization. PMID- 16667270 TI - Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa). AB - Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [(14)C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [(14)C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminase-directed modifications of ribulose 1,5-bisphosphate carboxylase/oxygenase. PMID- 16667271 TI - Inhibition of Loading of C Assimilates by p-Chloromercuribenzenesulfonic Acid : Localization of the Apoplastic Pathway in Vicia faba. AB - The apoplast of mature leaves excised from broadbean (Vicia faba L.) plants was infiltrated with 2 millimolar p-chloromercuribenzenesulfonic acid (PCMBS) via the transpiration stream, and the ability of the tissues to take up sugars was tested. An infiltration time of 75 minutes was sufficient to obtain a maximal (75%) inhibition of exogenous [(14)C]sucrose (1 millimolar) uptake. This infiltration affected neither CO(2) assimilation nor the transmembrane potential difference of leaf cells but strongly inhibited phloem loading of endogenous [(14)C] assimilates. The study of the symplastic relations between the different cell types of the mature leaf showed that the density of the plasmodesmata is generally very low in comparison with other species investigated so far, particularly when considering the mesophyll/bundle sheath and the bundle sheath/phloem cells connections, as well as the connections of the transfer cell sieve tube complex with the surrounding cells. These three successive barriers therefore strongly limit the possibilities of symplastic transit of the assimilates to the conducting cells. The comparison of the densities of plasmodesmata in an importing and an exporting leaf suggests that the maturation of the leaf is characterized by a marked symplastic isolation of the phloem, and, within the phloem itself, by the isolation of the conducting complex. As a consequence, these physiological and cytological data demonstrate the apoplastic nature of loading in the mature leaf of Vicia faba, this species undoubtedly presenting a typical model for apoplastic loading. PMID- 16667272 TI - Crown gall disease and hairy root disease : a sledgehammer and a tackhammer. AB - The neoplastic diseases crown gall and hairy root are incited by the phytopathogenic bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Although the molecular mechanism of T-DNA transfer to the plant most likely is the same for both species, the physiological basis of tumorigenesis is fundamentally different. Crown gall tumors result from the over production of the phytohormones auxin and cytokinin specified by A. tumefaciens T DNA genes. Although the T-DNA of some Riplasmids of A. rhizogenes contains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to endogenous auxin levels. An understanding of hairy root tumorigenesis will likely result in an increased knowledge of plant developmental processes. PMID- 16667273 TI - Nitrate Reduction in Response to CO(2)-Limited Photosynthesis : Relationship to Carbohydrate Supply and Nitrate Reductase Activity in Maize Seedlings. AB - The effects of CO(2)-limited photosynthesis on (15)NO(3) (-) uptake and reduction by maize (Zea mays, DeKalb XL-45) seedlings were examined in relation to concurrent effects of CO(2) stress on carbohydrate levels and in vitro nitrate reductase activities. During a 10-hour period in CO(2)-depleted air (30 microliters of CO(2)/ per liter), cumulative (15)NO(3) (-) uptake and reduction were restricted 22 and 82%, respectively, relative to control seedlings exposed to ambient air containing 450 microliters of CO(2) per liter. The comparable values for roots of decapitated maize seedlings, the shoots of which had previously been subjected to CO(2) stress, were 30 and 42%. The results demonstrate that reduction of entering nitrate by roots as well as shoots was regulated by concurrent photosynthesis. Although in vitro nitrate reductase activity of both tissues declined by 60% during a 10-hour period of CO(2) stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of (15)NO(3) (-) reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO(2) stress. Collectively, the results indicate that nitrate uptake and reduction were regulated by the supply of energy and carbon skeletons required to support these processes, rather than by the potential enzymatic capacity to catalyze nitrate reduction, as measured by in vitro nitrate reductase activity. PMID- 16667274 TI - Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts. AB - Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F(o)) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the ;high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F(o) quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll. PMID- 16667275 TI - Relative contributions of zeaxanthin-related and zeaxanthin-unrelated types of ;high-energy-state' quenching of chlorophyll fluorescence in spinach leaves exposed to various environmental conditions. AB - We have identified two rapidly relaxing components of non-photochemical fluorescence quenching which suggests that dissipative processes occur in two different sites in the photochemical system of leaves. Under a variety of treatment conditions involving different leaf temperatures, photon flux densities (PFD), exposure times, and in the presence of 5% CO(2) or 2% O(2), no CO(2), the components of nonphotochemical fluorescence quenching were characterized with respect to their sensitivity to dithiothreitol (DTT, which completely inhibits zeaxanthin formation), the effect on instantaneous fluorescence, and the rapidity of relaxation upon darkening. Under most circumstances the DTT-sensitive component (associated with a quenching of instantaneous fluorescence and correlated with zeaxanthin) represented the majority of the rapidly relaxing portion of fluorescence quenching. A DTT-insensitive (zeaxanthin-independent) component, which also relaxed rapidly upon darkening but was not associated with a quenching of instantaneous fluorescence, became proportionally greater in an atmosphere of 2% O(2) and no CO(2), at elevated leaf temperatures, and to some degree during the induction of photosynthesis (1 minute after the onset of illumination). A third component which was also DTT-insensitive and was sustained upon darkening, was largely suppressed in 2% O(2), O% CO(2). We conclude that, under conditions favorable for photosynthesis, energy dissipation occurred mainly in the chlorophyll antennae whereas, under conditions less favorable for photosynthesis, a second dissipation process, probably in or around the reaction center of photosystem II, also developed. Furthermore, evidence is presented that the zeaxanthin-associated dissipation process prevents sustained inactivation of photochemistry by excessive light. PMID- 16667276 TI - Structure of the Threonine-Rich Extensin from Zea mays. AB - Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the ;P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot. PMID- 16667277 TI - A chenopod extensin lacks repetitive tetrahydroxyproline blocks. AB - An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criteria for membership of the extensin family save one, notably, lack of the ;diagnostic' pentamer Ser-Hyp-Hyp-Hyp-Hyp. However, sequence analysis of the major tryptic peptides shows that sugar beet extensin shares a motif in common with tomato extensin P1 but differs by the position of an insertion sequence [X] or [Y] which, in sugar beet, splits the tetrahydroxyproline block: Ser-Hyp-Hyp-[X]-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, where [X] is [Val-His-Glu/Lys-Tyr Pro], while in tomato the insertion sequence [Y] = [Val-Lys-Pro-Tyr-His-Pro] and, when it occurs, immediately follows the tetrahydroxyproline block: Ser-Hyp-Hyp Hyp-Hyp-[Y]-Thr-Hyp-Val-Tyr-Lys. Based on these data we reinterpret three highly repetitive cDNA sequences, including nodulin N75 from soybean and wound-induced P33 of carrot, as extensins with split tetra(hydroxy)proline blocks. PMID- 16667278 TI - Transient gene expression in maize, rice, and wheat cells using an airgun apparatus. AB - An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The beta-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 x 10(-3). In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals. PMID- 16667279 TI - Solubilization and reconstitution of the oat root vacuolar h/ca exchanger. AB - Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H(+)/Ca(2+) antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [DeltapH] dependent). The first step toward purification and identification of the H(+)/Ca(2+) antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H(+)/Ca(2+) antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A DeltapH (acid inside) was generated in the proteoliposomes with an NH(4)Cl gradient (NH(4) (+) (in) >> NH(4) (+) (out)) as determined by methylamine uptake. Fundamental properties of DeltapH dependent calcium uptake such as the K(m) for calcium ( approximately 15 micromolar) and the sensitivity to inhibitors such as N,N'-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H(+)/Ca(2+) antiporter has been reconstituted in active form. PMID- 16667280 TI - Developmental regulation of enzymes of sucrose and hexose metabolism in effective and ineffective soybean nodules. AB - Soybean (Glycine max) nodules formed by inoculation with either an effective strain or an ineffective (noninvasive, nodule-forming) strain of Bradyrhizobium japonicum were assayed for changes in developmental patterns of carbon metabolic enzymes of the plant nodule cells. Of the enzyme activities measured, only sucrose synthase, glutamine synthetase, and alcohol dehydrogenase were altered in the ineffective nodules relative to the effective nodules. Sucrose synthase and glutamine synthetase activities were greatly reduced, whereas alcohol dehydrogenase activity was elevated. Dark-induced senescence severely affected sucrose synthase but had little, if any, effect on the other enzymes measured. The developmental patterns of the anaerobically induced enzymes, aldolase and alcohol dehydrogenase, were different from those expected, implying that their development is not regulated solely by oxygen deprivation. However, anaerobic treatment of nodules resulted in responses similar to those enzymes in maize. The developmental profiles of the carbon metabolic enzymes suggest that carbohydrates are metabolized via the sucrose synthase and pentose phosphate pathways. This route of carbon metabolism, compared to glycolysis, would reduce the requirement of ATP for carbohydrate catabolism, generate NADPH for biosynthetic reactions, and provide intermediates for plant secondary metabolism. PMID- 16667281 TI - Further studies on o(2)-resistant photosynthesis and photorespiration in a tobacco mutant with enhanced catalase activity. AB - The increase in net photosynthesis in M(4) progeny of an O(2)-resistant tobacco (Nicotiana tabacum) mutant relative to wild-type plants at 21 and 42% O(2) has been confirmed and further investigated. Self-pollination of an M(3) mutant produced M(4) progeny segregating high catalase phenotypes (average 40% greater than wild type) at a frequency of about 60%. The high catalase phenotype cosegregated precisely with O(2)-resistant photosynthesis. About 25% of the F(1) progeny of reciprocal crosses between the same M(3) mutant and wild type had high catalase activity, whether the mutant was used as the maternal or paternal parent, indicating nuclear inheritance. In high-catalase mutants the activity of NADH-hydroxypyruvate reductase, another peroxisomal enzyme, was the same as wild type. The mutants released 15% less photorespiratory CO(2) as a percent of net photosynthesis in CO(2)-free 21% O(2) and 36% less in CO(2)-free 42% O(2) compared with wild type. The mutant leaf tissue also released less (14)CO(2) per [1-(14)C]glycolate metabolized than wild type in normal air, consistent with less photorespiration in the mutant. The O(2)-resistant photosynthesis appears to be caused by a decrease in photorespiration especially under conditions of high O(2) where the stoichiometry of CO(2) release per glycolate metabolized is expected to be enhanced. The higher catalase activity in the mutant may decrease the nonenzymatic peroxidation of keto-acids such as hydroxypyruvate and glyoxylate by photorespiratory H(2)O(2). PMID- 16667282 TI - Differentiation between Phycobiliprotein and Colorless Linker Polypeptides by Fluorescence in the Presence of ZnSO(4). AB - Microcystis aeruginosa, a unicellular cyanobacterium, contains small phycobilisomes consisting of C-phycocyanin, allophycocyanin, and linker polypeptides. SDS-polyacrylamide gels of the phycobilisomes were examined for fluorescent bands before and after spraying with a solution of ZnSO(4), followed by Coomassie brilliant blue staining for protein. This procedure provides a rapid and sensitive method for detecting small amounts of phycobilin-containing polypeptides and distinguishing them from other tetrapyrrole-containing polypeptides and from ;colorless' ones. Three polypeptide bands, in addition to the alpha and beta phycobiliprotein subunits, have been detected under these conditions. An 85 kilodalton polypeptide was identified as a phycobiliprotein due to its enhanced fluorescence in the presence of ZnSO(4). The other polypeptides do not contain chromophores and are colorless. They are approximately 34.5 and 30 kilodaltons in size. PMID- 16667283 TI - Purification and thermal dependence of glutathione reductase from two forage legume species. AB - Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from ;Cimarron' alfalfa and ;PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45 degrees C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25 degrees C), and (c) the thermal kinetic windows (6-19 degrees C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID- 16667284 TI - Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa. AB - Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anolles, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells. PMID- 16667285 TI - The distribution of catalase activity, isozyme protein, and transcript in the tissues of the developing maize seedling. AB - The catalase activity, CAT-2 and CAT-3 isozyme protein levels, and the steady state mRNA levels for each of the three catalase genes were determined in the scutellum, root, epicotyl, and leaf of the developing maize (Zea mays L.) seedling. Catalase activity was highest in the scutellum, with 10-fold lower enzyme activity in the leaf and epicotyl. Very low levels of catalase activity were found in the root. The highest levels of CAT-2 protein were found in the scutellum, with about 10-fold lower levels in the green leaf. CAT-2 protein was present in trace amounts early in root development and no CAT-2 protein was detected in the epicotyl. Shortly after germination, CAT-3 protein was present at high levels in both the epicotyl and green leaf. With development, the amount of CAT-3 protein decreased slowly in the epicotyl and rapidly in the green leaf. Low levels of this isozyme were detected in the scutellum and root. The Cat1 transcript accumulated to low levels in all four tissues during the 14 day developmental period. High levels of the Cat2 transcript were found in the scutellum, with moderate levels of the mRNA in the green leaf. The Cat2 transcript levels were very low in the root and epicotyl. While the Cat3 mRNA level in the scutellum was low, high levels of the Cat3 transcript were detected in the root, epicotyl, and leaf. There was a positive correlation between the accumulation of a catalase isozyme and its transcript, indicating that the tissue specificity of maize catalase gene expression was regulated pretranslationally. PMID- 16667286 TI - Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes. AB - Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37 degrees C of 8.82 x 10(2) per molar per second and a dissociation rate constant of 3.3 x 10(-3) per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (K(i) of 2 x 10(-6) molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (K(i) of 6.23 x 10(-8) molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4. PMID- 16667287 TI - Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor. AB - Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M(r) 30,000). The enzyme had a K(m) for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37 degrees C for 1 hour, (NH(4))(2)SO(4) fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M(r) 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37 degrees C for 1 hour. PMID- 16667288 TI - Immunological characterization of in vitro forms of homoserine dehydrogenase from carrot suspension cultures. AB - Multiple forms of homoserine dehydrogenase (HSDH) from carrot (Daucus carota L.) have been identified. One form of HSDH (T-form) has a relative molecular weight of 240,000 and is strongly inhibited by threonine. Another form (K-form) has a relative molecular weight of 180,000 and is insensitive to inhibition by threonine. The interconversion of these two forms is dependent upon the presence or absence of threonine and potassium. Polyacrylamide electrophoretic gels stained for HSDH activity and protein, paralleled with Western blot analysis, verified the interconversion of the T- and K-forms in 5 millimolar threonine and 100 millimolar potassium, respectively. Carrot HSDH also aggregates to form higher molecular weight complexes of 240,000 up to 720,000 M(r.) Polyclonal antibody from mouse was raised against the T-form (240,000 M(r)) of carrot HSDH. Specificity of the mouse antisera to carrot HSDH was verified by immunoprecipitation and Western blot analysis. The T-form, K-form, and all of the higher molecular aggregates of carrot HSDH cross-reacted with the anti-HSDH antiserum. The antiserum also cross-reacted with soybean HSDH, but did not cross react with either of the two HSDH forms found in Escherichia coli. A model for the in vivo regulation of threonine biosynthesis in the chloroplast is presented. The model is based on the interconversion of the HSDH forms by potassium and threonine. PMID- 16667289 TI - The anaerobic response of soybean. AB - The effect of anoxia on roots of soybean (Glycine max [L.] Merr., variety ;Williams') was studied at various levels and the results compared to those from previously studied species. While alcohol dehydrogenase (ADH) activity is induced in a manner similar to other plant species, other aspects of the anaerobic response are unique to soybean. A variety of molecular clones was used to analyze changes in soybean and maize RNA levels. Increased RNA accumulation was observed in both species with a maize ADH clone, while a maize aldolase and one of the two different maize glyceraldehyde-3-phosphate dehydrogenase cDNA clones showed induction only in maize. A maize sucrose synthase 1 clone showed induction in maize but no hybridization to soybean RNA samples. The reduction in the number of anaerobically inducible soybean genes relative to maize is consistent with in vivo and in vitro protein synthesis results. Only four major proteins are labeled during anoxia in soybean, one corresponding to ADH, while maize has been reported to have about 20. In either species, in vitro translation yields similar products with RNA from anaerobic and pre-stress plants, indicative of translational control during anoxia. These results are discussed in relation to the differential tolerance of maize and soybean to anaerobic stress. PMID- 16667290 TI - Photosynthetic Potential and Accumulation of Assimilates in the Developing Chloroembryos of Cyamopsis tetragonoloba (L.) Taub. AB - The photosynthetic potential of leaves and chloroembryos of Cyamopsis tetragonoloba (L.) Taub as measured by (14)C-bicarbonate fixation, Hill activity, and in vivo fluorescence transients is compared. On a chlorophyll basis, dark fixation of NaH(14)CO(3) in chloroembryos was 1.5 times higher than that of the leaf, whereas carbon fixation under illumination was threefold higher in the leaf than in the embryos. Rates of O(2) evolution were four times more in embryo than in leaf chloroplasts. Shading of developing fruits on the day of anthesis for 10 days induced a 65% reduction in dry matter accumulation in the etiolated embryos, as compared to the normal green embryos of the same fruit half covered by a transparent Polythene sheet. The reduction in dry weight, size of the embryos, and levels of assimilates after shading the developing fruits may be ascribed to partial autotrophy of the chloroembryos. PMID- 16667291 TI - Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs. AB - Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with beta-glucosidase, beta-GIcNAcase, and alpha-mannosidase, respectively. Swainsonine specifically inhibited alpha-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited beta-N acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised. PMID- 16667292 TI - Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control. AB - The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38 degrees C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b deficient strain pg-113 at the same rate in the dark or the light at 38 degrees C, an indication that light did not affect translation of LHCP mRNA. Membrane associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells. PMID- 16667293 TI - Changes in the Composition and Synthesis of Proteins in Cellular Membranes of Echinochloa crus-galli (L.) Beauv. Seeds during the Transition from Dormancy to Germination. AB - The effect of alcohols which stimulate or have no effect on germination on the composition and synthetic pattern of proteins in the cellular membranes of Echinochloa crus-galli (L.) Beauv. seeds was studied. Imbibition of dry seeds was accompanied by an increase in the synthesis of proteins and by synthesis of new proteins in their intracellular membranes. The transition of the seeds from a dormant to a nondormant state was associated with synthesis of specific proteins and a decrease in content of others in the plasma membrane. The synthesis of a 23 kilodalton protein was strongly increased upon release from dormancy. The changes in the pattern of protein synthesis were not directly associated with the beginning of germination. The results suggest that the plasma membrane constitutes the first site in the seed cells, at which the stimulus from external factors affecting seed dormancy is detected. PMID- 16667294 TI - Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons. AB - Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids. PMID- 16667295 TI - Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures. AB - Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-beta-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression. PMID- 16667296 TI - Stress Responses in Alfalfa (Medicago sativa L.): II. Purification, Characterization, and Induction of Phenylalanine Ammonia-Lyase Isoforms from Elicitor-Treated Cell Suspension Cultures. AB - l-Phenylalanine ammonia-lyase has been purified from elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of M(r) 311,000; the intact subunit M(r) was about 79,000, although polypeptides of M(r) 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different K(m) values for l-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction. PMID- 16667297 TI - Inhibition of phosphoenolpyruvate carboxylase by malate. AB - Malate has been noted to be a ;mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examination of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC. PMID- 16667298 TI - Effect of polyethylene glycol exclusion on the water potential of solution saturated filter paper. AB - Solutions of high molecular weight polyethylene glycol are often used to control water potential in seed germination studies. There is an implicit assumption that the seed support materials do not alter the water potential of the osmotic solution. Filter paper, however, contains a hydrophylic volume fraction that is inaccessible to high molecular weight polymers. Water absorbed by filter paper fibers was found to concentrate polyethylene glycol and lower water potential in solution-filter paper mixtures. The magnitude of this concentration effect is a function of the original concentration of polyethylene glycol and the ratio of solution volume to filter paper weight. PMID- 16667299 TI - Induction of glutathione s-transferase isozymes in sorghum by herbicide antidotes. AB - Certain chemicals referred to as herbicide antidotes protect sorghum from injury by chloroacetanilide herbicides such as metolachlor. The effect of herbicide antidotes on the glutathione S-transferase isozyme complement of etiolated sorghum (Sorghum bicolor [L.] Moench) shoots was examined. Elution profiles of glutathione S-transferase isozymes from untreated and antidote-treated seedlings were generated by fast protein liquid chromatography utilizing an anion exchange (Mono Q) column. In untreated seedlings, there were two glutathione S-transferase isozymes, a major isozyme which exhibited activity toward 1-chloro-2,4 dinitrobenzene and a minor isozyme which exhibited activity toward metolachlor. Treating sorghum seedlings with various antidotes (flurazole, oxabetrinil, CGA 133205, naphthalic anhydride, dichlormid) resulted in the appearance of four to five additional glutathione S-transferase isozymes (de-pending on the particular antidote) which exhibited activity toward metolachlor as a substrate and little or no activity with 1-chloro-2,4-dinitrobenzene. Treating etiolated sorghum shoots with metolachlor was also found to induce at least four isozymes which exhibited activity toward the herbicide. An increase in glutathione S-transferase activity, measured with metolachlor as substrate, was detected within 4 h after treatment with 30 micromolar oxabetrinil, but 36 hours were required for maximum expression of activity. Addition of either the transcription inhibitor cordycepin or the translation inhibitor cycloheximide inhibited the appearance of glutathione S-transferase activity measured with metolachlor as substrate. The results are consistent with the hypothesis that antidotes confer protection against metolachlor injury in sorghum by inducing the de novo synthesis of glutathione S-transferase isozymes which catalyze the detoxification of the herbicide. PMID- 16667300 TI - Developmental staging of maize microspores reveals a transition in developing microspore proteins. AB - A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period. PMID- 16667301 TI - Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured frankia. AB - Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O(2) partial pressure (pO(2)) rise, to the proximity of the starting and ending oxygen tensions to the pO(2) optimum, and to the time for which the plant was exposed to the lower pO(2). Oxygen-induced transients, induced both by step jumps in pO(2) and by O(2) pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance. PMID- 16667302 TI - Photoinhibition in Vitis californica: The Role of Temperature during High-Light Treatment. AB - Leaves of Vitis californica Benth. (California wild grape) exposed to a photon flux density (PFD) equivalent to full sun exhibited temperature-dependent reductions in the rates or efficiencies of component photosynthetic processes. During high-PFD exposure, net CO(2) uptake, photon yield of oxygen evolution, and photosystem II chlorophyll fluorescence at 77 Kelvin (F(m), F(v), and F(v)/F(m)) were more severely inhibited at high and low temperatures than at intermediate temperatures. Sun leaves tolerated high PFD more than growth chamber-grown leaves but exhibited qualitatively similar temperature-dependent responses to high-PFD exposures. Photosystem II fluorescence and net CO(2) uptake exhibited different sensitivities to PFD and temperature. Fluorescence and gas exchange kinetics during exposure to high PFD suggested an interaction of multiple, temperature dependent processes, involving both regulation of energy distribution and damage to photosynthetic components. Comparison of F(v)/F(m) to photon yield of oxygen evolution yielded a single, curvilinear relationship, regardless of growth condition or treatment temperature, whereas the relationship between F(m) (or F(v)) and photon yield varied with growth conditions. This indicated that F(v)/F(m) was the most reliable fluorescence indicator of PSII photochemical efficiency for leaves of different growth conditions and treatments. PMID- 16667303 TI - Two distinct blue-light responses regulate epicotyl elongation in pea. AB - Blue light induces a long-term suppression of epicotyl elongation in red-light grown pea (Pisum sativum L.) seedlings. The fluence-response characteristics are bell-shaped, indicating the possibility of two different blue-light responses: a lower fluence response causing suppression and a higher fluence response alleviating the suppression. To determine if two responses are in effect, we have grown pea seedlings under dark conditions hoping to eliminate one or the other response. Under these growth conditions, only the lower fluence portion of the response (suppression of elongation) is apparent. The kinetics of suppression are similar to those observed for the lower fluence response of red-light-grown seedlings. The response to blue light in the dark-grown seedlings is not due to the excitation of phytochrome because a pulse of far-red light large enough to negate phytochrome-induced suppression has no effect on the blue-light-induced suppression. Furthermore, treatment of the dark-grown seedlings with red light immediately prior to treatment with high fluence blue light does not elicit the higher fluence response, indicating that the role of red light in the blue high fluence response is to allow the plant to achieve a specific developmental state in which it is competent to respond to the higher fluences of blue light. PMID- 16667304 TI - Chloramphenicol Acetyl Transferase (CAT) Protein Is Expressed in Transgenic Tobacco in Field Tests following Attack by Insects. AB - The expression of chloramphenical acetyl transferase (CAT) protein driven by the wound-inducible promoter from the proteinase inhibitor II K (pin2) gene was examined in whole tobacco (Nicotiana tabacum L.) plants under field conditions. Mechanical wounding of the field-grown leaves caused an accumulation of CAT protein in these leaves which begins several hours after wounding and continues to accumulate for about 36 hours. When sections of leaves were assayed for accumulation of CAT protein following wounding, the CAT protein was found to accumulate in the apical portions of the leaves. When endogenous insects attacked the leaves of transgenic plants grown in the field, the plants responded by inducing CAT protein. The mesophyll cells of the leaf were the site of expression of the CAT protein rather than the mid-vein or major veins within the leaf blade, indicating that the wound-inducible pin2 promoter specifically directs the synthesis of novel genes in tissues preferentially consumed by larval insects. PMID- 16667305 TI - Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes. AB - The effects of heat shock on the synthesis of alpha-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25 degrees C to 40 degrees C for 3 hours, inhibits the accumulation of alpha-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca(2+). When ER is isolated from heat-shocked aleurone layers, less newly synthesized alpha-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca(2+) transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers. PMID- 16667306 TI - Photoinhibition Resistance in the Red Alga Porphyra perforata: The Role of Photoinhibition Repair. AB - Photoinhibition resistance exhibited by the high intertidal red alga Porphyra perforata relative to its subtidal congener Porphyra nereocystis was examined using the protein synthesis inhibitor chloramphenicol to separate the damage and repair components of photoinhibition. Under photoinhibitory conditions, the rates of both damage to and replacement of photoinhibition-sensitive proteins was much higher in P. nereocystis than in P. perforata. Thus, photoinhibition resistance in P. perforata appears to be due to a reduced rate of photoinhibition damage rather than to an accelerated rate of photoinhibition repair. Reduction of photoinhibition damage in P. perforata may be by means of biophysical processes which increase the radiationless decay of excitation to heat in photosystem II. Alternatively, the photoinhibition-sensitive proteins in P. perforata may have slight structural alterations that improve their stability or they may be protected by enzyme systems that quench radicals formed by overexcitation of photosystem II. Reduction of the damage component of photoinhibition is a reasonable way to limit photoinhibition in P. perforata during the severe desiccation and exposure to full sun that occur simultaneously during daily low tides, conditions under which the protein synthesis required for photoinhibition repair could not occur. PMID- 16667307 TI - Tobacco Mesophyll Protoplasts Synthesize 1,3-beta-Glucanase, Chitinases, and "Osmotins" during in Vitro Culture. AB - Tobacco (Nicotiana tabacum) mesophyll protoplasts synthesize six basic proteins (a, a', a(1), b, b', and c) which are undetectable in the leaf and whose synthesis is reduced by auxin (Y Meyer, L Aspart, Y Chartier [1984] Plant Physiol 75: 1027-1033). Polypeptides a, a', and a(1) were shown to have similar mobilities on two-dimensional electrophoresis as one 1,3-beta-glucanase and two chitinases from tobacco mosaic virus-infected leaves. In immunoblotting experiments, polypeptide a was recognized by specific antibodies raised against the 1,3-beta-glucanase and a' and a(1) reacted with anti-chitinase antibodies. Similarly, b and b' comigrated with osmotin and its neutral counterpart, two proteins characteristic of salt-adapted tobacco cells, and reacted with anti osmotin antibodies. In addition it has been shown that 1,3-beta-glucanase and chitinase activities increased at the same time as a, a', and a(1) accumulated in cultivated protoplasts. Finally, polypeptide c was also detected in tobacco mosaic virus-infected leaves but could not be identified as any of the pathogenesis-related proteins characterized so far in tobacco. Thus, cultivated tobacco protoplasts synthesize and accumulate typical stress proteins. PMID- 16667308 TI - Potential importance of metal-ligand interactions in enzyme assays demonstrated with the assay cocktail for phosphoenolpyruvate carboxylase. AB - Assay cocktail modification such as addition of effector can cause inadvertent changes in the concentration of other metalligand and free species. In some cases, e.g. in the assay for phosphoenolpyruvate carboxylase under a limiting substrate condition, unintentional changes in substrate concentration are significant and confound an interpretation based solely on the total concentration of each component. A cautionary argument is developed on the basis of examples from the current literature. PMID- 16667309 TI - Circadian Rhythm of the Prokaryote Synechococcus sp. RF-1. AB - The prokaryotic Synechococcus sp. RF-1 exhibited a nitrogen fixation circadian rhythm with characteristics remarkably similar to the circadian rhythm of eukaryotes. The rhythm had a free-running period of about 24 hours when the length of the preen-trained cycle did not differ too much from 24 hours, and it was insensitive to changes in temperature from 22 degrees C to 33 degrees C. Because the endogenous rhythm of nitrogen fixation was not affected by a phase shift of its previous cycles, the circadian rhythm in Synechococcus sp. RF-1 was not considered to be controlled simply by a feedback mechanism. PMID- 16667310 TI - Carbon isotope discrimination varies genetically in c(4) species. AB - Carbon-isotope discrimination (Delta) is used to distinguish between different photosynthetic pathways. It has also been shown that variation in Delta occurs among varieties of C(3) species, but not as yet, in C(4) species. We now report that Delta also varies among genotypes of sorghum (Sorghum bicolor Moench), a C(4) species. The discrimination in leaves of field-grown plants of 12 diverse genotypes of sorghum was measured and compared with their grain yields. Discrimination varied significantly among genotypes, and there was a significant negative correlation between grain yield and Delta. The variation in Delta may be caused by genetic differences in either leakiness of the bundle-sheath cells or by differences in the ratio of assimilation rate to stomatal conductance. At the leaf level, the former should be related to light-use efficiency of carbon fixation and the latter should be related to transpiration efficiency. Both could relate to the yield of the crop. PMID- 16667311 TI - Globulin Gene Expression in Embryos of Maize viviparous Mutants : Evidence for Regulation of the Glb1 Gene by Abscissic Acid. AB - Expression of genes encoding the major Zea mays embryo globulins was examined in the maize precocious germination viviparous (vp) mutants. Comparison of globulin protein profiles of precociously germinating mutant embryos with those of normally germinating mature embryos revealed substantial differences with respect to the proteins encoded by the Glb1 gene. Analysis of Glb1 transcript levels in vp/vp embryos suggests that these mutants do not fully switch from a program of embryo maturation to one of germination. These preliminary studies indicate that the vp mutants provide an excellent system for the study of embryo maturation in maize. We also provide evidence for the positive regulation of Glb1 expression by the plant growth regulator abscisic acid. PMID- 16667312 TI - A Two-Dimensional Vibrating Probe Study of Currents around Lateral Roots of Raphanus sativus Developing in Culture. AB - A computer-assisted, two-dimensional vibrating probe was used to study the ionic currents around developing lateral roots of Raphanus sativus in vitro. This system allowed us to superimpose current vectors on the video image of the roots. In a young lateral root, current entered the cap, meristematic, and elongation zones and exited the primary root surface close to the base of the lateral root. As the lateral root grew, current began to exit from its basal (cell maturation zone) end. The densities of currents entering the apical portion of the faster growing lateral roots in a medium lacking indole 3-acetic acid were about twice as large as those entering the apical region of the slower-growing lateral roots in indole 3-acetic acid-supplemented medium. PMID- 16667313 TI - Polyamine levels and tomato fruit development: possible interaction with ethylene. AB - Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life. PMID- 16667314 TI - Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves. AB - The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9' cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an (18)O(2)-containing atmosphere resulted in the synthesis of ABA with levels of (18)O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA. PMID- 16667315 TI - Emission of volatile sulfur compounds from spruce trees. AB - Spruce (Picea Abies L.) trees from the same clone were supplied with different, but low, amounts of plant available sulfate in the soil (9.7-18.1 milligrams per 100 grams of soil). Branches attached to the trees were enclosed in a dynamic gas exchange cuvette and analyzed for the emission of volatile sulfur compounds. Independent of the sulfate supply in the soil, H(2)S was the predominant reduced sulfur compound continuously emitted from the branches with high rates during the day and low rates in the night. In the light, as well as in the dark, the rates of H(2)S emission increased exponentially with increasing water vapor flux from the needles. Approximately 1 nanomole of H(2)S was found to be emitted per mole of water. When stomata were closed completely, only minute emission of H(2)S was observed. Apparently, H(2)S emission from the needles is highly dependent on stromatal aperture, and permeation through the cuticle is negligible. In several experiments, small amounts of dimethylsulfide and carbonylsulfide were also detected in a portion of the samples. However, SO(2) was the only sulfur compound consistently emitted from branches of spruce trees in addition to H(2)S. Emission of SO(2) mainly proceeded via an outburst starting before the beginning of the light period. The total amount of SO(2) emitted from the needles during this outburst was correlated with the plant available sulfate in the soil. The diurnal changes in sulfur metabolism that may result in an outburst of SO(2) are discussed. PMID- 16667316 TI - Cytokinins and flower bud formation in vitro in tobacco: role of the metabolites. AB - Explants from flower stalks of Nicotiana tabacum L. were cultured on different cytokinins to induce flower bud formation. All cytokinins tested except zeatin and zeatin-riboside induced the same maximal number of flower buds. Benzyladenine, benzyladenosine, and dihydrozeatin were the most active compounds whereas isopentenyladenosine and isopentenyladenine acted at a 20-fold higher concentration. These data suggest that the active cytokinins bind to the same receptor with different affinities. The presence of benzyladenine in the medium was necessary only during the first 2 days of culture (initiation period). The equilibrium between benzyladenine and its conjugates (the riboside, glucoside, and nucleotides) after a 4-day pulse was independent of the benzyladenine concentration whether it was inductive or noninductive for bud formation. The level of all derivatives was proportional to the benzyladenine concentration in the medium. Isopentenyladenine was used as a competitive inhibitor of benzyladenine conjugation. Isopentenyladenine concentrations that were too low for bud formation led to a synergistic increase in bud number when applied together with benzyladenine. Isopentenyladenine decreased benzyladenine uptake and conjugation. In spite of the lower uptake, the concentration of free benzyladenine inside the explants was higher in the presence of isopentenyladenine than in its absence whereas the concentration of the 7 glucoside of benzyladenine was lower. It was concluded that the free cytokinin base is the main active compound. PMID- 16667317 TI - Phycobilisome Heterogeneity in the Red Alga Porphyra umbilicalis. AB - Phycobilisomes were isolated from Rhodophyceae brought from the field (Porphyra umbilicalis) or grown in culture under laboratory conditions (Antithamnion glanduliferum). In P. umbilicalis two kinds of well-coupled (ellipsoidal and hemidiscoidal) phycobilisomes were detected, in contrast to A. glanduliferum cultured algae in which only one kind of well-coupled, ellipsoidaltype phycobilisome appeared. The new phycobilisome-type particle detected in P. umbilicalis is characterized by an impoverishment in R-phycoerythrin and by sedimentation at lower density. The comparison between both phycobilisomes of P. umbilicalis allows determination of the presence of one colorless linker polypeptide (30 kilodaltons) associated with R-phycocyanin and allophycocyanin and two (40 and 38 kilodaltons) associated to R-phycoerythrin. The percentage of linker polypeptides associated with this pigment is low in the new phycobilisome like particle detected. This suggests that part of the R-phycoerythrin is less strongly bound to the phycobilisome than the other pigments. This feature could probably explain the existence of two kinds of phycobilisomes as intermediary steps of phycobilisome organization in algae exposed to rapid changes in environmental factors. In contrast, algae growing in culture and adapted to specific conditions do not present intermediary organization steps. Polypeptide composition and identification are given for this phycobilisome-like particle. PMID- 16667318 TI - Short-Term Effects of gamma-Irradiation on 1-Aminocyclopropane-1-Carboxylic Acid Metabolism in Early Climacteric Cherry Tomatoes : Comparison with Wounding. AB - gamma-Irradiation of early climacteric (breaker) cherry tomatoes (Lycopersicon pimpinellifollium L.) caused a sharp burst in ethylene production during the first hour. The extent of ethylene production was dose dependent and was maximum at about 3 kilograys. The content of 1-aminocyclopropane-1-carboxylic acid (ACC), followed the same evolution as ethylene production, while malonyl ACC increased steadily with time in irradiated fruits. The burst in ethylene production was accompanied by a sharp stimulation of ACC synthase activity which began 15 minutes after irradiation. The stimulation was completely prevented by cycloheximide, but not by actinomycin d or cordycepin. In contrast with irradiation, mechanical wounding continuously stimulated ethylene production over several hours. gamma-Irradiation and cordycepin applied to wounded tissues both caused the cessation of this continuous increase, but the initial burst was still persisting. These data suggest that gamma-irradiation, like wounding, stimulates the translation of preexisting mRNAs. It also reduces, at least temporarily, the subsequent transcription-dependent stimulation of ethylene production. gamma Irradiation greatly inhibited the activity of ethylene-forming enzyme at doses higher than 1 kilogray. Such sensitivity is in accordance with a highly integrated membranebound enzyme. PMID- 16667319 TI - Effects of the developmental state of the tissue on the competence for flower bud regeneration in pedicel explants of tobacco. AB - The competence of pedicel explants of tobacco (Nicotiana tabacum L. cv Samsun) to regenerate flower buds in response to auxin was manipulated by preincubating excised tissues in the absence of auxin. When exposed to 1 micromolar 1 naphthaleneacetic acid, these tissues formed fewer buds than controls that were not preincubated. The number of buds eventually formed correlated with the 1 naphthaleneacetic acid concentration in the tissue 6 hours after the start of hormone application. The internal concentrations in pretreated explants were lower than in tissues that were not pretreated due to diminished uptake per milligram fresh weight and increased hormone conjugation. The change in the developmental state induced by auxin deprivation had a dual effect on bud regeneration: (a) the pretreatment caused fewer buds to be formed at any 1 naphthaleneacetic acid concentration tested, and (b) a higher auxin concentration in the medium was required to get a maximum bud number on precultured explants. An increase of the 1-naphthaleneacetic acid concentration in the medium led to an elevated hormone level in freshly cut as well as in preincubated tissues. It was concluded that the developmental state of the tissue directly affects the maximum number of buds that can be regenerated. Apart from that there is an indirect effect exerted via modulation of the ratio between external and internal auxin concentration. The change in this ratio can be compensated for by an adjustment of the auxin concentration in the medium. PMID- 16667320 TI - Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures. AB - Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 +/- 5000, 105,000 +/- 5000, and 94,000 +/- 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M(r), as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K(m) values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and alpha-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism. PMID- 16667321 TI - Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions. AB - Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r(2) = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Psi(nod)) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C(2)H(2) reduction capacity of mildly water stressed nodules (Psi(nod) = -1.2 megapascals) was possible by increasing the external O(2) partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O(2) diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N(2) fixation activity with glucose than with succinate as an energy yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O(2), and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N(2) fixation under water restricted conditions. PMID- 16667322 TI - Foliar boron applications increase the final number of branches and pods on branches of field-grown soybeans. AB - Our previous work demonstrated that boron (B) supplied to soybeans (Glycine max [L.] Merrill cv ;Williams 82') by a stem infusion technique increased the number of pods on branches and led to a significant yield increase. Therefore, research was continued to determine whether soil or foliar applications of B could be used to achieve the same results. Field experiments were completed with both soil and foliar applications of B. Only the foliar applications of B resulted in a significant increase in the number of pods/branch. When split foliar treatments were applied twice during flowering, the total application of 0.56 kilograms of B per hectare was the optimal treatment for increasing pods/branch. In a second field experiment in 1987, soybeans were treated weekly from flowering through podfill with six split foliar applications of aqueous H(3)BO(3) solutions so that total applications were either 0, 1.1, or 2.24 kilograms of B per hectare. Foliar applications increased the number of branches/plant at the end of the season and significantly stimulated the formation of pods on branches, with 1.12 kilograms of B per hectare being the optimal treatment for these variables. This rate also tended to increase the number of seeds/plant and seed yield/plant. A duplicate experiment with minor modifications was conducted during the summer of 1988, and again the 1.12 kilograms of B per hectare application rate resulted in significant increases in number of branches at harvest as well as number of pods on branches. The 2.24 kilograms of B per hectare application rate also significantly increased these parameters. Foliar B applications induced increases in leaf B concentration far above the 60 micrograms per gram level that was previously accepted as the upper level of tolerance for soybeans. Since optimal branching and per plant yield parameters were achieved by plants with B leaf concentrations greater than 160 micrograms per gram, the accepted range of soybean tolerance for B must be reconsidered when B is foliarly applied. PMID- 16667323 TI - Effects of water vapor pressure deficit on photochemical and fluorescence yields in tobacco leaf tissue. AB - The relationship between photochemical quantum yield (phi(s)) and fluorescence yield have been investigated in leaf tissue from Nicotiana tabacum using CO(2) exchange and a modulated fluorescence measuring system. The quantum yield of CO(2) fixation at 1.6% (v/v) O(2) and limiting irradiance was reduced 20% by increasing the mean H(2)O vapor pressure deficit (VPD) from 9.2 to 18.6 mbars. As [CO(2)] and irradiance were varied, the intrinsic quantum yield of open photosystem II units (phi(s)/q(Q) where q(Q) is the photochemical fluorescence quenching coefficient) declined linearly with the degree of nonphotochemical fluorescence quenching. The slope and y-intercept values for this function were significantly reduced when the mean VPD was 18.4 millibars relative to 8.9 millibars. Susceptibility of the leaf tissue to photoinhibition was unaffected by VPD. Elevated O(2) concentrations (20.5% v/v) reduced the intrinsic quantum yield of net CO(2) uptake due to the occurrence of O(2)-reducing processes. However, the relative effect of high VPD compared to low VPD on intrinsic quantum yield was not dependent on the O(2) level. This suggests that the Mehler reaction does not mediate the response of quantum yield to elevated VPD. The results are discussed with regard to the possible role of transpiration stress in regulating dissipation of excitation by electron transport pathways other than noncyclic electron flow supporting reduction of CO(2) and/or O(2). PMID- 16667324 TI - Purification and Characterization of Pea Epicotyl beta-Amylase. AB - The most abundant beta-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this beta-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a beta-amylase by end product analysis and by its inability to hydrolyze beta-limit dextrin and to release dye from starch azure. Pea beta-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K(m) of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea beta amylase is competitively inhibited by its end product, maltose, with a K(i) of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with alpha-cyclohexaamylose being a stronger inhibitor than beta cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea beta-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea beta amylase is the multichain type. Possible roles of pea beta-amylase in cellular glucan metabolism are discussed. PMID- 16667325 TI - Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus. AB - Scenedesmus cells grown on high CO(2), when adapted to air levels of CO(2) for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO(2) from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O(2) evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO(2) pump with K(0.5) for O(2) evolution of less than 5 micromolar CO(2) was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO(3) (-) concentrating process between pH 8 to 10 lowered the K(0.5) (DIC) from 7300 micromolar HCO(3) (-) in high CO(2) grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO(3) (-) pump was inhibited by vanadate (K(i) of 150 micromolar), as if it involved an ATPase linked HCO(3) (-) transporter. The CO(2) pump was formed on low CO(2) by high CO(2) grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO(3) (-) pump was partially activated on low CO(2) within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO(3) (-) pump at pH 9 had requirements similar to the activation of the CO(2) pump. Air-grown or air adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO(2)-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO(2) pump and in some algae an additional vanadate-sensitive plasmalemma HCO(3) (-) transporter for a HCO(3) (-) pump. PMID- 16667326 TI - Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae. AB - Rates of photosynthetic O(2) evolution, for measuring K(0.5)(CO(2) + HCO(3) (-)) at pH 7, upon addition of 50 micromolar HCO(3) (-) to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K(1)(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO(2) uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O(2) evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO(3)), and the rate of (14)CO(2) fixation with 100 micromolar [(14)C] HCO(3) (-). Salicylhydroxamic acid inhibition of O(2) evolution and (14)CO(2)-fixation was reversed by higher levels of NaHCO(3). Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO(2) accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related. PMID- 16667327 TI - Subcellular compartmentation of uridine nucleotides and nucleoside-5' diphosphate kinase in leaves. AB - The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools. PMID- 16667328 TI - The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant. AB - A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue. PMID- 16667329 TI - Photosynthetic Decline from High Temperature Stress during Maturation of Wheat : I. Interaction with Senescence Processes. AB - Photosynthetic capacity decreases rapidly when temperate species are exposed to heat stress during reproductive development. We investigated whether injury in wheat (Triticum aestivum L.) resulted from general acceleration of senescence processes or specific heat-induced lesions. In situ photosynthetic capacity of leaf discs and thylakoid reactions were measured using flag leaf tissue from two cultivars maintained at 20 and 35 degrees C during maturation. Photosynthetic rates of leaf discs decreased faster at 35 than at 20 degrees C and were more photolabile in cv Len than in cv Waverly at high temperature. Patterns of thylakoid breakdown also differed in the two wheat genotypes at 20 degrees C: intersystem electron transport and photosystem II activity decreased linearly during postanthesis development in Len wheat, whereas coupling of photophosphorylation to electron transport declined late during senescence in Waverly wheat. Heat stress induced early loss of intersystem electron transport followed sequentially by decreased silicomolybdic acid, + 3-(3,4-dichlorophenyl) 1-dimethylurea-mediated photosystem II activity and 2,5-dichloro-p-benzoquinone mediated photosystem II activity in Len. Stress accelerated the uncoupling process, but loss of intersystem electron transport and photosystem II activities was slower in Waverly than in Len. We conclude that high temperature initially accelerated thylakoid component breakdown, an effect similar to normal senescence patterns. Thylakoid breakdown may induce a destabilizing imbalance between component reaction rates; an imbalance between photosystem II and cytochrome f/b(6)-mediated activities would be particularly damaging during heat stress. PMID- 16667330 TI - Photosynthetic Decline from High Temperature Stress during Maturation of Wheat : II. Interaction with Source and Sink Processes. AB - High temperature stress reduces grain growth in wheat (Triticum aestivum L.) by altering source activity and sink capacity. The impact of stress on source and sink interactions in two wheat cultivars of differing source thermotolerance was monitored by analysis of chlorophyll fluorescence transients, Fv (variable fluorescence) and PSM (peak, stationary, maximum), of attached flag leaves on intact and decapitated tillers grown at optimum (20 degrees C) and stress (35 degrees C) temperatures after anthesis. The thermotolerant cultivar Waverly had reduced Fv and PS quenching and a large increase of SM during heat stress. The less thermotolerant cultivar, Len, exhibited increased Fv and PS quenching and a small increase of SM. Fluorescence induction was similar in intact and decapitated tillers of Len, indicating diminished sinksource interaction during heat stress. The present results and previous observations of photosynthetic activities indicate that cyclic electron transport and photophosphorylation in flag leaves of the thermotolerant cultivar were stimulated by sink demand (increased SM in intact plants). Reduced grain development in the thermolabile cultivar resulted from limited capacity to support cyclic electron transport and photophosphorylation (slight increase in SM of intact plants and large reduction of Cytochrome f/b(6)-mediated electron transport capacity). It was concluded that heat stress injures the photosynthetic apparatus during reproductive growth of wheat and that diminished source activity and sink capacity may be equally important in reducing productivity. PMID- 16667331 TI - Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia. AB - Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed. PMID- 16667332 TI - Cryopreservation of rye protoplasts by vitrification. AB - A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20 degrees C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0 degrees C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN(2)); and (d) recovery of the protoplasts from LN(2) and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN(2) if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3+/-2%. In contrast, survival of ACC protoplasts following recovery from LN(2) was 34 +/- 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 +/- 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival. PMID- 16667333 TI - Evidence for and subcellular localization of a ca-stimulated phospholipase d from maize roots. AB - Autolytic lipid changes in corn (Zea mays L.) root crude homogenates and isolated membranes were examined by the use of high performance thin-layer chromatography. In the absence of added CaCl(2), losses in phosphatidylcholine and other phospholipids corresponds to increase in fatty acids without the accumulation of either phosphatidic acid or lyso-phosphatidylcholine. However, in the presence of 1 millimolar CaCl(2), phosphatidylcholine concentrations declined more rapidly with an immediate increase in phoshatidic acid, and slower rate of fatty acid accumulation. Autolytic phospholipid degradation yielded primarily free fatty acids in the absence of Ca and phosphatidic acid in the presence of 1 millimolar CaCl(2), suggesting the presence of an acyl hydrolase and phospholipase D activities. Differential centrifugation studies indicate that 50 to 80% of the crude homogenate's phospholipase D activity is membrane-bound. Density centrifugation experiments suggest that the membrane-bound phospholipase D activity is localized primarily on mitochondrial membranes. PMID- 16667334 TI - Purification and Partial Characterization of a Fructanase which Hydrolyzes Natural Polysaccharides from Sugarcane Juice. AB - A new sugarcane (Saccharum officinarum L.) fructanase which hydrolyzes both high molecular weight polysaccharides mid R:Fructose(4):Galactitol(5)mid R:(n) (SP) and moderate-sized carbohydrates mid R:Fructose(2):Galactitol(3)3mid R:(n) (MMWC) has been purified from sugarcane juice. The K(m) value has been estimated to be 33.7 micrograms per milliliter and 20 micrograms per milliliter for SP and MMWC, respectively. The optimal pH and temperature values are 6.0 and 30 degrees C, respectively. Purified protein has a pl value of 6.35 and a molecular weight of 13.2 kilodaltons. Fructanase activity appears to be Mn(2+)-dependent. PMID- 16667335 TI - On the regulation of spinach nitrate reductase. AB - A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin. PMID- 16667336 TI - Spermidine Uptake by Mitochondria of Helianthus tuberosus. AB - In the present work evidence is provided that spermidine, a polyamine largely present in plant tissues, may be transported, at physiological concentrations, into the matrix space of mitochondria isolated from tubers of Helianthus tuberosus L. cv OB1 (Jerusalem artichoke). It is concluded that the movement of spermidine strictly depends on membrane potential, since it is drastically blocked by valinomycin and only slightly sensitive to nigericin. Mg(2+) and K(+) inhibit the transport of spermidine in line with the general concept that these cations compete for the same binding sites on the mitochondrial membrane. In contrast to previous data on mammalian mitochondria, spermidine uptake by plant mitochondria does not depend on the presence of inorganic phosphate. This latter result, along with evidence that Ca(2+) does not affect accumulation of spermidine, indicates that the control of the polyamine uptake mechanism in plant mitochondria is distinct from that of mammalian systems. PMID- 16667337 TI - Senescence and stomatal aperture as affected by antibiotics in darkness and light. AB - In leaves of barley (Hordeum vulgare), as previously found with oats (Avena sativa), a group of six antibiotics that interfere in different ways with the sequence DNA --> mRNA --> protein all delay senescence in the dark, acting to conserve chlorophyll (Chl) and protein and also to open the stomata. Among the active compounds is chloramphenicol, which had previously been reported to act only on procaryotes. It is now shown that all these compounds with senescence delaying action in darkness have the opposite effect in light, accelerating Chl destruction and partially or completely closing the stomata. Leaves of the dicot Tropaeolum majus show most of the same responses, though the changes in protein and amino acids are more variable. The data as a whole support the previous conclusion that the synthesis of one or more proteins controls both the opening and the closing of the stomata. An additional compound, kanamycin, acts in the same way as the other six compounds on oats and barley, though its action on proteolysis is unclear. On Tropaeolum, however, it opens the stomata in both light and darkness. Anisomycin and ethidium bromide have comparably atypical effects. Thus, although changes in stomatal opening or closing in the majority of cases are closely linked to the breakdown or preservation of Chl, the occasional exception shows that the biochemical phenomena of senescence cannot be under the direct control of changes in stomatal aperture. PMID- 16667338 TI - Characterization of alpha-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris. AB - The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect alpha-amylases, but not of plant alpha-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, alpha-amylase inhibitor (alphaAl) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). Purified alphaAl can be resolved by SDS-PAGE into five bands (M(r) 15,000 19,000), four of which have covalently attached glycans. These bands represent glycoforms of two different polypeptides. All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it (LM Hoffman [1984] J Mol Appl Genet 2: 447-453; J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889). The primary translation product of alphaAl is a polypeptide of M(r) 28,000. Immunologically cross-reacting glycopolypeptides of M(r) 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M(r) 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that alphaAl is a typical bean lectin-type protein that is synthesized on the rough endoplasmlc reticulum, modified in the Golgi, and transported to the protein storage vacuoles. PMID- 16667339 TI - Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants. AB - NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C(3) plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C(4) plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C(4) plants, 0.5% in the CAM plant, and less than 0.01% in C(3) plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C(3) and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C(3) and CAM plants than to those of C(4) plants. PMID- 16667340 TI - The Formation and Distribution of Ice within Forsythia Flower Buds. AB - Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0 degrees C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between -10 and -25 degrees C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze fixed at -5 degrees C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil. PMID- 16667341 TI - Effects of the Fungal Endophyte Acremonium coenophialum on Nitrogen Accumulation and Metabolism in Tall Fescue. AB - Infection by the fungal endophyte Acremonium coenophialum affected the accumulation of inorganic and organic N in leaf blades and leaf sheaths of KY 31 tall fescue (Festuca arundinacea Schreb.) grown under greenhouse conditions. Total soluble amino acid concentrations were increased in either the blade or sheath of the leaf from infected plants. A number of amino acids were significantly increased in the sheath, but only asparagine increased in the blade. Infection resulted in higher sheath NH(4) (+) concentrations, whereas NO(3) (-) concentrations decreased in both leaf parts. The effects on amino acid, NO(3) (-), and NH(4) (+) concentrations were dependent upon the level of N fertilization and were usually apparent only at the high rate (10 millimolar) of application. Administration of (14)CO(2) to the leaf blades increased the accumulation of (14)C in their amino acid fraction but not in the sheaths of infected plants. This may indicate that infection increased amino acid synthesis in the blade but that translocation to the sheath, which is the site of fungal colonization, was not affected. Glutamine synthetase activity was greater in leaf blades of infected plants at high and low N rates of fertilization, but nitrate reductase activity was not affected in either part of the leaf. Increased activities of glutamine synthetase together with the other observed changes in N accumulation and metabolism in endophyte-infected tall fescue suggest that NH(4) (+) reassimilation could also be affected in the leaf blade. PMID- 16667342 TI - Correlation between the Maintenance of Photosynthesis and in Situ Protoplast Volume at Low Water Potentials in Droughted Wheat. AB - Studies were undertaken to examine the relationship between water deficit effects on photosynthesis and the extent of protoplast volume reduction which occurs in leaves at low water potential (Psi(w)). This relationship was monitored in two cultivars (;Condor' and ;Capelle Desprez') of cultivated wheat (Triticum aestivum) that differed in sensitivity to drought, and in a wild relative of cultivated wheat (Triticum kotschyi) that has been previously found to be ;drought resistant.' When subjected to periods of water stress, Condor and T. kotschyi plants underwent osmotic adjustment; Capelle plants did not. Photosynthetic capacity was maintained to different extents in the three genotypes as leaf Psi(w) declined during stress; Capelle plants were most severely affected. Calculations of internal leaf [CO(2)] and stomatal conductance from gas exchange measurements indicated that differences in photosynthetic inhibition at low Psi(w) among the genotypes were primarily due to nonstomatal effects. The extent of protoplast volume reduction that occurred in leaves at low Psi(w) was also found to be different in the three genotypes; maintenance of protoplast volume and photosynthetic capacity in stressed plants of the genotypes appeared to be correlated. When the extent of water stress-induced inhibition of photosynthesis was plotted as a function of declining protoplast volume, this relationship appeared identical for the three genotypes. It was concluded that there is a correlative association between protoplast volume and photosynthetic capacity in leaves of wheat plants subjected to periods of water stress. PMID- 16667343 TI - Ubiquitin Pool Modulation and Protein Degradation in Wheat Roots during High Temperature Stress. AB - Ubiquitin, a key component in an ATP-dependent proteolytic pathway, participates in the response of various eucaryotic organisms to high temperature stress. Our objective was to determine if ubiquitin serves a similar capacity for metabolizing altered proteins in higher plants during stress. Degradation of total proteins was measured, and ubiquitin pools (free versus conjugated) were extracted with an improved protocol from wheat (Triticum aestivum L. cv Len) roots treated at 22, 27, 32, 37, and 42 degrees C for 1 hour and assayed by western blots and radioimmunoassays. Heat-shock protein synthesis was detected by in vivo labeling and autoradiography. Mean half-life of total root proteins decreased from 51 hours at 22 degrees C to 23 hours at 40 degrees C. Ubiquitin pools were extracted better and proteolysis was slowed more by the improved protocol than by a conventional procedure for plant proteins. Amounts of high molecular mass conjugates were elevated and levels of low molecular mass conjugates and free ubiquitin were depressed when roots were treated at 37 or 42 degrees C than at lower temperatures; the same high temperatures also induced synthesis of heat-shock proteins. We concluded that high temperatures increase breakdown of root proteins, which are degraded via the ubiquitin proteolytic pathway. A conjugate with an apparent molecular mass of 23 kilodaltons was tentatively identified as an ubiquitinated histone. PMID- 16667344 TI - Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate. AB - The GTP-driven component of Ca(2+) uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca(2+)-translocating ATPase and assess its utility as a probe for this transport system. Uptake of (45)Ca(2+) in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca(2+) concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca(2+)-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of (45)Ca(2+)-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP driven (45)Ca(2+) uptake represents the capacity of the plasma membrane Ca(2+) translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [gamma-(32)P] GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca(2+)-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca(2+)-ATPases present in animal cells. PMID- 16667345 TI - Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures. AB - Respiration rates of Zea mays L. seedling tissues grown at 30 and 14 degrees C were measured at 25 degrees C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30 degrees C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14 degrees C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14 degrees C after growth at 30 degrees C for 2 days had the same alternative oxidase capacity as seedlings grown at 30 degrees C. Seedlings grown at 14 degrees C for 10 to 12 days, then transferred to 30 degrees C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20 degrees C. At 41 degrees C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41 degrees C was reversible. After incubation at 41 degrees C, the alternative oxidase capacity measured at 25 degrees C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41 degrees C as they did when incubated at 25 degrees C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration. PMID- 16667346 TI - Seedling growth, mitochondrial characteristics, and alternative respiratory capacity of corn genotypes differing in cold tolerance. AB - Four maize (Zea mays L.) inbreds representing genetic differences in seedling cold tolerance were used to determine the effect of growth temperatures on dry weight accumulation and mitochondrial properties, especially the alternative oxidase capacity. Seedlings were grown in darkness at 30 degrees C (constant), 14 degrees C (constant), and 15 degrees C for 16 hours and 8 degrees C for 8 hours. Inbreds B73 and B49 were characterized as cold tolerant while G50 and G84 were cold sensitive. Shoot growth rate of cold-sensitive inbreds in the lower temperatures was slower relative to the tolerant inbreds. Mesocotyl tissue was particularly sensitive to low temperatures during growth after germination. There were no significant differences in relative rates of mitochondrial respiration in the cold-tolerant compared to cold-sensitive inbreds measured at 25 degrees C. Mitochondria from all seedlings grown at all temperatures had the ability to phosphorylate as indicated by the observation of respiratory control. This result indicated that differences in low temperature growth were probably not related to mitochondrial function at low temperatures. Alternative oxidase capacity was higher in mitochondria from seedlings of all inbreds grown at 14 degrees C compared to 30 degrees C. Capacities in seedlings of 14 degrees C-grown B73 and G50 were higher than in B49 and G84. Capacities in seedlings grown for 16 hours at 15 degrees C and 8 hours at 8 degrees C were similar to those from 14 degrees C-grown except in G50 which was lower and similar to those grown at 30 degrees C. Mesocotyl tissue was the most responsive tissue to low growth temperature. Coleoptile plus leaf tissue responded similarly but contained lower capacities. Antibody probing of western blots of mitochondrial proteins confirmed that differences in alternative oxidase capacities were due to differences in levels of the alternative oxidase protein. Male sterile lines of B73 were also grown under the three different temperature regimes. These lines grew equally as well as the normal B73 at all temperatures and the response of alternative oxidase capacity and protein to low growth temperature was similar to normal B73. PMID- 16667347 TI - Fructan precipitation from a water/ethanol extract of oats and barley. AB - Fructan was precipitated from a water and ethanol extract of oat (Avena sativa L.) and barley (Hordeum vulgare L.). The degree of polymerization and response on a differential refractometer, based on peak area and height, was compared to fructan collected from a lead-based HPLC column and to commercially available inulin. Statistically significant differences are discussed. PMID- 16667348 TI - Effect of Light Quality and Vernalization on Late-Flowering Mutants of Arabidopsis thaliana. AB - We have analyzed the response to vernalization and light quality of six classes of late-flowering mutants (fb, fca, fe, fg, ft, and fy) previously isolated following mutagenesis of the early Landsberg race of Arabidopsis thaliana (L.) Heynh. When grown in continuous fluorescent illumination, four mutants (fca, fe, ft, and fy) and the Landsberg wild type exhibited a reduction in both flowering time and leaf number following 6 weeks of vernalization. A significant decrease in flowering time was also observed for all the mutants and the wild type when constant fluorescent illumination was supplemented with irradiation enriched in the red and far red regions of the spectrum. In the most extreme case, the late flowering phenotype of the fca mutant was completely suppressed by vernalization, suggesting that this mutation has a direct effect on flowering. The fe and fy mutants also showed a more pronounced response than wild type to both vernalization and incandescent supplementation. The ft mutant showed a similar response to that of the wild type. The fb and fg mutants were substantially less sensitive to these treatments. These results are interpreted in the context of a multifactorial pathway for induction of flowering, in which the various mutations affect different steps of the pathway. PMID- 16667349 TI - Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures. AB - Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I(50) values for growth, chlorophyll (Chl), beta-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [(14)C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action. PMID- 16667350 TI - Differential Regulation of Maize Homoserine Dehydrogenase under Physiological Conditions. AB - Homoserine dehydrogenase is associated with the multibranched pathway of amino acid biosynthesis originating with aspartic acid. Like most of the related pathway enzymes, this enzyme is localized in chloroplasts. The activity and regulatory properties of the threonine-sensitive isozyme of homoserine dehydrogenase isolated from Zea mays var earliking were examined under variable conditions that could exist within chloroplasts. Catalytic activity is not significantly altered within the range of pHs that occur within these organelles, but inhibition of the enzyme by the pathway product, l-threonine, is markedly diminished at the alkaline pHs characteristic of illuminated chloroplasts. Inhibition by threonine is also subject to modulation by physiological levels of NADPH. Under conditions considered to represent the environment within unilluminated chloroplasts, the enzyme is severely inhibited by micromolar concentrations of threonine, but significant enzyme activity is retained under conditions that are likely to occur during illumination, even in the presence of millimolar levels of threonine. These results indicate that homoserine dehydrogenase may be subject to environmentally mediated regulation in vivo. Other observations support this concept and suggest that the intrinsic catalytic and regulatory properties of key enzymes could facilitate a direct link between light-dependent carbon and nitrogen assimilation and amino acid biosynthesis in chloroplasts of higher plants. PMID- 16667351 TI - Xylem-to-Phloem Transfer of Organic Nitrogen in Young Soybean Plants. AB - Xylem-to-phloem transfer in young vegetative soybean (Glycine max [L.] Merr.) plants (V4 stage) was identified as the difference in the distribution of [(14)C]inulin, a xylem marker, and [(14)C]aminoisobutyric acid (AIB), a synthetic amino acid, fed via the transpiration stream. Since [(14)C]AIB was retained in the stem to some extent, whereas [(14)C]inulin was not, the distribution of these marker compounds in each leaf was expressed as a percentage of the total [(14)C] radioactivity recovered in the foliage. The developing third trifoliolate was a consistent and reliable indicator of xylem-to-phloem transfer. The phloem stream provided to the developing trifoliolate up to fourfold the relative proportion of solute received from the xylem stream; this was markedly reduced by increased light intensity and consequently water flow through the xylem. Evidence from heat girdling experiments is discussed with respect to the vascular anatomy of the soybean plant, and interpreted to suggest that direct xylem-to-phloem transfer in the stem, in the region of the second node, accounted for about one-half of the AIB supplied to the developing trifoliolate, with the remainder being provided from the second trifoliolate. Since AIB is not metabolized it seems likely that rapid transfer within the second trifoliolate occurred as direct veinal transfer rather than indirect cycling through the mesophyll. This study confirmed that xylem-to-phloem transfer plays a major role in the partitioning of nitrogen for early leaf development. PMID- 16667352 TI - Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized Euglena gracilis Cells. AB - Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called ;satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO(2)-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO(2)-fixation in photosynthesis. PMID- 16667353 TI - Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia. AB - Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out. PMID- 16667354 TI - The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray. AB - Washed rubber particles isolated from stem homogenates of Parthenium argentatum Gray by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain rubber transferase which catalyzes the polymerization of isopentenyl pyrophosphate into rubber polymer. The polymerization reaction requires Mg(2+) isopentenyl pyrophosphate, and an allylic pyrophosphate. The K(m) values for Mg(2+), isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 x 10( 4) molar, 8.3 x 10(-5) molar, and 9.6 x 10(-5) molar, respectively. The molecular characteristics of the rubber polymer synthesized from [(14)C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 x 10(6) to 500 Angstroms Ultrastyragel in a Waters 150C Gel Permeation Chromatograph. The peak molecular weight of the radioactive polymer increased from 70,000 in 15 minutes to 750,000 in 3 hours. The weight average molecular weight of the polymer synthesized over a 3 hour period was 1.17 x 10(6) compared to 1.49 x 10(6) for the natural rubber polymer extracted from the rubber particles. Over 90% of the in vitro formation of the rubber polymer was de novo from dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Treatment of the washed rubber particles with 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonate solubilized the rubber transferase. The solubilized enzyme(s) catalyzed the polymerization of isopentenyl pyrophosphate into rubber polymer with a peak molecular weight of 1 x 10(5) after 3 hours of incubation with Mg(2+) and dimethylallyl pyrophosphate. The data support the conclusion that the soluble preparation of rubber transferase is capable of catalyzing the formation of a high molecular weight rubber polymer from an allylic pyrophosphate initiator and isopentenyl pyrophosphate monomer. PMID- 16667355 TI - Red light regulates calcium-activated potassium channels in mougeotia plasma membrane. AB - The alga Mougeotia has a large central chloroplast whose positioning is regulated by photoactivation of phytochrome, possibly via modulation of cytosolic calcium (Serlin B, Roux SJ [1984] Proc Natl Acad Sci USA 81: 6368-6372). We used the patch clamp technique to examine the effects of red and far-red light on ion channel activity in the plasma membrane of Mougeotia protoplasts to determine if ion channels play a role in chloroplast movement. Patch clamping in the cell attached mode reveals two channels of about 2 and 4 picoamperes amplitude at 0 millivolt (inside pipette) and estimated conductances of 30 and 65 picosiemens. They are activated by red light irradiation after a lag period of about 2 to 5 minutes. Far-red light, when applied immediately after red light irradiation, reverses this activation; otherwise it has no effect. This result implicates phytochrome. The addition of the calcium ionophore, A23187, also activates ion channel activity after a lag of a few minutes. The channels are not specific for calcium since they are present when calcium is removed from the external and pipette media. They are inhibited by quaternary ammonium ions. Thus, we believe they are calcium-activated potassium channels. Their possible role in chloroplast positioning is discussed. PMID- 16667356 TI - Calcium activation of mougeotia potassium channels. AB - Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca(2+)] as low as 20 micromolar. However, external [Ca(2+)] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation. PMID- 16667357 TI - Interorgan translocation of 1-aminocyclopropane-1-carboxylic Acid and ethylene coordinates senescence in emasculated cymbidium flowers. AB - In Cymbidium flowers, emasculation by removal of the pollinia and the anther cap leads within 24 hours to red coloration of the labellum (lip). Lip coloration, being the first sign of senescence in these flowers, has been ascribed to the action of ethylene in the lip. When a small incision in the base of the lip is made prior to emasculation, or when the lip is excised and placed in water within 10 to 15 hours after emasculation, coloration is considerably delayed. This indicates that a coloration-associated factor is moving in or out of the lip. Measurements of ethylene production of excised flower parts, isolated at different times after emasculation, showed an increase only in the central column; the other flower parts, including the lip, did not show a measurable change. In contrast, in situ measurements of the ethylene production of the central column and the remaining portion of the flower revealed a simultaneous increase in all the flower parts following emasculation. Similarly, application of radiolabeled 1-aminocyclopropane-1-carboxylic acid (ACC) to the top of the central column in situ leads to the production of radiolabeled ethylene by all the flower parts. In addition, the ethylene production of isolated lips, measured immediately after excision, was initially high but ceased within 10 to 15 minutes. Treatment of the central column in situ with ethylene or ethephon did not stimulate ACC production but did stimulate lip coloration and this was accompanied by an increased internal ethylene concentration in the lip. The data indicate that endogenously produced as well as applied ACC is rapidly translocated from the site of production or application to all the other flower parts where it is immediately converted into ethylene. By excision of a flower organ, the influx of ACC is prevented, causing a rapid decrease in ethylene production. In addition, it was found that ethylene may also be translocated in physiologically significant amounts within the flower. The roles of ACC and ethylene as mobile senescence or wilting factors in emasculation- and pollination induced senescence is discussed. PMID- 16667358 TI - Oxidation versus Reductive Detoxification of SO(2) by Chloroplasts. AB - Intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) both oxidized and reduced added sulfite in the light. Oxidation was fast only when endogenous superoxide dismutase was inhibited by cyanide. It was largely suppressed by scavengers of oxygen radicals. After addition of O-acetylserine, chloroplasts reduced sulfite to cysteine and exhibited sulfite-dependent oxygen evolution. Cysteine synthesis from sulfite was faster than from sulfate. The results are discussed in relation to species-specific differences in the phytotoxicity of SO(2). PMID- 16667359 TI - Effect of Ca and Calmodulin on DeltapH Formation in Tonoplast Vesicles from Corn Roots. AB - The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (DeltapH) were studied in tonoplast membrane vesicles from corn (Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca(2+) and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H(+) efflux or due to inhibition of the H(+)-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed DeltapH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed DeltapH by 40% after the H(+) ATPase was inhibited with NO(3) (-). This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H(+)-ATPase by direct inhibition. PMID- 16667360 TI - Responses of transpiration and hydraulic conductance to root temperature in nitrogen- and phosphorus-deficient cotton seedlings. AB - Suboptimal N or P availability and cool temperatures all decrease apparent hydraulic conductance (L) of cotton (Gossypium hirsutum L.) roots. The interaction between nutrient status and root temperature was tested in seedlings grown in nutrient solutions. The depression of L (calculated as the ratio of transpiration rate to absolute value of leaf water potential [Psi(w)]) by nutrient stress depended strongly on root temperature, and was minimized at high temperatures. In fully nourished plants, L was high at all temperatures >/=20 degrees C, but it decreased greatly as root temperature approached the chilling threshold of 15 degrees C. Decreasing temperature lowered Psi(w) first, followed by transpiration rate. In N- or P-deficient plants, L approached the value for fully nourished plants at root temperatures >/=30 degrees C, but it decreased almost linearly with temperature as roots were cooled. Nutrient effects on L were mediated only by differences in transpiration, and Psi(w) was unaffected. The responses of Psi(w) and transpiration to root cooling and nutrient stress imply that if a messenger is transmitted from cooled roots to stomata, the messenger is effective only in nutrient-stressed plants. PMID- 16667361 TI - A Second Locus, Ixr B1 in Arabidopsis thaliana, that Confers Resistance to the Herbicide Isoxaben. AB - An isoxaben resistant mutant of Arabidopsis thaliana is described whose locus, Ixr B1, is unlinked genetically to the previously described resistance locus Ixr A (DR Heim, JL Roberts, PD Pike, IM Larrinua [1989] Plant Physiol 90: 146-150). A cross of strains each homozygous for one of these two resistance loci gives rise to some isoxaben sensitive F(2) progeny. Growth curves versus isoxaben of this mutant, its F(1) progeny and the wild-type parent strain showed that this locus displays a weakly codominant Mendelian phenotype. Callus cultures were established from plants homozygous as well as heterozygous for this locus. Growth inhibition curves done with these cultures mimic the data obtained in vivo. PMID- 16667362 TI - Nitrite photoreduction in vivo is inhibited by oxygen. AB - It was hypothesized previously that an O(2) inhibition of NO(2) (-) photoreduction would reflect a competition between O(2) and NO(2) (-) for electrons from ferredoxin at the site of plastid nitrite reductase. In order to test this in vivo, intact spinach (Spinacia oleracea L.) leaf chloroplast and mesophyll cell isolates held in high light were aerated with streams of 20% O(2)/80% N(2) (250 micromolar O(2) in aqueous solution) or, alternatively, streams of 100% N(2). Bicarbonate plus CO(2) and NO(2) (-) were supplied to reaction mixtures at levels just sufficient to promote maximal assimilations of CO(2) and NO(2) (-). In chloroplast isolates, there was a 9 to 30% O(2) inhibition of NO(2) (-) reduction while there were high rates of CO(2) fixation. In spinach and soybean (Glycine max) leaf cell isolates, NO(2) (-) photoreduction rates were 10 to 55% inhibited by O(2) at near ambient levels. It is possible that O(2) may compete, albeit weakly, with NO(2) (-) (nitrite reductase) for equivalents derived from reduced ferredoxin. Also, O(2) may oxidize sulfhydryl groups on nitrite reductase which are involved in substrate binding and/or activation. PMID- 16667363 TI - Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato. AB - A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome. PMID- 16667364 TI - Metabolism of Tryptophan and Tryptophan Analogs by Rhizobium meliloti. AB - The alfalfa symbiont Rhizobium meliloti Rm1021 produces indole-3-acetic acid in a regulated manner when supplied with exogenous tryptophan. Mutants with altered response to tryptophan analogs still produce indole-3-acetic acid, but are Fix(-) because bacteria do not fully differentiate into the nitrogen-fixing bacteriod form. These mutations are in apparently essential genes tightly linked to a dominant streptomycin resistance locus. PMID- 16667365 TI - Detection of Kestoses and Kestose-Related Oligosaccharides in Extracts of Festuca arundinacea, Dactylis glomerata L., and Asparagus officinalis L. Root Cultures and Invertase by C and H Nuclear Magnetic Resonance Spectroscopy. AB - A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that (13)C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose. PMID- 16667366 TI - Root hypoxia reduces leaf growth : role of factors in the transpiration stream. AB - This study examined the potential role of restricted phloem export, or import of substances from the roots in the leaf growth response to root hypoxia. In addition, the effects of root hypoxia on abscisic acid (ABA) and zeatin riboside (ZR) levels were measured and their effects on in vitro growth determined. Imposition of root hypoxia in the dark when transpirational water flux was minimal delayed the reduction in leaf growth until the following light period. Restriction of phloem transport by stem girdling did not eliminate the hypoxia induced reduction in leaf growth. In vitro growth of leaf discs was inhibited in the presence of xylem sap collected from hypoxic roots, and also by millimolar ABA. Disc growth was promoted by sap from aerated roots and by 0.1 micromolar ZR. The flux of both ABA and ZR was reduced in xylem sap from hypoxic roots. Leaf ABA transiently increased twofold after 24 hours of hypoxia exposure but there were no changes in leaf cytokinin levels. PMID- 16667367 TI - Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : III. Sensitivity of Germination to Water Potential and Abscisic Acid during Development. AB - Muskmelon (Cucumis melo L.) seeds are germinable 15 to 20 days before fruit maturity and are held at relatively high water content within the fruit, yet little precocious germination is observed. To investigate two possible factors preventing precocious germination, the inhibitory effects of abscisic acid and osmoticum on muskmelon seed germination were determined throughout development. Seeds were harvested at 5-day intervals from 30 to 65 days after anthesis (DAA) and incubated either fresh or after drying on factorial combinations of 0, 1, 3.3, 10, or 33 micromolar abscisic acid (ABA) and 0, -0.2, -0.4, -0.6, or -0.8 megapascals polyethylene glycol 8000 solutions at 30 degrees C. Radicle emergence was scored at 12-hour intervals for 10 days. In the absence of ABA, the water potential (Psi) required to inhibit fresh seed germination by 50% decreased from 0.3 to -0.8 megapascals between 30 and 60 DAA. The Psi inside developing fruits was from 0.4 to 1.4 megapascals lower than that required for germination at all stages of development, indicating that the fruit Psi is sufficiently low to prevent precocious germination. At 0 megapascal, the ABA concentration required to inhibit germination by 50% was approximately 10 micromolar up to 50 DAA and increased to >33 micromolar thereafter. Dehydration improved subsequent germination of immature seeds in ABA or low Psi. There was a linear additive interaction between ABA and Psi such that 10 micromolar ABA or -0.5 megapascal osmotic potential resulted in equivalent, and additive, reductions in germination rate and percentage of mature seeds. Abscisic acid had no effect on embryo solute potential or water content, but increased the apparent minimum turgor required for germination. ABA and osmoticum appear to influence germination rates and percentages by reducing the embryo growth potential (turgor in excess of a minimum threshold turgor) but via different mechanisms. Abscisic acid apparently increases the minimum turgor threshold, while low Psi reduces turgor by reducing seed water content. PMID- 16667368 TI - Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : IV. Characteristics of the Perisperm during Seed Development. AB - We previously reported that an apparent water potential disequilibrium is maintained late in muskmelon (Cucumis melo L.) seed development between the embryo and the surrounding fruit tissue (mesocarp). To further investigate the basis of this phenomenon, the permeability characteristics of the tissues surrounding muskmelon embryos (the mucilaginous endocarp, the testa, a 2- to 4 cell-layered perisperm and a single cell layer of endosperm) were examined from 20 to 65 days after anthesis (DAA). Water passes readily through the perisperm envelope (endosperm + perisperm), testa, and endocarp at all stages of development. Electrolyte leakage (conductivity of imbibition solutions) of individual intact seeds, decoated seeds (testa removed), and embryos (testa and perisperm envelope removed) was measured during imbibition of freshly harvested seeds. The testa accounted for up to 80% of the total electrolyte leakage. Leakage from decoated seeds fell by 8- to 10-fold between 25 and 45 DAA. Presence of the perisperm envelope prior to 40 DAA had little effect on leakage, while in more mature seeds, it reduced leakage by 2- to 3-fold. In mature seeds, freezing, soaking in methanol, autoclaving, accelerated aging, and other treatments which killed the embryos had little effect on leakage of intact or decoated seeds, but caused osmotic swelling of the perisperm envelope due to the leakage of solutes from the embryo into the space between the embryo and perisperm. The semipermeability of the perisperm envelope of mature seeds did not depend upon cellular viability or lipid membrane integrity. After maximum seed dry weight is attained (35-40 DAA), the perisperm envelope prevents the diffusion of solutes, but not of water, between the embryo and the surrounding testa, endocarp, and mesocarp tissue. PMID- 16667369 TI - Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : V. Water Relations of Imbibition and Germination. AB - The initiation of radicle growth during seed germination may be driven by solute accumulation and increased turgor pressure, by cell wall relaxation, or by weakening of tissues surrounding the embryo. To investigate these possibilities, imbibition kinetics, water contents, and water (Psi) and solute (psi(s)) potentials of intact muskmelon (Cucumis melo L.) seeds, decoated seeds (testa removed, but a thin perisperm/endosperm envelope remains around the embryo), and isolated cotyledons and embryonic axes were measured. Cotyledons and embryonic axes excised and imbibed as isolated tissues attained water contents 25 and 50% greater, respectively, than the same tissues hydrated within intact seeds. The effect of the testa and perisperm on embryo water content was due to mechanical restriction of embryo swelling and not to impermeability to water. The Psi and psi(s) of embryo tissues were measured by psychrometry after excision from imbibed intact seeds. For intact or decoated seeds and excised cotyledons, Psi values were >-0.2 MPa just prior to radicle emergence. The Psi of excised embryonic axes, however, averaged only -0.6 MPa over the same period. The embryonic axis apparently is mechanically constrained within the testa/perisperm, increasing its total pressure potential until axis Psi is in equilibrium with cotyledon Psi, but reducing its water content and resulting in a low Psi when the constraint is removed. There was no evidence of decreasing psi(s) or increasing turgor pressure (Psi-psi(s)) prior to radicle growth for either intact seeds or excised tissues. Given the low relative water content of the axes within intact seeds, cell wall relaxation would be ineffective in creating a Psi gradient for water uptake. Rather, axis growth may be initiated by weakening of the perisperm, thus releasing the external pressure and creating a Psi gradient for water uptake into the axis. The perisperm envelope contains a cap of small, thin-walled endosperm cells adjacent to the radicle tip. We hypothesize that weakening or separation of cells in this region could initiate radicle expansion. PMID- 16667370 TI - Light Energy Dissipation under Water Stress Conditions: Contribution of Reassimilation and Evidence for Additional Processes. AB - Using (14)CO(2) gas exchange and metabolite analyses, stomatal as well as total internal CO(2) uptake and evolution were estimated. Pulse modulated fluorescence was measured during induction and steady state of photosynthesis. Leaf water potential of Digitalis lanata EHRH. plants decreased to -2.5 megapascals after withholding irrigation. By osmotic adjustment, leaves remained turgid and fully exposed to irradiance even at severe water stress. Due to the stress-induced reduction of stomatal conductance, the stomatal CO(2) exchange was drastically reduced, whereas the total CO(2) uptake and evolution were less affected. Stomatal closure induced an increase in the reassimilation of internally evolved CO(2). This ;CO(2) recycling' consumes a significant amount of light energy in the form of ATP and reducing equivalents. As a consequence, the metabolic demand for light energy is only reduced by about 40%, whereas net photosynthesis is diminished by about 70% under severe stress conditions. By CO(2) recycling, carbon flux, enzymatic substrate turnover and consumption of light energy were maintained at high levels, which enabled the plant to recover rapidly after rewatering. In stressed D. lanata plants a variable fluorescence quenching mechanism, termed ;coefficient of actinic light quenching,' was observed. Besides water conservation, light energy dissipation is essential and involves regulated metabolic variations. PMID- 16667371 TI - Temperature-Induced Changes in the Fatty Acid Composition of the Cyanobacterium, Synechocystis PCC6803. AB - Changes in glycerolipid and fatty acid composition with a change in growth temperature were studied in the cyanobacterium, Synechocystis PCC6803. Under isothermal growth conditions, temperature did not significantly affect the composition of the various classes of lipids, but a decrease in temperature altered the degree of unsaturation of C(18) acids at the sn-1 position, but not that of C(16) acids at the sn-2 position of the glycerol moiety in each class of lipids. When the growth temperature was shifted from 38 degrees C to 22 degrees C, the desaturation of C(18) acids, but not that of C(16) acids, was stimulated. The desaturation of fatty acids occurred only in the light and was inhibited by chloramphenicol, rifampicin and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, but not by cerulenin, an inhibitor for fatty acid synthesis. These findings suggest that desaturase activities are induced after a shift from a higher to a lower temperature, and that the desaturation of fatty acids is connected with the reactions involved in photosynthetic electron transport. PMID- 16667372 TI - Changes in the Level of Peptidase Activities in Pea Ovaries during Senescence and Fruit Set Induced by Gibberellic Acid. AB - The activities and changes in the levels of exopeptidase and endopeptidase activities were characterized in unpollinated ovaries of Pisum sativum L. cv Alaska during senescence and early fruit development induced by gibberellic acid (GA(3)). Two aminopeptidases and one iminopeptidase were electrophoretically separated. These peptidases were sensitive to inhibitors of sulfhydryl proteases. Carboxypeptidase activity was inhibited by phenylmethyl sulfonyl fluoride. An azocasein-degrading endopeptidase, sensitive to thiol protease inhibitors, was also found. An increase in the specific activity of aminopeptidase during both fruit development and ovary senescence was observed. In contrast, the specific activity of carboxypeptidase and endopeptidase increased only during senescence of the ovary. Changes in exopeptidase activity in senescing ovaries could be mainly the consequence of a greater stability to proteolysis while the rise in endopeptidase activity appeared to be due to new or increased synthesis of the enzyme. These results suggest that endopeptidase, and not amino or carboxypeptidase, plays a key role in the senescence of pea ovaries and that the changes in unpollinated ovaries leading to ovary senescence or fruit development can be controlled by gibberellins. PMID- 16667373 TI - Developmental stages of cucumber seedlings: kinetics of chlorophyll accumulation and other growth parameters. AB - The changes in morphology during dark germination and subsequent growth of cucumber (Cucumis sativus) seedlings in the light go through three different phases described as latent, active, and steady-state. This pattern is consistently observed for several related developmental processes. The latent period lasts about 2 days following water imbibition after which the following capabilities appear in concert: (a) root and stem elongation, (b) pigment synthesis including protochlorophyll, chlorophyll, carotenoid, and phytochrome, (c) synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase, and (d) enhancement of greening by excision. Following the active phase, which lasts for another 2 to 3 days, these processes slow to a steady-state. Inhibition of chlorphyll accumulation by SO(2) was only observed for seedlings in the steady state phase. PMID- 16667374 TI - A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana. AB - A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS. PMID- 16667375 TI - Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves : implication of a transport function for cadmium-binding peptides. AB - The synthesis of Cd-binding peptides (CdBPs) was induced upon addition of 20 micromolar CdCl(2) (nonphytotoxic level) to the nutrient solution of hydroponically grown tobacco seedlings (Nicotiana rustica var Pavonii). Amino acid analysis showed that the main components were gamma-(Glu-Cys)(3)-Gly and gamma-(Glu-Cys)(4)-Gly. Seedlings exposed to the metal for 1 week contained similar glutathione levels as found in the controls (about 0.18 micromole per gram fresh weight). If, as has been proposed, CdBPs are involved in Cd detoxification by chelation, both metal and ligand must be localized in the same cellular compartment. To directly determine the localization of Cd and CdBPs, protoplasts and vacuoles were isolated from leaves of Cd-exposed seedlings. Purified vacuoles contained virtually all of the CdBPs and Cd found in protoplasts (104% +/- 8 and 110% +/- 8, respectively). CdBPs were associated with the vacuolar sap and not with the tonoplast membrane. Glutathione was observed in leaves and protoplasts but not in vacuoles. The probability that CdBPs are synthesized extravacuolarly and our finding that they and Cd are predominantly located in the vacuole suggest that these molecules might be involved in transport of Cd to the vacuole. Our results also suggest that a simple cytoplasmic chelator role for CdBPs in Cd tolerance cannot be assumed. PMID- 16667376 TI - Gibberellins and Stem Growth as Related to Photoperiod in Silene armeria L. AB - Stem growth and flowering in the long-day plant Silene armeria L. are induced by exposure to a minimum of 3 to 6 long days (LD). Stem growth continues in subsequent short days (SD), albeit at a reduced rate. The growth retardant tetcyclacis inhibited stem elongation induced by LD, but had no effect on flowering. This indicates that photoperiodic control of stem growth in Silene is mediated by gibberellins (GA). The objective of this study was to analyze the effects of photoperiod on the levels and distribution of endogenous GAs in Silene and to determine the nature of the photoperiodic after-effect on stem growth in this plant. The GAs identified in extracts from Silene by full-scan combined gas chromatography-mass spectrometry (GC-MS), GA(12), GA(53), GA(44), GA(17), GA(19), GA(20), GA(1), GA(29), and GA(8), are members of the early 13-hydroxylation pathway. All of these GAs were present in plants under SD as well as under LD conditions. The GA(53) level was highest in plants in SD, and decreased in plants transferred to LD conditions. By contrast, GA(19), GA(20), and GA(1) initially increased in plants transferred to LD, and then declined. Likewise, when Silene plants were returned from LD to SD, there was an increase in GA(53), and a decrease in GA(19), GA(20), and GA(1) which ultimately reached levels similar to those found in plants kept in SD. Thus, measurements of GA levels in whole shoots of Silene as well as in individual parts of the plant suggest that the photoperiod modulates GA metabolism mainly through the rate of conversion of GA(53). As a result of LD induction, GA(1) accumulates at its highest level in shoot tips which, in turn, results in stem elongation. In addition, LD also appear to increase the sensitivity of the tissue to GA, and this effect is presumably responsible for the photoperiodic after-effect on stem elongation in Silene. PMID- 16667377 TI - Mechanism of Nitrogenase Inhibition in Soybean Nodules : Pulse-Modulated Spectroscopy Indicates that Nitrogenase Activity Is Limited by O(2). AB - A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O(2) concentration (O(i)) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O(2) in the infected cells of the nodule (O(i)). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO(3) (-) treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O(i) and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H(2) evolution in Ar:O(2)) versus O(i) for the various treatments was consistent with the concept that O(i) limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O(i) to estimate nitrogenase activity in laboratory and field-grown legumes is discussed. PMID- 16667378 TI - Subcellular Localization of Chitinase and of Its Potential Substrate in Tomato Root Tissues Infected by Fusarium oxysporum f. sp. radicis-lycopersici. AB - Antiserum raised against a tomato (Lycopersicon esculentum Mill.) chitinase (molecular mass of 26 kilodaltons) was used as a probe to study the subcellular localization of this enzyme in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici. A time-course experiment revealed that chitinase accumulated earlier in the incompatible interaction than in the compatible one. However, in both systems, chitinase deposition was largely correlated with pathogen distribution. The enzyme was found to accumulate in areas where host walls were in close contact with fungal cells. In contrast, the enzyme could not be detected in vacuoles and intracellular spaces. The substantial amount of chitinase found at the fungus cell surface supports the view of an antifungal activity. However, the preferential association of the enzyme with altered fungal wall areas indicates that chitinase activity is either preceded by the hydrolytic action of other enzymes such as beta-1,3-glucanases or coincides with these enzymes. The possibility that fungal glucans released through the action of beta-1,3-glucanases may act as elicitors of chitinase production is discussed. PMID- 16667379 TI - Uptake and accumulation of the herbicide bentazon by cultured plant cells. AB - Cellular absorption of the herbicide bentazon, a weak acid with pK(a) 3.45, was investigated using suspension-cultured cells of velvetleaf (Abutilon theophrasti Medic.). Bentazon accumulated rapidly to concentrations approximately four times that of the external medium. Bentazon accumulation against a concentration gradient was not due to its conversion to metabolites, partitioning into lipids, or binding onto cellular constituents. Bentazon uptake was related linearly to the external bentazon concentration, implying that movement of the herbicide into cells was not carrier-mediated. Bentazon was able to diffuse freely and extensively out of the cells, indicating that bentazon can readily diffuse across cell membranes. Potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone inhibited bentazon accumulation as did nitrogen gas when bubbled through the uptake medium. Absorption was pH-dependent with the greatest amount of bentazon accumulating at acidic external pH. Calculations indicated that conversion of uncharged bentazon to bentazon anion in the cytoplasm accounts for cellular accumulation of bentazon. These results provide evidence that bentazon is absorbed across membranes via simple diffusion and that bentazon accumulates in plant cells via an energy-dependent, ion-trapping mechanism which results in bentazon accumulation in the cytoplasm. PMID- 16667380 TI - Potassium-dependent changes in the expression of membrane-associated proteins in barley roots : I. Correlations with k(rb) influx and root k concentration. AB - Barley (Hordeum vulgare L. cv Halcyon) seedlings which had been grown in full strength complete inorganic nutrient media (containing 6 millimolar K(+)) had high internal K(+) concentrations and low values of K(+) ((86)Rb(+)) influx when influx was measured from solutions containing 100 micromolar K(+). Transfer of these plants to solutions lacking K(+) resulted in significant reductions of root and shoot K(+) concentrations and values of K(+) ((86)Rb(+)) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K(+), the changes induced by K(+) deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K(+) ((86)Rb(+)) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K(+) status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washed in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar. PMID- 16667381 TI - Heat shock proteins are not required for the degradation of alpha-amylase mRNA and the delamellation of endoplasmic reticulum in heat-stressed barley aleurone cells. AB - When barley (Hordeum vulgare) aleurone layers are heat shocked, the synthesis and secretion of alpha-amylase and other secretory proteins is arrested and the synthesis of heat shock proteins (hsps) is induced. alpha-Amylase mRNA, normally a very stable mRNA, is actively degraded during heat shock. In addition, endoplasmic reticulum (ER) is delamellated during heat shock, possibly causing the destabilization of the mRNA for the secreted alpha-amylase. To ascertain whether or not hsps play any role in the destabilization of alpha-amylase mRNA or in the delamellation process of ER, heat shocked cells were treated with the transcription inhibitor cordycepin, which effectively inhibits the synthesis of hsps yet does not affect alpha-amylase synthesis after this enzyme has been fully induced by gibberellic acid (12 hours). In the absence of hsp expression, heat shock still causes the destabilization of alpha-amylase mRNA and the delamellation of ER. Alternatively, the synthesis of hsps may be induced in the absence of temperature increase by incubating cells in the presence of arsenite. Arsenite-induced expression of some hsps in the absence of increased temperature does not result in the destabilization of alpha-amylase mRNA or in the delamellation of ER. If cordycepin or cycloheximide are used to inhibit hsp synthesis during heat shock, the tissue recovers from heat shock with normal recovery kinetics. Although hsps have been implicated in the establishment of thermotolerance, our observations indicate that hsps do not play a role in the other heat shock-induced changes observable in aleurone cells. Furthermore, if the synthesis of hsp mRNA is inhibited during heat shock (by cordycepin) hsp mRNAs are synthesized later, during recovery, indicating that there is a stable inducer of hsp synthesis in aleurone tissues. PMID- 16667382 TI - Nitrogen fixation of nodulation mutants of soybean as affected by nitrate. AB - It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C(2)H(2) reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C(2)H(2) reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C(2)H(2) reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent. PMID- 16667383 TI - Flavonoid Accumulation Is Correlated with Adventitious Roots Formation in Eucalyptus gunnii Hook Micropropagated through Axillary Bud Stimulation. AB - Eucalyptus gunnii Hook microcuttings, obtained in vitro through axillary bud stimulation, show different rooting responses on the same rooting medium depending on the physiological state induced by cytokinins used in the previous multiplication medium. 6-Furfurylamino purine and 6-(4-hydroxy-3-methylbut-2 enylamino)purine induced a physiological state characterized by high sensitivity of microcuttings to the rooting stimulus exerted by the auxin 3-indolebutyric acid, but N(6)-benzyladenine did not produce the same effect. The former physiological state was characterized by an increased accumulation of two endogenous flavonoids (identified as quercetin glycosides) which may be markers of a well defined physiological state. They could have some direct influence on the rooting processes of the explants cultivated in vitro. PMID- 16667384 TI - Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings. AB - The most abundant alpha-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This alpha-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an alpha-amylase by polarimetry, substrate specificity, and end product analyses. The purified alpha-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This alpha amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. beta-glucan) are not substrates of the enzyme. A very low amount of this alpha-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or alpha-amylase which is integrally associated with the chloroplast. PMID- 16667385 TI - Involvement of Endogenous Abscisic Acid in Onset and Release of Helianthus annuus Embryo Dormancy. AB - Mature seeds of Helianthus annuus L. exhibit dormancy that is eliminated during storage in dry conditions. In vitro culture of immature embryos isolated at different times after anthesis showed that the youngest embryos are able to germinate, but within the third week after pollination, dormancy progressively affected most of the embryos. A radioimmunoassay showed that the endogenous abscisic acid (ABA) level, which increased sharply in the first half of the development period, fell at precisely the moment when embryo dormancy became established. An application of fluridone, before the increase of ABA level, prevented both ABA synthesis and development of embryo dormancy. Applied later, after the rise of the ABA level, fluridone could not prevent embryo dormancy development. Dormancy thus appears to be dependent on ABA synthesis but not concomitant with its accumulation; it must therefore be induced by ABA during maturation. Furthermore, a preincubation in water allowed dormant embryos to germinate. This acquisition of germinability could not be directly related to a leaching of free ABA. Possible effects of this treatment are discussed. PMID- 16667386 TI - Sodium Stimulates Growth of Panicum coloratum through Enhanced Photosynthesis. AB - A sodium-requiring C(4) plant, Panicum coloratum Walt. cv Kabulabula, was grown with and without sodium. Rate of nitrogen uptake and photosynthesis were measured during the recovery from sodium deficiency. The beneficial effect of sodium on growth was apparent irrespective of nitrogen source, ammonium- or nitrate nitrogen. The leaf photosynthetic rate ((14)CO(2) fixation) doubled by sodium within 1 hour of the application. PMID- 16667387 TI - Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies. AB - A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during post-germinative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattern typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue. PMID- 16667388 TI - Photosynthesis and Chlorophyll Fluorescence Characteristics in Relationship to Changes in Pigment and Element Composition of Leaves of Platanus occidentalis L. during Autumnal Leaf Senescence. AB - The loss of chlorophyll and total leaf nitrogen during autumnal senescence of leaves from the deciduous tree Platanus occidentalis L. was accompanied by a marked decline in the photosynthetic capacity of O(2) evolution on a leaf area basis. When expressed on a chlorophyll basis, however, the capacity for light-and CO(2)-saturated O(2) evolution did not decline, but rather increased as leaf chlorophyll content decreased. The photon yield of O(2) evolution in white light (400-700 nanometers) declined markedly with decreases in leaf chlorophyll content below 150 milligrams of chlorophyll per square meter on both an incident and an absorbed basis, due largely to the absorption of light by nonphotosynthetic pigments which were not degraded as rapidly as the chlorophylls. Photon yields measured in, and corrected for the absorptance of, red light (630-700 nanometers) exhibited little change with the loss of chlorophyll. Furthermore, PSII photochemical efficiency, as determined from chlorophyll fluorescence, remained high, and the chlorophyll a/b ratio exhibited no decline except in leaves with extremely low chlorophyll contents. These data indicate that the efficiency for photochemical energy conversion of the remaining functional components was maintained at a high level during the natural course of autumnal senescence, and are consistent with previous studies which have characterized leaf senescence as being a controlled process. The loss of chlorophyll during senescence was also accompanied by a decline in fluorescence emanating from PSI, whereas there was little change in PSII fluorescence (measured at 77 Kelvin), presumably due to decreased reabsorption of PSII fluorescence by chlorophyll. Nitrogen was the only element examined to exhibit a decline with senescence on a dry weight basis. However, on a leaf area basis, all elements (C, Ca, K, Mg, N, P, S) declined in senescent leaves, although the contents of sulfur and calcium, which are not easily retranslocated, decreased to the smallest extent. PMID- 16667389 TI - Interaction between Light Quality and Light Quantity in the Photoregulation of Anthocyanin Production. AB - The interaction between phytochrome photoequilibrium (phi) and photon flux in the photoregulation of anthocyanin production under prolonged irradiation was studied in seedlings of Brassica oleracea L. and Lycopersicon esculentum Mill. In cabbage, anthocyanin production increases with decreasing phi, reaching a maximum at the lowest value (phi = 0.13) used in this study; in tomato, the extent of the response is higher at intermediate values, reaching a maximum at phi = 0.46. In cabbage, the response increases with increasing photon flux at all phi values; however, the response to changes in photon flux is minimal at phi = 0.85, and, at phi = 0.13, minimal at photon fluxes higher than 5 micromolar per square meter per second. In tomato, the response increases with increasing photon flux at phi = 0.46, 0.65, and 0.85, the response to changes in photon fluxes being minimal at phi = 0.85; at phi = 0.13 and 0.29 the response first increases (significantly at phi = 0.29 and minimally at phi = 0.13) and then decreases with increasing photon fluxes, the transition occurring at about 1 micromolar per square meter per second at phi = 0.13, and at 5 micromolar per square meter per second at phi = 0.29. The patterns of light quality-quantity interaction in the photoregulation of anthocyanin production are significantly different in cabbage and tomato and are also significantly different than those observed for other photomorphogenic responses to prolonged irradiations. PMID- 16667390 TI - Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition. AB - The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII(alpha)) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q(b)-reducing centers). The smaller antenna (PSII(beta)) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q(b)-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII(beta) content remained the same or slightly increased during strong irradiance treatment, whereas the photochemical activity of PSII(alpha) decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII(beta) from a Q(b) nonreducing to a Q(b)-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII(alpha). The results suggest that PSII(beta) in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII(alpha). PMID- 16667391 TI - Genetic variation for gas exchange rates in grain sorghum. AB - Carbon assimilation rate (A) and stomatal conductance (g) are highly correlated. However, the slope of the A versus g relationship differs among species and environments resulting in differences in gas exchange efficiency which should reflect water use efficiency. The objective of this research was to determine the genetic variation for A and g in grain sorghum (Sorghum bicolor [L.] Moench.). Field experiments were conducted using 30 sorghum hybrids with four water supply treatments. A, g, and leaf water potential (Psi(w)) of individual leaves were monitored every 15 to 20 days. Significant genetic variation existed among the hybrids for A and g. Plant age and water supply also affected A and g as expected. When A was regressed on g for each hybrid, large and significant differences existed among the slopes, implying differences in intrinsic gas exchange efficiency. The regression analysis of A and g versus Psi(w) suggested that A was more sensitive than g to increasing water stress. Genetic differences in the rate of change in A as water stress increased were observed. Regression analysis was used to evaluate the individual hybrid response relative to other hybrids. Twofold difference in slopes existed for A. These results provide evidence for genetic variation in gas exchange rates which might directly contribute to whole plant water use efficiency and productivity. PMID- 16667392 TI - Characteristics of ribulose-1,5-bisphosphate carboxylase/oxygenase degradation by lysates of mechanically isolated chloroplasts from wheat leaves. AB - The lysate from intact chloroplasts mechanically isolated from primary leaves of 9 day old seedlings of wheat (Triticum aestivum L. var Aoba) was incubated in the pH range of 5.5 to 8.5 at 37 degrees C for 5 hours. Proteolytic activity against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was estimated by disappearance of the large subunit of Rubisco or the appearance of its degradation products. Although the activity in lysates was weak, the products were detected by applying Western blotting. The degradation products were similar to those obtained when Rubisco was incubated with the lysate of vacuoles isolated from like leaves. Although some of the products were similar to those from vacuole lysates, many were clearly different after incubation of Rubisco with trypsin, V-8 protease, or reactive oxygen (hydroxy radical). Lysates of chloroplasts, pretreated with thermolysin at 4 degrees C for 30 minutes, had no proteolytic activity against Rubisco after incubation at 37 degrees C for 5 hours. These results show that the proteolytic activity against Rubisco found in lysates of our mechanically isolated chloroplasts was mostly due to the contamination of vacuolar proteases adhering to the outer envelope of the chloroplasts during their isolation. PMID- 16667393 TI - Selection and characterization of sethoxydim- tolerant maize tissue cultures. AB - ;Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO(3) (-) incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [(14)C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [(14)C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme. PMID- 16667394 TI - Extended Host Range of Agrobacterium tumefaciens in the Genus Pinus. AB - Two-to 4-month-old seedlings of nine pine species (Pinus eldarica Medw., Pinus elliottii Engelm., Pinus jeffreyi Grev. & Balf., Pinus lambertiana Dougl., Pinus ponderosa Laws., Pinus radiata D. Don, Pinus sylvestris L., Pinus taeda L., Pinus virginiana Mill), Douglas fir (Pseudotsuaa menziesii (Mirb.) Franco) and incense cedar (Libocedrus decurrens Torr.) were inoculated with five strains of Agrobacterium tumefaciens. Transformation occurred in all conifer species tested as determined by gall formation and opine production. The frequency of gall formation varied by host species, by bacterial strain, and was related to the age of the stem when inoculated. Galls were visible 8 to 12 weeks after inoculation and were small (often less than 2.5 millimeters in diameter). Fewer than half (230 of 502) of the galls originally formed on the trees were present after 1 year, and 26 of these grew to diameters greater than 2 centimeters. The majority of these larger galls (18 of 26) were found in P. radiata. Bacterial strain specific opines were found in 67 of the 81 gall tissues sampled. PMID- 16667395 TI - Chloramphenicol Stimulates the Accumulation of Light-Harvesting Chlorophyll a/b Protein II by Affecting Posttranscriptional Events in the Chlorina CD3 Mutant Wheat. AB - The levels of total chlorophyll (Chl), total carotenoids, light-harvesting Chl a/b apoprotein of photosystem II (LHCPII), and light-harvesting Chl a/b apoprotein (LHCP) mRNA were examined in the CD3 chlorina mutant wheat (Triticum aestivum, L.) after 18 hours greening at either a low (3 micromoles of photons per square meter per second) or moderate (200 micromoles of photons per square meter per second) irradiance. The Chl b and LHCPII deficient mutant wheat accumulated significantly greater levels of Chl and LHCPII when greened under low irradiance than when greened under a moderate irradiance level. The level of LHCP mRNA, as measured by dot-blot and Northern hybridization analyses to a cDNA probe, increased in response to the irradiance level in the wheat. Applications of chloramphenicol (CAP) to the mutant wheat increased total Chl, Chl b, and LHCPII accumulations at both irradiance levels. Even though the CAP-treated CD3 mutant wheat accumulated similar levels of plastid pigments as those of CAP treated wild type, the LHCPII amounts were much higher in the wild type than in the CD3 mutant of wheat. CAP treatment did not significantly increase the LHCP mRNA level in either wheat. Applications of either benzyladenine or CAP to the mutant, greened under the moderate irradiance level for 72 hours, increased all plastid pigment levels except for beta-carotene. The benzyladenine plus CAP combination treatment had little effect on the LHCPII levels in the wild-type wheat. The combination treatment increased the LHCPII accumulation in the CD3 mutant of wheat by about twice that of the untreated mutant. Excess LHC pigment accumulation was promoted in each wheat line. We conclude that the regulation of LHCPII in the CD3 mutant of wheat is controlled by a posttranscriptional event. Furthermore, the accumulation of LHC bound pigments is not coupled with the accumulation of LHCPII in wheat thylakoid membranes. PMID- 16667396 TI - ChrA Is a Carotenoid-Binding Protein in Chromoplasts of Capsicum annuum. AB - Chromoplasts of Capsicum annuum var Albino contain a carotenoid-protein complex, which migrates as a brilliant orange band in gels under conditions of nondenaturing electrophoresis. In a second, denaturing separation, the complex resolves into a principal protein (ChrA) of 58 kilodaltons and several minor proteins of 20 to 55 kilodaltons, which may be adventitiously associated. Analysis of Western blots of both one- and two-dimensional gels showed that the principal protein component of the carotenoid complex is ChrA, a protein previously shown to be located specifically in chromoplast membranes. The identification of ChrA as a carotenoid-binding protein appears to be the first instance of a nonthylakoid, carotenoid-binding protein in higher plants. PMID- 16667397 TI - Differences in Proteins Synthesized in Needles of Unshaded and Shaded Pinus ponderosa var Scopulorum Seedlings during Prolonged Drought. AB - Proteins were radiolabeled and extracted from needles of Pinus ponderosa var scopulorum (Dougl. ex Laws.) seedlings progressively drought-stressed for about 1 month. A set of novel, low molecular weight proteins was detected in fluorographs of two-dimensional gels when relative water content of needles fell below 70%. Their synthesis was undetectable in the fully recovered seedlings within 48 hours after rewatering. In similarly stressed seedlings that were shaded to 10% full light, the low molecular weight polypeptides were not detected or appeared at very low levels. The shaded seedlings, in which drought tolerance was reduced, did not recover upon termination of the drought. The results suggest that protein synthesis induced by water deficit in drought-tolerant seedlings may contribute to resisting the effects of cellular dehydration. PMID- 16667398 TI - Inhibition of ribulose 1,5-bisphosphate carboxylase/oxygenase by 2 carboxyarabinitol-1-phosphate. AB - In some plants, 2-carboxy-d-arabinitol 1-phosphate (CA 1P) is tightly bound to catalytic sites of ribulose, 1,5-bisphosphate carboxylase/oxygenase (rubisco). This inhibitor's tight binding property results from its close resemblance to the transition state intermediate of the carboxylase reaction. Amounts of CA 1P present in leaves varies with light level, giving CA 1P characteristics of a diurnal modulator of rubisco activity. Recently, a specific phosphatase was found that degrades CA 1P, providing a mechanism to account for its disappearance in the light. The route of synthesis of CA 1P is not known, but could involve the branched chain sugar, hamamelose. There appear to be two means for diurnal regulation of the number of catalytic sites on rubisco: carbamylation mediated by the enzyme, rubisco activase, and binding of CA 1P. While strong evidence exists for the involvement of rubisco activase in rubisco regulation, the significance of CA 1P in rubisco regulation is enigmatic, given the lack of general occurrence of CA 1P in plant species. Alternatively, CA 1P may have a role in preventing the binding of metabolites to rubisco during the night and the noncatalytic binding of ribulose bisphosphate in the light. PMID- 16667399 TI - Sealed inside-out and right-side-out plasma membrane vesicles : optimal conditions for formation and separation. AB - Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H(+) pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H(+) pumping were both completely inhibited by vanadate (K(i) approximately 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation. PMID- 16667400 TI - Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits. AB - Activity of the enzyme ADPglucose pyrophosphorylase is known to be reduced in maize (Zea mays L.) endosperm mutants at two independent loci, Shrunken-2 (Sh(2)) and Brittle-2 (Bt(2)). Spinach leaf ADPglucose pyrophosphorylase has previously been shown to comprise two subunits of 51 and 54 kilodaltons. Anti-bodies raised to each of the two subunits of spinach leaf ADPglucose pyrophosphorylase were found to cross-react to different bands on Western blots prepared from polyacrylamide gel electrophoresis separated wild-type maize endosperm proteins. The anti-spinach leaf 51 kilodalton subunit antibody cross-reacted with a 55 kilodalton maize endosperm protein and the anti-spinach leaf 54 kilodalton subunit antibody cross-reacted with a 60 kilodalton maize endosperm protein. These immunological reactions were observed in maize endosperm extracts and with a highly purified preparation of maize endosperm ADPglucose pyrophosphorylase. Mutant bt(2) endosperm lacked the 55 kilodalton subunit while mutant sh(2) endosperm lacked the 60 kilodalton subunit on Western blots. These results suggest that the maize endosperm ADPglucose pyrophosphorylase is made up of two immunologically dissimilar subunits and that the bt(2) and sh(2) mutations cause reduction in ADPglucose pyrophosphorylase activity through the lack of one of these two subunits. An ADPglucose pyrophosphorylase cDNA clone antigenically selected from a rice seed cDNA expression library was found to hybridize strongly with a cDNA corresponding to a maize endosperm transcript which is absent in a W64A bt(2) mutant. Thus, the bt(2) mutant causes the absence not only of the small subunit but of the corresponding transcript. Bt(2) is implicated as the structural gene for the small (54 kilodalton) subunit of maize endosperm ADPglucose pyrophosphorylase. PMID- 16667401 TI - Ca requirement for aerobic nitrogen fixation by heterocystous blue-green algae. AB - The requirement of Ca(2+) for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N(2) under microaerobic conditions, Ca(2+) was not required for growth, at least in concentrations greater than traces. In contrast, Ca(2+) was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca(2+) to an aerobic culture without Ca(2+) promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca(2+) promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca(2+) or low oxygen tension was dependent on de novo protein synthesis. The role of Ca(2+) seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen. PMID- 16667402 TI - Adaptation of the Photosynthetic Apparatus to Irradiance in Dunaliella tertiolecta: A Kinetic Study. AB - The time course of adaptation from a high to a low photon flux density was studied in the marine chlorophyte Dunaliella tertiolecta. A one-step transition from 700 to 70 micromole quanta per square meter per second resulted in a reduction of doubling rate from 1.1 to 0.4 per day within 24 hours, followed by a slower accumulation of photosynthetic pigments, light harvesting antenna complexes, Photosystem II reaction centers and structural lipids that constitute the thylakoid membranes. Photoregulated changes in the biochemical composition of the thylakoid proteins and lipids were functionally accompanied by decreases in the minimal photosynthetic quantum requirement and photosynthetic capacity, and an increase in the minimal turnover time for in vivo electron transport from water to CO(2). Analysis of de novo synthesis of thylakoid membranes and proteins indicates that a high light to low light transition leads to a transient in carbon metabolism away from lipid biosynthesis toward the synthesis of the light harvesting antenna protein complexes, accompanied by a slower restoration rate of reaction centers and thylakoid membranes. This pattern of sequential synthesis of light harvesting complexes followed by reaction centers and membranes, appears to optimize light harvesting capabilities as cells adapt to low photon flux densities. PMID- 16667403 TI - Expression and Processing of an Arabidopsis 2S Albumin in Transgenic Tobacco. AB - 2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleavages. In order to determine if this process is correctly carried out in transgenic plants, the gene AT2S1 encoding an Arabidopsis thaliana 2S albumin isoform has been expressed in transgenic tobacco. Initial experiments using a reporter gene demonstrated that the AT2S1 promoter directs seed specific expression in both transgenic tobacco and Brassica napus plants. The entire AT2S1 gene was then transferred into tobacco plants, where it showed a tissue specific and developmentally regulated expression. Arabidopsis 2S albumin accumulates up to 0.1% of the total high-salt extractable seed protein. Protein sequencing demonstrated that the amino termini of the two Arabidopsis 2S albumin subunits were correctly processed, suggesting that the protease(s) necessary for posttranslational processing of 2S albumin precursors may display common specificities among different dicot plant species. Immunocytochemical studies showed that the Arabidopsis 2S albumin is localized in the protein body matrix of tobacco endosperm and embryo. Correct processing and targeting of the 2S albumin in transgenic plants suggests that modified versions could be expressed, allowing the study of 2S albumin processing and in particular the possible roles of the processed fragments in protein stability and/or targeting. PMID- 16667404 TI - Fe uptake mechanism in fe-efficient cucumber roots. AB - Fe-efficient plants respond to iron stress both by morphological and physiological modifications. In roots of a Fe-efficient plant (Cucumis sativus L.) grown in the presence or in the absence of iron, the capacity to acidify the external medium, change in the transmembrane electrical potential, and the ATPase activity have been determined. Roots from plants grown in the absence of iron showed a great capacity to acidify the external medium, a higher transmembrane electrical potential difference (-145 millivolts, versus -105 millivolts), and a higher ATPase activity (+30%). The administration of Fe(2+), but not Fe(3+), caused a block of the acidification capacity, a great decrease in the transmembrane electrical potential difference in root cells, and a large inhibition of the ATPase activity of isolated microsomal membrane vesicles. PMID- 16667405 TI - Inverse Relationship between Polyamine Levels and the Degree of Phenotypic Alteration Induced by the Root-Inducing, Left-Hand Transferred DNA from Agrobacterium rhizogenes. AB - Floral induction in plants is a paradigm for signal perception, transduction, and physiological response. The introduction of root-inducing, left-hand transferred DNA (Ri T-DNA) into the genomes of several plants results in modifications of flowering (D Tepfer [1984] Cell 47: 959-967), including a delay in flowering in tobacco (Nicotiana tabacum). Conjugated polyamines are markers for flowering in numerous species of plants. In tobacco their accumulation is correlated with the onset of flowering (F Cabanne et al. [1981] Physiol Plant 53: 399-404). Using tobacco, we have explored the possibility of a correlation between the expression of Ri TL-DNA and changes in polyamine metabolism. We made use of two levels of phenotypic change, designated T and T', that retard flowering by 5 to 10 and 15 to 20 days, respectively. We show that delay in flowering is correlated with a reduction in polyamine accumulation and with a delay in appearance of conjugated polyamines, and we propose that genes carried by the Ri TL-DNA intervene either directly in polyamine metabolism or that polyamine metabolism is closely linked to direct effects of Ri T-DNA expression. PMID- 16667406 TI - Biochemical Similarities between Soluble and Membrane-Bound Calcium-Dependent Protein Kinases of Barley. AB - The soluble and membrane-bound forms of the calcium-dependent protein kinase from barley leaves (Hordeum vulgare L. cv. Borsoy) have been partially purified and compared. Both forms showed an active polypeptide of 37 kilodaltons on activity gels with incorporated histone as substrate. They eluted from chromatofocusing columns at an identical isoelectric point of pH 4.25 +/- 0.2, and also comigrated on several other chromatographic affinity media including Matrex Gel Blue A, histone-agarose, phenyl-Sepharose, and heparin-agarose. Both activities comigrated with chicken ovalbumin during gel filtration through Sephacryl S-200, indicating a native molecular mass of 45 kilodaltons. The activities share a number of enzymatic properties including salt and pH dependence, free calcium stimulation profile, substrate specificity, and Km values. The soluble activity was shown to bind to artificial lipid vesicles. These data suggest strongly that the soluble and membrane-bound calcium-dependent protein kinases from barley are very closely related or even identical. PMID- 16667407 TI - Feruloylputrescine and Caffeoylputrescine Are Not Involved in Growth and Floral Bud Formation of Stem Explants from Nicotiana tabacum L. var Xanthi nc. AB - The role of feruloylputrescine (FP) and of caffeoylputrescine (CP) was investigated in an explant system of stem explants from day-neutral Nicotiana tabacum L. var Xanthi nc. Previously, a correlation between cortical callus formation and increase in FP content, as well as between in vitro flower formation and increase in CP content had been shown. During the explant growth in vitro, the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2 phenylethyl)phosphonic acid and 2-amino-indene-2-phosphonic acid, both inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5). dl-alpha-difluoromethylarginine, an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), prevented only the increase in FP, while dl-alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase (EC 4.1.1.17), reduced only that of CP. Increase in dry weight and the formation of cortical callus and of floral buds of explants were not affected by any of the inhibitors. We conclude, in contrast to earlier hypotheses, that FP and CP do not trigger growth and differentiation in the explants. It seems more likely that FP and CP increase in response to auxin and cytokinin in the media. PMID- 16667408 TI - Cell-Wall Carbohydrates of the Endosperm of the Seed of Gleditsia triacanthos. AB - The endosperm of the seed of Gleditsia triacanthos L. contains 18.55% of its dry weight as nonreserve, cell-wall carbohydrates. Of this carbohydrate material, comprising mainly mannose, galactose, and glucose, 76.1% was of low-molecular weight or highly hydrophilic. Mannose, galactose, and glucose were also the major sugar components of the polysaccharides extracted with alkali (23.1% of the cell wall), while the same sugars, with minor amounts of arabinose, form the residues. Methylation analysis of the polysaccharides and the borate-sodium hydroxide residue indicate that the cell walls are built up on a network of galactomannans, with high Man/Gal ratios, reinforced with minor amounts of cellulose. PMID- 16667409 TI - Chloroplast and mitochondrial DNA polymerases from cultured soybean cells. AB - DNA polymerases were purified from chloroplasts and mitochondria of cultured Glycine max cells. The chloroplast enzyme exists in two forms which are indistinguishable from each other biochemically. All three organellar enzymes have an estimated molecular weight of 85,000 to 90,000 and prefer poly(rA)dT(12 18) over activated DNA as a template in vitro. Maximum activity of the chloroplast and mitochondrial DNA polymerases requires KCl and a reducing agent, and the enzymes are completely resistant to inhibitors of DNA polymerase alpha. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases gamma. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides. PMID- 16667410 TI - Modulation of Host Gene Expression during Initiation and Early Growth of Nodules in Cowpea, Vigna unguiculata (L.) Walp. AB - Inoculation of 2-day-old cowpea (Vigna unguiculata [L.] Walp.) seedlings with Rhizobium fredii USDA257 results in proficient nodulation of the tap root. The most abundant nodulation occurs in a region roughly corresponding to the position of the root tip at the time of inoculation. We have examined plant gene expression in this region, after inoculation with either USDA257 or a nonnodulating mutant, 257B3. After isolation of mRNA and in vitro translation, the protein products were separated by two-dimensional gel electrophoresis. Seven proteins are induced within 2.5 days after inoculation with USDA257. One additional induced protein is detectable by 3.5 days after inoculation, and three more appear by day 6. Three of the proteins that are differentially expressed at 2.5 and 3.5 days after inoculation are produced at equivalent levels after 6 days, indicating transient induction of these genes during early stages of nodule development. Several proteins were more abundant in translations of mRNA from roots that had been inoculated with the nonnodulating mutant. This was particularly true after 6 days, when nine proteins were in this class. Thus, altered plant gene expression in carefully selected, highly responsive tissue can be detected 2 days before emerging nodules are visible on the roots, and 6 to 7 days before acetylene reduction is detectable. Additionally, comparisons of ionically bound cell wall proteins isolated 6 days after inoculation revealed four that were unique to nodulating roots, suggesting that some of the nodulation induced genes may code for structural proteins. PMID- 16667411 TI - Triazine Resistance in Senecio vulgaris Parental and Nearly Isonuclear Backcrossed Biotypes Is Correlated with Reduced Productivity. AB - Isonuclear triazine-susceptible and triazine-resistant Senecio vulgaris L. biotypes were developed by making reciprocal crosses between susceptible and resistant biotypes to obtain F(1) hybrids and backcrossing the hybrids to the appropriate pollen parent. The electrophoretic isozyme patterns of the enzyme aconitase obtained from leaf extracts of triazine-susceptible parental (S) and backcrossed (SxR(BC6)) biotypes, and triazine-resistant parental (R) and backcrossed (RxS(BC6)) biotypes verified that the biotypes had the expected nuclear genomes. Atrazine inhibition of chloroplast whole chain electron transport from water to methyl viologen was measured to verify susceptibility or resistance to triazine herbicides. The photosynthetic rate and biomass accumulation of greenhouse grown susceptible and resistant S. vulgaris biotypes were measured 28, 35, 42, 50, 57, and 64 days after planting to determine the effect of altered chloroplast function. S and SxR(BC6) biotypes had CO(2) assimilation rates of 16.2 and 16.6 micromoles CO(2) per square meter per second, respectively, and I(50) values (herbicide concentration producing 50% inhibition) of about 0.49 micromolar atrazine. The corresponding values for the R and RxS(BC6) biotypes were 14.7 and 14.6 micromoles CO(2) per square meter per second with I(50) values of 65.0 micromolar atrazine. The S biotype was larger and more productive than the R biotype at all harvests. At the harvest 57 days after planting, mean shoot dry weight was 33.2 and 8.7 grams for the S and R biotypes, respectively. The growth effect associated with chloroplast differences was shown in comparisons of the S biotype with the RxS(BC6) biotype and of the SxR(BC6) biotype with the R biotype. The RxS(BC6) biotype had 72% of the shoot dry weight of the S biotype while the R biotype had 55% of the shoot dry weight of the SxR(BC6) biotype. The RxS(BC6) and R biotypes produced about 73 and 62% of the leaf area of the S and SxR(BC6) biotypes, respectively. Relative growth rate was similar in biotypes with the same nuclear genome; however, instantaneous unit leaf rate was higher in the S compared to the RxS(BC6) biotype and in the SxR(BC6) compared to the R biotype. At 57 days after planting, the cumulative leaf area duration (i.e. photosynthetic opportunity) of the RxS(BC6) and R biotypes was 86 and 66% of that of the S and SxR(BC6) biotypes, respectively. Our data indicate that impaired chloroplast function in triazine resistant S. vulgaris biotypes limits growth and productivity at the whole plant level. PMID- 16667412 TI - Regulation of expression of carbon-assimilating enzymes by nitrogen in maize leaf. AB - We have utilized the cellular differentiation gradient of the developed, youngest leaf to examine the regulation by nitrogen of levels of phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase (PPDK), and ribulose 1,5 bisphosphate carboxylase in maize (Zea mays L.). The protein whose level regulated most preferentially by N availability was PEPCase, followed by PPDK, and the changes in level occurred most conspicuously at the photosynthetically maturing cells. Pulse and pulse-chase experiments to analyze photosynthetic fixation of [(14)C]CO(2) indicate that maize leaf primarily exploited a C(4)-mode of photosynthetic fixation of carbon dioxide even under a selective reduction in levels of these proteins. The effects of N on the synthesis of these proteins and the accumulation of corresponding mRNAs during recovery from a deficiency were examined by pulse and pulse-chase labeling with [(35)S]Met and by hybridization, respectively. The rate of turnover of PPDK was substantially higher than that of the other proteins. Results also showed that the reduced accumulation of PEPCase, as well as PPDK, under N deficiency could largely be accounted for a reduced level of synthesis of protein with a concomitant reduction in level of their mRNAs. This indicates that the N-dependent selective accumulation of these enzymes is primarily a consequence of level of its mRNAs. PMID- 16667413 TI - Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation. AB - Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl phosphoryl) uridine 3'(2') monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M(r) of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2',3'-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5' purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate. PMID- 16667414 TI - Increases in Phosphorus Requirements for CO(2)-Enriched Pine Species. AB - Pinus radiata D. Don (half-sib families 20010 and 20062) and Pinus caribaea var hondurensis (an open-pollinated family) were grown for 49 weeks at seven levels of phosphorus and at CO(2) concentrations of either 340 or 660 microliters per liter, to establish if the phosphorus requirements differed between the CO(2) concentrations and if mycorrhizal associations were affected. When soil phosphorus availability was low, phosphorus uptake was increased by elevated CO(2). This may have been related to changes in mycorrhizal competition. When the phosphorus concentration in the youngest fully expanded needles was above 600 milligrams per kilogram the shoot weight of all pine families was greater at high CO(2) due to increases in rates of photosynthesis. More dry weight was partitioned to the stems of P. radiata family 20010 and P. caribaea. At foliar phosphorus concentrations above 1000 milligrams per kilogram (P. radiata) and 700 milligrams per kilogram (P. caribaea), growth did not increase at 340 microliters of CO(2) per liter. Soluble sugar levels in the same needles mirrored the growth response, but the starch concentration declined with increasing phosphorus. At 660 microliters of CO(2) per liter, shoot weight and soluble sugar concentrations were still increasing up to a foliar P concentration of 1800 milligrams per kilogram for P. radiata and 1600 milligrams per kilogram for P. caribaea. The starch concentrations did not decline. These results indicate that higher foliar phosphorus concentrations are required to realize the maximum growth potential of pines at elevated CO(2). PMID- 16667415 TI - Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium. AB - Some Rhizobium bacteria have H(2)-uptake (Hup) systems that oxidize H(2) evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars ;JI1205' and ;Alaska' produce high Hup (Hup(++)) and moderate Hup (Hup(+)) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup(++) and Hup(+) phenotypes in R. leguminosarum 128C53 were identified in 173 F(2)-derived F(6) families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P /=5 hours before high germination percentages were observed. Such a requirement for a long term exposure to a steady state concentration of an inherently labile, exuded compound would provide an extra degree of resolution to signal detection and host commitment in Striga parasitism. PMID- 16667437 TI - Vegetative/Parasitic transition: control and plasticity in striga development. AB - Striga asiatica (Scrophulariaceae), an obligate parasite of grasses including many of the world's major grain crops, switches from vegetative to parasitic development by the differentiation of the root meristem into the host attachment organ, the haustorium. This change was induced in culture by the exposure to a single, low molecular weight signal molecule, 2,6-dimethoxy-p-benzo-quinone. A concentration of 10(-6) molar quinone and an exposure time of >/=6 hours were required before the developmental process could be completed. With shorter exposure times, haustorial development was prematurely aborted and meristematic elongation was reestablished. The new meristem was capable of developing a second haustorium if reexposed to the signal molecule. These results are discussed in terms of the transition to the parasitic phase and the general control of plant cellular development. PMID- 16667438 TI - Diamine Oxidase from Cultured Roots of Hyoscyamus niger: Its Function in Tropane Alkaloid Biosynthesis. AB - Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective K(m) values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants V(max)/K(m) for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis. PMID- 16667439 TI - Biophysics of the inhibition of the growth of maize roots by lowered temperature. AB - Roots of hydroponically grown maize (Zea mays cv LG11) have a greatly reduced growth rate at 5 degrees C (0.02 millimeters per hour) compared with those at 20 degrees C (1.2 millimeters per hour). Various physical parameters of roots growing at each temperature were compared. Turgor pressure of cells in the elongation zone increased from 0.59 +/- 0.05 megapascal at 20 degrees C to 0.82 +/- 0.04 megapascal after 70 hours at 5 degrees C; thus, growth rate was not limited by decreased pressure. On cooling, tissue plasticity (measured by Instron/tensiometer) decreased slowly over 70 hours. On rewarming to 20 degrees C from 5 degrees C, growth rate, turgor pressure, and tissue plasticity all returned concertedly to their original values over a period of days. However, immediately following cooling growth rate dropped rapidly from 1.8 to 0.12 millimeters per hour in 30 minutes but turgor pressure and tissue Instron plasticity remained unchanged. A plot of turgor pressure against growth rate indicated that, following cooling from 30 to 15 degrees C, the in vivo wall extensibility of the tissue was reduced by 75%. Yield threshold was unchanged. Cells whose expansion was arrested in the long-term cold treatment do not resume growth. Root growth recovers by the expansion of cells newly produced by the meristem. Cessation of extension growth is an effect on the individual expanding cell. Growth recovery is not a reverse of this effect but requires the generation of fresh cells. PMID- 16667440 TI - A Photorespiratory Mutant of Chlamydomonas reinhardtii. AB - A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO(2) concentration for photoautrophic growth in spite of the apparent induction of a functional CO(2) concentrating mechanism in air-adapted cells. In 2% O(2) the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O(2), photosynthetic O(2) evolution is severely inhibited in the mutant by preillumination in limiting CO(2), although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O(2) evolution measurements. Net CO(2) uptake is also inhibited when the cells are exposed to air (21% O(2), 0.035% CO(2), balance N(2)) for longer than a few minutes. [(14)C]Phosphoglycolate accumulates within 5 minutes of photosynthetic (14)CO(2) fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO(2) requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1. PMID- 16667441 TI - Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris. AB - Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of beta 1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of beta-1,3-glucanase was observed after inoculation of leaves with heat or rifampicin-killed X. c. vitians, Escherichia coli, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major beta-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pl] ~8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pl 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2. PMID- 16667442 TI - Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. AB - The properties of the tight and specific binding of 2-C-carboxy-d-arabinitol 1,5 bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5 bisphosphate carboxylase (Rubisco) that are activated by CO(2) and Mg(2+), were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [(14)C]CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported (S Johal, BE Partridge, R Chollet [1985] J Biol Chem 260: 9894-9904). The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg(2+). Addition of [(14)C]CABP and EDTA stopped binding of Mg(2+) and allowed tight binding of the radiolabel only to sites which were CO(2)/Mg(2+)-activated at that moment. This approach estimated the amount of CO(2)/Mg(2+)-activated sites in the presence of inactive sites and carbamylated sites lacking Mg(2+). The rate of CO(2) fixation was proportional to the CO(2)/Mg(2+)-activated sites. During light-dependent CO(2) fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg(2+) and CO(2). Lysis of chloroplasts in media with [(14)C]CABP plus EDTA estimated those carbamylated sites having Mg(2+). The loss of Rubisco activation during illumination was partially due to the lack of Mg(2+) to stabilize the carbamylated sites. PMID- 16667443 TI - Phosphatidylcholine biosynthesis in castor bean endosperm : purification and properties of cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase. AB - Cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been purified to near homogeneity (3350-fold) from castor bean (Ricinus communis L. var Hale) endosperm. The steps of purification included a differential solubilization of this enzyme with n-octyl beta-d-glucopyranoside (OGP) and column chromatography on sequential DEAE-sepharose, sepharose-6B, and second DEAE sepharose columns. The uses of appropriate concentrations of the detergent, OGP, in each step were crucial to obtain the highly purified enzyme. The purified enzyme gave a single protein band on nondenaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed one major protein band of 40 kilodaltons. Gel filtration chromatography indicated that native cytidylyltransferase was approximately 155 kilodaltons, suggesting that it exists naturally as a tetramer. The purified enzyme used methylethanolamine-phosphate as a substrate but not ethanolamine-phosphate and dimethylethanolamine-phosphate. ATP and other nucleotides tested showed little effect on the purified enzyme. The purified enzyme activity was stimulated by both phospholipids extracted from castor bean endosperm and phosphatidylcholineoleate vesicles. PMID- 16667444 TI - Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants : use of adenosine 5'-phosphosulfate and adenosine-3'-phosphate 5'-phosphosulfate as precursors. AB - Adenosine-5'-phosphosulfate (APS) and adenosine-3'-phosphate 5'-phosphosulfate (PAPS) have been used as precursors of sulfoquinovosyldiacylglycerol (SQDG) in intact chloroplasts incubated in the dark. Competition studies demonstrated APS was preferred over PAPS and SO(4) (2-). Rates of SQDG synthesis up to 3 nanomoles per milligram of chlorophyll per hour were observed when [(35)S]APS and appropriate cofactors were supplied to chloroplasts incubated in the dark. The pH optimum for utilization of APS was 7.0. The incorporation was linear for at least 30 minutes. ATP and UTP stimulated the incorporation of sulfur from APS into SQDG, but the most stimulatory additions were DHAP and glycerol-3-P. The concentration curve for APS showed a maximum at 20 micromolar in the absence of DHAP and 30 micromolar in the presence of DHAP. The optimum concentration of DHAP for conversion of APS into SQDG was 2 millimolar. Rates of synthesis up to 4 nanomoles per milligram of chlorophyll per hour were observed when [(35)S]PAPS was the sulfur donor and appropriate cofactors were supplied to chloroplasts. Optimal rates for conversion of sulfur from PAPS into SQDG occurred with concentrations of DHAP between 5 and 10 millimolar. DHAP was by far the most effective cofactor, although ATP and UTP also stimulated the utilization of PAPS for SQDG biosynthesis. In general, triose phosphates, including glycerol-3-P were not effective cofactors for SQDG biosynthesis. PMID- 16667445 TI - Comparison between NO(x) Evolution Mechanisms of Wild-Type and nr(1) Mutant Soybean Leaves. AB - The nr(1) soybean (Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NO(x) (NO + NO(2)). It was tested for possible nonenzymic NO(x) formation and evolution because of known chemical reactions between NO(2) (-) and plant metabolites and the instability of nitrous acid. It did not evolve NO(x) during the in vivo NR assay, but intact leaves did evolve small amounts of NO(x) under dark, anaerobic conditions. Experiments were conducted to compare NO(3) (-) reduction, NO(2) (-) accumulation, and the NO(x) evolution processes of the wild type (cv Williams) and the nr(1) mutant. In vivo NR assays showed that wild-type leaves had three times more NO(3) (-) reducing capacity than the nr(1) mutant. NO(x) evolution from intact, anerobic nr(1) leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr(1) mutant leaves was usually higher than wild type due to low NO(x) evolution. Lag times and threshold NO(2) (-) concentrations for NO(x) evolution were similar for the two genotypes. While only 1 to 2% of NO(x) from wild type is NO(2), the nr(1) mutant evolved 15 to 30% NO(2). The kinetic patterns of NO(x) evolution with time were completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NO(x) evolution by wild type is primarily an enzymic conversion of NO(2) (-) to NO. However, this report concludes that NO(x) evolution by the nr(1) mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO(2) (-) and/or decomposition of nitrous acid. Nonenzymic NO(x) evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process. PMID- 16667446 TI - Hypoxic stress inhibits multiple aspects of the potato tuber wound response. AB - Potato (Solanum tuberosum L.) tubers subjected to wounding under hypoxic stress do not synthesize RNA species that are induced in response to wounding in aerobic conditions. Further, wound-response proteins fail to be synthesized when wounded tubers are transferred to hypoxic conditions although messenger RNAs which encode them persist for many hours after transfer. Hypoxic stress also prevents the incorporation of [(3)H]thymidine by wounded tubers that occurs in aerobic conditions. In contrast, hypoxic tubers accumulate and translate transcripts of genes whose products are involved in anaerobic metabolism whether or not they are wounded. Both the hypoxic response and the aerobic wound response preclude the synthesis of proteins encoded by messenger RNAs which accumulated during the tuberization process and which can be translated in vitro. Finally, wounding elicits the degradation of a subset of these tuberization-associated transcripts. These data indicate a complex and precise regulation of gene expression at several levels of macromolecular synthesis. PMID- 16667447 TI - Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. AB - Neutral carrier-based liquid membrane ion-selective microelectrodes for NH(4) (+) and NO(3) (-) were developed and used to investigate inorganic nitrogen acquisition in two varieties of barley, Hordeum vulgare L. cv Olli and H. vulgare L. cv Prato, originating in cold and warm climates, respectively. In the present paper, the methods used in the fabrication of ammonium- and nitrate-selective microelectrodes are described, and their application in the study of inorganic nitrogen uptake is demonstrated. Net ionic fluxes of NH(4) (+) and NO(3) (-) were measured in the unstirred layer of solution immediately external to the root surface. The preference for the uptake of a particular ionic form was examined by measuring the net flux of the predominant form of inorganic nitrogen, with and without the alternative ion in solution. Net flux of NH(4) (+) into the cold adapted variety remained unchanged when equimolar concentrations (200 micromolar) of NH(4) (+) and NO(3) (-) were present. Similarly, net flux of NO(3) (-) into the warm-adapted variety was not affected when NH(4) (+) was also present in solution. The high temporal and spatial resolution afforded by ammonium- and nitrate-selective microelectrodes permits a detailed examination of inorganic nitrogen acquisition and its component ionic interactions. PMID- 16667448 TI - Evidence for cotransport of nitrate and protons in maize roots : I. Effects of nitrate on the membrane potential. AB - The electrical response of nitrate-grown maize (Zea mays L.) roots to 0.1 millimolar nitrate was comprised of two sequential parts: a rapid and transient depolarization of the membrane potential, followed by a slower, net hyperpolarization to a value more negative than the original resting potential. The magnitude of the response was smaller in roots of seedlings grown in the absence of nitrate, but, within 3 hours of initial exposure to 0.1 millimolar nitrate, increased to that of nitrate-grown roots. Chloride elicited a separate electrical response with a pattern similar to that of the nitrate response. However, the results presented in this study strongly indicate that the electrical response to nitrate reflects the activity of a nitrate-inducible membrane transport system for nitrate which is distinct from that for chloride. Inhibitors of the plasmalemma H(+)-ATPase (vanadate, diethylstilbestrol) completely inhibited both parts of the electrical response to nitrate, as did alkaline external pH. The magnitude of the initial nitrate-dependent, membrane potential depolarization was independent of nitrate concentration, but the subsequent nitrate-dependent hyperpolarization showed saturable dependence with an apparent K(m) of 0.05 millimolar. These results support a model for nitrate uptake in maize roots which includes a depolarizing NO(3) (-)/H(+) symport. The model proposes that the nitrate-dependent membrane potential hyperpolarization is due to the plasma membrane proton pump, which is secondarily stimulated by the operation of the NO(3) (-)/H(+) symport. PMID- 16667449 TI - Evidence for Cotransport of Nitrate and Protons in Maize Roots : II. Measurement of NO(3) and H Fluxes with Ion-Selective Microelectrodes. AB - We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO(3) (-) and H(+) within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO(3) (-) uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H(+) ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO(3) (-) (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO(3) (-) absorption. These results strongly indicate that the NO(3) (-) electrical response is due to the operation of a NO(3) (-) transport system in the plasmalemma of maize root cells. Furthermore, the results from the H(+)-ATPase inhibitor studies indicate that the NO(3) (-) transport system is linked to the H(+)-ATPase, presumably as a NO(3) ( )/H(+) symport. This is further supported by the pH response of the NO(3) (-) transport system (inhibition at alkaline pH values) and the change in net H(+) flux from a moderate efflux in the absence of NO(3) (-), to zero net H(+) flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO(3) (-)/H(+) flux data is a NO(3) (-)/H(+) symport with a NO(3) (-):H(+) flux stoichiometry >1, whose operation results in the stimulation of the H(+)-ATPase due to the influx of protons through the cotransport system. PMID- 16667450 TI - Respiration and mitochondrial biogenesis in germinating embryos of maize. AB - Function of the cyanide-sensitive mitochondrial electron transport system was required for germination of the Zea mays embryo. Respiration of the standard electron transport system (rather than the alternate oxidase) began immediately upon initiation of imbibition. This respiration depended upon cytochrome c oxidase and ATPase that were conserved in an active form in the quiescent embryo rather than upon newly synthesized or assembled enzyme complexes. Immunoprecipitation of radiolabeled subunits of these enzymes showed that the initiation of mitochondrial biogenetic activities, including de novo synthesis of nuclear- and mitochondrial-encoded enzyme subunit peptides, was strongly induced after 6 hours of embryo germination. Undetectable or very low levels of transcripts for subunits 1 and 2 of the F(1)-ATPase and subunit 2 of cytochrome c oxidase were present in the quiescent embryo; these transcripts accumulated rapidly between 6 and 12 hours of germination and their translation products were rapidly synthesized between 6 and 24 hours. An exception was the gene for subunit 9 of the ATPase; transcripts of this mitochondrial gene were abundant in the dry embryo and rapidly accumulated further upon initiation of imbibition; they were translated actively during the first 6 hours. We isolated and sequenced a near full-length cDNA for subunit 2 (beta) of the F(1)-ATPase, and we compared the deduced protein sequence with related sequences of other organisms. PMID- 16667451 TI - Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942. AB - A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO(2)-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO(2). The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild type cells in their growth rates under either low or high CO(2) conditions. The activity of inorganic carbon (C(i)) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO(2) was the main species of C(i) transported and the activities of CO(2) and HCO(3) (-) transport increased when high CO(2)-grown cells were exposed to low CO(2). We conclude that the 42 kilodalton protein is not directly involved in the C(i)-accumulating mechanism of Synechococcus PCC 7942. PMID- 16667453 TI - Effect of preincubation temperature on in vitro light saturated photosystem I activity in thylakoids isolated from cold hardened and nonhardened rye. AB - Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5 degrees C or 20 degrees C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol --> methylviologen) after dark preincubation at temperatures between 0 and 60 degrees C. Thylakoids isolated in the presence or absence of Na(+)/Mg(2+) from 20 degrees C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60 degrees C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20 degrees C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20 degrees C grown rye, thylakoids isolated from 5 degrees C grown rye in the presence of Na(+)/Mg(2+) exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60 degrees C. This was not due to damage to PSI electron transport in thylakoids isolated from 5 degrees C grown plants since light saturated PSI activity was 60% higher in 5 degrees C thylakoids than 20 degrees C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5 degrees C thylakoids could be observed after dark preincubation only when 5 degrees C thylakoids were initially isolated in the absence of Na(+)/Mg(2+). We suggest that 5 degrees C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20 degrees C thylakoids and hence cannot be increased further by dark preincubation. PMID- 16667452 TI - Alternative Oxidase of Potato Is an Integral Membrane Protein Synthesized de Novo during Aging of Tuber Slices. AB - The rise in alternative respiratory capacity upon aging of potato (Solanum tuberosum) tuber slices is correlated with changes in mitochondrial membrane protein composition and a requirement for cytoplasmic protein synthesis. However, the lack of an antibody specific to the alternative oxidase has, until recently, prevented examination of the alternative oxidase protein(s) itself. We have employed a monoclonal antibody raised against the Sauromatum guttatum alternative oxidase to investigate developmental changes in the alternative pathway of aging potato slice mitochondria and to characterize the potato alternative oxidase by one- and two-dimensional gel electrophoresis. The relative levels of a 36 kilodalton protein parallel the rise in alternative path capacity. A plausible interpretation is that this alternative oxidase protein is synthesized de novo during aging of potato slices. PMID- 16667454 TI - Oxygen and Hydrogen Isotope Fractionation during Cellulose Metabolism in Lemna gibba L. AB - Lemna gibba L. B3 was grown under heterotrophic, photoheterotrophic, and autotrophic conditions in water having a variety of hydrogen and oxygen isotopic compositions. The slopes of the linear regression lines between the isotopic composition of water and leaf cellulose indicated that under the three growth conditions about 40, 70, and 100% of oxygens and carbon-bound hydrogens of cellulose exchanged with those of water prior to cellulose formation. Using the equations of the linear relationships, we estimated the overall fractionation factors between water and the exchanged oxygen and carbon bound-hydrogen of cellulose. At least two very different isotope effects must determine the hydrogen isotopic composition of Lemna cellulose. One reflects the photosynthetic reduction of NADP, while the second reflects exchange reactions that occur subsequent to NADP reduction. Oxygen isotopic composition of cellulose apparently is determined by a single type of exchange reaction with water. Under different growth conditions, variations in metabolic fluxes affect the hydrogen isotopic composition of cellulose by influencing the extent to which the two isotope effects mentioned above are recorded. The oxygen isotopic composition of cellulose is not affected by such changes in growth conditions. PMID- 16667455 TI - Correlation of xylem sap cytokinin levels with monocarpic senescence in soybean. AB - Cytokinins (CKs) coming from the roots via the xylem are known to delay leaf senescence, and their decline may be important in the senescence of soybean (Glycine max) plants during pod development (monocarpic senescence). Therefore, using radioimmunoassay of highly purified CKs, we quantified the zeatin (Z), zeatin riboside (ZR), the dihydro derivatives (DZ, DZR), the O-glucosides, and DZ nucleotide in xylem sap collected from root stocks under pressure at various stages of pod development. Z, ZR, DZ, and DZR dropped sharply during early pod development to levels below those expected to retard senescence. Pod removal at full extension, which delayed leaf senescence, caused an increase in xylem sap CKs (particularly ZR and DZR), while depodding at late podfill, which did not delay senescence, likewise did not increase the CK levels greatly. The levels of the O-glucosides and the DZ nucleotide were relatively low, and they showed less change with senescence or depodding. The differences in the responses of individual CKs to senescence and depodding suggest differences in their metabolism. Judging from their activity, concentrations and response to depodding, DZR and ZR may be the most important senescence retardants in soybean xylem sap. These data also suggest that the pods can depress CK production by the roots at an early stage and this decrease in CK production is required for monocarpic senescence in soybean. PMID- 16667456 TI - Long-Lasting Light Effects in Imbibed Kalanchoe blossfeldiana Seeds in the Presence of Gibberellic Acid. AB - Two brief red (R) irradiations, separated by 24 hours, given to Kalanchoe blossfeldiana Poelln. cv Feuerblute seeds, made secondarily dormant by a prolonged dark incubation period on water and transferred to GA(3), induce very low germination. Some effect of these irradiations is preserved, however, during a long dark interval in fully imbibed seeds and greatly increases the germination induced by another brief R exposure. This long-lasting light effect is, at 20 degrees C, only lost after a dark interval of about 1 month. It can also be induced by two brief far-red (FR) exposures. Its preservation is temperature dependent, low temperatures being favorable. Light-induced changes in the ATP content were demonstrated during preservation and expression of the long-lasting light effect, indicating a long-lasting metabolic change. In seeds with primary dormancy sown on GA(3), an analogous long-lasting light effect is induced by one or two brief R or FR irradiations, even when they are given before germination can take place. The presence of GA(3), which was shown to induce a very low fluence germination response in Kalanchoe seeds, is required for the occurrence of the long-lasting light effect. The data suggest long-term preservation of some effect(s) of Pfr rather than persistent presence of Pfr itself. PMID- 16667457 TI - Replacement of Histidines of Light Harvesting Chlorophyll a/b Binding Protein II Disrupts Chlorophyll-Protein Complex Assembly. AB - Eukaryotic light harvesting proteins (LHCPs) bind pigments and assemble into complexes (LHCs) that channel light energy into photosynthetic reaction centers. The structures of several prokaryotic LHCPs are known and histidines are important for the binding of the associated pigments. It has been difficult to predict how the eukaryotic LHCPs associate with pigments as the structure of the major LHCP of photosystem II is not yet known. While each LHCPII binds approximately 13 chlorophylls the protein contains only three histidines, one in each putative transmembrane helix. Experiments that use isolated pea (Pisum sativum L.) chloroplasts and mutant LHCPII synthesized in vitro show that the substitution of either an alanine or an arginine for each histidine residue inhibits some aspect of LHCII assembly. The histidine of the first membrane helix, but not the second or third, may be involved in the transport across the chloroplast envelope. No histidine alone is essential for the insertion of LHCP into thylakoid membranes, yet arginine substitutions are more inhibitory than those of alanine. The histidine replacements have their most pronounced effect on the assembly of LHCP into LHCII. PMID- 16667458 TI - ATP-Regulated Ion Channels in the Plasma Membrane of a Characeae Alga, Nitellopsis obtusa. AB - Using the patch-clamp technique, we recorded single-channel currents across the excised patch of the plasma membrane of Nitellopsis. Both K(+) and Na(+) can pass this channel, but currents were not carried by Cl(-). Upon the addition of ATP or AMP to the cytoplasmic side, the frequency of channel opening decreased. This is the first report on an ATP-regulated channel in plant cells. PMID- 16667459 TI - Delta6 desaturase inhibition : a novel mode of action of norflurazone. AB - The herbicide Norflurazone was shown to be an inhibitor of fatty acid of the Delta6 desaturation system. Treating cultures of the microalgae Spirulina platensis or Monodus subterraneus with this herbicide brought about a reduction in the level of gamma-linolenic acid and in an increase in the level of linoleic acid. In Monodus, the increase in linoleic acid made it more available for omega3 desaturation, resulting in an increase in the proportion of eicosapentaenoic acid. The proportion of arachidonic acid did not decrease albeit the drastic decrease in its precursor gamma-linolenic acid. PMID- 16667460 TI - Regulation of tonoplast k channels by voltage in the range of physiological electric potentials. AB - The presence of tonoplast ion channels regulated by voltage in the physiological range of transtonoplast electric potential was studied in isolated vacuoles from Acer pseudoplatanus cultured cells. In symmetrical KCl or K-gluconate depolarizing pulses induced instantaneously developing, decaying outward currents, while in symmetrical tetramethylammonium chloride these currents were absent. The outward currents were reduced if the depolarizations were applied from a holding potential of +30 millivolts and increased upon depolarizations from a holding potential of -30 millivolts and even more from a holding potential of -50 millivolts. These results indicate that the outward currents are due to K(+) movement through channels which are open around 0 millivolt and close at positive potentials. These K(+) channels, regulated in the range of the physiological electric potentials reported for the vacuoles in situ, are likely the same K(+) channels activated by hyperpolarizations which we have previously described (R Colombo, R Cerana, P Lado, A Peres [1988] J Membr Biol 103: 227 236). PMID- 16667461 TI - Fructokinases from developing maize kernels differ in their specificity for nucleoside triphosphates. AB - A new form of fructokinase has been identified from developing maize (Zea mays L.) kernels that utilizes CTP, UTP, and GTP from four to eight times more effectively than ATP at nonlimiting concentrations. Ten millimolar dithiothreitol was necessary to stabilize activity. A second form of fructokinase was nonspecific for nucleoside triphosphate whereas a third form was fairly specific for ATP. PMID- 16667462 TI - Cytochrome and alternative pathway respiration in green algae : measurements using inhibitors and o(2) discrimination. AB - Inhibitor titration curves and discrimination against (18)O(2) by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities. PMID- 16667463 TI - Isolation and characterization of the cytosolic and chloroplastic 3 phosphoglycerate kinase from spinach leaves. AB - The cytosol and chloroplast 3-phosphoglycerate kinases (3-PGK) from spinach (Spinacia oleracea L.) were purifled to apparent homogeneity. The procedure included a conventional anion-exchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anion-exchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzmyes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of 800 units (per milligram of protein). The molecular weight for the two 3 PGKs under nondenaturing (size exclusion chromatography) and denaturing (SDS PAGE) conditions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP(2-) were biphasic. The presence of Na(2)SO(4) changed the MgATP(2-) dependence to linearity but not the glycerate-3-phosphate dependence. PMID- 16667464 TI - Distribution of Chlorophyll-Protein Complexes during Chilling in the Light Compared with Heat-Induced Modifications. AB - The effects of chilling in the light (4 days at 5 degrees C and 100-200 micromoles of photons per square meter per second) on the distribution of chlorophyll (Chl) protein complexes between appressed and nonappressed thylakoid regions of pumpkin (Cucurbita pepo L.) chloroplasts were studied and compared with the changes occurring during in vitro heat treatment (5 minutes at 40 degrees C) of isolated thylakoids. Both treatments induced an increase (18 and 65%, respectively) in the relative amount of the antenna Chl a protein complexes (CP47 + CP43) of photosystem II (PSII) in stroma lamellae vesicles. Freeze fracture replicas of light-chilled material revealed an increase in the particle density on the exoplasmic fracture face of unstacked membrane regions. These two treatments differed markedly, however, in respect to comigration of the light harvesting Chl a/b protein complex (LHCII) of PSII. The LHCII/PSII ratio in stroma lamellae vesicles remained fairly constant during chilling in the light, whereas it dropped during the heat treatment. Moreover, it was a minor light harvesting Chl a/b protein complex of PSII, CP29, that increased most in stroma lamellae vesicles during light-chilling. Changes in the organization of LHCII during chilling were suggested by a shift to particles of smaller sizes on the protoplasmic fracture face of stacked membrane regions and a decrease in the amount of trans-Delta(3)-hexadecenoic acid in the phosphatidyldiacylglycerol fraction. PMID- 16667465 TI - Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia. AB - Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I(50) to susceptible I(50)) was 5- to 28-fold, 2- to 6-fold, and 20 fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [(14)C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K(m) values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme. PMID- 16667466 TI - Partial characterization of ornithine carbamoyltransferase in three microalgae : anabolic role only. AB - Although the existence of isozymes of ornithine carbamoyltransferase (carbamoylphosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) in higher plants has been reported, and the possibility exists that one or more of these operates catabolically to produce ornithine and carbamoylphosphate from citrulline and inorganic phosphate, no proof has been forthcoming. In view of the fact that many unicellular algae degrade arginine via arginine deiminase to citrulline and ammonium, and that the pathway of utilization of citrulline is unknown, we decided to investigate the possibility of the presence of a catabolic form of ornithine carbamoyltransferase in three microalgae known to have arginine deiminase activity. These were Chlorella autotrophica, Chlorella saccharophila, and Dunaliella tertiolecta. Our results show that the properties of OCT from these three algae are similar to OCTs from many higher plants with respect to general kinetics (K(m) values for ornithine and carbamoylphosphate), substrate inhibition by ornithine at high pHs, apparent sequential ordered kinetic mechanisms and paucity of apparent regulatory properties. Our data indicate an exclusively anabolic role of ornithine carbamoyltransferase in these algae. PMID- 16667467 TI - Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii. AB - Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytoklnins from vegetative, female, and male buds of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The purified cytokinins were separated by reverse phase high performance liquid chromatography and analyzed by radioimmunoassay. Confirmation of cytokinin identities was by gas chromatography-mass spectrometry. Immediately prior to bud burst, all bud types contained three major cytokinins: isopentenyladenosine, zeatin riboside, and a hexose conjugate of zeatin riboside (not zeatin riboside O-glucoside). Zeatin-type cytokinins were present in relatively high concentration in vegetative and female buds. In male buds, however, relatively high levels of isopentenyladenosine were found together with low levels of zeatin-type cytokinins. PMID- 16667468 TI - Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification? AB - The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium +/- NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls. PMID- 16667469 TI - Warm growth temperatures decrease soybean cholinephosphotransferase activity. AB - The activity of cytidine 5'-diphosphate (CDP) choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) in developing soybean (Glycine max L. var Williams 82) seeds was 3 to 5 times higher in cotyledons grown at 20 degrees C than in those grown at 35 degrees C. Some characteristics of the enzyme from cotyledons cultured at 20 and 35 degrees C were compared. In preparations from both growth temperatures, the enzyme showed a pH optimum of 7, K(m) of 7.0 micromolar for CDP-choline, and an optimum assay temperature of 45 degrees C. Both enzyme preparations were stimulated by increasing concentrations of Mg(2+) or Mn(2+), up to 10 millimolar and 50 micromolar, respectively, though Mn(2+) produced lower activities than Mg(2+). Enzymes from both 20 and 35 degrees C show the same specificity for exogenous diacylglycerol. No metabolic effectors were detected by addition of heat treated extracts to the assay mixture. The above findings suggest that the higher enzyme activity at 20 degrees C can be attributed to a higher level of the enzyme rather than to the involvement of isozymes or metabolic effectors. Enzyme activity decreased rapidly during culture at 35 degrees C, indicating a rapid turnover of the enzyme. The level of temperature modulation was found to be a function of seed developmental stage. PMID- 16667470 TI - Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma). AB - The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or -3 degrees C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4 degrees C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4 degrees C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0 degrees C treatment than microtubules in root-tip cells of nonacclimated, 22 degrees C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and -3 degrees C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the -3 degrees C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and -3 degrees C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of -9 degrees C. Cold acclimation lowered this value to -14 degrees C. Treatment of 22 degrees C-grown seedlings for 24 h with the microtubule stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of -3 degrees C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye. PMID- 16667471 TI - Tubulin Isotypes in Rye Roots Are Altered during Cold Acclimation. AB - The cold stability of cortical microtubules in root-tip cells of winter rye (Secale cereale L. cv Puma) is altered by growth temperature (GP Kerr, JV Carter [1990] Plant Physiol 93:77-82). One hypothesis for the basis of this alteration is that different tubulin isotypes are present at different growth temperatures, and that the cold stability of microtubules is affected by these isotypic differences. We have explored the first part of this hypothesis by comparing protein extracts from roots of seedlings grown for 2 days at 22 degrees C (nonacclimated) or for an additional 2 or 4 days at 4 degrees C (cold acclimated). Immunoblots of two-dimensional polyacrylamide gels were probed with monoclonal antibodies to alpha- and beta-tubulin. At least six alpha- and seven beta-tubulins were present in the extracts from both the nonacclimated and cold acclimated roots. Changes in electrophoretic mobility and isotype number of both alpha- and beta-tubulin were observed after only 2 days at 4 degrees C. Further changes in tubulin were observed after 4 days at 4 degrees C. Changes in alpha tubulin were more pronounced than those in beta-tubulin. PMID- 16667472 TI - Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion. AB - The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination. PMID- 16667473 TI - Kinetic studies of lysine-sensitive aspartate kinase purified from maize suspension cultures. AB - Steady state substrate kinetics and feedback regulation properties were determined for lysine-sensitive aspartate kinase (AK) purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures. Two AK isoforms (AK Early and AK Late) were separated by two passages through an anion exchange column as the final steps in a procedure giving 1200-fold purification. Kinetic properties were determined for the major AK Late eluting isoform. Assays were conducted at the pH activity maximum (8.0) and with excess Mg(2+) to favor a two-substrate reaction involving aspartate and complexed MgATP. AK catalyzed a sequential reaction in which MgATP and aspartate both bind to the enzyme complex before the ADP and aspartyl-phosphate products are released. The K(m) value calculated for MgATP was 0.43 millimolar and for aspartate was 1.04 millimolar. Cooperativity in substrate binding was not observed and was not induced by lysine. The lysine concentration required for 50% inhibition of AK activity was 7 micromolar. An apparent Hill coefficient of 1.4 indicated a minimum of two lysine-binding sites on the active AK complex. At nonsaturating substrate concentrations, lysine inhibition was characteristic of an S-parabolic, I-parabolic noncompetitive allosteric inhibitor. The parabolic inhibitor replot, Hill coefficients > 1, and the lack of substrate cooperativity were consistent with a model for multiple lysine-binding sites per active AK subunit. Similar kinetic properties were observed for the AK Early isoform. PMID- 16667474 TI - Transmembrane Signaling via Phosphatidylinositol 4,5-Bisphosphate Hydrolysis in Plants. AB - Recent investigations have confirmed the presence of the polyphosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP(2)), as well as inositol phospholipid-specific phospholipase C in higher plant and microalgal cells. In addition, it has been shown that stimulation of some photosynthetic cell types by environmental or hormonal challenge is accompanied by degradation of the polyphosphoinositides. The products of phospholipase C-catalyzed PIP(2) hydrolysis, inositol 1,4,5-trisphosphate and diacylglycerol, appear to be capable of releasing organelle-bound Ca(2+) and stimulating protein kinase C-like activity in vitro. However, a direct cause and effect relationship between stimulated PIP(2) breakdown and changes in intracellular calcium, protein phosphorylation, or cell function has not yet been unequivocally established. Despite a number of technical difficulties slowing progress in this field, it is likely that photosynthetic organisms will soon be shown to transmit physiologically significant extracellular signals across their plasma membranes by a PIP(2)-mediated transduction mechanism. PMID- 16667475 TI - Respiration in Cells and Mitochondria of Male-Fertile and Male-Fertile and Male Sterile Nicotiana spp. AB - Three cytoplasmic male-sterile Nicotiana cultivars together with corresponding male-fertile progenitors and restored lines were investigated in order to find possible correlations between respiratory characteristics and male sterility. Oxygen consumption measurements were performed on cells from suspension cultures as well as on mitochondria isolated from green leaves. Inhibitors, which have been reported to specifically block either the cytochrome (KCN) or the alternative (propyl gallate and sali-cylhydroxamic acid [SHAM] respiratory pathways, were used in order to measure the capacity and activity of the two pathways. One of the inhibitors, SHAM, was found unsuitable to measure the activity of the alternative pathway due to the lack of specificity of SHAM for this pathway. A great difference in the capacity of the alternative pathway was detected between the two types of cell materials tested. Mitochondria isolated from green leaves showed a capacity of the alternative pathway of 5 to 20% of total mitochondrial repiration, while the capacity of cells from suspension cultures generally ranged from 50 to 80%. In addition to this, in organello synthesis of mitochondrial proteins revealed differences between mitochondria isolated from green leaves and from cell suspensions. No correlation, however, could be found between respiratory characteristics and male sterility. PMID- 16667476 TI - Separation, purification, and comparative properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley leaves. AB - The chloroplast and cytoplasmic isoenzymes of phosphoglycerate kinase (PGK) (EC. 2.7.2.3) from Hordeum vulgare leaves have been separated and purified for the first time to apparent homogeneity. The method for purifying the isoenzymes is described here and consists of DEAE Sephacel chromatography followed by affinity chromatography on ATP Sepharose. This consistently provided a 500- to 900-fold purification of each isoenzyme. Most of the total PGK in green barley leaves was found to be in the chloroplasts with only 10% in the cytoplasm. The immunological properties of the two isoenzymes were compared. The antisera raised to the separate isoenzymes showed cross-reactivity, although there is evidence that each isoenzyme possesses some distinct epitopes. The isoenzymes differ in overall charge with isoelectric points at 5.2 and 5.4 for the chloroplast and cytoplasmic isoenzymes, respectively. Molecular mass estimations by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis provided similar values of approximately 38 kilodaltons for each isoenzyme, some 4 to 5 kilodaltons less than the values calculated from the cDNA sequences of the wheat isoenzymes. The isoenzymes have broadly similar pH optima of pH 7 to 8. The cytoplasmic isoenzyme is more thermally stable than the chloroplast isoenzyme. Further studies are now in progress to compare both the regulatory properties of the isoenzymes and also their three-dimensional structures as compared with the yeast enzyme. PMID- 16667477 TI - Carbon Use Efficiency and Cell Expansion of NaCl-Adapted Tobacco Cells. AB - Carbon use efficiencies (gram cell organic dry weight accumulated per gram sugar assimilated from the medium) of unadapted and NaCl-adapted (428 millimolar) cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) were determined to evaluate metabolic costs associated with growth and survival in a saline environment. No net increase in carbon costs was associated with salt adaptation. At low substrate levels, carbon use efficiencies of unadapted and NaCl-adapted cells were not appreciably different (0.495 and 0.422, respectively) and at higher substrate levels carbon use efficiency of NaCl-adapted cells was clearly higher than that of unadapted cells. These results indicate that a homeostasis of metabolic efficiency is established after cells have adapted to NaCl. Altered carbon availability does not cause the reduced cell volume that results from adaptation to NaCl. This does not preclude, however, the possibility that altered intracellular partitioning of carbon affects cell expansion. PMID- 16667478 TI - Photoinduction of Massive beta-Carotene Accumulation by the Alga Dunaliella bardawil: Kinetics and Dependence on Gene Activation. AB - The massive accumulation of beta-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of beta-carotene accumulation, cells continuously grown under white light of approximately 27 microeinsteins per square meter per second were exposed to light of approximately 1650 microeinsteins per square meter per second. The exposed cells accumulate beta-carotene in two stages: the first stage, lasting for 24 hours, starts shortly after exposure, whereas the second stage starts concomitantly with the onset of the stationary phase and persists until the cells collapse. Actinomycin D, chloramphenicol, or cycloheximide added to low illuminated cultures abolish the subsequent induction of beta-carotene accumulation by high light intensity. These results, together with the early exponential kinetics of accumulation, point to the role of gene activation in the process. In vivo labeling of proteins and in vitro translation of poly(A)(+) mRNA revealed several pronounced differences between low-illuminated and high illuminated cells. A strongly light-induced protein of approximately 55 kilodaltons, as well as other light-induced proteins could possibly fulfill a carotenogenic function. PMID- 16667479 TI - Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : III. Structural Changes in the Feraxan during Coleoptile Elongation. AB - Changes in structural features of feraxan (feruloylated arabinoxylans) in cell walls during development of maize (Zea mays L.) coleoptiles were investigated by analysis of fragments released by feraxanase, a specific enzyme purified from Bacillus subtilis. The following patterns were identified: (a) The total quantity of carbohydrate dissociated from a given dry weight of cell wall by feraxanase remained rather constant throughout the initial 10 days of coleoptile development. However, during the same period the proportion of ferulic acid in the fraction increased 12-fold. The absolute amount of ferulic acid per coleoptile also increased rapidly during this developmental phase. (b) Fragments dissociated by the enzyme were resolved into feruloylated and nonferuloylated components by reversed phase chromatography. While the quantity of feruloylated fractions represented a small portion of the total arabinoxylan during the phase of maximum coleoptile elongation (days 2-4) this component increased in abundance to reach a plateau (after 8-10 days). In contrast, nonferuloylated fractions were most abundant during the stage of maximum elongation but declined to a constant level by day 6. (c) Glycosidic linkage analysis of carbohydrate reveals that substitution of the xylan backbone of feraxan by arabinosyl residues decreased during coleoptile growth. We conclude that significant incorporation of ferulic acid occurs and/or more feruloyated domains are added to the arabinoxylan during development. This augmentation in phenolic acids is accompanied by a concerted displacement of arabinosyl residues and/or a reduction in the incorporation of regions enriched in arabinosyl sidechains. PMID- 16667480 TI - Plasma membrane potential of the alga dunaliella, and its relation to osmoregulation. AB - A fluorescent dye sensitive to membrane potential was used to follow the plasma membrane potential in the unicellular halo-tolerant alga Dunaliella salina. The signal observed during dissipation of the plasma membrane potential by the addition of excess K(+) and valinomycin, or a protonophore, was taken as a measure of the preexisting potential. A resting potential of -85 to -100 millivolts (negative inside) was calculated. Following a hypertonic shock, the plasma membrane was rapidly hyperpolarized. This hyperpolarization was transient, and the algae resumed their resting potential about 30 minutes after the shock. The resting plasma membrane potential was decreased by vanadate and is concluded to be generated mostly by the plasma membrane ATPase of Dunaliella. The transient hyperpolarization following a hypertonic shock indicates, therefore, a transient activation of the ATPase. This is further corroborated by a rapid transient decrease in the intracellular ATP following a hypertonic shock and its inhibition by vanadate. It is suggested that activation of the plasma membrane ATPase may be the trigger for osmoregulation in Dunaliella. PMID- 16667481 TI - Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes. AB - Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH(v)) of 4.74 +/- 0.1 is generated in the presence of 0.1 millimolar PP(i), but only 4.93 +/- 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP(i). Cl(-)-stimulates the H(+)-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K((1/2)) approximately 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP(i). The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH(v) unit has been estimated. From the free energy (-11.42 kilojoules per mole) for the hydrolysis of PP(i) under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H(+) per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H(+)-pump rather than as a pyrophosphate synthetase. PMID- 16667482 TI - A Study of Phospholipids and Galactolipids in Pollen of Two Lines of Brassica napus L. (Rapeseed) with Different Ratios of Linoleic to Linolenic Acid. AB - The phospholipids and galactolipids of the pollen-coat and internal domains of two lines of Brassica napus, Wesroona and IXLIN, with different linoleic/linolenic acid ratios (18:2/18:3) have been characterized by normal phase silica high performance liquid chromatography and gas liquid chromatography. The polar lipids of the pollen-coat are similar to leaf lipids in the high proportion of galactolipids (almost 50%) and the fatty acids; 18:3, palmitic (16:0) and hexadecatrienoic (16:3). In contrast, the pollen internal domain, although rich in 18:3, 18:2 and 16:0, is composed primarily of phosphatidyl-choline, -ethanolamine, and -inositol whose 18:2/18:3 ratio is correlated with that of the seed generation. The difference between the two divergent 18:2/18:3 ratio lines is most evident in the internal domain phospholipids. The 18:2/18:3 ratio of the galactolipids of both pollen domains is not significantly effected by the line genotype. The results are interpreted in terms of the previously described ;prokaryotic' and ;eukaryotic' plant desaturation pathways (PG Roughan, CR Slack [1982] Annu Rev Plant Physiol 33: 97 132). We propose that the eukaryotic pathway is the major desaturation pathway providing polyunsaturated fatty acids to the haploid-specified internal domain in which the IXLIN genotype modifies the activity of the sn-2 linoleoyl phosphatidylcholine desaturase/s of the endoplasmic reticulum. In the diploid specified pollen-coat, our evidence suggests that a combination of the prokaryotic and eukaryotic pathways contribute polyunsaturated fatty acids. PMID- 16667483 TI - Entrainment and Phase-Shifting of the Circadian Rhythm of Cell Division by Calcium in Synchronous Cultures of the Wild-Type Z Strain and of the ZC Achlorophyllous Mutant of Euglena gracilis. AB - Cell division in exponentially increasing populations of the wild-type, photosynthetic Z strain of Euglena gracilis Klebs cultured autotrophically on an aerated, magnetically stirred, minimal mineral medium (pH 7.0) in constant light (LL) or in a light-dark 1 hour:1 hour cycle (LD:1,1) at 25 degrees C could be synchronized by a 10-hour:10-hour low (2 micromolar):normal (200 micromolar) cycle in the concentration of external calcium. Similar results were obtained with the photosynthesis-deficient, achlorophyllous ZC mutant cultured in darkness at 16 degrees C on mineral medium supplemented with 0.1% ethanol as a carbon source; even a single low-Ca(2+) (2 micromolar) pulse was effective in eliciting synchrony. In contrast, whereas the 20-hour entrained rhythm of cell division in ZC then free-ran with a circadian period (tau = 26 hours) for many cycles after the imposed calcium regimen was discontinued, division rhythmicity did not persist in the Z strain in LL. The rhythm in wild-type cultures (free-running in LD:1,1) could be phase-shifted by a single 2-hour increase (from 200 micromolar to 10 millimolar; HiCa) or decrease (from 200-2 micromolar; LoCa) in external Ca(2+) concentration (varied by the addition of CaCl(2) or EDTA, respectively, to the medium). Pulses were terminated by returning the cells to medium containing 200 micromolar Ca(2+) (the normal concentration), and the steady-state phase shifts engendered (if any) after transients had subsided were calculated with reference to an unperturbed culture. For both HiCa and LoCa pulses given at different circadian times, strong (type 0) phase-response curves (PRCs) were obtained, but although the LoCa PRC was the same as that obtained for light signals, the HiCa PRC was the opposite (a mirror image). These results implicate calcium in clock function, although it is likely that only a small portion of the total intracellular Ca(2+) ion is playing a role since the period of the division rhythm in cultures grown in the continuous presence of excess Ca(2+) or under LoCa was not altered significantly. PMID- 16667484 TI - Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation. AB - The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the ;early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred. PMID- 16667485 TI - Polyamines and Flower Development in the Male Sterile Stamenless-2 Mutant of Tomato (Lycopersicon esculentum Mill.) : I. Level of Polyamines and Their Biosynthesis in Normal and Mutant Flowers. AB - The levels of free putrescine, spermidine, and spermine, and the activities of ornithine decarboxylase and s-adenosylmethionine decarboxylase were determined in the floral organs of the normal and a male sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.). Under the intermediate temperature regime, all mutant floral organs possessed significantly higher levels of polyamines and enzyme activities than their normal counterparts. In the low temperature-reverted mutant stamens, the polyamine levels and the activity of PA biosynthetic enzymes were not significantly different from the normal. It is suggested that the abnormal stamen development in the sl-2/sl-2 mutant is, in part, related to elevated levels of endogenous PAs. PMID- 16667486 TI - Polyamines and Flower Development in the Male Sterile Stamenless-2 Mutant of Tomato (Lycopersicon esculentum Mill.) : II. Effects of Polyamines and Their Biosynthetic Inhibitors on the Development of Normal and Mutant Floral Buds Cultured in Vitro. AB - The floral organs of the male sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.) contain significantly higher level of polyamines than those of the normal (R Rastogi, VK Sawhney [1990] Plant Physiol 93: 439 445). The effects of putrescine, spermidine and spermine, and three different inhibitors of polyamine biosynthesis on the in vitro development of floral buds of the normal and sl-2/sl-2 mutant were studied. The polyamines were inhibitory to the in vitro growth and development of both the normal and mutant floral buds and they induced abnormal stamen development in normal flowers. The inhibitors of polyamine biosynthesis also inhibited the growth and development of floral organs of the two genotypes, but the normal flowers showed greater sensitivity than the mutant. The inhibitors also promoted the formation of normal-looking pollen in stamens of some mutant flowers. The effect of the inhibitors on polyamine levels was not determined. The polyamine-induced abnormal stamen development in the normal, and the inhibitor-induced production of normal-looking pollen in mutant flowers support the suggestion that the elevated polyamine levels contribute to abnormal stamen development in the sl-2/sl-2 mutant of tomato. PMID- 16667487 TI - H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips. AB - The relationship between changes in H(+) flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H(+) efflux was abolished in anoxia but not by the 2 deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H(+)-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H(+) extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H(+) secretion under the conditions of this investigation. PMID- 16667488 TI - Water Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells. AB - Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 x 10(-5) molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (23 degrees C) for 7 days acclimated to 28 degrees C (LT(50)) when treated with ABA, or to -5 degrees C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H(2)O) or as grams water per gram dry weight (gram H(2)O/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H(2)O/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H(2)O/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H(2)O/10(6) cells), did not differ in ABA treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/10(6) cells increased to 3.3 milligram/10(6) cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/10(6) cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur. PMID- 16667489 TI - Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust. AB - When highly resistant wheat (Triticum aestivum L.) varieties are infected by an avirulent race of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Erics. and E. Henn.), penetrated host cells undergo rapid necrotization. This hypersensitive cell death is correlated with cellular lignification which efficiently restricts further fungal growth. Three competitive inhibitors of phenylalanine ammonia-lyase, the first enzyme of the general phenylpropanoid pathway and, thus, of lignin biosynthesis, namely alpha-aminooxyacetate, alpha aminooxy-beta-phenylpropionic acid, and (1-amino-2-phenylethyl)phosphonic acid, and two highly specific irreversible suicide inhibitors of the lignification specific enzyme cinnamyl-alcohol dehydrogenase, namely N(O aminophenyl)sulfinamoyl-tertiobutyl acetate and N(O-hydroxyphenyl)sulfinamoyl tertiobutyl acetate, were applied to genetically resistant wheat plants prior to inoculation with stem rust. Treatment with any of these inhibitors decreased the frequency of lignified necrotic host cells and concomitantly led to increased fungal growth. The cinnamyl-alcohol dehydrogenase inhibitors were generally more effective than the phenylalanine ammonia-lyase inhibitors, occasionally allowing some sporulation to occur on the resistant wheat leaves. These results clearly point to a causal relationship between the formation of lignin precursors and the resistance of wheat to stem rust. PMID- 16667490 TI - Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress. AB - Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H(+)-transport activity were measured by quinacrine fluorescence (DeltapH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (DeltaPsi) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1 napthalenesulphonic acid (ANS) provided dissociation constants (K(d)) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Psi(o)) of-26.0 and -13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K(d) values and the calculated intrinsic affinity constant, K(i). The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Psi(o) values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K(+)-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl( ) ions to stimulate H(+)-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane. PMID- 16667491 TI - Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don. AB - Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H(+) efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H(+)/H(2)PO(4) (-) stoichiometry of the cotransport is most likely to be 4. PMID- 16667492 TI - Effect of glutathione on phytochelatin synthesis in tomato cells. AB - Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [gamma Glu-Cys](2)-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [(35)S]cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione. PMID- 16667493 TI - Soluble peroxidase from winter wheat seedlings with phenoloxidase-like activity. AB - Peroxidase isozymes from winter wheat (Triticum aestivum L. cv Orso) seedlings extracts showed phenoloxidase-like activity, becoming visible on polyacrylamide gels also in the absence of hydrogen peroxide. The results obtained after a characterization of the two activities, based on their substrate specificity, on their selective inhibition, and on the possible occurrence of artifacts, suggested the existence of polyfunctional peroxidase isozymes. Different isozymes possessing only phenol oxidase activity were not found in the same plant material. This appears to be the first evidence of phenoloxidase-acting isoperoxidases in winter wheat. PMID- 16667494 TI - Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods. AB - We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max [L.] Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using (15)N dilution. In one experiment with CB1809, plants were supplied throughout growth with either N-free nutrients or with nutrients supplemented with 1, 2, 4, or 8 millimolar (15)N-nitrate and harvested on eight occasions between V6 and R7 for root-bleeding sap, vacuum extracted sap, stems (including petioles), and whole plant dry matter. Analyses of the saps and stem extracts for ureides (allantoin plus allantoic acid), alpha amino-N, and nitrate, and of dry matter for N and (15)N, indicated a positive effect of nitrate supply on concentrations of nitrate in saps and extracts and a negative effect on ureides and on the proportion of plant N derived from nitrogen fixation. The relative abundance of ureide-N in root-bleeding sap, vacuum extracted sap (100 [ureide-N]/[ureide-N+ alpha-amino-N + nitrate-N]) and stem extracts (100 [ureide-N]/[ureide-N + nitrate-N]) and the proportion of plant N, derived from nitrogen fixation between successive samplings were highly correlated (r = 0.97-1.00). For each variable, two standard curves were prepared to account for the shifts in the compositions of N solutes of xylem saps and extracts after flowering which were not related to a change in nitrogen fixation. Relationships between relative ureide-N and the proportion of plant N derived from nitrogen fixation were not affected by plant genotype or by strain of rhizobia. Therefore, assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules. PMID- 16667495 TI - Response to Phosphate Deprivation in Brassica nigra Suspension Cells : Enhancement of Intracellular, Cell Surface, and Secreted Phosphatase Activities Compared to Increases in Pi-Absorption Rate. AB - Suspension cells of Brassica nigra responded to Pi deprivation by increasing their potential for Pi influx and by raising the active levels of intracellular, cell surface, and secreted acid phosphatases. These responses, however, were temporally distinct. Phosphate influx capacity increased 15-fold in parallel to a 10-fold decrease in endogenous Pi during 7 days of culture in basal growth medium. In contrast, intracellular and cell surface phosphatase activities changed only after alterations in cellular phosphorus status had been in place for a number of days. Even in nutrient sufficient cells the secretion of phosphatase remained relatively high as did the activities of the other phosphatases. The cell surface acid phosphatase had a K(m) of approximately 10 times that of the influx process and molybdate was a much stronger inhibitor of this phosphatase activity. From these results it appears that Pi absorption and the production or activation of phosphatases are regulated in a distinct manner. In addition, Pi uptake into Brassica nigra cells does not appear to directly involve the cell surface phosphatase under Pi-deficient conditions. PMID- 16667496 TI - P NMR Observations on the Effect of the External pH on the Intracellular pH Values in Plant Cell Suspension Cultures. AB - (31)P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the response of oil palm (Elaeis guineensis) and carrot (Daucus carota) cell suspensions to changes in the external pH. An airlift system was used to oxygenate the cells during the NMR measurements and a protocol was developed to enable a constant external pH to be maintained in the suspension when required. Phosphonoacetic acid was used as an external pH marker and the intracellular pH values were measured from the chemical shifts of the cytoplasmic and vacuolar orthophosphate resonances. In contrast to earlier studies the cytoplasmic pH was independent of the external pH over the range 5.5 to 8.0 and it was only below pH 5.5 that the cytoplasmic pH varied, falling at a rate of 0.12 pH unit per external unit. Loss of pH control was observed in response to sudden increases in external pH with the response of the cells depending on the conditions imposed. A notable feature of the recovery from these treatments was the transient acidification of the cytoplasm that occurred in a fraction of the cells and overshoot phenomena of this kind provided direct evidence for the time dependence of the regulatory mechanisms. PMID- 16667497 TI - Expression of Mammalian Metallothionein Suppresses Glucosinolate Synthesis in Brassica campestris. AB - Transfection of Brassica campestris leaves with cauliflower mosaic virus (CaMV) harboring a mammalian metallothionein (MT) cDNA at the ORFII position lowered the glucosinolate (GS) concentration to approximately one-half the level in leaves infected with wild-type CaMV. This suppression was independent of the plant's sulfate status, suggesting that the pathways for protein (MT) and GS biosynthesis were competing for S on an equal basis. The expression of MT may have lowered the endogenous levels of Cys, 3'-phosphoadenosine-5'-phosphosulfate, or Met, all of which are required for GS synthesis in B. campestris. These results indicate that the introduction of structural genes coding for high levels of specific amino acids can be used to alter the production of nonproteinaceous molecules within plants. PMID- 16667498 TI - Relationship between Growth and Electric Oscillations in Bean Roots. AB - Extracellular and intracellular electric potentials in bean roots are known to show electric oscillations along the longitudinal axis with a period of several minutes. The relationship between growth and the electric oscillations was studied using roots of adzuki (Phaseolus chrysanthos). We measured surface electric potentials with a multielectrode apparatus while simultaneously measuring elongation using a CCD camera and monitor. Roots having an electric oscillation grew faster than roots with no oscillation. Furthermore, elongation rate was higher in roots with higher oscillation frequency. Oscillation frequency had a strong dependence on temperature; i.e. Q(10) was estimated at 1.7. These results suggest a correlation between electric oscillation and elongation. PMID- 16667499 TI - Spinach Nitrate Reductase : Effects of Ionic Strength and pH on the Full and Partial Enzyme Activities. AB - Initial velocity studies of immunopurified spinach nitrate reductase have been performed under conditions of controlled ionic strength and pH and in the absence of chloride ions. Increased ionic strength stimulated NADH:ferricyanide reductase and reduced flavin:nitrate reductase activities and inhibited NADH:nitrate reductase, NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities. NADH:dichlorophenolindophenol reductase activity was unaffected by changes in ionic strength. All of the partial activities, expressed in terms of micromole 2 electron transferred per minute per nanomole heme, were faster than the overall full, NADH:nitrate reductase activity indicating that none of the partial activities included the rate limiting step in electron transfer from NADH to nitrate. The pH optimum for NADH:nitrate reductase activity was determined to be 7 while values for the various partial activities ranged from 6.5 to 7.5. Chlorate, bromate, and iodate were determined to be alternate electron acceptors for the reduced enzyme. These results indicate that unlike the enzyme from Chlorella vulgaris, intramolecular electron transfer between reduced heme and Mo is not rate limiting for spinach nitrate reductase. PMID- 16667500 TI - Regulation of ribulose-1,5-bisphosphate carboxylase expression in second leaves of maize seedlings from low and high yield populations. AB - Ribulose-1,5-bisphosphate carboxylase oxygenase (EC 4.1.1.39) (Rubisco) activity, Rubisco-protein, and Rubisco large and small subunit gene (rbcL and rbcS) transcripts were measured at seven stages of development in the second leaf of maize (Zea mays L.) seedlings belonging to low and high yield populations. During the three early stages of development, when the leaf has not yet expanded, it was determined that increments in Rubisco-activity were caused by increases in Rubisco-protein and its mRNAs. Afterward, the rbcS level decreased sharply down to nondetectable levels at the seventh stage, when the leaf was at the beginning of senescence. As a contrast, rbcL transcript decreased slowly and Rubisco protein accumulated up to the fifth stage, when the leaf reached its maximum expansion. A slight decrease in Rubisco-protein was then observed. These results suggest that at early stages of development Rubisco-activity and Rubisco-protein are regulated mainly at the transcriptional level. At the later phase the regulation seems to be at other biochemical levels. Neither Rubisco activity nor Rubisco-protein showed correlation with yield for both maize populations at this stage of development. Slightly higher levels of both transcripts were observed in the high yield population. PMID- 16667502 TI - Decreased growth temperature increases soybean stearoyl-acyl carrier protein desaturase activity. AB - Developing soybean (Glycine max) seeds respond to a change in growth temperature by changing the level of stearoyl acyl carrier protein desaturase activity in the tissue. After 20 hours in liquid culture, seeds grown at 20 degrees C show an increase in activity while seeds grown at 35 degrees C show a decrease in activity, relative to their preculture levels. Analysis of the enzyme from both growth conditions shows the change not to be due to induction of kinetically distinct iosenzymes; desaturase activities from both 20 degrees C and 35 degrees C have identical behavior with regard to pH, temperature optimum, substrate concentration and cofactor requirements. Experiments with boiled extracts indicate that the modulation is not caused by induction of metabolic effectors. From these data, it appears that stearoyl-acyl carrier protein desaturase responds to changes in growth temperature by altering the level of active enzyme present in the tissue. The magnitude of this response is a function of the developmental stage of the seed and not a function of the growth conditions of the parent plant. Changing the age of the seeds from early late R5 changed the ratio of 20:35 degrees C activity from 3.8:1 to 1.2:1. Changing the temperature at which the parent plants were grown over a range from 20/12 degrees C to 34/28 degrees C (day/night) produced only minor, and inconsistent, changes in the ratio of 20:35 degrees C activities. PMID- 16667501 TI - Effects of CO(2)-Enrichment and of Aminoacetonitrile on Growth and Photosynthesis of Photoautotrophic Calli of Nicotiana plumbaginifolia. AB - Photoautotrophic calli of Nicotiana plumbaginifolia were grown for 3 weeks under two CO(2) concentrations (500 and 20,000 microliters of CO(2) per liter). Calli cultured at high CO(2) exhibited a two-fold higher rate of growth. At CO(2) test levels, these calli were characterized by a lower net photosynthetic capacity than calli cultured at low CO(2). This diminution due to CO(2) adaptation could be ascribed to a 170% stimulation of dark respiration, a 40% decrease in total ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and also to a feedback inhibition of photosynthesis: high CO(2) grown calli contained about 5.5-fold more sucrose and three-fold less orthophosphate (Pi) than low CO(2) grown calli. Whether the decrease in Rubisco activity is related to the accumulation of sucrose and to the Pi limitation is discussed. Both calli exhibited a Warburg effect showing the existence of active photorespiration at low CO(2). In calli grown at low CO(2) with 5 millimolar aminoacetonitrile (AAN), an inhibitor of the glycolate pathway, fresh weight decreased by 25% and chlorophyll content by 40%, dark respiration increased by 50% and net CO(2) uptake decreased by about 60% at 340 microliters of CO(2) per liter and 35% at 10,000 microliters of CO(2) per liter. In these calli, glutamine and glutamate contents were half of control calli. In contrast, AAN did not provoke any noticeable effect in calli grown at high CO(2). In photoautotrophic calli, the inhibition of the glycolate pathway by AAN results in severe perturbations in glutamate metabolism and in chlorophyll biosynthesis. PMID- 16667503 TI - Effect of Boron Deficiency on Photosynthesis and Reductant Sources and Their Relationship with Nitrogenase Activity in Anabaena PCC 7119. AB - Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O(2) evolution and in CO(2) fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N(2) fixation is independent of its effects on photosynthesis and reductant supply. PMID- 16667504 TI - Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to k. AB - Although many studies have alluded to a role for boron (B) in membrane function, there is little evidence for a direct effect of B on the plasmalemma of higher plant cells. These studies were conducted to demonstrate, by electrophysiological techniques, a direct effect of B on the membrane potential (E(m)) of sunflower (Helianthus annuus [L.], cv Mammoth Grey Stripe) root tip cells and to determine if the response to B occurs rapidly enough to account for the previously observed effects of B on ion uptake. By inserting a glass microelectrode into an individual cell in the root tip, the E(m) of the cell was determined in basal salt medium (BSM), pH 6.0. The perfusion solution surrounding the root tissue was then changed to BSM + 50 micromolar H(3)BO(3), pH 6.0. The exposure to B induced a significant plasmalemma hyperpolarization in sunflower root cells within 20 minutes. After just 3 minutes of exposure to B, the change in E(m) was already significantly different from the negligible change in E(m) observed over time in root cells never exposed to B. Membrane hyperpolarization could be caused by a stimulation of the proton pump or by a change in the conductance of one or more permeable ions. Since B has been shown to affect K(+) uptake by plants, the electrophysiological techniques described above were used to determine if B has an effect on membrane permeability to K(+), and could thereby lead to an increased diffusion potential. When sunflower root tips were pretreated in 50 micromolar B for 2 hours, cell membranes exhibited a significantly greater depolarization with each 10-fold increase in external [K(+)] than minus-B cells. Subsequent studies demonstrated that the depolarization due to increased external [K(+)] was also significantly greater when tissue was exposed to B at the same time as the 10-fold increase in [K(+)], indicating that the effect of B on K(+) permeability was immediate. Analysis of sunflower root tips demonstrated that treatment in 50 micromolar B caused a significantly greater accumulation of K(+) after 48 hours. The B-induced increase in K(+) uptake may cause a subsequent stimulation of the H(+)-ATPase (proton pump) and lead to the observed hyperpolarization of root cell membranes. Alternatively, B may stimulate the proton pump, with the subsequent hyperpolarization resulting in an increased driving force for K(+) influx. PMID- 16667505 TI - Isolation and Characterization of a Ferredoxin Gene from Arabidopsis thaliana. AB - We report the cloning and characterization of an Arabidopsis thaliana (L.) Heynh. (Columbia ecotype) ferredoxin gene (Fed A). Sequence analysis of a genomic clone shows an intron-free, 444-base pair open reading frame which encodes a 96 amino acid mature ferredoxin polypeptide preceded by a 52 amino acid transit peptide. Comparison with other plant ferredoxin proteins suggests that Fed A encodes a leaf ferredoxin. Genomic Southern blot analysis indicates the presence of a second, weakly related gene, consistent with other reports of at least two ferredoxins in plants. The Fed A gene promoter contains two regions, ACGCCACGTGGTAGATAGGATT (G-I box) and CCACGCCATTTCCACAAGC (CCAC box), which are strongly conserved in both sequence and position between the Arabidopsis and pea ferredoxin genes. Similarities with other better characterized plant promoter elements are also discussed. PMID- 16667506 TI - X-ray photoelectron spectroscopy surface analysis of aluminum ion stress in barley roots. AB - X-ray photoelectron spectroscopy (XPS) has been used to analyze root surface changes when Dayton barley (Hordeum vulgare) (Al tolerant) and Kearney barley (Al sensitive) seedlings were grown in nutrient solution in the presence and absence of 37.0 micromolar Al. The electron spectra from root surfaces contained strong lines in order of decreasing intensity from organic forms of carbon, oxygen, and nitrogen and weak lines due to inorganic elements in the form of anions and cations on the surface. The surface composition of root tips from Kearney was C, 65.6%; 0, 26.8%; N, 4.4% and tips from Dayton was C, 72.7%; O, 23.6%; N, 1.9%, grown in the absence of aluminum. Electron lines characteristic of nitrate, potassium, chloride, phosphate were also present in the spectra from those roots. Dayton roots grown in the presence of 37.0 micromolar aluminum contained 2.1% aluminum while Kearney contained 1.3% aluminum. The ratio of aluminum to phosphate was close to 1.0. Dayton roots usually contained twice as much aluminum phosphate in the surface region as Kearney. Dayton may be less susceptible to Al toxic effects by accumulation of aluminum phosphate on the root surface which then acts as a barrier to the transport of aluminum into the interior of the roots. PMID- 16667507 TI - Induction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase by anoxia in rice seedlings. AB - Rice (Oryza sativa) seeds were imbibed for 3 days and the seedlings were further incubated for 8 days in the presence of either air or nitrogen. In aerobiosis, the specific activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase and that of the ATP-dependent phosphofructokinase increased about fourfold. In anaerobiosis, the specific activity of ATP-dependent phosphofructokinase remained stable, whereas that of pyrophosphate:fructose 6-phosphate 1-phosphotransferase increased as much as in the presence of oxygen and there was also a fourfold increase in the concentration of fructose 2,6-bisphosphate, a potent stimulator of that enzyme. These data suggest a preferential involvement of pyrophosphate:fructose 6-phosphate 1-phosphotransferase rather than of ATP dependent phosphofructokinase in glycolysis during anaerobiosis. PMID- 16667508 TI - Leaf Senescence in a Nonyellowing Mutant of Festuca pratensis: Metabolism of Cytochrome f. AB - In a mutant genotype of Festuca pratensis Huds., net degradation of a number of thylakoid membrane proteins during senescence is impaired. Previous studies have suggested that the highly hydrophobic intrinsic chlorophyll-binding proteins were the definitive subjects of the metabolic lesion. In the present study we find that cytochrome f, as determined by haem-staining, Western blotting, enzyme linked immunosorbent assay, and immunogold electron microscopy, is also abnormally stable in the mutant. The structural feature common to all the proteins in the mutant so far recognized to be abnormally stable is possession of a tetrapyrrole prosthetic group. It is suggested that degradation of chlorophyll and haem may regulate degradation of the associated apoproteins, and hence has an important role to play in membrane protein turnover and in mobilisation of amino acids during chloroplast disassembly. PMID- 16667509 TI - Characterization of the expression of the petunia glycine-rich protein-1 gene product. AB - We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178 181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels. PMID- 16667510 TI - Aspartate Aminotransferase in Alfalfa Root Nodules : II. Immunological Distinction between Two Forms of the Enzyme. AB - Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT 1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa. PMID- 16667511 TI - Evidence for Metabolic Domains within the Matrix Compartment of Pea Leaf Mitochondria : Implications for Photorespiratory Metabolism. AB - The simultaneous oxidation of malate and of glycine was investigated in pea (Pisum sativum) leaf mitochondria. Adding malate to state 4 glycine oxidation did not inhibit, and under some conditions stimulated, glycine oxidation. State 4 oxygen uptake with glycine is restricted because of the control exerted by the membrane potential but reoxidation of NADH by oxaloacetate reduction can still occur. Thus, malate addition stimulates glycine metabolism by producing oxaloacetate. The malate dehydrogenase (EC 1.1.1.37) enzyme fraction remote from glycine decarboxylase (EC 2.1.2.10) oxidizes malate whereas that closely associated with it produces malate, i.e. they function in opposite directions. It is shown that these opposing directions of malate dehydrogenase activity occur within the same mitochondrial matrix compartment and not in different mitochondrial populations. It is concluded that metabolic domains containing different complements of mitochondrial enzymes exist within the one mitochondrial matrix without physical barriers separating them. The differential spatial organization within the matrix may account for the previously reported limited access of some enzymes to the respiratory electron transport chain. The implications for leaf mitochondrial metabolism are discussed. PMID- 16667512 TI - Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth. AB - Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [(14)C]stachyose or [(14)C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [(14)C]stachyose, but some degree of vein labeling with [(14)C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and alpha-galactosidase and extensively metabolized exogenously supplied (14)C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species. PMID- 16667513 TI - The S-Methylmethionine Cycle in Lemna paucicostata. AB - The metabolism of S-methylmethionine has been studied in cultures of plants of Lemna paucicostata and of cells of carrot (Daucus carota) and soybean (Glycine max). In each system, radiolabeled S-methylmethionine was rapidly formed from labeled l-methionine, consistent with the action of S-adenosyl-l methionine:methionine S-methyltransferase, an enzyme which was demonstrated during these studies in Lemna homogenates. In Lemna plants and carrot cells radiolabel disappeared rapidly from S-methylmethionine during chase incubations in nonradioactive media. The results of pulse-chase experiments with Lemna strongly suggest that administered radiolabeled S-methylmethionine is metabolized initially to soluble methionine, then to the variety of compounds formed from soluble methionine. An enzyme catalyzing the transfer of a methyl group from S methylmethionine to homocysteine to form methionine was demonstrated in homogenates of Lemna. The net result of these reactions, together with the hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine, is to convert S-adenosylmethionine to methionine and adenosine. A physiological advantage is postulated for this sequence in that it provides the plant with a means of sustaining the pool of soluble methionine even when overshoot occurs in the conversion of soluble methionine to S-adenosylmethionine. The facts that the pool of soluble methionine is normally very small relative to the flux into S adenosylmethionine and that the demand for the latter compound may change very markedly under different growth conditions make it plausible that such overshoot may occur unless the rate of synthesis of S-adenosylmethionine is regulated with exquisite precision. The metabolic cost of this apparent safeguard is the consumption of ATP. This S-methylmethionine cycle may well function in plants other than Lemna, but further substantiating evidence is neeeded. PMID- 16667514 TI - Cyanogenic Lipids: Utilization during Seedling Development of Ungnadia speciosa. AB - Large amounts of cyanogenic lipids (esters of 1 cyano-2-methylprop-2-ene-1-ol with C:20 fatty acids) are stored in the seeds of Ungnadia speciosa. During seedling development, these lipids are completely consumed without liberation of free HCN to the atmosphere. At the same time, cyanogenic glycosides are synthesized, but the total amount is much lower (about 26%) than the quantity of cyanogenic lipids formerly present in the seeds. This large decrease in the total content of cyanogens (HCN-potential) demonstrates that at least 74% of cyanogenic lipids are converted to noncyanogenic compounds. Whether the newly synthesized cyanogenic glycosides are derived directly from cyanogenic lipids or produced by de novo synthesis is still unknown. Based on the utilization of cyanogenic lipids for the synthesis of noncyanogenic compounds, it is concluded that these cyanogens serve as storage for reduced nitrogen. The ecophysiological significance of cyanolipids based on multifunctional aspects is discussed. PMID- 16667515 TI - Regulation of Carbon Partitioning in Source and Sink Leaf Parts in Sweet Pepper (Capsicum annuum L.) Plants : Role of Fructose 2,6-Bisphosphate. AB - Area expansion rate, partitioning of photosynthetically fixed carbon, and levels of fructose 2,6-bisphosphate (fru-2,6-P(2)) were determined in individual parts of developing leaves of sweet pepper (Capsicum annuum L.). The base was rapidly expanding and allocated less carbon to sucrose synthesis in comparison to the leaf tip, where expansion had almost stopped. The change in leaf expansion rate and carbon partitioning happened gradually. During day time levels of fru-2,6 P(2) were consistently higher in the leaf base than in the leaf tip. Leaf expansion rate and carbon partitioning were closely related to day time levels of fru-2,6-P(2), suggesting that fru-2,6-P(2) is an important factor in adjustment of metabolism during sink-to-source transition of leaf tissue. The levels of fru 2,6-P(2) changed markedly after a dark-to-light transition in the leaf base, but not in the leaf tip, suggesting that regulatory systems based on fru-2,6-P(2) are different in sink and source leaf tissue. During the period upon dark-to-light transition the variations in level of fru-2,6-P(2) did not show a close correlation to changes in the carbon partitioning, until the metabolism had reached a steady state. PMID- 16667516 TI - Induction of nitrate transport in maize roots, and kinetics of influx, measured with nitrogen-13. AB - Unlike phosphate or potassium transport, uptake of nitrate by roots is induced, in part, by contact with the substrate ion. Plasmalemma influx of (13)N-labeled nitrate in maize roots was studied in relation to induction of the uptake system, and the influence of short-term N starvation. Maize (Zea mays) roots not previously exposed to nitrate had a constitutive transport system (state 1), but influx increased 250% during six hours of contact with 100 micromolar nitrate, by which time the transport mechanism appeared to be fully synthesized (state 2). A three-day period of N starvation prior to induction and measurement of nitrate influx resulted in a greater capacity to transport nitrate than in unstarved controls, but this was fully expressed only if roots were kept in contact with nitrate for the six hours needed for full induction (state 2E). A kinetic analysis indicated a 160% increase in maximum influx in N-starved, induced roots with a small decrease in K(m). The inducible component to nitrate influx was induced only by contact with nitrate. Full expression of the nitrate inducible transport system was dependent upon mRNA synthesis. An inhibitor of cytoplasmic protein synthesis (cycloheximide) eliminated the formation of the transport system while inhibition by chloramphenicol of mitochondrial- or plastid-coded protein synthesis had no effect. Poisoning of membrane-bound proteins effectively disabled both the constitutive and induced transport systems. PMID- 16667517 TI - Effects of low water potential on cortical cell length in growing regions of maize roots. AB - Roots growing under low water potential commonly exhibit a marked decrease in growth rate and in diameter. Using median longitudinal sections of fixed maize (Zea mays L. cv WF9 x Mo 17) seedling roots, we investigated the cellular basis for these effects. Cortical cells in the shortened elongation zone of water stressed roots were longer than cortical cells in the comparable location of well watered roots. Nearly twofold differences in cell length were seen in the region 2 to 4 millimeters behind the root apex. The shortened growth zone, however, leads to a final mean cortical cell length approximately 30% shorter in the stressed roots. These differences were present regardless of the age of the control roots. These data, and the slower growth rate seen in water-stressed roots, suggest that the water deficit causes a significant reduction in the rate of cell supply to the cortical cell files. PMID- 16667518 TI - Variation among Species in the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination. AB - The relationship between the thermal dependence of the reappearance of chlorophyll variable fluorescence following illumination and temperature dependence of the apparent Michaelis constant (K(m)) of NADH hydroxypyruvate reductase for NADH was investigated in cool and warm season plant species. Brancker SF-20 and SF-30 fluorometers were used to evaluate induced fluorescence transients from detached leaves of wheat (Triticum aestivum L. cv TAM-101), cotton (Gossypium hirsutum L. cv Paymaster 145), tomato (Lycopersicon esculentum cv Del Oro), bell pepper (Capsicum annuum L. cv California Wonder), and petunia (Petunia hybrida cv. Red Sail). Following an illumination period at 25 degrees C, the reappearance of variable fluorescence during a dark incubation was determined at 5 degrees C intervals from 15 degrees C to 45 degrees C. Variable fluorescence recovery was normally distributed with the maximum recovery observed at 20 degrees C in wheat, 30 degrees C in cotton, 20 degrees C to 25 degrees C in tomato, 30 to 35 degrees C in bell pepper and 25 degrees C in petunia. Comparison of the thermal response of fluorescence recovery with the temperature sensitivity of the apparent K(m) of hydroxypyruvate reductase for NADH showed that the range of temperatures providing fluorescence recovery corresponded with those temperatures providing the minimum apparent K(m) values (viz. the thermal kinetic window). PMID- 16667519 TI - Purification and Some Properties of a Daucus carota Lectin which Enhances the Activation of Prophenoloxidase by CaCl(2). AB - Carrot (Daucus carota) cell cultures were found to secrete a protein, with the molecular weight of 58,000, which could strongly enhance the activation of carrot prophenoloxidase by CaCl(2). The protein was isolated from the culture filtrate and purified to electrophoretic homogeneity. The purified protein did also show hemagglutinating activity toward rat as well as rabbit erythrocytes and this activity was inhibitied by N-acetylglucosamine or fetuin. PMID- 16667520 TI - Synthesis of abscisic Acid-responsive, heat-stable proteins in embryonic axes of dormant wheat grain. AB - Germination of embryonic axes from dormant grain is inhibited by low concentrations of abscisic acid (ABA) compared with axes from nondormant grain. Incubation of dormant grain axes in 0.05 to 50 micromolar ABA caused the prolonged synthesis of a set of heat-stable proteins. Two of these proteins were identified as dehydrins. In nondormant grain axes, 100- to 1000-fold greater ABA concentrations were required to produce similar results. When embryonic axes of dormant wheat (Triticum aestivum) grain were imbibed without ABA, endogenous ABA levels increased 2.5-fold by 4 hours and then gradually declined. Heat-stable proteins were continuously synthesized for at least 18 hours. No increase in endogenous ABA was observed when nondormant grain axes were imbibed. Endogenous ABA levels in nondormant grain axes remained constant at 4 hours and then declined. The nondormant grain axes initially synthesized the heat-stable proteins, but that synthesis disappeared between 8 and 12 hours. These results showing the prolonged synthesis of ABA-responsive, heat-stable proteins by dormant grain axes, demonstrate that biochemical differences occur when axes from dormant compared with nondormant grains are imbibed. PMID- 16667521 TI - Control of Seed Respiration and Growth in Vicia faba by Oxygen and Temperature: No Evidence for an Oxygen Diffusion Barrier. AB - The rate of dry matter accumulation by seeds of Vicia faba L. cv. Minica increases with temperature in the range of 16 to 26 degrees C. The duration of dry matter accumulation decreases with temperature, resulting in a decrease of final seed dry weight. In this study we test the hypothesis that a diffusion barrier for O(2), located in the seed coat, inhibits seed respiration and growth. The rate of O(2) uptake of intact seeds and of excised embryos and seed coats (separated seeds) was measured in air and buffer at 16, 20, and/or 26 degrees C at various O(2) concentrations and developmental stages. Oxygen uptake rates of intact seeds in buffer were only 9 to 15% of those in air. In buffer, the respiration rate of intact seeds decreased at a pO(2) below air saturation (21 kilopascals), whereas separated seeds showed a decline of O(2) uptake only below 80% of air saturation. In air, embryo excision had no effect on the sensitivity of seed respiration to pO(2), at both 20 and 26 degrees C. In air at 20 degrees C, separated and intact seeds showed similar rates of O(2) uptake. Oxygen uptake by intact seeds, both halfway and beyond the linear growth phase, showed a temperature coefficient Q(10) of 2.3 and was insensitive to pO(2) in the range of 80 to 100% of ambient. These results indicate that V. faba seed respiration in air is not limited by the diffusion of O(2) into the seed. PMID- 16667522 TI - Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor Releasing Factor, beta-1,3-Endoglucanase, in Soybean. AB - Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3 fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3 endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3 endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription. PMID- 16667523 TI - Control of Pyrophosphated-Fructose-6-Phosphate 1-Phosphotransferase Activity in the Cotyledons of Citrullus lanatus. AB - After initiation of radicle elongation, the pyrophosphate:d-fructose-6-phosphate 1-phosphotransferase (PFP) activity sharply increases in the cotyledons of Citrullus lanatus. Removal of the radicle early during incubation prevents the increase in PFP activity in the cotyledons evident in the control. Removal of the radicle at any stage after germination results in a decrease in PFP activity in the cotyledons. Application of kinetin (0.5 micromolar) or 2-chlorophosphonic acid (0.1 micromolar) to isolated cotyledons replaces the effect of the radicle. Gibberellic acid (0.09 micromolar GA(3)) also partially mimics the presence of the radicle. Anaerobic conditions, as well as cycloheximide application (0.18 micromolar) to intact embryos or to kinetin and ethrel treated isolated cotyledons prevent the increase in PFP activity evident in the control. PMID- 16667524 TI - Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves. AB - The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer. PMID- 16667525 TI - Isoxaben Inhibits the Synthesis of Acid Insoluble Cell Wall Materials In Arabidopsis thaliana. AB - The effect of the herbicide isoxaben on the incorporation of radiolabeled glucose, leucine, uracil, and acetate into acid insoluble cell wall material, protein, nucleic acids, and fatty acids, respectively, was measured. Dichlobenil, cycloheximide, actinomycin D, and cerulenin, inhibitors of the incorporation of these precursors into these macromolecular components, functioned as expected, providing positive controls. The incorporation of radiolabeled glucose into an acid insoluble cell wall fraction was severely inhibited by isoxaben at nanomolar concentrations. Amitrole, fluridone, ethalfluralin, and chlorsulfuron, as well as cycloheximide, actinomycin D, and cerulenin did not inhibit incorporation of glucose into this fraction, ruling out a general nonspecific effect of herbicides on glucose incorporation. The evidence thus suggests that isoxaben is an extremely powerful and specific inhibitor of cell wall biosynthesis. PMID- 16667526 TI - The magnitude of the stomatal response to blue light : modulation by atmospheric humidity. AB - The effect of leaf-air vapor pressure difference (VPD) on the magnitude of the stomatal response to blue light was investigated in soybean (Glycine max) by administering blue light pulses (22 seconds by 120 micromoles per square meter per second) at different levels of VPD and temperature. At 20 degrees C and 25 degrees C, the magnitude of the integrated conductance response decreased with increasing VPD (0.4 to 2.6 kiloPascals), due to an earlier onset of stomatal closure that terminated the pulse response. In contrast, at 30 degrees C this magnitude increased with rising VPD (0.9 to 3.5 kiloPascals), due to an increasing maximum excursion of the conductance response despite the accelerated onset of stomatal closure. When the feedforward response of stomata to humidity caused steady state transpiration to decrease with increasing VPD, the magnitude of the pulse-induced conductance response correlated with VPD rather than with transpiration. This suggests that water relations or metabolite movements within epidermal rather than bulk leaf tissue interacted with guard cell photobiological properties in regulating the magnitude of the blue light response. VPD modulation of pulse magnitude could reduce water loss during stomatal responses to transient illumination in natural light environments. PMID- 16667527 TI - Measurement of nitrogen fixation by soybean in the field using the ureide and natural N abundance methods. AB - Nitrogen fixation by field-grown soybean (Glycine max [L.] Merrill) was assessed by the natural (15)N abundance and ureide methods. The field sites (five) and genotypes (six, plus two levels of inoculation on Bragg) were chosen to provide a range of proportions of plant N derived from nitrogen fixation (P). Genotypes K466, K468, nts1007, and nts1116 and Davis were included on the basis of their reported tolerance of the suppressive effects of nitrate on nodulation and nitrogen fixation. Bragg was included as a ;nitrate-sensitive' genotype. Seeds of all genotypes were inoculated at sowing with Bradyrhizobium japonicum CB1809 (USDA136). Amounts of nitrate in the soil profile (0-1.2 meter depth) at sowing ranged from 70 (site 3) to 278 kilograms per hectare (site 5), resulting in large effects on plant nodulation, on the delta(15)N values of nodulated plants, on the relative abundance of ureide-N in vacuum-extracted sap (VES) and stem extracts, and finally on the estimates of P. There was no relationship between amount of soil nitrate at sowing and the delta(15)N of the plant-available soil N. Correlation matrices of the measured and calculated parameters indicated generally weak correlations between crop growth (dry matter and N) and the parameters of symbiotic activity (nodule weight, delta(15)N, relative ureide-N); correlations were strong and highly significant between nodulation and the measures of nitrogen fixation (delta(15)N, relative ureide-N; r = 0.79-0.92). Estimates of P ranged between 0 and 68% (delta(15)N) and between 6 and 56% (ureide) and were highly correlated (r = 0.97). Results indicated that the ureide method can be used with confidence to assess P by field-grown crops of soybean. PMID- 16667528 TI - Pleiotropic Effects of brz: A Mutation in Pisum sativum (L.) cv ;Sparkle' Conditioning Decreased Nodulation and Increased Iron Uptake and Leaf Necrosis. AB - Treatment of Pisum sativum (L.) cv ;Sparkle' with ethylmethane sulfonic acid produced a stable mutant, E107, which forms few nodules. The mutant allele exhibits other pleiotropic properties including bronze necrotic spots on the leaflets and high accumulation of iron in the shoot. The mutant phenotype is under monogenic recessive control. The gene, designated brz (bronze), is nonallelic with two other genes conditioning necrotic spots on leaves of other mutants of P. sativum. The brz allele was located on chromosome 4 by linkage with wax production controlled by alleles at the was locus. PMID- 16667529 TI - Physiological Characteristics of Fe Accumulation in the ;Bronze' Mutant of Pisum sativum L., cv ;Sparkle' E107 (brz brz). AB - The pea (Pisum sativum L.) mutant, E107 (brz, brz) accumulated extremely high concentrations of Fe in its older leaves when grown in light rooms in either defined nutrient media or potting mix, or outdoors in soil. Leaf symptoms (bronze color and necrosis) were correlated with very high Fe concentrations. When E107 plants were grown in nutrient solutions supplied 10 mum Fe, as the Fe(III)-N,N' ethylenebis[2-(2-hydroxyphenyl)glycine] chelate, their roots released higher concentrations of Fe(III) reducing substances to the nutrient media than did roots of the normal parent cv, ;Sparkle.' Reciprocal grafting experiments demonstrated that the high concentrations of Fe in the shoot was controlled by the genotype of the root. In short-term (59)Fe uptake studies, 15-day-old E107 seedlings exhibited higher rates of Fe absorption than did ;Sparkle' seedlings under Fe-adequate growth conditions. Iron deficiency induced accelerated short term Fe absorption rates in both mutant and normal genotypes. Iron-treated E107 roots also released larger amounts of both protons and Fe(III) reductants into their nutrient media than did iron-treated ;Sparkle' roots. Furthermore, the mutant translocated proportionately more Fe to its shoot than did the parent regardless of Fe status. PMID- 16667530 TI - Uptake and phytotoxicity of the herbicide metsulfuron methyl in corn root tissue in the presence of the safener 1,8-naphthalic anhydride. AB - Growth of Zea mays L. cv Potro roots was inhibited by the herbicide metsulfuron methyl (MSM) at the lowest concentration tested: 5 nanomoles per liter. Pretreatment of corn seeds with commercial 1,8-naphthalic anhydride (NA) at 1% (w/w) partially reversed MSM-induced root growth inhibition. MSM at a concentration of 52 nanomoles per liter was taken up rapidly by roots and accumulated in the corn tissue to concentrations three times those in the external medium; the safener NA increased MSM uptake up to 48 hours. The protective effect of NA was related to the ability of the safener to increase the metabolism of MSM; tenfold increases in the metabolic rates of MSM were observed in NA-pretreated corn seedlings grown for 48 hours on 52 nanomolar [(14)C]MSM solution. DNA synthesis determined by measurement of [(3)H]thymidine incorporation into DNA was inhibited by root MSM applications; after a 6-hour application period, 13 nanomolar MSM solution reduced DNA synthesis by 64%, and the same reduction was also observed with NA-pretreated seedlings. Pretreatment of corn seeds with safener NA did not increase the acetolactate synthase activity in the roots and did not change, up to 13 micromoles per liter, the in vitro sensitivity of roots to MSM. PMID- 16667531 TI - Immunodetection of protein glycoforms encoded by two independent genes of the self-incompatibility multigene family of brassica. AB - Glycoprotein products of two highly homologous Brassica S gene family members were studied: SLSG (S locus-specific glycoprotein), product of an SLG gene at the S locus, and SLR1 (S locus-related) protein, product of the SLR1 gene, a gene unlinked to the S locus. A polyclonal antibody directed against a trpE-SLR1 fusion protein facilitated study of the SLR1 protein. SLR1 protein was detected in a number of crucifer species. No variation in the level of this protein was found between self-compatible and self-incompatible plants. Both SLSG and SLR1 protein occurred as glycoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Each glycoform had several charge forms, indicated by elution patterns from a high performance liquid chromatography cation exchange column and behavior on two-dimensional gels. Deglycosylation of both SLSG and SLR1 protein caused loss of the glycoforms, which apparently arose from differences in glycosylation. Consistent with their apparent similar post translational processing, immunolocalization showed that SLR1 protein, like SLSG, accumulated in the stigma papillae cell walls. Thus, both SLSG and SLR1 protein are present at the site of pollen-stigma interaction. PMID- 16667532 TI - Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution. AB - The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca(2+) per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I(50) values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I(50) of 25 micromolar). These results suggest that direct Ca(2+) transport and Ca(2+)/H(+) antiport activities are present in both sucrose gradient fractions. PMID- 16667533 TI - Shikimate kinase from spinach chloroplasts : purification, characterization, and regulatory function in aromatic amino Acid biosynthesis. AB - Shikimate kinase was purified to near homogenity from spinach Spinacia oleracea L. chloroplasts and found to consist of a single 31 kilodalton polypeptide. The purified enzyme was unstable, but could be stabilized by a variety of added proteins, including oxidized and reduced thioredoxins. Whereas the isolated enzyme was stimulated by mono- and dithiol reagents, the enzyme in intact chloroplasts was unaffected by added thiols and showed only minor response to dark/light transitions. These results indicate that the previously reported stimulation of shikimate kinase activity by reduced thioredoxins is due to enzyme stabilization rather than to activation. In the current study, the purified enzyme was inhibited by added ADP and showed a strong response to energy charge. When intact chloroplasts were incubated in the dark in presence of shikimate, phosphoenolpyruvate and a source of ATP (dihydroxyacetone phosphate or ATP itself under appropriate conditions), aromatic amino acids were formed: phenylalanine and tyrosine. The data indicate that energy charge plays a role in regulating shikimate kinase, thereby controlling the shikimate pathway. An unidentified enzyme of the latter part of the pathway, leading from shikimate-3-phosphate to phenylalanine, appears to be activated by light. PMID- 16667534 TI - Inhibition by white light of rb absorption in the root apex of corn. AB - Measurements of cell lengths made at 0.5 millimeter intervals in median longitudinal sections of the primary roots of corn (Zea mays) were used to construct a growth curve. The region 1.5 to 4.0 millimeters from the apex contained the largest number of elongating cells. Absorption of (86)Rb(+) was measured using intact, dark-grown corn seedlings. Following uptake and exchange, the terminal 8.0 millimeters of each root was cut into four 2.0 millimeter segments. Maximum (86)Rb(+) uptake occurred in the region from 0.0 to 4.0 millimeter from the root tip. Washing the intact primary root in fresh 2.0 millimolar CaSO(4) for 2 hours prior to uptake augmented the rate of (86)Rb(+) uptake in all regions. Illumination with white light during washing caused a reduction of (86)Rb(+) uptake as compared with controls washed in darkness, and the region of greatest light response was the region of elongation. Removal of the coleoptile prior to washing did not prevent the light inhibition of subsequent (86)Rb(+) uptake. Removal of the root cap prior to washing in light partially reversed the light-induced inhibition of the washing response. PMID- 16667535 TI - Studies on Cyanidium caldarium Phycobiliprotein Pigment Mutants. AB - Phycobiliprotein biosynthesis was investigated in four strains of the unicellular rhodophyte, Cyandium caldarium, with different pigment phenotypes. All strains were incapable of synthesizing phycobiliproteins when grown in the dark. Western blotting experiments showed that dark-grown cells of the wild-type and mutant GGB synthesized the alpha and beta subunit polypeptides of allophyocyanin and phycocyanin after exposure to light for 24 hours, whereas cells of mutant IIIC and GGBY did not. Similarly, light promoted the appearance of allophycocyanin and phycocyanin mRNAs in the wild-type and GGB but not in IIIC and GGBY. However, Southern blots of restricted genomic DNA from the wild type, IIIC, GGBY, and GGB, all hybridized with heterologous phycobiliprotein gene probes and revealed that all four strains contained identical Pst, EcoRI, and Dral restriction fragments containing allophycocyanin and phycocyanin genes. Cells of the wild type and GGB incubated in the dark with the heme precursor. delta-aminolevulinate, synthesized allophycocyanin and phycocyanin apoproteins providing strong evidence for the role of a tetrapyrrole in regulation of phycobiliprotein gene expression. However, cells of IIIC and GGBY incubated in the dark with delta-aminolevulinate did not contain detectable quantities of allophycocyanin or phycocyanin apoproteins. The possible role of a tetrapyrrole in phycobiliprotein gene expression and basis for the genetic lesion in mutants IIIC and GGBY is discussed. PMID- 16667536 TI - Phosphate Uptake by Excised Maize Root Tips Studied by in VivoP Nuclear Magnetic Resonance Spectroscopy. AB - The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo(31)P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH approximately 6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N(2), the cytoplasmic Pi increased approximately 150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of approximately 10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H(+) ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells. PMID- 16667537 TI - The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase. AB - ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes. PMID- 16667538 TI - Photosynthesis at High Temperature in Tuber-Bearing Solanum Species : A Comparison between Accessions of Contrasting Heat Tolerance. AB - Differences in the photosynthetic performance between pairs of heat tolerant (HT) and heat sensitive (HS) accessions of tuber-bearing Solanum species were measured at 40 degrees C, after treating plants at 40/30 degrees C. After 1 to 9 days of heat treatment, both HT and HS accessions showed progressive inhibitory effects, primarily decreased rates of CO(2) fixation, and loss of leaf chlorophyll. These effects were most pronounced in the HS accessions. Stomatal conductivity and internal CO(2) concentrations were lower for both accessions at 40 degrees C especially for the HS accessions, suggesting that at ambient CO(2) concentrations, stomatal conductance was limiting CO(2) availability at the higher temperature. In the HT accessions, stomatal limitations were largely attributed to differences in vapor pressure deficit between 25 degrees and 40 degrees C, while the HS accessions exhibited significant nonstomatal limitations. The young expanding leaves of the HS accession showed some HT characteristics, while the oldest leaves showed severe senescence symptoms after 9 days at 40/30 degrees C. The data suggest that differences in heat sensitivity between HT and HS accessions are the result of accelerated senescence, chlorophyll loss, reduced stomatal conductance, and inhibition of dark reactions at high temperature. PMID- 16667539 TI - Anions activate the oxidation of indoleacetic Acid by peroxidases from tomato and other sources. AB - Anionic peroxidase from tomato (Lycopersicon esculentum) fruit oxidized indoleacetic acid (IAA) slowly in the presence of Mn(2+) and dichlorophenol in acetate buffers. The addition of certain anions to the reaction mixture increased the rate of oxidation. Phosphate was one of the effective anions and exerted maximal activation at 0.1 molar. The most effective activator of tomato peroxidase was nitrilotriacetate (NTA) at an optimum concentration of 60 micromolar. Only 0.17 nanomolar peroxidase was needed to oxidize 0.1 micromole IAA/5 minutes in the presence of NTA compared to 650 nanomolar peroxidase for the same rate in the absence of NTA. Other effective anions were oxalate, pyrophosphate, malate, and citrate. Each activator exhibited an optimum concentration and higher concentrations were inhibitory. Anionic peroxidase from horseradish was activated by the same anions. A cationic peroxidase from horseradish and lactoperoxidase oxidized IAA in acetate buffer although anions activated these enzymes severalfold. Microperoxidase and other hematoporphrins also catalyzed IAA oxidation in the presence of anions. It is proposed that IAA oxidation by peroxidase may be important when vacuolar contents mix with peroxidase as during plant injury. PMID- 16667540 TI - Tobacco Plants Transformed with the Bean alphaai Gene Express an Inhibitor of Insect alpha-Amylase in Their Seeds. AB - Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant alpha-amylases. We recently (J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889) presented strong circumstantial evidence that this alpha-amylase inhibitor (alphaAI) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5' and 3' flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M(r) 10,000-18,000) that cross react with antibodies to the bean alpha-amylase inhibitor. Most of these polypeptides bind to a pig pancreas alpha-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas alpha-amylase activity as well as the alpha-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called alphaai) into other leguminous plants may be a strategy to protect the seeds from the seed eating larvae of Coleoptera. PMID- 16667541 TI - Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a non-ethylene pathway for induction. AB - Antisera to acidic isoforms of pathogenesis-related proteins were used to measure the induction of these proteins in tobacco (Nicotiana tabacum) leaves. Endo-(1-4) beta-xylanase purified from culture filtrates of Trichoderma viride was a strong elicitor of pathogenesis-related protein synthesis in tobacco leaves. The synthesis of these proteins was localized to tissue at the area of enzyme application. The inhibitors of ethylene biosynthesis and ethylene action, 1 aminoethoxyvinylglycine and silver thiosulfate, inhibited accumulation of pathogenesis-related proteins induced by tobacco mosaic virus and alpha aminobutyric acid, but did not inhibit elicitation by xylanase. Likewise, the induction of these proteins by the tobacco pathogen Pseudomonas syringae pv. tabaci was not affected by the inhibitors of ethylene biosynthesis and action. The leaf response to tobacco mosaic virus and alpha-aminobutyric acid was dependent on light in normal and photosynthetically incompetent leaves. In contrast, the response of leaves to xylanase was independent of light. Tobacco mosaic virus and alpha-aminobutyric acid induced concerted accumulation of pathogenesis-related proteins. However, xylanase elicited the accumulation of only a subset of these proteins. Specifically, the plant (1-3)-beta-glucanases, which are normally a part of the concerted response, were underrepresented. These experiments have revealed the presence of a novel ethylene-independent pathway for pathogenesis-related protein induction that is activated by xylanase. PMID- 16667542 TI - Hordeum vulgare Seedlings Amine Oxidase: Purification and Properties. AB - Although no amine oxidase could be detected in crude extracts, the enzyme has been purified to apparent homogeneity from Hordeum vulgare seedlings using ammonium sulfate precipitation and chromatography on DEAE cellulose, Hydroxylapatite, and Sephadex G200 columns. Gel filtration experiments indicate a molecular weight of about 150,000. The pH optimum of the enzyme was found to be 7.5 in potassium phosphate buffer. The spectrum of ultraviolet and visible regions were similar to Cuamine oxidase from Leguminosae. PMID- 16667543 TI - Thermal Dependence of the Apparent K(m) of Glutathione Reductases from Three Plant Species. AB - The thermal dependencies of the apparent K(m) of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent K(m) of the enzymes were found to vary up to 9 fold between 12.5 and 45 degrees C. Values of the apparent K(m) in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term "thermal kinetic window" to describe to the range of temperatures over which the apparent K(m) of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16 degrees C for spinach, 23 to 32 degrees C for corn, and 35 to 41 degrees C for cucumber. PMID- 16667544 TI - Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis. AB - In C(4) plants carbonic anhydrase catalyzes the critical first step of C(4) photosynthesis, the hydration of CO(2) to bicarbonate. The maximum activity of this enzyme in C(4) leaf extracts, measured by H(+) production with saturating CO(2) and extrapolated to 25 degrees C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C(3) leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO(2) concentrations was apparently just sufficient to prevent the rate of conversion of CO(2) to HCO(3) (-) from limiting C(4) photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities. PMID- 16667545 TI - Expression of cab Genes in Douglas-Fir Is Not Strongly Regulated by Light. AB - Dark-grown Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings had approximately 30% of the major polypeptide of the light-harvesting chlorophyll a/b binding protein, 30% of cab mRNA, 54% of psbA mRNA, and 14% of total chlorophyll, in comparison with amounts in light-grown seedlings. Seedlings entrained under a 24-hour photoperiod of light and dark showed small diurnal fluctuations in cab and psbA mRNA levels and, when transferred to continuous conditions, no circadian rhythms in mRNA levels were apparent. These results suggest that regulation of cab gene expression in Douglas-fir differs from regulation in angiosperms, because in the latter, both light and circadian factors strongly influence the expression of cab genes. PMID- 16667546 TI - A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii. AB - The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO(2). Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO(2)-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO(2)-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO(2)-grown C. reinhardtii cells. PMID- 16667547 TI - Effect of d-myo-Inositol 1,4,5-Trisphosphate on the Electrical Properties of the Red Beet Vacuole Membrane. AB - The effect of channel opening in the tonoplast by d-myo-inositol 1,4,5 trisphosphate [Ins(1,4,5)P(3)] has been examined on red beet (Beta vulgaris) vacuoles. Patch-clamp measurements of the vacuolar potential and current were performed on vacuoles isolated in 0.1 micromolar free Ca(2+) medium. With vacuoles clamped at +30 millivolts, the Ins(1,4,5)P(3) induced changes in current were depending on the Ca(2+) buffer strength in the external medium. The spontaneous depolarization of vacuoles in which H(+)-pumps were activated by 5 millimolar MgATP was increased from +6 to +18 millivolts by 1 micromolar Ins(1,4,5)P(3). We have interpreted our data by assuming that even with 2 millimolar EGTA to buffer Ca(2+) at 0.1 micromolar in the external medium, Ins(1,4,5)P(3) released enough Ca(2+) from the vacuole to produce an accumulation of this ion near the tonoplast. Apart from their dependency with free Ca(2+) in the cytoplasm, the electrical properties of the tonoplast could be depending on the Ins(1,4,5)P(3) and Ca(2+) buffer values in the cytoplasm. PMID- 16667548 TI - Sulfhydryl reagents and energy-linked reactions in monocot thylakoids. AB - Monofunctional maleimides have been used to covalently modify the coupling factor protein of monocot thylakoid membranes. As with dicot thylakoids, incubation of the monocot thylakoids with maleimides in the light but not in the dark results in inhibition of both ATP synthesis and hydrolysis. In the dark, sites on the gamma and epsilon subunits of maize Zea mays coupling factor 1 are modified after incubation of maize mesophyll thylakoids with the fluorescent maleimide N (anilinonaphthyl-4) maleimide. A light accessible site localized solely to the gamma subunit has also been demonstrated. In contrast to the case with dicot thylakoids (spinach [Spinacia oleracea] and pea [Pisum sativum]) treatment of monocot thylakoids (maize, barley [Hordeum vulgare], crabgrass [Digitaria sanguinalis]) with bifunctional maleimides or thiol oxidants in the light does not result in functional uncoupling, i.e the bifunctional reagents act more like energy transfer inhibitors. The lack of functional uncoupling could be due either to a failure of the reagents to cross-link key sulfhydryl residues in the gamma subunit or to the continued ability of the gamma subunit to gate proton movements through the chloroplast coupling factor complex even though its conformation has been altered by sulfhydryl reagents. PMID- 16667549 TI - Influence of Endogenous Cytokinins on Reverse Mobilization in Cotyledons of Cicer arietinum L: Reproduction of Endogenous Levels of Total Cytokinins, Zeatin, Zeatin Riboside, and Their Corresponding Glucosides. AB - The embryonic axis plays an essential role in the mobilization of the main reserves of the cotyledons of seeds of Cicer arietinum L. cv Castellana. This control by the axis of the metabolism of the storage products of the cotyledons largely takes place through the cytokinins, which are transported from the embryonic axis to the cotyledons where the mobilization of reserves begins. The principal regulatory role of the endogenous cytokinins concerns the metabolism of carbohydrates and proteins; there is less influence on lipid metabolism. However, each cytokinin seems to have a different role in the mobilization processes. The glucosides, glucosyl zeatin riboside, and glucosyl zeatin act only as storage forms of the hormones. Zeatin riboside affects mainly the mobilization of carbohydrates and has less effect on protein mobilization. Zeatin regulates both the mobilization of carbohydrates and that of proteins and is more marked in the latter case. PMID- 16667550 TI - Reduction of Iron by Leaf Extracts and Its Significance for the Assay of Fe(II) Iron in Plants. AB - "Active Fe" in plants has generally been interpreted to be ferrous iron. This study evaluated some of the commonly followed active Fe determination procedures based on Fe(II) measurements. In each of 12 species examined, leaf extracts exhibited strong reducing activity and quickly reduced any added Fe(III) with or without light. The reducing activity was attributed to ascorbic acid and phenols in the plant extracts. The reliability of the Fe(II) iron determination procedures in plants and the interpretation that active Fe is Fe(II) are questionable. PMID- 16667551 TI - Cell Wall and Extensin mRNA Changes during Cold Acclimation of Pea Seedlings. AB - During exposure to 2 degrees C, pea (Pisum sativum) seedlings cold acclimated to a killing temperature of -6 degrees C. Associated with this increase in freezing resistance was an increase in the weight of cell walls and changes in wall composition. Arabinosyl content increased by 100%, while other cell wall glycosyl residues and cellulose increased by about 20%. The cell wall hydroxyproline content increased by 80%. Arabinose and hydroxyproline are both major components of the structural cell wall glycoprotein, extensin. The increase in these components indicates that the level of extensin in the cell wall increases during cold acclimation. Northern blot analysis, using the pDC5A1 genomic clone as a probe, revealed a more than three-fold increase in total extensin mRNA during exposure to cold temperature. Specific extensin transcripts of 6.0, 4.5, 3.5, 2.6, 2.3, 1.8, and 1.5 kilobases were identified. Those at 6.0, 2.6, and 1.5 kilobases were especially promoted by low temperature treatment. The rise in extensin during cold acclimation may be regulated, at least in part, at the gene level. The possible structural role of this protein in freezing protection is discussed. PMID- 16667552 TI - Oligomeric forms of plant acetolactate synthase depend on flavin adenine dinucleotide. AB - Acetolactate synthase (ALS, EC 4.1.3.18) has been extracted and partially purified from etiolated barley shoots (Hordeum vulgare L.). Multiple forms of this enzyme were separated by gel filtration and/or anion-exchange chromatography using fast protein liquid chromatography. It could be demonstrated that these two species are in equilibrium, which strongly depends on the structural role of flavin adenine dinucleotide and pyruvate. With 50 micromolar of flavin adenine dinucleotide in the medium most of the ALS aggregates as a high molecular weight form (M(r) = 440,000), while 50 millimolar pyruvate facilitates dissociation into the smaller form (M(r) = 200,000). Data are presented to show that two enzymatically active forms are not isozymes but different oligomeric species or aggregates of the basic 58-kilodalton subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine and isoleucine or in inhibition by the sulfonylurea herbicide chlorsulfuron. Both aggregation forms show a broad pH-optimum between 6.5 and 7. Furthermore, the affinity for pyruvate and the amount of directly-formed acetoin indicate similar properties of these separated ALS forms. PMID- 16667553 TI - In Vivo Gibberellin Biosynthesis in Endosperm of Sechium edule Sw. Seeds. AB - Biosynthesis of gibberellins (GAs) was studied in vivo in endosperms of Sechium edule Sw. Exogenous ent-[(14)C]kaurene was metabolized into four major products: GA(12), GA(4), GA(7) and 16, 17-dihydro-16-hydroxy-GA(15) alcohol glucoside. Other minor metabolites were also observed including ent-kaurenol and ent kaurenal. Conversion of ent-[(14)C]kaurene to ent-kaurenol glucoside by endosperm cell-free preparations in the presence of UDPG was observed. However, the finding was not confirmed in in vivo studies and is probably artifactual. Overall evidence coming from the analysis of endogenous GAs and in vitro and in vivo biosynthetic studies are discussed in relation to the possible existence in the Sechium seeds of a different route, along with the known pathway, branching from ent-kaurene or ent-7-alpha-hydroxykaurenoic acid and this also leading to biologically active GAs. PMID- 16667554 TI - Purification and characterization of abundant secreted protein in suspension cultured pumpkin cells : abundant secreted protein may be a chitinase. AB - The abundant secreted protein with molecular weight of 32,000 was purified from the culture medium of suspension-cultured pumpkin (Cucurbita sp.) cells. Two steps, ammonium sulfate fractionation and Sepharose 6B column chromatography, were sufficient for purification to homogeneity. Antibodies against the pure protein were used to show that a protein of the same size is made by callus cells. There is considerable homology between the amino-terminal amino acid sequence of this secreted protein and chitinase isolated from tobacco (Nicotiana tabacum L.) or bean (Phaseolus vulgaris L.). PMID- 16667555 TI - Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role of cellulase in cell expansion. AB - Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose(4). xylose(3) (XG7) core. The substituted oligosaccharides XG8 (glucose(4). xylose(3). galactose) and XG9n (glucose(4). xylose(3). galactose(2)) were more effective than XG7 itself and XG9 (glucose(4). xylose(3). galactose. fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10(-4) molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of [(3)H]xyloglucan to ethanol-soluble fragments. This suggest that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products). We suggest that the promotion of midchain xyloglucan cleavage, by loosening the primary cell wall matrix, explains the promotion of growth by the oligosaccharides. PMID- 16667556 TI - Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid. AB - The activation of H(+)-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H(+)-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H(+)-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides. PMID- 16667557 TI - A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism. AB - A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degrees C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury-leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of (14)CO(2) into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury. PMID- 16667558 TI - Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg. AB - To clarify the kinetic characteristics and ionic requirements of the tonoplast H(+)-translocating inorganic pyrophosphatase (H(+)-PPiase), PPi hydrolysis and PPi-dependent H(+) transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoe daigremontiana Hamet et Perrier de la Bathie. The tonoplast H(+)-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H(+) PPiase activity was maximal in the presence of K(+) (K(50) approximately 3 millimolar), with PPi-dependent H(+) transport being more selective for K(+) than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H(+)-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg(2+) for activation. At saturating concentrations of free Mg(2+), H(+)-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi(2 ) but not Mg(2)PPi, demonstrating that MgPPi(2-) was the true substrate of the enzyme. The apparent K(m) (MgPPi(2-)) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H(+) transport (7 micromolar). Free Mg(2+) was shown to be an allosteric activator of the H(+)-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H(+) transport. Half-maximal H(+)-PPiase activity occurred at a free Mg(2+) concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg(2+). In contrast, cytosolic concentrations of K(+) and MgPPi(2-) appear to be saturating for H(+)-PPiase activity. We propose that one function of the H(+) PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H(+)-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg(2+) would be released from the MgATP(2-) complex, thereby activating the tonoplast H(+)-PPiase. PMID- 16667559 TI - Boron deficiency in cultured pine cells : quantitative studies of the interaction with ca and mg. AB - A pronounced interaction between calcium, magnesium, and boron was found in growth studies with Pinus radiata cell cultures. Quantitative isoactivity data for the interaction was analyzed in terms of selected simple and plausible theoretical models. The data was found to be consistent with a model in which a critical acceptor molecule is activated only by binding both Ca and B at separate sites; Mg competitively displaces Ca to inactivate the acceptor. It was found that B is, surprisingly, not bound strongly (K(diss) = 450 +/- 80 micromolar) and that the affinity for Ca is two orders of magnitude stronger than for Mg. Therefore only a small proportion of the acceptor will be boronated under natural conditions. Moderate levels of mannitol were found to aggravate B deficiency due to its effective removal by direct chemical complexation. At higher concentrations of mannitol (or other sugars), where osmotic contribution is significant, little B was needed to overcome growth inhibition-a result consistent with B having a primary role in cell wall biosynthesis. PMID- 16667560 TI - Chlorophyll a fluorescence and carbon assimilation in developing leaves of light grown cucumber. AB - The development of photochemical activity and carbon assimilation in light-grown cucumber (Cucumis sativus L. cv Natsusairaku) leaves was studied to determine the pattern of acquisition and its relationship to leaf growth and expansion. Measurements of chlorophyll a fluorescence showed that leaves acquire photochemical function over a period of 6 or more days, and gas exchange studies showed increases in carbon assimilation over a parallel time period. As leaves expand and mature, they undergo a sequential, three-step series of changes in fluorescence response. The initial kinetics show the absence of wholly functional quenching mechanisms. Dynamic imaging of fluorescence kinetics showed that a temporal series of changes occurred within defined areas of individual developing leaves. The spatial acquisition of photochemical activity in leaves was basipetal as is their directional expansion, development of air spaces and stomata, and the cessation of imported carbon. PMID- 16667561 TI - Patterns of chlorophyll fluorescence kinetics in relation to growth and expansion in cucumber leaves. AB - Photochemical development was studied in developing cucumber (Cucumis sativus L. cv Hokushin) leaves to determine if the spatial pattern coincided with relative growth rates of expanding leaves, intercalary cell division, or position relative to the vascular tissue. Both leaf surfaces undergo a series of similar changes in chlorophyll a fluorescence kinetics, but the upper surface more quickly achieved the characteristic response. Imaging of fluorescence showed an individual developing leaf has four regions differing in kinetics, but these regions do not coincide with areas of increasing relative growth rate. Two of these regions lie at the leaf edge and their divergent kinetics may be related to structural and physiological features present at this position. A third area with different kinetics, in the basal region of the leaf, is spatially consistent with primordial regions that are clonal during development. The correspondence between areas of clonal growth and specific fluorescence kinetics indicates that cells of common ancestry show functional uniformity. No evidence was found that the proximity of the vascular tissue influenced development of photochemical function. PMID- 16667562 TI - Seed Development in Phaseolus vulgaris L. cv Seminole: II. Precocious Germination in Late Maturation. AB - Seeds of Phaseolus vulgaris L. cv Seminole in late maturation phase germinated precociously in vitro. Germination occurred in the absence of free water after 5 days but within 24 to 48 hours in contact with water. Excised axes germinated within 12 hours and embryos by 48 hours only if supplied with water. Ethylene accelerated the germination of seeds and embryos irrespective of water availability. There was no effect of ethylene on the rate of axis germination. Ethylene was equally effective within the range 0.5 to 1000 parts per million and 1 hour exposure was fully effective. Induction of precocious germination in vivo was observed by manipulating water content inside pods or by ethylene injection, whether pods were attached to the parent plant or not. These results demonstrate the importance of endogenous regulation of water supply in suppressing precocious germination. Ethylene is identified as a powerful antagonist to the natural control. PMID- 16667563 TI - ARF-B(2): A Protein Complex that Specifically Binds to Part of the Anaerobic Response Element of Maize Adh 1. AB - Crude whole cell extracts from maize (Zea mays L.) suspension cells were examined for DNA binding proteins that specifically interact with a portion of the maize Adh 1 promoter that was previously shown to be in contact with a trans-acting factor in vivo. A 17 base pair, double-stranded oligonucleotide probe was constructed that centered around a strong in vivo dimethylsulfate footprint (B(2)) that coincides with part of the anaerobic response element (ARE). Gel retardation assays were used to characterize a major, specific DNA binding protein activity found in the crude extracts. The activity is present in both aerobic and hypoxically treated cultures and has been designated ARF-B(2) (ARE binding factor). ARF-B(2) appears to be a multicomponent complex, with a 54 kilodalton subunit termed ARF-B(2)alpha in primary contact with the target DNA. PMID- 16667564 TI - Differential Inhibition of Tonoplast H-ATPase Activities by Fluorescamine and Its Derivatives. AB - Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H(+)-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H(+) leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane. PMID- 16667565 TI - Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones. AB - Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25 degrees C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO(3) (-)/NH(4) (+) ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba x P. tremula and of the poplar hybrid Populus trichocarpa x P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not. PMID- 16667566 TI - Influence of water deficits on the abscisic Acid and indole-3-acetic Acid contents of cotton flower buds and flowers. AB - A field experiment was conducted during the summer of 1988 to test the hypothesis that water deficit affects the abscisic acid (ABA) and indole acetic acid (IAA) concentrations in cotton (Gossypium hirsutum L.) flower buds in ways that predispose young fruits (bolls) that subsequently develop from them to increased abscission rates. Water deficit had little effect on the ABA content of flower buds but increased the ABA content of flowers as much as 66%. Water deficit decreased the concentrations of free and conjugated IAA in flower buds during the first irrigation cycle but increased them during the second cycle. Flowers contained much less IAA than buds. Water deficit slightly increased the conjugated IAA content of flowers but had no effect on the concentration of free IAA in flowers. Because water deficit slightly increased the ABA content but did not decrease the IAA content of flowers, any carry-over effect of water deficit on young boll shedding might have been caused by changes in ABA but not from changes in IAA. PMID- 16667567 TI - Plastidic Isoprenoid Synthesis during Chloroplast Development : Change from Metabolic Autonomy to a Division-of-Labor Stage. AB - The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO(2) --> pyruvate --> acetyl-coenzyme A (C(3) --> C(2)) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C(3) --> C(2) metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO(2) fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH(14)CO(3) decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl coenzyme A. PMID- 16667568 TI - An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings. AB - Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H(+)-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H(+)-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg(2+)-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H(+)-translocating PPase. PMID- 16667569 TI - Extensin and Phenylalanine Ammonia-Lyase Gene Expression Altered in Potato Tubers in Response to Wounding, Hypoxia, and Erwinia carotovora Infection. AB - Potato (Solanum tuberosum L.) tubers are susceptible to infection by Erwinia carotovora, causal agent of bacterial soft rot, when wounded and subjected to wet, hypoxic environments. The expression of two putative plant defense genes, extensin and phenylalanine ammonia-lyase (PAL), was examined by monitoring their respective mRNA levels and cell wall hydroxyproline levels in tuber tissues under various conditions leading to susceptibility or resistance and after inoculation with E. carotovora in order to assess the possible roles of these genes and their products in this plant-pathogen interaction. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels accumulated markedly in response to wounding and subsequent aerobic incubation. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels decreased in response to wounding and subsequent anaerobic incubation; these changes were correlated with high susceptibility of tuber tissue to E. carotovora infection. Inoculation of wound sites with E. carotovora caused some additional accumulation of the wound-regulated extensin and PAL mRNAs under certain aerobic conditions, but never under anaerobic conditions. PMID- 16667570 TI - Expression of the Heat Shock Response in a Tomato Interspecific Hybrid Is Not Intermediate between the Two Parental Responses. AB - While it is apparent that the heat shock response is ubiquitous, variabilities in the nature of the heat shock response between closely related species have not been well characterized. The heat shock response of three genotypes of tomato, Lycopersicon esculentum, Lycopersicon pennellii, and the interspecific sexual hybrid was characterized. The two parental genotypes differed in the nature of the heat shock proteins synthesized; the speciesspecific heat shock proteins were identified following in vivo labeling of leaf tissue with [(35)S]methionine and cysteine. The duration of, and recovery from, heat shock varied between the two species: L. esculentum tissue recovered more rapidly and protein synthesis persisted longer during a heat shock than in the wild species, L. pennellii. Both species induced heat shock protein synthesis at 35 degrees C and synthesis was maximal at 37 degrees C. The response of the F1 to heat shock was intermediate to the parental responses for duration of, and recovery from, heat shock. In other aspects, the response of the F1 to heat shock was not intermediate to the parental responses: the F1 induced only half of the L. esculentum specific heat shock proteins, and all of the L. pennellii specific heat shock proteins. A discussion of the inheritance of the regulation of the heat shock response is presented. PMID- 16667571 TI - Compartmentation of solutes and water in developing sugarcane stalk tissue. AB - Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from -0.6 to -1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from -1.0 to approximately -2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose. PMID- 16667572 TI - Location of transported auxin in etiolated maize shoots using 5-azidoindole-3 acetic Acid. AB - A study was undertaken using the photoaffinity labeling agent, tritiated 5 azidoindole-3-acetic acid ([(3)H],5-N(3)IAA), to identify cells in the etiolated maize (Zea mays L.) shoot which transport auxin. Transport of [(3)H],5-N(3)IAA was shown to be polar, inhibited by 2,3,5-triiodobenzoic acid (TIBA) and essentially freely mobile. There was no detectable radiodecomposition of [(3)H],5 N(3)IAA within tissue kept in darkness for 4 hours. Shoot tissue which had taken up [(3)H],5-N(3)IAA was irradiated with ultraviolet light to covalently fix the photoaffinity labeling agent within cells that contained it at the time of photolysis. Subsequent microautoradiography showed that all cells contained radioactivity; however, the amount of radioactivity varied among different cell types. Epidermal cells contained the most radioactivity per area, approximately twofold more than other cells. Parenchyma cells in the mature stelar region contained the next largest amount and cortical cells, sieve tube cells, tracheary cells, and all cells in the leaf base contained the least amount of the radioactive label. Two observations suggest that the auxin within the epidermal cells is transported in a polar manner: (a) the amount of auxin in the epidermal cells is greatly reduced in the presence of TIBA, and (b) auxin accumulates on the apical side of a wound in the epidermis and is absent on the basal side. While these results indicate that auxin in the epidermis is polarly transported, this tissue cannot be the only pathway since the epidermis is only a small fraction of the shoot volume. The greater than twofold difference between the concentration of auxin in the epidermal and subtending cells demonstrates that physiological differences in the concentration of auxin can occur between adjacent cells. PMID- 16667573 TI - Arrested Embryos from the bio1 Auxotroph of Arabidopsis thaliana Contain Reduced Levels of Biotin. AB - The bio1 auxotroph of Arabidopsis thaliana is a recessive embryonic lethal that forms normal plants in the presence of biotin. The purpose of this study was to determine whether aborted seeds produced by heterozygous plants grown without vitamin supplements contained reduced levels of biotin. Two methods were used to determine the biotin content of mutant and wild-type tissues: streptavidin binding in microtiter plates and growth of the biotin-requiring bacterium Lactobacillus plantarum. Total biotin was measured in extracts prepared from immature seeds prior to desiccation. Aborted seeds produced by heterozygous (bio1/BIO1) plants contained some biotin in the maternal seed coat but virtually no detectable biotin in the arrested embryo. This lack of biotin was not observed in arrested embryos from other mutants with similar patterns of abnormal development. These results are consistent with the model that bio1 tissues are defective in biotin synthesis. The alternative model of increased degradation is inconsistent with the recessive nature of the mutation and the ability of rescued plants to continue growing for several weeks following removal of supplemental biotin. PMID- 16667574 TI - A mutant gene that increases gibberellin production in brassica. AB - A single gene mutant (elongated internode [ein/ein]) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A(3) (GA(3)) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA(1) and GA(3) were estimated by gas chromatography-selected ion monitoring using [(2)H]GA(1), as a quantitative internal standard and at day 14 were 1.5- and 12.1 fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA(20) and GA(1), and the rate of GA(19) metabolism were simultaneously analyzed at day 7 by feeding [(2)H(2)]GA(19) and measuring metabolites [(2)H(2)]GA(20) and [(2)H(2)]GA(1) and endogenous GA(20) and GA(1), with [(2)H(5)]GA(20) and [(2)H(5)]GA(1) as quantitative internal standards. Levels of GA(1) and GA(20) were 4.6- and 12.9 fold higher, respectively, and conversions to GA(20) and GA(1) were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA(1) biosynthesis in ein, the conversion of [(3)H]GA(20) to [(3)H]GA(1) was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA(1) biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A(1) and A(3). The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation. PMID- 16667575 TI - Potassium stimulation of corn root plasmalemma ATPase : I. Hydrolytic activity of native vesicles and purified enzyme. AB - Potassium stimulation of the plasmalemma (Zea mays L. var Mona) was studied by using a constant ionic strength to prevent indirect stimulation by the electrostatic effect of K(+) salts. The transmembrane electrochemical H(+) gradient was eliminated by using gramicidin. In these conditions, K(+) stimulation was attributable to a direct effect of the cation on plasmalemma proteins. We used both native vesicles isolated on a sucrose cushion, and solubilized and purified ATPase from phase-partitioned plasmalemma, according to the method of T. Nagao, W. Sasakawa, and T. Sugiyama ([1987] Plant Cell Physiol 28: 1181-1186). The purified enzyme had a high specific activity (15 micromoles per minute per milligram protein), but was only about 20% stimulated by K(+). In both preparations, potassium (in the range around 1 millimolar) specifically decreased two-fold the vanadate inhibition constant, and increased the maximum rate of ATP hydrolysis. In plasmalemma vesicles, the Eadie-Scatchard graph of the K(+)-dependent ATPase activity as a function of K(+) concentration was linear only at constant ionic strength. The purified ATPase preparation appeared as two closely spaced bands in the 100 kilodalton region with isoelectric point about 6.5. Nevertheless, this biochemical heterogeneity seems unlikely to be related to K(+) stimulation, since K(+) modified neither the pH optimum of the activity (pH 6.5) nor the monophasic kinetics of the vanadate inhibition, in both native plasmalemma and purified enzyme preparation. PMID- 16667576 TI - Potassium Stimulation of Corn Root Plasmalemma ATPase : II. H-Pumping in Native and Reconstituted Vesicles with Purified ATPase. AB - The stimulation by K(+) of the initial rate of H(+)-pumping by ATPase was studied in native plasmalemma (Zea mays L. var Mona) vesicles and in reconstituted vesicles with enzyme purified on a glycerol gradient. In reconstituted vesicles, a very high H(+)-pumping rate (200,000% quenching per minute per milligram protein) was obtained with 9-amino-6-chloro-2-methoxyacridine provided that the pump was short-circuited by K(+)-valinomycin. A constant ionic strength was used to prevent indirect stimulation by the electrostatic effects of K(+) salts. Indirect stimulation of H(+)-pumping by the short-circuiting effect of internal K(+), could be abolished by using the permeant anions NO(3) (-) and Br(-) in native, but not in reconstituted vesicles. In both materials, half-stimulation of the H(+)-pumping by K(+) was observed at about 5 millimolar. The same stimulation was obtained when K(+) was present only in the external solution or when it was present both outside and inside the vesicles. It was concluded that the stimulating effect of K(+) on the H(+)-pumping evidenced in these experiments on both native and reconstituted vesicles was due to a direct effect of the cation on the cytoplasmic face of the ATPase. These results are discussed within the context of the hypothesis of an active K(+) transport driven by the ATPase through a direct H(+)/K(+) exchange mechanism. PMID- 16667577 TI - In Vitro Selection of Calli Resistant to a Triazole Cytochrome-P-450-Obtusifoliol 14-Demethylase Inhibitor from Protoplasts of Nicotiana tabacum L. cv Xanthi. AB - The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4 Chlorophenyl)-3-phenyl-1-(1H-1,2,4- triazol-1-yl)-2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P-450-obtusifoliol 14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell. PMID- 16667578 TI - Photoregulation of beta-Tubulin mRNA Abundance in Etiolated Oat and Barley Seedlings. AB - The effect of light on the abundance of beta-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L. seedlings. Slot blot analysis employing an oat beta-tubulin cDNA clone was used to measure beta-tubulin mRNA levels. White light induced a 45% decrease in oat beta-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in beta-tubulin mRNA levels in oats and barley, respectively. Recovery of beta tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat beta-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in beta-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the beta-tubulin genes. PMID- 16667579 TI - Isolation of transcriptionally regulated root-specific genes from tobacco. AB - Four root-specific cDNA clones and their corresponding genomic clones have been isolated from tobacco (Nicotiana tabacum) by a novel differential hybridization procedure. The genes are expressed at high levels in roots and are not detectable in leaves. The cDNAs are encoded by small gene families of two to four members. Transcription experiments with isolated nuclei demonstrate that the genes are, at least in part, transcriptionally regulated. Constructions in which 1.4 kilobase pairs of 5' flanking region of one of the root-specific genes was fused to a reporter gene (beta-glucuronidase) were transformed into tobacco. beta Glucuronidase activity in transgenic plants was localized in the roots, demonstrating the cis-acting sequences regulating root-specific expression are present on the 5' flanking region. PMID- 16667580 TI - Steady-State and Oscillating Photosynthesis by a Starchless Mutant of Nicotiana sylvestris. AB - The photosynthetic characteristics of wild type Nicotiana sylvestris (Speg. et Comes) were compared with those of a ;starch-less' mutant NS458 that contains a defective plastid phosphoglucomutase (EC 2.1.5.1) (KR Hanson, NA McHale [1988] Plant Physiol 88: 838-844). The steady-state rate of net CO(2) assimilation (A) was studied as a function of [CO(2)], [O(2)], irradiance, and temperature. At 30 degrees C with saturating light and [CO(2)] and low [O(2)], A for the mutant was half that for the wild type, whereas in normal air it was 90%. The irradiance and [CO(2)] at low [O(2)] required for saturation were lower than the values for the wild type. At 2000 microbars CO(2), 30 degrees C, and saturating irradiance A for both the mutant and wild type was stimulated on going from 4 to 25% O(2) by at least 13%. Slow oscillations in A were readily induced with the mutant but not the wild type, provided irradiance and [CO(2)] were saturating and [O(2)] was low. The period, which was about 5 minutes at 30 degrees C and decreased by about 0.67 minutes per degree, was an order of magnitude slower than periods reported for other plants at corresponding temperatures. To achieve the full oscillation amplitude both irradiance and [CO(2)] had to exceed the minimal levels for steady state saturation. The slowness and duration of the oscillations and the metabolic simplification introduced by deleting starch synthesis makes the mutant especially suitable for investigating the regulatory processes that generate such oscillations. PMID- 16667581 TI - Carbon metabolism in spinach leaves as affected by leaf age and phosphorus and sulfur nutrition. AB - Spinach (Spinacea oleracea) plants were grown either continuously on complete nutrient solutions or for 2 weeks on media deficient in phosphate or sulfate. To characterize leaf carbohydrate metabolism, levels of phosphorylated intermediates, activities of enzymes involved in photosynthetic carbon metabolism, contents of soluble and acid hydrolyzable sugars were measured in leaves differing in age and mineral status and related to leaf rates of photosynthesis and assimilate partitioning. Concentrations of metabolites particularly those which are preferentially compartmented in the cytosol decreased from young to old leaves and were lowest in old phosphate starved leaves. Nutrient deficiency showed comparable effects on stromal and cytosolic intermediates. Whole leaf ATP to ADP ratios were dependent on the growth regime, but did not much change with leaf age. The assimilatory force increased in all leaves suffering from mineral deficiency; the assimilatory force was low when photosynthesis was high and vice versa. Sugars accumulated although enzyme activities were decreased under deficiency. The results show that growth of P- and S-starved plants is not limited by photosynthetic reactions. PMID- 16667582 TI - Effect of acetylene on root respiration and acetylene reducing activity in nodulated soya bean. AB - Acetylene decreased root and nodule respiration, as measured by CO(2) evolution of nodulated or non-nodulated Glycine max. An inhibition of 25 to 35% in 15 to 30 minutes occurred when 13% C(2)H(2) was introduced in the gas flux which aerated the root nutrient solution. When the light intensity was doubled to 800 microeinsteins per square meter per second, the inhibition increased to 50% and nodule acetylene reduction activity was inhibited 50%. PMID- 16667583 TI - Purification and Properties of Arginase from Soybean, Glycine max, Axes. AB - Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K(m) value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine. PMID- 16667584 TI - Protein methylation in pea chloroplasts. AB - The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [(3)H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. One is a polypeptide with a molecular mass of 64 kD, a second has an M(r) of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyl-linkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [(3)H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [(3)H]methyl group. PMID- 16667585 TI - Phosphorylation of Plant H2A Histones. AB - Phosphorylation of wheat (Triticum aestivum) and alfalfa (Medicago sativa) H2A histone variants was examined during early seedling growth. The C-terminal regions of wheat H2A variants contain multiple S-P tetrapeptides (serine-proline adjacent to a pair of basic amino acids) which resemble known phosphorylation sites in histones from other species. Phosphorylation of nucleosomal core histones was assessed by autoradiography of proteins labeled in vivo with (32)Pi and resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylation sites were mapped by cleaving in vivo labeled H2A variants with N bromosuccinimide. Essentially all phosphorylation of nucleosomal core histones in wheat and alfalfa seedlings occurred within the C-terminal peptides obtained from wheat and alfalfa H2A variants. A hypothesis accounting for the presence of large H2A and H2B histone variants in plants and phosphorylation of plant H2A C terminal regions is proposed. The utility of S-P tetrapeptides for modulation of DNA-protein interactions is discussed. PMID- 16667587 TI - Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid. AB - The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C(3) pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation. PMID- 16667586 TI - Molecular Cloning and Expression of cor (Cold-Regulated) Genes in Arabidopsis thaliana. AB - We have previously shown that changes in gene expression occur in Arabidopsis thaliana. L. (Heyn) during cold acclimation (SJ Gilmour, RK Hajela, MF Thomashow [1988] Plant Physiol 87: 745-750). Here we report the isolation of cDNA clones of four cold-regulated (cor) genes from Arabidopsis and examine their expression in response to low temperature, abscisic acid (ABA), water stress, and heat shock. The results of Northern analysis indicated that the transcript levels for the four cor genes, represented by clones pHH7.2, pHH28, pHH29, and pHH67, increased markedly between 1 and 4 hours of cold treatment, reached a maximum at about 8 to 12 hours, and remained at elevated levels for as long as the plants were kept in the cold (up to 2 weeks). Returning cold acclimated plants to control temperature resulted in the levels of the cor transcripts falling rapidly to those found in nonacclimated plants; this occurred within 4 hours for the transcripts represented by pHH7.2 and pHH28, and 8 hours for those represented by pHH29 and pHH67. Nuclear run-on transcription assays indicated that the temperature regulated expression of the cor genes represented by pHH7.2, pHH28, and pHH29 was controlled primarily at the posttranscriptional level while the cor gene represented by pHH67 was regulated largely at the transcriptional level. Northern analysis also indicated that the levels of cor gene transcripts increased in response to both ABA application and water stress, but not to heat shock. The possible significance of cor genes being regulated by both low temperature and water stress is discussed. PMID- 16667588 TI - Competitive Al Inhibition of Net Mg Uptake by Intact Lolium multiflorum Roots : II. Plant Age Effects. AB - Rhizotoxicity of Al is more pronounced in younger plants. Effects of Al on nutrient uptake by plants of different age are poorly understood. The depletion technique was used to monitor net Mg(2+) uptake from nutrient solutions by intact 15- and 35-day-old plants of two ryegrass (Lolium multiflorum Lam.) cultivars. Lowering the pH from 6.0 to 4.2 decreased the maximum net ion influx without affecting K(m). Aluminum at 6.6 micromolar Al(3+) activity increased K(m) indicating competitive inhibition. The effects of pH and 6.6 micromolar Al(3+) on net Mg(2+) uptake were much larger in 15- than in 35-day-old plants. Aluminum at 26 micromolar Al(3+) activity competitively inhibited net Mg(2+) uptake by 35-day old plants, while causing time- and external Mg(2+) activity-dependent net Mg(2+) efflux from 15-day-old plants. The equilibrium constant (K(i)) of a reversible combination of postulated plasmalemma Mg(2+) transporter and Al(3+) was calculated to be 2 and 5 micromolar Al(3+) activity for 15-day-old plants of Wilo and Gulf ryegrass, respectively, and 21 micromolar Al(3+) activity for 35-day-old plants of both cultivars. The Al(3+)-mediated increase in K(m) was larger for 15 day-old plants of the Al-sensitive cultivar ;Wilo' than of the more Al-tolerant cultivar ;Gulf,' while Al(3+) affected 35-day-old plants of both cultivars to the same extent. PMID- 16667589 TI - Effects of (22S,23S)-Homobrassinolide and Related Compounds on Membrane Potential and Transport of Egeria Leaf Cells. AB - (22S,23S)-Homobrassinolide was tested for its effect on the electric cell potential, proton extrusion, ferricyanide reduction, and amino acid and sucrose uptake of leaves of Egeria densa Planchon. In the light, (22S,23S) homobrassinolide and its derivative, 2alpha-3alpha-dihydroxy-5alpha-stigmast-22 en-6-one, were similar to each other and similar to fusicoccin in causing hyperpolarization and proton extrusion, whereas stigmasterol was less effective. In darkness, the three sterols showed comparable effects. (22S,23S) Homobrassinolide slightly stimulated ferricyanide reduction and promoted uptake of sucrose and alpha-aminoisobutyric acid. The results are compatible with a stimulation of an electrogenic proton pump mechanism at the plasmalemma by (22S,23S)-homobrassinolide. PMID- 16667590 TI - Measuring intracellular ca levels in plant cells using the fluorescent probes, indo-1 and fura-2 : progress and prospects. AB - Recent advances in the development of methods for measuring cytoplasmic Ca(2+) levels in higher plant cells are discussed. Emphasis is placed on the new generation of Ca(2+)-sensitive fluorescent dyes particularly fura-2 and indo-1. These dyes offer many advantages for the measurement of cytosolic Ca(2+) levels. They can be introduced into cells in a nonintrusive manner, their K(d) for Ca(2+) matches plant cell cytoplasmic Ca(2+) levels, and shifts in their emission (indo 1) or excitation (fura-2) spectra following Ca(2+) binding permit accurate quantitation of Ca(2+) activities. Examples of cytoplasmic Ca(2+) levels measured in plants with fura-2 and indo-1 are presented, and the prospects for applying more advanced technologies to fluorescent dye measurement are discussed. PMID- 16667591 TI - Stabilization of nitrate reductase in maize roots by chymostatin. AB - Nitrate reductase (NR) in maize (Zea mays cv W64A x W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO(-) (2) produced per hour per gram fresh weight was measured at 4 days). PMID- 16667592 TI - Effects of CO(2) Concentration during Growth on Fatty Acid Composition in Microalgae. AB - The degree of unsaturation of fatty acids was higher in Chlorella vulgaris 11h cells grown with air (low-CO(2) cells) than in the cells grown with air enriched with 2% CO(2) (high-CO(2) cells). The change in the ratio of linoleic acid to alpha-linolenic acid was particularly significant. This change of the ratio was observed in four major lipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylcholine, and phosphatidylethanolamine). The relative contents of lipid classes were essentially the same both in high CO(2) and low-CO(2) cells. After high-CO(2) cells were transferred to low CO(2) condition, total amount of fatty acids remained constant but the relative content of alpha-linolenic acid increased during a 6-hour lag phase in growth with concomitant decreases in linoleic and oleic acids. When low-CO(2) cells were transferred to high CO(2) condition, total amount of fatty acids and relative content of oleic acid increased significantly. The amount of alpha-linolenic acid remained almost constant, while the amounts of palmitic, oleic, and linoleic acids increased. Similar, but smaller, changes in fatty acid compositions were observed in two species of green algae Chlamydomonas reinhardtii and Dunaliella tertiolecta. However, no difference was found in Euglena gracilis, Porphyridium cruentum, Anabaena variabilis, and Anacystis nidulans. PMID- 16667593 TI - A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants. AB - We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed. PMID- 16667594 TI - Salinity Induced Limitations on Photosynthesis in Prunus salicina, a Deciduous Tree Species. AB - The response of photosynthetic CO(2) assimilation to salinization in 19 year old Prunus salicina was evaluated under field conditions for a 3 year period. The observed decline in CO(2) assimilation capacity was apparently related to increasing leaf chloride (Cl(-)) content, and independent of changes in leaf carbohydrate status. The response of net CO(2) assimilation (A) to leaf intercellular CO(2) partial pressure (C(i)) indicated that the reduction in the capacity for A with Cl(-) was not the result of decreased stomatal conductance but a consequence of nonstomatal inhibition. The nonstomatal limitations to CO(2) assimilation capacity, as determined by the response of A to C(i) and biochemical assay, were related to a decline in the activity of ribulose 1,5-bisphosphate carboxylase (Rubpcase) and the pool size of triose phosphate, ribulose 1,5 bisphosphate (Rubp) and phosphoglycerate with increasing salinity. Lack of agreement between the initial slope of the A to C(i) response curve and Rubpcase activity suggests the occurrence of heterogeneous stomatal apertures with the high salinity treatment (28 millimolar). Prolonged exposure to chloride salts appeared to increase the Rubp or Pi regeneration limitation, decrease Rubpcase activity and reduce leaf chlorophyll content. Observed changes in the biochemical components of CO(2) fixation may, in turn, affect total leaf carbohydrates, which also declined with time and salinity. The reduction in Rubpcase activity was apparently a consequence of a reduced Rubpcase protein level rather than either a regulatory or inhibitory effect. PMID- 16667595 TI - Molecular, Kinetic, and Immunological Properties of the 6-Phosphofructokinase from the Green Alga Selenastrum minutum: Activation during Biosynthetic Carbon Flow. AB - The ATP:d-fructose-6-phosphate 1-phosphotransferase (PFK) from Selenastrum minutum was purified to homogeneity. The purified plastid enzyme had a specific activity of 180 micromoles per milligram of protein per minute. It is a homomer with a subunit molecular weight of 70,000. The smallest enzymatically active form of the protein is a homotetramer of 280,000 daltons. The enzyme can, however, aggregate into different active forms, the largest of which has a molecular weight of more than 6 x 10(6). The pH optimum, regardless of aggregation state, is 7.25. The enzyme exhibits sigmoidal kinetics with respect to fructose-6 phosphate and hyperbolic kinetics with respect to ATP. Phosphate changes the sigmoidal fructose-6-phosphate saturation kinetics to hyperbolic. Phosphoenolpyruvate, 3-phosphoglycerate, 2-oxoglutarate, malate, citrate and ATP all inhibit the enzyme. The ratios of phosphoenolpyruvate and/or 3-PGA to phosphate are probably the most important factors regulating PFK activity in vivo. The enzyme cross-reacts with several antisera against both cytosolic and plastidic PFKs as well as against native potato pyrophosphate dependent phosphofructokinase suggesting that the algal PFK represents an evolutionarily primitive form. PMID- 16667596 TI - Analysis of the state of posttranslational calmodulin methylation in developing pea plants. AB - A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [(3)H]methyl S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase (DM Roberts et al. [1986] J Biol Chem 261: 1491-1494) raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity. PMID- 16667597 TI - Growth under Red Light Enhances Photosystem II Relative to Photosystem I and Phycobilisomes in the Red Alga Porphyridium cruentum. AB - Acclimation of the photosynthetic apparatus to light absorbed primarily by photosystem I (PSI) or by photosystem II (PSII) was studied in the unicellular red alga Porphyridium cruentum (ATCC 50161). Cultures grown under green light of 15 microeinsteins per square meter per second (PSII light; absorbed predominantly by the phycobilisomes) exhibited a PSII/PSI ratio of 0.26 +/- 0.05. Under red light (PSI light; absorbed primarily by chlorophyll) of comparable quantum flux, cells contained nearly five times as many PSII per PSI (1.21 +/- 0.10), and three times as many PSII per cell. About 12% of the chlorophyll was attributed to PSII in green light, 22% in white light, and 39% in red light-grown cultures. Chlorophyll antenna sizes appeared to remain constant at about 75 chlorophyll per PSII and 140 per PSI. Spectral quality had little effect on cell content or composition of the phycobilisomes, thus the number of PSII per phycobilisome was substantially greater in red light-grown cultures (4.2 +/- 0.6) than in those grown under green (1.6 +/- 0.3) or white light (2.9 +/- 0.1). Total photosystems (PSI + PSII) per phycobilisome remained at about eight in each case. Carotenoid content and composition was little affected by the spectral composition of the growth light. Zeaxanthin comprised more than 50% (mole/mole), beta-carotene about 40%, and cryptoxanthin about 4% of the carotenoid pigment. Despite marked changes in the light-harvesting apparatus, red and green light-grown cultures have generation times equal to that of cultures grown under white light of only one third the quantum flux. PMID- 16667598 TI - Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues. AB - An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K(m) values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP(+) in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP(+) oxidoreductase. The effect of NaCl and MgCl(2) concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin NADP(+) oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues. PMID- 16667599 TI - Fructan metabolism in wheat in alternating warm and cold temperatures. AB - The objective of this research was to develop a system in which the direction of fructan metabolism could be controlled. Three-week-old wheat seedlings (Triticum aestivum L. cv Caldwell) grown at 25 degrees C were transferred to cold temperature (10 degrees C) to induce fructan synthesis and then were transferred to continuous darkness at 25 degrees C after defoliation and fructan degradation monitored. The total fructan content increased significantly 1 day after transferring from 25 degrees C to 10 degrees C in both leaf blades and the remainder of the shoot tissue, 90% of which was leaf sheath tissue. Leaf sheaths contained higher concentrations of fructan and greater portions of high molecular weight fructan than did leaf blades. Fructan content in leaf sheaths declined rapidly and was gone completely within 48 hours following transfer to 25 degrees C in darkness. In leaf blades the invertase activity fluctuated during cold treatment. The activity of sucrose:sucrose fructosyl transferase increased markedly during cold treatment, while fructan hydrolase activity decreased slightly. In leaf sheaths, however, the activity of invertase decreased rapidly upon transfer to cold temperature and remained low. Trends in sucrose:sucrose fructosyl transferase and hydrolase activity in sheaths were the same as those of leaf blades. Sheath invertase and hydrolase activity increased when plants were transferred back to darkness at 25 degrees C, while sucrose:sucrose fructosyl transferase activity decreased. These results indicate that changing leaf sheath temperature can be utilized to control the direction of fructan metabolism and thus provide a system in which the synthesis or degradation of fructan can be examined. PMID- 16667600 TI - Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana. AB - Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue. PMID- 16667601 TI - Abscisic (ABA)-Aldehyde Is a Precursor to, and 1',4'-trans-ABA-Diol a Catabolite of, ABA in Apple. AB - Previous (18)O labeling studies of abscisic acid (ABA) have shown that apple (Malus domestica Borkh. cv Granny Smith) fruits synthesize a majority of [(18)O]ABA with the label incorporated in the 1'-hydroxyl position and unlabeled in the carboxyl group (JAD Zeevaart, TG Heath, DA Gage [1989] Plant Physiol 91: 1594-1601). It was proposed that exchange of (18)O in the side chain with the medium occurred at an aldehyde intermediate stage of ABA biosynthesis. We have isolated ABA-aldehyde and 1'-4'-trans-ABA-diol (ABA-trans-diol) from (18)O labeled apple fruit tissue and measured the extent and position of (18)O incorporation by tandem mass spectrometry. (18)O-Labeling patterns of ABA aldehyde, ABA-trans-diol, and ABA indicate that ABA-aldehyde is a precursor to, and ABA-trans-diol a catabolite of, ABA. Exchange of (18)O in the carbonyl of ABA aldehyde can be the cause of loss of (18)O from the side chain of [(18)O]ABA. Results of feeding experiments with deuterated substrates provide further support for the precursor-product relationship of ABA-aldehyde --> ABA --> ABA-trans diol. The ABA-aldehyde and ABA-trans-diol contents of fruits and leaves were low, approximately 1 and 0.02 nanograms per gram fresh weight for ABA-aldehyde and ABA trans-diol, respectively, while ABA levels in fruits ranged from 10 to 200 nanograms per gram fresh weight. ABA biosynthesis was about 10-fold lower in fruits than in leaves. In fruits, the majority of ABA was conjugated to beta-d glucopyranosyl abscisate, whereas in leaves ABA was mainly hydroxylated to phaseic acid. Parallel pathways for ABA and trans-ABA biosynthesis and conjugation in fruits and leaves are proposed. PMID- 16667602 TI - Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport. AB - The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. PMID- 16667603 TI - Reexamination of the Acid growth theory of auxin action. AB - Some crucial arguments against the acid growth theory of auxin action (U Kutschera, P Schopfer [1985] Planta 163: 483-493) have been reinvestigated by simultaneous measurements of proton fluxes and growth of maize (Zea mays L.) coleoptiles. Special care was taken to obtain a mild, effective, and reproducible abrasion of the cuticle. Proton secretion rates were determined in a computer controlled pH-stat. In some experiments, equilibrium pH was measured. Growth rates were determined simultaneously in the same vessel using a transducer-type auxanometer. It was found that (a) the timing of auxin and fusicoccin-induced (FC) proton secretion and growth matches well, (b) the equilibrum external pHs in the presence of IAA and FC are lower than previously recorded and below the so called ;threshold-pH,' (c) neutral or alkaline unbuffered solutions partially inhibit FC and IAA-induced growth in a similar manner, (d) the action of pH, FC, and IAA on growth are not additive. It is concluded that the acid-growth-theory correctly describes incidents taking place in the early phases of auxin-induced growth. PMID- 16667604 TI - Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System. AB - The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with (45)Ca(2+) through the action of the plasma membrane Ca(2+)-ATPase. While the Ca(2+)-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC(50)] = 70 micromolar) and W-7 (EC(50) = 100 micromolar), but to a much lesser extent, W-5 (EC(50) = 500 micromolar) led to a rapid efflux of (45)Ca(2+) from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La(3+), but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in (45)Ca(2+) in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca(2+)-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca(2+) from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC(50)-values of these compounds for mobilizing membrane-associated Ca(2+) (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca(2+) release from Ca(2+)-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca(2+) channel in the plasma membrane of C. communis. The channel is obtained in a Ca(2+)-inactivated state after preparation and Ca(2+)-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca(2+)-mobilizing effect of these compounds. The activated Ca(2+) channel is La(3+) sensitive and, in the cell, would allow for passage of Ca(2+) into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca(2+) influx, displaying channel properties, in a higher plant. PMID- 16667605 TI - Effect of pO(2) on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.). AB - Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range of pO(2) (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N(2) fixation were largely unaffected by pO(2) from 10 to 40%. Decrease in fixation at pO(2) below 5% was due to lower nodulation and nodule mass and, at pO(2) above 60%, to a fall in specific N(2)-fixing activity of nodules. Root:shoot ratios were significantly lower at pO(2) below 2.5%. The effect of pO(2) on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supra- and subambient extremes of O(2). H(2) evolution showed a sharp optimum at 20% O(2) but was at most 4% of total nitrogenase activity. The ratio of CO(2) evolved to substrate (C(2)H(2)+H(+)) reduced by crown root nodulation zones was constant (6 moles CO(2) per mole substrate reduced) from 2.5 to 60% O(2) but at levels below 2.5 and above 80% O(2) reached values between 20 and 30 moles CO(2) per mole substrate reduced. Effects of long term growth with nonambient pO(2) on adaptation and efficiency of functioning of nodules are discussed. PMID- 16667606 TI - Effect of pO(2) during Growth on the Gaseous Diffusional Properties of Nodules of Cowpea (Vigna unguiculata L. Walp.). AB - Adaptations of nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium CB 756) to growth in pO(2) ranging from 1 to 80% O(2) (volume/volume) involved both readily reversible mechanisms of adjustment and more stable alterations which together resulted in nodules with widely ranging resistance to diffusion of gases. Those grown in subambient pO(2) (1-5% O(2) were altered such that rapid diffusional adjustment was unable to prevent irreversible loss of nitrogenase on their transfer to higher levels of O(2). Those cultured in 80% had adapted to over-supply of O(2) such that their transfer to lower levels of O(2) limited both nitrogenase and respiratory CO(2) release. There was also some evidence for ;protective respiration.' Measurement of diffusional properties based on gas exchange kinetics indicated that gaseous permeability values for nodules from 5 to 40% O(2) were relatively constant around 20 x 10(-3) millimeters per second, while those for nodules from 1% O(2) were as high as 67.7 x 10(-3) millimeter per second and from 80% as low as 6.8 x 10(-3) millimeters per second. Estimates of the thickness of the diffusion barrier ranged from 7.5 micrometers for nodules from 1% O(2) to 71.9 micrometers in those from 80% O(2). PMID- 16667607 TI - Cross-Resistance of a Chlorsulfuron-Resistant Biotype of Stellaria media to a Triazolopyrimidine Herbicide. AB - A biotype of Stellaria media (L.) Vill. has been identified that is highly resistant to the herbicide chlorsulfuron. Resistance is due to an altered acetolactate synthase (ALS) that is much less sensitive to chlorsulfuron than the ALS from the susceptible (S) biotype. The S biotype was extremely sensitive to D489 (N-[2,6-dichlorophenyl]-5,7-dimethyl-1,2,4-triazolo[1,5a] pyrimidine-2 sulfonamide), a member of a new class of triazolopyrimidine herbicides, while the chlorsulfuron-resistant biotype exhibited complete cross-resistance at both the whole plant and enzyme levels. ALS activity of the S biotype was reduced by approximately 90% in the presence of 0.1 micromolar D489, while that of the R biotype was reduced by less than 10%. This result suggests that the two herbicides share a common binding site on ALS. Only very slight cross-resistance at the ALS level was found to imazamethabenz, an imidazolinone herbicide. PMID- 16667608 TI - Effect of potato seed-tuber age on plant establishment and amelioration of age linked effects with auxin. AB - Studies were conducted to characterize the effect of advanced potato (Solanum tuberosum L.) seed-tuber age on plant growth potential and whole-plant morphology. Plant growth from single-eye seedcores from 5- to 18-month old ;Russet Burbank' seedtubers was compared. Loss in apical dominance was apparent with advanced age. On a per-core basis, the amount of plant dry weight was equal for the two ages at 30 days from planting. However, individual plants from older cores displayed reduced shoot, root and leaf dry weights, leaf area, and leaf number. These effects reflected altered dry-matter partitioning and contributed to an overall change in plant morphology with advanced age. On a total seedcore basis, relative growth rate of plants from older seedcores was greater than that from younger seedcores over the 30 day growth interval. Leaf area ratio was also greater for plants from the older seedcores; however, unit leaf rate was not affected by tuber age and plants from both young and old seedcores assimilated dry matter at the same rate. Age-induced differences in growth indices reflected differences in the degree of plant differentiation over the interval of study. Treating younger seedcores with 1-naphthaleneacetic acid (NAA) prior to planting inhibited overall plant growth. In older cores, NAA stimulated root growth, restored apical dominance, decreased leaf number per plant, and increased average leaf area per leaf. In short, NAA altered the morphology of plants growing from older seedcores to more closely resemble that of plants growing from younger seedcores. While auxin significantly altered plant form, vigor of plants from older seedcores was not fully restored by auxin treatment, indicating that age reduced vigor of potato seed-tubers is not solely mediated by auxin. PMID- 16667609 TI - Physiological Characterization of a Single-Gene Mutant of Pisum sativum Exhibiting Excess Iron Accumulation: I. Root Iron Reduction and Iron Uptake. AB - Root systems of mutant (E107) and parental (cv ;Sparkle') Pisum sativum genotypes were studied to determine the basis for excess Fe accumulation in E107. Plants were grown with (+Fe-treated) or without (-Fe-treated) added Fe(III)-N,N' ethylenebis[2-(2-hydroxyphenyl)glycine] in aerated nutrient solutions. Daily measurements of Fe(III) reduction indicated a four-to seven-fold higher reduction rate in +Fe- or -Fe-treated E107, and -Fe-treated Sparkle, when compared with +Fe treated Sparkle. An agarose-based staining technique used to localize Fe(III) reduction, revealed Fe(III) reduction over most of the length of the roots (but not at the root apices) in both E107 treatments and -Fe-treated Sparkle. In +Fe treated Sparkle, Fe(III) reduction was either nonexistent or localized to central regions of the roots. Measurements of short-term Fe influx (with 0.1 millimolar (59)Fe(III)-ethylenediaminetetraacetic acid) was also enhanced (threefold) in +Fe or -Fe-treated E107 and -Fe-treated Sparkle, relative to +Fe-treated Sparkle. The physiological characteristics of E107 root systems, which are similar to those seen in Fe-deficient Sparkle, have led us to conclude that the mutation causes E107 to act functionally as an Fe-deficient plant, and appears to explain the excess Fe accumulation in E107. PMID- 16667610 TI - The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes. AB - Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 mug/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 mug/mL and 20 mug/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 mug/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed. PMID- 16667611 TI - Photocontrol of the Expression of Genes Encoding Chlorophyll a/b Binding Proteins and Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase in Etiolated Seedlings of Lycopersicon esculentum (L.) and Nicotiana tabacum (L.). AB - Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5 bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A ;high irradiance response' also controls the induction of these genes. Genes encoding photosystem II- and I associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light. These distinct expression characteristics are shown to reflect differences at the level of transcription. PMID- 16667612 TI - Sugar Composition and Molecular Weight Distribution of Cell Wall Polysaccharides in Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls. AB - The elongation growth of stem segments is determined by the outer cell layers (epidermis and collenchyma). We measured the sugar composition and molecular weight distribution of pectin and hemicellulose fractions obtained from inner and outer tissues of squash (Cucurbita maxima Duch.) hypocotyls. In addition, we studied the changes in these parameters after a 9 hour period of incubation of the segments. The results show that outer tissues have higher molecular weight pectin and hemicellulose compared to inner tissues (2-3 times higher). Incubation results in a 13 to 25% decrease in the amount of pectin and hemicellulose in inner tissues and an increase of 11 to 32% in the outer tissues. This increase in the outer tissues is accompanied by a decrease in the molecular weight of some of the components. These results clearly show that cell wall metabolism during elongation growth differs markedly in inner and outer tissues, and that future studies on the effect of auxin need to take these differences into account. PMID- 16667613 TI - Biosynthesis of the Tetrapyrrole Pigment Precursor, delta-Aminolevulinic Acid, from Glutamate. AB - delta-Aminolevulinic acid (ALA), the common biosynthetic precursor of hemes, chlorophylls, and bilins, is synthesized by two distinct routes. Among phototrophic species, purple nonsulfur bacteria form ALA by condensation of glycine with succinyl-CoA, catalyzed by ALA synthase, in a reaction identical to that occurring in the mitochondria of animals, yeast, and fungi. Most or all other phototrophic species form ALA exclusively from the intact carbon skeleton of glutamic acid in a reaction sequence that begins with activation of the alpha carboxyl group of glutamate by an ATP-dependent ligation to tRNA(Glu), catalyzed by glutamyl-tRNA synthetase. Glutamyl-tRNA is the substrate for a pyridine nucleotide-dependent dehydrogenase reaction whose product is glutamate-1 semialdehyde or a similar reduced compound. Glutamate-1-semialdehyde is then transaminated to form ALA. Regulation of ALA formation from glutamate is exerted at the dehydrogenase step through end product feedback inhibition and induction/repression. In some species, end product inhibition of the glutamyl tRNA synthetase step and developmental regulation of tRNA(Glu) level may also occur. PMID- 16667614 TI - Evaluation of chlorophyll fluorescence as a probe for drought stress in willow leaves. AB - The effect of drought on the photosynthetic apparatus of leaves of Salix sp. was studied by measurements of the induction of chlorophyll fluorescence and the capacity for O(2) evolution. Using a multivariate analysis, a model was developed that could predict the degree of drought stress from the data of fluorescence kinetics. Even mild drought stress was detected with high precision; this was not always possible when the photosynthetic capacity was measured. The most clear discrimination between control and drought-stressed leaves was obtained if fluorescence induction was measured at high rather than normal CO(2) levels, and at low rather than high light levels. All information provided by fluorescence pertaining to drought was contained within the slow phase of the induction curve. It is suggested that rapid dehydration is different from drought at the mechanistic level as judged by the fluorescence characteristics. PMID- 16667615 TI - Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants. AB - Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6-(3)H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5'-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants. PMID- 16667616 TI - Distribution of Indole-3-Acetic Acid and the Occurrence of Its Alkali-Labile Conjugates in the Extraxylary Region of Pinus sylvestris Stems. AB - Free and conjugated indole-3-acetic acid (IAA) were measured by quantitative gas chromatography-selected ion monitoringmass spectrometry in the extraxylary region of the stem of large Pinus sylvestris (L.) trees during the annual cycle of cambial activity and dormancy. The extraxylary region at the stem top and bottom was divided into 3 and 4 fractions, respectively, for the free IAA measurements, while the entire extraxylary region was extracted when the IAA-conjugates were analyzed. The effect on the distribution pattern of expressing IAA level as a concentration (per gram fresh weight or dry weight) and as total amount (per square centimeter) was examined. The IAA level was much higher in the cambial region than in the fractions that contained the nonfunctional phloem and the periderm. The largest IAA concentration occurred in the fraction that included the cambium, whereas the total amount of IAA was greatest in the phloemcontaining fraction. The significance of the nonuniform radial distribution of IAA for estimating the IAA concentration in the cambial region is discussed in relation to how the cambial region is sampled. A slight Iongitudinal gradient in IAA concentration, decreasing from the top to the bottom of the stem, was observed in the cambial region when the cambium was in the grand period of activity, but not at the end of the cambial growing period. In all fractions, the total amount of IAA was highest when the cambium was active. However, the IAA concentration in the cambial region did not follow the same pattern, actually being lowest during the tracheid production period at the stem bottom. IAA conjugates were detected on all sampling dates except June 23, but their concentrations were always less than 14% of that of free IAA, and their occurrence did not obviously vary during the year. In general, there was a higher concentration of ester conjugates than of amide conjugates, and the ester conjugates were more abundant at the top of the stem than at the bottom. PMID- 16667617 TI - Regulation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum: Properties Associated with Replenishment of Tricarboxylic Acid Cycle Intermediates during Ammonium Assimilation. AB - Two isoforms of phosphoenolpyruvate carboxylase (PEPC) with very different regulatory properties were partially purified from the green alga Selenastrum minutum. They were designated PEPC(1) and PEPC(2). PEPC(1) showed sigmoidal kinetics with respect to phosphoenolpyruvate (PEP) whereas PEPC(2) exhibited a typical Michaelis-Menten response. The S(0.5)(PEP) of PEPC(1) was 2.23 millimolar. This was fourfold greater than the S(0.5)(PEP) of PEPC(2), which was 0.57 millimolar. PEPC(1) was activated more than fourfold by 2.0 millimolar glutamine and sixfold by 2.0 millimolar dihydroxyacetone phosphate (DHAP) at a subsaturating PEP concentration of 0.625 millimolar. In contrast, PEPC(2) showed only 8% and 52% activation by glutamine and DHAP, respectively. The effects of glutamine and DHAP were additive. PEPC(1) was more sensitive to inhibition by glutamate, 2-oxoglutarate, and aspartate than PEPC(2). Both isoforms were equally inhibited by malate. All of these metabolites affected only the S(0.5)(PEP) not the V(max). The regulatory properties of S. minutum PEPC in vitro are discussed in terms of (a) increased rates of dark carbon fixation (shown to be catalyzed predominantly by PEPC) and (b) changes in metabolite levels in vivo during enhanced NH(4+) assimilation. Finally, a model is proposed for the regulation of PEPC in vivo in relation to its role in replenishing tricarboxylic acid cycle intermediates consumed in NH(4+) assimilation. PMID- 16667618 TI - Properties of a Mutant from Synechocystis PCC6803 Resistant to Acetazolamide, an Inhibitor of Carbonic Anhydrase. AB - A spontaneous mutant of the cyanobacterium Synechocystis PCC6803 was isolated for its resistance to acetazolamide, an inhibitor of carbonic anhydrase. The mutant showed a deficiency in oxygen exchange between CO(2) and H(2)O, a lower level of stable internal CO(2) pool and a decreased capacity to adapt its photosynthetic affinity under limited inorganic carbon regime. The initial rate of uptake of inorganic carbon was identical to that of wild-type cells. It is demonstrated that the mutation affects the carbonic anhydrase activity. This could result from either of two impairments: a deficiency in the enzyme activity detectable by mass spectrometric determinations, or a modification of the cellular compartment in which the enzyme is located, preventing its activity. PMID- 16667619 TI - Oxygen-evolving activity of thylakoids from barley plants cultivated on different concentrations of jasmonic Acid. AB - The kinetics characteristics of oxygen evolution in thylakoids prepared from barley (Horeum vulgare) seedlings grown in the presence of different jasmonic acid (JA) concentrations were studied. In comparison to control preparations, 100 micromolar JA-treated samples show an inhibition of the Hill activity (46%) and of O(2)-flash yields to above 70%. A damping in the oscillations of O(2) yields, induced by a flash train, increases with increasing growth regulator concentration. After these treatments, the value of the total number of oxygen evolving centers (S(0) + S(1)), estimated according to the Kok scheme, shows a considerable decrease. In 100 micromolar JA-treated preparations, the turnover half-time of S(1)-states increases and the stability of the S(2)- and S(3)-states decreases. PMID- 16667621 TI - Increased endogenous abscisic Acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials. AB - Roots of maize (Zea mays L.) seedlings continue to grow at low water potentials that cause complete inhibition of shoot growth. In this study, we have investigated the role of abscisic acid (ABA) in this differential growth sensitivity by manipulating endogenous ABA levels as an alternative to external applications of the hormone. An inhibitor of carotenoid biosynthesis (fluridone) and a mutant deficient in carotenoid biosynthesis (vp 5) were used to reduce the endogenous ABA content in the growing zones of the primary root and shoot at low water potentials. Experiments were performed on 30 to 60 hour old seedlings that were transplanted into vermiculite which had been preadjusted to water potentials of approximately -1.6 megapascals (roots) or -0.3 megapascals (shoots). Growth occurred in the dark at near-saturation humidity. Results of experiments using the inhibitor and mutant approaches were very similar. Reduced ABA content by either method was associated with inhibition of root elongation and promotion of shoot elongation at low water potentials, compared to untreated and wild-type seedlings at the same water potential. Elongation rates and ABA contents at high water potential were little affected. The inhibition of shoot elongation at low water potential was completely prevented in fluridone-treated seedlings during the first five hours after transplanting. The results indicate that ABA accumulation plays direct roles in both the maintenance of primary root elongation and the inhibition of shoot elongation at low water potentials. PMID- 16667620 TI - Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize. AB - We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40 degrees C but not at 28 degrees C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40 degrees C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear encoded, plastid-localized protein in maize. PMID- 16667622 TI - Growth of the Maize Primary Root at Low Water Potentials : II. Role of Growth and Deposition of Hexose and Potassium in Osmotic Adjustment. AB - Primary roots of maize (Zea mays L. cv WF9 x Mo17) seedlings growing in vermiculite at various water potentials exhibited substantial osmotic adjustment in the growing region. We have assessed quantitatively whether the osmotic adjustment was attributable to increased net solute deposition rates or to slower rates of water deposition associated with reduced volume expansion. Spatial distributions of total osmotica, soluble carbohydrates, potassium, and water were combined with published growth velocity distributions to calculate deposition rate profiles using the continuity equation. Low water potentials had no effect on the rate of total osmoticum deposition per unit length close to the apex, and caused decreased deposition rates in basal regions. However, rates of water deposition decreased more than osmoticum deposition. Consequently, osmoticum deposition rates per unit water volume were increased near the apex and osmotic potentials were lower throughout the growing region. Because the stressed roots were thinner, osmotic adjustment occurred without osmoticum accumulation per unit length. The effects of low water potential on hexose deposition were similar to those for total osmotica, and hexose made a major contribution to the osmotic adjustment in middle and basal regions. In contrast, potassium deposition decreased at low water potentials in close parallel with water deposition, and increases in potassium concentration were small. The results show that growth of the maize primary root at low water potentials involves a complex pattern of morphogenic and metabolic events. Although osmotic adjustment is largely the result of a greater inhibition of volume expansion and water deposition than solute deposition, the contrasting behavior of hexose and potassium deposition indicates that the adjustment is a highly regulated process. PMID- 16667623 TI - A Nuclear Factor Recognizing a Positive Regulatory Upstream Element of the Antirrhinum majus Chalcone Synthase Promoter. AB - A positive regulatory element directing maximal expression of the Antirrhinum majus chalcone synthase promoter was characterized by protein-DNA-interaction studies and cis deletion analysis. The positive regulatory element consists of a 47 base pair direct repeat between positions -564 and -670 and provides three binding sites for nuclear protein factors from Nicotiana tabacum and Antirrhinum majus. Oligonucleotide competition assays revealed that the same factor(s) interact(s) with all three binding sites. Transient expression of chimeric chalcone synthase-neomycin phosphotransferase II genes in parsley protoplasts demonstrated that both halves of the 47 base pair repeat element are required for its in vivo function. A possible role of redundant binding sites for the positive regulatory function of the 47 base pair repeat element is discussed. PMID- 16667624 TI - Cause for dark, chilling-induced inactivation of photosynthetic oxygen-evolving system in cucumber leaves. AB - Effects on oxygen evolution of the storage of detached cucumber (Cucumis sativus) leaves at 0 degrees C in the dark were investigated with thylakoids and oxygen evolving photosystem II membranes isolated from stored leaves. The cold and dark treatment of leaves selectively inactivated electron transport on the oxidizing side of photosystem II. Photosystem II membranes isolated from treated leaves were largely depleted of two proteins of 20 and 14 kilodaltons, which correspond to the extrinsic 23- and 17- kilodalton proteins of spinach functioning in oxygen evolution. The manganese content of photosystem II membranes was also markedly reduced by the treatment. Thus, the inactivation of oxygen evolution induced by the dark, chilling treatment is ascribed to solubilization of the 20- and 14 kilodalton proteins and extraction of manganese. PMID- 16667625 TI - Malate Dehydrogenases in Guard Cells of Pisum sativum. AB - Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness. PMID- 16667626 TI - Preventing photochemistry in culture media by long-pass light filters alters growth of cultured tissues. AB - Exposure of plant tissue culture media to light from fluorescent bulbs changed the growth regulating properties of the media. The light caused nutrient medium dependent photosensitized degradation of the phytohormone indole-3-acetic acid and other media components. Photochemical changes in culture media were caused by light from 290 to 450 nanometers and were prevented with a yellow long-pass filter. The use of appropriately filtered light when culturing plant material can eliminate unnecessary variability by stabilizing the culture media composition. PMID- 16667627 TI - Regulation of senescence-related gene expression in carnation flower petals by ethylene. AB - Ethylene plays a regulatory role in carnation (Dianthus caryophyllus L.) flower senescence. Petal senescence coincides with a burst of ethylene production, is induced prematurely in response to exogenous ethylene, and is delayed by inhibitors of ethylene biosynthesis or action. We have investigated the role of ethylene in the regulation of three senescence-related cDNA clones isolated from a senescent carnation petal library (KA Lawton et al. [1989] Plant Physiol 90: 690-696). Expression of two of the cloned mRNAs in response to ethylene is floral specific, while the expression of another mRNA can be induced in both leaves and flowers exposed to ethylene. Although ethylene induces expression of these mRNAs in petals, message abundance decreases when flowers are removed from ethylene unless an autoenhancement of ethylene production is induced. This indicates continued perception of ethylene is required for their expression. Interruption of ethylene action following the onset of natural senescence results in a substantial decrease in transcript abundance of two of these mRNAs. However, the abundance of another mRNA remains unaffected, indicating this gene responds to temporal cues as well as to ethylene. As flowers age the dosage of exogenous ethylene required to induce expression of the cloned mRNAs decreases, indicating sensitivity to ethylene changes as the tissue matures. Nuclear run-on transcription experiments indicate that relative transcription rates of cloned mRNAs increase in response to exogenous ethylene. PMID- 16667628 TI - A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis. AB - The catalytic activity of ribulosebisphosphate carboxylase (Rubisco) declined as soon as catalysis was initiated by exposure to its substrate, d-ribulose-1,5 bisphosphate (ribulose-P(2)). The decline continued exponentially, with a half time of approximately 7 minutes until, eventually, a steady state level of activity was reached which could be as low as 15% of the initial activity. The ratio of the steady state activity to the initial activity was lower at low CO(2) concentration and at low pH. The inhibitors 6-phosphogluconate and H(2)O(2) alleviated the inactivation, increasing the final/initial rate ratio and the half time. Varying ribulose-P(2) concentration in the range above that required to saturate catalysis did not affect the kinetics of inactivation. The affinities for CO(2) and ribulose-P(2) were unaffected by the inactivation. The decline in activity occurred with preparations of ribulose-P(2) which contained no detectable d-xylulose-1,5-bisphosphate and also with ribulose-P(2) which had been generated enzymatically immediately before use. Inclusion of an aldolase system for removing d-xylulose-1,5-bisphosphate also did not alter the inactivation process. The inactivated Rubisco did not recover after complete exhaustion of ribulose-P(2). We conclude that the inactivation is not caused by readily reversible binding of ribulose-P(2) at a site different from the active site and that it is unlikely to be attributable to inhibitory contaminants in ribulose P(2) preparations. PMID- 16667629 TI - Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site. AB - An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. Carbamylation level was measured using the reaction-intermediate analogue 2' -carboxy-D-arabinitol-1, 5-bisphosphate (carboxyarabinitol-P(2)). A dual isotope procedure was used in which [(3)H]carboxyarabinitol-P(2) measured total active sites and (14)CO(2) reported the level of carbamylation. The efficacy of the procedure was verified both in the presence and in the absence of the substrate d-ribulose-1, 5-bisphosphate (ribulose-P(2)). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P(2) binding and sequestering the E form of the enzyme. Ribulose-P(2) did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg(2+)-bound active sites by an inhibitor. PMID- 16667630 TI - Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Caused by Accumulation of a Slow, Tight-Binding Inhibitor at the Catalytic Site. AB - The slow inactivation which accompanies catalysis by higher-plant ribulose-P(2) carboxylase-oxygenase (Rubisco) in vitro was only partially reversed when the enzyme was gel filtered to remove small molecules. However, gel filtration or dialysis in the presence of high SO(2-) (4) concentrations induced full recovery. This suggests that the inactivation is caused by a tight-binding inhibitor whose effective affinity is reduced by competition with SO(2-) (4) ions, which are known to bind at the catalytic site. The involvement of an inhibitor was confirmed by observations that supernatants obtained after acid-precipitation of inactivated Rubisco were inhibitory when applied to fresh enzyme. The inhibitor bound slowly and tightly and showed strong negative cooperativity. The inhibitor was moderately unstable at pH 8.3, decaying with a halflife of several hours, but was more stable at pH 2. It was destroyed by phosphatase treatment but not by H(2)O(2) or o-phenylenediamine, compounds which react with vicinal dicarbonyl groups. It did not contain a carbon atom derived from substrate CO(2). Possibilities concerning the identity, genesis, and physiological relevance of this inhibitor are discussed. PMID- 16667631 TI - Polyamines, hydroxycinnamoylputrescines, and root formation in leaf explants of tobacco cultivated in vitro: effects of the suicide inhibitors of putrescine synthesis. AB - In vitro formation of roots is obtained directly, without intermediate growth of callus, from foliar explants of a tobacco (Nicotiana tabacum) plant cultured on Murashige and Skoog medium containing IAA. Auxin-induced root formation was accompanied by significant changes in hydroxycinnamoylputrescine levels. Increasing levels were found in leaf explants during the first 14 days in culture; this was followed by a sharp decline after 20 days. Early changes in putrescine conjugates were detected in leaf explants before the visible appearance of roots. An early and transitory accumulation of hydroxycinnamoylputrescines was observed in the roots. Free polyamines (putrescine, spermidine, and spermine) in leaf explants and roots were always at a low level and only small changes in their concentrations were observed, alpha dl-difluoromethylarginine and alpha-dl-difluoromethylornithine, specific, irreversible inhibitors of arginine decarboxylase and ornithine decarboxylase, respectively, inhibited putrescine accumulation and root initiation and reduced the fresh and dry weights of leaf explants. These effects were reversed by free putrescine or hydroxycinnamoylputrescines. The results reported here suggest that hydroxycinnamoylputrescines are associated with root formation. The relationship among free polyamines, hydroxycinnamoylputrescines, cell division, and root formation is discussed. PMID- 16667632 TI - Postphloem, nonvascular transfer in citrus: kinetics, metabolism, and sugar gradients. AB - Postphloem, nonvascular assimilate transport occurs over an unusually long area in citrus fruit and thus facilitates investigation of this process relative to sugar entry into many sink structures. Labeled photosynthates moving into juice tissues of grapefruit (Citrus paradisi Macf.) slowed dramatically after entering the postphloem transport path (parenchyma cells, narrow portions of segment epidermis, and hair-like, parenchymatous stalks of juice sacs). Kinetic, metabolic, and compositional data indicated that transfer through the nonvascular area was delayed many hours by temporary storage and/or equilibration with sugars in compartments along the postphloem path. Labeled assimilates were generally recovered as sucrose throughout the path, and extent of hexose formation enroute bore no apparent relationship to the assimilate transfer process. Even after 24 hours, radiolabel was restricted to discrete, highly localized areas directly between vascular bundles and juice sacs. Postphloem transfer occurred against an ascending sucrose concentration gradient in young fruit, whereas a descending gradient (favoring diffusion/cytoplasmic streaming) developed only later in maturation. Involvement of a postphloem bulk flow is complicated in the present instance by the extremely limited water loss from juice sacs either via transpiration or fluid backflow. Nonetheless, tissue expansion can account for a collective water inflow of at least 1.0 milliliter per day throughout the majority of juice sac development, thus providing a modest, but potentially important means of nonvascular solution flow. Overall, data indicate postphloem transfer (a) can follow highly localized paths through sizable nonvascular areas (up to 3.0 centimeters total), (b) appears to involve temporary storage and/or equilibration with compartmentalized sugars enroute, (c) can occur either against an overall up-hill sugar gradient (young tissues) or along a descending gradient (near full expansion), and (d) appears to involve at least some contribution by nonvascular mass flow accommodated by tissue expansion. PMID- 16667633 TI - d-Galactose Uptake by Fenugreek Cotyledons : Effect of Water Stress. AB - The uptake of d-galactose was studied in detached fenugreek (Trigonella foenum graecum L.) cotyledons. Uptake kinetics and treatment with p-chloromercury benzenesulfonic acid indicated that at low concentrations d-galactose was taken up by a carrier. At higher concentrations a diffusion-like component existed. Proton flux and pH studies, treatment with alpha-naphthaleneacetic acid, and uptake experiments under water stress conditions suggested that d-galactose was not taken up via H(+) contransport. However, d-galactose uptake was under metabolic control. Uptake kinetics under water stress conditions suggested that moderate water stress either increased the K(m) of the carrier or decreased the V(max). However, prolonged stress transformed the carrier-mediated uptake into a diffusion uptake transport. The uptake of d-galactose by fenugreek cotyledons was very low before and just after germination, was maximum after 35 hours imbibition, and started decreasing thereafter. The different uptake rates of d galactose with imbibition times were attributed to the operation of the carrier. At low uptake rates the carrier did not operate. Treatment with cycloheximide suggested that the carrier was synthesized de novo just after germination and stopped operating when all galactomannan hydrolysis was over. Results were discussed in the context of control of endosperm galactomannan hydrolysis by the cotyledons of fenugreek embryo. PMID- 16667634 TI - Correlations between Carbon Isotope Discrimination and Leaf Conductance to Water Vapor in Common Beans. AB - Carbon isotope discrimination (Delta) was measured in the field on 10 cultivars of common bean (Phaseolus vulgaris L.). There was substantial variation (more than 2 per thousand) in leaf Delta values and these differences were maintained between vegetative and reproductive developmental stages. These bean lines also exhibited substantial differences in leaf conductance to water vapor, and again these differences were maintained across developmental stages. The differences in leaf conductance were positively correlated with Delta values, whether conductance was measured as total leaf conductance or as the individual conductances of either upper or lower leaf surfaces. The observed differences in leaf conductance were not associated with differences in stomatal density. There were small differences among bean lines in their leaf Kjeldahl nitrogen contents, which is interpreted as indicating that photosynthetic capacity among bean lines was similar. Thus, because Delta values and leaf conductance were positively correlated, these data suggested that there may have been differences among bean lines in the extent to which stomata limited photosynthetic gas exchange rates. PMID- 16667635 TI - Studies of the Uptake of Nitrate in Barley: I. Kinetics of NO(3) Influx. AB - (13)NO(3) (-) was used to investigate patterns of NO(3) (-) influx into roots of barley plants (Hordeum vulgare L. cv Klondike) previously grown with (;induced') or without (;uninduced') a source of external NO(3) (-) ([NO(3) (-)](0)). In both induced and uninduced plants, (13)NO(3) (-) influx was biphasic in the range from 0.005 to 50 moles per cubic meter [NO(3) (-)](0). In the low concentration range (<1 mole per cubic meter for induced plants and <0.3 mole per cubic meter for uninduced plants), influx was saturable and V(max) and K(m) values for influx either increased or decreased according to NO(3) (-) pretreatment. By contrast, (13)NO(3) (-) influx in the high concentration range revealed a strictly linear concentration dependence. These fluxes appeared to be mediated by a constitutive, rather than an inducible, transport system. PMID- 16667636 TI - Response of the Photosynthetic Apparatus in Dunaliella salina (Green Algae) to Irradiance Stress. AB - The response of the photosynthetic apparatus in the green alga Dunaliella salina, to irradiance stress was investigated. Cells were grown under physiological conditions at 500 millimoles per square meter per second (control) and under irradiance-stress conditions at 1700 millimoles per square meter per second incident intensity (high light, HL). In control cells, the light-harvesting antenna of photosystem I (PSI) contained 210 chlorophyll a/b molecules. It was reduced to 105 chlorophyll a/b in HL-grown cells. In control cells, the dominant form of photosystem II (PSII) was PSII(alpha)(about 63% of the total PSII) containing >250 chlorophyll a/b molecules. The smaller antenna size PSII(beta) centers (about 37% of PSII) contained 135 +/- 10 chlorophyll a/b molecules. In sharp contrast, the dominant form of PSII in HL-grown cells accounted for about 95% of all PSII centers and had an antenna size of only about 60 chlorophyll a molecules. This newly identified PSII unit is termed PSII(gamma). The HL-grown cells showed a substantially elevated PSII/PSI stoichiometry ratio in their thylakoid membranes (PSII/PSI = 3.0/1.0) compared to that of control cells (PSII/PSI = 1.4/1.0). The steady state irradiance stress created a chronic photoinhibition condition in which D. salina thylakoids accumulate an excess of photochemically inactive PSII units. These PSII units contain both the reaction center proteins and the core chlorophyll-protein antenna complex but cannot perform a photochemical charge separation. The results are discussed in terms of regulatory mechanism(s) in the plant cell whose function is to alleviate the adverse effect of irradiance stress. PMID- 16667637 TI - Herbicide Chlorsulfuron Decreases Assimilate Transport Out of Treated Leaves of Field Pennycress (Thlaspi arvense L.) Seedlings. AB - Treatment of field pennycress (Thlaspi arvense L.) leaves with the herbicide chlorsulfuron resulted in a decrease in the export of assimilate. Twelve hours after a spot application of 1 microgram, assimilate translocation was 70% of that in control leaves. In excised leaves treated with chlorsulfuron the total amounts of sugars and free amino acids were 150 and 170%, respectively, of the amounts in control leaves, 30 hours after herbicide treatment. The amount of sucrose was 247% of that in control leaves. The increase in the concentration of sucrose in the chlorsulfuron-treated leaves, combined with the absence of an effect of chlorsulfuron on carbon dioxide fixation, suggests that the decrease in assimilate transport is not due to an effect on the synthesis of assimilates, but rather to an effect on their movement out of the leaves. Supplying branched-chain amino acids to the field pennycress seedlings prior to the application of chlorsulfuron prevented the occurrence of the effects described. PMID- 16667638 TI - Localization of carboxypeptidase I in germinating barley grain. AB - Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain. PMID- 16667639 TI - Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development. AB - Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety "Red Kidney." PMID- 16667641 TI - Limiting Factors in Photosynthesis: VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at Low Photochemical Capacity. AB - Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimited by photosynthetic electron transport capacity, even at low intercellular CO(2) concentrations. Here we monitored leaf metabolites diurnally and the activities of key Calvin cycle enzymes in the leaves of three treatment groups of sugar beet (Beta vulgaris L.) plants representing three different in vivo photochemical capacities, i.e. Fe-sufficient (control) plants, moderately Fe-deficient, and severely Fe-deficient plants. The results show that the decrease in photosynthesis with Fe deficiency mediated reduction in photochemical capacity was through a reduction in ribulose 1,5 bisphosphate (RuBP) regeneration and not through a decrease in ribulose 1,5 bisphosphate carboxylase/oxygenase activity. Based on measurements of ATP and NADPH and triose phosphate/3-phosphoglycerate ratios in leaves, there was little evidence that photosynthesis and RuBP regeneration in Fe-deficient leaves were limited directly by the supply of ATP and NADPH. It appeared more likely that photochemical capacity influenced RuBP regeneration through modulation of enzymes in the photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency. Starch and sucrose levels changed independently of one another to some extent during the diurnal period (both increasing in the day and decreasing at night) but the average rates of starch or sucrose accumulation over the light period were each proportional to photochemical capacity and photosynthetic rate. PMID- 16667640 TI - Fructose 1,6-Bisphosphatase in the Green Alga Selenastrum minutum: I. Evidence for the Presence of Isoenzymes. AB - Two isoforms of fructose 1,6-bisphosphatase are present in the green alga Selenastrum minutum. The isoenzymes can be separated with ionexchange chromatography or acid precipitation. The stability of the two isoenzymes differ largely. The acid insoluble enzyme exhibits properties similar to that of the enzyme from the chloroplasts of higher plants, i.e. an alkaline pH optima in the absence of reductant, a lower affinity for substrate, strong inhibition by phosphate, and a low sensitivity to fructose-2,6-bisphosphate and AMP. The more abundant form of the enzyme exhibits several properties indicative of heterotrophic fructose 1,6 bisphosphatases, i.e. a high affinity for substrate and sensitivity toward fructose-2,6-bisphosphate and AMP. but is absolutely dependent on a reductant for stability and activity. Evidence is provided indicating that previously reported purification protocols cause inactivation of one of the isoenzymes which could lead to the erroneous conclusion that algae have a single fructose 1,6-bisphosphatase isoenzyme. PMID- 16667642 TI - Quantitation of Gibberellins A(1), A(3), A(4), A(9) and a Putative A(9)-Conjugate in Grafts of Sitka Spruce (Picea sitchensis) during the Period of Shoot Elongation. AB - The levels of endogenous gibberellin A(1) (GA(1)), GA(3), GA(4), GA(9), and a cellulase hydrolyzable GA(9) conjugate in needles and shoot stems of mature grafts of Sitka spruce (Picea sitchensis [Bong.] Carr.) grown under environmental conditions that were either inductive, hot, and dry, or noninductive, cool, and wet, for flowering, were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated [(2)H(2)]GA(1), GA(3), GA(4), and GA(9) as internal standards. The samples were taken when the shoots had elongated about 30, 70, and 95% of the final shoot length and 17 days after elongation had terminated. The concentration of putative GA(9)-conjugate, estimated by GCSIM of GA(9) after cellulase hydrolysis of the highly water soluble fraction, was 33 nanograms per gram fresh weight in the needles of both heat and drought- and cool and wet-treated plants sampled just after bud burst. The concentration gradually decreased to a final value of 13 nanograms per gram fresh weight in the heat and drought-treated grafts and 6 nanograms per gram fresh weight in the cool and wet-treated grafts. The stems contained no detectable putative GA(9) conjugate. Free GA(9) was highest in heat and drought treated material. For plants subjected to this treatment, GA(9) increased from 22 to 32 nanograms per gram fresh weight in needles and from 1 to 22 nanograms per gram fresh weight in stems during the rapid stem elongation phase. By day 17, after cessation of shoot elongation, GA(9) had decreased to 12 nanograms per gram fresh weight in needles and 9 nanograms per gram fresh weight in the shoot stems. The cool and wet-treated material also showed an increase in GA(9) concentration during shoot elongation. However, the concentration was not as high and was also delayed compared with heat and drought-treated material. By day 17, after cessation of shoot elongation, GA(9) concentration was 9 nanograms per gram fresh weight in needles and 5 nanograms per gram fresh weight in stems for cool and wet treatment plants. The concentration of GA(4) was very low in tissue from both treatments. Fluctuation in concentration of the more polar gibberellins, GA(1) and GA(3), showed the same pattern as fluctuations in the content of GA(9). However, the heat and drought-treated material had lower amounts of GA(1) and GA(3) during the later phases of shoot elongation, than the cool and wet-treated material. These results imply differential metabolism between clones treated with conditions inductive and noninductive for flowering. Higher concentrations of putative GA(9) conjugate and free GA(9) in the hot and dry treatment indicate a higher capacity of synthesizing, for flowering, the physiologically important GA(4) in the heat and drought-treated material. This synthesis does not, however, result in a buildup of the GA(4) pool, probably because of a high turnover rate of GA(4). The cool and wet-treated material had higher amounts of GA(1) and GA(3), indicating that the differentiation was preferentially directed toward vegetative growth. PMID- 16667643 TI - Inactivation of stress induced 1-aminocyclopropane carboxylate synthase in vivo differs from substrate-dependent inactivation in vitro. AB - The activity of 1-aminocyclopropane carboxylate (ACC) synthase increased rapidly in tomato (Lycopersicon esculentum Mill.) leaf discs after vacuum infiltration, reached a maximum after about 30 minutes, and subsequently decayed with an apparent half-life of about 20 minutes. Aminoethoxyvinylglycine, a known inhibitor of ACC synthase, did not alter the apparent turnover of ACC synthase in vivo although it efficiently blocked inactivation of the enzyme by its substrate S-adenosylmethionine in vitro. Similar results were obtained, using a novel assay with permeabilized cells, for ACC synthase in tomato cell cultures treated with a fungal elicitor. The results indicate that inactivation of ACC synthase in vivo differs from substrate-dependent inactivation in vitro. PMID- 16667644 TI - Transcriptional activation by heat and cold of a thiol protease gene in tomato. AB - We previously determined that low temperature induces the accumulation in tomato (Lycopersicon esculentum) fruit of a cloned mRNA, designated C14, encoding a polypeptide related to thiol proteases (MA Schaffer, RL Fischer [1988] Plant Physiol 87: 431-436). We now demonstrate that C14 mRNA accumulation is a response common to both high (40 degrees C) and low (4 degrees C) temperature stresses. Exposure of tomato fruit to 40 degrees C results in the accumulation of C14 mRNA, by 8 hours. This response is more rapid than that to 4 degrees C, but slower than the induction of many heat shock messages by 40 degrees C, and therefore unique. We have also studied the mechanism by which heat and cold exposure activate C14 gene expression. Both high and low temperature regulate protease gene expression through transcriptional induction of a single C14 gene. A hypothesis for the function of C14 thiol protease gene expression in response to heat and cold is discussed. PMID- 16667645 TI - Biotin-mediated delivery of exogenous macromolecules into soybean cells. AB - We have demonstrated that attachment of biotin to a variety of macromolecules allows the uptake of those macromolecules into cultured soybean cells (Glycine max Merr cv Kent). Macromolecules that were nondestructively delivered into intact cells in large numbers (>10(6)/cell) by this technique include bovine insulin (M(r) about 5,700), bovine ribonuclease (M(r) about 14,000), human hemoglobin (M(r) about 64,000), and bovine serum albumin (M(r) about 68,000). It is hypothesized that this methodology may be useful for delivering antibodies, toxins, enzymes, and genetic material into living plant cells without requiring prior removal of the cell wall or infection with Agrobacterium. PMID- 16667646 TI - Biosynthesis of Phytosiderophores : In Vitro Biosynthesis of 2'-Deoxymugineic Acid from l-Methionine and Nicotianamine. AB - 2' -Deoxymugineic acid (DMA), one of mugineic acid-family phytosiderophores (MAs), was synthesized in vitro both from l-methionine and from nicotianamine (NA) with a cell-free system derived from root tips of iron-deficient barley (Hordeum vulgare L.). The reactions producing DMA from NA needed an amino group acceptor (i.e. 2-oxoglutarate, pyruvate, or oxalacetic acid) and a reductant (i.e. NADH or NADPH). The activity of the enzymes to produce NA from l-methionine was the highest at about pH 9. This biosynthetic activity was markedly induced by iron-deficiency stress. The synthesis of NA from S-adenosyl-l-methionine was more efficient than from l-methionine. From the results with the cell-free system reported here, we propose a revised biosynthetic pathway of MAs. PMID- 16667647 TI - Molecular cloning and characterization of cold-regulated genes in barley. AB - Five different cDNA clones have been isolated which are homologous to cold regulated mRNAs in barley (Hordeum vulgare L.). The analyses of their hybridizations indicate that the transcripts accumulate to different levels during cold-treatment. Hybridization experiments using RNAs isolated from different plant tissues indicate that several cold-regulated genes are expressed in a tissue-specific manner. The expression studies suggest that in barley several different genes are involved in the cold hardening process depending on developmental stages and tissues involved. Homology has been found between the isolated cDNAs and cold-induced transcripts of related cereals. DNA sequence analysis of the clones pT59 and pA086 reveals that the proteins deduced from the longest open reading frame contain arginine rich basic domains. PMID- 16667648 TI - Activity of ribulose 1,5-bisphosphate carboxylase oxygenase as a function of storage conditions. AB - Optimal storage conditions to retain ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated. The soluble spinach (Spinacia oleracea) enzyme was pretreated with its activators, Mg(2+) and HCO(3) (-), and then stored for up to 30 days at 4 or -18 degrees C or in liquid N(2). Cold inactivation and conformational changes were suggested to be involved during Rubisco storage in the cold, leading to its inactivation. Pretreatment of the enzyme with Mg(2+) and CO(2) and subsequent storage at either 4 degrees C or in liquid N(2) or flushing the samples with N(2) and rapid freezing and storage in liquid N(2) are recommended as storage procedures. These storage treatments will prevent inactivation, so that full original specific activity will be preserved. PMID- 16667649 TI - Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes. AB - Characterization of sugar content and enzyme activity in germinating soybean (Glycine max L. Merrell) seeds led to the discovery of sorbitol accumulating in the axes during germination. The identity of sorbitol was confirmed by relative retention times on high-performance liquid chromatography and gas liquid chromatography and by mass spectra identical with authentic sorbitol. Accumulation of sorbitol in the axes started on day 1 of germination as sucrose decreased and glucose and fructose increased. Sucrose also decreased in the cotyledons, but there was no accumulation of sorbitol, glucose, or fructose. Accumulation of sorbitol and hexoses was highly correlated with increased invertase activity in the axes, but not with sucrose synthase and sucrose phosphate synthase activities. Sucrose synthase activity was relatively high in the axes, whereas the activity of sucrose phosphate synthase was relatively high in the cotyledons. Ketose reductase and aldose reductase were detected in germinating soybean axes, but not in cotyledons. Fructokinase and glucokinase were present in both axes and cotyledons. The data suggest a sorbitol pathway functioning in germinating soybean axes, which allows for the interconversion of glucose and fructose with sorbitol as an intermediate. PMID- 16667650 TI - Physiological investigations of a tobacco mutant with o(2)-resistant photosynthesis and enhanced catalase activity. AB - Experiments are described further indicating that O(2)-resistant photosynthesis observed in a tobacco (Nicotiana tabacum) mutant with enhanced catalase activity is associated with decreased photorespiration under conditions of high photorespiration relative to net photosynthesis. The effects on net photosynthesis of (a) increasing O(2) concentrations from 1% to 42% at low CO(2) (250 microliters CO(2) per liter), and (b) of increasing O(2) concentrations from 21% to 42% at high CO(2) (500 microliters CO(2) per liter) were investigated in M(6) progeny of mutant and wild-type leaf discs. The mutant displayed a progressive increase in net photosynthesis relative to wild type with increasing O(2) and the faster rate at 42% O(2) was completely reversed on returning to 21% O(2). The photosynthetic rate by the mutant was similar to wild type in 21% and 42% O(2) at 500 microliters CO(2) per liter, and a faster rate by the mutant was restored on returning to 250 microliters CO(2) per liter. The results are consistent with a lowered release of photorespiratory CO(2) by the mutant because greater catalase activity inhibits the chemical decarboxylation of alpha-keto acids by peroxisomal H(2)O(2). Higher catalase activity was observed in the tip and middle regions of expanding leaves than in the basal area. On successive selfing of mutant plants with enhanced catalase activity, the percent of plants with this phenotype increased from 60% in M(4) progeny to 85% in M(6) progeny. An increase was also observed in the percent of plants with especially high catalase activity (averaging 1.54 times wild type) on successive selfings suggesting that homozygosity for enhanced catalase activity was being approached. PMID- 16667651 TI - Inhibition of Cottonseed Choline- and Ethanolaminephosphotransferases by Calcium during Postgerminative Growth. AB - Activities of choline- and ethanolaminephosphotransferase (CPT and EPT) were reproducibly high in microsomes from imbibed seeds of cotton (Gossypium hirsutum, L.). Initial studies showed that both activities dramatically declined during postgerminative growth when demand for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis was high. Addition of CaCl(2) (0.1 millimolar) or aliquots of supernatant fractions (150,000g, 60 minutes) from cotyledons of 48-hour-old seedlings to imbibed-seed microsomes reduced the CPT and EPT activities to levels approximating those found in 48-hour microsomes. Inhibition by supernatants was completely reversed by adding EGTA (1.0 millimolar), but not by boiling the supernatants. EGTA (1.0 or 5.0 millimolar) relieved inhibition in cellular fractions whether it was added to the homogenization media or the assay reaction mixtures. A time course of CPT and EPT activities in cellular fractions prepared with 1.0 millimolar EGTA showed that activities were well developed in imbibed seeds, doubled coincidentally to a peak at 36 hours, then declined during the next 12 hours to levels approximating those in imbibed seeds. Greater than 90% of the CPT and EPT activities were pelletable (150,000g, 60 minutes) at all ages examined. Calcium apparently was artificially released upon homogenization, to a progressively greater extent in older cotyledons, and severely inhibited CPT and EPT activities. This is the only time course of CPT and EPT activities reported for cotyledons of any oilseed; it is substantially different from that in oil-storing endosperm. PMID- 16667652 TI - Genotypic variation in cytokinin oxidase from phaseolus callus cultures. AB - Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues. PMID- 16667653 TI - Comparative indole-3-acetic Acid levels in the slender pea and other pea phenotypes. AB - Free indole-3-acetic acid levels were measured by gas chromatography-mass spectrometry in three ultra-tall ;slender' Pisum sativum L. lines differing in gibberellin content. Measurements were made for apices and stem elongation zones of light-grown plants and values were compared with wild-type, dwarf, and nana phenotypes in which internode length is genetically regulated, purportedly via the gibberellin level. Indole-3-acetic acid levels of growing stems paralleled growth rates in all lines, and were high in all three slender genotypes. Growth was inhibited by p-chlorophenoxyisobutyric acid, demonstrating the requirement of auxin activity for stem elongation, and also by the ethylene precursor 1 aminocyclopropane-1-carboxylic acid. It is concluded that the slender phenotype may arise from constant activation of a gibberellin receptor or transduction chain event leading directly or indirectly to elevated levels of indole-3-acetic acid, and that increased indole-3-acetic acid levels are a significant factor in the promotion of stem elongation. PMID- 16667654 TI - Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters. AB - Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C(4) and C(5) acids, and branched and straight chain C(10), C(11), and C(12) acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [(14)C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C(4) and C(5) acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C(4) and C(5) branched acids were elongated by two carbon units to produce the branched C(10)-C(12) groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters. PMID- 16667655 TI - Concurrent Synthesis and Release of nod-Gene-Inducing Flavonoids from Alfalfa Roots. AB - Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium meliloti. Alfalfa roots release three major nod-gene inducers: 4',7-dihydroxyflavanone, 4',7-dihydroxyflavone, and 4,4' dihydroxy-2'-methoxychalcone. The objective of the present study was to define temporal relationships between synthesis and exudation for those flavonoids. Requirements for concurrent flavonoid biosynthesis were assessed by treating roots of intact alfalfa seedlings with [U-(14)C]-l-phenylalanine in the presence or absence of the phenylalanine ammonia-lyase inhibitor l-2-aminoxy-3 phenylpropionic acid (AOPP). In the absence of AOPP, each of the three flavonoids in exudates contained (14)C. In the presence of AOPP, (14)C labeling and release of all the exuded nod-gene inducers were reduced significantly. AOPP inhibited labeling and release of the strongest nod-gene inducer, methoxychalcone, by more than 90%. Experiments with excised cotyledons, hypocotyls, and roots incubated in solution showed that the flavonoids could be synthesized in and released from each organ. However, the ratio of the three flavonoids in exudates from intact plants was most similar to the ratio recently synthesized and released from excised roots. A portion of recently synthesized flavonoid aglycones was found conjugated, presumably as glycosides, in root extracts and may have been involved in the release process. Data from root extracts showed that formononetin, an isoflavonoid which does not induce nod genes, was present in conjugated and aglycone forms but was not released by normal intact roots. In contrast, roots stressed with CuCl(2) did release the aglycone formononetin. Thus, the release process responsible for exudation of nod-gene inducers appears to be specific rather than a general phenomenon such as a sloughing off of cells during root growth. The synthesis and specific concurrent release of flavonoid nod-gene inducers in this study is consistent with the physiological requirement for nodule formation of the 3-day-old seedlings used. PMID- 16667656 TI - Metabolism of Monoterpenes in Cell Cultures of Common Sage (Salvia officinalis) : Biochemical Rationale for the Lack of Monoterpene Accumulation. AB - Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes (<0.3 nanograms per gram fresh weight) at any stage of the growth cycle, even in the presence of a polystyrene resin trap. Monoterpene biosynthesis from [U-(14)C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+) camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism. PMID- 16667657 TI - Growth of the Cellular Slime Mold, Dictyostelium discoideum, Is Gravity Dependent. AB - The effect of artificial gravity on the growth of a microorganism, Dictyostelium discoideum, was studied and the following results were obtained: (a) Germination efficiency increased as gravity increased up to 3 gravities. (b) Cell differentiation was influenced by gravity. Retardation of spore formation or reduction in the spore fraction was observed at hypergravity. (c) Fruiting bodies were taller at hypergravity and smaller at simulated microgravity when compared at 1 gravity. It is suggested that modulation of gravity provides useful information on the mechanisms of life. PMID- 16667658 TI - Study of root uptake and xylem translocation of cinmethylin and related compounds in detopped soybean roots using a pressure chamber technique. AB - A pressure chamber technique was used to study the root uptake and xylem translocation of nonradiolabeled cinmethylin and its analogs in detopped soybean (Glycine max) roots. Quantifications of compounds were achieved by gas chromatography analysis using a mass spectrometry detector under selected ion monitoring. The compounds tested, with octanol-water partition coefficients (log Kow values) ranging from 0.96 to 5.3, were all nonionizable under the experimental conditions. Root efflux curves of all compounds exhibited a steady state kinetic profile. The time required to achieve the steady state efflux concentration in the xylem sap correlated with log Kow values in a manner very similar to the root binding profile reported previously by GG Briggs et al. ([1982] Pestic Sci 13: 495-504). After reaching the steady state efflux, the concentration ratio of each compound in the xylem sap to the final concentration in the pressure chamber was taken as the transpiration stream concentration factor (TSCF). A nonlinear relationship was observed between TSCF and log Kow values. The highest TSCF value was between 0.6 to 0.8 for compounds with log Kow between 2.5 to 3.5. The range of optimal log Kow values was slightly higher than that reported earlier by Briggs et al. ([1982] Pestic Sci 13: 495-504). After taking into account the binding of the compound to soil, the apparent optimal Kow value for best root-to-shoot translocation is lowered to around 1. The relationship of root-to-shoot and phloem translocation was also discussed to promote a better understanding at the whole plant level of the uptake and translocation of a soil-applied xenobiotic. PMID- 16667659 TI - Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L. AB - In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl(2) concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5'-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5'-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis. PMID- 16667660 TI - Studies of the Uptake of Nitrate in Barley : II. Energetics. AB - Q(10) values for (13)NO(3) (-) influx were determined in ;uninduced' (NO(3) (-) starved) and ;induced' (NO(3) (-)-pretreated) roots of barley (Hordeum vulgare L.) plants at various concentrations of external NO(3) (-) ([NO(3) (-)](0)). At 0.02 mole per cubic meter [NO(3) (-)](0), Q(10) values for influx were from 3 to 4 between 5 and 10 degrees C. As [NO(3) (-)](0) increased Q(10) values decreased, reaching values of 1.2 and 2.0, respectively, at 20 moles per cubic meter in uninduced and induced plants. The metabolic dependence of (13)NO(3) (-) influx at low and high [NO(3) (-)](0) (0.1 and 20.0 moles per cubic meter, respectively) in uninduced and induced plants was probed by the use of various inhibitors. These experiments confirmed the findings of the Q(10) studies, demonstrating that at low [NO(3) (-)](0) (13)NO(3) (-) influx was extremely sensitive to metabolic inhibition. By contrast, at high [NO(3) (-)](0), influx was relatively insensitive to the presence of inhibitors. PMID- 16667661 TI - Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport. AB - The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on DeltapH dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V(max) only with no apparent change in K(m). Sucrose transport rates driven by membrane potential only, i.e. in the absence of DeltapH, were comparable to DeltapH-driven flux. Both membrane potential and DeltapH-driven sucrose transport were used to examine proton binding to the symport and the apparent K(m) for H(+) was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane. PMID- 16667662 TI - Uptake of Inorganic Carbon by Isolated Chloroplasts of the Unicellular Green Alga Chlorella ellipsoidea. AB - Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO(2) and PGA-dependent rates of O(2) evolution of 64.5 to 150 micromoles per milligram of chlorophyll per hour, that is 30 to 70% of the photosynthetic activity of the parent cells. Intact chloroplasts showed no carbonic anhydrase activity, but it was detected in preparations of ruptured organelles. Rates of photosynthesis, measured in a closed system at pH 7.5, were twice the calculated rate of CO(2) supply from the uncatalyzed dehydration of HCO(3) (-) indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO(2) depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO(2) transport across the chloroplast envelope. PMID- 16667663 TI - Primary events regulating stem growth at low water potentials. AB - Cell enlargement is inhibited by inadequate water. As a first step toward understanding the mechanism, all the physical parameters affecting enlargement were monitored to identify those that changed first, particularly in coincidence with the inhibition. The osmotic potential, turgor, yield threshold turgor, growth-induced water potential, wall extensibility, and conductance to water were measured in the elongating region, and the water potential was measured in the xylem of stems of dark-grown soybean (Glycine max [L.] Merr.) seedlings. A stepdown in water potential was achieved around the roots by transplanting the seedlings to vermiculite of low water content, and each of the parameters was measured simultaneously in the same plants while intact or within a few minutes of being intact using a newly developed guillotine psychrometer. The gradient of decreasing water potential from the xylem to the enlarging cells (growth-induced water potential) was the first of the parameters to decrease to a growth-limiting level. The kinetics were the same as for the inhibition of growth. The decreased gradient was caused mostly by a decreased water potential of the xylem. This was followed after 5 to 10 hours by a similar decrease in cell wall extensibility and tissue conductance for water. Later, the growth-induced water potential recovered as a result of osmotic adjustment and a rise in the water potential of the xylem. Still later, moderate growth resumed at a rate apparently determined by the low wall extensibility and tissue conductance for water. The turgor did not change significantly during the experiment. These results indicate that the primary event during the growth inhibition was the change in the growth-induced water potential. Because the growth limitation subsequently shifted to the low wall extensibility and tissue conductance for water, the initial change in potential may have set in motion subsequent metabolic changes that altered the characteristics of the wall and cell membranes. PMID- 16667665 TI - Assessing the Rhizotoxicity of the Aluminate Ion, Al(OH)(4). AB - Dissolved aluminum (III) in acidic soils or culture media is often rhizotoxic (inhibitory to root elongation). Alkaline solutions of Al are also sometimes rhizotoxic, and for that reason toxicity has been attributed to the aluminate ion, Al(OH)(4) (-). In the present study, seedlings of wheat (Triticum aestivum L. cv Tyler) and red clover (Trifolium pratense L. cv Kenland) were cultured in aerated aluminate solutions at pH 8.0 to 8.9. The bulk phases of these solutions were free of reactive polynuclear hydroxy-Al (including the extremely toxic species AlO(4)Al(12)[OH](24)[H(2)O](7+) (12) [Al(13)]) according to the ferron (8 hydroxy-7-iodo-5-quinolinesulfonic acid) assay. At an aluminate concentration of 25 micromolar (23 micromolar activity) and a pH of 8, root elongation was less than 40% of Al-free controls, but at pH 8.9 elongation was 100% of controls. The hypothesis is offered that aluminate is nontoxic and that the inhibition at lower pH values is attributable to Al(13) postulated to have formed in the acidic free space of the roots where the ratio /{Al(3+)/}//{H(+)/}(3) may rise above 10(10). At this value hydroxy-Al in over-saturated, alkaline solutions begins to undergo rapid conversion to polynuclear species. PMID- 16667664 TI - Wall extensibility and cell hydraulic conductivity decrease in enlarging stem tissues at low water potentials. AB - Measurements with a guillotine psychrometer (H Nonami, JS Boyer [1990] Plant Physiol 94: 1601-1609) indicate that the inhibition of stem growth at low water potentials (low psi(w)) is accompanied by decreases in cell wall extensibility and tissue hydraulic conductance to water that eventually limit growth rate in soybean (Glycine max L. Merr.). To check this conclusion, we measured cell wall properties and cell hydraulic conductivities with independent techniques in soybean seedlings grown and treated the same way, i.e. grown in the dark and exposed to low psi(w) by transplanting dark grown seedlings to vermiculite of low water content. Wall properties were measured with an extensiometer modified for intact plants, and conductances were measured with a cell pressure probe in intact plants. Theory was developed to relate the wall measurements to those with the psychrometer. In the elongation zone, the plastic deformability of the walls decreased when measured with the extensiometer while growth was inhibited at low psi(w). It increased during a modest growth recovery. This behavior was the same as that for the wall extensibility observed previously with the psychrometer. Tissue that was killed before measurement with the extensiometer also showed a similar response, indicating that changes in wall extensibility represented changes in wall physical properties and not rates of wall biosynthesis. The elastic compliance (reciprocal of bulk elastic modulus) did not change in the elongating or mature tissue. The hydraulic conductivity of cortical cells decreased in the elongating tissue and increased slightly during growth recovery in a response similar to that observed with the psychrometer. We conclude that the plastic properties of the cell walls and the conductance of the cells to water were decreased at low psi(w) but that the elastic properties of the walls were of little consequence in this response. PMID- 16667666 TI - Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat. AB - Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25 degrees C growth chamber cultivated, or from plants given 38 degrees C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25 degrees C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42 degrees C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25 degrees C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25 degrees C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25 degrees C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times. PMID- 16667667 TI - Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia. AB - Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines. PMID- 16667668 TI - Purification and Characterization of Glutamyl-tRNA Synthetase : An Enzyme Involved in Chlorophyll Biosynthesis. AB - Chlorophyll biosynthesis starts with the synthesis of glutamyl-tRNA (glu-tRNA) by a glutamyl-tRNA synthetase (Glu RS). The glu-tRNA is subsequently transformed to delta-aminolevulinic acid (ALA), which is a committed and regulated precursor in the chlorophyll biosynthetic pathway. The Glu RS from a green alga, Chlamydomonas reinhardtii, was purified and shown to be able to synthesize glu-tRNA and to participate in ALA synthesis in a coupled enzyme assay. Physical and chemical characterization of the purified Glu RS indicated that the enzyme had been purified to homogeneity. The purified enzyme has a native molecular weight of 60,000, an isoelectric point of 4.6, and it formed a single band of 32,500 daltons when analyzed by a silver stained denaturing gel. The N-terminal amino acid sequence of the 32,500 dalton protein was determined to be Asn-Lys-Val-Ala Leu-Leu-Gly-Ala-Ala-Gly. The molecular weight analyses together with the unambiguous N-terminal amino acid sequence obtained from the purified enzyme suggested that the native enzyme was composed of two identical subunits. Polyclonal antibodies raised against the purified and denatured enzyme were able to inhibit the activity of the native enzyme and to interact specifically with the 32,500 dalton band on Western blots. Thus, the antibodies provided an additional linkage for the structural and functional identities of the enzyme. In vitro experiments showed that over 90% of the glu RS activity was inhibited by 5 micromolar heme, which suggested that Glu RS may be a regulated enzyme in the chlorophyll biosynthetic pathway. PMID- 16667669 TI - Many maize inbreds lack an endosperm cytosolic phosphoglucomutase. AB - Starch gel electrophoresis of extracts from developing maize (Zea mays L.) endosperms 22 days postpollination reveals only a single zone of phosphoglucomutase activity in the majority of the inbreds tested. The other inbreds had the expected two zones of activity. The activity that is present in all inbreds is the amyloplast isozyme while the absent form is a cytosolic enzyme. The lack of the cytosolic isozyme has no discernible phenotypic consequences. PMID- 16667670 TI - Immunocytolocalization of Plasma Membrane H-ATPase. AB - The localization of plasma membrane H(+)-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific anti-ATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a ;two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H(+) currents, which leave nongrowing parts and enter growing tips. PMID- 16667671 TI - Salt-soluble lectins of corn grain. AB - Lectins extracted from corn (Zea mays L.) kernel with Tris-HCl buffer pH 7.5 were isolated from the crude extract by affinity chromatography on Sepharose 6B-N acetyl-d-galactosamine and Sepharose 6B-methylalpha-d-mannoside, and also by lectin affinity chromatography using concanavalin A and Lens culinaris lectin as ligands. According to preferential monosaccharide specificity, salt-soluble lectins of corn seed comprise at least two distinct types: N-acetyl-d galactosamine-interactive and mannose-interactive lectins. The extracted lectins are unstable, with a tendency to form aggregates during storage. PMID- 16667672 TI - On ethylene and stem elongation in green pea seedlings. AB - Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10(-5) molar indoleacetic acid (IAA), with submaximal responses occurring at 10(-4) and 10(-3) molar. Accompanying elongation at concentrations of IAA of 10(-6) to 10(-3) molar was production of ethylene, with the amount increasing up to 10(-4) molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10(-5) molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable. PMID- 16667673 TI - Metabolism of l-Canavanine and l-Canaline in Leguminous Plants. AB - Massive accumulation of l-canavanine, the 2-amino-4-(guanidinooxy)butyric acid structural analog of l-arginine, occurs in many legumes. Accumulation of large amounts of this nonprotein amino acid results in large part from canavanine's protective efficacy; it forms an effective chemical barrier to predation, disease, and even competition with other plants. Diversion of metabolic resources for the synthesis and storage of appreciable canavanine does not place an inordinate burden on the plant. Catabolism of this nonprotein amino acid provides respiratory carbon, generates essential primary metabolites, and ammoniacal nitrogen for the developing plant. PMID- 16667674 TI - Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : III. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids. AB - Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5 trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-(14)C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia. PMID- 16667675 TI - Immunocharacterization of NADH-Glutamate Dehydrogenase from Vitis vinifera L. AB - Rabbit antiserum was raised against NADH-glutamate dehydrogenase (GDH) isoenzyme 1, purified from leaves of Vitis vinifera L. cv Soultanina and its specificity was tested. This antiserum was used for immunocharacterization of the GDH from leaf, shoot, and root tissues. The antiserum recognized the seven isoenzymes of NADH-GDH and precipitated all the enzyme activity from the three tissues tested. Western blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kilodaltons corresponding to NADH-GDH subunit. Results, based on the immunological studies, revealed that NADH-GDH from leaf, shoot, and root tissues are closely related proteins. Furthermore, addition of ammonium ions to the culture medium of in vitro grown explants resulted in a significant increase in NADH-GDH activity in root, shoot, and leaf tissues. PMID- 16667676 TI - Flowering Response of Pharbitis nil to Agents Affecting Cytoplasmic pH. AB - Permeant weak acids and auxins have been shown to reduce the cytosplasmic pH in several systems. Lactic, citric, formic, butyric, salicylic, parahydroxybenzoic, propionic acid, and sodium propionate inhibited the flowering response of Pharbitis nil seedlings when applied immediately before an inductive dark period. The acidic auxins IAA, indolebutyric, and alpha-naphtaleneacetic acid, as well as the nonacidic auxin alpha-naphtaleneaceteamid, also inhibited the flowering response. Inhibition was generally more pronounced with a 12-hour than with a 16 hour dark period. Salicylic acid and sodium propionate shifted the response curve of the dark period by about 2 hours. Salicyclic acid, sodium propionate, and indolebutyric acid were inhibitory when applied during the first few hours of the dark period. The permeant weak bases NH(4)Cl, procaine, and trisodium citrate enhanced the flowering response. NH(4)Cl reduced the length of the critical dark period. The inhibition of flowering by acids and auxins as well as the promotion of flowering by bases was obtained even when only the cotyledons had been treated. The inhibition of floral induction by auxins may not be dependent on their effect on the cytoplasmic pH. PMID- 16667677 TI - Movement of Cations through Cuticles of Citrus aurantium and Acer saccharum: Diffusion Potentials in Mixed Salt Solutions. AB - We examined some biophysical mechanisms of ion migration across leaf cuticles enzymatically isolated from Acer saccharum L. and Citrus aurantium L. leaves. Diffusion potential measurements were used to calculate the permeabilities of Cl( ), Li(+), Na(+), and Cs(+) ions all as a ratio with respect to the permeability of K(+) in cuticles. In 2 millimolar ionic strength solutions the permeability sequence from high to low was K = Cs > Na > Li >> Cl. When the outer and inner surfaces of cuticles were bathed in artificial precipitation and artificial apoplast, respectively, diffusion potentials ranging from -52 to -91 millivolts were measured (inside negative). The Goldman equation predicted that the measured potentials were enough to increase the driving force on the accumulation of heavy metals by a factor of 4 to 7. Other ions migrate with forces 3 to 10 times less than predicted by the Goldman equation for concentration differences alone. Our analysis showed that Ca(2+), and perhaps Mg(2+), might even be accumulated against concentration gradients under some circumstances. Their uptake was apparently driven by the diffusion potentials created by the outward migration of monovalent salts. We feel that future models predicting leaching of nutrients from trees during acid rain events must be modified to account for the probable influence of diffusion potentials on ion migration. PMID- 16667678 TI - Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L. AB - [17-(13)C,(3)H]-Labeled gibberellin A(20) (GA(20)), GA(5), and GA(1) were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). (13)C-Labeled GA(29), GA(8), GA(5), GA(1), and 3-epi-GA(1), as well as unmetabolized [(13)C]GA(20), were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-(13)C, (3)H]GA(20) to all three genotypes. (13)C-Labeled GA(8) and 3-epi G(1), as well as unmetabolized [(13)C]GA(1), were identified by GC-SIM from feeds of [17-(13)C, (3)H]GA(1) to all three genotypes. From feeds of [17-(13)C, (3)H]GA(5), (13)C-labeled GA(3) and the GA(3)-isolactone, as well as unmetabolized [(13)C]GA(5), were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-(13)C, (3)H]GA(5) to [(13)C]GA(1), either by full scan GC-mass spectrometry or by GC SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA(20), as follows: (a) GA(20) --> GA(1) --> GA(8); (b) GA(20) --> GA(5) --> GA(3); and (c) GA(20) --> GA(29). The in vivo biogenesis of GA(3) from GA(5), as well as the origin of GA(5) from GA(20), are conclusively established for the first time in a higher plant (maize shoots). PMID- 16667679 TI - Aluminum Ions Induce Oat Protoplasts to Produce an Extracellular (1-->3)beta-d Glucan. AB - Aluminum chloride induced mesophyll protoplasts of oat (Avena sativa) to produce an extracellular polysaccharide (EPS). EPS induced by AlCl(3) appeared identical to that produced in response to the phytotoxin victorin (JD Walton, ED Earle [1985] Planta 165: 407-415). Al ions at 1 millimolar were toxic to protoplasts, but maximum EPS production occurred at a sublethal concentration of 200 micromolar, assayed at pH 6.0. As measured by incorporation of [(14)C]glucose, AlCl(3) stimulated EPS production 10- to 15- fold. Pretreatment of protoplasts with cycloheximide prevented EPS production but not cell death in response to AlCl(3), indicating that protein synthesis was necessary for EPS production but not for the phytotoxicity of Al ions. The trivalent salts of Y, Yb, Gd, and In also induced EPS production but those of Sc, Fe, Ga, Cr, and La did not. Mesophyll protoplasts from an acid-soil tolerant oat cultivar, Coker 83-23, produced less EPS in response to AlCl(3) than the acid-soil sensitive cultivar Fla 501. EPS was also produced by wheat (Triticum aestivum) and barley (Hordeum vulgare) protoplasts in response to AlCl(3). An Al-tolerant cultivar of wheat, Atlas, produced less EPS than an Al-sensitive cultivar, Scout, but an Al-tolerant cultivar of barley, Dayton, produced more than the Al-sensitive cultivar Kearney. Therefore, production of EPS by protoplasts in response to Al ions did not appear to be related to Al ion tolerance at the level of whole plants. EPS fluoresced in the presence of Calcofluor and Sirofluor and was degraded by purified laminarinase [(1-->3)beta-d-glucanase] but not pectinase (polygalacturonase). EPS was composed solely of glucose in 1-->3 linkages; hence it is a (1-->3)beta-d glucan (callose). PMID- 16667680 TI - Biosynthetic Origin of Gibberellins A(3) and A(7) in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica. AB - Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A(9) (GA(9)) and 2,3-dehydroGA(9) to GA(7); GA(9) was also metabolized to GA(4) in a branch pathway. The preparation from Marah seeds also metabolized GA(5) to GA(3) in high yield; GA(6) was a minor product and was not metabolized to GA(3). Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA(5) to GA(3) and of 2,3-dehydroGA(9) to GA(7) occurs with the loss of the 1beta-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1beta,2beta-(2)H(2)]GA(4), was metabolized to [1,2-(2)H(2)]GA(3) with the loss of the 1alpha- and 2alpha hydrogens. These results provide further evidence that the biosynthetic origin of GA(3) and GA(7) in higher plants is different from that in the fungus Gibberella fujikuroi. PMID- 16667681 TI - Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L. AB - Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A(1), (GA(1)), GA(9), and GA(20) caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA(53), or GA(8). Moreover, of the biologically active compounds, GA(9) was the most active followed closely by GA(1). In thermoinduced plants (4-week cold treatment at 6 degrees C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA(9) was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA(20). No such increase in activity was observed for either GA([unk]) or GA(53). The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA(9) is the endogenous mediator of thermoinduced stem growth in field pennycress. PMID- 16667682 TI - Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde. AB - Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [(2)H]-ent-kaurenoic acid (KA) and [(14)C]-gibberellin A(12)-aldehyde (GA(12)-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [(2)H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [(2)H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A(9) (GA(9)) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA(9). In contrast, thermo- and noninduced leaves metabolized exogenous [(2)H]-KA into GA(20) equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [(14)C]-GA(12) aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA(12)-aldehyde. PMID- 16667683 TI - Use of chemical fractionation and proton nuclear magnetic resonance to probe the physical structure of the primary plant cell wall. AB - Proton magnetic resonance has been used to monitor the microscopic physical properties of etiolated hypocotyl cell walls from Phaseolus vulgaris L. at all stages in a series of chemical fractionations with ammonium oxalate and potassium hydroxide. Solid echo measurements indicate that 75% of the polymers in the intact cell wall, including the cellulose and most of the hemicelluloses, are arranged such that there is almost complete restraint of molecular motion. The chemical fractionations generally altered the physical structures of the remaining cell wall components. Digestion with 0.25% ammonium oxalate/oxalic acid solubilized the pectin and increased the mobility of the hemicellulose I component. Extraction with 4% potassium hydroxide removed the hemicellulose I component and loosened the hemicellulose II. Further extraction with 24% potassium hydroxide removed the hemicellulose II and loosened some of the cellulose. The cellulose crystallinity, as monitored by Jeener echo measurements decreased from 83% to 63% during these fractionations. We conclude that, while hemicellulose I is firmly attached to hemicellulose II, it is not in a closely packed structure. Hemicellulose II is strongly bound to cellulose and has a much more closely packed structure. PMID- 16667684 TI - Calcium and proton transport in membrane vesicles from barley roots. AB - Ca(2+) uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of (45)Ca(2+) was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca(2+) uptake coincided with the peak of proton transport by the tonoplast H(+)-ATPase. Depending on the concentration of Ca(2+) in the assay, Ca(2+) uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca(2+) uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that (45)Ca uptake was driven by the low affinity, high capacity tonoplast Ca(2+)/nH(+) antiporter and also by a high affinity, lower capacity Ca(2+)-ATPase. The Ca(2+)-ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca(2+) transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-man-nosyl synthase activities. A small shoulder of Ca(2+) uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca(2+)-ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca(2+) concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca(2+) created 800- to 2000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca(2+) on ATP-generated pH gradients are discussed and the suggestion is made that Ca(2+) dissipates pH gradients by a different mechanism than is responsible for Ca(2+) uptake into tonoplast vesicles. PMID- 16667685 TI - Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria. AB - In this article we compare the kinetic behavior toward pyridine nucleotides (NAD(+), NADH) of NAD(+)-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD(+) was the varied substrate. However, the NAD(+)-linked malic enzyme exhibited the weakest affinity for NAD(+) and the lowest sensitivity for NADH. It is suggested that NAD(+)-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I. PMID- 16667686 TI - Comparison of gibberellins in normal and slender barley seedlings. AB - Gibberellins A(1), A(3), A(8), A(19), A(20), and A(29) were identified by full scan gas chromatography-mass spectrometry in leaf sheath segments of 7-day-old barley (Hordeum vulgare L. cv Golden Promise) seedlings grown at 20 degrees C under long days. In a segregating population of barley, cv Herta (Cb 3014), containing the recessive slender allele, (sln 1) the concentration of GA(1) and GA(3) was reduced by 10-fold and 6-fold, respectively, in rapidly growing homozygous slender, compared with normal, leaf sheath segments. However, the concentration of the C(20) precursor, GA(19), was nearly 2-fold greater in slender than in normal seedlings. There was little difference in the ABA content of sheath segments between the two genotypes. The gibberellin biosynthesis inhibitor, paclobutrazol, reduced the final sheath length of normal segregants (50% inhibition at 15 micromolar) but had no effect on the growth of slender seedlings at concentrations below 100 micromolar. There was a 15-fold and 4-fold reduction in GA(1) and GA(3), respectively, in sheath segments of 8-day-old normal seedlings following application of 10 micromolar paclobutrazol. The same treatment also reduced the already low concentrations of these gibberellins in slender segregants. The results show that the pool sizes of gibberellins A(1) and A(3) are small in slender barley and that leaf sheath extension in this genotype appears to be gibberellin-independent. The relationship between gibberellin status and tissue growth-rate in slender barley is contrasted with other gibberellin nonresponsive, but dwarf, mutants of wheat (Triticum aestivum) and maize (Zea mays). PMID- 16667687 TI - Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings. AB - Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a K(m) for acetyl-CoA of 74 micromolar and a V(max) of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a K(is) of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate. PMID- 16667688 TI - Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric. AB - During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO(2) respired during ripening was positively correlated with sugar accumulation (R(2) = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO(2) was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP. PMID- 16667689 TI - Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts. AB - Intact spinach (Spinacia oleracea) chloroplasts, pulse-labeled with [(14)C]acetate, desaturate newly formed fatty acids as ester groups of monogalactosyl diacylglycerol in a subsequent chase in the dark. Rupture of pulse labeled chloroplasts by addition of a detergent solution 3-([3 cholamidopropyl]dimethylammonio)-1-propane sulfonate preserves part of this desaturation activity. Direct addition of different free fatty acids together with appropriate cofactors to detergent-ruptured chloroplasts results in fatty acid labeling of monogalactosyl diacylglycerol. During subsequent incubation these lipid-linked fatty acids are desaturated, i.e. 18:1 to 18:2 and 18:3 and to a small extent also 16:0 to 16:3. The formation of 18:2 was also observed after incorporation of 18:1 into sulfolipid and phosphatidyl choline. Density gradient centrifugation separated a membrane fraction from detergent-ruptured chloroplasts which in the presence of appropriate cofactors incorporated 18:1 and 18:2 into the above-mentioned lipids. In the light, desaturation was dependent on added ferredoxin, whereas in the dark, in addition to ferredoxin NAD(P)H was also required. Preliminary evidence for the involvement of membrane-bound ferredoxin:NADP oxidoreductase (FNR) as a third component of desaturation in the dark was obtained by inhibitor studies including antibodies against FNR. Desaturation of lipid-bound 18:1 and 18:2 resembles stearoyl-ACP desaturation with respect to its requirement of reduced ferredoxin and oxygen. PMID- 16667690 TI - Is there a third photoreceptor involved in the control of chloroplast movements in mougeotia? AB - The photometric method was used to test a possibility proposed recently that a new photoreceptor with maximum activity at 620 nm is involved in mediating chloroplast rotation in Mougeotia (Z Lechowski, J Bialczyk [1988] Plant Physiol 88: 189-193). The hypothesis was tested under conditions of continuous dichromatic unilateral or mutually perpendicular irradiation with red light of wavelengths 620 or 660 (680) nanometers and far-red. When the red light was polarized parallel to the long cell axis, chloroplast response could be monitored by changing the direction of far-red irradiation. The level of the response obtained with red and far-red applied from the same direction depended on far-red intensity: at higher fluence rates the maximum response was shifted to longer wavelengths of red light. A high fluence rate of far-red inhibited the response. The absorption coefficients of Mougeotia chloroplasts were measured for the studied wave-lengths using the microphotometric method. Possible impact of absorption by the chloroplast on photoreception has been discussed. Current and previous results can be interpreted in terms of phytochrome action and do not support the involvement of the hypothetical 620 nanometer photoreceptor. PMID- 16667691 TI - Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures. AB - The isoflavonoid conjugates medicarpin-3-O-glucoside-6''-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6''-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with [(14)C]phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, [(14)C]phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of (14)C-labeled, elicited cells with l-alpha aminooxy-beta-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures. PMID- 16667692 TI - Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide. AB - Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000 fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine. PMID- 16667693 TI - Activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) by rubisco activase : effects of some sugar phosphates. AB - The activation of purified ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) has been studied in the presence of sugar phosphates, and the effect of rubisco activase on this process determined. During an 11-minute time course at pH 7.7 and 11 micromolar CO(2), the activation of rubisco was strongly inhibited by ribulose-1,5-bisphosphate (4 millimolar), fructose-1,6-bisphosphate (1 millimolar) and ribose 5-phosphate (5 millimolar), but this inhibition was overcome by the addition of rubisco activase and activation then proceeded to a greater extent than spontaneous activation of rubisco. Glycerate 3-phosphate (20 millomolar) slowed the initial rate but not the extent of activation and rubisco activase had no effect on this. The activation of rubisco was shown to be affected by phosphoenolpyruvate (3 millimolar) but not by creatine phosphate (3 millimolar) or ATP (3 millimolar), and the creatine-phosphate/creatine phosphokinase system was used to generate the high ATP/ADP quotients required for rubisco activase to function. ATP was shown to be required for the rubisco activase-dependent rubisco activation in the presence of fructose-1,6 bisphosphate (1 millimolar). It is concluded that rubisco activase has a mixed specificity for some sugar phosphate-bound forms of rubisco, but has low or no activity with others. Some possible bases for these differences among sugar phosphates are discussed but remain to be established. PMID- 16667694 TI - A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization. AB - We previously described the purification and characterization of a 37,000 M(r) cysteine proteinase, designated EP-A, from gibberellic acid (GA(3))-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M(r) of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M(r) as determined by gradient SDS-PAGE. PMID- 16667695 TI - Photosynthesis and photosynthate partitioning in n(2)-fixing soybeans. AB - Leaf area, chlorophyll content, net CO(2) photoassimilation, and the partitioning of fixed carbon between leaf sucrose and starch and soluble protein were examined in Glycine max (L) Merr. cv Williams grown under three different nitrogen regimes. One group (Nod+/+) was inoculated with Bradyrhizobium and watered daily with a nutrient solution containing 6 millimolar NH(4)NO(3). A second set (Nod+/ ) was inoculated and had N(2) fixation as its sole source of nitrogen. A third group (Nod(-)) was not inoculated and was watered daily with a nutrient solution containing 6 millimolar NH(4)NO(3). The mean net micromole CO(2) uptake per square decimeter per hour of the most recently matured source leaves was similar among the three groups of plants, being about 310. Mean leaf area of the source leaves, monitored for net photosynthesis was also similar. However, the mean milligram of chlorophyll per square decimeter of Nod+/- test leaves was about 50% lower than the other groups' leaves and indicated nitrogen deficiency. Thus, Nod+/- utilized their chlorophyll more efficiently for photosynthetic CO(2) uptake than the plants of the other treatments. The ratio of foliar carbohydrate:protein content was high in Nod+/- but low in the plants from the other two treatments. This inverse relationship between foliar protein and carbohydrate content suggests that more fixed carbon is diverted to the synthesis of protein when nitrogen availability is high. It was also found that Nod+/- sequestered more storage protein in their paraveinal mesophyll than plants of the other treatments. This study indicates that when inorganic nitrogen regimes are used to control photosynthate partitioning, then both leaf carbohydrate and leaf protein must be considered as end products of carbon assimilate allocation. PMID- 16667696 TI - DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves: I. Evidence for Carrier-Mediated, Electrogenic Flux through Multiple Transport Systems. AB - Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. DeltapH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent K(m) values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. DeltapH-Dependent isoleucine and arginine transport exhibited biphasic kinetics, suggesting these amino acids may be transported by at least two transport systems. Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (DeltaPsi) on DeltapH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When DeltaPsi was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative DeltaPsi, alanine transport increased sixfold. These results are consistent with an electrogenic transport process which results in a net flux of positive charge into the vesicles. The effect of changing DeltaPsi on the kinetics of alanine transport altered V(max) with no apparent change in K(m). Amino acid transport was inhibited by the protein modifier diethyl pyrocarbonate, but was insensitive to N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, p chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N' dicyclohexylcarbodiimide. Four amino acid symport systems, two neutral, one acidic, and one basic, were resolved based on inter-amino acid competition experiments. One neutral system appears to be active for all neutral amino acids while the second exhibited a low affinity for isoleucine, threonine, valine, and proline. Although each symport was relatively specific for a given group of amino acids, each system exhibited some crossover specificity for amino acids in other groups. PMID- 16667697 TI - Determination of oxygen emission and uptake in leaves by pulsed, time resolved photoacoustics. AB - Pulsed, time resolved photoacoustics has sufficient sensitivity to determine oxygen emission and uptake by single turnover flashes to leaves. The advantage over previous methodologies is that when combined with single turnover flashes the kinetics of the thermal and the gas signals can be resolved to 0.1 millisecond and separated. The S-state oscillations of oxygen formation are readily observed. The gas signal from common spongy leaves such as spinach (Spinacia sp.), Japanese andromeda (Pieris japonica), mock orange (Philadelphus coronarius) and viburnum (Viburnum tomentosum), after correction for instrumental rise time, show a lag of only 1 millisecond and a rise time of 5 milliseconds in the formation of oxygen. Thus a recent proposal that the formation of oxygen requires over 100 milliseconds cannot be true for choroplasts in vivo. The rapid emission is correlated with structure of the leaf. At low light flash energies a rapid gas uptake is observed. The uptake has slightly slower kinetics than oxygen evolution, and its magnitude increases with damage to the leaf. The pulse methodology shows that the uptake begins with the very first flash after dark adaption, and allows the detection of a positive signal (oxygen) on the third flash. These observations, the long wavelength of excitation (695 nanometers) and the magnitude of the signal support the contention that the gas uptake is oxygen reduction by electrons from photosystem I. These results show that important physiological aspects of a leaf can be studied by pulsed, time resolved photoacoustics. PMID- 16667698 TI - Pattern of the Cyanide-Potential in Developing Fruits : Implications for Plants Accumulating Cyanogenic Monoglucosides (Phaseolus lunatus) or Cyanogenic Diglucosides in Their Seeds (Linum usitatissimum, Prunus amygdalus). AB - The absolute cyanide content of developing fruits was determined in Costa Rican wild lima beans (Phaseolus lunatus), oil flax (Linum usitatissimum), and bitter almonds (Prunus amygdalus). The cyanide potential (HCN-p) of the lima bean and the almond fruit began to increase shortly after anthesis and then stopped before fruit maturity. In contrast, the flax inflorescence had a higher HCN-p in absolute terms than the mature flax fruit. At all times of its development the bean fruit contained the monoglucosides linamarin and lotaustralin. The almond and the flax fruits contained, at anthesis, the monoglucosides prunasin, and linamarin and lotaustralin, respectively, while, at maturity, only the corresponding diglucosides amygdalin, and linustatin and neolinustatin, respectively, were present. PMID- 16667699 TI - Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum. AB - The rate of NH(4) (+) assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH(4) (+) assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H(14)CO(-) (3) into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C(3) organism. PMID- 16667700 TI - Isolation of chloroplastic phosphoglycerate kinase : kinetics of the two-enzyme phosphoglycerate kinase/glyceraldehyde-3-phosphate dehydrogenase couple. AB - We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts (J Macioszek, LE Anderson [1987] Biochim Biophys Acta 892: 185-190). Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast. PMID- 16667701 TI - Purification and characterization of zein-degrading proteases from endosperm of germinating maize seeds. AB - We have purified a group of four proteases (molecular mass 26-33 kilodalton) from germinating seeds of maize (Zea mays L. var W64A) using ammonium sulfate and isoelectric precipitations, anion exchange chromatography, and electroelution from preparative nondenaturing polyacrylamide gels. Their appearance in the endosperm of germinating seeds coincides with the onset of zein degradation. We have shown that these proteases degrade zeins dissolved in alcoholic solution as well as aggregated in protein bodies from developing maize kernels. The apparent molecular weights and net negative charges of each of these proteases are very similar. Additionally, they are inhibited by thiol-blocking agents and activated by reducing compounds. These characteristics suggest that they are a group of cysteine proteases involved in the first steps of storage protein degradation. PMID- 16667702 TI - Photoregulation of the Light-Harvesting Chlorophyll Protein Complex Associated with Photosystem II in Dunaliella tertiolecta: Evidence that Apoprotein Abundance but Not Stability Requires Chlorophyll Synthesis. AB - The marine chlorophyte Dunaliella tertiolecta Butcher responds to a one-step transition from a high growth irradiance level (700 micromoles quanta per square meter per second) to a low growth irradiance level (70 micromoles quanta per square meter per second) by increasing the total amount of light-harvesting chlorophyll (Chl) a/b binding protein associated with photosystem II (LHC II), and by modifying the relative abundance of individual LHC II apoproteins. When high light-adapted cells were incubated with gabaculine, which inhibits Chl synthesis, and transferred to low light, the LHC II apoproteins were still synthesized and the (35)S-labeled LHC II apoproteins remained stable after a 24 hour chase. These results suggest that Chl synthesis is not required for stability of the LHC II apoproteins in this alga. However, when the control cells are transferred from high light to low light, the amount of the four LHC II apoproteins per cell increases, whereas it does not in the presence of gabaculine. These results suggest that Chl synthesis is required for a photoadaptive increase in the cellular level of LHC II. PMID- 16667703 TI - Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.). AB - Hydrogen peroxide (H(2)O(2)) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50 degrees C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K(m) = 40 millimolar) and H(2)O(2) (apparent K(m) = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed. PMID- 16667704 TI - Degradation of Native Starch Granules by Barley alpha-Glucosidases. AB - The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to alpha-amylases. We report that barley (Hordeum vulgare L.) seed alpha-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant alpha-amylase isozymes. Two alpha-glucosidase charge isoforms were used individually and in combination with purified barley alpha-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl alpha-glucosidase was combined with either high or low pl alpha-amylase. Synergism was also found when low pl alpha-glucosidase was combined with alpha-amylases. Scanning electron micrographs revealed that starch granule degradation by alpha-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of alpha-glucosidases and alpha-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by alpha-amylase alone. As the presence of alpha-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the alpha-glucosidases to hydrolyze glucosidic bonds other than alpha-1,4- and alpha 1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by alpha-amylases. Since both alpha-glucosidase and alpha-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro. PMID- 16667705 TI - Phosphorus stress effects on assimilation of nitrate. AB - An experiment was conducted to investigate alterations in uptake and assimilation of NO(3) (-) by phosphorus-stressed plants. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were deprived of an external phosphorus (P) supply for 12 days. On selected days, plants were exposed to (15)NO(3) (-) during the 12 hour light period to determine changes in NO(3) ( ) assimilation as the P deficiency progressed. Decreased whole-plant growth was evident after 3 days of P deprivation and became more pronounced with time, but root growth was unaffected until after day 6. Uptake of (15)NO(3) (-) per gram root dry weight and translocation of absorbed (15)NO(3) (-) out of the root were noticeably restricted in -P plants by day 3, and effects on both increased in severity with time. Whole-plant reduction of (15)NO(3) (-) and (15)N incorporation into insoluble reduced-N in the shoot decreased after day 3. Although the P limitation was associated with a substantial accumulation of amino acids in the shoot, there was no indication of excessive accumulation of soluble reduced-(15)N in the shoot during the 12 hour (15)NO(3) (-) exposure periods. The results indicate that alterations in NO(3) (-) transport processes in the root system are the primary initial responses limiting synthesis of shoot protein in P stressed plants. Elevated amino acid levels evidently are associated with enhanced degradation of protein rather than inhibition of concurrent protein synthesis. PMID- 16667706 TI - Localization and quantitation of chloroplast enzymes and light-harvesting components using immunocytochemical methods. AB - Seven chloroplast proteins were localized in Porphyridium cruentum (ATCC 50161) by immunolabeling with colloidal gold on electron microscope sections of log phase cells grown under red, green, and white light. Ribulose bisphosphate carboxylase labeling occurred almost exclusively in the pyrenoid. The major apoproteins of photosystem I (56-64 kD) occurred mostly over the stromal thylakoid region and also appeared over the thylakoids passing through the pyrenoid. Labeling for photosystem II core components (D2 and a 45 kD Chl-binding protein), for phycobilisomes (allophycocyanin, and a 91 kD L(cm) linker) and for ATP synthase (beta subunit) were predominantly present in the thylakoid region but not in the pyrenoid region of the chloroplast. Red light cells had increased labeling per thylakoid length for polypeptides of photosystem II and of phycobilisomes, while photosystem I density decreased, compared to white light cells. Conversely, green light cells had a decreased density of photosystem II and phycobilisome polypeptides, while photosystem I density changed little compared with white light cells. A comparison of the immunogold labeling results with data from spectroscopic methods and from rocket immunoelectrophoresis indicates that it can provide a quantitative measure of the relative amounts of protein components as well as their localization in specific organellar compartments. PMID- 16667707 TI - Apparent processing of a soybean oil body protein accompanies the onset of oil mobilization. AB - The membrane surrounding the oil body contains several different specific polypeptides. To study the biosynthesis and posttranslational modification of these polypeptides we have prepared monoclonal antibodies against purified oil bodies of soybean (Glycine max). Three of the five monoclonals selected recognize a molecular mass 34 kilodalton protein (P34). Epitope mapping of CNBr and proteolytic fragments of P34 indicates that two of the anti-P34 monoclonal antibodies are directed at different epitopes. P34 is accumulated during seed maturation at the same time as the reserve proteins and oil. SDS/PAGE-immunoblots of germinating soybean seed cotyledons indicate that the protein is initially present as a molecular mass 34 kilodalton polypeptide and is processed to molecular mass 32 kilodalton on the fourth through sixth days of seedling growth simultaneously with the onset of oil mobilization. A comparison of reduced and carboxymethylated oil body proteins with nonreduced proteins by SDS/PAGE indicates that P34 exists in vivo as a dimer of molecular mass 58 kilodalton. Comparing the amino terminal sequences of P34 and P32 indicates that their difference is at least in part due to the removal of the amino terminus of P34. The amino terminal sequences of P34 and P32 were aligned to show that the transition of P34 to P32 was accompanied by the removal of a hydrophilic decapeptide (KKMKKEQYSC) at the amino terminus of P34. Hopp-Woods hydrophilicity analysis of the deleted amino terminus of P34 shows that it is more hydrophilic and charged than the sequence of the protein which immediately follows. PMID- 16667708 TI - Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942. AB - Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO(2) and HCO(3) (-) transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O(2) evolution studies. The K(i) for COS inhibition was 29 micromolar for CO(2) transport and 110 micromolar for HCO(3) (-) transport. These results support a model of inorganic carbon transport with a central CO(2) pump and an inducible HCO(3) (-) utilizing accessory protein which supplies CO(2) to the primary pump. PMID- 16667709 TI - Regulation of photosynthate partitioning into starch in soybean leaves : response to natural daylight. AB - Studies conducted in controlled environments indicate that daylength affects the proportion of photosynthate stored in leaves as starch or sucrose. To examine the response of partitioning to natural daylight, soybeans (Glycine max [L.] Merr. cv Williams) were grown at 12 different times between May and November in a constant temperature greenhouse without supplemental lighting. Plants were transferred from the greenhouse to a controlled environment chamber at the end of civil twilight at a set developmental stage (expanding seventh trifoliolate leaf, counting acropetally). Net photosynthesis and the accumulation of starch and sugar in fully expanded fourth trifoliolate leaves were determined the following day under standard conditions in the chamber (lights-on synchronized with sunrise). Photosynthesis on a leaf area basis decreased about 10% between midsummer and early autumn. Leaf soluble sugar accumulation was low at all harvests. However, a twofold increase in photosynthate partitioning into starch occurred over the same time period, resulting in an 80% increase in absolute starch accumulation rate. Starch was responsible for about 78% of the increase in leaf dry matter during the light at all harvests, indicating that starch accumulation as affected by prior daylight conditions will alter export of photosynthate during the light period. Photosynthate partitioning into starch was linearly correlated with daylength at harvest, prior average peak solar irradiance, and other parameters that correlated with daylength and solar radiation such as harvested top dry matter. The relation between growth and seasonal changes in daylight (including daylength, irradiance, and light integral) are discussed in relation to photosynthate partitioning under field conditions. PMID- 16667710 TI - Acclimation of Photosynthetic Light Reactions during Induction of Inorganic Carbon Accumulation in the Green Alga Chlamydomonas reinhardtii. AB - Cells of the unicellular green algae Chlamydomonas reinhardtii were grown in high dissolved inorganic carbon (DIC) concentrations (supplied with 50 milliliters per liter CO(2)[g]) and transferred to low DIC concentrations (supplied with Rb(+) > Na(+) approximately Cs(+) approximately Li(+). It is blocked by Cs(+) and by Ba(2+) in a voltage dependent manner, exhibiting a ;long-pore' behavior, similarly to various types of K(+) channels in animal systems. Cadmium, known for its blockage of Ca(2+) channels in animal systems, and Gd(3+), closely related to La(3+), which also blocks Ca(2+) channels in animal cells, both block K(+) currents in Samanea in a voltage-independent manner, and without interfering with the kinetics of the currents. The suggested mechanism of block is either (a) by a direct interaction with the K(+) channel, but external to its lumen, or, alternatively, (b) by blocking putative Ca(2+) channels, and preventing the influx of Ca(2+), on which the activation of the K(+) channels may be dependent. PMID- 16667731 TI - Genetic analysis of the role of gibberellin in the red light inhibition of stem elongation in etiolated seedlings. AB - Red light causes a reduction in the extension growth of dark-grown seedlings. The involvement of gibberellin in this process was tested by screening a number of gibberellin synthesis and gibberellin response mutants of Pisum sativum L. for the kinetic response of stem growth inhibition by red light. Gibberellin deficient dwarfs, produced by mutant alleles at the Le, Na, and Ls loci, and gibberellin response mutants produced by mutant alleles at the La and Cry(2), Lka, and Lkb loci were tested. Extension growth of expanding third internodes of dark-grown seedlings was recorded with high resolution using angular position transducers. Seedlings were treated with red light at a fluence rate of 4 micromoles per square meter per second either continuously or for 75 seconds, and the response was measured over 9 hours. With certain small exceptions, the response to the red light treatments was similar in all the mutants and wild types examined. The lag time for the response was approximately 1 hour and a minimum in growth rate was reached by 3 to 4 hours after the onset of the light treatment. Growth rate depression at this point was about 80%. Seedlings treated with 75 seconds red light recovered growth to a certain extent. Red/far-red treatments indicated that the response was mediated largely by phytochrome. The similar responses to red light among these wild-type and mutant genotypes suggest that the short-term (i.e. 9 hour) response to red light is not mediated by either a reduction in the level of gibberellin or a reduction in the level or affinity of a gibberellin receptor. PMID- 16667732 TI - Rapid and Specific Modulation of Stomatal Conductance by Blue Light in Ivy (Hedera helix) : An Approach to Assess the Stomatal Limitation of Carbon Assimilation. AB - Low intensity (0.015 millimole per square meter per second) blue light applied to leaves of Hedera helix under a high intensity red light background (0.50 millimole per square meter per second red light) induced a specific stomatal opening response, with rapid kinetics comparable to those previously reported for stomata with ;grass type' morphology. The response of stomatal conductance to blue light showed a transient ;overshoot' behavior at high vapor pressure difference (2.25 +/- 0.15 kiloPascals), but not at low vapor pressure difference (VPD) (0.90 +/- 0.10 kilo-Pascal). The blue light-induced conductance increase was accompanied by an increase in net photosynthetic carbon assimilation, mediated by an increase in the intercellular concentration of carbon dioxide. Values of assimilation once the blue light-stimulated conductance increase reached steady state were less than those at the peak of the overshoot, but the ratios of assimilation to transpiration (A/E) and blue light-stimulated DeltaA/DeltaE were greater during the steady-state response than during the overshoot. These results indicate that significant stomatal limitation of assimilation can occur, but that this limitation may improve water use efficiency under high VPD conditions. Under high intensity red light, the decline in A/E associated with an increase in VPD was minimized when conductance was stimulated by additional low intensity blue light. This effect indicates that the blue light response of stomata may be important in H. helix for the optimization of water use efficiency under natural conditions of high irradiance and VPD. PMID- 16667733 TI - Measurement of Phloem transport rates by an indicator-dilution technique. AB - An indicator-dilution technique for the measurement of flow rates, commonly used by animal physiologists for circulation measurements, was adapted to the measurement of phloem translocation rates in the wheat (Triticum aestivum L.) peduncle. The approach is based on the observation that, during the transport of a given amount of solute, its mean concentration will be inversely proportional to flow rate. For phloem transport in the wheat peduncle, the necessary measurements are (a) the time course of tracer kinetics in the peduncle phloem, (b) the volume of sieve tubes and companion cells in the monitored segment of the peduncle, and (c) the amount of tracer transported past that point. The method was evaluated by in situ monitoring of (32)PO(4) transport in pulse-labeling experiments. Specific activities (i.e.(32)P concentrations) of phloem exudate were in good agreement with those calculated from in situ count rates and measured phloem areas. Mass transport rates, calculated from volume flow rates and phloem exudate dry matter content, also agreed well with expected mass transport rates based on measurements of grain growth rate and net CO(2) exchange by the ear. The indicator-dilution technique appears to offer good precision and accuracy for short-term measurements of phloem transport rates in the wheast peduncle and should be useful for other systems as well. In contrast to velocities based on time-delay measurements, it is more precise, more accurate, and produces an estimate of mean, rather than maximum, velocity. Also, since only a single detector is required, it can be applied to very short transport paths. PMID- 16667734 TI - Photosynthetic and Respiratory Activity of Fruiting Forms within the Cotton Canopy. AB - The supply of photosynthates by leaves for reproductive development in cotton (Gossypium hirsutum L.) has been extensively studied. However, the contribution of assimilates derived from the fruiting forms themselves is inconclusive. Field experiments were conducted to document the photosynthetic and respiratory activity of cotton leaves, bracts, and capsule walls from anthesis to fruit maturity. Bracts achieved peak photosynthetic rates of 2.1 micromoles per square meter per second compared with 16.5 micromoles per square meter per second for the subtending leaf. However, unlike the subtending leaf, the bracts did not show a dramatic decline in photosynthesis with increased age, nor was their photosynthesis as sensitive as leaves to low light and water-deficit stress. The capsule wall was only a minor site of (14)CO(2) fixation from the ambient atmosphere. Dark respiration by the developing fruit averaged -18.7 micromoles per square meter per second for 6 days after anthesis and declined to -2.7 micromoles per square meter per second after 40 days. Respiratory loss of CO(2) was maximal at -158 micromoles CO(2) per fruit per hour at 20 days anthesis. Diurnal patterns of dark respiration for the fruit were age dependent and closely correlated with stomatal conductance of the capsule wall. Stomata on the capsule wall of young fruit were functional, but lost this capacity with increasing age. Labeled (14)CO(2) injected into the fruit interior was rapidly assimilated by the capsule wall in the light but not in the dark, while fiber and seed together fixed significant amounts of (14)CO(2) in both the light and dark. These data suggest that cotton fruiting forms, although sites of significant respiratory CO(2) loss, do serve a vital role in the recycling of internal CO(2) and therein, function as important sources of assimilate for reproductive development. PMID- 16667735 TI - Interaction of Elevated Ultraviolet-B Radiation and CO(2) on Productivity and Photosynthetic Characteristics in Wheat, Rice, and Soybean. AB - Wheat (Triticum aestivum L. cv Bannock), rice (Oryza sativa L. cv IR-36), and soybean (Glycine max [L.] Merr cv Essex) were grown in a factorial greenhouse experiment to determine if CO(2)-induced increases in photosynthesis, biomass, and yield are modified by increases in ultraviolet (UV)-B radiation corresponding to stratospheric ozone depletion. The experimental conditions simulated were: (a) an increase in CO(2) concentration from 350 to 650 microliters per liter; (b) an increase in UV-B radiation corresponding to a 10% ozone depletion at the equator; and (c) a and b in combination. Seed yield and total biomass increased significantly with elevated CO(2) in all three species when compared to the control. However, with concurrent increases in UV-B and CO(2), no increase in either seed yield (wheat and rice) or total biomass (rice) was observed with respect to the control. In contrast, CO(2)-induced increases in seed yield and total plant biomass were maintained or increased in soybean within the elevated CO(2), UV-B environment. Whole leaf gas exchange indicated a significant increase in photosynthesis, apparent quantum efficiency (AQE) and water-use-efficiency (WUE) with elevated CO(2) in all 3 species. Including elevated UV-B radiation with high CO(2) eliminated the effect of high CO(2) on photosynthesis and WUE in rice and the increase in AQE associated with high CO(2) in all species. Elevated CO(2) did not change the apparent carboxylation efficiency (ACE) in the three species although the combination of elevated CO(2) and UV-B reduced ACE in wheat and rice. The results of this experiment illustrate that increased UV-B radiation may modify CO(2)-induced increases in biomass, seed yield and photosynthetic parameters and suggest that available data may not adequately characterize the potential effect of future, simultaneous changes in CO(2) concentration and UV-B radiation. PMID- 16667736 TI - Bleaching herbicide flurtamone interferes with phytoene desaturase. AB - The mode of action of the furanone herbicide flurtamone and derivatives was investigated with cress seedlings and with the unicellular cyanobacterium Anacystis. Either in the light or in the dark these compounds inhibited the formation of alpha- and beta-carotene and all of the xanthophylls in the seedlings. Instead, phytoene, a precursor of colored carotenoids, was accumulated. In illuminated seedlings photooxidative destruction of chlorophyll was observed. The I(50) value of flurtamone inhibition of carotenoid biosynthesis in intact Anacystis cells and the K(1) value for interaction of flurtamone with phytoene desaturase with Anacystis thylakoids were 30 and 18 nanomoles, respectively. Concentrations of flurtamone which strongly inhibited carotenoid synthesis had no direct peroxidative activities and did not inhibit photosynthetic electron transport. PMID- 16667737 TI - Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase. AB - The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO(2) (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously. PMID- 16667738 TI - In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots. AB - Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl(2) and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [(3)H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl methanol. PMID- 16667739 TI - Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds. AB - Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-(14)C]oleoyl CoA and malonyl-CoA or from oleoyl-CoA and [2-(14)C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-(14)C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2 (14)C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols. PMID- 16667740 TI - Pyrophosphate Fructose-6-P 1-Phosphotransferase from Tomato Fruit : Evidence for Change during Ripening. AB - Three forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) were purified from both green and red tomato (Lycopersicon esculentum) fruit: (a) a classical form (designated Q(2)) containing alpha- (66 kilodalton) and beta- (60 kilodalton) subunits; (b) a form (Q(1)) containing a beta-doublet subunit; and (c) a form (Q(0)) that appeared to contain a beta-singlet subunit. Several lines of evidence suggested that the different forms occur under physiological conditions. Q(2) was purified to apparent electrophoretic homogeneity; Q(1) and Q(0) were highly purified, but not to homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q(2), 29% (90%); Q(1), 47% (6%); and Q(0), 24% (4%). The major difference distinguishing the red from the green tomato enzymes was the fructose-2,6-bisphosphate (Fru-2,6-P(2))-induced change in K(m) for fructose-6-phosphate (Fru-6-P), the ;green forms' showing markedly enhanced affinity on activation (K(m) decrease of 7-9-fold) and the ;red forms' showing either little change (Q(0), Q(1)) or a relatively small (2.5-fold) affinity increase (Q(2)). The results extend our earlier findings with carrot root to another tissue and indicate that forms of PFP showing low or no affinity increase for Fru 6-P on activation by Fru-2,6-P(2) (here Q(1) and Q(0)) are associated with sugar storage, whereas the classical form (Q(2)), which shows a pronounced affinity increase, is more important for starch storage. PMID- 16667741 TI - Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum). AB - Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID- 16667742 TI - Stamens and Gibberellic Acid in the Regulation of Flavonoid Gene Expression in the Corolla of Petunia hybrida. AB - Stamen removal at an early stage of flower development inhibits anthocyanin synthesis and chalcone flavanon isomerase (CHI) enzyme activity in corollas of Petunia hybrida. The inhibition can be overcome by gibberellic acid (GA(3)) application. Gibberellin also induces anthocyanin synthesis in detached, young green corollas, grown in vitro in a sucrose medium and promotes CHI enzyme activity. Western blot analysis indicates an increase in chalcone synthase (CHS) and CHI protein levels following GA(3) treatment in both the in vivo and the in vitro systems. Northern blot analysis shows a higher level of steady-state mRNAs for CHS and CHI 24 hours after GA(3) application. In corollas from a transgenic plant containing a beta-glucuronidase gene driven by a CHI promoter, a sixfold increase of beta-glucuronidase activity was measured following GA(3) application. The mode of action of stamens and GA(3) control over flavonoid gene expression is discussed. PMID- 16667743 TI - Sucrose Synthase in Developing Maize Leaves: Regulation of Activity by Protein Level during the Import to Export Transition. AB - The maize (Zea mays) leaf is a valuable system to study the sucrose import to sucrose export transition at the cellular level. Rapidly growing and fully heterotrophic cells in the basal part of the young leaf showed a high sucrose synthase (SS) activity. Leaf SS has been purified to homogeneity. By comparison with purified kernel SS isozymes, the leaf SS has been identified as SS(2). SS(1) protein and SS(2) protein were clearly separated by electrophoresis and the two monomers differed in size by 6 kilodaltons. Nevertheless, kinetic parameters of both enzymes were very similar. Immunodetection of SS protein showed that in young heterotrophic tissues SS(2) was a major protein accounting for 3% of the total protein. Concurrent with greening, SS activity decreased and the change of activity was explained by regulation of the protein level. In mature green tissues, which are synthetizing sucrose as evidenced by the presence of sucrose phosphate synthase activity, SS activity was almost completely absent. Results suggested that down regulation of SS(2) enzyme protein level was an early event in the transition from import to export status of the leaf. PMID- 16667744 TI - Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H ATPase after NaCl Adaptation. AB - Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H(+)-ATPase. These antibodies cross reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO(3) (-) sensitive H(+) transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H(+) transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H(+) transport capacity after adaptation was also observed for ATP hydrolysis. PMID- 16667745 TI - Ascorbate free-radical reduction by glyoxysomal membranes. AB - Glyoxysomal membranes from germinating castor bean (Ricinus communis L. cv Hale) endosperm contain an NADH dehydrogenase. This enzyme can utilize extraorganellar ascorbate free-radical as a substrate and can oxidize NADH at a rate which can support intraglyoxysomal demand for NAD(+). NADH:ascorbate free-radical reductase was found to be membrane-associated, and the activity remained in the membrane fraction after lysis of glyoxysomes by osmotic shock, followed by pelleting of the membranes. In whole glyoxysomes, NADH:ascorbate free-radical reductase, like NADH:ferricyanide reductase and unlike NADH:cytochrome c reductase, was insensitive to trypsin and was not inactivated by Triton X-100 detergent. These results suggest that ascorbate free-radical is reduced by the same component which reduces ferricyanide in the glyoxysomal membrane redox system. NADH:ascorbate free-radical reductase comigrated with NADH:ferricyanide and cytochrome c reductases when glyoxy-somal membranes were solubilized with detergent and subjected to rate-zonal centrifugation. The results suggest that ascorbate free-radical, when reduced to ascorbate by membrane redox system, could serve as a link between glyoxysomal metabolism and other cellular activities. PMID- 16667746 TI - Enzyme Sets of Glycolysis, Gluconeogenesis, and Oxidative Pentose Phosphate Pathway Are Not Complete in Nongreen Highly Purified Amyloplasts of Sycamore (Acer pseudoplatanus L.) Cell Suspension Cultures. AB - Differential centrifugation and Percoll-gradient centrifugation of protoplast lysates of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) yielded pure amyloplasts. Contamination of the final amyloplast preparation by foreign compartments was assessed by measuring marker enzyme activities. The activity of alkaline pyrophosphatase was taken as a 100% plastid marker; relative to this marker, mitochondria (cytochrome c oxidase) averaged 0.34%, microbodies (catalase) 0.61%, and cytosol (alcohol dehydrogenase) 0.09%. Enzymatic activities of the glycolytic, gluconeogenic, pentose phosphate and the starch degradation pathways were found to be present in these amyloplast extracts in appreciable amounts. But the pyrophosphate-dependent phosphofructokinase and phosphoglyceromutase were judged to be essentially absent from amyloplasts because the activities of these enzymes were not enriched above the level of contaminating enzymatic activities in the amyloplast fractions. Additionally, the in vitro activities of starch phosphorylase, ATP dependent phosphofructokinase, NAD dependent glyceraldehyde-3 phosphate dehydrogenase, and glucose-6 phosphate dehydrogenase did not seem to support carbon fluxes from starch to triose phosphates as calculated from the rate of starch disappearance during carbon starvation of the cells. These results provide additional, indirect evidence for the recently emerged view that, in addition to the well known phosphate triosephosphate translocator, another hexose phosphate and possibly also an ATP/ADP translocating system play major roles in nongreen plastids. PMID- 16667747 TI - Relationship between Photosynthetic Electron Transport and Stromal Enzyme Activity in Pea Leaves : Toward an Understanding of the Nature of Photosynthetic Control. AB - The responses of the quantum efficiencies of photosystem (PS) II and PSI measured in vivo simultaneously with estimations of the activities and activation states of NADP-malate dehydrogenase, chloroplast fructose-1,6-bisphosphatase, and ribulose-1,5-bisphosphate carboxylase were used to study the relationship between electron transport and carbon metabolism. The effects of varying irradiance and CO(2) partial pressure on the relationship between the quantum efficiencies of PSI and II, and the activity of these enzymes shows that the interrelationships vary according to the limitations placed on the system. The relationship between the quantum efficiencies of PSII and PSI was linear in most situations. In response to increasing irradiance, the activity of all three enzymes increased. In the case of NADP-malate dehydrogenase this increase was well correlated with the estimated flux of electrons through PSI and PSII. The other two enzymes showed a more complex relationship with the estimated flux of electrons through both photosystems. These relationships are consistent with the known interactions between these stromal enzymes and the thylakoids. The response to varying CO(2) partial pressure is more complex. The efficiencies of PSI and II declined with decreasing CO(2) partial pressure and the activity of each enzyme varied uniquely. However, there are clear correlations between the activities of the enzymes and the flux of electrons through the photosystems. In contrast to the data obtained under conditions of varying irradiance, there is clear evidence of photosynthetic control of electron transport when the CO(2) concentration is varied. PMID- 16667749 TI - Purification and characterization of a cysteine endopeptidase in cotyledons of germinated mung bean seeds. AB - A cysteine endopeptidase (EC 3.4.22.-) present in cotyledons of mung bean (Vigna radiata) seedlings was purified to homogeneity, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This proteinase has an apparent molecular mass of 33 kilodaltons as estimated by SDS-PAGE and belongs to the class of cysteine proteinases as judged by the effects of various proteinase inhibitors on the activity of the enzyme. When proangiotensin is used as a substrate, the enzyme preferentially hydrolyzes the peptide bonds formed by the amino group of Leu or lle in this oligopeptide chain; for the enzyme to cleave those bonds, peptide sequences consisting of at least three amino acid residues on the amino side of Leu or lle must be present. The proteinase readily digests globulin present in mung bean cotyledons to smaller polypeptides. PMID- 16667748 TI - High Sensitivity to Auxin is a Common Feature of Hairy Root. AB - The responses to auxin of Lycopersicon esculentum roots transformed by (T(l)+T(r))-DNA of the Ri plasmid of agropine-type Agrobacterium rhizogenes strain 15834 and Catharanthus trichophyllus roots transformed by the (T(l)+T(r)) DNA, and by T(l)- or T(r)- DNA alone of the same bacterial strain were compared to that of their normal counterparts. The transmembrane electrical potential difference of root protoplasts was measured as a function of the concentration of exogenous naphthalene acetic acid. The sensitivity to auxin expressed by this response was shown to be independent of the measurement conditions and of the basal polarization of isolated protoplasts. According to this electrical response, as well as to the modulation by auxin of proton excretion by root tips and root tip elongation, roots transformed by (T(l)+T(r)) DNA are 100 to 1000 times more sensitive to exogenous auxin than normal roots, as is the case with normal and transformed roots from Lotus corniculatus (WH Shen, A Petit, J Guern, J Tempe [1988] Proc Natl Acad Sci USA 85: 3417-3421). Further-more, transformed roots of C. trichophyllus are not modified in their sensitivity to fusicoccin, illustrating the specificity of the modification of the auxin sensitivity. Roots transformed by the T(r)-DNA alone showed the same sensitivity to auxin as normal roots, whereas the roots transformed by the T(l)-DNA alone exhibited an auxin sensitivity as high as the roots transformed by (T(l)+T(r))-DNA. It was concluded that the high sensitivity to auxin is controlled by the T(l)-DNA in agropine type Ri plasmids. PMID- 16667750 TI - Synthesis of early heat shock proteins in young leaves of barley and sorghum. AB - The in vivo synthesis of early heat-shock proteins in young leaves of barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) was studied by one- and two dimensional electrophoresis. Analysis of whole leaf protein patterns demonstrated clearly the enhanced resolution of heat-shock proteins, especially those of low molecular weight, when separated by two-dimensional electrophoresis. Comparison between the two cereals showed that a greater number and diversity of heat-shock proteins were induced in the subtropical C(4) (sorghum) species compared to the temperate C(3) (barley) species. Fractionation of whole leaf proteins into soluble and membrane fractions showed the majority of heat-shock proteins to be associated with the soluble fraction in both sorghum and barley. However, several low molecular mass (17-24 kilodalton) heat-shock proteins were clearly identified in the membrane fractions, indicating a likely association with thylakoid membranes in vivo during the early stages of a heat-shock response in both species. PMID- 16667751 TI - Kinetics of Aluminum Uptake in Triticum aestivum L: Identity of the Linear Phase of Aluminum Uptake by Excised Roots of Aluminum-Tolerant and Aluminum-Sensitive Cultivars. AB - The identity of a linear phase of aluminum (Al) uptake in Triticum aestivum was investigated by analysis of the kinetics of Al uptake by excised roots and purified cell wall fractions. Classical interpretation of kinetic data suggests that a linear phase of uptake with time reflects uptake across the plasma membrane; however, in studies with Al the possibility that the linear phase of uptake includes accumulation of Al in both the symplasm and the apoplasm has not been discounted. In our experiments, we observed a linear phase of Al uptake at both ambient and low temperatures, although the rate of uptake at 0 degrees C was 53 to 72% less than at 23 degrees C, depending on cultivars. This nonsaturable phase of uptake at low temperature suggests that a portion of the linear phase of Al uptake is nonmetabolic. Furthermore, analysis of Al in cell wall fractions isolated from excised roots pretreated with Al suggests that the linear phase of uptake includes a cell wall component. When excised roots were pretreated with Al, accumulation of Al in purified cell wall material included a linear phase that could not be desorbed with a 30 minute wash in citrate. The rates of linear phase accumulation of Al by cell wall material and cell contents were similar. In contrast, the linear phase of in vitro uptake of Al by purified cell wall material was completely desorbed by a 30 minute wash with citrate. These results suggest that the linear phase of Al uptake observed in excised roots of T. aestivum included metabolism-dependent binding of Al in apoplasm. PMID- 16667752 TI - Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato. AB - In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco. PMID- 16667753 TI - Distinction between the Responses of Developing Maize Kernels to Fluridone and Desiccation in Relation to Germinability, alpha-Amylase Activity, and Abscisic Acid Content. AB - Developing kernels of the maize (Zea mays) hybrid W64A x W182E germinated precociously following fluridone treatment. Likewise, following premature drying, the kernels germinated upon subsequent rehydration. Tolerance of the aleurone layer to premature desiccation considerably preceded that of the embryo. The increase in alpha-amylase activity following premature drying was substantial and was equal to, or exceeded, the increase which occurred following normal maturation drying. In contrast, there was only a small increase in enzyme activity, regardless of the concentration of the supplied gibberellic acid, following fluridone treatment. Both fluridone and drying cause a decrease in abscisic acid content within the developing kernels. While this decline in growth regulator may permit kernels to germinate, alone this is not sufficient to permit an increase in alpha-amylase activity. Thus drying is necessary to sensitize the aleurone layer to gibberellin, and thereby elicit enzyme synthesis. For this tissue to achieve its full potential to produce alpha-amylase, it must not only be free of the inhibitory effects of abscisic acid, but it must also be competent to respond to gibberellin. PMID- 16667754 TI - Purification and characterization of a type-I topoisomerase from cultured tobacco cells. AB - Cultured tobacco (Nicotiana tabacum, var Xanthi) cells contain a topoisomerase that removes positive and negative supercoils from DNA. The enzyme has an estimated molecular mass of 30,000 daltons under denaturing conditions, but may exist as a multimeric protein in the native state. Activity is enhanced significantly by either MgCl(2) or CaCl(2), but other divalent cations are much less effective in stimulating DNA relaxation. The purified enzyme acts by altering the linking number in topological steps of one and is inhibited by berenil or camptothecin, not novobiocin. Taken together, these data identify this enzyme as a type I topoisomerase. PMID- 16667755 TI - Characterization of the Xanthophyll Cycle and Other Photosynthetic Pigment Changes Induced by Iron Deficiency in Sugar Beet (Beta vulgaris L.). AB - In this work we characterize the changes induced by iron deficiency in the pigment composition of sugar beet (Beta vulgaris L.) leaves. When sugar beet plants were grown hydroponically under limited iron supply, neoxanthin and beta carotene decreased concomitantly with chlorophyll a, whereas lutein and the carotenoids within the xanthophyll cycle were less affected. Iron deficiency caused major increases in the lutein/chlorophyll a and xanthophyll cycle pigments/chlorophyll a molar ratios. Xanthophyll cycle carotenoids in Fe deficient plants underwent epoxidations and de-epoxidations in response to ambient light conditions. In dark adapted Fe-deficient plants most of the xanthophyll cycle pigment pool was in the epoxidated form violaxanthin. We show, both by HPLC and by in vivo 505 nanometers absorbance changes, that in Fe deficient plants and in response to light, the de-epoxidated forms antheraxanthin and zeaxanthin were rapidly formed at the expense of violaxanthin. Several hours after returning to dark, the xanthophyll cycle was shifted again toward violaxanthin. The ratio of variable to maximum chlorophyll fluorescence from intact leaves was decreased by iron deficiency. However, in iron deficient leaves this ratio was little affected by light conditions which displace the xanthophyll cycle toward epoxidation or de-epoxidation. This suggests that the functioning of the xanthophyll cycle is not necessarily linked to protection against excess light input. PMID- 16667756 TI - Acetohydroxy Acid Synthase Activity in Chlorella emersonii under Auto- and Heterotrophic Growth Conditions. AB - Acetohydroxyacid synthase (AHAS) activity was studied in the green unicellular alga Chlorella emersonii. This activity and its regulation was compared in the algae grown autotrophically and heterotrophically on glucose in the dark. No evidence for the existence of more than one enzyme was found. The activity in crude extracts from either heterotrophically or autotrophically grown cells showed a K(m) for pyruvate of 9 millimolar, a 22-fold preference for 2 ketobutyrate over pyruvate as the second substrate, 50% inhibition by 0.5 millimolar valine, and 50% inhibition by 0.3 micromolar sulfometuron methyl (SMM). Spontaneous mutants of the alga resistant to SMM were isolated, which appeared to be single gene mutants containing SMM-resistant AHAS activity. Hence, AHAS appears to be the sole direct target site of SMM in C. emersonii. The fact that the mutants had equivalent SMM resistance under auto- and heterotrophic conditions further supports the conclusion that the same enzyme functions under both physiological regimes. The addition of valine and isoleucine leads to partial relief of SMM inhibition of biomass increase, but not of SMM inhibition of cell division. PMID- 16667757 TI - Carbon and nitrogen economy of 24 wild species differing in relative growth rate. AB - The relation between interspecific variation in relative growth rate and carbon and nitrogen economy was investigated. Twentyfour wild species were grown in a growth chamber with a nonlimiting nutrient supply and growth, whole plant photosynthesis, shoot respiration, and root respiration were determined. No correlation was found between the relative growth rate of these species and their rate of photosynthesis expressed on a leaf area basis. There was a positive correlation, however, with the rate of photosynthesis expressed per unit leaf dry weight. Also the rates of shoot and root respiration per unit dry weight correlated positively with relative growth rate. Due to a higher ratio between leaf area and plant weight (leaf area ratio) fast growing species were able to fix relatively more carbon per unit plant weight and used proportionally less of the total amount of assimilates in respiration. Fast growing species had a higher total organic nitrogen concentration per unit plant weight, allocated more nitrogen to the leaves and had a higher photosynthetic nitrogen-use efficiency, i.e. a higher rate of photosynthesis per unit organic nitrogen in the leaves. Consequently, their nitrogen productivity, the growth rate per unit organic nitrogen in the plant and per day, was higher compared with that of slow growing species. PMID- 16667758 TI - Photosynthetic induction state of leaves in a soybean canopy in relation to light regulation of ribulose-1-5-bisphosphate carboxylase and stomatal conductance. AB - Photosynthetic induction state, stomatal conductance and light regulation of ribulose-1,5-bisphosphate carboxylase (rubisco) were examined for leaves in a mature, closed soybean (Glycine max) canopy (leaf area index approximately 5) with the objective to determine the extent to which these factors may be limiting the capacity to respond to light transients during sunflecks. When sampled along a vertical gradient, leaves near the bottom of the canopy had lower rubisco contents and chlorophyll a/b ratios as compared with upper leaves. Leaves sampled at midcanopy showed a wide variation in photosynthetic induction state (ratio of the photosynthetic rate achieved after 1 minute exposure to high light to the steady-state assimilation rate achieved after 20 minutes exposure). Both photosynthetic induction state and the initial rubisco activity varied in parallel with stomatal conductance. By contrast there was no correlation between total rubisco activity and stomatal conductance. The results indicate that induction state, as determined by the light regulation of both rubisco activity and stomatal conductance, is an important limitation to the ability of leaves in a soybean canopy to respond to light transients that occur during sunflecks. PMID- 16667759 TI - Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L. AB - The protein(s) that constitute(s) the ATP-driven Ca(2+)-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca(2+)-transport system was studied using ATP-driven (45)Ca(2+) import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K(m) for Ca(2+) (5.2 micromolar), inhibition by relatively high levels of vanadate (IC(50) = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC(50) = 0.01 micromolar) and had a low apparent K(m) for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca(2+) transport detectably in the reconstituted system. However, low levels of the Ca(2+)-ionophore A 23187 instantaneously discharged 90% of the Ca(2+) associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca(2+)-transport in the reconstituted system was thus primary active, through a Ca(2+)-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca(2+)-ATPase and for studying structural and functional aspects of the purified enzyme. PMID- 16667760 TI - Changes in Properties of Barley Leaf Mitochondria Isolated from NaCl-Treated Plants. AB - Treatment of barley (Hordeum vulgare) seedlings with 400 millimolar NaCl for 3 days resulted in a reduction in plant growth and an increase in the leaf content in ions (K(+) + Na(+)) and proline. Purified mitochondria were successfully isolated from barley leaves. Good oxidative and phosphorylative properties were observed with malate as substrate. Malate-dependent electron transport was found to be only partly inhibited by cyanide, the remaining oxygen uptake being SHAM sensitive. The properties of mitochondria from NaCl-treated barley were modified. The efficiency of phosphorylation was diminished with only a slight decrease in the oxidation rates. In both isolated mitochondria and whole leaf tissue of treated plants, the lower respiration rate was due to a lower cytochrome pathway activity. In mitochondria, the activity of the alternative pathway was not modified by salt treatment, whereas this activity was increased in whole leaf tissue. The possible participation of the alternative pathway in response to salt stress will be discussed. PMID- 16667761 TI - Kinetics of NH(4) Assimilation in Zea mays: Preliminary Studies with a Glutamate Dehydrogenase (GDH1) Null Mutant. AB - In higher plants it is now generally considered that glutamate dehydrogenase (GDH) plays only a small or negligible role in ammonia assimilation. To test this specific point, comparative studies of (15)NH(4) (+) assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDH1-positive wild type from which this mutant was derived. The kinetics of (15)NH(4) (+) assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% ((15)NH(4))(2)SO(4) via the aerated root medium in hydroponic culture over a 24-h period. The GDH1-null mutant, with a 10- to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of (15)NH(4) (+) assimilation into total reduced nitrogen. Observed rates of root ammonium assimilation were 5.9 and 3.1 micromoles per hour per gram fresh weight for the wild type and mutant, respectively. The lower rate of (15)NH(4) (+) assimilation in the mutant was associated with lower rates of labeling of several free amino acids (including glutamate, glutamine-amino N, aspartate, asparagine-amino N, and alanine) in both roots and shoots of the mutant in comparison to the wild type. Qualitatively, these labeling kinetics appear consistent with a reduced flux of (15)N via glutamate in the GDH1-null mutant. However, the responses of the two genotypes to the potent inhibitor of glutamine synthetase, methionine sulfoximine, and differences in morphology of the two genotypes (particularly a lower shoot:root ratio in the GDH1-null mutant) urge caution in concluding that GDH1 is solely responsible for these differences in ammonia assimilation rate. PMID- 16667762 TI - Purification and Properties of Glutamine Synthetase in Leaves and Roots of Pinus banksiana Lamb. AB - A method is described for the purification of glutamine synthetase (GS; EC. 6.3.1.2) from the leaves and roots of Pinus banksiana Lamb., a conifer which utilizes ammonium as its primary nitrogen source. The enzyme was purified to apparent homogeneity by a procedure involving salt fractionation as well as ion exchange, size exclusion, and affinity chromatography. Since the final preparation produced two bands on SDS polyacryamide gels but only one band on a nondenaturating gel, it is concluded that the two subunits (44 and 40 kilodaltons, respectively) are part of a single enzymatic protein which shows GS activity. The pH optimum for leaf GS ranged between 6.2 and 6.5, one pH unit lower than the values reported for higher plants which utilize primarily nitrate nitrogen. Magnesium requirements for GS in P. banksiana were different for leaves and roots, showing V(max)/2 values of 2.5 and 8 millimolar, respectively at 5 millimolar ATP. Furthermore, K(m) values for ammonium were higher for the enzyme in leaves (33.1 micromolar) than in roots (19.2 micromolar). K(m) values for ATP and for glutamate, on the other hand, were similar for the two tissues. A polyclonal antibody was produced against the purified leaf GS. Western blots of leaf homogenates produced two bands, the lighter one being more abundant. The same pattern was found when immunodetection was performed using an anti GS IgG produced against purified GS from Phaseolus nodules thus indicating common antigenic determinants. At least 30% of total GS was recovered in a plastid fraction of dark-grown calli produced from the basal part of P. banksiana hypocotyls. PMID- 16667763 TI - Influence of Temperature Stress on in Vitro Fertilization and Heat Shock Protein Synthesis in Maize (Zea mays L.) Reproductive Tissues. AB - This study was conducted to investigate the response of maize (Zea mays) male and female mature reproductive tissues to temperature stress. We have tested the fertilization abilities of the stressed spikelets and pollen using in vitro pollination-fertilization to determine their respective tolerance to stress. The synthesis of heat shock proteins (HSPs) was also analyzed in male and female tissues using electrophoresis of (35)S-labeled proteins and fluorography, to establish a relationship between the physiological and molecular responses. Pollen, spikelets, and pollinated spikelets were exposed to selected temperatures (4, 28, 32, 36, or 40 degrees C) and tested using an in vitro fertilization system. The fertilization rate is highly reduced when pollinated spikelets are exposed to temperatures over 36 degrees C. When pollen and spikelets are exposed separately to temperature stress, the female tissues appear resistant to 4 hours of cold stress (4 degrees C) or heat stress (40 degrees C). Under heat shock conditions, the synthesis of a typical set of HSPs is induced in the female tissues. In contrast, the mature pollen is sensitive to heat stress and is responsible for the failure of fertilization at high temperatures. At the molecular level, no heat shock response is detected in the mature pollen. PMID- 16667764 TI - Effects of trans-Cinnamic Acid on Expression of the Bean Phenylalanine Ammonia Lyase Gene Family. AB - Using DNA probes specific for the three members of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (PAL) gene family, we have studied the effects of the product of the PAL reaction, trans-cinnamic acid (CA), on the appearance of individual PAL transcripts in suspension cultured bean cells. Concentrations of CA in excess of 10(-4) molar inhibited appearance of elicitor-induced transcripts encoding PAL1, PAL2, and PAL3 when added to the cells at the same time as fungal elicitor. Addition of CA 4 hours postelicitation caused a major reduction in the levels of all three PAL transcripts, but with different kinetics and subsequent rates of recovery. The inhibition of accumulation of PAL1, PAL2, or PAL3 transcripts, measured 3 hours after exposure to elicitor, as a function of the time of addition of CA postelicitation reflected the different rates of appearance of the three PAL transcripts in the presence of elicitor alone. The inhibitory effects of CA as seen on PAL transcripts were not observed for the constitutively expressed transcript H1, or the elicitor-inducible 1,3-beta-D glucanase. Analysis of in vitro translated polypeptides showed that some elicitor induced mRNA activities were not down-regulated by CA, and that a number of other mRNA activities were induced by CA, thus providing further evidence for specificity in the action of CA on bean cells. Treatment of elicited cells with L alpha-aminooxy-beta-phenylpropionic acid, a potent and specific inhibitor of PAL activity, resulted in maintenance of elevated PAL transcript levels beyond 12 hours postelicitation, this effect being greatest for PAL transcripts 2 and 3. Our results are consistent with a model in which CA, or a derivative, could act as a component in a regulatory feedback system operating at the level of phenylpropanoid gene transcription. PMID- 16667765 TI - Soybean Roots Retain the Seed Urease Isozyme Synthesized during Embryo Development. AB - Roots of young soybean (Glycine max [L.] Merr.) plants (up to 25 days old) contain two distinct urease isozymes, which are separable by hydroxyapatite chromatography. These two urease species (URE1 and URE2) differ in: (a) electrophoretic mobility in native gels, (b) pH dependence, and (c) recognition by a monoclonal antibody specific for the seed ("embryo-specific") urease. By these parameters root URE1 urease is similar to the abundant embryo-specific urease isozyme, while root URE2 resembles the "ubiquitous" urease which has previously been found in all soybean tissues examined (leaf, embryo, seed coat, and cultured cells). The embryo-specific and ubiquitous urease isozymes are products of the Eu1 and Eu4 structural genes, respectively. Roots of the eu1 sun/eu1-sun genotype, which lacks the embryo-specific urease (i.e. ;seed urease null'), contain no URE1 urease activity. Roots of eu4/eu4, which lacks ubiquitous urease, lack the URE2 (leaflike) urease activity. From these genetic and biochemical criteria, then, we conclude that URE1 and URE2 are the embryo specific and ubiquitous ureases, respectively. Adventitious roots generated from cuttings of any urease genotype lack URE1 activity. In seedling roots the seedlike (URE1) activity declines during development. Roots of 3-week-old plants contain 5% of the total URE1 activity of the radicle of 4-day-old seedlings, which, in turn, has approximately the same urease activity level as the dormant embryonic axis. The embryo-specific urease incorporates label from [(35)S]methionine during embryo development but not during germination, indicating that there is no de novo synthesis of the embryo-specific (URE1) urease in the germinating root. We conclude that the seedlike urease (URE1) found in roots of young soybean plants is a remnant of the Eu1-encoded, abundant, embryo-specific urease which accumulates in the embryonic root axis during seed development. PMID- 16667766 TI - Abscisic Acid Regulation of DC8, A Carrot Embryonic Gene. AB - DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H) pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage enbryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 x 10(-7) molar ABA while 10(-6) molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA. PMID- 16667767 TI - Modification of the Red Beet Plasma Membrane H-ATPase by Diethylpyrocarbonate. AB - Incubation of the red beet (Beta vulgaris L.) plasma membrane H(+)-ATPase with micromolar concentrations of diethylpyrocarbonate (DEPC) resulted in inhibition of both ATP hydrolytic and proton pumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggesting specific modification of histidine residues. Kinetic analyses of DEPC inhibition performed on both membrane-bound and solubilized enzyme preparations suggested the presence of at least one essential histidine moiety per active site. Inclusion of either ATP (substrate) or ADP (product and competitive inhibitor) in the modification medium reduced the amount of inhibition observed in the presence of DEPC. However, protection was not entirely effective in returning activity to noninhibited control values. These results suggest that the modified histidine does not reside directly in the ATP binding region of the enzyme, but is more likely involved in enzyme regulation through subtle conformational effects. PMID- 16667768 TI - Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures. AB - Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [(14)C]mevalonate ([(14)C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [(14)C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [(14)C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase. PMID- 16667769 TI - High performance liquid chromatography resolution of ubiquitin pathway enzymes from wheat germ. AB - The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with (125)I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin (125)I-lysozyme conjugates (epsilon-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (alpha-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated. PMID- 16667770 TI - Transient accumulation of asparagine in sycamore cells after a long period of sucrose starvation. AB - The mobilization of stored carbohydrates (sucrose and starch) and proteins during sucrose starvation was studied with sycamore (Acer pseudoplatanus L.) cells. When almost all the intracellular carbohydrate pools had disappeared, the cell protein content declined progressively whereas asparagine determined by either (13)C nuclear magnetic resonance or reversed phase high performance liquid chromatography increased steadily. After a long period of sucrose starvation, the most intense resonances in the (13)C nuclear magnetic resonance spectra were from citrate and asparagine. The total amounts of asparagine (expressed as nitrogen) and free amino acids that appeared after a long period of sucrose deprivation corresponded roughly to the total amount of protein (expressed as nitrogen), that disappeared within the same period of time. Addition of sucrose in the culture medium after a long period of sucrose starvation led to a disappearance of asparagine. These results suggest therefore that the presence of asparagine in plant cells in large excess should be considered as a good marker of protein utilization after a long period of sucrose starvation and is very likely related to stress. PMID- 16667771 TI - Glycine-Glomus-Bradyrhizobium Symbiosis : X. Relationships between Leaf Gas Exchange and Plant and Soil Water Status in Nodulated, Mycorrhizal Soybean under Drought Stress. AB - Soybean (Glycine max [L.] Merr.) plants were colonized by the vesicular arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (VAM plants) or fertilized with KH(2)PO(4) (nonVAM plants) and grown for 50 days under controlled conditions. Plants were harvested over a 4-day period during which the soil was permitted to dry slowly. The harvest was terminated when leaf gas exchange was no longer measurable due to drought stress. Significantly different effects in shoot water content, but not in shoot water potential, were found in VAM and nonVAM plants in response to drought stress. Leaf conductances of the two treatments showed similar response patterns to changes in soil water and shoot water potential but were significantly different in magnitude and trend relative to shoot water content. The relationships between transpiration, CO(2) exchange and water-use efficiency (WUE) were the same in VAM and nonVAM plants in response to decreasing soil water and shoot water potential. As a function of shoot water content, however, WUE showed different response patterns in VAM and nonVAM plants. PMID- 16667772 TI - Flow Cytometric Fluorescence Anisotropy of Lipophilic Probes in Epidermal and Mesophyll Protoplasts from Water-Stressed Lupinus albus L. AB - The blue emission anisotropy, r, of two lipophilic probes, diphenylhexatriene (DPH) and its trimethyl-ammonium derivative (TMA-DPH), has been measured in foliar Lupinus albus L. protoplasts for the first time by flow cytometry. Distinctive values have been obtained for protoplasts of epidermal and mesophyll origin, identified by their intensities of chlorophyll fluorescence. Fluorescence microscopy confirmed that TMA-DPH remained in the plasma membrane while DPH penetrated into intracellular lipid domains. Typical emission anisotropy values at 22 degrees C for mesophyll and epidermal protoplasts, respectively, were 0.225 and 0.312 with TMA-DPH, and 0.083 and 0.104 with DPH. This indicates that epidermal cells-and notably their plasma membranes (TMA-DPH)-have higher lipid microviscosity and/or more ordered lipid structure. Two lupin genotypes characterized as resistant or susceptible to drought were analyzed with or without 9 days of water stress shown to increase ion leakage from foliar discs. Water stress greatly increased the apparent fluidity, and more so in the susceptible genotype; the effect was more pronounced in the chlorophyll containing mesophyll cells than in the epidermal cells. PMID- 16667773 TI - Partial Characterization and Subcellular Localization of Three alpha-Glucosidase Isoforms in Pea (Pisum sativum L.) Seedlings. AB - Three isoforms of alpha-glucosidase (EC 3.2.1.20) have been extracted from pea (Pisum sativum L.) seedlings and separated by DEAE-cellulose and CM-Sepharose chromatography. Two alpha-glucosidase isoforms (alphaG1 and alphaG2) were most active under acid conditions, and appeared to be apoplastic. A neutral form (alphaG3) was most active near pH 7, and was identified as a chloroplastic enzyme. Together, the activity of alphaG1 and alphaG2 in apoplastic preparations accounted for 21% of the total acid alpha-glucosidase activity recovered from pea stems. The vast majority (86%) of the apoplastic acid alpha-glucosidase activity was due to alphaG1. The apparent K(m) values for maltose of alphaG1 and alphaG2 were 0.3 and 1.3 millimolar, respectively. The apparent K(m) for maltose of alphaG3 was 33 millimolar. The respective native molecular weights of alphaG1, alphaG2, and alphaG3 were 125,000, 150,000, and 110,000. PMID- 16667774 TI - Essential arginine residues in the nitrate uptake system from corn seedling roots. AB - Three dicarbonyl reagents were used to demonstrate the presence of an essential arginine residue in the NO(3) (-) uptake system from corn seedling roots (Zea mays L., Golden Cross Bantam). Incubation of corn seedlings with 2,3-butanedione (0.125-1.0 millimolar) and 1,2-cyclohexanedione (0.5-4.0 millimolar) in the presence of borate or with phenylglyoxal (0.25-2.0 millimolar) at pH 7.0 and 30 degrees C resulted in a time-dependent loss of NO(3) (-) uptake following pseudo first-order kinetics. Second-order rate constants obtained from slopes of linear plots of pseudo-first-order rate constants versus reagent concentrations were 1.67 x 10(-2), 0.68 x 10(-2), and 1.00 x 10(-2) millimolar per minute for 2,3 butanedione, 1,2-cyclohexanedione, and phenylglyoxal, respectively, indicating the faster rate of inactivation with 2,3-butanedione at equimolar concentration. Double log plots of pseudo-first-order rate constants versus reagent concentrations yielded slope values of 1.031 (2,3-butanedione), 1.004 (1,2 cyclohexanedione), and 1.067 (phenylglyoxal), respectively, suggesting the modification of a single arginine residue. The effectiveness of the dicarbonyl reagents appeared to increase with increasing medium pH from 5.5 to 8.0. Unaltered K(m) and decreased V(max) in the presence of reagents indicate the inactivation of the modified carriers with unaltered properties. The results thus obtained indicate that the NO(3) (-) transport system possesses at least one essential arginine residue. PMID- 16667775 TI - Effect of Nitrogen Starvation on Polypeptide Composition, Ribulose-1,5 Bisphosphate Carboxylase/Oxygenase, and Thylakoid Carotenoprotein Content of Synechocystis sp. Strain PCC6308. AB - Synechocystis sp. strain PCC6308 cells were starved for nitrogen for 5 days. The polypeptide compositions of whole cell extracts and washed membranes of nitrogen replete and nitrogen-starved cells were compared by one- and two-dimensional electrophoresis. Immunoblotting of one-dimensional gels indicated that pelletable ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was depleted in cells starved for nitrogen, while levels of soluble Rubisco were comparable in nitrogen starved and nitrogen-replete cells. This is consistent with the hypothesis that pelletable Rubisco may serve as a nitrogen reserve in Synechocystis 6308. Other polypeptides were differentially enriched in the membrane or soluble fractions of nitrogen-replete cells or nitrogen-starved cells, suggesting nitrogen starvation may alter partitioning of polypeptides into soluble and membrane fractions. Degradation of abundant polypeptides during nitrogen starvation appeared to cause an effective magnification of less abundant polypeptides in the molecular mass range of 20 to 40 kilodaltons, as shown by two-dimensional electrophoresis. A 42 kilodalton thylakoid carotenoid protein identified by immunoblotting was conserved in membranes from nitrogen-starved cells. This may be functional for cells depleted of pigment and thus exposed to higher light levels because of decreased self-shading. PMID- 16667776 TI - Mutants of Synechocystis PCC6803 Defective in Inorganic Carbon Transport. AB - Eighty mutants of Synechocystis PCC6803 that require high CO(2) for growth were examined with a mass spectrometer for their ability to take up CO(2) in the light. Two of these mutants (type A) did not show any CO(2) uptake while the rest of the mutants (type B) took up CO(2) actively. Type A mutants (RKa and RKb) and one type B mutant (RK11) were partially characterized. At 3% CO(2), growth rates of the mutants and the wild type (WT) were similar. Under air levels of CO(2), growth of RKa and RKb was very slow, and RK11 did not grow at all. The photosynthetic affinities for inorganic carbon (C(i)) in these three mutants were about 100 times lower than the affinity in WT. The following characteristics of type A mutants indicated that the mutants have a defect in their CO(2)-transport system: (a) the activity of (13)C(18)O(2) uptake in RKa and RKb in the light was less than 5% the activity in WT, and (b) each mutant had only a low level of activity of (14)CO(2) uptake as measured by the method of silicone oil-filtering centrifugation. The HCO(3) (-)-transport system was also impaired in these mutants. The activity of H(14)CO(3) (-) uptake was negligibly low in RKb and was one-third the activity of WT in RKa. On the other hand, the type B mutant, RK11, transported CO(2) and HCO(3) (-) into the intracellular C(i) pool as actively as WT but was unable to utilize it for photosynthesis. Complementation analysis of type A mutants indicated that RKa and RKb have mutations in different regions of the genome. These results suggested that at least two kinds of proteins are involved in the C(i)-transport system. PMID- 16667777 TI - Conversion of monogalactosyldiacylglycerols to triacylglycerols in ozone fumigated spinach leaves. AB - Molecular species and fatty acid distribution of triacylglycerol (TG) accumulated in spinach (Spinacia oleracea L.) leaves fumigated with ozone (0.5 microliter per liter) were compared with those of monogalactosyldiacylglycerol (MGDG). Analysis of positional distribution of the fatty acids in MGDG and the accumulated TG by the enzymatic digestion method showed that hexadecatrienoate (16:3) was restricted to sn-2 position of the glycerol backbone in both MGDG and TG, whereas alpha-linolenate (18:3) was preferentially located at sn-1 position in MGDG, and sn-1 and/or sn-3 positions in TG, suggesting that 1,2-diacylglycerol moieties of MGDG are the direct precursor of TG in ozonefumigated leaves. Further analysis of TG molecular species by argentation chromatography and mass spectrometry showed that TG increased with ozone fumigation consisted of approximately an equal molar ratio of sn-1,3-18:3-2-16:3 and sn-1,2,3-18:3. Because the molecular species of MGDG in spinach leaves is composed of a similar molar ratio of sn-1-18:3-2-16:3 and sn-1,2-18:3, we concluded that MGDG was converted to 1,2-diacylglycerol and acylated with 18:3 to TG in ozone-fumigated spinach leaves. PMID- 16667778 TI - Pathway for the synthesis of triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated spinach leaves. AB - When the upper leaf surface of spinach (Spinacia oleracea L.) plants was treated with [1-(14)C]acetate and grown for 2 days, (14)C was effectively incorporated into acyl moieties of leaf lipids in ratios approximately their composition by mass. Fumigation of the plants with ozone (0.5 microliter per liter) caused a redistribution of (14)C among lipid classes, i.e. a marked increase of (14)C content in triacylglycerol (TG) and 1,2-diacylglycerol (1,2-DG) and a decrease of label in monogalactosyldiacylglycerol (MGDG) without affecting (14)C distribution in leaf fatty acids. Label in both TG and 1,2-DG was found predominantly in their polyene molecular species. Since MGDG consists of similar polyene molecular species, the results indicate the synthesis of TG from MGDG via 1,2-DG. Label was also accumulated in tri- and tetragalactosyldiacylglycerol, products of galactolipid:galactolipid galactosyltransferase (GGGT). Moreover, there was a close relation between increases in the amounts of TG and the oligogalactolipids in ozonetreated leaves. These results indicate that MGDG was converted to 1,2-DG by GGGT and then to TG. In intact chloroplasts isolated from ozone-treated leaves, there was an enhanced production of free fatty acid (FFA), which was diminished by the addition of coenzyme A (CoA) and ATP, indicating that ozone stimulated the hydrolysis of MGDG to liberate FFA, which was in turn converted to acyl-CoA. The final step of TG synthesis, acylation of 1,2-DG with acyl-CoA, was confirmed by feeding with [1-(14)C]linolenic acid in leaf discs excised from ozone-fumigated leaves; (14)C was effectively incorporated into TG but not into 1,2-DG. These results demonstrate the synthesis of TG from 1,2-DG and FFA which were liberated from MGDG in ozone-fumigated spinach leaves. PMID- 16667779 TI - Free Fatty acids regulate two galactosyltransferases in chloroplast envelope membranes isolated from spinach leaves. AB - Effects of MgCl(2) and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl(2) with a saturated concentration of more than 5 millimolar. Free alpha-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl(2), without affecting its maximum activity at higher MgCl(2) concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl(2) were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl(2), CaCl(2) and a high concentration of KCl in place of MgCl(2). UDGT activity was hyperbolically enhanced by MgCl(2) with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl(2)-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl(2) concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves. PMID- 16667780 TI - Ca transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure. AB - The influence of ozone on Ca(2+) transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca(2+) influx into the cell and (b) active Ca(2+) efflux driven by an ATP-dependent system, which has two components: a primary Ca(2+) transport directly linked to ATP which is partially activated by calmodulin and a H(+)/Ca(2+) antiport coupled to activity of a H(+)-ATPase. The passive Ca(2+) permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca(2+) than does a square wave, even though the total dose was the same (0.6 microliter per liter x hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca(2+) extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca(2+) out of the cell. PMID- 16667781 TI - Effects of heat shock on amino Acid metabolism of cowpea cells. AB - When cowpea (Vigna unguiculata) cells maintained at 26 degrees C are transferred to 42 degrees C, rapid accumulation of gamma-aminobutyrate (>10-fold) is induced. Several other amino acids (including beta-alanine, alanine, and proline) are also accumulated, but less extensively than gamma-aminobutyrate. Total free amino acid levels are increased approximately 1.5-fold after 24 hours at 42 degrees C. Heat shock also leads to release of amino acids into the medium, indicating heat shock damage to the integrity of the plasmalemma. Some of the changes in metabolic rates associated with heat shock were estimated by monitoring the (15)N labeling kinetics of free intracellular, extracellular and protein-bound amino acids of cultures supplied with (15)NH(4) (+), and analyzing the labeling data by computer simulation. Preliminary computer simulation models of nitrogen flux suggest that heat shock induces an increase in the gamma-aminobutyrate synthesis rate from 12.5 nanomoles per hour per gram fresh weight in control cells maintained at 26 degrees C, to as high as 800 nanomoles per hour per gram fresh weight within the first 2 hours of heat shock. This 64-fold increase in the gamma-aminobutyrate synthesis rate greatly exceeds the expected (Q(10)) change of metabolic rate of 2.5- to 3-fold due to a 16 degrees C increase in temperature. We suggest that this metabolic response may in part involve an activation of glutamate decarboxylase in vivo, perhaps mediated by a transient cytoplasmic acidification. Proline appears to be synthesized from glutamate and not from ornithine in cowpea cells. Proline became severalfold more heavily labeled than ornithine, citrulline and arginine in both control and heat-shocked cultures. Proline synthesis rate was increased 2.7-fold by heat shock. Alanine, beta-alanine, valine, leucine, and isoleucine synthesis rates were increased 1.6-, 3.5-, 2.0-, 5.0-, and 6.0-fold, respectively, by heat shock. In contrast, the phenylalanine synthesis rate was decreased by 50% in response to heat shock. The differential effects of heat stress on metabolic rates lead to flux and pool size redistributions throughout the entire network of amino acid metabolism. PMID- 16667782 TI - Partial Purification and Characterization of a 3'- Phosphoadenosine 5' Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum). AB - A 3' -phosphoadenosine 5' -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 +/- 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K(m) 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K(m) 82 micromolar), desulfo-p hydroxybenzylglucosinolate (K(m) 670 micromolar), and desulfoallylglucosinolate (K(m) 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl(2), MnCl(2) and reducing agents and inhibited by ZnCl(2), PbNO(3) NiCl(2) and the reaction product PAP. The thiol reagents N-ethylmaleimide, p chloromercuriphenylsulfonic acid, and 5,5' -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by beta-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second. PMID- 16667783 TI - Enzymology of the reduction of hydroxypyruvate and glyoxylate in a mutant of barley lacking peroxisomal hydroxypyruvate reductase. AB - The use of LaPr 88/29 mutant of barley (Hordeum vulgare), which lacks NADH preferring hydroxypyruvate reductase (HPR-1), allowed for an unequivocal demonstration of at least two related NADPH-preferring reductases in this species: HPR-2, reactive with both hydroxypyruvate and glyoxylate, and the glyoxylate specific reductase (GR-1). Antibodies against spinach HPR-1 recognized barley HPR-1 and partially reacted with barley HPR-2, but not GR-1, as demonstrated by Western immunoblotting and immunoprecipitation of proteins from crude leaf extracts. The mutant was deficient in HPR-1 protein. In partially purified preparations, the activities of HPR-1, HPR-2, and GR-1 could be differentiated by substrate kinetics and/or inhibition studies. Apparent K(m) values of HPR-2 for hydroxypyruvate and glyoxylate were 0.7 and 1.1 millimolar, respectively, while the K(m) of GR-1 for glyoxylate was 0.07 millimolar. The K(m) values of HPR-1, measured in wild type, for hydroxypyruvate and glyoxylate were 0.12 and 20 millimolar, respectively. Tartronate and P-hydroxypyruvate acted as selective uncompetitive inhibitors of HPR-2 (K(i) values of 0.3 and 0.4 millimolar, respectively), while acetohydroxamate selectively inhibited GR-1 activity. Nonspecific contributions of HPR-1 reactions in assays of HPR-2 and GR 1 activities were quantified by a direct comparison of rates in preparations from wild-type and LaPr 88/29 plants. The data are evaluated with respect to previous reports on plant HPR and GR activities and with respect to optimal assay procedures for individual HPR-1, HPR-2, and GR-1 rates in leaf preparations. PMID- 16667784 TI - Malate Metabolism in the Dark After CO(2) Fixation in the Crassulacean Plant Kalanchoe tubiflora. AB - The metabolism of [(13)C]malate was studied in the Crassulacean plant Kalanchoe tubiflora following exposure to (13)CO(2) for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [(13)C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4-(13)C]malate to [1- (13)C]malate by fumarase, varied substantially during the dark period. At both 15 and 25 degrees C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of (13)CO(2) uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired (12)CO(2) also served as a source of carbon for malate formation. At 15 degrees C, 15% of the total malate was formed from respired (12)CO(2), while at 25 degrees C, 49% of the accumulated malate was derived from respired (12)CO(2). Some of the malate synthesized from external (13)CO(2) was also respired during the night. The proportion of the total [(13)C]malate respired during the dark period was similar at 15 and 25 degrees C, and respiration of newly formed [(13)C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO(2) fixation. PMID- 16667785 TI - Interaction between the Component Enzymes of the Glycine Decarboxylase Multienzyme Complex. AB - The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein monomers:1 L-protein dimer. When the complex is diluted it tends to dissociate into its component enzymes. This prevents the purification of the intact complex by gel filtration or ultracentrifugation. In the dissociated state the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows typical substrate kinetics. When the complex is reformed, the H-protein no longer acts as a substrate but as an integrated part of the enzyme complex. PMID- 16667786 TI - A water relations analysis of seed germination rates. AB - Seed germination culminates in the initiation of embryo growth and the resumption of water uptake after imbibition. Previous applications of cell growth models to describe seed germination have focused on the inhibition of radicle growth rates at reduced water potential (Psi). An alternative approach is presented, based upon the timing of radicle emergence, to characterize the relationship of seed germination rates to Psi. Using only three parameters, a ;hydrotime constant' and the mean and standard deviation in minimum or base Psi among seeds in the population, germination time courses can be predicted at any Psi, or normalized to a common time scale equal to that of seeds germinating in water. The rate of germination of lettuce (Lactuca sativa L. cv Empire) seeds, either intact or with the endosperm envelope cut, increased linearly with embryo turgor. The endosperm presented little physical resistance to radicle growth at the time of radicle emergence, but its presence markedly delayed germination. The length of the lag period after imbibition before radicle emergence is related to the time required for weakening of the endosperm, and not to the generation of additional turgor in the embryo. The rate of endosperm weakening is sensitive to Psi or turgor. PMID- 16667787 TI - Changes in mRNA and Protein during Ripening in Apple Fruit (Malus domestica Borkh. cv Golden Delicious). AB - Poly(A)(+) RNA was extracted from cortical tissue of apple fruit (Malus domestica Borkh. cv Golden Delicious), and in vitro translation products were analyzed by two-dimensional gel electrophoresis. As fruit internal ethylene concentration increased from a basal level of 0.0 to 0.1 microliter per liter to 1 to 5 microliters/liter, substantial changes in the pattern of in vitro translation products were observed. More subtle changes were observed as fruit continued to ripen and internal ethylene concentration increased to 80 to 100 microliters/liter. Overall, the levels of at least six mRNAs were found to increase, while one mRNA decreased. Analysis of proteins extracted from ripening fruit indicated that the level of at least three proteins increased with ripening. PMID- 16667788 TI - Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction. AB - Malate oxidation supported C(2)H(2) reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O(2) tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (K(m) = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (K(m) = 2.32 millimolar). Both enzymes exclusively required NAD(+) as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N(2) fixation was demonstrated. The inhibition of O(2) consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O(2), is important in a general strategy of this legume to maintain N(2) fixation under O(2) limited conditions. PMID- 16667789 TI - Comparison of the effect of indoleacetic Acid and fusicoccin on the breakdown of phosphatidylinositol in maize coleoptiles. AB - The effect of indoleacetic acid (IAA) and fusicoccin (FC) on the breakdown of phosphatidylinositol in maize (Zea mays L.) coleoptiles has been studied. Coleoptiles were able to incorporate [(3)H] myo-inositol into the phospholipid fraction almost linearly for 8 hours. Thin layer chromatography analysis of total phospholipids showed that [(3)H]myo-inositol was incorporated only into phosphatidylinositol. Prelabeled coleoptiles treated with IAA showed a loss of the radioactivity incorporated in the phospholipid fraction, whose level decreased by 34% after 1 hour. Treatment with FC, on the contrary, did not modify the content of labelled phosphatidylinositol with respect to the control. The different effects of IAA and FC and a possible mechanism of IAA action on growth are discussed. PMID- 16667790 TI - NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria. AB - Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD(+)- and NADP(+)-isocitrate dehydrogenases, NAD(+)- and NADP(+)-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP(+)-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP(+)-malate dehydrogenase activity is probably due to unspecificity of the NAD(+)-malate dehydrogenase. NADP(+) specific isocitrate dehydrogenase had much lower K(m)s for NADP(+) and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD(+)-specific enzyme (101 micromolar for NAD(+) and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP(+)-specific isocitrate dehydrogenase whereas the NAD(+)-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP(+) stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP(+) is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP(+)-reducing activities of malate dehydrogenase and the NADP(+)-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes. PMID- 16667791 TI - Theoretical basis of protocols for seed storage. AB - The protocols presently established for optimum seed storage do not account for the chemical composition of different seed species, the physiological status of the seed, and the physical status of water within the seed. The physiological status of seeds from five species with varying chemical compositions was determined by measurements of rates of oxygen uptake and seed deterioration. The physical status of water was determined by water sorption characteristics. For each species studied, there was a specific moisture content for the onset of respiration, chemical reactions, and accelerated aging rates. The moisture contents at which these physiological levels were observed varied among the species and correlated with the lipid content of the seed. However, the changes in physiological activities and the physical status of water occurred at specific relative humidities: 91% for the onset of respiration, 27% for the increased rates of thermal-chemical reactions, and 19% for optimum longevity. Based on these observations, we propose that equilibrating seeds between 19 and 27% relative humidity provides the optimum moisture level for maintaining seed longevity during longterm storage. PMID- 16667792 TI - Intercellular Diffusion Limits to CO(2) Uptake in Leaves : Studies in Air and Helox. AB - We studied plants of five species with hypostomatous leaves, and six with amphistomatous leaves, to determine the extent to which gaseous diffusion of CO(2) among the mesophyll cells limits photosynthetic carbon assimilation. In helox (air with nitrogen replaced by helium), the diffusivities of CO(2) and water vapor are 2.3 times higher than in air. For fixed estimated CO(2) pressure at the evaporating surfaces of the leaf (p(i)), assimilation rates in helox ranged up to 27% higher than in air for the hypostomatous leaves, and up to 7% higher in the amphistomatous ones. Thus, intercellular diffusion must be considered as one of the processes limiting photosynthesis, especially for hypostomatous leaves. A corollary is that CO(2) pressure should not be treated as uniform through the mesophyll in many leaves. To analyze our helox data, we had to reformulate the usual gas-exchange equation used to estimate CO(2) pressure at the evaporating surfaces of the leaf; the new equation is applicable to any gas mixture for which the diffusivities of CO(2) and H(2)O are known. Finally, we describe a diffusion-biochemistry model for CO(2) assimilation that demonstrates the plausibility of our experimental results. PMID- 16667793 TI - Purification of a beta-Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism. AB - Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic beta-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the beta-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild type sources, their substrate specificities, and K(m) for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of beta-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified beta-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated beta-amylase protein. PMID- 16667794 TI - Quantification of Apoplastic Potassium Content by Elution Analysis of Leaf Lamina Tissue from Pea (Pisum sativum L. cv Argenteum). AB - K(+) content and concentration within the apoplast of mesophyll tissue of pea (Pisum sativum L., cv Argenteum) leaflets were determined using an elution procedure. Following removal of the epidermis, a 1 centimeter (inside diameter) glass cylinder was attached to the exposed mesophyll tissue and filled with 5 millimolar CaCl(2) solution (1 degrees C). From time-course curves of cumulative K(+) diffusion from the tissue, the amount of K(+) of extracellular origin was estimated. Apoplastic K(+) contents for leaves from plants cultured in nutrient solution containing 2 or 10 millimolar K(+) were found to range from 1 to 4.5 micromoles per gram fresh weight, comprising less than 3% of the total K(+) content within the lamina tissue. Assuming an apoplastic solution volume of 0.04 to 0.1 milliliters per gram fresh weight and a Donnan cation exchange capacity of 2.63 micromoles per gram fresh weight (experimentally determined), the K(+) concentration within apoplastic solution was estimated at 2.4 to 11.8 millimolar. Net movement of Rb(+) label from the extracellular compartment within mesophyll tissue into the symplast was demonstrated by pulse-chase experiments. It was concluded that the mesophyll apoplast in pea has a relatively low capacitance as an ion reservoir. Apoplastic K(+) content was found to be highly sensitive to changes in xylem solution concentration. PMID- 16667795 TI - Mechanism of Photosynthesis Decrease by Verticillium dahliae in Potato. AB - Young, visually symptomless leaves from potato (Solanum tuberosum) plants infected with Verticillium dahliae exhibited reduced carbon assimilation rate, stomatal conductance, and intercellular CO(2), but no increase in dark respiration, no change in the relationship between carbon assimilation rate versus intercellular CO(2), and no change in light use efficiency when intercellular CO(2) was held constant. Therefore, the initial decrease in photosynthesis caused by V. dahliae was caused by stomatal closure. Errors in the intercellular CO(2) calculation caused by uneven distribution of carbon assimilation rate across the leaf were tested by (14)CO(2) autoradiography. Patchiness was found at a low frequency. Low stomatal conductance was correlated with low leaf water potentials. Infection did not affect leaf osmotic potentials. PMID- 16667796 TI - Starch Grain Distribution in Taproots of Defoliated Medicago sativa L. AB - Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines. PMID- 16667797 TI - A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt. AB - The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the alpha- and beta-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in beta-hordothionin and Cys29-Arg30 of alpha-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P(2) position, not those immediately adjacent to the susceptible bonds. The P(2) amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P(1)-P(1)' in the general sequence NH(2)-P(2)-P(1)-P(1)'-COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P(2). The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity. PMID- 16667798 TI - Ethylene production and petiole growth in rumex plants induced by soil waterlogging: the application of a continuous flow system and a laser driven intracavity photoacoustic detection system. AB - Petiole growth of Rumex acetosa L., Rumex crispus L., and Rumex palustris Sm. in response to soil waterlogging was studied in relation to production of the gaseous plant hormone ethylene. Ethylene production was monitored in a flow through system and a recently developed laser driven photoacoustic detection system, which allowed ethylene measurements as low as 6 picoliters per liter. R. acetosa showed a two-fold increase in ethylene production correlated with a slight enhancement of the growth of the petiole that developed during the waterlogging treatment. Both R. crispus and R. palustris showed a strong petiole elongation of existing as well as newly formed petioles, which was correlated with a 20-fold increase in ethylene production after approximately 7 days. Increased rates of ethylene production in R. palustris were related to a strong increase in 1-aminocyclopropane-1-carboxylic acid (ACC) concentration and a slight, but detectable, increase in ethylene forming enzyme activity. In R. acetosa on the other hand, only a very small increase in ACC concentration was observed. Changes in ethylene production in Rumex are strongly correlated with variation in ACC content and ethylene forming enzyme activity. The interaction between ethylene production/internal concentration and ethylene sensitivity of the three Rumex species is discussed in relation to their field location in a flooding gradient and their differential resistance toward waterlogging and submergence. PMID- 16667799 TI - Cold acclimation in Arabidopsis and wheat : a response associated with expression of related genes encoding ;boiling-stable' polypeptides. AB - Changes in gene expression occur during cold acclimation in a wide variety of plant species. Here we show that a number of the polypeptides encoded by cold regulated (cor) genes of Arabidopsis thaliana L. (Heyn) and wheat share the unusual biochemical property that they remain soluble upon boiling in aqueous solution. Further, cDNA cloning in conjunction with Southern and Northern analyses indicate that wheat has a cor gene that is related to Arabidopsis cor47, a gene encoding a 47 kilodalton ;boiling-stable' COR polypeptide. We suggest it is likely that the boiling-stable COR polypeptides have a fundamental role in plants acclimating to cold temperatures and discuss the possibility that they may act as cryoprotectants. PMID- 16667800 TI - Dynamics of Endogenous Cytokinins during the Growth Cycle of a Hormone Autotrophic Genetic Tumor Line of Tobacco. AB - The profile of endogenous cytokinins in a genetic tumor line of tobacco, namely, Nicotiana glauca (Grah.) x Nicotiana langsdorffii (Weinm.), following 1 to 10 weeks of growth on solid medium was determined by radioimmunoassay. (3)H-labeled cytokinins of high specific activity were added during tissue extraction to correct for the purification losses. Following subculture (of 4-week-old tissues when their cytokinin content is high) onto fresh medium the total cytokinin content continued to be high during the first week (1470 picomoles per gram fresh weight) when the tissue fresh weight remained essentially unchanged (lag phase). The cytokinin levels then declined by about half in 2- and 3-week-old tissues (626 and 675 picomoles per gram fresh weight, respectively), a period when rapid increase in tissue fresh weight was recorded. Increments of 840% and 2780% over initial fresh weight were obtained in 2- and 3-week-old cultures, respectively. The cytokinin content then increased to initial high levels in 4-week-old tissues (1384 picomoles per gram fresh weight) after which it gradually declined with tissue age. The lowest cytokinin levels (432 picomoles per gram fresh weight) were observed in 10-week-old tissues. Maximal tissue fresh weight (4030% increase over initial fresh weight) was recorded in 5-week-old cultures after which it decreased slowly to 77.5% of the highest tissue fresh weight in 10-week-old cultures. Zeatin appeared to be the dominant endogenous cytokinin in tissues of all ages. Other cytokinins quantified were dihydrozeatin, zeatin riboside, and dihydrozeatin riboside; the values may include contributions from aglucones derived from the hydrolysis of corresponding O-glucosides, since the entire basic fraction was treated with beta-glucosidase before analysis. In addition the levels of isopentenyladenine, isopentenyladenosine, and the nucleotides of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine were also determined. PMID- 16667801 TI - Dose-Response Analysis of Factors Involved in Germination and Secondary Dormancy of Seeds of Sisymbrium officinale: I. Phytochrome. AB - The germination of seeds of Sisymbrium officinale is light- and nitrate dependent. A close interaction between the effects of light and nitrate on germination has been reported previously (HWM Hilhorst, CM Karssen [1988] Plant Physiol 86: 591-597). In this study, a detailed dose-response analysis of the light-induced germination during induction of secondary dormancy is presented. Germination in water dropped from 90 to 0% after a dark incubation of 15 degrees C of approximately 160 hours. In the presence of 25 millimolar KNO(3), the decrease in germination level was delayed. At 24-hour intervals fluence-response curves were obtained in the presence of 25 millimolar KNO(3). With increasing length of the preincubation period, fluence-response curves shifted along the abscissa to the right. After 120 hours the maximal germination level started to decline. The fluence-response curves were simulated by using formulations from receptor occupancy theory for a simple bimolecular reaction in which the reaction partners were Pfr and its tentative receptor X. A good simulation was obtained when cooperativity of the binding of Pfr to X was assumed. The experimental curve parameters could then be interpreted as binding parameters. PMID- 16667802 TI - Dose-Response Analysis of Factors Involved in Germination and Secondary Dormancy of Seeds of Sisymbrium officinale: II. Nitrate. AB - The role of nitrate as a promoter of germination of Sisymbrium officinale seeds was examined in optimal light conditions. It was shown that the requirement for nitrate was absolute. This was true for all seed lots used. The probit of germination in water was log-linearly related to the level of endogenous nitrate. Preincubation at 15 degrees C resulted in an immediate decrease in germination, whereas in 25 millimolar KNO(3) the decrease was delayed. The decline of germination in water was strongly correlated with the rate at which nitrate leached from the seeds. The germination response to a range of KNO(3) concentrations was followed during preincubation at 24-hour intervals. During the entire 264-hour preincubation period increasingly higher nitrate concentrations were required to maintain a response. This resulted in a right-hand shift of the dose-response curve parallel to the x axis. After 120 hours the high maximum germination level started to decline. The dose-response curves could be simulated by an equation from the receptor-occupancy theory. It is proposed that induction of secondary dormancy is a result of a decrease of the number of nitrate receptors. After 24 and 48 hours of preincubation, the nitrate-response curves were biphasic. The biphasic character could be related to the level of endogenous nitrate and to a differential requirement for nitrate of two fractions of the seed population. Similarities with the behavior of fluence-response curves after prolonged dark incubation led to the hypothesis that phytochrome and nitrate share the same site of action. PMID- 16667803 TI - Light-Induced Carbonic Anhydrase Expression in Chlamydomonas reinhardtii. AB - The effect of external inorganic carbon concentration and light on carbonic anhydrase (CA) protein accumulation and steady-state mRNA levels were examined in Chlamydomonas reinhardtii. When photoautotrophically grown high-CO(2) cells were transferred to low-CO(2) conditions, they exhibited a significant accumulation of the 2.0-kilobase CA transcript after 1 hour with the maximum level reached after 2 hours. An increase in the accumulation of the 37-kilodalton CA monomer was observed after 2-hour exposure to air. Cells allowed to adapt to air levels of CO(2) in the dark showed neither an increase in CA mRNA abundance nor in the accumulation of the enzyme. Similarly, addition of 10 micromole 3-(3,4 dichlorophenyl)-1, 1-dimethylurea immediately after transferring high-CO(2) cells to low-CO(2) condition did not cause an increase in CA transcript abundance and enzyme accumulation, suggesting that photosynthesis is absolutely required in the regulation of CA transcript abundance. In addition to the photosynthesis dependent process, a blue light stimulated mechanism is also involved in CA transcript regulation. Experiments with transcription inhibitors confirmed the notion that the gene for carbonic anhydrase enzyme is encoded by the nuclear genome and that the induction of the enzyme depends heavily on the transcription of the CA gene. PMID- 16667804 TI - Lack of Cross-Resistance of Imidazolinone-Resistant Cell Lines of Datura innoxia P. Mill. to Chlorsulfuron : Evidence for Separable Sites of Action on the Target Enzyme. AB - Two cell lines of Datura innoxia resistant to two imidazolinone herbicides, imazapyr and imazaquin, were isolated from mutagenized, predominantly haploid cell suspension cultures. Both of the resistant variants were >1000-fold more resistant than the wild-type to the two imidazolinones. The variant resistant to imazapyr showed cross-resistance to imazaquin and vice versa, but no cross resistance to a structurally different inhibitor, chlorsulfuron, a sulfonylurea herbicide, was observed. The target enzyme, acetolactate synthase, extracted from imidazolinone-resistant cell lines was not inhibited by imazapyr or imazaquin but was sensitive to chlorsulfuron indicating separable sites of action for these inhibitors. The variation in resistance and cross-resistance of chlorsulfuron resistant (PK Saxena, J King [1988] Plant Physiol 86: 863-867) and imidazolinone resistant cell lines of Datura innoxia demonstrates the possibility of separate mutations of acetolactate synthase gene resulting in specific phenotypes. PMID- 16667805 TI - Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product. AB - The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose 1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga. PMID- 16667806 TI - Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells. AB - The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH(4) (+) in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH(4) (+) to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6 bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose 2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6 phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6 bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH(4) (+) assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH(4) (+) assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH(4) (+) assimilation under anoxia was via glutamine synthetase glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH(4) (+) assimilation. PMID- 16667807 TI - Cytochrome and Alternative Pathway Respiration during Transient Ammonium Assimilation by N-Limited Chlamydomonas reinhardtii. AB - Mass spectrometric analysis of gas exchange in light and dark by N-limited cells of Chlamydomonas reinhardtii indicated that ammonium assimilation was accompanied by an increase in respiratory carbon flow to provide carbon skeletons for amino acid synthesis. Tricarboxylic acid (TCA) cycle carbon flow was maintained by the oxidation of TCA cycle reductant via the mitochondrial electron transport chain. In wild-type cells, inhibitor studies and (18)O(2) discrimination experiments indicated that respiratory electron flow was mediated entirely via the cytochrome pathway in both the light and dark, despite a large capacity for the alternative pathway. In a cytochrome oxidase deficient mutant, or in wild-type cells in the presence of cyanide, the alternative pathway could support the increase in TCA cycle carbon flow. These different mechanisms of oxidation of TCA cycle reductant were reflected by the much greater SHAM sensitivity of ammonium assimilation by cytochrome oxidase-deficient cells as compared to wild type. PMID- 16667808 TI - Developmental control of crassulacean Acid metabolism inducibility by salt stress in the common ice plant. AB - Ice plant (Mesembryanthemum crystallinum) is a facultative halophyte that responds to water stress in the form of drought or high salinity by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM), a physiological adaptation that increases water conservation. Although CAM is clearly environmentally controlled, and reversible upon removal of water stress, the competence to switch is developmentally determined. We have demonstrated this by measuring three parameters in the expression of a gene encoding a stress-specific isoform of a key enzyme of CAM, phosphoenolpyruvate carboxylase (PEPCase, Ppc1): (a) protein accumulation; (b) steady-state amounts of mRNA; and (3) transcriptional activity in isolated nuclei. Young plants (3 weeks of age) show little induction of PEPCase protein, mRNA, or transcription when stressed. In contrast, salt stress elicits a strong induction at all three levels of expression at 6 weeks of age. By 9 weeks of age, plants have already accumulated PEPCase protein and mRNA without being stressed. More importantly, transcriptional activation of Ppc1 by salt stress in 9-week-old plants is no longer observed despite an increase of both Ppc1 mRNA and protein. From these results we suggest that a developmental program exists that regulates PEPCase transcription and mRNA stability. This program appears to be synchronized with the climatic conditions in the plant's native environment. PMID- 16667809 TI - Effect of 2,4-Dichlorophenoxyacetic Acid on Endogenous Cyanide, beta-Cyanoalanine Synthase Activity, and Ethylene Evolution in Seedlings of Soybean and Barley. AB - Treatment of etiolated seedlings of barley (Hordeum vulgare) and soybean (Glycine max) with 1 millimolar 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in a 14 fold and greater than 100-fold increase in ethylene production, respectively. Simultaneous monitoring of endogenous cyanide and beta-cyanoalanine synthase (beta-CAS) (EC 4.4. 1.9) activity was also performed. Endogenous levels of cyanide did not change in barley. In soybean, endogenous cyanide increased within 3 hours, increased again 6 hours after exposure to 2,4-D, and continued to increase throughout the experimental period. The activity of beta-CAS increased in both barley and soybean 9 hours after herbicide treatment. The increase in cyanide preceded the increase in beta-CAS activity by 3 to 6 hours in soybean. The steady-state concentration of endogenous cyanide in soybean was 1 micromolar, based on rates of ethylene production and cyanide metabolism by beta-CAS. This agreed with the determination of endogenous cyanide by both distillation and isotope dilution. Given the apparent compartmentalization of beta-CAS in mitochondria and the localization of ethylene/HCN production at the plasmalemma and/or tonoplast, our results suggest that extra-mitochondrial accumulation of cyanide in the cytoplasm may occur. If so, the activity of cyanide-sensitive cytoplasmic enzymes could be adversely affected, thus possibly contributing to the toxicity of 2,4-D. PMID- 16667810 TI - Tonoplast vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis. AB - Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (T(A) and T(B)) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (T(B)) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (T(A)) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron, and Waring Blendor homogenization gave predominantly cytoplasmic side out vesicles, whereas mashing with a mortar and pestle gave nearly equal amounts of the two populations of membrane vesicles of different orientation. PMID- 16667811 TI - Effects of Glucose Feeding on Respiration and Photosynthesis in Photoautotrophic Dianthus caryophyllus Cells: Mass Spectrometric Determination of Gas Exchange. AB - When glucose (20 millimolar) was added to photoautotrophic cell suspension cultures of Dianthus caryophyllus, there was during the first 10 hours an accumulation of carbohydrates and phosphorylated compounds. These biochemical changes were accompanied by a progressive decrease of net photosynthesis and a twofold increase of the dark respiratory rate. The rise of respiration was associated with a rise of fumarase and cytochrome c oxidase activities, two mitochondrial markers. Gas exchange of illuminated cells were performed with a mass spectrometry technique and clearly established that during the first hours of glucose feeding, the decrease of net photosynthesis was essentially due to an increase of respiration in light, whereas the photosynthetic processes (gross O(2) evolution and gross CO(2) fixation) were almost not affected. However, after 24 hours of experiment, O(2) evolution and CO(2) fixation started to decline in turn. While ribulose-1,5-bisphosphate carboxylase activity was little affected during the first 48 hours of the experiment, the maximal light-induced phosphoribulokinase activity dramatically decreased with time and represented after 48 hours only 30% of its initial activity. It is postulated that the decrease in phosphoribulokinase activity was at least partially responsible for the decrease of CO(2) fixation and the metabolic events involved in this regulation are discussed. PMID- 16667812 TI - Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells. AB - We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5 (3)H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3'-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes. PMID- 16667813 TI - Three Classes of Abscisic Acid (ABA)-Insensitive Mutations of Arabidopsis Define Genes that Control Overlapping Subsets of ABA Responses. AB - Wild type and three abscisic acid (ABA)-insensitive mutants of Arabidopsis (ABI1, ABI2, and ABI3) were compared for their ability to respond to ABA for a variety of ABA-inducible responses throughout the life cycle of the plants. The responses tested included effects on seedling growth, proline accumulation in seedlings, ABA-regulated protein synthesis in plantlets, and seed storage protein and lipid synthesis and accumulation. The abi1 and abi2 mutants showed reduced sensitivity to ABA for inhibition of seedling growth, induction of proline accumulation, and alterations in protein synthesis patterns during vegetative growth, but had wild type levels of storage reserves. In contrast, the abi3 mutant had wild type sensitivity for induction of proline accumulation and was only slightly less responsive to ABA with respect to effects on seedling growth and changes in patterns of protein synthesis. The major effects of this mutation were on seed development. Seeds of the abi3 mutant had two-thirds of the wild type level of storage protein and one-third the wild type level of eicosenoic acid, the major fatty acid component of storage lipids in wild type seeds. These results show that none of the abi mutants is insensitive for all ABA-inducible responses and that the abi3 effects are not seed-specific. Comparison of the degree of ABA sensitivity of monogenic mutant lines with that of digenic mutant lines carrying pairwise combinations of the abi mutations suggests that ABA responses in mature seeds are controlled by at least two parallel pathways. PMID- 16667814 TI - Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): I. Properties of the Herbicide Target Enzymes Acetyl-Coenzyme A Carboxylase and Acetolactate Synthase. AB - Lolium rigidum biotype SR4/84 is resistant to the herbicides diclofop-methyl and chlorsulfuron when grown in the field, in pots, and in hydroponics. Similar extractable activities and affinities for acetyl-coenzyme A of carboxylase (ACCase), an enzyme inhibited by diclofop-methyl, were found for susceptible and resistant L. rigidum. ACCase activity from both biotypes was inhibited by diclofop-methyl, diclofop acid, haloxyfop acid, fluazifop acid, sethoxydim, and tralkoxydim but not by chlorsulfuron or trifluralin. Exposure of plants to diclofop-methyl did not induce any changes in either the extractable activities or the herbicide inhibition kinetics of ACCase. It is concluded that, in contrast to diclofop resistance in L. multiflorum and diclofop tolerance in many dicots, the basis of resistance to diclofop-methyl and to other aryloxyphenoxypropionate and cyclohexanedione herbicides in L. rigidum is not due to the altered inhibition characteristics or expression of the enzyme ACCase. The extractable activities and substrate affinity of acetolactate synthase (ALS), an enzyme inhibited by chlorsulfuron, from susceptible and resistant biotypes of L. rigidum were similar. ALS from susceptible and resistant plants was equally inhibited by chlorsulfuron. Prior exposure of plants to 100 millimolar chlorsulfuron did not affect the inhibition kinetics. It is concluded that resistance to chlorsulfuron is not caused by alterations in either the expression or inhibition characteristics of ALS. PMID- 16667815 TI - Isozymes of Superoxide Dismutase in Mitochondria and Peroxisomes Isolated from Petals of Carnation (Dianthus caryophyllus) during Senescence. AB - The balance between reactions involving free radicals and processes which ameliorate their effect plays an important role in the regulation of plant senescence. In this study a method was developed to isolate peroxisomes and mitochondria from carnation (Dianthus caryophyllus L. cv Ember) petals. Based on electron microscopy and marker enzyme levels, the proportion of peroxisomes to mitochondria increases during senescence. The superoxide dismutase (SOD) content of these fractions was examined. Mitochondria and peroxisomes were shown to contain two electrophoretically distinct SODs, a manganese-, and an ironcontaining SOD. The Mn- and Fe-SOD were found to have relative molecular weights of 75,000 and 48,000 and isoelectric points of 4.85 and 5.00, respectively. The presence of a Fe-SOD in mitochondria and peroxisomes is unique because this enzyme is usually located in chloroplasts. The activity of these two isoenzymes decreased during senescence in mitochondria but remained high in peroxisomes from senescent tissue. It is suggested that peroxisomes play a particular role in the process of senescence. PMID- 16667816 TI - Characterization of a cDNA Encoding Lens culinaris Glycolate Oxidase and Developmental Expression of Glycolate Oxidase mRNA in Cotyledons and Leaves. AB - mRNA obtained from green leaves of lentil (Lens culinaris) was used to construct a cDNA library in phage lambdagt11. The cDNA library was screened with antibodies raised against lentil glycolate oxidase and catalase. Clone CL 1 containing the full-length sequence complementary to glycolate oxidase mRNA was characterized and sequenced. In addition, a 800-base pair catalase cDNA clone was sequenced. To prove the correlation of cDNA insert in CL 1 with glycolate oxidase, the cDNA was transcribed in vitro. The mRNA was translated in vitro yielding a 43 kilodalton protein immunoprecipitable with anti-glycolate oxidase serum. Nucleotide sequences of lentil cDNA and spinach cDNA were 86% identical. Lentil glycolate oxidase was characterized by a C-terminal sequence -P-R-A-L-P-R-L. The expression of glycolate oxidase mRNA in cotyledons, leaves and roots was compared with that of catalase. In leaves, the relative amount of glycolate oxidase mRNA increased during the first 2 days of greening, but decreased later, and was hardly detectable during senescence. In cotyledons of germinating seeds, the level of glycolate oxidase mRNA was markedly lower than the catalase mRNA. PMID- 16667817 TI - Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba). AB - Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl(2), ZnCl(2), or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2 erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1 oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [(14)C]glycerol-3 phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering. PMID- 16667818 TI - Fatty acids, membrane permeability, and sugars of stored potato tubers. AB - The relationships of potato (Solanum tuberosum L.) tuber membrane permeability and membrane lipid composition to sugar accumulation were examined. Tubers from four potato cultivars were stored for 40 weeks at 3 degrees C and 9 degrees C. Rates of tuber membrane electrolyte leakage, total fatty acid composition, free fatty acid composition, and sugar content were measured throughout the storage period. Storage of tubers at 3 degrees C caused dramatic increases in total fatty acid unsaturation, membrane permeability, and sugar content compared to tubers stored at 9 degrees C. Cultivars with higher levels of fatty acid unsaturation had lower rates of membrane electrolyte leakage and lower sugar contents. We propose that high initial levels or high induced levels of membrane lipid unsaturation mitigate increases in tuber membrane permeability during storage, thus positively influencing the processing quality of stored potato tubers. PMID- 16667819 TI - Superoxide Dismutase, Catalase, and alpha-Tocopherol Content of Stored Potato Tubers. AB - Activated oxygen or oxygen free radical mediated damage to plants has been established or implicated in many plant stress situations. The extent of activated oxygen damage to potato (Solanum tuberosum L.) tubers during low temperature storage and long-term storage is not known. Quantitation of oxygen free radical mediated damage in plant tissues is difficult. However, it is comparatively easy to quantitate endogenous antioxidants, which detoxify potentially damaging forms of activated oxygen. Three tuber antioxidants, superoxide dismutase, catalase, and alpha-tocopherol were assayed from four potato cultivars stored at 3 degrees C and 9 degrees C for 40 weeks. Tubers stored at 3 degrees C demonstrated increased superoxide dismutase activities (up to 72%) compared to tubers stored at 9 degrees C. Time dependent increases in the levels of superoxide dismutase, catalase, and alpha-tocopherol occurred during the course of the 40 week storage. The possible relationship between these increases in antioxidants and the rate of activated oxygen production in the tubers is discussed. PMID- 16667820 TI - Turnover of dhurrin in green sorghum seedlings. AB - The turnover of dhurrin in green seedlings of Sorghum bicolor (Linn) Moench var Redland x Greenleaf, Sudan 70 has been investigated using glyphosate and pulse labeling studies with (14)C-tyrosine and [(14)C]shikimic acid. The rate of dhurrin breakdown was 4.8 nanomoles per hour in the shoot and 1.4 nanomoles per hour in the root. The rate of dhurrin accumulation in the shoot of 4- to 5-day old seedlings was high but decreased with age until at the peak period of dhurrin accumulation, the rates of dhurrin synthesis and breakdown were equal. Using a first order equation (an approximation) the rate of dhurrin synthesis (which equals accumulation plus breakdown rates) was 17.4 nanomoles per hour in the shoot and 4.1 nanomoles per hour in the root. In both tissues, the breakdown rate was between 27 and 34% of their synthetic capacity within the experimental period. Dhurrin synthesis in green sorghum seedlings occurred in both the light and dark photoperiods but was faster in the dark period. The result is discussed in relation to the possible metabolic roles of the turnover. PMID- 16667821 TI - Characteristics of a membrane-associated lipoxygenase in tomato fruit. AB - Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22 degrees C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2(*) phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 30 degrees C, the formation of fatty acid oxidation products from 16:0/18:2(*) phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 22 degrees C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2(*) phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same patterns of metabolite formation from radiolabeled linoleic acid and 16:0/18:2(*) phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent K(m) of 0.52 millimolar and an apparent V(max) of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons. PMID- 16667822 TI - Age-related changes in petal membranes from attached and detached rose flowers. AB - Changes in petal membrane properties during aging were studied in cut and in attached rose flowers (Rosa hybrida L., cv Mercedes). Both cut and attached flowers exhibited a growth phase characterized by an increase in fresh weight and an accumulation of membrane components. The growth phase, which was more pronounced in the attached than in the cut flowers, was followed by a senescence phase, characterized by a decrease in fresh weight and a decline in membrane components. In cut flowers, both the growth and the senescence phases were accompanied by a decrease in membrane fluidity and in the ratio of unsaturated to saturated fatty acids, but the ratio of sterol to phospholipid increased. In attached flowers, while both the membrane fluidity and the sterol-to-phospholipid ratio remained unchanged during the growth phase, the senescence phase was accompanied (as in cut flowers) by a decrease in membrane fluidity and an increase in the sterol-to-phospholipid ratio. Unlike in cut flowers, however, the age-related changes in the ratio of unsaturation of fatty acids were not correlated with those of fluidity. Changes in the saturation of phospholipid acyl chains are commonly thought to influence membrane fluidity. Our observations question this view and suggest instead that the ratio of sterol to phospholipid may play the major role in maintaining membrane lipid fluidity. PMID- 16667823 TI - Hypoxic stress-induced changes in ribosomes of maize seedling roots. AB - The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with [(35)S]methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with [(32)P]orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress. PMID- 16667824 TI - Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast. AB - Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier mediated transport of export sugar from the apoplast into the sieve element companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of (14)C photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to (14)CO(2). The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides. PMID- 16667825 TI - Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate. AB - By measuring (18)O exchange from doubly labeled CO(2) ((13)C(18)O(18)O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C(i)]) or air enriched with 5% CO(2) (high C(i)). Intact low C(i) protoplasts had a 10-fold higher carbonic anhydrase activity than did high C(i) protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of (18)O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C(i) cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C(i) than in high C(i) preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment. PMID- 16667826 TI - Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules. AB - Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N(2) fixation, and production of NADP(+) to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N(2) is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N(2)-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N(2) fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities. PMID- 16667827 TI - Hydroxyproline-Rich Glycoprotein Transcripts Exhibit Different Spatial Patterns of Accumulation in Compatible and Incompatible Interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum. AB - The distribution of transcripts encoding hydroxyproline-rich glycoproteins in hypocotyls of Phaseolus vulgaris L. infected with Colletotrichum lindemuthianum was examined by in situ hybridization to tissue sections. The expression of hypersensitive resistance in an incompatible interaction was accompanied by a massive early accumulation of transcripts in the epidermal, cortical, and perivascular parenchymal tissues immediately adjacent to the inoculation site. In a compatible interaction, there was no accumulation of transcripts in the epidermal and cortical tissues even though fungal hyphae ramified throughout these tissues. However, transcripts accumulated at a later stage in the perivascular tissue directly below the site of infection and in tissue several millimeters from the inoculation site. Thus, there is a spatial and tissue specific counterpart to the differential timing of transcript accumulation in incompatible versus compatible interactions (AM Showalter, JN Bell, CL Cramer, JA Bailey, CJ Lamb [1985] Proc Natl Acad Sci USA 82: 6551-6555). These differences in the spatial distribution and tissue specificity of transcript accumulation imply the differential induction of signaling systems involved in race:cultivar specific interactions. PMID- 16667828 TI - Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. AB - The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [(32)P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of (32)P incorporation and the electrophoretic patterns were dependent on (32)P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K(m) values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins. PMID- 16667829 TI - Nodulation of Soybean by a Transposon-Mutant of Rhizobium fredii USDA257 Is Subject to Competitive Nodulation Blocking by Other Rhizobia. AB - Rhizobium fredii USDA257 fails to nodulate the improved soybean [Glycine max (L.)Merr.] cultivar McCall in plastic growth pouches. Mutant 257DH4, which was derived from USDA257 by transposon mutagenesis, forms nitrogen fixing nodules under these conditions. If USDA257 is present in inocula containing the mutant, most infections are arrested prior to organization of the nodule meristem, and nodule number is reduced by 95%. The improved cultivars Essex, Harosoy, Hodgson 78, and Vicoja, as well as a supernodulating mutant of Williams, respond like McCall to inoculation with such mixtures of bacteria. Nodulation blocking on McCall can be elicited by rhizobia other than USDA257, provided that they meet two criteria: Blocking strains must themselves be able to induce cortical cells of McCall to divide, and such divisions must proceed to the stage of nodule meristem formation. Nodulation by the mutant remains sensitive to a challenge inoculation with USDA257 for only the first 6 to 12 hours after inoculation. Nodulation blocking involving mutant 257DH4 thus appears to be a rapid, generalized process. PMID- 16667830 TI - Oleosin isoforms of high and low molecular weights are present in the oil bodies of diverse seed species. AB - Oleosins are unique and major proteins localized on the surface of oil bodies in diverse seed species. We purified five different oleosins (maize [Zea mays L.] KD 16 and KD 18, soybean [Glycine max L.] KD 18 and KD 24, and rapeseed [Brassica campestris L.] KD 20), and raised chicken antibodies against them. These antibodies were used to test for immunological cross-reactivity among oleosins from diverse seed species. Within the same seed species, antibodies raised against one oleosin isoform did not cross-react with the other oleosin isoform (i.e. between maize oleosins KD 16 and KD 18, and between soybean oleosins KD 18 and KD 24). However, the respective antibodies were able to recognize oleosins from other seed species. Where interspecies cross-reactivity occurred, the results suggest that there are at least two immunologically distinct isoforms of oleosins present in diverse seed species, one of lower M(r), and another one of higher M(r). This suggestion is also supported by the relative similarities between the amino acid sequence of a small portion of rapeseed oleosin KD 20 and those of maize oleosins KD 16 and KD 18. In maize kernel, there was a tissue specific differential presentation of the three oleosins, KD 16, KD 18, and KD 19, in the oil-storing scutellum, embryonic axis, and aleurone layer. The phylogenetic relationship between the high and low M(r) isoforms within the same, and among diverse, seed species is discussed. PMID- 16667831 TI - A monoclonal antibody specific to zeatin o-glycosyltransferases of phaseolus. AB - Zeatin O-xylosyltransferase and zeatin O-glucosyltransferase occur in immature embryos of Phaseolus vulgaris and P. lunatus, respectively. Purified preparations of the xylosyltransferase were used as antigen to elicit the formation of antibodies in mice. Hybridoma clones were produced by fusion of mouse spleen cells with myeloma cell line Fox-NY. A clone secreting monoclonal antibody (MAb), XZT-1, capable of immunoprecipitating both enzymes was obtained. The MAb detected a unique protein band from crude embryo extracts of each species with the correct molecular mass (50 kilodaltons) and relative charge (R(F) = 0.5 and 0.3) of the respective enzymes. Competition experiments with substrates indicated that the glycosyl dinucleotide binding sites of the enzymes are probably not involved in MAb-enzyme recognition. Western blotting of samples from vegetative tissues of P. vulgaris detected a low level of O-glucosyltransferase but not O xylosyltransferase, in leaves. These findings suggest the occurrence of two genes in P. vulgaris coding for O-glycosylation enzymes with tissue-specific expression. The MAb will be used to screen expression libraries and to obtain pure enzymes for amino acid sequencing and for the production of additional MAbs. PMID- 16667832 TI - Similar Photosynthetic Performance in Low Light-Grown Isonuclear Triazine Resistant and -Susceptible Brassica napus L. AB - Triazine-resistant plants grown under moderate to high photon flux density (PFD) conditions exhibit decreased photon yield, decreased light-saturated O(2) evolution and slower growth than triazine-susceptible plants. In this study we tested the hypothesis that the comparable growth previously observed in resistant and susceptible Brassica napus L. lines grown under low PFD was accompanied by comparable photon yield and light-saturated O(2) evolution. We measured photon yield, O(2) flash yield, fluorescence decay kinetics, fluorescence transient kinetics, and quenching components, F(v)/F(m) and light saturated O(2) evolution in leaf disks of low PFD-grown triazine-resistant and susceptible B. napus isogenic lines. Results indicated that slow electron transfer from the primary to secondary quinone electron acceptors of photosystem II was still present in the resistant line but photon yield and light-saturated O(2) evolution were similar in the two B. napus lines. We conclude that the alteration in the D1 protein that confers resistance does not necessarily cause decreased photosynthetic performance. Decreased photon yield in resistant plants grown at high PFD is not a direct consequence of the alteration in D1, but represents secondary damage. PMID- 16667833 TI - High Light-Induced Reduction and Low Light-Enhanced Recovery of Photon Yield in Triazine-Resistant Brassica napus L. AB - Triazine-resistant and -susceptible Brassica napus L. plants grown under low photon flux density (PFD) have previously been shown to exhibit a similar photon yield. In contrast, high PFD-grown resistant plants have a lower photon yield than high PFD-grown susceptible plants (JJ Hart, A Stemler [1990] Plant Physiol 94: 1295-1300). In this work we tested the hypothesis that high PFD can induce a differential decrease in photon yield in low PFD-grown plants. We measured photon yield, variable fluorescence/maximum fluorescence, and O(2) flash yield in low PFD-grown resistant and susceptible leaf discs before and after exposure to high PFD exposure. The results demonstrated that high PFD exposure results in a greater decrease in photosystem II (PSII) activity in resistant plants. Characteristics of recovery and other evidence suggest that the differential decrease in PSII efficiency in resistant leaf discs is caused by photoinhibitory damage. We propose that the differential reduction in photon yield and photosynthesis often observed in resistant plants is the result of increased sensitivity to photoinhibition. PMID- 16667834 TI - Maturation in Larch : II. Effects of Age on Photosynthesis and Gene Expression in Developing Foliage. AB - The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO(2) per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS or cab expression. Long shoot foliage is the dominant foliar type on young trees and its lower specific leaf weight will permit production of more photosynthetic surface area per unit of leaf biomass. PMID- 16667835 TI - Photorespiratory mutants of the mitochondrial conversion of glycine to serine. AB - Two mutants of barley (Hordeum vulgare L.), LaPr 85/55 and LaPr 87/30, have been isolated that accumulate glycine, with a concomitant reduction in the aminodonors glutamate and alanine, when transferred to air. Studies have shown that these plants have wild-type levels of serine transhydroxymethylase (EC 2.1.2.1) activity. When supplied (14)CO(2), 48 and 66% of the supplied carbon was retained as glycine in LaPr 85/55 and LaPr 87/30, respectively, compared with a value of 11% for the wild type. In the short-term, both mutant plants are unable to metabolize [(14)C] glycine, but when fed the isotope for 2 hours, LaPr 85/55 was able to metabolize most (70%) of the supplied carbon into sugars with only 15% remaining in glycine. LaPr 87/30, however, was unable to metabolize more than 4% of the supplied carbon into sugars even after 2 hours. Measurement of glycine decarboxylase (EC 2.1.2.10) activity via the glycine-bicarbonate exchange reaction showed LaPr 85/55 to have approximately 70% wild-type activity with LaPr 87/30 having only 14% wild-type activity. The approximation of LaPr 85/55 to wild type activities was maintained for (14)CO(2) release from [(14)C]glycine feeding and ammonia accumulation in the presence of methionine sulphoximine with the equivalent rates for LaPr 87/30 being less than 40% and 10%, respectively. CO(2) fixation rates for the mutants fell to between 35 and 40% of wild-type rates within 10 min of transfer to air. This was shown to be partly due to a run down of aminodonors, because when 40 millimolar serine was supplied through the xylem stream these rates recovered for both mutants to 70% of the wild-type rate. These data suggested a mutation in a glycine transport system for LaPr 85/55 and in the proteins of glycine decarboxylase for LaPr 87/30. Western blotting with antisera to the P, H, T, and L proteins of glycine decarboxylase showed cross-reaction against all four proteins for LaPr 85/55 but little cross-reaction against P or H protein for LaPr 87/30, reaffirming the possibility of a transport mutation in LaPr 85/55. We also suggest that genes for P and H proteins could be either coordinately regulated or that one protein is undetectable or unstable in the absence of the other. PMID- 16667836 TI - Isolation and Characterization of a 2-Oxoglutarate Dependent Dioxygenase Involved in the Second-to-Last Step in Vindoline Biosynthesis. AB - Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2 oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus. PMID- 16667837 TI - Abscisic Acid inhibition of endosperm cell division in cultured maize kernels. AB - The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (+/-)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (+/-)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = -0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel. PMID- 16667838 TI - Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors. AB - The steady state kinetics of glyceraldehyde 3-phosphate:NADP(+) oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K(m) values of 2 to 5 micromolar for NADP(+) and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP(+) as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This ;G3P-inhibited state' is alleviated by saturating amounts of NADP(+) or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP(+) (K(i) 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP(+) and G3P (K(i) 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP(+) binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K(i) 7 micromolar) and Pi (K(i) 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP(+)) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts. PMID- 16667839 TI - Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves. AB - The intracellular compartmentation of ATP sulfurylase and O acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized. PMID- 16667840 TI - Does Iron Deficiency in Pisum sativum Enhance the Activity of the Root Plasmalemma Iron Transport Protein? AB - Roots of Fe-sufficient and Fe-Deficient pea (Pisum sativum L.) were studied to determine the effect of Fe-deficiency on the activity of the root-cell plasmalemma Fe(2+) transport protein. Rates of Fe(III) reduction and short-term Fe(2+) influx were sequentially determined in excised primary lateral roots using Fe(III)-ethylene-diaminetetraacetic acid (Fe[III]-EDTA). Since the extracellular Fe(2+) for membrane transport was generated by root Fe(III) reduction, rates of Fe(2+) influx for each root system were normalized on the basis of Fe(III) reducing activity. Ratios of Fe(2+) influx to Fe(III) reduction (micromole Fe(2+) absorbed/micromole Fe[III] reduced) revealed no enhanced Fe(2+) transport capacity in roots of Fe-deficient peas (from the parental genotype, Sparkle) or the functional Fe-deficiency pea mutant, E107 (derived from Sparkle), relative to roots of Fe-sufficient Sparkle plants. Data from studies using 30 to 100 micromolar Fe(III)-EDTA indicated a linear relationship between Fe(2+) influx and Fe(III) reduction (Fe(2+) generation), while Fe(2+) influx saturated at higher concentrations of Fe(III)-EDTA. Estimations based on current data suggest the Fe(2+) transport protein may saturate in the range of 10(-4.8) to 10(-4) molar Fe(2+). These results imply that for peas, the physiological rate limitation to Fe acquisition in most well-aerated soils would be the root system's ability to reduce soluble Fe(III)-compounds. PMID- 16667841 TI - Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules. AB - Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P(2) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N(2) fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P(2) but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P(1) isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P(1) was three orders of magnitude lower than for AAT-P(2). Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P(2). PMID- 16667842 TI - Light Regulation of delta-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis. AB - Chlorophyll synthesis in Euglena, as in higher plants, occurs only in the light. The key chlorophyll precursor, delta-aminolevulinic acid (ALA), is formed in Euglena, as in plants, from glutamate in a reaction sequence catalyzed by three enzymes and requiring tRNA(Glu). ALA formation from glutamate occurs in extracts of light-grown Euglena cells, but activity is very low in dark-grown cell extracts. Cells grown in either red (650-700 nanometers) or blue (400-480 nanometers) light yielded in vitro activity, but neither red nor blue light alone induced activity as high as that induced by white light or red and blue light together, at equal total fluence rates. Levels of the individual enzymes and the required tRNA were measured in cell extracts of light- and dark-grown cells. tRNA capable of being charged with glutamate was approximately equally abundant in extracts of light- and dark-grown cells. tRNA capable of supporting ALA synthesis was approximately three times more abundant in extracts of light-grown cells than in dark-grown cell extracts. Total glutamyl-tRNA synthetase activity was nearly twice as high in extracts of light-grown cells as in dark-grown cell extracts. However, extracts of both light- and dark-grown cells were able to charge tRNA(Glu) isolated from light-grown cells to form glutamyl-tRNA that could function as substrate for ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1 semialdehyde (GSA), was approximately fourfold greater in extracts of light-grown cells than in dark-grown cell extracts. GSA aminotransferase activity was detectable only in extracts of light-grown cells. These results indicate that both the tRNA and enzymes required for ALA synthesis from glutamate are regulated by light in Euglena. The results further suggest that ALA formation from glutamate in dark-grown Euglena cells may be limited by the absence of GSA aminotransferase activity. PMID- 16667843 TI - Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin. AB - Endo-beta-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-beta 1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-beta-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-beta-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in beta-1,3-glucanase activity and in the number of beta-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. beta-1,3-Glucanase was localized cytochemically with anti-barley-beta-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of beta-1,3 glucanases from wheat and germinated uredospores of the rust fungus with the anti barley-beta-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots. PMID- 16667844 TI - Partial Purification and Characterization of the Gibberellin A(20) 3beta Hydroxylase from Seeds of Phaseolus vulgaris. AB - The GA(20) 3beta-hydroxylase present in immature seeds of Phaseolus vulgaris has been partially purified and characterized. The physical characteristics of the enzyme are similar to those of the GA 2beta-hydroxylases present in mature and immature seeds of Pisum sativum. It is acid-labile, hydrophobic, and of M(r) 45,000. The enzyme catalyzes the synthesis of GA(1), GA(5), and GA(29) from GA(20). Activity is dependent upon the presence of Fe(2+), ascorbate, 2 oxoglutarate, and oxygen. 2-Oxoglutarate does not function as a cosubstrate; in the presence of the enzyme, succinate is not a reaction product. PMID- 16667845 TI - Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles. AB - Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2', 5'-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GR(A) and GR(B)). Purified GR(A) had a specific activity of 1.81 microkatals per milligram of protein and GR(B) had a specific activity of 6.08 microkatals per milligram of protein. GR(A) accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR(B) accounted for 83%. The native molecular mass for GR(A) was 103 to 104 kilodaltons and for GR(B) was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GR(A) and 57 kilodaltons for GR(B). The pH optimum for GR(A) was 7.25 to 7.75 and for GR(B) was 7.25. At 25 degrees C the K(m) for GSSG was 15.3 and 39.8 micromolar for GR(A) and GR(B), respectively. For NADPH, the K(m) was 3.7 and 8.8 micromolar for GR(A) and GR(B), respectively. Antibody produced from purified GR(B) was reactive with both native and denatured GR(B), but was cross-reactive with only native GR(A). PMID- 16667846 TI - High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase. AB - A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls. PMID- 16667847 TI - Measurement of heme efflux and heme content in isolated developing chloroplasts. AB - Hemes destined for cytosolic hemoproteins must originate in one of the cellular compartments which have the capacity for heme synthesis, namely the chloroplast or the mitochondria. Since developing chloroplasts from greening cucumber (Cucumis sativus, cv. Sumter) cotyledons are known to contain complete heme and chlorophyll biosynthetic pathways, they were tested for their capacity export hemes. Picomole quantities of heme were measured by reconstitution of the heme with apo-peroxidase and subsequent determination of peroxidase activity. The assay method was sensitive (as little as 0.7 picomole of heme could be detected in a volume of 100 microliters) and was linear with heme concentration. When intact plastids were incubated with apo-peroxidase, a steady-state rate of efflux between 0.12 and 0.45 picomole heme/minute/milligram plastid protein was measured. The efflux rate was not due to plastid breakage and could be enhanced by incubating with the heme precursor, delta-aminolevulinic acid. Cold acetone extraction removed 47 +/- 17 picomoles heme/milligram plastid protein from the total b-type heme pool in the chloroplasts (166 +/- 9 picomoles heme/milligram protein, by acid-acetone extraction). The reconstitution technique provided a similar estimate of readily exchangeable heme in the plastid, 37 +/- 8 picomoles heme/milligram protein (or 6 micromolar in the plastids). These values may be indicative of a ;free heme pool' which exists in the chloroplast. PMID- 16667848 TI - Influence of vesicular arbuscular mycorrhizae and leaf age on net gas exchange of citrus leaves. AB - The purpose of this study was to test the hypothesis that vesicular arbuscular mycorrhizal (VAM) fungi affect net assimilation of CO(2) (A) of different-aged citrus leaves independent of mineral nutrition effects of mycorrhizae. Citrus aurantium L., sour orange plants were grown for 6 months in a sandy soil low in phosphorus that was either infested with the VAM fungus, Glomus intraradices Schenck & Smith, or fertilized with additional phosphorus and left nonmycorrhizal (NM). Net CO(2) assimilation, stomatal conductance, water use efficiency, and mineral nutrient status for expanding, recently expanded, and mature leaves were evaluated as well as plant size and relative growth rate of leaves. Nutrient status and net gas exchange varied with leaf age. G. intraradices-inoculated plants had well-established colonization (79% of root length) and were comparable in relative growth rate and size at final harvest with NM plants. Leaf mineral concentrations were generally the same for VAM and NM plants except for nitrogen. Although leaf nitrogen was apparently sufficient for high rates of A, VAM plants did have higher nitrogen concentrations than NM at the time of gas exchange measurements. G. intraradices had no effect on A, stomatal conductance, or water use efficiency, irrespective of leaf age. These results show that well established VAM colonization does not affect net gas exchange of citrus plants that are comparable in size, growth rate, and nutritional status with NM plants. PMID- 16667849 TI - Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase. AB - Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K(m) (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K(m) (MgPEP) was found to be significantly lower than the respective K(m) (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose 1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism. PMID- 16667850 TI - Adaptation of the Photosynthetic Apparatus in Maize Leaves as a Result of Nitrogen Limitation : Relationships between Electron Transport and Carbon Assimilation. AB - In maize (Zea mays L., cv Contessa), nitrogen (NO(3) (-)) limitation resulted in a reduction in shoot growth and photosynthetic capacity and in an increase in the leaf zeaxanthin contents. Nitrogen deficiency had only a small effect on the quantum yield of CO(2) assimilation but a large effect on the light-saturated rate of photosynthesis. Linear relationships persisted between the quantum yield of CO(2) assimilation and that of photosystem II photochemistry in all circumstances. At high irradiances, large differences in photochemical quenching and nonphotochemical quenching of Chl a fluorescence as well as the ratio of variable to maximal fluorescence (Fv/Fm) were apparent between nitrogen-deficient plants and nitrogen-replete controls, whereas at low irradiances these parameters were comparable in all plants. Light intensity-dependent increases in nonphotochemical quenching were greatest in nitrogen-deficient plants as were the decreases in Fv/Fm ratio. In nitrogen-deficient plants, photochemical quenching decreased with increasing irradiance but remained higher than in controls at high irradiances. Thermal dissipative processes were enhanced as a result of nitrogen deficiency (nonphotochemical quenching was elevated and Fv/Fm was lowered) allowing PSII to remain relatively oxidised even when carbon metabolism was limited via nitrogen limitation. PMID- 16667851 TI - Biosynthesis of the Phytoalexin Pisatin : Isoflavone Reduction and Further Metabolism of the Product Sophorol by Extracts of Pisum sativum. AB - NADPH-dependent reduction of 2',7-dihydroxy-4',5'-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl(2). The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of Cucl(2)-treated seedlings was also observed; three products were radiolabeled when [(3)H]sophorol was the substrate, one of which is tentatively identified as maackiain. PMID- 16667852 TI - Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine. AB - The metabolism of [1,4-(14)C]putrescine and [terminal methylene-(3)H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The (14)C label from putrescine was incorporated into spermidine, gamma-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [(3)H]Spermidine was catabolized into putrescine and beta-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed. PMID- 16667853 TI - Sucrose synthase in rice plants : growth-associated changes in tissue specific distributions. AB - Different parts of the rice (Oryza sativa L.) plant at different growth stages were analyzed for sucrose synthase (SS) by enzyme activity assay and enzyme linked immunosorbent assay directly on the extracts or on the eluates from a gel filtration column. On a dry matter basis, the amount of soluble protein and SS activity decreased significantly, but the amount of enzyme protein changed little in growing leaves. In the grain, the SS activity was the highest at the early ripening stage and decreased later, but the amount of SS protein increased with the increase in maturity. In the root, a low activity of SS was detectable only in the tillering but not in other stages. Immunoblotting of SS protein extracted from different parts of rice showed two bands. Elution patterns of crude extracts from a gel filtration column showed the presence of several types of SS protein. Among them, two to three types with larger elution volumes had the SS activity but others with smaller elution volumes (considered as the aggregated forms) had no activity. The SS purified from different parts of the plant showed similar but distinctly different electrophoretic mobilities in a native gel. It has been concluded that different isozymes are expressed in different tissues at different growth stages. PMID- 16667854 TI - The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance. AB - Mutagenized populations of Arabidopsis thaliana seedlings were screened for plants capable of root growth on inhibitory concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Four of the mutant lines recovered from this screen display a defect in root gravitropism as well as hormone resistance. The aerial portions of these plants are similar to wild-type in appearance. Genetic analysis of these four mutants demonstrated that hormone resistance segregated as a recessive trait and that all four mutations were alleles of the auxin-resistant mutation aux1 [Maher HP, Martindale SJB (1980) Biochem Genet 18: 1041-1053]. These new mutants have been designated aux1-7, 1 12, 1-15, and 1-19. The sensitivity of wild-type and aux1-7 roots to indole-3 acetic acid, 2,4-dichlorophenoxyacetic acid, and ethylene was determined. The results of these assays show that aux1-7 plants require a 12-fold (indole-3 acetic acid) or 18-fold (2,4-dichlorophenoxyacetic acid) higher concentration of auxin than wild-type for a 50% inhibition of root growth. In addition, ethylene inhibition of root growth in aux1-7 plants is approximately 30% that of wild-type at saturating ethylene concentrations. These results indicate that aux1 plants are resistant to both auxin and ethylene. We have also determined the effect of ethylene treatment on chlorophyll loss and peroxidase activity in the leaves of aux1 and wild-type plants. No difference between mutant and wild-type plants was observed in these experiments, indicating that hormone resistance in aux1 plants may be limited to root growth. Our studies suggest that the AUX1 gene may have a specific function in the hormonal regulation of gravitropism. PMID- 16667855 TI - Isolation and characterization of a novel nuclear protein from pollen mother cells of lily. AB - Pollen mother cells of the lily (Lilium speciosum) were found to have a histone H1-like protein (PMCP) not detected in other tissues. The PMCP appears from the late S-G(2) period of premeiosis and is present in mature pollen. PMCP and H1 were extracted from pollen mother cells with 5% perchloric acid and isolated by reverse-phase high-performance liquid chromatography. The amino acid composition of PMCP differs from that of somatic H1. However, PMCP is similar to H1t in mammalian testis with regard to amino acid composition. PMID- 16667856 TI - Effect of Potassium Levels on the Stomatal Behavior of the Hemi-Parasite Striga hermonthica. AB - The hemi-parasite Striga hermonthica, exhibits an anomalous pattern of stomatal response, stomata remaining open in darkness and when subjected to water stress. This suggests irregularity in stomatal response due to malfunction of the stomatal mechanism. To test this suggestion guard cells were isolated from the effects of surrounding cells, by incubating epidermal strips at low pH. These stomata responded rapidly to low CO(2) concentrations, darkness, and ABA. Thus, a paradox exists between stomatal behavior observed in whole leaves and that in isolated guard cells. However, when incubated in the presence of high potassium concentrations (>200 millimolar KCl) stomatal responses in epidermal strips resembled those found in whole leaves, with enhanced opening and reduced closing responses. It is suggested that the anomalous behavior of stomata in Striga and other leafy hemiparasites can be explained by the modulatory effects of high potassium concentrations which accumulate in the leaves as a consequence of high transpiration rates and the lack of a retranslocation system. PMID- 16667857 TI - Reduction of Chloroplast DNA Content in Solanum nigrum Suspension Cells by Treatment with Chloroplast DNA Synthesis Inhibitors. AB - Suspension cell cultures of Solanum nigrum were grown in the presence of six different chloroplast DNA synthesis inhibitors in order to determine whether the pool size of chloroplast DNA (cpDNA) could be selectively reduced relative to the nuclear DNA content. One of the effects of the inhibitors was a reduction in cell growth and viability. Cell growth (fresh weight) was reduced 50% (in 8 day cultures) by: 100 micromolar bisbenzimide, 8 micromolar ethidium bromide, 0.3 micromolar 5-fluordeoxyuridine (Fudr), 200 micromolar nalidixic acid, 30 micromolar novobiocin, or 10 micrograms per milliliter rifampicin. At these concentrations, three of the inhibitors, ethidium bromide, Fudr, and rifampicin, also substantially reduced the viability of the cultures. Analyses of the chloroplast and nuclear DNA content per gram fresh weight by dot blot hybridizations indicated that the reduction of cpDNA content was greatest at inhibitor concentrations which reduced cell growth by more than 50% but this depended on the culture conditions. For example, the two DNA gyrase inhibitors, nalidixic acid and novobiocin, were more effective in lowering cpDNA content in cultures which were transferred (2 x 4 days) once during the eight day incubation. Because several inhibitors were toxic to cell growth, the DNA content of treated cells was also determined on the basis of cell (protoplasts) number. Analyses of nuclear and cpDNA content per cell for each treatment indicated that only the DNA gyrase inhibitors, nalidixic acid, and novobiocin reduced cpDNA content. Neither inhibitor reduced nuclear DNA content. These results suggest that DNA gyrases participate in cpDNA replication. The selective reduction of cpDNA content in regeneratable cultures may facilitate the generation and selection of cpDNA mutants or transformants from higher plants. PMID- 16667858 TI - Organ Specificity and Light Regulation of NADH-Dependent Hydroxypyruvate Reductase Transcript Abundance. AB - By probing total RNA blots with an NADH-dependent hydroxypyruvate reductase (HPR) cDNA clone, we have found that HPR transcript abundance is highly regulated in developing cucumber (Cucumis sativus) seedlings. HPR transcript levels in light grown seedlings are 50-fold more abundant in leaves and cotyledons than in roots. When 12-day light-grown seedlings are shifted to the dark for 4 days, the HPR transcript level in leaves drops 20-fold. Upon return of these dark-adapted plants to the light, HPR transcript levels rise to 50% of the previous light grown level within 2 hours. PMID- 16667859 TI - Isolation of a cDNA Clone for alpha-Amylase in Mung Bean Cotyledons : Analysis of alpha-Amylase mRNA Levels in Cotyledons during and following Germination of Mung Bean Seeds. AB - A cDNA was isolated that codes for alpha-amylase in mung bean (Vigna radiata) cotyledons, and the nucleotide sequence was determined. The deduced amino acid sequence (421 amino acid residues) is about 65% homologous with those of barley alpha-amylases. By comparing the deduced sequence with the sequence of the purified alpha-amylase, it was inferred that 23 N-terminal amino acids of a nascent polypeptide represent a signal peptide. Northern blot analysis showed that the levels of alpha-amylase mRNA are in parallel with the activities of alpha-amylase synthesis in cotyledons. Under the conditions where the solute leakage from cotyledons is accelerated during imbibition, a rapid increase in the amount of the alpha-amylase mRNA occurs. We postulate that a factor(s) which regulates in an inhibitory manner the alpha-amylase expression at the transcriptional level may be present in dry cotyledons and be removed by leakage. PMID- 16667860 TI - Conversion of l-Sorbosone to l-Ascorbic Acid by a NADP-Dependent Dehydrogenase in Bean and Spinach Leaf. AB - An NADP-dependent dehydrogenase catalyzing the conversion of l-sorbosone to l ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at -20 degrees C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. K(m) for sorbosone were 12 +/- 2 and 18 +/- 2 millimolar and for NADP(+), 0.14 +/- 0.05 and 1.2 +/- 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of l-ascorbic acid biosynthesis, had no effect on the reaction. PMID- 16667861 TI - d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves. AB - d-[6-(14)C]Glucosone that had been prepared enzymically from d-[6-(14)C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, (14)C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6 (14)C]glucose underwent considerable redistribution during AA formation, whereas (14)C from [6-(14)C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-(14)C]sorbosone was found to be equivalent to [6-(14)C]glucose as a source of (14)C for AA. In the presence of 0.1% d glucosone, conversion of [6-(14)C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d glucosone and l-sorbosone are putative intermediates in the conversion of d glucose to AA in higher plants. PMID- 16667862 TI - Novel occurrence of uncommon polyamines in higher plants. AB - Diamines and polyamines are ubiquitous components of living cells, and apparently are involved in numerous cellular and physiological processes. Certain "uncommon" polyamines have limited distribution in nature and have been associated primarily with organisms adapted to extreme environments, although the precise function of these polyamines in such organisms is unknown. This article summarizes current knowledge regarding the occurrence in higher plants of the uncommon polyamines related to and including norspermidine and norspermine. A putative biosynthetic pathway to account for the occurrences of these uncommon polyamines in higher plants is presented, with a summary of the supporting evidence indicating the existence of the requisite enzymatic activities in alfalfa, Medicago sativa L. PMID- 16667863 TI - Response of enzymes and storage proteins of maize endosperm to nitrogen supply. AB - To examine the effects of N nutrition upon endosperm development, maize (Zea mays) kernels were grown in vitro with either 0, 3.6, 7.1, 14.3, or 35.7 millimolar N. Kernels were harvested at 20 days after pollination for determination of enzyme activities and again at maturity for quantification of storage products and electrophoretic separation of zeins. Endosperm dry weight, starch, zein-N, and nonzein-N all increased in mature kernels as N supply increased from zero to 14.3 millimolar. The activities of sucrose synthase, aldolase, phosphoglucomutase, glutamate-pyruvate transaminase, glutamate oxaloacetate transaminase, and acetolactate synthase increased from 1- to 2.5 fold with increasing N supply. Adenosine diphosphate-glucose pyrophosphorylase and both ATP- and PPi-dependent phosphofructokinases increased to lesser extents, while no significant response was detected for hexose kinases and glutamine synthetase. Nitrogen-induced changes in enzyme activities were often highly correlated with changes in final starch and/or zein-N contents. Separation of zeins indicated that these peptides were proportionately enhanced by N supply, with the exception of C-zein, which was relatively insensitive to N. These data indicate that at least a portion of the yield increase in maize produced by N fertilization is induced by a modification of kernel metabolism in response to N supply. PMID- 16667864 TI - Early autoregulation of symbiotic root nodulation in soybeans. AB - Autoregulation of symbiotic root nodulation in soybean seedlings (Glycine max L. Merrill cv Pride 216) was studied following double inoculation of primary roots with Bradyrhizobium japonicum 110. When the second inoculation was given 10 or 17 hours after the first, the nodulation in the first-inoculated region of the root was suppressed. The effect was eliminated if B. japonicum 110 containing Tn5 insertions in the ;common' nod ABC genes was used for the second inoculation, indicating the requirement for changes in the root mediated by these bacterial genes. When the root cortex in the suppressed basal region was examined 3 days after inoculation, cell division centers were present in numbers not significantly different from the numbers in control roots given a sham second inoculation; their size distribution, however, showed a failure of enlargement compared with controls. PMID- 16667865 TI - Abscisic Acid Alters the Metabolism of Indole-3-Acetic Acid in Senescing Flowers of Cucumis melo L. AB - Experiments were conducted to investigate indole-3-acetic acid (IAA) and abscisic acid (ABA) metabolism associated with postanthesis senescence of ovaries from nonpollinated muskmelon (Cucumis melo L.) flowers. Flowers attached to the vine were allowed to senesce for 4 days after anthesis or were harvested at full anthesis and aged for the same time interval with or without access to water. The IAA ester, amide-linked forms of IAA, free ABA, and ABA esters increased in senescent ovaries from flowers left attached to the vine. Detaching flowers from the vine resulted in an accumulation of free and amide-linked IAA in the senescing ovary but suppressed accumulation of ester IAA. Free ABA failed to increase in ovaries detached from the vine. Subjecting detached flowers to water stress had no effect on the endogenous level of free ABA but resulted in the accumulation of ABA ester and suppression of any increase in free IAA. However, detached flowers treated with 0.1 millimolar ABA accumulated 75% less free IAA and initiated the synthesis of ester IAA. Detached flowers treated with ABA also accumulated high levels of ester ABA. These results suggest that the metabolism of free IAA in muskmelon ovary tissue is regulated in situ and not the consequence of external synthesis and importation. ABA appears to be transported into the senescing ovary from an external source and alters the IAA metabolism in such a manner as to suppress the level of free IAA while stimulating accumulation of the ester IAA. PMID- 16667866 TI - Effects of Abscisic Acid and High Osmoticum on Storage Protein Gene Expression in Microspore Embryos of Brassica napus. AB - Storage protein gene expression, characteristic of mid- to late embryogenesis, was investigated in microspore embryos of rapeseed (Brassica napus). These embryos, derived from the immature male gametophyte, accumulate little or no detectable napin or cruciferin mRNA when cultured on hormone-free medium containing 13% sucrose. The addition of abscisic acid (ABA) to the medium results in an increase in detectable transcripts encoding both these polypeptides. Storage protein mRNA is induced at 1 micromolar ABA with maximum stimulation occurring between 5 and 50 micromolar. This hormone induction results in a level of storage protein mRNA that is comparable to that observed in zygotic embryos of an equivalent morphological stage. Effects similar to that of ABA are noted when 12.5% sorbitol is added to the microspore embryo medium (osmotic potential = 25.5 bars). Time course experiments, to study the induction of napin and cruciferin gene expression demonstrated that the ABA effect occurred much more rapidly than the high osmoticum effect, although after 48 hours, the levels of napin or cruciferin mRNA detected were similar in both treatments. This difference in the rates of induction is consistent with the idea that the osmotic effect may be mediated by ABA which is synthesized in response to the reduced water potential. Measurements of ABA (by gas chromatography-mass spectrometry using [(2)H(6)]ABA as an internal standard) present in microspore embryos during sorbitol treatment and in embryos treated with 10 micromolar ABA were performed to investigate this possibility. Within 2 hours of culture on high osmoticum the level of ABA increased substantially and significantly above control and reached a maximum concentration within 24 hours. This elevated concentration was maintained for 48 hours after culturing and represents a sixfold increase over control embryos. The ABA-treated embryos accumulated the hormone very quickly, but ABA concentrations returned to basal levels within 72 hours after treatment. The possibility that embryo-synthesized ABA may be a mediator of effects of osmotic stress on gene expression in Brassica embryos is discussed. PMID- 16667867 TI - An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample. AB - A continuous spectrophotometric assay of H(+)-ATPase activity was developed by combining two well-known methods for measuring proton pumping and ATPase activity. Proton uptake into plasma membrane vesicles from Avena sativa L. (cv Rhiannon) was monitored as the absorbance decrease at 495 nm of the DeltapH probe acridine orange. Simultaneously, ATPase activity was measured by following the absorbance decrease at 340 nanometers by coupling ATP hydrolysis enzymatically to the oxidation of NADH. This H(+)-ATPase assay is convenient for determining the relative relationship between ATP hydrolysis and proton pumping. PMID- 16667868 TI - Concurrent synthesis and degradation of alcohol dehydrogenase in elicitor-treated and wounded potato tubers. AB - The accumulation of alcohol dehydrogenase (ADH) in arachidonic acid-elicited potato (Solanum tuberosum L.) tuber discs was studied. In accordance with our previous report of the accumulation of Adh mRNA beginning 2 hours after elicitor treatment (DP Matton, CP Constabel, N Brisson [1990] Plant Mol Biol 14: 775-783), immunoprecipitation of ADH from in vivo labeled discs indicated that ADH synthesis occurred as early as 12 hours after treatment. However, levels of ADH activity and protein, as shown by enzyme assay and immunoblot, did not rise in parallel but decreased during the first 24 hours of treatment. After 24 hours, ADH activity and protein began to increase, reaching a several-fold increase at 96 hours after elicitation. Water-treated control discs showed a similar though delayed and less pronounced pattern. These results imply a turnover of ADH following elicitor treatment of potato tuber discs. As shown by nondenaturing gel electrophoresis, the synthesis and degradation involved the same ADH isozyme. PMID- 16667869 TI - Effects of Irradiance on the in Vivo CO(2):O(2) Specificity Factor in Tobacco Using Simultaneous Gas Exchange and Fluorescence Techniques. AB - The effects of gas phase O(2) concentration (1%, 20.5%, and 42.0%, v/v) on the quantum yield of net CO(2) fixation and fluorescence yield of chlorophyll a are examined in leaf tissue from Nicotiana tabacum at normal levels of CO(2) and 25 to 30 degrees C. Detectable decreases in nonphotochemical quenching of absorbed excitation occurred at the higher O(2) levels relative to 1% O(2) when irradiance was nearly or fully saturating for photosynthesis. Photochemical quenching was increased by high O(2) levels only at saturating irradiance. Simultaneous measurements of CO(2) and H(2)O exchange and fluorescence yield permit estimation of partitioning of linear photosynthetic electron transport between net CO(2) fixation and O(2)-dependent, dissipative processes such as photorespiration as a function of leaf internal CO(2) concentration. Changes in the in vivo CO(2):O(2) ;specificity factor' (K(sp)) with increasing irradiance are examined. The magnitude K(sp) was found to decline from a value of 85 at moderate irradiance to 68 at very low light, and to 72 at saturating photon flux rates. The results are discussed in terms of the applicability of the ribulose bisphosphate carboxylase/oxygenase enzyme model to photosynthesis in vivo. PMID- 16667870 TI - sym 13-A Gene Conditioning Ineffective Nodulation in Pisum sativum. AB - Treatment of Pisum sativum (L.) cv. ;Sparkle' with ethyl methanesulfonic acid (EMS) produced a stable mutant, E135F, which forms small, white, ineffective nodules. These nodules exhibit histological zonation typical of an indeterminant nodule, e.g. meristematic, early symbiotic, late symbiotic, and senescent zones. Compared with the nitrogen fixing nodules of the parent, the zones are smaller and the nodules senesce prematurely. Bacteroids in E135F are less elongated and less differentiated than those in ;Sparkle.' The E135F mutant forms ineffective nodules when inoculated with nine different effective strains of Rhizobium leguminosarum and also when grown in a soil containing effective strains. The ineffective phenotype of E135F is under monogenic recessive control; the gene is designated sym 13. sym 13 was located on chromosome 2 by linkage with genes for shikimic dehydrogenase and esterase-2. The original selection E135F carried another mutation in heterozygous form at a separate locus, yielding some homozygous recessive nonnodulating progeny, E135N, in later generations. This indicates that EMS treatments may cause mutations at more than one sym gene. The gene conditioning non-nodulation in E135N was designated sym 14. It mapped to a locus on a different part of chromosome 2 by linkage to the gene for fumarase. The data demonstrate that sym genes are not necessarily closely linked. PMID- 16667872 TI - Differential expression of potato tuber protein genes. AB - Patatin and the 22-kilodalton protein complex make up more than 50% of the soluble protein present in potato (Solanum tuberosum) tubers and these two proteins are coordinately regulated during tuber development. Although genomic sequences related to these tuber genes exist in the genome of potato species that do not bear tubers, they cannot be induced into expression under the tested conditions. These genes are not expressed during substantial starch accumulation in petioles from a model petiole-leaf cutting system in nontuber-bearing plants, indicating that starch accumulation and synthesis of the major tuber proteins occur independently. Tuber protein gene expression also has been examined in hybrid potato plants that contain genomes from both tuberizing and nontuberizing species. One such triploid hybrid produced only stolons, whereas a pentaploid hybrid with an increased number of tuber genomes produced tubers. It was shown, using immunoblotting and Northern blot hybridization, that these two hybrids actively expressed both patatin and the 22-kilodalton tuber protein in induced petioles from the leaf-cutting system. The induced accumulation of patatin transcripts was consistent in all genotypes containing some tuberizing genome. The induced accumulation of the 22-kilodalton protein transcripts, however, was lower in genotypes containing some nontuberizing genome. Sucrose induction of these genes in leaves corroborates the induction patterns in petioles. A correlation exists between 22-kilodalton protein gene expression and a potato plant's ability to produce stolons or tubers. PMID- 16667871 TI - Chlorsulfuron modifies biosynthesis of acyl Acid substituents of sucrose esters secreted by tobacco trichomes. AB - Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID- 16667873 TI - Photoacoustic measurements in vivo of energy storage by cyclic electron flow in algae and higher plants. AB - Energy storage by cyclic electron flow through photosystem I (PSI) was measured in vivo using the photoacoustic technique. A wide variety of photosynthetic organisms were considered and all showed measurable energy storage by PSI-cyclic electron flow except for higher plants using the C-3 carbon fixation pathway. The capacity for energy storage by PSI-cyclic electron flow alone was found to be small in comparison to that of linear and cyclic electron flows combined but may be significant, nonetheless, under conditions when photosystem II is damaged, particularly in cyanobacteria. Light-induced dynamics of energy storage by PSI cyclic electron flow were evident, demonstrating regulation under changing environmental conditions. PMID- 16667874 TI - In Vitro Interaction of Nuclear Proteins with the Promoter of Soybean Heat Shock Gene Gmhsp17.5E. AB - Proteins present in crude nuclear extracts of soybean (Glycine max) plumules were shown to bind in vitro to the 5' flanking sequences of the soybean heat shock gene Gmhsp17.5E. The specificity of binding activity present in extracts from both control (28 degrees C) and heat shocked (40 degrees C) tissues was demonstrated by reciprocal competition experiments using gel mobility retardation assays. Footprinting experiments using DNase I with crude nuclear extracts indicated that a continuous stretch of 5' flanking sequences extending from -40 to -153 was protected from digestion in vitro. Nuclear proteins that were partially purified by heparin agarose chromatography were shown to bind specific TATA-proximal sequences containing the heat shock consensus elements (HSEs) (-73 to -49; -107 to -84) and AT-rich motifs (-119 to -153). Other binding sites within AT-rich sequences (-906 to -888, -868 to 863, -859 to 853, and -841 to 830), distal HSE elements (-568 to -532) and a TATA/dyad (-234 to -207) were also identified by DNase I footprinting of TATA-distal probes. DNA binding activities specific for the HSE and AT-rich sequences were present in nuclear extracts from both control and heat shocked tissues. Both types of binding activity were increased after heat shock treatment; HSE binding increased from 1.8- to 2.7 fold, and binding to AT-rich sequences showed an increase from 1.3- to 1.7-fold. PMID- 16667875 TI - Regulation of Chloroplast Development by Nitrogen Source and Growth Conditions in a Chlorella protothecoides Strain. AB - Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6 bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6 phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media. PMID- 16667876 TI - Involvement of na in active uptake of pyruvate in mesophyll chloroplasts of some c(4) plants : na/pyruvate cotransport. AB - An artificial Na(+) gradient across the envelope (Na(+) jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C(4) plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219:347). In the present study, (22)Na(+) and pyruvate uptake were examined in mesophyll chloroplasts of several species of C(4) plants. Enhancement of pyruvate uptake by a Na(+) jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in turn enhanced Na(+) uptake in the dark when added together with Na(+). When flux of endogenous Na(+) was measured in these mesophyll chloroplasts preincubated with (22)Na(+), pyruvate addition induced Na(+) influx, and the extent of the pyruvate-induced Na(+) influx positively correlated with that of pyruvate uptake. A Na(+)/H(+) exchange ionophore, monensin, nullified all the above mutual effects of Na(+) and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na(+) uptake and increased equilibrium level of chloroplast (22)Na(+). Measurements of initial uptake rates of pyruvate and Na(+) gave a stoichiometry close to 1:1. These results point to Na(+)/pyruvate cotransport into mesophyll chloroplasts of some C(4) plants. PMID- 16667877 TI - Polysaccharide Fraction from Higher Plants which Strongly Interacts with the Cytosolic Phosphorylase Isozyme : I. Isolation and Characterization. AB - From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by (14)C-labeling experiments in which the glucosyl transfer from [(14)C]glucose 1-phosphate to the polysaccharide preparation was monitored. PMID- 16667878 TI - Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants. AB - We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein. PMID- 16667879 TI - Adaptation and growth of tomato cells on the herbicide 2,6-dichlorobenzonitrile leads to production of unique cell walls virtually lacking a cellulose-xyloglucan network. AB - Suspension-cultured cells of tomato (Lycopersicon esculentum VF 36) have been adapted to growth on high concentrations of 2,6-dichlorobenzonitrile, an herbicide which inhibits cellulose biosynthesis. The mechanism of adaptation appears to rest largely on the ability of these cells to divide and expand in the virtual absence of a cellulose-xyloglucan network. Walls of adapted cells growing on 2,6-dichlorobenzonitrile also differ from nonadapted cells by having reduced levels of hydroxyproline in protein, both in bound and salt-elutable form, and in having a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. Most of these latter polymers are apparently cross-linked in the wall via phenolic-ester and/or phenolic ether linkages, and these polymers appear to represent the major load-bearing network in these unusual cell walls. The surprising finding that plant cells can survive in the virtual absence of a major load-bearing network in their primary cell walls indicates that plants possess remarkable flexibility for tolerating changes in wall composition. PMID- 16667880 TI - Regulation of the heat shock response in soybean seedlings. AB - The transcriptional response of soybean (Glycine max) seedlings during heat shock (HS) was investigated under two different treatment regimes. During prolonged heat treatment at 40 degrees C, active transcription of the HS genes (as measured by "runoff" transcription assays) occurs only during the first few hours. Nonetheless, mRNAs for these genes are present at relatively high abundance even after 9 hours of exposure to 40 degrees C. Because HS mRNAs have a fairly short half-life (less than 3 hours) at 28 degrees C, these results indicate that HS mRNAs are inherently more stable at 40 degrees C. During a second type of heat treatment regime-short pulses of high (45 degrees C) heat followed by 1 to 2 hours at 28 degrees C-transcription of HS genes is comparable to that achieved at 40 degrees C for the first few hours, even though the tissue is maintained at non HS temperatures. The transcriptional responses to these two different heat treatments indicate that regulatory controls for the transcription of the HS genes must involve more than a simple sensing of ambient temperature, since transcription of these genes can be turned off at 40 degrees C (in the case of prolonged exposure) and can continue at 28 degrees C (following a short, severe heat treatment). Additional results demonstrate that the response of soybean seedlings to a particular HS depends on their prior exposure to heat; seedlings given a preheat treatment (that is known to induce thermotolerance) respond more moderately to a short heat pulse at 45 degrees C. Overall, this research indicates that plants have mechanisms for both monitoring the severity of changes in temperature and for measuring the magnitude and duration of the stress. Such information is then used to regulate the plant's response to heat both transcriptionally and posttranscriptionally. PMID- 16667881 TI - Sugar uptake and starch biosynthesis by slices of developing maize endosperm. AB - (14)C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of (14)C among the soluble sugars extracted from endosperm slices incubated in (14)C-sugars. Competing hexoses reduced the incorporation of (14)C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue. PMID- 16667882 TI - Light as a signal influencing the phosphorylation status of plant proteins. AB - The phosphorylation-status of a number of plant enzymes has been shown to be altered in response to light. Phosphoenolpyruvate carboxylase is phosphorylated (more active) in C(4) plants in the light but CAM phosphoenolpyruvate carboxylase is phosphorylated (more active) in the dark. C(4) plant pyruvate, Pi dikinase is dephosphorylated (activated) in the light and sucrose phosphate synthase is less phosphorylated (more active) in the light. The mitochondrial pyruvate dehydrogenase is inactivated (phosphorylated) in the light. The reversal of these events occurs in the dark or when photosynthesis is inhibited. Phytochrome and blue light receptors also alter the phosphorylation-status of proteins. The evidence is rapidly increasing in support of signal transduction networks in plants that involve light reception. PMID- 16667883 TI - Distribution and metabolism of xylem-borne ureido and amino compounds in developing soybean shoots. AB - Pulse-chase feeding (30-120 minutes) of (14)C-labeled nitrogenous compounds to cut transpiring shoots was used to investigate the early fate of the major xylem borne solutes in N(2)-fixing soybean (Glycine max) plants at the V(4) growth stage. By comparison with the foliar distribution of [(14)C]inulin (a xylem marker), it was determined that the phloem supply of allantoin, allantoic acid, asparagine, glutamine, aspartate, and arginine, respectively, provided about 20, 10, three, two, five, and 20 times the (14)C delivered to the developing trifoliolate in the xylem stream. Recovery of unmetabolized asparagine, aspartate, and arginine in this indicator trifoliolate, and significant declines in the percentage of (14)C from allantoic acid and allantoin recovered in the first trifoliolate, provided some support for the direct xylem-to-phloem transfer of these compounds, but did not preclude the involvement of indirect transfer. Data on stem retention and foliar distribution, expressed as a function of the relative xylem sap composition, indicated that ureides provide the major sources of nitrogen to all plant parts. There was no consistent distinction in distribution patterns between pairs of similar anionic and neutral compounds. The extent of xylem-to-phloem transfer among the ureido or the amino compounds was inversely related to its prominence in xylem sap. PMID- 16667884 TI - Phosphorylation by Inorganic Phosphate of the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.). AB - The phosphorylation of plasma membrane proteins from red beet (Beta vulgaris L.) by radioactive inorganic phosphate was studied. Only few proteins were phosphorylated, among them was one polypeptide with an apparent molecular weight of about 100,000. The phosphorylation of this protein was decreased when orthovanadate was present in the reaction mixture, or when the phosphorylated protein was treated with hydroxylamine. These facts suggest that this protein is a transport ATPase which is phosphorylated in a carboxyl group during the catalytic cycle. This protein was identified immunologically as the plasma membrane H(+)-ATPase. The phosphorylation level of this enzyme was enhanced by dimethyl sulfoxide, whereas potassium ions did not have a significant effect on this level unless ATP was present. ATP stimulated the phosphorylation by inorganic phosphate. This stimulation was more apparent in the presence of potassium ions. PMID- 16667885 TI - Purification of leucoplast pyruvate kinase from developing castor bean endosperm. AB - Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases. PMID- 16667886 TI - Cytokinins and Differentiation Processes in Mercurialis annua: Genetic Regulation, Relations with Auxins, Indoleacetic Acid Oxidases, and Sexual Expression Patterns. AB - Cytokinins in apices of eight isogenic lines of Mercurialis annua were compared (high performance liquid chromatography-gas chromatography mass spectroscopy computer system). These apices develop normal staminate or pistillate differentiation processes (sex series lines) or empty (sterile), semiempty (semisterile), and full anthers (restored fertile male) in the sterility series in which a pistillate line was constructed. Both series developed two different cytokinin pathways: trans-cytokinins characterized the sex series, whereas the cis pathway characterized the sterility series. Drastic changes in the trans pathway (0/250 nanograms trans-zeatin and 166/0 nanograms zeatin nucleotide) induced staminate/pistillate differentiations. Less drastic quantitative changes in the cis pathway induced sterility or restored fertility compared to normal fertile anthers (192 or 669 nanograms/traces). The action of the complete cis pathway was morphologically effective in the sterility series when the ratio of cis to trans pathways was 1:2 or 1:1 instead of 1:3. A final diagram shows the action of each sex or sterility allele on the enzymes controlling specific metabolites in both pathways. The discussion provides insights on the regulation of cytokinin-auxin balances specific for each kind of reproductive differentiation. PMID- 16667887 TI - Photosynthesis in Ulva fasciata: V. Evidence for an Inorganic Carbon Concentrating System, and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase CO(2) Kinetics. AB - Evidence of an inorganic carbon concentrating system in a marine macroalga is provided here. Based on an O(2) technique, supported by determinations of inorganic carbon concentrations, of experimental media (as well as compensation points) using infrared gas analysis, it was found that Ulva fasciata maintained intracellular inorganic carbon levels of 2.3 to 6.0 millimolar at bulk medium concentrations ranging from 0.02 to 1.5 millimolar. Bicarbonate seemed to be the preferred carbon form taken up at all inorganic carbon levels. It was found that ribulose-1,5-bisphosphate carboxylase/oxygenase from Ulva had a K(m)(CO(2)) of 70 micromolar and saturated at about 250 micromolar CO(2). Assuming a cytoplasmic pH of 7.2 (as measured for another Ulva species, P Lundberg et al. [1988] Plant Physiol 89: 1380-1387), and given the high activity of internal carbonic anhydrase (S Beer, A Israel [1990] Plant Cell Environ [in press]) and the here measured internal inorganic carbon level, it was concluded that internal CO(2) in Ulva could, at ambient external inorganic carbon concentrations, be maintained at a high enough level to saturate ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation. It is suggested that this suppresses photorespiration and optimizes net photosynthetic production in an alga representing a large group of marine plants faced with limiting external CO(2) concentrations in nature. PMID- 16667888 TI - C and N Mobilization from Stalk and Leaves during Kernel Filling by C and N Tracing in Zea mays L. AB - The sink capacity of the stalk in Zea mays L. (cv DEA) during the elongation period was previously investigated with (13)C and (15)N tracing. The chase experiment described here demonstrates the different behavior of intermediary reserves for C and N remobilization until full maturity of the kernels. Carbon incorporated during stalk elongation participated mainly in cellulose formation in vegetative organs appearing after the labeling period; the remobilization to kernels was low (0.5%). Soluble carbohydrates and proteins were the main intermediary sink compounds, starch being little remobilized. N first incorporated in roots, sheaths, stalk, blades was translocated to the kernel; 42% of the labeled N were recovered in kernels where they represented 8% of the total N. Cob, husk, and shank acted first as N sinks and then as N sources during ear development. It appeared that aminoacids used for synthesis of kernel proteins have a common origin, except for glutelin G(3). PMID- 16667889 TI - Boron requirement in cyanobacteria : its possible role in the early evolution of photosynthetic organisms. AB - The effect of boron on heterocystous and nonheterocystous dinitrogen fixing Cyanobacteria was examined. The absence of boron in culture media inhibited growth and nitrogenase activity in Nodularia sp., Chlorogloeopsis sp., and Nostoc sp. cultures. Examinations of boron-deficient cultures showed changes in heterocyst morphology. However, cultures of nonheterocystous Cyanobacteria, Gloeothece sp. and Plectonema sp., grown in the absence of boron did not show any alteration in growth or nitrogenase activity. These results suggest a requirement of boron only by heterocystous Cyanobacteria. A possible role for this element in the early evolution of photosynthetic organisms is proposed. PMID- 16667890 TI - Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum. AB - Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl(-) and H(2)PO(4) (-) uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K(+) uptake. During fusicoccin dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K(+) absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl(-) FC induced intracellular acidification. Since H(+) fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K(+)/A(-) symport or HCO(3) (-)/A(-) antiport for anion uptake. Though not suitable to reveal the actual H(+)/A(-) stoichiometry, the results are consistent with an H(+)/A(-) cotransport mechanism. PMID- 16667891 TI - Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle. AB - The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G(2) phase, and underwent cell division simultaneously. Only 10% of the cells remained in G(0) or G(1). Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G(0) + G(1)/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G(2) phase. However, reversal of G(1) arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G(0) + G(1) phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G(0). The frequency of cells entering G(0) was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium. PMID- 16667892 TI - Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin. AB - The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoe daigremontiana leaves revealed gradients of activity. The results indicate that the auxin stimulated cell wall invertase is essential for the establishment of the tumor sink. PMID- 16667893 TI - Comparison of ripening processes in intact tomato fruit and excised pericarp discs. AB - Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L(*)a(*)b(*) color space, changing from green (a(*) < -5) to red (a(*) > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO(2) production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a(*) = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a(*) = -5), appeared as discs turned yellow-red (a(*) = 5), and increased another eight-fold as discs became full red (a(*) value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a(*) = 0) to red. PMID- 16667894 TI - Diurnal Rhythmicity in the Pattern of mRNAs in the Leaves of Sinapis alba. AB - Previous studies have shown that certain specific leaf mRNAs exhibit a diurnal rhythmicity in their quantity in higher plants. To determine whether this situation is restricted to a few mRNAs, or affects a large number, we have used in vitro translation and two-dimensional polyacrylamide gel electrophoresis to analyze the mRNA complement in leaves of Sinapis alba at different times during an 8-hour/16-hour day/night cycle. A method for the visual analysis of two dimensional polyacrylamide gel electrophoresis was also developed. This method selected, at each sampling time, spots that were significant. It then selected, between two sampling times, intensity changes that were significant at the 0.02 confidence level. During a day/night cycle, complex rhythmic changes affected about 10% of the mRNAs. Nineteen different rhythm patterns were found. These 19 patterns fell into four main classes: mRNAs that increase during the light period and decrease during the dark, mRNAs that increase and then decrease during the light period, mRNAs that decrease during the light period and increase during the dark period, and mRNAs that increase and then decrease during the dark period. PMID- 16667895 TI - Does water deficit stress promote ethylene synthesis by intact plants? AB - The effect of plant water deficit on ethylene production by intact plants was tested in three species, beans (Phaseolus vulgaris L.), cotton (Gossypium hirsutum L.) and miniature rose (Rosa hybrida L., cv Bluesette). Compressed air was passed through glass, plant-containing cuvettes, ethylene collected on chilled columns, and subsequently assayed by gas chromatography. The usual result was that low water potential did not promote ethylene production. When plants were subjected to cessation of irrigation, ethylene production decreased on a per plant or dry weight basis of calculation. No significant promotion of ethylene production above control levels was detected when water deficit-treated bean or cotton plants were rewatered. The one exception to this was for cotton subjected to a range of water deficits, plants subjected to deficits of -1.4 to -1.6 MPa exhibited a transient increase of ethylene production of 40 to 50% above control levels at 24 or 48 hours. Ethylene was collected from intact leaves while plants developed a water deficit stress of -2.9 megapascals after rewatering, and no significant promotion of ethylene production was detected. The shoots of fruited, flowering cotton plants produced less ethylene when subjected to cessation of irrigation. In contrast, the ability of bench drying of detached leaves to increase ethylene production several-fold was verified for both beans and cotton. The data indicate that detached leaves react differently to rapid drying than intact plants react to drying of the soil with regard to ethylene production. This result suggests the need for additional attention to ethylene as a complicating factor in experiments employing excised plant parts and the need to verify the relevance of shock stresses in model systems. PMID- 16667896 TI - Aspartate Aminotransferase in Alfalfa Root Nodules : III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species. AB - Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT 2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day old alfalfa plants contained about eight times more AAT-2 than did nodules of 7 day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N(2)-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non N(2)-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species. PMID- 16667897 TI - Photosynthetic characterization of photoautotrophic cells cultured in a minimal medium. AB - Photosynthetic properties of photoautotrophic suspensions cultured in a minimal growth medium have been evaluated to determine whether changes have occurred in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, phosphoenol pyruvate (PEP) carboxylase activity, chlorophyll content, or culture growth. Five photoautotrophic lines Amaranthus powellii, Datura innoxia, Glycine max, Gossypium hirsutum, and a Nicotiana tabacum-Nicotiana glutinosa fusion hybrid were grown in a medium without organic carbon other than phytohormones, and without vitamins. These photoautotrophic lines had total Rubisco activities ranging from 85 to 266 micromoles CO(2) fixed per milligram chlorophyll hour(-1), with percent activation of Rubisco ranging from 16 to 53%. Inclusion of protease inhibitors in the homogenization buffer did not result in higher Rubisco activity. PEP carboxylase activity for cells cultured in minimal medium was found to range from 16 to 146 micromoles CO(2) per milligram chlorophyll hour(-1), with no higher activity in the C(4)Amaranthus cells compared with PEP carboxylase activity in the C(3) species assayed. Rubisco-to-PEP carboxylase ratios ranged from 2.2 to 1 up to 9.4 to 1. Chlorophyll contents increased in all but the Nicotiana cell line, and all of the photoautotrophic culture lines were capable of growth in vitamin-free medium with the exception of SB-P, which requires thiamine. PMID- 16667898 TI - Comparison of increased expression of wild-type and herbicide-resistant acetolactate synthase genes in transgenic plants, and indication of posttranscriptional limitation on enzyme activity. AB - Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nicotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the native wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25 fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated. PMID- 16667899 TI - Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L. AB - Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR(1)) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA(3), was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA(3). Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress. PMID- 16667900 TI - Effects of High Light Stress on Carotenoid-Deficient Chloroplasts in Pisum sativum. AB - The effects of high light stress on chloroplast ultrastructure and protein and mRNA composition were investigated in carotenoid-deficient peas (Pisum sativum, L.). In low light, the thylakoid membrane polypeptide pattern was altered, with several prominent chlorophyll-binding proteins present in diminished amounts. This change was found to be reflected in the ultrastructural organization of internal chloroplast membranes. In contrast to the normal grana stacking found in the controls, carotenoid-deficient plastids contained long, unstacked lamellae. Exposure to photooxidative light that resulted in destruction of >70% of chlorophyll did not lead to changes in total RNA and total cellular protein patterns. This treatment did lead to gross alterations in the chloroplast structure. Within 24 hours the plastid was seen as a swollen vesicle with only a few membrane remnants still present. Accumulation of five plastid-encoded mRNAs encoding a diverse array of photosynthetic proteins was found to be affected in different ways. While psaA mRNA was rapidly reduced by more than 75%, levels of psbF/E and atpB/E were reduced by 50%. psbA and petA mRNAs, on the other hand, appeared to be more resistant to photobleaching and remained relatively unchanged during 24 hours of high fluence-rate light treatment. PMID- 16667901 TI - A flower-inducing substance of high molecular weight from higher plants. AB - The flower-inducing activities of aqueous extracts of several plants were fractionated by gel filtration. Three major peaks, corresponding to molecular weights of about 120, 20 to 30, and 5 to 10 kilodaltons, were detected in extracts of Lemna, Pharbitis, and Brassica. The latter two peaks may be degradation products generated during the extraction procedure. In extracts of soybean seeds, only the peak of material of 120 kilodaltons was detected. This is the first published report of a high molecular mass substance with florigenic activity in Lemna plants. The florigenic substance had some properties associated with proteins (or polypeptides), but the activity was unaffected by treatment with proteinase K. PMID- 16667902 TI - Characterization of Five Abscisic Acid-Responsive cDNA Clones Isolated from the Desiccation-Tolerant Plant Craterostigma plantagineum and Their Relationship to Other Water-Stress Genes. AB - Leaves of resurrection plants tolerate desiccation as do embryos of many higher plants. From the resurrection plant Craterostigma plantagineum a number of desiccation-related transcripts have recently been cloned; they are abundantly expressed in dried leaves and abscisic acid-treated dried callus (D Bartels, K Schneider, G Terstappen, D Piatkowski, F Salamini [1990] Planta 18: 27-34). Five distinct cDNA clones representing low copy number genes were selected for further characterization. Their nucleotide sequences were determined and proteins were predicted with a molecular mass between 16 and 34 kilodaltons. Three of these proteins have unusual amino acid compositions and extreme hydrophilic characters. Two of them contain a cluster of contiguous serine residues and lysine-rich repeats. These sequence motifs display homologies to desiccation-related genes expressed in embryos or dehydrated seedlings of several plants. A third cDNA clone contains tracts of sequences which are related to a cotton Lea (late embryogenesis abundant) gene (JC Baker, C Steele, L Dure III [1988] Plant Mol Biol II: 277-291). Secondary structure predictions are discussed and suggest that the deduced proteins could play a role in protecting core cell structures in a dehydrated cell. It is concluded that at least in part the gene products involved in the desiccation-induced pathways are common to leaves of resurrection plants and embryos. Two cDNA clones appear to code for Craterostigma-specific mRNAs. The expression patterns of all five transcripts were studied in comparison to desiccated leaves in dehydrated roots, in wound-stressed leaves and in salt stressed callus. The data obtained point to the possibility that not only specificity of induction but also the expression level of specific gene products may be of importance for osmoprotection. PMID- 16667903 TI - Sequence and Expression of a HSP83 from Arabidopsis thaliana. AB - A full-length cDNA encoding a heat shock protein (hsp) belonging to the 83 to 90 kilodalton hsp family of Arabidopsis thaliana has been isolated and sequenced. Truncated cDNA clones were isolated by nucleic acid hybridization to a truncated soybean HSP83 cDNA probe and a fragment generated from a Drosophila HSP83 gene. A single strand DNA vector/primer based extension procedure was employed to obtain the full-length cDNA. The level of transcripts homologous to this cDNA (AtHS83) is low in 2-week-old Arabidopsis plants but is rapidly enhanced by elevated temperatures. DNA sequence comparison between this cDNA and hsp83-90 sequences from human, yeast and Drosophila reveal amino acid identities of 63 to 69%, typical identities for interspecies comparisons between hsp83 to 90 kilodalton proteins. Genomic DNA blot analysis performed with probes derived from AtHS83 indicate the presence of a HSP83 gene family estimated to be comprised of at least three genes. PMID- 16667904 TI - Light-Stimulated Gibberellin Biosynthesis in Gibberella fujikuroi. AB - Gibberellins (GAs) are a group of plant growth hormones that were first isolated from the fungus Gibberella fujikuroi. The biosynthesis of GA in liquid cultures of the fungus has been examined using high-performance liquid chromatography and combined gas chromatography-mass spectrometry. GA(3) was the predominant GA in well-aerated cultures. GA(4) and GA(7), intermediates in GA(3) biosynthesis, accumulated in cultures with low levels of dissolved oxygen, but were not detectable in more highly aerated cultures. Light stimulated the production of GA(3) in G. fujikuroi cultures grown from young stock cultures. Cell-free enzyme studies revealed a significant stimulation in the levels of kaurenoic acid oxidation in cultures grown in the light in comparison with those grown in the dark. However, measurements of the relative rates of [(14)C]mevalonic acid incorporation into kaurene showed no effect of light on this early part of the pathway. Preliminary experiments indicated that blue light is most effective in enhancing kaurenoic acid oxidation. PMID- 16667905 TI - Cooperative regulation of cytoplasmic streaming and ca fluxes by pfr and photosynthesis in vallisneria mesophyll cells. AB - In mesophyll cells of Vallisneria gigantea Graebner, Ca(2+) regulates the induction and cessation of cytoplasmic streaming. Streaming is induced when the level of calcium in the cytoplasm is lowered through light-accelerated release of Ca(2+) from the cells (S Takagi, R Nagai [1988] Plant Physiol 88: 228-232). We have now initiated an investigation on the nature of the photoreceptor(s) that are involved in the regulation of Ca(2+) movements across the cell membrane and of streaming. Streaming is induced only when phytochrome exists in the phytochrome-far redabsorbing form (Pfr)-and photosynthesis is allowed to take place for at least 4 minutes. The former effect is typically photoreversible by red and far-red light, and phytochrome is spectro-photometrically detectable in the crude extract from the leaves. The latter effect is assessed in terms of the wavelength dependency and the effects of diuron and atrazine, two inhibitors of photosynthesis. A similar requirement for Pfr and photosynthesis is found to be associated with the acceleration of Ca(2+) efflux in the protoplasts. The results suggest that phytochrome and photosynthetic pigment(s) cooperatively regulate cytoplasmic streaming via modulation of the Ca(2+) transport in the cell membrane. PMID- 16667906 TI - Identification of Two Brassinosteroids from the Cambial Region of Scots Pine (Pinus silverstris) by Gas Chromatography-Mass Spectrometry, after Detection Using a Dwarf Rice Lamina Inclination Bioassay. AB - A simple and improved dwarf rice (Oryza sativa var Tan-ginbozu) lamina inclination bioassay for brassinosteroids (BRs) was developed based on a previously published method (K Takeno, RP Pharis [1982] Plant Cell Physiol 23: 1275-1281). The assay used 3-day-old intact seedlings, and detection of BR was made more sensitive by synergizing the response to BR with indole-3-acetic acid (IAA). The minimum detectable amount of BR was less than 0.1 nanogram/rice plant (brassinolide equivalents). Purification steps for isolation of BR from tissue scrapings taken from the cambial region of Scots pine (Pinus silverstris) harvested during the period of rapid wood production were guided by this assay. After column chromatography (silica gel and PrepPak C(18)) and reversed phase C(18) high performance liquid chromatography, the biologically active fractions were analyzed by gas chromatography-mass spectrometry (GC-MS) and/or GC-MS selected ion monitoring. Two BRs, castasterone (major) and brassinolide (minor) were identified. This is the first identification of BR from the cambial region of a conifer. PMID- 16667907 TI - Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.). AB - Cytokinin-like activity in samples of xylem and phloem sap collected from field grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C(18) reversed-phase high performance liquid chromatography --> NH(2) normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring. PMID- 16667908 TI - Tracheid production in response to changes in the internal level of indole-3 acetic Acid in 1-year-old shoots of scots pine. AB - Different concentrations of indole-3-acetic acid (IAA) were applied in lanolin to 1-year-old shoots of Pinus sylvestris (L.) in a manner known to stimulate cambial activity. The internal concentration of free IAA was measured at a distance below the application point by combined gas chromatography-selected ion monitoring-mass spectrometry using [(13)C(6)]IAA as a quantitative internal standard, and related to the production of tracheids at the same site. The experiment was performed with: (a) debudded cuttings, where the major source of endogenous IAA, the apical buds, were replaced with exogenous IAA, and (b) intact, attached shoots, where endogenous IAA was supplemented by applying IAA around the circumference of the shoot. In both experimental systems, an increase in the internal IAA level was positively related to increased tracheid production. It was also demonstrated that the concentration of internal IAA measured at the sampling site was comparable with endogenous IAA levels found in intact control shoots, and that a wide range of applied IAA concentrations was associated with a relatively small range of internal IAA levels. PMID- 16667909 TI - A Model Describing the Regulation of Ribulose-1,5-Bisphosphate Carboxylase, Electron Transport, and Triose Phosphate Use in Response to Light Intensity and CO(2) in C(3) Plants. AB - A model of the regulation of the activity of ribulose-1,5-bisphosphate carboxylase, electron transport, and the rate of orthophosphate regeneration by starch and sucrose synthesis in response to changes in light intensity and partial pressures of CO(2) and O(2) is presented. The key assumption behind the model is that nonlimiting processes of photosynthesis are regulated to balance the capacity of limiting processes. Thus, at CO(2) partial pressures below ambient, when a limitation on photosynthesis by the capacity of rubisco is postulated, the activities of electron transport and phosphate regeneration are down-regulated in order that the rate of RuBP regeneration matches the rate of RuBP consumption by rubisco. Similarly, at subsaturating light intensity or elevated CO(2), when electron transport or Pi regeneration may limit photosynthesis, the activity of rubisco is downregulated to balance the limitation in the rate of RuBP regeneration. Comparisons with published data demonstrate a general consistency between modelled predictions and measured results. PMID- 16667910 TI - Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Light Intensity and CO(2) in the C(3) Annuals Chenopodium album L. and Phaseolus vulgaris L. AB - The light and CO(2) response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (k(cat)) of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C(3) annuals Chenopodium album and Phaseolus vulgaris at 25 degrees C. The initial slope of the photosynthetic CO(2) response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO(2) response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO(2) partial pressures (C(1)) between the CO(2) compensation point and 500 microbars. Above a C(1) of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C(1), decreasing as the C(1) was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C(1) only under conditions when the activation state of rubisco was dependent on C(1). Otherwise, RuBP pool sizes increased as C(1) was reduced. ATP pools in C. album tended to increase as C(1) was reduced. In P. vulgaris, decreasing C(1) at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k(cat). These results support modelled simulations of the rubisco response to light and CO(2), where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration. PMID- 16667911 TI - The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems. AB - The hypothesis that auxin (IAA) and gibberellic acid (GA(3)) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA(3) (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA(3)/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA(3) induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA(3)/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed. PMID- 16667912 TI - Separation and Partial Purification of 1,3-beta-Glucan and 1,4-beta-Glucan Synthases from Saprolegnia. AB - Enriched 1,3-beta-glucan and 1,4-beta-glucan synthase fractions from the fungus Saprolegnia were isolated by rate zonal centrifugation on glycerol gradient. Purification was improved by entrapment of the enzymes in their reaction product, i.e. microfibrillar glucans. 1,3-beta-Glucan synthases were separated from 1,4 beta-glucan synthases following resuspension of entrapped enzymes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated that 1,3-beta-glucan and 1,4-beta-glucan synthases may have a different polypeptide composition because they were enriched for different protein subunits (34, 48, and 50 kD for the 1,3-beta-glucan synthase and 60 kD for the 1,4-beta-glucan synthase). PMID- 16667913 TI - Regeneration of Freezing-Tolerant Spring Wheat (Triticum aestivum L.) Plants from Cryoselected Callus. AB - A cryoselection protocol has been developed that provides freezing-tolerant callus that, in turn, can regenerate plants with enhanced cold hardiness. Tolerant calli were selected from spring wheat (Triticum aestivum L.) callus by immersion in liquid nitrogen without addition of cryoprotectants. Less than 15% of the calli survived the initial challenge, whereas 30 to 40% of previously selected calli survived subsequent exposure. Seed progeny from five of 11 regenerant (R2) lines tested exhibited significantly enhanced tolerance to freezing at -12 degrees C. Thus, cryoselection appears to involve at least in part, selection for genetic rather than epigenetic variants. Analysis of one callus line indicated that cryoselection did not induce significant alterations in lipid composition, adenylate energy charge, or freezing point. An increase in the soluble sugar component was detected. Changes were also detected in the protein complement of microsomal membrane and soluble protein extracts of cryoselected callus. In all, seven unique proteins ranging from 79 to 149 kilodaltons were identified. The results demonstrate that freezing tolerant callus can be isolated from a heterogeneous population by cryoselection, and factors that contribute to hardiness at the callus level are biologically stable and can contribute to tolerance at the whole plant level. PMID- 16667914 TI - Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles. AB - We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature. PMID- 16667915 TI - Pattern of Variations in Abscisic Acid Content in Suspensors, Embryos, and Integuments of Developing Phaseolus coccineus Seeds. AB - Free abscisic acid (ABA) content in suspensors, embryos, and integuments was determined during seed development of Phaseolus coccineus. A highly specific and sensitive solid-phase radioimmunoassay based on a monocional antibody raised against free (S)-ABA was used for ABA quantification. Very small amounts of ABA were detected in the suspensor during initial stages of development; later two peaks of ABA occurred. Levels of ABA in the embryo and integument show a coincident triphasic distribution: two maxima in ABA content occurred when the embryo was 11 to 12 and 15 to 16 millimeters in length; later, when the embryo was 19 to 20 millimeters long, a further increase was observed. The role of ABA in runner bean seeds is discussed in relation to the development of the different seed tissues. PMID- 16667916 TI - Leaf water relations and maintenance of gas exchange in coffee cultivars grown in drying soil. AB - Plant water status, leaf tissue pressure-volume relationships, and photosynthetic gas exchange were monitored in five coffee (Coffea arabica L.) cultivars growing in drying soil in the field. There were large differences among cultivars in the rates at which leaf water potential (Psi(L)) and gas exchange activity declined when irrigation was discontinued. Pressure-volume curve analysis indicated that increased leaf water deficits in droughted plants led to reductions in bulk leaf elasticity, osmotic potential, and in the Psi(L) at which turgor loss occurred. Adjustments in Psi(L) at zero turgor were not sufficient to prevent loss or near loss of turgor in three of five cultivars at the lowest values of midday Psi(L) attained. Maintenance of protoplasmic volume was more pronounced than maintenance of turgor as soil drying progressed. Changes in assimilation and stomatal conductance were largely independent of changes in bulk leaf turgor, but were associated with changes in relative symplast volume. It is suggested that osmotic and elastic adjustment contributed to maintenance of gas exchange in droughted coffee leaves probably through their effects on symplast volume rather than turgor. PMID- 16667917 TI - Tonoplast ion channels from sugar beet cell suspensions : inhibition by amiloride and its analogs. AB - The properties of the vacuolar membrane (tonoplast) ion channels of sugar beet (Beta vulgaries) cell cultures were studied using the patch-clamp technique. Tonoplast currents displayed inward rectification in the whole vacuole and isolated outside-out patch configurations and permeability ratios P(K+)/P(Na+) = 1 and P(K+)/P(Cl-) = 5. Amiloride and two of its analogs, 5-(N-methyl-N-isobutyl) amiloride and benzamil, inhibitors of Na(+) channels in animal systems, blocked inward currents by reducing single-channel openings. Concentrations for 50% inhibition of vacuolar currents of 730 nanomolar, 130 nanomolar, and 1.5 micromolar for amiloride, benzamil, and 5-(N-methyl-N-isobutyl)-amiloride, respectively, were obtained from whole-vacuole recordings. The high inhibitory action (affinity) of amiloride and its analogs for the tonoplast cation channel suggests that these compounds could be used for the isolation and biochemical characterization of this protein. PMID- 16667918 TI - Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton. AB - Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the DeltapH probe [(14)C]methylamine. Evidence is presented for the operation of Na(+)/H(+) and K(+)/H(+) antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H(+) ATPase or by imposing an artificial pH gradient) brings about dissipation of the DeltapH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl(-), using the DeltaPsi probe SCN(-). Cation/H(+) exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H(+)-ATPase; coupling of the opposed cation and H(+) fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na(+)/H(+) and K(+)/H(+) antiport in Atriplex. Moreover, additive effects are obtained when Na(+) is supplied together with saturating concentrations of K(+), and vice versa, suggesting that separate antiporters for Na(+) and for K(+) may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast. PMID- 16667919 TI - Stress Responses in Alfalfa (Medicago sativa L.): VI. Differential Responsiveness of Chalcone Synthase Induction to Fungal Elicitor or Glutathione in Electroporated Protoplasts. AB - Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by FE and GSH, which involve previously indentified cis-elements in the CHS promoter. PMID- 16667920 TI - Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L. AB - Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4 dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of (35)SO(4) (2-), and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using (35)SO(4) (2-), roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5'-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5'-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5'-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved. PMID- 16667921 TI - Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function. AB - Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or beta-limit dextrin containing gel and KI/I(2) staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar beta-amylase (EC 3.2.1.2) and the primarily apoplastic alpha-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily beta amylase) activity and considerably higher ratios of beta- to alpha-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and beta-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of alpha-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32 fold (protein basis) higher alpha-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in alpha-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the alpha-amylase that was greatly increased in activity was the primarily apoplastic alpha-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic alpha-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar beta-amylase activity. PMID- 16667922 TI - Short-term treatment with cell wall degrading enzymes increases the activity of the inositol phospholipid kinases and the vanadate-sensitive ATPase of carrot cells. AB - Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [(3)H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed. PMID- 16667923 TI - Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis. AB - The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5 degrees C or 16 degrees C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16 degrees C and 5 degrees C plants, respectively. Growth at 5 degrees C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO(2) exchange or O(2) evolution were not affected by the growth temperature. Furthermore, 5 degrees C expanded leaves were found to be more resistant to photoinhibition at 5 degrees C than were 16 degrees C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5 degrees C to 16 degrees C or from 16 degrees C to 5 degrees C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO(2) exchange and O(2) evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent. PMID- 16667924 TI - Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide. AB - Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure. PMID- 16667925 TI - Development of Endoplasmic Reticulum and Glyoxysomal Membrane Redox Activities during Castor Bean Germination. AB - Redox activities, NADH:ferricyanide reductase, NAD(P)H:cytochrome reductases, and NADH:ascorbate free-radical reductase, are present in endoplasmic reticulum (ER) and glyoxysomal membranes from the endosperm of germinating castor bean (Ricinus comminus L. var Hale). The development of these functions was followed in glyoxysomes and ER isolated on sucrose gradients from castor bean endosperm daily from 0 through 6 days of germination. On a per seed basis, glyoxysomal and ER protein, glyoxysomal and ER membrane redox enzyme activities, and glyoxylate cycle activities peaked at day 4 as did the ER membrane content of cytochrome P 450. NADH:ferricyanide reductase was present in glyoxysomes and ER isolated from dry seed. This activity increased only about twofold in glyoxysomes and threefold in ER during germination relative to the amount of protein in the respective fractions. The other reductases, NADH:cytochrome reductase and NADH:ascorbate free-radical reductase, increased about 10-fold in the ER relative to protein up to 4 to 5 days, then declined. NADPH:cytochrome reductase reached maximum activity relative to protein at day 2 in both organelles. The increases in redox activities during germination indicate that the membranes of the ER and glyoxysome are being enriched with redox proteins during their development. The development of redox functions in glyoxysomes was found to be coordinated with development of the glyoxylate cycle. PMID- 16667926 TI - An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves. AB - We have previously demonstrated that a protein purified from xylan-induced culture filtrates of Trichoderma viride contains beta-1,4-endoxylanase activity and induces ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs. When the ethylene biosynthesis-inducing xylanase (EIX) was applied to cut petioles of detached tobacco leaves, it induced ethylene biosynthesis within 1 hour and extensive electrolyte leakage and necrosis were observed in tobacco leaf tissue within 5 hours. Ethylene-pretreatment (120 microliters per liter ethylene for 14 hours) of tobacco leaves enhanced ethylene biosynthesis in response to EIX by more than threefold and accelerated development of cellular leakage and necrosis. In intact plants, similar symptoms could be induced in leaves that were distant from the point of the enzyme application. The evidence suggests that EIX is translocated via the vascular system and elicits plant responses similar to those observed in a hypersensitive response. PMID- 16667927 TI - Correlation of Pectolytic Enzyme Activity with the Programmed Release of Cells from Root Caps of Pea (Pisum sativum). AB - In many plant species, the daily release of hundreds to thousands of healthy cells from the root cap into the soil is a normal process, whose function is unknown. We studied the separation of the cells in pea (Pisum sativum) using an aeroponic system in which separated cells were retained on the root until they were washed off for counting. We found that cell separation is a developmentally regulated, temperature-sensitive process that appears to be regulated independently of root growth. No cells were released from very young roots. When plants were grown aeroponically, cell numbers increased with increasing root length to a mean of 3400 cells per root, at which point the release of new cells ceased. The process could be reset and synchronized by washing the root in water to remove shed cells. Cell separation from the root cap was correlated with pectolytic enzyme activity in root cap tissue. Because these cells that separate from the root cap ensheath the root as it grows and thus provide a cellular interface between the root surface and the soil, we propose to call the cells "root border cells." PMID- 16667928 TI - Regulation of a sesquiterpene cyclase in cellulase-treated tobacco cell suspension cultures. AB - The regulation of an elicitor-inducible sesquiterpene cyclase in tobacco (Nicotiana tabacum) cell suspension cultures was investigated. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 15 hours of cellulase addition to the cell cultures. The induction of the cyclase activity was correlated with an absolute amount of the cyclase protein as measured in immunoblots. Both the in vivo synthesis rate, measured as the incorporation of [(35)S]methionine by cell cultures into immunoprecipitable cyclase protein, and the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of isolated RNA, were maximal at that time when the increase in cyclase enzyme activity was maximal. Using thiouridine to selectively label and isolate de novo synthesized mRNA, the in vitro translation products encoded by the newly synthesized RNA from elicitor-treated, but not control, cell cultures contained immunoprecipitable cyclase protein. These results suggest that the induction of the sesquiterpene cyclase in elicitor treated cell cultures is primarily regulated by transcriptional control of the cyclase gene. PMID- 16667929 TI - Molecular Cloning of Tomato Plasma Membrane H-ATPase. AB - Two cDNA clones (LHA1 and LHA2) from tomato (Lycopersicon esculentum) which likely encode isoforms of the plasma membrane H(+)-ATPase were isolated. The longest cDNA (3229 base pairs), LHA1, comprises an open reading frame that encodes a 956 amino acid, 105 kilodalton polypeptide with several potential transmembrane domains. In vitro transcription and translation of LHA1 yields a major translation product of approximately 100 kilodaltons that is immunoprecipitable with antiserum to the corn root plasma membrane H(+)-ATPase. LHA2 encodes a portion of a coding sequence that is 96% identical to LHA1, suggesting that LHA2 encodes an isoform of the H(+)-ATPase. Genomic DNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to a common set of six to eight restriction fragments at moderate stringency and to single distinct fragments at high stringency. LHA1 and LHA2 map to distinct sites on chromosomes three and six, respectively. RNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to 3.4 kilobase pair transcripts present in both leaves and roots, although the LHA2 transcript is relatively more abundant in leaves than in roots. These results indicate that in tomato as many as six to eight genes may encode the plasma membrane H(+)-ATPase, two of which are expressed at the level of mRNA in both roots and leaves. PMID- 16667930 TI - Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit. AB - We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo. PMID- 16667931 TI - Purification and Characterization of Methionine:Glyoxylate Aminotransferase from Brassica carinata and Brassica napus. AB - The first step in the biosynthesis of allylglucosinolate from methionine in Brassica is thought to be the transamination of methionine to 2-keto-4 methylthiobutyrate. By using Q-Sepharose and Red Agarose, followed by high resolution anion exchange chromatography and chromatofocussing, a methionine:glyoxylate aminotransferase (MGAT) was purified to homogeneity from leaves of Brassica carinata var R-4218, and approximately 5000-fold from leaves of Brassica napus var Topas. The final purification was accomplished using nondenaturing polyacrylamide gel electrophoresis. The enzyme has a pl of 4.3, a native molecular mass of 230 to 290 kilodaltons, and a subunit molecular mass of approximately 50 kilodaltons. Four isozymes of the enzyme were identified in the six species of Brassica commonly cultivated. Nonglucosinolate producing species had only low levels of MGAT or an MGAT isozyme which was distinctly different from that in Brassica. PMID- 16667932 TI - Isolation and characterization of a proline-rich cell wall protein from soybean seedlings. AB - We have purified a cell wall protein from extracts of soybean (Glycine max) that was previously shown to be immunologically related to p33, a wound-induced carrot cell wall protein (Tierney ML, Wiechert J, Pluymers D [1988] Mol Gen Genet 211: 393-399). Amino acid composition analysis reveals that the protein is 36% proline and hydroxyproline and its amino-terminus was determined to be AsnTyrGluAsnProHypValTyrLysProHypThrGluLysProHypValTyr. In vivo [(3)H]proline labeling of soybean hypocotyl tissues and Western blot analysis indicate that the apical hook, a region characterized by active growth, is a predominant site for synthesis of this protein and that wounding does not significantly affect its synthesis or accumulation. Antibodies raised against the hook protein also cross reacted with a higher molecular weight protein in cell wall extracts of wounded hook tissue. These data indicate that there are at least two related, proline rich cell wall proteins in the hook region of soybean seedlings, one that appears to be developmentally regulated and another that is wound-inducible. RNA encoding the developmentally regulated seedling cell wall protein was detected in both the hook and elongating region of the hypocotyl. The presence of this RNA in the elongating region in the absence of detectable levels of the protein indicates either that the expression of this cell wall protein is subject to posttranscriptional control or that the rate of insolubilization of this protein increases in more mature regions of the hypocotyl. PMID- 16667933 TI - Effect of Temperature Alterations on the Diurnal Expression Pattern of the Chlorophyll a/b Binding Proteins in Tomato Seedlings. AB - In the leaves of plants that are grown in the natural environment, the accumulation of mRNAs encoding the chlorophyll a/b binding proteins (CAB) follow a circadian rhythm. It is generally accepted that the day/night (sunset, light/dark) or night/day (sunrise, dark/light) transitions play an important role in the synchronization of the rhythm and the determination of the accumulation amplitude. As the results of the experiments presented in this paper indicate, temperature alterations also support the setting and the arrangement of the rhythm. Apparently, simulating "day/night" temperature alternations influences the tomato (Lycopersicon esculentum) plants to express a typical circadian oscillation pattern of cab mRNAs. This rhythm was sustained in the plants after long-term exposure to an alternating temperature regime. In constant conditions, e.g. continuous illumination at either 18 degrees C or 24 degrees C or in continuous darkness at 24 degrees C, this diurnal fluctuation pattern with a period of about 24 hours remained present for at least 2 days. PMID- 16667934 TI - Cultivar-Specific Interactions of Soybean with Rhizobium fredii Are Regulated by the Genotype of the Root. AB - Rhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean cultivar Peking, but not on cultivar McCall. This pattern of nodulation persists when McCall and Peking seedlings are cultivated together in plastic growth pouches. Reciprocal grafting experiments confirm that the root genotype, and not that of the shoot, regulates such cultivar specificity. When Peking roots are grafted onto McCall seedlings, the nodulation responses of roots similarly remain unaffected. Transposon-mutant 257DH4, which is derived from USDA257, can form nitrogen-fixing nodules on McCall. Such nodulation is blocked by the presence of USDA257 in the inoculum. Grafting experiments indicate that blocking is not due to a translocatable inhibitor produced by McCall roots or triggered by their interaction with USDA257. Thus, neither freely diffusible nor graft-transmissible substances are involved in cultivar-specific interactions of soybean with R. fredii and its derivatives. PMID- 16667935 TI - Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin. AB - Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [(35)S]cysteine, in which [(35)S]sulfide and iron are incorporated into Fd to build up the (35)S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [(35)S]sulfide or iron, or in Fe-S cluster formation itself. [(35)S]Sulfide was liberated from [(35)S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [(35)S]cysteine occurred before the formation of the (35)S-labeled Fe-S cluster, and the amount of radioactivity in [(35)S]sulfide was greater than that in (35)S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na(2)S) inhibited competitively formation of the (35)S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [(35)S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed. PMID- 16667936 TI - Bacteriohopanetetrol: abundant lipid in frankia cells and in nitrogen-fixing nodule tissue. AB - An unusual class of lipid with amphiphilic properties has been detected in nodule tissue of Alnus and Ceanothus. High levels of the same lipid (20-50% of total cell lipids) were detected in solvent extracts of Frankia spp. cells. However, the lipid was absent in host roots. The lipid was purified and quantified by high performance liquid chromatography/flame ionization detector. Phenol-sulfuric acid determinations and proton nuclear magnetic resonance indicated that the purified lipid is not a glycolipid. Mass spectra of the predominant species are consistent with published spectra for bacteriohopanetetrol (C(35)H(62)O(4)), a pentacyclic triterpenoid, or hopanoid. PMID- 16667937 TI - Genetic Regulation of Development in Sorghum bicolor: V. The ma(3) Allele Results in Gibberellin Enrichment. AB - Sorghum bicolor genotypes, near isogenic with different alleles at the third maturity locus, were compared for development, for responsiveness to GA(3) and a GA synthesis inhibitor, and occurrence and concentrations of endogenous GAs, IAA, and ABA. At 14 days the genotype 58M (ma(3) (R)ma(3) (R)) exhibited 2.5-fold greater culm height, 1.75-fold greater total height, and 1.38-fold greater dry weight than 90M (ma(3)ma(3)) or 100M (Ma(3)Ma(3)). All three genotypes exhibited similar shoot elongation in response to GA(3), and 58M showed GA(3)-mediated hastening of floral initiation when harvested at day 18 or 21. Both 90M and 100M had exhibited hastening of floral initiation by GA(3) previously, at later application dates. Tetcyclacis reduced height, promoted tillering, and delayed flowering of 58M resulting in plants which were near phenocopies of 90M and 100M. Based on bioassay activity, HPLC retention times, cochromatography with (2)H(2) labeled standards on capillary column GC and matching mass spectrometer fragmentation patterns (ions [m/z] and relative abundances), GA(1), GA(19), GA(20), GA(53), and GA(3) were identified in extracts of all three genotypes. In addition, based on published Kovats retention index values and correspondence in ion masses and relative abundances, GA(44) and GA(17) were detected. Quantitation was based on recovery of coinjected, (2)H(2)-labeled standards. In 14 day-old plants, total GA-like bioactivity and GA(1) concentrations (nanograms GA/gram dry weight) were two- to six-fold higher in 58M than 90M and 100M in leaf blades, apex samples, and whole plants while concentrations in culms were similar. Similar trends occurred if data were expressed on a per plant basis. GA(1) concentrations for whole plants were about two-fold higher in 58M than 90M and 100M from day 7 to day 14. Concentrations of ABA and IAA did not vary between the genotypes. The results indicate the mutant allele ma(3) (R) causes a two- to six fold increase in GA(1) concentrations, does not result in a GA-receptor or transduction mutation and is associated with phenotypic characteristics that can be enhanced by GA(3) and reduced by GA synthesis inhibitor. These observations support the hypothesis that the allele ma(3) (R) causes an overproduction of GAs which results in altered leaf morphology, reduced tillering, earlier flowering, and other phenotypic differences between 58M and 90M or 100M. PMID- 16667938 TI - Distinction between Hypoxanthine and Xanthine Transport in Chlamydomonas reinhardtii. AB - Chlamydomonas reinhardtii cells consumed hypoxanthine and xanthine by means of active systems which promoted purine intracellular accumulation against a high concentration gradient. Both uptake and accumulation were also observed in mutant strains lacking xanthine dehydrogenase activity. Xanthine and hypoxanthine uptake systems exhibited very similar Michaelis constants for transport and pH values, and both systems were induced by either hypoxanthine or xanthine. However, they differed greatly in the length of the lag phase before uptake induction, which was longer for hypoxanthine than for xanthine. Cells grown on ammonium and transferred to hypoxanthine media consumed xanthine before hypoxanthine, whereas cells transferred to xanthine media did not take up hypoxanthine until 2 hours after commencing xanthine consumption. Metabolic and photosynthetic inhibitors such as 2,4-dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, and carbonylcyanide m-chlorophenylhydrazone inhibited to a different extent the hypoxanthine and xanthine uptake. Similarly, N-ethylmaleimide abolished xanthine uptake but slightly affected that of hypoxanthine. Hypoxanthine consumption was inhibited by adenine and guanine whereas that of xanthine was inhibited only by urate. We conclude that hypoxanthine and xanthine in C. reinhardtii are taken up by different active transport systems which work independently of the intracellular enzymatic oxidation of these purines. PMID- 16667939 TI - Acetyl-coenzyme a can regulate activity of the mitochondrial pyruvate dehydrogenase complex in situ. AB - In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon K(m) and K(i) relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition. PMID- 16667940 TI - Elicitor-Inducible Caffeoyl-Coenzyme A 3-O-Methyltransferase from Petroselinum crispum Cell Suspensions : Purification, Partial Sequence, and Antigenicity. AB - Parsley (Petroselinum crispum) cell cultures respond rapidly to treatment with fungal elicitor by the accumulation of coumarin phytoalexins in the culture fluid and by incorporation of ferulic esters into their cell walls. S-Adenosyl-l methionine:trans-caffeoyl-CoA 3-O-methyltransferase activity, specifically involved in the formation of ferulic esters, is induced under these conditions. Such an inducible methyltransferase activity has been found in plant cells of various species. The methyltransferase was purified to homogeneity from parsley cells that had been treated for 12 hours with crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea. It consists of two very similar or identical subunits of approximately 24 kilodaltons, which are N-terminally blocked. Attempts to generate antisera against the native enzyme in rabbit or mouse failed, but an antiserum, cross-reactive in enzyme-linked immunosorbent assays, was raised in mouse by intraperitoneal injection of the heat-denatured enzyme. Roughly 33% of the amino acid sequence was elucidated by microsequencing of tryptic peptides of the methyltransferase. The parsley enzyme may be related to adenine-specific methyltransferases known from bacterial sources. Antiserum generated in rabbit against a synthetic decapeptide, as inferred from one of the tryptic peptides and conjugated to ovalbumin, specifically cross-reacted with the methyltransferase protein in Western blots developed after SDS-polyacrylamide electrophoresis. This serum did not react, however, with native parsley methyltransferase. PMID- 16667941 TI - Purification and Characterization of Isoperoxidases Elicited by Aspergillus flavus in Cotton Ovule Cultures. AB - Two anionic isoperoxidases were isolated from media of Aspergillus flavus inoculated cotton (Gossypium hirsutum L.) ovule cultures and purified about 150 fold to apparent homogeneity by treatment with Cell Debris Remover and ion exchange chromatography on Accell QMA medium. These isoperoxidases were present in noninoculated cotton ovule cultures at low levels. The major activity peak (B) represented 90% of the recovered peroxidase activity and was electrophoretically homogeneous. The minor activity peak (A) was about 95% pure. Isoelectric focusing analysis showed that B was greater than 95% pure with respect to other peroxidase isozymes, while the enzyme in A was about 90% isozymically pure. Each isoperoxidase displayed a molecular mass of 56 kilodaltons by interpolation from denaturing gel electrophoresis. The B isozyme displayed a molecular mass of 55 kilodaltons by gel filtration chromatography. The pH optima for the cotton ovule isoperoxidases were similar, 5.0 for isozyme A and 6.0 for isozyme B. The isoelectric points for isozymes A and B were 4.2 and 4.4, respectively. Eugenol, guaiacol, and 3,3',5,5'-tetramethylbenzidine were good electron donor substrates, whereas 4-aminoantipyrine was a poor substrate. The absorption spectrum of the material in B revealed a major peak at 400 nanometers and a minor peak at 280 nanometers. The molar extinction coefficient at 400 nanometers (pH 7.0) was calculated to be 1.07 x 10(5) per square centimeter per mole. Amino acid analysis of isozyme B confirmed the acidic nature of this protein and identified a number of similarities to the anionic peroxidases from tobacco and potato. This glycoprotein was found to contain 12 to 14% sugar (by weight), mainly in the form of galactose and mannose. PMID- 16667942 TI - Phytochrome-mediated detection of changes in reflected light. AB - Measurements of phytochrome photoequilibria and photoconversion rates in vivo, in seedlings of Cucurbita pepo L. exposed to light in growth chambers, indicate that significant changes in the state of phytochrome can be brought about by changes in the quality and quantity of the light reflected from the walls of the growth chambers. The changes in reflected light, although large, were small in terms of the total radiation (direct light from the lamps plus wall-reflected light) to which the seedlings were exposed. The conditions used were approximate simulations of direct and reflected sunlight conditions in the natural environment. Keeping in mind the limitations imposed by the approximation of the simulations, the results from this study are consistent with the hypothesis that, in the natural environment, a plant might be capable of detecting the presence of nearby plants, before being shaded by them, through the phytochrome-mediated perception of changes in reflected light. PMID- 16667943 TI - Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni. AB - To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13 hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A. PMID- 16667944 TI - Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria. AB - The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K(+)) caused a decrease in the apparent K(m) and an increase in the apparent V(max) for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C(4+) > C(3+)). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent K(m) for Ca(2+) from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg(2+) and K(+). The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo. PMID- 16667945 TI - Isolation of a cDNA Clone for Spinach Lipid Transfer Protein and Evidence that the Protein Is Synthesized by the Secretory Pathway. AB - A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles. PMID- 16667946 TI - Rapid adjustment of guard-cell abscisic Acid levels to current leaf-water status. AB - Detached broad bean (Vicia faba L.) leaflets were water stressed; within 15 minutes, guard-cell abscisic acid (ABA) concentration increased ninefold. This result eliminates the apparent discrepancy raised by reports of no correlation between initial water-stress effects on stomata and leaf ABA concentration. Six hours after stress relief, guard-cell ABA concentration was near the prestress value, which would seem to implicate other factors in stress aftereffects on stomata. PMID- 16667947 TI - Specific Levels of DNA Methylation in Various Tissues, Cell Lines, and Cell Types of Daucus carota. AB - The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos. PMID- 16667948 TI - Simultaneous gas chromatography-mass spectrometry quantification of endogenous [C]- and applied [C]indole-3yl-acetic Acid levels in growing maize roots. AB - The use of stable indole-3yl-acetic acid (IAA) labeled by 6 atoms of (13)C allowed, after [(13)C]IAA treatment, simultaneous gas chromatography-mass spectrometry quantifications of both endogenous [(12)C]IAA and applied [(13)C]IAA levels in Zea mays L. roots. Root material was immersed for 1 hour in a buffered (pH 6.0) solution without or with [(13)C]IAA at 10(-7) molar. Both applied and endogenous IAA were thus measured for three zones of the roots (apical, elongating, differentiating) directly after treatment and also 2 hours later. Growth was followed over a 4 hour period. Roots not immersed elongated more than control roots (immersed in buffer), which grew more than IAA-treated roots. Immersion in buffer induced a large decrease (-68%) of [(12)C]IAA in the apical part of control roots, whereas immersion in [(13)C]IAA prevented most of it. No significant difference between control and treated roots occurred in the two other zones. Two hours after treatment, [(13)C]IAA had completely disappeared from the elongating zone even though [(12)C]IAA level was essentially stable. A direct relationship occurred between the level of IAA in the elongating zone and the growth of the root. This relationship was strongly disturbed if unmetabolized [(13)C]IAA was present. However, the relationship returned to its initial state when significant amounts of free [(13)C]IAA were no longer detectable. These results are discussed in terms of the stability of both types of compounds and the utility of the method of using stable isotopes of hormones, for the understanding of hormonal regulation of plant growth. PMID- 16667949 TI - Modifications to Thylakoid Composition during Development of Maize Leaves at Low Growth Temperatures. AB - The effects of reductions in growth temperature on the development of thylakoids of maize (Zea mays var LG11) leaves are examined. Thylakoids isolated from mesophyll cells of leaves grown at 17 degrees and 14 degrees C, compared with 25 degrees C, exhibited a decreased accumulation of many polypeptides, which was accompanied by a loss of activity of photosystems (PS) I and II. Probing the polypeptide profiles with a range of antibodies specific for thylakoid proteins demonstrated that a number of polypeptides encoded by the chloroplast genome failed to accumulate at low temperatures. Although thylakoid protein synthesis was reduced severely at 14 degrees C compared with 25 degrees C, major synthesis of both chloroplast and nuclear encoded polypeptides was detected. It is suggested that the lack of accumulation of some thylakoid proteins at low temperatures may be due to an inability to stabilize the proteins in the membranes. A number of thylakoid polypeptides were found to appear as the growth temperature was decreased. Analyses of pigments and polypeptides demonstrated that decreases in the photosystem reaction center core complexes occur relative to the light harvesting complex associated with PS II at reduced growth temperatures. Differential effects on the development of PSI and PSII were also observed, with PSII activity being preferentially reduced. Reductions in PSII content and activity occurred in parallel with decreases in the quantum yield and light-saturated rate of CO(2) assimilation. Fractionation of thylakoid pigment protein complexes showed that the ratio of monomeric:oligomeric form of the light harvesting complex associated with PSII increased at low growth temperature, which is consistent with a chill-induced modification of thylakoid organization. Many, but not all, of the characteristic changes in thylakoid protein metabolism, which were observed when leaves were grown at low temperatures in controlled environments, were identified in leaves of a field maize crop during the early growing season when low temperatures were experienced by the crop. Chill-induced perturbations of thylakoid development can occur in the field in temperate regions and may have implications for the photosynthetic productivity of the crop. PMID- 16667950 TI - Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum. AB - Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH(4) (+) assimilation on exogenous CO(2). N-sufficient cells were only able to assimilate NH(4) (+) maximally in the presence of CO(2) and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH(4) (+) assimilation. These results indicate that NH(4) (+) assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO(2) fixation. N-limited cells assimilated NH(4) (+) both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO(2) fixation was not required for NH(4) (+) assimilation. Using CO(2) removal techniques reported previously in the literature, we were unable to demonstrate CO(2)-dependent NH(4) (+) assimilation in N-limited cells. However, employing more stringent CO(2) removal techniques we were able to show a CO(2) dependence of NH(4) (+) assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO(2) requirements for NH(4) (+) assimilation. The first is as a substrate for photosynthetic CO(2) fixation, whereas the second is a nonphoto synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase. PMID- 16667951 TI - Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: I. Cells in Darkness. AB - We have developed protocols for phase shifting the circadian rhythm of Chlamydomonas reinhardtii by light pulses. This paper describes the photobiology of phase-resetting the Chlamydomonas clock by brief (3 seconds to 15 minutes) light pulses administered during a 24 hour dark period. Its action spectrum exhibited two prominent peaks, at 520 and 660 nanometers. The fluence at 520 nanometers required to elicit a 4 hour phase shift was 0.2 millimole photon per square meter, but the pigment that is participating in resetting the clock under these conditions is unknown. The fluence needed at 660 nanomoles to induce a 4 hour phase shift was 0.1 millimole photon per square meter, which is comparable with that needed to induce the typical low fluence rate response of phytochrome in higher plants. However, the phase shift by red light (660 nanometers) was not diminished by subsequent administration of far-red light (730 nanometers), even if the red light pulse was as short as 0.1 second. This constitutes the first report of a regulatory action by red light in Chlamydomonas. PMID- 16667952 TI - 4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme. AB - 4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000 fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower V(max) and a four-fold higher K(m) value copurifies with MeGln aminohydrolase activity. 4 Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4 methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components. PMID- 16667953 TI - Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles. AB - Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH(2)-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH(2)-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD 1 and SOD-3, respectively. PMID- 16667954 TI - Distribution of myrosinase in rapeseed tissues. AB - Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem. PMID- 16667955 TI - Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen fixing soybean nodules : evidence for phosphorylation of nodulin-26. AB - By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca(2+)-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca(2+) (K(0.5) = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2 dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca(2+)-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with (32)P-phosphate. Overall, the results show the existence of a Ca(2+)-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca(2+)-dependent phosphorylation. PMID- 16667956 TI - Carbon partitioning and export from mature cotton leaves. AB - The partitioning of carbon in intact, mature cotton (Gossypium hirsutum L.) leaves was examined by steady-state (14)CO(2) labeling. Plants were exposed to dark periods of varying lengths, followed by similar illuminated labeling periods. These treatments produced leaves with a range of starch and soluble sugar contents, carbon exchange, and carbon export rates. Export during the illuminated periods was neither highly correlated with photosynthesis nor was export during the illuminated periods significantly different among the treatments. In contrast, the rate of subsequent nocturnal carbon export from these leaves varied widely and was found to be highly correlated with leaf starch content at the end of the illumination period (r = 0.934) and with nocturnal leaf respiration (r = 0.954). Leaves which had accumulated the highest levels of starch (about 275 micrograms per square centimeter) by the end of the illumination period exhibited nocturnal export rates very similar to those during the daylight hours. Leaves which accumulated starch to only 50 to 75 micrograms per square centimeter virtually ceased nocturnal carbon export. For leaves with starch accumulations of between 50 and 275 micrograms per square centimeter, nocturnal export was directly proportional to leaf starch at the end of the illumination period. After the nocturnal export rate was established, it continued at a constant rate throughout the night even though leaf starch and sucrose contents declined. PMID- 16667957 TI - Induction of leaf abscission in cotton is a common effect of urea- and adenine type cytokinins. AB - Cytokinins of the urea and adenine type induced leaf abscission in young cotton (Gossypium hirsutum) plants in the following order of activity: N-phenyl-N'-1,2,3 thiadiazol-5-ylurea (thidiazuron) >> N-phenyl-N'-(2-chloro-4-pyridyl)urea > isopentenyladenine >/= 6-benzyladenine > zeatin = dihydrozeatin > kinetin. It is suggested that ethylene production is implicated in this response because it was stimulated by the compounds in cotton leaf discs with nearly the same effectiveness. Moreover, similar to thidiazuron (JC Suttle [1985] Plant Physiol 78: 272-276), isopentenyladenine-induced defoliation was inhibited by aminoethoxyvinylglycine, and the effect was restored by 1-aminocyclopropane-1 carboxylic acid. PMID- 16667958 TI - Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene. AB - We obtained carrot (Daucus carota) cells possessing the 5'-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial beta-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in beta-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4 dichlorophenoxyacetic acid, enhanced expression of beta-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of beta-glucuronidase activities, in concord with embryogenesis, were also noted. PMID- 16667959 TI - Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue. AB - The H(+)/ATP stoichiometry was determined for the plasma membrane H(+)-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H(+)/ATP stoichiometry utilized a kinetic approach where rates of H(+) influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H(+) influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H(+) leakage from a steadystate pH gradient after stopping the H(+)-ATPase or utilizing a mathematical model which describes the net transport of H(+) at any given point in time. When the rate of H(+) influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H(+)/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (DeltaG(atp)), this value for H(+)/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo. PMID- 16667960 TI - Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits. AB - 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48 kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S adenosyl-l-methionine. PMID- 16667961 TI - A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture. AB - The subcellular distribution of l-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and (35)S-methionine. Using antibodies raised against purified GDH, we identified a (35)S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH. PMID- 16667962 TI - Isolation and characterization of a cDNA coding for pea chloroplastic carbonic anhydrase. AB - Using a polyclonal antibody generated against the purified pea (Pisum sativum) carbonic anhydrase (CA) monomeric species, we have isolated and characterized a cDNA coding for this enzyme. Protein sequence analysis was used to confirm the identity of the clone. The presence of a large transit peptide suggests that CA is transported into the chloroplast and then processed to the mature size of approximately 26 kilodaltons. Northern hybridization, using the CA cDNA as a probe of total leaf RNA, revealed a single transcript of 1.45 kilobase pairs. This transcript was not detected in RNA extracted from root or etiolated leaf tissue. Comparison of the deduced amino acid sequence with that of spinach CA showed approximately 68% identity over the length of the nascent protein but with greater similarity observed within the mature protein sequences. In addition, regions of the pea and spinach CA proteins were found to be significantly similar to the Escherichia coli cyanate permease. PMID- 16667963 TI - Characterization of peroxidases in lignifying peach fruit endocarp. AB - Developing peach (Prunus persica L. Batsch ;Redskin') fruit were used to characterize the role of peroxidases in lignification. During development, the endocarp of these drupes becomes lignified while the mesocarp remains parenchymatous. Acidic peroxidase from lignifying endocarp were similar to those of the fleshy mesocarp. The endocarp had a larger amount and number of basic peroxidases than the mesocarp. Cultured peach leaf cells are thought to be lignified because their walls give a positive reaction with phloroglucinol-HCI. These cells also secreted a basic peroxidase. Peroxidases were difficult to extract from endocarp tissue as they lignified. This was also demonstrated by tissue printing on nitrocellulose. Flesh, but not endocarp peroxidase was evident in tissue prints. This suggests that tissue printing may fail to reveal the presence of enzymes which are firmly attached to the cell. PMID- 16667964 TI - Diel Patterns of Water Potential Components for the Crassulacean Acid Metabolism Plant Opuntia ficus-indica when Well-Watered or Droughted. AB - Under well-watered conditions, chlorenchyma acidity in cladodes of Opuntia ficus indica increased substantially at night, fully accounting for the 0.26-megapascal nocturnal increase in osmotic pressure in the outer 2 millimeters. Osmotic pressure in the inner part of the chlorenchyma and in the water-storage parenchyma did not change significantly over 24-hour periods. Three months of drought decreased nocturnal acid accumulation by 73% and essentially abolished transpiration; also, 27% of the chlorenchyma water and 61% of the parenchyma water was lost during such drought, but the average tissue osmotic pressure was little affected. Turgor pressure was maintained in the chlorenchyma after 3 months of drought, although it decreased sevenfold in the water-storage parenchyma compared with the well-watered condition. Moreover, the nocturnal increases in turgor pressure of about 0.08 megapascal in the outer part of the chlorenchyma was also unchanged by such drought. The water potential magnitudes favored water movement from the parenchyma to the chlorenchyma at the end of the night and in the reverse direction during the late afternoon. Experiments with tritiated water support this pattern of water movement, which is also in agreement with predictions based on electric-circuit analog models for Crassulacean acid metabolism plants. PMID- 16667965 TI - Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots. AB - The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8 hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO(3) (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes. PMID- 16667966 TI - Antigenic and calcium binding properties of a Peptide containing the essential cysteine in lima bean lectin. AB - Polyclonal antisera were raised against a peptide containing the cysteine residue required for carbohydrate binding activity in the lima bean lectin. The antisera were tested for cross-reactivity with (a) synthetic peptide analogs to the essential cysteine containing peptide, (b) proteolytic digests of related lectins, (c) native lectins. The antisera were specifically inhibited from binding to a peptide conjugate by free synthetic peptides. The degree of inhibition by lectin digests correlated approximately along evolutionary relationships and the degree of sequence conservation. One antiserum was found to cross-react with certain lectins in the native state. In a second set of experiments, the calcium binding properties of the synthetic peptides were investigated using metal ion-chelate chromatography and UV-difference spectroscopy. The nonapeptide and undecapeptide bound to a Ca(2+) iminodiacetic acid agarose column and were eluted with EDTA. Ultraviolet difference spectral titrations with Ca(2+) performed on the synthetic undecapeptide and a related favin derived peptide resulted in dissociation constants of approximately 6 x 10(3) per molar. PMID- 16667967 TI - Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity. AB - The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3 degrees ) leaves but had no effect on the fully expanded first true (1 degrees ) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3 degrees leaves occurred while <50% abscission of the 1 degrees leaves was observed. On a leaf basis, endogenous levels of free IAA in 1 degrees leaves were approximately twice those of 3 degrees leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [(14)C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3 degrees leaves abscised faster than 1 degrees leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3 degrees explants was not appreciably inhibited by exogenous IAA while 1 degrees explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1 degrees and 3 degrees explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves. PMID- 16667968 TI - Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation. AB - Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with [gamma-(32)P]ATP (JLA Huber, SC Huber, TH Nielsen [1989] Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25 degrees C before assay. The "spontaneous" inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg(2+) and was relatively insensitive to Ca(2+) and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [(32)P]Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 degrees C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo. PMID- 16667969 TI - Variability in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunits and Carboxylation Activity in Fern Gametophytes Grown under Different Light Spectra. AB - Two distinct ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) populations were observed in Pteris vittata gametophytes grown under different illumination conditions. Exposure of the fern gametophytes to continuous red light (R) resulted in Rubisco SSUs that were not recognized by polyclonal antibodies raised against SSUs from spinach. Unlike the R-induced SSUs, blue light (B) induced SSUs were well recognized. This difference in SSU composition also reflected in Rubisco activity. In vitro, B-induced Rubisco exhibits a significantly higher carboxylation activity as compared to the R induced Rubisco. Approximately a two- to threefold increase in the V(max) value of the B-induced carboxylase as compared to the R-induced one was measured. It thus seems very likely that certain domains in the SSU molecule affect enzyme activity. PMID- 16667970 TI - Water Transport across Maize Roots : Simultaneous Measurement of Flows at the Cell and Root Level by Double Pressure Probe Technique. AB - A double pressure probe technique was used to measure simultaneously water flows and hydraulic parameters of individual cells and of excised roots of young seedlings of maize (Zea mays L.) in osmotic experiments. By following initial flows of water at the cell and root level and by estimating the profiles of driving forces (water potentials) across the root, the hydraulic conductivity of individual cell layers was evaluated. Since the hydraulic conductivity of the cell-to-cell path was determined separately, the hydraulic conductivity of the cell wall material could be evaluated as well (Lp(cw) = 0.3 to 6.10(-9) per meter per second per megapascal). Although, for radial water flow across the cortex and rhizodermis, the apoplasmic path was predominant, the contribution of the hydraulic conductance of the cell-to-cell path to the overall conductance increased significantly from the first layer of the cortex toward the inner layers from 2% to 23%. This change was mainly due to an increase of the hydraulic conductivity of the cell membranes which was Lp = 1.9.10(-7) per meter per second per megapascal in the first layer and Lp = 14 to 9.10(-7) per meter per second per megapascal in the inner layers of the cortex. The hydraulic conductivity of entire roots depended on whether hydrostatic or osmotic forces were used to induce water flows. Hydrostatic Lp(r) was 1.2 to 2.3.10(-7) per meter per second per megapascal and osmotic Lp(r) = 1.6 to 2.8.10(-8) per meter per second per megapascal. The apparent reflection coefficients of root cells (sigma(s)) of nonpermeating solutes (KCI, PEG 6000) decreased from values close to unity in the rhizodermis to about 0.7 to 0.8 in the cortex. In all cases, however, sigma(s) was significantly larger than the reflection coefficient of entire roots (sigma(sr)). For KCI and PEG 6000, sigma(sr) was 0.53 and 0.64, respectively. The results are discussed in terms of a composite membrane model of the root. PMID- 16667971 TI - Ethylene Biosynthesis-Inducing Xylanase : II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride. AB - The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion. PMID- 16667972 TI - Respiratory Shifts in Developing Petals of Saxifraga cernua. AB - Changes in the oxygen uptake of petal slices by the cytochrome and alternative respiratory pathways were monitored during petal development in the arctic herb Saxifraga cernua. As the petals developed, rates of total respiration increased to a maximum rate during petal unfolding (day 4.5), and thereafter declined. Respiration in petals of all ages was at least partially resistant to cyanide, indicating the capacity for the alternative pathway. In all, except day 1 and senescing day 8 petals, respiration was inhibited by salicylhydroxamic acid, indicating engagement of the alternative pathway. In general, temporal changes in the respiratory activity along each pathway were similar and in parallel with changes in total respiration, although maximum rates along each pathway occurred at different times. Maximum cytochrome pathway activity occurred during petal expansion (day 4) whereas the alternative pathway peaked during petal unfolding at day 4.5. The control of respiration was also investigated. In the presence of salicylhydroxamic acid, the addition of the uncoupler carbonyl cyanide m chlorophenylhydrazone was never stimulatory, suggesting that the cytochrome pathway was not restricted by adenylate levels. The addition of sucrose stimulated respiration only in day 1 petals, suggesting substrate limitation at this developmental stage. Since the rate of alternative pathway respiration peaked during petal unfolding, a time of high energy requirement, we suggest that the alternative pathway may have been used as an inefficient energy source during petal development. PMID- 16667973 TI - Gene nadA, Encoding Quinolinate Synthetase, Is Located on the Cyanelle DNA from Cyanophora paradoxa. PMID- 16667974 TI - Identification and localization of carbonic anhydrase in two chlorella species. AB - Carbonic anhydrase (CA) activity and localization have been examined in two species of the eukaryotic green alga Chlorella. Mass spectrometric and potentiometric assays of CA activity indicate that C. ellipsoidea contains very little extracellular CA activity whereas C. saccharophila exhibits significant extracellular activity when grown at alkaline pH values. Extracellular CA activity appears to be correlated with the presence of a 36 kilodalton polypeptide that was detected immunologically using a polyclonal antibody directed against the 37 kilodalton Chlamydomonas CA monomer. Both Chlorella species and enzymatically isolated C. ellipsoidea chloroplasts also contain an immunologically similar 38 kilodalton polypeptide that may be a cytosolic or chloroplastic form of CA. PMID- 16667975 TI - Isoelectric focusing of cassava protoplasts. AB - Cassava (Manihot esculenta Crantz) protoplast was analyzed by using isoelectric focusing techniques. Two populations, representing 68 and 32% of the total sample, with mean isoelectric points of 4.48 and 4.60, were obtained using mesophyll protoplasts. The use of this technique allows demonstration of a discontinuous distribution of protoplast isoelectric point from one species according to their surface potential. PMID- 16667976 TI - Ethylene Directly Inhibits Foliar Gas Exchange in Glycine max. AB - Gas exchange of individual attached leaves of soybean, Glycine max (L.) Merr cv Davis, was monitored during exposure to exogenous ethylene (C(2)H(4)) to test the hypothesis that the effects of C(2)H(4) on net photosynthesis (P(N)) and stomatal conductance to H(2)O vapor (g(s)) are direct and not mediated by changes in leaf orientation to light. Leaflets were held perpendicular to incident light in a temperature-controlled cuvette throughout a 5.5 hour exposure to 10 microliters per liter C(2)H(4). Declines in both P(N) and g(s) were evident within 2 hours and became more pronounced throughout the exposure period. In C(2)H(4) treated plants, P(N) and g(s) decreased to 80 and 62%, respectively, of the rates in control plants. Because epinastic movement of the leaflets was prohibited by the cuvette, the observed declines in P(N) and g(s) were a direct effect of C(2)H(4) rather than the result of reduced light interception caused by changing leaf angle. PMID- 16667977 TI - Regulation of Electron Transport in Plant Mitochondria under State 4 Conditions. AB - The regulation of electron transport in pea (Pisum sativum L.) leaf mitochondria under state 4 conditions has been investigated by simultaneously monitoring oxygen uptake, the steady-state reduction level of ubiquinone, and membrane potential. Membrane potentials were measured using a methyltriphenylphosphonium electrode while a voltametric technique was used to monitor changes in the steady state reduction levels of quinone. It was found that the addition of glycine to mitochondria oxidising malate in state 4 led to a marked increase in the rate of O(2) uptake and increased both the membrane potential and reduction level of the quinone pool. Increases in the state 4 respiratory rate were attributed to both an increase in driving flux, due to increased Q-pool reduction, and in membrane potential. Due to the nonohmic behavior of the inner membrane, under these conditions, an increase in potential would result in a considerable rise in proton conductance. Measurement of dual substrate oxidation, in the presence of n propylgallate, revealed that the increase in respiratory activity was not mediated by the alternative oxidase. Similar increases in membrane potential and the level of Q-pool reduction were observed even in the presence of rotenone suggesting that the rotenone-insensitive pathway is a constitutive feature of plant mitochondria and may play a role in facilitating rapid state 4 rates even in the presence of a high energy charge. PMID- 16667978 TI - Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit. AB - The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c-a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-(14)C]putrescine and [terminal labeled-(3)H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase. PMID- 16667979 TI - Monoclonal antibody recognition of abscisic Acid analogs. AB - Specificities of three monoclonal antibodies (15-I-C5, DBPA 1, and MAC 62) raised against the plant hormone (S)-(+)-abscisic acid (ABA) have been compared. Immunological cross-reactivities against fifteen biologically active analogs of ABA were measured. The ABA analogs were altered at one or more of four positions: the double bonds in the ring, at C-2 C-3 and at C-4 C-5, and in the oxidation level at C-1. Several analogs were optically active with chiral centers at C-1' and C-2'. For cross-reactivity, all three monoclonal antibodies required the carboxylic acid group, and the cis configuration of the double bond at C-2 C-3 of the ABA molecule. Monoclonals 15-I-C5 and DBPA 1 required the entire ABA sidechain from the C-1 to C-1', but these monoclonals did cross-react with analogs with the ring double bond reduced and the C-2' methyl cis to the sidechain. Only MAC 62 recognized analogs containing an acetylene at C-4 C-5. MAC 62 had more strict requirements for the ring double bond, but gave some cross reactivity with acetylenic analogs having a saturated ring. All three monoclonals had higher specificity for analogs having the same absolute configuration at C-1' as (S)-(+)-ABA. This work provides new information about the spatial regions of the ABA molecule that elicit immunological recognition, and serves as a basis for future investigations of the ABA receptor using ABA analogs and anti-idiotypic antibodies. PMID- 16667980 TI - Inheritance of Resistance to Crown Gall in Pisum sativum. AB - We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 10(9) bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two-to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three ;supersusceptible' genotypes, which developed very large tumors, also were identified. PMID- 16667981 TI - Analysis of Glucocerebrosides of Rye (Secale cereale L. cv Puma) Leaf and Plasma Membrane. AB - Glucocerebrosides of whole rye (Secale cerale L. cv Puma) leaf and plasma membrane were analyzed using gas chromatography-mass spectrometry and gas chromatography following hydrolysis or as intact molecules purified by reverse phase high performance liquid chromatography. Fatty acids of acid-hydrolyzed leaf and plasma membrane glucocerebrosides consisted of >98 weight percent saturated and monounsaturated 2-hydroxy fatty acids which contained 16 to 26 carbon atoms. The major fatty acids detected were 2-hydroxynervonic acid (24:1h), 2 hydroxylignoceric acid (24:0h), 2-hydroxyerucic acid (22:1h), and 2 hydroxybehenic acid (22:0h). Long-chain bases of alkaline-hydrolyzed glucocerebrosides consisted primarily of cis-trans isomers of the trihydroxy base 4-hydroxysphingenine (t18:1) and the dihydroxy base sphingadienine (d18:2) with lesser amounts of 4-hydroxysphinganine (t18:0) and isomers of sphingenine (d18:1). Intact, underivatized glucocerebroside molecular species of rye leaf and plasma membrane were separated into more than 30 molecular species using reverse phase HPLC. The molecular species composition of leaf and plasma membrane were quantitatively and qualitatively similar. The major molecular species was 24:1h t18:1 which constituted nearly 40 weight percent of leaf and plasma membrane extracts. Several other species including 22:1h-t18:1, 24:1h-t18:1 (isomer), 22:0h-t18:1, 24:1h-d18:2, and 24:0h-t18:1 each comprised 4 to 8% of the total. It is anticipated that the high performance liquid chromatography procedure developed in this study to separate intact, underivatized lipid molecular species will be useful in future studies of the physical properties and biosynthesis of plant glucocerebrosides. PMID- 16667982 TI - NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase. AB - Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b(5) reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b(5) reductase). PMID- 16667983 TI - Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings. AB - Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (EC 2.7.8.2) (CPT) and ethanolaminephosphotransferase (EC 2.7.8.1) (EPT) activities, which equilibrated with the main peak of Antimycin A-insensitive NADH:cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities (less than 5% of the total) in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes (containing 83% of total CPT and EPT activities) in 0.2 millimolar MgCl(2) followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with (14)C-CDPcholine (or (14)C CDPethanolamine) resulted in synthesis and incorporation of (14)C phosphatidylcholine (PC) (or (14)C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg(2+) concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. Under these conditions, newly synthesized and incorporated (14)C-PC (or PE) was recovered in these membranes. Transmission electron microscopy of this fraction confirmed binding of ribosomes to membranes. Radiolabeling in vivo of cotyledons with [methyl-(14)C] choline chloride or [1,2 ethanolamine-(14)C] ethanolamine hydrochloride resulted in a linear incorporation of radiolabel into PC or PE in a time dependent manner. Subfractionation of homogenates of radiolabeled cotyledons on sucrose gradients showed that membranes sedimenting at 24% (w/w) sucrose (ER) contained the majority of radiolabeled PC and PE with a minor peak at 40% (w/w) sucrose (mitochondria), but no radioactive PC or PE was recovered in glyoxysomes. These results indicate that ER in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis. PMID- 16667984 TI - Similarity between Cytokinin and Blue Light Inhibition of Cucumber Hypocotyl Elongation. AB - The cytokinin benzyladenine inhibited endogenous hypocotyl elongation in intact etiolated seedlings of cucumber (Cucumis sativus L.). In hypocotyl segments, the inhibitory effect of benzyladenine on growth was clearly detectable in the presence of indoleacetic acid. Fusicoccin-induced elongation was unaffected by the presence of cytokinin. The effect of cytokinin on elongation of the segments was determined by measuring changes in fresh weight, a linear function of extension growth. The effect of benzyladenine on hypocotyl growth was at least as large in segments prepared from red-light-grown seedlings as in those from seedlings grown in total darkness. A comparison was made between the inhibitory effects of cytokinin and blue light. The use of the calcium chelator ethyleneglycol-bis(beta-aminoethyl ether)-N, N'-tetraacetic acid indicated that calcium ions are required for manifestation of benzyladenine-induced inhibition. PMID- 16667985 TI - Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii. AB - Chloroplastic respiration was monitored by measuring (14)CO(2) from (14)C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of (14)CO(2) evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K(m) for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of (14)CO(2) was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO(2) evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO(2) evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH(4)Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO(2) and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism. PMID- 16667986 TI - Cultured Ovules as Models for Cotton Fiber Development under Low Temperatures. AB - Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a "growth ring" for each time the temperature cycled to 22 or 15 degrees C. Rings were rarely detected when the low point was 28 degrees C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34 degrees C/22 degrees C cycling developed similarly to constant 34 degrees C controls, but 34 degrees C/22 degrees C and 34 degrees C/15 degrees C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34 degrees C/22 degrees C cycling, but both parameters were reduced at the end of the experiment under 34 degrees C/15 degrees C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response. PMID- 16667987 TI - Formation of the fe-s cluster of ferredoxin in lysed spinach chloroplasts. AB - In vitro formation of the (35)S-labeled Fe-S cluster of ferredoxin (Fd) has been achieved by incubating apo-Fd and [(35)S]cysteine with osmotically lysed chloroplasts of spinach (Spinacia oleracea). Correct integration of the (35)S labeled Fe-S cluster into Fd was verified on the basis of the following: (a) Under nondenaturing conditions, (35)S-labeled holo-Fd showed the same electrophoretic mobility as authentic holo-Fd; (b) (35)S-labeled holo-Fd showed an ability to bind Fd-NADP(+) reductase; (c) the (35)S-labeled moiety was removed from the Fd polypeptide by TCA treatment but not by 2-mercaptoethanol treatment; (d) externally added pea II apo-Fd was converted to (35)S-labeled holo-Fd. This reconstitution was dependent on both ATP and light, and formation of the (35)S labeled Fe-S cluster was observed upon addition of ATP or when an ATP generation system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substitute, indicating the requirement of ATP hydrolysis for cluster formation. GTP was able to substitute for ATP, but CTP and UTP were less effective. Fe-S cluster formation in lysed chloroplasts was stimulated by light even in the presence of added ATP. Light stimulation was inhibited by DCMU or methyl viologen but not by NH(4) (+). NADPH was able to substitute for light, indicating that light energy is required for the production of reducing compounds such as NADPH in addition to the generation of ATP. These results confirm the requirement of light for the Fe-S cluster formation observed previously in intact chloroplasts. PMID- 16667988 TI - Diversity of specificity and function of phosphate translocators in various plastids. AB - This report gives a comparison of the specificity of phosphate translocators in various plastids. Whereas the phosphate translocator of the C(3) plant spinach mediates a counter exchange between inorganic phosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate, the phosphate translocators in chloroplasts from C(4) and CAM plants transport phosphoenolpyruvate in addition to the above mentioned metabolites. In plastids from pea roots the phosphate translocator also transports glucose 6-phosphate. This diversity of phosphate translocators is discussed in view of the special functions of the various plastids. PMID- 16667989 TI - Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green. AB - Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [(13)C(6)]-IAA or [(2)H(6)]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation. PMID- 16667990 TI - Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal 5'-Phosphate-Dependent Enzyme. AB - Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5'-phosphate-containing enzyme, but the requirement of pyridoxal-5' phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5'-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5' phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5'-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5'-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5' phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5' phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5'-phosphate that is present in the mammalian and bacterial enzymes. PMID- 16667991 TI - Abscisic Acid and the developmental regulation of embryo storage proteins in maize. AB - The relationship of abscisic acid (ABA) inhibition of precocious germination and ABA-induced storage protein accumulation was examined over the course of embryogenesis in wild-type and viviparous mutants of maize (Zea mays L.). We show that a high level of embryo ABA and the product of the Viviparous-1 gene are both required in early maturation phase for germination suppression and the accumulation of storage globulins encoded by the gene Glb1. Suppressing precocious germination with a high osmoticum is not sufficient to initiate Glb1 protein synthesis, although continued accumulation is contingent upon this inhibition; germination of immature or mature embryos leads to a decline in synthesis and the degradation of stored globulins. Late in embryogenesis, fragments of Glb1 protein accumulate, coinciding with the loss of ABA sensitivity. These results suggest that ABA influences storage globulin accumulation by initiating synthesis, suppressing degradation, and inhibiting precocious germination. PMID- 16667992 TI - Alfalfa Controls Nodulation during the Onset of Rhizobium-induced Cortical Cell Division. AB - The formation of first nodules inhibits subsequent nodulation in younger regions of alfalfa (Medicago sativa L.) roots by a feedback regulatory mechanism that controls nodule number systemically (G Caetano-Anolles, WD Bauer [1988] Planta 175: 546-557). Following inoculation with wild-type Rhizobium meliloti, almost all infections associated with cortical cell division developed into mature nodules. While the distribution of Rhizobium- induced cell divisions closely paralleled the distribution of first emergent nodules, only 9 to 15% of total cell division foci failed to become functional nodules. Nodule formation was restricted to the primary root when plants were inoculated before lateral root emergence. Excision of these primary root nodules allowed nodules to reappear in lateral roots clustered around the location of the root tip at the time of nodule removal. Apparently, this region regained susceptibility to infection within the first hours after excision of primary nodules and suppression of nodulation was restored a day later probably due to the development of new infection foci. Our results suggest that alfalfa controls nodulation during the onset of cell division in the root cortex and not during infection development as in soybean. PMID- 16667993 TI - Blue Light, a Positive Switch Signal for Nitrate and Nitrite Uptake by the Green Alga Monoraphidium braunii. AB - Blue light was shown to regulate the utilization of oxidized nitrogen sources by green algae, both by activating nitrate reductase and promoting nitrite reductase biosysnthesis (MA Quinones, PJ Aparicio [1990] Inorganic Nitrogen in Plants and Microorganisms, Springer-Verlag, Berlin, pp 171-177; MA Quinones, PJ Aparicio [1990] Photochem Photobiol 51: 681-692). The data reported herein show that, when cells of Monoraphidium braunii at pH 8, containing both active nitrate reductase and nitrite reductase, were sparged with CO(2)-free air and irradiated with strong background red light, they took up oxidized nitrogen sources only when PAR comprised blue light. The activation of the transport system(s) of either both nitrate and nitrite was very quick and elicited by low irradiance blue light. In fact, blue light appears to act as a switch signal from the environment, since the uptake of these anions immediately ceased when this radiation was turned off. The requirement of blue light for nitrate uptake was independent of the availability of CO(2) to cells. However, cells under high CO(2) tensions, although they showed an absolute blue light requirement to initially establish the uptake of nitrite, as they gained carbon skeletons to allocate ammonia, gradually increased their nitrite uptake rates in the subsequent red light intervals. Under CO(2)-free atmosphere, cells irradiated with strong background red light of 660 nanometers only evolved oxygen when they were additionally irradiated with low irradiance blue light and either nitrate or nitrite was present in the media to provide electron acceptors for the photosynthetic reaction. PMID- 16667994 TI - Light-Independent Expression of cab and rbcS Genes in Dark-Grown Pine Seedlings. AB - In angiosperms, light has been shown to induce the expression of cab and rbcS genes, which encode the apoprotein of light-harvesting chlorophyll a/b binding protein (LHCP) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), respectively. By contrast, chlorophylls are synthesized in the cotyledons of pine seedlings even if seeds are germinated in the dark. We have examined the expression of cab and rbcS genes in the cotyledons of pine (Pinus thunbergii) seedlings grown in darkness. The proteins of LHCP and the large subunit and the small subunit of Rubisco were detected in the cotyledons of dark-grown seedlings. The transcripts of cab and rbcS genes were present at substantial levels in dark-grown seedlings. However, the transcripts and the translated products of the genes were not found in the embryos of dry seeds. These results indicate that light is not required for the expression of cab and rbcS genes during the course of development of the cotyledons of pine seedlings. The processing of the precursor polypeptides of the mature proteins also appears to take place even in the dark. PMID- 16667995 TI - Soybean Nodule-Specific Uricase (Nodulin-35) Is Expressed and Assembled into a Functional Tetrameric Holoenzyme in Escherichia coli. AB - A complete nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3) was isolated from a soybean (Glycine max L. var. Prize) nodule cDNA expression library using a previously isolated partial cDNA clone. The N-35 cDNA was expressed in Escherichia coli driven by the lacZ promoter and was found to be functionally active. The uricase activity was detected in the cytoplasmic fraction of E. coli with the same pH optimum and apparent K(m) values as that in the nodules. Because a stop codon is located 15 base pairs upstream of the N-35 initiation codon, it appears that a fusion protein with LacZ was not made, but reinitiation occurred due to the presence of a putative Shine-Dalgarno sequence in the appropriate region. The size of the N-35 polypeptide made in E. coli is identical to that present in soybean nodules and is able to assemble into a tetrameric holoenzyme with the same molecular weight as the native uricase. Thus, the presence of peroxisomes does not appear to be essential for the proper assembly of the holoenzyme in E. coli. These data also indicate that posttranslational modifications or membrane transport are not essential either for the assembly of N-35 into a holoenzyme or for the activity of uricase. PMID- 16667996 TI - Oxidative phosphorylation by mitochondria extracted from dry sunflower seeds. AB - The role of mitochondria in the phosphorylation of ADP to ATP in the early steps of seed germination has been studied. Mitochondria were extracted from dry sunflower (Helianthus annuus) seeds. Adenylate kinase-dependent ATP synthesis was inhibited by p(1),p(5)-di(adenosine-5')pentaphosphate. Synthesis of ATP was observed with the different substrates: citrate, alpha-ketoglutarate, succinate, malate, pyruvate or NADH. This synthesis was activated by cytochrome c, and inhibited by cyanide, oligomycin, carbonyl cyanide p (trifluoromethoxy)phenylhydrazone, and carboxyatractyloside. The ATP/O values with succinate were 0.85 and 1.2 in the absence or presence, respectively, of cytochrome c. Electron micrographs showed that mitochondria of dry tissues have different structures when observed in situ or in vitro after aqueous extraction, suggesting that profound changes occurred after the contact with the aqueous medium. These results confirm previous data obtained in vivo showing that mitochondria present in dry seeds are able to synthesize ATP as soon as the seeds are rehydrated. PMID- 16667997 TI - Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds. AB - A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed. PMID- 16667998 TI - Isolation of Genes that Are Preferentially Expressed at the G(1)/S Boundary during the Cell Cycle in Synchronized Cultures of Catharanthus roseus Cells. AB - A cDNA library was screened for genes that may be involved in the progression of the cell cycle of cells of higher plants. The Catharanthus roseus L. (G) Don. cells were synchronized by the double phosphate starvation method, and a lambdagt11 cDNA library was prepared using poly(A)(+) RNA from cells in the S phase of the cell cycle. Two independent sequences, cyc02 and cyc07, were identified by differential screening. The levels of cyc02 and cyc07 mRNAs increased dramatically, but transiently, at the G(1)/S boundary of the cell cycle. High levels of cyc02 mRNA, but not of cyc07 mRNA, were also present in cells arrested at the G(1) phase by phosphate starvation. In an asynchronous batch culture, cyc02 and cyc07 mRNAs accumulated transiently at different stages of the growth cycle, cyc02 mRNA early in the stationary phase, and cyc07 mRNA in the midlogarithmic phase. When the proliferation of cells was arrested by nutrient starvation, i.e. by sucrose or nitrogen starvation, the relative amounts of the cyc02 and cyc07 mRNAs decreased. These results indicate that cyc02 and cyc07 contain nucleotide sequences from growth-related genes. The analysis of nucleotide sequence of cyc02 shows that the predicted product of this gene is basic and is composed of 101 amino acids. No significant homology to other known proteins was detected. PMID- 16667999 TI - Metabolism of Inositol(1,4,5)trisphosphate by a Soluble Enzyme Fraction from Pea (Pisum sativum) Roots. AB - Metabolism of the putative messenger molecule d-myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] in plant cells has been studied using a soluble fraction from pea (Pisum sativum) roots as enzyme source and [5-(32)P]Ins(1,4,5)P(3) and [2 (3)H]Ins(1,4,5)P(3) as tracers. Ins(1,4,5)P(3) was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P(2)] whereas inositol(1,4)bisphosphate [Ins(1,4)P(2)] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P(4). Dephosphorylation of Ins(1,4,5)P(3) to Ins(4,5)P(2) was dependent on Ins(1,4,5)P(3) concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p nitrophenylphosphate. Conversion of Ins(1,4,5)P(3) to Ins(4,5)P(2) and Ins(1,4,5,X)P(4) was inhibited by 55 micromolar Ca(2+). This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P(3) and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom. PMID- 16668000 TI - Slow-growth phenotype of transgenic tomato expressing apoplastic invertase. AB - The growth of transgenic tomato (Lycopersicon esculentum) plants that express in their apoplast yeast invertase under the control of the cauliflower mosaic virus 35S promoter is severely inhibited. The higher the level of invertase, the greater the inhibition of growth. A second phenotypic characteristic of these transgenic plants is the development of yellow and necrotic spots on the leaves, and leaf curling. Again the severity of the symptoms is correlated with the level of invertase. These symptoms do not develop in shaded leaves indicating the need for photosynthesis. Keeping the plants in the dark for a prolonged period (24 hours) results in the disappearance of leaf starch from the control plants, but not from the plants with apoplastic invertase. These results are consistent with the interpretation that apoplastic invertase prevents photosynthate export from source leaves and that phloem loading includes an apoplastic step. PMID- 16668001 TI - Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex. AB - Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F(1) progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and beta-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F(1) generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F(1) progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes. PMID- 16668002 TI - Effect of inoculation and nitrogen on isoflavonoid concentration in wild-type and nodulation-mutant soybean roots. AB - The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizobium japonicum. The common nod genes of rhizobia are in turn responsible for stimulating root hair curling and cortical root cell division, the earliest steps in the host response. This study evaluated whether there was a relationship between root isoflavonoid production and the hypernodulation phenotype of selected soybean (Glycine max [L.] Merr.) mutants. Three independently selected hypernodulating soybean mutants (NOD1-3, NOD2-4, and NOD3-7) and a nonnodulating mutant (NN5) were compared with the Williams parent for isoflavonoid concentrations. High performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations throughout the 12-day growth period after transplanting of both inoculated and noninoculated plants; transplanting and inoculation were done 6 days after planting. No significant differences were detected in the concentration of these compounds among the three noninoculated hypernodulating mutants and the Williams parent. In response to inoculation, the three hypernodulating mutants had higher isoflavonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. However, the inoculated nonnodulating mutant also had higher isoflavonoid concentrations than did Williams. N application [urea, (NH(4))(2)SO(4) and NO(3) (-)] decreased the concentration of all three isoflavonoid compounds in all soybean lines. Application of NO(3) (-) was most inhibitory to isoflavonoid concentrations, and inhibition by NO(3) (-) was concentration dependent. These results are consistent with a conclusion that differential NO(3) (-) inhibition of nodulation may be partially due to changes in isoflavonoid levels, although the similar response of the nonnodulating mutant brings this conclusion into question. Alternatively, the nodulation control in the NN5 mutant may be due to factors totally unrelated to isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of lines genetically competent to nodulate. PMID- 16668003 TI - Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins. AB - Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11 day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37 degrees C for 6 hours followed by 4 days at 2.5 or 12.5 degrees C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5 degrees C, but not to 12.5 degrees C, a nonchilling temperature. Exposure to 37 degrees C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5 degrees C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37 degrees C and held at the nonchilling temperature of 12.5 degrees C. There was no relationship between abscisic acid content following exposure to 12.5 or 37 degrees C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42 degrees C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42 degrees C. PMID- 16668004 TI - Purification and Characterization of an Antifungal Chitinase from Arabidopsis thaliana. AB - Plants exhibit an altered pattern of protein synthesis in response to pathogen invasion and abiotic stress. One of these ;pathogenesis-related' proteins has been identified as chitinase, which is capable of inhibiting fungal growth in vitro. This observation has led to the suggestion that the in vivo role of chitinases is to protect plants against fungal invasion. Here, we report the purification and characterization of a basic chitinase from Arabidopsis thaliana (L.) Heynh. Columbia wild type. The purified enzyme has a molecular mass of approximately 32 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and an apparent pl of approximately 8.7 as determined by isoelectric focusing. The purified protein is an effective inhibitor of the growth of Trichoderma reesei in vitro but does not affect the growth of several other fungi. Amino acid composition analysis of the intact protein as well as amino acid composition analysis and automatic Edman degradation of isolated tryptic fragments of the enzyme indicate that it may be identical to the product of a chitinase gene isolated from an Arabidopsis genomic library (Samac DA, Hironaka CM, Yallaly PE, Shah DM [1990] Plant Physiol 93: 907 914). PMID- 16668005 TI - Chilling-Induced Inactivation and Its Recovery of Tonoplast H-ATPase in Mung Bean Cell Suspension Cultures. AB - The processes involved in adaptation to cold temperature were examined by growing suspension cultured cells of mung bean (Vigna radiata [L.] Wilczek) at 2 degrees C for various periods of time and assaying the activities of various membrane bound enzymes in vitro. The tonoplast H(+)-ATPase activity and the ATP-proton transport extracted from cells incubated at 2 degrees C declined rapidly and reached a minimum level after 10 hours. The inactivation was reversible within 24 hours of chilling. The recovery of the cold-inactivated H(+)-ATPase was found to proceed in two steps, a faster recovery of ATP hydrolysis activity and a slower recovery of the proton transport. The recovery was markedly inhibited by the presence of azide, but not affected by 0.578 millimolar cycloheximide. This suggested the involvement of an energy process that had no requirement for de novo synthesis of protein. The cold-induced inactivation of the H(+)-ATPase may be due to a structural alteration of the enzyme. The slower recovery of proton transport relative to ATP hydrolysis during warming suggests that the protogenic domains in the enzyme may be affected differently by chilling. PMID- 16668006 TI - alpha-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes. AB - alpha-dl-Difluoromethylarginine (DFMA) and alpha-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway. PMID- 16668007 TI - The Latex of Hevea brasiliensis Contains High Levels of Both Chitinases and Chitinases/Lysozymes. AB - The latex of the commercial rubber tree, Hevea brasiliensis, was fractionated by ultracentrifugation as described by G. F. J. Moir ([1959] Nature 184: 1626-1628) into a top layer of rubber particles, a cleared cytoplasm, and a pellet that contains primarily specialized vacuoles known as lutoids. The proteins in each fraction were resolved by two-dimensional gel electrophoresis. Both the pellet fraction and cleared cytoplasm contained large amounts of relatively few proteins, suggesting that laticifers serve a very specialized function in the plant. More than 75% of the total soluble protein in latex was found in the pellet fraction. Twenty-five percent of the protein in the pellet was identified as chitinases/lysozymes, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of bacterial cell walls. Both the chitinase and lysozyme activities were localized exclusively in the pellet or lutoid fraction. The chitinases/lysozymes were resolved into acidic and basic classes of proteins and further purified. An acidic protein (molecular mass 25.5 kD) represented 20% of the chitinase activity in latex; this protein lacked the low level of lysozyme activity that is associated with many plant chitinases. Six basic proteins, having both chitinase and lysozyme activities in various ratios and molecular mass of 27.5 or 26 kD, were resolved. Two of the basic proteins had very high lysozyme specific activities which were comparable to the specific activities reported for animal lysozymes. Like animal lysozymes, but unlike previously characterized plant chitinases/lysozymes, these basic chitinases/lysozymes were also capable of completely lysing or clearing suspensions of bacterial cell walls. These results suggest that laticifers may serve a defensive role in the plant. PMID- 16668008 TI - Oxidation of spermine by an amine oxidase from lentil seedlings. AB - Spermine is a substrate of lentil (Lens culinaris) seedling amine oxidase and the oxidation products are reversible inactivators of the enzyme. The spermine is oxidized at the terminal amino groups to a dialdehyde: 2 moles of hydrogen peroxide and 2 moles of ammonia per mole of spermine are formed. The pH optimum of the enzyme with spermine is 7.9 in TI-HCI buffer; the K(m) value is 4.4.10(-4) molar, similar to that found with other substrates (putrescine and spermidine). PMID- 16668009 TI - Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens. AB - The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development. PMID- 16668010 TI - Responses of Cultured Tobacco Cells to Cryptogein, a Proteinaceous Elicitor from Phytophthora cryptogea: Possible Plasmalemma Involvement. AB - In culture, the phytopathogenic fungus Phytophthora cryptogea secretes a protein which elicits hypersensitive-like necroses and protects tobacco plants against invasion by the pathogen Phytophthora parasitica var. nicotianae. This protein, named cryptogein, has been purified and its amino acid sequence determined. In this work, we studied the effect of cryptogein on tobacco cell suspension cultures. Cryptogein was lethal at about 0.10 micromolar. When added at sublethal doses, it elicited the production of ethylene and phytoalexins. It also induced a rapid increase in pH and conductivity of the extracellular medium without affecting the integrity of the plasma membrane. Cryptogein reduced the fusicoccin induced acidification of the extracellular medium. The concentration which inhibited the fusicoccin response by 50% was 0.8 nanomolar, while 1 micromolar erythrosine B, an ATPase inhibitor, was needed to produce the same inhibition. However, cryptogein did not inhibit the activity of a purified plasma membrane ATPase. Results of binding studies with whole cells suggested the presence of elicitor-binding sites with a high affinity for cryptogein. The involvement of the plasma membrane during the initial interaction between elicitor and cells is discussed. PMID- 16668011 TI - Effect of Light Quality on Phycobilisome Components of the Cyanobacterium Spirulina platensis. AB - Phycobilisomes from the nonchromatic adapting cyanobacterium Spirulina platensis are composed of a central core containing allophycocyanin and rods with phycocyanin and linker polypeptides in a regular array. Room temperature absorption spectra of phycobilisomes from this organism indicated the presence of phycocyanin and allophycocyanin. However, low temperature absorption spectra showed the association of a phycobiliviolin type of chromophore within phycobilisomes. This chromophore had an absorption maximum at 590 nanometers when phycobilisomes were suspended in 0.75 molar K-phosphate buffer (pH 7.0). Purified phycocyanin from this cyanobacterium was found to consist of three subparticles and the phycobiliviolin type of chromophore was associated with the lowest density subparticle. Circular dichroism spectra of phycocyanin subparticles also indicated the association of this chromophore with the lowest density subparticle. Absorption spectral analysis of alpha and beta subunits of phycocyanin showed that phycobiliviolin type of chromophore was attached to the alpha subunit, but not the beta subunit. Effect of light quality showed that green light enhanced the synthesis of this chromophore as analyzed from the room temperature absorption spectra of phycocyanin subparticles and subunits, while red or white light did not have any effect. Low temperature absorption spectra of phycobilisomes isolated from green, red, and white light conditions also indicated the enhancement of phycobiliviolin type of chromophore under green light. PMID- 16668012 TI - Nonsteady-State Photosynthesis following an Increase in Photon Flux Density (PFD) : Effects of Magnitude and Duration of Initial PFD. AB - The response of photosynthesis to an increase in photon flux density (PFD) from low to higher PFD was investigated using spinach (Spinacia oleracea L.). The time course for this response was qualitatively similar to that observed for a dark-to high-PFD transition, showing an initial, rapid increase in photosynthesis over the first minute or so, followed by a slower increase lasting 5 to 10 minutes. This slow increase was approximately exponential and could be linearized using a semilogarithmic plot. The relaxation time (tau) for this slow phase was found to be a function of the starting PFD value. At starting PFD values below approximately 135 micromoles per square meter per second (including darkness), tau for the slow phase was approximately twice that observed for starting PFD values above 135 micromoles per square meter per second. This indicates a slower approach to steady state for leaves starting at PFD values below this threshold and a greater loss of potential photosynthesis. tau was relatively insensitive to starting PFD values below or above this transition value. The contribution of the slow phase to the total increase in photosynthesis following a low-to-high-PFD transition increased approximately exponentially with time at the lower PFD. The tau for the increase in the contribution of slow phase was determined to be 10.1 minutes. The implications of these data for activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase and for the functioning of the leaf in a fluctuating light environment are discussed. PMID- 16668013 TI - Comparison of Temperature Dependency of Tonoplast Proton Translocation between Plants Sensitive and Insensitive to Chilling. AB - Proton transport activities in isolated tonoplast vesicles were measured as quenching of fluorescence of acridine orange. A marked difference in the temperature dependency of two types of tonoplast proton transports, i.e. ATP- and pyrophosphate-driven, was observed between two leguminous plants sensitive (mung bean, Vigna radiata [L.] Wilczek) and insensitive (pea, Pisum sativum L.) to chilling. In tonoplast vesicles isolated from hypcotyls of mung bean seedlings that were germinated for 3.5 days at 26 degrees C in the dark, the total amount of fluorescence quenching at the steady state in both types of proton pumps, as a measurement of the inside-acidic pH gradient across the membrane vesicles, was markedly suppressed under temperatures below 10 degrees C. In tonoplast vesicles isolated from epicotyls of pea seedlings, which were germinated for 7 days at 18 degrees to 23 degrees C in the dark, no suppression occurred in the formations of the pH gradient in either type of proton pump, even at 0 degrees C. The cause of the low temperature-induced suppression of the proton pumps in mung bean tonoplasts seems to be not an increased permeability of the membrane vesicles to protons or accompanying anions and cations, but instead a marked inhibition in the catalytic activity of both enzymes under low temperatures. PMID- 16668014 TI - The role of glutamate dehydrogenase in plant nitrogen metabolism. AB - In vivo nuclear magnetic resonance spectroscopy, in vitro gas chromatography-mass spectrometry, and automated (15)N/(13)C mass spectrometry have been used to demonstrate that glutamate dehydrogenase is active in the oxidation of glutamate, but not in the reductive amination of 2-oxogiutarate. In cell suspension cultures of carrot (Daucus carota L. cv Chantenay), primary assimilation of ammonium occurs via the glutamate synthase pathway. Glutamate dehydrogenase is derepressed in carbonlimited cells and in such cells the function of glutamate dehydrogenase appears to be the oxidation of glutamate, thus ensuring sufficient carbon skeletons for effective functioning of the tricarboxylic acid cycle. This catabolic role for glutamate dehydrogenase implies an important regulatory function in carbon and nitrogen metabolism. PMID- 16668015 TI - Dissipation of the proton electrochemical potential in intact and lysed chloroplasts : I. The electrical potential. AB - Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts, whereas gramicidin was the most potent ionophore in lysed chloroplasts. The significance of the ionophore:chlorophyll ratios required to collapse the electrical potential is discussed with regard to bioenergetic studies, especially those that examine the contribution of the transmembrane electrochemical potential to protein transport into chloroplasts. PMID- 16668016 TI - The Sequence of Change within the Photosynthetic Apparatus of Wheat following Short-Term Exposure to Ozone. AB - The basis of inhibition of photosynthesis by single acute O(3) exposures was investigated in vivo using analyses based on leaf gas exchange measurements. The fully expanded second leaves of wheat plants (Triticum aestivum L. cv Avalon) were fumigated with either 200 or 400 nanomoles per mole O(3) for between 4 and 16 hours. This reduced significantly the light-saturated rate of CO(2) uptake and was accompanied by a parallel decrease in stomatal conductance. However, the stomatal limitation, estimated from the relationship between CO(2) uptake and the internal CO(2) concentration, only increased significantly during the first 8 hours of exposure to 400 nanomoles per mole O(3); no significant increase occurred for any of the other treatments. Analysis of the response of CO(2) uptake to the internal CO(2) concentration implied that the predominant factor responsible for the reduction in light-saturated CO(2) uptake was a decrease in the efficiency of carboxylation. This was 58 and 21% of the control value after 16 hours at 200 and 400 nanomoles per mole O(3), respectively. At saturating concentrations of CO(2), photosynthesis was inhibited by no more than 22% after 16 hours, indicating that the capacity for regeneration of ribulose bisphosphate was less susceptible to O(3). Ozone fumigations also had a less pronounced effect on light-limited photosynthesis. The maximum quantum yield of CO(2) uptake and the quantum yield of oxygen evolution showed no significant decline after 16 hours with 200 nanomoles per mole O(3), requiring 8 hours at 400 nanomoles per mole O(3) before a significant reduction occurred. The photochemical efficiency of photosystem II estimated from the ratio of variable to maximum chlorophyll fluorescence and the atrazine-binding capacity of isolated thylakoids demonstrated that photochemical reactions were not responsible for the initial inhibition of CO(2) uptake. The results suggest that the apparent carboxylation efficiency appears to be the initial cause of decline in photosynthesis in vivo following acute O(3) fumigation. PMID- 16668017 TI - UV-B-Inducible and Temperature-Sensitive Photoreactivation of Cyclobutane Pyrimidine Dimers in Arabidopsis thaliana. AB - Removal of cyclobutane pyrimidine dimers (CBPDs) in vivo from the DNA of UV irradiated eight-leaf seedlings of Arabidopsis thaliana was rapid in the presence of visible light (half-life about 1 hour); removal of CBPDs in the dark, presumably via excision repair, was an order of magnitude slower. Extracts of plants contained significant photolyase in vitro, as assayed by restoration of transforming activity to UV-irradiated Escherichia coli plasmids; activity was maximal from four-leaf to 12-leaf stages. UV-B treatment of seedlings for 6 hours increased photolyase specific activity in extracts twofold. Arabidopsis photolyase was markedly temperature-sensitive, both in vitro (half-life at 30 degrees C about 12 minutes) and in vivo (half-life at 30 degrees C, 30 to 45 minutes). The wavelength dependency of the photoreactivation cross-section showed a broad peak at 375 to 400 nm, and is thus similar to that for maize pollen; it overlaps bacterial and yeast photolyase action spectra. PMID- 16668018 TI - Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules. AB - Nicotinate has been postulated to interfere with the binding of O(2) to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O(2). Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O(2) in soybean root nodules. PMID- 16668019 TI - Direct Photolabeling with [P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase. AB - We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1-->3)-beta-glucan (callose) synthase of developing fibers of Gossypium hirsutum (cotton). Such a polypeptide migrates coincident with callose synthase during glycerol gradient centrifugation in the presence of EDTA, and can be directly photolabeled with the radioactive substrate, alpha-[(32)P]UDP-glucose. Interaction with the labeled probe requires Ca(2+), a specific activator of callose synthase which is known to lower the K(m) of higher plant callose synthases for the substrate UDP-glucose. Using this probe and several other related ones, several other proteins which interact with UDP glucose were also identified, but none satisfied all of the above criteria for being components of the callose synthase. PMID- 16668020 TI - Partial purification and characterization of the mitochondrial and peroxisomal isozymes of enoyl-coenzyme a hydratase from germinating pea seedlings. AB - Distinct organellar forms of the beta-oxidation enzyme enoyl-coenzyme A (CoA) hydratase were partially purified and characterized from 2-day germinated pea (Pisum sativum L.) seedlings. The purification was accomplished by disruption of purified mitochondria or peroxisomes, (NH(4))(2)SO(4) fractionation, and gel permeation chromatography using a column of Sephacryl S-300. The organellar isozymes had distinct kinetic constants for the substrates 2-butenoyl-CoA and 2 octenoyl-CoA, and could be easily distinguished by differences in thermostability and salt activation. The peroxisomal isozyme had a native M(r) of 75,000 and appeared to be a typical bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, while the mitochondrial isozyme had a native M(r) of 57,000 and did not have associated dehydrogenase activity. Western blots of total pea mitochondrial proteins gave a positive signal when probed with anti-rat liver mitochondrial enoyl-CoA hydratase antibodies but there was no signal when blots of total peroxisomal proteins were probed. PMID- 16668021 TI - Isolation and Characterization of a UDPGlucose: Flavonol O-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings. AB - A UDPGlc:flavonol O(3)-glucosyltransferase (EC 2.4.1.91) that catalyzes the formation of quercetin and kaempferol O(3)-glucosides has been purified about 1450-fold from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings with a 3.3% yield. Purification of the enzyme was achieved by (NH(4))(2)SO(4)-precipitation, gel-filtration, ion-exchange chromatography on DEAE-Bio-Gel and Q-Sepharose, chromatofocusing, and electrophoresis in nondenaturing polyacrylamide (10%) gels. The enzyme preparation had a pH optimum between 5.8 and 6.2, isoelectric point in the pH range 4.25 to 4.55, a M(r) of 59,000, and it was composed of two similar subunits of M(r) 29,500. The glucosyltransferase reached half substrate saturation at 180 micromolar (UDPGlc) and 7 micromolar (quercetin) concentrations. Kaempferol, which was glucosylated at a relative rate of 87%, had a lesser affinity for the enzyme (K(m)~12 micromolar). Flavanones, flavanols, flavones, dihydroflavonols, and anthocyanidins were not readily utilized as substrates, suggesting that the enzyme is specific for flavonol glucoside biosynthesis. PMID- 16668022 TI - Evidence for a senescence-associated gene induced by darkness. AB - A nearly full-length cDNA was isolated from a cDNA library prepared from incipiently senescent radish (Raphanus sativus L.) cotyledons using a previously isolated cDNA clone for dark-inducible mRNA as a probe (A Watanabe, N Kawakami, Y Azumi [1989] In Cell Separation in Plants, NATO ASI Series, Vol H35, pp 31-38. Springer-Verlag, Berlin). The clone detected transcripts of 800 bases which increased more than 100-fold after 24 hours of darkness. The transcripts also accumulated under light when plants were exposed to ethylene or heat stress, and (6)N-benzyladenine partially repressed its accumulation in the dark. These responses of the gene to physiological stimuli closely paralleled the effects of the stimuli on the progress of senescence of the cotyledons. We have named the gene din 1 (dark inducible gene 1). The cDNA encodes a polypeptide of 20 kilodaltons, and its nucleotide sequence shows a high (49%) similarity to a subfamily of pathogenesis-related proteins of tobacco. The predicted amino acid sequence of the product, however, shows only 20% homology to the pathogenesis related protein. PMID- 16668023 TI - A rapid, high resolution high performance liquid chromatography profiling procedure for plant and microbial aromatic secondary metabolites. AB - High performance liquid chromatography protocols have been developed to allow the simultaneous analysis of a very wide range of soluble aromatic secondary metabolites in unfractionated biological extracts. The methods are simple, sensitive, and highly reproducible. They are applicable to a wide variety of natural product investigations in both plants and microorganisms. High resolution of metabolites is achieved in 25 minutes by chromatography on a reverse phase C18 column in a gradient of 0 to 55% acetonitrile in water at pH 3. For example, near baseline resolution of over 20 phenylpropanoid metabolites and 18 naturally occurring metabolites of indole-3-acetic acid can be obtained. The methods can be applied directly to whole tissue extracts without prepurification or enrichment. Moreover, the simplicity and sensitivity of the protocols allow their application to a large number of very small tissue samples, such as those encountered in research on host-microbe interactions. Such profiles allow one to monitor simultaneously the various alternative metabolic fates of a complex array of molecules. Examination of the profiles over time thus provides one with a powerful tool to correlate many concurrent molecular events that may relate to a given biological phenomenon. The final protocol requires as little as 1 milligram of tissue, which is extracted directly in a microfuge tube in 80% ethanol. With a variable wavelength detector, as little as 100 femtomoles of a given metabolite can be analyzed. Examples of the application of the protocols to a number of plant and microbial secondary product investigations and to screening for flavonoid mutants of Arabidopsis thaliana (L.) Heynh. are given. PMID- 16668024 TI - Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. AB - The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event. PMID- 16668025 TI - Determination of Apparent K(m) Values for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Using the Spectrophotometric Assay of Rubisco Activity. AB - The spectrophotometric assay for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used to determine the rate of increase in Rubisco activity over time in the presence or absence of Rubisco activase. Polynomial approximations to the raw data were used to smooth out minor fluctuations in the spectrophotometer readings, and Rubisco activase activity was expressed as nanomoles of activated Rubisco per minute. This assay was used to examine the effects of CO(2) and the inactive-Rubisco:ribulose 1,5-bisphosphate complex (ER) on the activase-catalyzed activation reaction. Double-reciprocal plots of activase activity and ER at several concentrations of CO(2) were consistent with two-substrate Michaelis Menton kinetics, and the apparent K(m) (CO(2)) and K(m)(ER) were determined to be 53 and 2.7 micromolar, respectively. These data do not prove that ER and CO(2) are substrates for the reaction catalyzed by activase, but they may be important to our understanding of the activation process in vivo. The implications of these data and their relation to previously published data on the effects of ER and CO(2) on activase are discussed. PMID- 16668026 TI - Infrared spectroscopy of plant cell cultures : noninvasive measurement of viability. AB - Infrared spectroscopy was used to examine suspension-cultured pear (Pyrus communis L.) and Spartina pectinata cells. Noninvasive measurements were made using internal reflectance sampling. Spectra of actively growing cells exhibited a pronounced absorbance at 2343 reciprocal centimeters. The absorbance peak was identified and verified as CO(2) dissolved in water. This peak was absent in nonviable cells. Peak height was directly proportional to percent viability in artificial mixtures of viable and nonviable cells, indicating that the level of intracellular CO(2) production could be used as a viability determinant for plant cells. Suspension-cultured cells were slowly cooled to subzero temperatures and analyzed for viability using infrared spectroscopy and tetrazolium staining. Both methods showed similar trends in viability assessment. Infrared spectroscopy could provide a more detailed understanding of cell viability and allow measurement on a noninvasive basis. PMID- 16668027 TI - Degradation of tobacco pathogenesis-related proteins : evidence for conserved mechanisms of degradation of pathogenesis-related proteins in plants. AB - Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663 669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants. PMID- 16668028 TI - Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl. AB - The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed. PMID- 16668029 TI - Effects of Salinity on Stomatal Conductance, Photosynthetic Capacity, and Carbon Isotope Discrimination of Salt-Tolerant (Gossypium hirsutum L.) and Salt Sensitive (Phaseolus vulgaris L.) C(3) Non-Halophytes. AB - The effects of salinity on growth, stomatal conductance, photosynthetic capacity, and carbon isotope discrimination (Delta) of Gossypium hirsutum L. and Phaseolus vulgaris L. were evaluated. Plants were grown at different NaCl concentrations from 10 days old until mature reproductive structures were formed. Plant growth and leaf area development were strongly reduced by salinity, in both cotton and bean. Stomatal conductance also was reduced by salinity. The Delta always declined with increasing external salinity concentration, indicating that stomatal limitation of photosynthesis was increased. In cotton plant dry matter, Delta correlated with the ratio of intercellular to atmospheric CO(2) partial pressures (p(l)/p(a)) calculated by gas exchange. This correlation was not clear in bean plants, although Delta showed a more pronounced salt induced decline in bean than in cotton. Possible effects of heterogeneity of stomatal aperture and consequent overestimation of p(l) as determined from gas exchange could explain these results. Significant differences of Delta between leaf and seed material were observed in cotton and bean. This suggests different patterns of carbon allocation between leaves and seeds. The photon yield of O(2) evolution determined at rate-limiting photosynthetic photon flux density was insensitive to salinity in both species analyzed. The light- and CO(2)-saturated rate of CO(2) uptake and O(2) evolution showed a salt induced decline in both species. Possible explanations of this observation are discussed. O(2) hypersensitivity was observed in salt stressed cotton plants. These results clearly demonstrate that the effect of salinity on assimilation rate was mostly due to the reduction of stomatal conductance, and that calculation of p(l) may be overestimated in salt stressed plants, because of heterogeneity of stomatal aperture over the leaf surface. PMID- 16668030 TI - Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L. AB - A mass spectrometric method combining (16)O/(18)O and (12)C/(13)C isotopes was used to quantify the unidirectional fluxes of O(2) and CO(2) during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O(2) uptake and CO(2) evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O(2) (61 micromoles of O(2) per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O(2) per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO(2) in darkness at a rate of 27 micromoles of CO(2) per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O(2) evolution and CO(2) fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO(2) evolution by guard cell protoplasts was sharply decreased (37%), while O(2) uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO(2) assimilation and may be dissipated for other purposes such as ion uptake. PMID- 16668031 TI - Substrate regulation of single potassium and chloride ion channels in Arabidopsis plasma membrane. AB - Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K(+) and/or Cl(-). The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations. PMID- 16668032 TI - Drought Stress, Enzymes of Glutathione Metabolism, Oxidation Injury, and Protein Synthesis in Tortula ruralis. AB - The activities of glutathione reductase (EC 1.6.4.2), glutathione peroxidase (EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) were found to increase during slow drying or during rehydration following rapid drying of the drought tolerant moss Tortula ruralis. Little change was observed in the activity of malate deydrogenase (NAD(+) oxidoreductase, EC 1.1.1.37) during dehydration or subsequent rehydration. When the tissue was treated with cycloheximide, actinomycin D, or cordycepin, the increase in the activities of glutathione reductase and glutathione S-transferase was largely prevented while effect on glutathione peroxidase was much smaller. Concomitantly, oxidized glutathione (GSSG) as percentage of total glutathione increased. GSSG level was correlated positively with the levels of lipid peroxidation and solute leakage and negatively with the rate of protein synthesis. The results show that GSSG level is a good indicator of oxidation stress and provide support to the suggestion that GSSG mediates, at least in part, the drought stress-induced inhibition of protein synthesis. PMID- 16668033 TI - Auxin inhibition of flower induction of pharbitis is not mediated by ethylene. AB - Treatment of whole Pharbitis nil seedlings or cotyledons with indole butyric acid (IBA) immediately before an inductive dark period greatly inhibited flowering. Treatment of the shoot tip alone with IBA had little or no effect. 1 Aminocyclopropane 1-carboxylic acid, which increased ethylene production by the seedlings much more than IBA, had no effect on the flowering response. Pretreatment of seedlings with the ethylene biosynthesis inhibitor aminooxyacetic acid or with the inhibitor of ethylene action silver thiosulfate did not reduce the inhibitory effect of IBA on flower induction. We concluded, therefore, that the auxin-induced inhibition of flowering of P. nil was not mediated by ethylene. PMID- 16668034 TI - Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: III. Alanine Is the Product of Anaerobic Ammonium Assimilation. AB - We have determined the flow of (15)N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of (15)NH(4) (+) to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH(4) (+) accumulates in alanine. This is a unique feature of anaerobic NH(4) (+) assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH(4) (+) assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH(4) (+) assimilation. PMID- 16668035 TI - Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II. AB - We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells. PMID- 16668036 TI - Photosynthate partitioning in Basal zones of tall fescue leaf blades. AB - Elongating grass leaves have successive zones of cell division, cell elongation, and cell maturation in the basal portion of the blade and are a strong sink for photosynthate. Our objective was to determine dry matter (DM) deposition and partitioning in basal zones of elongating tall fescue (Festuca arundinacea Schreb.) leaf blades. Vegetative tall fescue plants were grown in continuous light (350 micromoles per square meter per second photosynthetic photon flux density) to obtain a constant spatial distribution of elongation growth with time. Content and net deposition rates of water-soluble carbohydrates (WSC) and DM along elongating leaf blades were determined. These data were compared with accumulation of (14)C in the basal zones following leaf-labeling with (14)CO(2). Net deposition of DM was highest in the active cell elongation zone, due mainly to deposition of WSC. The maturation zone, just distal to the elongation zone, accounted for 22% of total net deposition of DM in elongating leaves. However, the spatial profile of (14)C accumulation suggested that the elongation zone and the maturation zone were sinks of equal strength. WSC-free DM accounted for 55% of the total net DM deposition in elongating leaf blades, but only 10% of incoming (14)C-photosynthate accumulated in the water-insoluble fraction (WIF approximately WSC-free DM) after 2 hours. In the maturation zone, more WSC was used for synthesis of WSC-free DM than was imported as recent photosynthate. PMID- 16668037 TI - Anaerobic stress induces the transcription and translation of sucrose synthase in rice. AB - Sucrose synthase activity increased in 2-day-old rice (Oryza sativa) seedlings submitted to anaerobic stress. Likewise, both denaturing and native Western blot analysis detected a rise in the cellular concentration of sucrose synthase protein. Significantly higher steady-state levels of sucrose synthase mRNA, as determined by Northern blots and by the ability of total RNA to direct in vitro synthesis of sucrose synthase, were also induced by anaerobic treatment. Analysis of run-on transcripts showed increased transcription of sucrose synthase genes as early as 60 minutes after initiation of anaerobic stress. Together, these results indicate that sucrose synthase is a typical anaerobic protein in rice. PMID- 16668038 TI - Electrical Membrane Properties of Leaves, Roots, and Single Root Cap Cells of Susceptible Avena sativa: Effect of Victorin C. AB - The effect of the purified host-selective toxin victorin C, a cyclized penta peptide, was compared to that of CCCP and vanadate on membrane functions of susceptible leaves, roots, and single root cap cells of Avena sativa with conventional electrophysiology. The plasmalemma depolarized irreversibly by about 80 millivolts and to below the diffusion potential within 1 hour. Concentrations as low as 12.5 picomolar were effective in the susceptible but not the resistant cultivar. Electrical membrane potential difference changes were independent of pH and could not be prevented by fusicoccin or Ca(2+). Membranes began to depolarize after a lag phase that never was shorter than 6.5 minutes, even with concentrations as high as 1.25 micromolar. Membrane depolarization was accompanied by a distinct decrease in specific membrane resistance from 4.5 to 1.0 ohm times square meter on average. These changes were followed by K(+) and Cl(-) efflux and extracellular alkalinization. ATP level and O(2) uptake did not decrease within 2 hours. It is concluded that the victorin-induced deleterious membrane alterations are not caused by direct interaction with the plasmalemma H(+)-ATPase, K(+) channels, lipid structure, nor energy metabolism, but they seem to be triggered by a cascade of events leading to an unspecific increase in membrane permeability. PMID- 16668039 TI - Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization. AB - A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia. PMID- 16668040 TI - Expression of DNA coding for diphtheria toxin chain a is toxic to plant cells. AB - DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. The introduction of this chimeric molecule into tobacco cells by electroporation lowered the level of the transient expression of the coelectroporated chloramphenicol acetyltransferase reporter gene indicating that expression of diphtheria toxin chain A in plant cells is toxic. We have developed a binary vector pGA987 which can be used for probing a variety of plant promoters. PMID- 16668041 TI - Purification and Characterization of Phosphoglycolate Phosphatase from the Cyanobacterium Coccochloris peniocystis. AB - The properties and role of the enzyme phosphoglycolate phosphatase in the cyanobacterium Coccochloris peniocystis have been investigated. Phosphoglycolate phosphatase was purified 92-fold and had a native molecular mass of approximately 56 kilodaltons. The enzyme demonstrated a broad pH optimum of pH 5.0 to 7.5 and showed a relatively low apparent affinity for substrate (K(m) = 222 micromolar) when compared to that from higher plants. The enzyme required both an anion and divalent cation for activity. Mn(2+) and Mg(2+) were effective divalent cations while Cl(-) was the most effective anion tested. The enzyme was specific for phosphoglycolate and did not show any activity toward a variety of organic phosphate esters. Growth of the cells on high CO(2) and transfer to air did not result in any significant change in phosphoglycolate phosphatase activity. Competitive inhibition of C. peniocystis triose phosphate isomerase by phosphoglycolate was demonstrated (K(i) = 12.9 micromolar). These results indicate the presence of a specific noninducible phosphoglycolate phosphatase whose sole function may be to hydrolyze phosphoglycolate and prevent phosphoglycolate inhibition of triose phosphate isomerase. PMID- 16668042 TI - Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings. AB - Low-temperature stress was shown to cause a rapid increase in steady-state levels of alcohol dehydrogenase-1 message (Adh1) and protein activity (ADH1) in maize (Zea mays) (B37N, A188) and rice (Oryza sativa) (Taipei 309, Calmochi 101) seedlings. Maize roots and rice shoots and roots from 7-day seedlings shifted to low temperature (10 degrees C) contained as much as 15-fold more Adh1 mRNA and 8 fold more ADH1 protein activity than the corresponding tissues from untreated seedlings. Time-course studies showed that these tissues accumulated Adh1 mRNA and ADH1 activity severalfold within 4- to 8-hour, levels plateaued within 20 to 24 hours, and remained elevated at 4 days of cold treatment. Within 24 hours of returning cold-stressed seedlings to ambient temperature, Adh1 mRNA and ADH1 activity decreased to pretreatment levels. Histochemical staining of maize and rice tissue imprints showed that ADH activity was enhanced along the lengths of cold-stressed maize primary roots and rice roots, and along the stems and leaves of rice shoots. Our observations suggest that short-term cold stress induces Adh1 gene expression in certain plant tissues, which, reminiscient of the anaerobic response, may reflect a fundamental shift in energy metabolism to ensure tissue survival during the stress period. PMID- 16668043 TI - N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots. AB - Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo N'(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H(+)-ATPase in microsomal preparations; however, the plasma membrane-type H(+)-ATPase was unaffected. The H(+)-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H(+)-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H(+)-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots. PMID- 16668044 TI - Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation. AB - We report the isolation of five cDNA clones whose corresponding mRNAs accumulate in cultured soybean cells (Glycine max cv Mandarin) during cytokinin or auxin starvation. The levels of three of these mRNAs decrease rapidly after addition of 5 micromolar zeatin to cytokinin-starved cells or after addition of 10 micromolar alpha-naphthaleneacetic acid to auxin-starved cells. These mRNAs also exhibit various patterns of accumulation in the tissues of intact soybean plants. Partial nucleotide sequence analysis demonstrates that one of the cDNAs in the collection, called SAM46, is 46% identical at the amino acid level to the iron superoxide dismutase gene of Escherichia coli. Expression of this cDNA in Escherichia coli cells results in detectable iron superoxide dismutase activity, confirming the identity of the cDNA. PMID- 16668045 TI - Water Relations of Growing Maize Coleoptiles : Comparison between Mannitol and Polyethylene Glycol 6000 as External Osmotica for Adjusting Turgor Pressure. AB - Water relations of growing segments of maize (Zea mays L.) coleoptiles were investigated with osmotic methods using either mannitol (MAN) or polyethylene glycol 6000 (PEG) as external osmotica. Segments were incubated in MAN or PEG solutions at 0 to - 15 bar water potential (Psi(o)) and the effects were compared on elongation growth, osmotic shrinkage, cell sap osmolality (OC), and osmotic pressure (pi(i)). The nonpenetrating osmoticum PEG affects pi(i) in agreement with Boyle-Mariotte's law, i.e. the segments behave in principle as ideal osmometers. There is no osmotic adjustment in the Psi(o) range permitting growth (0 to -5 bar) nor in the Psi(o) range inducing osmotic shrinkage (-5 to -10 bar). Promoting growth by auxin (IAA) has no effect on the osmotic behavior of the tissue toward PEG. In contrast to PEG, MAN produces an apparent increase in pi(i) accompanied by anomalous effects on segment elongation and shrinkage leading to a lower value for Psi(o) which establishes a growth rate of zero and to an apparent recovery from osmotic shrinkage after 2 hours of incubation. These effects can be quantitatively attributed to uptake of MAN into the tissue. MAN is taken up into the apoplastic space and the symplast as revealed by a large temperature dependent component of MAN uptake. It is concluded that MAN, in contrast to PEG, is unsuitable as an extemal osmoticum for the quantitative determination of water relations of growing maize coleoptiles. PMID- 16668046 TI - Effect of pO(2) on the Formation and Status of Leghemoglobin in Nodules of Cowpea and Soybean. AB - Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO(2) (1-80%, v/v) in N(2) for up to 28 days after planting. At the extremes of sub- and supra-ambient pO(2), the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO(2). Short-term changes in pO(2) (transferring plants grown with sub- or supra-ambient pO(2) in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO(2) was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO(2) with those for their structural adaptation to an under supply of O(2) indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O(2)) compared to those grown in air. Progressive decline in pO(2) resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O(2) level. PMID- 16668047 TI - Light Energy Distribution in the Brown Alga Macrocystis pyrifera (Giant Kelp). AB - The brown alga Macrocystis pyrifera (giant kelp) was studied by a combination of fluorescence spectroscopy at 77 kelvin, room temperature modulated fluorimetry, and photoacoustic techniques to determine how light energy is partitioned between photosystems I and II in states 1 and 2. Preillumination with farred light induced the high fluorescence state (state 1) as determined by fluorescence emission spectra measured at 77K and preillumination with green light produced a low fluorescence state (state 2). Upon transition from state 1 to state 2, there was an almost parallel decrease of all of the fluorescence bands at 693, 705, and 750 nanometers and not the expected decrease of fluorescence of photosystem II and increase of fluorescence in photosystem I. The momentary level of room temperature fluorescence (fluorescence in the steady state, F(s)), as well as the fluorescence levels corresponding to all closed (F(m)) or all open (F(o)) reaction-center states were measured following the kinetics of the transition between states 1 and 2. Calculation of the distribution of light 2 (540 nanometers) between the two photosystems was done assuming both the ;separate package' and ;spill-over' models. Unlike green plants, red algae, and cyanobacteria, the changes here of the light distribution were rather small in Macrocystis so that there was approximately an even distribution of the photosystem II light at 540 nanometers to photosystem I and photosystem II in both states 1 and 2. Photoacoustic measurements confirmed the conclusions reached as a result of fluorescence measurements, i.e. an almost equal distribution of light-2 quanta to both photosystems in each state. This conclusion was reached by analyzing the enhancement phenomenon by light 2 of the energy storage measured in far red light. The effect of light 1 in decreasing the energy storage measured in light 2 is also consistent with this conclusion. The photoacoustic experiments showed that there was a significant energy storage in light 1 which could be explained by cyclic electron transport around photosystem I. From a quantitative analysis of the enhancement effect of background light 2 (maximum enhancement of 1.4-1.5) it was shown that around 70% of light 1 was distributed to this cyclic photosystem I transport. PMID- 16668048 TI - Glutamate Oxaloacetate Transaminase in Pea Root Nodules : Participation in a Malate/Aspartate Shuttle between Plant and Bacteroid. AB - Glutamate oxaloacetate transaminase (l-glutamate: oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the K(m) values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle. PMID- 16668049 TI - Ethanol-induced injuries to carrot cells : the role of acetaldehyde. AB - Carrot (Daucus carota L.) cell cultures show high sensitivity to ethanol since both unorganized cell growth and somatic embryogenesis are strongly inhibited by ethanol at relatively low concentrations (10-20 millimolar). The role of acetaldehyde on ethanol-induced injuries to suspension cultured carrot cells was evaluated. When ethanol oxidation to acetaldehyde is prevented by adding an alcohol-dehydrogenase (EC 1.1.1.1) inhibitor (4-methylpyrazole) to the culture medium, no ethanol toxicity was observed, even if ethanol was present at relatively high concentrations (40-80 millimolar). Data are also presented on the effects of exogenously added acetaldehyde on both carrot cell growth and somatic embryogenesis. We conclude that the observed toxic effects of ethanol cannot be ascribed to ethanol per se but to acetaldehyde. PMID- 16668050 TI - N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn. AB - Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation ((15)N(2) fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and (15)N(2) fixation in light. The relationship between ethylene formation and N(2)-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C(2)H(4) per nanomole (15)N(2). Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O(2) in light, while in darkness there was a broad optimum around 5 to 8% O(2). Inhibition of nitrogenase, in light, by pO(2) above 0.6% was irreversible. Measurements of light-dependent O(2) evolution and (14)CO(2) fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O(2) levels above 6% O(2) and the inhibition was reversible. These cells showed rates of light-dependent O(2) evolution and (14)CO(2) fixation which were 100- to 200 fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O(2), and a direct dependence on the cycad for substrates to support nitrogenase activity. PMID- 16668051 TI - Response to Anoxia in Rice and Wheat Seedlings: Changes in the pH of Intracellular Compartments, Glucose-6-Phosphate Level, and Metabolic Rate. AB - (31)P nuclear magnetic resonance spectroscopy was used to measure intracellular pH in living tissues. Oxygen deprivation caused fast cytoplasmic acidification from pH 7.4 to 7.0 in shoots of rice, Oryza sativa L. var arborio, a species highly resistant to anoxia. Acidification was complete after 10 minutes of anoxia. Alkalinization of both cytosplasm and vacuole followed thereafter. In the anoxia intolerant wheat shoots, Triticum aestivum L. var MEK, the same treatment caused a sharper cytoplasmic acidification, from pH 7.4 to 6.6, which occurred during a period of 2 hours. Cytoplasmic acidification continued with progress of anoxia and there was no vacuolar alkalinization comparable to the one observed in rice. In wheat oxyen, withdrawal also caused the reduction of both glucose-6 phosphate level and of metabolic rate. It also induced heavy losses of inorganic phosphate from tissues. Conversely, in rice, glucose-6-phosphate level and metabolic rate were increased and inorganic phosphate leakage from tissues was completely absent. These results are discussed in relation to the mechanisms of plant resistance to anoxia. PMID- 16668052 TI - Daily Changes in CO(2) and Water Vapor Exchange, Chlorophyll Fluorescence, and Leaf Water Relations in the Halophyte Mesembryanthemum crystallinum during the Induction of Crassulacean Acid Metabolism in Response to High NaCl Salinity. AB - Simultaneous measurements of net CO(2) exchange, water vapor exchange, and leaf water relations were performed in Mesembryanthemum crystallinum during the development of crassulacean acid metabolism (CAM) in response to high NaCl salinity in the rooting medium. Determinations of chlorophyll a fluorescence were used to estimate relative changes in electron transport rate. Alterations in leaf mass per unit area, which-on a short-term basis-largely reflect changes in water content, were recorded continuously with a beta-gauge. Turgor pressure of mesophyll cells was determined with a pressure probe. As reported previously (K Winter, DJ von Willert [1972] Z Pflanzenphysiol 67: 166-170), recently expanded leaves of plants grown under nonsaline conditions showed gas-exchange characteristics of a C(3) plant. Although these plants were not exposed to any particular stress treatment, water content and turgor pressure regularly decreased toward the end of the 12 hour light periods and recovered during the following 12 hours of darkness. When the NaCl concentration of the rooting medium was raised to 400 millimolar, in increments of 100 millimolar given at the onset of the photoperiods for 4 consecutive days, leaf water content and turgor pressure decreased by as much as 30 and 60%, respectively, during the course of the photoperiods. These transient decreases probably triggered the induction of the biochemical machinery which is required for CAM to operate. After several days at 400 millimolar NaCl, when leaves showed features typical of CAM, overall turgor pressure and leaf mass per unit area had increased above the levels before onset of the salt treatment, and diurnal alterations in leaf water content were reduced. Net carbon gain during photoperiods and average intercellular CO(2) partial pressures at which net CO(2) uptake occurred, progressively decreased upon salinization. Reversible diurnal depressions in leaf conductance and net CO(2) uptake, with minima recorded in the middle of the photoperiods, preceded the occurrence of nocturnal net CO(2) uptake. During these reductions, intercellular CO(2) partial pressure and rates of photosynthetic electron transport decreased. With advancing age, leaves of plants grown under nonsaline conditions exhibited progressively greater diurnal reductions in turgor pressure and developed a low degree of CAM activity. PMID- 16668053 TI - Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography. AB - The major storage proteins of maize (Zea mays L.) endosperm are located in protein bodies, and may be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into two major classes and four minor classes of polypeptides. The two major classes (commonly known as zeins) have been separated previously into a large number of components by isoelectric focusing (IEF). Reversed-phase high performance liquid chromatography (HPLC) further separated the major classes into additional components, and gave distinctive peaks for each minor zein class. Some IEF bands produced two or more HPLC fractions, while some HPLC fractions produced two or more IEF bands. Apparently identical IEF bands from different inbreds may appear in different fractions after HPLC. Thus the total number of zeins revealed by separations based on apparent size (SDS-PAGE), net charge (IEF), and hydrophobicity (HPLC) is very large. Different laboratories have developed diverse nomenclatures which cause much confusion. A key is presented to provide a flexible and expandable nomenclature for this complex group of proteins. PMID- 16668054 TI - Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria. AB - L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780 784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K(m) for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD(+)-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments. PMID- 16668055 TI - Maize microsomal benzoxazinone N-monooxygenase. AB - The benzoxazinones occur in hydroxamic acid and lactam forms in maize (Zea mays L.) tissue. The hydroxamic acid forms which possess a N-hydroxyl group are found in the highest concentration while the lactam members which lack the N-hydroxyl group occur in lower concentrations. The hydroxamic acid 2,4-dihydroxy-1,4 benzoxazin-3-one (DIBOA) has as its lactam counterpart 2-hydroxy-1,4-benzoxazin-3 one (HBOA). An enzyme has been identified in maize microsomal preparations which catalyzes the N-hydroxylation of HBOA to form DIBOA. The enzyme is initially observed in seedlings 2 days after imbibition which coincides with the onset of hydroxamic acid accumulation. The enzyme requires NADPH and is inhibited by sulfhydryl reagents, NADP, cytochrome c, cations, carbon monoxide, and nitrogen gas. The effect of nitrogen can be reversed by exposing the enzyme to air, while the effect of carbon monoxide can be reversed by exposing the enzyme to 450 nanometer light during the incubation period. The apparent K(m) values for HBOA and NADPH are 13 and 5 micromolar, respectively. The pH optimum is 7.5 and the temperature optimum for the enzyme is 35 degrees C. A 450 nanometer absorbance peak is observed when reduced microsomal preparations are exposed to carbon monoxide which in combination with other data presented supports the hypothesis that the enzyme is a cytochrome P-450 dependent N-monooxygenase. PMID- 16668056 TI - Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti. AB - Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3',4',5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin 7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots. PMID- 16668057 TI - Release and Modification of nod-Gene-Inducing Flavonoids from Alfalfa Seeds. AB - Traces of luteolin, an important rhizobial nod gene inducer in Rhizobium meliloti, are released by alfalfa (Medicago sativa L.) seeds, but most luteolin in the seed exudate is conjugated as luteolin-7-O-glucoside (L7G). Processes affecting the production of luteolin from L7G in seed exudate are poorly understood. Results from this study establish that (a) seed coats are the primary source of flavonoids, including L7G, in seed exudate; (b) these flavonoids exist in seeds before imbibition; and (c) both the host plant and the symbiotic R. meliloti probably can hydrolyze L7G to luteolin. Glycolytic cleavage of L7G is promoted by glucosidase activity released from sterile seeds during the first 4 hours of imbibition. Thus, L7G from imbibing alfalfa seeds may serve as a source of the nod-gene-inducing luteolin and thereby facilitate root nodulation by R. meliloti. PMID- 16668058 TI - Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness. AB - Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-protein) of Rhodospirillum rubrum and against dinitrogenase (MoFe-protein) of Azotobacter vinelandii were used. Western blots were made transparent and nitrogenase proteins were quantified spectrophotometrically. Nitrogenase activity both in vivo and in vitro decreased after about 23 hours of darkness and continued to decrease to about 25% and 16% of initial activity, respectively, after 40 hours. The amount of Fe-protein and MoFe-protein in Frankia of the same plants decreased to 60% and 35%, respectively, after 40 hours of darkness. Loss of nitrogenase activity thus appeared to be largely explained by loss of MoFe-protein. PMID- 16668059 TI - Molecular cloning and expression of abscisic Acid-responsive genes in embryos of dormant wheat seeds. AB - Hydrated dormant cereal seeds do not germinate even when environmental conditions are favorable for germination. By using cDNA cloning and differential screening, we have identified mRNAs from five gene families that are abundant in the embryos of imbibed, but developmentally arrested wheat (Triticum aestivum L.) seeds. Gene transcript levels of these mRNAs are maintained and even increase in embryos of imbibed dormant seeds for as long as the seeds remain dormant. In contrast, transcript levels decline in nondormant seeds after imbibition and disappear as germination occurs. All the identified genes are ABA responsive. Based on these data we conclude that wheat seeds in the hydrated dormant state exhibit prolonged expression of ABA-responsive genes. PMID- 16668060 TI - A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina. AB - Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M(r) cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface. PMID- 16668061 TI - Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development. AB - Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from approximately 80% "stage 1" to approximately 55% "stage 2". When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K(m) approximately 9 moles per cubic meter; V(max) approximately 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k(i) approximately 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V(max) from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2 cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure. PMID- 16668062 TI - Alanine synthesis by bundle sheath cells of maize. AB - Because in the phloem sap of maize (Zea mays L.) leaves a quarter of the total amino nitrogen can be found as alanine, the capacity of a de novo synthesis of alanine from 3-phosphoglycerate (3-PGA) was studied with isolated bundle sheath (BS) strands of maize. Inasmuch as these cells have retained their plasmodesmatic openings, it was possible to study the formation of alanine from 3-PGA when glutamate and ADP were being added. Alanine synthesis required the existence of the intact cell structure. From the formation of the intermediates, partially released to the medium, the activities of the enzymes of the reaction chain from 3-PGA to alanine could be measured in the intact cells. The results show that in the BS cells the rate of alanine production from pyruvate (0.5 micromole/minute per milligram BS chlorophyll) is more than sufficient to produce one-fourth of the assimilated nitrogen as alanine. As the activity of pyruvate kinase in intact bundle sheath cells in the light was found to be only 0.2 micromole/minute per milligram BS chlorophyll, it is concluded that in the light part of the conversion of 3-PGA to pyruvate may not occur via pyruvate kinase reaction, but via phosphoeno/pyruvate carboxylase, NADP-malate dehydrogenase, and NADP-malic enzyme in the mesophyll and BS cells. PMID- 16668063 TI - A loss in the plasma membrane ATPase activity and its recovery coincides with incipient freeze-thaw injury and postthaw recovery in onion bulb scale tissue. AB - Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase. PMID- 16668064 TI - Endoglycanase-Catalyzed Degradation of Hemicelluloses during Development of Carnation (Dianthus caryophyllus L.) Petals. AB - Large molecular-size hemicelluloses, including xyloglucan, decreased in quantity during development of carnation (Dianthus caryophyllus L. cv White Sim) petals, along with a relative increase in polymers with an average size of 10 kilodaltons. An enzyme extract from senescing petal tissue depolymerized the large molecular-size hemicelluloses in a pattern similar to that occurring in vivo during petal development. The products generated in vitro were composed of polymeric and monomeric components, the latter consisting primarily of xylose, galactose, and glucose. The 10 kilodalton hemicelluloses were resistant to in vitro enzymic hydrolysis. Glycosyl-linkage composition of the large molecular size polymers provided evidence for the presence of xyloglucan with smaller amounts of arabinoxylan and arabinan. The 10 kilodalton polymers were enriched in mannosyl and 4-linked glucosyl residues, presumably derived from glucomannan. During petal development or enzymic hydrolysis, no change was observed in the relative glycosyl-linkage composition of the large molecular-size hemicelluloses. The in vitro activity of carnation petal enzymes active toward native hemicelluloses increased threefold at the onset of senescence and declined slightly thereafter. Gel chromatography revealed 23 and 12 kilodalton proteins with hemicellulase activity. The enzymes hydrolyzed the large molecular-size hemicelluloses extensively and without formation of monomers. Endoxylanase activity was detected in the partially purified enzyme preparation. Xyloglucan was depolymerized in the absence of cellulase activity, suggesting the presence of a xyloglucan-specific glucanase. These data indicate that the hemicellulose molecular-size changes observed during development of carnation petals are due, in part, to the enzymic depolymerization of large molecular-size hemicelluloses. PMID- 16668065 TI - T-URF 13 Protein from Mitochondria of Texas Male-Sterile Maize (Zea mays L.) : Its Purification and Submitochondrial Localization, and Immunogold Labeling in Anther Tapetum during Microsporogenesis. AB - The protein T-URF13 (URF13) is specific to mitochondria of maize (Zea mays L.) with Texas (T) male-sterile cytoplasm and has been implicated in causing male sterility and susceptibility to T-cytoplasm-specific fungal diseases. T-URF13 was purified from isolated mitochondria from maize (line B73) with T cytoplasm by gel filtration and a quasi two-dimensional polyacrylamide gel electrophoresis system. Antibodies to the purified and denatured protein were produced in rabbits. Anti-T URF13 antiserum was used to show that T-URF13 is in the inner membrane of mitochondria and behaves as an integral membrane protein when mitochondria are fractionated with sodium carbonate or Triton X-114. The antiserum and protein A tagged with 20-nanometer-gold particles were used to localize T-URF13 in T mitochondria by electron microscopy of sections of isolated mitochondria from etiolated shoots and sections of roots and of tapetal cells at pre-and post degeneration stages of microsporogenesis. The microscopic study confirms that T URF13 is specifically localized in the mitochondrial membranes of all of the T mitochondria tested, notably those in the tapetum from the meiocyte stage to the late-microspore stage. No change in the amount of labeled T-URF13 protein in the mitochondria of aging tapetal cells was detected. PMID- 16668066 TI - Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. AB - The objective of the current research was to examine the response of woody plant tissues to freezing stress by using scanning electron microscopy (SEM). Nonsupercooling species red osier dogwood (Cornus stolonifera Michx.), weeping willow (Salix babylonica L.), and corkscrew willow (Salix matsudana Koidz. f. tortuosa Rehd.) survived freezing stress as low as -60 degrees C. Cell collapse of ray parenchyma cells of these species was expected but did not occur. It was concluded that ray parenchyma cells of these species do not fit into either the supercooling or extracellular freezing classifications. Tissues from flowering dogwood (Cornus florida L.), apple (Malus domestica Borkh. cv "Starking III"), red oak (Quercus rubra L.), scarlet oak (Quercus coccinea Muench.), and red ash (Fraxinus pennsylvanica Marsh) were confirmed as supercooling species, and did not survive exposures below -40 degrees C. Ray parenchyma cells of these species did not collapse in response to freezing stress, as was expected. Cell collapse along the margins of voids were observed in bark of all seven species. Voids were the result of extracellular ice crystals formed in the bark during exposure to freezing stress. Tissues prepared by freeze substitution techniques were found to be adequately preserved when compared to those prepared by conventional fixation and low temperature SEM techniques. A freezing protocol for imposing freezing stress at temperatures lower than experienced naturally in the area where the study was conducted was developed that produced responses comparable to those observed in specimens collected in the field during natural freezing events. PMID- 16668067 TI - Biochemical plant responses to ozone : I. Differential induction of polyamine and ethylene biosynthesis in tobacco. AB - Polyamine metabolism was examined in tobacco (Nicotiana tabacum L.) exposed to a single ozone treatment (5 or 7 hours) and then postcultivated in pollutant-free air. The levels of free and conjugated putrescine were rapidly increased in the ozone-tolerant cultivar Bel B and remained high for 3 days. This accumulation was preceded by a transient rise of l-arginine decar-boxylase (ADC, EC 4.1.1.19) activity. The ozone-sensitive cultivar Bel W3 showed a rapid production of ethylene and high levels of 1-aminocyclopropane-1-carboxylic acid after 1 to 2 hours of exposure. Induction of putrescine levels and ADC activity was weak in this cultivar and was observed when necrotic lesions developed. Leaf injury occurred in both lines when the molar ratio of putrescine to 1-aminocyclopropane 1-carboxylic acid or ethylene fell short of a certain threshold value. Monocaffeoyl-putrescine, an effective scavenger for oxyradicals, was detected in the apo-plastic fluid of the leaves of cv Bel B and increased upon exposure to ozone. This extracellular localization could allow scavenging of ozone-derived oxyradicals at the first site of their generation. Induction of either polyamine or ethylene pathways may represent a control mechanism for inhibition or promotion of lesion formation and thereby contribute to the disposition of plants for ozone tolerance. PMID- 16668068 TI - A Quantification of the Significance of Assimilatory Starch for Growth of Arabidopsis thaliana L. Heynh. AB - These studies use starch synthesis mutants to quantify the contribution of assimilatory starch to whole plant growth and form. Arabidopsis thaliana (L.) Heynh plants were used with null plastid phosphoglucomutase (T Caspar, SC Huber, CR Sommerville, [1986] Plant Physiol 79; 1-7) or 7% of wild-type ADP-glucose pyrophosphorylase (T-P Lin, T Caspar, CR Sommerville, J Preiss [1988] Plant Physiol 88; 1175-1179). The daily turnover of starch and the rate of biomass increase in the mutants and the wild type were investigated during growth in a 14 hour light/10 hour dark cycle in high irradiance (600 micromoles per square meter per second) and nitrogen (6 millimolar NH(4)NO(3)), in high irradiance and low nitrogen (0.1 millimolar NH(4)NO(3)) or in low irradiance (80 micromoles per square meter per second) and high nitrogen. There is some variability in the data, but the following conclusions can be drawn. Growth was slow in the absence of starch turnover. In high nitrogen conditions, about 1 mole of carbon per gram dry weight per day was incorporated additionally into structural biomass for every one mole of carbon turned over as starch per gram dry weight per day. In low nitrogen, the gain was much lower. This indicates that temporary storage of photosynthate is important for rapid growth in high nitrogen, but not in low nitrogen when carbohydrate is in excess. Starch-deficient plants showed the usual decrease of the shoot/root ratio in low nitrogen and increase of the ratio in low light. This shows that adjustment of plant form to nitrogen nutrition and irradiance is not mediated via regulation of photosynthate partitioning in the leaf. Starch deficient plants had lower shoot/root ratios than the wild type and the nitrogen concentration in their leaves was increased. It is discussed how interactions between carbohydrate allocation, respiration and growth at the organ and whole plant level generate these changes. We conclude that mutants with a decreased capacity to carry out a particular partial process provide a powerful tool to disect complex mutually interacting systems, and define and quantify causal interactions at the level of whole plant growth. PMID- 16668069 TI - Nitrogen Fixation in Peanut Nodules during Dark Periods and Detopped Conditions with Special Reference to Lipid Bodies. AB - The peanut plant (Arachis hypogaea L.), unlike other known legumes, can sustain nitrogen fixation when prolonged periods of darkness or detopping curtail the supply of photosynthate to the nodule. This ability to withstand photosynthate stress is attributed to the presence of lipid bodies in infected nodule cells. In both dark-treated and detopped plants, the lipid bodies show a gradual decrease in numbers, suggesting their utilization as a source of energy and carbon for nitrogen fixation. Lipolytic activity can be localized in the lipid bodies, and the existence of beta-oxidation pathway and, glyoxylate cycle is shown by the release of (14)CO(2) from (14)C lineoleoyl coenzyme A by the nodule homogenate. PMID- 16668070 TI - Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity. AB - Formation of the riboside-5'-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N(6)-benzyladenine (BA). Formation of N(6)-benzyladenosine-5' monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and primarily converted to BAMP and to BA 7- and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N(6)-benzyladenosine was not affected in the mutant plantlets. In vitro conversion of BA to its nucleoside-5'-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5'-monophosphate in extracts of wild-type plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRT-deficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets. PMID- 16668071 TI - Acyl chain and head group regulation of phospholipid catabolism in senescing carnation flowers. AB - Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal. PMID- 16668072 TI - Immunological comparison of the in vitro and in vivo labeled victorin binding protein from susceptible oats. AB - The fungus Cochliobolus victoriae causes victoria blight of oats and produces the host-specific toxin victorin. The reaction of oats to the fungus and its toxin is controlled by a single dominant gene whose product has been hypothesized to function as the site of action (receptor) of the toxin in susceptible oat genotypes. Previously, using a biologically active (125)I derivative of the toxin, we identified a 100 kilodalton victorin-binding protein (VBP) which binds victorin in a ligand-specific manner and binds in vivo only in susceptible oat genotypes. However, a VBP in both the susceptible and resistant oat genotypes was identified by in vitro binding experiments. One interpretation of the lack of genotype-specific binding in vitro is that the 100 kilodalton protein detected in vitro is not the same 100 kilodalton protein detected in vivo. To clarify the relationship between the 100 kilodalton protein(s) labeled in vivo and in vitro, we developed antisera to the in vitro-labeled VBP from the susceptible genotype and demonstrated that these preparations react with the in vivo-labeled VBP from the susceptible genotype. This finding coupled with previous observations strongly suggest that the VBP observed in vivo is the same protein detected in vitro. Furthermore, the results support our previous observations which suggest that the VBPs labeled in vitro in susceptible and resistant genotypes are closely related or identical. PMID- 16668073 TI - The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit. AB - When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A(+)) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening. PMID- 16668074 TI - Sequence analysis and comparison of avocado fruit and bean abscission cellulases. AB - A 1700 nucleotide cDNA clone for a bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase (endo-(1,4)-beta-d-glucanase) has been identified and sequenced. This cDNA clone contains a 1485 nucleotide open reading frame which includes coding sequences for a putative signal peptide and mature protein. The nucleotide and deduced amino acid sequences for the bean abscission cellulase are compared to the previously reported sequences of an avocado fruit ripening cellulase. Optimal alignment of these sequences shows 64% and 50% identically matched nucleotides and amino acids, respectively. Analysis of the deduced amino acid sequences for the mature bean and avocado cellulases indicates that these two proteins share similar molecular weights, position of cysteine residues, and hydropathic character, but have very different isoelectric points and glycosylation. Genomic blot data suggest that the avocado fruit cellulase belongs to a small gene family, whereas the bean abscission cellulase appears to be encoded by a single gene or a few very closely related genes. PMID- 16668075 TI - Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria. AB - Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca(2+) and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH. PMID- 16668076 TI - Ethylene production by growing and senescing pear fruit cell suspensions in response to gibberellin. AB - A pear (Pyrus communis L. cv Passe Crassane) cell suspension was used as a model system to study the influence of gibberellin on processes related to fruit ripening. Growth of the cell cultures was inhibited and their loss of viability was accelerated when 0.5 millimolar gibberllic acid (GA(3)) was added to suspensions at two stages of cell development, namely, growth and quiescence. Cell respiration rate was unaffected up to 2 millimolar GA(3) but ethylene production, both basal and 1-aminocyclopropane-1-carboxylic acid-induced, was inhibited at all stages of cell development. However, the degree of inhibition decreased as the cell cultures aged. The site of ethylene inhibition by GA(3) appeared to be related to the ethylene-forming enzyme. The coincident acceleration of cell senescence and inhibition of ethylene production indicate that the pear cell suspension cannot serve as an analogous model for studying the mode of action of gibberellin in delaying ripening and senescence of fruits in its entirety, although certain specific effects might be relevant. PMID- 16668077 TI - Regulation of Alternative Pathway Activity in Plant Mitochondria : Deviations from Q-Pool Behavior during Oxidation of NADH and Quinols. AB - External NADH and succinate were oxidized at similar rates by soybean (Glycine max) cotyledon and leaf mitochondria when the cytochrome chain was operating, but the rate of NADH oxidation via the alternative oxidase was only half that of succinate. However, measurements of the redox poise of the endogenous quinone pool and reduction of added quinones revealed that external NADH reduced them to the same, or greater, extent than did succinate. A kinetic analysis of the relationship between alternative oxidase activity and the redox state of ubiquinone indicated that the degree of ubiquinone reduction during external NADH oxidation was sufficient to fully engage the alternative oxidase. Measurements of NADH oxidation in the presence of succinate showed that the two substrates competed for cytochrome chain activity but not for alternative oxidase activity. Both reduced Q-1 and duroquinone were readily oxidized by the cytochrome oxidase pathway but only slowly by the alternative oxidase pathway in soybean mitochondria. In mitochondria isolated from the thermogenic spadix of Philodendron selloum, on the other hand, quinol oxidation via the alternative oxidase was relatively rapid; in these mitochondria, external NADH was also oxidized readily by the alternative oxidase. Antibodies raised against alternative oxidase proteins from Sauromatum guttatum cross-reacted with proteins of similar molecular size from soybean mitochondria, indicating similarities between the two alternative oxidases. However, it appears that the organization of the respiratory chain in soybean is different, and we suggest that some segregation of electron transport chain components may exist in mitochondria from nonthermogenic plant tissues. PMID- 16668078 TI - Acetaldehyde stimulation of net gluconeogenic carbon movement from applied malic Acid in tomato fruit pericarp tissue. AB - Applied acetaldehyde is known to lead to sugar accumulation in fruit including tomatoes (Lycopersicon esculentum) (O Paz, HW Janes, BA Prevost, C Frenkel [1982] J Food Sci 47: 270-274) presumably due to stimulation of gluconeogenesis. This conjecture was examined using tomato fruit pericarp discs as a test system and applied i-[U-(14)C]malic acid as the source for gluconeogenic carbon mobilization. The label from malate was recovered in respiratory CO(2), in other organic acids, in ethanol insoluble material, and an appreciable amount in the ethanol soluble sugar fraction. In Rutgers tomatoes, the label recovery in the sugar fraction and an attendant label reduction in the organic acids fraction intensified with fruit ripening. In both Rutgers and in the nonripening tomato rin, these processes were markedly stimulated by 4000 ppm acetaldehyde. The onset of label apportioning from malic acids to sugars coincided with decreased levels of fructose-2,6-biphosphate, the gluconeogenesis inhibitor. In acetaldehyde treated tissues, with enhanced label mobilization, this decline reached one-half to one third of the initial fructose-2,6-biphosphate levels. Application of 30 micromolar fructose-2,6-biphosphate or 2,5-anhydro-d-mannitol in turn led to a precipitous reduction in the label flow to sugars presumably due to inhibition of fructose-1,6-biphosphatase by the compounds. We conclude that malic and perhaps other organic acids are carbon sources for gluconeogenesis occurring normally in ripening tomatoes. The process is stimulated by acetaldehyde apparently by attenuating the fructose-2,6-biphosphate levels. The mode of the acetaldehyde regulation of fructose-2,6-biphosphate metabolism awaits clarification. PMID- 16668079 TI - Xylanase and xylosidase activities in avocado fruit. AB - The activities of xylanase and xylosidase were demonstrated in mature avocado (Persea americana Mill.) fruits from different cultivars. When monitored on the day of harvest during the season at 1-month intervals, xylanase activity decreased and xylosidase activity increased between January and February and then remained stable until May. When monitored during the ripening process (January harvest), xylanase activity was constant, and xylosidase activity reached a peak at the climax of ethylene evolution and cellulase activity. Xylanase, which originated from Trichoderma viride and was added to the medium in which avocado discs were incubated, induced ethylene evolution. PMID- 16668080 TI - Short-term leaf elongation kinetics of maize in response to salinity are independent of the root. AB - The essentiality of roots to the short-term responses of leaf elongation to salinity was tested by removing the roots of maize (Zea mays L.) from the shoots and comparing the initial short-term response of leaf elongation to that with intact plants. Eightday-old seedlings growing in solution culture were treated with 80 millimolar NaCl and their leaf elongation rate (LER) was monitored with a linear variable differential transformer connected to a computerized data aquisition system. Initially, LER of intact plants was sharply reduced by salinity, then rose rapidly to reach a new steady-state rate about 1.5 hours after salinization. The new steady-state rate of salinized intact plants was about 80% of the control rate. When the roots of nonsalinized plants were excised under the surface of the nutrient solution, excision did not disturb the steady state LER. When these shoots were salinized, they responded in a manner nearly identical to that of intact plants, indicating that roots are not essential for the modulation of short-term LER of salt-stressed plants. PMID- 16668081 TI - Transient Gene Expression in Protoplasts of Phaseolus vulgaris Isolated from a Cell Suspension Culture. AB - A cell suspension culture of Phaseolus vulgaris cv Negro jamapa was established. Protoplasts isolated from this culture are suitable for transient gene expression studies after DNA transfer by either electroporation or treatment with polyethylene glycol. The optimized conditions for both methods were established, taking into consideration expression and cell viability. We have investigated two features of posttranscriptional gene regulation. We demonstrate that inclusion of the RNA leader sequence, omega (Omega), derived from tobacco mosaic virus, and a dicot intron in the leader region together enhance gene expression in this legume species about 10-fold. PMID- 16668082 TI - Nucleotide sequence of a cDNA clone corresponding to the maize globulin-2 gene. PMID- 16668083 TI - cDNA Sequences for Transcripts of the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunit Gene Family of Mesembryanthemum crystallinum. PMID- 16668084 TI - In Vivo Modulation of T-DNA Encoded Amidohydrolase Activity in Transformed Tobacco Cells. AB - Auxin autonomous growth of most crown gall tumor cells requires the expression of two auxin biosynthesizing genes (tms 1 and tms 2) from the T-DNA of Agrobacterium tumefaciens. The potential role of the tms 2 locus to affect auxin accumulation was studied by measuring the activity of its gene product, indoleacetamide hydrolase (AH), in cloned cells of tobacco (Nicotiana tabacum) transformed by the A6 strain of Agrobacterium tumefaciens. AH activity followed a consistent pattern over a 30 day culture cycle with a peak at 10 to 14 days. This same pattern was observed in a number of independently isolated clones as well as in uncioned tumor tissue, suggesting that AH activation is a regular process in wounded, transformed cells. Transfer of unwounded tissue to fresh media resulted in a similar pattern of AH activation, but with the peak activity only about 50 percent of the cut tissues. These results show that the tms 2 encoded AH activity is modulated over the culture cycle, and that the modulation is affected by wounding and supplying fresh nutrients in the medium. AH activity correlated closely with free indoleacetic acid levels which suggests that it can be an important determinant in controlling free IAA levels in transformed cells. PMID- 16668085 TI - Acid- and Enzyme-Mediated Solubilization of Cell-Wall beta-1.3,beta-1.4-d-Glucan in Maize Coleoptiles : Implications for Auxin-Mediated Growth. AB - The release of soluble carbohydrates from isolated cell wall of maize (Zea mays L.) was investigated in the range of pH 1 to 8.5. The pH profile demonstrated two peaks, a broad peak at pH 6 due to enzymatic breakdown of beta-glucan to monosaccharides (wall autolysis) and a sharp peak at pH 2.5 due to acid-mediated, nonenzymatic liberation of macromolecular beta-glucan from the wall. The pH dependence of acid-induced growth and cell-wall extensibility of coleoptile segments closely agrees with the pH dependence of acid-mediated beta-glucan solubilization in the isolated wall. However, there is no evidence that enzymatic or nonenzymatic beta-glucan solubilization is involved in the mechanism of auxin mediated growth. PMID- 16668086 TI - Evidence for Cytokinin Involvement in Rhizobium (IC3342)-Induced Leaf Curl Syndrome of Pigeonpea (Cajanus cajan Millsp.). AB - A uniquely abnormal shoot development (shoot tip-bending, leaf curling, release from apical dominance, and stunted growth) in pigeonpea (Cajanus cajan Millsp) induced by a nodulating Rhizobium strain, IC3342, is thought to be due to a hormonal imbalance. Amaranthus betacyanin bioassay indicated that xylem exudate and leaf extracts from pigeonpea plants with Rhizobium-induced leaf curl symptoms contained high concentrations of cytokinin relative to those in normal plants. Radioimmunoassay (RIA) of samples purified with high performance liquid chromatography revealed that zeatin riboside (ZR) and dihydrozeatin riboside (DZR) concentrations in xylem sap from plants with leaf curl symptoms were 7 to 9 times higher than those in the sap from symptomless, nodulated plants. The sap from symptomless plants nodulated by a Curl(-) mutant had ZR and DZR concentrations comparable to those in the normal plant sap. RIA indicated that the respective concentrations of zeatin and N(6)-isopenteny-ladenine in culture filtrates of the curl-inducing strain IC3342 were 26 and 8 times higher than those in filtrates of a related normal nodulating strain (ANU240). Gas chromatographic-mass spectrometric analyses revealed similar differences. Gene specific hybridization and sequence comparisons failed to detect any homology of IC3342 DNA to Agrobacterium tumefaciens or Pseudomonas savastanoi genetic loci encoding enzymes involved in cytokinin biosynthesis. PMID- 16668087 TI - Sink Metabolism in Tomato Fruit : IV. Genetic and Biochemical Analysis of Sucrose Accumulation. AB - Fruit of domesticated tomato (Lycopersicon esculentum) accumulate primarily glucose and fructose, whereas some wild tomato species, including Lycopersicon chmielewskii, accumulate sucrose. Genetic analysis of progeny resulting from a cross between L. chmielewskii and L. esculentum indicated that the sucrose accumulating trait could be stably transferred and that the trait was controlled by the action of one or two recessive genes. Biochemical analysis of progeny resulting from this cross indicated that the sucrose-accumulating trait was associated with greatly reduced levels of acid invertase, but normal levels of sucrose synthase. Invertase from hexose-accumulating fruit was purified and could be resolved into three isoforms by chromatofocusing, each with isoelectric points between 5.1 and 5.5. The invertase isoforms showed identical polypeptide profiles on sodium dodecyl sulfate polyacrylamide gel electrophoresis, consisting of a primary 52 kilodalton polypeptide and two lower molecular mass polypeptides that appear to be degradation products of the 52 kilodalton polypeptide. The three invertase isoforms were indistinguishable based on pH, temperature, and substrate concentration dependence. Immunological detection of invertase indicated that the low level of invertase in sucrose-accumulating fruit was due to low levels of invertase protein rather than the presence of an invertase inhibitor. Based on comparison of genetic and biochemical data we speculate that a gene either encoding tomato fruit acid invertase or one required for its expression, plays an important role in determining sucrose accumulation. PMID- 16668088 TI - Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : II. Chlorsulfuron Resistance Involves a Wheat-Like Detoxification System. AB - Lolium rigidum Gaud. biotype SLR31 is resistant to the herbicide diclofop-methyl and cross-resistant to several sulfonylurea herbicides. Wheat and the cross resistant ryegrass exhibit similar patterns of resistance to sulfonylurea herbicides, suggesting that the mechanism of resistance may be similar. Cross resistant ryegrass is also resistant to the wheat-selective imidazolinone herbicide imazamethabenz. The cross-resistant biotype SLR31 metabolized [phenyl-U (14)C]chlorsulfuron at a faster rate than a biotype which is susceptible to both diclofop-methyl and chlorsulfuron. A third biotype which is resistant to diclofop methyl but not to chlorsulfuron metabolized chlorsulfuron at the same rate as the susceptible biotype. The increased metabolism of chlorsulfuron observed in the cross-resistant biotype is, therefore, correlated with the patterns of resistance observed in these L. rigidum biotypes. During high performance liquid chromatography analysis the major metabolite of chlorsulfuron in both susceptible and cross-resistant ryegrass coeluted with the major metabolite produced in wheat. The major product is clearly different from the major product in the tolerant dicot species, flax (Linium usitatissimum). The elution pattern of metabolites of chlorsulfuron was the same for both the susceptible and cross resistant ryegrass but the cross-resistant ryegrass metabolized chlorsulfuron more rapidly. The investigation of the dose response to sulfonylurea herbicides at the whole plant level and the study of the metabolism of chlorsulfuron provide two independent sets of data which both suggest that the resistance to chlorsulfuron in cross-resistant ryegrass biotype SLR31 involves a wheat-like detoxification system. PMID- 16668089 TI - Role of Abscisic Acid in the Induction of Freezing Tolerance in Brassica napus Suspension-Cultured Cells. AB - Brassica napus suspension-cultured cells could be hardened in 6 days at 25 degrees C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LT(50) of -17.5 degrees C or -18 degrees C, respectively, while the LT(50) for the comparable nonhardened control (sucrose) was -10 degrees C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4- to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells. PMID- 16668090 TI - Inhibition of plant glutamine synthetases by substituted phosphinothricins. AB - Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal alpha- and gamma substituted analogs of phosphinothricin (l-2-amino-4 (hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS(1) and chloroplastic GS(2) competitively versusl-glutamate, with K(i) values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme inhibitor dissociation. Herbicidal phosphinothricins exhibit low K(i) values and slow enzyme turnover, as described by reactivation characteristics. Both the GS(1) and GS(2) isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series. PMID- 16668091 TI - Effects of diclofop and diclofop-methyl on membrane potentials in roots of intact oat, maize, and pea seedlings. AB - Growth and electrophysiological studies in roots of intact diclofop-methyl susceptible and resistant seedlings were conducted to test the hypothesis that the herbicide acts primarily as a proton ionophore. The ester formulation of diclofop, at 0.2 micromolar, completely inhibited root growth in herbicide susceptible oat (Avena sativa L.) after a 96 hour treatment, but induced only a delayed transient depolarization of the membrane potential in oat root cortical cells. Root growth in susceptible maize (Zea mays L.) seedlings was dramatically reduced by exposure to 0.8 micromolar diclofop-methyl, while the same diclofop methyl exposure hyperpolarized the membrane potential within 48 hours after treatment. Furthermore, exposure of maize roots to the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 nanomolar), inhibited growth by only 31%, 96 hours after treatment, while the same CCCP exposure depolarized the resting potential by an average of 32 millivolts. Thus, the protonophore hypothesis cannot account for a differential membrane response to phytotoxic levels of diclofop-methyl in two susceptible species. From the results of others, much of the evidence to support the protonophore hypothesis was obtained using high concentrations of diclofop acid (100 micromolar). At a similar concentration, we also report a rapid (3 minute) diclofop-induced depolarization of the membrane potential in roots of susceptible oat and maize, moderately tolerant barley (Hordeum vulgare L.), and resistant pea (Pisum sativum L.) seedlings. Moreover, 100 micromolar diclofop acid inhibited growth in excised cultured pea roots. In contrast, 100 micromolar diclofop-methyl did not inhibit root growth. Since the membrane response to 100 micromolar diclofop acid does not correspond to differential herbicide sensitivity under field conditions, results obtained with very high levels of diclofop acid are probably physiologically irrelevant. The results of this study suggest that the effect of diclofop-methyl on the membrane potentials of susceptible species is probably unrelated to the primary inhibitory effect of the herbicide on plant growth. PMID- 16668092 TI - Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls. AB - Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes. PMID- 16668093 TI - Purification and immunohistochemical detection of an embryogenic cell protein in carrot. AB - An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos. PMID- 16668094 TI - Heat Production in the Voodoo Lily (Sauromatum guttatum) as Monitored by Infrared Thermography. AB - The pattern of surface temperatures of the inflorescence of Sauromatum guttatum was investigated by using an infrared camera. The male flowers are weakly thermogenic on the first day of inflorescence opening (D-day) as well as on the next day (D + 1), reaching 0.5 to 1 degrees C above ambient temperature. The appendix (the upper sterile part of the inflorescence) is highly thermogenic on D day, reaching 32 degrees C, and is faintly thermogenic on D + 1, reaching 1 degrees C above ambient temperature. The lower part of the spadix, close to the female flowers, is also thermogenic on D-day and D + 1, reaching a temperature similar to that of the appendix only on D + 1. Salicylic acid does not induce heat production in the lower part of the spadix, as it does in the appendix. Respiration of tissue slices obtained from the appendix shows that the capacity for cyanide-insensitive respiration is present in young and mature appendices. This alternative respiratory pathway is not, however, utilized in young appendix tissue, but is engaged during the maturation of that tissue. PMID- 16668095 TI - Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum. AB - The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to "bypass" the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism. PMID- 16668096 TI - Metabolically driven self-restoration of energy-linked functions by avocado mitochondria: general characteristics and phosphorylative aspects. AB - To assess the restorative capacity of isolated avocado (Persea americana) fruit mitochondria, the organelles were first aged in the absence of an energy source at 25 degrees C for several hours until respiratory control and oxidative phosphorylation were greatly diminished or totally lost. Energy-linked functions were then gradually restored over a period of several hours after the addition of substrate. Restoration of respiratory control resulted from both an increase in state 3 and a decrease in state 4 respiratory rates. Either alpha-ketoglutarate or succinate served as restorants, each with distinctive rates of recovery in state 3 and state 4 respiration. ATP also served as a restorative agent but not as effectively as metabolizable substrate. ATP synthase activity was modulated by stress and restoration but neither the extent nor the rate of change was sufficient to constrain state 3 rates. Orthophosphate was released from the mitochondria during substrate-deprived stress. Restoration of phosphorylation preceded that of RC with phosphate uptake and phosphorylation being evident immediately upon the addition of substrate. During restoration [(32)P]orthophosphate was incorporated into several organic fractions: phospholipid, ATP, a trichloroacetic acid-precipitable mitochondrial fraction, and an organophosphate that accumulated in the medium in relatively large amounts. The organophosphate was tentatively identified as a hexosephosphate. Incorporation into ATP and the putative hexosephosphate continued unabated beyond the point of maximum restoration. Phosphate metabolism thus appears to be a necessary but not sufficient precondition for mitochondrial restoration and maintenance. Based on the recovery kinetics of the various phosphorylated components, the mitochondrial-bound fraction appears to be most directly linked with restoration. Results are discussed with reference to specific characteristics and components of self-restoration and to possible underlying mechanisms. We suggest that a degree of self-restoration is consistent with the quasi-autonomous nature of mitochondria and that this intrinsic capacity may be pivotal to the respiratory climacteric in senescent fruit cells and to cellular homeostasis in general. PMID- 16668097 TI - Effect of localized nitrate application on isoflavonoid concentration and nodulation in split-root systems of wild-type and nodulation-mutant soybean plants. AB - Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypernodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized NO(3) (-) application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypernodulating mutant (NOD1 3). Nitrate application markedly decreased isoflavonoid concentrations in non inoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar NO(3) (-) compared with the other root-half receiving 0 millimolar NO(3) (-). High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar NO(3) (-) was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of NO(3) (-) on root growth. The inoculated NOD1-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of NO(3) (-). These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of NO(3) (-) on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO(3) (-) also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that internal levels of root isoflavonoids may affect nodule development. PMID- 16668098 TI - Betaine deficiency in maize : complementation tests and metabolic basis. AB - Maize (Zea mays L.) is a betaine-accumulating species, but certain maize genotypes lack betaine almost completely; a single recessive gene has been implicated as the cause of this deficiency (D Rhodes, PJ Rich [1988] Plant Physiol 88: 102-108). This study was undertaken to determine whether betaine deficiency in diverse maize germplasm is conditioned by the same genetic locus, and to define the biochemical lesion(s) involved. Complementation tests indicated that all 13 deficient genotypes tested shared a common locus. One maize population (P77) was found to be segregating for betaine deficiency, and true breeding individuals were used to produce related lines with and without betaine. Leaf tissue of both betaine-positive and betaine-deficient lines readily converted supplied betaine aldehyde to betaine, but only the betaine-containing line was able to oxidize supplied choline to betaine. This locates the lesion in betaine-deficient plants at the choline --> betaine aldehyde step of betaine synthesis. Consistent with this location, betaine-deficient plants were shown to have no detectable endogenous pool of betaine aldehyde. PMID- 16668099 TI - Dependency of Iron Reduction on Development of a Unique Root Morphology in Ficus benjamina L. AB - The activity of the Fe(3+) reductase of excised adventitious roots of Ficus benjamina L., grown in hydroponic culture without iron, was determined by a colorometric assay simplified by the use of a microplate reader. Reductase activity remained the same from pH 4.5 to 6.5 and decreased sharply above pH 6.5. Acetate buffer inhibited reduction. During early stages of root growth, excised roots did not exhibit Fe(3+) reductase activity. After several weeks and extensive root system development, Fe(3+) reduction still was not detectable in primary roots, but intermediate and high rates of reduction occurred in lateral and newly formed root clusters, respectively. Clustered roots only developed on plants grown at 0 or very low (<1 micromolar) iron. Microscopic examination revealed the root cluster to be composed of up to 30 lateral roots, usually less than 1 millimeter in diameter and 1 centimeter in length, that were completely covered with root hairs. PMID- 16668100 TI - Nonrecirculating hydroponic system suitable for uptake studies at very low nutrient concentrations. AB - We describe the mechanical, electronic, hydraulic, and structural design of a nonrecirculating hydroponic system. The system is particularly suited to studies at very low nutrient concentrations, for which on-line concentration monitoring methods either do not exist or are costly and limited to monitoring relatively few individual plants. Solutions are mixed automatically to chosen concentrations, which can be set differently for every pump fed from a master supply of deionized water and nutrient concentrates. Pumping rates can be varied over a 50-fold range, up to 400 liters per day, which suffices to maintain a number of large, post-seedling plants in rapid growth at (sub)micromolar levels of N and P. The outflow of each pump is divided among as many as 12 separate root chambers. In each chamber one may monitor uptake by individual plant roots or segments thereof, by measuring nutrient depletion in batch samples of solution. The system is constructed from nontoxic materials that do not adsorb nutrient ions; no transient shifts of nitrate and phosphate concentrations are observable at the submicromolar level. Nonrecirculation of solutions limits problems of pH shifts, microbial contamination, and cumulative imbalances in unmonitored nutrients. We note several disadvantages, principally related to high consumption of deionized water and solutes. The reciprocating pumps can be constructed inexpensively, particularly by the researcher. We also report previously unattainable control of passive temperature rise of chambers exposed to full sunlight, by use of white epoxy paint. PMID- 16668101 TI - Redox Transfer across the Inner Chloroplast Envelope Membrane. AB - In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO(2) the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10(-3) is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved. From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane. PMID- 16668102 TI - Autonomous Local Area Control over Membrane Transport in Chara Internodal Cells. AB - Internodal cells of Chara were separated with a Plexiglas divider into two segments and the vibrating probe was used to investigate the extracellular current profiles that formed along these two surfaces. Treating one segment of the Chara cell with K(+) concentrations greater than 2 millimolar caused a dramatic reduction in the extracellular current pattern in this compartment. Concentrations of 5, 10, and 20 millimolar K(+) were used to establish that a normal current profile could be maintained along the cell surface in the control compartment, whereas the extracellular current profile was strongly reduced along the entire cell surface that was located in the second, high-K(+) compartment. Simultaneous measurements of the membrane potential in the two segments of the divided Chara cell established that, in the presence of elevated K(+) concentrations, a longitudinal voltage gradient of up to 60 millivolts was maintained. Experiments in which the pH value in one compartment was either decreased (pH 6.0) or increased (pH 11) gave rise to a reduced extracellular current profile along this segment of the cell, whereas the pattern in the control segment remained unaltered. These results are discussed in terms of the cellular spatial control system that must function to regulate the regions of outward and inward current, and the concept of autonomous local area (domain) control is presented. PMID- 16668103 TI - New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin. AB - The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyruvate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, an initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute assay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [(14)C]chlorsulfuron and [(14)C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency. PMID- 16668104 TI - Interactions between Glucose and Inorganic Carbon Metabolism in Chlorella vulgaris Strain UAM 101. AB - Chlorella vulgaris strain UAM 101 has been isolated from the effluent of a sugar refinery. This alga requires glucose to achieve maximal growth rate even under light saturating conditions. The growth rate of cultures grown on light + CO(2) + glucose (3.16 per day) reaches the sum of those grown on light + CO(2) (1.95 per day) and on dark + glucose (1.20 per day). Unlike other Chlorella strains, uptake of glucose (about 2 micromoles per milligram dry weight per hour) was induced to the same extent in the light and dark and was not photosensitive. The rate of dark respiration was not affected by light and was strongly stimulated by the presence of glucose (up to about 40% in 4 hours). The rate of photosynthetic O(2) evolution was measured as a function of the CO(2) concentration. These experiments were conducted with cells which experienced different concentrations of CO(2) or glucose during growth. The maximal photosynthetic rate was inhibited severely by growing the cells in the presence of glucose. A rather small difference in the apparent photosynthetic affinity for extracellular inorganic carbon (from 10-30 micromolar) was found between cells grown under low and high CO(2). Growth with glucose induced a reduction in the apparent affinity (45 micromolar) even though cells had not been provided with CO(2). Experiments performed at different pH values indicate CO(2) as the major carbon species taken from the medium by Chlorella vulgaris UAM 101. PMID- 16668105 TI - Rapid identification of cytokinins by an immunological method. AB - A method for rapid identification of bacterial cytokinins has been developed in which cultures are fed [(3)H]adenine, the cytokinins (including (3)H-labeled cytokinins) are isolated by immunoaffinity chromatography, and analyzed by HPLC with on-line scintillation counting. Analysis of Agrobacterium tumefaciens strains showed that some produced primarily trans-zeatin, whereas others produced primarily trans-zeatin riboside. Pseudomonas syringae pv savastanoi produced mixtures of trans-zeatin, dihydrozeatin, 1''-methyl-trans-zeatin riboside, and other unknown cytokinin-like substances. Corynebacterium fascians, produced cis zeatin, isopentenyladenine and isopentenyladenosine. The technique is designed for qualitative rather than quantitative studies and allows ready identification of bacterial cytokinins. It may also have utility in the study of plant cytokinins if adequate incorporation of label into cytokinin precursor pools can be achieved. PMID- 16668106 TI - Phenotypic characterization of lettuce dwarf mutants and their response to applied gibberellins. AB - Four monogenic, recessive dwarf mutants of lettuce (Lactuca sativa L.), previously isolated from a population induced by ethyl methanesulfonate, were compared with the normal genotype (E-1) for plant height, weight, leaf area, as well as hypocotyl length and root length. These nonallelic dwarfs (dwf1, dwf2, and dwf3) exhibited reduced hypocotyl length, smaller, dark green leaves, and reduced stem length. Another mutant, dwf2, allelic with dwf2, exhibited an intermediate phenotype. Epidermal cells on hypocotyls and mature leaves were counted for both normal E-1 and dwf2 plants. The total number of epidermal cells per unit area for hypocotyls and for leaves from these plants was very similar, implying the dwarf's smaller size was due to an inhibition of cell expansion and not due to decreased cell divisions. Both dwarf and normal hypocotyls elongated normally in response to exogenous gibberellin A(3) (GA(3)). In the rosette stage, only E-1 and dwf2 responded similarly to lower concentrations of GA(3), while the other dwarfs required higher concentrations to respond. Hypocotyls of dwf2 and E 1 elongated equally with applied ent-kaurenol, ent-kaurenoic acid, GA(53) aldehyde, GA(53), GA(19), GA(20), and GA(1) indicating that the biochemical block in dwf2 occurs at a very early step in the GA-biosynthetic pathway. PMID- 16668107 TI - The endogenous gibberellins of dwarf mutants of lettuce. AB - The gibberellin (GA) content of E-1, a tall genotype of early flowering lettuce (Lactuca sativa L.), and of three selected GA-responsive dwarfs, dwf1, dwf2, and dwf2(1), has been determined using (13)C-labeled internal standards and gas chromatographymass spectrometry (GC-MS). In the shoots of the E-1 parent, GA(1), 3-epi-GA(1), GA(3), GA(5), GA(8), GA(19), GA(20), GA(29), and GA(53) were identified by full scan GC-MS and Kovats retention indices. Purification by immunoaffinity chromatography selective for 13-hydroxy GAs, was necessary for GA identification. Relative to the parent E-1, the concentrations of GA(1), GA(8), GA(20), and GA(29) in the shoots of dwf2 plants were reduced to about 10% and in shoots of dwf2(1) plants to less than 50%. In dwf1 the levels of GA(1), GA(8), and GA(29) were also reduced to less than 50% of the parent E-1, but the level of GA(20) was fivefold higher than in E-1. Plant height was correlated with the endogenous levels of GA(1) and GA(8). PMID- 16668108 TI - 1-Naphthyl Acetate-Dependent Medium Acidification by Zea mays L. Coleoptile Segments. AB - Zea mays L. cv INRA 5a coleoptile segments ecidify the incubation medium on the addition of 1-naphthyl acetate (1-NA). The buffering capacity of the bathing solution increases during 1-NA stimulated medium acidification. The solution bathing the 1-NA treated coleoptile segment was analyzed by high performance liquid chromatography. A considerable amount of acetic acid was detected in the bathing solution used to measure 1-NA-dependent medium acidification. For the first time, the data demonstrate directly the release of acetic acid from 1-NA. The extent of medium acidification was proportional to 1-NA concentration. Simultaneous measurement of medium acidification and acetate content upon addition of 1-NA showed that both processes were temporally correlated. The stoichiometry of proton equivalents to acetate ion was 0.966. Addition of 50 micromolar N,N'-dicyclohexylcarbodiimide had little effect on 1-NA-dependent medium acidification. The results indicate that 1-NA is hydrolyzed in the extracellular space of coleoptile cells. PMID- 16668109 TI - Mutants of Arabidopsis with altered regulation of starch degradation. AB - Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes alpha-amylase, beta-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation. PMID- 16668110 TI - Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts : substrate specificity, regulation, intactness, and ATP requirements. AB - Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, beta,gamma-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I(50) 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N ethylmaleimide (I(50), 50 micromolar) and the metal ion chelators 2,2'-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations. PMID- 16668111 TI - Ethylene-Mediated Regulation of Gibberellin Content and Growth in Helianthus annuus L. AB - Elongation of hypocotyls of sunflower can be promoted by gibberellins (GAs) and inhibited by ethylene. The role of these hormones in regulating elongation was investigated by measuring changes in both endogenous GAs and in the metabolism of exogenous [(3)H]- and [(2)H(2)]GA(20) in the hypocotyis of sunflower (Helianthus annuus L. cv Delgren 131) seedlings exposed to ethylene. The major biologically active GAs identified by gas chromatography-mass spectrometry were GA(1), GA(19), GA(20), and GA(44). In hypocotyls of seedlings exposed to ethylene, the concentration of GA(1), known to be directly active in regulating shoot elongation in a number of species, was reduced. Ethylene treatment reduced the metabolism of [(3)H]GA(20) and less [(2)H(2)]GA(1) was found in the hypocotyls of those seedlings exposed to the higher ethylene concentrations. However, it is not known if the effect of ethylene on GA(20) metabolism was direct or indirect. In seedlings treated with exogenous GA(1) or GA(3), the hypocotyls elongated faster than those of controls, but the GA treatment only partially overcame the inhibitory effect of ethylene on elongation. We conclude that GA content is a factor which may limit elongation in hypocotyls of sunflower, and that while exposure to ethylene results in reduced concentration of GA(1) this is not sufficient per se to account for the inhibition of elongation caused by ethylene. PMID- 16668112 TI - Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid. AB - We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [(15)N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[(15)N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[(15)N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants. PMID- 16668113 TI - Abscisic Acid accumulates at positive turgor potential in excised soybean seedling growing zones. AB - Abscisic acid (ABA) accumulated in soybean (Glycine max [L.] Merr. cv Williams) hypocotyl elongating regions when seedlings were transferred to low water potential vermiculite (Psi = -0.3 megapascals) even though positive turgor is retained in this tissue. Accumulation of ABA in growing zones could occur from de novo biosynthesis within this tissue or transport from adjacent nongrowing zones. Both growing and nongrowing hypocotyl and root tissues accumulated significant levels of ABA when excised and dehydrated to reduce turgor. Surprisingly, excised growing zones (which experienced no water loss) also accumulated ABA when incubated in darkness for 4 hours at 100% relative humidity and 29 degrees C. Induction of ABA accumulation in the excised elongating region of the hypocotyl was not caused by disruption of root pressure or wounding. While excision of hypocotyl elongating regions induced ABA accumulation, no change in either extensin or p33 mRNA levels was observed. Accumulation of extensin or p33 mRNA required more severe wounding. This suggests that ABA is not involved in the response of these genes in wounded tissue and that wound signals are not causing ABA accumulation in excised tissue. Accumulation of ABA in excised elongating regions was correlated with growth inhibition and a decline in turgor to the yield threshold (Psi;(p) = 0.37 megapascals; R Matyssek, S Maruyama, JS Boyer [1988] Plant Physiol 86: 1163-1167). Inhibiting hypocotyl growth by transferring seedlings to lower temperatures or light did not cause ABA accumulation. We conclude that induction of ABA accumulation in growing zones is more sensitive to changes in turgor than the induction which occurs in mature tissues. PMID- 16668114 TI - Soybean leaves contain multiple lipoxygenases. AB - Chromatofocusing of soybean (Glycine max L.) leaf lipoxygenases revealed three distinct peaks of activity. Based on their isoelectric points (pls), pH optima, and mutant analysis it appears that the leaf isozymes are different from those described from mature soybean seed. At least one leaf lipoxygenase appears to differ from those found in hypocotyls. The pls of the main bands of the three leaf lipoxygenase peaks are 6.67, 5.91, and 5.67. The pH optima curves of three active fractions exhibit peaks at pH 6.2, 5.5, and 8.5, respectively. One of the fractions has two polypeptides with slightly different molecular weights, both of which react to soybean seed lipoxygenase antibodies. The other two fractions contain a polypeptide of unit molecular weight reacting with the lipoxygenase antibodies. PMID- 16668115 TI - In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots. AB - Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with gamma-[(32)P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg(2+) was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg(2+) concentrations and was greatly enhanced by Ca(2+). When roots of intact plants were labeled with [(32)P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect. PMID- 16668116 TI - Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis. AB - Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H(+) movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg(2+) causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg(2+) was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg(2+) and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6 dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H(+) fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg(2+) modulated) H(+) flux linked to monovalent cation antiport, and ATPase-dependent H(+) efflux. PMID- 16668117 TI - Effect of Diethylpyrocarbonate on the Allosteric Properties of Phosphoenolpyruvate Carboxylase from Crassula argentea. AB - Phosphoenolpyruvate carboxylase from the Crassulacean acid metabolism plant Crassula argentea was substantially desensitized to the effects of regulatory ligands by treatment with diethylpyrocarbonate, a reagent which selectively modifies histidyl residues. Desensitization of the enzyme to the inhibitor malate and the activator glucose 6-phosphate was accompanied by the appearance of a peak in the ultraviolet difference spectrum at 240 nanometers, indicating the formation of ethoxyformylhistidyl derivatives. Hydroxylamine reversed part of the spectral change under native conditions, and almost all of the change under denaturing conditions, but failed to restore sensitivity to effectors. The pH profiles of desensitization to malate and glucose 6-phosphate indicated the involvement of groups on the enzyme with pK, values of 6.8 and 6.4, respectively. Under denaturing conditions, a total of 15 histidine residues per subunit were modified by diethylpyrocarbonate, whereas for the native enzyme nine histidines were modified per subunit. Effector desensitization occurs after the modification of two to three histidyl residues per subunit. The presence of malate reduced the apparent rate constant for desensitization by 60%, suggesting that the modification occurred at the malate binding site. Diethylpyrocarbonate treatment also eliminated the kinetic lag caused by malate. Glucose 6-phosphate did not protect the enzyme against diethylpyrocarbonate-induced desensitization. PMID- 16668118 TI - Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje. AB - To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1 phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2 degrees C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4 degrees C. During storage at 8 degrees C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6 phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8 degrees C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8 degrees C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4 degrees C but remained the same throughout storage at 8 degrees C. After exposure to 2 degrees C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4 degrees C but decreased slightly at 8 degrees C. All observed changes became annulled after transfer of cold stored tubers to 18 degrees C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation. PMID- 16668119 TI - Primary structure of sweet potato starch phosphorylase deduced from its cDNA sequence. AB - Sweet potato (Ipomoea batatas) starch phosphorylase cDNA clones were isolated by screening an expression library prepared from the young root poly(A)(+) RNA successively with an antiserum, a monoclonal antibody, and a specific oligonucleotide probe. One cDNA clone had 3292 nucleotide residues in which was contained an open reading frame coding for 955 amino acids. This sequence was compared with those of potato (916 residues plus 50-residue putative transit peptide) and rabbit muscle (841 residues) phosphorylases. The sweet potato phosphorylase has an overall structural feature highly homologous to that reported for potato phosphorylase, in conformity with the finding that they belong to the same class of plant phosphorylase. High divergencies of the two enzymes are found in the about 70 residue N-termini each including a putative transit peptide, and the midchain 78 residue insert typical of type I plant phosphorylase. We consider that the very high dissimilarity found in the midchain inserts is related to the difference in proteolytic lability of the two plant phosphorylases. Some structural features of the cDNA clone were also discussed. PMID- 16668120 TI - Partial Purification and Some Properties of Flavonol 7-Sulfotransferase from Flaveria bidentis. AB - A novel flavonol-specific sulfotransferase was partially purified from the shoot tips of Flaveria bidentis var. Angustifolia O.K. (Asteraceae) by chromatography on 3'-phosphoadenosine 5'-phosphate-agarose affinity column and chromatofocusing on Mono P. The latter step resulted in the separation of two isoforms, both of which exhibited expressed specificity for position 7 of quercetin 3,3'- and quercetin 3,4'-disulfate. The 7-sulfotransferase isoforms I and II had a pH optimum of 7.5 in phosphate buffer, apparent pl values of 6.5 and 6.3, and an M(r) of 35,000. They had no requirement for divalent cations and were not inhibited by EDTA or SH group reagents. Their K(m) values for both the sulfate donor and flavonol acceptor were of the same order of magnitude (0.20-0.46 micromolar). This enzyme, together with the recently reported flavonol 3-, 3'-, and 4'-sulfotransferases from F. chloraefolia (L Varin, RK Ibrahim [1989] Plant Physiol 90: 977-981) form the complement involved in the biosynthesis of polysulfated flavonols in this genus. A proposed sequential order for the enzymatic sulfation in both species is described. PMID- 16668121 TI - Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration. AB - A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis. PMID- 16668122 TI - Enhanced Net K Uptake Capacity of NaCl-Adapted Cells. AB - Maintenance of intracellular K(+) concentrations that are not growth-limiting, in an environment of high Na(+), is characteristic of NaCl-adapted cells of the glycophyte, tobacco (Nicotiana tabacum/gossii). These cells exhibited a substantially greater uptake of (86)Rb(+) (i.e. an indicator of K(+)) relative to unadapted cells. Potassium uptake into NaCl-adapted cells was 1.5-fold greater than unadapted cells at 0 NaCl and 3.5-fold greater when cells were exposed to 160 millimolar NaCl. The difference in net K(+) uptake between unadapted and NaCl adapted cells was due primarily to higher rates of entry rather than to reduced K(+) leakage. Presumably, enhanced K(+) uptake into adapted cells is a result of electrophoretic flux, and a component of uptake may be linked to vanadate sensitive H(+) extrusion. PMID- 16668123 TI - On the Role of Mitochondrial Oxidative Phosphorylation in Photosynthesis Metabolism as Studied by the Effect of Oligomycin on Photosynthesis in Protoplasts and Leaves of Barley (Hordeum vulgare). AB - Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6 phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport. PMID- 16668124 TI - Effect of abscisic Acid on the linoleic Acid metabolism in developing maize embryos. AB - Partially purified protein extracts from maize (Zea mays L.) embryos, whether treated or not with abscisic acid (ABA), were incubated with linoleic acid (LA) and 1-[(14)C]LA. The resulting LA metabolites were monitored by high performance liquid chromatography with a radioactivity detector and identified by gas chromatography-mass spectrometry. alpha- and gamma-ketol metabolites arising from 9-lipoxygenase activity were the more abundant compounds detected in the incubates, although the corresponding metabolites produced by 13-lipoxygenase were also present in the samples. In addition, a group of stereoisomers originating from two isomeric trihydroxy acids (9, 12, 13-trihydroxy-10 octadecenoic and 9, 10, 13-trihydroxy-11-octadecenoic acids) are described. Important variations in the relative proportions of the LA metabolites were observed depending on the embryo developmental stage and on ABA treatment. Two new ABA-induced compounds have been detected. These compounds are present in embryos at all developmental stages, being more abundant in old (60 days) embryos. Furthermore, ABA induction of these compounds is maximum at very young developmental stages, decreasing as maturation progresses. A tentative structure for these compounds (10-oxo-9, 13-dihydroxy-11-octadecenoic acid and 12-oxo-9, 13 dihydroxy-10-octadecenoic acid) is also provided. This study revealed an early stage in maize embryogenesis characterized by a higher relative sensitivity to ABA. The physiological importance of ABA on LA metabolism is discussed. PMID- 16668125 TI - Proline is not the primary determinant of chilling tolerance induced by mannitol or abscisic Acid in regenerable maize callus cultures. AB - Chilling sensitive regenerable maize (Zea mays L.) callus cultures can be induced to survive prolonged exposure to 4 degrees C by treatments with mannitol, abscisic acid (ABA), and/or high levels of proline. Maize callus with a free proline content of about 122 micromoles/grain fresh weight survived longer exposures to 4 degrees C than did callus with a free proline content of about 68 micromoles/grain fresh weight. The addition of 0.53 molar mannitol or 0.1 millimolar ABA to culture medium produced a free proline content in maize callus of about 136 and 145 micromoles/grain fresh weight, respectively, if the medium contained 12 millimolar proline or about 36 and 1 micromoles/grain fresh weight, respectively, if no proline was in the medium. Although these mannitol and ABA treatments produced drastically different free proline levels in maize callus, callus grown on these media survived longer exposures to 4 degrees C than did maize callus grown on any proline treatment alone. Thus, the internal free proline level of treated callus is not the primary factor conferring chilling tolerance on these tissues. PMID- 16668126 TI - A Polypeptide That Induces Flowering in Lemna paucicostata at a Very Low Concentration. AB - A flower-inducing substance of high molecular mass, extracted from Lemna paucicostata, was purified to homogeneity. It had characteristics of a polypeptide, with an amino-terminal sequence of Leu-Val-Gly-Asn-Thr, and induced formation of flower buds of L. paucicostata 151 at a concentration of 10(-10) molar. PMID- 16668127 TI - Abscisic Acid in Developing Zygotic Embryos of Theobroma cacao. AB - Abscisic acid (ABA) levels were measured by enzyme-linked immunosorbent assay in developing zygotic embryos of Theobroma cacao. ABA was detected in all embryos tested, with a peak of ABA at levels of 1 to 3 micrograms per gram fresh weight during early maturation. This corresponded to embryos of 10 to 30% dry weight and to early stages of anthocyanin and lipid accumulation. PMID- 16668128 TI - Translational modification of an 18 kilodalton polypeptide by spermidine in rice cell suspension cultures. AB - When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-(3)H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis. PMID- 16668129 TI - Alkaline phytase activity in nonionic detergent extracts of legume seeds. AB - Alkaline phytase activity, with a pH optimum of 8, was recovered from detergent extracts of dormant seeds of nine varieties of Phaseolus vulgaris L., Pisum sativum L. var. Early Alaska, and Medicago sativa L. This alkaline phytase of legume seeds was activated by calcium and differed from most seed phytases in its relative insensitivity to inhibition by fluoride. PMID- 16668130 TI - Isoelectric Focusing of Plant Plasma Membrane Proteins : Further Evidence that a 55 Kilodalton Polypeptide Is Associated with beta-1,3-Glucan Synthase Activity from Pea. AB - Detergent-solubilized plasma membrane proteins from pea (Pisum sativum L.) stem tissue were separated by isoelectric focusing (IEF) using a Bio-Rad Rotofor cell, with the goal of identifying protein(s) involved in beta-1,3-glucan synthase (GS II) activity. Ordinary IEF procedures result in membrane protein precipitation. Inclusion of 10% glycerol mitigates this problem in digitonin-solubilized preparations, but not in those solubilized in 3-[( cholamidopropyl)dimethylammonio]-1-propanesulfonate. Loss of GS-II activity during IEF is minimized by improved cooling of the Rotofor cell. GS-II focuses at pH 5.1. Antiserum against a 55 kilodalton (kD) polypeptide that was recognized from other evidence as involved in GS-II activity, detects this polypeptide in exact correspondence with the GS-II activity peak. A presumptive P-type ATPase, detected using an antibody against corn root plasma membrane 97 kD ATPase, focuses at pH 5.3. In this digitonin/glycerol medium, most of the membrane proteins focus within the relatively narrow pH range of 4.5 to 6, compared to pH 5.5 to 8.5 for IEF in the presence of 9 molar urea, 2% Nonidet P-40 (NP-40), and 5% mercaptoethanol, a medium that inactivates GS-II. This latter medium increases the apparent isoelectric point (pl) values of the abovementioned 55 and 97 kD polypeptides to 5.8 and 7.3, respectively. In the digitonin/glycerol medium, membrane polypeptides apparently focus at pH values lower than their true pls, because of adhering negatively charged phospholipids, which can be at least partially removed by the detergent NP-40 in the presence of urea. These results provide independent evidence that the 55 kD polypeptide is associated with the GS II activity and indicate that inclusion of urea and a strong nonionic detergent such as NP-40 is necessary if membrane proteins are to be focused at pH values near their true pls. PMID- 16668131 TI - Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants. AB - Control of C(4) photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C(4) and CAM plants is both metabolite-related and posttranslational. Specificially, the regulatory properties of the enzyme vary in accord with the physiological activity of C(4) photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by l-malate under light (C(4) plants) or at night (CAM plants) than in darkness (C(4)) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currently in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C(4) plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C(4) and CAM plants, respectively. PMID- 16668132 TI - Triacontanol and Its Second Messenger 9-beta-l(+)-Adenosine as Plant Growth Substances. AB - Triacontanol (TRIA), a common constituent of plant waxes, was first shown in 1977 to be an active growth substance which at nanomolar concentrations increased the growth and yield of crops. TRIA is used to increase crop yields on millions of hectares, particularly in Asia. Many investigators have shown that it affects several basic metabolic processes including photosynthesis, nutrient uptake, and enzyme activity. However, the initial site of action has not been elucidated. TRIA rapidly elicits a second messenger (TRIM) in rice (Oryza sativa L.), which at nanomolar concentrations causes plants to respond in a manner similar to TRIA. TRIM has been identified as 9-beta-l(+)-adenosine (9H-purin-6-amine, 9-beta-l ribofuranosyl). During the process of isolating and identifying 9-beta-l(+) adenosine, it was shown that this enantiomer, which previously has not been reported as occurring in nature, made up about 1% of the total adenosine pool in roots from untreated rice seedlings. PMID- 16668133 TI - Carrier-Mediated Uptake of Abscisic Acid by Suspension-Cultured Amaranthus tricolor Cells. AB - Abscisic acid (ABA) uptake by Amaranthus tricolor cell suspensions was found to include both a nonsaturable component and a saturable part with K(m) of 3.74 +/- 0.43 micromolar and an apparent V(max) of 1.5 +/- 0.12 nanomoles per gram per minute. These kinetic parameters as well as the uptake by intact cells at 0 degrees C or by frozen and thawed cells, are consistent with operation of a saturable carrier. This carrier-mediated ABA uptake was partially energized by DeltapH: it increased as the external pH was lowered to pH 4.0; it decreased after the lowering of the DeltapH by the proton ionophore carbonylcyanide-m chlorophenylhydrazone or after the altering of metabolically maintained pH gradient by metabolic inhibitors (KCN, oligomycin). The carrier is specific for ABA among the plant growth regulators tested, is unaffected by (RS)-trans-ABA and was inhibited by (S)-ABA, (R)-ABA, and also by the ABA analog LAB 173711. PMID- 16668134 TI - Diurnal Fluctuations in the Content and Functional Properties of the Light Harvesting Chlorophyll a/b Complex in Thylakoid Membranes : Correlation with the Diurnal Rhythm of the mRNA Level. AB - Diurnal fluctuations were observed in the content and some structural and functional properties of the light-harvesting chlorophyll (Chl) a/b pigment protein complex of photosystem II (LHCII) in young developing wheat (Triticum aestivum) leaves grown under 16 hours light/8 hours dark illumination regime. The fluctuations could be correlated with the diurnal oscillation in the level of mRNA for LHCII. The most pronounced changes occurred in the basal segments of the leaves. They were weaker or hardly discernible in the middle and tip segments. As judged from the diurnal variations of the Chl-a/Chl-b molar ratio, the LHCII content of the thylakoid membranes peaked around 2 pm. This can be accounted for by the cumulative effect of the elevated level of mRNA in the morning and early afternoon. In the basal segment, the extent of the fluctuation in the LHCII content was approximately 25%, as determined from gel electrophoresis ("green gels"). The amplitude of the principal bands of the circular dichroism (CD) spectra of isolated chloroplasts paralleled the changes in the LHCII content. Our circular dichroism data suggest that the newly synthesized LHCII complexes are incorporated into the existing helically organized macrodomains of the pigment protein complexes or themselves form such macrodomains in the thylakoid membranes. Chl-a fluorescence induction kinetics also showed diurnal variations especially in the basal segments of the leaves. This most likely indicates fluctuations in the ability of membranes to undergo "state transitions." These observations suggest a physiological role of diurnal rhythm of mRNA for LHCII in young developing leaves. PMID- 16668135 TI - Redox-modulation of chloroplast enzymes : a common principle for individual control. AB - Assimilation of C, N, and S into organic compounds requires effective and flexible cooperation among the energy-converting, tightly coupled, thylakoid bound processes and stromal metabolism. Fluctuations of light, temperature, and changing concentrations of the various reducible substrates pose unique regulatory problems to photoautotrophic plant cells. Covalent redox modification of enzyme proteins as mediated by the ferredoxin/thiore-doxin-system is suited to provide short-term adaptation of various enzymatic activities in the chloroplast. This mode of regulation is based on the continuous turnover of interconvertible enzyme forms, as in the systems driven by protein phosphorylation/dephosphorylation, but is particularly adapted to the unique conditions of a compartment performing oxygenic photosynthesis by depending on the simultaneous presence of reducing power and of oxygen. Individual fine control of each of the enzymes subjected to redox modification is achieved by specific metabolites acting as additional positive or negative effectors of the reductive (and/or oxidative) modification reaction. The biochemical prerequisite for such a control is the presence of regulatory (extra) sequences carrying cysteine residues which are subjected to reversible redox changes. Although no common amino acid sequence has yet been identified among the known regulatory peptides, in all cases the evolution of autotrophy should be related to the presence of extrasequences in otherwise very conserved enzyme molecules. PMID- 16668136 TI - Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench. AB - The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P 450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration. PMID- 16668137 TI - Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity. AB - We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or "wild-type" velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K(m) values for glutathione, atrazine, or 1 chloro-2,4-dinitrobenzene. However, the V(max) for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V(max) for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation. PMID- 16668138 TI - In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina. AB - Na(+)/H(+) exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na(+) were followed. Following a mild intracellular acidification, intracellular Na(+) content increased dramatically and then decreased. We interpret the phase of Na(+) influx as due to the activation of the plasma membrane Na(+)/H(+) antiporter and the phase of Na(+) efflux as due to an active Na(+) extrusion process. The following observations are in agreement with this interpretation: (a) the Na(+) influx phase was sensitive to Li(+), which is an inhibitor of the Na(+)/H(+) antiporter, did not require energy, and was insensitive to vanadate; (b) the Na(+) efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na(+) concentration is due to a combination of Na(+) uptake via the Na(+)/H(+) antiporter and an active, ATPase dependent, Na(+) extrusion. The Na(+)/H(+) antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella. PMID- 16668139 TI - Antitranspirant-induced increases in leaf water potential increase tuber calcium and decrease tuber necrosis in water-stressed potato plants. AB - Experiments were undertaken with field-grown potato (Solanum tuberosum L.) plants to test the hypothesis that altering leaf:tuber water potential gradients within a plant subjected to low soil moisture will allow greater Ca accumulation in tubers and reverse Ca deficiency-related tuber necrosis. Antitranspirant formulations containing a wax emulsion and a spreader/sticker surfactant increased leaf water potential during a drought episode, significantly reducing the potential gradient that develops between leaf and tuber during a period of stress. Increased leaf water potential in treated plants was associated with decreased leaf Ca and increased tuber Ca. Tuber necrosis was found to be reduced in treated plants, thus increasing tuber quality. PMID- 16668140 TI - Physical, Chemical, Developmental, and Genetic Factors that Modulate the Agrobacterium-Vitis Interaction. AB - Tumor formation in Vitis species and hybrids, incited by Agrobacterium tumefaciens, was altered by chemical, physical, developmental, and genetic variables. Knowledge of the effect of these variables was used to develop a stringent in vitro assay system to select parents for a study of genetic factors that modulate tumor formation. Tumor formation was reduced by short day preconditioning of assay plants and by inoculation of the morphological apex of isolated stem segments. Pretreatment of plants with auxin or cytokinin altered specificity in various combinations of strains and host genotypes. All Vitis species and hybrids formed tumors in response to strains designated as limited host range, but some displayed a necrotic reaction (cell death at and below site of inoculation) or a null response (same as the response to inoculation with an avirulent strain) to strains designated as wide host range (VC Knauf, CG Panagopoulos, EW Nester [1982] Phytopathology 72: 1545-1549). Screens of F(1) progeny, derived from crosses of null, necrotic, and tumor-producing phenotypes, demonstrated that the null and the necrotic phenotypes were modulated by dominant and recessive host genes. The extent of cellular necrosis in the necrotic phenotype was modified by the morphological location of the inoculation site, by the presence of buds on the host stem, and by deletion of the tryptophane monooxygenase locus gene of the Ti-plasmid. PMID- 16668141 TI - Nitrogen and methyl jasmonate induction of soybean vegetative storage protein genes. AB - Vegetative storage protein (VSP) and VSP mRNA levels in soybean (Glycine max) leaves correlated with the amount of NH(4)NO(3) provided to nonnodulated plants. The mRNA level declined as leaves matured, but high levels of N delayed the decline. This is consistent with the proposed role for VSP in the temporary storage of N. Wounding, petiole girdling, and treatment with methyljasmonate (MeJA) increased VSP mRNA in leaves 24 hours after treatment. The magnitude of the response depended on leaf age and N availability. N deficiency essentially eliminated the response to wounding and petiole girdling. MeJA was almost as effective in N-deficient plants as in those receiving abundant N. Inhibitors of lipoxygenase, the first enzyme in the jasmonic acid biosynthetic pathway, blocked induction by wounding and petiole girdling but not by MeJA. This supports a role for endogenous leaf jasmonic acid (or MeJA) in the regulation of VSP gene expression. PMID- 16668142 TI - Measurement of legume nodule respiration and o(2) permeability by noninvasive spectrophotometry of leghemoglobin. AB - Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O(2) permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O(2) consumption) rate and internal O(2) concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N(2). The rate of increase of FOL under 100% O(2) was then used to calculate nodule O(2) permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O(2) for 15 minutes led to decreases in both permeability and O(2)-saturated respiration (V(max)), but the brief (<15 seconds) exposures to 100% O(2) required by the assay itself had little effect on either parameter. A gradual increase in external O(2) concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V(max). The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs. PMID- 16668143 TI - Biosynthesis and desaturation of prokaryotic galactolipids in leaves and isolated chloroplasts from spinach. AB - Mono- and digalactosyldiacylglycerol (MGDG and DGDG) were isolated from the leaves of sixteen 16:3 plants. In all of these plant species, the sn-2 position of MGDG was more enriched in C(16) fatty acids than sn-2 of DGDG. The molar ratios of prokaryotic MGDG to prokaryotic DGDG ranged from 4 to 10. This suggests that 16:3 plants synthesize more prokaryotic MGDG than prokaryotic DGDG. In the 16:3 plant Spinacia oleracea L. (spinach), the formation of prokaryotic galactolipids was studied both in vivo and in vitro. In intact spinach leaves as well as in chloroplasts isolated from these leaves, radioactivity from [1 (14)C]acetate accumulated 10 times faster in MGDG than in DGDG. After 2 hours of incorporation, most labeled galactolipids from leaves and all labeled galactolipids from isolated chloroplasts were in the prokaryotic configuration. Both in vivo and in vitro, the desaturation of labeled palmitate and oleate to trienoic fatty acids was higher in MGDG than in DGDG. In leaves, palmitate at the sn-2 position was desaturated in MGDG but not in DGDG. In isolated chloroplasts, palmitate at sn-2 similarly was desaturated only in MGDG, but palmitate and oleate at the sn-1 position were desaturated in MGDG as well as in DGDG. Apparently, palmitate desaturase reacts with sn-1 palmitate in either galactolipid, but does not react with the sn-2 fatty acid of DGDG. These results demonstrate that isolated spinach chloroplasts can synthesize and desaturate prokaryotic MGDG and DGDG. The finally accumulating molecular species, MGDG(18:3/16:3) and DGDG(18:3/16:0), are made by the chloroplasts in proportions similar to those found in leaves. PMID- 16668144 TI - Cytochemical Localization of ATPase Activity in Salt-Treated and Salt-Free Grown Lycopersicon esculentum Roots. AB - Adenosine-triphosphatase activity was localized by cytochemical methods in Lycopersicon esculentum Mill seedling roots. The identity of the enzyme was confirmed by its sensitivity to specific inhibitors. A differential distribution of ATPase activity was found depending on the region of the root. Under saline conditions, an increase of the tonoplast ATPase activity is observed, while the plasma membrane bound-ATPase activity decreases in the medial and basal regions of the root. PMID- 16668146 TI - Evidence for Hygrometric Pressurization in the Internal Gas Space of Spartina alterniflora. AB - Higher pressure, up to several hundred pascal relative to ambient, is generated by hygrometric pressurization within the central hollow space of the stem in Spartina alterniflora. Dilution of oxygen and nitrogen by water vapor within the plant's internal gas space results in an influx of nitrogen and oxygen from the air and a net increase in the internal gas pressure at steady state. The nature of the pressure gradient suggests that small pores exist in the plant tissues. Moreover, the compact arrangement of leaf mesophyll cells creates a high resistance for the mass flow of gases and contributes to the higher pressure within leaves. After experimentally venting the internal pressure, outside air diffused through the basal area of the adaxial side of the leaves into the internal space and elevated pressure was restored. PMID- 16668145 TI - Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.). AB - The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening." PMID- 16668147 TI - Analysis of changes in minimal and maximal fluorescence yields with irradiance and o(2) level in tobacco leaf tissue. AB - The responses of minimal and maximal fluorescence yields of chlorophyll a to irradiance of actinic white light were determined by pulse modulated fluorimetry in leaf discs from tobacco, Nicotiana tabacum, at 1.6, 20.5, and 42.0% (v/v) O(2). Steady-state maximal fluorescence yield (F(m)', measured during a saturating light pulse) declined with increasing irradiance at all O(2) levels. In contrast, the steady-state minimal fluorescence yield (F(o)', measured during a brief dark interval) increased with irradiance relative to that recorded for the fully dark-adapted leaf (F(o)) or that observed after 5 minutes of darkness (F(o) (*)). The relative magnitude of this increase was somewhat greater and extended to higher irradiances at the elevated O(2) levels compared with 1.6% O(2). Suppression of F(o)' was only observed consistently at saturating irradiance. The results are interpreted in terms of the occurrence of photosystem II units possessing exceedingly slow turnover times (i.e. "inactive" units). Inactive units play an important role, along with thermal deactivation of excited chlorophyll, in determining the response of in vivo fluorescence yield to changes in irradiance. Also, a significant interactive effect of O(2) concentration and the presence or absence of far red light on oxidation of photosystem II acceptors in the dark was noted. PMID- 16668148 TI - Role of Ethylene on de Novo Shoot Regeneration from Cotyledonary Explants of Brassica campestris ssp. pekinensis (Lour) Olsson in Vitro. AB - The promotive effect of AgNO(3) and aminoethoxyvinylglycine (AVG) on in vitro shoot regeneration from cotyledons of Brassica campestris ssp. pekinensis in relation to endogenous 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, ACC, and ethylene production was investigated. AgNO(3) enhanced ACC synthase activity and ACC accumulation, which reached a maximum after 3 to 7 days of culture. ACC accumulation was concomitant with increased emanation of ethylene which peaked after 14 days. In contrast, AVG was inhibitory to endogenous ACC synthase activity and reduced ACC and ethylene production. The promotive effect of AVG on shoot regeneration was reversed by 2-chloroethylphosphonic acid at 50 micromolar or higher concentrations, whereas explants grown on AgNO(3) medium were less affected by 2-chloroethylphosphonic acid. The distinctive effect of AgNO(3) and AVG on endogenous ACC synthase, ACC, and ethylene production and its possible mechanisms are discussed. PMID- 16668149 TI - The signal Peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein. AB - A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins. PMID- 16668150 TI - Auxin transport in suspension-cultured soybean root cells : I. Characterization. AB - The kinetic parameters of auxin transport in suspension-cultured soybean (Glycine max [L.] Merr.) root cells were investigated. The same processes that are responsible for polar indoleacetic acid (IAA) transport in other plant tissues were found to occur in soybean root cells. These include (a) passive diffusion of the undissociated auxin molecule across the plasma membrane, (b) uptake via a specific, saturable carrier, and (c) phytotropin-sensitive efflux. Metabolism of exogenously added IAA was rapid; at the end of a 15-minute uptake period >80% of the IAA taken up had been converted to other compounds. The time course of [(14)C]IAA uptake in the first 90 seconds revealed two phases, the first corresponding to a rate of uptake approximately twice as large as the second phase. The transition to the second phase was delayed in the presence of the phytotropins triiodobenzoic acid or naphthylphthalamic acid, suggesting that an increase in the efflux of label as IAA accumulates in the cytoplasm is responsible for the transition. Carrier-mediated uptake contributes between 50 and 60% to the total rate of auxin uptake from a 0.28 micromolar IAA solution, with passive diffusion accounting for the remainder. Kinetic analysis of carrier mediated uptake revealed a pH optimum of 5.0 and a Michaelis-Menten constant of 0.4 micromolar at pH 5.5. Because phytotropins had no effect on the initial rate of uptake, the efflux carrier does not appear to be involved in the uptake process. PMID- 16668151 TI - Auxin Transport in Suspension-Cultured Soybean Root Cells : II. Anion Effects on Carrier-Mediated Uptake. AB - To test the hypothesis that the carrier-mediated component of the indoleacetic acid (IAA) influx involves an electrogenic proton/IAA anion symport, the effects on the IAA influx of salts expected to depolarize the membrane potential were examined in suspension-cultured soybean (Glycine max [L.] Merr.) root cells. Although KCl does inhibit carrier-mediated uptake, the effect is specific to the anion at low concentrations and not due to more general processes such as changes in ionic or osmotic strength. Other anions such as bromide, iodide, and fluoride inhibit the carrier more strongly. Because potassium iminodiacetate, which is also expected to depolarize the membrane potential, has no inhibitory effect on the IAA influx, there is no evidence for the involvement of the membrane potential in carrier-mediated uptake. It is therefore most likely that in soybean cells, if carrier-mediated uptake occurs via a proton symport, the H(+):IAA- stoichiometry is 1:1. At concentrations greater than 70 millimolar, sorbitol, a nonionic osmoticum, inhibits carrier-mediated IAA uptake. The effects of specific anions and osmotic potential on the uptake carrier necessitates the reevaluation of other auxin transport studies in which KCl was routinely used as an agent with which to depolarize the membrane potential. PMID- 16668152 TI - The synergistic effect of drought and light stresses in sorghum and pearl millet. AB - The effects of drought stress and high irradiance and their combination were studied under laboratory conditions using young plants of a very drought resistant variety, ICMH 451, of pearl millet (Pennisetum glaucum) and three varieties of sorghum (Sorghum bicolor)-one drought-resistant from India, one drought-tolerant from Texas, and one drought-sensitive variety from France. CO(2) assimilation rates and photosystem II fluorescence in leaves were analyzed in parallel with photosynthetic electron transport, photosystem II fluorescence, and chlorophyll-protein composition in chloroplasts isolated from these leaves. High irradiance slightly increased CO(2) assimilation rates and electron transport activities of irrigated plants but not fluorescence. Drought stress (less than -1 megapascal) decreased CO(2) assimilation rates, fluorescence, and electron transport. Under the combined effects of drought stress and high irradiance, CO(2) assimilation rates and fluorescence were severely inhibited in leaves, as were the photosynthetic electron transport activities and fluorescence in chloroplasts (but not photosystem I activity). The synergistic or distinctive effect of drought and high irradiance is discussed. The experiments with pearl millet and three varieties of sorghum showed that different responses of plants to drought and light stresses can be monitored by plant physiological and biochemical techniques. Some of these techniques may have a potential for selection of stress-resistant varieties using seedlings. PMID- 16668153 TI - Measurement of 2-carboxyarabinitol 1-phosphate in plant leaves by isotope dilution. AB - The level of 2-carboxyarabinitol 1-phosphate (CA1P) in leaves of 12 species was determined by an isotope dilution assay. (14)C-labeled standard was synthesized from [2-(14)C]carboxyarabinitol 1,5-bisphosphate using acid phosphatase, and was added at the initial point of leaf extraction. Leaf CA1P was purified and its specific activity determined. CA1P was found in dark-treated leaves of all species examined, including spinach (Spinacea oleracea), wheat (Triticum aestivum), Arabidopsis thaliana, and maize (Zea mays). The highest amounts were found in bean (Phaseolus vulgaris) and petunia (Petunia hybrida), which had 1.5 to 1.8 moles CA1P per mole ribulose 1,5-bisphosphate carboxylase catalytic sites. Most species had intermediate amounts of CA1P (0.2 to 0.8 mole CA1P per mole catalytic sites). Such intermediate to high levels of CA1P support the hypothesis that CA1P functions in many species as a light-dependent regulator of ribulose 1,5-bisphosphate carboxylase activity and whole leaf photosynthetic CO(2) assimilation. However, CA1P levels in spinach, wheat, and A. thaliana were particularly low (less than 0.09 mole CA1P per mole catalytic sites). In such species, CA1P does not likely have a significant role in regulating ribulose 1,5 bisphosphate carboxylase activity, but could have a different physiological role. PMID- 16668154 TI - Purification and Developmental Analysis of the Major Anionic Peroxidase from the Seed Coat of Glycine max. AB - We show that the majority of peroxidase activity in soybean (Glycine max var Williams 82) seeds is localized to the seed coat. A single isozyme is responsible for this activity and has been purified to electrophoretic homogeneity by successive chromatography on DEAE Sepharose Fast Flow, concanavalin A-Sepharose, and Sephadex G-75. The peroxidase exhibits a pl of 4.1, an apparent molecular mass of 37 kilodaltons, and has properties characteristic of a glycoprotein. The enzyme begins to accumulate approximately 21 days after anthesis and continues to do so throughout the maturation of the seed coat where it can represent at least 5% of the soluble protein in dry seed coats. Due to its localization in the seed, we propose that this isozyme may play a role in the hardening of the seed coat. PMID- 16668155 TI - Effect of the Long-Term Elevation of CO(2) Concentration in the Field on the Quantum Yield of Photosynthesis of the C(3) Sedge, Scirpus olneyi. AB - CO(2) concentration was elevated throughout 3 years around stands of the C(3) sedge Scirpus olneyi on a tidal marsh of the Chesapeake Bay. The hypothesis that tissues developed in an elevated CO(2) atmosphere will show an acclimatory decrease in photosynthetic capacity under light-limiting conditions was examined. The absorbed light quantum yield of CO(2) uptake (o(abs) and the efficiency of photosystem II photochemistry were determined for plants which had developed in open top chambers with CO(2) concentrations in air of 680 micromoles per mole, and of 351 micromoles per mole as controls. An Ulbricht sphere cuvette incorporated into an open gas exchange system was used to determine o(abs) and a portable chlorophyll fluorimeter was used to estimate the photochemical efficiency of photosystem II. When measured in an atmosphere with 10 millimoles per mole O(2) to suppress photorespiration, shoots showed a o(abs) of 0.093 +/- 0.003, with no statistically significant difference between shoots grown in elevated or control CO(2) concentrations. Efficiency of photosystem II photochemistry was also unchanged by development in an elevated CO(2) atmosphere. Shoots grown and measured in 680 micromoles per mole of CO(2) in air showed a o(abs) of 0.078 +/- 0.004 compared with 0.065 +/- 0.003 for leaves grown and measured in 351 micromoles per mole CO(2) in air; a highly significant increase. In accordance with the change in o(abs), the light compensation point of photosynthesis decreased from 51 +/- 3 to 31 +/- 3 micro-moles per square meter per second for stems grown and measured in 351 and 680 micromoles per mole of CO(2) in air, respectively. The results suggest that even after 3 years of growth in elevated CO(2), there is no evidence of acclimation in capacity for photosynthesis under light-limited conditions which would counteract the stimulation of photosynthetic CO(2) uptake otherwise expected through decreased photorespiration. PMID- 16668156 TI - Characterization of Cytoplasmic and Nuclear Mutations Affecting Chlorophyll and Chlorophyll-Binding Proteins during Senescence in Soybean. AB - Soybean plants (Glycine max [L.] Merr. cv Clark) carrying nuclear and cytoplasmic "stay-green" mutations, which affect senescence, were examined. Normally, the levels of chlorophyll (Chl) a and b decline during seedfill and the Chl a/b ratio decreases during late pod development in cv Clark. Plants homozygous for both the d(1) and d(2) recessive alleles, at two different nuclear loci, respectively, retained most (64%) of their Chl a and b and exhibited no change in their Chl a/b ratio. Combination of G (a dominant nuclear allele in a third locus causing only the seed coat to stay green during senescence) with d(1)d(2) further inhibited the loss of Chl in the leaf. Whereas the thylakoid proteins seem to be degraded in normal Clark leaves during late pod development, they were not substantially diminished in d(1)d(2) and Gd(1)d(2) leaves. In plants carrying a cytoplasmic mutation, cytG, Chl declined in parallel with normal cv Clark; however, the cytG leaves had a much higher level of Chl b, and somewhat more Chl a, remaining at abscission, enough to color the leaves green. In cytG, most thylakoid proteins were degraded, but the Chl a/b-binding polypeptides of the light-harvesting complex in photosystem II (LHCII), and their associated Chl a and b molecules, were not. Thus, the combination of d(1) and d(2) causes broad preservation of the thylakoid proteins, whereas cytG appears to selectively preserve LHCII. The cytG mutation may be useful in elucidating the sequence of events involved in the degradation of LHCII proteins and their associated pigments during senescence. PMID- 16668157 TI - Chloroplast Structure and Function Is Altered in the NCS2 Maize Mitochondrial Mutant. AB - The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts. PMID- 16668158 TI - Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle. AB - The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20 degrees C. If inhibitors of ethylene formation or action (Co(2+), aminoethoxyvinylglycine, or Ag(+)) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag(+) is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag(+) for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1 carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle. PMID- 16668159 TI - Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase. AB - Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Delta(1) pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Delta(1)-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Delta(1)-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties. PMID- 16668160 TI - Fructosyl Transfer between 1-Kestose and Sucrose in Wheat Leaves. AB - The labeling pattern of the sugar moieties of 1-kestose after in vivo pulse labeling with (14)CO(2) was not the same as that after in vitro labeling with (14)C-sucrose. The two fructosyl residues of 1-kestose had similar specific radioactivities after in vitro synthesis, but after in vivo radiolabeling the specific radioactivity of the terminal fructosyl moiety was significantly less than the internal fructosyl moiety. Evidence is presented that the uneven specific radioactivity of in vivo radiolabeling results from enzymatic transfer of terminal fructosyl residue from 1-kestose to sucrose. PMID- 16668161 TI - Herbicide Resistance in Datura innoxia: Kinetic Characterization of Acetolactate Synthase from Wild-Type and Sulfonylurea-Resistant Cell Variants. AB - Acetolactate synthase (ALS, EC 4. 1.3. 18), the first enzyme in the biosynthesis of branched-chain amino acids, was isolated from wild-type and sulfonylurea resistant Datura innoxia cell variants and characterized. Apparent K(m) values of the ALS for pyruvate from three sulfonylurea-resistant variants (CSR2, CSR6, and CSR10) were manyfold greater than that of the wild type. The inhibition of wild type and herbicide-resistant ALS activity by chlorsulfuron (CS), a sulfonylurea herbicide, and l-leucine (l-Leu), one of the feedback inhibitors of the enzyme, was examined. ALS from two CS-resistant variants exhibited severalfold greater resistance to CS than did the wild-type enzyme. Inhibition of ALS by l-Leu fitted a partially competitive pattern most closely. It is proposed that the herbicide resistance mutation accentuated the partial inhibition characteristics of ALS by l-Leu. ALS from one of the two CS-resistant variants (CSR6) had a K(i) for l-Leu an order of magnitude greater than that of the wild-type enzyme. The alterations in kinetic properties observed in the ALS from sulfonylurea-resistant variants are discussed in relation to the possible evolutionary significance of the herbicide binding site of this enzyme, the physiological effects of such biochemical alterations, and their practical utility in genetic studies. PMID- 16668162 TI - Colocalization of Polyphenol Oxidase and Photosystem II Proteins. AB - Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti PPO antibody) and activity (based upon ability to oxidize di dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex. PMID- 16668163 TI - Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress. AB - Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O(2) regimes. Low O(2) concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O(2) did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O(2) but not in 7.5 or 10% O(2). The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O(2) tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O(2) levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O(2) levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O(2). PMID- 16668164 TI - Differential expression of the two Arabidopsis nitrate reductase genes. AB - The differential regulation of the two nitrate reductase (NR, EC 1.6.6.1) genes of Arabidopsis thaliana L. Heynh was examined. cDNAs corresponding to each of the NR genes (NR1 and NR2) were used to measure changes in the steady-state levels of NR mRNA in response to nitrate, light, circadian rhythm, and tissue specificity. Although nitrate-induction kinetics of the two genes are very similar, NR1 is expressed in the absence of nitrate at a higher basal level than NR2. Nitrate induction is transient both in the roots and leaves, however the kinetics are different: the induction and decline in the roots precede that in the leaves. Light induces the expression of each of the genes with significantly different kinetics: NR2 reached saturation more rapidly than did NR1. Both genes showed similar diurnal patterns of circadian rhythm, with NR2 mRNA accumulating earlier in the morning. PMID- 16668165 TI - Cloning and Inactivation of a Gene Essential to Inorganic Carbon Transport of Synechocystis PCC6803. AB - A clone (HP-1) which transforms the high CO(2)-requiring mutant (RKb) of Synechocystis PCC6803 defective in inorganic carbon transport to the wild-type (WT) phenotype was isolated from a WT genomic library. The clone contained a 5.4 kilobase-pair DNA insert. Complementation tests with subclones derived from HP-1 allowed the mutation in RKb to be located within 141 base-pair nucleotides. Sequencing of nucleotides in this region revealed an open reading frame encoding a hydrophobic protein consists of 80 amino acids. A defined mutant (M9) constructed by inactivating this putative inorganic carbon transport gene, designated ictA, was unable to transport CO(2) and HCO(3) (-) into the intracellular inorganic carbon pool. Cloning and sequence analysis of the respective RKb gene revealed a base substitution which generates a stop codon in the middle of ictA. PMID- 16668166 TI - Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls. AB - Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1-->3), (1-->4)-beta-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the beta-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles. PMID- 16668167 TI - Abscisic Acid Control of rbcS and cab Transcription in Tomato Leaves. AB - Leaves of tomato (Lycopersicon esculentum) plants grown in soil in which moisture was lowered from field capacity to levels approaching permanent wilting point show a 10-fold increase in abscisic acid (ABA) and a 60 to 70 percent decrease in rbcS and cab steady-state mRNA levels. As indicated by transcription run-on experiments, the effect occurs primarily at the transcriptional level. Similar water deficit had only a minor effect on ABA level and on rbcS and cab expression in leaves of sitiens, an ABA mutant of tomato. Expression of rbcL, the chloroplast gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, is not affected by water stress. Application of exogenous ABA results in decreased rbcS and cab expression in both wild-type and sitiens leaves. Analysis of the expression of individual members of the rbcS gene family indicates that under water-deficit conditions, expression derives primarily from only three of the five rbcS genes. Effects of dark adaptation and water deficit are additive for cab but not for rbcS expression. These results support the hypothesis that, at least under water-deficit conditions, ABA or a derivative thereof mediates a negative regulation of rbcS and cab transcription in tomato plants. PMID- 16668168 TI - In vivo regulatory phosphorylation site in c(4)-leaf phosphoenolpyruvate carboxylase from maize and sorghum. AB - Reversible seryl-phosphorylation contributes to the light/dark regulation of C(4) leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein serine kinase(s), leads to an increase in catalytic activity and a decrease in malate-sensitivity of the target enzyme has been recently identified as Ser-15 in (32)P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [(32)P]phosphopeptides were isolated and purified from in vivo(32)P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more (32)P radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residue (Ser-15 in maize PEPC and its structural homolog, Ser-8, in the sorghum enzyme) was found to be (32)P phosphorylated in the light or dark. These in vivo observations, combined with the results from our previous in vitro phosphorylation studies (J-A Jiao, R Chollet [1989] Arch Biochem Biophys 269: 526-535; [1990] Arch Biochem Biophys 283: 300-305), demonstrate that an N-terminal seryl residue in C(4) PEPC is, indeed, the regulatory site that undergoes light/dark changes in phosphorylation status and, thus, plays a major, if not cardinal role in the light-induced changes in catalytic and regulatory properties of this cytoplasmic C(4) photosynthesis enzyme in vivo. PMID- 16668169 TI - Glass formation and desiccation tolerance in seeds. AB - The formation of intracellular glass may help protect embryos from damage due to desiccation. Soluble sugars similar to those found in desiccation tolerant embryos were studied with differential scanning calorimetry. Those sugars from desiccation tolerant embryos can form glasses at ambient temperatures, whereas those from embryos that do not tolerate desiccation only form glasses at subzero temperatures. It is concluded that tolerant embryo cells probably contain sugar glasses at storage temperatures and water contents, but intolerant embryo cells probably do not. PMID- 16668170 TI - Physiological responses of soybean plants grown in a nitrogen-free or energy limited environment. AB - Soybean (Glycine max [L.] Merr.) seedlings grown in the absence of combined N and in an Ar:O(2) (79:21, volume/volume) atmosphere had greater seedling and nodule mass, threefold higher acetylene reducing activity per gram fresh weight nodules, no observable increase in nitrogenase Fe-protein, and a higher energy charge than did control plants. A sharp fall in acetylene reducing activity and energy charge accompanying stem-girdling was prevented by exogenous succinate, a result consistent with a path from the roots to the nodule other than via the phloem. PMID- 16668171 TI - Acetolactate synthase inhibiting herbicides bind to the regulatory site. AB - Acetolactate synthase from spontaneous mutants of tobacco (Nicotiana tabacum; KS 43 and SK-53) and cotton (Gossypium hirsutum; PS-3, PSH-91, and DO-2) selected in tissue culture for resistance to a triazolopyrimidine sulfonanilide showed varying degrees of insensitivity to feedback inhibitor(s) valine and/or leucine. A similar feature was evident in the enzyme isolated from chlorsulfuron-resistant weed biotypes, Kochia scoparia and Stellaria media. Dual inhibition analyses of triazolopyrimidine sulfonanilide, thifensulfuron, and imazethapyr versus feedback inhibitor leucine revealed that the three herbicides were competitive with the amino acid for binding to acetolactate synthase from wild-type cotton cultures. Acetolactate synthase inhibiting herbicides may bind to the regulatory site on the enzyme. PMID- 16668172 TI - Resistance to the herbicide paraquat and increased tolerance to photoinhibition are not correlated in several weed species. AB - Photoinhibition was examined in paraquat-resistant and paraquat-susceptible biotypes of Hordeum glaucum Steud., Hordeum leporinum Link., Arctotheca calendula (L.) Levyns., and Conyza bonariensis (L.) Cronq. Plants were photoinhibited at low temperature, and the extent of photoinhibition determined by O(2) evolution and 77 K fluorescence. No difference in the degree of photoinhibition was detected between paraquat-resistant and paraquat-susceptible biotypes for any of the species examined. C. bonariensis plants were also photoinhibited by treatment without CO(2) at either 21% (volume/volume) O(2) or 4% (volume/volume) O(2), and again no difference was observed between the paraquat-resistant and paraquat susceptible biotypes in reduction of the ratio of variable fluorescence to maximal fluorescence. This is in contrast to a recent report (MAK Jansen, Y Shaaltiel, D Kazzes, O Canaani, S Malkin, J Gressel, [1989] Plant Physiol 91: 1174-1178 in which it was claimed that a paraquat-resistant biotype of C. bonariensis was more tolerant of photoinhibition than a paraquat-susceptible biotype. We conclude that paraquat-resistant biotypes of these plant species are not more tolerant of photoinhibition when compared with the paraquat-susceptible biotypes. PMID- 16668173 TI - Protein Synthesis in Isolated Mitochondria of Rice (Oryza sativa L.) Seedlings. AB - For studies of in organello mitochondrial protein synthesis in rice, Oryza sativa L., conventional surface-sterilization procedures were demonstrated to be ineffective. Because of the over-whelmingly efficient [(35)S]methionine utilization by contaminating bacteria, even "essentially bacteria-free" rice mitochondria were shown to be unsuitable for the study of in organello protein synthesis. We developed a procedure to obtain a bacteria-free preparation of rice mitochondria. Such mitochondria favored a membrane-dependent ATP-generating system over an external ATP-generating system as the energy supplement for in organello protein synthesis. Two distinct classes of [(35)S]methionine-labeled, cycloheximide-insensitive products were detected: an electrophoretically unresolved population and a set of some 22 to 27 discrete polypeptide species, each with a characteristic electrophoretic mobility and relative abundance. PMID- 16668174 TI - Purification and characterization of soybean root nodule ferric leghemoglobin reductase. AB - A ferric leghemoglobin reductase from the cytosol of soybean (Glycine max) root nodules was purified to homogeneity and partially characterized. The enzyme is a flavoprotein with flavin adenine dinucleotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxido-reductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex. PMID- 16668175 TI - Setting of the Circadian N(2)-Fixing Rhythm of the Prokaryotic Synechococcus sp. RF-1 while Its nif Gene Is Repressed. AB - The N(2)-fixing activity of the prokaryotic Synechococcus sp. RF-1 was repressed in the presence of nitrate. When the cultures in nitrate-containing medium were exposed to diurnal light-dark cycles, an endogenous circadian N(2)-fixing rhythm developed after the cells were transferred to nitrate-free medium and incubated in continuous light. The N(2)-fixing phase of the rhythm coincided with the dark phase of the light-dark cycles that were imposed when the cells were in nitrate containing medium. The results indicate that after the endogenous N(2)-fixing rhythm has been set, it can be kept latent for at least 38 hours before first manifesting itself. PMID- 16668176 TI - Kinetic characterization of caffeoyl-coenzyme a-specific 3-o-methyltransferase from elicited parsley cell suspensions. AB - The activity of caffeoyl-coenzyme A (CoA) 3-O-methyltransferase, an enzyme widely distributed in plants and involved in cell wall reinforcement in a disease resistance response, appears to be subject to a complex type of regulation in vivo. In cultured parsley (Petroselinum crispum) cells treated with an elicitor from Phytophthora megasperma f.sp. glycinea, the enzyme activity is rapidly induced by a transient increase in the rate of de novo transcription. Parsley caffeoyl-CoA-specific methyltransferase differs in several aspects from other plant O-methyltransferases but shows limited homology to bacterial adenine specific DNA methyltransferases. Kinetic analysis revealed an Ordered Bi Bi mechanism for catalysis, with caffeoyl-CoA bound prior to S-adenosyl-l-methionine and feruloyl-CoA released last from the enzyme. The small inhibitory constant determined in vitro for feruloyl-CoA suggests that, in vivo, the enzyme activity is also under tight control by the steady-state product concentration in addition to the rate of transcription that becomes affected upon elicitor challenge. PMID- 16668177 TI - Sodium does not compete with calcium in saturating plasma membrane sites regulating na influx in salinized maize roots. AB - Half maximal inhibition of sodium ((22)Na(+)) influx into maize (Zea mays L.) root segments incubated in solutions containing from 0.25 to 100 millimolar NaCl was consistently attained with external calcium activity at 0.26 +/- 0.10 millimolar. Sodium ions do not appear to compete with calcium during initial binding to sites on the plasma membrane that participate in the regulation of sodium influx under saline conditions. PMID- 16668178 TI - Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis. AB - Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P(2) carboxylase under catalytic conditions (in the presence of CO(2), Mg(2+), and ribulose-P(2)) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO(2) and Mg(2+) only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle. PMID- 16668179 TI - Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity. AB - The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301). In this paper, we show the effect of various concentrations of phosphatidyinositol, PIP, and PIP(2) on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP(2) at concentrations of 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids. PMID- 16668180 TI - Nucleotide Sequence of a Complementary DNA Clone Encoding Stearoyl-Acyl Carrier Protein Desaturase from Castor Bean, Ricinus communis. PMID- 16668181 TI - Isolation and Characterization of a Gene Encoding a PR-1-Like Protein from Arabidopsis thaliana. PMID- 16668182 TI - Defense mechanisms of conifers : relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content. AB - Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue. PMID- 16668183 TI - Conjugation of ubiquitin to proteins from green plant tissues. AB - Conjugation of the polypeptide ubiquitin to endogenous proteins was studied in oat (Avena sativa L.) plants, and particularly in green tissues. Conjugating activity in leaf extracts was different from that in root extracts, and in both was less than in etiolated tissue. The conjugates were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their formation was both time- and ATP-dependent and had a pH optimum of about 8.2. The assay had a high affinity for ATP with a probable K(m) of less than 50 micromolar. The ubiquitin conjugating system was also shown to be present in isolated chloroplasts, and ubiquitin could be conjugated to endogenous proteins of lyzed chloroplasts in which the ATP concentrations were reduced by preincubation or desalting. SDS-PAGE analysis led to the suggestion that the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase) may be able to be ubiquitinated, and we have shown that ubiquitin can stimulate the in vitro breakdown of (125)I-labeled RuBPCase. These results invite the speculation that ubiquitin may be involved in the regulation of protein turnover in green plants. PMID- 16668184 TI - Defense mechanisms of conifers : differences in constitutive and wound-induced monoterpene biosynthesis among species. AB - Levels of monoterpene cyclase activity were determined in extracts from wounded and unwounded saplings of 10 conifer species to assess whether oleoresin biosynthesis is induced by stem wounding. Species of Abies and Picea, with low to moderate levels of constitutive monoterpene cyclase activity, exhibited a five- to 15-fold increase in cyclase activity 7 days after wounding relative to unwounded controls. In contrast, species of genera such as Pinus, with high levels of constitutive cyclase activity, did not significantly respond to wounding by alteration in the level of cyclase activity. The highest fold increase in monoterpene cyclase activity was consistently observed in Abies grandis, and the time-course of induction of activity following stem wounding in this species demonstrated a threefold increase at 2 days relative to unwounded controls, rising to a maximum increase in the response at 9 days (greater than 10 fold) followed by an apparent decline. The wound response was localized, and both bark (phloem) and wood (xylem) tissues displayed increased cyclase activity at the wound site. The magnitude of the increase in cyclase activity was dependent on the severity of the wound. PMID- 16668185 TI - Regulation of glycerol synthesis in response to osmotic changes in dunaliella. AB - Changes in phosphometabolites, following osmotic shock, were analyzed by two dimensional thin layer chromatography, in extracts of the halotolerant alga Dunaliella salina in order to clarify the regulation of glycerol synthesis from starch. The experiments were carried out in wild-type and in osmotically defective mutant cells. It is demonstrated that hyperosmotic shock induces a decrease in fructose 6-phosphate and an increase in fructose-1,6-bisphosphate indicating the activation of phosphofructokinase. Two mutants, which are specifically defective in their response to hyperosmotic shock, accumulate glucose 6-phosphate or phosphogluconate following shock, and have remarkably reduced activities of glucose-6-phosphate dehydrogenase and of phosphogluconate dehydrogenase, respectively. These results indicate that the pentose-phosphate oxidative pathway has a major role in glycerol synthesis. Hyperosmotic shock leads to a transient accumulation of phosphorylcholine and to a decrease of inositolbisphosphate in D. salina extracts. Accumulation of phosphorylcholine is not detected in osmotically defective mutants. Hypoosmotic shock induces an increase in inositolbisphosphate but not in phosphorylcholine. These results are consistent with previous indications for differential activations of phospholipases by hyper or hypoosmotic shock in Dunaliella. Based on these results we suggest that (a) phosphofructokinase is an important checkpoint enzyme in the regulation of glycerol production, and (b) that the pentose-phosphate pathway has a major role in keeping oxidation-reduction balance during glycerol synthesis. The possible role of lipid breakdown products as second messengers in regulating glycerol production in Dunaliella is discussed. PMID- 16668186 TI - Characterization of Ribonuclease Activity of Three S-Allele-Associated Proteins of Petunia inflata. AB - Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T(2) and RNase Rh. In this study, the S-proteins from P. inflata plants of S(1)S(2) and S(2)S(3) genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (S(1), S(2), and S(3)), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 50 degrees C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three S-proteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid. PMID- 16668187 TI - Mitochondria Isolated from NaCl-Adapted Tobacco Cell Lines (Nicotiana tabacum/gossii) Maintain Their Phosphorylative Capacity in Highly Saline Media. AB - The in vivo functioning of mitochondria isolated from two tobacco cell lines in suspension culture (Nicotiana tabacum/gossii), wild type, and NaCl-adapted (A190), has been compared in the face of rising external salinity. The O(2) uptake of both state 3 and state 4 mitochondria was progressively inhibited with increasing external NaCl concentration in the case of both lines. Phosphorylation, however, was maintained at a higher level in the case of A190 mitochondria, as indicated both by stability of ADP:O ratio and rate of incorporation of (32)Pi. The superior phosphorylation performance of A190 mitochondria also emerged when phosphorylation was calculated per reducing equivalent, but not per unit DeltamuH(+) (electrochemical potential gradient for protons). However, the overall DeltamuH(+) was maintained at a higher level in A190 mitochondria due to the fact that the depolarization accompanying increase in external NaCl concentration was compensated for in A190 mitochondria by an increase in the transmembrane pH gradient, but not in wild type mitochondria. Increased proton permeability of the inner membrane is among the probable causes suggested for the loss of phosphorylation ability in wild type mitochondria; in contrast, A190 mitochondria maintain better membrane integrity under saline stress. PMID- 16668188 TI - Molecular cloning and differential expression of the maize ferredoxin gene family. AB - In maize (Zea mays L.), four ferredoxin (Fd) isoproteins, Fd I to Fd IV, are differentially distributed in photosynthetic and nonphotosynthetic organs of young seedlings (Y Kimata, T Hase [1989] Plant Physiol 89: 1193-1197). To understand structural characteristics of the Fd isoproteins and molecular mechanism of the differential expression of their genes, we have cloned and characterized three different maize Fd cDNAs. DNA sequence analyses showed that two of the cDNAs encoded the entire precursor polypeptides of Fd I and Fd III, which were composed of 150 and 152 amino acid residues, respectively, and the other encoded a 135 amino acid precursor polypeptide of Fd not yet identified. High degrees of homologies were found in the deduced amino acid sequences of mature regions of these Fd isoproteins, but the transit peptide of Fd III differed considerably from those of other Fd isoproteins. Fd I and the unidentified Fd were encoded mainly with codons ending in C or G, but such strong codon bias was not seen in Fd III. Gene specific probes for each cDNA were used to probe Northern blots of RNA isolated from leaves, mesocotyls, and roots of maize seedlings. The gene transcripts for Fd I and the unidentified Fd were restricted to leaves and their levels increased markedly upon illumination of etiolated seedlings, whereas that for Fd III was detected in all organs and its accumulation was not light dependent. This organ specific accumulation of Fd mRNAs corresponds exactly to the distribution pattern of Fd isoproteins. PMID- 16668189 TI - Independent Regulatory Aspects and Posttranslational Modifications of Two beta Amylases of Rye : Use of a Mutant Inbred Line. AB - We have examined the occurrence/disappearance, tissue location, and posttranslational modification of beta-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete beta amylase deficiency. This deficiency corresponds to a lack of accumulation of beta amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct beta-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of beta amylases are discussed in relation to recent reports indicating the presence of two types of beta-amylase with different functions and gene loci in barley and wheat. PMID- 16668190 TI - Effect of Na(3)VO(4) on the P State of Nitella translucens. AB - The capacity of sodium orthovanadate to inhibit the plasmalemma H(+) ATPase of Nitella translucens internodal cells in vivo was tested. Here we show that 1 millimolar vanadate added externally depolarizes strongly and permanently the membrane potential, both in dark and light, to the Nernst potential for potassium consistent with pump inhibition by vanadate. From the results it is clear that the H(+) ATPase is always active, under light or dark conditions, in contradiction with the widespread idea of pump inactivation by darkness. The changes in conductance for light, dark, and vanadate-induced conditions are analyzed. The effect of dark on membrane passive permeabilities and on the possibility that some plasmalemma channels could be regulated by a phosphorylation-dephosphorylation process is discussed. PMID- 16668191 TI - The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum. AB - The seeds of Spartium junceum contained a large quantity of lectin-like protein that did not appear to be either a hemagglutinin or active lectin. The cross reactive material (CRM), like most legume seed lectins, was a tetrameric glycoprotein of about 130,000 M(r). The singlesized subunits of about 33,000 M(r) were not covalently associated. The amino acid composition was typical of legume lectins and was rich in hydroxy-amino acids and poor in sulfur-containing amino acids. The Spartium CRM contained about 3.5% covalently associated carbohydrate, most likely of the high-mannose type, since the CRM was precipitated by concanavalin A. The CRM was localized by electron-microscopic immunocytochemistry and found to be exclusively in protein-filled vacuoles (protein bodies). Because this protein was so similar immunologically, structurally, and in its physiology, to classic legume seed lectins, it is most likely a lectin homolog. Similar seed lectin CRMs appear to be both common and widespread in the Leguminosae. PMID- 16668192 TI - The evidence for symplastic Phloem loading. AB - Experimental protocols used to study the route(s) of phloem loading in source leaves are evaluated; they include the analysis of plasmodesmatal ultrastructure and distribution, mobile compounds in the free space, loading patterns of exogenous sugars, the site of sugar synthesis, and dye coupling. All these methods have drawbacks. There are indications that some plants, especially those that translocate the raffinose series of sugars, load by a symplastic (through plasmodesmata) pathway, but the evidence is weak. PMID- 16668193 TI - Effects of Nitrogen Nutrition on Nitrogen Partitioning between Chloroplasts and Mitochondria in Pea and Wheat. AB - Nitrogen partitioning among proteins in chloroplasts and mitochondria was examined in pea (Pisum sativum L.) and wheat (Triticum aestivum L.) grown hydroponically with different nitrogen concentrations. In pea leaves, chloroplast nitrogen accounted for 75 to 80% of total leaf nitrogen. We routinely found that 8% of total ribulose-1,5-bisphosphate carboxylase/oxygenase adhered to thylakoids during preparation and could be removed with Triton X-100. With this precaution, the ratio of stroma nitrogen increased from 53 to 61% of total leaf nitrogen in response to the nitrogen supply, but thylakoid nitrogen remained almost constant around 20% of total. The changes in the activities of the stromal enzymes and electron transport in response to the nitrogen supply reflected the nitrogen partitioning into stroma and thylakoids. On the other hand, nitrogen partitioning into mitochondria was appreciably smaller than that in chloroplasts, and the ratio of nitrogen allocated to mitochondria decreased with increasing leaf nitrogen content, ranging from 7 to 4% of total leaf nitrogen. The ratio of mitochondrial respiratory enzyme activities to leaf-nitrogen content also decreased with increasing leaf-nitrogen content. These differences in nitrogen partitioning between chloroplasts and mitochondria were reflected in differences in the rates of photosynthesis and dark respiration in wheat leaves measured with an open gas-exchange system. The response of photosynthesis to nitrogen supply was much greater than that of dark respiration, and the CO(2) compensation point decreased with increasing leaf-nitrogen content. PMID- 16668194 TI - Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis : I. Modulation in Vivo by CO(2) Availability. AB - It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Forster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO(2) in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO(2) supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in -CO(2)-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg(2+) or Ca(2+). Mg(2+) concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg(2+) concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V(max) changed. PMID- 16668195 TI - Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP. AB - Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO(2) or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg(2+) or Ca(2+), kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg(2+) and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO(2) compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (beta, gamma-imido ATP, beta, gamma-methyl-ATP) had no effect on NR, whereas gamma-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP. PMID- 16668196 TI - Effect of Increases in Oxygen Concentration during the Argon-Induced Decline in Nitrogenase Activity in Root Nodules of Soybean. AB - When intact nodulated roots of soybean (Glycine max L. Merr. nodulated with Bradyrhizobium japonicum strain USDA 16) were exposed to an atmosphere lacking N(2) gas (Ar:O(2) 80:20), total nitrogenase activity (measured as H(2) evolution) and respiration (CO(2) evolution) declined with time of exposure. In Ar-inhibited nodules, when the O(2) concentration in the rhizosphere was increased in a linear ;ramp' of 2.7% per minute, 93% of the original H(2) evolution and 99% of the CO(2) evolution could be recovered. The internal nodule O(2) concentration (estimated from leghemoglobin oxygenation) declined to 56% of its initial value after 60 minutes of Ar:O(2) exposure and could be partially recovered by the linear increases in O(2) concentration. Nodule gas permeability, as estimated from the lag in ethylene production following exposure of nodules to acetylene, decreased to 26% of its initial value during the Ar-induced decline. Collectively, the results provide direct evidence that the Ar-induced decline results from decreased nodule gas permeability and indicate that the decline in permeability, rather than being immediate, occurs gradually over the period of Ar:O(2) exposure. PMID- 16668197 TI - Spinach leaf acetyl-coenzyme a synthetase: purification and characterization. AB - Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per milligram protein. The average M(r) value of the native enzyme was about 73,000. The Michaelis constants determined for Mg-ATP, acetate, and coenzyme A were 150, 57, and 5 micromolar, respectively. The purified enzyme was sensitive to substrate inhibition by CoA with an apparent K(i) for CoA of 700 micromolar. The enzyme was specific for acetate; other short and long chain fatty acids were ineffective as substrates. Several intermediates and end products of fatty acid synthesis were examined as potential inhibitors of acetyl-CoA synthetase activity, but none of the compounds tested significantly inhibited acetyl-CoA synthetase activity in vitro. The properties of the purified enzyme support the postulated role of acetyl-CoA synthetase as a primary source of chloroplast acetyl-CoA. PMID- 16668198 TI - Differential Regulation of beta-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection. AB - The acidic, extracellular, glucan endo-1,3-beta-glucosidases (EC 3.2.1.39; beta 1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-O) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-O. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of beta-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the beta-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of beta-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level. PMID- 16668199 TI - Regulation of NO(3) Assimilation by Anion Availability in Excised Soybean Leaves. AB - The regulation of NO(3) (-) assimilation by xylem flux of NO(3) (-) was studied in illuminated excised leaves of soybean (Glycine max L. Merr. cv Kingsoy). The supply of exogenous NO(3) (-) at various concentrations via the transpiration stream indicated that the xylem flux of NO(3) (-) was generally rate-limiting for NO(3) (-) reduction. However, NO(3) (-) assimilation rate was maintained within narrow limits as compared with the variations of the xylem flux of NO(3) (-). This was due to considerable remobilization and assimilation of previously stored endogenous NO(3) (-) at low exogenous NO(3) (-) delivery, and limitation of NO(3) (-) reduction at high xylem flux of NO(3) (-), leading to a significant accumulation of exogenous NO(3) (-). The supply of (15)NO(3) (-) to the leaves via the xylem confirmed the labile nature of the NO(3) (-) storage pool, since its half-time for exchange was close to 10 hours under steady state conditions. When the xylem flux of (15)NO(3) (-) increased, the proportion of the available NO(3) (-) which was reduced decreased similarly from nearly 100% to less than 50% for both endogenous (14)NO(3) (-) and exogenous (15)NO(3) (-). This supports the hypothesis that the assimilatory system does not distinguish between endogenous and exogenous NO(3) (-) and that the limitation of NO(3) (-) reduction affected equally the utilization of NO(3) (-) from both sources. It is proposed that, in the soybean leaf, the NO(3) (-) storage pool is particularly involved in the short-term control of NO(3) (-) reduction. The dynamics of this pool results in a buffering of NO(3) (-) reduction against the variations of the exogenous NO(3) ( ) delivery. PMID- 16668200 TI - Water-stress-induced ethylene production in wheat : a fact or artifact? AB - Effects of water stress on ethylene evolution from excised leaf segments and intact plants of wheat (Triticum aestivum L. cv Katepwa) were studied. Excised leaf segments of 8 day or 6 week old plants were dried until they lost 8% of their fresh weight (water potential about -2.3 megapascals). These and nondried control leaf segments (water potential about -1.0 megapascal) were sealed in glass tubes, and their ethylene production rates were compared by head space analysis via gas-chromatography. The dried leaves of both ages produced significantly more ethylene than the corresponding controls. However, when 6 week old intact plants were water-stressed by withholding water supply, and their ethylene production measured using a continuousflow system, no increase in ethylene was deteceted despite a drop in water potential to -2.9 megapascals over 6 days. Even the leaf segments excised from plants that had been subjected to water stress for 2, 4, or 6 days produced no more ethylene (in sealed tubes) than the leaves from well-watered plants. In fact, the ethylene production by these segments decreased with the increase in the severity of stress experienced by the plants. The results show that the commonly reported overproduction of ethylene by excised leaves subjected to rapid drying represents an artifact, which has little relevance to the water stress responses of intact wheat plants. PMID- 16668201 TI - A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves. AB - Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of -7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose 1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants. PMID- 16668202 TI - Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system. AB - We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH approximately 7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression. PMID- 16668203 TI - Inhibition of auxin-induced cell elongation of maize coleoptiles by antibodies specific for cell wall glucanases. AB - Polyclonal antibodies were raised in rabbits in response to the administration of purified exo- and endoglucanases extracted from cell walls of maize (Zea mays L. B37 x Mo17) coleoptiles. Since the antibodies formed specific conjugates when challenged with the glucanase antigens in immunoblot assays they were employed to evaluate the participation of glucanases in tissue growth. Indole-3-acetic acid induced cell elongation of abraded coleoptile segments was inhibited when the antibodies were supplied as a short term pretreatment (25-200 microgram/milliliter of serum protein). The extent of inhibition of IAA induced cell elongation was additive when endo- and exoglucanase antibodies were applied together. The results suggest that both enzymes have a role in mediating IAA induced cell elongation. Pretreatment with exo- and endoglucanases antibodies also inhibited IAA induced degradation of noncellulosic beta-d-glucans and the increased level of cellulosic polymers in maize coleoptiles. Antibodies also inhibited the expression of the autohydrolytic degradation of glucans in isolated cell walls. The extent of inhibition was dependent on the antibody concentration applied. The results support the contention that enzymatic processes mediated by exo- and endoglucanases are responsible for cell wall autolytic reactions and that these reactions are linked to the mechanism for expressing auxin induced cell elongation in maize coleoptiles. PMID- 16668204 TI - Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides. AB - Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl] 3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2 chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl beta-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein. PMID- 16668205 TI - Spatial distribution of turgor and root growth at low water potentials. AB - Spatial distributions of turgor and longitudinal growth were compared in primary roots of maize (Zea mays L. cv FR27 x FRMo 17) growing in vermiculite at high ( 0.02 megapascals) or low (-1.6 megapascals) water potential. Turgor was measured directly using a pressure probe in cells of the cortex and stele. At low water potential, turgor was greatly decreased in both tissues throughout the elongation zone. Despite this, longitudinal growth in the apical 2 millimeters was the same in the two treatments, as reported previously. These results indicate that the low water potential treatment caused large changes in cell wall yielding properties that contributed to the maintenance of root elongation. Further from the apex, longitudinal growth was inhibited at low water potential despite only slightly lower turgor than in the apical region. Therefore, the ability to adjust cell wall properties in response to low water potential may decrease with cell development. PMID- 16668206 TI - Isolation and characterization of dihydrodipicolinate synthase from maize. AB - Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme specific to lysine biosynthesis in plants, was purified from maize (Zea mays L.) cell suspension cultures and leaves. The subunit molecular weight of maize dihydrodipicolinate synthase was estimated to be 38,000 based on SDS-PAGE. The condensation of l aspartate semialdehyde and pyruvate by highly purified dihydrodipicolinate synthase exhibited kinetics characteristic of a Ping Pong Bi Bi ordered reaction in which pyruvate binds first to the enzyme. Substrate inhibition evident at higher concentrations of l-aspartate semialdehyde was partially alleviated by increasing concentrations of pyruvate. Pyruvate binding exhibited cooperativity with an apparent number of 2 and 1.86 millimolar concentration required for 50% of maximal activity. The K(m) for aspartate semialdehyde was estimated to be 0.6 millimolar concentration. Lysine was an allosteric cooperative inhibitor of dihydrodipicolinate synthase with an estimated Hill number of 4 and 23 micromolar concentration required for 50% inhibition. The physical and kinetic data are consistent with a homotetramer model for the native enzyme. PMID- 16668207 TI - Multiplicity of histone h3 variants in wheat, barley, rice, and maize. AB - Histone H3 proteins were purified to near homogeneity from etiolated seedlings of wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oryza sativa), maize (Zea mays), and alfalfa (Medicago sativa) to determine the number of histone H3 variants. Five distinct histone H3 variants were identified by gradient gel electrophoresis and reversed phase chromatography. These variants occur in various combinations of two to four forms in each plant species. One minor histone variant form (variant III, named H3.2 in alfalfa) appeared present and identical in all mono- and dicotyledonous plant species tested to date. All H3 proteins were acetylated to multiple levels and in every species the variant III form was acetylated most extensively. The level of histone H3 acetylation showed an inverse correlation with plant genome size. These observations support the idea that acetylated histones and especially variant III proteins are an element of transcriptionally active chromatin. PMID- 16668208 TI - In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control. AB - When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of beta glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of beta-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4 dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating beta-glucanase in the presence of 16 micromolar 1 naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced beta glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 x 10( 5) molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco beta-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts. PMID- 16668209 TI - Short-term effects of carbon dioxide on carnation callus cell respiration. AB - The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O(2) uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO(2) in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO(2) again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO(2)/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO(2)/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO(2)/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO(2)) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited. PMID- 16668210 TI - Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants: protein purification from corn leaves and immunological detection. AB - We have purified the protein for the enzyme sucrose phosphate synthase (SPS) from corn (Zea mays) leaves. Partially purified SPS protein was used to generate specific monoclonal antibodies. The following immunoaffinity chromatography allowed the isolation of pure SPS protein. The apparent molecular mass of the SPS polypeptide is 138 kilodaltons. By immunoblot, an SPS antigen was found to accumulate in mature leaves. SPS protein levels remain constant during the day/night cycle. The observed diurnal fluctuation of extractable enzyme activity, therefore, must be caused by modification of the specific activity of SPS in vivo. PMID- 16668211 TI - Characterization of Two Gene Transcripts Modulated by Cytokinins in Micropropagated Apple (Malus domestica [L.] Borkh) Plantlets. AB - The micropropagation of apple (Malus domestica [L.] Borkh) cultivars is usually achieved by axillary bud stimulation and requires an exogenous cytokinin supply. Two cDNA libraries were constructed from mRNA isolated from plantlets grown in vitro on medium with or without benzyladenine. One cDNA clone (pSD3), corresponding to transcripts more abundant in plantlets grown on medium containing cytokinin than on medium lacking the hormone, was isolated. It corresponds to a mRNA of about 1,800 nucleotides which codes for a proline-rich protein with a predicted mass of 31,000 daltons. Its accumulation is restricted to roots and stems of in vivo grown apple plantlets and to stems of microcuttings cultivated on medium without cytokinin. Furthermore, it accumulates to comparable levels in leaves and stems when plantlets are grown on medium containing benzyladenine. A second cDNA clone (pSD4), corresponding to transcripts down regulated in the presence of cytokinin in the culture medium, was also characterized. Its corresponding mRNA is about 700 nucleotides in length and encodes a protein highly homologous to the precursor of the 10-kilodalton polypeptide of the photosystem II from spinach. This mRNA accumulates specifically in leaves of apple plantlets and is more abundant in leaves of plantlets grown in the absence of cytokinin compared with plantlets grown in the presence of benzyladenine. PMID- 16668212 TI - Tissue-Specific Expression and Anaerobically Induced Posttranscriptional Modulation of Sucrose Synthase Genes in Sorghum bicolor M. AB - We have used antibodies directed against the two sucrose synthase (SS) isozymes, and the cDNA clones corresponding to the two nonallelic genes in maize to describe sorghum (Sorghum bicolor) SS genes and their expressions at protein and RNA levels. Western blot analyses have shown evidence of two SS isozymes, SS1 and SS2, in sorghum; these were similar, but not identical, to maize isozymes in size, charge, subunit composition, and epitope specificities against both monoclonal and polyclonal antibodies. Tissue-specific distributions of isozymes and genomic Southern hybridization data are consistent with a hypothesis that the SS1 and SS2 isozymes are encoded by two nonallelic genes, designated here as Sus1 and Sus2, respectively. Northern blot hybridizations on root RNAs showed gene specific transcript patterns and, as in maize, the SS2-specific transcripts were slightly larger than the SS1-specific transcripts. Interestingly, no difference in the size of the SS1 and SS2 polypeptides was detected. Anaerobic induction led to significant elevations in steady-state levels of both SS1 and SS2 transcripts, but there was no detectable increase in the levels of the SS proteins. Thus, both the SS genes in sorghum were significantly regulated at the posttranscriptional level; whereas in maize, only one of the two SS genes was affected in this fashion. Another difference between maize and sorghum SS isozymes was in endosperm-specific polymerization among the SS subunits. Unlike maize endosperm where only the two SS homotetramers are seen, sorghum endosperm showed five SS isozymes attributable to a random copolymerization of SS1 and SS2 subunits, presumably due to a simultaneous expression of both genes in the endosperm cells. Physiological and molecular bases of these differences between these two crop plant species remains to be elucidated. PMID- 16668213 TI - Low growth temperature effects a differential inhibition of photosynthesis in spring and winter wheat. AB - In vivo room temperature chlorophyll a fluorescence coupled with CO(2) and O(2) exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (5 degrees C/5 degrees C, day/night). Plants of comparable physiological stage, but grown at nonhardening temperatures (20 degrees C/16 degrees C, day/night) were used in comparison. Winter wheat cultivars grown at 5 degrees C had light saturated rates of CO(2) exchange and apparent photon yields for CO(2) exchange and O(2) evolution that were equal to or greater than those of winter cultivars grown at 20 degrees C. In contrast, spring wheat cultivars grown at 5 degrees C showed 35% lower apparent photon yields for CO(2) exchange and 25% lower light saturated rates of CO(2) exchange compared to 20 degrees C grown controls. The lower CO(2) exchange capacity is not associated with a lower efficiency of photosystem II activity measured as either the apparent photon yield for O(2) evolution, the ratio of variable to maximal fluorescence, or the level of reduced primary quinone electron acceptor maintained at steady-state photosynthesis, and is most likely associated with carbon metabolism. The lower CO(2) exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following over-night exposure of nonhardened plants to 5 degrees C. PMID- 16668215 TI - Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L. AB - Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined. PMID- 16668214 TI - Transformation of Wall Deficient Cultured Tobacco Protoplasts by Agrobacterium tumefaciens. AB - Attachment of virulent Agrobacterium tumefaciens to plant cells is required for transformation. To further study the components of the plant cell wall that may be involved in the attachment process, tobacco (Nicotiana tabacum L.) protoplasts were cultured in the presence of 2,6 dichlorobenzonitrile (DB), an inhibitor of cellulose biosynthesis, and then assayed for their ability to be transformed by Agrobacterium. The DB treated protoplasts were deficient in wall production. Nevertheless, they were transformable at high frequency by wild type Agrobacterium strains but not by mutant strains that lack the ability to bind to normal, walled cells. Small quantities of calcofluor white positive material present on DB treated cells were correlated with their competence to be transformed. Further, the plant:bacterial association that leads to transformation is shown to become stable within 5 hours after bacterial co cultivation with either control or DB treated cells. PMID- 16668216 TI - Dark Ammonium Assimilation Reduces the Plastoquinone Pool of Photosystem II in the Green Alga Selenastrum minutum. AB - The impact of dark NH(4) (+) and NO(3) (-) assimilation on photosynthetic light harvesting capability of the green alga Selenastrum minutum was monitored by chlorophyll a fluorescence analysis. When cells assimilated NH(4) (+), they exhibited a large decline in the variable fluorescence/maximum fluorescence ratio, the fluorescence yield of photosystem II relative to that of photosystem I at 77 kelvin, and O(2) evolution rate. NH(4) (+) assimilation therefore poised the cells in a less efficient state for photosystem II. The analysis of complementary area of fluorescence induction curve and the pattern of fluorescence decay upon microsecond saturating flash, indicators of redox state of plastoquinone (PQ) pool and dark reoxidation of primary quinone electron acceptor (Q(A)), respectively, revealed that the PQ pool became reduced during dark NH(4) (+) assimilation. NH(4) (+) assimilation also caused an increase in the NADPH/NADP(+) ratio due to the NH(4) (+) induced increase in respiratory carbon oxidation. The change in cellular reductant is suggested to be responsible for the reduction of the PQ pool and provide a mechanism by which the metabolic demands of NH(4) (+) assimilation may alter the efficiency of photosynthetic light harvesting. NO(3) (-) assimilation did not cause a reduction in PQ and did not affect the efficiency of light harvesting. These results illustrate the role of cellular metabolism in the modulating photosynthetic processes. PMID- 16668217 TI - Photosynthetic and photorespiratory characteristics of flaveria species. AB - The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C(3), C(3)-C(4), C(4)-like, and C(4) plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C(3)-C(4) intermediates showed an inverse continuum in level of photorespiration and development of the C(4) syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C(4) photosynthesis to F. Among the intermediates, the photosynthetic CO(2) compensation points at 30 degrees C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C(4)-like species varied from 3 to 6 microbars, similar to those measured for the C(4) species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C(3) to C(3)-C(4) to C(4)-like and C(4) species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C(3) species, but generally lower activities compared to C(4)-like and C(4) species. The levels of these C(4) enzymes are correlated with the degree of C(4) photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C(4) syndrome in C(3)-C(4)Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C(4) photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C(4) pathway. In the progression from C(3) to C(4) species in Flaveria, the CO(2) compensation point decreased more rapidly than did the decrease in O(2) inhibition of photosynthesis or the increase in the degree of C(4) photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO(2) and prior to substantive reduction in O(2) inhibition and development of the C(4) syndrome. However, further reduction in O(2) inhibition in some intermediates and C(4)-like species is considered primarily due to the development of the C(4) syndrome. Thus, the evolution of C(3)-C(4) intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO(2) concentration first via refixation of photorespired CO(2), followed by development of the C(4) syndrome. PMID- 16668218 TI - Carbon gain and photosynthetic response of chrysanthemum to photosynthetic photon flux density cycles. AB - Most models of carbon gain as a function of photosynthetic irradiance assume an instantaneous response to increases and decreases in irradiance. High- and low light-grown plants differ, however, in the time required to adjust to increases and decreases in irradiance. In this study the response to a series of increases and decreases in irradiance was observed in Chrysanthemum x morifolium Ramat. "Fiesta" and compared with calculated values assuming an instantaneous response. There were significant differences between high- and low-light-grown plants in their photosynthetic response to four sequential photosynthetic photon flux density (PPFD) cycles consisting of 5-minute exposures to 200 and 400 micromoles per square meter per second (mumol m(-2)s(-1)). The CO(2) assimilation rate of high-light-grown plants at the cycle peak increased throughout the PPFD sequence, but the rate of increase was similar to the increase in CO(2) assimilation rate observed under continuous high-light conditions. Low-light leaves showed more variability in their response to light cycles with no significant increase in CO(2) assimilation rate at the cycle peak during sequential cycles. Carbon gain and deviations from actual values (percentage carbon gain over- or underestimation) based on assumptions of instantaneous response were compared under continuous and cyclic light conditions. The percentage carbon gain overestimation depended on the PPFD step size and growth light level of the leaf. When leaves were exposed to a large PPFD increase, the carbon gain was overestimated by 16 to 26%. The photosynthetic response to 100 mumol m(-2) s(-1) PPFD increases and decreases was rapid, and the small overestimation of the predicted carbon gain, observed during photosynthetic induction, was almost entirely negated by the carbon gain underestimation observed after a decrease. If the PPFD cycle was 200 or 400 mumol m(-2) s(-1), high- and low-light leaves showed a carbon gain overestimation of 25% that was not negated by the underestimation observed after a light decrease. When leaves were exposed to sequential PPFD cycles (200-400 mumol m(-2) s(-1)), carbon gain did not differ from leaves exposed to a single PPFD cycle of identical irradiance integral that had the same step size (200-400-200 mumol m(-2) s(-1)) or mean irradiance (200 300-200 mumol m(-2) s(-1)). PMID- 16668219 TI - Implication of Gibberellins in Head Smut (Sporisorium reilianum) of Sorghum bicolor. AB - The head smut fungus, Sporisorium reilianum ([Kuhn] Landon and Fullerton), was shown to reduce plant height in infected Sorghum bicolor ([L.] Moench) plants. The major reductions occurred in the internodes nearest the panicle and were more severe in naturally infected than in inoculated plants. Less affected plants developed reproductively sterile panicles, and eventually smutted panicles developed phyllodied growths which progressed into leafy shoots. Extracts of smutted, sterile, and healthy (control) panicles of field-grown plants exhibited gibberellin (GA)-like activity in the dwarf rice bioassay. When extracts were purified and assayed with deuterium-labeled GA standards by gas chromatography mass spectrometry-selected ion monitoring (GC-MS-SIM), GA(1), GA(3), GA(19), GA(20), and GA(53) were detected based on coelution with the standards, identical Kovats retention index values, and matching ion masses and relative abundances for three major ions. In addition, based on published Kovats retention index values, ion masses, and relative abundance values, GA(4), GA(7), GA(8), GA(14), GA(29), and GA(44) were tentatively identified. Quantitative analysis revealed that panicles of healthy control plants contained from 60 to 100% higher total concentrations of GAs than panicles of smutted plants. These comparisons were most striking for the early 13-hydroxylation pathway precursors GA(53), GA(44), and GA(19) but not for GA(20). Extracts of S. reilianum sporidia and culture medium exhibited GA-like bioactivity, and GA(1) and GA(3) were detected based on GC-MS-SIM assay with (2)H-labeled internal standards. Quantitative analysis of these GAs showed increasing concentrations from 4 to 7 to 10 days of culture and a decline at 20 days. This is the first GC-MS-SIM detection of GAs in a non Ascomycete fungus, and the disease symptoms and quantitative data suggested that fungal infection may interfere with biosynthesis of GAs by the host plant. PMID- 16668220 TI - Novel Technique for Measuring Tissue Firmness within Tomato (Lycopersicon esculentum Mill.) Fruit. AB - Developmental changes of tomato (Lycopersicon esculentum) fruit tissues during maturation were analyzed by a physically defined method (stress-relaxation analysis). The tip of a conical probe connected to a load sensor was positioned on the cut surface of a sliced tomato fruit, and the decay of the imposed stress was monitored. Stress-relaxation data thus obtained were used for the calculation of three stress-relaxation parameters. Different zones within tomato fruit harvested at six different ripening stages were analyzed. One of the stress relaxation parameters, minimum stress-relaxation time (T(0)), decreased as the fruits matured. The decrease in T(0) was first found in the core of the carpel junction within the endopericarp at the blossom end during the breaker stage. The decrease in T(0) progressed from the blossom end, through the equatorial region and finally throughout the shoulder, as the fruit matured. In mature green fruit, T(0) values within the placenta and the proximal carpel junction were lower than those by other parts of the fruit. For all measurements the maximum stress relaxation time was not substantially changed during maturation, nor were their changes observed in different regions of the fruit. The observed relaxation rate was therefore correlated with softening. The results indicate that fruit softening may be physically associated with the stress-relaxation parameter, T(0), and the extent of softening is a function of position within the fruit. Decreases in T(0) value appear to be correlated with the reported regional variation in the appearance of polygalacturonase. PMID- 16668221 TI - Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles. AB - Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl(1)Glc(1)), xyloglucan heptasaccharides (Xyl(3)Glc(4)), and octasaccharides (Gal(1)Xyl(3)Glc(4)). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin induced elongation or cell wall loosening of oat coleoptiles. PMID- 16668222 TI - Solubilization and reconstitution of ca pump from corn leaf plasma membrane. AB - The Ca(2+) transport system of corn (Zea mays) leaf plasma membrane is composed of Ca(2+) pump and Ca(2+)/H(+) antiporter driven by H(+) gradient imposed by a H(+) pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca(2+) transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca(2+) pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C(12)E(8)) was the most effective detergent for a series of extraction and functional reconstitution of the Ca(2+) pump among seven detergents examined. This was judged from activities of ATP dependent (45)Ca(2+) uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C(12)E(8)-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca(2+)-ATPase was separated from VO(4) (3-)-sensitive Mg(2+)-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca(2+) uptake was measured. The liposomes reconstituted with the Ca(2+)-ATPase, but not with the VO(4) (3-) sensitive Mg(2+)-ATPase, showed ATP-dependent Ca(2+) uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca(2+) uptake into liposomes reconstituted with the Ca(2+)-ATPase, suggesting that the Ca(2+)/H(+) antiporter was not present in the preparation. These results indicate that the Ca(2+)-ATPase actually functions as Ca(2+) pump in the corn leaf plasma membrane. PMID- 16668223 TI - Ethylene Biosynthesis-Inducing Xylanase : III. Product Characterization. AB - Induction of ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs by the ethylene biosynthesis-inducing xylanase (EIX) isolated from Cellulysin or xylan-grown cultures of Trichoderma viride was dependent upon the concentration of xylanase applied and upon the length of incubation. Arrhenius activation energies of 9,100 and 10,500 calories for the Cellulysin and T. viride EIX xylanase activities, respectively, were derived from the K(m) and V(max) values determined for each enzyme at several temperatures. The two xylanases digested xylan in a strictly endo fashion, releasing neither xylobiose nor free xylose, and no debranching activity was associated with either enzyme. The xylanases released polysaccharides from ground corn cobs, but little or no carbohydrate was released from tobacco mesophyll cell walls incubated with EIX. No heat-stable products capable of inducing ethylene biosynthesis in tobacco leaf discs were found in EIX digests of purified xylans. PMID- 16668224 TI - Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidase. AB - Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H(2)O(2) via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue. PMID- 16668225 TI - Effect of Gabaculine on the Synthesis of Heme and Cytochrome f in Etiolated Wheat Seedlings. AB - The effect of gabaculine (3-amino 2,3-dihydrobenzoic acid), an inhibitor of tetrapyrrole synthesis, on the accumulation of heme and cytochrome f in etiolated wheat (Triticum aestivum var Mardler) seedlings has been examined. Gabaculine treatment resulted in decreased amounts of heme and of holocytochrome f detected spectroscopically and by peroxidase activity after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amount of the cytochrome f polypeptide detected immunochemically on Western blots was much less affected by gabaculine treatment, indicating that apocytochrome f synthesis was not tightly coupled to heme availability. Gabaculine treatment did not affect the size of the cytochrome f polypeptide, indicating that heme addition is not required for proteolytic removal of the presequence. PMID- 16668226 TI - Comparison of Modeled and Observed Environmental Influences on the Stable Oxygen and Hydrogen Isotope Composition of Leaf Water in Phaseolus vulgaris L. AB - In this paper we describe how a model of stable isotope fractionation processes, originally developed by H. Craig and L. I. Gordon ([1965] in E Tongiorgi, ed, Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Paleotemperature, Spoleto, Italy, pp 9-130) for evaporation of water from the ocean, can be applied to leaf transpiration. The original model was modified to account for turbulent conditions in the leaf boundary layer. Experiments were conducted to test the factors influencing the stable isotopic composition of leaf water under controlled environment conditions. At steady state, the observed leaf water isotopic composition was enriched above that of stem water with the extent of the enrichment dependent on the leaf-air vapor pressure difference (VPD) and the isotopic composition of atmospheric water vapor (AWV). The higher the VPD, the larger was the observed heavy isotope content of leaf water. At a constant VPD, leaf water was relatively depleted in heavy isotopes when exposed to AWV with a low heavy isotope composition, and leaf water was relatively enriched in heavy isotopes when exposed to AWV with a large heavy isotope composition. However, the observed heavy isotope composition of leaf water was always less than that predicted by the model. The extent of the discrepancy between the modeled and observed leaf water isotopic composition was a strong linear function of the leaf transpiration rate. PMID- 16668227 TI - Xylem sap proteins. AB - Xylem sap from apple (Malus domestica Borkh), peach (Prunus persica Batsch), and pear (Pyrus communis L.) twigs was collected by means of pressure extrusion. This sap contained a number of acidic peroxidases and other proteins. Two other sources of xylem sap used in this study were stem exudates and guttation fluid. Similar peroxidases were also found in stem exudates and guttation fluids of strawberry (Fragaria x ananassa Duch.), tomato (Lycopersicum esculentum L.), and cucumber (Cucumis sativus L.). Isoelectric focusing activity gels showed that two peroxidases (isoelectric point [pl] 9 and pl 4.6) were present in initial stem exudates collected in the first 30 minutes after excision. Subsequent samples of stem exudate collected contained only the pl 4.6 isozyme. The pl 4.6 peroxidase isozyme was also found in root tissue and guttation fluid. These observations suggest that roots produce and secrete the pl 4.6 peroxidase into xylem sap. Cucumber seedlings were treated with 100 microliters per liter ethylene for 16 hours and the exudate from decapitated hypocotyl stumps was collected over a 3 hour period. Ethylene increased the peroxidase activity of stem exudates and inhibited the amount of exudate released. These observations suggest that xylem sap peroxidase may play a role in plugging damaged vascular tissue. PMID- 16668228 TI - Characterization of Fatty Acid biosynthesis in isolated pea root plastids. AB - Fatty acid biosynthesis from Na[1-(14)C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl(2), 1 millimolar each of MnCl(2) and glycerol-3-phosphate, 15 millimolar KHCO(3), 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35 degrees C. At the standard incubation temperature of 25 degrees C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg(2+) and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [(14)C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions. PMID- 16668229 TI - Evidence for a nonstatistical carbon isotope distribution in natural glucose. AB - The relative carbon isotope content (delta(13)C value) in each position of glucose from a C(4) plant (maize starch) and a C(3) plant (sugar beet sucrose) has been determined by stepwise chemical and biochemical degradation of the molecule and stable isotope ratio measurement of the fragments. The suitability of the degradation methods has been tested through their chemical yield and isotope balance. The results from both methods agreed perfectly, revealing a defined and reproducible (13)C distribution in glucose from both origins. Most prominent was a relative (13)C enrichment by 5 to 6 delta-units in position 4 and a depletion by about 5 delta-units in carbon 6. As possible reasons for these nonstatistical isotope distributions, isotope effects of the aldolase, the triose phosphate isomerase, and the transketolase reactions during carbohydrate biosynthesis are discussed. The practical importance of the results in regard to isotope distributions in secondary plant products as a means for food authenticity control is outlined. PMID- 16668230 TI - Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves. AB - The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K(+). The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (DeltapH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K(+)-selective ionophore, valinomycin, stimulated uptake in the presence of a DeltapH, and the protonophore, carbonyl cyanide m chlorophenylhydrazone (CCCP), greatly inhibited DeltapH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H(+)/sucrose symporter in PM vesicles isolated from spinach leaves. PMID- 16668231 TI - Study of glucose starvation in excised maize root tips. AB - Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4 dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. During starvation the sugar content sharply decreased for the first 20 hours and reached zero at 120 hours. Following root excision, proteins and lipids were continuously degraded and were virtually the only substrates for respiration and biosyntheses after 20 hours of starvation. Over the first 90 hours of starvation, enzymic activities related to sugar metabolic pathways and the Krebs cycle decreased to 20% or less of their initial activity. Starvation was reversible only for the first 80 to 90 hours. Between 80 and 100 hours, there was a sharp fall in intracellular osmolarity and a 25% loss in the dry weight. The irreversibility may be due, as in senescence, to a change in membrane selective permeability. PMID- 16668232 TI - Root restriction as a factor in photosynthetic acclimation of cotton seedlings grown in elevated carbon dioxide. AB - Interactive effects of root restriction and atmospheric CO(2) enrichment on plant growth, photosynthetic capacity, and carbohydrate partitioning were studied in cotton seedlings (Gossypium hirsutum L.) grown for 28 days in three atmospheric CO(2) partial pressures (270, 350, and 650 microbars) and two pot sizes (0.38 and 1.75 liters). Some plants were transplanted from small pots into large pots after 20 days. Reduction of root biomass resulting from growth in small pots was accompanied by decreased shoot biomass and leaf area. When root growth was less restricted, plants exposed to higher CO(2) partial pressures produced more shoot and root biomass than plants exposed to lower levels of CO(2). In small pots, whole plant biomass and leaf area of plants grown in 270 and 350 microbars of CO(2) were not significantly different. Plants grown in small pots in 650 microbars of CO(2) produced greater total biomass than plants grown in 350 microbars, but the dry weight gain was found to be primarily an accumulation of leaf starch. Reduced photosynthetic capacity of plants grown at elevated levels of CO(2) was clearly associated with inadequate rooting volume. Reductions in net photosynthesis were not associated with decreased stomatal conductance. Reduced carboxylation efficiency in response to CO(2) enrichment occurred only when root growth was restricted suggesting that ribulose-1,5-bisphosphate carboxylase/oxygenase activity may be responsive to plant source-sink balance rather than to CO(2) concentration as a single factor. When root-restricted plants were transplanted into large pots, carboxylation efficiency and ribulose 1,5-bisphosphate regeneration capacity increased indicating that acclimation of photosynthesis was reversible. Reductions in photosynthetic capacity as root growth was progressively restricted suggest sink-limited feedback inhibition as a possible mechanism for regulating net photosynthesis of plants grown in elevated CO(2). PMID- 16668233 TI - Zeaxanthin Formation and Energy-Dependent Fluorescence Quenching in Pea Chloroplasts under Artificially Mediated Linear and Cyclic Electron Transport. AB - Artificially mediated linear (methylviologen) and cyclic (phenazine methosulfate) electron transport induced zeaxanthin-dependent and independent (constitutive) nonphotochemical quenching in osmotically shocked chloroplasts of pea (Pisum sativum L. cv Oregon). Nonphotochemical quenching was quantitated as Stern-Volmer quenching (SV(N)) calculated as (F(m)/F'(m))-1 where F(m) is the fluorescence intensity with all PSII reaction centers closed in a nonenergized, dark-adapted state and F'(m) is the fluorescence intensity with all PSII reaction centers closed in an energized state. Reversal of quenching by nigericin and electron transport inhibitors showed that both quenching types were energy-dependent SV(N). Under light-induced saturating DeltapH, constitutive-SV(N) reached steady state in about 1 minute whereas zeaxanthin-SV(N) continued to develop for several minutes in parallel with the slow kinetics of violaxanthin deepoxidation. SV(N) above the constitutive level and relative zeaxanthin concentration showed high linear correlations at steady-state and during induction. Furthermore, F(o) quenching, also treated as Stern-Volmer quenching (SV(O)) and calculated as (F(o)/F'(o))-1, showed high correlation with zeaxanthin and consequently with SV(N) (F(o) and F'(o) are fluorescence intensities with all PSII reaction centers in nonenergized and energized states, respectively). These results support the view that zeaxanthin increases SV(N) above the constitutive level in a concentration-dependent manner and that zeaxanthin-dependent SV(N) occurs in the pigment bed. Preforming zeaxanthin increased the rate and extent of SV(N), indicating that slow events other than the amount of zeaxanthin also affect final zeaxanthin-SV(N) expression. The redox state of the primary electron acceptor of photosystem II did not appear to determine SV(N). Antimycin, when added while chloroplasts were in a dark-adapted or nonenergized state, inhibited both zeaxanthin-SV(N) and constitutive-SV(N) induced by linear and cyclic electron transport. These similarities, including possible constitutive F(o) quenching, suggest that zeaxanthin-dependent and constitutive SV(N) are mechanistically related. PMID- 16668234 TI - Aluminum and Temperature Alteration of Cell Membrane Permeability of Quercus rubra. AB - This report extends research on Al-induced changes in membrane behavior of intact root cortex cells of Northern red oak (Quercus rubra). Membrane permeability was determined by the plasmometric method for individual intact cells at temperatures from 2 or 4 to 35 degrees C. Al (0.37 millimolar) significantly increased membrane permeability to urea and monoethyl urea and decreased permeability to water. Al significantly altered the activation energy required to transport water (+32%), urea (+9%), and monoethyl urea (-7%) across cell membranes. Above 9 degrees C, Al increased the lipid partiality of the cell membranes; below 7 degrees C, Al decreased it. Al narrowed by 6 degrees C the temperature range over which plasmolysis occurred without membrane damage. These changes in membrane behavior are explainable if Al reduces membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains. PMID- 16668235 TI - Response of photosynthesis and cellular antioxidants to ozone in populus leaves. AB - Atmospheric ozone causes formation of various highly reactive intermediates (e.g. peroxyl and superoxide radicals, H(2)O(2), etc.) in plant tissues. A plant's productivity in environments with ozone may be related to its ability to scavenge the free radicals formed. The effects of ozone on photosynthesis and some free radical scavengers were measured in the fifth emergent leaf of poplars. Clonal poplars (Populus deltoides x Populus cv caudina) were fumigated with 180 parts per billion ozone for 3 hours. Photosynthesis was measured before, during, and after fumigation. During the first 90 minutes of ozone exposure, photosynthetic rates were unaffected but glutathione levels and superoxide dismutase activity increased. After 90 minutes of ozone exposure, photosynthetic rates began to decline while glutathione and superoxide dismutase continued to increase. Total glutathione (reduced plus oxidized) increased in fumigated leaves throughout the exposure period. The ratio of GSH/GSSG also decreased from 12.8 to 1.2 in ozone exposed trees. Superoxide dismutase levels increased twofold in fumigated plants. After 4 hours of ozone exposure, the photosynthetic rate was approximately half that of controls while glutathione levels and superoxide dismutase activity remained above that of the controls. The elevated antioxidant levels were maintained 21 hours after ozone exposure while photosynthetic rates recovered to about 75% of that of controls. Electron transport and NADPH levels remained unaffected by the treatment. Hence, elevated antioxidant metabolism may protect the photosynthetic apparatus during exposure to ozone. PMID- 16668236 TI - Peptidyl proline hydroxylation and the growth of a soybean cell culture. AB - Peptidyl proline hydroxylase inhibitors block the growth of cultured soybean (Glycine max) cells and bring about the disappearance of the major salt extractable hydroxyproline-rich protein, the 33 kilodalton repetitive proline rich protein (RPRP2). Three polypeptides of 28, 20, and 14 kilodalton that cross react with an antibody to RPRP2 accumulate in the culture during steady-state growth. In the presence of the proline hydroxylase inhibitors, all of these repetitive proline-rich proteins disappear. These results indicate that the hydroxyproline-rich proteins play a role in cell growth, and that hydroxylation may regulate the steady-state level of at least one of these proteins by influencing its turnover. PMID- 16668237 TI - Glass transitions in soybean seed : relevance to anhydrous biology. AB - We have investigated the mechanism by which anhydrobiotic organisms can survive severe dehydration. The method used was measurement of the rotational diffusion coefficient of a hydrophilic spin probe, inserted in the cytoplasm of soybean (Glycine max L.) axes, as a function of temperature and sample water content. Results indicate the existence of a hydration-dependent glass-like transition at physiological temperatures. No glass transitions have been observed in desiccation-intolerant samples, suggesting that the ability to withstand dehydration is associated with glass formation. PMID- 16668238 TI - Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases. AB - Activity levels of UDP-glucose: (1,3)-beta-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1 phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A(2), C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action. PMID- 16668239 TI - A Flower Senescence-Related mRNA from Carnation Shares Sequence Similarity with Fruit Ripening-Related mRNAs Involved in Ethylene Biosynthesis. PMID- 16668240 TI - Multiple forms of plant cytochromes p-450. AB - Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases are implicated in the metabolism of sterols, terpenes, gibberellins, isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 are involved in the detoxification of herbicides (chlorotoluron, primsulfuron, and diclofop) includes photoreversible CO inhibition of the reactions, and a requirement for O(2) and NADPH. Several cytochromes P-450, M(r) 45,000 to 65,000, have been isolated, including hydroxylases of cinnamic acid, 3,9-dihydroxypterocarpan, and digitoxin. In some cases the purified cytochrome P-450 has been successfully reconstituted with NADPH:cytochrome P-450 reductase (M(r) 72,000-84,000 protein). This reductase appears to be a nonspecific electron donor to different forms of cytochrome P 450. Immunological techniques and specific inhibitors (triazoles, imidazole derivatives) are being used to characterize plant cytochromes P-450 and the NADPH:cytochrome P-450 reductase. Specific cytochromes P-450 are induced by wounding or pathogens, others are expressed in specific cell types. Plant cytochromes P-450 are found in various subcellular locations, including endoplasmic reticulum, plasma membranes, glyoxysomes, and perhaps mitochondria. A cytochrome P-450 demethylase from avocado has recently been sequenced and found to have a hydrophobic N terminus similar to the membrane anchor of cytochromes P 450 from other organisms. The existence of cytochromes P-450 in different subcellular locations suggests that there are many genes for cytochromes P-450 in plants which have yet to be identified and classified. PMID- 16668241 TI - Secreting glandular trichomes: more than just hairs. AB - Secreting glandular plant trichome types which accumulate large quantities of metabolic products in the space between their gland cell walls and cuticle permit the plant to amass secretions in a compartment that is virtually outside the plant body. These structures not only accumulate and store what are often phytotoxic oils but they position these compounds as an apparent first line of defense at the surface of the plant. Recent advances in methods for isolation and study of trichome glands have allowed more precise analysis of gland cell metabolism and enzymology. Isolation of mutants with altered trichome phenotypes provides new systems for probing the genetic basis of trichome development. These advances and their continuation can pave the way for future attempts at modification of trichome secretion. The biochemical capability of glandular secreting trichomes and the potential for its future manipulation to exploit this external storage compartment is the focus of this review. PMID- 16668242 TI - Flavonoid evolution: an enzymic approach. AB - Flavonoid evolution in land plants is discussed from an enzymic point of view, based on the present day distribution of the major subgroups of flavonoids in bryophytes, lower and higher vascular plants. The importance of varied functions in the origin of pathways with a series of sequential steps leading to end products is considered; it is argued that the initial function is that of an internal regulatory agent, rather than as a filter against ultraviolet irradiation. The basic synthases, hydroxylases, and reductases of flavonoid pathways are presumed to have evolved from enzymes of primary metabolism. A speculative scheme is presented of flavonoid evolution within a primitive group of algae derived from a Charophycean rather than a Chlorophycean line, as a land environment was invaded. Flavonoid evolution was preceded by that of the phenylpropanoid and malonyl-coenzyme A pathways, but evolved prior to the lignin pathway. PMID- 16668243 TI - Photoperiod control of poplar bark storage protein accumulation. AB - Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32 kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP. PMID- 16668244 TI - Purification and characterization of galactinol synthase from mature zucchini squash leaves. AB - Galactinol synthase catalyzes the first committed step in the biosynthesis of raffinose sugars. Previous attempts to purify the enzyme have proven difficult and have resulted in low quantities of unpurified enzyme. Galactinol synthase was purified 752-fold from mature zucchini (Cucurbita pepo L. cv Burpee Hybrid) leaves using sequential liquid chromatography on DE 52, Octyl-Sepharose CL-4B, and Sephacryl S-200. This isolation scheme resulted in an 18.6% recovery of the initial activity. The purified enzyme had a specific activity of 23.3 micromoles per minute per milligram protein, a pH optimum of 7.5, and the activity was enhanced by dithiothreitol and MnCl(2). The enzyme was only half as active with MgCl(2) as with MnCl(2). Na(+), K(+), and Ca(2+) cations had little effect on the enzyme activity, while Co(2+), Zn(2+), Cu(2+), and Fe(3+) cations were strongly inhibitory at 10 millimolar concentrations. Purified galactinol synthase bound specifically to the substrates myo-inositol and UDP-galactose (K(m) = 6.5 and 1.8 millimolar, respectively), while exhibiting little affinity for an alternative glycosyl donor (UDP-glucose) or inositol epimers (epi- and scyllo-). Ten millimolar concentrations of UMP, UDP, UTP, AMP, ADP, ATP, NAD(+), NADH, NADP(+), UDP-xylose, and UDP-mannose, or 20 millimolar sucrose, talose, galactose, glucose, xylose, and melibiose exhibited various degrees of inhibitory effects. Twenty millimolar stachyose, raffinose, fructose, and mannose, and 10 millimolar UDP-glucuronic acid and UDP-galacturonic acid had little or no effect on the enzyme activity. The purified galactinal synthase is a monomer of M(r) 42,000 with an isoelectric point of 4.1. PMID- 16668245 TI - Immunocytochemical localization of nitrite reductase in green algae. AB - The distribution of nitrite reductase (EC 1.7.7.1) in the green algae Chlamydomonas reinhardtii, Monoraphidium braunii, Chlorella fusca, and Scenedesmus obliquus was studied by immunoelectron microscopy. The labeling of ultrathin cryosections was performed with anti-nitrite reductase antibodies followed by gold-labeled goat anti-rabbit antibodies. In C. reinhardtii sections, gold label was mainly associated with the pyrenoid, tonoplast, and plasmalemma. Significant labeling was also detected in the thylakoid region. In all other organisms, label density was lower but distributed in the same locations, except that the plasmalemma of S. obliquus was not significantly labeled. From estimates of the relative volume of different cell regions, we found that approximately 80% of the total enzyme is located in the chloroplastic region (thylakoids plus pyrenoid) of C. reinhardtii, M. braunii, and C. fusca, and 97% in the case of S. obliquus. PMID- 16668246 TI - Heterogeneity and cell type-specific localization of a cell wall glycoprotein from carrot suspension cells. AB - EP1, an extracellular protein from carrot (Daucus carota) cell suspensions, has been partially characterized by means of an antiserum and a cDNA clone. In both embryo and suspension cultures different molecular mass EP1 proteins were detected, some of which (31, 32, 52, and 54 kilodaltons) were bound to the cell wall and released into the medium, whereas others (49, 60, and 62 kilodaltons) were more firmly bound to the cell wall and could be extracted with a salt solution. Immunoprecipitation of in vitro translation products revealed a single primary translation product of 45 kilodaltons, suggesting that EP1 heterogeneity is due to differential posttranslational modification. In seedlings organ specific modification of EP1 proteins was observed, a phenomenon which did not persist in suspension cultures initiated from different seedling organs. In culture EP1 proteins were only found to be associated with vacuolated, nonembryogenic cells, and on these cells they were localized in loosely attached, pectin-containing cell wall material. Purified 52/54 kilodaltons EP1 proteins did not alleviate the inhibitory effect of the glycosylation inhibitor tunicamycin on somatic embryogenesis. PMID- 16668247 TI - CO(2)-Enhanced Yield and Foliar Deformation among Tomato Genotypes in Elevated CO(2) Environments. AB - Yield increases observed among eight genotypes of tomato (Lycopersicon esculentum Mill.) grown at ambient CO(2) (about 350) or 1000 microliters per liter CO(2) were not due to carbon exchange rate increases. Yield varied among genotypes while carbon exchange rate did not. Yield increases were due to a change in partitioning from root to fruit. Tomatoes grown with CO(2) enrichment exhibited nonepinastic foliar deformation similar to nutrient deficiency symptoms. Foliar deformation varied among genotypes, increased throughout the season, and became most severe at elevated CO(2). Foliar deformation was positively related to fruit yield. Foliage from the lower canopy was sampled throughout the growing season and analysed for starch, K, P, Ca, Mg, Fe, and Mn concentrations. Foliar K and Mn concentrations were the only elements correlated with deformation severity. Foliar K decreased while deformation increased. In another study, foliage of half the plants of one genotype received foliar applications of 7 millimolar KH(2)PO(4). Untreated foliage showed significantly greater deformation than treated foliage. Reduced foliar K concentration may cause CO(2)-enhanced foliar deformation. Reduced K may occur following decreased nutrient uptake resulting from reduced root mass due to the change in partitioning from root to fruit. PMID- 16668248 TI - Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2). AB - Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O(2) (volume/volume) in N(2). Plant dry matter yield was unaffected by partial pressure of oxygen (pO(2)) and N(2) fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O(2). Slight inhibition of nitrogenase activity occurred at 1% O(2) and as much as 50% inhibition occurred at 80% O(2). Low pO(2) (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO(2) used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO(2) was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO(2) and decreased permeability in those from plants cultured with their roots in pO(2) greater than ambient. A range of structural alterations in nodules resulting from low pO(2) were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO(2) comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O(2). Long-term adaptation to different external pO(2) involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier. PMID- 16668249 TI - Mechanism of aluminum tolerance in snapbeans : root exudation of citric Acid. AB - One proposed mechanism of aluminum (Al) tolerance in plants is the release of an Al-chelating compound into the rhizosphere. In this experiment, two cultivars of snapbeans (Phaseolus vulgaris L. "Romano" and "Dade") that differ in Al tolerance were grown hydroponically with and without Al under aseptic conditions. After growth in nutrient solutions for 8 days, aliphatic and phenolic organic acids were analyzed in the culture solutions with an ion chromatograph and a high pressure liquid chromatograph. The tolerant snapbean, "Dade", when exposed to Al, exuded citric acid into the rhizosphere in a concentration that was 70 times as great as that of "Dade" grown without Al, and 10 times as great as that of "Romano" grown with or without Al. The sensitive cultivar, "Romano", exuded only slightly more citric acid into the growing medium under Al-stress, compared to nonstressed conditions. Citric acid is known to chelate Al strongly and to reverse its phytotoxic effects. Also, citric acid has been shown previously to enhance the availability of phosphorus (P) from insoluble Al phosphates. Thus, one mechanism of Al-tolerance in snapbeans appears to be the exudation of citric acid into the rhizosphere, induced either by toxic levels of Al or by low P due to the precipitation of insoluble Al phosphates. Our experiment was not able to distinguish between these two factors; however, tolerance to both primary and secondary Al-stress injuries are important for plants growing in Al-toxic soils. PMID- 16668250 TI - Biochemical characterization of a spearmint mutant that resembles peppermint in monoterpene content. AB - A radiation-induced mutant of Scotch spearmint (Mentha x gracilis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the C6-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and C6 oxygenated families of monoterpenes from the common precursor (-)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (-)-limonene hydroxylase; the C6-hydroxylase producing (-)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (-)-trans isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the alpha,beta unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3 oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3- and C6-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha. PMID- 16668251 TI - Blue light-modulation of chlorophyll a fluorescence transients in guard cell chloroplasts. AB - Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO(2) concentration on the fluorescence transients was investigated in the presence of HCO(3) (-) at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO(2) concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations. PMID- 16668252 TI - 5-Aminolevulinic Acid Induced Photodynamic Damage of the Photosynthetic Electron Transport Chain of Cucumber (Cucumis sativus L.) Cotyledons. AB - Cucumber (Cucumis sativus L. cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid (ALA) and then incubated in dark for 14 hours. Upon transfer to sunlight ( approximately 800 watts per square meter) the plants died after 5 hours of exposure due to photosensitization reaction of metalloporphyrins. Due to the photodynamic damage, photosystem II (PSII), photosystem I (PSI), and whole chain reactions were impaired. PSII activity was more susceptible to photodynamic damage than PSI. The variable fluorescence was significantly reduced in ALA-treated plants within 1 hour of exposure to sunlight. At low temperature (77 degrees K), the PSI fluorescence peak height (F(734)) was drastically reduced and blue shifted by 6 nanometers. The photodynamic damage was irreversible; rather, it continued upon dark incubation of ALA-treated cucumber plants exposed to sunlight for 15 minutes. In the latter experiment, continued production of malondialdehyde during dark treatment suggests the degradation of unsaturated membrane lipids. PMID- 16668253 TI - Wheat Inhibitors of Heterologous alpha-Amylases : Characterization of Major Components from the Monomeric Class. AB - The four major components of the wheat monomeric alpha-amylase inhibitors (WMAI) from wheat, Triticum aestivum, endosperm have been isolated and characterized. Two of them, WMAI-1 and WMAI-2, are highly active against the alpha-amylase from the insect Tenebrio molitor and their N-terminal amino acid sequences indicate that they are closely related to each other (86% identical residues) and to the other members of the family (subunits of dimeric and tetrameric alpha-amylase inhibitors and trypsin inhibitors). WMAI-1, which is identical to the previously described 0.28 inhibitor, is encoded by a gene located in the short arm of chromosome 6D and WMAI-2 by a gene in the short arm of chromosome 6B. Components 3 and 4, which have blocked N-terminal residues, have identical internal amino acid sequences and are a separate class of proteins with respect to WMAI-1 and WMAI-2, although their amino acid composition and apparent molecular weights are quite similar. Their inhibitory activity versus alpha-amylases is either unstable during the purification process or due to contamination with other inhibitors. PMID- 16668254 TI - Characterization of Tobacco Expressing Functional Oat Phytochrome : Domains Responsible for the Rapid Degradation of Pfr Are Conserved between Monocots and Dicots. AB - Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome-far red-absorbing form (Pfr) to phytochrome-red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species. PMID- 16668255 TI - Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth. AB - Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation. PMID- 16668256 TI - Wounding Nicotiana tabacum Leaves Causes a Decline in Endogenous Indole-3-Acetic Acid. AB - We have previously observed that auxin can act as a repressor of the wound inducible activation of a chimeric potato proteinase inhibitor II-CAT chimeric gene (pin2-CAT) in transgenic tobacco (Nicotiana tobacum) callus and in whole plants. Therefore, this study was designed to examine endogenous levels of indole 3-acetic acid (IAA) in plant tissues both before and after wounding. Endogenous IAA was measured in whole plant tissues by gas chromatography-mass spectrometry using an isotope dilution technique. (13)C-Labeled IAA was used as an internal standard. The endogenous levels of IAA declined two- to threefold within 6 hours after a wound. The kinetics of auxin decline are consistent with the kinetics of activation of the pin2-CAT construction in the foliage of transgenic tobacco. PMID- 16668257 TI - Ethylene Promotes Elongation Growth and Auxin Promotes Radial Growth in Ranunculus sceleratus Petioles. AB - Submergence induces elongation in the petioles of Ranunculus sceleratus L., after a rise in endogenous ethylene levels in the tissue. Petioles of isolated leaves also elongate 100% in 24 hours when treated with ethylene gas, without a change in the radius. Application of silver thiosulfate, aminoethoxyvinylglycine (AVG), abscisic acid (ABA), or methyl jasmonate inhibits this elongation response. Gibberellic acid treatment promotes ethylene-induced elongation, without an effect on the radius. Indoelastic acid (IAA) induces radial growth in the petioles, irrespective of the presence or absence of added ethylene. High concentrations of IAA will also induce elongation growth, but this is largely due to auxin-induced ethylene synthesis; treatment with silver thiosulfate, AVG, ABA, or methyl jasmonate inhibit this auxin-promoted elongation growth. However, the radial growth induced by IAA is not affected by gibberellic acid, and not specifically inhibited by ABA, methyl jasmonate, silver thiosulfate, or AVG. These results support the idea that petiole cell elongation during "accommodation growth" can be separated from radial expansion. The radial expansion may well be regulated by IAA. However, effects of high levels of IAA are probably anomalous, since they do not mimic normal developmental patterns. PMID- 16668258 TI - Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules. AB - Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of O(2). The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high pO(2). Nodule contents of ascorbate and reduced glutathione were also greater in the high pO(2) treatments. Treatment of nodulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N(2) fixation and defenses against activated forms of oxygen. PMID- 16668259 TI - Two distinct steps for spontaneous generation of subprotoplasts from a disintegrated bryopsis cell. AB - The unusual nature of protoplasm to generate subprotoplasts spontaneously from disintegrated Bryopsis cells was examined. Protoplasm extruded from algal cells aggregated rapidly in cell sap which was derived mainly from huge central vacuoles of the cells. Electron microscopic observations revealed extensive agglutination of algal cellular membranes in the protoplasmic masses, suggesting that this is of primary importance for the wound-healing ability of the alga. Seawater caused spheration of the resultant protoplasmic aggregates. Gelatinous sheaths were formed temporarily surrounding the spherical protoplasmic masses before reformation of cell membrane. Staining with phosphotungstic and chromic acids suggested that new cell membrane was formed by fusion of the disintegrated original cell membrane with cytoplasmic vesicles on the surfaces of the protoplasmic masses. Both pH and salts were found to be essentially important at the two steps of subprotoplast generation. The newly formed cell membranes were responsible for subsequent notable plasmolysis of the wounded cells in seawater. Thus, it is suggested that unicellular marine algae Bryopsis spp. naturally contain effective materials for agglutinating and fusing particular cellular membranes through the sequential aid of acidic cell sap and alkaline seawater after disintegration of the giant cells. PMID- 16668260 TI - Lipid peroxidation in peribacteroid membranes from French-bean nodules. AB - Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H(2)O(2), membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H(2)O(2). Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (III) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation. PMID- 16668261 TI - Circadian Rhythms in Photosynthesis : Oscillations in Carbon Assimilation and Stomatal Conductance under Constant Conditions. AB - Net carbon assimilation and stomatal conductance to water vapor oscillated repeatedly in red kidney bean, Phaseolus vulgaris L., plants transferred from a natural photoperiod to constant light. In a gas exchange system with automatic regulation of selected environmental and physiological variables, assimilation and conductance oscillated with a free-running period of approximately 24.5 hours. The rhythms in carbon assimilation and stomatal conductance were closely coupled and persisted for more than a week under constant conditions. A rhythm in assimilation occurred when either ambient or intercellular CO(2) partial pressure was held constant, demonstrating that the rhythm in assimilation was not entirely the result of stomatal effects on CO(2) diffusion. Rhythms in assimilation and conductance were not expressed in plants grown under constant light at a constant temperature, demonstrating that the rhythms did not occur spontaneously but were induced by an external stimulus. In plants grown under constant light with a temperature cycle, a rhythm was entrained in stomatal conductance but not in carbon assimilation, indicating that the oscillators driving the rhythms differed in their sensitivity to environmental stimuli. PMID- 16668262 TI - Separation of Light-Induced [C]ent-Kaurene Metabolism and Light-Induced Germination in Grand Rapids Lettuce Seeds. AB - The effect of light on the metabolism of [(14)C]kaurene in light-requiring lettuce seeds (Lactuca sativa L. cv Grand Rapids) was investigated. Seeds were soaked in a solution of [(14)C]ent-kaurene in methylene chloride with 0.01% Tween 20, dried, and incubated in 20% polyethylene glycol (PEG) to prevent seedling development. Labeled metabolites were extracted and analyzed by high performance liquid chromatography and gas chromatography-radio counting. [(14)C]ent-Kaurenol and [(14)C]ent-kaurenal were identified in seeds incubated in constant white light, while no ethyl acetate-soluble metabolites were found in seeds incubated in the dark. In time course experiments using acid scarified seeds, metabolism began after 18 hours of incubation and greatly increased after 24 hours of incubation in 20% PEG. By 48 hours, several unidentified, more polar metabolites were found. Germination was induced in seeds imbibed in 20% PEG by 4 hours of red or 4 hours of white light following 20 hours in the dark, and was fully reversed by 2 hours of far red light. However, in metabolism experiments, [(14)C]ent kaurene oxidation was observed only with constant white light. These results indicate that although ent-kaurene oxidation is a light sensitive step in the biosynthesis of gibberellins in Grand Rapids lettuce seeds, ent-kaurene metabolism is not required for light-induced germination. PMID- 16668263 TI - Effect of fe-catalyzed photooxidation of EDTA on root growth in plant culture media. AB - Light from fluorescent lamps can induce formaldehyde production and iron deficiency in plant nutrient culture medium. Formaldehyde is produced from EDTA when it is oxidized by the photochemical reduction of ferric iron and it can accumulate to inhibitory levels. The medium becomes iron deficient because iron becomes unchelated and forms an unavailable precipitate as EDTA is oxidized. The combination of light-induced formaldehyde production and loss of available iron reduces the ability of the culture media to support growth of Arabidopsis thaliana roots. Removing ultraviolet and blue wavelengths with a yellow acrylic filter is a simple and effective means of preventing Fecatalyzed photooxidation of EDTA in plant culture media. PMID- 16668264 TI - Gum arabic glycoprotein is a twisted hairy rope : a new model based on o galactosylhydroxyproline as the polysaccharide attachment site. AB - Separation of the wound exudate from Acacia senegal (L.) Willd., "gum arabic," on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glycoprotein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogenous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large ( approximately 400 residue) hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small ( approximately 30 residue) hydroxyproline (Hyp)-polysaccharide substituents. After partial acid hydrolysis of the Hyp-polysaccharide fraction we identified O-galactosylhydroxyproline as the glycopeptide linkage, identical with that of hydroxyproline-rich arabinogalactan-proteins (AGPs). However, unlike the acidic alanine-rich AGPs, GAGP is basic and notably deficient in alanine. Thus, while the GAGP polypeptide backbone more closely resembles that of the Hyp-rich cell wall protein extensin, the GAGP polysaccharide sidechains resemble AGPs. Possibly all three proteins comprise a phylogenetically related extensin superfamily of extended rod-like macromolecules. The "wattle-blossom" model for AGP and gum arabic predicts a few large polysaccharide substituents along the polypeptide backbone of a spheroidal macromolecule. On the contrary, our data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptide backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide-peptide subunit of about 7 kilodaltons. The small polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain how such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall. PMID- 16668265 TI - Effect of Exogenous Indole-3-Acetic Acid and Indole-3-Butyric Acid on Internal Levels of the Respective Auxins and Their Conjugation with Aspartic Acid during Adventitious Root Formation in Pea Cuttings. AB - The influence of exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on the internal levels of these auxins was studied during the first 4 days of adventitious root formation in cuttings of Pisum sativum L. The quantitations were done by high performance liquid chromatography with spectrofluorometric detection. IBA, identified by combined gas chromatography-mass spectrometry (GC MS), was found to naturally occur in this plant material. The root inducing ability of exogenous IBA was superior to that of IAA. The IAA level in the tissue increased considerably on the first day after application of IAA, but rapidly decreased again, returning to a level twice the control by day 3. The predominant metabolic route was conjugation with aspartic acid, as reflected by the increase in the level of indole-3-acetylaspartic acid. The IBA treatment resulted in increases in the levels of IBA, IAA, and indole-3-acetylaspartic acid. The IAA content rapidly returned to control levels, whereas the IBA level remained high throughout the experimental period. High amounts of indole-3-butyrylaspartic acid were found in the tissue after feeding with IBA. The identity of the conjugate was confirmed by (1)H-nuclear magnetic resonance and GC-MS. IBA was much more stable in solution than IAA. No IAA was detected after 48 hours, whereas 70% IBA was still recovered after this time. The relatively higher root inducing ability of IBA is ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. Adventitious root formation is discussed on the basis of these findings. PMID- 16668266 TI - Isoenzymes of glucose 6-phosphate dehydrogenase from the plant fraction of soybean nodules. AB - Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH(4))(2)SO(4) gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP(+) and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP(+). The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP(+) and double this size in the presence of NADP(+). The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP(+) at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP(+) and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP(+) and 70 to 100 micromolar glucose 6 phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6 phosphate dehydrogenases. PMID- 16668267 TI - Maturation proteins associated with desiccation tolerance in soybean. AB - A set of proteins that accumulates late in embryogenesis (Lea proteins) has been hypothesized to have a role in protecting the mature seed against desiccation damage. A possible correlation between their presence and the desiccation tolerant state in soybean seeds (Glycine max L. Chippewa) was tested. Proteins that showed the same temporal pattern of expression as that reported for Lea proteins were identified in the axes of soybean. They were distinct from the known storage proteins and were resistant to heat coagulation. The level of these "maturation" proteins was closely correlated with desiccation tolerance both in the naturally developing and in the germinating seed: increasing at 44 days after flowering, when desiccation tolerance was achieved, and decreasing after 18 hours of imbibition, when desiccation tolerance was lost. During imbibition, 100 micromolar abscisic acid or Polyethylene glycol-6000 (-0.6 megapascals) delayed disappearance of the maturation proteins, loss of desiccation tolerance, and germination. During maturation, desiccation tolerance was prematurely induced when excised seeds were dried slowly but not when seeds were held for an equivalent time at high relative humidity. In contrast, maturation proteins were induced under both conditions. We conclude that maturation proteins may contribute to desiccation tolerance of soybean seeds, though they may not be sufficient to induce tolerance by themselves. PMID- 16668268 TI - Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls: Changes in IAA Movement, Net IAA Uptake, and Phytotropin Binding. AB - Basipetal transport of [(14)C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [(3)H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [(3)H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age. PMID- 16668269 TI - Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit. AB - Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K(m) values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K(m) value for sucrose, and substrate specificity. PMID- 16668270 TI - Sucrose Synthase Expression during Cold Acclimation in Wheat. AB - When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4 degrees C) above 0 degrees C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23 degrees C to 4 degrees C. The level of SS diminished when plants were moved back to 23 degrees C. Northern blots of poly(A)(+) RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress. PMID- 16668271 TI - Betaines of alfalfa : characterization by fast atom bombardment and desorption chemical ionization mass spectrometry. AB - Leaf tissue of alfalfa (Medicago sativa L.) was found to contain prolinebetaine, pipecolatebetaine, hydroxyprolinebetaine, and glycinebetaine. As n-butyl esters, these chemical species exhibit molecular cations at mass/charge ratio (m/z) 200, 214, 216, and 174, respectively, when analyzed by fast atom bombardment mass spectrometry. The underivatized betaines exhibit protonated molecular ions at m/z 144, 158, 160, and 118, respectively, when analyzed by desorption chemical ionization mass spectrometry. Extensive (>45-fold) genotypic variation for hydroxyprolinebetaine level was identified in alfalfa. Because a significant inverse correlation between prolinebetaine and hydroxyprolinebetaine levels was observed among 15 alfalfa genotypes evaluated, it is possible that these compounds may be derived from a common intermediate. Birdsfoot trefoil (Lotus corniculatus L.) contained prolinebetaine, but only traces of glycinebetaine, pipecolatebetaine, and hydroxyprolinebetaine. Red clover (Trifolium pratense L.) lacked prolinebetaine, pipecolatebetaine, and hydroxyprolinebetaine, but contained appreciable levels of both glycinebetaine and trigonelline. Trigonelline was not detectable in the leaf tissue of any alfalfa genotype or cultivar evaluated. PMID- 16668272 TI - Nitrate-dependent o(2) evolution in intact leaves. AB - Evolution of O(2) by illuminated intact detached leaves from barley (Hordeum vulgare L. cv Athos) and pea (Pisum sativum L. cv Lincoln) in a CO(2)-saturating atmosphere was enhanced when KNO(3) (1-2.5 millimolar) had been previously supplied through the transpiration stream. The extra O(2) evolution observed after feeding KNO(3) increased with the light intensity, being maximal at near saturating photon flux densities and resulting in no changes in the initial slope of the O(2)versus light-intensity curve. No stimulation of O(2) evolution was otherwise observed after feeding KCl or NH(4)Cl. The data indicate that nitrate assimilation uses photosynthetically generated reductant and stimulates the rate of non-cyclic electron flow by acting as a second electron-accepting assimilatory process in addition to CO(2) fixation. PMID- 16668273 TI - Sucrose-Induced Accumulation of beta-Amylase Occurs Concomitant with the Accumulation of Starch and Sporamin in Leaf-Petiole Cuttings of Sweet Potato. AB - beta-Amylase of sweet potato (Ipomoea batatas L.), which constitutes about 5% of the total soluble protein of the tuberous root, is absent or is present in only small amounts in organs other than the tuberous roots of the normal, field-grown plants. However, when leaf-petiole cuttings from such plants were supplied with a solution that contained sucrose, the accumulation of beta-amylase was induced in both leaf and petiole portions of the explants. The sucrose-induced accumulation of beta-amylase in leaf-petiole cuttings occurred concomitant with the accumulation of starch and of sporamin, the most abundant storage protein of the tuberous root. The accumulation of beta-amylase, of sporamin and of starch in the petioles showed similar dependence on the concentration of sucrose, and a 6% solution of sucrose gave the highest levels of induction when assayed after 7 days of treatment. The induction of mRNAs for beta-amylase and sporamin in the petiole could be detected after 6 hours of treatment with sucrose, and the accumulation of beta-amylase and sporamin polypeptides, as well as that of starch, continued for a further 3 weeks. In addition to sucrose, glucose or fructose, but not mannitol or sorbitol, also induced the accumulation of beta amylase and sporamin, suggesting that metabolic effects of sucrose are important in the mechanism of this induction. Treatment of leaf-petiole cuttings with water under continuous light, but not in darkness, also caused the accumulation of small amounts of these components in the petioles, probably as a result of the endogenous supply of sucrose by photosynthesis. These results suggest that the expression of the gene for beta-amylase is under metabolic control which is coupled with the expression of sink function of cells in the sweet potato. PMID- 16668274 TI - Intraplastidic Localization of the Enzymes That Convert delta-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons. AB - The intraplastidic localization of the enzymes that catalyze the conversion of delta-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five- to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not. PMID- 16668275 TI - Light regulation of sink metabolism in tomato fruit : I. Growth and sugar accumulation. AB - Light/dark effects on growth and sugar accumulation in tomato (Lycopersicon esculentum) fruit during early development were studied on intact plants (in vivo) and in tissue culture (in vitro). Through the use of an in vitro culture of tomato fruit, it was possible to investigate the direct effects of light on sink metabolism by eliminating the source tissue. Similar growth patterns were found in vivo and in vitro. Fruit growth in different sugars indicated that sucrose was the best source of carbon for in vitro fruit growth. Fruit growth increased as sucrose concentration increased up to 8%. Darkening the fruit decreased fruit dry weight about 40% in vivo and in vitro. The differences in the CO(2) exchange rate between light and dark grown fruit indicated that light stimulation of fruit growth was due to mechanisms other than photosynthesis. Supporting this conclusion was the fact that light intensities ranging from 40 to 160 micromoles per square meter per second had no significant influence on fruit growth, and light did not increase growth of fruit cultured with glucose or fructose as a carbon source. However, light stimulated fruit growth significantly when sucrose was used as the carbon source. Light-grown fruit took up 30% more sucrose from the same source and accumulated almost twice as much hexose and starch as dark grown fruit. A possible expansion of an additional sink for carbon by light stimulation of starch synthesis during early development will be discussed. PMID- 16668276 TI - Light Regulation of Sink Metabolism in Tomato Fruit : II. Carbohydrate Metabolizing Enzymes. AB - Effects of light on carbohydrate levels and certain carbon metabolizing enzyme activities were studied during the early development of tomato (Lycopersicon esculentum) fruit. Sucrose levels were low and continued to decline during development and were unaffected by light. Starch was significantly greater in light. Invertase activity was similar in both light- and dark-grown fruit. Sucrose synthase activity was much lower than invertase and showed a slight decrease in light-grown fruit between days 21 and 28. Light-grown fruit also had higher ADP glucose pyrophosphorylase activity than dark-grown fruit, which was correlated with higher starch levels. The rapidly decreasing activity of ADP glucose pyrophosphorylase during early fruit development in the dark in conjunction with reduced starch levels and rates of accumulation indicates that ADP glucose pyrophosphorylase is crucial for carbon import and storage in tomato. The differential stimulation of ADP glucose pyrophosphorylase activity from light and dark-grown tissue by 3-phosphoglycerate suggests that this enzyme may be allosterically altered by light. PMID- 16668277 TI - Growth Enhancement and Developmental Modifications of in Vitro Grown Potato (Solanum tuberosum spp. tuberosum) as Affected by a Nonfluorescent Pseudomonas sp. AB - A plant growth-promoting rhizobacterium, designated Ps JN and isolated from onion roots, was identified as a nonfluorescent Pseudomonas sp. The percentage of similarity of Ps JN to P. gladioli (NCPPB 1891), P. cichorii (NCPPB 943), and P. viridiflava (NCPPB 635), as determined from 135 biochemical and physiological tests was 77, 70, and 66%, respectively. Ps JN persisted through successive generations of in vitro cultured potato plantlets, both as endophytic and epiphytic populations. In vitro inoculated potato (Solanum tuberosum) nodal explants produced plantlets with significant increases in root number (24-196%), root dry weight (44-201%), haulm dry weight (14-151%), and stem length (26-28%) as compared with noninoculated control plants. Bacterization also enhanced leaf hair formation (55-110%), secondary root branching, and total plant lignin content (43%). Other root colonizing bacteria or heat-killed cells of Ps JN had no significant effect on plant growth. Detached leaves from in vitro grown control plants, when exposed to 19 degrees C and 50% relative humidity, lost 55% of their moisture content in 2.5 hours. Moisture loss by leaves of in vitro grown, bacterized plants, as well as greenhouse-acclimated, bacterized plants, and control plants, was less than 20%. Changes in stomatal closure appear to account for this difference. PMID- 16668278 TI - Acyl carrier protein is conjugated to glutathione in spinach seed. AB - Acyl carrier protein (ACP) contains an essential sulfhydryl group in its phosphopantetheine prosthetic group. We have investigated the state of this sulfhydryl in developing and mature spinach seed (Spinacia oleracea). Seed extracts were separated on sodium dodecyl sulfate or native polyacrylamide gels, blotted to nitrocellulose, and probed with antibodies raised against spinach ACP I. In extracts of mature seeds prepared with reducing agents, ACP-II migrated as a single major band, whereas extracts prepared without reducing agents gave two major bands. The additional band was identified as a conjugate of ACP-II to glutathione (ACP-S-S-G) on the basis of its sensitivity to reducing agents and its comigration with standards in both native and sodium dodecyl sulfate gel electrophoresis. In developing spinach seeds ACP-II exists primarily in its free sulfhydryl form or as acyl derivatives, with essentially no ACP-S-S-G present. During later stages of seed development, as seed water content declines, ACP-S-S G accumulates to approximately 50% of the total ACP. Seed imbibition results in a rapid decline in ACP-S-S-G levels. The ACP-S-S-G:ACP-SH ratio of seeds during storage was found to be a function of seed water content and this could be manipulated by controlling the relative humidity under which the seeds were stored. We speculate that conjugation of ACP to glutathione protects the ACP from sulfhydryl oxidative damage in dry seeds. PMID- 16668279 TI - Ethylene and a Wound Signal Modulate Local and Systemic Transcription of win2 Genes in Transgenic Potato Plants. AB - The transcriptional regulation of a win2-beta-glucuronidase gene fusion in transgenic potato (Solanum tuberosum) plants by wounding and ethylene has been analyzed. In common with other genes that are expressed in response to mechanical or chemical stress, win2 is transcribed at the site of injury and also in distant undamaged parts of the wounded plant. Similar kinetics of induction and patterns of transcription were observed in response to mechanical, wounding, elicitor, or arachidonic acid application. Experiments involving the use of chemicals that inhibited ethylene action, and those that increased ethylene production, showed that local induction of win2 transcription did not have an absolute requirement for ethylene, but ethylene was necessary for high levels of expression. In contrast, systemic expression of win2 required both a putative wound signal and ethylene. Ethylene alone had no direct effect on win2 gene expression in the absence of wounding. PMID- 16668280 TI - Changes to the Stoichiometry of Glycine Decarboxylase Subunits during Wheat (Triticum aestivum L.) and Pea (Pisum sativum L.) Leaf Development. AB - Changes in the levels of the four subunits of the mitochondrial enzyme glycine decarboxylase (EC 2.1.2.10) have been investigated during development in the 8 day old primary leaf of wheat (Triticum aestivum L.). Proteins were extracted from wheat leaf sections between the basal meristem and 8.5 centimeters. The individual glycine decarboxylase subunits were detected by Western blotting, using subunit-specific polyclonal antibodies, and quantified by laser densitometry. P, T, and H subunits showed similar developmental patterns along the leaf. All were below the level of detection up to 1.5 centimeters from the meristem, but then increased over the leaf length examined. In contrast, the increase in the L protein (lipoamide dehydrogenase) was more gradual, and levels in the youngest regions of the leaf were maintained at approximately 14% of those at 8.5 centimeters. In a complementary study, levels of the four subunits in light-grown leaf tissues were compared to those in etiolated leaves from wheat and pea (Pisum sativum L.), using the activity of the mitochondrial marker enzyme fumarase as the basis for comparison. For both wheat and pea, levels of P, T, and H proteins in etiolated tissues were between 25 and 30% of those in lightgrown tissue. However, in etiolated tissues L protein was present at levels of 60 to 70% of that in light-grown tissues. The results indicate that discrete mechanisms may control the synthesis of L, as compared to P, T, and H proteins. PMID- 16668281 TI - Characterization of heat injury in grapes using h nuclear magnetic resonance methods : changes in transverse relaxation times. AB - Transverse relaxation times (T(2)) of tissue water ((1)H) in leaves and suspension cultured cells of grape hybrids (Vitis spp. cv ;Venus' and ;Veeblanc') were measured by nuclear magnetic resonance at various temperatures. The tissue water was characterized by two T(2) time constants. A sharp decrease in T(2) for the major fraction of tissue water was observed in association with heat injury, as measured by electrolyte leakage and triphenyltetrazolium chloride reduction in both leaves and suspension cultured cells. The changes in T(2) as a result of heat injury were irreversible, as indicated by a temperature dependent hysteresis of T(2). Studies using a paramagnetic probe (Mn(+2)) indicated that the plasma membrane was irreversibly damaged at the killing temperature, resulting in a loss of cell compartmentalization. Tissue water in heat-killed samples was characterized by only a single T(2). PMID- 16668282 TI - Light-Induced Chloroplast Differentiation in Soybean Cells in Suspension Culture : Ultrastructural Changes during the Bleaching and Greening Cycles. AB - Suspension cultures of SB-P cells of soybean (Glycine max) provide a novel, reproducible, and readily manipulable greening system useful for inducing chloroplast differentiation. The cells are subcultured and grown heterotrophically (3% sucrose) in the dark for at least three successive 14-day periods, subcultured and grown in the dark for 7 days more, and finally placed under white light and grown photoautotrophically. Chlorophyll begins to accumulate by 1 hour of light and continues up to 12 days. The chlorophyll a:chlorophyll b ratio is 3:1. Dark-grown cells contain a small amount of total carotenoids which increase 10-fold during greening. Chloroplast differentiation is strictly light dependent, with photosynthetic pigments accumulating in the light and being lost from cells returned to the dark. In the dark, the chloroplasts dedifferentiate to amyloplasts as the organized thylakoid network is lost and starch accumulates. Under continuous light, the amyloplasts differentiate into mature chloroplasts as the organelle elongates, becomes spanned by several bands of thylakoids, and undergoes grana formation. Chloroplast differentiation in SB-P cells is similar to that in intact angiosperms developing under normal light-dark cycles. PMID- 16668284 TI - Genes with Homology to Fungal and S-Gene RNases Are Expressed in Arabidopsis thaliana. AB - The only RNase genes that have been cloned from higher plants are those expressed in species that are known to exhibit self-incompatibility, such as the S genes of Nicotiana alata. In this report, we investigated whether the expression of this particular type of RNase gene is restricted to self-incompatible species, or if similar genes are expressed in other plants. Using a polymerase chain reaction approach we have identified a set of three putative RNase genes in the self compatible plant Arabidopsis thaliana (L.) Heynh. ecotype Columbia. These A. thaliana genes, designated RNS1, RNS2, and RNS3, do not cross-hybridize to each other, and their products are homologous to both the S-gene RNases of N. alata and a set of related fungal enzymes. The A. thaliana RNS1, RNS2, and RNS3 genes encode transcripts of 1.1, 1.3, and 1.1 kilobases, respectively, and of the three genes, RNS2 is the most highly expressed in whole plants. The identification of the RNS genes in a self-compatible species suggests that this class of RNases is of general significance in RNA catabolism in higher plants. PMID- 16668283 TI - Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane. AB - In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling. PMID- 16668285 TI - Systemic Endopolyploidy in Arabidopsis thaliana. AB - Microfluorometric analysis of the nuclear DNA contents of the somatic tissues of Arabidopsis thaliana has revealed extensive endoreduplication, resulting in tissues that comprise mixtures of polyploid cells. Endoreduplication was found in all tissues except those of the inflorescences and was developmentally regulated according to the age of the tissues and their position within the plant. PMID- 16668286 TI - Downward Regulation of Photosynthesis and Growth at High CO(2) Levels : No Evidence for Either Phenomenon in Three-Year Study of Sour Orange Trees. AB - Numerous photosynthesis and growth measurements of sour orange (Citrus aurantium L.) trees maintained in ambient air and air enriched with an extra 300 microliters per liter of CO(2) have revealed the CO(2)-enriched trees to have consistently sequestered approximately 2.8 times more carbon than the control trees over a period of three full years. Under field conditions in the natural environment, plants may not experience the downward regulation of photosynthetic capacity typically observed in long-term CO(2) enrichment experiments with plants growing in pots. PMID- 16668287 TI - Alpha-amylase inhibitor, not phytohemagglutinin, explains resistance of common bean seeds to cowpea weevil. AB - There are claims that phytohemagglutinin (PHA), the lectin of common bean, Phaseolus vulgaris, is toxic when fed to the cowpea weevil, Callosobruchus maculatus, and that PHA serves as the chemical defense against this seed-feeding bruchid beetle (DH Janzen, HB Juster, IE Liener [1976] Science 192: 795-796; AMR Gatehouse, FM Dewey, J Dove, KA Fenton, A Pusztai [1984] J Sci Food Agric 35: 373 380). However, our studies indicate that neither PHA nor its isolectins have detrimental effects when fed to the cowpea weevil. To explain these contradictory results we characterized the commercial lectin source used by A. M. R. Gatehouse, F. M. Dewey, J. Dove, K. A. Fenton, A. Pusztai (1984, J Sci Food Agric 35: 373 380). We demonstrate here that the toxic effects of PHA to cowpea weevil are due to an alpha-amylase inhibitor contaminant in the commercial preparation. PMID- 16668288 TI - Cloning and nucleotide sequence of a s-adenosylmethionine synthetase cDNA from carnation. PMID- 16668289 TI - Partial Purification and Reconstitution of the alpha-Ketoglutarate Carrier from Corn (Zea mays L.) Mitochondria. AB - The alpha-ketoglutarate carrier from corn shoot mitochondria (Zea mays L., B 73) was solubilized in Triton X-114 and partially purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. On SDS-gel electrophoresis, the hydroxyapatite/celite eluate showed various protein bands between 12 and 70 kilodaltons. When reconstituted into liposomes, the alpha ketoglutarate transport protein catalyzed a phthalonate-sensitive alpha ketoglutarate/alpha-ketoglutarate exchange. The protein was purified 60-fold with a recovery of 88% with respect to the mitochondrial extract. The protein yield was 0.6%. The properties of the reconstituted carrier, i.e. requirement for a counter-anion, substrate specificity, and inhibitor sensitivity, were similar to those of the alpha-ketoglutarate transport system as characterized in plant and animal mitochondria. PMID- 16668290 TI - Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture. AB - Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G(1) phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G(1) to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs. PMID- 16668291 TI - Sucrose Phosphate Is Not Transported into Vacuoles or Tonoplast Vesicles from Red Beet (Beta vulgaris) Hypocotyl. AB - Tonoplast vesicles and vacuoles isolated from red beet (Beta vulgaris L.) hypocotyl accumulated externally supplied [(14)C]sucrose but not [(14)C]sucrose phosphate despite the occurrence of sucrose phosphate phosphohydrolytic activity in the vacuole. The activities of sucrose synthase and sucrose phosphate synthase in whole cell extracts were 960 and 30 nanomoles per milligram protein per minute, respectively; whereas, no sucrose synthesizing activity was measured in tonoplast preparations. The results obtained in this investigation are incompatible with the involvement of sucrose phosphate synthase in the process of sucrose synthesis and accumulation in the storage cells of red beet. PMID- 16668292 TI - Photophosphorylation in Attached Leaves of Helianthus annuus at Low Water Potentials. AB - The in situ response of photophosphorylation and coupling factor activity to low leaf water potential (psi(L)) was investigated using kinetic spectroscopy to measure the flash-induced electrochromic absorption change in attached sunflower (Helianthus annuus L. cv IS894) leaves. The electrochromic change is caused by the formation of an electric potential across the thylakoid membrane associated with proton uptake. Since depolarization of the thylakoid membrane following flash excitation is normally dominated by proton efflux through the coupling factor during ATP formation, this measurement can provide direct information about the catalytic activity of the coupling factor. Under low psi(L) conditions in which a clear nonstomatal limitation of net photosynthesis could be demonstrated, we found a strong inhibition of coupling factor activity in dark adapted leaves which was probably caused by an increase in the energetic threshold for the activation of the enzyme at low psi(L). While this result supported earlier in vitro findings, we further discovered that the light dependent reduction of coupling factor reversed any observable effect of low psi(L) on the energetics of activation or on photophosphorylation competence. Furthermore, coupling factor was reduced, even in severely droughted sunflower, almost immediately upon illumination. Based on these measurements, we conclude that the nonstomatal limitation of photosynthesis observed by us and others in droughted plants cannot be explained by impaired coupling factor activity. PMID- 16668293 TI - Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves. AB - Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6 bisphosphate in the presence of either Mg(2+) or Mn(2+), and was free of interfering enzyme activities. Ca(2+) was an effector of fructose-1,6 bisphosphatase activity, and showed different kinetics, depending on whether Mg(2+) or Mn(2+) was used as cofactor. In the presence of 5 millimolar Mg(2+), Ca(2+) appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6 bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca(2+). However, with Mn(2+) (60 micromolar) as cofactor, Ca(2+) exhibited the Michaelis Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca(2+) behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg(2+) or Mn(2+) as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg(2+) as cofactor, but this effect was low with Mn(2+). From these results we suggest that Ca(2+) plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase. PMID- 16668294 TI - Plasmalemma redox activity and h extrusion in roots of fe-deficient cucumber plants. AB - Cucumber plants (Cucumis sativus L.) with incipient Fe deficiency showed increased root capacity to reduce chelated Fe(3+) compared to Fe-sufficient plants. When Fe-ethylenediaminete-traacetate was added to the root medium of the Fe-deficient plants, the reductase activity was associated with acidification of the medium and an increase in the net apparent K(+) efflux. In the presence of the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide the net apparent H(+) efflux was completely suppressed, though some reductase activity was preserved, and the net apparent K(+) efflux was significantly increased. The inhibition of the reductase activity by N,N'-dicyclohexylcarbodiimide was similar whether the pH of the medium was buffered or not. Anoxia and the protonophore carbonyl cyanide m-chlorophenyl hydrazone also caused a similar inhibition of the reductase activity. It is proposed that this redox system transports electrons only and that its activity is inhibited by plasmamembrane depolarization and anoxia. The H(+) and K(+) efflux associated with the reductase activity may be a result of the plasmamembrane depolarization it causes. PMID- 16668295 TI - Expression of a Conserved Family of Cytoplasmic Low Molecular Weight Heat Shock Proteins during Heat Stress and Recovery. AB - Plants synthesize several families of low molecular weight (LMW) heat shock proteins (HSPs) in response to elevated temperatures. We have characterized two cDNAs, HSP18.1 and HSP17.9, that encode members of the class I family of LMW HSPs from pea (Pisum sativum). In addition, we investigated the expression of these HSPs at the mRNA and protein levels during heat stress and recovery. HSP18.1 and HSP17.9 are 82.1% identical at the amino acid level and are 80.8 to 92.9% identical to class I LMW HSPs of other angiosperms. Heat stress experiments were performed using intact seedlings subjected to a gradual temperature increase and held at a maximum temperature of 30 to 42 degrees Celsius for 4 hours. HSP18.1 and HSP17.9 mRNA levels peaked at the beginning of the maximum temperature period and declined rapidly after the stress period. Antiserum against a HSP18.1 fusion protein recognized both HSP18.1 and HSP17.9 but not members of other families of LMW HSPs. The accumulation of HSP18.1-immunodetected protein was proportional to the severity of the heat stress, and the protein had a half-life of 37.7 +/- 8 hours. The long half-life of these proteins supports the hypothesis that they are involved in establishing thermotolerance. PMID- 16668296 TI - Heat Shock Causes Selective Destabilization of Secretory Protein mRNAs in Barley Aleurone Cells. AB - The aleurone layer of GA(3)-stimulated barley (Hordeum vulgare L., cv Himalaya) grains is normally devoted to the synthesis and secretion of hydrolytic enzymes. Heat shock, however, suppresses the synthesis of the main hydrolytic enzyme, alpha-amylase, by destabilizing its otherwise highly stable mRNA (FC Belanger, MR Brodl, T-hD Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358). In this paper we document that heat shock causes the suppression of the synthesis of some normal cellular proteins, while the synthesis of other normal cellular proteins is unaffected by heat shock. There are two major isozymic forms of alpha-amylase encoded by distinct mRNAs. The mRNA levels for both isozymic forms and the mRNA levels of two other secretory proteins, a protease and an endochitinase, were markedly reduced during heat shock. However, the levels of actin and beta-tubulin mRNAs, both nonsecretory proteins, were not diminished during heat shock. In addition, the levels of three other mRNA species detected by a set of unidentified cDNA clones (the sequence of one shows that it lacks a signal sequence) remained unchanged during heat shock. These data indicate that there are two classes of normal cellular protein mRNAs with regard to the effect of heat shock upon their persistence in the cell, and suggest that the distinction between them is whether or not they encode secretory proteins. PMID- 16668297 TI - Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon. AB - The levels of polyamines in leaves of Gynura aurantiaca DC and tomato, Lycopersicon esculentum Mill. cv Rutgers, infected with citrus exocortis viroid (CEVd) or treated with silver nitrate or ethephon (2-chloroethylphosphonic acid) were measured by HPLC in relation to development of symptoms. Previously it had been demonstrated that treatment of G. aurantiaca plants with silver nitrate or ethephon closely mimicked the effects of viroid infection in the plants. In the studies reported here, a marked decrease in putrescine level was observed in plants infected by CEVd or treated with silver ions or ethephon. There was no significant change in either spermidine or spermine levels. Treatment of G. aurantiaca plants with specific inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, Co(2+)) or ethylene action (norbornadiene) prevented the decrease of putrescine associated with silver nitrate treatment and had no effect on spermidine or spermine levels. The development of viroid-like symptoms, the production of associated pathogenesis-related proteins, and the rise in protease activity induced by silver nitrate, were all suppressed by exogenous application of putrescine. The decreased level of putrescine as an ethylene mediated step in the transduction of the viroid and silver or ethephon signaling is discussed. PMID- 16668298 TI - Solubilization, partial purification, and reconstitution of the glycolate/glycerate transporter from chloroplast inner envelope membranes. AB - The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d glycerate and, usually, [(3)H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [(14)C]d glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [(14)C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [(14)C]d-glycerate was strongly inhibited by HgCl(2), which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [(14)C]glycolate accumulation under similar conditions was much greater than that of [(14)C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [(14)C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly. PMID- 16668299 TI - Evidence of zein-bound indoleacetic Acid using gas chromatography-selected ion monitoring-mass spectrometry analysis and immunogold labeling. AB - Commercial zein was base-hydrolyzed and purified extracts were subjected to gas chromatography-selected ion monitoring-mass spectrometry analysis. Indoleacetic acid (IAA) was shown to be released from this storage protein of corn (Zea mays). Isotope dilution using [(13)C(6)]IAA as an internal standard revealed a conservative ratio of 1 mole IAA to 175 moles zein. Immunoelectron micrographs of isolated protein bodies also showed IAA or an IAA-like molecule associated with zein and deposited within these organelles. PMID- 16668300 TI - Western blot analysis of cereal grain prolamins using an antibody to carboxyl linked indoleacetic Acid. AB - A monoclonal antibody raised against carboxyl-linked IAA was used in Western blot analysis of storage proteins from kernels of Avena sativa, Pennisetum americanum, Sorghum bicolor, and Zea mays. IAA or an IAA-like molecule is associated with the ethanolsoluble protein fraction of the seed. Western blotting of commercial zein, the major storage protein of maize, along with physicochemical evidence reported by Leverone et al. ([1991] Plant Physiol, 96: 1070-1075) indicated that IAA is linked with this prolamin. Results suggest that an IAA-prolamin association may be widespread throughout the Poaceae. PMID- 16668301 TI - Cryptochrome, phytochrome, and anthocyanin production. AB - Anthocyanin production in cabbage (Brassica oleracea L.) and tomato (Lycopersicon esculentum Mill.) seedlings exposed to prolonged irradiations was studied under conditions that allowed discrimination, within certain limits, between the contribution of cryptochrome and phytochrome in the photoregulation of the response. The results of the study provide confirming evidence for the involvement of cryptochrome and direct evidence for a significant contribution of cryptochrome to the fluence rate dependence of the response to blue. The results provide some preliminary, direct indication for an interaction between cryptochrome and phytochrome in the photoregulation of anthocyanin production in seedlings exposed to the prolonged irradiations required for a high level of expression of the response. The type and degree of interaction between the two photoreceptors vary significantly, depending on the species and experimental conditions. PMID- 16668302 TI - Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease. AB - Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti(a), to Ti(a) (m), the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the alpha, alpha', and beta subunits of soybean beta conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon. PMID- 16668303 TI - Homeohydrous (Recalcitrant) Seeds: Dehydration, the State of Water and Viability Characteristics in Landolphia kirkii. AB - Differential scanning calorimetry was used to study the relationships among drying rate, desiccation sensitivity, and the properties of water in homeohydrous (recalcitrant) seeds of Landolphia kirkii. Slow drying of intact seeds to axis moisture contents of approximately 0.9 to 0.7 gram/gram caused lethal damage, whereas very rapid (flash) drying of excised embryonic axes permitted removal of water to approximately 0.3 gram/gram. The amount of nonfreezable water in embryonic axes (0.28 gram H(2)O/gram dry mass) did not change with drying rate and was similar to that of desiccation-tolerant seeds. These results suggest that the amount of nonfreezable water per se is not an important factor in desiccation sensitivity. However, flash drying that removed all freezable water damaged embryonic axes. Differences between desiccation-sensitive and -tolerant seeds occur at two levels: (a) tolerant seeds naturally lose freezable water, and sensitive seeds can lose this water without obvious damage only if it is removed very rapidly; (b) tolerant seeds can withstand the loss of a substantial proportion of nonfreezable water, whereas sensitive seeds are damaged if nonfreezable water is removed. PMID- 16668304 TI - ent-Kaurene Biosynthesis in Germinating Barley (Hordeum vulgare L., cv Himalaya) Caryopses and Its Relation to alpha-Amylase Production. AB - ent-Kaurene biosynthesis as a prerequisite for gibberellin (GA) biosynthesis was studied in germinating Hordeum vulgare L., cv Himalaya caryopses and correlated, in time, with the appearance of alpha-amylase activity. The rate of ent-kaurene biosynthesis was estimated by inhibiting its further metabolism with plant growth retardants (triapenthenol or tetcyclacis) and measuring its accumulation by isotope dilution using combined gas chromatographymass spectrometry. In the inhibitor-treated caryopses, ent-kaurene accumulation began approximately 24 hours after imbibition and proceeded at a rate of about 1 to 2 picomoles per hour per caryopsis, depending on the batch of seeds. In the absence of inhibitor, ent kaurene did not accumulate, indicating that it is normally turned over rapidly, presumably to further intermediates of the GA biosynthesis pathway and eventually to GAs. ent-Kaurene accumulation occurred almost exclusively in the shoot, which is, therefore, probably the site of biosynthesis. alpha-Amylase production began between 30 and 36 hours after imbibition and, thus, correlated well with de novo GA biosynthesis, as estimated from ent-kaurene accumulation. However, inhibition of ent-kaurene oxidation by plant growth retardants did not reduce the alpha amylase production significantly, although it did reduce shoot elongation. We conclude that ent-kaurene is produced in the shoot and is continuously converted to GA, which is essential for normal shoot elongation, but not for the production of alpha-amylase in the aleurone layer. PMID- 16668305 TI - Water Relations and Hydraulic Architecture of a Tropical Tree (Schefflera morototoni) : Data, Models, and a Comparison with Two Temperate Species (Acer saccharum and Thuja occidentalis). AB - The water relations and hydraulic architecture of a tropical tree (Schefflera morototoni) and of two temperate species (Acer saccharum and Thuja occidentalis) are reported. Among the water relations parameters measured were leaf and stem water storage capacity, leaf water potential, transpiration, and vulnerability of stems to cavitation and loss of hydraulic conductivity by embolisms. Among the hydraulic architecture parameters measured were hydraulic conductivity per unit pressure gradient, specific conductivity, leaf-specific conductivity, and Huber value. In terms of vulnerability of stems to cavitation, stem and leaf capacitances, and leaf-specific conductivity, all three species followed the same sequence: Schefflera > Acer > Thuja. It is argued here that the high stem capacitance and high leaf-specific conductivity of Schefflera are necessary to compensate for its high vulnerability to cavitation. Extractable water storage per unit leaf area in Schefflera stems is >100 times that of Acer and may permit the species to survive unusually long, dry seasons in Panama. Although Schefflera frequently grows >20 meters, the biggest resistance to water flow in the shoots resides in the leaves. PMID- 16668306 TI - Effects of temperature on the coupled activities of the vanadate-sensitive proton pump from maize root microsomes. AB - The mechanism by which proton transport is coupled to ATP hydrolysis by vanadate sensitive pumps is poorly understood. The effects of temperature on the activities of the vanadate-sensitive ATPase from maize (Zea mays) roots were assessed to provide insight into the coupling mechanism. The initial rate of proton transport had a bell-shaped dependence on temperature with an optimal range between 20 and 30 degrees C. However, the rate of vanadate-sensitive ATP hydrolysis increased as the temperature was raised from 4 to 43 degrees C. The differential sensitivity of proton transport to temperatures above 30 degrees C was also observed when the ATPase was reconstituted into dioleoylphosphatidylcholine vesicles. Inhibition of proton transport with temperatures above 30 degrees C was associated with higher rates of proton leakage from the membranes. In addition, proton transport was more inhibited than ATP hydrolysis at temperatures below 10 degrees C. Reduced rates of proton transport at lower temperatures were not associated with higher rate of proton conductivity across the membranes. Therefore, the preferential inhibition of proton transport at temperatures below 10 degrees C may reflect an effect of temperature on the coupling between proton transport and ATP hydrolysis within the vanadate-sensitive ATPase. PMID- 16668307 TI - Correlation between CAM-Cycling and Photosynthetic Gas Exchange in Five Species of Talinum (Portulacaceae). AB - Photosynthetic gas exchange and malic acid fluctuations were monitored in 69 well watered plants from five morphologically similar species of Talinum in an investigation of the ecophysiological significance of the Crassulacean acid metabolism (CAM)-cycling mode of photosynthesis. Unlike CAM, atmospheric CO(2) uptake in CAM-cycling occurs exclusively during the day; at night, the stomata are closed and respiratory CO(2) is recaptured to form malic acid. All species showed similar patterns of day-night gas exchange and overnight malic acid accumulation, confirming the presence of CAM-cycling. Species averages for gas exchange parameters and malic acid fluctuation were significantly different such that the species with the highest daytime gas exchange had the lowest malic acid accumulation and vice versa. Also, daytime CO(2) exchange and transpiration were negatively correlated with overnight malic acid fluctuation for all individuals examined together, as well as within one species. This suggests that malic acid may effect reductions in both atmospheric CO(2) uptake and transpiration during the day. No significant correlation between malic acid fluctuation and water-use efficiency was found, although a nonsignificant trend of increasing water-use efficiency with increasing malic acid fluctuation was observed among species averages. This study provides evidence that CO(2) recycling via malic acid is negatively correlated with daytime transpirational water losses in well-watered plants. Thus, CAM-cycling could be important for survival in the thin, frequently desiccated soils of rock outcrops on which these plants occur. PMID- 16668308 TI - Growth of the Maize Primary Root at Low Water Potentials : III. Role of Increased Proline Deposition in Osmotic Adjustment. AB - Seedlings of maize (Zea mays L. cv WF9 x Mo 17) growing at low water potentials in vermiculite contained greatly increased proline concentrations in the primary root growth zone. Proline levels were particularly high toward the apex, where elongation rates have been shown to be completely maintained over a wide range of water potentials. Proline concentration increased even in quite mild treatments and reached 120 millimolal in the apical millimeter of roots growing at a water potential of -1.6 megapascal. This accounted for almost half of the osmotic adjustment in this region. Increases in concentration of other amino acids and glycinebetaine were comparatively small. We have assessed the relative contributions of increased rates of proline deposition and decreased tissue volume expansion to the increases in proline concentration. Proline content profiles were combined with published growth velocity distributions to calculate net proline deposition rate profiles using the continuity equation. At low water potential, proline deposition per unit length increased by up to 10-fold in the apical region of the growth zone compared to roots at high water potential. This response accounted for most of the increase in proline concentration in this region. The results suggest that osmotic adjustment due to increased proline deposition plays an important role in the maintenance of root elongation at low water potentials. PMID- 16668309 TI - Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes. AB - The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO(3) to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants. PMID- 16668310 TI - Lipids of plasma membranes prepared from oat root cells : effects of induced water-deficit tolerance. AB - Plasma membranes were isolated from oat (Avena sativa) roots by the phase partitioning method. The membranes were exposed to repeated periods of moderate water-deficit stress, and a water-deficit tolerance was induced (acclimated plants). The plasma membranes of the controls (nonacclimated plants) were characterized by a high phospholipid content, 79% of total lipids, cerebrosides (9%) containing hydroxy fatty acids (>90% 24:1-OH) and free sterols, acylated sterylglucosides, sterylglucosides, and steryl esters, together amounting to 12%. Major phospholipids were phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. After the membranes were acclimated to dehydration, the lipid to protein ratio decreased from 1.3 to 0.7 micromoles per milligram. Furthermore, the cerebrosides decreased to 5% and free sterols increased from 9% (nonacclimated plants) to 14%. Because the total phospholipids did not change significantly, the free sterol to phospholipid ratio increased from 0.12 to 0.19. There was no change in the relative distribution of sterols after acclimation. The ratio of phosphatidylcholine to phosphatidylethanolamine changed from 1.1 in the nonacclimated plants to 0.69 in the acclimated plants. The results show that acclimation to dehydration implies substantial alterations in the lipid composition of the plasma membrane. PMID- 16668311 TI - Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.). AB - Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues. PMID- 16668312 TI - Occurrence of an Inhibitor of Tissue-Type Plasminogen Activator in Seeds and in Vitro Cultures of Erythrina caffra Thunb. AB - The level of an inhibitor of tissue-type plasminogen activator (t-PA) increased slowly during the early developmental stage of seeds of Erythrina caffra Thunb. Thereafter, the inhibitor increased exponentially until the seeds reached maturity. At maturity, the t-PA inhibitor levels in the cotyledons were 38 times higher than the levels at the onset of seed development. The t-PA inhibitor accumulated at a faster rate than the storage proteins, which reached a concentration 15 times higher than the protein concentration at the onset of seed development. During the imbibition and germination process, the t-PA inhibitor decreased gradually. The inhibitor kept on decreasing during the growth of the seedlings until the 10th day after imbibition, when it leveled off at 4.1% of that of the initial inhibitor concentration. The inhibitor remained at this level until the cotyledons were shed at day 22. The total protein in the cotyledons decreased at a slower rate than the inhibitor and reached a minimum concentration at day 20 of 3.6% of the initial protein concentration in the cotyledons. Callus cultures of root, shoot, leaf, and cotyledonary tissue was established and maintained on Murashige-Skoog medium supplemented with 3% sucrose, 10 micromolar benzyladenine, and 5 micromolar 2,4-dichlorophenoxyacetic acid. A shoot cell suspension culture was established on Murashige-Skoog medium supplemented with 3% sucrose, 1 micromolar benzyladenine, and 0.5 micromolar 2,4-dichlorophenoxyacetic acid (pH 5.7) and shaken at 60 revolutions per minute. The level of t-PA inhibitor in root, shoot, leaf, and cotyledonary callus was substantially lower than in the corresponding intact tissue. The t-PA inhibitor levels in the linear growth phase was higher than in the lag or stationary growth phases of the cell suspension culture. A hydrolysate of the cell walls of tomato and E. caffra Thunb, as well as polyamines and organic acids, did not increase the concentration of t-PA inhibitor in suspension cultures or intact leaf tissue of E. caffra. The t-PA inhibitor levels of suspension cultures were increased by Na(2)SO(4) but not by I-cysteine in the nutrient medium. PMID- 16668313 TI - Use of Dimethyl Sulfoxide to Detect Hydroxyl Radical during Bacteria-Induced Hypersensitive Reaction. AB - Excess active oxygen is generated during the hypersensitive reaction (HR), an incompatible reaction of plants to bacterial pathogens. During HR, lipid peroxidation correlates chronologically with production of the oxygen species, superoxide (O(2) (.-)). However, O(2) (.-) may not be the active oxygen species that initiates lipid peroxidation. Evidence from other systems suggest that O(2) (.-) is converted to the hydroxyl radical (HO(.)) before lipid peroxidation is initiated. Until recently, HO(.) could not be detected directly in vivo. This study utilizes a newly reported method to directly detect and quantify the formation of HO(.)in vivo. Dimethyl sulfoxide (DMSO), used as a molecular probe, is oxidized by HO(.), forming the stable compound methanesulfinic acid. The methanesulfinic acid can be easily extracted from plant tissues and measured with a colorimetric assay. This study demonstrates significant increases in HO(.) concentration after simultaneous infiltration of cucumber (Cucumis sativa L.) plants with paraquat and DMSO. The concentration of HO(.) did not increase significantly when cucumber plants were infiltrated simultaneously with the HR inducing bacteria, Pseudomonas syringae pv. pisi, and with DMSO. Lipid peroxidation, however, could be measured at times when HO(.) was not detectable. It appears that HO(.) is not generated during bacteria-induced HR; therefore, HO(.) is not responsible for the initiation of lipid peroxidation. PMID- 16668314 TI - Binding form of pollen mother cell protein in the nucleosomes of lily. AB - We have previously reported the existence of pollen mother cell nuclear protein (PMCP) which appears during microsporogenesis in lily (Lilium spesiosum). It is very similar to mammalian testis specific H1 histone, H1t. In this paper, we describe the PMCP distribution in lily nucleosomes. Isolated nuclei were treated with micrococcal nuclease, and template active and inactive chromatin fractions were prepared. The nucleosome repeat length of pollen mother cells was determined to be 210 base pairs. The majority of the PMCP was found in the template inactive chromatin fraction, similar to other histones. PMCP was contained in the nucleosome monomer, but not in the core particle. However, PMCP was mainly found in the nucleosome dimer when slightly digested. Salt extraction from isolated nuclei indicated that PMCP and H1 histone share similar binding affinities to DNA. Judging from our results, it seems probable that PMCP links two core particles more strongly than H1 histone does. Since it is known that meiotic chromatin includes nick transferase and nuclease activity, one possible role of PMCP is the protection of its own chromatin. Other possible functions of PMCP are also discussed. PMID- 16668315 TI - Oxidized oligogalacturonides activate the oxidation of indoleacetic Acid by peroxidase. AB - Partial hydrolysis of polygalacturonic acid with a purified alpha-1,4 endopolygalacturonase yielded oligogalacturonides and trace amounts of a series of modified oligogalacturonides. Three of the minor products were isolated and identified as oxidized oligogalacturonides possessing termini of galactaric acid. Oxidation of indole-3-acetic acid by peroxidases was activated by oxidized oligogalacturonides but not by normal analogs. PMID- 16668316 TI - Ethylene Evolution from Maize (Zea mays L.) Seedling Roots and Shoots in Response to Mechanical Impedance. AB - The effect of mechanical impedance on ethylene evolution and growth of preemergent maize (Zea mays L.) seedlings was investigated by pressurizing the growth medium in triaxial cells in a controlled environment. Pressure increased the bulk density of the medium and thus the resistance to growth. The elongation of maize primary roots and preemergent shoots was severely hindered by applied pressures as low as 10 kilopascals. Following a steep decline in elongation at low pressures, both shoots and roots responded to additional pressure in a linear manner, but shoots were more severely affected than roots at higher pressures. Radial expansion was promoted in both organs by mechanical impedance. Primary roots typically became thinner during the experimental period when grown unimpeded. In contrast, pressures as low as 25 kilopascals caused a 25% increase in root tip diameter. Shoots showed a slight enhancement of radial expansion; however, in contrast to roots, the shoots increased in diameter even when growing unimpeded. Such morphological changes were not evident until at least 3 hours after initiation of treatment. All levels of applied pressure promoted ethylene evolution as early as 1 hour after application of pressure. After 1 hour, ethylene evolution rates had increased 10, 32, 70, and 255% at 25, 50, 75, and 100 kilopascals respectively, and continued to increase linearly for at least 10 hours. When intact corn seedlings were subjected to a series of hourly cycles of pressure, followed by relaxation, ethylene production rates increased or decreased rapidly, illustrating tight coupling between mechanical impedance and tissue response. Seedlings exposed to 1 microliter of ethylene per liter showed symptoms similar to those shown by plants grown under mechanical impedance. Root diameter increased 5 times as much as the shoot diameter. Pretreatment with 10 micromolar aminoethoxyvinyl glycine plus 1 micromolar silver thiosulfate maintained ethylene production rates of impeded seedlings at basal levels and restored shoot and root extension to 84 and 90% of unimpeded values, respectively. Our results support the hypothesis that ethylene plays a pivotal role in the regulation of plant tissue response to mechanical impedance. PMID- 16668317 TI - Transmembrane Electron Transport in Plasma Membrane Vesicles Loaded with an NADH Generating System or Ascorbate. AB - Sugar beet (Beta vulgaris L.) leaf plasma membrane vesicles were loaded with an NADH-generating system (or with ascorbate) and were tested spectrophotometrically for their ability to reduce external, membrane-impermeable electron acceptors. Either alcohol dehydrogenase plus NAD(+) or 100 millimolar ascorbate was included in the homogenization medium, and right-side-out (apoplastic side-out) plasma membrane vesicles were subsequently prepared using two-phase partitioning. Addition of ethanol to plasma membrane vesicles loaded with the NADH-generating system led to a production of NADH inside the vesicles which could be recorded at 340 nanometers. This system was able to reduce 2,6-dichlorophenolindophenol-3' sulfonate (DCIP-sulfonate), a strongly hydrophilic electron acceptor. The reduction of DCIP-sulfonate was stimulated severalfold by the K(+) ionophore valinomycin, included to abolish membrane potential (outside negative) generated by electrogenic transmembrane electron flow. Fe(3+)-chelates, such as ferricyanide and ferric citrate, as well as cytochrome c, were not reduced by vesicles loaded with the NADH-generating system. In contrast, right-side-out plasma membrane vesicles loaded with ascorbate supported the reduction of both ferric citrate and DCIP-sulfonate, suggesting that ascorbate also may serve as electron donor for transplasma membrane electron transport. Differences in substrate specificity and inhibitor sensitivity indicate that the electrons from ascorbate and NADH were channelled to external acceptors via different electron transport chains. Transplasma membrane electron transport constituted only about 10% of total plasma membrane electron transport activity, but should still be sufficient to be of physiological significance in, e.g. reduction of Fe(3+) to Fe(2+) for uptake. PMID- 16668318 TI - Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits. AB - During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (alpha-subunit) and 65 (beta-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in beta-subunit and twofold increase in alpha-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total beta-subunit to alpha-subunit content. The beta- to alpha subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6 phosphate is similar. Both molecular forms are activated by fructose-2,6 bisphosphate. PMID- 16668319 TI - Rapid Image Analysis Screening Procedure for Identifying Chloroplast Number Mutants in Mesophyll Cells of Arabidopsis thaliana (L.) Heynh. AB - To analyze the genetic control of the process of chloroplast division, a direct image analysis screening procedure has been developed in which mutants of Arabidopsis thaliana (L.) Heynh. var Landsberg erecta are selected on the basis of abnormal chloroplast number. The selection procedure is based on image analysis thresholding after iodine staining, which facilitates the automatic counting of chloroplasts in isolated mesophyll cells. M2 seedlings are screened for significant deviation from the wild type relationship between mesophyll cell size and chloroplast number. Mutants with both abnormally high and abnormally low chloroplast numbers were identified. Of 3500 individual M2 seedlings screened, 18 mutant lines have been isolated and shown to be stably inherited in three subsequent generations. The most extreme phenotypes show an 80% reduction or a 50% increase in chloroplast number per mesophyll cell. PMID- 16668320 TI - Primary structures of Arabidopsis calmodulin isoforms deduced from the sequences of cDNA clones. AB - Complementary DNA (cDNA) clones encoding calmodulin isoforms were isolated from an Arabidopsis leaf lambdagt10 library by screening with cloned barley calmodulin cDNA probes. Two cDNAs, one a 626-base pair partial-length clone (ACaM-1) and one a 1400-base pair full-length clone (ACaM-2), encode calmodulin polypeptides that differ by four conservative amino acid substitutions. None of the amino acid sequence differences occur within the four Ca(2+)-binding domains of the proteins. Whereas the deduced amino acid sequences of the two Arabidopsis calmodulin isoforms share 97% identity, the nucleotide sequences encoding the two isoforms share 87% sequence identity. Most of these nucleotide sequence differences (80%) occur in codon wobble positions. ACaM-1 and ACaM-2 both hybridize with a distinct set of restriction fragments of Arabidopsis total DNA, indicating that they were derived from transcripts of separate genes; these genes are single- or very low-copy in the Arabidopsis genome. Both cDNAs hybridize to messenger RNA (mRNA) species of 0.8 kilobases that are expressed to a greater extent in developing siliques compared with leaves, flowers, and stems. Northern blot and polymerase chain reaction assays both indicate that ACaM-1 mRNA is more highly expressed than ACaM-2 mRNA in developing siliques. The steady-state levels of both isoform mRNAs increase as a result of touch stimulation; the kinetics and extent of increase are comparable for the two mRNAs. PMID- 16668321 TI - Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.). AB - Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N(6)-(delta(2)-isopentenyl)adenine, and N(6)-(delta(6)-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation. PMID- 16668322 TI - Biochemical and tissue print analyses of hydroxyproline-rich glycoproteins in cell walls of sporophytic maize tissues. AB - In an effort to understand the role of hydroxyproline-rich glycoproteins (HRGPs) in plant cell wall structure, we studied the distribution and physical properties of PC-1-like proteins (PC-1 being the major pericarp HRGP) throughout sporophytic tissues of two maize (Zea mays L.) varieties. We determined total amounts of hydroxyproline, an indicator of HRGPs, and did tissue print and Western blot analysis. We found hydroxyproline in cell walls of stems, leaves, roots, tassels, and silks. We also observed reactivity of anti-PC-1 monoclonal antibodies with anatomical prints of these tissues on nitrocellulose paper. Stem nodes and silks contained the most hydroxyproline and exhibited the strongest reaction with the antibody. PC-1 was localized in vascular bundles and the epidermis of stem tissue. However, localization to a specific cell type in the silk could not be determined at the resolution of the tissue print. The stem node protein had the same electrophoretic mobility as the pericarp protein as determined on Western blots prepared from cationic neutral gels. Protein extracts from silk tissues of both varieties studied contained one protein of the same size/charge as that found in pericarp, as well as some minor variant bands. The data presented here document that cell wall proteins are present in many tissues of the maize plant, although they are primarily in cell types contributing to support. PMID- 16668323 TI - Soluble Chloroplast Enzyme Cleaves preLHCP Made in Escherichia coli to a Mature Form Lacking a Basic N-Terminal Domain. AB - We have investigated the specificity of a chloroplast soluble processing enzyme that cleaves the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP). The precursor of LHCP (preLHCP) was synthesized in Escherichia coli and recovered from inclusion-like bodies. It was found to be a substrate for proteolytic cleavage by the soluble enzyme in an organelle-free reaction, yielding a 25 kilodalton peptide. This peptide co-migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the smaller of the forms (25 and 26 kilodalton) produced when either the E. coli-synthesized precursor, or preLHCP made in a reticulocyte lysate, was imported into chloroplasts. N-Terminal sequence analysis of the E. coli-generated precursor showed that it lacked an N terminal methionine. N-Terminal sequencing of the 25 kilodalton peptide produced in the organelle-free reaction indicated that processing occurred between residues 40 and 41, removing a basic domain (RKTAAK) thought to be at the N terminus of all LHCP molecules of type I associated with photosystem II. To determine if the soluble enzyme involved also cleaves other precursor polypeptides, or is specific to preLHCP, it was partially purified, and the precursors for Rubisco small subunit, plastocyanin, Rubisco activase, heat shock protein 21, and acyl carrier protein were tested as substrates. All of these precursors were cleaved by the same chromatographic peak of activity that processes preLHCP in the organelle-free reaction. PMID- 16668324 TI - Effects of Salt Stress on Amino Acid, Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of Alfalfa (Medicago sativa L.). AB - Ethanol-soluble organic acid, carbohydrate, and amino acid constituents of alfalfa (Medicago sativa) roots and nodules (cytosol and bacteroids) have been identified by gas-liquid chromatography and high performance liquid chromatography. Among organic acids, citrate was the predominant compound in roots and cytosol, with malonate present in the highest concentration in bacteroids. These two organic acids together with malate and succinate accounted for more than 85% of the organic acid pool in nodules and for 97% in roots. The major carbohydrates in roots, nodule cytosol, and bacteroids were (descending order of concentration): sucrose, pinitol, glucose, and ononitol. Maltose and trehalose appeared to be present in very low concentrations. Asparagine, glutamate, alanine, gamma-aminobutyrate, and proline were the major amino acids in cytosol and bacteroids. In addition to these solutes, serine and glutamine were well represented in roots. When alfalfa plants were subjected to 0.15 m sodium chloride stress for 2 weeks, total organic acid concentration in nodules and roots were depressed by more than 40%, whereas lactate concentration increased by 11, 27, and 94% in cytosol, roots, and bacteroids, respectively. In bacteroids, lactate became the most abundant organic acid and might contribute partly to the osmotic adjustment. On the other hand, salt stress induced a large increase in the amino acid and carbohydrate pools. Within the amino acids, proline showed the largest increase, 11.3-, 12.8-, and 8.0-fold in roots, cytosol, and bacteroids, respectively. Its accumulation reflected an osmoregulatory mechanism not only in roots but also in nodule tissue. In parallel, asparagine concentration was greatly enhanced; this amide remained the major nitrogen solute and, in bacteroids, played a significant role in osmoregulation. On the contrary, the salt treatment had a very limited effect on the concentration of other amino acids. Among carbohydrates, pinitol concentration was increased significantly, especially in cytosol and bacteroids (5.4- and 3.4-fold, respectively), in which this cyclitol accounted for more than 35% of the total carbohydrate pool; pinitol might contribute to the tolerance to salt stress. However, trehalose concentration remained low in both nodules and roots; its role in osmoregulation appeared unlikely in alfalfa. PMID- 16668325 TI - Isolation and Characterization of Three Genes Negatively Regulated by Phytochrome Action in Lemna gibba. AB - We have isolated three distinct cDNA clones from Lemna gibba representing mRNAs that increase in abundance during dark treatment. All three mRNAs showed reduced expression in response to red or white light. These mRNAs range from approximately 680 to 800 nucleotides in length and thus encode relatively small proteins (maximum relative molecular weight 17,000 to 19,000). The genes corresponding to these dark-abundant mRNAs are designated NPR (negatively phytochrome regulated) 1, 2, and 3. Differences in the rapidity of mRNA accumulation during dark treatment were observed for each of the genes in both mature green plants and in etiolated plants. Differences in accumulation pattern were also observed in etiolated plants, depending on whether the plants received a far-red light treatment prior to darkness. Transcription of all three genes, assayed in nuclei isolated from either green or etiolated plants, increased during dark treatment. In etiolated plants, a single 2 minute red light treatment caused a detectable decrease in the transcription of the genes after the dark treatment, and 10 minutes of far-red light given immediately after the red light resulted in a reversal of the effect of red light. Additionally, treatment of the plants with far-red light prior to darkness resulted in greater rates of transcription of the NPR genes. Therefore, we conclude that phytochrome action results in decreased transcription of these NPR genes. Each of the NPR mRNAs are encoded by one to two genes. PMID- 16668326 TI - Limitations of Photosynthesis in Pinus taeda L. (Loblolly Pine) at Low Soil Temperatures. AB - The relative importance of stomatal and nonstomatal limitations to net photosynthesis (A) and possible signals responsible for stomatal limitations were investigated in unhardened Pinus taeda seedlings at low soil temperatures. After 2 days at soil temperatures between 13 and 7 degrees C, A was reduced by 20 to 50%, respectively. The reduction in A at these moderate root-chilling conditions appeared to be the result of stomatal limitations, based on the decrease in intercellular CO(2) concentrations (c(i)). This conclusion was supported by A versus c(i) analysis and measurements of O(2) evolution at saturating CO(2), which suggested increases in stomatal but not biochemical limitations at these soil temperatures. Nonuniform stomatal apertures, which were demonstrated with abscisic acid, were not apparent 2 days after root chilling, and results of our A versus c(i) analysis appear valid. Bulk shoot water potential (psi) declined as soil temperature dropped below 16 degrees C. When half the root system of seedlings was chilled, shoot psi and gas-exchange rates did not decline. Thus, nonhydraulic root-shoot signals were not implicated in stomatal limitations. The initial decrease in leaf conductance to water vapor after root chilling appeared to precede any detectable decrease in bulk fascicle psi, but may be in response to a decrease in turgor of epidermal cells. These reductions in leaf conductance to water vapor, which occurred within 30 minutes of root chilling, could be delayed and temporarily reversed by reducing the leaf-to-air vapor-pressure deficit, suggesting that hydraulic signals may be involved in initiating stomatal closure. By independently manipulating the leaf-to-air vapor-pressure deficit of individual fascicles, we could induce uptake of water vapor through stomata, suggesting that nonsaturated conditions occur in the intercellular airspaces. There was an anomaly in our results on seedlings maintained for 2 days at soil temperatures below 7 degrees C. Lower A appeared primarily the result of nonstomatal limitations, based on large increases in calculated c(i) and A versus c(i) analysis. In contrast, measurements of O(2) evolution at saturating CO(2) concentrations implied nonstomatal limitations per se did not increase at these temperatures. One explanation for this paradox is that calculations of c(i) are unreliable at very low gas-exchange rates because of inadequate measurement resolution, and limitations of A are predominantly stomatal. An alternative interpretation is that increases in c(i) are real and the results from O(2) evolution measurements are in error. The high CO(2) concentration used in O(2) evolution measurements (15%) may have overcome nonstomatal limitations by enzymes that were down-regulated by a feedback mechanism. In this scenario, carbohydrate feedback limitations may be responsible for nonstomatal reductions in A after 2 days at soil temperatures below 7 degrees C. PMID- 16668327 TI - Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana. AB - A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant. PMID- 16668328 TI - Effect of Elicitors on the Plasmamembrane of Petunia hybrida Cell Suspensions : Role of DeltapH in Signal Transduction. AB - Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia lyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and N,N' dicyclohexylcarbodiimide and the ionophores carbonyl cyanide-4 trifluoromethoxyphenylhydrazone and nigericin were all effective elicitors. Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin elicit also when used in combination with N,N'-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia lyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by N,N'-dicyclohexylcarbodiimide, carbonyl cyanide-4 trifluoromethoxyphenylhydrazone, and nigericin was observed directly with the use of a sensitive pH electrode and internal acidification was deduced from the changes in emission intensity of the fluorescent probe bis[3-propyl-5-oxoisoxazol 4-yl] pentamethineoxonol. These data indicate that changes in the activity of the plasmamembrane H(+)-ATPase, and subsequent decrease of the proton gradient (particularly of the pH gradient) by itself are sufficient to influence phenylalanine ammonia lyase activity of P. hybrida cells and are therefore important intermediates in signal transduction. PMID- 16668329 TI - Isolation and characterization of a small heat shock protein gene from maize. AB - A maize (Zea mays L.) genomic clone (Zmempr 9') was isolated on the basis of its homology to a meiotically expressed Lilium sequence. Radiolabeled probe made from the maize genomic clone detected complementary RNA at high fidelity. Furthermore, it hybridized to RNA isolated from staged (an interval that is coincident with meiotic prophase) maize tassel spikelets. Complimentary RNA was strongly (at least 50-fold) induced during heat shock of maize somatic tissue and appeared as a single size class in Northern blot hybridizations. Sequencing of the complete coding region of Zmempr 9' confirmed the homology of the inferred amino acid sequence to other small heat shock proteins. Consensus sequences found in the flanking regions corresponded to the usual signals for initiation of RNA transcription, polyadenylate addition, and the induction of heat shock genes. The latter sequences conferred heat shock-specific transient expression in electroporated protoplasts when cloned into promoterless reporter gene plasmid constructs. Hybrid-selected translations revealed specific translation products ranging from 15 to 18 kilodaltons, providing evidence that this gene is a member of a related multigene family. We therefore conclude that this maize genomic DNA clone, recovered through its homology to clones for meiotic transcripts in lily, represents a genuine maize small heat shock protein gene. PMID- 16668330 TI - Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants. AB - It was previously reported that the hypernodulating soybean (Glycine max [L.] Merr.) mutants, derived from the cultivar Williams, had higher root concentration of isoflavonoid compounds (daidzein, genistein, and coumestrol) than did Williams at 9 to 12 days after inoculation with Bradyrhizobium japonicum. These compounds are known inducers of nod genes in B. japonicum and may be involved in subsequent nodule development. The current study involving reciprocal grafts between NOD1-3 (hypernodulating mutant) and Williams showed that root isoflavonoid concentration and content was more than twofold greater when the shoot genotype was NOD1-3. When grafted, NOD1-3 shoots also induced hypernodulation on roots of both Williams and NOD1-3, while Williams shoots induced normal nodulation on both root genotypes. This shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by the shoot. In contrast, the nonnodulating characteristic of the NN5 mutant was strictly root controlled, based on reciprocal grafts. Delayed inoculation (7 days after planting) resulted in greater nodule numbers on both NOD1-3 and Williams, compared with a seed inoculation treatment. The nodulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or uninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed. This observation, coupled with the fact that no difference existed in nodule number of NOD1-3 and Williams until after 9 days from seed inoculation, indicated that if isoflavonoids play a role in differential nodulation of the hypernodulating mutant and the wild type, the effect is on advanced stages of nodule ontogeny, possibly related to autoregulation, rather than on initial infection stages. PMID- 16668331 TI - Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources. AB - Two cDNA clones, PK(p)alpha and PK(p)beta, for the leucoplast isozyme of pyruvate kinase have been isolated and characterized. A Southern blot of castor (Ricinus communis) DNA probed with PK(p)alpha indicates the presence of a single gene for PK(p). Most (1610 base pairs) of the sequence of both cDNAs is identical. These 1610 base pairs begin with an ATG translation initiation codon, and have 248 base pairs of 3'-untranslated and 1362 base pairs of coding sequence. The sequences of the two clones 5'- to the identical regions are different but both encode peptides with a high percentage of hydrophobic amino acids. The derived sequence of PK(p)alpha encodes eight amino acid residues which have been identified as the amino-terminus of one subunit of PK(p) from castor seed leucoplasts when the enzyme is purified in the absence of cysteine endopeptidase inhibitors. The sequence upstream of these amino acids is possibly the transit peptide for this protein. When PK(p) is extracted under conditions that eliminate its proteolytic degradation, its alpha-subunit has a relative molecular weight equal to the full length coding sequence of PK(p)alpha. The data indicate that the transit peptide for the subunit of leucoplast pyruvate kinase encoded by PK(p)alpha is not cleaved until the protein is released from the plastid. The derived amino acid sequences of PK(p)alpha and PK(p)beta are most closely related to Escherichia coli pyruvate kinase. Although the residues involved in substrate binding are conserved in leucoplast pyruvate kinase, there is no phosphorylation site and only 5 of 15 amino acids in the E. coli fructose-1,6-bisphosphate binding site are conserved. PMID- 16668332 TI - Growth Yields and Maintenance Coefficients of Unadapted and NaCl-Adapted Tobacco Cells Grown in Semicontinuous Culture. AB - Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y(g)) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y(g) (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity. PMID- 16668333 TI - Metabolic evidence for stelar anoxia in maize roots exposed to low o(2) concentrations. AB - This investigation presents metabolic evidence to show that in 4- to 5-day-old roots of maize (Zea mays hybrid GH 5010) exposed to low external O(2) concentrations, the stele receives inadequate O(2) for oxidative phosphorylation, while the cortex continues to respire even when the external solution is at zero O(2) and the roots rely solely on aerenchyma for O(2) transport. Oxygen uptake rates (micromoles per cubic centimeter per hour) declined at higher external O(2) concentrations in excised segments from whole roots than from the isolated cortex; critical O(2) pressures for respiration were greater than 0.26 moles per cubic meter O(2) (aerated solution) for the whole root and only 0.075 moles per cubic meter O(2) for the cortex. For plants with their shoots excised and the cut stem in air, ethanol concentrations (moles per cubic meter) in roots exposed to 0.06 moles per cubic meter O(2) were 3.3 times higher in the stele than in the cortex, whereas this ethanol gradient across the root was not evident in roots exposed to 0 moles per cubic meter O(2). Alanine concentrations (moles per cubic meter) in the stele of roots exposed to 0.13 and 0.09 moles per cubic meter O(2) increased by 26 and 44%, respectively, above the levels found for aerated roots, whereas alanine in the cortex was unchanged; the increase in stelar alanine concentration was not accompanied by changes in the concentration of free amino acids other than alanine. For plants with their shoots intact, alcohol dehydrogenase and pyruvate decarboxylase activities (micromoles per gram protein per minute) in roots exposed to 0.13 moles per cubic meter O(2) increased in the stele by 40 to 50% over the activity in aerated roots, whereas there was no appreciable increase in alcohol dehydrogenase and pyruvate decarboxylase activity in the cortex of these roots. More convincingly, for roots receiving O(2) solely from the shoots via the aerenchyma, pyruvate decarboxylase in the cortex was in an "inactive" state, whereas pyruvate decarboxylase in the stele was in an "active" state. These results suggest that for roots in O(2)-free solutions, the aerenchyma provides adequate O(2) for respiration in the cortex but not in the stele, and this was supported by a change in pyruvate decarboxylase in the cortex to an active state when the O(2) supply to the roots via the aerenchyma was blocked. PMID- 16668334 TI - Glyceollin I in soybean-cyst nematode interactions : spatial and temporal distribution in roots of resistant and susceptible soybeans. AB - Accumulation of the phytoalexin glyceollin I in roots of soybean (Glycine max [L.] Merr.) following inoculation with race 1 of Heterodera glycines Ichinohe, the soybean cyst nematode (SCN), was determined in a whole-root system by high performance liquid chromatography (HPLC) and in a cross-section system by a radioimmunoassay procedure. In the whole-root system, roots were harvested from controls and nematode-inoculated seedlings immediately after inoculation and at 2 day intervals for 8 days. The roots were extracted with ethanol, and the extracts were subjected to HPLC. Glyceollin I was not detected in roots of either resistant cultivar Centennial or susceptible cultivar Ransom immediately after inoculation with SCN but steadily accumulated in large quantity in roots of Centennial. Accumulation of glyceollin I in roots of Ransom following nematode inoculation was minimal. In the cross-section system, 3-day-old soybean seedlings were inoculated with juvenile nematodes, and root segments containing a single nematode were dissected from inoculated plants at 4-hour intervals under a dissecting microscope. The root segments were embedded in ice and cut into 16 micrometer sections with a cryostat microtome. The spatial and temporal distribution of glyceollin I was determined with a radioimmunoassay procedure specific for the phytoalexin. Glyceollin I was found to accumulate in tissues immediately adjacent to the head region of the nematode in Centennial but not in Ransom. Glyceollin I was detected 8 hours after nematode penetration, and the concentration increased steadily up to 0.3 micromole per milliliter in Centennial 24 hours after penetration. PMID- 16668335 TI - Relationship of endogenous abscisic Acid to sucrose level and seed growth rate of soybeans. AB - It has been proposed that abscisic acid (ABA) may stimulate sucrose transport into filling seeds of legumes, potentially regulating seed growth rate. The objective of this study was to determine whether the rate of dry matter accumulation in seeds of soybeans (Glycine max L.) is correlated with the endogenous levels of ABA and sucrose in those sinks. The levels of ABA and sucrose in seed tissues were compared in nine diverse Plant Introduction lines having seed growth rates ranging from 2.5 to 10.0 milligrams dry weight per seed per day. At 14 days after anthesis (DAA), seeds of all genotypes contained less than 2 micrograms of ABA per gram fresh weight. Levels of ABA increased rapidly, however, reaching maxima at 20 to 30 DAA, depending upon tissue type and genotype. ABA accumulated first in seed coats and then in embryos, and ABA maxima were higher in seed coats (8 to 20 micrograms per gram fresh weight) than in embryos (4 to 9 micrograms per gram fresh weight. From 30 to 50 DAA, ABA levels in both tissues decreased to less than 2 micrograms per gram fresh weight. Levels of sucrose were also low early in development, less than 10 milligrams per gram fresh weight at 14 DAA. However, by 30 DAA, sucrose levels in seed coats had increased to 20 milligrams per gram fresh weight and remained fairly constant for the remainder of the filling period. In contrast, sucrose accumulated in embryos throughout the filling period, reaching levels greater than 40 milligrams per gram fresh weight by 50 DAA. Correlation analyses indicated that the level of ABA in seed coats and embryos was not directly correlated to the level of sucrose measured in those tissues or to the rate of seed dry matter accumulation during the linear filling period. Rather, the ubiquitous pattern of ABA accumulation early in development appeared to coincide with water uptake and the rapid expansion of cotyledons occurring at that time. Whole tissue sucrose levels in embryos and seed coats, as well as sucrose levels in the embryo apoplast, were generally not correlated with the rate of dry matter accumulation. Thus, it appears that, in this set of diverse soybean genotypes, seed growth rate was not limited by endogenous concentrations of ABA or sucrose in reproductive tissues. PMID- 16668336 TI - Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A. AB - An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K(m) of the NADH oxidase after activation by lipids was four- to fivefold less than the K(m) before activation. The V(max) was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A(2) block the stimulation of the oxidase by auxins. PMID- 16668337 TI - Origin of Thylakoid Membranes in Chlamydomonas reinhardtii y-1 at 38 degrees C. AB - The origin of thylakoid membranes was studied in Chlamydomonas reinhardtii y-1 cells during greening at 38 degrees C. Previous studies showed that, when dark grown cells are exposed to light under these conditions, the initial rates of accumulation of chlorophyll and the chlorophyll a/b-binding proteins in membranes are maximal (MA Maloney JK Hoober, DB Marks [1989] Plant Physiol 91: 1100-1106; JK Hoober MA Maloney, LR Asbury, DB Marks [1990] Plant Physiol 92: 419-426). As shown in this paper, photosystem II activity, which was nearly absent in dark grown cells, also increased at a linear rate in parallel with chlorophyll. As compared with those made at 25 degrees C, photosystem II units assembled during greening at 38 degrees C were photochemically more efficient, as judged by saturation at a lower fluence of light and a negligible loss of excitation energy as fluorescence. Electron microscopy of cells in light for 5 or 15 minutes at 38 degrees C showed that these initial, functional thylakoid membranes developed in association with the chloroplast envelope. PMID- 16668338 TI - Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds. AB - Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by alpha-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity. PMID- 16668339 TI - DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves: II. Evidence for Multiple Aliphatic, Neutral Amino Acid Symports. AB - Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent K(m) and K(i) values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the beta-carbon position that places a dense electron cloud close to the alpha amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated volumes did not account for the observed differences in transport specificity. PMID- 16668340 TI - Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots. AB - The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K(m) values of about 160 and 240 micromolar and with V(max) values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K(m) of about 700 micromolar and a V(max) of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K(m) values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme. PMID- 16668341 TI - Effect of Macerase, Oxalic Acid, and EGTA on Deep Supercooling and Pit Membrane Structure of Xylem Parenchyma of Peach. AB - The object of this study was to determine if calcium cross-linking of pectin in the pit membrane of xylem parenchyma restricts water movement which results in deep supercooling. Current year shoots of ;Loring' peach (Prunus persica) were infiltrated with oxalic acid or EGTA solutions for 24 or 48 hours and then either prepared for ultrastructural analysis or subjected to differential thermal analysis. The effect of 0.25 to 1.0% pectinase (weight/volume) on deep supercooling was also investigated. The use of 5 to 50 millimolar oxalic acid and pectinase resulted in a significant reduction (flattening) of the low temperature exotherm and a distinct swelling and partial degradation of the pit membrane. EGTA (10 millimolar) for 24 or 48 hours shifted the low temperature exotherm to warmer temperatures and effected the outermost layer of the pit membrane. A hypothesis is presented on pectin-mediated regulation of deep supercooling of xylem parenchyma. PMID- 16668342 TI - Transport and Metabolism of 1-Aminocyclopropane-1-carboxylic Acid in Sunflower (Helianthus annuus L.) Seedlings. AB - Transport and metabolism of [2,3-(14)C] 1-aminocyclopropane-1-carboxylic acid (ACC) from roots to shoots in 4-day-old sunflower (Helianthus annuus L.) seedlings were studied. [(14)C]ACC was detected in, and (14)C(2)H(4) was evolved from, shoots 0.5 hours after [(14)C]ACC was supplied to roots. Ethylene emanation from the shoots returned to normal levels after 6 hours. The roots showed a similar pattern, although at 24 hours ethylene emanation was still slightly higher than in those plants that did not receive ACC. [(14)C]N-malonyl-ACC (MACC) was detected in both tissues at all times sampled. [(14)C]MACC levels surpassed [(14)C]ACC levels in the shoot at 2 hours, whereas [(14)C]MACC levels in the root remained below [(14)C]ACC levels until 6 hours, after which they were higher. Thin-layer chromatography analysis identified [(14)C] ACC in 1-hour shoot extracts, and [(14)C]MACC was identified in root tissues at 1 and 12 hours after treatment. [(14)C]ACC and [(14)C] MACC in the xylem sap of treated seedlings were identified by thin-layer chromatography. Xylem transport of [(14)C]ACC in treated seedlings, and transport of ACC in untreated seedlings, was confirmed by gas chromatography-mass spectrometry. Some evidence for the presence of [(14)C]MACC in xylem sap in [(14)C]ACC-treated seedlings is presented. A substantial amount of radioactivity in both ACC and MACC fractions was detected leaking from the roots over 24 hours. A second radiolabeled volatile compound was trapped in a CO(2)-trapping solution but not in mercuric perchlorate. Levels of this compound were highest after the peak of ACC levels and before peak MACC levels in both tissues, suggesting that an alternate pathway of ACC metabolism was operating in this system. PMID- 16668343 TI - Light-enhanced dark respiration in mesophyll protoplasts from leaves of pea. AB - The respiratory oxygen uptake by mesophyll protoplasts of pea (Pisum sativum cv Arkel) was stimulated up to threefold after 15 minutes of illumination at an intensity of 1250 microeinsteins per square meter per second in the presence of 5 millimolar bicarbonate at 30 degrees C. The extent of light-enhanced dark respiration (LEDR) increased progressively with duration of preillumination. The LEDR exhibited two phases. The initial high rate of respiration decreased in about 10 minutes to a lower steady value similar to that before illumination. The promotion of LEDR by the presence of bicarbonate and inhibition by glyceraldehyde or 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that LEDR was dependent on products of photosynthetic carbon assimilation/electron transport. Thus, the photosynthetic products exert a markedly quick influence on dark respiration in mesophyll protoplasts. PMID- 16668344 TI - Complete Amino Acid Sequence of a Polypeptide from Zea mays Similar to the Pathogenesis-Related-1 Family. AB - A polypeptide serologically related to the tobacco pathogenesis-related-1 family of proteins has been purified from the root tissue of maize (Zea mays L.), and the complete amino acid sequence has been determined. The mature protein has a calculated molecular weight of 14,970 and isoelectric point of 4.2. The maize protein shows 66 to 68% amino acid identity with the tobacco pathogenesis-related 1 family and 55% identity with the tomato p14 protein. PMID- 16668345 TI - Methyl jasmonate and transpiration in barley. AB - Transpirational water loss from barley (Hordeum vulgare L. var Morrison) leaves was determined gravimetrically. In contrast to the known effects of other growth regulating compounds, it is shown that methyl jasmonate-used either as an airborne vapor or in the transpiration stream-is not acting as a primary short term modulator of stomatal behavior in this species. PMID- 16668346 TI - Comparison of the Cat2 Complementary DNA Sequences of a Normal Catalase Activity Line (W64A) and a High Catalase Activity Line (R6-67) of Maize. PMID- 16668347 TI - Complete Nucleotide Sequence of a Hordeum vulgare Gene Encoding (1-->3, 1-->4) beta-Glucanase Isoenzyme II. PMID- 16668348 TI - Nucleotide sequence of a cDNA encoding malic enzyme from poplar. PMID- 16668349 TI - Isolation, Characterization, and Chromosomal Location of a New cab Gene from Arabidopsis thaliana. PMID- 16668350 TI - Nucleotide Sequence of a Chymotrypsin Inhibitor-2 Gene of Barley (Hordeum vulgare L.). PMID- 16668351 TI - Nucleotide sequence of a complementary DNA encoding pea cytosolic copper/zinc superoxide dismutase. PMID- 16668352 TI - Nucleotide sequence of an iron superoxide dismutase complementary DNA from soybean. PMID- 16668353 TI - Genomic Nucleotide Sequence of a Brassica napus 20-Kilodalton Oleosin Gene. PMID- 16668354 TI - Regulation of 5-Aminolevulinic Acid Synthesis in Developing Chloroplasts : IV. An Endogenous Inhibitor from the Thylakoid Membranes. AB - 5-Aminolevulinic acid synthesis in isolated, intact, developing chloroplasts from greening cucumber (Cucumis sativus) cotyledons was inhibited by broken chloroplast fragments. It was shown that the inhibitory constituent was associated with the thylakoid membrane system. The inhibitor was resistant to boiling, was not a form of ribonuclease, and did not inhibit Mg-chelatase, indicating that massive organelle destruction was not involved. The inhibitor was also found in etioplast and mature chloroplasts; and it was found in barley as well as cucumber. 5-Aminolevulinate synthesis in the dark with exogenous ATP and NADPH, or in the light without added cofactors, were inhibited approximately equally. In the dark, 5-aminolevulinate synthesis and protochlorophyllide synthesis from glutamate were inhibited to about equal extent. The inhibition was decreased when the membranes were washed with aqueous acetone prior to incubation. The inhibition by the unknown factor was compared to the inhibition by gabaculine, 4-amino-5-hexynoic acid, protoheme, and glutathione. The unknown inhibitor appeared to have a number of similarities with protoheme. PMID- 16668355 TI - Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio. AB - The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, alpha and beta, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of alpha- and beta-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of beta-polypeptide and of the more cathodal isoenzymes, whereas alpha-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the alpha- and the catabolic reaction with the beta-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions. PMID- 16668356 TI - Growth and Photosynthetic Characteristics of Solanum tuberosum Plantlets Cultivated in Vitro in Different Conditions of Aeration, Sucrose Supply, and CO(2) Enrichment. AB - Growth characteristics, oxygen exchange, and carbohydrate and chlorophyll contents were determined 30 days after subculturing of single node-derived plantlets of Solanum tuberosum cv Haig cultivated in vitro. Cultivation conditions were: (a) photomixotrophy in closed vessel, (b) photomixotrophy in closed vessel on medium supplemented with silver thiosulfate, (c) photomixotrophy in aerated vessel, (d) photoautotrophy in air, (e) photoautotrophy in CO(2) enriched air. In photomixotrophic conditions, aeration of the vessel enhanced sucrose utilization and had a positive effect on plantlet growth. In photoautotrophic conditions, growth of the plantlets was slow in air and was strongly enhanced by CO(2) enrichment of the atmosphere. Starch to sucrose ratios were higher in plants grown photoautotrophically than in plants grown with sucrose in the medium. Oxygen exchange characteristics on a chlorophyll basis were similar between the plantlets when measured under moderate light, and resembled those of greenhouse plant leaves. In high light, however, plantlets grown photoautotrophically in a CO(2)-enriched atmosphere had higher oxygen exchange rates. We concluded from these results that potato plantlets in vitro in conditions (c), (d), and (e) developed C3-plant photosynthetic characteristics, which were in photoautotrophically grown plantlets comparable to those of field grown plants. PMID- 16668357 TI - Action Spectrum for the Light-Dependent Step in Gametic Differentiation of Chlamydomonas reinhardtii. AB - Differentiation of Chlamydomonas reinhardtii vegetative cells to gametes requires two environmental signals: nitrogen starvation and light. Vegetative cells incubated without nitrogen differentiate into pregametes. Pregametes can be converted into sexually mature gametes by irradiation with light. The action spectrum for the light-dependent step in gamete formation showed two maxima at 370 and 450 nanometers. This is similar to the spectrum of other blue light/ultraviolet light-A-absorbing photoreceptors. PMID- 16668358 TI - Ethylene Production and 1-Aminocyclopropane-1-Carboxylic Acid Conjugation in Thermoinhibited Cicer arietinum L. Seeds. AB - The effect of supraoptimal temperatures (30 degrees C, 35 degrees C) on germination and ethylene production of Cicer arietinum (chick-pea) seeds was measured. Compared with a 25 degrees C control, these temperatures inhibited both germination and ethylene production. The effect of supraoptimal temperatures could be alleviated by treating the seeds with ethylene. It was concluded that one effect of high temperature on germination was due to its negative effect on ethylene production. This inhibitory effect of high temperature was due to increased conjugation of 1-aminocyclopropane-1-carboxylic acid to 1 (malonylamino)cyclopropane-1-carboxylic acid and to an inhibition of ethylene forming enzyme activity. PMID- 16668359 TI - Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants. AB - The levels of cysteine (Cys), gamma-glutamylcysteine (gammaEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6 day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of gammaEC, as if the activity of GSH synthetase was reduced in vivo. [(35)S]Cys injected into endosperms of seedlings was partly metabolized to [(35)S]sulfate. The scutella absorbed both [(35)S]sulfate and [(35)S]Cys and transformed 68 to 87% of the radioactivity into [(35)S]GSH. [(35)S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves. PMID- 16668360 TI - Acid phosphatase-1 from nematode resistant tomato : isolation and characterization of its gene. AB - Aps-1 encodes acid phosphatase-1, one of the many acid phosphatases present in tomato (Lycopersicon esculentum Mill.). Aps-1 is closely linked to Mi, a gene conferring resistance against nematodes. Thus, a clone of Aps-1 would provide access to the region of the genome containing Mi. Acid phosphatase-1 was purified from tomato suspension culture cells. Fragmentary amino acid sequences were derived from the purified protein and from its proteolytic and chemical digestion products. One of these amino acid sequences was used to design an oligodeoxyribonucleotide probe expected to hybridize to acid phosphatase-1 cDNA. This probe identified, in a cDNA library, a clone encoding the carboxyl-terminal sequence of a protein that is very similar, but not identical, to acid phosphatase-1. Using this clone, we discovered a second cDNA clone that corresponds in its carboxyl terminal sequence to acid phosphatase-1 but, surprisingly, retains sequences of an Aps-1 intron. The second cDNA clone was used to detect both a cDNA clone and a genomic clone corresponding to Aps-1. The identity of these clones was confirmed by sequence analysis and by the correlation of a restriction fragment length polymorphism with two Aps-1 alleles in a segregating tomato population. The deduced amino acid sequence of the Aps-1 open reading frame predicts a hydrophobic animoterminal signal sequence and a mature protein with a molecular weight of 25,000. The amino acid sequence of this protein has a strong similarity in size and sequence to a vegetative storage protein of soybean. PMID- 16668361 TI - Light Intensity-Induced Changes in cab mRNA and Light Harvesting Complex II Apoprotein Levels in the Unicellular Chlorophyte Dunaliella tertiolecta. AB - During a transition from high growth irradiance (700 micromoles quanta per square meter per second) to low growth irradiance (70 micromoles quanta per square meter per second), the unicellular marine chlorophyte Dunaliella tertiolecta Butcher increases the cellular pool size of the light-harvesting complex of photosystem II (LHC II). We showed that the increase in LHC II apoproteins and in chlorophyll content per cell is preceded by an approximately fourfold increase in cab mRNA. The increase in cab mRNA is detectable within 1.5 hours following a shift from high to low light intensity. An increase in the relative abundance of cab mRNA was also found following a shift from high light to darkness and from high light to low light in the presence of gabaculine, a chlorophyll synthesis inhibitor. However, the LHC II apoproteins did not accumulate in the latter experiments, suggesting that LHC II apoprotein synthesis is coupled to chlorophyll synthesis at or beyond translation. We propose that changes in energy balance brought about by a change in light intensity may control a regulatory factor acting to repress cab mRNA expression in high light. PMID- 16668362 TI - Two kunitz-type proteinase inhibitors from potato tubers. AB - Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et al. [1989] FEBS Lett 251:94-98). Potato Kunitz inhibitor-2 (molecular weight 20,500, isoelectric point 8.6) is an inhibitor of the microbial proteinase subtilisin Carlsberg; its amino terminus is almost identical to an abundant 22 kilodalton protein from potato tubers (Suh et al. [1990] Plant Physiol 94:40-45) and has significant homology to other Kunitz-type subtilisin inhibitors from small grains. Both Kunitz inhibitors are abundant proteins of the cortex of potato tubers. PMID- 16668363 TI - Influence of water deficit on maize endosperm development : enzyme activities and RNA transcripts of starch and zein synthesis, abscisic Acid, and cell division. AB - In maize (Zea mays L.), drought during the post-pollination stage decreases kernel growth and often leads to grain yield losses. Kernels in the apical region of the ear are more severely affected than basally positioned kernels. We hypothesized that water deficit during early endosperm development might inhibit kernel growth by decreasing endosperm cell division, and that this response might be mediated by changes in endosperm abscisic acid (ABA) levels. Greenhouse-grown maize, cultivar Pioneer 3925, was subjected to water limitation from 1 to 15 days after pollination (DAP), spanning the period of endosperm cell division and induction of storage product accumulation. Water deficit decreased the number of endosperm nuclei during the treatment period; the most substantial effect was in the apical region of ears. Correspondingly, endosperm fresh weight, starch accumulation and dry mass at maturity were decreased by water limitation. Abscisic acid concentrations in endosperm were quantified by enzyme-linked immunosorbent assay. Water deficit increased ABA concentration in apical-region endosperm by four-fold compared to controls. ABA concentrations were also increased in middle and basal regions of the ear, but to a lesser extent. Two key enzymes in the starch synthesis pathway, sucrose synthase and granule-bound ADP glucose starch synthase, and zein, the major storage protein in maize endosperm, were studied as markers of storage product synthesis. Water deficit did not affect sucrose synthase enzyme activity or RNA transcript abundance relative to total RNA. However, ADP-glucose starch synthase activity and RNA transcript abundance decreased slightly in apical-region endosperm of water-limited plants by 15 DAP, compared with well-watered controls. In contrast to starch, there was no treatment effect on the accumulation of zein, evaluated at either the polypeptide or RNA level. We conclude that under the conditions tested, the establishment of starch and zein synthetic potential in endosperm was only slightly affected by plant water deficit during the early phase of kernel growth, and that capacity for growth and starch accumulation was affected by the extent to which cell division was inhibited. Based on correlative changes in ABA concentration and cell division we suggest that ABA may play a role in inhibiting endosperm cell division during water limitation. PMID- 16668364 TI - Relevance of amadori and maillard products to seed deterioration. AB - The possible role of Amadori and Maillard reactions in the deterioration of dry seeds was investigated using model systems and whole soybean seeds, Glycine max cv Hodgson. In model systems of glucose plus an enzyme (lysozyme), the production of Amadori products was accelerated by higher temperature and relative humidity. The reaction between glucose and lysozyme at 50 degrees C, 75% relative humidity, leads to a progressive decline in enzymatic activity. During accelerated aging of soybean seeds (40 degrees C, 100% relative humidity), a sequence is observed in which the Amadori products increase with time and then decline under conditions in which the Maillard products increase in the axes. Loss of germinability occurs at the time when the Maillard products increase in the soybean axes. These results are suggestive of a role for nonenzymic glycation in soybean seed deterioration during accelerated aging. PMID- 16668365 TI - Delayed Onset of Isoprene Emission in Developing Velvet Bean (Mucuna sp.) Leaves. AB - Isoprene (2-methyl-1,3-butadiene) is one of the major volatile hydrocarbons emitted by plants, but its biosynthetic pathway and role in plant metabolism are unknown. Mucuna sp. (velvet bean) is an isoprene emitter, and leaf isoprene emission rate increased as much as 125-fold as leaves developed, and declined in older leaves. Net CO(2) assimilation and stomatal conductance, under different growth and environmental conditions, increased 3 to 5 days prior to an increase in isoprene emission rate, indicating that photosynthetic competence develops before significant isoprene emission occurs. PMID- 16668366 TI - Microtubules in mesophyll cells of nonacclimated and cold-acclimated spinach : visualization and responses to freezing, low temperature, and dehydration. AB - Responses of cortical microtubules in spinach (Spinacia oleracea L. cv Bloomsdale) mesophyll cells to freezing, thawing, supercooling, and dehydration were assessed. Microtubules were visualized using a modified procedure for indirect immunofluorescence microscopy. Leaf sections of nonacclimated and cold acclimated spinach were slowly frozen to various temperatures, fixed while frozen, and microtubules immunolabelled. Both nonacclimated and cold-acclimated cells exhibited nearly complete microtubule depolymerization after ice formation. After 1 hour thawing at 23 degrees C, microtubules in both nonacclimated and cold acclimated cells repolymerized. With time, however, microtubules in nonacclimated cells again depolymerized. Since microtubules in cells of leaf tissue frozen slowly are subjected to dehydration as well as subzero temperatures, these stresses were applied separately and their effects on microtubules noted. Supercooling induced microtubule depolymerization in both nonacclimated and cold acclimated cells, but to a smaller extent than did freezing. Exposing leaf sections to solutions of sorbitol (a cell wall-penetrating osmoticum) or polyethylene glycol 10,000 (a nonpenetrating osmoticum) at room temperature caused microtubule depolymerization. The effects of low temperature and dehydration are roughly additive in producing the observed microtubule responses during freezing. Only small differences in microtubule stability were resolved between nonacclimated and cold-acclimated cells. PMID- 16668367 TI - Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach. AB - Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT(50) (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury. PMID- 16668368 TI - Influence of Water and Temperature Stress on the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination. AB - The temperature dependence of the rate and magnitude of the reappearance of photosystem II (PSII) variable fluorescence following illumination has been used to determine plant temperature optima. The present study was designed to determine the effect of a plant's environmental history on the thermal dependency of the reappearance of PSII variable fluorescence. In addition, this study further evaluated the usefulness of this fluorescence technique in identifying plant temperature optima. Laboratory and greenhouse grown potato (Solanum tuberosum L. cv "Norgold M") plants had a thermal kinetic window between 15 and 25 degrees C. The minimum apparent K(m) of NADH hydroxypyruvate reductase for NADH occurred at 20 degrees C. This temperature was also the temperature providing maximal reappearance of variable fluorescence. Soybean (Glycine max [L.] Merrill cv "Wayne") plants had a thermal kinetic window between 15 and 30 degrees C with a minimum apparent K(m) at 25 degrees C. Maximal reappearance of variable fluorescence was seen between 20 and 30 degrees C. To determine if increasing environmental temperatures increased the temperature optimum provided from the fluorescence response curves, potato and soybean leaves from irrigated and dryland field grown plants were evaluated. Although the absolute levels of PSII variable fluorescence declined with increasing thermal stress, the temperature optimum of the dryland plants did not increase with increased exposure to elevated temperatures. Because of variability in the daily period of high temperature stress in the field, studies were initiated with tobacco plants grown in controlled environment chambers. The reappearance of PSII variable fluorescence in tobacco (Nicotiana tabacum L. cv "Wisconsin 38") leaves that had experienced continuous leaf temperatures of 35 degrees C for 8 days had the same 20 degrees C optima as leaves from plants grown at room temperature. The results of this study suggest that the temperature optimum for the reappearance of variable fluorescence following illumination is not altered by the plant's previous exposure to variable environmental temperatures. These findings support the usefulness of this procedure for the rapid identification of a plant's temperature optimum. PMID- 16668369 TI - Elicitor-induced ethylene biosynthesis in tomato cells: characterization and use as a bioassay for elicitor action. AB - The induction of ethylene biosynthesis by an elicitor partially purified from yeast extract was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells. Unstimulated cells produced little ethylene during exponential growth and even less in stationary phase. Treatment with elicitor stimulated ethylene biosynthesis 10-fold to 20-fold in the exponentially growing cells and more than 100-fold in stationary cells. Activities of both 1-aminocyclopropane-1 carboxylate (ACC) synthase, measured in vitro, and ethylene-forming enzyme (EFE), measured in vivo, increased strongly in response to elicitor treatments. During exponential growth, cells contained large pools of ACC, and the elicitor stimulated ethylene biosynthesis primarily through induction of EFE. In the stationary phase, cells contained almost no ACC, and the elicitor stimulated ethylene biosynthesis primarily through its effect on ACC synthase activity. Cordycepin did not affect the increase in activity of ACC synthase but blocked that of EFE, indicating that the former was posttranscriptionally regulated, the latter transcriptionally regulated. Removal of elicitor by washing or inactivation of a biotinylated derivative of the elicitor by complexation with avidin caused a rapid cessation of the increase in ACC synthase activity, suggesting that continuous presence of stimulus is necessary for the response. Using induction of ethylene production to measure amounts of elicitor, it was found that the elicitor disappeared from the incubation medium during the course of the treatment. PMID- 16668370 TI - Electrical Potentials during Gravitropism in Bean Epicotyls. AB - An apparatus for measuring simultaneously the surface electrical potentials along epicotyl was developed to study the largely bending situation by gravity. Potentials increased on the upper side and decreased on the lower side after the horizontal placement. The time course of electrical changes consisted of two components which correspond to growth movements observed during gravitropism. PMID- 16668371 TI - Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley. AB - In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides. PMID- 16668372 TI - Characterization and comparison of arcelin seed protein variants from common bean. AB - Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L., were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and M(r)s were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M(r)s, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins-3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-1d and 2) could be distinguished by M(r)s and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-1t and arcelin-4 also could be distinguished from each other based on M(r)s and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean alpha-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family. PMID- 16668373 TI - Single rol Genes from the Agrobacterium rhizogenes T(L)-DNA Alter Some of the Cellular Responses to Auxin in Nicotiana tabacum. AB - Two kinds of cellular responses to auxin, the hyperpolarization of protoplasts and the division of protoplast-derived cells, were compared in Nicotiana tabacum plants transformed by different T-DNA fragments of Agrobacterium rhizogenes strain A4. Using transmembrane potential difference measurements to characterize hormonal sensitivity of mesophyll protoplasts, we found a 30-fold increase in sensitivity to auxin in protoplasts transformed by the whole Ri A4 T-DNA. Furthermore, the rol genes of the Ri A4 T(L)-DNA, together or as single genes, were able to increase the sensitivity to auxin by factors up to 10(4). The different effects of the single rol genes on the sensitivity of mesophyll protoplasts to auxin, rolB being the most powerful, were consistent with their respective rhizogenic effects on leaf fragments (A Spena, T Schmulling, C Koncz, J Schell [1987] EMBO J 6: 3891-3899). No difference was seen concerning the effects of auxin on division of cells derived from normal or transformed protoplasts. These results suggest that only some cellular responses to auxin could be selectively altered by rol genes. They also show that rol-transformed tobaccos can be a model system to study auxin action in plants. PMID- 16668374 TI - Biochemical Genetics of Plant Secondary Metabolites in Arabidopsis thaliana: The Glucosinolates. AB - Mutants of Arabidopsis thaliana with a glucosinolate content different from wild type were isolated by screening a mutagenized population of plants. Six mutants were detected out of a population of 1200 screened. One of these mutants, TU1, was analyzed in detail. Leaf and seed tissues of line TU1 lack or have reduced amounts of many of the aliphatic glucosinolates found in the wild type due to a recessive allele, gsm1, of a single nuclear gene, GSM1. The seed phenotype is inherited as a maternal effect suggesting that the embryo is dependent on the maternal tissue for its glucosinolates. Experiments involving feeding of (14)C labeled intermediates suggested that the gsm1 allele results in a metabolic block which decreases the availability of several amino acid substrates required for glucosinolate biosynthesis: 2-amino-6-methylthiohexanoic acid, 2-amino-7 methylthioheptanoic acid, and 2-amino-8-methylthiooctanoic acid. The mutation does not result in any obvious changes in morphology or growth rate. A pathway for the biosynthesis of glucosinolates in A. thaliana is proposed. PMID- 16668375 TI - Amino Acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the Phloem sap of spinach leaves. AB - Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells. PMID- 16668376 TI - Cell Surface Interactions between Bean Leaf Cells and Colletotrichum lindemuthianum: Cytochemical Aspects of Pectin Breakdown and Fungal Endopolygalacturonase Accumulation. AB - After a brief period of biotrophic growth, the anthracnose fungus Colletotrichum lindemuthianum (Sacc. et Mgn.) Bri et Cav. develops extensively in bean leaf cells, causing severe wall alterations and death of the host protoplast. Aplysia gonad lectin, a polygalacturonic acid-binding agglutinin, was complexed to gold and used to study the extent of pectin breakdown during the necrotrophic phase of the infection process. In view of its specific binding properties for the endopolygalacturonase produced by C. lindemuthianum, a polygalacturonase inhibiting protein isolated from bean cell walls was successfully tagged with gold particles and used for localizing the sites of enzyme accumulation in infected host tissues. The basal level of endopolygalacturonase produced by C. lindemuthianum grown in culture was found to increase severalfold when the fungus developed in host plant tissues. The enzyme was able to diffuse freely in the host cell wall, causing drastic degradation of the pectic material of primary walls and middle lamella matrices. The enzymatic alteration of plant cell walls was accompanied by the release of pectic fragments and by the accumulation of pectic molecules at specific sites, such as intercellular spaces and aggregated cytoplasm of infected host cells. The occurrence of pectic molecules at those sites where fungal growth is likely to be restricted is discussed in relation to their origin and their implication in the plant's defense system. PMID- 16668377 TI - Response of soybean photosynthesis and chloroplast membrane function to canopy development and mutual shading. AB - The effect of natural shading on photosynthetic capacity and chloroplast thylakoid membrane function was examined in soybean (Glycine max. cv Young) under field conditions using a randomized complete block design. Seedlings were thinned to 15 plants per square meter at 20 days after planting. Leaves destined to function in the shaded regions of the canopy were tagged during early expansion at 40 days after planting. To investigate the response of shaded leaves to an increase in available light, plants were removed from certain plots at 29 or 37 days after tagging to reduce the population from 15 to three plants per square meter and alter the irradiance and spectral quality of light. During the transition from a sun to a shade environment, maximum photosynthesis and chloroplast electron transport of control leaves decreased by two- to threefold over a period of 40 days followed by rapid senescence and abscission. Senescence and abscission of tagged leaves were delayed by more than 4 weeks in plots where plant populations were reduced to three plants per square meter. Maximum photosynthesis and chloroplast electron transport activity were stabilized or elevated in response to increased light when plant populations were reduced from 15 to three plants per square meter. Several chloroplast thylakoid membrane components were affected by light environment. Cytochrome f and coupling factor protein decreased by 40% and 80%, respectively, as control leaves became shaded and then increased when shaded leaves acclimated to high light. The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers were not affected by light environment or leaf age in field grown plants, resulting in a constant PSII/PSI ratio of 1.6 +/- 0.3. Analysis of the chlorophyll-protein composition revealed a shift in chlorophyll from PSI to PSII as leaves became shaded and a reversal of this process when shaded leaves were provided with increased light. These results were in contrast to those of soybeans grown in a growth chamber where the PSII/PSI ratio as well as cytochrome f and coupling factor protein levels were dependent on growth irradiance. To summarize, light environment regulated both the photosynthetic characteristics and the timing of senescence in soybean leaves grown under field conditions. PMID- 16668378 TI - Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L. AB - The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5' phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5'-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5'-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O acetyl-l-serine at the time of the transfer caused a 100% increase in acid soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of (35)SO(4) (2-) into organic compounds decreased by 80% without O acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5'-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from (35)SO(4) (2-) into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation. PMID- 16668379 TI - Spatial Distribution of Flavonoid Conjugates in Relation to Glucosyltransferase and Sulfotransferase Activities in Flaveria bidentis. AB - The spatial distribution of sulfated and glucosylated flavonols as well as of the enzymes involved in the later steps of their biosynthesis, sulfotransferase and glucosyltransferase, were investigated in the shoots of Flaveria bidentis. The highest amounts of both types of flavonoid conjugates (as micromole per gram fresh weight) and the highest activities of their enzymes (as picokat per milligram) were detected in the terminal bud and the first pair of leaves. Sulfotransferase activity was also highest in the upper stem segments and in the basal section of the leaves. Western blot analysis of protein extracts showed that variations in sulfotransferase activity in different tissues correlate well with the amounts of immunodetected enzyme protein. These results were discussed in relation to the possible role of conjugated flavonoids in plant growth. PMID- 16668380 TI - Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L. AB - Metabolism of [(14)C]gibberellin (GA) A(12) (GA(12)) and [(14)C]gibberellin A(12) aldehyde (GA(12)-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13 hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [(14)C]GA(12) was slower in seed coats than in cotyledons. [(14)C]GA(12)-aldehyde was also metabolized to conjugates in seed coats. Seed coat [(14)C]-metabolites produced from [(14)C]GA(12)-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [(14)C]GA(53)-aldehyde, [(14)C]GA(12)-aldehyde conjugate, and [(14)C]GA(53)-aldehyde conjugate were major metabolites produced from [(14)C]GA(12)-aldehyde by seed coats aged 20-22 days or older. The dilution of (14)C in these compounds by (12)C, as compared to the supplied [(14)C]GA(12) aldehyde, indicated that they are endogenous. Feeding [(14)C]GA(53)-aldehyde led to the production of [(14)C]GA(53)-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [(14)C]GA(12), [(14)C]GA(12)-aldehyde, or [(3)H]GA(9), were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA(15), GA(24), GA(9), GA(51), GA(51)-catabolite, GA(20), GA(29), and GA(29)-catabolite were detected in seed coats, whereas GA(9), GA(53), GA(44), GA(19), GA(20), and GA(29) were found in cotyledons. The highest GA levels were for GA(20) and GA(29) in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA(29) and GA(29)-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively). PMID- 16668381 TI - Characterization of tonoplast polypeptides isolated from corn seedling roots. AB - Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg(2+) from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions. PMID- 16668382 TI - Diffusion and Electric Mobility of Ions within Isolated Cuticles of Citrus aurantium: Steady-State and Equilibrium Values. AB - We report a new method for measuring cation and anion permeability across cuticles of sour orange, Citrus aurantium, leaves. The method requires the measurement of two electrical parameters: the diffusion potential arising when the two sides of the cuticle are bathed in unequal concentrations of a Cl(-) salt; and the electrical conductance of the cuticle measured at a salt concentration equal to the average of that used in the diffusion-potential measurement. The permeabilities of H(+), Li(+), Na(+), K(+), and Cs(+) ranged from 2 x 10(-8) to 0.6 x 10(-8) meters per second when cuticles were bathed in 2 moles per cubic meter Cl(-) salts. The permeability of Cl(-) was 3 x 10(-9) meters per second. The permeability of Li(+), Na(+), and K(+) was about five times less when measured in 500 moles per cubic meter Cl(-) salts. We also report an asymmetry in cuticle-conductance values depending on the magnitude and the direction of current flow. The asymmetry disappears at low current-pulse magnitude and increases linearly with the magnitude of the current pulse. This phenomenon is explained in terms of transport-number effects in a bilayer model of the cuticle. Conductance is not augmented by current carried by exchangeable cations in cuticles; conductance is rate limited by the outer waxy layer of the cuticle. PMID- 16668383 TI - Physiological basis for differential sensitivities of plant species to protoporphyrinogen oxidase-inhibiting herbicides. AB - With a leaf disc assay, 11 species were tested for effects of the herbicide acifluorfen on porphyrin accumulation in darkness and subsequent electrolyte leakage and photobleaching of chlorophyll after exposure to light. Protoporphyrin IX (Proto IX) was the only porphyrin that was substantially increased by the herbicide in any of the species. However, there was a wide range in the amount of Proto IX accumulation caused by 0.1 millimolar acifluorfen between species. Within species, there was a reduced effect of the herbicide in older tissues. Therefore, direct quantitative comparisons between species are difficult. Nevertheless, when data from different species and from tissues of different age within a species were plotted, there was a curvilinear relationship between the amount of Proto IX caused to accumulate during 20 hours of darkness and the amount of electrolyte leakage or chlorophyll photobleaching caused after 6 and 24 hours of light, respectively, following the dark period. Herbicidal damage plateaued at about 10 nanomoles of Proto IX per gram of fresh weight. Little difference was found between in vitro acifluorfen inhibition of protoporphyrinogen oxidase (Protox) of plastid preparations of mustard, cucumber, and morning glory, three species with large differences in their susceptibility at the tissue level. Mustard, a highly tolerant species, produced little Proto IX in response to the herbicide, despite having a highly susceptible Protox. Acifluorfen blocked carbon flow from delta-aminolevulinic acid to protochlorophyllide in mustard, indicating that it inhibits Protox in vivo. Increasing delta-aminolevulinic acid concentrations (33-333 micromolar) supplied to mustard with 0.1 millimolar acifluorfen increased Proto IX accumulation and herbicidal activity, demonstrating that mustard sensitivity to Proto IX was similar to other species. Differential susceptibility to acifluorfen of the species examined in this study appears to be due in large part to differences in Proto IX accumulation in response to the herbicide. In some cases, differences in Proto IX accumulation appear to be due to differences in activity of the porphyrin pathway. PMID- 16668384 TI - Activity and accumulation of cell division-promoting phenolics in tobacco tissue cultures. AB - Dehydrodiconiferyl alcohol glucosides (DCGs) are derivatives of the phenylpropanoid pathway that have been isolated from Catharansus roseus L. (Vinca rosea) crown gall tumors. Fractions containing purified DCGs have been shown previously to promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins. In this study, we utilized synthetic DCG isomers to confirm the cell division-promoting activity of DCG isomers A and B and show that they neither promote shoot meristem initiation on Nicotiana tabacum L., cv Havana 425, leaf explants nor induce betacyanin synthesis in amaranth seedlings. Analysis of cultured tobacco pith tissue demonstrated that DCG accumulation was stimulated by cytokinin treatment and correlated with cytokinin-induced cell division. Thus, the accumulation of metabolites that could replace cytokinin in cell division bioassays is stimulated by cytokinins. These data support the model that DCGs are a component of a cytokinin-mediated regulatory circuit controlling cell division. PMID- 16668385 TI - Effects of Mild Water Stress and Diurnal Changes in Temperature and Humidity on the Stable Oxygen and Hydrogen Isotopic Composition of Leaf Water in Cornus stolonifera L. AB - In this paper we make comparisons between the observed stable isotopic composition of leaf water and the predictions of the Craig-Gordon model of isotopic enrichment when plants (Cornus stolonifera L.) were exposed to natural, diurnal changes in temperature and humidity in a glasshouse. In addition, we determined the effects of mild water stress on the isotopic composition of leaf water. The model predicted different patterns of diurnal change for the oxygen and hydrogen isotopic composition of leaf water. The observed leaf water isotopic composition followed qualitatively similar patterns of diurnal change to those predicted by the model. At midday, however, the model always predicted a higher degree of heavy isotope enrichment than was actually observed in leaves. There was no effect of mild water stress on the hydrogen isotopic composition of leaf water. For the oxygen isotopic composition of leaf water, there was either no significant difference between control and water-stressed plants or the stressed plants had lower delta(18)O values, despite the enriched stem water isotopic composition observed for the stressed plants. PMID- 16668386 TI - Phytochelatin accumulation and cadmium tolerance in selected tomato cell lines. AB - Four cell lines of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, were selected for their ability to grow in the presence of up to 6 millimolar CdCl(2). The intracellular Cd concentration in these cells was at least 2.3 times higher than in the medium. Growth in media containing higher concentrations of Cd was accompanied by increased production of Cd-binding phytochelatins and a trend toward accumulation of higher molecular weight phytochelatins. At least 90% of the Cd in the most tolerant cells was associated with Cd-phytochelatin complexes. Cell lines maintained an increased tolerance of Cd in the absence of continuous selection pressure. PMID- 16668387 TI - Influence of Ozone on the Stable Carbon Isotope Composition, deltaC, of Leaves and Grain of Spring Wheat (Triticum aestivum L.). AB - The relative composition of stable carbon isotopes, delta(13)C, was determined in flag leaves and grain of spring wheat (Triticum aestivum L. cv Albis) grown in open-top field fumigation chambers and exposed to different O(3) levels during the growing season. The aim of the study was to establish exposure-response relationships for the radiation-weighted seasonal mean O(3) concentration and delta(13)C (relative deviation of the (13)C/(12)C ratio) values of the two plant parts. Samples were collected at harvest in 1986, 1987, and 1988. With increasing O(3) concentration, delta(13)C values increased (became less negative) proportionally. Year to year delta(13)C differences at equivalent O(3) concentrations were small. The shift in delta(13)C caused by O(3) was more pronounced in grain than in leaves. According to models of (13)C discrimination in C(3) plants, these results indicate increasing limitation of photosynthesis by CO(2) diffusion relative to limitation by carboxylation with increasing O(3) exposure. This conclusion is not in agreement with results from gas exchange analysis. Water use efficiency in green flag leaves tended to decrease with increasing O(3), indicating a dominating effect of O(3) on CO(2) carboxylation. PMID- 16668388 TI - Cloning and Sequencing of the cDNA Encoding the Rubber Elongation Factor of Hevea brasiliensis. AB - In Hevea brasiliensis, the rubber particle in the laticiferous vessel is the site of rubber (cis-1-4-polyisoprene) biosynthesis. A 14 kilodalton protein, rubber elongation factor (REF), is associated with the rubber particle in a ratio of one REF to one rubber molecule (Dennis M, Henzel W, Bell J, Kohr W, Light D [1989] J Biol Chem 264: 18618-18628; Dennis M, Light D [1989] J Biol Chem 264: 18608 18617). To obtain more information concerning the function of REF and its synthesis and assembly in the rubber particle, we isolated cDNA clones encoding REF. We used antibodies to REF to screen a Hevea leaf gammagt11 cDNA expression library and obtained several positive clones. Sequence analysis of the REF cDNA clones showed that the REF mRNA contains 121 nucleotides of 5'-nontranslated sequences and a 205 nucleotide 3'-nontranslated region. The open reading frame encodes the entire 14 kilodalton REF protein without any extra amino acids (Dennis M, Henzel W, Bell J, Kohr W, Light D [1989] J Biol Chem 264: 18618 18628). The REF cDNA was subcloned in pGEM-3Z/-4Z and expressed in vitro. The translation product is a 14 kilodalton protein that can be immunoprecipitated with antibodies to REF. Addition of microsomal membranes to the in vitro translation product did not alter the mobility of the REF protein. This, and the sequence data, indicate that REF is not made as a preprotein. Our results suggest that REF is synthesized on free polysomes in the laticifer cytoplasm and that assembly of the rubber particles is likely to occur in the cytosol. PMID- 16668389 TI - Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus. AB - Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance. PMID- 16668390 TI - Photoacoustic Study of Changes in the Energy Storage of Photosystems I and II during State 1-State 2 Transitions. AB - Using photoacoustic spectroscopy, state 1-state 2 transitions were demonstrated in vivo in intact sugar maple leaves (Acer saccharum Marsh.) by following the changes in energy storage of photosystems (PS) I and II. Energy storage measured with 650 nm modulated light (light 2) in the presence of background white light indicated the total energy stored by both photosystems (ES(t)), and in the presence of background far-red light showed the energy stored by PSI (ES(psi)). The difference between ES(t) and ES(psi) gave the energy stored by PSII (ES(psii)). While ES(t) remained nearly constant during state transitions, both ES(psi) and ES(psii) changed considerably. The ratio of ES(psii) to ES(psi), an indicator of the energy distribution between the two photosystems, decreased or increased during transition to state 2 or state 1, respectively. State transitions were completed in about 20 min and were fully reversible. During transition from state 1 to state 2, the fraction of excitation energy gained by PSI was nearly equal to that lost by PSII. This fraction of excitation energy transferred from PSII to PSI accounted for about 5% of the absorbed light (fluorescence is not considered), 19% of ES(t), 34% of ES(psii), and 43% of ES(psi) in state 2. NaF treatment inhibited the transition to state 1. Data in the present study confirm the concept of changes in absorption cross-section of photosystems during state transitions. PMID- 16668391 TI - Regulation of light-induced chloroplast transcription and translation in eight day-old dark-grown barley seedlings. AB - Plastid transcription and translation are light-activated in 8-day-old dark-grown barley (Hordeum vulgare L.) seedlings. Pretreatment of dark-grown seedlings with cycloheximide (inhibitor of cytoplasmic protein synthesis) abolished the activation of rbcL, psbA, and psaA-B transcription by light. In contrast, inhibition of plastid protein synthesis by chloramphenicol stimulated light activated transcription of rbcL, psbA, and psaA-B. Light-induced transcription of the plastid genome occurred normally in the chlorophyll-deficient mutant xan J(64). These results suggest that although the light-induced activation of plastid transcription is modulated by cytoplasmic and organellar protein synthesis, transcriptional activation is not dependent on the absorption of light by protochlorophyllide or the attainment of photosynthetic competence. In addition, plastid translation increased dramatically when 8-day-old dark-grown seedlings were illuminated and activation was dependent on cytoplasmic protein synthesis. Blockage of light-activated plastid transcription by Tagetin treatment (inhibitor of plastid RNA polymerase) did not attenuate the activation of plastid translation by light. These results suggest that while light simultaneously activates plastid transcription and translation, the rapid burst in plastid protein synthesis is due mainly to cytoplasmic-derived changes that regulate the rate of translation of pre-existing mRNAs. PMID- 16668392 TI - Superoxide Dismutase in the Symbiont Anabaena azollae Strasb. AB - Superoxide dismutase was investigated in the symbiont Anabaena azollae Strasb. living in Azolla filiculoides Lam. In vegetative cells, three isoenzymatic forms of superoxide dismutase, containing manganese, iron, and the hybrid iron manganese, respectively, were present. Hybrid superoxide dismutase, detected for the first time in cyanobacteria, was 7% of the total superoxide dismutase present in vegetative cells. All three superoxide dismutase forms increased in the Anabaena vegetative cells obtained from irradiated plants grown in winter. In heterocysts, only an iron superoxide dismutase was present, which amounted to 25% of total vegetative cell superoxide dismutase activity. Hybrid superoxide dismutase appeared in heterocysts after irradiation. In vegetative cells of Anabaena from plants grown in summer, the basal level of total superoxide dismutase increased by 60% as compared with winter, and was unaffected by irradiation. The levels of superoxide dismutase in heterocysts from control and exposed plants grown in summer were comparable to those observed in heterocysts obtained from the plants grown during winter. No direct correlation was found between nitrogenase activity and superoxide dismutase in heterocysts. The presence of cyanophycin granules, either within the heterocyst pore channel or close to the transversal septum of vegetative cells, suggested a mechanism to stop communications between vegetative cells and heterocysts. PMID- 16668393 TI - Dihydroflavonol Reductase Activity in Relation to Differential Anthocyanin Accumulation in Juvenile and Mature Phase Hedera helix L. AB - Juvenile phase English ivy (Hedera helix L.) plants accumulate anthocyanin pigment in the hypodermis of stems and petioles, whereas genetically identical plants of the mature phase do not. The objective of this work was to assess which enzyme(s) might limit anthocyanin accumulation in mature phase ivy. Leaf discs of both juvenile and mature phase ivy accumulated comparable levels of the flavonols kaempferol and quercetin, whereas only juvenile phase discs accumulated anthocyanin. The accumulation of quercetin, but lack of accumulation of leucocyanidin or anthocyanin in mature phase discs, suggested that mature discs lacked dihydroflavonol reductase activity. There was no detectable dihydroflavonol reductase activity in mature phase discs, whereas there was an induction of activity in juvenile phase discs in response to sucrose, or photosynthetically fixed carbon, and light as a photomorphogenic signal. Phenylalanine ammonia-lyase, an enzyme early in the anthocyanin biosynthetic pathway, was induced above its basal level by sucrose and light in discs of both phases of ivy, with greater activity in mature phase discs. Phenylpropanoids, a class of compounds that are precursors to flavonoids, accumulated in leaf discs of both phases, with greater levels in mature phase discs. These results indicate that the lack of dihydroflavonol reductase activity limits the accumulation of anthocyanin in mature phase tissue. PMID- 16668394 TI - Germin-Like Polypeptides Increase in Barley Roots during Salt Stress. AB - The 26 kilodalton, isoelectric point 6.3 and 6.5 (Gs1 and Gs2) polypeptides that increase in barley (Hordeum vulgare L.) roots during salt stress were isolated and identified. Both Gs1 and Gs2 had high sequence similarity to germin, a protein that increases significantly in germinating wheat seeds. Like germin, Gs1 and Gs2 were resistant to proteases and were glycosylated. Immunoblots were probed with antibodies to Gs1 and Gs2 to determine the distribution of these polypeptides among organs and cell-free fractions. Gs1 and Gs2 were present in roots and coleoptiles, but absent from leaves. In roots, Gs1 and Gs2 were present in the mature region, but not the tip. Gs1 and Gs2 increased in roots, but decreased in coleoptiles in response to salt stress. Gs1 and Gs2 were distributed among the soluble, microsomal, and cell wall fractions of roots, but the majority of Gs1 and Gs2 was present in the soluble fraction. Although Gs1 and Gs2 were heat stable, their synthesis was not affected by abscisic acid treatment. Gs2 accumulated during abscisic acid treatment, whereas Gs1 did not. However, a 25.5 kilodalton, isoelectric point 6.1 polypeptide that was immunologically related to Gs1 did accumulate with abscisic acid treatment. PMID- 16668395 TI - Plastid import and iron-sulfur cluster assembly of photosynthetic and nonphotosynthetic ferredoxin isoproteins in maize. AB - We have previously isolated and characterized two cDNAs of maize (Zea mays) ferredoxin (Fd) isoproteins, which are differentially expressed in photosynthetic and nonphotosynthetic organs, and in response to illumination of the developing seedlings (Hase T, Kimata Y, Yonekura K, Matsumura T, Sakakibara H [1991] Plant Physiol 96: 77-83). To clarify the biosynthetic pathway of Fds present in the different organs, precursors of the two Fd isoproteins synthesized by in vitro transcription and translation were examined to determine whether they are imported to chloroplasts and etioplasts. Precursors for both Fd I and Fd III, a leaf-specific, photosynthetic isoprotein and a constitutive, nonphotosynthetic one, respectively, were imported into chloroplasts and processed to the mature size. Some of the mature-sized molecules inside the organelles were found to be assembled with an iron-sulfur cluster. The cluster assembly occurred without tight coupling to the translocation and processing steps of the protein import, and the process was time and temperature dependent and did not require light. Etioplasts were also capable of importing the precursor of Fd III and assembling the cluster. These combined data show that the constitutive, nonphotosynthetic Fd has the ability to become localized in plastids as a functional molecule. PMID- 16668396 TI - Thylakoid Organization in the Chromophyte Alga Ochromonas danica: Isolation and Characterization of a New Pigment-Protein Complex. AB - This report describes the isolation and preliminary characterization of a new pigment-protein complex from the chromophyte alga, Ochromonas danica. The pigment protein complex was obtained by extracting a thylakoid membrane preparation with the zwitterionic detergent lauryldimethylamine oxide followed by ultracentrifugation on sucrose gradients. The pigment-protein complex has been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, absorption spectroscopy, and low temperature (77 Kelvin) chlorophyll fluorescence spectroscopy. A polypeptide with a monomeric molecular weight of 31,000 as determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the major constituent of this pigment-protein complex. The major pigment in this complex was chlorophyll a, although an as yet unidentified carotenoid was also present. There was no evidence for the presence of chlorophyll c. PMID- 16668397 TI - In Vivo and In Vitro Protein Phosphorylation Studies on Ochromonas danica, an Alga with a Chlorophyll a/c/Fucoxanthin Binding Protein. AB - The phosphorylation of thylakoid membranes in the Chromophyte alga Ochromonas danica was studied in whole cells and in vitro. Protein kinase activity was observed in the thylakoid fraction, and several membrane-bound polypeptides were found to be phosphorylated. The thylakoid protein kinase demonstrated several unusual regulatory properties. Both the polypeptides that were phosphorylated and the rate of protein phosphorylation were independent of illumination. Protein kinase activity was also unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron. The kinase activity was inhibited under strong reducing conditions. Whole cells labeled with (32)PO(4) (3-) were converted to light states I and II by pre illumination favoring photosystem I or photosystem II, respectively. Analysis of the phosphoproteins from cells in state I and state II showed that no changes in phosphorylation accompanied the change in energy redistribution. PMID- 16668398 TI - Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP Glucose:Dolicholphosphate Glucosyltransferase. AB - UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl(2). The K(m) for UDP-glucose was determined as 27 micromolar, and the K(m) for dolichol-P was 2 micromolar. Using the UDP glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [(32)P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins. PMID- 16668399 TI - In vitro flower bud formation in tobacco: interaction of hormones. AB - External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate. PMID- 16668400 TI - Characterization of the Arrest in Anther Development Associated with Gibberellin Deficiency of the gib-1 Mutant of Tomato. AB - The role of gibberellins in flower bud development was investigated by studying the gib-1 mutant of tomato, Lycopersicon esculentum. This gibberellin-deficient mutant initiates flower buds, but floral development is not completed unless the mutant is treated with gibberellin. Treatment with other plant growth regulators does not induce normal flower development. Development of gib-1 flower buds, as measured by progress toward anthesis, ceases at a bud length of 2.5 millimeters; however, increase in size of the bud continues. Buds between 2.5 and 3.7 millimeters are developmentally arrested but still are capable of developing normally after treatment with gibberellic acid. Anthers of these developmentally arrested buds contain pollen mother cells that are in the G1 phase of premeiotic interphase. Following treatment of developmentally arrested buds with gibberellic acid, premeiotic DNA synthesis and callose accumulation in pollen mother cells are evident by 48 hours posttreatment, and within 66 hours, prophase I of meiosis and meiosis-related changes in tapetum development are observable. PMID- 16668401 TI - Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO(2)-Free Air : Evidence for Cyclic Electron Flow in Vivo. AB - The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO(2) compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO(2) had been removed. P700 was more oxidized at any measured irradiance in CO(2)-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO(2)-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO(2)-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO(2)-free air, with an activation state 50% of maximum. We conclude that, at the CO(2) compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane. PMID- 16668402 TI - Biochemical Correlates of the Circadian Rhythm in Photosynthesis in Phaseolus vulgaris. AB - A circadian rhythm in photosynthesis occurs in Phaseolus vulgaris after transfer from a natural or artificial light:dark cycle to constant light. The rhythm in photosynthesis persists even when intercellular CO(2) partial pressure is held constant, demonstrating that the rhythm in photosynthesis is not entirely due to stomatal control over the diffusion of CO(2). Experiments were conducted to attempt to elucidate biochemical correlates with the circadian rhythm in photosynthesis. Plants were entrained to a 12-hour-day:12-hour-night light regimen and then monitored or sampled during a subsequent period of constant light. We observed circadian oscillations in ribulose-1,5-bisphosphate (RuBP) levels, and to a lesser extent in phosphoglyceric acid (PGA) levels, that closely paralleled oscillations in photosynthesis. However, the enzyme activity and activation state of the enzyme responsible for the conversion of RuBP to PGA, ribulose-1,5-bisphosphate carboxylase/oxygenase, showed no discernible circadian oscillation. Hence, we examined the possibility of circadian effects on RuBP regeneration. Neither ribulose-5-phosphate kinase activity nor the level of ATP fluctuated in constant light. Oscillations in triose-phosphate levels were out of phase with those observed for RuBP and PGA. PMID- 16668403 TI - Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii. AB - The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO(2)-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO(2). Adaptation to air levels of CO(2) by Chlamydomonas involves induction of a CO(2)-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO(2)-enriched cells. Unlike WT, the high-CO(2)-requiring mutant, cia-5, does not respond to limiting CO(2) conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO(2) which are different from WT and cia 5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved. PMID- 16668404 TI - Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth. AB - Root temperature strongly affects shoot growth, possibly via "nonhydraulic messengers" from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25 degrees and 35 degrees C, respectively. Hydraulic conductance (L(p)) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25 degrees C, remaining high with further warming. In sorghum, the L(p) of intact plants and of detached roots peaked at 35 degrees C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L(p). Extreme temperatures greatly decreased ion flux, particularly K(+) and NO(3) (-), to the xylem of detached roots of both species. Removing external K(+) did not alter short-term K(+) flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently "uncoupled" from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions. PMID- 16668405 TI - Regulation of a chitinase gene promoter by ethylene and elicitors in bean protoplasts. AB - Chitinase gene expression has been shown to be transcriptionally regulated by a number of inducers, including ethylene, elicitors, and pathogen attack. To investigate the mechanism(s) responsible for induction of chitinase gene expression in response to various stimuli, we have developed a transient gene expression system in bean (Phaseolus vulgaris) protoplasts that is responsive to ethylene and elicitor treatment. This system was used to study the expression of a chimeric gene composed of the 5' flanking sequences of a bean endochitinase gene fused to the reporter gene beta-glucuronidase linked to a 3' fragment from nopaline synthase. Addition of 1-aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene, or elicitors such as chitin oligosaccharides or cell wall fragments derived from Colletotrichum lagenarium, to transformed protoplasts resulted in a rapid and marked increase in the expression of the chimeric gene. The kinetics and dose response for these treatments were similar to those observed for the native gene in vivo. Analyses of 5' deletion mutants in the protoplast system indicated that DNA sequences located between -305 and -236 are important for both ethylene and elicitor induction of the reporter gene. PMID- 16668406 TI - Carbon Transfer and Partitioning between Vegetative and Reproductive Organs in Pisum sativum L. AB - Assimilate partitioning was studied in the common pea (Pisum sativum L.) by feeding (14)CO(2) to whole plants and measuring radioactivity in different organs 48 hours after labeling. Two experimental protocols were used. For the first, one reproductive node was darkened with an aluminum foil, to prevent photosynthesis during labeling. The aim was to study assimilate translocation among nodes. The second was carried out to assess any priority among sinks. Whole plants were shaded, during labeling, to reduce carbon assimilation. Various developmental stages between the onset of flowering and the final stage in seed abortion of the last pod were chosen for labeling. When all photosynthetic structures at the first reproductive node were darkened at any stage of development after the formation of the first flower, the first pod was supplied with assimilates from other nodes. In contrast, later developed pods, when photosynthetic structures at their node were darkened, received assimilates from other nodes only when they were beyond their final stage in seed abortion. Reducing illumination to 30% did not change distribution of assimilated carbon between vegetative and reproductive structures, nor among pods. It appears that the relative proportion of (14)C allocated to any one pod, compared to other pods, depends on the dry weight of that pod as a proportion of the total reproductive dry weight. When the plant was growing actively, following the start of the reproductive phase until a few days before the end of flowering, the top of the plant (i.e., all the organs above the last opened flower) had a higher sink strength and a higher relative specific activity than pods, suggesting that it was a more competitive sink for assimilates. The pattern of assimilate distribution described here provides an explanation for pod and seed abortion. PMID- 16668407 TI - Overproduction of petunia chloroplastic copper/zinc superoxide dismutase does not confer ozone tolerance in transgenic tobacco. AB - Transgenic tobacco (Nicotiana tabacum cultivar W38) plants that overproduce petunia chloroplastic Cu/Zn superoxide dismutase were exposed to ozone dosages that injure control tobacco plants. Based on foliar injury ratings, there was no consistent protection provided to the transgenic plants. These data indicate that an increase in the chloroplastic Cu/Zn superoxide dismutase alone is not sufficient to reduce ozone toxicity. PMID- 16668408 TI - Gibberellic Acid Regulates the Level of a BiP Cognate in the Endoplasmic Reticulum of Barley Aleurone Cells. AB - The isolation of a 70-kilodalton protein from barley (Hordeum vulgare L.) aleurone layers that cross-reacts with an antibody against yeast binding protein (BiP) is reported. Endoplasmic reticulum isolated from aleurone layers treated with gibberellic acid contain much higher levels of the BiP cognate than do membranes isolated from layers treated with abscisic acid. PMID- 16668409 TI - Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco. AB - The complete coding sequence for the trichodiene synthase gene from Fusarium sporotrichioides was introduced into tobacco (Nicotiana tabacum) under the regulation of the cauliflower mosiac virus 35S promoter. Expression of trichodiene synthase was demonstrated in the leaves of transformed plants. Leaf homogenates incubated with [(3)H]farnesyl pyrophosphate produced trichodiene as a major product. Trichodiene was detected in the leaves of a transformed plant at a level of 5 to 10 nanograms per gram fresh weight. The introduction of a fungal sesquiterpene cyclase gene into tobacco has resulted in the expression of an active enzyme and the accumulation of low levels of its sesquiterpenoid product. PMID- 16668410 TI - Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene. AB - The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in (13)C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO(2). beta-Carotene, an isoprenoid plant constituent, was depleted in (13)C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in (13)C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less (13)C) than C(3) plant material but when plants are stressed the isotopic composition could vary. PMID- 16668411 TI - Sequence of a Complementary DNA from Cucumis sativus L. Encoding the Stearoyl Acyl-Carrier Protein Desaturase. PMID- 16668412 TI - Amino Acid Sequence of an Algal Peptide Elongation Factor EF-2 Deduced from the Complementary DNA Sequence. PMID- 16668413 TI - Barley Acyl Carrier Protein II: Nucleotide Sequence of cDNA Clones and Chromosomal Location of the Acl2 Gene. PMID- 16668414 TI - SO(2) Effect on Photosynthetic Activities of Intact Sugar Maple Leaves as Detected by Photoacoustic Spectroscopy. AB - Short-term (4 hours) effect of different concentrations of SO(2) fumigation on in vivo photochemical activities of sugar maple (Acer saccharum Marsh.) leaves was investigated using photoacoustic spectroscopy. The relative quantum yield of O(2) evolution (ratio of O(2) signal to the photothermal signal) and photochemical energy storage are increased by 0.05 microliter per liter of SO(2). This increase is more pronounced in 5 to 7 year old saplings than in 3 month old seedlings. Both oxygen-relative quantum yield and energy storage of seedlings are inhibited by increased concentrations of SO(2) and the inhibition is concentration dependent. The inhibition is greater in seedlings than in saplings at 2 microliters per liter of SO(2), indicating the more susceptible nature of seedlings. The present study indicates a concentration dependent differential effect of SO(2) on photochemical activities of sugar maple leaves. PMID- 16668415 TI - Circadian Rhythm in Amino Acid Uptake by Synechococcus RF-1. AB - In the prokaryote Synechococcus RF-1, circadian changes in the uptake of l leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in l-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a free-running period of about 24 hours in L/L at 29 degrees C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0 degrees C). The free-running periodicity was "temperature-compensated" from 21 to 37 degrees C. A 24 hour depletion of extracellular Ca(2+) before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO(3). The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L. PMID- 16668416 TI - Modulation of auxin-binding proteins in cell suspensions : I. Differential responses of carrot embryo cultures. AB - This paper shows that the level of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium determines the level of auxin-binding proteins in the membranes of carrot, Daucus carota, cells grown in suspension. This induction takes slightly more than 2 hours to complete and can be elicited by natural as well as synthetic auxins. The auxin binding sites thus generated, which are pronase-sensitive, bind 2,4-D, indoleacetic acid, and naphthalene-acetic acid (NAA) equally well. However both alpha- and beta-NAA bind, whereas only alpha-NAA is effective in the inductive process. Cells committed to embryogeny (proembryogenic masses) do not respond to auxin, i.e. their level of auxin-binding proteins remains very low, and they do not seem to synthesize the hormone, as indicated by inhibitor studies. Sensitivity to, and production of, auxin, begins when the embryo becomes polarized, i.e. at postglobular stage. PMID- 16668417 TI - Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston. AB - Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of (14)C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1 (14)C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3 phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-(14)C]18:1-CoA and G-3-P indicated that (14)C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-(3)H(N)]G-3-P and [1-(14)C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn 3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-(14)C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [(3)H]phosphatidylcholines to [(3)H]diacylglycerols, which subsequently acted as acceptors for (14)C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs. PMID- 16668418 TI - Stress Responses in Alfalfa (Medicago sativa L.): X. Molecular Cloning and Expression of S-Adenosyl-l-Methionine:Caffeic Acid 3-O-Methyltransferase, a Key Enzyme of Lignin Biosynthesis. AB - S-Adenosyl-l-methionine:caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the conversion of caffeic acid to ferulic acid, a key step in the biosynthesis of lignin monomers. We have isolated a functionally active cDNA clone (pCOMT1) encoding alfalfa (Medicago sativa L.) COMT by immunoscreening a lambdaZAPII cDNA expression library with anti-(aspen COMT) antibodies. The derived amino acid sequence of pCOMT1 is 86% identical to that of COMT from aspen. Southern blot analysis indicates that COMT in alfalfa is encoded by at least two genes. Addition of an elicitor preparation from bakers' yeast to alfalfa cell suspension cultures resulted in a rapid accumulation of COMT transcripts, which reached a maximum level around 19 hours postelicitation. Northern blot analysis of total RNA from different organs of alfalfa plants at various developmental stages showed that COMT transcripts are most abundant in roots and stems. Transcripts encoding ATP: i-methionine-S-adenosyl transferase (AdoMet synthetase, EC 2.5.1.6), the enzyme responsible for the synthesis of the methyl donor for the COMT reaction, were coinduced with COMT transcripts in elicitor-treated cells and exhibited a similar pattern of expression to that of COMT in different organs of alfalfa plants at various stages of development. PMID- 16668419 TI - Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits. AB - We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a lambdagtll expression library constructed from MG pericarp poly(A)(+) RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits. PMID- 16668420 TI - Metabolic bases for differences in sensitivity of two pea cultivars to sulfur dioxide. AB - An oxidative chain reaction of sulfite initiated by the superoxide ion produced in the Mehler reaction has been implicated in the damage of plants exposed to sulfur dioxide. The toxicity of SO(2) may be alleviated by free radical scavenging systems acting to terminate this chain reaction. Hence, the relative sensitivity of plants to SO(2) toxicity could depend on differences in the responses of the levels of antioxidant metabolites and enzymes. The effect of SO(2) exposure on glutathione and ascorbic acid contents, glutathione reductase, and superoxide dismutase activities was assayed in two cultivars (Progress, Nugget) of pea (Pisum sativum L.) in which apparent photosynthesis showed a differential sensitivity to 0.8 microliter per liter SO(2) (R. Alscher, J. L. Bower, W. Zipfel [1987] J Exp Bot 38:99-108). Total and reduced glutathione increased more rapidly and to a greater extent in the insensitive Progress than in the sensitive Nugget, as did glutathione reductase activities. Superoxide dismutase activities increased significantly in Progress, whereas no such change was observed in Nugget as a result of SO(2) exposure. This increase in superoxide dismutase activity was observed at 210 minutes after 0.8 microliter per liter SO(2) concentration had been reached, in marked contrast to the increases in reduced glutathione content and glutathione reductase activity, which were apparent at the 90 minute time point. These data suggest that one basis for the relative insensitivity of the apparent photosynthesis of the pea cultivar Progress to SO(2) is the enhanced response of glutathione reductase, superoxide dismutase activities, and glutathione content. PMID- 16668421 TI - A Lipoxygenase Pathway Is Activated in Rice after Infection with the Rice Blast Fungus Magnaporthe grisea. AB - Lipoxygenase (LOX) and lipid hydroperoxide-decomposing activity (LHDA) markedly increased in the fifth leaves of rice (Oryza sativa cv Aichiasahi) after infection with the rice blast fungus, Magnaporthe grisea. The increases in the enzyme activities were significantly higher in response to infection with an incompatible strain (race 131) compared with infection with a compatible strain (race 007) of the fungus. Using ion-exchange chromatography, we isolated three LOX activities (leaf LOX-1, -2, -3) from both uninoculated and infected leaves. The activity of leaf LOX-3, in particular, increased in the incompatible race infected leaves. The leaf LOX-3 had a pH optimum of 5.0 and produced preferentially 13-l-hydroperoxy-9,11 (Z,E)-octadecadienoic acid (13-HPODD) from linoleic acid. 13-HPODD and 13-l-hydroxy-9,11 (Z,E)-octadecadienoic acid, one of the reaction products from 13-HPODD by LHDA, were highly inhibitory to the germination of conidia of the fungus. The present study provides correlative evidence for important roles of LOX and LHDA in the resistance response of rice against the blast fungus. PMID- 16668422 TI - Oxidation of External NAD(P)H by Purified Mitochondria from Fresh and Aged Red Beetroots (Beta vulgaris L.). AB - Mitochondria were isolated from fresh beetroots (Beta vulgaris L. cvs Rubria and Nina) by differential centrifugation followed by Percoll gradient centrifugation. These purified mitochondria oxidized external NADH, although relatively slowly (20-40 versus 100-120 nanomoles oxygen per minute times milligram protein for NADH and succinate oxidation, respectively), with respiratory control ratios of two to three and ADP/O ratios of 1.2 to 1.6. NADPH was also oxidized, but even more slowly and with little or no coupling. The optimum for both NADH and NADPH oxidation by fresh beetroot mitochondria was pH 6. The rate of external NADH oxidation by isolated mitochondria was enhanced threefold during storage of the intact tubers at 10 degrees C for 12 weeks. The optimum of the induced NADH oxidation was approximately pH 6.8. Succinate and malate oxidation only increased by 30% during the same period and NADPH oxidation was constant. This is strong evidence that NADH and NADPH oxidation are catalyzed by different enzymes at least in beetroots. Activity staining of nondenaturing polyacrylamide gels with NADH and Nitro Blue Tetrazolium did not show differences in banding pattern between mitochondria isolated from fresh and stored beetroots. The induction is discussed in relation to physiological aging processes. PMID- 16668423 TI - Small light-harvesting antenna does not protect from photoinhibition. AB - High-light-induced decrease in photosystem II (PSII) electron transfer activity was studied in high- and low-light-grown pumpkin (Cucurbita pepo L.) plants in vivo and in vitro. The PSII light-harvesting antenna of the low-light leaves was estimated to be twice as big as that of the high-light leaves. The low-light leaves were more susceptible to photoinhibition in vivo. However, thylakoids isolated from these two plant materials were equally sensitive to photoinhibition when illuminated in the absence of external electron acceptors. Only the intensity of the photoinhibitory light and the chlorophyll concentration of the sample, not the size of the light-harvesting antenna, determined the rate of PSII photoinhibition in vitro. Because excitation of the reaction center and not only the antenna chlorophylls is a prerequisite for photoinhibition of PSII activity, independence of photoinhibition on antenna size provides support for the hypothesis (Schatz EH, Brock H, Holzwarth AR [1988] Biophys J 54: 397-405) that the excitations of the antenna chlorophylls are in equilibrium with the excitations of the reaction centers. Better tolerance of the high-light leaves in vivo was due to a more active repair process and more powerful protective mechanisms, including photosynthesis. Apparently, some protective mechanism of the high-light-grown plants is at least partially active at low temperature. The protective mechanisms do not appear to function in vitro. PMID- 16668424 TI - Enhanced Photosynthesis and Stomatal Conductance of Pima Cotton (Gossypium barbadense L.) Bred for Increased Yield. AB - Yield of Pima cotton (Gossypium barbadense L.) has tripled over the last 40 years with the development of new cultivars. Six genetic lines representing successive stages in the breeding process (one primitive noncultivated accession, four cultivars with release dates from 1949 to 1983, and one unreleased breeding line) were grown in a greenhouse, and their gas exchange properties were compared. Among the cultivated types, genetic advances were closely associated with increasing single-leaf photosynthetic rate (A) and stomatal conductance (g(s)), especially in the morning. The A and g(s) of the primitive line approached those of the cultivated types early in the morning, but were much lower for the rest of the day. In both morning and afternoon, A was correlated with g(s) across genotypes but was not correlated with leaf thickness, concentrations of chlorophyll or starch, or intercellular CO(2) concentration (c(i)). In the oldest cultivar, the relationship of A to c(i) did not change between morning and afternoon. In the two most recent lines, the slopes of the A:c(i) curves at limiting c(i) exceeded that of the oldest cultivar by 25 to 50% in the morning, but the differences were much smaller in the afternoon. The maximum A of the newer lines at high c(i) exceeded that of the oldest cultivar only in the morning. Breeding for increasing yield has enhanced the photosynthetic capacity and stomatal conductance of Pima cotton and altered the diurnal regulation of photosynthesis. PMID- 16668425 TI - Tissue and Cellular Localization of Proteinase Inhibitors I and II in the Fruit of the Wild Tomato, Lycopersicon peruvianum (L.) Mill. AB - The cellular and subcellular localization of proteinase inhibitor I and inhibitor II proteins in the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill., LA 107 was determined by immunoanalysis of tissue blots and protein-A gold immunocytochemistry. Tissue blot analysis showed that the proteinase inhibitor I proteins were located throughout the fruit tissue, with the exception of the seeds. Light microscopy, using immunocytochemical labeling, indicated that all the parenchyma cells of the pericarp contained inhibitor I and II proteins in dense vacuolar protein aggregates that were not membrane bound. The size, number, and morphology of the aggregates within individual cells varied greatly. The funiculus, ovule, and early embryonic tissues were devoid of inhibitor I and II. Immunocytochemical analysis using transmission electron microscopy confirmed that the proteinase inhibitor I proteins were principally located and stored in protein aggregates within the vacuole of the fruit parenchyma cells. Some cytoplasmic protein-A gold immunolabeling of inhibitor I proteins was evident, which may be related to the synthesis and intermediate transport steps preceding storage of the inhibitor I proteins in the vacuoles. PMID- 16668426 TI - Uniquely regulated proteinase inhibitor I gene in a wild tomato species : inhibitor I family gene is wound-inducible in leaves and developmentally regulated in fruit. AB - A uniquely regulated proteinase inhibitor I gene was isolated from the wild tomato species Lycopersicon peruvianum (L.) Mill. (LA 107) and characterized. The inhibitor gene is wound-inducible in leaves and is expressed in unripe fruit during development. The gene (lambda clone 1) is present on a 15.5 kilobase pair Sal 1-SalI genomic DNA fragment. Southern blot analysis of L. peruvianum genomic DNA shows only one strongly hybridizing DNA fragment to probes derived from lambda clone 1. S1 nuclease protection experiments and Northern analyses confirm that this gene is both wound-inducible in leaves and developmentally regulated in young unripe fruit. These observations are supported by comparisons of the 5' flanking DNA sequences of the L. peruvianum inhibitor I gene with known elicitor responsive cis-acting sequences. The transcriptional regulation of the lambda clone 1 inhibitor I gene in leaves of wounded plants and in developing unripe fruit indicates that the gene contains unique complex regulating elements. These elements respond to both environmental and developmental tissue-specific signals that can regulate proteinase inhibitor synthesis to protect the tissues of this wild species of tomato against predators and pathogens. PMID- 16668427 TI - Nonsedimentable microvesicles from senescing bean cotyledons contain gel phase forming phospholipid degradation products. AB - A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7 day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca(2+) is added to activate membrane-associated lipolytic enzymes. The Ca(2+) treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc Natl Acad Sci USA 88: 2269-2273), the term "deteriosome" connotes this putative function and would serve to distinguish these microvesicles from other cytoplasmic microvesicles unrelated to deterioration. PMID- 16668428 TI - Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall. AB - The physicochemical nature of the cell wall was determined in the fourth internode of three isogenic brittle mutants of barley (Hordeum vulgare L.) and corresponding nonbrittle strains. Cellulose contents of the brittle culms were 17.5 to 20.3% of those of corresponding nonbrittle strains. No major difference was found in lignin and noncellulose components (except glucose) between brittle and nonbrittle strains. Maximum bending stresses of brittle culms were 38.0 to 54.2% of those of corresponding nonbrittle strains. The degree of polymerization of cellulose, measured by viscometry, was similar between the brittle and the nonbrittle strains. Mole number of cellulose molecules in a unit length of brittle culms, calculated by dividing cellulose mass by molecular weight, was 7.7 to 17.3% of those of the nonbrittle strains. These results indicate that brittleness of mutant culms is due to fewer numbers of cellulose molecules in the cell walls. PMID- 16668429 TI - Acclimation of CO(2) Assimilation in Cotton Leaves to Water Stress and Salinity. AB - Cotton (Gossypium hirsutum L. cv Acala SJ2) plants were exposed to three levels of osmotic or matric potentials. The first was obtained by salt and the latter by withholding irrigation water. Plants were acclimated to the two stress types by reducing the rate of stress development by a factor of 4 to 7. CO(2) assimilation was then determined on acclimated and nonacclimated plants. The decrease of CO(2) assimilation in salinity-exposed plants was significantly less in acclimated as compared with nonacclimated plants. Such a difference was not found under water stress at ambient CO(2) partial pressure. The slopes of net CO(2) assimilation versus intercellular CO(2) partial pressure, for the initial linear portion of this relationship, were increased in plants acclimated to salinity of -0.3 and 0.6 megapascal but not in nonacclimated plants. In plants acclimated to water stress, this change in slopes was not significant. Leaf osmotic potential was reduced much more in acclimated than in nonacclimated plants, resulting in turgor maintenance even at -0.9 megapascal. In nonacclimated plants, turgor pressure reached zero at approximately -0.5 megapascal. The accumulation of Cl(-) and Na(+) in the salinity-acclimated plants fully accounted for the decrease in leaf osmotic potential. The rise in concentration of organic solutes comprised only 5% of the total increase in solutes in salinity-acclimated and 10 to 20% in water stress-acclimated plants. This acclimation was interpreted in light of the higher protein content per unit leaf area and the enhanced ribulose bisphosphate carboxylase activity. At saturating CO(2) partial pressure, the declined inhibition in CO(2) assimilation of stress-acclimated plants was found for both salinity and water stress. PMID- 16668430 TI - Organ-specific invertase deficiency in the primary root of an inbred maize line. AB - An organ-specific invertase deficiency affecting only the primary root system is described in the Oh 43 inbred maize (Zea mays). Invertases (acid and neutral/soluble and insoluble) were assayed in various tissues of hybrid (NK 508) and inbred (Oh 43, W 22) maize lines to determine the basis for an early report that Oh 43 root tips were unable to grow on sucrose agar (27). Substantial acid invertase activity (7.3 to 16.1 micromoles of glucose per milligram of protein per hour) was evident in extracts of all tissues tested except the primary root system of Oh 43. This deficiency was also evident in lateral roots arising from the primary root. In contrast, morphologically identical lateral roots from the adventitious root system had normal invertase levels. These results suggest that ontogenetic origin of root tissues is an important determinant of invertase expression in maize. Adventitious roots (including the seminals) arise above the scutellar node and are, therefore, of shoot origin. The Oh 43 deficiency also demonstrated that invertase activity was not essential for maize root growth. Sucrose synthase was active in extracts from all root apices and theoretically provided the only available avenue for sucrose degradation in primary root tips of Oh 43. The deficiency described here will provide a useful avenue of investigation into the expression and significance of root invertase. PMID- 16668431 TI - Chemical characterization of stress-induced vascular coating in tomato. AB - Indirect evidence suggests that vascular coatings formed by plants in response to stress consist of suberin-like substances containing lipid and phenolic compounds. To provide more direct chemical evidence that coatings are suberin, we used a natural pathogen, Verticillium albo-atrum, or a stress-responsive hormone, abscisic acid, to induce coating in two isolines of tomato (Lycopersicon esculentum L. cultivar Craigella) that are resistant or susceptible to the pathogen. Using treated petioles that had been monitored cytologically, chemical depolymerization followed by combined gas-liquid chromatography-mass spectrometry analysis of alkane-alpha,omega-diol levels confirmed the presence of suberin after induction of coating and showed quantitative differences between the isolines that correlated with cytological measurements of the coating response. Northern analysis of suberization-associated anionic peroxidase mRNA showed corresponding increases, and tissue blot analysis further indicated that induction of the mRNA was localized in the responding vascular bundles, as determined by suberin histochemistry. Taken together, these results provide chemical evidence that the coatings are mainly suberin. PMID- 16668432 TI - Fe-Chelate Reductase Activity of Plasma Membranes Isolated from Tomato (Lycopersicon esculentum Mill.) Roots : Comparison of Enzymes from Fe-Deficient and Fe-Sufficient Roots. AB - Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (-Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3-))(2). NADH and Fe(3+)(citrate(3-))(2)K(m) values for reductase in PM from +Fe and -Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from -Fe tomatoes. The pH optimum for Fe chelate reductase was 6.5. Fe-chelate reductases from -Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n octyl beta-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from -Fe PM. We conclude that PM from roots of +Fe and -Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM. PMID- 16668433 TI - Induction of Senescence-Like Deterioration of Microsomal Membranes from Cauliflower by Free Radicals Generated during Gamma Irradiation. AB - Membrane deterioration differs in aging and senescent tissues. Involvement of free radicals in the process is generally recognized. Little is known about the physiological effects of gamma irradiation on plant tissues. Degradation of microsomal membranes by the action of free radicals, generated in vivo by gamma rays, was investigated. Cauliflower florets (Brassica oleracea L., Botrytis group) were exposed to 2 or 4 kiloGray of gamma radiation. Membrane deterioration was assessed during 8-day storage at 13 degrees C. Some senescence was indicated in nonirradiated controls by a parallel depletion of lipid phosphate and protein. Irradiation caused an immediate increase in tissue electrolyte leakage and a small increase in the free fatty acid content of membranes. In irradiated samples, leakage of electrolytes and the ratios of sterol to phospholipid and of free fatty acid to phospholipid increased with storage. During this period, membrane protein was progressively lost and the lipid phosphate-to-protein ratio increased markedly. Polyunsaturated fatty acids were selectively depleted from the free fatty acid fraction for all treatments, suggesting lipoxygenase activity. No change in lipid saturation was observed in the polar lipid fraction. The results suggest an enzyme-catalyzed senescence-like membrane deterioration, probably induced by chemical deesterification of phospholipids by free radicals generated during irradiation. PMID- 16668434 TI - Tracing cell wall biogenesis in intact cells and plants : selective turnover and alteration of soluble and cell wall polysaccharides in grasses. AB - Cells of proso millet (Panicum miliaceum L. cv Abarr) in liquid culture and leaves of maize seedlings (Zea mays L. cv LH51 x LH1131) readily incorporated d [U-(14)C]glucose and l-[U-(14)C]arabinose into soluble and cell wall polymers. Radioactivity from arabinose accumulated selectively in polymers containing arabinose or xylose because a salvage pathway and C-4 epimerase yield both nucleotide-pentoses. On the other hand, radioactivity from glucose was found in all sugars and polymers. Pulse-chase experiments with proso millet cells in liquid culture demonstrated turnover of buffer soluble polymers within minutes and accumulation of radioactive polymers in the cell wall. In leaves of maize seedlings, radioactive polymers accumulated quickly and peaked 30 hours after the pulse then decreased slowly for the remaining time course. During further growth of the seedlings, radioactive polymers became more tenaciously bound in the cell wall. Sugars were constantly recycled from turnover of polysaccharides of the cell wall. Arabinose, hydrolyzed from glucuronoarabinoxylans, and glucose, hydrolyzed from mixed-linkage (1-->3, 1-->4)beta-d-glucans, constituted most of the sugar participating in turnover. Arabinogalactans were a large portion of the buffer soluble (cytoplasmic) polymers of both proso millet cells and maize seedlings, and these polymers also exhibited turnover. Our results indicate that the primary cell wall is not simply a sink for various polysaccharide components, but rather a dynamic compartment exhibiting long-term reorganization by turnover and alteration of specific polymers during development. PMID- 16668435 TI - NaCl Regulation of Tonoplast ATPase 70-Kilodalton Subunit mRNA in Tobacco Cells. AB - A cDNA clone encoding the 70-kilodalton subunit of the tobacco (Nicotiana tabacum var Wisconsin 38) tonoplast ATPase has been isolated. The 1.656 kilobase insert contains only open reading frame that represents more than 80% of the carrot cDNA coding region. The deduced amino acid sequence has greater than 95% sequence identity with the homologous carrot sequence. A transcript of approximately 2.7 kilobase was detected on Northern blots of tobacco poly(A)(+) selected or total RNA using labeled probe produced from this clone. The gene was expressed throughout the growth cycle in unadapted and 428 millimolar NaCl adapted cells. Transcription of the 70-kilodalton subunit gene or mRNA stability was induced by short-term NaCl treatment in NaCl adapted cells or by abscisic acid treatment in both adapted and unadapted cells. Southern analysis indicated the presence of up to four genes encoding the 70-kilodalton subunit. PMID- 16668436 TI - Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants. AB - A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study beta-1,3 glucan synthase activity changes. All tissues contained an active beta-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active beta-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in beta-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that beta-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development. PMID- 16668437 TI - Evidence for light-dependent recycling of respired carbon dioxide by the cotton fruit. AB - Conservation of respired CO(2) by an efficient recycling mechanism in fruit could provide a significant source of C for yield productivity. However, the extent to which such a mechanism operates in cotton (Gossypium hirsutum L.) is unknown. Therefore, a combination of CO(2) exchange, stable C isotope, and chlorophyll (Chl) fluorescence techniques were used to examine the recycling of respired CO(2) in cotton fruit. Respiratory CO(2) losses of illuminated fruit were reduced 15 to 20% compared with losses for dark-incubated fruit. This light-dependent reduction in CO(2) efflux occurred almost exclusively via the fruit's outer capsule wall. Compared with the photosynthetic activity of leaves, CO(2) recycling by the outer capsule wall was 35 to 40% as efficient. Calculation of (14)CO(2) fixation on a per Chl basis revealed that the rate of CO(2) recycling for the capsule wall was 62.2 micromoles (14)CO(2) per millimole Chl per second compared with an assimilation rate of 64.6 micromoles (14)CO(2) per millimole Chl per second for leaves. During fruit development, CO(2) recycling contributed more than 10% of that C necessary for fruit dry weight growth. Carbon isotope analyses (delta(13)C) showed significant differences among the organs examined, but the observed isotopic compositions were consistent with a C(3) pathway of photosynthesis. Pulse-modulated Chl fluorescence indicated that leaves and fruit were equally efficient in photochemical and nonphotochemical dissipation of light energy. These studies demonstrated that the cotton fruit possesses a highly efficient, light-dependent CO(2) recovery mechanism that aids in the net retention of plant C and, therein, contributes to yield productivity. PMID- 16668438 TI - Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis. AB - Studies of Spinacia oleracea L. were undertaken to characterize further how Mg(2+) external to the isolated intact chloroplast interacts with stromal K(+), pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg(2+) result in lower stromal K(+), which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg(2+) interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg(2+) was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg(2+) effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg(2+) caused lower stromal K(+) by restricting the rate of K(+) influx; Mg(2+) did not affect K(+) efflux from the stroma. Mg(2+) restriction of K(+) influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg(2+) with the chloroplast envelope likely mediated external Mg(2+) effects on chloroplast metabolism. PMID- 16668439 TI - Postanoxic Injury in Soybean (Glycine max) Seedlings. AB - The postanoxic injury, also known as reperfusion injury, is associated with the returning of anoxic tissues to normal atmosphere. Using tetrazolium chloride staining, ATP content, and seedling growth rate as indicators, we found that postanoxic injuries in soybean (Glycine max) seedlings were more severe after 1 and 2 hours of anoxia than after longer anoxic durations (3 to 5 hours). Anaerobic incubation of root tips in the presence of 100 mm ascorbate, an antioxidant and free radical-scavenging compound, alleviated the postanoxic injury associated with the short durations of anoxia. Extracts from soybean seedling roots returned to air from 1 hour of anoxia had an elevated capacity to produce superoxide radicals over extracts from postanoxic roots stressed for 3 or 5 hours. Activity of superoxide dismutase in soybean roots returned to air from 1 and 2 hours of anoxia was 30 to 50% lower than activities in roots returned to air from 5 hours of anoxia. Superoxide dismutase-specific transcripts were also lower in postanoxic roots stressed for 1 hour than in roots stressed for longer anoxic durations. The evidence suggested that the postanoxic injury of soybean roots after a short anoxic stress was associated with an increased superoxide radicals production capacity coupled with a reduced superoxide dismutase activity. Periods of anoxia of at least 3 hours were necessary for soybean seedlings to develop the ability to cope with postanoxic stress. PMID- 16668440 TI - Abscisic Acid induces anaerobiosis tolerance in corn. AB - Flooding is a frequently occurring environmental stress that can severely affect plant growth. This study shows that treatment of corn (Zea mays L.) seedlings with abscisic acid (ABA) increases their tolerance to anoxia 10-fold over untreated seedlings and twofold over seedlings treated with water. Corn seedlings stressed anoxically for 1 day showed only 8% survival when planted in vermiculite. Pretreatment of root tips with 100 micromolar ABA or water for 24 hours before the 1 day anoxic stress increased the anoxic survivability of seedlings to 87% and 47%, respectively. Cycloheximide (5 milligrams per liter), added together with ABA, reduced the seedling survival rate, indicating that the induction of anoxic tolerance in corn by ABA was partly a result of the synthesis of new proteins. ABA treatment induced a threefold increase in alcohol dehydrogenase enzyme activity in corn roots. However, after 24 h of anoxia, alcohol dehydrogenase enzyme activity between the ABA-pretreated and non pretreated corn roots was not significantly different. The results indicated that ABA played an important role in inducing anoxic tolerance in corn and that the induced tolerance was probably mediated by an increase in alcohol dehydrogenase enzyme activity before the anoxic stress. PMID- 16668441 TI - The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species. AB - The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 +/- 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside out detached patches. The K(+)/Na(+) permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na(+) accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K(+)/Na(+) permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K(+)/Cl(-) was greater than 50:1. The K(+)/Na(+) permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca(2+) concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na(+) had no significant effect on the K(+)/Na(+) permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K(+)/Na(+) selectivity of these channels. PMID- 16668442 TI - Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures. AB - Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography. PMID- 16668443 TI - Characterization of Satellite DNA from Three Marine Dinoflagellates (Dinophyceae): Glenodinium sp. and Two Members of the Toxic Genus, Protogonyaulax. AB - Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA(1), hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA(2) and satellite DNA(3), recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical. PMID- 16668444 TI - Differential effects of elicitors on the viability of rice suspension cells. AB - We have compared the effects of two elicitors of defense-related processes on rice (Oryza sativa L.) suspension cells. Both chitosan and salicylic acid induced the accumulation of extracellular chitinase, thickening of the cell wall, and a variety of cytological changes in treated cells. Chitosan also induced the production of a brown pigment and cell death. Both of these effects depended on the availability of reactive oxygen species, because the damage was greatly reduced by either catalase or free-radical scavengers. Pretreating cells with salicylic acid also protected them from the cytotoxic effects of chitosan. This type of induced tolerance persisted when salicylic acid was removed and was not simply due to the release of extracellular substances, because salicylic acid treated cells did not protect untreated cells from chitosan-induced death. Salicylic acid also stimulated the production of a 10-kilodalton subtilisin inhibitor that was not produced by chitosan-treated cells. Most of these changes are associated with the hypersensitive response of many plant species, including monocotyledons, and may serve as an in vitro model for investigating the biochemistry of some diseases. PMID- 16668445 TI - Determination of the Cellular Mechanisms Regulating Thermo-Induced Stem Growth in Thlaspi arvense L. AB - Field pennycress (Thlaspi arvense L.) is a species with a cold requirement for the initiation of reproductive development (thermoinduction). Work in this laboratory has been focused on elucidating the biochemical and molecular mechanisms underlying the bolting or rapid stem elongation response that is an intricate part of reproductive development in this species. In the present paper the cellular basis for thermo-induced stem growth was determined. Evidence is presented indicating that bolting results from the production of new cells that elongate to their original length before thermoinduction. This increase in cell division occurs in the pith and cortex approximately 0.5 to 5.0 millimeters below the stem apex. For at least the early stages of thermo-induced stem growth, enhanced cell elongation does not appear to be a factor because average lengths of pith cells from stems of thermo-induced plants were similar or less than noninduced controls. In addition, both the amount of increase in the production of new pith cells and stem growth were positively correlated with the length of the cold treatment. Two other lines of evidence are presented corroborating previous assertions (JD Metzger [1985] Plant Physiol 78: 8-13) that gibberellins mediate thermo-induced stem growth in field pennycress. First, treatment of noninduced plants with gibberellin A(3) completely mimicked the effects of a 4 week cold treatment on mitotic activity in the pith and cortex. Second, very little increase in the production of new cells was observed in the pith and cortex of thermo-induced plants of a gibberellin-deficient dwarf mutant of field pennycress. It is also shown that the influence of photoperiod on stem growth is mediated by an effect on the final length that cells ultimately attain. PMID- 16668446 TI - Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation. AB - Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3 phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6 phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of (13)C-labeling [NMR] in sucrose when tissue was supplied with [2-(13)C]glucose). Fluoride inhibited the incorporation of [U (14)C] glucose, [U-(14)C]sucrose, [U-(14)C]glucose 1-phosphate, and [U-(14)C] glycerol into starch. The incorporation of [U-(14)C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of (14)C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions. PMID- 16668447 TI - Transport of arginine and aspartic Acid into isolated barley mesophyll vacuoles. AB - The transport of arginine into isolated barley (Hordeum vulgare L.) mesophyll vacuoles was investigated. In the absence of ATP, arginine uptake was saturable with a K(m) of 0.3 to 0.4 millimolar. Positively charged amino acids inhibited arginine uptake, lysine being most potent with a K(i) of 1.2 millimolar. In the presence of free ATP, but not of its Mg-complex, uptake of arginine was drastically enhanced and a linear function of its concentration up to 16 millimolar. The nonhydrolyzable adenylyl imidodiphosphate, but no other nucleotide tested, could substitute for ATP. Therefore, it is suggested that this process does not require energy and does not involve the tonoplast ATPase. The ATP-dependent arginine uptake was strongly inhibited by p chloromercuriphenylsulfonic acid. Furthermore, hydrophobic amino acids were inhibitory (I(50) phenylalanine 1 millimolar). Similar characteristics were observed for the uptake of aspartic acid. However, rates of ATP-stimulated aspartic acid transport were 10-fold lower as compared to arginine transport. Uptake of aspartate in the absence of ATP was negligible. PMID- 16668448 TI - Two Apoplastic alpha-Amylases Are Induced in Tobacco by Virus Infection. AB - alpha-Amylase activity (EC 3.2. 1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two alpha amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extra-cellular localization was demonstrated by comparing the alpha-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both alpha-amylases produced in tobacco upon TMV infection. PMID- 16668449 TI - Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli: Production of Polyclonal Antibodies, and Immunological Characterization of the Enzyme. AB - Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine. This enzyme is the target site of several classes of structurally unrelated herbicides. The conventional method of antibody production using purified protein has not been successful with this enzyme. Two separate fragments of a gene encoding a portion of the mature region of AHAS from Arabidopsis were fused with the trpE gene from Escherichia coli using the pATH1 vector. E. coli cells transformed with each respective plasmid expressed a fusion protein at levels greater than 10% of the total cell protein. The fusion protein was purified and used to immunize rabbits. Antisera obtained from the immunized rabbits immunoprecipitated AHAS activity from Arabidopsis cell free extracts. The anti-AHAS antisera reacted with a 65 kilodalton protein band in electrophoretically resolved extracts of Arabidopsis. In cross-reactivity tests, this antibody was able to immunoprecipitate AHAS activity from various plant species. Furthermore, a protein band with a molecular mass of 65 kilodaltons was detected in the crude extracts of all plant species tested on a Western blot. These results indicate that the 65 kilodalton protein represents AHAS in various plant species. The wide spectrum of cross-reactivity for the antisera supports the view that the AHAS enzyme is highly conserved across all plant species. PMID- 16668450 TI - Effects of chilling on the biochemical and functional properties of thylakoid membranes. AB - The mechanism of chilling resistance was investigated in 4-week-old plants of the chilling-sensitive cultivated tomato, Lycopersicon esculentum Mill. cv H722, and rooted cuttings of its chilling-resistant wild relative, L. hirsutum Humb. and Bonpl., which were chilled for 3 days at 2 degrees C with a 14-hour photoperiod and light intensity of 250 micromoles per square meter per second. This chilling stress reduced the chlorophyll fluorescence ratio, stomatal conductance, and dry matter accumulation more in the sensitive L. esculentum than in the resistant L. hirsutum. Photosynthetic CO(2) uptake at the end of the chilling treatment was reduced more in the resistant L. hirsutum than in L. esculentum, but recovered at a faster rate when the plants were returned to 25 degrees C. The reduction of the spin trap, Tiron, by isolated thylakoids at 750 micromoles per square meter per second light intensity was taken as a relative indication of the tendency for the thylakoids to produce activated oxygen. Thylakoids isolated from the resistant L. hirsutum with or without chilling treatment were essentially similar, whereas those from chilled leaves of L. esculentum reduced more Tiron than the nonchilled controls. Whole chain photosynthetic electron transport was measured on thylakoids isolated from chilled and control leaves of the two species at a range of assay temperatures from 5 to 25 degrees C. In both species, electron transport of the thylakoids from chilled leaves was lower than the controls when measured at 25 degrees C, and electron transport declined as the assay temperature was reduced. However, the temperature sensitivity of thylakoids from chilled L. esculentum was altered such that at all temperatures below 20 degrees C, the rate of electron transport exceeded the control values. In contrast, the thylakoids from chilled L. hirsutum maintained their temperature sensitivity, and the electron transport rates were proportionately reduced at all temperatures. This sublethal chilling stress caused no significant changes in thylakoid galactolipid, phospholipid, or protein levels in either species. Nonchilled thylakoid membranes from L. hirsutum had fourfold higher levels of the fatty acid 16:1, than those from L. esculentum. Chilling caused retailoring of the acyl chains in L. hirsutum but not in L. esculentum. The chilling resistance of L. hirsutum may be related to an ability to reduce the potential for free radical production by close regulation of electron transport within the chloroplast. PMID- 16668451 TI - Abscisic alcohol is an intermediate in abscisic Acid biosynthesis in a shunt pathway from abscisic aldehyde. AB - It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples [1988] Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate trans-ABA and trans-ABA glucose ester and that this accumulation is due to trans-ABA biosynthesis. (18)O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [(18)O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [(18)O]ABA with two (18)O atoms in the carboxyl group. We further show, by feeding experiments with [(2)H(6)]ABA-alcohol and (18)O(2), that this doubly-carboxyl-labeled ABA is synthesized from [(18)O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [(2)H(6)]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Likewise, carbon monoxide inhibits the synthesis of doubly-carboxyl-labeled ABA in (18)O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant. PMID- 16668452 TI - Isolation and expression of a maize type 1 protein phosphatase. AB - The dephosphorylation of phosphoproteins by protein phosphatases represents an important mechanism for regulating specific cellular processes in eukaryotic cells. The aim of the present study was to examine the structural and biochemical characteristics of a specific class of protein Ser/Thr phosphatases (type 1 protein phosphatases) which have received very little attention in higher plants. A cDNA clone (ZmPP1) was isolated from a maize (Zea mays L.) cDNA library. The deduced amino acid sequence is 80% identical with a 292-amino acid core region of rabbit and yeast type 1 protein phosphatase catalytic subunit. Southern blot analysis indicates that ZmPP1 may belong to a family of related genes in maize. ZmPP1 RNA was present in all maize tissues examined, indicating that it may play a fundamental role in cellular homeostasis. To demonstrate that ZmPP1 encodes an active protein phosphatase and, in an effort to characterize this gene product biochemically, high levels of ZmPP1 were expressed in Escherichia coli. Active ZmPP1 enzyme dephosphorylates rabbit phosphorylase a and is strongly inhibited by okadaic acid and by the mammalian inhibitor-2. These data show that ZmPP1 is structurally and biochemically very similar to the corresponding enzyme in animal cells. These results also suggest that the function and regulation of the higher plant type 1 protein phosphatases may be similar to the mammalian protein phosphatases. PMID- 16668453 TI - Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization. AB - Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-beta-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg(2+), 1 millimolar Ca(2+)) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi subunit enzyme complex. PMID- 16668454 TI - Elicitor-inducible 3-hydroxy-3-methylglutaryl coenzyme a reductase activity is required for sesquiterpene accumulation in tobacco cell suspension cultures. AB - Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [(14)C]acetate and [(3)H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation. PMID- 16668455 TI - Induction and regulation of ethylene biosynthesis by pectic oligomers in cultured pear cells. AB - Pectic oligomers induced a rapid, transient increase in ethylene biosynthesis when added to pear cells in suspension culture. The rate of ethylene biosynthesis increased within 30 to 40 minutes after oligomer addition, reached a maximum between 90 and 120 minutes after addition, and then decreased to basal rates of synthesis. Both the rapid increase and decrease in biosynthesis appear to be precisely regulated components of the ethylene response to oligomers. Induction of ethylene biosynthesis by pectic oligomers resulted in a reduced sensitivity of cells to further ethylene induction. This reduction in sensitivity occurred within 90 minutes after an oligomer treatment, slightly preceding the decline in ethylene synthesis. The degree of insensitivity induced was proportional to the concentration of oligomer in the first treatment. Induced insensitivity to elicitors appears to represent a novel mechanism which may limit continued ethylene biosynthesis after ethylene induction. Ethylene was produced by pear cells throughout the cell growth cycle, as cells increased in density over a 6 day period. Endogenous ethylene biosynthesis was at a maximum during the first 4 days of rapid cell growth, then declined to half the peak rate through day 10. Pectic oligomers could induce an increase in ethylene biosynthesis above this background rate only after day 5, as endogenous biosynthesis declined. Changes in sensitivity to added oligomer during the growth cycle may result from insensitivity to elicitors induced by growth processes. PMID- 16668456 TI - Induction and regulation of ethylene biosynthesis and ripening by pectic oligomers in tomato pericarp discs. AB - The effect of pectic oligomers and 1-aminocyclopropane carboxylic acid on ethylene biosynthesis and color change was studied in ripening tomato pericarp discs excised from mature-green tomato fruit (Lycopersicon esculentum Mill.). Pectic oligomers induced at least four distinct responses when added to pericarp discs: (a) a short-term, transient increase in ethylene biosynthesis; (b) a long term, persistent increase in climacteric ethylene in discs excised from mature green fruit; (c) an advance in ripening processes, as indicated by increased reddening of the disc surfaces; and (d) a darkening of the treated endocarp surface. Pectic oligomers appear to affect the ripening of exocarp and endocarp tissues by different mechanisms. In exocarp tissues, the acceleration of reddening by pectic oligomers might simply be a consequence of induced ethylene biosynthesis. In endocarp tissues, the acceleration of reddening appears to be a direct effect of oligomers on ripening processes. We suggest that the rate of ripening of endocarp tissues may be regulated, in part, by the release of pectic oligomers from the cell walls of adjacent exocarp tissues. Exocarp and endocarp tissues of pericarp discs appear to differ in their sensitivity to ethylene at each maturity stage, and to exhibit independent changes in sensitivity to ethylene as ripening progresses. The tissue-specific pattern of reddening in tomato pericarp may result from this differential sensitivity to endogenous ethylene concentrations. PMID- 16668457 TI - Genetic Regulation of Development in Sorghum bicolor: VI. The ma(3) Allele Results in Abnormal Phytochrome Physiology. AB - Physiological processes controlled by phytochrome were examined in three near isogenic genotypes of Sorghum bicolor, differing at the allele of the third maturity gene locus. Seedlings of 58M (ma(3) (R)ma(3) (R)) did not show phytochrome control of anthocyanin synthesis. In contrast, seedlings of 90M (ma(3)ma(3)) and 100M (Ma(3)Ma(3)) demonstrated reduced anthocyanin synthesis after treatment with far red and reversal of the far red effect by red. De etiolation of 48-hour-old 90M and 100M dark-grown seedlings occurred with 48 hours of continuous red. Dark-grown 58M seedlings did not de-etiolate with continuous red treatment. Treatment of seedlings with gibberellic acid or tetcyclacis, a gibberellin synthesis inhibitor, did not alter anthocyanin synthesis. Levels of chlorophyll and anthocyanin were lower in light-grown 58M seedlings than in 90M and 100M. Etiolated seedlings of all three genotypes have similar amounts of photoreversible phytochrome. Crude protein extracts from etiolated seedlings were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Phytochrome was visualized with Pea-25, a monoclonal antibody directed to phytochrome from etiolated peas. The samples from all three genotypes contained approximately equivalent amounts of a prominent, immunostaining band at 126 kD. However, the sample from 58M did not show a fainter, secondary band at 123 kD that was present in 90M and 100M. The identity and importance of this secondary band at 123 kD is unknown. We propose that 58M is a phytochrome-related mutant that contains normal amounts of photoreversible phytochrome and normal phytochrome protein when grown in the dark. PMID- 16668458 TI - Involvement of wound and climacteric ethylene in ripening avocado discs. AB - Avocado (Persea americana Mill. cv Hass) discs (3 mm thick) ripened in approximately 72 hours when maintained in a flow of moist air and resembled ripe fruit in texture and taste. Ethylene evolution by discs of early and midseason fruit was characterized by two distinct components, viz. wound ethylene, peaking at approximately 18 hours, and climacteric ethylene, rising to a peak at approximately 72 hours. A commensurate respiratory stimulation accompanied each ethylene peak. Aminoethoxyvinyl glycine (AVG) given consecutively, at once and at 24 hours following disc preparation, prevented wound and climacteric respiration peaks, virtually all ethylene production, and ripening. When AVG was administered for the first 24 hours only, respiratory stimulation and softening (ripening) were retarded by at least a day. When AVG was added solely after the first 24 hours, ripening proceeded as in untreated discs, although climacteric ethylene and respiration were diminished. Propylene given together with AVG led to ripening under all circumstances. 2,5-Norbornadiene given continuously stimulated wound ethylene production, and it inhibited climacteric ethylene evolution, the augmentation of ethylene-forming enzyme activity normally associated with climacteric ethylene, and ripening. 2,5-Norbornadiene given at 24 hours fully inhibited ripening. When intact fruit were pulsed with ethylene for 24 hours before discs were prepared therefrom, the respiration rate, ethylene-forming enzyme activity buildup, and rate of ethylene production were all subsequently enhanced. The evidence suggests that ethylene is involved in all phases of disc ripening. In this view, wound ethylene in discs accelerates events that normally take place over an extended period throughout the lag phase in intact fruit, and climacteric ethylene serves the same ripening function in discs and intact fruit alike. PMID- 16668459 TI - Purification and characterization of pea chloroplastic phosphoriboisomerase. AB - Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K(m) values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose 5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase. PMID- 16668460 TI - Effects of the gibberellin biosynthetic inhibitor uniconazol on mutants of Arabidopsis. AB - Using the gibberellin (GA) biosynthetic inhibitor Uniconazol, we determined that det1, a mutant that no longer requires light to be germinated, still requires GA synthesis for germination. This result suggests that dark inhibition of germination in Arabidopsis may be due to inhibition of GA synthesis by the DET1 gene product in mature wild-type seeds. Similar experiments with mutants that lack seed dormancy due to a reduced sensitivity to abscisic acid (abi) have shown that abi1 and abi3 no longer require GA for germination. Furthermore, by shifting wild-type seeds to inhibitor at 6-hour intervals during imbibition, we determined that GA synthesis is only required during the first 24 hours of the imbibition process to reverse abscisic acid-induced dormancy in Arabidopsis. PMID- 16668461 TI - Accumulation of beta-Fructosidase in the Cell Walls of Tomato Roots following Infection by a Fungal Wilt Pathogen. AB - Active defense in plants is associated with marked metabolic alterations, but little is known about the exact role of the reported changes in specific activity of several enzymes in infected plant tissues. beta-Fructosidase (invertase), the enzyme that converts sucrose into glucose and fructose, increases upon infection by fungi and bacteria. To understand the relationship between fungal growth and beta-fructosidase accumulation, we used an antiserum raised against a purified deglycosylated carrot cell wall beta-fructosidase to study by immunogold labeling the spatial and temporal distribution of the enzyme in susceptible and resistant tomato (Lycopersicon esculentum) root tissues infected with the necrotrophic fungus, Fusarium oxysporum f. sp. racidis-lycopersici. In susceptible plants, the enzyme started to accumulate in host cell walls about 72 hours after inoculation. Accumulation occurred only in colonized cells and was mainly restricted to areas where the walls of both partners contacted each other. In resistant plants, accumulation of beta-fructosidase was noticeable as soon as 48 hours after inoculation and appeared to reach an optimum by 72 hours after inoculation. Increase in wall-bound beta-fructosidase was not restricted to infected cells but occurred also, to a large extent, in tissues that remained uncolonized during the infection process. The enzyme also accumulated in wall appositions (papillae) and intercellular spaces. This pattern of enzyme distribution suggests that induction of beta-fructosidase upon fungal infection is part of the plant's defense response. The possible physiological role(s) of this enzyme in infected tomato plants is discussed in relation to the high demand in energy and carbon sources during pathogenesis. PMID- 16668462 TI - Anthocyanidins and Flavonols, Major nod Gene Inducers from Seeds of a Black Seeded Common Bean (Phaseolus vulgaris L.). AB - Eleven compounds released from germinating seeds of a black-seeded bean (Phaseolus vulgaris L., cv PI165426CS) induce transcription of nod genes in Rhizobium leguminosarum biovar phaseoli. Aglycones from 10 of those compounds were identified by spectroscopic methods (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy), and their biological activities were demonstrated by induction of beta-galactosidase activity in R. leguminosarum strains containing nodA-lacZ or nodC-lacZ fusions controlled by R. leguminosarum biovar phaseoli nodD genes. By making comparisons with authentic standards, the chemical structures for aglycones from the 10 molecules were confirmed as being anthocyanidins (delphinidin, petunidin, and malvidin) and flavonols (myricetin, quercetin, and kaempferol). All anthocyanidins and flavonols had 3-O glycosylation and free hydroxyl groups at the 4', 5, and 7 positions. Hydrolysis experiments showed that the mean concentration required for half-maximum nod gene induction (I(50)) by the 10 glycosides was about half that of the corresponding aglycones. The mean I(50) value for the three anthocyanidins (360 nanomolar) was less (P 25% of the total soluble proteins in these tissues. Nitrate concentration in the leaves and nitrate uptake rate decreased dramatically when LD plants were transferred to short-day conditions; the protein content in leaves was unaffected. A decrease of the 32- and 36-kilodalton polypeptides occurs after transferring induced plants back to LD conditions. Both polypeptides are glycosylated and can be efficiently purified by affinity chromatography using concanavalin A-Sepharose 4B. The 32- and the 36-kilodalton polypeptides have identical basic isoelectric points and both consist of at least three isoforms. The storage proteins show a loss in apparent molecular mass after deglycosylation with trifluoromethanesulfonic acid. It is concluded that the 32- and 36-kilodalton polypeptides are glycoforms differing only in the extent of glycosylation. The relative molecular mass of the native storage protein was estimated to be 58 kilodalton, using gel filtration. From the molecular mass and the elution pattern it is supposed that the storage protein occurs as a heterodimer composed of one 32- and one 36-kilodalton subunit. Preliminary data suggest the involvement of the phytochrome system in the induction process of the 32- and 36-kilodalton polypeptides. PMID- 16668486 TI - Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : III. On the Mechanism of Resistance to Diclofop-Methyl. AB - Annual ryegrass (Lolium rigidum) biotype SLR 31 is resistant to the postemergent graminicide methyl-2-[4-(2,4-dichlorophenoxy)phenoxy]-propanoate (diclofop methyl). Uptake of [(14)C](U-phenyl)diclofop-methyl and root/shoot distribution of radioactivity in susceptible and resistant plants were similar. In both biotypes, diclofop-methyl was rapidly demethylated to the biocidal metabolite diclofop acid which, in turn, was metabolized to ester and aryl-O-sugar conjugates. Susceptible plants accumulated 5 to 15% more radioactivity in dicloflop acid than did resistant plants. Resistant plants had a slightly greater capacity to form nonbiocidal sugar conjugates. Despite these differences, resistant plants retained 20% of (14)C in the biocidal metabolite diclofop acid 192 hours after treatment, whereas susceptible plants, which were close to death, retained 30% in diclofop acid. The small differences in the pool sizes of the active and inactive metabolites are by themselves unlikely to account for a 30 fold difference in sensitivity to the herbicide at the whole plant level. Similar high-pressure liquid chromatography elution patterns of conjugates from both susceptible and resistant biotypes indicated that the mechanisms and the products of catabolism in the biotypes are similar. It is suggested that metabolism of diclofop-methyl by the resistant biotype does not alone explain resistance observed at the whole-plant level. Diclofop acid reduced the electrochemical potential of membranes in etiolated coleoptiles of both biotypes; 50% depolarization required 1 to 4 mum diclofop acid. After removal of diclofop acid, membranes from the resistant biotype recovered polarity, whereas membranes from the susceptible biotype did not. Internal concentrations of diclofop acid 4 h after exposing plants to herbicide were estimated to be 36 to 39 micromolar in a membrane fraction and 16 to 17 micromolar in a soluble fraction. Such concentrations should be sufficient to fully depolarize membranes. It is postulated that differences in the ability of membranes to recover from depolarization are correlated with the resistance response of biotype SLR 31. PMID- 16668487 TI - Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): IV. Correlation between Membrane Effects and Resistance to Graminicides. AB - The herbicidally active aryloxyphenoxypropionates diclofop acid, haloxyfop acid, and fluazifop acid and the cyclohexanedione sethoxydim depolarized membranes in coleoptiles of eight biotypes of herbicide-susceptible and herbicide-resistant annual ryegrass (Lolium rigidum). Membrane polarity was reduced from -100 millivolts to -30 to -50 millivolts. Membranes repolarized after removal of the compounds only in biotypes with resistance to the compound added. Repolarization was not observed in herbicide-susceptible L. rigidum, nor was it observed in biotypes resistant to triazine, triazole, triazinone, phenylurea, or sulfonylurea herbicides but not resistant to aryloxyphenoxypropionates and cyclohexanediones. Chlorsulfuron, a sulfonylurea herbicide, at a saturating concentration of 1 micromolar, reduced membrane polarity in all biotypes studied by only 15 millivolts. The recovery of membrane potential following the removal of chlorsulfuron was restricted to chlorsulfuron-susceptible and -resistant biotypes that did not exhibit diclofop resistance. These differences in membrane responses are correlated with resistance to dicloflop rather than with resistance to chlorsulfuron. It is suggested that the differences may reflect altered membrane properties of diclofop-resistant biotypes. Further circumstantial evidence for dissimilarity of properties of membranes from diclofop-resistant and diclofop susceptible ryegrass is provided by observations that K(+)/Na(+) ratios were significantly higher in coleoptiles from diclofop-resistant biotypes than in coleoptiles from susceptible plants. Intact and excised roots from susceptible biotypes were capable of acidifying the external medium, whereas roots from resistant biotypes were unable to do so. The ineluctable conclusion is that in L. rigidum the phenomena of membrane repolarization and resistance to aryloxyphenoxypropionate and cyclohexanedione herbicides are correlated. PMID- 16668488 TI - Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia. AB - Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants. PMID- 16668489 TI - Oilbody Proteins in Microspore-Derived Embryos of Brassica napus: Hormonal, Osmotic, and Developmental Regulation of Synthesis. AB - A number of treatments were tested for their ability to affect the synthesis of oilbody proteins in microspore-derived embryos of rapeseed (Brassica napus). Synthesis of the oilbody proteins was determined by [(35)S]methionine incorporation in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of washed oilbody fractions. Oilbody proteins of approximately 19, 23, and 32 kilodaltons were found to be prominent. These proteins showed differential patterns of regulation. The 19 and 23 kilodalton proteins (oleosins) were greatly enhanced by treatments with abscisic acid, jasmonic acid, and osmotic stress imposed using sorbitol (12.5%). Synthesis of the 32 kilodalton protein was inhibited by abscisic acid and by sorbitol (12.5%), but unaffected by jasmonates. The strong promotion of synthesis of the 19 and 23 kilodalton oilbody proteins appeared to be specific as they are not seen with gibberellic acid treatment or with a stress such as heat shock. Time course experiments revealed that the abscisic acid stimulation of oleosin synthesis is quite rapid (less than 2 hours), reaching a maximum at 6 to 8 hours. The response of the oleosins to abscisic acid is found in all stages of embryogenesis, with a major increase in synthetic rates even in globular embryos on abscisic acid treatment. This suggests that these proteins may accumulate much earlier in embryogenesis than has previously been believed. The 32 kilodalton oilbody-associated protein appears different from the oleosins in several ways, including its distinct pattern of regulation and its unique property, among the oilbody proteins, of undergoing phosphorylation. PMID- 16668490 TI - Hydroperoxide Lyase and Other Hydroperoxide-Metabolizing Activity in Tissues of Soybean, Glycine max. AB - Hydroperoxide lyase (HPLS) activity in soybean (Glycine max) seed/seedlings, leaves, and chloroplasts of leaves required detergent solubilization for maximum in vitro activity. On a per milligram of protein basis, more HPLS activity was found in leaves, especially chloroplasts, than in seeds or seedlings. The total yield of hexanal from 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13S HPOD) from leaf or chloroplast preparations was 58 and 66 to 85%, respectively. Because of significant competing hydroperoxide-metabolizing activities from other enzymes in seed/seedling preparations, the hexanal yields from this source were lower (36-56%). Some of the products identified from the seed or seedling preparations indicated that the competing activity was mainly due to both a hydroperoxide peroxygenase and reactions catalyzed by lipoxygenase. Different HPLS isozyme compositions in the seed/seedling versus the leaf/chloroplast preparations were indicated by differences in the activity as a function of pH, the K(m) values, relative V(max) with 13S-HPOD and 13(S)-hydroperoxy-cis-9,trans 11,cis-15-octadecatrienoic acid (13S-HPOT), and the specificity with different substrates. With regard to the latter, both seed/seedling and chloroplast HPLS utilized the 13S-HPOD and 13S-HPOT substrates, but only seeds/seedlings were capable of metabolizing 9(S)-hydroperoxy-trans-10,cis-12-octadecadienoic acid into 9-oxononanoic acid, isomeric nonenals, and 4-hydroxynonenal. From 13S-HPOD and 13S-HPOT, the products were identified as 12-oxo-cis-9-dodecenoic acid, as well as hexanal from 13S-HPOD and cis-3-hexenal from 13S-HPOT. In seed preparations, there was partial isomerization of the cis-3 or cis-9 into trans-2 or trans-10 double bonds, respectively. PMID- 16668491 TI - Effect of Sucrose on Phase Behavior of Membranes in Intact Pollen of Typha latifolia L., as Measured with Fourier Transform Infrared Spectroscopy. AB - In previous studies, we have shown that the temperature dependent vibrational frequency of the CH(2) stretch in hydrocarbons in intact pollen grains can be recorded with Fourier transform infrared spectroscopy and used to measure phase transition temperatures (T(m)) in these hydrocarbons. Circumstantial evidence was provided that the major contribution to the signal seen in these samples was from membrane phospholipids, and that sucrose in the dry pollen grains reduced T(m) of those phospholipids. In the current study, we clarify why a major constituent of the pollen grains, neutral lipids contained in discrete lipid droplets, does not contribute significantly to the signal. Further, we have isolated membranes from the pollen and show that T(m) in the isolated membranes rises from -6 degrees C in the hydrated membranes to 58 degrees C when the membranes are dried without the addition of sucrose. However, when the isolated membranes are dried in the presence of increasing amounts of sucrose, T(m) fell steadily, reaching a minimal value of 31 degrees C, a figure in good agreement with that seen in the intact pollen grains. The amount of sucrose required to depress T(m) maximally in these membranes is also apparently in agreement with that found in the intact pollen, suggesting that sucrose depresses T(m) in the pollen. PMID- 16668492 TI - Enzymatic Detoxification of HC-toxin, the Host-Selective Cyclic Peptide from Cochliobolus carbonum. AB - Resistance to the fungal plant pathogen Cochliobolus carbonum race 1 and to its host-selective toxin, HC-toxin, is determined by Hm, a single dominant gene in the host plant maize, (Zea mays L). Radiolabeled HC-toxin of specific activity 70 milliCuries per millimole, prepared by feeding tritiated d,l-alanine to the fungus, was used to study its fate in maize leaf tissues. HC-toxin was converted by resistant leaf segments to a single compound, identified by mass spectrometry and nuclear magnetic resonance as the 8-hydroxy derivative of HC-toxin formed by reduction of the 8-keto group of 2-amino-9, 10-epoxy-8-oxo-decanoic acid, one of the amino acids in HC-toxin. Reduction of HC-toxin occurred in cell-free preparations from etiolated (Hm/hm) maize shoots, and the activity was sensitive to heat and proteolytic digestion, dependent on NADPH, and inhibited by p hydroxymercuribenzoate and disulfiram. The enzyme (from the Hm/hm genotype) was partially purified by ammonium sulfate precipitation and diethylaminoethyl-ion exchange chromatography. By gel filtration chromatography, the enzyme had a molecular weight of 42,000. NADH was approximately 30% as effective as NADPH as a hydride donor, and flavin-containing cofactors had no effect on activity. When HC toxin was introduced to maize leaf segments through the transpiration stream, leaf segments from both resistant and susceptible maize inactivated toxin equally well over a time-course of 9 hours. Although these data suggest no relationship between toxin metabolism and host selectivity, we discuss findings in apparent conflict with the current data and describe why the relationship between enzymatic reduction of HC-toxin and Hm remains unresolved. PMID- 16668493 TI - Mutant of Arabidopsis deficient in xylem loading of phosphate. AB - A mutant of Arabidopsis thaliana deficient in the accumulation of inorganic phosphate has been isolated by screening directly for plants with altered quantities of total leaf phosphate. The mutant plants accumulate approximately 5% as much inorganic phosphate, and 24 to 44% as much total phosphate, as wild-type plants in aerial portions of the plant. Growth of the mutant is reduced, relative to wild type, and it exhibits other symptoms normally associated with phosphate deficiency. The phosphate deficiency is caused by a single nuclear recessive mutation at a locus designated pho1. The rate of phosphate uptake into the roots was similar between mutant and wild-type plants over a wide range of external phosphate concentrations. In contrast, when plants were grown in media containing 200 micromolar phosphate or less, phosphate transfer to the shoots of the mutant was reduced to 3 to 10% of the wild-type levels. The defect in phosphate transfer to the shoots could be overcome by providing higher levels of phosphate. Transfer of sulfate to the shoots was essentially normal in the mutant, indicating that the pho1 lesion was not a general defect in anion transport. Movement of phosphate through the xylem of the shoots was not impaired. The results suggest that the mutant is deficient in activity of a protein required to load phosphate into the xylem. PMID- 16668494 TI - delta-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis: Photocontrol of Enzyme Levels in a Chlorophyll-Free Mutant. AB - Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, delta aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNA(Glu) (tRNA(Glu)) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Non-greening mutant Euglena strain W(14)ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA(Glu). Other cellular tRNA(Glu)s present in the mutant cells were capable of being charged with glutamate, but the resulting glutamyl-tRNAs did not support ALA synthesis. Surprisingly, the mutant cells contain all three of the enzymes, and their cell extracts can convert glutamate to ALA when supplemented with tRNA(Glu) obtained from wild-type cells. Activity levels of the three enzymes were measured in extracts of cells grown under a number of light conditions. All three activities were diminished in extracts of cells grown in complete darkness, and full induction of activity required 72 hours of growth in the light. A light intensity of 4 microeinsteins per square meter per second was sufficient for full induction. Blue light was as effective as white light, but red light was ineffective, in inducing extractable enzyme activity above that of cells grown in complete darkness, indicating that the light control operates via the nonchloroplast blue light receptor in the mutant cells. Of the three enzyme activities, the one that is most acutely affected by light is glutamate-1 semialdehyde aminotransferase, as has been previously shown for wild-type Euglena cells. These results indicate that the enzymes required for ALA synthesis from glutamate are present in an active form in the nongreening mutant cells, even though they cannot participate in ALA formation in these cells because of the absence of the required tRNA(Glu), and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNA(Glu) precludes the synthesis of proteins within the plastids, the three enzymes must be synthesized in the cytoplasm and their genes encoded in the nucleus in Euglena. PMID- 16668495 TI - Carbon Assimilation and Leaf Water Status in Sugar Beet Leaves during a Simulated Natural Light Regimen. AB - Carbon assimilation and leaf water status were studied in sugar beet (Beta vulgaris L., Klein E-type multigerm) leaves during a light period in which illumination either increased rapidly to full irradiance or changed gradually in a sinusoidal manner as generally occurs during a natural day. A light regimen that simulated the light of a natural day was produced by adjusting irradiance with a neutral-density filter under the control of a computer. Under this light regimen, photosynthesis, transpiration, and stomatal conductance followed the irradiance pattern very closely and ribulose bisphosphate carboxylase was nearly fully activated. When illumination was increased rapidly at the beginning of a light period, transpiration also increased quickly, causing leaves to wilt to some extent. The activation state of ribulose bisphosphate carboxylase increased to only 52%, but ribulose bisphosphate level was nearly twice as high as during the simulated natural day. In spite of the differences in activation state and ribulose bisphosphate levels, photosynthesis rates were very similar under both regimens. Nevertheless, differences in parameters between leaves under the two irradiance regimens can affect how a plant responds to internal or external factors, and therefore, the rate at which irradiance increases at the beginning of a light period is an important consideration when interpreting data. PMID- 16668496 TI - Effect of N-(Phosphonomethyl)glycine on Carbon Assimilation and Metabolism during a Simulated Natural Day. AB - The effects of N-(phosphonomethyl)glycine (glyphosate) on the regulation of carbon assimilation, metabolism, and translocation were studied in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under a light regimen that began with gradually increasing irradiance as generally occurs on a natural day. Soon after application, glyphosate caused a marked increase in ribulose bisphosphate and a decrease in phosphoglyceric acid. The response is most simply explained by direct inhibition of ribulose bisphosphate carboxylase activity. The extent of inhibition was small, and the carbon assimilation rate did not decrease. As predicted, photosynthesis declined within an hour after glyphosate was applied to leaves under gradually increasing light. Inhibition resulted from a decrease in ribulose bisphosphate due to depletion of carbon from the photosynthetic carbon reduction cycle. Photoinhibition, a light-dependent limitation of photosynthetic capacity, appeared to be necessary for marked glyphosate-induced inhibition of photosynthesis. As a result, photosynthesis rate increased with irradiance until it exceeded 400 micromoles per square meter per second but then declined as the light level increased beyond 500 micromoles per square meter per second. Glyphosate changed the allocation of newly fixed carbon between starch and sucrose for export. Changes in the levels of ribulose bisphosphate and phosphoglyceric acid produced important effects on the regulation of carbon assimilation and metabolism. PMID- 16668497 TI - Regulation of photosynthetic carbon reduction cycle by ribulose bisphosphate and phosphoglyceric Acid. AB - The activation states of a number of chloroplastic enzymes of the photosynthetic carbon reduction cycle and levels of related metabolites were measured in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under slowly changing irradiance during a day. The activation states of both phosphoribulokinase and NADP(+)-glyceraldehyde-3-phosphate dehydrogenase increased early in the light period and remained constant during the middle of the day. Initial ribulose 1,5 bisphosphate carboxylase activity was already about one third of the midday level, did not change for the first 2 hours, but then increased in parallel with the rate of carbon fixation. Because the activation states increased by turns, first phosphoribulokinase and NADP(+)-glyceraldehyde-3-phosphate dehydrogenase and later ribulose 1,5-bisphosphate carboxylase, the ratios of the activation states changed remarkably. Levels of ribulose bisphosphate and phosphoglycerate, which were high enough to affect enzyme reaction rates and changed in concert with activation state, indicate that these metabolites are involved in feedback/feedforward regulation of enzymes of carbon assimilation. This regulatory sequence is able to explain how the reaction rates for the enzymes of carbon assimilation are adjusted to maintain their activities in balance with each other and with the flux of carbon fixation. PMID- 16668498 TI - Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: II. Illuminated Cells. AB - The action spectrum for resetting the phase of the circadian clock in Chlamydomonas reinhardtii is different depending upon whether the light stimuli are presented to cells that were in darkness versus dim illumination before stimulation. In this report, we show that phase resetting of illuminated cells appears to be mediated by components of the photosynthetic apparatus. This conclusion is based upon the action spectrum for phase-shifting illuminated cells (which looks like that for photosynthesis) and upon the fact that inhibitors of photosynthetic electron transport also inhibit the light-induced phase shift of illuminated cells. Both of these characteristics differ from that of cells taken from darkness. We, therefore, believe that at least two resetting pathways for this circadian clock exist and that both of these pathways are ecologically significant. PMID- 16668499 TI - Inward Rectifying K Channels in the Plasma Membrane of Arabidopsis thaliana. AB - Ion channels in the plasma membrane of protoplasts isolated from cultured cells of Arabidopsis thaliana were studied by means of the patch-clamp technique applied in the whole-cell configuration. In some protoplasts, depolarizing pulses and, in other protoplasts, hyperpolarizing pulses elicited time-dependent currents; both kinds of current were only rarely observed in the same protoplast. The hyperpolarization-activated inward rectifying currents, the focus of this paper, appeared to be due to the relatively slow opening of channels (activation time constant = 150 to 300 milliseconds), which closed at positive potentials. The reversal potential of this current, measured in the presence of different ion concentrations (symmetrical or asymmetrical K(+) and Cl(-) or gluconate), was always close to the electrochemical equilibrium potential of K(+). The currents were inhibited by 10 millimolar tetraethylammonium, a K(+) channel blocker. These data show that the hyperpolarization-activated currents flow through K(+) channels, which can provide a pathway for the passive diffusion of K(+) down its electrochemical gradient. PMID- 16668500 TI - Effects of Salinity on Water Transport of Excised Maize (Zea mays L.) Roots. AB - The root pressure probe was used to determine the effects of salinity on the hydraulic properties of primary roots of maize (Zea mays L. cv Halamish). Maize seedlings were grown in nutrient solutions modified by additions of NaCl and/or extra CaCl(2) so that the seedlings received one of four treatments: Control, plus 100 millimolar NaCl, plus 10 millimolar CaCl(2), plus 100 millimolar NaCl plus 10 millimolar CaCl(2). The hydraulic conductivities (Lp(r)) of primary root segments were determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. Exosmotic hydrostatic Lp(r) for the different treatments were 2.8, 1.7, 2.8, and 3.4.10(-7) meters per second per megapascals and the endosmotic hydrostatic Lp(r) were 2.4, 1.5, 2.7, and 2.3.10(-7) meters per second per megapascals, respectively. Exosmotic Lp(r) of the osmotic experiments were 0.55, 0.38, 0.68, and 0.60.10(-7) meters per second per megapascals and the endosmotic Lp(r) were 0.53, 0.21, 0.56, and 0.54.10(-7) meters per second per megapascals, respectively. The osmotic Lp(r) was significantly smaller (4-5 times) than hydrostatic Lp(r). However, both hydrostatic and osmotic Lp(r) experiments showed that salinization of the growth media at regular (0.5 millimolar) calcium levels decreased the Lp(r) significantly (30-60%). Addition of extra calcium (10 millimolar) to the salinized media caused ameliorative effects on Lp(r). The low Lp(r) values may partially explain the reduction in root growth rates caused by salinity. High calcium levels in the salinized media increased the relative availability of water needed for growth. The mean reflection coefficients of the roots using NaCl were between 0.64 and 0.73 and were not significantly different for the different treatments. The mean values of the root permeability coefficients to NaCl of the different treatments were between 2.2 and 3.5.10(-9) meters per second and were significantly different only in one of four treatments. Cutting the roots successively from the tip and measuring the changes in the hydraulic resistance of the root as well as staining of root cross-sections obtained at various distances from the root tip revealed that salinized roots had mature xylem elements closer to the tip (5-10 millimeters) compared with the controls (30 millimeters). Our results demonstrate that salinity has adverse effects on water transport and that extra calcium can, in part, compensate for these effects. PMID- 16668501 TI - Water Relations of Pachysandra Leaves during Freezing and Thawing : Evidence for a Negative Pressure Potential Alleviating Freeze-Dehydration Stress. AB - The evergreen herb Pachysandra terminalis becomes moderately frost-hardy in winter. The water relations of its frost-hardy leaves were studied during a freeze-thaw cycle. Leaf water potentials, measured by psychrometry at subfreezing temperatures, were identical with those of ice, indicating equilibrium freezing. Microscopic observations showed extracellular freezing of tissue water. As evidenced by thermal analysis, the freezing process starts with the crystallization of a minor volume which was identified as apoplasmic water. The following long-lasting exotherm indicated slow export of water from the protoplasts driven by extracellular crystallization. In partially frozen leaves, the fractions of liquid water were measured at several subfreezing temperatures by nuclear magnetic resonance spectroscopy. They were consistently greater than those calculated from the osmotic potentials of cellular fluid, and the differences increased with decreasing temperature. About 50% of the differences could be abolished by freeze-killing of the leaf and was thus ascribed to the effect of a (negative) pressure reinforcing the osmotic potential. The persistent part of the differences may have reflected a matric component. At -7 degrees C, the absolute values of both potentials were -1.7 megapascals each. The water relations of Pachysandra leaves clearly indicate nonideal equilibrium freezing where negative pressures and matric potentials contribute to the leaf water potential and thus alleviate freeze-dehydration of the tissue. PMID- 16668502 TI - Computer-simulated evaluation of possible mechanisms for quenching heavy metal ion activity in plant vacuoles: I. Cadmium. AB - Various mechanisms have been suggested for the quenching of Cd ion activity in plant vacuoles. These include solution complexation with organic acids and sulfhydryl-containing peptides and precipitation as sulfides. Because direct experimental support for these mechanisms is lacking and difficult to obtain, we have used a computer model to evaluate the quenching role of possible organic and inorganic ligands of tobacco cultured cells exposed to Cd. Results of this thermodynamic evaluation, which assumes that a chemical equilibrium state is met in the vacuole, support the conclusion that sulfhydryl-containing peptides and certain organic acids may form soluble Cd complexes. Although complexation of malate and oxalate with Cd is predicted to be less significant, citrate in the concentration range encountered in the tobacco cultured cell vacuoles has high potential for forming soluble complexes with Cd over the entire possible vacuolar pH range, especially 4.3 to 7.0, even in the presence of low levels of Cd-binding peptides. In addition, results show that inorganic chloride, sulfide (if present), and phosphate may also act to sequester Cd ion activity in the vacuole by forming soluble Cd-Cl and insoluble CdS and Cd-phosphate. PMID- 16668503 TI - Induction by Electric Currents of Ethylene Biosynthesis in Cucumber (Cucumis sativus L.) Fruit. AB - The effects of an electric current on ethylene biosynthesis were investigated in cucumber (Cucumis sativus L.) fruit that were producing almost no ethylene. Direct currents at 0.5 to 3.0 milliamperes induced much ethylene synthesis, with a rapid continuous increase in the rate, which reached a peak within 5 to 6 hours and then decreased. The rate of production was greater with a stronger current. Ethylene production was not observed after the use of a sine-wave alternating current (60 hertz) at 3 milliamperes, the magnitude at which a direct current had the greatest effect. The activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ethylene forming enzyme (EFE) increased before the rise in ethylene production. ACC synthase and EFE were activated sixfold and fourfold, respectively, by 2 hours. The concentration of ACC increased linearly up to 6 hours and then decreased. Ethylene induction by an electric current was suppressed almost completely by the infiltration of the cucumbers with 5 millimolar aminooxyacetic acid, an inhibitor of ACC synthase, and was also suppressed 70% by 5 millimolar salicylic acid, an inhibitor of EFE. The results indicate that the ethylene induced by the direct current was synthesized via the ACC-ethylene pathway as a result of electrical stress, a new kind of stress to be identified. PMID- 16668504 TI - Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana. AB - gamma-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms. (gram fresh weight)(-1) free indoleacetic acid (IAA), 150 nanograms. (gram fresh weight)(-1) ester-conjugated IAA, and 10 to 20 micrograms. (gram fresh weight)(-1) amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms. (gram fresh weight)(-1) of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls. PMID- 16668505 TI - Localization of a protease in protoplast preparations in infected cells of French bean nodules. AB - Protoplasts from infected and uninfected cells were isolated from the central nitrogen fixing tissue of French bean (Phaseolus vulgaris L. cv Contender) root nodules. Successive filtrations allowed the separation of the infected cells, whereas the small uninfected cells were isolated on a discontinuous Percoll gradient. Higher yields of intact protoplasts were obtained from young (4-week old) nodules whereas no protoplasts could be isolated from the oldest nodules. When proteolysis was determined in the cytosolic fraction of both infected and uninfected cells, at pH 5.0 and 8.0, with leghemoglobin or azocasein as substrate, activity was present only in infected cell protoplasts and increased with nodule age. A protease with an acidic pH optimum, mainly responsible for this increasing activity, was highly purified from senescing nodules by electro elution after nondenaturing polyacrylamide gel electrophoresis and used to produce polyclonal antibodies. Western blots of nodule protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with purified anti-protease immunoglobulin G showed the molecular mass of the protease to be 58 kilodaltons. Blots also confirmed that protease protein was located in infected cell protoplasts only, regardless of nodule age. PMID- 16668506 TI - Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants. AB - Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen. PMID- 16668507 TI - Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases. AB - ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A(0.5) = 0.81 millimolar; n(H) = 2.0). Activation by 3 phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A(0.5) = 0.12 millimolar; n(H) = 1.0), having a maximal stimulation of 17-fold. I(0.5) values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A(0.5) = 10 micromolar; n(H) = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3 phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase. PMID- 16668508 TI - Autotrophy in maize husk leaves : evaluation using natural abundance of stable isotopes. AB - The natural abundance of carbon and hydrogen isotopic composition, expressed as a delta(13)C value of plant dry matter and cellulose in the hypsophylls (husk leaves) of maize (Zea mays L.) was measured and compared with that of leaves and cobs. The delta(13)C values of outer hypsophylls were usually 2 to 3% per thousand more negative than leaves or other tissues, and became more negative with increasing chlorophyll content, indicating significant local C(3) pathway fixation of CO(2) in the outer hypsophylls. The deltaD values indicated a significant part of hypsophyll cellulose was derived from heterotrophic sources (sucrose from C(4) photosynthesis in other tissues). Isotopic mass balance calculations allowed quantitative estimation of these carbon sources and, in the samples examined, about 16% of hypsophyll cellulose was derived from local C(3) photosynthesis, about 62% from local C(4) photosynthesis, and about 22% from sucrose imported from other leaves. PMID- 16668509 TI - Comparative Physiological Evidence that beta-Alanine Betaine and Choline-O Sulfate Act as Compatible Osmolytes in Halophytic Limonium Species. AB - The quaternary ammonium compounds accumulated in saline conditions by five salt tolerant species of Limonium (Plumbaginaceae) were analyzed by fast atom bombardment mass spectrometry. Three species accumulated beta-alanine betaine and choline-O-sulfate; the others accumulated glycine betaine and choline-O-sulfate. Three lines of evidence indicated that beta-alanine betaine and choline-O-sulfate replace glycine betaine as osmo-regulatory solutes. First, tests with bacteria showed that beta-alanine betaine and choline-O-sulfate have osmoprotective properties comparable to glycine betaine. Second, when beta-alanine betaine and glycine betaine accumulators were salinized, the levels of their respective betaines, plus that of choline-O-sulfate, were closely correlated with leaf solute potential. Third, substitution of sulfate for chloride salinity caused an increase in the level of choline-O-sulfate and a matching decrease in glycine betaine level. Experiments with (14)C-labeled precursors established that beta alanine betaine accumulators did not synthesize glycine betaine and vice versa. These experiments also showed that beta-alanine betaine synthesis occurs in roots as well as leaves of beta-alanine betaine accumulators and that choline-O-sulfate and glycine betaine share choline as a precursor. Unlike glycine betaine, beta alanine betaine synthesis cannot interfere with conjugation of sulfate to choline by competing for choline and does not require oxygen. These features of beta alanine betaine may be advantageous in sulfate-rich salt marsh environments. PMID- 16668510 TI - Light Microscopy Study of Nodule Initiation in Pisum sativum L. cv Sparkle and in Its Low-Nodulating Mutant E2 (sym 5). AB - We compared nodule initiation in lateral roots of Pisum sativum (L.) cv Sparkle and in a low-nodulating mutant E2 (sym 5). In Sparkle, about 25% of the infections terminated in the epidermis, a similar number stopped in the cortex, and 50% resulted in the formation of a nodule meristem or an emerged nodule. The mutant E2 (sym 5) was infected as often as was the parent, and it formed a normal infection thread. In the mutant, cell divisions rarely occurred in advance of the infection thread, and few nodule primordia were produced. Growing the mutant at a low root temperature or adding Ag(+) to the substrate increased the number of cell divisions and nodule primordia. We conclude that, in the E2 line, the infection process is arrested in the cortex, at the stage of initial cell divisions before the establishment of a nodule primordium. PMID- 16668511 TI - Evidence for a highly specific k/h antiporter in membrane vesicles from oil-seed rape hypocotyls. AB - We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substrate-specific, K(+)/H(+) antiport in both tonoplast and plasma membrane-enriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K(+) (with Cl(-) or SCN( )) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H(+)-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K(+)/H(+) exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCl was supplied, even in the absence of 100 millimolar Cl(-) and, second, efflux of K(+) from K(+)-loaded vesicles drives intravesicular accumulation of H(+) against the electrochemical potential gradient. Neither was the exchange due to competition between K(+) and quinacrine for membrane sites, nor to inhibition of the H(+)-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K(+) (at micromolar concentrations); Na(+)/H(+) antiport was detected only at concentrations two orders of magnitude higher. Rb(+), Li(+), or Cs(+) were ineffective. Dependence of tonoplast K(+)/H(+) antiport on K(+) concentration was complex, showing saturation at 10 millimolar K(+) and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar NO(3) (-) and showed no sensitivity to vanadate or oligomycin) and with plasma membrane enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol sensitive but showed no sensitivity to NO(3) (-) or oligomycin). The possible physiological role of such a K(+)/H(+) exchange mechanism is discussed. PMID- 16668512 TI - Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine. AB - Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO(3) (-) on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO(3) (-)-treated roots. These results support our hypothesis that the inhibitory effect of NO(3) (-) is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO(3) (-)-fed plants probably as a result of the very high stimulation of nodulation. PMID- 16668513 TI - Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress. AB - Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH(4) (+) are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K(+) efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic-a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis. PMID- 16668514 TI - Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina. AB - The location and mobilization of polyphosphates in response to an amine-induced alkaline stress were studied in the halotolerant alga Dunaliella salina. The following observations suggest that polyphosphates accumulate in acidic vacuoles: (a) Accumulation of large amounts of polyphosphates is manifested as intravacuolar dense osmiophilic bodies in electron micrographs. (b) Uptake of amines into the vacuoles induces massive hydrolysis of polyphosphates, demonstrated by in vivo(31)P-nuclear magnetic resonance, and by analysis of hydrolytic products on thin layer chromatograms. The analysis indicates that: (a) Polyphosphate hydrolysis is kinetically correlated with amine accumulation and with the recovery of cytoplasmic pH. (b) The major hydrolytic product is tripolyphosphate. (c) The peak position of the tripolyphosphate terminal phosphate in nuclear magnetic resonance spectra is progressively shifted as the cells recover, indicating that the pH inside the vacuoles increases while the pH in the cytoplasm decreases. (d) In lysed cell preparations, in which vacuoles become exposed to the external pH, mild alkalinization in the absence of amines induces polyphosphate hydrolysis to tripolyphosphates. It is suggested that amine accumulation within vacuoles activates a specific phosphatase, which hydrolyzes long-chain polyphosphates to tripolyphosphates. The hydrolysis increases the capacity of the vacuoles to sequester amines from the cytoplasm probably by releasing protons required to buffer the amine, and leads to recovery of cytoplasmic pH. Thus, polyphosphate hydrolysis provides a high-capacity buffering system that sustains amine compartmentation into vacuoles and protects cytoplasmic pH. PMID- 16668515 TI - Hydrolysis of polyphosphates and permeability changes in response to osmotic shocks in cells of the halotolerant alga dunaliella. AB - The effects of osmotic shocks on polyphosphates and on the vacuolar fluorescent indicator atebrin have been investigated to test whether acidic vacuoles in the halotolerant alga Dunaliella salina have a role in osmoregulation. Upshocks and downshocks induce different patterns of polyphosphate hydrolysis. Upshocks induce rapid formation of new components, tentatively identified as 5 or 6 linear polyphosphates, formed only after upshocks with NaCl and not with glycerol, indicative of compartmentation of Na(+) into the vacuoles. Conversely, downshocks induce a slower transient accumulation of tripolyphosphates, indicating activation of a different hydrolytic process within the vacuoles. Osmotic shocks do not lead to release of atebrin from acidic vacuoles, indicating that they do not induce a major intravacuolar alkalinization. However, osmotic shocks induce transient permeability changes measured by amine-induced atebrin release from vacuoles. Hypoosmotic shocks transiently increase the permeability (up to 20 fold), whereas hyperosmotic shocks induce a rapid drop in permeability. Electron micrographs of osmotically shocked cells also reveal transient changes in the surface and internal organelles of D. salina cells. It is suggested that hyperosmotic and hypoosmotic shocks induce different changes within acidic vacuoles and in the organization and/or composition of the plasma membrane in Dunaliella. PMID- 16668516 TI - Sucrose synthase and invertase in isolated vascular bundles. AB - Vascular bundles were isolated from grapefruit (Citrus paradisi Macf.) during periods of rapid sucrose translocation into fruit. Invertase and sucrose synthase activities were assayed in these strands and compared with immediately adjacent tissues (inner most peel and segment epidermis) and phloem-free juice sacs during four growing seasons. Although sucrose synthase was present in sink cells, the significantly greater activity in vascular strands (per unit fresh weight and protein) indicated that the role of this enzyme in translocation may include a vascular function in addition to its proposed involvement in metabolism of importing cells. PMID- 16668517 TI - Jasmonic Acid induces tuberization of potato stolons cultured in vitro. AB - The aim of the study was to assess the potential in vitro effects of jasmonic acid and kinetin on tuberization of potato (Solanum tuberosum). Of the two, the former was by far the stronger in vitro promoter of stolon tuberization. Number of tubers induced per stolon, tuberization rate, and final tuber weight were higher by factors of 2.8, 2.3, and 6.4, respectively. Bioassay sensitivity of jasmonic acid, measured in terms of the point at which the concentration for inducing tuberization was saturating, was more than 20 times greater than that of kinetin. Tuberization in both cases was associated with a decrease in rooting ability. Jasmonic acid also triggered a general state of induction throughout the stolon. PMID- 16668518 TI - Effect of Hydrogen Cyanamide on Amino Acid Profiles in Kiwifruit Buds during Budbreak. AB - Buds, and resultant shoots, were collected from kiwifruit (Actinidia deliciosa [A. Chev.] CF Liang et AR Ferguson var deliciosa cv Hayward) vines from late autumn until late spring with and without hydrogen cyanamide treatment. Those samples were weighed and analyzed for total nitrogen and free amino acids. By 7 days after hydrogen cyanamide treatment, the amount of proline had risen to nearly one-quarter of the total amino acid pool in the treated buds and that proportion was maintained for at least 14 days before it declined. The maximum concentration detected in treated buds was 25.8 micromoles per gram dry weight, 6.6 times that detected in untreated buds. By 95 days after treatment, the relative amounts of proline were not significantly different. PMID- 16668519 TI - Abscisic Acid Negatively Regulates Expression of Chlorophyll a/b Binding Protein Genes during Soybean Embryogeny. AB - The levels of abscisic acid (ABA) during embryogenesis in the soybean (Glycine max) cultivar Dare were quantitated. An increase in the quantity of ABA per cotyledon was correlated with a decrease in the chlorophyll a/b binding (Cab) protein gene mRNA population. Soybean cotyledons were cultured in vitro in the presence or absence of ABA. Quantitation of cotyledonary ABA levels and Cab mRNA levels indicated that the application of 5 x 10(-5) molar and 5 x 10(-6) molar exogenous ABA decreased Cab mRNA prevalences. S1 nuclease protection experiments demonstrated that exogenous ABA modulated the level of Cab3 mRNA. These data strongly suggest that one of the developmental regulators of Cab gene expression during soybean embryogeny is the plant hormone, ABA; ABA negatively regulates Cab mRNA accumulation. PMID- 16668520 TI - Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment. AB - After cold treatment of seedlings of winter wheat (Triticum aestivum L.), levels of hydrogen peroxide in the leaves were measured. The concentration of hydrogen peroxide increased to about three times the control level within a few minutes, and returned to the normal level in 15 to 20 minutes. The elevated level of hydrogen peroxide was found to be equivalent to 1.5 micromoles per gram fresh weight tissues of leaves. PMID- 16668521 TI - Sequence of the Gossypium hirsutum D-Genome Alloallele of Legumin A and Its mRNA. PMID- 16668522 TI - Physiological and molecular aspects of the inorganic carbon-concentrating mechanism in cyanobacteria. AB - This paper reviews progress made in elucidating the inorganic carbon concentrating mechanism in cyanobacteria at the physiological and molecular levels. Emphasis is placed on the mechanism of inorganic carbon transport, physiological and genetical analysis of high-CO(2)-requiring mutants, the polypeptides induced during adaptation to low CO(2), the functional significance of carboxysomes, and the role of carbonic anhydrase. We also make occasional reference to the green algal inorganic carbon-concentrating mechanism. PMID- 16668523 TI - Purification and Characterization of Two Major Lectins from Araucaria brasiliensis syn. Araucaria angustifolia Seeds (Pinhao). AB - Two major lectins (lectin I and lectin II) were purified to homogeneity from the seeds of Araucaria brasiliensis (Gymnospermae). The purity of the lectins was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and high performance liquid chromatography. They are glycoproteins in nature containing 6.3 and 2.9%, respectively, of neutral sugar and have absorption coefficients of 3.8 and 4.7, respectively, at 280 nanometers. The molecular weights of both lectins obtained by gel filtration on Sephacryl S-400 were equal: 200,000. After dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular weights were 20,000 and 34,000, respectively, for lectin I and lectin II, suggesting they are decameric and hexameric in nature. The amino acid composition of both lectins showed little difference, but both had high amounts of acidic amino acids and lacked methionine in their molecule. The carbohydrate binding specificity of lectins was directed towards mannose, glucose, and their oligomers. High inhibitory activity was also found with thyroglobulin. The erythroagglutinating activity of the lectins was enhanced in the presence of high molecular-weight substances both at 37 and 4 degrees C. Divalent cations do not appear to be essential for activity. They maintained their agglutinating activity over a broad but different range of pH: 5.5 to 7.5 and 6.5 to 7.5, respectively. Both lectins agglutinated erythrocytes of human ABO blood types equally well. PMID- 16668524 TI - Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions. AB - Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO(2) assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway. PMID- 16668525 TI - Effect of Chlorate Treatment on Nitrate Reductase and Nitrite Reductase Gene Expression in Arabidopsis thaliana. AB - The herbicide chlorate has been used extensively to isolate mutants that are defective in nitrate reduction. Chlorate is a substrate for the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Because NR is a substrate (NO(3) (-))-inducible enzyme, we investigated the possibility that chlorate may also act as an inducer. Irrigation of ammonia-grown Arabidopsis plants with chlorate leads to an increase in NR mRNA in the leaves. No such increase was observed for nitrite reductase mRNA following chlorate treatment; thus, the effect seems to be specific to NR. The increase in NR mRNA did not depend on the presence of wild-type levels of NR activity or molybdenum-cofactor, as a molybdenum-cofactor mutant with low levels of NR activity displayed the same increase in NR mRNA following chlorate treatment. Even though NR mRNA levels were found to increase after chlorate treatment, no increase in NR protein was detected and the level of NR activity dropped. The lack of increase in NR protein was not due to inactivation of the cells' translational machinery, as pulse labeling experiments demonstrated that total protein synthesis was unaffected by the chlorate treatment during the time course of the experiment. Chlorate-treated plants still retain the capacity to make functional NR because NR activity could be restored by irrigating the chlorate-treated plants with nitrate. The low levels of NR protein and activity may be due to inactivation of NR by chlorite, leading to rapid degradation of the enzyme. Thus, chlorate treatment stimulates NR gene expression in Arabidopsis that is manifested only at the mRNA level and not at the protein or activity level. PMID- 16668526 TI - Accumulation of Scoparone in Heat-Treated Lemon Fruit Inoculated with Penicillium digitatum Sacc. AB - Phytoalexin scoparone (6,7-dimethoxycoumarin) generally was not detected in noninoculated lemon fruit (Citrus limon [L.] Burm., cv Eureka) but accumulated in fruit after inoculation with Penicillium digitatum Sacc. A much greater increase in the amount of scoparone was found in fruit exhibiting an incompatible response to Penicillium after heat treatment at 36 degrees C for 3 days. Heat treatment prevented development of decay in the inoculated fruit. The concentration of the compound after inoculation continued to increase during and after the heat treatment period, reaching 178 micrograms per gram fresh weight of the flavedo 6 days after the heat treatment. Changes in scoparone concentration in fruit were closely correlated with the changes in the antifungal activity of the fruit extract. A low concentration of the phytoalexin was detected in fruit injured mechanically. Scoparone also accumulated in the fruit following ultraviolet illumination; the concentration of the compound was dose-dependent. Median effective dose values of the inhibition of germ tube elongation and spore germination of P. digitatum were 29 and 46 micrograms per milliliter, respectively. Our findings suggest that the rapid increase in scoparone concentration plays an important role in the increased resistance of heat-treated lemon fruit to infection by P. digitatum. PMID- 16668527 TI - Chlorophyll Fluorescence and Photon Yield of Oxygen Evolution in Iron-Deficient Sugar Beet (Beta vulgaris L.) Leaves. AB - The response of sugar beet (Beta vulgaris L.) leaves to iron deficiency can be described as consisting of two phases. In the first phase, leaves may lose a large part of their chlorophyll while maintaining a roughly constant efficiency of photosystem II photochemistry; ratios of variable to maximum fluorescence decreased by only 6%, and photon yields of oxygen evolution decreased by 30% when chlorophyll decreased by 70%. In the second phase, when chlorophyll decreased below a threshold level, iron deficiency caused major decreases in the efficiency of photosystem II photochemistry and in the photon yield of oxygen evolution. These decreases in photosystem II photochemical efficiency were found both in plants dark-adapted for 30 minutes and in plants dark-adapted overnight, indicating that photochemical efficiency cannot be repaired in that time scale. Decreases in photosystem II photochemical efficiency and in the photon yield of oxygen evolution were similar when measurements were made (a) with light absorbed by carotenoids and chlorophylls and (b) with light absorbed only by chlorophylls. Leaves of iron-deficient plants exhibited a room temperature fluorescence induction curve with a characteristic intermediate peak I that increases with deficiency symptoms. PMID- 16668528 TI - Regulation of 2-carboxyarabinitol 1-phosphatase. AB - The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A(0.5) value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A(0.5) was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3 phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased V(max) but did not appreciably alter K(m) (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a K(i) of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35 degrees C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30 degrees C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole. PMID- 16668529 TI - Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato. AB - The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression. PMID- 16668530 TI - Maintenance of turgor by rapid sealing of puncture wounds in leaf epidermal cells. AB - When leaf epidermal cells are puncture wounded with a glass microcapillary tip, a small droplet of fluid is discharged and then evaporates, leaving a solid residue on the cell surface. For puncture wounds of about 3.5 micrometers in diameter, this process is complete within 2 to 3 seconds. A second puncture wound also exhibits a similar discharge, indicating the persistence of some turgor pressure within the cell, despite damage to the cell wall. Direct measurement of turgor on the large epidermal cells of Tradescantia virginiana L. demonstrated that turgor was substantially maintained (91-96%) after puncture wounding. Anatomical and histochemical evidence suggests that the damaged portion of the cell wall was sealed with an amorphous plug of material comprised of pectinaceous polysaccharides. Rapid sealing of puncture wounds and the maintenance of turgor in epidermal cells may be an important functional component of plant adaptation to physical damage such as that caused by insect feeding. PMID- 16668531 TI - Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase. AB - The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose P(2) carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-(3)H]-l methionine to spinach ribulose-P(2) carboxylase. (3)H-Methyl groups incorporated into spinach ribulose-P(2) carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P(2) carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose P(2) carboxylase LS (lysine) ;N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P(2) carboxylase and exhibited apparent K(m) values of 10 micromolar for S-adenosyl-l methionine and 18 micromolar for ribulose-P(2) carboxylase, a V(max) of 700 picomoles CH(3) groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P(2) carboxylase LS (lysine)(epsilon)N-methyltransferase was capable of incorporating 24 (3)H-methyl groups per spinach ribulose-P(2) carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P(2) carboxylase LS, but was inactive on ribulose-P(2) carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg(2+) and CO(2)) and unactivated forms of ribulose-P(2) carboxylase as substrates. However, complexes of activated ribulose-P(2) carboxylase with the reaction-intermediate analogue 2'-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P(2) carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS (epsilon)N-methyltransferase. PMID- 16668532 TI - Diacylglycerol Metabolism in the Green Alga Dunaliella salina under Osmotic Stress : Possible Role of Diacylglycerols in Phospholipase C-Mediated Signal Transduction. AB - The sn-1,2-diacylglycerol (DAG) content and molecular species composition of Dunaliella salina whole cells and cell fractions were measured by complementary high performance liquid chromatography and gas chromatography techniques. At 4.2 nanomoles per 100 nanomoles lipid phosphorus, the whole cell DAG level was high in comparison with most animal tissues. The DAG concentration was highest in the microsome-enriched fraction, followed by that in the chloroplast and in the plasma membrane fractions. The predominant DAG molecular species in all cell fractions contained oleic (18:1), linoleic (18:2), or linolenic (18:3) acid in the sn-1 position and palmitate (16:0) in the sn-2 position. Recent studies have raised the possibility of DAG serving a signal transducing function in osmotically stressed D. salina cells. During the first 30 seconds following hypoosmotic shock, there was a 40% increase in the plasma membrane DAG content, whereas the DAG content of the microsome-enriched fraction was unchanged. On a nanomole per 100 nanomoles phospholipid basis, the rise in plasma membrane DAG nearly matched the previously reported (KJ Einspahr, TC Peeler, GA Thompson Jr [1988] J Biol Chem 263: 5775-5779) transient fall in phosphatidylinositol 4,5 bisphosphate. Furthermore, 18:1/16:0 DAG, one of the major plasma membrane DAG molecular species increasing in amount after hypoosmotic shock, was the characteristic molecular species of plasma membrane phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate, but no other lipid of that membrane. Evidence was found for a rise in 16:0/18:2 and 16:0/18:3 DAG as well following hypoosmotic shock. This pattern suggested that phosphatidylcholine hydrolysis also contributed to the stress-induced production of DAG in the D. salina plasma membrane. The extent of the sudden DAG increase was sufficient to consider it a potential second messenger in phospholipase C mediated signal transduction. PMID- 16668533 TI - Energy status and functioning of phosphorus-deficient soybean nodules. AB - Characterization of the effects of long-term P deficiency and of onset and recovery from P deficiency on bacteroid mass and number per unit nodule mass and energy status of soybean (Glycine max L. Merr.) nodules was used to investigate the mechanisms by which P deficiency decreases symbiotic N(2) fixation. The continuous P deficiency treatment (0.05 millimolar P) significantly decreased the whole plant dry mass, P, and N by 62, 90, and 78%, respectively, relative to the P-sufficient control (1.0 millimolar) at 44 days after transplanting. Specific nitrogenase activity was decreased an average of 28% over a 16-day experimental period by P deficiency. Whole nodules of P-deficient controls contained 70 to 75% lower ATP concentrations than nodules of P-sufficient controls. Energy charge and ATP concentrations in the bacteroid fraction of nodules were not significantly affected by P treatment. However, ATP and total adenylate concentrations and energy charge in the plant cell fraction of nodules were significantly decreased 91, 62, and 50%, respectively, by the P deficiency treatment. Specific nitrogenase activity, energy charge, and ATP concentration in the plant cell fraction increased to the levels of nonstressed controls within 2, 2, and 4 days, respectively, after alleviation of external P limitation, whereas bacteroid mass per unit nodule mass and bacteroid N concentration did not increase to the level of nonstressed controls until 7 days after alleviation of external P limitation. All of these parameters except bacteroid mass per unit nodule mass decreased to the levels of the P-deficient controls by 11 days after onset of external P limitation. Concentration of ATP in the bacteroid fraction was not significantly affected by alteration in the external P supply. Energy charge in the bacteroid fraction from plants recovering from P deficiency was decreased to a small (10%) but significant extent (P < 0.05) at two sampling dates relative to P-sufficient controls. These ATP concentration and energy charge measurements indicate that P deficiency impaired oxidative phosphorylation in the plant cell fraction of nodules to a much greater extent than in the bacteroids. The concurrence of significant changes in specific nitrogenase activity (2 days) and in the energy charge (2 days) and ATP concentration (4 days) in the plant cell fraction during recovery from external P limitation is consistent with the conclusion that P deficiency decreases the specific nitrogenase activity by inhibiting an energy dependent reaction(s) in the plant cell fraction of the nodules. PMID- 16668534 TI - Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro. AB - Posttranslational modifications that give rise to multiple forms of alpha-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley alpha-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). alpha-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and alpha-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic alpha-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley alpha-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify alpha-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of alpha-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase. PMID- 16668535 TI - High Affinity Transport of CO(2) in the Cyanobacterium Synechococcus UTEX 625. AB - The active transport of CO(2) in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO(3) (-) transport. The substrate concentration required to give one half the maximum rate for whole cell CO(2) transport was determined to be 0.4 +/- 0.2 micromolar (mean +/- standard deviation; n = 7) with a range between 0.2 and 0.66 micromolar. The maximum rates of CO(2) transport ranged between 400 and 735 micromoles per milligram of chlorophyll per hour with an average rate of 522 for seven experiments. This rate of transport was about three times greater than the dissolved inorganic carbon saturated rate of photosynthetic O(2) evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO(2) transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO(2) transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO(2) transport was caused by HCO(3) (-) or by Na(+)-dependent HCO(3) (-) transport. However, (12)CO(2) readily interfered with (13)CO(2) transport. CO(2) transport and Na(+)-dependent HCO(3) (-) transport are separate, independent processes and the high affinity CO(2) transporter is not only responsible for the initial transport of CO(2) into the cell but also for scavenging any CO(2) that may leak from the cell during ongoing photosynthesis. PMID- 16668536 TI - Changes in Osmotic Pressure and Mucilage during Low-Temperature Acclimation of Opuntia ficus-indica. AB - Opuntia ficus-indica, a Crassulacean acid metabolism plant cultivated for its fruits and cladodes, was used to examine chemical and physiological events accompanying low-temperature acclimation. Changes in osmotic pressure, water content, low molecular weight solutes, and extracellular mucilage were monitored in the photosynthetic chlorenchyma and the water-storage parenchyma when plants maintained at day/night air temperatures of 30/20 degrees C were shifted to 10/0 degrees C. An increase in osmotic pressure of 0.13 megapascal occurred after 13 days at 10/0 degrees C. Synthesis of glucose, fructose, and glycerol accounted for most of the observed increase in osmotic pressure during the low-temperature acclimation. Extracellular mucilage and the relative apoplastic water content increased by 24 and 10%, respectively, during exposure to low temperatures. These increases apparently favor the extracellular nucleation of ice closer to the equilibrium freezing temperature for plants at 10/0 degrees C, which could make the cellular dehydration more gradual and less damaging. Nuclear magnetic resonance studies helped elucidate the cellular processes during ice formation, such as those revealed by changes in the relaxation times of two water fractions in the chlorenchyma. The latter results suggested a restricted mobility of intracellular water and an increased mobility of extracellular water for plants at 10/0 degrees C compared with those at 30/20 degrees C. Increased mobility of extracellular water could facilitate extracellular ice growth and thus delay the potentially lethal intracellular freezing during low-temperature acclimation. PMID- 16668537 TI - Purification and characterization of pea cytosolic ascorbate peroxidase. AB - The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14 day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves. PMID- 16668538 TI - Effects of exogenous auxins on expression of lipoxygenases in cultured soybean embryos. AB - The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either alpha-naphthaleneacetic acid or indoleacetic acid but not by 2,4 dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by alpha-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos. PMID- 16668539 TI - Ectomycorrhizin Synthesis and Polypeptide Changes during the Early Stage of Eucalypt Mycorrhiza Development. AB - In functioning eucalypt ectomycorrhizas, biochemical alterations are accompanied by a differential accumulation of polypeptides including the synthesis of symbiosis-related proteins (JL Hilbert, Martin FM [1988] New Phytol 110: 339 346). In the present study, protein biosynthesis in the early stages of ectomycorrhiza formation on Eucalyptus globulus subsp. bicostata Kirkp. was examined using compatible and incompatible isolates of the basidiomycete Pisolithus tinctorius (Coker & Couch). Changes in polypeptide composition were observed within hours following contact of the compatible mycelium with the roots, well before the differentiation of typical symbiotic tissues. At this stage, at least seven symbiosis-related proteins (ectomycorrhizins) accumulated in root tissues. In vivo incorporation of [(35)S]methionine by ectomycorrhizas followed by electrophoresis of the labeled proteins revealed that most of these differences in polypeptide concentrations, including the ectomycorrhizin accumulation, are the result of differential protein biosynthesis rather than posttranslational modifications of the polypeptides. The initial development of eucalypt ectomycorrhizas, therefore, coincides with the synthesis of symbiosis related proteins and the data presented here provide essential evidence to ascribe a functional developmental role to these proteins. PMID- 16668540 TI - Assessing the degree of c(4) photosynthesis in c(3)-c(4) species using an inhibitor of phosphoenolpyruvate carboxylase. AB - An analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2 propenoate (DCDP), was used to inhibit phosphoenolpyruvate carboxylase and, therefore, assess the contribution of the C(4) pathway to photosynthesis in detached leaves of several C(3)-C(4) intermediate species. There was no effect of 4 millimolar DCDP on apparent photosynthesis or on inhibition of apparent photosynthesis by 210 milliliters per liter of O(2) for the C(3)-C(4) species Panicum milioides, Moricandia arvensis, and Neurachne minor or the C(3) species Flaveria pringlei. However, when leaves of Flaveria linearis (C(3)-C(4)), Flaveria brownii (C(4)-like), and Flaveria trinervia (C(4)) were fed 4 millimolar DCDP, photosynthesis was reduced 32, 60, and 90%, respectively. Photosynthetic inhibition by 210 milliliter per liter of O(2) was also significantly increased in 4 millimolar DCDP-fed leaves of F. linearis and F. brownii but not in F. trinervia when compared with control values. These results with DCDP clearly demarcate C(3)-C(4) species into species including Panicum, Moricandia, and Neurachne whose reduced photorespiration occurs without any C(4) photosynthetic involvement and species of Flaveria in which C(4) photosynthesis contributes to CO(2) assimilation. PMID- 16668541 TI - Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. under Salinity. AB - Salt tolerant spinach (Spinacia oleracea) and salt sensitive pea (Pisum sativum) plants were exposed to mild salinity under identical growth conditions. In order to compare the ability of the two species for extra- and intracellular solute compartmentation in leaves, various solutes were determined in intercellular washing fluids and in aqueously isolated intact chloroplasts. In pea plants exposed to 100 millimolar NaCl for 14 days, apoplastic salt concentrations in leaflets increased continuously with time up to 204 (Cl(-)) and 87 millimolar (Na(+)), whereas the two ions reached a steady concentration of only 13 and 7 millimolar, respectively, in spinach leaves. In isolated intact chloroplasts from both species, sodium concentrations were not much different, but chloride concentrations were significantly higher in pea than in spinach. Together with data from whole leaf extracts, these measurements permitted an estimation of apoplastic, cytoplasmic, and vacuolar solute concentrations. Sodium and chloride concentration gradients across the tonoplast were rather similar in both species, but spinach was able to maintain much steeper sodium gradients across the plasmamembrane compared with peas. Between day 12 and day 17, concentrations of other inorganic ions in the pea leaf apoplast increased abruptly, indicating the onset of cell disintegration. It is concluded that the differential salt sensitivity of pea and spinach cannot be traced back to a single plant performance. Major differences appear to be the inability of pea to control salt accumulation in the shoot, to maintain steep ion gradients across the leaf cell plasmalemma, and to synthesize compatible solutes. Perhaps less important is a lower selectivity of pea for K(+)/Na(+) and NO(3) (-)/Cl(-) uptake by roots. PMID- 16668542 TI - Environmental Control of Phosphoenolpyruvate Carboxylase Induction in Mature Mesembryanthemum crystallinum L. AB - Mesembryanthemum crystallinum L. plants shift the mode of carbon assimilation from C(3) to Crassulacean acid metabolism when stressed by high salinity. A prerequisite for Crassulacean acid metabolism induction is the synthesis of phosphoenolpyruvate carboxylase (PEPCase). A moderate increase in the abundance of PEPCase transcripts and activity is observed in 7-week-old, well-watered plants. This increase in PEPCase coincides in time with a decrease in the growth rate of the shoots. The steady-state level of PEPCase activity is uniform along the leaves of well-watered plants, as can be shown by comparing leaves of different age from individual 7-week-old plants. In contrast, the rate of induction in response to salt stress varies with the age of plants and to a lesser extent with the age of the leaves. Two-week-old seedlings induce PEPCase slowly under a moderate salt stress regimen, whereas older plants induce faster. When individual leaves from a seven-week-old plant are compared with respect to induction velocity, no clear-cut correlation with leaf age is apparent. The highest induction rate is observed in leaves from node five that are about 2 weeks old at the beginning of the experiment. PEPCase transcripts are readily down-regulated to minute levels when detached leaves are hydrated. The levels reached after 8 hours of rehydration are very similar, regardless of whether the leaves were cut from young or old plants or whether the plants were previously salt-stressed or well-watered. It is concluded that environmental rather than developmental factors are predominant in determining abundance of PEPCase activity and transcripts in leaves of mature M. crystallinum plants. PMID- 16668543 TI - Purification and Characterization of Chorismate Synthase from Euglena gracilis: Comparison with Chorismate Synthases of Plant and Microbial Origin. AB - Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli. PMID- 16668544 TI - Biochemical Plant Responses to Ozone : II. Induction of Stilbene Biosynthesis in Scots Pine (Pinus sylvestris L.) Seedlings. AB - Formation of the stilbenes pinosylvin and pinosylvin 3-methyl ether, as well as the activity of the biosynthetic enzyme stilbene synthase (pinosylvin-forming), were induced several hundred- to thousandfold in primary needles of 6-week-old pine (Pinus sylvestris L.) seedlings upon exposure to a single pulse of ozone of at least 0.15 microliters per liter. The seedlings required 4 hours of exposure as a minimum for the induction of stilbene biosynthesis when exposed to 0.2 microliters per liter ozone. Both stilbene synthase activity and stilbene accumulation increased with the duration of ozone treatment. The activity of phenylalanine ammonia-lyase and the activity of chalcone synthase, a key enzyme of the flavonoid pathway that uses the same substrates as stilbene synthase, were also stimulated about twofold by ozone. Stilbene biosynthesis appears to represent the first example of a dose-dependent biochemical response to ozone in a conifer species and may serve as a useful biomarker to study stress impacts on pine trees. PMID- 16668545 TI - Photosynthetic Characteristics of Rice Leaves Aged under Different Irradiances from Full Expansion through Senescence. AB - Effects of irradiance on photosynthetic characteristics were examined in senescent leaves of rice (Oryza sativa L.). Two irradiance treatments (100 and 20% natural sunlight) were imposed after the full expansion of the 13th leaf through senescence. The photosynthetic rate was measured as a function of intercellular CO(2) pressure with a gas-exchange system. The amounts of cytochrome f, coupling factor 1, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and chlorophyll were determined. The coupling factor 1 and cytochrome f contents decreased rapidly during senescence, and their rates of decrease were much faster from the 20% sunlight treatment than from the full sunlight treatment. These changes were well correlated with those in the photosynthetic rate at CO(2) pressure = 600 microbars, but not with those under the ambient air condition (350 microbars CO(2)) and 200 microbars CO(2). This suggested that the amounts of coupling factor 1 and cytochrome f from the full sunlight treatment cannot be limiting factors for the photosynthetic rate at ambient air conditions. The Rubisco content also decreased during senescence, but its decrease from the 20% sunlight treatment was appreciably retarded. However, this difference was not reflected in the photosynthetic rates at the ambient and 200 microbars CO(2). This implied that in vivo Rubisco activity may be regulated in the senescent leaves from the 20% sunlight treatment. The chlorophyll content decreased most slowly. In the 20% sunlight treatment, it remained apparently constant with a decline in chlorophyll a/b ratio. These photosynthetic characteristics of the senescent rice leaves under low irradiance were discussed in relation to acclimation of shade plants. PMID- 16668546 TI - Immunological characterization of two dominant tonoplast polypeptides. AB - At least 14 distinct polypeptides reside in the tonoplast of barley (Hordeum vulgare) mesophyll vacuoles. Two of the polypeptides were isolated from two dimensional separations of vacuoplast membrane proteins and used for immunization. With the antisera, the localization on the membrane and the distribution of the polypeptides in the plant kingdom and in various tissues of barley plants was studied. The polypeptides have an apparent molecular mass of 31 and 40 kilodaltons. After freeze-thaw cycles or washing of the membranes with 4.5 millimolar NaCl, the polypeptides were still sedimented with the membranes, suggesting an intrinsic localization. The antiserum against the 31-kilodalton polypeptide bound to the outer surface of isolated intact vacuoles. In chromatographic separations of Triton X-100-solubilized membrane fractions, the residual activities of various acid hydrolases eluted distinct from the 31- and 40-kilodalton polypeptides. Both polypeptides tend to form larger aggregates, however smaller than the tonoplast ATPase. Cross-reactive polypeptides were present in higher and lower plants (the green alga Chara corallina and the liverwort Conocephalum) and in liver tissue from rat and beef, but were not detected in other animal tissues tested so far. The results indicate a wide distribution of these tonoplast polypeptides in vacuole-containing organisms. PMID- 16668547 TI - Role of the root apoplasm for iron acquisition by wheat plants. AB - The role of the root apoplasm for iron acquisition was studied in wheat (Triticum aestivum L. cv Ares) grown in nutrient solution under controlled environmental conditions. To obtain different levels of Fe in the root apoplasm, plants were supplied in the dark for 5 hours (preloading period) with various (59)Fe-labeled Fe compounds [Fe(III) hydroxide; microbial siderophores: Fe rhodotorulic acid (FeRDA) and ferrioxamin (FeDesferal(3)), and synthetic Fe chelate (FeEDDHA)], each at a concentration of 5 micromolar. Large pools of apoplasmic Fe were formed after supplying Fe(III) hydroxide or FeRDA, but no such pools were observed after supplying FeDesferal or FeEDDHA. Depending on plant Fe nutritional status (preculture +/- 0.1 millimolar FeEDTA), apoplasmic Fe was used to different extent for translocation to the shoot. Under Fe deficiency, a much greater fraction of the apoplasmic Fe was utilized than in Fe-sufficient plants, as a result of the different rates of phytosiderophore release. Because of the diurnal rhythm in release of phytosiderophores in Fe-deficient plants, the utilization of the apoplasmic Fe for translocation into the shoot started 2 hours after onset of the light period and was dependent on the concentration of Fe in the apoplasm, which followed the order: Fe(III) hydroxide >> FeRDA >> FeDesferal = FeEDDHA. From these results, it can be concluded that in soil-grown plants the apoplasmic Fe pool loaded by various indigenous Fe compounds such as siderophores in the soil solution can be an important Fe source in graminaceous species, particularly during periods of limited Fe supply from the soil. PMID- 16668548 TI - Protein compositions of mesophyll and paraveinal mesophyll of soybean leaves at various developmental stages. AB - Mesophyll and paraveinal mesophyll protoplasts (PVMP) were isolated from leaves of soybean (Glycine max) at various stages of physiological development, and protein compositions of the two protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypeptides of 27, 29 (previously shown to be storage proteins), and 94 kilodaltons were found to be PVMP-specific proteins and were present in both nodulated and nonnodulated plants. The 27 and 94 kilodalton polypeptides were major PVMP constituents. All three polypeptides accumulate as early as one-quarter leaf expansion. Immunoblotting and immunocytochemical studies using antibodies against the 27/29 kilodalton proteins confirmed that they are specific to the paraveinal mesophyll (PVM) and that they are localized in the PVM vacuole. The 27 kilodalton polypeptide increased significantly by two weeks depodding, and this accumulation was restricted to the PVM vacuole. Radiolabeling experiments showed that the difference in relative amounts of the 27 and 29 kilodalton polypeptides was due to a greater rate of synthesis of the 27 kilodalton polypeptide. The 94 kilodalton polypeptide accumulated to a maximum at anthesis, but was absent at 2 weeks postanthesis in both depodded and podded nodulated plants, probably because they were nitrogen limited. In nonnodulated plants, it was present through 2 weeks postanthesis. The results confirm that the 27 and 29 kilodalton proteins of soybean leaf are stored in the PVM vacuole and show that they are accumulated early during leaf development while they are still strong sinks for nitrogen. The 94 kilodalton protein, previously found to accumulate in leaves after depodding, is also a PVM protein and is likely a third vegetative storage protein, although its accumulation appears to be more dependent on excess nitrogen availability. The results further support the hypothesis that the PVM is a specialized leaf tissue that functions in synthesis and compartmentation of storage proteins. PMID- 16668549 TI - Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria. AB - Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the beta form of NADH. The other two activities, peaks II and III, oxidize only beta-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca(2+) or Mg(2+). Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca(2+) and Mg(2+). As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca(2+) and Mg(2+), but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase. PMID- 16668550 TI - Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities. AB - We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I homoserine dehydrogenase I. PMID- 16668551 TI - Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans. AB - The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP. PMID- 16668552 TI - Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase. AB - Leucoplast pyruvate kinase (PK(p); EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4 degrees C. Purified castor seed PK(p) was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK(p)) immunoglobulin G. Proteolytic activity against PK(p) was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4 degrees C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK(p). The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nalpha-p-tosyl l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK(p). This endopeptidase is present throughout all stages of endosperm development. Its PK(p)-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4 degrees C for up to 21 hours, no degradation of PK(p) was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK(p) remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK(p). PMID- 16668553 TI - Imidazolinone-induced loss of acetohydroxyacid synthase activity in maize is not due to the enzyme degradation. AB - Acetohydroxyacid synthase (AHAS), the first enzyme leading to the biosynthesis of valine, leucine, and isoleucine, is inhibited by different chemical classes of herbicides. There is a loss in the extractable AHAS activity in imidazolinone treated plants. Immunological studies using a monoclonal antibody against AHAS revealed no degradation of AHAS protein in imidazolinone-treated maize (Zea mays) plants. Therefore, the loss in AHAS activity is not due to the loss of AHAS protein. PMID- 16668554 TI - Systemic Induction of Salicylic Acid Accumulation in Cucumber after Inoculation with Pseudomonas syringae pv syringae. AB - Inoculation of one true leaf of cucumber (Cucumis sativus L.) plants with Pseudomonas syringae pathovar syringae results in the systemic appearance of salicylic acid in the phloem exudates from petioles above, below, and at the site of inoculation. Analysis of phloem exudates from the petioles of leaves 1 and 2 demonstrated that the earliest increases in salicylic acid occurred 8 hours after inoculation of leaf 1 in leaf 1 and 12 hours after inoculation of leaf 1 in leaf 2. Detaching leaf 1 at intervals after inoculation demonstrated that leaf 1 must remain attached for only 4 hours after inoculation to result in the systemic accumulation of salicylic acid. Because the levels of salicylic acid in phloem exudates from leaf 1 did not increase to detectable levels until at least 8 hours after inoculation with P. s. pathovar syringae, the induction of increased levels of salicylic acid throughout the plant are presumably the result of another chemical signal generated from leaf 1 within 4 hours after inoculation. Injection of salicylic acid into tissues at concentrations found in the exudates induced resistance to disease and increased peroxidase activity. Our results support a role for salicylic acid as an endogenous inducer of resistance, but our data also suggest that salicylic acid is not the primary systemic signal of induced resistance in cucumber. PMID- 16668555 TI - Xylulose 1,5-Bisphosphate Synthesized by Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase during Catalysis Binds to Decarbamylated Enzyme. AB - Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to Rubisco during catalysis with the purified enzyme and from celery (Apium graveolens var Utah) leaf extracts. XuBP does not bind tightly to carbamylated sites, but does bind tightly to decarbamylated sites. Upon incubation of fully activated Rubisco with 5 micromolar XuBP, loss of activator CO(2) occurred before XuBP bound to the enzyme catalytic sites, even in the presence of excess CO(2) and Mg(2+). Binding of XuBP to decarbamylated Rubisco sites was highly pH dependent. At pH 7.0 and 7.5 with 10 millimolar MgCl(2) and 10 millimolar KHCO(3), the apparent dissociation constant for XuBP, K(d), was 0.03 micromolar, whereas at pH 8.0 and 8.5, the apparent K(d) was 0.35 and 2.0 micromolar, respectively. This increase in K(d) with pH was a result of a decrease in the association rate constant and an increase in the dissociation rate constant of XuBP bound to decarbamylated sites on Rubisco. The K(d) of 2-carboxyarabinitol 1-phosphate binding to carbamylated sites was only slightly pH dependent. PMID- 16668556 TI - Fallover of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity : Decarbamylation of Catalytic Sites Depends on pH. AB - Loss of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity during CO(2) fixation, called fallover, occurred with or without loss of activator CO(2) from catalytic sites depending on pH. At pH 7.5, but not at pH 8.5, the fraction of Rubisco sites that were carbamylated decreased during fallover. Inhibitors which formed during fallover were identified following NaBH(4) reduction and separation of the products by high performance anion-exchange chromatography and pulsed amperometric detection. They were xylulose 1,5-bisphosphate (XuBP) and 3 ketoarabinitol 1,5-bisphosphate. During fallover at pH 8.5, 3-ketoarabinitol-P(2) was the only inhibitor binding to Rubisco and this binding was at carbamylated sites, although both inhibitors were made. At pH 7.5, both inhibitors were bound to catalytic sites of Rubisco with XuBP bound tightly to decarbamylated sites, whereas 3-ketoarabinitol-P(2) bound to carbamylated sites. The pH during fallover also influenced the ratio of 3-ketoarabinitol-P(2) to XuBP formed. When fallover occurred at pH 7.5, both the formation of XuBP and its binding affinity to decarbamylated Rubisco sites were increased compared with those at pH 8.5. 3 Ketoribitol-P(2) was not found at either pH. PMID- 16668557 TI - [C]GA(12)-Aldehyde, [C]GA(12), and [H]- and [C]GA(53) Metabolism by Elongating Pea Pericarp. AB - Gibberellins (GAs) are either required for, or at least promote, the growth of the pea (Pisum sativum L.) fruit. Whether the pericarp of the pea fruit produces GAs in situ and/or whether GAs are transported into the pericarp from the developing seeds or maternal plant is currently unknown. The objective of this research was to investigate whether the pericarp tissue contains enzymes capable of metabolizing GAs from [(14)C]GA(12)-7-aldehyde ([(14)C]GA(12)ald) to biologically active GAs. The metabolism of GAs early in the biosynthetic pathway, [(14)C]GA(12) and [(14)C]GA(12)ald, was investigated in pericarp tissue isolated from 4-day-old pea fruits. [(14)C]GA(12)ald was metabolized primarily to [(14)C]GA(12)ald-conjugate, [(14)C]GA(12), [(14)C]GA(53), and polar conjugate like products by isolated pericarp. In contrast, [(14)C]GA(12) was converted primarily to [(14)C]GA(53) and polar conjugate-like products. Upon further investigations with intact 4-day-old fruits on the plant, [(14)C]GA(12) was found to be converted to a product which copurified with endogenous GA(20). Lastly, [(2)H]GA(20) and [(2)H]GA(1) were recovered 48 hours after application of [(2)H]- and [(14)C]GA(53) to pericarp tissue of intact 3-day-old pea fruits. These results demonstrate that pericarp tissue metabolizes GAs and suggests a function for pericarp GA metabolism during fruit growth. PMID- 16668558 TI - Organ-Specific and Environmentally Regulated Expression of Two Abscisic Acid Induced Genes of Tomato : Nucleotide Sequence and Analysis of the Corresponding cDNAs. AB - The cDNAs, pLE4 and pLE25, represent mRNAs that accumulate in response to water deficit and elevated levels of endogenous abscisic acid in detached leaves of drought-stressed tomato (Lycopersicon esculentum Mill., cv Ailsa Craig) (A Cohen, EA Bray [1990] Planta 182: 27-33). DNA sequence analysis of pLE4 and pLE25 showed that the deduced polypeptides were 13.9 and 9.3 kilodaltons, respectively. Each polypeptide was hydrophilic, cysteine- and tryptophan-free, and found to be similar to previously identified proteins that accumulate during the late stages of embryogenesis. pLE4 and pLE25 mRNA accumulated in a similar organ-specific pattern in response to specific abiotic stresses. Yet, expression patterns of the corresponding genes in response to developmental cues were not similar. pLE25 mRNA accumulated to much higher levels in developing seeds than in drought stressed vegetative organs. pLE4 mRNA accumulated predominantly in drought stressed leaves. The similarities and differences in the accumulation characteristics of these two mRNAs indicates that more than one mechanism exists for the regulation of their corresponding genes. PMID- 16668559 TI - Nitrogen metabolism in the stalk tissue of maize. AB - During ear development in maize (Zea mays L.), nitrogenous compounds are translocated from vegetative organs to the kernels. At anthesis, the stalk contains approximately 40% of the total plant N, and contributes 45% of the N remobilized to the ear. Therefore, the stalk has an important function as a temporary reservoir for N. Little is known of the metabolism of maize stalks, and this paper describes initial studies of enzymes of N metabolism. High in vitro activity of glutamine synthetase (GS) in maize stalk samples throughout ear development contrasted with a peak in activity of glutamate synthase soon after anthesis and negligible nitrate reductase. With fresh sections of stalk tissue collected at anthesis, (15)N-feeding experiments confirmed high GS and low nitrate reductase activities. Two isoforms of GS were separated from extracts from stalk tissue: GS1, the cytoplasmic form, increased to maximum levels at 2 weeks postanthesis and remained fairly high thereafter; whereas the plastidic form, GS2, declined progressively during kernel development. Western blot analysis confirmed the presence of constantly high levels of GS protein after anthesis. The levels of GS proteins decreased after transfer of N-starved, hydroponically grown plants to N-rich conditions in order to restrict remobilization of N. In contrast, transfer of plants grown under abundant N conditions to N-free medium, which encourages N remobilization, resulted in a relative increase in GS protein. Because glutamine is the major form of N transported in maize, the results indicate that GS, specifically the GS1 isoform, has a central role in the remobilization on nitrogenous compounds from the stalk to the ear. PMID- 16668560 TI - Mitochondrial malate dehydrogenase from corn : purification of multiple forms. AB - A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels. PMID- 16668561 TI - Effects of O(2) and CO(2) Concentrations on Quantum Yields of Photosystems I and II in Tobacco Leaf Tissue. AB - The interactive effects of irradiance and O(2) and CO(2) levels on the quantum yields of photosystems I and II have been studied under steady-state conditions at 25 degrees C in leaf tissue of tobacco (Nicotiana tabacum). Assessment of radiant energy utilization in photosystem II was based on changes in chlorophyll fluorescence yield excited by a weak measuring beam of modulated red light. Independent estimates of photosystem I quantum yield were based on the light-dark in vivo absorbance change at 830 nanometers, the absorption band of P700(+). Normal (i.e. 20.5%, v/v) levels of O(2) generally enhanced photosystem II quantum yield relative to that measured under 1.6% O(2) as the irradiance approached saturation. Photorespiration is suspected to mediate such positive effects of O(2) through increases in the availability of CO(2) and recycling of orthophosphate. Conversely, at low intercellular CO(2) concentrations, 41.2% O(2) was associated with lower photosystem II quantum yield compared with that observed at 20.5% O(2). Inhibitory effects of 41.2% O(2) may occur in response to negative feedback on photosystem II arising from a build-up in the thylakoid proton gradient during electron transport to O(2). Covariation between quantum yields of photosystems I and II was not affected by concentrations of either O(2) or CO(2). The dependence of quantum yield of electron transport to CO(2) measured by gas exchange upon photosystem II quantum yield as determined by fluorescence was unaffected by CO(2) concentration. PMID- 16668562 TI - Expression of Maize Ferredoxin cDNA in Escherichia coli: Comparison of Photosynthetic and Nonphotosynthetic Ferredoxin Isoproteins and their Chimeric Molecule. AB - Maize (Zea mays L.) has two types of ferredoxin (Fd) differentially expressed in photosynthetic and nonphotosynthetic organs. A cDNA fragment encoding the mature polypeptide of Fd III, an Fd isoprotein of the nonphotosynthetic type, was expressed in Escherichia coli, and the Fd was synthesized as a holo-form assembled with the [2Fe-2S] cluster, which was completely identical with authentic Fd III prepared from maize roots. This expression system made it possible to prepare Fd present at fairly low levels in plants in amounts sufficient for functional and structural studies. Comparison of electron transfer activity of Fd III with that of Fd I, an Fd isoprotein of the photosynthetic type, showed that Fd III was superior as an electron acceptor from NADPH, and Fd I was superior as an electron donor for NADP(+), in reactions catalyzed by Fd NADP(+) reductase from maize leaf. The circular dichronism spectra of the two Fds also indicated a subtle difference in the geometry of their iron-sulfur clusters. These results are consistent with the view that photosynthetic and nonphotosynthetic Fds may be functionally differentiated. An artificial chimeric Fd, Fd III/Fd I, whose amino-terminal and carboxylterminal halves are derived from the corresponding regions of Fd III and Fd I, respectively, showed an activity and CD spectrum significantly similar to those of Fd I. This suggests that 18 amino acid substitutions between Fd III and Fd III/Fd I alter the properties of Fd III so that they resemble those of Fd I. PMID- 16668563 TI - Three RNases in Senescent and Nonsenescent Wheat Leaves : Characterization by Activity Staining in Sodium Dodecyl Sulfate-Polyacrylamide Gels. AB - We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WL(A)), and two apparently novel enzymes, designated RNases WL(B) and WL(C). RNase WL(B) activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl(2). RNase WL(C) activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl(2), and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WL(A), WL(B), and WL(C), which are present in senescent and nonsenescent leaves, during the course of leaf senescence. PMID- 16668564 TI - Expression of Three RNase Activities during Natural and Dark-Induced Senescence of Wheat Leaves. AB - We have monitored the activities of RNases WL(A), WL(B), and WL(C) (A Blank, TA McKeon [1991] Plant Physiol 97: 1402-1408) during leaf senescence in wheat (Triticum aestivum L. cv Chinese Spring). When seedlings were induced to senesce in darkness, protein loss from primary leaves began immediately. RNase WL(B) activity was unchanged for 2 days and then rose linearly, reaching a sixfold elevation in 7 days. RNase WL(C) activity declined for 2 days and then rose linearly, reaching a twofold elevation in 7 days. RNase WL(A) activity declined in the first 2 days and was unchanged thereafter. Although differentially expressed, these RNase activities may respond to a common regulatory mechanism(s) which, at 2 days of darkness, signals progression into a more advanced stage of senescence. The RNase activities were also differentially expressed during light induced recovery, returning to normal levels in dissimilar patterns. In flag leaves of greenhouse-grown wheat, the three RNase activities increased during the early postanthesis period when protein content was stable and underwent further, accelerated accumulation during senescence. RNase WL(B) activity showed the largest overall senescence-associated elevation (sixfold), followed by RNase WL(C) (fourfold) and RNase WL(A) (threefold). PMID- 16668565 TI - Sugar-Dependent Expression of the CHS-A Gene for Chalcone Synthase from Petunia in Transgenic Arabidopsis. AB - Transgenic Arabidopsis thaliana plants were constructed by introduction of a fusion of the gene for beta-glucuronidase (GUS) to the CHS-A gene, which is one of the two genes for chalcone synthase that are actively expressed in the floral organs of petunia. The expression of the fusion gene CHS-A::GUS was low in transgenic Arabidopsis plantlets, but it was enhanced when plantlets or detached leaves were transferred to a medium that contained 0.3 molar sucrose, glucose, or fructose. No enhancement was observed when plantlets were transferred to a medium that contained 0.3 molar mannitol. Measurements of cellular levels of sugars revealed a tight linkage between the level of expression of the CHS-A::GUS gene and the level of accumulation of exogenously supplied sugars, in particular sucrose. The parallelism between the organ-specific accumulation of sugar and the organ-specific expression of the CHS-A::GUS gene was also observed in petunia and A. thaliana plants grown under normal conditions in soil. The consensus sequences for sugar responses, such as boxes II and III in members of the family of sporamin genes from the sweet potato, were found in the promoter region of the CHS-A gene that was used for fusion to the GUS gene. It is suggested that the expression of the CHS-A gene is regulated by sugars, as is the expression of other sugar-responsive genes, such as the genes for sporamin. A putative common mechanism for the control of expression of "sugar-related" genes, including the CHS-A gene, is discussed. PMID- 16668566 TI - Turnover of catalase heme and apoprotein moieties in cotyledons of sunflower seedlings. AB - The turnover of catalase apoprotein and catalase heme was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings by density labeling of apoprotein and radioactive labeling of heme moieties. The heavy isotope (50% (2)H(2)O) and the radioactive isotope ([(14)C]5-aminolevulinic acid) were applied either during growth in the dark (day 0-2.5) or in the light (day 2.5 and 5). Following isopycnic centrifugation of catalase purified from cotyledons of 5-day-old seedlings, superimposition curve fitting was used to determine the amounts of radioactive heme moieties in native and density-labeled catalase. Data from these determinations indicated that turnover of catalase heme and apoprotein essentially was coordinate. Only small amounts of heme groups were recycled into newly synthesized apoprotein during growth in the light, and no evidence was found for an exchange of heme groups in apoprotein moieties. It followed from these observations that degradation of catalase apoprotein was slightly faster than that of catalase heme. A degradation constant for catalase apoprotein of 0.263 per day was determined from the data on heme recycling and the degradation constant of catalase heme determined previously to be 0.205 per day (R Eising, B Gerhardt [1987] Plant Physiol 84: 225-232). PMID- 16668567 TI - Heat Shock Proteins in Two Lines of Zea mays L. That Differ in Drought and Heat Resistance. AB - Synthesis of heat shock proteins (HSPs) in the leaves of a drought- and heat resistant (line ZPBL 1304), and a drought- and heat-sensitive (line ZPL 389) line of maize (Zea mays L.) was studied under two environmental stress treatments: (a) soil drying and high temperature and (b) high temperature. In the first treatment 13-day-old plants were exposed to 7-day soil drying followed by high temperature stress (45 degrees C), and in the second treatment 20-day-old plants were exposed to high temperature stress (45 degrees C). Second leaves were labeled with [(35)S]methionine. During the labeling period line ZPBL 1304 showed no signs of leaf dehydration under soil drying and high temperature stress conditions. In contrast, line ZPL 389 was dehydrated 23%, as determined by relative water content. Incorporation of [(35)S]methionine into protein was greater in the resistant than in the sensitive line in both treatments. The pattern of synthesis of HSPs in the two lines was similar in treatments 1 and 2. Both lines synthesized a high molecular mass set and a low molecular mass set of HSPs. Proteins from both sets from both lines of maize appeared similar to each other, with respect to the molecular mass. Heated plants of the drought- and heat resistant line ZPBL 1304 synthesized a band of HSP(s) of approximately 45 kilodaltons which was not found in heated plants of the drought and heat sensitive line ZPL 389. This is the first report on qualitative intraspecific difference in the synthesis of HSPs in maize. PMID- 16668568 TI - Role of Ethylene in the Germination of the Hemiparasite Striga hermonthica. AB - Seed germination of the hemiparasitic angiosperm Striga hermonthica (Del.) Benth is elicited by compounds present in the root exudates of the host plant. Although a variety of compounds can substitute for the host-derived signal, the mechanism through which these act is unknown. In the present study, an inhibitor of ethylene biosynthesis, aminoethoxyvinyl glycine, was found to inhibit germination. Addition of an intermediate in ethylene biosynthesis, 1 aminocyclopropane-1-carboxylic acid, was found to override this inhibition and to act as a substitute for the host-derived signal. 2,5-Norbornadiene, an inhibitor of ethylene action, was also found to inhibit germination. Ethylene is rapidly produced by Striga seeds after treatment with host root exudates. These results are consistent with a model for Striga seed germination in which host-derived signals and other compounds act by eliciting the synthesis of ethylene and in which ethylene itself initiates the biochemical changes leading to germination. PMID- 16668569 TI - Utilization of Inorganic Carbon by Ulva lactuca. AB - Thalli discs of the marine macroalga Ulva lactuca were given inorganic carbon in the form of HCO(3) (-), and the progression of photosynthetic O(2) evolution was followed and compared with predicted O(2) evolution as based on calculated external formation of CO(2) (extracellular carbonic anhydrase was not present in this species) and its carboxylation (according to the K(m)(CO(2)) of ribulose-1,5 bisphosphate carboxylase/oxygenase), at two different pHs, assuming a photosynthetic quotient of 1. The K(m)(inorganic carbon) was some 2.5 times lower at pH 5.6 than at the natural seawater pH of 8.2, whereas V(max) was similar under the two conditions, indicating that the unnaturally low pH per se had no adverse effect on U. lactuca's photosynthetic performance. These results, therefore, could be evaluated with regard to differential CO(2) and HCO(3) (-) utilization. The photosynthetic performance observed at the lower pH largely followed that predicted, with a slight discrepancy probably reflecting a minor diffusion barrier to CO(2) uptake. At pH 8.2, however, dehydration rates were too slow to supply CO(2) for the measured photosynthetic response. Given the absence of external carbonic anhydrase activity, this finding supports the view that HCO(3) (-) transport provides higher than external concentrations of CO(2) at the ribulose-1,5-bisphosphate carboxylase/oxygenase site. Uptake of HCO(3) (-) by U. lactuca was further indicated by the effects of potential inhibitors at pH 8.2. The alleged band 3 membrane anion exchange protein inhibitor 4,4' diisothiocyanostilbene-2,2'disulphonate reduced photosynthetic rates only when HCO(3) (-) (but not CO(2)) could be the extracellular inorganic carbon form taken up. A similar, but less drastic, HCO(3) (-)-competitive inhibition of photosynthesis was obtained with Kl and KNO(3). It is suggested that, under ambient conditions, HCO(3) (-) is transported into cells at defined sites either via facilitated diffusion or active uptake, and that such transport is the basis for elevated internal [CO(2)] at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation. PMID- 16668570 TI - Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan. AB - Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells. PMID- 16668571 TI - Cell Wall Metabolism in Ripening Fruit : V. Analysis of Cell Wall Synthesis in Ripening Tomato Pericarp Tissue Using a d-[U-C]Glucose Tracer and Gas Chromatography-Mass Spectrometry. AB - A gas chromatographic-mass spectrometric technique utilizing d-[U-(13)C]glucose as a density label tracer was used to follow the synthesis of cell wall polysaccharides in pericarp discs that were excised from mature green tomato fruit (Lycopersicon esculentum) and allowed to ripen in culture. The biosynthetic capacity of discs from four different maturity stages was examined. Label was differentially incorporated into wall polysaccharides as the discs matured, indicating a change in the nature of wall polymers being synthesized. These differential rates of incorporation are consistent with descriptions of ripening related cell wall compositional changes previously reported by other authors. Specific changes in wall biosynthesis noted include increased incorporation of xylosyl and mannosyl residues into hemicellulosic cell wall fractions as the discs mature and decreased incorporation of galactosyl residues into chelator soluble pectins. PMID- 16668572 TI - Isolation and characterization of a tomato Acid phosphatase complementary DNA associated with nematode resistance. AB - The tomato (Lycopersicon esculentum) acid phosphatase-1 (Apase-1(1), EC 3.1.3.2) isozyme variant, genetically linked to the root-knot nematode resistance locus (Mi) on chromosome 6, has been purified by a rapid procedure from tomato cell suspension cultures. Peptide fragments of the purified enzyme were generated from trypsin and Lys-C endoprotease digests and separated by reverse-phase high performance liquid chromatography. Amino acid sequences derived from the purified peptide fragments represented >50% of the total amino acid content of the protein and enabled the construction of degenerate oligonucleotide probes that were used to screen a tomato cell culture complementary DNA library. Clones corresponding to full-length coding sequences for Apase-1 have been isolated and sequenced. Southern blot analysis of DNA isolated from a number of tomato cultivars shows that the Apase-1(1) gene (aps1) is present at one copy per genome and that genotypes containing the aps1(1) allele have restriction fragment length polymorphisms that distinguish them from cultivars having the aps1(+) allele. Segregation analysis demonstrates that the restriction fragment length polymorphisms are associated with the aps1 locus. Tomato Apase-1(1) is also found to have significant homology at the amino acid sequence level to a class of vegetative storage proteins characterized in soybean. PMID- 16668573 TI - Effect of Light and NO(3) on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity: Evidence for Covalent Modulation of the C(3) Enzyme. AB - Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO(3) (-) leaves) or 40 millimolar KNO(3) (high-NO(3) (-) leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO(3) (-). In contrast, the lower rate of increase recorded for low-NO(3) (-) leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short term NO(3) (-) effect increased linearly with the level of NO(3) (-) uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO(3) (-) leaves, illuminated high-NO(3) ( ) leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, (32)P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO(3) (-) conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO(3) (-), an interpretation of the role of NO(3) (-) as either an inhibitor of P protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO(3) (-), the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO(3) (-) modulates the relative protein kinase/protein phosphatase ratio to favor increased phosphorylation of both enzymes in order to redirect carbon flow away from sucrose synthesis and toward amino acid synthesis. PMID- 16668574 TI - Vacuolar Release of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid, the Conjugated Form of the Ethylene Precursor. AB - The mechanisms underlying the vacuolar retention or release of 1 (malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, has been studied in grape (Vitis vinifera) cells grown in vitro using the technique of compartmental analysis of radioisotope elution. Following its accumulation in the vacuole, M[2,3-(14)C]ACC could be released from cells when the vacuolar pH was artificially lowered by external buffers from its initial value of 6.2 to below the critical pH of 5.5. Successive release and retention of vacuolar MACC could be achieved by switching the vacuolar pH from values lower and higher than 5.5. The rate constant of efflux was highly correlated with the vacuolar pH. In plant tissues having low vacuolar pH under natural conditions, e.g. apple fruits (pH 4.2) and mung bean hypocotyls (pH 5.3), an efflux of M[2,3-(14)C]ACC also occurred. Its rate constant closely corresponded to the theorical values derived from the correlation established for grape cells. Evidence is presented that the efflux proceeded by passive lipophilic membrane diffusion only when MACC was in the protonated form. In contrast to other organic anions like malic acid, the mono and diionic species could not permeate the tonoplast, thus indicating the strict dependence of MACC retention upon the ionic status of the molecule and the absence of carrier mediated efflux. PMID- 16668575 TI - Identification and Properties of the Major Ribonucleases of Arabidopsis thaliana. AB - The profile of major ribonuclease (RNase) activities of Arabidopsis thaliana has been identified and characterized using a substrate-based gel assay. Following sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as many as 16 RNases, varying in size from 9 to 41 kilodaltons can be detected. Most of the RNase activities exhibit a pH optimum of about 6.5; however, the activity of a 22.6 kilodalton RNase is greatly enhanced at low pH. A number of the RNases in the 30- to 41-kilodalton range are sensitive to ethylenediaminetetraacetic acid, and their activities are enhanced by the presence of a low concentration of zinc during renaturation. At least one RNase appears to comigrate with a major DNase activity. The differential accumulation of several RNases in stems versus leaves indicates that some RNases are controlled in an organ-specific manner in A. thaliana. PMID- 16668576 TI - Phenylalanine Ammonia-Lyase from Tomato Cell Cultures Inoculated with Verticillium albo-atrum. AB - Tomato (Lycopersicon esculentum Mill.) cell suspension cultures accumulated wall bound phenolic materials in response to inoculation with Verticillium albo-atrum Reinke et Berth. in a fashion analogous to that observed in whole plants. Both monomeric and polymeric materials were recovered. Deposition of phenolics into the cell walls of inoculated tomato cell cultures was inhibited by the phenylalanine ammonia-lyase (PAL) inhibitor, 2-amino-2-indanephosphate. Tomato PAL activity was induced over 12-fold by fungal inoculation, with a concomitant increase in the corresponding mRNA. The enzyme was purified >3400-fold, to apparent homogeneity, by anion-exchange chromatography, chromatofocusing, and gel filtration. The holoenzyme had a molecular mass of 280 to 320 kilodaltons, comprising 74-kilodalton subunits, and displayed an isoelectric point of 5.6 to 5.7. Induced PAL displayed apparent Michaelis-Menten kinetics (K(m) = 116 micromolar) and was not appreciably inhibited by its product cinnamic acid. Chromatographic analysis did not reveal multiple forms of the enzyme in either inoculated or uninoculated cultures. PMID- 16668577 TI - Influence of Protoplasmic Water Loss on the Control of Protein Synthesis in the Desiccation-Tolerant Moss Tortula ruralis: Ramifications for a Repair-Based Mechanism of Desiccation Tolerance. AB - Desiccation tolerance of the moss Tortula ruralis is characterized by a desiccation-induced change in gene expression that becomes evident upon rehydration. As reported earlier, this change in gene expression is apparently brought about by a change in the control of translation and does not include a major shift in mRNA abundance. A full qualitative and quantitative analysis of the alteration in gene expression, which is characterized by the loss of (or greater than fivefold decrease in) the synthesis of 25 hydration (h) proteins and initiation (or greater than fivefold increase) of the synthesis of 74 rehydration (r) proteins, is given in this report. Exposure to a desiccating atmosphere, for times that result in varying levels of water loss, enabled the determination that the control of synthesis of r proteins is different from the control of synthesis of h proteins. The r and h protein synthesis responses are internally coordinate, however. Similarly, the return to normal levels of h protein synthesis differs from that of the r proteins. The return to normal synthetic levels for all h proteins is synchronous, but the rate of loss of r protein synthesis varies with each individual r protein. Run-off translation of polysomes isolated from gametophytes during the drying phase demonstrates that there are no novel mRNAs recruited and no particular mRNA is favored for translation during desiccation. These findings add credence to the argument that translational control is the major component of the desiccation-induced alteration in gene expression in this plant, as discussed. Aspects of the response of protein synthesis to desiccation are consistent with the hypothesis that T. ruralis exhibits a repair-based mechanism of desiccation tolerance. PMID- 16668578 TI - Methyl jasmonate treatment eliminates cell-specific expression of vegetative storage protein genes in soybean leaves. AB - Soybean (Glycine max) plants accumulate a vacuolar glycoprotein in the parenchymal cells of leaves, petioles, stems, seed pods, and germinating cotyledons that acts in temporary nitrogen storage during vegetative growth. In situ immunolocalization of this vegetative storage protein (VSP) revealed that it accumulates in those parenchymal cells in close proximity to existing and developing vasculature, as well as in epidermal and cortical cells. The protein was more prevalent in younger, nitrogen-importing tissues before pod and seed development. Removal of actively growing seed pods greatly enhanced VSP accumulation, primarily in bundle sheath and paraveinal mesophyll cells. In situ hybridization of a VSP RNA probe to mRNA in leaf sections demonstrated that cell specific mRNA accumulation corresponded with the pattern of protein localization. Treatment of leaf explants with 50 micromolar methyl jasmonate resulted in accumulation of VSP mRNA and protein in all cell types. PMID- 16668579 TI - Identification of Gibberellins in Spinach and Effects of Light and Darkness on their Levels. AB - The endogenous gibberellin (GA) content of spinach (Spinacia oleracea) was reinvestigated by combined gas chromatography-mass spectrometry analysis. The 13 hydroxy GAs: GA(53), GA(44), GA(19), GA(17), GA(20), GA(5), GA(1), GA(29), and GA(8); the non-3, 13-hydroxy GAs: GA(12), GA(15), GA(9), and GA(51); and the 3beta-hydroxy GAs: GA(4), GA(7), and GA(34), were identified in spinach extracts by comparing full-scan mass spectra and Kovats retention indices with those of reference GAs. In addition, spinach plants contained GA(7)-isolactone, 16,17 dihydro-17-hydroxy-GA(53), GA(29)-catabolite, 3-epi-GA(1), and 10 uncharacterized GAs with mass spectra indicative of mono- and dihydroxy-GA(12), monohydroxy GA(25), dihydroxy-GA(24), and dihydroxy-GA(g). The effect of light-dark conditions on the GA levels of the 13-hydroxylation pathway was studied by using labeled internal standards in selected ion monitoring mode. In short day, the GA levels were higher at the end of the light period than at the end of the dark period. Levels of GAs at the end of each short day were relatively constant. During the first supplementary light period of long day treatment, GA(53) and GA(19) declined dramatically, GA(44) and GA(1) decreased slightly, and GA(20) increased. During the subsequent high-intensity light period, the GA(20) level decreased and the levels of GA(53), GA(44), GA(19), and GA(1) increased slightly. Within 7 days after the beginning of long day treatment, similar patterns for GA(53) and GA(19) occurred. Furthermore, when these plants were transferred to darkness, an increase in the levels of GA(53) and GA(19) was observed. These results are compatible with the idea that in spinach, the flow through the GA biosynthetic pathway is much enhanced during the high-intensity light period, although GA turnover occurs also during the supplementary period of long day, both effects being responsible for the increase of GA(20) and GA(1) in long day. PMID- 16668580 TI - Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels. AB - Ion transport, measured using double-barreled micropipettes to obtain current voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to "drive" cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range -200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques. PMID- 16668581 TI - Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme. AB - Ca(2+)-ATPases keep cytoplasmic [Ca(2+)] low by pumping Ca(2+) into intracellular compartments or out of the cell. The transport properties of Ca(2+)-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca(2+) transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase) partially inhibited oxalate-stimulated Ca(2+) transport activity; however, it had no effect on calmodulin-stimulated Ca(2+) uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca(2+)-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma(32)P]ATP resulted in the formation of a single acyl [(32)P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca(2+), but independent of Mg(2+). Its enhancement by La(3+) is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca(2+) transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca(2+) transport, similar to their effect on the erythrocyte plasma membrane Ca(2+)-ATPase. These results would indicate that the calmodulin-stimulated Ca(2+) transport originated in large part from a plasma membrane-type Ca(2+) pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca(2+)-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested. PMID- 16668582 TI - Alternative respiration and heat evolution in plants. AB - The alternative respiratory pathway dissipates most of the chemical energy of respiratory substrates as heat. We have shown that this heat can be quantified by microcalorimetry and is a measure of alternative pathway activity in vivo. The alternative pathway is known to increase in aged potato (Solanum tuberosum) slices and in chill-stressed leaves. Aging of potato slices for 24 hours was accompanied by an almost fourfold increase in the rate of heat evolution. This heat increase was resistant to KCN but could be blocked by an alternative pathway inhibitor, salicylhydroxamic acid (SHAM). In cucumber (Cucumis sativus) leaves subjected to chilling stress (between 4 and 16 degrees C), the rate of heat evolution was inversely related to temperature. As in aged potato slices, the increased rate of heat evolution in cucumber leaves was blocked by SHAM, but not by KCN. Nitrogen or the combination of SHAM and KCN blocked most of the heat evolution in both aged potato slices and chill-stressed cucumber leaves. Calorimetric measurements of the alternative pathway corresponded to respiration measurements performed using an oxygen electrode. PMID- 16668583 TI - Lidocaine and ATPase inhibitor interaction with the chloroplast envelope. AB - Photosynthetic capacity of isolated intact chloroplasts is known to be sensitive to K(+) fluxes across the chloroplast envelope. However, little is known about the system of chloroplast envelope proteins that regulate this K(+) movement. The research described in this report focused on characterizing some of the components of this transport system by examining inhibitor effects on chloroplast metabolism. Digitoxin, an inhibitor of membrane-bound Na(+)/K(+) ATPases, was found to reduce stromal K(+) at a range of external K(+) and inhibit photosynthesis. Scatchard plot analysis revealed a specific protein receptor site with a K(m) for digitoxin binding of 13 nanomolar. Studies suggested that the receptor site was on the interior of the envelope. The effect of a class of amine anesthetics that are known to be K(+) channel blockers on chloroplast metabolism was also studied. Under conditions that facilitate low stromal pH and concomitant photosynthetic inhibition, the anesthetic, lidocaine, was found to stimulate photosynthesis. This stimulation was associated with the maintenance of higher stromal K(+). Comparison of the effects on photosynthesis of lidocaine analogs which varied in lipophilicity suggested a lipophilic pathway for anesthetic action. The results of experiments with lidocaine and digitoxin were consistent with the hypothesis that a K(+) channel and a K(+)-pumping envelope ATPase contribute to overall K(+) flux across the chloroplast envelope. Under appropriate assay conditions, photosynthetic capacity of isolated chloroplasts was shown to be much affected by the activity of these putative envelope proteins. PMID- 16668584 TI - Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope. AB - Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18 degrees C (optimum at 12 degrees C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves. PMID- 16668585 TI - ADP-Glucose Transport by the Chloroplast Adenylate Translocator Is Linked to Starch Biosynthesis. AB - In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-alpha-d-glucan 4-alpha-d glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol. PMID- 16668586 TI - Patch clamping protoplasts from vascular plants : method for the quick isolation of protoplasts having a high success rate of gigaseal formation. AB - A method is described for the isolation of protoplasts (Pisum sativum, Phaseolus vulgaris, Avena sativa, Arabidopsis thaliana) in preparation for ion flux studies using patch clamp electrophysiology. Protoplasts that have been exposed to hydrolytic, cell wall degrading, enzymes for as little as 5 minutes form gigaseals (seal resistance higher than 10 giga Ohm) with the patch pipette with success rates greater than 40%. Sealing of these protoplasts is fast, averaging less than 2 minutes. This method yields high rates of gigaseal formation in a variety of tissues from both monocots and dicots and will enhance data collection in ion flux studies of plasma membranes of vascular plants. PMID- 16668587 TI - Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii. AB - A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by differential centrifugation and Percoll gradient centrifugation. This procedure generates relatively high yields of chloroplasts capable of CO(2) fixation. These chloroplasts were characterized by electron microscopy, marker enzyme analysis, and ferricyanide exclusion. Transmission electron microscopy indicates that these chloroplasts retain their pyrenoids and eyespots. Scanning electron microscopy confirms that the characteristic cup shape of C. reinhardtii chloroplasts persists in vitro. This rapid, inexpensive procedure produces chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts. PMID- 16668588 TI - Transient down-regulation of phytochrome mRNA abundance in etiolated cucumber cotyledons in response to continuous white light. AB - Phytochrome mRNA abundance decreased to 20% of the initial level in etiolated cucumber (Cucumis sativus L.) cotyledons exposed to continuous white light. Unexpectedly, by 12 hours of continuous white light, phytochrome mRNA had reaccumulated to 60% of the control level. High stringency RNA blot analyses suggest that it is the mRNA encoding type I phytochrome that reaccumulates. PMID- 16668589 TI - Efficiency of particle-bombardment-mediated transformation is influenced by cell cycle stage in synchronized cultured cells of tobacco. AB - Plasmid DNA pB1221 harboring beta-glucuronidase gene was delivered to synchronized cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells of different cell cycle stages by a pneumatic particle gun. The cells bombarded at M and G(2) phases gave 4 to 6 times higher transformation efficiency than those bombarded at the S and G(1) phases. PMID- 16668590 TI - Enzymatic synthesis of isoprene from dimethylallyl diphosphate in aspen leaf extracts. AB - Aspen (Populus tremuloides Michx.) leaf extracts contain a newly discovered enzyme activity that catalyzes the magnesium ion-dependent elimination of diphosphate from dimethylallyl diphosphate with rearrangement to form isoprene (2 methyl, 1-3-butadiene). This isoprene synthase activity has been partially purified. The nonenzymatic reaction of dimethylallyl diphosphate to isoprene, known to be acid catalyzed, may be insignificant at physiological pH. In contrast, the enzymatic reaction may be responsible for the majority of light dependent isoprene production by isoprene-emitting plants. PMID- 16668591 TI - Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis. AB - Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O(2)-resistant photosynthesis. The apparent K(m) for H(2)O(2) was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level. PMID- 16668592 TI - Nucleotide Sequence for the Genomic DNA Encoding the Chloroplast ATP Synthase gamma-Subunit Gene (atpC) from Chlamydomonas reinhardtii. PMID- 16668593 TI - Nucleotide Sequence of a cDNA Clone Encoding a beta-Amylase from Arabidopsis thaliana. PMID- 16668594 TI - Korean Ginseng Mitochondrial DNA Encodes an Intact rps12 Gene Downstream of the nad3 Gene. PMID- 16668595 TI - Nucleotide Sequence of a cDNA Clone Encoding gamma-Coixin from Coix lacryma-jobi Seeds. PMID- 16668596 TI - Nucleotide Sequence of a cDNA Clone Encoding gamma-Kafirin Protein from Sorghum bicolor. PMID- 16668597 TI - Function of Photosynthetic Apparatus of Intact Wheat Leaves under High Light and Heat Stress and Its Relationship with Peroxidation of Thylakoid Lipids. AB - Effects of high light and temperature stress on the structure and function of the photosynthetic apparatus of wheat (Triticum aestivum) were studied. There was a decrease in the electron transport activity of chloroplasts isolated from photoinhibited and heat-stressed leaves. Chlorophyll fluorescence was measured in photoinhibited and heat-stressed leaves and the decrease in variable fluorescence and variable to maximum fluorescence ratio of the stressed leaves indicated a loss in the quantum yield of photosynthesis. The decrease in electron transport activity was accompanied by an increase in peroxidation of thylakoid lipids. Lipid peroxidation indicated the oxidative degradation of polyunsaturated fatty acyl residues of the thylakoid lipids. A negative correlation was observed between electron transport activity and lipid peroxidation. The electron transport activity was completely lost as the peroxidation level reached a threshold equivalent to 0.6 micromoles malondialdehyde. The threshold of lipid peroxidation for complete loss of activity was the same for both photoinhibition and heat treatment, suggesting that the nature of the environmental stress may be less important with respect to the relationship between electron transport and lipid peroxidation. Thus, it seems likely that lipids are required for sustaining the photosynthetic activity under environmental stress, and a loss in activity is observed as the lipids are degraded either by high light or high temperature stress. PMID- 16668598 TI - Auxin transport and the interaction of phytotropins: probing the properties of a phytotropin binding protein. AB - We have described the inhibition of polar auxin transport by several phytotropins including 1-N-naphthylphthalamic acid (NPA) and quercetin. Semicarbazones (substituted phenylsemicarbazones of 2-acetylarylcarboxylic acids) are inhibitors consistent with previously predicted general structural requirements for auxin transport inhibitors. The best semicarbazone derivative tested to date, hereafter called SCB-I, binds to the NPA binding protein with high affinity, K(b) = 4 nanomolar. Quantification of the binding of various phytotropins allows us to make some general statements concerning the structure/properties of the NPA binding protein. The data suggest that the ligand binding region of this protein is multifaceted, a conclusion supported by the chemical predictions of Katekar and Geissler ([1977] Plant Physiol 60: 826-829). Although the data do not allow us to make specific conclusions on the structure of the binding site, they do show that both NPA and SCB-I could each occupy two regions of the protein. At least one of these binding regions appears to be common for both inhibitors of auxin transport. We suggest that the diversity of the binding site structure reflects the possible existence of more than one type of natural ligand controlling the process of auxin transport. PMID- 16668599 TI - Solute Accumulation and Compartmentation during the Cold Acclimation of Puma Rye. AB - During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection. PMID- 16668600 TI - Expression of foreign genes in transgenic yellow-poplar plants. AB - Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding beta-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-beta-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-beta-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves. PMID- 16668601 TI - Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems. AB - Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2'5' ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K(m) = 12 micromolar) and coniferaldehyde (K(m) = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical. PMID- 16668602 TI - Covalent binding sites of victorin in oat leaf tissues detected by anti-victorin polyclonal antibodies. AB - Polyclonal antibodies against victorin, the host-specific toxin produced by Cochliobolus victoriae, were raised in rabbits immunized with a victorin-bovine serum albumin conjugate. The antibodies were purified from serum by protein A column chromatography and characterized by indirect and direct enzymelinked immunosorbent assays (ELISA). The concentration of victorin that inhibited anti victorin antibody binding by 50% was 10 nanograms per milliliter in an indirect ELISA. The lowest concentration of victorin detectable was 10 picograms per milliliter. In a direct ELISA, 25 nanograms per milliliter of victorin inhibited binding of victorin-horseradish peroxidase conjugate by 50%. In vivo and in vitro covalent binding of victorin to proteins in susceptible and resistant oat (Avena sativa) tissue was examined by western blotting assays using anti-victorin antibody and a second antibody conjugated with (125)I or alkaline phosphatase. In vivo binding of victorin to proteins of 100 and 45 kilodaltons was observed in both susceptible and resistant cultivars of oats. Victorin also bound in vitro to proteins of 100, 65, and 45 kilodaltons in both susceptible and resistant oats. The data indicate that victorin binds covalently to the same sites in susceptible and resistant genotypes of oats. PMID- 16668603 TI - Heavy Metal-Activated Synthesis of Peptides in Chlamydomonas reinhardtii. AB - In this study, we have addressed the capacity of the green alga Chlamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd(2+), Hg(2+), and Ag(+). Cells cultured in the presence of sublethal concentrations of Cd(2+) synthesized and accumulated oligopeptides consisting solely of glutamic acid, cysteine, and glycine in an average ratio of 3:3:1. Cadmium-induced peptides were isolated in their native form as higher molecular weight peptide-metal complexes with an apparent molecular weight of approximately 6.5 x 10(3). The isolated complex bound cadmium (as evidenced by absorption spectroscopy) and sequestered (with a stoichiometry of 0.7 moles of cadmium per mole of cysteine) up to 70% of the total cadmium found in extracts of cadmium-treated cells. In Hg(2+)-treated cells, the principal thiol-containing compound induced by Hg(2+) ions was glutathione. It is possible that glutathione functions in plant cells (as it does in animal cells) to detoxify heavy metals. Cells treated with Ag(+) ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd(2+)-induced peptides. But, in contrast to the results obtained using Cd(2+) as an inducer, these molecules did not accumulate to significant levels in Ag(+)-treated cells. The presence of physiological concentrations of Cu(2+) in the growth medium blocked the synthesis of the Ag(+)-inducible component(s) and rendered cells resistant to the toxic effects of Ag(+), suggesting competition between Cu(2+) and Ag(+) ions, possibly at the level of metal uptake. PMID- 16668604 TI - Enhanced Sensitivity to Ethylene in Nitrogen- or Phosphate-Starved Roots of Zea mays L. during Aerenchyma Formation. AB - Adventitious roots of maize (Zea mays L. cv TX 5855), grown in a well-oxygenated nutrient solution, were induced to form cortical gas spaces (aerenchyma) by temporarily omitting nitrate and ammonium (-N), or phosphate (-P), from the solution. Previously this response was shown (MC Drew, CJ He, PW Morgan [1989] Plant Physiology 91: 266-271) to be associated with a slower rate of ethylene biosynthesis, contrasting with the induction of aerenchyma by hypoxia during which ethylene production is strongly stimulated. In the present paper, we show that aerenchyma formation induced by nutrient starvation was blocked, under noninjurious conditions, by addition of low concentrations of Ag(+), an inhibitor of ethylene action, or of aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis. When extending roots were exposed to low concentrations of ethylene in air sparged through the nutrient solution, N or P starvation enhanced the sensitivity to exogenous ethylene at concentrations as low as 0.05 microliters ethylene per liter air, promoting a more rapid and extensive formation of aerenchyma than in unstarved roots. We conclude that temporary deprivation of N or P enhances the sensitivity of ethylene-responsive cells of the root cortex, leading to cell lysis and aerenchyma. PMID- 16668605 TI - Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem. AB - Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed. PMID- 16668606 TI - Light activation of maize phosphoenolpyruvate carboxylase protein-serine kinase activity is inhibited by mesophyll and bundle sheath-directed photosynthesis inhibitors. AB - C(4) phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4 dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (dl glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays L.) PEPC-PK and the ensuing regulatory phosphorylation of its target enzyme in vivo. Based on these and related observations, we propose that the Calvin cycle supplies the C(4) mesophyll cell with (a) a putative signal (e.g. phosphorylated metabolite, amino acid) that interacts with the cytoplasmic protein synthesis event to effect the light activation of PEPC-PK and the concomitant phosphorylation of PEPC, and (b) high levels of known positive effectors (e.g. triose-phosphate, glucose-6-phosphate) that interact directly with the carboxylase. The combined result of this complex regulatory cascade is to effectively desensitize PEPC to feedback inhibition by the millimolar levels of l-malate required for rapid diffusive transport to the bundle sheath during high rates of C(4) photosynthesis. PMID- 16668607 TI - Differential Protein Accumulation in Banana Fruit during Ripening. AB - Banana (Musa acuminata, cv Dwarf Cavendish) proteins were extracted from pulp tissue at different stages of ripening and analyzed by two-dimensional electrophoresis. The results provide evidence of differential protein accumulation during ripening. Two sets of polypeptides have been detected that increase substantially in ripe fruit. These polypeptides were characterized as glycoproteins by western blotting and concanavalin A binding assays. Antibodies againts tomato polygalacturonase cross-react with one of these sets of proteins. PMID- 16668608 TI - Complete amino Acid sequence of soybean leaf p21 : similarity to the thaumatin like polypeptides. AB - A polypeptide structurally related to the thaumatin family of proteins has been purified from soybean (Glycine max) leaves and the complete amino acid sequence has been determined. The mature protein, which we have termed P21, has a calculated molecular weight of 21,461 and an isoelectric point of 4.6. The soybean protein shows 64% amino acid identity with thaumatin, a sweet-tasting protein found in the West African shrub Thaumatococcus danielli, and as much as 71% identity with thaumatin-like polypeptides present in tobacco and maize. PMID- 16668609 TI - Rejuvenation of Sequoia sempervirens by Repeated Grafting of Shoot Tips onto Juvenile Rootstocks in Vitro: Model for Phase Reversal of Trees. AB - Repeated grafting of 1.5-centimeter long shoot tips from an adult Sequoia sempervirens tree onto fresh, rooted juvenile stem cuttings in vitro resulted in progressive restoration of juvenile traits. After four successive grafts, stem cuttings of previously adult shoots rooted as well, branched as profusely, and grew with as much or more vigor as those of seedling shoots. Reassays disclosed retention for 3 years of rooting competence at similar levels as originally restored. Adventitious shoot formation was remanifested and callus development was depressed in stem segments from the repeatedly grafted adult. The reversion was associated with appearance and disappearance of distinctive leaf proteins. Neither gibberellic acid nor N(6)-beneyladenine as nutrient supplements duplicated the graft effects. PMID- 16668610 TI - Selective Inhibition of Active Uptake of Sucrose into Plasma Membrane Vesicles by Polyclonal Sera Directed against a 42 Kilodalton Plasma Membrane Polypeptide. AB - Several polyclonal sera were raised in rabbits and in mice against putative sucrose carrier proteins, i.e. a 42 kilodalton (O Gallet, R Lemoine, C Larsson, S Delrot [1989] Biochim Biophys Acta 978: 56-64) and a 62 kD (KG Ripp, PV Viitanen, WD Hitz, VR Fransceschi [1988] Plant Physiol 88: 1435-1445) polypeptide of the plasma membrane. The effects of these sera on the active uptake of sucrose and of valine into purified plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves and roots were studied. At a dilution of 1/50, the anti-42 kilodalton sera consistently inhibited sucrose uptake in plasma membranes from leaves or from roots. They had no effect on valine uptake. Under the same experimental conditions, the anti-62 kilodalton sera had no effect on active uptake of sucrose. The data further support the view that a 42 kilodalton polypeptide is a component of the transport system mediating sucrose uptake across the plasma membrane of plant cells. PMID- 16668611 TI - Purification and Partial Characterization of Maize (Zea mays L.) beta Glucosidase. AB - Maize (Zea mays L.) beta-glucosidase (beta-d-glucoside glucohydrolase, EC 3.2.1.21) was extracted from the coleoptiles of 5- to 6-day-old maize seedlings with 50 millimolar sodium acetate, pH 5.0. The pH of the extract was adjusted to 4.6, and most of the contaminating proteins were cryoprecipitated at 0 degrees C for 24 hours. The pH 4.6 supernatant from cryoprecipitation was further fractionated by chromatography on an Accell CM column using a 4.8 to 6.8 pH gradient of 50 millimolar sodium acetate, which yielded the enzyme in two homogeneous, chromatographically different fractions. Purified enzyme was characterized with respect to subunit molecular weight, isoelectric point, amino acid composition, NH(2)-terminal amino acid sequence, pH and temperature optima, thermostability, and activity and stability in the presence of selected reducing agents, metal ions, and alkylating agents. The purified enzyme has an estimated subunit molecular mass of 60 kilodaltons, isoelectric point at pH 5.2, and pH and temperature optima at 5.8 and 50 degrees C, respectively. The amino acid composition data indicate that the enzyme is rich in Glx and Asx, the sum of which approaches 25%. The sequence of the first 20 amino acids in the N-terminal region was H(2)N-Ser-Ala-Arg-Val-Gly-Ser-Gln-Asn-Gly-Val-Gln-Met-Leu-Ser-Pro (Ser?) -Glu-Ile-Pro-Gln, and it shows no significant similarity to other proteins with known sequence. The enzyme is extremely stable at 0 to 4 degrees C up to 1 year but loses activity completely at and above 55 degrees C in 10 minutes. Likewise, the enzyme is stable in the presence of or after treatment with 500 millimolar 2-mercaptoethanol, and it is totally inactivated at 2000 millimolar 2 mercaptoethanol. Such metal ions as Hg(2+) and Ag(+) reversibly inhibit the enzyme at micromolar concentrations, and inhibition could be completely overcome by adding 2-mercaptoethanol at molar excess of the inhibitory metal ion. The alkylating agents iodoacetic acid and iodoacetamide irreversibly inactivate the enzyme and such inactivation is accelerated in the presence of urea. PMID- 16668612 TI - Sidedness of plant plasma membrane vesicles altered by conditions of preparation. AB - Right-side-out vesicles of plasma membrane from soybean (Glycine max Merr.) were isolated by aqueous two-phase partition. Inside-out vesicles were formed when these preparations were diluted or frozen and thawed. Sidedness (orientation) was determined by preparative free-flow electrophoresis, concanavalin A binding, and ATPase latency. Under usual conditions of aqueous two-phase partition, the bulk of the vesicles were strongly reactive with concanavalin A-peroxidase and showed a high level of structure-linked latency as expected of a right-side-out (cytoplasmic-side-in) orientation. The vesicles migrated as a single electrophoretic peak. When frozen and thawed, vesicle diameters were reduced and a second population of vesicles of increased electrophoretic mobility was obtained. This second population of vesicles was weakly reactive with concanavalin A-peroxidase and showed low latency as expected of an inside-out (cytoplasmic-side-out) orientation. If the plasma membrane vesicles were diluted with water, a mixture of right-side-out and inside-out vesicles again was obtained. However, some of the cytoplasmic-side-out vesicles that were concanavalin A-unreactive and had low ATPase latency migrated more slowly as a second, less electronegative peak, upon free-flow electrophoresis. The results suggest that right-side-out and inside-out plasma membrane vesicles differ in electrophoretic mobility but that both the orientation and the absolute electrophoretic mobility of the differently oriented vesicles may be influenced by the preparative conditions. PMID- 16668613 TI - Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida. AB - The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA(3)). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA(3) in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA(3) in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA(3) was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA(3) controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA(3) resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA(3). The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA(3) suggest that GA(3) controls chs transcription in an indirect manner. Our data support a model in which GA(3) induces the production of a regulatory protein such as a receptor or a trans acting factor that is directly involved in chs transcription. PMID- 16668614 TI - An improved method for using electrolyte leakage to assess membrane competence in plant tissues. AB - A new expression for ion leakage from plant tissue, the tissue ionic conductance (g(Ti)), is compared with electrical conductivity (EC) and a commonly used damage index (I(d)) to test the ability of each expression to correctly describe leakiness in two model systems representing examples of physiological processes with well-known effects on membrane permeability. In experiments in which drought acclimated leaves were compared with nonacclimated leaves and senescing leaves were compared with nonsenescing leaves, I(d) contradicted our expectation that acclimated tissue would be less leaky than nonacclimated tissue, and g(Ti) and EC confirmed this expectation. In a comparison of senescing and nonsenescing tissue, I(d) again contradicted our expectation that senescing tissue would be more leaky than nonsenescing, and EC and g(Ti) were confirming. Using a diffusion analysis approach, we show that I(d) fails to account for variation in the concentration gradient between the tissue and the bathing solution and variation in the surface area through which efflux occurs. Furthermore, because I(d) is a parameter that relates treatment performance to control performance as a percentage value, it distorts the actual differences among treatments. The resulting artifacts lead to a presentation of membrane integrity which is probably incorrect. EC is a more direct measurement of net ion efflux and appears to be less vulnerable to artifact. However, because g(Ti) is the only expression that explicitly includes chemical driving force and tissue surface area, it is the most reliable of the three expressions. PMID- 16668615 TI - Expression of constitutive and tissue-specific acyl carrier protein isoforms in Arabidopsis. AB - We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hlousek-Radojcic, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP 2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms. PMID- 16668616 TI - Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures. AB - Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH. PMID- 16668617 TI - Gibberellins and the Legume-Rhizobium Symbiosis : I. Endogenous Gibberellins of Lima Bean (Phaseolus lunatus L.) Stems and Nodules. AB - The content of gibberellin-like substances in nodules formed by Bradyrhizobium species strain 127E14 on roots of lima bean (Phaseolus lunatus L.) has been previously found to be relatively high. The objectives of the present study were to purify and identify the endogenous gibberellins from the stems and nodules of lima bean. By sequential silica gel partition column chromatography, C(18) reverse-phase high performance liquid chromatography, and combined gas chromatography-mass spectrometry, the gibberellins A(1), A(3), A(19), A(20), A(29), and A(44) were identified from root nodules. Gibberellins A(1), A(3), A(19), A(20), and A(44) were also identified from lima bean stem tissue. These data provide the first mass spectral-based evidence that gibberellins are present in leguminous root nodules. The presence of the gibberellins identified indicates that the early 13-hydroxylation gibberellin biosynthetic pathway predominates in stem and nodule tissue. However, it is not known if the gibberellins within the nodules are produced in situ, or if they are imported from some remote host plant tissue. PMID- 16668618 TI - Cadmium-Sulfide Crystallites in Cd-(gammaEC)(n)G Peptide Complexes from Tomato. AB - Hydroponically grown tomato plants (Lycopersicon esculentum P. Mill. cv Golden Boy) exposed to 100 micromolar cadmium sulfate produced metal-(gammaEC)(n)G peptide complexes containing acid-labile sulfur. The properties of the complexes resemble those of the cadmium-(gammaEC)(n)G peptide complexes from Schizosaccharomyces pombe and Candida glabrata known to contain a cadmium sulfide crystallite core. The crystallite is stabilized by a sheath of peptides of general structure (gammaGlu-Cys)(n)-Gly. The cadmium-peptide complexes of tomato contained predominantly peptides of n(3), n(4), and n(5). spectroscopic analyses indicated that the tomato cadmium-sulfide-peptide complex contained CdS crystallite core particles smaller than 2.0 nanometers in diameter. PMID- 16668619 TI - Aluminum effects on calcium fluxes at the root apex of aluminum-tolerant and aluminum-sensitive wheat cultivars. AB - The role of Ca(2+) transport in the mechanism of Al toxicity was investigated, using a Ca(2+)-selective microelectrode system to study Al effects on root apical Ca(2+) fluxes in two wheat (Triticum aestivum L.) cultivars: Al-tolerant Atlas 66 and Al-sensitive Scout 66. Intact 3-day-old low-salt-grown (100 micromolar CaCl(2), pH 4.5) wheat seedlings were used, and it was found that both cultivars maintained similar rates of net Ca(2+) uptake in the absence of Al. Addition of Al concentrations that were toxic to Scout (5-20 micromolar AlCl(3)) immediately and dramatically inhibited Ca(2+) uptake in Scout, whereas Ca(2+) transport in Atlas was relatively unaffected. The Al-induced inhibition of Ca(2+) uptake in Scout 66 was rapidly reversed following removal of Al from the solution bathing the roots. Similar studies with morphologically intact root cell wall preparations indicated that the Al effects did not involve Al-Ca interactions in the cell wall. These results suggest that Al inhibits Ca(2+) influx across the root plasmalemma, possibly via blockage of calcium channels. The differential effect of Al on Ca(2+) transport in Al-sensitive Scout and Al-tolerant Atlas suggests that Al blockage of Ca(2+) channels could play a role in the cellular mechanism of Al toxicity in higher plants. PMID- 16668620 TI - Identification and Analysis of a Barley cDNA Clone Encoding the 31-Kilodalton LHC IIa (CP29) Apoprotein of the Light-Harvesting Antenna Complex of Photosystem II. AB - The light-harvesting complex (LHC) of photosystem II is composed of several different pigment-binding apoproteins. We have identified a cDNA clone LHCIIa-1 encoding the 31-kilodalton LHC IIa (CP29, Chl a/b-P1) apoprotein of barley (Hordeum vulgare). Direct protein microsequencing of an internal peptide fragment from the LHC IIa apoprotein has been used to identify unequivocally the cDNA clone as that coding for the LHC IIa apoprotein. Microsequencing of the 28 kilodalton LHC IIc protein (CP26) showed only minor sequence similarity to the LHC IIa protein, indicating that they are two different gene products. LHCIIa-1 codes for a protein of 286 amino acid residues (molecular weight, 31,308), which displays strong similarities to other pigment-binding LHC proteins, and yet contains an additional 42 amino acid residue segment. Two regions of strong intramolecular sequence similarity are also observed. PMID- 16668621 TI - Immunochemical Analysis of the Temporal and Tissue-Specific Expression of an Avena sativa Plasma Membrane Determinant. AB - An immunoglobulin Mk monoclonal (F8IVE9) antibody raised against oat (Avena sativa cv Garry) root homogenate has been produced and characterized. The predominant target bound by this antibody is a 62-kilodalton protein (p62) that is expressed in both oat root and oat shoot cells. Treatment of the oat antigen with periodate, or with recombinant N-glycanase, affects the F8IVE9 binding to the antigen, suggesting that the specific epitope for this monoclonal antibody involves a carbohydrate determinant. Levels of p62 present in cells of the oat root increase approximately twofold as the root tissue matures during the first 11 days postgermination. In contrast, levels of expression in shoot tissue remain relatively constant during the same period. The p62 antigen has been shown to be expressed at the plasma membrane by immunohistochemical means, by immunofluorescent labeling of protoplasts, and by enzyme-linked immunosorbent assay analysis of purified plasma membrane. The F8IVE9 antigenic target appears to be uniformly distributed through root tissue but is differentially expressed in specific sections of the shoot. F8IVE9 antibody also binds to antigens expressed in a number of other species, including clover, corn, pea, broccoli, mustard, and bean, and has been shown to bind to Samanea protoplast plasma membranes. This monoclonal antibody may prove to be useful for a variety of investigations, including an analysis of the specific patterns of cellular differentiation that occur during early morphogenesis, and the characterization of plasma membrane-associated elements in plants. PMID- 16668622 TI - The Shibata Shift and the Transformation of Etioplasts to Chloroplasts in Wheat with Clomazone (FMC 57020) and Amiprophos-Methyl (Tokunol M). AB - The Shibata shift is a change in the absorption maximum of chlorophyllide from 684 to 672 nanometers that occurs within approximately 0.5 hour of phototransformation of protochlorophyllide to chlorophyllide. Two compounds, clomazone and amiprophos-methyl, which previously have been shown to inhibit the Shibata shift in vivo, were used to look for correlations between the Shibata shift and other processes that occur during etioplast to chloroplast transformation. Leaf sections from 6-day-old etiolated wheat seedlings (Triticum aestivum L. cv Walde) were treated with 0.5 millimolar clomazone or 0.1 millimolar amiprophos-methyl in darkness. In addition to the Shibata shift, the esterification of chlorophyllide to chlorophyll and the relocation of protochlorophyllide reductase from the prolamellar bodies to the developing thylakoids were inhibited by these treatments. Prolamellar body transformation did not appear to be affected by amiprophos-methyl and was only slightly affected by clomazone. The results indicate that: (a) there is a strong correlation between the occurrence of the Shibata shift and esterification activity; (b) transformation of the prolamellar bodies does not depend on the Shibata shift; and (c) the occurrence of the Shibata shift may be a prerequisite to the relocation of protochlorophyllide reductase from prolamellar bodies to thylakoids. PMID- 16668623 TI - Arabinogalactan-rich glycoproteins are localized on the cell surface and in intravacuolar multivesicular bodies. AB - We investigated the subcellular distribution of antigenic sites immunoreactive to the monoclonal antibody 16.4B4 (PM Norman, VPM Wingate, MS Fitter, CJ Lamb [1986] Planta 167: 452-459) in tobacco (Nicotiana tabacum) leaf cells. This antibody is directed against a glycan epitope in a family of plasma membrane arabinogalactan proteins of 135 to 180 kilodaltons, elaborated from a polypeptide of relative molecular mass 50 kilodaltons (PM Norman, P Kjellbom, DJ Bradley, MG Hahn, CJ Lamb [1990] Planta 181: 365-373). We demonstrated by immunogold electron microscopy that the epitope reactive with monoclonal antibody 16.4B4 is localized on the cell surface in the leaf parenchyma cell periplast. The 16.4B4 antigen is also localized in multivesicular invaginations of the plasma membrane also known as plasmalemmasomes, implying a biochemical and, hence, functional interrelationship between these structures. Monoclonal antibody 16.4B4 also labels intracellular multivesicular bodies that appear to represent internalized plasmalemmasomes. Antibody reactivity was also observed in partially degraded multivesicular bodies sequestered within the central vacuole. We propose that the subcellular distribution of the epitope reactive with monoclonal antibody 16.4B4 defines a plasmalemmasome (or multivesicular body-mediated) pathway for the internalization of the periplasmic matrix for vacuolar mediated disposal. The multivesicular bodies appear to be equivalent to the well-characterized endosomes and multivesicular bodies of animal cells involved in the internalization and lysosome-mediated degradation of extracellular materials. PMID- 16668624 TI - Variation of the polypeptide composition of mitochondria isolated from different potato tissues. AB - The protein contents of mitochondria from different potato (Solanum tuberosum L.) tissues (tubers, dark-grown shoots, and green leaves) grown in a greenhouse or in vitro were compared by two-dimensional polyacrylamide gel electrophoresis. Two different methods were used: using the method that gave the highest resolution, an average number of 360 polypeptides was revealed on the mitochondrial patterns after silver staining. The mitochondrial protein patterns of etiolated tissues (tubers, dark-grown shoots) are roughly similar but distinct from those of green leaves. The four subunits of the glycine decarboxylase complex (involved in photorespiration) and a few other polypeptides are very abundant in green tissues, compared with nonphotosynthetic tissues. Conversely, some other polypeptides that are abundant in tubers and dark-grown shoots are hardly detectable in green leaf mitochondria. A rabbit antiserum was raised against a 40 kilodalton polypeptide that is among the most characteristic of these nonphotosynthetic tissue-specific polypeptides, and the N-terminal sequence of this polypeptide was determined. No effect of in vitro culture was observed on the protein composition of mitochondria isolated from differentiated tissues. However, the protein patterns of callus and cell suspension mitochondria are distinct from those of any differentiated tissues, although their basic pattern is clearly mitochondrial. PMID- 16668625 TI - Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria. AB - The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the alpha, beta, and [ill] mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria. PMID- 16668627 TI - Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants. AB - A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO(2) assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO(2), and 25 degrees C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO(2) partial pressures (p(i)). In control leaves, Rubisco activity only limited the rate of CO(2) assimilation below a p(i) of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p(i). The unchanged conductance and lower CO(2) assimilation resulted in a higher p(i), which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity. PMID- 16668626 TI - Sucrose Metabolism in Tubers of Potato (Solanum tuberosum L.): Effects of Sink Removal and Sucrose Flux on Sucrose-Degrading Enzymes. AB - Excision of developing potato (Solanum tuberosum L.) tubers from the mother plant, followed by storage at 10 degrees C, resulted in a rapid, substantial decrease in sucrose synthase activity and considerable increases in hexose content and acid invertase activity. A comparison of the response of three genotypes, known to accumulate different quantities of hexoses in storage, showed that both sucrose synthase activity and the extent to which activity declined following excision were similar in all cases. However, there was significant genotypic variation in the extent to which acid invertase activity developed, with tubers accumulating the highest hexose content also developing the highest extractable activity of invertase. Similar effects were found in nondetached tubers when growing plants were maintained in total darkness for a prolonged period. Furthermore, supplying sucrose to detached tubers through the cut stolon surface prevented the decline in sucrose synthase activity. Maltose proved to be ineffective. Western blots using antibodies raised against maize sucrose synthase showed that the decline in sucrose synthase activity was associated with the loss of protein rather than the effect of endogenous inhibitors. Although there were indications that maintaining a flux of sucrose into isolated tubers could prevent the increase in acid invertase activity, the results were not conclusive. PMID- 16668628 TI - Isolation, Purification, and Identification of 2-(p-Hydroxyphenoxy)-5, 7 Dihydroxychromone: A Fungal-Induced Phytoalexin from Cassia obtusifolia. AB - A single phytoalexin was isolated and purified from 12- to 14-day-old leaves of Cassia obtusifolia L. inoculated with Alternaria cassiae Jurair & Khan. The structure was elucidated by (1)H- and (13)C-nuclear magnetic resonance and mass spectrometry as 2-(p-hydroxyphenoxy)-5,7-dihydroxychromone. The compound was shown to be derived in part from phenylalanine. Radial growth of A. cassiae was inhibited 50% by the compound at 0.3 millimolar. This moderate toxicity is compensated for by the relatively high levels (3 millimolar) accumulated. Phenoxychromones have been previously reported only as constitutive secondary metabolites in Artemisia capillaris Thunb., in which their function is unknown. PMID- 16668629 TI - Nitrate-regulated growth and cytokinin responses in seminal roots of barley. AB - The influence of nitrate availability on growth of seminal roots, and root cytokinin levels, was studied in barley (Hordeum vulgare L. cv Golf). Nitrate was continuously supplied to initially N-starved seedlings at relative addition rates (RA) of 0.03 to 0.21 per day (standard cultures) or at RA 0.09 per day in split root cultures with the nitrate additions distributed in ratios of 100:0 or 80:20 to the two subroots. Data were collected both during a phase of acclimation (first 10 days of N additions) and in the acclimated stage (>10 days after onset of N additions). Limitation of whole-plant growth was observed at RA <0.15 per day. The lateral root frequency increased with RA in plants of equal chronological age. However, the lateral root frequency was related to root size rather than to RA; roots of uneven age but having comparable total root lengths also had comparable lateral root frequencies. Growth of individual subroots in split root systems during acclimation was proportional to the fraction of the total N addition that was fed to the root. All subroots had comparable relative growth rates in acclimated plants, and their lateral root frequency correlated with total root length in the same manner as in standard cultures. Onset of N additions in a 80:20 split root culture resulted in doublings of zeatin riboside (ZR) levels in shoots and in the "80" root, whereas the response of the "20" root was small. No effect of perturbed nitrate availability on xylem translocation of ZR was observed. The ZR levels remained higher in the "80" root during acclimation but returned to the level of the "20" root after acclimation. Root cytokinin levels and xylem translocation in acclimated standard cultures were unaffected by RA in the lower range but increased at high RA. Arguments for involvement of cytokinins in the nitrate-regulated growth response are discussed. PMID- 16668630 TI - Diurnal starch accumulation and utilization in phosphorus-deficient soybean plants. AB - The effects of phosphorus deficiency on carbohydrate accumulation and utilization in 34-day-old soybean (Glycine max L. Merr.) plants were characterized over a diurnal cycle to evaluate the mechanisms by which phosphorus deficiency restricts plant growth. Phosphorus deficiency decreased the net CO(2) exchange rate throughout the light period. The decrease in the CO(2) exhange rate was associated with a decrease in stomatal conductance and an increase in the internal CO(2) concentration. These observations indicate that phosphorus deficiency increased mesophyll resistance. Assimilate export rate from the youngest fully expanded leaves was decreased by phosphorus deficiency, whereas starch concentrations in these leaves were increased. Higher starch concentrations in phosphorus-deficient youngest fully expanded leaves resulted from a longer period of net starch accumulation and a shorter period of net starch degradation relative to those for phosphorus-sufficient controls. Phosphorus deficiency decreased sucrose-P synthase activity by 27% (averaged over the diurnal cycle), and essentially eliminated diurnal variation in sucrose-P synthase activity. Diurnal variations in nonstructural carbohydrate concentrations in leaves and stems were also less pronounced in phosphorus deficient plants than in controls. In phosphorus-deficient plants, only 30% of the whole plant starch present at the end of a light phase was utilized during the subsequent 12-hour dark phase as compared with 68% for phosphorus-sufficient controls. Although phosphorus deficiency decreased the CO(2) exchange rate and whole plant leaf area, accumulation of high starch concentrations in leaves and stems and restricted starch utilization in the dark indicate that growth processes (i.e. sink activities) were restricted to a greater extent than photosynthetic capacity. Further experimentation is required to determine whether decreased starch utilization in phosphorus-deficient plants is the cause or the result of restricted growth. PMID- 16668631 TI - Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene. AB - The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L 3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in cotyledons. PMID- 16668632 TI - Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad). AB - A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases. PMID- 16668633 TI - Immunological Evidence of Thaumatin-Like Proteins during Tobacco Floral Differentiation. AB - Tobacco proteins that share homology with thaumatin, a sweet protein of Thaumatococcus daniellii Benth., are produced in various physiological situations such as pathogenesis-related stress or water deficit stress. Using purified polyclonal anti-thaumatin antibodies, we have detected other thaumatin-like proteins in tobacco (Nicotiana tabacum var Samsun) that have been related with floral differentiation. Thaumatin-like proteins with apparent molecular masses of 42.6, 31.6, and 26.3 kilodaltons were found in immature and mature flower organs in vivo, and others of 46.7, 41.7, and 27.5 kilodaltons were exclusively detected in thin cell layer explants forming flowers. In situ immunolocalization revealed their synthesis in newly differentiated floral meristems, in tracheids, and in parenchyma cells. PMID- 16668634 TI - Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L. AB - Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H(+) extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O(2) uptake with a 4H(+) to O(2) ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3 (3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light. PMID- 16668635 TI - Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth. AB - The dehydrodiconiferyl alcohol glucosides A and B are factors isolated from transformed Vinca rosea tumor cells that can replace the cytokinin requirement for growth of tobacco (Nicotiana tabacum) pith and callus cells in culture. These factors, present in tobacco pith cells, have their concentrations elevated approximately 2 orders of magnitude after cytokinin exposure. Biosynthesis experiments showed that these compounds are not cell wall fragments, as previously suggested, but are produced directly from coniferyl alcohol. Their synthesis is probably associated with the existing pathway for cell wall biosynthesis in both Vinca tumors and tobacco pith explants. The pathway requires only two steps, the dimerization of coniferyl alcohol by a soluble intracellular peroxidase and subsequent glycosylation. Biosynthetic experiments suggested that dehydrodiconiferyl alcohol glucoside breakdown was very slow and control of its concentration was exerted through restricted availability of coniferyl alcohol. PMID- 16668636 TI - Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants. AB - To determine whether abscisic acid (ABA) accumulation in endosperms of water limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/-/-; +/+/-; +/+/+) or incapable (-/-/-) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit. PMID- 16668637 TI - Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure. AB - Relative molecular size distributions of pectic and hemicellulosic polysaccharides of pea (Pisum sativum cv Alaska) third internode primary walls were determined by gel filtration chromatography. Pectic polyuronides have a peak molecular mass of about 1100 kilodaltons, relative to dextran standards. This peak may be partly an aggregate of smaller molecular units, because demonstrable aggregation occurred when samples were concentrated by evaporation. About 86% of the neutral sugars (mostly arabinose and galactose) in the pectin cofractionate with polyuronide in gel filtration chromatography and diethylaminoethyl-cellulose chromatography and appear to be attached covalently to polyuronide chains, probably as constituents of rhamnogalacturonans. However, at least 60% of the wall's arabinan/galactan is not linked covalently to the bulk of its rhamnogalacturonan, either glycosidically or by ester links, but occurs in the hemicellulose fraction, accompanied by negligible uronic acid, and has a peak molecular mass of about 1000 kilodaltons. Xyloglucan, the other principal hemicellulosic polymer, has a peak molecular mass of about 30 kilodaltons (with a secondary, usually minor, peak of approximately 300 kilodaltons) and is mostly not linked glycosidically either to pectic polyuronides or to arabinogalactan. The relatively narrow molecular mass distributions of these polymers suggest mechanisms of co- or postsynthetic control of hemicellulose chain length by the cell. Although the macromolecular features of the mentioned polymers individually agree generally with those shown in the widely disseminated sycamore cell primary wall model, the matrix polymers seem to be associated mostly noncovalently rather than in the covalently interlinked meshwork postulated by that model. Xyloglucan and arabinan/galactan may form tightly and more loosely bound layers, respectively, around the cellulose microfibrils, the outer layer interacting with pectic rhamnogalacturonans that occupy interstices between the hemicellulose coated microfibrils. PMID- 16668638 TI - Changes in molecular size of previously deposited and newly synthesized pea cell wall matrix polysaccharides : effects of auxin and turgor. AB - Effects of indoleacetic acid (IAA) and of turgor changes on the apparent molecular mass (M(r)) distributions of cell wall matrix polysaccharides from etiolated pea (Pisum sativum L.) epicotyl segments were determined by gel filtration chromatography. IAA causes a two- to threefold decline in the peak M(r) of xyloglucan, relative to minus-auxin controls, to occur within 0.5 hour. IAA causes an even larger decrease in the peak M(r) concurrently biosynthesized xyloglucan, as determined by [(3)H]fucose labeling, but this effect begins only after 1 hour. In contrast, IAA does not appreciably affect the M(r) distributions of pectic polyuronides or hemicellulosic arabinose/galactose polysaccharides within 1.5 hours. However, after epicotyl segments are cut, their peak polyuronide M(r) increases and later decreases, possibly as part of a wound response. Xyloglucan also undergoes IAA-independent changes in its M(r) distribution after cutting segments. In addition, the peak M(r) of newly deposited xyloglucan increases from about 9 kilodaltons shortly after deposition to about 30 kilodaltons within 0.5 hour. This may represent a process of integration into the cell wall. A step increase in turgor causes the peak M(r) of previously deposited xyloglucan (but not of the other major polymers) to increase about 10-fold within 0.5 hour, returning to its initial value by 1.5 hours. This upshift may comprise a feedback mechanism that decreases wall extensibility when the rate of wall extension suddenly increases. IAA-induced reduction of xyloglucan M(r) might cause wall loosening that leads to cell enlargement, as has been suggested previously, but the lack of a simple relation between xyloglucan M(r) and elongation rate indicates that loosening must also involve other wall factors, one of which might be the deposition of new xyloglucan of much smaller size. Although the M(r) shifts in polyuronides may represent changes in noncovalent association, and for xyloglucan this cannot be completely excluded, xyloglucan seems to participate in a dynamic process that can both decrease and increase its chain length, possible mechanisms for which are suggested. PMID- 16668639 TI - Phenylalanine ammonia-lyase from loblolly pine : purification of the enzyme and isolation of complementary DNA clones. AB - Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from differentiating secondary xylem of loblolly pine (Pinus taeda L.). Native molecular weight of the enzyme was estimated to be 280,000, with a subunit molecular weight of 74,000; isoelectric point, 5.8; and Michaelis constant for i-phenylalanine, 27 micromolar. No evidence was obtained for the existence of isoforms of the enzyme, nor for negative cooperativity of substrate binding. Polyclonal antibodies were raised against the phenylalanine ammonia-lyase subunit and used to identify a pal clone in an expression library of xylem complementary DNA (cDNA). Polymerase chain reaction, using oligonucleotide primers made from N-terminal amino acid sequence and from the 5' end of the clone isolated from the expression library, was also used to isolate cDNA clones. These methods yielded cDNA clones covering the protein coding region of the pal messenger RNA. Comparisons of nucleotide sequence of pal cDNAs from pine, bean, sweet potato, and rice showed 60 to 62% identity between the pine clone and the angiosperm clones. PMID- 16668640 TI - 5'CATGCAT-3' Elements Modulate the Expression of Glycinin Genes. AB - Promoters of most seed proteins in legumes contain one or more 5'-CATGCAT-3' elements. To test if these elements have a function in the expression of these genes, the 2.3 kilobase pairs Gy2 glycinin promoter was ligated to a beta glucuronidase reporter sequence and transformed into tobacco. Elimination of a 5' CATGCAT-3' element 101 base pairs upstream from the transcription start site in the construction caused about a 10-fold reduction in the amount of beta glucuronidase activity compared with when the element was present in the gene. Elimination of 1.9 kilobase pairs from the 5'-end of the promoter caused a two-to threefold reduction in activity. The results show that the 5'-CATGCAT-3' element plays a role in regulating the amount of expression from the gene, but that there are also other factors farther upstream from the gene that affect the level of expression. PMID- 16668641 TI - Biochemical characteristics of thylakoid membranes in chloroplasts of dark-grown pine cotyledons. AB - Japanese black pine (Pinus thunbergii) cotyledons were found to synthesize chlorophylls in complete darkness during germination, although the synthesis was not as great as that in the light. The compositions of thylakoid components in plastids of cotyledons grown in the dark and light were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides and spectroscopic determination of membrane redox components. All thylakoid membrane proteins found in preparations from light-grown cotyledons were also present in preparations from dark-grown cotyledons. However, levels of photosystem I, photosystem II, cytochrome b([ill])/f, and light-harvesting chlorophyll-protein complexes in dark-grown cotyledons were only one-fourth of those in light-grown cotyledons, on a fresh weight basis. These results suggest that the low abundance of thylakoid components in dark-grown cotyledons is associated with the limited supply of chlorophyll needed to assemble the two photosystem complexes and the light-harvesting chlorophyll-protein complex. PMID- 16668642 TI - Occurrence of Temperature-Sensitive Phenotypic Plasticity in Chlorophyll Deficient Mutants of Arabidopsis thaliana. AB - A collection of 75 putative mutants with alterations in leaf pigmentation was visually selected from Arabidopsis thaliana plants (M(2) generation) grown at 26 degrees C from seeds treated with the mutagen ethylmethanesulfonate. Fifty-eight of the plants were found to have chlorophyll contents decreased by at least 10% from the parental Columbia ecotype. These plants were screened for chlorophyll content and the ratio of chlorophyll b/a after growth at 20 or 26 degrees C. Relative to the parental type, a significant number of individuals in which the chlorophyll-deficient phenotype was exacerbated at one of the growth temperatures were identified. We conclude that temperature-sensitive phenotypic plasticity for chlorophyll content is relatively common in mutant populations of higher plants. PMID- 16668643 TI - nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii. AB - Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits. PMID- 16668644 TI - Mannosyl- and Xylosyl-Containing Glycans Promote Tomato (Lycopersicon esculentum Mill.) Fruit Ripening. AB - The oligosaccharide glycans mannosylalpha1-6(mannosylalpha1-3)mannosylalpha1 6(mannosylalpha1-3) mannosylbeta1-4-N-acetylglucosamine and mannosylalpha1 6(mannosylalpha1-3)(xylosylbeta1-2) mannosylbeta1-4-N acetylglucosaminyl(fucosylalpha1-3) N-acetylglucosamine were infiltrated into mature green tomato fruit (Lycopersicon esculentum Mill., cv Rutgers). Coinfiltration of 1 nanogram per gram fresh weight of the glycans with 40 micrograms per gram fresh weight galactose, a level of galactose insufficient to promote ripening, stimulated ripening as measured by red coloration and ethylene production. PMID- 16668645 TI - Nucleotide Sequence of the Photoregulated Maize Chloroplast psal Gene, Encoding a 4.0-Kilodalton Component of Photosystem I. PMID- 16668646 TI - Nucleotide Sequence of S-Adenosyl-l-Methionine: Magnesium Protoporphyrin Methyltransferase from Rhodobacter capsulatus. PMID- 16668647 TI - Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP. AB - The 2',3'-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H(+)-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH(4). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K(i) of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s(-1). The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [(3)H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested. PMID- 16668648 TI - Expression of a Brassica napus Malate Synthase Gene in Transgenic Tomato Plants during the Transition from Late Embryogeny to Germination. AB - To study gene regulation during the transition from late embryogeny to germination, we have analyzed the expression of a gene encoding the glyoxylate cycle enzyme malate synthase in transgenic tomato (Lycopersicon esculentum) plants. We have shown that although there are at least four classes of malate synthase genes in Brassica napus L., one gene is expressed at a high level during both late embryogeny and postgermination. Analyses of transgenic tomato plants containing the expressed B. napus gene along with 4.7 and 1.0 kilobase pairs of 5' and 3' flanking sequences, respectively, confirmed that a single gene is expressed at both stages of development. Furthermore, localization studies have shown that mRNA encoded by the B. napus gene is distributed throughout the tissues of a mature embryo but is not detected in the vascular cylinder of a seedling. We conclude that the sequences required to qualitatively regulate the gene correctly over the plant life cycle are present within the transferred gene and/or flanking regions. Moreover, the malate synthase gene is regulated differently during late embryogeny and postgermination in the developing vascular cylinder. PMID- 16668649 TI - Fungal Elicitor-Mediated Responses in Pine Cell Cultures : III. Purification and Characterization of Phenylalanine Ammonia-Lyase. AB - Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is involved in the lignification of pine suspension cultures in response to an elicitor prepared from an ectomycorrhizal fungus. To elucidate the molecular basis of this response, PAL was purified to homogeneity from jack pine (Pinus banksiana) suspension cultures using anion-exchange and chromatofocussing fast protein liquid chromatography. Physical characterization of the enzyme revealed that pine PAL was similar to PAL from other plant sources. Pine PAL had a pH optimum of 8.8, an isoelectric point of 5.75, and a native molecular mass of 340 kilodaltons. The enzyme appears to be a tetramer composed of 77 kilodalton subunits. Chromatographic and western blot analyses were used to identify possible isoenzymic changes in pine PAL in response to elicitation and to determine the nature of the increase in PAL activity associated with inducible lignification in these cultures. Only one species of PAL was detected in P. banksiana cell cultures and increased quantities of this protein were correlated with the enhanced enzyme activity observed in elicited cultures. P. banksiana PAL was not feedback-inhibited by a wide range of phenolic compounds at micromolar concentrations, including the reaction product cinnamic acid. Our data suggest that a different set of metabolic and molecular controls must be in place for the regulation of PAL in pine. PMID- 16668650 TI - Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts. AB - Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen ((1)O(2)) scavengers, histidine and sodium azide (NaN(3)) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of (1)O(2) monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the (1)O(2) quencher, NaN(3). Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN(3) and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer. PMID- 16668651 TI - Cell Wall Dissolution in Ripening Kiwifruit (Actinidia deliciosa) : Solubilization of the Pectic Polymers. AB - Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa ;Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na(2)CO(3), 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na(2)CO(3)-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na(2)CO(3) soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation. Pectolytic enzymes such as endopolygalacturonase and beta-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo. PMID- 16668652 TI - Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum. AB - A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two different-sized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by western blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH(2)-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH(2)-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene. PMID- 16668653 TI - Growth stimulation by catecholamines in plant tissue/organ cultures. AB - Addition of catecholamines at micromolar concentrations caused a dramatic stimulation of growth of tobacco (Nicotiana tabacum) thin cell layers (TCLs) and Acmella oppositifolia "hairy" root cultures. A threefold increase in the rate of ethylene evolution was observed in the catecholamine-treated explants. Aminooxyacetic acid and silver thiosulfate, inhibitors of ethylene biosynthesis and action, respectively, reduced the growth-promoting effect of dopamine. However, these compounds alone could also inhibit the growth of the TCL explants. When ethylene in the culture vessel was depleted by trapping with mercuric perchlorate, dopamine-stimulated growth was still obtained, suggesting that ethylene does not mediate the dopamine effect. Dopamine potentiated the growth of TCLs grown in Murashige and Skoog medium supplemented with indoleacetic acid (IAA) and kinetin. When IAA was replaced by 2,4-dichlorophenoxyacetic acid, dopamine addition showed no growth-promoting effect. Instead, 2,4 dichlorophenoxyacetic acid stimulated the growth of TCL explants to the same extent as that obtained with IAA plus dopamine. Because synthetic auxins do not appear to be substrates for IAA oxidizing enzymes, we hypothesized that catecholamines exert their effect by preventing IAA oxidation. Consistent with this explanation, dopamine (25 micromolar) inhibited IAA oxidase activity by 60 to 100% in crude enzyme extracts from tobacco roots and etiolated corn coleoptiles, but had no effect on peroxidase activity in the same extracts. Furthermore, addition of dopamine to TCL cultures resulted in a fourfold reduction in the oxidative degradation of [1-(14)C]IAA fed to the explants. Because the growth enhancement by catecholamines is observed in both IAA requiring and IAA-independent cultures, we suggest that these aromatic amines may have a role in the regulation of IAA levels in vivo. PMID- 16668654 TI - Effects of Low O(2) Root Stress on Ethylene Biosynthesis in Tomato Plants (Lycopersicon esculentum Mill cv Heinz 1350). AB - Low O(2) conditions were obtained by flowing N(2) through the solution in which the tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were growing. Time course experiments revealed that low O(2) treatments stimulated 1 aminocyclopropane-1-carboxylate (ACC) synthase production in the roots and leaves. After the initiation of low O(2) conditions, ACC synthase activity and ACC content in the roots increased and reached a peak after 12 and 20 hours, respectively. The conversion of ACC to ethylene in the roots was inhibited by low levels of O(2), and ACC was apparently transported to the leaves where it was converted to ethylene. ACC synthase activity in the leaves was also stimulated by low O(2) treatment to the roots, reaching a peak after 24 hours. ACC synthase levels were enhanced by cobalt chloride and aminooxyacetic acid (AOA), although they inhibited ethylene production. Cobalt chloride enhanced ACC synthase only in combination with low O(2) conditions in the roots. Under aeration, AOA stimulated ACC synthase activity in both the roots and leaves. However, in combination with low O(2) conditions, AOA caused a stimulation in ACC synthase activity in the leaves and no effect in the roots. PMID- 16668655 TI - Effects of anoxia on mitochondrial biogenesis in rice shoots: modification of in organello translation characteristics. AB - Shoots of germinating rice (Oryza sativa L.) seedlings are able to grow under anoxia and to withstand long periods of anoxic treatment. Mitochondria were purified from aerobically germinated and anaerobically treated rice shoots by differential and isopycnic centrifugation and were found to consist of two subpopulations. The mitochondrial subpopulation of higher density was used for further characterization. Ultrastructural studies showed anaerobic mitochondria to be significantly different from aerobic mitochondria, with a matrix of lower density and more developed cristae. Aerobic and anaerobic mitochondria also differed in their specific activities for fumarase and succinate dehydrogenase, which were significantly lower after the anoxic treatment. In vivo labeling of seedlings with l-[(35)S]methionine and subsequent isolation of the mitochondria indicated that anoxia induced a drastic decrease, but not a total inactivation, of the synthesis of mitochondrial proteins. In organello protein synthesis showed that anaerobic mitochondria were able to synthesize most of the polypeptides synthesized by aerobic mitochondria, although only in the presence of exogenous ATP, as would occur under anoxia. Anaerobic mitochondria, but not aerobic mitochondria, could carry out protein synthesis without a functional respiratory chain. Thus, mitochondrial protein synthesis was found to be potentially functional in the rice shoot under anoxia. PMID- 16668656 TI - Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii. AB - In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit 3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID- 16668657 TI - Herbicide clomazone does not inhibit in vitro geranylgeranyl synthesis from mevalonate. AB - Clomazone reduced the chlorophyll and carotenoid contents of spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), velvetleaf (Abutilon theophrasti Medik.), and soybean (Glycine max L. Merr.) seedlings. The order of species sensitivity was velvetleaf > spinach > barley > soybean. Clomazone (100 micromolar) did not affect the in vitro activities of spinach isopentenyl pyrophosphate isomerase or prenyl transferase. Clomazone also did not affect the synthesis of isopentenyl pyrophosphate from mevalonic acid. Thus, clomazone had no direct in vitro effect on the synthesis of geranylgeranyl pyrophosphate from mevalonic acid. Greening seedlings of both soybean and velvetleaf metabolized clomazone. No qualitative differences in the metabolites were detected between soybean and velvetleaf. Thus, differential metabolism of clomazone to a toxic chemical that inhibits terpenoid synthesis is unlikely. Clomazone has either a mode of action not yet identified or a metabolite that is selective in that it is much more active in sensitive than tolerant species. PMID- 16668658 TI - Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits. AB - The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited. PMID- 16668659 TI - Transport of Stachyose and Sucrose by Vacuoles of Japanese Artichoke (Stachys sieboldii) Tubers. AB - Vacuoles are the stores for large amounts of stachyose [alphagal (1,6) alphagal (1,6) alphaglc (1,2) betafru] in tubers of Japanese artichoke (Stachys sieboldii). The uptake of stachyose by these vacuoles was examined and compared with that of sucrose. The uptake mechanisms of both sugars were quite similar. The kinetics showed a single saturable response to increasing external concentrations of (14)C-sugars with similar apparent K(m) values of about 50 and 30 millimolar for stachyose and sucrose, respectively. The uptake rates, however, were always higher for stachyose than for sucrose. Stachyose and sucrose uptake was inhibited by fructose and raffinose, and, reciprocally, by sucrose and stachyose, but not by glucose or galactose. The main structural feature common to all sugars recognized by the uptake systems seems to be a terminal fructosyl residue. The uptake of both sugars was stimulated by Mg-ATP and inorganic pyrophosphate, suggesting a proton-sugar antiport system. The possibility that stachyose and sucrose might be transported by the same carrier is discussed. PMID- 16668660 TI - Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina. AB - A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [(35)S]GTPgammaS or [alpha-(32)P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [(32)P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5'-O-(2-thiodiphosphate) (added in 3 x 10(4)-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na(2)CO(3), but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [(3)H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants. PMID- 16668661 TI - Calcium transport by corn mitochondria : evaluation of the role of phosphate. AB - Mitochondria from some plant tissues possess the ability to take up Ca(2+) by a phosphate-dependent mechanism associated with a decrease in membrane potential, H(+) extrusion, and increase in the rate of respiration (AE Vercesi, L Pereira da Silva, IS Martins, CF Bernardes, EGS Carnieri, MM Fagian [1989] In G Fiskum, ed, Cell Calcium Metabolism. Plenum Press, New York, pp 103-111). The present study reexamined the nature of the phosphate requirement in this process. The main observations are: (a) Respiration-coupled Ca(2+) uptake by isolated corn (Zea mays var Maya Normal) mitochondria or carbonyl cyanide p trifluoromethoxyphenylhydrazone-induced efflux of the cation from such mitochondria are sensitive to mersalyl and cannot be dissociated from the silmultaneous movement of phosphate in the same direction. (b) Ruthenium red induced efflux is not affected by mersalyl and can occur in the absence of phosphate movement. (c) In Ca(2+)-loaded corn mitochondria, mersalyl causes net Ca(2+) release unrelated to a decrease in membrane potential, probably due to an inhibition of Ca(2+) cycling at the level of the influx pathway. It is concluded that corn mitochondria (and probably other plant mitochondria) do possess an electrophoretic influx pathway that appears to be a mersalyl-sensitive Ca(2+)/inorganic phosphate-symporter and a phosphate-independent efflux pathway possibly similar to the Na(2+)-independent Ca(2+) efflux mechanism of vertebrate mitochondria, because it is not stimulated by Na(+). PMID- 16668662 TI - Expression of maize phosphoenolpyruvate carboxylase in transgenic tobacco : effects on biochemistry and physiology. AB - The expression of maize (Zea mays) phophoenolpyruvate carboxylase (PPC) gene constructions was studied in transgenic tobacco plants (Nicotiana tabacum). Where transcription was under the control of a maize PPC gene promoter, a low level of aberrantly large PPC transcript was detected. Analysis of this PPC transcript indicated that transcription initiation occurs upstream of the normal site. Despite the aberrant transcription initiation, expression of the PPC transcript was still light-regulated. Higher levels of maize PPC transcript of the correct size were obtained with a chimeric gene construction containing a tobacco (Nicotiana plumbaginifolia) chlorophyll a/b binding protein gene promoter. The PPC activities in the leaves of these transgenic plants were up to twofold higher than those of nontransformed plants. Two forms of PPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form with a high apparent K(m) for phosphoenolpyruvate (maize isozyme), and a second form exhibiting a low apparent K(m) (tobacco isozyme). Biochemical analyses of these plants indicated that the transgenic plants had significantly elevated levels of titratable acidity and malic acid. These biochemical differences did not produce any significant physiological changes with respect to photosynthetic rate or CO(2) compensation point. PMID- 16668663 TI - Induction of 1-aminocyclopropane-1-carboxylate synthase mRNA by auxin in mung bean hypocotyls and cultured apple shoots. AB - Auxin is known to promote ethylene production in vegetative tissues by increasing the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase; therefore, we have studied the effect of auxins on ACC synthase mRNA expression. Total RNA was isolated from auxin-incubated cultured apple (Malus sylvestris Mill.) shoots or mung bean (Vigna radiata L.) hypocotyls. These RNAs and a set of oligonucleotide primers corresponding to two conserved amino acid sequences (SNPLGTT and MSSFGLV) found in ACC synthases isolated from other species were used for polymerase chain reaction-based amplification of DNA fragments encoding the ACC synthase-active site domain. We obtained and sequenced a 290-base pair cDNA fragment (pAA1) from cultured apple shoots and a 328-base pair cDNA clone (pMBA1) from mung bean hypocotyls. Comparisons of their deduced amino acid sequences with those of previously characterized ACC synthase cDNAs indicate that both fragments are, indeed, closely related to ACC synthase cDNA. Northern blot analyses further showed that the expression of these transcripts is regulated by auxin treatment. These data indicate that auxin induces ethylene production transcriptionally by increasing the ACC synthase transcripts. The pAA1 shares 46% amino acid sequence homology with ripening-regulated apple fruit ACC synthase, indicating that ripening-regulated and auxin-regulated ACC synthases are encoded by different genes. In mung bean hypocotyls, aminooxyacetic acid, a potent inhibitor of ACC synthase activity, promoted the expression of auxin-induced ACC synthase mRNA, but cycloheximide inhibited this induction. PMID- 16668664 TI - Microheterogeneity in purified broad bean polyphenol oxidase. AB - Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin lectins. Under native isoelectric focusing, several charged isoforms were present in the pH range of 4 to 6. Many, if not all, of the isoforms separated by isoelectric focusing were glycosylated and bound concanavalin A. All these isoforms shared a 65 kilodalton protein in common, and some of the isoforms were associated with both a 65 and 68 kilodalton protein. Isoforms separated by isoelectric focusing in the presence of 9 molar urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a similar pattern of proteins within a slightly higher pH range from 5 to 6.5. PMID- 16668665 TI - Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy. AB - (31)P-, (13)C-, and (15)N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and gamma aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state. PMID- 16668666 TI - Auxin and growth regulation of rice coleoptile segments : molecular analysis. AB - Auxin-stimulated elongation of apical segments of rice (Oryza sativa L. cv Arborio) coleoptiles occurring in the first 4 hours of treatment has been studied. Cell extension promoted in the first 2 hours by 10 micromolar indole-3 acetic acid (IAA) is specifically auxin-dependent, whereas after 4 hours, elongation also depends on endogenous production of ethylene. Similar to other systems, rice coleoptile cell elongation stimulated by auxin requires continuous synthesis of RNA and protein. Two-dimensional gel analysis of the in vitro translation products obtained from polyadenylated RNAs extracted from treated and untreated segments after 1 or 4 hours from the initial addition of IAA shows few transcriptional differences. At 60 minutes of treatment, the level of three mRNAs coding for proteins of 22.5, 25, and 33 kilodaltons was moderately enhanced while the disappearance of a 38 kilodalton translation product was observed. Additional repression of another mRNA coding for a 28 kilodalton product begins to show by this time, but becomes more evident after 4 hours treatment. At 4 hours, four IAA specific mRNA enhancements coding for proteins with molecular masses ranging between 35 to 40 kilodaltons were also observed. We discuss these data in relation to the possible involvement of IAA-mediated transcriptional regulation in growth promotion of rice coleoptiles and, more widely, in control of cell elongation. PMID- 16668667 TI - Wounding Induces One of Two Isoenzymes of 3-Deoxy-d-arabino-Heptulosonate 7 Phosphate Synthase in Solanum tuberosum L. AB - Potato (Solanum tuberosum L.) tubers contain two isoenzymes of 3-deoxy-d-arabino heptulosonate 7-phosphate synthase (EC 4.1.2.15), the enzyme that catalyzes the first step of aromatic amino acid biosynthesis. One of the isoenzymes is specifically activated by Mn(2+), and the other requires Co(2+), Mg(2+), or another divalent cation for activity. Monospecific polyclonal antibodies against the Mn(2+)-activated isoenzyme do not cross-react with the other isoenzyme. Wounding of potato tubers induces the Mn(2+)-activated form but not the other. We conclude that two different genes encode two different isoenzymes that catalyze the first step in the shikimate pathway. PMID- 16668668 TI - Seasonal variation in the antioxidant system of eastern white pine needles : evidence for thermal dependence. AB - Antioxidant metabolites in eastern white pine (Pinus strobus L.) needles increased two- to fourfold from the summer to the winter season. Antioxidant enzymes in needle tissue increased between 2- and 122-fold during this same period. These seasonal changes were determined by monitoring ascorbate and glutathione concentrations and the activity of ascorbate peroxidase, glutathione reductase (GR), and superoxide dismutase. Levels of antioxidant metabolites and enzymes were observed always to be lowest during the summer, or active growing season, and highest during the winter, or dormant season. These data correlated well with the thermal kinetic window for purified GR obtained from summer needles. The minimum, apparent K(m,NADPH) for two isoforms of GR (GR(A) and GR(B)) occurred at 5 and 10 degrees C, respectively. The upper limit of the thermal kinetic window (200% of the minimum K(m)) for GR(A) and GR(B) was 20 and 25 degrees C, respectively, indicating that needle temperatures exceeding 25 degrees C may result in impairment of antioxidant metabolism. The needle content and kinetic properties of GR, the increased activities of other enzymes, and the high substrate concentrations observed during the winter are consistent with the protective function this pathway may provide against photooxidative, winter injury. PMID- 16668669 TI - Distribution of napin and cruciferin in developing rape seed embryos. AB - The distribution of napin and cruciferin, the two major storage proteins in rape seed, Brassica napus, has been visualized during seed development by antibody staining of paraffin-embedded and sectioned seeds. The results indicate that the synthesis of both proteins during embryogenesis is strictly regulated with respect to time and tissue. Although the synthesis of napin started a few days earlier than that of cruciferin, both proteins displayed similar patterns in their spatial distributions. They were first detected in the axis, then in the outer cotyledon, and finally in the cells of the inner cotyledon. Both proteins are also present in the endosperm, although in lower amounts. In germinating seeds, napin and cruciferin were rapidly degraded. Within 2 days the amounts had decreased dramatically, and after 4 days hardly any cells contained napin or cruciferin. Biochemical analyses of dissected embryos showed that, for napin as well as for cruciferin, similar levels of polypeptides were found in the axis and cotyledons. PMID- 16668670 TI - Regulation of Photosynthetic Rate of Two Sunflower Hybrids under Water Stress. AB - The effect of short-term water stress on photosynthesis of two sunflower hybrids (Helianthus annuus L. cv Sungro-380 and cv SH-3622), differing in productivity under field conditions, was measured. The rate of CO(2) assimilation of young, mature leaves of SH-3622 under well-watered conditions was approximately 30% greater than that of Sungro-380 in bright light and elevated CO(2); the carboxylation efficiency was also larger. Growth at large photon flux increased assimilation rates of both hybrids. The changes in leaf composition, including cell numbers and sizes, chlorophyll content, and amounts of total soluble and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein, and in Rubisco activity and amount of ribulose-1,5-bisphosphate (RuBP) were determined to assess the factors regulating the differences in assimilation of the hybrids at high and low water potentials. The amounts of chlorophyll, soluble protein, Rubisco protein and the initial activity of Rubisco and its activation state did not differ significantly between hybrids. However, unstressed leaves of SH-3622 had more, smaller cells per unit area and 60% more RuBP per unit leaf area than that of Sungro-380. Water stress developing over 4 days decreased the assimilation of both hybrids similarly. Changes in the amounts of chlorophyll, soluble and Rubisco protein, and Rubisco activity and activation state were small and were not sufficient to explain the decrease in photosynthesis; neither was decreased stomatal conductance (or stomatal "patchiness"). Reduction of photosynthesis per unit leaf area from 25 to 5 micromoles CO(2) per square meter per second in both hybrids was caused by a decrease in the amount of RuBP from approximately 130 to 40 micromoles per square meter in SH-3622 and from 80 to 40 micromoles per square meter in Sungro. Differences between hybrids and their response to water stress is discussed in relation to control of RuBP regeneration. PMID- 16668671 TI - Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.). AB - Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs. PMID- 16668672 TI - Cellulase occurs in multiple active forms in ripe avocado fruit mesocarp. AB - The existence of multiple forms of avocado (Persea americana Mill. cv Hass) cellulase in crude protein extracts of ripe avocado fruit is reported. Cellulase was separated into at least 11 multiple forms by native isoelectric focusing in the pH range between 4 and 7 and visualized by both activity staining using Congo red and immunostaining. The enzyme components were acidic proteins with isoelectric points in the range of pH 5.10 to 6.80, the predominant forms having isoelectric points of 5.60, 5.80, 5.95, and 6.20. All 11 forms were immunologically related with molecular masses of 54 kilodaltons. PMID- 16668673 TI - Some Enzymes and Properties of the Reductive Carboxylic Acid Cycle Are Present in the Green Alga Chlamydomonas reinhardtii F-60. AB - The reductive carboxylic acid cycle, the autotrophic pathway of CO(2) assimilation in prokaryotes (photosynthetic and nonphotosynthetic autotrophic bacteria), was investigated in Chlamydomonas reinhardtii F-60, an algal mutant lacking a complete photosynthetic carbon reduction pathway (C(3)) due to a deficiency in phosphoribulokinase. Evidence was obtained consistent with the presence of the reductive carboxylic acid cycle in F-60. This conclusion is based on the fact that: (a) acetate approximately doubled CO(2) fixation in whole cells (4 micromoles per milligram chlorophyll per hour) and in chloroplasts (32 nanomoles per milligram chlorophyll per hour); and (b) pyruvate synthase, alpha ketoglutarate synthase, and ATP-citrate lyase, three indicators of the cycle, were found in cell-free extracts. PMID- 16668674 TI - Stomatal response to abscisic Acid is a function of current plant water status. AB - We investigated, under laboratory and field conditions, the possibility that increasing abscisic acid (ABA) concentrations and decreasing water potentials can interact in their effects on stomata. One experiment was carried out with epidermal pieces of Commelina communis incubated in media with a variety of ABA and polyethylene glycol concentrations. In the media without ABA, incubation in solutions with water potentials between -0.3 and -1.5 megapascals had no significant effect on stomatal aperture. Conversely, the sensitivity of stomatal aperture to ABA was trebled in solutions at -1.5 megapascals compared with sensitivity at -0.3 megapascals. The effect of the change in sensitivity was more important than the absolute effect of ABA at the highest water potential. In a field experiment, sensitivity of maize stomatal conductance to the concentration of ABA in the xylem sap varied strongly with the time of the day. We consider that the most likely explanation for this is the influence of a change in leaf or epidermal water potential that accompanies an increase in irradiance and saturation deficit as the day progresses. These observations suggest that epidermal water relations may act as a modulator of the responses of stomata to ABA. We argue that such changes must be taken into account in studies or modeling of plant responses to drought stress. PMID- 16668675 TI - Photoaffinity Labeling of Mature and Precursor Forms of the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase after Expression in Escherichia coli. AB - The small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) possesses a binding site that can be photoaffinity labeled with [(32)P]8-azidoadenosine 5' triphosphate (N(3)ATP). In the present study, photoaffinity labeling was used to compare the nucleotide analog binding properties of SSU in the Rubisco holoenzyme complex (holoE SSU) with the properties of isolated SSU and the precursor form (pSSU) that contains a transit peptide. To facilitate these studies, the complete coding regions of tobacco (Nicotiana tabacum L.) SSU and pSSU were cloned into pET expression vectors and the polypeptides were synthesized in Escherichia coli. Protein import studies showed that cloned pSSU polypeptides were imported into intact chloroplasts, where they were processed to the mature form and assembled into the Rubisco holoenzyme. Cloned SSU and pSSU isolated from E. coli were photoaffinity labeled with N(3)ATP. The apparent K(d) value for SSU and pSSU, 18 micromolar N(3)ATP, was identical to the value determined for holoE SSU. However, differences in photolabeling between cloned SSU or pSSU and holoE SSU were apparent in the level of protection afforded by ATP and UTP, in the response of photolabeling to free Mg(2+), and in the higher photolabeling efficiency that characterized the cloned SSU. Treatment of the Rubisco holoenzyme with a concentration of urea sufficient to disassociate the subunits markedly increased photoincorporation into SSU, indicating that intersubunit associations within the holoenzyme complex may be the major factor influencing photolabeling efficiency of SSU. Thus, differences in SSU conformation between the isolated and assembled states affect photolabeling efficiency and other nucleotide analog binding properties of the SSU, but not the apparent affinity for N(3)ATP. PMID- 16668676 TI - Ripening-related gene from avocado fruit : ethylene-inducible expression of the mRNA and polypeptide. AB - Fruit ripening involves a series of changes in gene expression regulated by the phytohormone ethylene. AVOe3, a ripening-related gene in avocado fruit (Persea americana Mill. cv Hass), was characterized with regard to its ethylene-regulated expression. The AVOe3 mRNA and immunopositive protein were induced in mature fruit within 12 hours of propylene treatment. The AVOe3 mRNA levels reached a maximum 1 to 2 days before the ethylene climacteric, whereas the immunopositive protein continued to accumulate. RNA selected by the pAVOe3 cDNA clone encoded a polypeptide with molecular mass of 34 kilodaltons, corresponding to the molecular mass of the AVOe3 protein determined by immunoblots. The protein was soluble, remaining in solution at 100,000 gravity and eluted as a monomer on gel filtration. Because of its pattern of induction and relationship to an ethylene related gene of tomato, the possible involvement of AVOe3 in ethylene biosynthesis is discussed. PMID- 16668677 TI - Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625. AB - The active transport and intracellular accumulation of HCO(3) (-) by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na(+).Na(+)-dependent HCO(3) (-) accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O(2) evolution and CO(2) fixation. However, when Synechococcus was grown in standing culture, high rates of HCO(3) (-) transport and photosynthesis were observed in the absence of added Na(+). The internal HCO(3) (-) pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO(3) (-) transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air grown cells. The ability of standing culture cells to utilize HCO(3) (-) from the medium in the absence of Na(+) was lost within 16 hours after transfer to air grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO(2) transport involving a high-affinity transport system which was reversibly inhibited by H(2)S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO(2) transport system, whereas Na(+)-dependent and Na(+)-independent HCO(3) (-) transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO(3) (-) was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO(2) fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO(3) (-) + CO(2). The pattern of fluorescence quenching observed in response to added HCO(3) (-) and Na(+) in air-grown and standing culture cells was highly characteristic for Na(+)-dependent and Na(+)-independent HCO(3) (-) accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO(3) (-) transport and the dynamics of intracellular HCO(3) (-) accumulation and dissipation. PMID- 16668678 TI - Overproduction of gamma-Linolenic and Eicosapentaenoic Acids by Algae. AB - The pharmaceutical interest and limited availability of gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA) prompted the search for genetic means for increasing the production of these fatty acids from algal sources. Cell lines of Spirulina platensis and Porphyridium cruentum resistant to the growth inhibition of the herbicide Sandoz 9785 were selected by serial transfers of the culture in the presence of increasing concentrations of the herbicide. The resistant cell lines of S. platensis overproduced GLA and those of P. cruentum overproduced EPA and were stable for at least 50 generations in the absence of the inhibitor. PMID- 16668679 TI - Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition. AB - In leaves of spinach plants (Spinacia oleracea L.) performing CO(2) and NO(3) (-) assimilation, at the time of sudden darkening, which eliminates photosystem I dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells. PMID- 16668680 TI - Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae. AB - Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O(2) evolution, dissolved inorganic carbon accumulation, and photosynthetic CO(2) fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO(2) concentrating process. This COS inhibition can be attributed to inhibition of external HCO(3) (-) conversion to CO(2) and OH(-) by the carbonic anhydrase component of an active CO(2) pump. At a low pH of 5 to 6, COS stimulated O(2) evolution during photosynthesis by algae with low CO(2) in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO(2). Although COS had less effect on HCO(3) (-) accumulation at pH 9 by a HCO(3) (-) pump in Scenedesmus, COS reduced O(2) evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO(2) and H(2)S, its inhibition of the CO(2) pump activity and photosynthesis is not accurate, when measured by O(2) evolution, by NaH(14)CO(3) accumulation, or by (14)CO(2) fixation. PMID- 16668681 TI - Regulation of Nodule Glutamine Synthetase by CO(2) Levels in Bean (Phaseolus vulgaris L.). AB - Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO(2) and from day 8 to 17 in high (0.1%) CO(2) in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO(2) compared with plants grown in 0.02% CO(2) and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO(2), particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO(2) did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the gamma polypeptide was significantly reduced in response to high CO(2), whereas the beta polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper. PMID- 16668682 TI - Partial Purification and Characterization of Complex I, NADH:Ubiquinone Reductase, from the Inner Membrane of Beetroot Mitochondria. AB - A NADH dehydrogenase was isolated from an inner membrane-enriched fraction of beetroot mitochondria (Beta vulgaris L.) by solubilization with sodium deoxycholate and purified using gel filtration and affinity chromatography. The NADH dehydrogenase preparation contained a minor ATPase contamination. Beetroot mitochondria were chosen as the isolation material for purifying the enzymes responsible for oxidizing matrix NADH due to the absence of the externally facing NADH dehydrogenase in the variety we have used. The purified NADH dehydrogenase complex catalyzed the reduction of various electron acceptors with NADH as the electron donor, was not sensitive to rotenone inhibition, and had a slow NADPH ubiquinone 5 reductase activity. The isolated complex contained 14 major polypeptides. It was concluded that the dehydrogenase represented a form of the plant mitochondrial complex I and not the internally facing rotenone-insensitive NADH dehydrogenase found in plant mitochondria because of its complex structure, its cross-reactivity with antisera raised against bovine heart mitochondrial complex I, and the similarity of its kinetics and inhibitor responses to rotenone sensitive NADH oxidation by beetroot submitochondrial particles. PMID- 16668683 TI - Processing of the Precursors for the Light-Harvesting Chlorophyll-Binding Proteins of Photosystem II and Photosystem I during Import and in an Organelle Free Assay. AB - We have investigated whether the precursors for the light-harvesting chlorophyll a/b binding proteins (LHCP) of photosystems II and I (PSII and PSI) are cleavable substrates in an organelle-free reaction, and have compared the products with those obtained during in vitro import into chloroplasts. Representatives from the tomato (Lycopersicon esculentum) LHCP family were analyzed. The precursor for LHCP type I of PSII (pLHCPII-1), encoded by the tomato gene Cab3C, was cleaved at only one site in the organelle-free assay, but two sites were recognized during import, analogous to our earlier results with a wheat precursor for LHCPII-1. The relative abundance of the two peptides produced was investigated during import of pLHCPII-1 into chloroplasts isolated from plants greened for 2 or 24 hours. In contrast to pLHCPII-1, the precursors for LHCP type II and III of PSI were cleaved in both assays, giving rise to a single peptide. The precursor for LHCP type I of PSI, encoded by gene Cab6A, yielded two peptides of 23.5 and 21.5 kilodaltons during import, whereas in the organelle-free assay only the 23.5 kilodalton peptide was found. N-terminal sequence analysis of this radiolabeled peptide has tentatively identified the site cleaved in the organelle-free assay between met40 and ser41 of the precursor. PMID- 16668684 TI - Metabolism of ent-Kaurene to Gibberellin A(12)-Aldehyde in Young Shoots of Normal Maize. AB - Young shoots of normal maize (Zea mays L.) were used to determine both the stepwise metabolism of ent-kaurene to gibberellin A(12)-aldehyde and the endogenous presence of the members in this series. Each of the five steps in the sequence was established by feeds of 17-(13)C, (3)H-labeled kauranoids to cubes from the cortex of elongating internodes, to homogenates from the cortex of elongating internodes, and/or to homogenates from dark-grown seedlings. The (13)C metabolites were identified by Kovats retention indices (KRI) and full-scan capillary gas chromatography-mass spectrometry (GC-MS). Five substrates and the final product in this sequence were shown to be native by the isotopic dilution of 17-(13)C, (3)H-labeled substrates added as internal standards to extracts obtained from elongating internodes. Evidence for the isotopic dilution was obtained by KRI and full-scan capillary GC-MS. Thus, we document the presence in young maize shoots of the metabolic steps, ent-kaurene --> ent-kaurenol --> ent kaurenal --> ent-kaurenoic acid --> ent-7 alpha-hydroxykaurenoic acid --> gibberellin A(12)-aldehyde. PMID- 16668685 TI - Transport kinetics and metabolism of exogenously applied putrescine in roots of intact maize seedlings. AB - Putrescine metabolism, uptake, and compartmentation were studied in roots of hydroponically grown intact maize (Zea mays L.) seedlings. In vivo analysis of exogenously applied putrescine indicated that the diamine is primarily metabolized by a cell wall-localized diamine oxidase. Time-dependent kinetics for putrescine uptake could be resolved into a rapid phase of uptake and binding within the root apoplasm, followed by transport across the plasma membrane that was linear for 30 to 40 minutes. Concentration-dependent kinetics for putrescine uptake (between 0.05 and 1.0 millimolar putrescine) appeared to be nonsaturating but could be resolved into a saturable (V(max) 0.397 micromoles per gram fresh weight per hour; K(m) 120 micromolar) and a linear component. The linear component was determined to be cell wall-bound putrescine that was not removed during the desorption period following uptake of [(3)H]putrescine. These results suggest that a portion of the exogenously applied putrescine can be metabolized in maize root cell walls by diamine oxidase activity, but the bulk of the putrescine is transported across the plasmalemma by a carrier-mediated process, similar to that proposed for animal systems. PMID- 16668686 TI - Differential Expression of Histone H3 Gene Variants during Cell Cycle and Somatic Embryogenesis in Alfalfa. AB - Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [(3)H]thymidine incorporation. Histone H3 11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4 dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belongs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription. PMID- 16668687 TI - Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures. AB - The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy dependent programs. PMID- 16668688 TI - Mechanism of aluminum inhibition of net ca uptake by amaranthus protoplasts. AB - Calcium ions serve as a second messenger in signal transduction and metabolic regulation. Effects of Al on calcium homeostasis remain to be elucidated. Short term net (45)Ca(2+) uptake by Amaranthus tricolor protoplasts was monitored from uptake media prepared to test the influence of pH, Al, and various inhibitors. Accumulation of (45)Ca(2+) increased during the first 3 to 6 minutes and then leveled off or declined. Al and Ca(2+) channel blockers (verapamil and bepridil) decreased net (45)Ca(2+) uptake. This decrease was more pronounced when Al and bepridil were both present in uptake media, but Al did not aggravate verapamil induced reduction of net (45)Ca(2+) uptake. Erythrosin B and calmidazolium each increased net (45)Ca(2+) uptake, probably by interfering with Ca(2+) efflux. This effect was undetectable in the presence of Al. Mycophenolic acid decreased net (45)Ca(2+) uptake; guanosine alleviated this effect. Al-induced reduction of net (45)Ca(2+) uptake was not aggravated by mycophenolic acid. Net (45)Ca(2+) uptake was generally less at pH 4.5 than at 5.5 for all treatments. It is concluded that Al ions affect net (45)Ca(2+) uptake by binding to the verapamil-specific channel site that is different from the bepridil-specific one, as well as by interfering with the action of guanosine 5'-triphosphate-binding proteins. PMID- 16668689 TI - Differential Inhibition by Ferulic Acid of Nitrate and Ammonium Uptake in Zea mays L. AB - The influence of the allelopathic compound ferulic acid (FA) on nitrogen uptake from solutions containing both NO(3) (-) and NH(4) (+) was examined in 8-day-old nitrogen-depleted corn (Zea mays L.) seedlings. Concurrent effects on uptake of Cl(-) and K(+) also were assessed. The presence of 250 micromolar FA inhibited the initial (0-1 hours) rate of NO(3) (-) uptake and also prevented development of the NO(3) (-)-inducible accelerated rate. The pattern of recovery when FA was removed was interpreted as indicating a rapid relief of FA-restricted NO(3) (-) uptake activity, followed by a reinitiation of the induction of that activity. No inhibition of NO(3) (-) reduction was detected. Ammonium uptake was less sensitive than NO(3) (-) uptake to inhibition by FA. An inhibition of Cl(-) uptake occurred as induction of the NO(3) (-) transport system developed in the absence of FA. Alterations of Cl(-) uptake in the presence of FA were, therefore, a result of a beneficial effect, because NO(3) (-) uptake was restricted, and a direct inhibitory effect. The presence of FA increased the initial net K(+) loss from the roots during exposure to the low K, ammonium nitrate uptake solution and delayed the recovery to positive net uptake, but it did not alter the general pattern of the response. The implications of the observations are discussed for growth of plants under natural conditions and cultural practices that foster periodic accumulation of allelopathic substances. PMID- 16668690 TI - Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles. AB - Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall. PMID- 16668691 TI - Glyphosate Suppression of an Elicited Defense Response : Increased Susceptibility of Cassia obtusifolia to a Mycoherbicide. AB - The major effort in developing pathogenic fungi into potential mycoherbicides is aimed at increasing fungal virulence to weeds without affecting crop selectivity. Specific suppression of biosynthesis of a phytoalexin derived from the shikimate pathway in Cassia obtusifolia L. by a sublethal dose (50 micromolar) of glyphosate increased susceptibility to the mycoherbicide Alternaria cassiae Jurair & Khan. Glyphosate applied with conidia suppressed phytoalexin synthesis beginning at 12 hours, but not an earlier period 8 to 10 hours after inoculation. The phytoalexin synthesis elicited by fungal inoculation was also suppressed by darkness. The magnitudes of virulence of the mycoherbicide in the dark or with glyphosate in the light were both higher than after inoculation in the light with the same concentration of conidia in the absence of glyphosate. Five times less inoculum was needed to cause disease symptoms when applied with glyphosate than without. Glyphosate did not render A. cassiae virulent on soybean (Glycine max), a crop related to the host. These results suggest that a specific inhibition of a weed's elicited defense response can be a safe way to enhance virulence and improve the efficacy of the mycoherbicide. PMID- 16668692 TI - Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon. AB - The extent and occurrence of water stress-induced "patchy" CO(2) uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [(14)C]CO(2) before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to -1.1 megapascals during a 4-day period did result in heterogenous CO(2) assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO(2) assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO(2)] in stressed bean leaves. When extent of heterogenous CO(2) uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to -1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO(2) assimilation patterns when stressed to a leaf water potential of -2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits. PMID- 16668693 TI - Stromal pH and Photosynthesis Are Affected by Electroneutral K and H Exchange through Chloroplast Envelope Ion Channels. AB - Potassium movement across the limiting membrane of the chloroplast inner envelope is known to be linked to counterex-change of protons. For this reason, K(+) efflux is known to facilitate stromal acidification and the resultant photosynthetic inhibition. However, the specific nature of the chloroplast envelope proteins that facilitate K(+) fluxes, and the biophysical mechanism which links these cation currents to H(+) counterflux, is not characterized. It was the objective of this work to elucidate the nature of the system regulating K(+) flux linked to H(+) counterflux across the chloroplast envelope. In the absence of external K(+), exposure of spinach (Spinacia oleracea) chloroplasts to the K(+) ionophore valinomycin was found to increase the rate of K(+) efflux and H(+) influx. These data were interpreted as suggesting that H(+) counterexchange must be indirectly linked to movement of K(+) across the envelope. Studies using the K(+) channel blocker tetraethylammonium indicated that K(+) likely moves, in a uniport fashion, into or out of the stroma through a monovalent cation channel in the envelope. Blockage of K(+) efflux from the stroma by exposure to tetraethylammonium was found to restrict H(+) influx, further substantiating an indirect linkage of these cation currents. Further studies comparing the effect of exogenous H(+) ionophores and K(+)/H(+) exchangers suggested that K(+) uniport through this ion channel likely is the main endogenous pathway for K(+) currents across the envelope. These experiments were also consistent with the presence of a proton channel in the envelope. Movement of H(+) through this channel was speculated to be regulated and rate limited by an electroneutral requirement for K(+) countercurrents through the separate K(+) uniport pathway. K(+) and H(+) fluxes across the chloroplast envelope were envisioned to be interrelated via this mechanism. The significant effect of cation currents across the envelope, as mediated by these channels, on photosynthetic capacity of the isolated chloroplast was also demonstrated. PMID- 16668694 TI - Characterization of parameters influencing receptor-mediated endocytosis in cultured soybean cells. AB - In a recent publication, we were able to demonstrate that biotin enters plant cells by receptor-mediated endocytosis and that impermeable macromolecules can be cotransported into cells by the same pathway if they are first covalently linked to biotin. In the present study, we have exploited the biotin endocytosis pathway to evaluate the variables in the cell wall and surrounding growth medium that influence the efficiency of endocytosis in plants. Under normal growth conditions, the major constraint limiting macromolecule endocytosis was found to be the size of the internalized macromolecule. Thus, a log-linear relationship with a negative slope exists between the molecular weight of the biotin conjugated macromolecule and its rate of internalization by cultured soybean cells. This relationship, which extends from insulin (M(r) approximately 5700) to immunoglobulin G (M(r) approximately 160,000), is characterized by a slope of 1.04 x 10(5) molecules/cell/min per log M(r) unit and an x intercept (no endocytosis detectable) of approximately log 160,000 daltons. Unfortunately, mild digestion with cell wall-degrading enzymes is unable to increase significantly the upper size limit of molecules that can be internalized, but uptake of lower molecular weight proteins can be enhanced by mild cell wall digestion. The optimal extracellular pH for endocytosis was found to be 4.6, i.e. near the normal pH of the cell culture medium. Furthermore, the osmotic strength at which endocytosis occurs most rapidly was observed to be isotonic to slightly hypotonic, suggesting that turgor pressure within the plant cell must not be a major determinant of endocytosis rates by cultured soybean (Glycine max) cells. Finally, cell age was found to impact significantly on the rate of macromolecule internalization, with maximal uptake rates occurring during early exponential growth and decreasing by a factor of 2 when the cells reach stationary growth phase. PMID- 16668695 TI - Effect of Elicitation and Changes in Extracellular pH on the Cytoplasmic and Vacuolar pH of Suspension-Cultured Soybean Cells. AB - We have employed both (31)P nuclear magnetic resonance spectroscopy and two intracellular fluorescent pH indicator dyes to monitor the pH of the vacuole and cytoplasm of suspension-cultured soybean cells (Glycine max Merr cv Kent). For the (31)P nuclear magnetic resonance studies, a flow cell was constructed that allowed perfusion of the cells in oxygenated growth medium throughout the experiment. When the perfusion medium was transiently adjusted to a pH higher than that of the ambient growth medium, a rapid elevation of vacuolar pH was observed followed by a slow (approximately 30 minute) return to near resting pH. In contrast, the concurrent pH changes in the cytoplasm were usually fourfold smaller. These data indicate that extracellular pH changes are rapidly communicated to the vacuole in soybean cells without significantly perturbing cytoplasmic pH. When elicitors were dissolved in a medium of altered pH and introduced into the cell suspension, the pH of the vacuole, as above, quickly reflected the pH of the added elicitor solution. In contrast, when the pH of either a polygalacturonic acid or Verticillium dahliae elicitor preparation was adjusted to the same pH as the ambient medium, no significant change in either vacuolar or cytoplasmic pH was observed during the 35 minute experiment. These results were confirmed in experiments with pH-sensitive fluorescent dyes. We conclude that suspension-cultured soybean cells do not respond to elicitation by significantly changing the pH of their vacuolar or cytoplasmic compartments. PMID- 16668696 TI - Complementary DNA cloning of poplar bark storage protein and control of its expression by photoperiod. AB - Bark storage proteins accumulate in the bark of many woody plants during autumn and winter. In poplar (Populus deltoides Bartr. ex Marsh), the accumulation of the 32-kilodalton bark storage protein is controlled by photoperiod. We have isolated a full-length cDNA encoding for the poplar 32-kilodalton bark storage protein and determined its nucleotide sequence. The derived amino acid sequence shows that poplar bark storage protein is rich in serine, leucine, phenylalanine, and lysine. Poplar bark storage protein is similar to the poplar wound-induced cDNA clone 4 and clone 16 (TJ Parsons, HD Bradshaw, MP Gordon [1989] Proc Natl Acad Sci USA 86: 7895-7899). DNA gel blot analysis suggests that poplar bark storage protein is encoded by a multigene family of about five genes. Poplar plants grown in long days contained low levels of mRNA for the bark storage protein. Exposure to short days resulted in an increase in bark storage protein mRNA within 7 days. After 21 days of short day exposure, high levels of mRNA were detected. The accumulation of bark storage protein mRNA in response to short days was also observed in plants exposed to natural shortening daylengths. Our results indicate that the accumulation of poplar bark storage protein mRNA is controlled by photoperiod. This finding will provide a useful system for investigating photoperiodism in woody plants. PMID- 16668697 TI - Photosynthesis and photorespiration in presenescent, senescent, and rejuvenated soybean cotyledons. AB - Various growth and physiological parameters were measured in germinating, presenescent, and senescing soybean (Glycine max [L.] Merr.) cotyledons and in cotyledons rejuvenated by epicotyl removal 18 days after planting. The maximal measured carbon dioxide exchange rates (CER) in the cotyledons were in the range of those reported for field-grown soybean leaves. Rejuvenated cotyledons accumulated total chlorophyll in excess of the maximum observed in presenescent cotyledons. When photosynthetic rates were expressed per cotyledon, the CER in rejuvenated tissue recovered to the maximal rates observed in presenescent cotyledons. Ribulose-1,5-bisphosphate carboxylase/oxygenase in rejuvenated cotyledons also recovered to the maximal amount seen in presenescent cotyledons so that CER appeared to be a function of ribulose-1,5-bisphosphate carboxylase/oxygenase content during most of the period studied. Observations of the postillumination outburst of CO(2) and (14)C label in glycine indicated that photorespiration was occurring in the cotyledons and that photorespiration relative to photosynthesis was different in rejuvenated compared with presenescent cotyledons. PMID- 16668698 TI - Changes in the polypeptide patterns of barley seedlings exposed to jasmonic Acid and salinity. AB - Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins. PMID- 16668699 TI - beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis. AB - In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of beta-furfuryl-beta-glucoside (FG), an endogenous activator of plant UDP-glucose:(1-->3)-beta-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized alpha-furfuryl-beta glucoside also stimulates callose synthase, exhibiting a slightly higher K(a) of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using beta-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca(2+), is proposed. PMID- 16668700 TI - Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit. AB - This work was undertaken in an effort to reconcile the conflicting proline accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings. PMID- 16668701 TI - Energy requirements for Fatty Acid and glycerolipid biosynthesis from acetate by isolated pea root plastids. AB - Fatty acid and glycerolipid biosynthesis from [(14)C]acetate by isolated pea root plastids is completely dependent on exogenously supplied ATP. CTP, GTP, and UTP are ineffective in supporting fatty acid biosynthesis, all resulting in <3% of the activity obtained with ATP. However, ADP alone or in combination with inorganic phosphate (Pi) or pyrophosphate (PPi) gave up to 28% of the ATP control activity, whereas AMP + PPi, PPi alone, or Pi alone were ineffective in promoting fatty acid biosynthesis. The components of the dihydroxyacetonephosphate (DHAP) shuttle (DHAP, oxaloacetate, and Pi), which promote intraplastidic ATP synthesis, restored 41% of the control ATP activity, whereas the omission of any of the shuttle components abolished this activity. When the DHAP shuttle components were supplemented with ADP, the rate of fatty acid biosynthesis was completely restored to that observed in the presence of ATP. Under the conditions of ADP + DHAP shuttle-driven fatty acid biosynthesis, exogenously supplied ATP gave only a 6% additional stimulation of activity. In general, variations in the energy source had only small effects on the proportions of radioactive fatty acids and glycerolipids synthesized. Most notably, higher amounts of radioactive oleic acid, free fatty acids, and diacylglycerol and lower amounts of phosphatidic acid were observed when ADP and/or the DHAP shuttle were substituted for ATP. The results presented here indicate that, although isolated pea root plastids readily utilize exogenously supplied ATP for fatty acid biosynthesis, these plastids can also synthesize sufficient ATP when provided with the appropriate cofactors. PMID- 16668702 TI - Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells. AB - Elicitor induction of phenylpropanoid metabolism was investigated in suspension cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4 coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin beta glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in beta-glucosidase activity capable of hydrolyzing 4 nitrophenyl-beta-glucoside was observed. Subsequent to increases in l phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds. PMID- 16668703 TI - Oxidation and reduction of sulfite by chloroplasts and formation of sulfite addition compounds. AB - After exposing intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) and capable of photoreducing CO(2) at high rates to different concentrations of radioactive sulfite in the light or in the dark, (35)SO(2) and H(2) (35)S were removed from the acidified suspensions in a stream of nitrogen. Remaining activity could be fractionated into sulfate, organic sulfides, and sulfite addition compounds. When chloroplast suspensions contained catalase, superoxide dismutase and O-acetylserine, the oxidation of sulfite to sulfate was slower in the light than the reductive formation of sulfides that exhibited a maximum rate of about 2 micromoles per milligram chlorophyll per hour, equivalent to about 1% of maximum carbon assimilation. Botht the oxidative and the reductive detoxification of sulfite were very slow in the dark. Oxidation was somewhat, but not much, accelerated in the light in the absence of O-acetylserine, which caused a dramatic decrease in the formation of organic sulfides and an equally dramatic increase in the concentration of sulfite addition compounds whose formation was light-dependent. The sulfite addition compounds were not identified. Addition compounds did not accumulate in the dark. In the light, the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, decreased not only the reduction, but also the oxidation of sulfite and the formation of addition compounds. PMID- 16668704 TI - Abscisic Acid increases terrestrial plant cell resistance to hydrostatic pressure. AB - Cells of the terrestrial plant species bromegrass (Bromus inermis L.) are not naturally adapted to withstand the hydrostatic pressures encountered in aquatic environments. However, after treatment with the natural plant growth hormone abscisic acid (75 micromolar), bromegrass cells survived a hydrostatic pressure of 101.3 megapascals, approximating the limits of ocean depth (10,860 m). The increased resistance to hydrostatic pressure from 1 to 7 days of abscisic acid treatment paralleled the induced elevation of cell tolerance to freezing stress. PMID- 16668705 TI - Low measuring temperature induced artifactual increase in chlorophyll a fluorescence. AB - Measurement of in vivo chlorophyll a fluorescence at temperatures lower than 20 degrees C can cause an artifactual, nonphotochemically related overestimation of variable fluorescence leading to the calculation of negative values for the nonphotochemical quenching parameter and an underestimation of the photochemical quenching parameter. This artifact is observed only upon exposure of the leaf sample to actinic light. We suggest that a temperature differential between the fiber-optic probe and the leaf sample results in the deposition of water vapor on the probe that distorts the light path such that an increased modulated fluorescence signal is observed. This artifact is eradicated by ensuring that the end of the fiber-optic probe is kept free of condensation. PMID- 16668706 TI - High Performance Liquid Chromatography Analysis of Carbohydrates of Cotton-Phloem Sap and of Honeydew Produced by Bemisia tabaci Feeding on Cotton. AB - Phloem sap from cotton (Gossypium hirsutum L.) was collected from young and mature leaves by the aphid-stylet technique. Exudate was analyzed for carbohydrates by HPLC using sensitive pulsed amperometric detection. The predominant carbohydrate present (>90%) was identified as sucrose. A second, unidentified compound that was not one of the more commonly translocated sugars was detected in mature leaves. Carbohydrates in honeydew produced by the sweet potato whitefly (Bemisia tabaci [Genn.]) feeding on cotton were sucrose, glucose, fructose, trehalulose, and a series of oligosaccharides. PMID- 16668707 TI - CO(2) Inhibits Respiration in Leaves of Rumex crispus L. AB - Curly dock (Rumex crispus L.) was grown from seed in a glasshouse at an ambient CO(2) partial pressure of about 35 pascals. Apparent respiration rate (CO(2) efflux in the dark) of expanded leaves was then measured at ambient CO(2) partial pressure of 5 to 95 pascals. Calculated intercellular CO(2) partial pressure was proportional to ambient CO(2) partial pressure in these short-term experiments. The CO(2) level strongly affected apparent respiration rate: a doubling of the partial pressure of CO(2) typically inhibited respiration by 25 to 30%, whereas a decrease in CO(2) elicited a corresponding increase in respiration. These responses were readily reversible. A flexible, sensitive regulatory interaction between CO(2) (a byproduct of respiration) and some component(s) of heterotrophic metabolism is indicated. PMID- 16668708 TI - Light and Fungal Elicitor Induce 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase mRNA in Suspension Cultured Cells of Parsley (Petroselinum crispum L.). AB - Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7 phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated. PMID- 16668709 TI - Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate. AB - A new method is presented for measurement of the CO(2)/O(2) specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [(14)C]3 phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. (14)CO(2) fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO(2) in O(2)-saturated water and carboxylase only with 160 micromolar CO(2) under N(2). Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high performance anion exchange chromatography and the radioactive counts of the [(14)C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 +/- 4), from the green alga Chlamydomonas reinhardtii (66 +/- 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods. PMID- 16668710 TI - Behavior of Etiolated Peas (Pisum sativum cv Alaska) When Obstructed by a Mechanical Barrier. AB - Etiolated pea (Pisum sativum cv Alaska) seedlings growing against a horizontal barrier in the soil will assume a horizontal orientation and continue to grow for prolonged periods of time. With removal of the barrier or after seedlings grow out from underneath the obstruction, seedlings immediately return to normal vertical growth. Ethylene production increased several hours after the seedlings began to grow horizontally and not at the first contact with a barrier. Increases in ethylene production from horizontally growing seedlings were associated with decreased rates of elongation and increased stem diameter. The data suggest that increased ethylene production does not play a mediating role in the horizontal growth of pea seedlings when obstructed during emergence. We conclude that seedlings follow a path of least resistance when they grow against a barrier in the soil. PMID- 16668711 TI - Hysteresis and cooperative behavior of a latent plant polyphenoloxidase. AB - Appearance of a lag period dependent on pH in the expression of the catecholase activity of a polyphenoloxidase extracted in a latent state from Airen grape (Vitis vinifera L.) berries, is revealed, suggesting the hysteretic nature of the enzyme. The lag time was independent of enzyme concentration, indicating that slow pH-induced conformational changes in the protein must occur during assay. Results obtained by varying substrate concentration show that the system presents hyperbolic or cooperative kinetics depending on the pH of the assay. PMID- 16668712 TI - Induction of Pseudoactinorhizae by the Plant Pathogen Agrobacterium rhizogenes. AB - Infection of Elaeagnus angustifolia cotyledonary wounds by Agrobacterium rhizogenes strain NCPPB 2659 resulted in the formation of pseudoactinorhizae on roots differentiated from callus. These pseudoactinorhizal root nodules were anatomically indistinguishable from the actinorhizae induced by the plant's microsymbiont Frankia. This unusual hairy root phenotype provides support for the concept that the genetic program for actinorhiza morphogenesis resides in the plant's genome. PMID- 16668713 TI - Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase. AB - The effect of 5-5'-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V(max) is found to be independent of the presence of this thiol reagent. The K(m) is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K(m) (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K(m) for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT. PMID- 16668714 TI - Mannityl opine accumulation and exudation by transgenic tobacco. AB - Three genes from the T(R) region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants. PMID- 16668715 TI - Cloning and Nucleotide Sequence of a Complementary DNA Encoding the Cytosolic Fructose-1,6-Bisphosphatase of Sugar Beet (Beta vulgaris L.). PMID- 16668716 TI - Nucleotide Sequence of a Soybean Vegetative Storage Protein vspA Gene. PMID- 16668717 TI - Nucleotide Sequence of a Soybean Vegetative Storage Protein vspB Gene. PMID- 16668718 TI - Nucleotide sequence of a complementary DNA encoding o-methyltransferase from poplar. PMID- 16668719 TI - Molecular Analysis of the psaC Gene Encoding the F(A)/F(B) Apoprotein of Photosystem I in the Filamentous Cyanobacterium Anabaena variabilis ATCC 29413. PMID- 16668720 TI - Regulation of phycobilisome structure and gene expression by light intensity. AB - The cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp PCC 7002) was grown turbidostatically in white light at three levels of irradiance: 20, 200, and 1260 microeinsteins per square meter per second. Phycobilisomes were isolated from each culture and analyzed by absorbance, gel electrophoresis, and electron microscopy. The ratio of phycocyanin to allophycocyanin decreased 1.8-fold from the lowest to highest irradiance. This change was due entirely to an approximately 2.5-fold decrease in one structural unit of rod domains, the complex of phycocyanin, and a 33-kilodalton linker polypeptide (LR33). For a given irradiance, phycobilisomes from cells grown on ammonium as the nitrogen source had 10 to 20% more phycocyanin than those from nitrate cultures. Total RNA was isolated from all cultures and probed with gene fragments specific to phycocyanin and allophycocyanin subunits and LR33. The relative level of RNAs encoding phycocyanin and allophycocyanin was found to vary with light intensity in parallel with the phycobiliprotein ratio. Hence, the light-harvesting capacity of phycobilisomes is directly regulated by relative levels of phycobiliprotein mRNA. The LR33 transcript occurs as a 3' extension on about 10% of phycocyanin transcripts. The ratio of RNA encoding LR33 to that encoding phycocyanin did not vary with irradiance, although the protein ratio changed 1.7- to twofold between extremes. Based on these and other observations, we propose that the LR33 protein is constitutively synthesized at a rate higher than that required to complex with available phycocyanin. PMID- 16668721 TI - Differential Synthesis of Photosystem Cores and Light-Harvesting Antenna during Proplastid to Chloroplast Development in Spirodela oligorrhiza. AB - Proplastids and etioplasts are common starting points for monitoring chloroplast development in higher plants. Although proplastids are the primary precursor of chloroplasts, most proplastid to chloroplast systems are cumbersome to study temporally. Conversely, the etioplast to chloroplast transition is initiated by light and is readily examined as a function of time. Etioplasts, however, are found mostly in plants germinated in the dark and are not an obligatory step in chloroplast development. We have chosen to study chloroplast ontogeny in Spirodela oligorrhiza (Kurtz) Hegelm (a C(3)-monocot) because of its unique ability to grow indefinitely in the dark. Ultrastructural, physiological, and molecular evidence is presented in support of a temporal, light-triggered proplastid to chloroplast transition in Spirodela. The dark-grown plants are devoid of chlorophyll, and upon illumination synchronously green over a 3- to 5 day period. Synthesis of chloroplast proteins involved in photosynthesis is coincident with thylakoid assembly, chlorophyll accumulation, and appearance of CO(2) fixation activity. Interestingly, the developmental sequence in Spirodela was slow enough to reveal that biosynthesis of the D1 photosystem II reaction center protein precedes biosynthesis of the major light-harvesting antenna proteins. This, coupled with the high chlorophyll a/b ratio observed early in development, indicated that reaction center assembly occurred prior to accumulation of the light-harvesting complexes. Thus, with Spirodela one can study proplastid to chloroplast conversions temporally in higher plants and follow the process on a time scale that enables a detailed dissection of plastid maturation processes. PMID- 16668722 TI - Proline fed to intact soybean plants influences acetylene reducing activity and content and metabolism of proline in bacteroids. AB - Supplying l-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding l-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylene reducing activity. Twenty-four hours after irrigating with l-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-(14)C]l-proline was supplied to uprooted, intact plants incubated in aerated solution, (14)C-labeled products of proline metabolism, as well as [(14)C]proline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-(3)H]l-proline, (3)H-labeled proline was found in host cells and bacteroids. [(3)H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-(3)H]l-proline. When [U-(14)C]l-proline was supplied for 24 hours, a significant amount of [(14)C] pyrroline-5 carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-(3)H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the in vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules. PMID- 16668723 TI - Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin. AB - Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H(+) translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V(max) of the enzyme. Fusicoccin stimulation of maize H(+)-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.). PMID- 16668724 TI - Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis. AB - The uptake and metabolism of the cytokinin benzyl adenine (BA) was compared in two lines of Petunia hybrida Vilm. differing in their shoot organogenic response. Leaf transfer experiments using shoot induction medium containing 4.4 micromolar BA showed that leaf explants from petunia line St40 required a shoot induction period of 6 to 10 days for commitment to shoot organogenesis; whereas leaf explants from petunia TLV1 required 12 to 28 days. The short induction period of petunia St40 and the higher organogenic response was positively associated with a threefold higher absorption of BA from the medium, an increased BA ribotide metabolite pool, the presence of BA within the explant during the shoot induction period, and the production of an unidentified metabolite C. However, the study of petunia TLV1 leaf explants showed that neither BA nor metabolite C are required during the shoot induction period for eventual shoot development. The longer shoot induction period of TLV1 was associated with low BA uptake during 24 days, a decreasing ribotide metabolite pool, the absence of benzyl adenosine triphosphate and metabolite C throughout the study, and the absence of BA within the explant during the shoot induction period. Differences in the shoot organogenic response of these related plant lines have been shown to be associated with differences in exogenous cytokinin uptake and the subsequent metabolism of that hormone. PMID- 16668725 TI - Physiological changes accompanying senescence in the ephemeral daylily flower. AB - The daylily flower, Hemerocallis hybrid cv Cradle Song, develops from the opening bud to full senescence in 36 hours. Unlike other ephemeral flowers studied to date, it does not respond to ethylene, but other senescence phenomena are similar. There was a small respiration climacteric coinciding with early flower senescence, and it was also observed in isolated petals and petal slices. Cycloheximide abolished the climacteric and delayed senescence in all three systems. Petal apparent free space increased from 30% at bud opening to 38% at the onset of senescence, and sugar efflux increased from 0.2 to 2.8 milligrams per gram of fresh weight per hour during the same period. A sharp increase in ion efflux from 0.8 to 4.0 micromoles of NaCl equivalents per gram of fresh weight per hour, coinciding with the climacteric, was abolished by cycloheximide. Uptake of radiolabeled inorganic phosphate by petal slices from 100 micromolar solution increased during onset of senescence from 6 to 10 nmoles per gram of fresh weight per hour. Half was esterified; of this, 14% went into ATP, and the cellular energy charge remained high at 0.86 during senescence. The proportion incorporated into phospholipid (2.2%) did not change during senescence, but the proportion in phosphatidyl choline increased and in phosphatidyl glycerol decreased during senescence. The general phosphate ester pattern in presenescent slices closely resembled that in other plant tissues except that phospholipid precursors were more prominent (approximately 20% of total organic (32)P versus 5%). In senescent slices, the proportion of hexose phosphates decreased from 40 to 15% of total organic (32)P and that of phospholipid precursors increased to approximately 50%, suggesting that phospholipid synthesis was blocked early in senescence. PMID- 16668726 TI - Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. AB - A cell suspension culture of tobacco (Nicotiana tabacum L.) was used as a model to study injury to cells during biolistic transformation. Lawns of cells were bombarded with tungsten particles that were coated with a plasmid containing the beta-glucuronidase and the neomycin phosphotransferase II genes. When a gunpowder driven biolistic device was used, numerous transiently expressing cells were focused around the epicenter of the blast which was manifested by a hole blown in the filter paper supporting the cells. However, transformed cells nearest the blast epicenter were injured and could not be recovered as stable transformants. The injury was primarily caused by physical trauma to the cells from gas blast and acoustic shock generated by the device. Postlaunch baffles or meshes placed in the gunpowder device reduced cell injury and increased the recovery of kanamycin-resistant colonies 3.5- and 2.5-fold, respectively. A newly developed helium-driven device was more gentle to the cells and also increased the number of transformants. Cell injury could be further moderated by using a mesh and a prelaunch baffle in the helium device. Toxicity of the tungsten microprojectiles also contributed to cell injury. Gold microprojectiles were not toxic and resulted in fourfold more kanamycin-resistant colonies than when similar quantities of similarly sized tungsten particles were used. PMID- 16668727 TI - Quantitative models characterizing seed germination responses to abscisic Acid and osmoticum. AB - Mathematical models were developed to characterize the physiological bases of the responses of tomato (Lycopersicon esculentum Mill. cv T5) seed germination to water potential (psi) and abscisic acid (ABA). Using probit analysis, three parameters were derived that can describe the germination time courses of a seed population at different psi or ABA levels. For the response of seed germination to reduced psi, these parameters are the mean base water potential ( psi(b), MPa), the standard deviation of the base water potential among seeds in the population (sigma(psib), MPa), and the "hydrotime constant" (theta(H), MPa.h). For the response to ABA, they are the log of the mean base ABA concentration ([unk]ABA(b), m), the standard deviation of the base ABA concentration among seeds in the population (sigma(ABA) (b), log[m]), and the "ABA-time constant" (theta(ABA), log[m].h). The values of psi(b) and [unk]ABA(b) provide quantitative estimates of the mean sensitivity of germination rate to psi or ABA, whereas sigma(psi) (b) and sigma(ABA) (b) account for the variation in sensitivity among seeds in the population. The time constants, theta(H) and theta(ABA), indicate the extent to which germination rate will be affected by a given change in psi or ABA. Using only these parameters, germination time courses can be predicted with reasonable accuracy at any medium psi according to the equation probit(g) = [psi (theta(H)/t(g)) - psi(b)]/sigma(psib), or at any ABA concentration according to the equation probit(g) = [log[ABA] - (theta(ABA)/t(g)) - log[[unk]ABA(b)]]/sigma(ABA) (b), where t(g) is the time to radicle emergence of percentage g, and ABA is the ABA concentration (m) in the incubation solution. In the presence of both ABA and reduced psi, the same parameters can be used to predict seed germination time courses based upon strictly additive effects of psi and ABA in delaying the time of radicle emergence. Further analysis indicates that ABA and psi can act both independently and interactively to influence physiological processes preparatory for radicle growth, such as the accumulation of osmotic solutes in the embryo. The models provide quantitative values for the sensitivity of germination to ABA or psi, allow evaluation of independent and interactive effects of the two factors, and have implications for understanding how ABA and psi may regulate growth and development. PMID- 16668728 TI - Regulation of Photosynthesis in Triazine-Resistant and -Susceptible Brassica napus. AB - The response of photosynthetic carbon assimilation and chlorophyll fluorescence quenching to changes in intercellular CO(2) partial pressure (C(i)), O(2) partial pressure, and leaf temperature (15-35 degrees C) in triazine-resistant and susceptible biotypes of Brassica napus were examined to determine the effects of the changes in the resistant biotype on the overall process of photosynthesis in intact leaves. Three categories of photosynthetic regulation were observed. The first category of photosynthetic response, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-limited photosynthesis, was observed at 15, 25, and 35 degrees C leaf temperatures with low C(i). When the carbon assimilation rate was Rubisco-limited, there was little difference between the resistant and susceptible biotypes, and Rubisco activity parameters were similar between the two biotypes. A second category, called feedback-limited photosynthesis, was evident at 15 and 25 degrees C above 300 microbars C(i). The third category, photosynthetic electron transport-limited photosynthesis, was evident at 25 and 35 degrees C at moderate to high CO(2). At low temperature, when the response curves of carbon assimilation to C(i) indicated little or no electron transport limitation, the carbon assimilation rate was similar in the resistant and susceptible biotypes. With increasing temperature, more electron transport limited carbon assimilation was observed, and a greater difference between resistant and susceptible biotypes was observed. These observations reveal the increasing importance of photosynthetic electron transport in controlling the overall rate of photosynthesis in the resistant biotype as temperature increases. Photochemical quenching of chlorophyll fluorescence (q(P)) in the resistant biotype never exceeded 60%, and triazine resistance effects were more evident when the susceptible biotype had greater than 60% q(P), but not when it had less than 60% q(P). PMID- 16668729 TI - Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules. AB - Formation of the heme precursor delta-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60 degrees C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[(14)C]glutamate and 3,4-[(3)H]glutamate but not from 1 [(14)C]glutamate, and thus, operation of the C(5) pathway could not be confirmed. PMID- 16668730 TI - alpha-Amylase Isoforms are Posttranslationally Modified in the Endomembrane System of the Barley Aleurone Layer. AB - The subcellular site of the posttranslational modification of alpha-amylase was investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya). Aleurone layers of Himalaya barley synthesize and secrete two groups of alpha amylase isoforms, referred to as low-isoelectric point (low-pl) or HAMY1 and high pl or HAMY2, when incubated in gibberellic acid and CaCl(2). Whereas homogenates of aleurone layers contain four isoforms of HAMY1 with pls 4.90, 4.72, 4.64, and 4.56, incubation media contain predominantly isoforms 4.72 and 4.56. Microsomal membranes isolated from aleurone layers contain all four isoforms of HAMY1. Microsomal membranes can be resolved into two peaks by isopycnic density gradient centrifugation: a peak of heavy membranes with endoplasmic reticulum and Golgi apparatus (GApp) marker enzyme activities and a peak of light membranes with characteristics of the GApp. The heavy membranes contain proportionally more HAMY1 pl 4.90 and 4.64 isoforms, whereas light membranes contain a higher proportion of pl 4.72 and 4.56 isoforms. Experiments with the ionophore monensin show that membranes of the GApp as well as the endoplasmic reticulum are involved in the posttranslational modification of HAMY1 isoforms. Monensin inhibits the secretion of alpha-amylase and causes the enzyme to accumulate within the cell. Precursor forms of HAMY1 accumulate in light membranes isolated from monensin treated aleurone layers indicating that the GApp is involved in the conversion of the precursor to the secreted forms of the enzyme. PMID- 16668731 TI - K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa. AB - In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K(+)](o). When the membrane voltage was more positive than -50 millivolts, the membrane conductance in the whole-cell mode was low, and K(+) channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than -50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K(+) compared with Cs(+), Na(+), choline or Cl( ). Low concentrations of [Cs(+)](o) or [Na(+)](o) blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K(+) channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K(+)](o) about 10 millimolar, the influx through these channels is sufficient to increase the internal [K(+)] by 2 millimolar per minute. These K(+) channels are activated by membrane voltages in the normal physiological range and could contribute to K(+) uptake whenever the membrane is more negative than the K(+) equilibrium potential. PMID- 16668732 TI - Endosperm acidification and related metabolic changes in the developing barley grain. AB - The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoeno/pyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation. PMID- 16668733 TI - Changes in Activities of Enzymes of Carbon Metabolism in Leaves during Exposure of Plants to Low Temperature. AB - The aim of this study was to determine the response of photosynthetic carbon metabolism in spinach and bean to low temperature. (a) Exposure of warm-grown spinach and bean plants to 10 degrees C for 10 days resulted in increases in the total activities of a number of enzymes, including ribulose 1,5-bisphosphate carboxylase (Rubisco), stromal fructose 1,6 bisphosphatase (Fru 1,6-P(2)ase), sedoheptulose 1,7-bisphosphatase (Sed 1,7-P(2)ase), and the cytosolic Fru 1,6 P(2)ase. In spinach, but not bean, there was an increase in the total activity of sucrose-phosphate synthase. (b) The CO(2)-saturated rates of photosynthesis for the cold-acclimated spinach plants were 68% greater at 10 degrees C than those for warm-acclimated plants, whereas in bean, rates of photosynthesis at 10 degrees C were very low after exposure to low temperature. (c) When spinach leaf discs were transferred from 27 to 10 degrees C, the stromal Fru 1,6-P(2)ase and NADP-malate dehydrogenase were almost fully activated within 8 minutes, and Rubisco reached 90% of full activation within 15 minutes of transfer. An initial restriction of Calvin cycle fluxes was evident as an increase in the amounts of ribulose 1,5-bisphosphate, glycerate-3-phosphate, Fru 1,6-P(2), and Sed 1,7-P(2). In bean, activation of stromal Fru 1,6-P(2)ase was weak, whereas the activation state of Rubisco decreased during the first few minutes after transfer to low temperature. However, NADP-malate dehydrogenase became almost fully activated, showing that no loss of the capacity for reductive activation occurred. (d) Temperature compensation in spinach evidently involves increases in the capacities of a range of enzymes, achieved in the short term by an increase in activation state, whereas long-term acclimation is achieved by an increase in the maximum activities of enzymes. The inability of bean to activate fully certain Calvin cycle enzymes and sucrose-phosphate synthase, or to increase nonphotochemical quenching of chlorophyll fluorescence at 10 degrees C, may be factors contributing to its poor performance at low temperature. PMID- 16668734 TI - Purification and Characterization of a 70-Kilodalton Polyadenylate-Binding Protein from Pea (Pisum sativum). AB - A polyadenylate-binding protein (PABP) was purified from cell-free extracts prepared from pea seedlings (Pisum sativum) by ammonium sulfate precipitation and Affi-Gel Blue and polyadenylate-Sepharose 4B affinity chromatography. The final preparation from polyadenylate-Sepharose 4B columns contained a single 70 kilodalton polypeptide with high polyadenylate-binding activity. The purified protein was active over a broad range of ionic strengths and showed temperature and pH optima of 37 degrees C and pH 6.5, respectively. Specificity studies indicated that the pea PABP was most active with polyadenylic acids, showed some activity with polyguanylic acid, and did not bind to polycytidylic acid. Moreover, longer polyadenylate molecules were bound more effectively than shorter ones. Because these properties are similar to PABPs isolated from other sources, we conclude that we have identified, purified, and characterized a plant PABP analogous to those described in yeast and animal systems. PMID- 16668735 TI - Modification by ethylene of the cell growth pattern in different tissues of etiolated lupine hypocotyls. AB - The influence of ethylene on growth in etiolated lupine (Lupinus albus L.) hypocotyls was studied in ethephon-treated plants. Ethephon reduced the length and increased the diameter of hypocotyls. At the end of the hypocotyl growth period (14 days), the fresh weight was reduced by 53%, and the dry weight was reduced by 16%. Thus, ethylene reduced water uptake in the tissues to a greater extent than the incorporation of new materials. Light microscopic measurements showed that the thickness of tissues was stimulated by ethylene, the vascular cylinder and cortex exhibiting greater increases (55 and 45%, respectively) than pith (26%) or epidermis (12%). Ethephon modified the cell growth pattern, stimulating lateral cell expansion and cell wall thickness, while reducing cell elongation. The response to ethylene varied in the different tissues and was higher in cortex and pith cells than in the epidermis cells. The ethylene-induced cell expansion in the cortex varied according to the localization of cells in the tissue: the central and subepidermal layers showed little change, whereas the innermost layers exhibited the greatest increase. Electron microscopy revealed that ethylene increased both the rough endoplasmic reticulum and dictyosomes, suggesting that ethylene stimulated the secretion of cell wall materials. In untreated seedlings, the pattern of cell growth was similar in cells from the epidermis, cortex, and pith. The final cell size varied along the hypocotyl, the cells becoming shorter and broader the closer to the basal zones of the organ. PMID- 16668736 TI - Phosphorus nutrition influence on leaf senescence in soybean. AB - Remobilization of mineral nutrients from leaves to reproductive structures is a possible regulatory factor in leaf senescence. The relationship between P remobilization from leaves of soybean (Glycine max [L.] Merr. cv McCall) during reproductive development and leaf senescence was determined by utilizing soil P treatments that supplied deficient, optimum, and supraoptimum soil P levels. The soil P treatments simulated field conditions, being initiated at the time of planting with no subsequent addition or removal of P. It was hypothesized that P deficiency would accelerate leaf senescence and that supraoptimum P nutrition would delay the timing or rate of leaf senescence relative to plants grown with optimum P. Supraoptimum soil P led to a two- to fourfold increase in leaf P concentration compared with optimum P, and during senescence there was no net P remobilization from leaves for this treatment. Leaf P concentration was similar for plants grown at optimum or deficient soil P, and there was significant net P remobilization from leaves of both treatments in one of the two experiments. As indicated by changes in leaf N, carbon dioxide exchange rate, ribulose 1,5 bisphosphate carboxylase/oxygenase activity, and chlorophyll concentration, leaf senescence patterns were similar for all soil P treatments. Thus, it can be concluded that leaf senescence was not affected by either P deficiency or enhanced leaf P concentration resulting from supraoptimum soil P. The results suggest that P nutrition in general, and specifically P remobilization from leaves, does not exert any regulatory control on the process of leaf senescence. PMID- 16668737 TI - Phosphorus Nutrition Influence on Starch and Sucrose Accumulation, and Activities of ADP-Glucose Pyrophosphorylase and Sucrose-Phosphate Synthase during the Grain Filling Period in Soybean. AB - Several lines of evidence indicate that the partitioning of photosynthate between starch and sucrose is influenced by the relative concentrations of inorganic phosphate (Pi) in the cytosol and chloroplast. Two greenhouse experiments were conducted to determine the influence of long-term differences in soil P levels, ranging from deficient to supraoptimum, on leaf starch and sucrose concentrations, and activities of adenosine diphosphate glucose (ADPG) pyrophosphorylase and sucrose-phosphate synthase (SPS) during the grain filling period in soybean (Glycine max [L.] Merr.). It was hypothesized that, compared with optimum P nutrition, leaf starch and sucrose concentrations would be increased and decreased, respectively, for P deficiency and visa versa for supraoptimum P nutrition. Relative to the optimum soil P level, leaf Pi concentration was not altered by P deficiency but was increased two- to fourfold for the supraoptimum soil P treatment. The concentrations of leaf starch and sucrose were not markedly affected by any of the P fertility treatments and were not closely related to the activities of ADPG pyrophosphorylase and SPS. P deficiency resulted in increased activity of both enzymes in one of the experiments. The results indicated that long-term soil P treatments, that caused either large decreases in plant growth (P deficiency) or large increases in leaf Pi concentration (supraoptimum P), did not markedly alter starch and sucrose metabolism. Furthermore, it can be inferred that the method of plant culture and/or imposition of the P treatments is a critical factor in interpreting results of P nutrition studies. PMID- 16668738 TI - Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms. AB - Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and alpha ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to alpha ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, alpha-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque 2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm. PMID- 16668739 TI - Diacylglycerol kinase from suspension cultured plant cells : characterization and subcellular localization. AB - Diacylglycerol kinase (adenosine 5'-triphosphate:1,2-diacylglycerol 3 phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5'-triphosphate and guanosine 5'-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane. PMID- 16668740 TI - Characterization of the Expression of the Photosystem II-Oxygen Evolving Complex in C(4) Species of Flaveria. AB - We have determined the levels of photosystem II activity and polypeptide abundance in whole leaves and isolated bundle sheath and mesophyll cells of C(4), "C(4)-like," and C(3) species of the genus Flaveria (Asteraceae). On a chlorophyll basis, the whole leaf levels of the D1, D2, and 34-kilodalton photosystem II polypeptides were similar for each Flaveria species. Photosystem II activity varied twofold, but was not correlated with photosynthetic type (C(3) or C(4)). The bundle sheath cell levels of photosystem II activity and associated polypeptides in C(4)-like and C(4)Flaveria species were approximately one-half those observed in mesophyll cells but equivalent to those in bundle sheath cells of the C(3) species, Flaveria cronquistii. Analyses of the steady-state levels of transcripts encoding photosystem II polypeptides indicated that there were no differences in transcript abundance between mesophyll and bundle sheath cells of the C(4)Flaveria species. This pattern was in contrast to the three- to tenfold higher levels of transcripts encoding photosystem II polypeptides in mesophyll versus bundle sheath cells of maize. It is apparent that the higher mesophyll cell to bundle sheath ratio of photosystem II polypeptides in C(4)- and C(4)-like species of Flaveria is the result of higher levels of photosystem II expression in mesophyll cells rather than lower levels of expression in bundle sheath cells. PMID- 16668741 TI - Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength. AB - Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH(4))(2)SO(4) fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K(m) values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50 degrees C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits. PMID- 16668742 TI - Posttranslational modifications in the amino- terminal region of the large subunit of ribulose- 1,5-bisphosphate carboxylase/oxygenase from several plant species. AB - A combination of limited tryptic proteolysis, reverse phasehigh performance liquid chromatography, Edman degradative sequencing, amino acid analysis, and fast-atom bombardment mass-spectrometry was used to remove and identify the first 14 to 18 N-terminal amino acid residues of the large subunit of higher plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas reinhardtii, Marchantia polymorpha, pea (Pisum sativum), tomato (Lycopersicon esculentum), potato (Solanum tuberosum), pepper (Capsicum annuum), soybean (Glycine max), petunia (Petunia x hybrida), cowpea (Vigna sinensis), and cucumber (Cucumis sativus) plants. The N-terminal tryptic peptide from acetylated Pro-3 to Lys-8 of the large subunit of Rubisco was identical in all species, but the amino acid sequence of the penultimate N-terminal tryptic peptide varied. Eight of the 10 species examined contained a trimethyllysyl residue at position 14 in the large subunit of Rubisco, whereas Chlamydomonas and Marchantia contained an unmodified lysyl residue at this position. PMID- 16668743 TI - Relationships among Isoprene Emission Rate, Photosynthesis, and Isoprene Synthase Activity as Influenced by Temperature. AB - Isoprene emissions from the leaves of velvet bean (Mucuna pruriens L. var utilis) plants exhibited temperature response patterns that were dependent on the plant's growth temperature. Plants grown in a warm regimen (34/28 degrees C, day/night) exhibited a temperature optimum for emissions of 45 degrees C, whereas those grown in a cooler regimen (26/20 degrees C, day/night) exhibited an optimum of 40 degrees C. Several previous studies have provided evidence of a linkage between isoprene emissions and photosynthesis, and more recent studies have demonstrated that isoprene emissions are linked to the activity of isoprene synthase in plant leaves. To further explore this linkage within the context of the temperature dependence of isoprene emissions, we determined the relative temperature dependencies of photosynthetic electron transport, CO(2) assimilation, and isoprene synthase activity. When measured over a broad range of temperatures, the temperature dependence of isoprene emission rate was not closely correlated with either the electron transport rate or the CO(2) assimilation rate. The temperature optima for electron transport rate and CO(2) assimilation rate were 5 to 10 degrees C lower than that for the isoprene emission rate. The dependence of isoprene emissions on photon flux density was also affected by measurement temperature in a pattern independent of those exhibited for electron transport rate and CO(2) assimilation rate. Thus, despite no change in the electron transport rate or CO(2) assimilation rate at 26 and 34 degrees C, the isoprene emission rate changed markedly. The quantum yield of isoprene emissions was stimulated by a temperature increase from 26 to 34 degrees C, whereas the quantum yield for CO(2) assimilation was inhibited. In greenhouse-grown aspen leaves (Populus tremuloides Michaux.), the high temperature threshold for inhibition of isoprene emissions was closely correlated with the high temperature-induced decrease in the in vitro activity of isoprene synthase. When taken together, the results indicate that although there may be a linkage between isoprene emission rate and photosynthesis, the temperature dependence of isoprene emission is not determined solely by the rates of CO(2) assimilation or electron transport. Rather, we propose that regulation is accomplished primarily through the enzyme isoprene synthase. PMID- 16668744 TI - Growth and Movement of Spot Inoculated Rhizobium meliloti on the Root Surface of Alfalfa. AB - Inoculum droplets of approximately 10 nanoliter volume and containing about 10 Rhizobium meliloti cells were placed onto the root surface of alfalfa seedlings in plastic growth pouches at either the root tip, the position of the smallest emergent root hairs, or at a site midway between these points. The droplets were initially confined to an area of about 0.2 square millimeter at the point of application. By 48 and 96 hours after inoculation, the inoculum bacteria and their progeny were distributed over several centimeters of the root between the initial site of deposition and the growing root tip, reaching densities of 10(3) to 10(4) bacteria per centimeter near the site of initial deposition and decreasing exponentially from that point toward the root tip. Graphite particles deposited on the root surface close to the growing tip were similarly distributed along the root length by 48 and 96 hours, suggesting that passive displacement by root cell elongation was primarily responsible for the spread of bacteria. A nonmotile mutant of R. meliloti colonized alfalfa roots to the same extent as the wild type and was usually distributed in the same manner, indicating that bacterial motility contributed little under these conditions to long distance spread of the bacteria. However, when applied in low numbers, R. meliloti mutants defective in motility or chemotaxis were considerably less efficient in initiating nodules near the point of inoculation than the wild type. This implies that motility and/or chemotaxis contribute significantly to local exploration for suitable infection sites. Almost all nodules on the primary root formed within a few millimeters of the spot-inoculation site, indicating that, under our experimental conditions, movement and multiplication of R. meliloti on the root surface were not sufficient to maintain an adequate population in the infectible region of the root during root growth. PMID- 16668745 TI - Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures. AB - Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity. PMID- 16668746 TI - Plasma Membrane Ca-ATPase of Radish Seedlings : I. Biochemical Characterization Using ITP as a Substrate. AB - In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The majority of the ITPase activity of plasma membrane was Ca(2+)-dependent. The Ca(2+)-dependent ITPase activity was Mg(2+)-dependent and was stimulated by the calcium ionophore A23187. It was inhibited by erythrosin B (concentration giving 50% inhibition, 50 nanomolar) and by vanadate (concentration giving 50% inhibition, 3 micromolar) and displayed a broad pH optimum around pH 7.2 to 7.5. Both the hydrolytic and the transport activity of the plasma membrane Ca-ATPase were half-saturated by Ca(2+) in the micromolar concentration range. No major effect of EGTA on the saturation kinetics of the enzyme was observed. The affinity of the plasma membrane Ca-ATPase for Ca(2+) was about fourfold higher at pH 7.5 than at pH 6.9. The Ca(2+)-dependent ITPase activity was stimulated about twofold by polyoxyethylene 20 cetyl ether, although it was inhibited by Triton X-100 and by lysolecithin. PMID- 16668747 TI - Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin. AB - The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca(2+) concentration. Stimulation was marked at low, nonsaturating Ca(2+) concentrations and decreased increasing Ca(2+), so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca(2+). The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca(2+), stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca ATPase activity to comparable extents. PMID- 16668748 TI - Changes in Soluble Carbohydrates during Seed Storage. AB - The soluble sugars present in the maize (Zea mays L.) embryo may serve as important components of protection or may contribute to the deteriorative changes occurring during seed storage. Examination of the changes in sugars during accelerated aging of maize seeds indicates that the decline in vigor is associated with a marked decline in monosaccharides and in raffinose. Sucrose content remains relatively stable. The depletion of raffinose may have special relevance to the decline in seed vigor. PMID- 16668749 TI - Novel light-regulated chloroplast thylakoid membrane protein. AB - A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known. PMID- 16668750 TI - Genomic Nucleotide Sequence of a Wild-Type Shrunken-2 Allele of Zea mays. PMID- 16668751 TI - Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5 bisphosphate pool size. AB - Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO(2) and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO(2)-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5 bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3 phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP binding sites may have maintained the Rubisco-specific activity in phosphate deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize. PMID- 16668752 TI - Photo and Nutritional Regulation of the Light-Harvesting Chlorophyll a/b-Binding Protein of Photosystem II mRNA Levels in Euglena. AB - In Euglena gracillis var bacillaris, light exposure increases the level of mRNA encoding the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) approximately twofold. LHCPII mRNA levels increased in the dark upon either malate or ethanol addition. LHCPII mRNA is present but LHCPII is not synthesized in the bleached mutants W(3)BUL and W(10)BSmL, which lack protochlorophyll(ide) and most if not all of the chloroplast genome. Light exposure increased LHCPII mRNA levels in W(3)BUL but not in W(10)BSmL. Carbon availability and light acting through a nonchloroplast photoreceptor appear to regulate LHCPII mRNA levels. A chloroplast photoreceptor and/or a product produced by the chloroplast appear to regulate LHCPII mRNA translation. PMID- 16668753 TI - Purification and characterization of dihydrodipicolinate synthase from pea. AB - Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-beta-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-beta semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-alpha-(2 aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-alpha-(2-aminoethoxyvinyl)glycine is noncompetitive towards l aspartate-beta-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-beta semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase. PMID- 16668754 TI - Gametic Differentiation of Chlamydomonas reinhardtii: Control by Nitrogen and Light. AB - Gametic differentiation of the unicellular green alga Chlamydomonas reinhardtii proceeds in two steps controlled by the extrinsic signals nitrogen deficiency and light. Nitrogen deprivation induces the differentiation of vegetative cells to sexually immature pregametes. A light signal is required to convert the pregametes to gametes. Both signals are also required for the maintenance of mating competence. Two converging signal transduction chains are proposed to control gamete formation. For the differentiation of pregametes to gametes, a fluence rate-dependent reaction, requiring continuous irradiation, is suggested by photobiological experiments. PMID- 16668755 TI - Gradients of turgor, osmotic pressure, and water potential in the cortex of the hypocotyl of growing ricinus seedlings : effects of the supply of water from the xylem and of solutes from the Phloem. AB - To evaluate the possible role of solute transport during extension growth, water and solute relations of cortex cells of the growing hypocotyl of 5-day-old castor bean seedlings (Ricinus communis L.) were determined using the cell pressure probe. Because the osmotic pressure of individual cells (pi(i)) was also determined, the water potential (psi) could be evaluated as well at the cell level. In the rapidly growing part of the hypocotyl of well-watered plants, turgor increased from 0.37 megapascal in the outer to 1.04 megapascal in the inner cortex. Thus, there were steep gradients of turgor of up to 0.7 megapascal (7 bar) over a distance of only 470 micrometer. In the more basal and rather mature region, gradients were less pronounced. Because cell turgor approximately pi(i) and psi approximately 0 across the cortex, there were also no gradients of psi across the tissue. Gradients of cell turgor and pi(i) increased when the endosperm was removed from the cotyledons, allowing for a better water supply. They were reduced by increasing the osmotic pressure of the root medium or by cutting off the cotyledons or the entire hook. If the root was excised to interrupt the main source for water, effects became more pronounced. Gradients completely disappeared and turgor fell to 0.3 megapascal in all layers within 1.5 hours. When excised hypocotyls were infiltrated with 0.5 millimolar CaCl(2) solution under pressure via the cut surface, gradients in turgor could be restored or even increased. When turgor was measured in individual cortical cells while pressurizing the xylem, rapid responses were recorded and changes of turgor exceeded that of applied pressure. Gradients could also be reestablished in excised hypocotyls by abrading the cuticle, allowing for a water supply from the wet environment. The steep gradients of turgor and osmotic pressure suggest a considerable supply of osmotic solutes from the phloem to the growing tissue. On the basis of a new theoretical approach, the data are discussed in terms of a coupling between water and solute flows and of a compartmentation of water and solutes, both of which affect water status and extension growth. PMID- 16668756 TI - Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus. AB - The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake. PMID- 16668757 TI - Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars. AB - The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves. PMID- 16668758 TI - Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels. AB - Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C(4) species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT 2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT 2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness. PMID- 16668759 TI - Circadian rhythms of chemotaxis to ammonium and of methylammonium uptake in chlamydomonas. AB - Chlamydomonas reinhardtii expresses a well-documented circadian rhythm of phototaxis, which peaks in the subjective daytime. We find that vegetative cells also express circadian rhythms of chemotaxis to ammonium and ammonium uptake (as gauged by uptake of [(14)C]methylammonium). The chemotaxis rhythm peaks in the subjective night. Methylammonium uptake is light dependent, and its rhythm peaks at subjective dawn. Unlike vegetative cells, gametes are not attracted to ammonium. We believe this to be the first report of a circadian rhythm of chemotaxis. PMID- 16668760 TI - Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves. AB - Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O(2) or H(2)O(2) and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K(m) 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O(2) as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves. PMID- 16668761 TI - Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil. AB - Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H(2) evolution in Ar:O(2) (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO(2) production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O(2) permeability decreased 50% within 5 hours in each species. A similar decrease in the O(2)-saturated respiration rate (V(max)) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V(max) in each species. V(max) may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O(2) inactivation of nitrogenase. PMID- 16668762 TI - Mathematical modeling of oxygen diffusion and respiration in legume root nodules. AB - The O(2) permeability of legume root nodules is under physiological control; decreases in permeability are triggered by various forms of stress. Two linked mathematical models were used to explore several hypotheses concerning the physical nature of the variable diffusion barrier in nodules. Respiration and diffusion of dissolved O(2) and oxygenated leghemoglobin were simulated for the nodule cortex and the nodule interior. Measured nodule permeabilities were shown to be inconsistent with the hypothesis that large numbers of air-filled pores penetrate the diffusion barrier. Changes in the affinity of leghemoglobin for O(2) or in the rate of cytoplasmic streaming in diffusion barrier cells did not result in the large changes in O(2) permeability reported for real nodules. The presence or absence, but not the thickness, of aqueous plugs in radial pores through the cortex was found to have a large effect on permeability. Flooding of intercellular spaces, either between layers of cells in the cortex or in the nodule interior, also caused large changes in simulated permeability. The unsteady-state O(2) method for determining nodule permeability was tested using data generated by the model. The accuracy of the method was confirmed, provided that certain assumptions (full oxygenation of leghemoglobin under pure O(2) and uniform conditions in the nodule interior) are met. PMID- 16668763 TI - Bound Water in Durum Wheat under Drought Stress. AB - To study drought stress effects on bound water, adsorption isotherms and pressure volume curves were constructed for two durum wheat (Triticum durum Desf.) cultivars: Capeiti 8 (drought tolerant) and Creso (drought sensitive). Plants were grown under well-watered and water-stressed conditions in a controlled environment. Differential enthalpy (DeltaH) was calculated through van't Hoff analysis of adsorption isotherms at 5 and 20 degrees C, which allowed us to determine the strength of water binding. DeltaH reached the most negative values at approximately 0.06 gram H(2)O/gram dry weight and then increased rapidly for well-watered plants (until 0.10 gram H(2)O/gram dry weight) or more slowly for drought-stressed plants (until 0.15-0.20 gram H(2)O/gram dry weight). Bound water values from pressure-volume curves were greater for water-stressed (0.17 gram H(2)O/gram dry weight) than for well-watered plants (0.09 gram H(2)O/gram dry weight). They may be estimates of leaf moisture content where DeltaH reaches the less negative values and hence some free water appears. With respect to the well watered plants, tightly bound water tended to be less bound during drought, and more free water was observed in cv Creso compared to cv Capeiti 8 at moisture contents >0.10 gram H(2)O/gram dry weight. PMID- 16668764 TI - Immunological detection of acetaldehyde-protein adducts in ethanol-treated carrot cells. AB - Polyclonal antibodies able to recognize protein-acetaldehyde conjugates were produced and characterized. The antibodies react with sodium cyanoborohydride reduced Schiff's bases between acetaldehyde and a protein, independently of the nature of the macromolecule binding the acetaldehyde moiety. Only conjugates between acetaldehyde or propionaldehyde and a protein are recognized; conjugates obtained with other aldehydes are not reactive. Results concerning the formation of acetaldehyde adducts with carrot (Daucus carota L.) proteins are presented as well as the presence of such conjugates in ethanol-treated carrot cell cultures, a system highly sensitive to the presence of ethanol in the culture medium. PMID- 16668765 TI - A repetitive proline-rich protein from the gymnosperm douglas fir is a hydroxyproline-rich glycoprotein. AB - Intact cell elution of suspension cultures derived from Douglas fir, Pseudotsuga menziesii (Mirbel) Franco, yielded two extensin monomers, the first hydroxyproline-rich glycoproteins (HRGPs) to be isolated from a gymnosperm. These HRGPs resolved on Superose-6 gel filtration. The smaller monomer was compositionally similar to angiosperm extensins like tomato P1. The larger monomer had a simple composition reminiscent of repetitive proline-rich proteins (RPRPs) from soybean cell walls and contained proline, hydroxyproline, and sugar; hence designated a proline-hydroxyproline-rich glycoprotein (PHRGP). The simple composition of the PHRGP implied a periodic structure which was confirmed by the simple chymotryptic map and 45-residue partial sequence of the major proline hydroxyproline-rich glycoprotein chymotryptide 5: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val- Tyr-Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp Val-Val-Lys-Pro-Hyp-Hyp- Val-Tyr-Lys-Ile-Pro-Pro(Hyp)-Val-Ile-Lys-Pro. Proline hydroxyproline-rich glycoprotein chymotryptide 5 contained an 18-residue tandem repeat devoid of tetra(hydroxy)-proline or serine; it also contained two instances of the five-residue motif Hyp-Hyp-Val-Tyr-Lys and five of the general Pro-Pro-X-X-Lys motif, thereby establishing its homology with typical angiosperm RPRPs and extensins from tomato, petunia, carrot, tobacco, sugar beet, and Phaseolus. Unlike the nonglycosylated soybean RPRP, the highly purified Douglas fir PHRGP was lightly glycosylated, confirmed by a quantitative hydroxyproline glycoside profile, indicating that extensins can range from highly glycosylated hydroxyproline to little or no glycosylated hydroxyproline. Comparison of extensin sequence data strongly indicates that a major determinant of hydroxyproline glycosylation specificity is hydroxyproline contiguity: extensins with tetrahydroxyproline blocks are very highly arabinosylated (>90% hydroxyproline glycosylated), tri- and dihydroxyproline are less so, and single hydroxyproline residues perhaps not at all. Despite high yields of extensins eluted from intact cells, the Douglas fir cell wall itself was hydroxyproline poor yet remarkably rich in protein (>20%), again emphasizing the existence of other structural cell wall proteins that are neither HRGPs nor glycine-rich proteins. PMID- 16668766 TI - Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence. AB - The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs. PMID- 16668767 TI - Biosynthesis and Secretion of Cryptogein, a Protein Elicitor Secreted by Phytophthora cryptogea. AB - The phytopathogenic fungi Phytophthora subspecies elicit hypersensitive-like necroses on their nonhost tobacco (Nicotiana tabacum), with the exception of the tobacco pathogen Phytophthora nicotianae. In culture, these fungi-except P. nicotianae-secrete proteins, called elicitins, that cause these remote leaf necroses and are responsible for the incompatible reaction. These proteins protect tobacco against invasion by the agent of the tobacco black shank, P. nicotianae, which is unable to produce such an elicitor. Cryptogein, secreted by Phytophthora cryptogea, has been purified, sequenced, and characterized as an elicitin, a novel family of 10 kilodalton holoproteins. In the present paper, we examined the secretion and biosynthesis of this protein elicitor from P. cryptogea culture. Results showed that the secretion of cryptogein began later than its synthesis and stopped earlier, simultaneously with mycelium growth, when the nitrogen source in the culture medium was nearly exhausted. Electrophoretic patterns of total protein from mycelium extracts and N-terminal sequence analysis showed that cryptogein accumulated in the mycelium in its mature form. The comparison of the immunoselected in vitro translation products with (35)S in vivo labeled cryptogein showed that cryptogein was synthesized as a preprotein with a signal peptide removed cotranslationally before the secretion into the culture medium. Immunoselected in vitro-synthesized products were subjected to radiosequencing to clearly determine the N-terminal position and the size (20 amino acids) of the signal peptide. Cryptogein did not undergo any other posttranslational modification. PMID- 16668768 TI - Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants. AB - Mutation at either of two genetic loci (Eu2 or Eu3) in soybean (Glycine max [L.] Merr.) results in a pleiotropic elimination of the activity of both major urease isozymes. Surprisingly, the phenotype of a phylloplane bacterium, Methylobacterium mesophilicum, living on the leaves of eu2/eu2 or eu3-e1/eu3-e1 mutants is also affected by these plant mutations. The bacteria isolated from leaves of these soybean mutants have transient urease- and hydrogenase-deficient phenotypes that can be corrected by the addition of nickel to free-living cultures. The same bacterium growing on wild-type soybeans or on urease mutants eu1-sun/eu1-sun or eu4/eu4, each deficient in only one urease isozyme, are urease positive. These results suggest that the bacterium living on the eu2/eu2 or eu3 e1/eu3-e1 mutant is unable to produce an active urease or hydrogenase because it is effectively starved for nickel. We infer that mutations at Eu2 or Eu3 result in defects in nickel metabolism but not in Ni(2+) uptake or transport, because eu2/eu2 and eu3-e1/eu3-e1 mutants exhibit normal uptake of (63)NiCl(2). Moreover, wild-type plants grafted on mutant rootstocks produce seeds with fully active urease, indicating unimpeded transport of nickel through mutant roots and stems. PMID- 16668769 TI - Leaf Photosynthesis and Respiration of High CO(2)-Grown Tobacco Plants Selected for Survival under CO(2) Compensation Point Conditions. AB - Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO(2) atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO(2) levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO(2) assimilation rate per unit area measured in the growth room at high CO(2) was not higher in the selected plants. The CO(2) assimilation rate versus intercellular CO(2) curve and the CO(2) compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO(2) compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The alternative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO(2) survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon. PMID- 16668770 TI - Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones. AB - A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of beta-1,3 glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission. PMID- 16668771 TI - Nondwarf rice seedling bioassay for gibberellins. AB - The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10 fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities. PMID- 16668772 TI - Alteration of Components of Leaf Water Potential and Water Content in Velvetleaf under the Effects of Long-Term Humidity Difference. AB - Velvetleaf (Abutilon theophrasti Medik.) was grown in growth chambers set at 45 or 85% relative humidity at 30 degrees C, CO(2) 350 microliters per liter and 1000 micromoles per square meter per second of photosynthetically active radiation. Soil water potential was maintained at -0.05 megapascal by subirrigation with half strength Hoagland solution. The third, fourth, and fifth leaves from the base of 21- and 25-day-old plants were used for pressure-volume measurements. Components of leaf water status including water potential (osmotic and potential associated with the apoplast), leaf water content (apoplasmic and symplasmic water), and elastic modulus of leaf tissue were determined. Results indicate: (a) persistent dry air generated leaves with lower water potential at a given relative water content than did humid air; (b) the higher total leaf water content in plants grown in dry air was related to an increase in apoplasmic water, whereas symplasmic water remained similar in both humidity treatments; (c) difference in leaf water potential between low and high humidity treatments was related to decreased potential associated with the apoplast but not to a change in cell wall elasticity. PMID- 16668773 TI - Identification and characterization of lipoxygenase isoforms in senescing carnation petals. AB - A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Reve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X 114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents. PMID- 16668774 TI - Hormone-resistant mutants of Arabidopsis have an attenuated response to agrobacterium strains. AB - We have examined the response of the hormone-resistant mutants axr1 and axr2 of Arabidopsis thaliana to inoculation by Agrobacterium tumefaciens and Agrobacterium rhizogenes. Our results indicate that recessive mutations in the axr1 gene affect the frequency of tumor formation after inoculation with either Agrobacterium strain. In addition, tumors produced on axr1 plants were smaller than those growing on wild-type plants. These results indicate that the product of the AXR1 gene is important for both crown gall and hairy root tumor formation. In contrast, the dominant axr2 mutation has a more severe effect on the development of crown gall tumors than on hairy root tumors. Crown gall tumors produced on axr2 plants had a different morphology than wild-type tumors and did not grow when they were removed from the explant. In contrast, a large number of hairy root tumors were produced on wild-type and axr2 plants, and both types of tumors grew when they were removed from the explant. Like the roots of axr2 plants, roots produced on axr2 explants lacked root hairs. PMID- 16668775 TI - Differential carbon monoxide sensitivity of cytochrome C oxidase in the leaves of c(3) and c(4) plants. AB - CO sensitivity of cytochrome a(3) in the leaves of a number of C(3) and C(4) plants was monitored by the nitrate reductase assay under differing CO to O(2) ratios. All the C(3) plants were relatively insensitive to CO and required a high CO to O(2) ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to O(2) ratios of 10 or less. On the other hand, the leaves of all C(4) plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to O(2) ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from mesophyll or bundle sheath cells or both, lacked tight respiratory control. PMID- 16668776 TI - Relationship between Electron Transport across the Plasmalemma and a pH Decrease in the Bulk Medium. AB - The hypothesis is tested that acidification of the bulk medium during transplasmalemma electron transport to ferricyanide is due solely to a requirement for charge balance. According to this hypothesis, reduction of the trivalent anion, ferricyanide, to the tetravalent anion, ferrocyanide, results in a charge difference that is balanced by protons. A coulometric device is used that rapidly and efficiently reoxidizes ferrocyanide to ferricyanide, thus maintaining a constant charge in the bulk medium. Oat (Avena sativa L. cv Garry) mesophyll protoplasts are chosen as experimental material to facilitate ferricyanide reduction and the concomitant ferrocyanide reoxidation by the coulometric device. The kinetics of ferricyanide reduction and proton excretion by protoplasts are similar to those of other cell types and tissues. Rates of net proton excretion are identical regardless of whether the ferrocyanide is simultaneously reoxidized. We conclude that acidification may occur during transplasmalemma electron transport when there is no change in negative charge of the bulk medium. PMID- 16668777 TI - Regulation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic Acid. AB - Gel electrophoretic analysis of the proteinase inhibitor proteins induced in tomato leaves by airborne methyl jasmonate (EE Farmer, CA Ryan [1990] Proc Natl Acad Sci USA 87: 7713-7716) revealed the new appearance of inhibitors I and II and two other, higher molecular mass proteins (63.5 and 87 kilodaltons). Northern analysis of methyl jasmonate-induced inhibitors I and II mRNAs in tomato (Lycopersicon esculentum) leaves, and of alfalfa trypsin inhibitor (a Bowman-Birk family inhibitor) mRNA in alfalfa (Medicago sativa) leaves, indicated that nascent inhibitor mRNAs were regulated in a manner similar to wounding, that is, at the transcriptional level. In tobacco (Nicotiana tabacum), transformed with a fused gene composed of the 5' and 3' regions of a wound-inducible potato inhibitor II and a chloramphenicol acetyl transferase (CAT) gene coding region, CAT activity was induced in leaves by methyl jasmonate, consistent with a transcriptional regulation of the inhibitor II gene. In tomato leaves, inhibitor I and II mRNAs and proteins accumulated in leaves distal to those exposed to methyl jasmonate or jasmonic acid to similar levels as in exposed leaves. We suggest that in response to wound signals generated by insect or pathogen attacks, linolenic acid is released into the cytoplasm from plant cell membrane lipids and is rapidly converted in cells to jasmonic acid (or perhaps a closely related derivative such as methyl jasmonate), which serves as a signal to regulate the expression of proteinase inhibitor genes. PMID- 16668778 TI - Oxidation and reduction of leghemoglobin in root nodules of leguminous plants. AB - Reactions involving changes that affect the function of leghemoglobin (Lb) are reviewed. The chemical nature of Lb and conditions inside nodules, such as slightly acid pH and the presence of metal ions, chelators, and toxic metabolites (nitrite, superoxide radical, peroxides), are conducive for oxidation of ferrous Lb (Lb(2+)) or its oxygenated form (LbO(2)) to nonfunctional ferric Lb (Lb(3+)) and ferryl Lb. Because Lb(3+) is nearly nonexistent in nodules and undergoes observable reduction in vivo, mechanisms must operate in nodules to maintain Lb in the Lb(2+) state. Redox reactions of Lb are mediated, for the most part, by activated oxygen species: (a) oxidation of LbO(2) to Lb(3+) involves superoxide; (b) excess peroxide oxidizes LbO(2) and Lb(3+) to ferryl Lb and may cause breakdown of heme, release of iron, and generation of hydroxyl radicals (protein radicals may be formed in this process); (c) enzymatic reduction of Lb(3+) requires active flavin and thiol groups and involves formation of peroxide; and (d) direct reduction of Lb(3+) by NADH is mediated by superoxide and peroxide. Transition metal ions and certain small molecules of nodules such as flavins may act as intermediate electron carriers between NADH and Lb(3+), increasing the rate of reaction, which then proceeds via superoxide or flavin radicals, respectively. PMID- 16668779 TI - Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. AB - The influence of varied Mg supply (10-1000 micromolar) and light intensity (100 580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H(2)O(2) scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H(2)O(2) scavenging enzymes increased with light intensity, in particular in Mg deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O(2) (.-) and H(2)O(2) scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O(2) (.-) and H(2)O(2) scavenging enzymes in chloroplasts. PMID- 16668780 TI - Effects of abscisic Acid treatment on the thermostability of the photosynthetic apparatus in barley chloroplasts. AB - Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley (Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10(-5) molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F(740)/F(685) was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F(o)) was virtually eliminated in ABA-treated (10(-5) molar) chloroplasts up to 45 degrees C and slightly increased at 50 degrees C, relative to control chloroplasts where F(o) increased even at 35 degrees C and reached its maximal value at 45 degrees C. In control chloroplasts, F(o) increased with a 5-minute pretreatment temperature, an effect observed as low as 35 degrees C. F(o) was maximal at 45 degrees C. In contrast, chloroplasts treated with 10(-5) molar ABA did not exhibit a heat-induced increase in F(o) until 50 degrees C. PMID- 16668781 TI - Malate- and pyruvate-dependent Fatty Acid synthesis in leucoplasts from developing castor endosperm. AB - Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoll gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction. PMID- 16668782 TI - Glycopeptide elicitors of stress responses in tomato cells: N-linked glycans are essential for activity but act as suppressors of the same activity when released from the glycopeptides. AB - Induction of ethylene, an early symptom of the stress response in tomato (Lycopersicon esculentum [L.] Mill) cells, was used as a bioassay to purify elicitor activity from yeast extract. The purified elicitor preparation consisted of small glycopeptides (mean relative molecular weight of approximately 2500) and induced ethylene biosynthesis and phenylalanine ammonia-lyase activity half maximally at 15 nanograms per milliliter. Elicitor activity was partially abolished by pronase and almost completely by endo-beta-N-acetylglucosaminidase H, alpha-mannosidase, or periodate. The oligosaccharides released upon treatment with endo-beta-N-acetylglucosaminidase H competitively inhibited the elicitor activity of the glycopeptides. This suppressor activity was abolished by periodate oxidation and alpha-mannosidase treatment. The suppressors were chromatographically separated into four active fractions with sizes corresponding to 7 to 10 monosaccharides. They consisted predominantly of mannose and contained also N-acetylglucosamine and glucose. The suppressors had no effect on the response of the tomato cells to a different elicitor, derived from cell walls of Phytophthora megasperma f. sp. glycinea. This strongly suggests that different recognition sites exist for different elicitors in tomato cells, and that the oligosaccharide suppressors act specifically on the perception of just one elicitor. The hypothesis is put forward that the suppressors bind to one of the elicitor recognition sites nonproductively, i.e. without producing a signal, thereby preventing induction of the stress responses by the corresponding elicitor. PMID- 16668783 TI - Characterization of the major integral protein of vacuolar membrane. AB - The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N'-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H(+)-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane. PMID- 16668784 TI - Transcriptional regulation of nitrate reductase mRNA levels by cytokinin-abscisic Acid interactions in etiolated barley leaves. AB - To investigate the mechanisms by which the expression of a specific gene may be modulated by multiple hormones, the regulation of nitrate reductase (NR) mRNA levels by cytokinin and abscisic acid (ABA) was studied in etiolated barley (Hordeum vulgare L. cv Robust) leaves using a barley NR cDNA as a probe. Northern blot analyses of the levels of NR mRNA indicate that accumulation of the mRNA was enhanced by the cytokinin, benzyladenine (BA), and suppressed by ABA and that the hormonal effects on NR gene expression were responsive to dose concentrations (10(-7) to 10(-4) molar). The NR mRNA levels were influenced by the BA/ABA concentration ratios, and the inhibition of the NR transcript levels by ABA was not totally reversed by addition of equal concentrations (10(-7) to 10(-4) molar) of BA. Nuclear runoff transcription studies showed that the level of transcription was suppressed by ABA, and addition of an equal concentration of BA partially reversed the ABA action. Thus, the interaction of cytokinin and ABA on NR gene expression is, at least in part, at the level of transcription. PMID- 16668785 TI - RNA Editing of Cytochrome Oxidase Subunit III in Sunflower Mitochondria. AB - Direct sequencing of cytochrome oxidase subunit III (coxIII) mRNA with a specific primer confirms RNA editing in sunflower (Helianthus annus) mitochondria. Six instances of mRNA editing could be verified, one of these specific to this species. All the editing events involve C to U transitions in the coxIII mRNA causing codon changes that lead to amino acids better conserved in evolution than those encoded in the genomic DNA. This observation confirms RNA editing to be widespread in higher plant mitochondria. PMID- 16668786 TI - Purification and Properties of Putrescine Hydroxycinnamoyl Transferase from Tobacco (Nicotiana tabacum) Cell Suspensions. AB - The enzyme putrescine hydroxycinnamoyl transferase (PHT) was purified 400-fold in 7.1% yield from tobacco (Nicotiana tabacum L. cv Xanthi) cell suspensions to a final specific activity of 45 nanokatal per milligram protein. The purification procedure involved conventional chromatography techniques (anion exchange chromatography, gel permeation, and hydroxylapatite chromatography) followed by chromatography on caffeoyl-cysteamine-Sepharose. This procedure led to considerable enrichment of a 50 kilodalton protein that could be further purified to near homogeneity by chromatofocalization (apparent isoelectric point = 8). PHT activity was repeatedly found associated with this protein, although approximately 66% of the enzymic activity was lost during chromatofocalization. Purified PHT exhibited the same properties as in the unpurified extract. It was not specific for putrescine and used other aliphatic diamines (mainly diaminopropane and cadaverine) as substrates. The most efficient phenolic substrate was caffeoyl-CoA, but cinnamoyl-, feruloyl-, sinapoyl-, and p-coumaroyl CoA were also conjugated to putrescine, in decreasing order of activity. PHT could also use the artificial substrate p-fluorocinnamoyl-CoA. PMID- 16668787 TI - Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast DeltapH within Hevea Latex Cells. AB - The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the "sink effect" and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast DeltapH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H(+)-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel. PMID- 16668788 TI - Characterization and subcellular localization of debranching enzyme and endoamylase from leaves of sugar beet. AB - Sugar beet leaves (Beta vulgaris L.) contained up to five endoamylases, two exoamylases, and a single debranching enzyme. Four of the endoamylases and the debranching enzyme were present in the chloroplast. The chloroplastic starch debranching enzyme and an apoplastic endoamylase were copurified from mature leaves of sugar beet by 35 to 50% ammonium sulfate precipitation and chromatography on diethylaminoethyl-Sephacryl, beta-cyclodextrin Sepharose 6B, and Sephadex G-150. The debranching enzyme, which was purified to homogeneity, had a molecular mass of 100 kilodaltons and a pH optimum of 5.5. It showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with native starch grains isolated from sugar beet leaves. The endoamylase, which was partially purified, had a molecular mass of 43,000 kilodaltons, a pH optimum of 6.5, required calcium for activity and thermal stability, and showed an ability to hydrolyze native starch grains. PMID- 16668789 TI - Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase. AB - Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K(m) value for ribulose 5 phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K(m) values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V(max) of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate. PMID- 16668790 TI - Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids. AB - The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity. PMID- 16668791 TI - Comparison of the outer and inner epidermis : inhibition of auxin-induced elongation of maize coleoptiles by glucan antibodies. AB - Polyclonal antibodies, raised against beta-d-glucans prepared from oat (Avena sativa L.) caryopses, cross-reacted specifically with (1-->3),(1-->4)-beta-d glucans when challenged in a dot blot analysis of related polymers bound to a cellulose thin layer chromatography plate. The antibodies suppressed indoleacetic acid (IAA)-induced elongation of segments from maize (Zea mays L.) coleoptiles when the outer surface was abraded. However, IAA-induced elongation of nonabraded segments or segments with abrasion restricted to the interior of the cylinder was not influenced by the antibodies. Fab fragments prepared from the antibodies gave similar results. The capacity for IAA to overcome outward curvature of split coleoptile segments was partially reversed by treatment of the segments with the antibodies. Fluorescence microscopy revealed that antibody penetration was largely restricted to the epidermal cell wall region. These results support the view that the degradation of (1-->3),(1-->4)-beta-d-glucans in the outer epidermal cell wall serves an essential role in auxin-induced elongation of Poaceae coleoptiles. PMID- 16668792 TI - Phytoalexin Accumulation in Arabidopsis thaliana during the Hypersensitive Reaction to Pseudomonas syringae pv syringae. AB - Inoculation of leaves of Arabidopsis thaliana (L.) Heynh. with the wheat pathogen, Pseudomonas syringae pv syringae, resulted in the expression of the hypersensitive reaction and in phytoalexin accumulation. No phytoalexin accumulation was detected after infiltration of leaves with a mutant of P. s. syringae deficient in the ability to elicit a hypersensitive reaction; with the crucifer pathogen, Xanthomonas campestris pv campestris; or with 10 millimolar potassium phosphate buffer (pH 6.9). Phytoalexin accumulation was correlated with the restricted in vivo growth of P. s. syringae. A phytoalexin was purified by a combination of reverse phase flash chromatography, thin layer chromatography, followed by reverse phase high performance liquid chromatography. The Arabidopsis phytoalexin was identified as 3-thiazol-2'-yl-indole on the basis of ultraviolet, infrared, mass spectral, (1)H-nuclear magnetic resonance, and (13)C-nuclear magnetic resonance data. PMID- 16668793 TI - Internal CO(2) Measured Directly in Leaves : Abscisic Acid and Low Leaf Water Potential Cause Opposing Effects. AB - Observations of nonuniform photosynthesis across leaves cast doubt on internal CO(2) partial pressures (p(i)) calculated on the assumption of uniformity and can lead to incorrect conclusions about the stomatal control of photosynthesis. The problem can be avoided by measuring p(i) directly because the assumptions of uniformity are not necessary. We therefore developed a method that allowed p(i) to be measured continuously in situ for days at a time under growth conditions and used it to investigate intact leaves of sunflower (Helianthus annuus L.), soybean (Glycine max L. Merr.), and bush bean (Phaseolus vulgaris L.) subjected to high or low leaf water potentials (psi(w)) or high concentrations of abscisic acid (ABA). The leaves maintained a relatively constant differential (Deltap) between ambient CO(2) and measured p(i) throughout the light period when water was supplied. When water was withheld, psi(w) decreased and the stomata began to close, but measured p(i) increased until the leaf reached a psi(w) of -1.76 (bush bean), -2.12 (sunflower) or -3.10 (soybean) megapascals, at which point Deltap = 0. The increasing p(i) indicated that stomata did not inhibit CO(2) uptake and a Deltap of zero indicated that CO(2) uptake became zero despite the high availability of CO(2) inside the leaf. In contrast, when sunflower leaves at high psi(w) were treated with ABA, p(i) did not increase and instead decreased rapidly and steadily for up to 8 hours even as psi(w) increased, as expected if ABA treatment primarily affected stomatal conductance. The accumulating CO(2) at low psi(w) and contrasting response to ABA indicates that photosynthetic biochemistry limited photosynthesis at low psi(w) but not at high ABA. PMID- 16668794 TI - Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures. AB - To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K(m) values for l-glutamine, 2 oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells. PMID- 16668795 TI - Changes in Cytosolic Glutamine Synthetase Polypeptide and its mRNA in a Leaf Blade of Rice Plants during Natural Senescence. AB - Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of their corresponding mRNAs have been investigated in segments of the 13th leaf of hydroponically grown rice (Oryza sativa L.) plants during natural senescence. The leaf blade on the main stem at early (0 day), middle (15 days), and late (25 days) stages of senescence was harvested and cut into 18 or 19 segments, 2 centimeters in length from the base to the tip. The amount of GS1 polypeptide, detected with specific antibody for the GS1, was greatest near the middle of the leaf blade (segments 11-13). There was little difference in the GS1 content between corresponding leaf segments obtained at the early and middle stages of senescence. At the late senescence stage, all segments had lost some GS1 polypeptide, but more than 50% of GS1 detected at both the early and middle stages was still detectable in segments. The relative content of mRNA for GS1 in the total RNA in all segments was very low during early senescence but increased in all leaf segments during later senescence. At the late stage of senescence, GS1 mRNA in the total RNA increased about 4.2- to 4.6-fold in segments 12 to 16 in the day-25 samples compared with those in the early stage. The content of the GS2 polypeptide, as well as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein, was highest in segment 17 in the 0-day samples. During senescence, this peak became lower and broader, and finally disappeared, i.e. approximately 80% of GS2 polypeptide and Rubisco protein in segment 17 were lost by day 25. In contrast with GS2 polypeptide, the relative level of GS2 mRNA increased 1.8- to 2.9-fold in individual segments at the middle stage of senescence. Even at the late stage, the transcript signals remained slightly higher than those at the early stage in all segments. Thus, GS1 and GS2 polypeptides and corresponding mRNAs responded in a different manner within an attached rice leaf during natural senescence. The contents of GS1 and GS2 polypeptides were not simply determined by the abundance of their corresponding mRNAs in the rice leaf blades during natural senescence. PMID- 16668796 TI - Factors affecting expression of enhanced catalase activity in a tobacco mutant with o(2)-resistant photosynthesis. AB - Tobacco (Nicotiana tabacum) mutants with 40 to 50% more catalase activity than wild type show O(2)-resistant photosynthesis under conditions of high photorespiration. More than 90% of the population of mutant plants of an M(7) and M(8) generation had enhanced catalase activity, and nearly 40% had activities >3 standard deviations above the mean of wild type. Superoxide dismutase activity was the same in mutant and wild-type leaves. The greater photosynthetic rate of mutant leaves previously observed in the laboratory was confirmed with field grown plants that showed significantly higher rates (8%) than wild type during 8 days of measurements during a 19-day period of active growth. The tip region of expanding mutant leaves had higher catalase activity than the base of the lamina, and photosynthesis was O(2) resistant in 42% O(2) in the tip compared with the base, thus further supporting the hypothesis that there is a biochemical linkage between these traits. Plants grown in high light (270 micromole photons per square meter per second) had greater catalase activity and an activity ratio of mutant to wild type of 1.45 compared with 1.22 for those grown in low light (130 micromole photons per square meter per second). After acclimation for 3 weeks, plants transferred from low to high light showed increasing activities, and after 5 days the activity ratio of mutant to wild type was the same as in plants acclimated in higher light. The role of enhanced catalase activity in reducing photorespiratory CO(2) is discussed. PMID- 16668797 TI - Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light Regulated Peroxidase Activity. AB - Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6-phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related. PMID- 16668798 TI - Metabolism of 1-Aminocyclopropane-1-Carboxylic Acid in Etiolated Maize Seedlings Grown under Mechanical Impedance. AB - We investigated the metabolism of 1-aminocyclopropane-1-carboxylic acid (ACC) in etiolated maize (Zea mays L.) seedlings subjected to mechanical impedance by applying pressure to the growing medium. Total concentrations of ACC varied little in unimpeded seedlings, but impeded organs accumulated ACC. Roots had consistently higher concentrations of ACC than shoots or seeds, regardless of treatment. The concentration of ACC in the roots increased more than 100% during the first hour of treatment irrespective of the pressure applied; in shoots, total ACC concentration increased 46% at either low or high pressure during the first hour of treatment. The bulk of ACC synthesized under impeded and unimpeded conditions was present in a conjugated form, presumably, 1-(malonylamino) cyclopropane-1-carboxylic acid. However, 1-(malonylamino)-cyclopropane-1 carboxylic acid increased 73% over controls after 10 hours at 25 kilopascals of pressure. Unimpeded tissue had about 77% ACC as the conjugate and 17% as free ACC, and less than 6% was used in ethylene production. Increased amounts of ACC were converted into ethylene under stress. In vivo ACC synthase activity in roots became six and seven times higher only 1 hour after initiation of treatment at 25 and 100 kilopascals of pressure, respectively, and remained high for at least 6 hours. However, the immediate and massive conjugation of mechanically induced ACC suggests that ACC N-malonyltransferase may play an important role in the regulation of mechanically induced ethylene production. After 8 hours, in vivo activity of the ethylene-forming enzyme complex increased 100 and 50% above normal level at 100 and 25 kilopascals, respectively. Furthermore, ethylene forming enzyme complex activity was significantly greater at 100 kilopascals than in controls as early as 1 hour after treatment initiation. These data suggest that regulation of ethylene production under mechanical impedance involves the concerted action of ACC synthase, the ethylene-forming enzyme complex, and ACC N malonyltransferase. PMID- 16668799 TI - Synergistic effect of light and fusicoccin on stomatal opening : epidermal peel and patch clamp experiments. AB - Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K(+) channel blocker, tetraethyl-ammonium(+) (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (-60 millivolts) at which K(+) channels are inactivated. These results are indicative of FC activation of a H(+) pump. FC effects on the magnitude of inward and outward K(+) currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H(+) ATPase of guard cells is a primary locus for the FC effect on stomatal apertures. PMID- 16668800 TI - Regulation of Em Gene Expression in Rice : Interaction between Osmotic Stress and Abscisic Acid. AB - Expression of the Em gene was characterized in rice (Oryza sativa L.) suspension cultures following exposure of the cultures to various combinations of abscisic acid (ABA) and salt. Response-saturating concentrations of either ABA (50 micromolar) or NaCl (0.4 molar) rapidly induced (by 60 minutes) the accumulation of Em mRNA, with a maximum accumulation occurring 12 to 24 hours after treatment. NaCl-induced Em expression was accompanied by a doubling of endogenous ABA levels as determined by immunoassay. Inhibition of ABA biosynthesis by fluridone during NaCl treatment reduced the levels of endogenous ABA by fourfold and Em expression by 50%. Desiccation of the cultures to 12 to 15% of their initial fresh weight increased endogenous ABA more than twofold and was accompanied by an increase in Em mRNA levels. Exposure of the cultures to heat shock temperatures, chilling, or ultraviolet light neither increased endogenous ABA levels nor induced Em expression. When a subthreshold or saturating level of NaCl was added in combination with increasing levels of ABA, Em transcripts were detected at ABA concentrations that alone did not induce expression of Em. Treatment with saturating levels of both NaCl and ABA resulted in a doubling of Em transcript levels over the maximum signal for each treatment alone. Hence, our data suggested that salt interacted synergistically with ABA, in part because of the increased sensitivity of rice cells to ABA. The effect of salt stress on Em gene expression in rice suspension cells appeared to operate through two pathways: one is mediated through increases in the level of ABA; the other is via a unique salt response pathway that includes an intermediate that is common to both the salt and ABA response chains. PMID- 16668801 TI - Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.). AB - Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K(m) = 1.7 micromolar) compared with sinapaldehyde (K(m) in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the lambdaCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme. PMID- 16668802 TI - Inhibition of violaxanthin deepoxidation by ultraviolet-B radiation in isolated chloroplasts and intact leaves. AB - The effect of pretreatment with ultraviolet-B (UV-B) light (280-320 nanometers) on the enzymatic conversion of the diepoxyxanthophyll violaxanthin to the epoxy free zeaxanthin occurring in thylakoid membranes was investigated. When isolated chloroplasts of pea (Pisum sativum) were exposed to UV-B, a biologically effective fluence of 7000 joules per square meter caused about 50% inhibition of the activity of the violaxanthin deepoxidase, measured as the first order rate constant of the absorbance change at 505 nanometers. The dose requirement for the inhibition of the deepoxidase in intact leaves, however, was about 2 orders of magnitude higher. The inhibition of the rate constant was observed for both the dark deepoxidation at pH 5, and for the light-driven deepoxidation induced by the lumen acidification due to electron transport from H(2)O to methylviologen or due to a photosystem I partial reaction with duroquinol as the electron donor. The availability of violaxanthin was not directly affected by UV-B radiation, as shown for UV-B-treated chloroplasts by the final extent of the 505 nanometer change measured in the dark at pH 5 or by the partial photosystem I reaction. A significant decrease in the violaxanthin availability was observed when lumen acidification was caused by electron transport from H(2)O to methylviologen. That effect was probably caused by the wellknown UV-B inhibition of photosystem II with a subsequent decreased ability to reduce the plastoquinone pool, the redox state of which is believed to regulate the final amount of converted violaxanthin. PMID- 16668803 TI - Phase shift in leaf movements of xanthium attributed to age and rhythm patterns. AB - Leaves of cockelbur (Xanthium strumarium L.) have been reported to be in either an upright or downward position during the dark span (night) of a 24-hour cycle. Results from our studies clearly indicate that such differences in leaf position are not related to differences in ecotypes but can be attributed to age of the leaf, pattern of the waveform of the rhythm at various stages of the light-dark synchronizer regimen, and the statistical model used for the analysis of the waveform. Younger leaves reached a maximum upright position closer to the middle of the dark span, whereas older leaves reached this position closer to the end of the dark span. A phase shift of up to 6 to 10 hours may occur as the leaf ages. Results from the examination of the pattern of the waveform at four different times showed that the pattern of a younger leaf was different from that of an older leaf during the middle of the dark span, during the light-to-dark transition, and during the middle of the light span, but not during the dark-to light transition. Linear regression, statistical analyses, and the fitting of harmonics clearly indicate that it is the trough, more than the peak, that differs with the age of the leaf. PMID- 16668804 TI - In Situ Abscisic Acid Synthesis : A Requirement for Induction of Embryo Dormancy in Helianthus annuus. AB - When applied to young nondormant embryos of sunflower (Hellanthus annus) (7-10 day[s] after pollination [DAP]), abscisic acid (ABA) inhibited germination as long as it was present. However, whatever the dose used and the duration of its application, ABA was unable to induce dormancy because after transfer of treated embryos to control (without ABA) medium, germination occurred. Thereafter, exogenous ABA became effective and allowed the dormancy to develop in 13 and 17 DAP embryos, i.e. in embryos which after isolation were still able to germinate in high percentage. After embryo dormancy was well established (21 DAP), application of fluridone allowed the germination to occur very quickly on control medium. Isolated dormant axes were also induced to germinate by an application of fluridone. Radioimmunological analysis showed that 24 hours after these treatments, endogenous ABA levels were drastically reduced in the axes. When these fluridone-treated embryos were cultured on ABA medium, germination was again inhibited as long as exogenous ABA was present but germination occurred as soon as embryos were transferred to control medium. Such behavior suggested that in situ ABA synthesis is necessary to impose and maintain the embryo dormancy. PMID- 16668805 TI - Abscisic Acid and the maturation of cacao embryos in vitro. AB - Abscisic acid (ABA) was tested for its ability to affect development of immature zygotic embryos of cacao (Theobroma cacao) in vitro, by adding exogenous ABA, fluridone, or mefluidide to cultured embryos. Endogenous ABA levels, measured by enzyme-linked immunosorbent assay, were increased by exogenous ABA or by culture on sucrose increasing to 21%, and were decreased by fluridone and, to a lesser extent, by mefluidide. The effects of these on maturation were measured as effects on anthocyanins, lipids, and fatty acid saturation, all of which increase with maturation of the cacao embryo. Maturation was stimulated by increasing sucrose and, to a lesser degree, the addition of ABA, but decreasing endogenous ABA by treating with fluridone significantly inhibited all maturation parameters. Although desiccation tolerance does not develop in cacao embryos, these results suggest that ABA and sucrose are both needed for the initiation of events associated with maturation in vitro. PMID- 16668806 TI - Mannitol Synthesis in Higher Plants : Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase. AB - Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase (14)C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K(m) (M6P) was 15.8 millimolar, but the apparent K(m) (mannitol 1 phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1 phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg(2+), Ca(2+), Zn(2+), and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants. PMID- 16668807 TI - Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress. AB - We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells. PMID- 16668808 TI - Translational Regulation of the Large and Small Subunits of Ribulose Bisphosphate Carboxylase/Oxygenase during Induction of the CO(2)-Concentrating Mechanism in Chlamydomonas reinhardtii. AB - In conditions of limiting external inorganic carbon, the unicellular alga Chlamydomonas reinhardtii induces a mechanism to actively transport and accumulate inorganic carbon within the cell. A high internal inorganic carbon concentration enables the cell to photosynthesize efficiently with little oxygen inhibition, even in conditions of limiting external inorganic carbon. A correlation between limiting inorganic carbon-induced induction of the CO(2) concentrating mechanism and decreased synthesis of the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase has been observed. Cells that had been transferred from elevated CO(2) to limiting CO(2) exhibit transient declines of label incorporation into both subunit polypeptides. We have found that this decrease in synthesis of large and small subunits results from specific and coordinated down-regulation of translation of both subunits possibly resulting, at least in part, from modification of large and small subunit transcripts. PMID- 16668809 TI - Effect of diclofop on the membrane potentials of herbicide-resistant and susceptible annual ryegrass root tips. AB - Electrophysiological measurements were made on root tip cells in the elongation zone of diclofop-methyl-resistant (SR4/84) and -susceptible (SRS2) biotypes of annual ryegrass (Lolium rigidum Gaud.) from Australia. The phytotoxic action of diclofop-methyl (methyl 2-[4-(2',4'-dichlorophenoxy)phenoxy]propanoate) on susceptible whole plants was completely reversed by a simultaneous application of 2,4-dichlorophenoxyacetic acid (dimethylamine salt). The phytotoxic acid metabolite, diclofop (50 micromolar), depolarized membrane potentials of both biotypes to a steady-state level within 10 to 15 minutes. Repolarization of the membrane potential occurred only in the resistant biotype following removal of diclofop. The resistant biotype has an intrinsic ability to reestablish the electrogenic membrane potential, whereas the susceptible biotype required an exogeneous source of IAA to induce partial repolarization. Both biotypes were susceptible to depolarization by carbonylcyanide-m-chlorophenylhy-drazone (CCCP), and their membrane potentials recovered upon removal of CCCP. A 15-minute pretreatment with p-chloromercuribenzenesulphonic acid (PCMBS) blocked the depolarizing action of diclofop in both biotypes. However, PCMBS had no effect on the activity of CCCP. The action of diclofop appears to involve a site-specific interaction at the plasmalemma in both Lolium biotypes to cause the increased influx of protons into sensitive cells. The differential response of membrane depolarization and repolarization to diclofop treatment may be a significant initial reaction in the eventual phytotoxic action of the herbicide. PMID- 16668810 TI - Development of the Potential for Cyanogenesis in Maturing Black Cherry (Prunus serotina Ehrh.) Fruits. AB - Biochemical changes related to cyanogenesis (hydrogen cyanide production) were monitored during maturation of black cherry (Prunus serotina Ehrh.) fruits. At weekly intervals from flowering until maturity, fruits (or selected parts thereof) were analyzed for (a) fresh and dry weights, (b) prunasin and amygdalin levels, and (c) levels of the catabolic enzymes amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. During phase I (0-28 days after flowering [DAF]), immature fruits accumulated prunasin (mean: 3 micromoles/fruit) but were acyanogenic because they lacked the above enzymes. Concomitant with cotyledon development during mid-phase II, the seeds began accumulating both amygdalin (mean: 3 micromoles/seed) and the catabolic enzymes and were highly cyanogenic upon tissue disruption. Meanwhile, prunasin levels rapidly declined and were negligible by maturity. During phases II (29-65 DAF) and III (66-81 DAF), the pericarp also accumulated amygdalin, whereas its prunasin content declined toward maturity. Lacking the catabolic enzymes, the pericarp remained acyanogenic throughout all developmental stages. PMID- 16668811 TI - Theoretical Considerations when Estimating the Mesophyll Conductance to CO(2) Flux by Analysis of the Response of Photosynthesis to CO(2). AB - The conductance for CO(2) diffusion in the mesophyll of leaves can limit photosynthesis. We have studied two methods for determining the mesophyll conductance to CO(2) diffusion in leaves. We generated an ideal set of photosynthesis rates over a range of partial pressures of CO(2) in the stroma and studied the effect of altering the mesophyll diffusion conductance on the measured response of photosynthesis to intercellular CO(2) partial pressure. We used the ideal data set to test the sensitivity of the two methods to small errors in the parameters used to determine mesophyll conductance. The two methods were also used to determine mesophyll conductance of several leaves using measured rather than ideal data sets. It is concluded that both methods can be used to determine mesophyll conductance and each method has particular strengths. We believe both methods will prove useful in the future. PMID- 16668812 TI - Estimation of Mesophyll Conductance to CO(2) Flux by Three Different Methods. AB - The resistance to diffusion of CO(2) from the intercellular airspaces within the leaf through the mesophyll to the sites of carboxylation during photosynthesis was measured using three different techniques. The three techniques include a method based on discrimination against the heavy stable isotope of carbon, (13)C, and two modeling methods. The methods rely upon different assumptions, but the estimates of mesophyll conductance were similar with all three methods. The mesophyll conductance of leaves from a number of species was about 1.4 times the stomatal conductance for CO(2) diffusion determined in unstressed plants at high light. The relatively low CO(2) partial pressure inside chloroplasts of plants with a low mesophyll conductance did not lead to enhanced O(2) sensitivity of photosynthesis because the low conductance caused a significant drop in the chloroplast CO(2) partial pressure upon switching to low O(2). We found no correlation between mesophyll conductance and the ratio of internal leaf area to leaf surface area and only a weak correlation between mesophyll conductance and the proportion of leaf volume occupied by air. Mesophyll conductance was independent of CO(2) and O(2) partial pressure during the measurement, indicating that a true physical parameter, independent of biochemical effects, was being measured. No evidence for CO(2)-accumulating mechanisms was found. Some plants, notably Citrus aurantium and Simmondsia chinensis, had very low conductances that limit the rate of photosynthesis these plants can attain at atmospheric CO(2) level. PMID- 16668813 TI - Protein Differences between Two Isogenic Cultivars of Barley (Hordeum vulgare L.) that Differ in Sensitivity to Photoperiod and Far-Red Light. AB - A photoperiodically sensitive cultivar of barley (Hordeum vulgare L. Shabet) (BMDR-8) and an isogenic, single-gene recessive mutant of this genotype that is insensitive to photoperiod (BMDR-1) were grown under continuous cool white light with or without supplemental far-red fluorescent light. BMDR-1 initiates flowers 6 days after germination, irrespective of light treatment, whereas BMDR-8 remains vegetative for at least a week longer, even in continuous light. When far-red light is added, the delay of flowering in BMDR-8 is overcome and both genotypes initiate floral primordia at the same time. Total phenol extracted proteins of seedlings of both genotypes were resolved by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No protein differences were found between the genotypes when isoelectric focusing gels were run in the first dimension. Two qualitative genotypic differences were found when nonequilibrium pH gradient gel electrophoresis was run in the first dimension. An 85-kilodalton polypeptide (A) and a 26-kilodalton polypeptide (B) were always present in BMDR-8 but never found in BMDR-1. The levels of A appeared to decrease from the BMDR-8 during the first 3 days of far-red treatment but did not disappear completely even after 6 days of growth in the presence of farred. Polypeptide B decreases rapidly in continuous cool white light but is stabilized by far-red. The phytochrome content of BMDR-1 was found to be greater than that for BMDR-8. This increase appears to be caused by the type I (etiolated-tissue abundant) phytochrome pool, even in plants grown in continuous light. PMID- 16668814 TI - Factors Affecting the Acetylene-Induced Decline during Nitrogenase Assays in Root Nodules of Myrica gale L. AB - Our goal was to determine why the rate of acetylene reduction by nodules of actinorhizal plants declines after an initial peak value. The decline was eliminated by pretreatment with argon, indicating that the decline is initiated by cessation of ammonia synthesis. When O(2) concentration was decreased during the decline, the rate of acetylene reduction increased. This shows that during the decline there is either O(2) toxicity or competition between respiration and nitrogenase for reductant. The decline was not eliminated when uptake hydrogenase was inactivated by pretreatment with acetylene, showing that cessation of H(2) oxidation is not the primary cause of the decline. The effects of a variety of other treatments on the decline were also studied. Overall, we conclude that the cessation of ammonia formation is the primary cause of the acetylene-induced decline. We hypothesize that the supply of reductant for nitrogenase depends on amino acids that are depleted following cessation of ammonia formation. We also conclude that the initial peak rate of acetylene reduction provides the best measure of nitrogenase activity. PMID- 16668815 TI - Sugar Concentrations in Guard Cells of Vicia faba Illuminated with Red or Blue Light : Analysis by High Performance Liquid Chromatography. AB - Concentrations of soluble sugars in guard cells in detached, sonicated epidermis from Vicia faba leaves were analyzed quantitatively by high performance liquid chromatography to determine the extent to which sugars could contribute to changes in the osmotic potentials of guard cells during stomatal opening. Stomata were illuminated over a period of 4 hours with saturating levels of red or blue light, or a combination of red and blue light. When stomata were irradiated for 3 hours with red light (50 micromoles per square meter per second) in a solution of 5 millimolar KCl and 0.1 millimolar CaCl(2), stomatal apertures increased a net maximum of 6.7 micrometers and the concentration of total soluble sugar was 289 femtomoles per guard cell (70% sucrose, 30% fructose). In an identical solution, 2.5 hours of irradiation with 25 micromoles per square meter per second of blue light caused a maximum net increase of 7.1 micrometers in stomatal aperture and the total soluble sugar concentration was 550 femtomoles per guard cell (91% sucrose, 9% fructose). Illumination with blue light at 25 micromoles per square meter per second in a solution lacking KCl caused a maximum net increase in stomatal aperture of 3.5 micrometers and the sugar concentration was 382 femtomoles per guard cell (82% sucrose, 18% fructose). In dual beam experiments, stomata irradiated with 50 micromoles per square meter per second of red light opened steadily with a concomitant increase in sugar production. Addition of 25 micromoles per square meter per second of blue light caused a further net gain of 3.7 micrometers in stomatal aperture and, after 2 hours, sugar concentrations had increased by an additional 138 femtomoles per guard cell. Experiments with 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) were performed with epidermis illuminated with 50 micromoles per square meter per second of red light or with 25 micromoles per square meter per second of blue light in solutions containing or lacking KCl. DCMU completely inhibited sugar production under red light, had no effect on guard cell sugar production under blue light when KCl was present, and inhibited sugar production by about 50% when guard cells were illuminated with blue light in solutions lacking KCl. We conclude that soluble sugars can contribute significantly to the osmoregulation of guard cells in detached leaf epidermis of V. faba. These results are consistent with the operation of two different sugar producing pathways in guard cells: a photosynthetic carbon reduction pathway and a pathway of blue light-induced starch degradation. PMID- 16668816 TI - Phosphate-Regulated Induction of Intracellular Ribonucleases in Cultured Tomato (Lycopersicon esculentum) Cells. AB - Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nurnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems. PMID- 16668817 TI - Inhibition of Sucrose Enhancer Effect of the Potato Proteinase Inhibitor II Promoter by Salicylic Acid. AB - Effect of salicylic acid (SA) on the expression of the potato proteinase inhibitor (PI) II promoter was studied with transgenic tobacco plants (Nicotiana tabacum) carrying a gene fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) reporter. As previously observed, the PI II promoter was inducible by wounding and the promoter activity was further enhanced by sucrose. Addition of SA did not influence the wound induction of the PI-II promoter but significantly inhibited the sucrose response. The 5'-deletion mutant -573 was unable to respond to wounding but did respond to sucrose and SA. The 3'-deletion analysis indicated the presence of a sucrose-responsive element between -574 and -520. A study of the insertion mutants revealed the function of another sucrose-responsive element between -522 and -500. Enhancer effects of these sucrose-responsive elements were inhibited by SA. These studies suggest that SA inhibits PI-II promoter activity by decreasing the sucrose response. Analysis of SA-related chemicals revealed that only acetyl-SA showed a similar inhibitory effect, and other hydroxybenzoic acids had little or no effect on the sucrose enhancer activity. Therefore, it seems that the interaction between SA and the receptor molecule is specific. PMID- 16668818 TI - Role of Abscisic Acid in the Induction of Desiccation Tolerance in Developing Seeds of Arabidopsis thaliana. AB - In contrast to wild-type seeds of Arabidopsis thaliana and to seeds deficient in (aba) or insensitive to (abi3) abscisic acid (ABA), maturing seeds of recombinant (aba,abi3) plants fail to desiccate, remain green, and lose viability upon drying. These double-mutant seeds acquire only low levels of the major storage proteins and are deficient in several low mol wt polypeptides, both soluble and bound, and some of which are heat stable. A major heat-stable glycoprotein of more than 100 kilodaltons behaves similarly; during seed development, it shows a decrease in size associated with the abi3 mutation. In seeds of the double mutant from 14 to 20 days after pollination, the low amounts of various maturation specific proteins disappear and many higher mol wt proteins similar to those occurring during germination are induced, but no visible germination is apparent. It appears that in the aba,abi3 double mutant seed development is not completed and the program for seed germination is initiated prematurely in the absence of substances protective against dehydration. Seeds may be made desiccation tolerant by watering the plants with the ABA analog LAB 173711 or by imbibition of isolated immature seeds, 11 to 15 days after pollination, with ABA and sucrose. Whereas sucrose stimulates germination and may protect dehydration-sensitive structures from desiccation damage, ABA inhibits precocious germination and is required to complete the program for seed maturation and the associated development of desiccation tolerance. PMID- 16668819 TI - Two Types of Channels Involved in the Malate Ion Transport across the Tonoplast of a Crassulacean Acid Metabolism Plant. AB - Ion channels in tonoplast of leaf cells of a Crassulacean acid metabolism plant, Graptopetalum paraguayense, using the patch clamp technique were investigated. Results showed the existence of two types of channels involved in the malate ion transport across the tonoplast. One type corresponded to the slow-activating vacuolar-type (R Hedrich, E Neher [1987] Nature 329: 833-836), probably taking part in the malate efflux from vacuoles. Another showed the membrane potential dependent channel current of malate flux over a wide range of cytoplasmic free Ca(2+) concentration (10(-8)-10(-5) molar), a property favoring the malate uptake. This type seems to be different from the fast-activating vacuolar-type. PMID- 16668820 TI - Evidence of two isomers of phosphatidylinositol in plant tissue. AB - The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-(3)H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2 diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group. PMID- 16668821 TI - Pathogenesis-related proteins and polyamines in a developmental mutant of tomato, epinastic. AB - The polyamine level and the accumulation of pathogenesis-related (PR) proteins were studied in the ethylene overproducing Epinastic (Epi) tomato (Lycopersicon esculentum Mill.) mutant, as compared with its parent, cv VFN8. Neither a decreased putrescine level nor an enhanced production of PR proteins were detected in Epi, contrary to what could be expected from our previous studies (JM Belles, J Carbonell, V Conejero [1991] Plant Physiol 96: 1053-1059). However, treatment with the ethylene-releasing compound 2-chloroethylphosphonic acid (ethephon) or silver nitrate at high doses induced a decrease in putrescine content and an enhancing of the synthesis of PR proteins in Epi as ascertained by immunoblot analysis using antisera raised against Rutgers tomato PR proteins. PMID- 16668822 TI - Amino Acid Sequence and Molecular Weight of Native NADP Malate Dehydrogenase from the C(4) Plant Zea mays. AB - N-terminus amino acid analysis of purified corn (Zea mays) NADP malate dehydrogenase showed that the mature protein begins at serine-41 of the preprotein sequence and not threonine-58 as previously concluded; therefore, the transit peptide consists of 40 amino acids. The theoretical molecular weight of the mature subunit protein (392 amino acids) is 42,564, agreeing with an experimental value of about 43,000. The molecular weight of the native unactivated (dark form) and activated (light form) of NADP malate dehydrogenase, determined by analytical ultracentrifugation analysis, was about 84,000, indicating that both forms are dimers. However, conventional and high performance liquid chromatography gel filtration procedures indicated apparent molecular weights of about 110,000 to 120,000 for the unactivated native enzyme and about 143,000 to 150,000 for the active enzyme; in these cases, the molecular weight may be overestimated due to the effect of an unusual molecular conformation on the mobility of the enzyme. PMID- 16668823 TI - Phytochrome activation of k channels and chloroplast rotation in mougeotia: action spectra. AB - The action spectra for K(+) channel activation and chloroplast rotation are shown to be similar. Both phenomena exhibit activation at 660 nanometers, inhibition at 740 nanometers, and partial activation at 460 to 500 nanometers. This confirms that K(+) channels in Mougeotia are regulated by phytochrome, and indicates that both phenomena share at least part of the same transduction pathway. PMID- 16668824 TI - Clomazone does not inhibit the conversion of isopentenyl pyrophosphate to geranyl, farnesyl, or geranylgeranyl pyrophosphate in vitro. AB - Clomazone, an herbicide that reduces the levels of leaf carotenoids and chlorophylls, is thought to act by inhibiting isopentenyl pyrophosphate isomerase or the prenyltransferases responsible for the synthesis of geranylgeranyl pyrophosphate. Cell-free extracts prepared from the oil glands of common sage (Salvia officinalis) are capable of converting isopentenyl pyrophosphate to geranylgeranyl pyrophosphate. Clomazone at 250 micromolar (a level that produced leaf bleaching) had no detectable effect on the activity of the relevant enzymes (isopentenyl pyrophosphate isomerase and the three prenyltransferases, geranyl, farnesyl, and geranylgeranyl pyrophosphate synthases). Thus, inhibition of geranylgeranyl pyrophosphate biosynthesis does not appear to be the mode of action of this herbicide. PMID- 16668825 TI - H-Protein of the Glycine Decarboxylase Multienzyme Complex : Complementary DNA Encoding the Protein from Arabidopsis thaliana. PMID- 16668826 TI - Nucleotide Sequences of cDNAs Encoding Two Members of the Brazil Nut Methionine Rich 2S Albumin Gene Family. PMID- 16668827 TI - Sequences of the Cotton Group 2 LEA/RAB/Dehydrin Proteins Encoded by Lea3 cDNAs. PMID- 16668828 TI - Isolation and Characterization of a Gene Encoding a Carboxypeptidase Y-Like Protein from Arabidopsis thaliana. PMID- 16668829 TI - Sequence of a cDNA coding for a 1-aminocyclopropane-1-carboxylate oxidase homolog from apple fruit. PMID- 16668830 TI - Complementary DNA Sequence of a Low Temperature-Induced Brassica napus Gene with Homology to the Arabidopsis thaliana kin1 Gene. PMID- 16668831 TI - Tomato Acid phosphatase-1 gene from a nematode resistant cultivar. PMID- 16668832 TI - Barley (Hordeum vulgare) Gene for CP29, a Core Chlorophyll a/b Binding Protein of Photosystem II. PMID- 16668833 TI - Nucleotide Sequence of the psbB Gene of Chlamydomonas reinhardtii Chloroplasts. PMID- 16668834 TI - A rapid method for isolating glandular trichomes. AB - A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium xhortorum L.H. Bailey. These purified cells contained high concentrations of unusual omega5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no omega5-fatty acids, thereby unequivocally localizing omega5-fatty acids to the trichomes. Because omega5-fatty acids are unique precursors for the biosynthesis of omega5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes. PMID- 16668835 TI - Light-Dependent Changes of the Cytoplasmic H and Cl Activity in the Green Alga Eremosphaera viridis. AB - Ion-sensitive microelectrodes were used to measure Cl(-) and H(+) activities in the cytoplasm of the unicellular green alga Eremosphaera viridis de Bary. In the light, cytoplasmic Cl(-) activity was 2.2 millimolar at most and cytoplasmic H(+) activity was about 5.4.10(-8) molar (pH 7.3). Darkening resulted in a permanent increase of the Cl(-) activity to 3.2 millimolar and in a transient acidification, which was compensated within 3 to 5 minutes. Switching light on again decreased the Cl(-) activity to the light level (2.2 millimolar). Simultaneously, a transient alkalization of the cytoplasm was observed. The transient character of the light-dependent pH changes was probably caused by pH stat mechanisms, whereas the light-dependent Cl(-) activity changes were compensated to a much smaller degree. Studies with different inhibitors (3-(3,4 dichlorophenyl)-1, 1-dimethylurea, piretanide, venturicidin) indicated a direct relation between the light-driven H(+) flow across the thylakoid membrane and the observed light-dependent Cl(-) and H(+) activity changes in the cytoplasm. It is suggested that light-driven H(+) flux across the thylakoid membrane was in part electrically compensated by a parallel Cl(-) flux. The resulting Cl(-) and H(+) activity changes in the stroma were compensated by Cl(-) and H(+) fluxes across the chloroplast envelope giving rise to the observed Cl(-) and H(+) activity changes in the cytoplasm. PMID- 16668836 TI - Induction of somatic embryogenesis using side chain and ring modified forms of phenoxy Acid growth regulators. AB - The induction of somatic embryo development in cell cultures of alfalfa (Medicago sativa), celery (Apium graveolens), and lettuce (Lactuca sativa) was compared for 2,4-dichlorophenoxy-acetic acid (2,4-D) and various phenoxy acid growth regulators. Tests using a series of straight chain extensions to the phenoxy acid side chain indicate that phenoxybutanoic acid is active, whereas the phenoxypropanoic and phenoxypentanoic analogs are inactive for the induction of alfalfa embryogenesis. Side branching on the carbon adjacent to the phenoxy group results in optically active compounds. Racemic mixtures and the (+) enantiomers of the compounds are active for alfalfa embryo induction, whereas the (-) enantiomers are inactive and apparently do not inhibit embryogenesis in any way. Development of alfalfa embryos, as measured by plantlet formation from individual embryos, is improved by 4-(2,4-dichlorophenoxy)butanoic acid and with side branching at the carbon adjacent to the phenoxy group compared with induction with 2,4-D. Similarly, substituted phenoxy acids also enhance somatic embryo development in celery and lettuce when compared with 2,4-D. These results are discussed with reference to earlier studies on the structure activity of various synthetic auxins during cell elongation and with reference to the possible importance of auxin metabolism on subsequent somatic embryo development. PMID- 16668837 TI - Pyrroline-5-Carboxylate Reductase in Soybean Nodules : Comparison of the Enzymes in Host Cytosol, Bradyrhizobium japonicum Bacteroids, and Cultures. AB - Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD(+)-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP(+) and proline. The K(1) for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable. PMID- 16668838 TI - Changes in fluorescence quenching brought about by feeding dithiothreitol to illuminated leaves. AB - When CO(2) is abruptly removed from the atmosphere surrounding an illuminated leaf, the primary electron-accepting plastoquinone of photosystem II (Q(A)) (as measured by photochemical quenching, q(p)) is rapidly reduced and then, after some seconds, becomes more oxidized. The reoxidation of Q(A) (-) is accompanied by an increase in DeltapH (as measured by nonphotochemical quenching, q(N)) with kinetics consistent with a causal relationship. The fact that, in such circumstances, Q(A) can become more oxidized in the absence of CO(2) than in its presence indicates a diminished rate of reduction of Q(A), consequent upon impaired photosystem II efficacy. Dithiothreitol (DTT) feeding, which does not affect quantum yield or the maximum rate of photosynthesis, inhibits the reoxidation of Q(A) (-) but not the increase in the proton gradient. When leaves are reilluminated in high light following a dark interval of several minutes, DTT also abolishes the separation in time between the first maximum in q(P) and the first maximum in the rate of O(2) evolution. It also diminishes subsequent oscillations. These results are held to demonstrate DeltapH control of photosystem II and are consistent with DTT inhibition of the xanthophyll cycle and hydrogen peroxide reduction. They support the concept that oxygen and hydrogen peroxide are involved, as Hill oxidants, in a pH-related manner, during oscillatory behavior. PMID- 16668839 TI - Occurrence of acetylcholine-hydrolyzing activity at the stele-cortex interface. AB - Acetylcholine (ACh) is a chemical transmitter serving to propagate an electrical perturbation across the synaptic junctions of animals. ACh and AChE have previously been demonstrated to occur in plants. In this work, we detected AChE at the interface between stele and cortex of the mesocotyl of Zea mays by measuring hydrolysis of acetylthiocholine and by liberation of labeled acetate from [1-(14)C]ACh. AChE activity was also detected in a crude membrane fraction. The hydrolytic activity is inhibited by neostigmine. Hydrolysis of ACh was also measured after injection of [1-(14)C]ACh into kernels of Zea mays and the radioactivity transported into the mesocotyl cortex. A gravity stimulus was then given by placing the plants in a horizontal position. Significantly more radioactivity was found in the lower cortex of horizontally placed seedlings. A working hypothesis is presented for the involvement of ACh and AChE in the tropic response of Z. mays seedlings. PMID- 16668840 TI - Glycine uptake into barley mesophyll vacuoles is regulated but not energized by ATP. AB - [U-(14)C]glycine uptake into barley (Hordeum vulgare cv Hasso) vacuoles was investigated. Glycine (2 millimolar) transport was stimulated two- to fourfold by NaATP. Stimulation was saturable with respect to ATP (1 millimolar) and linear up to 20 millimolar glycine. Stimulation by NaATP was suppressed by Mg(2+) in equimolar amounts. Neither MgATP nor Mg-inorganic pyrophosphate had any effect on basal transport rate. Thus, the proton motive force can be excluded as the driving force. Uncouplers (valinomycine/carbonylcyanide-m-chlorophenylhydrazone) inhibited the basal rate up to 30% but had no influence on NaATP-stimulated uptake. Vanadate had no effect on either basal or NaATP-stimulated uptake. Nonhydrolyzable ATP analogs (adenylyl(beta, gamma-methylen)-diphosphate or adenylyl-imidodiphosphate) stimulated comparable to NaATP. Other nucleotides (UTP, ADP) had no effect. Some evidence exists that other amino acids (arginine, alanine, isoleucine, phenylalanine) are transported to a certain extent by a similar mechanism. The results indicate a high capacity channel-like translocator that is regulated but not energized by ATP. PMID- 16668841 TI - Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose 1,6-Bisphosphate in Maize (Zea mays L.) Kernels. AB - The significance of the glycolytic and gluconeogenic conversion of fructose-6 phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates. PMID- 16668842 TI - Alterations in Carbohydrate Intermediates in the Endosperm of Starch-Deficient Maize (Zea mays L.) Genotypes. AB - Metabolite levels in kernels of selected starch-deficient mutants of maize (Zea mays L.) were investigated to gain insight into partitioning of carbohydrate metabolism during kernel development. Several free sugars, hexose phosphates, triose phosphates, fructose-2,6-bisphosphate, and pyrophosphate were measured in normal, shrunken, shrunken-2, amylose extender dull waxy, and brittle genotypes, which were in a near-isogenic W64A background. These mutants were selected to include at least one lesion in both the cytosolic (shrunken) and amyloplastic (shrunken-2) compartments. All the starch-deficient genotypes contained elevated levels of fructose-2,6-bisphosphate and triose phosphates but reduced levels of pyrophosphate, indicating an enhanced glycolytic utilization of carbohydrates in response to the reduced utilization of sugars for starch synthesis. The shrunken kernels (sucrose synthase deficient) contained reduced levels of glucose-1 phosphate, glucose-6-phosphate, and fructose-6-phosphate, and this reduction paralleled the reduction in starch accumulation, but levels of triose phosphates were elevated. In shrunken-2 kernels, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate, dihydroxyacetone phosphate, and glyceraldehyde-3 phosphate were increased, but fructose-1,6-bisphosphate was lower. These findings support the view that hexose phosphate transport across the amyloplast envelope is more important for starch biosynthesis than transport of triose phosphates. The amylose extender dull waxy mutation showed less dramatic effects on hexose phosphates, but the triose phosphates were greatly increased. The brittle mutation, which has an unknown lesion, showed distinctly similar changes in metabolite levels with shrunken-2, suggesting that the lesion may be associated with the amyloplast. PMID- 16668843 TI - Characterization of the Activation of Membrane-Bound and Soluble CF(1) by Thioredoxin. AB - The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF(1)) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF(1). Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the gamma subunit of CF(1) to reduced ThR. The proton gradient substantially accelerates the activation of CF(1) compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF(1) with ThR was studied using fluorescent probes. CF(1) in solution, with and without its associated epsilon-subunit, was labeled at Cys-322 of the gamma subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF(1) showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF(1) form an intermolecular complex. When the epsilon-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the gamma subunit of CF(1). The distance between the fluorescent probes on CF(1) and ThR was calculated to be approximately 65 A when epsilon-subunit was present and 52 A when epsilon was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF(1). Whereas the extent of quenching increased by removal of the epsilon-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the epsilon-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching. PMID- 16668844 TI - Inhibition of Water Splitting Increases the Susceptibility of Photosystem II to Photoinhibition. AB - Photosystem II (PSII)-enriched membrane particles were isolated from peas (Pisum sativum L.) and treated in several different ways to inhibit the water oxidation reactions, but not reaction center function itself, as judged by the light induced rate of reduction of 2,6-dichlorophenol indophenol with and without the artificial electron donor, diphenyl carbazide. It was shown that such treatments increased the susceptibility of the PSII-enriched membranes to photoinhibition. This trend was further observed if 2,6-dichlorophenol indophenol was present during the illumination with photoinhibitory light. On the other hand, protection against the enhanced photoinhibition was found when the water-splitting activity was reconstituted or when the artificial electron donor diphenyl carbazide was present during the preillumination. The results indicate that irreversible photodamage occurred within the PSII reaction center as a consequence of illumination with strong light and that the rate of this damage was enhanced under conditions that are expected to give rise to a photoaccumulation of oxidizing species such as P680(+) on the donor side of PSII. This mechanism of photoinhibitory damage occurred under both aerobic and anaerobic conditions. PMID- 16668845 TI - Dissociation and Reassembly of the Vacuolar H-ATPase Complex from Oat Roots. AB - Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H(+)-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H(+)-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg(2+) and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H(+)-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO(3), or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO(3) > KBr > K-acetate > K(2)SO(4) > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H(+)-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H(+) pumping as seen from the recovery of H(+) transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H(+) ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H(+)-ATPase to form a functional H(+) pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H(+) ATPase in vivo. PMID- 16668846 TI - Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots. AB - The vacuolar H(+)-translocating ATPase (H(+)-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A(1), a specific vacuolar type ATPase inhibitor. The purified oat H(+)-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H(+)-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H(+) pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H(+)-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H(+)-ATPase. These results demonstrate that the vacuolar H(+)-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H(+) ATPases exist in plants. PMID- 16668847 TI - Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin stimulated growth. AB - The structural features required for xyloglucan oligosaccharides to inhibit 2,4 dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-beta-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8') was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific alpha-xylosidase from pea seedlings. XG8' was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4 dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth. PMID- 16668848 TI - Radial Turgor and Osmotic Pressure Profiles in Intact and Excised Roots of Aster tripolium: Pressure Probe Measurements and Nuclear Magnetic Resonance-Imaging Analysis. AB - High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems. PMID- 16668849 TI - A role for membrane lipid polyunsaturation in chloroplast biogenesis at low temperature. AB - Two different mutants of Arabidopsis thaliana deficient in chloroplast membrane lipid polyunsaturation were indistinguishable in appearance from the wild-type when grown at 22 degrees C. By contrast, leaf tissues of the mutants that developed during growth at 5 degrees C were chlorotic, whereas the wild type was not. This is the first direct evidence that chloroplast lipid polyunsaturation contributes to low-temperature fitness. Chloroplasts from mutant lines grown at 5 degrees C were much smaller than those of the wild-type, and the thylakoid membrane content was reduced by up to 70%. However, there was no discernible effect of low temperature on chloroplasts that developed prior to exposure to low temperatures. These and related observations suggest that the high degree of chloroplast membrane lipid polyunsaturation is required for some aspect of chloroplast biogenesis. PMID- 16668850 TI - Structural Changes and Associated Reduction of Hydraulic Conductance in Roots of Sorghum bicolor L. following Exposure to Water Deficit. AB - The effects of a severe water deficit on total root (L(t)) and axial (L(x)) hydraulic conductances and on the development of the hypodermis, endodermis, and xylem were studied in sorghum (Sorghum bicolor L.). Water deficit was imposed in the upper rooting zone while the lower zones were kept moist. L(t) and L(x) were based on water flow rates obtained by applying suction to proximal xylem ends of excised roots. The development of the hypodermis, endodermis, and other tissues were examined by staining with fluorescent berberine hemisulfate and phloroglucinol-HCl. The L(t) value (x 10(-8) meters per second per megapascal) for unstressed control roots was 22.0 and only 5.9 for stressed roots. The low L(t) in stressed roots was attributed, in part, to accelerated deposition of lignin and suberin in the hypodermis and endodermis. Calcofluor, an apoplastic tracer that binds to cellulose, was blocked in stressed roots at the lignified and suberized outer tangential walls of the hypodermis but readily penetrated the cortical walls of similar root regions in controls where the casparian band was not developed. L(x) per unit root length was about 100 times lower in stressed roots than in controls because of the persistence of late metaxylem cross-walls and the smaller diameter and lower number of conductive protoxylem and early metaxylem vessels. PMID- 16668851 TI - Effect of Cold Treatments on the Binding Stability of Photosystem II Extrinsic Proteins and an Associated Increase in Susceptibility to Photoinhibition. AB - When pea plants (Pisum sativum L. cv Feltham First) are subjected to freezing conditions (-18 degrees C) followed by a thaw to 18 degrees C, there is a significant inhibition of water-splitting capacity judged by the rate of light induced reduction of 2,6-dichlorophenol indophenol using isolated thylakoid membrane fragments enriched in photosystem II (PSII). The freeze-thaw-induced inhibition of water-splitting activity has been correlated with the loss of the 17- and 23-kilodalton extrinsic protein of PSII and with a weakening of the binding of the 33-kilodalton protein. There was no apparent loss of bound manganese. Addition of 10 millimolar CaCl(2), however, allowed a full recovery of the water-splitting activity of these modified PSII-enriched particles. The freeze-thaw-induced changes in the organization and functional capacity of PSII was found to increase its susceptibility to photoinhibition in agreement with the concepts presented in the accompanying paper, that oxidative damage can occur within the PSII reaction center as a consequence of extending the lifetime of P680(+). PMID- 16668852 TI - Enhancement of Anaerobic Respiration in Root Tips of Zea mays following Low Oxygen (Hypoxic) Acclimation. AB - Root tips (10-millimeter length) were excised from hypoxically pretreated (HPT, 4% [v/v] oxygen at 25 degrees C for 16 hours) or nonhypoxically pretreated (NHPT, 40% [v/v] oxygen) maize (Zea mays) plants, and their rates of respiration were compared by respirometry under aerobic and anaerobic conditions with exogenous glucose. The respiratory quotient under aerobic conditions with 50 millimolar glucose was approximately 1.0, which is consistent with glucose or other hexose sugars being utilized as the predominant carbon source in glycolysis. Under strictly anaerobic conditions (anoxia), glycolysis was accelerated appreciably in both HPT and NHPT root tips, but the rate of anaerobic respiration quickly declined in NHPT roots. [U-(14)C]Glucose supplied under anaerobic conditions was taken up and respired by HPT root tips up to five times more rapidly than by NHPT roots. When anaerobic ethanol production was measured with excised root tips in 50 millimolar glucose, HPT tissues consistently produced ethanol more rapidly than NHPT tissues. These data suggest that a period of low oxygen partial pressure is necessary to permit adequate acclimation of the root tip of maize to subsequent anoxia, resulting in more rapid rates of fermentation and generation of ATP. PMID- 16668853 TI - Stomatal and Nonstomatal Components to Inhibition of Photosynthesis in Leaves of Capsicum annuum during Progressive Exposure to NaCl Salinity. AB - Young bell pepper (Capsicum annuum L.) plants grown in nutrient solution were gradually acclimated to 50, 100, or 150 moles per cubic meter NaCl, and photosynthetic rates of individual attached leaves were measured on several occasions during the salinization period at external CO(2) concentrations ranging from approximately 70 to 1900 micromoles per mole air. Net CO(2) assimilation (A) was plotted against computed leaf internal CO(2) concentration (C(i)), and the initial slope of this A-C(i) curve was used as a measure of photosynthetic ability. During the 10 to 14 days after salinization began, leaves from plants exposed to 50 moles per cubic meter NaCl showed little change in photosynthetic ability, whereas those treated to 100 or 150 moles per cubic meter NaCl had up to 85% inhibition, with increase in CO(2) compensation point. Leaves appeared healthy, and leaf chlorophyll content showed only a 14% reduction at the highest salinity levels. Partial stomatal closure occurred with salinization, but reductions in photosynthesis were primarily nonstomatal in origin. Photosynthetic ability was inversely related to the concentration of either Na(+) or Cl(-) in the leaf laminas sampled at the end of the experimental period. However, the concentration of Cl(-) expressed on a tissue water basis was greater, exceeding 300 moles per cubic meter, and Cl(-) was more closely associated (R(2) = 0.926) with the inhibition of photosynthetic ability. Leaf turgor was not reduced by salinization and leaf osmotic potential decreased to a slightly greater extent than the osmotic potential decreases of the nutrient solutions. Concentration of accumulated Na(+) and Cl(-) (on a tissue water basis) accounted quantitatively for maintenance of leaf osmotic balance, assuming that these ions were sequestered in the vacuoles. PMID- 16668854 TI - The Role of Ribulose-1,5-Bisphosphate Regeneration in the Induction Requirement of Photosynthetic CO(2) Exchange under Transient Light Conditions. AB - The temporally variable light environment of natural plant canopies presents distinct limitations to carbon assimilation, partially as a result of the photosynthetic induction requirement that develops when leaves are shaded. This study was undertaken with soybean (Glycine max L.) leaves to further identify factors contributing to the activation state of the fast component of induction during low photosynthetic photon flux density (PPFD) periods. Determination of pool sizes of carbon reduction cycle intermediates at low light and upon return to saturating light indicated that different limitations to photosynthetic activity arise over the time course of a 10-minute low PPFD period. Photosynthetic activity upon reillumination was limited by the regeneration of ribulose 1,5-P(2). There was an increase in the levels of fructose 1,6-P(2), sedoheptulose 1,7-P(2), triose-P, ribose 5-P, and ribulose 5-P pools, indicating inactivation of stromal enzymes, most notably fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and ribulose 5-P kinase. The fast-induction component was the most important factor limiting assimilation during rapid, brief light transients, during which the decay of the slow component was minimal. This may be particularly significant for upper leaves in soybean canopies that generally experience very rapid light transients. PMID- 16668855 TI - Changes in the Ascorbate System during Seed Development of Vicia faba L. AB - Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H(2)O(2) produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts. PMID- 16668856 TI - Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes. AB - Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I. PMID- 16668857 TI - Deletion Mutants of Chlorophyll a/b Binding Proteins Are Efficiently Imported into Chloroplasts but Do Not Integrate into Thylakoid Membranes. AB - Chlorophyll a/b binding polypeptides (CABp) are integral thylakoid membrane proteins containing three membrane-spanning helices. We have created a series of mutations in tomato CABp to test whether individual membrane helices with hydrophilic flanking sequences, when fused to a transit peptide, can be imported into chloroplasts and correctly targeted to thylakoid membranes. All of the mutated precursors, including those with large C-terminal and internal deletions, were imported successfully, showing that these regions of the mature CABp are not required for import into chloroplasts. All mutants tested, containing either one or two membrane helices, were found primarily in the stroma and not in the thylakoids. The small amount of protein found associated with the thylakoids was largely resistant to alkali extraction but was sensitive to protease, unlike wild type protein, which is resistant to both treatments. When incubated with thylakoids in the absence of stroma and/or ATP, a significant amount of wild-type protein assumes a form that is resistant to alkali extraction but is protease sensitive, like the imported deletion proteins. This form of the wild-type protein is not chased into a protease-resistant form by adding stroma and/or ATP. These results suggest that CABp can spontaneously associate with membranes as an aberrant species that is not an intermediate in the process of integration. The inability of the deletion forms of CABp to assume a protease-resistant conformation suggests that correct integration is afforded by elements within the entire protein that collectively contribute to the proper conformation of the protein. The ability of deletion mutants to associate with thylakoids in a nonphysiological way suggests that the study of such mutants may not be useful in elucidating thylakoid-targeting signals. PMID- 16668858 TI - Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : IV. Induction of Vanillic Acid Formation. AB - Kinetin is used as an elicitor to induce vanillic acid formation in cell suspension cultures of Vanilla planifolia. Maximal induction is observed at a kinetin concentration of 20 micrograms per gram of fresh weight of cells. Vanillic acid synthesis is observed a few hours after elicitation. The effects of kinetin on the activity of some enzymes of the phenylpropanoid pathway, i.e. phenylalanine ammonia-lyase, 4-hydroxycinnamate:coenzyme A ligase and uridine 5' diphosphate-glucose:trans-cinnamic acid glucosyltransferase, are reported and compared to the effects of chitosan. The former two enzymes are induced by chitosan with a maximum activity of approximately 25 to 40 hours after elicitation. All three enzymes are induced by kinetin with maximum activities for phenylalanine ammonia lyase and 4-hydroxycinnamate:coenzyme A ligase at approximately 50 hours after induction, whereas maximum glucosyltransferase activity is seen already after 24 hours. Furthermore, both elicitors induced the formation of lignin-like material, whereas only kinetin induced vanillic acid biosynthesis. Finally, kinetin but not chitosan induces catechol-4-O methyltransferase activity, catalyzing the formation of 4-methoxycinnamic acids, which were shown to be intermediates of hydroxybenzoic acid biosynthesis within cells of V. planifolia. It is suggested that this methyltransferase is directly involved in the biosynthesis of vanillic acid. PMID- 16668859 TI - Effect of inhibition of abscisic Acid accumulation on the spatial distribution of elongation in the primary root and mesocotyl of maize at low water potentials. AB - Previous work showed that accumulation of endogenous abscisic acid (ABA) acts both to maintain primary root growth and inhibit shoot growth in maize seedlings at low water potentials (psi(w)) (IN Saab, RE Sharp, J Pritchard, GS Voetberg [1990] Plant Physiol 93: 1329-1336). In this study, we have characterized the growth responses of the primary root and mesocotyl of maize (Zea mays L. cv FR27 x FRMo 17) to manipulation of ABA levels at low psi(w) with a high degree of spatial resolution to provide the basis for studies of the mechanism(s) of ABA action. In seedlings growing at low psi(w) and treated with fluridone to inhibit carotenoid (and ABA) biosynthesis, ABA levels were decreased in all locations of the root and mesocotyl growing zones compared with untreated seedlings growing at the same psi(w). In the root, low psi(w) (-1.6 megapascals) caused a shortening of the growing zone, as reported previously. The fluridone treatment was associated with severe inhibition of root elongation rate, which resulted from further shortening of the growing zone. In the mesocotyl, low psi(w) (-0.3 megapascal) also resulted in a shortened growing zone. In contrast with the primary root, however, fluridone treatment prevented most of the inhibition of elongation and the shortening of the growing zone. Final cell length measurements indicated that the responses of both root and mesocotyl elongation to ABA manipulation at low psi(w) involve large effects on cell expansion. Measurements of the relative changes in root and shoot water contents and dry weights after transplanting to a psi(w) of -0.3 megapascal showed that the maintenance of shoot elongation in fluridone-treated seedlings was not attributable to increased water or seed-reserve availability resulting from inhibition of root growth. The results suggest a developmental gradient in tissue responsiveness to endogenous ABA in both the root and mesocotyl growing zones. In the root, the capacity for ABA to protect cell expansion at low psi(w) appears to decrease with increasing distance from the apex. In the mesocotyl, in contrast, the accumulation of ABA at low psi(w) appears to become increasingly inhibitory to expansion as cells are displaced away from the meristematic region. PMID- 16668860 TI - Effects of sulfur nutrition on expression of the soybean seed storage protein genes in transgenic petunia. AB - The 7S seed storage protein (beta-conglycinin) of soybean (Glycine max [L]. Merr.) has three major subunits; alpha, alpha', and beta. Accumulation of the beta-subunit, but not the alpha- and alpha'-subunits, has been shown to be repressed by exogenously applied methionine to the immature cotyledon culture system (LP Holowach, JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) and to be enhanced under sulfate deficiency in soybean plants (KR Gayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenic petunia (Petunia hybrida) harboring either the alpha'- or beta-subunit gene were constructed to test whether the patterns of differential expression were retained in petunia. Petunia regulates these genes in a similar way as soybean in response to sulfur nutritional stimuli, i.e. (a) expression of the beta-subunit gene is repressed by exogenous methionine in in vitro cultured seeds, whereas the alpha'-subunit gene expression is not affected; and (b) accumulation of the beta-subunit is enhanced by sulfur deficiency. The pattern of accumulation of major seed storage protein of petunia was not affected by these treatments. These results indicate that this mechanism of gene regulation in response to sulfur nutrition is conserved in petunia even though it is not used to regulate its own major seed storage proteins. PMID- 16668861 TI - Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to alpha-Methyltryptophan. AB - Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by alpha-methyltryptophan (aMT). One mutant, amt 1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 +/- 2.7 and 4.7 +/- 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 +/- 2.6 and 31.8 +/- 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 +/- 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 +/- 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 +/- 2.05 nanomoles per milligram protein per hour and 7.74 +/- 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I(50)) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I(50) for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I(50) greater than the average for the entire extract. PMID- 16668862 TI - Evidence for Mitochondrial Regulation of Photosynthesis by a Starchless Mutant of Nicotiana sylvestris. AB - Mutant NS458 of Nicotiana sylvestris (Speg. et Comes) contains a defective plastid phosphoglucomutase and accumulates only trace amounts of starch. Determinations of carbon partitioning using tracer d-[3-(14)C]glyceric acid showed that the maximal CO(2) assimilation by mature leaves of the mutant at saturating [CO(2)] and light and low [O(2)] was close to the flux for sucrose formation in the wild type. The mutant is characterized by exceptionally slow oscillations in maximal CO(2) assimilation. The postulate that these slow oscillations follow changes in the cytosolic rate of sucrose phosphate synthesis has been investigated. Studies with wild-type and mutant leaf discs subjected to various treatments failed to indicate that any significant activation inactivation cycle in sucrose-P synthase activity can occur. The rate of sucrose phosphate synthesis, however, might be altered by variations in the supply of uridine UDP-glucose which is controlled by the rate of ATP regeneration (via UTP regeneration). Treating mutant leaf protoplasts and young leaves with oligomycin, an inhibitor of mitochondrial ATP regeneration, reduced photosynthesis by as much as 25 and 40%, respectively. The wild type failed to show inhibition by oligomycin, i.e. its effect is masked when starch and sucrose synthesis can interact. It is concluded that maximal CO(2) assimilation in the mutant is fine tuned by mitochondrial metabolism such that interactions between sucrose synthesis and mitochondrial processes may generate the observed oscillations. PMID- 16668863 TI - An Arabidopsis thaliana Gene with Sequence Similarity to the S-Locus Receptor Kinase of Brassica oleracea: Sequence and Expression. AB - Primary signal transduction plays a vital role in the way plants react to environmental and developmental signals. We report the sequence and expression of a putative receptor kinase gene, ARK1, in Arabidopsis thaliana that may be important in this regard. This Arabidopsis gene encodes a transmembrane protein with a cytoplasmic kinase catalytic domain, a transmembrane region, and an extracellular domain with sequence similarity to the secreted S-locus glycoprotein (SLG) gene of Brassica oleracea. This structure is similar to the S locus receptor kinase (SRK) gene of Brassica and to the receptor kinase ZmPK1 gene of maize. RNA blots indicate that transcripts accumulate predominantly in leaf tissue, with limited amounts in stem and floral bud tissue and no detectable transcripts accumulating in root tissue. A smaller transcript that could be an alternative transcript of ARK1 also accumulates in leaf tissue. This transcript possibly encodes a secreted SLG-like glycoprotein that lacks transmembrane and kinase domains. The predominantly vegetative expression of ARK1 indicates that this gene is not primarily involved in pollen/pistil interactions in Arabidopsis. PMID- 16668864 TI - Quantitation of Rates of Transport, Metabolic Fluxes, and Cytoplasmic Levels of Inorganic Carbon in Maize Root Tips during K Ion Uptake. AB - Our aim was to determine whether fixation of inorganic carbon (C(i)), due to phosphoenolpyruvate carboxylase activity, is limited by the availability of C(i) in the cytoplasm of maize (Zea mays L.) root tips. Rates of C(i) uptake and metabolism were measured during K(2)SO(4) treatment, which stimulates dark C(i) fixation. (13)C(i) uptake was followed by (13)C-nuclear magnetic resonance (NMR); 5 millimolar K(2)SO(4) had no significant effect on (13)C(i) influx. The contribution of respiratory CO(2) production to cytoplasmic HCO(3) (-) was measured using in vivo(13)C-NMR and (1)H-NMR of cell extracts; K(2)SO(4) treatment had no effect on respiratory CO(2) production. The concentration of cytoplasmic HCO(3) (-) was estimated to be approximately 11 millimolar, again with K(2)SO(4) having no significant effect. These experiments allowed us to determine the extent to which extracellularly supplied (14)C(i) was diluted in the cytoplasm by respiratory CO(2) and thereby measure phosphoenolpyruvate (PEP) carboxylase activity in vivo using (14)C(i). PEP carboxylase activity in root tips was enhanced approximately 70% over controls within 12 minutes of the addition of 5 millimolar K(2)SO(4). The activity of carbonic anhydrase, which provides PEP carboxylase with C(i), was determined by saturation transfer (13)C NMR to be more than 200 times that of PEP carboxylase in vivo. The regulation of PEP carboxylase in K(2)SO(4)-treated roots is discussed. PMID- 16668865 TI - Biphasic Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves as Determined from Nonsteady-State CO(2) Exchange. AB - The activation kinetics of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) following an increase in photon flux density (PFD) were studied by analyzing CO(2) assimilation time courses in spinach leaves (Spinacia oleracea). When leaves were exposed to 45 minutes of darkness before illumination at 690 micromoles per square meter per second, Rubisco activation followed apparent first-order kinetics with a relaxation time of about 3.8 minutes. But when leaves were illuminated for 45 minutes at 160 micromoles per square meter per second prior to illumination at 690 micromoles per square meter per second the relaxation time for Rubisco activation was only 2.1 minutes. The kinetics of this change in relaxation times were investigated by exposing dark-adapted leaves to 160 micromoles per square meter per second for different periods before increasing the PFD to 690 micromoles per square meter per second. It was found that the apparent relaxation time for Rubisco activation changed from 3.8 to 2.1 minutes slowly, requiring at least 8 minutes for completion. This result indicates that at least two sequential, slow processes are involved in light mediated activation of Rubisco in spinach leaves and that the relaxation times characterizing these two processes are about 4 and 2 minutes, respectively. The kinetics of the first process in the reverse direction and the dependence of the relaxation time for the second process on the magnitude of the increase in PFD were also determined. Evidence that the first slow process is activation of the enzyme Rubisco activase and that the second slow process is the catalytic activation of Rubisco by activase is discussed. PMID- 16668866 TI - Light-dependent changes in ribulose bisphosphate carboxylase activase activity in leaves. AB - An assay for the activity of ribulose bisphosphate carboxylase (Rubisco) activase in crude leaf extracts was developed. The assay is based on a spectrophotometric assay of Rubisco, and activase activity (in nanomoles activated Rubisco per minute per milligram chlorophyll) was calculated from the rate of increase in Rubisco activity over time. Activase activity measurements were made using samples from spinach (Spinacia oleracea) leaves undergoing (a) steady-state photosynthesis at various photon flux density (PFD) values and (b) nonsteady state photosynthesis following an increase from darkness to a high PFD. Analysis of these samples showed that steady-state Rubisco activase activity was relatively low in darkness, increased with PFD, and saturated below 300 micromoles per square meter per second. Rubisco activity (measured spectrophotometrically) was also found to be low in darkness and to increase with PFD, but it saturated at much higher PFD values (approximately 1000 micromoles per square meter per second) along with the rate of photosynthesis. Following an increase in PFD from darkness to 650 micromoles per square meter per second, activase activity increased more or less linearly over a period of 5 to 6 minutes, after which it was constant. Rubisco activity, however, increased more slowly. The light-dependence of Rubisco activase is consistent with previous gas exchange data showing two interdependent processes in the activation of Rubisco following an increase in PFD. PMID- 16668867 TI - A Calorimetric Study of the Changes in Lipids during Seed Storage under Dry Conditions. AB - Changes in the lipid constituents of seeds are believed to be associated with losses of seed viability during storage. The physical properties of lipids in seeds of genetically similar backgrounds but varying ages were compared using differential scanning calorimetry. The technique measures the temperature and energy associated with lipid-melting transitions and is noninvasive. Although differences in the temperature at which lipids melted were sometimes observed among deteriorated and fresh seeds, the direction of the change was inconsistent among species tested. For all species tested except tomato, there was a decrease in the energy associated with the lipid melt in deteriorated samples, and the change occurred at a similar rate as the loss of seed vigor. The data suggest that there are changes in the lipid components of seeds that are associated with seed deterioration and that these changes can be measured using differential scanning calorimetry. PMID- 16668868 TI - Broad bean leaf polyphenol oxidase is a 60-kilodalton protein susceptible to proteolytic cleavage. AB - Polyphenol oxidase (PPO) in leaves is generally considered to be a 45-kilodalton protein. PPO purified to homogeneity from broad bean (Vicia faba L.) leaves in the presence of protease inhibitors had an apparent molecular mass of 60 kD determined by denaturing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Under partially denaturing conditions, the protein had an apparent molecular mass of 43 kilodaltons, but this was shifted to 60 kilodaltons in the presence of sulfhydryl reductants, suggesting the presence of disulfide bonding. The purified protein was totally latent; i.e. PPO activity was only detected when assayed with sodium dodecyl sulfate. Treatment with proteases in the presence of 0.1% SDS inhibited enzyme activity, but in the absence of SDS the 60-kilodalton PPO was proteolytically cleaved with no loss of PPO activity. This yielded a 42-kilodalton peptide that had PPO activity and inactive peptides of 12 to 18 kilodaltons. Amino acid sequencing established that the 42-kilodalton protein was derived from the N-terminal end of the 60-kilodalton form of PPO. By comparison with the sequence of a cDNA clone for broad bean leaf PPO, the 18 kilodalton peptide was located at the C-terminal end of the 60-kilodalton protein. Northern analysis of mRNA from broad bean leaves probed with a cDNA clone of PPO identified a transcript of 2.2 kilobase pairs, which is more than sufficient to encode the 60-kilodalton protein. It is concluded that PPO is a 60 kilodalton protein in broad bean leaves but that it is susceptible to proteolysis during extraction, removing a peptide of 15 to 18 kilodaltons from the C-terminal end without decreasing enzyme activity. PMID- 16668869 TI - Enzymic Components of Sucrose Accumulation in the Wild Tomato Species Lycopersicon peruvianum. AB - Sugar and soluble solids content and invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13), and sucrose phosphate synthase (EC 2.4.1.14) enzyme activities were measured throughout fruit development in tomato (Lycopersicon esculentum Mill.) and the green fruited species Lycopersicon peruvianum. Fruit of L. peruvianum accumulated predominantly sucrose, in contrast with hexose accumulation, which is characteristic of L. esculentum. The percentage of soluble solids in ripe L. peruvianum fruit was more than twice that present in L. esculentum and attributed primarily to the high level of sucrose accumulated in L. peruvianum. Low levels of invertase and sucrose synthase activity were associated with the period of significant sucrose accumulation and storage in L. peruvianum. Increased sucrose phosphate synthase activity was observed during the latter stages of fruit development in sucrose-accumulating fruit but was not coincident with maximum rates of sucrose accumulation. PMID- 16668870 TI - Expression of Osmotin-Like Genes in the Halophyte Atriplex nummularia L. AB - A peptide (molecular mass 50 kilodaltons) that is immunologically related to tobacco osmotin was detected in cells of the halophyte Atriplex nummularia. This protein was constitutively expressed in both unadapted and NaCl-adapted cells. A predominant osmotin-like peptide (molecular mass 24 kilodaltons) was also found in culture media after cell growth. Two unique A. nummularia cDNA clones, pA8 and pA9, encoding osmotin-like proteins have been isolated. The pA8 and pA9 inserts are 952 and 792 base pairs and encode peptides of 222 and 224 amino acids, respectively. The peptide deduced from pA8 has a molecular mass of 23,808 daltons and theoretical isoelectric point of 8.31, whereas the peptide derived from pA9 has a molecular mass of 23,827 daltons and an isoelectric point of 6.88. Unique transcripts were detected by the inserts of the cDNA clones, two (1.2 and 1.0 kilobases) by pA8 and one (0.9 kilobase) by pA9. The pA8 transcripts were constitutively accumulated in unadapted and NaCl-adapted cells, whereas the mRNA levels were up-regulated by abscisic acid treatment. The level of pA9 mRNA was induced by NaCl treatment and increased in cells as a function of NaCl adaptation. Southern analysis of the genomic DNA indicated the presence of osmotin-like multigene families in A. nummularia. PMID- 16668871 TI - Group 3 LEA Gene HVA1 Regulation by Cold Acclimation and Deacclimation in Two Barley Cultivars with Varying Freeze Resistance. AB - The level of expression of the group 3 late embryogenesis abundant abscisic acid regulated gene (HVA1) to cold treatment has been studied in winter barley (Hordeum vulgare) seedling tissue. The cDNA clone (pHVA1) encoding this late embryogenesis abundant protein was used as a hybridization probe to detect the corresponding mRNA. Expression of the HVA1 gene was determined after the tissue had been subjected to a regimen of 2 degrees C exposure (cold acclimation), followed by a return to 25 degrees C growth conditions (deacclimation). Accumulation of HVA1 mRNA occurred upon cold acclimation of the tissue and disappeared as early as 2 hours after exposure to deacclimation conditions. A comparison of the response to cold acclimation and deacclimation was made between seedling tissue of a freeze-resistant and less freeze-resistant cultivar. In both cultivars, the HVA1 gene was expressed and modulated by cold treatment. Within 2 hours of deacclimation HVA1 mRNA was no longer detectable in either cultivar independently of freeze resistance. The level of expression of HVA1 appeared to be greater in the less freeze-resistant cultivar (Winter Malt). PMID- 16668872 TI - Temperature-Dependent Water and Ion Transport Properties of Barley and Sorghum Roots : II. Effects of Abscisic Acid. AB - Water flux through excised roots (J(v)) is determined by root hydraulic conductance (L(p)) and the ion flux to the xylem (J(i)) that generates an osmotic gradient to drive water movement. These properties of roots are strongly temperature dependent. Abscisic acid (ABA) can influence J(v) by altering L(p), J(i), or both. The effects of root temperature on responses to ABA were determined in two species differing in their temperature tolerances. In excised barley (Hordeum vulgare L.) roots, J(v) was maximum at 25 degrees C; 10 micromolar ABA enhanced J(v), primarily by increasing L(p), at all temperatures tested (15-40 degrees C). In sorghum (Sorghum bicolor L.) roots, J(v) peaked at 35 degrees C; ABA reduced this optimum temperature for J(v) to 25 degrees C by increasing L(p) at low temperatures and severely inhibiting J(i) (dominated by fluxes of K(+) and NO(3) (-)) at warm temperatures. The inhibition of K(+) flux by ABA at high temperature was mostly independent of external K(+) availability, implying an effect of ABA on ion release into the xylem. In sorghum, ABA enhanced water flux through roots at nonchilling low temperatures but at the expense of tolerance of warm temperatures. These effects imply that ABA may shift the thermal tolerance range of roots of this heat-tolerant species toward cooler temperatures. PMID- 16668873 TI - Effects of Atmospheric CO(2) Enrichment on Photosynthesis, Respiration, and Growth of Sour Orange Trees. AB - Numerous net photosynthetic and dark respiratory measurements were made over a period of 4 years on leaves of 24 sour orange (Citrus aurantium) trees; 8 of them growing in ambient air at a mean CO(2) concentration of 400 microliters per liter, and 16 growing in air enriched with CO(2) to concentrations approaching 1000 microliters per liter. Over this CO(2) concentration range, net photosynthesis increased linearly with CO(2) by more than 200%, whereas dark respiration decreased linearly to only 20% of its initial value. These results, together with those of a comprehensive fine-root biomass determination and two independent aboveground trunk and branch volume inventories, suggest that a doubling of the air's current mean CO(2) concentration of 360 microliters per liter would enhance the growth of the trees by a factor of 3.8. PMID- 16668874 TI - NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid. AB - The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO(3) (-) uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO(3) (-) leaves) or 40 mm NO(3) (-) solution (high NO(3) (-) leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO(3) (-)-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low NO(3) (-) and high-NO(3) (-) leaves, indicating that NO(3) (-) had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO(3) (-) compared with low NO(3) (-) leaves showed evidence that NO(3) (-) enhanced the protein kinase activity. These results support the concept that NO(3) (-), or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s). PMID- 16668875 TI - Spatial d/h heterogeneity of leaf water. AB - The mean deltaD value of petiole water of Pterocarpus indicus Willd (deltaD = 9.0 +/- 2.5 per thousand, n = 3) was not significantly different from the mean value of stem water (-8.3 +/- 2.8 per thousand, n = 3). deltaD values of main vein water ranged from -11.1 to + 12.0 per thousand (n = 14) and increased along the main vein from petiole to the tip of leaves. Mesophyll water was highly enriched in deuterium (mean deltaD = +32.0 +/- 2.0 per thousand, n = 19) when compared with stem, petiole, and vein water. deltaD values of mesophyll water for different areas of the lamina, however, were not homogenous and could differ by as much as 20 per thousand. PMID- 16668876 TI - Tomato fruit Acid invertase complementary DNA : nucleotide and deduced amino Acid sequences. PMID- 16668877 TI - Nucleotide sequences of a soybean complementary DNA encoding a 50-kilodalton late embryogenesis abundant protein. PMID- 16668878 TI - Comparison of the rbcL Gene Sequence of Two Potato Cultivars with Differential Sensitivity to Ozone. PMID- 16668879 TI - Molecular Cloning and Sequencing of ADP-Glucose Pyrophosphorylase from Synechocystis PCC 6803. PMID- 16668880 TI - Nucleotide Sequence of a Complementary DNA Clone Encoding Stearoyl-Acyl Carrier Protein Desaturase from Simmondsia chinensis. PMID- 16668881 TI - Ethylene Synthesis and Floral Senescence following Compatible and Incompatible Pollinations in Petunia inflata. AB - Ethylene production and floral senescence following compatible and incompatible pollinations were studied in a self-incompatible species, Petunia inflata. Both compatible and incompatible pollinations resulted in a burst of ethylene synthesis that peaked 3 hours after pollination. P. inflata pollen was found to carry large amounts of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The amount of pollen-held ACC varied in different genetic backgrounds, and the magnitude of the peak correlated with the amount of ACC borne by the pollen. Aminooxyacetic acid (AOA), an inhibitor of ACC synthesis, had no inhibitory effect on this ethylene response, indicating that pollen-borne ACC was largely responsible for the early synthesis of ethylene. After compatible pollination, a second increase in ethylene synthesis began at 18 hours, and the first sign of senescence appeared at 36 hours. Upon treatment with AOA, the second phase of ethylene production was reduced by 95%, indicating that endogenous ACC synthesis was required for this phase of ethylene synthesis. AOA treatment also delayed senescence to 6 days after anthesis. After incompatible pollination, a second increase in ethylene production did not occur until 3 days, and the first sign of senescence occurred 12 hours later. Unpollinated flowers showed an increase in ethylene production 3 to 4 days after anthesis and displayed signs of senescence 1 day later. The significance of the early and late phases of pollination-induced ethylene synthesis is discussed. PMID- 16668882 TI - Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.). AB - During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media, yet that protein is not evident on stained gels until day 10. Thus, both transcriptional and posttranscriptional events appear to be important in determining the protein complement of these seed tissues. On the basis of storage protein and mRNA accumulation, mature (14 days) somatic embryos most closely resemble stage VI zygotic embryos. The results of the developmental comparison also suggest that the patterns of synthesis of the individual storage proteins (7S, 11S, or 2S) are regulated independently of each other during embryogenesis in alfalfa. PMID- 16668883 TI - Flow Cytometric Characteristics of Sperm Cells Isolated from Pollen of Zea mays L. AB - Sperm cells have been isolated from pollen of maize (Zea mays L.) and purified with Percoll density centrifugation. Their flow cytometric characteristics were determined on a FACScan flow cytometer with the fluorescent dyes, fluorescein diacetate and propidium iodide. Freshly isolated sperm cells appeared as a dot cluster on the forward scatter and side scatter dot plot. This dot cluster contained 85 to 95% of the 10 thousand counts collected. More than 98% of cells from the cluster were fluorescein diacetate positive, with no propidium iodide positivity, indicating high cell viability. After 5 hours in 15% (w/v) sucrose at room temperature (23 degrees C), scattering properties, cell number, and percentage of fluorescein diacetate-positive cells remained the same. In contrast, Brewbaker and Kwack salts in 15% sucrose resulted in the emergence of a new cell population, as well as a decrease in cell number at 5 hours. Further investigations with individual components of the Brewbaker and Kwack salts showed that calcium was mainly responsible for the deleterious effects. These results demonstrate the utility of flow cytometry as a tool to determine viability and to monitor morphological changes of plant sperm cells and to challenge current views on the ability of Brewbaker and Kwack salts to maintain viability of isolated sperm cells. PMID- 16668884 TI - Regulation of Mitochondrial Function and Biogenesis in Cucumber (Cucumis sativus L.) Cotyledons during Early Seedling Growth. AB - The aim of this work was to characterize the respiratory metabolism of the greening cotyledons of cucumber (Cucumis sativus L.) during early seedling growth and to investigate how this is integrated with changes in mitochondrial biogenesis and function. In light-grown cotyledons, lipid mobilization extended from germination to 6 days postimbibition, reaching a maximum at 3 to 4 days postimbibition. The rate of dark oxygen uptake reached a maximum at 2 days postimbibition in dark-grown and 3 days postimbibition in light-grown cotyledons. Development of photosynthetic capacity occurred from 4 to 7 days postimbibition. In dark-grown cotyledons, lipid mobilization extended beyond 7 days postimbibition, and there was no greening or acquisition of photosynthetic competence. Measurements of mitochondrial function indicated that the respiratory capacity of the tissue changed such that during lipid mobilization there was a much greater capacity for the operation of the nondecarboxylating portion of the citric acid cycle (succinate to oxaloacetate), whereas during the development of photosynthetic function the activity of the remainder of the cycle (oxaloacetate to succinate) was induced. Comparison of the maximum capacities for mitochondrial substrate oxidations in vitro with the rates of in vivo substrate oxidations, predicted from the rate of lipid breakdown, indicated that mitochondria in this tissue operate at or below state 4 rates, suggesting limitation by both availability of ADP and substrate. PMID- 16668885 TI - Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus aurantium. AB - The distribution of the flavanones naringin and neohesperidin has been analyzed during the development of the leaves, flower buds, and fruits of Citrus aurantium. These flavonoids are at maximum concentration in the organs studied during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the naringin and neohesperidin concentration in leaves, flower buds, and fruits is due to a dilution of the flavonoids caused by cell growth, because total content per organ continues to increase. The levels of neohesperidin are always greater than those of naringin, although the ratio between the relative concentrations is different in the three organs studied. Leaves have the highest ratios, varying between 8.83 and 5.18, followed by flowers (3.15-1.85), and fruits (2.23-1.02). These observations suggest different relationships between the respective enzymic activities in their biosynthetic pathway. PMID- 16668886 TI - Immunodetection and Identification of N-(o-Hydroxybenzylamino)Purine as a Naturally Occurring Cytokinin in Populus x canadensis Moench cv Robusta Leaves. AB - A highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for 9-beta-d-ribofuranosyl-N(6)-(o-hydroxybenzylamino)purine [(oOH)[9R]BAP] and structurally related cytokinins. As little as 3 femtomoles of the compound could be detected by this method. Cross-reactivity studies demonstrated the specificity of four polyclonal antibodies for (oOH)[9R] BAP and its free base in preference to a range of natural cytokinins and other purines. After evaluating the method by internal standardization employing [2 (3)H](oOH)[9R]BAP of high specific radioactivity as recovery marker by dilution analyses and by immunohistograms, it was possible to apply ELISA to quantify (oOH)[9R]BAP in plant extracts. In addition to (oOH)[9R]BAP, an unknown cytokinin reacting with the same antibody was detected in high performance liquid chromatography-fractionated extracts of mature Populus x canadensis Moench cv Robusta. The structure of the new compound was determined by gas chromatography mass spectrometry and finally confirmed by synthesis as N(6)-(o hydroxybenzylamino)purine. PMID- 16668887 TI - Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost. AB - Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, gamma-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of gamma-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development. PMID- 16668888 TI - Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts. AB - Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants. PMID- 16668889 TI - Growth Kinetics, Carbohydrate, and Leaf Phosphate Content of Clover (Trifolium subterraneum L.) after Transfer to a High CO(2) Atmosphere or to High Light and Ambient Air. AB - Intact air-grown (photosynthetic photon flux density, 400 microeinsteins per square meter per second) clover plants (Trifolium subterraneum L.) were transfered to high CO(2) (4000 microliters CO(2) per liter; photosynthetic photon flux density, 400 microeinsteins per square meter per second) or to high light (340 microliters CO(2) per liter; photosynthetic photon flux density, 800 microeinsteins per square meter per second) to similarly stimulate photosynthetic net CO(2) uptake. The daily increment of net CO(2) uptake declined transiently in high CO(2), but not in high light, below the values in air/standard light. After about 3 days in high CO(2), the daily increment of net CO(2) uptake increased but did not reach the high light values. Nightly CO(2) release increased immediately in high light, whereas there was a 3-day lag phase in high CO(2). During this time, starch accumulated to a high level, and leaf deterioration was observed only in high CO(2). After 12 days, starch was two- to threefold higher in high CO(2) than in high light, whereas sucrose was similar. Leaf carbohydrates were determined during the first and fourth day in high CO(2). Starch increased rapidly throughout the day. Early in the day, sucrose was low and similar in high CO(2) and ambient air (same light). Later, sucrose increased considerably in high CO(2). The findings that (a) much more photosynthetic carbon was partitioned into the leaf starch pool in high CO(2) than in high light, although net CO(2) uptake was similar, and that (b) rapid starch formation occurred in high CO(2) even when leaf sucrose was only slightly elevated suggest that low sink capacity was not the main constraint in high CO(2). It is proposed that carbon partitioning between starch (chloroplast) and sucrose (cytosol) was perturbed by high CO(2) because of the lack of photorespiration. Total phosphate pools were determined in leaves. Concentrations based on fresh weight of orthophosphate, soluble esterified phosphate, and total phosphate markedly declined during 13 days of exposure of the plants to high CO(2) but changed little in high light/ambient air. During this time, the ratio of orthophosphate to soluble esterified phosphate decreased considerably in high CO(2) and increased slightly in high light/ambient air. It appears that phosphate uptake and growth were similarly stimulated by high light, whereas the coordination was weak in high CO(2). PMID- 16668890 TI - Ion channels in Arabidopsis plasma membrane : transport characteristics and involvement in light-induced voltage changes. AB - White light (25 watts per square meter) induced an increase in plasma membrane K(+)-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K(+) on the cytoplasmic side of the patch and 50 millimolar K(+) in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K(+) over Na(+) and Cl(-). One (named PKC1) had a maximum conductance (G(max)) of 44 picosiemens at a membrane voltage (V(m)) of -65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had G(max) = 66 picosiemens at V(m) = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K(+) and Na(+) about equally well but not Cl(-). It had G(max) = 109 picosiemens at V(m) = 55 millivolts in (250/50 K) with 10 millimolar Ca(2+) on the cytoplasmic side. Reducing Ca(2+) to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar beta-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types. PMID- 16668891 TI - Electrofusion and electroporation of plants. AB - The utilization of electrofusion and electroporation techniques has had a major impact on the genetic manipulation of plants within the last decade. This review of the development of electrofusion and electroporation, as it applies to plants, highlights major developmental aspects of this technology. These include mechanisms for cell fusion, molecular exchange, and parameters that affect the efficiency of fusion and electroporation. PMID- 16668892 TI - The Distribution of Gibberellins in Vegetative Tissues of Pisum sativum L. : I. Biological and Biochemical Consequences of the le Mutation. AB - The concentrations of endogenous gibberellin (GA) 1, 5, 8, 19, 20, and 29 in the component tissues of maturing tall (Le) and dwarf (le) pea (Pisum sativum) plants have been determined. The following conclusions were drawn from the data obtained: (a) GA(20) and its metabolites accumulate only in the growing regions of Le and le plants; (b) the le mutation is biochemically expressed in all immature tissues of the dwarf plants; (c) the quantitative composition of the GA metabolites in the various immature tissues is variable; (d) the total GA concentration in apical buds, unexpanded leaves, and tendrils is considerably higher than in GA(1)-responsive stem tissue; and (e) there is very little GA accumulation of the inactive 2beta-hydroxylated GAs (GA(8) and GA(29)) in either the mature vegetative tissues or the roots of pea plants. PMID- 16668893 TI - Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation. AB - A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA(1) or its immediate precursors. The primary site of synthesis of GA(20) from GA(19) was immature leaflets and tendrils, and the synthesis of bioactive GA(1) and its inactive catabolite GA(8) occurred predominantly in stem tissue. GA(29), the inactive catabolite of GA(20), was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA(1), GA(19), or even GA(20). During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [(3)H]GA(1) (and [(3)H]GA(29)) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA(20) 3beta-hydroxylase protein. PMID- 16668894 TI - In vitro processing of tomato proteinase inhibitor I by barley microsomal membranes: a system for analysis of cotranslational processing of plant endomembrane proteins. AB - A plant-derived in vitro system for the study of cotranslational processing of plant endomembrane proteins has been developed and used to investigate cotranslational proteolytic processing of tomato proteinase inhibitor I. Translation of the inhibitor I precursor in wheat germ lysate supplemented with barley aleurone microsomal membranes resulted in cotranslational import of the protein into microsomal vesicles and cleavage of the signal sequence. NH(2) terminal sequence analysis of the translocated inhibitor I processing intermediate showed that the signal sequence was cleaved between Ala(23) and Arg(24) of the precursor protein. Parallel experiments using dog pancreas microsomal membranes indicated an identical site of cleavage, suggesting that the substrate determinants for signal sequence processing are conserved across kingdoms. The plant-derived processing system used for this study may be valuable for analysis of cotranslational processing of other plant preproteins and for characterizing the components of the cotranslational import machinery in plants. PMID- 16668895 TI - Isolation and analysis of the expression of two genes for the 81-kilodalton heat shock proteins from Arabidopsis. AB - We have cloned and characterized two members of the family of genes for the 81 kilodalton heat-shock proteins from Arabidopsis thaliana, HSP81-1 and HSP81-2. Comparison of the entire genomic sequence of the HSP81-1 gene with the corresponding full-length cDNA previously reported as AtHS83 (TW Conner, PR LaFayette, RT Nagao, JL Key [1990] Plant Physiol 94: 1689-1695) reveals the presence of three introns of 315, 83, and 88 base pairs. By contrast, analysis of the HSP81-2 genomic and partial cDNA sequences suggests that the HSP81-2 gene is interrupted by only two introns of 304 and 106 base pairs. The 5'-initiation sites of the two corresponding mRNAs were mapped from results of experiments with S1 nuclease. The deduced amino acid sequences of the proteins encoded by these two genes show 88% identity, and they also show striking similarities to the hsp90 family of proteins in yeast and animal cells. From the results of northern blot analysis of transcripts, it appears that the expression of the HSP81-1 gene occurs at only very low levels in the absence of heat shock and is strongly induced by heat (35 degrees C). The HSP81-2 gene is constitutively expressed at much higher levels, and its expression is moderately enhanced by elevated temperatures. Severe heat shock appears to block the splicing of the pre-mRNA transcribed from HSP81-1. We also examined the effects of arsenite and cadmium on the expression of the HSP81 genes, as well as on other groups of hsp genes in Arabidopsis. Treatment with cadmium was marginally effective in inducing hsp genes, whereas arsenite stress strongly stimulated the accumulation of each mRNA in a coordinated fashion. PMID- 16668896 TI - Purification and characterization of a secreted purple phosphatase from soybean suspension cultures. AB - We purified and partially sequenced a purple (lambda(max) = 556 nanometers) acid phosphatase (APase; EC 3.1.3.2) secreted by soybean (Glycine max) suspension culture cells. The enzyme is a metalloprotein with a Mn(2+) cofactor. This APase appears to be a glycoprotein with a monomer subunit molecular weight of 58,000 and an active dimer molecular weight of approximately 130,000. The protein has an isoelectric point of about 5.0 and a broad pH optimum centered near 5.5. The purified enzyme, assayed with p-nitrophenyl phosphate as the substrate, has a specific activity of 512 units per milligram protein and a K(m) of approximately 0.3 millimolar; phosphate is a competitive inhibitor with a K(i) of 0.7 millimolar. This APase is similar to one found in soybean seed meal but dissimilar to that found in soybean seedlings. PMID- 16668897 TI - Expression of Chloroplast and Mitochondrial Genes during Microsporogenesis in Maize. AB - Mitochondrial and plastid gene expression has been examined during maize (Zea mays) microsporogenesis. Accumulation of transcripts was found for three mitochondrial genes studied (cob, atp6, and atp9) at the mid-term of pollen development. In contrast, these mitochondrial transcripts were undetectable in mature pollen. Southern and DNA gel blot experiments showed that the copy number of mitochondrial genes was amplified in microspores at stages preceding the accumulation of these transcripts. Plastid transcripts of the photosynthetic psbA and rbcL genes could not be detected after the two mitoses, whereas precursors of the 16S rRNA are detected at low levels. PMID- 16668898 TI - Dissection of Nodule Development by Supplementation of Rhizobium leguminosarum biovar phaseoli Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside. AB - Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4 aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 10(5) to 10(6) bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule. PMID- 16668899 TI - DNA methylation is involved in maintenance of an unusual expression pattern of an introduced gene. AB - In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced beta-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited. PMID- 16668900 TI - Effect of Low Root Medium pH on Net Proton Release, Root Respiration, and Root Growth of Corn (Zea mays L.) and Broad Bean (Vicia faba L.). AB - The effect of low pH on net H(+) release and root growth of corn (Zea mays L.) and broad bean (Vicia faba L.) seedlings was investigated in short-term experiments at constant pH. Broad bean was more sensitive to low pH than corn: the critical values (pH values below which net H(+) release and root growth ceased) were pH 4.00 (broad bean) and pH 3.50 (corn) at 1 millimolar Ca(2+). Both proton release and root growth were progressively inhibited as the medium pH declined. Additional Ca(2+) in the root medium helped to overcome the limitations of low pH for net H(+) release and root growth. Potassium (for corn) and abscisic acid (for broad bean) increased both net H(+) release and root growth rate at the critical pH value. It is concluded that poor root growth at low pH is caused by a lack of net H(+) release that may decrease cytoplasmic pH values. Inhibited net H(+) release at high external H(+) activity is not due to a shortage of energy supply to the H(+) ATPase. Instead, a displacement of Ca(2+) by H(+) at the external side of the plasmalemma may enhance reentry of H(+) into root cells. PMID- 16668901 TI - Induction of Expression of Genes Coding for Sporamin and beta-Amylase by Polygalacturonic Acid in Leaf-Petiole Cuttings of Sweet Potato. AB - Sporamin and beta-amylase are two major proteins of tuberous storage root of sweet potato (Ipomoea batatas) and their accumulation can be induced concomitantly with the accumulation of starch in leaves and petioles by sucrose (K Nakamura, M Ohto, N Yoshida, K Nakamura [1991] Plant Physiol 96: 902-909). Although mechanical wounding of leaves of sweet potato only occasionally induced the expression of sporamin and beta-amylase genes, their expression could be reproducibly induced in leaf-petiole cuttings when these explants were dipped in a solution of polygalacturonic acid or chitosan at their cut edges. Polygalacturonic acid seemed to induce expression of the same genes coding for sporamin and beta-amylase that are induced by sucrose. Because polygalacturonic acid and chitosan are known to mediate the induction of wound-inducible defense reactions, these results raise an interesting possibility that beta-amylase, in addition to sporamin, may have some role in the defense reaction. Expression of sporamin and beta-amylase genes could also be induced by abscisic acid, and this induction by abscisic acid, as well as induction by polygalacturonic acid or sucrose, was repressed by gibberellic acid. By contrast, methyl jasmonate did not cause the significant induction of either sporamin or beta-amylase mRNAs. Induction of expression of sporamin and beta-amylase genes by polygalacturonic acid or sucrose was inhibited by cycloheximide, suggesting that de novo synthesis of proteins is required for both of the induction processes. PMID- 16668902 TI - Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings. AB - Cysteine, gamma-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, gamma-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl(2) concentrations up to 200 micromolar. At 50 micromolar Cd(2+), gamma-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd(2+), the concentration of gamma-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd(2+) concentrations, a significant increase in gamma-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd(2+). Labeling of isolated roots with [(35)S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of gamma-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd. PMID- 16668903 TI - Evidence for the involvement of sucrose phosphate synthase in the pathway of sugar accumulation in sucrose-accumulating tomato fruits. AB - To better understand the mechanism of sugar unloading and sugar concentration in hexose- and sucrose-accumulating tomato fruits (Lycopersicon chmielewskii and L. esculentum, respectively) and to determine the causes of the late accumulation of sucrose present in sucrose-accumulating tomato fruits, the assimilation of [(3)H](fructosyl)-sucrose was studied. Key enzymes involved in carbohydrate metabolism were also assayed. The results demonstrated that the low level of sucrose present in young fruits accumulates directly without undergoing hydrolysis, suggesting a symplastic pathway for sucrose unloading. By contrast, the large quantity of the sucrose present in ripe sucrose-accumulating fruits originates from hydrolysis and resynthesis, suggesting an apoplastic pathway for sucrose unloading. The increase in sucrose level observed in sucrose-accumulating fruits is associated with a gradual decline in invertase activity and an increase in sucrose phosphate synthase activity. This latter enzyme seems to play a key biochemical role in the accumulation of sucrose and the establishment of a high sugar content in tomato fruits. PMID- 16668904 TI - Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides. AB - Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H(+)-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-alpha-d-glucopyranoside, phenyl-alpha-d thioglucopyranoside, and phenyl-alpha-d-4-deoxythioglucopyranoside (PDTGP; K(i) = 67, 180, and 327 micromolar, respectively). The K(m) for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-alpha-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-alpha-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-alpha-d-4 deoxy-4-fluorothioglucopyranoside, and phenyl-alpha-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with K(i) values ranging from 600 to 800 micromolar, and phenyl-alpha-d-thiomannopyranoside, phenyl-beta-d glucopyranoside, and phenylethyl-beta-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-alpha-d-thioglucopyranoside and PDTGP, were transported. PMID- 16668905 TI - Phosphoenolpyruvate carboxylase activity and concentration in the endosperm of developing and germinating castor oil seeds. AB - Monospecific polyclonal antibodies against maize leaf phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were utilized to examine the subunit composition and developmental profile of endosperm PEPC in developing and germinating castor oil seeds (Ricinus communis L. cv Baker 296). PEPC from developing endosperm consists of a single type of 100-kilodalton subunit, whereas the enzyme from 2- to 5-day germinated endosperm appears to contain equal proportions of immunologically related 103- and 108-kilodalton subunits. The maximal activity of PEPC in developing endosperms (2.67 micromoles oxaloacetate produced per minute per gram fresh weight) is approximately 20-fold and threefold greater than that of fully mature (dry seed) and germinating endosperms, respectively. The most significant increase in the activity and concentration of endosperm PEPC occurs during the middle cotyledon to full cotyledon stage of seed development; this period coincides with the most active phase of storage oil accumulation by ripening castor oil seeds. The data are compatible with the recent proposal (RG Smith, DA Gauthier, DT Dennis, DH Turpin [1992] Plant Physiol 1233-1238) that PEPC plays a fundamental role in vivo in the cytosolic production of an important substrate (malate) for fatty acid biosynthesis by developing castor oil seed leucoplasts. Immediately following seed imbibition, PEPC activity and concentration increase in parallel, with the greatest levels attained by the third day of germination. It is suggested that during this early phase of seed germination PEPC has a critical function to build up cellular dicarboxylic acid pools required to initiate significant activities of both the tricarboxylic acid and glyoxylate cycles. PMID- 16668906 TI - Characterization of biotin and 3-methylcrotonyl-coenzyme a carboxylase in higher plant mitochondria. AB - Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3 methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg(2+). Kinetic constants (apparent K(m) values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO(3) (-), 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed. PMID- 16668907 TI - Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology. AB - Transmembrane electrical potential differences (Deltapsi) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO(3) (-) on Deltapsi (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO(3) (-) uptake. Both transport systems caused depolarization of Deltapsi, demonstrating that the LATS (like the HATS) for NO(3) (-) uptake is probably mediated by an electrogenic cation (H(+)?) cotransport system. Membrane depolarization by the HATS was "inducible" by NO(3) (-), and saturable with respect to exogenous [NO(3) (-)]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO(3) (-)]. H(+) fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO(3))(2) solutions, failed to alkalinize external media as anticipated for a 2 H(+):1 NO(3) (-) symport. However, switching from K(2)SO(4) solutions (which were strongly acidifying) to KNO(3) solutions at the same K(+) concentration caused marked reductions in H(+) efflux. These observations are consistent with NO(3) ( ) uptake by the HATS and the LATS via 2 H(+):1 NO(3) (-) symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO(3) (-). PMID- 16668908 TI - Deletion Analysis of the 5'-Upstream Region of the Agrobacterium rhizogenes Ri Plasmid rolC Gene Required for Tissue-Specific Expression. AB - The cis-acting elements located in the -848 and +23 region of the 5'-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid were investigated. The cis-acting DNA region required for phloem-specific expression was found within the -153 region, whereas a minimum region needed for the expression in the seed embryo was located at position -120. PMID- 16668909 TI - Plant growth environment effects on rapeseed microspore development and culture : a flow cytometric study. AB - The influence of donor plant growth conditions on microspore embryogenesis in rapeseed (Brassica napus) was studied for plants grown at 23/18 degrees C (16/8 hours) under continuous light, 23/18 degrees C (16/8 hours) with a light/dark (16/8 hours) cycle, 15/12 degrees C (16/8 hours) under continuous light and 15/12 degrees C (16/8 hours) with a light/dark (16/8 hours) cycle. Significantly higher embryo yields were obtained from microspore cultures initiated from donor plants grown at 15/12 degrees C instead of 23/18 degrees C. Flow cytometric measurements of the microspores isolated from 2.5- to 5.0-millimeter buds showed that the microspores isolated from low-temperature-grown plants had significantly lower log 90-degree light scatter to forward angle light scatter and log 90-degree light scatter to time of flight ratios than those isolated from high-temperature grown plants, suggesting that the former are more translucent than the latter. Thus, the effect of donor plant growth temperature on microspore embryogenesis may be mediated by a change in the physiology of the microspore cell, which results in the reduction of its cytoplasmic granularity and/or exine density. PMID- 16668910 TI - CO(2) Enhancement of Growth and Photosynthesis in Rice (Oryza sativa) : Modification by Increased Ultraviolet-B Radiation. AB - Two cultivars of rice (Oryza sativa L.) IR-36 and Fujiyama-5 were grown at ambient (360 microbars) and elevated CO(2) (660 microbars) from germination through reproduction in unshaded greenhouses at the Duke University Phytotron. Growth at elevated CO(2) resulted in significant decreases in nighttime respiration and increases in photosynthesis, total biomass, and yield for both cultivars. However, in plants exposed to simultaneous increases in CO(2) and ultraviolet-B (UV-B) radiation, CO(2) enhancement effects on respiration, photosynthesis, and biomass were eliminated in IR-36 and significantly reduced in Fujiyama-5. UV-B radiation simulated a 25% depletion in stratospheric ozone at Durham, North Carolina. Analysis of the response of CO(2) uptake to internal CO(2) concentration at light saturation suggested that, for IR-36, the predominant limitation to photosynthesis with increased UV-B radiation was the capacity for regeneration of ribulose bisphosphate (RuBP), whereas for Fujiyama-5 the primary photosynthetic decrease appeared to be related to a decline in apparent carboxylation efficiency. Changes in the RuBP regeneration limitation in IR-36 were consistent with damage to the photochemical efficiency of photosystem II as estimated from the ratio of variable to maximum chlorophyll fluorescence. Little change in RuBP regeneration and photochemistry was evident in cultivar Fujiyama-5, however. The degree of sensitivity of photochemical reactions with increased UV-B radiation appeared to be related to leaf production of UV-B absorbing compounds. Fujiyama-5 had a higher concentration of these compounds than IR-36 in all environments, and the production of these compounds in Fujiyama 5 was stimulated by UV-B fluence. Results from this study suggest that in rice alterations in growth or photosynthesis as a result of enhanced CO(2) may be eliminated or reduced if UV-B radiation continues to increase. PMID- 16668911 TI - Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis. AB - Euglena gracilis cells synthesize the key tetrapyrrole precursor, delta aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain alpha-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, alpha-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and alpha-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP(+), and was inhibited by NAD(+). Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of alpha-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products. PMID- 16668912 TI - Expression of "Dehydrin-Like" Proteins in Embryos and Seedlings of Zizania palustris and Oryza sativa during Dehydration. AB - Proteins inducible by dehydration and abscisic acid (ABA), termed dehydrins or RAB (Responsive to ABA) proteins, have been identified in a number of species and have been suggested to play a role in desiccation tolerance, particularly during seed development. Seeds (caryopses) of North American wild rice (Zizania palustris var interior [Fassett] Dore) are tolerant of dehydration to <10% moisture content (fresh weight basis) only under restricted dehydration and rehydration conditions. In comparison, seeds of paddy rice (Oryza sativa L.) readily tolerate desiccation to <5% water content. Expression of "dehydrin-like" proteins in Zizania and Oryza seedlings and embryos was examined to investigate the relationship between the presence of such proteins and desiccation tolerance. [(35)S]Methionine labeling of newly synthesized proteins showed that seedlings (first leaf stage) of both Zizania and Oryza synthesized a novel "heat-stable" protein of apparent molecular weight = 20,000 when dehydrated to <75% of their initial fresh weight. ABA (100 micromolar) induced synthesis of a protein with similar electrophoretic mobility in both species. Western blots using antiserum raised against maize (Zea mays L.) dehydrin detected a protein band from dehydrated Zizania shoots and mature embryonic axes that comigrated with the labeled 20-kilodalton polypeptide. Northern blots using a cDNA for an ABA responsive protein from Oryza (rab 16a) showed that both seedlings and excised embryonic axes of Zizania accumulated RNA similar in sequence to rab 16a in response to water loss. Zizania seedlings and embryonic axes were also capable of ABA accumulation during dehydration. The intolerance of Zizania seeds to dehydration at low temperature is apparently not due to an absence of dehydrin like proteins or an inability to accumulate ABA. PMID- 16668913 TI - Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum. AB - Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO(3) (-) or NH(4) (+) assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH(4) (+) was added to N limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO(3) (-) addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO(3) (-) assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO(3) (-) assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH(4) (+) assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO(3) (-) assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis. PMID- 16668914 TI - Optically pure abscisic Acid analogs-tools for relating germination inhibition and gene expression in wheat embryos. AB - We report an examination of the structural requirements of the abscisic acid (ABA) recognition response in wheat dormant seed embryos using optically pure isomers of ABA analogs. These compounds include permutations to the ABA structure with either an acetylene or a trans bond at C-4 C-5, and either a single or double bond at the C-2' C-3' double bond. (R)-ABA and the three isomers with the same configuration at C-1' as natural ABA were found to be effective germination inhibitors. The biologically active ABA analogs exhibited differential effects on ABA-responsive gene expression. All the ABA analogs that inhibited germination induced two ABA-responsive genes, wheat group 3 lea and dhn (rab). However, (R) ABA and (S)-dihydroABA were less effective in inducing the ABA-responsive gene Em within the time that embryonic germination was inhibited. PMID- 16668915 TI - Effect of inorganic cations and metabolic inhibitors on putrescine transport in roots of intact maize seedlings. AB - The specificity and regulation of putrescine transport was investigated in roots of intact maize (Zea mays L.) seedlings. In concentration-dependent transport studies, the kinetics for putrescine uptake could be resolved into a single saturable component that was noncompetitively inhibited by increasing concentrations of Ca(2+) (50 micromolar to 5 millimolar). Similarly, other polyvalent cations, including Mg(2+) (1.8 millimolar) and La(3+) (200 micromolar), almost completely abolished the saturable component for putrescine uptake. This suggests that putrescine does not share a common transport system with other divalent or polyvalent inorganic cations. Further characterization of the putrescine transport system indicated that 0.3 millimolar N-ethyl-maleimide had no effect on putrescine uptake, and 2 millimolar p-chloromercuribenzene sulfonic acid only partially inhibited transport of the diamine (39% inhibition). Metabolic inhibitors, including carbonylcyanide-m-chlorphenylhydrazone (20 micromolar) and KCN (0.5 millimolar), also partially inhibited the saturable component for putrescine uptake (V(max) reduced 48-60%). Increasing the time of exposure to carbonylcyanide-m-chlorphenylhydrazone from 30 minutes to 2 hours did not significantly increase the inhibition of putrescine uptake. Electrophysiological evidence indicates that the inhibitory effect on putrescine uptake by these inhibitors is correlated to a depolarization of the membrane potential, suggesting that the driving force for putrescine uptake is the transmembrane electrical potential across the plasmalemma. PMID- 16668916 TI - Ethylene Inhibitors Partly Restore Nodulation to Pea Mutant E 107 (brz). AB - E107 (brz) is a pleiotropic mutant of pea (Pisum sativum L. cv Sparkle) characterized by low nodulation, leaf necrosis, excessive ion accumulation, and decreased plant size. The defective nodulation of E107 was studied by light microscopy of lateral roots. The number of infections per centimeter of lateral root was only a third that of Sparkle. Moreover, most of the infections were aborted early; i.e. in only 14% of the infections did the infection thread penetrate beyond the epidermis. Nodulation of E107 was partly restored by treating the plant with the ethylene inhibitors aminoethoxyvinylglycine (AVG) or Ag(+). Treatment with Ag(+) did not increase the number of infections, but half of the infections went to completion. Ag(+) and AVG did not alter the size of the mutant, the accumulation of cations in its shoots, nor the leaf necrosis. Thus, in E107, nodule development can be uncoupled from other pleiotropic characteristics. PMID- 16668917 TI - DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide. AB - Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the "boiling-stable" polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed. PMID- 16668918 TI - Expression of ethylene biosynthetic pathway transcripts in senescing carnation flowers. AB - We have examined the expression of mRNAs for S-adenosylmethionine synthetase (EC 2.5.1.6), 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14), and the ethylene-forming enzyme (EFE) in various floral organs of carnation (Dianthus caryophyllus) during the increase in ethylene biosynthesis associated with petal senescence. The abundance of ACC synthase and EFE mRNAs increased and S adenosylmethionine synthetase transcripts decreased concomitantly with the ethylene climacteric in senescing petals. The increase in abundance of ACC synthase and EFE mRNAs in aging flowers was prevented by treatment with the ethylene action inhibitor 2,5-norbornadiene. Furthermore, an increase in ACC synthase and EFE transcripts was detected in petals from presenescent flowers within 3 to 6 hours of exposure to 2 microliters per liter of ethylene. The increase in ethylene production by senescing petals was associated with a concomitant increase in ethylene biosynthesis in styles, ovary, and receptacle tissues. In all tissues, this increase was associated with increased activities of ACC synthase and EFE. The increase in EFE activities by all floral organs examined was correlated with increased abundance of EFE transcripts. In contrast, the level of ACC synthase mRNA, as detected by the cDNA probe pCARACC3, did not always reflect enzyme activity. The combined tissues of the pistil exhibited high rates of ACC synthase activity but contained low levels of ACC synthase mRNAs homologous to pCARACC3. In addition, pollinated styles exhibited a rapid increase in ethylene production and ACC synthase activity but did not accumulate detectable levels of ACC synthase mRNA until several hours after the initiation of ethylene production. These results suggest that transcripts for ACC synthase leading to the early postpollination increase in ACC synthase activity and ethylene production are substantially different from the mRNA for the ethylene responsive gene represented by pCARACC3. PMID- 16668919 TI - The in Vivo and in Vitro Inhibition of Catalase from Leaves of Nicotiana sylvestris by 3-Amino-1,2,4-Triazole. AB - Seedlings of tobacco (Nicotiana sylvestris) were treated in vivo with 0.03 to 20 millimolar 3-amino-1,2,4-triazole (aminotriazole). There was a rapid loss of catalase (EC 1.11.1.6) activity over the first 5 hours followed by a slower decrease for the next 4 hours to a level that was 15 to 20% of the initial activity, with little or no change for periods up to 3 days. Fifty percent loss of catalase activity occurred at 0.10 to 0.15 millimolar inhibitor (18-hour incubation). The isozymes of tobacco catalase differed in sensitivity to the inhibitor. Enhanced-peroxidatic catalase (EP-CAT) (Havir EA, McHale NA, [1989] Plant Physiol 91: 812-815) decreased 35% under conditions in which the major isozyme decreased 85%. The resistance to aminotriazole inhibition demonstrated in vivo by EP-CAT was also observed in vitro. The times for 50% inhibition at 0.67, 3.33, 5.0, 10.0, and 15 millimolar aminotriazole were 15, 5, 2.6, 2.5, and 1.5 minutes, respectively, for the major isozyme of catalase and 60, 18.5, 5.1, 4, and 3.0 minutes, respectively, for EP-CAT. Increasing H(2)O(2) concentration did not change the sensitivity of EP-CAT to aminotriazole. The major form of catalase contained 4.0 +/- 0.4 moles of heme per mole enzyme and EP-CAT 3.4 +/- 0.3. Thus, the resistance of EP-CAT to aminotriazole is probably not due to lowered affinity for H(2)O(2) or alteration in heme content but to structural changes that impair inhibitor binding. PMID- 16668920 TI - A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein. AB - Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize cell suspension cultures yield a new extensin rich in histidine (HHRGP) that also has characteristics of arabinogalactan proteins (AGPs). Thus, chymotryptic peptide maps of anhydrous hydrogen fluoride (HF)-deglycosylated HHRGP showed repetitive motifs related to both extensins and AGPs as follows. HHRGP contains Ala-Hyp(3) and Ala-Hyp(4) repeats that may be related to the classical dicot Ser-Hyp(4) extensin motif by the single T --> G (Ser --> Ala) base change. Furthermore, HHRGP also contains the repetitive motif Ala-Hyp-Hyp Hyp-His-Phe-Pro-Ser-Hyp-Hyp related to the Ser-Hyp(4)-Ser-Hyp-Ser-Hyp(4) motif of P3-type dicot extensin. However, HHRGP also has AGP characteristics, notably an elevated alanine content, near sequence identity with the known Lolium AGP peptide Ser-Hyp-Hyp-Ala-Pro-Ala-Pro, the putative presence of glucuronoarabinogalactan, and precipitation by Yariv antigen, but beta elimination of arabinogalactan indicates its O-linkage to serine rather than the characteristic O-hydroxyproline link of other AGPs. Although HHRGP might be a "chimera" of two different proteins, i.e. an extensin and an AGP, this is unlikely because one can account for the apparent chimera by the codon relationships of the five common hydroxyproline-rich glycoprotein amino acid residues, Ser, Pro, Thr, Ala (TCx, CCx, ACx, GCx) and histidine (CAT or CAC), which facilitate interconversion of major motifs by single point mutations. Thus, we propose that the extensin family of wall proteins consists of a highly diversified phylogenetic series ranging from basic minimally glycosylated repetitive pro-rich proteins to the highly glycosylated acidic AGPs. To relate this diversity of form and function at the molecular level, we identified putative functional domains hypothetically involved in properties such as reptation, recognition, adhesion, intermolecular cross-linkage, and self assembly. Not previously noted, peptide palindromes feature prominently in HHRGP: Hyp-Hyp-Ala-Ala-Asn-Ala-Ala-Hyp-Hyp and Hyp-Hyp-Hyp-His-His-His-Hyp-Hyp-Hyp; in P3: Hyp(4)-Ser-Hyp-Ser-Hyp(4), and in other extensins. Such palindromes would enhance glycoprotein stereoregularity, thereby possibly promoting quasicrystalline interactions between wall components. PMID- 16668921 TI - A gymnosperm extensin contains the serine-tetrahydroxyproline motif. AB - The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser Hyp(4) motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp(4) motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp(4) motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp(4), in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp(3)-Thr-Hyp-Tyr, Ser-Hyp(4)-Lys, and (Ala-Hyp)(n) repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp(4) is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins. PMID- 16668922 TI - K stimulation of ATPase activity associated with the chloroplast inner envelope. AB - Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg(2+) and Mg.ATP complex effects on ATPase activity revealed that any Mg(2+) stimulation of activity was likely a function of the use of the Mg.ATP complex as a substrate by the enzyme; free Mg(2+) may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K(+). This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K(+). K(+) stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K(+) effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K(m) increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K(+)-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K(+) effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H(+)-ATPase). Such ATPases are thought to be indirectly involved in active K(+) uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K(+) effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo. PMID- 16668923 TI - Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana. AB - Reports from a number of laboratories describe the presence of a family of proteins (the major intrinsic protein family) in a variety of organisms. These proteins are postulated to form channels that function in metabolite transport. In plants, this family is represented by the product of NOD26, a nodulation gene in soybean that encodes a protein of the peribacteroid membrane, and tonoplast intrinsic protein (TIP), an abundant protein in the tonoplast of protein storage vacuoles of bean seeds (KD Johnson, H Hofte, MJ Chrispeels [1990] Plant Cell 2: 525-532). Other homologs that are induced by water stress in pea and in Arabidopsis thaliana and that are expressed in the roots of tobacco have been reported, but the location of the proteins they encode is not known. We now report the presence and derived amino acid sequences of two different TIP proteins in A. thaliana. alpha-TIP is a seed-specific protein that has 68% amino acid sequence identity with bean seed TIP; gamma-TIP is expressed in the entire vegetative body of A. thaliana and has 58% amino acid identity with bean seed TIP. Both proteins are associated with the tonoplast. Comparisons of the derived amino acid sequences of the seven known plant proteins in the major intrinsic protein family show that genes with similar expression patterns (e.g. water stress-induced or seed specific) are more closely related to each other than the three A. thaliana homologs are related. We propose that the nonoverlapping gene expression patterns reported here, and the evolutionary relationships indicated by the phylogenetic tree, suggest a functional specialization of these proteins. PMID- 16668924 TI - A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant Pathogenic Pseudomonads. AB - Lipoxygenase (LOX) mRNA, enzyme protein, and enzyme activity were found to be induced in leaves of tomato (Lycopersicon esculentum Mill. cv Moneymaker) on inoculation with plant pathogenic bacteria. The rate of enzyme activity with linoleic or linolenic acid as substrate was approximately 10 times greater than that with arachidonic acid. Optimum activity was at pH 7.0. In the incompatible interaction, which was associated with a hypersensitive reaction (HR), a single band with relative molecular weight approximately 100,000 was revealed by probing western blots of enzyme extracts with antiserum raised against a pea lipoxygenase. Changes in the intensity of this band reflected the changes observed in LOX enzyme activity after bacterial inoculations. In the hypersensitive reaction, i.e. after inoculation with Pseudomonas syringae pv syringae, LOX mRNA was induced by 3 hours and enzyme activity began to increase between 6 and 12 hours and had reached maximum levels by 24 to 48 hours. In tomato leaves inoculated with P. syringae pv tomato (compatible interaction), LOX mRNA was induced later and enzyme activity changed only marginally in the first 24 hours, then increased steadily up to 72 hours, reaching the levels seen in the HR. PMID- 16668925 TI - Osmotic adjustment in sorghum: I. Mechanisms of diurnal osmotic potential changes. AB - Osmotic adjustment, defined as a lowering of osmotic potential (psi(pi)) due to net solute accumulation in response to water stress, has been considered to be a beneficial drought tolerance mechanism in some crop species. The objective of this experiment was to determine the relative contribution of passive versus active mechanisms involved in diurnal psi(pi) changes in sorghum (Sorghum bicolor L. Moench) leaf tissue in response to water stress. A single sorghum hybrid (cv ATx623 x RTx430) was grown in the field under variable water supplies. Water potential, psi(pi), and relative water content were measured diurnally on expanding and the uppermost fully expanded leaves before flowering and on fully expanded leaves during the grain-filling period. Diurnal changes in total osmotic potential (Deltapsi(pi)) in response to water stress was 1.1 megapascals before flowering and 1.4 megapascals during grain filling in comparison with 0.53 megapascal under well-watered conditions. Under water-stressed conditions, passive concentration of solutes associated with dehydration accounted for 50% (0.55 megapascal) of the diurnal Deltapsi(pi) before flowering and 47% (0.66 megapascal) of the change during grain filling. Net solute accumulation accounted for 42% (0.46 megapascal) of the diurnal Deltapsi(pi) before flowering and 45% (0.63 megapascal) of the change during grain filling in water-stressed leaves. The relative contribution of changes in nonosmotic volume (decreased turgid weight/dry weight) to diurnal Deltapsi(pi) was less than 8% at either growth stages. Water stress did not affect leaf tissue elasticity or partitioning of water between the symplasm and apoplasm. PMID- 16668926 TI - Osmotic Adjustment in Sorghum: II. Relationship to Gas Exchange Rates. AB - Lowering of the solute potential by osmotic adjustment (OA) has been proposed to allow maintenance of leaf turgor potential (Psi(p)), stomatal conductance (g), and photosynthesis (A) at low leaf water potential. However, literature concerning the role of OA in the maintenance of g and A under water stress is limited and often contradictory. The objective of this experiment was to examine the association of OA with g and A in grain sorghum (Sorghum bicolor L. Moench). A single sorghum hybrid (cv ATx623 x RTx430) was studied under field conditions using four different water supplies. Diurnal and midday water potential, solute potential, Psi(p), OA, g, and A were measured during preflowering and grain filling growth stages. A second experiment was conducted under greenhouse conditions. Two sorghum genotypes (BTx623 and BTx378) differing in their g and A responses to plant water stress were compared for their OA capacity during a water deficit cycle imposed from the beginning of panicle initiation through flowering. Under both field and greenhouse conditions, g and A rapidly declined with increased water stress despite the occurrence of OA. Under greenhouse conditions, BTx623 maintained significantly higher g and A than BTx378 during the water stress cycle. However, no significant differences in OA or Psi(p) existed between the two genotypes, indicating that OA was not associated with differences observed in g and A between these genotypes. We conclude that the response of g and A to water stress was not directly associated with OA and certainly was not maintained by OA. PMID- 16668927 TI - Active Translation of the D-1 Protein of Photosystem II in Senescing Leaves. AB - Primary leaves of bean (Phaseolus vulgaris L.) show pronounced symptoms of senescence within 21 days of planting. Total RNA levels decline by approximately fourfold, and pulse-labeling studies with [(35)S]methionine have indicated that synthesis of all thylakoid proteins except the D-1 protein of photosystem II is sharply curtailed. Measurements of transcript levels for D-1, which is encoded by psbA, and for two other thylakoid proteins, the 68- to 70-kilodalton protein of photosystem I encoded by psaA/B and the beta subunit of ATPase encoded by atpB/E, have indicated that the continued strong synthesis of D-1 relative to other proteins in senescing thylakoids does not reflect differential changes in message abundance. Specifically, although transcript quantity for each of these proteins decreases by approximately fourfold with advancing senescence, only the 68- to 70 kilodalton protein of photosystem I and the beta-subunit of ATPase show decreased synthesis. As well, the proportion of total psbA transcript associated with polysomes is higher in senescent leaves than in young leaves, whereas for each of psaA/B and atpB/E, there is less message in polysome formation in the senescent leaves than in young leaves. Thus, the continued strong synthesis of D-1 in senescent leaves appears to reflect preferential sequestering of ribosomes by psbA transcript from a dwindling ribosome pool. This ability to preferentially sequester ribosomes may be related to the unusually high turnover rate that characterizes D-1. PMID- 16668928 TI - Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips. AB - The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO(2) by the root tips was increased 2.5 fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the beta-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall beta oxidation activity was assayed as the formation of [(14)C]acetyl-CoA from [(14)C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the beta-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues. PMID- 16668929 TI - Bicarbonate Concentration by Synechocystis PCC6803 : Modulation of Protein Phosphorylation and Inorganic Carbon Transport by Glucose. AB - The ability of the cyanobacterium Synechocystis PCC6803 to transport inorganic carbon in the form of bicarbonate rapidly decreased following a shift from bicarbonate-limited growth to either excess bicarbonate supply or to photoheterotrophic growth on glucose. Nonmetabolizable analogs of glucose did not exert this effect. The rate at which the bicarbonate uptake rate declined was too rapid to be accounted for by dilution of the activity by culture growth and suggested that posttranslational modification may be involved. Several proteins that were unphosphorylated during bicarbonate-limited growth became phosphorylated during the shifts to high CO(2) conditions and to photoheterotrophic growth. A similar alteration in the profile of phosphopolypeptides was observed following a shift into the dark. The changes in protein phosphorylation were not blocked by chloramphenicol or rifampicin. PMID- 16668930 TI - Accumulation of Group 3 Late Embryogenesis Abundant Proteins in Zea mays Embryos : Roles of Abscisic Acid and the Viviparous-1 Gene Product. AB - Several different types of proteins that are modulated by abscisic acid (ABA) accumulate in developing embryos of maize (Zea mays L.). Some of these proteins are specific to the developing seed, such as the storage globulin, GLB1, whereas others are involved in general responses to water deficit. Here we describe a maize protein family of this second type, a Group 3 late embryogenesis abundant (MLG3). Like other proteins of this class, MLG3 polypeptides are ABA-responsive. They are found in maturing seeds and in dehydrating plant tissues. Antigenically related proteins are found in other cereals. To distinguish the regulation of developmentally programmed ABA responses from those that are environmentally induced, we compared the ontological pattern and accumulation requirements of MLG3 polypeptides with those we previously described for GLB1. GLB1 accumulation begins early in the maturation phase and specifically requires high levels of ABA and the participation of the Viviparous-1 (Vp1) gene product. Vp1 is required for other ABA-modulated events in maize seed development as well. In experiments using vp1 mutants and mutants deficient in ABA synthesis (vp5 mutation), we show that MLG3 accumulation also is dependent upon ABA, but it shows striking differences from GLB1. MLG3 accumulates much later in embryogenesis, coincident with the onset of dehydration. In contrast to GLB1, MLG3 proteins can be induced by de novo ABA synthesis in response to culturing in high osmoticum. Unlike GLB1, MLG3 has no specific requirement for the Vp1 gene product. PMID- 16668931 TI - Protein Synthesis in Maize during Anaerobic and Heat Stress. AB - Protein accumulation and protein synthesis were investigated during anaerobic stress and heat shock in maize seedlings (Zea mays L.). Antibodies against alcohol dehydrogenase (ADH) and cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) were used to investigate the expression of the genes encoding these proteins during stress treatment. ADH1 protein accumulation is shown to increase about 10-fold in the root after 24 hours of anaerobic treatment. The Gpc gene products are separable into two size classes: the slow mobility GAPC1 and GAPC2 (GAPC1/2), and the faster GAPC3 and GAPC4 (GAPC3/4). The GAPC1/2 antigen did not increase at all, whereas the GAPC3/4 antigen increased less than fourfold. The proteins synthesized in the root during aerobic and anaerobic conditions were compared, and GAPC3/4 was identified as an anaerobic polypeptide. In vitro translations were used to estimate the levels of different mRNAs in roots following anaerobiosis, recovery from anaerobiosis, and heat shock. This was compared with the in vivo protein synthesis rates in roots labeled under identical conditions. In vivo labeling indicates that GAPC and ADH are not heat shock proteins. Although both GAPC3/4- and ADH1-translatable mRNA levels increase about 10-fold during anaerobiosis, in vivo labeling of these proteins (relative to total protein synthesis) is further enhanced, leading to a selective translation effect for ADH1 of threefold, and for GAPC3/4 of sixfold. In contrast, anoxia causes no change in GAPC1/2-translatable mRNA levels or in vivo labeling. As an additional comparison, beta-glucosidase mRNA levels are found to be constant during anoxia, but in vivo synthesis decreases. PMID- 16668932 TI - Computer, Simulated Evaluation of Possible Mechanisms for Sequestering Metal Ion Activity in Plant Vacuoles: II. Zinc. AB - Various mechanisms have been suggested for sequestering Zn ion activity in vacuoles of Zn-tolerant plants. One of these mechanisms, complexation in the vacuole with organic acids, has received some support in the recent literature. However, the lack of experimental evidence for anticipated vacuolar compartmentation and concerning the nature of metal-ligand species occurring in the vacuole has been criticized. In this study we have used computer modeling of chemical equilibria to predict the metalligand species in vacuoles of tobacco (Nicotiana tabacum) cultured cells. Results of this thermodynamic evaluation support the conclusion that citrate in the concentration range encountered in tobacco cultured cells exposed to 300 or 2000 mum Zn has high potential for forming soluble complexes with Zn, over the entire probable range of vacuolar pH 4 to 7. Complexation of Zn with oxalate is also predicted, especially in cells exposed to high Zn levels. Malate, though the most abundant acid present, showed little potential for competing with other ligands for Zn. Overall, results suggest that vacuolar sequestration of Zn by high levels of vacuolar citrate may be a central mechanism in the accumulation of Zn in plants exposed to either low or high levels of this metal. PMID- 16668933 TI - Identification of G-Box Sequence as an Essential Element for Methyl Jasmonate Response of Potato Proteinase Inhibitor II Promoter. AB - The potato proteinase inhibitor II promoter was studied to identify cis-acting regulatory sequences involved in methyl jasmonate (MJ) response using transgenic tobacco plants carrying various lengths of the promoter fused to a chloramphenicol acetyltransferase reporter gene. An internal fragment between 625 and -520 was sufficient to confer a response to MJ, wounding, or sucrose when it was placed upstream of the nos promoter -101, which contains the CAAT-TATA region. Deletion of the proteinase inhibitor II promoter sequence upstream of 611 did not affect the MJ response, but a further deletion to -573 eliminated the response. The 3'-deletion study showed that the DNA sequence downstream from -520 is dispensable. However, 3'-deletion mutant -574 did not respond to the MJ treatment. These results indicated that an element essential for the MJ response is located at the -574/-573 region where the G-box sequence (CACGTGG) is located. The G-box sequence was not required for the sucrose enhancer effect, suggesting that the MJ response mechanism is different from that of sucrose. PMID- 16668934 TI - Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : I. The Use of Steady-State Labeling. AB - The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with (14)CO(2). The continuous assimilation of (14)CO(2) at constant specific activity produced stable tracer fluxes that facilitated straightforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of "empty" seed coats for perfusion. PMID- 16668935 TI - Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity. AB - Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats. PMID- 16668936 TI - Binding of 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole to an Essential Cysteine Residue(s) in the Tonoplast H-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls. AB - Vacuolar-type H(+)-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [(14)C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of (14)C incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [(14)C]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H(+)-ATPase and that the modification closely correlates with the loss of ATPase activity. PMID- 16668937 TI - The production and efflux of 4-aminobutyrate in isolated mesophyll cells. AB - The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. l-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with l-[U-(14)C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular l-Glu metabolites. Incubation with l-[1-(14)C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with l-[U-(14)C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 x 10(-1) nanocuries per nanomole. In the presence of exogenous l Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that l-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA. PMID- 16668938 TI - Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells. AB - Addition of l-[U-(14)C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-(14)C]glutamate, and (c) efflux of [(14)C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H(+)/l-glutamate contransport is dependent upon GAD activity, (b) the pH dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H(+)/l-Glu symport may involve both H(+) consumption during 4-aminobutyrate production and ATP-driven H(+) efflux. PMID- 16668939 TI - Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells: Measurement with Fluorescent pH Indicator and P-Nuclear Magnetic Resonance. AB - Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2',7'-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and (31)P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by (31)P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested. PMID- 16668940 TI - Photooxidative Destruction of Chloroplasts Leads to Reduced Expression of Peroxisomal NADH-Dependent Hydroxypyruvate Reductase in Developing Cucumber Cotyledons. AB - Photooxidative destruction of chloroplasts by exposure of norflurazon-treated cucumber (Cucumis sativus L.) seedlings to white light leads to reduced levels of the nuclear-encoded, peroxisomal enzyme hydroxypyruvate reductase. The partial reduction in hydroxypyruvate reductase activity under photooxidative conditions is accompanied by reductions in levels of hydroxypyruvate reductase protein and transcript. The low level of hydroxypyruvate reductase gene expression in the dark is not affected by norflurazon, and nonphotooxidizing far-red light is able to induce significant increases in hydroxypyruvate reductase expression even in the presence of norflurazon. We conclude that intact plastids are required for maximal expression of hydroxypyruvate reductase in the light and that the plastids affect hydroxypyruvate reductase gene expression at a pretranslational level. PMID- 16668941 TI - Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA. AB - A cDNA clone was isolated from a maize (Zea mays L. cv W64AxW183E) scutellum lambdagt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH dependent cytochrome c and ferricyanide reduction. PMID- 16668942 TI - Frost, Abscisic Acid, and Desiccation Hasten Embryo Development in Brassica napus. AB - Seed development in canola (Brassica napus) following a mild nonlethal freeze was examined with respect to abscisic acid (ABA) levels, desiccation, and expression of LEA.76 and isocitrate lyase (ICL) transcripts. Plants with seed of 70 and 55% moisture contents were frozen to -5 degrees C for 3 hours, and seed development followed after thawing. In addition, similar processes were compared during induction of extreme desiccation tolerance by application of ABA in Brassica microspore-derived haploid embryos in culture. A mild freeze/thaw caused a premature switch in seed developmental direction from predesiccation to desiccation as indicated by an immediate and accelerated loss of seed moisture to levels similar to the mature seed in 7 instead of 35 days, and by elevated ABA levels and induction of low levels of LEA.76 and ICL transcripts. Similarly, addition of ABA to haploid embryos in culture resulted in the induction of desiccation tolerance and low levels of late embryogenesis-abundant (LEA) but not ICL transcripts. In contrast, normal seed development and desiccation of ABA treated (desiccation-tolerant) embryos resulted in the induction of ICL and very high levels of LEA.76 transcripts. Similarly, desiccation of control (desiccation sensitive) embryos resulted in very high levels of LEA.76 transcripts. These results indicate that although LEA-type proteins have been implicated in the development of desiccation tolerance, high transcript levels of LEA.76 were not observed in the induction of desiccation tolerance either by a hastening of the maturation process in the developing Brassica seed, or by the exogenous application of ABA to Brassica haploid embryos in culture. PMID- 16668943 TI - Abscisic Acid-induced chilling tolerance in maize suspension-cultured cells. AB - The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7 day exposure to 4 degrees C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 28 degrees C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 28 degrees C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins. PMID- 16668944 TI - Tissue-specific expression of the alternative oxidase in soybean and siratro. AB - Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro. PMID- 16668945 TI - Evidence for the presence of two different types of protein bodies in wheat endosperm. AB - Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route. PMID- 16668946 TI - Characterization of the Major Protease Involved in the Soybean beta-Conglycinin Storage Protein Mobilization. AB - Protease C1, the protease responsible for the initial degradation of the alpha' and alpha subunits of the soybean beta-conglycinin storage protein (Glycine max [L.] Merrill), has been purified. The enzyme was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to have a molecular weight of 70,000 and a pH optimum of 3.5 to 4.5. Susceptibility to protease inhibitors indicates that protease C1 is a serine protease. Study of the proteolytic intermediates generated suggests that the cleavage of the alpha' and alpha subunits of beta conglycinin by protease C1 results in intermediates that are 1 or 2 kilodaltons smaller than the native alpha' and alpha subunits. Following that, a succession of intermediates exhibiting molecular masses of 70.0 and 58.0 kilodaltons, then 63.0, 61.0, 55.0, and 53.5 kilodaltons, are observed. A 50.0- and a 48.0- kilodalton intermediate are the final products of protease C1 action. Comparison of these intermediates with the prominent anti-beta-conglycinin cross-reacting bands that increase during the first few days of germination and early growth show that protease C1 plays an important physiological role, but not an exclusive one, in the living plant. PMID- 16668947 TI - Measurement of Net Fluxes of Ammonium and Nitrate at the Surface of Barley Roots Using Ion-Selective Microelectrodes : II. Patterns of Uptake Along the Root Axis and Evaluation of the Microelectrode Flux Estimation Technique. AB - Net fluxes of NH(4) (+) and NO(3) (-) into roots of 7-day-old barley (Hordeum vulgare L. cv Prato) seedlings varied both with position along the root axis and with time. These variations were not consistent between replicate plants; different roots showed unique temporal and spatial patterns of uptake. Axial scans of NH(4) (+) and NO(3) (-) net fluxes were conducted along the apical 7 centimeters of seminal roots of intact barley seedlings in solution culture using ion-selective microelectrodes in the unstirred layer immediately external to the root surface. Theoretically derived relationships between uptake and concentration gradients, combined with experimental observations of the conditions existing in our experimental system, permitted evaluation of the contribution of bulk water flow to ion movement in the unstirred layer, as well as a measure of the spatial resolution of the microelectrode flux estimation technique. Finally, a method was adopted to assess the accuracy of this technique. PMID- 16668948 TI - A Developmental Analysis of the Enolase Isozymes from Ricinus communis. AB - Enolase activity was measured in clarified homogenates of various tissues during the life cycle of the castor oil plant (Ricinus communis L. cv Baker 296). The proportions of total activity due to the plastid and cytosolic isozymes were determined after separation by ion-exchange chromatography. The contribution of the plastid isozyme varied from more than 30% of the total at the midpoint of endosperm development to less than 1% in mature leaves and roots. During endosperm development, enolase activity increased to a peak coincident with the maximum rate of storage lipid accumulation, then decreased to nearly undetectable levels in the mature seed. Plastid enolase protein, measured using an enzyme linked immunosorbent assay, increased in parallel with the increase in activity but decreased less rapidly and was still easily detectable in mature seeds. PMID- 16668949 TI - Extraction and partial characterization of the ethylene-forming enzyme from apple fruit. AB - Ethylene-forming enzyme (EFE) was isolated from apple (Malus domestica Borkh. cv Golden Delicious) fruit tissue. The enzyme activity in the homogenate is associated with the pellet fraction and can be solubilized with Triton X-100 or polyvinylpolypyrrolidone. The solubilized enzyme system resembles the in vivo system in that it exhibits a low K(m) (17 micromolar) for its substrate 1 aminocyclopropane-1-carboxylic acid (ACC), is stereospecific toward 2-ethyl-ACC stereoisomers for 1-butene production, and is inhibited by cobalt ions and alpha aminoisobutyric acid. Intact preclimacteric fruits treated with exogenous ethylene showed a marked increase in in vivo EFE activity and this increase was accompanied by a parallel increase in in vitro EFE activity. These results support the notion that the isolated EFE represents the authentic in vivo activity. PMID- 16668950 TI - Oligomerization and regulation of higher plant phosphoenolpyruvate carboxylase. AB - The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6 phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K(m), and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed. PMID- 16668951 TI - Induction of mRNA for Phosphoenolpyruvate Carboxylase Is Correlated with a Decrease in Shoot Water Content in Well-Irrigated Mesembryanthemum crystallinum. AB - The abundance of mRNA specific for phosphoenolpyruvate carboxylase (PEPCase) was measured in leaves from well-watered plants of Mesembryanthemum crystallinum. Plants grown side by side in pots of four different volumes (0.16, 0.74, 2.6, 6.5 liters) were compared. The time of increase in the steady-state level of PEPCase mRNA in well-watered plants was dependent on soil volume. The larger the pot, the later PEPCase transcripts were increased. PEPCase mRNA induction started when shoot water content decreased to well below 4000% of dry weight. No positive correlation with the developmental status of the plants could be found. The data indicate that PEPCase mRNA induction in well-watered plants up to 10 weeks of age is controlled environmentally rather than developmentally. PMID- 16668952 TI - Effect of Exogenous Abscisic Acid on Proline Dehydrogenase Activity in Maize (Zea mays L.). AB - Plant responses to drought stress include proline and abscisic acid (ABA) accumulation. Proline dehydrogenase (PDH) (EC 1.4.3) is the first enzyme in the proline oxidation pathway, and its activity has been shown to decline in response to water stress (PJ Rayapati, CR Stewart [1991] Plant Physiol 95: 787-791). In this investigation, we determined whether ABA treatment affects PDH activity in a manner similar to drought stress in maize (Zea mays L.) seedlings. Four exogenous ABA treatments (0, 11, 33, and 100 micromolar ABA) were applied to well-watered maize seedlings. Mitochondria were isolated and PDH was solubilized using Nonidet P-40. PDH activity was measured by the reduction of iodonitrotetrazolium violet under proline-dependent conditions. There was no effect of ABA on PDH activity at 33 and 100 micromolar ABA, but there was a 38% decline at 11 micromolar. This decline was less than the 69% reduction in activity under drought stress. Endogenous ABA determinations and plant growth rate showed that ABA entered the plant and was affecting metabolic processes. ABA treatments had a small effect on shoot and root proline concentration, whereas drought stress caused a 220% increase in root tissues. We conclude that ABA is not part of the pathway linking drought stress and decreased PDH activity. PMID- 16668953 TI - Genetic Regulation of Development in Sorghum bicolor: VII. ma(3) Flowering Mutant Lacks a Phytochrome that Predominates in Green Tissue. AB - Phytochrome content of three near-isogenic genotypes of Sorghum bicolor was analyzed using immunological and spectrophotometric means. Seedlings of the photoperiodically sensitive genotypes 90M (Ma(1)Ma(1), Ma(2)Ma(2), ma(3)ma(3)) and 100M (Ma(1)Ma(1), Ma(2)Ma(2), Ma(3)Ma(3)) contain 126- and 123-kilodalton phytochromes. The 126-kilodalton protein is immunostained by antibodies Oat-16 and Pea-25. The 123-kilodalton phytochrome is immunostained by antibodies Pea-25 and Green-Oat-7. Seedlings of the photoperiodically insensitive genotype 58M (Ma(1)Ma(1), Ma(2)Ma(2), ma(3) (r)ma(3) (r)) contain only the 126-kilodalton phytochrome. In 58M seedlings, 123-kilodalton phytochrome is not detected by either Pea-25 or Green-Oat-7. Deetiolation by white light causes the 126 kilodalton phytochrome to disappear but does not greatly affect the abundance of the 123-kilodalton phytochrome. In 58M, 90M, and 100M seedlings, the 126 kilodalton phytochrome is the most abundant in etiolated tissue, whereas the 123 kilodalton phytochrome of 90M and 100M seedlings predominates in green tissue. Spectrophotometric assays show that the bulk phytochrome of etiolated tissues of all three genotypes degrades similarly upon exposure to light. At least two phytochromes are detected in sorghum: a light-labile 126-kilodalton phytochrome that predominates in etiolated tissue and a 123-kilodalton phytochrome that predominates in green tissue. Photoperiodic control of flowering in sorghum is correlated with the presence of the 123-kilodalton phytochrome. PMID- 16668954 TI - Nucleotide sequence of acyl-acyl carrier protein: glycerol-3-phosphate acyltransferase from cucumber. PMID- 16668955 TI - Nucleotide sequences of a soybean cDNA encoding an 18 kilodalton late embryogenesis abundant protein. PMID- 16668956 TI - A wound-inducible member of the hydroxyproline-rich glycoprotein gene family in sunflower. PMID- 16668957 TI - Plant cDNA similar to a bacterial plasmid partition locus. PMID- 16668958 TI - Cotton Mat5-A (C164) Gene and Mat5-D cDNAs Encoding Methionine-Rich 2S Albumin Storage Proteins. PMID- 16668959 TI - Cotton Lea4 (D19) and LeaA2 (D132) Group 1 Lea Genes Encoding Water Stress Related Proteins Containing a 20-Amino Acid Motif. PMID- 16668960 TI - Nucleotide Sequence of a cDNA Encoding a Constitutively Expressed Glutathione S Transferase from Cell Suspension Cultures of Silene cucubalus. PMID- 16668961 TI - Sequence of the cDNA encoding the 17-kilodalton protein of the photosynthetic oxygen-evolving complex of pea. PMID- 16668962 TI - Nucleotide Sequence of a Complementary DNA Encoding Plasma Membrane H-ATPase from Rice (Oryza sativa L.). PMID- 16668963 TI - Chloroplast Division and Expansion Is Radically Altered by Nuclear Mutations in Arabidopsis thaliana. AB - We have isolated three mutants of Arabidopsis thaliana in which there is a sevenfold change in chloroplast number in fully expanded leaf mesophyll cells and increases and decreases in chloroplast number are compensated for by changes in chloroplast size. The changes are stably inherited in sexual crosses for three generations and mutant phenotypes are effected by changes at single recessive nuclear loci, termed arc loci. This is the first report of large, stably inherited changes in chloroplast number in higher plants, and represents a major advance toward the genetic dissection of the control of chloroplast division. PMID- 16668964 TI - Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition. AB - Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium. PMID- 16668965 TI - Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption. AB - The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic beta-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, beta-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther beta-1,3-glucanase is serologically unrelated to the basic beta-1,3 glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as beta-1,3-glucanase are specific to each process. PMID- 16668966 TI - Aluminum Partitioning in Intact Roots of Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars. AB - Aluminum (Al) partitioning in intact roots of wheat (Triticum aestivum L.) cultivars that differ in sensitivity to Al was investigated. Roots of intact seedlings were exposed to Al for up to 24 hours and distribution of Al was assessed visually by hematoxylin staining or by direct measurement of concentration of Al by atomic absorption spectrophotometry or ion chromatography. Major differences in Al accumulation between Al-tolerant (Atlas 66) and Al sensitive (Tam 105) cultivars were found in the growing regions 0 to 2 and 2 to 5 millimeters from the root apex. Al content was 9 to 13 times greater in the 0 to 2 millimeters root tips of cv Tam 105 than in the tips of cv Atlas 66 when exposed to 50 micromolar Al for 19 to 24 hours. The oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone and the protein synthesis inhibitor cycloheximide increased Al uptake by intact root tips of cv Atlas 66. Also, loss of Al from the roots of both cultivars was measured after the roots were "pulsed" with 50 micromolar Al for 2 hours and then placed in an Al-free nutrient solution for 6 hours. The 0 to 2 millimeter root tips of cv Tam 105 lost 30% of the absorbed Al, whereas the tips of cv Atlas 66 lost 60%. In light of these results, we conclude that the differential Al sensitivity in wheat correlates with the concentration of Al in the root meristems. The data support the hypothesis that part of the mechanism for Al tolerance in wheat is based on a metabolism-dependent exclusion of Al from the sensitive meristems. PMID- 16668967 TI - Isolation and characterization of an alkaline phosphatase from pea thylakoids. AB - Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme. PMID- 16668968 TI - Oxidation of External NAD(P)H by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria : A Kinetic and Inhibitor Study. AB - The functional interaction between the externally located NAD(P)H dehydrogenase and the Q-pool acceptor site(s) in Percoll-purified mitochondria from Jerusalem artichoke (Helianthus tuberosus L. cv OB1) mitochondria has been investigated. Oxidation of exogenous NADH is stimulated by ubiquinone (UQ(1)) with a parallel decrease of the apparent K(m) for NADH. In the presence of saturating amounts of UQ(1) as electron acceptor, the K(m) (NADH) is not affected by variations of the ionic strength. Conversely, the K(m) for UQ(1) is decreased by the screening effect of negative charges on the outer membrane surface. Under low-ionic strength, the hydroxyflavone platanetin progressively inhibits NADH oxidation with a mean inhibition dose of approximately 3 nanomoles of inhibitor per milligram of protein. Interestingly, under high-ionic strength, oxidation of NADH proceeds through two platanetin binding sites, one of which has a lower affinity for the inhibitor (mean inhibition dose = 20 nanomoles per milligram protein), because it is located near the outer surface of the membrane. This latter site is the one involved in the oxidation of external NADPH and, possibly, also affected by spermine and spermidine. Similarly to NADH, oxidation of NADPH is fully sensitive to micromolar concentrations of free Ca(2+) ions; in addition, similar concentrations of the sulfhydryl reagent mersalyl are required to inhibit both NADH and NADPH oxidative activities. The results are interpreted as evidence for the presence of a single nonspecific NAD(P)H dehydrogenase. PMID- 16668969 TI - Increased Activity of a Cationic Peroxidase Associated with an Incompatible Interaction Between Xanthomonas oryzae pv oryzae and Rice (Oryza sativa). AB - Rice (Oryza sativa L.) cultivar Cas 209 carries the gene Xa-10 for resistance to race 2 of Xanthomonas oryzae pv oryzae, the bacterial blight pathogen. When seedling leaves of Cas 209 plants were infiltrated with bacterial cell suspensions of strain PXO86(Rif) (race 2, incompatible), total peroxidase activity in extracts from extracellular spaces increased almost threefold between 16 and 24 hours after inoculation. The increase in total peroxidase activity in extracellular extracts was correlated with the appearance of a 43-kilodalton peroxidase isoenzyme with an isoelectric point of 8.6. Increases in the activities of two anionic peroxidase isoenzymes also were associated with the incompatible interaction. Later during the interactions, total peroxidase activities increased in both compatible (cv Cas 209 infiltrated with race 1, PXO61(Sm)) and control (Cas 209 infiltrated with water) treatments, but final activity levels were less than that observed in the incompatible combination. Similarly, the cationic peroxidase was detected in all three treatments by 48 hours after infiltration, but at reduced levels in compatible and water infiltrated control treatments relative to the incompatible combination. Accumulation of this peroxidase in extracellular spaces thus may play a role in the defense response in cultivar Cas 209. PMID- 16668970 TI - Endosperm cell division in maize kernels cultured at three levels of water potential. AB - The influence of osmoticum treatments on early kernel development of maize (Zea mays L.) was studied using an in vitro culture method. Kernels with subtending cob sections were placed in culture at 5 days after pollination. Sucrose (0.29, 0.44, or 0.58 molar) and sorbitol (0, 0.15, or 0.29 molar) were used to obtain six media with water potentials of -1.1, -1.6, or -2.0 megapascals. Kernel water potential declined in correspondence with the water potential of the medium; however, fresh weight growth was not significantly inhibited from 5 to 12 days after pollination. In stress treatments with media water potentials of -1.6 or 2.0 megapascals, endosperm tissue accumulated water and solutes from 10 and 12 days after pollination at a rate similar to or greater than that of the control ( 1.1 megapascals). In contrast, endosperm cell division was inhibited in all treatments relative to control. At 10 days after pollination, endosperm sucrose concentration was greater in two of the -2.0 megapascal treatments with 0.44 or 0.58 molar media sucrose compared to control kernels cultured in 0.29 molar sucrose at -1.1 megapascals. Significant increases in abscisic acid content per gram of fresh weight were detected in two -2.0 megapascal treatments (0.29 molar sucrose plus 0.29 molar sorbitol and 0.58 molar sucrose) at 10 days after pollination. We conclude that in cultured maize kernels, endosperm cell division was more responsive than fresh weight accumulation to low water potential treatments. Data were consistent with mechanisms involving abscisic acid or lowered tissue water potential, or an interaction of the two factors. PMID- 16668971 TI - Diffusion and Electric Mobility of KCI within Isolated Cuticles of Citrus aurantium. AB - Fick's second law has been used to predict the time course of electrical conductance change in isolated cuticles following the rapid change in bathing solution (KCI) from concentration C to 0.1 C. The theoretical time course is dependent on the coefficient of diffusion of KCI in the cuticle and the cuticle thickness. Experimental results, obtained from cuticles isolated from sour orange (Citrus aurantium), fit with a diffusion model of an isolated cuticle in which about 90% of the conductance change following a solution change is due to salts diffusing from polar pores in the wax, and 10% of the change is due to salt diffusion from the wax. Short and long time constants for the washout of KCI were found to be 0.11 and 3.8 hours, respectively. These time constants correspond to KCI diffusion coefficients of 1 x 10(-15) and 3 x 10(-17) square meters per second, respectively. The larger coefficient is close to the diffusion coefficient for water in polar pores of Citrus reported elsewhere (M Becker, G Kerstiens, J Schonherr [1986] Trees 1: 54-60). This supports our interpretation of the washout kinetics of KCI following a change in concentration of bathing solution. PMID- 16668972 TI - Transgenic Tobacco Plants Coexpressing the Agrobacterium tumefaciens iaaM and iaaH Genes Display Altered Growth and Indoleacetic Acid Metabolism. AB - Transgenic tobacco (Nicotiana tabacum) SR1 plants expressing the Agrobacterium tumefaciens nopaline transferred DNA iaaH gene were transformed with a 35S-iaaM construct. The transformants displayed several morphological aberrations, such as adventitious root formation on stem and leaves, dwarfism, epinastic leaf growth, increased apical dominance, and an overall retardation in development. In addition, xylem lignification was higher than in wild type. Free and conjugated indoleacetic acid (IAA) levels were quantified by gas chromatography-multiple ion monitoring-mass spectrometry in leaves and internodes of wild-type plants and two transformed lines with different phenotypes. Both transformed lines contained elevated levels of free and conjugated IAA, which was associated with increased transcription of the iaaM gene. The line with the highest IAA level also had the most altered pattern of growth and development. These IAA-overproducing plants will provide a model system for studies on IAA metabolism, IAA interactions with other phytohormones, and IAA roles in regulating plant growth and development. PMID- 16668973 TI - Functional compartmentation of the Golgi apparatus of plant cells : immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells. AB - The Golgi apparatus of plant cells is engaged in both the processing of glycoproteins and the synthesis of complex polysaccharides. To investigate the compartmentalization of these functions within individual Golgi stacks, we have analyzed the ultrastructure and the immunolabeling patterns of high-pressure frozen and freeze-substituted suspension-cultured sycamore maple (Acer pseudoplatanus L.) cells. As a result of the improved structural preservation, three morphological types of Golgi cisternae, designated cis, medial, and trans, as well as the trans Golgi network, could be identified. The number of cis cisternae per Golgi stack was found to be fairly constant at approximately 1, whereas the number of medial and trans cisternae per stack was variable and accounted for the varying number of cisternae (3-10) among the many Golgi stacks examined. By using a battery of seven antibodies whose specific sugar epitopes on secreted polysaccharides and glycoproteins are known, we have been able to determine in which types of cisternae specific sugars are added to N-linked glycans, and to xyloglucan and polygalacturonic acid/rhamnogalacturonan-I, two complex polysaccharides. The findings are as follows. The beta-1,4-linked d glucosyl backbone of xyloglucan is synthesized in trans cisternae, and the terminal fucosyl residues on the trisaccharide side chains of xyloglucan are partly added in the trans cisternae, and partly in the trans Golgi network. In contrast, the polygalacturonic/rhamnogalacturonan-I backbone is assembled in cis and medial cisternae, methylesterification of the carboxyl groups of the galacturonic acid residues in the polygalacturonic acid domains occurs mostly in medial cisternae, and arabinose-containing side chains of the polygalacturonic acid domains are added to the nascent polygalacturonic acid/rhamnogalacturonan-I molecules in the trans cisternae. Double labeling experiments demonstrate that xyloglucan and polygalacturonic acid/rhamnogalacturonan-I can be synthesized concomitantly within the same Golgi stack. Finally, we show that the xylosyl residue-linked beta-1,2 to the beta-linked mannose of the core of N-linked glycans is added in medial cisternae. Taken together, our results indicate that in sycamore maple suspension-cultured cells, different types of Golgi cisternae contain different sets of glycosyl transferases, that the functional organization of the biosynthetic pathways of complex polysaccharides is consistent with these molecules being processed in a cis to trans direction like the N-linked glycans, and that the complex polysaccharide xyloglucan is assembled exclusively in trans Golgi cisternae and the trans Golgi network. PMID- 16668974 TI - Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees. AB - Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate. PMID- 16668975 TI - A new bioassay for auxins and cytokinins. AB - The authors have developed a sensitive bioassay that can be used to detect auxins as well as cytokinins. The bioassay is based on the expression in transformed tobacco (Nicotiana tabacum) mesophyll protoplasts of a chimeric gene, consisting of the upstream sequences of the Agrobacterium tumefaciens gene 5, coupled to the coding sequence of the beta-glucuronidase. The expression of this gene is induced by the presence of both auxin and cytokinin in the culture medium. Using this assay, indole-3-acetic acid was detected at 5 x 10(-8) molar, whereas trans zeatin could be detected at 5 x 10(-11) molar. The assay can be performed in microtiter plates, allowing numerous samples to be analyzed simultaneously. Only 2.5 x 10(5) protoplasts are required for one individual assay in 250 microliters of culture medium and for qualitative results, the reaction is readily visualized by ultraviolet light. PMID- 16668976 TI - Influence of the degree of polymerization of oligogalacturonates and of esterification pattern of pectin on their recognition by monoclonal antibodies. AB - The ability of galacturonic and oligogalacturonic acids with degrees of polymerization (DP) from 2 to 10 to inhibit the recognition of homopolygalacturonic acid by a monoclonal antibody specific for dimers of pectin (F Liners, J-J Letesson, C Didembourg, P Van Cutsem [1989] Plant Physiol 91: 1419 1424) has been tested by enzyme-linked immunosorbent assays. Oligomers of DP9 and above preincubated with the antibodies clearly inhibited the association between the antibodies and immobilized pectin. A minimum DP of nine consecutive galacturonic residues is thus necessary to be associated through calcium cations to form dimers. Randomly deesterified pectin was recognized by the antibody if its degree of methylesterification was <30%, whereas blockwise deesterified pectin was recognized up to 40% of methylesterification. The replacement of calcium ions by magnesium prevented the recognition of polygalacturonic acid by the antibody. PMID- 16668977 TI - Photosynthate metabolism in the source leaves of n(2)-fixing soybean plants. AB - Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N(2) fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N(2)-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO(3) (-) and NH(4) (+) (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N(2)-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6 bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N(2)-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N(2)-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose 6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K(m) values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N(2)-fixing as well as N-supplemented plants, and G1,6BP levels in N(2)-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N(2)-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N(2)-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO(3) (-) and NH(4) (+), the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N(2)-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N(2)-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis. PMID- 16668978 TI - Effect of Phloem-Translocated Malate on NO(3) Uptake by Roots of Intact Soybean Plants. AB - In soybean (Glycine max L. Merr. cv Kingsoy), NO(3) (-) assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO(3) (-) uptake by roots. The net NO(3) ( ) uptake rate by roots of soybean plants was stimulated by the addition of K malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO(3) (-) uptake and C excretion rates by roots. These results suggest that NO(3) (-) uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO(3) ( ) uptake, and excretion of HCO(3) (-) ions was probably released by malate decarboxylation. NO(3) (-) uptake rate increased when the supply of NO(3) (-) to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO(4) (2-). We conclude that in situ, NO(3) (-) reduction rate in the shoot may control NO(3) (-) uptake rate in the roots via the translocation rate of malate in the phloem. PMID- 16668979 TI - Chloroplast Biogenesis 65 : Enzymic Conversion of Protoporphyrin IX to Mg Protoporphyrin IX in a Subplastidic Membrane Fraction of Cucumber Etiochloroplasts. AB - The preparation from Percoll-purified cucumber (Cucumis sativus)etiochloroplasts of a subplastidic membrane fraction that is capable of high rates of Mg insertion into protoporphyrin IX is described. The plastid stroma was inactive when used either alone or in combination with the membrane fraction. Successful preparation of the subplastidic membrane fraction required that Mg-protoporphyrin chelatase was first stabilized by its substrate. This was achieved by lysing Percoll purified plastids in a fortified hypotonic medium containing protoporphyrin IX prior to ultracentrifugation and separation of the stroma from the plastid membranes. Protoporphyrin IX became membrane bound. Other additives needed for enzyme activity fell into two groups: (a) those needed for enzyme stabilization during membrane preparation and (b) those involved in the primary mechanism of Mg insertion into protoporphyrin IX. Ethylenediaminetetraacetate belonged to the first group, magnesium belonged to the second group, and ATP belonged to both groups. PMID- 16668980 TI - Developmental history affects the susceptibility of spinach leaves to in vivo low temperature photoinhibition. AB - Room temperature chlorophyll a fluorescence was used to determine the effects of developmental history, developmental stage, and leaf age on susceptibility of spinach to in vivo low temperature (5 degrees C) induced photoinhibition. Spinach (Spinacia oleracea cv Savoy) leaves expanded at cold hardening temperatures (5 degrees C day/night), an irradiance of 250 micromoles per square meter per second of photosynthetic proton flux density, and a photoperiod of 16 hours were less sensitive than leaves expanded at nonhardening temperatures (16 or 25 degrees C day/night) and the same irradiance and photoperiod. This differential sensitivity to low-temperature photoinhibition was observed at high (1200) but not lower (500 or 800 micromoles per square meter per second) irradiance treatment. In spite of a differential sensitivity to photoinhibition, both cold-hardened and nonhardened spinach exhibited similar recovery kinetics at either 20 or 5 degrees C. Shifting plants grown at 16 degrees C (day/night) to 5 degrees C (day/night) for 12 days after full leaf expansion did not alter the sensitivity to photoinhibition at 5 degrees C. Conversely, shifting plants grown at 5 degrees C (day/night) to 16 degrees C (day/night) for 12 days produced a sensitivity to photoinhibition at 5 degrees C similar to control plants grown at 16 degrees C. Thus, any resistance to low-temperature photoinhibition acquired during growth at 5 degrees C was lost in 12 days at 16 degrees C. We conclude that leaf developmental history, developmental stage, and leaf age contribute significantly to the in vivo photoinhibitory response of spinach. Thus, these characteristics must be defined clearly in studies of plant susceptibility to photoinhibition. PMID- 16668981 TI - Molecular cloning and characterization of genes related to chilling tolerance in rice. AB - Polyadenylated RNA was isolated and a cDNA library constructed from seedlings of a chilling-tolerant rice cultivar (Oryza sativa L. subsp. Japonica cv Nipponbare). Four clones were isolated by differential screening. Northern blot hybridization using RNAs from chilling-tolerant (Nipponbare) and -sensitive (IR36) cultivars revealed higher steady-state levels of transcripts for the four genes in Nipponbare than in IR36 maintained at the same low temperatures. The accumulation of transcripts homologous to selected cDNA sequences during chilling were tissue-specific. The nucleotide and deduced amino acid sequences of three clones, pBC121, pBC442, and pBC591, were determined, and no homology was identified by comparison with the latest version of EMBL and LASL gene data bases. The deduced protein sequences from the longest open reading frame of the clones pBC121 and pBC442 are rich in leucine and serine, whereas that of the clone pBC591 contains arginine-rich basic domains. PMID- 16668982 TI - Purification and characterization of actin from maize pollen. AB - Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold. PMID- 16668983 TI - On the role of abscisic Acid and gibberellin in the regulation of growth in rice. AB - Submergence induces rapid elongation of rice coleoptiles (Oryza sativa L.) and of deepwater rice internodes. This adaptive feature helps rice to grow out of the water and to survive flooding. Earlier, we found that the growth response of submerged deepwater rice plants is mediated by ethylene and gibberellin (GA). Ethylene promotes growth, at least in part, by increasing the responsiveness of the internodal tissue to GA. In the present work, we examined the possibility that increased responsiveness to GA was based on a reduction in endogenous abscisic acid (ABA) levels. Submergence and treatment with ethylene led, within 3 hours, to a 75% reduction in the level of ABA in the intercalary meristem and the growing zone of deepwater rice internodes. The level of GA(1) increased fourfold during the same time period. An interaction between GA and ABA could also be shown by application of the hormones. ABA inhibited growth of submerged internodes, and GA counteracted this inhibition. Our results indicate that the growth rate of deepwater rice internodes is determined by the ratio of an endogenous growth promoter (GA) and a growth inhibitor (ABA). We also investigated whether ABA is involved in regulating the growth of rice coleoptiles. Rice seedlings were grown on solutions containing fluridone, an inhibitor of carotenoid and, indirectly, of ABA biosynthesis. Treatment with fluridone reduced the level of ABA in coleoptiles and first leaves by more than 75% and promoted coleoptile growth by more than 60%. Little or no enhancement of growth by fluridone was observed in barley, oat, or wheat. The involvement of ABA in determining the growth rate of rice coleoptiles and deepwater rice internodes may be related to the semiaquatic growth habit of this plant. PMID- 16668984 TI - Laccase from Sycamore Maple (Acer pseudoplatanus) Polymerizes Monolignols. AB - Current understanding of the final oxidative steps leading to lignin deposition in trees and other higher plants is limited with respect to what enzymes are involved, where they are localized, how they are transported, and what factors regulate them. With the use of cell suspension cultures of sycamore maple (Acer pseudoplatanus), an in-depth study of laccase, one of the oxidative enzymes possibly responsible for catalyzing the dehydrogenative polymerization of monolignols in the extracellular matrix, was undertaken. The time course for secretion of laccase into suspension culture medium was determined with respect to age and mass of the cells. Laccase was completely separated from peroxidase activity by hydrophobic interaction column chromatography, and its purity was assessed with different types of gel electrophoresis (isoelectric focusing-, native-, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Amino acid and glycosyl analyses of the purified enzyme were compared with those reported from previous studies of plant and fungal laccases. The specific activity of laccase toward several common substrates, including monolignols, was determined. Unlike a laccase purified from the Japanese lacquer tree (Rhus vernicifera), laccase from sycamore maple oxidized sinapyl, coniferyl, and p coumaryl alcohols to form water-insoluble polymers (dehydrogenation polymers). PMID- 16668985 TI - Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin. AB - Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched beta-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice. PMID- 16668986 TI - Sequencing and characterization of the soybean leaf metalloproteinase : structural and functional similarity to the matrix metalloproteinase family. AB - A novel zinc endoproteinase has been sequenced and characterized from soybean leaves (Glycine max var Williams 82) and has been designated as Protein Identification Resource accession No. A41820 SMEP1 (soybean metalloendoproteinase 1). Comparison of the primary amino acid sequence with other zinc proteinases revealed the enzyme to be a new member of the matrix metalloproteinase (MMP) family of enzymes. SMEP was found to have MMP cleavage specificity toward peptide substrates and the enzyme is specifically inhibited by naturally occurring tissue inhibitors of MMPs through a high-affinity interaction (inhibitor concentration resulting in an approximate 50% decrease in enzyme activity = 23 x 10(-9) molar). Together, these results suggest that the origin of the MMP family of enzymes and their cognate inhibitors predates the divergence of plants and animals. PMID- 16668987 TI - Characteristics of alpha-Amylase during Germination of Two High-Sugar Sweet Corn Cultivars of Zea mays L. AB - The role of the scutellum and the aleurone in alpha-amylase production in the high-sugar sweet corn cultivars Illini X-tra Sweet (shrunken-2, sh2) and Illinois 677a (sugary, sugary enhancer; su se) was compared to that in the starchy (Su) hybrid Funks G4646 with the use of alpha-amylase enzyme assays, isoelectric focusing, electron microscopy, and laser scanning confocal microscopy. The scutellum of Illinois 677a had low levels of alpha-amylase activity compared to that of Funks G4646 through 10 days after imbibition, and the aleurone of Illini X-tra Sweet had negligible activity. On the isoelectric focusing gels, the Illinois 677a scutellum had fewer alpha-amylase isozymes at 7 days compared to the Funks G4646 scutellum. The Illini X-tra Sweet aleurone had no alpha-amylase isozymes. Funks G4646 scutellar epithelial and aleurone cells contained abundant rough endoplasmic reticulum, polysomes, and dictyosomes at 5 and 7 days, respectively. The scutellar epithelial cells of Illinois 677a contained fewer of these structures by 5 days, and the Illini X-tra Sweet aleurone contained mostly lipid bodies through 7 days. Few cytoplasmic membranes and little RNA were detected with laser scanning confocal microscopy in the Illini X-tra Sweet aleurone compared to Funks G4646 at 7 days. These data suggest that the scutellum of Illinois 677a and the aleurone of Illini X-tra Sweet have impaired abilities to produce alpha-amylase. PMID- 16668988 TI - Aluminum Toxicity in Roots : Correlation among Ionic Currents, Ion Fluxes, and Root Elongation in Aluminum-Sensitive and Aluminum-Tolerant Wheat Cultivars. AB - The inhibition of root growth by aluminum (Al) is well established, yet a unifying mechanism for Al toxicity remains unclear. The association between cell growth and endogenously generated ionic currents measured in many different systems, including plant roots, suggests that these currents may be directing growth. A vibrating voltage microelectrode system was used to measure the net ionic currents at the apex of wheat (Triticum aestivum L.) roots from Al-tolerant and Al-sensitive cultivars. We examined the relationship between these currents and Al-induced inhibition of root growth. In the Al-sensitive cultivar, Scout 66, 10 micromolar Al (pH 4.5) began to inhibit the net current and root elongation within 1 to 3 hours. These changes occurred concurrently in 75% of experiments. A significant correlation was found between current magnitude and the rate of root growth when data were pooled. No changes in either current magnitude or growth rate were observed in similar experiments using the Al-tolerant cultivar Atlas 66. Measurements with ion-selective microelectrodes suggested that H(+) influx was responsible for most of the current at the apex, with smaller contributions from Ca(2+) and Cl(-) fluxes. In 50% of experiments, Al began to inhibit the net H(+) influx in Scott 66 roots at the same time that growth was affected. However, in more than 25% of cases, Al-induced inhibition of growth rate occurred before any sustained decrease in the current or H(+) flux. Although showing a correlation between growth and current or H(+) fluxes, these data do not suggest a mechanistic association between these processes. We conclude that the inhibition of root growth by Al is not caused by the reduction in current or H(+) influx at the root apex. PMID- 16668989 TI - Alteration of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase activities by site-directed mutagenesis. AB - Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser 112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild type enzyme with mutant enzymes. PMID- 16668990 TI - Effect of Specific Elicitors of Cladosporium fulvum on Tomato Suspension Cells : Evidence for the Involvement of Active Oxygen Species. AB - Intercellular fluid (IF) obtained from tomato (Lycopersicon esculentum L.) leaflets colonized by Cladosporium fulvum Cooke contains specific elicitors that induce necrosis in tomato cultivars resistant to the race of C. fulvum used to produce the IF. The responses of cell-suspension cultures produced from tomato lines near-isogenic for resistance genes Cf 4 and Cf 5 to IF produced from leaves infected by races 4 (virulent on Cf 4 but not Cf 5 plants), 2.4.5, and 2.4.5.9 (both virulent on Cf 4 and Cf 5 plants) were used to investigate the possibility that active oxygen (AO) species were involved in the initial host reaction to these elicitors. Concurrently, the same assays were used to determine if the cell lines retained the elicitor specificity of the original plants. An IF/cell combination that gives an incompatible reaction in leaves (race 4 IF and Cf 5 cells) showed reduced oxygen uptake and increases in malonaldehyde (a product of lipid peroxidation); cytochrome c reducing activity, which was inhibited by superoxide dismutase (SOD) (an assay for superoxide); luminol-dependent chemiluminescence (an assay for several AO species); activity of extracellular peroxidases; and extracellular phenolic compounds. In contrast, compatible combinations (IF from races 2.4.5 or 2.4.5.9 and Cf 4 or Cf 5 cells; race 4 IF and Cf 4 cells) did not exhibit any of these changes. The addition of catalase, SOD, ascorbate (a scavenger of superoxide), mannitol (a scavenger of the hydroxyl radical), KCN, or salicyl hydroxamic acid (both inhibitors of peroxidases) prior to IF treatment reduced the IF-induced increases in malonaldehyde and extracellular phenolics. Catalase was an effective inhibitor of the IF-induced changes in oxygen uptake and cytochrome c reducing activity. These results demonstrate the specificity of the IF-induced cell responses and confirm that AO species are involved in the initial cell response. PMID- 16668991 TI - Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum. AB - Uridine 5'-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mmN-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5'-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5'-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations. PMID- 16668992 TI - Developmental Regulation of (1-->3, 1-->4)-beta-Glucanase Gene Expression in Barley : Tissue-Specific Expression of Individual Isoenzymes. AB - Two genes encode (1-->3,1-->4)-beta-d-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzymes EI and EII in barley (Hordeum vulgare L.). Specific DNA probes have been used in Northern analyses to examine the developmental regulation of individual (1-->3,1-->4)-beta-glucanase genes in the aleurone and scutellum of germinated grain and in young leaves and young roots. In aleurone and scutella excised from germinated grain, mRNAs encoding both isoenzymes are present but developmental patterns differ between the two tissues. Thus, levels of both isoenzyme EI and EII mRNA increase significantly in the aleurone between 1 and 3 days after the initiation of germination. In the scutellum, isoenzyme EI mRNA predominates and decreases as germination proceeds. Isoenzyme EI mRNA appears in young leaves approximately 8 days after the initiation of germination and levels rise until about 20 days. Enzyme activity in leaf extracts parallels the development of isoenzyme EI mRNA. No isoenzyme EII mRNA is detected in the leaves in this period. Analysis of RNA from different leaf segments indicates that the isoenzyme EI mRNA is distributed relatively evenly along the length of the leaf. In young roots, mRNA encoding (1-->3,1-->4)-beta-glucanase isoenzyme EI is detected at high levels 3 to 6 days after the initiation of germination; again, little or no isoenzyme EII mRNA is found. Overall, transcription of the (1-->3,1- >4)-beta-glucanase isoenzyme EII gene appears to be restricted to the germinating grain, whereas isoenzyme EI is expressed in a wider range of tissues during seedling development. PMID- 16668993 TI - Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum). AB - The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition. PMID- 16668994 TI - Effects of O(2) and CO(2) Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora: Evidence for Rubisco-Mediated CO(2) Fixation and Photorespiration in Guard Cells. AB - A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (F(s)) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, F(s) is a very sensitive indirect measure of the balance of adenosine 5'-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light generated products. We found that F(s) under constant light is sensitive to manipulation of ambient CO(2) concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO(2) fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O(2) concentration had a strong effect on fluorescence yield, and that O(2) sensitivity was only evident when the concentration of CO(2) was low. This finding provides evidence that both O(2) and CO(2) can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C(3) plants. Indeed, Rubisco has been detected at low levels in guard cell chloroplasts, and our studies indicate that it is active in the pathways for photosynthetic carbon reduction and photorespiration in guard cells. PMID- 16668995 TI - Expression of the Genes for the alpha- and beta-Subunits of Pyrophosphate Dependent Phosphofructokinase in Germinating and Developing Seeds from Ricinus communis. AB - Various tissues from both germinating and developing castor seeds (Ricinus communis L.) have been analyzed for the level of expression of the genes for the alpha- and beta-subunits of pyrophosphate-dependent phosphofructokinase (PFP). In tissues in which PFP is expressed, there is a single mRNA species of approximately 2 kilobases for each of the subunits. In germinating endosperm, the gene for the alpha-subunit is expressed at an earlier time after imbibition than that for the beta-subunit, whereas in developing castor seed endosperm, both genes are highly and coordinately expressed. During seedling development, there is tissue-specific expression of the two genes. Tissues in which there is a high level of mRNA correspond with tissues in which both subunits of PFP can be detected. The differential expression of the two subunit genes in germinating endosperm does not result in the presence of the alpha-subunit polypeptide in the absence of the beta-subunit polypeptide. Southern analysis of castor genomic DNA indicates the presence of a single gene for both the alpha- and beta-subunits of PFP in contrast with potato, in which there are at least two genes for each subunit. PMID- 16668996 TI - Galactinol Synthase is an Extravacuolar Enzyme in Tubers of Japanese Artichoke (Stachys sieboldii). AB - Galactinol synthase (GS, UDP-alpha-d-galactose:1l-myo-inositol-1-O- alpha-d galactopyranosyltransferase) is a key enzyme in the biosynthetic pathway of the raffinose family of oligosaccharides. The subcellular location of GS was studied in the parenchyma of stachyose-storing tubers of Japanese artichoke (Stachys sieboldii) by isolation of protoplasts and vacuoles. A comparison of the activities of GS, malate dehydrogenase, and alcohol dehydrogenase (extravacuolar markers) and alpha-mannosidase and beta-N-acetylglucosaminidase (vacuolar markers) in parenchyma protoplasts with those of vacuoles isolated from them showed that GS was an extravacuolar enzyme. PMID- 16668997 TI - Primary Structure of Cytochrome b(5) from Cauliflower (Brassica oleracea L.) Deduced from Peptide and cDNA Sequences. AB - Cytochrome b(5) is a microsomal protein that functions as an intermediate electron donor in fatty acid desaturation and other oxidation/reduction reactions. cDNA clones were isolated from cauliflower (Brassica oleracea L.) by using oligonucleotides based on the partial amino acid sequence of the protein. The deduced amino acid sequence of the polypeptide exhibited approximately 30% sequence identity with the homologous protein from vertebrates. PMID- 16668998 TI - Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis. AB - Several lines of evidence indicate that abscisic acid (ABA) is derived from 9' cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties. PMID- 16668999 TI - Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum. AB - In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C(3) photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte. PMID- 16669001 TI - Changing of the guard. PMID- 16669000 TI - Nucleotide sequence of a cDNA encoding starch-branching enzyme, or q-enzyme I, from rice endosperm. PMID- 16669002 TI - Salicylate, a new plant hormone. PMID- 16669003 TI - Jasmonate, genes, and fragrant signals. PMID- 16669004 TI - Adaptation to dim-red light leads to a nongradient pattern of stem elongation in cucumis seedlings. AB - Relative growth rate determinations on 5-millimeter regions of cucumber (Cucumis sativus L.) hypocotyls show that dim-red light-grown seedlings have an even distribution of growth along the stem axis. This contrasts with the apical to basal graded decline in growth rate seen in dark-grown seedlings, including dark grown cucumber seedlings used as controls in this study. Dark-grown seedlings convert to the nongradient pattern when transferred to dim-red light. The small amount of light required suggests that the change in developmental pattern may happen in the natural light environment. PMID- 16669005 TI - Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors. AB - Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli. PMID- 16669006 TI - Brassica juncea Produces a Phytochelatin-Cadmium-Sulfide Complex. AB - Phytochelatins (PCs) are enzymically synthesized peptides produced in higher plants and some fungi upon exposure to heavy metals. We have examined PC production in the Se-tolerant wild mustard Brassica juncea and found that it produces two types of PC-Cd complexes with the same characteristics as those from fission yeast Schizosaccharomyces pombe, including a high molecular weight PC-Cd sulfide form. PMID- 16669007 TI - Immunological Characterization of a Tapetal Protein in Developing Anthers of Lilium longiflorum. AB - Antiserum was raised in rabbits against a lily (Lilium longiflorum) anther specific protein (LLA-15). Monospecific anti-LLA-15 antibodies were prepared to investigate the distribution of LLA-15 during anther development in a variety of flowering plants. Immunoblot analyses of total protein from floral and vegetative organs confirmed that LLA-15 or LLA-15-like proteins accumulated to detectable levels only in a discrete stage of anther development. In situ localization using anti-rabbit immunoglobulin G conjugated with gold particles confirmed that LLA-15 was specifically localized in the tapetal tissue of lily anthers. The maximal level of LLA-15 was strictly coincident with the peak of tapetal secretory functions. Immunoblots of two-dimensional polyacrylamide gels of lily anther proteins indicated that the seven LLA-15 isoforms ranged from isoelectric point 5.6 to 6.1. In vitro translation of lily anther mRNAs showed that four of these isoforms were primary products, the additional three being a result of posttranslational processing of the primary translation products. PMID- 16669008 TI - Differential Detergent Stability of the Major Light-Harvesting Complex II in Thylakoids Isolated from Monocotyledonous and Dicotyledonous Plants. AB - A survey of isolated thylakoids from 11 different higher plant species (Spinacia oleracea L., Pisum sativum L., Vicia faba L., Brassica napus L., Vigna sinensis L., Vinca minor L., Secale cereale L., Triticum aestivum L., Triticosecale Wittn., Hordeum vulgare L., Zea mays L.) indicated that the ratio of the oligomeric:monomeric form of the light-harvesting complex II was twofold higher for the dicots (3.16 +/- 0.35) than the monocots (1.64 +/- 0.25) examined under identical separation procedures. Under conditions specifically designed to stabilize the oligomeric form in vitro, we show that the oligomeric form of dicot light-harvesting complex II is twice as stable to solubilization in the presence of sodium dodecyl sulfate (SDS) than that observed for monocots. This decreased stability of monocot light-harvesting complex II is associated with a twofold increase in the trienoic fatty acid level of thylakoid phosphatidylglycerol but with no significant changes in the trienoic fatty acid levels in the major galactolipids. In addition, SDS polyacrylamide gel electrophoresis and western blot analyses with monoclonal antibodies indicated that monocots exhibited greater heterogeneity in the polypeptide complements associated with subfractions of light-harvesting complex II than the dicots examined. The data indicate that the oligomeric form of the light-harvesting complex II is not the result of a simple oligomerization of a common monomeric unit. We suggest that the difference in stability of the oligomeric form of light-harvesting complex II in isolated thylakoids of monocots and dicots is probably due to a differential accessibility to SDS. The differential SDS accessibility may be due to differences in thylakoid protein-protein and/or protein-lipid interactions. PMID- 16669009 TI - Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula. AB - The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7.1) was investigated in leaves and roots from European white birch seedlings (Betula pendula Roth). Induction by nitrate and light of both enzymes was analyzed by in vitro assays and by measuring NR- and NiR-encoding mRNA pools with homologous cDNAs as probes. When birch seedlings were grown on a medium containing ammonium as the sole nitrogen source, low constitutive expression of NR and NiR was observed in leaves, whereas only NiR was significantly expressed in roots. Upon transfer of the seedlings to a nitrate-containing medium, mRNA pools and activities of NR and NiR dramatically increased in leaves and roots, with a more rapid induction in leaves. Peak accumulations of mRNA pools preceded the maximum activities of NR and NiR, suggesting that the appearance of both activities can be mainly attributed to an increased expression of NR and NiR genes. Expression of NR was strictly light dependent in leaves and roots and was repressed by ammonium in roots but not in leaves. In contrast with NR, constitutive expression of NiR was not affected by light, and even a slight induction following the addition of nitrate was found in the dark in roots but not in leaves. No effect of ammonium on NiR expression was detectable in both organs. In leaves as well as in roots, NiR was induced more rapidly than NR, which appears to be a safety measure to prevent nitrite accumulation. PMID- 16669010 TI - Migration of the Fungal Protein Cryptogein within Tobacco Plants. AB - Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea. PMID- 16669011 TI - Purification and characterization of aleurain : a plant thiol protease functionally homologous to Mammalian cathepsin h. AB - Barley (Hordeum vulgare L. cv Himilaya) aleurain is a vacuolar thiol protease originally isolated as a cDNA with 65% derived amino acid sequence identity with cathepsin H (JC Rogers, D Dean, GR Heck [1985] Proc Natl Acad Sci USA 82: 6512 6516). We purified aleurain from barley leaves to homogeneity (>1000-fold) and characterized its activity against a number of substrates. Aleurain is best described as an aminopeptidase; it hydrolyzes three different aminopeptidase substrates with similar catalytic efficiency but is less efficient at hydrolyzing an NH(2)-blocked substrate analog and azocasein. Our values for K(m) and k(cat) for three substrates (arginine 4-methyl-7-coumarylamide, l-arginine beta naphthylamide, and N-alpha-benzoyl-l-arginine beta-naphthylamide) and specific activity with azocasein are all within a threefold range of those previously reported for human cathepsin H for these substrates (WN Schwartz, AJ Barrett [1980] Biochem J 191: 487-497). Aleurain also shows a number of other similarities to cathepsin H including heterogeneity of charge forms, position of the NH(2)-terminus of the mature protein, and pH-activity profile. The similar properties of aleurain and cathepsin H suggest that these enzymes have a similar function(s) that is required by both plant and animal cells. The availability of a plant system may permit functional ablation experiments in the future to clarify the role of this enzyme in higher eukaryotes. PMID- 16669012 TI - Metabolic Response of Maize Roots to Hyperosmotic Shock : An in VivoP Nuclear Magnetic Resonance Study. AB - (31)P nuclear magnetic resonance spectroscopy was used to study the response of maize (Zea mays L.) root tips to hyperosmotic shock. The aim was to identify changes in metabolism that might be relevant to the perception of low soil water potential and the subsequent adaptation of the tissue to these conditions. Osmotic shock was found to result in two different types of response: changes in metabolite levels and changes in intracellular pH. The most notable metabolic changes, which were produced by all the osmotica tested, were increases in phosphocholine and vacuolar phosphate, with a transient increase in cytoplasmic phosphate. It was observed that treatment with ionic and nonionic osmotica produced different effects on the concentrations of bioenergetically important metabolites. It is postulated that these changes are the result of hydrolysis of phosphatidylcholine and other membrane phospholipids, due to differential activation of specific membrane-associated phospholipases by changes in the surface tension of the plasmalemma. These events may be important in the detection of osmotic shock and subsequent acclimatization. A cytoplasmic alkalinization was also observed during hyperosmotic treatment, and this response, which is consistent with the activation of the plasmalemma H(+)-ATPase, together with the other metabolic changes, may suggest the existence of a complex and integrated mechanism of osmoregulation. PMID- 16669013 TI - Properties and partial protein sequence of plant annexins. AB - We have examined the characteristics of Ca(2+)-dependent phospholipid-binding proteins (annexins) in maize (Zea mays L.) coleoptiles and tip-growing pollen tubes of Lilium longiflorum. In maize, there are three such proteins, p35, p33, and p23. Partial sequence analysis reveals that peptides from p35 and p33 have identity to members of the annexin family of animal proteins and to annexins from tomato. Interestingly, multiple sequence alignments reveal that the domain responsible for Ca(2+) binding in animal annexins is not conserved in these plant peptide sequences. Although p33 and p35 share the annexin characteristic of binding to membrane lipid, unlike annexins II and VI they do not associate with detergent-insoluble cytoskeletal proteins or with F-actin from either plants or animals. Immunoblotting with antiserum raised to p33/p35 from maize reveals that cross-reactive polypeptides of 33 to 35 kilodaltons are also present in protein extracts from pollen tubes of L. longiflorum. Immunolocalization at the light microscope level suggests that these proteins are predominantly confined to the nongranular zone at the tube tip, a region rich in secretory vesicles. Our hypothesis that plant annexins mediate exocytotic events is supported by the finding that p23, p33, and p35 bind to these secretory vesicles in a Ca(2+) dependent manner. PMID- 16669014 TI - Peroxidase activity in the leaf elongation zone of tall fescue : I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone. AB - Cessation of cell expansion has been associated with cell wall cross-linking reactions catalyzed by peroxidase. This study utilized two genotypes of tall fescue (Festuca arundinacea Schreb.) that differ in length of the leaf elongation zone to investigate the relationship between ionically bound peroxidase activity and the spatial distribution of leaf elongation. Peroxidase activity was also localized histochemically in transverse sections of the leaf blade using 3,3' diaminobenzidine. Soluble or soluble plus ionically bound peroxidase activities were extracted from homogenized segments of the elongating leaf blade and assayed spectrophotometrically. Activity of the ionically bound fraction, expressed per milligram fresh weight or per microgram protein, increased as cells were displaced through the distal half of the elongation zone, corresponding to the region in which the elongation rate declined. In both genotypes, the initial increase in activity preceded the onset of growth deceleration by about 10 hours. In the basal region where elongation began, histochemical localization showed that peroxidase activity was found only in vascular tissues. As cells were displaced farther through the elongation zone, peroxidase activity appeared in walls of other longitudinally continuous tissues such as the epidermis and bundle sheaths. Increase in ionically bound peroxidase activity and changes in localization of peroxidase activity occurred at comparable developmental stages in the two genotypes. The results indicate that cessation of elongation followed an increase in cell wall peroxidase activity. PMID- 16669015 TI - Peroxidase Activity in the Leaf Elongation Zone of Tall Fescue : II. Spatial Distribution of Apoplastic Peroxidase Activity in Genotypes Differing in Length of the Elongation Zone. AB - Previous work suggested that cell wall peroxidase activity increased as cells were displaced through the elongation zone in leaf blades of tall fescue (Festuca arundinacea Schreb.). In this study, two genotypes that differ in length of the elongation zone were used to examine the relationship between peroxidase activity in apoplastic fluid of intact leaf blade segments and the spatial distribution of leaf growth. Apoplastic fluid was extracted by vacuum infiltration and centrifugation, and peroxidase activity was assayed spectrophotometrically. Isoelectric focusing was used to characterize the isoforms of apoplastic peroxidase within the region of elongation and in the region of secondary cell wall deposition, which is distal to the elongation zone. A striking correlation was found in each genotype between both the location and timing of increase in apoplastic peroxidase activity and the onset of growth deceleration. Only cationic isoforms of apoplastic peroxidase could be identified in the elongation zone, whereas additional anionic isoforms appeared in the region of secondary cell wall deposition. We conclude that cessation of elongation growth in tall fescue leaf blades is likely to be related to the secretion of cationic isoforms of peroxidase into the cell wall. PMID- 16669016 TI - Effects of NaCl and CaCl(2) on Water Transport across Root Cells of Maize (Zea mays L.) Seedlings. AB - The effect of salinity and calcium levels on water flows and on hydraulic parameters of individual cortical cells of excised roots of young maize (Zea mays L. cv Halamish) plants have been measured using the cell pressure probe. Maize seedlings were grown in one-third strength Hoagland solution modified by additions of NaCl and/or extra calcium so that the seedlings received one of four treatments: control; +100 millimolar NaCl; +10 millimolar CaCl(2); +100 millimolar NaCl + 10 millimolar CaCl(2). From the hydrostatic and osmotic relaxations of turgor, the hydraulic conductivity (Lp) and the reflection coefficient (sigma(s)) of cortical cells of different root layers were determined. Mean Lp values in the different layers (first to third, fourth to sixth, seventh to ninth) of the four different treatments ranged from 11.8 to 14.5 (Control), 2.5 to 3.8 (+NaCl), 6.9 to 8.7 (+CaCl(2)), and 6.6 to 7.2 . 10( 7) meter per second per megapascal (+NaCl + CaCl(2)). These results indicate that salinization of the growth media at regular calcium levels (0.5 millimolar) decreased Lp significantly (three to six times). The addition of extra calcium (10 millimolar) to the salinized media produced compensating effects. Mean cell sigma(s) values of NaCl ranged from 1.08 to 1.16, 1.15 to 1.22, 0.94 to 1.00, and 1.32 to 1.46 in different root cell layers of the four different treatments, respectively. Some of these sigma(s) values were probably overestimated due to an underestimation of the elastic modulus of cells, sigma(s) values of close to unity were in line with the fact that root cell membranes were practically not permeable to NaCl. However, the root cylinder exhibited some permeability to NaCl as was demonstrated by the root pressure probe measurements that resulted in sigma(sr) of less than unity. Compared with the controls, salinity and calcium increased the root cell diameter. Salinized seedlings grown at regular calcium levels resulted in shorter cell length compared with control (by a factor of 2). The results demonstrate that NaCl has adverse effects on water transport parameters of root cells. Extra calcium could, in part, compensate for these effects. The data suggest a considerable apoplasmic water flow in the root cortex. However, the cell-to-cell path also contributed to the overall water transport in maize roots and appeared to be responsible for the decrease in root hydraulic conductivity reported earlier (Azaizeh H, Steudle E [1991] Plant Physiol 97: 1136-1145). Accordingly, the effect of high salinity on the cell Lp was much larger than that on root Lp(r). PMID- 16669017 TI - Repression of the Plastidic Isoenzymes of Aldolase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase in the Barley Mutant "albostrians". AB - White leaves of the mutant line albostrians and green leaves of the wild-type cultivar Salome of barley (Hordeum vulgare L.) were screened for the presence of plastidic and cytosolic isoenzymes of sugar-phosphate metabolism. Isoenzyme separation was achieved by anion-exchange chromatography on Fractogel TSK DEAE 650(S). The mutant tissue had a markedly reduced level of plastidic 3 phosphoglycerate kinase, triosephosphate isomerase, and aldolase activity. In contrast, the activity of plastidic glucosephosphate isomerase, fructose 1,6 bisphosphatase, 6-phosphogluconate dehydrogenase, starch phosphorylase, and ADP glucose pyrophosphorylase was in the same range as in wild-type leaf tissue. The activity of the corresponding cytosolic isoenzymes (including UDP-glucose pyrophosphorylase) showed essentially no differences in mutant and wild type. The same trend was observed in dark-grown mutant and wild-type leaves. Interestingly, the total activity levels of all isoenzymes were about the same when comparing dark-grown and light-grown mutant or wild-type plants. From these data, it is concluded that mutant leaves exhibit a selective decrease of a subgroup of plastidic isoenzymes associated with the Calvin cycle. PMID- 16669018 TI - Organization and topology of photosystem I subunits. AB - Intact spinach (Spinacia oleracea) thylakoid membranes were treated with various proteases and photosystem I (PSI) complexes were isolated from these membranes to define the membrane topology of specific PSI subunits. Trypsin treatment caused cleavage of the PSI-D and E subunits. Thermolysin treatment cleaved the PSI-D, E, H, and K subunits, and also caused limited degradation of the reaction center core PSI-A and B subunits. Pronase treatment produced the most dramatic results as the PSI-A and B subunits were cleaved to 47-, 45-, 26-, and 24-kilodalton products. In addition, pronase degraded the PSI-D, E, H, K, and L subunits. Proteolytic cleavage sites for several of the products were identified by amino acid sequencing. The results indicate that PSI-A, B, D, E, H, K, and L subunits all have stroma-exposed regions, and these findings are summarized in a model describing the subunit organization of PSI. PMID- 16669019 TI - Effects of temperature on the phase behavior and permeability of thylakoid lipid vesicles : relevance to chilling stress. AB - Large unilamellar vesicles composed of thylakoid glycolipids, phosphatidylglycerol, and varying proportions of dipalmitoylphosphatidylglycerol (DPPG) have been examined for the temperature dependence of their permeability to (86)Rb(+) and for the occurrence of liquid-crystalline to gel (L(alpha)-to L(beta)) phase separations. In vesicles in which the normal 12 mole percent of moderately unsaturated thylakoid phosphatidylglycerol was partially or completely replaced by DPPG, analysis by differential scanning calorimetry indicated that an L(alpha)-to-L(beta) phase separation did not occur between 0 and 60 degrees C. However, in similar vesicle dispersions that were first subjected to a freeze thaw cycle, L(alpha)-to-L(beta) phase separations were observed to occur between 17 and 53 degrees C. The temperature and enthalpy of these phase separations were closely related to the proportion of DPPG in the original lipid mixture. In parallel experiments, large unilamellar vesicles were measured for their permeability to (86)Rb(+) between 7 and 30 degrees C. There were no systematic increases in permeability to (86)Rb(+) as a function of DPPG content at the temperatures relevant to chilling stress in higher plants. It is concluded that (a) L(alpha)-to-L(beta) phase separations do not occur in well-defined galactolipid vesicles containing 50%). On the other hand, spinach (Spinacia oleracea L.), a typical sucrose storer (up to 160 milligrams of sucrose per square decimeter in girdled leaves), showed only a slight reduction in CER and almost no change in A(max). Intermediate plants like tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuus L.), broad bean (Vicia faba L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.), which upon girdling store both starch and sucrose, responded to the girdle by a considerable reduction in CER but only moderate inhibition of A(max), indicating that the observed reduction in CER was primarily a stomatal response. Both the wild-type tobacco (Nicotiana sylvestris) (which upon girdling stored starch and hexoses) and the starchless mutant (which stored only hexoses, up to 90 milligrams per square decimeter) showed 90 to 100% inhibition of CER and approximately 50% inhibition of A(max). In general, excised leaves (6 days) behaved like girdled leaves of the respective species, showing 50% reduction of A(max) in wild-type and starchless N. sylvestris but only slight decline of A(max) in spinach. The results of the present study demonstrate the possibility of the occurrence of end-product inhibition of photosynthesis in a large number of crop plants. The long-term inhibition of photosynthesis in girdled leaves is not confined to stomatal responses since the A(max) declined up to 50%. The inhibition of A(max) by girdling was strongest in starch storers, but starch itself cannot be directly responsible, because the starchless mutant of N. sylvestris was also strongly inhibited. Similarly, the inhibition cannot be attributed to hexose sugars either, because soybean, cotton, and cucumber are among the plants most strongly inhibited although they do not maintain a large hexose pool. Spinach, a sucrose storer, showed the least inhibition in both girdled and excised leaf systems, which indicates that sucrose is probably not directly responsible for the end product inhibition of photosynthesis. The occurrence of strong end-product inhibition appears to be correlated with high acid-invertase activity in fully expanded leaves. The inhibition may be related to the nature of soluble sugar metabolism in the extrachloroplastic compartment and may be caused by a metabolite that has different rates of accumulation and turnover in sucrose storers and other plants. PMID- 16669057 TI - Carbon Partitioning and Growth of a Starchless Mutant of Nicotiana sylvestris. AB - We have further characterized the photosynthetic carbohydrate metabolism and growth of a starchless mutant (NS 458) of Nicotiana sylvestris that is deficient in plastid phosphoglucomutase (Hanson KR, McHale NA [1988] Plant Physiol 88: 838 844). In general, the mutant had only slightly lower rates of photosynthesis under ambient conditions than the wild type. However, accumulation of soluble sugars (primarily hexose sugars) in source leaves of the mutant compensated for only about half of the carbon stored as starch in the wild type. Therefore, the export rate was slightly higher in the mutant relative to the wild type. Starch in the wild type and soluble sugars in the mutant were used to support plant growth at night. Growth of the mutant was progressively restricted, relative to wild type, when plants were grown under shortened photoperiods. When grown under short days, leaf expansion of the mutant was greater during the day, but was restricted at night relative to wild-type leaves, which expanded primarily at night. We postulate that restricted growth of the mutant on short days is the result of several factors, including slightly lower net photosynthesis and inability to synthesize starch in both source and sink tissues for use at night. In short-term experiments, increased "sink demand" on a source leaf (by shading all other source leaves) had no immediate effect on starch accumulation during the photoperiod in the wild type or on soluble sugar accumulation in the mutant. These results would be consistent with a transport limitation in N. sylvestris such that not all of the additional carbon flux into sucrose in the mutant can be exported from the leaf. Consequently, the mutant accumulates hexose sugars during the photoperiod, apparently as the result of sucrose hydrolysis within the vacuole by acid invertase. PMID- 16669058 TI - Molecular and physiological analysis of a heat-shock response in wheat. AB - We have isolated two cDNA clones from wheat (Triticum aestivum L. var Stephens), designated WHSP16.8 and WHSP16.9, that are highly similar in sequence to the low molecular weight heat-shock protein genes previously isolated from soybean. RNA blot analysis confirms that these sequences are present in heat-shocked wheat seedlings, but not in control tissues. The WHSP16.8 and WHSP16.9 cDNAs were isolated by screening a lambda gt11 expression library with antibodies to HMGc (a chromosomal protein of wheat). Immunoblot analysis has demonstrated that the antibodies raised against HMGc also recognize a group of proteins that are induced by heat shock and have molecular weights (estimated by sodium dodecyl sulfate electrophoresis) consistent with the molecular weights of the proteins deduced from the sequences of the cDNAs. PMID- 16669059 TI - Interactive effects of Al, h, and other cations on root elongation considered in terms of cell-surface electrical potential. AB - The rhizotoxicities of Al(3+) and of La(3+) to wheat (Triticum aestivum L.) were similarly ameliorated by cations in the following order of effectiveness: H(+) approximately C(3+) > C(2+) > C(1+). Among tested cations of a given charge, ameliorative effectiveness was similar except that Ca(2+) was slightly more effective than other divalent cations and H(+) was much more effective than other monovalent cations. H(+) rhizotoxicity was also ameliorated by cations in the order C(3+) > C(2+) > C(1+). These results suggest a role for cell-surface electrical potential in the rhizotoxicity of Al(3+), La(3+), H(+), and other toxic cations: negatively charged cell surfaces of the root accumulate the toxic cations, and amelioration is effected by treatments that reduce the negativity of the cell-surface electrical potential by charge screening or cation binding. Membrane-surface activities of free Al(3+) or La(3+) computed according to a Gouy Chapman-Stern model correlated well with growth inhibition, which correlated only poorly with Al(3+) or La(3+) activities in the external medium. The similar responses of Al-intoxicated and La-intoxicated roots to ameliorative treatments provide evidence that Al(3+), rather than AlOH(2+) or Al(OH)(2) (+), is the principal toxic species of mononuclear Al. Comparisons of the responses of Al sensitive and Al-tolerant wheats to Al(3+) and to La(3+) did not support the hypothesis that varietal sensitivity to Al(3+) is based upon differences in cell surface electrical potential. PMID- 16669060 TI - Wheat vegetative nitrogen compositional changes in response to reduced reproductive sink strength. AB - N redistribution patterns and the N composition of vegetative tissues above the peduncle node of wheat (Triticum aestivum L.) plants with altered reproductive sink strength were evaluated to determine the role of vegetative storage proteins in the temporary storage of excess N destined for export. The degree of leaf senescence symptoms (loss of chlorophyll, total N, and ribulose-1,5-bisphosphate carboxylase/oxygenase) were initially reduced, but the complete senescence of vegetative tissues proceeded even for plants completely lacking reproductive sinks. Plants with 50% less sink strength than control plants with intact spikes redistributed vegetative N to the spike almost as effectively as the control plants. Plants without reproductive sinks exported less N from the flag leaf and had flag leaf blades and peduncle tissues with higher soluble protein and alpha NH(2) amino acid levels than control plants. An abundant accumulation of polypeptides in the soluble protein profiles of vegetative tissues was not evident in plants with reduced sink strength. Storage of amino acids apparently accommodates any excess N accumulated by vegetative tissues during tissue reproductive growth. Any significant role of vegetative storage proteins in the N economy of wheat is unlikely. PMID- 16669061 TI - Are phytochelatins involved in differential metal tolerance or do they merely reflect metal-imposed strain? AB - Plants from nontolerant and copper-tolerant populations of Silene vulgaris both produce phytochelatins upon exposure to copper. The threshold copper concentration for induction of phytochelatin and the copper concentration at which maximum phytochelatin contents occurs increase proportionally with the level of tolerance to copper. When exposed to their own highest no-effect concentration or 50%-effect concentration of copper for root growth, tolerant and nontolerant plants exhibit equal phytochelatin contents in the root apex, which is the primary copper target. This also holds for distinctly tolerant nonsegregating F(3) families, derived from a single cross of a nontolerant plant to a tolerant one. Therefore, the phytochelatin content of the root apex can be used as a quantitative tolerance-independent measure of the degree of toxicity experienced by the plant. Differential copper tolerance in S. vulgaris does not appear to rely on differential phytochelatin production. PMID- 16669062 TI - Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. AB - Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants. PMID- 16669063 TI - Substrate specificity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato tuber. AB - The aim of this work was to establish the precise ionic form of the reactants used by pyrophosphate:fructose-6-phosphate phosphotransferase. The enzyme was purified to near-homogeneity from potato (Solanum tuberosum L.) tubers. Changes in enzyme activity when the pH of the assay and the concentration of fructose 6 phosphate, pyrophosphate, and magnesium are varied independently indicate that fructose 6-phosphate(2-) and MgP(2)O(7) (2-) are the reacting species in the glycolytic direction. Analogous experiments with fructose 1,6-bisphosphate, inorganic phosphate, and magnesium demonstrate that the enzyme uses fructose 1,6 bisphosphate(4-), HPO(4) (2-), and Mg(2+) in the gluconeogenic direction. The ionic species used in the glycolytic direction are comparable with those required by bacterial ATP-dependent phosphofructokinase. This is consistent with the proposal that the active site of pyrophosphate:fructose-6-phosphate phosphotransferase in plants is equivalent to that of the bacterial phosphofructokinase (SM Carlisle et al. [1990] J Biol Chem 265: 18366-18371). PMID- 16669064 TI - Cloning and Characterization of an Arabidopsis thaliana Topoisomerase I Gene. AB - cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana lambdagt11 and lambdaFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOP1 probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody. PMID- 16669065 TI - Stomatal and nonstomatal limitations to net photosynthesis in seedlings of woody angiosperms. AB - Comparative responses of net photosynthesis (A) to water stress in woody species from a variety of habitats were studied to assess the relationship between photosynthetic attributes and drought tolerance. Stomatal and nonstomatal limitations to A were compared in three-month-old white oak (Quercus alba L.), post oak (Quercus stellata Wangenh.), sugar maple (Acer saccharum Marsh.), and black walnut (Juglans nigra L.) seedlings during a drying cycle. Relative stomatal limitation of photosynthesis (I) was less than 50% in all species except for Q. stellata seedlings subjected to severe water stress. No significant changes in I were observed in Q. alba and J. nigra before, during, and after drought. In A. saccharum, I was generally low and decreased significantly under water stress. Under well-watered conditions, A was highest in Q. stellata, intermediate in Q. alba, and lower in A. saccharum and J. nigra. High A in well watered Q. stellata was associated with high stomatal conductance and carboxylation efficiency, whereas low A was associated with low stomatal conductance and carboxylation efficiency in A. saccharum and low stomatal conductance, low carboxylation efficiency, and high CO(2) compensation point in J. nigra. Under severe water stress, A, carboxylation efficiency, and stomatal conductance decreased substantially in all species; however, Q. stellata had the highest carboxylation efficiency and lowest CO(2) compensation point under these conditions. After 5 days at high soil moisture after drought, stomatal and mesophyll components of A in A. saccharum and J. nigra had not recovered to predrought levels, whereas they had completely recovered in Q. stellata and Q. alba. The photosynthetic apparatus, especially mesophyll components, of drought tolerant Quercus species showed either less inhibition under water stress, superior recovery to predrought capacity, or both. Exposure of the leaves to (14)CO(2) indicated apparent asymmetric stomatal closure for mildly water stressed seedlings, but not for leaves of well-watered, severely stressed, or rehydrated plants. These results suggest that patchy stomatal closure under mild water stress might be important for water stress-induced inhibition of photosynthesis, but not under the more severe water stress imposed in this study. PMID- 16669066 TI - Immunofluorescent Localization of Plasma Membrane H-ATPase in Barley Roots and Effects of K Nutrition. AB - The plasma membrane H(+)-ATPase (PM-H(+)-ATPase) of barley (Hordeum vulgare L. cv Klondike) roots was assayed by cross-reaction on western blots and cryosections with an antibody against the PM-H(+)-ATPase from corn roots. Under conditions of reduced K availability, which have previously been shown to increase K influx by greater than 25-fold, there were only minor changes detected in PM-H(+)-ATPase levels. Antibody labeling of cryosections showed the relative distribution of PM H(+)-ATPase among cell types in root tips and mature roots. Epidermal cells, both protoderm and mature root epidermis, including root hairs, had high levels of antibody binding. In mature roots, the stelar tissue showing the highest antibody binding was the companion cells of the phloem, followed by pericycle, xylem parenchyma, and endodermis. PMID- 16669067 TI - Dry pea seed proteasome : purification and enzymic activities. AB - Proteasomes were isolated from mature, dry pea seeds (Pisum sativum L.). They appear to be similar to proteasomes from other sources in that they are cylindrical (shown by negative staining), have a molecular mass greater than 600 kilodaltons (by gel permeation chromatography), and consist of several subunits between 25 and 31 kilodaltons. The seed proteasomes possess three characteristic partial activities (trypsin-like, chymotrypsin-like, and peptidyl glutamyl peptidase) as determined with fluorogenic peptide substrates. Activation and inhibition by various effectors, and particularly sensitivity to porphyrins, also match characteristics of proteasomes described for other organisms. The potential role of the proteasome in seed biology is discussed. PMID- 16669068 TI - Purification and characterization of an anaerobically induced alanine aminotransferase from barley roots. AB - Alanine aminotransferase (AlaAT, EC 2.6.1.2) is an enzyme that is induced under anaerobic conditions in cereal roots. In barley (Hordeum vulgare L.) roots, there are a number of isoforms of AlaAT. We have identified the anaerobically induced isoform and have purified it to homogeneity. The isolation procedure involved a two-step ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, and chromatofocusing. The enzyme was purified approximately 350 fold to a specific activity of 2231 units/milligram protein. The apparent molecular masses of the native and sodium dodecyl sulfate-denatured AlaAT proteins are 97 and 50 kilodaltons, respectively, indicating that the native enzyme is probably a homodimer. AlaAT has a number of interesting characteristics when compared with other plant aminotransferases. AlaAT does not require the presence of pyridoxyl-5-phosphate to retain its activity, and it appears to be very specific in the reactions that it will catalyze. PMID- 16669069 TI - Trigonelline and Stachydrine Released from Alfalfa Seeds Activate NodD2 Protein in Rhizobium meliloti. AB - Spectroscopic data (nuclear magnetic resonance, mass spectrometry, ultraviolet visible) in this study identify trigonelline and stachydrine as major components of alfalfa (Medicago sativa L.) seed rinse. Moreover, biological assays show that these natural products induce nodulation (nod) gene transcription in Rhizobium meliloti by activating the regulatory protein NodD2, but not the homologous NodD1 protein. These findings contrast with the fact that the only previously identified NodD2 activator, 4,4' -dihydroxy-2' -methoxychalcone (MCh), also activates NodD1 protein. Trigonelline and stachydrine induce nod genes only at much higher concentrations than MCh, but they are released from seeds in correspondingly greater amounts. The existence of these amphoteric, nonflavonoid nod gene inducers broadens our understanding of the biochemical processes and ecological mechanisms that a legume host uses to regulate its microbial symbiont. PMID- 16669070 TI - Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase. AB - Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K(+) resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K(+) following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K(+) ions. These results suggest that K(+) channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K(+) -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H(+) -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K(+) channels. PMID- 16669071 TI - Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize. AB - Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew (14)C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. (14)C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development. PMID- 16669072 TI - Identification and levels of 2'-carboxyarabinitol in leaves. AB - 2'-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C(3), C(4), and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants. PMID- 16669073 TI - Metabolism of 2'-carboxyarabinitol in leaves. AB - Results presented here indicate that 2'-carboxyarabinitol (CA) is the in vivo precursor and product of 2'-carboxyarabinitol 1-phosphate (CA1P) metabolism in leaves. When [2-(14)C]CA was fed in the light to leaves of five species known to be highly active in CA1P metabolism (Phaseolus vulgaris, Lycopersicon esculentum, Helianthus annuus, Petunia hybrida, and Beta vulgaris), [(14)C]CA1P was formed in the dark. Reillumination of a Phaseolus leaf caused this [(14)C]CA1P to be rapidly metabolized to [(14)C]CA (t((1/2)) = 1 min). The epimer 2'-carboxyribitol could not substitute for CA in the dark synthesis of CA1P, and CA in the anionic form was a better substrate than CA in the lactone form. In leaves of Phaseolus vulgaris, the active CA pool size used in the dark synthesis of CA1P is between about 70 and 110 nanomoles per milligram of chlorophyll. The photosynthetic electron transport inhibitor diuron did not affect the dark synthesis of [(14)C]CA1P, but did greatly reduce the rate of its subsequent light degradation (t((1/2)) = approximately 10 min). Dark synthesis of [(14)C]CA1P was inhibited by dithiothreitol and NaF. From the present data, we suggest that CA1P and CA participate in a metabolic substrate cycle in vivo. PMID- 16669074 TI - Cold Acclimation in Genetically Related (Sibling) Deciduous and Evergreen Peach (Prunus persica [L.] Batsch): I. Seasonal Changes in Cold Hardiness and Polypeptides of Bark and Xylem Tissues. AB - Seasonal patterns of proteins and of cold hardiness were characterized in bark and xylem tissues of genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). In contrast with deciduous trees, which entered endodormancy and abscised leaves in the fall, evergreen trees retained their leaves and exhibited shoot elongation under favorable environmental conditions. A successive increase in the cold hardiness of bark and xylem was observed during the fall in both genotypes. This was followed by a subsequent decrease from midwinter to spring. Xylem tissue in both genotypes exhibited deep supercooling and a significant correlation (r = 0.99) between the midpoint of the low temperature exotherm and the subzero temperature at which 50% injury occurred (assessed by electrolyte leakage) was noted. The maximum hardiness level attained in deciduous trees was more than twofold that of evergreens. Seasonal pattern of proteins from bark and xylem of the sibling genotypes was characterized by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among other qualitative and quantitative changes, accumulation of a 19-kilodalton polypeptide in the bark of both genotypes was observed during fall followed by a decrease in spring. This polypeptide accumulated to higher levels in the deciduous peach compared with the evergreen. Additionally, a 16-kilodalton protein exhibited the same pattern in deciduous trees but not in the evergreen trees. Both the 19- and a 16-kilodalton bark proteins conform to the criteria of a bark storage protein. The relationship of seasonal changes in protein to cold hardiness and dormancy in these genetically related peach genotypes is discussed. PMID- 16669075 TI - A Single Genetic Locus, Ckr1, Defines Arabidopsis Mutants in which Root Growth Is Resistant to Low Concentrations of Cytokinin. AB - Arabidopsis mutants resistant to cytokinin (benzyladenine [BA]) have been isolated with the intent to find plants defective in cytokinin perception or response. At low concentrations, BA produces a "cytokinin root syndrome" in which primary root elongation is inhibited, but root hair elongation is stimulated. Five independent mutants that did not express this syndrome in the presence of BA were selected. All five mutants were recessive, and crosses between them indicated that they were in the same complementation group. The genetic locus represented by these mutations has been designated ckr1 and mapped to chromosome 5. PMID- 16669076 TI - Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins. AB - Satsuma (Citrus unshiu [Mak] Marc.) and Clementine (Citrus reticulata [Hort.] Ex. Tanaka, cv Oroval) are two species of seedless mandarins differing in their tendency to develop parthenocarpic fruits. Satsuma is a male-sterile cultivar that shows a high degree of natural parthenocarpy and a high fruit set. Seedless Clementine varieties are self-incompatible, and in the absence of cross pollination show a very low ability to set fruit. The gibberellins (GAs) GA53, putative 17-OH-GA(53), GA(44), GA(17), GA(19), GA(20), GA(29), GA(1), 3-epi GA(1), GA(8), GA(24), GA(9), and GA(4) have been identified from developing fruits of both species by full-scan combined gas chromatography-mass spectrometry. Using selected ion monitoring with [(2)H(2)]- and [(13)C]-labeled internal standards, the levels of GA(53), GA(44), GA(19), GA(20), GA(1), GA(8), GA(4), and GA(9) were determined in developing ovaries at anthesis and 7 days before and after anthesis, from both species. Except for GA8, levels of the 13 hydroxy-GAs were higher in Satsuma than in Clementine, and these differences were more prominent for developing young fruits. At petal fall, Satsuma had, on a nanograms per gram dry weight basis, higher levels of GA(53) (10.4x), GA(44) (13.9x), GA(19) (3.0x), GA(20) (11.2x), and GA(1) (2.0x). By contrast, levels of GA(8) were always higher in Clementine, whereas levels of GA(4) did not differ greatly. Levels of GA(9) were very low in both species. At petal fall, fruitlets of Satsuma and Clementine contained 65 and 13 picograms of GA(1), respectively. At this time, the application of 25 micrograms of paclobutrazol to fruits increased fruit abscission in both varieties. This effect was reversed by the simultaneous applications of 1 microgram of GA(3). GA(3) alone improved the set in Clementine (13x), but had little influence on Satsuma. Thus, seedless fruits of the self-incompatible Clementine mandarin may not have adequate GA levels for fruit set. Collectively, these results suggest that endogenous GA content in developing ovaries is the limiting factor controlling the parthenocarpic development of the fruits. PMID- 16669077 TI - Studies of the uptake of nitrate in barley : v. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity-implications for nitrate influx. AB - The cytoplasmic NO(3) (-) concentration ([NO(3) (-)](c)) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO(3) (-) concentration ([NO(3) (-)](o)), [NO(3) (-)](c) was estimated to be 0.66 mm for plants previously grown in 100 mum NO(3) (-). It increased linearly with [NO(3) (-)](o) between 2 and 20 mm, up to 3.9 mm at 20 mm [NO(3) (-)](o). The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO(3) (-)](c) for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO(3) (-)](c) and cell membrane potentials of -200 to -300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO(3) (-) is possible via the high-concentration, low-affinity transport system for NO(3) ( ). This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO(3) (-) against its electrochemical potential gradient. PMID- 16669078 TI - Distribution of Cytosolic mRNAs Between Polysomal and Ribonucleoprotein Complex Fractions in Alfalfa Embryos : Stage-Specific Translational Repression of Storage Protein Synthesis during Early Somatic Embryo Development. AB - Cell-free translational and northern blot analyses were used to examine the distribution of storage protein messages in the cytoplasmic polysomal and mRNA protein complex (mRNP) fractions during development of somatic and zygotic embryos of alfalfa (Medicago sativa cv Rangelander RL-34). No special array of messages was identified in the mRNP fraction; however, some messages were selectively enriched in either the polysome or mRNP fractions, and their distribution pattern varied quantitatively during development of the embryos. During the earliest stages of somatic embryo development, storage protein messages already were present, but there was no detectable accumulation of the proteins. Selective enrichment of messages for the 11S, 7S, and 2S storage proteins occurred in the mRNP fraction during the globular, heart, and torpedo stages of somatic embryogenesis, but the distribution pattern was shifted toward the polysomal fraction at the beginning of cotyledon development. Thus, there was translational repression of storage protein synthesis at the early stage of somatic embryo development that was relieved later. During the cotyledonary development stages in the somatic and zygotic embryos, storage protein synthesis and distribution of the messages were similar in that these specific messages were predominantly in the polysomal fraction. PMID- 16669079 TI - Purification and Characterization of Glutamate 1-Semialdehyde Aminotransferase from Barley Expressed in Escherichia coli. AB - The immediate precursor in the synthesis of tetrapyrroles is Delta aminolevulinate (ALA). ALA is synthesized from glutamate in higher plants, algae, and certain bacteria. Glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) (GSA AT), the third enzyme involved in this metabolic pathway, catalyzes the transamination of GSA to form ALA. The gene encoding this aminotransferase has previously been isolated from barley (Hordeum vulgare) and inserted into an Escherichia coli expression vector. We describe herein the purification of this recombinant barley GSA-AT expressed in Escherichia coli. Coexpression of GroEL and GroES is required for isolation of active aminotransferase from the soluble protein fraction of Escherichia coli. Purified GSA-AT exhibits absorption maxima characteristic of vitamin B(6)-containing enzymes. GSA-AT is primarily in the pyridoxamine form when isolated and can be interconverted between this and the pyridoxal form by addition of 4,5-dioxovalerate and 4,5-diaminovalerate. The conversion of GSA to ALA under steady-state conditions exhibited typical Michaelis-Menten kinetics. Values for K(m) (d,l-GSA) and k(cat) were determined to be 25 micromolar and 0.11 per second, respectively, by nonlinear regression analysis. Stimulation of ALA synthesis by increasing concentrations of d,l-GSA at various fixed concentrations of 4,5-diaminovalerate supports the hypothesis that 4,5-diaminovalerate is the intermediate in the synthesis of ALA. PMID- 16669080 TI - Identification and Characterization of the ictA/ndhL Gene Product Essential to Inorganic Carbon Transport of Synechocystis PCC6803. AB - The ictA gene, renamed ndhL in this paper, essential to inorganic carbon transport of Synechocystis PCC6803, was expressed in Eschericia coli as a fusion protein with glutathione S-transferase. An antibody was raised against this fusion protein. Western analysis of the thylakoid membrane of wild-type (WT) Synechocystis revealed that a protein with an apparent molecular mass of 6.7 kilodaltons cross-reacted with this antibody. No immunoreactive protein was present in the thylakoid membranes of the Synechocystis mutants, RKb and M9, which have defects in the ictA/ndhL gene, or in the cytoplasmic membranes of the WT and mutant cells. Thus, the protein reacted with the antibody is the ictA gene product (IctA) and is localized in the thylakoid membrane of WT cells. IctA was absent in the thylakoid membranes of the M55 mutant, in which the ndhB gene is inactivated, and was poorly immunostained in the membranes of the mutants (M-ndhC and M-ndhK) constructed by inactivating the ndhC and ndhK genes of WT Synechocystis, respectively. The carbon dioxide uptake activity was nearly zero in M-ndhK and was about 40% of the activity of WT cells in M-ndhC. The RKb, M ndhC, and M-ndhK mutants were unable to grow or grew very slowly under photoheterotrophic conditions. These results indicated that NADH dehydrogenase is essential to inorganic carbon transport and photoheterotrophic growth of Synechocystis and that IctA is one of the subunits of this enzyme. PMID- 16669081 TI - Biosynthesis of Acyl Lipids Containing Very-Long Chain Fatty Acids in Microspore Derived and Zygotic Embryos of Brassica napus L. cv Reston. AB - Biosynthesis of very long chain (>C(18)) fatty acids (VLCFAs) and the pathway for their incorporation into acyl lipids was studied in microspore-derived (MD) and zygotic embryos of Brassica napus L. cv Reston. In the presence of [1 (14)C]oleoyl-coenzyme A or [1-(14)C] eicosenoyl-coenzyme A, malonyl-coenzyme A, and reducing equivalents, maximal in vitro elongation activity was expressed in protein preparations from early-mid cotyledonary stage MD embryos (17-20 days in culture), when endogenous eicosenoic (20:1) and erucic (22:1) acids were just beginning to accumulate (approximately 1.5 milligrams per gram dry weight). The biosynthesis of VLCFAs and their incorporation into glycerolipids in vitro in the MD embryo system occurred at rates comparable to those measured in developing zygotic Reston embryos at about 20 days postanthesis. When glycerol-3-phosphate was supplied as acyl acceptor in time-course experiments using homogenates prepared from 18-day MD embryos, newly synthesized [(14)C]20:1 and [(14)C]22:1 were incorporated primarily into triacylglycerols (TAGs) and, to a lesser extent, into lyso-phosphatidic/phosphatidic acids, diacylglycerols, and phosphatidylcholines as well as the acyl-coenzyme A and free fatty acid pools. [(14)C]24:1 was not detected in any acyl lipid. Stereospecific analyses of the radiolabeled TAGs indicated that [(14)C]20:1 and [(14)C]22:1 moieties were esterified predominantly at the sn-3 position, but were also found at the sn-1 position. [(14)C]20:1, but not [(14)C]22:1, was detected at the sn-2 position. Similar patterns of (14)C-labeled VLCFA distribution were obtained in experiments conducted using a 15,000g pellet fraction from 18-day MD embryos. All trends observed in the formation of TAGs containing VLCFAs in the Reston MD embryo system were also confirmed in studies of zygotic embryos of the same cultivar. The data support the biosynthesis of 20:1 and then 22:1 via successive condensations of malonyl-coenzyme A with oleoyl-coenzyme A and, for the first time in B. napus, demonstrate the incorporation of newly synthesized VLCFAs into TAGs via the Kennedy pathway. PMID- 16669082 TI - Aberrant processing of polyphenol oxidase in a variegated grapevine mutant. AB - Bruce's Sport is a mutant grapevine (Vitis vinifera L.) with green and white variegated fruit derived from the Sultana variety. The white regions of tissue have decreased polyphenol oxidase (PPO) activity resulting in a reduced capacity for browning. Active PPO from Sultana grapes was purified and had an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots indicated that mature Sultana grapes contained a single 40-kilodalton PPO, and young Sultana berries also had small quantities of a 60-kilodalton protein. Bruce's Sport grapes had much less of the 40-kilodalton PPO and greater amounts of the 60-kilodalton band. Protease digestion of Bruce's Sport extracts decreased the proportion of the 60-kilodalton protein and increased the 40-kilodalton band. A cDNA clone of grape PPO was used to probe a northern blot of Sultana and Bruce's Sport RNA and hybridized to a 2.2-kilobase transcript in both grapevines. The level of PPO mRNA was high in the early stages of berry development but then declined. The results suggest that in grapevine the active 40-kilodalton form of PPO is synthesized as a precursor protein of at least 60 kilodaltons, and normal processing is interrupted in Bruce's Sport resulting in the accumulation of the 60-kilodalton inactive preform of PPO. PMID- 16669083 TI - The rb Mutation of Peas Causes Structural and Regulatory Changes in ADP Glucose Pyrophosphorylase from Developing Embryos. AB - A mutation at the rb locus of pea (Pisum sativum L.) alters the shape, reduces the starch content, and increases the lipid and sucrose contents of the seed. These effects are probably all consequences of a reduction of up to 40-fold in the maximum catalytic activity of ADP glucose pyrophosphorylase in the developing embryo of the mutant relative to the wild type. We have investigated how the mutation brings about this reduction in activity. The purified enzyme from mutant embryos has a specific activity about 10-fold lower than that from wild-type embryos, and it is much more sensitive to the effectors inorganic phosphate and 3 phosphoglycerate than the wild-type enzyme. Both wild-type and mutant enzymes consist of polypeptides of around 50 kilodaltons. One of the polypeptides of the purified wild-type enzyme is missing from the mutant enzyme. We deduce that in the wild-type embryo this protein may interact with other subunits to confer a high specific activity and a low susceptibility to effectors on the enzyme. PMID- 16669085 TI - Subcellular location of delta-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean. AB - The expression of Delta(1)-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated (AJ Delauney, DPS Verma [1990] Mol Gen Genet 221: 299-305). P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCl). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate(+) inhibited P5CR activity, whereas nicotinamide adenine dinucleotide(+) did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production. PMID- 16669084 TI - Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor. AB - The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H(+) transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one- and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.). PMID- 16669086 TI - Effects and Absorption of Sethoxydim in Cell Cycle Progression of Corn (Zea mays) and Pea (Pisum sativum). AB - The mechanisms of selective herbicidal action of sethoxydim were investigated by using cultured root tips of corn (Zea mays L. cv Goldencrossbantam) and pea (Pisum sativum L. cv Alaska). Meristematic cells in the cultured roots were arrested in G(1) and G(2) of the cell division cycle by sucrose starvation and resumed growth and cell division (proliferation) when sucrose was provided. Corn root growth after sucrose addition was inhibited by sethoxydim at concentrations of 0.01 micromolar and greater when roots were treated in the presence of sucrose but was not inhibited at 10 micromolar sethoxydim when they were treated during sucrose starvation. Greater absorption of [(14)C]sethoxydim into the meristematic region of corn roots was observed when cells were in proliferative condition but not when they were arrested by sucrose starvation, whereas no greater absorption of the herbicide into pea meristems was observed in either growth condition. In the cell cycle study, greater absorption of [(14)C]sethoxydim into the corn root meristem was observed at a certain limited time before S (DNA synthesis) stage. The physiological effects and the greater absorption of sethoxydim clearly depended on cell cycle progression of corn root meristem, whereas fatty acid synthesis, as well as its inhibition by sethoxydim, was not associated with either cell cycle progression or greater absorption of the herbicide. PMID- 16669087 TI - A cold environment is a prerequisite for formation of "plastid initials" in winter buds of poplar. AB - The "plastid initial," the presumed precursor of eoplasts and proplastids, is present in the cells of the apical meristem of winter buds of poplar (Populus euramericana). The formation of the plastid initial in the cells of winter buds is initiated soon after the breaking of the innate or resting stage of dormancy, even in winter under nongrowing conditions in mid-January or early February. From this stage to March, the conglomeration of the plastid initial and the formation of prolamellar bodies is evident. In contrast to the poplar samples kept outdoors, the cells of the apical meristem of the indoor winter buds did not show any indication of the formation of the plastid initial and the buds of the latter sample did not flush even at the end of May. These results suggest that the sequence of reactions involved in the onset of regrowth may be closely related to the formation of the plastid initial. PMID- 16669088 TI - Regulation of Phosphoenolpyruvate Carboxylase and Crassulacean Acid Metabolism Induction in Mesembryanthemum crystallinum L. by Cytokinin : Modulation of Leaf Gene Expression by Roots? AB - Phosphoenolpyruvate carboxylase (PEPCase), the key enzyme of Crassulacean acid metabolism, is induced by water stress in leaves of Mesembryanthemum crystallinum. In water-stressed plants or excised leaves, exogenous cytokinin suppresses PEPCase transcript accumulation in the leaves. Cytokinin (6 benzylaminopurine) used in concentrations from 5 to 500 micromolar (a) inhibits the upregulation of PEPCase transcripts, enzyme activity, and Crassulacean acid metabolism induction in salt-stressed intact plants when sprayed once daily during the stress period, (b) inhibits the accumulation of PEPCase mRNA in leaves from well-watered plants, (c) down-regulates PEPCase transcripts within 8 hours in prestressed, intact plants after a single spraying of an individual leaf, (d) inhibits accumulation of PEPCase transcripts in excised, wilting leaves, and (e) accelerates the net decrease of PEPCase transcripts in excised leaves from prestressed plants under rehydration conditions. When roots, the main site of cytokinin biosynthesis, are excised, PEPCase induction under drought stress is intensified. We propose that roots, acting as sensors of soil water status, may regulate PEPCase gene expression in the leaves with cytokinin as a signal transducer. PMID- 16669089 TI - Asparagine and boric Acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase. AB - Our previous work demonstrated substantial accumulation of allantoate in leaf tissue of nodulated soybeans (Glycine max L. Merr., cv Williams) in response to nitrogen fertilization. Research was continued to determine the effect of nitrate and asparagine on ureide assimilation in soybean leaves. Stem infusion of asparagine into ureide-transporting soybeans resulted in a significant increase in allantoate concentration in leaf tissue. Accumulation of allantoate was also observed when asparagine was supplied in the presence of allopurinol, an inhibitor of xanthine dehydrogenase in the pathway of ureide biosynthesis. In vitro, asparagine was found to have an inhibitory effect on the activity of allantoate amidohydrolase, a Mn(2+)-dependent enzyme catalyzing allantoate breakdown in soybean leaves. The inhibition was partially overcome by supplemental Mn(2+) in enzyme assays. Another inhibitor of allantoate amidohydrolase, boric acid, applied foliarly on field-grown nodulated soybeans, caused up to a 10-fold increase in allantoate content of leaf tissue. Accumulation of allantoate in response to boric acid was either eliminated or greatly reduced in plants presprayed with Mn(2+). We conclude that elevated levels of allantoate in leaves of ureide-transporting soybeans fertilized with ammonium nitrate result from inhibition of allantoate degradation by asparagine and that Mn(2+) is a critical factor in this inhibition. Furthermore, our studies with asparagine and boric acid indicate that availability of Mn(2+) has a direct effect on ureide catabolism in soybean. PMID- 16669090 TI - Photographic survey of the occurrence of bundle-sheath extensions in deciduous dicots. AB - In a survey of over 300 nonevergreen dicots in 69 families, many species were found to have translucent patterns attributed to the presence of bundle-sheath extensions (BSE) on the small and ultimate veinlets. The BSE have been shown by others to inhibit transverse air movement within leaves, and it has been suggested that they are important passageways between vascular tissue and the palisade. The only characteristic found to be associated with prominent BSE is that more trees have such features than herbaceous plants. However, many important herbs have them also, including soybeans and sunflowers. PMID- 16669091 TI - Phosphate Deficiency in Maize : III. Changes in Enzyme Activities during the Course of Phosphate Deprivation. AB - The effects of low concentrations of phosphate (low-P) on soluble protein content, the activities of 12 different enzymes, and the rates of photosynthesis and respiration on the basis of leaf area were investigated in maize (Zea mays L.) leaves 16 to 24 days after planting (DAP). With low-P treatment, a drastic decrease in the rate of photosynthesis to only 6% of the maximum rate in control plants was observed by 24 DAP. Low-P treatment had almost no effect on the rate of respiration until 21 DAP, but then the rate of respiration decreased progressively to about 55% of the maximum rate in control plants. The soluble protein content in low-P plants decreased to 56% of the maximum content in control plants. The changes in the activities of enzymes in low-P plants showed several different patterns. The activities of pyruvate, orthophosphate dikinase, 3-phosphoglycerate kinase, phosphoenolpyruvate carboxylase (PEPC), ribulose 1,5 bisphosphate carboxylase, fructose 1,6-bisphosphate aldolase, catalase, phosphohexose isomerase, chloroplastic fructose 1,6-bisphosphatase, and ADP glucose-pyrophosphorylase decreased steadily from 85 to 100% of the maximum activity found in 18- to 21-day-old control plants (V(max)) to 30 to 70% of V(max). The activity of sucrose phosphate synthase remained virtually constant at approximately 85 to 100% of V(max). The activity of UDP-glucose-pyrophosphorylase remained almost constant up to 21 DAP and then decreased to 80% of V(max) by 24 DAP. The activity of cytochrome c oxidase increased slightly up to 21 DAP but then decreased to 50% of V(max) by 24 DAP. As indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins, the subunit of PEPC stained less intensely in 24-d-old low-P plants. The possibility is discussed that during low-P treatment there is selective degradation of PEPC without a concomitant degradation of sucrose phosphate synthase, both of which are known to be localized in the cytoplasmic compartment of mesophyll cells. PMID- 16669092 TI - Localized Patch Clamping of Plasma Membrane of a Polarized Plant Cell : Laser Microsurgery of the Fucus spiralis Rhizoid Cell Wall. AB - We used an ultraviolet laser to rupture a small region of cell wall of a polarized Fucus spiralis rhizoid cell and gained localized access to the plasma membrane at the growing apex. Careful control of cell turgor enabled a small portion of plasma membrane-bound cytoplasm to be exposed. Gigaohm seals allowing single-channel recordings were obtained with a high success rate using this method with conventional patch clamp techniques. PMID- 16669093 TI - A Wound-Inducible Glycine-Rich Protein from Daucus carota with Homology to Single Stranded Nucleic Acid-Binding Proteins. AB - A cDNA clone was isolated that encodes a wound-inducible glycine-rich protein. The homology of the carrot (Daucus carota) protein with the A1 protein of the heterogeneous ribonucleoprotein complex suggests that the polypeptide plays a role in the biosynthesis and processing of heterogeneous nuclear RNA and in the maturation of specific mRNAs in response to wounding. PMID- 16669094 TI - Methyl jasmonate, calcium, and leaf senescence in rice. AB - The possible involvement of calcium in the regulation of methyl jasmonate promoted senescence of detached rice (Oryza sativa) leaves was investigated. Calcium effectively reduced methyl jasmonate-promoted senescence of detached rice leaves. The effect of methyl jasmonate on the senescence was also significantly reduced by calcium ionophore A23187. Methyl jasmonate-promoted senescence of detached rice leaves may be mediated through blocking the entrance of calcium ions into the cytosol. PMID- 16669095 TI - Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction. AB - The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3 phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte. PMID- 16669096 TI - Differential Accumulation of Biotin Enzymes during Carrot Somatic Embryogenesis. AB - The activities of four biotin enzymes, acetyl-coenzyme A (CoA) carboxylase, 3 methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase, and the accumulation of six biotin-containing polypeptides were determined during development of somatic embryos of carrot (Daucus carota). Acetyl-CoA carboxylase activity increased more than sevenfold, whereas the activities of 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase were relatively unaltered. An increase also occurred in the accumulation of three of the biotin-containing polypeptides (molecular masses of 220, 62, and 34 kilodaltons). Of these, the most dramatic change was in the accumulation of the 62-kilodalton biotin-containing polypeptide, which increased by at least 50-fold as embryogenic cell clusters developed into torpedo embryos. PMID- 16669097 TI - Morphoregulatory role of thidiazuron : substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures. AB - Somatic embryogenesis was induced in hypocotyl explants of geranium (Pelargonium x hortorum) cultured on media supplemented with various concentrations of N phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron). In less than 2 weeks, somatic embryos were observed in treatments containing levels of thidiazuron (TDZ) ranging from 0.2 to 1.0 micromolar. The use of N(6)-benzylaminopurine in combination with indole-3-acetic acid also evoked embryogenesis, but the efficiency of somatic embryo production was significantly lower than that obtained with TDZ. Hypocotyl culture for only 2 days on TDZ-supplemented medium before transfer to a basal medium was sufficient for inducing somatic embryogenesis. This distinction between the induction and expression of embryogenesis may provide an experimental system for studying the developmental biology of somatic embryogenesis. Substitution of the auxin-cytokinin requirement for the induction of somatic embryogenesis by TDZ suggests the possibility of a novel mode of its action by modulation of endogenous growth regulators. PMID- 16669098 TI - Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations. AB - The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance. PMID- 16669099 TI - Organ- and development-specific acyl coenzyme a lysophosphatidate acyltransferases in palm and meadowfoam. AB - Of the three acyl coenzyme A acyltransferases (AT) directly involved in the assembly of fatty acids into triacylglycerols (TAG) in maturing seed, lysophosphatidate (LPA) AT has the highest substrate stringence and dictates which fatty acids can be used. We studied LPA-AT in the microsomes from various organs of palm (Syragrus cocoides) and found that only the microsomes from maturing seed could act on 1-lauroyl-LPA and lauroyl-coenzyme A to produce dilauroyl-phosphatidate. Similarly, of the microsomes from various organs of meadowfoam (Limnanthes alba), only those from maturing seed were active with 1 erucoyl-LPA and erucoyl-coenzyme A to generate dierucoyl-phosphatidate. During maturation of the seeds of both species, the pattern of appearance of LPA-AT that produced dioleoyl phosphatidate was different from that of LPA-AT that generated dilauroyl or dierucoyl phosphatidate. The results show that in seeds, at least those that contain unusual fatty acids in the storage TAG, LPA-AT for the synthesis of TAG is different from the enzyme(s) for the synthesis of membrane lipids. They also suggest that there may be distinct pathways and/or compartments for the synthesis of TAG and membrane phospholipids. PMID- 16669100 TI - Lithium decreases cold-induced microtubule depolymerization in mesophyll cells of spinach. AB - Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter [1991] Plant Physiol 97: 175-181). The objective of this study was to gain insight into the question of whether microtubules depolymerize as a direct response to environmental stresses or as an indirect response to cellular changes that accompany the stresses. Leaf sections of spinach were treated with Li(+) before and during exposure to low temperature. Treatment with Li(+) decreased the amount of microtubule depolymerization in cells subjected to low temperature, relative to a nontreated control, raising the possibility that the microtubules in these cells may not be inherently cold labile. Rather, microtubule depolymerization may be in response to cold-induced changes in concentration of cytoplasmic components. PMID- 16669101 TI - Measurement of indolebutyric Acid in plant tissues by isotope dilution gas chromatography-mass spectrometry analysis. AB - An internal standard, [(13)C][indole-2]-indole-3-butyric acid, was synthesized from indole-2[(13)C] and was shown to be effective for the quantitative determination of indole-3-butyric acid from plant tissue. When this standard was used along with [(13)C(6)]indole-3-acetic acid, both indolic auxins could be quantified from the same tobacco (Nicotiana tabacam) leaf sample by isotope dilution analysis using selected ion monitoring gas chromatography-mass spectrometry for detection. PMID- 16669102 TI - Two Oryza sativa Genomic DNA Clones Encoding 16.9-Kilodalton Heat-Shock Proteins. PMID- 16669103 TI - Nucleotide Sequence of a cDNA for Catalase from Arabidopsis thaliana. PMID- 16669104 TI - Nucleotide Sequence of the Gene for a Glutathione S-Transferase from Cell Suspension Cultures of Silene cucubalus. PMID- 16669105 TI - A polyubiquitin cDNA from a red alga. PMID- 16669106 TI - An Atriplex nummularia cDNA with Sequence Relatedness to the Algal Caltractin Gene. PMID- 16669107 TI - New vaccine for flu season. PMID- 16669108 TI - Protecting the nation's food supply. PMID- 16669109 TI - Safe use of acne drug. PMID- 16669110 TI - Alert on meningococcal vaccine. PMID- 16669111 TI - More U.S. adults at risk of becoming overweight or obese. PMID- 16669112 TI - Paving the way for safer, more effective drugs, food, and medical products. PMID- 16669113 TI - Genomics and personalized medicine. PMID- 16669114 TI - Proteomics: moving beyond the human genome. PMID- 16669115 TI - Metabolomics: working toward personalized medicine. PMID- 16669116 TI - Genomics and medical devices: a new paradigm for health care. PMID- 16669117 TI - Nanotechnology: the size of things to come. PMID- 16669118 TI - Pharmacogenetics: on the road to 'personalized medicine'. PMID- 16669119 TI - [Ability of actin-regulatory protein, gelsolin in induction of monocytic leukemia cell differentiation]. PMID- 16669120 TI - Surveillance for laboratory-confirmed influenza requiring hospital admission in adults, metropolitan Toronto and Peel region, 2004-2005 influenza season. PMID- 16669121 TI - Locally acquired histoplasmosis cluster, Alberta, 2003. PMID- 16669123 TI - An investigation of two simultaneous E. coli O157:H7 outbreaks in Health Region 3, New Brunswick, August to September 2003. PMID- 16669122 TI - Bacterial meningitis in Canada: hospitalizations (1994-2001). PMID- 16669124 TI - Pertussis in Newfoundland and Labrador: 1991-2004. PMID- 16669125 TI - Mind the gap: on common ground and true pluralism. PMID- 16669126 TI - Oculo-respiratory syndrome following influenza vaccination: review of post marketing surveillance through four influenza seasons in Canada. PMID- 16669127 TI - Outbreaks of Salmonella infections associated with eating Roma tomatoes--United States and Canada, 2004. PMID- 16669128 TI - On: whose Bion? Who is Bion? PMID- 16669129 TI - Highly pathogenic H5N1 avian influenza virus confirmed in a dead swan in Great Britain. PMID- 16669130 TI - Management of a tuberculosis exposure in an oncology hospital. PMID- 16669131 TI - A snapshot of immunotherapy for rheumatoid arthritis. PMID- 16669132 TI - Effectiveness of vaccine against medical consultation due to laboratory-confirmed influenza: results from a sentinel physician pilot project in British Columbia, 2004-2005. PMID- 16669133 TI - Translating the concept of suppressor/regulatory T cells to clinical applications. AB - The in vivo expansion of suppressor/regulatory T cells (Tregs) is a desirable event in autoimmunity and transplantation. Here we summarize the general rules involved in antigen recognition by T cells and describe Tregs and their requirements, discussing different levels of immune intervention. PMID- 16669134 TI - [Iatrogenic tracheal rupture: a case report and indications for conservative management]. AB - Tracheal rupture after endotracheal intubation requires immediate intervention. There have been an increasing number of reports that describe nonsurgical management of this issue. We report the case of a 47-year-old woman who experienced an iatrogenic tracheal rupture during endotracheal intubation for a surgical procedure with general anaesthesia. She was successfully managed conservatively with a broad-spectrum antibiotic. We managed it non-operatively, because the patient had a small tear, was hemodynamically stable, show no evidence of infection or respiratory failure, and the diagnosis was not immediate. Bronchoscopy was a good diagnostic tool and it was used to make decisions regarding conservative management, and to detect granulation tissue and rule out any tracheal stenosis after treatment. We review available literature on conservative management of tracheal rupture. Immediate recognition and adequate treatment are very important in managing this potentially fatal situation. The final decision should be based on clinical, radiologic and bronchoscopic findings. PMID- 16669136 TI - A day in the life. How PAs live and work. PMID- 16669135 TI - [Hydroxychloroquine in pediatric idiopathic pulmonary hemosiderosis--a case report]. AB - The authors present the case of a child with severe idiopathic pulmonary hemosiderosis who after having begun treatment with hydroxychloroquine had a significant and lasting improvement. The efficacy of this therapeutic is pointed out. They also report the occurrence of clinical exacerbation, with hemoptysis, after receiving an influenza vaccine. PMID- 16669137 TI - [Brain aging, biological and psychosocial aspect]. AB - The world population is aging The frontier between physiological and pathological brain aging is somewhat unclear. Pathological aging can cause both mild cognitive impairments and dementias (vascular and Alzheimer). It is important to understand brain aging in order to identify risk factors and develop protective measures. Proper lifetime brain protection could improve quality of life in old age. PMID- 16669138 TI - [Early clinical signs of dementia in the elderly]. AB - Clinical signs and symptoms preceding the onset of dementia are sometimes acute, such as mood disorders often associated with hypochondriacal traits and cognitive slowing, sudden and serious suicide attempts, character and conduct disorders contrasting with the previous state, and psychotic disorders presenting as pathological mistrust, or ill-structured prejudice or persecution. Most forerunning symptoms reflect a progressive deterioration of cognitive functions over a long period, that have been masked by various coping strategies used by the patient with the spouse's help. Progressive cognitive deficits may develop over years before dementia can be diagnosed with confidence. Quantitative tools can help to detect dementia incipiens, such as the Folstein Mini Mental Test, the Mattis Dementia Scale, the five-word learning test, the clock drawing test, and the brief cognitive battery. The profile of early cognitive deterioration varies according to the type of dementia (Alzheimer's disease, fronto-temporal dementia, Lewy body dementia, and vascular dementia). The symptoms of dementia may be interlinked with symptoms of other disorders. Neuropsychological tests and brain imaging are needed to validate the diagnosis. PMID- 16669139 TI - [Geriatric surgery]. AB - Modern medicine, which is evidence-based and overly scientific, has forgotten its artistic component, which is very important for surgery in general and for geriatric surgery in particular. The surgeon treating an old patient must be a politician more than a technician, more an artist than a scientist. Like Leonardo da Vinci, he or she must use scientific knowledge with intelligence and sensitivity, transforming the elderly patient's last days of life into a beautiful and harmonious painting and not into something like an atomic power station which, while no doubt useful, is deprived of beauty and sometimes very dangerous. PMID- 16669140 TI - [Mini-invasive fixation of proximal femoral fractures: what benefit for elderly patients?]. AB - Fixation is used to treat more than two-thirds of proximal femur fractures. The mortality rate is about 25% at one year in these patients, who have an average age of about 80 years. This is mainly due to aging, but also to a gradual deterioration of general health (especially if the operation has been delayed, or after a long stay in the surgical ward) and to local complications (displacement, infection, hematoma). Two fixation devices (a sliding screw plate and a trochanteric nail) have been designed for mini-invasive treatment with fluoroscopic guidance and an incision smaller than 50 mm. The aim is to respect the soft tissues and thereby to avoid local complications, diminish pain, and facilitate early weight-bearing Hospital discharge is possible after 3 or 4 days. Laboratory experiments have shown the satisfactory resistance of the implant and bone at full weight bearing A preliminary series of 30 patients showed the feasibility of these techniques. Primary fusion was achieved in 27 cases. There were no infections and no bleeding, despite antiplatelet treatment. The techniques have now been optimized and multicenter studies are held to determine their real benefit. Fracture fusion and hip motion should be at least as good as with open surgery (90 to 96% fusions, albeit influenced by the precise position of the implants and by osteoporosis). Mortality may be slightly reduced, thanks to immediate operation, early discharge, and fewer local complications. The cost of treatment could also be significantly reduced by the shorter hospital stay. In a few years' time, mini-invasive treatment may become the standard for elderly patients with proximal femur fractures. PMID- 16669142 TI - [Epidemiology of asthma and allergies. The prevalence of allergies increases worldwide, and asthma has reached his highest-ever prevalence in Europe: why?]. AB - Up to 10% of adults and 35% of children worldwide suffer from asthma. The incidence of asthma is also higher in children than in adults, possibly owing to a "cohort" effect. Recent population-based studies show that the increase in the prevalence of asthma noted worldwide in the past 30 years has started to subside in industrialised countries. This phenomenon has been accompanied by a parallel increase in the use of asthma medications. Possible explanations for this increase include the "hygiene hypothesis", which implicates diminished exposure to microbial pathogens but was recently challenged by new biological and immunological findings. Asthma might result from the interactions between individual and environmental factors (diet, air pollution, etc.), but further investigations are needed to understand the precise pathophysiological mechanisms of asthma. PMID- 16669143 TI - [Genetics of asthma and atopy: how many genes?]. AB - Asthma is a complex and heterogeneous disease involving many genes and environmental factors. Numerous genetic association studies of asthma, atopy and bronchial hyper-responsiveness have consistently implicated a small number of genes, most of which are involved in immune responses. Genome-wide screens of various populations in the past three years have identified six asthma and/or atopy susceptibility genes by means of positional cloning. However, the precise functions of these genes are unclear. Recent advances in molecular methods for genotyping thousands of polymorphisms in candidate regions (and, very soon, across the entire genome), together with new statistical methods applicable to complex biological systems, will rapidly identify new culprit genes as well as gene-gene and gene-environment interactions. A better knowledge of asthma susceptibility will lead to better diagnosis, prevention and treatment of this increasingly frequent disease. PMID- 16669144 TI - [Pathophysiological mechanisms of asthma and atopy: old and new concepts]. AB - After the discovery of IgE, fundamental immunology studies done during the 1980s and 1990s established that allergic reactions were a particular form of inflammatory reaction governed by Th2 lymphocytes. However, this model, while relevant to atopy, is insufficient to explain allergies, and notably asthma, in which Th1 inflammation is associated. The recent concept that natural tolerance is deficient in atopic subjects, increased in allergic subjects, and maximal in symptomatic allergic subjects, reconciles these conflicting findings by proposing a model in which three axes of T cell activation are involved, namely Th2, Th1 and regulatory T cells. PMID- 16669145 TI - [Nasal allergy and asthma: one or two diseases?]. AB - Epidemiological studies have shown that rhinitis and asthma are associated. At least four out of five asthmatics suffer from rhinitis and one out of four rhinitic subjects develop asthma. Recent progress in cellular and molecular biology confirms that the same inflammatory cells and similar mechanisms participate in the pathophysiology of the two diseases. Histological findings show that although nasal inflammation resembles bronchial inflammation, specific remodelling features that are present in asthma are lacking in rhinitis. This may be due to the different embryologic origin of the two organs. These similarities explain certain clinical interactions and are the basis of the ARIA (Allergic Rhinitis and its Impact on Asthma)--WHO guidelines recommending that symptoms of rhinitis be sought in asthmatic subjects and symptoms of asthma in rhinitic subjects. PMID- 16669146 TI - [Specific immunotherapy and therapeutic strategies in allergic diseases. What's new?]. AB - Specific immunotherapy (SIT) can induce tolerance of airborne allergens in patients with rhinitis, conjunctivitis and asthma. SIT is also effective for venom anaphylaxis. In contrast, SIT is not recommended in severe asthma, food allergy or skin allergy. The mechanisms of SITare unclear, but attention has recently focused on regulatory T cells and their capacity to induce tolerance via IL10 and TGF-alfa production. Recent studies show that SIT not only reduces symptom scores but also modifies the natural history of allergic diseases, preventing sensitization to new allergens and/or limiting the onset of asthma in children with rhinitis. Another recent advance is sublingual IT which is allergic effective and suppresses the risk of systemic anaphylactic reactions. Alternatives to SIT are in the pipeline, such as recombinant allergens, nude DNA vaccines, and immunostimulatory sequence/allergen complexes. PMID- 16669147 TI - [Cross reactivity of food allergens and its clinical relevance]. AB - Cross-reactions between food allergens and other allergens are a major focus of interest. They include cross-allergies between Betulaceae and Compositae pollen, and also between fruits and vegetables (Prunoideae and Apiaceae). Cross-allergies between animal allergens include mites, cockroaches and crustaceans, milk and meat, animal epithelia, meat and egg. Cross-reactivity results from homology between protein sequences, and is highly likely when this homology reaches about 70%. Phylogenetically similar proteins occur in all species and are known as pan allergens. Profilins, Bet v1 homologues, and lipid transfer proteins have varying degrees of clinical relevance. The involvement of cross-reactivity in the persistence of sensitization and in allergic disorders is unclear. The consequences of cross-reactivity during specific immunotherapy with total allergenic extracts are random. Interpretation of biological tests of IgE binding is also biased by cross-reactivity. The use of panels of major recombinant allergens should help to identify specific sensitization profiles as well as clinically relevant sensitization. Cross-reactivity between epitopes of inhalants and of food allergens may perpetuate and intensify allergic disorders. The consequences of cross-reactivity between allergens and autologous proteins are unknown. PMID- 16669148 TI - [Eye movement: from basic semiology to cognitive neuroscience]. AB - Eye movements are an integral part of visual function. They are particularly well developed in frontal-eyed predator species. The abducens nuclei ensure lateral ocular motor synergy, while the oculomotor nuclei control vertical eye movements and convergence. The two main types of conjugate eye movement--saccades (image switching) and slow movements (image stabilization)--are controlled by specific brainstem premotor structures (respectively reticular and vestibular). Upstream of the brainstem, the cortical and subcortical pathways involved in eye movements are gradually being elucidated. Saccades are being used as a motor model to study the different cognitive processes, such as spatial memory, prediction and decision, involved in general movement preparation. The classical approaches based on lesion analysis, combined with transcranial magnetic stimulation and functional cerebral imaging, are contributing to our understanding of how the brain functions. Eye movements can be used for bedside diagnostic purposes, but also for sophisticated laboratory studies designed to explore the universe of cognition. PMID- 16669149 TI - [Results of laparoscopic antireflux surgery at five years and beyond]. AB - Laparoscopic antireflux surgery was first introduced in 1991. As with any new technique, there was a learning curve during which surgeons had a unique opportunity to prospectively document the evolution of the technique and its results. We have regularly assessed our results and conducted randomised prospective studies with the aim of obtaining solid long-term outcome data. We now have at least 5 years of follow-up for two groups of patients. Group I consists of the first 178 patients who underwent fundoplicature, i.e. during the learning curve. Group II consists of 107 patients who were enrolled in a randomised trial comparing total fundoplicature with anterior partial fundoplication. The re-operation rate was 15% in Group I and only 6% in Group II. Overall, about 85% of patients were free of reflux symptoms 5-8 years after fundoplication, indicating that laparoscopic surgery provides a durable benefit in the vast majority of patients. PMID- 16669150 TI - [The anterior cingulate cortex in error detection and conflict monitoring. Unitary neuronal activity in monkeys]. AB - Using a Stroop-like visuo-motor task in monkeys, we studied the unitary neuronal activity of the anterior cingulate cortex (ACC, CMAr : 24c) during error detection and conflict monitoring. A high percentage of cingular neurons modified their firing frequency during both attentional and evaluation periods. During the latter period, however, changes in discharge rates were always much more pronounced for erroneous responses than for successful movements that induced reward delivery. Moreover, some neurons responded more markedly to the warning stimulus when a previous trial failed In this case, the probability of a subsequent correct response increased. Such a neuronal apparatus could represent an error compensation system that serves to adapt a subject's behavioral response following an unfavorable and unexpected outcome. Abnormal activity of these neurons, which appear to take advantage of past errors, could contribute to the genesis of obsessive-compulsive disorders (OCD). PMID- 16669151 TI - [Case management of pelvic floor disorders]. AB - Pelvic floor disorders can involve all three parts of the pelvic floor, and must be managed holistically. They are often associated with one another, because they all involve an imbalance between counterbalancing forces, namely abdominal pressure on the one hand and mooring forces on the other hand. The mooring forces consist of 1) the visceral ligaments, which are fibrous cellular condensations around vessels and nerves, connecting the pelvic wall to the organs; 2) the endopelvic fascia; and 3) the pelvic muscles, including the levator ani. The physical examination must be both functional and anatomical, in order to detect obvious and occult disorders of the three parts of the pelvic floor. When physical examination is inadequate, standard radiography or MRI may be used. Radiographic explorations include colpocystography and defecography. The first is carried out in the standing position with a "blocked" perineum, and the second is performed in the defecation position with a "relaxed" perineum. Treatment must take into account dysfunctions, incontinence, and ptosis of the three parts. The author discusses the various examinations and treatments (by the abdominal or vaginal approach), based on published data and personal experience. The choice of treatment always depends on the patient's age, anatomical defects and functional disorders, and the surgeon's expertise PMID- 16669152 TI - [Recommendations of the National Academy of Medicine for improvement of the clinical training of medical students]. AB - Young medical students' initiation into clinical activity and clinical responsabilities could be improved by developing theoretical courses and clinical activities under the strict control of senior medical staff. Such teaching activities should be taken into account in career advancement. PMID- 16669153 TI - [Some considerations about the AIE Congress held in Pisa and the future of the Italian Association of Epidemiology]. PMID- 16669154 TI - [Policies threatening population health?]. PMID- 16669155 TI - [Epidemiology and politics: an issue to think about]. PMID- 16669156 TI - [Past and future of International Classifications of Diseases]. PMID- 16669157 TI - [Answers to Panizza e Ricci about Brescia]. PMID- 16669158 TI - [Caffaro pollution, the affaire approaches the end]. PMID- 16669159 TI - [European researchers comment the new draft directive on air quality]. PMID- 16669160 TI - [Preliminary study of cause-specific mortality of a population exposed to 50 Hz magnetic fields, in a district of Rome municipality]. AB - OBJECTIVE: To investigate cause-specific mortality in a population exposed to high levels of 50 Hz magnetic fields, in a district of Rome. DESIGN: A cohort of 357 subjects that are resident in an area of about 100 meters on the two sides of a 60 kV power line was studied. Exposure to 50 Hz magnetic fields was evaluated by spot and long-term measurements and by adopting a model based on current load, characteristics of the line and distance of dwellings from conductors. The mortality experience of the cohort was observed from January 1st, 1980 through December 31st, 2003. Analysis took into account duration of residence and latency. Separate analyses were performed for three subcohorts characterized by different distances from the line and different estimated and measured field levels. MAIN OUTCOMES: Cause-specific standardized mortality ratios (SMRs) were computed comparing the observed number of deaths with the corresponding expected figures derived from the Latium Region mortality rates. This was done both in the overall cohort and in the subcohorts. RESULTS: All-causes mortality of the cohort did not differ from expected values (SMR 0.99; 95% CI 0.73-1.35; 40 observed). All cancers mortality showed a non-significant increase (SMR 1.34; 95% CI 0.82 2.18; 16 observed), that reached statistical significance in the analyses limited to subjects with more than 30 years of residence (SMR 2.09; 95% CI 1.05-4.19; 8 observed). Mortality from lymphohaematopoietic malignancies was increased, based on two cases of leukemia. All cancers mortality was highest in the subcohort closest to the power line and exposed to highest levels of magnetic fields. An increased risk for digestive tract neoplasms (SMR 3.57; 95% CI 1.49-8.58; 5 observed) was observed, which was mainly caused by pancreatic cancer. DISCUSSION AND CONCLUSION: Some increase in mortality of the overall cohort and especially of the highly exposed sub cohort was detected, notwithstanding the low power of the study. Other studies are currently on-going in the same area: a cross sectional investigation on health status, an analysis of hospital discharge cards, a project for estimating individual exposure levels and a research in veterinary epidemiology. It is recommended to replicate the present study in areas presenting similar exposure patterns, in order to be able to pool data with the aim of estimating risk associated with the exposure levels of interest. PMID- 16669161 TI - [The active search for occupational cancer cases: bladder cancer in Lombardy Region]. AB - OBJECTIVE: A study called OCCAM (OCcupational CAncer Monitoring) has been carried out in order to establish a nationwide surveillance system for occupational cancer. This project consists of population-based case control studies where information on past occupations are automatically gathered National Institute for Social Security's archive, cancer cases are obtained from Cancer Registries (CR) and controls are sampled from population files of the National Health Service. As previous results obtained using CR were encouraging, we tried to use regional hospital discharge records as a source of incident cases. DESIGN: We have conducted a population based case-control study with 1568 male bladder cancer cases occurred in the years 1999 and 2000 and 18818 controls randomly sampled from resident population in Lombardy region (Italy). RESULTS: Despite the limits of this approach the following industries were found at increased risk of bladder cancer: leather and shoes industry (OR=1.83; CI 90%: 1.01-3.33; observed: 10); transports (OR=1.28; CI 90%: 0.94-1.76; observed: 37), rubber industry (OR=1.22 CI 90% 0.80-1.85; observed: 19) and printing industry (OR=1.5 CI 90%: 1.10-2.05; observed: 38). CONCLUSION: The ability of OCCAM surveillance system to find known associations using routinely available data offers new opportunities to detect cancer cases likely to be of occupational origin. PMID- 16669162 TI - [Monitoring microbiological safety of small systems of water distribution. Comparison of two sampling programs in a town in central Italy]. AB - OBJECTIVE: To determine the frequency of sampling in small water distribution systems (<5,000 inhabitants) and compare the results according to different hypotheses in bacteria distribution. DESIGN AND SETTING: We carried out two sampling programs to monitor the water distribution system in a town in Central Italy between July and September 1992; the Poisson distribution assumption implied 4 water samples, the assumption of negative binomial distribution implied 21 samples. Coliform organisms were used as indicators of water safety. The network consisted of two pipe rings and two wells fed by the same water source. The number of summer customers varied considerably from 3,000 to 20,000. RESULTS: The mean density was 2.33 coliforms/100 ml (sd= 5.29) for 21 samples and 3 coliforms/100 ml (sd= 6) for four samples. However the hypothesis of homogeneity was rejected (p-value <0.001) and the probability of II type error with the assumption of heterogeneity was higher with 4 samples (beta= 0.24) than with 21 (beta= 0.05). CONCLUSION: For this small network, determining the samples' size according to heterogeneity hypothesis strengthens the statement that water is drinkable compared with homogeneity assumption. PMID- 16669163 TI - [Residential facilities for the elderly in five Italian regions. The Progres Anziani Project]. AB - OBJECTIVE: To survey all Residential Facilities (RFs) for the elderly in five Italian Regions (Calabria, Sardinia, Sicily. Umbria and Veneto), and to evaluate their logistic and organizational characteristics, staff and residents' features. DESIGN: Structured interviews were conducted with RF managers. SETTING: All RFs operating in the five Italian Regions. RESULTS: In the five Regions there are 853 RFs with an average of 198.0 beds per 10000 elderly inhabitants; this rate shows a marked variability between different Regions. The mean number of beds in the RFs is 59.8 (median: 34). The large majority (96%) have a 24-hour staff cover. In terms of management, the RFs are handled by local municipalities (29%), religious non-profit associations (24%), and other non-profit organizations (21%). In the 754 RFs surveyed (91.1% of the sample) there were 24.456 workers employed, even the number of staff in each facility shows a great variability. The mean number of workers directly involved in residents' care is 27.8, and the ratio residents/staff is 2.1, which becomes lower (1.8) if we consider the ratio non independent residents/staff. In the 754 RFs there were 42,687 residents, with an average of 53.5 elderly residents for each facility and 3.2 residents below the age of 65; the mean age of the resident sample was 79.3 years, and there is an high proportion of residents with neurological (including dementia), psychiatric or medical disorders. CONCLUSION: There is marked variability in the provision of residential places between different Regions; many other characteristics of RFs for the elderly, including staff/residents ratios, show a similar variability Most RFs host elderly with a variety of neurological, psychiatric and medical disorders. The future waves of the project will shed light on many features of these institutions, which care for 2.1% of the elderly population in Italy and may serve larger proportions of the elderly in the future. PMID- 16669164 TI - [Mortality among workers in a cigarette factory in Lucca (Tuscany)]. AB - Aim of the present cohort study was to evaluate the mortality pattern among workers in a cigar and cigarette factory in Lucca, Italy. The study followed 2341 workers (1585 women and 756 men) registered in the company payrolls and employed for at least six months from 1 January 1960 through 1 January 1994. Follow-up was between the start of employment in the factory and 1 June 2002 (totally 74363,5 person-years). For both sexes, all-causes mortality was lower than expected (men: SMR= 0.8; CI95% 0.7-0.9; 158 deaths; women: SMR= 0.9; CI95% 0.8-1.0; 584 deaths) and no excess of mortality was reported for all malignant neoplasms. Among female workers, the frequency of deaths from pleural cancer was elevated at a statistically significant level (SMR= 6.0; CI95% 2.4-12.6; 5 deaths). One death for pleural cancer also occurred among men versus 0.4 expected. All women deceased from pleural cancer had been working in tobacco manufacturing for at least 30 years. The excess of pleural neoplasms reported in this study suggests the opportunity to evaluate the risk due to asbestos use in many manifacturing industries, especially where steam was used for extractive or warming purpose. PMID- 16669165 TI - [Use of ISTAT mortality records for follow up study and local mortality data base]. AB - The aim of the present study is to assess the quality of the variables "name" and "surname" recorded in the mortality records that the Italian Institute of Statistics (ISTAT) releases to its regional offices since 1999. These records could both constitute a regional mortality database in those regions that do not have one, and be a useful tool for ascertainment of vital status in follow-up studies. The study was conducted in Turin, North West Italy, through record linkage between ISTAT database and the local population register that records all deaths occurred among residents. This was considered as the gold standard. Firstly the concordance of name and surname was studied; this was 92% if the full length of name and surname was used raising to 97% using substrings of the two information. Secondly the cohort of 1999-2001 residents in Turin was linked to the ISTAT database using a four step record linkage with four different keys. 94,3% of subjects were correctly found. Within those not linked there was a higher proportion of subjects born abroad, women, young and unmarried. We conclude that the quality of ISTAT database should be further improved before implementing its use for follow-up studies. PMID- 16669166 TI - [Information technology for the management of health care data: the EPIweb project]. AB - In the US, the Center for Disease Control and Prevention has produced has increased the permeability of the computer science technologies, in order to achieve a better and more efficient management of health care data. In this context, the present paper proposes a discussion regarding a web-based information system, called EPIweb. This system allows researchers to select the centers for the data entry, collect and elaborate health care data, produce technical reports and discuss results. Such a system aims to be easy-to-use, totally configurable and particularly suitable for the management of multicenter studies. The paper shows the EPIweb features, proposes a sample system run, and concludes with a discussion regarding both the advantages and the possible improvements and extensions. PMID- 16669167 TI - [A teaching experience of Evidence Based Prevention]. AB - One year after a 3-day training course on EBP (Evidence Based Prevention), the 130 participants were sent a follow-up questionnaire intended to estimate the persistence of knowledge and the extent of the use of evaluation techniques in EBP. Although respondents to the questionnaire (73%) and participants to the course were not representative of workers in the health field area, results clearly indicate that major drawbacks the use of EBP are the organization of daily work in prevention activities and the limited knowledge of English (i.e. the language in which most scientific literature related to EBP is available). More education on EBP is advisable, including the application of distant learning techniques into the frame of Continuous Medical Education for all Italian professionals involved in Prevention. PMID- 16669168 TI - [Asbestos industry: an incomplete mosaic]. PMID- 16669169 TI - [Comments on the appeal verdict of the Porto Marghera trial]. PMID- 16669170 TI - [Judge and expert in penal proceedings: a debate]. PMID- 16669171 TI - [Scientific knowledge in Court]. PMID- 16669172 TI - [Science, democracy and participation]. PMID- 16669173 TI - [The Longarina case: an oasis steeped in vegetation and in EMF]. PMID- 16669174 TI - A reappraisal of infarct ECG patterns based on CMR. PMID- 16669175 TI - CINE-MR imaging of the normal and infarcted rat heart using an 11.7 T vertical bore MR system. AB - MR imaging is uniquely placed to non-invasively study rodent cardiac structure and function. High-field MR scanners commonly have a vertical bore, and the purpose of this work was to demonstrate CINE-MR imaging in normal and infarcted rat hearts after determining hemodynamic stability when positioned vertically for imaging. Optimisation of imaging parameters was carried out prior to assessment of cardiac function in a group of normal and infarcted rat hearts. Rat hemodynamics were unaltered when vertical for 90 minutes, compared with horizontal measurements and rat cardiac parameters were measured accurately and reproducibly with our optimized CINE-MR protocol. A flip angle of 17.5 degrees was shown to provide optimal contrast for the assessment of structure and function, and, in contrast to our findings in mice, respiratory gating was not found to be essential. Hence, we conclude that vertical bore MR systems can be used to measure in vivo cardiac function in normal and infarcted rat hearts. PMID- 16669176 TI - Utility of contrast-enhanced cardiovascular magnetic resonance (CE-CMR) to assess how likely is an infarct to produce a typical ECG pattern. AB - OBJECTIVES: For over 50 years, Q-wave myocardial infarction (MI) location has been based on pathologic ECG studies. Although contrast-enhanced magnetic resonance (CE-CMR) is currently the "gold standard" technique for location and quantification of necrotic areas, we found no large study in the literature devoted to establish which ECG patterns corresponds to different MI location detected by CE-CMR. We hypothesized that CE-CMR would be very accurate for evaluating different ECG patterns and its sensitivity (SE) and specificity (SP) for locating MI in different LV areas. METHODS AND RESULTS: CE-CMR/ECG correlation was studied in 48 patients who presented a first MI due to acute coronary syndrome (ACS) with ST-segment elevation and in whom CE-CMR was performed in chronic phase. We evaluated the ECG patterns that best correlated with the 7 prespecified necrotic areas assessed by CE-CMR, 4 in anteroseptal zone (septal, apical/anteroseptal, extensive anterior, and limited anterolateral) and 3 in inferolateral zone (inferior, lateral and inferolateral). The global concordance between CE-CRM and ECG was of 75% and 7 ECG patterns were stablished. CONCLUSION: The capacity of CE-CMR to detect ECG patterns for necrotic area location presents highly acceptable concordance. Thanks to CE-CMR, we defined 7 ECG patterns for MI detection according to the 7 areas of the LV studied. The areas that present more cases with normal ECG are limited anterolateral and the areas of the inferolateral zone. PMID- 16669177 TI - T1-weighted cine FLASH is superior to IR imaging of post-infarction myocardial viability at 4.7T. AB - PURPOSE: Data are unavailable for rational selection of pulse sequences to assess postinfarction myocardial viability in rodents at high field strength. We implemented a widely used clinical inversion recovery (IR) sequence at 4.7T and compared the results to a heavily T1-weighted cine FLASH sequence (T1-CF) for assessment of infarction size. MATERIALS AND METHODS: Eleven infarcted rats were examined within 24 h of infarction after injection of Gadophrin-3 contrast agent. Images were acquired using both pulse sequences and a standard cine (SC) sequence. Estimates of infarct size were compared to TTC. Global LV function was compared between the T1-CF and SC sequences. RESULTS: SNR, relative SNR, and CNR for the infarcted and normal myocardium were significantly greater for the IR sequence. Infarction size was overestimated by both sequences, but correlated highly and showed very close agreement with TTC. Global function revealed no significant differences between T1-CF and SC. CONCLUSION: Both IR and T1-CF produced reliable results for assessment of infarction size at 4.7T. While the IR sequence delivers better overall SNR and CNR, the T1-CF allows concomitant assessment of global cardiac function with a much shorter acquisition time. PMID- 16669178 TI - Cardiac magnetic resonance appearance of myocarditis caused by high dose IL-2: similarities to community-acquired myocarditis. AB - The purpose of this study was to describe and compare the cardiac magnetic resonance (CMR) characteristics of myocarditis caused by high dose interleukin-2 (7 patients) with community-acquired myocarditis (14 patients). A total of 21 patients with suspected myocarditis and elevated cardiac enzymes underwent cine CMR followed by delayed enhancement. The mean ejection fraction was mildly decreased in both groups. The location, pattern, and extent of DE were similar in both groups of patients. The CMR similarities between these two populations suggest that cytokine-mediated cytotoxicity may play an important role in community-acquired myocarditis. PMID- 16669179 TI - Value of repeated cardiac magnetic resonance imaging in patients with suspected arrhythmogenic right ventricular cardiomyopathy. AB - AIM: Diagnosis of early stages of arrhythmogenic right ventricular cardiomyopathy (ARVC) with minimal structural abnormalities is challenging. The purpose of this study was to assess the value of repeated cardiac magnetic resonance imaging (CMR) in patients referred for right ventricular arrhythmias and clinical suspicion of ARVC. METHODS AND RESULTS: Prospective follow-up study of 18 patients (8 females) studied with CMR for suspected ARVC. Patients with implanted defibrillators (ICD) were excluded. Mean follow-up was 37 +/- 16 (12-59) months. Patients were assigned to 2 categories (ARVC likely or ARVC unlikely) according to a CMR-score based on right ventricular abnormalities. Clinical follow-up revealed no disease progression in 17 patients (94%). In 1 patient, an ICD was implanted because of disease progression. Of 9 patients with initial findings suggestive of ARVC, follow-up CMR remained positive in 3 and was diagnosed as normal in 6, mainly due to the inability to confirm the presence of fatty infiltrates at follow-up (5 of 6 patients). Initially, 9 patients had a normal CMR and 8 of those remained normal during follow-up. CONCLUSION: Repeated CMR after an average follow-up of 3 years was normal in 6 of 9 patients with clinical findings consistent with early stages of ARVC at the time of baseline CMR. Thus, CMR diagnosis of early stage ARVC is difficult and should be made with caution. PMID- 16669180 TI - Value of gadolinium-enhanced magnetic resonance imaging in patients with Tako Tsubo-like left ventricular dysfunction. AB - We present gadolinium-enhanced cardiac magnetic resonance imaging (CMRI) in Tako Tsubo-like left ventricular dysfunction showing the findings in acute phase and in follow-up. Gadolinium-enhanced CMRI allows to distinguish between myocardial infarction and other myocardial alterations, e.g., myocarditis. CMRI may thus permit to non-invasively identify patients with Tako-Tsubo syndrome by ruling out myocardial infarction or myocarditis in the setting of wall motion abnormalities (WMA). PMID- 16669181 TI - Limited flow reserve in non-obstructed bypass grafts supplying infarcted myocardium: implications for cardiovascular magnetic resonance imaging protocols. AB - We evaluated flow reserve in non-obstructed bypass grafts supplying infarcted and noninfarcted myocardium. Bypass grafts were examined by phase-contrast flow measurements and myocardial viability was assessed with late enhancement technique. Flow reserve was higher in bypasses supplying normal myocardium compared to those supplying infarcted myocardium (2.9 vs. 1.5, p<.0001). This difference remained significant after adjusting for co-variables. Bypass grafts supplying infarcted myocardium were more likely to have lower flow reserve than those supplying normal myocardium (flow reserve < or =2, 84% vs 18%, p = .0003). Flow reserve is reduced in non-stenosed bypasses supplying infarcted myocardium, likely due to altered microcirculation. Thus, cardiovascular magnetic resonance based bypass assessment must include myocardial viability testing. PMID- 16669182 TI - Cardiovascular magnetic resonance imaging for non-invasive assessment of vascular function: validation against ultrasound. AB - Ultrasound is an established modality for quantification of vascular function in clinical studies of cardiovascular disease. We determined whether cardiovascular magnetic resonance imaging (CMR) can provide a comparable assessment of vascular function. In seventeen control subjects, we used CMR to quantify endothelium dependent (flow mediated dilatation, FMD) and endothelium-independent dilatation of the brachial artery, brachial and carotid distensibility, aortic compliance, and pulse wave velocity. These were compared to brachial and carotid measurements obtained by established ultrasound protocols. Twelve of the volunteers then underwent repeated measurements with both modalities. There was good agreement between imaging modalities for measures of endothelial function and arterial structure in the same subjects (difference between CMR and ultrasound for FMD = 0.14 +/- 6.8%, and brachial artery area = - 0.7 +/- 2.2 mm2, correlation between modalities for FMD = 0.62, p = 0.01 and for area = 0.87, p = < 0.0001). Inter study reproducibility was also similar (coefficient of variation (CV) for FMD: CMR = 0.3, ultrasound = 0.3, CV for brachial artery area: CMR = 0.1, ultrasound = 0.1). Comparability and reproducibility were not as strong for functional measures if repeated studies were several days apart (CV for FMD by ultrasound on the same day = 0.1 and several days apart = 0.4). CMR and ultrasound show good agreement for quantitative measures of vascular structure and function with good reproducibility for both modalities. The major advantage of CMR is that it allows one-stop integrated assessment of both peripheral and central measures of vascular function. PMID- 16669183 TI - Co-registration of cardiac MRI and rest gated SPECT in the assessment of myocardial perfusion, function and viability. AB - PURPOSE: Myocardial perfusion is routinely measured by SPECT--this technique has a rather low spatial resolution but covers the whole myocardium and is equipped with efficient image analysis software. Cardiac MRI has higher spatial resolution than SPECT and excellent sequences for myocardial function and viability detection but the lack of easy-to-use methods of acquisition and post-processing of perfusion images prevents this method from being used for perfusion evaluation in clinical practice. The aim of the study was to explore whether the 3-D co registration of "cine" MRI (cine MRI), delayed enhancement MRI (DE MRI) and gated SPECT (GSPECT) images might be used for differentiating all reversible and irreversible effects of ischemia in anatomically matched myocardial regions. METHODS: We analyzed 685 segments of the heart (6 segments in each short axis slice)-obtained as a result of MRI and GSPECT studies performed in 18 patients. In each segment, myocardial function, perfusion and viability were analyzed. Myocardial wall function was evaluated using the matched images of diastolic and systolic phases of cine MRI. Perfusion as MIBI uptake per volume (MIV) (counts/mm3) in each myocardial segment was evaluated by co-registration of diastolic phases of cine MRI and GSPECT. Transmural extent of infarction was determined by co-registration of DE MRI and diastolic phase of cine MRI. RESULTS: We have found a close correlation between regional perfusion and function at rest in matched MRI and SPECT images: dysfunctional segments had significantly less MIV (MIV = 4.63 SD 1.58) than normal segments (MIV = 8.86 SD 2.77) (p < .05). There was no significant difference in MIV between viable and non-viable dysfunctional segments defined by DE MR due to a small number of nonviable segments in our study (18/685). CONCLUSION: Matching rest perfusion and function in anatomically co-registered myocardial segments in our study confirms that 3-D image co-registration of cine MRI, DE MRI and gated SPECT could be a precise method of integrated visualization of perfusion, function and viability helping in differentiating all forms of reversible and irreversible effects of myocardial ischemia. PMID- 16669184 TI - Detection of Anderson-Fabry cardiomyopathy with CMR in a patient with chest pain and elevated cardiac biomarkers. AB - This case illustrates the utility of CMR in evaluating a patient with undiagnosed Anderson-Fabry disease who presented with chest pain, elevated cardiac biomarkers, normal coronary arteries, and an abnormal echocardiogram. PMID- 16669185 TI - Unusual fatty infiltration of the basal left ventricle. PMID- 16669186 TI - [Pathological and histopathological findings in the joints of fattening turkeys]. AB - This paper reports on pathological and histopathological findings in the joints of male turkeys (B.U.T. Big 6) reared in two different housing systems. Pathological investigations were conducted on all animals which died between the 6th week of life and the end of fattening (585 birds, 1170 hip, knee and intertarsal joints) from four fattening periods with 3000 birds each. This study included 297 birds kept in a conventional housing system (barn 2) with a stocking density of 2.8 birds/m2, and 288 birds from a barn (1) from which the animals had access to an outdoor scratching area (stocking density 2.5 birds/m2). In all, 157 (26.5%) of the 585 fallen turkeys (barn 1:93; barn 2:70) showed at least one pathological joint alteration. and there were macroscopically detectable changes in 274 (7.8%) of the 3510 investigated joints: 152 (7.8%) of the 1728 joints from barn 1, and 122 (8.8%) of the 1782 from barn 2. In both housing systems the most frequent pathological alterations were in the intertarsal joint affecting 16% (188) of these 1170 joints (barn 1:106; barn 2:82); next most frequent were changes in the knee joint with 4.5% (53) of the 1170 joints affected (barn 1:26; barn 2:27). In both barns, synovial alterations occurred predominantly in the intertarsal and the knee joints. Bacteriological investigation of altered synovial fluids (83) most frequently showed an infection with Staphylococcus spp. (44) and E. coli (15). Macroscopic investigation showed alterations in 31 (2.7%) of the 1170 hip joints (barn 1:19; barn 2:12); 2% (22) of these showed cartilage degenerations of the femoral head (barn 1:15; barn 2:7). Histological investigation of these cartilage lesions showed that they were predominantly (70%) due to osteochondrosis with complete epiphyseolysis affecting 16 femoral heads (barn 1:10; barn 2:6). Partial epiphysiolysis was found in 24% (5) of the femoral heads (barn 1:4; barn 2:1). Furthermore, 43% (9) of the 22 femoral heads with cartilage degeneration also had osteomyelitis and osteochondritis, as well (barn 1:6; barn 2:3). Histological investigation of the 104 femoral heads with no macroscopic findings (barn 1:56; barn 2:48), revealed osteochondrosis in 11 cases (10.6%) (barn 1:6; barn 2:5). Osteomyelitis and osteochondritis were found in 17 (16.3%) of these femoral heads (barn 1:9; barn 2:8). There were no significant (p < 0.05) differences between the two housing systems in the incidence and frequency of joint alterations. It is to be assumed that these joint alterations were the cause of acute, chronic pain. PMID- 16669187 TI - [Water supply for Peking ducks--possible alternatives for bathing]. AB - In order to investigate possible advantages of open water drinkers such as troughs and bells in the intensive husbandry of ducks, we evaluated in this study the effect of open water systems on the behavior and health of peking ducks. In this investigation six fattening trials each with 1152 Cherry Valley Peking ducks were analyzed. The average fattening period was 47 to 49 days. The ducks were kept on straw in six standardized compartments each 32 qm with 192 ducks. 25% of the compartment with perforated grid, with one of the tested water systems. In contrast to pens with nipple-drinkers only, the ducks in pens with open water troughs/showers had the opportunity to exhibit their natural drinking behavior and water associated activities in accordance with the animal welfare requirements. In the free-choice pens the open water systems were significantly preferred, and over a 24-hour period the ducks in watering areas with open water troughs showed higher activity. Limiting the access to the open water systems to eight, four or two hours per day led to an increased use per time unit. Ducks with access to nipple drinkers only showed a significantly higher percentage of plugged up nostrils than animals from pens with open water drinkers. Open water drinkers had also a positive impact on the plumage condition. PMID- 16669188 TI - [Alternatives for castration of suckling piglets, determination of catecholamines and wound healing after castration of suckling piglets at different points of time]. AB - According to the applicable animal welfare legislation, the surgical castration of pigs is allowed up until the age of 4 weeks, without anaesthesia. According to the european guideline (2001/93/EG) it is only permitted in the first week after birth. The investigation should show, whether the castration of young piglets takes a milder course and to what extent stress reactions occur in different age groups. The healing process of castration wounds in piglets that were castrated at the age of four days progressed more rapidly and with less complications than those piglets that were castrated at 28 days of age. The catecholamine levels in younger piglets rose significantly after the operation, while these levels virtually stayed the same in piglets castrated at 28 days. As alternatives to castration without anaesthesia, several methods are in discussion: (1) Castration under general anaesthesia can only be practiced by a veterinary surgeon. The sole use of Azaperon and Ketamine has insufficient pain sedating effect. Isofluran anaesthesia is apparatively extravagant. (2) CO2-Anaesthesia in piglets leads to high strain. (3) Castration under local anaesthesia must be practiced by a veterinary surgeon. The application and the pain after the castration are not taken into consideration. (4) Jung boar fattening up until the slaughtering weight of 80 kg is not transformable, because of boar taste. (5) The breeding of slaughter pigs with little boar taint is not yet transformable. (6) Sperm sexing is not and will not, in the near future, be mature for practice. (7) Immunocastration is an active immunisation against GnRH. The immunological elimination of GnRH suppresses the development of sex hormones, such as testosterone, as well as the substance responsible for boar taint, Androstenone. To consider is the acceptance of the consumer. The preparation has the same effect in humans as it has in swine. "Self injections" have the same effect in humans as it has in swine. "Self injections" are therefore risky. PMID- 16669189 TI - [Recognizing and assessing aggressive behaviour in dogs]. AB - Within the population the sensitivity to aggressive behaviour in dogs has increased. The authorities are confronted with a problem: if any incident occurs it is their task to decide whether the dogs involved constitute a threat to other people or whether the charge is only the result of a quarrel between neighbours. For this reason, an examination of the dogs with regard to their aggressive behaviour is necessary. Seen from the biological point of view, aggressive behaviour is one of four possibilities a dog can chose from to solve a conflict. The dog's intention in showing aggressive behaviour is to eliminate disturbances and to maintain a distance in space and time. Aggressive behaviour might also be necessary to acquire or defend resources essential to the dog's life. This is to secure its survival and its success in reproduction. One can see from this that aggressive behaviour is a very important and biologically necessary adjustment factor. However, when living together with man aggressive behaviour might become a problem. For the assessment and the therapy of the problem it is necessary to exa-mine the behaviour shown by the dog with regard to its cause. To be able to do this an exact anamnesis, a medical check, and an examination of the dog on the basis of its display in special situations are necessary. For this reason, exclusively veterinarians with a special further education in the field of behaviour should carry out the examination of dogs. PMID- 16669190 TI - [Prohibition to exhibit docked dogs--problems of execution]. AB - In Germany it is illegal to exhibit dogs when parts of their body are amputated to meet certain distinctions of their race except there has been a medical reason for the operation. Experiences in practice with the inspection of dog exhibitions reveal that this exceptional rule is not effective. Many breeders and owners of dogs, especially of the race "Doberman", try to escape the prohibition to exhibit docked dogs with the help of dubious veterinarian certificates. Therefore it is proposed to eliminate this exception from section 10 of the regulation on the welfare of dogs. PMID- 16669191 TI - [Analysis of veterinary certificates on tail docking in dogs]. AB - Despite the fact that docking of dogs has been banned in Germany for years, many animals are still docked, mainly owing to overcome traditional thinking. This is possible by some exceptions made by the law: the import of dogs that have been docked abroad, and medical indications for docking. In order to substantiate the necessity of such medical indications, the patient material of the Small Animal Clinic of the Veterinary School Hanover was analysed and compared with a number of veterinary certificates for docked dogs that were to be shown at exhibitions, stating a medical indication. It shows that the latter predominantly cover dogs belonging to breeds that are traditionally docked, the Doberman in the first line. Contrasted with this, dogs from our own patient material showed an even breed distribution, with no Doberman at all within a five year period. Moreover, the fact that certain breeds were predominantly docked by the same veterinarian as well as the medical reasons stated were suspicious. Consequently, doubts should be raised in cases of accumulations of tail amputations in dogs belonging to a traditionally docked breed, when the reasons given are the same in several animals, and when the same veterinarian performs the operation in several dogs belonging to the same breeder. Because of the scientifically proven pain that is caused to the animal both at the time of the procedure and later, as well as the possibility of long lasting chronic health impairment owing to docking, it is necessary to consequently fight this malpractice. In order to prove the stated medical indication in individual cases, either a radiograph or photography must be submitted. PMID- 16669192 TI - [Problems and solutions in giving an expert evaluation]. AB - According to the law for the prevention of cruelty to animals, the officially employed veterinarians are the experts per se. All administrative legal steps are based on their reports. Such reports are also of major significance at court hearings. However, veterinary colleagues working in private practices or clinics are often requested to submit an expert evaluation in cases of cruelty to animals. The problems which occur in putting together an expert's report are often based on a lack of knowledge about the clear structure of such a report (preliminary details, description of the situation as found, assessments of results, evaluation of findings, justification). The situation is made more complicated by the two different languages of lawyers (that of administrative officials, advocates of district courts and higher district courts or public prosecutors) and of veterinarians. A detailed description and a clear assessment together with a detailed justification of the concluding findings are absolutely essential. By providing examples of the correct way to write an expert's report, both the problems and solutions to improve the situation can be pointed out. This will be of the utmost importance in the future when quality management of animal rights is introduced. Specific training should be introduced during studies, in training courses for district examinations and in practical, further education. In addition, the range of problems caused by the constantly rising number of expert's reports made as a favour should also be considered. PMID- 16669193 TI - [Influence of a turkey stable with a veranda on performance, behaviour and health of male turkeys]. AB - Throughout 4 fattening periods (summer and winter) the influence of a veranda (roofed outside run) on the performance, behaviour and health of tom turkeys was investigated in a pilot study. A second conventional naturally ventilated stable without a veranda was also included in the investigations for special aspects. The aim of this study was to investigate this first turkey stable with a veranda and to give first recommendation for the practical use. The use of the veranda and the differences in behaviour between inside the stable with a veranda and inside the veranda as well as inside both stables were analysed. From 100 turkeys in each period we examined feather condition, walking ability and leg posture in the 9th, 13th and 17th week of age in both stables. In addition, body weight, food consumption and mortality were recorded. The results of this pilot study indicate that the tom turkeys of the line BUT Big 6 used the veranda without negative effects on health and performance parameters. There was some indication for an improved health of animals or a reduced mortality in the stable with veranda. The decrease of walking ability and normal leg posture with increasing age and body weight in both stables suggests that the genetic influence seems to have a higher influence than the enrichment of environment in this study using the fast growing line BUT Big 6. On the other hand, in this pilot study only one stable with a veranda and another commercial stable without a veranda at a different location were included. For this reason the results should be interpreted carefully with respect to possible effects in the management between the two farms. PMID- 16669194 TI - [Beak trimming in turkeys]. AB - In Germany, Bio-beaker and infra-red irradiation (PSP) are used for beak trimming in turkey chicks as routine methods to prevent feather pecking and cannibalism. By both procedures the animals suffer from extensive damage and considerable continuous pain. Beak trimming is allowed by animal welfare legislation, but the operation requires an official permission and must be carried out under anesthesia. The official intention, due to animal welfare reasons, to decrease this operation and to question it permanently fails because beak trimming without anesthesia is tolerated. Elaboration of alternative programs has been demanded for a long time. Therefore selection of stocks tending less to cause feather pecking and cannibalism as well as adequate keeping and housing conditions during hatching and rearing are proposed. PMID- 16669195 TI - The new E.U. Animal Transport Regulation: improved welfare and health or increased administration? AB - There is public discussion of the new E.U. Animal Transport Regulation No 1/2005 of Dec. 2004 and its advantages and draw-backs. This Regulation is no longer a Directive, so that it is directly applicable in the Members States. Although the Regulation is recognised to have great potential to improve welfare and health of transported animals, it will also increase administrative work. Most improvements will come through better education and the increased responsibilities of animal attendants, drivers, keepers and transport organisers, and through the stricter control mechanisms (log book, training, instructions etc.) and the introduction of the GPS control systems to further enhance the transparency of animal movements. The formats of the transport certificates used in all Member States will be harmonised. Technical records will be kept on air temperature and water consumption. Contact offices in all member states for transport affairs will improve the exchange of data between the responsible authorities and harmonise control and surveillance practice. Specific regulations are now in place for horses (broken, unbroken, registered) and for the transport age of young animals (piglets, lambs, calves, foals). In spite of some substantial improvements there are still significant gaps in our knowledge of both normal and long transports, for example optimal journey times, food and water supply on long transports, environmental factors such as vibration, motion, light and ventilation requirements in different European geographical regions. The same is true for the epidemiological aspects of the prevention of disease transmission; for example, very little is known about the bacterial and particulate emissions of the animal transport vehicles which travel across Europe. A serious drawback of the regulation is the fact that it does not abolish the unloading of animals on long transports to rest for 24 h at staging points, so that the concomitant risks to health and welfare remain, as do the opportunities for the transmission of infectious diseases at these stations. Two examples are given that demonstrate the physiological (heart rate) and biochemical (cortisol) reactions of cattle during transport. It seems useful to observe how the new Regulation affects practice and to assess the usefulness and practicability of the new rules after one or two years of operation. At the same time, more detailed research should be carried out in order to further adapt the Regulation to the needs of the animal species and gender in terms of travel time periods, food supply and resting schemes. This should also include reconsideration of loading and unloading rules in staging points on long journeys for all animals, particularly in view of risks of injury and transmission of infectious diseases. Investigations should be carried out to improve our understanding of the bio-aerosol emissions from driving and standing vehicles. Transport schemes on long journeys should be more closely adapted to the needs of the animals. The new Regulation represents another step forward toward improved animal welfare during transport in spite of some increase in the administrative measures required. It is necessary to bring together veterinary and animal science, engineering, logistics and practical and technical experience in order to improve the Regulation and the health and welfare of animals during transport. PMID- 16669196 TI - [Black flies--for horses a underestimated risk]. PMID- 16669197 TI - Specialist nurses must not be undervalued. PMID- 16669198 TI - How can the NHS function safely if nurse numbers are cut? PMID- 16669199 TI - A prescription for success. PMID- 16669200 TI - Bringing a new peace. PMID- 16669201 TI - The development of new treatments for lung cancer. AB - Lung cancer is the UK's biggest cancer killer and the most common type, non-small cell lung cancer, usually presents at an advanced stage. This article discusses the socio-political factors influencing the perception of the disease and outlines the latest treatments, including biological agents. PMID- 16669202 TI - Attention deficit hyperactivity disorder. PMID- 16669203 TI - Homeostasis part 3: temperature regulation. PMID- 16669204 TI - Assessing, diagnosing and managing neuropathic pain. AB - Neuropathic pain and its co-morbidities can be detrimental to a patient's quality of life, affecting both physical and psychological well-being. Diagnosing neuropathic pain can be difficult and some patients may have to wait up to 12 months to see a pain specialist. The nursing contribution to recognising and managing neuropathic pain is vital if patient care is to be improved. It is therefore necessary for nurses to understand the assessment, diagnosis and management of neuropathic pain. PMID- 16669205 TI - Safer medicine administration through the use of e-learning. AB - The administration of medicines to patients in acute hospitals can generate significant clinical risk. Nurses who undertake this must be competent and confident to practise. This article describes the development and implementation of an e-learning programme to educate and assess nurses in safe drug administration practice. PMID- 16669206 TI - Benefits of patient pathways in rheumatoid arthritis care. AB - Rheumatoid arthritis is a painful, inflammatory, chronic condition that often results in irreversible joint damage. This article uses the condition to highlight the benefits of patient pathways and illustrate how they can be developed. PMID- 16669207 TI - Mefloquine. PMID- 16669208 TI - Peripheral arterial disease: why screen in primary care? PMID- 16669209 TI - Pain management of mixed aetiology ulcers. PMID- 16669211 TI - How to recruit staff effectively. PMID- 16669210 TI - Mixed aetiology leg ulcers. AB - Irene Anderson and Brenda King outline the causes of mixed aetiology leg ulcers and the treatment of these complex wounds. PMID- 16669212 TI - A varied and dynamic environment. PMID- 16669213 TI - Rear view. PMID- 16669214 TI - [Latency of recognition of geometrical figures with sides or corners masked to a different degree in humans]. PMID- 16669215 TI - [The effects of simulated microgravity on characteristics of slow presaccadic potentials]. PMID- 16669216 TI - [Spatial organization of EEG activity during active hyperventilation (cyclic breath). I. General patterns of changes in brain functional state and the effect of paroxysmal activity]. PMID- 16669217 TI - [Changes in the reflex excitability of the soleus spinal center in humans performing various motor tasks]. PMID- 16669218 TI - [Measuring the distribution of plantar pressures during walking in patients with multiple sclerosis to evaluate treatment efficiency]. PMID- 16669219 TI - [Patellar reflex: I. Studies in healthy subjects]. PMID- 16669220 TI - [Endogenous attention in 5-month-old full-term and premature infants]. PMID- 16669221 TI - [Spectral analysis and interpretation of spectral components of heart rate variability]. PMID- 16669222 TI - [Adaptive changes in the myocardium of patients with ischemic heart disease]. PMID- 16669223 TI - [Heart rate variability as an autonomic index of conflict-induced behavior of individuals under emotional stress]. PMID- 16669224 TI - [Estimation of the local fraction of cardiac output using a two-phase model of pulsatile hemodynamics]. PMID- 16669225 TI - [Significance of chronotypic specificity of healthy individuals for the variability of cardiac rhythm]. PMID- 16669226 TI - [The glutathione-dependent system of placenta antioxidant defense in miscarriage]. PMID- 16669227 TI - [Intestinal endotoxin as a universal factor of adaptation and pathogenesis of general adaptation syndrome]. PMID- 16669228 TI - [Analysis of a series of significant genetic polymorphisms in athletes]. PMID- 16669229 TI - [Morphological and functional characteristics of 9- to 22-year-old female athletes involved in sports aerobics]. PMID- 16669230 TI - [Gastric emptying and motility in athletes with different psychological features]. PMID- 16669231 TI - [Adaptation potential of students and smoking]. PMID- 16669232 TI - [Immunophysiology of epithelial cells and pattern-recognition receptors]. PMID- 16669233 TI - [Method and computer-aided system of intensive health recovery for estimation and correction of the psychophysical condition of an individual]. PMID- 16669234 TI - [The state of the cardiovascular system in children aged 8-9 in the norm and in impairments of intellectual development]. PMID- 16669235 TI - [Parameters of immune status and axes of morphological typology]. PMID- 16669236 TI - [Activity of leukocyte lactate dehydrogenase isozymes in subjects of different ages]. PMID- 16669237 TI - [The IV All-Russian Symposium on Slow Oscillatory Processes in the Human Body and in the II School/Seminar on Nonlinear Dynamics in Physiology and Medicine (Novokuznetsk, Russia, May 24-27, 2005)]. PMID- 16669238 TI - [Cytophysiology of spiny stellate cells of striate cortex and their role in excitatory mechanisms of intracortical synaptic circulation]. AB - This review describes the spiny stellate cells (SCC) which constitute a unique category of cortical interneurons and represent a major component of intracolumnar, lateral and collosal excitation of neocortical pyramidal and nonpyramidal neurons. SCC axons establish contacts with apical dendrites of pyramidal neurons forming cartridge-type synapses en passant with them. SCC establish recurrent connections with inhibitory interneurons and other SCC and also form contacts on themselves through autapses. SCC subtypes are identified, their structure, neurochemical specialization and spatial organization in human and animal neocortex are described. On the basis of the results of author's own investigations and literature data, critical analysis of up-to-date concepts of the histophysiology of recurrent excitatoty and inhibitory neurocirculation in the cortical module, is presented. PMID- 16669239 TI - [Aging of the osseous tissue. Theoretical basis for new directions of mechanotransduction optimization]. AB - On the basis on literature studies and the results of personal studies, the authors describe ultra- and micro-structural changes in the osseous tissue associated with the process of aging. Functional disturbances in tissue processes causing osseous tissue loss and osteoporosis development are characterized. It is suggested that the described changes are induced by age-dependent hypermineralization of the osseous tissue with the consequent disturbances of mechano-transduction. A hypothesis is discussed concerning a novel approach to pharmacological treatment of osteoporosis. Development of a new group of drugs controlling the calcium exchange between the osseous compartment and the blood is considered. Presumed pathogenetic and clinical effects of these drugs are described. PMID- 16669240 TI - [Changes of the structure of neuronal connections in visual cortex of cats with experimentally induced bilateral strabismus]. AB - The spatial distribution of neuronal connections in cortical area 17 in cats with bilateral convergent strabismus, surgically induced on postnatal days 10-14, was investigated. Single cortical columns of areas 17 and 18 were microiontophoretically injected with horseradish peroxidase and retrogradely labeled cells in both hemispheres were detected. The intrahemispheric connections in area 17 along the mediolateral direction (the projection of visual field horizontal meridian) were found to be either dilated or reduced. For most columns, the interhemispheric connections in this direction were increased, which may provide for more reliable unification of two visual hemifields. Moreover, the extension of intra- and interhemispheric connections in rostrocaudal direction (along the visual field vertical meridian projection) was detected in some cortical columns. Thus bilateral strabismus induced during the developmental critical period, modifies the structure of both of intra- and inter-hemispheric spatial neuronal connections of individual cortical columns in areas 17 and 18. PMID- 16669241 TI - [The effect of streptokinase on the development of rat cerebral cortex cells in vitro]. AB - The aim of this study was to determine the effect of streptokinase (SK) on the ultrastructure of cellular elements in the cerebral cortex of newborn rats in vitro. Three series of cell cultures grown on DMEM were used, including those grown on the medium enriched with 15% fetal calf serum (control 1), cultures transferred to the depleted medium containing only 0.5% of this serum (control 2), and the experimental cultures, to which SK (2000 IU/ml) was added. Addition of SK to the medium prevented a reduction of the viability of mature (14 days) dissociated neocortical cell culture from 1-2-day-old rats, induced by a transfer of the culture to a blood serum protein-deficient medium. In a 7-day culture SK potentiated the decrease in the cell viability. In organotypical cultures, with the use of electron microscopy, it was found that SK in concentration used prevented the development of destructive changes in astrocytes, oligodendrocytes, and neurons of explants, induced by a deficit in serum proteins. The neurons contained numerous mitochondria, some of which had only a few cristae. Signs of destruction were observed only in neuronal nuclei. After exposure to SK for 48 hours an activation of oligodendrocytes (containing numerous myelin bodies) was noted which was accompanied by astrocyte disintegration (with hyperchromatic nuclei in the remaining cells). The neurons were resistant to SK exposure. PMID- 16669242 TI - [Structural and functional reorganization of the nonapeptidergic hypothalamic neurosecretory nuclei in experimental acute pancreatitis]. AB - The structures of pancreas and hypothalamic magnocellular nuclei (supraoptic and paraventricular) were studied using light and electron microscopes in acute pancreatitis induced experimentally in mature male rats. Histo- and organotypic transformations of the pancreatic parenchymal and stromal elements were analyzed with a simultaneous cytological assessment of the hypothalamic nonapeptidergic neurosecretory centers. A phenomenon of the inhibition of hypothalamic nonapeptide release at the level in axo-vasal complexes was established, that aggravated the outcome of the destructive and necrobiotic processes in pathologically changed organ. PMID- 16669243 TI - [Immunocytochemical characteristic of neurons of the mouse truncus sympaticus stellate ganglion in postnatal ontogenesis]. AB - Neurotransmitter content in stellate ganglion neurons was studied immunocytochemically in mice of different ages (newborns, 10-, 20-, 30- and 60 days-old). Majority of the stellate ganglion neurons in mice of all the groups studied were tyrosine hydroxylase (TH)-positive. Most of neurons with the immunore-activity to choline acetyltransferase (ChAT) were shown to be also TH positive in the newborn and 10-day-old animals. The percentage of neurons containing TH and neuropeptide Y increased from birth throughout all the age periods studied. The proportion of vasoactive intestinal peptide (VIP)- and ChAT positive neurons was maximal in 10-day-old animals and then decreased up to 60 days of age. The somatostatin- and galanin-reactive cells were absent in all the mice. Thus, the maturation of neurotransmitter composition is complete in the mouse stellate ganglion by the end of the second month of life. PMID- 16669244 TI - [Architecture of the rat ventricular myocardium]. AB - The working myocardium architecture was studied in heart ventricles of Wistar rats using the method of layer-by-layer dissection. Ventricle myocardium in the rat was found to be formed by three layers: superficial (subepicardial), middle and deep (subendocardial). The superficial and deep layers were present in both ventricles, while the middle one was found only in the left ventricle. The greatest variations were detected in the orientation of superficial layer muscle fibers: in the left ventricle subepicardial fibers had mainly a longitudinal arrangement, while in the right ventricle they were arranged transversely to the heart longitudinal axis. Most of the myocardial mass in the left ventricle, with the exception of the apex, was represented by a middle layer of circular fibers. In the deep layer, the fibers were oriented longitudinally, forming the trabeculae and papillary muscles. PMID- 16669245 TI - [Structural and functional changes in the right atrium of the rats induced by short- and long-term heat stress]. AB - In acute experiments performed on rats placed in heat-chamber (40 degrees C, 30- and 60-minutes-long exposure), an increase in NADPH-diaphorase (NADPH-d) and succinate dehydrogenase (SDH) activities were found in the neurons of intramural ganglia of the right atrium, while SDH and lactate dehydrogenase (LDH) activities were increased in cardiomyocytes. Hormone-producing cardiomyocytes, containing atrial natriuretic peptide, had numerous large secretory granules under the sarcolemma close to the microvessels. In contractile cardiomyocytes, the signs of early stage of apoptosis were observed including the subsarcolemmal accumulation of small mitochondria and cytoplasmic invaginations into the nucleus. Prolonged heating (37 degrees C, 4 h daily) led to an increased activities of NADPH-d and SDH in neurons of intramural ganglia of the right atrium and of NADPH-d and LDH in cardiomyocytes. Secretory granules in hormone-producing cardiomyocytes were small and reduced in number. Cardiomyocytes showing the signs of the late stage of apoptosis (with nuclear and cytoplasmic condensation) were demonstrated. PMID- 16669246 TI - [Intestinal microvessels in patients of different ages after an appendectomy]. AB - The numbers and diameters of microcirculatory bed vessels located in the wall of various regions of small and large intestine, were determined in 43 patients of lirst and second mature ages and in 19 aged and senile patients less than one year - more than ten years after an appendectomy. In was established that during the first year following the operation, the number of arterioles, capillaries and venules per standard area of the section in middle-aged patients was increased in the wall of caecum and ileo-caecal transition with a concomitant increase in the lumen of these vessels. These changes were most pronounced in capillaries, while arterioles were less affected. Vascular changes were expressed in the wall of caecum close to post-operational scar and in the wall of ileo-caecal transition, but in the wall of middle part of small intestine and middle part of colon transversum they disappeared. Three years following the operation, the numbers of microcirculatory bed vessels and their luminal diameters were similar to those found in people with an intact appendix. The changes described were not detected in aged and senile persons. PMID- 16669247 TI - [Morphological and functional characteristics of thyroid gland in the offspring of female rats with experimental chronic liver disease of various etiology]. AB - The peculiarities of structural and functional development of the thyroid gland were studied in the offspring of female rats with experimental chronic liver disease. Morphometric methods as well as immune enzyme analysis were used. Impairment in the development of thyroid gland structure was found in experimental animals, as evidenced by lower magnitudes of volume density of follicular and intrafollicular epithelium, as well as by reduced values of thyrocyte height. Morphological changes of the thyroid gland in the offspring of female rats with chronic liver disease were accompanied by the impairment of the gland endocrine function (decreased serum levels of T3 and T4). PMID- 16669248 TI - [Mast cells of the mammary gland and of the regional lymph node in rats with breast cancer induced by N-methyl-N-nitrosourea]. AB - Using the methods of light and electron microscopy, morphometric and functional characteristics of mast cell (MC) populations were studied in the mammary gland (MG) and regional lymph node (RLN) at different stages of breast cancer (BC) induced in 120 outbred female albino rats with the body mass of 120-150 g. BC was induced by subcutaneous injections of chemical cancerogen N-methyl-N-nitrosourea. MC numbers and activity in MG and RLN were found to increase at all stages of BC formation studied (64-180 days of cancerogen exposure). Gradual increase in MC degranulation rate and degree, MC disintegration closer to the center of the tumor, together with the appearance in the peritumorous tissues, in the tumor itself, and in RLN of numerous small, immature MC, by their morphometric parameters identical to the intestinal mucosal MC, may presumably indicate the decompensation phenomena in the system and of the approaching of process terminal stage. PMID- 16669249 TI - [Reparative bone regeneration after aseptic necrosis of the femoral head as influenced by 1-chloromethylsilatrane treatment]. AB - The aim of this work was to study the effect of 1-chloromethyisilatranum on the processes of reparative bone regeneration in experimentally induced aseptic femoral head necrosis. Aseptic femoral head necrosis was modeled in 4 Chinchilla rabbits by destruction of spongy substance of femoral neck and cutting of ligamentum teres of its head. Rabbits of the experimental group were injected with 0.4 ml of 1% solution of 1-chloromethylsilatranum on postoperational days 5, 10, 15 and 20 into the damaged femoral head and close to the damaged joint. Animal were sacrificed by air embolism 5 weeks from the start of an experiment. Necrotic and repair processes in hip joints from damaged and intact sides were assessed radiologically and histologically prior to sacrifice. In rabbits of both control and experimental groups extensive aseptic necrosis of hip joints was found. The osseous tissue of damaged hip joints of experimental animals contained the chains of osteoblasts actively producing new bone matrix. Foci of bone regeneration were found both in the deep and peripheral regions of the epiphysis, which is indicative of active processes of bone repair. The results obtained suggest that administration of organic-silicon drug 1-chloromethylsilatranum stimulates (optimizes) the processes of repair osteogenesis following aseptic femoral head necrosis in experimental animals. Application of this preparation appears to be very promising. PMID- 16669250 TI - [Experimental histological study of the articular cartilage of the external femoral condyle in tibial elongation in dogs]. AB - The tibia was elongated in 18 adult mongrel dogs with the rate of 1 mm per day produced with 60 increments (1st series, autodistraction) or 8 increments (2nd series, manual distraction). On completion of 28 days of distraction, followed by 30 days of fixation and 30 days without the fixator, the articular cartilage of the external femoral condyle was studied in the experimental and 3 intact animals using the methods of light and scanning electron microscopy, computer morphometry and stereometry. It was established that the structural changes in the cartilage during the experiment included destruction of the superficial area. The degree of destruction severity was directly related to the duration of the stage when the fixator was on. In a month after the fixator removal the return to the motor stereotype resulted in restoration of the cartilage surface in the 1st series, whereas in the second one the changes persisted. The studies performed revealed general features of the morpho-functional state of the articular cartilage in tibial lengthening in both experimental series, and showed that the autodistraction mode was less traumatic for the articular cartilage. PMID- 16669251 TI - [How to measure the structures, or new stereology. I. Sampling and orientation of samples]. AB - This article presents a brief review of newly developed methods of modern stereology for measurement of biostructures, which allow to avoid problems of uncertainty and technical difficulties peculiar to old stereology. In the first part of the review, a brief historical report on the development of new stereological approaches and their practical application to the selection and orientation of samples, is given. In particular, Cavalieri's principle, random systematic isotropic sampling, fractionator, isector, orientator and principle of vertical sections are described. Some details for practical aspects of measurements have been provided. PMID- 16669252 TI - [Immunocytochemical demonstration of brain neurons using NeuN as a selective marker]. AB - The aim of the present study was to find optimal protocol of immunocytochemical reaction to NeuN nuclear protein for light and laser confocal microscopy, which would exclude the antigen heat unmasking procedure that results in deteriorating the state of the tissue and requires expensive adhesive-coated slides. Best preservation of the antigen was found to be provided by fixation in zinc-formalin and Bouin's fixative (for a period of less than 24 hrs). Two protocols allowing to avoid antigen heat unmasking procedure during demonstration of the NeuN neuronal label in paraffin sections by the light and confocal microscopy are presented. PMID- 16669253 TI - [Methodological approaches to study characteristics of surgical anatomy of episublingual area and carotid triangle securing neck dissection within fascial sheaths]. AB - The studies performed in 91 non-fixed human cadavers included: layer-by-layer preparation of the tissues to the depth of the bottom of operation wound after cervical lymphadenectomy within the whole anterior triangle of the neck; layer-by layer preparation within the limits of anatomical zones and structures of the anterior triangle of the neck; study of the number and completeness of fascial sheaths in the same locations using the developed method. The anatomical findings obtained necessitate re-consideration of the conventionally described submandibular triangle medial and lateral borders in favor of their expansion. The study has defined the fascial plate dividing the contents of submandibular and carotid triangles and has specified the lines of tissue dissection with the purpose to improve the ablasticity of neck dissection operations for metastases of maxillofacial malignant tumors. PMID- 16669254 TI - [Conflict of cultures?]. PMID- 16669255 TI - ["Quality must be learned"]. PMID- 16669256 TI - [From patient to patient]. PMID- 16669257 TI - [Do you speak German?]. PMID- 16669258 TI - [Addressing needs individually]. PMID- 16669259 TI - [The focal point is the person]. PMID- 16669260 TI - [What foreign patients should be like]. PMID- 16669261 TI - ["The values have shifted"]. PMID- 16669262 TI - [Changes due to acculturation]. PMID- 16669263 TI - [Cultural sensitivity eases reciprocal relations]. PMID- 16669264 TI - [From the culture of dying to individual terminal care]. PMID- 16669265 TI - [Invisible danger from water]. PMID- 16669266 TI - [Disability aids provide safety at critical moments]. PMID- 16669267 TI - [Successful is the nurse who reaches the client]. PMID- 16669269 TI - [Like a tsunami: diabetes wave floods the whole world]. PMID- 16669270 TI - [More modest use in secondary prevention, risky in primary prevention]. PMID- 16669268 TI - [Fall incident reports in theory and practice: recommendations for developing of practice-oriented fall incident reports in nursing homes]. AB - Nursing home residents are known to be subject to a special risk for falls and fall-related injuries. The collection and analysis of root-causes and conditions of a fall in fall incident reports represent a vital instrument for secondary prevention and quality management. Preceding falls are proven to be one of the most important risk factors for repeated falls. Therefore it is recommended to report and evaluate the situation, in which the fall happened, in order to develop strategies for preventing repeated falls and for minimising the consequences of a fall. The question arises, how a fall incident report should look like, to meet both the scientific requirements and the expectations of the user in practice. To answer this question, six fall incident reports selected from theoretical and practical sources were described and analysed. After systematic analysis of the related German-language literature and conduction of some partly-structured interviews with different user groups the goals and requirements for fall incident reports were identified. Based on these targets and requirements as well as on the recommendations of national experts and scientific literature, criteria for evaluating fall incident reports were developed. None of the selected reports meets all the criteria. Finally, based on the theoretical and practical recommendations, components for fall incident reports were developed which meet both the criteria found. These components are guidelines for healthcare action based on the care process and, if followed properly, an important tool for fall prevention and for protecting nursing homes against liability claims. PMID- 16669271 TI - [Odyssey with severely injured patients]. PMID- 16669272 TI - [Good emergency medicine is expensive (interview by Dr. med. H. Gross)]. PMID- 16669273 TI - [A cancer--or perhaps not?]. PMID- 16669274 TI - [What is recommended in rosacea?]. PMID- 16669275 TI - ["Tubinger bomb" is now personally mixed]. PMID- 16669276 TI - [Secondary dyslipoproteinemia--genes and lifestyle are not always responsible for high cholesterol levels]. PMID- 16669278 TI - [Treatment of dyslipoproteinemia in chronic renal failure]. AB - In patients with chronic kidney disease, treatment with statins presumably not only has an influence on cardiovascular endpoints, but also delays the progression of the renal disease. Recommendations for the treatment of lipid metabolism disorders in the general population with normal renal function are based on numerous prospective, randomized and placebo-controlled studies. In contrast, recommendations for patients with pathological renal function can merely be extrapolated from the results of those studies. Post hoc analyses from the large pravastatin studies confirm a significant risk reduction of primary cardiovascular and cerebrovascular end points for the CDK stages 2 and 3. For CKD stages 4 and 5, available data are merely rudimentary, so that the results of ongoing studies will have to be awaited. PMID- 16669277 TI - [Diabetic dyslipoproteinemia]. AB - Diabetic dyslipoproteinemia is considered to be an integral component of type 2 diabetes mellitus and the metabolic syndrome. Major pathogenetic factors include abdominal obesity, insulin resistance and hyperglycemia with increased hepatic secretion of triglyceride-rich lipoproteins. Elevated concentrations of triglycerides and cholesterol, together with decreased HDL cholesterol are therefore found. LDL cholesterol is either normal or only slightly increased, but at the same time, the composition of the LDL particles is altered. This metabolic disorder contributes considerably to the clearly elevated cardiovascular risk. Dyslipoproteinemia, usually in the form of hypertriglyceridemia, may also occur in type 1 diabetes mellitus. Since general measures and good blood sugar control often fail to achieve the desired lipid levels, many patients require medication, initially usually statins, but, where necessary, combination treatment. In patients with isolated hypertriglyceridemia, treatment with fibrates may also be considered. PMID- 16669279 TI - [Secondary dyslipoproteinema in liver disease]. AB - By far the most common dyslipoproteinema in patients with liver disease is hypertriglyceridemia with decreased HDL cholesterol occurring in fatty liver diseases. Since these latter frequently occur in patients with a metabolic syndrome, it must be assumed that lipid metabolism disorder is associated with a pronounced atherogenic effect. This means that--in contrast to cholestatic liver disease--treatment is almost always indicated. General therapeutic measures (weight reduction, optimization of blood sugar) are to the fore. As medication, metformin, fibrates and insulin sensitizers may be considered since, apart from improving glucose metabolism and hypertriglyceridemia, they also have a direct hepatic effect. It must, however, be noted that the available study data are not yet satisfactory, so that an individual decision must be made as to whether medical treatment of a lipid metabolism disorder makes good lipidological sense and is hepatologically justified. PMID- 16669280 TI - [Stalking--what does the family doctor need to know?]. AB - Stalking is a widespread phenomenon, which for the stalked person is associated with a not inconsiderable risk of becoming the victim of an act of violence. Furthermore, stalked persons may develop significant psychological problems, and therefore often seek out a doctor as their first contact. Another reason for this is the still unsatisfactory legal situation and widespread ignorance of the stalking problem on the part of the police and the courts. In the light of the above, it is of great importance that the physician should thoroughly familiarize himself with the problems associated with stalking. PMID- 16669282 TI - [Benefits of antihypertensive treatment. How much damage does it really prevent?]. PMID- 16669285 TI - [AOK pays for bureaucracy relief]. PMID- 16669283 TI - [AIDA--results of a large post-marketing product surveillance study of maintenance asthma therapy with once-daily ciclesonide]. PMID- 16669287 TI - [Drug combination helps in goiter caused by iodine deficiency]. PMID- 16669281 TI - [Laparoscopic resection of the colon]. AB - In terms of achievable outcome, laparoscopic resection of the colon is equally as good as the conventional open procedure. The sole contraindications are patients with severe cardiopulmonary disease, massive adhesions, and peritonitis following a perforation (relative contraindication). The question as to whether the minimally invasive approach is justifiable from the oncological point of view remains to be clarified in further studies. PMID- 16669288 TI - [Melancholy in artistic diversity and expression]. PMID- 16669289 TI - [Too much sun to the ear]. PMID- 16669286 TI - [Assess the patients' necks!]. PMID- 16669284 TI - [Help for the mistreated ear canal]. PMID- 16669291 TI - [Gagging during oral health care treatments. Available diagnostic and treatment tools]. AB - The aim of this paper was to explore the diagnostic and treatment tools for patients who gag during oral health care treatment. Reference to such tools is not available in scientific literature. For diagnostic purposes, a questionnaire was developed. In due course the results of its reliability and validity will be published. As yet there is no preferred method of dealing with the problem of patients' gagging. A recommended option to treat the problem is based on a cognitive behavioural perspective with gradual exposure in vivo. In cases of extreme gagging, teamwork of oral health care provider and psychologist seems to be a prerequisite for success. PMID- 16669290 TI - [Maxillofacial growth and dental implants in the maxillary anterior region]. AB - Single tooth replacement with a dental implant is an increasingly popular solution in patients with loss of an upper anterior tooth. There is a risk, however, of placing the implant prematurely in youths. In a case series the effects of residual maxillary growth on the results of implant treatment in the anterior maxillary region were measured. The results confirm findings from other studies on this subject and indicate that cessation and degree of vertical growth are unpredictable. This may result in infraposition of the implant and lead to periodontal bone loss around the implant region and its neighbouring teeth. Clinicians should be aware of this and inform their patients that these changes may eventually compromise the aesthetic result of the treatment. PMID- 16669293 TI - [Modernisation of the design of clinical trials for caries preventive agents]. AB - In January 2002 the International Consensus Workshop on Caries Clinical Trials was organised in Scotland. The meeting was an initiative of both academic and industrial partners, in order to arrive at a consensus about ways to modernise the design of clinical trials for caries preventive agents (Caries Clinical Trials or CCTs). All presentations delivered at the workshop and the consensus statements formulated at the end of the workshop were published last year in a special issue of the Journal of Dental Research. In this paper some important aspects are highlighted and the workshop conclusions are presented. PMID- 16669292 TI - [Children's distress during application of local anaesthesia. Computerized tool versus traditional syringe]. AB - This study compared the level of distress in children during the application of local anaesthesia by a computerized tool (Wand) and by a traditional syringe. The influence of children's mild or extreme anxiety regarding the dental procedure was also determined. 125 children between the ages of 4 and 11 years old participated in this study. Their discomfort was recorded on videotape and marked on the Venham scale and the Observational Scale of Behavioral Distress at 15 second intervals. Parents rated their child's level of anxiety on the Dental Subscale of the Children's Fear Survey Schedule. The results indicate that mildly anxious children suffered less distress and showed less muscle tension, verbal protest and body movement during the first 2 intervals when the computerized tool was used. However, the maximum distress score at the moment of injection was similar in both methods. There was no difference found in the level of distress in the highly anxious children. PMID- 16669294 TI - [Macroglossia]. AB - A 63-year-old woman was referred to a department of oral and maxillofacial surgery because of slowly increasing enlargement of the tongue. Histopathologic examination of a biopsy showed the presence of amyloid. Further physical and laboratory studies disclosed the presence of multiple myeloma (Kahler's disease). A few years before, the patient had been operated for metacarpol syndrome of both hands, most likely because of amyloid deposition. The patient underwent a stam cell transplantation. After one year follow-up the patient is still alive. There was no distinct regression of the macroglossia. PMID- 16669295 TI - [Non ordinary radiopacity on a panoramic radiograph]. AB - A dental panoramic radiograph of a 56-years-old Sudanese woman revealed calcifications, which could be attributed to atherosclerotic lesions of the carotid artery. The occurence of these lesions is related to the incidence of cardiovascular and cerebrovascular diseases, such as transient ischemic attack or cerebrovascular accident. Carotid calcifications can be detected in 2 to 3.8% of panoramic radiographs of patients over 50 years of age. If detected early, treatment of the disease and the underlying risk factors can significantly reduce vascular related complications. There is a significant role to be played for dentists in early referral of patients. PMID- 16669296 TI - [Coagulation abnormalities and the dentist]. AB - Defects in the coagulation system increase the risk of excessive bleeding during and after invasive dental procedures. This increased risk of bleeding is irrespective whether the coagulation disorder is inherited or acquired, for example by using anticoagulant medication. However, the cause of the coagulation disorder dictates preventive measures to be taken. Therefore, a careful medical history is crucial to identify and characterize patients with coagulation abnormalities. Recently, the insight in the mechanism of coagulation has increased considerably. This has resulted in specific procedures to adapt the level of coagulation required for specific invasive dental procedures. The dentist must discuss this temporary alteration of the coagulation level with the medical specialist of the patient. PMID- 16669297 TI - Interface pressure measurement during surgery: a comparison of four operating table surfaces. AB - OBJECTIVE: To compare the pressure-reducing and pressure-redistributing characteristics of four operating room (OR) table mattresses using interface pressure measurements, in two positions adopted for surgical procedures. METHOD: Support surfaces were randomly assigned to 80 patients. These were: an overlay pad filled with fibres (the standard OR mattress), a custom-made viscoelastic polyurethane foam mattress, an inflatable mattress with air-filled cells and a fluid mattress. An XSENSOR full-body pressure-mapping pad was used to record interface pressures of 40 patients in the supine position and 40 patients in the lithotomy position. Measurements were analysed for peak pressure, peak pressure index, total contact surface area and the occurrence of a significant increase in interface pressure during the surgical procedure. RESULTS: The highest interface pressures were measured on the standard mattress, in both the supine and lithotomy position. Overall, the fluid mattress showed the best pressure-reducing and pressure-redistributing capacities. CONCLUSION: As long as no reference values are available for interface pressures under which no pressure-related damage will occur, clinical testing of OR table surfaces is still necessary. PMID- 16669298 TI - Ingredient-based wound dressing classification: a paradigm that is passe and in need of replacement. AB - Wound dressings are classified according to their ingredients, but in many cases dressings within the same group have different recommended uses and even ingredients. Should future classifications be based on dressing functions? PMID- 16669300 TI - Let's put education before sponsorship. PMID- 16669301 TI - The clinical and psychosocial challenges associated with bilateral hand traumas. AB - This case study highlights the importance of providing holistic and multidisciplinary care when managing patients with complex bilateral traumatic hand wounds. Such collaboration can achieve optimum results. PMID- 16669302 TI - Can transcutaneous oxygen tension measurement determine re-amputation levels? AB - OBJECTIVE: To review the preoperative transcutaneous oxygen tension (TcPO2) measurements in patients having major lower leg amputation, and also consider the re-amputation rate, wound infection and the definitive level of amputation. METHOD: A case-control study was performed in a consecutive cohort of 170 patients (1999-2003). Fifty-two patients underwent preoperative TcPO2 measurements (cases) and 118 patients did not (control). Multiple logistic regression analysis was performed to analyse independent risk factors associated with re-amputations. RESULTS: Primary and definitive (in case of a re-amputation) amputation levels were lower in the TcPO2 group, although this did not reach statistical significance. The number of re-amputations in the TcPO2 group was significantly higher: 15 versus 18 patients (p=0.039). Selection of an amputation level with aTcPO2 of 30mmHg resulted in a positive predictive value of re amputation of 41% and a negative predictive value of 90%. A cut off value of 20mmHg resulted in 41% and 77% respectively. CONCLUSION: The use of TcPO2 measurements for major amputation level selection resulted in an increased rate of re-amputation. However, there was a trend in gaining a more distal definitive amputation level. Selection of an amputation level solely based on a TcPO2 value is unreliable. PMID- 16669303 TI - Late presentation of abdominal aortic occlusion in a patient with spinal cord injury. AB - Although pressure ulcers are a common problem in patients with paraplegia, pressure is not the only cause of them. This case report illustrates that distal abdominal aortic obstruction resulted in multiple ulcers in a paraplegic patient. PMID- 16669304 TI - Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers. AB - OBJECTIVE: Hypochlorous acid (HOCl) is a highly microbiocidal agent active against bacteria, viruses and fungi. Using quantitative microbiology, preliminary studies showed it achieved an appreciable reduction in the bacterial burden in chronic venous leg ulcers. The study aimed to determine whether it has a role as an additional treatment for chronic venous ulcers that have not healed with conventional treatment. METHOD: On the basis of previous reports we designed a study in which patients acted as their own controls, in that only patients who failed to achieve a 44% reduction in wound size with standard treatment (compression bandaging) received HOCl washes. RESULTS: Of 30 patients admitted to the study, 10 achieved a 44% ulcer reduction after three weeks of standard treatment. In addition to the standard compression treatment, the remaining 20 patients were given HOCl washes over 12 weeks. Of the 20 ulcers, nine (45%) healed and five (25%) reduced in size by over 60%. All patients became free of pain. CONCLUSION: These findings confirm the clinical efficacy of treating venous leg ulcers with hypochlorous washes. Use of HOCl washes as an adjunctive therapy for recalcitrant venous leg ulcers appreciably increases healing and rapidly relieves pain. PMID- 16669299 TI - Microbial ecology associated with soft-tissue infections. PMID- 16669305 TI - An exploration of the use of ultrasound in the treatment of chronic venous leg ulcers. AB - A nurse-led multicentre randomised controlled trial will compare the clinical effectiveness of weekly ultrasound combined with standard care in the treatment of 'hard-to-heal' venous leg ulcers. Recruitment started last autumn. PMID- 16669308 TI - Accumulation of heavy metals in some species of lichens in south Tamilnadu, India. AB - Lichens are lower group of plants. They accumulate heavy metals and have been used as bio-indicators in pollution monitoring. Nine lichen species were collected from urban, rural and hilly areas in the southern part of Tamilnadu. Accumulation of metals differed with species and also with respect to their locations. Among the nine species, Pyxine petricola collected from urban areas showed greater accumulation of the heavy metals i.e., copper, zinc, lead, nickel, chromium and cadmium. PMID- 16669306 TI - Epidemiology, aetiology and management of abnormal scarring: a review of the literature. AB - Keloid and hypertrophic scars are the result of abnormal processes in scar formation. This paper reviews the literature and the many debates concerning the processes that cause abnormal scarring. PMID- 16669307 TI - Adsorption of Ni (II) using agrowaste, rice husk. AB - The present investigation was carried out to study the removal of nickel using a non-conventional adsorbent, rice husk. The effects of contact time, adsorbent dosage, pH, concentration of the metal ion and isotherm models are studied in a batch process. A steady trend was observed for all variables. The adsorption percentage increased with increase in contact time, adsorbent dosage and pH, but there was a decrease in adsorption with increase in concentration of the metal ion. K, the adsorbent capacity and 1/n, the adsorption intensity values showed that rice husk, a non-conventional adsorbent is efficient in removing Ni (II) from an aqueous solution. The adsorption data fitted well into Freundlich adsorption isotherm. PMID- 16669309 TI - Effect of Cr in irrigation water on germination and growth of French beans (Dolichos lablab L.). AB - The waters containing chromium (Cr) mainly from industries enter into the sewers, municipal treatment plant, streams and lakes that are used for irrigation purposes. The effect of Cr in irrigation water on seed germination and growth of French bean (Dolichos lablab L.) was investigated. Germination studies were conducted in the lab and field conditions. Seeds were treated with different concentrations (0 to 25 mg/1) of Cr in irrigation water. The percentage germination of seeds and plant growth showed a gradual decrease with increase in Cr concentration. Stimulatory effect in growth of plant was observed at lower concentration (1 to 2 mg/l). Accumulation of Cr was in the order of Root > Leaf > Stem > Pod. There was an increase in the total concentration of Cr content of soil with increase in Cr concentration in irrigation water. PMID- 16669310 TI - Leaching study of trace elements from coal ashes: A case study of Bokaro Thermal Power Station "B". AB - Electricity generation has been increasing at a rapid rate due to rapid industrialization and changing life styles. In our Country coal is the major source of energy since India has vast reserves of thermal power grade coal. Nearly every naturally occurring element is likely to be present in coal and these get entertained in the resultant coal ash. Indian coals contain high ash coupled with low calorific value and consequently resulting huge amount of coal ashes. This ash accumulates in on site piles and ponds thereby resulting in serious environmental problems particularly trace elements contamination of ground and surface waters. This study envisages the environmental assessment of coal ashes from Bokaro Thermal Power Station- Leaching study of coal ashes was made through analysis of leachates from open percolation leaching column experiments over a period of 300 days. Trace elements were observed within the regulatory limits. Many of the trace elements evaluated viz. Ni., Co, Se, Al, As, B, Ba, Sb, Hg, were observed at below detection limits of AAS. Na, K, Ca, Fe, Pb, Cd and other dissolved ions leached at significant concentration levels. This study suggests low cost high volume utilisation of bottom ash as fill material for reclaiming surrounding abandoned mined out areas in an environmentally acceptable manner. PMID- 16669312 TI - Removal of phenol and chlorophenols from water by coir pith carbon: equilibrium and rate studies. AB - Batch mode studies were conducted to study the removal of phenol, 2,4,6 Trichlorophenol (TCP) and Pentachlorophenol (PCP) from aqueous solution on coir pith carbon by adsorption process under varying experimental conditions such as agitation time, adsorbent dose, pH and temperature. Kinetics of adsorption obeyed second order rate equation and the rate constant was found to be in the range 0.0098-0.0672, 0.0949-0.8801 and 0.172-0.305 g/mg/min for phenol, TCP and PCP respectively. Equilibrium adsorption data follow Langmuir isotherm for phenol and PCP and the adsorption capacities were found to be 48.3 mg and 3.7 mg/g, respectively. For TCP, adsorption followed Freundlich isotherm only. Acidic pH was favorable for the adsorption of all the chlorophenols. Studies on pH effect and desorption show that chemisorption seems to play a major rule in the adsorption process. The positive values of H0 24.99, 18.69, and 8.907 kJ/mol for phenol, TCP and PCP respectively, confirm the endothermic nature of adsorption. PMID- 16669314 TI - An environmental profile of brick kilns in Lucknow. AB - Twenty two kilns in Lucknow were studied to identify the environmental hazards posed by the brick kilns and to quantify the environmental degradation which could be attributed to this industry. Study variables comprised SPM and RSPM levels, metal concentrations and total silica content in soil, temperature, humidity and noise levels in the work environment. Information's about the nature and type of kiln, fuel and water sources was also collected. 18.18 per cent of the kilns had moving steel chimneys with less than prescribed height. Approximately 1.89 + 0.87 acre of fertile agricultural land was used for manufacturing of clay brick per kiln, resulting in land degradation decrease in herb density and nutrient disorders in plants/trees in immediate vicinity. SPM level (93.3 mg/m3) was found to be much higher than the prescribed limit. The workers engaged in these kilns were at risk from dust and heat related diseases. PMID- 16669311 TI - Nutrient concentration in groundwater of Pondicherry region. AB - The surface and groundwater samples were collected from urban, rural areas, shallow and deep aquifers in Pondicherry region. The samples were analysed for various nutrients such as Nitrate Nitrogen, Nitrite Nitrogen, Ammonia Nitrogen, Phosphate and Dissolved silica. The elevated concentrations often fold increase against a background concentration of less than 2 mg/L of nitrates was observed in the study area. The maximum nitrate concentration (22 mg/L) in water is well within the limits of 45 mg/L prescribed by World Health Organization. The nutrients were observed about 2 to 3 times higher in groundwater of urban areas than in rural areas, indicating abundant leaching of nutrients from municipal waste and sewage effluent. The nutrients displayed a trend of higher concentration in shallow groundwater and lower in the water in deep aquifer and tank water. The higher concentrations of dissolved silica (13 mg/L to 100 mg/L) against an average concentration of 5 mg/L in the ground water of unreactive aquifers shows the presence of amorphous silica in the subsurface formations, slightly acidic to neutral nature of water in aquifers and also interaction between formation material and water. PMID- 16669313 TI - Diurnal and monthly variation of indoor radon and thoron progeny concentrations at a hillside place of northern India. AB - The natural radioactivity levels all over the world can create health problems due to the inhalation of radioactive aerosols. Radon and thoron progeny content of indoor air have major contribution to natural radiation dose. In the present study, simultaneous indoor measurements of radon and thoron progeny concentrations have been carried out over a period of four months at a low activity hilly area of northern Punjab, India to see their diurnal and monthly variations. The method used for this purpose is based on the defined solid angle absolute beta counting of radioactive aerosols sampled on a filter. The average values of equilibrium equivalent radon and thoron concentrations were 5.20 Bq m( 3) and 0.235 Bq m(-3), respectively, over the period August to November 2003. Both, EECRn and EECTh show a negative correlation with temperature. PMID- 16669316 TI - Removal of chromium by two aquatic pteridophytes. AB - In the recent years, the use of phytoremediation for ameliorating metal pollution has been gaining ground. This study explores the. possibility of utilising two common aquatic ferns, Salvinia molesta Mitchell and Azolla rubra R.Br. for scavenging chromium from solutions. Chromium removal from spiked solutions ranged from 40-99% during the seven day exposure. Results reveal their bioremoval capabilities and potentialfor use in wastewater decontaminant PMID- 16669315 TI - Polycyclic aromatic hydrocarbons in the ambient air of Jeedimetla Industrial Development Area-Hyderabad. AB - Polycyclic aromatic hydrocarbons (PAHs) have received increased attention in recent years in air pollution studies, as some of these compounds are highly carcinogenic or mutagenic. PAHs are mainly associated with respirable suspended particulate matter (RSPM) (diameter <10 m in size). Hence, it is important to determine the concentration of PAHs adsorbed on RSPM to know the extent of human exposure to these potentially carcinogenic substances. The study was carried out at 10 locations in and around the Jeedimetla industrial development area, Hyderabad during April 2000. Higher concentrations of B(a)A and B(b)F were observed at Nandanagar, Py and B(a)P at Kukatpally while, F1 concentrations at Allwyn Colony. These variations in the concentration can be attributed to their source and climatic conditions during sampling. PMID- 16669318 TI - Manage the disease (not cost) of addiction. PMID- 16669319 TI - A treatment planning reality check. PMID- 16669320 TI - Putting schizophrenia's demons to sleep. PMID- 16669317 TI - Improving environmental conditions of a slum in Chandigarh by an awareness campaign. AB - An intervention project, in the form of an environmental awareness campaign was conducted in a slum of Chandigarh with a population of about 40,000, from December 1998-April 1999. The activities enforced during this campaign included focus group discussions with general public and mothers, household visit by medical students, distribution of literature, use of mass media by involving local residents, primary child care workers, teachers and school children. Evaluation of this campaign was carried out in May 1999 and compared with the baseline data obtained from a survey done in 1997. The study revealed that the method of storing water in covered buckets and utensils had improved significantly from 14.3% and 4.7% in the baseline survey to 35.2% (P < 0.001) and 16.2% (P < 0.01) respectively. Refuse disposal in the community bin and own bin had significantly improved to 22.2% (P < 0.001) and 19% (P < 0.01) from 8.4% and 11.2% respectively in the baseline survey. 51.2% of the residents using own bins disposed the refuse finally into the community bin. The use of community latrines had significantly increased among males (50.0%), females (47.7%) and children (41.2%) after intervention as compared to 32.8%, 32.5% and 16.7% respectively as found in the baseline survey (P < 0.001) and similarly open defaecation had significantly reduced among them. Further the study shows that the awareness regarding diarrhoea as hazard of unsafe water had improved significantly from 28.7% in baseline survey to 55.6% after intervention (P < 0.001). Knowledge of the residents regarding hazards of refuse disposal in the open and open defaecation had also improved significantly. Simple environmental awareness campaign can change the knowledge and behavioural practices of slum dwellers and could be a model for launching similar projects in other slums of India and other developing countries. PMID- 16669321 TI - Missouri's experiment to increase quality--and decrease costs. PMID- 16669322 TI - Diving into a 'Blue Ocean Strategy'. PMID- 16669323 TI - Caring for today's elderly--and preparing for tomorrow's. PMID- 16669325 TI - Ending the cycle of abuse. PMID- 16669324 TI - Normalizing the patient environment. PMID- 16669326 TI - Make sure a vendor can get the job done. PMID- 16669328 TI - Evaluation and analysis of the noise quality of Ambur, Tamil Nadu, India. AB - The noise levels of Ambur town were studied in silence, residential, commercial and industrial zones. Noise levels were assessed in 22 locations in a typical peak and non-peak hours of a day. In non-peak hours, a gradual decrease in noise levels is detected. The results show that the noise pollution in the city is widespread throughout most of its area. The noise in these areas is composite in nature and generated from many sources near and far with no particular sound predominance. Based on the results, some remedial measures were suggested. Public participation, education, traffic management, structural designing play a major role in noise management. PMID- 16669329 TI - Assessment and mapping of water pollution indices in zone-III of municipal corporation of hyderabad using remote sensing and geographic information system. AB - A preliminary survey of area under Zone-III of MCH was undertaken to assess the ground water quality, demonstrate its spatial distribution and correlate with the land use patterns using advance techniques of remote sensing and geographical information system (GIS). Twenty-seven ground water samples were collected and their chemical analysis was done to form the attribute database. Water quality index was calculated from the measured parameters, based on which the study area was classified into five groups with respect to suitability of water for drinking purpose. Thematic maps viz., base map, road network, drainage and land use/land cover were prepared from IRS ID PAN + LISS III merged satellite imagery forming the spatial database. Attribute database was integrated with spatial sampling locations map in Arc/Info and maps showing spatial distribution of water quality parameters were prepared in Arc View. Results indicated that high concentrations of total dissolved solids (TDS), nitrates, fluorides and total hardness were observed in few industrial and densely populated areas indicating deteriorated water quality while the other areas exhibited moderate to good water quality. PMID- 16669327 TI - Removal of nickel ions from industrial plating effluents using activated alumina as adsorbent. AB - Removal of nickel ions from industrial effluents has been studied using activated alumina prepared by the galvanic oxidation of aluminum metal at ambient temperature as the adsorbent. The effect of various factors, such as initial concentration of nickel, contact time, dose of adsorbent and pH of the solution has been investigated. Batch and column type of adsorption studies have been made. The results indicate that the adsorption process is favored at pH 9. The adsorption data were fitted with suitable adsorption isotherm. The optimum conditions for the best adsorption have been evaluated for the following factors: dosage, contact time, pH, initial concentration of nickel ions and temperature. The process of regeneration of the adsorbent has also been studied. PMID- 16669330 TI - Electrochemical oxidation of pharmaceutical effluent using cast iron electrode. AB - Electrochemical oxidation of low (BOD/COD) ratio pharmaceutical wastewater was investigated in this study, using cast iron electrode. The batch experimental results were assessed in terms of COD and BOD concentration while the recalcitrance was monitored in terms of change in the (BOD/COD) ratio during the process. The effects of operating parameters like pH, electrolysis duration and current density were studied on the treatment efficiency and their operating ranges were experimentally determined. The efficiency and energy consumption of anode were estimated. Cast iron electrode has been found to be effective in removing 72% COD after 2hours of electrolysis. In particular, it was found that the (BOD/COD) ratio had improved from 0.18 to 0.3 after 120 min. of electrolysis indicating improvement of biodegradability of wastewater. It has been found, the pharmaceutical wastewater could be effectively pretreated by anodic oxidation. PMID- 16669331 TI - Water quality aspects of some wells, springs and rivers in parts of the Udhampur District (J & K). AB - Water samples were collected from wells (9 Nos.), springs (11 Nos.) and rivers/streams (6 Nos.) during pre and post monsoon seasons in the months of June and October 1999, respectively. Various physico-chemical parameters and trace elements (viz., Cu, Mn, Zn, Co, Fe, Ni, Cr and Pb) were analysed to evaluate drinking water quality on the basis of BIS and irrigation water quality on the basis of salinity, sodicity, residual sodium carbonate, and concentration of toxic elements. The study showed alkaline nature of surface and ground water. Calcium and magnesium are dominating cations and bicarbonate is major anion in the study area. At some locations the concentration of TDS, Mg, Ca, total hardness, Fe, Mn and Cr exceeded the limits set up for drinking purposes. Water quality evaluation for irrigation purposes on the basis of SAR values indicates excellent category of water. PMID- 16669332 TI - Study of dust pollution caused by traffic in Aligarh city. AB - In the present study the dust pollution caused by vehicles in Aligarh city was estimated. Four major national roads viz. Anoopshahar road, Delhi road, Agra road and Kanpur road of Aligarh city were selected. The dust pollution was maximum on Kanpur road (46.44 gm/m2/ month) followed by Agra road (38.94 gm/m2/month) and Delhi road (34.52 gm/m2/month). The least dust pollution was recorded on Anoopshahar road (20.10gm/m2/month). In general, the dust fall rate per unit area was highest at 3 km inside city (38.66 gm/m2/month) closely followed by starting point (37.27gm/m2/month) and least at 3 km outside city (29.09 gm/m2/ month). The average dust fall rate per unit area was estimated to be about 35 gm/m2/month in Aligarh city. PMID- 16669333 TI - Studies on the sorption of As(III) on the synthetic gel sodium potassium fluorophlogopite. AB - Sorption of As(III) has been studied on the synthesized gel close to Sodium potassium fluorophlogopite [Na0.5K0.5Mg3 (Si3 AlO10) F2] as a function of initial solution concentration at pH 2,4 and 7. The effect of parameters like equilibration time (0.5 - 24.0 hr), weight of the exchanger (30-150 mg) and temperature (25 degrees - 45 degrees c) has been investigated. The uptake of metal ion, As(III) has been expressed in terms of distribution coefficient i.e. (kd) values. PMID- 16669335 TI - Rectangular surface aerators. AB - Aeration experiments were conducted in two rectangular surface aeration tanks of L/B ratios 1.5 and 2 along with a square tank (L/B=1) to study their relative performance due to shape on oxygen transfer process while re-aerating the same volume of water such that the cross-sectional area of all the three tanks is the same. An identical rotor with six flat blades was used in all the three tanks. The geometric dimensions, which were developed to produce maximum aeration in square tanks by a previous study, were used in the present study as well. Results have confirmed that at lower dynamic conditions of the rotor the oxygen transfer coefficient is maximum in square tanks followed by rectangular tank of L/B=2 and it is the least in rectangular tank of L/B=1.5. However, the rectangular tank of L/B=1.5 produces always lower k values for any given rotor speed when compared to square tank, whereas the performance of rectangular tank of L/B=2 is marginally better than square tanks when rotor speeds are higher. PMID- 16669334 TI - Quality control in use of laboratory glasswares for environmental applications. PMID- 16669336 TI - Determination of trace elements in dairy milk collected from the environment of coal-fired power plant. AB - In the present study the environmental effects on herbivores mammals in and around Coal-fired power plant were studied by collecting the various milk samples of Cow and Buffalo in clean polyethylene bottles. Milk samples collected at five different locations along the banks of the Paravanaru river in and around Neyveli area. These samples were prepared for trace metal determination. The concentration of trace metals (Cu, Zn, Ni, Cd, Cr, Mn, Co and Hg) were determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) and Cold Vapour Atomic Absorption Spectrometry (CVAAS). It is observed that the samples contain greater amounts of trace metals than that in the unexposed areas. Obviously the milk samples are contaminated with these metals due to fly ash released in such environment. PMID- 16669337 TI - Hair as an indicator for assessing adverse effect of cadmium on human health. AB - Occupational health problems of 240 male workers of roadways workshop, carriage workshop and battery factory were investigated. Cadmium concentrations in the hair were determined using atomic absorption spectrophotometer. Health effects, such as acidity, hypotension, hypertension, dermatitis, ophthalmic problems, cardiac disease, indigestion, diabetes, tuberculosis, hepatitis B, respiratory troubles, psychological and liver problems were found in subjects. Significant levels between the unhealthy and normal subjects were determined by applying a parametric test, the student's t test at a significant level of p < 0.05. The results of our studies indicate that the significant difference in cadmium levels were observed in subjects suffering from acidity, ophthalmic problems, hypertension, psychological problems, tuberculosis and their respective controls. No correlation with cadmium was obtained with cardiac diseases, diabetes, hepatitis B, respiratory problems, dermatitis and hypotension. PMID- 16669338 TI - Changes in selected biomarkers in freshwater teleost fish, Cyprinus carpio var. communis exposed to sublethal concentrations of chromium sulphate toxicity. AB - The toxicity of chromium sulphate on plasma electrolytes (Na+, K+, Cl-) level and Na+, K(+)-ATPase activity of an economically important fish, Cyprinus carpio var. communis was evaluated. During sublethal treatment, plasma sodium level was increased, whereas plasma chloride level decreased throughout the experimental period. Plasma potassium level increased upto 10th day and then declined in the rest of the study period. The Na+, K(+)-ATPase activity decreased upto 15th day of treatment and slowly recovered showing significant increase upto 25th day of treatment. The significant changes in the plasma electrolytes levels and Na+, K(+)-ATPase activity can serve as a valuable biomarker of pollutant exposure and effects. PMID- 16669339 TI - Assessment of groundwater quality status in Amini Island of Lakshadweep. AB - Amini Island is one of the 10 inhabited islands in Lakshadweep. Built on the ancient volcanic formations Lakshadweep is the the tiniest Union Territory of India. The major problem experienced by the islanders is the acute scarcity of fresh drinking water. Groundwater is the only source of fresh water and the availability of the same is very restricted due to peculiar hydrologic, geologic, geomorphic and demographic features. Hence, proper understanding of the groundwater quality, with reference to temporal and spatial variations, is very important to meet the increasing demand and also to formulate future plans for groundwater development. In this context, the assessment of groundwater quality status was carried out in Amini Island. All the available information on water quality, present groundwater usage pattern, etc. was collected and analyzed. Total hardness and salinity are found to be the most critical water quality parameters exceeding the permissible limits of drinking water standards. Spatial variation diagrams of salinity and hardness have been prepared for different seasons. It is also observed from these maps that the salinity and hardness are comparatively better on the lagoon side compared to the seaside. These maps also suggest that the salinity and the hardness problem is more in the southern tip compared to northern portion. PMID- 16669340 TI - Hepatorenal dysfunctions in lead pollution. AB - Environmental and occupational lead pollution is a common problem in both developing and industrialised countries. Both hepatotoxicity and nephrotoxicity are known to occur in persons with exposure to heavy metals. We studied both liver function and renal function and blood lead concentraton in random population sample of 372 men (age range, 24 to 55 years). In all the subjects we measured both liver and renal function tests and both blood lead and urinary concentration of lead. Raised blood and urinary lead concentrations were associated with moderate changes in liver function and abnormal renal function, reflected in decrease of albumin and increased levels of liver enzymes and raised urea and creatinine concentrations, and with a reduction in creatinine clearance rate as compared to apparently normal subjects. These findings emphasis the importance of measurement of blood lead concentrations in adults in the genereal population to combat the effects of lead toxicity before the clinical signs predominate. PMID- 16669342 TI - Comparison of free iodine as a function of the dilution of two commercial povidone-iodine formulations. AB - Binding studies by means of equilibrium dialysis on two different Povidone-lodine solutions reveal that the amount of available iodine and free iodine is very different as such and after dilution. The free iodine concentration in the Braunol concentrate was found to be ca. 22 mg/L and in the iso-Betadine concentrate only ca. 2.1 mg/L, despite the total amount of available iodine in iso-Betadine being higher than that of Braunol. As the bactericidal level of free iodine is characterised by concentrations >5 ppm, Braunol can be employed as a disinfectant as such, iso-Betadine has to be diluted before use. In both concentrates more than 99% of available iodine is present as reservoir for free iodine. Concerning the results as a function of dilution, it was demonstrated that, for both solutions, free iodine reaches a maximum after a 50-fold dilution (ca. 31 mg/L and ca. 51 mg/L for iso-Betadine and Braunol respectively). After dilution, a more constant level of free iodine was observed in the Braunol than in the iso-Betadine solution, and this is attributed to the present molar ratio of I(2)/I- and the addition of iodate in the former. The pH for both solutions approximates that of the skin, as such and after dilution. In summary, it can be stated that Braunol is superior to iso-Betadine as to the release of free iodine in both the undiluted as well as in the diluted form. PMID- 16669341 TI - [Study of the performances of Unguator mixing equipments for the pharmaceutical compounding of dermatological dosage forms]. PMID- 16669343 TI - [Neurobiology and psychology of addictions]. AB - The clinical observation of patients that are addicted to drugs allows the distinction between physical dependence and psychic dependence (addiction). Empirical research has made important breakthrough in the understanding of the mechanisms involved in the development of dependence and opens new therapeutic options. Neurobiologic findings concerning dopaminergic brain reward circuit provide a better insight on the incentiv aspects of drug seeking. The present article is a review of current models of addiction with a specific focus on the link between biological aspects and psychological observations. PMID- 16669344 TI - [Tobacco use treatment]. PMID- 16669345 TI - [Alcoholism treatments]. AB - The problem of the alcoholic dependence entails often dramatic consequences: problems of health, acts of violence, traffic accidents, absenteeism at work and lost of social insertion. The alcohol has on the other hand a sociocultural registration in our way of life often associated with conviviality. The alcoholism is often defined by some as a disease, by the others as a social plague. It also differs according to the forms which it takes, its previous history and the functions which it performs. What definition can we accept? What are the therapeutic options to be implemented? Some answers will be considered through condiderations on the concept of motivation to change. PMID- 16669346 TI - [Drawing the alcoholic out of his isolation]. AB - Alcohol is by far the most frequent addiction across industrial countries. Associated with unrest, rejection and other negative counter-attitudes, alcohol dependence often confines the patient to isolation. The objective of this article is to describe potential obstacles to access to health care, the strategies enabling this access and finally different programs and therapeutic modalities of a long-term treatment. This article focuses on these issues in term of level of intervention. PMID- 16669347 TI - Role of selenium on antioxidant capacity in methomyl-treated mice. AB - Methomyl carbamate is a pesticide widely used in the control of insects. The present work aims at studying the effect of selenium on the antioxidant system of methomyl-treated mice. Swiss albino mice were intraperitoneally administered a single dose of methomyl (7 mg/Kg body weight). Mice of another group were injected with sodium selenite (5 pmole/Kg b.wt.) 7 days before methomyl intoxication. After 24 hours, methomyl exposure resulted in significant increase in lactic dehydrogenase activity (LDH). The antioxidant capacity of hepatic cells in terms of the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) content was diminished. It appears that methomyl exerts its toxic effect via peroxidative damage to hepatic, renal and splenic cell membranes. Also, methomyl induced DNA damage in these organs as detected by alkaline filter elution technique. The distribution of methomyl in different organs of mice was detected by HPLC. Selenium administration prior to methomyl injection produced pronounced protective action against methomyl effects. It is observed that selenium enhances the endogenous antioxidant capacity of the cells by increasing the activities of SOD, CAT, GR and GST as well as increasing GSH content. The activity of LDH was decreased in blood and the damage of DNA was suppressed comparable to controls. In conclusion, the adverse effects of methomyl in mice could be ameliorated by selenium. PMID- 16669348 TI - Collaborative effect of SERCA and PMCA in cytosolic calcium homeostasis in human platelets. AB - Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c. PMID- 16669351 TI - Key developments in respiratory medicine. PMID- 16669352 TI - Managing bronchiectasis. PMID- 16669349 TI - Production of nitric oxide and self-nitration of proteins during monocyte differentiation to dendritic cells. AB - Nitric oxide (NO) can stimulate dendritic cells to a more activated state. However, nitric oxide and peroxynitrites production by dendritic cells has been usually associated with pathological situations such as autoimmunity or inflammatory diseases. This study was designed to determine if dendritic cells obtained from healthy volunteers produce nitric oxide and peroxynitrites, which results in protein nitration. The expression of arginase II, but not arginase I, isoform was detected in monocytes and dendritic cells. There was higher inducible nitric oxide synthase (iNOS) protein expression and lower arginase activity both in immature and mature dendritic cells, compared to monocytes. This caused nitric oxide production, and maturation of dendritic cells which provoked a significative increase of nitrites and nitrates compared to immature dendritic cells. There was also peroxynitrites synthesis during monocyte differentiation as shown by the nitration of proteins. Immunoblot revealed a pattern of nitrated proteins in cell extracts obtained from monocytes and dendritic cells, however there were bands that appeared only in human dendritic cells, in particular an intense 90 kDa band. Nitric oxide production and nitrotyrosine formation could affect the antigen presentation and modify the immune response. PMID- 16669350 TI - The role of GLUT2 in dietary sugar handling. AB - GLUT2 is a facilitative glucose transporter located in the plasma membrane of the liver, pancreatic, intestinal, kidney cells as well as in the portal and the hypothalamus areas. Due to its low affinity and high capacity, GLUT2 transports dietary sugars, glucose, fructose and galactose in a large range of physiological concentrations, displaying large bidirectional fluxes in and out the cells. This review focuses on the roles of GLUT2. The first identified function of GLUT2 is its capacity to fuel metabolism and to provide metabolites stimulating the transcription of glucose sensitive genes. Recently, two other functions of GLUT2 are uncovered. First, the insertion of GLUT2 into the apical membrane of enterocytes induces the acute regulation of intestinal sugar absorption after a meal. Second, the GLUT2 protein itself initiates a protein signalling pathway triggering a glucose signal from the plasma membrane to the transcription machinery. PMID- 16669353 TI - Frequently-asked questions: brittle asthma. PMID- 16669354 TI - Diagnosing pleural conditions in practice. PMID- 16669355 TI - Managing sleep disorders. PMID- 16669357 TI - Grading revisited. PMID- 16669358 TI - Diathermy plate issue. PMID- 16669359 TI - Evidence-based measures. PMID- 16669360 TI - Cricoid pressure--increased interest. PMID- 16669361 TI - Sold down the river (yet again). PMID- 16669362 TI - Making a difference: Visits to Bangladesh. PMID- 16669363 TI - Timesheet fraud: A nurse's responsibility. AB - A nurse is more important than ever in today's NHS. They not only have more responsibility when it comes to the treatment of patients but a greater say in the administration of wards and departments. With this responsibility comes a duty to protect the NHS from those who try to abuse it. PMID- 16669364 TI - Preoperative assessment and intraoperative care planning. AB - We interviewed ten theatre nurses about their contribution to patient care. Their assessment strategy usually involved meeting patients on arrival in the department and did not include accessing the Trust's preoperative assessment document. In this paper we discuss the nursing assessment of surgical patients in the context of the nursing process as it was described in our research interviews. PMID- 16669365 TI - Facilitating benefit, minimising risk: Responsibilities of the surgical practitioner during electrosurgery. AB - This article will explore the role of the surgical practitioner in providing best care for the patient, by discussing electrosurgical intervention (ESI). It will exemplify the technological aspect of the role. Management of ESI equipment, and its inherent risks, will be shown to relate directly to the practitioner's role. Some safety claims for ESI equipment will be analysed, demonstrating the need for a theatre practitioner to establish the validity of sources that inform practice. The roles of circulating practitioner and scrubbed practitioner will be elucidated, with exploration of professional and ethical responsibilities to the patient, themselves and colleagues. PMID- 16669366 TI - Achieving level three of the Clinical Negligence Scheme. AB - This article discusses my experience, from a theatre nurse's perspective, of meeting Level Three of the Clinical Negligence Scheme for Trusts (CNST). As the practice development sister for Theatres, Day Unit and Endoscopy and as a member of the CNST group, it is my responsibility to gather evidence for my areas. PMID- 16669368 TI - IHF and The Health Research and Education Trust enter into new collaboration to encourage cross-national health research. PMID- 16669369 TI - Getting to know the president: an interview with Gerard Vincent. PMID- 16669367 TI - The Foley catheter. AB - In the third of our biographical series tracing the origination of some of today's most commonly used surgical instruments and the people behind them, Professor Ellis explores the history of the catheter, and the breakthrough design by American urologist Frederick Foley. PMID- 16669370 TI - The healthcare system in Taiwan. PMID- 16669371 TI - Quality measurement across borders: needs and options. AB - It is very clear that, when given a choice, patients will travel across national borders to obtain healthcare. Cost, access and proximity are the most frequently sited reasons. It is less clear however, to policy makers, the payer for the services and health professionals, what the level of quality will be when patients cross borders. The traditional regulatory approaches such as licensure, accreditation and certification vary within and between countries, and private sector approaches to quality vary as frequently. The source of this variation is that private and public quality evaluation systems vary widely and produce non comparable quality data. A survey conducted by Joint Commission International demonstrated wide country variation exists in use of the electronic medical record, the best source of quality data and in the collection of adverse event information, a rich source for understanding the frequency and type of unwanted events. The use of international standards that focus on the processes that most directly determine outcomes is described for understanding risks and quality across borders. PMID- 16669372 TI - Cross border access to healthcare services within the European Union. AB - This article focuses on the issue of "medical tourism" and the extent to which patients resident in EU member states have a legally enforceable right to access healthcare services in other EU member states which are paid for by such patients' home member states. The article explains the various relevant legislation and explores the tensions within it and also focuses on various recent cases which have discussed whether prior authorization systems in relation to cross border medical treatment are justifiable in the objective public interest. The article also looks at the most recent developments in the case law concerning EU based national health services. PMID- 16669373 TI - Determining the agent factors related with time management of responsible doctors and nurses in clinics at Ankara University hospitals. AB - This research has been planned and conducted as a descriptive scanning model field study in order to determine the agent factors related with time management of doctors and nurses in positions of responsibility at Ankara University hospitals. As data collection instruments; the "Personal Information Form" which has been developed to determine the socio-demographical characteristics of the research group, the questionnaire of "Determining the Time Management Attitudes and Behaviour of Managers, Time Management Opportunities of the Managers, Prodcutive Working Times of the Managers and the Factors Causing Them to Lose Time", developed by Erdem has been used. It has been determined that the time management attitudes and behaviour of doctors, nurses and nurse assistants responsible for clinics are all different. It was found that nurse assistants graduated from pre-undergraduate or high schools are the least conscious of time management. In particular, nurse assistants of 36 years old and over with 21 years of work experience and 11 years of management experience show little awareness of time management. The time losing factors of the research group were found to be unnecessary visitors, lack of materials and the excessive amount of time spent on obtaining the necessary equipment. PMID- 16669374 TI - Recognition and valuation of hospital personnel in Italy. AB - After the 1993 healthcare reform, all public hospitals in Italy were changed into public firms. The increasing commitment to pursue efficiency has introduced the need for a managerial responsibility in healthcare organizations. However, activities and resource use in hospitals driven by professionals and senior management often has a limited insight on business processes. We developed a three-dimensional evaluation system based on: (a) personal support to the overall team output, (b) assessment of organizational behavour and (c) individual goals setting (MBO). According to the position held, personal goals are negotiated with direct reports across three domains: managerial attitude, professional skills and organizational behaviours. PMID- 16669375 TI - Opportunities for overcoming tuberculosis: new treatment regimens. AB - TB is mainly concentrated in developing countries and global control efforts have been hampered by the deficiencies of diagnostic, prophylactic and therapeutic tools. This article looks at the need for new TB therapies and how the Stop TB Partnership's Working Group on New TB Drugs has positively influenced the TB drugs pipeline. This has involved developing a revolutionary concept for TB drug development and leveraging the potential of the existing drugs pipeline. As part of the process, there is also a need for broader collaboration to identify new candidate compounds, expand clinical trial capacity, advance new technologies and ensure regulatory harmonization. PMID- 16669376 TI - Computerizing a health service: the potential barriers to success. AB - Any project to computerise the complex health services will take time and be beset with problems. However, some of these problems arise from barriers which could have been anticipated. There are genuine benefits of supporting the delivery of clinical care with technology, but unless these potential barriers are appreciated and understood, the well intentioned objectives will never be realised. This article describes how the National Health Service in England is approaching such a complex subject. It also describes what could prevent it from happening and suggests a strategy for overcoming them. PMID- 16669377 TI - Integrated care for people with disabilities: an international perspective. PMID- 16669380 TI - Metal-on-metal hip joint tribology. AB - The basic tribological features of metal-on-metal total hip replacements have been reviewed to facilitate an understanding of the engineering science underpinning the renaissance of these hard-on-hard joints. Metal-on-polymer hip replacements operate in the boundary lubrication regime, thus leading to the design guidance to reduce the femoral head diameter as much as is feasible to minimize frictional torque and volumetric wear. This explains why the gold standard implant of this form from the past half-century had a diameter of only 22.225 mm (7/8 in). Metal-on-metal implants can operate in the mild mixed lubrication regime in which much of the applied load is supported by elastohydrodynamic films. Correct tribological design leads to remarkably low steady state wear rates. Promotion of the most effective elastohydrodynamic films calls for the largest possible head diameters and the smallest clearances that can reasonably be adopted, consistent with fine surface finishes, good sphericity and minimal structural elastic deformation of the cup on its foundations. This guidance, which is opposite in form to that developed for metal-on-polymer joints, is equally valid for solid (monolithic) metallic heads on metallic femoral stems and surface replacement femoral shells. Laboratory measurements of friction and wear in metal-on-metal joints have confirmed their potential to achieve a very mild form of mixed lubrication. The key lies in the generation of effective elastohydrodynamic lubricating films of adequate thickness compared with the composite roughness of the head and cup. The calculation of the film thickness is by no means easy, but the full procedure is outlined and the use of an empirical formula that displays good agreement with calculations based upon the full numerical solutions is explained. The representation of the lambda ratio, lambda, embracing both film thickness and composite roughness, is described. PMID- 16669379 TI - Hip resurfacing arthroplasty: the evolution of contemporary designs. AB - Metal-on-metal hip resurfacing is considered by many as the most significant recent development in hip arthroplasty. It preserves proximal femoral bone stock, optimizes stress transfer to the proximal femur, and offers inherent stability and optimal range of movement. The early results of hip resurfacing in the 1970s and 1980s were poor and the procedure was largely abandoned by the mid-1980s. The expectation that these prostheses would be easy to revise was not often fulfilled. The large diameter of the articulation combined with thin polyethylene cups or liners resulted in accelerated wear and the production of large volumes of biologically active particulate debris, leading to bone loss and implant loosening. Failure has been attributed to other factors, mainly avascular necrosis of the femoral head. However, this concern has not been confirmed by retrieval studies. The failure of early hip resurfacings was essentially a consequence of the use of inappropriate materials, poor implant design, inadequate instrumentation, and crude surgical technique. It was not an inherent problem with the procedure itself. The renaissance of metal-on-metal articulations for total hip arthroplasty enabled the introduction of new hip resurfacings and most of the major implant manufacturers have already introduced such systems. Early results are encouraging and complications commonly seen in the 1970s and 1980s, such as early implant loosening and femoral neck fracture, now appear to be rare. Whilst early results should be regarded with caution, modern metal-on-metal hip resurfacing potentially offers the ultimate bone preservation and restoration of function in appropriately selected young patients. PMID- 16669381 TI - Metal-on-metal bearings surfaces: materials, manufacture, design, optimization, and alternatives. AB - When first introduced, total hip replacements offered pain relief and improved mobility in elderly patients. The success of this procedure in terms of long-term durability and restoration of function has led to its use in younger, more active patients. This has resulted in a commensurate increase in patient expectation regarding longevity and the degree to which function and lifestyle is restored. The bearing surface is a key feature of the performance of replacement joints. It is generally accepted that excessive amounts of wear debris preclude their long term survivorship and hence there is an ongoing requirement for bearing surfaces which minimize debris generation. The purpose of this paper is to review the factors which affect the performance of so-called metal-on-metal bearings, to compare their performance with that of the other commonly used contemporary alternatives, metal and ceramic articulating against highly cross-linked polyethylene, and ceramic-on-ceramic, and finally to consider the potential solutions offered by new developments such as ceramic-on-metal and coatings applied to metal-on-metal bearings. PMID- 16669384 TI - Tribological principles in metal-on-metal hip joint design. AB - An analysis of some 100 published and communicated findings on running-in volumetric wear and steady state wear rates from simulator tests carried out in eight laboratories in three countries has been undertaken. Powerful indications have emerged of the dominant role of mixed lubrication in current metal-on-metal hip replacements, with elastohydrodynamic film thickness controlling wear. The background to the calculation of film thickness in the elastic-isoviscous mode of lubrication has been outlined and graphs of representative film thickness and lambda ratio have been presented. For minimum wear and wear rate the diameter of the femoral component should be as large as possible, while the clearance should be as small as is practicable. The findings are valid for both monolithic and surface replacement implants. A tentative proposal is made for the prediction of lifetime wear in metal-on-metal total hip replacements. PMID- 16669378 TI - Background of metal-on-metal resurfacing. AB - Hip resurfacing is not a new concept and attempts to treat hip arthritis without resecting the femoral head and neck have been made since the 1950s. The resurgence of new and better-engineered metal-on-metal bearings has provided the means to develop a viable prosthetic solution from a concept that was once abandoned. The lessons drawn from the early resurfacing era led to modern designs all using a cementless fixation of the acetabular component and a short metaphyseal stem designed for component alignment on the femoral side. Currently, only metallic devices can be manufactured with sufficient strength as a thin one piece shell, combining excellent wear properties for large femoral heads and a bone-conserving device on the acetabular side. The early results of these new designs are extremely encouraging but the rapid development of the procedure needs to be controlled by appropriate training programmes to ensure its future success. PMID- 16669383 TI - The effect of microstructure on the wear of cobalt-based alloys used in metal-on metal hip implants. AB - The influence of microstructure on the wear of cobalt-based alloys used in metal on-metal hip implants was investigated in a boundary lubrication regime designed to represent the conditions that occurred some of the time in vivo. These cobalt chromium-molybdenum alloys were either wrought, with a total carbon content of 0.05 or 0.23 wt %, cast with a solution-annealing procedure or simply as-cast but not solution annealed. Bars of these different alloy grades were subjected to various heat treatments to develop different microstructures. The wear was evaluated in a linear-tracking reciprocating pin-on-plate apparatus with a 25 per cent bovine serum lubricant. The wear was found to be strongly affected by the dissolved carbon content of the alloys and mostly independent of grain size or the carbide characteristics. The increased carbon in solid solution caused reductions in volumetric wear because carbon helped to stabilize a face-centred cubic crystal structure, thus limiting the amount of strain-induced transformation to a hexagonal close-packed crystal structure. Based on the observed surface twining in and around the contact zone and the potentially detrimental effect of the hexagonal close-packed phase, it was postulated that the wear of cobalt-based alloys in the present study was controlled by a deformation mechanism. PMID- 16669382 TI - 'Severe' wear challenge to 'as-cast' and 'double heat-treated' large-diameter metal-on-metal hip bearings. AB - The wear generation of double-heat-treated and as-cast large-diameter metal-on metal (MOM) hip bearings was investigated using standard- and 'severe'-gait simulations. The test hypothesis was that double heat treatment would change MOM hip wear compared with the as-cast condition. Two groups of high-carbon MOM bearings of 40 mm diameter were manufactured and subjected to either hot isostatic pressing (HIP) and solution annealing (SA) or no heat treatment (as cast). The results showed no statistical difference between the two groups under both running-in and steady state conditions. Even under the most 'severe'-gait simulation published to date, the mean volumetric wear rates were 2.9 and 3.9 mm3 per 10(6) cycles for the HIP-SA and as-cast bearings respectively, showing a ten fold increase in wear compared with walking. These differences were not statistically different; therefore our hypothesis was negated. Changes in alloy microstructure do not appear to influence the wear behaviour of high-carbon cast MOM articulations with similar chemical compositions. This is in sharp contrast with the published significance of bearing diameter and radial clearance on the wear of MOM hip bearings. PMID- 16669385 TI - Understanding the role of corrosion in the degradation of metal-on-metal implants. AB - In metal-on-metal joints the primary concerns in terms of long-term durability relate to corrosion, wear, and their joint (tribocorrosion) effects. The release of ions through corrosion processes and nanoscale debris from wear processes can seriously affect joint integrity and can lead to an adverse biological reaction by the host. In this paper an integrated study of corrosion-wear interactions in serum, Dulbecco's Modified Eagle's Medium and 0.3 per cent NaCl has demonstrated that the biological nature of the fluid affects the total degradation rate and also the level of wear-corrosion interactions. The specific action of proteins in corrosion and tribocorrosion for high-carbon Co-Cr-Mo and low-carbon Co-Cr-Mo alloys is discussed. PMID- 16669386 TI - The tribology of metal-on-metal total hip replacements. AB - Total hip surgery is an effective way of alleviating the pain and discomfort caused by diseased or damaged joints. However, in the majority of cases, these joints have a finite life. The main reason for failure is osteolysis (bone resorption). It is well documented that an important cause of osteolysis, and therefore the subsequent loosening and failure of conventional metal- or ceramic on-ultra-high molecular weight polyethylene joints, is the body's immunological response to the polyethylene wear particles. To avoid this, interest has been renewed in metal-on-metal joints. The intention of this paper is to review the studies that have taken place within different laboratories to determine the tribological performance of new-generation metal-on-metal total hip replacements. These types of joint offer a potential solution to enhance the longevity of prosthetic hip systems; however, problems may arise owing to the effects of metal ion release, which are, as yet, not fully understood. PMID- 16669387 TI - Wear particles from metal-on-metal total hip replacements: effects of implant design and implantation time. AB - Detailed characterization of wear particles is necessary to understand better the implant wear mechanisms and the periprosthetic tissue response. The purposes of the present study were to compare particle characteristics of current with older designs of metal-on-metal (MM) total hip replacements (THRs), and to determine the effect of implantation time on wear particle characteristics. Metal wear particles isolated from periprosthetic tissues from 19 patients with MM THRs of current and older designs and at different implantation times (very short, longer, and very long) were studied using transmission electron microscopy and energy dispersive X-ray analysis. The particles from the current design implants with implantation times of not more than 15 months (very short-term) were almost exclusively round to oval chromium oxide particles. In all other cases, although the predominance was still round to oval chromium oxide particles, greater proportions of cobalt-chromium-molybdenum (Co-Cr-Mo) particles, mainly needle shaped, were detected. Very long-term THRs implanted for more than 20 years had the highest percentage of needle-shaped Co-Cr-Mo particles. Particle lengths were not markedly different between the different designs and implantation times except for the current design implants of not more than 15 months, which had a significantly smaller mean length of 39 nm. In conclusion, the implant design did not seem to have a significant influence on particle characteristics whereas the implantation time appeared to have the most effect on the particles. It should be noted that, because of the limited number of tissue retrievals available, some uncertainty remains regarding the generality of these findings. PMID- 16669389 TI - Investigation on stick phenomena in metal-on-metal hip joints after resting periods. AB - Insufficient understanding of tribological behaviour in total joint arthroplasty is considered as one of the reasons for prosthesis failure. Contrary to the continuous motion input profiles of hip simulators, human locomotion contains motion interruptions. These occurring resting periods can cause stick phenomena in metal-on-metal hip joints. The aim of the present study was to investigate the tribological sensitivity of all-metal bearings to motion interruptions on in vitro test specimens and retrieved implants. Friction and wear with and without resting periods were quantified. Unlike the metal-on-polyethylene joints, the static friction of metal-on-metal joints increased up to micros = 0.3 with rest, while wear appeared to be unaffected. This effect is caused by the interlocking of firmly adhered carbon layers, which were generated from the protein-containing lubricant through tribochemical reactions. Since more than 80 per cent of the retrieved implants exhibited macroscopically visible carbon layers, the increase in friction presumably also occurs under physiological conditions, which is then transferred to the bone-implant interface. These recurrent tangential stress peaks should be considered for the design features of the cup-bone interface, in particular when larger-sized implant heads are used. PMID- 16669388 TI - Head replacement, head rotation, and surface damage effects on metal-on-metal total hip replacements: a hip simulator study. AB - The possibility of replacing the femoral head alone, in either solid or articular surface replacement form, during revision operations on metal-on-metal total hip replacements remains an attractive feature of such implants. In the present investigation, laboratory simulator studies of the influence upon volumetric wear of inserting a new femoral head, of introducing some head rotation, and of damaging the femoral head by scratches have all been explored. New and rotated heads both involve an additional running-in period, but the experimental studies show that the volumetric wear associated with this process is less than the initial running-in wear. The beneficial effects upon volumetric wear of small clearances have been confirmed, while the processing of high-carbon Co-Cr-Mo materials appears to be much less influential. Scratches did not affect wear as much as head replacement or head rotation, but the ongoing wear rates were somewhat higher. PMID- 16669390 TI - Clinical experience with metal-on-metal total joint replacements: indications and results. AB - Survivorship of total joint arthroplasty depends on the durability of fixation and durability of articulation. The metal-on-polyethylene articular couple has been the most widely used. Polyethylene wear (and the associated cytochemical events that culminate in osteolysis) has been identified as a major factor adversely influencing the durability of joint replacement. This stimulated the orthopaedic community to explore the possibility of using alternative bearings with lower wear rates. Hard-on-hard bearings have been shown to be associated with reduced wear. Metal-on-metal bearings have wear rates that are 20-100 times lower than metal on conventional polyethylene. However, patients with metal-on metal articulations have increased levels of cobalt and chromium in the serum and urine, and this has raised concerns about toxicity, mutagenesis, and hypersensitivity. At this stage there is no epidemiological evidence to suggest that the risk of carcinogenesis is anything more than theoretical. Successful long-term results have been reported with the cast cobalt-chromium metal-on-metal couples of the mid-1960s. Tissues retrieved at revision of these implants did not show the giant-cell inflammatory response associated with polyethylene particles. Several researchers have reported excellent mid-term results with the current generation of high-precision metal-on-metal bearings. PMID- 16669393 TI - The effect of 'running-in' on the tribology and surface morphology of metal-on metal Birmingham hip resurfacing device in simulator studies. AB - It is well documented that hard bearing combinations show a running-in phenomenon in vitro and there is also some evidence of this from retrieval studies. In order to investigate this phenomenon, five Birmingham hip resurfacing devices were tested in a hip wear simulator. One of these (joint 1) was also tested in a friction simulator before, during, and after the wear test and surface analysis was conducted throughout portions of the testing. The wear showed the classical running in with the wear rate falling from 1.84 mm3 per 10(6) cycles for the first 10(6) cycles of testing to 0.24 mm3 per 10(6) cycles over the final 2 x 10(6) cycles of testing. The friction tests suggested boundary lubrication initially, but at 1 x 10(6) cycles a mixed lubrication regime was evident. By 2 x 10(6) cycles the classical Stribeck curve had formed, indicating a considerable contribution from the fluid film at higher viscosities. This continued to be evident at both 3 x 10(6) and 5 x 10(6) cycles. The surface study complements these findings. PMID- 16669391 TI - History and modern concepts in surface replacement. AB - Hip resurfacing is not a modern concept. Professor Sir John Charnley attempted the first hip resurfacing procedure in the 1950s. This implant failed as a result of unsatisfactory materials and the same problem has troubled hip resurfacing developments over the subsequent forty years. Modern hip resurfacing using metal on-metal bearings has shown good medium-term results in the most challenging subgroup--young active patients. PMID- 16669395 TI - Finite element analysis of the resurfaced femoral head. AB - Failure of the resurfaced femoral head may occur in the short term owing to femoral neck fracture or in the long term owing to aseptic loosening as a result of strain shielding. Resurfacing arthroplasties are not all the same. In particular, there is considerable debate regarding the role of the metaphyseal stem and cementing technique. This study examines the influence of various metaphyseal stem configurations (diameter, percentage length in contact with bone, and bonded versus debonded) and cement mantle thickness on the load transfer within the femoral head. Resurfacing resulted in significant strain shielding in the superior femoral head and elevated strain in the superior femoral neck. Although the increase in strain in the femoral neck was significant, the mean strains were below the yield strain for cancellous bone. Peak strains were observed above the yield strain, but they accounted for less than 1 per cent of the total head-neck bone volume and therefore were unlikely to result in femoral neck fracture. Increasing the stem diameter and increasing the percentage stem length in contact with bone both increased the degree of strain shielding. Bonding the metaphyseal stem produced the most dramatic strain shielding, which also extended into the head-neck junction. In contrast, varying the cement mantle thickness had a negligible effect on the load transfer. PMID- 16669392 TI - Development rationale for an articular surface replacement: a science-based evolution. AB - Hip resurfacing has an enduring appeal because of the advantages of bone conservation and maximal joint stability. However, a far from satisfactory experience with earlier resurfacing designs led to its virtual disappearance in the 1980s. The concept was reintroduced in the late 1990s. The current generation of resurfacing devices generally consisted of a large-diameter metal-on-metal articulation, the femoral components being cemented and the acetabular components utilizing various forms of cementless fixation. The encouraging medium-term results, with a follow-up of up to 8 years using the current generation of surface replacement joints, combined with favourable reports related to long-term performance of some metal bearings have led to a rapid increase in the use of such components with these devices. This trend is most marked in younger, more active patients who have expectations of restoration of lifestyle in addition to improved mobility and pain relief and in whom failure with conventional total hip replacement is much higher than previously reported with more sedentary patients. The aim of this paper is, firstly, to highlight a number of areas of improvement and, secondly, to explain how these may be addressed by making modifications to the design of both implants and instrumentation and to the surgical technique. The areas identified for improvement were tissue preservation (thinner components, and reduced steps between sizes), acetabular cup issues (fixation, insertion, and positioning), femoral component issues (design, loading, and cementation), improved bearing surface characteristics, and simplified precise instrumentation with a low-trauma surgical technique. PMID- 16669394 TI - Metal-on-metal hip simulator study of increased wear particle surface area due to 'severe' patient activity. AB - This study investigated changes in metal-on-metal (MOM) hip wear and wear particle characteristics arising from a more aggressive patient activity level compared with normal walking. The test hypothesis was that 'severe'-gait conditions will change wear, wear particle sizes, and morphology owing to a decline in joint lubrication. Four carbon MOM hip bearings 40 mm high were subjected to normal-walking and fast-jogging simulations in an orbital hip joint simulator with 25 per cent alpha-calf serum as a lubricant. Co-Cr-Mo wear particles were extracted using an enzymatic method, and prolate ellipsoid equations were used to estimate particle volume and surface area. Fast-jogging simulations generated a sevenfold increase in volumetric wear, a 33 per cent increase in mean wear particle size, and a threefold increase in the number of larger (needle) particles compared with walking. This resulted in a twentyfold increase in total wear particle surface area per 10(6) cycles compared with walking, thereby confirming our hypothesis. The clinical significance of this result suggests that highly active MOM patients may exhibit greater ion release than less active patients. PMID- 16669398 TI - Cementing techniques in hip resurfacing. AB - The subject of the cementing technique in hip resurfacing has been poorly studied to date. The hip resurfacing prosthesis is unique in the family of cemented prostheses because the cement mantle is blind (hidden underneath the implant) and is radiographically obscured. This presents an immediate challenge to the surgeon at the time of surgery, but also has a longer-term implication in terms of lack of post-operative clinical observation. This should be compared with total hip replacement or total knee replacement where the cement mantle can at least be partially observed both intra- and post-operatively. With this in mind, the objective of this review is, firstly, to understand the cement mantles typically achieved in current clinical practice and, secondly, to identify those factors affecting the cement mantle and to consolidate them into an improved and reproducible cementing technique. The outcome of this work shows that the low viscosity technique can commonly lead to excessive cement penetration in the proximal femoral head and an incompletely seated component, whereas a more consistent controlled cement mantle can be achieved with a high-viscosity cementing technique. Consequently, it is recommended that a high-viscosity technique should be used to minimize the build-up of excessive cement, to reduce the temperature created by the exothermic polymerization, and to help to ensure correct seating of the prosthesis. A combination of these factors is potentially critical to the clinical success of some articular surface replacement (ASR) procedures. It is important to note that we specifically studied the DePuy ASR system; therefore only the general principles (and not the specifics) of the cementing technique may apply to other resurfacing prostheses, because of differences in internal geometry, clearance, and surgical technique. PMID- 16669396 TI - Deformation of press-fitted metallic resurfacing cups. Part 1: Experimental simulation. AB - The interference press fit of a metallic one-piece acetabular cup employed for metal-on-metal hip resurfacing procedures was investigated experimentally under laboratory conditions in the present study, in particular regarding the cup deformation. Tests were carried out in cadavers as well as polyurethane foams of various grades with different elastic moduli to represent different cancellous bone qualities. The cadaver test was used to establish the most suitable configuration of the foam model representing realistic support and geometrical conditions at the pelvis. It was found that a spherical cavity, with two identical areas relieved on opposite sides, was capable of creating a two-point pinching action of the ischeal and ilial columns on the cup as the worst-case scenario. Furthermore, the cup deformation produced from such a two-point loading model with a grade 30 foam was similar to that measured from the cadaver test. Therefore, such a protocol was employed in subsequent experimental tests. For a given size of the outside diameter of the cup of 60 mm, the cup deflection was shown to be dependent largely on the cup wall thickness and the diametral interference between cup and prepared cavity at implantation. For a relatively thin cup with a wall thickness between 2.3 mm (equator) and 4 mm (pole) and with a modest nominal diametral interference of 1 mm, which corresponds to an actual interference of approximately 0.5 mm, the maximum diametral cup deflection (at the rim) was around 60 microm, compared with a diametral clearance of 80-120 microm between the femoral head and the acetabular cup, generally required for fluid-film lubrication and tribological performances. Stiffening of the cup, by both thickening and lateralizing by 1 mm, reduced the cup deformation to between 30 and 50 microm with actual diametral interferences between 0.5 and 1 mm. PMID- 16669397 TI - Deformation of press-fitted metallic resurfacing cups. Part 2: Finite element simulation. AB - The deformation of metallic acetabular cups employed for metal-on-metal hip resurfacing procedures was considered theoretically using the finite element method in the present study, following on the experimental investigation reported in Part 1. Three representative cups, characterized by the cup wall thickness as thin, intermediate, and thick, were considered. For the intermediate cup, the effects of both the size and the diametral interference on the cup deformation were investigated. Both two-dimensional axisymmetric and three-dimensional finite element models were developed to examine the important parameters during and after the press-fit procedure, and in particular the deformation of the metallic cup. The theoretical prediction of the cup deformation was in reasonable agreement with the corresponding experimental measurement reported in Part 1. The most significant factor influencing the cup deformation was the cup wall thickness. Both the size and the diametral interference were also shown to influence the cup deformation. It is important to ensure that the cup deformation does not significantly affect the clearance designed and optimized for tribological performances of metal-on-metal hip resurfacing prostheses. Furthermore the contact parameters at the cup and bone interface associated with the press fit were also discussed. PMID- 16669399 TI - Biomechanical, morphological, and histological analysis of early failures in hip resurfacing arthroplasty. AB - The present revival of hip resurfacing arthroplasty may be related to an increase in early failures owing to the challenging technique of the procedure. Fifty-five retrieved implants were analysed with respect to wear, cement mantle and cement penetration, fracture and head morphology, as well as standard histology. Femoral neck fractures occurred in median after 102 days. The time to failure was shorter for older women. Major deviations from the suggested cement mantle thickness and cement penetration were found. Indications for high trauma during implantation leading to early failure due to weakening of the femoral neck were also observed. Some failures had signs of pseudarthrosis beneath the implant. Four different fracture patterns with different mean survival times were identified. Observed wear was minor with the exception of that due to alignment mistakes (rim loading). The cups were not damaged by the failures. Histological results indicate that avascular necrosis is not necessarily connected with this kind of endoprosthetic surgery. Most of the failures analysed can probably be attributed to the 'learning curve' effect, which is an unsatisfactory situation. PMID- 16669402 TI - Development and problems of metal-on-metal hip arthroplasty. AB - For over 40 years, the metal-on-polyethylene bearing has dominated the field of total hip replacement. Problems of wear, osteolysis (dissolution of bone), and ultimately failure of prostheses have led to the development of alternative bearing surfaces. Metal-on-metal hip resurfacing has taken current orthopaedic surgery almost by storm. However, metal ion release following metal-on-metal hip resurfacing remains a major cause for concern. This article looks into the development and examines problems and issues surrounding metal-on-metal resurfacing arthroplasty. PMID- 16669400 TI - Lessons learned from early clinical experience and results of 300 ASR hip resurfacing implantations. AB - Between August 2003 and April 2005, 300 ASR metal-on-metal resurfacing hip endoprostheses were implanted by the first author and a fellow surgeon. The mean age at surgery was 56.8 years (18-75.9 years) and mean body mass index was 27.6 kg/m2 (range, 19-41 kg/m2). The mean follow-up time was 202 days. The mean Harris hip score improved from 44 pre-operatively to 89 at 3 months post-operatively. In total, eight (2.7 per cent) cases [five neck fractures (1.66 per cent) and three cup revisions (1 per cent)] were revised. Two neck fractures occurred within a group of seven cases of femoral neck notching detected postoperatively; one neck fracture occurred out of two cases of incomplete seating of the femoral implant. A significantly higher (p < 0.001) failure rate was observed for patients who had undergone a previous osteosynthesis of the proximal femur (three revisions in a group of 15 patients). Revision cases had a significantly greater body mass index (p = 0.031). A learning curve was evident from the reduction in revisions from 5 in the first 100 surgical procedures to 2 in the next 100 and 1 in the last 100. These results show the importance of accurate surgical technique and careful patient selection for fourth-generation hip resurfacing implants, PMID- 16669403 TI - Biological effects of metal-on-metal hip replacements. AB - The advantages seen by patients receiving total hip arthroplasties and the implications of the release of both metal particles and soluble metal ions are discussed. This paper describes some of the early changes that have been observed in metal-on-metal hip arthroplasties, in terms of both changes in metal levels in blood and chromosome changes. PMID- 16669404 TI - The clinical significance of metal ion release from cobalt-chromium metal-on metal hip joint arthroplasty. AB - Metal-on-metal (MOM) bearings offer extremely low wear and the avoidance of polyethylene but generate metallic wear particles. Although their total volume is dramatically smaller than polyethylene debris, these particles are in the nanometre size range and are many times more numerous. Metallic particles are ingested by macrophages or may be disseminated via lymphatics to the reticuloendothelial system. They corrode, and metal ions are present in the circulation and concentrated in erythrocytes. Excretion of metal ions via the kidneys seems to balance their generation in patients with MOM implants. However, highly sensitive detection methods can be used to show that levels of circulating cobalt and chromium ions are several times the normal level. These concentrations are well within the limits identified as dangerous to health in workers exposed to industrial chemicals, and also considerably lower than the levels found to cause cell toxicity in vitro. The local concentrations of particles and metal ions in the synovial tissue may occasionally exceed these limits and cause tissue necrosis. Clinical experience of lysis is rare in association with MOM bearings, as are hypersensitivity reactions and MOM bearings have had an excellent record over four decades and have a favourable benefit to risk ratio. Further reduction in risk will be achieved by improvement of materials, engineering, and accuracy of insertion. PMID- 16669401 TI - Biological effects of clinically relevant wear particles from metal-on-metal hip prostheses. AB - The problems of osteolysis and late aseptic loosening associated with ultra-high molecular weight polyethylene (UHMWPE) particles has lead to a renewed interest in metal-on-metal prostheses. Wear particles generated by modern Co-Cr-on-Co-Cr prostheses are nanometre in size (range: 10-120 nm; mean: about 40 nm), an order of magnitude smaller than the size of UHMWPE known to be critical for activation of osteolytic cytokines by macrophages. Co-Cr wear particles will induce osteolytic cytokine production by human macrophages, but only at high volumetric concentrations. Unlike UHMWPE, Co-Cr particles are not inert. Co-Cr particles have the potential to release metal ions; they may be toxic to cells, induce deoxyribonucleic acid damage or cause host hypersensitivity. The nanometre size range of Co-Cr wear particles means that they may be disseminated widely in the body. The potential for metal-on-metal bearings to induce adverse effects clinically will be dependent upon the rate of wear. What constitutes a safe wear rate for modern metal-on-metal bearings is unknown. However, the wear of metal-on metal prostheses is critically dependent upon the design and, in particular, the carbon content of the alloy, the radial clearance, and the head diameter. Thus, the potential for adverse biological reactions associated with metal-on-metal bearings can be reduced by selection of appropriately designed implants. PMID- 16669405 TI - Cancer incidence and causes of death among total hip replacement patients: a review based on Nordic cohorts with a special emphasis on metal-on-metal bearings. AB - All patients with total hip arthroplasty (THA) are exposed to soluble or particulate forms of Co and Cr. Adverse effects of these wear products are not known. Data from Nordic registries is used to estimate adverse effects on a large scale, based mostly on metal-on-polyethylene bearings. Cancer incidence was in line with the general population when the patients were operated on for all indications and significantly decreased when the indication was primary osteoarthritis. Stomach cancer and colorectal cancers were significantly reduced and prostate cancer and skin melanoma significantly increased. There was no significant excess of cancer in target organs, i.e. liver, kidney, or haematopoietic cancers. THA patients had reduced mortality and extended life expectancy compared with standard Nordic populations. All-site cancer incidence of the first-generation metal-on-metal McKee-Farrar patients operated on for primary osteoarthritis was in line with the general population after follow-up for up to 28 years. General mortality of these patients was also reduced and they also had an extended life expectancy. Temporary increases in haematopoietic cancers at different follow-up periods were seen in some cohorts. This malignancy deserves a special record linkage monitoring while large numbers of young patients are provided with the second generation of metal-on-metal prostheses. PMID- 16669406 TI - Variability of flow-mediated dilation measurements with repetitive reactive hyperemia. AB - To capture the response of an acute intervention, multiple post intervention measurements of flow-mediated dilation (FMD) must be performed. The effect of repetitive reactive hyperemia on endothelial function and the measurement of FMD are unknown. The purpose of this investigation was (1) to examine the effect of repetitive reactive hyperemia on brachial artery FMD and (2) to determine whether brachial artery FMD is stable during a 2-h morning period. We investigated FMD in 20 apparently healthy college students on three randomized treatment days every 30 min (T30), 60 min (T60), and 120 min (T120) throughout a 2-h morning period (08.00 h to 10.00 h). An ANOVA (p > 0.05) and ICC (> 0.40) were both needed to confirm no difference among repetitive reactive hyperemia treatments. In response to repetitive reactive hyperemia, there was no difference (p = 0.307; ICC > 0.40) within the first and last FMD measurements of each treatment condition or between treatment conditions (p = 0.344; ICC > 0.40). FMD was similar (p = 0.348) throughout the 2-h morning period. In conclusion, repetitive reactive hyperemia over a 2-h period has no effect on FMD measurements in apparently healthy college students. In addition, this study found no time trends for FMD measurements during the 2-h morning period to allow for pre/post intervention FMD measurements. PMID- 16669407 TI - Plasma markers of NO synthase activity in women after ovarian hyperstimulation: influence of estradiol on ADMA. AB - The beneficial effects of estrogen on vasculature are partially explained by an estrogen-induced increase in nitric oxide (NO) synthesis. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase. In the present study, the effect of 17beta-estradiol (E2) on ADMA and NO synthesis was investigated both in vivo and in vitro. Plasma NO and ADMA levels were measured in healthy women at a low menstrual estrogenic stage (E2 < 100 pg/ml) and in women who were undergoing ovarian hyperstimulation (E2 > 2000 pg/ml) before in vitro fertilization embryo transfer. Primary human umbilical vein endothelial cell (HUVEC) cultures were incubated with and without 100 nM E2 for 24 hours and the NO and ADMA levels of the media were measured. A nitric oxide analyzer was used for the detection of NO metabolites. ADMA and L-arginine were measured by high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde. The IVF patients with high plasma E2 concentrations had significantly lower (48%, n = 12) plasma ADMA and higher (56%, n = 14) NO levels than the women at a low estrogenic stage. The incubation of HUVEC cultures with estradiol resulted in a significant decrease (47%, n = 10) in ADMA and an increase (46%, n = 10) in NO concentration in the culture media. Estradiol, by reducing ADMA, may therefore facilitate NO synthesis in endothelial cells. The protective effects of estradiol on vasculature may partly be related to a reduction in ADMA levels. PMID- 16669408 TI - Does the association between serum endostatin, an endogenous anti-angiogenic protein, and acute myocardial infarction differ by race? AB - Endostatin, an endogenous anti-angiogenic protein, has been linked to reduced atherosclerosis in animal models. We conducted a nested case-control study to ascertain whether decreased circulating endostatin might be associated with increased odds of acute myocardial infarction (AMI) and whether this association varied by sex or race. Cases were 211 subjects who subsequently developed AMI, and controls were 173 subjects free of cardiovascular disease matched on age, sex, race and follow-up time. In conditional logistic regression adjusting for traditional risk factors, the odds ratio of AMI per 1 SD increment in endostatin was 0.85 (95% confidence interval, 0.73-1.00). This association varied by race (but not by sex) such that a statistically significant inverse relation was found among Asians and white individuals and a significant positive relation among black individuals. Further research is needed to replicate these findings and to elucidate potential mechanisms for these race/ethnic differences. PMID- 16669410 TI - Limitation of the resting ankle-brachial index in symptomatic patients with peripheral arterial disease. AB - Peripheral arterial disease (PAD) has been demonstrated to be prevalent in the primary care setting. However, it has also been shown to be unrecognized and under-treated. Owing to the association with cardiovascular disease it has been recommended to screen high-risk patients for PAD in the primary care setting using the ankle-brachial index (ABI). ABI has been demonstrated to be highly sensitive and specific in diagnosing PAD in patients with significant stenosis. However, the utility in patients with less severe stenosis and calcified vessels is in question. The aims of this study were to determine the diagnostic utility of measuring the ABI at rest in patients referred to the vascular laboratory for evaluation of suspected PAD, and to assess the added value of pulse volume recordings and post-exercise studies in patients with a normal ABI. A computerized vascular diagnostic laboratory database was queried for symptomatic outpatients referred for measurement of segmental blood pressure, the ABI or pulse volume recordings by physicians not specialized in the evaluation and management of patients with peripheral vascular disease. Of 707 patients undergoing outpatient physiologic arterial evaluations between February 1, 2003 and July 31, 2004, 396 met these inclusion criteria. Data recorded included resting ABI, ABI following treadmill exercise test and the presence of abnormal pulse volume recordings. The study population (n = 396) consisted of equal numbers of men and women (mean age 69 years, range 19-100 years). Among 396 studies, resting ABI values were normal in 183 (46.2%) and abnormal in 159 (40.2%). Of the 138 patients who underwent exercise testing, 84 had normal ABI readings at rest. In the 84 patients who had a normal ABI at rest and underwent exercise testing, the ABI fell below 0.9 after exercise in 26 (31%). Arterial non compressibility was detected in 54 (13.6%) patients, whose average age was 67 years. Thirteen (24%) of those with non-compressible vessels had abnormal pulse volume recording (PVR) results, compared to five with normal resting ABI who had abnormal PVR findings (2.7%). In conclusion, this study demonstrated that nearly half of patients referred to the outpatient vascular laboratory because of suspected arterial disease had a normal resting ABI. While it is recommended that the ABI be measured at rest in patients at risk of PAD in primary care practice, these findings suggest that patients with symptoms of PAD should be more completely evaluated in a vascular laboratory. Furthermore, when the ABI is normal at rest in patients with symptoms of intermittent claudication, exercise testing is recommended to enhance the sensitivity for detection of PAD. PMID- 16669409 TI - Markers of vascular inflammation are associated with the extent of atherosclerosis assessed as angiographic score and treadmill walking distances in patients with peripheral arterial occlusive disease. AB - The importance of inflammation in atherosclerosis is well established in cardiovascular disease. However, limited data exist on the relationship between vascular inflammation and the severity of peripheral arterial occlusive disease (PAD). We investigated the relationship between biochemical markers of vascular inflammation and the diagnostic measures of PAD: ankle-brachial pressure index (ABI), maximum treadmill walking distance and angiographic score. In 127 patients (mean age 66 years; 64% males) with angiographically verified PAD, fasting blood samples were drawn for determination of selected soluble cell adhesion molecules, cytokines and chemokines. Tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and CD40 ligand (CD40L) were all significantly correlated with the angiographic score (p < 0.05 for all). After adjustment for relevant co-variates, MCP-1 and CD40L remained statistically significant (p < 0.01 for both). IL-6 was, independent of other risk factors, inversely correlated with the maximum treadmill walking distance (p < 0.01). Our cross-sectional study in PAD patients showed that the vascular inflammatory markers MCP-1, CD40L and IL-6 were significantly associated with the extent of atherosclerosis, assessed by angiographic score and maximum treadmill walking distance. These findings indicate that vascular inflammation is implicated in PAD, which might be of importance in future diagnosis and treatment of the disease. PMID- 16669411 TI - An unusual case of toe ulceration. AB - We describe an unusual presentation of toe ulceration in a 39-year-old otherwise healthy man. The left fifth toe was painful for several months with ulceration at the tip. The other toes appeared cyanotic and discolored. Foot pulses were normal. Laboratory testing revealed the platelet count at 1208 thousand/microliter, with hemoglobin 19.2g/dl and hematocrit 57%. The erythropoietin level was found to be markedly decreased at 1 mU/ml. The patient was given the diagnosis of polycythemia vera with iron deficiency. Phlebotomy was performed and aspirin and cytoreductive therapy with hydroxyurea was prescribed with resolution of the toe ulceration. PMID- 16669412 TI - Cellular adhesion molecules and peripheral arterial disease. AB - Cellular adhesion molecules (CAMs), by mediating the recruitment of circulating leukocytes to the blood vessel wall and their subsequent migration into the subendothelial spaces, play a crucial role in all stages of atherosclerosis. Soluble forms of CAMs, probably derived from proteolytic shedding, are present in the circulation and their blood levels parallel the amount expressed on the cell surface. In patients with peripheral arterial disease (PAD), increased levels of soluble CAMs have been found during exercise-induced claudication, are associated with the presence, the severity and the extent of atherosclerosis in the arteries of the lower limbs, and portend a worse outcome. These findings have provided new insights into the pathophysiology of PAD and its consequences. However, further large population studies are needed to firmly establish whether increased levels of circulating CAMs give additive information to current risk assessment approaches, and to verify whether PAD patients with elevated levels of circulating CAMs would benefit from any specific therapy. PMID- 16669413 TI - Images in vascular medicine. Giant coronary aneurysm due to Kawasaki's disease. PMID- 16669414 TI - Images in vascular medicine. Jugular vein aneurysm--ultrasonographic evaluation. PMID- 16669415 TI - Images in vascular medicine. Morphologic and functional assessment of extensive pulmonary arteriovenous malformations using MRI. PMID- 16669416 TI - Masterclass series in peripheral arterial disease. Antiplatelet therapy for peripheral arterial disease and claudication. AB - Peripheral arterial disease (PAD) of the lower extremities is a common and potentially life-threatening manifestation of systemic atherosclerosis. Significant PAD is identified by an ankle brachial index (ABI) < 0.90; its presence is strongly associated with the major modifiable cardiac risk factors. Early detection and treatment of asymptomatic PAD is a current focus of numerous cardiovascular guideline organizations as less than a third of patients report typical claudication symptoms. This has created an ever-increasing treatment gap, whereby millions of eligible patients are inadequately treated. Risk factor management including exercise, smoking cessation, and aggressive treatment of lipids and blood pressure are essential in PAD patients. However, life-long antiplatelet therapy provides additional reductions in vascular events beyond aggressive risk factor management. The use of aspirin as well as more potent antiplatelet therapies such as thienopyridines holds promise for reducing atherothrombosis in this very high-risk population. PMID- 16669417 TI - Coronary-artery revascularization before elective major vascular surgery. McFalls EO, ward HB, Moritz TE, Goldman S, Krupski WC, Littooy F, Pierpont G, Santilli S, Rapp J, Hattler B, Shunk K, Jaenicke C, Thottapurathu L, Ellis N, Reda DJ, Henderson WG. N Engl J Med. 2004; 351: 2795-804. PMID- 16669419 TI - An article of faith. PMID- 16669418 TI - The brains of babes. PMID- 16669420 TI - Back pain: more to the story. PMID- 16669421 TI - Back pain: don't overlook evidence. PMID- 16669422 TI - The school of hard knocks. PMID- 16669423 TI - Fostering healthy brains. PMID- 16669424 TI - Field research. PMID- 16669425 TI - Taking teens to task. PMID- 16669426 TI - The brain-boosting benefits of breastfeeding. PMID- 16669427 TI - Keeping a cool head. PMID- 16669430 TI - Desperately seeking solutions. PMID- 16669428 TI - The pursuit of excellence. PMID- 16669429 TI - Translating care on the farm and in the city. PMID- 16669431 TI - Betwixt and between a new view of the adolescent brain. PMID- 16669432 TI - Assessment and diagnosis of attention-deficit/hyperactivity disorder by family physicians. AB - In this study, our aim was to determine the extent to which family physicians in Minnesota follow the American Academy of Pediatrics (AAP) guideline in the assessment and diagnosis of attention-deficit/hyperactivity disorder (ADHD) in school-aged children and to identify barriers to using the guideline. We surveyed 1,000 randomly chosen members of the Minnesota Academy of Family Physicians. Of 303 respondents, 36% always referred children for a diagnosis and 99.7% referred children for a diagnosis of ADHD some of the time. Fifty-four percent were unaware of the AAP guideline. However, among those who said they evaluate children for ADHD, most followed the criteria in the AAP guideline. For example, most family physicians (97%) used a child's response to stimulant medication in their assessment. Respondents also said that barriers to ADHD assessment included lack of reimbursement and training. PMID- 16669433 TI - The effect of maternal diabetes during pregnancy on the neurodevelopment of offspring. AB - As many as 10% of pregnancies are complicated by maternal glucose intolerance. With the risk of diabetes and gestational diabetes rising because of the obesity epidemic, that figure is likely to rise. Many obese individuals suffer from the metabolic syndrome, which makes them more prone to glucose intolerance when they are pregnant. Among the potential risks posed by poor maternal glucose control are those to the developing fetal brain. The goal of this article is to acquaint physicians with the results and clinical implications of studies conducted at the University of Minnesota on outcomes of infants of diabetic mothers and, in particular, on the role of iron deficiency in differential brain processing. PMID- 16669434 TI - Child maltreatment and brain development. AB - A growing body of research has linked childhood experiences of maltreatment with a host of physical conditions that manifest in adulthood. In addition, newer neuroimaging techniques have documented structural changes that occur in the brains of individuals who suffer early maltreatment. This article briefly reviews the literature on these topics and outlines the connection between abuse in childhood and health problems in adulthood. PMID- 16669436 TI - [Amoxicillin may be a cause of enamel hypomineralization]. PMID- 16669437 TI - [Karl Adolf Hallstrom founding member of Duodecim Society]. PMID- 16669435 TI - [Epigenetic mechanism underlying psychiatric disorders]. PMID- 16669438 TI - [Calcified heart]. PMID- 16669439 TI - [Postmenopausal hormone therapy]. PMID- 16669440 TI - [Munchausen syndrome]. PMID- 16669441 TI - [Chronic fatigue syndrome]. PMID- 16669442 TI - [Towards total evaluation of occupational performance]. PMID- 16669443 TI - [Severe acute pancreatitis as a systemic disease--can multiorgan failure be predicted and prevented?]. PMID- 16669444 TI - [The development of continuing basic medical education in Espoo Finland]. PMID- 16669445 TI - [Use of pad gives accuracy to the diagnosis of urinary tract infections in children]. PMID- 16669446 TI - [Duplication of external auditory canal of a four year old girl]. PMID- 16669448 TI - [Care of drug abusers]. PMID- 16669447 TI - [Recurrent aphthous ulcers of oral mucosa]. PMID- 16669449 TI - X-ray examination of the stomach bubble after frequent experimental swallowing of saliva: the mechanism of aerophagia. AB - BACKGROUND: While the mechanism of aerophagia remains unclear, the frequency of clenching has been reported to be increased when under stress. We hypothesized that, via the swallowing reflex, chronic air swallowing was induced through a "learned habit" of the oral cavity, which was acquired through psychological factors. This study examined whether the habitual repeated swallowing in the oral cavity was a process of aerophagia. METHODS: After continuous experimental saliva swallowing, changes in the stomach bubble were examined by abdominal X-rays in a standing position. The subjects included 9 males and 10 females aged 20 to 36 years that were without organic disease in the pharynx and nasal cavity. X-ray images were digitized, and the area of the stomach bubble was measured by tracing. RESULTS: A close correlation was revealed in the stomach bubble area between posteroanterior and lateral views in six males (p<0.001, r = 0.910). In 3 males and 10 females, the area of the stomach bubble by posteroanterior view after 30 swallows was significantly increased compared with that before swallowing (p = 0.004). CONCLUSIONS: In the present study, abdominal X-rays confirmed that frequent saliva swallowing expanded the stomach bubble. PMID- 16669450 TI - Current state and need for improvement of system for antibody testing and counseling for HIV infection at public health centers in Japan. AB - BACKGROUND: Public Health Centers (PHCs) throughout Japan have been playing a role in preventing HIV in Japan. The number of HIV seropositive persons and AIDS patients is increasing from year to year in Japan. METHODS: A survey regarding the HIV antibody test was sent out to 594 PHCs between January 1997 and December 2001. The response rate was 73.9%. In order to assess the HIV testing system at PHCs, Pearson's correlation coefficient, an analysis of variance (ANOVA) and multiple regression analysis were used. RESULTS: There was a difference in the demand for HIV antibody tests according to area. The change in number of HIV antibody tests per 100,000 persons was high in metropolitan areas during the 5 year period. It was influenced by two factors; population density and ratio of daytime population to nighttime population. Rate of examinees who did not return to receive their test results was influenced by the area where the PHC was located. CONCLUSIONS: PHCs need to establish an HIV testing system which reflects the characteristics of the area and meets the needs of people who want to get tested. PMID- 16669451 TI - Changes in salivary components by drug administration in patients with heart diseases. AB - In this study, patients with heart diseases were classified into 2 groups: Warfarin user and Warfarin non-user, and six salivary components were determined to assess intraoral pathologic conditions. Groups of healthy subjects and patients with periodontal disease without receiving any medication were set as control groups, and they were compared with those of the 2 groups with heart diseases. In patients with heart diseases in both the groups, albumin (ALB) level was found to be significantly higher compared to that in the control groups, and it was significantly higher in the patient group receiving Warfarin user and Warfarin non-user compared to that in the patient group with periodontal disease. C-reactive protein (CRP) levels were found to be higher in both the groups with heart diseases than those in the healthy group. Correlations between various salivary components and the clinical parameters were examined, showing significant correlations between ALB and gingival index (GI) and clinical attachment level (CAL), and between alanine aminotransferase (ALT) and GI, probing depth (PlI), bleeding on probing (BOP) and CAL. Significant correlations were also found between creatine kinase (CK) and PlI, GI and BOP. Thus, it was suggested that ALB and CRP might serve as the markers of intraoral pathologic conditions, and CK and ALT might serve as those alternative to GI. PMID- 16669452 TI - Elevated cyclooxygenase-2 expression is associated with histological grade in invasive ductal breast carcinoma. AB - PURPOSE: The aim of this study was to evaluate the expression of cyclooxygenase-2 in human breast cancer using immunohistochemistry and to determine whether the expression of cyclooxygenase-2 is associated with clinicopathological factors in invasive ductal breast carcinoma. METHODS: Cyclooxygenase-2 expression was investigated by immunohistochemistry in 30 invasive ductal breast carcinoma specimens and relationships between cyclooxygenase-2 expression and age, histological grade, histological type, nodal status, and hormone receptor status were evaluated. RESULTS: Cyclooxygenase-2 expression was found in 56.7% of the tumor samples and was related to histological grade (P<0.01) and histological type (P<0.001). CONCLUSIONS: Our results suggest that cyclooxygenase-2 expression has an important role in tumor differentiation in invasive ductal breast carcinoma. PMID- 16669453 TI - The effect of occlusal contacts on adjacent tooth. AB - PURPOSE: The purpose of this study was to clarify the influence of changes of occlusal contacts on adjacent tooth displacement during articulation. MATERIALS AND METHODS: The amount and direction of displacement of the maxillary left first molar and adjacent teeth were measured using a displacement transducer, Type M-3. Seven subjects were included in this study. On the maxillary left first molar, 8 experimental occlusal contact points were established using platinum foil and these were divided into 2 groups, the buccal and lingual groups, which corresponded to the buccal and lingual cusps, respectively. RESULTS: During clenching, the buccal group showed differences in the direction of the tooth displacement paths among the 3 teeth (second premolar, first and second molar), but there was no difference in the lingual group and the direction of displacement of these 3 teeth was similar to the natural teeth. CONCLUSION: It is necessary to make at least one lingual occlusal contact point to obtain displacement of not only the abutment tooth but also the adjacent teeth. PMID- 16669454 TI - Identification of genes preferentially expressed in articular cartilage by suppression subtractive hybridization. AB - Suppression subtractive hybridization is very effective to enrich differentially expressed genes in two different tissues or cells. We therefore used the technique to identify characteristic genes expressed in rat knee joint articular cartilage as compared to rat costal cartilage. In this study, we revealed that several genes were enriched in a subtracted articular cartilage cDNA library. The most enriched gene is lubricin that is a putative key molecule for joint lubrication. The second gene is milk fat globule epidermal growth factor (EGF) factor 8, MFG-E8 whose expression has never been observed in cartilage. Other enriched genes are known to be expressed in cartilage, however their differential expressions in cartilages have not been necessarily common. The preferential expression of characteristic genes in articular cartilage would provide unique properties to the tissue. Our findings will provide a new view of articular cartilage. PMID- 16669455 TI - Critical contribution of sanitation infrastructure and primary medical care to child health status in Japan from 1955 to 2000. AB - We examined the associations between prefectural health status of children and sanitation infrastructure, medical care services and socioeconomic status in Japan during 1955-2000. Four indicators of child health status: infant mortality rate (IMR), Ascaris lumbricoides prevalence and height and weight of elementary school children, and five prefectural social indicators: water supply, sewerage, number of physicians, income and women's education, were calculated over five year intervals and examined by correlation and panel data analyses. Associations between paediatric health and five social indicators were significant (p<0.001) until 1970. After adjustment for income or education, water supply diffusion rate showed a significantly negative association with IMR until the 1980s. A significantly positive correlation was observed between the number of physician and paediatric health from 1955-70 after adjustment for income. Water supply development showed a positive association with paediatric health after adjustment for economic or educational impact during the phases of rapid and steady economic growth, while medical care showed a positive correlation with paediatric health after adjustment for income during only the phase of rapid economic growth. Relationships between child health status and social indicators have changed over time with stage of economic development. PMID- 16669456 TI - Antifungal effects of a tissue conditioner coating agent with TiO2 photocatalyst. AB - Tissue conditioners are susceptible to colonization by microorganisms. Therefore, the prevention of biofilm formation are important for oral hygiene. However, mechanical and chemical cleaning methods may cause clinical problems such as deformation or surface degradation of tissue conditioners. The objective of this study is to evaluate the antifungal effects of coating agents with a TiO2 photocatalyst. Photocatalytic antifungal effects on C. albicans biofilms and photodegradation effects of adsorbed protein were measured by colorimetric assays. Scanning electron microscopy was used to examine morphological changes in C. albicans. Viscosities of coating agents increased with incorporation of TiO2. However, both of coating agents with TiO2 were acceptable to the application by brush. The antifungal and protein degradation effects increased with the concentration of TiO2 in the coating agents. These effects also increased with radiation time. After 90 min radiation, the viability of C. albicans was reduced to 16.2+/-3.3 %. Scanning electron microscopy observation showed C. albicans remained on the coated surfaces even after 90 min radiation. These results suggest that coating agents with TiO2 photocatalyst can be effective for the maintenance of tissue conditioners when dentures are removed; during sleep. PMID- 16669457 TI - Toward secure distribution of electronic health records: quantitative feasibility study on secure E-mail systems for sharing patient records. AB - If the quality and efficiency of medical services are to be ensured, electronic health records (EHR) and EHR-supporting infrastructure must be prevalent. Many hospitals, however, have EHR systems for their internal use only, and the standardization process for the exchange of medical information is still in process. This standardization process addresses information security and is considering public key infrastructure (PKI) as one security measure, but PKI is rarely used by medical practioners because of its poor user-friendliness. Here we propose an effective use of the identity-based encryption (IBE) system as a security measure. This system enables us to send encrypted and signed messages without requiring the receiver to get a public key, and it enables us to deliver secured messages to ambiguous receivers like those to whom letters of reference are sent. We evaluated the feasibility of this technology by using the analytic hierarchy process, which is an effective analysis tool when selection and judgment depend on nonquantitative psychological factors, to analyze the results of an experiment in which medical workers used E-mail agents with and without PKI and IBE. We found that medical practioners and researchers avoid using PKI because of its poor user-friendliness and instead use IBE even though it is harder to install. We therefore think IBE would encourage medical institutions to share patient records. PMID- 16669458 TI - Pilot study of nitric oxide-donating aspirin in patients with pancreatic cancer pain. PMID- 16669459 TI - Cancer survivors and infertility: a review of a new problem and novel answers. AB - Improvements in cancer treatment have changed the way in which cancer is viewed and experienced. However, these same treatments have led to numerous early and late effects, including the loss of fertility. Infertility can influence the biologic and psychologic health of both male and female survivors. Reproductive science can now offer methods to address this concern and provide promising new approaches that may eliminate or mitigate this treatment-related outcome. For current and future reproductive options to serve the needs of survivors more fully, health providers must understand the complexities of infertility as well as their role in delivering answers their patients require. This review will discuss what is known about the causes and experience of infertility among cancer survivors as well as the forms of fertility preservation available. PMID- 16669460 TI - An individualized approach to fertility preservation in women with cancer. PMID- 16669461 TI - Cancer survivors and infertility: where do we go from here? PMID- 16669462 TI - The financial requirements and time commitments of caregivers for autologous stem cell transplant recipients. AB - This study is a prospective evaluation of the time commitment and financial requirements of caregivers of autologous stem cell recipients during the period of inpatient hospitalization. Eligible patients identified one caregiver, and a one-page survey addressing the necessary time commitment and out-of-pocket expenses was completed by the caregiver at each visit. The caregivers of 40 patients participated (non-Hodgkin's lymphoma [n = 19], multiple myeloma [n = 18], Hodgkin's lymphoma [n = 2], or acute myelogenous leukemia [n = 1]). Caregivers included spouses (n = 35), partners/friends (n = 2), or family members (n = 3). Results were summarized for the patient's total length of stay. Each caregiver traveled a median of 829 miles over 17.8 hours. Out-of-pocket expenses varied greatly depending on whether a caregiver stayed in local accommodations (cohort 1; n = 11) or in the patient's hospital room (cohort 2; n = 29). Total expenses (median) for each caregiver in cohort 1 were dollar 849.35, including accommodations (dollar 560), gasoline (dollar 87.35), and food (dollar 202). Total expenses (median) for each caregiver in cohort 2 were dollar 181.15, including gasoline (dollar 70) and food (dollar 111.15). Each caregiver in cohort 1 lost a median of 43.5 hours of work compared with 8 hours for each caregiver in cohort 2. The results from this prospective study demonstrate that there is a significant financial and time requirement on the part of the caregiver when a family member or significant other is hospitalized for an autologous stem cell transplant. PMID- 16669463 TI - Development and validation of the Functional Assessment of Cancer Therapy-Kidney Symptom Index (FKSI). AB - The Functional Assessment of Cancer Therapy (FACT)-Kidney Symptom Index (FKSI) was developed and validated to enhance treatment decision-making, practice guidelines, symptom management, and treatment efficacy for kidney cancer patients. Thirty-four symptoms related to the disease were identified and tested. An equal weighting of patient and clinician ratings of the relative importance of each of these items led to production of a 15-item index (FKSI-15) and a 10-item abbreviated option (FKSI-10). To assess psychometric properties, patients completed the FKSI, Functional Assessment of Cancer Therapy-General (FACT-G), Eastern Cooperative Oncology Group-Performance Status Rating (ECOG-PSR), and a Global Rating of Change Scale (GRCS). Patient responses to the FKSI were analyzed for internal consistency, test-retest reliability, convergent and discriminant validity, and responsiveness to change in clinical status. The FKSI-10 showed high internal consistency; correlations between both FKSI-10 and the physical and functional well being domains of the FACT-G were high. The FKSI-10 differentiated patients grouped by ECOG-PSR (all P < 0.001) and discriminated patients based on their GRCS rating. The minimally important difference (MID) range estimate for the FKSI-10 was 2-4 points; the psychometric properties of the FKSI-15 were very similar (MID range, 3-5 points).Thus, the FKSI-15 and FKSI-10 are reliable and valid symptom indices for evaluating kidney cancer patients. PMID- 16669464 TI - Communicating a prognosis in advanced cancer. PMID- 16669465 TI - Recent Supreme Court decisions. PMID- 16669466 TI - Michigan health care leaders define the future of medicine and health care. Results of the future of medicine and health care survey. PMID- 16669467 TI - Implementing EMRs in the medical office--one doctor's story. Interview by Matthew Sabin. PMID- 16669468 TI - The final voyage. PMID- 16669469 TI - Cryotherapy for treatment of oral lesions. AB - Cryotherapy is the deliberate destruction of tissue by application of extreme cold. It is well received by patients due to a relative lack of discomfort, the absence of bleeding and minimal to no scarring after healing. It has many applications in oral medicine and clinical oral pathology, and is extremely usefu in patients for whom surgery is contra-indicated due to either age or medical history. In this paper we outline the principles, mechanisms of action, and current applications of cryotherapy in the treatment of oral lesions, and present some clinical cases. PMID- 16669470 TI - Dental therapy in Western Australia: profile and perceptions of the workforce. AB - BACKGROUND: In 2002, the Centre for Rural and Remote Oral Health (CRROH) completed a rural oral health workforce survey which indicated that a high number of therapists, although registered, were not working as therapists. The aim of the present study was to develop a profile of the dental therapy workforce and analyse the perceptions of therapists. METHODS: In 2004, a postal questionnaire survey was undertaken amongst all registered dental and school dental therapists for 1999, 2000, 2001, 2002 and 2003. RESULTS: Valid information was obtained from 253 therapists (55 per cent response rate). The therapy workforce are almost exclusively female, have an average age of 40 years, are working in urban areas, obtained their qualification on average 20 years ago, work for the School Dental Service and qualified in Western Australia. More than a quarter no longer worked as therapists. Perceptions regarding the advantages and disadvantages of dental therapy as a career were identified. CONCLUSIONS: When trying to promote dental therapy and school dental therapy as a career, retain therapists and recruit new graduates, the opportunities identified in this survey should be embraced. A clear focus on the issues will be required to facilitate meeting the workforce objectives as outlined in Australia's National Oral Health Plan. PMID- 16669471 TI - Guided self diagnosis: an innovative approach to triage for emergency dental care. AB - BACKGROUND: The basic model of triage, developed more than 200 years ago by the French, is based on categorization of patient needs and thus the urgency and time required for care. This model in various forms is used in most hospitals throughout the world. METHODS: In this study, a fully computerized guided self diagnosis system (based on a neural network design) was designed, prototyped, developed and trialled by front line non-clinically trained personnel in emergency dentistry. RESULTS: A total of 699 patients were seen as a result of the triage assessment within 14 days of their initial phone call and/or first contact. Patients categorized as requiring care on the day (Category 1) were provided more items of care (50 per cent of all items) and the greatest number of extractions (76 per cent) and endodontic treatments (78 per cent). Other categories were found to require less urgent care. CONCLUSIONS: The triage system developed in this study clearly holds significant promise in reducing the impact of emergency dental patients on dental health care systems. However, it is recognized that the system still requires some adjustment to ensure all Category 1 patients are examined before less urgent categories. PMID- 16669472 TI - Epidemiological analysis of tongue cancer in South Australia for the 24-year period, 1977-2001. AB - BACKGROUND: Tongue cancer (141 ICD-9) is the most common intra-oral malignancy in Western countries. In recent decades, reported tongue cancer incidence and mortality rates have increased both in Europe and in the United States, whilst survival has not improved. This study aimed to determine the epidemiology and survival trends of tongue cancer in South Australia over the 24-year period from 1977 to 2001. METHODS: Population-based data for tongue cancer were provided by the Central Cancer Registry Unit of the Epidemiology Branch of the South Australian Department of Health. Age-standardized incidence and mortality rates for males and females were calculated. Kaplan-Meier survival analysis was conducted according to time periods, age, sex and tongue sub-sites. Cox regression analysis was used to determine factors that influenced survival. RESULTS: During this 24-year period, 611 cases of tongue cancer (398 males, 213 females) were reported, the majority of which were squamous cell carcinomas. The most common age of diagnosis was 65-69 years in males and 60-64 years in females. Fifty cases (8.18 per cent of all tongue cancer cases) occurred in patients 40 years or younger. The most common cancer sub-sites reported were 'unspecified site' (48.45 per cent), lateral border (25.53 per cent) and base (18.49 per cent) of the tongue. The age-standardized incidence and mortality rates for males and females in South Australia were relatively low and stable, and there was no significant improvement in survival of tongue cancer over this period. Significant predictors for survival were sex, age and tongue sub-sites, with male, advanced age and base of tongue associated with poorer survival. CONCLUSIONS: Tongue cancer is an important health issue associated with poor survival. Early detection and diagnosis is important in order to improve survival rate for this malignancy. PMID- 16669473 TI - The comprehensive evaluation of temporomandibular disorders seen in rheumatoid arthritis. AB - BACKGROUND: We studied clinical signs and symptoms of temporomandibular disorders and radiological changes in the temporomandibular joint from patients with rheumatoid arthritis (RA) compared to patients with myofascial pain dysfunction of the temporomandibular system and control patients to evaluate clinical and radiological relationships. METHODS: A cross-sectional, controlled, clinical and radiological study was planned and 99 subjects (69 patients and 30 controls) were included in the study. RESULTS: Twenty-three patients with RA (69.7 per cent) had painful temporomandibular joint. Fifty-five per cent had myofascial pain dysfunction according to the research diagnostic criteria for temporomandibular disorders (TMD). Nearly all of our patients with RA (93.9 per cent) had symptoms, and almost all of them had positive findings of TMD in high resolution computed tomography. Condylar head resorption, joint space narrowing and degeneration were statistically more prominent features in patients with rheumatoid arthritis compared with controls (p < 0.05). The pain score on active palpation correlated with the number of the mandibular subchondral cysts on high resolution computed tomography (r = 0.6, p < 0.05). CONCLUSION: Although the myofascial pain of the temporomandibular system is an important cause of pain in rheumatoid arthritis, prospective controlled studies are needed to develop effective therapeutic strategies for these patients. PMID- 16669474 TI - Accessing government subsidized specialist orthodontic services in Western Australia. AB - BACKGROUND: In Australia there is growing demand for dental services. This leads to more pressure on the oral health service providers, and in particular government subsidized dental care. Against this backdrop it is important that government dental services (rationed to health care cardholders) are provided equitably on a basis of need, not access. The primary hypothesis investigated in this study was that there would be an even distribution of patients referred for government subsidized orthodontic care across Western Australia when regionally adjusted for socio-economic status. METHODS: Data were obtained as a de identified waiting list for orthodontic treatment at The University of Western Australia. The data included all patients on the orthodontic waiting list as at December 2003. RESULTS: Significant differences between ARIA categories were detected when all waiting list entries were distributed. However, the trend was towards more entries (adjusted for population) in highly accessible areas. It was found that people from relatively wealthy areas tended to be more likely to be on the waiting list than those from more disadvantaged regions. CONCLUSION: In summary, the results of this study indicate that there is an uneven distribution of demand for orthodontics waiting list positions across Western Australian postcode areas by remoteness (ARIA) and by socio-economic disadvantage (IRSD). The results suggest that demand for subsidized orthodontic care may be influenced by the general demand for treatment of that region and not on what would be expected to be an even need for treatment across all health care cardholders. PMID- 16669475 TI - The influence of the leaf gauge and anterior jig on jaw muscle electromyography and condylar head displacement: a pilot study. AB - BACKGROUND: A leaf gauge and an anterior jig may be used to assist the recording of a reproducible jaw position for restorative and prosthodontic treatment. This study investigated possible condylar displacement using an opto-electronic jaw tracking device and a leaf gauge or anterior jig. The effect of a leaf gauge and anterior jig on jaw muscle electromyography was also examined. METHODS: Five healthy adults without symptoms of temporomandibular disorders were selected. Condylar displacement during clenching were recorded simultaneously with electromyographic activity of superior and inferior heads of the lateral pterygoid, anterior and posterior temporalis, masseter, and suprahyoid muscles. Subjects were trained to bite at maximum and half-maximum bite-force using an anterior jig incorporating a force transducer. RESULTS: No consistent condylar displacement was observed in x, y and z axes between different bite-forces although there was a trend towards superior displacement. Comparison of maximum intercuspal clench and maximum clench on a leaf gauge and an anterior jig produced significant decrease in anterior temporalis activity (p < 0.05), whilst an anterior jig with maximum clench significantly decreased posterior temporalis muscle activity. CONCLUSION: Within the limits of this pilot study, no consistent change in condylar position was identified with these appliances. PMID- 16669476 TI - The effect of silver fluoride and potassium iodide on the bond strength of auto cure glass ionomer cement to dentine. AB - BACKGROUND: Diamine silver fluoride (Ag(NH3)2F), referred to as AgF, has been shown to reduce the incidence of caries in primary dentitions. The clinical application of this material has been limited by staining associated with both teeth and restorative materials. The application of potassium iodide (KI) after AgF eliminates stain formation. There is a lack of information as to how the addition of AgF followed by KI may affect the bond strength to dentine. The purpose of this study was to compare the bond strengths of auto cure glass ionomer cement to dentine surfaces that had been treated with AgF and KI and without treatment. METHODS: Ten recently extracted human third molars were embedded into methyl methacrylate resin and sliced to form a square block of exposed dentine surfaces. Each of the four surfaces were treated by one of the following procedures: (a) etching with 37 per cent phosphoric acid; (b) applying GC dentine conditioner; (c) etching, followed by application of AgF/KI then washing off the precipitate and air drying; and (d) etching, applying AgF/KI and air drying the reaction products on the surface. Fuji VII auto cure glass ionomer cement was bonded onto each sample and fracture tested. RESULTS: The dentine samples treated with AgF/KI followed by washing away the precipitate and air drying had bond strengths (2.83 MPa) not significantly different from samples that had been conditioned (2.40 MPa). Samples where the AgF/KI precipitate had been air dried onto the dentine surface had significantly lower bond strengths (1.49 MPa) than the washed samples. Samples that were etched had significantly lower bond strengths (1.91MPa) than the conditioned samples. CONCLUSIONS: This study found that the application of AgF/KI to etched dentine samples followed by washing off the precipitate, created bond strengths that were not significantly different to conditioned samples. Leaving the AgF/KI precipitate on the dentine surface significantly reduced the bond strength of auto cured glass ionomer cement to dentine. Washing away the reaction products and air drying is recommended as the clinical protocol for using AgF and KI on dentine surfaces prior to application of an auto cure glass ionomer cement. PMID- 16669478 TI - Flexural properties of glass fibre reinforced acrylic resin polymers. AB - BACKGROUND: In recent years, glass fibres have been used to strengthen denture base resins. A major difficulty in using reinforcing fibres with multiphase acrylic resins, such as powder liquid resins, is inadequate impregnation of the fibres with the resin. METHODS: This investigation examined the reinforcing effect of glass fibres on the fracture resistance and flexural strength of acrylic denture base resins. Eighty identical specimens were formed in specially designed moulds in accordance with the manufacturer's recommendations. The four experimental groups were prepared and these consisted of conventional acrylic resin and the same resin reinforced with glass fibres. Ten specimens were fabricated in a standardized fashion for each experimental group. Flexural strength was tested using a 3-point universal testing machine. RESULTS: In this study, statistically significant differences were found in the flexural strength of the specimens (P < 0.05). The injection-moulded, fibre-reinforced group had significantly lower flexural strength than the injection-moulded group (P < 0.001), strength than the microwave-moulded, fibre-reinforced group (P < 0.001), and the microwave-moulded, fibre-reinforced group had lower flexural strength than the microwave-moulded group. The fracture resistance was significantly higher in the injection-moulded, fibre-reinforced group than in the injection moulded group (P < 0.05), and the fracture resistance was significantly higher in the microwave-moulded, fibre-reinforced group than in the microwave-moulded group. CONCLUSION: Within the limitations of this study, the flexural strength of heat-polymerized PMMA denture resin was improved after reinforcement with glass fibres. It may be possible to apply these results to distal extension partial and complete denture bases. PMID- 16669477 TI - Factors influencing recent dental graduates' location and sector of employment in Victoria. AB - BACKGROUND: Recruitment and retention of dentists in the public sector and rural areas in Victoria has become increasingly difficult in recent years. There are little available data on the factors that influence the sector and location of practice of new dental graduates. The objective of this study was to investigate the factors considered by new graduates in determining the location and sector of employment after graduation, and influencing any early changes in career path. METHODS: Questionnaires were sent to dentists who Mgraduated from The Univrersity of Melbourne from graduated from The University of Melbourne from 2000-2003 who were currently practising in Victoria. There were 154 subjects to whom questionnaires were sent and 109 useable questionnaires were returned, a response rate of 74 per cent. RESULTS: Upon graduation, 53 per cent of the new graduates chose to work in the private sector only, compared to 15 per cent in public sector only and 33 per cent in both. At present, 71 per cent work in the private sector only, 17 per cent in the public sector only and 12 per cent in both. The most important factors for choosing to work in the private sector were receiving broad range of clinical experience, opportunities to familiarize with practice management and providing a continuity of care. The principal factors for practising in the public sector were clinical mentoring and advice, consolidating clinical skills and work environment. Initially, 48 per cent of the sample chose to work in metropolitan areas only, 39 per cent in rural areas only and 13 per cent in both. Factors that influenced the decision to work in rural areas were the broad range of clinical experience and remuneration, while the main factors for choosing to work in metropolitan areas were lifestyle and proximity to family and friends. CONCLUSIONS: This study found that a large proportion of new dental graduates initially chose to work in the public sector and rural areas on graduation primarily as a means of consolidating their clinical skills. However, retention of dentists in both these areas appears to be a problem, with less than 10 per cent of 2000-2001 graduates still than 10 per cent of 2000-2001 graduates still working in the public sector and only 20 per cent of 2000-2001 graduates still working in rural areas. PMID- 16669479 TI - Reaction of rat pulp tissue to Carisolv 'new gel'--a histocytological evaluation. AB - BACKGROUND: This study was an histological examination of pulp tissue exposed to Carisolv 'new gel' after 1 to 28 days. METHODS: An occlusal cavity was prepared in 64 caries-free molar teeth of 16 Wistar rats. The roofs of the pulp chambers were perforated and Carisolv 'new gel' solution was placed onto the exposed pulps of 32 molar teeth for 20 minutes. Thirty-two contralateral molar teeth served as controls and were coated with an inert liquid containing isotonic saline solution and carmellose for 20 minutes as well. The pulps of all teeth were capped with Ca(OH)2 and the cavities were filled with a flowable composite in combination with a self-etching dentine adhesive. The animals were sacrificed after 1, 3, 7 and 28 days. Eight teeth per group and the time period were histologically examined, scored, and statistically evaluated (Wilcoxon-test). RESULTS: The results showed no statistically significant differences between the Carisolv group and the control group (p > 0.05). The observed pulp reaction was essentially the same as those reported in the past being typical for the effect of calcium hydroxide as a direct pulp capping agent. CONCLUSION: Compared to Ca(OH)2, Carisolv 'new gel' did not cause any different or additional pulp reaction in healthy teeth. PMID- 16669480 TI - Methods of determining the relationship of the mandibular canal and third molars: a survey of Australian oral and maxillofacial surgeons. AB - BACKGROUND: Surgical extraction of third molars is one of the most common oral and maxillofacial surgical procedures performed and may have a number of associated complications. One of these complications is inferior alveolar nerve (IAN) dysaesthesia or impairment of sensory perception (including paraesthesia and/or anaesthesia). Previous studies assume that most clinicians use various combinations of nine radiologic criteria on panoramic radiographs as indicators of the relationship and, therefore, predictors of the risk of postoperative dysaesthesia. Our study assessed both the current radiologic modalities and assessment criteria used by Australian oral and maxillofacial surgeons when determining the proximity of mandibular canal to third molars. METHODS: A survey of all surgeon members of the Australian and New Zealand Association of Oral and Maxillofacial Surgeons (ANZOMS) practising in Australia was undertaken. RESULTS: Of the 105 questionnaires sent to surgeons, 72 responses (68 per cent) were returned. All surgeons reported using the panoramic radiograph but only 25 per cent considered it sufficiently accurate in determining the relationship between the mandibular canal (MC) and the third molar root, while 61 per cent of surgeons use CT for this purpose but the average frequency of use was very low (five per cent). This study also revealed that the nine radiologic criteria on a panoramic radiograph are used to varying extents by Australian surgeons. Nearly all surgeons use 'change in MC direction' and 'MC narrowing' to determine and close relationship. Thirty-one per cent used superimposition of the MC and the root of the third molar alone and 24 per cent used appearance of contact of the root with the MC alone in the absence of any other radiologic criteria to indicate close or intimate relationship. CONCLUSION: Further research is required to determine the accuracy and observer agreement or reliability of using the nine panoramic characteristics, to determine this relationship and whether the presurgical determination of proximity and position (buccal or lingual) of the canal utilizing CT has any usefulness in determining the surgical protocol or affect on postoperative morbidity. PMID- 16669481 TI - Analysis of the acute postoperative pain experience following oral surgery: identification of 'unaffected', 'disabled' and 'depressed, anxious and disabled' patient clusters. AB - BACKGROUND: Pain is defined as both a sensory and an emotional experience. Acute postoperative tooth extraction pain is assessed and treated as a physiological (sensory) pain while chronic pain is a biopsychosocial problem. The purpose of this study was to assess whether psychological and social changes occur in the acute pain state. METHODS: A biopsychosocial pain questionnaire was completed by 438 subjects (165 males, 273 females) with acute postoperative pain at 24 hours following the surgical extraction of teeth and compared with 273 subjects (78 males, 195 females) with chronic orofacial pain. Statistical methods used a k means cluster analysis. RESULTS: Three clusters were identified in the acute pain group: 'unaffected', 'disabled' and 'depressed, anxious and disabled'. Psychosocial effects showed 24.8 per cent feeling 'distress/suffering' and 15.1 per cent 'sad and depressed'. Females reported higher pain intensity and more distress, depression and inadequate medication for pain relief (p < 0.001). Distress and depression were associated with higher pain intensity. The developed questionnaire had tested reliability (test-retest r = 0.89) and estimated validity. CONCLUSION: Cluster analysis showed constituent groups with a range of psychosocial effects in acute postoperative dental extraction pain and is associated with an increase in pain intensity. PMID- 16669482 TI - Dental fear in Australia: who's afraid of the dentist? AB - BACKGROUND: This study aimed to describe both the prevalence of dental fear in Australia and to explore the relationship between dental fear and a number of demographic, socio-economic, oral health, insurance and service usage variables. METHODS: A telephone interview survey of a random sample of 7312 Australian residents, aged five years and over, from all states and territories. RESULTS: The prevalence of high dental fear in the entire sample was 16.1 per cent. A higher percentage of females than males reported high fear (HF). Adults aged 40 64 years old had the highest prevalence of high dental fear with those adults aged 80+ years old having the least. There were also differences between low fear (LF) and HF groups in relation to socioeconomic status (SES), with people from higher SES groups generally having less fear. People with HF were more likely to be dentate, have more missing teeth, be covered by dental insurance and have a longer time since their last visit to a dentist. CONCLUSIONS: This study found a high prevalence of dental fear within a contemporary Australian population with numerous differences between individuals with HF and LF in terms of socioeconomic, socio-demographic and self-reported oral health status characteristics. PMID- 16669483 TI - Failure to obtain adequate anaesthesia associated with a bifid mandibular canal: a case report. AB - The inferior alveolar nerve (IAN) block is the most common method for obtaining mandibular anaesthesia in dental practice but it is estimated to have a success rate of only 80 to 85 per cent. Causes of failure include problems with operator technique and anatomical variation between individuals. This case report involves a patient who received IAN blocks on two separate occasions that resulted in only partial anaesthesia of the ipsilateral side of the mandible. Radiographic assessment disclosed the presence of bifid mandibular canals that were present bilaterally and that may have affected the outcomes of the local anaesthetic procedures. Previous studies of bifid mandibular canals are reviewed and suggestions provided that should enable clinicians to differentially diagnose, and then manage, cases where IAN blocks result in inadequate mandibular anaesthesia. PMID- 16669484 TI - Practice profiles of Australian private general dental practitioners. PMID- 16669485 TI - [Healed war injuries of the cranium in the osteologic collection from the Broumov Ossuary (13th-18th century)]. AB - Traumatic bone lesions form an important part of the study of human paleopathology. Injuries of the skull are frequent in the history of the human race. 647 adult dry skulls of both sexes from the "Broumov Ossuary" (13th-18th century) were examined for the incidence of cranium injuries. In this paper, an extensive collection (n=122) of healed dry skull injuries is presented. In the neuro- and splanchnocranium of skulls (n=78), linear and depressed fractures, slash and stab wounds have been seen but no gunshot wound. In part of the skulls (n=29), multiple injuries have been observed. Many detected traumatic lesions seem to have been caused by traditional weapons of the last centuries: war swords, axes, or pole-arms with metal spikes. All the skull injuries show clear signs of well-healing with bone remodelling that indicates the survival of victims for a long period after the cranial trauma. PMID- 16669486 TI - [Glutathione and glutathione assays]. AB - Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule. PMID- 16669487 TI - [The tumor supressor gene p53]. AB - p53 is known as a tumor suppressor gene important for maintenance of genomic integrity. p53 is a short lived protein that is maintained at very low levels in cells. p53 is regulated by Mdm2 protein which participate in p53 rapid degradation. The activation and accumulation of p53 is a response to cellular stress such as DNA damage. Activated p53 is a sequence specific DNA-binding transcription factor and some target genes of its transcriptional activity are important for cell-cycle arrest or for inducing apoptosis. p53 is also used for cancer therapy by p53 gene therapy when p53 is applied into tumors or by reactivation of mutant p53. PMID- 16669488 TI - [Prognostics in idiopathic scoliosis]. AB - Prognostics in idiopathic scoliosis is aimed at finding rules for predicting the progression of the curve. The authors report certain prognostic methods as well as the result of the latest Japanese method applied to our own group of patients. The calculated prediction was true in 50 % of the cases. Therefore, the tested Yamauchi's prognostic system proves to be reliable only to a small extent. PMID- 16669489 TI - [Correction of idiopathic scoliotic curves by means of conservative treatment using spinal bracing]. AB - Three groups of patients were selected for this retrospective study. Each group underwent treatment with spinal bracing. The aim was to identify the correction of curves during several stages of treatment. Results were statistically processed. The best initial correction was shown in Cheneau hypercorrective brace. However, at the end effect the statistical significant difference between the braces was not proved. PMID- 16669490 TI - [Indication and radicality of surgical treatment in spinal myeloma]. AB - In our set of 19 operated patients with spinal myeloma it was in 17 patients (89%) the first manifestation of malignant haematological disease. Indication for admission for surgery were in 18 patients (92%) graphical signs of spinal compression of osteolytic affliction of the vertebra of unclear aetiology, which in 17 (63%) of patients were demonstrated by neurological deficit. On the basis of graphical examinations the most frequent suspicion was on metastatic affliction of the spine at unknown primary focus and the basic diagnosis was determined after the operation at most of cases. In decision about the choice of radicalness of surgery intervention important data about the type of tumour in the spine were usually missing and it especially did not make possible to estimate the life expectation of the patient. When deciding about the radicalness of the surgery, we started from the degree of spinal compression and surgical character of pathological process in the spine. The aim of the operation is to decompress the spinal cord in tumour and to stabilize the afflicted part of the spine. Radical extirpation of tumour followed with stabilization and spine reconstruction we decided to carry out in 7 patients, i.e. 37%, all of them clinically improved and from this group only 2 patients (29%) died till now. We carried out only palliative surgery on the spine in next 12 patients, i.e. in 63%, only 6 patients improved neurologically, i.e. 50% and 10 patients, i.e. 83% died till now. PMID- 16669491 TI - [Surgical treatment of brain abscess--retrospective analysis of the group of patients with brain abscess, treated at our department in last 12 years]. AB - The authors retrospectively evaluated group of patiens treated at the Department of neurosurgery in Hradec Kralove from 10/1993 to 10/2004 with the diagnosis of brain abscess. During this period, we treated 23 patients, 15 males and 8 women with the median age 48 years. Patiens with the iatrogenic etiology and those with pyocefalus and subdural and epidural empyema were excluded from this group. We provided 45 surgical procedures with total mortality 17,4 %. PMID- 16669492 TI - [Two interesting methods of estimation of the diagnosis uncertainty]. AB - It's very important to estimate the measure of uncertainty of data in the case of diagnosis of disease. We recommend two methods with help fuzzy interpretation of disease data. PMID- 16669493 TI - Wanless: public health reform has fallen victim to pay rises. PMID- 16669494 TI - Cuts threaten 18-week wait target. PMID- 16669495 TI - Chancellor delays senior management pay deal. PMID- 16669496 TI - HIV/AIDS centre closure threat. PMID- 16669497 TI - PbR under the microscope: what the South Yorks boffins found. PMID- 16669498 TI - On 'real men' and trust. PMID- 16669499 TI - Breaking the time barrier. AB - The DoH must deliver annual savings of pound 6.5bn by 2008. Productive time will help achieve these savings through increased efficiency. Patient care should also improve as a result. Obstacles include the current NHS restructuring, which could cause a loss of mementum. PMID- 16669500 TI - Lean healthcare. Think yourself thin. AB - Lean thinking looks at whole processes, not steps within them in isolation. It does not provide simplistic answers but streamlines processes and reduces waits. Managing and synchronising different processes can improve productivity while reducing delays and errors. Lean modelling can improve performance and PMID- 16669501 TI - Best in show. PMID- 16669502 TI - Management theory. Flying the flag. AB - The NHS Institute for Innovation and Improvement says costs will fall and patient care will improve if the service adopts techniques pioneered in the industry. The private sector practice of listening to customers is vital to progress in the NHS. Reducing sickness absence needs to include the promotion of flexible working practices. PMID- 16669503 TI - Performance measures. Problem of the scale. AB - Current productivity measurements do not cover quality of care and any changes in this, or whether patients' health has improved. The productive time initiative will measure changes in key indicators rather than overall output. Observers believe payment by results will provide an impetus for trusts to improve data collection. PMID- 16669504 TI - Frontline engagement. Leap of faith. AB - Making jobs interesting is a good motivator for nursing staff. The initiative requires effective leaders at all levels in the health and social system. Lack of time is often a major barrierto engaging people in productive time work. PMID- 16669505 TI - Data briefing. Are ITCs cost-effective? PMID- 16669506 TI - Primary care. Settle for super. AB - The healthcare outside hospitals white paper laid emphasis on provision of a range of services in a primary care setting. But there is no specific commitment from the Department of Health to 'polyclinics' in the white paper. Many of the new-style surgeries are being funded via the local improvement finance trust programme. PMID- 16669507 TI - On primary care. PMID- 16669508 TI - New PPS on the way. New prospective payment system expected this week. PMID- 16669509 TI - Business allies with the AHA. PMID- 16669510 TI - Diversity in the executive suite. Modern Healthcare salutes the Top 25 Minority Executives in Healthcare, who say their colleagues still face numerous obstacles. AB - With minorities expected to swell to half of all U.S. residents by 2050, the healthcare C-suite needs to reflect that diversity to better meet patients' needs. We honor the Top 25 Minority Executives in Healthcare, who have found ways to help others along the way. "I've learned to navigate within a system and work with people for the greater good, says Christopher Mosley, left, who made the Top 25. PMID- 16669511 TI - Second mistrial for Alvarado. No word if Tenet will face another trial on allegations. PMID- 16669512 TI - Universal solution? Mass. hospitals welcome bill to cover nearly all. PMID- 16669514 TI - Speeding up IT. Lawmakers frustrated by delays in creating network. PMID- 16669513 TI - The second wave. Report unveils another national IT plan. PMID- 16669515 TI - Fighting for privacy. Protect patients in rush to health IT: coalition. PMID- 16669516 TI - Initiatives join to expand P4P. PMID- 16669517 TI - Drug lawsuits building. Merck, Pfizer facing charges of misrepresentation. PMID- 16669518 TI - N.J. hospitals to fight tax-me, pay-me plan. PMID- 16669519 TI - Through the teflon ceiling. A helping hand is needed if ranks of the influential are to become more diverse. PMID- 16669520 TI - High priority, low progress. Safety gets more attention, but 'incidents' climb. PMID- 16669521 TI - Bacillus (corynebacterium) xerosis and the ocular surface. PMID- 16669522 TI - Cell markers and the side population phenotype in ocular surface epithelial stem cell characterization and isolation. AB - The ocular surface is covered by tworapidly renewing and embryologically-related linings, the corneal and conjunctival epithelia. The long-term survival of thesetissues is ultimately dependent on their respective resident stem cells. In the corneal epithelium, the stem cells and their early precursors are exclusively circumscribed to the narrow vasscularize limbal rim that provides epithelial precursor cells to the critically transparent central cornea. Limbal damage causes an interruption of this essential cell supply and allows the invasion of the corneal surface by the conjunctival epithelium, an event that ultimately leads to corneal scarring. The limited supply of immunocompatible tissue is a major hindrance to efforts to develop effective procedures for ocular surface reconstruction. This review describes some of the current work and strategies being developed to achieve the isolation of the limbal stem cell and define its genetic, biochemical, and functional make-up. The study of isolated ocular surface stem cells will foster basic understanding of the environmentalrequisites for their survival and proliferation in a self-replicative mode, leading eventually to advances in therapeutic approaches. PMID- 16669523 TI - Silicone hydrogel contact lenses and the ocular surface. AB - For 30 years, contact lens research focused on the need for highly oxygen permeable (Dk) soft lens materials. High Dk silicone hydrogel contact lenses, made available in 1999, met this need. The purpose of this review is to examine how silicone hydrogel lens wear affects the ocular surfaces and to highlight areas in which further research is needed to improve biocompatibility. Silicone hydrogel lenses have eliminated lens-induced hypoxia for the majority of wearers and have a less pronounced effect on corneal homeostasis compared to other lens types; however, mechanical interaction with ocular tissue and the effects on tear film structure and physiology are similar to that found with soft lens wear in general. Although the ocular health benefits of silicone hydrogel lenses have increased the length of time lenses can be worn overnight, the risk of infection is similar to that found with other soft lens types, and overnight wear remains a higher risk factor for infection than daily wear, regardless of lens material. Future contact lens research will focus on gaining a better understanding of the way in which contact lenses interact with the corneal surface, upper eyelid, and the tear film, and the lens-related factors contributing to infection and inflammatory responses. PMID- 16669524 TI - Deborah Pavan-Langston. PMID- 16669525 TI - Risks and benefits. PMID- 16669526 TI - When Medicaid pays the bills. PMID- 16669527 TI - From consumer to caregiver. Individuals and systems benefit from use of peer support models. PMID- 16669528 TI - EMRs bring all of healthcare together. PMID- 16669529 TI - The 411 on choosing cellular service. PMID- 16669530 TI - Managing risk means ensuring quality care. PMID- 16669531 TI - Crossing the field's quality chasm. PMID- 16669532 TI - CATIE renews debate over old versus new antipsychotics. PMID- 16669533 TI - Planning for disasters after Katrina and Rita. PMID- 16669534 TI - Ten steps to leaving an organization in good hands. PMID- 16669535 TI - The times are a-changin'. Three recent conferences on behavioral health IT highlight the technologic challenges ahead for the field. PMID- 16669536 TI - Lead--don't manage--EHR adoption. PMID- 16669537 TI - What do customers really want? PMID- 16669538 TI - The quest for rest. PMID- 16669539 TI - To sleep? That's a dream. PMID- 16669540 TI - The war on HPV. PMID- 16669542 TI - New secrets for youthful skin. PMID- 16669541 TI - Is estrogen for you? PMID- 16669543 TI - Not always 'the happiest time'. PMID- 16669544 TI - Real-life answers. PMID- 16669545 TI - [Artificial nutrition in the patient with hyperglycemia]. AB - The stress of critical illness evokes insulin resistance and hyperglycemia. Artificial nutrition is commonly considered one of the main causes of hyperglycemia in hospitalized patients. Patients with newly diagnosed hyperglycemia had a significantly higher mortality rate and a lower functional outcome than patients with a known history of diabetes or normoglycemia Intensive insulin treatment to normalize blood glucose during feeding has been shown to improve morbidity and mortality in patients in intensive care. Insulin glargine may contribute to improving the glycemic values in patients receiving artificial nutrition with hyperglycemia. PMID- 16669546 TI - Do hypertension and diabetes mellitus influence the site of atherosclerotic plaques? AB - BACKGROUND: The concomitant occurrence of atherosclerotic plaques in carotid, coronary and peripheral vessels has been described in a number of studies. A few studies were, on the contrary, done for determining the role of hypertension and/or type 2 diabetes mellitus for the occurrence of the atherosclerotic plaques in different anatomical sites. Moreover these studies deal with atherosclerotic lesions that are generally considered, without differentiating their morphology as a function of the underlying disease, territory, and risk factors. Primary aim of this study is, thus, to verify whether the two most common causes for atherosclerotic disease, i.e., hypertension and type 2 diabetes mellitus, may influence the site of appearance of the atherosclerotic plaque. A second aim is to verify if the anatomical site of the plaque influences plaque morphology and vulnerability. PATIENTS AND METHODS: A retrospective study of 244 patients affected with type 2 diabetes mellitus or hypertension was performed; 114 subjects were affected by moderate-severe and drugs-treated hypertension (Group A); 55 were affected by type 2 diabetes mellitus in treatment with oral antidiabetic drugs (Group B); 75 were diagnosed as affected by the association hypertension and diabetes (Group C). The inclusion criteria were: exhaustive images of the cardiovascular system (coronary angiography, colour Doppler ultrasound of lower limb arteries and carotid arteries, transthoracic Doppler echocardiography ) and a serum lipid profile (total serum cholesterol, HDL cholesterol, LDL-cholesterol, triglycerides). Three different anatomical sites: carotid axis, ilio-femoral arteries and coronary district, were considered. In each site a plaque lesion-classification was performed to describe the morphology of the plaque. RESULTS: In patients with hypertension, carotid district seems to be the preferential site of onset of atherosclerotic plaques even if a statistical significant association between the two conditions was not found. Statistical evaluation didn't show significant association between different risk factors and coronary district too. On the opposite, a significant association (p < 0.001) between diabetes and the presence of atherosclerotic plaques into lower limb district was found. A very significant association (p < 0.001) between type 2-diabetes and the presence of non-ulcerative plaques was found too. CONCLUSIONS: Our study underlines the relationship between vessel plaques localization and concomitant risk factors for atherosclerosis and suggests a possible difference in plaque morphology and biological behaviour related to different anatomical site. PMID- 16669547 TI - [Efficacy and distribution pattern of the medical device Prima Difesa on rhinosinusal area]. AB - AIM: Common cold is the commonest form of acute rhinitis and the first pathology of the upper airways. Viruses, the main responsible of this pathology, carry out their cytopathic effect on the ciliated cells of the airways mucosa. Mucociliary transport shows their dangerous effect. The aim of our study is to evaluate the effect of the medical device Prima Difesa on the functioning of the mechanism thanks to its composition and its distribution modality in the nasal cavities. MATERIALS AND METHODS: The present study has been performed on three groups of subjects with their informed consent: Group A (30 healthy subjects: 19 M, 11 F; 18-36 yrs, mean age: 29.1 yrs), Gruppo B (30 patients affected by rhinosinusal pathology: 7 M, 13 F; 19-38 yrs, mean age 30.7 yrs) and Group C (20 healthy subjects: 12 M, 8 F; 20-40 yrs, mean age: 32.2 yrs). The protocol of administration for the Group A and B, in order to evaluate the efficacy of Prima Difesa, consisted of 2 puff per nostril of Prima Difesa 4 times a day over 15 days. The follow-up of patients belonging to Grup A and Group B was articulated in three consecutive visits, each consisting of: anterior rhinoscopy, active anterior rhinomanometry, measurement of mucociliary transport (TMCt). The C patients underwent only one administration of the device to the extent of evaluating through endoscopy its distribution modality in the nasal cavities. RESULTS: The results show that clinical effects, evaluated in term of improvement of MCTt and in term of decreasing of nasal resistance, are particularly clear in patients with alterations of these parameters caused by rhinosinusal pathologies. The device can reach the "key" areas of rhinosinusal pathologies that are: middle turbinate and osteomeatal complex. Beside, the percentage of diffusion, about 30% of the middle turbinate head, justifies its clinical effect. CONCLUSIONS: The medical device Prima Difesa has resulted to be able in interfering with the correct maintenance and performance of the Mucociliary Transport mechanism, that represents an important aspecific machinery for the local defence of nasal mucosa by microbial injuries. PMID- 16669549 TI - [Ethics of care and new profiles of the woman]. AB - INTRODUCTION: In health professions listening represents an essential condition for any cure relation. Learning to listen is thus neither easy nor granted. It requires the ability to put oneself in somebody else's place, maintaining self possession of judgement and the ability of evaluating events. PROBLEM ANALYSIS: In health professions women have always occupied a privileged position for their sensitiveness and their ability of giving meaning even to everyday life. Empathy is the privileged way women have of getting to know, understand and take care of people. They follow a feminine style which accepts and welcomes other people even if they are not able to explain their intimate contradictions, their anxieties, their difficulties, their pain and weakness. Women know how to be near people, even if they understand they are not up to the situation, not expecting to solve pain: neither personal nor other people's. They act in a different way from men who instead are attached to a reductionism that assumes a theoretical scheme to understand the issue. Man's need to dominate the problem with knowledge makes him sacrifice the complexity of reality in which he lives. Men's simplicity, their tendency to generalize processes and easily find guidelines to proceed with no useless wastes, often sacrifices elements which find no place into the chosen standards. For women this way of behaving, apparently reassuring, is not possible because they are aware that such aspects while escaping their understanding express the personality of the people involved in this process, their uniqueness which does not accept generalisations. Behind interpretative schemes, behind apparently definitive diagnosis, women perceive vagueness and interpretative inadequacy. CONCLUSIONS: Female rationality is inclined to live with one's own and other's contradictions just because it is strongly attached to a reality which avoids simplification of understanding. It refuses interpretative schemes, theories which try to offer themselves as exhaustive models which can explain all events, lessening the value or even denying elements which do not fall within the interpretation model assumed. Women appear contradictory because they do not refuse confrontation with life contradictions and its perennial changes. PMID- 16669548 TI - Glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factor differently modifies actin polymerization in neutrophils. AB - AIM: Several neutrophil functions can be modified by rhG-CSF administration. Neutrophil morphology changes in the course of treatment with Filgrastim (nonglycosylated rhG-CSF), along with impairment of chemotaxis. Both morphology and chemotaxis are not affected by treatment with Lenograstim (glycosylated rhG CSF). Thus, we evaluated actin polymerization in neutrophils induced by treatment with the two forms of rhG-CSF. In fact, actin polymerization is crucial for neutrophil motility. MATERIALS AND METHODS: We evaluated twelve healthy subjects undergoing peripheral blood stem cells (PBSC) mobilization for allogeneic transplantation to HLA-identical siblings. Neutrophils were isolated by peripheral venous blood before and after administration of either Filgrastim (six PBSC donors) or Lenograstim (six PBSC donors). Actin polymerization was investigated by a flow cytometric assay, using FITC-phalloidin as a specific probe for F-actin, and two parameters were measured: spontaneous actin polymerization in resting neutrophils; fMLP-stimulated actin polymerization. Results were expressed as relative F-actin content. Fifteen blood donors were studied as a control group. RESULTS: Filgrastim administration induced an increased relative F-actin content in resting neutrophils; however, no further actin polymerization was observed after fMLP stimulation. Neutrophils from subjects treated with Lenograstim showed a normal behaviour in terms of both spontaneous and stimulated actin polymerization. CONCLUSIONS: Glycosylated and nonglycosylated rhG-CSF differently affect actin polymerization in newly generated neutrophils. Such effects may explain some previous findings concerning both morphology and chemotactic properties and may be due to different effects of the two forms of rhG-CSF on proteins involved in neutrophil motility regulation. PMID- 16669550 TI - Circadian distribution of iron and ferritin in serum of healthy and type 2 diabetic males. AB - AIM: We examined the circulating levels of iron and ferritin in serum of seven healthy and three insulin non-dependent diabetic (Type 2) males in order to compare their circadian characteristics. METHODS: Blood samples were collected every 3h over a 24h period and were analyzed for serum iron and ferritin. RESULTS: The mean Fe level was significantly higher in healthy than in diabetic subjects: 80.0 +/- 3.3 vs. 63.0 +/- 3.7 microg/dL. The ferritin level was significantly lower in healthy than in diabetic men: 79.8 +/- 4.7 vs. 186.3 +/- 110.5 microg/L. A significant (p < 0.001) time-effect was found by ANOVA and circadian rhythm was detected at p < 0.001 in all data sets when a 24h cosine was fitted to the normalized data. Acrophases were located in mid to late morning for Fe (11:30, vs. 09:22h) and for ferritin (11:10 vs. 11:46h). DISCUSSION: We concluded that there is significant circadian variation in both serum Fe and ferritin, with predictable peaks in the mid to late morning. PMID- 16669551 TI - Clinical chronopharmacology of the cardiovascular system: hypertension and coronary heart disease. AB - There is sound evidence that the cardiovascular and the renal systems are well organized in time. Mechanisms of regulation and pathophysiological events are not evenly distributed over the 24-h scale. Moreover, certain diseases may even alter the physiological circadian pattern both in the cardiovascular system and in the kidneys. This observation bares implications for drug treatment, e.g., regarding drug formulations and dosing time intervals. Pitfalls may arise from neglecting circadian phase-dependency in pharmacokinetics and in the concentration-effect relationship. Moreover, different types of drugs may be superior to others when circadian time-related symptoms are concerned. No doubt, "time-of-day" has to be included in our diagnostic and therapeutic strategies. PMID- 16669552 TI - [The association of asbestos and cigarette smoke in lung cancer. Note 2]. AB - This work follows up the previous edited in the year 2004, and deals with the still serious and discussed problem of work risks even though the use of asbestos has been forbidden since 10 years, and the tobacco's smoke since one year. The Authors discuss the question related to the genetic point of view as a first and multiplying action caused at the some time by the tobacco's smoke and asbestos dust in the working places. This work report an up-to-date list of law about environmental prevention. PMID- 16669553 TI - Role of hemochromatosis genes in chronic hepatitis C. AB - PURPOSE: Hereditary hemochromatosis is commonly associated with iron overload and hepatitis C virus (HCV). Association between hemochromatosis C282Y or H63D mutation has been observed, although not uniformly, and iron overload is also commonly found in chronic HCV hepatitis. This study explored the contribution of genetic hemochromatosis to iron accumulation in hepatitis C. DESIGN: Review of current literature. RESULTS: The prevalence of increased serum iron stores in patients with HCV infection is 28% (patients having an elevated ferritin or transferrin saturation). Patients with elevated serum iron markers have more active chronic hepatitis with more liver fibrosis. In the opinion of the experts HFE mutations are not associated with a high hepatic iron content. No relation was detected between hepatic iron stores and HFE gene mutation. Significant iron deposition in the liver was uncommon and overall the quantity of iron that was detectable histologically and biochemically was unrelated to the grade and stage of HCV related liver injury. The mechanism by which liver iron accumulates in patients is unclear. CONCLUSIONS: Carriage of HFE mutations does not have a role in the accumulation of iron or the liver disease in HCV. These findings do not support a role for iron depletion in patients with chronic HCV infection, including these with elevated serum studies. PMID- 16669554 TI - [Latent autoimmune diabetes in adults]. AB - Latent autoimmune diabetes in adults (LADA) is a disorder with onset after age 30, insulin independence for at least 6 months after diagnosis, and the presence of circulating pancreatic islet autoantibodies. The prevalence of LADA varies substantially across ethnic groups and ranges approximately from 1% to 10% among patients with type 2 diabetes. In this review we discuss the nomenclature, diagnostic criteria, immunologic and genetic markers, metabolic alterations and therapy of this form of diabetes. PMID- 16669555 TI - Level of hCG for considering an ectopic pregnancy. PMID- 16669556 TI - Pain management is part of advance directives discussion. PMID- 16669557 TI - Bariatric surgery: too many unanswered questions. PMID- 16669558 TI - Religion, spirituality, and their relevance to medicine: an update. PMID- 16669559 TI - Amenorrhea: evaluation and treatment. AB - A thorough history and physical examination as well as laboratory testing can help narrow the differential diagnosis of amenorrhea. In patients with primary amenorrhea, the presence or absence of sexual development should direct the evaluation. Constitutional delay of growth and puberty commonly causes primary amenorrhea in patients with no sexual development. If the patient has normal pubertal development and a uterus, the most common etiology is congenital outflow tract obstruction with a transverse vaginal septum or imperforate hymen. If the patient has abnormal uterine development, mullerian agenesis is the likely cause and a karyotype analysis should confirm that the patient is 46,XX. If a patient has secondary amenorrhea, pregnancy should be ruled out. The treatment of primary and secondary amenorrhea is based on the causative factor. Treatment goals include prevention of complications such as osteoporosis, endometrial hyperplasia, and heart disease; preservation of fertility; and, in primary amenorrhea, progression of normal pubertal development. PMID- 16669560 TI - Information from your family doctor. Amenorrhea: what you should know. PMID- 16669561 TI - Arrhythmogenic right ventricular dysplasia. AB - Arrhythmogenic right ventricular dysplasia (ARVD) is a disorder in which normal myocardium is replaced by fibrofatty tissue. This disorder usually involves the right ventricle, but the left ventricle and septum also may be affected. Although the exact prevalence of ARVD is unknown, it is thought to occur in six per 10,000 persons in certain populations. After hypertrophic heart disease, it is the number one cause of sudden cardiac death in young persons, especially athletes. Patients with ARVD are usually men younger than 35 years who complain of chest pain or rapid heart rate. In some cases, sudden cardiac death is the first presentation. The initial diagnosis of ARVD is based on the presence of major and minor criteria established in 1994. Further confirmation of the diagnosis includes noninvasive studies, such as echocardiography and magnetic resonance imaging of the heart, and invasive studies such as ventricular angiography and endomyocardial biopsy. Patients with ARVD are treated initially with antiarrhythmic agents with serious consideration for automatic implantable cardioverter-defibrillator placement. In patients with persistent symptomatic arrhythmias, radiofrequency ablation, ventriculotomy, or even cardiac transplant may be necessary. PMID- 16669562 TI - Information from your family doctor. Arrhythmogenic right ventricular dysplasia: what you should know. PMID- 16669563 TI - Caring for patients after bariatric surgery. AB - Bariatric surgery leads to sustainable long-term weight loss and may be curative for such obesity-related comorbidities as diabetes and obstructive sleep apnea in severely obese patients. The Roux-en-Y gastric bypass has become the most common procedure for patients undergoing bariatric surgery. The procedure carries a mortality risk of up to 1 percent and a serious complication risk of up to 10 percent. Indications include body mass index of 40 kg per m2 or greater, or 35 kg per m2 or greater with serious obesity-related comorbidities (e.g., diabetes, obstructive sleep apnea, coronary artery disease, debilitating arthritis). Pulmonary emboli, anastomotic leaks, and respiratory failure account for 80 percent of all deaths 30 days after bariatric surgery; therefore, appropriate prophylaxis for venous thrombo-embolism (including, in most cases, low-molecular weight heparin) and awareness of the symptoms of common complications are important. Some of the common short-term complications of bariatric surgery are wound infection, stomal stenosis, marginal ulceration, and constipation. Symptomatic cholelithiasis, dumping syndrome, persistent vomiting, and nutritional deficiencies may present as long-term complications. PMID- 16669564 TI - Diagnosis and treatment of Chlamydia trachomatis infection. AB - Chlamydia trachomatis infection most commonly affects the urogenital tract. In men, the infection usually is symptomatic, with dysuria and a discharge from the penis. Untreated chlamydial infection in men can spread to the epididymis. Most women with chlamydial infection have minimal or no symptoms, but some develop pelvic inflammatory disease. Chlamydial infection in newborns can cause ophthalmia neonatorum. Chlamydial pneumonia can occur at one to three months of age, manifesting as a protracted onset of staccato cough, usually without wheezing or fever. Treatment options for uncomplicated urogenital infections include a single 1-g dose of azithromycin orally, or doxycycline at a dosage of 100 mg orally twice per day for seven days. The recommended treatment during pregnancy is erythromycin base or amoxicillin. The Centers for Disease Control and Prevention and the U.S. Preventive Services Task Force recommend screening for chlamydial infection in women at increased risk of infection and in all women younger than 25 years. PMID- 16669565 TI - Outpatient vs. inpatient treatment of community-acquired pneumonia. PMID- 16669566 TI - Papules and plaques on the nose. Lupus pernio. PMID- 16669567 TI - Putting aging into perspective for our profession. PMID- 16669568 TI - Rebuilding an interdisciplinary rehabilitation team. PMID- 16669569 TI - Facilitating a connection with a rehabilitation patient who has schizophrenia. PMID- 16669571 TI - Developing a staffing matrix using CMI as acuity indicator. AB - Adequate staffing levels on an acute rehabilitation unit may not be maintained because staffing needs fluctuate according to the needs of patient groupings. Acuity regulations from some state agencies and the Joint Commission on Accreditation of Healthcare Organizations require that staffing address patient acuity needs. Proposed ratio laws either require concrete patient-to-nurse ratios or neglect to consider acute rehabilitation. Neither have practical working tools to support their proposals. The questions of what to measure and how to translate this into effective nursing staffing remain unanswered. At the same time, nursing dissatisfaction grows with increased workloads, overtime, and perceptions of ineffectiveness. This article describes one effort to define and use a working tool for staffing acute rehabilitation units. The study used case mix index as an indicator of nursing time, integrated into a shift staffing matrix. Early results have shown it to be effective, quick, flexible, and efficient. Using this tool, quality patient outcomes within national length of stay benchmarks were maintained and staff satisfaction on this unit improved. PMID- 16669570 TI - Inadequate cardiovascular disease prevention in women with physical disabilities. AB - Health promotion and screening tests are important in persons with disability to avert secondary conditions that can lead to suboptimal functioning or premature death. Conversely, the existence of a primary disability can increase a person's susceptibility to secondary conditions. Cardiovascular disease (CVD) is the major cause of death in the United States, and its prevalence has been underinvestigated in persons with disability. This descriptive study used survey research to compare the risk of CVD in samples of 100 physically disabled women with 50 nondisabled women in the community. Participants, recruited from health fairs, completed questionnaires that explored the participants' knowledge of CVD risk factors, possession of specific CVD risk factors, and experience with CVD preventive screening procedures. Data revealed that compared with women without disability, women with disability were less knowledgeable about CVD risk factors and experienced marked deficiencies in CVD preventive screening. Body weight measurement, baseline electrocardiograms, family history, and smoking queries were performed less often in women with disabilities than in women without disabilities of similar age. Physical inactivity and postmenopausal status were specific CVD risk factors found to be more prevalent in the sample of women with disability. These findings suggest that risk of CVD is underrecognized and underassessed in women with a physical disability. PMID- 16669573 TI - CPAP devices: encouraging patients with sleep apnea. AB - Continuous positive airway pressure devices (CPAP) used at night prevent apnea, hypoxia, and sleep disturbance. Although CPAP is more effective than placebo in improving sleepiness and quality of life measures in people with obstructive sleep apnea, patients often prefer a less-effective oral appliance. This article examines help-seeking experiences in support groups of individuals with sleep apnea who use CPAP devices. To understand patients' experiences and difficulties using CPAP, an urban medical center and a rural hospital shared data collected from 17 individuals with sleep apnea who use CPAP for treatment and attend a support group. Four related themes emerged including (a) becoming motivated to persist with help from the group, (b) accommodating to the device, (c) listening and telling stories to gain practical knowledge, and (d) implementing a support group as a caring community. Healthcare providers could recommend support groups on CPAP use while nurses guide discussion, provide technical information, and promote empowerment. PMID- 16669572 TI - Using the Cancer Rehabilitation Questionnaire in patients with colorectal cancer. AB - This article describes the development and testing of the Cancer Rehabilitation Questionnaire (CRQ) in patients with colorectal cancer. A descriptive, correlational survey was conducted in a sample of patients with colon or rectal cancer who received care at an outpatient cancer clinic at a large, northeastern U.S. academic medical center. Patients were identified from the Tumor Registry (N = 327) and received a mailed questionnaire. One hundred and three patients with colorectal cancer completed the demographic questionnaire, the CRQ developed by the investigators, and the Quality of Life Index (QLI). Significant differences were found between individuals with colon cancer and individuals with rectal cancer on the total CRQ (p < .005) and the physical, future orientation, and role relationship subscales. For the QLI, significantly lower scores were reported on the family subscale for patients with rectal cancer in comparison with those with colon cancer. More attention to rehabilitation issues and quality of life is required across the trajectory of the cancer experience. With the increasing incidence of colorectal cancer, nurses in rehabilitation practice must address the issues affecting patients with this disease as they progress from diagnosis to treatment and rehabilitation. PMID- 16669575 TI - Characterization of metals emitted from motor vehicles. AB - A systematic approach was used to quantify the metals present in particulate matter emissions associated with on-road motor vehicles. Consistent sampling and chemical analysis techniques were used to determine the chemical composition of particulate matter less than 10 microm in aerodynamic diameter (PM10*) and particulate matter less than 2.5 microm in aerodynamic diameter (PM2.5), including analysis of trace metals by inductively coupled plasma mass spectrometry (ICP-MS). Four sources of metals were analyzed in emissions associated with motor vehicles: tailpipe emissions from gasoline- and diesel powered vehicles, brake wear, tire wear, and resuspended road dust. Profiles for these sources were used in a chemical mass balance (CMB) model to quantify their relative contributions to the metal emissions measured in roadway tunnel tests in Milwaukee, Wisconsin. Roadway tunnel measurements were supplemented by parallel measurements of atmospheric particulate matter and associated metals at three urban locations: Milwaukee and Waukesha, Wisconsin, and Denver, Colorado. Ambient aerosol samples were collected every sixth day for one year and analyzed by the same chemical analysis techniques used for the source samples. The two Wisconsin sites were studied to assess the spatial differences, within one urban airshed, of trace metals present in atmospheric particulate matter. The measurements were evaluated to help understand source and seasonal trends in atmospheric concentrations of trace metals. ICP-MS methods have not been widely used in analyses of ambient aerosols for metals despite demonstrated advantages over traditional techniques. In a preliminary study, ICP-MS techniques were used to assess the leachability of trace metals present in atmospheric particulate matter samples and motor vehicle source samples in a synthetic lung fluid. PMID- 16669574 TI - Evaluating functional activity in older Thai adults. AB - The purpose of this study was to translate the original English version of the Self-Efficacy and Outcome Expectations Scales for Exercise and Functional Activity and to establish their reliability in older Thai adults in geriatric rehabilitation. This study used a correlational design with repeated measures. Reliability of the Self-Efficacy for Exercise Scale (SEES), the Self-Efficacy for Functional Activity Scale (SEFAS), the Outcome Expectations for Exercise Scale (OEES), and the Outcome Expectations for Functional Activity Scale (OEFAS) developed by Resnick was measured in 20 older adults aged 60 years or older after hip or knee replacement or another orthopedic surgery of their lower extremity. All scales were translated into Thai and back translated into English according to the process described by Marin and Marin. The instruments were administered twice--the 1st and 2nd day in the participants' geriatric rehabilitation program (i.e., the 4th and 5th postoperative day). Results indicated that there was sufficient evidence for internal consistency of the SEES, SEFAS, OEES, and OEFAS with alpha coefficients of 0.84, 0.86, 0.70, and 0.86 respectively. Test-retest reliability of the tools was also demonstrated with Spearman correlation coefficients of 0.84 for the SEES, 0.87 for the SEFAS, 0.61 for the OEES, and 0.54 for the OEFAS. The findings from this study provide important information for instrument adaptation and the applicability of these scales for further studies of older Thai adults. PMID- 16669576 TI - Developing an ambulatory surgery center: understanding the key legal issues and the essential elements of an ASC operating agreement. PMID- 16669577 TI - Nursing and IT: an encumbered strategic resource. PMID- 16669578 TI - New dynamics of payment for care: consumer plans are coming. PMID- 16669579 TI - The new alphabet soup: models of data integration, part 2. PMID- 16669580 TI - Early experiences with E-prescribing. AB - Most physicians understand that e-prescribing will reduce medical errors and will be perceived by patients as making the prescription process easier. However, they are skeptical about a number of things. They worry whether their office processes will be improved or streamlined; e-prescribing will interface seamlessly with their existing practice management software; training and support will be available; e-prescribing data will be seamlessly transferable to an electronic health record when they implement a more advanced clinical record system for their practice; and if they will achieve a return on investment. Early adopting clinicians in Massachusetts can convince the majority of clinicians to adopt e prescribing by sharing their motivations for adopting e-prescribing, the challenges that they needed to overcome, the hardware and software requirements, and integration into their office workflow. Finally, interaction with the physicians and practice managers in the audience makes the adoption of e prescribing seem both reasonable and exciting. Resources such as vendor lists, questions to ask, and hardware and software requirements also need to be readily available and in a form that non-technical staff can read and understand. Physicians who know the "why" would also like to know PMID- 16669581 TI - Marketing 101--A refresher for management engineers. PMID- 16669582 TI - Progress and challenges in nursing documentation, part I. PMID- 16669583 TI - How healthcare IT can address the nursing shortage. AB - But if applied correctly, IT can play an important role in redefining the way nursing is conducted. It can do so by introducing flexible models of nursing education to increase the pool of qualified nursing professionals; automating and standardizing nursing workflows; eliminating paperwork; supporting patient care delivery models that address the needs of an older workforce; establishing point of-care clinical decision support tools to improve patient safety; and making health, wellness, and patient education information available. If healthcare IT solutions are deployed in direct response to nursing's ills, particularly job dissatisfaction, the positive image of nursing can be restored, once again making it a career of choice. IT can further reverse the tide of nursing defections, which continually drain the healthcare industry of experienced and dedicated professionals. Finally, both individually and collectively, healthcare IT technologies can reorient nursing activity back towards its principal role of delivering quality patient care. PMID- 16669584 TI - Virtual house calls: legal challenges to remote caregiving. AB - Medicine in the digital age requires new thinking to bring cost-effective and quality medical care to patients in home settings. Along with the promise of remote treatment advances come many challenges relating to technology, regulation, and the building of new virtual teams and social structures to support the remote environment. For remote caregiving to succeed, the legal and ethical issues of home monitoring and ubiquitous computing must be addressed side by-side with the technology advances. PMID- 16669585 TI - Transforming IT--governing for value realization. PMID- 16669586 TI - Case study: factors in defining the nurse informatics specialist role. AB - Healthcare organizations, consultant groups, vendor companies, and academic institutions feel the challenge to enhance user experiences with information systems. To meet this challenge, organizations and companies are looking to better understand and utilize a variety of informatics roles to further marketing, business, or healthcare goals. Nursing is one practice area that can support the successful integration of information systems development, implementation, support, and user experience. However, the definition and development of such a role or position has met with mixed success. This article explores some of the issues and influences related to the role's development. The issues, impacts, and influences have been identified based on healthcare business assessment, job description analysis, employment and project evaluations, and professional standards set by the American Nurses Association. PMID- 16669588 TI - Effects of implementing computerized practitioner order entry and nursing documentation on nursing workflow in an emergency department. AB - Nurses'perceptions of effective use of their time are critical to the successful implementation of information system changes. We examined the effects of implementing computerized practitioner order entry and nursing documentation in our emergency department with an anonymous survey of nurses and repeated time motion studies. Emergency care nurses were positive about effects of CPOE, reporting needing less time to complete medication, laboratory, and radiology orders and less time spent clarifying orders. Their perceptions of time spent were congruent with observations from time-motion studies where combined computer and-paper time and direct-patient-care time did not change significantly. Nurses also reported supplementing template options with free text, and those who were more comfortable using computers reported supplementing template options more often than their counterparts, suggesting that assessments of users' expertise in computer use may influence their ability to maximize their use of the functionality of emergency department information systems. PMID- 16669587 TI - Surveying acute care providers in the U.S. to explore the impact of HIT on the role of nurses and interdisciplinary communication in acute care settings. AB - In 2005, the Healthcare Information and Management Systems Society's Nursing Informatics Task Force surveyed acute care providers across the United States to explore the impact of health information technology on the role of nurses and interdisciplinary communication in acute care settings. More than 1,700 healthcare professionals responded to the online survey and provided numerous insights into the impact that clinical information systems have in their daily communication and workflow patterns. This paper provides an overview of survey results from four large healthcare delivery systems with a focus on how the results will be employed to support an evidence-based approach to improving acute care IT systems across the following healthcare delivery systems: Partners and Lifespan Health Systems in the Northeast, Baptist Health South Florida in the Southeast, and Providence HealthSystem in the Northwest.A case study approach is employed to describe survey findings at the healthcare system level and then provide a set of recommendations to leverage findings to capitalize on the strengths of existing IT applications and tools, and work towards overcoming barriers to interdisciplinary communication or acute care workflows. PMID- 16669589 TI - Nursing admissions process redesigned to leverage EHR. AB - Among the challenges facing nurses in acute care facilities are duplicate documentation, complex rules for coordinating care, and pressure to accelerate patient discharge. Christiana Care, a two-hospital, 900-bed health system in Delaware, redesigned its nursing admissions process to address these issues and provide a centralized source of assessment information. An interdisciplinary team redesigned the assessment process to leverage the clinical documentation, decision support, and workflow tools of Christiana Care's evolving electronic health record. Nurses use a wireless device at the patient's bedside to enter admission information. A series of decision-support rules evaluate the information and send electronic referrals to appropriate ancillary departments. Departments electronically document interventions that can be tracked by nursing, closing the loop.A dedicated rollout coordinator and system-generated audit reports were critical to early identification and resolution of several implementation problems. The new process has not only simplified the work of nursing but led to more appropriate referrals to the ancillary departments. More than 25 percent of the patients have information imported from a previous encounter, eliminating duplicate documentation and improving patient satisfaction. Admission information also is being used to monitor and support a variety of quality efforts, including core measures. PMID- 16669590 TI - Maintaining excellence in physician nurse communication with CPOE: A nursing informatics team approach. AB - Designing and implementing CPOE systems is difficult, presenting many challenges to overcome related to workflow redesign. As part of the Mayo Clinic Arizona CPOE design and implementation, it was recognized that physicians would be placing inpatient orders from multiple locations; therefore nurses no longer would have the usual visual clues to identify new physician orders. This could easily lead to a delay in implementing orders that require immediate action. To address this, a multidisciplinary team evaluated various communication options and designed two in-house Web-based applications to solve the communication gap. One application will provide staff nurses with visual alerts for stat and routine orders as they are processed in real time. A second application is a Web-based link to the nursing assignment sheets, which will give physicians and support staff access to staff nurse assignments and phone numbers, specific to each nursing unit. PMID- 16669591 TI - Banking on health: Personal records and information exchange. AB - Consumer demand for personal health records (PHRs) and the capabilities provided by regional health information organizations (RHIOs) will change healthcare, just as automatic teller machines have changed banking. The PHR is predicated on the existence of electronic medical records (EMRs) and electronic health records (EHRs). Patient and consumer principles guiding the development of the PHR reflect issues of access, control, privacy, and security. Working models illustrate the variations of RHIOs and PHRs possible and suggest the benefits that electronic information exchange can accrue for healthcare and healthcare consumers. Today both the private and public sectors are working to define the issues involved in efforts that are now taking place and that will transform healthcare. Consumers are ready for the type of changes that will improve healthcare quality. PMID- 16669592 TI - Using an evidence-based approach for system selection at a large academic medical center: lessons learned in selecting an ambulatory EMR at Mount Sinai Hospital. AB - The experience of Mount Sinai Hospital is representative of the challenges and problems facing large academic medical centers in selecting an ambulatory EMR. The facility successfully revived a stalled process in a challenging financial climate, using a framework of science and rigorous investigation. The process incorporated several innovations: 1) There was a thorough review of medical informatics literature to develop a mission statement, determine practical objectives and guide the demonstration process; 2) The process involved rigorous investigation of vendor statements, industry statements and other institution's views of vendors; 3) The initiative focused on user-centric selection, and the survey instrument was scientifically and specifically designed to assess user feedback; 4) There was scientific analysis of validated findings and survey results at all steering meetings; 5) The process included an assessment of vendors' ability to support research by identifying funded and published research; 6) Selection involved meticulous total cost of ownership analysis to assess and compare real costs of implementing a vendor solution; and finally, 7) There were iterative meetings with stakeholders, executives and users to understand needs, address concerns and communicate the vision. PMID- 16669593 TI - Enhancing operational workflow and patient tracking in an ambulatory setting. AB - As Massachusetts General Hospital (MGH) was forging ahead with the completion of the new Yawkey Center for Outpatient Care, administrative groups began to struggle with how they would be able to function in the new building. They would be moving from smaller, isolated practice areas into much larger state-of-the-art facilities with shared common spaces. The affected practices were challenged with developing new workflows and communication processes. After an unsuccessful vendor search, teams from Partners/MGH Information Systems built a solution in house, using the vendor search requirements as their functional specifications. The application, and the new building have been live since October 2004. The application now is managing 9,000 patients per appointments a day from all of the appointment scheduling systems used throughout the building. This case study will review how Massachusetts General used information systems to ensure success in its new 10-story ambulatory care center, and met the needs of clinicians, patients, staff, and practice managers. PMID- 16669594 TI - Compliance with HIPAA security standards in U.S. Hospitals. AB - With the widespread use of computer networks, the amount of information stored electronically has grown exponentially, resulting in increased concern for privacy and security of information. The healthcare industry has been put to the test with the federally mandated Health Insurance Portability and Accountability Act (HIPAA) of 1996. To assess the compliance status of HIPAA security standards, a random sample of 1,000 U.S. hospitals was surveyed in January 2004, yielding a return rate of 29 percent. One year later, a follow-up survey was sent to all previous respondents, with 50 percent replying. HIPAA officers'perceptions of security compliance in 2004 and 2005 are compared in this article. The security standards achieving the highest level of compliance in both 2004 and 2005 were obtaining required business associate agreements and physical safeguards to limit access to electronic information systems. Respondents indicated least compliance both years in performing periodic evaluation of security practices governed by the Security Rule. Roadblocks, threats, problems and solutions regarding HIPAA compliance are discussed. This information may be applied to current and future strategies toward maintaining security of information systems throughout the healthcare industry. PMID- 16669595 TI - How automated access verification can help organizations demonstrate HIPAA compliance: A case study. AB - This case study of Sharp HealthCare takes an in depth look at how the organization has embedded security policies into its business process and automated workflow to ensure users are granted only the IT access that is necessary for them to perform their jobs and to ensure patient privacy Some of the most pressing audit and compliance concerns in healthcare organizations today revolve around the need to constantly review and give an account for users' IT access. The need for this at Sharp is exacerbated because of the high rate of change within the organization and the large percentage of non-employee staff such as traveling nurses moving throughout hospital departments on their rotations. By implementing a software solution to verify the accuracy of user access rights or automatically initiate appropriate corrective actions, Sharp is now able to extend the responsibility and accountability for compliance to the most appropriate resources. PMID- 16669596 TI - Saving time, improving satisfaction: the impact of a digital radiology system on physician workflow and system efficiency. AB - The objective of the study was to assess UC Davis Health System's transition to digital radiology. The study involved pre- and post-PACS analyses of workflow and costs, physician satisfaction surveys, and self-recorded radiology interactions by on-call residents. The study revealed significant results. Before the PACS implementation, physicians spent one to three hours searching for films daily and were dissatisfied with radiology services. After implementation, images were readily available, physicians were more likely to view and interpret images themselves, and they reported increased satisfaction. From real-time reporting, residents viewed studies with radiologists 90.2 percent less often. Average image search time decreased, from 16 to 2 minutes, saving 21.5 physician years, worth dollar 1,034,150 annually. Reductions in film printing (73.4 percent) and file clerk full-time equivalents (50.3 percent) saved dollar 1,001,452 annually, and freed up 1,218 hospital and 8,108 warehouse square feet, worth dollar 2,018,320. As a result, UCDHS's digital radiology system improved clinician satisfaction and workflow, increased clinician image viewing, and decreased clinician engagement with radiologists. System implementation saved 21 physician years and dollar 2 million annually. PMID- 16669597 TI - Implications of an animal model of sugar addiction, withdrawal and relapse for human health. AB - The effect of intermittent glucose administration on the circadian rhythm of body temperature was studied in rats to provide evidence of sugar addiction, withdrawal and relapse. Metabolic and behavioral phenomena were also observed. Biotelemetry transmitters recorded body temperature for the duration of the 4 week experiment. Rats were divided into an experimental and a control group, which were maintained on the same habituation conditions for the duration of the experiment, with the exception of weeks 2 and 4, when the experimental group was presented with a 25% glucose solution. Experimental animals displayed a precipitous drop in body temperature and behavioral changes associated with withdrawal during week 3, when sugar was removed. There was an increase in kilocalories (kcal) consumed during weeks 2 and 4 by experimental animals and, by the end of the experiment, these animals showed a greater percent increase in body weight. Elevated blood glucose levels were found in experimental animals. The study demonstrates that the effects of sugar addiction, withdrawal and relapse are similar to those of drugs of abuse. Implications of the rewarding and addicting effects of sugar are related to weight gain, obesity and Type II diabetes. Furthermore, pitfalls related to dieting are elucidated. PMID- 16669598 TI - Perinatal nutritional iron deficiency reduces hippocampal synaptic transmission but does not impair short- or long-term synaptic plasticity. AB - Studies show that perinatal nutritional iron deficiency (ID) produces learning and memory impairments in humans and animals. This suggests that the functional physiology of learning and cognitive centers in the brain, such as the hippocampus, may be compromised by developmental ID. The present study used electrophysiological brain slice methods to examine multiple measures of hippocampal synaptic efficacy from rats that were subjected to perinatal ID diets or control (CN) diets. Measures of synaptic efficacy were obtained from the first and last synaptic regions of the hippocampal tri-synaptic loop (i.e. the dentate gyrus (DG) and CA1). Rats were placed on ID or CN diets on gestational day 11, and hippocampal brain slices were prepared between postnatal day 25 and 37. Results show that ID slices were not impaired in short-term (i.e. paired-pulse facilitation (PPF)) or long-term measures (i.e. long-term potentiation (LTP)) of synaptic plasticity in either the DG or CA1 areas. Input-output (IO) measures showed that synaptic transmission was reduced in both of these areas in the ID slices when compared with the CN slices. This suggests that ID-induced learning deficits may be the result of reductions in synaptic transmission throughout the hippocampus, and possibly in other learning and memory centers. PMID- 16669600 TI - Lesions of the lateral parabrachial area block the aversive component and induced flavor preference for the delayed intragastric administration of nutrients in rats: effects on subsequent food and water intake. AB - The aim of this study was to examine the function of the lateral parabrachial area (LPB) in relation to the intragastric administration of nutrients. The consumption of flavors associated with intragastric nutrient administration and the subsequent food and water intake were measured in rats with lesions in the LPB. The results showed that bilateral LPB lesions prevented development of aversions and induced flavor preference when there was a delay between the presentation of a flavor and the intragastric administration of nutrients. However, these lesions did not disrupt development of the aversive process when there was no delay between the presentations. Likewise, the LPB lesions increased subsequent food intake when there was a delay but not when there was no delay between the presentations. In contrast, the water intake was reduced in both situations. These results are interpreted in terms of a dual visceral system for processing the intragastric effects of foods. PMID- 16669599 TI - Peripheral ghrelin participates in glucostatic feeding mechanisms and in the anorexigenic signalling mediated by CART and CRF neurons. AB - Ghrelin is upregulated under negative energy balance conditions, including starvation and hypoglycemia, while it is downregulated under situations of positive energy balance, such as feeding, hyperglycemia and obesity. The aims of this study were to assess potential ghrelin interactions with glucose levels in appetite control and to identify potential mechanisms involving orexigenic and anorexigenic ghrelin mediated signals by using a specific anti-ghrelin antibody. Our results confirm that peripheral ghrelin is an important signal in meal initiation and food intake stimulation. C-fos positive neurons in the PVN increased after insulin or 2-deoxyglucose administration. Moreover, we also demonstrate that peripheral ghrelin blockade with a specific anti-ghrelin antibody reduces, in part, the orexigenic signal induced by insulin and 2-DG administration. Furthermore, when we blocked peripheral ghrelin, c-fos positive CRF neurons and CART expression increased in the PVN, both under hypoglycemia or cytoglycopenia conditions, suggesting a neuronal activation (anorexigenic signalling) in this hypothalamic region. In summary, our findings imply that peripheral ghrelin plays an important role in regulatory "glucostatic" feeding mechanisms due to its role as a "hunger" signal affecting the PVN area, which may contribute to energy homeostasis through both orexigenic/anorexigenic pathways. PMID- 16669601 TI - Mediation of cognitive function by high fat diet following stress and inflammation. AB - In addition to commonly advertised hazards of obesity contributed by excess dietary fat, evidence of alterations in brain chemistry and structure are well documented. This brief review examines the role of nutrients, minerals and certain lipids, primarily the essential fatty acids (FA), that are beneficial to the maintenance of good health and that may offer therapeutic options by dietary supplementation. The review also considers the damaging effects of stress, especially in pre-existing conditions of obesity and diabetes, as studied in both animals and humans. The main focus of this brief review is to examine the effects of a high fat diet on stress and the immune system with particular emphasis on brain and cognitive function. PMID- 16669602 TI - Evidence for growth-promoting effects of omega n - 3 fatty acids alone and in combination with a specific vitamin and mineral complex in rat neuroblastoma cells. AB - The beneficial effects of Omega n - 3 polyunsaturated fatty acids (n - 3 PUFA) in situations of cognitive impairment may be associated with enhanced neuronal growth. Since neuronal growth is impaired in n - 3 PUFA deficiency, and enhanced by certain vitamins and trace elements, the effects of n - 3 PUFA, vitamin and mineral cell complex (VMC) and their association on neuronal growth were investigated in cultured rat neuroblastoma cells. Treatment of cells for 3 days with n - 3 PUFA significantly enhanced neurite length without affecting the number of neurites or cells. VMC significantly increased cell number without affecting neurite length or their number. Combined n - 3 PUFA and VMC significantly enhanced all three growth parameters. The data confirm the growth promoting effects of n - 3 PUFA and VMC in cultured neurons over a relatively short time frame. PMID- 16669603 TI - Chronic malnutrition does not influence the development of ethylnitrosourea induced brain tumors. AB - There is controversy over the subject of malnutrition as a potential risk factor for cancer; we studied the effect of chronic malnutrition on the development of tumors in rats prenatally exposed to the carcinogenic ethylnitrosourea. Twelve pregnant Wistar rats were administered on the 19th day of gestation with a single i.v. dose of 30 mg/kg of ethylnitrosourea. Immediately after weaning, at 23 days of age, half of the offspring were nourished with a protein-deficient diet (less than 6%), which consisted mostly of a corn-based diet with high calorie and low fiber contents. In the adult age, 83 rats (74%) developed a tumor of the nervous system; in comparison with controls, we found no differences in time of development, site and histological characteristics of the tumors that developed in animals subjected to chronic malnutrition. PMID- 16669604 TI - Niacin improved rigidity and bradykinesia in a Parkinson's disease patient but also caused unacceptable nightmares and skin rash--a case report. AB - A patient with Parkinson's disease taking levodopa/carbidopa, selegiline, buproprion, aspirin and niacin had decreased rigidity and bradykinesia when his niacin dose was steadily escalated for treatment of high triglycerides, but ultimately the patient could not tolerate niacin because of severe nightmares and skin rash. If further research can reproduce this patient's initial beneficial experience while avoiding the adverse effects, niacin could be a useful adjunctive agent for Parkinson's disease, either population-wide or in a pharmacogenomically defined set of responders. PMID- 16669605 TI - Anticipating clinical resistance to target-directed agents : the BCR-ABL paradigm. AB - The deregulated tyrosine kinase activity of BCR-ABL is necessary and sufficient to induce chronic myelogenous leukemia (CML). This observation has paved the way for the development of small-molecule inhibitors specifically targeting the kinase activity of the BCR-ABL protein. Indeed, the amazing success of imatinib has revolutionized the whole area of targeted cancer therapeutics. However, enthusiasm for the striking efficacy of imatinib has been tempered by the development of clinical resistance. In essentially all cases, resistance results from kinase domain mutations and/or overexpression of the BCR-ABL gene. To overcome resistance, several novel BCR-ABL inhibitors have been developed and are in clinical trials, though it is inevitable that resistance to second-generation inhibitors will occur as well. Nonetheless, kinases represent an attractive target for therapeutic intervention in several diseases and, at present, some 50 different kinase inhibitors are in clinical trials. We anticipate that resistance to these compounds will follow mechanisms similar to those observed with imatinib. Resistance mutations cause their effect either by direct steric hindrance to drug binding or by allosterically modulating kinase dynamics. This review highlights the principal mechanisms underlying point mutations from these two different classes to confer drug resistance. PMID- 16669606 TI - New strategies for blood donor screening for hepatitis B virus: nucleic acid testing versus immunoassay methods. AB - Serologic testing for hepatitis B virus (HBV) surface antigen (HBsAg) and antibody to HBV core antigen (anti-HBc) has historically been the foundation of blood screening, while HBV nucleic acid testing (NAT) was recently developed to detect HBsAg-negative, anti-HBc-negative blood units donated during early acute infection. Comparison data on seroconversion panels using HBsAg assays of varying sensitivities and pooled- or single-sample NAT, along with viral load estimates corresponding to HBsAg assay detection limits, have provided information on the theoretical benefits of NAT relative to HBsAg. Model-derived estimates have generally been predictive of the yields of DNA-positive, HBsAg-negative window period blood units detected in a number of studies from Europe, Japan, and the US. Studies indicate that the added benefit of pooled-sample NAT is relatively small in areas of low endemicity, with greater yields in areas highly endemic for HBV. Single-sample NAT would offer more significant early window period closure and could prevent a moderate number of residual HBV transmissions not detected by HBsAg assays; however, no fully automated single-sample HBV NAT systems are currently available.Even single-sample HBV NAT may not substitute for anti-HBc screening, as indicated by studies of donors with isolated anti-HBc who have extremely low DNA levels undetectable by standard single-sample NAT and who have been associated with transfusion-transmitted HBV. Moreover, HBsAg testing may still be needed even in the setting of combined anti-HBc and NAT screening. HBsAg positive units from donors in the chronic stage of infection may contain very low or intermittently detectable DNA levels that single-sample NAT would miss. Although such donors are usually anti-HBc reactive and would be interdicted by anti-HBc screening, some lack anti-HBc. Extensive parallel testing will be needed to determine whether single-sample NAT in combination with anti-HBc might be sufficient to detect all the infectious donors currently interdicted by HBsAg testing. In countries that do not screen for anti-HBc, HBsAg testing would be the only means of detecting donations from chronically infected individuals with low/intermittently detectable DNA, since even single-donor NAT would not identify these potentially infectious blood units. In the future, the current fully automated HBsAg assays may incorporate significant sensitivity improvements, and automated single-sample HBV NAT may become a reality. Each country will need to develop its blood screening strategy based on HBV endemicity, yields of infectious units detected by different serologic/NAT screening methods, and cost effectiveness of test methods in ensuring blood safety. PMID- 16669607 TI - Role of cholesterol 7alpha-hydroxylase (CYP7A1) in nutrigenetics and pharmacogenetics of cholesterol lowering. AB - The relationship between dietary composition/cholesterol-lowering therapy and final plasma lipid levels is to some extent genetically determined. It is clear that these responses are under polygenic control, with multiple variants in many genes participating in the total effect (and with each gene contributing a relatively small effect). Using different experimental approaches, several candidate genes have been analyzed to date.Interesting and consistent results have been published recently regarding the A-204C promoter variant in the cholesterol 7alpha-hydroxylase (CYP7A1) gene. CYP7A1 is a rate-limiting enzyme in bile acid synthesis and therefore plays an important role in maintaining cholesterol homeostasis. CYP7A1-204CC homozygotes have the greatest decrease in total cholesterol level in response to dietary changes in different types of dietary intervention studies. In contrast, one study has reported that the effect of statins in lowering low-density lipoprotein (LDL)-cholesterol levels was slightly greater in -204AA homozygotes. The CYP7A1 A-204C variant accounts for a significant proportion of the genetic predisposition of the response of plasma cholesterol levels. PMID- 16669608 TI - Nutrigenomics: integrating genomic approaches into nutrition research. AB - It has been suggested that the supermarket of today will be the pharmacy of tomorrow. Such statements have been derived from recognition of our increasing ability to optimize nutrition, and maintain a state of good health through longer periods of life. The new field of nutrigenomics, which focuses on the interaction between bioactive dietary components and the genome, recognizes that current nutritional guidelines may be ideal for only a relatively small proportion of the population. There is good evidence that nutrition has significant influences on the expression of genes, and, likewise, genetic variation can have a significant effect on food intake, metabolic response to food, individual nutrient requirements, food safety, and the efficacy of disease-protective dietary factors. For example, a significant number of human studies in various areas are increasing the evidence for interactions between single nucleotide polymorphisms (SNPs) in various genes and the metabolic response to diet, including the risk of obesity. Many of the same genetic polymorphisms and dietary patterns that influence obesity or cardiovascular disease also affect cancer, since overweight individuals are at increased risk of cancer development. The control of food intake is profoundly affected by polymorphisms either in genes encoding taste receptors or in genes encoding a number of peripheral signaling peptides such as insulin, leptin, ghrelin, cholecystokinin, and corresponding receptors. Total dietary intake, and the satiety value of various foods, will profoundly influence the effects of these genes. Identifying key SNPs that are likely to influence the health of an individual provides an approach to understanding and, ultimately, to optimizing nutrition at the population or individual level. Traditional methods for identification of SNPs may involve consideration of individual variants, using methodologies such as restriction fragment length polymorphisms or quantitative real-time PCR assays. New developments allow identification of up to 500,000 SNPs in an individual, and with increasingly lowered pricings these developments may explode the population-level potential for dietary optimization based on nutrigenomic approaches. PMID- 16669609 TI - Endogenous histamine and cortisol levels in subjects with different histamine N methyltransferase C314T genotypes : a pilot study. AB - BACKGROUND: Histamine N-methyltransferase (HNMT) catalyzes the methylation of histamine and plays an important role in histamine biotransformation in bronchial epithelium. Enzymatic activity of HNMT has been shown to be regulated by genetic factors, including polymorphisms in the HNMT gene. In this pilot study we determined endogenous levels of histamine and cortisol in plasma and whole blood samples from subjects with different genotypes for the HNMT C314T polymorphism, and investigated whether these parameters differed between individuals with the HNMT CC genotype and those with the CT genotype. METHODS: Blood samples were collected from 48 unrelated volunteers (36 males, 12 females), aged 21-40 years, who participated in the study. PCR-restriction fragment length polymorphism analysis was used to determine HNMT C314T genotypes. Erythrocyte HNMT activity was determined as well as plasma and whole blood levels of histamine and cortisol. Two-group comparisons of the various parameters were analyzed by Blocked Wilcoxon test and Wilcoxon Rank Sum test as appropriate. RESULTS: Thirty seven subjects (24 Caucasians, three African Americans, one Middle Eastern, five Indians, three Chinese, and one Filipino) were found to have the homozygous CC genotype. Ten subjects (eight Caucasians, one Middle Eastern, and one Chinese) were heterozygous and one individual (Pakistani) was homozygous for the variant 314T allele. The frequency of HNMT CT heterozygotes in the small Caucasian cohort was 0.125. Median enzyme activity was significantly lower in subjects with the heterozygous CT genotype than in those with the homozygous CC genotype (485 vs 631 U/mL of red blood cells; p=0.023). A broad range of histamine levels in plasma and whole blood was observed for all subjects. Whereas the median plasma histamine level was found to be higher in heterozygotes for the wild-type 314C allele than homozygotes (3.32 vs 2.30 nmol/L; p=0.021), there was no difference between the two groups in histamine levels in whole blood. Cortisol levels were similar between individuals with the homozygous CC genotype and those with the heterozygous CT genotype. CONCLUSION: Wide variability of plasma and whole-blood histamine levels was observed in subjects with different HNMT C314T genotypes. Endogenous levels of histamine are likely to be affected by various genes and polymorphisms. PMID- 16669610 TI - Population haplotypes of exon ORF15 of the retinitis pigmentosa GTPase regulator gene in Germany : implications for screening for inherited retinal disorders. AB - BACKGROUND: Mutations in exon ORF15 of the retinitis pigmentosa GTPase regulator gene (RPGR) within chromosomal region Xp21.1 are a significant cause of a number of retinal disorders. The high mutation rate is ascribed to the highly repetitive, purine-rich tracts within the exon ORF15 sequence. Importantly, all exon ORF15 mutations observed to date represent protein-truncating mutations (nonsense and frameshift mutations). Because of its repetitive motifs, mutation screening of the hot-spot region by direct DNA sequencing is a technically challenging task. METHODS: We devised a screening strategy for exon ORF15 mutations that reserves DNA sequencing for precise sizing and base-order assessment of detected mutations. The screening strategy is based on a PCR/restriction fragment length polymorphism (RFLP) analysis of exon ORF15 and comparison with population-specific RFLP haplotypes. The latter were constructed from PCR/RFLP analysis of DNA samples from 100 healthy German male individuals. Mutational alterations of normal RFLP haplotype patterns were predicted. RESULTS: Six distinct RFLP haplotypes (founder alleles H1-H6) were observed with frequencies ranging from 2% to 63%. All natural variations of exon ORF15 were in frame alterations ranging in size between 3bp and 36bp. Prediction of mutation specific RFLP patterns indicated a high detection rate of mutations. CONCLUSION: A new strategy has been developed using routine protocols for mutation screening of difficult-to-sequence, highly repetitive exon ORF15 of the RPGR gene in a German population. PMID- 16669611 TI - Simultaneous detection of precore/basal core promoter mutations in hepatitis B virus using arrayed primer extension. AB - BACKGROUND: Hepatitis B is a major disease that causes serious public health problems worldwide. The loss of HBeAg expression due to point mutations or single nucleotide polymorphisms (SNPs) in the precore/basal core promoter region of the hepatitis B virus (HBV) is associated with hepatocellular cirrhosis and carcinoma. Simultaneous screening for these mutations is strongly advocated for monitoring disease development in HBV-infected patients. The aim of this study is to apply arrayed primer extension (APEX) for the detection of HBV SNPs at the precore/basal core promoter. METHODS AND RESULTS: We optimized APEX for simultaneous detection of eight potential sites of SNPs in the precore/basal core promoter region of HBV. The precore/basal core promoter regions of HBV from 36 HBV-infected patients were amplified by PCR. After purification and DNA fragmentation, the short, single-stranded HBV DNA fragments were allowed to hybridize with the oligonucleotides corresponding to the sites of SNPs immobilized on glass slides, followed by incorporation of different fluorescently labeled dideoxynucleotides. This allows fast and unequivocal discrimination between wild-type and mutant genotypes with high dideoxy-nucleotide incorporation efficiency, sensitivity, and specificity. The coexistence of both genotypes was also detected; this was undetected by DNA sequencing. CONCLUSION: The simultaneous detection of SNPs in HBV precore/basal core promoter by APEX enables large-scale diagnostic analysis, which can be extended to the whole HBV genome. PMID- 16669612 TI - Secretory phospholipase A2 from Arabidopsis thaliana: insights into the three dimensional structure and the amino acids involved in catalysis. AB - A low-molecular weight phospholipase A2 from Arabidopsis thaliana, isoform phospholipase A2-alpha, has been expressed in Escherichia coli in the form of inclusion bodies, refolded, and purified to homogeneity to yield the active mature enzyme. The enzyme was characterized with respect to pH, temperature optimum, and Ca2+ ion requirement. The enzyme has been shown to be a true secretory phospholipase A2 that requires Ca2+ ions in the millimolar range and belongs to group XIB. On the basis of the three-dimensional structures of secretory phospholipase A2 forms (sPLA2s) from bee venom and bovine pancreas, a homology model was generated. Analysis of this model and alignments of different plant sPLA2s showed that the common His-Asp dyad of animal sPLA2s does not exist in plant sPLA2s. In place of the aspartate residue of the dyad, the plant enzymes of group XIA contain a histidine residue, and the enzymes of group XIB contain a serine or an asparagine residue. Mutagenesis of amino acids supposed to be involved in catalysis has shown that His62, the calcium-coordinating Asp63, and the above-mentioned Ser79 residue are essential for activity. PMID- 16669613 TI - Discovery of different types of inhibition between the human and thermotoga maritima alpha-fucosidases by fuconojirimycin-based derivatives. AB - An efficient method for examining the selectivity of inhibitors on two alpha fucosidases, one from Thermotoga maritima and the other from human, was established. The X-ray crystal structure of the former enzyme makes possible the homology modeling of the human alpha-fucosidase, indicating the major difference between both enzymes in the periphery of the catalytic site. To investigate the difference at the molecular level, a variety of fuconojirimycin (FNJ) derivatives with substitution at C1, C2, C6, or N were rapidly prepared in microplates and screened without purification for the inhibition activities of the two alpha fucosidases. Among the molecules that were tested, only the substitution at C1 can significantly enhance the inhibitory potency, in contrast to the control (no substitution) and compounds with substitution at other positions. The majority of C1-substituted FNJs were found to be slow tight-binding inhibitors of the Thermotoga enzyme, while acting as the reversible inhibitors of the human fucosidase. The best inhibitor exhibited 13,700-fold difference in affinity between the two enzymes, which was attributed to the dissimilar aglycon binding site. Further investigations were carried out, including site-directed mutagenesis, the comparison of K(i) values among the wild type and mutants, and the intrinsic fluorescence change upon inhibitor titration, all supporting the idea that Tyr64 and Tyr267 of the Thermotoga alpha-fucosidase are critically involved in closely interacting with the aglycon of inhibitors. The increased level of contact thus induced conformational change, leading to the observed slow tight-binding inhibition. PMID- 16669614 TI - Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase. AB - Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action. PMID- 16669616 TI - Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins. AB - Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types. PMID- 16669617 TI - Residues from transmembrane helices 3 and 5 participate in leukotriene B4 binding to BLT1. AB - Leukotrienes are inflammatory mediators that bind to seven transmembrane, G protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val105 and Ile108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs. PMID- 16669615 TI - The pleckstrin homology domain of phospholipase Cbeta transmits enzymatic activation through modulation of the membrane-domain orientation. AB - Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also unclear are observations that the extent of Gbetagamma activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of the PLCbeta2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to that with Gbetagamma subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic headgroups. Also, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gbetagamma activatable mutant, PLCbeta2/delta1(C193S). We find an increase in the level of FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral headgroups. These results suggest that enzymatic activation can be conferred through optimal association of the PHbeta71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gbetagamma activation on different membrane surfaces. PMID- 16669618 TI - Substitution of tyrosine residues at the aromatic cluster around the betaA-betaB loop of rubisco small subunit affects the structural stability of the enzyme and the in vivo degradation under stress conditions. AB - Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) plays a central metabolic role in photosynthetic eukaryotes, and its catabolism is a crucial process for the nutrient economy of higher plants. The rubisco holoenzyme is assembled from eight chloroplast-encoded large subunits and eight nuclear-encoded small subunits. We have identified a cluster of conserved tyrosines at the interface between subunits (comprising Y67, Y68, and Y72 from the betaA-betaB loop of the small subunit and Y226 from the large subunit) that may contribute to holoenzyme stability. To investigate the role of these tyrosines in rubisco structure and in vivo degradation, we have examined site-directed mutants of these residues (Y67A, Y68A, Y72A, and Y226L) in Chlamydomonas reinhardtii. Even if all mutant strains were able to grow photoautotrophically, they exhibited a reduction in rubisco activity and/or the level of expression, especially the Y67A and Y72A mutants. Besides, all mutant rubiscos were inactivated at a lower temperature than the wild type. The kinetics of proteolysis of the mutant enzymes with subtilisin revealed structural alterations, leading to facilitated disassembly (in the cases of Y67A and Y72A) or aggregation propensity (for Y68A and Y226L). When subjected to oxidative stress in vivo through exposure of liquid cultures to hydrogen peroxide, all mutant strains degraded rubisco at a faster rate than the wild type. These results demonstrate that the tyrosine cluster around the betaA-betaB loop of rubisco small subunit plays a stabilizing role by affecting the catalytic activity and the degradation rate of the enzyme in stressed cells. PMID- 16669619 TI - Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme. AB - The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented. PMID- 16669620 TI - Electron exchange between Fe(II)-horse spleen ferritin and Co(III)/Mn(III) reconstituted horse spleen and Azotobacter vinelandii ferritins. AB - Azotobacter vinelandii bacterioferritin (AvBF) containing 800-1500 Co or Mn atoms as Co(III) and Mn(III) oxyhydroxide cores (Co-AvBF, Mn-AvBF) was synthesized by the same procedure used previously for horse spleen ferritin (HoSF). The kinetics of reduction of Co-AvBF and Mn-AvBF by ascorbic acid are first-order in each reactant. The rate constant for the reduction of Mn-AvBF (8.52 M(-1) min(-1)) is approximately 12 times larger than that for Co-AvBF (0.72 M(-1) min(-1)), which is consistent with a previous observation that Mn-HoSF is reduced approximately 10-fold faster than Co-HoSF [Zhang, B. et al. (2005) Inorg. Chem. 44, 3738-3745]. The rates of reduction of M-AvBF (M = Co and Mn) are more than twice that for the reduction of the corresponding M-HoSF. HoSF containing reduced Fe(II) cores (Fe(II)-HoSF), prepared by methyl viologen and CO, also reduces M-HoSF and M-AvBF species, with both cores remaining within ferritin, suggesting that electrons transfer through the ferritin shell. Electron transfer from Fe(II)-HoSF to Co AvBF occurs at a rate approximately 3 times faster than that to Co-HoSF, indicating that the Co cores in AvBF are more accessible to reduction than the Co cores in HoSF. The presence of nonconductive (SiO2) or conductive (gold) surfaces known to bind ferritins enhances the rate of electron transfer. A more than approximately 4-fold increase in the apparent reaction rate is observed in the presence of gold. Although both surfaces (SiO2 and gold) enhance reaction by providing binding sites for molecular interaction, results show that ferritins with different mineral cores bound to a gold surface transfer electrons through the gold substrate so that direct contact of the reacting molecules is not required. PMID- 16669621 TI - Major groove interactions of vaccinia Topo I provide specificity by optimally positioning the covalent phosphotyrosine linkage. AB - Vaccinia DNA topoisomerase (vTopo) is a prototypic eukaryotic type I topoisomerase that shows high specificity for nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5' (C/T)+5 C+4 C+3 T+2 T+1 p / N(-1). This reaction involves reversible transesterification where the active site tyrosine of the enzyme and a 5' hydroxyl nucleophile of DNA compete for attack at the phosphoryl group. The finite lifetime of the covalent phosphotyrosine adduct allows the enzyme to relax multiple supercoils by rotation of the 5'-OH strand before the DNA backbone is religated. To dissect the nature of the unique sequence specificity, subtle modifications to the major groove of the GGGAA 5'-sequence of the nonscissile strand were introduced and their effects on each step of the catalytic cycle were measured. Although these modifications had no effect on noncovalent DNA binding (K(D)) or the rate of reversible DNA cleavage (k(cl)), significant decreases in the cleavage equilibrium (K(cl) = k(cl)/k(r)) arising from increased rates of 5' hydroxyl attack (k(r)) at the phosphotyrosine linkage were observed. These data and other findings support a model in which major groove interactions are used to position the phosphotyrosine linkage relative to the mobile 5'-hydroxyl nucleophile. In the absence of native sequence interactions, the phosphotyrosine has a higher probability of encountering the 5'-hydroxyl nucleophile, leading to an enhanced rate of ligation and a diminished equilibrium constant for cleavage. By this unusual specificity mechanism, the enzyme prevents formation of stable covalent adducts at nonconsensus sites in genomic DNA. PMID- 16669622 TI - A study of the regional effects of alpha-synuclein on the organization and stability of phospholipid bilayers. AB - Associations between the protein alpha-synuclein (alpha-syn) and presynaptic vesicles have been implicated in synaptic plasticity and neurotransmitter release and may also affect how the protein aggregates into fibrils found in Lewy bodies, the cellular inclusions associated with neurodegenerative diseases. This work investigated how alpha-syn interacts with model phospholipid membranes and examined what effect protein binding has upon the physical properties of lipid bilayers. Wide line 2H and 31P NMR spectra of phospholipid vesicles revealed that alpha-syn associates with membranes containing lipids with anionic headgroups and can disrupt the integrity of the lipid bilayer, but the protein has little effect on membranes of zwitterionic phosphatidylcholine. A peptide, alpha-syn(10-48), which corresponds to the lysine-rich N-terminal region of alpha-syn, was found to associate with lipid headgroups with a preference for a negative membrane surface charge. Another peptide, alpha-syn(120-140), which corresponds to the glutamate rich C-terminal region, also associates weakly with lipid headgroups but with a slightly higher affinity for membranes with no net surface charge than for negatively charged membrane surfaces. Binding of alpha-syn(10-48) and alpha syn(120-140) to the lipid vesicles did not disrupt the lamellar structure of the membranes, but both peptides appeared to induce the lateral segregation of the lipids into clusters of acidic lipid-enriched and acidic lipid-deficient domains. From these findings, it is speculated that the N-terminal and C-terminal domains of full-length alpha-syn might act in concert to organize the membrane components during normal protein function and perhaps play a role in presynaptic vesicle synthesis, maintenance, and fusion. PMID- 16669623 TI - Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy. AB - Magainins are antimicrobial peptides that selectively disrupt bacterial cell membranes. In an effort to determine the propensity for oligomerization of specific highly active magainin analogues in membrane mimetic systems, we studied the structures and lipid interactions of two synthetic variants of magainins (MSI 78 and MSI-594) originally designed by Genaera Corp. Using NMR experiments on these peptides solubilized in dodecylphosphocholine (DPC) micelles, we found that the first analogue, MSI-78, forms an antiparallel dimer with a "phenylalanine zipper" holding together two highly helical protomers, whereas the second analogue, MSI-594, whose phenylalanines 12 and 16 were changed into glycine and valine, respectively, does not dimerize under our experimental conditions. In addition, magic angle spinning solid-state NMR experiments carried out on multilamellar vesicles were used to corroborate the helical conformation of the peptides found in detergent micelles and support the existence of a more compact structure for MSI-78 and a pronounced conformational heterogeneity for MSI-594. Since magainin activity is modulated by oligomerization within the membrane bilayers, this study represents a step forward in understanding the role of self association in determining magainin function. PMID- 16669625 TI - Interaction of alpha-and beta-oligoarginine-acids and amides with anionic lipid vesicles: a mechanistic and thermodynamic study. AB - The interaction of alpha- and beta-oligoarginine amides and acids and of alpha polyarginine with anionic lipid vesicles was studied. The beta-oligoarginines used were beta3-homologues of the alpha-oligoarginines. Lipid bilayers were composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1 palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) containing 5 mol % pyrene-PG (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-[phospho-rac-1 glycerol]). Kinetic analysis of the binding process onto large unilamellar POPC/POPG (3:7, molar ratio) vesicles (100 nm diameter) shows biphasic time courses for all tested peptides. The first binding step is fast and takes place within approximately 10 s with no disruption of the membrane as indicated by corresponding calcein release measurements. The second binding phase is slow and occurs within the next 30-300 s with substantial membrane disruption. In this context, beta-hexa- and octaarginine amides possess higher second half-times than the beta-hexa- and octaarginine acids of the same chain length. Furthermore beta octaarginine amide induces a calcein release approximately twice as large as that of the beta-octaarginine acid. Thermodynamic analysis of the binding process, using the complex formation model that assumes that each peptide binds independently to n POPG lipids, reveals apparent binding constants (K(app1)) of approximately 5 x 10(6)-10(8) M(-1) and n-values from 3.7 for beta-hexaarginine acid up to 24.8 for alpha-polyarginine. Although the K(app1)-values are similar, the number of binding sites clearly depends on the chemical nature of the oligoarginine: beta-oligoarginine amides and alpha-oligoarginine acids interact with more lipids than beta-oligoarginine acids of the same length. Calculation of the electrostatic contribution to the total free energy of binding reveals that for all oligoarginines only 25-30% has electrostatic origin. The remaining approximately 70-75% is nonelectrostatic, corresponding to hydrogen bonding and/or hydrophobic interactions. From the obtained data, a mechanism is suggested by which oligoarginines interact with anionic vesicles: (1) initial electrostatic interaction that is fast, nonspecific, and relatively weak; (2) nonelectrostatic interaction that is rate-limiting, stronger, and induces bilayer rigidification as well as release of aqueous contents from the vesicles. PMID- 16669626 TI - Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism. AB - The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin. PMID- 16669624 TI - Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity. AB - Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID- 16669627 TI - Using protein design to dissect the effect of charged residues on metal binding and protein stability. AB - Ca2+ controls biological processes by interacting with proteins with different affinities, which are largely influenced by the electrostatic interaction from the local negatively charged ligand residues in the coordination sphere. We have developed a general strategy for rationally designing stable Ca2+- and Ln3+ binding proteins that retain the native folding of the host protein. Domain 1 of cluster differentiation 2 (CD2) is the host for the two designed proteins in this study. We investigate the effect of local charge on Ca2+-binding affinity based on the folding properties and metal-binding affinities of the two proteins that have similarly located Ca2+-binding sites with two shared ligand positions. While mutation and Ca2+ binding do not alter the native structure of the protein, Ca2+ binding specifically induced changes around the designed Ca2+-binding site. The designed protein with a -5 charge at the binding sphere displays a 14-, 20-, and 12-fold increase in the binding affinity for Ca2+, Tb3+, and La3+, respectively, compared to the designed protein with a -3 charge, which suggests that higher local charges are preferred for both Ca2+ and Ln3+ binding. The localized charged residues significantly decrease the thermal stability of the designed protein with a -5 charge, which has a T(m) of 41 degrees C. Wild-type CD2 has a T(m) of 61 degrees C, which is similar to the designed protein with a -3 charge. This decrease is partially restored by Ca2+ binding. The effect on the protein stability is modulated by the environment and the secondary structure locations of the charged mutations. Our study demonstrates the capability and power of protein design in unveiling key determinants to Ca2+-binding affinity without the complexities of the global conformational changes, cooperativity, and multibinding process found in most natural Ca2+-binding proteins. PMID- 16669628 TI - Phosphorylation of STAT3 serine-727 by cyclin-dependent kinase 1 is critical for nocodazole-induced mitotic arrest. AB - Signal transducer and activator of transcription 3 (STAT3) mediates cellular responses to diverse cytokines and growth factors by modulating the expression of specific target genes. While phosphorylation of STAT3 at Tyr-705 has been demonstrated to be a prerequisite for STAT3 dimerization, nuclear translocation, and activation of gene transcription, the role of Ser-727 in regulation of STAT3 activity is controversial. Kinetworks KPSS-1.1 phospho-site screening of nocodazole-treated HeLa cells revealed that STAT3 Ser-727 phosphorylation was enhanced during mitosis, and this correlated with a reduction of Tyr-705 phosphorylation. Overexpression of STAT3 mutants in which these phosphorylation sites were separately abolished revealed that phosphorylation at these sites appeared to be mutually antagonistic. The nocodazole-induced STAT3 Ser-727 phosphorylation was reduced by selective inhibition of CDK1 phosphotransferase activity, and CDK1 could directly phosphorylate GST-STAT3 Ser-727 in vitro and co immunoprecipitate with STAT3 in vivo. Blocking Ser-727 phosphorylation enhanced STAT3 DNA-binding activity toward its target gene promoters, implying a negative effect of Ser-727 phosphorylation on its transcriptional activity. Interference of Ser-727 phosphorylation resulted in an exit from mitotic arrest induced by nocodazole treatment and a cell cycle arrest at the G1 phase, as indicated by the accumulation of 2N cell population and enhanced expression of G1 cell cycle regulators including p21(CIP1/WAF1), p27(Kip1), and cyclin E. Taken together, our observations point to a novel role of STAT3 Ser-727 phosphorylation in control of the onset and maintenance of the M phase during the cell cycle through downregulation of CDK inhibitors. PMID- 16669629 TI - Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149. AB - Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1 binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope. PMID- 16669630 TI - Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease. AB - Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme. PMID- 16669631 TI - Testing the steric exclusion model for hexameric helicases: substrate features that alter RNA-DNA unwinding by the transcription termination factor Rho. AB - Typical hexameric helicases form ring-shaped structures involved in DNA replication. These enzymes have been proposed to melt forked DNA substrates by binding to, and pulling, one strand within their central channel, while the other strand is forced outside of the hexamer by steric exclusion and specific contacts with the outer ring surface. Transcription termination factor Rho also assembles into ring-shaped hexamers that are capable to use NTP-derived energy to unwind RNA and RNA-DNA helices. To delineate the potential relationship between helicase structural organization and unwinding mechanism, we have performed in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. We show that a physical discontinuity (nick) inhibits RNA-DNA unwinding when present in the RNA but not in the DNA strand. Moreover, the presence of a 3'-overhanging DNA tail (Y-shaped substrate) does not affect initial Rho binding but can impair helicase activity. This inhibitory effect varies with the length of the tail, is independent of the identity (A or U) of the tail residues, and is also obtained when a biotin-streptavidin complex replaces the single-stranded DNA arm. However, it is readily relaxed upon moving the reporter RNA-DNA helix farther from the Rho loading site. The data indicate that the Rho helicase uses a steric exclusion mechanism whereby the initial formation of a productive Rho-transcript complex is a crucial rate-limiting event, while no specific interactions with the displaced strand are required. These results outline significant similarities as well as some differences in the mechanism of unwinding between Rho and other hexameric helicases which are discussed in relation with the biological function of the Rho helicase. PMID- 16669633 TI - Early and late cytotoxic effects of external application of the Alzheimer's Abeta result from the initial formation and function of Abeta ion channels. AB - Extracellular application of the Alzheimer's beta-amyloid (Abeta) peptide evokes a series of cellular responses that leads to the death of cells by apoptosis. Some responses to freshly prepared Abeta occur immediately, including changes in intracellular calcium concentration and changes in membrane permeability and phosphatidylserine asymmetry. We show here that the cytotoxic action of externally applied Abeta, such as caspase activation and apoptotic loss of cell viability, occurs and persists even several days after Abeta is removed from the medium. We find that the mechanism for this persistent cytotoxic action of extracellular Abeta is based on the sustained activity of active Abeta ion channels that remain incorporated in the cell membrane. To confirm this assessment, we blocked the late cytotoxic action of Abeta with the classically known Abeta channel blockers zinc and tromethamine. To further validate this conclusion, we developed a specific peptide segment from the sequence forming the mouth of the Abeta channel to block Abeta Ca2+ channels acutely and to block late Abeta effects on caspase activation and apoptosis. This is the first report of a specific Abeta channel blocker compound, NA4, which efficaciously and potently blocks the most known cellular responses to Abeta. PMID- 16669634 TI - Conformation of a clathrin triskelion in solution. AB - A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R(g), and hydrodynamic radius, R(H), of isolated triskelia, under conditions where cage assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending the legs and a puckering at the vertex. We also show that the values of R(g) and R(H) measured for clathrin triskelia in solution are qualitatively consistent with the conformation of a triskelion in a "D6 barrel" cage assembly measured by cryoelectron microscopy. PMID- 16669632 TI - Extrinsic interactions dominate helical propensity in coupled binding and folding of the lactose repressor protein hinge helix. AB - A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short alpha-helices when bound to DNA but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge-helix folding and/or hinge-hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality. PMID- 16669635 TI - Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue. AB - ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments. PMID- 16669637 TI - Nucleophilic addition to (3-methylpentadienyl)iron(1+) cations: counterion control of regioselectivity; application to the enantioselective synthesis of 4,5 disubstituted cyclohexenones. AB - The regioselectivity of malonate addition to (3-methylpentadienyl)Fe(CO)3+ is controlled by the malonate-counterion association. The Li+ salt of malonate proceeds via C1 nucleophilic attack to afford the 1,3Z-diene complex 4a, while reaction of highly dissociated ion pair (i.e., Na+ or Li+/12-crown-4) salt proceeds at the C2 internal carbon to eventually afford cyclohexenone products 6. Reaction of 1a with the sodium salt of bis(8-phenylmenthyl)malonate proceeds with excellent diastereocontrol to afford a single diastereomeric cyclohexenone. PMID- 16669638 TI - Cellular uptake of PNA--terpyridine conjugates and its enhancement by Zn2+ ions. AB - Conjugation of usually impermeable peptide nucleic acids (PNA) to the chelator 2,2':6',2' '-terpyridine strongly promotes cellular and nuclear uptake by cultured HeLa cells. Cellular accumulation is further enhanced in the presence of extracellular Zn2+. PMID- 16669639 TI - Semipermeable polymer vesicle (PICsome) self-assembled in aqueous medium from a pair of oppositely charged block copolymers: physiologically stable micro /nanocontainers of water-soluble macromolecules. AB - A new entity of polymer vesicle with a polyion complex (PIC) membrane, a PICsome, was prepared by simple mixing of a pair of oppositely charged block copolymers, composed of biocompatible PEG and poly(amino acid)s, in an aqueous medium. Flow particle image analysis revealed the formation of spherical particles with a size range up to 10 mum. Observation by dark-field and confocal laser scanning microscopes clearly confirmed that the PICsome has a hollow structure with an inner-water phase, in which FITC-dextran emitting green fluorescence was successfully encapsulated simply by the simultaneous mixing with the block copolymers. Confocal laser scanning microscopic observation and spectral analysis revealed the smooth penetration of a low molecular weight fluorescent dye (TRITC; MW = 443.5) emitting red fluorescence into the FITC-dextran encapsulated PICsome to give the PICsome image with a merged color of yellows, indicating the semipermeable nature of the PICsome membrane. The PICsomes showed appreciable physiological stability even in the presence of serum proteins, suggesting their feasibility in biomedical fields such as carriers of therapeutic compounds and compartments for diagnostic enzymes. PMID- 16669640 TI - Synthesis and characterization of a bisadduct of La@C82. AB - A bisadduct of La@C82 has been synthesized in a good yield by a Bingel-Hirsch reaction. Its structure has been well-defined by X-ray crystallographic analysis. A pair of enantiomers of the adduct form a dimer in the single crystal. PMID- 16669641 TI - Stabilization of G-quadruplex DNA and inhibition of telomerase activity by square planar nickel(II) complexes. AB - Two new alkylamine-substituted nickel(II)-salphen complexes have been prepared and their interactions with DNA investigated. FRET studies have shown that these complexes have a remarkable ability to stabilize G-quadruplex DNA. Furthermore, TRAP/Taq assays have shown that these complexes inhibit telomerase at low micromolar concentrations. PMID- 16669642 TI - The importance of dynamics in substrate-assisted catalysis and specificity. AB - The QM/MM MD and free energy simulations show that the dynamics involving a His residue at the P1 site of the substrate may play an important role in substrate assisted catalysis and specificity for a serine-carboxyl peptidase. PMID- 16669643 TI - Acquisition of a "Group A"-selective Src kinase inhibitor via a global targeting strategy. AB - A "global" strategy for the acquisition of selective high affinity inhibitors for the Src kinase subfamily of tyrosine kinases is described. Members of the Src family exhibit a strong amino acid sequence homology. However, recent studies have revealed differences in the relative spatial relationships of the three distinct protein-binding domains present in these enzymes. We have constructed an inhibitor, using an amalgamation of combinatorial methods and directed design, which simultaneously associates with the active site and an ancillary protein binding region (SH2 domain). The inhibitor exhibits high inhibitory potency and selectivity for the Group A versus Group B subset of Src kinases. PMID- 16669644 TI - Pt and Pd 1,4-shifts at the edge of dibenz[a,c]anthracene. AB - 1,4-Shifts of the carbon-bonded metal are observed on heating solutions of trans bromo(dibenz[a,c]anthracen-9-yl)bis(triethylphosphine)platinum or trans bromo(dibenz[a,c]anthracen-9-yl)bis(triethylphosphine)palladium. The shift does not occur from the cis isomer and is more rapid for the Pt complex. Solvent effects suggest polar species, while the absence of deuterium incorporation in the presence of D2O indicates nonacidic intermediates. PMID- 16669645 TI - Conformational analysis of chiral helical perfluoroalkyl chains by VCD. AB - Vibrational circular dichroism (VCD) technique successfully revealed the absolute configuration of the biased helix of perfluoroalkyl chains in solution with the aid of theoretical calculations, which was supported by an X-ray crystallographic study. PMID- 16669646 TI - Chemical routes to nanocrystalline thermoelectrically relevant AgPb(m)SbTe(m+2) materials. AB - A direct synthesis of nanoparticles of the thermoelectrically relevant AgPbmSbTem+2 materials (m = 0, 1, 2) was accomplished in reverse micelles. The procedure offers several distinct advantages and opens the field for experimentation of thermoelectric properties for nanoparticle-derived materials. PMID- 16669647 TI - A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells. AB - We have found a specific reaction between ketone 1 and peroxynitrite (ONOO-), rather than other reactive oxygen species and reactive nitrogen species generated in the biological system. On the basis of this reaction, we have successfully developed a new fluorescent probe HKGreen-1, which is highly selective for the detection of peroxynitrite in living cells. Before the oxidation with peroxynitrite, the dichlorofluorescein part is masked and the probe is nonfluorescent. However, upon reaction with peroxynitrite, the fluorophore is released, resulting in strong enhancement in fluorescence intensity. PMID- 16669648 TI - The influence of macromolecular crowding on HIV-1 protease internal dynamics. AB - High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close-packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease flaps are open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle. PMID- 16669649 TI - Chemisorbed benzoate-to-benzene conversion via phenyl radicals on Cu(110): kinetic observation of conformational effects. AB - The adsorption and decomposition of benzoic acid on the Cu(110) surface has been investigated using temperature-programmed reaction (TPR) spectroscopy and scanning tunneling microscopy (STM). The benzoate species is found to exist in two conformations: a phase containing upright species at monolayer saturation and a phase containing many tilted species at lower coverages. Thermal decomposition begins to occur near 500 K, yielding benzene and CO2. It is found that phenyl radicals, generated preferentially from the tilted benzoate species, efficiently abstract H atoms from undecomposed benzoate species to produce benzene in a rate controlling process with an activation energy of about 29 kcal/mol. Using deuterium atom substitution at the 4-C position on the benzoate ring, it is found that the hydrogen abstraction reaction is selective for 2-,3- and 5-,6-C-H bonds. This observation indicates that the mobile phenyl radical is surface bound and preferentially attacks C-H bonds which are nearest the Cu surface binding the benzoate species, either as an upright species or as a tilted species. PMID- 16669650 TI - Transmission of extramembrane conformational change into current: construction of metal-gated ion channel. AB - Interaction with Fe(III) induces the reversible conformational switch of the extramembrane segment in the artificial receptor channel, which is transmitted into membranes as an increase in channel current (ion flux). PMID- 16669651 TI - Searching for cyclin-dependent kinase inhibitors using a new variant of the cope elimination. AB - beta-Piperidinoethylsulfides are oxidized by m-chloroperbenzoic acid to intermediates containing both N-oxide and sulfone functions. These undergo a Cope type elimination to a vinylsulfone that can be captured by amines to afford beta aminoethylsulfones. When a beta-aminoethylsulfone group is linked to the 4 position of a phenyl group attached at N-2 of O6-cyclohexylmethylguanine, the resulting derivatives are inhibitors of the cyclin-dependent kinase CDK2. One of the most potent inhibitors (IC50 = 45 nM) contained a N-3-hydroxypropyl group on the aminoethylsulfonyl substituent. The crystal structure of this inhibitor bound to CDK2/cyclin A was determined and shows an unusual network of hydrogen bonds. The synthetic methodology developed can be utilized in multiple-parallel format and has numerous potential applications in medicinal chemistry. PMID- 16669652 TI - High amplification rates from the association of two enzymes confined within a nanometric layer immobilized on an electrode: modeling and illustrating example. AB - Electrochemical responses (e.g., chronoamperometric) obtained with an immobilized enzyme that produces an electroactive species may be used to quantitate the amount of enzyme or the concentration of its substrate. It is shown, on theoretical and experimental bases, that product-to-substrate coupling with a second enzyme co-immobilized with the first within one or within a small number of monolayers, allows high amplification rates (higher than 1000), avoids membrane transport limitations, and lends itself to precise kinetic analyses that provide guidelines for optimization of the analytical sensitivity. Very large amplification factors, as large as several thousands, can be reached experimentally, in agreement with appropriately derived theoretical predictions, thus opening the route to the rational design of high-performance substrate sensing or affinity assays applications. PMID- 16669653 TI - A one-dimensional coordination polymer, BBDTA.InCl4: possible spin-Peierls transition with high critical temperature of 108 K. AB - We have studied the crystal structure and magnetic properties of the organic radical cation salt, BBDTA.InCl4. This material formed a one-dimensional coordination polymer. Magnetic measurements indicated the spin-Peierls transition like behavior at 108 K, which was higher than those typically observed for the other organic spin-Peierls materials. The structural aspects of the lattice distortion from X-ray diffraction measurements at 50 K have been discussed. PMID- 16669654 TI - Mono(dinitrogen) and carbon monoxide adducts of bis(cyclopentadienyl) titanium sandwiches. AB - The silyl-substituted titanocene complex, (eta5-C5Me4SiMe2Ph)2Ti, coordinates dinitrogen upon cooling to -35 degrees C to yield an unprecedented example of a mono(dinitrogen) complex of a substituted bis(cyclopentadienyl) titanium compound, (eta5-C5Me4SiMe2Ph)2Ti(N2). Analogous monocarbonyl derivatives, (eta5 C5Me4R)2Ti(CO) (R = SiMe3, SiMe2Ph, CHMe2), have been prepared by mixing the dicarbonyl compounds with the corresponding sandwiches. Both (eta5 C5Me4SiMe2Ph)2Ti(N2) and (eta5-C5Me4SiMe2Ph)2Ti(CO) have been characterized by X ray diffraction, and mixed N2-CO titanocene complexes have also been observed by in situ IR spectroscopy. PMID- 16669655 TI - Hyperdimensional NMR spectroscopy. AB - Hyperdimensional NMR describes the multiple interactions between a large number, N, of coupled nuclear spins. We show how to construct a hypothetical N dimensional spectrum from a limited set of low-dimensional measurements, exploiting the concept of projection-reconstruction. In the process, many correlation spectra are obtained that were not investigated directly. As an illustration, the hypothetical ten-dimensional spectrum of a small protein (agitoxin) has been reconstructed, and a large number of pairwise correlation spectra have been recorded. PMID- 16669656 TI - Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase. AB - The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3). PMID- 16669657 TI - Site-selective binding and dual mode recognition of serum albumin by a squaraine dye. AB - With the objective of developing small molecule based probes for proteins, interactions of polyhydroxyl-substituted squaraine dye (SQ) with bovine serum albumin (BSA) have been investigated by absorption, steady-state and time resolved fluorescence, circular dichroism (CD), cyclic voltammetry (CV), 1H NMR, scanning electron, and tapping mode atomic force microscopic techniques. Increase in addition of BSA resulted in increase in absorbance and fluorescence quantum yields (80-fold) of SQ, along with significant bathochromic shifts in the absorption and fluorescence maxima. Half-reciprocal analysis of the absorption data gave a 1:1 stoichiometry for the complex between BSA and SQ with high association (Kass) constant of (1.4 +/- 0.1) x 106 M-1 and change in free energy of -35 kJ/mol. The complex formation was further confirmed by observation of induced CD signal corresponding to the SQ chromophore at 610 nm, upfield shift (about Deltadelta 0.1 ppm) of aromatic protons of SQ in 1H NMR spectra, and decrease in current intensity (CV) of SQ when bound to BSA. The picosecond time resolved fluorescence studies indicated that the BSA-SQ complex exhibits biexponential decay with significantly enhanced lifetimes of 0.5 and 1.5 ns when compared to the lifetime of SQ (tau = 121 ps) in the absence of BSA. Employing displacement cum fluorimetry using site-specific binding ligands, such as dansylproline and dansylamide, indicated that SQ binds with protein selectively at site II involving hydrophobic, hydrogen bonding, and electrostatic interactions. The uniqueness of this molecular system is that it interacts with BSA selectively at site II and signals the binding event through dual mode recognition of "visual color" change and "turn on" fluorescence mechanism. PMID- 16669658 TI - Hydrogenation and hydrocarbonation and etching of single-walled carbon nanotubes. AB - We present a systematic experimental investigation of the reactions between hydrogen plasma and single-walled carbon nanotubes (SWNTs) at various temperatures. Microscopy, infrared (IR) and Raman spectroscopy, and electrical transport measurements are carried out to investigate the properties of SWNTs after hydrogenation. Structural deformations, drastically reduced electrical conductance, and an increased semiconducting nature of SWNTs upon sidewall hydrogenation are observed. These changes are reversible upon thermal annealing at 500 degrees C via dehydrogenation. Harsh plasma or high temperature reactions lead to etching of nanotubes likely via hydrocarbonation. Smaller SWNTs are markedly less stable against hydrocarbonation than larger tubes. The results are fundamental and may have implications to basic and practical applications including hydrogen storage, sensing, band gap engineering for novel electronics, and new methods of manipulation, functionalization, and etching of nanotubes. PMID- 16669659 TI - Efforts toward developing direct probes of protein dynamics. AB - We report the first IR characterization of a single C-D bond within a protein, methyl-d1 Met80 of horse heart cytochrome c. A comparison was made to methyl d1/d3 methionine as well as methyl-d3 Met80. We found that for methyl-d1 and the asymmetric stretches of methyl-d3, line widths/line shapes are dominated by inhomogeneous broadening, whereas the symmetric stretch of methyl-d3 has a significant homogeneous component. Vibrational energy relaxation calculations found that a significantly stronger Fermi resonance exists for the symmetric stretch than for the asymmetric stretches, thereby suggesting that a difference in intramolecular vibrational relaxation (IVR) causes the observed line width/line shape difference between the symmetric and asymmetric stretches. PMID- 16669660 TI - Structure and bonding between an aryl group and metal surfaces. AB - Modifying solid surfaces with aryl groups has many potential applications. Using first principles density functional theory methods, we investigated the trend of the structure and bonding of the phenyl group (C6H5, the simplest aryl group) on selected transition metals across the periodic table. We found that the bond between C6H5 and metal surfaces is chemical in nature. Decreasing bond strength is found from left to right, concurrent with a switching of the preferred orientation for C6H5 from the flat-lying configuration to the upright configuration. This switching is attributed to the increasing of d-electrons; that is, early transition metals, lacking d-electrons, favor the carbon-metal pi bond and therefore the flat-lying configuration, while late transition metals rich in d-electrons prefer the carbon-metal sigma-bond and thus the upright fashion. C6H5 is also found to undergo beta-dehydrogenation on early transition metals. This work invites further theoretical and experimental research on the aryl-solid interface. PMID- 16669661 TI - Chiral discrimination of cryptochiral saturated quaternary and tertiary hydrocarbons by asymmetric autocatalysis. AB - Chiral discrimination of saturated hydrocarbons has been very difficult to establish, or has not been possible at all. The first chiral discrimination of cryptochiral 5-ethyl-5-propylundecane 1, that is, (n-butyl)ethyl(n-hexyl)(n propyl)methane, a chiral saturated quaternary hydrocarbon, which is known to exhibit practically no detectable value of optical rotation between 280 and 580 nm, has been accomplished by asymmetric autocatalysis of pyrimidyl alkanol. The absolute configuration of 1 has been determined. In the presence of (R)- or (S) 1, the reaction between pyrimidine-5-carbaldehyde and diisopropylzinc affords (S) and (R)-pyrimidyl alkanol with 91-97% ee, respectively. Thus, asymmetric autocatalysis serves as a powerful tool for the chiral discrimination of saturated hydrocarbons. PMID- 16669662 TI - Pressure-induced sequential magnetic pole inversion and antiferromagnetic ferromagnetic crossover in a trimetallic prussian blue analogue. AB - The flexibility of the structure of Prussian blue analogues and its ability to incorporate a variety of competing magnetic interactions have allowed the design of mixed ferro-ferrimagnets, which span the whole spectrum of magnetic behavior, including the rare phenomenon of magnetization reversal in response to a change in temperature. Hydrostatic pressure is used here to induce multiple reversals of the direction of the spontaneous magnetization in the trimetallic Prussian blue analogue, Rb0.64Ni0.31Mn0.87[Fe(CN)6].2.8H2O. Remarkably, the magnetic response is extremely sensitive to pressure, and the magnetization flips from positive to negative and back to positive in a very narrow pressure range (0 < P < 0.6 kbar). A further increase in pressure to 4.0 kbar induces an internal redox reaction, and the magnetic order switches from ferrimagnetic to bulk ferromagnetism. PMID- 16669663 TI - Chromatographic isolation of "missing" Au55 clusters protected by alkanethiolates. AB - We report on the first synthesis of alkanethiolate-protected Au55 (11 kDa), which has been a "missing" counterpart of Schmid's Au55(PR3)12Cl6. Au:SCx clusters (x = 12, 18) were prepared by the reaction of alkanethiol (CxSH) with polymer stabilized Au clusters ( approximately 1.3 nm) and subsequently incubated in neat CxSH. The resulting clusters were successfully fractionated by recycling gel permeation chromatography into Au approximately 38:SCx and Au approximately 55:SCx and identified by laser-desorption ionization mass spectrometry. The Au approximately 55:SCx clusters exhibited structured optical spectra, suggesting molecular-like properties. The thiolate monolayers were found to be liquid-like on the basis of the IR spectrum and the monolayer thickness, which was estimated from the hydrodynamic diameter. PMID- 16669664 TI - Synthesis and structure of ruthenium-silylene complexes: activation of Si-Cl bonds in N-heterocyclic silanes. AB - Ru(0) complexes of bis(imino)pyridine ligands, [eta2-N3]Ru(eta6-Ar) and {[N3]Ru}2(mu-N2), where Ar = C6H6 or C6H5Me and [N3] = 2,6-(MesN=CMe)2C5H3N, react with N-heterocyclic silicon(IV) compounds to yield Ru(II) silylene complexes of the type [N3]Ru(X)(Cl){Si(NN)} (X = H, Cl, and Si(NN) = N,N' bis(neopentyl)-1,2-phenylenedi(amino)silylene). The activation of two groups on the silane occurs in a stepwise fashion: initial oxidative addition of a Si-X bond, followed by 1,2-migration (alpha-elimination) of the Si-Cl group to the metal. Reversible dissociation from the Ru(II) center leads to free silylene, which can be preferentially trapped with Ru(0) complexes to generate a zero valent silylene complex, [N3]Ru(N2){Si(NN)}, which also contains a terminal dinitrogen ligand. PMID- 16669665 TI - Measuring amide nitrogen quadrupolar coupling by high-resolution 14N/13C NMR correlation under magic-angle spinning. AB - The measurement of amide nitrogen 14N quadrupolar coupling by two-dimensional 14N/13C correlation experiment is presented with a natural abundant polypeptide. Directly bonded 14N/13C pairs are correlated through J and residual dipolar coupling under magic-angle spinning using a HMQC-type pulse sequence. The 14N quadrupolar coupling is measured from the isotropic second-order quadrupolar shift obtained by comparing the 14N peak positions with the 15N chemical shifts. The high spectral resolution and sensitivity through 13C detection make this method applicable to many organic, inorganic, and biological molecules for the measurement and the use of 14N quadrupolar coupling as a probe for molecular structure and dynamics. PMID- 16669666 TI - Rhodium-catalyzed intramolecular, anti-Markovnikov hydroamination. Synthesis of 3 arylpiperidines. AB - The intramolecular anti-Markovnikov hydroamination of 1-(3 aminopropyl)vinylarenes in the presence of a readily available rhodium catalyst to form 3-arylpiperidines is reported. In contrast to intermolecular hydroamination of vinylarenes, which occurred in high yields in the presence of rhodium catalysts containing DPEphos, the intramolecular reaction occurred in high yield in the presence of [Rh(COD)(DPPB)]BF4 as catalyst. Reactants with substituents beta to the nitrogen occurred in high yield, and these reactions formed 3,5-disubstituted piperidines with high diastereomeric excess. The regiochemistry of these cyclizations contrasts with the regiochemistry of intramolecular hydroaminations catalyzed by lanthanide complexes, group III metal complexes, and platinum complexes, all of which have been reported to form cyclization products from Markovnikov addition. PMID- 16669668 TI - Direct asymmetric hydroxyamination reaction catalyzed by an axially chiral secondary amine catalyst. AB - A direct asymmetric hydroxyamination reaction of aldehydes with nitrosobenzene was found to be catalyzed by the novel axially chiral secondary amine catalyst (S)-1d. The resulting optically enriched hydroxyamination products were readily converted to beta-amino alcohols or 1,2-diamines in one pot. PMID- 16669667 TI - Palladium-catalyzed asymmetric ring expansion of allenylcyclobutanols: an asymmetric Wagner-Meerwein shift. AB - In this study, we developed a palladium-catalyzed atom economic asymmetric Wagner Meerwein shift of allenylcyclobutanol substrates. It is an excellent method for creating functionalized cyclopentanones with an alpha-chiral O-tertiary center by ring expansion of allenylcyclobutanols. This reaction was initiated by hydropalladation and afforded excellent enantioselectivity as well as atom economy. This method provides an efficient route toward the synthesis of natural products such as trans-kumausyne's family, spiro ring systems. In addition, we obtained excellent diastereoselectivity and enantioselectivity at the same time by using 3-monosubstituted allenylcyclobutanol. PMID- 16669669 TI - The Mannich reaction of malonates with simple imines catalyzed by bifunctional cinchona alkaloids: enantioselective synthesis of beta-amino acids. AB - We describe the first efficient, direct asymmetric Mannich reactions with malonates and N-Boc aryl and alkyl imines by cooperative hydrogen-bonding catalysis with a cinchona alkaloid bearing a thiourea functionality. We have also extended the scope of this reaction to beta-ketoesters. The synthetic value of this new reaction is demonstrated in the establishment of a convergent enantioselective route toward the biologically important beta-amino acids under mild and air- and moisture-tolerant conditions. PMID- 16669670 TI - A direct, one step synthesis of imidazoles from imines and acid chlorides: a palladium catalyzed multicomponent coupling approach. AB - A palladium-catalyzed one-step synthesis of imidazoles from imines and acid chlorides is described. A plausible mechanism for this multicomponent reaction is provided, which explains the selective incorporation of two different imines into the final product with perfect regiocontrol. Overall, this catalytic process provides a modular method to prepare imidazoles directly from building blocks that are all either commercially available or readily generated. PMID- 16669671 TI - Optically transparent Au{111} substrates: flat gold nanoparticle platforms for high-resolution scanning tunneling microscopy. AB - We demonstrate a new type of Au{111} substrate that is both atomically flat and optically transparent, which consists of solution-grown flat gold nanoparticles (FGNPs) deposited on indium tin oxide (ITO)-coated glass. We show that FGNPs are atomically flat single-crystal plates with large {111} faces that expose only 2-4 atomic layers. These FGNPs are excellent platforms for alkanethiol self-assembled monolayers (SAMs) and for high-resolution scanning tunneling microscopy (STM). Our supported FGNPs are also low-cost Au{111} substrates, employing only basic wet chemical techniques in preparation. This approach should be broadly applicable to other types of substrates for scanning probe microscopies. PMID- 16669672 TI - New class of nucleophiles for palladium-catalyzed asymmetric allylic alkylation. Total synthesis of agelastatin A. AB - New classes of nucleophiles, pyrroles, and N-methoxyamides were developed for Pd catalyzed AAA reactions. By varying the functional groups at the 2-position of pyrroles, either regioisomer of the piperazinone is available. Using one regioisomer, the total synthesis of (+)-agelastatin A in 10 total steps is accomplished. For this synthesis, a new copper-catalyzed aziridination and an indium-catalyzed oxidative ring opening of a N-tosylaziridine were developed. The feasibility of accessing (-)-agelastatin A from the same enantiomer of the chiral catalyst from the other regioisomeric piperazinone is indicated. PMID- 16669673 TI - "Click" chemistry in a supramolecular environment: stabilization of organogels by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. AB - Organogels are thermoreversible, viscoelastic (soft) materials consisting of low molecular weight compounds which self-assemble into fibers, often of micrometer lengths and nanometer diameters. The installation of terminal azide and alkyne functional groups on the end of a standard alkylamide-based organogelator was found to cause a modest disruption in the gelation properties of the molecule. Cross-linking of those groups by the copper(I)-catalyzed azide-alkyne cycloaddition reaction produced thermoreversible materials of substantially greater gelation temperatures and mechanical rigidity. These results highlight the ability of azides and alkynes-participants in the most commonly used "click" reaction-to function as innocuous precursors to meaningful covalent interactions in materials science. PMID- 16669674 TI - Enantioselective alpha-hydroxylation of 2-arylacetic acid derivatives and buspirone catalyzed by engineered cytochrome P450 BM-3. AB - Here we report that an engineered microbial cytochrome P450 BM-3 (CYP102A subfamily) efficiently catalyzes the alpha-hydroxylation of phenylacetic acid esters. This P450 BM-3 variant also produces the authentic human metabolite of buspirone, R-6-hydroxybuspirone, with 99.5% ee. PMID- 16669675 TI - Origin of the relative stereoselectivity of the beta-lactam formation in the Staudinger reaction. AB - The relative (cis, trans) stereoselectivity of the beta-lactam formation is one of the critical issues in the Staudinger reaction. Although many attempts have been made to explain and to predict the stereochemical outcomes, the origin of the stereoselectivity remains obscure. We are proposing a model that explains the relative stereoselectivity based on a kinetic analysis of the cis/trans ratios of reaction products. The results were derived from detailed Hammett analyses. Cyclic imines were employed to investigate the electronic effect of the ketene substituents, and it was found that the stereoselectivity could not be simply attributed to the torquoelectronic model. Based on our results, the origin of the relative stereoselectivity can be described as follows: (1) the stereoselectivity is generated as a result of the competition between the direct ring closure and the isomerization of the imine moiety in the zwitterionic intermediate; (2) the ring closure step is most likely an intramolecular nucleophilic addition of the enolate to the imine moiety, which is obviously affected by the electronic effect of the ketene and imine substituents; (3) electron-donating ketene substituents and electron-withdrawing imine substituents accelerate the direct ring closure, leading to a preference for cis-beta-lactam formation, while electron-withdrawing ketene substituents and electron-donating imine substituents slow the direct ring closure, leading to a preference for trans-beta-lactam formation; and (4) the electronic effect of the substituents on the isomerization is a minor factor in influencing the stereoselectivity. PMID- 16669676 TI - Emergent functionality of nucleobase radical cations in duplex DNA: prediction of reactivity using qualitative potential energy landscapes. AB - The one-electron oxidation of a series of DNA oligonucleotides was examined. Each oligomer contains a covalently linked anthraquinone (AQ) group. Irradiation of the AQ group with near-UV light results in a one-electron oxidation of the DNA that generates a radical cation (electron "hole"). The radical cation migrates through the DNA by a hopping mechanism and is trapped by reaction with water or molecular oxygen, which results in chemical reaction at particular nucleobases. This reaction is revealed as strand cleavage when the irradiated oligonucleotide is treated with piperidine. The specific oligomers examined reveal the existence of three categories of nucleobase sequences: charge shuttles, charge traps, and barriers to charge migration. The characterization of a sequence is not independent of the identity of other sequences in the oligonucleotide, and for this reason, the function of a particular sequence emerges from an analysis of the entire structure. Qualitative potential energy landscapes are introduced as a tool to assist in the rationalization and prediction of the reactions of nucleobases in oxidized DNA. PMID- 16669678 TI - CB11Me11 boronium ylides: carba-closo-dodecaboranes with a naked boron vertex. AB - In pentane solution, 2 equiv of the icosahedral CB(11)Me(12)(*) radical cleaves the Si-Si bond of hexaalkyldisilanes by boron to silicon methyl transfer with formation of 2 equiv of methyltrialkylsilanes. The loss of a methyl radical converts the CB(11)Me(12)(*) radical into an internally charge-compensated "boronium ylide" CB(11)Me(11) with a naked vertex, which can be formally viewed as a deprotonated hypercloso carborane. It has been isolated as an air-sensitive solid, stable only below approximately -60 degrees C. The naked vertex appears to be in position 12 since the material reacts instantaneously with alcohols and ethers to form the 12-alkoxy anions 12-CB(11)Me(11)OR. It reacts with many other nucleophiles to yield more complex mixtures containing similar products. DFT calculations for the four CB(11)Me(11) isomers give closed-shell ground-state electronic structures. For the isomer with naked vertex 12, a DFT computational search failed to reveal any skeletal dimers, apparently due to excessive methyl methyl repulsions, and only a cyclic dimer bound through weak interactions of one of the 7-methyl hydrogen atoms on each cage with the empty exocyclic orbitals on B12 of the other cage was found. Natural hybrid orbital populations suggest that the three possible isomers of monomeric boronium ylides are close to true singlet ylides, with triplet states approximately 50 kcal/mol higher. The calculated electronic structure of the carbonium ylide is close to a singlet carbene, with a triplet state approximately 16 kcal/mol higher. An attempted preparation of Me(3)C(+)CB(11)Me(12)(-) yielded neopentane and products consistent with a sequential loss of methyl groups from the carborane cage with a transient formation of similar boronium ylides. Probable mechanisms of these methyl transfer reactions are considered, and the possibly quite general role of "ylide" structures in Lewis acid induced substitution reactions on the boron vertices of carboranes and boranes is noted. PMID- 16669677 TI - Concerted proton-electron transfer in the oxidation of hydrogen-bonded phenols. AB - Three phenols with pendant, hydrogen-bonded bases (HOAr-B) have been oxidized in MeCN with various one-electron oxidants. The bases are a primary amine ( CPh(2)NH(2)), an imidazole, and a pyridine. The product of chemical and quasi reversible electrochemical oxidations in each case is the phenoxyl radical in which the phenolic proton has transferred to the base, (*)OAr-BH(+), a proton coupled electron transfer (PCET) process. The redox potentials for these oxidations are lower than for other phenols, predominately from the driving force for proton movement. One-electron oxidation of the phenols occurs by a concerted proton-electron transfer (CPET) mechanism, based on thermochemical arguments, isotope effects, and DeltaDeltaG(++)/DeltaDeltaG degrees . The data rule out stepwise paths involving initial electron transfer to form the phenol radical cations [(*)(+)HOAr-B] or initial proton transfer to give the zwitterions [(-)OAr BH(+)]. The rate constant for heterogeneous electron transfer from HOAr-NH(2) to a platinum electrode has been derived from electrochemical measurements. For oxidations of HOAr-NH(2), the dependence of the solution rate constants on driving force, on temperature, and on the nature of the oxidant, and the correspondence between the homogeneous and heterogeneous rate constants, are all consistent with the application of adiabatic Marcus theory. The CPET reorganization energies, lambda = 23-56 kcal mol(-)(1), are large in comparison with those for electron transfer reactions of aromatic compounds. The reactions are not highly non-adiabatic, based on minimum values of H(rp) derived from the temperature dependence of the rate constants. These are among the first detailed analyses of CPET reactions where the proton and electron move to different sites. PMID- 16669679 TI - Minimization and optimization of designed beta-hairpin folds. AB - Minimized beta hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, DeltaG(U)(298) >or= +7 kJ/mol) and 12 residue (KTWNPATGKWTE, DeltaG(U)(298) = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-pi interaction between Lys(1) and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hepsilon3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the -TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small DeltaC(p) for unfolding (+420 J K(-)(1)/mol). In the case of Asx-Pro-Xaa Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH(2)) with the fold stability reduced to DeltaG(U)(278) = -0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also form the same hairpin-stabilizing W/W interaction. PMID- 16669680 TI - Hydrogen and deuterium atoms in octasilsesquioxanes: experimental and computational studies. AB - The rate of detrapping of atomic hydrogen from several octasilsesquioxanes is the same for dissolved and solid samples and is independent of the presence of other species such as free radicals or oxygen; varying the cage substituents leads to only minor differences in the activation parameters. Hydrogen atoms are found to be more strongly stabilized in homosubstituted octasilsesquioxanes compared with singly Ge-substituted cages. A kinetic isotope effect observed for the detrapping of H and D from MeT(8) is ascribed to the difference in the zero-point energies of the trapped atoms. There is a secondary H/D isotope effect in the temperature dependence of the (29)Si-superhyperfine splitting constants in the range 228-353 K. Cage relaxation has a substantial effect on the detrapping barrier but little influence on the intracage potential. Calculations using a rigid cage approximation give satisfactory agreement with zero-point parameters extracted from experimental data. Different model chemistries yield qualitatively different pictures of the dependence of the hyperfine coupling constant of the trapped H atom upon the detrapping coordinate. Within an isotropic approximation of the vibrational displacements, the B3LYP data give fairly close agreement with the experimental temperature dependence, subject to a shift of the absolute value related to known weaknesses of the method. For the Si(7)Ge cage, it is found that the transition state in which the H atom passes through a Ge-containing face is strongly favored, accounting for the larger detrapping rate parameters observed experimentally for this species. PMID- 16669681 TI - A thermodynamically stable nanophase material. AB - Nanoparticles are metastable relative to the equivalent bulk material due to the positive excess interfacial free energy (IFE). Previous studies have shown that, with increasing surface interaction strength, the IFE diminishes but remains positive. We describe an experimental multicomponent system in which a nanoscale ZnS material is thermodynamically favored and can be formed at the expense of bulk ZnS. In 17 M sodium hydroxide solution, at 230 degrees C, both 3 nm ZnS nanoparticles and bulk ZnS are transformed into sheetlike nanocrystals with a ZnS polytype structure. Our results are theoretically compatible with the concept of a negative IFE, although not with the assumption of constant interface composition. We clarify the meaning of an effective negative IFE and present the necessary conditions for strong chemical surface interactions to stabilize nanoscale inorganic materials relative to bulk matter. Our results show that synthesis methods employing thermodynamic controls can produce nanomaterials with novel morphology. PMID- 16669682 TI - Implications of protonation and substituent effects for C-O and O-P bond cleavage in phosphate monoesters. AB - A recent study of phosphate monoesters that broke down exclusively through C-O bond cleavage and whose reactivity was unaffected by protonation of the nonbridging oxygens (Byczynski et al. J. Am. Chem. Soc. 2003, 125, 12541) raised several questions about the reactivity of phosphate monoesters, R-O-P(i). Potential catalytic strategies, particularly with regard to selectively promoting C-O or O-P bond cleavage, were investigated computationally through simple alkyl and aryl phosphate monoesters. Both C-O and O-P bonds lengthened upon protonating the bridging oxygen, R-O(H(+))-P(i), and heterolytic bond dissociation energies, DeltaH(C)(-)(O) and DeltaH(O)(-)(P), decreased. Which bond will break depends on the protonation state of the phosphoryl moiety, P(i), and the identity of the organosubstituent, R. Protonating the bridging oxygen when the nonbridging oxygens were already protonated favored C-O cleavage, while protonating the bridging oxygen of the dianion form, R-O-PO(3)(2)(-), favored O-P cleavage. Alkyl R groups capable of forming stable cations were more prone to C-O bond cleavage, with tBu > iPr > F(2)iPr > Me. The lack of effect on the C-O cleavage rate from protonating nonbridging oxygens could arise from two precisely offsetting effects: Protonating nonbridging oxygens lengthens the C-O bond, making it more reactive, but also decreases the bridging oxygen proton affinity, making it less likely to be protonated and, therefore, less reactive. The lack of effect could also arise without bridging oxygen protonation if the ratio of rate constants with different protonation states precisely matched the ratio of acidity constants, K(a). Calculations used hybrid density functional theory (B3PW91/6 31++G) methods with a conductor-like polarizable continuum model (CPCM) of solvation. Calculations on Me-phosphate using MP2/aug-cc-pVDZ and PBE0/aug-cc pVDZ levels of theory, and variations on the solvation model, confirmed the reproducibility with different computational models. PMID- 16669683 TI - Cope elimination: elucidation of solvent effects from QM/MM simulations. AB - The Cope elimination reactions for threo- and erythro-N,N-dimethyl-3-phenyl-2 butylamine oxide have been investigated using QM/MM calculations in water, THF, and DMSO. The aprotic solvents provide up to million-fold rate accelerations. The effects of solvation on the reactants, transition structures, and rates of reaction are elucidated here using two-dimensional potentials of mean force (PMF) derived from free-energy perturbation calculations in Monte Carlo simulations (MC/FEP). The resultant free energies of activation in solution are in close agreement with experiment. Ab initio calculations at the MP2/6-311+G-(2d,p) level using the PCM continuum solvent model were also carried out; however, only the QM/MM methodology was able to reproduce the large rate increases in proceeding from water to the dipolar aprotic solvents. Solute-solvent interaction energies and radial distribution functions are also analyzed and show that poorer solvation of the reactant in the aprotic solvents is primarily responsible for the observed rate enhancements. It is found that the amine oxide oxygen is the acceptor of three hydrogen bonds from water molecules for the reactant but only one to two weaker ones at the transition state. The overall quantitative success of the computations supports the present QM/MM/MC approach, featuring PDDG/PM3 as the QM method. PMID- 16669684 TI - Evidence for two ferryl species in chloroperoxidase compound II. AB - Using a combination of density functional calculations and Mossbauer spectroscopy, we have examined chloroperoxidase compound II (CPO-II). The Mossbauer spectrum of CPO-II suggests the presence of two distinct ferryl species in an approximately 70:30 ratio. Density functional calculations and cryogenic reduction and annealing experiments allow us to assign the major species as an Fe(IV)OH intermediate. The Mossbauer parameters of the minor component are indicative of an authentic iron(IV)oxo species, but we have found the 70:30 ratio to be pH invariant. The unchanging ratio of component concentrations is in agreement with CPO-II's visible absorption spectrum, which shows no change over the enzyme's range of pH stability. PMID- 16669685 TI - Clay-fulleropyrrolidine nanocomposites. AB - In this work, we describe the insertion of a water-soluble bisadduct fulleropyrrolidine derivative into the interlayer space of three layered smectite clays. The composites were characterized by a combination of powder X-ray diffraction, transmission electron microscopy, X-ray photoemission and FTIR spectroscopies, and laser flash photolysis measurements. The experiments, complemented by computer simulations, give insight into the formation process, structural details, and properties of the fullerene/clay nanocomposites. The reported composite materials constitute a new hybrid system, where C(60) differs from its crystals or its solutions, and open new perspectives for the design and construction of novel C(60)-based organic/clay hybrid materials. PMID- 16669686 TI - Sensing metal ions with DNA building blocks: fluorescent pyridobenzimidazole nucleosides. AB - We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2 benzimidazole and 2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown heterocyclic precursors and Hoffer's chlorosugar, yielding alpha anomers as the chief products. X-ray crystal structures confirmed the geometry and showed that the pyridine and benzimidazole ring systems deviated from coplanarity in the solid state by 154 degrees and 140 degrees , respectively. In methanol compounds 1 and 2 had absorption maxima at 360 and 370 nm, respectively, and emission maxima at 494 and 539 nm. Experiments revealed varied fluorescence responses of the nucleosides to a panel of 17 monovalent, divalent, and trivalent metal ions in methanol. One or both of the nucleosides showed significant changes with 10 of the metal ions. The most pronounced spectral changes for ligand-nucleoside 1 included red shifts in fluorescence (Au(+), Au(3+)), strong quenching (Cu(2+), Ni(2+), Pt(2+)), and substantial enhancements in emission intensity coupled with red shifts (Ag(+), Cd(2+), Zn(2+)). The greatest spectral changes for ligand-nucleoside 2 included a red shift in fluorescence (Ag(+)), a blue shift (Cd(2+)), strong quenching (Pd(2+), Pt(2+)), and substantial enhancements in emission intensity coupled with a blue shift (Zn(2+)). The compounds could be readily incorporated into oligodeoxynucleotides, where an initial study revealed that they retained sensitivity to metal ions in aqueous solution and demonstrated possible cooperative sensing behavior with several ions. The two free nucleosides alone can act as differential sensors for multiple metal ions, and they are potentially useful monomers for contributing metal ion sensing capability to DNAs. PMID- 16669687 TI - Theoretical studies on farnesyl cation cyclization: pathways to pentalenene. AB - In this article, we describe studies, using quantum chemical computations, on possible polycyclization pathways of the farnesyl cation leading to the complex sesquiterpene pentalenene. Two distinct pathways to pentalenene with similar activation barriers are described, each differing from previous mechanistic proposals, and each involving unusual and unexpected intermediates. Direct deprotonation of intermediates on these pathways leads to sesquiterpene byproducts, such as humulene, protoilludene, and asteriscadiene, supporting the notion that a key function of pentalenene synthase, the enzyme that produces pentalenene in Nature, is to regulate the timing and location of proton removal. The implications of the computational results for experimental studies on pentalenene synthase are discussed. PMID- 16669688 TI - Photoinduced electron transfer in 2-tert-butyl-3-(anthracen-9-yl)-2,3 diazabicyclo[2.2.2]octane. AB - Intramolecular photoinduced electron transfer from a hydrazine unit to an aromatic group is studied by resonance Raman spectroscopy and electronic absorption spectroscopy. Substituted hydrazine functional groups have played an important role in studies of electron-transfer reactions, photoinduced intramolecular electron transfer, and of mixed valence. A prototypical compound, 2-tert-butyl-3-(anthracen-9-yl)-2,3-diazabicyclo[2.2.2]octane, that has the hydrazine-to-anthracene charge-transfer band in a region of the visible spectrum suitable for detailed resonance Raman spectroscopy is studied in detail. Excitation profiles are obtained, calculated quantitatively by using time dependent theoretical methods, and interpreted with the assistance of molecular orbital calculations. Excited-state distortions are calculated. The largest distortions occur on the hydrazine unit; the normal mode showing the largest distortion (659 cm(-1), calculated at 665 cm(-1)) involves an out-of-plane C-N-N C bend consistent with removing an electron from the N-N pi antibonding orbital. Anthracene ring-centered C-C stretches also are enhanced, consistent with populating an antibonding pi orbital centered on the ring. Excellent fits to all of the excitation profiles and to the absorption band are obtained using one set of excited-state potential surfaces. PMID- 16669689 TI - Coupling between protein and reaction dynamics in enzymatic processes: application of Grote-Hynes Theory to catechol O-methyltransferase. AB - The generalized Langevin equation (GLE)-based Grote-Hynes (GH) theory is used to calculate the transmission coefficients, kappa, for the methyl transfer from S adenosylmethionine to catecholate both in aqueous solution and in the catechol O methyltransferase active site. Values of kappa, which measures the deviation of the rate constants from the Transition State Theory (TST) predictions, are obtained by means of rare event molecular dynamics simulations. The results are 0.62 +/- 0.04 and 0.83 +/- 0.03 for the aqueous and enzymatic environments, respectively, while the Grote-Hynes predictions are 0.58 +/- 0.09 and 0.89 +/- 0.03, respectively. The Kramers theory estimates are much smaller, about 0.01 and 0.1, respectively. Thus, the enzymatic transmission coefficient is closer to TST predictions than the value obtained in solution. In addition, our results show that the enzymatic coefficient is also closer to its nonadiabatic (or frozen environment) limit than is the solution coefficient. These findings can be understood considering that, during the passage over the barrier top, there is a smaller coupling between the reactive system and the environment in the enzyme than in solution, as well as a smaller reorganization suffered by the enzyme. Analysis of the transition state friction kernel leads to the identification of some key vibrational modes governing the coupling between the two different environments and the reacting solute in the transition state region and insights on their relevance for the reaction dynamics' influence on the transmission coefficient. PMID- 16669690 TI - X-Shaped electro-optic chromophore with remarkably blue-shifted optical absorption. Synthesis, characterization, linear/nonlinear optical properties, self-assembly, and thin film microstructural characteristics. AB - A novel type of "X-shaped" two-dimensional electro-optic (EO) chromophore with extended conjugation has been synthesized and characterized. This chromophore is found to exhibit a remarkably blue-shifted optical maximum (357 nm in CH(2)Cl(2)) while maintaining a very large first hyperpolarizability (beta). Hyper-Rayleigh Scattering (HRS) measurements at 800 nm provide a beta(zzz) value of 1840 x 10( 30) esu. Self-assembled thin films of this chromophore were fabricated via a layer-by-layer chemisorptive siloxane-based approach. The chromophoric multilayers have been characterized by transmission optical spectroscopy, advancing contact angle measurements, synchrotron X-ray reflectivity, atomic force microscopy, and angle-dependent polarized second harmonic generation spectroscopy. The self-assembled chromophoric films exhibit a dramatically blue shifted optical maximum (325 nm) while maintaining a large EO response (chi(2)(333) approximately 232 pm/V at 1064 nm; r(33) approximately 45 pm/V at 1310 nm). This work demonstrates an attractive approach to developing EO materials offering improved nonlinearity-transparency trade-offs. PMID- 16669691 TI - Mechanism-based cofactor derivatization of a copper amine oxidase by a branched primary amine recruits the oxidase activity of the enzyme to turn inactivator into substrate. AB - The copper amine oxidases (CAOs) have evolved to catalyze oxidative deamination of unbranchedprimary amines to aldehydes. We report that a branched primary amine bearing an aromatization-prone moiety, ethyl 4-amino-4,5-dihydrothiophene-2 carboxylate (1), is recognized enantioselectively (S >> R) by bovine plasma amine oxidase (BPAO) both as a temporary inactivator and as a substrate. Substrate activity results from an O(2)-dependent turnover of the covalently modified enzyme, with release of 4-aminothiophene-2-carboxylate (2) as ultimate product. Interaction of (S)-1 with BPAO occurs within the enzyme active site with a dissociation constant of 0.76 microM. Evidence from kinetic and spectroscopic studies, and HPLC analysis of stoichiometric reactions of BPAO with (S)-1, combined with a model study using a quinone cofactor mimic, establishes that the enzyme metabolizes 1 according to a transamination mechanism. Following the initial isomerization of substrate Schiff base to product Schiff base, a facile aromatization of the latter results in a metastable N-aryl derivative of the reduced cofactor aminoresorcinol, which is catalytically inactive. The latter derivative is then slowly oxidized by O(2), apparently facilitated partially by the active-site Cu(II), to form a quinonimine of the native cofactor that releases 2 upon hydrolysis or transimination with substrate amine. Preferential metabolism of (S)-1 is consistent with the preferential removal of the pro-Salpha proton in metabolism of benzylamine by BPAO. This study represents the first report of product identification in metabolism of a branched primary amine by a copper amine oxidase and suggests a novel type of reversible mechanism-based (covalent) inhibition where inhibition lifetime can be fine-tuned independently of inhibition potency. PMID- 16669692 TI - Computational study of carbon atom (3P and 1D) reaction with CH2O. Theoretical evaluation of 1B1 methylene production by C (1D). AB - Singlet and triplet free energy surfaces for the reactions of C atom ((3)P and (1)D) with CH(2)O are studied computationally to evaluate the excited singlet ((1)B(1)) methylene formation from deoxygenation of CH(2)O by C ((1)D) atom as suggested by Shevlin et al. Carbon atoms can react by addition to the oxygen lone pair or to the C=O double bond on both the triplet and singlet surfaces. Triplet C ((3)P) atoms will deoxygenate to give CO plus CH(2) ((3)B(1)) as the major products, while singlet C ((1)D) reactions will form ketene and CO plus CH(2) ((1)A(1)). No definitive evidence of the formation of excited singlet ((1)B(1)) methylene was found on the singlet free energy surface. A conical intersection between the (1)A' and (1)A' ' surfaces located near an exit channel may play a role in product formation. The suggested (1)B(1) state of methylene may form via the (1)A' ' surface only if dynamic effects are important. In an effort to interpret experimental observation of products trapped by (Z)-2-butene, formation of cis- and trans-1,2-dimethylcyclopropane is studied computationally. The results suggests that "hot" ketene may react with (Z)-2-butene nonstereospecifically. PMID- 16669693 TI - Structural analysis of alanine tripeptide with antiparallel and parallel beta sheet structures in relation to the analysis of mixed beta-sheet structures in Samia cynthia ricini silk protein fiber using solid-state NMR spectroscopy. AB - The structural analysis of natural protein fibers with mixed parallel and antiparallel beta-sheet structures by solid-state NMR is reported. To obtain NMR parameters that can characterize these beta-sheet structures, (13)C solid-state NMR experiments were performed on two alanine tripeptide samples: one with 100% parallel beta-sheet structure and the other with 100% antiparallel beta-sheet structure. All (13)C resonances of the tripeptides could be assigned by a comparison of the methyl (13)C resonances of Ala(3) with different [3-(13)C]Ala labeling schemes and also by a series of RFDR (radio frequency driven recoupling) spectra observed by changing mixing times. Two (13)C resonances observed for each Ala residue could be assigned to two nonequivalent molecules per unit cell. Differences in the (13)C chemical shifts and (13)C spin-lattice relaxation times (T(1)) were observed between the two beta-sheet structures. Especially, about 3 times longer T(1) values were obtained for parallel beta-sheet structure as compared to those of antiparallel beta-sheet structure, which could be explicable by the difference in the hydrogen-bond networks of both structures. This very large difference in T(1) becomes a good measure to differentiate between parallel or antiparallel beta-sheet structures. These differences in the NMR parameters found for the tripeptides may be applied to assign the parallel and antiparallel beta-sheet (13)C resonances in the asymmetric and broad methyl spectra of [3 (13)C]Ala silk protein fiber of a wild silkworm, Samia cynthia ricini. PMID- 16669694 TI - Water soluble cryptophanes showing unprecedented affinity for xenon: candidates as NMR-based biosensors. AB - Cryptophanes bearing OCH(2)COOH groups in place of the methoxy groups represent a new class of xenon-carrier molecules soluble in water at biological pH. By using (1)H and (129)Xe NMR (thermally- and laser-polarized dissolved gas), the structural and dynamical behaviors of these host molecules as well as their interaction with xenon are studied. They are shown to exist in aqueous solution under different conformations in very slow exchange. A saddle form present for one of these conformations could explain the (1)H NMR spectra. Whereas the cryptophanes in such a conformation are unable to complex xenon, unprecedented high binding constants are found for cryptophanes in the other canonical crown crown conformation. These host molecules could therefore be valuable candidates for biosensing using (129)Xe MRI. PMID- 16669695 TI - High hydride count rhodium octahedra, [Rh6(PR3)6H12][BArF4]2: synthesis, structures, and reversible hydrogen uptake under mild conditions. AB - A new class of transition metal cluster is described, [Rh(6)(PR(3))(6)H(12)][BAr(F)(4)](2) (R = (i)Pr (1a), Cy (2a); BAr(F)(4) = [B{C(6)H(3)(CF(3))(2)}(4)](-)). These clusters are unique in that they have structures exactly like those of early transition metal clusters with edge bridging pi-donor ligands rather than the structures expected for late transition metal clusters with pi-acceptor ligands. The solid-state structures of 1a and 2a have been determined, and the 12 hydride ligands bridge each Rh-Rh edge of a regular octahedron. Pulsed gradient spin-echo NMR experiments show that the clusters remain intact in solution, having calculated hydrodynamic radii of 9.5(3) A for 1a and 10.7(2) A for 2a, and the formulation of 1a and 2a was unambiguously confirmed by ESI mass spectrometry. Both 1a and 2a take up two molecules of H(2) to afford the cluster species [Rh(6)(P(i)Pr(3))(6)H(16)][BAr(F)(4)](2) (1b) and [Rh(6)(PCy(3))(6)H(16)][BAr(F)(4)](2) (2b), respectively, as characterized by NMR spectroscopy, ESI-MS, and, for 2b, X-ray crystallography using the [1-H CB(11)Me(11)](-) salt. The hydride ligands were not located by X-ray crystallography, but (1)H NMR spectroscopy showed a 15:1 ratio of hydride ligands, suggesting an interstitial hydride ligand. Addition of H(2) is reversible: placing 1b and 2b under vacuum regenerates 1a and 2a. DFT calculations on [Rh(6)(PH(3))(6)H(x)()](2+) (x = 12, 16) support the structural assignments and also show a molecular orbital structure that has 20 orbitals involved with cluster bonding. Cluster formation has been monitored by (31)P{(1)H} and (1)H NMR spectroscopy, and mechanisms involving heterolytic H(2) cleavage and elimination of [HP(i)Pr(3)](+) or the formation of trimetallic intermediates are discussed. PMID- 16669698 TI - Climate, deer, rodents, and acorns as determinants of variation in lyme-disease risk. AB - Risk of human exposure to vector-borne zoonotic pathogens is a function of the abundance and infection prevalence of vectors. We assessed the determinants of Lyme-disease risk (density and Borrelia burgdorferi-infection prevalence of nymphal Ixodes scapularis ticks) over 13 y on several field plots within eastern deciduous forests in the epicenter of US Lyme disease (Dutchess County, New York). We used a model comparison approach to simultaneously test the importance of ambient growing-season temperature, precipitation, two indices of deer (Odocoileus virginianus) abundance, and densities of white-footed mice (Peromyscus leucopus), eastern chipmunks (Tamias striatus), and acorns (Quercus spp.), in both simple and multiple regression models, in predicting entomological risk. Indices of deer abundance had no predictive power, and precipitation in the current year and temperature in the prior year had only weak effects on entomological risk. The strongest predictors of a current year's risk were the prior year's abundance of mice and chipmunks and abundance of acorns 2 y previously. In no case did inclusion of deer or climate variables improve the predictive power of models based on rodents, acorns, or both. We conclude that interannual variation in entomological risk of exposure to Lyme disease is correlated positively with prior abundance of key hosts for the immature stages of the tick vector and with critical food resources for those hosts. PMID- 16669699 TI - Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells. AB - Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER. PMID- 16669700 TI - How a lateralized brain supports symmetrical bimanual tasks. AB - A large repertoire of natural object manipulation tasks require precisely coupled symmetrical opposing forces by both hands on a single object. We asked how the lateralized brain handles this basic problem of spatial and temporal coordination. We show that the brain consistently appoints one of the hands as prime actor while the other assists, but the choice of acting hand is flexible. When study participants control a cursor by manipulating a tool held freely between the hands, the left hand becomes prime actor if the cursor moves directionally with the left-hand forces, whereas the right hand primarily acts if it moves with the opposing right-hand forces. In neurophysiological (electromyography, transcranial magnetic brain stimulation) and functional magnetic resonance brain imaging experiments we demonstrate that changes in hand assignment parallels a midline shift of lateralized activity in distal hand muscles, corticospinal pathways, and primary sensorimotor and cerebellar cortical areas. We conclude that the two hands can readily exchange roles as dominant actor in bimanual tasks. Spatial relationships between hand forces and goal motions determine hand assignments rather than habitual handedness. Finally, flexible role assignment of the hands is manifest at multiple levels of the motor system, from cortical regions all the way down to particular muscles. PMID- 16669701 TI - X chromosomes alternate between two states prior to random X-inactivation. AB - Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells. PMID- 16669702 TI - A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcgamma receptor-mediated phagocytosis. AB - Fcgamma receptor (FcgammaR)-mediated phagocytosis of IgG-coated particles is regulated by 3'-phosphoinositides (3'PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcgammaR mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcgammaR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome. PMID- 16669703 TI - Qualitative and quantitative analysis of phosphorylated ATM foci induced by low dose ionizing radiation. AB - We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose. PMID- 16669704 TI - Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts. AB - In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities. PMID- 16669705 TI - Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks. AB - One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness. PMID- 16669706 TI - Synergistic effects of radiation and beta-lapachone in DU-145 human prostate cancer cells in vitro. AB - It has been reported that beta-lapachone (beta-lap), a bioreductive anti-cancer drug, synergistically interacts with ionizing radiation and that the sensitivity of cells to beta-lap is closely related to the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here we report the results of our studies of mechanisms underlying the synergistic interaction of beta-lap and radiation in killing cancer cells using the DU-145 human prostate cancer cell line. The clonogenic cell death caused by the combination of radiation and beta-lap was synergistic when beta-lap was administered 0-10 h after irradiation but not when it was given before irradiation. The expression and activity of NQO1 increased significantly and remained elevated for longer than 12 h after 4 Gy irradiation, suggesting that the long-lasting elevation of NQO1 sensitized the cells to beta-lap. Studies with split-dose irradiation demonstrated that beta-lap given immediately after irradiation effectively inhibited sublethal radiation damage (SLD) repair. Taken together, these results lead us to conclude that the synergistic interaction between beta-lap and radiation in killing cells is the result of two distinct mechanisms: First, radiation sensitizes cells to beta-lap by up-regulating NQO1, and second, beta-lap sensitizes cells to radiation by inhibiting SLD repair. The combination of beta-lap and radiotherapy is potentially promising modality for the treatment of cancer in humans. PMID- 16669707 TI - Effects of XeCl UV308 nm laser radiation on survival and mutability of recA proficient and recA-defective Escherichia coli strains. AB - recA1, recA13 and recA56 are considered null alleles of the Escherichia coli recA gene because they were shown to have essentially no activity in vivo. In this study, we used strains harboring the recA null alleles and their recA-proficient congenic counterpart to assess the lethal and the mutagenic effects elicited by near-UV(308 nm) coherent radiation generated by a XeCl excimer laser. We compared these effects with those produced by a conventional far-UV(254 nm) germicidal lamp. Compared to the germicidal lamp, the excimer laser was able to better discriminate the different recA-defective strains on the basis of their UV radiation sensitivity, which was progressively higher in the strains with the alleles in the order recA1, recA56 and recA13. This finding was consistent with previous data on residual biochemical activities of the respective mutated RecA proteins in vitro. The discrepancy between the results obtained with the lamp and laser irradiation suggested that the biological response to the two radiations involves distinct mechanisms. This hypothesis was supported by the evidence that exposure to near-UV(308 nm) radiation induced mutagenesis in recA-defective strains at an extent considerably greater than in recA-proficient strains. In contrast, far-UV(254 nm)-radiation-induced mutagenesis was reported to be largely dependent on a functional recA allele. PMID- 16669708 TI - Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice. AB - The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to gamma rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80 degrees C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase-luminol-p iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against gamma-radiation-induced death. PMID- 16669709 TI - Angelica sinensis down-regulates hydroxyproline and Tgfb1 and provides protection in mice with radiation-induced pulmonary fibrosis. AB - Pulmonary fibrosis is a common delayed side effect of radiation therapy, and it has a poor prognosis. Tgfb1 is a potent chemoattractant for fibroblasts and stimulates the production of collagen, the protein that contains hydroxyproline. Since collagen is by far the most abundant protein in the lung, comprising 60-70% of the tissue mass, analysis of the hydroxyproline content in lung tissues provides a reliable quantitative index for pulmonary fibrosis. Thus hydroxyproline and Tgfb1 may be involved in the development of fibrosis. In this study, we investigated radiation-induced pulmonary fibrosis in a mouse model. C57BL/6 mice were assigned into four groups: no treatment, treated with Angelica sinensis treated only, X-irradiated only (a single fraction of 12 Gy to the thorax), and Angelica sinensis treatment plus radiation. We assayed expression of hydroxyproline and the mRNA and protein of Tgfb1 in the four groups. We found that Angelica sinensis down-regulated the production of Tgfb1 and hydroxyproline in mice with radiation-induced pulmonary fibrosis. This study has demonstrated for the first time that Angelica sinensis inhibits the progress of radiation induced pulmonary fibrosis, possibly by down-regulating the expression of the proinflammatory cytokine Tgfb1. These data suggest that Angelica sinensis may be useful in preventing and/or treating radiation-induced pulmonary fibrosis in the clinic. PMID- 16669710 TI - Ret, Abl1 (cAbl) and Trp53 gene fragmentations in comet-FISH assay act as in vivo biomarkers of radiation exposure in C57BL/6 and CBA/J mice. AB - The International Commission on Radiation Protection (ICRP) has lowered the dose limits for workers and for the general public exposed to ionizing radiation. Consequently, a reliable dosimetric method for monitoring possible radiation induced damage is of great importance in radioprotection. The counting of dicentric chromosomal aberrations and of micronuclei in peripheral blood lymphocytes is unreliable when it is applied to in vivo biopsies and for low-dose exposures. Single-cell gel electrophoresis (SCGE or comet assay), although sensitive and rapid, shows high variability when applied in vivo, probably due to prompt repair of the DNA breaks and confounding environmental factors. In this paper, we describe specific in situ hybridization of Ret, Abl1 (cAbl), and Trp53 gene fragmentations on SCGE slides (comet-FISH assay) in peripheral blood cells from C57BL/6 and CBA/J mice as an indicator of radiation-induced DNA damage. The results obtained from four mice for each experimental point (0, 1, 2 and 4 Gy of X rays) discriminated in a statistically significant way the effects of all doses when fragmentations were analyzed for the Ret, Ab1 and Trp53 genes. SCGE alone, when applied to the same specimens, produced no significant results because of interindividual and experimental variability. PMID- 16669711 TI - Organ-specific gene expressions in C57BL/6 mice after exposure to low-dose radiation. AB - The possibility that radiation-induced alterations in gene expression are tissue specific and are related to apoptosis was examined using samples from brain, heart, lung, spleen and intestine from female C57BL6 mice after exposure to 0.2 Gy radiation. Apoptosis was the highest in spleen and intestine, moderate in lung, and absent in brain and heart. However, the mRNA expression of Trp53 and Cdkn1a (p21) after irradiation was not different among the organ types, and immunohistochemistry revealed that all the organs expressed these two proteins after irradiation. When expression patterns of 23 genes in the organs were examined by RT-PCR, neogenine, Apo1, nuclease sensitive element binding protein 1, syntaxin, cyclin G1, hNOP56, paraoxonase and glutathione peroxidase were overexpressed after irradiation in all the organs sampled, suggesting them as universal exposure markers for low-dose radiation. Sialyltransferase may be a candidate for radiation detection in spleen and intestine, which are radiosensitive organs. Because Sod1 (Cu/ZnSOD) and alphaB crystalline were expressed only in spleen, and protein tyrosine kinase and platelet membrane glycoprotein lib were expressed in both spleen and lung, these genes may also be potential markers for detection of radiation exposure, especially low-dose radiation, in these tissues. These data suggested possible tissue-specific markers of low-dose radiation exposure and suggested potential novel genetic modifiers of radiation response. PMID- 16669712 TI - Alterations in gene expression in rat skin exposed to 56Fe ions and dietary vitamin A acetate. AB - The purpose of the present work was to examine gene expression patterns in rat skin exposed to a beam of (56)Fe ions, a surrogate for the high-energy, heavy-ion galactic radiation background, as a basis for obtaining a better understanding of the possible mechanism(s) behind the radioprotective activity of vitamin A. A 2 x 4-cm rectangle of dorsal rat skin was exposed to 1.01 GeV/nucleon (56)Fe ions generated by the Alternating Gradient Synchrotron at Brookhaven National Laboratory. Gene expression patterns were monitored in either the presence or absence of a 250-ppm dietary supplement of vitamin A acetate in powdered lab chow. Although vitamin A and other retinoids show anti-carcinogenic activity in several animal models, the underlying changes in gene expression have not been examined extensively. At either 1 or 7 day after irradiation, a 1-cm square of irradiated and control rat skin was excised and analyzed using the Affymetrix rat microarray (RG_U34A) system. Microarray responses were displayed and processed by GeneSpring 7.0 and GOTree software. At 1 day after 3 Gy of (56)Fe-ion irradiation, the expression of 110 genes was significantly up-regulated (P < = 0.05) in comparison to levels in control rat skin, while no genes were altered by the vitamin A acetate supplement alone. Combined with (56)Fe-ion radiation, the vitamin A acetate supplement blocked the expression of 88 (80%) of the 110 genes and eliminated 16 of 18 gene categories that were significantly altered (all increased) by the (56)Fe-ion radiation. Categories with large numbers of genes eliminated by the retinoid included response to stress, 33 genes; response to biotic stimulus, 38 genes; signal transduction, 35 genes; and regulation of cellular/physiological process, 40 genes. Even for immune response and response to biotic stimulus, the only two categories that remained significantly altered in the presence of the vitamin, the combined number of altered genes was reduced from 74 to 13. No significant alterations in gene expression were found at 7 days relative to the numbers in controls. The results indicate that at 1 day dietary vitamin A acetate strongly interfered with (56)Fe-ion-induced gene expression within the broad categories of stimulus- and stress-related genes, implying that the latter gene categories likely play a role in the radioprotective action of the vitamin. PMID- 16669713 TI - Depletion of neural precursor cells after local brain irradiation is due to radiation dose to the parenchyma, not the vasculature. AB - The underlying mechanisms associated with radiation-induced cognitive impairments remain elusive but may involve changes in hippocampal neural precursor cells. Proliferating neural precursor cells have been shown to be extremely sensitive to X rays, either from damage to the cells themselves and/or through microenvironmental factors, including the anatomical relationship with the microvasculature, which is altered by radiation. The neutron capture reaction in boron was used to determine whether the sensitivity of neural precursor cells was dominated by direct radiation effects or was mediated through changes in the microvasculature. Young adult rats were irradiated with X rays, neutrons only, or neutrons plus either mercapto-undecahydro-dodecaborane (BSH) or p-dihydroxyboryl phenylalanine (BPA). BSH remains inside cerebral vessels, thereby limiting the neutron capture intravascularly; BPA readily passes into the parenchyma. One month after irradiation, cell proliferation and numbers of immature neurons were determined using immunohistochemistry. Results showed that (1) neural precursor cells and their progeny were decreased in a dose-dependent manner by mixed high- and low-LET radiation, and (2) selective irradiation of the microvasculature resulted in less loss of neural precursor cells than when the radiation dose was delivered uniformly to the parenchyma. This information, and in particular the approach of selectively irradiating the vasculature, may be useful in developing radioprotective compounds for use during therapeutic irradiation. PMID- 16669714 TI - FISH chromosome analysis of plutonium workers from the Sellafield nuclear facility. AB - Chromosome analysis using a single-color FISH technique to paint three pairs of chromosomes was undertaken on a group of 46 retired plutonium workers with assessed bone marrow doses >60 mSv, 34 of whom were categorized as having robust dosimetry and 12 for whom internal doses were considered less reliable. Comparisons were made with a group of 34 workers with negligible radiation exposure and a group of 34 workers with similar recorded external gamma-ray doses but negligible internal dose. The simple translocation frequency of 17.65 +/- 1.96 x 10(-3) per genome equivalent for the 34 plutonium workers with robust dosimetry was significantly increased in comparison with that of 10.06 +/- 1.16 x 10(-3) per genome equivalent for the unirradiated control group (P = <0.001) and that of 13.55 +/- 1.43 x 10(-3) per genome equivalent for the group with similar external gamma-ray exposure (P = 0.012). Thus, although in vitro studies have indicated that the majority of alpha-particle-irradiated cells suffer complex non transmissible chromosome damage, in vivo a significant proportion survive with simple exchanges that can be passed on to descendant cells. In contrast, the three groups demonstrated no significant differences in stable complex aberrations. No evidence of an increase in dicentrics or unstable complex aberrations associated with plutonium exposure was observed, and it can therefore be assumed that there is little, if any, ongoing irradiation of mature lymphocytes. The translocation frequency of 12.08 +/- 1.92 x 10(-3) per genome equivalent for the group of 12 plutonium workers with less reliable internal dosimetry could adequately be accounted for by age and external dose and indicates that the internal bone marrow doses are likely to have been overestimated. Cytogenetic analysis can therefore make a valuable contribution to the validation of internal doses from plutonium deposition. PMID- 16669715 TI - Atypical antipsychotics in the treatment of mania: a meta-analysis of randomized, placebo-controlled trials. AB - BACKGROUND: Randomized, controlled trials have demonstrated efficacy for atypical antipsychotics in the treatment of mania in bipolar disorder, either as monotherapy or adjunctive treatment. However, there are no published comparisons of individual atypical antipsychotics for mania. DATA SOURCES AND STUDY SELECTION: We conducted a systematic review and meta-analysis of randomized, placebo-controlled monotherapy and adjunctive therapy trials of atypical antipsychotics for acute bipolar mania. Studies published through 2004 were identified using searches of PubMed/MEDLINE with the search terms mania, placebo, and each of the atypical antipsychotics, limited to randomized, controlled clinical trials; review of abstracts from the 2003 meetings of the American College of Neuropsychiatry, American Psychiatric Association, and International Conference on Bipolar Disorder; and consultations with study investigators and representatives of pharmaceutical companies that market atypical antipsychotics. DATA EXTRACTION: Analyses were performed on the changes in Young Mania Rating Scale or Mania Rating Scale total scores from baseline to endpoint, using last observation carried forward and computing the difference in change scores between each drug and its corresponding placebo arm. A random-effects model with fixed drug effects was used to combine the studies and make comparisons of the antipsychotics to each other and to placebo. DATA SYNTHESIS: Data from 12 placebo controlled monotherapy and 6 placebo-controlled adjunctive therapy trials involving a total of 4304 subjects (including 1750 placebo-treated subjects) with bipolar mania were obtained. Aripiprazole, olanzapine, quetiapine, risperidone, and ziprasidone all demonstrated significant efficacy in monotherapy (i.e., all confidence intervals exclude zero). However, after adjusting for multiple comparisons, pairwise comparisons of individual effects identified no significant differences in efficacy among antipsychotics. Magnitude of improvement was similar whether the antipsychotic was utilized as monotherapy or adjunctive therapy. CONCLUSIONS: The 5 newer atypical antipsychotics were all superior to placebo in the treatment of bipolar mania. For monotherapy and add-on therapy, cross-trial comparisons suggest that differences in acute efficacy between the drugs, if any, are likely to be small. PMID- 16669716 TI - Suicide rates in relation to health care access in the United States: an ecological study. AB - OBJECTIVE: We tested the hypothesis that suicide rates in the United States are associated with indicators of access to health care services. METHOD: With an ecological study design, we compared age-adjusted suicide rates for men and women with demographic, socioeconomic, and other indices of access to health care, by state (N = 51, including the District of Columbia). The most recently available information from the National Statistics Reports at the U.S. Census Bureau, the U.S. Centers for Disease Control and Prevention National Center for Health Statistics, and the American Board of Medical Specialties was used. Data on suicide are from 2001; other measures were matched for the closest available year, except that state-based data on psychiatrists and physicians are from 2004. RESULTS: Positive bivariate associations with state suicide rates (all p < or = .005) are ranked as follows: male sex, Native American ethnicity, and higher proportion of uninsured residents. Negative bivariate associations (all p < or = .002) are ranked as follows: higher population density, higher annual per capita income, higher population density of psychiatrists, higher population density of physicians, higher federal aid for mental health, and higher proportion of African Americans. All factors were associated with state suicide rates in expected directions. In multivariate models of associations between suicide rates and indices of access to health care, the state rate of federal aid for mental health was the strongest indicator, followed by the rate of uninsured persons and population density of psychiatrists and physicians and by population density. DISCUSSION: Such aggregate analyses cannot specify risk indices for individual persons. Nevertheless, the methods employed detected several factors with well established associations with suicide. They also yielded strong correlations of state-based suicide rates with proposed indicators of access to health care. The findings support the view that clinical intervention is a crucial element in the prevention of suicide. PMID- 16669717 TI - Functional impairments in adults with self-reports of diagnosed ADHD: A controlled study of 1001 adults in the community. AB - OBJECTIVE: The objective of this study was to evaluate functional impairments in a nonreferred sample of adults identifying themselves as having been diagnosed with attention-deficit/hyperactivity disorder (ADHD) by a clinician in their community. METHOD: We completed a survey in April and May 2003 of a community sample of 500 adults who reported having received a diagnosis of ADHD in the community and 501 gender- and age-matched comparisons from a national sample representative of the U.S. population. RESULTS: Adults with self-reports of diagnosed ADHD in the community were significantly less likely to have graduated high school (83% vs. 93% of controls; p < or = .001) or obtain a college degree (19% vs. 26%; p < .01), were less likely to be currently employed (52% vs. 72%; p < or = .001), and had significantly more mean job changes over 10 years (5.4 vs. 3.4 jobs; p < or = .001). They also were significantly more likely to have been arrested (37% vs. 18% of controls; p < or = .001) or divorced (28% vs. 15%; p < or = .001) and were significantly less satisfied (p < or = .001) with their family, social, and professional lives. CONCLUSION: Adults who reported having received a diagnosis of ADHD in the community had significant impairment in multiple domains of functioning compared with age- and gender-matched controls without this diagnosis, highly consistent with findings derived from carefully diagnosed referred samples. PMID- 16669718 TI - Neurologic soft signs in borderline personality disorder. AB - OBJECTIVE: Borderline personality disorder is a disabling and dramatic psychiatric condition. To date, its pathophysiology remains unclear. Scientific evidence seems to have found underlying, nonfocal, central nervous system dysfunction in borderline personality disorder. Neurologic soft signs are anomalies only evidenced by specific motor, sensory, or integrative testing when no other sign of a neurologic lesion is present. Neurologic soft signs have been proposed to be nonfocal in origin and to reflect central nervous system failure. The assessment of neurologic soft signs now appears reliable and stable. Assuming that neurologic soft signs reflect nonfocal central nervous system dysfunction, we hypothesized that patients with borderline personality disorder should have an increased frequency of neurologic soft signs, therefore enhancing the possibility of the existence in borderline personality disorder of a nonlocalized brain dysfunction. METHOD: To test this hypothesis, we compared 29 neurologic soft signs in 20 drug-free patients with DSM-III-R borderline personality disorder and 20 controls, using an examination adapted from the literature on neurologic soft signs. The study was conducted from February 1991 to March 1993. RESULTS: Thirteen neurologic soft signs were significantly more frequent in the borderline group. Patients with borderline personality disorder showed more left side, right side, and total neurologic soft signs than controls (p = .0001). All patients in the borderline group exhibited at least 1 neurologic soft sign, while only 7 controls did (p = .0001). CONCLUSION: Our hypothesis was confirmed. These results add evidence to the possibility of the existence of a nonfocal central nervous system failure in borderline personality disorder. PMID- 16669719 TI - Weight management program for treatment-emergent weight gain in olanzapine treated patients with schizophrenia or schizoaffective disorder: A 12-week randomized controlled clinical trial. AB - BACKGROUND: The main objective was to assess the efficacy of a weight management program designed for outpatients taking olanzapine for schizophrenia or schizoaffective disorder and to compare these patients with a randomized control group. The effects of the weight management program were also assessed with regard to safety and quality of life. METHOD: Forty-eight patients were enrolled in a 12-week, randomized, multicenter weight management study. Thirty-three patients were randomly allocated to an intervention group in which they received olanzapine within a weight management program. Fifteen patients were allocated to a control group in which they were given olanzapine treatment as usual outpatients. Weight, body mass index (BMI), and measurements of safety and quality of life were evaluated. The study was conducted from January 7, 2003, to September 16, 2003. RESULTS: Thirty-six patients (75%) completed this study. We found significant differences in weight (-3.94 +/- 3.63 kg vs. -1.48 +/- 1.88 kg, p = .006) and BMI (-1.50 +/- 1.34 vs. -0.59 +/- 0.73, p = .007) change from baseline to endpoint between the intervention and control groups, respectively. Significant differences in weight reduction were initially observed at week 8 (p = .040). No significant differences were found with regard to the safety outcomes. When the ratio of low-density lipoproteins to high-density lipoproteins was calculated, change from baseline was greater in the intervention group than the control group (-0.19 vs. -0.04), but the difference was not statistically significant (p = .556). After the completion of the weight management program, there was a trend toward statistical difference in the physical health score changes between the weight management and control groups (1.12 in the intervention group vs. -0.93 in the control group, p = .067). CONCLUSION: The weight management program was effective in terms of weight reduction in patients with schizophrenia or schizoaffective disorder taking olanzapine and was also found to be safe in terms of psychiatric symptoms, vital signs, and laboratory data. In addition, such a weight management program might improve quality of life in patients with schizophrenia or schizoaffective disorder with respect to their physical well-being. PMID- 16669720 TI - A systematic review of modafinil: Potential clinical uses and mechanisms of action. AB - BACKGROUND: Modafinil is a novel wake-promoting agent that has U.S. Food and Drug Administration approval for narcolepsy and shift work sleep disorder and as adjunctive treatment of obstructive sleep apnea/hypopnea syndrome. Modafinil has a novel mechanism and is theorized to work in a localized manner, utilizing hypocretin, histamine, epinephrine, gamma-aminobutyric acid, and glutamate. It is a well-tolerated medication with low propensity for abuse and is frequently used for off-label indications. The objective of this study was to systematically review the available evidence supporting the clinical use of modafinil. DATA SOURCES: The search term modafinil OR Provigil was searched on PubMed. Selected articles were mined for further potential sources of data. Abstracts from major scientific conferences were reviewed. Lastly, the manufacturer of modafinil in the United States was asked to provide all publications, abstracts, and unpublished data regarding studies of modafinil. DATA SYNTHESIS: There have been 33 double-blind, placebo-controlled trials of modafinil. Additionally, numerous smaller studies have been performed, and case reports of modafinil's use abound in the literature. CONCLUSIONS: Modafinil is a promising drug with a large potential for many uses in psychiatry and general medicine. Treating daytime sleepiness is complex, and determining the precise nature of the sleep disorder is vital. Modafinil may be an effective agent in many sleep conditions. To date, the strongest evidence among off-label uses exists for the use of modafinil in attention-deficit disorder, postanesthetic sedation, and cocaine dependence and withdrawal and as an adjunct to antidepressants for depression. PMID- 16669721 TI - Characterizing impaired driving in adults with attention-deficit/hyperactivity disorder: A controlled study. AB - OBJECTIVE: We sought to confirm previously documented findings that individuals with attention-deficit/hyperactivity disorder (ADHD) demonstrate impaired driving behavior when compared with controls. METHOD: Subjects were adults with (N = 26) and without (N = 23) DSM-IV ADHD ascertained through clinical referrals to an adult ADHD program and through advertisements in the local media. Driving behavior was assessed using the Manchester Driving Behavior Questionnaire (DBQ) and 10 questions from a driving history questionnaire. Neuropsychological testing and structured interviews were also administered to all subjects. RESULTS: Substantially more ADHD subjects had been in an accident on the highway (35% vs. 9%, p = .03) or had been rear-ended (50% vs. 17%, p = .02) compared with controls. Analysis of the DBQ findings showed that ADHD subjects had significantly higher mean +/- SD scores than control subjects on the total DBQ (34.1 +/- 15.2 vs. 18.0 +/- 8.6, p < .001) and in all 3 subscales of the DBQ: errors (9.3 +/- 5.4 vs. 4.6 +/- 3.5, p < .001), lapses (12.4 +/- 6.2 vs. 6.1 +/- 3.5, p < .001), and violations (12.4 +/- 5.2 vs. 7.4 +/- 4.1, p < .001). Using the score that separated ADHD from control drivers on the DBQ as a cutoff, ADHD drivers at high risk for poor driving outcomes had more severe rates of comorbidity and exhibited more impaired scores on neuropsychological testing. CONCLUSIONS: Our results confirm and extend previous work documenting impaired driving behavior in subjects with ADHD. Results also suggest that ADHD individuals at high risk for poor driving behavior might be distinguishable from other ADHD individuals on DBQ scores, neuropsychological deficits, and patterns of comorbidities. PMID- 16669722 TI - Metabolic syndrome and the risk of coronary heart disease in 367 patients treated with second-generation antipsychotic drugs. AB - OBJECTIVE: To examine the relationship between presence of metabolic syndrome and the risk of coronary heart disease (CHD) events (angina pectoris, myocardial infarction, and sudden cardiac death) in patients treated with second-generation antipsychotic medications. METHOD: 367 adults treated with second-generation antipsychotics randomly selected from consecutive psychiatric admissions to a single hospital between August 1, 2004, and March 1, 2005, underwent assessments evaluating the presence of metabolic syndrome. The 10-year risk of CHD events was calculated according to the Framingham scoring system for age, smoking, total cholesterol, high-density lipoprotein (HDL)-cholesterol, blood pressure, and history of diabetes and was compared in patients with and without the metabolic syndrome. RESULTS: Metabolic syndrome, present in 137 patients (37.3%), was associated with a significantly greater age- and race-adjusted 10-year risk of CHD events, i.e., 11.5% vs. 5.3% for men (risk ratio = 2.18, 95% CI = 1.88 to 2.48, p < .0001) and 4.5% vs. 2.3% for women (risk ratio = 1.94, 95% CI = 1.65 to 2.23, p = .0005). The increased risk of CHD events in patients with metabolic syndrome remained significant after the exclusion of diabetic patients. In a logistic regression analysis of variables independent of the Framingham scoring system, triglyceride levels (p < .0001), waist circumference (p = .035), and white race (p = .047) were significantly associated with the 10-year risk of CHD events (R2 = 0.134; p < .0001). CONCLUSIONS: These data confirm the high prevalence of metabolic syndrome in patients receiving second-generation anti psychotics, indicate that metabolic syndrome doubles the 10-year risk of CHD events in this population, and emphasize the importance of the "hypertriglyceridemic waist" for the identification of psychiatric patients at high risk of CHD. PMID- 16669723 TI - Differences in Axis I and II comorbidity between bipolar I and II disorders and major depressive disorder. AB - OBJECTIVE: To obtain a comprehensive view of differences in current comorbidity between bipolar I and II disorders (BD) and (unipolar) major depressive disorder (MDD), and Axis I and II comorbidity in BD in secondary-care psychiatric settings. METHOD: The psychiatric comorbidity of 90 bipolar I and 101 bipolar II patients from the Jorvi Bipolar Study and 269 MDD patients from the Vantaa Depression Study were compared. We used DSM-IV criteria assessed by semistructured interviews. Patients were inpatients and outpatients from secondary-care psychiatric units. Comparable information was collected on clinical history, index episode, symptom status, and patient characteristics. RESULTS: Bipolar disorder and MDD differed in prevalences of current comorbid disorders, MDD patients having significantly more Axis I comorbidity (69.1% vs. 57.1%), specifically anxiety disorders (56.5% vs. 44.5%) and cluster A (19.0% vs. 9.9%) and C (31.6% vs. 23.0%) personality disorders. In contrast, BD had more single cluster B personality disorders (30.9% vs. 24.6%). Bipolar I and bipolar II were similar in current overall comorbidity, but the prevalence of comorbidity was strongly associated with the current illness phase. CONCLUSIONS: Major depressive disorder and BD have somewhat different patterns in the prevalences of comorbid disorders at the time of an illness episode, with differences particularly in the prevalences of anxiety and personality disorders. Current illness phase explains differences in psychiatric comorbidity of BD patients better than type of disorder. PMID- 16669724 TI - Psychiatric comorbidity in binge-eating disorder as a function of smoking history. AB - OBJECTIVE: To examine the comorbidity of psychiatric disorders in obese women with binge-eating disorder (BED) as a function of smoking history. METHOD: A consecutive series of 103 obese treatment-seeking women with current DSM-IV diagnoses of BED were administered structured diagnostic interviews to assess all DSM-IV Axis I psychiatric disorders. Participants were classified as "never" or "daily" smokers, and lifetime rates of comorbid psychopathology were compared across smoking groups using logistic regression. The study was conducted from February 2003 to March 2005. RESULTS: Smokers were significantly more likely to meet criteria for co-occurring diagnoses of major depressive disorder (p = .03), panic disorder (p = .01), posttraumatic stress disorder (p < .05), and substance abuse or dependence (p = .01). Even after excluding participants with substance use disorders, significant differences remained, with lifetime smokers having significantly higher rates of co-occurring anxiety disorders. CONCLUSIONS: It is possible that for some obese women with BED, binge eating and cigarette smoking share common functions, i.e., both behaviors may serve to modulate negative affect and/or anxiety. Although the current findings are consistent with a view of a common diathesis for the development of impulsive eating, cigarette or other substance use, and additional Axis I psychopathology, prospective longitudinal studies are needed to elucidate the nature of potential pathways. PMID- 16669725 TI - Serotonin-norepinephrine reuptake inhibitors in the treatment of obsessive compulsive disorder: A critical review. AB - OBJECTIVE: To critically review the antiobsessional properties of serotonin norepinephrine reuptake inhibitors (SNRIs) (venlafaxine and clomipramine) in the treatment of obsessive-compulsive disorder (OCD) as an alternative to selective serotonin reuptake inhibitors (SSRIs), which are currently considered the first line treatment of OCD. DATA SOURCES: A MEDLINE search was performed to identify clinical trials with the SNRIs venlafaxine and clomipramine published from 1996 to 2004 (keywords: SNRIs, venlafaxine, duloxetine, and clomipramine, each matched individually with the term OCD), focusing on the best-designed studies for inclusion. DATA SYNTHESIS: Much of the literature about SNRIs in OCD supports the efficacy of these compounds in the treatment of OCD. However, double-blind, placebo-controlled studies with venlafaxine are lacking, and the most relevant studies consist of active comparison trials between SNRIs and SSRIs. In these studies, SNRIs seem to be as effective as SSRIs in OCD; SNRIs might be preferred for patients with certain types of treatment-resistant OCD or those with particular comorbid conditions. A large number of placebo-controlled and active comparison trials with clomipramine document efficacy in OCD, and meta-analytic studies suggest a small superiority over SSRIs. Compared with clomipramine, the SNRI venlafaxine showed fewer side effects and better tolerability. CONCLUSION: The SNRIs may represent a valid alternative to the SSRIs, particularly in specific cases. Double-blind, placebo-controlled studies are, however, needed to confirm the positive findings reported by several studies with venlafaxine. PMID- 16669726 TI - A randomized double-blind trial of paroxetine and/or dextroamphetamine and problem-focused therapy for attention-deficit/hyperactivity disorder in adults. AB - OBJECTIVE: To determine the effect of psychotherapy, dextroamphetamine, and/or paroxetine on attention-deficit/hyperactivity-disorder (ADHD) in adults. METHOD: Ninety-eight adults with DSM-IV ADHD were randomly assigned to receive psychotherapy and dextroamphetamine, paroxetine, both, or placebo for 20 weeks. A 2 x 2 factorial design compared patients who received dextroamphetamine versus no dextroamphetamine with patients who received paroxetine versus no paroxetine. Data were collected from August 2000 until May 2002. RESULTS: One half of the 98 enrolled subjects were found to have at least 1 lifetime mood or anxiety disorder on the Structured Clinical Interview for DSM-IV. Sixty percent of patients who received medication and 80% of those who received placebo completed the 5-month trial. ADHD symptoms were significantly (p = .012) lower in patients in the completer group who received dextroamphetamine. Paroxetine had no effect on ADHD. Hamilton Rating Scales for Anxiety (HAM-A) and Depression (HAM-D) scores were low to start, and no treatment differences were evident at endpoint. Significantly (p < .001) more patients in the completer group were rated by clinicians as ADHD responders if they received dextroamphetamine (85.7%) or combined treatment (66.7%) versus paroxetine (20.0%) or placebo (21.1%). Significantly (p = .003) more patients in the completer group were rated by clinicians as mood/anxiety responders if they received paroxetine (100%) or combined treatment (73.3%) versus those receiving dextroamphetamine (57.15%) or placebo (47.4%). Clinicians rated any patient who received medication and psychological therapy as significantly more improved overall than those who received placebo and psychological therapy (intent to treat: p = .033; completers: p = .001). CONCLUSION: ADHD symptoms improved with dextroamphetamine. Mood and internalizing symptoms were seen as improved with paroxetine by clinicians, despite absence of response on the HAM-A and HAM-D. The presence of a lifetime internalizing disorder attenuated the response to dextroamphetamine. Patients who received both dextroamphetamine and paroxetine had more severe adverse events but did not show greater improvement overall than patients treated with 1 medication. Clinical Trials Registry #GSK707. PMID- 16669727 TI - Moral objections to suicide: Can they counteract suicidality in patients with cluster B psychopathology? AB - OBJECTIVE: Patients with cluster B personality disorder (CBPD) are particularly prone to suicidal behavior, yet possible protective mechanisms are not often studied. The present study investigated a possible protective role of moral objections to suicide (MOS) against suicidal behavior in patients with CBPD and current depression. The effect of MOS was then examined in relation to other mechanisms affecting suicide risk including trait aggressivity and the presence of effective coping strategies. METHOD: 147 depressed patients with comorbid CBPD (DSM-III-R) were compared with 210 depressed patients without CBPD in terms of their history of suicide attempts and clinical and demographic characteristics. The relationship of MOS to suicide attempt was examined by logistic regression controlling for demographic and clinical differences between the groups as well as presence of comorbid CBPD. Data were collected from 1990 to 2003. RESULTS: Subjects with comorbid CBPD had fewer MOS and reported more previous suicide attempts. In logistic regression, fewer MOS/religious beliefs, lower coping potential, and higher aggression level were associated with suicide attempt. A CBPD diagnosis did not affect the relationship between MOS and suicide attempts. CONCLUSIONS: The results of this study suggest that the presence of MOS/religious beliefs may have a protective effect against suicidal behavior in depressed patients with comorbid CBPD and may be a target for therapeutic intervention. PMID- 16669728 TI - A randomized, double-blind, placebo-controlled 26-week trial of aripiprazole in recently manic patients with bipolar I disorder. AB - OBJECTIVE: To investigate the safety and efficacy of aripiprazole in preventing relapse of a mood episode in recently manic- or mixed-episode patients with bipolar I disorder stabilized on aripiprazole. METHOD: This randomized, double blind, parallel-group, placebo-controlled, multicenter study enrolled patients from 76 centers in 3 countries (Argentina, Mexico, United States) from March 2000 to June 2003. Bipolar I disorder (DSM-IV) patients who had recently been hospitalized and treated for a manic or mixed episode entered an open-label stabilization phase (aripiprazole monotherapy: 15 or 30 mg/day, 6-18 weeks). After meeting stabilization criteria (Young Mania Rating Scale score of < or = 10 and Montgomery-Asberg Depression Rating Scale score of < or = 13 for 6 consecutive weeks), 161 patients were randomly assigned to aripiprazole or placebo for the 26-week, double-blind phase. The primary endpoint was time to relapse for a manic, mixed, or depressive episode (defined by discontinuation caused by lack of efficacy). RESULTS: Aripiprazole was superior to placebo in delaying the time to relapse (p = .020). Aripiprazole-treated patients had significantly fewer relapses (25%) than placebo patients (43%; p = .013). Aripiprazole was superior to placebo in delaying the time to manic relapse (p = .01); however, no significant differences were observed in time to depressive relapse (p = .68). Weight gain (> or = 7% increase) occurred in 7 (13%) aripiprazole-treated and 0 placebo-treated patients. Adverse events (> or = 5% incidence and twice that of placebo) reported by aripiprazole-treated patients were akathisia, pain in the extremities, tremor, and vaginitis. CONCLUSIONS: Aripiprazole, 15 or 30 mg/day, was superior to placebo in maintaining efficacy in patients with bipolar I disorder with a recent manic or mixed episode who were stabilized and maintained on aripiprazole treatment for 6 weeks, as shown by a longer time to relapse. PMID- 16669729 TI - Efficacy of ziprasidone against hostility in schizophrenia: Post hoc analysis of randomized, open-label study data. AB - OBJECTIVE: The objective was to determine the effects of sequential intramuscular/oral ziprasi-done on hostility. METHOD: A total of 572 inpatients diagnosed with DSM-IV schizophrenia or schizoaffective disorder were the subjects in a randomized, rater-blinded, 6-week, open-label study comparing sequential intramuscular and oral ziprasidone with haloperidol. The Brief Psychiatric Rating Scale (BPRS) was the principal outcome measure. To determine the effect of ziprasidone on hostility, post hoc analyses of scores on the hostility item from the BPRS were conducted. Introducing positive symptoms and akathisia as covariates tested specific antihostility effect. The study was conducted from October 23, 1998, to August 15, 2000. RESULTS: Ziprasidone demonstrated specific antihostility effects over time throughout the 42-day study period and statistically significant superiority to haloperidol on this measure in the first week of treatment (p = .0149 at first evaluation [day 1, 2, or 3]; p = .0358 at day 7). CONCLUSION: Ziprasidone is an effective treatment for hostility in patients with schizophrenia or schizoaffective disorder. PMID- 16669730 TI - Factors associated with temporal priority in comorbid bipolar I disorder and alcohol use disorders: Results from the national epidemiologic survey on alcohol and related conditions. AB - OBJECTIVE: To compare illness characteristics, comorbidities, treatment utilization, and family history among individuals with comorbid bipolar I disorder and alcohol use disorders (AUD) based on temporal priority of onset. METHOD: The 2001-2002 National Epidemiologic Survey on Alcohol and Related Conditions identified respondents with lifetime comorbid bipolar I disorder and AUD for whom AUD were antecedent (Alcohol First; N = 311), the onset of the 2 conditions occurred in the same year (Same Year; N = 113), or bipolar I disorder was antecedent (Bipolar First; N = 233). Diagnoses were generated using the National Institute on Alcohol Abuse and Alcoholism Alcohol Use Disorder and Associated Disabilities Interview Schedule-DSM-IV Version. This study examined between-group differences in bipolar I- and AUD-related variables. RESULTS: Bipolar First respondents were most likely to experience prolonged manic episodes. There were no differences in the 12-month prevalence of bipolar I disorder among respondents with prior history of bipolar I disorder. The 12-month prevalence of AUD among respondents with prior history of AUD was lower among Alcohol First respondents compared to Same Year or Bipolar First respondents. Same Year respondents were most likely to seek AUD treatment and reported comparatively short latency between onset and treatment of both bipolar I disorder and AUD. The prevalence of family history of comorbid depression and AUD was greatest among Same Year respondents. Same Year respondents also showed the lowest prevalence of anxiety disorders. Overall psychosocial functioning was similar across groups. CONCLUSION: Temporal priority in comorbid bipolar I disorder and AUD is associated with several significant between-group differences in features of bipolar I disorder and AUD severity, treatment utilization, other comorbidities, and family history. Same-year onset of bipolar I disorder and AUD may be a marker of a specific subtype of bipolar I-AUD comorbidity. Potential implications of these findings are discussed. PMID- 16669731 TI - In this issue: The genetic, transcriptomic, and proteomic study of Alzheimer's disease. PMID- 16669733 TI - Novel transdermal delivery formulation of the monoamine oxidase inhibitor selegiline nearing release for treatment of depression. PMID- 16669734 TI - The emergence of darkness phobia in a bipolar patient during quetiapine treatment. PMID- 16669735 TI - Simultaneous true seizures and pseudoseizures. PMID- 16669736 TI - High-dose aripiprazole in treatment-resistant schizophrenia. PMID- 16669737 TI - Clozapine augmentation with aripiprazole for negative symptoms. PMID- 16669732 TI - Genetics, transcriptomics, and proteomics of Alzheimer's disease. AB - OBJECTIVE: To provide an updated overview of the methods used in genetic, transcriptomic, and proteomic studies in Alzheimer's disease and to demonstrate the importance of those methods for the improvement of the current diagnostic and therapeutic possibilities. DATA SOURCES: MEDLINE-based search of 233 peer reviewed articles published between 1975 and 2006. DATA SYNTHESIS: Alzheimer's disease is a genetically heterogeneous disorder. Rare mutations in the amyloid precursor protein, presenilin 1, and presenilin 2 genes have shown the importance of the amyloid metabolism for its development. In addition, converging evidence from population-based genetic studies, gene expression studies, and protein profile studies in the brain and in the cerebrospinal fluid suggest the existence of several pathogenetic pathways such as amyloid precursor protein processing, beta-amyloid degradation, tau phosphorylation, proteolysis, protein misfolding, neuroinflammation, oxidative stress, and lipid metabolism. CONCLUSIONS: The development of high-throughput genotyping methods and of elaborated statistical analyses will contribute to the identification of genetic risk profiles related to the development and course of this devastating disease. The integration of knowledge derived from genetic, transcriptomic, and proteomic studies will greatly advance our understanding of the causes of Alzheimer's disease, improve our capability of establishing an early diagnosis, help define disease subgroups, and ultimately help to pave the road toward improved and tailored treatments. PMID- 16669738 TI - Early bicarbonate loading and dantroline for ziprasidone/haloperidol-induced neuroleptic malignant syndrome. PMID- 16669742 TI - Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo. AB - We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4 (dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation. PMID- 16669743 TI - The effects of pulsed 860 MHz radiofrequency radiation on the promotion of neurogenic tumors in rats. AB - In a previous study, this laboratory reported a statistically nonsignificant trend for shortened latency of ethylnitrosourea (ENU)-induced brain tumors in Sprague-Dawley rats exposed to an 860 MHz pulsed radiofrequency (RF) signal. The present study was designed to investigate further any promoting effect of the pulsed RF signal on latency and other characteristics of neurogenic tumors in the progeny of pregnant rats treated with 6.25 or 10 mg/kg ENU. The resulting 1080 offspring were randomized equally by number, sex and ENU dose into pulsed RF, sham and cage control groups. The rats were exposed to the pulsed RF signal 6 h per day 5 days per week; the sham-exposed group was similarly confined for the same periods, and the cage controls were housed in standard cages. An essentially equal number of rats from each group were killed humanely every 30 days between the ages of 171 and 325 days; 32 rats died and 225 rats were killed when they were moribund. Postmortem examinations on the 1080 rats revealed 38 spinal cord tumors, 191 spinal nerve tumors, 232 cranial nerve tumors, and 823 brain tumors. A methodical study of the tumor characteristics disclosed no evidence that exposure to the pulsed RF signal affected the incidence, malignancy, volume, multiplicity, latency or fatality associated with any kind of neurogenic tumor. PMID- 16669744 TI - Point-source irradiation with eccentric rotation causes inhomogeneous dose distribution. AB - Radioactive point sources are regularly used for irradiating cell culture and other biological materials. Eccentric rotation is often used to minimize dose disparities that arise from irradiating samples that span a distance from the point source. Rotation provides a great improvement in dose homogeneity compared to inert irradiation yet still presents an obvious shortcoming for exposures in which the sample completes only partial rotation or fractional rotation. In such cases, certain areas of the sample have a closer average distance to the radiation source than other areas within the same sample. This obstacle can be partially overcome by adjusting rotation speed so the sample traverses a full rotation (or multiple thereof) throughout the total irradiation time. Here we investigate the effects of irradiation with eccentric rotation on dose homogeneity. We show that due to the inverse square law that governs dose, even exposures with full rotation result in inhomogeneous dose distributions. This dose inhomogeneity can be substantial, especially for large samples and small source- sample distances. We observed a 33% difference in survival across 100-mm dishes and a 400% difference for 150-mm dishes. The dose inhomogeneity inherent to eccentric rotation increases the actual average dose delivered across the sample compared to that delivered at sample center. We offer a table of correction factors that account for this dose increase and correct the dose delivered at center to the actual average dose delivered across the entire sample. PMID- 16669745 TI - Comments on "Dose-response relationships for radicals trapped in irradiated solids" by W. H. Nelson (Radiat. Res. 163, 673-680, 2005). PMID- 16669746 TI - Making it Work 2: using a virtual community to focus on rural health issues. AB - Between 21 and 23 September 2005, over 200 delegates from eight countries gathered in Tromso, within the Arctic Circle, to discuss challenges and solutions to rural health issues. This conference was a sequel to a previous event entitled 'Making it Work', held in Scotland in 2003, in which it was identified that service delivery in remote and rural areas needed to be innovative to ensure equity. A major aim of this event was to move the debate forward to describe specific examples of practice that could be adopted in participating countries. The delegates included clinicians, managers and administrators, senior policymakers and educationalists, elected local and national politicians, patients and their representatives. In order to focus debate, the organisers provided an outline of a virtual remote community ('Hope'), including some geographic and demographic information, together with four case studies of individual health problems faced by residents of the community. During the introductory session, a short film was shown featuring the 'residents' of this community, introducing delegates to the specific problems they faced. Throughout the conference, delegates were asked to reflect back to how any recommendations made might apply to the citizens of Hope. The clinical scenarios presented included: (1) a 37 year old pregnant woman in labour during adverse weather conditions; (2) a 17 year old island resident with acute psychosis who attempts suicide; (3) an 80 year old woman living alone who suffers a stroke; and (4) a family of four with a complex range of chronic health issues including smoking, alcoholism, diabetes, teenage pregnancy, asthma and depression on a background of deprivation and unemployment. Parallel discussions and workshops focussed on a number of key themes linked to the examples highlighted in the 'Hope' scenario. These included: maternity services; mental health; chronic disease management; health improvement and illness prevention; supporting healthy rural communities; and education for rural health staff. This approach to targeting discussion is valuable in rural health conferences where the participants may be from diverse backgrounds and the issues discussed are multi-faceted. PMID- 16669747 TI - Ostomy care and the consultant pharmacist. AB - Many long-term care residents have ostomies. Three types of ostomy are most common: ostomies of the small bowel (ileostomies/jejunostomies), ostomies of the large bowel (colostomy), and urostomies. Ostomates (people who have had an ostomy) must deal with their physical and psychological repercussions. Complications may include dermatological problems, short-bowel syndrome, and vitamin deficiencies. When the ostomy involves the bowel, medication absorption may be affected. Pharmacists should be aware of contraindicated medications, as well as those that change the color or odor of effluent or stool. PMID- 16669748 TI - Practical considerations of new drugs: new choices for old problems. AB - As drug therapy experts, pharmacists are often called upon for facts and opinions about recently introduced drugs. New drugs are introduced at a rate that exceeds the reading time of many busy practitioners. This article is intended to provide an objective information and broad-based discussion about new drug options that have particular relevance for the elderly. Drugs that will be reviewed include: acamprosate, darifenacin, eszopiclone, exenatide, ibandronate, nitazoxanide, pramlintide, pregabalin, ramelteon, tigecycline, tinidazole, trospium, and solifenacin. PMID- 16669749 TI - Job satisfaction and stress among pharmacists in the long-term care sector. AB - OBJECTIVE: To provide normative information regarding levels of job satisfaction and stress between consulting and dispensing pharmacists in the long-term care arena. DESIGN: Cross-sectional. SETTING: United States. PARTICIPANTS: All current members of the American Society of Consultant Pharmacists as of March 2005. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Health Professional Stress Inventory. RESULTS: More than 90% of consultant pharmacists were satisfied with their jobs. The most frequently reported source of stress among the pharmacists was short staffing, affecting their ability to perform their duties. More than one-third of dispensing pharmacists were often stressed, frequently because of the need to keep up with developments to maintain professional competence. CONCLUSION: Pharmacists serving the long-term care sector are very satisfied. Methods to assist dispensing pharmacists to keep up with practice developments must be explored. Whether or not new models of long-term care pharmacy improve job satisfaction must be explored. PMID- 16669750 TI - Medication sleuth: an important role for pharmacists in determining the etiology of delirium. AB - Delirium is characterized by disturbances of consciousness, attention, cognition, and perception and is the most common reason for acute cognitive dysfunction in hospitalized elderly patients. Causes of delirium can be multifactorial, and a careful medical and medication history can help determine the underlying cause of behavioral disturbances. A 65-year-old patient with a history of chronic pain, insomnia, and multiple medical problems, who presented with altered mental status and aggressive behavior, is described. The patient had taken an overdose of zolpidem prior to admission, and she required chemical and physical restraints and one-on-one care for safety. With time and washout of the zolpidem, the patient's behavior did not improve. On the second day of admission, medication reconciliation of this patient's medication profile helped to reveal a medication cause for this patient's delirium. A pharmacist should be included early in the process of obtaining a medication history. Recommendations for the management of chronic pain and insomnia in the elderly are presented. PMID- 16669753 TI - Looking at life: from binoculars to the electron microscope. PMID- 16669754 TI - MicroRNAS and their regulatory roles in plants. AB - MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these approximately 21-nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although plant miRNAs have some conserved functions extending beyond development, the importance of miRNA-directed gene regulation during plant development is now particularly clear. Identified in plants less than four years ago, miRNAs are already known to play numerous crucial roles at each major stage of development-typically at the cores of gene regulatory networks, targeting genes that are themselves regulators, such as those encoding transcription factors and F-box proteins. PMID- 16669755 TI - Chlorophyll degradation during senescence. AB - The catabolic pathway of chlorophyll (Chl) during senescence and fruit ripening leads to the accumulation of colorless breakdown products (NCCs). This review updates an earlier review on Chl breakdown published here in 1999 ( 69 ). It summarizes recent advances in the biochemical reactions of the pathway and describes the characterization of new NCCs and their formation inside the vacuole. Furthermore, I focus on the recent molecular identification of three chl catabolic enzymes, chlorophyllase, pheophorbide a oxygenase (PAO), and red Chl catabolite reductase (RCCR). The analysis of Chl catabolic mutants demonstrates the importance of Chl breakdown for plant development and survival. Mutants defective in PAO or RCCR develop a lesion mimic phenotype, due to the accumulation of breakdown intermediates. Thus, Chl breakdown is a prerequisite to detoxify the potentially phototoxic pigment within the vacuoles in order to permit the remobilization of nitrogen from Chl-binding proteins to proceed during senescence. PMID- 16669756 TI - Quantitative fluorescence microscopy: from art to science. AB - A substantial number of elegant experimental approaches have been developed to image the distribution and dynamics of DNA, mRNA, proteins, organelles, metabolites, and ions in living plant cells. Although the human brain can rapidly assimilate visual information, particularly when presented as animations and movies, it is much more challenging to condense the phenomenal amount of data present in three-, four-, or even five-dimensional images into statistically useful measurements. This review explores a range of in vivo fluorescence imaging applications in plants, with particular emphasis on where quantitative techniques are beginning to emerge. PMID- 16669757 TI - Control of the actin cytoskeleton in plant cell growth. AB - Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the activity of actin-binding proteins is proving to be essential for proper cell morphogenesis. Actin-nucleating proteins participate in cell expansion and cell plate formation whereas the recycling of actin monomers is required to maintain actin dynamics and controlled growth. Coordination of actin-binding protein activity and other aspects of cytoskeletal behavior during cell development maintains cohesive cell expansion. Emerging plant signaling networks are proving to be powerful regulators of morphology-shaping cytoskeletal activity, and in this review we highlight current research in actin network regulation. PMID- 16669758 TI - Responding to color: the regulation of complementary chromatic adaptation. AB - The acclimation of photosynthetic organisms to changes in light color is ubiquitous and may be best illustrated by the colorful process of complementary chromatic adaptation (CCA). During CCA, cyanobacterial cells change from brick red to bright blue green, depending on their light color environment. The apparent simplicity of this spectacular, photoreversible event belies the complexity of the cellular response to changes in light color. Recent results have shown that the regulation of CCA is also complex and involves at least three pathways. One is controlled by a phytochrome-class photoreceptor that is responsive to green and red light and a complex two-component signal transduction pathway, whereas another is based on sensing redox state. Studies of CCA are uncovering the strategies used by photosynthetic organisms during light acclimation and the means by which they regulate these responses. PMID- 16669759 TI - Seasonal control of tuberization in potato: conserved elements with the flowering response. AB - Fluctuations in day length determine the time to flower in many plants and in potato are critical to promote differentiation of tubers. Day length is perceived in the leaves and under inductive conditions these synthesize a systemic signal that is transported to the underground stolons to induce tuber development. Flowering tobacco shoots grafted into potato stocks promote tuberization in the stocks, indicating that the floral and tuber-inducing signals might be similar. We describe recent progress in the identification of the molecular mechanisms underlying day-length recognition in potato. Evidence has been obtained for a conserved function of the potato orthologs of the CONSTANS (CO) and FLOWERING LOCUS T (FT) proteins in tuberization control under short days (SDs). These observations indicate that common regulatory pathways are involved in both flowering and tuberization photoperiodic responses in plants. PMID- 16669760 TI - Laser microdissection of plant tissue: what you see is what you get. AB - Laser microdissection (LM) utilizes a cutting or harvesting laser to isolate specific cells from histological sections; the process is guided by microscopy. This provides a means of removing selected cells from complex tissues, based only on their identification by microscopic appearance, location, or staining properties (e.g., immunohistochemistry, reporter gene expression, etc.). Cells isolated by LM can be a source of cell-specific DNA, RNA, protein or metabolites for subsequent evaluation of DNA modifications, transcript/protein/metabolite profiling, or other cell-specific properties that would be averaged with those of neighboring cell types during analysis of undissected complex tissues. Plants are particularly amenable to the application of LM; the highly regular tissue organization and stable cell walls of plants facilitate the visual identification of most cell types even in unstained tissue sections. Plant cells isolated by LM have been the starting point for a variety of genomic and metabolite studies of specific cell types. PMID- 16669761 TI - Integrative plant biology: role of phloem long-distance macromolecular trafficking. AB - Recent studies have revealed the operation of a long-distance communication network operating within the vascular system of higher plants. The evolutionary development of this network reflects the need to communicate environmental inputs, sensed by mature organs, to meristematic regions of the plant. One consequence of such a long-distance signaling system is that newly forming organs can develop properties optimized for the environment into which they will emerge, mature, and function. The phloem translocation stream of the angiosperms contains, in addition to photosynthate and other small molecules, a variety of macromolecules, including mRNA, small RNA, and proteins. This review highlights recent progress in the characterization of phloem-mediated transport of macromolecules as components of an integrated long-distance signaling network. Attention is focused on the role played by these proteins and RNA species in coordination of developmental programs and the plant's response to both environmental cues and pathogen challenge. Finally, the importance of developing phloem transcriptome and proteomic databases is discussed within the context of advances in plant systems biology. PMID- 16669762 TI - The role of root exudates in rhizosphere interactions with plants and other organisms. AB - The rhizosphere encompasses the millimeters of soil surrounding a plant root where complex biological and ecological processes occur. This review describes recent advances in elucidating the role of root exudates in interactions between plant roots and other plants, microbes, and nematodes present in the rhizosphere. Evidence indicating that root exudates may take part in the signaling events that initiate the execution of these interactions is also presented. Various positive and negative plant-plant and plant-microbe interactions are highlighted and described from the molecular to the ecosystem scale. Furthermore, methodologies to address these interactions under laboratory conditions are presented. PMID- 16669763 TI - Genetics of meiotic prophase I in plants. AB - During meiotic prophase I, traits are reassorted as a result of a highly organized process involving sister chromatid cohesion, homologous chromosome alignment, pairing, synapsis, and recombination. In the past two years, a number of components involved in this pathway, including Structure Maintenance of Chromosomes (SMC), MRE11, the RAD51 homologs, BRCA2, MSH4, MER3, and ZIP1, have been characterized in plants; in addition, several genes that encode components unique to plants, such as POOR HOMOLOGOUS SYNAPSIS 1 and AMEIOTIC 1, have been cloned. Based on these recent data, essentially from maize and Arabidopsis, we discuss the conserved and plant-specific aspects of meiosis commitment and meiotic prophase I features. PMID- 16669764 TI - Biology and biochemistry of glucosinolates. AB - Glucosinolates are sulfur-rich, anionic natural products that upon hydrolysis by endogenous thioglucosidases called myrosinases produce several different products (e.g., isothiocyanates, thiocyanates, and nitriles). The hydrolysis products have many different biological activities, e.g., as defense compounds and attractants. For humans these compounds function as cancer-preventing agents, biopesticides, and flavor compounds. Since the completion of the Arabidopsis genome, glucosinolate research has made significant progress, resulting in near-complete elucidation of the core biosynthetic pathway, identification of the first regulators of the pathway, metabolic engineering of specific glucosinolate profiles to study function, as well as identification of evolutionary links to related pathways. Although much has been learned in recent years, much more awaits discovery before we fully understand how and why plants synthesize glucosinolates. This may enable us to more fully exploit the potential of these compounds in agriculture and medicine. PMID- 16669765 TI - Bioinformatics and its applications in plant biology. AB - Bioinformatics plays an essential role in today's plant science. As the amount of data grows exponentially, there is a parallel growth in the demand for tools and methods in data management, visualization, integration, analysis, modeling, and prediction. At the same time, many researchers in biology are unfamiliar with available bioinformatics methods, tools, and databases, which could lead to missed opportunities or misinterpretation of the information. In this review, we describe some of the key concepts, methods, software packages, and databases used in bioinformatics, with an emphasis on those relevant to plant science. We also cover some fundamental issues related to biological sequence analyses, transcriptome analyses, computational proteomics, computational metabolomics, bio ontologies, and biological databases. Finally, we explore a few emerging research topics in bioinformatics. PMID- 16669766 TI - Leaf hydraulics. AB - Leaves are extraordinarily variable in form, longevity, venation architecture, and capacity for photosynthetic gas exchange. Much of this diversity is linked with water transport capacity. The pathways through the leaf constitute a substantial (>or=30%) part of the resistance to water flow through plants, and thus influence rates of transpiration and photosynthesis. Leaf hydraulic conductance (K(leaf)) varies more than 65-fold across species, reflecting differences in the anatomy of the petiole and the venation architecture, as well as pathways beyond the xylem through living tissues to sites of evaporation. K(leaf) is highly dynamic over a range of time scales, showing circadian and developmental trajectories, and responds rapidly, often reversibly, to changes in temperature, irradiance, and water supply. This review addresses how leaf structure and physiology influence K(leaf), and the mechanisms by which K(leaf) contributes to dynamic functional responses at the level of both individual leaves and the whole plant. PMID- 16669767 TI - Plant uncoupling mitochondrial proteins. AB - Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide sensitive free fatty acid-activated H(+) flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (Deltamu(H)+) and production of reactive oxygen species. PMID- 16669768 TI - Genetics and biochemistry of seed flavonoids. AB - Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks. PMID- 16669769 TI - Cytokinins: activity, biosynthesis, and translocation. AB - Cytokinins (CKs) play a crucial role in various phases of plant growth and development, but the basic molecular mechanisms of their biosynthesis and signal transduction only recently became clear. The progress was achieved by identifying a series of key genes encoding enzymes and proteins controlling critical steps in biosynthesis, translocation, and signaling. Basic schemes for CK homeostasis and root/shoot communication at the whole-plant level can now be devised. This review summarizes recent findings on the relationship between CK structural variation and activity, distinct features in CK biosynthesis between higher plants and Agrobacterium infected plants, CK translocation at whole-plant and cellular levels, and CKs as signaling molecules for nutrient status via root-shoot communication. PMID- 16669770 TI - Global studies of cell type-specific gene expression in plants. AB - Technological advances in expression profiling and in the ability to collect minute quantities of tissues have come together to allow a growing number of global transcriptional studies at the cell level in plants. Microarray technology, with a choice of cDNA or oligo-based slides, is now well established, with commercial full-genome platforms for rice and Arabidopsis and extensive expressed sequence tag (EST)-based designs for many other species. Microdissection and cell sorting are two established methodologies that have been used in conjunction with microarrays to provide an early glimpse of the transcriptional landscape at the level of individual cell types. The results indicate that much of the transcriptome is compartmentalized. A minor but consistent percentage of transcripts appear to be unique to specific cell types. Functional analyses of cell-specific patterns of gene expression are providing important clues to cell-specific functions. The spatial dissection of the transcriptome has also yielded insights into the localized mediators of hormone inputs and promises to provide detail on cell-specific effects of microRNAs. PMID- 16669771 TI - Mechanism of leaf-shape determination. AB - Biodiversity of plant shape is mainly attributable to biodiversity of leaf shape and the shape of floral organs, the modified leaves. However, the exact mechanisms of leaf-shape determination remain unclear due to the complexity of flat-structure organogenesis that includes the simultaneous cell cycling and cell enlargement in primordia. Recent studies in developmental and molecular genetics have revealed several important aspects of leaf-shape control mechanisms. For example, understanding of polar control in leaf-blade expansion has advanced greatly. A curious phenomenon called "compensated cell enlargement" found in leaf organogenesis studies should also provide interesting clues regarding the mechanisms of multicellular organ development. This paper reviews recent research findings with a focus on leaf development in Arabidopsis thaliana. PMID- 16669772 TI - Mosses as model systems for the study of metabolism and development. AB - The haploid gametophyte stage of the moss life cycle is amenable to genetic and biochemical studies. Many species can be cultured on simple defined media, where growth is rapid, making them ideal material for metabolic studies. Developmental responses to hormones and to environmental inputs can be studied both at the level of individual cells and in multicellular tissues. The protonemal stage of gametophyte development comprises cell filaments that extend by the serial division of their apical cells, allowing the investigation of the generation and modification of cell polarity and the role of the cytoskeleton in these processes. Molecular techniques including gene inactivation by targeted gene replacement or by RNA interference, together with the nearly completed sequencing of the Physcomitrella patens genome, open the way for detailed study of the functions of genes involved in both development and metabolism. PMID- 16669773 TI - Structure and function of photosystems I and II. AB - Oxygenic photosynthesis, the principal converter of sunlight into chemical energy on earth, is catalyzed by four multi-subunit membrane-protein complexes: photosystem I (PSI), photosystem II (PSII), the cytochrome b(6)f complex, and F ATPase. PSI generates the most negative redox potential in nature and largely determines the global amount of enthalpy in living systems. PSII generates an oxidant whose redox potential is high enough to enable it to oxidize H(2)O, a substrate so abundant that it assures a practically unlimited electron source for life on earth. During the last century, the sophisticated techniques of spectroscopy, molecular genetics, and biochemistry were used to reveal the structure and function of the two photosystems. The new structures of PSI and PSII from cyanobacteria, algae, and plants has shed light not only on the architecture and mechanism of action of these intricate membrane complexes, but also on the evolutionary forces that shaped oxygenic photosynthesis. PMID- 16669774 TI - Glycosyltransferases of lipophilic small molecules. AB - Glycosyltransferases of small molecules transfer sugars to a wide range of acceptors, from hormones and secondary metabolites to biotic and abiotic chemicals and toxins in the environment. The enzymes are encoded by large multigene families and can be identified by a signature motif in their primary sequence, which classifies them as a subset of Family 1 glycosyltransferases. The transfer of a sugar onto a lipophilic acceptor changes its chemical properties, alters its bioactivity, and enables access to membrane transporter systems. In vitro studies have shown that a single gene product can glycosylate multiple substrates of diverse origins; multiple enzymes can also glycosylate the same substrate. These features suggest that in a cellular context, substrate availability is a determining factor in enzyme function, and redundancy depends on the extent of coordinate gene regulation. This review discusses the role of these glycosyltransferases in underpinning developmental and metabolic plasticity during adaptive responses. PMID- 16669775 TI - Protein degradation machineries in plastids. AB - Plastids undergo drastic morphological and physiological changes under different developmental stages and in response to environmental conditions. A key to accomplishing these transitions and maintaining homeostasis is the quality and quantity control of many plastid proteins by proteases and chaperones. Although a limited number of plastid proteases have been identified by biochemical approaches, recent progress in genome information revealed various plant proteases that are of prokaryotic origin and that are localized in chloroplasts. Of these, ATP-dependent proteases such as Clp, FtsH, and Lon are considered the major enzymes involved in processive degradation (gradual degradation to oligopeptides and amino acids). The basic architecture of plant ATP-dependent proteases is very similar to the architechture of bacterial enzymes, such as those in Escherichia coli, but plastid enzymes apparently have extraordinary numbers of isomers. Recent molecular genetic characterization in Arabidopsis has identified differential roles of these isomers. This review covers what is currently known about the types and function of plastid proteases together with our new observations. PMID- 16669776 TI - Molybdenum cofactor biosynthesis and molybdenum enzymes. AB - The molybdenum cofactor (Moco) forms the active site of all eukaryotic molybdenum (Mo) enzymes. Moco consists of molybdenum covalently bound to two sulfur atoms of a unique tricyclic pterin moiety referred to as molybdopterin. Moco is synthesized from GTP by an ancient and conserved biosynthetic pathway that can be divided into four steps involving the biosynthetic intermediates cyclic pyranopterin monophosphate, molybdopterin, and adenylated molybdopterin. In a fifth step, sulfuration or bond formation between Mo and a protein cysteine result in two different catalytic Mo centers. There are four Mo enzymes in plants: (1) nitrate reductase catalyzes the first and rate-limiting step in nitrate assimilation and is structurally similar to the recently identified, (2) peroxisomal sulfite oxidase that detoxifies excessive sulfite. (3) Aldehyde oxidase catalyzes the last step of abscisic acid biosynthesis, and (4) xanthine dehydrogenase is essential for purine degradation and stress response. PMID- 16669777 TI - Peptide hormones in plants. AB - In recent years, numerous biochemical and genetic studies have demonstrated that peptide signaling plays a greater than anticipated role in various aspects of plant growth and development. A substantial proportion of these peptides are secretory and act as local signals mediating cell-to-cell communication. Specific receptors for several peptides were identified as being membrane-localized receptor kinases, the largest family of receptor-like molecules in plants. These findings illustrate the importance of peptide signaling in the regulation of plant growth, functions that were previously ascribed to the combined action of small lipophilic compounds referred to as "traditional plant hormones." Here, we outline recent advances in the current understanding of biologically active peptides in plants, currently regarded as a new class of plant hormones. PMID- 16669778 TI - Sugar sensing and signaling in plants: conserved and novel mechanisms. AB - Sugars not only fuel cellular carbon and energy metabolism but also play pivotal roles as signaling molecules. The experimental amenability of yeast as a unicellular model system has enabled the discovery of multiple sugar sensors and signaling pathways. In plants, different sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues to modulate growth, development, and stress responses. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase (HXK) as a conserved glucose sensor. Diverse sugar signals activate multiple HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability and enzymatic activity. Important and complex roles for Snf1-related kinases (SnRKs), extracellular sugar sensors, and trehalose metabolism in plant sugar signaling are now also emerging. PMID- 16669779 TI - Vitamin synthesis in plants: tocopherols and carotenoids. AB - Carotenoids and tocopherols are the two most abundant groups of lipid-soluble antioxidants in chloroplasts. In addition to their many functional roles in photosynthetic organisms, these compounds are also essential components of animal diets, including humans. During the past decade, a near complete set of genes required for the synthesis of both classes of compounds in photosynthetic tissues has been identified, primarily as a result of molecular genetic and biochemical genomics-based approaches in the model organisms Arabidopsis thaliana and Synechocystis sp. PCC6803. Mutant analysis and transgenic studies in these and other systems have provided important insight into the regulation, activities, integration, and evolution of individual enzymes and are already providing a knowledge base for breeding and transgenic approaches to modify the types and levels of these important compounds in agricultural crops. PMID- 16669780 TI - Plastid-to-nucleus retrograde signaling. AB - Plant cells store genetic information in the genomes of three organelles: the nucleus, plastid, and mitochondrion. The nucleus controls most aspects of organelle gene expression, development, and function. In return, organelles send signals to the nucleus to control nuclear gene expression, a process called retrograde signaling. This review summarizes our current understanding of plastid to-nucleus retrograde signaling, which involves multiple, partially redundant signaling pathways. The best studied is a pathway that is triggered by buildup of Mg-ProtoporphyrinIX, the first intermediate in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In addition, there is evidence for a plastid gene expression-dependent pathway, as well as a third pathway that is dependent on the redox state of photosynthetic electron transport components. Although genetic studies have identified several players involved in signal generation, very little is known of the signaling components or transcription factors that regulate the expression of hundreds of nuclear genes. PMID- 16669781 TI - The genetics and biochemistry of floral pigments. AB - Three major groups of pigments, the betalains, the carotenoids, and the anthocyanins, are responsible for the attractive natural display of flower colors. Because of the broad distribution of anthocyanins (synthesized as part of the flavonoid pathway) among the flowering plants, their biosynthesis and regulation are best understood. However, over the past few years, significant progress has been made in understanding the synthesis and participation of carotenoids (derived from isoprenoids) and betalains (derived from tyrosine) in flower pigmentation. These three families of pigments play important ecological functions, for example in the attraction of pollinating animals. Anthocyanins in particular have also been the target of numerous biotechnological efforts with the objective of creating new, or altering the properties of existing, coloring compounds. The focus of this review is to examine the biosynthesis, regulation, and contribution to flower coloration of these three groups of pigments. PMID- 16669782 TI - Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. AB - Plant growth and productivity are greatly affected by environmental stresses such as drought, high salinity, and low temperature. Expression of a variety of genes is induced by these stresses in various plants. The products of these genes function not only in stress tolerance but also in stress response. In the signal transduction network from perception of stress signals to stress-responsive gene expression, various transcription factors and cis-acting elements in the stress responsive promoters function for plant adaptation to environmental stresses. Recent progress has been made in analyzing the complex cascades of gene expression in drought and cold stress responses, especially in identifying specificity and cross talk in stress signaling. In this review article, we highlight transcriptional regulation of gene expression in response to drought and cold stresses, with particular emphasis on the role of transcription factors and cis-acting elements in stress-inducible promoters. PMID- 16669783 TI - Pyrimidine and purine biosynthesis and degradation in plants. AB - Nucleotide metabolism operates in all living organisms, embodies an evolutionarily ancient and indispensable complex of metabolic pathways and is of utmost importance for plant metabolism and development. In plants, nucleotides can be synthesized de novo from 5-phosphoribosyl-1-pyrophosphate and simple molecules (e.g., CO(2), amino acids, and tetrahydrofolate), or be derived from preformed nucleosides and nucleobases via salvage reactions. Nucleotides are degraded to simple metabolites, and this process permits the recycling of phosphate, nitrogen, and carbon into central metabolic pools. Despite extensive biochemical knowledge about purine and pyrimidine metabolism, comprehensive studies of the regulation of this metabolism in plants are only starting to emerge. Here we review progress in molecular aspects and recent studies on the regulation and manipulation of nucleotide metabolism in plants. PMID- 16669785 TI - Microtubule dynamics and organization in the plant cortical array. AB - Live-cell studies have brought fresh insight into the organizational activities of the plant cortical array. Plant interphase arrays organize in the absence of a discrete microtubule organizing center, having plus and minus ends distributed throughout the cell cortex. Microtubule nucleation occurs at the cell cortex, frequently followed by minus-end detachment from origin sites. Microtubules associate tightly with the cell cortex, resisting lateral and axial translocation. Slow, intermitant loss of dimers from minus ends, coupled with growth-biased dynamic instability at the plus ends, results in the migration of cortically attached microtubules across the cell via polymer treadmilling. Microtubule-microtubule interactions, a direct consequence of treadmilling, result in polymer reorientation and creation of polymer bundles. The combined properties of microtubule dynamics and interactions among polymers constitute a system with predicted properties of self-organization. PMID- 16669784 TI - Phytochrome structure and signaling mechanisms. AB - Phytochromes are a widespread family of red/far-red responsive photoreceptors first discovered in plants, where they constitute one of the three main classes of photomorphogenesis regulators. All phytochromes utilize covalently attached bilin chromophores that enable photoconversion between red-absorbing (P(r)) and far-red-absorbing (P(fr)) forms. Phytochromes are thus photoswitchable photosensors; canonical phytochromes have a conserved N-terminal photosensory core and a C-terminal regulatory region, which typically includes a histidine kinase-related domain. The discovery of new bacterial and cyanobacterial members of the phytochrome family within the last decade has greatly aided biochemical and structural characterization of this family, with the first crystal structure of a bacteriophytochrome photosensory core appearing in 2005. This structure and other recent biochemical studies have provided exciting new insights into the structure of phytochrome, the photoconversion process that is central to light sensing, and the mechanism of signal transfer by this important family of photoreceptors. PMID- 16669786 TI - The von Hippel-Lindau tumour-suppressor protein interaction with protein kinase Cdelta. AB - The VHL (von Hippel-Lindau) tumour-suppressor protein forms a multi-protein complex [VCB (pVHL-elongin C-elongin B)-Cul-2 (Cullin-2)] with elongin C, elongin B, Cul-2 and Rbx1, acting as a ubiquitin-ligase (E3) and directing proteasome dependent degradation of targeted proteins. The alpha-subunit of Hif1alpha (hypoxia-inducible factor 1alpha) is the principal substrate for the VCB-Cul-2 complex; however, other substrates such as aPKC (atypical protein kinase C) have been reported. In the present study, we show with FRET (fluorescence resonance energy transfer) analysis measured by FLIM (fluorescence lifetime imaging microscopy) that PKCdelta and pVHL (VHL protein) interact directly in cells. This occurs through the catalytic domain of PKCdelta (residues 432-508), which appears to interact with two regions of pVHL, residues 113-122 and 130-154. Despite this robust interaction, analysis of the PMA-induced proteasome-dependent degradation of PKCdelta in different RCC (renal cell carcinoma) lines (RCC4, UMRC2 and 786 O) shows that there is no correlation between the degradation of PKCdelta and the presence of active pVHL. Thus, in contrast with aPKC, PKCdelta is not a conventional substrate of the ubiquitin-ligase complex, VCB-Cul-2, and the observed interaction between these two proteins must underlie a distinct signalling output. PMID- 16669787 TI - Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1. AB - The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4). PMID- 16669788 TI - Integrin beta3-mediated Src activation regulates apoptosis in IEC-6 cells via Akt and STAT3. AB - Intestinal epithelial (IEC-6) cells are resistant to apoptosis following the inhibition of ODC (ornithine decarboxylase) and subsequent polyamine depletion. The depletion of polyamines rapidly activates NF-kappaB (nuclear factor kappaB) and STAT3 (signal transducer and activator of transcription 3), which is responsible for the observed decrease in apoptosis. Since both NF-kappaB and STAT3 signalling pathways can be activated by Src kinase, we examined its role in the antiapoptotic response. Inhibition of ODC by DFMO (alpha difluoromethylornithine) increased the activity of Src and ERK1/2 (extracellular signal-regulated kinase 1/2) within 30 min, which was prevented by exogenous polyamines added to the DFMO-containing medium. Conversely, epidermal growth factor-mediated Src and ERK1/2 activation was not prevented by the addition of polyamines. Inhibition of Src with PP2 {4-amino-5-(4-chlorophenyl)-7-(t butyl)pyrazolo[3,4-d]pyrimidine} and a DN-Src (dominant-negative Src) construct prevented the activation of Akt, JAK (Janus kinase) and STAT3. Spontaneous apoptosis was increased in DN-Src-expressing cells and the protective effect of polyamine depletion was lost. Polyamine depletion by DFMO increased integrin beta3 Tyr785 phosphorylation. Cells plated on fibronectin had significantly higher beta3 phosphorylation and Src activation compared with plastic. Exogenous polyamines added to the fibronectin matrix prevented Src activation. Arg-Gly-Asp Ser inhibited beta3, Src and Akt phosphorylation and sensitized polyamine depleted cells to tumour necrosis factor alpha/cycloheximide-mediated apoptosis. Fibronectin activated Src and subsequently protected cells from apoptosis. Together, these results suggest that the inhibition of ODC rapidly removes a small pool of available polyamines triggering the activation of beta3 integrin, which in turn activates Src. The subsequent Akt and JAK activation is accompanied by translocation of NF-kappaB and STAT3 to the nucleus and the synthesis of antiapoptotic proteins. PMID- 16669789 TI - Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase. AB - Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases. PMID- 16669790 TI - Names in frames: infants interpret words in sentence frames faster than words in isolation. AB - In child-directed speech (CDS), adults often use utterances with very few words; many include short, frequently used sentence frames, while others consist of a single word in isolation. Do such features of CDS provide perceptual advantages for the child? Based on descriptive analyses of parental speech, some researchers argue that isolated words should help infants in word recognition by facilitating segmentation, while others predict no advantage. To address this question directly, we used online measures of speech processing in a looking-while listening procedure. In two experiments, 18-month-olds were presented with familiar object names in isolation and in a sentence frame. Infants were 120 ms slower to interpret target words in isolation than when the same words were preceded by a familiar carrier phrase, suggesting that the sentence frame facilitated word recognition. Familiar frames may enable the infant to 'listen ahead' more efficiently for the focused word at the end of the sentence. PMID- 16669792 TI - Dyslexia: what's the problem? PMID- 16669791 TI - The role of sensorimotor impairments in dyslexia: a multiple case study of dyslexic children. AB - This study attempts to investigate the role of sensorimotor impairments in the reading disability that characterizes dyslexia. Twenty-three children with dyslexia were compared to 22 control children, matched for age and non-verbal intelligence, on tasks assessing literacy as well as phonological, visual, auditory and motor abilities. The dyslexic group as a whole were significantly impaired on phonological, but not sensorimotor, tasks. Analysis of individual data suggests that the most common impairments were on phonological and visual stress tasks and the vast majority of dyslexics had one of these two impairments. Furthermore, phonological skill was able to account for variation in literacy skill, to the exclusion of all sensorimotor factors, while neither auditory nor motor skill predicted any variance in phonological skill. Visual stress seems to account for a small proportion of dyslexics, independently of the commonly reported phonological deficit. However, there is little evidence for a causal role of auditory, motor or other visual impairments. PMID- 16669793 TI - Sensorimotor impairments in dyslexia: getting the beat. PMID- 16669794 TI - Do cerebellar deficits underlie phonological problems in dyslexia? PMID- 16669795 TI - What happens when 'dyslexic' subjects do not meet the criteria for dyslexia and sensorimotor tasks are too difficult even for the controls? PMID- 16669797 TI - Are preschoolers sensitive to configural information in faces? AB - Several researchers have proposed that developmental improvements in children's face recognition abilities might reflect an increasing reliance on configural information (i.e. spatial relations between features) in faces (Carey & Diamond, 1994; Mondloch, Le Grand & Maurer, 2002). We investigated 4- and 5-year-olds' use of configural information for upright and inverted faces using Tanaka and Sengco's (1997) configural change paradigm. Participants saw a photograph of a child's face (e.g. Luke). Memory for features (e.g. Luke's mouth) was then tested in a face with the same configuration as the study face (e.g. Luke's face), in a face with a novel spatial configuration (e.g. Luke's face with the eyes shifted further apart), and presented on their own (e.g. Luke's mouth alone). We found that preschoolers and adults recognized target features from upright faces better when tested in the context of a face with the same configuration as the study face, than when they were embedded in a face with a new spatial configuration or when they were presented in isolation. This effect was lost when faces were inverted. The results suggest that adult-like styles of face processing are present from 4 years of age. PMID- 16669798 TI - Switching between tasks and responses: a developmental study. AB - Task switching requires the ability to flexibly switch between task rules and responses, and is sensitive to developmental change. We tested the hypothesis that developmental changes in task switch performance are associated with changes in the facilitating or interfering effect of the previously retrieved stimulus response (S-R) association. Three age groups (7-8-year-olds, 10-12-year-olds and 20-25-year-olds) performed a two-choice reaction time (RT) task in which spatially compatible or incompatible responses were required. The RT costs associated with switching between tasks were larger when responses were repeated than when responses were alternated. Younger children showed a greater cost than adults when switching between tasks but repeating responses. This age difference decreased when the interval between the previous response and the upcoming stimulus increased. Switch costs were larger when switching to the compatible task than to the incompatible task, but this effect did not differ between age groups. These findings suggest that young children build up stronger transient associations between task sets and response sets, which interfere with their ability to switch to currently intended actions. A similar pattern has previously been observed for older adults (Mayr, 2001), suggesting a common contributor to task switching deficits across the life span. PMID- 16669799 TI - The face behind the mask: a developmental study. AB - Faces are a rich and available source of social information, and the representation for faces is robust in adults (i.e. the face detection effect; Purcell & Stewart, 1988). The current study compared the developmental trajectory of the robustness of face perception against the trajectory for a non-face object. Participants (5-35 years old) were presented with rapid (17 and 33 millisecond) presentations of face and house stimuli and were instructed to identify the object category of the stimulus (face or house). There was an interaction between object type and age such that the developmental slope for face identification was steeper than the slope for house identification for the 17-millisecond presentation. These data show that faces are processed in a different way than a non-face object during the period from middle childhood through adolescence and adulthood, and this differential processing may involve the massive amount of exposure we have to faces. PMID- 16669800 TI - When the social mirror breaks: deficits in automatic, but not voluntary, mimicry of emotional facial expressions in autism. AB - Humans, infants and adults alike, automatically mimic a variety of behaviors. Such mimicry facilitates social functioning, including establishment of interpersonal rapport and understanding of other minds. This fundamental social process may thus be impaired in disorders such as autism characterized by socio emotional and communicative deficits. We examined automatic and voluntary mimicry of emotional facial expression among adolescents and adults with autistic spectrum disorders (ASD) and a typical sample matched on age, gender and verbal intelligence. Participants viewed pictures of happy and angry expressions while the activity over their cheek and brow muscle region was monitored with electromyography (EMG). ASD participants did not automatically mimic facial expressions whereas the typically developing participants did. However, both groups showed evidence of successful voluntary mimicry. The data suggest that autism is associated with an impairment of a basic automatic social-emotion process. Results have implications for understanding typical and atypical social cognition. PMID- 16669801 TI - The effect of divided attention on inhibiting the gravity error. AB - Children who could overcome the gravity error on Hood's (1995) tubes task were tested in a condition where they had to monitor two falling balls. This condition significantly impaired search performance with the majority of mistakes being gravity errors. In a second experiment, the effect of monitoring two balls was compared in the tubes task and a spatial transposition task not involving gravity. Again, monitoring two objects produced impaired search performance in the gravity task but not in the spatial transposition task. These findings support the view that divided attention disrupts the ability to exercise inhibitory control over the gravity error and that the performance drop on this task is not due to the additional task demands incurred by divided attention. PMID- 16669802 TI - Crossmodal integration of emotional information from face and voice in the infant brain. AB - We examined 7-month-old infants' processing of emotionally congruent and incongruent face-voice pairs using ERP measures. Infants watched facial expressions (happy or angry) and, after a delay of 400 ms, heard a word spoken with a prosody that was either emotionally congruent or incongruent with the face being presented. The ERP data revealed that the amplitude of a negative component and a subsequent positive component in infants' ERPs varied as a function of crossmodal emotional congruity. An emotionally incongruent prosody elicited a larger negative component in infants' ERPs than did an emotionally congruent prosody. Conversely, the amplitude of infants' positive component was larger to emotionally congruent than to incongruent prosody. Previous work has shown that an attenuation of the negative component and an enhancement of the later positive component in infants' ERPs reflect the recognition of an item. Thus, the current findings suggest that 7-month-olds integrate emotional information across modalities and recognize common affect in the face and voice. PMID- 16669803 TI - The shape of boubas: sound-shape correspondences in toddlers and adults. AB - A striking demonstration that sound-object correspondences are not completely arbitrary is that adults map nonsense words with rounded vowels (e.g. bouba) to rounded shapes and nonsense words with unrounded vowels (e.g. kiki) to angular shapes (Kohler, 1947; Ramachandran & Hubbard, 2001). Here we tested the bouba/kiki phenomenon in 2.5-year-old children and a control group of adults (n =20 per age), using four pairs of rounded versus pointed shapes and four contrasting pairs of nonsense words differing in vowel sound. Overall, participants at both ages matched words with rounded vowels to the rounder shapes and words with unrounded vowels to the pointed shapes (both ps < .0005), with no significant difference between the two ages (p > .10). Such naturally biased correspondences between sound and shape may influence the development of language. PMID- 16669804 TI - Choose and choose again: appearance-reality errors, pragmatics and logical ability. AB - In the Appearance/Reality (AR) task some 3- and 4-year-old children make perseverative errors: they choose the same word for the appearance and the function of a deceptive object. Are these errors specific to the AR task, or signs of a general question-answering problem? Preschoolers completed five tasks: AR; simple successive forced-choice question pairs (QP); flexible naming of objects (FN); working memory (WM) span; and indeterminacy detection (ID). AR errors correlated with QP errors. Insensitivity to indeterminacy predicted perseveration in both tasks. Neither WM span nor flexible naming predicted other measures. Age predicted sensitivity to indeterminacy. These findings suggest that AR tests measure a pragmatic understanding; specifically, different questions about a topic usually call for different answers. This understanding is related to the ability to detect indeterminacy of each question in a series. AR errors are unrelated to the ability to represent an object as belonging to multiple categories, to working memory span, or to inhibiting previously activated words. PMID- 16669805 TI - Constructing health and sickness in the context of motherhood and paid work. AB - Changes in the labour market, especially the rise in the employment of women (lone or partnered) with children, alongside an increased policy emphasis on work as a component of active citizenship for men and women, have stimulated the development of research examining the balance between work and home. Although sociologists have long been interested in the interface between the spheres of paid work and domestic life, understandings of the subjective experience of health and illness have tended to keep the domains of family and work separate. This paper addresses the construction of health and illness as operating at the interface between the worlds of work and home. Interviews were conducted with 30 mothers in paid work and having primary school aged children; the study was located in Edinburgh, Scotland. Through an analysis of the interview accounts, this paper examines respondents' experiences and constructions of health, sickness and wellbeing in themselves and in their children. Four areas are discussed: respondents' accounts of the effects of caring and providing on their own health; respondents' accounts of the influence of workplace relationships in the construction of sickness; respondents' accounts of negotiating absence for their children's sickness and how they made sense of and defined child sickness. We argue that managing sickness, itself an anticipated but unpredictable event, gives analytical purchase to understanding the values and practices that characterise the interrelationship between work and family life. The intersections of home and work operate powerfully in respondents' constructions of health and sickness, and the analysis demonstrates how these are played out in everyday life, at home and at work. PMID- 16669806 TI - Embodied masculinity and androgen deprivation therapy. AB - This paper describes the findings from an ethnographic study of 16 Anglo Australian men treated with androgen deprivation therapy (ADT) for advanced prostate cancer. Utilising a social constructionist gendered analysis, participants' experiences, particularly in relation to embodied masculinity, are described in the context of reduced testosterone that accompany ADT. The findings indicated that participants reformulated many ideals of hegemonic masculinity in response to functional body changes. However, hegemonic masculinity strongly influenced participants' philosophical resolve to "fight" prostate cancer. The findings are considered in broader ongoing debates about essentialist sex and the social construction of gender. PMID- 16669807 TI - "I've been like a coiled spring this last week": embodied masculinity and health. AB - This paper draws on research exploring lay men's and community health professionals' attitudes towards 'masculinity' and 'preventative health care' conducted in the northwest of England. It is specifically concerned with the findings from the men's narratives that relate to male embodiment and the relevance of this to health and wellbeing. Whilst there is a burgeoning interest in the 'sociology of the body' it is only relatively recently that debates about embodiment have been supported by empirical research. Empirical work on male embodiment remains minimal and even less work has been done in considering the links between masculinity, embodiment and health. A series of focus groups and interviews were conducted with men that captured narratives about the lived (male) body in everyday life and its relation to health. These data allowed for greater insight into Watson's (2000) previous empirical work on the 'male body schema'. Current findings presented here suggest that male bodies need to be considered as both material and representational, and that these two modes are not distinct but continually interact, with each other and with men's conceptualisations of health, to influence health practices and wider social interactions that impact on health and wellbeing. PMID- 16669808 TI - E-dating, identity and HIV prevention: theorising sexualities, risk and network society. AB - This paper addresses how London gay men use the internet to meet sexual partners, or for e-dating. Based on qualitative interviews conducted face-to-face or via the internet, this research develops an account of how information technologies mediate the negotiation of identity and risk in connection with sexual practice. E-dating itself is a bricolage, or heterogeneous DIY practice of internet-based communication (IBC). A central aspect of IBC is "filtering" in and out prospective e-dates based on the images and texts used to depict sexual identities. Interpretations and depictions of personal HIV risk management approaches in IBC are framed by the meanings of different identities, such as the stigma associated with being HIV positive. This paper argues for a sexualities perspective in a theory of network society. Further, HIV prevention in e-dating can potentially be addressed by considering the interplay of the HIV prevention imperatives associated with different HIV serostatus identities. There is a case for encouraging more explicit IBC about risk in e-dating and incorporating the expertise of e-daters in prevention activity. There is also a need to rethink traditional conceptions of risk management in HIV prevention to make space for the risk management bricolage of network society. PMID- 16669809 TI - Risk, life extension and the pursuit of medical possibility. AB - With increasing frequency, the oldest members of US society are undergoing medical interventions aimed at prolonging life. Using cardiac care as a case study, this paper explores how a discourse of risk infuses and legitimates high tech clinical treatments in late life. In particular, we examine how the diminishing risks associated with biomedical procedures produce a sense of medical possibility regarding life extension, and push the definition of "old age" into a receding future. Simultaneously, physicians, patients and families come to understand the management and reduction of future cardiac risks to be germane for individuals even near the end of life. Driven by the logic and language of risk, decisions to intervene are experienced as incremental and largely unremarkable, and the pursuit of an open-ended future via biomedical means is perceived as an ethical imperative, trumping deliberation or discussion of the utility of intervention and the ultimate ends being pursued. For practitioners and patients alike, the engagement of risk, the preservation of hope it facilitates and the routinisation of intervention it produces all contribute to the emerging mandate to treat at ever-older ages. PMID- 16669821 TI - Reducing recurrence and costs in superficial bladder cancer: preclinical evaluation of osmotic cytolysis by distilled water vs. mitomycin. AB - Transurethral resection followed by instillation of chemotherapeutic agents such as mitomycin is considered as standard therapy in recurrent superficial bladder cancer. However, incidence of bladder cancer is increasing, and contrariwise resources to finance the health care systems are decreasing. Therefore, effective alternatives to expensive chemotherapeutics are necessary. Recurrence of bladder cancer after transurethral resection is mostly promoted by reimplantation of dissolved tumour cells which are therapeutical purpose of any intravesically instillated agent. Monolayer cultures of human RT112, RT4, T24 and TCC SUP bladder cancer cells were incubated and exposed to mitomycin or distilled water. Cell survival was determined by microculture tetrazolium assay. Distilled water led to significant cytolysis in all tumour cells. This effect was comparable to exposition to mitomycin. Distilled water and mitomycin have comparable in vitro effects in bladder cancer cells. These findings have to be substantiated by an animal model emphasising the aspect of larger tumour cell compounds or possible damage to healthy bladder tissue. PMID- 16669822 TI - Prevalence of hepatitis A virus infection among municipal solid-waste workers. AB - A study was conducted among 151 municipal workers (72 solid-waste workers, and 79 workers not exposed to waste). Total antibodies against Hepatitis A virus (HAV) were measured, and socio-demographic information was collected using a self administered questionnaire. Univariate analysis has shown that occupational exposure to waste, age, duration of employment and educational status were significantly associated with the prevalence of anti-HAV(+). Municipal Solid Waste Workers had a higher prevalence of anti-HAV(+) in comparison with municipal workers not exposed to waste. Duration of employment was significantly associated anti-HAV(+). Multivariate analysis revealed an independent association of anti HAV(+) with occupational exposure to waste and ageing. Our results suggest a potential causal role of occupational exposure to waste, in the development of HAV infection. PMID- 16669823 TI - Diuretic-induced hypokalaemia in patients with hypertension. AB - We describe a 55 year-old woman with longstanding hypertension who developed hypokalaemia following diuretic treatment. Investigations revealed primary hyperaldosteronism due to an adrenal adenoma, and normal blood pressure was restored after surgical removal of the tumour. Primary hyperaldosteronism is a potentially curable cause of hypertension and should be considered in hypertensive patients who present with diuretic - induced hypokalaemia. PMID- 16669824 TI - Intra-aortic stent graft in oesophageal carcinoma invading the aorta. Prophylaxis for fatal haemorrhage. AB - In patients with advanced oesophageal carcinoma with aortic invasion, any therapy potentially causes fatal haemorrhage. We describe here the successful application of intra-aortic stent graft to prevent haemorrhage before radical oesophagectomy for advanced oesophageal cancer. Four patients with advanced oesophageal cancer complicated by invasion of the aorta. Under general anaesthesia, aortic invasion is evaluated by an intravascular sonography. The stent graft is passed through the right femoral artery into the descending aorta. Subsequently, the stent graft is released to expand in the thoracic aorta during an artificial cardiac arrest. Aortography is performed to check for any stent migration or endoleakage. This procedure was successful in all four patients without any complications. All patients underwent radical oesophagectomy following aortic stent-grafting. One patient survived more than 2 years after stent grafting and operation. This procedure is safe and applicable for the patient with aortic invasion before radiochemotherapy or operation. PMID- 16669825 TI - Diagnostic value of leptin in tuberculous pleural effusions. AB - It is suggested that leptin may be involved in inflammation. Although relation between leptin levels and active pulmonary tuberculosis has been studied, there is no information about relation between leptin levels and tuberculous pleural effusions (TPE). We evaluated the diagnostic value of pleural fluid and serum leptin levels in TPE and compared them with adenosine deaminase (ADA). Forty-five patients, 17 tuberculous effusion and 28 nontuberculous effusion, with exudative pleural effusions were included. Leptin and ADA levels were measured from serum and pleural fluid in all patients. There were no statistically significant differences between tuberculous and nontuberculous groups with respect to the serum ADA activity and pleural fluid/serum leptin ratio. On the contrary, pleural fluid leptin level, pleural fluid ADA activity, serum leptin level and pleural fluid/serum ADA activity ratio were statistically different between tuberculous and nontuberculous groups. When leptin levels were corrected for body mass index, serum leptin levels did not reach statistical significance. Cut-off points to predict tuberculosis were calculated as 9.85 ng/ml and 35.55 U/l for pleural fluid leptin level and pleural fluid ADA activity, respectively. Sensitivity, specificity and area under the curve +/- standard error were 82.4%, 82.1%, 0.83 +/- 0.07 for pleural fluid leptin levels and 100%, 100%, 1.00 +/- 0.00 for pleural fluid ADA activity, respectively; the difference between these curves was significant (p = 0.01). Pleural fluid leptin levels were lower in tuberculous effusions than in other exudates. Pleural fluid leptin has a diagnostic value for TPE but not as good as that of ADA. PMID- 16669826 TI - Does Ramadan fasting affect sleep? AB - Experimental fasting has been shown to alter the sleep-wakefulness pattern in various species. As fasting during Ramadan is distinct from experimental fasting, the physiological and behavioural changes occurring during Ramadan fasting may differ from those occurring during experimental fasting. There has been increased interest in recent years in sleep changes and daytime sleepiness during Ramadan. Moreover, many of those who fast during Ramadan associate this fasting with increased daytime sleepiness and decreased performance. This raises the question of whether Ramadan fasting affects sleep. In this review, we discuss the findings of research conducted to assess changes in sleep pattern, chronobiology, circadian rhythms, daytime sleepiness and function and sleep architecture during the month of Ramadan. Where applicable, these findings are compared with those obtained during experimental fasting. PMID- 16669827 TI - Visceral fat thickness determined using ultrasonography is associated with anthropometric and clinical parameters of metabolic syndrome. AB - The aim of this study is to find out the relation between the ultrasonographic (USG) measurements of the abdominal fat thickness and cardiovascular risk factors in metabolic syndrome. The thickness of subcutaneous fat (SF), visceral fat (VF) and preperitoneal fat (PF) was measured using USG in 75 subjects (35 women and 40 men) with metabolic syndrome. The body mass index (BMI), waist circumference (WC), systolic (SBP) and diastolic (DBP) blood pressures, fasting plasma glucose, fasting insulin and lipid parameters of all participants were recorded. Insulin resistance was estimated using HOMA-IR formula. BMI (p < 0.05), WC (p < 0.01), SBP (p < 0.001), DBP (p < 0.05), fasting insulin (p < 0.05), total cholesterol (p < 0.001) and triglyceride (p < 0.001) levels were found in correlation with VF thickness in the female group. There was a positive association between WC and SF thickness (p < 0.05) in the same group. In the male patients, BMI (p < 0.001), WC (p < 0.01), SBP (p < 0.05), DBP (p < 0.05) and triglyceride level (p = 0.01) were significantly correlated with VF thickness. SF thickness was associated with BMI (p < 0.001) and WC (p < 0.01) in this group. There was no relation between PF thickness and clinical variables in both groups (p > 0.05). It can be concluded that VF thickness may have a significant pathophysiological role in the development of the metabolic syndrome. PMID- 16669828 TI - The validity of a hyperventilation test for an investigation of autonomic failure: assessment in patients with multiple system atrophy and Parkinson's disease. AB - Although heart rate (HR) responses to hyperventilation (HV) have been used as a cardiovascular autonomic function test, autonomic involvement during HV remains uncertain. To clarify the relationship between autonomic activity and cardiovascular changes during HV, we compared cardiovascular responses during HV among subjects with different autonomic function, namely 16 patients with probable multiple system atrophy (MSA), 16 with possible MSA, 28 with Parkinson's disease (PD) and 28 healthy controls. Abnormalities of cardiovascular responses to head-up postural change and the Valsalva maneuver were definitely present in the order of probable MSA, possible MSA and PD, and abnormal HR and blood pressure (BP) responses during HV were observed in probable MSA and possible MSA, but not in PD. Unlike the significant difference in standard cardiovascular autonomic function tests, the HR and BP responses during HV were equivalent between probable and possible MSA. These findings suggest that cardiovascular control during HV may be affected not only by autonomic activity but also by other factors. PMID- 16669829 TI - The role of fluoroscopy in the removal of tracheobronchial pin aspiration. AB - In this study, we investigated the role of fluoroscopy in the bronchoscopic removal of aspirated pins. Of 373 patients who underwent bronchoscopy for presumed tracheobronchial foreign body aspiration, 56 pin aspiration cases were selected and divided into two groups according to whether fluoroscopic guidance was required (group I) or not (group II). The localisation of foreign bodies, mortality and morbidity ratios and the duration of the procedures were investigated. Pin aspiration percentage was 15. Pins were mostly located in peripheral airways in group I, and in central airways in group II (p < 0.05). The mean duration of the procedure was 42 +/- 30 min in group I and 17 +/- 13 min in group II (p < 0.01). There was no mortality. Morbidity percentage was 7 in group I and 12 in group II (p > 0.05). No thoracotomy was required in any cases. Fluoroscopic guidance is safe and carries no additional morbidity and mortality and can be used for pins in the tracheobronchial tree. PMID- 16669830 TI - The consistency of emergency physicians' and cardiologists' ECG interpretation and likelihood classification of chest pain patients. AB - Patients presenting to the emergency department with chest pain are evaluated by emergency physicians in hospitals without cardiology cover 24 h a day. The purpose of this study is to determine the consistency of electrocardiography (ECG) interpretation and chest pain likelihood classification between emergency physicians and cardiologists. This randomised prospective cross-sectional study was performed in a tertiary care university hospital emergency department. The study form included ECG interpretation and chest pain likelihood classification according to American College of Cardiology (ACC)/American Heart Association (AHA) guideline which were recorded by emergency physicians and cardiologists separately in a blinded fashion. All chest pain patients who consulted with a cardiologist were enrolled into the study during the study period. The consistency between the two groups and the kappa value were calculated. Recorded study forms of 133 patients with cardiology consultations were evaluated. The consistency in the interpretation of ECG between the emergency physicians and cardiologists was found to be 94.6% (kappa = 0.85) for ST segment elevation, 78.6% (kappa = 0.57) for ischaemic ECG findings and 79.3% (kappa = 0.36) for dynamic ECG changes. The consistency for the likelihood classification between two groups for predicting the pain as angina or non-cardiac was 90.8% (kappa = 0.30), for classifying as acute coronary syndrome or stable angina pectoris (SAP) was 95.6% (kappa = 0.26) and for classifying patients as low likelihood or intermediate-high likelihood was 86.3% (kappa = 0.61). A strong consistency was shown between the emergency physicians' and cardiologists' ECG interpretation especially in determining the ST segment elevation. And also, there is a strong concordance in the likelihood classification of chest pain patients. PMID- 16669831 TI - Thrombin-antithrombin complex and alpha-plasmin inhibitor-plasmin complex levels in singleton and twin pregnancies. AB - Thrombin-antithrombin complex (TAT) is a sensitive marker for thrombin formation, and alpha2-plasmin inhibitor-plasmin complex (PIC) is a marker for fibrinolysis. Previous studies have shown that blood coagulation and fibrinolysis are activated in normal pregnancies. Thus, the present study examined changes in TAT and PIC levels during the course of singleton and twin pregnancies and compared these markers between the two pregnant groups. The subjects were 148 pregnant women, of whom 84 were women carrying single fetuses and 64 were women with twin fetuses. Their average gestational age at entry was 10, 20, 30 and 38 weeks of gestation in singleton pregnancies and 10, 20, 30 and 35 weeks in twin pregnancies. Peripheral blood was collected, and plasma was separated after centrifugation and then stored at -20 degrees C. The TAT levels increased significantly with the progression of pregnancy in both pregnant groups, whereas the PIC levels increased significantly only in singleton pregnancies between 30 and 38 weeks of gestation. The TAT levels were significantly higher in twin pregnancies than in singleton pregnancies. The PIC levels were significantly lower in twin pregnancies than in singleton pregnancies at 35-38 weeks. Thrombin formation might be enhanced with the progression of pregnancy in both pregnant groups. This enhancement might be more intensified in twin pregnancies. Fibrinolysis might increase only in singleton pregnancies from 30 to 38 weeks of gestation. Fibrinolysis might be less intensified in twin pregnancies than in singleton pregnancies only at 35-38 weeks. PMID- 16669832 TI - Seizure development after stroke. AB - Although there have been many studies on seizures following stroke, there is still much we do not know about them. In this study, we evaluated the characteristics of seizures in stroke patients. There were 2267 patients with a first-ever stroke, and after excluding 387 patients, 1880 were available for analysis. Of these 1880 patients, we evaluated 200 patients with seizures and 400 patients without seizures. We investigated the seizures according to age, gender, stroke type, the aetiology of ischaemic stroke and the localisation of the lesion. The seizures were classified as early onset and late onset and the seizure type as partial, generalised or secondarily generalised. Seizures occurred in 200 (10.6%) of 1880 strokes. The number of patients with seizures were 138 (10.6%) in ischaemic stroke group and 62 (10.7%) in haemorrhagic stroke group. Patients with ischaemic strokes had 41 embolic (29.7%) and 97 thrombotic (70.3%) origin, and these were not statistically significant in comparison with controls. Cortical involvement for the development of seizures was the most important risk factor (odds ratios = 4.25, p < 0.01). It was concluded that embolic strokes, being younger than 65 years old, and cortical localisation of stroke were important risks for developing seizures. PMID- 16669833 TI - Evaluation and analysis of colonoscopy in the diagnosis of 186 cases of ulcerative colitis patients. AB - The objective of this study was to evaluate the diagnostic value of colonoscopy plus biopsy in patients with ulcerative colitis (UC). Retrospective analysis was performed on clinical data in 186 cases. Erosions, or ulcers, together with mucosal hyperemia and oedema were the most common manifestations of colonoscopy in 87% of patients. In about 56.4% of 186 cases, such manifestations occurred in the rectum and the sigmoid colon. Nearly 65.6% of the patients had a chronic intermittent clinical course. One case developed colon cancer, and another case had toxic megacolon; each case represents 0.05% of the total 186 patients. Therefore, prevalence of both malignancy and complication is low. Colonoscopy plus biopsy is considered to be the major means of the diagnosis of UC, demonstrating its value in differential diagnosis. PMID- 16669834 TI - Frequencies of serum antibodies to Helicobacter pylori CagA and VacA in a Turkish population with various gastroduodenal diseases. AB - Infection with Helicobacter pylori (H. pylori) strains secreting cytotoxin associated gene A (CagA) and vacuolating cytotoxin A (VacA) proteins is associated with more severe gastroduodenal pathologies. However, this association varies among geographical regions and ethnic groups. We investigated the frequencies of antibodies to CagA and VacA proteins in 131 H. pylori-infected dyspeptic patients [40 duodenal ulcer (DU), 19 gastric ulcer (GU), 28 gastric cancer (GC), and 44 non-ulcer dyspepsia (NUD)] across 30 H. pylori-infected and endoscopically normal asymptomatic subjects (AS). Anti-CagA and anti-VacA antibodies were detected by Western blotting. The positivity rates of anti-CagA and anti-VacA antibodies were higher in patients with DU (92.5 and 75%), GU (89.5 and 84.2%) and GC (96.4 and 85.7%) than patients with NUD (70.5 and 50%) and AS (50 and 23.3%) (p < 0.05). CagA+ VacA+ phenotype was more frequent in patients with DU, GU and GC than patients with NUD and AS (75, 84.2, 85.7 vs. 47.7 and 20%, respectively) (p < 0.01). Our results showed that there is a significantly positive association between the presence of anti-CagA and anti-VacA antibodies and DU, GU and GC in our region. PMID- 16669835 TI - Restenosis after carotid endarterectomy. AB - Multiple randomised trials over the last decade for both symptomatic and asymptomatic carotid stenosis have proven the efficacy of carotid endarterectomy (CE) in reducing the risk of stroke. The long-term patency of the carotid artery after CE is an important factor in the success of the operation. The incidence of recurrent carotid stenosis (excluding residual lesions) ranges from 1 to 37% with only 0-8% of patients having restenosis-related symptoms (1). Generally, recurrent carotid stenosis is attributed to myointimal hyperplasia during the early postoperative period (within 3 years) or recurrent atherosclerosis thereafter. The management of recurrent carotid stenosis after CE remains a dilemma. It is generally accepted that operation for significant recurrent carotid stenosis is indicated for symptomatic patients, and several authors also recommend CE for >80% asymptomatic recurrent stenosis. Treatment of recurrent carotid stenosis involves repeat endarterectomy with patch angioplasty, although more recently endovascular techniques have been used. PMID- 16669836 TI - Abdominal malignancy masquerading as gastro-oesophageal reflux. AB - Gastro-oesophageal reflux disease is a common condition, and the typical symptoms of postprandial heartburn and upper abdominal pain usually respond well to acid suppressive medication. Occasionally, the gastro-oesophageal reflux is secondary to gastric outlet obstruction, and rarely, it is due to more distal intestinal obstruction. Two patients are described who seemed to have a primary diagnosis of gastro-oesophageal reflux but who were subsequently found to have disseminated intra-abdominal malignancy. Further investigation beyond the endoscopic confirmation of oesophagitis should be considered in patients who have typical gastro-oesophageal reflux symptoms but who also have any associated worrying clinical features. PMID- 16669837 TI - Parotid mass due to cat scratch disease. AB - Cat scratch disease (CSD), due to Bartonella henselae, is a self-limited chronic lymphadenopathy. A previously healthy 22-year-old woman presented with a palpable painful swelling in the right submandibular region accompanied by enlarged cervical lymph nodes. A diagnosis of B. henselae infection was made according to her personal history that divulged frequent contacts with cats and to a high titre of immunoglobulin G (IgG) and IgM antibodies for this agent. The patient improved within 1 month without the requirement of antibiotic treatment or surgery. The CSD should always be included in the differential diagnosis of all equivocal masses in the neck, especially in young individuals. In addition, it is important that a meticulous personal history is obtained. PMID- 16669838 TI - Brucellar spondylodiscitis: a case report. AB - Brucellosis is a common zoonosis which still remains as a major health problem in certain parts of the world. Osteoarticular involvement is the most frequent complication of brucellosis, in which the diagnosis of brucellar spondylodiscitis is often difficult since the clinical presentation may be obscured by many other conditions. Herein, we reported an uncommon case of spondylodiscitis due to brucella in an elderly male who had diabetes mellitus and degenerative spinal disease as underlying conditions. The diagnosis was established by using magnetic resonance imaging after the brucella-agglutination test was found to be positive. The diagnosis was also confirmed by positive blood culture. A high degree of suspicion in the diagnosis of brucellar spondylitis is essential to reduce the delay for the treatment. Thus, it should be essentially included in the differential diagnosis of longstanding back pain particularly in regions where brucellosis is endemic. Screening serologic tests for brucella should be used more widely in cases with low index of suspicion, especially in endemic areas. PMID- 16669839 TI - A prescription for better prescribing. PMID- 16669841 TI - The implications of a growing evidence base for drug use in elderly patients. Part 1. Statins for primary and secondary cardiovascular prevention. AB - Although elderly patients represent a rapidly growing population often requiring multiple drug treatment, the evidence of effectiveness is limited for many interventions and therapies in this age group. Only during the last 30 years has a requirement to incorporate evidence into the treatment of older subjects become part of the pre- and postmarketing regulatory process in Europe and the United States. Recently, elderly patients have been shown to benefit comparably from several treatments. These studies have supported the validity of an increasingly interventional approach to disorders common in late life. However, an important issue is the applicability of the growing body of clinical trials to 'real life' patients. This is particularly true in very old (i.e. >80 years) patients and those with significant comorbidities. We review the current evidence and controversies related to the effectiveness and safety of several therapeutic strategies in cardiovascular disease (i.e. statins, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, beta-adrenoceptor blockers, and thrombolytic agents) and bone health (i.e. vitamin D and bisphosphonates). PMID- 16669842 TI - The implications of a growing evidence base for drug use in elderly patients Part 2. ACE inhibitors and angiotensin receptor blockers in heart failure and high cardiovascular risk patients. AB - Traditionally, angiotensin converting enzyme (ACE) inhibitors have been used for the management of patients with congestive cardiac failure. Studies performed over the last decade have demonstrated that (1) angiotensin receptor blockers (ARBs) are as effective as ACE inhibitors in reducing morbidity and mortality in cardiac failure; and (2) inhibition of the renin-angiotensin system provides beneficial effects in patients at high cardiovascular risk without cardiac failure. This review focuses on the applicability of the results of the main trials with ACE inhibitors and ARBs to the elderly population. PMID- 16669843 TI - The implications of a growing evidence base for drug use in elderly patients. Part 3. Beta-adrenoceptor blockers in heart failure and thrombolytics in acute myocardial infarction. AB - Beta-adrenoceptor blockers and thrombolytic agents are of established value in the pharmacological management of heart failure and ST-elevation myocardial infarction, respectively. However, there is uncertainty as to whether these therapeutic strategies can be safely and effectively adopted in elderly patients with comorbidities, particularly in old-old individuals. This review focuses on these trials and the age-related efficacy and safety of these drugs. PMID- 16669844 TI - The implications of a growing evidence base for drug use in elderly patients. Part 4. Vitamin D and bisphosphonates for fractures and osteoporosis. AB - Fractures are common in elderly subjects, disabling and occasionally fatal. Their incidence increases exponentially with age, with the commonest affected sites being the wrist, vertebrae, hip and humerus. Of these, hip fractures are the most relevant in terms of morbidity and financial cost. The increase in fracture rate with age is believed to result predominantly from age-related increases in the incidence of osteoporosis and falls. This article reviews the evidence for the use of vitamin D and bisphosphonates for the prevention of bone fractures and osteoporosis in elderly patients. PMID- 16669845 TI - Stereoselective interaction of manidipine and grapefruit juice: a new twist on an old tale. PMID- 16669846 TI - Effect of grapefruit juice on the disposition of manidipine enantiomers in healthy subjects. AB - AIM: To examine the effect of grapefruit juice, an inhibitor of CYP3A4 in the small intestine, on the disposition of manidipine enantiomers in healthy subjects. METHODS: A randomized cross-over study with at least a 2-week wash-out period was performed. Seven healthy male volunteers received an oral 40-mg dose of racemic manidipine after an overnight fast with either grapefruit juice (GFJ) or water, as a control study. Plasma concentrations of (S)- and (R)-manidipine were monitored up to 10 h after the dosing. RESULTS: The plasma concentrations of (S)-manidipine were significantly higher (P<0.001) than those of (R)-manidipine in the control phase with an S/R ratio for the AUC0-infinity of 1.62 (95% confidence interval 1.52, 1.73). GFJ significantly increased Cmax and AUC0 infinity of (S)-manidipine by 2.4-fold (P<0.01) and 2.3-fold (P<0.01), respectively, and Cmax and AUC0-infinity of (R)-manidipine were increased by 3.4 fold (P<0.01) and 3.0-fold (P<0.01), respectively. There were significant differences (P<0.01) in GFJ-mediated percentage increases in Cmax and AUC0 infinity of (S)-manidipine compared with those of (R)-manidipine. The S/R ratio for AUC0-infinity was significantly decreased from 1.6 to 1.2 during the GFJ phase (P<0.01). CONCLUSION: These results indicate that the stereoselective disposition of manidipine was altered by GFJ, as an inhibitor of CYP3A4. GFJ appears to affect this metabolic disposal of (R)-manidipine to a greater extent than that of (S)-manidipine. PMID- 16669847 TI - Effects of itraconazole and diltiazem on the pharmacokinetics of fexofenadine, a substrate of P-glycoprotein. AB - AIMS: Fexofenadine is a substrate of several drug transporters including P glycoprotein. Our objective was to evaluate the possible effects of two P glycoprotein inhibitors, itraconazole and diltiazem, on the pharmacokinetics of fexofenadine, a putative probe of P-glycoprotein activity in vivo, and compare the inhibitory effect between the two in healthy volunteers. METHODS: In a randomized three-phase crossover study, eight healthy volunteers were given oral doses of 100 mg itraconazole twice daily, 100 mg diltiazem twice daily or a placebo capsule twice daily (control) for 5 days. On the morning of day 5 each subject was given 120 mg fexofenadine, and plasma concentrations and urinary excretion of fexofenadine were measured up to 48 h after dosing. RESULTS: Itraconazole pretreatment significantly increased mean (+/-SD) peak plasma concentration (Cmax) of fexofenadine from 699 (+/-366) ng ml-1 to 1346 (+/-561) ng ml-1 (95% CI of differences 253, 1040; P<0.005) and the area under the plasma concentration-time curve [AUC0,infinity] from 4133 (+/-1776) ng ml-1 h to 11287 (+/-4552) ng ml-1 h (95% CI 3731, 10575; P<0.0001). Elimination half-life and renal clearance in the itraconazole phase were not altered significantly compared with those in the control phase. In contrast, diltiazem pretreatment did not affect Cmax (704+/-316 ng ml-1, 95% CI -145, 155), AUC0, infinity (4433+/-1565 ng ml-1 h, 95% CI -1353, 754), or other pharmacokinetic parameters of fexofenadine. CONCLUSIONS: Although some drug transporters other than P-glycoprotein are thought to play an important role in fexofenadine pharmacokinetics, itraconazole pretreatment increased fexofenadine exposure, probably due to the reduced first pass effect by inhibiting the P-glycoprotein activity. As diltiazem pretreatment did not alter fexofenadine pharmacokinetics, therapeutic doses of diltiazem are unlikely to affect the P-glycoprotein activity in vivo. PMID- 16669848 TI - Prediction of drug clearance in children from adults: a comparison of several allometric methods. AB - AIMS: In recent years with the advent of paediatric exclusivity and requirements to conduct clinical studies in children, the current emphasis is to find a safe and efficacious dose of a drug in children. It has been suggested that one can predict the clearance of a drug in children according to the equation: CL in the child=adult CLx(weight of the child/70)0.75. Considering the controversy surrounding the exponent of 0.75 for the prediction of clearance and lack of any systematic evaluation of the aforementioned proposal, the objectives of the study were as follows: (i) to determine if indeed the exponent 0.75 is the most suitable exponent for the prediction of clearance in children from adult data; (ii) to explore and search for other exponents that are more accurate or as good as 0.75; and (iii) to propose a new approach (if any) based on the findings of the current evaluation. METHODS: Six methods were used to predict clearance of drugs in children from adult data. Besides evaluating the exponent of 0.75, exponents of 0.80, 0.85 and 1.0 were also evaluated. An empirical approach based on kidney and liver weights was also examined. Based on the results of five methods, a sixth method was introduced. RESULTS: The results of the study indicate that no single method is suitable for all drugs or for all age groups. The exponents 0.75, 0.80, and 0.85 provided the same degree of accuracy or error in the prediction of clearance in children. CONCLUSIONS: Since no single method is suitable for all drugs or for all age groups. A combination of approaches is suggested which may help in improving the prediction of clearance in children from adult data. PMID- 16669849 TI - Paroxetine: population pharmacokinetic analysis in late-life depression using sparse concentration sampling. AB - AIM: To develop a population pharmacokinetic (PK) model using sparse sampling of long-term treatment with paroxetine in elderly depressed subjects, incorporating CYP2D6 genotype as well as other covariates. METHODS: Elderly subjects (age>or=70 years) with nonpsychotic, nonbipolar major depressive disorder from the inpatient and outpatient clinic were treated with paroxetine in a 5-year clinical trial investigating 'Maintenance Therapies in Late-Life Depression' (MTLD-2). Plasma concentrations were collected during regular visits. CYP2D6 genotype was determined using polymerase chain reaction (PCR) for each individual. A nonlinear mixed-effects model was developed with NONMEM for these subjects who received 10 40 mg day-1 of paroxetine during treatment. One- and two-compartment models with linear and nonlinear elimination (Michaelis-Menten) were evaluated. PK parameters as well as interindividual and residual variability were estimated. The effects of age, weight, sex, race and CYP2D6 genotypes on the pharmacokinetics of paroxetine were evaluated. RESULTS: One hundred and seventy-one subjects with a mean age of 77 years (range 69-95) and a mean weight of 72.0 kg (range 32.9 137.0) were enrolled in the MTLD-2 clinical trial. A total of 1970 paroxetine concentrations were available for population PK analyses. Approximately 10 samples were taken per subject. A two-compartment nonlinear PK model with additive and proportional error provided the best base model for description of the data. Weight and CYP2D6 polymorphisms were found to have a significant effect on maximal velocity (Vm), whereas sex had an effect on volume of distribution of the central compartment. The Vm estimates in each of the CYP2D6 phenotypic groups were: 125 microg h-1 in poor metabolizer (n=1), 182 microg h-1 in intermediate metabolizers (n=28), 454 microg h-1 in extensive metabolizers (n=36) and 3670 microg h-1 in ultra-rapid metabolizers (n=5). CONCLUSIONS: The population PK model adequately described paroxetine data in this elderly depressed population. The data indicate that female and male subjects with different CYP2D6 polymorphisms have different elimination rates and therefore may need to be dosed differently based on metabolizer genotype. PMID- 16669850 TI - An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid. AB - AIMS: To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. METHODS: Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (KI and kinact) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. RESULTS: Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (KI and kinact) for isoniazid were 48.6 microm and 0.042 min-1 and 79.3 microm and 0.039 min-1. Clorgyline was a selective inactivator of CYP1A2 (6.8 microm and 0.15 min-1). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. CONCLUSIONS: These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors. PMID- 16669851 TI - Discrepancies between prescribed daily doses and WHO defined daily doses of antibacterials at a university hospital. AB - AIMS: The defined daily doses (DDD) defined by the WHO are widely used as an indicator to measure antibiotic use in the hospital setting. However, discrepancies exist between countries in terms of antibiotic dosage. The aim of the present study was to compare, for each antibacterial agent available at our university hospital, the prescribed daily doses (PDD) with the DDD. METHODS: Data were extracted from the pharmacy computer system. Antibiotic use was expressed in DDD per 1000 patient days. We also calculated the ratio of number of DDD:number of treatment-days and estimated the average PDD for each antibiotic and route of administration. RESULTS: The average PDD did not correspond to the DDD for many classes of antibiotics. If fluoroquinolones and cephalosporins were prescribed at a dosage close to the DDD, other antimicrobial classes such as penicillins, aminoglycosides or macrolides were not. Overall, the number of DDD overestimated the number of treatment days by 40%. For the most consumed antibiotic at our hospital, i.e. oral amoxicillin-clavulanic acid, the PDD was three times the DDD. CONCLUSIONS: Our study shows that, except for the fluoroquinolones and the cephalosporins, the number of DDD did not correctly reflect the number of antibiotic treatment days at our hospital. This does not invalidate the systematic approach of the WHO and hospitals should use the DDDs to make national and international comparisons of their antibiotic use. However, each hospital should define and validate its own indicators to describe the local exposures to antibiotics and to study the relationship with resistance. PMID- 16669852 TI - Prescribing behaviour according to Dutch and European guidelines on the management of hypercholesterolaemia (1992-1999). AB - BACKGROUND: The success of the full implementation of a new guideline may depend on the observed discrepancy between daily medical practice developed before the release of the guideline and new treatment recommendations issued by the guideline. AIM: To assess whether the initiation of statin treatment for primary prevention of cardiovascular disease in an elderly population was in agreement with guidelines. METHODS: Data were obtained from the Rotterdam Study, a prospective population-based cohort study consisting of 7983 subjects aged>or=55 years. In the period 1992-1999, all patients starting statins for primary prevention were selected. Treatment eligibility was established according to Dutch guidelines based on considerations of cost effectiveness (1998) and European guidelines based on clinical effectiveness (1998 and 2003). RESULTS: Only 5.7% [95% confidence interval (CI) 3.1, 8.3] of the 299 subjects starting statins for primary prevention met the eligibility criteria of the Dutch guidelines. Most patients (92.0%, 95% CI 88.9, 95.1) met the criteria of the 2003 European guidelines. Patients who did not meet any eligibility criteria were female and had one or less cardiovascular risk factor, except for two patients with total cholesterol levels<5 mmol l-1 prior to start of statin therapy. CONCLUSIONS: The use of statins was in agreement with the most recent European guidelines in over 90% of elderly patients who started statins for primary prevention, but in only 6% of these patients according to the Dutch guidelines. As long as existing guidelines are as discrepant as they are now, variation in agreement between physicians' prescribing and guideline recommendations is unavoidable. PMID- 16669854 TI - Access to emergency hormonal contraception from community pharmacies and family planning clinics. AB - AIMS: To evaluate differences in the time taken to access progestogen-only emergency hormonal contraception (EHC) by young women from family planning (FP) or community pharmacy settings. METHODS: An observational study of 203 women requesting EHC from FP clinics and community pharmacies in South-west Kent Primary Care Trust (PCT) from December 2002 to October 2003. RESULTS: Access to EHC from community pharmacy was significantly faster than from FP clinics (16 h vs. 41 h, P<0.001). Older teenagers tended to seek EHC more quickly and were more likely to have had a contraceptive failure rather than have used no contraception at all. CONCLUSION: The results provide further support for pharmacist involvement in the supply of EHC. PMID- 16669853 TI - Long-term and concentration-dependent beneficial effect of efavirenz on HDL cholesterol in HIV-infected patients. AB - AIMS: To investigate the long-term effects of efavirenz on cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL-C) and triglycerides (TG). METHODS: Thirty-four HIV-infected patients who commenced efavirenz therapy were monitored for 36 months. RESULTS: In patients with baseline HDL-C<40 mg.dL-1 an increase in HDL-C from 31+/-1 mg.dL-1 to 44+/-2 mg.dL-1 (95% confidence interval 5.9, 21.9, P<0.01) was observed and remained throughout the follow-up period. Median efavirenz plasma concentration was 1.98 mg.L-1 and a direct correlation between percentage of HDL-C variation or TC/HDL-C ratio and efavirenz plasma concentrations was found. CONCLUSIONS: There is evidence of a long-term and concentration-dependent beneficial effect of efavirenz on HDL-C in HIV-infected patients. PMID- 16669855 TI - Bilateral plantar tendinitis during levofloxacin therapy. PMID- 16669860 TI - Pharmacokinetics of the calcium-channel blocker diltiazem after a single intravenous dose in horses. AB - The pharmacokinetics of diltiazem were determined in eight healthy horses. Diltiazem HCl, 1 mg/kg i.v., was administered over 5 min. Venous blood samples were collected at regular intervals after administration. Plasma concentrations of diltiazem and desacetyldiltiazem were determined by high-performance liquid chromatography. A second, putative metabolite was detected, but could not be identified due to the lack of an authentic standard. Data were analyzed by nonlinear least-squares regression analysis. The median (minimum-maximum) peak plasma concentration of diltiazem was 727 (539-976) ng/mL. Plasma diltiazem concentration vs. time data were best described by a two-compartment model with first-order drug elimination. The distribution half-life was 12 (6-23) min, the terminal half-life was 93 (73-161) min, the mean residence time was 125 (99-206) min, total plasma clearance was 14.4 (10.4-18.6) mL/kg/min, and the volume of distribution at steady-state was 1.84 (1.46-2.51) L/kg. The normalized ratio of the area under the curve (AUC) of desacetyldiltiazem to the AUC of diltiazem was 0.088 (0.062-0.179). The disposition of diltiazem in horses was characterized by rapid distribution and elimination and a terminal half-life shorter than reported in humans and dogs. Because of the reported low pharmacologic activity, plasma diltiazem metabolite concentrations were not considered clinically important. PMID- 16669859 TI - Dexmedetomidine or medetomidine premedication before propofol-desflurane anaesthesia in dogs. AB - The objective of this study was to evaluate dexmedetomidine as a premedicant in dogs prior to propofol-desflurane anaesthesia, and to compare it with medetomidine. Six healthy dogs were anaesthetized. Each dog received intravenously (i.v.) five preanaesthetic protocols: D1 (dexmedetomidine, 1 microg/kg, i.v.), D2 (dexmedetomidine, 2 microg/kg, i.v.), M1 (medetomidine, 1 microg/kg, i.v.), M2 (medetomidine, 2 microg/kg, i.v.), or M4 (medetomidine, 4 microg/kg, i.v.). Anaesthesia was induced with propofol (2.3-3.3 mg/kg) and maintained with desflurane. The following variables were studied: heart rate (HR), mean arterial pressure, systolic arterial pressure, diastolic arterial pressure, respiratory rate (RR), arterial oxygen saturation, end-tidal CO2, end tidal concentration of desflurane (EtDES) required for maintenance of anaesthesia and tidal volume. Arterial blood pH (pHa) and arterial blood gas tensions (PaO2, PaCO2) were measured during anaesthesia. Time to extubation, time to sternal recumbency and time to standing were also recorded. HR and RR decreased significantly during sedation in all protocols. Cardiorespiratory variables during anaesthesia were statistically similar for all protocols. EtDES was significantly different between D1 (8.1%) and D2 (7.5%), and between all doses of medetomidine. Desflurane requirements were similar for D1 and M2, and for D2 and M4 protocols. No statistical differences were observed in recovery times. The combination of dexmedetomidine, propofol and desflurane appears to be effective for induction and maintenance of general anaesthesia in healthy dogs. PMID- 16669861 TI - Flow cytometric evaluation of selected antimicrobial efficacy for clearance of Anaplasma marginale in short-term erythrocyte cultures. AB - The tick-borne rickettsia, Anaplasma marginale, causes the economically important cattle disease anaplasmosis. Once infected, cattle remain lifelong carriers. Herein, we used flow cytometry to test the efficacy of three antimicrobials; oxytetracycline, imidocarb and enrofloxacin against Virginia (VGN) or Oklahoma (OK) A. marginale isolates in short-term erythrocyte cultures. Parasite viability was assessed using the vital dye hydroethidine (HE), which is detectable when living organisms convert HE to ethidium bromide. Viability of A. marginale in selected cultures was determined by subinoculation into susceptible calves. Data were analyzed by MANOVA, Tukey-Kramer honest significant difference and Wilcoxon rank sum tests. Receiver operating characteristic (ROC) analysis was used to correlate results with culture infectivity. Enrofloxacin inhibited A. marginale in a dose dependent manner. Surprisingly, higher concentrations of imidocarb were less effective than lower concentrations against A. marginale with significant differences (P < 0.05) observed between the two isolates. Oxytetracycline was the least active drug tested. Cultures infected with the OK isolate exposed to 4.0 microg/mL enrofloxacin and those of the VGN and OK isolates exposed to 1.0 microg/mL imidocarb were sterilized. This is the first in vitro study demonstrating the efficacy of enrofloxacin against A. marginale. Furthermore, these data indicate that flow cytometry is a useful assay for screening antimicrobials against A. marginale. PMID- 16669862 TI - Pharmacokinetics of ofloxacin in broiler chicken. AB - Pharmacokinetics of ofloxacin, a fluoroquinolone antimicrobial agent, was determined in broiler chickens after intravenous or oral administration of a single dose (10 mg/kg). Ofloxacin concentrations in plasma were determined using a high-performance liquid chromatography assay. Plasma concentration profiles were analyzed by the noncompartmental method. Elimination half-life and mean residence time of ofloxacin in plasma were 4.46 and 5.48 h after intravenous administration and 5.85 and 7.43 h, respectively, after oral administration. Maximal plasma concentration of 3.65 microg/mL was achieved at 1.25 h after oral administration. Apparent volume of distribution of 1.76 and 2.16 L/kg and total body clearance of 4.96 and 4.5 mL/min/kg were obtained following intravenous and oral administration, respectively. The oral bioavailability of ofloxacin was 110.01%. Ofloxacin was found to be more rapidly absorbed, widely distributed and more quickly eliminated than other fluoroquinolones in broilers. Based on these kinetic parameters, a dosage of 10 mg/kg given orally every 24 h can be recommended for the treatment of bacterial infections with MIC90 < 0.3 microg/mL. PMID- 16669863 TI - The pharmacokinetics of orbifloxacin in the horse following oral and intravenous administration. AB - The purpose of this study was to determine the pharmacokinetics and physicochemical characteristics of orbifloxacin in the horse. Six healthy adult horses were administered oral and intravenous orbifloxacin at a dose of 2.5 mg/kg. Plasma samples were collected and analyzed by high-pressure liquid chromatography with ultraviolet detection. Plasma protein binding and lipophilicity were determined in vitro. Following i.v. administration, orbifloxacin had a terminal half-life (t1/2) of 5.08 h and a volume of distribution (V(d(SS))) of 1.58 L/kg. Following oral administration, the average maximum plasma concentration (Cmax) was 1.25 microg/mL with a t1/2 of 3.42 h. Systemic bioavailability was 68.35%. Plasma protein binding was 20.64%. The octanol:water partition coefficient (pH 7.4) was 0.2 +/- 0.11. No adverse reactions were noted during this study. Dosage regimens were determined from the pharmacokinetic-pharmacodynamic parameters established for fluoroquinolone antibiotics. For susceptible bacteria, an oral dose of approximately 5 mg/kg once daily will produce plasma concentrations within the suggested range. This dose is suggested for further studies on the clinical efficacy of orbifloxacin for treatment of susceptible bacterial infections in the horse. PMID- 16669864 TI - Levamisole synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating ileal Peyer's patch T cells in weaned pigs. AB - Recent findings demonstrate that priming by levamisole of weaned pigs experimentally vaccinated against postweaning colibacillosis (PWC) contributes to immune protection from challenge-induced clinical disease through stimulation of the mesenteric lymph node cells that participate in cell-mediated immunity. With the objective of better understanding the mechanisms by which levamisole induces protective mucosal cell-mediated immune response to vaccination against PWC, it was tested whether the drug synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating T cells also in the jejunal lamina propria (JLP) and ileal Peyer's patch (IPP) upon virulent challenge. Commercial crossbred pigs weaned at 4 weeks were allocated into two equal groups. The experimental group was i.m. primed with levamisole at an immunostimulatory dose of 2.5 mg/kg once daily, for 3 consecutive days, and controls received saline. Both groups were vaccinated orally with the vaccinal E. coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. The results determined by immunophenotyping of isolated cells indicate that priming by levamisole of the vaccinated weaned pigs selectively recruited and activated T cells in the IPP, a lymphoid organ-generating B lymphocytes. The pig IPP is normally populated with up to 5% of CD3+ T cells and CD6 is an activation antigen expressed exclusively by T cells in swine. Therefore, a significantly higher number of CD3+ (P < 0.01) and CD6+ (P < 0.001) cells observed within the IPP follicles of the primed-vaccinated vs. unprimed-vaccinated challenge-infected pigs suggest enhanced T cell-mediated immunity in this B-cell compartment induced by the potentiating action of the drug and vaccine. The ability of levamisole to influence interaction between activated T cells and B cells in the IPP of primed vaccinated weaned pigs, and the possibility that this interaction plays a role in regulating B-cell maturation within the IPP follicles, are discussed. PMID- 16669865 TI - Pharmacokinetics of a novel closantel/ivermectin injection in cattle. AB - Two studies are described on the pharmacokinetics of a combination anthelmintic consisting of ivermectin and closantel for use in cattle. In the first, the pharmacokinetics of both active drugs in the combination were compared with the formulation with either ivermectin or closantel removed. No differences in the pharmacokinetics were observed, indicating that neither the absorption nor distribution of ivermectin or closantel in the combination were influenced by the presence of the other. In the second study the pharmacokinetics of ivermectin and closantel in the combination product were compared with control formulations of each. No difference was found between the closantel formulations. With ivermectin it was noted that absorption and excretion were more rapid and Cmax higher in the combination, although the AUC of both formulations were not significantly different. PMID- 16669866 TI - Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep. AB - Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity. PMID- 16669867 TI - Benazepril increases feed intake and body weight in healthy growing cats. PMID- 16669868 TI - Comparative pharmacokinetics of marbofloxacin after a single intramuscular administration at two dosages to camels (Camelus dromedarius). PMID- 16669869 TI - Exposure-to-treatment interval and clinical severity in canine poisoning: a retrospective analysis at a Portland veterinary emergency center. PMID- 16669871 TI - Ovarian epithelial tumors of low malignant potential: are they precursors of ovarian carcinoma? AB - Ovarian tumors of low malignant potential (LMP) are intermediate between benign adenoma and frank ovarian carcinoma, and are characterized by the absence of destructive stromal invasion, or microinvasion. If LMP did not develop into invasive carcinoma, then a minimally invasive simple treatment could be chosen. However, histological diagnosis cannot completely predict the prognosis of patients with ovarian tumors. It was found that mucinous LMP is most frequent in Japan, whereas serous LMP is the most common in Western countries. Mucinous LMP with loss of heterozygosity (LOH) at chromosomes 5q14-21 and 17q11.2, and serous LMP were suggested to be precursor lesions of ovarian carcinomas. Follow-up study revealed that patients with mucinous LMP who had LOH at 19q12 and/or Xq11-12 were at the greatest risk of progression. It is concluded that molecular genetic analysis such as LOH study could predict prognosis and aid in treatment decision making. The present review describes molecular genetic changes of LMP and presents problems on LMP that remain to be elucidated. PMID- 16669872 TI - Immunohistochemical analysis of thyroid-specific transcription factors in thyroid tumors. AB - Thyroid transcription factor 1 (TTF1), thyroid transcription factor 2 (TTF2) and paired box gene 8 (Pax8) are demonstrated to play a crucial role for the differentiation and organogenesis of thyroid follicular cells. Their roles in thyroid carcinogenesis are not very clear. Because dedifferentiation is a common process in thyroid carcinogenesis, thyroid-specific transcription factors seem also to be involving in thyroid carcinogenesis. The purpose of the present paper was to investigate their expression in a broad spectrum of follicular cell tumors in different degrees of differentiation, from well-differentiated benign follicular adenoma to anaplastic carcinoma. Medullary (C cell) carcinoma was also included in the investigation. Results of immunohistochemical staining showed that nuclear localization of these transcription factors was gradually decreased corresponding to the progressive dedifferentiation of thyroid tumors. Also, abnormal cytoplasmic accumulation of TTF2 and Pax8 was detected in many tumors samples, which may indicate a subtle regulation mechanism on the function of these transcription factors. In conclusion, abnormal expression of TTF1, TTF2 and Pax8 was closely related to thyroid tumorigenesis. PMID- 16669874 TI - Expression of vascular endothelial growth factor-C and its receptor in invasive micropapillary carcinoma of the breast. AB - Invasion to lymphatic vessels and metastasis to lymph nodes are frequent complications in invasive micropapillary carcinoma (IMPC) of human breast cancer. Vascular endothelial growth factor-C (VEGF-C) and its receptor, VEGFR-3 have been implicated as the important factors in the formation of lymphatic vessels and recent experimental evidence strongly suggests that lymphangiogenesis in tumor promotes lymphatic metastasis. To clarify the mechanism of its occurrence, the expression of VEGF-C, VEGFR-3 and lymphatic vessel density (LVD) was examined in 40 cases of IMPC (pure and mixed type) and in 40 cases of pseudo-IMPC. Cytoplasmic expression of VEGF-C and VEGFR-3 were more frequent in tumor cells of IMPC compared to those of pseudo-IMPC. A significant positive correlation was found between the expression of VEGF-C and VEGFR-3 in both IMPC and pseudo-IMPC. The expression of VEGF-C was also significantly associated with higher peritumoral LVD, lymphatic invasion and number of lymph node metastasis in IMPC. These findings suggest that VEGF-C promotes the proliferation of peritumoral lymphatic vessels and that lymphatic invasion and metastasis to lymph nodes are frequently induced in IMPC of breast. PMID- 16669873 TI - Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays. AB - Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD-A was treated with 12-O-tetradecanoylphorbol-13 acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time-course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease. PMID- 16669876 TI - Carcinoid tumor arising in a duplication cyst of the duodenum. AB - Reported herein is a case of carcinoid tumor arising in a duplication cyst of the duodenum in a 34-year-old woman. Ultrasonography at a health check-up revealed a 10 cm cystic mass located in the retroperitoneum adjacent to the third portion of the duodenum. Macroscopically, it had a smooth surface without communication with other organs and was filled with brownish necrotic fluid. More than half of the inner wall was occupied by a white, irregular, and solid nodule, which protruded into the lumen. The nodule was diagnosed as a carcinoid tumor invasive to the deep muscular layer. The non-neoplastic cyst wall had bowel structures including mucosa, submucosa and double layers of smooth muscle, indicating that the lesion was a duodenal duplication cyst. Most of the mucosa was eroded by marked inflammation. The remaining mucosa consisted of various types of epithelium, the major type having the nature of primitive gastric mucosa. Of interest was the presence of hamartomatous components in the wall. Furthermore, the non-neoplastic mucosal lining cells around the carcinoid tumor expressed neuroendocrine antigens and had minimal proliferative activity, suggesting that part of the cyst wall provided a microenvironment for accelerated differentiation of epithelial cells to an endocrine phenotype and transformation to neoplasia. PMID- 16669875 TI - Serum-dependence of AMPA receptor-mediated proliferation in glioma cells. AB - Glutamate may cause Ca(2+) entry through Ca(2+)-permeable glutamate receptors, which in turn stimulates the anti-apoptotic signaling cascade in glioma cells. It was found that a human glioma cell line, U-87 MG, expressed subunits of alpha amino-3-hydroxy-5-methyl-4-isoxazolepropionate acid-type glutamate receptors (AMPAR). Ca(2+) entry through AMPAR was detected in approximately 40% of U-87 MG cells. AMPAR agonists facilitated cell proliferation in low-serum medium containing 0.5% fetal calf serum (FCS). Unexpectedly, cell proliferation by the activation of AMPAR was not detected in serum-rich medium containing 10% FCS. Overexpression of Ca(2+)-permeable AMPAR facilitated proliferation of U-87 MG cells in the low-serum condition, whereas it had again no effect in the serum rich condition. Cell proliferation of U-87 MG cells is likely to be under the regulation of both growth factors contained in the serum and Ca(2+) entry through AMPAR, and that the latter regulation becomes evident only when serum factors are deprived of culture medium. PMID- 16669877 TI - Concomitant hepatocellular carcinoma and non-Hodgkin's lymphoma in a patient with nodular regenerative hyperplasia. AB - Hepatocellular carcinoma (HCC) is the most common primary cancer in the liver. Liver invasion of non-Hodgkin's lymphoma (NHL) is also often observed. But simultaneous existence of HCC and NHL in a liver is extremely rare. Such patients reported previously had cirrhotic livers. Herein is reported a patient who simultaneously had HCC and NHL in a liver without cirrhosis, but with nodular regenerative hyperplasia (NRH). NHL was of the diffuse large B-cell type. Lymphoma cells invaded the portal vein, and formed thrombi. These thrombi would contribute to the development of NRH by decreasing portal vein blood flow. HCC was of the well-differentiated type and there was a 2 cm-sized nodule at the lateral segment. There is the possibility that NRH was associated with the HCC because NRH is reported as a premalignant lesion. HCC and NHL were colocalized in the liver without hepatic virus infection or cirrhosis, although common cause(s) of development of these malignancies remain unclear in the present case. PMID- 16669878 TI - Hemosiderotic fibrohistiocytic lipomatous lesion: early pleomorphic hyalinizing angiectatic tumor? AB - Hemosiderotic fibrohistiocytic lipomatous lesion (HFLL) and early pleomorphic hyalinizing angiectatic tumor (PHAT) are characterized histologically by an admixture of fat, moderately cellular fascicles of hemosiderin-laden spindle cells growing in a perivascular, periadipocytic and septal pattern, as well as the presence of macrophages and chronic inflammatory cells. In contrast to a suggested reactive nature of HFLL, PHAT is regarded as a non-metastasizing tumor of uncertain lineage in the recent World Health Organization classification of soft tissue tumors. Reported herein is the case of a 47-year-old woman with an unencapsulated and irregularly circumscribed recurring lesion in the ankle/foot region that developed following ankle distortion and that fulfills histological criteria for both HFLL and early PHAT. In summary, the present case suggests a reactive over-neoplastic nature of HFLL and confirms profound histological similarities with early PHAT. Until more data become available on the biological potential of HFLL/early PHAT, radical surgical excision and follow up of the patient remains the best treatment option. PMID- 16669879 TI - The trials of Alcoholics Anonymous. PMID- 16669880 TI - Isn't it time we found out more about what the heck happens around American liquor stores? PMID- 16669881 TI - Heroin supply in the long-term and the short-term perspectives: comments on Wood et Al. 2006. PMID- 16669882 TI - A little (more) knowledge: comments on Wood et Al. (2006). PMID- 16669884 TI - Sri Lanka: alcohol now and then. PMID- 16669885 TI - Addiction: a journal and its Invisible College. AB - PROVENANCE: This paper derives from a lecture given before the Society for the Study of Addiction in November 2004, on the author's retirement from the position of Editor-in-Chief of Addiction, one of the Society's journals. AIM: To identify the live processes which have influenced the journal's evolution since its foundation in 1884. CONCLUSIONS: Over the 120-year period a strong, continuing historical thread has been the fluctuating success of the journal's engagement with its 'Invisible College', the community which it seeks to serve. It is argued that the journal's future success will depend on its capacity to explore and nurture further this two-way relationship. Addiction is a journal with an active and clearly articulated, multiple vision of it purpose, and this vision is outlined. It dares to try to influence its future with the authority so to do rooted in its ability to reflect the aspirations and concerns of its readers. It is 'a journal of the second kind'. PMID- 16669886 TI - It is about people. PMID- 16669887 TI - Bridging the invisible boundaries. PMID- 16669888 TI - ADDICTION: a publisher's perspective. PMID- 16669889 TI - Publish or perish: the art of managing a successful scientific journal. PMID- 16669890 TI - The Invisible College of non-English addiction journals. PMID- 16669891 TI - History is useful. PMID- 16669892 TI - The making of a successful addiction speciality journal; vision, passion and courage: comments on Edwards (2006). PMID- 16669893 TI - Some day hence. PMID- 16669895 TI - Allocating treatment options to patient profiles: clinical art or science? AB - BACKGROUND: For many researchers, the disappointing results of Project MATCH were the death blow for any further activities in the field of patient-treatment interactions. On the other hand, we have an increased knowledge of patient heterogeneity and a greater variety of treatment options than before, and allocation guidelines for an ongoing process of patient-placement decisions are of high practical relevance. AIMS: To analyse deficits in the current research and to provide suggestions for future action. FINDINGS: It is argued that (1) certain major design aspects of Project MATCH and other research studies--such as stringent patient exclusion criteria and low treatment 'dosage'-minimize the chances of detecting possible patient-treatment interactions and (2) Project MATCH obscures our view of previous treatment-allocation research findings. CONCLUSIONS: Several research strategies and specific research topics are suggested for (1) improving the theoretical and methodological basis for detecting possible patient-treatment interactions and (2) stimulating research on major treatment decision needs, such as site, setting, time in treatment (extensiveness and intensity), service components and specific treatment modules. More international research cooperation is needed to clarify the inconsistent findings. PMID- 16669896 TI - Is treatment-matching dead? Comments on Buhringer (2006). PMID- 16669897 TI - To profit from the variability of the system: comments on Buhringer (2006). PMID- 16669899 TI - Opiate addiction in China: current situation and treatments. AB - BACKGROUND: Historically, China has had extraordinarily high rates of opiate dependence. These rates declined drastically following the 1949 revolution; however, opiate abuse has re-emerged in the late 1980's and has spread quickly since then. AIMS: To describe the current situation of opiate addiction and treatments in China and make some suggestions. DESIGN: A descriptive study based on literature searched from Medline and the China National Knowledge Infrastructure database (1996 to 2004) and hand-picked references. FINDINGS: The number of registered addicts in 2004 was 1.14 million (more than 75% of them heroin addicts), but the actual number is probably far higher. Opiate abuse contributes substantially to the spread of HIV/AIDS in China, with intravenous drug use the most prevalent route of transmission (51.2%). Currently, the main treatments for opiate dependence in China include short-term detoxification with opiate agonists or non-opiate agents, such as clonidine or lofexidine; Chinese herbal medicine and traditional non-medication treatments are also used. Methadone maintenance treatment (MMT) has not been officially approved by the Chinese government for widespread implementation, but some pilot studies are currently underway. CONCLUSION: China faces substantial drug abuse problems that appear to be worsening with time. Opiate dependence is a major threat to the public health and social security of China because of its devastating medical effects, its impact on risk for HIV/AIDS and criminal behaviors, low rates of recovery and high rates of relapse. There is an urgent need to implement MMT and other modern treatments for opiate dependence more widely in China. PMID- 16669900 TI - Ecological models of alcohol outlets and violent assaults: crime potentials and geospatial analysis. AB - AIMS: Empirical tests of relationships between alcohol outlets and violence are generally conducted with statistical controls for correlates related to characteristics of people and the places in which they live. Crime potentials theory asserts that certain subpopulations are disposed to participate in criminal activities (population potentials) and certain neighborhoods are more likely to be places where crimes occur (place potentials). The current study assesses the degree to which measures of the different geographic distributions of these potentials contribute to violent crime. DESIGN: Cross-sectional data on hospital discharges for violent assaults were obtained for residents of 1637 zip code areas in California. Assault rates were related to measures of population and place characteristics using spatial statistical models corrected for spatial autocorrelated error. FINDINGS: Rates of assault were related to population and place characteristics within zip code areas, and with characteristics of populations living in adjacent zip code areas. Assault rates were greater in densely populated, poor minority urban areas with greater residential instability. Assault rates were also greater in zip code areas adjacent to densely populated urban areas. Assault rates were related significantly to local densities of off-premise alcohol retail establishments, not bars. However, densities of bars moderated substantially effects related to local population characteristics. Bars were related significantly to violence in unstable poor minority areas and in rural middle-income areas of the state. CONCLUSION: Population and place characteristics are associated with rates of violence across spatial areas. Alcohol outlets directly affect and moderate potentials for violence associated with socio-demographic groups. PMID- 16669901 TI - Intensive referral to 12-Step self-help groups and 6-month substance use disorder outcomes. AB - AIMS: This study implemented and evaluated procedures to help clinicians make effective referrals to 12-Step self-help groups. DESIGN: Randomized controlled trial. Setting Out-patient substance use disorder treatment. Participants Individuals with substance use disorders (SUDs) entering a new treatment episode (n = 345) who were assigned randomly to a standard referral- or an intensive referral-to-self-help condition. MEASUREMENTS: Self-reports of 12-Step group attendance and involvement and substance use at baseline and a 6-month follow-up. INTERVENTION: The intensive referral intervention focused on encouraging patients to attend 12-Step meetings by connecting them to 12-Step volunteers. FINDINGS: Among patients with relatively less previous 12-Step meeting attendance, intensive referral was associated with more meeting attendance during follow-up than was standard referral. Among all patients, compared with those who received standard referral, those who received intensive referral were more likely to be involved with 12-Step groups during the 6-month follow-up (i.e. had provided service, had a spiritual awakening and currently had a sponsor). Intensive referral patients also had better alcohol and drug use outcomes at 6 months. Twelve-Step involvement mediated part of the association between referral condition and alcohol outcomes. CONCLUSIONS: The brief intensive referral intervention was associated with improved 12-Step group involvement and substance use outcomes even among patients with considerable previous 12-Step group exposure and formal treatment. Future 12-Step intensive referral procedures should focus on encouraging 12-Step group involvement in addition to attendance to benefit patients most effectively. PMID- 16669902 TI - Changes in Canadian heroin supply coinciding with the Australian heroin shortage. AB - AIMS: Previous studies have largely attributed the Australian heroin shortage to increases in local law enforcement efforts. Because western Canada receives heroin from similar source nations, but has not measurably increased enforcement practices or funding levels, we sought to examine trends in Canadian heroin related indices before and after the Australian heroin shortage, which began in approximately January 2001. METHODS: During periods before and after January 2001, we examined the number of fatal overdoses and ambulance responses to heroin related overdoses that required the use of naloxone in British Columbia, Canada. As an overall marker of Canadian supply reduction, we also examined the quantity of heroin seized during this period. Lastly, we examined trends in daily heroin use among injection drug users enrolled in the Vancouver Injection Drug Users Study (VIDUS). RESULTS: There was a 35% reduction in overdose deaths, from an annual average of 297 deaths during the years 1998-2000 in comparison to an average of 192 deaths during 2001-03. Similarly, use of naloxone declined 45% in the period coinciding with the Australian heroin shortage. Interestingly, the weight of Canadian heroin seized declined 64% coinciding with the Australian heroin shortage, from an average of 184 kg during 1998-2000 to 67 kg on average during 2001-03. Among 1587 VIDUS participants, the period coinciding with the Australian heroin shortage was associated independently with reduced daily injection of heroin [adjusted odds ratio: 0.55 (95% CI: 0.50-0.61); P < 0.001]. CONCLUSIONS: Massive decreases in three independent markers of heroin use have been observed in western Canada coinciding with the Australian heroin shortage, despite no increases in funding to Canadian enforcement efforts. Markedly reduced Canadian seizure activity also coincided with the Australian heroin shortage. These findings suggest that external global heroin supply forces deserve greater investigation and credence as a potential explanation for the Australian heroin shortage. PMID- 16669903 TI - Changes in cannabis use and its consequences over 3 years in a remote indigenous population in northern Australia. AB - BACKGROUND: Few studies describe cannabis use in indigenous populations, and no longitudinal studies are available in Australia. We conducted 3-year follow-up interviews and assessments in Aboriginal communities in Arnhem Land (Northern Territory, NT). METHODS: A randomly selected sample (n = 161; 80 males, 81 females aged 13-36 years) was assessed in October 2001 and then reassessed in September 2004. An opportunistically recruited sample (n = 104; 53 males, 51 females aged 13-36 years) was also interviewed in 2001 and followed-up in 2004. Cannabis and other substance use were determined by combining proxy assessments by local Aboriginal health workers, medical records and data from interviews. Changes in cannabis use and symptoms of misuse were assessed using McNemar's test for paired proportions and the Wilcoxon signed rank test. Logistic regression assessed associations between clinical presentations and cannabis use at both time-points. RESULTS: Those who used cannabis at both baseline and follow-up were at greater risk than those who never used it to have suffered: auditory hallucinations; suicidal ideation; and imprisonment. In the randomly selected cohort there were fewer cannabis users at follow-up than at baseline (P = 0.003). The reduction was evident in females generally (P = 0.008) and older males (aged = 16 at baseline) (P = 0.007). In those interviewed at both baseline and follow up we measured no statistically significant reduction in frequency and levels of use, although fewer cannabis users reported symptoms of misuse such as: fragmented thought processes; memory disruption; difficulties controlling use; and auditory and visual hallucinations. CONCLUSIONS: Modest reductions in cannabis use and its consequences in this population were demonstrated. These may be the result of enhanced supply control and broader socio-political changes. PMID- 16669904 TI - Declared and undeclared substance use among emergency department patients: a population-based study. AB - AIMS: To estimate both self-reported and corrected prevalences of substance use in a population-based study of general hospital emergency department (ED) patients and predict undeclared use. DESIGN: A state-wide cross-sectional, two stage probability sample survey that incorporates toxicological screening. SETTING: Seven Tennessee EDs in acute care, adult, civilian, non-psychiatric hospitals. PARTICIPANTS: A total of 1502 Tennessee residents, 18 years of age and older, possessing intact cognition, able to give informed consent and not in police custody. Measurements Prevalence of self-reported current substance use by age, sex and type with correction for under-reporting based on toxicological screening. Covariates in the multivariate analysis of undeclared use were socio demographics, ED visit circumstances, health-care coverage, prior health status and treatment history and tobacco addiction. FINDINGS: Declared current use was highest for alcohol (females 26%, males 47%), marijuana (males 11%, females 6%) and benzodiazepines (females 10%, males 7%). After correction for under reporting, overall use for any of the eight targeted substances rose from 44% to 56% for females and 61% to 69% for males. Largest absolute changes involved opioids, benzodiazepines, marijuana, amphetamines and/or methamphetamine, with little change for alcohol. Patients aged 65 years and older manifested excess undeclared use relative to patients aged 18-24 years, as did patients not reporting tobacco addiction or receiving substance abuse treatment. CONCLUSION: Adjustment for under-reporting produced minimal change in the estimated prevalence of alcohol use. However, toxicological screening markedly increased estimates of other drug use, especially for the elderly, who may under-report medication use. Screening tests are useful tools for detecting undeclared substance use. PMID- 16669905 TI - Subjective responses to initial experience with cocaine: an exploration of the incentive-sensitization theory of drug abuse. AB - AIMS: This study investigated the relationship between positive and negative subjective responses at the time of initial cocaine use with adult cocaine dependence and life-time use rates. Psychostimulant pre-exposure, regular smoking or stimulant treatment before initiation were examined to explore the incentive sensitization theory of addiction. PARTICIPANTS: A total of 202 adult participants who had tried cocaine on at least one occasion were studied prospectively from childhood into adulthood. The cocaine-initiated group included 89 who met Diagnostic and Statistical Manual version IV (DSM-IV) criteria for attention deficit hyperactive disorder (ADHD) and 113 age-matched controls. DESIGN: Five childhood and three adulthood interviews provided data on ages of initiation into cocaine and life-time use of cocaine from ages 16-40 years. Correlations of each subjective response and analyses of variance (ANOVAs) of cocaine 'liking' and 'wanting' with DSM-III-R cocaine dependence and life-time use provided support for the validity of the measures. ANOVA provided evidence of the effect of psychostimulant pre-exposure on 'liking' and 'wanting'. Logistic regression modeled the prediction of dependence and life-time use with the independent variables of 'liking' and 'wanting', psychostimulant pre-exposure and participant characteristics. RESULTS: When cocaine was first tried, 'liking' and 'wanting' were significant predictors of cocaine dependence and life-time use. Mean 'liking' or 'wanting' responses did not differ by participant characteristics. Those who were pre-exposed by regular smoking or stimulant treatment had higher 'liking' and 'wanting' scores; but participants who were pre exposed by both stimulant treatment and regular smoking reported the lowest liking and the highest wanting responses, consistent with the incentive sensitization theory. Logistic regression showed that the 'liking' and 'wanting' responses increased significantly the odds of DSM-III-R cocaine dependence and life-time use. CONCLUSION: In this sample, subjective 'liking' and 'wanting' measured risk for cocaine abuse. PMID- 16669906 TI - An outbreak of hepatitis B virus infection among methamphetamine injectors: the role of sharing injection drug equipment. AB - AIM: To identify risk factors for acute hepatitis B virus (HBV) infection among Wyoming methamphetamine injectors. DESIGN: A case-control study conducted in the setting of an outbreak. SETTING: A county in central Wyoming, United States. PARTICIPANTS: Cases were identified through surveillance and contact tracing and were defined as Natrona County, Wyoming, residents who were either symptomatic or confirmed serologically to be acutely infected with HBV during January-August, 2003. Controls were susceptible to HBV infection. All participants identified themselves as methamphetamine injectors. MEASUREMENTS: Participants were administered a survey that inquired about risk factors for HBV infection, including drug use practices and sexual behaviors. Controls were also tested serologically for acute HBV infection. FINDINGS: Among the 18 case-patients and 49 controls who participated in the study, sharing water used to prepare injections and/or rinse syringes was associated with HBV infection (94% of case participants versus 44% of controls; OR = 21.9, 95% CI: 2.7, 177.8), as was sharing cotton filters (89% of case-participants versus 52% of controls; OR = 7.4, 95% CI: 1.5, 35.6); sharing syringes was not statistically associated. In logistic regression models adjusted for age, sex, and interview site, sharing rinse water and sharing cotton remained statistically associated. CONCLUSIONS: Methamphetamine use has become increasingly prevalent in the United States. Our findings highlight the need for awareness of risks associated with injection drug use and sharing behaviors. Enhanced hepatitis B vaccination programs and educational campaigns that target methamphetamine injectors specifically, including those living in rural areas, should be developed and implemented. PMID- 16669907 TI - Can home-made injectable opiates contribute to the HIV epidemic among injection drug users in the countries of the former Soviet Union? AB - AIMS: Home-made preparation of heroin is common in countries of the former Soviet Union (FSU), and the addition of blood during its preparation and the use of contaminated syringes to distribute it may play a role in the rapid spread of HIV 1 among injecting drug users (IDUs). This study was designed to determine the viability of HIV-1 during these procedures. SETTING: Field observations of home made opiate manufacture in four FSU countries were used to develop a consensus protocol to replicate manufacture in the laboratory that included the addition of human blood contaminated with HIV-1. DESIGN AND MEASUREMENT: Following the addition of HIV-1-contaminated blood during manufacture or storage, we attempted to recover viable HIV-1. The recovery was measured by propagation of the virus in stimulated white blood cells from uninfected donors. FINDINGS: In experiments in which HIV-1 contaminated blood was added during manufacture, no viable HIV-1 was recovered. In experiments in which chornaya was introduced into HIV-contaminated syringes, the percentage of syringes containing viable HIV-1 was reduced. The reduction appeared to be related to the interaction of HIV-1 contaminated blood with a component of the poppies. While HIV-contaminated syringes used to dispense or inject home-made opiates might transmit HIV, the ability of chornaya to reduce HIV viability seems to make this route of transmission less efficient. CONCLUSIONS: The epidemic of HIV-1 among IDUs in the FSU resulted more probably from recognized injection risk behaviors-including sharing syringes and drug solutions--than from opiate solutions harboring viable HIV-1. PMID- 16669908 TI - The economic impact of a smoke-free bylaw on restaurant and bar sales in Ottawa, Canada. AB - AIMS: On 1 August 2001, the City of Ottawa (Canada's Capital) implemented a smoke free bylaw that completely prohibited smoking in work-places and public places, including restaurants and bars, with no exemption for separately ventilated smoking rooms. This paper evaluates the effects of this bylaw on restaurant and bar sales. DATA AND MEASURES: We used retail sales tax data from March 1998 to June 2002 to construct two outcome measures: the ratio of licensed restaurant and bar sales to total retail sales and the ratio of unlicensed restaurant sales to total retail sales. Restaurant and bar sales were subtracted from total retail sales in the denominator of these measures. DESIGN AND ANALYSIS: We employed an interrupted time-series design. Autoregressive integrated moving average (ARIMA) intervention analysis was used to test for three possible impacts that the bylaw might have on the sales of restaurants and bars. We repeated the analysis using regression with autoregressive moving average (ARMA) errors method to triangulate our results. FINDINGS: Outcome measures showed declining trends at baseline before the bylaw went into effect. Results from ARIMA intervention and regression analyses did not support the hypotheses that the smoke-free bylaw had an impact that resulted in (1) abrupt permanent, (2) gradual permanent or (3) abrupt temporary changes in restaurant and bar sales. CONCLUSIONS: While a large body of research has found no significant adverse impact of smoke-free legislation on restaurant and bar sales in the United States, Australia and elsewhere, our study confirms these results in a northern region with a bilingual population, which has important implications for impending policy in Europe and other areas. PMID- 16669909 TI - Genevieve Knupfer, 1914-2005: turning presumption into researchable questions. PMID- 16669911 TI - Antisocial personality disorder and gambling: comments on Pietrzak & Petry (2005). PMID- 16669914 TI - Abstracts of the 39th Annual Scientific Meeting of the Australasian College of Dermatologists. May 14-17, 2006 Melbourne, Victoria, Australia. PMID- 16669915 TI - Can prostate biopsies affect erectile function? AB - The impact of transrectal ultrasound (TRUS)-guided prostate biopsies on erectile function was prospectively studied. Forty-six men (median age: 67.51 years) who underwent TRUS-guided prostate biopsies completed the International Index of Erectile Function (IIEF)-5 questionnaire at the day of the biopsy, 1 and 3 months later. Erectile dysfunction (ED) severity was classified into five categories. Concomitant ED-related systemic diseases and/or medications that could affect erectile function were also recorded. The paired t-test was used for statistical analysis. The median IIEF-5 score was 15.91 prior to biopsies, while 1 and 3 months after, the median IIEF-5 score was 14.33 and 14.81 respectively (P > 0.05). Prior to prostate biopsies, ED was reported by 38 patients (82.60%): mild ED in 39.13%, mild to moderate in 19.56%, moderate in 15.21% and severe ED in 8.69%. Concomitant ED-related systemic diseases and/or medications were recorded in 28 patients (60.86%). One month after, ED was revealed in 42 patients (91.30%): mild ED in 26.08%, mild to moderate in 30.43%, moderate in 19.56%, and severe ED in 15.21%. Three months post-biopsy, ED was reported by 41 patients (89.13): mild ED in 21.73%, mild to moderate in 28.26%, moderate in 21.73%, and severe ED in 17.39%. Overall, three and two patients (6.52% and 4.34%) had prostate biopsy attributed ED (i.e. without concomitant ED-related disease or medication) 1 and 3 months after prostate biopsies. TRUS-guided prostate biopsies did not induce ED in a statistically significant manner. Evaluating potency at referral for TRUS-guided prostate biopsies is advisable. PMID- 16669916 TI - Changes in peak systolic velocity induced by chronic therapy with phosphodiesterase type-5 inhibitor. AB - The aim of this study was to assess the influence of chronic therapy with phosphodiesterase type-5 inhibitor on penile haemodynamics at colour Doppler ultrasound. Thirty patients affected by erectile dysfunction (ED) of different aetiology tested with the International Index of Erectile Function (IIEF-5) were evaluated with penile colour Doppler ultrasound during basic and dynamic phases (10 microg PGE1) before and after chronic self-administration of sildenafil citrate (dosage: 100 mg as required, two to three times a week) for a period of 5 20 months (mean: 12.3). Treatment was interrupted 14-21 days before the second ultrasound evaluation. Peak systolic velocity (PSV) and end-diastolic velocity (EDV) were recorded by means of colour Doppler; cut off values were 25 and 5 cm s(-1) respectively. Data were compared by nonparametric tests. Twenty-two of the 30 patients showed normal pre-treatment PSV, while eight of 30 had an insufficient arterial flow. Mean pre-treatment EDV was 4.7 +/- 0.5. After chronic therapy with sildenafil, a global improvement of 10.5% on PSV was seen (P < 0.001), without any statistical difference between patients with normal pre treatment peak and those with a borderline one. No statistically significant changes were found for EDV (P = 0.98). It is concluded that chronic therapy with phosphodiesterase-5 inhibitor results in a significant improvement in PSV values, probably due to a penile chronic vasoactive enhancement. PMID- 16669917 TI - Oligozoospermia: recent prognosis and the outcome of 73 pregnancies in oligozoospermic couples. AB - The minimum value for each of the five main semen parameters, below which conception rarely occurred or did not occur at all, was calculated in a group of 1884 couples complaining of primary and secondary infertility: 304 conceptions including first as well as consecutive conceptions, occurred. The parameters evaluated were (minimum value calculated in this study between brackets) volume (1.0 ml), sperm count ml(-1) (2.0 million), total sperm count (4.0 million), motility (10%), forward progression (2.0 MacLeod units: scale 1-4) and normal sperm morphology (3%). The pregnancy rate in the group of 308 oligozoospermic men and the minimum value of semen parameters were the cornerstones in determining the prognosis for oligozoospermic patients. A sperm count of >2.0 million ml(-1) was considered relatively adequate for eventual conception judged by the 68 of 308 (22.1%) pregnancies that occurred among oligozoospermic men in this study, provided that the other five semen parameters showed values above the minimum value. In cases where the average sperm count was <2 million ml(-1), the chances for conception became rare, viz five of 308 (1.6%). The total number of pregnancies in the group classified as oligozoospermic was 73 (23.7%). With these pregnancies there was no increase in the rate of foetal wastage and congenital abnormalities. Abortion occurred in 15.09% and ectopic pregnancy in 0.9% among first and consecutive pregnancies. One infant among the 56% boys and 44% girls was born with congenital abnormalities. Most of these infants had a normal birth mass of >2500 g. PMID- 16669918 TI - Can sperm protamine deficiency induce sperm premature chromosomal condensation? AB - Sperm premature chromatin condensation (PCC) has been considered as the second cause of failed fertilization post-intracytoplasmic sperm injection (post-ICSI). Cytoplasmic factors, including oocyte cytoplasmic immaturity have been suggested to induce PCC sperm. However, recent studies suggest that sperm chromatin anomaly might also lead to PCC sperm. During this study, human sperm from infertile patients with protamine deficiency or with adequate amount of protamine assessed by chromomycin A3 were injected into metaphase II mouse oocyte, treated with colcemid. Chromatin analysis was carried out on the injected oocyte. The results of this study show that contrary to the percentage of intact sperm, percentage of PCC sperm was significantly higher in oocytes injected with protamine deficient sperm (36.43 +/- 4.46) compared to oocytes injected with sperm with an adequate amount of protamine (11.99 +/- 3.54, P < 0.001). A significant correlation was also observed between percentage of PCC sperm and protamine deficiency (r = 0.46, P = 0.004). Therefore, it can be suggested that oocytes injected with protamine deficient sperm have a higher chance of forming PCC sperm and may result in failed fertilization post-ICSI. PMID- 16669919 TI - Measurement of induced acrosome reactions in human sperm using physiologic stimuli--relevance for the prediction of fertilization outcome. AB - Fertilization failure following standard in vitro fertilization in couples with normozoospermic men is an as yet unexplained phenomenon. A wide range of gametic disorders as well as environmental factors might contribute to this pathologic condition. One crucial condition appears to be the inability of the spermatozoa to undergo the acrosome reaction (AR). A discriminative test to distinguish fertile from non-fertile spermatozoa would be of utmost interest. In a prospective study, semen samples from men with normal semen parameters and fertilization failure were compared with semen samples from men with normal semen parameters and normal fertilization as to their capacity to undergo the AR. AR was induced using calcium ionophore as well as the physiologic stimuli progesterone and prostaglandin E(1). Discriminance analyses were undertaken to help identify patients with probable fertilization failure. Our data show that in patients with fertilization failure, the capacity of spermatozoa to undergo induced AR is greatly reduced using both unphysiologic and physiologic stimuli. However, physiologic stimuli are more suitable to identify patients with fertilization failure. Using physiologic stimuli, a formula was established to identify patients likely to fail at fertilization. PMID- 16669920 TI - The Estonian version of the National Institutes of Health chronic prostatitis symptom index. AB - Our aim was to establish the Estonian version of the National Institutes of Health chronic prostatitis symptom index (NIH-CPSI), and to examine its validity and applicability in chronic prostatitis patients as well as in community-based study. The questionnaire was tested in 54 category III prostatitis patients. In addition, 452 men were enrolled into a community-based study that resulted in 83 completed questionnaires. The discriminatory power, psychometric properties and internal correlations of the questionnaire were tested. The questionnaire demonstrated good internal consistency, with Cronbach's alpha coefficient 0.82. The pain and quality of life domains demonstrated high correlations with each other and with the entire questionnaire. The total score and the scores of pain and quality of life domains of the Estonian NIH-CPSI differed significantly between the chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) patients and the men without CP/CPPS, and so the index proved a good discriminant validity. We can conclude that the Estonian NIH-CPSI is easily understood and reflects accurately the clinical symptoms and the quality of life in patients with chronic prostatitis. The similarity of the psychometric properties of the English and Estonian versions of the NIH-CPSI supports their measurement equivalence. PMID- 16669921 TI - Relationship between sexual abstinence duration and the acrosome index in teratozoospermic semen: analysis of 1800 semen samples. AB - A high acrosome index (percentage of sperm with normal acrosome morphology- cutoff value > or =10%) is known to be associated with an improved fertilization rate in conventional IVF. A retrospective evaluation of the relationship between duration of sexual abstinence and acrosome index among oligozoospermic and normozoospermic semen samples with teratozoospermia was undertaken. A significant (P = 0.001) decrease in the acrosome index was observed among the normozoospermic samples (n = 1264) between the peak value of 10.2 +/- 3.6% on day 2 and 8.5 +/- 4.0% on day 5 of abstinence, while for the oligozoospermic samples (n = 536) the peak value of 8.7 +/- 3.5% was observed on day 1 and the lowest values of 6.8 +/- 3.7% (P = 0.04) on day 5 of abstinence. The results suggest that an optimal acrosome index will be obtained following a short sexual abstinence. PMID- 16669924 TI - Clinical experience in the management of community-acquired pneumonia: lessons from the use of fluoroquinolones. AB - Community-acquired pneumonia (CAP) remains a major cause of morbidity and mortality worldwide. The treatment of CAP has been complicated by several factors, including the expanding spectrum of causative organisms and the rising prevalence of antibiotic resistance among respiratory pathogens. Initial antimicrobial treatment for patients with CAP is usually selected empirically and should provide appropriate coverage against the most common causative organisms, including resistant strains. Respiratory fluoroquinolones, such as levofloxacin, are the only antimicrobials that are highly active against the pathogens most frequently implicated in CAP, including macrolide-resistant and penicillin resistant pneumococci, Haemophilus influenzae, Legionella spp., and atypical agents. This paper reviews recent studies involving adult patients with CAP that suggest that levofloxacin, as compared with other conventional antibiotic treatments, may be associated with better clinical outcomes. PMID- 16669925 TI - The atypical pneumonias: clinical diagnosis and importance. AB - The most common atypical pneumonias are caused by three zoonotic pathogens, Chlamydia psittaci (psittacosis), Francisella tularensis (tularemia), and Coxiella burnetii (Q fever), and three nonzoonotic pathogens, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella. These atypical agents, unlike the typical pathogens, often cause extrapulmonary manifestations. Atypical CAPs are systemic infectious diseases with a pulmonary component and may be differentiated clinically from typical CAPs by the pattern of extrapulmonary organ involvement which is characteristic for each atypical CAP. Zoonotic pneumonias may be eliminated from diagnostic consideration with a negative contact history. The commonest clinical problem is to differentiate legionnaire's disease from typical CAP as well as from C. pneumoniae or M. pneumonia infection. Legionella is the most important atypical pathogen in terms of severity. It may be clinically differentiated from typical CAP and other atypical pathogens by the use of a weighted point system of syndromic diagnosis based on the characteristic pattern of extrapulmonary features. Because legionnaire's disease often presents as severe CAP, a presumptive diagnosis of Legionella should prompt specific testing and empirical anti-Legionella therapy such as the Winthrop-University Hospital Infectious Disease Division's weighted point score system. Most atypical pathogens are difficult or dangerous to isolate and a definitive laboratory diagnosis is usually based on indirect, i.e., direct flourescent antibody (DFA), indirect flourescent antibody (IFA). Atypical CAP is virtually always monomicrobial; increased IFA IgG tests indicate past exposure and not concurrent infection. Anti-Legionella antibiotics include macrolides, doxycycline, rifampin, quinolones, and telithromycin. The drugs with the highest level of anti Legionella activity are quinolones and telithromycin. Therapy is usually continued for 2 weeks if potent anti-Legionella drugs are used. In adults, M. pneumoniae and C. pneumoniae may exacerbate or cause asthma. The importance of the atypical pneumonias is not related to their frequency (approximately 15% of CAPs), but to difficulties in their diagnosis, and their nonresponsiveness to beta-lactam therapy. Because of the potential role of C. pneumoniae in coronary artery disease and multiple sclerosis (MS), and the role of M. pneumoniae and C. pneumoniae in causing or exacerbating asthma, atypical CAPs also have public health importance. PMID- 16669926 TI - Legionella: macrolides or quinolones? AB - Following the first outbreaks of legionnaire's disease, erythromycin emerged as the treatment of choice without the foundation of rigorous clinical trials. The number of therapeutic failures with erythromycin, as well as the side-effects and drug interactions, led to the consideration of other drugs such as the new macrolides and quinolones for the treatment of legionnaire's disease in the 1990s. In this article, 19 studies in in-vitro intracellular models and seven animal studies that compared macrolides to quinolones were reviewed. Quinolones were found to have greater activity in intracellular models and improved efficacy in animal models compared with macrolides. No randomised trials comparing the clinical efficacy of the new macrolides and new quinolones have ever been performed. Three observational studies totalling 458 patients with legionnaire's disease have compared the clinical efficacy of macrolides (not including azithromycin) and quinolones (mainly levofloxacin). The results suggested that quinolones may produce a superior clinical response compared with the macrolides (erythromycin and clarithromycin) with regard to defervescence, complications, and length of hospital stay. Little data exist for direct comparison of quinolones and azithromycin. PMID- 16669927 TI - Clinical implications and treatment of multiresistant Streptococcus pneumoniae pneumonia. AB - Streptococcus pneumoniae is the leading bacterial cause of community-acquired respiratory tract infections. Prior to the 1970s this pathogen was uniformly susceptible to penicillin and most other antimicrobials. However, since the 1990s there has been a significant increase in drug-resistant Streptococcus pneumoniae (DRSP) due, in large part, to increased use of antimicrobials. The clinical significance of this resistance is not definitely established, but appears to be most relevant to specific MICs for specific antimicrobials. Certain beta-lactams (amoxicillin, cefotaxime, ceftriaxone), the respiratory fluoroquinolones, and telithromycin are among several agents that remain effective against DRSP. Continued surveillance studies, appropriate antimicrobial usage campaigns, stratification of patients based on known risk factors for resistance, and vaccination programmes are needed to appropriately manage DRSP and limit its spread. PMID- 16669928 TI - Should patients with acute exacerbation of chronic bronchitis be treated with antibiotics? Advantages of the use of fluoroquinolones. AB - The pathological changes in chronic bronchitis (CB) produce airflow obstruction, reduce the effectiveness of the mucocilliary drainage system and lead to bacterial colonisation of bronchial secretion. The presence of bacteria induces an inflammatory response mediated by leukocytes. There is a direct relationship between the degree of impairment of the mucocilliary drainage system, the density of bacteria in mucus and the number of leukocytes in the sputum. Purulent sputum is a good marker of a high bacterial load. Eventually, if the number of leukocytes is high, their normal activity could decrease the effectiveness of the drainage system, increase the bronchial obstruction and probably damage the lung parenchyma. Whenever the density of bacteria in the bronchial lumen is >or=10(6) CFU/mL, there is a high probability that the degree of inflammatory response will lead to a vicious cycle which in turn tends to sustain the process. This situation can arise during the clinical course of any acute exacerbation of CB, independently of its aetiology, provided the episode is sufficiently severe and/or prolonged. Fluoroquinolones of the third and fourth generation are bactericidal against most microorganisms usually related to acute exacerbations of CB. Their diffusion to bronchial mucus is adequate. When used in short (5-day) treatment they reduce the bacterial load in a higher proportion than is achieved by beta-lactam or macrolide antibiotics given orally. Although the clinical cure rate is similar to that obtained with other antibiotics, the time between exacerbations could be increased. PMID- 16669929 TI - Antibiotic consumption and generation of resistance in Streptococcus pneumoniae: the paradoxical impact of quinolones in a complex selective landscape. AB - The development of resistance to the different antibiotics by the majority of bacterial species of clinical importance seems unavoidable. However, not all drugs have the same efficiency to select for resistance. Large differences in the qualitative and quantitative consumption of antibiotics among countries are known to exist and several authors have consistently reported the direct relationship between consumption and selection of resistance for Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Escherichia coli and beta lactams and macrolides use. In Spain, extensive surveillance started in 1996, and the Willow (SAUCE) Project, to monitor and update resistance in respiratory pathogens and to couple those data with data concerning national antibiotic consumption (IMS) from both a temporal and geographical approach. Temporally, despite a continuous increase of 16% in quinolone consumption from 1997 to 2001, basically due to the arrival of respiratory quinolones, levofloxacin and moxifloxacin, a continuous linear increase in the resistance rates to ciprofloxacin in S. pneumoniae was not observed. There also was an inverse correlation between provincial consumption of quinolones and resistance to ciprofloxacin. Several hypotheses are proposed and discussed to explain these apparent paradoxical observations, such as the replacement of ciprofloxacin by more potent antipneumococcal quinolones, the possibility of an antibiotic pressure threshold, the influence of other nonquinolone drugs on the expression of ciprofloxacin-resistance biological costs, and the influence of changes in temporal or spatial prevalence of particular clones. PMID- 16669930 TI - Current challenges in the treatment of complicated urinary tract infections and prostatitis. AB - Serious urinary tract infections (UTIs) and acute bacterial prostatitis in adults cause significant morbidity and economic burden. Chronic bacterial prostatitis is a rather rare condition seen in urological practice, however, in certain occasions difficult to treat. In this paper, we review the bacterial etiologies and the resistance patterns found in adults with serious UTIs and bacterial prostatitis, and discuss considerations for selecting optimal antimicrobial therapy. The role of fluoroquinolones as targeted therapy for serious UTIs is highlighted. The use of effective antimicrobial therapy is the foundation of management of serious UTIs and bacterial prostatitis. Selection of the optimal antimicrobial agent must take into account patient-specific factors; infection characteristics (e.g., severity, community- vs. institutional- or hospital acquired, need for IV agent, UTI, prostatitis); local resistance pattern; pharmacokinetic and pharmacodynamic principles; and cost. Fluoroquinolones are among the alternatives for empirical antibiotic treatment of serious UTIs and acute bacterial prostatitis. In serious UTIs activity of the antimicrobial agent against Pseudomonas aeruginosa needs to be taken into account. In chronic bacterial prostatitis fluoroquinolones are the first choice because of their favourable pharmacokinetic properties at the site of infection. Targeted antimicrobial therapy--emphasising the correct antibacterial spectrum and correct dosage--is likely to provide important benefits, such as reduced morbidity and associated costs, reduced emergence of resistance and maintenance of class efficacy. PMID- 16669931 TI - Levofloxacin in the treatment of ventilator-associated pneumonia. AB - The use of levofloxacin in critically ill patients has progressively increased since commercial marketing of the drug in 1999, despite the fact that few studies have been designed to assess the use of levofloxacin in this population. Pharmacological characteristics, broad spectrum of activity, and tolerability account for the high interest in the drug for the treatment of different infectious diseases, including ventilator-associated pneumonia (VAP), and the recommendation of levofloxacin in guidelines developed by a number of scientific societies. According to pharmacokinetic-pharmacodynamic data, it seems reasonable to assume that an increase in activity follows from a larger dose, so that 500 mg/12 h is adequate in patients with VAP. In critically ill patients with VAP, levofloxacin monotherapy is indicated for empirical treatment of patients with early onset pneumonia without risk factors for multiresistant pathogens, and in combination therapy for late onset VAP or for patients at risk for multiresistant pathogens. The use of levofloxacin in combination therapy is supported by multiple reasons, including: increased empirical coverage in infections with suspected intracellular pathogens; substitution for more toxic antimicrobial agents (e.g., aminoglycosides) in patients with renal dysfunction and in those at risk for renal insufficiency; and severity of systemic response to infection (septic shock) that justifies multiple treatment with better tolerated antibiotics. The availability of the oral formulation allows sequential therapy, switching from the intravenous route to the oral route. Levofloxacin is well tolerated by critically ill patients, with few adverse events of mild to moderate severity. PMID- 16669932 TI - Management of infections of osteoarticular prosthesis. AB - Prosthetic joint infections are an uncommon complication of joint replacement surgery, but are associated with significant morbidity and costs when they do occur. Gram-positive cocci, in particular Staphylococcus aureus and Staphylococcus epidermidis, are the most commonly recovered microorganisms (>or=50% of all isolates). About 60% of prosthetic joint infections probably occur by direct contamination during the operative procedure. Certain systemic conditions in the patients, as well as foreign material, have been identified as risk factors for prosthetic joint infection. The clinical diagnosis is only certain when there are sinus tracts that reach the prosthesis or purulent secretion is obtained from joint aspiration or during open surgery. The treatment of an infected joint prosthesis must be individualised, but it generally involves both systemic antibiotics and surgical intervention. Exchange arthroplasty in one or two stages continues to be the standard approach to management. Prosthesis retention, in conjunction with debridement and prolonged (for at least 3 months) oral antibiotic therapy, can be an alternative for early postoperative or late acute haematogenous infections, when the duration of symptoms is less than 1 month, the implant is stable, and the pathogen is relatively avirulent and sensitive to an orally well absorbed antibiotic. Good results have been achieved under these conditions in staphylococcal infections with rifampin associated with quinolones and other antibiotics, e.g., cotrimoxazole, fusidic acid, and linezolid. PMID- 16669933 TI - Anaphylaxis: persistent enigma. PMID- 16669934 TI - Alcohol misuse: positive response. Alcohol Health Work for every acute hospital saves money and reduces repeat attendances. PMID- 16669935 TI - Injury severity: role of alcohol, substance use and risk-taking. AB - OBJECTIVE: The objective of the present study was to quantify the relationship between acute alcohol consumption and injury severity. METHODS: A cross-sectional study was conducted at the Gold Coast Hospital, Gold Coast, Queensland, Australia between October 2000 and October 2001. Data were collected from a systematic sample of patients greater than 15 years of age who presented to the ED for treatment of an injury sustained less than 24 h prior to presentation. Study participants were interviewed face to face on-site. Information obtained included: demographics details; situational variables relative to time of injury (i.e. location, activity and companions at time of injury); self-reported alcohol consumption in the 6 and 24 h prior to time of injury; usual alcohol consumption patterns; self-reported substance use in the 6 and 24 h prior to time of injury; and risk-taking behaviour. Injury severity was coded from patient medical records using the New Injury Severity Score. RESULTS: Of 789 eligible patients presenting during the study periods, 593 were interviewed (75.2%). Patients who reported drinking above low-risk levels (odds ratio [OR] = 3.35; 95% confidence interval [CI] 1.2-9.6) or who drank beer (OR = 3.54; 95% CI 1.1-11.1) in 6 h prior to injury were significantly more likely to sustain serious than minor injury. Drinking setting and usual drinking patterns were not significantly associated with injury severity, either in crude analyses, or after adjusting for relevant variables. CONCLUSION: The results of the present study support the conclusion that among injured patients who presented for treatment at a large metropolitan ED, although acute alcohol consumption does not appear to be associated with minor or moderate injury, there is some evidence to suggest that acute alcohol consumption is associated with serious injury. PMID- 16669936 TI - Content analysis of Australian newspaper portrayals of emergency medicine. AB - OBJECTIVE: To evaluate portrayals of emergency medicine (EM) in Australian newspapers. METHOD: Systematic content analysis of articles about the ED in four major Australian newspapers. RESULTS: From July 2003 to June 2004 176 articles about the ED were published. Articles about ED overload were dominant (37.9% of all subjects identified). Articles about solutions to ED problems (16%) and errors (12.7%) were also prominent. Emergency physicians (EP) provided 13.5% of all of the commentary in the articles. Health administrators, spokespersons and politicians collectively constituted 44.3% of voices. The Australasian College for Emergency Medicine (ACEM) provided little commentary (4%). Overall 18% of articles portrayed EM in a positive light, 37% neutral and 45% negative. The ratio of negative to positive articles was 2.5:1. Page-one stories were mostly concerned with ED overload and errors and were predominantly negative. Positive articles (18%) most commonly covered solutions to problems, 'Life in the Emergency Department' or descriptions of staff involvement with interesting diseases. CONCLUSIONS: There is a significant media focus on ED problems and errors. The majority of comment comes from politicians and bureaucrats not EP or the ACEM and, overall, the articles examined portrayed EM in a negative light. EP and the ACEM should evaluate ways to improve the interface between the specialty and the media. PMID- 16669937 TI - Alcohol use among young adults presenting to the emergency department. AB - OBJECTIVES: To determine the prevalence of young ED patients at risk from hazardous alcohol consumption, to identify high-risk patient subgroups and evaluate the feasibility of use of the Alcohol Use Disorders Identification Test (AUDIT) in this setting. METHODS: We undertook a cross-sectional survey of 336 ED patients aged 18-30 years, inclusive. All were breathalysed prior to self administering the AUDIT. A 'positive' AUDIT score (> or = 8) defined hazardous alcohol consumption. AUDIT scores were correlated with sex and trauma diagnosis. RESULTS: One hundred and thirty-one (39.0%, 95% confidence interval [CI] 33.8 44.5) patients were classified as AUDIT-positive. Men were significantly more likely to be AUDIT-positive (49% vs 23%, P < 0.001) and had significantly higher total AUDIT scores (P < 0.001) than women. Trauma patients were significantly more likely to be AUDIT-positive (P < 0.001) and had significantly higher AUDIT scores than non-trauma patients (P < 0.001). Of the six patients who recorded a positive breath alcohol reading, all were AUDIT-positive. One hundred (76.3%, 95% CI 68.0-83.1) AUDIT-positive patients did not report others being concerned about their drinking or had not been given advice to cut down. CONCLUSION: It is feasible to use the AUDIT screening tool in the ED to identify those at risk from hazardous drinking. In our ED there is a high prevalence of hazardous alcohol consumption in young adult patients, many of whom have not previously received advice to cut down on their drinking. PMID- 16669938 TI - Profiling patients suspected of drug seeking in an adult emergency department. AB - OBJECTIVES: (i) To profile ED consultations where drug seeking is considered; (ii) to clarify if an Australian patient cohort shares the characteristics identified in the literature, that is, high rate of psychiatric, chronic pain and drug dependency problems; and (iii) to quantify the extent of missed organic disease in suspected drug-seeking presentations. METHODS: Prospective descriptive study with an initial enrolment period of 3 months, follow up at 3 and 5 years. Tertiary hospital ED doctors voluntarily identified suspected drug-seeking behaviour. Patients' demographic information, past history, presenting features, investigations, management and missed diagnoses of organic pathology were collected. Recurrent presentations of drug seeking, self-harming, psychiatric, chronic pain and drug dependency complaints were documented. RESULTS: Thirty seven presentations (31 patients) of 10,958 total attendances were analysed. All patients were less than 65 years. Twenty-one patients (68%, 95% confidence interval [CI 49.7-85.8]) were on unemployment or disability pension. Twenty-six presentations (70% [95% CI 54.2-86.3]) described psychiatric problems, whereas three presentations (8% [95% CI 0.0-18.3]) had chronic pain and 10 presentations (27% [95% CI 11.4-42.7]) had drug dependency problems. Twenty-six patients (84% [95% CI 69.3-98.4]) recurrently presented with self-harming and drug-seeking behaviour at 3 years. The risk of missing organic pathology was 8/37 (22% [95% CI 7.0-36.2]). CONCLUSIONS: Further characterization of this population would help accurate diagnosis of this aberrant behaviour and decrease the risk of missing organic pathology. The management of frequently presenting patients should prompt formulation of departmental plans to effectively assess and manage these people. PMID- 16669939 TI - Diagnosing drug-seeking behaviour in an adult emergency department. AB - OBJECTIVE: The objective of the present study were to determine factors used by ED doctors to diagnose drug seeking and their attitude towards management of this patient group. METHODS: Prospective, descriptive study, of doctors working in a tertiary hospital ED. Doctors voluntarily identified patients they suspected were drug seeking and completed a written survey. RESULTS: Thirty-seven surveys were completed for presentations involving 31 patients. A patient's specific request for narcotics, previous documented episodes of suspected drug seeking, an unwillingness to try simple analgesia and demanding or aggressive behaviour were the most frequently used factors for diagnosing drug seeking. These factors are similar to, but not the same, as the criteria in the literature. Doctors commonly found consultations involving drug seeking to be unsatisfying. CONCLUSIONS: Factors, such as those used by doctors in the present study, should not be used as diagnostic markers. Further study is warranted in order to improve the effectiveness of consultations involving drug seeking. PMID- 16669940 TI - Paediatric Research in Emergency Departments International Collaborative (PREDICT): first steps towards the development of an Australian and New Zealand research network. AB - Paediatric emergency research is hampered by a number of barriers that can be overcome by a multicentre approach. In 2004, an Australia and New Zealand-based paediatric emergency research network was formed, the Paediatric Research in Emergency Departments International Collaborative (PREDICT). The founding sites include all major tertiary children's hospital EDs in Australia and New Zealand and a major mixed ED in Australia. PREDICT aims to provide leadership and infrastructure for multicentre research at the highest standard, facilitate collaboration between institutions, health-care providers and researchers and ultimately improve patient outcome. Initial network-wide projects have been determined. The present article describes the development of the network, its structure and future goals. PMID- 16669941 TI - Blood pressure measurements on children in the emergency department. AB - BACKGROUND: There is no consensus on routine blood pressure (BP) measurement for children presenting to the ED. By contrast, BP measurements are performed routinely in adult ED. With increasing childhood obesity, and the knowledge that elevated BP in childhood correlates with hypertension in adulthood, routine BP measurement in children should be considered. OBJECTIVE: To review the current practice of BP measurement in children presenting to the ED of Princess Margaret Hospital for Children, Perth, Western Australia, a tertiary referral paediatric centre. METHOD: A prospective observational study of 504 children presenting to the ED over a 6 month period in 2003. RESULTS: In total, 114 children (22.6%) had their BP measured. A total of 35 (30.7%) had a BP above the 95th percentile for age and sex. In total, 19 (54.3%) of 35 with abnormal BP had their BP remeasured. Only one child of 10 with consecutive hypertensive measurements was followed up. BP measurement was more common in children aged 2 years and above, and in higher triage category patients. CONCLUSIONS: The present study demonstrates that BP measurement in the ED is both relatively infrequent and inconsistent. Further research into this area is required to determine the usefulness and feasibility of routine BP measurements in children presenting to the ED. This might improve the quality of health care and positively impact on public health for the future. PMID- 16669942 TI - Anaphylaxis: clinical concepts and research priorities. AB - Anaphylaxis is a severe immediate-type hypersensitivity reaction characterized by life-threatening upper airway obstruction bronchospasm and hypotension. Although many episodes are easy to diagnose by the combination of characteristic skin features with other organ effects, this is not always the case and a workable clinical definition of anaphylaxis and useful biomarkers of the condition have been elusive. A recently proposed consensus definition is ready for prospective validation. The cornerstones of management are the supine position, adrenaline and volume resuscitation. An intramuscular dose of adrenaline is generally recommended to initiate treatment. If additional adrenaline is required, then a controlled intravenous infusion might be more efficacious and safer than intravenous bolus administration. Additional bronchodilator treatment with continuous salbutamol and corticosteroids are used for severe and/or refractory bronchospasm. Aggressive volume resuscitation, selective vasopressors, atropine (for bradycardia), inotropes that bypass the beta-adrenoreceptor and bedside echocardiographic assessment should be considered for hypotension that is refractory to treatment. Management guidelines continue to be opinion- and consensus-based, with retrospective studies accounting for the vast majority of clinical research papers on the topic. The clinical spectrum of anaphylaxis including major disease subgroups requires clarification, and validated scoring systems and outcome measures are needed to enable good-quality prospective observational studies and randomized controlled trials. A systematic approach with multicentre collaboration is required to improve our understanding and management of this disease. PMID- 16669943 TI - B-type natriuretic peptide in the diagnosis of heart failure in the emergency department. AB - B-type natriuretic peptide (BNP) is increasingly being used as a diagnostic marker in the diagnosis of heart failure. Here we evaluate the evidence base for its utility in the ED. Clinical trials suggest that it is more accurate than clinical acumen especially when emergency physicians have diagnostic problems. BNP appears more accurate than any clinical findings or radiological signs. In conjunction with considered clinical judgement, knowledge of its limitations and variable cut-off points, BNP can be of considerable utility to the emergency physician. PMID- 16669944 TI - Prevalence of use, epidemiology and toxicity of 'herbal party pills' among those presenting to the emergency department. AB - The aim of the present study was to establish the prevalence of use, epidemiology and toxicity of 'herbal party pills' in ED presenters. This was an analytical cross-sectional survey of patients and relatives presenting to a large tertiary ED. Consenting participants completed a specifically designed questionnaire. A total of 1043 people completed the questionnaire (participation rate of 97.2%). One hundred and twenty-five (11.9%) had taken herbal party pills and subgroup analysis showed that use was most prevalent in those aged 14-25 years (30%). The majority had taken pills between two and five times (56%). Eighty-three (66.4%) had been drinking alcohol when they first took party pills. Only 80 (64%) had read the product directions, and 48 (38.4%) had, at some stage, taken more pills than recommended. One hundred and six (84.8%) had felt effects from party pills, but only 63 (59% of those feeling effects or 50.4% of total) described these as 'good'. Six (5.7% of those with effects or 4.8% of total) had sought medical attention for effects. Seventy-four (59.2%) would take herbal party pills again. In conclusion, the use of herbal party pills is common in presenters to the ED, particularly in those aged 14-25 years. These people are at risk for toxicity from the pills because there is a tendency for them not to read the instructions before ingestion, to take more pills than recommended and to coingest alcohol. Emergency physicians need to be aware of the use and potential adverse effects of herbal party pills to enable them to recognize signs of toxicity in ED presenters and thus provide appropriate supportive care. PMID- 16669945 TI - Truth hurts--hard lessons from Australia's largest mass casualty exercise with contaminated patients. AB - In response to the increasing threat of a mass casualty incident involving chemical, biological or radiological agents, and concern over the preparedness of our hospital system to cope with patients from such an incident, we conducted the largest hospital-based field exercise involving contaminated patients that has been held in Australia. In the present paper, we outline the background to, and methodology of, Exercise Supreme Truth, and the efforts made to increase its realism. We focus our discussion on three issues highlighted by the exercise, which we believe have enormous implications for the development of hospital chemical, biological or radiological plans and the likelihood of their success- hospital security, crowd control and decontamination. PMID- 16669946 TI - Trauma response to the Asian tsunami: Krabi Hospital, Southern Thailand. AB - The date 26 December 2004 saw a massive tidal wave propagated from a 9.0 Richter scale suboceanic earthquake off the coast of Sumatra in South-East Asia. It swept across the Indian Ocean over a matter of hours leaving destruction in its wake. In Southern Thailand, Krabi Hospital, that province's major tertiary health centre, received the majority of the region's tsunami victims. Well-rehearsed contingency plans were in place to cope with 10, 20 and 40 trauma victims in the case of an extreme event. By the end of 26 December some 500 injured people had been treated at Krabi Hospital: well in excess of the 'worst case scenario' planning. Over the following days a total of 1357 tsunami victims were treated. Over the course of the day victims were able to move through the hospitals' system and gain appropriate treatment. This was achieved through the almost superhuman dedication of the hospitals' well-trained nursing and medical staff. In addition to this were large numbers of both Thai and foreign volunteers, who aided people with basic necessities such as providing them with food, water and clothing as well as simple human comfort, some also acting as translators for the health-care workers and the masses of injured and displaced people. Makeshift wards were constructed in halls and little used areas of the hospital, using army style stretchers to accommodate the wounded. Even though the disaster contingency plans at Krabi Hospital were utterly overrun, the fact that well-thought out and practised strategies were in place saved incalculable lives. The message is clear: practised responses to mass trauma situations will save lives and allows health-care teams to coordinate well in the face of overwhelming odds without panic. All centres should routinely practise disaster response through scenario based training. PMID- 16669947 TI - On the ground in Nias in response to an earthquake--an emergency team's experience. AB - Within a few short months of the tsunami, an earthquake measuring 8.7 on the Richter scale devastated the Indonesian island of Nias. The present paper describes the experiences of two emergency medicine staff deployed to the island operating in small teams on the remote western part of the island. It discusses the benefits of utilizing experienced emergency medicine staff and looks at lessons learnt from the deployment. PMID- 16669950 TI - Making the case for USCOM. PMID- 16669951 TI - Early goal-directed therapy in the emergency department. PMID- 16669953 TI - Review article: uncomplicated diverticular disease of the colon. AB - Diverticular disease of the colon is the fifth most important gastrointestinal disease in terms of direct and indirect health care costs in western countries. Uncomplicated diverticular disease is defined as the presence of diverticula in the absence of complications such as perforation, fistula, obstruction and/or bleeding. The distribution of diverticula along the colon varies worldwide being almost always left-sided and directly related to age in western countries and right-sided where diet is rich in fibre. The pathophysiology of diverticular disease is complex and relates to abnormal colonic motility, changes in the colonic wall, chronic mucosal low-grade inflammation, imbalance in colonic microflora and visceral hypersensitivity. Moreover, there can be genetic factors involved in the development of colonic diverticula. The use of non-absorbable antibiotics is the mainstay of therapy in patients with mild to moderate symptoms, and the effect of fibre-supplementation alone does not appear to be significantly different from placebo, although no definite data are available. More recently, alternative treatments have been reported. Mesalazine acts as a local mucosal immunomodulator and has been shown to improve symptoms and prevent recurrence of diverticulitis. In addition, probiotics have also been shown to be beneficial by re-establishing a normal gut microflora. In this study, the current literature on uncomplicated diverticular disease of the colon is reviewed. PMID- 16669954 TI - Review article: oesophageal spasm - diagnosis and management. AB - Oesophageal spasm is a common empiric diagnosis clinically applied to patients with unexplained chest pain. In contrast it is an uncommon manometric abnormality found in patients presenting with chest pain and/or dysphagia and diagnosed by >or=20% simultaneous oesophageal contractions during standardized motility testing. Using Medline we searched for diagnostic criteria and treatment options for oesophageal spasm. While the aetiology of this condition is unclear, studies suggest the culprit being a defect in the nitric oxide pathway. Well-known radiographic patterns have low sensitivities and specificities to identify intermittent simultaneous contractions. Recognizing that simultaneous contractions may result from gastro-oesophageal reflux this diagnosis should be investigated or treated first. Studies have documented improvements with proton pump inhibitors, nitrates, calcium-channel blockers and tricyclic antidepressants or serotonin reuptake inhibitors. Small case series reported benefits after botulinium toxin injections, dilatations and myotomies. Uncertainties persist regarding the optimal management of oesophageal spasm and recommendations are based on controlled studies with small numbers of patients or on case series. Acid suppression, muscle relaxants and visceral analgetics should be tried first. Botulinium toxin injections should be reserved for patients who do not respond. Pneumatic dilatations or myotomies represent rather heroic approaches for non responding patients. PMID- 16669955 TI - Safety and efficacy of two dose formulations of alicaforsen enema compared with mesalazine enema for treatment of mild to moderate left-sided ulcerative colitis: a randomized, double-blind, active-controlled trial. AB - BACKGROUND: Alicaforsen is an antisense oligonucleotide inhibitor of intercellular adhesion molecule 1 protein expression with activity in subjects with ulcerative colitis and pouchitis. AIM: To compare the effects of alicaforsen enema to standard of care mesalazine (mesalamine) enema in subjects with mild to moderate active left-sided ulcerative colitis. METHOD: A randomized, double blind, active-controlled multicentre clinical trial. Subjects received a nightly enema of 120 mg alicaforsen (n=55), 240 mg alicaforsen (n=50), or 4 g mesalazine (n=54) for 6 weeks, followed by a 24-week monitoring period. The primary end point was Disease Activity Index at week 6. Clinical improvement, remission and relapse were secondary end points. RESULTS: No significant difference was observed between treatment arms in the primary end point. However, the median duration of response to alicaforsen enema treatment was two- to threefold longer (128 and 146 days) in comparison with mesalazine (54 days). Complete mucosal healing occurred in 24% of the 240 mg alicaforsen group, when compared with 17% in the mesalazine. CONCLUSIONS: Alicaforsen enema demonstrated an acute response and safety profile similar to mesalazine enema, but was differentiated by a more durable response. The extended length of remission suggests that alicaforsen enema treatment may have a disease modifying effect. PMID- 16669956 TI - A phase II dose ranging, double-blind, placebo-controlled study of alicaforsen enema in subjects with acute exacerbation of mild to moderate left-sided ulcerative colitis. AB - BACKGROUND: Alicaforsen is an antisense oligonucleotide designed to inhibit expression of human intercellular adhesion molecule 1. Previous clinical studies have demonstrated activity of alicaforsen enema in ulcerative colitis and pouchitis. AIM: To determine the minimally effective dosing regimen of alicaforsen enema in subjects with mild to moderate left-sided ulcerative colitis. METHODS: Randomized, placebo-controlled, double-blind, two-dose ranging multicentre study. One hundred and twelve subjects were equally randomized to receive one of four alicaforsen enema regimens or placebo daily for 6 weeks. Primary end point was Disease Activity Index at week 6. Secondary end points included evaluation of clinical improvement, relapse rates and durability of response. Analysis of data were performed on the intent-to-treat population. RESULTS: No significant difference was observed between treatment arms and placebo in the primary end point. A prolonged reduction in mean% Disease Activity Index relative to baseline was observed in the daily 240 mg alicaforsen enema treatment arm in comparison with placebo from week 18 (51% vs. 18%, P=0.04) to week 30 (50% vs. 11%, P=0.03). CONCLUSIONS: Alicaforsen enema was safe and well tolerated at all doses studied. The durability of the response to alicaforsen enema treatment may suggests a disease-modifying effect. PMID- 16669957 TI - Bioavailability and therapeutic activity of alicaforsen (ISIS 2302) administered as a rectal retention enema to subjects with active ulcerative colitis. AB - BACKGROUND: Alicaforsen is a phosphorothioate-modified antisense oligodeoxynucleotide designed to sequence-specifically reduce intercellular adhesion molecule 1 messenger RNA levels. AIMS: To determine the systemic and local bioavailability of alicaforsen, and its activity when administered as a once daily enema in subjects with active ulcerative colitis. METHODS An open label study was conducted to assess the relative absorption (local and systemic pharmacokinetics) and pharmacologic activity of alicaforsen enema in subjects with active ulcerative colitis. Fifteen subjects received nightly enemas of alicaforsen (240 mg) for a treatment period of 6 weeks. Alicaforsen concentrations in plasma and colonic tissue biopsies were determined. Disease activity index and multiple measurements including endoscopy were used to assess alicaforsen activity in these subjects. RESULTS: Plasma concentrations of parent alicaforsen represented < 0.6% mean bioavailability when compared with historical intravenous area under the plasma concentration-time curves. Concentrations of the intact oligonucleotide in mucosal colonic tissue biopsies were orders of magnitude higher than those observed in plasma. A 46% reduction in mean Disease Activity Index and 33% rate of remission as defined by complete mucosal healing were observed at the end of treatment. Conclusion These data confirm that alicaforsen enema provides local treatment for a local disease with little meaningful systemic exposure. PMID- 16669958 TI - Basiliximab for the treatment of steroid-resistant ulcerative colitis: further experience in moderate and severe disease. AB - BACKGROUND: Preliminary data have suggested that interleukin-2 receptor blockade with basiliximab may increase steroid sensitivity. We have previously reported a small case series demonstrating the potential of basiliximab as a novel agent for the treatment of steroid-resistant ulcerative colitis. AIM: To report further experience of the efficacy and safety of treatment with the interleukin-2 receptor blocking monoclonal antibody basiliximab, in addition to steroids, for the treatment of severe and moderate steroid-resistant ulcerative colitis. METHODS: Twenty patients were enrolled - 13 patients with moderate steroid resistant ulcerative colitis (Ulcerative Colitis Symptom Score: >or=6) and seven patients with severe steroid-resistant ulcerative colitis. All were given a single dose of 40 mg basiliximab plus standard steroid therapy in an open-label, uncontrolled trial. Primary end point was clinical remission within 8 weeks (Ulcerative Colitis Symptom Score: 60 min before meals, after meals, as needed, or at bedtime. RESULTS: Forty-six percent dosed optimally. Fifty-four percent dosed sub-optimally with 21 of 54 (39%) dosing >60 min before meals, 16 (30%) after meals, 15 (28%) at bedtime and two (4%) as needed. Only 6% of the subjects on once-daily proton pump inhibitor regimens and 33% of subjects taking proton pump inhibitors two- to three times daily dosed in a manner that maximized acid suppression (15-30 min before a meal). CONCLUSIONS: In this study, 54% of patients dosed proton pump inhibitors sub-optimally and only 12% dosed in a manner that maximized acid suppression. As sub-optimal proton pump inhibitor dose timing can limit efficacy, patients with refractory symptoms should be asked about dose timing to avoid inappropriate and costly dose escalations. PMID- 16669963 TI - Efficacy and safety of bisacodyl in the acute treatment of constipation: a double blind, randomized, placebo-controlled study. AB - BACKGROUND: Although laxatives are a first-line treatment for constipation, there are few randomized placebo-controlled trials assessing their efficacy. AIM: To determine the effect and safety of oral bisacodyl on stool frequency and consistency in patients with idiopathic constipation. METHODS: 55 patients (age 19-89 years) with idiopathic constipation were recruited from eight primary care practices and randomized to receive bisacodyl, 10 mg once daily, or placebo, on three successive days following a 3-day run-in period. Patients recorded stool frequency and consistency and adverse events. RESULTS; In each treatment group, 27 patients were evaluable for efficacy. The mean number of stools per day was significantly greater in the bisacodyl-treated group (1.8/day) compared with placebo (0.95/day) over the treatment phase (P=0.0061). Mean stool consistency score improved from 'hard' (run-in) to between 'soft' and 'well-formed' during bisacodyl treatment, remaining between 'moderately hard' and 'hard' for placebo treatment (P<0.0001). The investigator's global efficacy score was superior for the bisacodyl group compared with placebo. Both treatments were well tolerated. Serum electrolyte levels and incidence of adverse events were comparable between treatment groups. CONCLUSIONS: Bisacodyl is effective and safe in improving stool frequency and consistency in acute treatment of idiopathic constipation. PMID- 16669964 TI - The impact of low-dose aspirin on endoscopic gastric and duodenal ulcer rates in users of a non-selective non-steroidal anti-inflammatory drug or a cyclo oxygenase-2-selective inhibitor. AB - BACKGROUND: The effect of low-dose aspirin on endoscopic ulcer incidence in cyclo oxygenase-2-selective inhibitor or non-selective non-steroidal anti-inflammatory drug users remains controversial. AIM: To compare prospectively the incidence of endoscopic ulcers in healthy subjects receiving low-dose aspirin plus celecoxib or naproxen. METHODS: In this double-blind, placebo-controlled, 1-week study, subjects (50-75 years) were randomized to receive aspirin 325 mg o.d. plus either celecoxib 200 mg o.d., naproxen 500 mg b.d., or placebo. Baseline and end of study endoscopies were performed. The primary end point was incidence of one or more gastric and duodenal ulcers. RESULTS: A lower incidence of gastric and duodenal ulcers was seen in celecoxib/aspirin-treated subjects (19%) vs. naproxen/aspirin (27%; RR: 0.63, 95% CI: 0.44-0.92). Both naproxen/aspirin and celecoxib/aspirin groups demonstrated a higher incidence of gastric and duodenal ulcers vs. placebo/aspirin (8%; RR: 3.7, 95% CI: 1.8-7.6 and RR: 2.6, 95% CI: 1.2 5.8, respectively). CONCLUSIONS: Fewer endoscopic ulcers were observed in patients treated with celecoxib/aspirin vs. naproxen/aspirin. However, celecoxib/aspirin was associated with a significantly higher incidence of gastric and duodenal ulcers than aspirin alone. Further studies are required to determine the generalizability of these findings in the aspirin users and to determine the appropriate strategy to minimize risk in susceptible patients. PMID- 16669965 TI - Rare case of Alstrom syndrome without obesity and with short stature, diagnosed in adulthood. AB - Alstrom syndrome is a rare autosomal recessive disorder characterized by retinal degeneration, sensorineural hearing loss, obesity, type 2 diabetes mellitus and chronic nephropathy. It may be associated with acanthosis nigricans, hypergonadotropic hypogonadism, hepatic dysfunction, hepatic steatosis, hyperlipidaemia, dilated cardiomyopathy and short stature. We report a patient with Alstrom syndrome who had hypergonadotropic hypogonadism, hepatic dysfunction, hepatic steatosis and short stature with normal body weight, all of which are seen infrequently with this syndrome. PMID- 16669966 TI - Rethinking the peritoneal dialysis prescription: results of recent studies. AB - Peritoneal dialysis treats uraemia in a way different from hemodialysis. The continuous nature of peritoneal dialysis optimises the removal of uraemic toxins of larger molecular weight, the so-called 'middle molecules'. Initially, there was an appreciation of the efficacy of this kind of slow, continuous dialysis. However, with the growing emphasis on adequacy as defined by small solute kinetics, blood purification by peritoneal dialysis was considered to be inferior to that performed with hemodialysis. With the subsequent publication of studies showing a lack of correlation of small solute clearance parameters with outcome in peritoneal dialysis, attention is again being paid to the benefits of continuous dialysis that treats renal failure in a way not quantifiable by small solute kinetics. PMID- 16669967 TI - National haemodialysis guidelines: an assessment of compliance from 2001-2003. AB - AIM: The Caring for Australians with Renal Impairment (CARI) clinical practice guidelines were established in 2000 to provide recommended ranges for parameters associated with anaemia management in patients with chronic kidney disease. This study used data from the Renal Anaemia Management (RAM) database to determine the level of compliance with the CARI Guidelines in Australia since their implementation. METHODS: De-identified data from haemodialysis patients at 15 dialysis centres were obtained from the RAM database provided by Janssen-Cilag Pty Ltd, Australia. Validated data were extracted over 6 months (April-September) in 2001 (n = 2586 patients) and 2003 (n = 3190 patients). The percentage of patients with biochemical and haematological parameters that were within the ranges recommended in the CARI Guidelines was compared from 2001-2003. RESULTS: There was a significant increase in the number of patients with values within the recommended ranges for serum ferritin, phosphate, calcium phosphate product and urea reduction ratio from 2001-2003. There was no change in the proportion of patients with values within the recommended ranges for haemoglobin, transferrin saturation, calcium or parathyroid hormone. There was considerable variation in compliance with recommended ranges between and within individual dialysis centres. Compliance to the target haemoglobin level (> or =110 g/L) ranged from 42-78% of patients at different centres in 2003. CONCLUSION: Although the number of patients with values within those recommended in the CARI guidelines has increased for some parameters, many patients in Australia have clinical parameters outside the ranges recommended in the CARI Guidelines. PMID- 16669968 TI - Pantoea peritonitis in a patient receiving chronic ambulatory peritoneal dialysis. AB - Pantoea agglomerans is usually the most common organism transmitted through plant thorn injuries. This report is of a female patient maintained on chronic ambulatory peritoneal dialysis (CAPD) who developed peritonitis attributed to P. agglomerans. Peritonitis is an uncommon complication of P. agglomerans and there is no previous report of peritonitis associated with this organism in a CAPD patient. The source of infection was thought to be due to rose-thorn injury. Antibiotic therapy with ceftazidime and amikacin i.p. led to a clinical improvement, with disappearance of the organism in the peritoneal fluid. PMID- 16669969 TI - Blood pressure control in haemodialysis patients: an audit. AB - OBJECTIVE: This audit was conducted to study the level of achievement of some criteria relevant to blood pressure control in haemodialysis patients and to evaluate if auditing process improves the quality of medical care given to these patients. METHODS: The records of all 105 patients on maintenance haemodialysis were included in the study. Five criteria relevant to blood pressure control were selected for auditing. The criteria were: predialysis blood pressure control <140/80, dry weight, dialysis adequacy, salt and water intake restriction, and patient education. The auditing process was conducted at two different times 6 months apart. RESULTS: The first data collected in the auditing process showed that there were only 40 patients (38%) with controlled predialysis blood pressure. In the second data collection the number of patients with controlled predialysis blood pressure had risen significantly to 78 (74.3%), P < 0.0001. The same improvement was achieved for dry weight 39 (37%) to 73 (69.5%) P < 0.0001, dialysis adequacy 38 (36.2%) to 75 (71.4%) P = 0.043, salt and water intake restriction 39 (37%) to 71 (67.6%) P = 0.045, and patient education 30 (28.6%) to 55 (52.4%) P < 0.0001. CONCLUSION: Setting up audit cycles to evaluate achievement of required standard in relevant criteria contribute to better blood pressure control and leads to improvement in the care of patients on dialysis. Auditing can be an essential tool in identification of poorly controlled blood pressure, its cause and is useful in control of hypertension in the haemodialysis population. PMID- 16669970 TI - Aldose reductase regulates TGF-beta1-induced production of fibronectin and type IV collagen in cultured rat mesangial cells. AB - AIM: To study the effects of aldose reductase (AR) on production of fibronectin and type IV collagen in rat mesangial cells (MsC). METHODS: The vector, pcDNA3 AR, was constructed based on pET-15b-AR. Lipofect AMINE was used for stable transfection and G418 was used for selecting positive clones. Sorbinil and zopolrestat were added for suppressing the activity of AR, respectively. The production of fibronectin and type IV collagen and the activation of Smads and MAPK signal transduction pathway were analysed by western blot and AP-1 activity was analysed by electrophoretic mobility shift assays (EMSA). RESULTS: The normal MsC showed increased expression of fibronectin and type IV collagen with stimulation of TGF-beta1. Compared with the normal MsC, the MsC pre-incubated with ARI showed reduced expression (P < 0.05) and the AR-transfected MsC showed increased expression (P < 0.05). The normal MsC showed activation of ERK, JNK and p38 with stimulation of TGF-beta1, while the activation of JNK and p38 was inhibited in the MsC pre-incubated with ARI and only the activation of JNK was enhanced in the AR-transfected MsC. The normal MsC showed enhanced AP-1 activity with the stimulation of TGF-beta1, and similarly the activity was inhibited in the MsC pre-incubated with ARI and was more enhanced in the AR transfected MsC. CONCLUSION: AR can regulate the expression of fibronectin and type IV collagen with the stimulation of TGF-beta1 in MsC, which may have relations with the activation of JNK-MAPK and p38-MAPK signalling pathways and AP-1. PMID- 16669971 TI - Randomised controlled trial of leflunomide in the treatment of immunoglobulin A nephropathy. AB - AIM: To investigate the effect of leflunomide for treatment of immunoglobulin A (IgA) nephropathy. METHODS: Sixty IgA nephropathy patients were divided into two groups at random. Patients in the test group received leflunomide and patients in the control group received fosinopril. Clinical data were obtained at weeks 2, 4, 6, 8, 12, 16, 20, 24 and 28. RESULTS: The complete remission rate was 62.1% and the total effectiveness rate was 72.4%. In the leflunomide group, proteinuria significantly decreased from 1.66 +/- 0.42 g to 0.60 +/- 0.68 g (P < 0.05). The efficacy rate of leflunomide compared with fosinopril in treating IgA nephropathy was not statistically different (P > 0.05). Side-effects were mild in both treatment groups. CONCLUSION: These preliminary results are encouraging, but further randomised studies are required before leflunomide can be recommended for the treatment of IgA nephropathy. PMID- 16669972 TI - Ehlers-Danlos syndrome coexisting with juvenile nephronophtisis. AB - Ehlers-Danlos syndrome (EDS), a heterogeneous disease of the connective tissues, is diagnosed by a triad of symptoms that include skin hyperextensibility, joint hypermobility and connective tissue fragility. Nephronophtisis (NPH) is an autosomal recessive interstitial nephritis leading to terminal renal insufficiency around puberty. The occurrence of these two rare diseases together is unusual. A review of the literature discloses no case of this association. We report here on a 16-year-old man with undiagnosed EDS, who was referred to our hospital because of renal insufficiency, history of polyuria and polydipsia. Renal ultrasound showed normal kidney size, with a lack of corticomedullary differentiation. Renal biopsy specimen disclosed chronic tubulointerstitial nephritis resembling NPH. Further evaluation identified hypermobile joints and hyperextensible skin, which led to the diagnosis of the EDS. These data suggest that patients with EDS need to be evaluated carefully for the presence of renal anomalies. PMID- 16669973 TI - Synchronous carcinomas of stomach and bladder together with AA amyloidosis. AB - Although the association and causality between chronic inflammatory states and systemic AA amyloidosis have been well established, the evidence linking solid malignancies to reactive AA amyloidosis is scarce. Here, a case of diagnosed AA amyloidosis associated with synchronous carcinomas of stomach and bladder complicated with nephrotic syndrome and renal failure is reported. PMID- 16669974 TI - Optimal care of autosomal dominant polycystic kidney disease patients. AB - Autosomal dominant polycystic kidney disease (ADPKD) is the most common life threatening, hereditary disease. The prevalence of ADPKD is more common than Huntington disease, haemophilia, sickle cell disease, cystic fibrosis, myotonic dystrophy and Down syndrome combined. In recent years there have not only been advances in the understanding of the genetic and molecular events involved in ADPKD, but some diagnostic and therapeutic advances have also emerged. In the genetics area, the gene for PKD1 was localised to chromosome 16, is associated with polycystin-2 protein, and found to account for approximately 85% of patients with ADPKD. The gene for PKD2, found in chromosome 4, accounts for approximately 15% of ADPKD, and is associated with the polycystin-2 protein. While these genetic and molecular biology findings have stimulated a great deal of exciting basic research in ADPKD, therapies to decrease morbidity and mortality in ADPKD patients have yet to emerge from these findings. In contrast, the early diagnosis and treatment of hypertension with inhibitors of the renin-angiotensin aldosterone system have the potential to decrease or prevent left ventricular hypertrophy cardiac complications and slow the progression of the renal disease. PMID- 16669975 TI - Hyperlipidaemia, lack of sleep and smoking as risk factors for proteinuria among high altitude mountain trekkers. AB - AIM: The number of alpine accidents has been increasing among trekkers in Japan. Some of the alpine accidents have been related to acute mountain sickness (AMS). At present, the mechanism of AMS is poorly clarified, however, it is well known that hypoxia at high altitudes plays an important role. A good relation exists between proteinuria, altitude and hypoxia. However, no reports are available concerned with the risk factors of proteinuria for trekkers at high altitudes. METHODS: The authors evaluated 41 trekkers and 13 highlanders (cabin employees) through physical examinations and interviews held in a mountain cabin (2300 m above sea level) located in a mountain (2926 m above sea level) in the Japanese Alps. First, urine samples were collected and urinalysis was performed. Second, non-invasive blood pressure, heart rate, and arterial oxygen saturation (SpO2) were measured using a portable life monitor. RESULTS: Proteinuria was present in 20 (49%) participants. Past history of hyperlipidaemia, amount of sleep, and smoking history are independently associated with proteinuria. Among the physiological examinations conducted at the cabin, only systolic blood pressure demonstrated strong association with proteinuria. Receiver operating characteristic curve analysis using a score combining past history of hyperlipidaemia, amount of sleep and smoking history revealed that the diagnostic accuracy of proteinuria at the cut-off score of 2 was 80%. CONCLUSIONS: This study indicated that these three variables were significantly and independently correlated with proteinuria: hyperlipidaemia, amount of sleep (night before) and smoking history. Improvement of these three variables might decrease the risk of alpine accidents including AMS. PMID- 16669976 TI - Folic acid 5 or 15 mg/d similarly reduces plasma homocysteine in patients with moderate-advanced chronic renal failure. AB - BACKGROUND: Hyperhomocysteinaemia is an independent risk factor for cardiovascular disease with a remarkable prevalence in patients with chronic renal failure (CRF). Low doses of folic acid (FA) with or without vitamin B6 and B12 has been shown to effectively reduce plasma homocysteine (Hcy). The aim of this study was to compare the short-term effects of two different oral doses of FA (5 vs 15 mg/d) on plasma Hcy levels in subjects suffering from moderate-severe CRF. METHODS: A double-blind, double-dummy, comparative, two-stage randomised study was performed. Seventeen patients aged 45-71 years, with glomerular filtration rates between 15.4-50 mL/min 1.73/m2 were randomly assigned to receive FA 5 mg/d (FA-5, n: 8) or FA 15 mg/d (FA-15, n: 9) for 30 days. At the end of this 30-day double-blind period, all the participants were placed on FA 5 mg/d (open period), for 5 additional months. Both groups were also supplemented with vitamins B1, B6 and B12 throughout the trial. Blood samples were drawn at 0, 15, 30, 90 and 180 days to assess Hcy, complete blood count (CBC) and sequential multichannel analysis (SMA). Chest X-ray and a 12-lead electrocardiogram (ECG) were also performed. RESULTS: Plasma Hcy (mean +/- SEM) decreased from 27.9 +/- 1.4 (baseline) to 15.1 +/- 0.6, 13.3 +/- 0.9, 14.1 +/- 0.5 and 13.8 +/- 0.5 micromol/L (FA-5) and from 28.8 +/- 2.7 to 15.6 +/- 1.2, 14.4 +/- 1.3, 13.0 +/- 0.7 and 13.1 +/- 0.6 micromol/L (FA-15) at days 15, 30, 90 and 180, respectively. (P < 0.01 from day 15 to 180 vs baseline for both groups with no differences between them). Renal function remained stable throughout the entire period of the study in all but one patient in whom it deteriorated to pre-end stage disease. No adverse cardiovascular events developed during the trial. CONCLUSION: Both folic acid doses induced a significant and similar decrease in plasma Hcy in subjects with moderate-severe chronic renal failure. The possible dose-related effect of this approach in reducing the risk of accelerated sclerotic vascular disease and cardiovascular events in this especially vulnerable population should be a matter of further investigation. PMID- 16669977 TI - Long-term comparison of sevelamer hydrochloride to calcium-containing phosphate binders. AB - AIM: In patients with end-stage renal disease (ESRD), hyperphosphataemia and an elevated calcium-phosphorus (Ca-P) product contribute to morbidity and mortality. Suggested target goals for serum phosphorus concentration and calcium-phosphorus product have recently been lowered. As a result, long-term comparative studies of the efficacy of phosphate binders are critical. This study compares the long-term efficacy of sevelamer hydrochloride to calcium-containing binders (CCB). METHODS: A retrospective chart review was conducted in 30 patients receiving sevelamer hydrochloride for >1 years and 25 patients receiving CCB. RESULTS: Patients on sevelamer hydrochloride had lower serum bicarbonate concentration than those on CCB, 18.6 +/- 2.7 versus 20.3 +/- 1.8 mmol/L (P = 0.0017). Serum phosphorus concentration was higher in patients on sevelamer hydrochloride compared to CCB 2.10 +/- 0.87 versus 1.74 +/- 0.28 mmol/L (P = 0.0013), as was the Ca-P product 4.97 +/- 0.94 mmol2L2 (62.1 +/- 11.8 mg2/dL2) versus 3.97 +/- 1.18 mmol2/L2 (49.7 +/- 14.7 mg2/dL2), P = 0.0009). Only 36% of patients on sevelamer hydrochloride compared with 68% on CCB (P = 0.015) met the serum phosphorus goal of < or =1.78 mmol/L. CONCLUSION: Patients on sevelamer hydrochloride for >1 years compared to those on CCB had a lower serum bicarbonate concentration, a higher serum phosphorus concentration and a higher Ca-P product. Clinicians should balance the increase in calcium load with CCB versus the cost and effectiveness of sevelamer hydrochloride in choosing a phosphate binder for ESRD patients. PMID- 16669978 TI - Colchicine myoneuropathy in chronic renal failure patients with gout. AB - Colchicine myoneuropathy is a rare and often underdiagnosed disease. It often presents as painless subacute muscle weakness. We present a case of painful colchicine myoneuropathy in a 76-year-old man with chronic renal failure and gout. Published work about clinical presentations of colchicine myoneuropathy in gouty arthritis patients are reviewed. During the previous year, the patient had a drug regimen of colchicine 0.5 mg three times per day for a 3 day course each month. He developed bilateral lower leg weakness and severe myalgia. His serum creatinine level was 680.7 micromol/L and creatinine kinase was 959 IU/L on admission. Laboratory findings included decreasing amplitude of motor and sensory nerve conduction velocity and an electromyogram showed small amplitude, short duration polyphasic waves over the right biceps. A muscle biopsy disclosed vacuolar changes in the cytoplasm. These results all supported a diagnosis of colchicine myoneuropathy. After cessation of colchicine, the creatinine kinase level decreased approximately 50% in 6 days, myalgia subsided and his muscle weakness improved gradually over the next 2 weeks. PMID- 16669979 TI - Preliminary experience with mycophenolate mofetil for preservation of renal function in cardiac transplant patients with documented cyclosporine nephrotoxicity. AB - BACKGROUND: Cyclosporine (CyA) has positively impacted on the outcome of cardiac transplantation; however, the nephrotoxicity associated with CyA has been a major drawback. METHODS: In an effort to reduce exposure to CyA and possibly alleviate its nephrotoxic effects, we undertook a therapeutic strategy to switch cardiac transplant patients with biopsy-proven CyA nephrotoxicity from azathioprine (AZA) to mycophenolate mofetil (MMF) with subsequent CyA dose reduction or elimination. RESULTS: MMF was substituted for AZA in five cardiac transplant patients (four males; mean age, 60 +/- 6 years old; average time from transplant was 7 +/- 3 years) who had biopsy proven evidence of CyA nephrotoxicity, and in whom CyA dose was reduced (3/5) or discontinued (2/5). At the time of the therapeutic intervention, four patients had an average serum creatinine of 230 +/- 62 micromol/L and one patient had just been started on haemodialysis (HD). During an average follow-up period of 42 months, the slope of the inverse serum creatinine significantly improved in three patients and continued to deteriorate in one patient. The patient on HD could be transiently taken off HD. However, he developed a severe episode of cardiac rejection requiring antirejection therapy and increase in the dose of CyA. The patient was subsequently returned back on HD. CONCLUSION: In this preliminary report, we show that AZA to MMF switch with subsequent CyA dose reduction or discontinuation may slow down the progression of kidney disease in some patients. However, the patients should be followed closely for evidence of cardiac rejection. PMID- 16669980 TI - Miliary tuberculosis in a Caucasian male transplant recipient and the role of intravenous immunoglobulin as an immunosuppressive sparing agent. AB - Opportunistic infections are a common and anticipated accompaniment of transplantation, but are generally somewhat predictable in their timing and epidemiology. The authors report here a case of miliary tuberculosis occurring within 3 weeks of transplantation, in a patient not expected to be significantly at risk, and with a normal chest X-ray at the time of transplantation. A 25-year old Caucasian male dialysis patient who received two paediatric kidneys as an en bloc renal transplant developed fever 3 weeks following transplantation; this eventually proved to be miliary tuberculosis. As well as antituberculous therapy and a significant reduction in the patient's conventional immunosuppression, intravenous immunoglobulin was used as anti-rejection prophylaxis. The case highlights the immunosuppressed status of dialysis patients prior to transplantation and the need for broad differential diagnosis in transplant recipients even in the absence of recognized epidemiological factors. The case also emphasizes the role of intravenous immunoglobulin as an anti-rejection therapy that does not add to the patient's immunosuppressive burden. PMID- 16669981 TI - The changing nature of the educational process. PMID- 16669983 TI - Beyond dualism: leading out of oppression. AB - PURPOSE: To reexamine our beliefs about our gender identity in order to identify new possibilities for leading in nursing. SOURCES OF INFORMATION: Leadership is complex. This article is the result of a lengthy iterative process of exploring the empowerment, image, leadership, feminist, and oppression literature. All of this was distilled in the context of the author's experience as a nurse and nurse leader. CONCLUSIONS: Moving beyond dualism creates new possibilities for leading nurses out of oppression. PMID- 16669984 TI - Self-healing: a concept analysis. AB - Complementary and alternative medicine (CAM) is a rapidly growing specialty within the healthcare field. One concept that appears central to the notion of CAM therapies is the concept of self-healing. Although "self-healing" is addressed within several bodies of literature, the concept is ill-defined within the context of CAM therapies, specifically energy-based healing modalities such as reiki therapy. The purpose of this paper is to investigate the concept of self healing through a concept analysis using Walker and Avant's technique (1995). The resultant operational definition of self-healing was the result of 6 weeks of study and is not considered to be a final product, but merely a beginning step to understanding this unique phenomenon. PMID- 16669985 TI - Mother nature's disasters and their health effects: a literature review. AB - Each year global warming and climate changes are affecting Mother Nature, causing extreme weather events to occur (Patz & Khaliq, 2002). The impact of these disasters uproots populations, causes massive infrastructure destruction, and sets the stage for an increase of population mortality and morbidity. Before, during, and after the disaster, nurses provide health care to those in need. Nurses should be aware of the anticipated diseases, traumas, and short- and long term illnesses that occur following the different types of natural disasters. The purpose of this article is to review existing data and alert nurses to the potential threats they may face following a natural disaster. PMID- 16669986 TI - Experiences of teaching nursing in four countries. AB - This article presents reflections on personal experiences of teaching nursing in the United Kingdom, Ghana, Uganda, and South Africa. The experiences related focus on differences and similarities in institutional cultures, teaching strategies, categories of students, students' expectations, and learning opportunities. The four institutions could be described as different; yet because of the hierarchical nature of the nursing profession, even within the training or university settings, a new staff member regardless of his/her qualifications and experiences is frequently relegated to the level of a novice. Discussions on some of the challenges facing Ghana and Uganda nursing training institutions and suggestions on possible approaches to address those challenges are presented. PMID- 16669987 TI - Said another way. Our obligation to float. AB - TOPIC: Floating is one method that can help alleviate the burden and consequences of the nursing shortage. PURPOSE: The devastating effects that occur as a result of the nursing shortage are discussed. As a result, floating is argued as a viable solution. The practice of floating is explored from an ethical approach. SOURCES OF INFORMATION: Professional literature, electronic resources, nursing textbooks, from statements provided from the American Association of Colleges of Nursing, Joint Commission on Accreditation of Healthcare Organizations, Six Sigma, legal cases, and personal experience. CONCLUSION: Although many nurses oppose the practice of floating, it remains not only necessary, but it is also our obligation. It is acknowledged that floating has its opposition, but the arguments posed are not justifiable when they are weighed against the consequences of not floating. PMID- 16669989 TI - The differential diagnosis of atopic dermatitis in childhood. AB - Atopic is the most common of the dermatitides seen in infancy and childhood, but there are numerous other diseases that can mimic the skin findings. These include seborrheic dermatitis, immunodeficiency, and psoriasis in infancy; scabies, tinea corporis infection, perioral, nummular, contact, and molluscum dermatitis in childhood. It is sometimes extremely difficult to differentiate between ichthyosis and AD, and it is also important to differentiate AD from erythrodermic conditions including acrodermatitis enteropathica, biotin deficiency, and Netherton syndrome. A rare condition in children that may mimic AD is mycosis fungoides. PMID- 16669990 TI - Pediatric atopic dermatitis: should we treat it differently? AB - Atopic dermatitis is an extremely common childhood skin disease that can have far reaching impact on patients and families. Pediatric patients, particularly infants, pose special concerns for parents and providers, and equal emphasis must be placed on both nonpharmacologic and prescription interventions. Concerns for adverse effects of prescription therapies and a universal parental fear of an undetected allergy are hallmarks of pediatric atopic dermatitis care. The purpose of the present study is to highlight important educational and therapeutic strategies designed to optimally care for this patient population. PMID- 16669991 TI - Patterns of care and referral in children with atopic dermatitis and concern for food allergy. AB - Although many providers believe that up to 30% of atopic dermatitis (AD) is food induced, food challenge studies show that food-induced eczematous reactions are rare. When food allergy is suggested to cause AD, it often leads to allergy testing with a high false-positivity rate, in turn further focusing parents on food allergy. Study subjects were children less than 11 years old with AD and food allergy suspicion. Prior diagnoses, provider, and testing patterns were assessed by questionnaire given to the parents. Thirty-eight patients with AD were enrolled. Most subject's parents suspected food allergy induced AD. Initial skin diagnoses were made by pediatricians (79%) and family practitioners (18%) as eczema. Allergy was suggested by providers as cause for AD in 63% of the present study's patients. Seventy-nine percent had allergy testing. Greater than 90% of parents claimed their children had food allergy and food-induced AD. Sixty-six percent had positive food allergy tests and 37% had definite history of immediate IgE reactions to food. The majority of this population had allergy suggested as causative for eczema by their primary care provider and were subsequently evaluated by allergist and allergy testing. Consensus about the role of food allergy between the different providers of AD in children would result in more effective, efficient, and less costly health care. PMID- 16669992 TI - Does food allergy cause atopic dermatitis? Food challenge testing to dissociate eczematous from immediate reactions. AB - The objective is to evaluate and diagnose, in a controlled setting, suspected food allergy causation in patients hospitalized for management of severe, unremitting atopic dermatitis (AD). Nineteen children were hospitalized at Oregon Health and Science University with atopic dermatitis from 1986 to 2003 for food restriction, then challenge, following standard recommendations. Challenges were prioritized by categories of (a) critical foods (e.g., milk, wheat, egg, soy); (b) important foods; and (c) other suspected foods. Patients were closely observed for evidence of pruritus, eczematous responses, or IgE-mediated reactions. If results were inconsistent, double-blind, placebo-controlled food challenge was performed. A total of 17 children with atopic dermatitis were assessed. Two could not be fully evaluated, thus were excluded from data tabulations. Only one positive eczematous food response was observed of 58 challenges. Three children had well-documented histories of food-induced IgE mediated anaphylactoid or urticaria reactions to seafood and/or nuts and were not challenged with those foods. Atopic dermatitis, even in the highest-risk patients, is rarely induced by foods. Undocumented assumptions of food causation detract from proper anti-inflammatory management and should be discouraged. Immediate IgE-mediated food reactions are common in atopic dermatitis patients; such reactions are rapid onset, typically detected outside the clinic, and must be distinguished from eczematous reactions. Diagnosis of food-induced eczema cannot be made without food challenge testing. Such tests can be practical and useful for dispelling unrealistic assumptions about food allergy causation of atopic dermatitis. PMID- 16669993 TI - The psychosocial burden of childhood atopic dermatitis. AB - Atopic dermatitis is an extremely common childhood disease of increasing prevalence that greatly affects the quality of life of afflicted children and of their families. The disease alters the emotional and social functioning of the affected child and their family. The complex multidimensional effects of atopic dermatitis in children and families have been described qualitatively and measured quantitatively with quality of life instruments. Emotional effects on both the child and parents are predominant. The burden of atopic dermatitis can be improved by targeting parents and caregivers with education, psychosocial support, and specialty care. PMID- 16669994 TI - Atopic dermatitis prevention. AB - A shift in focus from disease therapy toward disease prevention is occurring in many medical specialties, including dermatology. There are no generally accepted strategies for the prevention of atopic dermatitis. Most research has focused on allergen avoidance and has not produced consistently effective interventions. Immune cell modulation and skin barrier protection are examples of new approaches that hold promise for preventing, or modifying the course of, this common disease. PMID- 16669995 TI - Topical tacrolimus in the management of atopic dermatitis in Japan. AB - Atopic dermatitis (AD) is a common, chronic, relapsing, severely pruritic, eczematous skin disease. Topical steroids are the mainstay of treatment. However, the adverse effects of steroids on hormonal function are the major obstacle for their use as long-term topical therapy. Topical calcineurin inhibitors, such as tacrolimus, not only complement existing treatment options but also overcome some of the drawbacks of topical steroid therapy and fulfill the long-term needs of patients in preventing disease progression. Short- and long-term efficacy and safety of topical tacrolimus has been widely recognized and it is also accepted as a first-line treatment for the inflammation of AD. In order to reduce the possible long-term adverse effects, it is important to monitor the clinical dose in daily clinics. PMID- 16669996 TI - Integrated maps in quail (Coturnix japonica) confirm the high degree of synteny conservation with chicken (Gallus gallus) despite 35 million years of divergence. AB - BACKGROUND: By comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps. RESULTS: The integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied. CONCLUSION: Integrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail. PMID- 16669997 TI - Relationship of serum adiponectin and resistin to glucose intolerance and fat topography in South-Asians. AB - OBJECTIVES: South-Asians have lower adiponectin levels compared to Caucasians. It was not clear however, if this intrinsic feature is related to aspects of glucose metabolism. This study aims to determine the relationship between body fat distribution and adipocytokine in South-Asian subjects by measuring serum adipocytokines, adiposity, insulinemia, and glucose tolerance levels. METHODS: In this cross-sectional study, 150 South-Asians (80 males, 70 females) were included, 60 had NGT (Control group, Age 51.33 +/- 11.5, BMI 27 +/- 2.3), 60 had IGT (Age 57.7 +/- 12.5, BMI 27.2 +/- 2.7), 30 had type 2 DM (Age 49.5 +/- 10.9, BMI 28 +/- 1.7). Measures of adiposity, adipocytokines and other metabolic parameters were determined. Parameters were measured using the following: a) Plasma glucose by glucose oxidase method b) CRP by immunoturbidimetric method (Roche/Hitachi analyser) c) insulin by Medgenix INS-ELISA immunoenzymetric assay by Biosource (Belgium) d) Leptin, Adiponectin by radioimmunoassay kits by Linco Research (St. Charles MO) e) Resistin by immunoassay kits by Phoenix Pharmaceuticals INC (530 Harbor Boulevard, Belmont CA 94002, USA). RESULTS: Adiponectin concentrations were highest in NGT, decreased in IGT and lowest in DMT2, (both p < 0.01). Leptin was significantly higher in DMT2 than IGT and NGT p = 0.02 and 0.04 respectively. There was a significant positive relationships between log adiponectin and 2-hr insulin values, p = 0.028 and history of hypertensions and a ischemic heart disease p = 0.008 with R = 0.65. There was a significant inverse correlation between log adiponectin and resistin, p < 0.01. CONCLUSION: Resistin levels had an inverse correlation with adiponectin levels, indicating an inverse relationship between pro-inflammatory cytokines and adiponectin. Adiponectin levels were related to glucose tolerance. PMID- 16669998 TI - Council tax valuation bands, socio-economic status and health outcome: a cross sectional analysis from the Caerphilly Health and Social Needs Study. AB - Council tax valuation bands (CTVBs) are a categorisation of household property value in Great Britain. The aim of the study was to assess the CTVB as a measure of socio-economic status by comparing the strength of the associations between selected health and lifestyle outcomes and CTVBs with two measures of socio economic status: the National Statistics Socio-Economic Classification (NS-SEC) and the 2001 UK census-based Townsend deprivation index. METHODS: Cross-sectional analysis of data on 12,092 respondents (adjusted response 62.7%) to the Caerphilly Health and Social Needs Study, a postal questionnaire survey undertaken in Caerphilly county borough, south-east Wales, UK. The CTVB was assigned to each individual by matching the sampling frame to the local authority council tax register. Crude and age-gender adjusted odds ratios for each category of CTVB, NS-SEC and fifth of the ward distribution of Townsend scores were estimated for smoking, poor diet, obesity, and limiting long-term illness using logistic regression. Mean mental (MCS) and physical (PCS) component summary scores of the Short-Form SF-36 health status questionnaire were estimated in general linear models. RESULTS: There were significant trends in odds ratios across the CTVB categories for all outcomes, most marked for smoking and mental and physical health status. The adjusted odds ratio for being a smoker in the lowest versus highest CTVB category was 3.80 (95% CI: 3.06, 4.71), compared to 3.00 (95% CI: 2.30, 3.90) for the NS-SEC 'never worked and long-term unemployed' versus 'higher managerial and professional' categories, and 1.61 (95% CI: 1.42, 1.83) for the most deprived versus the least deprived Townsend fifth. The difference in adjusted mean MCS scores was 5.9 points on the scale for CTVB, 9.2 for NS-SEC and 3.2 for the Townsend score. The values for the adjusted mean PCS scores were 6.3 points for CTVB, 11.3 for NS-SEC, and 2.5 for the Townsend score. CONCLUSION: CTVBs assigned to individuals were strongly associated with the health and lifestyle outcomes modelled in this study. CTVBs are readily available for all residential properties and deserve further consideration as a proxy for socio-economic status in epidemiological studies in Great Britain. PMID- 16669999 TI - Chronic Obstructive Pulmonary Disease, inflammation and co-morbidity--a common inflammatory phenotype? AB - Chronic Obstructive Pulmonary Disease (COPD) is and will remain a major cause of morbidity and mortality worldwide. The severity of airflow obstruction is known to relate to overall health status and mortality. However, even allowing for common aetiological factors, a link has been identified between COPD and other systemic diseases such as cardiovascular disease, diabetes and osteoporosis. COPD is known to be an inflammatory condition and neutrophil elastase has long been considered a significant mediator of the disease. Pro-inflammatory cytokines, in particular TNF-alpha (Tumour Necrosis Factor alpha), may be the driving force behind the disease process. However, the roles of inflammation and these pro inflammatory cytokines may extend beyond the lungs and play a part in the systemic effects of the disease and associated co-morbidities. This article describes the mechanisms involved and proposes a common inflammatory TNF-alpha phenotype that may, in part, account for the associations. PMID- 16670000 TI - Secular trends of blood isolates in patients from a rural area population hospitalized in a tertiary center in a small city in Greece. AB - BACKGROUND: Most of the studies evaluating the secular trends of blood isolates come from tertiary hospitals in urban areas. We sought to study the trends of the antimicrobial resistance of blood isolates in patients from a rural population hospitalized in a tertiary hospital in a small city in Greece. METHODS: We retrospectively collected and analysed data for the first positive blood culture obtained for each admission for each patient hospitalized in General Hospital of Tripolis, Tripolis, Peloponnesus, Greece during a 5 year period (16/05/2000 15/05/2005). RESULTS: Sixty-seven thousand and seventy patients were hospitalized during the study period from whom 3,206 blood cultures were obtained. A higher increase of the number of obtained blood cultures than the number of admissions was noted during the study period (p < 0.001). Three hundred and seventy-three (11.6%) blood cultures were positive. Coagulase-negative staphylococci (35.9%), Escherichia coli (29%), and Staphylococcus aureus (18.2%) were the most commonly isolated pathogens. Among the Staphylococcus aureus isolates, the proportion of methicillin-resistant Staphylococcus aureus (MRSA) was 17.2% (5/29). The proportion of Escherichia coli resistant to trimethoprim and sulfamethoxazole, ampicillin and cefuroxime was 29.6% (32/108), 25.0% (27/108), and 8.3% (9/108) respectively. Imipenem-resistance was noted in 3.4% (1/29) of Pseudomonas aeruginosa isolates. There were only 6 (1.6%) Acinetobacter baummanii blood isolates during the study period. CONCLUSION: The antimicrobial resistance of isolates from patients receiving care at the studied tertiary hospital in a small city in Greece is considerably less compared to that noted in tertiary hospitals in larger cities of the country. PMID- 16670001 TI - Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase. AB - BACKGROUND: In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. RESULTS: Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a gamma-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. gamma-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG gamma-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong gamma-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. CONCLUSION: We have applied biochemical approaches, not used previously, to characterise prominent D. melanogaster accessory gland products. Of the thirteen accessory gland secreted proteins reported in this study, six were represented in a D. simulans male accessory gland EST library that was biased for male-specific genes. Therefore, the present study has identified seven new secreted accessory gland proteins, including GGT-1, which was not recognised previously as a secreted accessory gland product. PMID- 16670002 TI - Circadian rhythm of the autonomic nervous system in insulin resistant subjects with normoglycemia, impaired fasting glycemia, impaired glucose tolerance, type 2 diabetes mellitus. AB - BACKGROUND: In type 2 diabetes mellitus both insulin resistance and hyperglycemia are considered responsible for autonomic dysfunction. The relation between the autonomic activity, impaired fasting glycemia and impaired glucose tolerance is, however, unclear. The purpose of this study was to evaluate and compare the circadian autonomic activity expressed as heart rate variability (HRV) measured by 24-hours ECG recording in insulin resistant subjects (IR) with characteristics as follow: IR subjects with normal oral glucose tolerance test results, IR subjects with impaired fasting glucose, IR subjects with impaired glucose tolerance and subjects with type 2 diabetes mellitus. METHODS: Eighty Caucasian insulin resistant subjects (IR) and twenty five control subjects were recruited for the study. IR subjects were divided into four groups according to the outcoming results of oral glucose tests (OGTTs): IR subjects with normal glucose regulation (NGR), IR subjects with impaired fasting glycemia (IFG), IR subjects with impaired glucose tolerance (IGT) and subjects with type 2 diabetes mellitus (DM). Autonomic nervous activity was studied by 24-hours ECG recording. Heart rate variability analysis was performed in time and frequency domains: SDNN, RMS SD, low frequency (LF) and high frequency (HF) were calculated. RESULTS: The total SDNN showed statistically significant reduction in all four groups with insulin resistant subjects (IR) when compared to the control group (p <0,001). During night LF normalized units (n.u.) were found to be higher in all four groups including IR subjects than in the control group (all p < 0,001) and subjects with normal glucose regulation (NGR), with impaired fasting glycemia (IFG) and with impaired glucose tolerance (IGT) were found to have higher LF n.u. than those in the type 2 diabetes mellitus group. The linear regression model demonstrated direct association between LF values and the homeostasis model assessment-index (HOMA-I), in the insulin resistant group (r = 0,715, p <0,0001). CONCLUSION: The results of our study suggest that insulin resistance might cause global autonomic dysfunction which increases along with worsening glucose metabolic impairment. The analysis of sympathetic and parasympathetic components and the sympathovagal balance demonstrated an association between insulin resistance and sympathetic over-activity, especially during night. The results indicated that the sympathetic over-activity is directly correlated to the grade of insulin resistance calculated according to the HOMA-I. Since increased sympathetic activity is related to major cardiovascular accidents, early diagnosis of all insulin resistant patients should be contemplated. PMID- 16670003 TI - AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines. AB - BACKGROUND: The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. METHODS: The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. RESULTS: Significant amplifications (5-23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB 330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)3CAA(CAG)9(CAACAG)3(CAACAGCAG)2CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. CONCLUSION: These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification. PMID- 16670005 TI - Knee complaints vary with age and gender in the adult population. Population based reference data for the Knee injury and Osteoarthritis Outcome Score (KOOS). AB - BACKGROUND: Self-reported knee complaints may vary with age and gender. Reference data from the adult population would help to better interpret the outcome of interventions due to knee complaints. The objectives of the present study were to describe the variation of self-reported knee pain, function and quality of life with age and gender in the adult population and to establish population-based reference data for the Knee injury and Osteoarthritis Outcome Score (KOOS). METHODS: Population-based cohort retrieved from the national population register. The knee-specific Knee injury and Osteoarthritis Outcome Score (KOOS) was mailed to 840 subjects aged 18-84 yrs. RESULTS: 68% response rate. Women in the age group 55-74 reported more knee-related complaints in all the KOOS subscales than age-matched men. The differences were significant for the subscales Pain (p = 0.027), Symptoms (p = 0.003) and ADL function (p = 0.046). In men, worse ADL and Sport and Recreation function was seen in the oldest age group 75-84 years compared to the younger age groups (p < 0.030). In women, worse Pain (p < 0.007), ADL (p < 0.030), Sport and Recreation (p < 0.001) and QOL (p < 0.002) were seen already in the age group 55-74 compared to the younger age groups. CONCLUSION: We found pain and other symptoms, physical function, and knee-related quality of life to vary with age and gender implying the use of age- and gender matched reference values for improved understanding of the outcome after interventions due to knee injury and knee OA. PMID- 16670004 TI - Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21). AB - BACKGROUND: The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. RESULTS: In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. CONCLUSION: The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID- 16670006 TI - Short-term sequence evolution and vertical inheritance of the Naegleria twin ribozyme group I intron. AB - BACKGROUND: Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA. RESULTS: To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus. CONCLUSION: The Nae.S516 twin-ribozyme intron was gained early in the Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%), leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle. PMID- 16670007 TI - A comparison of univariate and multivariate gene selection techniques for classification of cancer datasets. AB - BACKGROUND: Gene selection is an important step when building predictors of disease state based on gene expression data. Gene selection generally improves performance and identifies a relevant subset of genes. Many univariate and multivariate gene selection approaches have been proposed. Frequently the claim is made that genes are co-regulated (due to pathway dependencies) and that multivariate approaches are therefore per definition more desirable than univariate selection approaches. Based on the published performances of all these approaches a fair comparison of the available results can not be made. This mainly stems from two factors. First, the results are often biased, since the validation set is in one way or another involved in training the predictor, resulting in optimistically biased performance estimates. Second, the published results are often based on a small number of relatively simple datasets. Consequently no generally applicable conclusions can be drawn. RESULTS: In this study we adopted an unbiased protocol to perform a fair comparison of frequently used multivariate and univariate gene selection techniques, in combination with a range of classifiers. Our conclusions are based on seven gene expression datasets, across several cancer types. CONCLUSION: Our experiments illustrate that, contrary to several previous studies, in five of the seven datasets univariate selection approaches yield consistently better results than multivariate approaches. The simplest multivariate selection approach, the Top Scoring method, achieves the best results on the remaining two datasets. We conclude that the correlation structures, if present, are difficult to extract due to the small number of samples, and that consequently, overly-complex gene selection algorithms that attempt to extract these structures are prone to overtraining. PMID- 16670008 TI - Characterizing disease states from topological properties of transcriptional regulatory networks. AB - BACKGROUND: High throughput gene expression experiments yield large amounts of data that can augment our understanding of disease processes, in addition to classifying samples. Here we present new paradigms of data Separation based on construction of transcriptional regulatory networks for normal and abnormal cells using sequence predictions, literature based data and gene expression studies. We analyzed expression datasets from a number of diseased and normal cells, including different types of acute leukemia, and breast cancer with variable clinical outcome. RESULTS: We constructed sample-specific regulatory networks to identify links between transcription factors (TFs) and regulated genes that differentiate between healthy and diseased states. This approach carries the advantage of identifying key transcription factor-gene pairs with differential activity between healthy and diseased states rather than merely using gene expression profiles, thus alluding to processes that may be involved in gene deregulation. We then generalized this approach by studying simultaneous changes in functionality of multiple regulatory links pointing to a regulated gene or emanating from one TF (or changes in gene centrality defined by its in-degree or out-degree measures, respectively). We found that samples can often be separated based on these measures of gene centrality more robustly than using individual links. We examined distributions of distances (the number of links needed to traverse the path between each pair of genes) in the transcriptional networks for gene subsets whose collective expression profiles could best separate each dataset into predefined groups. We found that genes that optimally classify samples are concentrated in neighborhoods in the gene regulatory networks. This suggests that genes that are deregulated in diseased states exhibit a remarkable degree of connectivity. CONCLUSION: Transcription factor-regulated gene links and centrality of genes on transcriptional networks can be used to differentiate between cell types. Transcriptional network blueprints can be used as a basis for further research into gene deregulation in diseased states. PMID- 16670009 TI - Reconciling disparate prevalence rates of PTSD in large samples of US male Vietnam veterans and their controls. AB - BACKGROUND: Two large independent studies funded by the US government have assessed the impact of the Vietnam War on the prevalence of PTSD in US veterans. The National Vietnam Veterans Readjustment Study (NVVRS) estimated the current PTSD prevalence to be 15.2% while the Vietnam Experience Study (VES) estimated the prevalence to be 2.2%. We compared alternative criteria for estimating the prevalence of PTSD using the NVVRS and VES public use data sets collected more than 10 years after the United States withdrew troops from Vietnam. METHODS: We applied uniform diagnostic procedures to the male veterans from the NVVRS and VES to estimate PTSD prevalences based on varying criteria including one-month and lifetime prevalence estimates, combat and non-combat prevalence estimates, and prevalence estimates using both single and multiple indicator models. RESULTS: Using a narrow and specific set of criteria, we derived current prevalence estimates for combat-related PTSD of 2.5% and 2.9% for the VES and the NVVRS, respectively. Using a more broad and sensitive set of criteria, we derived current prevalence estimates for combat-related PTSD of 12.2% and 15.8% for the VES and NVVRS, respectively. CONCLUSION: When comparable methods were applied to available data we reconciled disparate results and estimated similar current prevalences for both narrow and broad definitions of combat-related diagnoses of PTSD. PMID- 16670010 TI - Policy makers ignoring science and scientists ignoring policy: the medical ethical challenges of heroin treatment. AB - A decade of research in Switzerland, The Netherlands, Germany, and Spain now constitutes a massive body of work supporting the use of heroin treatment for the most difficult patients addicted to opiates. These trials concur on this method's safety and efficacy and are now serving as a prelude to the institution of heroin treatment in clinical practice throughout Europe. While the different sampling and research protocols for heroin treatment in these studies were important to the academic claims about specific results and conclusions that could be drawn from each study, the overall outcomes were quite clear--and uniformly positive. They all find that the use of prescribed pharmaceutical heroin does exactly what it is intended to do: it reaches a treatment refractory group of addicts by engaging them in a positive healthcare relationship with a physician, it reduces their criminal activity, improves their health status, and increases their social tenure through more stable housing, employment, and contact with family. The Canadian trial (NAOMI), now underway for over a year, but not yet completed, now faces a dilemma about what to do with its patients who have successfully completed 12 months of heroin and must be withdrawn from heroin and transferred to other treatments in accordance with the research protocol approved by Government of Canada, federal granting body and host institutions. The problem is that the principal criterion for acceptance to NAOMI was their history of repeated failure in these very same treatment programs to which they will now be referred. The existence of the results from abroad (some of which were not yet available when NAOMI was designed and initiated) now raises a very important question for Canada: is it ethical to continue to prohibit the medical use of heroin treatment that has already been shown to be feasible and effective in numerous medical studies throughout the world? And while this is being worked out, is it acceptable to require patients who have been successfully treated with heroin in Canada, to be forced to move back to less effective treatments (treatments that failed to be efficacious in the past)? This essay discusses this dilemma and places it in the broader context of ethics, science, and health policy. It makes the case for continuation of the current successful patients in heroin treatment and the institution of heroin treatment to all Canadian patients living with active addictions who qualify. PMID- 16670011 TI - Candidaemia and antifungal therapy in a French University Hospital: rough trends over a decade and possible links. AB - BACKGROUND: Evidence for an increased prevalence of candidaemia and for high associated mortality in the 1990s led to a number of different recommendations concerning the management of at risk patients as well as an increase in the availability and prescription of new antifungal agents. The aim of this study was to parallel in our hospital candidemia incidence with the nature of prescribed antifungal drugs between 1993 and 2003. METHODS: During this 10-year period we reviewed all cases of candidemia, and collected all the data about annual consumption of prescribed antifungal drugs. RESULTS: Our centralised clinical mycology laboratory isolates and identifies all yeasts grown from blood cultures obtained from a 3300 bed teaching hospital. Between 1993 and 2003, 430 blood yeast isolates were identified. Examination of the trends in isolation revealed a clear decrease in number of yeast isolates recovered between 1995-2000, whereas the number of positive blood cultures in 2003 rose to 1993 levels. The relative prevalence of Candida albicans and C. glabrata was similar in 1993 and 2003 in contrast to the period 1995-2000 where an increased prevalence of C. glabrata was observed. When these quantitative and qualitative data were compared to the amount and type of antifungal agents prescribed during the same period (annual mean defined daily dose: 2662741; annual mean cost: 615,629 euros) a single correlation was found between the decrease in number of yeast isolates, the increased prevalence of C. glabrata and the high level of prescription of fluconazole at prophylactic doses between 1995-2000. CONCLUSION: Between 1993 and 2000, the number of cases of candidemia halved, with an increase of C. glabrata prevalence. These findings were probably linked to the use of Fluconazole prophylaxis. Although it is not possible to make any recommendations from this data the information is nevertheless interesting and may have considerable implications with the introduction of new antifungal drugs. PMID- 16670012 TI - A cross-sectional survey of complementary and alternative medicine use by children and adolescents attending the University Hospital of Wales. AB - BACKGROUND: A high prevalence of CAM use has been documented worldwide in children and adolescents with chronic illnesses. Only a small number of studies, however, have been conducted in the United Kingdom. The primary aim of this study was to examine the use of CAM by children and adolescents with a wide spectrum of acute and chronic medical problems in a tertiary children's hospital in Wales. METHODS: Structured personal interviews of 100 inpatients and 400 outpatients were conducted over a 2-month period in 2004. The yearly and monthly prevalence of CAM use were assessed and divided into medicinal and non-medicinal therapies. This use was correlated with socio-demographic factors. RESULTS: There were 580 patients approached to attain 500 completed questionnaires. The use of at least one type of CAM in the past year was 41% (95% CI 37-46%) and past month 26% (95% CI 23-30%). The yearly prevalence of medicinal CAM was 38% and non-medicinal 12%. The users were more likely to have parents that were tertiary educated (mother: OR = 2.3, 95%CI 1.6-3.3) and a higher family income (Pearson chi-square for trend = 14.3, p < 0.001). The most common medicinal types of CAM were non-prescribed vitamins and minerals (23%) and herbal therapies (10%). Aromatherapy (5%) and reflexology (3%) were the most prevalent non-medicinal CAMs. None of the inpatient medical records documented CAM use in the past month. Fifty-two percent of medicinal and 38% of non-medicinal CAM users felt their doctor did not need to know about CAM use. Sixty-six percent of CAM users did not disclose the fact to their doctor. Three percent of all participants were using herbs and prescription medicines concurrently. CONCLUSION: There is a high prevalence of CAM use in our study population. Paediatricians need to ensure that they ask parents and older children about their CAM usage and advise caution with regard to potential interactions.CAM is a rapidly expanding industry that requires further evidence based research to provide more information on the effectiveness and safety of many CAM therapies. Statutory or self-regulation of the different segments of the industry is important. Integration of CAM with allopathic western medicine through education and better communication is slowly progressing. PMID- 16670013 TI - Experiences and perceptions of people with headache: a qualitative study. AB - BACKGROUND: Few qualitative studies of headache have been conducted and as a result we have little in-depth understanding of the experiences and perceptions of people with headache. The aim of this paper was to explore the perceptions and experiences of individuals with headache and their experiences of associated healthcare and treatment. METHODS: A qualitative study of individuals with headache, sampled from a population-based study of chronic pain was conducted in the North-East of Scotland, UK. Seventeen semi-structured interviews were conducted with adults aged 65 or less. Interviews were analysed using the Framework approach utilising thematic analysis. RESULTS: Almost every participant reported that they were unable to function fully as a result of the nature and unpredictability of their headaches and this had caused disruption to their work, family life and social activities. Many also reported a negative impact on mood including feeling depressed, aggressive or embarrassed. Most participants had formed their own ideas about different aspects of their headache and several had searched for, or were seeking, increased understanding of their headache from a variety of sources. Many participants reported that their headaches caused them constant worry and anguish, and they were concerned that there was a serious underlying cause. A variety of methods were being used to manage headaches including conventional medication, complementary therapies and self-developed management techniques. Problems associated with all of these management strategies emerged. CONCLUSION: Headache has wide-ranging adverse effects on individuals and is often accompanied by considerable worry. The development of new interventions or educational strategies aimed at reducing the burden of the disorder and associated anxiety are needed. PMID- 16670014 TI - The added value of C-reactive protein to clinical signs and symptoms in patients with obstructive airway disease: results of a diagnostic study in primary care. AB - BACKGROUND: To evaluate the diagnostic accuracy of clinical signs and symptoms, C reactive protein (CRP) and spirometric parameters and determine their interrelation in patients suspected to have an obstructive airway disease (OAD) in primary care. METHODS: In a cross sectional diagnostic study, 60 adult patients coming to the general practitioner (GP) for the first-time with complaints suspicious for obstructive airway disease (OAD) underwent spirometry. Peak expiratory flow (PEF)-variability within two weeks was determined in patients with inconspicuous spirometry. Structured medical histories were documented and CRP was measured. The reference standard was the Tiffeneau ratio (FEV1/VC) in spirometry and the PEF-variability. OAD was diagnosed when FEV1/VC < or = 70% or PEF-variability > 20%. RESULTS: 37 (62%) patients had OAD. The best cut-off value for CRP was found at 2 mg/l with a diagnostic odds ratio (OR) of 4.4 (95% CI 1.4-13.8). Self-reported wheezing was significantly related with OAD (OR 3.4; CI 1.1-10.3), whereas coughing was inversely related (OR 0.2; CI 0.1 0.7). The diagnostic OR of CRP increased when combined with dyspnea (OR 8.5; 95% CI 1.7-42.3) or smoking history (OR 8.4; 95% CI 1.5-48.9). CRP (p = 0.004), FEV1 (p = 0.001) and FIV1 (p = 0.023) were related with the severity of dyspnea. CRP increased with the number of cigarettes, expressed in pack years (p = 0.001). CONCLUSION: The diagnostic accuracy of clinical signs and symptoms was low. The diagnostic accuracy of CRP improved in combination with dyspnea and smoking history. Due to their coherence with the severity of dyspnea and number of cigarettes respectively, CRP and spirometry might allow risk stratification of patients with OAD in primary care. Further studies need to be done to confirm these findings. PMID- 16670015 TI - Genome-wide isolation of growth and obesity QTL using mouse speed congenic strains. AB - BACKGROUND: High growth (hg) modifier and background independent quantitative trait loci (QTL) affecting growth, adiposity and carcass composition were previously identified on mouse chromosomes (MMU) 1, 2, 5, 8, 9, 11 and 17. To confirm and further characterize each QTL, two panels of speed congenic strains were developed by introgressing CAST/EiJ (CAST) QTL alleles onto either mutant C57Bl/6J-hg/hg (HG) or wild type C57Bl/6J (B6) genetic backgrounds. RESULTS: The first speed congenic panel was developed by introgressing four overlapping donor regions spanning MMU2 in its entirety onto both HG and B6 backgrounds, for a total of eight strains. Phenotypic characterization of the MMU2 panel confirmed the segregation of multiple growth and obesity QTL and strongly suggested that a subset of these loci modify the effects of the hg deletion. The second panel consisted of individual donor regions on an HG background for each QTL on MMU1, 5, 8, 9, 11 and 17. Of the six developed strains, five were successfully characterized and displayed significant differences in growth and/or obesity as compared to controls. All five displayed phenotypes similar to those originally attributed to each QTL, however, novel phenotypes were unmasked in several of the strains including sex-specific effects. CONCLUSION: The speed congenic strains developed herein constitute an invaluable genomic resource and provide the foundation to identify the specific nature of genetic variation influencing growth and obesity. PMID- 16670016 TI - Allelic drop-out may occur with a primer binding site polymorphism for the commonly used RFLP assay for the -1131T>C polymorphism of the Apolipoprotein AV gene. AB - Apolipoprotein AV (ApoAV) gene variant, -1131T>C, is associated with increased triglyceride concentrations in all ethnic groups studied. An MseI based RFLP analysis is the most commonly used method for genotyping this SNP. We genotyped a large cohort comprising 1185 Asian Indians and 173 UK Caucasians for -1131T>C using an ARMS-PCR based tetra-primer method. For quality control, we re-genotyped approximately 10% random samples from this cohort utilizing the MseI RFLP, which showed a 2.9% (3/102) genotyping error rate between the two methods. To investigate further, we sequenced the 900 bp region around the -1131T>C polymorphism in 25 Asian Indians and 15 UK Caucasians and found a number of polymorphisms including the -987C>T polymorphism. Further analysis of the -987C>T SNP showed a higher rare allele frequency of 0.23 in Asian Indians (n = 158) compared to 0.09 in the UK Caucasians (n = 157). This SNP is located 4 bp from the 3' end of the RFLP forward primer and is in weak linkage disequilibrium with 1131T>C variant (r2 = 0.084 and D' = 1). Repeated RFLP analysis of seven subjects heterozygous for -987C>T (seven times), showed discordant results with the sequence at -1131T>C SNP nearly one third (15/49) of the time. We conclude that presence of -987C>T polymorphism in the forward primer of the MseI RFLP assay may lead to allelic drop-out and generate unforeseen errors in genotyping the 1131T>C polymorphism. Our results also emphasise the need for careful quality control in all molecular genetic studies, particularly while transferring genotyping methods between various ethnic groups. PMID- 16670018 TI - Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells. AB - BACKGROUND: Long interspersed nuclear elements (LINEs), Alu and endogenous retroviruses (ERVs) make up some 45% of human DNA. LINE-1 also called L1, is the most common family of non-LTR retrotransposons in the human genome and comprises about 17% of the genome. L1 elements require the integration into chromosomal target sites using L1-encoded endonuclease which creates staggering DNA breaks allowing the newly transposed L1 copies to integrate into the genome. L1 expression and retrotransposition in cancer cells might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome instability. There is however little evidence on the mechanism of L1-induced genetic instability and its impact on cancer cell growth and proliferation. RESULTS: We report that L1 has genome destabilizing effects indicated by an accumulation of gamma-H2AX foci, an early response to DNA strand breaks, in association with an abnormal cell cycle progression through a G2/M accumulation and an induction of apoptosis in breast cancer cells. In addition, we found that adjuvant L1 activation may lead to supra additive killing when combined with radiation by enhancing the radiation lethality through induction of apoptosis that we have detected through Bax activation. CONCLUSION: L1 retrotransposition is sensed as a DNA damaging event through the creation DNA breaks involving L1-encoded endonuclease. The apparent synergistic interaction between L1 activation and radiation can further be utilized for targeted induction of cancer cell death. Thus, the role of retrotransoposons in general, and of L1 in particular, in DNA damage and repair assumes larger significance both for the understanding of mutagenicity and, potentially, for the control of cell proliferation and apoptosis. PMID- 16670017 TI - Transcriptome coexpression map of human embryonic stem cells. AB - BACKGROUND: Human embryonic stem (ES) cells hold great promise for medicine and science. The transcriptome of human ES cells has been studied in detail in recent years. However, no systematic analysis has yet addressed whether gene expression in human ES cells may be regulated in chromosomal domains, and no chromosomal domains of coexpression have been identified. RESULTS: We report the first transcriptome coexpression map of the human ES cell and the earliest stage of ES differentiation, the embryoid body (EB), for the analysis of how transcriptional regulation interacts with genomic structure during ES self-renewal and differentiation. We determined the gene expression profiles from multiple ES and EB samples and identified chromosomal domains showing coexpression of adjacent genes on the genome. The coexpression domains were not random, with significant enrichment in chromosomes 8, 11, 16, 17, 19, and Y in the ES state, and 6, 11, 17, 19 and 20 in the EB state. The domains were significantly associated with Giemsa-negative bands in EB, yet showed little correlation with known cytogenetic structures in ES cells. Different patterns of coexpression were revealed by comparative transcriptome mapping between ES and EB. CONCLUSION: The findings and methods reported in this investigation advance our understanding of how genome organization affects gene expression in human ES cells and help to identify new mechanisms and pathways controlling ES self-renewal or differentiation. PMID- 16670019 TI - "Sleepiness" is serious in adolescence: two surveys of 3235 Canadian students. AB - BACKGROUND: Evidence is growing that sleep problems in adolescents are significant impediments to learning and negatively affect behaviour, attainment of social competence and quality of life. The objectives of the study were to determine the level of sleepiness among students in high school, to identify factors to explain it, and to determine the association between sleepiness and performance in both academic and extracurricular activities METHODS: A cross sectional survey of 2201 high school students in the Hamilton Wentworth District School Board and the Near North District School Board in Ontario was conducted in 1998/9. A similar survey was done three years later involving 1034 students in the Grand Erie District School Board in the same Province. The Epworth Sleepiness Scale (ESS) was used to measure sleepiness and we also assessed the reliability of this tool for this population. Descriptive analysis of the cohort and information on various measures of performance and demographic data were included. Regression analysis, using the generalised estimating equation (GEE), was utilized to investigate factors associated with risk of sleepiness (ESS>10). RESULTS: Seventy per cent of the students had less than 8.5 hours weeknight sleep. Bedtime habits such as a consistent bedtime routine, staying up late or drinking caffeinated beverages before bed were statistically significantly associated with ESS, as were weeknight sleep quantity and gender. As ESS increased there was an increase in the proportion of students who felt their grades had dropped because of sleepiness, were late for school, were often extremely sleepy at school, and were involved in fewer extracurricular activities. These performance measures were statistically significantly associated with ESS. Twenty-three percent of the students felt their grades had dropped because of sleepiness. Most students (58-68%) reported that they were "really sleepy" between 8 and 10 A.M. CONCLUSION: Sleep deprivation and excessive daytime sleepiness were common in two samples of Ontario high school students and were associated with a decrease in academic achievement and extracurricular activity. There is a need to increase awareness of this problem in the education and health communities and to translate knowledge already available to strategies to address it. PMID- 16670022 TI - Genetic evaluation of Addison's disease in the Portuguese Water Dog. AB - BACKGROUND: Addison's disease, also known as hypoadrenocorticism, has been reported in many individual dogs, although some breeds exhibit a greater incidence than the population as a whole. Addison's is presumed to be an autoimmune mediated hereditary defect but the mode of inheritance remains unclear. In particular, the heritability and mode of inheritance have not been defined for the Portuguese Water Dog although Addison's is known to be prevalent in the breed. RESULTS: The analyses present clear evidence that establishes Addison's disease as an inherited disorder in the Portuguese Water Dog with an estimate of heritability of 0.49 (+/- 0.16); there were no differences in risk for disease across sexes (p > 0.49). Further, the complex segregation analysis provides suggestive evidence that Addison's disease in the Portuguese Water Dog is inherited under the control of a single, autosomal recessive locus. CONCLUSION: The high heritability and mode of inheritance of Addison's disease in the Portuguese Water Dog should enable the detection of segregating markers in a genome-wide scan and the identification of a locus linked to Addison's. Though the confirmation of Addison's disease as an autosomal recessive disorder must wait until the gene is identified, breeders of these dogs may wish to keep the present findings in mind as they plan their breeding programs to select against producing affected dogs. PMID- 16670020 TI - Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation. AB - BACKGROUND: Activation of naive B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology annotation. RESULTS: CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated. CONCLUSION: These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation. PMID- 16670021 TI - Non-hormonal systemic therapy in men with hormone-refractory prostate cancer and metastases: a systematic review from the Cancer Care Ontario Program in Evidence based Care's Genitourinary Cancer Disease Site Group. AB - BACKGROUND: Prostate cancer that has recurred after local therapy or disseminated distantly is usually treated with androgen deprivation therapy; however, most men will eventually experience disease progression within 12 to 20 months. New data emerging from randomized controlled trials (RCTs) of chemotherapy provided the impetus for a systematic review addressing the following question: which non hormonal systemic therapies are most beneficial for the treatment of men with hormone-refractory prostate cancer (HRPC) and clinical evidence of metastases? METHODS: A systematic review was performed to identify RCTs or meta-analyses examining first-line non-hormonal systemic (cytotoxic and non-cytotoxic) therapy in patients with HRPC and metastases that reported at least one of the following endpoints: overall survival, disease control, palliative response, quality of life, and toxicity. Excluded were RCTs of second-line hormonal therapies, bisphosphonates or radiopharmaceuticals, or randomized fewer than 50 patients per trial arm. MEDLINE, EMBASE, the Cochrane Library, and the conference proceedings of the American Society of Clinical Oncology were searched for relevant trials. Citations were screened for eligibility by four reviewers and discrepancies were handled by consensus. RESULTS: Of the 80 RCTs identified, 27 met the eligibility criteria. Two recent, large trials reported improved overall survival with docetaxel-based chemotherapy compared to mitoxantrone-prednisone. Improved progression-free survival and rates of palliative and objective response were also observed. Compared with mitoxantrone, docetaxel treatment was associated with more frequent mild toxicities, similar rates of serious toxicities, and better quality of life. More frequent serious toxicities were observed when docetaxel was combined with estramustine. Three trials reported improved time-to disease progression, palliative response, and/or quality of life with mitoxatrone plus corticosteroid compared with corticosteroid alone. Single trials reported improved disease control with estramustine-vinblastine, vinorelbine hydrocortisone, and suramin-hydrocortisone compared to controls. Trials of non cytotoxic agents have reported equivocal results. CONCLUSION: Docetaxel-based chemotherapy modestly improves survival and provides palliation for men with HRPC and metastases. Other than androgen deprivation therapy, this is the only other therapy to have demonstrated improved overall survival in prostate cancer in RCTs. Further investigations to identify more effective therapies for HRPC including the use of systemic therapies earlier in the natural history of prostate cancer are warranted. PMID- 16670023 TI - Childhood trauma fatality and resource allocation in injury control programs in a developing country. AB - BACKGROUND: Only a few studies have addressed the trimodal distribution of childhood trauma fatalities in lesser developed countries. We conducted this study to evaluate pre-hospital, Emergency Department (ED) and in-hospital distribution of childhood injury-related death for each mechanism of injury in Tehran, Iran. This information will be used for the efficient allocation of the limited injury control resources in the city. METHODS: We used Tehran's Legal Medicine Organization (LMO) database. This is the largest and the most complete database that receives information about trauma fatalities from more than 100 small and large hospitals in Tehran. We reviewed all the medical records and legal documents of the deceased registered in LMO from September 1999 to September 2000. Demographic and injury related characteristics of the children 15 years old or younger were extracted from the records. RESULTS: Ten percent of the 4,233 trauma deaths registered in LMO occurred among children 15 years old or younger. Motor vehicle crashes (MVCs) (50%), burns (18%), falls (6%) and poisonings (6%) were the most common mechanisms of unintentional fatal injuries. Prehospital, emergency department and hospital deaths comprised 42%, 20% and 37% of the trauma fatalities, respectively. While, more than 80% of fatal injuries due to poisoning and drowning occurred in prehospital setting, 92% of burn related fatalities happened after hospital admission. CONCLUSION: Injury prevention is the single most important solution for controlling trauma fatalities due to poisoning and drowning. Improvements in the quality of care in hospitals and intensive care units might substantially alleviate the magnitude of the problem due to burns. Improvements in prehospital and ED care might significantly decrease MVC and falls-related fatalities. PMID- 16670024 TI - Utility of the detection of Plasmodium parasites for the diagnosis of malaria in endemic areas. AB - BACKGROUND: In populations where the prevalence of infection with Plasmodium parasites is high, blood tests that identify Plasmodium parasites in patients with fever may lead to false positive diagnosis of malaria-disease. We characterised the diminishing value of the parasite detection test as a function of the prevalence of infection. METHODS: We computed the ability of the parasite detection test to identify malaria at various levels of prevalence (0% to 90%), assuming plausible estimates of sensitivity (95% and 85%) and specificity (99% and 95%) for the detection of parasites. In each situation, we computed likelihood ratios of malaria (or absence of malaria) for positive and negative parasite detection tests. Likelihood ratios were classified as clinically useful (> or = 10), intermediate (5-10), or unhelpful (<5). RESULTS: Likelihood ratios of positive tests were strongly related to the prevalence of infection in the general population: a positive test was unhelpful when the prevalence was 20% or more, and useful only when prevalence was 5% or less. The sensitivity and specificity of the test had little influence on these results. Likelihood ratios of negative tests were clinically useful when prevalence was 70% or less, but only for high levels of sensitivity (95%). If sensitivity was low (85%), the negative test was at best of intermediate utility, and was unhelpful if the prevalence of asymptomatic infection exceeded 30%. CONCLUSION: Identification of Plasmodium parasites supports a diagnosis of malaria only in areas where the prevalence of Plasmodium infection is low. Wherever this prevalence exceeds about 20%, a positive test is clinically unhelpful. PMID- 16670025 TI - Understandings of reproductive tract infections in a peri-urban pueblo joven in Lima, Peru. AB - BACKGROUND: Control programs for Reproductive Tract Infections (RTIs) typically focus on increasing awareness of risks associated with different forms of sexual contact, and pay little attention to how or why people may link RTIs to other features of their physical or social environments. This paper describes how women in a peri-urban pueblo joven located in the coastal desert surrounding Lima, Peru conceptualize the links between RTIs, sexual behaviour, personal hygiene, and the adverse environment in which they live. METHODS: We combined qualitative interviews and a participatory voting exercise to examine social and physical environmental influences on RTIs and gynaecologic symptom interpretation. RESULTS: Knowledge of RTIs in general was limited, although knowledge of AIDS was higher. Perceived causes of RTIs fell into three categories: sexual contact with infected persons, personal hygiene and exposure to the contaminated physical environment, with AIDS clearly related to sexual contact. The adverse environment is thought to be a major contributor to vaginal discharge, "inflamed ovaries" and urinary tract infection. The more remote parts of this periurban squatter settlement, characterized by blowing sand and dust and limited access to clean water, are thought to exhibit higher rates of RTIs as a direct result of the adverse environment found there. Stigma associated with RTIs often keeps women from seeking care or obtaining information about gynaecologic symptoms, and favours explanations that avoid mention of sexual practices. CONCLUSION: The discrepancy between demonstrated disease risk factors and personal explanations influenced by local environmental conditions and RTI-related stigma poses a challenge for prevention programs. Effective interventions need to take local understandings of RTIs into account as they engage in dialogue with communities about prevention and treatment of RTIs. PMID- 16670026 TI - High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell. AB - BACKGROUND: The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. RESULTS: We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved CelF mutants and corresponding optimized clones in expression-ready plasmids. CONCLUSION: The multiplex method using an integrated automated platform for high throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines. PMID- 16670027 TI - Randomized pilot study to disseminate caries-control services in dentist offices. AB - BACKGROUND: To determine whether education and financial incentives increased dentists' delivery of fluoride varnish and sealants to at risk children covered by capitation dental insurance in Washington state (U.S.). METHODS: In 1999, 53 dental offices in Washington Dental Service's capitation dental plan were invited to participate in the study, and consenting offices were randomized to intervention (n = 9) and control (n = 10) groups. Offices recruited 689 capitation children aged 6-14 and at risk for caries, who were followed for 2 years. Intervention offices received provider education and fee-for-service reimbursement for delivering fluoride varnish and sealants. Insurance records were used to calculate office service rates for fluoride, sealants, and restorations. Parents completed mail surveys after follow-up to measure their children's dental utilization, dental satisfaction, dental fear and oral health status. Regression models estimated differences in service rates between intervention and control offices, and compared survey measures between groups. RESULTS: Nineteen offices (34%) consented to participate in the study. Fluoride and sealant rates were greater in the intervention offices than the control offices, but the differences were not statistically significant. Restoration rates were lower in the intervention offices than the control offices. Parents in the intervention group reported their children had less dental fear than control group parents. CONCLUSION: Due to low dentist participation the study lacked power to detect an intervention effect on dentists' delivery of caries-control services. The intervention may have reduced children's dental fear. PMID- 16670028 TI - Increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection. AB - BACKGROUND: Exacerbations of asthma are associated with viral respiratory tract infections, of which rhinoviruses (RV) are the predominant virus type. Airway smooth muscle is important in asthma pathogenesis, however little is known about the potential interaction of RV and human airway smooth muscle cells (HASM). We hypothesised that rhinovirus induction of inflammatory cytokine release from airway smooth muscle is augmented and differentially regulated in asthmatic compared to normal HASM cells. METHODS: HASM cells, isolated from either asthmatic or non-asthmatic subjects, were infected with rhinovirus. Cytokine production was assayed by ELISA, ICAM-1 cell surface expression was assessed by FACS, and the transcription regulation of IL-6 was measured by luciferase activity. RESULTS: RV-induced IL-6 release was significantly greater in HASM cells derived from asthmatic subjects compared to non-asthmatic subjects. This response was RV specific, as 5% serum- induced IL-6 release was not different in the two cell types. Whilst serum stimulated IL-8 production in cells from both subject groups, RV induced IL-8 production in only asthmatic derived HASM cells. The transcriptional induction of IL-6 was differentially regulated via C/EBP in the asthmatic and NF-kappaB + AP-1 in the non-asthmatic HASM cells. CONCLUSION: This study demonstrates augmentation and differential transcriptional regulation of RV specific innate immune response in HASM cells derived from asthmatic and non-asthmatics, and may give valuable insight into the mechanisms of RV-induced asthma exacerbations. PMID- 16670029 TI - K-SPMM: a database of murine spermatogenic promoters modules & motifs. AB - BACKGROUND: Understanding the regulatory processes that coordinate the cascade of gene expression leading to male gamete development has proven challenging. Research has been hindered in part by an incomplete picture of the regulatory elements that are both characteristic of and distinctive to the broad population of spermatogenically expressed genes. DESCRIPTION: K-SPMM, a database of murine Spermatogenic Promoters Modules and Motifs, has been developed as a web-based resource for the comparative analysis of promoter regions and their constituent elements in developing male germ cells. The system contains data on 7,551 genes and 11,715 putative promoter regions in Sertoli cells, spermatogonia, spermatocytes and spermatids. K-SPMM provides a detailed portrait of promoter site components, ranging from broad distributions of transcription factor binding sites to graphical illustrations of dimeric modules with respect to individual transcription start sites. Binding sites are identified through their similarities to position weight matrices catalogued in either the JASPAR or the TRANSFAC transcription factor archives. A flexible search function allows sub populations of promoters to be identified on the basis of their presence in any of the four cell-types, their association with a list of genes or their component transcription-factor families. CONCLUSION: This system can now be used independently or in conjunction with other databases of gene expression as a powerful aid to research networks of co-regulation. We illustrate this with respect to the spermiogenically active protamine locus in which binding sites are predicted that align well with biologically foot-printed protein binding domains. AVAILABILITY: http://klab.med.wayne.edu/kspmm/ PMID- 16670030 TI - Alcohol consumption, physical activity, and chronic disease risk factors: a population-based cross-sectional survey. AB - BACKGROUND: Whether the association of alcohol consumption and cardiovascular disease is the product of confounding and the degree to which this concern applies to other behaviors are unclear. METHODS: Using the 2003 Behavioral Risk Factor Surveillance System, a population-based telephone survey of adults in the US, we compared chronic disease risk factors between 123,359 abstainers and 126,674 moderate drinkers, defined as intake of < or = 2 drinks per day among men and < or = 1 drink per day among women, using age- and sex- and multivariable adjusted models. We also compared sedentary and active individuals, defined as moderate physical activity > or = 30 minutes per day for > or = 5 days per week, or vigorous activity for > or = 20 minutes per day on > or = 3 days. RESULTS: Chronic disease risk factors and features of unhealthy lifestyle were generally more prevalent among abstainers than drinkers in age- and sex-adjusted analyses, but these differences were generally attenuated or eliminated by additional adjustment for race and education. For low fruit and vegetable intake, divorced marital status, and absence of a personal physician, adjustment for race and education reversed initially positive age- and sex-adjusted associations with abstention. Comparison of sedentary and active individuals produced similar findings, with generally lower levels of risk factors among more physical active individuals. CONCLUSION: The differences between abstainers and drinkers are attenuated after adjustment for limited sociodemographic features, and sedentary and active individuals share a similar pattern. Although observational studies of both factors may be susceptible to uncontrolled confounding, our results provide no evidence that moderate drinking is unique in this regard. Ultimately, randomized trials of all such lifestyle factors will be needed to answer these questions definitively. PMID- 16670031 TI - Antenatal HIV testing in rural eastern Uganda in 2003: incomplete rollout of the prevention of mother-to-child transmission of HIV programme? AB - BACKGROUND: Uganda began to implement the prevention of mother-to-child transmission (PMTCT) of HIV programme in 2000, and by the end of 2003 it had expanded to cover 38 of the 56 districts including Mbale District. However, reports from Mbale Hospital showed that less than 10% of pregnant women accepted antenatal HIV testing. We therefore conducted a study to determine the proportion of pregnant women who tested for HIV and the gaps and barriers in PMTCT implementation. METHODS: The study was a cross sectional household survey of women aged 18 years or more, with children aged one year or less, who resided in Mbale Town or in the surrounding Bungokho County. We also conducted in-depth interviews with six health workers in Mbale Hospital. RESULTS: In 2003, we interviewed 457 women with a median age of 24 years. The prevalence of antenatal HIV testing was 10 percent. The barriers to antenatal HIV testing were unavailability of voluntary counselling and testing services (44%), lack of HIV counselling (42%) and perceived lack of benefits for HIV infected women and their infants. Primipara (OR 2.6, 95% CI 1.2-5.8), urban dwellers (OR 2.7, 95% CI 1.3 5.8), women having been counselled on HIV (OR 6.2, 95% CI 2.9-13.2), and women with husbands being their primary confidant (OR 2.3, 95% CI 1.0-5.5) were independently associated with HIV testing. CONCLUSION: The major barriers to PMTCT implementation were unavailability of PMTCT services, particularly in rural clinics, and poor antenatal counselling and HIV testing services. We recommend that the focus of the prevention of mother-to-child transmission of HIV programme should shift to the district and sub-district levels, strengthen community mobilization, improve the quality of antenatal voluntary counselling and HIV testing services, use professional and peer counsellors to augment HIV counselling, and ensure follow-up care and support for HIV positive women and their infants. PMID- 16670034 TI - Simple cellulitis or a more serious infection? PMID- 16670033 TI - Ectopic pregnancy: forget the "classic presentation" if you want to catch it sooner. PMID- 16670032 TI - Surveillance of Salmonella spp. and Campylobacter spp. in poultry production flocks in The Netherlands. AB - In The Netherlands, a national programme for the surveillance of zoonotic bacteria in farm animals has been operative since 1997. We describe the results of the surveillance of Salmonella spp. in flocks of laying hens and broilers and of Campylobacter spp. in broiler flocks in the period 1999-2002. The prevalence of Salmonella spp. in laying-hen flocks has significantly decreased from 21.1% in 1999 to 13.4% in 2002. This decreasing trend might indicate that the control measures taken by the poultry industry were effective. S. Enteritidis was the predominant serovar in laying hens accounting for one third of the positive flocks. Although prevalence estimates for Salmonella spp. in broiler flocks did not yield a significant decreasing trend in 1999-2002, a decrease in Salmonella prevalence to 11% was measured in 2002. During the study period, S. Paratyphi B var. Java emerged in broilers to become the predominant serovar in 2002 accounting for one third of the positive flocks. The prevalence of Campylobacter spp. in broiler flocks did not increase nor decrease continuously between 1999 and 2002, which roughly corresponds with the monitoring results from the poultry industry. In this period, the estimated flock prevalence roughly averaged around 20%, with C. jejuni being the predominant species. The approach of monitoring presented in this paper can serve as a blueprint for monitoring schemes in farm animal populations to be developed in the context of the EC Zoonoses Directive. PMID- 16670035 TI - A practical 3-step model for managing treatment refusal. PMID- 16670036 TI - How should we manage GERD? PMID- 16670037 TI - Screening for developmental delay: reliable, easy-to-use tools. PMID- 16670038 TI - Clarifying the US Preventive Services Task Force's 2005 recommendations. PMID- 16670039 TI - Clinical inquiries. How can you prevent migraines during pregnancy? PMID- 16670040 TI - Clinical inquiries. Does treatment with donepezil improve memory for patients with mild cognitive impairment? PMID- 16670041 TI - Clinical inquiries. What is the appropriate use of sunscreen for infants and children? PMID- 16670042 TI - Clinical inquiries. What are the risks to the fetus associated with diagnostic radiation exposure during pregnancy? PMID- 16670043 TI - Clinical inquiries. What is the appropriate evaluation and treatment of children who are "toe walkers"? PMID- 16670044 TI - Clinical inquiries. What is the role of herpes virus serology in sexually transmitted disease screening? PMID- 16670045 TI - Adjunctive treatment of acute mania with risperidone versus typical antipsychotics: a retrospective study. AB - Few studies have directly compared atypical antipsychotics (e.g. risperidone) with typical antipsychotics as adjunctive therapy in patients hospitalized for acute mania, especially during a lengthy hospital stay. Our retrospective, case controlled study is a chart review of 64 patients with Diagnostic and Statistical Manual of Mental Disorders, 4th edition, defined bipolar I disorder (current episode, mania). Patients were divided into two groups according to the adjunctive medications used: the risperidone group (mood stabilizers plus risperidone) and the control group (mood stabilizers plus typical antipsychotics). Outcome at discharge, medications, adverse drug effects, and length of hospital stay were compared between groups, controlling for gender, age, number of prior admissions, and duration of illness. Results indicated no statistically significant differences between groups in the controlled factors, Global Assessment of Functioning and Clinical Global Impression-Improvement scores, and adverse drug events. Patients in the risperidone group used significantly lower doses of trihexyphenidyl than those in the control group (p < 0.05). Patients treated with risperidone had a shorter hospital stay than those treated with typical antipsychotics (p < 0.01). In conclusion, antipsychotics are effective as adjunctive agents in the treatment of acute mania. The use of risperidone, in particular, decreases the need for anticholinergics and may lead to a shorter hospital stay compared with typical antipsychotics. PMID- 16670046 TI - Screening for Internet addiction: an empirical study on cut-off points for the Chen Internet Addiction Scale. AB - The aim of this study was to establish the optimal cut-off points of the Chen Internet Addiction Scale (CIAS), to screen for and diagnose Internet addiction among adolescents in the community by using the well-established diagnostic criteria of Internet addiction. This survey of 454 adolescents used screening (57/58) and diagnostic (63/64) cut-off points of the CIAS, a self-reported instrument, based on the results of systematic diagnostic interviews by psychiatrists. The area under the curve of the receiver operating characteristic curve revealed that CIAS has good diagnostic accuracy (89.6%). The screening cut off point had high sensitivity (85.6%) and the diagnostic cut-off point had the highest diagnostic accuracy, classifying 87.6% of participants correctly. Accordingly, the screening point of the CIAS could provide a screening function in two-stage diagnosis, and the diagnostic point could serve as a diagnostic criterion in one-stage massive epidemiologic research. PMID- 16670047 TI - Evaluation of life and death studies course on attitudes toward life and death among nursing students. AB - The aim of this study was to evaluate attitudes toward life and death among nursing students after attending the life and death studies (LDS) program. Both qualitative and quantitative methods were used to collect data. The pretest posttest control group design randomly assigned students to an experimental (n = 47) or control group (n = 49). The 13-week course included lectures, video appraisal, games, simulations, films, books, assignments and group sharing. Statistical and content analysis were used to analyze qualitative and quantitative data. The findings showed a significant improvement in perception of the meaningfulness of life in four categories of improvement: expanded viewpoint, sadness about death, treating life sincerely, and instilling hope in life. The qualitative data indicated that a positive change in meaning of life was associated with interaction with others and self-reflection. PMID- 16670048 TI - Poor outcomes in patients with primary malignant mediastinal germ-cell tumors. AB - Primary mediastinal germ-cell tumors (GCTs) without gonadal involvement are rare and can be divided into benign mature teratoma and malignant seminoma or nonseminoma. We describe our experience of malignant mediastinal GCTs and compare the presentations and outcome with those of benign teratomas. Four malignant GCTs (1 seminoma, 1 choriocarcinoma, and 2 yolk-sac tumors) have been treated in our hospital. All patients were men with obvious symptoms before diagnosis. The patient with seminoma was treated with surgery and radiation, while those with nonseminoma tumors were treated with chemotherapy and/or surgery. Two patients died, one with extended pulmonary metastasis and the other with relapsed disease and high levels of tumor markers. Compared with the nine cases of benign teratomas, the four malignant GCTs showed overwhelming male dominance, advanced symptoms at presentation, and poor outcome. These cases highlight the important role of disease staging and tumor-marker levels in malignant GCTs, and suggest that new treatment strategies for malignant GCTs await further investigation. PMID- 16670049 TI - Successful retrieval of dislodged paclitaxel-eluting stent with a nitinol loop snare: a case report. AB - Coronary stent dislodgment or embolization before deployment is a rare but challenging complication in interventional cardiology. Intracoronary embolization of the dislodged stent is associated with a high risk of coronary occlusion, due to thrombus formation and subsequent myocardial infarction. Furthermore, systemic embolization may cause severe cerebrovascular events. Nonsurgical retrieval strategies for this complication have been suggested, but bailout cardiac surgery may be indicated if percutaneous retrieval attempts fail. To our knowledge, this is the first case report of intracoronary drug-eluting stent dislodgment, and successful retrieval was accomplished by a loop snare technique. With the increasing trend of using drug-eluting stents in percutaneous coronary intervention, the likelihood of stent dislodgment or embolization may increase. It should be kept in mind, especially by coronary interventionists, how to manage this complication. PMID- 16670050 TI - Embolization of arteriovenous fistula after radiosurgery for multiple cerebral arteriovenous malformations. AB - Cerebral arteriovenous malformation (CAVM) associated with arteriovenous fistula (AVF) is rare. It may be difficult to identify hemodynamic details of mixed CAVM and AVF, even when using x-ray cerebral angiography (digital subtraction angiography). We report on a 37-year-old male patient with headache that led to an initial diagnosis of deep frontotemporal CAVM. The first DSA revealed engorged, tortuous, and high-flow venous drainage in addition to clusters of vasculature niduses. The patient was initially treated using gamma-knife radiosurgery (GKRS), which resulted in partial nidus obliteration, documented by a series of follow-up magnetic resonance imaging (MRI). However, the high-flow venous drainage remained, seen on MRI as engorged venous pouches. Clinically, the patient was bothered by persistent headache and bruits after GKRS. Follow-up DSA 3 years after GKRS confirmed a small remnant CAVM nidus and a nearby AVF, separated from and lateral to the original CAVM nidus in the ipsilateral deep temporal lobe. When the initial DSA was reviewed, it revealed that the AVF was difficult to define because of superimposition of the nidus and engorged drainage vessels. Embolization of the AVF using electrodetachable coils resulted in total occlusion of the AVF. The patient's symptoms resolved immediately after embolization. This case suggests that superselective angiography using a microcatheter may be necessary for the initial diagnosis of CAVM associated with AVF with high-flow and engorged venous drainage. For CAVM patients with persistent symptoms after radiosurgery and engorged venous drainage when CAVM is expected to be cured, a microcatheter and superselective endovascular approach may offer diagnosis. Immediate embolization for associated AVF in the same angiographic session may thereby improve neurologic deficits and reduce hemorrhagic risk during the latency after GKRS. PMID- 16670051 TI - Spontaneous perirenal hematoma: a case report. AB - Spontaneous perirenal hematoma is relatively uncommon but may be life threatening. There are some challenges in early diagnosis due to the lack of specific presentations. We report a case of spontaneous perirenal hematoma in a patient who had histories of systemic lupus erythematosus, hypertension, and uremia with hemodialysis, and initially presented with non-specific flank pain. Dizziness and unstable vital signs were noted in the emergency room. Computed tomography and abdominal ultrasonography revealed a large perirenal hematoma over the left retroperitoneal cavity. The patient received conservative treatment without surgical intervention and had an uneventful recovery. PMID- 16670052 TI - Giant rhinolith: a case report. AB - A rhinolith is a stone that forms in the nose. It occurs as a result of the solidification of mucus and nasal debris by mineral salts, calcium, magnesium phosphate and carbonate. It can be seen on radiographs as a radiopaque object in the nasal fossa and may be confused with several pathologic entities that will call for more invasive surgical procedures. We present the first case of a giant rhinolith, possibly arising from aspergillosis, and discuss its clinical and radiologic features. PMID- 16670061 TI - Determining which patients with myelodysplastic syndrome will respond to immunosuppressive treatment. PMID- 16670062 TI - Strategies for widening the use of cord blood in hematopoietic stem cell transplantation. PMID- 16670063 TI - Edoardo Storti (1909-2006). PMID- 16670064 TI - Neutrophil elastase and granulocyte colony-stimulating factor receptor mutation analyses and leukemia evolution in severe congenital neutropenia patients belonging to the original Kostmann family in northern Sweden. AB - BACKGROUND AND OBJECTIVES: Severe congenital neutropenia (SCN) or Kostmann syndrome was originally reported to be an autosomal recessive disease of neutrophil production causing recurrent, life-threatening infections. Mutations in the neutrophil elastase gene (ELA-2) have previously been identified in patients with sporadic or autosomal dominant SCN. DESIGN AND METHODS: We studied 14 individuals (four patients with SCN and ten close relatives) belonging to the original Kostmann family in northern Sweden for mutations in the ELA-2 and the granulocyte colony-stimulating factor (G-CSF) receptor genes. RESULTS: One patient belonging to the original Kostmann family harbored a novel heterozygous ELA-2 mutation (g.2310T-->A;Leu92His) that was not inherited from her parents. The mutation was identified in DNA isolated from both whole blood and skin fibroblasts, suggesting a sporadic de novo mutation. As a young adult this patient sequentially acquired two mutations in the gene for the G-CSF receptor (G CSFR) and therefore recently received a hematopoietic stem cell transplant, due to the risk of evolution to leukemia. Moreover, another patient developed acute leukemia and was treated with transplantation. No pathogenic ELA-2 or G-CSFR gene mutations were found in this patient or the other two patients, nor in any healthy relative. INTERPRETATION AND CONCLUSIONS: Our data are the first to document leukemia evolution and G-CSFR gene mutations in the original Kostmann kindred. In addition, our findings indicate that ELA-2 mutations are not the primary cause of SCN in the Swedish Kostmann family. PMID- 16670065 TI - Common features and differences in the transcriptome of large cell anaplastic lymphoma and classical Hodgkin's lymphoma. AB - BACKGROUND AND OBJECTIVES: Anaplastic large cell lymphoma (ALCL) and classical Hodgkin's lymphoma (HL) are derived from different cell types, namely T cells and B cells, respectively. However, both lymphomas share a similar cytological and immunohistochemical tumor cell phenotype with little resemblance to their cells of origin. DESIGN AND METHODS: In this study, the transcriptional profiles of ALCL cell lines, primary ALCL tumor cells from peripheral blood and HL cell lines were compared to each other and to normal B-cell subsets, B non-Hodgkin's lymphomas (NHL) and B NHL- and Epstein-Barr virus (EBV)-transformed B-cell lines in order to establish their relationship at the transcriptional level and to identify genes with possible pathobiological impact. Expression of some of the genes identified was confirmed in microdissected primary tumor cells by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: HL samples clustered separately from ALCL samples, but HL and ALCL were found to be more closely related to each other than to any normal or malignant B cell sample in the dataset. Their relationship was determined to a large extent, but not exclusively, by lack of expression of B-cell antigens and by the over expression of mRNA encoding activation markers and structural proteins. Apart from established differences between HL and ALCL, further genes of interest could be identified that distinguish both entities from each other and from the other samples. The differential expression of PRAME, DDR2, SOCS3 and CEBPD in HL and ALCL was confirmed in primary tumor tissue by immunohistochemistry and/or RT-PCR. INTERPRETATION AND CONCLUSIONS: At a transcriptional level HL is more closely related to Alk+ ALCL than to the B-NHL or B-cell samples investigated, although it is a B-cell derived lymphoma. The newly identified genes discriminating HL and ALCL may be pathobiologically important and may serve as possible therapeutic targets. PMID- 16670066 TI - The mechanisms of vitamin K2-induced apoptosis of myeloma cells. AB - BACKGROUND AND OBJECTIVES: Physiologically, vitamin K compounds act as co-factors for g-carboxylation of selected glutamates at the N-terminus of prothrombin and some other coagulation factors. These congeners have some growth inhibitory effects of human neoplastic cells. Furthermore, vitamin K2 (VK2) cause apoptosis of some leukemic cells. In search for a new candidate agent to use in the maintenance treatment of myeloma, we analyzed the growth inhibitory effects and apoptosis-inducing capacity of VK2 in human myeloma cells. DESIGN AND METHODS: The growth of myeloma, lymphoma and non-lymphoid cells cultured with various concentrations of VK2 with or without dexamethasone or allopurinol was assayed. Flow cytometry was used to detect apoptotic cells, activated caspase-3 and -9, the generation of superoxide by hydroethidine, and mitochondrial membrane potential (E centym). In addition, the activation of apoptosis-inducing MAPK, p38 and JNK, release of cytochrome c from mitochondria, and change in the relative Bcl-XL/Xs expression balance were analyzed by Western blotting. RESULTS: Myeloma cells and B-cell lymphoma cells were sensitive to VK2. The growth inhibition was caused by apoptosis and activation of caspase-3. The generation of superoxide, and inhibitory effects of the xanthine oxidase inhibitor allopurinol, were demonstrated in myeloma cells. The phosphorylation of MAPK was increased by VK2 in myeloma cells. In addition, the mitochondrial apoptotic pathway was activated. INTERPRETATION AND CONCLUSIONS: VK2 may be a good candidate for myeloma patients, particularly patients who are not suitable candidates for intensive cytoreductive chemotherapy due to age and/or complications. PMID- 16670068 TI - Fibrinogen Mumbai: intracellular retention due to a novel G434D mutation in the Bbeta-chain gene. AB - BACKGROUND AND OBJECTIVES: Afibrinogenemia and hypofibrinogenemia are rare inherited coagulation disorders characterized by hemorrhagic manifestations of variable entity and by plasma fibrinogen deficiency. So far, 57 mutations have been associated with these disorders, and 18 of these are missense mutations. The aim of this study was to characterize the molecular mechanism underlying severe hypofibrinogenemia in a proband from India. DESIGN AND METHODS: The mutational screening was accomplished by DNA sequencing of the three fibrinogen genes. The mutant protein was expressed in COS-1 cells, and intracellular and secreted mutant fibrinogen was analyzed by means of pulse-chase experiments. RESULTS: A novel homozygous G-->A transition in exon 8 (nucleotide position 8017) was found in the proband's fibrinogen Bbeta-chain gene. The resulting G434D missense mutation (fibrinogen Mumbai) involves a highly conserved amino acid residue, located in the C-terminal globular D domain. In vitro expression experiments demonstrated intracellular retention of the mutant fibrinogen and marked reduction of its secretion. INTERPRETATION AND CONCLUSIONS: The G434D substitution causes severe hypofibrinogenemia by impairing fibrinogen secretion. Expression data confirm the importance of Bbeta-chain D domain folding in the intracellular processing of fibrinogen. PMID- 16670069 TI - Solvent/detergent plasma for prevention of bleeding in recessively inherited coagulation disorders: dosing, pharmacokinetics and clinical efficacy. AB - BACKGROUND AND OBJECTIVES: This open-label multicenter trial of solvent/detergent (SD) plasma involving 17 patients with recessively inherited coagulation disorders (one afibrinogenemia, four FV, six combined FV and FVIII, one FX and five FXI deficiencies) evaluated the pharmacokinetics of the deficient factors and hemostatic efficacy. DESIGN AND METHODS: In vivo recovery (IVR) of the deficient coagulation factor was determined in a non-bleeding state in all patients and the mean values for FV, FVIII, FX, FXI and fibrinogen were 1.3, 1.2, 1.5, 1.3 and 1.5 dL/Kg, respectively. The mean plasma half-life of FV, FVIII and FX was 18, 43 and 33 hours, respectively. All patients underwent replacement therapy for elective procedures at risk of bleeding (surgery in 14 cases and vaginal delivery in two patients), except one treated for a central nervous system surgical emergency. RESULTS: Treatment courses with SD plasma were judged fully effective in 13/16 cases (81%). In the remaining three cases, mild bleeding occurred after major surgery in a FV deficient patient with a factor level of 43% and in a FXI deficient patient when factor levels were between 20% and 41%; and after minor surgery in a patient with FV and FVIII deficiency when factor levels were 41% and 18%, respectively. Bleeding was controlled by continuing or increasing treatment with SD plasma. INTERPRETATION AND CONCLUSIONS: These results suggest that, even though the current absolute risk of blood-borne infections associated with fresh-frozen plasma is relatively small, SD plasma should be preferred in patients with recessively inherited coagulation disorders who need replacement therapy when virus-inactivated single-factor concentrates are not available. PMID- 16670070 TI - Early hematopoietic recovery after single unit unrelated cord blood transplantation in adults supported by co-infusion of mobilized stem cells from a third party donor. AB - BACKGROUND AND OBJECTIVES: Our objective was to improve the outcome of cord blood (CB) transplantation in adults, by overcoming the limitations imposed by the low number of stem cells present in CB units. DESIGN AND METHODS: We combined single CB units and co-infusion of third party donor (TPD)-derived peripheral blood mobilized hematopoietic stem cells (MHSC) following myeloablative conditioning with reduced extra-hematologic toxicity. RESULTS: Twenty-seven eligible patients with high-risk hematologic malignancies (age 16-60 years, median 29, weight 43-78 Kg, median 67) received CB units (median nucleated cell count 2.37x10(7)/Kg, median CD34+ cells 0.11x10(6)/Kg) co-infused with TPD-derived MHSC (2.30x10(6)/Kg CD34+ cells; <1x10(4)/Kg CD3+ cells). Neutrophil engraftment (>0.5x10(9)/L) occurred 10 days (9-36) post-transplant and was initially of TPD-origin in all patients except for four who received maternal MHSC, and then became of stable CB origin. Median times to CB-derived neutrophil count >0.5x10(9)/L and full CB chimerism were 22 and 55 days, respectively. The maximum cumulative incidence for engraftment, CB-engraftment and full CB-chimerism was 0.93 (95%CI: 0.83-1.00). The median time to reach unsupported platelet counts >20x10(9)/L was 33 days, with a maximum cumulative incidence of 0.74 (95%CI: 0.59-0.93). Transplant related morbidity was associated primarily with non-neutropenic phase infections. Co-infusion of TPD-cells was well tolerated, with only 14.8% of recipients developing acute graft-versus-host disease (grade III-IV) and 20% developing a chronic (limited) form. The predicted 4-year overall survival was 69% for the whole group and 77% for the 23 patients receiving non-maternal TPD. INTERPRETATION AND CONCLUSIONS: Our strategy offers prompt engraftment with a low rate of complications in a feasible alternative protocol that overcomes the current limitations of a single CB-transplant in adults. PMID- 16670071 TI - The contribution of Ca+ calmodulin activation of human erythrocyte AMP deaminase (isoform E) to the erythrocyte metabolic dysregulation of familial phosphofructokinase deficiency. AB - Erythrocyte membrane leakage of Ca2+ in familial phosphofructokinase deficiency results in a compensatory increase of Ca2+-ATPase activity that depletes ATP and leads to diminished erythrocyte deformability and a higher rate of hemolysis. Lowered ATP levels in circulating erythrocytes are accompanied by increased IMP, indicating that activated AMP deaminase plays a role in this metabolic dysregulation. Exposure to a calmodulin antagonist significantly slows IMP accumulation during experimental energy imbalance in patients' cells to levels that are similar to those in untreated controls, implying that Ca2+-calmodulin is involved in erythrocyte AMP deaminase activation in familial phosphofructokinase deficiency. Therapies directed against activated isoform E may be beneficial in this compensated anemia. PMID- 16670072 TI - Antithymocyte globulin and cyclosporine A as combination therapy for low-risk non sideroblastic myelodysplastic syndromes. AB - The present study evaluated the combination of antithymocyte globulin (ATG) and cyclosporine A (CsA) in patients with low-risk myelodysplastic syndromes. Twenty patients (17 with refractory anemia and 3 with refractory anemia with excess blasts) received treatment with rabbit-ATG plus CsA. The overall response rate was 30% (6/20); three of the six responders had a complete response. The responses lasted 2-58 months, with two patients still being in complete remission at 42 and 58 months. Short-lasting cytogenetic remissions were achieved in two patients. ATG was poorly tolerated in patients over 70 years of age. Four out of 20 patients progressed to acute myeloid leukemia within a year. We conclude that immunosuppressive treatment may be a therapeutic option for selected patients with myelodysplastic syndrome. PMID- 16670073 TI - The impact of P53 and P21(waf1) expression on the survival of patients with the germinal center phenotype of diffuse large B-cell lymphoma. AB - Immunohistochemically detected over-expression of P53-related protein (P53+++) and absence of P21(waf1) expression (P21-) correspond to loss of function of the P53-gene in diffuse large B-cell lymphoma (DLBCL) patients. Using immunohistochemistry we examined 80 patients with DLBCL and found that 23% had the P53+++/P21- phenotype while 51% had a germinal center (GC) pattern. Both the P53+++/P21- phenotype and the non-GC pattern were associated with inferior outcome. Notably, the prognostic power of the P53+++/P21- phenotype was restricted to patients with a GC pattern, without effect on outcome of patients with a non-GC phenotype. Our results show that immunohistochemistry can parallel gene expression profiling in addressing clinical variability of DLBCL patients. PMID- 16670074 TI - Atypical lymphoproliferation progressing into B-cell lymphoma in rheumatoid arthritis treated with different biological agents: clinical course and molecular characterization. AB - A patient with rheumatoid arthritis (RA) developed an atypical lymphoproliferative disorder (LPD) after methotrexate and cyclosporine A, which regressed after suspension of both drugs. After subsequent treatment with rituximab, the LPD was still undetectable. Anti-tumor necrosis factor a therapy was used when the arthritis relapsed, but an aggressive B-cell non Hodgkin's lymphoma developed. Molecular analyses showed an oligoclonal B-cell expansion at the LPD step. A minor clone with significant sequence homology to B-cell lymphomas arising in Sjogren's syndrome and mixed cryoglobulinemia syndrome, given rise to the non-Hodgkin's lymphoma. Treatment of rheumatoid arthritis associated with lymphoproliferation represents a clinical challenge, and common pathogenetic pathways to lymphoma may occur in different autoimmune diseases. PMID- 16670075 TI - The risk of recurrent venous thromboembolism in patients with inherited deficiency of natural anticoagulants antithrombin, protein C and protein S. AB - Few data are available on the risk of recurrent venous thromboembolism (VTE) associated with the rare inherited deficiencies of natural anticoagulants. We studied 602 patients with previous VTE: the incidence of first recurrence in the absence of anticoagulation was retrospectively estimated in 64 patients with deficiency of antithrombin (AT, n=14), protein C (PC, n=28), or protein S (PS, n=22) and 538 with no known defect, who acted as the reference group. After adjustment for sex, age, and circumstances of the first event, AT deficiency resulted an independent risk factor for recurrence (hazard ratio 1.9, 95% CI 1.0 3.9); the carriers of PC or PS deficiency had a marginal increase in risk (hazard ratio 1.4, 95% CI 0.9-2.2). In conclusion, patients with AT deficiency are potential candidates for long-term oral anticoagulation. PMID- 16670076 TI - TERC mutations in children with refractory cytopenia. AB - Mutations in the human telomerase RNA gene (TERC) cause autosomal dominant dyskeratosis congenita and have been detected in individuals with bone marrow failure. Here, we screened for TERC mutations in a cohort of 80 children with hypocellular myelodysplastic syndrome and detected TERC alterations in two of them. PMID- 16670077 TI - Predicting erythroid response to recombinant erythropoietin plus granulocyte colony-stimulating factor therapy following a single subcutaneous bolus in patients with myelodysplasia. AB - We randomized 21 patients with low-risk myelodysplastic syndromes (MDS) to receive a single subcutaneous bolus of recombinant erythropoietin (epoietin) +/- granulocyte-colony stimulating factor (G-CSF), or placebo and monitored erythropoietic response over 7 days. In this small study, the reticulocyte response at day 7 was highly predictive of subsequent response to a therapeutic trial of epoietin + G-CSF. PMID- 16670078 TI - The effects of saquinavir on imatinib-resistant chronic myelogenous leukemia cell lines. AB - We evaluated the effect of the human immunodeficiency virus (HIV) protease inhibitor saquinavir on the imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia cell lines. Saquinavir, which is also a proteasome blocker, showed dose- and time-related anti-proliferative activity, particularly on the imatinib-resistant lines and a pro-apoptotic effect. Association with imatinib caused a significant increase of activity. PMID- 16670079 TI - Expression and prognostic significance of heat-shock proteins in myelodysplastic syndromes. AB - Using flow cytometry, we investigated the clinical and hematologic relevance of expression of heat-shock proteins (HSP) HSP27, HSP60, HSP70, HSP90 and HSP110 in bone marrow of 142 patients with newly diagnosed myelodysplastic syndromes, together with that of the membrane differentiation antigen CD34 and the drug resistance related protein, P170 (Pgp). PMID- 16670080 TI - Estimating the impact of rituximab on bcl-2-associated resistance to CHOP in elderly patients with diffuse large B-cell lymphoma. AB - Rituximab plus CHOP (R-CHOP) has been proven to increase overall survival in aggressive bcl-2-positive lymphoma patients. Using competing risk analysis, we studied the long-term impact of this treatment in patients from a GELA trial: R CHOP prevented from progression or relapse in both bcl-2-positive and bcl-2 negative patients without increasing the risk of death in complete remission. PMID- 16670081 TI - Candidemia in patients with hematologic malignancies: analysis of 7 years' experience in a single center. AB - We report 45 incidents of candidemia in 45 patients diagnosed with hematologic malignancies between 1997 and 2004. A large majority of species isolated were non albicans and there was an unexpectedly high incidence of Candida tropicalis. The attributable mortality (15%) was interestingly low in this population of severely immunocompromised patients. PMID- 16670082 TI - Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis. AB - The JAK2/V617F mutation has been noted in essential thrombocytemia. We investigated 19 cases with refractory anemia with ringed sideroblasts (RARS), including three RARS with thrombocytosis (RARS-T). Only the RARS-T patients showed this mutation. More cases need to be analyzed to determine the prevalence of the JAK2/V617F mutation in RARS-T. PMID- 16670083 TI - Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair. AB - Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-alpha-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound fructosamine 6 phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6 phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on fructosamine 3-phosphates. MDP-1 dephosphorylated up to approximately 75% of the fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP 1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P. PMID- 16670084 TI - Ets-1 and runx2 regulate transcription of a metastatic gene, osteopontin, in murine colorectal cancer cells. AB - Osteopontin (OPN) is a sialic acid-rich phosphoprotein secreted by a wide variety of cancers. We have shown previously that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. Although a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis- and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC (young adult mouse colon) epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nucleotide (nt) 107 to -174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared with YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt 101 to -123 and nt -121 to -145, which specifically bind CT26 nuclear proteins. Competition with unlabeled mutant oligonucleotides revealed that the regions nt 115 to -118 and nt -129 to -134 were essential for protein binding. Subsequent supershift and chromatin immunoprecipitation assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in a >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein. PMID- 16670085 TI - Mechanisms of helicases. PMID- 16670086 TI - Differential gel electrophoresis and transgenic mitochondrial calcium reporters demonstrate spatiotemporal filtering in calcium control of mitochondria. AB - Mitochondria must adjust both their intracellular location and their metabolism in order to balance their output to the needs of the cell. Here we show by the proteomic technique of time series difference gel electrophoresis that a major result of neuroendocrine stimulation of the Drosophila renal tubule is an extensive remodeling of the mitochondrial matrix. By generating Drosophila that were transgenic for both luminescent and fluorescent mitochondrial calcium reporters, it was shown that mitochondrial calcium tracked the slow (minutes) but not the rapid (<1 s) changes in cytoplasmic calcium and that this resulted in both increased mitochondrial membrane polarization and elevated cellular ATP levels. The selective V-ATPase inhibitor, bafilomycin, further enhanced ATP levels, suggesting that the apical plasma membrane V-ATPase is a major consumer of ATP. Both the mitochondrial calcium signal and the increase in ATP were abolished by the mitochondrial calcium uniporter blocker Ru360. By using both mitochondrial calcium imaging and the potential sensing dye JC-1, the apical mitochondria of principal cells were found to be selectively responsive to neuropeptide signaling. As the ultimate target is the V-ATPase in the apical plasma membrane, this selective activation of mitochondria is clearly adaptive. The results highlight the dynamic nature and both spatial and temporal heterogeneity of calcium signaling possible in differentiated, organotypic cells and provide a new model for neuroendocrine control of V-ATPase. PMID- 16670087 TI - Novel allosteric activation site in Escherichia coli fructose-1,6-bisphosphatase. AB - Fructose-1,6-bisphosphatase (FBPase) governs a key step in gluconeogenesis, the conversion of fructose 1,6-bisphosphate into fructose 6-phosphate. In mammals, the enzyme is subject to metabolic regulation, but regulatory mechanisms of bacterial FBPases are not well understood. Presented here is the crystal structure (resolution, 1.45A) of recombinant FBPase from Escherichia coli, the first structure of a prokaryotic Type I FBPase. The E. coli enzyme is a homotetramer, but in a quaternary state between the canonical R- and T-states of porcine FBPase. Phe(15) and residues at the C-terminal side of the first alpha helix (helix H1) occupy the AMP binding pocket. Residues at the N-terminal side of helix H1 hydrogen bond with sulfate ions buried at a subunit interface, which in porcine FBPase undergoes significant conformational change in response to allosteric effectors. Phosphoenolpyruvate and sulfate activate E. coli FBPase by at least 300%. Key residues that bind sulfate anions are conserved among many heterotrophic bacteria, but are absent in FBPases of organisms that employ fructose 2,6-bisphosphate as a regulator. These observations suggest a new mechanism of regulation in the FBPase enzyme family: anionic ligands, most likely phosphoenolpyruvate, bind to allosteric activator sites, which in turn stabilize a tetramer and a polypeptide fold that obstructs AMP binding. PMID- 16670088 TI - Deoxycholic acid blocks vibrio cholerae OmpT but not OmpU porin. AB - OmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open channel blocker, which may relate to deoxycholic acid permeation. PMID- 16670089 TI - C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1. AB - Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1. PMID- 16670090 TI - Clustering and coupled gating modulate the activity in KcsA, a potassium channel model. AB - Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na(+), and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na(+) blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses. PMID- 16670091 TI - FOXO1 represses peroxisome proliferator-activated receptor-gamma1 and -gamma2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity. AB - FOXO1 and peroxisome proliferator-activated receptor-gamma (PPARgamma) are crucial transcription factors that regulate glucose metabolism and insulin responsiveness in insulin target tissues. We have shown that, in primary rat adipocytes, both factors regulate transcription of the insulin-responsive GLUT4 gene and that PPARgamma2 detachment from the GLUT4 promoter upon thiazolidinedione binding up-regulates GLUT4 gene expression, thus increasing insulin sensitivity (Armoni, M., Kritz, N., Harel, C., Bar-Yoseph, F., Chen, H., Quon, M. J., and Karnieli, E. (2003) J. Biol. Chem. 278, 30614-30623). However, the mechanisms regulating PPARgamma gene transcription are largely unknown. We studied the effects of FOXO1 on human PPARgamma gene expression in primary rat adipocytes and found that both genes are endogenously expressed. FOXO1 coexpression dose-dependently repressed transcription from either the PPARgamma 1 or PPARgamma2 promoter reporter by 65%, whereas insulin (100 nm, 20-24 h) either partially or completely reversed this effect. Phosphorylation-defective FOXO1 mutants T24A, S256A, S319A, and T24A/S256A/S319A still repressed the PPARgamma1 promoter and partially lost their effects on the PPARgamma2 promoter in either basal or insulin-stimulated cells. Use of DNA binding-defective FOXO1 (H215R) indicated that this domain is crucial for FOXO1 repression of the PPARgamma2 (but not PPARgamma1) promoter. Progressive 5'-deletion and gel retardation analyses revealed that this repression involves direct and specific binding of FOXO1 to the PPARgamma2 promoter; chromatin immunoprecipitation analysis confirmed that this binding occurs in cellulo. We suggest a novel paradigm to increase insulin sensitivity in adipocytes in which FOXO1 repression of PPARgamma, the latter being a repressor of the GLUT4 promoter, consequently leads to GLUT4 derepression/up-regulation, thus enhancing cellular insulin sensitivity. The newly identified FOXO1-binding site on the PPARgamma2 promoter may serve as a therapeutic target for type 2 diabetes. PMID- 16670092 TI - Transcriptional activation of the SALL1 by the human SIX1 homeodomain during kidney development. AB - SALL1 is a member of the SAL gene family that encodes a group of putative developmental transcription factors. SALL1 plays a critical role during kidney development as mutations of the human SALL1 gene cause Townes-Brocks syndrome, which is associated with kidney malformation. Deletion of the mouse Sall1 gene results in renal agenesis or severe dysgenesis. To date, little is known about the molecular mechanisms controlling the regulation of SALL1 expression. This report describes the cloning and characterization of the human SALL1 gene promoter. Consensus binding sites were identified for several transcription factors, with multiple sites for WT1 and SIX1. In transient transfection assays, SALL1 promoter activity was higher in HEK-293 human kidney cells and COS-7 monkey kidney cells than in NIH-3T3 fibroblasts, consistent with its role in kidney development. Transcription from the SALL1 promoter was strikingly activated by the SIX1 protein. Utilizing a luciferase reporter gene assay, endogenous or exogenously added SIX1 activated the SALL1 promoter. Overexpression of SIX1 induced a significant increase in the endogenous SIX1 protein. In addition, co expression of SIX1 and Eya1 resulted in a significant increase in the SALL1 promoter activity when compared with either SIX1 or Eya1 alone. Finally, we demonstrate that SIX1 was able to bind to the SALL1 promoter by retardation assays and that deletion of the putative element of SIX1 significantly diminishes the SALL1 promoter activity response to SIX1 stimulation. Our findings, when taken together, indicate that SALL1 is a likely target gene for SIX1 during kidney development. PMID- 16670093 TI - Diminished hepatic gluconeogenesis via defects in tricarboxylic acid cycle flux in peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC 1alpha)-deficient mice. AB - The peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha) is a highly inducible transcriptional coactivator implicated in the coordinate regulation of genes encoding enzymes involved in hepatic fatty acid oxidation, oxidative phosphorylation, and gluconeogenesis. The present study sought to assess the effects of chronic PGC-1alpha deficiency on metabolic flux through the hepatic gluconeogenic, fatty acid oxidation, and tricarboxylic acid cycle pathways. To this end, hepatic metabolism was assessed in wild-type (WT) and PGC-1alpha(-/-) mice using isotopomer-based NMR with complementary gene expression analyses. Hepatic glucose production was diminished in PGC-1alpha(-/-) livers coincident with reduced gluconeogenic flux from phosphoenolpyruvate. Surprisingly, the expression of PGC-1alpha target genes involved in gluconeogenesis was unaltered in PGC-1alpha(-/-) compared with WT mice under fed and fasted conditions. Flux through tricarboxylic acid cycle and mitochondrial fatty acid beta-oxidation pathways was also diminished in PGC-1alpha(-/-) livers. The expression of multiple genes encoding tricarboxylic acid cycle and oxidative phosphorylation enzymes was significantly depressed in PGC-1alpha(-/-) mice and was activated by PGC-1alpha overexpression in the livers of WT mice. Collectively, these findings suggest that chronic whole-animal PGC-1alpha deficiency results in defects in hepatic glucose production that are secondary to diminished fatty acid beta-oxidation and tricarboxylic acid cycle flux rather than abnormalities in gluconeogenic enzyme gene expression per se. PMID- 16670094 TI - Constitutive ERK1/2 activation by a chimeric neurokinin 1 receptor-beta-arrestin1 fusion protein. Probing the composition and function of the G protein-coupled receptor "signalsome". AB - The beta-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of beta arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 is a G protein-uncoupled "constitutively desensitized" receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-betaArr1 expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-arrestin binding to GPCRs nucleates the formation of a stable "signalsome" that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment. PMID- 16670095 TI - Discrimination of esterase and peptidase activities of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 by a single mutation. AB - It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg(526)) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is approximately 7 times higher than the peptidase activity with Ac-Leu-p nitroanilide as substrate. However, with the same substrates, this difference was increased to approximately 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on k(cat) and K(m) of the enzyme were discussed based on molecular dynamics simulation studies. PMID- 16670096 TI - Consumer decision making in the individual health insurance market. AB - This paper summarizes the results from a study of consumer decision making in California's individual health insurance market. We conclude that price subsidies will have only modest effects on participation and that efforts to reduce nonprice barriers might be just as effective. We also find that there is substantial pooling in the individual market and that it increases over time because people who become sick can continue coverage without new underwriting. Finally, we show that people prefer more-generous benefits and that it is difficult to induce people in poor health to enroll in high-deductible health plans. PMID- 16670097 TI - Attenuation in risk estimates in logistic and Cox proportional-hazards models due to group-based exposure assessment strategy. AB - In occupational epidemiology, it is often possible to obtain repeated measurements of exposure from a sample of subjects (workers) who belong to exposure groups associated with different levels of exposure. Average exposures from a sample of workers can be assigned to all members of that group including those who are not sampled, leading to a group-based exposure assessment. We discuss how this group-based exposure assessment leads to approximate Berkson error model when the number of subjects with exposure measurements in each group is large, and how the error variance approximates the between-worker variability. Under the normality assumption of exposures and with moderately large number of workers in each group, there is attenuation in the estimate of the association parameter, the magnitude of which depends on the sizes of the between-worker variability and the true association parameter. Approximate equations for attenuation have been derived in logistic and Cox proportional-hazards models. These equations show that the attenuation in Cox proportional-hazards models is generally more severe than in logistic regression. Furthermore, when the between worker variability is large, our simulation study found that the approximation by equation is poor for the Cox proportional-hazards model. If the number of subjects is small, the approximation does not hold for either model. PMID- 16670098 TI - Isolated ventricular non-compaction of the myocardium in adults. AB - Isolated ventricular non-compaction (IVNC) in adults is a genetic cardiac disease of emerging importance with a distinct clinical and pathophysiological presentation. The body of evidence for the underlying genetic basis of the disease has also grown. Prognosis remains poor for patients with impaired systolic left ventricular function, as treatment options are very limited. The diagnosis of IVNC, however, is often missed, most often as a consequence of ignorance of the condition. The relevant clinical issues and the emerging concepts of the aetiology of IVNC are summarised. PMID- 16670099 TI - Aspiration thrombectomy and primary percutaneous coronary intervention. AB - How beneficial is the adjunctive use of thrombectomy devices in STEMI patients undergoing primary angioplasty? PMID- 16670100 TI - Oxidative stress and redox signalling in cardiac hypertrophy and heart failure. AB - Substantial evidence suggests the involvement of oxidative stress in the pathophysiology of congestive heart failure and its antecedent conditions such as cardiac hypertrophy and adverse remodelling after MI. Oxidative stress describes an imbalance between antioxidant defences and the production of reactive oxygen species (ROS), which at high levels cause cell damage but at lower levels induce subtle changes in intracellular signalling pathways (termed redox signalling). ROS are derived from many sources including mitochondria, xanthine oxidase, uncoupled nitric oxide synthases and NADPH oxidases. The latter enzymes are especially important in redox signalling, being implicated in the pathophysiology of hypertension and atherosclerosis, and activated by diverse pathologically relevant stimuli. We review the contribution of ROS to heart failure pathophysiology and discuss potential therapies that may specifically target detrimental redox signalling. Indeed, drugs such as ACE inhibitors and statins may act in part through such mechanisms. A better understanding of redox signalling mechanisms may enable the development of new targeted therapeutic strategies rather than the non-specific antioxidant approaches that have to date been disappointing in clinical trials. PMID- 16670101 TI - The pressure wire in practice. AB - Fractional flow reserve (FFR) is increasingly used to evaluate the functional significance of epicardial coronary disease. When compared with non-invasive techniques this index has superior sensitivity and specificity, is largely independent of prevailing haemodynamic conditions and has an unequivocal normal value. Furthermore, it can be used at the time of invasive coronary assessment. FFR measurements are invaluable for evaluation of intermediate coronary lesions but clinical decisions must be based on accurate measurements and an understanding of the limitations of the data supporting its use. This article identifies procedural and clinical issues pertaining to measurement of FFR and identifies potential pitfalls and situations where interpretation can be difficult; for example, the presence of serial stenoses and left main stem disease, its use in specific conditions such as diffuse epicardial disease, microvascular abnormalities, left ventricular dysfunction and acute coronary syndromes. PMID- 16670102 TI - The origins and sequelae of abnormal neuroendocrine function in polycystic ovary syndrome. AB - Polycystic ovary syndrome (PCOS) is a common clinical disorder characterized by ovulatory dysfunction and hyperandrogenaemia. A neuroendocrine hallmark of PCOS is persistently rapid LH (GnRH) pulsatility, which favours pituitary synthesis of LH over that of FSH and contributes to the increased LH concentrations and LH : FSH ratios typical of PCOS. Inadequate FSH levels contribute to impaired follicular development, whereas elevated LH levels augment ovarian androgen production. Whereas luteal phase elevations in progesterone normally slow GnRH pulse frequency, women with PCOS do not experience normal progesterone-mediated slowing, due in part to impaired hypothalamic progesterone sensitivity. This reduction in hypothalamic progesterone sensitivity appears to be mediated by elevated androgens because sensitivity can be restored with the androgen receptor blocker flutamide. The ovulatory and hormonal abnormalities associated with PCOS generally present during puberty, typically associated with hyperandrogenaemia. Along with elevated LH concentration and pulsatility, some girls with hyperandrogenaemia have impaired hypothalamic progesterone sensitivity similar to that seen in adult women with PCOS. We propose that peripubertal hyperandrogenaemia may lead to persistently rapid GnRH pulse frequency via impaired hypothalamic feedback inhibition. The subsequent abnormalities in gonadotropin secretion, androgen production and ovulatory function may support progression towards the adult PCOS phenotype. PMID- 16670104 TI - Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS. AB - By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains. PMID- 16670103 TI - Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling. AB - Profiling of the four known galactose-binding receptors in the C-type lectin family has been undertaken in parallel on a glycan array. The results are generally consistent with those of previous assays using various different formats, but they provide a direct comparison of the properties of the four receptors, revealing that they fall into two distinct groups. The major subunit of the rat asialoglycoprotein receptor and the rat Kupffer cell receptor show similar broad preferences for GalNAc-terminated glycans, while the rat macrophage galactose lectin and the human scavenger receptor C-type lectin (SRCL) bind more restricted sets of glycans. Both of these receptors bind to Lewis x-type structures, but the macrophage galactose lectin also interacts strongly with biantennary galactose- and GalNAc-terminated glycans. Although the similar glycan binding profiles for the asialoglycoprotein receptor and the Kupffer cell receptor might suggest that these receptors are functionally redundant, analysis of fibroblasts transfected with full-length Kupffer cell receptor reveals that they fail to endocytose glycosylated ligand. PMID- 16670105 TI - Ultrahigh-resolution immunofluorescence microscopy using ultrathin cryosections: subcellular distribution of caveolin-1alpha and CD31 in human placental endothelial cells. AB - We compared the z-axis resolutions achieved by immunofluorescence (IF) microscopic imaging of tissue sections of different thicknesses (ultrathin cryosections, optical sections of cryostat sections and conventional cryostat sections). We used these images to determine the distribution of caveolin-1alpha (CAV-1alpha) and CD31 in endothelial cells of full-term, human placenta. Anti-CAV 1alpha antibody was used to visualize caveolae, which are among the smallest organelles. By using ultrathin cryosections as substrates for IF microscopy, we were able to resolve discrete caveolae that were primarily present immediately beneath the endothelial cell surface. In contrast, neither conventional nor confocal images from cryostat sections were able to resolve individual caveolae, despite dramatic reductions in the confocal image degradation that arises from out-of-focus fluorescence signals. Anti-CD31 antibody labeled the endothelial cell surface exclusively. Quantitative analysis of ultrathin cryosections showed that about 2.5 times more CD31 was expressed on the luminal surface of cells than on the abluminal surface. Our results demonstrate that ultrathin cryosections can serve as excellent substrates for ultrahigh-resolution IF microscopy. PMID- 16670106 TI - Application of a quick-freezing and deep-etching method to pathological diagnosis: a case of elastofibroma. AB - A case of elastofibroma in a middle-aged Japanese woman was examined by the quick freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient. PMID- 16670107 TI - Emergence of PER and VEB extended-spectrum beta-lactamases in Acinetobacter baumannii in Belgium. AB - OBJECTIVES: Strains of Acinetobacter baumannii producing the extended-spectrum beta-lactamase (ESBL) PER-1 are widespread in Turkey and have also been reported from Korea and France. In contrast, A. baumannii producing the ESBL VEB-1 have only been reported from France, where one strain was responsible for a nationwide outbreak in 2003-2004. Here we describe the emergence of strains of A. baumannii producing VEB-1 and PER- 1 in Belgium. METHODS: Belgian hospitals were alerted in December 2003 to the emergence in France of VEB-1-producing A. baumannii susceptible only to meropenem and colistin. Isolates with a compatible susceptibility profile were sent to a single central laboratory for VEB-1 confirmation, molecular characterization and typing. RESULTS: From December 2003 to March 2005, three hospitals located close to the French border and one in the Brussels area reported isolation of eight A. baumannii isolates compatible with the French epidemic clone. Using PCR, six were identified as VEB-1-positive and two as PER-1-positive. All the VEB-1-positive isolates were clonally related by PFGE and by integron analysis to the French epidemic strain. The PER-1-positive strains were indistinguishable by PFGE but not related to the known French isolate or to several Turkish isolates. Both genes were chromosomally encoded. CONCLUSIONS: This work illustrates the inter-country spread of VEB-1-producing A. baumannii isolates as well as the emergence of PER-1-producing A. baumannii strains in Belgium. PMID- 16670108 TI - Plasmid-mediated florfenicol resistance in Pasteurella trehalosi. AB - OBJECTIVES: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR. METHODS: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization. RESULTS: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events. CONCLUSIONS: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae. PMID- 16670109 TI - Human intravenous immunoglobulin for experimental streptococcal toxic shock: bacterial clearance and modulation of inflammation. AB - OBJECTIVES: Polyclonal human intravenous immunoglobulin (IVIG) has been advocated as an adjunct to therapy in severe invasive streptococcal toxic shock because of its ability to neutralize superantigen toxins. The aim of this study was to assess IVIG therapeutic efficacy in an experimental model of streptococcal toxic shock. METHODS: To confirm the in vitro activity of IVIG against the Streptococcus pyogenes strain used in the study, IVIG was tested for superantigen neutralizing and bacterial opsonizing activity prior to in vivo studies. To evaluate the in vivo effects of IVIG in terms of microbiological outcome and disease severity in a superantigen-sensitive transgenic model of streptococcal shock, HLA-DQ transgenic mice were treated with IVIG either at the time of infection or after infection with S. pyogenes. Antibiotics were included in some studies. RESULTS: The IVIG preparation neutralized superantigenicity of S. pyogenes in vitro and enhanced bacterial killing in a whole blood assay. When given to mice at the time of S. pyogenes infection, IVIG neutralized circulating superantigens and reduced systemic inflammatory response. Remarkably, IVIG enhanced systemic clearance of bacteria and enhanced neutrophil infiltrate into the infected tissues. However, when used in combination with penicillin and clindamycin in a delayed treatment setting, IVIG did not confer additional therapeutic benefit, in terms of inflammatory response, bacterial clearance or survival. CONCLUSIONS: IVIG monotherapy can confer benefit in experimental streptococcal shock, but extension of these findings to the clinical situation will require further evaluation. PMID- 16670111 TI - Emergence delirium in adults in the post-anaesthesia care unit. AB - BACKGROUND: Emergence delirium in the post-anaesthesia care unit (PACU) is poorly understood. The goal of this prospective study was to determine frequency and risk factors of emergence delirium in adults after general anaesthesia. METHODS: In this prospective study, 1,359 consecutive patients were included. Contextual risk factors and occurrence of delirium according to the Riker sedation scale were documented. Groups were defined for the analysis according to the occurrence or not of agitation, then after exclusion of patients with preoperative anxiety and neuroleptics, or both, and antidepressants or benzodiazepines treatments. RESULTS: Sixty-four (4.7%) patients developed delirium in the PACU, which can go from thrashing to violent behaviour and removal of tubes and catheters. Preoperative anxiety was not found to be a risk factor. Preoperative medication by benzodiazepines (OR=1.910, 95% CI=1.101-3.315, P=0.021), breast surgery (OR=5.190, 95% CI=1.422-18.947, P=0.013), abdominal surgery (OR=3.206, 95% CI=1.262-8.143, P=0.014), and long duration of surgery increased the risk of delirium (OR=1.005, 95% CI=1.002-1.008, P=0.001), while a previous history of illness and long-term treatment by antidepressants decreased the risk (respectively, OR=0.544, 95% CI=0.315-0.939, P=0.029 and OR=0.245, 95% CI=0.084 0.710, P=0.010). CONCLUSIONS: Preoperative benzodiazepines, breast and abdominal surgery and surgery of long duration are risk factors for emergence delirium. PMID- 16670110 TI - Nitrous oxide does not change the incidence of postoperative delirium or cognitive decline in elderly surgical patients. AB - BACKGROUND: Postoperative delirium and cognitive decline are common in elderly surgical patients after non-cardiac surgery. Despite this prevalence and clinical importance, no specific aetiological factor has been identified for postoperative delirium and cognitive decline. In experimental setting in a rat model, nitrous oxide (N(2)O) produces neurotoxic effect at high concentrations and in an age dependent manner. Whether this neurotoxic response may be observed clinically has not been previously determined. We hypothesized that in the elderly patients undergoing non-cardiac surgery, exposure to N(2)O resulted in an increased incidence of postoperative delirium than would be expected for patients not receiving N(2)O. METHODS: Patients who were >or=65 yr of age, undergoing non cardiac surgery and requiring general anaesthesia were randomized to receive an inhalational agent and either N(2)O with oxygen or oxygen alone. A structured interview was conducted before operation and for the first two postoperative days to determine the presence of delirium using the Confusion Assessment Method. RESULTS: A total of 228 patients were studied with a mean (range) age of 73.9 (65 95) yr. After operation, 43.8% of patients developed delirium. By multivariate logistic regression, age [odds ratio (OR) 1.07; 95% confidence interval (CI) 1.02 1.26], dependence on performing one or more independent activities of daily living (OR 1.54; 95% CI 1.01-2.35), use of patient-controlled analgesia for postoperative pain control (OR 3.75; 95% CI 1.27-11.01) and postoperative use of benzodiazepine (OR 2.29; 95% CI 1.21-4.36) were independently associated with an increased risk for postoperative delirium. In contrast, the use of N(2)O had no association with postoperative delirium. CONCLUSIONS: Exposure to N(2)O resulted in an equal incidence of postoperative delirium when compared with no exposure to N(2)O. PMID- 16670112 TI - Magnesium sulphate as a technique of hypotensive anaesthesia. AB - BACKGROUND: This randomized, double-blind, placebo-controlled study was designed to assess the effect of perioperatively administered i.v. magnesium sulphate as a technique of hypotensive anaesthesia. METHODS: Sixty patients (25 female) undergoing functional endoscopic sinus surgery were included in two parallel groups. The magnesium group received magnesium sulphate 40 mg kg(-1) i.v. as a bolus before induction of anaesthesia and 15 mg kg(-1) h(-1) by continuous i.v. infusion during the operation. The same volume of isotonic solution was administered to the control group. Intraoperative bleeding was evaluated using a quality scale. RESULTS: In the magnesium group, there was a reduction in surgical time [68.1 (15.6) min vs 88.1 (10.7) min], although the anaesthetic time was 10 min longer and thus presuming a prolongation in anaesthetic emergence. There was a significant reduction of blood loss [165 (19) ml vs 257 (21) ml]. The anaesthetic requirements (fentanyl, vercuronium and sevoflurane), mean arterial blood pressure (P<0.005) and heart rate (P<0.005) were also significantly reduced. CONCLUSION: Magnesium sulphate led to a reduction in arterial pressure, heart rate, blood loss and duration of surgery. Furthermore, magnesium infusion alters anaesthetic dose requirements and emergence time. PMID- 16670113 TI - Coronary artery stenting and non-cardiac surgery--a prospective outcome study. AB - BACKGROUND: A 45% complication rate and a mortality of 20% were reported previously in patients undergoing non-cardiac surgery after coronary artery stenting. Discontinuation of antiplatelet drugs appeared to be of major influence on outcome. Therefore we undertook a prospective, observational multicentre study with predefined heparin therapy and antiplatelet medication in patients undergoing non-cardiac procedures after coronary artery stenting. METHODS: One hundred and three patients from three medical institutions were enrolled prospectively. Patients received coronary artery stents within 1 yr before non cardiac surgery (urgent, semi-urgent or elective). Antiplatelet drug therapy was not, or only briefly, interrupted. Heparin was administered to all patients. All patients were on an intensive/intermediate care unit after surgery. Main outcome was the combined (cardiac, bleeding, surgical, sepsis) complication rate. RESULTS: Of 103 patients, 44.7% (95% CI 34.9-54.8) suffered complications after surgery; 4.9% (95% CI 1.6-11.0) of the patients died. All but two (bleeding only) adverse events were of cardiac nature. The majority of complications occurred early after surgery. The risk of suffering an event was 2.11-fold greater in patients with recent stents (<35 days before surgery) as compared with percutaneous cardiac intervention more than 90 days before surgery. CONCLUSIONS: Despite heparin and despite having all patients on intensive/intermediate care units, cardiac events are the major cause for new perioperative morbidity/mortality in patients undergoing non-cardiac surgery after coronary artery stenting. The complication rate exceeds the re-occlusion rate of stents in patients without surgery (usually <1% annually). Patients with coronary artery stenting less than 35 days before surgery are at the greatest risk. PMID- 16670114 TI - Lessons learned from neutral ICD trials. AB - Multiple prospective randomized trials with implantable cardioverter defibrillators (ICDs) over the past decade have convincingly established the efficacy of ICD therapy in reducing all-cause mortality, by significantly reducing sudden cardiac death. Nevertheless, four trials have failed to show improved survival. Analysing these, in comparison with the positive trials, provides important information concerning the type of patients not likely to receive benefit from ICDs: (i) those with relatively low mortality (< or = 18% within 2 years of follow-up; (ii) those whose mechanism of death is predominantly non-arrhythmic; (iii) patients early (within 6 weeks) after infarction. PMID- 16670115 TI - Efficacy of patient activated antitachycardia pacing therapy using the Medtronic AT500 pacemaker. AB - A 28-year-old female with corrected anomalous pulmonary venous drainage presented with multiple atrial arrhythmias many years later. Ablation techniques eliminated most of the arrhythmias except atrial tachycardia with 1:1 AV conduction. A dual chamber pacemaker with antiatrial tachycardia features was implanted and was shown to be effective in arrhythmia control when the standard algorithm was overridden by an external patient activation device. PMID- 16670116 TI - Surgical management of inflammatory bowel disease. AB - AIM: To evaluate the outcome and morbidity after major surgical interventions for inflammatory bowel disease (IBD). METHODS: Retrospective case note analysis of 227 children referred to a tertiary referral centre between 1994 and 2002 for treatment of IBD. RESULTS: 26 of 125 children with Crohn's disease (21%) required surgical management. 13 with disease proximal to the left colon underwent limited segmental resections and primary anastomosis, without significant morbidity. Primary surgery for 13 children with disease distal to the transverse colon included 6 subtotal-colectomies or panprocto-colectomies. All seven children undergoing conservative segmental resections (three with primary anastomosis, four with stoma formation), required further colonic resection or defunctioning stoma formation. All three children undergoing primary anastomosis developed a leak or fistula formation. 22 of 102 children with ulcerative colitis (22%) required surgery. Definitive procedures (n = 17) included J-pouch ileoanal anastomosis (n = 11), ileorectal anastomosis (n = 2), straight ileoanal anastomosis (n = 3), and proctectomy/ileostomy (n = 1). Five children await restorative surgery after subtotal colectomy. Median daily stool frequency after J-pouch surgery was 5 (range 3-15), and 10 of 11 children reported full daytime continence. All three children with straight ileoanal anastomosis had unacceptable stool frequency and remain diverted. CONCLUSION: The complication rate after resectional surgery for IBD was 57% for Crohn's disease, and 31% for ulcerative colitis. In children with Crohn's disease, limited resection with primary anastomosis is safe proximal to the left colon. Where surgery is indicated for disease distal to the transverse colon, subtotal or panproctocolectomy is indicated, and an anastomosis should be avoided. Children with ulcerative colitis had a good functional outcome after J-pouch reconstruction. However, the overall failure rate of attempted reconstructive surgery was 24%, largely owing to the poor results of straight ileoanal anastomosis. PMID- 16670117 TI - Genital warts in children: what do they mean? AB - Human papillomaviruses (HPVs) are a diverse family of viruses, of which 30-40 genotypes specifically infect the genital tract. Genital HPVs are largely transmitted sexually, with most infections being asymptomatic and transient. In contrast, persistent infection with oncogenic genotypes in a minority is a strong risk factor, for subsequent development of high grade dysplasia, the precursor lesion to cervical neoplasia, which generally occurs after a long latency period. It is unknown whether there is a disease correlate in children chronically infected with oncogenic HPVs. Low risk HPV genotypes 6 and 11 are the primary cause of condylomata acuminata, although in children non-genital genotypes are also found in a proportion, with the mode of transmission being either perinatal, horizontal, or sexual. The finding of asymptomatic HPV DNA in children, and correlation with live virus, infectivity, or disease is unclear. Long term follow up for children with anogenital warts is recommended, although there are no longitudinal studies available to clarify whether they are at risk of developing carcinoma in young adulthood. PMID- 16670118 TI - Psychiatric, somatic, and gastrointestinal disorders, and Helicobacter pylori infection in children with recurrent abdominal pain. AB - AIMS: To examine the utility of the Rome II criteria in children with recurrent abdominal pain (RAP) and compare them to those who met Apley's criteria and those who met neither criteria. METHODS: Prospective study in general paediatric clinics in Komagane, Japan. Children with abdominal pain were classified into those who met Rome II criteria, those who met Apley's criteria, and those who met neither. RESULTS: A total of 182 children with RAP participated; 45 children met Rome II criteria, 55 met Apley's criteria, and 82 met neither. Children who met Rome II criteria had a significantly higher prevalence of psychiatric and somatic disorders compared to the group met neither (36% v 6%, 22% v 10%, respectively). The overall prevalence of H pylori was 7%; prevalence increased with age from 3% at age < or = 10 to 10% for children >10 years. Children who met Rome II criteria had a significantly higher prevalence of H pylori infection than the reference group (18% v 4%). In a logistic regression model, all the study variables were included in the model specifying first the Rome II criteria group as the independent variable; psychiatric disorders, H pylori infection, and older age group were independent risk factors. CONCLUSIONS: More than half the children suffering from recurrent abdominal pain met neither Apley's nor Rome II criteria. Children who meet Rome II criteria should be evaluated for psychiatric disorders and should be tested for H pylori infection. Despite the overall trend for a fall in the prevalence of H pylori infection among children in Japan, there are subpopulations of sick children where the prevalence of the infection is relatively high. PMID- 16670119 TI - WHO guidelines for severe malnutrition: are they feasible in rural African hospitals? AB - AIMS: To assess the feasibility of implementing and sustaining the WHO guidelines for inpatient management of severe malnutrition in under-resourced rural South African hospitals, and to identify any constraints. INTERVENTION: Three 2-day training workshops were held in 1998, followed by monthly 1-day visits for 5 months, ending in March 1999, in two rural district hospitals with limited resources in Eastern Cape Province, South Africa. METHODS: A 12-month observational study was conducted from April 2000 to April 2001 in Mary Theresa and Sipetu hospitals (Eastern Cape Province, South Africa), including 1011 child hours of observation on the wards, medical record reviews, interviews with carers and staff, and inventories of essential supplies. All admissions (n = 193) for severe malnutrition to the two hospitals were studied. The main outcomes were the extent to which the 10 steps for routine care of severely malnourished children were implemented, proficiency of performance and constraining factors. RESULTS: The hospitals made the changes required in clinical and dietary management, but the tasks were not always performed fully or with sufficient care. Play and stimulation and an effective system of follow-up were not implemented. Doctors' poor knowledge, nurses' inattentiveness and insufficient interaction with carers were constraints to optimal management. The underlying factors were inadequate undergraduate training, understaffing, high doctor turnover and low morale. CONCLUSIONS: Guidelines for severe malnutrition are largely feasible but training workshops are insufficient to achieve optimal management as staff turnover and an unsupportive health system erode the gains made and doctors treat cases without having being trained. Medical and nursing curricula in Africa must include treatment of severe malnutrition. PMID- 16670120 TI - Independent risk factors for chronic cyclosporine induced nephropathy in children with nephrotic syndrome. AB - BACKGROUND: Cyclosporine A (CsA) has been widely used in children with steroid dependent and steroid resistant nephrotic syndrome (NS) because of its efficacy in relieving these patients from systemic side effects of steroids. However, its long term use is controversial, since chronic CsA induced nephropathy (CsAN) may develop in a considerable number of patients. AIMS AND METHODS: In order to clarify the risk factors for the development of CsAN, the clinical characteristics of children with steroid dependent or steroid resistant NS taking CsA (target blood trough levels 50-150 ng/ml) for more than six months, managed at a single centre, were retrospectively analysed. RESULTS: Thirteen of 30 children (24 boys and 6 girls) taking CsA (mean duration 43 months, range 6-144) had CsAN defined as the presence of CsA associated arteriopathy with or without striped tubulointerstitial lesions. The multivariate analysis revealed that CsA treatment for more than 36 months and an age younger than 5 years at the start of CsA treatment were independent risk factors for the development of CsAN. The univariate analysis also showed that patients with CsAN had more frequent relapses during CsA treatment than those without CsAN. CONCLUSION: An alternative treatment should be seriously considered after a 36 month administration of CsA in order to prevent CsAN. Data also suggest that CsA treatment in children younger than 5 years should be avoided if possible. PMID- 16670121 TI - Evaluation of a diphtheria-tetanus-acellular pertussis-inactivated poliovirus Haemophilus influenzae type b vaccine given concurrently with meningococcal group C conjugate vaccine at 2, 3 and 4 months of age. AB - BACKGROUND AND OBJECTIVE: In view of the possible introduction of diphtheria tetanus-acellular pertussis-inactivated poliovirus-Haemophilus influenzae type b (DTaP-IPV-Hib, eg Pediacel) vaccine in the UK, a study of the immunogenicity of Pediacel when given with one of two different meningococcal group C conjugate (MCC) vaccines at 2, 3 and 4 months of age was conducted. METHODS: Randomised controlled study in 241 infants. RESULTS: Post vaccination, the proportion of infants with anti-polyribosylribitol phosphate (PRP) levels > or =0.15 microg/ml was 93.2% (95% confidence interval (CI) 86.6 to 96.7) in the Pediacel group compared with 100% (95% CI 96.4 to 100) in the diphtheria-tetanus-whole-cell pertussis-Haemophilus influenzae type b (DTwP-Hib) group. The anti-PRP response was lower in infants receiving either Pediacel or DTwP-Hib when these vaccines were given concomitantly with meningococcal group C conjugate with diphtheria derived protein CRM(197) as conjugate protein (MCC-CRM) compared with meningococcal group C conjugate with tetanus toxoid as conjugate protein (MCC TT). For group C meningococcus, the proportion of infants with serum bactericidal antibody (SBA) titre > or =1:8 in the Pediacel group was 99.0% compared with 100% in the DTwP-Hib group. The MCC SBA geometric mean titre (GMT) was lower in those receiving Pediacel with MCC-TT than in those receiving DTwP-Hib with MCC-TT, although all titres were well above the protective threshold. The MCC SBA GMT was similar in those receiving Pediacel and DTwP-Hib and MCC-CRM. Responses to all other vaccine components were equivalent in the two groups. CONCLUSIONS: Pediacel is immunogenic when given at 2, 3 and 4 months of age. Coadministration of MCC vaccine can influence the Hib response, and the MCC response to a tetanus conjugate can be influenced by the nature of the coadministered DTP-Hib vaccine. PMID- 16670122 TI - Short-term mortality and implementation of antiretroviral treatment for critically ill HIV-infected children in a developing country. AB - OBJECTIVE: To describe the short-term outcome of critically ill HIV-infected children with access to highly active antiretroviral therapy (HAART) in a developing region. METHODS: Prospective observational study conducted in a paediatric teaching hospital in Cape Town, South Africa. All children admitted to the paediatric intensive care unit (PICU) with suspected HIV infection were screened. Data are n (%) with 95% confidence intervals. RESULTS: Sixty eight of 96 HIV antibody-positive children, median age 3 months, were confirmed HIV infected. Predicted PICU mortality was 0.42. Fifty one children (75%; 95% CI 65 to 85%) survived to PICU discharge, but hospital survival was only 51% (95% CI 40 to 63%). Limitation of intervention (LOI) decisions were a factor in the majority of PICU and ward deaths. Twenty one PICU survivors (31%; 95% CI 20 to 42%) commenced HAART, and two children were already on treatment. Nineteen children (28%) were considered to be established on HAART after 1 month. Thirteen HIV infected children (19%; 95% CI 10 to 28%), representing 25% (95% CI 14 to 37%) of all PICU survivors, and 68% (95% CI 48 to 89%) of those PICU survivors who were established on HAART remain well on treatment after median 350 days. CONCLUSION: The majority of HIV-infected children survived to discharge from PICU, but only half survived to hospital discharge. LOI decisions, usually made in PICU, directly influenced short-term survival and the opportunity to commence HAART. Although few critically ill HIV-infected children survived to become established on HAART, the long-term outcome of children on HAART is encouraging and warrants further investigation. PMID- 16670123 TI - Activation of kinin B1 receptors induces chemotaxis of human neutrophils. AB - Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)] bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site. PMID- 16670124 TI - Nitric oxide-dependent killing of Cryptococcus neoformans by B-1-derived mononuclear phagocyte. AB - The role of B lymphocytes in protecting the host against pulmonary Cryptococcus neoformans infection is until now, uncertain. A recent study using B lymphocyte deficient mice suggests that B lymphocytes play a protective role in cryptococcal infection. It has been well established that at least three B cell subsets, B-1a, B-1b, and B-2, are present in the mouse periphery. B-1 cells constitute a minor fraction of the B cell population in the spleen and are not detected in lymph nodes of mice. We demonstrated that B-1 cells migrate to a nonspecific, inflammatory focus and differentiate into macrophage-like cells. However, the role these cells might play on the kinetics and evolution of the inflammatory response and on fungal infection has not yet been established. Based on these data, we decided to investigate the interaction of B-1-derived mononuclear phagocytes (BDMP) with C. neoformans to elucidate the possible influence of this cell in the progression of the disease. In this study, we demonstrated that the BDMP cell internalized C. neoformans and that this process was mediated by complement receptor 3. Thus, our results showed that the BDMP cell was more fungicidal than a macrophage and up-regulated major histocompatibility complex type II and the CD86 costimulatory molecule with the production of proinflammatory cytokines. The phagocytosis of C. neoformans results in the nitric oxide (NO)-mediated death of the fungus, as demonstrated by experiments using NO synthase 2 knockout and aminoguanidine-treated, wild-type mice. PMID- 16670125 TI - Effects of glucocorticoids on STAT4 activation in human T cells are stimulus dependent. AB - Glucocorticoids affect the immune system by a number of mechanisms, including modulation of cytokine production in lymphocytes. Glucocorticoids suppress T helper cell type 1 immune responses by decreasing the ability of T cells to respond to interleukin (IL)-12, a major inducer of interferon (IFN)-gamma. IFN beta increases the expression of the anti-inflammatory cytokine IL-10 and suppresses IL-12. Signaling pathways through IFN-beta and the IL-12 receptor (IL 12R) involve activation by phosphorylation of signal transducer and activator of transcription 4 (STAT4). Our aim was to investigate the effects of dexamethasone on STAT4 activation by IFN-beta and IL-12 in human T cell blasts. We report that dexamethasone decreases IL-12-induced STAT4 phosphorylation and IFN-gamma production and enhances IFN-beta-induced STAT4 activation and IL-10 production. These effects are associated with a down-regulation of IL-12Rbeta1 expression but an up-regulation of IFN-betaR. These results indicate that the effect of glucocorticoids on the STAT4 signaling pathway depends on the stimulus activating that pathway. PMID- 16670126 TI - Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6 long glycoside side-chain-branched beta-glucan. AB - Polysaccharide beta-1,3-D-glucans (beta-glucans) are components of the cell wall of various fungi and show immunomodulatory activities. beta-Glucans have been reported to enhance neutrophil accumulation during pathogenic fungi-induced lung inflammation. Therefore, we examined whether beta-glucans themselves possess chemotactic activities for human neutrophils. Among several kinds of beta glucans, beta-1,6-long glucosyl side-chain-branched beta-glucan, isolated from Candida albicans [Candida soluble beta-D-glucan (CSBG)], dose-dependently induced neutrophil migration in a Boyden chamber system. In contrast, 1,6-monoglucosyl branched beta-glucans, such as Sparassis crispa-derived beta-glucan (SCG) and grifolan (GRN), which were derived from nonpathogenic fungi, hardly induced neutrophil migration. Moreover, CSBG-induced neutrophil migration was inhibited completely by liposomes containing neutral glycosphingolipid lactosylceramide (LacCer; Galbeta1-4Glc-ceramide) but not NeuAcalpha2-3Galbeta1-4Glcbeta1-1'-Cer ganglioside. Furthermore, binding experiments demonstrated that CSBG bound to glycosphingolipids (such as LacCer) with a terminal galactose residue; however, SCG and GRN (1,6-monoglucosyl-branched beta-glucans) did not bind to LacCer. It is important that a Src kinase inhibitor protein phosphatase 1, a phosphatidylinositol-3 kinase (PI-3K) inhibitor wortmannin, and a Galpha(i/o) inhibitor pertussis toxin inhibited neutrophil migration toward CSBG. Taken together, our results suggest that beta-1,6-long glucosyl side-chain-branched beta-glucan CSBG binds to LacCer and induces neutrophil migration through the activation of Src family kinase/PI-3K/heterotrimeric G-protein signal transduction pathways. PMID- 16670127 TI - Summaries for patients. Treating obesity: laparoscopic gastric banding versus a nonsurgical weight loss program. PMID- 16670128 TI - Summaries for patients. Treatment outcomes for patients with multidrug-resistant tuberculosis. PMID- 16670129 TI - Summaries for patients. Ramipril improves walking ability in patients with peripheral arterial disease. PMID- 16670130 TI - Summaries for patients. Patient ratings of the overall quality of care in 2 managed care organizations were not associated with measures of the technical quality of care. PMID- 16670131 TI - Treatment of mild to moderate obesity with laparoscopic adjustable gastric banding or an intensive medical program: a randomized trial. AB - BACKGROUND: Obesity is a major, growing health problem. Observational studies suggest that bariatric surgery is more effective than nonsurgical therapy, but no randomized, controlled trials have confirmed this. OBJECTIVE: To ascertain whether surgical therapy for obesity achieves better weight loss, health, and quality of life than nonsurgical therapy. DESIGN: Randomized, controlled trial. SETTING: University departments of medicine and surgery and an affiliated private hospital. PATIENTS: 80 adults with mild to moderate obesity (body mass index, 30 kg/m2 to 35 kg/m2) from the general community. INTERVENTIONS: Patients were assigned to a program of very-low-calorie diets, pharmacotherapy, and lifestyle change for 24 months (nonsurgical group) or to placement of a laparoscopic adjustable gastric band (LAP-BAND System, INAMED Health, Santa Barbara, California) (surgical group). MEASUREMENTS: Outcome measures were weight change, presence of the metabolic syndrome, and change in quality of life at 2 years. RESULTS: At 2 years, the surgical group had greater weight loss, with a mean of 21.6% (95% CI, 19.3% to 23.9%) of initial weight lost and 87.2% (CI, 77.7% to 96.6%) of excess weight lost, while the nonsurgical group had a loss of 5.5% (CI, 3.2% to 7.9%) of initial weight and 21.8% (CI, 11.9% to 31.6%) of excess weight (P < 0.001). The metabolic syndrome was initially present in 15 (38%) patients in each group and was present in 8 (24%) nonsurgical patients and 1 (3%) surgical patient at the completion of the study (P < 0.002). Quality of life improved statistically significantly more in the surgical group (8 of 8 subscores of Short Form-36) than in the nonsurgical group (3 of 8 subscores). LIMITATIONS: The study included mildly and moderately obese participants, was not powered for comparison of adverse events, and examined outcomes only for 24 months. CONCLUSIONS: Surgical treatment using laparoscopic adjustable gastric banding was statistically significantly more effective than nonsurgical therapy in reducing weight, resolving the metabolic syndrome, and improving quality of life during a 24-month treatment program. PMID- 16670132 TI - Withholding resuscitation: a new approach to prehospital end-of-life decisions. AB - BACKGROUND: Emergency medical services (EMS) personnel often are not permitted to honor requests to withhold resuscitation at the end of life, particularly if there is no written do-not-resuscitate (DNR) order. OBJECTIVE: To determine whether EMS personnel from agencies implementing new guidelines would be more likely to withhold resuscitation from persons having out-of-hospital cardiac arrests than would personnel from agencies that did not implement the guidelines. DESIGN: Observational study in which 16 of 35 local EMS agencies volunteered to implement new guidelines for withholding resuscitation. SETTING: King County, Washington. PATIENTS: 2770 patients with EMS-attended cardiac arrest. INTERVENTION: New guidelines adopted by participating agencies permitted EMS personnel to withhold resuscitation if the patient had a terminal condition and if the patient, family, or caregivers indicated, in writing or verbally, that no resuscitation was desired. MEASUREMENTS: Proportion of resuscitations withheld in agencies that implemented new guidelines compared with those that did not. RESULTS: Emergency medical services personnel from agencies implementing new guidelines withheld resuscitation in 11.8% of patients (99 of 841 patients) having cardiac arrests, compared with an average of 5.3% (range, 4.2% to 5.9%) of patients (103 of 1929 patients) in 3 historical and contemporary control groups. Honoring verbal requests alone accounted for 53% of withheld resuscitations in the intervention group (52 of 99 patients) compared with an average of 8% (range, 7% to 9%) in the control groups (8 of 103 patients). LIMITATIONS: The study was not a randomized, controlled trial; individual agencies chose whether to implement the guidelines. CONCLUSIONS: Implementation of new guidelines was associated with an increase in the number of resuscitations withheld by EMS personnel. This increase was primarily due to honoring verbal requests. PMID- 16670133 TI - Regional variations in health care intensity and physician perceptions of quality of care. AB - BACKGROUND: Research has documented dramatic differences in health care utilization and spending across U.S. regions with similar levels of patient illness. Although patient outcomes and quality of care have been found to be no better in regions of high health care intensity, it is unknown whether physicians in these regions feel more capable of providing good patient care than those in low-intensity regions. OBJECTIVE: To determine whether physicians in high intensity regions feel better able to care for patients than physicians in low intensity regions. DESIGN: Physician telephone survey. SETTING: 51 metropolitan and 9 nonmetropolitan areas of the United States and a supplemental national sample. PARTICIPANTS: 10,577 physicians who provided care to adults in 1998 or 1999 were surveyed for the Community Tracking Study (response rate, 61%). MEASUREMENTS: The End-of-Life Expenditure Index, a measure of spending that reflects differences in the overall quantity of medical services provided rather than differences in illness or price, was used to determine health care intensity in the physicians' community. Outcomes included physicians' perceived availability of clinical services, ability to provide high-quality care to patients, and career satisfaction. RESULTS: Although the highest-intensity regions have substantially more hospital beds and specialists per capita, physicians in these regions reported more difficulty obtaining needed services for their patients. The proportion of physicians who felt able to obtain elective hospital admissions ranged from 50% in high-intensity regions to 64% in the lowest-intensity region (P < 0.001 for the relationship between intensity and perceived ability to obtain hospital admissions); the proportion of physicians who felt able to obtain high-quality specialist referrals ranged from 64% in high intensity regions to 79% in low-intensity regions (P < 0.001). Compared with low intensity regions, fewer physicians in high-intensity regions felt able to maintain good ongoing patient relationships (range, 62% to 70%; P < 0.001) or able to provide high-quality care (range, 72% to 77%; P = 0.009). In most cases, differences persisted but were attenuated in magnitude after adjustment for physician attributes, practice characteristics, and local market factors (for example, managed care penetration); the difference in perceived ability to provide high-quality care was no longer statistically significant (P = 0.099). LIMITATIONS: The cross-sectional design prevented demonstration of a causal relationship between intensity and physician perceptions of quality. CONCLUSION: Despite more resources, physicians in regions of high health care intensity did not report greater ease in obtaining needed services or greater ability to provide high-quality care. PMID- 16670134 TI - Time to sputum culture conversion in multidrug-resistant tuberculosis: predictors and relationship to treatment outcome. AB - BACKGROUND: Conversion of sputum mycobacterial cultures from positive growth to negative growth of Mycobacterium tuberculosis in patients with pulmonary tuberculosis (TB) is considered the most important interim indicator of the efficacy of anti-TB pharmacologic treatment for multidrug-resistant disease. OBJECTIVE: To evaluate and compare time to and predictors of initial sputum culture conversion with predictors of treatment outcome for patients with multidrug-resistant TB. DESIGN: Retrospective cohort study. SETTING: Latvia. PATIENTS: All civilian patients with multidrug-resistant TB treated with the DOTS Plus strategy between 1 January and 31 December 2000. INTERVENTION: Individualized treatment for confirmed sputum culture-positive pulmonary multidrug-resistant TB. MEASUREMENTS: Time to initial sputum culture conversion and treatment outcome. RESULTS: Among 167 patients who were sputum culture positive at initiation of second-line therapy, 129 (77%) converted in a median time of 60 days (range, 4 to 462 days) and 38 (23%) did not convert. Independent predictors of a longer sputum culture conversion time, using an accelerated failure time regression model, included previous treatment for multidrug resistant TB, high initial sputum culture colony count, bilateral cavitations on chest radiography, and the number of drugs the initial isolate was resistant to at treatment initiation. Treatment outcomes were statistically significantly worse for patients who did not convert their sputum culture within 2 months. LIMITATIONS: Twenty-five percent of patients missed 5 or more monthly sputum collections. CONCLUSIONS: Under program conditions in Latvia, most patients with multidrug-resistant TB achieved sputum culture conversion within 12 weeks of starting treatment. Chest radiography and sputum culture drug susceptibility testing can assist physicians in predicting which patients will convert more slowly. Sputum culture conversion is a useful and appropriate interim indicator of treatment outcome in patients with multidrug-resistant TB. PMID- 16670135 TI - Brief communication: ramipril markedly improves walking ability in patients with peripheral arterial disease: a randomized trial. AB - BACKGROUND: Peripheral arterial disease (PAD) affects up to 12% of adults older than 50 years of age. Conventional therapies have only modest effects in improving symptoms. OBJECTIVE: To examine the effects of angiotensin-converting enzyme inhibition on walking ability in patients with PAD. DESIGN: Randomized, double-blind, placebo-controlled trial initiated in March 2003 and completed in January 2005. SETTING: The Alfred Hospital, Melbourne, Australia. PARTICIPANTS: 40 older adults with symptomatic PAD and no history of diabetes or hypertension. INTERVENTION: 10 mg of ramipril (n = 20) or placebo (n = 20) once daily for 24 weeks. All patients completed the trial. MEASUREMENTS: Pain-free and maximum walking time were recorded during a standard treadmill test, and the standard Walking Impairment Questionnaire was administered. RESULTS: After adjustment for the baseline pain-free walking time, mean pain-free walking time after ramipril treatment was 227 seconds (95% CI, 175 seconds to 278 seconds; P < 0.001) longer than that after placebo treatment. Similarly, maximum walking time improved by 451 seconds in the ramipril group (CI, 367 seconds to 536 seconds; P < 0.001) but did not change in the placebo group. Ramipril improved the Walking Impairment Questionnaire median distance score from 5% (range, 1% to 39%) to 21% (range, 12% to 58%; P < 0.001), speed score from 3% (range, 3% to 39%) to 18% (range, 8% to 50%; P < 0.001), and stair-climbing score from 17% (range, 4% to 80%) to 67% (range, 38% to 88%; P < 0.001). No adverse events were reported. LIMITATIONS: The sample size is modest, and the strict inclusion criteria limit the applicability of the results to patients with claudication and infrainguinal disease and those without diabetes. CONCLUSION: Ramipril improved pain-free and maximum walking time in some adults with symptomatic PAD. PMID- 16670136 TI - Patients' global ratings of their health care are not associated with the technical quality of their care. AB - BACKGROUND: Patient global ratings of care are commonly used to assess health care. However, the extent to which these assessments of care are related to the technical quality of care received is not well understood. OBJECTIVE: To investigate the relationship between patient-reported global ratings of health care and the quality of providers' communication and technical quality of care. DESIGN: Observational cohort study. SETTING: 2 managed care organizations. PATIENTS: Vulnerable older patients identified by brief interviews of a random sample of community-dwelling adults 65 years of age or older who received care in 2 managed care organizations during a 13-month period. MEASUREMENTS: Survey questions from the second stage of the Consumer Assessment of Healthcare Providers and Systems program were used to determine patients' global rating of health care and provider communication. A set of 236 quality indicators, defined by the Assessing Care of Vulnerable Elders project, were used to measure technical quality of care given for 22 clinical conditions; 207 quality indicators were evaluated by using data from chart abstraction or patient interview. RESULTS: Data on the global rating item, communication scale, and technical quality of care score were available for 236 vulnerable older patients. In a multivariate logistic regression model that included patient and clinical factors, better communication was associated with higher global ratings of health care. Technical quality of care was not significantly associated with the global rating of care. LIMITATIONS: Findings were limited to vulnerable elders who were enrolled in managed care organizations and may not be generalizable to other age groups or types of insurance coverage. CONCLUSIONS: Vulnerable elders' global ratings of care should not be used as a marker of technical quality of care. Assessments of quality of care should include both patient evaluations and independent assessments of technical quality. PMID- 16670137 TI - Meta-analysis: low-molecular-weight heparin and bleeding in patients with severe renal insufficiency. AB - BACKGROUND: Dose adjustment or laboratory monitoring of low-molecular-weight heparin (LMWH) is commonly recommended for patients with severe renal insufficiency (creatinine clearance < or =30 mL/min), but the basis for this recommendation is unclear. PURPOSE: To compare levels of anti-Xa heparin and risk for major bleeding in LMWH-treated patients with a creatinine clearance of 30 mL/min or less versus those with a creatinine clearance greater than 30 mL/min by using standard weight-adjusted therapeutic doses, empirically adjusted doses, or prophylactic doses of LMWH. DATA SOURCES: Electronic databases (MEDLINE, EMBASE, and the Cochrane Library) searched to December 2005 with no language restrictions. The authors also searched reference lists and contacted experts. STUDY SELECTION: Observational or subgroups of randomized studies that included non-dialysis-dependent patients with varying degrees of renal function who were treated with LMWH and reported creatinine clearance and anti-Xa levels or major bleeding. DATA EXTRACTION: Two reviewers independently selected studies and extracted data on patient characteristics, renal function, LMWH treatment, anti Xa levels, and major bleeding. The pooled odds ratio of major bleeding in patients with a creatinine clearance of 30 mL/min or less was calculated by using the Peto method. DATA SYNTHESIS: Eighteen studies using 3 preparations of LMWH (15 studies using enoxaparin, 2 using tinzaparin, and 1 using dalteparin) were included. Peak anti-Xa levels measured 4 hours after a subcutaneous injection were statistically significantly higher in patients with a creatinine clearance of 30 mL/min or less compared with those with a creatinine clearance greater than 30 mL/min in studies that used a standard therapeutic dose of enoxaparin (4 studies) but not in studies of empirically dose-adjusted enoxaparin (3 studies). Data were insufficient to assess the relationship between anti-Xa and renal function for prophylactic doses of enoxaparin and therapeutic doses of tinzaparin or dalteparin. In 12 studies involving 4971 patients, LMWH was associated with a statistically significant increase in the risk for major bleeding in patients with a creatinine clearance of 30 mL/min or less compared with those with a creatinine clearance greater than 30 mL/min (5.0% vs. 2.4%; odds ratio, 2.25 [95% CI, 1.19 to 4.27]; P = 0.013). When analyzed according to LMWH preparation, major bleeding was increased when a standard therapeutic dose of enoxaparin was used (8.3% vs. 2.4%; odds ratio, 3.88 [CI, 1.78 to 8.45]) but may not be increased when an empirically adjusted dose of enoxaparin is used (0.9% vs. 1.9%; odds ratio, 0.58 [CI, 0.09 to 3.78]; P = 0.23 for heterogeneity). There were insufficient studies to assess the risk for major bleeding with tinzaparin, dalteparin, and prophylactic doses of enoxaparin. LIMITATIONS: The data for tinzaparin and dalteparin were limited. Data are observational, and the potential for confounding cannot be excluded. CONCLUSIONS: Non-dialysis-dependent patients with a creatinine clearance of 30 mL/min or less who are treated with standard therapeutic doses of enoxaparin have elevated levels of anti-Xa and an increased risk for major bleeding. Empirical dose adjustment of enoxaparin may reduce the risk for bleeding and merits additional evaluation. No conclusions can be made regarding other LMWHs. PMID- 16670138 TI - Informed consent for research and authorization under the Health Insurance Portability and Accountability Act Privacy Rule: an integrated approach. AB - Researchers have found that implementation of the Health Insurance Portability and Accountability Act (HIPAA) Privacy Rule is having a negative impact on clinical research. This impact traces, in part, to many research institutions complying with HIPAA by adding lengthy, complex language to their research consent documents. The addition of extensive language burdens institutional review boards and may undermine participants' understanding of the research in which they take part. Comparative analysis reveals, however, that the addition of lengthy text often is unnecessary. The U.S. federal requirements for informed consent for human subjects research and the HIPAA Privacy Rule's requirements for individual authorization overlap substantially. Hence, consent forms that satisfy the U.S. federal regulations for human subjects research need only minimal additional text to also satisfy the authorization requirements under the HIPAA Privacy Rule. PMID- 16670139 TI - Bariatric surgery: crossing a body mass index threshold. PMID- 16670140 TI - Withholding resuscitation in prehospital care. PMID- 16670141 TI - Does more health care spending produce better health and happier doctors? PMID- 16670142 TI - Working against the grain. PMID- 16670143 TI - Exercise and peripheral arterial disease. PMID- 16670144 TI - Exercise and peripheral arterial disease. PMID- 16670145 TI - Body mass index and risk for end-stage renal disease. PMID- 16670146 TI - Body mass index and risk for end-stage renal disease. PMID- 16670147 TI - Laboratory infection of a technician by mimivirus. PMID- 16670148 TI - Saving (internal) medicine. PMID- 16670149 TI - Saving (internal) medicine. PMID- 16670150 TI - Octreotide-induced manic episodes in a patient with acromegaly. PMID- 16670151 TI - Increases in serum estrogen levels during major illness are caused by increased peripheral aromatization. AB - Although serum testosterone levels decrease acutely in critically ill patients, estrogen levels rise. We hypothesized that increased rates of aromatization of androgens to estrogens underlie the increase in serum estrogen levels. Eleven men and three women (age 42-69 yr) were prospectively studied before and again after elective coronary artery bypass graft surgery (CABG). Each patient received priming doses of [(14)C]androgen and [(3)H]estrogen that were immediately followed by peripheral infusions for 210 min. Eight men and three women received androstenedione (A(4))/estrone (E(1)) and three men received testosterone (T)/estradiol (E(2)). Adipose tissue biopsies were obtained in another six men before and after CABG to evaluate levels of P450 aromatase mRNA. Serum T levels decreased postoperatively in all 17 men (P < 0.001), whereas E(1) levels rose (P = 0.004), with a trend toward a rise in E(2) (P = 0.23). Peripheral aromatization rates of androgens to estrogens rose markedly in all 14 patients (P < 0.0001). Estrogen clearance rates rose (P < 0.002). Mean serum A(4) levels increased slightly postoperatively (P = 0.04), although no increase in A(4) production rates (PRs) was observed. T PRs decreased in two of three men, whereas clearance rates increased in all three. Adipose tissue P450 aromatase mRNA content increased postoperatively (P < 0.001). We conclude that the primary cause of increased estrogen levels in acute illness is increased aromatase P450 gene expression, resulting in enhanced aromatization of androgens to estrogens, a previously undescribed endocrine response to acute illness. Both increased T clearance and decreased T production contribute to decreased serum T levels. Animal studies suggest that these opposing changes in circulating estrogen and androgen levels may be important to reduce morbidity and mortality in critical illness. PMID- 16670152 TI - Behavioral, physiological, and molecular differences in response to dietary restriction in three inbred mouse strains. AB - Food restriction paradigms are widely used in animal studies to investigate systems involved in energy regulation. We have observed behavioral, physiological, and molecular differences in response to food restriction in three inbred mouse strains, C57BL/6J, A/J, and DBA/2J. These are the progenitors of chromosome substitution and recombinant inbred mouse strains used for mapping complex traits. DBA/2J and A/J mice increased their locomotor activity during food restriction, and both displayed a decrease in body temperature, but the decrease was significantly larger in DBA/2J compared with A/J mice. C57BL/6J mice did not increase their locomotor activity and displayed a large decrease in their body temperature. The large decline in body temperature during food restriction in DBA/2J and C57BL/6J strains was associated with a robust reduction in plasma leptin levels. DBA/2J mice showed a marked decrease in white and brown adipose tissue masses and an upregulation of the antithermogenic hypothalamic neuropeptide Y Y(1) receptor. In contrast, A/J mice showed a reduction in body temperature to a lesser extent that may be explained by downregulation of the thermogenic melanocortin 3 receptor and by behavioral thermoregulation as a consequence of their increased locomotor activity. These data indicate that genetic background is an important parameter in controlling an animal's adaptation strategy in response to food restriction. Therefore, mouse genetic mapping populations based on these progenitor lines are highly valuable for investigating mechanisms underlying strain-dependent differences in behavioral physiology that are seen during reduced food availability. PMID- 16670153 TI - Coimmunoprecipitation of FAT/CD36 and CPT I in skeletal muscle increases proportionally with fat oxidation after endurance exercise training. AB - Although the increase in fatty acid oxidation after endurance exercise training has been linked with improvements in insulin sensitivity and overall metabolic health, the mechanisms responsible for increasing fatty acid oxidation after exercise training are not completely understood. The primary aim of this study was to determine the effect of adding endurance exercise training to a weight loss program on fat oxidation and the colocalization of the fatty acid translocase FAT/CD36 with carnitine palmitoyltransferase I (CPT I) in human skeletal muscle. We measured postabsorptive fat oxidation and acquired a muscle sample from abdominally obese women before and after 12% body weight loss through either dietary intervention with endurance exercise training (EX + DIET) or dietary intervention without endurance exercise training (DIET). Immunoprecipitation techniques were used on these muscle samples to determine whether the association between FAT/CD36 and CPT I is altered after DIET and/or EX + DIET. FAT/CD36 was found to coimmunoprecipitate with CPT I, and the amount of FAT/CD36 that coimmunoprecipitated with CPT I increased by approximately 25% after EX + DIET (P < 0.005) but was unchanged after DIET. In addition, the increase in the amount of FAT/CD36 that coimmunoprecipitated with CPT I in EX + DIET was strongly correlated with the increase in whole body fat oxidation (R2 = 0.857, P < 0.003). In conclusion, the findings from this study indicate that exercise training alters the localization of FAT/CD36 and increases its association with CPT I, which may help augment fat oxidation. PMID- 16670154 TI - Carbohydrate ingestion does not alter skeletal muscle AMPK signaling during exercise in humans. AB - There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPKalpha2 during exercise in humans. Similarly, increasing glucose levels decreases AMPKalpha2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 +/ 1% V(O2 peak). In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8 fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPKalpha2 activity, AMPKalpha2 Thr(172) phosphorylation and acetyl-CoA Ser(222) phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo. PMID- 16670155 TI - Bone oedema predicts erosive progression on wrist MRI in early RA--a 2-yr observational MRI and NC scintigraphy study. AB - OBJECTIVES: To investigate if disease assessment by contrast-enhanced dynamic and static magnetic resonance imaging (MRI) and quantitative nanocolloid (NC) scintigraphy gives useful additional information in early rheumatoid arthritis (RA). METHODS: Twenty-seven patients with early RA (disease duration < or =12 months) were followed up for 1 yr and 24 of them for 2 yrs with contrast-enhanced MRI and NC scintigraphy of the wrist joint. Synovial inflammation was assessed by measuring time-dependent enhancement rates (E-rate) from dynamic MRI scans and technetium(99m)-labelled nanocolloid ((99m)Tc-NC) uptake from scintigraphy scans. Synovial membrane hypertrophy, bone oedema and erosions were semiquantitatively scored according to the Outcome Measures in Rheumatology Clinical Trials RA-MRI scoring system from static MR images. Response to the treatment was evaluated based on whether or not > or = 50% improvement was achieved in the tender and swollen joint scores and the Health Assessment Questionnaire score, with normal C reactive protein (CRP) or erythrocyte sedimentation rate (ESR) levels. Progression of the erosion score on wrist MRI was evaluated as the outcome. RESULTS: The baseline MRI bone oedema score (rho= 0.67), MRI synovitis score (rho= 0.57), ESR (rho= 0.56), CRP (rho= 0.48), E-rate (rho= 0.47) and (99m)Tc-NC uptake (rho= 0.45) were related with the change in the MRI erosion score from baseline to 2 yrs (rho= Spearman's correlation). In the multivariate logistic regression model, the bone marrow oedema score was the only baseline variable that predicted erosive progression at 2 yrs' follow-up (OR 4.2, 95% CI 1.3-13.8). The median (interquartile range) change in the erosion score from baseline to 2 yrs was 0 (0, 0) and 4 (2, 5) in the patients with (n= 9) and without (n= 15) a persistent clinical response over the 2 yrs, respectively (P= 0.001). The non responders who presented with erosive progression from 1 yr to 2 yrs had higher MRI synovitis scores, bone oedema scores, E-rate and (99m)Tc-NC uptake at 1-yr follow-up than the non-responders without progressive bone damage. CONCLUSION: The degree of local synovial inflammation at baseline, evaluated by dynamic and static MRI and quantitative NC scintigraphy, is closely related to the progression of wrist joint erosions during the first 2 yrs of the disease. Furthermore, at follow-up, if no persistent clinical response is achieved, these imaging methods may help to predict future erosiveness and help in clinical therapeutic decision making. PMID- 16670156 TI - Measuring functioning in patients with hand osteoarthritis--content comparison of questionnaires based on the International Classification of Functioning, Disability and Health (ICF). AB - OBJECTIVE: When selecting a questionnaire, researchers and clinicians need to know whether or not a questionnaire covers the relevant outcomes. The aim of this study was to analyse and compare the content of questionnaires that have been used to assess functioning in patients with hand osteoarthritis (OA) based on the International Classification of Functioning, Disability and Health (ICF). METHOD: Questionnaires were identified in a structured literature search. All concepts included in the items of the questionnaires were linked to the ICF categories according to the 10 established linking rules by two health professionals. The degree of agreement between the two health professionals was determined by means of kappa statistic. On the basis of the linking, the content of the instruments was compared. For each concept, it was examined whether functioning is measured on the level of activity or participation or both activity and participation. Indicators for content density, content diversity and the percentage of linked ICF categories addressing participation were calculated. RESULTS: Health Assessment Questionnaire, AUSCAN, Cochin scale, FIHOA, SACRAH and AIMS2-SF were analysed. The result of the kappa statistic for agreement between the two investigators was 0.74. 163 concepts were identified in the 113 items of all instruments, which were then linked to seven ICF categories of the component body functions, 45 categories of the component activities and participation and six categories of the component environmental factors. AUSCAN and SACRAH had the lowest and AIMS2-SF showed the highest diversity ratio and the highest percentage of linked ICF categories that addressed participation. CONCLUSION: When selecting instruments for comprehensive measurements of functioning in hand OA, researchers and clinicians are advised to include both one instrument with a low diversity ratio (for disease-specific aspects) and another instrument with a high diversity ratio (for broader aspects of functioning including some aspects of participation). PMID- 16670157 TI - A case of chondromatosis indicates a synovial stem cell aetiology. AB - OBJECTIVE: To evaluate cell cultures derived from intrasynovial nodules from a patient with primary synovial chondromatosis (PSC) for aberrant numbers/differentiation of osteochondroprogenitor cells. METHODS: Cell cultures were established from PSC synovial nodules, or normal bovine or human osteoarthritis (OA) synovia (for comparison). Multi-lineage potential was determined using well-characterized in vitro culture systems to assess osteogenic, chondrogenic and adipogenic capability. RESULTS: Primary PSC cell cultures were typically fibroblastic but contained islands of dense cell clusters/nodules, some of which were isolated and cultured separately [putative osteochondroprogenitris (pOCP) cultures]. OA synovial cultures had barely detectable levels of alkaline phosphatase (AP) that increased (0.006+/-0.008 to 0.141+/-0.000 nmol p-nitrophenol/min/cm(2)) with dexamethasone treatment. AP activity was higher in primary PSC cell cultures and further enhanced by dexamethasone (from 0.076+/-0.022 to 5.735+/-0.000 nmol p-nitrophenol/min/cm(2)). Histochemically, AP was localized as discreet areas within PSC cultures. No AP activity was detected histochemically in OA or normal bovine synovial cultures. The pOCP cultures had high basal AP (5.036+/-0.439 nmol p-nitrophenol/min/cm(2)) and spontaneously formed mineralized nodules, which increased in number under standard osteogenic conditions. Under chondrogenic conditions, micromass or pellet-cultured pOCP cells spontaneously synthesized a matrix containing glycosaminoglycans and collagen II. In adipogenic medium, the number of lipid containing cells was increased. CONCLUSIONS: Compared with cultures established from OA or normal synovia, cell cultures established from PSC synovial nodules were enriched in osteochondroprogenitors, which, unlike normal mesenchymal cells, differentiated along chondrogenic and osteogenic lineages in the absence of dexamethasone. PMID- 16670158 TI - Common genetic variants of the FADS1 FADS2 gene cluster and their reconstructed haplotypes are associated with the fatty acid composition in phospholipids. AB - Fatty acid composition in membranes plays an important role in cellular processes and has shown to be associated with the aetiology of several complex diseases in humans. We report strong associations between variants in the human delta-5 and delta-6 desaturase genes FADS1 FADS2 and fatty acid composition in serum phospholipids. Eighteen polymorphisms located in this gene cluster were genotyped in 727 adults from Erfurt, a German centre of the European Community Respiratory Health Survey. The cluster is located at chromosome 11q12-11q13.1, a region repeatedly found to be linked with atopy and other complex diseases. Polymorphisms and statistically reconstructed haplotypes of FADS1 and the upstream region of FADS2 showed strongest associations with the level of the direct precursor of inflammatory eicosanoids, the n-6 fatty acid arachidonic acid (C20:4n-6), also strong associations with levels of the n-6 fatty acids C18:2n-6, C18:3n-6, C20:2n-6, C20:3n-6, C22:4n-6 and of the n-3 fatty acids C18:3n-3, C20:5n-3 and C22:5n-3 (P-values < 1.0 x 10(-13)). Carriers of the rare alleles of several SNPs and their respective haplotypes had a lower prevalence of allergic rhinitis and atopic eczema. No association was found for total and specific IgE levels. PMID- 16670159 TI - Proliferative activity of human thyroid cells in various age groups and its correlation with the risk of thyroid cancer after radiation exposure. AB - CONTEXT: The thyroid gland is vulnerable to the carcinogenic effects of ionizing radiation, and there is a well-documented inverse correlation between thyroid cancer and age at exposure, particularly for ages less than 20 yr. One of the factors responsible for this phenomenon may be more rapid cell proliferation in children. OBJECTIVE: The objective of this study was to determine the proliferative rate of normal human thyroid cells in different age groups. DESIGN: We used immunohistochemical analysis to determine the Ki-67 proliferative index in 117 thyroid glands obtained at autopsy, including 25 fetal thyroids (11-40 wk gestation), 55 childhood thyroids (0-19 yr), and 37 adult thyroids (20-60 yr). RESULTS: The rate of Ki-67 labeling in the three groups was 7.4 +/- 6.10, 0.23 +/ 0.15, and 0.08 +/- 0.04% respectively, demonstrating an overall trend for diminishing proliferative activity of thyroid cells with increasing age. However, a lack of correlation was noted between the slopes of cancer risk calculated from previous studies of irradiated populations and proliferative rate in the pediatric age intervals of 0-4 and 5-9 yr, suggesting that other factors are likely to be responsible for the particularly high sensitivity to radiation induced thyroid cancer among the youngest children. CONCLUSIONS: Our findings of a general decrease in proliferative activity of thyroid cells with age may explain, at least in part, the higher risks of radiation-related thyroid cancer in children compared with adults. However, the variation in the rate of cell proliferation is unlikely to be responsible entirely for this phenomenon and other factors may also be involved. PMID- 16670160 TI - Insulin-like growth factor-I and growth in height, leg length, and trunk length between ages 5 and 10 years. AB - OBJECTIVE: IGF-I, a major regulator of childhood growth, is also associated with the risk of several cancers in adult life. Adult height and particularly leg length are also associated with cancer risk. Prepubertal growth is more in leg than trunk length, and it has been suggested that leg length might be a biomarker of childhood IGF-I. However, there is little information on the association between childhood IGF-I and subsequent leg and trunk growth. In this study, we investigated the association of IGF-I measured at 5 and 7-8 yr with growth in height and the components of height (leg and trunk length) from 5 yr to 9-10 yr. PARTICIPANTS: A total of 675 children participated in the Avon Longitudinal Study of Parents and Children. RESULTS: IGF-I was strongly positively associated with growth in height in both sexes. Among boys, IGF-I was strongly associated with subsequent growth in both leg and trunk length, but there was no evidence that IGF-I was more strongly associated with one component of growth than the other. Among girls, IGF-I was strongly positively associated with growth in trunk but not leg length, although there was only weak evidence that these two associations differed in strength (P = 0.058). CONCLUSIONS: These results support the contention that the associations between height and cancer may be mediated by variation in childhood IGF-I. However, they provide no evidence to support the hypothesis that leg length is a better biomarker of childhood IGF-I levels than trunk length. PMID- 16670161 TI - Small changes in thyroxine dosage do not produce measurable changes in hypothyroid symptoms, well-being, or quality of life: results of a double-blind, randomized clinical trial. AB - CONTEXT: In patients with primary hypothyroidism, anecdotal evidence suggests that well-being is optimized by fine adjustment of T(4) dosage, aiming for a serum TSH concentration in the lower reference range. This has not been tested in a clinical trial. OBJECTIVE: Our objective was to test whether adjustment of T(4) dosage aiming for a serum TSH concentration less than 2 mU/liter improves well being compared with a serum TSH concentration in the upper reference range. DESIGN: We conducted a double-blind, randomized clinical trial with a crossover design. PARTICIPANTS: Fifty-six subjects (52 females) with primary hypothyroidism taking T(4) (>/=100 microg/d) with baseline serum TSH 0.1-4.8 mU/liter participated. INTERVENTIONS: Each subject received three T(4) doses (low, middle, and high in 25-microg increments) in random order. OUTCOME MEASURES: Outcome measures included visual analog scales assessing well-being (the primary endpoint) and hypothyroid symptoms, quality of life instruments (General Health Questionnaire 28, Short Form 36, and Thyroid Symptom Questionnaire), cognitive function tests, and treatment preference. RESULTS: Mean (+/- sem) serum TSH concentrations were 2.8 +/- 0.4, 1.0 +/- 0.2, and 0.3 +/- 0.1 mU/liter for the three treatments. There were no significant treatment effects on any of the instruments assessing well-being, symptoms, quality of life, or cognitive function and no significant treatment preference. CONCLUSIONS: Small changes in T(4) dosage do not produce measurable changes in hypothyroid symptoms, well being, or quality of life, despite the expected changes in serum TSH and markers of thyroid hormone action. These data do not support the suggestion that the target TSH range for the treatment of primary hypothyroidism should differ from the general laboratory range. PMID- 16670162 TI - Comparison of confirmatory tests for the diagnosis of primary aldosteronism. AB - CONTEXT: Primary aldosteronism (PA) is the most frequent form of secondary hypertension, accounting for up to 5-10% of all hypertensive patients, and the diagnosis of PA can present an important challenge for the clinician. After a positive screening test, the diagnosis is confirmed by a suppression test, often an iv saline load test (SLT) or a fludrocortisone suppression test (FST). The FST is considered by many to be the most reliable but is more complex and expensive. OBJECTIVE AND DESIGN: Our objective was to compare the specificity of SLT with FST for the diagnosis of PA. PATIENTS AND SETTING: The study included 100 hypertensive patients referred to hypertension units with suspected PA after the screening test. INTERVENTION: All patients underwent FST and SLT. MAIN OUTCOME MEASURES: We assessed plasma aldosterone concentrations (PAC) before and after FST and SLT. RESULTS: After iv SLT, 10.4% of the PA patients were negative and 16.1% of patients with essential hypertension were positive after SLT; that is, a correct diagnosis with SLT was obtained in 88% of patients compared with FST. PAC after SLT and PAC after FST were highly correlated (P < 0.0001). Receiver operator characteristic curve analysis demonstrated that the best cutoff for PAC after SLT was 5 ng/dl. Patients with aldosterone-producing adenoma displayed a smaller reduction of PAC compared with patients with bilateral adrenal hyperplasia; a PAC after SLT greater than 6 ng/dl identified all patients eventually diagnosed as having aldosterone-producing adenoma. CONCLUSIONS: This study demonstrates that the iv SLT is a reasonably good alternative to the more expensive and complex FST for the diagnosis of PA after a positive screening test. PMID- 16670163 TI - Heritability of serum resistin and its genetic correlation with insulin resistance-related features in nondiabetic Caucasians. AB - CONTEXT: Serum levels of resistin are believed to modulate insulin resistance in humans. OBJECTIVE: The aim of this study was to investigate whether serum resistin levels are genetically controlled and whether this control is shared with other insulin resistance traits. DESIGN AND METHODS: The study cohort included 264 nondiabetic probands, Caucasian from Italy, and their 473 adult family members. Phenotypic characterization included anthropometric variables, blood pressure, fasting glucose and insulin, lipid profile, and resistin levels. Genotypes were determined at position g.-420C-->G (rs1862513), IVS2+181G-->A (rs3745367), and GAT((n)) polymorphisms of the resistin (RETN) gene. RESULTS: In the 264 unrelated probands, resistin levels were significantly (P < 0.01) correlated with adiposity, blood pressure, C-reactive protein, and the metabolic syndrome score. In a variance component analysis of the 264 probands and their 473 relatives, about 70% of the observed variation of serum resistin levels was heritable (P < 0.0001). A small, but significant (P = 0.004) proportion of this variance was explained by the G-->A variation at position IVS2+181 of the RETN gene. Significant genetic correlations (P < 0.05) were observed between resistin and body mass index (rho(g) = 0.30), waist circumference (rho(g) = 0.32), the insulin resistance index HOMA(IR) (rho(g) = 0.28), and the metabolic syndrome score (rho(g) = 0.35). CONCLUSIONS: These data indicate that serum resistin is highly heritable and has some common genetic background with traits related to insulin resistance, reinforcing the hypothesis that this adipokine may play a pathogenic role in insulin resistance-related abnormalities, including type 2 diabetes and cardiovascular disease. PMID- 16670164 TI - The relationship between libido and testosterone levels in aging men. AB - CONTEXT: Although it is known that serum testosterone (T) concentrations are related to libido, the strength of that relationship in community-dwelling men has not yet been determined. OBJECTIVE: Our objective was to assess the strength and significance of the association between aging men's self-reports of libido and serum T concentrations. DESIGN: Our study was a community-based evaluation of men's health and aging, including three data collection waves: baseline (T1, 1987 1989) and follow-ups (T2, 1995-1997; T3, 2002-2004). Libido was measured on a 14 point scale assessing self-reported frequency of desire and thoughts/fantasies; low libido was defined as a score of less than 7 of 14. SETTING: We conducted an epidemiological study in greater Boston, Massachusetts. PARTICIPANTS: There were 1632 men aged 40-70 yr at baseline, with follow-up on 922 (56%) at 9 yr (T2) and 623 (38%) at 15 yr (T3). MAIN OUTCOME MEASURES: We assessed total and calculated bioavailable T . RESULTS: Three hundred eighteen (19%) subjects reported low libido at baseline. Libido and T displayed a significant association. However, the difference in mean T levels between those subjects with low libido and those without was small; analyses indicated a 3.4 ng/dl (0.12 nmol/liter) increase in total T per unit increase in libido. Subjects reporting low libido exhibited an increased but modest probability of exhibiting low T. Dividing T concentrations by the number of androgen receptor gene cytosine, adenine, guanine repeats did not enhance associations. CONCLUSIONS: Libido and T concentrations are strongly related at the population level. However, the value of individual patient reports of reduced libido as indicators of low T levels is open to question. PMID- 16670165 TI - Comparison of the dexamethasone-suppressed corticotropin-releasing hormone test and low-dose dexamethasone suppression test in the diagnosis of Cushing's syndrome. AB - CONTEXT: The low-dose dexamethasone suppression test (LDDST) is widely used in confirming a diagnosis of Cushing's syndrome. CRH administration at the end of an LDDST has been reported to improve the diagnostic accuracy of this test. OBJECTIVE: Our objective was to assess whether CRH administration after a standard LDDST (LDDST-CRH test) improves diagnostic accuracy in Cushing's syndrome. DESIGN, SETTING, AND PARTICIPANTS: Thirty-six individuals with a clinical suspicion of Cushing's syndrome each completed a standard LDDST and an LDDST-CRH test at Hammersmith Hospitals NHS Trust, London. The LDDST involved administration of 0.5 mg oral dexamethasone given 6-hourly for 48 h. Serum cortisol was measured 6 h after the last dose of dexamethasone, with a value of 50 nmol/liter or below excluding Cushing's syndrome. Immediately after this, the LDDST-CRH test commenced with administration of a ninth dose of 0.5 mg dexamethasone. Exactly 2 h later, 100 mug human-sequence CRH was administered. Serum cortisol was measured 15 min after the CRH injection, with a value of less than 38 nmol/liter also excluding Cushing's syndrome. MAIN OUTCOME MEASURE: Diagnosis or exclusion of Cushing's syndrome was the main outcome measure. RESULTS: Twelve subjects were diagnosed with Cushing's syndrome (eight Cushing's disease and four primary adrenal). The sensitivity of the LDDST in diagnosing Cushing's syndrome was 100%, with a specificity of 88%. In contrast, although the sensitivity of the LDDST-CRH test was also 100%, specificity was reduced at 67%. These results give a positive predictive value of 80% for the LDDST and 60% for the LDDST-CRH test. CONCLUSION: This small study suggests that the addition of CRH to the LDDST does not improve the diagnostic accuracy of the standard LDDST in Cushing's syndrome. PMID- 16670166 TI - Thyroxine-triiodothyronine combination therapy versus thyroxine monotherapy for clinical hypothyroidism: meta-analysis of randomized controlled trials. AB - CONTEXT: In some patients symptoms of hypothyroidism persist despite therapy with T(4). OBJECTIVE: The objective of the study was to compare the effectiveness of T(4)-T(3) combination vs. T(4) monotherapy for the treatment of clinical hypothyroidism in adults. DATA SOURCES: PubMed, EMBASE, LILACS, and the Cochrane Central Register of Controlled Trials (CENTRAL) databases were searched in September 2005. References of all included trials were scanned for additional studies. We put no restrictions on language, year of publication, or publication status. STUDY SELECTION: All randomized trials that compared the effectiveness of T(4)-T(3) combination vs. T(4) monotherapy for the treatment of clinical hypothyroidism in adults were included. DATA EXTRACTION: The data were extracted by two independent reviewers. DATA SYNTHESIS: We included 11 studies, in which 1216 patients were randomized. No difference was found in the effectiveness of combination vs. monotherapy in any of the following symptoms: bodily pain [standardized mean difference (SMD) 0.00, 95% confidence interval (CI) -0.34, 0.35], depression (SMD 0.07, 95% CI -0.20, 0.34), anxiety (SMD 0.00, 95% CI 0.12, 0.11), fatigue (SMD -0.12, 95% CI -0.33, 0.09), quality of life (SMD 0.03, 95% CI -0.09, 0.15), body weight, total serum cholesterol, triglyceride levels, low-density lipoprotein, and high-density lipoprotein. Adverse events did not differ between regimens. CONCLUSIONS: T(4) monotherapy should remain the treatment of choice for clinical hypothyroidism. PMID- 16670167 TI - Analyzing the functional and structural consequences of two point mutations (P94L and A368D) in the CYP11B1 gene causing congenital adrenal hyperplasia resulting from 11-hydroxylase deficiency. AB - CONTEXT: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause. OBJECTIVE: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function. RESULTS: The in vitro expression studies in COS 7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11 hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo. CONCLUSION: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods. PMID- 16670168 TI - Vitamin D metabolism in peripheral blood mononuclear cells is influenced by chewing "betel nut" (Areca catechu) and vitamin D status. AB - CONTEXT: Vitamin D deficiency, common in South Asians, is a risk factor for metabolic syndrome, type 2 diabetes, and ischemic heart disease. Vitamin D receptor (VDR) activation depends on activated vitamin D [1,25-dihydroxyvitamin D (1,25(OH)(2)D)] concentration, reflecting opposing actions of 25-hydroxyvitamin D 1alpha-hydroxylase [1-alpha(OH)ase] for formation and 25(OH)D-24-hydroxylase [24(OH)ase] for catabolism. We previously reported that circulating 1,25(OH)(2)D contributed to determination of VDR-protein levels and VDR genotype was a determinant of both VDR mRNA and VDR-protein in South Asians. OBJECTIVE: We hypothesized that chewing betel nut, an addictive habit common throughout South Asian communities, contributes to hypovitaminosis-D by modulating the enzymes regulating circulating 1,25(OH)(2)D concentration. DESIGN: Peripheral blood mononuclear cell (PBMC) 1-alpha(OH)ase and 24(OH)ase mRNA concentrations were measured and examined in relation to cross-sectional data on the vitamin-D axis, diet, smoking, and betel usage, including PBMC VDR-RNA and VDR-protein content in a pilot study of 33 healthy British Bangladeshis. RESULTS: PBMC 24(OH)ase mRNA correlated positively and serum 1,25(OH)(2)D negatively with betel quids per day (r = 0.49, P = 0.006 and r = -0.486, P = 0.006, respectively). Independent determinants for 24(OH)ase included betel quids per day (P < 0.0001) and serum 25 OHD (P = 0.024). Independent determinants for serum 1,25(OH)(2)D were gender, smoking, and betel quids per day. PBMC 1-alpha(OH)ase mRNA correlated inversely with VDR mRNA (r = -0.44; P = 0.013); its independent determinants were serum 1,25(OH)(2)D and VDR TaqI and BsmI polymorphisms (P = 0.03-0.0001). CONCLUSIONS: Betel chewing is a more powerful independent determinant of increased 24(OH)ase expression and of decreased serum calcitriol than serum 25-OHD, supporting the hypothesis that this habit could aggravate the effects of vitamin-D deficiency. PMID- 16670169 TI - Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. AB - CONTEXT: Adrenocortical carcinomas (ACC) are rare tumors with a poor prognosis. Few reports concerning large and homogeneous series are available. OBJECTIVE: We aimed to determine the clinical characteristics and outcome of ACC and to identify prognostic factors. DESIGN AND SETTING: This study is a descriptive and multivariate analysis of a cohort from a single endocrinology center. PATIENTS: A total of 202 consecutive patients with ACC were studied. RESULTS: The sex ratio (female to male) was 2.7. Mean age at diagnosis was 44 +/- 16 yr (range, 11-88 yr). We found that 154 patients (76%) had hypersecreting tumors [mostly cortisol and androgens (47%), cortisol alone (27%), or androgens alone (6%)] and 43 patients (21%) had metastases at diagnosis. At initial staging or during follow up, 85 patients (42%) had liver metastases, 79 patients (39%) had lung metastases, and 20 patients had bone metastases (10%). The survival rate was 37% at 5 yr. Multivariate analysis identified the following independent prognostic factors associated with shorter survival: older age at diagnosis [hazard ratio (HR), 1.03; P < 0.0001], initial MacFarlane extension stages 3 (HR, 4.42; P = 0.005) and 4 (HR, 7.93; P < 0.0001), and cortisol hypersecretion (HR, 3.90; P < 0.0001). Treatment with 1,1-dichlorodiphenildichloroethane (o,p'DDD) in the 3 months after surgery increased the survival rate of patients with cortisol secreting tumors (HR, 0.40; P = 0.04). CONCLUSION: This study highlights the better prognosis of ACC diagnosed at a noninvasive local stage, the particularly poor prognosis of patients with cortisol-secreting tumors, and the beneficial effect of o,p'DDD therapy in this subgroup of patients. PMID- 16670170 TI - Bronchiolitis obliterans following lung transplantation: early detection using computed tomographic scanning. AB - BACKGROUND: Computed tomographic (CT) scanning may enable earlier diagnosis of chronic lung allograft dysfunction than forced expiratory volume in 1 second (FEV1). A study was undertaken to determine intra-observer and inter-observer agreement of composite and air trapping CT scores, to examine the association of FEV1 with the composite and air trapping CT score, and to relate the baseline composite CT score to changes in FEV1 and changes in the composite CT score over 1 year. METHODS: Lung function and baseline CT scans following transplantation and at subsequent annual follow ups were analysed in 38 lung transplant recipients. Scans were randomly scored by two observers for bronchiectasis, mucus plugging, airway wall thickening, consolidation, mosaic pattern, and air trapping, and re-scored after 1 month. CT scores were expressed on a scale of 0 100 and correlated with FEV1 as a percentage of the post-transplant baseline value. RESULTS: The mean (SD) interval between baseline and follow up CT scans was 11.2 (4.7) months. Inter-observer and intra-observer agreement was good for both the composite and air trapping CT scores. There was a significant association between FEV1 and the composite CT score, with each unit of worsening in the baseline composite CT score predicting a 1.55% and 1.37% worsening in FEV1 over the following year (p<0.0001) and a 1.25 and 1.12 unit worsening in the composite CT score (p<0.0001) for observers 1 and 2, respectively. CONCLUSION: These findings indicate a potential role for a composite CT scoring system in the early detection of bronchiolitis obliterans. PMID- 16670172 TI - FEV1 decline in occupational asthma. AB - BACKGROUND: In occupational asthma continued workplace exposure to the causative agent is associated with a poor prognosis. However, there is little information available on how rapidly lung function declines in those who continue to be exposed, nor how removal from exposure affects lung function. METHODS: Forced expiratory volume in 1 second (FEV1) was studied in 156 consecutive subjects with occupational asthma (87% due to low molecular weight agents) using simple regression analyses to provide estimates of the decline in FEV1 before and after removal from exposure. RESULTS: In 90 subjects in whom FEV1 measurements had been performed for at least 1 year before removal from exposure (median 2.9 years), the mean (SE) rate of decline in FEV1 was 100.9 (17.7) ml/year. One year after removal from exposure FEV1 had improved by a mean (SE) of 12.3 (31.6) ml. The mean (SE) decline in FEV1 was 26.6 (18.0) ml/year in 86 subjects in whom measurements were made for at least 1 year (median 2.6 years) following removal from exposure. The decline in FEV1 was not significantly worse in current smokers than in never smokers, nor was it affected by the use of inhaled corticosteroids. CONCLUSION: FEV1 declines rapidly in exposed workers with occupational asthma. Following removal from exposure, FEV1 continued to decline but at a slower rate, similar to the rate of decline in healthy adults. PMID- 16670171 TI - Utility of signs and symptoms of chronic cough in predicting specific cause in children. AB - BACKGROUND: Paediatricians rely on cough descriptors to direct them to the level of investigations needed for a child presenting with chronic cough, yet there is a lack of published data to support this approach. A study was undertaken to evaluate (1) whether historical cough pointers can predict which children have a specific cause for their cough and (2) the usefulness of chest radiography and spirometry as standard investigations in children with chronic cough. METHODS: This was a prospective cohort study of children referred to a tertiary hospital with a cough lasting >3 weeks between June 2002 and July 2004. All included children completed a detailed history and examination using a standardised data collection sheet and followed a pathway of investigation until a diagnosis was made. RESULTS: In 100 consecutively recruited children of median age 2.8 years, the best predictor of specific cough observed was a moist cough at the time of consultation with an odds ratio (OR) of 9.34 (95% CI 3.49 to 25.03). Chest examination or chest radiographic abnormalities were also predictive with OR 3.60 (95% CI 1.31 to 9.90) and 3.16 (95% CI 1.32 to 7.62), respectively. The most significant historical pointer for predicting a specific cause of the cough was a parental history of moist cough (sensitivity 96%, specificity 26%, positive predictive value 74%). CONCLUSIONS: The most useful clinical marker in predicting specific cough is the presence of a daily moist cough. Both chest examination and chest radiographic abnormalities are also useful in predicting whether children have a specific cause of their cough. PMID- 16670174 TI - Cough frequency in children with mild asthma correlates with sputum neutrophil count. AB - BACKGROUND: A study was undertaken to measure cough frequency in children with stable asthma using a validated monitoring device, and to assess the correlation between cough frequency and the degree and type of airway inflammation. METHODS: Thirty six children of median age 11.5 years (interquartile range (IQR) 9-14) with stable asthma were recruited. They underwent spirometric testing, exhaled nitric oxide (eNO) measurement, sputum induction for differential cell count, and ambulatory cough monitoring for 24 hours. Coughing episodes were counted both as individual spikes and as clusters. RESULTS: All children had mild intermittent asthma and their median forced expiratory volume in 1 second and eNO were 83.3% (IQR 81.1-97.6) and 56.1 ppb (IQR 37.4-105), respectively. The median number of cough episodes per day was 25.5 (IQR 16-42.8). Sputum induction was successful in 69% of the subjects and cough frequency was found to have a significant positive correlation with sputum neutrophil count (r = 0.833, p = 0.0001). CONCLUSIONS: Children with stable mild asthma have increased cough frequency that might be driven by a neutrophilic inflammatory pathway. PMID- 16670173 TI - Bronchial hyperresponsiveness and the development of asthma and COPD in asymptomatic individuals: SAPALDIA cohort study. AB - BACKGROUND: Bronchial hyperresponsiveness (BHR) is a common feature of asthma. However, BHR is also present in asymptomatic individuals and its clinical and prognostic significance is unclear. We hypothesised that BHR might play a role in the development of chronic obstructive pulmonary disease (COPD) as well as asthma. METHODS: In 1991 respiratory symptoms and BHR to methacholine were evaluated in 7126 of the 9651 participants in the SAPALDIA cohort study. Eleven years later 5825 of these participants were re-evaluated, of whom 4852 performed spirometric tests. COPD was defined as an FEV1/FVC ratio of <0.70. RESULTS: In 1991 17% of participants had BHR, of whom 51% were asymptomatic. Eleven years later the prevalence of asthma, wheeze, and shortness of breath in formerly asymptomatic subjects with or without BHR was, respectively, 5.7% v 2.0%, 8.3% v 3.4%, and 19.1% v 11.9% (all p<0.001). Similar differences were observed for chronic cough (5.9% v 2.3%; p = 0.002) and COPD (37.9% v 14.3%; p<0.001). BHR conferred an adjusted odds ratio (OR) of 2.9 (95% CI 1.8 to 4.5) for wheezing at follow up among asymptomatic participants. The adjusted OR for COPD was 4.5 (95% CI 3.3 to 6.0). Silent BHR was associated with a significantly accelerated decline in FEV1 by 12 (5-18), 11 (5-16), and 4 (2-8) ml/year in current smokers, former smokers and never smokers, respectively, at SAPALDIA 2. CONCLUSIONS: BHR is a risk factor for an accelerated decline in FEV1 and the development of asthma and COPD, irrespective of atopic status. Current smokers with BHR have a particularly high loss of FEV1. PMID- 16670175 TI - Differential involvement of hypothalamic vasopressin neurons in multiple system atrophy. AB - We sought to determine whether there is differential involvement of different groups of hypothalamic arginine-vasopressin (AVP) synthesizing neurons in multiple system atrophy (MSA). Hypothalamus was obtained from five subjects with clinical diagnosis of MSA confirmed neuropathologically and five age-matched controls. Sections were immunostained for AVP, and cells with visible nuclei were counted in the posterior portion of the paraventricular nucleus (PVNp), supraoptic nucleus (SON), magnocellular PVN and suprachiasmatic nucleus (SCN). Sections of the hypothalamus and medulla were also immunostained for tyrosine hydroxylase (TH). There was a significant loss of AVP neurons in the PVNp in MSA compared with controls (17 +/- 3 versus 59 +/- 10 cells/section, P < 0.01). There was preservation of AVP- and TH-immunoreactive neurons in the SON and magnocellular PVN in all MSA cases. In contrast, there was marked depletion of TH immunoreactive fibres innervating these magnocellular AVP neurons, coincident with a loss of neurons in the A1 area (6 +/- 1 versus 13 +/- 1 cells/section, P < 0.01). There was loss of AVP neurons in the SCN in MSA compared with control cases (14 +/- 3 versus 71 +/- 16 cells/section, P < 0.02). Our results indicate that, in MSA, loss of AVP neurons in the PVNp may contribute to sympathetic failure, whereas loss of catecholaminergic input from the brainstem to the magnocellular AVP neurons may contribute to impaired AVP secretion in response to orthostatic stress. Loss of AVP neurons in the SCN may contribute to impaired circadian regulation of endocrine and autonomic functions. PMID- 16670176 TI - Histological growth patterns and genotype in oligodendroglial tumours: correlation with MRI features. AB - Oligodendroglial neoplasms with the -1p/-19q genotype are more indolent with longer survival and increased therapeutic responsiveness than those with intact 1p/19q, but the biological basis for these clinical differences is unclear. Recent research suggests that oligodendrogliomas with and without the -1p/-19q genotype may be distinguished by their magnetic resonance imaging (MRI) appearance, suggesting possible differences in growth characteristics. This study examined the relationship between genotype and histological growth patterns of oligodendroglial neoplasms in association with MR imaging characteristics. Tumour imaging features assessed on MRI included sharp-versus-indistinct border, smooth versus-irregular contour, homogeneous-versus-heterogeneous signal, contrast enhancement and paramagnetic susceptibility effect. Growth patterns (solid : mixed : infiltrative), tumour-margin transitions in cellularity and calcification were determined histopathologically. Allelic imbalance in chromosomes 1p36 and 19q13 was determined. Thirty-three oligodendrogliomas (25 with 1p/19q loss) and 53 oligoastrocytomas (18 with 1p/19q loss) were investigated. Solid, mixed or infiltrative growth patterns were seen in grade II and grade III tumours with or without 1p/19q loss, but infiltrative growth was more common in tumours with intact 1p/19q (chi2: P = 0.029). Grade III tumours were more likely to have a solid growth pattern (chi2: P = 0.046) associated with contrast enhancement (chi2: P = 0.011). Transition in cellularity at the radiological margin did not differ according to genotype. All cases with T1 or T2 signal homogeneity had intact 1p/19q. Tumours with sharp/smooth borders were more likely to have intact 1p/19q than those with indistinct/irregular borders (chi2: P < 0.001), but this was not related to histological growth characteristics. This study identified a group of oligodendroglial tumours with intact 1p/19q displaying distinctive MR imaging features that were unrelated to the histopathology characteristics. PMID- 16670177 TI - Cathepsin D deficiency underlies congenital human neuronal ceroid-lipofuscinosis. AB - Congenital neuronal ceroid-lipofuscinosis (NCL) is a devastating inherited neurodegenerative disorder of unknown metabolic basis. Eight patients with this rare disorder, all with similar clinical and neuropathological findings, have been reported, and here we describe two further patients. Previously, we showed that a mutation in the cathepsin D gene causes congenital NCL in sheep. On the basis of the neuropathological and ultrastructural similarities between the sheep and patients affected with congenital NCL, we screened the cathepsin D gene for mutations in a patient of Pakistani origin. We identified a nucleotide duplication, c.764dupA, in the cathepsin D gene in homozygous form in the patient, and in heterozygous form in his father. This duplication is likely to be disease-causing, as it creates a premature stop codon, predicting a truncation of the protein. When transiently expressed in cell cultures, the mutant protein was enzymatically inactive, but stable. In paraffin-embedded brain tissue samples of two affected siblings of the Pakistani patient, cathepsin D was absent, suggesting rapid degradation of the c.764dupA mutant cathepsin D at mRNA or protein level in vivo. Further, we were able to confirm lack of cathepsin D in the brain tissue of yet another, unrelated, patient of English origin with congenital NCL. On the basis of the present data, and the nearly identical clinical and/or pathological phenotype of the other reported cases of congenital NCL, it is reasonable to suggest that cathepsin D deficiency caused by mutations in the corresponding gene may underlie all cases of congenital NCL. The present observations also suggest that cathepsin D deficiency should be considered as a possible diagnosis in microcephalic neonates, who present with seizures at or before birth. PMID- 16670178 TI - What does transient global amnesia really mean? Review of the literature and thorough study of 142 cases. AB - Since the first reports of transient global amnesia (TGA) were published in 1956, several neuropsychological and functional imaging studies have shed light on different aspects of this neurological syndrome. By establishing diagnostic criteria, Hodges and Warlow (1990b) have made it far easier to identify clinical TGA-related features. However, no comprehensive survey has been yet carried out in order to validate their criteria/findings or provide information about previously unknown features. In the present paper, (i) we review the literature published since Hodges and Warlow's study and seek to characterize the demographic and clinical features of TGA more accurately, (ii) we report 142 personal TGA cases, with supplementary information regarding both episodes and patients, such as precipitating events, associated symptoms and personality, and (iii) we suggest the existence of different groups of TGA patients, on the basis of a hierarchical cluster analysis. This revealed that in women, episodes are mainly associated with an emotional precipitating event, a history of anxiety and a pathological personality. In men, they occur more frequently after a physical precipitating event. In younger patients, a history of headaches may constitute an important risk factor. No link was found with vascular risk factors. The relevance of each of the above-mentioned variables is discussed in the light of our classification. An extensive description of cases from both the literature and our patient population allows us to refine the characterization of clinical TGA features. PMID- 16670179 TI - The first ALS2 missense mutation associated with JPLS reveals new aspects of alsin biological function. AB - Primary lateral sclerosis (PLS) is a rare progressive paralytic disorder that results from dysfunction of the upper motoneurons. Although PLS is a sporadic disorder of adult middle age, it has also been described in children as juvenile PLS or JPLS. The causative gene for JPLS was found to be ALS2, which is also responsible for a recessive form of amyotrophic lateral sclerosis, for infantile onset ascending hereditary spastic paralysis (IAHSP) and for a form of complicated hereditary spastic paraplegia (cHSP). ALS2 gene encodes a protein termed alsin, containing multiple guanine nucleotide exchange factor domains, specifically binding to small GTPase Rab5 and acting as a GEF for Rab5. In vitrostudies performed with full-length and truncating forms of alsin protein support its role in endosomal dynamics and trafficking of mitochondria. All ALS2 mutations so far reported generate alsin protein truncation. Here, we describe the first homozygous missense mutation in ALS2, p.G540E. The mutation, which falls within the RCC1 domain, was identified in a 34-year-old patient with typical signs of JPLS such as ascending generalized and severe spasticity involving the limbs and the bulbar region, dysphagia, limb atrophy, preserved cognition and sensation. The father and two proband's sisters were found to be heterozygous carriers of the mutation with no signs of the disease. Studies in the neuronal cell line SK-N-BE indicated that the known subcellular localization of wild-type alsin with the early endosome antigen 1, in enlarged endosomal structures, and transferrin receptor is completely lost by the mutant protein, thus indicating that this mutation leads to protein delocalization. Mutant alsin induced neuronal death itself and also significantly enhanced the apoptogenic effect of NMDA and staurosporine. This effect was associated with decreased Bcl xL : Bax ratio. In contrast, wild-type alsin was neuroprotective and increased Bcl-xL : Bax ratio. Our results provide the first demonstration that a missense mutation in alsin is cytotoxic. In addition, the identification of Bcl-xL/Bax as target of protection by alsin and of cytotoxicity by the mutant form provides a new signalling event regulated by alsin protein that may be important to define its role in neuronal physiology and neurodegeneration. Finally, the phenotype genotype correlation in our patient, in view of all other ALS2 mutant cases reported previously, suggests a functional interplay of long and short forms of alsin in relation to disease onset and progression. PMID- 16670180 TI - Differences between Alzheimer's disease and dementia with Lewy bodies: an fMRI study of task-related brain activity. AB - We investigated whether previously reported differences between Alzheimer's disease and dementia with Lewy bodies (DLB) in resting occipital activity lead to activation differences within functionally specialized visual cortical areas and deactivation differences in the default network. Patients with Alzheimer's disease (n = 10; 5 male), DLB (n = 9; 4 male) and controls (n = 13; 5 male) performed three functional MRI (fMRI) scanning experiments involving visual colour, face or motion stimuli. Reaction time or accuracy in DLB and Alzheimer's disease differed significantly from controls but not between patient groups, with the exception of accuracy in the face task (DLB < Alzheimer's disease; P = 0.038). The most significant fMRI activations in the pooled data set were in left V4alpha for the colour task (Talairach coordinate: -30, -52, -24; P = 0.002 corrected), the right fusiform face area (FFA) for the face task (34, -48, -22; P = 0.005 corrected) and right intra-parietal sulcus (30, -66, 42; P = 0.003 corrected) for the motion task, with additional activity in right V5 (48, -64, 0; P = 0.015 corrected). Each task was associated with decreases in activity within the default network with prominent deactivation foci bilaterally in the posterior cingulate gyrus (+/-8, -48, 26; left P < 0.001; right P < 0.001 corrected) and medial frontal cortex (+/-18, 42, 32; left P < 0.001; right P < 0.001 corrected). Comparing patterns of task-related activity across groups, DLB patients showed more activation than Alzheimer patients within the superior temporal sulcus (STS) for the motion task (right STS: 44, 0, -20; P = 0.004 corrected; left STS: -40, 4, -26; P = 0.07 corrected). This difference could not be attributed to task performance, cognitive score or age [analysis of covariance (ANCOVA)F (2, 18) = 8.44, P = 0.003]. Within regions of interest, group activation differences were found for the face task (Alzheimer's disease > DLB P = 0.05; Alzheimer's disease > controls P = 0.14) and the motion task (DLB < Alzheimer's disease P = 0.031 and DLB < control P = 0.048). However, these differences could be explained by behavioural performance, failing to reach significance in the ANCOVA analysis. In the default network, group deactivation differences between controls and both patient groups were found for the colour and motion task (colour: control < Alzheimer's disease P = 0.02; control < DLB P = 0.019; motion: control < Alzheimer's disease P = 0.118; control < DLB P = 0.118) but could be accounted for by behavioural performance. The results suggest that cognitive fMRI can be used to detect both performance-dependent and performance-independent differences between Alzheimer's disease and DLB, reflecting the distribution of functional pathology in the two conditions. PMID- 16670181 TI - Gender differences in the association between religious involvement and depression: the Cache County (Utah) study. AB - We examined the relation between religious involvement, membership in the Church of Jesus Christ of Latter-Day Saints, and major depression in a population-based study of aging and dementia in Cache County, Utah. Participants included 4,468 nondemented individuals between the ages of 65 and 100 years who were interviewed in person. In logistic regression models adjusting for demographic and health variables, frequent church attendance was associated with a reduced prevalence of depression in women but increased prevalence in men. Social role loss and the potential impact of organizational power differential by sex are discussed. Though causality cannot be determined here, these findings suggest that the association between religious involvement and depression may differ substantially between men and women. PMID- 16670182 TI - Sex differences after all those years? Heritability of cognitive abilities in old age. AB - We investigated sex differences in genetic and environmental effects on cognitive abilities among older adult twins. We drew participants from the Swedish Twin Registry; our sample included 647 twin pairs. Our cognitive measures included Synonyms, Block Design, Digit Span, Thurstone's Picture Memory, Symbol Digit, and general cognitive ability tests. Higher age was related to lower performance in all cognitive measures, except synonyms. For digit span forward, symbol digit, and general cognitive ability tasks, there was a Sex x Age interaction, with greater deficits in the performance of women compared with those of men at higher ages. We found no sex-specific genetic influences. In other words, the same genetic effects were operating for men and women. Furthermore, the magnitude of genetic effect was similar for men and women. PMID- 16670183 TI - Short-term variability in cognitive performance and the calibration of longitudinal change. AB - Recent studies have documented that normal adults exhibit considerable variability in cognitive performance from one occasion to another. We investigated this phenomenon in a study in which 143 adults ranging from 18 to 97 years of age performed different versions of 13 cognitive tests in three separate sessions. Substantial within-person variability was apparent across 13 different cognitive variables, and there were also large individual differences in the magnitude of within-person variability. Because people differ in the amount of short-term variability, we propose that this variability might provide a meaningful basis for calibrating change in longitudinal research. Correlations among the measures of within-person variability were very low, even after we adjusted for reliability, and there was little evidence that increased age was associated with a larger amount of within-person variability. PMID- 16670184 TI - Ambivalent reactions in the parent and offspring relationship. AB - Theory suggests that aging parents and their adult children experience ambivalence (conflicting emotions) as a result of unclear norms governing the tie between them. This study investigated personality differences and relationship context differences in ambivalence, as well as the reactions of parents and offspring to each other. As part of the Adult Family Study, 474 individuals from 158 family triads consisting of a mother, father, and son or daughter aged 22 to 49 years completed telephone interviews, in-person interviews, and questionnaires. Multilevel models revealed that poor parental health and neuroticism in parents and offspring were associated with greater ambivalence. Surprisingly, investment in competing roles was associated with less ambivalence. Parents also experienced greater ambivalence when offspring scored higher on neuroticism, rated the parent as less important, or were less invested in their own spousal role. Parents' characteristics were not associated with offspring's ambivalence. Parents appear to react to their children's personality and achievements even after children are grown. PMID- 16670185 TI - Influence of personality and health behaviors on fatigue in late and very late life. AB - There is limited understanding about variables associated with fatigue in late and very late life. The purpose of this study was to explore the longitudinal influences of personality and health behaviors on changes in fatigue. One hundred and seventy-eight participants of the Georgia Centenarian Study were part of this study. Multiple regression analyses were computed to assess the influence of personality, physical activity, stress management, and nutritional risk on fatigue after initial fatigue, self-reported health, and psychosocial variables were controlled for. Anxiety, physical activity, and nutritional risk had significant longitudinal effects on fatigue. Older adults who were anxious, less engaged in physical activity, and who had higher levels of nutritional risk were more likely to show increased fatigue levels. The results suggest that more clinical and research attention should be paid to changes of fatigue in older populations. PMID- 16670186 TI - Verbal learning and everyday functioning in dementia: an application of latent variable growth curve modeling. AB - This study used latent variable growth curve modeling to identify predictors and correlates of verbal learning over trials on a list-learning task in patients with dementia. Data from 116 patients evaluated at the Detroit satellite of the Michigan Alzheimer's Disease Research Center were incorporated in the present analyses. Patients were administered the Fuld Object Memory Evaluation, examined independently by a geriatrician, and, if appropriate, given a diagnosis of probable Alzheimer's disease according to criteria from the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association. The presence of dementia significantly predicted both the intercept (i.e., level of performance) and the slope (i.e., learning over trials), with dementia patients demonstrating lower overall levels of performance and less verbal learning over trials. Rate of verbal learning over trials was a significant predictor of everyday functioning (instrumental activities of daily living) above and beyond general cognitive impairment and demographics. PMID- 16670188 TI - Specific effects of aging on proper name retrieval: now you see them, now you don't. AB - Existing research is equivocal as to whether aging negatively affects the retrieval of proper names more than other types of words. To clarify previous results, this study had young and older participants name and provide specific biographical information in response to pictures of familiar celebrities; it used two different measures of recall errors. An analysis of tip-of-the-tongue (TOT) responses indicated specific age-related increases in TOTs for names compared with words representing biographical information. An analysis including all other (non-TOT) recall errors did not indicate age-related deficits in name retrieval. These findings highlight the importance of the measures adopted in forming conclusions about age effects on the retrieval of names. PMID- 16670187 TI - Social cognition in nursing home residents with and without cognitive impairment. AB - Dementia affects the specific cognitive abilities underlying social functioning in ways that are just beginning to be understood. This pilot study compared the performances of 15 nursing home residents with cognitive impairment and 25 without cognitive impairment on a broad range of measures of social-cognitive functioning. The cognitively impaired group scored significantly lower than the unimpaired group on tests of face processing, person perception, and social reasoning but not on tests of affect recognition and the representation of social situations. Individuals' deficits in working memory and executive function did not fully account for their impaired performance on tests of social cognition. An improved understanding and assessment of the basic cognitive mechanisms underlying the ability of persons with dementia to interact with others would inform the design of interventions to optimize their social functioning. PMID- 16670189 TI - Cosmic transcendence and framework of meaning in life: patterns among older adults in the Netherlands. AB - OBJECTIVES: Gerotranscendence has been conceptualized as a potential development accompanying normal aging. Gerotranscendence is defined as a shift in metaperspective from a materialistic and pragmatic world view to a more cosmic and transcendent one. In the past decade, population-based studies have tested Tornstam's Gerotranscendence Scale. Its Cosmic Transcendence subscale, in particular, emerged as consistent. The aim of the present study was to examine (a) how cosmic transcendence relates to having a framework of meaning in life and (b) whether religiousness and demographic characteristics influence possible relationships. METHODS: Participants were 928 older Dutch adults who responded to a questionnaire that included the Cosmic Transcendence scale, aspects of religiousness, and the Framework of Meaning in Life subscale of the Life Regard Index. RESULTS: A substantial, positive association between cosmic transcendence and framework of meaning in life was observed. This association was much more pronounced among participants who were less involved in religion, who were women, who were age 75 or older, or who were widowed. DISCUSSION: The current study indicates that the personal relevance of cosmic transcendence depends on cultural factors such as secularization. Furthermore, cosmic transcendence seems to unfold as an important domain in the life view of women, the older old, and the widowed. PMID- 16670190 TI - Mind frames towards dying and factors motivating their adoption by terminally ill elders. AB - OBJECTIVE: This study was designed to advance the understanding of the physical and psychosocial factors that motivate terminally ill elders not only to consider a hastened death but also not to consider such a death. METHODS: I conducted face to-face in-depth qualitative interviews with 96 terminally ill elders. An inductive approach was taken to locating themes and patterns regarding factors motivating terminally ill elders to consider or not to consider hastening death. RESULT: Six mind frames towards dying emerged: (a) neither ready nor accepting; (b) not ready but accepting; (c) ready and accepting; (d) ready, accepting, and wishing death would come; (e) considering a hastened death but having no specific plan; and (f) considering a hastened death with a specific plan. From the data emerged approaches towards dying and accompanying emotions characterizing each mind frame, as well as factors motivating their adoption by elders. The results showed that psychosocial factors served more often than physical factors as motivators. DISCUSSION: The results demonstrate the importance of assessing the mind frame adopted by a terminally ill elder and his or her level of satisfaction with it. Terminally ill elders may experience a higher quality dying process when a traditional medical care approach is replaced by a holistic approach that addresses physical, spiritual, emotional, and social needs. PMID- 16670191 TI - Church-based social support and mortality. AB - OBJECTIVES: The purpose of this study was to see if support provided and received from fellow church members reduced the deleterious effects of financial strain on mortality in late life. METHODS: Interviews were conducted with a nationwide sample of 1,500 older adults in 2001 and 2004. Participants were asked in 2001 about financial strain, church-based social support, and a range of private and public religious practices. Mortality status was determined at the follow-up interview in 2004. RESULTS: The findings indicated that providing social support to fellow church members reduced the effects of support providers' own financial problems on mortality. In contrast, the data suggested that receiving support from people at church did not have the same stress-buffering effect. DISCUSSION: Finding ways to help older adults become more involved in providing support to others at church may form the basis for developing interventions aimed at improving their quality of life. PMID- 16670192 TI - Perceived social support and mortality in older people. AB - OBJECTIVES: This study examines the effect of perceived social support on all cause mortality at a 10-year follow-up as well as the plausible mediating factors in this association. METHODS: We measured perceived social support in 206 Finnish men and women aged 80 years old by using the Social Provision Scale, which consists of six dimensions: attachment, social integration, opportunity for nurturance, reassurance of worth, reliable alliance, and guidance. RESULTS: By using a theoretical framework that divided perceived social support into assistance-related and non-assistance-related support, we found that the risk of death was almost 2.5 times higher in women in the lowest tertile of non assistance-related social support (comprising infrequent experiences of reassurance of worth, emotional closeness, sense of belonging and opportunity for nurturance) than in women in the highest tertile. The risk remained strong even when we controlled for the indicators of baseline sociodemographics and psychological and physiological health and functioning. Among men, none of the perceived social support dimensions showed a significant association with mortality. DISCUSSION: The results of this study present a challenge for society to find and develop new social innovations and interventions in order to promote a sense of emotional social support in older people, thereby contributing to their health and welfare. PMID- 16670193 TI - Neighborhood effects on the self-rated health of elders: uncovering the relative importance of structural and service-related neighborhood environments. AB - OBJECTIVES: The purpose of this study was to investigate the independent relationship between neighborhood context (characterized through age structure, economic conditions, service provision, and residential stability) and self reported health among elders in one U.S. city. METHODS: By using multilevel statistical models, we examined the cross-sectional relationships between markers of neighborhood environment (derived from the 1980 U.S. Census and the Yellow Pages of the 1985 New Haven, Connecticut, telephone book) and self-rated health among elders. We used survey data from the 1985 New Haven Established Populations for Epidemiologic Studies of the Elderly, which comprised 1,926 elders nested within 28 census tracts. RESULTS: When controlled for individual age, gender, race, marital status, education, and income, neighborhood measures of percent poverty were positively associated with poor self-rated health (odds ratio [OR] = 1.09; 95% confidence interval [CI] = 1.02-1.17), whereas residential stability (OR = 0.90; 95% CI = 0.84-0.96) and concentration of elders (OR = 0.82; 95% CI = 0.72-0.94) were inversely associated with poor self-rated health. Neighborhood service density was not associated with self-rated health. DISCUSSION: We found support for the role of neighborhood structural context (reflected through measures of poverty, residential stability, and age-based demographic concentration) in predicting the health of elders. Density of neighborhood services did not appear to have an independent effect on the self-rated health of elders. PMID- 16670194 TI - Work-family conflict and retirement preferences. AB - OBJECTIVES: This study investigates relationships between retirement preferences and perceived levels of work-family conflict. METHODS: Using the large sample of 52-54-year-old respondents to the 1992 Wisconsin Longitudinal Study, we estimated multinomial logistic regression models of preferences for partial and full retirement within the next 10 years. We examined the association between retirement preferences and perceived work-family conflict, evaluated the extent to which work-family conflict was a mediating mechanism between stressful work and family circumstances and preferences to retire, and explored potential gender differences in the association between work-family conflict and preferring retirement. RESULTS: Work-family conflict was positively related to preferences for both full and partial retirement. Yet work-family conflict did not appear to mediate relationships between stressful work and family environments and retirement preferences, nor did significant gender differences emerge in this association. DISCUSSION: Our analyses provide the first direct evidence of the role played by work-family conflict in the early stages of the retirement process, although we were not able to identify the sources of conflict underlying this relationship. Identifying the sources of this conflict and the psychological mechanisms linking work-family conflict to retirement preferences is an important task for future researchers. PMID- 16670195 TI - Caspase-3, TUNEL and ultrastructural studies of small follicles in adult human ovarian biopsies. AB - BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles. PMID- 16670196 TI - Profile of David W. Schindler. PMID- 16670197 TI - Na(+)/monocarboxylate transport (SMCT) protein expression correlates with survival in colon cancer: molecular characterization of SMCT. AB - We report an extensive characterization of the Na(+)/monocarboxylate transporter (SMCT), a plasma membrane protein that mediates active transport of monocarboxylates such as propionate and nicotinate, and we show that SMCT may play a role in colorectal cancer diagnosis. SMCT, the product of the SLC5A8 gene, is 70% similar to the Na(+)/I(-) symporter, the protein that mediates active I(-) uptake in the basolateral surface of thyrocytes and other cells. SMCT was reported in the apical surface of thyrocytes and formerly proposed also to transport I(-) and was called the apical I(-) transporter. However, it is now clear that SMCT does not transport I(-). Here we demonstrate a high-affinity Na(+)-dependent monocarboxylate transport system in thyroid cells, which is likely to be SMCT. We show that, whereas thyroidal Na(+)/I(-) symporter expression is thyroid-stimulating hormone (TSH)-dependent and basolateral, SMCT expression is TSH-independent and apical not only in the thyroid but also in kidney and colon epithelial cells and in polarized Madin-Darby canine kidney cells. We determine the kinetic parameters of SMCT activity and show its inhibition by ibuprofen (K(i) = 73 +/- 9 microM) in Xenopus laevis oocytes. SMCT was proposed to be a tumor suppressor in colon cancer. Significantly, we show that higher expression of SMCT in colon samples from 113 colorectal cancer patients correlates with longer disease-free survival, suggesting that SMCT expression may be a favorable indicator of colorectal cancer prognosis. PMID- 16670198 TI - XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice. AB - Inactivation of the XRCC4 nonhomologous end-joining factor in the mouse germ line leads to embryonic lethality, in association with apoptosis of newly generated, postmitotic neurons. We now show that conditional inactivation of the XRCC4 in nestin-expressing neuronal progenitor cells, although leading to no obvious phenotype in a WT background, leads to early onset of neuronally differentiated medulloblastomas (MBs) in a p53-deficient background. A substantial proportion of the XRCC4/p53-deficient MBs have high-level N-myc gene amplification, often intrachromosomally in the context of complex translocations or other alterations of chromosome 12, on which N-myc resides, or extrachromosomally within double minutes. In addition, most XRCC4/p53-deficient MBs harbor clonal translocations of chromosome 13, which frequently involve chromosome 6 as a partner. One copy of the patched gene (Ptc), which lies on chromosome 13, was deleted in all tested XRCC4/p53-deficient MBs in the context of translocations or interstitial deletions. In addition, Cyclin D2, a chromosome 6 gene, was amplified in a subset of tumors. Notably, amplification of Myc-family or Cyclin D2 genes and deletion of Ptc also have been observed in human MBs. We therefore conclude that, in neuronal cells of mice, the nonhomologous end-joining pathway plays a critical role in suppressing genomic instability that, in a p53-deficient background, routinely contributes to genesis of MBs with recurrent chromosomal alterations. PMID- 16670200 TI - Local molecular field theory for effective attractions between like charged objects in systems with strong Coulomb interactions. AB - Strong, short-ranged positional correlations involving counterions can induce a net attractive force between negatively charged strands of DNA and lead to the formation of ion pairs in dilute ionic solutions. However, the long range of the Coulomb interactions impedes the development of a simple local picture. We address this general problem by mapping the properties of a nonuniform system with Coulomb interactions onto those of a simpler system with short-ranged intermolecular interactions in an effective external field that accounts for the averaged effects of appropriately chosen long-ranged and slowly varying components of the Coulomb interactions. The remaining short-ranged components combine with the other molecular core interactions and strongly affect pair correlations in dense or strongly coupled systems. We show that pair correlation functions in the effective short-ranged system closely resemble those in the uniform primitive model of ionic solutions and illustrate the formation of ion pairs and clusters at low densities. The theory accurately describes detailed features of the effective attraction between two equally charged walls at strong coupling and intermediate separations of the walls. Analytical results for the minimal coupling strength needed to get any attraction and for the separation at which the attractive force is a maximum are presented. PMID- 16670199 TI - Transcription regulatory elements are punctuation marks for DNA replication. AB - Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription initiation complex inhibits replication fork progression in an orientation dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as "punctuation marks" for DNA replication in vivo. PMID- 16670201 TI - Growth hormone promotes skeletal muscle cell fusion independent of insulin-like growth factor 1 up-regulation. AB - Growth hormone (GH) participates in the postnatal regulation of skeletal muscle growth, although the mechanism of action is unclear. Here we show that the mass of skeletal muscles lacking GH receptors is reduced because of a decrease in myofiber size with normal myofiber number. GH signaling controls the size of the differentiated myotubes in a cell-autonomous manner while having no effect on size, proliferation, and differentiation of the myoblast precursor cells. The GH hypertrophic action leads to an increased myonuclear number, indicating that GH facilitates fusion of myoblasts with nascent myotubes. NFATc2, a transcription factor regulating this phase of fusion, is required for GH action because GH is unable to induce hypertrophy of NFATc2-/- myotubes. Finally, we provide three lines of evidence suggesting that GH facilitates cell fusion independent of insulin-like growth factor 1 (IGF-1) up-regulation. First, GH does not regulate IGF-1 expression in myotubes; second, GH action is not mediated by a secreted factor in conditioned medium; third, GH and IGF-1 hypertrophic effects are additive and rely on different signaling pathways. Taken together, these data unravel a specific function of GH in the control of cell fusion, an essential process for muscle growth. PMID- 16670203 TI - Does low-molecular-weight heparin influence cancer-related mortality? PMID- 16670202 TI - Cost effectiveness of bisphosphonates in the management of breast cancer patients with bone metastases. AB - BACKGROUND: Bisphosphonates are recommended to prevent skeletal related events (SREs) in patients with breast cancer and bone metastases (BCBM). However, their clinical and economic profiles vary from one agent to the other. MATERIALS AND METHODS: Using modeling techniques, we simulated from the perspective of the UK's National Health Service (NHS) the cost and quality adjusted survival (QALY) associated with five commonly-used bisphosphonates or no therapy in this patient population. The simulation followed patients into several health states (i.e. alive or dead, experiencing an SRE or no SRE, and receiving first or second line therapy). Drugs costs, infusion costs, SREs costs, and utility values were estimated from published sources. Utilities were applied to time with and without SREs to capture the impact on quality of life. RESULTS: Compared to no therapy, all bisphosphonates are either cost saving or highly cost-effective (with a cost per QALY < or = 6126 pounds sterlings). Within this evaluation, zoledronic acid was more effective and less expensive than all other options. CONCLUSIONS: Based on our model, the use of bisphosphonates in breast cancer patients with bone metastases should lead to improved patient outcomes and cost savings to the NHS and possibly other similar entities. PMID- 16670204 TI - Improved incidence of pT0 downstaged surgical specimens in locally advanced rectal cancer (LARC) treated with induction oxaliplatin plus 5-fluorouracil and preoperative chemoradiation. AB - PURPOSE: To compare efficacy in terms of pathologic response in LARC patients treated with preoperative chemoradiation, with or without a short-intense course of induction oxaliplatin. PATIENTS AND METHODS: From 05/98 to 10/02, 114 patients were treated with preoperative chemoradiation (4500-5040 cGy + oral Tegafur 1200 mg/day) for cT(3)-(4)N(+/x)M(0) rectal cancer. Starting 05/01, 52 consecutive patients additionally received induction FOLFOX-4, oxaliplatin (85 mg/m(2) iv d1), 5-FU (400 mg/m(2) iv bolus d1) and 600 mg/m(2) iv continuous infusion in 22 h with leucovorin (200 mg iv) d1 and d2, every 15 days (2 cycles), followed by the previously described Tegafur chemoradiation regime. Surgery was performed in 5-6 weeks. Pathological assessment investigated post-treatment T and N status in the rectal wall and peri-rectal tissues. RESULTS: Patients, tumor and treatment characteristics were comparable between groups. Incidence of pT(0) specimens was significantly increased by induction FOLFOX-4 (P = 0.006). Total T and N downstaging were 58% versus 75% and 42% versus 40%, respectively (P = ns). T downstaging of > or =2 categories was significantly superior in FOLFOX-4 group (P = 0.029). CONCLUSIONS: Short-intense induction FOLFOX-4 significantly improves pathologic complete response in LARC patients treated with tegafur-sensitized preoperative chemoradiation. The 44% rate of pT(0)-(1) specimens observed in the oxaliplatin group should impulse innovative surgical approaches to promote ano rectal sphincter conserving protocols. PMID- 16670205 TI - A multicentre randomised phase II study of carboplatin in combination with gemcitabine at standard rate or fixed dose rate infusion in patients with advanced stage non-small-cell lung cancer. AB - BACKGROUND: Intracellular gemcitabine triphosphate (dFdCTP) levels can be optimised by administering gemcitabine at a fixed dose rate infusion. PATIENTS AND METHODS: Patients with chemonaive advanced non-small cell lung cancer (NSCLC) were randomised to receive gemcitabine at a fixed dose rate gemcitabine 750 mg/m(2) over 75 min (arm A) or gemcitabine 1000 mg/m(2) over 30 min (arm B) on days 1 and 8 every three week cycle. Carboplatin at AUC of 5 was administered in both treatment arms on day 1 of each cycle. End points were activity, tolerability and pharmacokinetics of plasma and intracellular gemcitabine. RESULTS: 76 patients were randomised. Response rate was 34% in arm A and 42% in arm B. Toxicity and quality of life scores were similar for both treatment arms. Mean plasma Cmax(gemcitabine) and mean dFdCTP AUC in arm A was 20.8 microM +/- 17.2 microM and 35,079 +/- 18,216 microM*min respectively and in arm B, 41.2 +/- 13.9 microM and 32 249 +/- 11 267 microM*min respectively. dFdCTP saturation was reached in Arm B but not in Arm A. CONCLUSION: The saturability of dFdCTP accumulation in Arm A suggests optimal delivery of gemcitabine is achieved using fixed rate infusion compared to 30-min infusion. Fixed dose rate gemcitabine is active and feasible, supporting the concept of fixed dosing rate of gemcitabine in advanced NSCLC. However, this entails a longer infusion time with associated higher costs involved. PMID- 16670206 TI - HIV counseling and testing: less targeting, more testing. PMID- 16670207 TI - "Let the record show . . .": art activism and the AIDS epidemic. PMID- 16670208 TI - A quarter century of AIDS. PMID- 16670209 TI - The AIDS memorial quilt. PMID- 16670211 TI - Independent research on tobacco control in Germany. PMID- 16670212 TI - Bad advice: how not to have sex in an epidemic. PMID- 16670213 TI - Safety net lessons from the Veterans Health Administration. PMID- 16670214 TI - Significant psychological distress and contacts with mental health professionals. PMID- 16670215 TI - What's missing from the weathering hypothesis? PMID- 16670216 TI - Perpetration of intimate partner violence associated with sexual risk behaviors among young adult men. AB - OBJECTIVE: We assessed the association between intimate partner violence (IPV) perpetration and sexual risk behaviors and fatherhood (having fathered children) among young men. METHODS: Sexually active men aged 18 to 35 years who visited an urban community health center and who reported having sexual intercourse with a steady female partner during the past 3 months (N = 283) completed a brief self administered survey about sexual risk behaviors, IPV perpetration, and demographics. We conducted logistic regression analyses adjusted for demographics to assess associations between IPV and sexual risk behaviors and fatherhood. RESULTS: Participants were predominantly Hispanic (74.9%) and Black (21.9%). Participants who reported IPV perpetration during the past year (41.3%) were significantly more likely to report (1) inconsistent or no condom use during vaginal and anal sexual intercourse, (2) forcing sexual intercourse without a condom, (3) having sexual intercourse with other women, and (4) having fathered 3 or more children. CONCLUSION: IPV perpetration was common among our sample and was associated with increased sexual risk behaviors. Urban community health centers may offer an important venue for reaching this at-risk population. PMID- 16670217 TI - Assessing the impact of federal HIV prevention spending on HIV testing and awareness. AB - OBJECTIVES: The United States allocates more than $900 million annually for the prevention of HIV infection. We assessed the impact of this funding on HIV testing and knowledge. METHODS: We linked data from the Behavioral Risk Factor Surveillance System with tracking of Centers for Disease Control and Prevention (CDC) HIV prevention funding. We developed and validated regression models of the relation between HIV prevention funding to a respondent's state and the odds that the respondent (1) had been tested for HIV, and (2) was aware of methods to prevent mother-to-child HIV transmission (MTCT). RESULTS: The odds of having been tested for HIV increased with increased CDC funding to states (P=.009), as did awareness of prevention of MTCT (P=.002). We estimate that CDC HIV prevention funds led to 12.8 million more people being tested for HIV between 1998 and 2003 than would have been tested had all states received funds equal to the lowest quintile of funding. CONCLUSIONS: Federal HIV prevention funds independently correlate with increased HIV testing and knowledge of prevention of MTCT. Proposed reductions in HIV prevention spending would likely have adverse public health consequences. PMID- 16670218 TI - Recruiting minority men who have sex with men for HIV research: results from a 4 city campaign. AB - We describe the efforts of a 4-city campaign to recruit Black and Hispanic men who have sex with men into an established HIV epidemiological study. The campaign used community organizing principles and a social marketing model that focused on personnel, location, product, costs and benefits, and promotion. The campaign was developed at the community, group, and individual levels to both increase trust and reduce barriers.The proportion of Hispanic men recruited during the 2002-2003 campaign doubled compared with the 1987 campaign, and the proportion and number of White men decreased by 20%. The proportion of Black men decreased because of the large increase in Hispanic men, although the number of Black men increased by 56%. Successful recruitment included training recruitment specialists, involving knowledgeable minority community members during planning, and having an accessible site with convenient hours. PMID- 16670219 TI - Computerized HIV preventive intervention for adolescents: indications of efficacy. AB - We tested the hypothesis that a computerized intervention would be as efficacious as an in-person, small-group intervention in reducing sexual risk behaviors. The sexual behavior of high-risk adolescents in 3 intervention conditions was examined: (1) computer based, (2) small groups, and (3) control. Adolescents in the computerized intervention were significantly less likely to engage in sexual activity and reported significantly fewer partners. For some youths, computers are a viable way to deliver prevention information and promote skill development. PMID- 16670220 TI - Using a multisectoral approach to assess HIV/AIDS services in the western region of Puerto Rico. AB - The Enhancing Care Initiative of Puerto Rico assessed services available to people living with HIV/AIDS in the western region of Puerto Rico. Participants were 212 people living with HIV/AIDS and 116 employees from 6 agencies providing HIV/AIDS services in the region. Two main findings were that depression symptoms were present in 98.1% of people living with HIV/AIDS, and 7 of the 15 municipalities in the region did not provide any specific services to this population. Most urgent needs identified by people living with HIV/AIDS were economic support, housing, mental and psychological services, medicines, medical treatment, and transportation. The Enhancing Care Initiative provides an example of a successful multisectoral, multidimensional volunteer team effectively overcoming challenges while translating research into interventions to enhance HIV/AIDS care. PMID- 16670221 TI - Patterns and correlates of deliberate abstinence among men and women with HIV/AIDS. AB - OBJECTIVES: We examined correlates of deliberate sexual abstinence among gay/bisexual men, heterosexual men, and women in a national probability sample of adults with HIV. METHODS: Participants in the HIV Cost and Services Utilization Study (HCSUS; n=1339) answered questions about oral, anal, or vaginal sexual intercourse in the past 6 months; those who reported none of these behaviors (n=415) were asked about their reasons for abstinence. Of these, 201 participants (11% of gay/bisexual men, 18% of women, 18% of heterosexual men) indicated that their abstinence was deliberate. Multivariate models were used to predict deliberate abstinence. RESULTS: In multivariate analyses, not having a primary relationship partner/spouse was a significant correlate of deliberate abstinence for all 3 groups. Higher perceived responsibility for limiting disease transmission and nondrinking status were related to deliberate abstinence only among gay/bisexual men. Worse health was associated with deliberate abstinence only among heterosexual men. CONCLUSIONS: Perhaps because HIV is more common in gay communities, abstinence choices may be more closely linked to a higher sense of responsibility for reducing transmission among gay/bisexual men, and their illness may be less of an impediment to sexual activity. PMID- 16670222 TI - Physical and sexual violence during pregnancy and after delivery: a prospective multistate study of women with or at risk for HIV infection. AB - OBJECTIVES: We sought to describe and compare prevalence rates of and risk factors for violence against women during pregnancy and postpartum. METHODS: Physical and sexual violence and violence risk factors were assessed during late pregnancy and 6 months postpartum in a prospective study of pregnant women with (n=336) and without (n=298) HIV in 4 US states. RESULTS: Overall, 10.6% of women reported having experienced violence, 8.9% during pregnancy and 4.9% after delivery. Of these women, 61.7% were abused only during their pregnancy, 21.7% were repeatedly abused, and 16.7% were abused only after their delivery. Sexual violence rarely occurred in the absence of physical violence. The strongest predictor of violence was engaging in bartered sex (adjusted odds ratio [OR]=5.54; 95% confidence interval [CI] =2.0, 15.4). Other predictors included frequent changes in residence (adjusted OR=1.57; 95% CI=1.1, 2.2), financial support from family or partners (adjusted OR=0.42; 95% CI=0.2, 0.8), and HIV diagnosis during current pregnancy (adjusted OR=0.30; 95% CI=0.1, 0.7). CONCLUSIONS: Women more commonly experienced violence during than after their pregnancy, but violence was best predicted by socioeconomic and behavioral indicators whose influence did not vary over time. PMID- 16670224 TI - Sexual orientation, sexual abuse, and HIV-risk behaviors among adolescents in the Pacific Northwest. AB - OBJECTIVES: We explored HIV risk behaviors, sexual orientation, and sexual abuse among 5 school-based cohorts in Seattle, Wash (SEA95 and SEA99:N=7477 and N=6590), and British Columbia (BC92, BC98, and BC03 [weighted]: N=239975, N=281576, and N=265132). METHODS: An HIV risk scale of 7 items assessed risky sexual behaviors and injection drug use. Self-identified sexual orientation included heterosexual, bisexual, gay/lesbian, and, in British Columbia only, mostly heterosexual. Analyses of covariance were conducted separately by gender and were adjusted for age and sexual abuse when comparing means. RESULTS: Gay/lesbian and bisexual adolescents had higher mean age-adjusted risk scores compared with heterosexual and mostly heterosexual adolescents. After we controlled for sexual abuse history, mean scores were 2 to 4 times higher among abused students than among nonabused students in each sexual orientation group. Age/abuse-adjusted models better explained the variance in risk scores (R(2)=0.10 0.31), but sexual orientation remained an independent predictor. CONCLUSION: Sexual minority adolescents who attended school reported higher HIV risk behaviors, and higher prevalence of sexual victimization may partially explain these risks. PMID- 16670223 TI - Greater risk for HIV infection of black men who have sex with men: a critical literature review. AB - HIV rates are disproportionately higher for Black men who have sex with men (MSM) than for other MSM. We reviewed the literature to examine 12 hypotheses that might explain this disparity. We found that high rates of HIV infection for Black MSM were partly attributable to a high prevalence of sexually transmitted diseases that facilitate HIV transmission and to undetected or late diagnosis of HIV infection; they were not attributable to a higher frequency of risky sexual behavior, nongay identity, or sexual nondisclosure, or to reported use of alcohol or illicit substances. Evidence was insufficient to evaluate the remaining hypotheses.Future studies must address these hypotheses to provide additional explanations for the greater prevalence of HIV infection among Black MSM. PMID- 16670225 TI - Reborn a virgin: adolescents' retracting of virginity pledges and sexual histories. AB - OBJECTIVES: We examined retractions of virginity pledges and of sexual histories among adolescents taking part in waves 1 and 2 of the National Longitudinal Study of Adolescent Health. METHODS: Logistic regression analyses were used to compare respondents' reports of virginity pledges and sexual histories at waves 1 and 2.Results. Among wave 1 virginity pledgers, 53% denied having made a pledge at wave 2; after control for confounders, pledgers who subsequently initiated sexual activity were 3 times as likely to deny having made a pledge as those who did not initiate sexual activity (odds ratio [OR] = 3.21; 95% confidence interval [CI] = 2.04, 5.04). Among wave 1 nonvirgins who subsequently took virginity pledges, 28% retracted their sexual histories at wave 2; respondents who took virginity pledges were almost 4 times as likely as those who did not to retract reports of sexual experience (OR=3.88; 95% CI=1.87, 8.07). CONCLUSIONS: Adolescents who initiate sexual activity are likely to recant virginity pledges, whereas those who take pledges are likely to recant their sexual histories. Thus, evaluations of sexual abstinence programs are vulnerable to unreliable data. In addition, virginity pledgers may incorrectly assess the sexually transmitted disease risks associated with their prepledge sexual behavior. PMID- 16670226 TI - Prevalence of childhood sexual abuse and physical trauma in an HIV-positive sample from the deep south. AB - We examined prevalence and predictors of trauma among HIV-infected persons in the Deep South using data from the Coping with HIV/AIDS in the Southeast (CHASE) study. Over 50% of CHASE participants were abused during their lives, with approximately 30% experiencing abuse before age 13, regardless of gender. Caregiver characteristics were associated with childhood abuse. Abuse is related to increases in high-HIV-risk activities. The findings help explain why people engage in such high-risk activities and can provide guidance in designing improved care and prevention messages. PMID- 16670227 TI - HIV testing among young adults in the United States: associations with financial resources and geography. AB - We estimated prevalence and odds ratios for self-reported HIV testing among sexually experienced young adults using nationally representative data obtained from Wave III of the National Longitudinal Study of Adolescent Health (Add Health). The prevalence of testing in the past year was 18.8%. Young adults who had private or no health insurance were less likely to report testing than were young adults who had public health insurance, particularly in the South. Respondents with functional income were less likely to report testing than were those without functional income, particularly in the South and Northeast. Variable HIV testing based on finances and insurance should be addressed. PMID- 16670228 TI - HIV Infection and AIDS in the Deep South. AB - We examine epidemiological and demographic data documenting the HIV/AIDS epidemic in the Deep South region of the United States. These data document substantial increases in AIDS cases in the Deep South from 2000 to 2003. In contrast, other US regions are experiencing stable rates or small increases in new AIDS cases. Furthermore, the AIDS epidemic in the Deep South is more concentrated than in other regions among African Americans, women, and rural residents. The Deep South also has some of the highest levels of poverty and uninsured individuals, factors that complicate the prevention and treatment of HIV infection. Further research is needed to determine the cause of the disproportionate rise in AIDS incidence and to develop effective means of preventing HIV infection and providing care of those infected in this region. PMID- 16670229 TI - Association of cigarette smoking with HIV prognosis among women in the HAART era: a report from the women's interagency HIV study. AB - OBJECTIVE: We assessed the association of cigarette smoking with the effectiveness of highly active antiretroviral therapy (HAART) among low-income women. METHODS: Data were analyzed from the Women's Interagency HIV Study, a multisite longitudinal study up to 7.9 years for 924 women representing 72% of all women who initiated HAART between July 1, 1995, and September 30, 2003. RESULTS: When Cox's regression was used after control for age, race, hepatitis C infection, illicit drug use, previous antiretroviral therapy, and previous AIDS, smokers on HAART had poorer viral responses (hazard ratio [HR]=0.79; 95% confidence interval [CI]=0.67, 0.93) and poorer immunologic response (HR=0.85; 95% CI=0.73, 0.99). A greater risk of virologic rebound (HR=1.39; 95% CI=1.06, 1.69) and more frequent immunologic failure (HR=1.52; 95% CI=1.18, 1.96) were also observed among smokers. There was a higher risk of death (HR=1.53; 95% CI=1.08, 2.19) and a higher risk of developing AIDS (HR=1.36; 95% CI=1.07, 1.72) but no significant difference between smokers and nonsmokers in the risk of death due to AIDS. CONCLUSIONS: Some of the benefits provided by HAART are negated in cigarette smokers. PMID- 16670231 TI - Lessons for management of sexually transmitted infection treatment programs as part of HIV/AIDS prevention strategies. AB - We analyzed the treatment schedule of a long-running HIV/AIDS prevention program in Managua, Nicaragua, that targets sex workers through mass and specific clinic based treatment of sexually transmitted infections and confirmed the role of frequency of treatment in generating a sustained reduction in measured prevalence of sexually transmitted infections. Unplanned variations in the time between treatments, a situation common to public health programs, provided the basis for attributing changes in measured levels of sexually transmitted infections to the program and for testing the statistical significance of the relationship. This information is critical to program design and funding and to resource allocation. PMID- 16670230 TI - Health care access among individuals involved in same-sex relationships. AB - OBJECTIVES: We used data from the National Health Interview Survey to compare health care access among individuals involved in same-sex versus opposite-sex relationships. METHODS: We conducted descriptive and logistic regression analyses from pooled data on 614 individuals in same-sex relationships and 93418 individuals in opposite-sex relationships. RESULTS: Women in same-sex relationships (adjusted odds ratio [OR]=0.60; 95% confidence interval [CI]=0.39, 0.92) were significantly less likely than women in opposite-sex relationships to have health insurance coverage, to have seen a medical provider in the previous 12 months (OR=0.66; 95% CI=0.46, 0.95), and to have a usual source of health care (OR=0.50; 95% CI=0.35, 0.71); they were more likely to have unmet medical needs as a result of cost issues (OR=1.85; 95% CI=1.16, 2.96). In contrast, health care access among men in same-sex relationships was equivalent to or greater than that among men in opposite-sex relationships. CONCLUSIONS: In this study involving a nationwide probability sample, we found some important differences in access to health care between individuals in same-sex and opposite-sex relationships, particularly women. PMID- 16670232 TI - Longitudinal relationships between use of highly active antiretroviral therapy and satisfaction with care among women living with HIV/AIDS. AB - OBJECTIVES: We used longitudinal data to examine the roles of 4 dimensions of patient satisfaction as both predictors and outcomes of use of highly active antiretroviral therapy (HAART) among women in the United States with HIV/AIDS. METHODS: Generalized estimating equations were used to analyze time-lagged satisfaction-HAART relationships over 8 years in the Women's Interagency HIV Study. RESULTS: Multivariate models showed that, over time, HAART use was associated with higher patient satisfaction with care in general, with providers, and with access/convenience of care; however, patient satisfaction was not associated with subsequent HAART use. Symptoms of depression and poor health related quality of life were associated with less satisfaction with care on all 4 dimensions assessed, whereas African American race/ethnicity, illegal drug use, and fewer primary care visits were associated with less HAART use. CONCLUSIONS: Our findings suggest that dissatisfaction with care is not a reason for underuse of HAART among women with HIV and that providers should not be discouraged from recommending HAART to dissatisfied patients. Rather, increasing women's access to primary care could result in both increased HAART use and greater patient satisfaction. PMID- 16670233 TI - Blood transfusions in the early years of AIDS in sub-Saharan Africa. AB - Blood transfusions transmit HIV more effectively than other means, yet there has been little examination of their role in the origins and early course of AIDS in sub-Saharan Africa. We review historical data in archives, government reports, and medical literature from African and European sources documenting the introduction, establishment, use, and growth of blood transfusions in sub-Saharan Africa. These data allow estimation of the geographic diffusion and growth of blood transfusions between 1940 and 1990. By 1955, 19 African colonies and countries reported transfusion programs-with national rates of 718 to 1372 per 100 000 by 1964, and urban rates similar to those in developed countries. We estimated 1 million transfusions per year in sub-Saharan Africa by 1970 and 2 million per year by the 1980s, indicating that transfusions were widely used throughout sub-Saharan Africa during the crucial period of 1950-1970, when all epidemic strains of HIV first emerged in this region. PMID- 16670234 TI - Sexual and drug risk behaviors among women who have sex with women. AB - OBJECTIVES: We examined risk behaviors of female drug users, comparing those who reported recently having had sex with women (recent WSW), those who reported previously having had sex with women (former WSW), and those who reported never having had sex with women (never WSW). METHODS: We used data from the Risk Evaluation and Assessment of Community Health III Study. Adjusted odds for predictors of WSW status were determined via multinomial logistic regression analyses. RESULTS: Of the participants, 75% were never WSW, 12% were former WSW, and 13% were recent WSW. In comparison with never WSW status, significant predictors of recent WSW status were living away from one's parents as a child (adjusted odds ratio [OR]=3.05; 95% confidence interval [CI]=1.07, 8.67) and recently having been paid for sex by men (adjusted OR=4.02; 95% CI=1.67, 9.68). Also, recently having been paid for sex by men was a significant predictor of former WSW status as opposed to never WSW status (adjusted OR=3.97; 95% CI=1.65, 9.59). CONCLUSIONS: The recency with which they had sex with women is one of the facets influencing the risk profile of WSW. The diverse characteristics of the WSW population need to be incorporated into future studies and risk interventions targeting this group. PMID- 16670235 TI - Sexual orientation and mental and physical health status: findings from a Dutch population survey. AB - OBJECTIVES: We sought to determine whether sexual orientation is related to mental and physical health and health behaviors in the general population. METHODS: Data was derived from a health interview survey that was part of the second Dutch National Survey of General Practice, carried out in 2001 among an all-age random sample of the population. Of the 19685 persons invited to participate, 65% took part in the survey. Sexual orientation was assessed in persons aged 18 years and older and reported by 98.2% of 9684 participants. The respondents' characteristics are comparable with those of the Dutch general population. RESULTS: Gay/lesbian participants reported more acute mental health symptoms than heterosexual people and their general mental health also was poorer. Gay/lesbian people more frequently reported acute physical symptoms and chronic conditions than heterosexual people. Differences in smoking, alcohol use, and drug use were less prominent. CONCLUSIONS: We found that sexual orientation was associated with mental as well as physical health. The causal processes responsible for these differences by sexual orientation need further exploration. PMID- 16670236 TI - Prevalence of HIV infection among young adults in the United States: results from the Add Health study. AB - OBJECTIVES: We estimated HIV prevalence rates among young adults in the United States. METHODS: We used survey data from the third wave of the National Longitudinal Study of Adolescent Health, a random sample of nearly 19000 young adults initiated in 1994-1995. Consenting respondents were screened for the presence of antibodies to HIV-1 in oral mucosal transudate specimens. We calculated prevalence rates, accounting for survey design, response rates, and test performance. RESULTS: Among the 13184 participants, the HIV prevalence rate was 1.0 per 1000 (95% confidence interval [CI] = 0.4, 1.7). Gender-specific prevalence rates were similar, but rates differed markedly between non-Hispanic Blacks (4.9 per 1000; 95% CI=1.8, 8.7) and members of other racial/ethnic groups (0.22 per 1000; 95% CI=0.00, 0.64). CONCLUSIONS: Racial disparities in HIV in the United States are established early in the life span, and our data suggest that 15% to 30% of all cases of HIV occur among individuals younger than 25 years. PMID- 16670238 TI - Efficacy of an HIV prevention program among female adolescents experiencing gender-based violence. AB - OBJECTIVES: We examined the efficacy of an HIV prevention intervention among African American female adolescents reporting a history of gender-based violence. METHODS: In this analysis of a subgroup of participants involved in a randomized controlled trial, consistent condom use, psychosocial mediators associated with HIV-preventive behaviors, and presence of sexually transmitted diseases were assessed at 6- and 12-month follow-ups. The intervention emphasized ethnic and gender pride, HIV knowledge, condom attitudes, healthy relationships, communication, and condom use skills. RESULTS: Relative to the comparison condition, participants randomized to the intervention reported using condoms more consistently, had fewer episodes of unprotected vaginal sex, engaged in a greater proportion of protected intercourse acts, were more likely to have used a condom during their most recent intercourse, were less likely to have a new sexual partner, were less likely to have a sexually transmitted disease, and demonstrated more proficient condom skills. CONCLUSIONS: Given the substantial prevalence of gender-based violence among female adolescents and the associations observed between gender-based violence, HIV risk, and HIV infection, it is essential that HIV interventions involving young women address partner violence. PMID- 16670237 TI - Victimization, substance use, and HIV risk behaviors among gay/bisexual/two spirit and heterosexual American Indian Men in New York City. AB - OBJECTIVES: secondary aims included describing condom-use attitudes, beliefs about HIV/AIDS in the Indian community, HIV knowledge, HIV status, and preference for and access to HIV prevention services in this population. METHODS: A survey was mailed to all members of an American Indian community organization in New York City. RESULTS: The 20 men self-identifying as gay, two-spirit, or bisexual (hereafter, "two-spirit") were more likely to report being victimized and engaging in HIV risk behaviors than the 51 heterosexual respondents, although they reported comparable levels of recent substance use. Overall, victimization was associated with lifetime HIV risk behaviors (even after control for sexual orientation) but not with substance use or unsafe sex in the past 12 months. The percentage of HIV infection was surprisingly high (10% of two-spirit men and 6% of heterosexual men). CONCLUSIONS: Two-spirit men are a vulnerable population whose victimization must be understood within an appropriate historical and political context. PMID- 16670239 TI - "We have our protector": misperceptions of protection against HIV among participants in a microbicide efficacy trial. AB - OBJECTIVES: We examined perceptions of the effectiveness and acceptability of a candidate microbicide among 94 South African female sex workers who had participated in a phase 3 microbicide trial for HIV prevention. METHODS: Sixteen focus groups were conducted in 2001, 12 to 15 months after participants were informed that the candidate microbicide had been determined to be ineffective in preventing HIV and other sexually transmitted infections (STIs). RESULTS: Participants clearly indicated that they understood the experimental nature of the candidate microbicide, and they recognized that they had been informed after the trial that the product was ineffective. Nevertheless, most continued to believe that the candidate microbicide helped prevent HIV and other STIs, alleviated reproductive tract pain and STI symptoms, and helped to clean the vagina. CONCLUSIONS: These findings underscore the importance of understanding women's perceptions of the efficacy of candidate microbicides and the rationale for these beliefs. These issues need to be addressed in counseling throughout microbicide trials for HIV prevention. These results also demonstrate how desperate many women at high risk of HIV infection may be for new HIV prevention technologies. PMID- 16670240 TI - A semiparametric approach for the nonparametric transformation survival model with multiple covariates. AB - The nonparametric transformation model makes no parametric assumptions on the forms of the transformation function and the error distribution. This model is appealing in its flexibility for modeling censored survival data. Current approaches for estimation of the regression parameters involve maximizing discontinuous objective functions, which are numerically infeasible to implement with multiple covariates. Based on the partial rank (PR) estimator (Khan and Tamer, 2004), we propose a smoothed PR estimator which maximizes a smooth approximation of the PR objective function. The estimator is shown to be asymptotically equivalent to the PR estimator but is much easier to compute when there are multiple covariates. We further propose using the weighted bootstrap, which is more stable than the usual sandwich technique with smoothing parameters, for estimating the standard error. The estimator is evaluated via simulation studies and illustrated with the Veterans Administration lung cancer data set. PMID- 16670243 TI - What is the best dementia screening instrument for general practitioners to use? AB - OBJECTIVE: The objective of this study was to review existing dementia screening tools with a view to informing and recommending suitable instruments to general practitioners (GPs) based on their performance and practicability for general practice. METHOD: A systematic search of pre-MEDLINE, MEDLINE, PsycINFO, and the Cochrane Library Database was undertaken. Only available full-text articles about dementia screening instruments written in English or with an English version were included. Articles using a translation of an English language instrument were excluded unless validated in a general practice, community, or population sample. RESULTS: The General Practitioner Assessment of Cognition (GPCOG), Mini-Cog, and Memory Impairment Screen (MIS) were chosen as most suitable for routine dementia screening in general practice. The GPCOG, Mini-Cog, and MIS were all validated in community, population, or general practice samples, are easy to administer, and have administration times of 5 minutes or less. They also have negative predictive validity and misclassification rates, which do not differ significantly from those of the Mini-Mental Status Examination. CONCLUSIONS: It is recommended that GPs consider using the GPCOG, Mini-Cog, or MIS when screening for cognitive impairment or for case detection. PMID- 16670244 TI - Cognitive impairment and depression outcomes in the IMPACT study. AB - INTRODUCTION: It is unclear whether cognitive impairment affects acute and long term treatment response in geriatric depression. In addition, little is known about the long-term outcome of depression among older individuals who experience cognitive decline during a course of treatment for depression. The authors examined both of these issues using data from the IMPACT trial. METHODS: The sample consisted of 1,684 participants in the IMPACT study who had baseline and two-year follow-up data. Subjects were randomized to one year of active intervention with a depression care manager or usual care. After one year, all subjects had usual care for another year. Data were collected for two years. The authors used the Six-Item Cognitive Screener to examine acute and long-term effects on depression outcome of baseline cognitive impairment and of cognitive decline. Depression measures included the HSCL-20 and an estimation of depression free days. Outcomes were determined using both linear regression and repeated measures analyses. RESULTS: Depressed subjects in the active intervention group had better depression outcomes at one year regardless of baseline cognitive impairment. Cognitively impaired subjects within each treatment group had similar outcomes to subjects without cognitive impairment. Subjects who experienced decline in cognitive score over two years had worse 24-month depression outcomes compared with subjects whose cognitive score did not change. CONCLUSIONS: Cognitively impaired depressed patients can experience significant improvement in depression with appropriate acute and continuation-phase management. Older depressed adults experiencing ongoing cognitive decline may be at higher risk for poor depression outcomes and may require more careful clinical monitoring and management of both cognitive and affective symptoms. PMID- 16670245 TI - Misunderstanding the intentions of others: an exploratory study of the cognitive etiology of persecutory delusions in very late-onset schizophrenia-like psychosis. AB - OBJECTIVE: The objective of this study was to explore the cognitive etiology of persecutory delusion formation and maintenance in very late-onset schizophrenia like psychosis (SLP). METHOD: Probabilistic reasoning, causal attributional style, and mentalizing ability were examined in 29 patients with SLP, 30 with onset of depression after the age of 60 years and 30 healthy comparison subjects. RESULTS: Patients with SLP made significantly more errors than the healthy comparison group in deception, but not false belief, mentalizing tasks. There were no significant performance differences between groups on the probabilistic reasoning task or the attributional style task. CONCLUSIONS: Mentalizing errors may contribute to the development and maintenance of persecutory delusions in SLP. These patients do not appear to show the wider range of cognitive biases described in deluded patients with schizophrenia with onset in younger adult life. PMID- 16670246 TI - Persistence of neuropsychologic deficits in the remitted state of late-life depression. AB - OBJECTIVE: Cognitive impairment in late-life depression (LLD) is prevalent, disabling, and persists despite the remission of depressive symptoms. This article characterizes neuropsychologic functioning during remission in LLD. METHODS: The authors examined longitudinal performance on a comprehensive neuropsychologic battery in 56 nondemented subjects age 60 or older who initially presented with an episode of nonpsychotic unipolar major depression and 40 nondemented, age- and education-equated comparison subjects with no history of depression. Subjects were assessed at baseline (in a depressed state) and one year later (when remitted). RESULTS: After one year, 45% of the LLD subjects were cognitively impaired despite remission of depression. Visuospatial ability, information-processing speed, and delayed memory were most frequently impaired; 94% of the patients who were impaired at baseline remained impaired one year later. Twenty-three percent of the patients who were cognitively normal while depressed developed impairment one year later. CONCLUSIONS: Most older individuals who are cognitively impaired during a depressive episode remain impaired when their depression remits. In addition, a substantial proportion of older depressed individuals who are cognitively intact when depressed are likely to be impaired one year later, although their depression has remitted. PMID- 16670247 TI - A responder analysis of memantine treatment in patients with Alzheimer disease maintained on donepezil. AB - OBJECTIVE: The objective of this study was to examine the clinical utility of memantine for moderate-to-severe Alzheimer disease (AD) using responder analyses. METHOD: Data from a previously published 24-week, randomized, double-blind, placebo-controlled trial of 10 mg memantine twice a day in patients with moderate to-severe AD (N = 404) on stable donepezil therapy were evaluated using three sets of responder criteria. Response rates were calculated and analyzed for the intention-to-treat population using a generalized estimating equations model. The following outcomes were examined separately and in combination: the Alzheimer's Disease Cooperative Study-Activities of Daily Living 19-Item Inventory (ADCS ADL19), Severe Impairment Battery (SIB), Clinician's Interview-Based Impression of Change Plus Caregiver Input (CIBIC-Plus), and Neuropsychiatric Inventory (NPI). RESULTS: When treatment response required cognitive improvement relative to baseline, memantine yielded higher response rates than placebo. When treatment response was defined as stabilization of individual outcomes, memantine resulted in significantly higher response rates than placebo for all outcomes, with number needed to treat (NNT) ranging from 8-10. More conservative definitions of response that required simultaneous stabilization on multiple outcome measures again favored memantine treatment for six of 10 combinatorial definitions. CONCLUSIONS: These responder analyses may assist clinicians in evaluating the impact of memantine in a relevant clinical scenario, i.e., in patients with AD previously stabilized on a cholinesterase inhibitor. The current results indicate that in this setting, memantine produces both improvement and stabilization of symptoms, across multiple outcomes, and thus provides a clinically important treatment benefit for patients with moderate-to-severe AD. PMID- 16670248 TI - Neuropsychiatric symptoms in Alzheimer disease and cognitively impaired, nondemented elderly from a community-based sample in Brazil: prevalence and relationship with dementia severity. AB - OBJECTIVES: The objectives of this study were to describe the prevalence of neuropsychiatric symptoms of dementia in Alzheimer disease (AD) and cognitively impaired nondemented (CIND) subjects from a community-based Brazilian sample and to correlate these symptoms with severity of cognitive deficits. METHOD: A total of 1,563 randomly selected subjects were evaluated with the following screening tests: Mini-Mental Status Examination, Fuld Object Memory Evaluation, Informant Questionnaire on Cognitive Decline in the Elderly, and Activities of Daily Living International Scale. Screen positives were submitted to a workup for dementia, physical and neurologic examination, cranial computed tomography or cerebral magnetic resonance imaging, the Cambridge Examination for Mental Disorders, Clinical Dementia Rating Scale (CDR), and the Neuropsychiatric Inventory (NPI). Diagnosis was made according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria. RESULTS: Sixty patients with AD, 25 CIND, and 78 healthy elderly subjects were evaluated. Informants reported that 78.33% of patients with AD had one or more neuropsychiatric symptoms. Apathy (53.33%), depression (38.33%), sleep alterations (38.33%), and anxiety (25%) were the most prevalent disturbances in AD subjects. These disturbances were more prevalent in patients with AD than in the comparison group and CIND individuals. In the CIND group, the most frequent neuropsychiatric symptoms were anxiety and sleep alterations (both with 24%) followed by depression (16%). Total NPI scores were significantly different between AD and CIND groups, AD and comparison groups, and CIND and the comparison group. Apathy was the only neuropsychiatric symptom that was significantly different between the groups divided according to the CDR being more frequent in subjects with moderate to severe dementia. CONCLUSIONS: Neuropsychiatric symptoms seem to be as common in patients living in a developing country as they are in demented patients from the developed world. Indeed, the fact that some of our results are similar to other population-based studies may suggest that cultural factors play a minor role in the emergence of these symptoms, at least in a Latin American country like Brazil. PMID- 16670249 TI - Discrepancies in information provided to primary care physicians by patients with and without dementia: the Steel Valley Seniors Survey. AB - OBJECTIVE: The objective of this study was to examine associations between discrepancies in health information provided to primary care providers and severity of impairment in older patients with and without dementia. METHODS: This study included brief assessment and medical record review of 1,107 patients with a mean (standard deviation) age of 76.3 (6.6) years (range: 65-100 years) in seven small-town primary care practices. In 358 patients, detailed in-home assessment included demographics; dementia by Clinical Dementia Rating (CDR) scale; and frequencies of memory complaints, falls, and inadvertent medication nonadherence determined from medical records and standardized in-home research assessments. Main outcome variables were trends in discrepancies between chart reviews and research assessments. Main explanatory variable was CDR box total scores. RESULTS: Proportions of patients reporting memory complaints and falls, and evidence of inadvertent nonadherence, in the charts and by research assessment increased with CDR. Discrepancies between medical record and research assessment, were also associated with CDR, showing linear trends for memory complaints and inadvertent nonadherence and a quadratic trend for falls. CONCLUSION: Memory complaints, falls, and inadvertent medication nonadherence increase with dementia severity. The levels of discrepancy between information patients provided to their physicians and information they provided in response to detailed, standardized assessments, also varied with dementia severity. Physicians should be alert to the possibility of receiving unreliable health information from even mildly demented patients, whether or not dementia has been detected. PMID- 16670250 TI - Outcome after traumatic brain injury sustained in older adulthood: a one-year longitudinal study. AB - OBJECTIVE: The objective of this study was to explore the effects of traumatic brain injury (TBI) on cognition and functioning in older adults in a one-year longitudinal study. METHODS: Participants with mild-to-moderate TBI were compared with an age-, gender-, and education-matched healthy comparison group on aspects of cognition. Neuropsychologic tests were administered at one year. Self-reported measures of functioning were completed at baseline, six months, and one year. Informants rated instrumental functioning at one year. RESULTS: Sixty-nine subjects aged 50 years and over (mean: 67 years; standard deviation: 7.9) and a comparison group of 79 participants were assessed. Patients with TBI had poorer processing speed, verbal memory, language, and executive function; they self reported more psychologic distress, psychosocial dysfunction, and postconcussive symptoms; and they were rated as more impaired in functioning than the comparison group. TBI of moderate severity accounted for most of the between-group differences. CONCLUSION: TBI, particularly of moderate severity, led to poorer cognitive and psychosocial functioning one year postinjury among older adults. The clinical significance of this may become more evident with time in this vulnerable population. PMID- 16670251 TI - Outcomes of depressed patients undergoing inpatient pulmonary rehabilitation. AB - OBJECTIVE: Approximately 30% of patients with chronic obstructive pulmonary disease (COPD) experience depression. Pulmonary rehabilitation for COPD focuses on physical conditioning, but includes behavioral interventions that may address depressive symptoms. This study tested the hypothesis that brief inpatient pulmonary rehabilitation is followed by improvement in both depressive symptoms and function in patients with COPD with major depression. METHODS: The subjects, who were recruited from the pulmonary rehabilitation unit of the Burke Rehabilitation Hospital in White Plains, NY, who had COPD and major depression were consecutively admitted patients to a pulmonary rehabilitation unit. Symptoms of depression, disability, medical burden, the experience of support, and satisfaction with treatment were systematically ascertained on admission and before discharge. RESULTS: Three hundred sixty-one patients were screened and 63 met criteria for COPD and major depression. Depressive symptoms improved by discharge (z = -6.785, p <0.0001); median length of stay was 16 days. Approximately 51% of subjects met criteria for response (50% or greater reduction in depressive symptoms scores from baseline), and 39% met criteria for remission (final Hamilton Depression scale score equal to or less than 10). History of treatment for depression was associated with limited change in depressive symptoms, whereas social support and satisfaction with treatment were predictors of improvement. All disability domains were lower at discharge compared to baseline (z = -3.928, p <0.0001). Subjects with pronounced disability at baseline had the greatest improvement if their depression improved by discharge. CONCLUSIONS: Acute inpatient rehabilitation is followed by improvement of depressive symptoms and disability in older patients with COPD and major depression. Improvement of depression may be the result of behavioral interventions rather than the use of antidepressant drugs. PMID- 16670252 TI - Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii. AB - Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation. PMID- 16670253 TI - Reporting protein identification data: the next generation of guidelines. PMID- 16670254 TI - Differential regional gene expression from cardiac dyssynchrony induced by chronic right ventricular free wall pacing in the mouse. AB - Routine clinical right ventricular pacing generates left ventricular dyssynchrony manifested by early septal shortening followed by late lateral contraction, which, in turn, reciprocally stretches the septum. Dyssynchrony is disadvantageous to cardiac mechanoenergetics and worsens clinical prognosis, yet little is known about its molecular consequences. Here, we report the influence of cardiac dyssynchrony on regional cardiac gene expression in mice. Mice were implanted with a custom-designed miniature cardiac pacemaker and subjected to 1 wk overdrive right ventricular free wall pacing (720 beats/min, baseline heart rate 520-620 beats/min) to generate dyssynchrony (pacemaker: 3-V lithium battery, rate programmable, 1.5 g, bipolar lead). Electrical capture was confirmed by pulsed-wave Doppler and dyssynchrony by echocardiography. Gene expression from the left ventricular septal and lateral wall myocardium was assessed by microarray (dual-dye method, Agilent) using oligonucleotide probes and dye swap. Identical analysis was applied to four synchronously contracting controls. Of the 22,000 genes surveyed, only 18 genes displayed significant (P < 0.01) differential expression between septal/lateral walls >1.5 times that in synchronous controls. Gene changes were confirmed by quantitative PCR with excellent correlations. Most of the genes (n = 16) showed greater septal expression. Of particular interest were seven genes coding proteins involved with stretch responses, matrix remodeling, stem cell differentiation to myocyte lineage, and Purkinje fiber differentiation. One week of iatrogenic cardiac dyssynchrony triggered regional differential expression in relatively few select genes. Such analysis using a murine implantable pacemaker should facilitate molecular studies of cardiac dyssynchrony and help elucidate novel mechanisms by which stress/stretch stimuli due to dyssynchrony impact the normal and failing heart. PMID- 16670255 TI - Nox2-containing NADPH oxidase and Akt activation play a key role in angiotensin II-induced cardiomyocyte hypertrophy. AB - Angiotensin II (ANG II) has profound effects on the development and progression of pathological cardiac hypertrophy; however, the intracellular signaling mechanisms are not fully understood. In this study, we used genetic tools to test the hypothesis that increased formation of superoxide (O2-*) radicals from a Rac1 regulated Nox2-containing NADPH oxidase is a key upstream mediator of ANG II induced activation of serine-threonine kinase Akt, and that this signaling cascade plays a crucial role in ANG II-dependent cardiomyocyte hypertrophy. ANG II caused a significant time-dependent increase in Rac1 activation and O2-* production in primary neonatal rat cardiomyocytes, and these responses were abolished by adenoviral (Ad)-mediated expression of a dominant-negative Rac1 (AdN17Rac1) or cytoplasmic Cu/ZnSOD (AdCu/ZnSOD). Moreover, both AdN17Rac1 and AdCu/ZnSOD significantly attenuated ANG II-stimulated increases in cardiomyocyte size. Quantitative real-time PCR analysis demonstrated that Nox2 is the homolog expressed at highest levels in primary neonatal cardiomyocytes, and small interference RNA (siRNA) directed against it selectively decreased Nox2 expression by >95% and abolished both ANG II-induced O2-* generation and cardiomyocyte hypertrophy. Finally, ANG II caused a time-dependent increase in Akt activity via activation of AT(1) receptors, and this response was abolished by Ad-mediated expression of cytosolic human O2-* dismutase (AdCu/ZnSOD). Furthermore, pretreatment of cardiomyocytes with dominant-negative Akt (AdDNAkt) abolished ANG II-induced cellular hypertrophy. These findings suggest that O2-* generated by a Nox2-containing NADPH oxidase is a central mediator of ANG II induced Akt activation and cardiomyocyte hypertrophy, and that dysregulation of this signaling cascade may play an important role in cardiac hypertrophy. PMID- 16670256 TI - What are the etiology and epidemiology of out-of-hospital pediatric cardiopulmonary arrest in Ontario, Canada? AB - BACKGROUND: Pediatric cardiopulmonary arrest (CPA) outside of the hospital has a very high mortality rate. OBJECTIVES: To evaluate the etiology and initial compromise of pediatric CPA cases in hopes of developing strategies to improve out-of-hospital resuscitation. METHODS: The Ontario Prehospital Advanced Life Support (OPALS) study was a large multicenter initiative to evaluate the impact of emergency medical services (EMS) programs on 17 communities with 40,000 critically ill and injured patients who were older than 11 years. As part of this study, the authors conducted a retrospective observational cohort study that included all children younger than 18 years of age with out-of-hospital CPA, during an 11-year period from 1991-2002. CPA was defined as patient being pulseless, apneic, and requiring chest compressions. Data were collected from ambulance call reports and centralized dispatch data and were reviewed by two independent investigators. RESULTS: There were 503 children with CPA in the sample. Mean age was 5.6 years (range, 0-17 yr); 58.4% of patients were male, and 37.8% were younger than 1 year of age. Cardiopulmonary resuscitation (CPR) first was started by a bystander in 32.4% of cases, whereas 66.0% were unwitnessed arrests. Initial rhythms were asystole 77.2% of the time, pulseless electrical activity 16.4% of the time, and ventricular fibrillation or ventricular tachycardia 4% of the time. Annual incidence was 9.1/100,000 children. CPA was witnessed in 34.0% of cases; 80.7% of these were bystander-witnessed, and 18.1% were EMS-witnessed. Primary pathogenic cause of arrest was medical in 61.2% of cases, trauma in 37.2% of cases, and indeterminate in 1.6% of cases. Initial underlying physiologic compromise of witnessed arrests was judged to be respiratory in 39.8% of cases, sudden collapse (presumed electrical) in 16.4% of cases, progressive shock in 1.2% of cases, and indeterminate in 42.6% of cases. Presumed etiology was trauma, 37.6%; sudden infant death syndrome (SIDS), 20.3%; and respiratory disease, 11.6%, most commonly. Survival to hospital discharge was 2.0%. CONCLUSIONS: This is one of the largest population-based, prospective cohorts of pediatric CPA reported to date, and it reveals that most pediatric arrests are unwitnessed and receive no bystander CPR. Those that are witnessed most often are caused by respiratory arrests or trauma. Trauma, SIDS, and respiratory disease are the most common etiologies overall. These data are vital to planning large resuscitation trials looking at specific interventions (i.e., increasing bystander CPR) and highlight the need for better strategies for prevention and early recognition. PMID- 16670257 TI - Evaluation of a pediatric-sedation service for common diagnostic procedures. AB - BACKGROUND: Pediatric patients often require sedation for diagnostic procedures such as magnetic resonance imaging and computed tomography scanning. In October 2002, a dedicated sedation service was started at a tertiary care pediatric facility as a joint venture between pediatric emergency medicine and pediatric critical care medicine. Before this service, sedation was provided by the department of radiology by using a standard protocol, with high-risk patients and failed sedations referred for general anesthesia. OBJECTIVES: To describe the initial experience with a dedicated pediatric-sedation service. METHODS: This was a retrospective analysis of quality-assurance data collected on all sedations in the radiology department for 23-month periods before and after sedation-service implementation. Study variables were number and reasons for canceled or incomplete procedures, rates of referral for general anesthesia, rates of hypoxia, prolonged sedation, need for assisted ventilation, apnea, emesis, and paradoxical reaction to medication. Results are reported in odds ratios (OR) with 95% confidence intervals (95% CI). RESULTS: Data from 5,444 sedations were analyzed; 2,148 before and 3,296 after sedation-service activation. Incomplete studies secondary to inadequate sedation decreased, from 2.7% before the service was created to 0.8% in the post-sedation-service period (OR, 0.29; 95% CI = 0.18 to 0.47). There also were decreases in cancellations caused by patient illness (3.8% vs. 0.6%; OR, 0.16; 95% CI = 0.10 to 0.27) and rates of hypoxia (8.8% vs. 4.6%; OR, 0.50; 95% CI = 0.40 to 0.63). There were no significant differences between the groups in rates of apnea, need for assisted ventilation, emesis, or prolonged sedation. The implementation of the sedation service also was associated with a decrease in both the number of patients referred to general anesthesia without a trial of sedation (from 2.1% to 0.1%; OR, 0.33; 95% CI = 0.06 to 1.46) and the total number of general anesthesia cases in the radiology department (from 7.5% to 4.4% of all patients requiring either sedation or anesthesia; OR, 0.56; 95% CI = 0.45 to 0.71). CONCLUSIONS: The implementation of a dedicated pediatric-sedation service resulted in fewer incomplete studies related to inadequate sedation, in fewer canceled studies secondary to patient illness, in fewer referrals for general anesthesia, and in fewer recorded instances of sedation-associated hypoxia. These findings have important implications in terms of patient safety and resource utilization. PMID- 16670258 TI - Characteristics of patients who leave emergency departments without being seen. AB - OBJECTIVES: Patients leaving the emergency department (ED) without being seen (LWBS) by a physician have become a growing concern in overcrowded EDs. The purpose of this study was to determine the acuity level, reasons, and outcomes of LWBS cases. METHODS: LWBS patients (or their guardians) from two linked Canadian EDs (one adult, one pediatric), identified during 11 sampling periods of seven days' duration each, were contacted by telephone. Descriptive statistics are provided. RESULTS: A total of 711 (4.5%) of 15,660 registered emergency patients left without being seen (50% male; median age, 33 years). Triage-matched controls waited a median of 87 minutes before seeing a physician. Of the 711 LWBS cases, 512 (72%) were contacted and 498 agreed to participate. The most common major reason for leaving was "fed up with waiting" (44.8%). Overall, 60% of LWBS cases sought medical attention within one week; 14 patients were hospitalized, and one required urgent surgery. Triage level was not associated with the probability of subsequently seeking medical attention (61%, 61%, and 60% in triage levels 3, 4, and 5, respectively). Of the 198 (39%) who did not subsequently seek medical attention, 50 patients (26%) had been triaged as urgent and one patient died six days after ED registration. CONCLUSIONS: The most common reason for LWBS is impatience during peak ED periods. Many of these patients seek medical care within one week. Complications occurred rarely; however, "high-risk" patients who leave without being seen do experience adverse health outcomes. Further research is required to examine ways to reduce LWBS cases. PMID- 16670259 TI - The use of performance improvement methods to enhance emergency department patient satisfaction in the United States: a critical review of the literature and suggestions for future research. AB - OBJECTIVES: The authors reviewed the evidence on performance improvement methods for increasing emergency department (ED) patient satisfaction to provide evidence based suggestions for clinical practice. METHODS: Data sources consisted of searches through MEDLINE, CINAHL, PSYCHINFO, Cochrane Library, and Emergency Medicine Abstracts and a manual search of references. Articles were included if they reported a performance improvement intervention targeting patient satisfaction in the ED setting. Articles on studies not conducted in the United States or that failed to provide enough details to allow critical evaluation of the study were excluded. Two authors used structured evaluation criteria to independently review each retained study. RESULTS: Nineteen articles met all selection criteria. Three studies found varying levels of support for multicomponent interventions, predominantly focused on implementation of clinical practice guidelines for specific presenting complaints and process redesign. Sixteen studies evaluated single-component interventions, with the following having at least one supportive study: using alternating patient assignment to provider teams rather than "zone"-based assignment, enhancing provider communication and customer service skills, incorporating information delivery interventions (e.g., pamphlets, video) that target patient expectations, using preformatted charts, and establishing ED-based observation units for specific conditions such as asthma and chest pain. CONCLUSIONS: There is modest evidence supporting a range of performance improvement interventions for improving ED patient satisfaction. Further work is needed before specific, evidence-based recommendations can be made regarding which process changes are most effective. Recommendations are made for improving the quality of performance improvement efforts in the ED setting. PMID- 16670260 TI - The relationship of plasma glucose and HbA1c Levels among emergency department patients with no prior history of diabetes mellitus. AB - OBJECTIVES: Patients without a history of diabetes mellitus may be incidentally found to be hyperglycemic in the emergency department (ED). If the hyperglycemia is due to undiagnosed diabetes, then an opportunity for detection exists. Hemoglobin A1c (HbA1c) provides a weighted average of blood glucose levels over the past several months; high HbA1c levels could indicate diabetes. The objective of this study was to determine whether hyperglycemia in ED patients without a history of diabetes was associated with higher HbA1c levels. METHODS: This was a prospective nonconsecutive case series of adults aged 18 years or older presenting to the ED with acute illness for whom a plasma glucose sample was drawn for clinical management. A history of diabetes/hyperglycemia or current symptoms of diabetes excluded patients. HbA1c levels were analyzed for a glucose cutoff of 110 mg/dL; the data were further analyzed using additional glucose cutoffs. Based on the Third National Health and Nutrition Examination Survey outpatient screening data, an HbA1c level > or =6.2% was considered elevated (sensitivity of 63% and specificity of 97% for identifying diabetes). RESULTS: There were 541 patients enrolled; the glucose level correlated with the HbA1c level (r = 0.60, p < 0.001). Among the 331 patients with a glucose level > or =110 mg/dL, 22.4% had an elevated HbA(1c) level; among the 210 patients with a glucose level < 110 mg/dL, 7.6% had an elevated HbA1c level. There were few patients (n = 13) with a glucose level > or =200 mg/dL, but most (85%) had an elevated HbA1c level. Among the 140 patients with a mildly elevated glucose level (110-125 mg/dL), 16.4% had an elevated HbA(1c) level. CONCLUSIONS: Elevated HbA1c levels are found in ED patients with elevated random plasma glucose values. ED patients with hyperglycemia may warrant referral for diabetes testing. PMID- 16670261 TI - T-bet is a critical determinant in the instability of the IL-17-secreting T helper phenotype. AB - IL-23, an IL-12-related cytokine, induces an IL-17-secreting T-helper phenotype that is involved in autoimmune diseases and host defense against certain pathogens. Although the transcription factors required for development of IL-23 stimulated cells are unknown, we show that T-bet is a critical negative regulator of the IL-23-primed T-cell phenotype, which we term Th1beta. Th1 or Th1beta Tbx21 /- cultures secrete higher than WT levels of IL-17 in response to T-cell receptor (TCR) or IL-23 + IL-18 stimulation. Ectopic T-bet expression in Th1beta cells promotes IFN-gamma secretion but decreases IL-17 production. Although antigen receptor stimulation of Th1beta cells stimulates IL-17 production, it also induces the IFN-gamma-independent expression of T-bet and progression to a Th1 cytokine secretion pattern. T-bet is required for the progression to the Th1 phenotype, because Tbx21-/- Th1beta cultures maintain the IL-17-secreting phenotype after 2 weeks of culture. Addition of IFN-gamma to Tbx21-/- Th1beta cultures cannot recover the progression to the Th1 phenotype, suggesting T-bet, rather than IFN-gamma, mediates Th1beta to Th1 progression. The transient nature of the Th1beta phenotype suggests that these cells are a component of type I immunity and that T-bet expression is a critical determinant of Th1 versus Th1beta cell fate. PMID- 16670262 TI - Leukemogenesis induced by wild-type and STI571-resistant BCR/ABL is potently suppressed by C/EBPalpha. AB - Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571 sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPalpha, which were more potent than those of K298E C/EBPalpha, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation. PMID- 16670263 TI - Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia. AB - Drug development in human chronic lymphocytic leukemia (CLL) has been limited by lack of a suitable animal model to adequately assess pharmacologic properties relevant to clinical application. A recently described TCL-1 transgenic mouse develops a chronic B-cell CD5(+) leukemia that might be useful for such studies. Following confirmation of the natural history of this leukemia in the transgenic mice, we demonstrated that the transformed murine lymphocytes express relevant therapeutic targets (Bcl-2, Mcl-1, AKT, PDK1, and DNMT1), wild-type p53 status, and in vitro sensitivity to therapeutic agents relevant to the treatment of human CLL. We then demonstrated the in vivo clinical activity of low-dose fludarabine in transgenic TCL-1 mice with active leukemia. These studies demonstrated both early reduction in blood-lymphocyte count and spleen size and prolongation of survival (P = .046) compared with control mice. Similar to human CLL, an emergence of resistance was noted with fludarabine treatment in vivo. Overall, these studies suggest that the TCL-1 transgenic leukemia mouse model has similar clinical and therapeutic response properties to human CLL and may therefore serve as a useful in vivo tool to screen new drugs for subsequent development in CLL. PMID- 16670264 TI - A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation. AB - Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase. PMID- 16670265 TI - Independent confirmation of a prognostic gene-expression signature in adult acute myeloid leukemia with a normal karyotype: a Cancer and Leukemia Group B study. AB - Patients with acute myeloid leukemia (AML) and normal karyotype are classified in an intermediate-risk group, albeit this subset is heterogeneous for clinical outcome. A recent complementary DNA microarray study identified a gene-expression signature that--when used to cluster normal karyotype patients--separated them into 2 prognostically relevant subgroups. We sought the first independent validation of the prognostic value of this signature. Using oligonucleotide microarrays to measure gene expression in samples from uniformly treated adults with karyotypically normal AML, we performed cluster analysis based on the previously identified signature. We also developed a well-defined classification rule using the signature to predict outcome for individual patients. Cluster analysis confirmed the prognostic utility of the signature: patient clusters differed in overall (P = .001) and disease-free (P = .001) survival. The signature-based classifier identified groups with differences in overall (P = .02) and disease-free (P = .05) survival. A strong association of the outcome classifier with the prognostically adverse FLT3 internal tandem duplication (FLT3 ITD) potentially explained the prognostic significance of the signature. However, in the subgroup of patients without FLT3 ITD there was a moderate difference in survival for the classifier-derived groups. Our analysis confirms the applicability of the gene-expression profiling strategy for outcome prediction in cytogenetically normal AML. PMID- 16670266 TI - JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV with secondary myelofibrosis. AB - A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders, we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months, mice developed polycythemia, macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months, polycythemia regressed, abnormally shaped red blood cells and platelets were seen in circulation, and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia, thrombocytopenia, high neutrophilia, and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders. PMID- 16670267 TI - Endothelial catabolism of extracellular adenosine during hypoxia: the role of surface adenosine deaminase and CD26. AB - Extracellular levels of adenosine increase during hypoxia. While acute increases in adenosine are important to counterbalance excessive inflammation or vascular leakage, chronically elevated adenosine levels may be toxic. Thus, we reasoned that clearance mechanisms might exist to offset deleterious influences of chronically elevated adenosine. Guided by microarray results revealing induction of endothelial adenosine deaminase (ADA) mRNA in hypoxia, we used in vitro and in vivo models of adenosine signaling, confirming induction of ADA protein and activity. Further studies in human endothelia revealed that ADA-complexing protein CD26 is coordinately induced by hypoxia, effectively localizing ADA activity at the endothelial cell surface. Moreover, ADA surface binding was effectively blocked with glycoprotein 120 (gp120) treatment, a protein known to specifically compete for ADA-CD26 binding. Functional studies of murine hypoxia revealed inhibition of ADA with deoxycoformycin (dCF) enhances protective responses mediated by adenosine (vascular leak and neutrophil accumulation). Analysis of plasma ADA activity in pediatric patients with chronic hypoxia undergoing cardiac surgery demonstrated a 4.1 +/- 0.6-fold increase in plasma ADA activity compared with controls. Taken together, these results reveal induction of ADA as innate metabolic adaptation to chronically elevated adenosine levels during hypoxia. In contrast, during acute hypoxia associated with vascular leakage and excessive inflammation, ADA inhibition may serve as therapeutic strategy. PMID- 16670269 TI - Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability. AB - The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease, expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis, little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture, resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology, and in defining its contribution to the development of leukemia. PMID- 16670268 TI - Alpha-4 integrins and VCAM-1, but not MAdCAM-1, are essential for recruitment of mast cell progenitors to the inflamed lung. AB - Normal mouse lungs lack appreciable numbers of mast cells (MCs) or MC progenitors (MCp's), yet the appearance of mature MCs in the tracheobronchial epithelial surface is a characteristic of allergic, T-cell-dependent pulmonary inflammation. We hypothesized that pulmonary inflammation would recruit MCp's to inflamed lungs and that this recruitment would be regulated by distinct adhesion pathways. Ovalbumin-sensitized and challenged mice had a greater than 28-fold increase in the number of MCp's in the lungs. In mice lacking endothelial vascular cell adhesion molecule 1 (VCAM-1) and in wild-type mice administered blocking monoclonal antibody (mAb) to VCAM-1 but not to mucosal addressin CAM-1 (MadCAM 1), recruitment of MCp's to the inflamed lung was reduced by greater than 75%. Analysis of the integrin receptors for VCAM-1 showed that in beta7 integrin deficient mice, recruitment was reduced 73% relative to wild-type controls, and in either BALB/c or C57BL/6 mice, mAb blocking of alpha4, beta1, or beta7 integrins inhibited the recruitment of MCp's to the inflamed lung. Thus, VCAM-1 interactions with both alpha4beta1 and alpha4beta7 integrins are essential for the recruitment and expansion of the MCp populations in the lung during antigen induced pulmonary inflammation. Furthermore, the MCp is currently unique among inflammatory cells in its partial dependence on alpha4beta7 integrins for lung recruitment. PMID- 16670270 TI - Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3. AB - Mature megakaryocytes form structures called proplatelets that serve as conduits for platelet packaging and release at vascular sinusoids. Since the megakaryocyte expresses abundant levels of integrin alpha IIb beta3, we have examined a role for fibrinogen in proplatelet development and platelet release alongside that of other matrices. Primary mature murine megakaryocytes from bone marrow aspirates readily formed proplatelets when plated on fibrinogen at a degree that was significantly higher than that seen on other matrices. In addition, alpha IIb beta3 was essential for proplatelet formation on fibrinogen, as megakaryocytes failed to develop proplatelets in the presence of alpha IIb beta3 antagonists. Interestingly, inhibition of Src kinases or Ca2+ release did not inhibit proplatelet formation, indicating that alpha IIb beta3-mediated outside-in signals are not required for this response. Immunohistochemical studies demonstrated that fibrinogen is localized to the bone marrow sinusoids, a location that would allow it to readily influence platelet release. Further, thrombopoietin-stimulated alpha IIb-/- mice had a reduced increase in platelet number relative to controls. A similar observation was not observed for platelet recovery in alpha IIb-/- mice in response to antibody-induced thrombocytopenia, indicating the existence of additional pathways of regulation of proplatelet formation. These results demonstrate that fibrinogen is able to regulate proplatelet formation via integrin alpha IIb beta3. PMID- 16670271 TI - Comment on "Human white blood cells synthesize morphine: CYP2D6 modulation". PMID- 16670273 TI - TCR trans-rearrangements: biological significance in antigen recognition vs the role as lymphoma biomarker. AB - V(D)J rearrangements occur within loci of TCR and BCR genes, thus generating the diversity of the AgR repertoire. In addition, interlocus V(D)J rearrangements occur, giving rise to so-called "trans-rearrangements." Such trans-rearrangements increase the diversity of the immune receptor repertoire and can be expressed as functional chimeric TCR proteins on the surface of T cells. Although chimeric receptors are not pathogenic per se, the frequency of AgR trans-rearrangements correlates with the level of genetic instability and thus could be used as a predictive biomarker for lymphoma risk. PMID- 16670274 TI - Cutting edge: constitutive B cell receptor signaling is critical for basal growth of B lymphoma. AB - B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igalpha or Igbeta-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas. PMID- 16670275 TI - Cutting edge: apoptosis-regulating signal kinase 1 is required for reactive oxygen species-mediated activation of IFN regulatory factor 3 by lipopolysaccharide. AB - IFN regulatory factor (IRF) 3 participates in the transcriptional induction of IFN-alpha, IFN-beta, and a subset of IFN-stimulated genes (ISGs) as a result of viral infection. In addition, bacterial cell wall components such as LPS activate IRF3 in a p38-dependent manner. In this study we show that IRF3-mediated ISG induction by LPS requires the production of reactive oxygen species (ROS) by the NADPH-dependent oxidase NOX4. Furthermore, we present evidence that LPS-mediated ROS production leads to activation of apoptosis-regulating-signal kinase (ASK) 1, a MAPK kinase kinase family member capable of activating the MAP kinase 6/p38 axis. ASK1 kinase activity proved essential for IRF3-mediated ISG induction by LPS. Thus, our results presented here suggest a novel role for ROS and ASK1 in the innate immune response as signaling intermediates in the IRF3 activation pathway. PMID- 16670276 TI - Cutting edge: monovalency of CD28 maintains the antigen dependence of T cell costimulatory responses. AB - CD28 and CTLA-4 are the major costimulatory receptors on naive T cells. But it is not clear why CD28 is monovalent whereas CTLA-4 is bivalent for their shared ligands CD80/86. We generated bivalent CD28 constructs by fusing the extracellular domains of CTLA-4 or CD80 with the intracellular domains of CD28. Bivalent or monovalent CD28 constructs were ligated with recombinant ligands with or without TCR coligation. Monovalent CD28 ligation did not induce responses unless the TCR was coligated. By contrast, bivalent CD28 ligation induced responses in the absence of TCR engagement. To extend these findings to primary cells, we used novel superagonistic and conventional CD28 Abs. Superagonistic Ab D665, but not conventional Ab E18, predominantly ligates CD28 bivalently at low CD28/Ab ratios and induces Ag-independent T cell proliferation. Monovalency of CD28 for its natural ligands is thus essential to provide costimulation without inducing responses in the absence of TCR engagement. PMID- 16670277 TI - Cutting edge: CD1a+ antigen-presenting cells in human dermis respond rapidly to CCR7 ligands. AB - Recent data from murine models have confirmed that Langerhans cells are not the only population of APCs in the skin involved in initiating immune responses. In healthy human skin, we identify CD1a(+) dermal APCs located close to the lymphatic vessels in the upper layers of the dermis that are unequivocally distinct from migrating Langerhans cells but exhibit both potent allostimulatory capacity and a chemotactic response to CCR7 ligands. In contrast, CD14(+) dermal APCs are distributed throughout the dermis and lack a chemotactic response to CCR7 ligands. CD1a(+) dermal APCs therefore represent an APC population distinct from Langerhans cells that are capable of migrating to lymph nodes and stimulating naive T cells. In humans, CD1a(+) dermal APCs may fulfill some of the roles previously ascribed to Langerhans cells. PMID- 16670278 TI - Cutting edge: nonproliferating mature immune cells form a novel type of organized lymphoid structure in idiopathic pulmonary fibrosis. AB - Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. In this study, we describe a novel type of organized lymphoid structure in the lung of human idiopathic pulmonary fibrosis, with key features of lymphoid neogenesis, including: 1) recently activated CD40 ligand (CD40L)+ T cells; 2) variable numbers of activated CD40+/CD40L+ B cells, sometimes organized in follicles; 3) fully mature dendritic cells (DC) expressing CD40, CD83, CD86, and DC-lysosome-associated membrane protein; 4) the expression of the chemokine CCL21; 5) the presence of vessels with characteristics of high endothelial venules; and 6) a dense network of follicular DC. Surprisingly, these structures are devoid of CCR7+ naive T cells, proliferating lymphocytes, and germinal centers, suggesting that newly recruited activated DC and Ag-experienced lymphocytes can drive lymphoid neogenesis and that factors present within the lymphoid aggregates, such as CD40L, are essential to induce DC maturation. PMID- 16670279 TI - Regulation of T cell responses in the developing human fetus. AB - Although human T cells enter the peripheral lymphoid tissues early during fetal development, the adaptive immune system in the fetus has largely been regarded as functionally immature and unresponsive to stimulation. In this study, we show that depletion of fetal CD4+CD25(high) T regulatory (T(Reg)) cells, which are present at high frequency in fetal lymphoid tissues, results in vigorous T cell proliferation and cytokine production in vitro, even in the absence of exogenous stimulation. Analysis of CD4+ and CD8(+) T cell populations revealed a large subset of cells that expressed the early activation Ag, CD69. We show that this population represents a subset of highly reactive fetal T cells actively suppressed by fetal CD4+CD25(high) T(Reg) cells during development. These findings indicate that fetal T cells are, in the absence of CD4+CD25(high) T(Reg) cells, highly responsive to stimulation and provide evidence for an important role for CD4+CD25(high) T(Reg) cells in controlling T cell responses in utero. PMID- 16670280 TI - Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA+ plasmablast recruitment to the intestinal lamina propria after rotavirus infection. AB - Rotaviruses (RV) are the most important cause of severe childhood diarrheal disease. In suckling mice, infection with RV results in an increase in total and virus-specific IgA(+) plasmablasts in the small intestinal lamina propria (LP) soon after infection, providing a unique opportunity to study the mechanism of IgA(+) cell recruitment into the small intestine. In this study, we show that the increase in total and RV-specific IgA(+) plasmablasts in the LP after RV infection can be blocked by the combined administration of Abs against chemokines CCL25 and CCL28, but not by the administration of either Ab alone. RV infection in CCR9 knockout mice still induced a significant accumulation of IgA(+) plasmablasts in the LP, which was blocked by the addition of anti-CCL28 Ab, confirming the synergistic role of CCL25 and CCL28. The absence of IgA(+) plasmablast accumulation in LP following combined anti-chemokine treatment was not due to changes in proliferation or apoptosis in these cells. We also found that coadministration of anti-CCL25 and anti-CCL28 Abs with the addition of anti alpha(4) Ab did not further inhibit IgA(+) cell accumulation in the LP and that the CCL25 receptor, CCR9, was coexpressed with the intestinal homing receptor alpha(4)beta(7) on IgA(+) plasmablasts. Finally, we showed that RV infection was associated with an increase in both CCL25 and CCL28 in the small intestine. Hence, our findings indicate that alpha(4)beta(7) along with either CCR9 or CCR10 are sufficient for mediating the intestinal migration of IgA(+) plasmablasts during RV infection. PMID- 16670282 TI - Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration. AB - CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein alpha, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47(-/-) mice exhibit a selective reduction of splenic CD11c(high)CD11b(high)CD8alpha(-)CD4(+) DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein alpha, possibly explaining their selective deficiency in CD47(-/-) mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47(-/-) mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47(-/-) mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL 5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs. PMID- 16670281 TI - CD16+ monocyte-derived macrophages activate resting T cells for HIV infection by producing CCR3 and CCR4 ligands. AB - The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells. PMID- 16670283 TI - Regulatory T cells inhibit protein kinase C theta recruitment to the immune synapse of naive T cells with the same antigen specificity. AB - The precise mechanisms by which regulatory T cells operate, particularly their effect on signaling pathways leading to T cell activation, are poorly understood. In this study we have used regulatory T (Treg) cells of known Ag specificity, generated in vivo, to address their effects on early activation events occurring in naive T cells of the same Ag specificity. We found that the Treg cells need to be present at the moment of priming to suppress activation and proliferation of the naive T cell. Furthermore, the Treg cells significantly inhibit the recruitment of protein kinase Ctheta (PKCtheta) to the immune synapse of the naive T cell as long as both T cells are of the same Ag specificity and are contacting the same APC. Finally, naturally occurring CD4(+)25(+) T cells seem to have the same effect on PKCtheta recruitment in CD25(-) T cells of the same Ag specificity. These results suggest that although additional mechanisms of regulation are likely to exist, inhibition of PKCtheta recruitment in the effector T cell may be a common regulatory pathway leading to the absence of NF kappaB activation and contributing to the block of IL-2 secretion characteristic of immune suppression. PMID- 16670284 TI - ERK1-/- mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis. AB - Activation of MAPK ERK1/2 has been shown to play an important role in Th1/Th2 polarization and in regulating cytokine production from APCs. The ERK family consists of two members ERK1 and ERK2, which share approximately 84% identity at the amino acid level and can compensate for each other for most functions. Despite these features, ERK1 and ERK2 do serve different functions, but there is very little information on the contribution of individual forms of ERK on innate and adaptive immune responses. In this study, we describe that ERK1(-/-) mice display a bias toward Th1 type immune response. Consistent with this observation, dendritic cells from ERK1(-/-) mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation. Furthermore, serum from ERK1(-/-) mice had 100-fold higher total IgG2b and 10-fold higher total IgG2a and IgG1 Ab isotype titers, and enhanced levels of Ag-specific IgG2b Ab titers, compared with wild-type mice. Consistent with this enhanced Th1 bias, ERK1(-/-) mice showed enhanced susceptibility to myelin oligodendrocyte glycoprotein (MOG)35-55 peptide induced experimental autoimmune encephalomyelitis (EAE) and developed EAE earlier, and with increased severity, compared with wild-type mice. Importantly, there was a profound skewing toward Th1 responses in ERK1(-/-) mice, with higher IFN-gamma production and lower IL-5 production in MOG35-55-primed T cells, as well as an augmentation in the MOG-specific IgG2a and IgG2b Th1 Ab isotypes. Finally, increased infiltrating cells and myelin destruction was observed in the spinal cord of ERK1(-/-) mice. Taken together, our data suggest that deficiency of ERK1 biases the immune response toward Th1 resulting in increased susceptibility to EAE. PMID- 16670285 TI - Highly oligomeric procyanidins ameliorate experimental autoimmune encephalomyelitis via suppression of Th1 immunity. AB - Extracts of Jatoba, a South American herb, when injected i.p. into a mouse model of experimental autoimmune encephalomyelitis (EAE), inhibited the aggravation of clinical symptoms. At the same time, production of myelin oligodendrocyte glycoprotein Ag-specific IFN-gamma and TNF-alpha by spleen cells was markedly suppressed. After administration of Jatoba there was minimal evidence of the demyelination that is characteristic of the EAE model. Decreases in clinical scores were observed when Jatoba extracts were injected just before Ag. The purified active compounds are likely to be polyphenols that are absorbable to polyvinylpolypyrrolidone. The active compounds were polymerized polyphenol polymers (procyanidins) and at least five degrees of polymerization were necessary for activity. In addition, extracts of other plant materials containing such procyanidins had similar activity. After administration of highly polymerized procyanidins, there was a decrease in both dendritic and CD4(+) T cells. Although macrophages were increased in number, the expression of CD80 and MHC class II molecules was depressed indicating that the macrophages were immature. The results indicate that the suppression of development of EAE by the highly polymerized procyanidins resulted from an inhibition of Th1 and the effects might be associated with depression of Ag-presenting capability. PMID- 16670286 TI - Th2 cytokines down-regulate TLR expression and function in human intestinal epithelial cells. AB - TLRs serve important immune and nonimmune functions in human intestinal epithelial cells (IECs). Proinflammatory Th1 cytokines have been shown to promote TLR expression and function in IECs, but the effect of key Th2 cytokines (IL-4, IL-5, IL-13) on TLR signaling in IECs has not been elucidated so far. We stimulated human model IECs with Th2 cytokines and examined TLR mRNA and protein expression by Northern blotting, RT-PCR, real-time RT-PCR, Western blotting, and flow cytometry. TLR function was determined by I-kappaBalpha phosphorylation assays, ELISA for IL-8 secretion after stimulation with TLR ligands and flow cytometry for LPS uptake. IL-4 and IL-13 significantly decreased TLR3 and TLR4 mRNA and protein expression including the requisite TLR4 coreceptor MD-2. TLR4/MD 2-mediated LPS uptake and TLR ligand-induced I-kappaBalpha phosphorylation and IL 8 secretion were significantly diminished in Th2 cytokine-primed IECs. The down regulatory effect of Th2 cytokines on TLR expression and function in IECs also counteracted enhanced TLR signaling induced by stimulation with the hallmark Th1 cytokine IFN-gamma. In summary, Th2 cytokines appear to dampen TLR expression and function in resting and Th1 cytokine-primed human IECs. Diminished TLR function in IECs under the influence of Th2 cytokines may protect the host from excessive TLR signaling, but likely also impairs the host intestinal innate immune defense and increases IEC susceptibility to chronic inflammation in response to the intestinal microenvironment. Taken together, our data underscore the important role of Th2 cytokines in balancing TLR signaling in human IECs. PMID- 16670287 TI - Features of medullary thymic epithelium implicate postnatal development in maintaining epithelial heterogeneity and tissue-restricted antigen expression. AB - Although putative thymic epithelial progenitor cells have been identified, the developmental potential of these cells, the extent of medullary thymic epithelium (mTEC) heterogeneity, and the mechanisms that mediate the expression of a wide range of peripheral tissue-restricted Ags (TRAs) by mTECs remain poorly defined. Here we have defined several basic properties of the mTEC population that refine our understanding of these cells and impose important constraints for any model of mTEC differentiation and function. We report here that mTECs from adult mice are mitotically active, implying continual turnover, differentiation, and replacement of mTEC populations in the adult thymus. The mTEC population in adult thymus expresses transcription factors implicated in the maintenance of multipotential progenitor cell populations, suggesting that epithelial progenitors in the adult thymus may not be restricted to a thymic fate. mTECs also express multiple transcription factors required for the specification of multiple epithelial lineages in peripheral tissues. Thus, expression of some TRAs by mTECs may represent coordinated gene expression that reflects alternate programs of epithelial differentiation among mTECs. Analysis of TRA expression in individual and small pools of sorted mTECs show that mTECs are highly heterogeneous; each individual mTEC expresses a limited spectrum of TRAs, and the frequency of mTECs that express any individual TRA is quite low (>0.4-2%). Collectively, these findings suggest that the differentiation of mTECs can involve some of the developmental programs used by other epithelial lineages and that expression of some TRAs by mTECs may reflect this activity. PMID- 16670288 TI - Novel function of alternatively activated macrophages: stabilin-1-mediated clearance of SPARC. AB - The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1 mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin 1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin 1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration. PMID- 16670289 TI - CD64-directed immunotoxin inhibits arthritis in a novel CD64 transgenic rat model. AB - Macrophages are known to play a key role during inflammation in rheumatoid arthritis (RA). Inflammatory macrophages have increased expression of CD64, the high-affinity receptor for IgG. Targeting this receptor through a CD64-directed immunotoxin, composed of an Ab against CD64 and Ricin A, results in effective killing of inflammatory macrophages. In this study, we show elevated levels of CD64 on synovial macrophages in both synovial lining and synovial fluid in RA patients. The CD64-directed immunotoxin efficiently eliminates activated synovial macrophages in vitro, while leaving quiescent, low CD64-expressing macrophages unaffected. To examine whether killing of CD64 macrophages results in therapeutic effects in vivo, we established an adjuvant arthritis (AA) model in newly generated human CD64 (hCD64) transgenic rats. We demonstrate that hCD64 regulation in this transgenic rat model is similar as in humans. After AA induction, treatment with CD64-directed immunotoxin results in significant inhibition of disease activity. There is a direct correlation between immunotoxin treatment and decreased macrophage numbers, followed by diminished inflammation and bone erosion in paws of these hCD64 transgenic rats. These data support synovial macrophages to play a crucial role in joint inflammation in AA in rats and in human RA. Selective elimination of inflammatory macrophages through a CD64 directed immunotoxin may provide a novel approach for treatment of RA. PMID- 16670290 TI - Regulatory T cell responses develop in parallel to Th responses and control the magnitude and phenotype of the Th effector population. AB - Host survival during schistosomiasis requires the development of a tightly regulated and Th2-polarized immune response against parasite egg Ags. In this system, Th1 response suppression has been thought to be enforced through the production of IL-10 by Th2 cells and natural T regulatory (Treg) cells. By comparing Th responses in schistosome egg-injected mice that lack IL-10, IL-4, and/or Treg cells, we have been able to build a detailed picture of the relative contributions of Treg cells, Th2 cells, and IL-10 to regulation of the egg induced response. Our data indicate that eggs induce a marked Treg cell response, evident as the extensive proliferation of Foxp3(+) cells that is proportionally as great as the response occurring within the Th compartment. Furthermore, we show that Treg cells prevent Th1 response development and limit the magnitude of the Th2 response. Although Treg cells are able to produce IL-10 after egg injection, we found no evidence for a role for IL-10 in Treg-mediated suppression of Th cell responses, nor did we find evidence for an inhibitory effect of Th2 cells on Th1 response development. Thus, the magnitude and phenotype of the egg induced effector Th response are controlled by a parallel response within the Treg population. PMID- 16670291 TI - Human metapneumovirus elicits weak IFN-gamma memory responses compared with respiratory syncytial virus. AB - Human metapneumovirus (MPV) is a recently discovered pathogen that causes repeated lower respiratory tract infections beginning in infancy. The prevalence, nature and control of human regulatory responses to MPV are unknown. In this study, we develop and optimize systems to evaluate MPV-driven cytokine responses. Using primary culture of human PBMC from previously exposed adults, MPV stimulated responses were directly compared with those elicited by genetically and clinically similar respiratory syncytial virus (RSV). Intense IL-6 production was evident following culture with infectious or inactivated RSV. MPV elicited IL 6 responses averaging 3.5-fold more intense (p < 0.001). Virus-dependent expression of IL-11, IL-12, IFN-alpha, and other innate immunity cytokines differed little between MPV and RSV. When examining adaptive immunity, RSV infection elicited strong IFN-gamma responses by all 60 adults. In marked contrast, MPV elicited IFN-gamma in a lower frequency of adults (p < 0.002) and at levels averaging 6-fold weaker (p < 0.001). These Th1-dominated responses were CD4, CD8, CD86 dependent, and were closely paralleled by strong virus-driven IL 10 and CCL5 production. For MPV and RSV, Th2 (IL-5, IL-13) responses were sporadic, occurring in 10-40% of the population. Thus, MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion, and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Although both viruses yield Th1 dominated responses with strong IL-10 and CCL5 production, MPV restimulation results in markedly more robust IL-6 and significantly weaker adaptive cytokine responses, in both prevalence and intensity, than does RSV. PMID- 16670292 TI - TLR4 signaling attenuates ongoing allergic inflammation. AB - The relationship between LPS exposure and allergic asthma is poorly understood. Epidemiologic studies in humans have found that exposure to LPS can protect, have no effect, or exacerbate allergic asthma. Similarly, LPS has had variable effects on allergic pulmonary inflammation in the mouse, depending on the model used. In the present study, we studied the effect of very low doses of LPS in models of both short-term and long-term allergen challenge. When challenged with allergen for short periods, wild-type and tlr4-deficient mice had similar responses. However, when challenged for periods of 1 wk or longer, tlr4-deficient mice developed dramatically increased airway eosinophils, serum IgE, and Th2 cytokines compared with similarly challenged, genetically matched C57BL/6 mice. The relative attenuation of allergic responses seen in C57BL/6 mice was dependent on bone marrow-derived cell-specific expression of tlr4, and was not associated with an increase in Th1 responses. The number of dendritic cells in lungs of challenged tlr4-deficient mice was significantly increased compared with those in challenged C57BL/6 mice. No differences were seen in the abilities of naive C57BL/6 and tlr4-deficient mice to develop allergen-specific tolerance after exposure to similar preparations of OVA, suggesting that tolerance and regulation of existing inflammation develop through different mechanisms. The attenuation of eosinophilic inflammation in C57BL/6 mice was abolished when these mice were challenged with OVA supplemented with additional LPS. Together, these findings show that low doses of endotoxin can have regulatory effects on allergic inflammation, particularly in the setting of ongoing allergen exposure. PMID- 16670293 TI - Increased expression of Ifi202, an IFN-activatable gene, in B6.Nba2 lupus susceptible mice inhibits p53-mediated apoptosis. AB - Increased expression of p202 protein (encoded by the Ifi202 gene) in splenocytes derived from B6.Nba2 mice (congenic for the Nba2 interval derived from the New Zealand Black mice) was correlated with defects in apoptosis of splenic B cells and increased susceptibility to develop systemic lupus erythematosus. We have now investigated the molecular mechanisms by which increased expression of p202 in B6.Nba2 cells contributes to defects in apoptosis. In this study, we report that increased expression of p202 in the B6.Nba2 splenocytes, as compared with cells derived from the parental C57BL/6 (B6) mice, was correlated with increased levels of p53 protein and inhibition of p53-mediated transcription of target genes that encode proapoptotic proteins. Conversely, knockdown of p202 expression in B6.Nba2 cells resulted in stimulation of p53-mediated transcription. We found that p202 bound to p53 in the N-terminal region (aa 44-83) comprising the proline-rich region that is important for p53-mediated apoptosis. Consistent with the binding of p202 to p53, increased expression of p202 in B6.Nba2 mouse embryonic fibroblasts inhibited UV-induced apoptosis. Taken together, our observations support the idea that increased expression of p202 in B6.Nba2 mice increases the susceptibility to develop lupus, in part, by inhibiting p53-mediated apoptosis. PMID- 16670294 TI - Expression of TCR alpha beta partly rescues developmental arrest and apoptosis of alpha beta T cells in Bcl11b-/- mice. AB - Bcl11b(-/-) mice show developmental arrest at the CD44(-)CD25(+) double-negative 3 (DN3) or immature CD8(+)single-positive stage of alphabeta T cell. We have performed detailed analysis of sorted subsets of Bcl11b(-/-) thymocytes, DN3 and CD44(-)CD25(-) double-negative 4 (DN4) cells. Surface expression of TCRbeta proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRbeta or TCRalphabeta expression on the cell surface failed to promote transition from the DN3 to CD4(+)CD8(+) double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b(-/-) DN3 thymocytes showed normal DNA rearrangements between Dbeta and Jbeta segments but limited DNA rearrangements between Vbeta and DJbeta without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b(-/-) DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vbeta and Dbeta gene locus. Analysis of Bcl11b(-/-) DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-x(L) and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRalphabeta in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests. PMID- 16670295 TI - CD25+Foxp3+ regulatory T cells facilitate CD4+ T cell clonal anergy induction during the recovery from lymphopenia. AB - T cell clonal anergy induction in lymphopenic nu/nu mice was found to be ineffective. Exposure to a tolerizing peptide Ag regimen instead induced aggressive CD4(+) cell cycle progression and increased Ag responsiveness (priming). Reconstitution of T cell-deficient mice by an adoptive transfer of mature peripheral lymphocytes was accompanied by the development of a CD25(+)Foxp3(+)CTLA-4(+)CD4(+) regulatory T cell population that acted to dampen Ag-driven cell cycle progression and facilitate the induction of clonal anergy in nearby responder CD25(-)CD4(+) T cells. Thus, an early recovery of CD25(+) regulatory T cells following a lymphopenic event can prevent exuberant Ag stimulated CD4(+) cell cycle progression and promote the development of clonal anergy. PMID- 16670296 TI - Differential effects of IL-27 on human B cell subsets. AB - IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL 12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation. PMID- 16670297 TI - CD2BP1 modulates CD2-dependent T cell activation via linkage to protein tyrosine phosphatase (PTP)-PEST. AB - Human CD2 regulates T cell activation and adhesion via mechanisms yet to be fully understood. This study focuses on CD2BP1, a CD2 cytoplasmic tail-binding protein preferentially expressed in hematopoetic cells. Structural and functional analyses suggest that CD2BP1 acts as a scaffold protein, participating in regulation of the actin cytoskeleton. In this study, using a murine Ag-specific primary T cell transduction system to assess CD69, IL-2, and IFN-gamma expression, we provide evidence that CD2BP1 directly and negatively impacts T cell activation via isolated CD2 triggering or TCR stimulation dependent on coordinate CD2 engagement. Disruption of protein tyrosine phosphatase-PEST and/or CD2BP1 association with the CD2 signalsome rescues T cells from the inhibitory effect of CD2 crosslinking. The overexpression of CD2BP1 selectively attenuates phospholipase Cgamma1, ERK1/2, and p38 phosphorylation without abrogating CD2 independent TCR stimulation. This study provides new insight on the regulation of T cell activation and may have implications for autoimmune processes known to be associated with CD2BP1 mutations. PMID- 16670298 TI - Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1. AB - Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject. PMID- 16670299 TI - Arginine transport via cationic amino acid transporter 2 plays a critical regulatory role in classical or alternative activation of macrophages. AB - Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases L-arginine transport only through the y(+) system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in L-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages. PMID- 16670300 TI - Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture. AB - Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8( ). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp fluoromethyl ketone or by the use of apoptotic protease-activating factor-1 deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint. PMID- 16670301 TI - Annexin V associates with the IFN-gamma receptor and regulates IFN-gamma signaling. AB - Many of the biological activities of IFN-gamma are mediated through the IFN gammaR3-linked Jak-Stat1alpha pathway. However, regulation of IFN-gamma signaling is not fully understood, and not all responses to IFN-gamma are Stat1alpha dependent. To identify novel elements involved in IFN-gamma cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-gammaR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-gammaR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-gammaR2 Abs showed that AxV interacted with IFN-gammaR2 to form a stable complex following incubation of cells with IFN-gamma. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1alpha in response to IFN-gamma was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-gamma response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-gammaR, AxV modulates cellular responses to IFN-gamma by modulating signaling through the Jak-Stat1 pathway. PMID- 16670302 TI - Class I and III phosphatidylinositol 3'-kinase play distinct roles in TLR signaling pathway. AB - PI3K involvement has been implicated in the TLR signal pathway. However, the precise roles of the different classes of PI3K in the pathway remain elusive. In this study, we have explored the functions of class I and class III PI3K in the TLR signal pathway using specific kinase mutants and PI3K lipid products. Our results reveal that class III PI3K specifically regulates CpG oligodeoxynucleotide (ODN)-induced cytokine and NO production as well as NF kappaB activation, whereas class I PI3K regulates both CpG ODN- and LPS-induced IL-12 production and NF-kappaB activation. Additional studies of CpG ODN uptake with flow cytometric analysis show that class III PI3K, but not class I, regulates cellular CpG ODN uptake. Furthermore, experiments with MyD88 overexpressing fibroblast cells transfected with dominant-negative mutants of PI3K demonstrate that class III PI3K regulates CpG ODN-mediated signaling upstream of MyD88, while class I PI3K regulation is downstream of MyD88. These results suggest that class I and class III PI3K play distinct roles in not only the uptake of CpG ODN, but also responses elicited by CpG ODN and LPS. PMID- 16670303 TI - Peripheral tolerance via the anterior chamber of the eye: role of B cells in MHC class I and II antigen presentation. AB - Ags introduced into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed AC-associated immune deviation (ACAID). ACAID mitigates ocular autoimmune diseases and promotes corneal allograft survival. Ags injected into the AC are processed by F4/80(+) APCs, which migrate to the thymus and spleen. In the spleen, ocular APCs induce the development of Ag-specific B cells that act as ancillary APCs and are required for ACAID induction. In this study, we show that ocular-like APCs elicit the generation of Ag-specific splenic B cells that induce ACAID. However, direct cell contact between ocular-like APCs and splenic B cells is not necessary for the induction of ACAID B cells. Peripheral tolerance produced by ACAID requires the participation of ACAID B cells, which induce the generation of both CD4(+) regulatory T cells (Tregs) and CD8(+) Tregs. Using in vitro and in vivo models of ACAID, we demonstrate that ACAID B cells must express both MHC class I and II molecules for the generation of Tregs. These results suggest that peripheral tolerance induced through the eye requires Ag-presenting B cells that simultaneously present Ags on both MHC class I and II molecules. PMID- 16670304 TI - Adiponectin is a negative regulator of NK cell cytotoxicity. AB - NK cells are a key component of innate immune systems, and their activity is regulated by cytokines and hormones. Adiponectin, which is secreted from white adipose tissues, plays important roles in various diseases, including hypertension, cardiovascular diseases, inflammatory disorders, and cancer. In this study the effect of adiponectin on NK cell activity was investigated. Adiponectin was found to suppress the IL-2-enhanced cytotoxic activity of NK cells without affecting basal NK cell cytotoxicity and to inhibit IL-2-induced NF kappaB activation via activation of the AMP-activated protein kinase, indicating that it suppresses IL-2-enhanced NK cell cytotoxicity through the AMP-activated protein kinase-mediated inhibition of NF-kappaB activation. IFN-gamma enhances NK cell cytotoxicity by causing an increase in the levels of expression of TRAIL and Fas ligand. The production of IFN-gamma, one of the NF-kappaB target genes in NK cells, was also found to be suppressed by adiponectin, accompanied by the subsequent down-regulation of IFN-gamma-inducible TRAIL and Fas ligand expression. These results clearly demonstrate that adiponectin is a potent negative regulator of IL-2-induced NK cell activation and thus may act as an in vivo regulator of anti-inflammatory functions. PMID- 16670305 TI - Inducible costimulator: a modulator of IFN-gamma production in human tuberculosis. AB - Effective host defense against Mycobacterium tuberculosis requires the induction of Th1 cytokine responses. We investigated the regulated expression and functional role of the inducible costimulator (ICOS), a receptor known to regulate Th cytokine production, in the context of human tuberculosis. Patients with active disease, classified as high responder (HR) or low responder (LR) patients according to their in vitro T cell responses against the Ag, were evaluated for T cell expression of ICOS after M. tuberculosis-stimulation. We found that ICOS expression significantly correlated with IFN-gamma production by tuberculosis patients. ICOS expression levels were regulated in HR patients by Th cytokines: Th1 cytokines increased ICOS levels, whereas Th2-polarizing conditions down-regulated ICOS in these individuals. Besides, in human polarized Th cells, engagement of ICOS increased M. tuberculosis IFN-gamma production with a magnitude proportional to ICOS levels on those cells. Moreover, ICOS ligation augmented Ag-specific secretion of the Th1 cytokine IFN-gamma from responsive individuals. In contrast, neither Th1 nor Th2 cytokines dramatically affected ICOS levels on Ag-stimulated T cells from LR patients, and ICOS activation did not enhance IFN-gamma production. However, simultaneous activation of ICOS and CD3 slightly augmented IFN-gamma secretion by LR patients. Together, our data suggest that the regulation of ICOS expression depends primarily on the response of T cells from tuberculosis patients to the specific Ag. IFN-gamma released by M. tuberculosis-specific T cells modulates ICOS levels, and accordingly, ICOS ligation induces IFN-gamma secretion. Thus, ICOS activation may promote the induction of protective Th1 cytokine responses to intracellular bacterial pathogens. PMID- 16670306 TI - OX40-OX40 ligand interaction through T cell-T cell contact contributes to CD4 T cell longevity. AB - Signals through the OX40 costimulatory receptor on naive CD4 T cells are essential for full-fledged CD4 T cell activation and the generation of CD4 memory T cells. Because the ligand for OX40 is mainly expressed by APCs, including activated B cells, dendritic cells, and Langerhans cells, the OX40-OX40 ligand (OX40L) interaction has been thought to participate in T cell-APC interactions. Although several reports have revealed the expression of OX40L on T cells, the functional significance of its expression on them is still unclear. In this study, we demonstrate that Ag stimulation induced an increase in the surface expression and transcript levels of OX40L in CD4 T cells. Upon contact with OX40 expressing T cells, the cell surface expression of OX40L on CD4 T cells was markedly down-regulated, suggesting that OX40-OX40L binding occurs through a novel T cell-T cell interaction. To investigate the function of this phenomenon, we examined the proliferative response and survival of OX40L-deficient CD4 T cells when challenged with Ag. In vitro studies demonstrated markedly less CD3 induced proliferation of OX40L-deficient CD4 T cells compared with wild-type CD4 T cells. When using TCR transgenic CD4 T cells upon Ag stimulation, survival of OX40L-deficient T cells was impaired. Furthermore, we show that upon antigenic stimulation, fewer OX40L-deficient CD4 T cells than wild-type cells survived following transfer into wild-type and sublethally irradiated recipient mice. Taken together, our findings indicate that OX40L-expressing T cells have an autonomous machinery that provides OX40 signals through a T cell-T cell circuit, creating an additional mechanism for sustaining CD4 T cell longevity. PMID- 16670307 TI - CD4+ T regulatory cell induction and function in transplant recipients after CD154 blockade is TLR4 independent. AB - Although the role of CD4(+) T regulatory cells (Treg) in transplantation tolerance has been established, putative mechanisms of Treg induction and function in vivo remain unclear. TLR4 signaling has been implicated in the regulation of CD4(+)CD25(+) Treg functions recently. In this study, we first examined the role of recipient TLR4 in the acquisition of operational CD4(+) Treg following CD154 blockade in a murine cardiac transplant model. Then, we determined whether TLR4 activation in allograft tolerant recipients would reverse alloimmune suppression mediated by CD4(+) Treg. We document that donor-specific immune tolerance was readily induced in TLR4-deficient recipients by a single dose of anti-CD154 mAb, similar to wild-type counterparts. The function and phenotype of CD4(+) Treg in both wild-type and TLR4 knockout long-term hosts was demonstrated by a series of depletion experiments examining their ability to suppress the rejection of secondary donor-type test skin grafts and to inhibit alloreactive CD8(+) T cell activation in vivo. Furthermore, TLR4 activation in tolerant recipients following exogenous LPS infusion in conjunction with donor type skin graft challenge, failed to break Treg-mediated immune suppression. In conclusion, our data reveals a distinctive property of CD4(+) Treg in tolerant allograft recipients, whose induction and function are independent of TLR4 signaling. PMID- 16670308 TI - In vivo and in vitro regulation of type I IFN synthesis by synergistic effects of CD40 and type II IFN. AB - During cognate interaction with CD40 ligand (CD154)-expressing T cells, Ag presenting accessory cells are activated for increased cytokine synthetic and costimulatory function. We examined whether CD40 modulates in vivo innate immune function over time, hypothesizing that distinct cytokine responses evolve to delayed microbial exposure. C3H/HeN mice pretreated with activating anti-CD40 Ab (FGK45) produced 10-fold more serum IFN-gamma and IL-12 p70 to delayed, but not synchronous, challenge with LPS. A novel finding was that LPS-induced IFN-alpha increased by 20-fold in mice pretreated for 24 h, but not 6 h or less, with anti CD40. Anti-CD40-pretreated C57BL/6 RAG-2(-/-) mice similarly increased IFN-alpha responses to delayed LPS challenge, confirming mediation by innate immunity. Type I IFNR- and IFN-gamma-deficient mice treated with anti-CD40 failed to expand serum IFN-alpha responses to LPS challenge. Combined pretreatment with anti-CD40 and anti-IFN-gamma mAb showed that IFN-gamma produced after anti-CD40 pretreatment, but before LPS challenge, was necessary for IFN-alpha synthetic enhancement. Anti-CD40 also increased polyinosinic-polycytidylic acid (poly(I:C)) inducible IFN-alpha by 5-fold in an IFN-gamma-dependent fashion, but did not significantly increase IFN-alpha production to CpG or Pam(3)Cys challenges. Poly(IC)-stimulated splenocytes from anti-CD40-pretreated mice produced 4-fold more IFN-alpha than controls and production associated with CD11c(+) cells. Finally, rIFN-gamma and anti-CD40 combined synergistically to increase poly(IC) inducible IFN-alpha synthetic capacity in bone marrow dendritic cells. We conclude that innate immune production of IFN-alpha is cooperatively regulated by CD40 and IFN-gamma acting on dendritic cells, suggesting a unique mechanism by which innate immune function evolves in response to specific adaptive immune signals. PMID- 16670309 TI - Defective IgG2a/2b class switching in PKC alpha-/- mice. AB - Using model tumor T cell lines, protein kinase C (PKC) alpha has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCalpha null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3(+) PKCalpha deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-kappaB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-gamma production are severely impaired in PKCalpha(-/-) CD3(+) T cells. Consistently, PKCalpha-deficient CD3(+) T cells from OVA-immunized PKCalpha deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCalpha-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCalpha(-/-) B cell proliferation are unimpaired. Our genetic data, thus, define PKCalpha as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell dependent IFN-gamma production and IgG2a/2b Ab responses. PMID- 16670310 TI - Trem-like transcript 2 is expressed on cells of the myeloid/granuloid and B lymphoid lineage and is up-regulated in response to inflammation. AB - The triggering receptor expressed on myeloid cells (TREM) gene cluster encodes a group of transmembrane proteins that are emerging as important components in innate and adaptive immunity. In both mice and humans, the TREM gene cluster encodes eight receptors; only four of these, however, are direct homologs: TREM 1, TREM-2, TREM-like transcript 1 (TLT1), and TLT2. Of the transmembrane receptors encoded by the four conserved genes within this cluster, TLT2 has not been studied previously. Data presented in this study demonstrate that TLT2 is expressed early in B cell development in conjunction with B220 and is detected on all developing mouse B cell populations as well as B cells in the periphery. TLT2 expression on B cells in the periphery exhibits a distinct hierarchy with the highest detectable levels observed on B1 B cells in the peritoneum. The overall gradation of TLT2 expression on B cells is: B1 > marginal zone/transitional 2 > transitional 1 > follicular. Additionally, TLT2 expression was observed on mouse neutrophils throughout the body. Although monocytes were not observed to express TLT2, resident peritoneal and lung macrophages do express TLT2, suggesting that it is up-regulated in association with terminal differentiation of monocytes. Finally, both neutrophils and macrophages were observed to up-regulate TLT2 expression in vivo in response to inflammatory stimuli, whereas TLT2 expression on B cells remained unchanged. In conclusion, the data suggest that TLT2 may be involved in the innate immune response based on its expression profile and the fact that it is up-regulated in response to inflammation. PMID- 16670311 TI - Costimulation of chemokine receptor signaling by matrix metalloproteinase-9 mediates enhanced migration of IFN-alpha dendritic cells. AB - Type I IFNs induce differentiation of dendritic cells (DCs) with potent Ag presenting capacity, termed IFN-alpha DCs, that have been implicated in the pathogenesis of systemic lupus erythematosus. In this study, we found that IFN alpha DCs exhibit enhanced migration across the extracellular matrix (ECM) in response to chemokines CCL3 and CCL5 that recruit DCs to inflammatory sites, but not the lymphoid-homing chemokine CCL21. IFN-alpha DCs expressed elevated matrix metalloproteinase-9 (MMP-9), which mediated increased migration across ECM. Unexpectedly, MMP-9 and its cell surface receptors CD11b and CD44 were required for enhanced CCL5-induced chemotaxis even in the absence of a matrix barrier. MMP 9, CD11b, and CD44 selectively modulated CCL5-dependent activation of JNK that was required for enhanced chemotactic responses. These results establish the migratory phenotype of IFN-alpha DCs and identify an important role for costimulation of chemotactic responses by synergistic activation of JNK. Thus, cell motility is regulated by integrating signaling inputs from chemokine receptors and molecules such as MMP-9, CD11b, and CD44 that also mediate cell interactions with inflammatory factors and ECM. PMID- 16670312 TI - Dual effects of Sprouty1 on TCR signaling depending on the differentiation state of the T cell. AB - Sprouty (Spry) is known to be a negative feedback inhibitor of growth factor receptor signaling through inhibition of the Ras/MAPK pathway. Several groups, however, have reported a positive role for Spry involving sequestration of the inhibitory protein c-Cbl. Thus, Spry may have various functions in the regulation of receptor-mediated signaling depending on the context. In the immune system, the function of Spry is unknown. In this study, we investigated the role of Spry1 in T cell activation. Spry1, among the four mammalian homologs, was specifically induced by TCR signaling of CD4(+) murine T cells. In fully differentiated Th1 clones, overexpressed Spry1 inhibited TCR signaling and decreased IL-2 production while reducing expression with specific siRNA transfection had the opposite effect, increasing IL-2 production. In contrast, in naive T cells, Spry1 overexpression enhanced TCR signaling, and increased proliferation and IL-2 production, while siRNA transfection again had the opposite effect, reducing IL-2 production following activation. The enhancing effect in naive cells was abrogated by preactivation of the T cells with Ag and APC, indicating that the history of exposure to Ag is correlated with a hierarchy of T cell responsiveness to Spry1. Furthermore, both the NF-AT and MAPK pathways were influenced by Spry1, implying a different molecular mechanism from that for growth factor receptor signaling. Thus, Spry1 uses a novel mechanism to bring about differential effects on TCR signaling through the same receptor, depending on the differentiation state of the T cell. PMID- 16670313 TI - Targeting melanoma cells with human high molecular weight-melanoma associated antigen-specific antibodies elicited by a peptide mimotope: functional effects. AB - Human high molecular weight-melanoma associated Ag (HMW-MAA) mimics have been shown to elicit HMW-MAA-specific humoral immune responses that appear to be clinically beneficial. This finding has stimulated interest in characterizing the mechanism(s) underlying the ability of the elicited Abs to exert an anti-tumor effect. To address this question, in the present study, we have generated HMW-MAA specific Abs by sequentially immunizing rabbits with the peptide P763.74, which mimics the HMW-MAA determinant recognized by mAb 763.74, and with HMW-MAA(+) melanoma cells. HMW-MAA-specific Abs isolated from immunized rabbits mediated cell-dependent cytotoxicity but did not mediate complement-dependent cytotoxicity of HMW-MAA(+) melanoma cells. These Abs also effectively inhibited spreading, migration and Matrigel invasion of HMW-MAA(+) melanoma cells. Besides contributing to our understanding of the role of HMW-MAA in the biology of melanoma cells, these results suggest that both immunological and nonimmunological mechanisms underlie the beneficial clinical effects associated with the induction of HMW-MAA-specific Abs in melanoma patients immunized with a HMW-MAA mimic. PMID- 16670314 TI - Advanced intercross line mapping of Eae5 reveals Ncf-1 and CLDN4 as candidate genes for experimental autoimmune encephalomyelitis. AB - Eae5 in rats was originally identified in two F(2) intercrosses, (DA x BN) and (E3 x DA), displaying linkage to CNS inflammation and disease severity in experimental autoimmune encephalomyelitis (EAE), respectively. This region overlaps with an arthritis locus, Pia4, which was also identified in the (E3 x DA) cross. Two congenic strains, BN.DA-Eae5 and BN.DA-Eae5.R1, encompassing the previously described Eae5 and Pia4, were established. DA alleles within the chromosome 12 fragment conferred an increase in disease susceptibility as well as increased inflammation and demyelination in the CNS as compared with BN alleles. To enable a more precise fine mapping of EAE regulatory genes, we used a rat advanced intercross line between the EAE-susceptible DA strain and the EAE resistant PVG.1AV1 strain. Linkage analysis performed in the advanced intercross line considerably narrowed down the myelin oligodendrocyte glycoprotein-EAE regulatory locus (Eae5) to a approximately 1.3-megabase region with a defined number of candidate genes. In this study we demonstrate a regulatory effect of Eae5 on MOG-EAE by using both congenic strains as well as fine mapping these effects to a region containing Ncf-1, a gene associated with arthritis. In addition to structural polymorphisms in Ncf-1, both sequence polymorphisms and expression differences were identified in CLDN4. CLDN4 is a tight junction protein involved in blood-brain barrier integrity. In conclusion, our data strongly suggests Ncf-1 to be a gene shared between two organ-specific inflammatory diseases with a possible contribution by CLDN4 in encephalomyelitis. PMID- 16670315 TI - Induction of primary human T cell responses against hepatitis C virus-derived antigens NS3 or core by autologous dendritic cells expressing hepatitis C virus antigens: potential for vaccine and immunotherapy. AB - Hepatitis C virus (HCV)-specific T cell responses have been suggested to play significant role in viral clearance. Dendritic cells (DCs) are professional APCs that play a major role in priming, initiating, and sustaining strong T cell responses against pathogen-derived Ags. DCs also have inherent capabilities of priming naive T cells against given Ags. Recombinant adenoviral vectors containing HCV-derived Core and NS3 genes were used to endogenously express HCV Core and NS3 proteins in human DCs. These HCV Ags expressing DCs were used to prime and stimulate autologous T cells obtained from uninfected healthy donors. The DCs expressing HCV Core or NS3 Ags were able to stimulate T cells to produce various cytokines and proliferate in HCV Ag-dependent manner. Evidence of both CD4(+) and CD8(+) T cell responses against HCV Core and NS3 generated in vitro were obtained by flow cytometry and Ab blocking experiments. Further, in secondary assays, the T cells primed in vitro exhibited HCV Ag-specific proliferative responses against recombinant protein Ags and also against immunodominant permissive peptide epitopes from HCV Ags. In summary, we demonstrate that the dendritic cells expressing HCV Ags are able to prime the Ag specific T cells from uninfected healthy individuals in vitro. These studies have implications in designing cellular vaccines, T cell adoptive transfer therapy or vaccine candidates for HCV infection in both prophylactic and therapeutic settings. PMID- 16670316 TI - Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth. AB - All members of the Bacillus genus produce endospores as part of their life cycle; however, it is not possible to determine the identity of spores by casual or morphological examination. The 2001 anthrax attacks demonstrated a need for fast, dependable methods for detecting Bacillus anthracis spores in vitro and in vivo. We have developed a variety of isotypes and specificities of mAbs that were able to distinguish B. anthracis spores from other Bacillus spores. The majority of Abs were directed toward BclA, a major component of the exosporium, although other components were also distinguished. These Abs did not react with vegetative forms. Some Abs distinguished B. anthracis spores from spores of distantly related species in a highly specific manner, whereas others discriminated among strains that are the closest relatives of B. anthracis. These Abs provide a rapid and reliable means of identifying B. anthracis spores, for probing the structure and function of the exosporium, and in the analysis of the life cycle of B. anthracis. PMID- 16670317 TI - Cross-neutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein. AB - The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a protective Ag for vaccine design. We previously demonstrated that the receptor-binding domain (RBD) of S protein contains multiple conformational epitopes (Conf I-VI) that confer the major target of neutralizing Abs. Here we show that the recombinant RBDs derived from the S protein sequences of Tor2, GD03, and SZ3, the representative strains of human 2002-2003 and 2003 2004 SARS-CoV and palm civet SARS-CoV, respectively, induce in the immunized mice and rabbits high titers of cross-neutralizing Abs against pseudoviruses expressing S proteins of Tor2, GD03, and SZ3. We also demonstrate that the Tor2 RBD induced-Conf I-VI mAbs can potently neutralize both human SARS-CoV strains, Tor2 and GD03. However, only the Conf IV-VI, but not Conf I-III mAbs, neutralize civet SARS-CoV strain SZ3. All these mAbs reacted significantly with each of the three RBD variants (Tor2-RBD, GD03-RBD, and SZ3-RBD) that differ at several amino acids. Regardless, the Conf I-IV and VI epitopes were completely disrupted by single-point mutation of the conserved residues in the RBD (e.g., D429A, R441A, or D454A) and the Conf III epitope was significantly affected by E452A or D463A substitution. Interestingly, the Conf V epitope, which may overlap the receptor binding motif and induce most potent neutralizing Abs, was conserved in these mutants. These data suggest that the major neutralizing epitopes of SARS-CoV have been apparently maintained during cross-species transmission, and that RBD-based vaccines may induce broad protection against both human and animal SARS-CoV variants. PMID- 16670318 TI - The proteasome pathway destabilizes Yersinia outer protein E and represses its antihost cell activities. AB - Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein. PMID- 16670319 TI - Defective adhesion in tumor infiltrating CD8+ T cells. AB - CD8(+) tumor-infiltrating lymphocytes (TIL) are defective in cytolysis due to tumor-induced inhibition of proximal TCR-mediated signaling, a defect that is relieved upon purification and brief culture. We show in this study that frequency of conjugation in vitro of nonlytic TIL with tumor cells is low in comparison with their lytic counterparts, and the strength of interaction and duration of conjugation are also reduced. Previous reports show that p56(lck) activation is required for TCR-initiated LFA-1 avidity up-regulation, raising the question: is low LFA-1 avidity the basis of reduced TIL conjugation frequency? When stimulated with phorbol ester, nonlytic TIL bind purified ICAM-1 equivalently as lytic TIL, suggesting that LFA-1 can be activated if proximal TCR signaling is bypassed. However, when treated with phorbol ester, the conjugation frequency of nonlytic TIL does not increase. CD2 and CD8 also mediate T cell adhesion to cognate target cells and are both expressed at lower levels in nonlytic TIL in addition to being excluded from the immune synapse formed upon conjugation. Collectively, these results imply that adhesion defects in nonlytic TIL result from a combination of decreased cell surface levels of adhesion molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the membrane contact site formed with cognate target cells. PMID- 16670320 TI - Roles of the alternative complement pathway and C1q during innate immunity to Streptococcus pyogenes. AB - Complement is important for innate immunity to the common bacterial pathogen Streptococcus pyogenes, but the relative importance of the alternative and classical pathways has not been investigated. Using mice and human serum deficient in either C1q, the first component of the classical pathway, or factor B, an important component of the alternative pathway, we have investigated the role of both pathways for innate immunity to S. pyogenes. C3b deposition on four different strains of S. pyogenes was mainly dependent on factor B. As a consequence opsonophagocytosis of S. pyogenes was reduced in serum from factor B deficient mice, and these mice were very susceptible to S. pyogenes infection. In contrast, C3b deposition was not dependent on C1q for two of the strains investigated, H372 and H305, yet opsonophagocytosis of all four S. pyogenes strains was impaired in serum deficient in C1q. Furthermore, infection in C1q deficient mice with strain H372 resulted in a rapidly progressive disease associated with large numbers of bacteria in target organs. These results demonstrate the important role of the alternative pathway and C1q for innate immunity to S. pyogenes and suggest that C1q-mediated innate immunity to at least some strains of S. pyogenes may involve mechanisms that are independent of C3b on the bacteria. PMID- 16670321 TI - CD94/NKG2A expression is associated with proliferative potential of CD8 T cells during persistent polyoma virus infection. AB - Memory CD8 T cells comprise a critical component of durable immunity because of their capacity to rapidly proliferate and exert effector activity upon Ag rechallenge. During persistent viral infection, memory CD8 T cells repetitively encounter viral Ag and must maintain a delicate balance between limiting viral replication and minimizing immunopathology. In mice infected by polyoma virus, a natural mouse pathogen that establishes long-term persistent infection, the majority of persistence-phase antiviral CD8 T cells express the inhibitory NK cell receptor CD94/NKG2A. In this study, we asked whether CD94/NKG2A expression is associated with Ag-specific recall of polyoma virus-specific CD8 T cells. During the persistent phase of infection, polyoma virus-specific CD8 T cells that express CD94/NKG2A were found to preferentially proliferate; this proliferation was dependent on cognate Ag both in vitro and in vivo. In addition, CD94/NKG2A(+) polyoma-specific CD8 T cells have a markedly enhanced capacity to produce IL-2 upon ex vivo Ag stimulation compared with CD94/NKG2A(-) polyoma-specific CD8 T cells. Importantly, CD94/NKG2A(+) anti-polyoma virus CD8 T cells appear to be essential for Ag-specific recall responses in mice persistently infected by polyoma virus. Because of its higher proliferative potential and capacity to produce IL-2, we propose that the CD94/NKG2A(+) subpopulation represents a less differentiated state than the CD94/NKG2A(-) subpopulation. Identification of proliferation-competent subpopulations of memory CD8 T cells should prove valuable in designing therapeutic vaccination strategies for persistent viral infections. PMID- 16670322 TI - HIV-1 epitope-specific CD8+ T cell responses strongly associated with delayed disease progression cross-recognize epitope variants efficiently. AB - The ability of HIV-1-specific CD8(+) T cell responses to recognize epitope variants resulting from viral sequence variation in vivo may affect the ease with which HIV-1 can escape T cell control and impact on the rate of disease progression in HIV-1-infected humans. Here, we studied the functional cross reactivity of CD8 responses to HIV-1 epitopes restricted by HLA class I alleles associated with differential prognosis of infection. We show that the epitope specific responses exhibiting the most efficient cross-recognition of amino acid substituted variants were those strongly associated with delayed progression to disease. Not all epitopes restricted by the same HLA class I allele showed similar variant cross-recognition efficiency, consistent with the hypothesis that the reported associations between particular HLA class I alleles and rate of disease progression may be due to the quality of responses to certain "critical" epitopes. Irrespective of their efficiency of functional cross-recognition, CD8(+) T cells of all HIV-1 epitope specificities examined showed focused TCR usage. Furthermore, interpatient variability in variant cross-reactivity correlated well with use of different dominant TCR Vbeta families, suggesting that flexibility is not conferred by the overall clonal breadth of the response but instead by properties of the dominant TCR(s) used for epitope recognition. A better understanding of the features of T cell responses associated with long term control of viral replication should facilitate rational vaccine design. PMID- 16670323 TI - B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection. AB - B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs. PMID- 16670324 TI - Anthrax lethal toxin has direct and potent inhibitory effects on B cell proliferation and immunoglobulin production. AB - Protective host immune responses to anthrax infection in humans and animal models are characterized by the development of neutralizing Abs against the receptor binding anthrax protective Ag (PA), which, together with the lethal factor (LF) protease, composes anthrax lethal toxin (LT). We now report that B cells, in turn, are targets for LT. Anthrax PA directly binds primary B cells, resulting in the LF-dependent cleavage of the MAPK kinases (MAPKKs) and disrupted signaling to downstream MAPK targets. Although not directly lethal to B cells, anthrax LT treatment causes severe B cell dysfunction, greatly reducing proliferative responses to IL-4-, anti-IgM-, and/or anti-CD40 stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have a markedly diminished capacity to proliferate and produce IgM in response to TLR-2 and TLR-4 ligands. The suppressive effects of anthrax LT on B cell function occur at picomolar concentrations in vitro and at sublethal doses in vivo. These results indicate that anthrax LT directly inhibits the function of B cells in vitro and in vivo, revealing a potential mechanism through which the pathogen could bypass protective immune responses. PMID- 16670325 TI - MyD88-dependent IFN-gamma production by NK cells is key for control of Legionella pneumophila infection. AB - Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium in aquatic systems and an opportunistic intracellular pathogen in immunocompromised humans causing a severe pneumonia known as Legionnaires' disease. Using a mouse model, we investigated molecular and cellular players in the innate immune response to infection with Lpn. We observed robust levels of inflammatory cytokines in the serum upon intranasal or i.v. infection with live, virulent Lpn, but not with inactivated or avirulent bacteria lacking the Icm/Dot type IV secretion system. Interestingly, Lpn-induced serum cytokines were readily detectable regardless of the capacity of Icm/Dot-proficient Lpn to replicate in host cells and the Lpn permissiveness of the host mice. We found NK cell-derived IFN-gamma to be the key cytokine in the resolution of Lpn infection, whereas type I IFNs did not appear to play a major role in our model. Accordingly, NK cell-depleted or IFN-II-R deficient mice carried severely increased bacterial burdens or failed to control Lpn infection, respectively. Besides the dependence of inflammatory cytokine induction on Lpn virulence, we also demonstrate a strict requirement of MyD88 for this process, suggesting the involvement of TLRs in the recognition of Lpn. However, screening of several TLR-deficient hosts did not reveal a master TLR responsible for the sensing of an Lpn infection, but provided evidence for either redundancy of individual TLRs in Lpn recognition or TLR-independent induction of inflammatory responses. PMID- 16670326 TI - Immature human dendritic cells infected with Leishmania infantum are resistant to NK-mediated cytolysis but are efficiently recognized by NKT cells. AB - Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-gamma. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania. PMID- 16670327 TI - MyD88-dependent signaling for IL-15 production plays an important role in maintenance of CD8 alpha alpha TCR alpha beta and TCR gamma delta intestinal intraepithelial lymphocytes. AB - Interaction between commensal bacteria and intestinal epithelial cells (i-ECs) via TLRs is important for intestinal homeostasis. In this study, we found that the numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta intestinal intraepithelial lymphocytes (i-IELs) were significantly decreased in MyD88 deficient (-/-) mice. The expression of IL-15 by i-ECs was severely reduced in MyD88(-/-) mice. Introduction of IL-15 transgene into MyD88(-/-) mice (MyD88(-/-) IL-15 transgenic mice) partly restored the numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta i-IELs. The i-IEL in irradiated wild-type (WT) mice transferred with MyD88(-/-) bone marrow (BM) cells had the same proportions of i-IEL as WT mice, whereas those in irradiated MyD88(-/-) mice transferred with WT BM cells showed significantly reduced proportions of CD8alphaalpha TCRalphabeta and TCRgammadelta i-IELs, as was similar to the proportions found in MyD88(-/-) mice. However, irradiated MyD88(-/-) IL-15 transgenic mice transferred with WT BM cells had increased numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta subsets in the i-IEL. These results suggest that parenchymal cells such as i-ECs contribute to the maintenance of CD8alphaalpha TCRalphabeta and gammadelta i-IELs at least partly via MyD88-dependent IL-15 production. PMID- 16670328 TI - IL-17 receptor knockout mice have enhanced myelotoxicity and impaired hemopoietic recovery following gamma irradiation. AB - IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline, the hemopoietic system of IL-17R knockout mice (IL-17Ra(-/-)) is, with the exception of increased splenic progenitor numbers, indistinguishable from normal control mice. However, when challenged with gamma irradiation, hemopoietic toxicity is significantly more pronounced in IL-17Ra(-/-) animals, with the gamma irradiation-associated LD(50) being reduced by 150 rad. In spleen-derived T cells, gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad), the infusion of purified CD4(+) T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra(-/-) animals or after in vivo blockade of IL 17Ra in normal mice, resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression, in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte, CFU-high proliferative potential), as well as 2- and 3-fold increases of bone marrow precursors, respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury. PMID- 16670329 TI - TLR5-mediated phosphoinositide 3-kinase activation negatively regulates flagellin induced proinflammatory gene expression. AB - Epithelial cells detect motile pathogens via TLR5 ligation of flagellin, resulting in rapid induction of antibacterial/proinflammatory gene expression. Although such flagellin-induced gene expression is quite transient, likely to avoid the negative consequences of inflammation, little is known regarding the molecular mechanisms that mediate its shutdown. We hypothesized that, analogous to the case for TLR4, phosphoinositide 3-kinase (PI3K) might negatively regulate TLR5 signaling. However, because PI3K is an essential positive mediator of some pathways of TLR-mediated gene expression, the opposite hypothesis was also considered. Herein, we observed that flagellin stimulation of epithelial cells indeed induced rapid (<30 min) PI3K activation, as evidenced by Akt phosphorylation, via a TLR5-mediated mechanism. Blockade of PI3K with wortmannin resulted in marked enhancement of flagellin-induced gene expression as assessed by measuring levels of inducible NO synthase, IL-6, and IL-8. Such enhancement of gene expression by PI3K inhibition correlated with prolonged activation of MAPK (p38 and ERK1/2) and was ablated under MAPK inhibition. Such effect of inhibiting PI3K with wortmannin was mimicked by the PI3K inhibitor LY294002, and, conversely, a constitutively active PI3K prevented p38 activation in response to flagellin. Last, to test the significance of these results in vivo, we measured flagellin-induced gene expression in PI3K knockout mice. PI3K-null mice displayed increased levels of flagellin-induced serum IL-6, KC (IL-8 homolog), and nitrite as compared with heterozygous littermates. Thus, TLR5's rapid activation of PI3K serves to limit MAPK signaling, thus limiting proinflammatory gene expression and reducing the potential negative consequences of proinflammatory gene expression. PMID- 16670330 TI - Modulation of dendritic cell function by naive and regulatory CD4+ T cells. AB - The consequences of interactions between dendric cells (DCs) and either naive CD4+ T cells or regulatory CD4+CD25+ T cells on the expression of proinflammatory IL-6 and anti-inflammatory IL-10 in DC were examined over a period of 12 h, spanning the time frame during which stable T cell-DC interactions shape the development of tolerance and immunity in vivo. We demonstrate that the basal production of IL-6 and IL-10, which is initiated following DC stimulation with LPS, is modified in distinctly different ways by interaction with the two T cell populations. Naive CD4 T cells skew DC cytokine production toward IL-6 and suppress IL-10, whereas CD4+CD25+ T cells have the opposite effect. CD8 T cells or memory CD4 T cells do not influence basal cytokine production by stimulated DC. The effect of CD4+CD25+ T cells is dominant in coculture with naive CD4 T cells as long as inflammatory LPS is absent; the addition of LPS abrogates the suppression of IL-6. However, the modulating influence of CD4+CD25+ T cells remains evident in the enhancement of IL-10 production. Thus, mutual interactions between DC and CD4+ T cell subpopulations following contact with pathogens are likely to influence the strength and quality of incipient immune responses in the local microenvironment. PMID- 16670331 TI - Regulatory roles for MD-2 and TLR4 in ligand-induced receptor clustering. AB - LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe(119) to Lys(132) (Arg(132) in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly(59) was found to be a novel critical amino acid for LPS binding outside the region 119-132. LPS signaling is thought to be triggered by ligand induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe(126) or Gly(129) impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition. PMID- 16670332 TI - TLRs mediate IFN-gamma production by human uterine NK cells in endometrium. AB - The human endometrium (EM) contains macrophages, NK cells, T cells, B cells, and neutrophils in contact with a variety of stromal and epithelial cells. The interplay between these different cell types and their roles in defense against pathogen invasion in this specialized tissue are important for controlling infection and reproduction. TLRs are a family of receptors able to recognize conserved pathogen-associated molecular patterns. In this study, we determined the expression of TLRs on uterine NK (uNK) cells from the human EM and the extent to which uNK cells responded to TLR agonist stimulation. uNK cells expressed TLRs 2, 3, and 4, and produced IFN-gamma when total human endometrial cells were stimulated with agonists to TLR2 or TLR3 (peptidoglycan or poly(I:C), respectively). Activated uNK cell clones produced IFN-gamma upon stimulation with peptidoglycan or poly(I:C). However, purified uNK cells did not respond directly to TLR agonists, but IFN-gamma was produced by uNK cells in response to TLR stimulation when cocultured with APCs. These data indicate that uNK cells express TLRs and that they can respond to TLR agonists within EM by producing IFN-gamma. These data also indicate that the uNK cells do not respond directly to TLR stimulation, but rather their production of IFN-gamma is dependent upon interactions with other cells within EM. PMID- 16670333 TI - Reevaluation of P-selectin and alpha 4 integrin as targets for the treatment of experimental autoimmune encephalomyelitis. AB - There has been a great deal of interest in adhesion molecules as targets for the treatment of multiple sclerosis and other inflammatory diseases. In this study, we systematically evaluate alpha(4) integrin and P-selectin as targets for therapy in murine models of multiple sclerosis-for the first time directly measuring the ability of their blockade to inhibit recruitment and relate this to clinical efficacy. Experimental autoimmune encephalomyelitis was induced in C57BL/6 or SJL/J mice and intravital microscopy was used to quantify leukocyte interactions within the CNS microvasculature. In both strains, pretreatment with blocking Abs to either alpha(4) integrin or P-selectin reduced firm adhesion to a similar extent, but did not block it completely. The combination of the Abs was more effective than either Ab alone, although the degree of improvement was more evident in SJL/J mice. Similarly, dual blockade was much more effective at preventing the subsequent accumulation of fluorescently labeled leukocytes in the tissue in both strains. Despite evidence of blockade of leukocyte recruitment mechanisms, no clinical benefit was observed with anti-adhesion molecule treatments or genetic deletion of P-selectin in the C57BL/6 model, or in a pertussis toxin-modified model in SJL/J mice. In contrast, Abs to alpha(4) integrin resulted in a significant delay in the onset of clinical signs of disease in the standard SJL/J model. Despite evidence of a similar ability to block firm adhesion, Abs to P-selectin had no effect. Importantly, combined blockade of both adhesion molecules resulted in significantly better clinical outcome than anti-alpha(4) integrin alone. PMID- 16670334 TI - Fas ligand is responsible for CXCR3 chemokine induction in CD4+ T cell-dependent liver damage. AB - Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4(+) T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core x TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4(+) T cells into the liver of the immunized core(+) TCR mice but not their core(-) TCR littermates. Strikingly, the inflammatory process in the liver of core(+) TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-gamma production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3(+) lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-gamma and cellular infiltration into the liver. These findings suggest that activated CD4(+) T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines. PMID- 16670335 TI - C/EBP homologous protein (CHOP) is crucial for the induction of caspase-11 and the pathogenesis of lipopolysaccharide-induced inflammation. AB - C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 is a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-mediated apoptosis. To determine whether the ER stress-CHOP pathway is involved in the pathogenesis of the lung inflammation, mice were given LPS intratracheally. Treatment with LPS induced mRNAs for CHOP and BiP. The LPS induced inflammation in lung, including the IL-1beta activity in bronchoalveolar lavage fluid, was attenuated in the Chop knockout mice. Caspase-11, which is needed for the activation of procaspase-1 and pro-IL-1beta, was induced by LPS treatment in the lung and primary cultured macrophages. The induction of caspase 11 by LPS was suppressed in Chop knockout mice. Caspase-11 was also induced by such ER stress inducers as thapsigargin or tunicamycin. These results show that CHOP plays a crucial role in the pathogenesis of inflammation through the induction of caspase-11. PMID- 16670336 TI - Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice. AB - Leukotriene B(4) mediates diverse inflammatory diseases through the G protein coupled receptors BLT1 and BLT2. In this study, we developed mice deficient in BLT1 and BLT2 by simultaneous targeted disruption of these genes. The BLT1/BLT2 double-deficient mice developed normally and peritoneal exudate cells showed no detectable responses to leukotriene B(4) confirming the deletion of the BLT1/BLT2 locus. In a model of collagen-induced arthritis on the C57BL/6 background, the BLT1/BLT2(-/-) as well as the previously described BLT1(-/-) animals showed complete protection from disease development. The disease severity correlated well with histopathology, including loss of joint architecture, inflammatory cell infiltration, fibrosis, pannus formation, and bone erosion in joints of BLT1/BLT2(+/+) animals and a total absence of disease pathology in leukotriene receptor-deficient mice. Despite these differences, all immunized BLT1(-/-) and BLT1/BLT2(-/-) animals had similar serum levels of anti-collagen Abs relative to BLT1/BLT2(+/+) animals. Thus, BLT1 may be a useful target for therapies directed at treating inflammation associated with arthritis. PMID- 16670337 TI - Interleukin-17 acts independently of TNF-alpha under arthritic conditions. AB - The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-alpha. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-alpha-deficient mice that TNF-alpha is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/BxN serum transfer arthritis to a similar degree in TNF-alpha-deficient mice and their wild type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17 enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions. PMID- 16670338 TI - Control of metastasized pancreatic carcinomas in SCID/beige mice with human IL 2/TKD-activated NK cells. AB - Pancreatic carcinoma, the fifth leading cause of cancer-related mortality, frequently presents the stress-inducible heat shock protein 70 (Hsp70) on the cell membrane. Therefore, we explored an immunological approach exploiting the efficacy of NK cells activated either with low dose IL-2 plus Hsp70-peptide TKDNNLLGRFELSG (TKD; IL-2/TKD) or with IL-2 alone in a xenograft pancreatic carcinoma model. An orthotopic injection of either 2.5 x 10(6) or 1 x 10(6) Colo357 cells in SCID/beige mice resulted in rapidly growing primary tumors and the development of hepatic metastases on days 5 and 10, respectively. In line with results of in vitro migration assays, these NK cells also had the capacity to infiltrate pancreatic tumors and liver metastases in tumor-bearing mice. In vitro, a combined treatment of NK cells with IL-2/TKD but neither of the two treatments alone causes a profound increase in the lytic capacity against Hsp70 membrane-positive Colo357 cells. In vivo, a single i.v. injection of these NK cells on day 15 post-tumor inoculation resulted in a significant reduction in tumor weights, a delayed onset of hepatic metastases, and a prolonged life expectancy. In contrast, identically treated T cells and NK cells treated with IL 2 alone were significantly less efficient in controlling pancreatic tumors and metastases. Most importantly, four repeated i.v. infusions of IL-2/TKD-activated NK cells eradicated primary tumors and prevented hepatic metastases. In summary, our mouse data have implicated that NK cells preactivated with IL-2/TKD might provide a novel therapeutic tool for the treatment of aggressive, Hsp70-positive pancreatic carcinoma. PMID- 16670339 TI - The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death. AB - Patients affected by Sjogren's syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sjogren's syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sjogren's syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients. PMID- 16670340 TI - Involvement of CCL18 in allergic asthma. AB - Allergic asthma is associated with a pulmonary recruitment of Th type 2 cells, basophils, and eosinophils, mainly linked to chemokine production. CCL18 is a chemokine preferentially expressed in the lung, secreted by APCs, induced by Th2 type cytokines, and only present in humans. Therefore, CCL18 may be involved in allergic asthma. PBMC from asthmatics allergic to house dust mite cultured in the presence of Dermatophagoides pteronyssinus 1 (Der p 1) allergen secreted CCL18, 48 and 72 h after stimulation, whereas those from healthy donors did not. Part of CCL18 was directly derived from Der p 1-stimulated plasmacytoid dendritic cells, whereas the other part was linked to monocyte activation by IL-4 and IL-13 produced by Der p 1-stimulated T cells. In bronchoalveolar lavages from untreated asthmatic allergic patients, CCL18 was highly increased compared with controls. Functionally, CCL18 preferentially attracted in vitro-polarized Th2 cells and basophils, but not eosinophils and Th1 cells, and induced basophil histamine and intracellular calcium release. These data show a new function for CCL18, i.e., the recruitment of Th2 cells and basophils, and suggest that CCL18 may play a predominant role in allergic asthma. PMID- 16670341 TI - TNF-alpha is necessary for induction of coronary artery inflammation and aneurysm formation in an animal model of Kawasaki disease. AB - Kawasaki disease is the most common cause of multisystem vasculitis in childhood. The resultant coronary artery lesions make Kawasaki disease the leading cause of acquired heart disease in children in the developed world. TNF-alpha is a pleiotropic inflammatory cytokine elevated during the acute phase of Kawasaki disease. In this study, we report rapid production of TNF-alpha in the peripheral immune system after disease induction in a murine model of Kawasaki disease. This immune response becomes site directed, with migration to the coronary arteries dependent on TNF-alpha-mediated events. Production of TNF-alpha in the heart is coincident with the presence of inflammatory infiltrate at the coronary arteries, which persists during development of aneurysms. More importantly, inflammation and elastin breakdown in the coronary vessels are completely eliminated in the absence of TNF-alpha effector functions. Mice treated with the TNF-alpha-blocking agent etanercept, as well as TNFRI knockout mice, are resistant to development of both coronary arteritis and coronary aneurysm formation. Taken together, TNF alpha is necessary for the development of coronary artery lesions in an animal model of Kawasaki disease. These findings have important implications for potential new therapeutic interventions in children with Kawasaki disease. PMID- 16670342 TI - Proteasome inhibition to maximize the apoptotic potential of cytokine therapy for murine neuroblastoma tumors. AB - Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-gamma plus TNF-alpha, we hypothesized that bortezomib also could sensitize these cells to IFN-gamma plus TNF-alpha. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-gamma and TNF-alpha on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN gamma plus TNF-alpha in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-gamma/TNF-alpha-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma. PMID- 16670343 TI - CXCR3+CD4+ T cells mediate innate immune function in the pathophysiology of liver ischemia/reperfusion injury. AB - Ischemia-reperfusion injury (IRI), an innate immune-dominated inflammatory response, develops in the absence of exogenous Ags. The recently highlighted role of T cells in IRI raises a question as to how T lymphocytes interact with the innate immune system and function with no Ag stimulation. This study dissected the mechanism of innate immune-induced T cell recruitment and activation in rat syngeneic orthotopic liver transplantation (OLT) model. Liver IRI was induced after cold storage (24-36 h) at 4 degrees C in University of Wisconsin solution. Gene products contributing to IRI were identified by cDNA microarray at 4-h posttransplant. IRI triggered increased intrahepatic expression of CXCL10, along with CXCL9 and 11. The significance of CXCR3 ligand induction was documented by the ability of neutralizing anti-CXCR3 Ab treatment to ameliorate hepatocellular damage and improve 14-day survival of 30-h cold-stored OLTs (95 vs 40% in controls; p < 0.01). Immunohistology analysis confirmed reduced CXCR3+ and CD4+ T cell infiltration in OLTs after treatment. Interestingly, anti-CXCR3 Ab did not suppress innate immune activation in the liver, as evidenced by increased levels of IL-1beta, IL-6, inducible NO synthase, and multiple neutrophil/monokine targeted chemokine programs. In conclusion, this study demonstrates a novel mechanism of T cell recruitment and function in the absence of exogenous Ag stimulation. By documenting that the execution of innate immune function requires CXCR3+CD4+ T cells, it highlights the critical role of CXCR3 chemokine biology for the continuum of innate to adaptive immunity in the pathophysiology of liver IRI. PMID- 16670344 TI - Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses. AB - Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology. PMID- 16670345 TI - T cell responses are better correlates of vaccine protection in the elderly. AB - It is commonly held that increased risk of influenza in the elderly is due to a decline in the Ab response to influenza vaccination. This study prospectively evaluated the relationship between the development of influenza illness, and serum Ab titers and ex vivo cellular immune responses to influenza vaccination in community dwelling older adults including those with congestive heart failure (CHF). Adults age 60 years and older (90 subjects), and 10 healthy young adult controls received the 2003-04 trivalent inactivated influenza vaccine. Laboratory diagnosed influenza (LDI) was documented in 9 of 90 older adults. Pre- and postvaccination Ab titers did not distinguish between subjects who would subsequently develop influenza illness (LDI subjects) and those who would not (non-LDI subjects). In contrast, PBMC restimulated ex vivo with live influenza virus preparations showed statistically significant differences between LDI and non-LDI subjects. The mean IFN-gamma:IL-10 ratio in influenza A/H3N2-stimulated PBMC was 10-fold lower in LDI vs non-LDI subjects. Pre-and postvaccination granzyme B levels were significantly lower in CHF subjects with LDI compared with subjects without LDI. In non-CHF subjects with LDI, granzyme B levels increased to high levels at the time of influenza infection. In conclusion, measures of the ex vivo cellular immune response to influenza are correlated with protection against influenza while serum Ab responses may be limited as a sole measure of vaccine efficacy in older people. Ex vivo measures of the cell-mediated immune response should be incorporated into evaluation of new vaccines for older adults. PMID- 16670346 TI - Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination. AB - We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1 specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors. PMID- 16670347 TI - IL-21 enhances tumor-specific CTL induction by anti-DR5 antibody therapy. AB - Tumor cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful anti-tumor immunotherapies. In this study, we show that induction of tumor cell apoptosis by agonistic mAb against DR5, combined with delayed IL-21 treatment, suppressed tumor growth and pre-established tumor metastases. Synergistic effects of the combination were observed in several tumor models where the target tumor was sensitive to DR5 mediated apoptosis. IL-21 promoted tumor-specific CTL activity and enhanced memory responses to tumor rechallenge. These results indicate that a rational combination of Ab-based therapy that causes tumor cell apoptosis and a cytokine that promotes T cell memory is a useful new strategy for cancer immunotherapy. PMID- 16670348 TI - Efficient gene transfer into macrophages and dendritic cells by in vivo gene delivery with mannosylated lipoplex via the intraperitoneal route. AB - In this study, we developed an antigen-presenting cell (APC)-selective intraperitoneal (i.p.) gene delivery system with mannosylated cationic liposomes (Man-liposomes)/plasmid DNA complex (Man-lipoplex). An in vitro study using cultured peritoneal macrophages demonstrated that Man-liposomes could transfect luciferase-encoding plasmid DNA (pCMV-Luc) more efficiently than cationic liposomes via a mannose receptor-mediated mechanism. In vivo gene transfection studies revealed that Man-lipoplex showed a higher gene expression in the liver, spleen, peritoneal exuded cells, and mesenteric lymph nodes than cationic liposomes/plasmid DNA complex (lipoplex) or naked pCMV-Luc after i.p. administration, and this gene expression lasted for at least 24 h. The transfection activity of Man-lipoplex after i.p. administration was significantly higher than that after i.v. gene delivery with the Man-liposomes we developed previously, indicating that gene delivery via the i.p. route seems to be an efficient approach for in vivo gene delivery to APCs. Furthermore, it was demonstrated that Man-lipoplex could enhance gene expression in both F4/80+ and CD11c+ cells in the spleen. These results show that gene delivery with Man liposomes via the i.p. route could be an effective approach for APC-selective gene transfection. PMID- 16670349 TI - An overview of drug combination analysis with isobolograms. AB - Drugs given in combination may produce effects that are greater than or less than the effect predicted from their individual potencies. The historical basis for predicting the effect of a combination is based on the concept of dose equivalence; i.e., an equally effective dose (a) of one will add to the dose (b) of the other in the combination situation. For drugs with a constant relative potency, this leads to linear additive isoboles (a-b curves of constant effect), whereas a varying potency ratio produces nonlinear additive isoboles. Determination of the additive isobole is a necessary procedure for assessing both synergistic and antagonistic interactions of the combination. This review discusses both variable and constant relative potency situations and provides the mathematical formulas needed to distinguish these cases. PMID- 16670350 TI - The cationic host defense peptide rCRAMP promotes gastric ulcer healing in rats. AB - Cathelicidin, a cationic host defense peptide, has been shown to promote cutaneous wound repair and reaches high levels in the gastric mucosa during infection and inflammation. Therefore, we investigated whether this peptide contributes to gastric ulcer healing in rats. Ulcer induction increased the expression of rat cathelicidin rCRAMP in the gastric mucosa. Further increase in expression of rCRAMP by local injection of rCRAMP-encoding plasmid promoted ulcer healing by enhancing cell proliferation and angiogenesis. rCRAMP directly stimulated proliferation of cultured rat gastric epithelial cells (RGM-1), which was abolished by inhibitors of matrix metalloproteinase (MMP), epidermal growth factor receptors (EGFR) tyrosine kinase, or mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase. rCRAMP also increased EGFR and ERK1/2 phosphorylation via an MMP-dependent mechanism. Knockdown of transforming growth factor alpha (TGFalpha), which is a ligand of EGFR, by small interfering RNA completely nullified the mitogenic signals evoked by rCRAMP in RGM-1 cells. These findings suggest that rCRAMP exhibits prohealing activity in stomachs through TGFalpha-dependent transactivation of EGFR and its related signaling pathway to induce proliferation of gastric epithelial cells. PMID- 16670351 TI - Comment: Epoetin alfa versus darbepoetin alfa for cancer patients with treatment related anemia. PMID- 16670353 TI - Special strengths of the woman practitioner. 1976. PMID- 16670354 TI - The sleeping women. 1975. PMID- 16670355 TI - Women's role in pharmacy practice in the year 2000. 1975. PMID- 16670356 TI - Salute to women pharmacists. 1975. PMID- 16670357 TI - Drug utilization review in an extended care facility. 1974. AB - Clinical pharmacy has expanded the scope of pharmaceutical services and one of the newer areas of involvement is the extended care facility. The author discusses the need, because of a limited budget, for better financial control of drug usage in Lions Gate Hospital's new 170 bed extended care unit. A need for pharmacy involvement in geriatrics because of the rising population of elderly people in extended care facilities and nursing homes and the complexities of using drugs in the elderly is discussed. The new extended care unit was provided with traditional pharmacy services and a pharmacist to participate in a patient review committee. This pharmacist monitored the patients' drug usage and made recommendations to the committee where applicable. The lowered costs of drug therapy in the extended care unit and the more efficacious use of certain medications are discussed in terms of pharmacy involvement. The author concludes that the pharmacist can make a significant contribution to the more economical use of drugs in extended care facilities and has a potential valuable role in improving patient care in this area. PMID- 16670358 TI - Specialization in pharmacy. 1974. PMID- 16670359 TI - Estimation of creatinine clearance in end-stage liver disease. AB - BACKGROUND: Estimation of renal function in patients with end-stage liver disease (ESLD) is complicated by several factors. OBJECTIVE: To develop a practical and relatively inexpensive method for estimating creatinine production and clearance in patients with ESLD. METHODS: Serum creatinine concentrations and urinary excretion of creatinine were measured in 27 patients with moderate-to-severe liver disease with the goal of developing equations to predict creatinine clearance from serum creatinine. Subjects were studied during an initial evaluation for a liver transplant program. Two 24 hour urine specimens were collected along with 3 serum samples over a 2 day evaluation period. Serum and urine creatinine concentrations were determined using both a modified Jaffe (autoanalyzer) method and an HPLC method. The data were analyzed using nonlinear mixed-effects modeling. RESULTS: Considering both statistical criteria and physiological conventions through allometric scaling theory, creatinine clearance (mL/min) in males can be estimated as (80/serum creatinine) x (actual body weight/70)0.75. For females, the same equation is valid, but the result is multiplied by 0.661. A simplified equation without the exponent is presented, along with equations that are appropriate when an HPLC assay is used for greater specificity. CONCLUSIONS: These equations offer potential for improved estimation of creatinine clearance in patients with liver impairment; however, they need further validation using an independent group of subjects. PMID- 16670360 TI - Meta-analysis of the safety of 5-HT3 antagonists with dexamethasone or droperidol for prevention of PONV. AB - BACKGROUND: Antiemetic guidelines recommend a combination of serotonin (5-HT3) with a second agent such as droperidol or dexamethasone. Physicians have been reluctant to employ these guidelines due to concerns over the black-box warning of droperidol and safety concerns with a steroid. OBJECTIVE: To assess the safety profiles of 5-HT3 receptor antagonist (5-HT3RA) monotherapy and combination therapy with a steroid or droperidol for prophylaxis of postoperative nausea and vomiting (PONV). METHODS: A MEDLINE search of English-language reports of randomized controlled trials (RCTs) was conducted (1966-September 2005) using the key terms 5-HT3, granisetron, ondansetron, dolasetron, tropisetron, PONV, postoperative, vomiting, emesis, and nausea. RCTs with treatment arms comparing 5 HT3RA monotherapy (granisetron, ondansetron, dolasetron, or tropisetron) with dexamethasone or droperidol or 5-HT3RA combinations and providing incidence data on adverse events were identified and reviewed. Within-study odds ratios with 95% confidence intervals were calculated to determine the incidence rates of all adverse events in RCTs using 5-HT3RA monotherapy and combination therapies. Overall effect sizes for frequently reported adverse events were estimated by pooling ORs using fixed- and random-effect models. RESULTS: Pooled ORs (OR(pooled)) for adverse events with 5-HT3RA/dexamethasone versus 5-HT3RA for PONV prophylaxis were not significant for any reported adverse events or the overall incidence of adverse events; 5-HT3RA/droperidol versus 5-HT3RA was significant only for decreased headache incidence (fixed model: OR(pooled) 0.35; 95% CI 0.18 to 0.69). The OR(pooled) for 5-HT3RA/dexamethasone versus dexamethasone was not significant for any reported adverse events except headaches (fixed model OR(pooled) 1.75; 95% CI 1.01 to 3.03), none of which was serious. OR(pooled) for 5-HT3RA/droperidol versus droperidol was not significant for any reported adverse events. Avascular necrosis, occult infection, and delayed wound healing were not observed with either combination therapy. Cardiac abnormalities were observed with 5-HT3RA/droperidol therapy. CONCLUSIONS: This meta-analysis indicates that either therapy has a safety profile similar to that of dexamethasone, droperidol, or 5-HT3RA. PMID- 16670361 TI - Meta-analysis of the use of rescue antiemetics following PONV prophylactic failure with 5-HT3 antagonist/dexamethasone versus single-agent therapies. AB - OBJECTIVE: To assess the use of rescue antiemetic medication following 5-HT3 receptor antagonist (5-HT3RA) plus dexamethasone therapy versus monotherapy with a 5-HT3RA for prophylaxis of postoperative nausea and vomiting (PONV). DATA SOURCES: Reports of randomized, controlled trials were identified via a MEDLINE search (1966-September 2005) using the key terms ondansetron, dolasetron, tropisetron, granisetron, 5-HT3, PONV, vomiting, emesis, and nausea. STUDY SELECTION AND DATA EXTRACTION: Randomized, controlled trials of adult populations that had treatment arms comparing 5-HT3RA/dexamethasone combination therapy with 5-HT3RA or dexamethasone monotherapies versus placebo or 5-HT3RA versus dexamethasone or placebo were selected for analysis. Another criterion was that a proportion of patients required rescue medication 48 hours or less following surgery. DATA SYNTHESIS: Odds ratios (ORs) with 95% confidence interval were calculated to determine incidence rates for use of rescue medications within early (0-6 h), late (6-24 h), and overall (0-24 or 48 h) postoperative periods. Overall effect sizes were calculated by pooling ORs within fixed and random effects models. CONCLUSIONS: Prophylaxis with 5-HT3RA/dexamethasone was associated with lower use of rescue antiemetics than 5-HT3RA (OR(pooled) = 0.48; 95% CI 0.29 to 0.77) or dexamethasone (OR(pooled) = 0.26; 95% CI 0.12-0.57) monotherapy during the overall postoperative period. Insufficient data were available to assess rescue use during early or late postoperative periods. It appears that patients at high risk of PONV who are treated prophylactically with combination 5-HT3RA/dexamethasone therapy are overall less likely to require rescue medication than if treated with 5-HT3RAs or dexamethasone alone. Additional large prospective studies are needed to determine the optimal regimen and timing of administration of prophylactic antiemetic therapy for different surgical populations. PMID- 16670362 TI - Guillain-Barre syndrome among recipients of Menactra meningococcal conjugate vaccine. PMID- 16670363 TI - Clozapine-induced systemic lupus erythematosus. AB - OBJECTIVE: To report a case of classic clozapine-induced systemic lupus erythematosus that also developed on rechallenge. CASE SUMMARY: A 32-year-old white woman diagnosed with schizophrenia presented in 1996 with clinical characteristics and laboratory markers consistent with drug-induced lupus (DIL). Clozapine, started 1 year prior, was withdrawn, with complete biological and clinical remission within 3 months. In 2004, 1 week after rechallenge with clozapine for uncontrolled schizophrenia, the patient developed clinical and biological signs and symptoms consistent with the diagnosis of DIL. Again, discontinuation of clozapine was followed by full remission within 2-3 months. DISCUSSION: DIL was first described more than 50 years ago, with multiple drugs implicated in the causation. Clozapine-induced lupus was reported recently, but does not meet the usual criteria for a diagnosis of DIL. We report a classic case of clozapine-induced lupus that, according to the Naranjo probability scale, demonstrates a highly probable relationship between DIL and clozapine. CONCLUSIONS: DIL demands a high index of suspicion for diagnosis. Although clozapine has an extensive safety profile, DIL must be considered as one of its serious adverse effects. PMID- 16670365 TI - Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs. PMID- 16670364 TI - Natural health products in the prevention and treatment of osteoporosis: systematic review of randomized controlled trials. AB - BACKGROUND: Consumers are increasingly looking to natural health products to manage specific diseases such as osteoporosis. As a result, healthcare providers need evidence-based information on which to base recommendations regarding use and efficacy. OBJECTIVE: To identify natural health products (NHPs, ie, dietary supplements) advocated for the prevention and treatment of osteoporosis and systematically review the evidence from randomized controlled trials for the effect of NHPs on bone mineral density (BMD)/fracture rate in women. METHODS: MEDLINE, Natural Medicines Comprehensive Database, and the Internet were initially searched to identify NHPs advocated for prevention and treatment of osteoporosis. For NHPs having evidence to support their claim, the aforementioned sources, along with International Pharmaceutical Abstracts, the Cochrane Library, the International Bibliographic Information on Dietary Supplements, the Cumulative Index to Nursing & Allied Health, and HerbMed, were searched to locate randomized controlled trials published in English between 1966 and October 2004. Bibliographies of identified articles were also searched. Randomized controlled trials were selected if they evaluated the use of a single NHP in women, using BMD/fracture rate as the outcome measure. NHPs were excluded from further evaluation if a review had already been published. Data were extracted using predetermined criteria and studies appraised using the Jadad scale. Forty-five NHPs were identified that the authors claimed to be beneficial in prevention and treatment of osteoporosis, with 15 having evidence to support their claim. Calcium; copper; evening primrose oil; fish oils; fluoride; magnesium; manganese; strontium; vitamin D; and black, green, and oolong tea did not meet study criteria. RESULTS: Results from randomized controlled trials evaluating dehydroepiandrosterone (DHEA), phytoestrogens, and vitamin K2 (menaquinone or menatetrenone) were promising; however, study limitations suggest the need for confirmatory evidence. CONCLUSIONS: Although no definitive conclusions can be drawn, the relative safety of phytoestrogens, DHEA, and vitamin K2 at the studied doses, as well as preliminary positive results from randomized controlled trials, provides some initial support for the use of these NHPs in the prevention and treatment of osteoporosis in women. PMID- 16670366 TI - Comparison of the Achilles Express ultrasonometer with central dual-energy X-ray absorptiometry. AB - BACKGROUND: Quantitative ultrasound (QUS) devices provide portable, easy-to operate, low-cost options for point-of-care screening of bone mineral density (BMD). Community pharmacists should be aware of the precision, sensitivity, and specificity of these devices prior to their purchase. OBJECTIVE: To determine the precision, sensitivity, and specificity of the Achilles Express ultrasonometer compared with central dual-energy X-ray absorptiometry (cDXA) as well as its utility as a bone density screening device in the community pharmacy setting. METHODS: A prospective study in a community pharmacy and outpatient ambulatory clinic was conducted with 2 groups of white women. Group 1 participants were 25 35 years of age (young, healthy), and those in Group 2 were 45 years of age or older (postmenopausal). BMD assessments of the spine and the nondominant wrist and hip were performed using cDXA. Assessments of the heel were performed using the Achilles Express, a QUS device. The main outcome measures were correlation of t-scores between cDXA and QUS measurements using the Pearson correlation test. RESULTS: Twenty-two (30 +/- 4 years of age) and 31 (55 +/- 17 years of age) women were enrolled into Groups 1 and 2, respectively. Significant correlations between QUS and hip and spine cDXA t-scores were found in both groups. Correlation coefficients for QUS versus hip cDXA were 0.51 (95% CI 0.11 to 0.77) and 0.70 (95% CI 0.46 to 0.85) in Groups 1 and 2, respectively. Correlation coefficients for the QUS versus spine cDXA were 0.64 (95% CI 0.31 to 0.84) and 0.60 (95% CI 0.31 to 0.79) in Groups 1 and 2, respectively. The QUS device has a sensitivity level of 88% and specificity of 71% to detect a hip cDXA t-score of less than-1. CONCLUSIONS: The Achilles Express ultrasonometer is a reasonable screening tool to detect low BMD in postmenopausal women. PMID- 16670367 TI - Suspected suppression of the INR by thiazolidinediones: interaction between warfarin and TZDs. PMID- 16670368 TI - Reflections on geriatric pharmacy practice: the times they are a changin'. PMID- 16670369 TI - Quality assessment of published health economic analyses from South America. AB - BACKGROUND: Health economic analyses have become important to healthcare systems worldwide. No studies have previously examined South America's contribution in this area. OBJECTIVE: To survey the literature with the purpose of reviewing, quantifying, and assessing the quality of published South American health economic analyses. METHODS: A search of MEDLINE (1990-December 2004), EMBASE (1990-December 2004), International Pharmaceutical Abstracts (1990-December 2004), Literatura Latino-Americana e do Caribe em Ciencias da Saude (1982 December 2004), and Sistema de Informacion Esencial en Terapeutica y Salud (1980 December 2004) was completed using the key words cost-effectiveness analysis (CEA), cost-utility analysis (CUA), cost-minimization analysis (CMA), and cost benefit analysis (CBA); abbreviations CEA, CUA, CMA, and CBA; and all South American country names. Papers were categorized by type and country by 2 independent reviewers. Quality was assessed using a 12 item checklist, characterizing scores as 4 (good), 3 (acceptable), 2 (poor), 1 (unable to judge), and 0 (unacceptable). To be included in our investigation, studies needed to have simultaneously examined costs and outcomes. RESULTS: We retrieved 25 articles; one duplicate article was rejected, leaving 24 (CEA = 15, CBA = 6, CMA = 3; Brazil = 9, Argentina = 5, Colombia = 3, Chile = 2, Ecuador = 2, 1 each from Peru, Uruguay, Venezuela). Variability between raters was less than 0.5 point on overall scores (OS) and less than 1 point on all individual items. Mean OS was 2.6 (SD 1.0, range 1.4-3.8). CBAs scored highest (OS 2.8, SD 0.8), CEAs next (OS 2.7, SD 0.7), and CMAs lowest (OS 2.0, SD 0.5). When scored by type of question, definition of study aim scored highest (OS 3.0, SD 0.8), while ethical issues scored lowest (OS 1.5, SD 0.9). By country, Peru scored highest (mean OS 3.8) and Uruguay had the lowest scores (mean OS 2.2). A nonsignificant time trend was noted for OS (R2 = 0.12; p = 0.104). CONCLUSIONS: Quality scores of health economic analyses articles published in South America were rated poor to acceptable and lower than previous research from other countries. Thus, efforts are needed to improve the reporting quality of these analyses in South America. Future research should examine the region's level of expertise and educational opportunities for those in the field of health economics. PMID- 16670370 TI - Montelukast treatment of urticaria. AB - OBJECTIVE: To review clinical trial data to determine the benefits of using montelukast alone or as combination therapy in the treatment of urticaria. DATA SOURCES: MEDLINE (1966-March 2006) and International Pharmaceutical Abstracts (1970-October 2005) were searched to find clinical trial publications that addressed the use of montelukast in urticaria. DATA SYNTHESIS: Six clinical trials were identified. Montelukast was compared alone and as combination therapy with nonsedating histamine1-receptor antagonists to determine efficacy and safety. Patients had chronic or physical urticaria. The results were mixed. Some studies demonstrated that montelukast can decrease urticarial symptoms with minimal adverse effects, while others found no differences. CONCLUSION: Large scale, controlled trials are needed to determine which patients would likely benefit from treatment with montelukast. PMID- 16670371 TI - Where do you think you are going? The NMDA-D1 receptor trap. AB - The number and outcomes of reciprocal interactions between dopamine (DA) D1 receptors and N-methyl-D-aspartate (NMDA)-type glutamate receptors continue to increase. Recent studies have demonstrated close physical interactions in which activation of one receptor affects the function of the other. In one physical interaction, the activation of NMDA receptors alters the topography and movement of D1 receptors by trapping them in dendritic spines and thus altering their distribution. In a second physical interaction, D1 and subunits of NMDA receptors form heterodimers, which are translocated from the cell interior to the surface. Finally, a third physical interaction posits that the C terminus of D1 receptors makes contact with subunits of the NMDA receptor. These physical interactions can attenuate or potentiate receptor function. In contrast, the more traditional interactions mediated by second messengers generally cause NMDA receptor function to be potentiated through the activation of D1 receptors and the cAMP-PKA-DARPP 32 [adenosine 3',5'-monophosphate (cAMP)-protein kinase A-cAMP-regulated phosphoprotein of 32 kD] or PKC (protein kinase C) cascades. Together, these mechanisms provide a basis for understanding the increasing complexity of D1-NMDA receptor interactions and their importance in physiological and pathological processes. PMID- 16670372 TI - Teaching Resources. Assembly and organization of macromolecular complexes. AB - This Teaching Resource provides lecture notes and slides for a class covering the roles of scaffold proteins in signal transduction and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture includes an overview of scaffold proteins in signal integration and focuses on the following scaffold proteins: A-kinase anchoring proteins (AKAPs), WAVE-1, and proteins that contain PDZ domains. PMID- 16670373 TI - Hepatocyte nuclear factor (HNF) 1 and HNF4 mediate hepatic multidrug resistance protein 2 up-regulation during hepatitis C virus gene expression. AB - Hepatitis C virus (HCV) is known to induce hepatic oxidative stress that is implicated in the up-regulation of multidrug resistance proteins (MRPs). The relationship between increased prooxidant production, MRPs, and HCV has not been investigated. Here, we report that a homeodomain-containing transcription factor, hepatocyte nuclear factor (HNF) 1, plays a central role in liver gene regulation during HCV gene expression and/or subgenome replication. MRP2 protein and mRNA expression were increased and MRP2 promoter activity was increased 7-fold. Mutations within the putative HNF1 binding site of the human MRP2 promoter abrogated HCV-induced activation, implicating HNF1 in the induction of MRP2 by HCV. The mechanism by which HNF1-mediated activation occurs seems to be transcriptional, because the regulated expression of HNF4, which is known to control HNF1 expression, was also increased. Consistent with this finding, HNF1 mRNA was increased 10-fold. A promoter-luciferase construct of the human HNF1 gene was activated in an HNF4-dependent manner, and a mutant construct lacking the HNF4 binding site was not activated in HCV-positive cells. Consistent with this hypothesis, HNF4 protein and mRNA levels as well as HNF4 promoter activity and DNA binding activity were increased. The expression of HNF1 seems to play a critical role in the induction of hepatic MRP2 secondary to HCV subgenomic replication. The ability of HCV to induce HNF1 and HNF4 is attributed to 1) increased oxidative stress and 2) direct protein-protein interactions between HCV nonstructural component (NS) 5A and HNF1, leading to enhanced HNF1 DNA binding. In conclusion, we describe a novel mechanism by which HCV gene expression may induce adaptive responses involving MRP2 via HNF1 activation. This may constitute, in part, the cellular detoxification task force during HCV infection. PMID- 16670374 TI - Polysaccharide purified from Ganoderma lucidum induces gene expression changes in human dendritic cells and promotes T helper 1 immune response in BALB/c mice. AB - Ganoderma lucidum is a medicinal mushroom in China and other Asian countries. The polysaccharide from G. lucidum (PS-G) is a branched (1-->6)-beta-d-glucan moiety. In this study, we examined the effects of PS-G on human monocyte-derived dendritic cells (DCs) with microarray analysis by Human Genome U133 Plus 2.0 GeneChip. In comparing mean signal values between PS-G-treated DCs with untreated DCs, 3477 (17%) probe sets were up-regulated, and 4418 (19%) probe sets were down regulated after PS-G treatment. These results demonstrate that genes associated with phagocytosis (CD36, CD206, and CD209) are decreased and genes associated with proinflammatory chemokines (CCL20, CCL5, and CCL19), cytokines [interleukin (IL)-27, IL-23A, IL-12A, and IL-12B], and costimulatory molecules (CD40, CD54, CD80, and CD86) are increased. To confirm the microarray data, we further investigated the effect of PS-G on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with ovalbumin (OVA)/PS-G showed that the anti-OVA IgG2a levels were significantly increased compared with OVA alone in BALB/c mice. Together, our data demonstrate that PS-G could effectively promote the activation and maturation of immature DCs, preferring a T helper 1 response. Furthermore, the results also demonstrate that the data from microarray analysis could be correlated with the in vivo effect of the immune-enhancing compound. PMID- 16670375 TI - Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain. AB - Once bound to methylated CpG sites, methyl-CpG-binding protein 2 (MeCP2) is thought to silence transcription of downstream genes by recruiting a histone deacetylase (HDAC). Mutations within the MeCP2 gene have been found to cause Rett syndrome, a disorder of arrested neuronal development. Using immunohistochemistry, we found that Mecp2, as well as the methyl-CpG-binding protein MBD1, were significantly induced in normal adult rat brain after repeated injections of fluoxetine or cocaine for 10 days (one injection per day). Mecp2 was not induced by repeated injections of 1-(2-bis(4-fluorphenyl)-methoxy)-ethyl) 4-(3-phenyl-propyl)piperazine (GBR-12909) or nortriptyline. Together, the data indicate that the serotonergic system is predominantly involved. Using real-time reverse transcription-polymerase chain reaction experiments, MBD1 mRNA and both Mecp2_e1 and Mecp2_e2 transcripts were found to be induced by fluoxetine. Induction of the methylbinding proteins was accompanied with enhanced HDAC2 labeling intensity and mRNA synthesis in response to fluoxetine. In tandem, acetylated forms of histone H3 were found to be decreased. The effect was characterized in three serotonin projection areas, the caudate-putamen, the frontal cortex, and the dentate gyrus subregion of hippocampus. Our data highlight GABAergic neurons as major target cells expressing Mecp2 in response to the serotonin-elevating agents and suggest that serotonin signaling enhances gene silencing in postmitotic neurons. PMID- 16670376 TI - Sentinel lymph node biopsy in early breast cancer: has its time come? PMID- 16670377 TI - Completion rates of adjuvant chemotherapy for colon cancer: a historical perspective. PMID- 16670378 TI - Research foundations find strength in numbers. PMID- 16670379 TI - Foundation drives research agenda for multiple myeloma. PMID- 16670380 TI - Immune system's Toll-like receptors have good opportunity for cancer treatment. PMID- 16670381 TI - CAMs are stopping cancer in its metastatic tracks. PMID- 16670382 TI - Stat bite: Effect of scientific journal requirement on clinical trial registration. PMID- 16670383 TI - Experts wrestle with problems developing biomarkers, search for new tests. PMID- 16670384 TI - Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesis testing clinical trials of innovative therapies. AB - Clinical trials of new cancer drugs should ideally include measurements of parameters such as molecular target expression, pharmacokinetic (PK) behavior, and pharmacodynamic (PD) endpoints that can be linked to measures of clinical effect. Appropriate PK/PD biomarkers facilitate proof-of-concept demonstrations for target modulation; enhance the rational selection of an optimal drug dose and schedule; aid decision-making, such as whether to continue or close a drug development project; and may explain or predict clinical outcomes. In addition, measurement of PK/PD biomarkers can minimize uncertainty associated with predicting drug safety and efficacy, reduce the high levels of drug attrition during development, accelerate drug approval, and decrease the overall costs of drug development. However, there are many challenges in the development and implementation of biomarkers that probably explain their disappointingly low implementation in phase I trials. The Pharmacodynamic/Pharmacokinetic Technologies Advisory committee of Cancer Research UK has found that submissions for phase I trials of new cancer drugs in the United Kingdom often lack detailed information about PK and/or PD endpoints, which leads to suboptimal information being obtained in those trials or to delays in starting the trials while PK/PD methods are developed and validated. Minimally invasive PK/PD technologies have logistic and ethical advantages over more invasive technologies. Here we review these technologies, emphasizing magnetic resonance spectroscopy and positron emission tomography, which provide detailed functional and metabolic information. Assays that measure effects of drugs on important biologic pathways and processes are likely to be more cost-effective than those that measure specific molecular targets. Development, validation, and implementation of minimally invasive PK/PD methods are encouraged. PMID- 16670385 TI - Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC Trial. AB - BACKGROUND: Sentinel lymph node biopsy in women with operable breast cancer is routinely used in some countries for staging the axilla despite limited data from randomized trials on morbidity and mortality outcomes. We conducted a multicenter randomized trial to compare quality-of-life outcomes between patients with clinically node-negative invasive breast cancer who received sentinel lymph node biopsy and patients who received standard axillary treatment. METHODS: The primary outcome measures were arm and shoulder morbidity and quality of life. From November 1999 to October 2003, 1031 patients were randomly assigned to undergo sentinel lymph node biopsy (n = 515) or standard axillary surgery (n = 516). Patients with sentinel lymph node metastases proceeded to delayed axillary clearance or received axillary radiotherapy (depending on the protocol at the treating institution). Intention-to-treat analyses of data at 1, 3, 6, and 12 months after surgery are presented. All statistical tests were two-sided. RESULTS: The relative risks of any lymphedema and sensory loss for the sentinel lymph node biopsy group compared with the standard axillary treatment group at 12 months were 0.37 (95% confidence interval [CI] = 0.23 to 0.60; absolute rates: 5% versus 13%) and 0.37 (95% CI = 0.27 to 0.50; absolute rates: 11% versus 31%), respectively. Drain usage, length of hospital stay, and time to resumption of normal day-to-day activities after surgery were statistically significantly lower in the sentinel lymph node biopsy group (all P < .001), and axillary operative time was reduced (P = .055). Overall patient-recorded quality of life and arm functioning scores were statistically significantly better in the sentinel lymph node biopsy group throughout (all P < or = .003). These benefits were seen with no increase in anxiety levels in the sentinel lymph node biopsy group (P > .05). CONCLUSION: Sentinel lymph node biopsy is associated with reduced arm morbidity and better quality of life than standard axillary treatment and should be the treatment of choice for patients who have early-stage breast cancer with clinically negative nodes. PMID- 16670386 TI - Completion of therapy by Medicare patients with stage III colon cancer. AB - BACKGROUND: Certain factors, such as race or age, are known to be associated with variation in initiation of adjuvant chemotherapy for stage III colon cancer, but little is known about what factors are associated with completion of adjuvant therapy. To determine whether predictors of initiation also predict completion, we analyzed Surveillance, Epidemiology, and End Results (SEER) program data linked to Medicare claims. We investigated mortality as a means to testing the validity of the completion measure that we created. METHODS: We studied 3193 stage III colon cancer patients whose diagnosis was recorded in 1992-1996 SEER program data linked to 1991-1998 Medicare claims and who initiated adjuvant chemotherapy after colon cancer resection. We defined a measure of adjuvant chemotherapy completion as one chemotherapy administration claim in a month. We tested the validity of the created measure and its relation to 3-year cancer mortality adjusted for demographic, clinical, and environmental variables. We explored the association of patient characteristics and treating physician characteristics with chemotherapy completion by use of multivariable logistic regression modeling. RESULTS: Of the 3193 patients, 2497 (78.2%) completed the course. Risk of cancer-related mortality was statistically significantly lower among those completing chemotherapy (relative risk = 0.79, 95% confidence interval = 0.69 to 0.89) than those with no adjuvant therapy. Patients who were female, widowed, increasingly elderly, rehospitalized, and living in certain regions were less likely to complete adjuvant chemotherapy than other patients. Race and other clinical, environmental, and physician characteristics were not associated with completion of therapy. CONCLUSIONS: Factors associated with incomplete adjuvant chemotherapy may represent physical frailty, treatment complications, and lack of social and psychological support. Interventions to mitigate these influences are a logical next step toward increasing chemotherapy completion rates. PMID- 16670387 TI - Reporting of randomized controlled trials in Hodgkin lymphoma in biomedical journals. AB - BACKGROUND: Randomized controlled trials (RCTs) are the best tool to evaluate the effectiveness of clinical interventions. The Consolidated Standards for Reporting Trials (CONSORT) statement was introduced in 1996 to improve reporting of RCTs. We aimed to determine the extent of ambiguity and reporting quality as assessed by adherence to the CONSORT statement in published reports of RCTs involving patients with Hodgkin lymphoma from 1966 through 2002. METHODS: We analyzed 242 published full-text reports of RCTs in patients with Hodgkin lymphoma. Quality of reporting was assessed using a 14-item questionnaire based on the CONSORT checklist. Reporting was studied in two pre-CONSORT periods (1966-1988 and 1989 1995) and one post-CONSORT period (1996-2002). RESULTS: Only six of the 14 items were addressed in 75% or more of the studies in all three time periods. Most items that are necessary to assess the methodologic quality of a study were reported by fewer than 20% of the studies. Improvements over time were seen for some items, including the description of statistics methods used, reporting of primary research outcomes, performance of power calculations, method of randomization and concealment allocation, and having performed intention-to-treat analysis. CONCLUSIONS: Despite recent improvements, reporting levels of CONSORT items in RCTs involving patients with Hodgkin lymphoma remain unsatisfactory. Further concerted action by journal editors, learned societies, and medical schools is necessary to make authors even more aware of the need to improve the reporting RCTs in medical journals to allow assessment of validity of published clinical research. PMID- 16670388 TI - Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus. AB - BACKGROUND: Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy. METHODS: To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad-infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan-Meier method and the Cox-Mantel log-rank analysis. All statistical tests were two-sided. RESULTS: hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad-induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P < .001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P < .001). CONCLUSIONS: hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death. PMID- 16670389 TI - Validation of a model of lung cancer risk prediction among smokers. AB - The Bach model was developed to predict the absolute 10-year risk of developing lung cancer among smokers by use of participants in the Carotene and Retinol Efficacy Trial of lung cancer prevention. We assessed the validity of the Bach model among 6239 smokers from the placebo arm of the Alpha-Tocopherol, Beta Carotene Cancer Prevention (ATBC) Study. The expected numbers of lung cancer cases and deaths without lung cancer were calculated from the Bach model and compared with the observed numbers of corresponding events over 10 years. We found that the risk model slightly underestimated the observed lung cancer risk (number of lung cancers expected/number observed = 0.89, 95% confidence interval [CI] = 0.80 to 0.99) over 10 years. The competing risk portion of the model substantially underestimated risk of non-lung cancer mortality (number of non lung cancer deaths expected/number observed = 0.61, 95% CI = 0.57 to 0.64) over 10 years. The age-specific concordance indices for 10-year predictions were 0.77 (95% CI = 0.70 to 0.84), 0.59 (95% CI = 0.53 to 0.65), 0.62 (95% CI = 0.57 to 0.67), and 0.57 (95% CI = 0.49 to 0.67) for the age groups 50-54, 55-59, 60-64, and 65-69 years, respectively. Periodic radiographic screening in the ATBC Study may explain why slightly more cancers were observed than expected from the Bach model. PMID- 16670390 TI - Re: Risk of thyroid cancer after exposure to (131)I in childhood. PMID- 16670391 TI - Re: Tamoxifen for the prevention of breast cancer: current status of the National Surgical Adjuvant Breast and Bowel Project P-1 study. PMID- 16670392 TI - Re: Carbonated soft drink consumption and risk of esophageal adenocarcinoma. PMID- 16670393 TI - Re: Carbonated soft drink consumption and risk of esophageal adenocarcinoma. PMID- 16670394 TI - Reflecting on governance for CMAJ. PMID- 16670395 TI - Persistent pulmonary hypertension of the newborn and maternal use of SSRIs. PMID- 16670398 TI - The cover. Bend in the Epte River near Giverney. PMID- 16670396 TI - Time variations in the risk of myocardial infarction among elderly users of COX-2 inhibitors. AB - BACKGROUND: The timing of cardiovascular risks associated with the use of cyclooxygenase-2 (COX-2) inhibitors is unclear. Using data collected in a previous population-based cohort study of elderly people starting nonsteroidal anti-inflammatory drug (NSAID) therapy, we evaluated the temporal nature of the risk of a first myocardial infarction (MI) associated with the use of rofecoxib and celecoxib. METHODS: We identified people 66 years of age or older without previous MI who were currently taking rofecoxib and celecoxib using Quebec's computerized health databases (January 1999 to June 2002). Data on use and MI outcome were analyzed using a time-matched, nested case-control approach with rate ratios, comparing current users and non-users of rofecoxib and celecoxib in the year preceding the index date, estimated using conditional logistic regression. RESULTS: The risk of MI was highest following first-time use of rofecoxib (adjusted rate ratio [RR] 1.67, 95% confidence interval [CI] 1.21 2.30), with events occurring within a median of 9 (6-13) days after therapy was started. The risk increase for first-time use of celecoxib was not statistically significant (RR 1.29, 95% CI 0.90-1.83). Repeated exposure to rofecoxib was associated with a small but statistically nonsignificant delayed risk (RR 1.17, 95% CI 0.98-1.40), but no risk was seen with celecoxib (RR 0.97, 95% CI 0.82 1.14). Treatment duration was not associated with increasing risk for either agent. The risk remained elevated for the first 7 days after rofecoxib was discontinued (RR 1.23, 95% CI 1.05-1.44) but appeared to return to baseline between day 8 and 30 (RR 0.82, 95% CI 0.61-1.09). INTERPRETATION: A small proportion of patients using rofecoxib for the first time had their first MI shortly after starting the drug. This risk did not increase with the length of treatment and returned to baseline shortly after treatment was discontinued. More research is needed to identify those most susceptible to cardiotoxicity mediated by COX-2 inhibitor therapy. PMID- 16670399 TI - A piece of my mind. A glimpse backstage. PMID- 16670400 TI - Animal-human diseases targeted to stop pandemics before they start. PMID- 16670401 TI - CT angiography: clearer picture, Fuzzier reception. PMID- 16670402 TI - Drug shows promise for marfan syndrome. PMID- 16670403 TI - Adverse events related to muraglitazar use in diabetes. PMID- 16670404 TI - Adverse events related to muraglitazar use in diabetes. PMID- 16670405 TI - Erectile dysfunction and incidence of cardiovascular disease. PMID- 16670406 TI - Erectile dysfunction and incidence of cardiovascular disease. PMID- 16670407 TI - Erectile dysfunction and incidence of cardiovascular disease. PMID- 16670408 TI - Akathisia with combined use of midodrine and promethazine. PMID- 16670409 TI - Combined pharmacotherapies and behavioral interventions for alcohol dependence: the COMBINE study: a randomized controlled trial. AB - CONTEXT: Alcohol dependence treatment may include medications, behavioral therapies, or both. It is unknown how combining these treatments may impact their effectiveness, especially in the context of primary care and other nonspecialty settings. OBJECTIVES: To evaluate the efficacy of medication, behavioral therapies, and their combinations for treatment of alcohol dependence and to evaluate placebo effect on overall outcome. DESIGN, SETTING, AND PARTICIPANTS: Randomized controlled trial conducted January 2001-January 2004 among 1383 recently alcohol-abstinent volunteers (median age, 44 years) from 11 US academic sites with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, diagnoses of primary alcohol dependence. INTERVENTIONS: Eight groups of patients received medical management with 16 weeks of naltrexone (100 mg/d) or acamprosate (3 g/d), both, and/or both placebos, with or without a combined behavioral intervention (CBI). A ninth group received CBI only (no pills). Patients were also evaluated for up to 1 year after treatment. MAIN OUTCOME MEASURES: Percent days abstinent from alcohol and time to first heavy drinking day. RESULTS: All groups showed substantial reduction in drinking. During treatment, patients receiving naltrexone plus medical management (n = 302), CBI plus medical management and placebos (n = 305), or both naltrexone and CBI plus medical management (n = 309) had higher percent days abstinent (80.6, 79.2, and 77.1, respectively) than the 75.1 in those receiving placebos and medical management only (n = 305), a significant naltrexone x behavioral intervention interaction (P = .009). Naltrexone also reduced risk of a heavy drinking day (hazard ratio, 0.72; 97.5% CI, 0.53-0.98; P = .02) over time, most evident in those receiving medical management but not CBI. Acamprosate showed no significant effect on drinking vs placebo, either by itself or with any combination of naltrexone, CBI, or both. During treatment, those receiving CBI without pills or medical management (n = 157) had lower percent days abstinent (66.6) than those receiving placebo plus medical management alone (n = 153) or placebo plus medical management and CBI (n = 156) (73.8 and 79.8, respectively; P<.001). One year after treatment, these between-group effects were similar but no longer significant. CONCLUSIONS: Patients receiving medical management with naltrexone, CBI, or both fared better on drinking outcomes, whereas acamprosate showed no evidence of efficacy, with or without CBI. No combination produced better efficacy than naltrexone or CBI alone in the presence of medical management. Placebo pills and meeting with a health care professional had a positive effect above that of CBI during treatment. Naltrexone with medical management could be delivered in health care settings, thus serving alcohol-dependent patients who might otherwise not receive treatment. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00006206. PMID- 16670410 TI - Association of long-distance corridor walk performance with mortality, cardiovascular disease, mobility limitation, and disability. AB - CONTEXT: Aerobic fitness, an important predictor of cardiovascular disease and mortality, is difficult to assess by maximal exercise testing in older adults. Extended walking tests have been examined as outcome predictors in medically ill populations but not in community-dwelling older adults. OBJECTIVE: To determine whether an extended walking test predicts poor outcomes in older adults. DESIGN, SETTING, AND PARTICIPANTS: Observational cohort study enrolling 3075 community dwelling adults aged 70 to 79 years living in Pittsburgh, Pa, or Memphis, Tenn. Of those participating in the Health, Aging, and Body Composition Study, 1584 (52%) were women and 1281 (42%) were black. Participants enrolled from March 1997 to April 1998. Ability to complete the long-distance corridor walk and total performance time was assessed at the baseline examination. MAIN OUTCOME MEASURES: Total mortality, incident cardiovascular disease, incident mobility limitation, and mobility disability were ascertained after a mean (SD) of 4.9 (0.9) years. RESULTS: Among patients eligible to exercise, 351 died, 308 had episodes of incident cardiovascular disease, 1116 had occurrences of mobility limitation, and 509 had occurrences of mobility disability. Inability to complete walking 400 m tended to be associated with a higher risk of mortality and incident cardiovascular disease and, after accounting for potential confounders, was associated with incident mobility limitation (212.6 vs 79.1 events/1000 person years; adjusted hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.58-2.18; P<.001) and mobility disability (85.2 vs 28.8 events/1000 person-years; adjusted HR, 1.95; 95% CI, 1.56-2.44; P<.001). Of those who completed 400 m, each additional minute of performance time was associated with an adjusted HR of 1.29 (95% CI, 1.12-1.48) for mortality, 1.20 (95% CI, 1.01-1.42) for incident cardiovascular disease, 1.52 (95% CI, 1.41-1.63) for mobility limitation, and 1.52 (95% CI, 1.37-1.70) for disability after adjustment for demographics, health behaviors, clinical and subclinical disease, and cardiovascular disease risk factors. Findings were consistent in both men and women and blacks and whites. Among participants who completed the test and after adjusting for potential confounders, those in the poorest quartile of functional capacity (walk time >362 seconds) had a higher risk of death than those in the best quartile (walk time <290 seconds; adjusted HR, 3.23; 95% CI, 2.11-4.94; P<.001). CONCLUSIONS: Older adults in the community who reported no difficulty walking had a wide range of performance on this extended walking test. Ability to do the test and performance were important prognostic factors for total mortality, cardiovascular disease, mobility limitation, and mobility disability in persons in their eighth decade. PMID- 16670411 TI - Use of health care services by lower-income and higher-income uninsured adults. AB - CONTEXT: More than 45 million individuals in the United States lack health insurance, potentially limiting their access to and use of appropriate health care services. Although the uninsured comprise a range of income levels, little attention has been directed at higher-income uninsured adults and their patterns of care. OBJECTIVE: To examine whether having higher income attenuates the association between being uninsured and using fewer recommended health care services. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional analysis of data from the 2002 Behavioral Risk Factor Surveillance System, drawn from a nationally representative sample of households. Participants were community-dwelling adults (n = 194 943; 50% women) aged 18 to 64 years in 2002. MAIN OUTCOME MEASURES: Self reported use of screening for cervical, breast, and colorectal cancer; serum cholesterol screening and measurement, aspirin use, and tobacco cessation and weight loss counseling for cardiovascular risk reduction; and serum cholesterol and glycosylated hemoglobin measurement, eye and foot examination, and influenza and pneumococcal vaccination for diabetes management. RESULTS: Among eligible adults, use of cancer prevention services ranged from 51% for colorectal cancer screening to 88% for cervical cancer screening, while use of cardiovascular risk reduction services ranged from 38% for weight loss counseling to 81% for aspirin use, and use of services for diabetes management ranged from 33% for pneumococcal vaccination to 88% for serum glycosylated hemoglobin measurement. In bivariate analyses, health insurance and annual household income were both strongly associated with use of nearly all examined health care services (P values <.01). Using multivariable analysis, increased annual household income did not significantly increase the likelihood of uninsured compared with insured adults receiving recommended health care services for cancer prevention, cardiovascular risk reduction, or diabetes management (P values >.05). CONCLUSIONS: Even among higher-income adults, lack of health care insurance was associated with significantly decreased use of recommended health care services; increased income did not attenuate the difference in use between uninsured and insured adults. Efforts to improve the use of recommended health care services among the uninsured should focus on patient education and expanding insurance eligibility for both lower-income and higher-income adults. PMID- 16670412 TI - Disease and disadvantage in the United States and in England. AB - CONTEXT: The United States spends considerably more money on health care than the United Kingdom, but whether that translates to better health outcomes is unknown. OBJECTIVE: To assess the relative heath status of older individuals in England and the United States, especially how their health status varies by important indicators of socioeconomic position. DESIGN, SETTING, AND PARTICIPANTS: We analyzed representative samples of residents aged 55 to 64 years from both countries using 2002 data from the US Health and Retirement Survey (n = 4386) and the English Longitudinal Study of Aging (n = 3681), which were designed to have directly comparable measures of health, income, and education. This analysis is supplemented by samples of those aged 40 to 70 years from the 1999-2002 waves of National Health and Nutrition Examination Survey (n = 2097) and the 2003 wave of the Health Survey for England (n = 5526). These surveys contain extensive and comparable biological disease markers on respondents, which are used to determine whether differential propensities to report illness can explain these health differences. To ensure that health differences are not solely due to health issues in the black or Latino populations in the United States, the analysis is limited to non-Hispanic whites in both countries. MAIN OUTCOME MEASURE: Self reported prevalence rates of several chronic diseases related to diabetes and heart disease, adjusted for age and health behavior risk factors, were compared between the 2 countries and across education and income classes within each country. RESULTS: The US population in late middle age is less healthy than the equivalent British population for diabetes, hypertension, heart disease, myocardial infarction, stroke, lung disease, and cancer. Within each country, there exists a pronounced negative socioeconomic status (SES) gradient with self reported disease so that health disparities are largest at the bottom of the education or income variants of the SES hierarchy. This conclusion is generally robust to control for a standard set of behavioral risk factors, including smoking, overweight, obesity, and alcohol drinking, which explain very little of these health differences. These differences between countries or across SES groups within each country are not due to biases in self-reported disease because biological markers of disease exhibit exactly the same patterns. To illustrate, among those aged 55 to 64 years, diabetes prevalence is twice as high in the United States and only one fifth of this difference can be explained by a common set of risk factors. Similarly, among middle-aged adults, mean levels of C reactive protein are 20% higher in the United States compared with England and mean high-density lipoprotein cholesterol levels are 14% lower. These differences are not solely driven by the bottom of the SES distribution. In many diseases, the top of the SES distribution is less healthy in the United States as well. CONCLUSION: Based on self-reported illnesses and biological markers of disease, US residents are much less healthy than their English counterparts and these differences exist at all points of the SES distribution. PMID- 16670413 TI - Effects of early treatment with statins on short-term clinical outcomes in acute coronary syndromes: a meta-analysis of randomized controlled trials. AB - CONTEXT: The short-term effects of early treatment with statins in patients after the onset of acute coronary syndromes (ACS) for the outcomes of death, myocardial infarction (MI), and stroke are unclear. OBJECTIVE: To evaluate relevant outcomes of patients from randomized controlled trials comparing early statin therapy with placebo or usual care at 1 and 4 months following ACS. DATA SOURCES AND STUDY SELECTION: Systematic search of electronic databases (MEDLINE, EMBASE, PASCAL, Cochrane Central Register) from their inception to August 2005, which was supplemented by contact with experts in the field. Two reviewers independently determined the eligibility of randomized controlled trials that compared treatment with statins with a control, were initiated within 14 days after onset of ACS, and had a minimal follow-up of 30 days. Trials with cerivastatin were only included in a sensitivity analysis. DATA EXTRACTION: Information on baseline characteristics of included trials and patients, reported methodological quality, lipid levels, and clinical outcome was independently extracted by 2 investigators. Investigators from each included trial contributed additional data if necessary. DATA SYNTHESIS: Twelve trials involving 13 024 patients with ACS were included in the meta-analysis. The risk ratios for the combined end point of death, MI, and stroke for patients treated with early statin therapy compared with control therapy were 0.93 (95% confidence interval [CI], 0.80-1.09; P = .39) at 1 month and 0.93 (95% CI, 0.81-1.07; P = .30) at 4 months following ACS. There were no statistically significant risk reductions from statins for total death, total MI, total stroke, cardiovascular death, fatal or nonfatal MI, or revascularization procedures (percutaneous coronary intervention or coronary artery bypass graft surgery). Sensitivity analyses with restriction to trials of high quality or with additional data from a large trial using cerivastatin indicated summary risk ratios even closer to 1. CONCLUSION: Based on available evidence, initiation of statin therapy within 14 days following onset of ACS does not reduce death, MI, or stroke up to 4 months. PMID- 16670415 TI - How the US drug safety system should be changed. PMID- 16670414 TI - Nonhormonal therapies for menopausal hot flashes: systematic review and meta analysis. AB - CONTEXT: Concern regarding the adverse effects of estrogen and other hormones for treating menopausal symptoms has led to demand for other options; however, the efficacy and adverse effects of nonhormonal therapies are unclear. OBJECTIVE: To assess the efficacy and adverse effects of nonhormonal therapies for menopausal hot flashes by reviewing published randomized controlled trials. DATA SOURCES: MEDLINE (1966-October 2005), PsycINFO (1974-October 2005), and the Cochrane Controlled Clinical Trials Register Database (1966-October 2005) were searched for relevant trials that provided data on treatment of menopausal hot flashes using 1 or more nonhormonal therapies. STUDY SELECTION: All English-language, published, randomized, double-blind, placebo-controlled trials of oral nonhormonal therapies for treating hot flashes in menopausal women measuring and reporting hot flash frequency or severity outcomes. DATA EXTRACTION: Trials were identified, subjected to inclusion and exclusion criteria, and reviewed. Data on participants, interventions, and outcomes were extracted and trials were rated for quality based on established criteria. A meta-analysis was conducted for therapies with sufficient trials reporting hot flash frequency outcomes. DATA SYNTHESIS: From 4249 abstracts, 43 trials met inclusion criteria, including 10 trials of antidepressants, 10 trials of clonidine, 6 trials of other prescribed medications, and 17 trials of isoflavone extracts. The number of daily hot flashes decreased compared with placebo in meta-analyses of 7 comparisons of selective serotonin reuptake inhibitors (SSRIs) or serotonin norepinephrine reuptake inhibitors (SNRIs) (mean difference, -1.13; 95% confidence interval [CI], -1.70 to -0.57), 4 trials of clonidine (-0.95; 95% CI, -1.44 to -0.47), and 2 trials of gabapentin (-2.05; 95% CI, -2.80 to -1.30). Frequency was not reduced in meta-analysis of trials of red clover isoflavone extracts and results were mixed for soy isoflavone extracts. Evidence of the efficacy of other therapies is limited due to the small number of trials and their deficiencies. Trials do not compare different therapies head-to-head and relative efficacy cannot be determined. CONCLUSION: The SSRIs or SNRIs, clonidine, and gabapentin trials provide evidence for efficacy; however, effects are less than for estrogen, few trials have been published and most have methodological deficiencies, generalizability is limited, and adverse effects and cost may restrict use for many women. These therapies may be most useful for highly symptomatic women who cannot take estrogen but are not optimal choices for most women. PMID- 16670416 TI - Evidence-based treatments for alcohol dependence: new results and new questions. PMID- 16670417 TI - Alternatives to estrogen for treatment of hot flashes: are they effective and safe? PMID- 16670424 TI - JAMA patient page. Alcohol abuse and alcoholism. PMID- 16670425 TI - A large field CCD system for quantitative imaging of microarrays. AB - We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities <<1 molecule/microm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation approximately +/-4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from approximately 450 to approximately 750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K x 2K pixel, cooled CCD chip with a well depth of approximately 10(5) electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 microm, sufficient for obtaining quantitative multicolor images from >30,000 array elements in an 18 mm x 18 mm square. PMID- 16670426 TI - Novel method for high throughput DNA methylation marker evaluation using PNA probe library hybridization and MALDI-TOF detection. AB - The methylation of CpG dinucleotides has become a topic of great interest in cancer research, and the methylation of promoter regions of several tumor suppressor genes has been identified as a marker of tumorigenesis. Evaluation of DNA methylation markers in tumor tissue requires hundreds of samples, which must be analyzed quantitatively due to the heterogeneous composition of biological material. Therefore novel, fast and inexpensive methods for high throughput analysis are needed. Here we introduce a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. This method is multiplexable, allows the use of standard 384 well automated pipetting, and is more specific and flexible than established methods, such as microarrays and MS SNuPE. The approach was used to evaluate three candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Ade-nomatous polyposis coli (APC), glycogen synthase kinase-beta-3 (GSK3beta) and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. APC and EYA4 were confirmed as being differentially methylated in colon cancer patients whereas GSK3beta did not show differential methylation. PMID- 16670427 TI - Design of LNA probes that improve mismatch discrimination. AB - Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for G-T mismatches, where discrimination decreased when the guanine nucleotide at the mismatch site or even the flanking nucleotides were modified. Fluorescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs. LNAs do not change the amount of counterions (Na+) that are released when duplexes denature. New guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes. PMID- 16670428 TI - siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells. PMID- 16670429 TI - Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1. AB - Recombination due to template switching during reverse transcription is a major source of genetic variability in retroviruses. In the present study we forced a recombination event in human immunodeficiency virus type 1 (HIV-1) by electroporation of T cells with DNA from a molecular HIV-1 clone that has a 300 bp long hairpin structure in the Nef gene (HIV-lhNef). HIV-lhNef does not replicate, but replication-competent escape variants emerged in four independent cultures. The major part of the hairpin was deleted in all escape viruses. In three cases, the hairpin deletion was linked to patch insertion of tRNA(asp), tRNA(glu) or tRNA(trp) sequences. The tRNAs were inserted in the viral genome in the antisense orientation, indicating that tRNA-mediated recombination occurred during minus-strand DNA synthesis. We here propose a mechanistic model for this hairpin-induced tRNA-mediated (HITME) recombination. The transient role of the cellular tRNA molecule as enhancer of retroviral recombination is illustrated by the eventual removal of inserted tRNA sequences by a subsequent recombination/deletion event. PMID- 16670430 TI - A clustering property of highly-degenerate transcription factor binding sites in the mammalian genome. AB - Transcription factor binding sites (TFBSs) are short DNA sequences interacting with transcription factors (TFs), which regulate gene expression. Due to the relatively short length of such binding sites, it is largely unclear how the specificity of protein-DNA interaction is achieved. Here, we have performed a genome-wide analysis of TFBS-like sequences for the transcriptional repressor, RE1 Silencing Transcription Factor (REST), as well as for several other representative mammalian TFs (c-myc, p53, HNF-1 and CREB). We find a nonrandom distribution of inexact sites for these TFs, referred to as highly-degenerate TFBSs, that are enriched around the cognate binding sites. Comparisons among human, mouse and rat orthologous promoters reveal that these highly-degenerate sites are conserved significantly more than expected by random chance, suggesting their positive selection during evolution. We propose that this arrangement provides a favorable genomic landscape for functional target site selection. PMID- 16670431 TI - Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo. AB - To be effective, antisense molecules should be stable in biological fluids, non toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2 phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms. PMID- 16670432 TI - Homologous recombination prevents methylation-induced toxicity in Escherichia coli. AB - Methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action. PMID- 16670433 TI - Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase. AB - The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIalpha to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an 'anti recombinase'. PMID- 16670434 TI - The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco. AB - We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential. PMID- 16670435 TI - Dietary Cl(-) restriction upregulates pendrin expression within the apical plasma membrane of type B intercalated cells. AB - Pendrin, encoded by Slc26a4, is a Cl(-)/HCO(3)(-) exchanger expressed in the apical region of type B and non-A, non-B intercalated cells, which regulates renal NaCl excretion. Dietary Cl(-) restriction upregulates total pendrin protein expression. Whether the subcellular expression of pendrin and whether the apparent vascular volume contraction observed in Slc26a4 null mice are Cl(-) dependent, but Na(+) independent, is unknown. Thus the subcellular distribution of pendrin and its role in acid-base and fluid balance were explored using immunogold cytochemistry and balance studies of mice ingesting a NaCl-replete or a Na(+)-replete, Cl(-)-restricted diet, achieved through substitution of NaCl with NaHCO(3). Boundary length and apical plasma membrane pendrin label density each increased by approximately 60-70% in type B intercalated cells, but not in non-A, non-B cells, whereas cytoplasmic pendrin immunolabel increased approximately 60% in non-A, non-B intercalated cells, but not in type B cells. Following either NaCl restriction or Cl(-) restriction alone, Slc26a4 null mice excreted more Cl(-) and had a higher arterial pH than pair-fed wild-type mice. In conclusion, 1) following dietary Cl(-) restriction, apical plasma membrane pendrin immunolabel increases in type B intercalated cells, but not in non-A, non B intercalated cells; and 2) pendrin participates in the regulation of renal Cl( ) excretion and arterial pH during dietary Cl(-) restriction. PMID- 16670437 TI - Renal oxidative vulnerability due to changes in mitochondrial-glutathione and energy homeostasis in a rat model of calcium oxalate urolithiasis. AB - Calcium oxalate monohydrate (COM) crystals are the commonest component of kidney stones. Oxalate and COM crystals in renal cells are thought to contribute to pathology via prooxidant events. Using an in vivo rat model of crystalluria induced by hyperoxaluria plus hypercalciuria [ethylene glycol (EG) plus 1,25 dihydroxycholecalciferol (DHC)], we measured glutathione and energy homeostasis of kidney mitochondria. Hyperoxaluria or hypercalciuria without crystalluria was also investigated. After 1-3 wk of treatment, kidney cryosections were analyzed by light microscopy. In kidney subcellular fractions, glutathione and antioxidant enzymes were measured. In mitochondria, oxygen consumption and superoxide formation as well as cytochrome c content were measured. EG plus DHC treatment increased formation of renal birefringent crystal. Histology revealed increased renal tubular pathology characterized by obstruction, distension, and interstitial inflammation. Crystalluria at all time points led to oxidative stress manifest as decreased cytosolic and mitochondrial glutathione and increased activity of the antioxidant enzymes glutathione reductase and peroxidase (mitochondria) and glucose-6-phosphate dehydrogenase (cytosol). These changes were followed by a significant decrease in mitochondrial cytochrome c content at 2-3 wk, suggesting the involvement of apoptosis in the renal pathology. Mitochondrial oxygen consumption was severely impaired in the crystalluria group without increased mitochondrial superoxide formation. Some of these changes were also evident in hyperoxaluria at week 1 but were absent at later times and in all calciuric groups. Our data indicate that impaired electron flow did not cause superoxide formation; however, mitochondrial dysfunction contributes to pathological events when tubular crystal-cell interactions are uncontrolled, as in kidney stones disease. PMID- 16670439 TI - Comparative sequencing of plant genomes: choices to make. PMID- 16670443 TI - Initial experience with a novel coronary rinsing and thrombectomy system: "Rinspiration". AB - BACKGROUND: Intracoronary thrombus is often problematic during percutaneous coronary intervention. Rinspiration is a new thrombectomy catheter system designed to mechanically disrupt and remove intravascular thrombus. METHODS: Rinspiration thrombectomy was performed in 15 patients with angiographically visible thrombus in native coronary arteries (10 cases) and in saphenous grafts (5 cases). Indications included acute myocardial infarction in 11 patients and postinfarction ischemia in 4 patients. RESULTS: The Rinspiration system appeared relatively simple to use. There were no device-related complications. Thrombectomy resulted in an improvement in thrombus grade from 3.8 +/- 1.6 to 2.5 +/- 1.7 (p = 0.02), and TIMI flow from 1.5 +/- 1.3 to 2.2 +/- 1.0 (p = 0.047). Stenting further improved thrombus grade to 0.8 +/- 1.6 and TIMI flow to 2.6 +/- 0.8. At 30-day follow up all patients remained free of repeat revascularization and reinfarction; 1 patient died from hemorrhagic stroke. CONCLUSIONS: The Rinspiration system appears capable of safely removing intracoronary thrombus and warrants further evaluation. PMID- 16670442 TI - Phototropism: bending towards enlightenment. PMID- 16670445 TI - A new proposed simplified classification of coronary artery bifurcation lesions and bifurcation interventional techniques. AB - Current classification systems of coronary bifurcation lesions are confusing and difficult to memorize. As coronary revascularization techniques become increasingly complex, it is important to establish a universal classification system. This manuscript proposes a simplified classification system that uses a combination of letters and numbers to provide a clinically relevant anatomic description of a given coronary artery bifurcation lesion. This classification consists of the prefix B (for Bifurcation lesion), followed by the addition of 4 separate suffixes. The first suffix consists of one of the letters C, N, S, or L. C = Close to the bifurcation: the lesion is close to a bifurcation, but the distance from the carina is more than the width of the plaque protruding into the lumen; N = Bifurcation lesion with one branch being Nonsignificant: nonsignificant being defined as less than 2.0 mm vessel diameter; S = Small proximal segment; or L = Large proximal segment: large defined as more than two thirds of the sum of the diameters of both branch vessels. The second suffix describes the number of diseased ostia. 1M = only the Main vessel ostium is involved; 1S = only the Side branch ostium is involved; or 2 = both ostia are involved. The third suffix classifies the angle between the bifurcation vessels and uses the letters V or T; V = the angle between the two branches is less than 70 degrees, T = angle more than 70 degrees. The fourth suffixes are optional: CA for calcified, LM for left main involvement. PMID- 16670444 TI - Evaluation of a new polymer-coated paclitaxel-eluting stent for treatment of de novo lesions: six-month clinical and angiographic follow-up results of the APPLAUSE trial. AB - BACKGROUND: This trial assesses the safety and efficacy of the PicoElite stent (amg International GmbH, Raesfeld-Erle, Germany), a new paclitaxel-eluting polymer-based stent. METHODS: This is a first-in-man, single-center pilot trial that randomized 30 patients with 35 de novo coronary lesions in a 2:1 proportion to either the paclitaxel-eluting PicoElite stent (PES group: 20 patients with 24 lesions) or the bare metal ArthosPico stent (BMS group: 10 patients with 11 lesions). RESULTS: Baseline characteristics were similar in both groups, apart from younger patients in the PES group (mean age 63.5 years vs. 71.7 years in the BMS group; p = 0.032) and shorter lesion length in the PES group (9.56 mm vs. 14.25 mm in the BMS group; p = 0.02). In both groups, there was no difference in the primary endpoint of major adverse cardiac events (MACE) at 30-day follow up. There was a nonsignificant trend towards lower 6-month MACE and target lesion revascularization (TLR) rates in the PES group (10.5% PES vs. 40.0% in the BMS group for both; p = 0.143), and no stent thrombosis in the PES group. At 6-month angiographic follow up, the PES group demonstrated a significantly reduced in stent binary restenosis rate (5.0% vs. 40.0%; p = 0.031), in-stent late loss (0.47 mm vs. 1.10 mm; p = 0.004), and in-segment late loss (0.72 mm vs. 1.16 mm; p = 0.016) as compared to the BMS group. CONCLUSIONS: The 6-month results of the APPLAUSE study demonstrate the safety and efficacy of the paclitaxel-eluting PicoElite stent for de novo lesions, with a significant reduction in late loss and a trend towards less TLR compared to a bare metal control. PMID- 16670446 TI - Comparison between sirolimus- and Paclitaxel-eluting stents for the treatment of chronic total occlusions. AB - OBJECTIVES: This study evaluated the clinical and angiographic effectiveness of sirolimus-eluting stent (SES) and paclitaxel-eluting stent (PES) implantation for chronic total occlusion (CTO) lesions. BACKGROUND: It has not been known which drug-eluting stent, SES vs. PES, is superior for the treatment of CTO lesions. METHODS: This study comprises 107 CTO lesions which were successfully treated with SES implantation and 29 CTO lesions with PES implantation. Six-month angiographic restenosis rates and major adverse cardiac events (MACE) including death, nonfatal myocardial infarction and target lesion revascularization were compared between the two groups. RESULTS: Baseline clinical characteristics were similar between the two groups. The procedural success rate was 98.1% in the SES group and 100% in the PES group. There was 1 sudden death due to in-hospital acute thrombosis in the PES group. There were no significant differences in baseline angiographic measures between both groups, except larger postprocedural minimal luminal diameter and acute gain in the PES group. At 6-month angiographic follow up, the restenosis rate was significantly higher in the PES group (28.6% vs. 9.4%; p = 0.020). Similarly, the late loss was significantly higher in the PES group (0.8 +/- 0.8 mm vs. 0.4 +/- 0.8 mm; p = 0.025). At one-year follow up, the MACE-free survival rate was significantly higher in the SES group (95.8% vs. 85.8%; p = 0.049). CONCLUSIONS: The implantation of SES in the treatment of CTO lesions showed more favorable results regarding restenosis and clinical outcomes compared with PES implantation. PMID- 16670447 TI - Sirolimus or paclitaxel drug-eluting stents for treatment of chronic total occlusions of coronary arteries: much ado about very little. PMID- 16670448 TI - The safety of autologous intracoronary stem cell injections in a porcine model of chronic myocardial ischemia. AB - BACKGROUND: Intracoronary mononuclear cell therapy may produce angiogenesis in chronic myocardial ischemia. Potential complications include periprocedural infarction secondary to: reduced coronary blood flow; hyperviscosity from the cell preparation; or microvascular dysfunction. To date, no studies to evaluate these potential complications have been reported. The objective of this report was to study the safety and feasibility of intracoronary injections of autologous bone marrow mononuclear cells in a porcine chronic myocardial ischemia model. METHODS: Domestic pigs (n = 5) underwent ameroid cuff placement of the left circumflex artery. Bone marrow-derived mononuclear cells [15 x 10(6) cells] labeled with CM dioctadecyl tetramethylindocarbocyanine were given by intracoronary injection. Animals were sacrificed, and hearts and vital organs were inspected grossly and by histopathology, and bone marrow underwent immunofluorescence microscopy. RESULTS: Troponin I levels, gross inspection and histopathology did not reveal evidence of myocardial infarction. Labeled cells were observed in perivascular structures in myocardium at the injection site in all animals and in the spleen from one animal. Bone marrow aspirates indicated labeled cells. CONCLUSIONS: Intracoronary injection of autologous mononuclear cells in a porcine chronic myocardial ischemia model appears safe. Intracoronary injection resulted in cell localization in the perivascular areas of myocardium supplied by the injected vessel. Cell localization was observed only in the spleen in just one animal. Labeled cells were identified in bone marrow aspirates from three animals following injection, suggesting a role for bone marrow engraftment and repopulation as a possible mechanism for progenitor cell localization in myocardium. PMID- 16670449 TI - Angioscopically-determined extent of coronary atherosclerosis is associated with severity of acute coronary syndrome. AB - OBJECTIVE: Some patients with acute coronary syndrome (ACS) have large myocardial infarction but others have small or no infarction. However, what makes this difference has not been clarified. We compared the angioscopic findings between those two categories of ACS patients and examined the association between the severity of ACS and the morphology of both culprit lesion and nonculprit coronary segments. METHODS: Prospectively and consecutively enrolled patients with ACS were classified as CK-elevation-ACS (CKE-ACS; n = 54) or non-CK-elevation-ACS (NCKE-ACS; n = 22). Patients were diagnosed as CKE-ACS when the elevation (greater than twice the normal upper limit) of CK-MB was detected; otherwise, patients were diagnosed as NCKE-ACS. They all underwent emergent catheterization and PCI of the culprit lesion. The entire culprit artery was observed by angioscopy, and the prevalence of thrombus and the color grade of yellow plaques were evaluated. The color grade of yellow plaques were classified as 0 (white), 1 (slight yellow), 2 (yellow), or 3 (intense yellow) according to the standard colors. The color grade of culprit plaque (CC), number (NP) and maximum (MC) color grade of yellow plaques in the nonculprit segments, plaque index (PI = N x MC), and prevalence of thrombus at the culprit lesion (CT) and in the nonculprit segments (NT) were compared between CKE-ACS and NCKE-ACS patients. RESULTS: CC (1.9 +/- 0.9 vs. 1.7 +/- 0.8; p = 0.3) and CT (93% vs. 77%; p = 0.06) were not significantly different between CKE-ACS and NCKE-ACS patients, however, NP (2.2 +/- 1.6 vs. 1.4 +/- 1.2; p = 0.03), MC (1.8 +/- 0.9 vs. 1.2 +/- 0.9; p = 0.008), PI (4.8 +/- 4.4 vs. 2.4 +/- 3.1; p = 0.03), and NT (39% vs. 11%; p = 0.02) were significantly higher in CKE-ACS than in NCKE-ACS patients. CONCLUSION: Although the culprit lesions of CKE- and NCKE-ACS had similar yellow color grades, the culprit lesions of CKE-ACS showed a trend towards a higher prevalence of thrombus. A greater number of yellow plaques of higher color grades and a higher prevalence of thrombosis in the nonculprit segments were detected in CKE-ACS compared to NCKE-ACS patients. The angioscopically-determined extent of coronary atherosclerosis appeared advanced in CKE-ACS patients compared to NCKE-ACS patients. PMID- 16670450 TI - Intracardiac echocardiography in cardiovascular catheter-based interventions: different devices for different purposes. PMID- 16670451 TI - A case of hypertrophic obstructive cardiomyopathy complicated by a single coronary artery treated by transcoronary septal ablation. PMID- 16670452 TI - Routine pressure-derived fractional flow reserve guidance: from diagnostic to everyday practice. AB - The pressure-derived fractional flow reserve (FFR) is a valuable and well validated index for assessing the ischemic significance of coronary lesions. The 0.75 cutoff value of FFR discriminates between lesions with and without ischemic potential, helps decision making as to whether to revascularize a coronary stenosis and assists in evaluating the results of catheter-based treatment. Recent data show that the FFR index is also useful in managing patients with complex coronary disease. The aim of this paper is to provide an overview of the theoretical background of this index and its clinical applicability in the catheterization laboratory. PMID- 16670453 TI - Disruption of in-stent neointima causing acute myocardial infarction. PMID- 16670454 TI - Injecting lubricant into the guiding catheter enables stent deployment. AB - BACKGROUND: The majority of percutaneous artery interventional failures is due to the inability to deploy stents in patients with tortuous arteries, fibrotic lesions and/or calcified plaque. Recent reports of lubricant-facilitated stent delivery have described the use of either dipping the stent into solution or applying the solution to the stent surface prior to insertion into the guiding catheter. This report demonstrates that injection of undiluted RotaGlide into the guiding catheter can better facilitate stent delivery into patients. Following unsuccessful stent placement, 2 cc of undiluted RotaGlide was injected into the guiding catheter by syringe with the undeployed stents just proximal to the lesion site. The stents were advanced and deployed without difficulty at the lesion site. This is the first report of RotaGlide being injected into the guiding catheter, resulting in what may be decreased friction of the buddy wire and/or stent. RotaGlide-facilitated stenting may be the best option when conventional techniques have failed. PMID- 16670455 TI - Percutaneous aspiration of inferior vena cava thrombus. PMID- 16670456 TI - Emergent primary PCI of anomalous LAD. AB - Approximately 0.3% to 2% of patients may have anomalous origins of the coronary arteries. Anomalous origin of the left coronary artery (LCA) or left anterior descending (LAD) artery from the right sinus has been well described. In persons in whom the course involves an interarterial track between the aorta (Ao) and pulmonary artery (PA), an increased incidence of sudden death has been reported, particularly during or shortly after exercise. This has been felt to be due to transient occlusion of the anomalous LAD from increased blood flow through the Ao and PA as the anomalous LAD courses between them, possibly causing myocardial ischemia. In an elective setting, further anatomic delineation with other methodologies such as cardiac magnetic resonance (MR) imaging is recommended. In this case report we present an emergent percutaneous coronary intervention (PCI) of an anomalous LAD arising from the right sinus of Valsalva and coursing between the Ao and PA in a nonsurgical candidate. PMID- 16670457 TI - Successful percutaneous coronary intervention for severe stenosis of an anomalous left coronary artery originating from the proximal right coronary artery. AB - An isolated, single coronary artery is a rare congenital anomaly with an incidence of 0.03% to 0.4% and an uncommon finding (0.6% to 1.3%) in patients undergoing coronary angiography. In this report we describe a rare case of a patient who had an anomaly of the left and right coronary arteries with a single coronary ostium in the right sinus of Valsalva, in which percutaneous coronary intervention (PCI) was successfully performed in both arteries. PMID- 16670458 TI - Combined peripheral and coronary artery percutaneous intervention in patients with significant coronary and peripheral vascular disease. Case reports and review. AB - Combined percutaneous coronary and peripheral intervention in patients with coronary and peripheral vascular disease can be time and cost saving. Despite the potential benefit, such hybrid procedures have been rarely reported. We report two cases of hybrid peripheral and coronary intervention that were performed at our institution with excellent outcomes. This is followed by a review of the literature. PMID- 16670459 TI - Systolic compression of bypass grafts. AB - Myocardial bridging, systolic compression of an intramyocardial segment of an epicardial coronary artery, is well known to involve native coronary arteries. Systolic compression of bypass grafts has been rarely described. We present two cases of dynamic systolic compression of bypass grafts to native coronary arteries: one of a vein graft to the right coronary artery, and another of a left internal mammary artery to the left anterior descending artery. PMID- 16670460 TI - Microcarcinoma of the thyroid. AB - Thyroid microcarcinoma, defined as a thyroid tumor measuring 1 cm or less, is an extremely indolent tumor. Papillary microcarcinoma, the most common subtype, is often identified incidentally in a thyroid removed for benign clinical nodules or diffuse processes (eg, thyroiditis). In this clinical situation, over 99% are cured by simple lobectomy. In the less common scenario, the microcarcinoma is the primary lesion to a lymph node metastasis presenting clinically as a neck mass; in this situation, the tumor should be treated as a clinical cancer. Other rare microcarcinomas can occur and, of these, the most recently described is micromedullary carcinoma. In the familial setting, these lesions are identified in prophylactic thyroidectomies and are not unexpected findings. However, when found as sporadic tumors, their implications are still unknown. The histologic features, pathologic mimics, and molecular facets of these microscopic neoplasms are discussed in this review. PMID- 16670461 TI - OCT4: a sensitive and specific immunohistochemical marker for metastatic germ cell tumors. PMID- 16670462 TI - Clear cell hidradenoma of the skin--a third tumor type with a t(11;19)-associated TORC1-MAML2 gene fusion: Genes Chromosomes Cancer. 2005;43:202-205. PMID- 16670463 TI - Podoplanin: a novel diagnostic immunohistochemical marker. AB - Podoplanin is a transmembrane mucoprotein recognized by the recently commercially available D2-40 monoclonal antibody. Recent investigations have shown that podoplanin is selectively expressed in lymphatic endothelium as well as lymphangiomas, Kaposi sarcomas, and in a subset of angiosarcomas with probable lymphatic differentiation. Podoplanin has also been shown to be strongly expressed in seminomas, epithelioid mesotheliomas, and hemangioblastomas, and immunostaining for this marker can assist in the diagnosis of these tumors. This article reviews the current information on the applications of podoplanin immunostaining in surgical pathology. PMID- 16670464 TI - Metastasizing "benign" fibrous histiocytoma of the skin: a report of two additional cases and review of the literature. PMID- 16670465 TI - Pathology and the internet: Washington University neuromuscular website. PMID- 16670471 TI - Promising results using the Hanna Corneal Trephine System in penetrating keratoplasty. AB - PURPOSE: To evaluate the outcome of penetrating keratoplasties using the Hanna Corneal Trephine System. METHODS: Fifty-five patients out of 70, who had penetrating keratoplasty performed by a single surgeon (KHO), from 1995 to 2001, were reviewed in 2002, at Department of Ophthalmology, Hospital of Hilleroed, Denmark. The reviews were performed by a single observer (NJ). All donor corneas were trephined from the epithelial side. Visual acuity, refraction, corneal astigmatism, and graft survival were main outcome measures. RESULTS: Seventy-two percent of the patients achieved a visual acuity > or =0.5. In major subgroups, that is, Fuchs endothelial dystrophy, secondary endothelial dystrophy, and keratoconus, a best corrected visual acuity of > or =0.5 was achieved in 57, 67, and 100%, respectively. The mean (+/-SD) postoperative spherical refraction was +0.84 D (+/-3.72), whereas mean keratometric and refractive astigmatism were 3.24 D (+/-1.51) and -3.06 D (+/-1.95), respectively. The overall graft survival rate was 96.7% after 2 years and 81.2% after 5 years. At least 90% of all patients were content with the result and course of the operation. CONCLUSIONS: The best corrected visual acuity was better, and the degree of astigmatism was less compared to previously reported studies. We suggest that these findings can be partly explained by the operative technique, that is, cutting the donor and the recipient cornea from the epithelial side by the use of an artificial chamber. PMID- 16670472 TI - Changes observed in keratolimbal allograft. AB - OBJECTIVE: To study the incidence and clinical characteristics of presumed immunologic reactions in limbal grafts after keratolimbal allograft(KLAL). METHODS: A total of 121 KLALs performed in consecutive 85 eyes of 78 patients with total limbal stem-cell deficiency were analyzed retrospectively. Mean follow up period was 46.6 months. Three types of limbal graft changes classified as epithelial defects, acute edema, and vascular engorgement were analyzed as probable signs of immunologic rejection. RESULTS: One or more changes in limbal grafts were observed after 16 KLALs (13.1%). All but 1 eye used systemic and local immunosuppression using corticosteroid and cyclosporine A, and most of the changes developed within 3 months postoperatively. All but 2 eyes developed epithelial breakdown in the central cornea, and 12 corneas underwent conjunctivalization. The rate of corneal epithelialization in eyes with limbal graft changes was significantly worse than those without the changes (P < 0.0001). Histologic examination in eyes with the limbal graft changes revealed high incidence of lymphocyte infiltration. CONCLUSIONS: Presumed limbal graft rejection occurred in approximately 13% of cases after KLAL. Suppression of the immune reaction remains a key to success in KLAL. PMID- 16670473 TI - An evaluation of keratometry in 6-year-old children. AB - PURPOSE: To evaluate the repeatability and comparability of keratometry measured by both the IOLMaster and RK-F1 AutoRef-Keratometer in children. METHODS: Keratometry results from a sample (n = 447) of 6-year-old children who were examined in the Sydney Myopia Study were analyzed. Corneal power was analyzed along the flattest and steepest meridians to determine if there were any systematic differences between repeat measurements or between the two instruments. The 95% limits of repeatability (LR) and 95% limits of agreement (LA) (mean difference +/- 1.96 x standard deviation of differences) were calculated. RESULTS: There were no systematic differences in repeat measurements for each instrument. For the IOLMaster, mean difference of the flattest corneal meridian was -0.01 (D) (P = 0.3, 95% LR, -0.22, 0.21 D) and of the steepest corneal meridian, 0.01 D (P = 0.3, 95% LR, -0.35, 0.38 D). For the RK-F1, mean difference of the flattest corneal meridian was -0.02 D (P = 0.3, 95% LR, -0.25, 0.21 D); and of the steepest corneal meridian, 0.00 D (P = 0.9, 95% LR, -0.39, 0.39 D). Systematic differences, however, were found between the two instruments. The IOLMaster gave significantly (P < 0.0001) steeper readings than the RK-F1 for both the flattest corneal meridian, 0.29 D (95% LA, -0.08, 0.66 D), and the steepest corneal meridian, 0.18 D (95% LA, -0.29, 0.65 D). CONCLUSIONS: Keratometry was highly repeatable for both the IOLMaster and RK-F1 instruments when used in young children. These instruments would be suitable for use in monitoring changes of corneal curvature over time. Small significant systematic differences in keratometry between the two instruments were also found. PMID- 16670474 TI - Corneal ectasia after laser in situ keratomileusis in patients without apparent preoperative risk factors. AB - PURPOSE: To evaluate patients who developed ectasia with no apparent preoperative risk factors. METHODS: Potential cases of patients who developed ectasia without apparent risk factors were identified by contacting participants in the Kera-Net (n = 580), ASCRS-Net (n = 450), and ISRS/AAO ISRS-Net (n = 525) internet bulletin boards from April to October 2003. Cases were included if ectasia developed after laser in situ keratomileusis in the absence of apparent preoperative risk factors. Reported cases were excluded for the following reasons: (1) calculated residual stromal bed less than 250 microm, (2) preoperative central pachymetry less than 500 microm, (3) any keratometry reading greater than 47.2 diopters (D), (4) a calculated inferior-superior value greater than 1.4, (5) more than 2 retreatments, (6) attempted initial correction greater than -12.00 D, (7) an Orbscan II "posterior float" (if obtained) greater than 50 microm, and (8) surgical/flap complications. RESULTS: A total of 27 eyes of 25 patients were submitted for consideration. Eight eyes (8 patients) met our inclusion criteria. Mean age was 27.7 years (range, 18-41 years). Preoperative manifest refraction spherical equivalent was -4.61 D (range, -2.00 to -8.00 D); steepest keratometric reading was 43.86 D (range, 42.50-46.40 D); keratometric astigmatism was 0.93 D (range, 0.25-1.90 D); and preoperative central pachymetry was 537 microm (range, 505-560 microm). The mean calculated ablation depth was 82.8 microm (range, 21 125.4 microm), and mean calculated residual stromal bed was 299.5 microm (range, 254-373 microm). Mean time to recognition of ectasia onset was 14.2 months (range, 3-27 months) postoperatively. At the time of ectasia diagnosis, the mean manifest refraction spherical equivalent was -1.23 D (range, +0.125 to -3.00) with a mean of 2.72 D (range, 0.75-4.00 D) of astigmatism. CONCLUSIONS: Ectasia can occur after an otherwise uncomplicated laser in situ keratomileusis procedure, even in the absence of apparent preoperative risk factors. PMID- 16670475 TI - The effects of eye drops on corneal thickness in adult myopia. AB - PURPOSE: To study the effects of Complex Tropicamide (0.5% Tropicamide and 0.5% phenylephrine HCI) and Saline solution on corneal thickness in adult myopic patients with the Orbscan II system. SETTING: Department of Ophthalmology, Yantai Yuhuangding Hospital. DESIGN: Prospective, nonrandomized, clinical trial. METHODS: The thinnest pachymetry of the cornea was obtained before and 1.5 hours after administration of three drops of Complex Tropicamide to the left eyes of 58 patients (58 eyes) and Saline solution to their 31 right eyes, respectively. The corneal thickness of the other 27 right eyes before and 1.5 hours after eyelid closure without exposure to eye drops was used as the control group. RESULTS: The thinnest pachymetry of the cornea was significantly higher after exposure to eye drops in the Tropicamide group (23.36 +/- 15.01 microm; t = -11.855, P < 0.001). Similar findings were also noted in the Saline group (7.13 +/- 8.11 microm, t = 4.894, P < 0.001). The difference between the two groups was also significant (t = 6.737, P < 0.001). There were no statistically changes in corneal thickness in control group. The drops tested have no effect on the location of the thinnest corneal site and its distance form the visual axis. CONCLUSION: Eye drops including Saline solutions may have significant effects on the corneal thickness in myopia, and this may have implications for corneal refractive surgery. PMID- 16670476 TI - Deep anterior lamellar keratoplasty for keratoconus. AB - PURPOSE: To investigate surgical outcomes after deep anterior lamellar keratoplasty (DALKP) in eyes with keratoconus. METHODS: Retrospective review of 127 eyes of 118 keratoconus patients who underwent DALKP at King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia between January 1, 1998 and November 30, 2001. The mean age of all patients was 25.9 years (range 11-70 years). The mean follow-up for all patients was 10.9 months (range 4-61 months). Baring of Descemet's membrane (DM) during DALKP was achieved in 47 (37%) eyes; some stroma was left during operation in the remaining 80 (63%) eyes. Snellen visual acuity, intraoperative complications, and postoperative graft status were assessed. RESULTS: At the last follow-up visit, 74% of all eyes were able to achieve a best corrected visual acuity of 20/50 or better. This level of improvement was more statistically significant with baring of DM (P = 0.005). Intraoperative perforation of DM occurred in 16 (13%) cases and was statistically significant in eyes with deep corneal scars (P = 0.012). However, this perforation did not seem to have an impact on the final visual acuity (P = 0.48). Main graft-related complications included graft-host vascularization (7/127), stromal graft rejection (4/127), graft infection (1/127), and persistent epithelial defect (1/127). CONCLUSIONS: Deep lamellar keratoplasty provides a safer and successful alternative to penetrating keratoplasty for keratoconus patients but remains a challenging procedure. Endothelial graft rejection is absent after DALKP. However, stromal graft rejection, although very rare, does occur. PMID- 16670477 TI - No pathogenic mutations identified in the TGFBI gene in polymorphic corneal amyloid deposition. AB - PURPOSE: To determine whether primary, polymorphic, corneal amyloid deposition is associated with a mutation of the TGFBI gene. METHODS: Interventional case series of 8 patients. Slit lamp examination of all patients and photodocumentation of 5 patients were performed. Genomic DNA was isolated from buccal mucosal swabs obtained from all patients and all 17 exons of the TGFBI gene were amplified and sequenced. RESULTS: Multiple polymorphic, refractile deposits were noted throughout the central corneal stroma in all patients. The deposits appeared gray white on direct illumination and translucent on retroillumination, characteristic of amyloid. In 2 patients, linear, branching opacities, reminiscent of lattice corneal dystrophy, were identified. Histopathologic examination confirmed the presence of stromal amyloid in the cornea of 1 patient who required corneal transplantation for pseudophakic corneal edema. Screening of the entire coding region of the TGFBI gene revealed 4 previously described synonymous substitutions, Leu217Leu, Val327Val, Leu472Leu, and Phe540Phe. A previously unreported missense change, Asp299Asn, was identified in one affected patient but not in her affected sister. No pathogenic mutations, including the Ala546Asp missense mutation previously associated with polymorphic corneal amyloidosis, were identified in any of the patients. CONCLUSIONS: TGFBI gene mutations were not identified in a series of patients with polymorphic corneal amyloid deposition. As bilateral, discrete stromal amyloid deposits may be dystrophic or degenerative, differentiation between these phenotypically similar conditions is facilitated with the use of molecular genetic analysis. PMID- 16670478 TI - Femtosecond laser preparation of donor tissue from the endothelial side. AB - PURPOSE: The femtosecond laser (Intralase) may provide advantages for dissecting a thin, uniform thickness posterior lamellar disk of donor tissue to be used for endothelial transplantation. We investigated the use of the Intralase to dissect the donor cornea from the posterior side to better obtain a thin and uniform lamellar disk. We investigated the use of a viscoelastic "cushion" to protect the endothelium during applanation and laser delivery. METHODS: Human eye bank donor buttons were placed endothelial side up, covered with a thin coat of viscoelastic, and brought into contact with the Intralase applanation lens. A 7 mm diameter, 100-microm lamellar disk was cut from the endothelial side. The endothelial viability after these procedures was determined using a live cell/dead cell assay. Controls were designed to assess the endothelial viability after applanation and laser application using only a balanced salt solution (BSS) cushion instead of viscoelastic material. Additionally, applanation without lasering using either BSS or a viscoelastic cushion was studied. RESULTS: The average endothelial cell loss in the laser experiment sets were 10% (n = 5, range of 4-17%, Sodium Hyaluronate), 14% (n = 5, range of 7-19%, Sodium Hyaluronate Sodium Chondroitin) and 6% (n = 5, range of 3-11%, Hydroxypropylmethyl cellulose). In the controls, laser and applanation with BSS resulted in an average endothelial loss of 18% (n = 5, range of 14-26%). Applanation alone without laser dissection resulted in cell loss of 9% (n = 5, range of 7-12%) using BSS and 9% (n = 6, range 1-42%) Hydroxypropylmethyl-cellulose. CONCLUSIONS: The technique of using a viscoelastic "cushion" to protect endothelial cells from damage during posterior laser dissection prior to transplantation is promising. Viscoelastic coating protects the endothelial layer from damage from the coupling lens better than a layer of BSS. The lasering process, however, causes damage in addition to applanation with the laser lens. Further studies are warranted to optimize reproducibility of endothelial cell survival and evaluate the smoothness of stromal dissections in the posterior cornea. PMID- 16670479 TI - Efficacy of contact lens storage solutions against different acanthamoeba strains. AB - PURPOSE: This study was undertaken in order to determine the susceptibility of acanthamoebae to various contact lens disinfecting systems, with respect to type of disinfectant and exposure time. METHODS: Ten contact lens solutions, including one new system, were tested for their effectivities against cysts of three Acanthamoeba strains. Of main interest was an incubation time of 8 hours (overnight disinfection) and cyst quantities of 10 and 10, respectively. RESULTS: Boston Advance (polyaminopropyl biguanide 0.0005%, chlorhexidine 0.003%) for the storage of rigid gas permeable contact lenses destroyed the cysts of all strains at either concentration within one hour of exposure. Meni Care Plus (polyhexamethylene biguanide 0.0005%) also destroyed 10 cysts of all strains, however, 10 cysts only of two strains within one hour; after an exposure time of 8 hours all cysts were killed. The disinfecting solutions for soft contact lenses did not kill 10 cysts of either strain after 8 hours of incubation time. CONCLUSION: Two of the tested contact lens solutions (the ones for rigid gas permeable lenses) destroyed the cysts of all strains. One of these, containing a combination of two amoebicidal ingredients even after only one hour of exposure time. PMID- 16670480 TI - Antimicrobial activity of antibiotic-treated amniotic membrane: An in vitro study. AB - PURPOSE: To investigate if amniotic membrane incubated with antibiotics could inhibit bacterial growth in vitro. METHODS: Amniotic membrane fragments were incubated with the antibiotics (netilmicin) solution; the washed and drained fragments were either tested after treatment or further incubated in antibiotic free medium. The antibacterial activity of both amniotic membrane and elution media was carried out by the Agar diffusion method, with Staphylococcus epidermidis as indicator, measuring the inhibition zone after overnight incubation. RESULTS: The amniotic membrane fragments soaked in antibiotics inhibited bacterial growth. Antibiotic uptake was dose-dependent and occurred rapidly. The drug was released from the membrane, and the antibacterial effect was present in the elution media at least 3 days after treatment. CONCLUSIONS: Our preliminary in vitro data show that amniotic membrane can absorb the antibiotic netilmicin and in the future may be used to deliver antibiotics, as reported for collagen shields and other medical prosthetic devices. PMID- 16670481 TI - Are multidose over-the-counter artificial tears adequately preserved? AB - PURPOSE: To investigate artificial tears containing different preservatives for antimicrobial efficacy. Based on the challenge test outlined in the European Pharmacopoeia, products were tested in their original containers to see whether their component preservative had sufficient activity. METHODS: Five brands of over-the-counter artificial tears each containing different preservatives (benzalkonium chloride/EDTA, parabens, chlorobutanol, silver chloride complex, and Purite-stabilized oxychloro complex) were inoculated with test microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans). Changes in the microbial start concentration with time were followed by plating onto growth media to provide a measure of the preservative efficacy. In another test, artificial tears were applied to paper disks that were then placed onto agar growth media seeded with microorganisms. Zones of inhibition were measured after incubation. RESULTS: Only the brand of artificial tears containing benzalkonium chloride/EDTA satisfied the major criteria for antimicrobial preservation for all the test microorganisms. Only a benzalkonium chloride/EDTA-containing disk placed on agar seeded with S. aureus produced a zone of inhibition in the agar diffusion test. CONCLUSION: The brand of artificial tears containing benzalkonium chloride/EDTA is suitable for sale in countries adopting the monographs of the European Pharmacopoeia. Other brands would only be suitable for sale if justified reasons for not meeting the major criteria for preservative efficacy can be provided. PMID- 16670482 TI - Identification of steroidogenic enzyme mRNAs in the human lacrimal gland, meibomian gland, cornea, and conjunctiva. AB - PURPOSE: Sex steroids exert a significant influence on the health and well-being of the ocular surface and adnexa. These hormones affect multiple aspects of the lacrimal and meibomian glands, conjunctiva, and cornea, and have been linked to the development of many ocular surface pathologies. We hypothesize that these hormone actions, as in other tissues, occur predominantly after the local synthesis of androgens and estrogens from adrenal precursors. To begin to test this hypothesis, we analyzed whether human ocular surface and adnexal tissues and cells contain the steroidogenic enzyme mRNAs necessary for the intracrine synthesis and metabolism of sex steroids. METHODS: Total RNA was isolated from human lacrimal and meibomian glands and immortalized corneal and conjunctival epithelial cells. Samples were reverse transcribed to cDNA and analyzed for the presence of enzyme mRNAs by real-time PCR. Positive and negative controls included human placental cDNA and the absence of template, respectively. RESULTS: Our results show that human lacrimal and meibomian glands and corneal and conjunctival epithelial cells contain the mRNAs for steroid sulfatase, 3beta hydroxysteroid dehydrogenase (HSD)-Delta-Delta-isomerase type 1, 17beta-HSD types 1 and 3, aromatase, and glucuronosyltransferase. In contrast, only lacrimal and meibomian tissues appeared to contain detectable mRNA for sulfotransferase. CONCLUSIONS: If the corresponding mRNAs are translated, our results indicate that human ocular surface and adnexal tissues contain the enzymatic machinery necessary for the intracrine synthesis and metabolism of sex steroids. PMID- 16670483 TI - Time course of angiogenesis and lymphangiogenesis after brief corneal inflammation. AB - PURPOSE: To study the time course of angiogenesis and lymphangiogenesis in the cornea after a short inflammatory insult. This might be helpful for the timing of corneal transplantation in high-risk eyes. METHODS: The mouse model of suture induced inflammatory corneal neovascularization was used. After placement of 3 interrupted 11-0 sutures into the corneal stroma of BALB/c mice (left in place for 14 days), corneas were excised 2, 3, 5, 7, 14, and 21 days as well as 1, 2, 3, 6, and 8 months after surgery. Hem- and lymphangiogenesis were evaluated using double immunohistochemistry of corneas with CD31/PECAM1 as panendothelial and LYVE-1 as lymphatic endothelial marker. RESULTS: Both blood and lymphatic vessels grew into the cornea as early as day 2 after suture placement. The outgrowth was initially parallel. Hem- and lymphangiogenesis peaked around day 14. Thereafter, both vessel types started to regress. Regression of lymphatic vessels started earlier and was more pronounced than that of blood vessels. Whereas at 6 and 8 months (partly) perfused CD31+++/LYVE-1(-) blood vessels and (nonperfused) ghost vessels could still be observed, there were no CD31+/LYVE-1+++ lymphatic vessels detectable beyond 6 months after this short inflammation. CONCLUSIONS: After a temporary inflammatory insult to the cornea, there is initially parallel outgrowth of both blood and lymphatic vessels. But thereafter, lymphatic vessels regress earlier than blood vessels and are completely regressed by 6 months. Earlier regression of pathologic corneal lymph versus blood vessels suggests that corneal graft survival in high-risk eyes might best be delayed for a prolonged interval following an inflammatory insult. PMID- 16670484 TI - Altered expression of type XIII collagen in keratoconus and scarred human cornea: Increased expression in scarred cornea is associated with myofibroblast transformation. AB - PURPOSE: Type XIII collagen (ColXIII) is a transmembrane protein thought to be involved in cell-cell and cell-matrix interactions. We report here on its presence in the normal human cornea and compare the results for keratoconus and scarred corneas. METHODS: Immunohistochemistry and in situ hybridization were applied to human corneal samples obtained by penetrating keratoplasty. RESULTS: In the normal human cornea, ColXIII was immunolocalized to the corneal epithelial cells, and to a lesser degree to the stromal keratocytes. The keratoconus cases showed otherwise similar results, but in areas containing Bowman membrane disruptions showed thinned epithelial cells reduced immunostaining for ColXIII, whereas occasionally pronounced immunoreactivity was seen in the stromal keratocytes. The corneal scar samples contained highly increased ColXIII immunostaining by stromal cells in the fibrotic foci, whereas the peripheral areas showed less intense immunostaining. In situ hybridization confirmed that the corneal epithelium and keratocytes actively synthesize the transcript. Immunostaining with alphaSMA revealed that a substantial proportion of the ColXIII mRNA-expressing cells in the stromal scar tissues was myofibroblasts and that these areas lack CD34 immunoreactivity. CONCLUSIONS: The results indicate that ColXIII, which is predominantly confined to the basal corneal cells in the normal cornea, may have a role in the adhesion of corneal epithelial cells to each other and to the underlying basement membrane. Additionally, highly increased expression in scarred corneas suggests that it participates in the corneal wound healing process. PMID- 16670485 TI - Comparative effects of estrogen on matrix metalloproteinases and cytokines in immortalized and primary human corneal epithelial cell cultures. AB - PURPOSE: Recently, we found that 17beta-estradiol induces cytokine and MMP gene expression in SV40 immortalized human corneal epithelial cells (SV40 HCEs). The purpose of this study was to determine if 17beta-estradiol stimulates MMP secretion by cultures of SV40 HCEs and primary human corneal epithelial cells. Further, we determined if estrogen influenced cytokine and MMP gene expression in the primary cultures. METHODS: Gelatin zymography was used to identify MMP-2 and MMP-9 gelatinase activity in the culture medium from SV40 HCEs and primary human corneal epithelial cell cultures exposed to vehicle or 17beta-estradiol for 6 or 24 hour. In addition, 17beta-estradiol effects on cytokine and MMP gene expression in primary cultures were evaluated by real-time PCR methods. RESULTS: 17beta-estradiol had no significant effects on MMP activity in the culture media of either SV40 HCEs or primary corneal epithelial cell cultures. SV40 HCEs secreted predominantly MMP-2, whereas the primary cultures secreted predominantly MMP-9. In primary cultures, 17beta-estradiol caused a significant decrease of IL 6 and IL-8 gene expression, but had no effect on the levels of IL-1beta, MMP-2, and MMP-9 gene expression. CONCLUSIONS: Our findings suggest that 17beta estradiol effects on gelatinase gene expression in SV40 HCEs are not translated into changes in secreted MMP activity. Estrogen influences on gene expression of cytokine and MMPs in primary cultures are different from those in SV40 HCEs. PMID- 16670486 TI - Sutureless amniotic membrane fixation using fibrin glue for ocular surface reconstruction in a rabbit model. AB - PURPOSE: Amniotic membrane transplantation has become an important treatment option for corneal surface reconstruction. However, suture fixation of the transplant has various disadvantages like corneal irritation, scarring, graft loss due to membrane shrinkage, and the need for subsequent suture removal. Replacement of sutures by bioadhesives might be an advantageous alternative. This controlled study was designed to evaluate a new sutureless technique for amniotic membrane fixation onto the corneal surface by using fibrin glue. METHODS: Standardized disks of cryopreserved amniotic membranes were transplanted onto the deepithelialized cornea of 12 rabbits using either conventional suture fixation or a new fibrin glue technique. The rabbits were followed-up with slit-lamp examination and fluorescein staining until epithelialization was completed. Consecutively, the rabbits were killed and the eyes processed for histology and immunohistochemistry for cytokeratin-3. RESULTS: All membranes of both groups stayed in place throughout the follow-up time and showed a progressive graft epithelialization that was completed after 12 days. Whereas suture-fixated membranes showed progressive tissue shrinkage, fibrin-glued sheets remained unaltered. In the bioadhesive group, histology revealed a smooth fibrin layer in the graft-host interface and a continuous, stratified layer of cytokeratin-3 expressing corneal epithelial cells on the membrane surface. In contrast, suture fixated membranes showed contracted and prominent membrane edges with epithelial ingrowth into the submembrane interface. CONCLUSION: Our results demonstrate the general feasibility of reproducible and reliable sutureless amniotic membrane fixation onto the corneal surface in rabbits. Stable adherence is maintained until epithelialization is completed. The sutureless technique gives sufficient manipulation time for the sheet before the final cross-linking process is completed. Furthermore, several advantageous characteristics could be demonstrated as increased biocompatibility, better epithelialization pattern and the lack of membrane shrinkage. PMID- 16670487 TI - Keratoplasty after mustard gas injury: clinical outcome and histology. AB - PURPOSE: Ocular injury by mustard gas can lead to severe eye damage with a delayed course. We report the corneal histology and follow-up after keratoplasty in a patient with mustard gas injury. METHODS: The patient presented with recurrent painful corneal inflammation in both eyes not improving under local therapy. Visual acuity impaired to handmovements. A penetrating keratoplasty was performed on the left eye and afterwards an autorotation keratoplasty on the right eye with a later corneal graft. RESULTS: After the operation of the left eye the patient was immediately painfree and the visual acuity improved to 0,4. So far there have been no signs for transplant rejection or inflammation. Histology of the cornea revealed massive stromal necrosis, and signs of chronic inflammation. Despite denervation of the cornea after autorotation keratoplasty the right eye was still painful and became only painfree after corneal transplantation. CONCLUSION: There has been not much experience with corneal transplantation after mustard gas injury and there is a high risk for transplant rejection due to inflammation and vascularisation of the cornea. Successful and painfree healing with keratoplasty seems only possible after complete removal of the necrotic material. PMID- 16670488 TI - Severe toxic effect of methylene blue 1% on iris epithelium and corneal endothelium. AB - PURPOSE: To report a case of corneal endothelial decompensation and iris pigment dispersion following the inadvertent use of methylene blue 1% for capsular staining during cataract surgery. DESIGN: Case report. METHODS: During an otherwise routine phacoemulsification cataract surgery, inadvertent anterior capsule staining with methylene blue 1% instead of trypan blue 0.025% was performed. Copious irrigation of the anterior chamber with balanced salt solution was initiated upon identification of the wrong dye. The operation was completed with minimal ultrasound energy without complications. RESULTS: Iris discoloration and severe corneal edema developed in the early postoperative period, resulting in severe visual loss. The patient developed bullous keratopathy and underwent penetrating keratoplasty 16 months later. CONCLUSIONS: In vivo intracameral injection of methylene blue 1% induces extreme cytotoxicity, primarily on the corneal endothelium and iris epithelium. PMID- 16670489 TI - Rapid healing of vernal shield ulcer after surgical debridement: A case report. AB - PURPOSE: This study was designed to report a patient with a vernal shield ulcer that did not respond to medical therapy but healed in 1 week after surgical debridement of the ulcer base. METHODS: Case report and literature review. RESULTS: A 12-year-old girl with a history of asthma and eczema was seen with a 5.7- x 5.7-mm corneal epithelial defect in the right eye. Her uncorrected visual acuity was 20/200 OD and 20/70 OS. Slit-lamp examination revealed giant papillae on the tarsal conjunctivae, diffuse punctate epitheliopathy, and a thick mucoid discharge in both eyes. A diagnosis of vernal keratoconjunctivitis both eyes (OU) with a shield ulcer OD was made and prednisolone 1% was started 4 times per day OD. Topical ofloxacin 0.3% 3 times per day and topical cyclosporine 0.05% every 2 hours were added OD 2 days later. On minimal improvement in the epithelial defect in 1 week, surgical debridement of the ulcer base was performed. The epithelial defect healed smoothly in 1 week with an underlying stromal scar and the uncorrected visual acuity improved to 20/70 OD at 1 month and 20/40 at 10 months. CONCLUSION: Corneal shield ulcers and plaques are rare but serious complications of vernal keratoconjunctivitis, which may be unresponsive to standard medical therapy. Surgical debridement is a fast and effective procedure yielding rapid healing of the ulcer and minimizing complications, such as infections. PMID- 16670490 TI - Mucous membrane pemphigoid with fatal bronchial involvement in a seventeen-year old girl. AB - PURPOSE: This study was designed to report a case of biopsy-proven mucous membrane pemphigoid with severe bronchial involvement in a young woman. METHODS: Case report of a 17-year-old girl who presented with worsening dyspnea, skin rash, and bilateral ocular injection, symblepharon, and fornix foreshortening. Conjunctival, skin, and bronchial biopsies were performed along with imaging and serological tests in an effort to establish a diagnosis for this unusual constellation of findings. The surprising occurrence of a cerebrovascular accident during her hospitalization also prompted a search for a concurrent coagulation disorder. RESULTS: Immunofluorescence studies of conjunctival, skin, and bronchial tissue specimens revealed deposition of multiple antibody classes at the basement membrane zone. The patient also possessed circulating basement membrane zone antibodies in her serum and a significant titer of antiphospholipid antibodies. She underwent dilation and stent placement for subglottic tracheal and left bronchial stenosis and was treated with immunosuppressive agents. After a favorable initial response, the patient experienced progressive bronchial stenosis and respiratory compromise, culminating in her death from bronchospasm and cardiopulmonary arrest. CONCLUSION: To our knowledge, this is the first report of mucous membrane pemphigoid involving the lower airways that was confirmed by immunofluorescence analysis. It highlights the potentially lethal, systemic nature of mucous membrane pemphigoid and underscores the need to question patients about symptoms of respiratory dysfunction. PMID- 16670491 TI - Management of intracorneal epithelial cysts with ethanol irrigation and cyst wall excision: A clinicopathologic report. AB - PURPOSE: The clinical and pathologic characteristics and the surgical treatment of 2 patients with intrastromal corneal epithelial cysts are described. METHODS: This interventional case report presents 2 young patients with decreased vision and cosmetic impairment caused by intracorneal epithelial cysts. The first patient had history of strabismus surgery, and the second patient had undergone corneal laceration repair. The cysts had enlarged gradually and dissected into the cornea, threatening the visual axis. Both patients were treated with drainage of cyst contents, chemical cytodestruction with 96% ethanol for 1 minute in a closed system, followed by cyst wall excision. RESULTS: Corneal clarity and appearance improved significantly after surgery. In the first patient, best corrected visual acuity was 20/20 before the operation and remained stable. In the second patient, best-corrected visual acuity improved from 20/400 to 20/60 after the procedure. Histopathologic findings revealed that the cyst walls were composed of nonkeratinized stratified squamous epithelium. No recurrence was noted up to 8 and 3 months in the first and second patients, respectively. No complication occurred during the follow-up period. CONCLUSION: Intracorneal epithelial cysts may be treated safely and effectively with a combination of drainage of cyst contents, irrigation with 96% ethanol, and cyst wall excision with good visual and cosmetic results. PMID- 16670492 TI - Exacerbation of Avellino corneal dystrophy after LASIK in North America. AB - PURPOSE: To report the first case of Avellino corneal dystrophy exacerbation after LASIK in a white or North American patient. METHODS: Case report and literature review. RESULTS: A 25-year-old white female developed progressive corneal opacities after LASIK. Preoperative examination had revealed only subtle white corneal opacities in each eye. The patient's mother had similar corneal opacities. DNA analysis of the patient revealed a heterozygous mutation at the R124H location in the BIGH3 gene. CONCLUSIONS: LASIK can exacerbate Avellino corneal dystrophy and should be avoided in patients with this condition. A careful history and genetic analysis can identify affected patients and those at risk. PMID- 16670493 TI - Analysis and documentation of progression of Fuchs corneal dystrophy with retroillumination photography. AB - PURPOSE: Fuchs corneal dystrophy (FCD) is a degenerative disorder of the cornea that is characterized by the progressive accumulation of guttae, which are small excrescences of Descemet's membrane. We describe a method for documenting the location and number of guttae, and ask whether disease progression can be observed during relatively short periods. METHODS: Patients with FCD were imaged by standard retroillumination photography with a slit lamp. Scanned photographs were analyzed by using NIH ImageJ software to determine the number of individual guttae and areas of confluence. RESULTS: In 4 FCD patients, photographs taken 23 to 30 months apart revealed that, once formed, individual guttae and their relative positions persisted during this period. Very few guttae disappeared, and the emergence of many new guttae was observed. Determination of the area with confluent guttae was used to quantify disease stage. CONCLUSIONS: Computer assisted analysis of retroillumination photographs is proposed as an effective way to document the number and distribution of individual guttae. Although the disease typically progresses slowly during decades, we have been able to detect the formation of new guttae within only 2 years. This rapid assessment of disease progression could be used to measure phenotypic differences between genetic subtypes of FCD. It also could provide important baseline information and methodology for clinical trials of therapeutic options, should these become available. PMID- 16670494 TI - Solitary eyelid Kaposi sarcoma in an HIV-negative patient. AB - PURPOSE: To describe a case of localized Kaposi sarcoma (KS) of the eyelid in an HIV-seronegative patient. METHODS: An 80-year-old man developed an ulcerated nodular tumor-like mass that grew rapidly on his left upper eyelid. There were no similar lesions elsewhere. The eyelid lesion was completely excised and histopathologically examined. Serological analyses and molecular biologic techniques, including polymerase chain reaction, were used. RESULTS: Laboratory examinations were within normal limits, and serology for HIV was negative. Histological sections revealed a vascular proliferation composed predominantly of small slit-like blood vessels and epithelioid spindle cells, supporting the diagnosis of KS. Polymerase chain reaction was positive for human herpesvirus 8. During a 2-year follow-up, no recurrences, development of new lesions, or HIV seroconversions were observed. CONCLUSION: This is a classic KS involving only the eyelid in an HIV-negative patient. Location in the eyelid is a possible, albeit rare, initial solitary manifestation of KS in elderly HIV-negative patients. Surgery is both safe and effective. PMID- 16670495 TI - Pyogenic granuloma formation following placement of the Medennium SmartPLUG punctum plug. AB - PURPOSE: To report a complication, not previously described, of the Medennium SmartPLUG, which is a new punctal plug with a novel design. METHODS: Two case reports are detailed, including clinical and pathologic photographs. RESULTS: Case 1 is a 58-year-old woman with Sjogren syndrome. Nearly 2 years following placement of a SmartPLUG in her right lower canaliculus, the patient presented with an erythematous, papillomatous growth overlying her punctum. Biopsy demonstrated pyogenic granuloma. Case 2 is a 62-year-old woman with dry eye. Exactly 2 years after a SmartPLUG was place in her right lower canaliculus, she also developed a pyogenic granuloma. After irrigation to remove the plug, the growth decreased in size. CONCLUSION: Pyogenic granuloma can develop as a late complication of the Medennium SmartPLUG punctum plug. PMID- 16670496 TI - Corneal lamellar flap retraction after LASIK following penetrating keratoplasty. PMID- 16670497 TI - Preserved peripheral corneal clarity after surgical trauma in patients with Avellino corneal dystrophy. PMID- 16670500 TI - Reis-Bucklers dystrophy. PMID- 16670502 TI - Outcome measures: linking science and ethics in clinical research. PMID- 16670503 TI - Why do we treat allergies with antibiotics? PMID- 16670504 TI - Primary prevention of childhood asthma and allergic disorders. AB - PURPOSE OF REVIEW: The prevalence of asthma and allergic disorders in childhood is increasing in many parts of the world. This review focuses on recent findings with regard to environmental risk factors and their manipulation in the primary prevention of these diseases in childhood. RECENT FINDINGS: Recent studies have failed to resolve the controversy on the subject of exposure to indoor aeroallergens and the risk of sensitization and asthma in childhood. Bronchiolitis due to respiratory syncytial virus in infancy is associated with a significant increase in risk of asthma, but not atopy. In the prevention of these diseases, the effects of breastfeeding are controversial, with studies showing a protective effect in children without allergic predisposition, but other studies showing no effect or even the potential for an increased asthma risk. A significant reduction in the prevalence of doctor-diagnosed asthma at 7 years was found in a randomized controlled multifaceted (reduction of inhaled and ingested allergens) intervention study of high-risk infants. Other intervention measures, such as an education program on how to reduce exposure, the use of probiotics, and immunization against infections, all showed some protective effects. SUMMARY: More research is required, especially with regard to longer periods of follow-up for all current intervention studies aimed at reducing exposure, the onset and duration of intervention, and other novel intervention measures in the primary prevention of asthma and allergic diseases in childhood. PMID- 16670505 TI - Treatment with inhaled corticosteroids improves pulmonary function in children under 2 years old with risk factors for asthma. AB - PURPOSE OF REVIEW: To report on recent studies on the effect of inhaled corticosteroids on pulmonary function in young children with asthma. RECENT FINDINGS: Inhaled corticosteroids are considered the most effective treatment for persistent asthma in children. Appropriate control of childhood asthma may prevent more serious disease or irreversible obstruction in later years. While some authors have described an improvement with the use of inhaled corticosteroids in young children, others found no clinical or functional benefit. Various studies have shown that inhaled corticosteroids ameliorate clinical outcomes, and recently a study demonstrated improvement in pulmonary function in young children with asthma. The use of different study designs may explain the lack of consistent results and disagreement regarding the efficacy of inhaled corticosteroids in these patients. SUMMARY: Based on the preponderance of evidence, treatment with inhaled corticosteroids in infants and young children with recurrent wheeze and risk factors of developing asthma appears to allow better control of the illness and improve the pulmonary function. PMID- 16670506 TI - Noninvasive monitoring of airway inflammation and steroid reduction in children with asthma. AB - PURPOSE OF REVIEW: Management of pediatric asthma is currently based on symptoms (often a second-hand report from parents) and lung function. Inhaled steroids are the mainstay of asthma management targeted at controlling airway inflammation. They should be used in the lowest possible doses. A number of noninvasive methods to assess inflammation have been developed in an effort to optimize anti inflammatory treatment. RECENT FINDINGS: The first longitudinal studies have been published demonstrating an improvement in asthma control in children by adding noninvasive monitoring of inflammation into the clinical management. New methods include exhaled nitric oxide measurements, induced sputum and markers in exhaled breath condensate. SUMMARY: Further studies will show the practicability of including these measurement methods into everyday clinical practice. Their addition to the conventional assessment of asthma control appears promising. Using these methods to evaluate the current inflammatory state seems obligatory in research into new asthma therapeutics and management strategies. Managing asthma in children in specialist practice relying only on symptoms and lung function is no longer state of the art. PMID- 16670507 TI - Aspirin desensitization in aspirin intolerance: update on current standards and recent improvements. AB - PURPOSE OF REVIEW: This review provides an overview of sensitivity to aspirin (acetylsalicylic acid) and its management. In particular, it focuses on current standards and recent improvements in aspirin desensitization. Recent publications on various desensitization protocols and routes of administration are discussed. RECENT FINDINGS: The incidence of aspirin hypersensitivity in the general population ranges from 0.6-2.5%, but that in adult asthmatics ranges from 4.3 11%. Carefully controlled challenge tests with aspirin or other non-steroidal anti-inflammatory drugs are performed as the diagnostic tool of choice. Aspirin desensitization therapy has demonstrated therapeutic effects. Various protocols and routes of administration have been elaborated in the last two decades. Oral administration by means of an initial desensitization with incremental doses of aspirin, followed by daily high-dose therapy, has proven clinical efficacy and safety. Immunological mechanisms of aspirin desensitization therapy are also discussed. SUMMARY: The full clinical picture of aspirin intolerance--the association of aspirin-induced bronchial asthma (with severe acute asthma attacks), aspirin sensitivity and nasal polyps--is commonly summarized as the 'Samter triad'. This condition is related to the abnormal metabolism of arachidonic acid, implicating both the lipoxygenase and the cyclooxygenase pathways. Knowledge concerning mechanisms and clinical features of aspirin intolerance has grown rapidly in recent years. Research has focused on new strategies of aspirin desensitization therapy, especially oral administration using high-dosage protocols. PMID- 16670508 TI - Clinical outcomes after endoscopic sinus surgery. AB - PURPOSE OF REVIEW: Chronic rhinosinusitis continues to present diagnostic and therapeutic challenges for clinicians. Recently, attention has been focused on reliable and valid measures of outcomes for many sinonasal diseases. As a significant fraction of patients with chronic rhinosinusitis will come to endoscopic sinus surgery, there has been a dedicated focus on determining objective clinical outcomes after medical and surgical treatment for CRS. This has been aided by validated instruments for measuring both symptom outcomes and quality of life after endoscopic sinus surgery. RECENT FINDINGS: Recent studies have demonstrated that endoscopic sinus surgery provides statistically and clinically significant improvements in most sinonasal symptoms. These benefits are maintained more than 1 year after surgery. In addition, the significant adverse effects of chronic rhinosinusitis on quality of life can be reversed with appropriate endoscopic sinus surgery. Certain sub-populations do not fare as well with endoscopic sinus surgery, however; adjunctive medical management is required to maintain symptomatic and quality-of-life improvements for patients with nasal polyposis. Unfortunately, paranasal sinus computed tomography scan findings do not predict symptomatic or quality-of-life improvements after endoscopic sinus surgery. SUMMARY: With reliable objective evidence of the health benefits of endoscopic sinus surgery, patients should be considered for such surgery after failure of medical management. Further work is required to characterize outcomes for sub-populations, and to stage and select patients who will derive appropriate benefit from endoscopic sinus surgery. PMID- 16670509 TI - Outcome evaluation of early discharge after hospitalization for asthma. AB - PURPOSE OF REVIEW: This review is designed to assess the evidence around the criteria used to decide when it is appropriate for an individual with asthma to be discharged from hospital. RECENT FINDINGS: There has been scanty recent published research on this subject, and no strong evidence-based discharge guidelines exist. The limited data available suggest that clinical outcome in children is similar when the timing of discharge is the need for 3-hourly rather than 4 hourly bronchodilator. In children, the adoption of this policy would shorten the average length of hospital stay by 5-6 h. SUMMARY: The available data for adults with acute asthma on the appropriate end points for discharge from hospital are inadequate to provide firm conclusions. Children with acute asthma should be considered ready for discharge when clinically stable on 3-hourly bronchodilator. PMID- 16670510 TI - Does acupuncture or Chinese herbal medicine have a role in the treatment of allergic rhinitis? AB - PURPOSE OF REVIEW: Complementary medicines, including acupuncture and Chinese herbal medicine, are being used increasingly for the management of allergies such as allergic rhinitis. Until relatively recently, however, evidence for the efficacy and safety of these therapies in allergic conditions has been lacking. RECENT FINDINGS: A limited number of well conducted studies, all with small sample sizes, have demonstrated the promising therapeutic potential of acupuncture and Chinese herbal medicine for allergic rhinitis. The possible additional benefit of combining the two therapies, however, is yet to be confirmed. There are concerns about the appropriateness of the sham/placebo controls that have been used in acupuncture studies and also about the safety evaluation of individual herbs and herbal formulations. In addition to well established symptom scores and specific quality of life questionnaires, the concurrent use of conventional anti-allergy medications has been utilized as an outcome measure in a number of trials that have evaluated the effectiveness of acupuncture and Chinese herbal medicine for allergic rhinitis. SUMMARY: Tentatively, it appears that acupuncture and Chinese herbal medicine can be effective treatments for allergic rhinitis. Confirmatory evidence, however, is needed from large and, ideally, multi-centre trials. PMID- 16670511 TI - Pediatric health-related quality of life questionnaires in clinical trials. AB - PURPOSE OF REVIEW: This review summarizes recent studies on the feasibility, reliability and validity of pediatric health-related quality of life questionnaires and gives an overview of recent applications of these measures in pediatrics. RECENT FINDINGS: The often-applied short form of the Child Health Questionnaire (CHQ-PF28) provides reliable physical and psychosocial summary measures, but reliable estimates for each scale require the longer version (CHQ PF50). In addition to this questionnaire, the Pediatric Quality of Life Inventory is another reliable and valid measure. The TNO-AZL Preschool Children Quality of Life questionnaire is a feasible and reliable measure for preschool children. Generally, generic questionnaires are less sensitive to the impact of specific diseases than are disease-specific questionnaires. Parent and self-reports provide different outlooks on quality of life, which complement each other. SUMMARY: There are several feasible, reliable and validated pediatric quality of life questionnaires that can be used in clinical trials. These include generic and disease-specific questionnaires and health profile measures, as well as preference-based measures in pediatric settings. Generally, a combination of these types of questionnaires would be the most appropriate approach. Moreover, a combination of parent and self-reports should be applied. Appropriate selection of outcome measures will enhance the quality of pediatric studies and the ability to assess treatment effects in clinical trials. PMID- 16670512 TI - Food allergen labeling in the USA and Europe. AB - PURPOSE OF REVIEW: The ingredient statement on the label of packaged foods is an important source of information for food-allergic consumers. New legislation in the USA and European Union will increase the amount of information available to food-allergic consumers. RECENT FINDINGS: The USA has implemented the Food Allergen Labeling & Consumer Protection Act, which mandates use of clear labeling and source labeling of ingredients derived from commonly allergenic sources. Similarly, the European Union is implementing new regulations that mandate the source labeling of ingredients derived from commonly allergenic foods and fuller disclosure of allergenic food components, even when present in small amounts. The food industry is providing consumers with information relating to the presence of allergenic residues by adding voluntary advisory statements, such as 'may contain peanuts'. SUMMARY: Food-allergic consumers are advised to follow specific strict avoidance diets that exclude allergenic foods and ingredients derived from these foods. Thus individuals must avoid potentially hazardous residues of the allergenic food that might occur as the result of common practices in the food industry. The ingredient statement on packaged food labels now contains more information than ever before. PMID- 16670513 TI - Update on the evaluation and diagnosis of celiac disease. AB - PURPOSE OF REVIEW: Our understanding of the pathophysiology of celiac disease has advanced with associated improvement in diagnostic modalities. Recent studies have placed the prevalence of celiac disease in Western populations at between 1:250 and 1:67. Celiac disease is common throughout the world and most cases go undiagnosed. Understanding the risk factors, clinical presentations and diagnostic modalities is necessary to identify and treat patients with this commonly misdiagnosed disorder. RECENT FINDINGS: Increased prevalence of celiac disease in individuals with autoimmune diseases, reduced bone mineral density and undiagnosed liver disease have been confirmed. However, celiac disease may not be associated with Down's syndrome or epilepsy. Evidence supports high sensitivity and specificity of endomysial- and tissue transglutaminase-based tests in most settings. In children, high or low tissue transglutaminase levels may preclude the need for duodenal biopsy. Cost-effectiveness studies suggest using tissue transglutaminase or endomysial initially, while distal duodenal or jejunal biopsy may confirm celiac disease in the absence of proximal changes. SUMMARY: There is insufficient evidence to support mass screening for celiac disease. However, case finding in individuals with risk factors for celiac disease is recommended. Further study is necessary to define diagnostic algorithms and target populations likely to benefit from testing. PMID- 16670514 TI - Food allergy in Asia. AB - PURPOSE OF REVIEW: Food allergy is increasing in prevalence in Western populations, but little is known about it in Asia. The perception is that the prevalence in this region is low, but is likely to increase with the global increase in allergy. Asia is unique because of the many different cultures and eating habits, with the resulting occurrence of unique food allergens. This review describes the epidemiology and clinical features of food allergy, and introduces some of the unusual food allergens in Asia. RECENT FINDINGS: Recent studies describing the pattern of anaphylaxis and the role of food triggers show that food is an important cause of severe allergic reactions in Asia. Progress has been made on the characterization of unique food allergens from the region. Peanut and tree nuts are rarely the cause of allergic reactions in Asia. The lack of availability of epinephrine auto-injectors in many countries is an important issue that needs to be addressed. SUMMARY: The pattern of food allergy in Asia is unique. Unfortunately, data from many parts of Asia are still lacking. Large, well-designed epidemiological studies are needed so that the scale of the problem can be understood, public awareness can be increased and important food allergens in the region can be identified. PMID- 16670515 TI - Current developments in peanut allergy. AB - PURPOSE OF REVIEW: Peanut allergy is among the most serious, life-threatening food sensitivities, and recent studies indicate increasing prevalence, particularly among children. Our objective is to highlight recent advances in the immunology and treatment of peanut allergy. RECENT FINDINGS: Peanut sensitization may be both a Th1- and Th2-driven process, and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) may play a role in regulating the response intensity. Preliminary work shows that the food matrix is important in the immune response to peanut and that purified peanut allergens may have little intrinsic stimulatory capacity. Studies characterizing peanut allergens have revealed Ara h 1 and Ara h 2 as the most potent allergens, but Ara h 3 may be more allergenic than previously thought. There appears to be a relationship between the diversity of IgE-binding patterns and the severity of clinical symptoms. Multiple novel approaches to treatment are being investigated, which include traditional Chinese medicine, various forms of modified immunotherapy and the use of adjuvants in modified immunotherapy. SUMMARY: By understanding the immunologic response to peanut and the roles of the major peanut allergens, it may be possible to predict those at risk for severe reactions, prevent peanut sensitization and effectively treat those already sensitized. PMID- 16670516 TI - Oral tolerance and allergic responses to food proteins. AB - PURPOSE OF REVIEW: The default response to protein antigens in the intestine is the induction of systemic and local hyporesponsiveness, ensuring the prevention of coeliac disease and food allergies. Interest is increasing in the role of dietary manipulation and probiotics in treating allergic and other diseases, but less is known about how these regimens might influence systemic and local immune responses. This paper addresses the mechanisms at the interface of innate and adaptive immunity that determine how the body responds to orally administered proteins and how local bacteria modify these. RECENT FINDINGS: This paper discusses evidence that dendritic cells in the intestinal mucosa are the critical cells that take up dietary proteins and migrate to the draining mesenteric lymph node, where they induce regulatory CD4 T-cell differentiation. The properties of tolerized T cells are discussed and it is proposed that the gut microenvironment maintains homeostasis by conditioning dendritic cells to remain in a quiescent state. Inhibitory signalling by commensal bacteria possibly contributes to this process. SUMMARY: A regulatory network controls how dietary antigens are taken up and presented to T lymphocytes by specialized antigen-presenting cells. Elucidating their nature and how they are influenced by external factors such as probiotics may help develop novel therapies for allergy and help understand diseases such as coeliac disease. PMID- 16670517 TI - The effect of gastric digestion on food allergy. AB - PURPOSE OF REVIEW: The role of the stomach as the primary location of protein digestion is very well recognized, leading to classification of proteins as digestion-resistant or digestion-labile. This review analyses the role of gastric digestion in food allergy. RECENT FINDINGS: Hindrance of gastric digestion by elevation of the gastric pH, the therapeutic goal of anti-ulcer medication, was recently shown to trigger food allergy via oral sensitization in a murine food allergy model. The relevance in humans was assessed in an observational study of 152 gastroenterological patients who were medicated with anti-ulcer drugs due to dyspeptic disorders. Twenty-five percent of all patients developed a boost or de novo IgE formation towards regular constituents of the daily diet. The clinical relevance of the induced antibodies was confirmed by positive skin and oral provocation tests. Moreover, the importance of gastric digestion was also proven for food-allergic patients, as the allergenicity of allergens were reduced up to a 10,000-fold by gastric digestion. SUMMARY: These recent studies indicate for the first time the important gate-keeping function of gastric digestion, both in the sensitization and the effector phases of food allergy. PMID- 16670518 TI - Gastroesophageal reflux disease, colic and constipation in infants with food allergy. AB - PURPOSE OF REVIEW: This review assesses the role of food allergy in the pathophysiology of gastroesophageal reflux disease, colic and constipation in infancy. RECENT FINDINGS: Frequent regurgitation, persistent crying and constipation are common clinical problems in infancy. A subgroup of infants with these conditions may respond to hypoallergenic diets, but only few randomized clinical trials have been conducted. Skin prick testing and food-specific antibody levels are usually not elevated in these infants, whereas atopy patch testing may diagnostic. The mechanisms by which cow's milk and other food allergens induce gastrointestinal motility disorders are not understood. Apart from cell-mediated reactions, non-immunological effects of food constituents on gastrointestinal motility and gut microbiota may be involved in the pathogenesis. In the absence of reliable diagnostic tests, dietary elimination and re-challenge are usually required to confirm food allergy. A trial of amino acid-based formula or an oligoantigenic maternal elimination diet may be indicated in infants who have failed conventional medical treatment. SUMMARY: Food allergy may contribute to gastroesophageal reflux disease, colic or constipation in infancy. Infants with these conditions often respond to hypoallergenic formula or a maternal elimination diet. Further research is needed to define the mechanisms and clinical markers of gastrointestinal food allergy in infancy. PMID- 16670519 TI - Evaluation of basophil activation in food allergy: present and future applications. AB - PURPOSE OF REVIEW: The diagnosis of immediate hypersensitivity relies on specific IgE and history. Because of low specificity, however, provocation challenges are often necessary. Furthermore, IgE testing does not predict features such as reaction severity; nor can it discriminate cross-reactivity from multiple sensitizations. Direct and passive basophil activation tests may address these needs. In addition, measuring basophil activation ex vivo may be useful for monitoring patients with food allergies. RECENT FINDINGS: Several papers using basophil activation tests demonstrate comparable sensitivity and specificity to current testing for food allergy. Flow-based basophil activation tests have also been used to assess functional characteristics of patient IgE. Finally, several activation phenotypes have been identified as markers of allergic inflammation in vivo; these phenotypes appear to correspond to earlier reports of spontaneous histamine-releasing basophils in patients with active allergic inflammation. SUMMARY: Although in their early stages, direct basophil activation tests may prove to be useful in the clinic. Indirect basophil activation studies are useful when applied to compare functional aspects of IgE. Identification of basophil activation ex vivo is a promising approach for monitoring allergic inflammation. PMID- 16670521 TI - Bibliography. Current world literature. Outcome measures. PMID- 16670520 TI - Component-resolved diagnostics in food allergy. AB - PURPOSE OF REVIEW: The purpose of this paper is to review and discuss recent studies on component-resolved diagnostics in food allergy, involving panels of pure allergen molecules or arrays of peptides derived from allergen sequences, and to summarize the reporting of new food allergens during the past 2 years. RECENT FINDINGS: Several component-resolved diagnostic studies in food allergy suggest that the use of panels of allergen molecules may allow refined clinical information to be obtained on the likelihood or severity of an allergic food reaction and regarding diagnostic specificity. Further, in some studies the use of pure allergen molecules has led to a clearly higher sensitivity of the immunoglobulin E immunoassay compared with conventional allergen extracts. SUMMARY: While common diagnostic methods in allergy assess the presence or absence of allergen-specific sensitization, to date, no in-vitro or in-vivo test exists which exhibits full correlation with clinical food allergy. A multitude of recently reported findings and observations indicate that molecular analysis of allergen sensitization pattern may serve to enhance the clinical utility of immunoglobulin E antibody-based allergy diagnostics. Pure natural and recombinant allergen molecules as well as panels of synthetic peptides have been used for this purpose. PMID- 16670522 TI - Immunostimulatory oligonucleotides inhibit colonic proinflammatory cytokine production in ulcerative colitis. AB - BACKGROUND: We previously showed that Toll-like receptor-9 (TLR-9) ligands ameliorate experimental colitis. In this study, we evaluated the effect of TLR-9 ligands on the generation of proinflammatory cytokines by human colonic mucosa. MATERIALS AND METHODS: Colonoscopic biopsies were obtained from patients with active ulcerative colitis (UC) and from normal subjects. The tissue was organ cultured for 24 hours in the presence or absence of different types of immunostimulatory (ISS) (CpG)-oligonucleotides (ODNs). Tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels in the medium were determined by enzyme-linked immunosorbent assay. RESULTS: In active UC, hTNF alpha and hIL-lbeta generation by inflamed colonic mucosa is 7- and 3-fold higher, respectively, than their generation by normal mucosa. Class B CpG ODNs inhibited colonic TNF-alpha and IL-1beta generation by 50%, whereas class A or C ODNs had a partial or no effect, respectively. A novel class of ODNs that is based on multiple TCG repeats was as effective as class B ODNs. This inhibition resulted from the transcriptional suppression of IL-1beta that occurred within the first 2 hours after ISS-ODN incubation. The addition of chloroquine abolished the inhibitory effects of ISS-ODNs on colonic TNF-alpha and IL-1beta generation. CONCLUSIONS: Only certain classes of ISS-ODNs inhibit the enhanced TNF-alpha and IL-1beta generated ex vivo by inflamed colonic mucosa of patients with UC. The effect of ISS-ODNs is mediated by triggering of TLR-9. These results suggest a potential therapeutic value for ISS-ODNs in UC. PMID- 16670523 TI - Relationship between CARD15, SLC22A4/5, and DLG5 polymorphisms and early-onset inflammatory bowel diseases: an Italian multicentric study. AB - BACKGROUND: Inflammatory bowel disease (IBD) has been associated with several polymorphisms in genes likely involved in innate immune responses and integrity of epithelial mucosal barrier. A major role in adult Crohn's disease (CD) has been defined for 3 polymorphisms in the CARD15 gene, whereas variants in the SLC22A4, SLC22A5, and DLG5 genes could have a minor contribution to IBD susceptibility. METHODS: We analyzed a panel of 6 polymorphisms within these genes in 227 Italian early-onset IBD patients (134 CD, 93 ulcerative colitis [UC]; age at diagnosis or=118 bp) of UC was higher than that in healthy controls (26% vs 4%, P = 0.0001/Pc = 0.0002, OR = 7.644, 95%CI 3.950 14.792), but was not associated with location and severity of the disease. Furthermore, the longer alleles were not associated with haplotypes of C 318T/A+49G of the CTLA-4 gene in Chinese patients with UC. The longer alleles of the CTLA-4 gene microsatellite polymorphism were strongly associated with UC in Chinese patients. PMID- 16670526 TI - CXC chemokine receptor 3 expression increases the disease-inducing potential of CD4+ CD25- T cells in adoptive transfer colitis. AB - BACKGROUND: CD4CD25 T cells induce severe colitis when injected into immunodeficient recipients. The migration of disease-inducing cells to the bowel is controlled by adhesion molecules and chemotactic proteins. Chemokine receptors expressed on the T cells are therefore potential targets for anti-inflammatory therapy in inflammatory bowel disease. In this study, we have investigated the role of the chemokine receptor CXCR3 in the development of chronic colitis in a murine model. METHOD: Expression of CXCR3 on CD4 T cell from normal and colitic mice was assessed by flow cytometry. Development of colitis was followed after transfer of either normal or CXCR3CD4CD25T cell into immunodeficient host. In addition, the ability of regulatory T cell to function in vivo in the absence of CXCR3 was tested. RESULTS: We find CXCR3 to be expressed on 80% to 90% of CD4 T cells isolated from colitic mice compared with only 4% to 10% of CD4 T cells in normal naive mice. Injecting CD4CXCR3CD25 T cells into immunodeficient hosts results in an ameliorated form of colitis with a lack of clinical symptoms, suggesting that CXCR3 expression is important for enteroantigen priming of CD4 T cells and/or subsequent migration into the gut wall. In contrast, CXCR3 expression does not affect the function of regulatory T cells because CXCR3 regulatory T cells are just as capable as their wild-type counterpart of controlling disease development. The diminished disease-inducing capability of CXCR3 T cells is not caused by the absence of enteroantigen specificity; we also tested the enteroantigen-specific proliferative ability of CD4CD25 T cells from CXCR3 mice in vitro and found that they respond even more strongly than wild-type cells. CONCLUSIONS: The present data indicate that CXCR3 plays an important role in controlling the migration of disease-inducing CD4CD25 T cells into the gut wall. In contrast, lack of CXCR3 expression by regulatory T cells does not compromise their function in this model of colitis. PMID- 16670527 TI - Critical role of IL-17 receptor signaling in acute TNBS-induced colitis. AB - BACKGROUND: Inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis are characterized by recurrent inflammation in the gastrointestinal tract. Infiltration of CD4 lymphocytes and neutrophils is one of the predominant features of IBD. MATERIALS AND METHODS: Recently, interleukin (IL)-23 and the downstream T cell-derived cytokine IL-17 have been found to be elevated in intestinal tissue and serum of IBD patients. However, the role of IL 17 and IL-17R signaling in gut inflammation is unknown. To examine this role, we investigated gut inflammation in wild-type or IL-17R knockout mice. RESULTS: Using a model of acute trinitrobenzenesulfonic acid (TNBS)-induced colitis, we found that IL-17 was produced in colon tissue at 24 and 48 hours and that IL-17R knockout mice were significantly protected against TNBS-induced weight loss, IL-6 production, colonic inflammation, and local macrophage inflammatory protein-2 induction. This protection occurred in the presence of equivalent induction of local IL-23 and higher levels of IL-12p70 and interferon-gamma in IL-17R knockout mice compared with wild-type mice. Moreover, IL-17R knockout mice showed reduced tissue myeloperoxidase activity. Furthermore, overexpression of an IL-17R IgG1 fusion protein significantly attenuated colonic inflammation after acute TNBS. CONCLUSIONS: These results demonstrate that IL-17R signaling plays a critical role in the development of TNBS-induced colitis and may represent a target for therapeutic intervention for IBD. PMID- 16670528 TI - Elevated serum anti-I2 and anti-OmpW antibody levels in children with IBD. AB - BACKGROUND: Bacteria are implicated as important factors in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to seek evidence of possible bacterial targets of the immune response related to IBD in children. METHODS: Seventy-eight children referred to the Department of Paediatrics at Tampere University Hospital on suspicion of IBD were included in the study. Upper and lower gastrointestinal endoscopies with biopsies were performed on all children. Sera from 75 children were tested for antibodies to the Pseudomonas fluorescens-associated sequence I2, a Bacteroides caccae TonB-linked outer membrane protein, OmpW, anti-Saccharomyces cerevisiae, and perinuclear anti neutrophil cytoplasmic antibodies. RESULTS: The IBD diagnosis was confirmed in 35 children (18 with Crohn's disease [CD], 12 with ulcerative colitis [UC], and 5 with indeterminate colitis [IC]); 43 children were found to have no inflammation in the gut. Forty-three percent (15 of 35) of those with IBD evinced positive seroreactivity to I2 and 46% (16 of 35) to OmpW. In CD, seroreactivity to I2 and OmpW was 50% (9 of 18) and 61% (11 of 18), respectively. Serum anti-I2 and anti OmpW immunoglobulin A levels were significantly elevated in children with CD in comparison with the non-IBD group (P = 0.007 and P = 0.001, respectively). A combination of OmpW, I2, and anti-S cerevisiae tests identified 94% of CD patients, and a combination of OmpW, I2, and perinuclear anti-neutrophil cytoplasmic antibodies detected 83% of UC cases. CONCLUSIONS: Among children with IBD, strong serological responses to microbial antigens can be found, suggesting that P fluorescens and B caccae antigens have a potential role in the microbiology and immunology of the disease. Furthermore, serologic reactivity to the set of antigens studied here seems to be applicable in the initial differential diagnosis of children with CD and UC. PMID- 16670529 TI - Probiotic treatment of collagenous colitis: a randomized, double-blind, placebo controlled trial with Lactobacillus acidophilus and Bifidobacterium animalis subsp. Lactis. AB - BACKGROUND: Probiotic treatment may be effective in diseases involving gut microflora and intestinal inflammation. In collagenous colitis (CC), a potential pathogenic role of the gut microflora has been proposed. The effect of probiotic treatment in CC is unknown. Our aim was to investigate the clinical effect of treatment with Lactobacillus acidophilus LA-5 and Bifidobacterium animalis subsp. lactis BB-12 (AB-Cap-10) in patients with CC. MATERIALS AND METHODS: Patients with CC and diarrhea were in a double-blind placebo-controlled study randomized (2:1) to AB-Cap-10 or placebo for 12 weeks. The primary end point was reduction in bowel frequency per week of >or=50%. Secondary end points were changes in bowel frequencies, stool consistency, stool weight, histopathology, and abdominal bloating and pain. RESULTS: Twenty-nine patients were randomized: 21 to probiotics and 8 to placebo. Reduction in bowel frequency per week of >or=50% occurred in 6 of 21 (29%) and in 1 of 8 (13%) patients receiving probiotic and placebo, respectively (P = 0.635). No differences between treatments were observed regarding the secondary end points. Post hoc analysis showed a median reduction in bowel frequency per week from 32 (range 18-84) to 23 (range 11-56; P < 0.005), a reduction in number of days with liquid stools per week from 6 days (range 0-7 days) to 1 day (range 0-7 days; P < 0.005), and an increase in number of days with solid stools per week (P < 0.05) in the AB-Cap-10 group. CONCLUSIONS: AB-Cap-10 had no significant effect on the chosen end points. Post hoc analysis demonstrated amelioration of clinical symptoms in the AB-Cap-10 group, indicating that probiotic treatment may potentially influence the disease course of CC. PMID- 16670530 TI - Relationship between sick leave, unemployment, disability, and health-related quality of life in patients with inflammatory bowel disease. AB - BACKGROUND: The goal of this study was to determine the rate of work disability, unemployment, and sick leave in an unselected inflammatory bowel disease (IBD) cohort and to measure the effect of working status and disability on the patient's health-related quality of life (HRQOL). MATERIALS AND METHODS: All eligible patients were clinically examined and interviewed at the 5-year follow up visit. In addition, they completed the 2 HRQOL questionnaires, the Short Form 36 Health Survey (SF-36) and the Inflammatory Bowel Disease Questionnaire validated for use in Norway (N-IBDQ). Data regarding sick leave, unemployment, and disability pension (DP) also were collected. RESULTS: All together, 495 patients were or had been in the workforce during the 5-year follow-up period since diagnosis. Forty-two patients (8.5%) were on DP compared with 8.8% in the background population. Women with Crohn's disease (CD) had the highest probability of receiving DP (24.6%). A total of 58 patients (11.7%) reported they were unemployed at 5 years. This was equally distributed between men and women but was more frequent in patients with ulcerative colitis. Sick leave for all causes was reported in 47% with ulcerative colitis and 53% with CD, whereas IBD related sick leave was reported in 18% and 23%, respectively. A majority (75%) had been sick <4 weeks, and a relatively small number of patients (25%) contributed to a large number of the total sick leave days. Both unemployment and DP reduced HRQOL scores, but the most pronounced effect on HRQOL was found in patients reporting IBD-related sick leave, measured with SF-36 and N-IBDQ. The observed differences also were highly clinically significant. Multiple regression analysis confirmed that IBD-related sick leave was the independent variable with the strongest association to the observed reduction in HRQOL scores. CONCLUSIONS: Unemployment or sick leave is more common in IBD patients than in the Norwegian background population. The number of patients receiving DP is significantly increased in women with CD but not in the other patient groups. Unemployment, sick leave, and DP are related to the patient's HRQOL in a negative way, but this effect is most pronounced in patients reporting IBD-related sick leave. PMID- 16670531 TI - Regulation of apoptosis during homeostasis and disease in the intestinal epithelium. AB - A single epithelial layer serves as the interface between the organism and the contents of the gastrointestinal tract, underlining the importance of regulating cellular viability despite an onslaught of pathogens, toxins, waste by-products, and cytokines. A balance between cellular proliferation and apoptosis is necessary to maintain this critical barrier. Recent findings have begun to explain the mechanisms by which intestinal epithelial cells are able to survive in such an environment and how loss of normal regulatory processes may lead to inflammatory bowel disease (IBD) and predispose to inflammation-associated neoplasia. This review focuses on the regulation of physiological apoptosis in development and homeostasis and on pathological apoptosis in intestinal disease, inflammation, and neoplasia, identifying remaining questions and areas of needed investigation. PMID- 16670532 TI - FOSter your intestinal flora: prebiotics for Crohn's disease. PMID- 16670533 TI - Interstitial pneumonitis after infliximab therapy for Crohn's disease. PMID- 16670534 TI - Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: a key to the regulation of epithelial apoptosis in Crohn's disease. AB - OBJECTIVES: Imbalanced apoptosis of enterocytes is likely to be 1 of the mechanisms underlying Crohn's disease (CD). Apoptosis of enterocytes is regulated by glial-derived neurotrophic factor (GDNF), which is increased in CD. The cellular source of GDNF during gut inflammation is unclear. The aim of the study was to identify the source of GDNF in CD during gut inflammation. MATERIALS AND METHODS: Glial fibrillary acidic protein (GFAP), GDNF, and smooth muscle actin (SMA) was detected in the gut from patients with CD by immunohistochemistry. Cultured enteric glia cells (EGC) were labeled with anti-GFAP, anti-GDNF, and antibodies and a Golgi marker (anti-58K antibodies) after blocking Golgi export with monensin. Cultured EGCs were treated with interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and lipopolysaccharides. Secretion of neurotrophic factors was detected by enzyme-linked immunosorbent assay. RESULTS: Mucosal GFAP positive EGCs are increased in the colon of patients with CD. This type of glia but not subepithelial myofibroblasts expresses significant amounts of GDNF. In vitro GDNF is continuously secreted from cultured EGCs. The neurotrophic factor secretion could be stimulated by IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharides in a time- and dose-dependent manner. The increased GDNF secretion by EGCs sustained for>12 hours after withdrawal of the proinflammatory cytokines. CONCLUSIONS: A mucosal GFAP expressing EGC population is dramatically increased in CD. This population is a major cellular source of the upregulated GDNF in the inflamed gut. Therefore, mucosal EGC may play a key role in protecting the gut epithelium and may contribute to reestablish the integrity of the injured epithelium. PMID- 16670535 TI - Early results of treatment of hip impingement syndrome in slipped capital femoral epiphysis and pistol grip deformity of the femoral head-neck junction using the surgical dislocation technique. AB - Pistol grip deformity of the femoral head-neck junction and slipped capital femoral epiphysis can cause anterior impingement leading to pain, cartilage damage and eventual osteoarthritis. Osteoplasty of this metaphyseal prominence, with or without concomitant intertrochanteric osteotomy, using a surgical dislocation approach, can effectively treat this problem. Clinical and radiographic outcomes were assessed in 19 patients who underwent osteoplasty or osteoplasty/intertrochanteric osteotomy via Ganz-type surgical dislocation with average 12-month follow-up. Preoperative and postoperative Western Ontario and McMaster Osteoarthritis Index (WOMAC) questionnaires and radiographs were obtained and evaluated. In the osteoplasty group, 7 patients improved, 4 were unchanged, and 2 worsened. In the osteoplasty/osteotomy group, 5 of 6 patients improved. Patients with chondral flaps had less improvement. No avascular necrosis was noted postoperatively, and all trochanteric osteotomies healed. The surgical dislocation approach, combined with osteoplasty and/or bony realignment, is a safe efficacious treatment option for symptomatic pistol grip deformity. Outcomes are worse if there is preexisting cartilage damage. PMID- 16670536 TI - The epidemiology of slipped capital femoral epiphysis: an update. AB - The Kids' Inpatient Database, reflecting 6.70 million pediatric discharges in 1997 and 7.30 million in 2000, was coupled with the US Census Bureau data and was used to elicit the epidemiology of idiopathic slipped capital femoral epiphysis (SCFE) that occurred in children 9-16 years. It was found that the overall incidence of SCFE in the United States for these years was 10.80 cases/100,000 children. The relative incidence of SCFE was 3.94 times higher in black children and 2.53 times higher in Hispanic children than in white children. The incidence rate was significantly higher in boys (13.35 cases/100,000 children) than in girls (8.07 cases/100,000 children). Higher incidence rates of SCFE were found in the Northeast and West when compared with rates in the Midwest and the South, suggesting that climate plays a role in the onset of SCFE. Increased incidence of SCFE was noted north of 40 degrees latitude during the summer and south of 40 degrees latitude during the winter. Age of onset was also lower than previously reported and seems to be on a downward trend. This study suggests that the relative incidences of SCFE in blacks and Hispanics are higher than previously reported in the United States. Geographic, racial, and seasonal variations suggest that both environmental and genetic factors may influence the development of SCFE. PMID- 16670537 TI - Proximal femoral physis shear in slipped capital femoral epiphysis--a finite element study. AB - The following finite element study was conducted to determine whether increased body weight, femoral retroversion, and varus hip loading could sufficiently raise physeal shear strains and stresses above the yield point and predispose an adolescent hip to a slip. A computer tomography scan of a 13-year-old child with slipped capital femoral epiphysis was used to generate a solid model of the proximal femur and physis. The model was parameterized using 3-dimensional software to generate three difference angles of femoral neck version-neutral, 15 degrees retroversion, and 15 degrees anteversion. Loads of 2.7 times body weight in a 46- and 86-kg child were applied to the proximal femur to model stance on one leg. In addition, the loading vector was reoriented at various degrees of varus to study the effect of varus loading on physis shear. The results demonstrated that physis stress, strain, and displacement increased with greater body weight, retroversion, and varus displacement of the loading vector. Physis shear strain in hips with a combination of varus loading and femoral neck retroversion exceeded the reported ultimate strain values for cartilaginous soft tissues. The finite element models suggest that in an overweight child, the combination of retroversion and varus hip load may be sufficient to increase physeal strains above the yield point and result in a slip. PMID- 16670538 TI - Torsional strength of double- versus single-screw fixation in a pig model of unstable slipped capital femoral epiphysis. AB - Slipped capital femoral epiphysis was produced in 9 matched pairs of fresh-frozen pig femurs (1-year-old pigs) by twisting the femoral head about the femoral neck axis. Each matched pair was fixed with a single steel cannulated screw on one side and 2 screws contralaterally, after which the perichondrium was removed before retesting. Double-screw fixation without the perichondrium provided 43% of the stiffness and 74% of the strength of the intact physis in torsion and approximately double the stiffness and strength of single-screw fixation (P<0.004). This information may be of use to the surgeon concerned about rotational stability in unstable slipped capital femoral epiphysis. PMID- 16670539 TI - The narrow window of bone age in children with slipped capital femoral epiphysis: a reassessment one decade later. AB - A narrow window of bone age (BA) in slipped capital femoral epiphysis (SCFE) was described a decade ago. Children now mature younger. Does the BA narrow window still exist? It is the purpose of this study to investigate this question. A retrospective review of children with idiopathic SCFE (1998-2003) was performed. The initial study data ("past") were used as a comparison. Anteroposterior pelvis radiographs were scored for BA (Oxford method) in 108 children. Only the first radiograph was used for children with sequential bilateral SCFEs. There were 67 boys and 41 girls. There were 73 children with unilateral and 35 with bilateral SCFEs. Chronological age (CA) was 12.4+/-1.8 years; Oxford score, 39+/-3 years; and Oxford BA (OXBA), 13.2+/-1.3 years. The recent children were younger (12.1 and 13.2 years; P=0.001), with lower OXBA (29.0 and 31.4; P=0.000003). When converted into years, there was no significant difference between the recent and past children (recent data, 13.1 years; past data, 13.5 years; P=0.18). Chronological age was higher in boys than in girls (12.7 and 11.6 years, respectively; P=0.0008), and so was BA (13.9+/-1.0 and 12.0 years, respectively; P<10). There were no differences in BA score between boys and girls (boys, 29.5; girls, 29.9; P=0.49). The recent data demonstrated the same narrow window of BA. The CA range for the boys' SCFE was 8.4 years, whereas the OXBA was 4.0 years. The CA range for girls was 5.3 years, whereas the OXBA was 2.3 years. Those with unilateral SCFEs were older than those with bilateral SCFEs in CA (12.7 and 11.7 years, respectively; P=0.013), but not in BA (29.8 and 29.4; P=0.51). All SCFEs are present, on average, at an OXBA of 29, regardless of sex, symptom duration, or unilateral/bilateral nature. This study has reaffirmed the narrow window of BA in SCFE, with a BA range approximating 50% of CA range. PMID- 16670540 TI - Lateral growth disturbances of the capital femoral epiphysis after nonoperative treatment of late developmental dislocation of the hip: thirty-five cases followed to skeletal maturity. AB - The purpose of this study was to evaluate the effect of type 2 growth disturbances in the developing hip and compare it with other treated hips without evidence of growth arrest. Radiographic data of 117 children (155 hips) with late developmental dislocation of the hip treated by closed reduction at an average age of 14.9 months and followed to skeletal maturity were retrospectively reviewed. Depending on the presence of lateral growth disturbances, our patients were divided into 2 groups similar with regard to age and treatment methods. Lateral physeal arrest was evident at an average age of 8.9 years in 23% of these hips. Seventy-six percent of these hips with type 2 avascular necrosis were classified as Severin classes 1 and 2. One quarter of them had no deformity of femoral head, 8 had deformity of 2 mm or less, and 18 more than 2 mm. There was no statistical difference in satisfactory and unsatisfactory results or most parameters describing the acetabulum between the 2 groups. Lateral tilting of the proximal femoral epiphysis in most cases does not affect final radiologic results. It is a mild form of avascular necrosis and indications for secondary operative procedures are mostly related to natural history of the disease rather than to lateral physeal arrest. PMID- 16670541 TI - The assessment of acetabular index: is there still a place for plain radiography? AB - This is a series of 7 children (14 hips) with a mean age of 7.3 years (range, 3.3 10.5 years) and an underlying diagnosis of developmental dysplasia of the hips and no previous open-hip surgery who underwent plain radiographic and magnetic resonance imaging (MRI) measures of bony acetabular index. There was a significant correlation between the measurement of acetabular index using plain radiography and MRI, with a Spearman correlation coefficient of 0.88 (95% confidence interval, 0.61-0.96; P<0.001) and a mean difference between the 2 measures of 0.36+/-6.5 degrees. Furthermore, the bony and cartilaginous acetabular indexes as measured by MRI had a significant correlation with a Spearman correlation coefficient of 0.88 (95% confidence interval, 0.80-0.98; P<0.001). We suggest that plain radiography is still an appropriate tool for follow-up for the nonoperated hip with developmental dysplasia and may be a good indicator of hip cartilaginous development. PMID- 16670542 TI - The role of the acetabulum in Perthes disease. AB - The purpose of this study is to evaluate the changes in the shape of acetabulum from the onset of the disease to the skeletal maturity in Legg-Calve-Perthes disease. The study population consisted of 243 patients with unilateral involvement who had reached skeletal maturity at last follow-up. All hips were treated by containment methods (which included bed rest and traction in abduction, Petri cast, brace, varus osteotomy, Salter osteotomy, and shelf acetabuloplasty). Necrosis of the femoral head was estimated according to the lateral pillar classification and late results according to the Stulberg classification. Changes in the shape of the acetabulum were divided into 3 groups: type I-normal, concave lateral acetabulum margin; type II-flat, horizontal lateral acetabulum margin; and type III-convex, sloping acetabular margin. Roentgenograms performed during the fragmentation stage revealed type I changes in 78 (32.1%) hips, type II in 136 (55.7%), and type III in 29 (12.2%). At the last follow-up, there were type I changes in 124 (51.2%) hips, type II in 81 (33.5%), and type III in 38 (15.3%). The statistical analysis revealed no significant statistical difference between the age at the onset and gender with regard to acetabular changes. There was significant statistical correlation between the lateral pillar classification, subluxation of the femoral head, the range of hip abduction, and the Stulberg classification with regard to acetabular changes. A type III was statistically associated with lateral pillar classification group C, major subluxation of the femoral head, limited hip abduction, and in Stulberg group 3, 4, or 5. Only surgical methods of treatment improved the acetabular shape at the last follow-up (P<0.05). In our opinion, the lateral acetabulum shape plays a very important role during the remodeling of the deformed proximal femoral epiphysis. PMID- 16670543 TI - Salter-Harris I and II fractures of the distal tibia: does mechanism of injury relate to premature physeal closure? AB - INTRODUCTION: The distal tibial physis is the second most commonly injured physis in long bones. Recent reports demonstrate a high rate of premature physeal closure (PPC) in Salter-Harris (SH) type I or II fractures of the distal tibia. METHODS: At our institution, 137 distal tibial SH type I or II fractures were treated from 1994 to 2002. Reviews were performed on all patients and 91 fractures met inclusion criteria. Patients were categorized according to treatment. RESULTS: We report a PPC rate of 39.6% in SH type I or II fractures of the distal tibial physis. We found a difference in PPC based on injury mechanism. The rate of PPC in patients with a supination-external-rotation-type injury was 35%, whereas patients with pronation-abduction-type injuries developed PPC in 54% of cases. Type of treatment may prevent PPC in some fractures. The most important determinant of PPC is the fracture displacement following reduction. DISCUSSION AND CONCLUSION: PPC is a common problem following SH type I or II fractures of the distal tibia. Operative treatment may decrease the frequency of PPC in some fractures. Regardless of treatment method, we recommend anatomic reduction to decrease the risk of PPC. PMID- 16670544 TI - Tarsal shape, size, and articulating surface morphology in adolescent surgically treated clubfoot and their contralateral normal foot. AB - Despite the inability of radiographic measurements to quantify the 3-dimensional (3D) shape and size of the hindfoot bones affected by the clubfoot pathology, radiographs continue to be used to evaluate treatment efficacy. Advancements in imaging and image analysis allow new quantitative insights to be obtained into bone shape and size. Therefore, this study sought to quantify and compare the 3D size, shape, and articulating surface morphology of the tibia, talus, calcaneus, navicular, and cuboid bones in the adolescent surgically treated unilateral clubfoot and the contralateral normal foot. Anatomic measurements were obtained by geometrically modeling 3D reconstructed magnetic resonance images of the hindfoot tarsals in the feet of 7 adolescents (mean age, 13.0+/-2.8 years). The results showed that the tarsal bones in the surgically treated clubfoot were smaller in volume (20%-36%) and smaller in surface area (16%-28%) than those in the contralateral normal foot. Correspondingly, the size and shape of the articulating surfaces of these bones in the surgically treated clubfoot were also smaller and flatter than those in the contralateral normal foot. Specifically, the mean talar articular surface area was 25% to 40% smaller, the mean talar tibiotalar articular surface length was 26% smaller, the mean tibiotalar articular surface length difference was 78% smaller, and the mean navicular "flattening index" was 86% larger in the surgically treated clubfoot. These data offer an objective standard that will advance the knowledge of the clubfoot pathology and aid treatment efficacy evaluation. PMID- 16670545 TI - Outcome of patients after Achilles tendon lengthening for treatment of idiopathic toe walking. AB - Fifteen children who were diagnosed with idiopathic toe walking that cannot be corrected by nonoperative treatment were assessed by clinical examination and computer-based gait analysis preoperatively and approximately 1 year after Achilles tendon lengthening. Passive dorsiflexion improved from a mean plantarflexion contracture of 8 degrees to dorsiflexion of 12 degrees after surgery. Ankle kinematics normalized, with mean ankle dorsiflexion in stance improving from -8 to 12 degrees and maximum swing phase dorsiflexion improving from -20 to 2 degrees. Peak ankle power generation increased from 2.05 to 2.37 W/kg but did not reach values of population norms. No patient demonstrated clinically relevant triceps surae weakness or a calcaneal gait pattern. Seven patients had a stance phase knee hyperextension preoperatively, and 6 of these corrected after surgery. Achilles tendon lengthening improves ankle kinematics without compromising triceps surae strength; however, plantarflexion power does not reach normal levels at 1 year after surgery. PMID- 16670546 TI - Characteristics of congenital scoliosis in a mouse model. AB - Pregnant mice were exposed to 600 ppm of carbon monoxide on gestation day 9. The carbon monoxide exposure produced 38 spinal anomalies in 17 offspring with congenital scoliosis. Microcomputer tomography improved the identification of spinal anomalies and detected more affected animals, as compared when magnified radiographs were used. The curves were measured and ranged from mild to moderate at an age that corresponds to childhood and, on average, remained at the range of mild to moderate through near-maturity. The group of mice with congenital anomalies near maturity contained severe curves. The bone mineral density (BMD) determined by microcomputer tomography in the mice with congenital scoliosis was the same as that in the controls. The cervical spine BMD was higher than the thoracic BMD, which was higher than the lumbar region in the controls and affected animals. The BMD was highest in the apex of the congenital curves in the animals that were near maturity. This relationship was not seen in the younger mice with congenital curves. Histology revealed growth rates in the hemivertebrae, which, over the last half of the growth period measured, had one third less growth than the adjacent vertebrae. The hemivertebrae were wedged, but the adjacent disks were not. PMID- 16670547 TI - Cervical spinal stenosis in metatropic dysplasia. AB - Metatropic dysplasia is a rare skeletal dysplasia characterized by rapid collapse of the thoracolumbar spine into kyphoscoliosis. Other spinal anomalies associated with metatropic dysplasia include odontoid hypoplasia and atlantoaxial instability leading to cervical myelopathy. Children with metatropic dysplasia evaluated at our institution for spinal deformity showed evidence of cervical stenosis with or without associated cord compression. Magnetic resonance imaging was found to demonstrate these changes. The association of cervical spinal stenosis and metatropic dysplasia has not been previously described. This has significant treatment implications, because decompression over the stenotic segments should be considered in conjunction with spinal fusion for treatment of odontoid hypoplasia or atlantoaxial instability. A retrospective review of 13 cases of metatropic dysplasia was performed. Despite the challenges provided by this patient population, the chance to halt or reverse neurological dysfunction and improve deformity necessitates prompt surgical intervention. PMID- 16670548 TI - Evaluation of an algorithmic approach to pediatric back pain. AB - Pediatric patients require a systematic approach to treating back pain that minimizes the number of diagnostic studies without missing specific diagnoses. This study reviews an algorithm for the evaluation of pediatric back pain and assesses critical factors in the history and physical examination that are predictive of specific diagnoses. Eighty-seven pediatric patients with thoracic and/or lumbar back pain were treated utilizing after this algorithm. If initial plain radiographs were positive, patients were considered to have a specific diagnosis. If negative, patients with constant pain, night pain, radicular pain, and/or an abnormal neurological examination obtained a follow-up magnetic resonance imaging. Patients with negative radiographs and intermittent pain were diagnosed with nonspecific back pain. Twenty-one (24%) of 87 patients had positive radiographs and were treated for their specific diagnoses. Nineteen (29%) of 66 patients with negative radiographs had constant pain, night pain, radicular pain, and/or an abnormal neurological examination. Ten of these 19 patients had a specific diagnosis determined by magnetic resonance imaging. Therefore, 31 (36%) of 87 patients had a specific diagnosis. Back pain of other 56 patients was of a nonspecific nature. No specific diagnoses were missed at latest follow-up. Specificity for determining a specific diagnosis was very high for radicular pain (100%), abnormal neurological examination (100%), and night pain (95%). Radicular pain and an abnormal neurological examination also had high positive predictive value (100%). Lumbar pain was the most sensitive (67%) and had the highest negative predictive value (75%). This algorithm seems to be an effective tool for diagnosing pediatric back pain, and this should help to reduce costs and patient/family anxiety and to avoid unnecessary radiation exposure. PMID- 16670549 TI - Back pain and backpacks in school children. AB - OBJECTIVE: Back pain in adults is common and well studied. In contrast, back pain in children has received comparatively little scientific study, despite recent media attention. The purpose of this study is to see what factors influence the prevalence of back pain in middle school children, with particular attention to the weight of children's backpacks and the availability of school lockers. METHODS: A population-based sample of 1540 children ages 11-14 years in a large metropolitan area was studied. A questionnaire was used to determine presence and severity of back pain, availability of lockers, backpack use, use of 1 or 2 straps to carry backpack, activity limitations due to back pain, and use of pain medication for back pain. Gender, age, weight of the child, and weight of his or her backpack were recorded. Results of scoliosis screening were evaluated with regard to the above information. Data were analyzed using the chi test and univariate or multivariate logistic regression analysis as appropriate. RESULTS: Overall, 37% of the children reported back pain. Backpacks were used by 97% of children, hence there were too few students not using backpacks to treat backpack use as an independent variable. Multivariate analysis found back pain associated with use of a heavier backpack (P=0.001), younger age (P<0.001), female sex (P<0.001), and a positive screening examination for scoliosis (P=0.009). Children with lockers available reported less back pain (P=0.016). The use of 1 or 2 straps to carry the backpack did not have a significant association with back pain (P=0.588). Of the children who reported back pain, 34% limited their activity due to the pain, 14% use medication for pain relief, and 82% believed their backpack either caused or worsened their pain. CONCLUSIONS: The incidence of back pain in early adolescence approaches that seen in adults. Recommendations for an "acceptable" weight of backpacks cannot be made from this study, as the weights of students' backpacks seem directly proportional to the likelihood of back pain. This study identifies 2 factors associated with self-reported back pain in early adolescents that are amenable to change: availability of school lockers and lighter backpacks. These findings may be useful in advising families and influencing school policies. PMID- 16670550 TI - The definition and treatment of pediatric subtrochanteric femur fractures with titanium elastic nails. AB - PURPOSE: Titanium elastic nailing (TEN) has become more common in the treatment of pediatric femur fractures in many European centers and in North America over the past several years. Prior studies have shown that the use of TEN for midshaft femur fractures results in excellent outcomes with an earlier return to activity, earlier mobilization, and a shortened hospital stay. However, subtrochanteric femur fractures continue to remain a difficult subset of fractures to care for, with loss of reduction and nonunion being significant complications. Studies have differed regarding the definition of pediatric subtrochanteric femur fractures. The purpose of this study is to establish a reproducible method of defining pediatric subtrochanteric fractures and then apply that definition in a retrospective review of 13 patients who sustained subtrochanteric femur fractures treated with TEN at North Carolina Baptist Hospital using a modified technique that allows for improved fracture stability. METHODS: Charts and radiographs were retrospectively reviewed for all pediatric patients sustaining subtrochanteric femur fractures treated with TEN from the period of 2000 to 2004 at Wake Forest University. The TEN outcome measures scale was applied to determine their results. RESULTS: TEN allowed rapid mobilization with excellent or satisfactory clinical and radiographic results in all patients. CONCLUSIONS: Results suggest that the use of TEN for subtrochanteric femur fractures is a safe and effective method of fixation that benefits patients through early mobilization, shorter hospital stays, and fewer complications. SIGNIFICANCE: By applying the definition of subtrochanteric femur fractures described by the authors, results of future studies can be objectively compared and classified. TEN is a safe and effective alternative for treating most pediatric subtrochanteric fractures by decreasing the morbidity that occurs with other treatment modalities. PMID- 16670551 TI - Proximal femoral epiphysiolysis during reduction of hip dislocation in adolescents. AB - Traumatic hip dislocation is an uncommon injury in children. The urgency of closed reduction to prevent possible osteonecrosis may present some pitfalls. Adolescents with open proximal femoral physis may have sustained trauma to the physis at the time of dislocation that can lead to displacement of the epiphysis during the reduction maneuver. The purpose of this study is to report 5 cases with this complication and discuss potential etiology and management. All of the 5 patients were between 12 and 16 years old and underwent closed reduction under conscious sedation. Epiphysiolysis of the femoral head was diagnosed after reduction in all 5 patients. Every patient underwent emergent open reduction and internal fixation of the femur and open hip reduction. Avascular necrosis was identified in all 5 patients within 3 to 15 months postinjury. If there is any suspicion of associated physeal injury or if there is any physeal instability noted under fluoroscopy, an open reduction is recommended in the operating room under radiograph guidance to prevent displacement. PMID- 16670552 TI - Genu varum in achondroplasia. AB - The etiology of genu varum in achondroplasia is the subject of much speculation. The purpose of the current study was to investigate the association between fibular overgrowth and genu varum in achondroplasia. A retrospective analysis was performed on the long-leg radiographs of 48 pediatric patients with achondroplasia. All patients were skeletally immature, and the average age was 7.7 years (range, 3-16 years). Boys were significantly more likely to have varus mechanical tibiofemoral angles than girls (Fisher exact test, P=0.038 for the right leg and P=0.008 for the left leg). The distance from the proximal and distal fibula to the proximal tibia knee and ankle joint orientation lines, respectively, was not associated with the alignment of the lower extremity. We conclude that fibular overgrowth does not correlate with the severity of genu varum. PMID- 16670554 TI - Hemiepiphysiodesis for posttraumatic tibial valgus. AB - Posttraumatic tibial valgus is a recognized complication of proximal tibial metaphyseal fractures in children. There is no consensus regarding management of this malalignment; approaches range from repeated osteotomy to therapeutic nihilism. The authors present 12 patients with an average age at fracture of 4 years 11 months, each of whom who was successfully managed by temporary hemiepiphysiodesis. An additional patient (age 33 years) is included to illustrate the potential long-term outcome of "benign neglect."Using staples or, more recently, a 2-hole plate for guided growth, we have demonstrated correction of posttraumatic tibial valgus by all criteria; including mechanical axis deviation, lateral tibial metaphyseal-diaphyseal angle, tibiofemoral angle, and tibial length. We conclude that hemiepiphysiodesis is a safe and effective method of treatment with a high degree of patient/parent satisfaction. There have been no permanent growth arrests. PMID- 16670553 TI - The effect of tibial rotation on varus deformity measurement. AB - PURPOSE: Tibial osteotomy is used to treat a variety of orthopedic conditions, including reduction in pain and improvement of deformity and function. In templating for surgery, accurate radiographic measurement aids in planning for correction. The purpose of our study was to examine the effect of internal rotation and external rotation on measurement of tibial sawbone models with 5 closing wedge varus deformities at 10-degree increments between 10 and 50 degrees. One sawbone without deformity served as the control. STUDY DESIGN: A total of 66 radiographs were measured by 5 individuals. Measurement deviations from the control in each of the 10 positions of rotation were assessed. RESULTS: In the analysis of variance models, increased rotation and varus angulation were associated with increased deviation compared with the control. In external rotation for every 10 degrees of varus angulation, the degree deviation increased 0.69 degrees. In IR for every 10 degrees of angulation, the degree deviation increased by 0.84 degrees. Internal rotation had the greatest impact. Mean differences between different rater's measurements were small (1.1+/-1.2 degrees) and correlations suggested high interrater reliability. CONCLUSIONS: As the degree of rotation from neutral increased and varus angulation increased, deviation from neutral increased. CLINICAL RELEVANCE: Clinically, the results of this study support the importance of proper alignment of the tibia during radiography to more precisely template surgery. PMID- 16670555 TI - Residual deformities following successful initial bone union in congenital pseudoarthrosis of the tibia. AB - The pathology of congenital pseudoarthrosis of tibia is an enigma and the treatment is challenging. Despite achieving union of the pseudoarthrosis, these patients may have compromised function secondary to residual deformities. The purpose of this study is to analyze the prevalence of these deformities, the morbidity caused by them, and the methods to overcome the problems. Sixteen patients who had a successful union were retrospectively reviewed. Clinically, the following parameters were assessed: pain, joint stiffness, and limb length discrepancy (LLD). Anteroposterior and lateral lower extremity radiographs were performed to analyze (1) union of the tibia and fibula, (2) deformity of the tibia, (3) degree of ankle valgus, (4) degree of calcaneus of the os calcis, (5) LLD, and (4) refracture. The average follow-up was 16 years from the first and 8 years from the last surgical procedure. Residual deformities included valgus of tibia (average 11.4 degrees) and procurvatum (average 19.4 degrees) deformities of the tibia or valgus deformity of the ankle (average 21.3 degrees) and calcaneus of the os calcis (average 46 degrees). There were 9 refractures in 8 patients. Nine patients had an LLD (average 3.5 cm). The study demonstrates that careful follow-up of these patients is necessary and residual problems are to be treated as they occur. PMID- 16670556 TI - Access to orthopaedic care for children with medicaid versus private insurance: results of a national survey. AB - BACKGROUND: It has been documented that children insured by Medicaid in California have significantly less access to orthopedic care than children with private insurance. Low Medicaid physician reimbursement rates have been hypothesized to be a major factor. The first objective of this study was to examine whether children insured by Medicaid have limited access to orthopedic care in a national sample. The second objective was to determine if state variations in Medicaid physician reimbursement rates correlate with access to orthopedic care. METHODS: Two-hundred fifty orthopedic surgeon's offices, 5 randomly chosen in each of 50 states, were telephoned. Each office called was asked to answer questions to an anonymous, disclosed survey. The survey asked whether the office accepted pediatric patients, whether they accepted children with Medicaid, and whether they limited the number of children that they accepted with Medicaid, and if so why. Each state sets its own rate of physician reimbursement rates that were collected from individual state Medicaid agencies for 3 different CPT codes. The relationship between acceptance of patients with Medicaid and the individual state's Medicaid reimbursement rate was examined. RESULTS: Children with Medicaid insurance had limited access to orthopedic care in 88 of 230 (38%) offices that treat children, and 18% (41/230) of offices would not see a child with Medicaid under any circumstances. Reimbursement rates for CPT codes widely varied by state: 99243 for an outpatient consultation (range, $20-$176.38), 99213 for an established follow-up outpatient visit (range, $6 $77.76), and 25560 for global treatment of a nondisplaced radius and ulna shaft fracture without manipulation (range, $50-$403.94). There was a statistically significant relationship between access to medical care for Medicaid patients and physician reimbursement rates for all 3 CPT codes. CONCLUSIONS: Children insured with Medicaid have limited access to orthopedic care in this nationwide sample. Medicaid physician reimbursement significantly correlates with patient access to medical care. These data may be of value in the ongoing efforts to improve access to medical care for children on Medicaid. The logical inference from this study is that increasing physician reimbursement rates will improve access. In the authors' opinion, reimbursement rates should be made higher than office overhead to effect meaningful change. PMID- 16670557 TI - Function and upright time following limb salvage, amputation, and rotationplasty for pediatric sarcoma of bone. AB - We wished to determine the relative physical and psychosocial merits of limb sparing reconstruction, above-knee amputation, and rotationplasty in survivors of childhood and adolescent lower extremity bone sarcoma. In comparing minimum 5 year disease-free survivors, we found that outcome was somewhat dependent on the measuring tool administered. A superior score of statistical significance on the system of evaluation of the Musculoskeletal Tumour Society and a trend toward higher Toronto Extremity Salvage Scores were found among patients with a limb sparing reconstruction compared with those with an above-knee amputation. Short Form 36 results and the amount of time spent in the upright position (uptime), as assessed with a remote activity monitor, were similar between these groups. Uptime was highest in patients who had undergone rotationplasty, although statistical assessment of this group was not feasible. Psychosocial outcome was similar between all groups. This information may be useful in discussing reconstruction with patients and their families. PMID- 16670558 TI - Double or single gloves: which is safer in pediatric orthopedic surgery. AB - BACKGROUND AND AIM: Surgical gloves should form an efficient barrier between surgeons and patients to prevent cross infection. Single gloves (SGs) have long been reported unsafe, and usage of double gloves (DGs) is still not universal. No study has reported the usage of DGs in pediatric orthopedic operations. The aim of this study was to assess the efficacy of DGs versus SGs in prevention of body fluid contact between patients and surgeons during pediatric orthopedic surgery. METHODOLOGY: After 150 pediatric orthopedic operations, DGs and SGs were collected and tested for perforations. Gloves were tested for size, site, and number of perforations among principal surgeons, assistant surgeons, and scrub nurses. Gloves were not changed during long surgical procedures and were changed only if perforations were identified and recorded. The DGs used were Maxitex Duplex, powder-free indicator gloves and the SGs were of Gammex-Ansell. One hundred unused gloves of each group were tested as controls. Medical records of the patients were reviewed for age, sex, type of operation, duration of operation, and any postoperative wound infection. The data were entered in database and analyzed using SPSS package. The data were compared between double and SGs using t test with a level of statistical significance at P less than 0.05. RESULTS: Five hundred twenty-six DGs and 316 SGs were tested. Forty-three perforations were detected in DGs (8.1%). Outer gloves were breached in 7.8% and inner in 0.3% as compared with SGs in which 28 (8.7%) were perforated. In DGs, 4% had multiple perforations compared with 11.9% in SGs. There was a statistical significance (P<0.001) when the perforations of inner gloves were compared with the SGs. None of the inner perforations were recognized during surgery, but the outer gloves of the DGs were recognized in 71% as compared with 9% in SGs (P<0.001). The majority of perforations were seen in the nondominant hand in surgeons and assistants hands, whereas scrub nurses had 85% of perforations in the dominant hand. The index finger was the site of perforations in DGs (53.4%; SGs, 43%). The inner gloves were breached only when the outer glove was found to be perforated. The duration of surgery had a direct impact on the number of perforations. There were no perforations in DGs in less than 60 minutes as compared with 3 (10.7%) in SGs. Between 60 and 120 minutes, the perforations in the DGs were 11, and in SGs, 21. During the study period, 4 patients had surgical site infection. Three were superficial and one deep-seated infection. In 3 patients with infection, the gloves were found to be perforated, and 1 patient with infection had no perforations in the gloves. CONCLUSION: Our study confirms that DGs are safer than SGs during pediatric orthopedic operations. In the event of nonavailability of DGs, SGs should be changed on an hourly basis during long procedures. Lastly, there exists a relationship between surgical site infection and glove perforations. PMID- 16670559 TI - Letter to the JPO editors re: article by Andreacchio et al entitled "lateral column lengthening as treatment for planovalgus foot deformity in ambulatory children with spastic cerebral palsy" (J Pediatr Orthop 2000;20:501-505). PMID- 16670560 TI - Neurofibromatosis update. AB - Type 1 neurofibromatosis (NF-1), also known as von Recklinghausen disease, is one of the most common human single-gene disorders, affecting at least 1 million persons throughout the world. It encompasses a spectrum of multifacted disorders and may present with a wide range of clinical manifestations, including abnormalities of the skin, nervous tissue, bones, and soft tissues. The condition can be conclusively diagnosed when 2 of 7 criteria established by the National Institutes of Health Consensus Development Conference are met. Most children with NF-1 have no major orthopedic problems. For those with musculoskeletal involvement, the most important issue is early recognition. Spinal deformity, congenital tibial dysplasia (congenital bowing and pseudarthrosis), and disorders of excessive bone and soft-tissue growth are the three types of musculoskeletal manifestitations that require evaluation. Statistics gathered from the Cincinnati Children's Hospital Neurofibromatosis Center database of 588 patients show the incidence of spinal deformity in children with NF-1 to be 21%; pectus deformity, 4.3%; limb-length inequality, 7.1%; congenital tibial dysplasia, 5%; hemihypertrophy, 1.4%; and plexiform neurofibromas, 25%. The orthopedic complications can be managed, but only rarely are they cured. Current developments in molecular genetics are exciting and give hope to more positive outcomes. PMID- 16670561 TI - "Informed" ??? consent. PMID- 16670571 TI - What is secondary adrenal insufficiency? PMID- 16670574 TI - Pinning down pinworms. PMID- 16670575 TI - Documenting discharge planning. PMID- 16670576 TI - Heeding TIAs: stroke's early warning system. PMID- 16670581 TI - Treating pain after a total joint replacement. PMID- 16670577 TI - Breaking the link between I.V. therapy and HIT. PMID- 16670586 TI - Myths and facts...about multiple endocrine neoplasia type 1. PMID- 16670597 TI - Patient Education Series. Heart attack. PMID- 16670595 TI - Suppressing the scourge of AMI. PMID- 16670598 TI - Recalling a legacy of service. PMID- 16670599 TI - Your patient's receiving noninvasive positive-pressure ventilation. PMID- 16670600 TI - Spin control: caring for a patient with inner ear disease. PMID- 16670601 TI - Understanding the new AHA guidelines, part II. PMID- 16670602 TI - Nursing 2006. Patient-safety survey report. PMID- 16670611 TI - Facing up to prostate cancer. PMID- 16670613 TI - Black widow spider bite. PMID- 16670615 TI - Impact of body overweight and class I, II and III obesity on the outcome of acute biliary pancreatitis. AB - OBJECTIVES: Body overweight and obesity have been associated with an increased morbidity in acute pancreatitis, but conflicting results were reported in the literature with regard to the type and frequency of complications. We investigated the occurrence of complications in different classes of overweight in a homogeneous group of patients with gallstone pancreatitis. METHODS: Data were collected prospectively from 250 patients with biliary pancreatitis to allow calculation of the Blamey (Glasgow) and Acute Physiology and Chronic Health Evaluation (APACHE II) scores. According to their body mass index (BMI), the patients were allocated to different categories of body overweight. The outcome for each category was measured by the components of the Atlanta criteria. Secondary end points of the study were the length of hospital stay, the length of stay at the intensive care unit, and the number of abdominal operations. RESULTS: When compared with normal-weight patients (BMI 18.5-24.9), all categories with BMI > or =25 had an increased risk of developing the "severe" form of acute pancreatitis [odds ratio (OR): 3.55, 95% confidence interval (CI): 1.50-8.40]. Patients with class I obesity (BMI 30-34.9) developed significantly more organ failure and local complications (OR: 3.469, 95% CI: 1.15-10.43). Patients with class II and III obesity (BMI 35-49.9) had, in addition to more organ failure and local complications, also more metabolic complications (OR: 7.33, 95% CI: 1.62 33.24) than did their normal-weight counterparts. They needed also more frequently intensive care and had a longer total hospital stay. CONCLUSION: In acute biliary pancreatitis, body overweight and obesity represent a risk of more "severe" disease and the number and type of complications increase in categories of increasing BMI. PMID- 16670614 TI - Beneficial effects of vasodilators in preventing severe acute pancreatitis shock. AB - OBJECTIVES: To investigate the effect of treatment with several vasodilatory substance on the changes in mean arterial pressure (MAP) of severe acute pancreatitis. METHODS: Pancreatitis was induced in rats by 5% sodium taurocholate retrograde infusion through the pancreatic duct, which produces a significant decrease in arterial blood pressure. RESULTS: Three hours after the induction of pancreatitis, a fall of approximately 25 mm Hg in MAP was observed, with no changes of MAP in untreated controls. The administration of the nitric oxide synthesis inhibitor, N-nitro-L-arginine methyl ester (25 mg/kg), previously to the induction of pancreatitis, produced a marked fall in MAP leading to the death of all the animals. When several vasodilatory substances, S-nitroso-N acetylpenicillamine (200 microg x kg x h), calcitonin gene-related peptide (10 microg/kg), and vasoactive intestinal polypeptide (8 microg x kg x h) were administered previously to the induction of pancreatitis, the MAP fall induced by pancreatitis was not observed. The improvement of physiological conditions observed in vasodilator-treated animals is in agreement with histological data, which show only minor structural changes in the pancreas from these animals, in contrast with the severe alterations observed in untreated pancreatitic rats. CONCLUSION: : Vasodilation confers protection against the systemic circulatory derangement derived from the development of severe acute pancreatitis. PMID- 16670616 TI - Evaluation of somatostatin inhibitory effect on pancreatic exocrine function using secretin-enhanced dynamic magnetic resonance cholangiopancreatography: a crossover, randomized, double blind, placebo-controlled study. AB - OBJECTIVES: Somatostatin inhibitory effect on the exocrine pancreas has been demonstrated by clinical and experimental studies performed with invasive investigative methods. The aim of this study was to quantify the inhibitory effect of low doses of somatostatin (62.5, 125, and 250 microg) on secretin stimulated pancreatic exocrine secretions using magnetic resonance cholangiopancreatography (MRCP). METHODS: Ten healthy volunteers underwent 4 MRCP at a 1-week interval. At each MRCP, 1 of the 3 doses of Somatostatin or the placebo was given by the intravenous route for a period of 40 minutes. After 20 minutes from the beginning of drug infusion, secretin was injected (0.3 CU/kg). MRCP was performed before and every 30 to 45 seconds for 15 minutes after secretin administration. Pancreatic exocrine secretions were quantified by the measurements of pancreatic flow output and total excreted volume, derived from a linear regression between MRCP calculated volumes and time. RESULTS: For the 3 doses of somatostatin, pancreatic flow output was significantly reduced compared to placebo (P < 0.05). Total excreted volume was significantly reduced only for the doses of 62.5 and 250 microg. No statistical significant differences were observed among the 3 doses. CONCLUSIONS: Low doses of somatostatin inhibit pancreatic exocrine secretions as demonstrated noninvasively with MRCP. PMID- 16670617 TI - Ethanol feeding alters death signaling in the pancreas. AB - OBJECTIVES: Alcohol abuse is a major cause of pancreatitis, which is associated with death of parenchymal cells. The goal of this study was to explore the effects of ethanol on cell death pathways in the pancreas. METHODS: Adult male Wistar rats were fed with ethanol diets using the Lieber-DeCarli method. Caspase 8, caspase-3, and cathepsin B expression and activity in the pancreas of these animals as well as the signals that regulate their expression were studied using Western blot analysis and specific assays for biochemical enzyme activity. RESULTS: In the pancreas from rats fed with ethanol, the protein expression and activity of caspase-8 decreased by 48% and 45%, respectively, and caspase-3 activity decreased by 39%. In contrast, cathepsin B protein expression and activity increased with ethanol feeding by 189% and 143%, respectively. Evaluation of the transcriptional regulatory system for caspase-8 and cathepsin B showed that the ethanol effects on these pathways were largely transcriptional. CONCLUSIONS: Our findings show effects of ethanol on the expression of several signals involved in cell death in the pancreas through alteration of transcriptional regulators. The decrease in caspase expression and increase in cathepsin B expression indicate that ethanol feeding may prevent apoptosis and promote necrosis of pancreatic tissue with stresses that cause pancreatitis. PMID- 16670618 TI - Defined localization of nestin-expressing cells in L-arginine-induced acute pancreatitis. AB - OBJECTIVE: Nestin is a stem cell marker originally described as an intermediate filament protein expressed in neuroepithelial stem cells. In the pancreas, a small number of nestin-expressing cells, which are believed to represent either stem cells or progenitor cells, are known to be present in islets, as well as in some stellate cells, pericytes, and endothelial cells. We monitored pancreatic nestin expression to delineate the location of stem cells/progenitor cells in the pancreas after L-arginine-induced pancreatitis. METHODS: Male Wistar rats received 2 intraperitoneal injections of L-arginine, each consisting of 250 mg/100 g of body weight, and were killed 3, 6, and 12 hours and 1, 4, 7, and 14 days later. RESULTS: Serum amylase and lipase levels increased after L-arginine injection, maximal levels occurring at 3 and 12 hours postinjection, respectively. Six hours after L-arginine injection, interstitial edema was observed in the pancreas, whereas on day 4 postinjection, there was severe pancreatic necrosis. Neovascularization and ductal-ductular proliferation were also present in the pancreas. Immunohistochemical analysis revealed increased Ki 67 labeling in acinar cells and capillary endothelial cells. Immunoblotting using antinestin antibody revealed increased nestin expression after L-arginine injection. In the control rat pancreas, nestin immunoreactivity was detected in a few capillary endothelial cells in some islets. After L-arginine injection, nestin was expressed in proliferating capillary endothelial cells, in stellate cells surrounding ductular structures and in submesothelial cells. CONCLUSIONS: Transient nestin expression occurs in specific cell types during the proliferative stage after recovery from L-arginine-induced pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the regenerative capacity of the pancreas. PMID- 16670619 TI - Cholesterol crystals enhance and phospholipids protect against pancreatitis induced by hydrophobic bile salts: a rat model study. AB - OBJECTIVES: The role of bile composition in the pathogenesis of biliary pancreatitis is unknown. The objective of this experiment was to explore the potential role of bile salts, phospholipids, and cholesterol crystals in the pathogenesis of biliary pancreatitis in a rat model. METHODS: Model systems composed of taurodeoxycholate (TDC), mixed bile salts (MBS), or tauroursodeoxycholate (TUDC) [in 10 mM phosphate-buffered saline (PBS), pH 7.4], with or without cholesterol crystals or phosphatidylcholine, were infused into bile ducts of male Sprague-Dawley rats. Twenty-four hours later, animals were killed for histopathologic scoring of (peri)pancreatic inflammation. RESULTS: : Severity of acute pancreatitis depended on bile salt hydrophobicity (TDC > MBS >> TUDC = PBS; histopathologic scores: 25.6 +/- 0.5, 23.0 +/- 1.5, 14.4 +/- 2.2, 14.8 +/- 1.0, respectively; P < 0.001), with corresponding differences in serum lipase concentration. Phosphatidylcholine protected against detrimental effects of TDC at physiological, but not at low, concentrations (scores: 19.5 +/- 2.3 vs 28.3 +/- 1.9 in case of Phosphatidycholine/(TDC + Phosphatidycholine) ratios 0.25 or 0.05, respectively). Cholesterol crystals increased severity of pancreatitis in model systems containing TDC or MBS, but not TUDC or PBS (33.2 +/- 0.4, 29.6 +/- 1.2, 18.6 +/- 1.5, 18.5 +/- 2.2, respectively; P < 0.001). CONCLUSIONS: In the rat model, hydrophobic bile salts and cholesterol crystals aggravate biliary pancreatitis, whereas phospholipids have a protective effect. PMID- 16670620 TI - Aberrant methylation of secreted apoptosis-related protein 2 (SARP2) in pure pancreatic juice in diagnosis of pancreatic neoplasms. AB - OBJECTIVE: Secreted apoptosis-related protein (SARP) families are considered to counteract the oncogenic Wnt signaling pathway, and inactivation of this gene may aid cancer development and progression. Recently, the aberrant methylation of SARP2 was detected frequently in pancreatic carcinoma (PCa) tissue, but not in normal pancreatic tissue. We evaluated the hypermethylation of SARP2 in pure pancreatic juices (PPJ) aspirated endoscopically from patients with PCa, intraductal papillary mucinous neoplasm of the pancreas (IPMN), chronic pancreatitis (CP), and a control group who were consequently free of pancreatic diseases according to the differential diagnosis of PCa. METHODS: We investigated the aberrant methylation of SARP2 in 9 human PCa cell lines and in the PPJ samples from 33 patients with PCa, 20 with IPMN, 19 with CP, and 10 control patients. DNAs extracted from not only sediment, but also the supernatant of the PPJ and PCa cell lines were treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction (PCR) (MSP). Moreover, real-time MSP was also performed for the melting curve analysis and the quantitative analysis of SARP2 in the PPJ. RESULTS: The incidence of the aberrant methylation of SARP2 using MSP was 8/9 (89%) in PCa cell lines, 26/33 (79%) in the PPJ with PCa, and 17/20 (85%) with IPMN. However, it was only 1/19 (5%) in the PPJ with CP, and 0/10 (0%) in the PPJ of the control patients, respectively. The incidences of aberrant methylation of SARP2 in the PPJ with PCa and IPMN were significantly higher than that in the PPJ with CP (P < 0.001, P < 0.001). Melting curve analysis by real-time MSP revealed that the incidences of aberrant methylation of SARP2 in PPJ was 28/33 (85%) with PCa, 9/11 (82%) with the malignant group of IPMN, 5/9 (56%) with the benign group of IPMN and 5/19 (26%) with CP. In this analysis, there were significant differences between PCa and CP (P < 0.001), and between the malignant group of IPMN and CP (P < 0.005). In the quantitative analysis by real-time MSP with a suitable cut-off value, the incidences of aberrant methylation of SARP2 in the PPJ with PCa, the malignant group of IPMN, the benign group of IPMN, and CP were 19/33 (58%), 6/11 (55%), 3/9 (33%), and 2/19 (11%), respectively. The incidence of the aberrant methylation of SARP2 in the PPJ was significantly different between PCa and CP and between the malignant group of IPMN and CP (P < 0.005 and P < 0.05, respectively). CONCLUSIONS: Hypermethylation of SARP2 in the PPJ may be a highly sensitive and useful marker for the detection of pancreatic neoplasms, including PCa and the malignant group of IPMN. PMID- 16670621 TI - Long-term survival and recurrence patterns in ampullary cancer. AB - OBJECTIVE: Ampullary cancers are associated with high resectability rates and good long-term survival. However, the small number of patients in various series has hampered survival analysis. METHODS: One hundred thirteen patients with ampullary cancer underwent pancreaticoduodenectomy between 1989 and 2000, with 48% morbidity and 8% mortality. One hundred four patients who survived the operation were analyzed to identify predictors of long-term survival. RESULTS: The overall median survival was 30.1 (1.6-140.0) months with actuarial 1-, 3-, and 5-year survival rates of 79%, 43%, and 33%, respectively. Lymph node metastasis (P = 0.002) and vascular invasion (P = 0.008) were 2 independent factors adversely influencing survival. Perioperative blood transfusion (P = 0.001) and vascular invasion (P = 0.026) were important factors predicting recurrent disease. CONCLUSIONS: Lymph node metastasis and vascular invasion were 2 important factors, which adversely influenced survival in patients with ampullary cancer. Perioperative blood transfusion and vascular invasion were associated with recurrent disease. PMID- 16670622 TI - The expression of adhesion molecules and the influence of inflammatory cytokines on the adhesion of human pancreatic carcinoma cells to mesothelial monolayers. AB - OBJECTIVES: Pancreatic cancer has a tremendously deplorable prognosis. Peritoneal dissemination frequently occurs after surgical resection of the tumor. Specific adhesion molecules may be of great importance in local tumor recurrence. These adhesion molecules may be influenced by inflammatory cytokines produced during surgery. The aim of this study was to investigate the effects of inflammatory cytokines, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), on the interaction between pancreatic tumor cells and mesothelial cells. METHODS: An experimental in vitro model was designed using Panc-1, MiaPaCa-2, and BxPC-3 pancreatic carcinoma cell lines. Primary cultures of mesothelial cells were incubated with the inflammatory cytokines, and after the incubation, the adherence of the different pancreatic cell lines was measured. By means of immunocytochemical staining and enzyme immunoassay, the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) and counterparts (LFA 1 and VLA-4) was investigated. RESULTS: Preincubation of the mesothelial monolayer with IL-1beta or TNF-alpha resulted in enhanced tumor cell adhesion of the MiaPaCa-2 and BxPC-3 cells. The amount of stimulation for the MiaPaCa-2 cells was more than 100% versus the control situation and for BxPC-3 cells between 20% to 35%. IL-6 did not affect the tumor cell adhesion of the MiaPaCa-2 and BxPC-3 cells. The adherence of Panc-1 was not enhanced after preincubation of the mesothelial monolayers with the inflammatory cytokines. Mesothelial cells show a significant enhancement of expression of ICAM-1, VCAM-1, and CD44 after stimulation with IL-1beta and TNF-alpha. CONCLUSIONS: The presented results prove that IL-1beta and TNF-alpha are significant stimulating factors in pancreatic tumor cell adhesion in vitro and may therefore account for tumor recurrence to the peritoneum in vivo. The immunocytochemical staining results demonstrate that ICAM-1 and CD44 important adhesion molecules and interference with their function may decrease the incidence of peritoneal tumor recurrence after curative resection of pancreatic cancer. PMID- 16670623 TI - Pretransplant culture selects for high-quality porcine islets. AB - OBJECTIVES: The pig is generally considered a suitable alternative donor for clinical islet transplantation. However, adult pig islets are difficult to isolate and culture, often behave variably in in vitro assays, and do not consistently cure diabetic nude mice. In this study, we compared the in vivo function of freshly isolated and cultured adult porcine islets by transplantation in diabetic nude mice. METHODS: Freshly isolated and cultured islets were transplanted in different doses to diabetic nude mice (N = 48). RESULTS: Average islet yield was 1924 islet-equivalents per gram of pancreas, purity 96%, and the viability that was measured by acridine orange and propidium iodide was greater than 80% in all freshly isolated islet preparations. Grafts of freshly isolated islets failed to reduce hyperglycemia in 17 of 18 recipients. Although after 1 day of culture islet recovery was only 21%, grafts of these islets cured 12 of 17 mice. After 7 to 14 days of culture, the recovery had decreased to 11%; however, these islets reversed hyperglycemia in all mice (13/13) and showed shorter time to-normoglycemia and more tightly regulated blood glucose. CONCLUSIONS: Although freshly isolated adult porcine islets survive culture and transplantation poorly, islets selected by prolonged culture are of high potential. PMID- 16670625 TI - Morphological changes of pancreatic ducts in Otsuka Long-evans Tokushima Fatty rats. AB - OBJECTIVE: To determine if pancreatic ductal changes characteristic of chronic pancreatitis occur in male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the 3-dimensional structure of the pancreatic ducts in those rats. METHODS: Male OLETF rats aged 10, 20, and 40 weeks were used. The pancreas was injected with a 120% barium sulfate suspension into the pancreatic ducts, after which radiograph images of the injected pancreas were taken using radiography of the soft parts. Conventional fixed pancreatic tissues were observed by the use of light and scanning electron microscopy, whereas corrosion casts of the pancreatic ducts were observed by the use of scanning electron microscopy. RESULTS: In 20- and 40-week-old male OLETF rats, deletion of acini and formation of tubular complexes and tortuous or helical, deformed, dilated pancreatic ducts were found, whereas the inner surfaces of pancreatic ducts had many craterlike depressions and long cilia. CONCLUSIONS: Male OLETF rats demonstrated the same morphological changes in pancreatic ductal lumina and histological changes in pancreatic tissues as the rats were subjected to pancreatic duct ligation. These results suggested that increased pancreatic duct pressure occurs in male OLETF rats and induces the chronic pancreatitis-like lesions, including the above-mentioned findings. PMID- 16670624 TI - Potential roles of large mafs in cell lineages and developing pancreas. AB - OBJECTIVES: Maf is a family of transcription factor proteins characterized by a typical bZip structure, and mafA, a member of the large-maf family, is a strong transactivator of insulin in cell lines. The present study investigated the expression profiles of the large-maf family proteins in porcine pancreatic tissue and in primary culture cells. METHODS: Immunohistochemical staining was performed to localize each maf protein. Messenger RNA expression was quantitated by real time polymerase chain reaction, and protein expression was assessed by Western blotting. RESULTS: Islet formation was not as clear in newborn pancreatic tissue as in adult pancreatic tissue. MafA- and c-maf-positive cells were more diffusely localized in pancreatic tissue with fewer mafB-positive cell clusters scattered throughout. By contrast, islet formation was clearer, and positive staining for mafA and c-maf tended to be more prominent in the islets of adult pancreatic tissue. Messenger RNA and protein expressions were consistent with the immunohistochemical findings. MafA, mafB, and c-maf coexpressed with insulin positive cells, and c-maf coexpressed with glucagon-positive cells in adult porcine pancreas based on the results of a double-staining study. CONCLUSIONS: Large mafs were identified in normal porcine and human pancreas, and the expression levels and localizations of the large mafs in newborn and adult pancreatic tissues differed. Mafs may play important roles in establishing endocrine function during pancreatic cell differentiation. PMID- 16670626 TI - Acute pancreatitis due to pancreatic arteriovenous malformation: 2 case reports and review of the literature. AB - Pancreatic arteriovenous malformation (AVM) is a relatively rare disease. Based on our literature search, 51 cases of pancreatic AVM have been reported since 1968. The gastrointestinal bleeding is the most common presenting symptom (24/51 cases [47%]). There were only 6 cases of pancreatitis in these cases. We describe 2 cases of acute pancretitis with pancreatic AVM. The patients who were diagnosed with acute pancreatitis were admitted to our hospital. Pancreatitis was considered to be caused by pancreatic AVM by some modalities of diagnostic imaging. The respective pancreatic AVM lesions of patients were resected to prevent the recurrence of pancreatitis. They are asymptomatic after the surgery. Pancreatic AVM is thought to be the one of the reasons for acute pancreatitis. PMID- 16670627 TI - Three-dimensional computed tomography pancreatography of an annular pancreas with special reference to embryogenesis. AB - OBJECTIVES: Several hypotheses have been proposed to explain the embryogenesis of an annular pancreas. Three-dimensional (3D) imaging of the annular pancreas may be useful to elucidate the embryogenesis. The aim of this case report is to clarify the pancreatic ducts in the annular pancreas by 3D computed tomography pancreatography (3D-CTP), stereographically. METHODS: Three-dimensional CTP under endoscopic retrograde pancreatography using a balloon catheter was performed with a helical CT scanner. Three-dimensional images of the pancreatic duct were obtained with simple threshold, shaped surface display. RESULTS: Wirsung duct that surrounds the ventral side of the descending portion of the duodenum and Santorini duct that surrounds the dorsal side of the descending portion of the duodenum were reconstructed by 3D-CTP, stereographically, and the presence of an annular pancreas was confirmed. These findings suggest that the formation of the annular pancreas in the current case may be attributable not only to abnormal development of the right lobe of the ventral pancreatic bud, but also to overgrowth of the dorsal pancreatic bud toward the ventral aspect of the duodenum or excessive rotation of the duodenum following fusion of the ventral bud to the dorsal bud. CONCLUSION: Three-dimensional CTP is expected to contribute usefully toward clarifying the embryogenesis of an annular pancreas. PMID- 16670628 TI - Pancreatic islet autotransplantation with completion pancreatectomy in the management of uncontrolled pancreatic fistula after whipple resection for ampullary adenocarcinoma. PMID- 16670629 TI - Insulin autoimmune syndrome (Hirata disease) as differential diagnosis in patients with hyperinsulinemic hypoglycemia. PMID- 16670630 TI - High-level Skp2 expression in pancreatic ductal adenocarcinoma: correlation with the extent of lymph node metastasis, higher histological grade, and poorer patient outcome. AB - OBJECTIVES: Recent studies have shown that overexpression of S-phase kinase associated protein 2 (Skp2) occurs in many cancers at an advanced stage. We examined the clinicopathologic significance and prognostic implication of Skp2 expression in pancreatic invasive ductal carcinoma. METHODS: Tissue samples from 46 pancreatic carcinomas were examined immunohistochemically for Skp2. The proportion of constituent tumor cells with Skp2 expression was analyzed and classified as high-level nuclear expression when more than 20% of the cancer cells were positive, or low-level nuclear expression otherwise. RESULTS: High level Skp2 overexpression was detected in 13 (28.3%) of the 46 tumors. The incidence of high-level Skp2 was correlated with higher histological grade (P = 0.0056), the extent of lymph node metastasis (P = 0.0086), the level of lymphatic permeation (P = 0.0024), and poorer patient outcome (P = 0.0189). Multivariate analysis showed that high-level Skp2 expression was an independent predictor of overall patient survival (P = 0.0140). CONCLUSIONS: It is suggested that examination of Skp2 expression might be clinically useful for prognostication in patients with pancreatic carcinoma and that Skp2 protein might be a novel therapeutic molecular target. PMID- 16670631 TI - What's new in Shock, April 2006? PMID- 16670632 TI - Issues of concern regarding the use of hypertonic/hyperoncotic fluid resuscitation of hemorrhagic hypotension. AB - Small volume resuscitation fluids continue to be of interest to the military and limited volume resuscitation is becoming more common in the treatment of hemorrhage in the civilian community. With renewed interest to undertake a large US-Canada multi-center clinical trial of hypertonic saline alone or combined with dextran (HSD) possibly in 2006, concerns related to the safe use of this product continue to surface. This review addresses the use of these products in uncontrolled hemorrhage models, in dehydration and addresses perceived risks associated with hypernatremia, dextran-associated anaphylactoid reactions and effects on coagulation and renal function. PMID- 16670633 TI - Cytokines in necrotizing enterocolitis. AB - Necrotizing enterocolitis (NEC) is a devastating intra-abdominal emergency in the newborn period. The disease involves bowel wall inflammation, ischemic necrosis, eventual perforation, and the need for urgent surgical intervention. Unrecognized or left untreated, the neonate can decompensate quickly, often progressing to shock, multisystem organ failure, and eventual death. During the past several years, a number of basic science and clinical trials have been established in an attempt to understand the pathophysiology of NEC. As many researchers feel that NEC develops as an uncontrolled inflammatory response that leads to intestinal ischemia, a large number of studies have been focused on the inflammatory cascade and the role that cytokines play within that cascade. Although a large amount of data has been generated from these studies, the events leading to the ischemic injury of the intestine are still not fully understood. This article will therefore focus on the key cytokines involved with NEC, in an attempt to present the current literature and studies that support their involvement. PMID- 16670634 TI - Left ventricular diastolic filling characteristics are not impaired but systolic performance was augmented in the early hours of experimental endotoxemia in humans. AB - This study was performed to determine whether endotoxemia causes diastolic cardiac dysfunction. Eleven healthy volunteers, 30 +/- 6 years of age, underwent comprehensive transthoracic echocardiographic assessment including two dimensional, M-mode transmitral and tissue Doppler of systolic and diastolic function at baseline and at 3 and 5 h after intravenous administration of purified Escherichia coli endotoxin (4 ng/kg). Data were analyzed by analysis of variance; P values of less than 0.05 were considered significant. Endotoxin administration resulted in a hyperdynamic state characterized by decreased mean arterial pressure and significant increase in cardiac index. This was accompanied by increases in several load-dependent systolic performance indices (3 and 5 h). Robust increases in peak systolic blood pressure/end-systolic volume index, one of the relatively load-independent contractility parameter, were also observed at 3 h after endotoxin administration. Transmitral peak early velocity (E), which represents early filling, significantly increased at 3 h after infusion. Late diastolic velocity (A), which represents atrial contraction, significantly increased at 3 and 5 h after infusion. The E/A ratio indicative of delayed relaxation significantly decreased due to increases in A (transmitral) and A (tissue Doppler) at 3 and 5 h after infusion. As expected, endotoxin infusion resulted in a hyperdynamic state associated with increases in systolic function indices including endocardial systolic velocities. The observed decreases in E/A (transmitral) and E/A (tissue Doppler) ratio were primarily due to increases in A and A. Moreover, isovolumic relaxation time and time constant for left ventricular relaxation, a load-independent parameter for ventricular relaxation, remained unchanged at 3 and 5 h after endotoxin infusion. Therefore, our findings are more likely due to enhanced atrial contractility resulting from increased sympathetic activity in response to reduction in left ventricular afterload and not due to altered diastolic filling characteristics. PMID- 16670636 TI - TLR2, TLR4, CD14, CD11B, and CD11C expressions on monocytes surface and cytokine production in patients with sepsis, severe sepsis, and septic shock. AB - Bacterial recognition and induced cellular activation are fundamental for the host control of infection, yet the limit between protective and harmful response is still inexact. Forty-one patients were enrolled in this study: 14 with sepsis, 12 with severe sepsis, and 15 with septic shock. Seventeen healthy volunteers (HV) were included as control. The expression of TLR2, TLR4, CD14, CD11b, and CD11c was analyzed on monocytes surface in whole blood. sCD14 was measured in serum, and TNF-alpha, IL-6, and IL-10 cytokine levels were measured in PBMC supernatants after LPS, IL-1beta, and TNF-alpha stimuli by ELISA. An increase in sCD14 and a decreased mCD14 were found in patients as compared with HV (P < 0.001). However, no differences in the expression of TLR2, TLR4, and CD11c were found among the groups. A trend toward differential expression of CD11b was observed, with higher values found in patients with sepsis as compared with HV. A negative regulation of the inflammatory cytokine production was observed in patients with severe sepsis and shock septic in relation to sepsis and HV, regardless of the stimulus. No significant difference in IL-10 production was found among the groups. In this study, we show that the inflammatory response is associated with the continuum of clinical manifestations of sepsis, with a strong inflammatory response in the early phase (sepsis) and a refractory picture in the late phases (severe sepsis and septic shock). Correlation between cell surface receptors and cytokine production after IL-1beta and TNF-alpha stimuli and the observation of a single and same standard response with the different stimulus suggest a pattern of immunology response that is not dependent only on the expression of the evaluated receptors and that is likely to have a regulation in the intracellular signaling pathways. PMID- 16670635 TI - Peripheral hemophagocytosis: An early indicator of advanced systemic inflammatory response syndrome/hemophagocytic syndrome. AB - Peripheral hemophagocytosis (PHP) is seen in patients with hemophagocytic syndrome (HPS), a clinical status in which activated macrophages play a role in its pathogenesis. The inflammatory state, systemic inflammatory response syndrome (SIRS), is also associated with activated macrophages. However, the link between HPS and SIRS and the clinical implications of PHP remain to be determined. In the present work, we examined the clinical utility and impact of the detection of PHP and the link between HPS and SIRS. We studied the clinical and laboratory profiles of 322 SIRS patients (174 men; mean age, 68 +/- 22 years; range, 16-99 years) who visited an urban hospital specializing in respiratory, cardiovascular, digestive, renal diseases, general surgery, and orthopedics in Japan. Peripheral hemophagocytosis was detected in 40 (23 men; mean age, 81.3 +/- 8.7 years; range, 63-98 years) of 322 patients on 3 +/- 2 days after SIRS diagnosis as determined with a "blunt-edged-smear" method differing from the conventional "feather-edged smear" method. The incidence of advanced SIRS and ensuing death in the SIRS+ PHP- group (37 and 21 of 40, respectively) was significantly greater than in the SIRS+ PHP- group (82 and 17 of 282) (P < 0.01). The duration from SIRS diagnosis to recovery in 19 SIRS+ PHP- surviving patients (26 +/- 18 days) was longer than that in 19 age-matched SIRS+ PHP- surviving patients who initially presented comparable clinical profiles (6 +/- 3 days) (P < 0.001). Bone marrow analysis in all 7 patients having PHP and SIRS showed no HPS initially (<3% hemophagocytes), but all subsequently developed HPS at 7 +/- 1 days after the diagnosis, confirmed by the presence of 9% +/- 13% hemophagocytes in the bone marrow. Electron microscopic and immunohistochemical analyses revealed that PHP was derived from hemophagocytes in the bone marrow. The present data strongly suggest that PHP detection could serve as an early indicator for advanced SIRS and/or HPS and that the use of the blunt-edged method is preferable for PHP detection. PMID- 16670637 TI - Plasma obtained during human endotoxemia increases endothelial albumin permeability in vitro. AB - To gain insight in the pathogenesis of increased vascular permeability during sepsis, we studied the effect of plasma obtained during human experimental endotoxemia on the permeability of cultured endothelial monolayers. Eight healthy subjects received an i.v. dose of 2 ng/kg Escherichia coli O:113 lipopolysaccharide (LPS). The concentration of various plasma mediators that supposedly induce vascular permeability was measured over time. Plasmas that were obtained before, and 2 and 4 h after the administration of LPS were added to human umbilical venular endothelial cells that were cultured on semipermeable membranes.The permeability of the endothelial monolayers to fluorescein isothiocyanate-labeled bovine serum albumin was determined and expressed as the relative concentration of fluorescein isothiocyanate-bovine serum albumin when compared with that measured across empty Transwell-COL (Corning Life Sciences B.V., Schiphol-Rijk, The Netherlands) membranes (i.e., without endothelial monolayers). The permeability levels were correlated with the concentrations of various mediators.Experimental endotoxemia resulted in elevated levels of tumor necrosis factor alpha, interleukin (IL) 1beta, IL-6, IL-8, IL-10, and vascular endothelial growth factor and a moderate increase of IL-12 and IFN-gamma (all P values < 0.01). Incubation of human umbilical venular endothelial cells with plasma obtained 2 and 4 h after the administration of LPS increased the relative permeability from a baseline level (median) of 17% (range, 14% - 31%) to 23% (range, 12% - 39%; P = not significant) and 28% (range, 11% - 40%; P < 0.05), respectively. Plasma levels of vascular endothelial growth factor and IL-10, but not TNF-alpha or any other mediators, significantly correlated with the increase in endothelial permeability (r = 0.47, P = 0.038; r = 0.43, P = 0.038, respectively). The data presented here demonstrate that plasmas obtained from experimental human endotoxemia increase endothelial albumin permeability in vitro. Thus, cultured human endothelial monolayers provide a model to study sepsis-associated vascular changes. PMID- 16670638 TI - Echocardiography assessment of myocardial function after burn injury. AB - INTRODUCTION: Echocardiography provides noninvasive and clinically relevant assessment of left ventricle (LV) function in injury and disease. In this present study, we hypothesized that application of transthoracic echocardiography in awake mice would provide in vivo assessment of myocardial performance that correlates with in vitro assessment of LV function using isolated perfused hearts (Langendorff). METHODS: Burn over 40% of the total body surface area (or sham burn) was given in C57/BL6 mice 22 to 24 g; lactated Ringer fluid resuscitation was given intraperitoneally. Transthoracic echocardiography was performed at baseline and at designated intervals over 7 days postburn. Subgroups of mice were killed at intervals and hearts perfused to assess LV pressure and +/-dP/dt max responses to inotropic challenge (n = 8/group per time). RESULTS: Burn produced myocardial depression, evidenced by a fall in fractional shortening from 69% +/- 3% at baseline to 50% +/- 2% (12 h postburn), 58% +/- 3% (24 h), 59% +/- 2% (48 h), and 62% +/- 2% (72 h) (P < 0.05). Burn-related changes in in vivo LV performance were paralleled by in vitro evidence of myocardial contractile depression; LV pressure progressively fell from 97 +/- 2 mmHg at baseline to 84 +/- 7 mmHg (12 h), 63 +/- 2 (24 h), 79 +/- 4 mmHg (48 h postburn) (P < 0.05). Analysis of variance and multiple comparison procedure. Myocardial recovery occurred by day 8 postburn. CONCLUSIONS: Echocardiography provides measures of cardiac function in a conscious animal, which correlate with contractile depression measured in vitro using Langendorff preparations. Echocardiography is a valuable tool for noninvasive assessment of postburn myocardial function. PMID- 16670639 TI - The pressure-volume loop revisited: Is the search for a cardiac contractility index a futile cycle? AB - Our previous studies indicate that left ventricular end-systolic pressure-volume relations (ESPVRs) or elastance (Ees) are not reduced in studies where expected reductions of contractility should be found (i.e., heart failure, stunning, and endotoxemia). The present study was done to assess whether this phenomenon is due to a particular load sensitivity of elastance, rendering this index inappropriate as a measurement of contractility in pathologic states in vivo. METHODS AND RESULTS: Analysis of previously generated data revealed an increased ESPVR in stunned hearts, in pigs made endotoxemic, and in hearts rapidly paced. After inducing acute heart failure by microembolization, the ESPVR was increased when assessed using linear relations but reduced when assessing ESPVR by a curvilinear algorithm. To further evaluate the effect of different load alterations on ESPVR, this relation was generated by (i) inferior vena caval occlusions (VCOs); (ii) gradually occluding the descending aorta (pressure interventions); and (iii) rapidly infusing blood (120 mL) into the left atrium (volume increments). The load protocol was applied in 5 pigs, before and after the left ventricle was stunned by 11 brief left main coronary artery occlusions/reperfusions (accumulated ischemia 20 min affecting 81% of the left ventricle). Correlation coefficients for left ventricular elastance ranged from 0.93 to 0.99 in all the 3 types of loading interventions. Despite significant reductions in stroke volume, stroke work, and dP/dtmax, VCO-calculated linear and curvilinear Ees increased 90 min after stunning (55% +/- 4% and 94% +/- 6%, respectively). Linear Ees during pressure interventions decreased 36% +/- 1%, whereas curvilinear Ees decreased 33% +/- 3%. During volume infusions, linear Ees decreased 27% +/- 2%. We achieved the same results after blocking the baroreceptor reflexes using hexamethonium. CONCLUSIONS: The Ees is particularly load dependent and will reflect load interventions more than the inotropic state of the cardiac muscle. A VCO generated Ees increase could be an unmasking of a pronounced preload sensitivity in failing myocardium. PMID- 16670640 TI - 5-lipoxygenase modulates the alteration of paracellular barrier function in mice ileum during experimental colitis. AB - Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJs) and adherence. Recently, it has been shown that 5 lipoxygenase (5-LO) plays an important role in the development of various inflammatory conditions like inflammatory bowel disease. In the present study, by comparing the responses in wild-type mice (5-LOWT) with those of mice lacking the 5-lipoxygenase (5-LOKO), we investigated the role played by this enzyme in the permeability and structure of small intestine TJs in an animal model of experimental colitis. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). Four days after colitis induction by DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin and ZO-1. When compared with DNBS-treated 5-LOWT mice, DNBS-treated 5-LOKO mice experienced a reduced rate of the extent and severity of the histological signs of colon injury. After administration of DNBS, 5-LOWT mice showed a significant increase of ileal permeability (88.3% +/- 1.2%) compared with sham (5.6% +/- 0.5%). In colitis, the percentage of "leaky" junctions in terminal ilea correlated positively with the macroscopic colon damage score. Distal colitis in 5-LOWT mice induces an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. On the contrary, a significant reduction of (1) the degree of colon injury, (2) the alteration of ZO 1 and occludin localization (immunohistochemistry), and (3) ileal permeability (8.1% +/- 0.7%) caused by DNBS in the colon was observed in 5-LOKO mice. Similarly, the treatment of 5-LOWT with zileuton (50 mg/kg per oral gavage twice a day), a 5-LO inhibitor, resulted in a significant reduction of all the previously described parameters. Taken together, our results clearly demonstrate that 5-LO modulates small intestinal permeability in experimental colitis through the regulation of TJ protein. PMID- 16670641 TI - Endotoxin tolerance enhances interleukin-10 renal expression and decreases ischemia-reperfusion renal injury in rats. AB - The potential implication of interleukin (IL) 6, tumor necrosis factor alpha (TNF alpha), and IL-10 in the protective effect of low-dose lipopolysaccharide (LPS) administration against renal ischemia-reperfusion injury was evaluated in a rat model. Eighteen male Sprague-Dawley rats were injected intravenously with either 0.5 mg/kg of LPS (tolerant group) or saline (control group) 2 days before surgery. Ischemic renal injury was induced by clamping the left renal artery for 60 min on rats immediately after right-side nephrectomy. Reperfusion was obtained by clamp removal and was studied at R0 (no reperfusion), 2H (R2), and 24H (R24) by renal tubular disorder characterization and by plasma creatinine as well as renal cytokine (IL-6, IL-10, and TNF-alpha) studies. No differences were observed between the two groups as concerns the period immediately after renal ischemia (R0). The endotoxin-tolerant group was associated with a significantly lower creatinine level at R24 (231 +/- 28 vs 315 +/- 36 micromol/L; P = 0.007). Pretreatment with LPS significantly reduced the degree of proximal tubule necrosis and outer medulla congestion. In such tolerant animals, renal IL-6 production was decreased, whereas IL-10 production was significantly increased at R2 and R24. There were no differences in TNF-alpha renal production. In this study, we demonstrated that administration of low doses of LPS to rats had a protective effect from renal reperfusion injury, and our data suggest that IL-10 might play a role in this phenomenon. PMID- 16670642 TI - Infusion of increasing doses of endotoxin induces progressive acute lung injury but prevents early pulmonary hypertension in pigs. AB - Administration of a single bolus of endotoxin is a model of sepsis response in experimental animal studies. Large animal species, such as pigs and sheep, are more sensitive to endotoxin administration due to an initial excessive pulmonary hypertensive response frequently resulting in acute right heart dysfunction. We investigated whether infusion of high-dose endotoxin in pigs but administered in an increasing dose results in inflammatory response without excessive pulmonary hypertension and right heart dysfunction. Piglets of both sexes weighing 25 to 30 kg were anesthetized and mechanically ventilated. After instrumentation and baseline measurements, animals received an infusion of total 500 microg kg(-1) i.v. endotoxin (Escherichia coli LPS) over 2 h in an increasing dose of 0.5 to 12 microg kg(-1) min(-1). Hemodynamic, respiratory, and oxygenation parameters were measured every hour. At 1 and 5 h following endotoxin, plasma levels of inflammatory and organ damage parameters were measured. Endotoxin infusion induced progressive arterial hypoxemia, an increase in peak inspiratory pressure, sustained pulmonary hypertension, and systemic hypotension that persisted throughout the experiment. Endotoxin plasma levels peaked at 1 h following infusion and declined toward baseline values at 5 h thereafter. In contrast, plasma levels of nitrite/nitrate, IL-1ra (as marker of cytokine response), remained markedly increased at 5 h after endotoxin infusion as compared with baseline values. Plasma markers of organ damage were significantly increased. Our data show that the dosing of endotoxin in an increasing manner in pigs produces a reliable model of an experimental sepsis response and organ dysfunction without immediate overwhelming pulmonary hypertension resulting in cardiovascular failure. PMID- 16670643 TI - Valproic acid prevents hemorrhage-associated lethality and affects the acetylation pattern of cardiac histones. AB - Pharmacological inhibitors of histone deacetylases (HDAC) demonstrate cytoprotective effects both in vitro and in vivo. In this study, we investigated whether valproic acid (VPA), a known mood stabilizer and anticonvulsant with HDAC inhibiting activity, improves survival following otherwise lethal hemorrhage in rats. We found that preinsult injection of VPA (300 mg/kg, twice) prolonged the survival of severely hypotensive animals up to 5 times. VPA treatment increased the acetylation of nonhistone and histone proteins in the rat heart. The pattern of modifications of individual histones revealed hyperacetylation of histones H2A, H3, and H4, indicating the presence of active genes. Expression of HSP70 and superoxide dismutase, implicated in the modulation of vitality, was increased by VPA. Our results reveal that VPA offers considerable protection in the hemorrhagic shock model and suggest a role for HDAC inhibition in mediating VPA actions. PMID- 16670644 TI - Effects of low abdominal blood flow and dobutamine on blood flow distribution and on the hepatic arterial buffer response in anaesthetized pigs. AB - Low cardiac output impairs the hepatic arterial buffer response (HABR). Whether this is due to low abdominal blood flow per se is not known. Dobutamine is commonly used to increase cardiac output, and it may further modify hepatosplanchnic and renal vasoregulation. We assessed the effects of isolated abdominal aortic blood flow changes and dobutamine on hepatosplanchnic and renal blood flow. Twenty-five anesthetized pigs with an abdominal aorto-aortic shunt were randomized to 2 control groups [zero (n = 6) and minimal (n = 6) shunt flow], and 2 groups with 50% reduction of abdominal blood flow and either subsequent increased abdominal blood flow by shunt reduction (n = 6) or dobutamine infusion at 5 and 10 microg kg(-1) min(-1) with constant shunt flow (n = 7). Regional (ultrasound) and local (laser Doppler) intra-abdominal blood flows were measured. The HABR was assessed during acute portal vein occlusion. Sustained low abdominal blood flow, by means of shunt activation, decreased liver, gut, and kidney blood flow similarly and reduced local microcirculatory blood flow in the jejunum. Shunt flow reduction partially restored regional blood flows but not jejunal microcirculatory blood flow. Low-but not high-dose dobutamine increased gut and celiac trunk flow whereas hepatic artery and renal blood flows remained unchanged. Neither intervention altered local blood flows. The HABR was not abolished during sustained low abdominal blood flow despite substantially reduced hepatic arterial blood flow and was not modified by dobutamine. Low-but not high-dose dobutamine redistributes blood flow toward the gut and celiac trunk. The jejunal microcirculatory flow, once impaired, is difficult to restore. PMID- 16670645 TI - Leptin and host defense against Gram-positive and Gram-negative pneumonia in mice. AB - Leptin is a pleiotrophic protein mainly produced by adipocytes that has been implicated as a link between nutritional status and immune function. Severe bacterial infection is associated with elevated plasma levels of leptin. To determine the role of leptin in the host response to bacterial pneumonia leptin deficient ob/ob mice and normal wild-type (WT) mice were intranasally infected with different doses of the Gram-positive pathogen Streptococcus (S.) pneumoniae or the Gram-negative bacterium Klebsiella (K.) pneumoniae. After infection with lower doses of either pathogen ob/ob mice displayed lower pulmonary levels of proinflammatory cytokines, in particular tumor necrosis factor-alpha and chemokines. However, after infection with a higher dose of S. pneumoniae or K. pneumoniae the lung concentrations of these inflammatory mediators did not differ between ob/ob and WT mice. In addition, the extent and severity of lung inflammation, as assessed by semi-quantitative histopathology scores, were similar in both mouse strains. Finally, leptin deficiency did not impact on the bacterial outgrowth in the lungs during either Gram-positive or Gram-negative pneumonia irrespective of the infective dose. These data suggest that although leptin may play a modest role in the regulation of inflammation during bacterial pneumonia, it does not contribute to host defense mechanisms that act to limit the outgrowth of S. pneumoniae or K. pneumoniae in the lower airways. PMID- 16670646 TI - An extract of the mushroom Agaricus blazei Murill protects against lethal septicemia in a mouse model of fecal peritonitis. AB - Bacterial septicemia is frequently occurring during gastroenterological surgery. Because of increasing problems in hospitals with bacteria developing multiresistance against antibiotics, prophylactic treatment using immunomodulators is interesting. We have examined the putatively anti-infective immunomodulatory action of the edible mushroom, Agaricus blazei Murill (AbM), in an experimental peritonitis model in BALB/c mice. The mice were orally given an extract of AbM or phosphate-buffered saline 1 day before the induction of peritonitis with various concentrations of feces from the mice. The state of septicemia, as measured by the number of colony-forming units of bacteria in blood, and the survival rate of the animals were compared between the groups. Mice that were orally treated with AbM extract before bacterial challenge showed significantly lower levels of septicemia and improved survival rates. Our findings suggest that the AbM extract, when given prophylactically, may improve health. Further studies are needed on humans when considering whether AbM could be used as an alternative treatment modality for patients at risk of contracting serious bacterial peritonitis. PMID- 16670647 TI - Sleep hygiene and melatonin treatment for children and adolescents with ADHD and initial insomnia. AB - OBJECTIVE: To evaluate the efficacy of sleep hygiene and melatonin treatment for initial insomnia in children with attention-deficit/hyperactivity disorder (ADHD). METHOD: Twenty-seven stimulant-treated children (6-14 years of age) with ADHD and initial insomnia (>60 minutes) received sleep hygiene intervention. Nonresponders were randomized to a 30-day double-blind, placebo-controlled, crossover trial of 5-mg pharmaceutical-grade melatonin provided by the study's sponsor. RESULTS: Sleep hygiene reduced initial insomnia to <60 minutes in 5 cases, with an overall effect size in the group as a whole of 0.67. Analysis of the trial data able to be evaluated showed a significant reduction in initial insomnia of 16 minutes with melatonin relative to placebo, with an effect size of 0.6. Adverse events were generally mild and not different from those recorded with placebo treatment. The effect size of the combined sleep hygiene and melatonin intervention from baseline to 90 days' posttrial was 1.7, with a mean decrease in initial insomnia of 60 minutes. Improved sleep had no demonstrable effect on ADHD symptoms. CONCLUSION: Combined sleep hygiene and melatonin was a safe and effective treatment for initial insomnia in children with ADHD taking stimulant medication. PMID- 16670649 TI - Does prolonged therapy with a long-acting stimulant suppress growth in children with ADHD? AB - OBJECTIVE: To investigate whether prolonged therapy with a long-acting stimulant affects growth in children with attention-deficit/hyperactivity disorder (ADHD). METHOD: One hundred seventy-eight children ages 6 to 13 years received OROS methylphenidate (OROS MPH, CONCERTA) for at least 21 months. Height and weight were measured monthly during the first year and every 3 months thereafter. RESULTS: At baseline, subjects were approximately the expected height for their age and somewhat heavier than expected. Subjects gained height steadily throughout the study and were on average 0.23 cm less than expected at month 21. Weight did not increase and BMI decreased slightly in the first 4 months. Thereafter, weight Z score and BMI Z score remained relatively constant and children were on average 1.23 kg less than expected at month 21. Previous stimulant therapy tended to be associated with a smaller decrease in Z score during the study compared with no previous stimulant therapy. Drug holidays did not significantly affect growth. CONCLUSIONS: The effects of prolonged OROS MPH therapy on growth were clinically insignificant and limited to slight decreases in weight during the first months of therapy. Drug holidays did not reduce any impact on growth and are thus of questionable utility for limiting potential effects of treatment on growth. PMID- 16670648 TI - Comparative effects of methylphenidate and mixed salts amphetamine on height and weight in children with attention-deficit/hyperactivity disorder. AB - OBJECTIVE: To determine whether methylphenidate (MPH) and mixed salts amphetamine (MSA) have different effects on growth in children with attention deficit/hyperactivity disorder. METHOD: Patients treated for at least 1 year with MPH or MSA were identified. A linear regression was performed to determine the effect of stimulant type, patient gender, cumulative stimulant dose, and length of time in treatment on change in Z scores for height. A subset of patients was identified who had 3 years of consistent stimulant treatment on either MSA or MPH. Repeated-measures analyses of variance were performed to examine the effects of time and medication type on Z scores for weight, height, and body mass index. RESULTS: The linear regression showed no effect of stimulant type, drug holidays, or length of time of treatment on change in height Z score. Cumulative dose of stimulant had a small (-0.26) relationship to change in height Z scores. For patients treated for 3 years, there were no effects of stimulant or time on height Z scores. MSA produced more decrease in weight and body mass index Z scores than MPH; all of the subjects were heavier than average at baseline. CONCLUSION: MSA and MPH did not differ in their effects on height. MSA had more of an effect on weight than MPH, although the effect was modest in magnitude and may be of limited clinical significance. PMID- 16670650 TI - Effects on parental mental health of an education and skills training program for parents of young children with autism: a randomized controlled trial. AB - OBJECTIVE: To determine the impact of a parent education and behavior management intervention (PEBM) on the mental health and adjustment of parents with preschool children with autism. METHOD: A randomized, group-comparison design involving a parent education and counseling intervention to control for nonspecific therapist effects and a control sample was used. Two metropolitan and two rural regions were randomly allocated to intervention groups (n = 70) or control (n = 35). The parents of consecutive children with autism (2(1/2)-5 years old) from the autism assessment services for the intervention regions were then randomly allocated to either a 20-week manual-based parent education and behavior management intervention (n = 35) or a manual-based parent education and counseling intervention (n = 35). The main outcome measure of parental mental health was the General Health Questionnaire used pre- and postintervention and at 6-month follow up. RESULTS: Both treatments resulted in significant and progressive improvement in overall mental health at follow-up (F = 2, 97, p =.007) and mental health significantly improved over time in the 54% of principal caregivers who had the highest levels of mental health problems. The parent education and behavior management intervention was effective in alleviating a greater percentage of anxiety, insomnia, and somatic symptoms and family dysfunction than parent education and counseling at 6-month follow-up. CONCLUSIONS: A 20-week parent education and skills training program for parents of young children newly diagnosed with autism provides significant improvements in parental mental health and adjustment, justifying its addition to early intervention programs at least for parents with mental health problems. PMID- 16670651 TI - Common and specific genetic influences on aggressive and nonaggressive conduct disorder domains. AB - OBJECTIVE: To explore the genetic and environmental influences on DSM-IV conduct disorder (CD) aggressive and nonaggressive subscales, taking into account age and sex differences. METHOD: A community sample of 1,100 twin pairs (ages 11-18) was interviewed using the Diagnostic Interview Schedule for Children. Bivariate analyses, using variable threshold models accounting for age and sex differences, were used to determine the extent to which the genetic and environmental influences on aggressive and nonaggressive CD domains are shared or unique. RESULTS: The phenotypic correlation between aggressive and nonaggressive CD domains was 0.32. The most parsimonious bivariate model included additive genetic effects and nonshared environmental effects only (AE model). CONCLUSIONS: The results of behavior genetic model fitting suggest that the DSM-IV CD domains are influenced by unique genetic and environmental factors, but also share some common genetic and environmental influences. A large percentage of the covariation (61%) is caused by genetic factors. These results are consistent with a previous report on the bivariate heritability of aggressive and nonaggressive antisocial behavior, but extend the findings to DSM-IV domains. PMID- 16670652 TI - Childhood predictors of male criminality: a prospective population-based follow up study from age 8 to late adolescence. AB - OBJECTIVE: To study childhood predictors for late adolescence criminality. METHOD: The follow-up sample included 2,713 Finnish boys born in 1981. Information about the 8-year-old boy' problem behavior was obtained from parents, teachers, and the children themselves. The follow-up information about criminal offenses was based on the national police register between the years 1998 and 2001 when the subjects were 16 to 20 years old. RESULTS: According to the national police register, 22.2% of boys had at least one criminal offense other than a minor traffic violation during the 4-year study period. Living in nonintact family, low parental education level, parent reports of conduct problems, and teacher reports of hyperkinetic problems when the child was 8 independently predicted a high level (more than five) of offenses. Living in nonintact family at age 8 predicted all types of criminal offenses. Low parental education level and parent or teacher reports of conduct problems independently predicted violence, property, traffic, and drunk driving offenses. Teacher reports of hyperkinetic problems independently predicted all types of criminal offenses except drunk driving. Self-reports of bullying others independently predicted violent offenses. CONCLUSIONS: Living in a broken home, low parental education level, conduct problems, and hyperactivity in middle childhood predict criminal offenses in late adolescence. Efforts to prevent later criminality already in childhood are emphasized. PMID- 16670653 TI - Temporal lobe anatomy and psychiatric symptoms in velocardiofacial syndrome (22q11.2 deletion syndrome). AB - OBJECTIVE: To investigate the association between mesial temporal lobe morphology, ratios of prefrontal cortex to amygdala and hippocampus volumes, and psychiatric symptomatology in children and adolescents with velocardiofacial syndrome (VCFS). METHOD: Scores on behavioral rating scales and volumetric measures of the amygdala, hippocampus, and prefrontal cortex based on high resolution magnetic resonance imaging were compared among 47 children with VCFS, 15 of their siblings, and 18 community controls. RESULTS: After covarying for whole brain volume, children with VCFS exhibited 11% greater volume of the left amygdala (p =.002) and 8% greater volume of the right amygdala (p =.01). Children with VCFS exhibited smaller volumes of the hippocampus, but not disproportionately to reductions in whole brain volume. Children with VCFS exhibited smaller volumetric ratios of prefrontal and orbitofrontal cortex to amygdala, but not prefrontal cortex to hippocampus. For children with VCFS, but not for the comparison sample, larger volumes of the amygdala and smaller ratios of prefrontal cortex to amygdala were associated with higher scores on the Internalizing, Externalizing, Anxiety, and Aggression scales of the Child Behavior Checklist and on the parent version of the Young Mania Rating Scale. CONCLUSIONS: These findings suggest that the prefrontal cortex-amygdala circuit that underlies emotional processing is disrupted in children with VCFS and may be an important neurobiological substrate of psychiatric disorder in these children. PMID- 16670654 TI - ADHD, major depressive disorder, and simple phobias are prevalent psychiatric conditions in youth with velocardiofacial syndrome. AB - OBJECTIVE: To examine prevalence rates of psychopathology in children with velocardiofacial syndrome (VCFS). METHOD: One hundred fifty-four children ages 6 to 15 participated in our between-group design with three samples, 84 children with VCFS (37 girls, 47 boys), 32 sibling controls (18 girls, 14 boys), and 38 community controls (12 girls, 26 boys). The Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version and several other parent report measures were used to assess for psychopathology. RESULTS: Compared to both control samples, children with VCFS had higher prevalence rates of major depressive disorder, attention-deficit/hyperactivity disorder, simple phobias, and enuresis. Additional findings from our analyses include (1) no gender differences in VCFS psychopathology prevalence rates, (2) children with VCFS who have comorbid psychopathology were rated by their parents as having less well-developed executive functions, and (3) across all three samples, the higher the IQ was, the higher the level of global functioning. CONCLUSIONS: These findings are consistent with previous research and suggest that major depressive disorder, attention-deficit/hyperactivity disorder, and simple phobias are salient features of the VCFS psychiatric phenotype. PMID- 16670655 TI - Playful "moments" in psychotherapy. PMID- 16670656 TI - Evidence-based assessment of attention-deficit/hyperactivity disorder: using multiple sources of information. PMID- 16670657 TI - Less is more: inpatient management of a child with complex pharmacotherapy. PMID- 16670659 TI - [Structure and physiology of the pleura and the pleural space]. AB - The pleural space, derived from the intraembryonic coelom, is limited by a serous membrane including the mesothelium formed by cells possessing not only the characteristic features of epithelial cells but also the potential of secretory cells (cytokines and growth factor). Blood supply to visceral pleurae differs depending on the species while the lymphatic circulation is directly connected to the pleural space via pores in the parietal pleura. Pleural physiology and movement of pleural fluid are directly related to the particular structures of the pleura. PMID- 16670660 TI - [Pathology of primary tumors and pseudotumors of the pleura]. AB - Primary tumors are relatively rare in the pleura. Histological types include mesothelioma, epitheliod, biphasic or sarcomatoid tumors as well as primary lymphoma and mesenchymatous tumors which include solitary fibrous tumor, epithelioid hemangioendothelioma and angiosarcoma and synovialosarcoma. We detail here the new WHO classification 2004 explaining the different entities, excluding metastatic tumors which are the most frequent tumors of the pleura. PMID- 16670661 TI - [Screening for benign pleural disease in adults: why and how?]. AB - Benign diseases of the pleura are clearly dominated by asbestos-related conditions. It is important to distinguish diseases affecting the parietal pleura (pleural plaques) and those affecting the visceral pleura (pleurisy and diffuse thickening). The social benefits which could be obtained by identifying affected persons warrants screening in France, even more so than non-demonstrated medical benefits. Thoracic computed tomography without contrast is the examination of choice. A rigorous protocol is required for proper execution and interpretation. PMID- 16670662 TI - [Computed tomographic anatomy of the intercostal space and the pleura. Pleural plaques: positive and differential diagnosis]. AB - Diagnosis of pleural plaques depends on adequate knowledge of the normal anatomy of the pleura and intercostals spaces which is indispensable for understanding the normal computed tomographic images of the thorax and anatomic variants. PMID- 16670663 TI - [Inflammation and pleural fibrosis]. AB - Pleural effusions are frequent. There are many etiologies of inflammatory pleural effusions and of pleural fibrosis. Imaging (standard radiography, ultrasound, scanner, MRI) is important for visualization, localization, and assessment of abundance. Exsudative (inflammatory) effusion can sometimes be distinguished from hydrothorax. Imaging signs generally remain nonspecific but can contribute to the etiological diagnosis. In particular, a CT scan of the chest, without and with contrast injection (tissue impregnation) can, in addition to the study of pleural space, enable an visual assessment of the visceral and parietal pleura, the adjacent chest wall, and the adjacent pulmonary parenchyma or vessels, which may provide diagnostic clues. Imaging can also contribute to therapeutic guidance. PMID- 16670664 TI - [Imaging benign tumors of the pleura]. AB - Five percent of pleural tumors are benign. Solitary fibrous tumors are the most frequent. We recall the clinical and pathological features and present the radiographic, computed tomographic and magenetic resonance imaging results obtained for the main types of benign tumors of the pleura. PMID- 16670665 TI - [Pleural mesothelioma: imaging contribution]. AB - Imaging plays an essential role in management of patients of with pleural mesothelioma. In this article, we discuss the respective roles for ultrasonography, computed tomography, magnetic resonance imaging, and positon emission tomography for the diagnosis, staging, and postherapeutic evaluation of pleural mesothelioma. PMID- 16670666 TI - [Other malignant tumors of the pleura]. AB - Malignant tumors of the pleura are most often diffuse, nethertheless they are sometimes localized. There is an overlap of the radiologic features of the benign and malignant pleural lesions. The differential diagnosis may be difficult, even on histological sample. Imaging allows the diagnosis of pleural involvement, suggests the malignity, guides percutaneous or thoracoscopic biopsies of the pleura, defines extent of the tumor and follows the course of the disease. We will describe the malignant pleural tumors: pleural metastases, pleural involvement of broncho-pulmonary cancer, of lymphoma and leukaemia. Then the rare pleural tumors will be described: malignant pleural fibroma, sarcoma, histiocytoma and hemangiopericytoma. PMID- 16670667 TI - [18-Fluoro-deoxy-glucose (15FDG) positon emission tomography (PET) for the evaluation of malignant pleural disease]. AB - Use of 18FDG-PET for malignant tumors of the pleura raises certain technical difficulties because of the small size of the tumors and their diffuse distribution, but hybrid PET/CT machines offer a better localization of FDG uptake. FDG-PET can discriminate between malignant and benign pleural tumors FDG uptake in the pleura is the best diagnostic criteria of malignancy. The presence of FDG uptake in pleural effusion is less discriminate between benign and malignant disease. For mesotheliomas, FDG-PET can difference malignant tumors from benign tumors of the pleura on the basis of the SUV value ( 2). It does not enable detection of mediastinal node involvement and regional extension but is effective in detecting extra-thoracic regional spread not identified with other imaging methods. FDG-PET can thus affect the therapeutic strategy. The intensity of uptake has prognostic value. SUV<4 associated with an epithelial tumor is a sign of good prognosis at three years. SUV > 4 associated with a non epithelial tumor is a sign of poor prognosis. For mesotheliomas, FDG uptake can be used to assess the effect of chemotherapy and determine whether patients are good responders or not. PMID- 16670668 TI - [Pleural biopsies and pleural drainage: interventional radiology]. AB - Interventional radiography performed for diagnostic and therapeutic purposes enables specific orientation of the diagnostic and/or therapeutic interention under radiographic guidance. This minimally invasive approach reduces intervention related morbidity. Mortality remains exceptional. Pleural biopsies and pleural drainage are two interventional procedures performed as routine practices in many centers. PMID- 16670669 TI - [Surgical diagnosis of pleural disease]. AB - Videothoracoscopy is a minimally invasive techinique providing a direct view of the pleural cavity. It enables complete exploration of the cavity with biopsies of pathological zones. The indication, based on clinical or radiographic findings, is retained when less invasive methods have been unable to establish the diagnosis and a specific pleural disease (tumor or other) is suspected. Depending on the observations at videothoracoscopy and the suspected disease, deep biopsies to the subpleural fat can be made with a forceps if nodules have been identified. If the pleura is uniformly thin, a small flap can be detached for the biopsy. Several pleura sites are biopsied and a direct pathology examination can be performed on certain specimens. Videothoracoscopy enables careful hemostasis of biopsied zones and symphysis (generally with talc) if needed. The perioperative mortality is low (<0.5%) with good sensitivity greater than 90% and excellent specificity at 100%. The presence of complete pleural symphysis counterindicates videothoracoscopy. In such patients, direct access via an intercostal incision is needed to obtain localized biopsies. PMID- 16670670 TI - ["Therapeutic" pleural surgery]. AB - Renewed interest in carcinological pleura surgery for the treatment of mesothelioma has resulted from an increased incidence of the tumor and also better control of postoperative mortality for an operation with a dramatic reputation. Techniques include pleurectomy, pleurodecrotication and wide pleuropneumectomy. To achive isolated resection of the parietal pleura or a combined resection of the parietal and visceral pleura with more or less wide resection of the diaphragm and pericardium. Indications depend on the tumor extension and the patient's status. Mortality, particularly for wide pleuropneumectomy is no well controlled by well-trained teams and is to the order of 5%. The rate of local recurrence is to the order of 10% and can warrant use of local treatments such as intrathoracic hyperthermic chemotherapy. Median survival for operated mesothelioma is 19 months with a 46% five-year survival for the tumors with the best prognosis. At the present time, radical surgical resection is the basis of local treatment for pleural mesothelioma. PMID- 16670671 TI - Ghrelin concentrations in acromegalic patients in relation to the administered therapy. AB - OBJECTIVES: Very little is still known about ghrelin in cases of GH hypersecretion. We decided to evaluate whether ghrelin concentrations in acromegalic patients differ according to the administered therapy (surgical and/or with long acting octreotide). We wanted to assess the correlation between serum ghrelin levels and 1) the treatment applied in the past or being applied at the time of our study 2) GH, IGF-1 and insulin concentrations. MATERIAL: Serum ghrelin, GH, IGF-1 and insulin were measured in 42 acromegalic patients who had received surgical and/or long acting octreotide therapy or who hadn't receive any treatment at all. According to the applied treatment, we divided the patients into groups, as follows: 1) LAO/+/, surgery /-/, 2) LAO /+/, surgery /+/, 3) LAO /-/, surgery /-/, 4) LAO /-/, surgery /+/. Ghrelin was also evaluated in 18 control healthy subjects. RESULTS: The difference between mean ghrelin level in the healthy subjects and acromegalic patients was not statistically significant (p=0.08). LAO /-/, surgery /-/ patients showed fasting ghrelin levels indistinguishable from those seen in healthy subjects (p=0.1). There wasn't any statistically significant difference between LAO /-/, surgery /-/ patients and LAO /-/, surgery /+/ group (p=0.14). Serum ghrelin levels in LAO /+/, surgery /+/ group were significantly lower from those observed in LAO /-/, surgery /+/ patients (p=0.006). A similar reduction in ghrelin levels was also found in patients LAO /+/ (both after and without surgery) compared to LAO /-/ patients (both after or without surgery) (p=0.001). Serum ghrelin levels showed a significant negative correlation with IGF-1 concentration and a tendency to a statistically significant correlation with GH in LAO /-/ group (p<0,05). CONCLUSIONS: Ghrelin concentrations were significantly lower in acromegalic patients who had been receiving long acting somatostatin analogue treatment; there was no significant difference in serum ghrelin levels between patients who had and who had not undergone surgery. A significant negative correlation between ghrelin and IGF-1 and a tendency to such correlation between ghrelin and GH was observed in LAO /-/ group of patients. PMID- 16670672 TI - Leptin affects oligodendroglial development in the mouse embryonic cerebral cortex. AB - OBJECTIVE: Leptin, which is an obese gene product, decreases appetite and increases energy expenditure in adults. In our previous study, leptin was found to maintain neural stem cells and/or progenitor cells, preferentially astrocyte/oligodendrocyte progenitor cells, whereas it reduces the proportion of oligodendrocyte lineage-restricted precursor cells. It has been reported that leptin-deficient (ob/ob) mice have lower levels of glial proteins than wild-type mice. These findings suggest that leptin affects the development of glial cells. In this study, therefore, we investigated oligodendrocyte development in the cerebral cortex of ob/ob and wild-type mouse embryos by histochemistry. METHODS: We obtained ob/ob or wild-type (C57BL/6J) embryos on embryonic day (E) 18. We performed immunohistochemistry in the embryonic cerebrum with antibodies against NG2, platelet-derived growth factor receptor-alpha (PDGFR-alpha) and leptin receptor (Ob-R). In the cerebral cortex, we compared the number of the oligodendrocyte precursor cells (OPCs), which are immunopositive for NG2 and/or PDGFR-alpha, between ob/ob and wild-type embryos. RESULTS: We revealed that ob/ob embryos had significantly more OPCs than wild-type embryos on E18. PDGFR-alpha positive OPCs did not coexpress leptin receptor in the cerebral cortex. CONCLUSION: These findings suggest that leptin inhibits differentiation of multipotent and/or glial progenitor cells into OPCs in the mouse embryonic cerebral cortex, but it does not directly act on OPCs. PMID- 16670673 TI - Wanted: a strategy for European science. PMID- 16670674 TI - Integrating new countries into the European Research Area. Croatian reflections on joining the European Union. PMID- 16670675 TI - Western science and traditional knowledge. Despite their variations, different forms of knowledge can learn from each other. PMID- 16670676 TI - Seeing is understanding. Improvements in computer software and hardware are revolutionizing three-dimensional imaging in biology. PMID- 16670677 TI - The infection connection. Helicobacter pylori is more than just the cause of gastric ulcers--it offers an unprecedented opportunity to study changes in human microecology and the nature of chronic disease. PMID- 16670678 TI - Robots emulating children. Scientists are developing robots using biology as their inspiration. Will they succeed in building cognitive agents? PMID- 16670681 TI - Copy-number control of the Escherichia coli chromosome: a plasmidologist's view. AB - The homeostatic system that sets the copy number, and corrects over-replication and under-replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy-number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative-feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number. PMID- 16670682 TI - T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity. AB - The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range. PMID- 16670683 TI - Yeast Gal4: a transcriptional paradigm revisited. AB - During the past two decades, the yeast Gal4 protein has been used as a model for studying transcriptional activation in eukaryotes. Many of the properties of transcriptional regulation first demonstrated for Gal4 have since been shown to be reiterated in the function of several other eukaryotic transcriptional regulators. Technological advances based on the transcriptional properties of this factor--such as the two-hybrid technology and Gal4-inducible systems for controlled gene expression--have had far-reaching influences in fields beyond transcription. In this review, we provide an updated account of Gal4 function, including data from new technologies that have been recently applied to the study of the GAL network. PMID- 16670685 TI - Transfer from table to wheelchair in men and women with spinal cord injury: coordination of body movement and arm forces. AB - STUDY DESIGN: A complex set-up was used to investigate kinematics and ground reaction forces. SETTING: Motor Control and Physical Therapy Research Laboratory, Neurotec Department, Karolinska Institutet, Huddinge, Sweden. OBJECTIVE: To investigate how men and women with spinal cord injury (SCI) perform transfers from table to wheelchair with regard to timing and magnitude of force generation beneath the hands and associated body movements. METHODS: A total of 13 subjects (seven men, six women) with thoracic SCI. Kinematics of body movement were recorded (Elite 2000 system) simultaneously with the signals from three force plates (AMTI) placed beneath the buttocks and hands. Temporal and spatial parameters regarding head, trunk and trailing arm displacement, loading amplitudes and loading torque directions of both hands were analyzed for each trial and subject and compared between genders. RESULTS: Men and women used similar amplitudes of head bending and forward displacement of the trailing shoulder, while female subjects had significantly larger trunk rotation. Both genders applied significantly more weight on the trailing hand. Differences between genders were seen in direction and timing of peak torque beneath the hands. CONCLUSIONS: The forces beneath the trailing hand were larger than those in the leading, if there is weakness or pain in one arm, this arm should be selected as the leading. To avoid excessive load on the arms, technical aids and environmental factors should be very well adapted. SPONSORSHIP: This project was funded by the Swedish Research Council and the Health Care Science Committee of Karolinska Institutet. PMID- 16670686 TI - Somatosensory- and motor-evoked potential monitoring during spine and spinal cord surgery. AB - STUDY DESIGN: Prospective, observational study. SETTING: Regional Trauma Center, Torino, Italy. OBJECTIVES: Complex spinal surgery carries a significant risk of neurological damage. The aim of this study is to determine the reliability and applicability of multimodality motor-evoked potentials (MEPs) and somatosensory evoked potentials (SEPs) monitoring during spine and spinal cord surgery in our institute. METHODS: Recordings of MEPs to multipulse transcranial electrical stimulation (TES) and cortical SEPs were made on 52 patients during spine and spinal cord surgery under propofol/fentanyl anaesthesia, without neuromuscular blockade. RESULTS: Combined MEPs and SEPs monitoring was successful in 38/52 patients (73.1%), whereas only MEPs from at least one of the target muscles were obtained in 12 patients (23.1%); both MEPs and SEPs were absent in two (3.8%). Significant intraoperative-evoked potential changes occurred in one or both modalities in five (10%) patients. Transitory changes were noted in two patients, whereas three had persistent changes, associated with new deficits or a worsening of the pre-existing neurological disabilities. When no postoperative changes in MEP or MEP/SEP modalities occurred, it was predictive of the absence of new motor deficits in all cases. CONCLUSION: Intraoperative combined SEP and MEP monitoring is a safe, reliable and sensitive method to detect and reduce intraoperative injury to the spinal cord. Therefore, the authors suggest that a combination of SEP/MEP techniques could be used routinely during complex spine and/or spinal cord surgery. PMID- 16670687 TI - Surgical outcomes of spinal cord astrocytomas. AB - STUDY DESIGN: Retrospective case series. OBJECTIVES: To analyze prognostic factors for patients with spinal cord astrocytomas. SETTING: Department of Orthopaedic Surgery, Keio University, Japan. METHODS: Seven patients received total excisions (group T), eight underwent partial excisions (group P), and 15 had excisional biopsies (group B). Impacts of the tumor histological grade, the level of the tumor, the types of surgical interventions, and the use of adjuvant radiotherapies on the survival and functional outcomes of 30 patients (18 in low grade and 12 high-grade malignancy tumors) were analyzed. RESULTS: The survival rate of the low-grade malignancy group was significantly higher than that of the high-grade group. The survival rate of the patients with thoracic astrocytomas was significantly higher than those with cervical astrocytomas. In both the low- and high-grade groups, the survival rates in groups P/T were significantly higher than those in group B. In the low-grade group, five patients, whose preoperative functional statuses were classified as 'fair' or better, remained 'fair' or better after surgery. In the high-grade group, the postoperative functional statuses were classified as 'no change' or 'aggravated' in all except two patients. No significant difference in the survival rates was detected between patients with and without adjuvant radiotherapy. CONCLUSIONS: The tumor grade and the extent of tumor resection were significant prognostic factors for survival rate. In low-grade malignancy group, good motor function was retained when surgeries were performed before substantial neurological deterioration. The efficacy of postoperative radiotherapy has yet to be determined and needs further study. PMID- 16670688 TI - KCNJ11 activating mutations are associated with developmental delay, epilepsy and neonatal diabetes syndrome and other neurological features. AB - Heterozygous activating mutations in the gene encoding for the ATP-sensitive potassium channel subunit Kir6.2 (KCNJ11) have recently been shown to be a common cause of permanent neonatal diabetes. Kir6.2 is expressed in muscle, neuron and brain as well as the pancreatic beta-cell, so patients with KCNJ11 mutations could have a neurological phenotype in addition to their diabetes. It is proposed that some patients with KCNJ11 mutations have neurological features that are part of a discrete neurological syndrome termed developmental Delay, Epilepsy and Neonatal Diabetes (DEND), but there are also neurological consequences of chronic or acute diabetes. We identified KCNJ11 mutations in four of 10 probands with permanent neonatal diabetes and one affected parent; this included the novel C166F mutation and the previously described V59M and R201H. Four of the five patients with mutations had neurological features: the patient with the C166F mutation had marked developmental delay, severe generalised epilepsy, hypotonia and muscle weakness; mild developmental delay was present in the patient with the V59M mutation; one patient with the R201H mutation had acute and chronic neurological consequences of cerebral oedema and another had diabetic neuropathy from chronic hyperglycaemia. In conclusion, the clinical features in these patients support the existence of a discrete neurological syndrome with KCNJ11 mutations. The severe DEND syndrome was seen with the novel C166F mutation and mild developmental delay with the V59M mutation. These features differ markedly from the neurological consequences of acute or chronic diabetes. PMID- 16670689 TI - Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses. AB - Mucopolysaccharidoses (MPS) are inherited, severe, progressive, metabolic disorders caused by deficiencies in different enzymes involved in degradation of glycosaminoglycans (GAGs). Although enzyme replacement therapy (ERT) has recently been available for MPS type I, and clinical trials have been performed in ERT for MPS II and MPS VI, there is little chance that this kind of treatment may be effective for neurodegenerative forms of MPS (due to inefficient delivery of enzymes to central nervous system through the blood-brain barrier), hence currently there is no effective therapy available for them. Therefore, we aim to develop an alternative therapy for these diseases. We found that genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran 4-one) inhibits synthesis of GAGs considerably in cultures of fibroblasts of MPS patients (types I, II, IIIA and IIIB were tested). Prolonged cultivation of these cells in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells as estimated by biochemical tests and electron microscopic analysis, respectively. As genistein inhibits kinase activity of epidermal growth factor receptor, which is required for full expression of genes coding for enzymes involved in GAG production, we propose to consider a substrate reduction therapy for MPS, which is referred to as 'gene expression-targeted isoflavone therapy'. PMID- 16670690 TI - Mosaicism for mitochondrial DNA polymorphic variants in placenta has implications for the feasibility of prenatal diagnosis in mtDNA diseases. AB - Women who have had a child with mitochondrial DNA (mtDNA) disease need to know the risk of recurrence, but this risk is difficult to estimate because mutant and wild-type (normal) mtDNA coexist in the same person (heteroplasmy). The possibility that a single sample may not reflect the whole organism both impedes prenatal diagnosis of most mtDNA diseases, and suggests radical alternative strategies such as nuclear transfer. We used naturally occurring mtDNA variants to investigate mtDNA segregation in placenta. Using large samples of control placenta, we demonstrated that the level of polymorphic heteroplasmic mtDNA variants is very similar in mother, cord blood and placenta. However, where placental samples were very small (< 10 mg) there was clear evidence of variation in the distribution of mtDNA polymorphic variants. We present the first evidence for variation in mutant load, that is, mosaicism for mtDNA polymorphic variants in placenta. This suggests that mtDNA mutants may segregate in placenta and that a single chorionic villous sample (CVS) may be unrepresentative of the whole placenta. Duplicates may be necessary where CVS are small. However, the close correlation of mutant load in maternal, fetal blood and placental mtDNA suggests that the average load in placenta does reflect the load of mutant mtDNA in the baby. Provided that segregation of neutral and pathogenic mtDNA mutants is similar in utero, our results are generally encouraging for developing prenatal diagnosis for mtDNA diseases. Identifying mtDNA segregation in human placenta suggests studies of relevance to placental evolution and to developmental biology. PMID- 16670691 TI - Vitamin B6 supplementation increases immune responses in critically ill patients. AB - OBJECTIVE: To investigate whether vitamin B6 supplementation has a beneficial effect on immune responses in critically ill patients. DESIGN: A single-blind intervention study. SETTING: The study was performed at the Taichung Veterans General Hospital, the central part of Taiwan. SUBJECTS: Fifty-one subjects who stayed over 14 days in the intensive care unit completed the study. Subjects were not treated with any vitamin supplement before the intervention. INTERVENTIONS: Patients were randomly assigned to one of three groups, control (n = 20), a daily injection of 50 mg vitamin B-6 (B6 -50, n=15), or 100 mg vitamin B-6 (B6 -100, n = 16) for 14 days. MAIN OUTCOME MEASURES: Plasma pyridoxal 5'-phosphate (PLP), pyridoxal (PL), 4-pyridoxic acid (4-PA), erythrocyte alanine (EALT-AC) and aspartate (EAST-AC) aminotransaminase activity coefficient, and urinary 4-PA were measured. The levels of serum albumin, hemoglobin, hematocrit, high-sensitivity C reactive protein (hs-CRP) and immune responses (white blood cell, neutrophils, total lymphocytes count (TLC), T- (CD3) and B-(CD19) lymphocytes, T-helper (CD4) and suppressor (CD8) cells) were determined. RESULTS: Plasma PLP, PL, 4-PA and urinary 4-PA concentrations significantly increased in two treated groups. T lymphocyte and T-helper cell numbers and the percentage of T-suppressor cell significantly increased on day 14 in the B6 -50 group. Total lymphocyte count, T helper and T-suppressor cell numbers, the percentage of T-lymphocyte cells and T suppressors significantly increased in the B6 -100 group at the 14th day. There were no significant changes with respect to immune responses in the control group over 14 days. CONCLUSIONS: A large dose of vitamin B6 supplementation (50 or 100 mg/day) could compensate for the lack of responsiveness of plasma PLP to vitamin B6 intake, and further increase immune response of critically ill patients. SPONSORSHIP: This study was supported by the National Science Council, Taiwan, Republic of China (NSC-92-2320-B-040-026). PMID- 16670692 TI - Effect of a novel dietary supplement on skin aging in post-menopausal women. AB - OBJECTIVE: The aim of the present study was to quantify the effects on skin in post-menopausal women of a novel dietary supplement (Imedeen Prime Renewal) that contained soy extract, fish protein polysaccharides, extracts from white tea, grape seed and tomato, vitamins C and E as well as zinc and chamomile extract. DESIGN: The study was a 6-month double blind, placebo controlled, randomized study on healthy post-menopausal females. SETTING: The study was performed at a commercial Contract Research Organisation (TJ Stephens & Associates Inc., TX, USA). INTERVENTIONS: Two tablets of Imedeen Prime Renewal or placebo were given twice daily for 6 months. SUBJECTS: Thirty-eight (active group) and 42 (placebo group) subjects completed the study out of 100. RESULTS: Clinical grading showed that the active group had a significantly greater improvement (P < 0.05) compared to placebo for the face after 6 months treatment for: forehead, periocular and perioral wrinkles, mottled pigmentation, laxity, sagging, under eye dark circles and overall apperance; skin on the decolletage after 2, 3 and 6 months treatment and skin on the hand after 3 and 6 months treatment. Photo evaluation showed that the active group had a significantly greater improvement (P < 0.05) on the face after 3 and 6 months for several parameters. Ultrasound measurements showed that the active group had a significantly greater improvement (P < 0.0001) for density measurements after 6 months treatment. CONCLUSION: In summary, this novel dietary supplement, Imedeen Prime Renewal, provides improved condition, structure and firmness of the skin in post-menopausal women after 6 months. PMID- 16670693 TI - Cereal grains and legumes in the prevention of coronary heart disease and stroke: a review of the literature. AB - A number of reviewers have examined studies investigating the relationship between coronary heart disease and stroke prior to 2000. Since then, several key studies have been published. Five studies have examined the relationship between wholegrain consumption, coronary heart disease (CHD) and cardiovascular (CVD) disease and found protection for either or both diseases. The researchers concluded that a relationship between wholegrain intake and CHD is seen with at least a 20% and perhaps a 40% reduction in risk for those who eat wholegrain food habitually vs those who eat them rarely. Notwithstanding the fact that fibre is an important component of wholegrains, many studies have not shown an independent effect of fibre alone on CHD events. Thus in terms of CHD prevention, fibre is best obtained from wholegrain sources. Wholegrain products have strong antioxidant activity and contain phytoestrogens, but there is insufficient evidence to determine whether this is beneficial in CHD prevention. Soluble fibre clearly lowers cholesterol to a small but significant degree and one would expect that this would reduce CHD events. There have been a small number of epidemiological studies showing soy consumption is associated with lower rates of heart disease. Countering the positive evidence for wholegrain and legume intake has been the Nurses Health Study in 2000 that showed women who were overweight or obese consuming a high glycaemic load (GL) diet doubled their relative risk of CHD compared with those consuming a low GL diet. Although the literature relating GL with CHD events is somewhat mixed, the relationship with risk factors such as HDL cholesterol, triglyceride and C reactive protein is relatively clear. Thus, carbohydrate-rich foods should be wholegrain and, if they are not, then the lowest glycaemic index (GI) product should be used. Promotion of carbohydrate foods should be focused on wholegrain cereals because these have proven to be associated with health benefits. There is insufficient evidence about whether the addition of other components of wholegrains such as polyphenolics or minerals (such as magnesium or zinc) would improve the health benefits of refined grain foods and this needs investigation. Whether adding bran to refined carbohydrate foods can improve the situation is also not clear, and it was found that added bran lowered heart disease risk in men by 30%. This persisted after full adjustment (including GL) suggesting, at least in men, that fibre may be more important than GI. Thus there are two messages: The intake of wholegrain foods clearly protects against heart disease and stroke but the exact mechanism is not clear. Fibre, magnesium, folate and vitamins B6 and vitamin E may be important. The intake of high GI carbohydrates (from both grain and non-grain sources) in large amounts is associated with an increased risk of heart disease in overweight and obese women even when fibre intake is high but this requires further confirmation in normal-weight women. RECOMMENDATION: Carbohydrate-rich foods should be wholegrain and if they are not, then the lowest GI product available should be consumed. Glycemic index is largely irrelevant for foods that contain small amounts of carbohydrate per serve (such as most vegetables). PMID- 16670694 TI - Sugar-, acid- and phenol contents in apple cultivars from organic and integrated fruit cultivation. AB - OBJECTIVE: This study was carried out to obtain data about the sugar-, acid- and phenol content of apple cultivars from organic and integrated fruit cultivation, with reference to their role in human health and especially for diet recommendations. SETTING: Styria (Austria) and Slovenia. INTERVENTIONS: HPLC, Spectral Photometry, organoleptic and olfactory tests. RESULTS: The total sugar content of most cultivars from integrated cultivation ranged between 115 and 160 g/kg. Some cultivars from organic growing reached higher values. The acid content of both cultivar types was similar. The phenol content in organically grown cultivars was much higher than that of the ones from integrated cultivation. CONCLUSION: Knowledge of the sugar content is very important for diabetic patients, owing to the assumption of general diet recommendations that 100 g fruit contain 12 g carbohydrates. This applies to most well-known cultivars like Golden Delicious or Gala, but not to most of the regional cultivars. For diabetics, it is necessary to know the carbohydrate content of food precisely, in order to adapt the amount of insulin to the ingestion. So, it is helpful to know the sugar content of each regional cultivar. Moreover, very high levels of phenolic compound in organically grown cultivars, and with it its importance for human health leads to the recommendation to eat regional fruits from organic fruit growing instead of those grown under integrated cultivation. PMID- 16670695 TI - Effects of breakfast meal composition on second meal metabolic responses in adults with Type 2 diabetes mellitus. AB - OBJECTIVE: We tested the relative importance of a low-glycemic response versus a high glycemic response breakfast meal on postprandial serum glucose, insulin and free fatty acid (FFA) responses after consumption of a standardized mid-day meal in adult individuals with Type 2 diabetes mellitus (DM). DESIGN: Following an overnight fast of 8-10 h, a randomized crossover intervention using control and test meals was conducted over a 3-week-period. A fasting baseline measurement and postprandial measurements at various time intervals after the breakfast and mid day meal were taken. SUBJECTS: Forty-five Type 2 DM subjects completed the requirements and were included in the study results. INTERVENTIONS: Two different breakfast meals were administered during the intervention: (A) a high glycemic load breakfast meal consisting of farina (kJ 1833; carbohydrate (CHO) 78 g and psylium soluble fiber 0 g), (B) a low-glycemic load breakfast meal consisting of a fiber-loop cereal (kJ 1515; CHO 62 g and psyllium soluble fiber 6.6 g). A standardized lunch was provided approximately 4 h after breakfast. Blood plasma concentrations and area under the curve (AUC) values for glucose, insulin and FFA were measured in response to the breakfast and mid-day lunch. Statistical analyses were performed using SAS software (8.02). Comparisons between diets were based on adjusted Bonferroni t-tests. RESULTS: In post-breakfast analyses, Breakfast B had significantly lower area under the curve (AUC) values for plasma glucose and insulin compared to Breakfast A (P<0.05) (95% confidence level). The AUC values for FFA were higher for Breakfast B than for Breakfast A (P<0.05) (95% confidence level). Post-lunch analyses indicated similar glucose responses for the two breakfast types. Insulin AUC values for Breakfasts B were significantly lower than Breakfast A (P<0.05) (95% confidence level). The AUC values for FFA were unaffected by breakfast type. CONCLUSIONS: These data indicate that ingesting a low-glycemic load meal containing psyllium soluble fiber at breakfast significantly improves the breakfast postprandial glycemic, insulinemic and FFA responses in adults with Type 2 DM. These data revealed no residual postprandial effect of the psyllium soluble fiber breakfast meal beyond the second meal consumed. Thus, there was no evidence of an improvement postprandially in the glycemic, insulinemic and FFA responses after the consumption of the lunch meal. PMID- 16670696 TI - A simple method to induce focal brain hypothermia in rats. AB - Hypothermia reduces cell death and promotes recovery in models of cerebral ischemia, intracerebral hemorrhage and trauma. Clinical studies report significant benefit for treating cardiac arrest and studies are investigating hypothermia for stroke and related conditions. Both local (head) and generalized hypothermia have been used. However, selective brain cooling has fewer side effects than systemic cooling. In this study, we developed a method to induce local (hemispheric) brain hypothermia in rats. The method involves using a small metal coil implanted between the Temporalis muscle and adjacent skull. This coil is then cooled by flushing it with cold water. In our first experiment, we tested whether this method induces focal brain hypothermia in anesthetized rats. Brain temperature was assessed in the ipsilateral cortex and striatum, and contralateral striatum, while body temperature was kept normothermic. Focal, ipsilateral cooling was successfully produced, while the other locations remained normothermic. In the second experiment, we implanted the coil, and brain and body temperature telemetry probes. The coil was connected via overhead swivel to a cold-water source. Brain hypothermia was produced for 24 h, while body temperature remained normothermic. A third experiment measured brain and body temperature along with heart rate and blood pressure. Brain cooling was produced for 24 h without significant alterations in pressure, heart rate or body temperature. In summary, our simple method allows for focal brain hypothermia to be safely induced in anesthetized or conscious rats, and is, therefore, ideally suited to stroke and trauma studies. PMID- 16670697 TI - Fructose metabolism in the adult mouse optic nerve, a central white matter tract. AB - Our recent report that fructose supported the metabolism of some, but not all axons, in the adult mouse optic nerve prompted us to investigate in detail fructose metabolism in this tissue, a typical central white matter tract, as these data imply efficient fructose metabolism in the central nervous system (CNS). In artificial cerebrospinal fluid containing 10 mmol/L glucose or 20 mmol/L fructose, the stimulus-evoked compound action potential (CAP) recorded from the optic nerve consisted of three stable peaks. Replacing 10 mmol/L glucose with 10 mmol/L fructose, however, caused delayed loss of the 1st CAP peak (the 2nd and 3rd CAP peaks were unaffected). Glycogen-derived metabolic substrate(s) temporarily sustained the 1st CAP peak in 10 mmol/L fructose, as depletion of tissue glycogen by a prior period of aglycaemia or high-frequency CAP discharge rendered fructose incapable of supporting the 1st CAP peak. Enzyme assays showed the presence of both hexokinase and fructokinase (both of which can phosphorylate fructose) in the optic nerve. In contrast, only hexokinase was expressed in cerebral cortex. Hexokinase in optic nerve had low affinity and low capacity with fructose as substrate, whereas fructokinase displayed high affinity and high capacity for fructose. These findings suggest an explanation for the curious fact that the fast conducting axons comprising the 1st peak of the CAP are not supported in 10 mmol/L fructose medium; these axons probably do not express fructokinase, a requirement for efficient fructose metabolism. PMID- 16670698 TI - Cerebral perfusion response to hyperoxia. AB - Graded levels of supplemental inspired oxygen were investigated for their viability as a noninvasive method of obtaining intravascular magnetic resonance image contrast. Administered hyperoxia has been shown to be effective as a blood oxygenation level-dependent contrast agent for magnetic resonance imaging (MRI); however, it is known that high levels of inspired fraction of oxygen result in regionally decreased perfusion in the brain potentially confounding the possibility of using hyperoxia as a means of measuring blood flow and volume. Although the effects of hypoxia on blood flow have been extensively studied, the hyperoxic regime between normoxia and 100% inspired oxygen has been only intermittently studied. Subjects were studied at four levels of hyperoxia induced during a single session while perfusion was measured using arterial spin labelling MRI. Reductions in regional perfusion of grey matter were found to occur even at moderate levels of hyperoxia; however, perfusion changes at all oxygen levels were relatively mild (less than 10%) supporting the viability of hyperoxia-induced contrast. PMID- 16670699 TI - Whisker stimulation enhances angiogenesis in the barrel cortex following focal ischemia in mice. AB - Post-ischemia angiogenesis and vascular plasticity help to restore blood flow to ischemic tissue and likely benefit long-term functional recovery. Physical activity has been shown to cause morphologic and functional effects, including promoting angiogenesis in normal or injured animals. A therapeutic effect of peripheral activity on central angiogenesis after cerebral ischemia, however, has not been studied. In the present study of whisker-barrel cortex ischemia in the mouse model, we tested the hypothesis that enhancing whisker activity and sensory input to the ischemic barrel cortex might promote post-ischemia cerebral angiogenesis. Three days after focal ischemia in adult mice, the whiskers corresponding to the ischemic barrel cortex were stimulated by two methods: (1) whiskers on the right side of the mouse face were trimmed away, so the left whiskers were overused by the animals, (2) left whiskers were manually stimulated to enhance input signals to the ischemic barrel cortex. Western blot analysis showed that whisker stimulation increased expression of the angiogenic factors vascular endothelial growth factor, basic fibroblast growth factor, Tie-1, angiopoietin-2 (Ang-2), and possibly Ang-1. Co-immunostaining with markers for proliferation (5-bromo-2'-deoxyuridine (BrdU)) and vascular endothelial cells (Glut-1/CD-31) identified vessel proliferation in the penumbra region. Whisker stimulation increased BrdU-positive endothelial cells and vessels in this region 7 and 14 days after ischemia. Whisker stimulation also attenuated endothelial cell death and increased local cerebral blood flow. Our data suggest that appropriately enhanced peripheral activity and afferent signals to the ischemic cortex can promote post-ischemic angiogenesis, which may imply beneficial effects of specific physical therapy on long-term recovery from ischemic stroke. PMID- 16670700 TI - Topical issues in unrelated donor haematopoietic stem cell transplants: a report from a workshop convened by the Anthony Nolan Trust in London - 2005. AB - Over more than three decades, The Anthony Nolan Trust (ANT) has provided an unrelated donor (UD) for over 4000 children and adults lacking a suitable family member donor, and has remained at the forefront of developments in haematopoietic stem cell transplantation (HSCT) and bone marrow register management. These three decades have seen major changes in clinical practice of UD-HSCT, including new indications, increased use of alternative haematopoietic cell sources, significant improvement of the outcome as a result of better support care, less toxic conditioning regimens, and better donor selection, and expansion to older patients with higher comorbidities. In order to foster our goal of improving UD HSCT availability and outcome in a progressively more complex clinical scenario, a new initiative from ANT was launched in 2005 to convene an experts workshop to address the topical issues in this field. Four consecutive panels addressed factors influencing donor selection and transplant outcome, the use of cord blood, regulatory and accreditation issues, and future developments in this field. This report summarizes the discussions held in this workshop, which will likely develop into a periodic event where transplant clinicians, scientists and registry members will meet to share their experience and vision in the field of UD-HSCT. PMID- 16670701 TI - High-dose melphalan (200 mg/m2) supported by autologous stem cell transplantation is safe and effective in elderly (>or=65 years) myeloma patients: comparison with younger patients treated on the same protocol. AB - Limited information is available on the feasibility and efficacy of autologous stem cell transplantation (ASCT) in multiple myeloma (MM) patients >65 years of age. In 1995-2005, 22 myeloma patients >or=65 years (median 68, eight >or=70) and 79 patients <65 years (median 57) were included in an identical treatment protocol. The first progenitor cell mobilization with cyclophosphamide plus granulocyte-colony stimulating factor (G-CSF) was successful in 95 and 96% of the patients, respectively. To date, 92 patients have received MEL (melphalan) 200 mg/m2 supported by ASCT. No early treatment-related deaths were observed among 22 elderly patients, whereas one younger patient died early. Engraftment and the need for supportive care were comparable between groups. The elderly patients tended to have more WHO grade 3-4 oral or gastrointestinal toxicity when compared to the younger patients (45 vs 23%, P=0.06). After ASCT, a complete response was observed in 44% of the elderly patients and 36% of the younger patients, respectively. No difference was observed between these age groups in progression free survival (23 vs 21 months) or overall survival (57 vs 66 months) after ASCT. We conclude that MEL200 is a safe and efficacious treatment in selected elderly myeloma patients. PMID- 16670702 TI - Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads. AB - Adult mesenchymal stem cells (MSC) that are able to differentiate into various mesenchymal cell types are typically isolated from bone marrow, but their significant presence in human peripheral blood (PB) is controversial. Fibrin microbeads (FMB) that bind matrix-dependent cells were used to isolate MSC from the mononuclear fraction of mobilized PB of adult healthy human donors treated with a granulocyte colony-stimulating factor. Isolation by plastic adherence resulted in a negligible number of MSC in all samples tested, whereas FMB-based isolation yielded spindle-shaped cell samples that could further expand on plastic or on FMB in eight out of the 11 samples. The yield of these cells at days 17-18 after the harvest was approximately 0.5% of the initial cell number. The isolated cells were grown on plastic and characterized by FACS analysis and immunohistochemistry for specific markers. Following culturing and first passage, the FMB-isolated cells stained positive for mesenchymal stromal cell markers CD90 and CD105, expressed vimentin and fibronectin and were negative for hematopoietic markers CD45 and CD34. These cells could differentiate into osteoblasts, adipocytes and chondrocytes. This study indicates that FMB may have special advantage in isolating MSC from sources such as mobilized PB, where the number of such cells is scarce. PMID- 16670703 TI - Increase in allergy following donor lymphocyte infusions. PMID- 16670704 TI - Baseline psychosocial predictors of survival in localised breast cancer. AB - Despite the large number of studies on the impact of psychosocial factors on breast cancer progression, there is no certainty about the contributing factors or processes involved. We investigated the relative impacts of socioeconomic, psychological, and psychosocial factors on survival in breast cancer. A consecutive sample of 102 patients (participation 82%) under 72 years of age with locoregional breast cancer completed validated questionnaires on coping with cancer, emotional expression (anger), perceived available support, noncancer life stresses, and quality of life 3-4 months after diagnosis. Survival times were measured from the date of diagnosis to the date of relapse and further to the date of death or date of last follow-up. Cumulative Cox regression analyses were carried out. After controlling for biological prognostic factors, age, and baseline treatment, longer survival was predicted by a long education and a minimising-related coping, while shorter survival was predicted by emotional defensiveness (antiemotionality), behavioural-escape coping, and a high level of perceived support. A shorter event-free time was also predicted by unemployment and depressive symptoms. Cancer survival is affected by a complex combination of psychosocial factors, among which minimising predicts a favourable prognosis and anger nonexpression and escape behaviour an unfavourable prognosis. Higher socioeconomic status is associated with longer survival. High scores in well being scales may reflect emotional nonexpression. PMID- 16670705 TI - Use of postmenopausal hormone replacement therapy and risk of non-Hodgkin's lymphoma: a Danish population-based cohort study. AB - Use of postmenopausal hormone replacement therapy (HRT) has been hypothesised to be associated with a reduced risk of non-Hodgkin's lymphoma (NHL), but the epidemiologic evidence is conflicting. To examine the risk of NHL in HRT users aged 40 and older, we conducted a cohort study in the County of North Jutland, Denmark (population 0.5 million) using data from population-based health registries for the period 1989-2002. We computed age-standardised NHL incidence rates and used Cox regression analysis to compute the relative risk (RR) and corresponding 95% confidence intervals (CI) of NHL among HRT users compared with non-users, adjusting for age and calendar period. The number of prescriptions redeemed (1, 2-4, 5-9, 10-19, or 20 or more prescriptions) was used as a proxy for duration of HRT. We identified 40 NHL cases among HRT users during 179 838 person-years of follow-up and 310 NHL cases among non-users during 1 247 302 person-years of follow-up. The age-standardised incidence rates of NHL were 25.7 per 100,000 among HRT users and 24.2 per 100,000 among non-users, yielding an adjusted RR of 0.99 (95% CI: 0.71-1.39). Our data did not support an association between HRT use and risk of NHL. PMID- 16670708 TI - Molecular phylogeny: My sister is a sea squirt? PMID- 16670709 TI - Bayesian mapping of genotype x expression interactions in quantitative and qualitative traits. AB - A novel Bayesian gene mapping method, which can simultaneously utilize both molecular marker and gene expression data, is introduced. The approach enables a quantitative or qualitative phenotype to be expressed as a linear combination of the marker genotypes, gene expression levels, and possible genotype x gene expression interactions. The interaction data, given as marker-gene pairs, contains possible in cis and in trans effects obtained from earlier allelic expression studies, genetical genomics studies, biological hypotheses, or known pathways. The method is presented for an inbred line cross design and can be easily generalized to handle other types of populations and designs. The model selection is based on the use of effect-specific variance components combined with Jeffreys' non-informative prior--the method operates by adaptively shrinking marker, expression, and interaction effects toward zero so that non-negligible effects are expected to occur only at very few positions. The estimation of the model parameters and the handling of missing genotype or expression data is performed via Markov chain Monte Carlo sampling. The potential of the method including heritability estimation is presented using simulated examples and novel summary statistics. The method is also applied to a real yeast data set with known pathways. PMID- 16670710 TI - Evaluation of the hypothesis that Balkan endemic nephropathy is caused by drinking water exposure to contaminants leaching from Pliocene coal deposits. AB - Balkan endemic nephropathy (BEN) is a kidney disease that has been reported in only certain rural villages in Serbia, Bulgaria, Romania, Croatia and Bosnia. The cause of BEN remains a mystery, but researchers seem to agree that exposure to one or more environmental agents is at least partially responsible. The Pliocene lignite hypothesis suggests the disease is due to long-term exposure to polycyclic aromatic hydrocarbons (PAHs) or other toxic organic compounds that have leached into drinking water supplies from low-rank coals. Although this hypothesis has been promoted by some researchers, efforts to substantiate it have been inconclusive due to limitations in sample size and methodology. The present study was designed to further examine this hypothesis by analyzing PAHs, which were implicated in the original hypothesis, in a larger number of water samples from endemic and nonendemic villages in Bulgaria and for other chemical differences between the villages. Results show that levels of all PAHs were low, with none exceeding the drinking water standard for benzo-[a]-pyrene, the most toxic PAH, and the only one for which a maximum contaminant level (MCL) has been set for drinking water. Comparison of additional unidentified chromatographic peaks from high-pressure liquid chromatography (HPLC) technique designed to detect dissolved organic compounds (DOCs) that leach from coal failed to show higher levels in BEN villages. This study finds no basis to connect PAHs or other unknown DOCs to the etiology of BEN, and suggests that the evidence in support of the Pliocene lignite hypothesis is limited to the spatial association originally proposed. PMID- 16670711 TI - Predictors of use and consumption of public drinking water among pregnant women. AB - Disinfection by-products (DBPs) in drinking water may be associated with adverse pregnancy outcomes. However, the results from previous epidemiological studies are not consistent, perhaps in part due to individual variation in water use and consumption. This study was performed to evaluate and describe demographic and behavioral characteristics as predictors of ingested water, showering, bathing, and swimming among pregnant women. Water use and consumption data were collected through telephone interviews with 2297 pregnant women from three geographical sites in the southern United States. The data were analyzed according to demographic, health, and behavioral variables expected to be predictors of water use and thus potential confounding factors relating water use to pregnancy outcome. The candidate predictors were evaluated using backward elimination in regression models. Demographic variables tended to be more strongly predictive of the use and consumption of water than health and behavior-related factors. Non Hispanic white women drank 0.4 (95% confidence interval (CI) 0.2; 0.7) liters more cold tap water per day than Hispanic women and 0.3 (95% CI 0.1; 0.4) liters more than non-Hispanic black women. Non-Hispanic white women also reported drinking a higher proportion of filtered tap water, whereas Hispanic women replaced more of their tap water with bottled water. Lower socioeconomic groups reported spending a longer time showering and bathing, but were less likely to use swimming pools. The results of this study should help researchers to anticipate and better control for confounding and misclassification in studies of exposure to DBPs and pregnancy outcomes. PMID- 16670712 TI - Field performance of a nephelometer in rural kitchens: effects of high humidity excursions and correlations to gravimetric analyses. AB - Rural kitchens of solid-fuel burning households constitute the microenvironment responsible for the majority of human exposures to health-damaging air pollutants, particularly respirable particles and carbon monoxide. Portable nephelometers facilitate cheaper, more precise, time-resolved characterization of particles in rural homes than are attainable by gravitational methods alone. However, field performance of nephelometers must contend with aerosols that are highly variable in terms of chemical content, size, and relative humidity. Previous field validations of nephelometer performance in residential settings explore relatively low particle concentrations, with the vast majority of 24-h average gravitational PM2.5 concentrations falling below 40 microg/m3. We investigate relationships between 24-h gravitational particle measurements and nephelometric data logged by the personal DataRAM (pDR) in highly polluted rural Chinese kitchens, where gravitationally determined 24-h average respirable particle concentrations were as high as 700 microg/m3. We find that where relative humidity remained below 95%, nephelometric response was strongly linear despite complex mixtures of aerosols and variable ambient conditions. Where 95% relative humidity was exceeded for even a brief duration, nephelometrically determined 24-h mean particle concentrations were nonsystematically distorted relative to gravitational data, and neither concurrent relative humidity measurements nor use of robust statistical measures of central tendency offered means of correction. This nonsystematic distortion is particularly problematic for rural exposure assessment studies, which emphasize upper quantiles of time resolved particle measurements within 24-h samples. Precise, accurate interpretation of nephelometrically resolved short-term particle concentrations requires calibration based on short-term gravitational sampling. PMID- 16670713 TI - Call-related factors influencing output power from mobile phones. AB - Mobile phone use is increasing but there is also concern for adverse health effects. Well-designed prospective studies to assess several health outcomes are required. In designing a study of mobile phone use, it is important to assess which factors need to be considered in classifying the exposure to radiofrequency fields (RF). A pilot study was performed in Sweden and in the UK 2002 to 2003 to test the feasibility of recruiting a cohort of mobile phone users from a random population sample and from mobile phone subscription lists for a prospective study. As one part of this pilot study, different factors were evaluated regarding possible influence on the output power of the phones. By local switch logging, information on calls made from predefined subscriptions or dedicated handsets were obtained and the output power of phones during calls made indoors and outdoors, in moving and stationary mode, and in rural as well in urban areas were compared. In this experiment, calls were either 1, 1.5 or 5 min long. The results showed that high mobile phone output power is more frequent in rural areas whereas the other factors (length of call, moving/stationary, indoor/outdoor) were of less importance. Urban and rural area should be considered in an exposure index for classification of the exposure to RF from mobile phones and may be assessed by first base station during mobile phone calls or, if this information is not available, possibly by using home address as a proxy. PMID- 16670714 TI - A phase II study of single-agent gefitinib as first-line therapy in patients with stage IV non-small-cell lung cancer. AB - The aim of this study was to evaluate the efficacy and tolerability of gefitinib ('IRESSA') in Japanese patients with previously untreated stage IV non-small-cell lung cancer (NSCLC). This was a multi-institutional phase II study. Thirty-four patients with previously untreated stage IV NSCLC were enrolled between May 2003 and September 2004. Gefitinib was administered orally 250 mg once a day and was continued until there was either disease progression or severe toxicity. Objective tumour response rate was 26.5% (95% confidence interval, 11.7-41.3%). Adverse events were generally mild (National Cancer Institute-Common Toxicity Criteria grade 1 or 2) and consisted mainly of skin rash, fatigue and liver dysfunction. No pulmonary toxicity was observed. The global health status revealed that there was no change in quality of life during the study. This study found that single-agent gefitinib is active and well tolerated in chemo-naive Japanese patients with advanced NSCLC. PMID- 16670715 TI - Distinguishing blood and lymph vessel invasion in breast cancer: a prospective immunohistochemical study. AB - Recently, peritumoural (lympho)vascular invasion, assessed on haematoxylin-eosin (HE)-stained slides, was added to the St Gallen criteria for adjuvant treatment of patients with operable breast cancer (BC). New lymphatic endothelium-specific markers, such as D2-40, make it possible to distinguish between blood (BVI) and lymph vessel invasion (LVI). The aim of this prospective study was to quantify and compare BVI and LVI in a consecutive series of patients with BC. Three consecutive sections of all formalin-fixed paraffin-embedded tissue blocks of 95 BC resection specimens were (immuno)histochemically stained in a fixed order: HE, anti-CD34 (pan-endothelium) and anti-D2-40 (lymphatic endothelium) antibodies. All vessels with vascular invasion were marked and relocated on the corresponding slides. Vascular invasion was assigned LVI (CD34 [plus sign in circle] or [minus sign in circle]/D2-40 [plus sign in circle]) or BVI (CD34 [plus sign in circle]/D2-40 [minus sign in circle]) and intra- (contact with tumour cells or desmoplastic stroma) or peritumoural. The number of vessels with LVI and BVI as well as the number of tumour cells per embolus were counted. Results were correlated with clinico-pathological variables. Sixty-six (69.5%) and 36 (37.9%) patients had, respectively, LVI and BVI. The presence of 'vascular' invasion was missed on HE in 20% (peritumourally) and 65% (intratumourally) of cases. Although LVI and BVI were associated intratumourally (P=0.02), only peritumoural LVI, and not BVI, was associated with the presence of lymph node (LN) metastases (p(peri)=0.002). In multivariate analysis, peritumoural LVI was the only independent determinant of LN metastases. Furthermore, the number of vessels with LVI was larger than the number of vessels with BVI (P=0.001) and lymphatic emboli were larger than blood vessel emboli (P=0.004). We demonstrate that it is possible to distinguish between BVI and LVI in BC specimens using specific lymphatic endothelium markers. This is important to study the contribution of both processes to BC metastasis. Furthermore, immunohistochemical detection of lymphovascular invasion might be of value in clinical practice. PMID- 16670716 TI - Performance of cytology and human papillomavirus testing in relation to the menstrual cycle. AB - Cervical smears prepared around the time of menses have been linked to unsatisfactory specimens and false negative results; however, it is unclear whether liquid-based cytology is similarly affected and data relating date of last menstrual period (LMP) to human papillomavirus (HPV) DNA testing are conflicting. Accordingly, we evaluated liquid-based cytology and HPV test results using Hybrid Capture 2 and PCR by LMP (days 0-10; 11-21; 22-28). We studied 5060 participants in ALTS, the Atypical Squamous Cells of Undetermined Significance (ASCUS) Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study. On average, women had 3.4 examinations (median 4, range 1-5) during a 2-year period of observation permitting an examination of intra-individual variation in cytology and HPV by LMP. Although uncommon, unsatisfactory cytology specimens were most likely on days 0-10. For satisfactory specimens, the frequency with which cytologic categories were reported varied by time since LMP, although differences were modest and did not affect the chance of abnormal cytology or its severity among women diagnosed with CIN2+. The frequency of positive HC2 tests did not vary with date of LMP. Among HPV infected women, independent of eventual diagnosis and the number of viral genotypes present, mid-cycle specimens yielded the highest frequency of LSIL cytologic interpretations and the highest HPV load; however, the magnitude of these effects were small. Intraindividual correlations of cytology or HPV by LMP were generally weak. We conclude that mid-cycle specimens yield slightly higher HPV DNA loads and slightly increased LSIL interpretations, but the clinical impact is marginal. Standardizing collection times would slightly improve interpretation of trends in HPV load. Finally, these data are consistent with the view that the biological properties of the HPV infected cervix vary with the date of the LMP. PMID- 16670717 TI - MYCN gene amplification is a powerful prognostic factor even in infantile neuroblastoma detected by mass screening. AB - MYCN is the most powerful prognostic factor in cases of older children. However, how MYCN is related to the prognosis of infantile cases is not clear. A mass screening program was carried out by measuring urinary catecholamine metabolites (VMA and HVA) from 6-month-old infants. Of 2084 cases detected by the screening program, MYCN amplification (MNA) was examined by Southern blot analyses in 1533 cases from 1987 to 2000. Of the 1533 cases examined, 1500 (97.8%) showed no MNA, 20 cases (1.3%) showed MNA from three to nine copies, and 13 (0.8%) cases showed more than 10 copies. The 4-year overall survival rates of these three groups (99, 89 and 53%, respectively) were significantly different (P<0.001), indicating that MYCN copy number correlates with the prognosis. Cases with MNA more than 10 copies were more advanced than those without amplification (stage III, IV vs I, II, IVs; P<0.001). Patients with MNA more than 10 copies had significantly higher serum levels of neuron-specific-enolase (NSE) and ferritin than non-amplified patients (P=0.049, P=0.025, respectively). MYCN amplification was strongly correlated with a poor prognosis in infantile neuroblastoma cases. Therefore, for the selection of appropriate treatment, an accurate determination of MNA is indispensable. PMID- 16670718 TI - Reliability of cyclin A assessment on tissue microarrays in breast cancer compared to conventional histological slides. AB - Cyclin A has in some studies been associated with poor breast cancer survival, although all studies have not confirmed this. Its prognostic significance in breast cancer needs evaluation in larger studies. Tissue microarray (TMA) technique allows a simultaneous analysis of large amount of tumours on a single microscopic slide. This makes a rapid screening of molecular markers in large amount of tumours possible. Because only a small tissue sample of each tumour is punched on an array, the question has arisen about the representativeness of TMA when studying markers that are expressed in only a small proportion of cells. For this reason, we wanted to compare cyclin A expression on TMA and on traditional large sections. Two breast cancer TMAs were constructed of 200 breast tumours diagnosed between 1997-1998. TMA slides and traditional large section slides of these 200 tumours were stained with cyclin A antibody and analysed by two independent readers. The reproducibility of the two readers' results was good or even very good, with kappa values 0.71-0.87. The agreement of TMA and large section results was good with kappa value 0.62-0.75. Cyclin A overexpression was significantly (P<0.001) associated with oestrogen receptor and progesterone receptor negativity and high grade both on TMA and large sections. Cyclin A overexpression was significantly associated with poor metastasis-free survival both on TMA and large sections. The relative risks for metastasis were similar on TMA and large sections. This study suggests that TMA technique could be useful to study histological correlations and prognostic significance of cyclin A on breast cancer on a large scale. PMID- 16670719 TI - Nucleostemin mRNA is expressed in both normal and malignant renal tissues. AB - Nucleostemin (NS), a p53-binding protein, has been shown essential for stem and cancer cell proliferation and implicated in oncogenesis. To explore potential contributions of NS to the development of clear cell renal cell carcinomas (ccRCCs), we determined NS expression in ccRCC cell lines, and in paired normal and malignant renal tissues from 31 patients with ccRCC. Nucleostemin mRNA and/or protein expression was observed in all four cell lines and 27 of 31 (87%) tumour specimens. Surprisingly, 16 of 31 (52%) adjacent normal renal samples also expressed NS mRNA and its levels in four of them were comparable with those in paired tumour tissues. Three of the patients had detectable NS mRNA in their normal renal tissues whereas lacked its expression in the matched tumours. Compared to the oncogene c-MYC expression in these same samples, NS expression showed a much less specificity for ccRCC. We further demonstrated that NS mRNA expression was closely associated with cellular proliferation in normal fibroblasts or T lymphocytes and renal cell carcinoma cell lines. Collectively, NS expression widely occurs in normal and malignant renal tissues, and is likely a proliferation marker rather than a unique regulator of cell proliferation and survival in stem and cancer cells. PMID- 16670720 TI - A simple, reproducible method for monitoring the treatment of tumours using dynamic contrast-enhanced MR imaging. AB - Dynamic contrast-enhanced MR imaging (DCE-MRI) may act as a biomarker for successful cancer therapy. Simple, reproducible techniques may widen this application. This paper demonstrates a single slice imaging technique. The image acquisition is performed in less than 500 ms making it relatively insensitive to respiratory motion. Data from phantom studies and a reproducibility study in solid human tumours are presented. The reproducibility study showed a coefficient of variation (CoV) of 19.1% for K(trans) and 15.8% for the initial area under the contrast enhancement curve (IAUC). This was improved to 16 and 13.9% if tumours of diameter less than 3 cm were excluded. The individual repeatability (the range within which individual measurements are expected to fall) was 30.6% for K(trans) and 26.5% for IAUC for tumours greater than 3 cm diameter. This approach to DCE MRI image acquisition can be performed with standard clinical scanners, and data analysis is straightforward. For treatment trials with 10 patients in a cohort, the CoV implies that the method would be sensitive to a treatment effect of greater than 18%. The individual repeatability is well inside the 40% change shown to be important in clinical studies using this DCE-MRI technique. PMID- 16670721 TI - Blood vessel density correlates with the effects of targeted intra-arterial carboplatin infusion with concurrent radiotherapy for squamous cell carcinomas of the oral cavity and oropharynx. AB - Our aim was first to evaluate the association between blood vessel density (BVD) and free platinum concentration in experimentally induced tumours in rabbits. We also investigated the association between tumour BVD and the clinical response of patients who had undergone targeted carboplatin intra-arterial (i.a.) chemoradiotherapy. VX2 carcinoma cells were transplanted into 46 inbred female Japanese white rabbits. In the i.a. group, carboplatin was infused into the lingual artery, and in the intravenous (i.v.) group, carboplatin was infused through the auricular vein. In the clinical study, we evaluated 19 patients with squamous cell carcinomas of the oral cavity and oropharynx, who had undergone targeted carboplatin i.a. chemoradiotherapy and had been administered i.a. tegafur/uracil chemotherapy before surgery. We quantified angiogenesis in both studies. Increased BVD was associated with a higher free platinum concentration in the tumour region in the i.a. group of rabbits. In the clinical study, using multivariate logistic regression analysis, only the BVD was related independently to the treatment effect. Therefore, BVD is a valid predictor of the effects of i.a. targeted carboplatin chemotherapy and concurrent radiotherapy for treating human oral and oropharyngeal squamous cell carcinomas. PMID- 16670723 TI - The relevance of RT-PCR markers for metastatic tumour cell detection. PMID- 16670722 TI - Neoadjuvant concurrent chemoradiation with weekly paclitaxel and carboplatin for patients with oesophageal cancer: a phase II study. AB - This study was performed to assess the efficacy and safety of preoperative chemoradiation consisting of carboplatin and paclitaxel and concurrent radiotherapy for patients with resectable (T2-3N0-1M0) oesophageal cancer. Treatment consisted of paclitaxel 50 mg m(-2) and carboplatin AUC=2 on days 1, 8, 15, 22 and 29 and concurrent radiotherapy (41.4 Gy in 23 fractions, 5 days per week), followed by oesophagectomy. All 54 entered patients completed the chemoradiation without delay or dose-reduction. Grade 3-4 toxicities were: neutropaenia 15%, thrombocytopaenia 2%, and oesophagitis 7.5%. After completion of the chemoradiotherapy 63% had a major endoscopical response. Fifty-two patients (96%) underwent a resection. The postoperative mortality rate was 7.7%. All patients had an R0-resection. The pathological complete response rate was 25%, and an additional 36.5% had less than 10% vital residual tumour cells. At a median follow-up of 23.2 months, the median survival time has not yet been reached. The probability of disease-free survival after 30 months was 60%. In conclusion, weekly neoadjuvant paclitaxel and carboplatin with concurrent radiotherapy is a very tolerable regimen and can be given on an outpatient basis. It achieves considerable down staging and a subsequent 100% radical resection rate in this series. A phase III trial with this regimen is now ongoing. PMID- 16670725 TI - [Avian flu: the next pandemic?]. PMID- 16670726 TI - [Present situation and future perspectives of the epidemic of HIV and AIDS in Spain]. AB - Until 1997 Spain was the European country with the highest incidence of AIDS, due mainly to transmission between users of injected drugs. Since early 1990 there has been a fall in the rate of diagnoses of HIV infection in the Spanish autonomous communities where this information is available, and in 2004 this rate was situated below that of several western European countries. New infections in users of intravenous drugs have declined, and although heterosexual transmission has not undergone significant changes, it has become the prime cause of new HIV infections. The rate of diagnoses of HIV has fallen in both the indigenous population and immigrants; however, demographic changes have meant an increase in the percentage of HIV diagnoses in immigrants. In homosexual men there have been descriptions of a recent increase in the incidence of syphilis and gonococcus, which are a warning of possible increases in the transmission of HIV in this group. The number of people who live with HIV in Spain remains between 100,000 and 150,000 (2.4 to 3.6 per 1,000 inhabitants). In spite of the improvement in prognosis due to antiretroviral treatments, there are annually in Spain over 2,000 cases of AIDS (4.8 per 100,000 inhabitants) and over 1,600 deaths from AIDS (3.8 per 100,000). One third of the people who developed AIDS in 2004 had not until then been diagnosed with HIV, which prevented starting the antiretroviral treatment in time. PMID- 16670727 TI - [Aetiology and antibiotic sensitivity of the most frequent outpatient infections]. AB - The diffusion of the outlines of antibiotic sensitivity is a practice that is recommended for improving the prescription of antibiotics and for controlling the appearance and spread of resistances. During the years 2003 and 2004, data was gathered on the sensitivity to antibiotics of the main bacteria isolated in outpatient urine cultures, coprocultures, faringoamygdal and otic smears in the Pamplona health area, which attends to a population of approximately 400,000 inhabitants. Of the 9,495 uropathogens isolated, 90% were sensitive to amoxicillin-clavulanate, 96.4% to 2nd generation cephalosporins, 95.3% to phosphomycin and 97.7% to nitrofurantoin. One point eight percent (1.8%) of the strains of Escherichia coli isolated in urine were producers of extended spectrum b-lactamases, while 0.5% of the urinary isolations of Klebsiella pneumoniae were producers of this type of b-lactamases. Ninety-six point five percent of the 797 isolations of Salmonella serotipo Enteritidis were sensitive to cotrimoxazol, and 97.5% of the 1,057 isolations of Campylobacter jejuni were sensitive to eritromycine. Of the 319 bacteria isolated in otitis media, 96.5% were sensitive to amoxyline. The Gram negative bacillae producers of otitis externa showed a sensitivity above 90% to ciprofloxacin, gentamycin and polimixin B. Twenty-nine point seven percent of the isolations of Streptococcus pyogenes were resistant to eritromycine. Fourteen point three percent and 17.4% of the Staphylococcus aureus isolated in the years 2003 and 2004 respectively were resistant to methicillin. Knowledge of the map of antibiotic sensitivity of the microorganisms in circulation in each geographical area is information that is necessary for the empirical choice of antibiotic treatment. PMID- 16670728 TI - [Disparities in food consumption and nutrient intake among in relation to smoking]. AB - BACKGROUND: Nutritional habits and smoking are well known to be two of the most important risk factors for the development of cardiovascular diseases and cancer. The aim of this study was to assess disparities in food consumption and nutrient intake in relation to smoking in the population of Pamplona, Spain. METHODS: A cross study by postal survey was carried out on a representative sample (n=875; 29.3% of the theoretical sample) of Pamplona's population aged between 18 and 65 years. Food consumption was evaluated by a food consumption frequency questionnaire. The survey also contained questionnaires about consumption of tobacco and alcohol, physical activity, as well as a sociodemographic record. A bivariant analysis was carried out (p<0.05) by Kruskal Wallis and chi2 Pearson tests. RESULTS: Male smokers consume less fruit, vegetables and chicken than other groups, while male ex-smokers seem to have healthier food consumption patterns. Female smokers have a less healthy diet as well, although differences are less marked. In both genders, smokers consume more alcohol. With respect to nutrients, smokers consume less folate, niacin and antioxidants (vitamin C). Female non-smokers consume more fibre than other groups. CONCLUSIONS: This study highlights the prevalence of a healthier diet among non-smokers and ex-smokers, who share similar diet patterns that are healthier than those of smokers. It would also be desirable for smokers to have healthier food consumption habits in order to reduce the effects of tobacco and to meet the additional demand for some nutrients required by this group. PMID- 16670730 TI - [Genetic mechanisms in the hereditary predisposition to colorectal cancer]. AB - A proportion of colorectal cancers shows some type of genetic predisposition that can be recognised in clinical practice. From the classical dominant inheritance pattern of familial adenomatous polyposis or hereditary non-polyposis colorectal cancer, through the recessive transmission of the MYH associated polyposis, to the new syndromes of the "serrated pathway" or low-penetrance alleles, the discovery of new genes and a deeper understanding of the mechanisms of action of already-known ones are enabling us to understand new aspects of the colorectal carcinogenesis. This is throwing a new light on some of the observed familial aggregation patterns which had remained unexplained. PMID- 16670729 TI - [Frequent users and difficult patients: how do they feel about their treatment by doctors?]. AB - BACKGROUND: To determine the opinions and expectations of difficult, frequent user, patients about their relationship with their GP. MATERIAL AND METHODS: Qualitative design. Discussion groups. Invitation to a meeting at the Chantrea Health Centre (Pamplona). The participants were frequent users of the health centre in the year 2003, who had been defined as "difficult"--according to previously defined criteria--by each of the 12 doctors of the health centre. Excluded were patients with mental retardation, severe hearing problems, severe mental illness, difficulties in mobility and travelling, and over 70 years of age. Groups were formed from amongst the 112 preselected patients. Four groups were designed: "older" persons (GMA) of 46 to 70 years (14); "women" (GMU) of 31 to 45 years (14); "men" (GHO) of 31 to 45 years (13) and "youths" (GJO) of 16 to 30 years (12). The patients were invited by letter and by a subsequent telephone confirmation to a meeting, the content of which was not specified. Sessions of 2 hours duration were held with each of the groups formed. Audio recording, with prior authorization, and verbatim transcription of sessions. Discussion and content analysis by the research group resulting in lines of consensus. RESULTS: The three groups of older patients were formed with a total of 16 participants. The group of youths was not formed since the figure for attendance did not reach the number of 3 or more after three appointments. CONCLUSIONS: From the perspective of the patient it is very important that there should be efficient communication with the doctor. Awareness of a state of frequent use was not detected in those attending. They feel themselves to be chronic patients who need frequent attention. Satisfaction at the treatment received was observed as well as an understanding attitude towards the organisational limitations of the health centre. They did not give verbal expression to the existence of conflictive relations with their GPs. PMID- 16670732 TI - [Incidence of influenza and influenza vaccine effectiveness in the 2004-2005 season]. AB - BACKGROUND: To quantify the incidence of flu in different groups of the population of Navarra in the 2004-2005 season, and to evaluate the effectiveness of anti-flu vaccination. METHODS: The analysis of influenza cases in the system of compulsory notificable diseases was complemented by the individualised notifications in the network of sentinel doctors that covers a population of 22,339 inhabitants. The coverage and effectiveness of the vaccine was studied. RESULTS: Vaccine coverage in (3)65 year olds reached 62%. The incidence of influenza was 42.6 cases per 1,000 inhabitants. It reached a maximum in mid January, exceeding 750 weekly cases per 100,000 inhabitants and 1,900 cases per 100,000 children. The highest rate of influenza was observed in under-15 year olds (49.4 cases per 1,000 inhabitants) and the lowest in non-institutionalised (3)65 year olds (2.6 per 1,000 inhabitants), although it was higher in geriatric residences (62.1 per 1,000; p<0.0001). Seventy-nine percent of the cases from 5 to 64 years resulted in absenteeism from school or work. The flu virus was identified in 42/65 (65%) nasopharyngeal smears, 90% being influenza virus A(H3). The incidence of influenza was 3.08% in the unvaccinated and 0.45% in the vaccinated (p<0.001). The global effectiveness of the anti-flu vaccine was 65%, and in (3)65 years old it was 73%. CONCLUSION: Although its effectiveness is not total, the vaccine is the main measure for preventing influenza. The network of sentinel doctors provide useful information for the coordination of care and public health activities against flu. PMID- 16670733 TI - [Interdisciplinary health project: a model based in home care makes the difference]. AB - OBJECTIVE: To propose an interdisciplinary approach in Community Health through a model based on Home Health Care. DESIGN: A prospective study programmed in two periods: a cross sectional study for data collection followed by home assistance for the health problems detected. METHODS: 100 families (570 inhabitants) were randomly submitted to a clinical, nutritional and laboratory examination in order to detect major health problems. Afterwards the same professional team took care of 10 families in health promotion counseling and illness treatment. RESULTS: This study shows that 84% of the patients in whom a health abnormality was detected were not aware of their illness. The population was mainly affected by hypertension, hypercholesterolemia, hypertrigliceridemia, and anemia. None of these problems were listed as the main causes of consultation in a local center belonging to the traditional health services. Almost 70% of the inhabitants consumed a daily diet with less nutrients and calories than recommended. The project included health care for all family members and free drugs provision for all illnesses detected. Ninety-eight percent of patients had a proper and continuous follow up of their illness for a 3 year period, compared with a 31% follow up provided by the traditional health care organization. CONCLUSIONS: This work shows an integrated effort by the University, health centers, and all health care disciplines to propose an alternative program that personalizes attention and moves health care from Institutions to people's own homes. This model improved community health conditions, had a high impact on illness follow up and increased health care accessibility. PMID- 16670731 TI - [The immunotherapy potential of agonistic anti-CD137 (4-1BB) monoclonal antibodies for malignancies and chronic viral diseases]. AB - Pharmacological intervention on the immune system to achieve more intense lymphocyte responses has potential application in tumour immunology and in the treatment of chronic viral diseases. Immunostimulating monoclonal antibodies are defined as a new family of drugs that augment cellular immune responses. They interact as artificial ligands with functional proteins of the immune system, either activating or inhibiting their functions. There are humanized monoclonal antibodies directed to the inhibitory receptor CD152 (CTLA-4) that are being tested in clinical trials with evidence of antitumoural activity. As a drawback, anti-CTLA-4 monoclonal antibodies induce severe autoimmunity reactions in a fraction of the patients. Anti-CD137 monoclonal antibodies have the ability to induce potent immune responses mainly mediated by cytotoxic lymphocytes with the result of frequent complete tumour eradications in mice. Comparative studies in experimental models indicate that the antitumour activity of anti-CD137 monoclonal antibodies is superior to that of anti-CD152. CD137 (4-1BB) is a leukocyte differentiation antigen selectively expressed on the surface of activated T and NK lymphocytes, as well as on dendritic cells. Monoclonal antibodies acting as artificial stimulatory ligands of this receptor (anti-CD137 agonist antibodies) enhance cellular antitumoural and antiviral immunity in a variety of mouse models. Paradoxically, anti-CD137 monoclonal antibodies are therapeutic or preventive in the course of model autoimmune diseases in mice. In light of these experimental results, a number of research groups have humanized antibodies against human CD137 and early clinical trials are about to start. PMID- 16670734 TI - [Implementation of the early care service in the rural area of Bortziriak]. PMID- 16670735 TI - [Pulmonary alveolar proteinosis and crazy paving pattern in high resolution CT]. AB - We present a case of pulmonary alveolar proteinosis in a 27 year old male, in which high resolution computerised tomography shows a typical crazy paving pattern. This test led to a specific differential diagnosis, in spite of the lack of specificity in the clinical presentation. The bronchoalveolar wash was not conclusive and the definitive diagnosis was made with a transbronchial biopsy. We review the most characteristic aspects of this rare disease and of the crazy paving pattern in high resolution CT. PMID- 16670736 TI - [Consequences of heroin consumption: compartmental syndrome and rhabdomyolysis]. AB - Compartment syndrome is a condition in which increased pressure within a limited space compromises tissue perfusion with the development of rhabdomyolysis. The causes can be physical, such as the pressure exerted on an anatomical region, or it can be associated with the consumption of illegal drugs, especially heroin. In this latter case, toxic and immunological mechanisms are involved in the physiopathology. Acute renal failure develops in 30% of cases of rhabdomyolysis. Its treatment includes efficient volume replacement, forcing urinary alkalinization and the administering of furosemide and mannitol, with certain precautions. No benefit has been shown in dopamine. When a heroin addict patient shows a clinical pattern of tumescence of his extremities, with or without a period of unconsciousness, the presence of compartment syndrome is to be suspected. PMID- 16670737 TI - [Optimism and realism in ethical borderland]. PMID- 16670738 TI - [Mouth--odontology or medicine?]. PMID- 16670739 TI - [Attitudes to estrogen replacement therapy among Norwegian women]. AB - BACKGROUND: The aim of the study was to investigate women's attitudes towards hormone treatment (HT), their judgment of the various sources of information, and their knowledge of indications for HT. METHODS: We posted questionnaires to 1000 Norwegian women between 45 and 60 years of age, randomly selected from the National Register. We received replies from 386 women (38.6%). RESULTS: Of those who replied, 28% reported that they were currently using HT, 22% were previous users, 24% were considering starting, whereas 26% refused ever to use HT. One half of current and previous users had obtained their prescriptions from a gynaecologist, the other half from their general practitioner. Mean age at start of HT was 47 years. 65% of the women started the medication before menopause. The women thought HT to be useful for treatment of hot flushes, prevention of osteoporosis, well-being, improved mood and sex life. The women were also asked about the rating of importance that they would give to the various sources of information. The highest ratings were given to their gynaecologist or general practitioner. Also reading about HT and the opinion of other health personnel obtained high ratings. CONCLUSIONS: The indications for HT given by the women correspond well with the prevailing guidelines. The recent observation about a possible negative effect of HT on cardiovascular disease seems to have reached the respondents. Many women begin to use HT several years before menopause. PMID- 16670740 TI - [Staging and measurement of renal function in chronic renal conditions]. AB - Kidney failure has traditionally been categorised as mild, moderate or severe. Other poorly defined terms like uraemia and end-stage kidney disease have commonly been applied. During the last few years, an international consensus has emerged staging chronic kidney disease into 5 stages: stage 1 being glomerular filtration rate (GFR) > 90 ml/min and signs of kidney damage, stage 2 GFR 60-89 ml/min and signs of kidney damage, stage 3 being GFR 30-59 ml/min, stage 4 being GFR 15-29 ml/min, and stage 5 GFR < 15 ml/min. Kidney function should be calculated from creatinine-based formulas, or measured by isotope or iohexol clearances rather than as renal clearance of creatinine in timed urine samples. We address the importance and implications of these new definitions. PMID- 16670741 TI - [Bone metabolism in patients with newly diagnosed caeliac disease]. AB - BACKGROUND: Caeliac disease is an immune-mediated enteropathy induced by gluten in genetically predisposed individuals. Malabsorption of vitamins and minerals is a common finding in untreated patients and disturbance in bone metabolism is therefore a suspected complication. We wanted to assess vitamin D status, parathyroid hormone (PTH) and bone mineral density (BMD) in patients with newly diagnosed caeliac disease. MATERIAL AND METHODS: Altogether 118 patients (93 females) were investigated. Median age was 42.5 years (range 20-87 years). Vitamin D metabolites, PTH and biochemical markers of bone metabolism were measured in blood. Lumbar spine, femoral neck and total body BMD were measured by dual x-ray absorptiometry (DXA); Z scores were obtained by comparison with locally derived age- and sex matched reference values. RESULTS: Vitamin D deficiency (25-hydroxyvitamin D < 30 nmol/l) was present in 20 % of the patients; 30 % of the patients had secondary hyperparathyroidism (PTH > or =7.0 pmol/l). BMD was significantly reduced (p < 0.001) at all skeletal sites measured. There was no relationship between vitamin D status and BMD, but PTH was negatively correlated to BMD at all skeletal sites (p < 0.005). Body mass index (BMI) was positively correlated to BMD in these patients (p < 0.001). CONCLUSION: Hypovitaminosis D and secondary hyperparathyroidism are common in patients with newly diagnosed caeliac disease in addition to low BMD. PMID- 16670742 TI - [Impact of Scandinavian guidelines on management of mild head injuries]. AB - BACKGROUND: A national survey in 1996 showed insufficient routines for management of patients with mild head injuries in Norwegian hospitals. Since then, the Scandinavian guidelines for management of mild head injuries have been published. METHODS: A cross-sectional questionnaire survey of management practice was performed in all 59 hospitals in 2002. We compared the results with figures from 1996 and evaluated guideline compliance. RESULTS: The proportion of noncompliant hospitals was reduced (p = 0.02) from 52% to 31%. The proportion assessing patient consciousness according to the Glasgow Coma Scale increased (p = 0.001) from 49% to 80%. The proportion requiring a normal computed tomographic scan if a patient with a history of loss of consciousness was to be discharged from the emergency department increased (p < 0.001) from 2% to 19%. CONCLUSION: The Scandinavian guidelines have had a significant impact on management practice in Norwegian hospitals. PMID- 16670743 TI - [Assessment of traumatic tooth injuries in the emergency room]. AB - Many patients with facial injuries are first seen by doctors in the emergency room. Injuries affecting teeth and alveolar process are common in children; approximately half of all children have sustained such an injury before adulthood. Dentoalveolar trauma does not pose a significant morbid risk for the trauma patient. However, failure to recognise or obtain appropriate consultation can result in premature tooth or alveolar bone loss, resulting in problematic prosthetic rehabilitation. Emergency room doctors should know the initial treatment guidelines for traumatic dental injuries to provide optimal treatment before the patient can seen by a dentist. An avulsed tooth should be replanted immediately, or kept moist until it can be replanted. Prognosis is related to storage media and the length of the extra-alveolar period. Teeth replanted within 5 minutes have the best prognosis. If the primary consultation is by phone the patient, or the parent, should be informed to replant the avulsed tooth. If this is not feasible the tooth should be stored in milk, saliva (oral cavity) or physiologic saline until replanted. Primary teeth are not replanted. PMID- 16670744 TI - [Diseases that affect both skin and oral mucosa]. AB - BACKGROUND: Several diseases may affect both skin and oral mucosa. This article presents hereditary, autoimmune and other inflammatory conditions within this group. MATERIAL AND METHODS: Review of the literature as well as the authors' own experience in the field. RESULTS AND INTERPRETATION: Lichen planus, erythema multiforme and lupus erythematosus are relatively common disorders with generally favourable prognosis. The much less frequent bullous diseases pemphigoid and pemphigus are characterized by a high morbidity, often requiring a deliberate use of immunosuppressants. Stevens-Johnson syndrome, toxic epidermal necrolysis and the more severe forms of epidermolysis bullosa are potentially life-threatening conditions. It is recommendable that medical doctors and dentists are familiar with the clinical presentations and diagnostic procedures of diseases affecting skin and oral mucosa. PMID- 16670745 TI - [Oral manifestations in HIV infection]. AB - In this review article, oral lesions in relation to HIV infection are presented and discussed. Lesions such as oral candidiasis, hairy leukoplakia and necrotising gingivitis or periodontitis may be the first sign of an HIV infection or of its progression. Almost all HIV-infected patients will contract oral diseases. Dentists and physicians play an essential role in early recognition of signs and symptoms of HIV disease or of its progression. Only through such recognition can appropriate definitive diagnostic testing be conducted and appropriate therapeutic intervention for the condition be considered. It is pivotal that both dentists and physicians are familiar with the most frequently occurring oral symptoms of HIV infection. PMID- 16670746 TI - [Trust in physicians is under pressure]. PMID- 16670747 TI - [Latin medical terminology--sure]. PMID- 16670748 TI - [Do we really want to concentrate on research?]. PMID- 16670752 TI - Why we think it is important to discuss intelligent design. AB - Belief in God and belief in the science of evolution are not mutually exclusive concepts. Thousands of scientists who believe in God are able to separately study and teach evolution. As scientists and parents, we owe it to our children to ensure that public school science curricula teach the science of evolution and not promote a particular religious faith or belief system. PMID- 16670753 TI - Defending science education against intelligent design: a call to action. AB - We review here the current political landscape and our own efforts to address the attempts to undermine science education in Wisconsin. To mount an effective response, expertise in evolutionary biology and in the history of the public controversy is useful but not essential. However, entering the fray requires a minimal tool kit of information. Here, we summarize some of the scientific and legal history of this issue and list a series of actions that scientists can take to help facilitate good science education and an improved atmosphere for the scientific enterprise nationally. Finally, we provide some model legislation that has been introduced in Wisconsin to strengthen the teaching of science. PMID- 16670754 TI - Nothing but skin and bone. AB - Skin and bone - what comes to mind at hearing this phrase? While certainly a metaphor for disease, it also defines two very different tissues, one a flexible and contiguous outer covering, the other a morphologically diverse hard tissue distributed at over 200 sites in the body. As the accompanying series of Reviews highlights, these tissues are indeed diverse, but there are also surprising similarities. Skin is the interface between the internal organs and the environment, and as such plays a crucial role in the body's defense mechanism. The skin and its many appendages are responsible for functions as diverse as epidermal barrier and defense, immune surveillance, UV protection, thermoregulation, sweating, lubrication, pigmentation, the sensations of pain and touch, and, importantly, the protection of various stem cell niches in the skin. Bone serves a number of purposes: it provides protection for vital organs, a lever for locomotion, a reservoir for calcium, and the site of adult hematopoiesis. The tissue is composed of osteoblasts, osteoclasts, and their individual precursors plus a complex mixture of mesenchymal, myeloid, and lymphoid cells in the marrow space. Finally, the endothelial microenvironment provides nutrition and is a conduit for the influx and emigration of cells that impact bone biology in several important ways. This Review series guides the reader through these various facets of 2 diverse, yet interdependent, tissues. PMID- 16670755 TI - Epidermal barrier formation and recovery in skin disorders. AB - Skin is at the interface between the complex physiology of the body and the external, often hostile, environment, and the semipermeable epidermal barrier prevents both the escape of moisture and the entry of infectious or toxic substances. Newborns with rare congenital barrier defects underscore the skin's essential role in a terrestrial environment and demonstrate the compensatory responses evoked ex utero to reestablish a barrier. Common inflammatory skin disorders such as atopic dermatitis and psoriasis exhibit decreased barrier function, and recent studies suggest that the complex response of epidermal cells to barrier disruption may aggravate, maintain, or even initiate such conditions. Either aiding barrier reestablishment or dampening the epidermal stress response may improve the treatment of these disorders. This Review discusses the molecular regulation of the epidermal barrier as well as causes and potential treatments for defects of barrier formation and proposes that medical management of barrier disruption may positively affect the course of common skin disorders. PMID- 16670756 TI - T cell control in autoimmune bullous skin disorders. AB - Autoimmune bullous disorders are a group of severe skin diseases characterized clinically by blisters and erosions of skin and/or mucous membranes. A hallmark of these disorders is the presence of IgG and occasionally IgA autoantibodies that target distinct adhesion structures of the epidermis, dermoepidermal basement membrane, and anchoring fibrils of the dermis. This Review focuses on the potential role of autoreactive T cells in the pathogenesis of these disorders. Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are the best characterized bullous disorders with regard to pathogenesis and T cell involvement. Activation of autoreactive T cells in PV and BP is restricted by distinct HLA class II alleles that are prevalent in individuals with these disorders. Autoreactive T cells are not only present in patients but can also be detected in healthy individuals. Recently, a subset of autoreactive T cells with remarkable regulatory function was identified in healthy individuals and to a much lesser extent in patients with PV, suggesting that the occurrence of autoimmune bullous disorders may be linked to a dysfunction of Tregs. PMID- 16670757 TI - Prophylactic human papillomavirus vaccines. AB - Human papillomavirus (HPV) infection causes virtually all cases of cervical cancer, the second most common cause of death from cancer among women worldwide. This Review examines prophylactic HPV subunit vaccines based on the ability of the viral L1 capsid protein to form virus-like particles (VLPs) that induce high levels of neutralizing antibodies. Following preclinical research by laboratories in the nonprofit sector, Merck and GlaxoSmithKline are developing commercial versions of the vaccine. Both vaccines target HPV16 and HPV18, which account for approximately 70% of cervical cancer. The Merck vaccine also targets HPV6 and HPV11, which account for approximately 90% of external genital warts. The vaccines have an excellent safety profile, are highly immunogenic, and have conferred complete type-specific protection against persistent infection and associated lesions in fully vaccinated women. Unresolved issues include the most critical groups to vaccinate and when the vaccine's cost may be low enough for widespread implementation in the developing world, where 80% of cervical cancer occurs. PMID- 16670758 TI - Frontiers in pruritus research: scratching the brain for more effective itch therapy. AB - This Review highlights selected frontiers in pruritus research and focuses on recently attained insights into the neurophysiological, neuroimmunological, and neuroendocrine mechanisms underlying skin-derived itch (pruritogenic pruritus), which may affect future antipruritic strategies. Special attention is paid to newly identified itch-specific neuronal pathways in the spinothalamic tract that are distinct from pain pathways and to CNS regions that process peripheral pruritogenic stimuli. In addition, the relation between itch and pain is discussed, with emphasis on how the intimate contacts between these closely related yet distinct sensory phenomena may be exploited therapeutically. Furthermore, newly identified or unduly neglected intracutaneous itch mediators (e.g., endovanilloids, proteases, cannabinoids, opioids, neurotrophins, and cytokines) and relevant receptors (e.g., vanilloid receptor channels and proteinase-activated, cannabinoid, opioid, cytokine, and new histamine receptors) are discussed. In summarizing promising new avenues for managing itch more effectively, we advocate therapeutic approaches that strive for the combination of peripherally active antiinflammatory agents with drugs that counteract chronic central itch sensitization. PMID- 16670759 TI - Estrogen deficiency and bone loss: an inflammatory tale. AB - Estrogen plays a fundamental role in skeletal growth and bone homeostasis in both men and women. Although remarkable progress has been made in our understanding of how estrogen deficiency causes bone loss, the mechanisms involved have proven to be complex and multifaceted. Although estrogen is established to have direct effects on bone cells, recent animal studies have identified additional unexpected regulatory effects of estrogen centered at the level of the adaptive immune response. Furthermore, a potential role for reactive oxygen species has now been identified in both humans and animals. One major challenge is the integration of a multitude of redundant pathways and cytokines, each apparently capable of playing a relevant role, into a comprehensive model of postmenopausal osteoporosis. This Review presents our current understanding of the process of estrogen deficiency-mediated bone destruction and explores some recent findings and hypotheses to explain estrogen action in bone. Due to the inherent difficulties associated with human investigation, many of the lessons learned have been in animal models. Consequently, many of these principles await further validation in humans. PMID- 16670760 TI - The stem cell niches in bone. AB - The stem cell niche is composed of a specialized population of cells that plays an essential role in regulating adult stem cell self-renewal and differentiation. In adults, osteoblasts, responsible for osteogenesis, and hematopoietic cells, responsible for hematopoiesis, are closely associated in the bone marrow, suggesting a reciprocal relationship between the two. It was recently discovered that a subset of osteoblasts functions as a key component of the HSC niche (namely, the osteoblastic niche), controlling HSC numbers. HSCs interact not only with osteoblasts but also with other stromal cells, including endothelial cells. Sinusoidal endothelial cells in bone marrow have been revealed as an alternative HSC niche called the vascular niche. In this Review we compare the architecture of these 2 HSC niches in bone marrow. We also highlight the function of osteoblasts in maintaining a quiescent HSC microenvironment and the likely role of the vascular niche in regulating stem cell proliferation, differentiation, and mobilization. In addition, we focus on studies of animal models and in vitro assays that have provided direct insights into the actions of these osteoblastic and vascular niches, revealing central roles for numerous signaling and adhesion molecules. Many of the discoveries described herein may contribute to future clinical treatments for hematopoietic and bone-related disorders, including cancer. PMID- 16670762 TI - Receptor heterodimerization: a new level of cross-talk. AB - Most G protein-coupled receptors (GPCRs) probably exist as homodimers, but it is increasingly recognized that GPCRs may also dimerize with other types of GPCRs and that this physical interaction may affect the function of either receptor. A study in this issue of the JCI demonstrates how heterodimerization between prostaglandin E receptors and beta(2)-adrenergic receptors (beta(2)ARs) in airway smooth muscle cells results in uncoupling of beta(2)ARs and a diminished bronchodilator response to beta(2)AR agonists (see the related article beginning on page 1400). This illustrates what we believe to be a novel mechanism of receptor cross-talk and highlights the potential importance of GPCR heterodimerization in diseases such as asthma and how this could lead to the development of more specific therapies in the future. PMID- 16670761 TI - Regulation of bone mass by Wnt signaling. AB - Wnt proteins are a family of secreted proteins that regulate many aspects of cell growth, differentiation, function, and death. Considerable progress has been made in our understanding of the molecular links between Wnt signaling and bone development and remodeling since initial reports that mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) are causally linked to alterations in human bone mass. Of the pathways activated by Wnts, it is signaling through the canonical (i.e., Wnt/beta-catenin) pathway that increases bone mass through a number of mechanisms including renewal of stem cells, stimulation of preosteoblast replication, induction of osteoblastogenesis, and inhibition of osteoblast and osteocyte apoptosis. This pathway is an enticing target for developing drugs to battle skeletal diseases as Wnt/beta-catenin signaling is composed of a series of molecular interactions that offer potential places for pharmacological intervention. In considering opportunities for anabolic drug discovery in this area, one must consider multiple factors, including (a) the roles of Wnt signaling for development, remodeling, and pathology of bone; (b) how pharmacological interventions that target this pathway may specifically treat osteoporosis and other aspects of skeletal health; and (c) whether the targets within this pathway are amenable to drug intervention. In this Review we discuss the current understanding of this pathway in terms of bone biology and assess whether targeting this pathway might yield novel therapeutics to treat typical bone disorders. PMID- 16670763 TI - An immunologic homunculus for type 1 diabetes. AB - Autoimmune diseases such as the diabetes that develops in NOD mice depend on immunologic recognition of specific autoantigens, but recognition can result in a pathogenic or protective T cell response. A study by Du et al. in this issue of the JCI demonstrates that TGF-beta signaling by T cells recognizing the insulin peptide B:9-23 is essential for such protection and that this inhibitory cytokine functions in both a paracrine and an autocrine manner (see the related article beginning on page 1360). We propose that the insulin peptide B:9-23 and a conserved TCR motif form an "immunologic homunculus" underlying the relatively common targeting of insulin by T cells that, as demonstrated by the study of Du and coworkers, results in a protective T cell response, or diabetes, as shown by other investigators, for related T cell receptors. PMID- 16670764 TI - Bypassing complement: evolutionary lessons and future implications. AB - Lectins like mannan-binding protein are part of the innate immune system. They circulate in association with serine proteases. Upon binding oligosaccharides, they activate the complement cascade analogous to the more familiar but evolutionarily more recent classical pathway, which is triggered by antibody binding to antigen. In this issue of the JCI, Selander et al. developed a sensitive and specific ELISA employing Salmonella-specific sugars to assess the activity of the lectin pathway of complement activation (see the related article beginning on page 1425). This more physiologic assay system allowed the investigators to rigorously define the requirements for lectin pathway activation. Furthermore, they uncovered an unsuspected means for this pathway to reach the desired critical step of activation of the opsonin C3. These types of functional assays will eventually replace the more laborious, less physiologic, and less informative approaches currently in use to monitor complement activation. PMID- 16670765 TI - The IL-23/IL-17 axis in inflammation. AB - IL-23 induces the differentiation of naive CD4(+) T cells into highly pathogenic helper T cells (Th17/Th(IL-17)) that produce IL-17, IL-17F, IL-6, and TNF-alpha, but not IFN-gamma and IL-4. Two studies in this issue of the JCI demonstrate that blocking IL-23 or its downstream factors IL-17 and IL-6, but not the IL-12/IFN gamma pathways, can significantly suppress disease development in animal models of inflammatory bowel disease and MS (see the related articles beginning on pages 1310 and 1317). These studies suggest that the IL-23/IL-17 pathway may be a novel therapeutic target for the treatment of chronic inflammatory diseases. PMID- 16670766 TI - Role of caveolin-1 in the regulation of the vascular shear stress response. AB - In blood vessels, endothelia are submitted to constant shear effects and are, under normal conditions, capable of responding to any variation in hemodynamic forces. Caveolae - 50- to 100-nm plasma membrane invaginations present at the surface of terminally differentiated cells and particularly enriched in ECs - are composed of a high sphingolipid and cholesterol content and the protein caveolin 1 (Cav-1). Previous studies have suggested that caveolae and endothelial Cav-1 may regulate the vascular response to altered shear stress. In this issue of the JCI, Yu et al. have examined the role of Cav-1/caveolae in the regulation of flow induced alterations (i.e., mechanotransduction) in vessels from wild-type mice, Cav-1-deficient mice, and Cav-1-deficient mice re-expressing Cav-1 only in ECs. Their data suggest that caveolae/Cav-1 may act as sensors of altered shear stress and that they also organize the signaling response in stimulated ECs (see the related article beginning on page 1284). PMID- 16670767 TI - Putting cholesterol in its place: apoE and reverse cholesterol transport. AB - To avoid toxic overload of cholesterol in peripheral cells, the reverse cholesterol transport pathway directs excess cholesterol through HDL acceptors to the liver for elimination. In this issue of the JCI, a study by Matsuura et al. reveals new features of this pathway, including the importance of the ATP-binding cassette transporter G1 in macrophages and apoE in cholesteryl efflux from cells to cholesterol ester-rich (CE-rich) HDL(2) acceptors (see the related article beginning on page 1435). One proposal for boosting reverse cholesterol transport has been to elevate plasma HDL levels by inhibiting CE transfer protein (CETP), which transfers CE from HDL to lower-density lipoproteins. However, there has been concern that large, CE-rich HDL(2) generated by CETP inhibition might impair reverse cholesterol transport. ApoE uniquely facilitates reverse cholesterol transport by allowing CE-rich core expansion in HDL. In lower species, these large HDLs are not atherogenic. Thus, CETP might not be essential for reverse cholesterol transport in humans, raising hope of using a CETP inhibitor to elevate HDL levels. PMID- 16670768 TI - Adenosine metabolism and murine strain-specific IL-4-induced inflammation, emphysema, and fibrosis. AB - To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic inflammation and mild airway fibrosis and did not shorten survival. In C57BL/6 mice, IL-4 caused profound eosinophilic inflammation, airway fibrosis, emphysematous alveolar destruction, and premature death. These differences could not be accounted for by changes in Th2 or Th1 cytokines, receptor components, STAT6 activation, MMPs, or cathepsins. In contrast, in C57BL/6 mice, alveolar remodeling was associated with decreased levels of tissue inhibitors of metalloproteinase 2, -3, and -4 and alpha1-antitrypsin, and fibrosis was associated with increased levels of total and bioactive TGF-beta1. Impressive differences in adenosine metabolism were also appreciated, with increased tissue adenosine levels and A(1), A(2B), and A(3) adenosine receptor expression and decreased adenosine deaminase (ADA) activity in C57BL/6 animals. Treatment with ADA also reduced the inflammation, fibrosis, and emphysematous destruction and improved the survival of C57BL/6 Tg animals. These studies demonstrate that genetic influences control IL-4 effector pathways in the murine lung. They also demonstrate that IL-4 has different effects on adenosine metabolism in Balb/c and C57BL/6 mice and that these differences contribute to the different responses that IL-4 induces in these inbred animals. PMID- 16670769 TI - Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels. AB - Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared long- and short-term flow mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav 1 into the endothelium. In conclusion, these results showed that endothelial Cav 1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels. PMID- 16670770 TI - IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL 17 and IL-6. AB - Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10-deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases. PMID- 16670772 TI - TGF-beta signaling is required for the function of insulin-reactive T regulatory cells. AB - We have previously isolated insulin-reactive Tregs from diabetic NOD mice designated 2H6, from which TCR transgenic mice were generated. The T cells from these 2H6 transgenic mice recognize insulin but have suppressive properties in vitro. They protect NOD mice in vivo from spontaneous development of diabetes and adoptive transfer of disease caused by polyclonal diabetogenic spleen cells as well as the highly diabetogenic monoclonal BDC2.5 TCR transgenic T cells that recognize an islet granule antigen. Using cells from both NOD and BDC2.5 mice that express a dominant-negative TGF-beta receptor type II (TGF-betaDNRII), we show that 2H6 T cells protected from disease by producing TGF-beta and that the ability of the target diabetogenic T cells to respond to TGF-beta was crucial. We further demonstrate that TGF-beta signaling in 2H6 cells was important for their protective properties, as 2H6 cells were unable to protect from adoptive transfer induced diabetes if they were unable to respond to TGF-beta. Thus, our data demonstrate that insulin-specific regulatory cells protect from diabetes by virtue of their production of TGF-beta1 that acts in an autocrine manner to maintain their regulatory function and acts in a paracrine manner on the target cells. PMID- 16670771 TI - Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis. AB - IL-23 is a member of the IL-12 cytokine family that drives a highly pathogenic T cell population involved in the initiation of autoimmune diseases. We have shown that IL-23-dependent, pathogenic T cells produced IL-17 A, IL-17 F, IL-6, and TNF but not IFN-gamma or IL-4. We now show that T-bet and STAT1 transcription factors are not required for the initial production of IL-17. However, optimal IL-17 production in response to IL-23 stimulation appears to require the presence of T bet. To explore the clinical efficacy of targeting the IL-23 immune pathway, we generated anti-IL-23p19-specific antibodies and tested to determine whether blocking IL-23 function can inhibit EAE, a preclinical animal model of human multiple sclerosis. Anti-IL-23p19 treatment reduced the serum level of IL-17 as well as CNS expression of IFN-gamma, IP-10, IL-17, IL-6, and TNF mRNA. In addition, therapeutic treatment with anti-IL-23p19 during active disease inhibited proteolipid protein (PLP) epitope spreading and prevented subsequent disease relapse. Thus, therapeutic targeting of IL-23 effectively inhibited multiple inflammatory pathways that are critical for driving CNS autoimmune inflammation. PMID- 16670773 TI - Airway smooth muscle prostaglandin-EP1 receptors directly modulate beta2 adrenergic receptors within a unique heterodimeric complex. AB - Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane spanning receptors activated during asthma, or by treatment with bronchodilators such as beta(2)-adrenergic receptor (beta(2)AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP(1) receptor, since its endogenous agonist prostaglandin E(2) is abundant in the airway, but its functional implications are poorly defined. Activation of EP(1) failed to elicit ASM contraction in mouse trachea via this G(alphaq)-coupled receptor. However, EP(1) activation markedly reduced the bronchodilatory function of beta(2)AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP(1) reduced beta(2)AR-stimulated cAMP in ASM but did not promote or augment beta(2)AR phosphorylation or alter beta(2)AR trafficking. Bioluminescence resonant energy transfer showed EP(1) and beta(2)AR formed heterodimers, which were further modified by EP(1) agonist. In cell membrane [(35)S]GTPgammaS binding studies, the presence of the EP(1) component of the dimer uncoupled beta(2)AR from G(alphas), an effect accentuated by EP(1) agonist activation. Thus alone, EP(1) does not appear to have a significant direct effect on airway tone but acts as a modulator of the beta(2)AR, altering G(alphas) coupling via steric interactions imposed by the EP(1):beta(2)AR heterodimeric signaling complex and ultimately affecting beta(2)AR-mediated bronchial relaxation. This mechanism may contribute to beta-agonist resistance found in asthma. PMID- 16670774 TI - Mannan-binding lectin activates C3 and the alternative complement pathway without involvement of C2. AB - Lectin pathway activation of C3 is known to involve target recognition by mannan binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP 2). We investigated C3 activation in C2-deficient human sera and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S. thompson, and S. enteritidis corresponding to serogroups B, C, and D (BO, CO, and DO). MBL bound to CO, but not to BO and DO, and efficiently supported C3 deposition in the absence of C2, C4, or MASP-2. The existence of an MBL-dependent C2 bypass mechanism for alternative pathway-mediated C3 activation was clearly demonstrated using CO, solid-phase mannan, and E. coli LPS. MASP-1 might contribute, but was not required for C3 deposition in the model used. Independent of MBL, specific antibodies to CO supported C3 deposition through classical and alternative pathways. MBL-dependent C2 bypass activation could be particularly important in various inherited and acquired complement deficiency states. PMID- 16670776 TI - Noise in a Small Genetic Circuit that Undergoes Bifurcation. AB - Based on the consideration of Boolean dynamics, it has been hypothesized that cell types may correspond to alternative attractors of a gene regulatory network. Recent stochastic Boolean network analysis, however, raised the important question concerning the stability of such attractors. In this paper a detailed numerical analysis is performed within the framework of Langevin dynamics. While the present results confirm that the noise is indeed an important dynamical element, the cell type as represented by attractors can still be a viable hypothesis. It is found that the stability of an attractor depends on the strength of noise related to the distance of the system to the bifurcation point and it can be exponentially stable depending on biological parameters. PMID- 16670775 TI - HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoE- and ABCG1-dependent pathway. AB - Genetic deficiency or inhibition of cholesteryl ester transfer protein (CETP) leads to a marked increase in plasma levels of large HDL-2 particles. However, there is concern that such particles may be dysfunctional in terms of their ability to promote cholesterol efflux from macrophages. Recently, the ATP-binding cassette transporter ABCG1, a macrophage liver X receptor (LXR) target, has been shown to stimulate cholesterol efflux to HDL. We have assessed the ability of HDL from subjects with homozygous deficiency of CETP (CETP-D) to promote cholesterol efflux from macrophages and have evaluated the role of ABCG1 and other factors in this process. CETP-D HDL-2 caused a 2- to 3-fold stimulation of net cholesterol efflux compared with control HDL-2 in LXR-activated macrophages, due primarily to an increase in lecithin:cholesterol acyltransferase-mediated (LCAT-mediated) cholesteryl ester formation in media. Genetic knockdown or overexpression of ABCG1 showed that increased cholesterol efflux to CETP-D HDL was ABCG1 dependent. LCAT and apoE contents of CETP-D HDL-2 were markedly increased compared with control HDL-2, and increased cholesterol esterification activity resided within the apoE-HDL fraction. Thus, CETP-D HDL has enhanced ability to promote cholesterol efflux from foam cells in an ABCG1-dependent pathway due to an increased content of LCAT and apoE. PMID- 16670778 TI - Taiwan, global epidemics and the WHO. PMID- 16670777 TI - An Erythroid-Specific Chromatin Opening Element Increases beta-Globin Gene Expression from Integrated Retroviral Gene Transfer Vectors. AB - Gene therapy strategies requiring long-term high-level expression from integrated genes are currently limited by inconsistent levels of expression. This may be observed as variegated, silenced or position-dependent gene expression. Each of these phenomena involve suppressive chromatin structures. We hypothesized that by actively conferring an open chromatin structure on integrated vectors would increase transgene expression. To test this idea we used a 100bp element from the beta-globin locus control region (LCR) which is able to independently open local chromatin structure in erythroid tissues. This element includes binding sites for GATA-1, NF-E2, EKLF and Sp-1 and is evolutionarily conserved. We constructed a series of MSCV-based vectors containing the beta-globin gene driven by a minimal beta-globin promoter with combinations of the HSFE and LCR derived enhancer elements. Pools of MEL clones containing integrated vectors were analyzed for chromatin structure and beta-globin gene expression. The HSFE increased the extent of nuclease sensitive chromatin over the promoters of the constructs. The most effective vector included tandem copies of the HSFE and produced a 5-fold increase in expression compared to the promoter alone. These results indicate that the HSFE is able to augment the opening of beta-globin promoter chromatin structure and significantly increase gene expression in the context of an integrated retroviral vector. PMID- 16670779 TI - The Padayachee question. PMID- 16670781 TI - NTproBNP--a role to play. PMID- 16670782 TI - How equitable is the scaling up of HIV service provision in South Africa? PMID- 16670783 TI - Prominent doctors and scientists reject Lancet report on homoeopathy. PMID- 16670784 TI - Winning the war against HIV/AIDS. PMID- 16670785 TI - Dangers of DDT. PMID- 16670786 TI - Emergency medicine. PMID- 16670787 TI - Concurrent use of clopidogrel (Plavix) and atorvasatin (Lipitor) is contraindicated. PMID- 16670788 TI - Guidelines for medical practitioners on living wills prepared by the South African Medical Association. PMID- 16670789 TI - The fallacy of 'risk management'. PMID- 16670790 TI - Saving lives amid bullets and bombs. PMID- 16670791 TI - U-Mass re-investing where it matters. PMID- 16670792 TI - Proud legacy of a provincial AIDS chief. PMID- 16670793 TI - Expert witness system 'favours defendants'. PMID- 16670794 TI - Illegal topical corticosteroids in KwaZulu-Natal--abuse continues. PMID- 16670795 TI - Cystic fibrosis in black patients: Western Cape experiences. PMID- 16670796 TI - Ear-invading arthropods: a South African survey. PMID- 16670797 TI - The failure of HIV prevention is South Africa's biggest health crisis. PMID- 16670798 TI - The Criminal Law (Sexual Offences) Amendment Bill--implications of criminalising non-disclosure of HIV status. PMID- 16670799 TI - A case for integrating human rights in public health policy. PMID- 16670800 TI - Diagnosing cystic fibrosis in South Africa. PMID- 16670801 TI - Using multiple-cause mortality data to resolve conflicting information on trends in maternal mortality in South Africa. PMID- 16670802 TI - Contraceptive failure with the Mirena intrauterine system. PMID- 16670803 TI - Criminalising deliberate HIV transmission--is this good public health? PMID- 16670804 TI - Rapid scale-up of a community-based HIV treatment service: programme performance over 3 consecutive years in Guguletu, South Africa. AB - BACKGROUND: Despite rapid expansion of antiretroviral therapy (ART) in sub Saharan Africa there are few longitudinal data describing programme performance during rapid scale-up. METHODS: We compared mortality, viral suppression and programme retention in 3 consecutive years of a public sector community-based ART clinic in a South African township. Data were collected prospectively from establishment of services in October 2002 to the censoring date in September 2005. Viral load and CD4 counts were monitored at 4-monthly intervals. Community based counsellors provided adherence and programme support. RESULTS: During the study period 1139 ART-naive patients received ART (161, 280 and 698 in the 1st, 2nd and 3rd years respectively). The median CD4 cell counts were 84 cells/microl (interquartile range (IQR) 42-139), 89 cells/microl (IQR 490-149), and 110 cells/microl (IQR 55-172), and the proportions of patients with World Health Organization (WHO) clinical stages 3 and 4 were 90%, 79% and 76% in each sequential year respectively. The number of counsellors increased from 6 to 28 and the median number of clients allocated to each counsellor increased from 13 to 33. The overall loss to follow-up was .9%. At the date of censoring, the Kaplan-Meier estimates of the proportion of patients still on the programme were 82%, 86% and 91%, and the proportion who were virally suppressed (< 400 copies/ml) were 100%, 92% and 98% for the 2002, 2003 and 2004 cohorts respectively. CONCLUSIONS: While further operational research is required into optimal models of care in different populations across sub-Saharan Africa, these results demonstrate that a single community-based public sector ART clinic can extend care to over 1000 patients in an urban setting without compromising programme performance. PMID- 16670805 TI - Performance of a trained traditional bonesetter in primary fracture care. AB - BACKGROUND: In developing nations traditional bonesetters (TBSs) play a significant role in primary fracture care. However, despite high patronage the TBS remains an untrained quack whose practice is often associated with high morbidity. This study evaluated the performance of a trained TBS in primary fracture care. METHODS: Between 2002 and 2004 a prospective study was undertaken comparing the performance of a trained TBS with that of an untrained TBS at two separate locations. The two centres selected were both popular in traditional bone setting. A 1-day instructional course was given to the TBS at Afuje study centre, while the TBS at Ogua control centre received no instruction. The outcome of treatment of tibial shaft fractures at the two centres was evaluated and compared to assess the success of the course. RESULTS: There was a considerable decrease in the rate of gangrenous limbs, infection, non-union and malunion at the trained TBS centre compared with the untrained TBS centre (2.5% v. 10%, 5% v. 12.5%, 7.5% v. 15%, and 20.0% v. 30%, respectively). The observed difference between the trained and untrained TBSs was statistically significant (p < 0.05). CONCLUSION: It appears that training TBSs can reduce morbidity rates following TBS treatment. PMID- 16670806 TI - Socio-economic inequalities as a predictor of health in South Africa--the Yenza cross-sectional study. AB - OBJECTIVES: To describe the demographic, environmental and health characteristics of the rural Eastern Cape and to explore demographic and environmental predictors of health. DESIGN: Cross-sectional study. SETTING: Engcobo and Umtata in the Eastern Cape, South Africa. SUBJECTS: 12,049 people from 4608 households. OUTCOME MEASURES: (i) Children: immunisation history and being breastfed; (ii) women aged 15-45 years: use of contraception; (iii) women aged 20-25 years: total numbers of dead children; and (iv) all adults: smoking status, body mass index and blood pressure. RESULTS: 2741 houses (59.5%) were structurally adequate, 1795 (39.0%) had access to clean water, and 1174 (25.6%) had access to gas or electric energy. Of children up to 5 years of age 1436 (44.2%) were fully immunised and 2472 (76.1%) were breastfed in their first year of life. Among women aged 15-45 years, 903 (37.8%) used contraceptives, and among women aged 20-25 years, 56 (11.8%) had lost at least 1 child. Self-report of chronic illness and disability was low. Current smokers include 264 men (12.7%) and 325 women (6.0%), and 315 men (15.2%) and 1439 women (26.7%) had a body mass index > 30. Adequate housing (odds ratio (OR) 1.31, 95% confidence interval (CI): 1.12-1.53) and access to refuse disposal (OR 1.65, 95% CI: 1.36-2.00) were predictive of complete immunisation in children. Children living in houses with access to clean water (OR 0.8, 95% CI: 0.64-0.99) and a fitted toilet (OR 0.56, 95% CI: 0.38-0.83) were less likely to be breastfed in the first year of their life. Women aged 15-45 years were likely to use contraception if they were well educated (OR 2.75, 95% CI: 1.76-4.28) and lived in houses with access to refuse disposal (OR 1.46, 95% CI: 1.20-1.78). Better education was associated with a reduced likelihood of loss of a child (OR 0.11, 95% CI: 0.02-0.51) and being a smoker (OR 0.52, 95% CI: 0.38-0.73). Obesity was associated with being female (OR 1.88, 95% CI: 1.62-2.18) and living in a house with electricity (OR 1.46, 95% CI: 1.26-1.68). High blood pressure was associated with living in a house with electricity (OR 1.36, 95% CI: 1.10-1.70) and with monthly household income exceeding R2 000 (OR 1.38, 95% CI: 1.07-1.78). CONCLUSIONS: High socio-economic deprivation in the Eastern Cape is associated with health status comparable to that of poorer regions in sub-Saharan Africa. The educational level of the population, access to electricity, clean water and refuse disposal facilities are important predictors of child, maternal and adult health. PMID- 16670807 TI - Beta-lactamases in Enterobacteriaceae--an ever-present threat. PMID- 16670808 TI - South African hypertension guideline 2006. PMID- 16670809 TI - Pulmonary responses to nutritional restriction and hyperoxia in premature rabbits. AB - OBJECTIVES: To analyze the effects of nutritional restriction and hyperoxia on lung weight and pulmonary morphometry in premature rabbits during the first 11 days of life. METHODS: New Zealand White rabbits were delivered by C-section at 28 days' gestational age and randomized into four groups: control diet and room air, control diet and hyperoxia (> or = 95% O2), nutritional restriction and room air and nutritional restriction and hyperoxia (> or = 95% O2). Nutritional restriction was achieved by reducing all nutrients by 30% in comparison with the control diet. Lung tissue slides were stained with hematoxylin-eosin, modified resorcin-orcein and picrosirius, before morphometric analysis was performed. RESULTS: From the fourth day onwards, less weight was gained by the nutritional restriction and hyperoxia group (p < 0.001), and from the sixth day on, by the nutritional restriction and room air group (p < 0.001), in comparison with their respective control groups. Nutritional restriction decreased alveoli number (p < 0.001) and collagen deposition (p < 0.001). Hyperoxia was responsible for reductions in number of alveoli (p < 0.001) and collagen deposition (p < 0.001), in addition to higher mean linear intercept values (p < 0.05) and thickening of alveolar septa (p < 0.001). When nutritional restriction was associated with hyperoxia, the reductions in number of alveoli (p < 0.001) and of collagen deposition (p < 0.001) intensified. CONCLUSIONS: Nutritional restriction intensified the changes of pulmonary architecture findings caused by hyperoxia, in particular through alterations to alveolarization and collagen deposition. PMID- 16670810 TI - [Rare autoimmune rheumatic illnesses during pregnancy. Systemic sclerosis, polymyositis/dermatomyositis and vasculitis]. AB - Autoimmune rheumatic diseases (ARD) affect young females during childbearing age. Over the last decades, improvements in survival as well as quality of life in patients affected with ARD have led to an increased number of pregnancies observed during the course of such diseases. Systemic lupus erythematosus (SLE) is the most frequently observed ARD during pregnancy, and the immunoendocrine changes occurring during pregnancy may influence the course of this disease. Pregnancy can also occur in patients with rare ARD, namely systemic sclerosis, polymyositis/dermatomyositis, systemic vasculitis including Wegener's granulomatosis, Churg-Strauss syndrome, polyarteritis nodosa, microscopic polyangiitis, Takayasu arteritis and Behcet disease. This review focuses on the complications during pregnancy caused by these rare ARD, and we briefly discuss the data published on these disorders. Some guidelines for the management of these conditions during pregnancy will also be provided. However, it is important to note that data on pregnancy outcome are very limited and, in the absence of prospective studies, most of the information derives from case reports and retrospective studies. PMID- 16670811 TI - [Systematic lupus erythematosus and antiphospholipid syndrome during pregnancy]. AB - Women with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are subject to several complications during pregnancy, including reactivation of SLE, thrombosis, miscarriage, neonatal lupus, pregnancy-induced hypertension, pulmonary hypertension and drug toxicity. Correct management of these patients requires combined medical-obstetric care, close surveillance of baby's growth and well-being, control of SLE activity and correct thromboprophylaxis. With good care, most pregnancies in women with SLE and APS end successfully. PMID- 16670812 TI - [Pregnancy in patients with rheumatoid arthritis and inflammatory spondylarthropathies]. AB - The activity of a rheumatic disease can be influenced by pregnancy and puerperium. Prospective studies have shown an improvement in joint involvement in rheumatoid arthritis in two thirds to three quarters of pregnancies. After birth, an exacerbation is common. In spondylarthropathies there is no relevant change in disease activity. The fetal outcome is not impaired in patients with rheumatoid arthritis and inflammatory spondylarthropathies. Every pregnancy in women with a rheumatic disease should be considered as high-risk, and such pregnancies require close collaboration between rheumatologists and obstetricians. PMID- 16670814 TI - CSNB1 in Chinese families associated with novel mutations in NYX. AB - X-linked congenital stationary night blindness (CSNB) and NYX mutation have not been reported in Chinese. Here, two Chinese families with the complete form of CSNB (CSNB1) are presented. Linkage analysis of one family mapped the disease to Xp11-Xq13 where NYX is located. Sequence analysis of NYX identified two novel mutations, c.281G>C and c.302T>C, which would result in missense changes of p.Arg94Pro and p.Ile101Thr in the encoded protein. These two mutations were not found in 96 controls. The c.281G>C mutation cosegregated with nyctalopia and myopia. Our results expand the mutation spectrum of NYX and enrich the clinical information related to NYX mutation. The importance of associated myopia with NYX mutations is discussed. PMID- 16670813 TI - [Antirheumatic therapy and reproduction. The influence on fertility, pregnancy and breast feeding]. AB - Antirheumatic drugs can have a negative effect on reproduction in both men and women. Possible negative effects are impairment of fertility, harmful effects on the fetus and adverse effects on the breastfed child. In women non-steroidal antiinflammatory drugs (NSAID) and cyclophosphamide can impair fertility. In men infertility can result from the use of salazopyrine and cyclophosphamide. A desire for children should be taken into account before the start of disease modifying drugs (DMARD). Treatment with NSAID is possible at some stages of pregnancy as well as during lactation. A limited number of DMARD is compatible with pregnancy and is presented. Cytostatic drugs and leflunomide must be prophylactically withdrawn before a planned pregnancy. TNF alpha antagonists should be discontinued at the start of pregnancy. Safe birth control must be practised during therapy with drugs that are gonadotoxic or teratogenic. Treatment with immunosuppressive drugs during lactation is limited because of insufficient documentation of safety for the breastfed child. PMID- 16670815 TI - Genetic analysis of skeletal dysplasia: recent advances and perspectives in the post-genome-sequence era. AB - Skeletal dysplasia is a group of disorders of the skeleton that result from derangement of growth, development and/or differentiation of the skeleton. Nearly 300 disorders are included; most of them are monogenic diseases. Responsible genes for skeletal dysplasia have been identified in more than 150 diseases mainly through positional cloning. Identification of disease genes would improve patient care through genetic diagnosis as well as improving our understanding of the diseases and molecular mechanism of skeletal tissue formation. Studies of skeletal dysplasia would also help identify disease genes for common diseases affecting bones and joints. In this study, the author reviews recent advances and the current status of the genetic analysis of skeletal dysplasia and its impacts on research into skeletal biology. PMID- 16670816 TI - The development of wild immature Sumatran orangutans (Pongo abelii) at Ketambe. AB - Orangutans have the longest immature period and inter-birth interval of all ape species. This may be explained by a slow life history, the need to develop skills or by their relatively solitary lifestyle, which prevents a mother from associating with two offspring. This study of wild immature orangutans at the Ketambe Research Station, Indonesia, describes, with partly cross-sectional, partly longitudinal data, their development to independence. The study subjects ranged from 1 to 11 years of age. Data on their activity budget, diet, mother offspring proximity and maintenance of proximity, association with conspecifics and play behavior were collected. The results indicate that immature orangutans can provide for their own food and transport, and therefore were independent of direct maternal care, at an age of possibly 3 but more clearly 6 years. This is similar to chimpanzees, and refutes the slow life history hypothesis. Immature orangutans remain within their mother's vicinity until the age of 8 years, indicating a dependence on indirect maternal care, and this coincides with the period during which the mother does not produce another offspring. A female orangutan seems unable to associate with an older immature while caring for a new infant. This is consistent with the solitary-lifestyle hypothesis and corroborates the results obtained with the Sumatran orangutan population at Suaq Balimbing. However, why an immature depends indirectly on its mother for such a long period remains unclear. It is possible that it needs to develop ecological or social skills or needs the protection of its mother. Unfortunately, no data were available to distinguish between these possibilities. PMID- 16670817 TI - Distribution of Assamese macaques (Macaca assamensis) in the inner Himalayan region of Bhutan and their mtDNA diversity. AB - The distributions of Assamese macaques (Macaca assamensis) and rhesus macaques (M. mulatta) in Bhutan have been only partially documented. In order to investigate the distribution patterns of these species, we conducted field observation and genetic assessment with mitochondrial DNA (mtDNA) typing of macaques in the Inner Himalayas of Bhutan. There were 24 sightings of macaque groups, and all were visually identified as Assamese macaques. No groups of rhesus macaques were sighted in this survey area, in contrast with the survey results in the Nepalese Himalayas. Molecular phylogenetic analysis revealed that the Bhutan macaques are closer in proximity to their counterparts in the Indo Chinese region (Thailand and Laos) than to rhesus macaques in China, Laos and India. However, clustering results suggested the marked differentiation of the macaques in Bhutan from the Assamese macaques in Indo-China. We tentatively conclude that the macaques of the Inner Himalayan regions in Bhutan are Assamese macaques and that they appear to be of a lineage distinct from Assamese macaques in the Indo-Chinese region (subspecies M. a. assamensis). The degree of mtDNA diversity suggests that the Assamese macaques in Bhutan are of a more ancient ancestry than M. a. assamensis, thereby supporting the speciation hypothesis of the expansion of a sinica-group of macaques from South Asia to Southeast and then to East Asia (Fooden; Fieldiana Zool 45:1-44, 1988). Assignment of Assamese macaques in Bhutan to M. a. pelops is premature due to the lack of molecular data and recent taxonomic controversy. The mtDNA diversity of Assamese macaques was greater than that of rhesus macaques, suggesting the earlier speciation of Assamese macaques. The significance of the ecogeographic segregation model of macaque distribution is discussed in relation to the evolutionary range expansion into the Himalayan regions in South Asia. PMID- 16670818 TI - Regulation of cardiac energy metabolism in newborn. AB - Energy in the form of ATP is supplied from the oxidation of fatty acids and glucose in the adult heart in most species. In the fetal heart, carbohydrates, primarily glucose and lactate, are the preferred sources for ATP production. As the newborn matures the contribution of fatty acid oxidation to overall energy production increases and becomes the dominant substrate for the adult heart. The mechanisms responsible for this switch in energy substrate preference in the heart are complicated to identify due to slight differences between species and differences in techniques that are utilized. Nevertheless, our current knowledge suggests that the switch in energy substrate preference occurs due to a combination of events. During pregnancy, the fetus receives a constant supply of nutrients that is rich carbohydrates and poor in fatty acids in many species. Immediately after birth, the newborn is fed with milk that is high in fat and low in carbohydrates. The hormonal environment is also different between the fetal and the newborn. Moreover, direct subcellular changes occur in the newborn period that play a major role in the adaptation of the newborn heart to extrauterin life. The newborn period is unique and provides a very useful model to examine not only the metabolic changes, but also the effects of hormonal changes on the heart. A better understanding of developmental physiology and metabolism is also very important to approach certain disorders in energy substrate metabolism. PMID- 16670819 TI - Physiological and pathological roles of a multi-ligand receptor CD36 in atherogenesis; insights from CD36-deficient patients. AB - Oxidized low density lipoprotein (LDL) (Ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Oxidized LDL is taken up by macrophages via scavenger receptors. CD36 is an 88 kDa glycoprotein expressed on platelets, monocyte-macrophages, microvascular endothelial cells, adipose tissue, skeletal muscles and heart. We found patients with CD36 deficiency and identified several mutations in the CD36 gene. We also reported that CD36-deficient macrophages showed a 50% reduction in the binding of Ox-LDL, suggesting that CD36 is one of the major receptors for Ox-LDL. CD36 was expressed on macrophages in the atherosclerotic lesions of human aorta and coronary arteries especially on foamed macrophages. The distribution of CD36 expression was slightly different from that of scavenger receptor class A types I and II. The expression of CD36 on macrophages was up-regulated by Ox-LDL and down-regulated by interferon gamma. Since CD36 is a transporter of long-chain fatty acids (LCFA), CD36-deficient patients showed a defect in the uptake of an LCFA analog, BMIPP, by the heart. Furthermore, the secretion of IL-1beta and TNF-alpha from monocyte-derived macrophages induced by Ox-LDL was markedly reduced and the activation of NF kappaB was attenuated in CD36-deficient subjects compared with controls, suggesting that CD36-mediated signaling is also impaired in CD36 deficiency. To elucidate the roles of CD36 in vivo, we characterized the clinical profile of CD36-deficient patients. Most of them were accompanied by hyperlipidemia (mainly hypertriglyceridemia), increased remnant lipoproteins and mild elevation of fasting plasma glucose level and blood pressure. Glucose clamp technique revealed mean whole body glucose uptake was reduced in CD36-deficient patients, indicating the presence of insulin resistance. The frequency of CD36 deficiency was higher in patients with coronary heart disease (CHD) than in control subjects. Taken together, CD36 deficiency is accompanied by (1) hyperlipidemia and increased remnant lipoproteins, (2) impaired glucose metabolism based upon insulin resistance, and (3) mild hypertension, and comprises one of the genetic backgrounds of the metabolic syndrome, leading to the development of CHD. PMID- 16670820 TI - Expression of inhibin/activin subunits alpha (-alpha), beta A (-beta (A)) and beta B (-beta (B)) in placental tissue of normal and intrauterine growth restricted (IUGR) pregnancies. AB - During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (beta (A) or beta (B)). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -alpha, -beta (A), -beta (B). Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-alpha subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-alpha immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-beta (B) subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin alpha and the beta-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling. PMID- 16670823 TI - Bilateral spontaneous pneumothorax in a patient with pulmonary rheumatoid nodules, secondary infected by Aspergillus. AB - This case report describes a 50-year-old woman with rheumatoid arthritis (RA) in whom nodular opacities were found on chest X-ray. She developed a bilateral spontaneous pneumothorax treated with surgical pleurodesis. Cultures remained negative. Histological examination of specimens confirmed the clinical diagnosis of rheumatoid granulomata. Therefore, corticosteroid therapy was started, after which the nodules decreased slightly in size and inflammatory parameters normalized. Three months later, she presented with respiratory insufficiency based on pulmonary fungus infection. Differential diagnosis between rheumatoid nodules and granulomas caused by Aspergillus is difficult in RA patients with pulmonary nodular lesions; in this case, both complications appeared subsequently. PMID- 16670821 TI - Parity violating energetic difference and enantiomorphous crystalsp-caveats; reinvestigation of tyrosine crystallization. AB - The present article challenges reports claiming to have demonstrated the Parity Violating Energetic Difference (PVED) between enantiomorphous D- and L-crystals. Apart from PVED, the presence of minute quantities and differing profiles of impurities incorporated during their different history of preparation will affect the physical properties of D- and L-crystals. These impurities are anticipated to play a much greater role in affecting crystallization behavior than PVED. The effect of impurities on the growth and dissolution of enantiomorphous crystals is illustrated with some representative examples. Shinitzky et al. (2002) reported recently dramatic differences in the growth and dissolution properties of the D- and L-crystals of tyrosine. We have repeated these experiments using commercial samples from different sources and employing a validated enantioselective gas chromatographic technique. We attribute Shinitzky's findings either to the use of inappropriate analytical techniques for the determination of enantiomeric composition and/or to the presence of unidentified contaminants in the commercial tyrosine samples. Related caveats hold also for the recently published claims by Shinitzky (2006) and Scolnik et al. (2006) to have observed experimentally PVED between enantiomeric helices of poly-glutamic acid composed of 24 repeating units. PMID- 16670824 TI - Radiographic study of joint destruction patterns in the rheumatoid elbow. AB - Knowledge of the pattern of joint destruction is important for planning the therapeutic approach to rheumatoid arthritis (RA) of the elbow. Accordingly, we carried out a large-scale radiographic study with the objective of elucidating the joint destruction pattern in rheumatoid elbows. From 2001 through 2003, we examined and took plain X-rays of both elbows of 193 RA patients (i.e., 386 elbows), consisting of 18 men and 175 women, with a mean age of 57.0 years. Radiographic images of the elbow joints were used to classify the degree of bone loss in various zones on the elbow joint surface into four grades of severity, and joint destruction was compared between the left and right elbows. In addition, correlation in the extent of bone loss between each of the zones of the same elbow and differences in the extent of bone loss were analyzed statistically. The results showed direct correlations for destruction of the elbow joint surface among the zones for the left and right elbow joints and in the same elbow joint. However, more severe destruction was observed on the radial side of the humeral trochlea, and it was surmised that destruction of the elbow joint must begin at that site and gradually spread mediolaterally. In addition, in the same elbow joint, the correlation in the degree of bone loss between the trochlea of humerus and the trochlear notch was especially strong, indicating that the bone destruction at both sites represented mirror lesions. We conclude that when performing radiographic diagnosis of the joint damage in the rheumatoid elbow, knowledge of this pattern of joint destruction will be useful for assessing whether there is joint destruction in the initial stage and for deciding the therapeutic approach. PMID- 16670826 TI - Strategies for the prevention and control of osteoporosis in developing countries. AB - The frequency of osteoporosis and fragility fractures has been studied to a very limited extent in few developing countries. The aim of this paper is to review briefly the burden of osteoporosis and fragility fractures in these countries and to propose some strategies for the prevention and control of those conditions, considering barriers and facilitators for their implementation. The evolution of the demographic composition in most regions with developing countries shows a considerable increase in life expectancy and therefore, a significant growth in elderly population can be expected. Reports on the incidence of fragility fractures show figures in many of those countries that are comparable to those found in developed nations. Health resources (for acute treatment of fractures, their rehabilitation and chronic management, for diagnostic centers and drug therapy for osteoporosis) are limited in most of those regions and are allocated to other health priorities. Internationally accepted guidelines can be adapted to the realities of developing nations and may be promoted by organizations of health professionals and patients, but require endorsement and support by health authorities. The steps should include: (a) campaigns to increase awareness, both among the population at risk and relevant health workers; (b) the promotion of a preventive lifestyle in the general population; (c) the development of national or regional, evidence-based guidelines for the diagnosis and treatment of osteoporosis; (d) development and implementation of guidelines for the treatment of fragility fractures, their rehabilitation and prevention of falls; (e) collection of economic data on fractures and osteoporosis; and (f) development of country-specific fracture databases. These steps may help in reducing the increasing burden of osteoporotic fractures. Their implementation will require solid scientific basis and commitment from policy makers, health professionals, patient organizations, and ultimately the general population. PMID- 16670825 TI - Prevention and treatment strategies for glucocorticoid-induced osteoporotic fractures. AB - Glucocorticoids are the most common cause of drug-related osteoporosis. We reviewed current evidence on risk factors for glucocorticoid-induced osteoporosis (GIOP) and prevention and treatment of GIOP-related fractures. Guidelines for GIOP management published since 2000 were also reviewed. Significant bone loss and increased fracture risk is seen with daily prednisone doses as low as 5 mg. Alternate-day glucocorticoid therapy can lead to similar bone loss. No conclusive evidence exists for a safe minimum dose or duration of glucocorticoid exposure. Physicians should consider risk factors for involutional osteoporosis such as older age, postmenopausal status, and baseline bone density measurements as they assess patients for prevention or treatment of GIOP. Bisphosphonates were reported to reduce GIOP-related vertebral fractures, but inconclusive data exist for hip fractures associated with glucocorticoid use. Hormone replacement therapy and parathyroid hormone analogs are effective in preserving bone density in GIOP. The risk of osteoporosis and fractures should be routinely assessed in patients receiving glucocorticoid therapy. Effective prevention and treatment options are available and can result in meaningful reduction of GIOP-related morbidity and mortality. Current guidelines for GIOP management recommend bisphosphonates, especially alendronate and risedronate, as first-line agents for GIOP, and these guidelines propose the preventive use of bisphosphonates early in the course of glucocorticoid therapy in high-risk patient subgroups. PMID- 16670827 TI - Use of antibodies recognizing cyclic citrullinated peptide in the differential diagnosis of joint involvement in systemic sclerosis. AB - OBJECTIVE: To determine the prevalence of anti-cyclic citrullinated peptide (CCP) antibodies in systemic sclerosis (SSc) and to assess any association between the presence of anti-CCP, radiographic features, and clinical manifestations. MATERIALS AND METHODS: Anti-CCP antibodies and rheumatoid factor (RF) were tested in serum samples from 75 patients with SSc (64 women and 11 men), with a mean age of 59.4 years (range 24-85) with either diffuse (dcSSc) and limited (lcSSc) cutaneous involvement. As a control group, 22 age- and sex-matched healthy controls (HCs) were examined. Standard radiographs of the hands and wrists were examined in each patient. RESULTS: The presence of anti-CCP was found in sera of 10.6% (8/75) patients with SSc (lcSSc 3 of 44, 6.8%; dcSSc 5 of 31, 16.1%). None of the HCs was positive for anti-CCP. The positivity of RF was observed in 19 of 75 (25.3%) SSc patients (lcSSc 10 of 44, 22.7%; dcSSc 9 of 31, 29%). Statistically significant association was found between anti-CCP positivity and the presence of arthritis (p<0.0001) and marginal erosions (p=0.001). CONCLUSION: Our data show that joint involvement is a common presenting feature of SSc. In this report, we show that anti-CCP antibodies can be detected also in patients with SSc, but they are generally less commonly present than in adults with rheumatoid arthritis (RA). Thus, the finding of high titers of anti-CCP antibodies may help to define the diagnosis of overlap syndrome SSc/RA and facilitate diagnosis and appropriate treatment. PMID- 16670828 TI - Predictive value of antibodies to cyclic citrullinated peptide in patients with early arthritis. AB - The objective of this study was to determine the diagnostic value for rheumatoid arthritis (RA) of antibodies to cyclic citrullinated peptides (anti-CCP) in patients with early arthritis and vasculitis. Sixty-four adult patients with early arthritis and disease duration of less than 4 months were clinically diagnosed by an experienced rheumatologist as having RA (n=27), spondyloarthropathy (n=11), and undifferentiated arthritis (n=26). Eighteen patients with vasculitis were also included in the study. The patients with early arthritis were followed up for 9 months. After the follow-up period, five of 26 patients with undifferentiated arthritis were diagnosed as having RA. All serum samples were tested for anti-CCP and IgM rheumatoid factor (IgM-RF). The anti-CCP positivity in RA patients (44.4%) was significantly more frequent than in patients with undifferentiated arthritis (3.8%), spondyloarthropathy (0%), and vasculitis (5.6%) (p=0.001, p<0.01, and p<0.01, respectively). The frequency of IgM-RF positivity was 40.7% in RA, 7.7% in undifferentiated arthritis, 0% in spondyloarthropathy, and 22.2% in vasculitis groups. The respective specificity of anti-CCP and IgM-RF tests for early RA were 97.3 and 94.6%, and the respective sensitivity of them were 44.4 and 40.7%, respectively. The combination of anti CCP and IgM-RF positivity had a very high specificity and positive predictive value (100%) but a rather low sensitivity (33.3%). When either anti-CCP or IgM-RF positivity combined into one criterion, the sensitivity became high (51.9%) but the specificity decreased to 91.9%. Overall performance of anti-CCP test alone for the early RA was higher than IgM-RF and the combination of anti-CCP and IgM RF (p<0.05), and was similar to the combination of anti-CCP or IgM-RF. The specificity of positive anti-CCP test for diagnosis of established RA reached up to 100%. In conclusion, the anti-CCP test is a new diagnostic test with extremely high specificity for RA. Anti-CCP antibody testing combined with IgM-RF testing has additional value over IgM-RF testing alone in patients with early arthritis. PMID- 16670829 TI - Rheumatic manifestations of psoriasis. AB - Psoriatic arthritis was described as a distinct rheumatic disease in the 1960s, and subsequently grouped among the spondyloarthropathies. Recently, other rheumatic manifestations of psoriasis, such as enthesopathy and osteoperiostitis, were recognized. This study attempts to examine the rheumatological and radiological manifestations of Psoriasis and their association with skin and nail disease. Eighty-one psoriatic outpatients were interviewed consecutively during 6 months. Questionnaires and indices were carried out to assess the extent and severity of skin and nail involvement, as well as the activity and severity of peripheral and axial rheumatic manifestations. Radiological examination of the hands, feet, spine and pelvis was also done for all patients. Fifty-nine psoriatic outpatients (73%) had rheumatic manifestations clinically and/or radiologically (Psoriatic arthropathy "PsA"). Clinical peripheral arthritis was found in 14 (23.7%) of the patients with PsA, being oligoarticular in 11, polyarticular in two, and exclusively of the distal interphalangeal (DIP) joints in one patient. Sacroiliitis and/or spondylitis were found in 38 (64.4%), enthesopathy in 36 (61%), dactylitis in two (3.3%), radiological DIP involvement in 24 (40.6%), and radiological osteoperiostitis in 49 (83%) of patients with PsA. Most PsA patients had more than one rheumatic manifestation, while four patients (6.7%) had isolated enthesopathy without any other rheumatic manifestations. Subungual hyperkeratosis of the nails was significantly correlated with PsA (p<0.05), as well as with clinical arthritis, enthesopathy, and DIP involvement (p<0.01), while other types of skin and nail lesions were correlated with selected rheumatic manifestations. The performance of existing criteria for PsA was poor, as individual sets favored either sensitivity or specificity. Psoriatic arthropathy (PsA), occurring in about three-quarters of hospital outpatients with psoriasis, is more common than previously thought. More sensitive and specific criteria for the diagnosis and classification of PsA need to be developed, taking into account the recently described clinical and radiological manifestations. PMID- 16670830 TI - Action potential simulation (APS) in patients with fibromyalgia syndrome (FMS): a controlled single subject experimental design. AB - OBJECTIVES: Action potential simulation (APS) is becoming a popular method of pain reduction. Nevertheless, little is known about the efficacy of this relatively new treatment. The aim of this study was to investigate whether APS helps to reduce pain, improves patients' perception of daily functioning and social participation in patients with fibromyalgia syndrome (FMS). MATERIALS AND METHODS: Ten patients with FMS according to the American College of Rheumatology (ACR) criteria entered this double blind crossover single-case study. In a period of 20 weeks, the patients underwent two treatment periods of 4 weeks, one with verum and one with placebo, at random, in a double blind fashion. Outcome measures were evaluated on a weekly basis. Primary outcome measure was pain measured with the Fibromyalgia Impact Questionnaire (FIQ) questions 4 and 5, the number of tender points and the total tender point pain intensity score. Both visual inspection and statistical analysis were done to analyse the data from this single-subject design. RESULTS: Performing visual inspection and statistical analysis, no positive results of the APS treatment were found in this study. Remarkable is the fact that placebo APS had significantly better results than verum APS. CONCLUSIONS: In this single-case study with ten patients (all female), APS was not a helpful method to reduce pain, to improve patients' perception of daily functioning and social participation in patients with FMS. PMID- 16670831 TI - Intravenous immunoglobulin in the treatment of resistant subacute cutaneous lupus erythematosus: a possible alternative. AB - Subacute cutaneous lupus erythematosus (SCLE) is a common manifestation of systemic lupus erythematosus. In many cases it appears to be resistant to various systemic or topical treatments. Three cases of resistant SCLE with good response to intravenous immunoglobulin (IVIG) are described here suggesting that IVIG could be an alternative treatment in these patients. PMID- 16670838 TI - Intrasellar germinoma treated with low-dose radiation. AB - A radiation dose lower than what had previously been recommended was given to a case of intrasellar germinoma in a 17-year-old patient who suffered from migraine like headache for 2 years, amenorrhoea for 3 months and a body-weight loss of 10 kg over 4 months. Baseline assessment of pituitary hormone reserve was within the lower limits of the normal range, except for an elevated serum prolactin level (PRL). Magnetic resonance imaging (MRI) showed an abnormal, slightly enhanced mass in the pituitary fossa, extending along the pituitary stalk and to the hypothalamus. Transsphenoidal removal of the intrasellar part of the tumour was performed and microscopic sections of the surgical specimens revealed a pure germinoma. Adjunctive radiation therapy (RT) was given 3 weeks after surgery. A total dose of only 27 Gy was delivered to this patient. The patient's menstrual cycles resumed in 4 months. Repeated MRI follow-up showed no recurrence of the tumour. PMID- 16670839 TI - Somatomotor functional MRI in a hypertensive arachnoid cyst. AB - In this article the authors report the study by functional MRI, before and after surgery, of the motor cerebral cortex surrounding a large hypertensive arachnoid cyst. They stress that the functional modifications due to surgery are more relevant than suggested by the simple morphological data. PMID- 16670840 TI - Reliability and responsiveness of the Dutch version of the Neck Disability Index in patients with acute neck pain in general practice. AB - A prospective cohort study with a 1 week follow-up. To examine the reliability and responsiveness of the Dutch version of the Neck Disability Index (NDI) in patients with acute neck pain in general practice. An increasing number of studies on treatment options is published in which the NDI is used. Reports of the ability of the NDI to detect change over time, often called responsiveness, however have not yet been published. At baseline 187 patients (119 women, 68 men) were included. They completed a questionnaire on demographic variables, self reported cause of their complaints and the NDI. After 1 week, 86 patients were sent the NDI again together with the perceived recovery scale which was used as our external criterion. The scale ranged from 1 (complete recovery) to 7 (complaints are worse than ever). Response rate was 93%. Test-retest scores on reliability were good (ICC = 0.90). A Bland and Altman plot and a graph of total sum score differences showed no visible tendency towards unequal spreading of the data. For patients that reported on the perceived recovery scale that they were "stable" we found a responsiveness ratio of 1.82. The standard error of measurement (SEM) was 0.60 what resulted in a minimal detectable change (MDC) of 1.66. The NDI has shown to be a reliable and responsive instrument in patients with acute neck pain in general practice. PMID- 16670846 TI - Catheterization of the hepatic artery via the left common carotid artery in rats. AB - The commonly used approach for rat hepatic artery catheterization is via the gastroduodenal artery, which is ligated after the procedure. A new method of rat hepatic artery catheterization via the left common carotid artery (LCCA) is described. The LCCA is repaired after catheterization. The catheterization procedures included the following: (1) opening the rat's abdominal cavity and exposing the portion of abdominal aorta at the level of the celiac trunk; (2) separating and exposing the LCCA; inserting a microguidewire and microcatheter set into the LCCA via an incision; after placement into the descending aorta, the microguidewire and microcatheter are maneuvered into the hepatic artery under direct vision; (3) after transcatheter therapy, the catheter is withdrawn and the incision at the LCCA is repaired. This technique was employed on 60 male Sprague Dawley rats with diethylnitrosamine-induced liver cancer, using a 3F microguidewire and microcatheter set. Selective hepatic artery catheterization was successfully performed in 57 rats. One rat died during the operation and five rats died within 7 days after the procedure. It is envisaged that as experience increases, the catheterization success rate will increase and the death rate will decrease. A new approach for selective hepatic artery catheterization via the LCCA in rats is introduced, which makes repeat catheterization of this artery possible and allows large embolization particles to be delivered by using a 3F catheter. PMID- 16670849 TI - Intra-abdominal erosion of artificial bowel sphincter reservoir. PMID- 16670842 TI - Mesocortical dopamine modulation of executive functions: beyond working memory. AB - RATIONALE: Dopamine (DA) neurotransmission in the prefrontal cortex (PFC) is known to play an essential role in mediating executive functions such as the working memory. DA exerts these effects by acting on D1 receptors because blockade or stimulation of these receptors in the PFC can impair performance on delayed response tasks. However, comparatively less is known about dopaminergic mechanisms that mediate other executive functions regulated by the PFC. Furthermore, the functional importance of other DA receptor subtypes that reside on PFC neurons (D2 and D4) is unclear. OBJECTIVES: This review will summarize previous findings and previously unpublished data addressing the contribution of PFC DA to higher-order cognition. We will compare the DA receptor mechanisms, which regulate executive functions such as working memory, behavioral flexibility, and decision-making. RESULTS AND CONCLUSIONS: Whereas PFC D1 receptor activity is of primary importance in working memory, D1 and D2 receptors act in a cooperative manner to facilitate behavioral flexibility. We note that the principle of the "inverted U-shaped" function of D1 receptor activity mediating working memory does not necessarily apply to other PFC functions. DA in different subregions of the PFC also mediates decision-making assessed with delay discounting or effort-based procedures, and we report that D1, D2, and D4 receptors in the medial PFC contribute to decision-making when animals must bias the direction of behavior to avoid aversive stimuli, assessed with a conditioned punishment procedure. Thus, mesocortical DA modulation of distinct executive functions is subserved by dissociable profiles of DA receptor activity in the PFC. PMID- 16670850 TI - Diagnostic significance of gently sloping depression and irregular margin in superficial elevated colorectal tumors. AB - INTRODUCTION: Recently, superficial elevated colorectal tumors have been increasingly diagnosed after improvements in endoscopic instruments and techniques. However, their biological characteristics remain obscure and it is difficult to predict malignant potential. The aim of this study is to clarify the characteristics of superficial elevated tumors in endoscopic examination for the evaluation of malignant potential. MATERIALS AND METHODS: Sixty-three resected superficial elevated colorectal tumors more than 10 mm in diameter were analyzed with regard to their morphological characteristics and histological findings. The samples were classified according to the presence of a gently sloping depression and irregular margin at the edge. Their depth of vertical invasion and the degree of depression were examined. RESULTS: The rate of carcinoma in 27 lesions with a gently sloping depression was significantly higher than in 36 lesions with an even surface. The rate of carcinoma in 46 lesions with irregular margin was significantly higher than in 17 lesions without irregular margin. A multivariate analysis revealed that the coexistence of both IM and GSD was significantly associated with submucosal invasion. Statistical associations of age, tumor location, gender, and pathological grade with submucosal invasion were not observed. CONCLUSIONS: In superficial elevated colorectal tumors, a gently sloping depression and irregular margin at the edge when viewed endoscopically may be a predictor of malignant potential. These characteristics should be given priority when deciding on treatment. PMID- 16670851 TI - Patient and caregiver time burden associated with anaemia treatment in different patient populations. AB - GOALS: Cancer patients treated with chemotherapy often develop anaemia. This cross-sectional analysis examined the effect of anaemia treatment on patient and caregiver time and activities. MATERIALS AND METHODS: The analysis included 9,920 patients from 646 US outpatient oncology centres. Patients completed a survey that contained questions about travel time, total time for the visit and other impacts. RESULTS: The mean time taken for a single clinic visit to receive anaemia treatment was 2.2 h. On average, patients receiving epoetin alfa required 17.6 h more than patients receiving darbepoetin alfa to complete a course of anaemia treatment. All patients in the study reported that they had to adjust at least one activity as a result of clinic visits. Older patients, women and patients from low-income areas were more likely to be accompanied during clinic visits. CONCLUSIONS: Reducing the number of clinic visits needed for anaemia treatment by using darbepoetin alfa may benefit patients. PMID- 16670857 TI - Clinical evidence for lead-induced inhibition of nitric oxide formation. AB - Lead exposure has been associated with increased cardiovascular risk, which may result, at least in part, from lead-induced increases in oxidative stress and depressed nitric oxide (NO) availability. However, no previous clinical study has examined whether lead exposure is associated with significant effects on biomarkers of NO activity (plasma nitrites, nitrates, and cyclic guanosine 3',5' monophosphate; cGMP). We investigated whether there is an association between the circulating concentrations of nitrites, nitrates, and cGMP and the concentrations of lead in whole blood (B-Pb) or plasma (P-Pb) from 62 lead-exposed subjects (30 men and 32 women). P-Pb was determined by inductively coupled plasma mass spectrometry and B-Pb by graphite furnace atomic absorption spectrometry. Plasma nitrite and nitrate concentrations were measured using an ozone-based chemiluminescence assay. Plasma cGMP concentrations were measured using a commercial enzyme immunoassay. We found a negative correlation between plasma nitrite and B-Pb concentrations (r = -0.358; P = 0.004), and between plasma nitrite and P-Pb concentrations (r = -0.264; P = 0.038), thus suggesting increased inhibition of NO formation with increasing B-Pb or P-Pb concentrations. However, no significant correlations were found between plasma nitrate or cGMP and B-Pb or P-Pb concentrations (all P > 0.05). These findings suggest a significant inhibitory effect of lead exposure on NO formation and provide clinical evidence for a biological mechanism possibly involved the association between lead exposure and increased cardiovascular risk. PMID- 16670858 TI - Flexible flatfoot and related factors in primary school children: a report of a screening study. AB - The aim of this study was to analyze the longitudinal arch morphology and related factors in primary school children. Five hundred and seventy-nine primary school children were enrolled in the study. Generalized joint laxity, foot progression angle, frontal hindfoot alignment, and longitudinal arch height in dynamic position were evaluated. The footprints were recorded by Harris and Beath footprint mat and arch index of Staheli was calculated. The mean age was 9.23 +/- 1.66 years. Four hundred and fifty-six children (82.8%) were evaluated as normal and mild flexible flatfoot, and 95 children (17.2%) were evaluated as moderate and severe flexible flatfoot. The mean arch indices of the feet was 0.74 +/- 0.25. The percentage of flexible flatfoot in hypermobile and non-hypermobile children was found 27.6 and 13.4%, respectively. There was a statistically significant difference in dynamic arch evaluation between hypermobile and non hypermobile children. There was a significant negative correlation between arch index and age, and a significant negative correlation between hypermobility score and age. Our study confirms that the flexible flatfoot and the hypermobility are developmental profiles. PMID- 16670859 TI - Telemedical support to improve glycemic control in adolescents with type 1 diabetes mellitus. AB - INTRODUCTION: In this paper, we evaluated the feasibility of a telemedical (TM) support program and its effect on glycemic control in adolescents with type 1 diabetes mellitus (T1DM). Thirty-six adolescents (m=20, median age at the start of the study: 15.3 years (range: 10.7-19.3 years), median age at diagnosis: 9.3 years (2.1-13.8 years), median duration of disease: 6.4 years (1.0-12.8 years), HbA1c>8%, all on intensified insulin therapy) were randomized in a crossover trial over 6 months (3 months with TM, 3 months with conventional support and paper diary (PD)). During the TM phase, the patients sent their data (date, time, blood glucose, carbohydrate intake, insulin dosage) via mobile phone, at least daily, to our server and diabetologists sent back their advice via short message service (SMS) once a week. RESULTS: Glycemic control improved during the TM phase, while it deteriorated during the PD phase: TM-PD group HbA1c (%, median (range)): 9.05 (8-11.3) (at 0 months), 8.9 (6.9-11.3) (at 3 months), and 9.2 (7.4 12.6) (at 6 months), and PD-TM group: 8.9 (8.3-11.6), 9.9 (8.1-11), and 8.85 (7.3 11.7) (p<0.05). Patients rated the TM support program to be a good idea. Technical problems with General Packet Radio Service (GPRS) data transmission led to data loss and decreased patient satisfaction. CONCLUSION: Our telemedical support program, VIE-DIAB, proved to be feasible in adolescents and helped to improve glycemic control. PMID- 16670860 TI - Brugada syndrome in childhood: a potential fatal arrhythmia not always recognised by paediatricians. A case report and review of the literature. AB - We report on a youngster followed by his paediatrician from birth until 14 years of age for premature beats, most likely of ventricular origin. The sudden death of his sister provoked a re-assessment of his electrocardiograms (ECG), resulting in the diagnosis of Brugada syndrome and the subsequent implantation of a cardioverter defibrillator. This syndrome is a well known entity in adult cardiology, first described by Brugada and Brugada in 1992. It is considered to be the second most common cause of death in young adults after road traffic accidents. In children, however, the Brugada syndrome is not well known and we believe to be certainly underdiagnosed. PMID- 16670861 TI - Netherton syndrome: report of identical twins presenting with severe atopic dermatitis. AB - We report the cases of 4-year-old identical twin sisters who presented with severe atopic dermatitis with intractable skin manifestations and multiple food allergies. Netherton syndrome (NS) (OMIM 256500) was suspected due to very high serum IgE levels, growth retardation, severe food allergies and typical hair finding (trichorrhexis invaginata). A definite diagnosis was made by genetic analysis. Our cases are unique in being the first identical twins with NS diagnosed by a novel mutation in the SPINK5 gene. NS should be considered in differential diagnosis in children who have generalized erythema with intractable eczematous lesions and elevated levels of IgE. PMID- 16670863 TI - Breast imaging. Preoperative breast cancer staging: comparison of USPIO-enhanced MR imaging and 18F-fluorodeoxyglucose (FDC) positron emission tomography (PET) imaging for axillary lymph node staging--initial findings. AB - Magnetic resonance (MR) imaging after ultra-small super paramagnetic iron oxide (USPIO) injection and 18F-fluorodeoxyglucose positron emission tomography (FDG PET) for preoperative axillary lymph node staging in patients with breast cancer were evaluated using histopathologic findings as the reference standard. USPIO enhanced MR and FDG-PET were performed in ten patients with breast cancer who were scheduled for surgery and axillary node resection. T2-weighted fast spin echo, T1-weighted three-dimensional (3D) gradient echo, T2*-weighted gradient echo and gadolinium-enhanced T1-weighted 3D gradient echo with spectral fat saturation were evaluated. MR imaging before USPIO infusion was not performed. The results were correlated with FDG-PET (acquired with dedicated PET camera, visual analysis) and histological findings. The histopathologic axillary staging was negative for nodal malignancy in five patients and positive in the remaining five patients. There was one false positive finding for USPIO-enhanced MR and one false negative finding for FDG-PET. A sensitivity (true positive rate) of 100%, specificity (true negative rate) of 80%, positive predictive value of 80%, and negative predictive value of 100% were achieved for USPIO-enhanced MR and of 80%, 100%, 100%, 80% for FDG-PET, respectively. The most useful sequences in the detection of invaded lymph nodes were in the decreasing order: gadolinium enhanced T1-weighted 3D gradient echo with fat saturation, T2*-weighted 2D gradient echo, T1-weighted 3D gradient echo and T2-weighted 2D spin echo. In our study, USPIO-enhanced T1 gradient echo after gadolinium injection and fat saturation emerged as a very useful sequence in the staging of lymph nodes. The combination of USPIO-enhanced MR and FDG-PET achieved 100% sensitivity, specificity, PPV and NPV. If these results are confirmed, the combination of USPIO MR with FDG-PET has the potential to identify the patient candidates for axillary dissection versus sentinel node lymphadenectomy. PMID- 16670864 TI - Dissection of the V4 segment of the vertebral artery: clinicoradiologic manifestations and endovascular treatment. AB - Intracranial vertebral artery (VA) dissection has three clinical presentations: ischemia, hemorrhage, and mass effect. Imaging findings of intracranial VA dissections vary according to clinical presentation. Irregular stenosis or occlusion of the VA is the most common finding in patients with posterior fossa infarction, whereas a dissecting aneurysm is the main feature in those with acute subarachnoid hemorrhage. A chronic, giant, dissecting aneurysm can cause mass effect on the brain stem or cranial nerves, as well as distal embolism. Magnetic resonance imaging is useful for detection of intramural hematomas and intimal flaps, both of which are diagnostic of VA dissection. Multidetector computed tomography angiography is increasingly used for diagnosis of VA dissection. Catheter angiography is still beneficial for evaluation of precise endoluminal morphology of the dissection before surgical or endovascular intervention. Endovascular treatment is now considered a major therapeutic option for patients with a ruptured dissecting aneurysm or a chronic dissecting aneurysm. Anticoagulation therapy is currently considered the initial treatment of choice in patients with posterior circulation ischemic symptoms. Endovascular treatment, such as stent-assisted angioplasty or coil occlusion at the dissection site, can be performed in selected patients with posterior fossa ischemic symptoms. PMID- 16670865 TI - Magnetic resonance cardiac perfusion imaging-a clinical perspective. AB - Coronary artery disease (CAD) with its clinical appearance of stable or unstable angina and acute myocardial infarction is the leading cause of death in developed countries. In view of increasing costs and the rising number of CAD patients, there has been a major interest in reliable non-invasive imaging techniques to identify CAD in an early (i.e. asymptomatic) stage. Since myocardial perfusion deficits appear very early in the "ischemic cascade", a major breakthrough would be the non-invasive quantification of myocardial perfusion before functional impairment might be detected. Therefore, there is growing interest in other, target-organ-specific parameters, such as relative and absolute myocardial perfusion imaging. Magnetic resonance (MR) imaging has been proven to offer attractive concepts in this respect. However, some important difficulties have not been resolved so far, which still causes uncertainty and prevents the broad application of MR perfusion imaging in a clinical setting. This review explores recent technical developments in MR hardware, software and contrast agents, as well as their impact on the current and future clinical status of MR imaging of first-pass myocardial perfusion imaging. PMID- 16670866 TI - Coronary artery bypass grafts: assessment of graft patency and native coronary artery lesions using 16-slice MDCT. AB - The objective of this study was to evaluate the accuracy of electrocardiography (ECG)-gated 16-slice multidetector-row computed tomography (MDCT) in detection of stenosis of bypass grafts and native coronary arteries in patients who have undergone coronary artery bypass grafting (CABG). ECG-gated contrast-enhanced MDCT using 12 x 0.75-mm collimation was performed in 20 patients with recurrent angina 4.75 years after undergoing CABG. A total of 50 grafts, 16 arterial and 34 venous, were examined. All graft and coronary segments were evaluated for stenosis in comparison with conventional coronary angiography (CCA). Among the 80 arterial graft segments, 62 could be assessed (77.5%). Sensitivity, specificity, and positive and negative predictive values for stenosis were 96.2%, 97.2%, 96.2%, and 97.2%, respectively. In a total of 180 venous graft segments, 167 could be assessed. Sensitivity, specificity, and positive and negative predictive values for stenosis were 98.5%, 93.9%, 91.8%, and 98.9%, respectively. MDCT could assess 179 of 260 native coronary artery segments (68.8%). Sensitivity, specificity, and positive and negative predictive values for stenosis were 92.1%, 76.9%, 87.5%, and 84.7%, respectively. Sixteen-slice MDCT provides excellent image quality and diagnostic accuracy in detection of graft and coronary artery lesions in patients with suspected graft dysfunction. PMID- 16670867 TI - Soft tissue masses with "cyst-like" appearance on MR imaging: Distinction of benign and malignant lesions. AB - The purpose of this study was to assess the reliability of MR imaging features in distinguishing between benign and malignant soft tissue masses that have a "cyst like" appearance (hypointense to muscle on T1-weighted and hyperintense on T2 weighted or fluid-sensitive MR sequences). MR imaging features of 40 patients with histologically proven soft tissue masses (27 benign and 13 malignant lesions) that were hypointense to muscle on T1-weighted and hyperintense on T2 weighted or fluid-sensitive MR sequences were analysed to see if a distinction could be made between benign and malignant lesions. There were 23 females and 17 males (mean age, 56 years; age range, 23-82 years). MR imaging features analysed for statistical significance included lesion size, lesion homogeneity, presence of lobulation, depth in relation to fascia and border definition. Clinical assessment included the age and sex of the patient and location of the lesion. Statistically significant MR imaging features favouring a diagnosis of a malignant lesion included larger average dimension of the mass (P < or = 0.004), larger greatest dimension of the mass (P < or = 0.028) and heterogeneity of the lesion on T1-weighted sequences (P = 0.017). The most statistically significant predictor of malignancy was the larger average dimension of the mass. PMID- 16670868 TI - Qualitative and quantitative evaluation of solitary thyroid nodules with contrast enhanced ultrasound: initial results. AB - To assess the feasibility of contrast-enhanced ultrasound (CEUS) of the thyroid gland and to evaluate the potential of this method for characterising solitary thyroid nodules.18 patients affected by solitary thyroid nodules (size range: 0.6 to 3.6 cm; mean: 1.8 cm) confirmed by surgery (nine papillary carcinomas, four follicular carcinomas, three hyperplasias, one follicular adenoma and one Plummer's adenoma) underwent pulse inversion US at low M.I. (0.06 to 0.08) after i.v. injection of a 2.4-mL bolus of SonoVue. Baseline echogenicity and the dynamic enhancement pattern of each nodule, in comparison with adjacent thyroid parenchyma, were assessed. Signal intensity values on grey-scale images were also calculated at baseline, 30 s, 60 s and 120 s after SonoVue administration. Following administration of SonoVue, malignant nodules showed absent (4 out of 13), faint dotted (4 out of 13) and diffuse (5 out of 13) contrast enhancement, in this last case inhomogeneous (4 out of 5 cases) or homogeneous (1 out of 5). Benign nodules showed diffuse contrast enhancement, both homogeneous (3 out of 5) and heterogeneous (2 out of 5). Quantitative data have confirmed subjective findings, but CEUS never modified precontrast analysis. CEUS of thyroid gland is a feasible technique, but overlapping findings seem to limit the potential of this technique in the characterization of thyroid nodules. PMID- 16670869 TI - Xanthogranulomatous pseudotumor of stomach induced by perforated peptic ulcer mimicking a stromal tumor. AB - Perforation is a serious complication of peptic ulcer disease occurring in 5% of such patients. Occasionally, the perforation may be sealed off by the omentum or the adjacent organs. Sealed perforated ulcer with pseudotumor formation is very rarely encountered. Here we present a case of gastric pseudotumor induced by perforation of a peptic ulcer. The imaging features in a barium sulfate study and computed tomography mimic an intramural tumor of the stomach. PMID- 16670870 TI - Laser ablation of lung metastases: results according to diameter and location. AB - Lung tumour ablation with a thin-calibre laser applicator system was evaluated. We quantified feasibility, technical success and complication rates in relation to lesion diameter and location. Forty-two patients with 64 lung tumours were treated (39 patients with metastases and three with primary tumours). Mean follow up was 7.6 months (range 6 weeks to 39 months). Eighty-six percent of treatments were technically successful in the first session. Pneumothorax was the main complication and occurred in 50% of the first 20 patients and in 35% of the rest. Two patients required a chest tube. Fourteen lesions were central and 50 were peripheral. It took several weeks for the effect of the therapy to become apparent on follow-up CT. Thirty-nine percent of all lesions increased in size immediately after treatment. Gross reduction in size with scar formation was seen in 50% of the lesions and cavitation in 13%. Local tumour control was achieved in 51 lesions. Progression after therapy was seen in 9% of lesions <1.5 cm but in more than 11% of larger lesions. Progression was also more frequent in lesions located in the basal parts of the lung (47%). Sixteen patients died due to systemic progression. Our results suggest that successful laser ablation of lung lesions is possible with a miniaturized applicator. PMID- 16670872 TI - Conservation of upstream regulators of scute on the notum of cyclorraphous Diptera. AB - Bristles on the notum of many cyclorraphous flies are arranged into species specific stereotyped patterns. Differences in the spatial expression of the proneural gene scute correlate with the positions of bristles in those species looked at so far. However, the examination of a number of genes encoding trans regulatory factors, such as pannier, stripe, u-shaped, caupolican and wingless, indicates that they are expressed in conserved domains on the prospective notum. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera, and that many differences are likely to be due to changes in cis-regulatory sequences of scute. In contrast, in Anopheles gambiae, a basal species with no stereotyped bristle pattern, the expression patterns of pannier and wingless are not conserved, and expression of AgASH, the Anopheles proneural gene, does not correlate in a similar manner with the bristle pattern. We discuss the possibility that independently acting cis-regulatory sequences at the scute locus may have arisen in the lineage giving rise to cyclorraphous flies. PMID- 16670873 TI - Regulation and function of tailless in the long germ wasp Nasonia vitripennis. AB - In the long germ insect Drosophila, the gene tailless acts to pattern the terminal regions of the embryo. Loss of function of this gene results in the deletion of the anterior and posterior terminal structures and the eighth abdominal segment. Drosophila tailless is activated by the maternal terminal system through Torso signaling at both poles of the embryo, with additional activation by Bicoid at the anterior. Here, we describe the expression and function of tailless in a long germ Hymenoptera, the wasp Nasonia vitripennis. Despite the morphological similarities in the mode of development of these two insects, we find major differences in the regulation and function of tailless between Nasonia and Drosophila. In contrast to the fly, Nasonia tll appears to rely on otd for its activation at both poles. In addition, the anterior domain of Nasonia tll appears to have little or no segmental patterning function, while the posterior tll domain has a much more extensive patterning role than its Drosophila counterpart. PMID- 16670874 TI - From embryo to adult--beyond the conventional periodization of arthropod development. AB - The traditional framework for the description of arthropod development takes the molt-to-molt interval as the fundamental unit of periodization, which is similar to the morphological picture of the main body axis as a series of segments. Developmental time is described as the subdivision into a few major stages of one or more instars each, which is similar to the subdivision of the main body axis into regions of one to many segments each. Parallel to recent criticisms to the segment as the fundamental building block of arthropod anatomy, we argue that, while a firm subdivision of development in stages is useful for describing arthropod ontogeny, this is limiting as a starting point for studying its evolution. Evolutionary change affects the association between different developmental processes, some of which are continuous in time whereas others are linked to the molting cycle. Events occurring but once in life (hatching; first achieving sexual maturity) are traditionally used to establish boundaries between major units of arthropod developmental time, but these boundaries are quite labile. The presence of embryonic molts, the 'gray zone' of development accompanying hatching (with the frequent delivery of an immature whose qualification as 'free-embryo' or ordinary postembryonic stage is arbitrary), and the frequent decoupling of growth and molting suggest a different view. Beyond the simple comparison of developmental schedules in terms of heterochrony, the flexible canvas we suggest for the analysis of arthropod development opens new vistas into its evolution. Examples are provided as to the origin of holometaboly and hypermetaboly within the insects. PMID- 16670875 TI - The structure of 5a,6-anhydrotetracycline and its Mg2+ complexes in aqueous solution. AB - Semiempirical molecular orbital theory has been used for a systematic scan of the binding positions for a Mg2+ ion with 5a,6-anhydrotetracycline taking both conformational flexibility and possible different tautomeric forms into account. The magnesium ion has been calculated alone and with four or five complexed water molecules in order to simulate the experimental situation more closely. The results are analyzed by comparing the behavior of the title compound with that of tetracycline itself and possible causes for the stronger induction of the Tetracycline Receptor (TetR) by 5a,6-anhydrotetracycline than by tetracycline are considered. Energetically favored 3D-structure of the zwitteranionic 5a,6 anhydrotetracycline magnesium complex in solution. PMID- 16670877 TI - [Silicone as a foreign body in the middle ear: a rare complication after taking an impression of the auditory canal]. AB - For the individual design of ear moulds, silicone is generally used for taking the impression of the external meatus and the cavum conchae. Complications are rare with only a few accounts in the literature. We report on three cases of accidental displacement of silicone into the middle ear cavity during impression taking. Clinical symptoms and detection of silicone as a rare middle ear foreign body are presented and discussed. PMID- 16670876 TI - [On the diagnosis and treatment of parotid gland tumors. Results of a nationwide survey of ENT hospitals in Germany]. AB - BACKGROUND: It seems that the choice of the treatment modalities for parotid gland tumors frequently depends on personal experience more than on evidence based criteria. A nationwide survey was conducted to obtain an overview of current practice in German ENT hospitals. METHODS: A standardised questionnaire comprising 19 questions on the treatment and diagnostic methods for parotid gland tumors was sent to all ENT hospitals in Germany. RESULTS: The overall return rate of the questionnaires was 128 of 170 (75%). The results confirmed highly variable strategies for the treatment of parotid gland tumors in Germany. CONCLUSION: On the basis of our survey and the upcoming establishment of a German database for salivary gland cancer in Erlangen, Germany, we hope to be able to clarify controversial topics on the treatment of parotid gland tumors in the near future. PMID- 16670878 TI - [Chronic eustachian tube dysfunction and its sequelae in adult patients with cleft lip and palate]. AB - BACKGROUND: While almost all children with cleft palate develop eustachian tube dysfunction, this tends to normalize with age although it remains impaired in a number of patients. METHODS: Eustachian tube function was evaluated by tympanometry in 40 patients with an average age of 19.9 years. The number of patients with chronic otitis media, a sequelae of chronic eustachian tube dysfunction, was determined microscopically and from the patient's history. Hearing was assessed by pure tone RESULTS: Eustachian tube dysfunction was found bilaterally in 25% of the patients and unilaterally in 6%, while chronic otitis media was found in 32.5% and 12.5%, respectively. The hearing level did not differ between the side of the cleft and the opposite side (P=0.562). CONCLUSION: Nearly a third of the adult patients still suffered from eustachian tube dysfunction. Pathological tympanograms and chronic otitis media usually occurred bilaterally. A relationship between the side of the cleft and the side on which the eustachian tube dysfunction or its sequelae occurred was not apparent. The main reason seems to be the continuing bilateral muscular insufficiency in opening the eustachian tube. PMID- 16670879 TI - [Silent mastoiditis in a 5-month-old infant]. AB - We report the case of a 5-month-old female infant who developed a progressive unilateral retroauricular swelling without further symptoms in the first 5 months of life. The otherwise healthy infant was breast fed and had no history of previous otitis media. The clinical suspicion of silent mastoiditis was confirmed by CT scans and the intraoperative finding of an abscess due to Streptococcus pneumoniae. The onset is unusual, since mastoid pneumatization develops only after birth, and it is presumed that maternal antibodies should protect the infant from serious infections within the first months of life. PMID- 16670880 TI - Cholesterol inhibits the insertion of the Alzheimer's peptide Abeta(25-35) in lipid bilayers. AB - The physiological relationship between brain cholesterol content and the action of amyloid beta (Abeta) peptide in Alzheimer's disease (AD) is a highly controversially discussed topic. Evidences for modulations of the Abeta/membrane interaction induced by plasma membrane cholesterol have already been observed. We have recently reported that Abeta(25-35) is capable of inserting in lipid membranes and perturbing their structure. Applying neutron diffraction and selective deuteration, we now demonstrate that cholesterol alters, at the molecular level, the capability of Abeta(25-35) to penetrate into the lipid bilayers; in particular, a molar weight content of 20% of cholesterol hinders the intercalation of monomeric Abeta(25-35) completely. At very low cholesterol content (about 1% molar weight) the location of the C-terminal part of Abeta(25 35) has been unequivocally established in the hydrocarbon region of the membrane, in agreement with our previous results on pure phospholipids membrane. These results link a structural property to a physiological and functional behavior and point to a therapeutical approach to prevent the AD by modulation of membrane properties. PMID- 16670881 TI - Prevalence of blood parasites in different local populations of reed warbler (Acrocephalus scirpaceus) and great reed warbler (Acrocephalus arundinaceus). AB - Blood parasite prevalence in two related warbler species (Acrocephalus arundinaceus and A. scirpaceus) was studied at three Bulgarian sites that differed mainly in altitude and weather. The prevalence of Haemoproteus, the most common parasite genus, was significantly lower at the highest altitude site (730 m above sea level, asl). Such a pattern was not found in ectoparasite-transmitted Hepatozoon. This can be explained with worsening conditions for the development of free-flying vectors with altitude and suggests loosening of the host-parasite conflict at higher breeding sites. The total heamatozoan prevalence was significantly higher in great reed warbler, compared with reed warbler especially as regards Haemoproteus parasites. PMID- 16670882 TI - The effect of temperature on the development of Angiostrongylus cantonensis (Chen 1935) in Pomacea canaliculata (Lamarck 1822). AB - Angiostrongyliasis cantonensis, clinically presented as eosinophilic meningitis, is a snail-borne parasitic disease. We studied the effects of different temperatures on the larval development of Angiostrongylus cantonensis in the freshwater snail Pomacea canaliculata. Six groups of snails were infected and each group was cultured under different temperature conditions. At predefined intervals, four snails from each group were dissected to examine the larval development. The development-time curve of each group was drawn according to the fraction of third-stage larvae present. The developmental time was defined as the time needed until 50% of the first-stage larvae developed into third-stage larvae. A linear regression model was established based on the time (D; in days) and the corresponding temperature (T; in degrees Celsius): DT = 15.04 x D + 262.53. The threshold temperature for larval development was 15.04 degrees C and the thermal constant was 262.53 degree-days. These parameters could be helpful in estimating the number of parasite generations in a year and the impact of climate change on the distribution of A. cantonensis. PMID- 16670883 TI - Larval trematodes in freshwater molluscs from the Elbe to Danube rivers (Southeast Germany): before and today. AB - Studies on life cycles of trematodes have a long tradition in Germany; (Odening 1978) listed a total of 177 trematodes, which can potentially complete their life cycles in German inland waters. However, almost no recent data on the occurrence of larval stages in molluscs are available. Therefore, a survey of trematodes in Southeast Germany was carried out in 2004. A total of 31 species of ten families (29 species of cercariae, seven species of metacercariae, and five species found of both) were found in 311 (4.9%) molluscs of 15 species. The dominant cercariae were Plagiorchis elegans, Echinoparyphium aconiatum, Opisthioglyphe ranae, and Diplostomum pseudospathaceum. Echinoparyphium pseudorecurvatum is reported, for the first time, under its valid scientific name from Germany. In previous studies from the same region, 88 species of cercariae of 16 families were found in 19 species of molluscs (52 cercariae with valid names and 36 not identified to species level). It is proposed that there is still a very similar spectrum of the most common species of cercariae typical for Central Europe as found 20, but also 100-150 years ago. PMID- 16670884 TI - A new Paravortex (Platyhelminthes, Dalyellioida) endoparasite of Mesodesma mactroides (Bivalvia, Mesodesmatidae) from Uruguay. AB - Many species of turbellarians (Platyhelminthes) are known to live associated with other organisms, especially invertebrates, as commensals or parasites. The family Graffillidae (Rhabdocoela) includes two genera that parasitize mollusks, Graffilla and Paravortex. Within the latter genus, six species were described as associated with mollusks. In other instances, unnamed Paravortex species were mentioned as parasites of other bivalves and of the body surface of fishes. In the present work, a new Paravortex species that was found in the intestine of Mesodesma mactroides from the Atlantic coast of Uruguay is described. In addition, a bibliographical revision of the known Paravortex species with their respective hosts, location, and distribution is made. Paravortex nicolli, described by Szidat for the Argentinean coast, is mentioned for the first time after the original description, and the authorship and date of description of Paravortex tapetis Noury-Srairi 1989 are elucidated. PMID- 16670892 TI - Comparison of two quantitative GC-MS methods for analysis of tomato aroma based on purge-and-trap and on solid-phase microextraction. AB - Two analytical procedures, one based on purge-and-trap and the other on solid phase microextraction, both followed by GC-MS measurement using an ion-trap mass spectrometer in the electron impact mode, have been developed for determination and quantitation of up to 39 aroma compounds in fresh tomatoes. The method based on purge-and-trap for isolation of the volatile compounds uses Tenax as adsorbent and a hexane-diethyl ether mixture as solvent for elution. The method was validated for linearity, precision (better than 20% for most compounds), and limit of detection, which was approximately 1 ng g(-1). This method enabled identification of up to 30 compounds in real samples. Use of SPME was considered as an alternative, to simplify sample treatment while maintaining the information level for the samples (e.g. the number of compounds detected) and quality of quantitation. A procedure based on SPME using a Carboxen/polydimethylsiloxane fibre was developed and validated for determination of 29 aroma compounds; precision was better than 20% and limits of detection ranged from 4 to 30 ng g( 1). PMID- 16670899 TI - Montelukast and zafirlukast do not affect the pharmacokinetics of the CYP2C8 substrate pioglitazone. AB - OBJECTIVE: Pioglitazone, a thiazolidinedione antidiabetic drug, is metabolised mainly by the cytochrome P450 (CYP) 2C8 enzyme. The leukotriene receptor antagonists montelukast and zafirlukast have potently inhibited CYP2C8 activity and the metabolism of pioglitazone in vitro. Our objective was to determine whether montelukast and zafirlukast increase the plasma concentrations of pioglitazone in humans. METHODS: In a randomised, double-blind crossover study with three phases and a washout period of 3 weeks, 12 healthy volunteers took either 10 mg montelukast once daily and placebo once daily, or 20 mg zafirlukast twice daily, or placebo twice daily, for 6 days. On day 3, they received a single oral dose of 15 mg pioglitazone. The plasma concentrations of pioglitazone and its metabolites M-IV, M-III, M-V and M-XI were measured for 96 h. RESULTS: The total area under the plasma concentration-time curve of pioglitazone during the montelukast and zafirlukast phases was 101% (range 71-143%) and 103% (range 78 146%), respectively, of that during the placebo phase. Also, the peak plasma concentration and elimination half-life of pioglitazone remained unaffected by montelukast and zafirlukast. There were no statistically significant differences in the pharmacokinetics of any of the metabolites of pioglitazone between the phases. CONCLUSIONS: Montelukast and zafirlukast do not increase the plasma concentrations of pioglitazone, indicating that their inhibitory effect on CYP2C8 is negligible in vivo, despite their strong inhibitory effect on CYP2C8 in vitro. The results highlight the importance of in vivo interaction studies and of the incorporation of relevant pharmacokinetic properties of drugs, including plasma protein binding data, to in vitro-in vivo interaction predictions. PMID- 16670900 TI - The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm. AB - Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g(-1) DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g(-1) DW) and IBS (0.307 mg g(-1) DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots. PMID- 16670901 TI - Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue. AB - We report the investigation of an endosperm-specific promoter from the rsus3 gene from rice (Oryza sativa). The promoter was characterized by deletion analysis and transient expression in guar (Cyamopsis tetragonoloba) seed-tissue. Transient expression was monitored by histochemical GUS assay, and quantitative dual reporter assays comprising firefly luciferase as a test reporter, and Renilla luciferase and GUS as reference reporters. These revealed high expression levels of the reporter genes directed by the rsus3 promoter in guar endosperm. Specificity for this tissue in seeds was apparent from a virtual absence of reporter activity in guar cotyledons. Removal of a putative intron region only slightly raised the expression level, whereas duplication of the minimal promoter region, in a tandem-repeat rsus3 promoter construct, retained endosperm specificity in guar, and displayed three times the reporter activity observed with the single copy construct. PMID- 16670902 TI - [Airway management in the spontaneously breathing child]. PMID- 16670903 TI - [Theophylline (aminophylline) and asystole. Case report and a brief review of the literature]. AB - We report the use of the non-specific adenosine antagonist theophylline (aminophylline) during a prolonged intraoperative cardiopulmonary resuscitation (CPR) due to myocardial infarction. In the 2005 guidelines of the European Resuscitation Council the general use of theophylline during CPR is not recommended, but in the case of an atropine and epinephrine resistant asystole, especially as a result of inferior myocardial infarction, theophylline might be a useful adjunct during CPR. PMID- 16670905 TI - First nonavian dinosaur from Lebanon: a brachiosaurid sauropod from the Lower Cretaceous of the Jezzine District. AB - Two sauropod teeth from an Early Cretaceous (Neocomian) fluviodeltaic sandstone near Jezzine (Southern Lebanon) are the first nonavian dinosaur remains to be reported from Lebanon. Their distinctive character places them within Brachiosauridae. The sauropod teeth from Lebanon are a significant addition to the very scanty dinosaur record from the Levant, which hitherto consisted mainly of very poorly preserved and not easily identifiable specimens. The Basal Cretaceous Sandstone of Lebanon, thus, appears to be a potentially important source of fossil vertebrate material. PMID- 16670906 TI - Do honeybees, Apis mellifera scutellata, regulate humidity in their nest? AB - Honeybees are highly efficient at regulating the biophysical parameters of their hive according to colony needs. Thermoregulation has been the most extensively studied aspect of nest homeostasis. In contrast, little is known about how humidity is regulated in beehives, if at all. Although high humidity is necessary for brood development, regulation of this parameter by honeybee workers has not yet been demonstrated. In the past, humidity was measured too crudely for a regulation mechanism to be identified. We reassess this issue, using miniaturised data loggers that allow humidity measurements in natural situations and at several places in the nest. We present evidence that workers influence humidity in the hive. However, there are constraints on potential regulation mechanisms because humidity optima may vary in different locations of the nest. Humidity could also depend on variable external factors, such as water availability, which further impair the regulation. Moreover, there are trade-offs with the regulation of temperature and respiratory gas exchanges that can disrupt the establishment of optimal humidity levels. As a result, we argue that workers can only adjust humidity within sub-optimal limits. PMID- 16670907 TI - A simple method to discriminate diapause from non-diapause pupae in large and small white butterflies, Pieris brassicae and P. rapae crucivora. AB - Differences in colour and shape have been used to discriminate diapause pupae from non-diapause pupae in butterflies. In the present study, we describe a simple discrimination method based on differences in the specific gravity of diapause and non-diapause pupae of large and small white butterflies, Pieris brassicae and Pieris rapae crucivora. When put into water, diapause pupae sink to the bottom (specific gravity is above 1.0), whereas non-diapause pupae float on the surface (specific gravity is below 1.0). Nuclear magnetic resonance microimaging revealed that this difference in specific gravity is due to a difference in the volume of an internal cavity located between the thorax and the abdomen in the pupae. The cavity appears quite early in development. We also examined the cavity in pupae of the unrelated swallowtail butterfly, Papilio xuthus, and found a similar cavity. However, in this species, the cavity is very small and, as a consequence, non-diapause as well as diapause pupae sink in water. PMID- 16670908 TI - The asteroid and comet impact hazard: risk assessment and mitigation options. AB - The impact of extraterrestrial matter onto Earth is a continuous process. On average, some 50,000 tons of dust are delivered to our planet every year. While objects smaller than about 30 m mainly disintegrate in the Earth's atmosphere, larger ones can penetrate through it and cause damage on the ground. When an object of hundreds of meters in diameter impacts an ocean, a tsunami is created that can devastate coastal cities. Further, if a km-sized object hit the Earth it would cause a global catastrophe due to the transport of enormous amounts of dust and vapour into the atmosphere resulting in a change in the Earth's climate. This article gives an overview of the near-Earth asteroid and comet (near-Earth object NEO) impact hazard and the NEO search programmes which are gathering important data on these objects. It also points out options for impact hazard mitigation by using deflection systems. It further discusses the critical constraints for NEO deflection strategies and systems as well as mitigation and evacuation costs and benefits. Recommendations are given for future activities to solve the NEO impact hazard problem. PMID- 16670909 TI - New remains of the enigmatic cetartiodactyl Bugtitherium grandincisivum Pilgrim, 1908, from the upper Oligocene of the Bugti Hills (Balochistan, Pakistan). AB - Newly discovered fossil material of the enigmatic cetartiodactyl Bugtitherium grandincisivum from the upper Oligocene of the Bugti Member of the Chitarwata Formation in the Bugti Hills (Balochistan, Pakistan) is reported. These new specimens consist of two fragmentary muzzles (one preserving the first incisors and belonging to a juvenile) and a fragmentary right mandible with m3. The morphologies of the anterior dentition and m3 provided by these new specimens confirm the validity of the genus Bugtitherium and advocate probable anthracotheriid affinity for the genus rather than entelodontid or suoid affinities, but do not definitively close the debate about Bugtitherium's familial affinities within Cetartiodactyla. Although still poorly documented, this large-sized anthracotheriid-like cetartiodactyl is a possible key form for understanding the early evolution of hippos, and, in turn, the ancestry of whales, because of both its morphological similarities with hippos and primitive Paleogene whales and its Tethysian distribution. PMID- 16670912 TI - Cost-effectiveness of memantine in community-based Alzheimer's disease patients: An adaptation in Spain. AB - Several clinical trials have demonstrated the efficacy and safety of the NMDA antagonist memantine in moderately severe to severe Alzheimer's disease (AD) patients. A 28-week pharmacoeconomic study conducted in the US also showed a reduction of total healthcare costs and informal care compared to placebo. Long term implications of memantine treatment were modelled in the UK and Finland and revealed reductions in dependency, institutionalization and costs. However, these conclusions were not directly applicable to the Spanish setting where patients are mainly treated within the community. The objective of this study was to estimate the long-term implications in terms of costs and health benefits of memantine therapy compared to standard care using a Spanish adaptation of previous models over a 2-year time horizon. As in previous adaptations, Markov health states were defined as a combination of severity (mild-moderate, moderately severe, severe) and dependency plus death as the absorbing state. Spain-specific data (costs, mortality and epidemiological data) were obtained from local and recently published cohorts of AD patients. Data on the effectiveness of memantine were derived from a randomized double-blind placebo controlled clinical trial of 252 moderately severe to severe AD patients. Effectiveness was measured as the time spent in a non-dependent health state. The evaluation was conducted over 2 years, while the efficacy of memantine was applied for 1 year only in order to ensure a conservative approach. The robustness of the model was tested by conducting stochastic analyses and various sensitivity analyses on the key assumptions. Patients receiving standard care were estimated to spend 6 months in a non-dependent state and to incur average total costs of Euro 24,700 over 2 years. The memantine strategy was associated with an additional 2.5 months in a non-dependent state and a Euro 700 cost reduction. Monte-Carlo simulations and sensitivity analyses supported these findings. Memantine appears to be cost-effective compared with standard care in moderately severe to severe AD patients in a Spanish setting. The prolonged independence provided by memantine treatment translated into cost reductions which offset drug costs and resulted in overall cost-savings. PMID- 16670917 TI - A case of achondroplasia with severe pulmonary hypertension due to obstructive sleep apnea. AB - Achondroplasia is the most common skeletal dysplasia in children. Achondroplasic patients have a short cranial face and midface hypoplasia. They often have sleep related respiratory disturbances that lead to hypoxemia caused by midfacial hypoplasia, a small upper airway, hypotonia of airway muscles, or brain stem compression. It has been well described that obstructive sleep apnea can cause pulmonary hypertension (PH) through the mechanism of chronic hypoxemia. However, severe PH due to obstructive-type sleep disorder is rare in patients with achondroplasia. In this report, we describe a 5-year-old girl with achondroplasia whose severe PH was caused by upper-airway obstruction and was resolved gradually after adenotonsillectomy. PMID- 16670918 TI - Ballooning-induced bradycardia during carotid stenting in primary stenosis and restenosis. AB - INTRODUCTION: We compared the incidence of intraprocedural bradycardia and hypotension during carotid artery stenting in patients with primary carotid artery stenosis and those with prior ipsilateral carotid endarterectomy. METHODS: A total of 213 carotid stenting procedures were performed in our institution in a 4-year period. The mean degree of stenosis was 78% (range 60-99%). Of these 213 procedures, 43 were performed for carotid restenosis, 9 after stenting and 34 after endarterectomy, and 170 for primary stenosis. Atropine was selectively administrated if patients suffered bradycardia (a decrease in heart rate to <50% or an absolute heart rate of <40 bpm) or hypotension (systolic blood pressure <90 mmHg). We compared the group of patients with primary stenosis (n=170) and the group of patients with restenosis after carotid endarterectomy (n=34) in relation to intraprocedural hypotension or bradycardia/need for atropine administration. RESULTS: Hypotension occurred in 49 patients with primary stenosis and 2 patients with restenosis. The difference was statistically significant. Atropine was administered for bradycardia to 58 patients with primary stenosis and 3 patients with restenosis. The difference was statistically significant. CONCLUSION: Intraprocedural bradycardia and hypotension occur more frequently in patients with primary carotid artery stenosis. PMID- 16670919 TI - Development and maturation of thymic dendritic cells during human ontogeny. AB - Thymic dendritic cells (TDC) are dendritic cells situated mainly in the cortico medullary zone and in the medullary region of the thymus. However, the phenotype of TDC during ontogeny is poorly documented. The aim of this study has been to investigate the development and maturation of TDC during human ontogeny. Immunohistochemical analyses and immunoelectron-microscopic investigation of 21 human thymus specimens have been performed to detect the subtypes of TDC by using various DC-related and DC-development-related markers. TDC express a Langerhans cell-like phenotype during human ontogeny. Cells expressing thymic stromal lymphopoietin receptor have been observed in Hassal's corpuscles of the thymus. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is also expressed in thymic epithelial cells (TEC) localized in Hassal's corpuscles. During human ontogeny, GM-CSF is produced by TEC of Hassal's corpuscles and might play a key role in the differentiation of TDC having Langerhans-cell-like phenotypes. PMID- 16670920 TI - Elevated gene expression of MMP-1, MMP-10, and TIMP-1 reveal changes of molecules involved in turn-over of extracellular matrix in cyclosporine-induced gingival overgrowth. AB - In humans, pathogenesis in cyclosporine A (CsA)-induced gingival overgrowth (GO) includes the accumulation of extracellular matrix (ECM) constituents, viz., collagen type-1 and type-3 and proteoglycans, in subgingival connective tissue. However, whether this increase is associated with alterations of molecules pivotal for the turn-over of collagens and proteoglycans remains unclear. The present study explores the status of matrix metalloproteinase MMP-1 and MMP-10, which are important for fibrillar collagen and proteoglycan turn-over, and their tissue inhibitor TIMP-1, on their gene expression and protein levels in frozen sections derived from GO and matched normal tissue. In situ hybridization (ISH) revealed elevated levels of MMP-1 gene expression in the connective tissue of GO compared with normal tissue. This elevation also applied to MMP-10 and TIMP-1, the latter exhibiting the strongest gene transcription in the deep connective tissue. These differences detected by ISH were corroborated by quantitative reverse transcription/polymerase chain reaction; relative gene expression analysis indicated a 1.9-fold increase for MMP-1, a 2.3-fold increase for MMP-10, and a 4.8-fold increase for TIMP-1. Detection of the protein by indirect immunofluorescence showed that normal gingival tissue was devoid of all three proteins, although they were detectable in GO tissue, with emphasis on TIMP-1. Analysis of our data indicates elevated levels of MMP-1 and-10, and particularly TIMP-1. With respect to TIMP-1, this elevation may in turn lead to alterations in ECM turn-over by abrogating MMP-1 and MMP-10, thereby contributing to ECM accumulation associated with GO. PMID- 16670921 TI - Parathyroid hormone(1-34) mediates proliferative and apoptotic signaling in human periodontal ligament cells in vitro via protein kinase C-dependent and protein kinase A-dependent pathways. AB - Periodontal ligament (PDL) cells exhibit several osteoblastic traits and are parathyroid hormone (PTH)-responsive providing evidence for a role of these cells in dental hard-tissue repair. To examine the hypothesis that PDL cells respond to PTH stimulation with changes in proliferation and apoptotic signaling through independent but convergent signaling pathways, PDL cells were cultured from human bicuspids obtained from six patients. PDL cells at different states of maturation were challenged with PTH(1-34) intermittently for 0, 1, or 24 h/cycle or exposed continuously. Specific inhibitors to protein kinases A and C (PKA, PKC) and the mitogen-activated protein kinase cascade (MAPK) were employed. At harvest, the cell number, BrdU incorporation, and DNA fragmentation were determined by means of cell counting and immunoassays. Intermittent PTH(1-34) caused a significant increase in cell number in confluent cells as opposed to a reduction in pre confluent cells. In confluent cells, the effect resulted from a significant increase in proliferation, whereas DNA fragmentation was reduced when PTH(1-34) was administered for 1 h/cycle but increased after PTH(1-34) for 24 h/cycle. Inhibition of PKC inhibited PTH(1-34)-induced proliferation but enhanced apoptosis. Inhibition of PKA enhanced proliferation and DNA fragmentation. Similar results were obtained in less mature cells, although, in the presence of the PKA inhibitor, the PTH(1-34)-induced changes were more pronounced than in confluent cells. In the presence of the MAPK inhibitor, all of the parameters examined were reduced significantly in both maturation states. Thus, PTH(1-34) mediates proliferative and apoptotic signaling in human PDL cells in a maturation state-dependent manner via PKC-dependent and PKA-dependent pathways. PMID- 16670922 TI - [Neuropsychological outcome following intrauterine exposure to valproate]. AB - A number of recent studies suggest a link between in utero exposure to valproate (VPA) and low IQ and behavioural disorders in children of mothers with epilepsy. In this review, a commission of the German Section of the International League Against Epilepsy discusses the evidence in the literature and practical recommendations for the use of VPA in women of childbearing potential. It is concluded that despite methodological shortcomings--largely due to the complexity of the problem and small case numbers in prospective studies--the existing data are sufficiently alarming to require great caution in the use of VPA in women who could become pregnant. The underlying mechanisms of how antiepileptic drugs may lead to neurodevelopmental problems are unclear. Further prospective studies are urgently needed to clarify this clinically important issue, and a collaborative study is suggested based on the international network established by the European Registry of Antiepileptic Drugs and Pregnancy. PMID- 16670926 TI - [Chronic lupoid leishmaniasis. A rare differential diagnosis in Germany for erythematous infiltrative facial plaques]. AB - Lupoid leishmaniasis is a unique form of cutaneous leishmaniasis characterized by unusual clinical features and a chronic relapsing course. Clinically and histologically it is similar to lupus vulgaris, which is thus the most important differential diagnostic consideration. All patients with granulomatous facial lesions coming from endemic areas or with a positive travel history should be suspected of having leishmaniasis. We describe a 59-year-old woman with facial lupoid leishmaniasis. PMID- 16670927 TI - [Symmetrical bullous acral erythema in a 58-year-old female alcoholic]. PMID- 16670928 TI - [Ulcerated plaques and papules limited to one leg]. PMID- 16670929 TI - Normocalcemic primary hyperparathyroidism in patients with recurrent kidney stones: pathological analysis of parathyroid glands. AB - The lack of overt elevation of serum calcium concentration in some patients suffering from primary hyperparathyroidism is an intriguing clinical phenomenon. Previous studies have substantiated abnormal parathyroid tissue in these patients, but the extent and mode of derangements remained largely undefined. The parathyroid tissues from patients of normocalcemic primary hyperparathyroidism (NCPHPT) and those having normal parathyroid glands, hypercalcemic primary hyperplasia, secondary hyperplasia, and adenoma were compared by undertaking quantitative immunohistochemistry analysis on tissue microarray. The statistic results suggested that the parathyroid tissue of NCPHPT approximates more to normal gland than to its counterpart in other groups of parathyroid proliferative diseases in terms of the lack of significant alterations of calcium-sensing receptor (CaSR), chromogranin A (CGA), parathyroid hormone (PTH), and proliferation index (Ki67). On the other hand, the depressed vitamin D receptor (VitDR) and elevated cyclin D1 (CyD1) of NCPHPT indicated the inherent functional abnormalities in parathyroid cells. Our results imply that inherent functional disengagement may exist between CaSR and CyD1 or between CaSR and VitDR or both in parathyroid cells of symptomatic NCPHPT. Lack of enhanced release of CGA and PTH and discordance between proliferative activity and CyD1 expression in parathyroid cells may further hinder the development of hypercalcemia. PMID- 16670930 TI - Well-differentiated endocrine tumor of the distal common bile duct: a case study and literature review. AB - Primary carcinoid tumors of the extrahepatic biliary tree are exceedingly rare, accounting for 0.2-2% of all digestive carcinoids. The authors in this study describe a case of biliary duct primary well-differentiated endocrine tumor in a 30-year-old man with symptoms of biliary obstruction and watery diarrhoea. Abdominal ultrasound showed a 2-cm solid lesion in the head of the pancreas, compressing the distal common bile duct. A computed tomography scan confirmed these findings, revealing the hypervascular pattern of the tumor. Gastrointestinal hormonal screening demonstrated an increase in plasma serotonin. The patient underwent standard pylorus-preserving pancreatoduodenectomy. Pathological examination showed a neuroendocrine tumor of the distal common bile duct measuring 1.8 cm in greatest dimension. The tumor cells were immunopositive for neuron-specific enolase (NSE), chromogranin A, synaptophysin, serotonin, and cytokeratin. Stains for gastrin and somatostatin were negative. Seven years later, the patient is well, with no evidence of disease. Given the site of these tumors and the difficulty in differentiating them from periampullary lesions, decisions as to the appropriate surgical approach may be problematic. After an exhaustive review of the literature, the authors conclude that pancreatoduodenectomy is the treatment of choice. PMID- 16670936 TI - Oxidative renal tubular injuries induced by aminocarboxylate-type iron (III) coordination compounds as candidate renal carcinogens. AB - Oxidative renal tubular injuries and carcinogenesis induced by Fe(III) nitrilotriacetate (NTA) and Fe(III)-ethylenediamine-N,N'-diacetate (EDDA) have been reported in rodent kidneys, but the identity of iron coordination structure essential for renal carcinogenesis, remains to be clarified. We compared renal tubular injuries caused by various low molecular weight aminocarboxylate type chelators with injuries due to NTA and EDDA. We found that Fe(III)-iminodiacetate (IDA), a novel iron-chelator, induced acute tubular injuries and lipid peroxidation to the same extent. We also prepared Fe(III)-IDA solutions at different pHs, and studied resultant oxidative injuries and physicochemical properties. The use of Fe(III)-IDA at pH 5.2, 6.2, and 7.2 resulted in renal tubular necrosis and apoptotic cell death, but neither tubular necrosis nor apoptosis was observed at pH 8.2. Spectrophotometric data suggested that Fe(III) IDA had the same dimer structure from pH 6.2 to 7.2 as Fe(III)-NTA; but at a higher pH, iron polymerized and formed clusters. Fe(III)-IDA was crystallized, and this was confirmed by X-ray analysis and magnetic susceptibility measurements. These data indicated that Fe(III)-IDA possessed a linear mu-oxo bridged dinuclear iron (III) around neutral pH. PMID- 16670937 TI - Relationship between body iron stores and diquat toxicity in male Fischer-344 rats. AB - The effects of body iron stores on diquat (DQ)-induced toxicity were examined in male Fischer-344 rats, which are sensitive to this herbicide. The rats (5 weeks old) were fed diets containing 40 (lower iron storage [LIS] group) or 320 ppm iron (higher iron storage [HIS] group) for 5 weeks. The concentrations of nonheme iron and ferritin in the liver and kidney were significantly higher in the HIS group than in the LIS group (P<0.0001), although there was no significant differences between the HIS and LIS groups in hematological parameters, including red blood cell count, hemoglobin concentration, and mean corpuscular volume. Three hours after administration of 0.1 mmol DQ/kg, serum alanine aminotransferase and urea nitrogen were significantly higher than in controls (saline injection) for both the LIS and HIS groups (P<0.01), and, after DQ injection, these parameters were significantly higher in the HIS group than in the LIS group (P<0.01). When the rats were injected with 0.075 or 0.1 mmol DQ/kg, the survival time was significantly shorter in the HIS group than in the LIS group (P<0.05). These findings suggest that higher body iron stores result in more severe DQ toxicity in Fischer-344 rats. PMID- 16670938 TI - Hepatocellular carcinoma occurring in a patient with Crohn's disease treated with both azathioprine and infliximab. AB - Hepatocellular carcinoma is known to be associated with underlying liver diseases, such as cirrhosis, hemochromatosis, and chronic viral hepatitis. All reported cases of hepatocellular carcinoma in association with Crohn's disease involve patients treated previously with azathioprine or both azathioprine and steroids. However, hepatocellular carcinoma associated with the use of azathioprine and infliximab has not been reported. In this report, we describe an unusual case of hepatocellular carcinoma and focal hepatic glycogenosis (FHG) occurring in a non-cirrhotic Crohn's disease patient who has been treated with both azathioprine and infliximab. PMID- 16670939 TI - Heparin and insulin in the treatment of hypertriglyceridemia-induced severe acute pancreatitis. AB - Heparin and insulin stimulate lipoprotein lipase activity. Therefore, they reduce serum triglyceride levels. However, heparin and insulin's efficacy in treatment of hypertriglyceridemia-induced acute pancreatitis is not well established. We report a patient in whom heparin and insulin were used successfully. PMID- 16670940 TI - A survey of irritable bowel syndrome in Vietnam using the Rome criteria. AB - Prevalence estimates for irritable bowel syndrome from surveys in Western countries are 4.4% to 22%, generally higher in women than men, and only a minority seek health care. There are few studies of bowel patterns in Asian countries. We conducted a survey of a nonpatient population in Ho Chi Minh City, Vietnam, to determine bowel patterns and the prevalence of bowel dysfunction. A forced-choice, self-report questionnaire was distributed to 738 predominantly health care workers, as well as patient relatives, at Cho Ray Hospital in Ho Chi Minh City and returned by 411 (response rate of 55.7%). Results were analyzed for men and women using Student's t-test for continuous variables and chi-square test for categorical variables. Subjects were 53.6% female, with a mean age of 27.7+/ 6.9 years. Overall perception of health was excellent/very good in 13.6%, good in 54.2%, and fair/poor in 32.1% (males, 17.1%, 51.3%, and 31.5%, vs. females, 10.6%, 56.7%, and 32.7%; P=NS). The mean number of stools reported per week was 6.5 (males, 6.6, vs. females, 6.4; P=NS) and ranged between 3 and 21 stools per week in 95.5%. The frequency of irritable bowel syndrome symptoms (using Rome I criteria) was 7.2% (95% CI=4.8-10.1), with males at 4.8% (95% CI=2.2-8.9) vs. females at 9.2% (95% CI=5.7-13.9) (P=0.08). Of the subjects with irritable bowel syndrome symptoms, 6 of 29 (20.7%) had seen a physician for bowel symptoms. There were no gender differences in reported infrequent stool (12.0%), frequent stool (11.3%), hard stool (17.5%), loose stool (6.5%), straining (14.5%), incomplete emptying (16.2%), bloating (15.0%), urgency (10.0%), or mucus (2.7%). In conclusion, this survey of a nonpatient population in Vietnam showed that irritable bowel syndrome symptoms as defined by Rome criteria were common and that there were no significant differences between sexes in either stool frequency or prevalence of irritable bowel syndrome, unlike previous studies from the United States. The prevalence of irritable bowel syndrome in Vietnam in this study was in the lower range of reported data from Western countries, possibly in part related to the use of the Rome criteria. Only a minority of subjects with irritable bowel syndrome symptoms reported seeking health care for these symptoms. PMID- 16670941 TI - Prognostic significance of changes in CA 15-3 serum levels during chemotherapy in metastatic breast cancer patients. AB - Tumor response to first-line chemotherapy in advanced breast cancer offers prognostic information and may be used as a surrogate marker for evaluating treatment efficacy. With this study we wanted to determine whether changes in circulating serum CA 15-3 levels during chemotherapy provided additional information for prognostic prediction. Serum CA 15-3 was measured at baseline and after 3 and 6 months during anthracycline-based first-line chemotherapy in 526 patients with advanced breast cancer prospectively enrolled in five phase II-III trials. Changes in marker levels were correlated with disease response, time to progression and overall survival. In all, 336 patients attained a disease response. A significant relationship was found between disease response and CA 15 3 variations, although many individual discrepancies were also observed. At the 6 month time point, the median time to progression was 15.3 months in patients with normal marker levels throughout the study, 11.7 months in those with a CA15-3 reduction >25%, 9.6 months in those with elevated baseline CA 15-3 levels which did not change during therapy and 8.6 months in those with increased marker levels (p < 0.001). The median survival was 42.3, 29.7, 28.5, and 24.8 months, respectively (p < 0.002). The prognostic role of changes in CA 15-3 levels was maintained in the patient subset attaining disease response or stabilization to treatment (p < 0.001) and after adjusting for clinical response and major prognostic parameters in the multivariate analysis (p < 0.001). In conclusion, monitoring serum CA 15-3 levels during first-line chemotherapy in advanced breast cancer patients provides prognostic information independently from tumor response. PMID- 16670944 TI - A case of relapsing glioblastoma multiforme responding to vinorelbine. AB - Childhood malignant gliomas are rare and their clinical behavior is almost as aggressive as in adults: they resist treatment, progress rapidly and often spread. Therapeutic strategies at relapse deserve an experimental approach, since none of the conventional-dose treatments have demonstrated a clear superiority over the others and no randomized trials have proved that high-dose chemotherapy is better than conventional treatment. Vinorelbine is a semi-synthetic vinca alkaloid with an in vitro and in vivo experimentally proven broad spectrum of activity, including against malignant brain glioma. We report our experience with a 19-year-old girl with glioblastoma multiforme (GBM) of the deep temporal region recurring 6 months after completing an intensive treatment that included preradiation chemotherapy (chemotherapy as a preradiation "sandwich" phase) with a myeloablative course of thiotepa, tumor bed radiotherapy and postradiation maintenance chemotherapy. The GBM proved fully responsive to intravenous vinorelbine, with a subsequent progression-free interval lasting more than 24 months. This case report suggests that vinorelbine is effective against high grade pediatric glioma and, since this evidence has only one precedent in the literature (and given the generally poor prognosis for this tumor), even this single success seems worth reporting. PMID- 16670946 TI - The structure and diversity of alpha1-acid glycoprotein/orosomucoid gene in Africans. AB - Human orosomucoid (ORM), or alpha(1)-acid glycoprotein, is known to be controlled by duplicated and triplicated genes on chromosome 9, encoding ORM1 and ORM2 proteins. In this study, the structure and diversity of the ORM gene were investigated in 16 Sub-Saharan Africans, who originated from widely dispersed locations in Africa. The duplicated ORM1-ORM2 gene was observed in all 16 samples. ORM1*S1(2), characterized by an ORM2 gene-specific sequence in intron 5, was common in Africans. Three Africans showed the duplication of the ORM1 gene. The organization of the triplicated ORM1A-ORM1B-ORM2 gene was established in two Africans. The recombination breakpoints resulting in the ORM1 duplication lay within a small genomic interval around exon 1 of the ORM1B gene. The duplication of the ORM2 gene reported previously was not detected in this population sample. Several single-nucleotide polymorphisms were observed in the ORM2 gene. The rearrangement of the ORM gene is likely to occur often in Africans. PMID- 16670947 TI - A study of conservation genetics in Cupressus chengiana, an endangered endemic of China, using ISSR markers. AB - ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (He) was 0.3120, effective number of alleles (Ae) was 1.5236, and Shannon's information index (I) was 0.4740. At the population level, PPB = 48%, Ae = 1.2774, He = 0.1631, and I = 0.2452. Genetic differentiation (Gst) detected by Nei's genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (Nm) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r = 0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity. PMID- 16670948 TI - Extract EGb 761 pretreatment limits ubiquitin positive aggregates in rabbit spinal cord neurons after ischemia-reperfusion. AB - 1. Ubiquitin immunohistochemistry was used for investigation of time dependent changes of ubiquitin in the nerve cells reacting to ischemic/reperfusion damage. In the rabbit spinal cord ischemia model a period of 30 min ischemia followed by 24 and 72 h of reperfusion caused neuronal degeneration selectively in the ventral horn motor neurons as well as interneurons of the intermediate zone. 2. Ubiquitin aggregates were accumulated in the neurons of lamina IX and the neurons of intermediate zone destined to die 72 h after 30 min of the spinal cord ischemia. 3. The activation of ubiquitin hydrolytic system is related to a defective homeostasis and could trigger different degenerative processes. Having in mind this, we used EGb 761 to rescue the motor neurons and interneurons against ischemia/reperfusion damage. Our results show that after 30 min of ischemia and 24 or 72 h of reperfusion with EGb 761 pre-treatment for 7 days the vulnerable neurons in the intermediate zone and lamina IX exhibit marked elevation of ubiquitin-positive granules in the cytoplasm, dendrites and nuclei. Abnormal protein aggregates have not been observed in these cells. 4. The rabbits were completely paraplegic after 30 min of ischemia and 24 or 72 h of reperfusion. However, after 7 days EGb 761 pre-treatment, 30 min of ischemia and 24 or 72 h of reperfusion the animals did not show paraplegia. 5. Evaluated ubiquitin-positive neurons of the L(5)-L(6) segments showed significant decrease in number and significant increase of density after 30 min of ischemia followed by 24 h and mainly 72 h of reperfusion. Ubiquitin immunohistochemistry confirmed the protective effect of EGb 761 against ischemia/reperfusion damage in the rabbit spinal cord. PMID- 16670962 TI - Initial depression severity and the trajectory of recovery following cognitive behavioral intervention for work disability. AB - INTRODUCTION: The present study examined the recovery trajectories of a group of mildly depressed and moderately-severely depressed injured workers enrolled in a 10-week community-based rehabilitation program. METHODS: A sample of 168 individuals (75 women, 93 men) with a disabling musculoskeletal pain condition participated in the research. On the basis of BDI-II (1) scores at pre-treatment assessment, patients were classified as either mildly (BDI-II =9-16; N=68) or moderately-severely depressed (BDI-II >16; N: 100). RESULTS: Both groups showed significant reductions in depression, but individuals in the (initially) moderately-severely depressed group were more likely to score in the depressed range of the BDI-II at post-treatment than individuals who were initially mildly depressed. For the mildly depressed group, early treatment reductions in pain catastrophizing, and perceived disability predicted improvement in depression scores. For the moderately-severely depressed group, none of the early treatment changes predicted improvement in depression; only late treatment reductions in pain catastrophizing and fear of movement/re-injury predicted improvement in depression. Chi-square analysis revealed that patients who were no longer depressed at post-treatment had the highest probability of returning to work (91%), followed by (post-treatment) mildly depressed patients (60%), and finally (post-treatment) moderately-severely depressed patients (26%), chi(2)=38.9, p < 0.001. CONCLUSION: In order to maximize return to work potential, the content, structure and duration of rehabilitation programs requires modification as a function of the injured workers level of the depression severity. PMID- 16670964 TI - Rat models of bone metastases. AB - Bone metastases occur frequently in patients with advanced breast or prostate cancer. Bone metastases can be predominantly osteolytic, osteoblastic or mixed. Studies with animal models allow advances in understanding the molecular basis for bone metastases and provide new targets for therapy. Several animal models have been developed in rat with different pathophysiologies; they required injection or implantation of neoplastic cells into orthotopic locations, bones or the left ventricle of the heart. Several specific strains of rat have an increased incidence of spontaneous tumors. Carcinomas can be induced by either chemicals or physical agents. However, the most used and convenient way to induce bone metastases is a syngeneic transmission. MAT-Ly-Lu cells have been used in several models using Copenhagen rats to induce osteoblastic bone lesions. PA-III cells derived from Pollard tumors can also produce a combination of osteolytic and osteoblastic reactions at the site of transplantation. Osteolytic bone lesions can be obtained with an injection of Walker cells. The use of 13762 or c SST2 cells allows also leads to osteolysis. Human xenografts can only be used in nude animals. It is essential to validate and correctly interpret the lesions in several models of bone metastasis. No animal model is sufficient by itself to represent the clinical findings observed in humans. The use of models developed in different species should be more predictive and bring a beam of arguments for a better knowledge of pathophysiological and therapeutic mechanisms. PMID- 16670965 TI - Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups. AB - The Actinobacteria constitute one of the main phyla of Bacteria. Presently, no morphological and very few molecular characteristics are known which can distinguish species of this highly diverse group. In this work, we have analyzed the genomes of four actinobacteria (viz. Mycobacterium leprae TN, Leifsonia xyli subsp. xyli str. CTCB07, Bifidobacterium longum NCC2705 and Thermobifida fusca YX) to search for proteins that are unique to Actinobacteria. Our analyses have identified 233 actinobacteria-specific proteins, homologues of which are generally not present in any other bacteria. These proteins can be grouped as follows: (i) 29 proteins uniquely present in most sequenced actinobacterial genomes; (ii) 6 proteins present in almost all actinobacteria except Bifidobacterium longum and another 37 proteins absent in B. longum and few other species; (iii) 11 proteins which are mainly present in Corynebacterium, Mycobacterium and Nocardia (CMN) subgroup as well as Streptomyces, T. fusca and Frankia sp., but they are not found in Bifidobacterium and Micrococcineae; (iv) 8 proteins that are specific for T. fusca and Streptomyces species, plus 2 proteins also present in the Frankia species; (v) 13 proteins that are specific for the Corynebacterineae or the CMN group; (vi) 14 proteins only found in Mycobacterium and Nocardia; (vii) 24 proteins unique to different Mycobacterium species; (viii) 8 proteins specific to the Micrococcineae; (ix) 85 proteins which are distributed sporadically in actinobacterial species. Additionally, many examples of lateral gene transfer from Actinobacteria to Magnetospirillum magnetotacticum have also been identified. The identified proteins provide novel molecular means for defining and circumscribing the Actinobacteria phylum and a number of subgroups within it. The distribution of these proteins also provides useful information regarding interrelationships among the actinobacterial subgroups. Most of these proteins are of unknown function and studies aimed at understanding their cellular functions should reveal common biochemical and physiological characteristics unique to either all actinobacteria or particular subgroups of them. The identified proteins also provide potential targets for development of drugs that are specific for actinobacteria. PMID- 16670967 TI - Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species. AB - In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production. PMID- 16670968 TI - Studies on two closely related species of octocorallians: biochemical and molecular characteristics of the organic matrices of endoskeletal sclerites. AB - Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein-polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37 kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22 kDa for S. polydactyla. A major protein band of about 67 kDa in L. crassum and two bands of proteins of about 70 and 63 kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca(2+) overlay analysis was conducted in the extract via (45)Ca autoradiography. The 102- and 67 kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca(2+) binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated. PMID- 16670969 TI - High-yielding enzymatic alpha-glucosylation of pyridoxine by marine alpha glucosidase from Aplysia fasciata. AB - We recently succeeded in the identification and purification of an interesting marine exo-alpha-glucosidase (EC 3.2.1.20) from the anaspidean mollusc Aplysia fasciata. The enzyme was characterized by good transglycosylation activity toward different acceptors using maltose as donor. High-yielding enzymatic alpha glycosylation of pyridoxine using this marine enzyme is reported here; the reaction has been optimized, reaching 80% molar yield of products (pyridoxine monoglucosides 24 g/l; pyridoxine isomaltoside 35 g/l). High selectivity toward the 5' position is observed for both monoglucoside and disaccharide formation. This is the first report describing the enzymatic production of pyridoxine isomaltoside. PMID- 16670970 TI - Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific mitochondrial primers. AB - This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working. PMID- 16670974 TI - Evolutionary feedback mediated through population density, illustrated with viruses in chemostats. AB - A cornerstone of evolutionary ecology is that population density affects adaptation: r and K selection is the obvious example. The reverse is also appreciated: adaptation impacts population density. Yet, empirically demonstrating a direct connection between population density and adaptation is challenging. Here, we address both evolution and ecology of population density in models of viral (bacteriophage) chemostats. Chemostats supply nutrients for host cell growth, and the hosts are prey for viral reproduction. Two different chemostat designs have profoundly different consequences for viral evolution. If host and virus are confined to the same chamber, as in a predator-prey system, viral regulation of hosts feeds back to maintain low viral density (measured as infections per cell). Viral adaptation impacts host density but has a small effect on equilibrium viral density. More interesting are chemostats that supply the viral population with hosts from a virus-free refuge. Here, a type of evolutionary succession operates: adaptation at low viral density leads to higher density, but high density then favors competitive ability. Experiments support these models with both phenotypic and molecular data. Parallels to these designs exist in many natural systems, so these experimental systems may yield insights to the evolution and regulation of natural populations. PMID- 16670975 TI - The confusion effect in predatory neural networks. AB - A simple artificial neural network model of image reconstruction in sensory maps is presented to explain the difficulty predators experience in targeting prey in large groups (the confusion effect). Networks are trained to reconstruct multiple randomly conformed "retinal" images of prey groups in an internal spatial map of their immediate environment. They are then used to simulate prey targeting by predators on groups of specific conformation. Networks trained with the biologically plausible associative reward-penalty method produce a more realistic model of the confusion effect than those trained with the popular but biologically implausible backpropagation method. The associative reward-penalty model makes the novel prediction that the accuracy-group size relationship is U shaped, and this prediction is confirmed by empirical data gathered from interactive computer simulation experiments with humans as "predators." The model further predicts all factors known from previous empirical work (and most factors suspected) to alleviate the confusion effect: increased relative intensity of the target object, heterogeneity of group composition, and isolation of the target. Interestingly, group compaction per se is not predicted to worsen predator confusion. This study indicates that the relatively simple, nonattentional mechanism of information degradation in the sensory mapping process is potentially important in generating the confusion effect. PMID- 16670976 TI - Three-way interactions among mutualistic mycorrhizal fungi, plants, and plant enemies: hypotheses and synthesis. AB - A number of studies have shown that an association with mycorrhizal fungi can alter the outcome of interactions between plants and their enemies. While the directions of these effects vary, their strength suggests the need for greater attention to multispecies interactions among plant enemies, plants, and mycorrhizal fungi. We recognize that mycorrhizal fungi could effect plant enemies by improving plant nutrition, modifying plant tolerance, or modifying plant defenses. In addition, mycorrhizal fungi could directly interfere with pathogen infection, herbivory, or parasitism by occupying root space. We formalize these alternative outcomes of multispecies interactions and explore the long-term dynamics of the plant-enemy interactions based on these different scenarios using a general model of interactions between plants and plant enemies. We then review the literature in terms of the assumptions of the alternative mechanisms and the predictions of these models. Through this effort, we identify new directions in the study of tritrophic interactions between enemies, plants, and soil mutualists. PMID- 16670977 TI - Is evolution necessary for range expansion? Manipulating reproductive timing of a weedy annual transplanted beyond its range. AB - Ecologists often consider how environmental factors limit a species' geographic range. However, recent models suggest that geographic distribution also may be determined by a species' ability to adapt to novel environmental conditions. In this study, we empirically tested whether further evolution would be necessary for northern expansion of the weedy annual cocklebur (Xanthium strumarium) in its native North American range. We transplanted seedlings beyond the northern border and photoperiodically manipulated reproductive timing, a trait important for adaptation to shorter growing seasons at higher latitudes within the range, to determine whether further evolution of this trait would result in a phenotype viable beyond the range. Earlier reproductive induction enabled plants to produce mature seeds beyond the range and to achieve a reproductive output similar to those grown within the range. Therefore, evolution of earlier reproduction in marginal populations would be necessary for northward range expansion. This study is the first to empirically show that evolution in an ecologically important trait would enable a species to survive and reproduce beyond its current range. These results suggest that relatively few traits may limit a species' range and that identifying evolutionary constraints on such traits could be important for predicting geographic distribution. PMID- 16670978 TI - Resource-dependent dispersal and the speed of biological invasions. AB - Many mobile organisms exhibit resource-dependent movement in which movement rates adjust to changes in local resource densities through changes in either the probability of moving or the distance moved. Such changes may have important consequences for invasions because reductions in resources behind an invasion front may cause higher dispersal while simultaneously reducing population growth behind the front and thus lowering the number of dispersers. Intuiting how the interplay between population growth and dispersal affects invasions is difficult without mathematical models, yet most models assume dispersal rates are constant. Here we present spatial-spread models that allow for consumer-resource interactions and resource-dependent dispersal. Our results show that when resources affect the probability of dispersal, then the invasion dynamics are no different than if resources did not affect dispersal. When resources instead affect the distance dispersed, however, the invasion dynamics are strongly affected by the strength of the consumer-resource interaction, and population cycles behind the wave front lead to fluctuating rates of spread. Our results suggest that for actively dispersing invaders, invasion dynamics can be determined by species interactions. More practically, our work suggests that reducing invader densities behind the front may be a useful method of slowing an invader's rate of spread. PMID- 16670979 TI - Metapopulation extinction thresholds in rain forest remnants. AB - Although habitat fragmentation is a major threat to global biodiversity, the demographic mechanisms underlying species loss from tropical forest remnants remain largely unexplored. In particular, no studies at the landscape scale have quantified fragmentation's impacts on colonization, extinction, and local population growth simultaneously. In central Amazonia, we conducted a multiyear demographic census of 292 populations of two leaf-inhabiting (i.e., epiphyllous) bryophyte species transplanted from continuous forest into a network of 10 study sites ranging from 1, 10, and 100 to > 10,000 ha in size. All populations experienced significantly positive local growth (lambda > 1) and a nearly constant per-generational extinction probability (15%). However, experimental leaf patches in reserves of > or = 100 ha experienced nearly double (48%) the colonization probability observed in small reserves (27%), suggesting that the proximate cause of epiphyll species loss in small fragments (< or = 10 ha) is reduced colonization. Nonetheless, populations of small fragments exhibit rates of colonization above patch extinction, positive local growth, and low temporal variation, which are features that should theoretically reduce the probability of extinction. This result suggests that for habitat-tracking metapopulations subject to frequent and stochastic turnover events, including epiphylls, colonization/extinction ratios must be maintained well above unity to ensure metapopulation persistence. PMID- 16670980 TI - Intraspecific competitive divergence and convergence under assortative mating. AB - Ecologically driven sympatric speciation has received much attention recently. We investigate a multilocus model of a quantitative trait that is under frequency dependent selection caused by intraspecific competition and acts as mating character for assortment. We identify the conditions that lead to the establishment of reproductively isolated clusters. This may be interpreted as evolutionary splitting or sympatric speciation. In our model, there are parameters that independently determine the strength of assortment, the costs for being choosy, and the strength of frequency-dependent natural selection. Sufficiently strong frequency dependence leads to disruptive selection on the phenotypes. The population consists of (sexual) haploid individuals. If frequency dependence is strong enough to induce disruptive selection and costs are absent or low, the result of evolution depends in a distinctive nonlinear way on the strength of assortment: under moderately strong assortment, less genetic variation is maintained than under weak or strong assortment, and sometimes there is none at all. Evolutionary splitting occurs only if frequency dependence and assortment are both strong enough and costs are low. Even then, the evolutionary outcome depends on the genetics and the initial conditions. The roles of the number of loci, of linkage, and of asymmetric selection are also explored. PMID- 16670981 TI - Context-dependent intergenerational effects: the interaction between past and present environments and its effect on population dynamics. AB - Intergenerational effects arise when parents' actions influence the reproduction and survival of their offspring and possibly later descendants. Models suggest that intergenerational effects have important implications for both population dynamical patterns and the evolution of life-history traits. However, these will depend on the nature and duration of intergenerational effects. Here we show that manipulating parental food environments of soil mites produced intergenerational effects that were still detectable in the life histories of descendents three generations later. Intergenerational effects varied in different environments and from one generation to the next. In low-food environments, variation in egg size altered a trade-off between age and size at maturity and had little effect on the size of eggs produced in subsequent generations. Consequently, intergenerational effects decreased over time. In contrast, in high-food environments, variation in egg size predominantly influenced a trade-off between fecundity and adult survival and generated increasing variation in egg size. As a result, the persistence and significance of intergenerational effects varied between high- and low-food environments. Context-dependent intergenerational effects can therefore have complex but important effects on population dynamics. PMID- 16670982 TI - Flight performance and competitive displacement of hummingbirds across elevational gradients. AB - Hummingbirds, with their impressive flight ability and competitive aerial contests, make ideal candidates for applying a mechanistic approach to studying community structure. Because flight costs are influenced by abiotic factors that change systematically with altitude, elevational gradients provide natural experiments for hummingbird flight ecology. Prior attempts relied on wing disc loading (WDL) as a morphological surrogate for flight performance, but recent analyses indicate this variable does not influence either territorial behavior or competitive ability. Aerodynamic power, by contrast, can be derived from direct measurements of performance and, like WDL, declines across elevations. Here, I demonstrate for a diverse community of Andean hummingbirds that burst aerodynamic power is associated with territorial behavior. Along a second elevational gradient in Colorado, I tested for correlated changes in aerodynamic power and competitive ability in two territorial hummingbirds. This behavioral analysis revealed that short-winged Selasphorus rufus males are dominant over long-winged Selasphorus platycercus males at low elevations but that the roles are reversed at higher elevations. Several lines of evidence support the hypothesis that the burst rather than sustained aerodynamic performance mediates competitive ability at high elevation. A minimum value for burst power may be required for successful competition, but other maneuverability features gain importance when all competitors have sufficient muscle power, as occurs at low elevations. PMID- 16670983 TI - Are fluctuating asymmetry studies adequately sampled? Implications of a new model for size distribution. AB - Previous work on fluctuating asymmetry (FA) has highlighted its controversial relationship with environmental stress and genetic architecture. While size-based measures of FA have been assumed to have half-normal distributions within populations, studies of model developmental mechanisms have suggested other plausible distributions for FA. We investigated the distribution of FA in large empirical data sets of wing shape and wing size asymmetry from three species of insects (cotton aphid Aphis gossipyii Glover, honeybee Apis mellifera, and long legged fly Chrysosoma crinitus). Regardless of measurement method, FA was best described by a double Pareto-lognormal (DPLN) distribution or one of its limiting functional forms. To investigate convergence of mean sample FA to the population mean at various sample sizes, we sampled repeatedly under a DPLN distribution using parameter values that best fitted our data. Sample variances are much larger, and hence, convergence is slowed considerably with univariate or multivariate size-based measures of FA in contrast to a multivariate shape-based measure of FA. We suggest that much of the past work on FA may be undersampled, and we recommend using multivariate shape-based approaches or collecting larger data sets in future studies. We also discuss the implications of the DPLN distribution for understanding the developmental mechanisms underlying FA. PMID- 16670984 TI - Plugging space into predator-prey models: an empirical approach. AB - Extrapolating ecological processes from small-scale experimental systems to scales of natural populations usually entails a considerable increase in spatial heterogeneity, which may affect process rates and, ultimately, population dynamics. We demonstrate how information on the heterogeneity of natural populations can be taken into account when scaling up laboratory-derived process functions, using the technique of moment approximation. We apply moment approximation to a benthic crustacean predator-prey system, where a laboratory derived functional response is made spatial by including correction terms for the variance in prey density and the covariance between prey and predator densities observed in the field. We also show how moment approximation may be used to incorporate spatial information into a dynamic model of the system. While the nonspatial model predicts stable dynamics, its spatial equivalent also produces bounded fluctuations, in agreement with observed dynamics. A detailed analysis shows that predator-prey covariance, but not prey variance, destabilizes the dynamics. We conclude that second-order moment approximation may provide a useful technique for including spatial information in population models. The main advantage of the method is its conceptual value: by providing explicit estimates of variance and covariance effects, it offers the possibility of understanding how heterogeneity affects ecological processes. PMID- 16670985 TI - Distributions of habitat suitability and the abundance-occupancy relationship. AB - Positive abundance-occupancy relationships (a relationship between the number of sites a species occupies and the average density of individuals in occupied sites) are widespread through a range of taxa. The simplest model for this is the "vital rates" model, which proposes that habitat suitability varies spatially; increasing average habitat quality thus leads to simultaneous increases in average densities within occupied areas, as well as the total area that is habitable. This model has not been tested. We develop a general analytical version of this model and show that it predicts that the skewness of population size or aggregation of individuals within sites should vary systematically with density and occupancy, depending on the distribution of habitat suitability, and that the variance in occupancy should be highest at low densities. We compare these predictions with data from the British Trust for Ornithology's Common Birds Census, and we find systematic changes in both variance and skewness of density, both intra- and interspecifically. PMID- 16670986 TI - A phylogenetic analysis of the allometry of diving. AB - The oxygen store/usage hypothesis suggests that larger animals are able to dive for longer and hence deeper because oxygen storage scales isometrically with body mass, whereas oxygen usage scales allometrically with an exponent <1 (typically 0.67-0.75). Previous tests of the allometry of diving tend to reject this hypothesis, but they are based on restricted data sets or invalid statistical analyses (which assume that every species provides independent information). Here we apply information-theoretic statistical methods that are phylogenetically informed to a large data set on diving variables for birds and mammals to describe the allometry of diving. Body mass is strongly related to all dive variables except dive:pause ratio. We demonstrate that many diving variables covary strongly with body mass and that they have allometric exponents close to 0.33. Thus, our results fail to falsify the oxygen store/usage hypothesis. The allometric relationships for most diving variables are statistically indistinguishable for birds and mammals, but birds tend to dive deeper than mammals of equivalent mass. The allometric relationships for all diving variables except mean dive duration are also statistically indistinguishable for all major taxonomic groups of divers within birds and mammals, with the exception of the procellariiforms, which, strictly speaking, are not true divers. PMID- 16670988 TI - A test of the "sexy son" hypothesis: sons of polygynous collared flycatchers do not inherit their fathers' mating status. AB - According to the original "sexy son" hypothesis, a female may benefit from pairing with an already-mated male despite a reduction in fecundity because her sons inherit their father's attractiveness. We used data from a long-term study of collared flycatchers (Ficedula albicollis) collected during 24 years to test this prediction. Our results show that the sons of polygynously mated females fledged in poor condition and therefore did not inherit their father's large forehead patch (a condition-dependent display trait) or mating status. From the female's perspective, polygynous pairing resulted in fewer recruited grandchildren than did a monogamous pairing. The reproductive value of sons did not outweigh the fecundity costs of polygyny because the low paternal care reduced the attractiveness of sons. When there are long-lasting parental effects on offspring attractiveness, costs of polygyny may include the production of nonsexy sons. PMID- 16670987 TI - Anther evolution: pollen presentation strategies when pollinators differ. AB - Male-male competition in plants is thought to exert selection on flower morphology and on the temporal presentation of pollen. Theory suggests that a plant's pollen dosing strategy should evolve to match the abundance and pollen transfer efficiency of its pollinators. Simultaneous pollen presentation should be favored when pollinators are infrequent or efficient at delivering the pollen they remove, whereas gradual dosing should optimize delivery by frequent and wasteful pollinators. Among Penstemon and Keckiella species, anthers vary in ways that affect pollen release, and the morphology of dried anthers reliably indicates how they dispense pollen. In these genera, hummingbird pollination has evolved repeatedly from hymenopteran pollination. Pollen production does not change with evolutionary shifts between pollinators. We show that after we control for phylogeny, hymenopteran-adapted species present their pollen more gradually than hummingbird-adapted relatives. In a species pair that seemed to defy the pattern, the rhythm of anther maturation produced an equivalent dosing effect. These results accord with previous findings that hummingbirds can be more efficient than bees at delivering pollen. PMID- 16670989 TI - Complex multivariate sexual selection on male acoustic signaling in a wild population of Teleogryllus commodus. AB - Mate choice may impose both linear (i.e., directional) and nonlinear (i.e., quadratic and correlational) sexual selection on advertisement traits. Traditionally, mate recognition and sensory tuning have been thought to impose stabilizing (i.e., negative quadratic) sexual selection, whereas adaptive mate choice effects directional selection. It has been suggested that adaptive choice may exert positive quadratic and/or correlational sexual selection. Earlier, we showed that five structural components of the advertisement call of male field crickets (Teleogryllus commodus) were under multivariate stabilizing selection under laboratory conditions. Here we experimentally estimate selection on these five traits plus a measure of calling activity (the number of repeats in a looped bout of calling) in the field. There was general support for multivariate stabilizing selection on call structure, and calling activity was under strong positive directional selection, as predicted for a signal of genetic quality. There was, however, also appreciable correlational selection, suggesting an interaction between male call structure and calling effort. Interestingly, selection for short interbout durations of silence favored longer intercall durations in the field, in contrast to results from continuous looped call playback in the laboratory. We discuss the general importance of nonlinear selection in the honest signaling of genetic quality. PMID- 16670990 TI - Polyploids with different origins and ancestors form a single sexual polyploid species. AB - Polyploidization is one of the few mechanisms that can produce instantaneous speciation. Multiple origins of tetraploid lineages from the same two diploid progenitors are common, but here we report the first known instance of a single tetraploid species that originated repeatedly from at least three diploid ancestors. Parallel evolution of advertisement calls in tetraploid lineages of gray tree frogs has allowed these lineages to interbreed, resulting in a single sexually interacting polyploid species despite the separate origins of polyploids from different diploids. Speciation by polyploidization in these frogs has been the source of considerable debate, but the various published hypotheses have assumed that polyploids arose through either autopolyploidy or allopolyploidy of extant diploid species. We utilized molecular markers and advertisement calls to infer the origins of tetraploid gray tree frogs. Previous hypotheses did not sufficiently account for the observed data. Instead, we found that tetraploids originated multiple times from extant diploid gray tree frogs and two other, apparently extinct, lineages of tree frogs. Tetraploid lineages then merged through interbreeding to result in a single species. Thus, polyploid species may have complex origins, especially in systems in which isolating mechanisms (such as advertisement calls) are affected directly through hybridization and polyploidy. PMID- 16670991 TI - Transactional skew and assured fitness return models fail to predict patterns of cooperation in wasps. AB - Cooperative breeders often exhibit reproductive skew, where dominant individuals reproduce more than subordinates. Two approaches derived from Hamilton's inclusive fitness model predict when subordinate behavior is favored over living solitarily. The assured fitness return (AFR) model predicts that subordinates help when they are highly likely to gain immediate indirect fitness. Transactional skew models predict dominants and subordinates "agree" on a level of reproductive skew that induces subordinates to join groups. We show the AFR model to be a special case of transactional skew models that assumes no direct reproduction by subordinates. We use data from 11 populations of four wasp species (Polistes, Liostenogaster) as a test of whether transactional frameworks suffice to predict when subordinate behavior should be observed in general and the specific level of skew observed in cooperative groups. The general prediction is supported; in 10 of 11 cases, transactional models correctly predict presence or absence of cooperation. In contrast, the specific prediction is not consistent with the data. Where cooperation occurs, the model accurately predicts highly biased reproductive skew between full sisters. However, the model also predicts that distantly related or unrelated females should cooperate with low skew. This prediction fails: cooperation with high skew is the observed norm. Neither the generalized transactional model nor the special-case AFR model can explain this significant feature of wasp sociobiology. Alternative, nontransactional hypotheses such as parental manipulation and kin recognition errors are discussed. PMID- 16670992 TI - Geographical variation in selection, from phenotypes to molecules. AB - Molecular technologies now allow researchers to isolate quantitative trait loci (QTLs) and measure patterns of gene sequence variation within chromosomal regions containing important polymorphisms. I develop a simulation model to investigate gene sequence evolution within genomic regions that harbor QTLs. The QTLs influence a trait experiencing geographical variation in selection, which is common in nature and produces obvious differentiation at the phenotypic level. Counter to expectations, the simulations suggest that selection can substantially affect quantitative genetic variation without altering the amount and pattern of molecular variation at sites closely linked to the QTLs. Even with large samples of gene sequences, the likelihood of rejecting neutrality is often low. The exception is situations where strong selection is combined with low migration among demes, conditions that may be common in many plant species. The results have implications for gene sequence surveys and, perhaps more generally, for interpreting the apparently weak connection between levels of molecular and quantitative trait variation within species. PMID- 16670993 TI - Manipulation of bee behavior by inflorescence architecture and its consequences for plant mating. AB - Angiosperms display flowers in many three-dimensional arrangements, but the functional significance of this diversity is largely unknown. We examined influences of inflorescence architecture on pollination and mating by quantifying the responses of bumblebees to three architectures and then using these observations as the basis of a model that simulated pollen dispersal. On artificial panicles, racemes, and umbels, each with 12 identical flowers, bees visited one more flower, on average, on umbels than on panicles (with racemes being intermediate). In contrast to this weak response, the consistency of foraging paths among flowers differed strongly among architectures (raceme > panicle > umbel). The simulation model revealed limited differences in self pollination and pollen export among the three inflorescence designs when all flowers presented and received pollen, because mating differences depended on only the number of flowers visited. In contrast, in simulations of inflorescences on which pollen receipt and presentation were segregated so as to minimize interference among flowers, the consistency of movement paths governed mating. In this case, racemes self-pollinated much less than umbels (with panicles being intermediate), and racemes exported much more pollen than umbels and panicles. These effects have diverse consequences for the evolution of inflorescence architecture, flower design, and sexual segregation. PMID- 16670994 TI - The temperature-size rule in ectotherms: may a general explanation exist after all? AB - The majority of ectotherms mature at a larger size at lower rearing temperatures. Although this temperature-size rule is well established, a general explanation for this phenomenon has remained elusive. In this article, we address the problem by exploring the proximate and ultimate reasons for why a temperate grasshopper, Chorthippus brunneus, is an exception to the temperature-size rule. Using a complete set of life-history data to parameterize an established life-history model, we show that it is optimal for this species to mature at a larger size at higher temperatures. We also show that plasticity in adult size is determined by the relative difference between the minimum temperature thresholds for growth and development rates. The mechanism relates to aspects of the biophysical model of van der Have and de Jong. Ectotherms that obey the temperature-size rule are identified as having a higher temperature threshold for development rate than for growth rate; exceptions are identified as having a lower temperature threshold for development rate than for growth rate. The latter scenario may arise broadly in two ways. These are discussed in reference to the thermal biology of temperate grasshoppers and ectotherms in general. PMID- 16670995 TI - Diet, morphology, and interspecific killing in carnivora. AB - Interspecific killing is a key determinant of the abundances and distributions of carnivores, their prey, and nonprey community members. Similarity of body size has been proposed to lead competitors to seek similar prey, which increases the likelihood of interference encounters, including lethal ones. We explored the influence of body size, diet, predatory habits, and taxonomic relatedness on interspecific killing. The frequency of attacks depends on differences in body size: at small and large differences, attacks are less likely to occur; at intermediate differences, killing interactions are frequent and related to diet overlap. Further, the importance of interspecific killing as a mortality factor in the victim population increases with an increase in body size differences between killers and victims. Carnivores highly adapted to kill vertebrate prey are more prone to killing interactions, usually with animals of similar predatory habits. Family-level taxonomy influences killing interactions; carnivores tend to interact more with species in the same family than with species in different families. We conclude that although resource exploitation (diet), predatory habits, and taxonomy are influential in predisposing carnivores to attack each other, relative body size of the participants is overwhelmingly important. We discuss the implications of interspecific killing for body size and the dynamics of geographic ranges. PMID- 16670996 TI - A hydrodynamics approach to the evolution of multicellularity: flagellar motility and germ-soma differentiation in volvocalean green algae. AB - During the unicellular-multicellular transition, there are opportunities and costs associated with larger size. We argue that germ-soma separation evolved to counteract the increasing costs and requirements of larger multicellular colonies. Volvocalean green algae are uniquely suited for studying this transition because they range from unicells to multicellular individuals with germ-soma separation. Because Volvocales need flagellar beating for movement and to avoid sinking, their motility is modeled and analyzed experimentally using standard hydrodynamics. We provide comparative hydrodynamic data of an algal lineage composed of organisms of different sizes and degrees of complexity. In agreement with and extending the insights of Koufopanou, we show that the increase in cell specialization as colony size increases can be explained in terms of increased motility requirements. First, as colony size increases, soma must evolve, the somatic-to-reproductive cell ratio increasing to keep colonies buoyant and motile. Second, increased germ-soma specialization in larger colonies increases motility capabilities because internalization of nonflagellated germ cells decreases colony drag. Third, our analysis yields a limiting maximum size of the volvocalean spheroid that agrees with the sizes of the largest species known. Finally, the different colony designs in Volvocales reflect the trade-offs between reproduction, colony size, and motility. PMID- 16670997 TI - Range size: disentangling current traits and phylogenetic and biogeographic factors. AB - The range size of a species can be determined by its current traits and by phylogenetic and biogeographic factors. However, only rarely have these factors been studied in combination. We use data on the geographic range sizes of all 26 Sylvia warblers to explicitly test whether range size was determined by current species-specific traits (e.g., body size, dispersal ability), phylogenetic factors (e.g., age of the lineage), or environmental, biogeographic factors (e.g., latitudinal position of the range). The results demonstrated that current traits and phylogenetic and biogeographic factors were interrelated. While a number of factors were significant in simple regression analyses, only one factor determined range size in the multiple regression analyses--dispersal ability. Species with better dispersal ability had larger ranges than species with poorer dispersal ability. Apparent increases of range size with latitude or with the age of the species resulted from correlations with dispersal ability. While the most significant factor that influences the range size of a group of species might differ from one group to the next, these results demonstrate that studies that focus only on a single, for example, phylogenetic, factor might yield misleading results. PMID- 16670999 TI - The offspring-size/clutch-size trade-off in mammals. AB - The Smith-Fretwell model for optimal offspring size assumes the existence of an inverse proportional relationship (i.e., trade-off) between the number of offspring and the amount of resources invested in an individual offspring; virtually all of the many models derived from theirs make the same trade-off assumption. Over the last 30 years it has become apparent that the predicted proportionality is often not observed when evaluated across species. We develop a general allometric approach to correct for size-related differences in the resources available for reproduction. Using data on mammals, we demonstrate that the predicted inverse proportional relationship between number of offspring and offspring size is closely approached after correcting for allocation, though there is a slight curvature in the relationship. We discuss applications for this approach to other organisms, possible causes for the curvature, and the usefulness of allometries for estimating life-history variables that are difficult to measure. PMID- 16670998 TI - The costs and benefits of genetic heterogeneity in resistance against parasites in social insects. AB - The occurrence of polygyny and polyandry in social insects has long puzzled evolutionary biologists. If cooperation requires genetic relatedness, how do we explain the occurrence and maintenance of mechanisms that reduce the degree of relatedness among colony members? A much-discussed hypothesis states that genetically diverse colonies are more resistant to parasitism than homogenous colonies because genetic diversity reduces the spread of a disease within a colony. However, as we will argue in this note, a necessary condition for the parasite hypothesis is that genetically heterogeneous colonies have a larger suite of parasites that are capable of infecting them. This implicit relationship is important because it implies that even if the cost per infection is reduced, this may not be sufficient to offset the increased rate of acquiring infections. The advantages of genetic heterogeneity as a defense against parasites thus may not be as big as commonly thought. PMID- 16671000 TI - Changes over time in the spatiotemporal dynamics of cyclic populations of field voles (Microtus agrestis L.). AB - We demonstrate changes over time in the spatial and temporal dynamics of an herbivorous small rodent by analyzing time series of population densities obtained at 21 locations on clear cuts within a coniferous forest in Britain from 1984 to 2004. Changes had taken place in the amplitude, periodicity, and synchrony of cycles and density-dependent feedback on population growth rates. Evidence for the presence of a unidirectional traveling wave in rodent abundance was strong near the beginning of the study but had disappeared near the end. This study provides empirical support for the hypothesis that the temporal (such as delayed density dependence structure) and spatial (such as traveling waves) dynamics of cyclic populations are closely linked. The changes in dynamics were markedly season specific, and changes in overwintering dynamics were most pronounced. Climatic changes, resulting in a less seasonal environment with shorter winters near the end of the study, are likely to have caused the changes in vole dynamics. Similar changes in rodent dynamics and the climate as reported from Fennoscandia indicate the involvement of large-scale climatic variables. PMID- 16671001 TI - Herbivory and competition interact to affect reproductive traits and mating system expression in Impatiens capensis. AB - As a step toward understanding how community context shapes mating system evolution, we investigated the combined role of two plant antagonisms, vegetative herbivory and intraspecific competition, for reproduction and mating system expression (relative production of selfing, cleistogamous and facultatively outcrossing, chasmogamous flowers and fruits) of Impatiens capensis. In a survey of I. capensis populations, we found that vegetative herbivory and intraspecific competition were positively correlated. In a greenhouse experiment where leaf damage and plant density were manipulated, multispecies interactions had dramatic effects on reproductive and mating system traits. Despite having additive effects on growth, herbivory and competition had nonadditive effects for mating system expression, chasmogamous fruit production, flower number and size, and cleistogamous flower production. Our results demonstrate that competitive interactions influence the effect of herbivory (and vice versa) on fitness components and mating system, and thus antagonisms may have unforeseen consequences for mating system evolution, population genetic diversity, and persistence. PMID- 16671002 TI - Circadian rhythmicity and photoperiodism in the pitcher-plant mosquito: can the seasonal timer evolve independently of the circadian clock? AB - The two major rhythms of the biosphere are daily and seasonal; the two major adaptations to these rhythms are the circadian clock, mediating daily activities, and the photoperiodic timer, mediating seasonal activities. The mechanistic connection between the circadian clock and the photoperiodic timer remains unresolved. Herein, we show that the rhythmic developmental response to exotic light:dark cycles, usually used to infer a causal connection between the circadian clock and the photoperiodic timer, has evolved independently of the photoperiodic timer in the pitcher-plant mosquito Wyeomyia smithii across the climatic gradient of eastern North America from Florida to Canada and from the coastal plain to the mountains. We conclude that the photoperiodic timing of seasonal events can evolve independently of the daily circadian clock. PMID- 16671003 TI - A matter of measurements: challenges and approaches in the comparative analysis of static allometries. AB - Comparisons of static allometries are frequently used to gain insights into patterns and processes underlying morphological and developmental evolution. A study by J. L. Tomkins and coworkers, recently published in the American Naturalist, examined complex nonlinear allometries in three insect species in which males are dimorphic in the expression of secondary sexual traits. Employing a novel approach to analyzing male allometries in these organisms, the authors were able to show that developmental reprogramming of trait primordia is not necessary to explain allometric scaling in two of the species examined, contrary to several previous studies on the same species. Instead, male dimorphisms could be explained by simple exponential growth, an important result that carries with it major evolutionary and developmental implications. Using this study as an example, I highlight some of the methodological challenges involved in analyzing and comparing static allometries and in inferring the developmental processes that underlie them. I end by discussing how correct application of hypothesis testing, on one side, and basic anatomy and developmental biology, on the other, should guide how morphology is measured. PMID- 16671005 TI - The structures of letters and symbols throughout human history are selected to match those found in objects in natural scenes. AB - Are there empirical regularities in the shapes of letters and other human visual signs, and if so, what are the selection pressures underlying these regularities? To examine this, we determined a wide variety of topologically distinct contour configurations and examined the relative frequency of these configuration types across writing systems, Chinese writing, and nonlinguistic symbols. Our first result is that these three classes of human visual sign possess a similar signature in their configuration distribution, suggesting that there are underlying principles governing the shapes of human visual signs. Second, we provide evidence that the shapes of visual signs are selected to be easily seen at the expense of the motor system. Finally, we provide evidence to support an ecological hypothesis that visual signs have been culturally selected to match the kinds of conglomeration of contours found in natural scenes because that is what we have evolved to be good at visually processing. PMID- 16671006 TI - Top-down and bottom-up control of life-history strategies in coho salmon (Oncorhynchus kisutch). AB - Sexual maturation profoundly affects population dynamics, but the degrees to which genetic, top-down, and bottom-up controls affect age at maturity are unclear. Salmonid fishes have plastic age at maturity, and we consider genetic and environmental effects on this trait by developing fitness functions for coho salmon (Oncorhynchus kisutch). The functions are based on size-specific survival and reproductive success, where reproductive success is the product of fecundity and ability to defend nests (females) or the product of sperm volume and ability to mate (males). We model genetic and bottom-up controls (e.g., food availability) with an environmentally explicit growth function and top-down control (predation mortality) with survival functions that consider both size dependent and size-independent mortality. For females, we predict that early maturation rarely maximizes fitness, but males can maximize fitness by maturing early if they grow well in freshwater. We predict that early maturation is most affected by the bottom-up effects of resource distribution at sea, followed by bottom-up and genotypic effects in freshwater. Top-down processes are predicted to have strong effects on the likelihood of delayed maturation. PMID- 16671007 TI - Brown ground: a soil carbon analogue for the green world hypothesis? AB - For many decades, ecologists have asked what prevents herbivores from consuming most of the plant biomass in terrestrial ecosystems, or "Why is the world green?" Here I ask the analogous question for detritivores: what prevents them from degrading most of the organic material in soils, or "Why is the ground brown?" For fresh plant detritus, constraints on decomposition closely parallel constraints on herbivory: both herbivore and decomposer populations may be controlled by plant tissue chemistry from the bottom up and predators from the top down. However, the majority of soil carbon is not plant litter but carbon that has been consumed by detritivores and reprocessed into humic compounds with complex and random chemical structures. This carbon persists mainly because the chemical properties of humic compounds and interactions with soil minerals constrain decomposition by extracellular enzymes in soil. Other constraints on decomposers, such as nutrient limitation of enzyme production and competition with opportunistic microbes, also contribute to brown ground. Ultimately, the oldest soil carbon persists via transformation into complex molecules that are impervious to enzymatic attack and effectively decoupled from processing by the soil food web. PMID- 16671008 TI - Inedible producers in food webs: controls on stoichiometric food quality and composition of grazers. AB - Ecological stoichiometry and food web theories focus on distinct mechanisms that shape communities. These mechanisms, however, likely interact in ways that neither theory alone addresses. To illustrate, we show how a model that tracks flow of energy and nutrients through two producers and two grazers reveals two indirect, interrelated roles for "neutrally inedible" producers. First, inedible producers can exert controls over the nutrient content of edible producers and indirectly influence whether grazers are nutrient or energy limited. Second, through these controls, inedible producers can shape community assembly by excluding grazers that are weak competitors for nutrients contained in edible producers. A mesocosm experiment revealed patterns consistent with both predictions: high abundances of inedible algae were accompanied by low phosphorus contents of edible algae and low abundances of the grazer Daphnia. Both lines of inference suggest that interactions between stoichiometry and plant heterogeneity may shape plankton communities. PMID- 16671009 TI - Divergent outcomes of reinforcement speciation: the relative importance of assortative mating and migration modification. AB - Most studies of reinforcement speciation focus on the evolution of assortative mating, but R. A. Fisher argued that migration modification is likely to be a common alternative mechanism. Despite previous models showing that assortative mating and migration modification may both be involved in reinforcement, no one has determined their relative evolutionary importance. This is surprising because understanding the biological conditions favoring these mechanisms may explain why certain pairs of species exhibit abutting, nonoverlapping geographical ranges with habitat fidelity while other pairs coexist in sympatry with sexual isolation. In this article, we explicitly model the evolution of both mechanisms simultaneously. First, we explore how these mechanisms differ in their evolutionary dynamics. Second, we ask how they affect each other's evolution and whether the interaction alters their relative importance in reinforcement. Our results reveal that assortative mating may evolve faster and under a broader range of biological conditions than migration modification. However, direct evolutionary interactions favor migration modification when populations experience strong divergent selection. Depending on the nature of postmating isolation, these mechanisms may either interfere with each other's evolution or coevolve in the same system. These results illustrate the importance of studying multiple mechanisms of speciation simultaneously in future speciation models. PMID- 16671010 TI - Phylogeographic lineages and species comparisons in conservation analyses: a case study of california herpetofauna. AB - Many phylogeographic studies have revealed strongly diverged lineages within species that are masked by a lack of congruent morphological differentiation. To assess the extent to which the genetic component of diversity affects conservation assessments, we compared spatial patterns of endemism and conservation value for 22 species of Californian amphibians and reptiles with the 75 phylogeographic lineages that they contain. We used bioclimatic distribution modeling with environmental layers to generate 5-km spatial-resolution maps of predicted distribution for each species and lineage. We found concentrations of lineage breaks across the Central Valley, San Francisco Bay, the Sierra Nevada, and the Tehachapi and Trinity ranges. Subdivision of the ranges of species into phylogeographic units revealed novel areas of endemism. Several areas of very high conservation value for lineages were not evident in the species-level analysis. These observations illustrate the importance of considering multiple levels of biodiversity in conservation assessments. PMID- 16671011 TI - Physiological effects on demography: a long-term experimental study of testosterone's effects on fitness. AB - Understanding physiological and behavioral mechanisms underlying the diversity of observed life-history strategies is challenging because of difficulties in obtaining long-term measures of fitness and in relating fitness to these mechanisms. We evaluated effects of experimentally elevated testosterone on male fitness in a population of dark-eyed juncos studied over nine breeding seasons using a demographic modeling approach. Elevated levels of testosterone decreased survival rates but increased success of producing extra-pair offspring. Higher overall fitness for testosterone-treated males was unexpected and led us to consider indirect effects of testosterone on offspring and females. Nest success was similar for testosterone-treated and control males, but testosterone-treated males produced smaller offspring, and smaller offspring had lower postfledging survival. Older, more experienced females preferred to mate with older males and realized higher reproductive success when they did so. Treatment of young males increased their ability to attract older females yet resulted in poor reproductive performance. The higher fitness of testosterone-treated males in the absence of a comparable natural phenotype suggests that the natural phenotype may be constrained. If this phenotype were to arise, the negative social effects on offspring and mates suggest that these effects might prevent high-testosterone phenotypes from spreading in the population. PMID- 16671012 TI - Historical contingency and animal diets: the origins of egg eating in snakes. AB - Evolutionary changes in animal diets must often begin through the inclusion of a novel food type as a minor component of the diet. An aspect of this initial change that has rarely been studied is the relationship between the existing diet and the use of specific novel foods. We used comparative analyses to test the hypothesis that, in snakes, feeding on squamate (lizard and snake) eggs or bird eggs--items that represent evolutionarily derived and, in most cases, minor components of the diet--is associated with feeding on squamates or birds, respectively. Phylogenetic concentrated-changes tests indicate a significant tendency for predation on eggs to arise in snake lineages characterized by feeding on the corresponding animals. These results also generally hold for analyses including only snake species that are likely to encounter eggs and are large enough to ingest them. The inferred histories of specialized egg eaters also support the hypothesis. Because snakes often use chemical cues to recognize prey, the observed phylogenetic patterns might be explained by chemical similarities between eggs and adult animals. Our results suggest broad effects of predispositions on snake diets and thus illustrate how historical contingencies can shape the ecology of organisms. PMID- 16671013 TI - Delayed density-dependent season length alone can lead to rodent population cycles. AB - Studies of cyclic microtine populations (voles and lemmings) have suggested a relationship between the previous year's population density and the subsequent timing of the onset of reproduction by overwintered breeding females. No studies have explored the importance of this relationship in the generation of population cycles. Here we mathematically examine the implications of variation in reproductive season length caused by delayed density-dependent changes in its start date. We demonstrate that when reproductive season length is a function of past population densities, it is possible to get realistic population cycles without invoking any changes in birth rates or survival. When parameterized for field voles (Microtus agrestis) in Kielder Forest (northern England), our most realistic model predicts population cycles of similar periodicity to the Kielder populations. Our study highlights the potential importance of density-dependent reproductive timing in microtine population cycles and calls for investigations into the mechanism(s) underlying this phenomenon. PMID- 16671014 TI - A theory of associating food types with their postingestive consequences. AB - Animals often face complex and changing food environments. While such environments are challenging, an animal should make an association between a food type and its properties (such as the presence of a nutrient or toxin). We use information theory concepts, such as mutual information, to establish a theory for the development of these associations. In this theory, associations are assumed to maximize the mutual information between foods and their consequences. We show that associations are invariably imperfect. An association's accuracy increases with the length of a feeding session and the relative frequency of a food type but decreases as time delay between consumption and postingestive consequence increases. Surprisingly, the accuracy of an association is independent of the number of additional food types in the environment. The rate of information transfer between novel foods and a forager depends on the forager's diet. In light of this theory, an animal's diet may have two competing goals: first, the provision of an appropriate balance of nutrients, and second, the ability to quickly and accurately learn the properties of novel foods. We discuss the ecological and behavioral implications of making associational errors and contrast the timescale and mechanisms of our theory with those of existing theory. PMID- 16671015 TI - Logic for designing nature reserves for multiple species. AB - We examine the logic of designing nature reserves to understand better how to integrate the concepts of representativeness and persistence. Simple models of viability are used to evaluate how the expected number of species in the reserve system changes with variation in the risk of extinction among species, their rate of occurrence, and the distribution of species. The optimal size of individual reserves increased with the mean and variance of the probability of extinction among species and with the rate at which the risk of extinction declines with the cost of each reserve. In contrast, the rate of occurrence of species within reserves and their rate of accumulation with increasing reserve area had a relatively minor influence on the optimal size of reserves. Patterns of endemism were most important for the location of reserves. Including differences among species in the analysis reduced the optimal number of individual reserves (and increased the size of each) when operating under a fixed budget compared with reserve designs based on single species. A case study in the city of Melbourne, Australia, demonstrates the conservation value of small (approximately 1 ha) grassland reserves and the underrepresentation of Melbourne's volcanic plains in the region's conservation network. PMID- 16671016 TI - The adaptive significance of sexually dimorphic scale rugosity in sea snakes. AB - In terrestrial snakes, rugose scales are uncommon and (if they occur) generally are found on both sexes. In contrast, rugose scales are seen in most sea snakes, especially in males. Why has marine life favored this sex-specific elaboration of scale rugosity? We pose and test alternative hypotheses about the function of rugose scales in males of the turtle-headed sea snake (Emydocephalus annulatus) and conclude that multiple selective forces have been involved. First, rugosities may aid male positioning during courtship, because histology shows that tubercles are more highly innervated than adjacent flat areas of each scale and hence are presumably more sensitive to tactile cues, and because biomechanical tests show that rugosities enhance friction between the bodies of males and females. Second, the occurrence of rugosities over the entire body of males and (albeit less well developed) in females as well suggests that rugosities also play a hydrodynamic role by modifying water flow across the snake's surface. Flow tank tests show that rugosities reduce the thickness of the boundary layer by almost 50% and create turbulent flow that should massively enhance rates of cutaneous oxygen uptake and hence prolong maximal courtship duration by males. PMID- 16671017 TI - The strength of direct selection against female promiscuity is associated with rates of extrapair fertilizations in socially monogamous songbirds. AB - A costs-benefits approach has frequently been used to understand the evolutionary origin and maintenance of promiscuity in animal populations. Recent meta-analyses suggest that direct costs to unfaithful females outweigh indirect benefits from infidelity in socially monogamous songbirds, suggesting that in this taxa, extrapair fertilization (EPF) evolved primarily as a self-interest male tactic. Here we present results of comparative analysis to show that standardized selection gradients acting against female infidelity (direct costs of promiscuity) explain variation in EPF rates at an interspecific level in passerines. This result confirms that costs to females resulting from reduced parental care from cheated males constrain promiscuity in this group. Our data indicate that females exert resistance over EPFs when the costs of infidelity are high and, conversely, that the rate of EPFs increases when selection on females to defend themselves against EPF attempts by males is weak and costs of infidelity are low. PMID- 16671018 TI - Independent and competing disease risks: implications for host populations in variable environments. AB - Disease models usually assume disease to act independently of other mortality- and morbidity-causing factors. Alternatively, disease may function as a competing risk factor, for example, killing already moribund hosts. Using tuberculosis (TB) in African buffalo as a model system, we explore consequences of competing or independent disease effects for host population dynamics. We include scenarios with density-dependent and density-independent effects of environmental variation, exemplified by variable food availability (driven by rainfall) and catastrophic droughts, respectively. Independent disease effects reduce population size linearly with prevalence, irrespective of the nature of environmental variation. Competing disease risks alter population size only if density-independent variation is present; then, disease reduces population size nonlinearly. Field data indicate that the net effect of TB on buffalo likely falls between the extremes of total independence and competition with other risk factors: TB increases mortality and decreases fecundity in some prime-aged buffalo, suggesting independent disease risks in these individuals, while similar disease effects in senescent buffalo may act as competing risks. Moreover, increased survival and fecundity of TB-negative buffalo may compensate for some disease-related losses. Model assumptions on independent or competing disease risks and environmental variability should be considered explicitly when assessing disease effects on wildlife populations. PMID- 16671019 TI - Leaf traits determine the growth-survival trade-off across rain forest tree species. AB - A dominant hypothesis explaining tree species coexistence in tropical forest is that trade-offs in characters allow species to adapt to different light environments, but tests for this hypothesis are scarce. This study is the first that uses a theoretical plant growth model to link leaf trade-offs to whole-plant performances and to differential performances across species in different light environments. Using data of 50 sympatric tree species from a Bolivian rain forest, we observed that specific leaf area and photosynthetic capacity codetermined interspecific height growth variation in a forest gap; that leaf survival rate determined the variation in plant survival rate under a closed canopy; that predicted height growth and plant survival rate matched field observations; and that fast-growing species had low survival rates for both field and predicted values. These results show how leaf trade-offs influence differential tree performance and tree species' coexistence in a heterogeneous light environment. PMID- 16671020 TI - Age-dependent traits: a new statistical model to separate within- and between individual effects. AB - Evolutionary questions regarding aging address patterns of within-individual change in traits during a lifetime. However, most studies report associations between age and, for example, reproduction based on cross-sectional comparisons, which may be confounded with progressive changes in phenotypic population composition. Unbiased estimation of patterns of age-dependent reproduction (or other traits) requires disentanglement of within-individual change (improvement, senescence) and between-individual change (selective appearance and disappearance). We introduce a new statistical model that allows patterns of variance and covariance to differ between levels of aggregation. Our approach is simpler than alternative methods and can quantify the relative contributions of within- and between-individual changes in one framework. We illustrate our model using data on a long-lived bird species, the oystercatcher (Haematopus ostralegus). We show that for different reproductive traits (timing of breeding and egg size), either within-individual improvement or selective appearance can result in a positive association between age and reproductive traits at the population level. Potential applications of our methodology are manifold because within- and between-individual patterns are likely to differ in many biological situations. PMID- 16671021 TI - Tuberculosis contacts, concerns, and controls: what matters for healthcare workers? PMID- 16671022 TI - High rate of negative results of tuberculin and QuantiFERON tests among individuals with a history of positive skin test results. AB - OBJECTIVES: To evaluate individuals at high risk for tuberculosis exposure who had a history of a positive tuberculin skin test (TST) result in order to determine the prevalence of unsuspected negative TST results. To confirm these findings with the QuantiFERON-TB test (QFT), an in vitro whole-blood assay that measures tuberculin-induced secretion of interferon-gamma. METHODS: This survey was conducted from November 2001 through December 2003 at 3 sites where TST screening is regularly done. Detailed histories and reviews of medical records were performed. TSTs were placed and read by 2 experienced healthcare workers, and blood was drawn for QFT. Any subject with a negative result of an initial TST during the study (induration diameter, <10 mm) underwent a second TST and a second QFT. The TST-negative group comprised individuals for whom both TSTs had an induration diameter of <10 mm. The confirmed-negative group comprised individuals for whom both TSTs yielded no detectable induration and results of both QFTs were negative. RESULTS: A total of 67 immunocompetent subjects with positive results of a previous TST were enrolled in the study. Of 56 subjects who completed the TST protocol, 25 (44.6%; 95% confidence interval [CI], 31.6%-57.6%) were TST negative (P<.001). Of 31 subjects who completed the TST protocol and the QFT protocol, 8 (25.8%; 95% CI, 10.4%-41.2%) were confirmed negative (P<.005). CONCLUSIONS: A significant proportion of subjects with positive results of a previous TST were TST negative in this study, and a subset of these were confirmed negative. These individuals' TST status may have reverted or may never have been positive. It will be important in future studies to determine whether such individuals lack immunity to tuberculosis and whether they should be considered for reentry into tuberculosis screening programs. PMID- 16671024 TI - Tuberculosis in healthcare workers at a general hospital in Mexico. AB - OBJECTIVE: To determine the incidence rate of tuberculosis (TB) disease among healthcare workers (HCWs) at a general hospital. DESIGN: Retrospective analysis of TB cases among HCWs over the course of 5 years. SETTING: A 140-bed general hospital in Tijuana, Mexico. PARTICIPANTS: All hospital employees who developed TB during the 5-year period. RESULTS: From 1 January 1999 through 31 December 2003, 18 TB cases were diagnosed among the hospital personnel. During that period, the mean (+/- standard deviation) annual work force of the hospital was 819+/-21.7 employees. The TB incidence rate was 439.56 cases per 100,000 employees; this rate was 10.98 times higher than the rate for the general population of the city. The TB incidence rate for physicians was 860.21 cases per 100,000 employees, that for nurses was 365.85 cases per 100,000 employees, and that for physicians in training was 1,846.15 cases per 100,000 employees. Physicians in training had a higher risk of acquiring TB than did either physicians (relative risk, 2.14 [95% confidence interval, 1.34-35.66) or nurses (relative risk, 5.04 [95% confidence interval, 3.16-83.33). Three of the HCWs with TB disease were infected with a drug-resistant strain of Mycobacterium tuberculosis, and one of the infecting strains was multidrug resistant. Asymptomatic TB infection among HCWs was not addressed during this study. CONCLUSIONS: The TB incidence rate among the HCWs at the hospital is extremely high, compared with that in the general population. The implementation of infection control measures is an urgent priority, to reduce this occupational hazard. PMID- 16671023 TI - Screening for tuberculosis infection using whole-blood interferon-gamma and Mantoux testing among Japanese healthcare workers. AB - OBJECTIVE: To examine the hypothesis that results of the QuantiFERON-TB Gold assay (QFT-G), a whole-blood test for detection of tuberculosis infection, are more significantly related to known risk factors for tuberculosis infection in healthcare workers (HCWs) who have received bacille Calmette-Guerin vaccine than are results of the Mantoux tuberculin skin test (TST). DESIGN: All HCWs (approximately 510) from a 370-bed general hospital in Tokyo where patients with and patients without tuberculosis are treated were invited to participate in the study. All study participants completed a questionnaire about their Mycobacterium tuberculosis infection risk factors as HCWs at the general hospital. They were then tested for LTBI by means of the QFT-G, followed by the TST. Statistical analyses were performed to compare results of each test with M. tuberculosis infection risk factors (age, length of employment in the healthcare industry, history of working with tuberculosis-positive patients in a tuberculosis ward or in the outpatient department of the hospital's tuberculosis clinic for more than 1 year, chest radiograph evidence of healed tuberculosis, history of performing bronchoscope procedures, and job classification), and for TST-positive HCWs, to compare the QFT-G result with the TST induration diameter. RESULTS: A total of 332 HCWs (95% of whom had been vaccinated with BCG) participated in the study, and 33 had positive QFT-G results, suggesting a prevalence of LTBI of 9.9%. Of 304 HCWs who underwent TST, 283 (93.1%) had an induration diameter of 10 mm or more. Multiple logistic regression analysis revealed that positive QFT-G results were significantly associated with age and with a history of working in a tuberculosis ward or an outpatient department of a tuberculosis clinic. TST results were not correlated with any of the tuberculosis infection risk factors we evaluated. CONCLUSIONS: Positive QFT-G results were closely associated with the presence of risk factors for LTBI in a hospital setting, suggesting that the QFT-G can detect LTBI in a population composed predominantly of BCG vaccinees. Because most HCWs worldwide have been vaccinated with BCG, the QFT-G offers a significant improvement over the TST in tuberculosis screening programs and minimizes unwarranted use of tuberculosis prophylaxis. PMID- 16671025 TI - Tuberculosis in healthcare workers: a molecular epidemiologic study in San Francisco. AB - OBJECTIVE: Healthcare workers (HCWs) are at risk of becoming infected with Mycobacterium tuberculosis through occupational exposure. To identify HCWs who became infected and developed tuberculosis as a result of their work, we studied the molecular epidemiology of tuberculosis in HCWs. DESIGN: Eleven-year prospective cohort molecular epidemiology study. SETTING: City and County of San Francisco, California. PATIENTS: All persons reported with tuberculosis between 1993 and 2003. HCWs were identified from the San Francisco Tuberculosis Control Section's database, and mycobacterial isolates from culture-positive subjects were analyzed by IS6110-based genotyping. RESULTS: Of 2510 cases of tuberculosis reported during the study period, 31 (1.2%) occurred in HCWs: the median age of the HCWs was 37 years, and 11 (35%) were male. HCWs were more likely than non HCWs to be younger (P=.0036), born in the United States (P=.0004), and female (P=.0003) and to not be homeless (P=.010). The rate of tuberculosis among HCWs remained constant during the study period, despite a significant decrease in the overall case rate in San Francisco. Work-related transmission was documented in at least 10 (32%) of 31 HCWs, including 4 of 8 HCWs whose isolates were part of genotypically determined clusters. Only 1 of 7 cases of tuberculosis in HCWs after 1999 was documented as being work-related. CONCLUSIONS: Although most cases of tuberculosis in HCWs, as in non-HCWs, developed as a result of endogenous reactivation of latent infection, at least half of clustered cases of tuberculosis in HCWs were related to work. The number of work-related cases of tuberculosis in HCWs decreased during the study period. PMID- 16671026 TI - Prospective 3-year surveillance for nosocomial and environmental Legionella pneumophila: implications for infection control. AB - OBJECTIVES: To perform a 3-year, prospective surveillance program for legionnaires disease (LD) in a large university hospital in Rome, and to assess the usefulness of the hospital water monitoring program in predicting the risk of nosocomial LD. METHODS: Samples from patients with new cases of nosocomial pneumonia were sent for legionella laboratory investigations. Meanwhile, water samples for bacteriological analysis were collected every 6 months from high- and medium-risk hospital wards (10 in total). Legionella pneumophila isolates collected were serotyped and analyzed by pulsed-field gel electrophoresis. RESULTS: From June 2001 through May 2004, the pneumonia surveillance identified one case of nosocomial LD among 43 cases of nosocomial pneumonia (2.3%). Environmental investigations detected L. pneumophila in 12 (18.7%) of the 64 water samples, of which 50% belonged to serogroup 1. The L. pneumophila count and the percentage of positive locations never exceeded 10(2) colony-forming units/L and 20%, respectively, except when the LD nosocomial case occurred (positive water samples, 40%; L. pneumophila count, <10(2) colony-forming units/L). Genotyping showed 3 prevalent clones of L. pneumophila in the water distribution network, of which one persisted over the 3 years. One clone contained 3 different L. pneumophila serogroups (2, 4, and 6). CONCLUSIONS: The low incidence of nosocomial cases of LD appears to be associated with a low percentage (<20%) of positive water samples per semester and with a low contamination level (<10(2) colony-forming units/L). An infection control system for nosocomial LD should, therefore, be based on both environmental and clinical surveillance, together with the appropriate maintenance of the hospital water distribution system. PMID- 16671027 TI - Epidemiologic study and containment of a nosocomial outbreak of severe acute respiratory syndrome in a medical center in Kaohsiung, Taiwan. AB - OBJECTIVE: We conducted an epidemiologic investigation at the beginning of a nosocomial outbreak of severe acute respiratory syndrome (SARS) to clarify the dynamics of SARS transmission, the magnitude of the SARS outbreak, and the impact of the outbreak on the community. METHODS: We identified all potential cases of nosocomially acquired SARS, linked them to the most likely infection source, and described the hospital containment measures. SETTING: A 2,300-bed medical center in Kaohsiung, Taiwan. RESULTS: A total of 55 cases of SARS were identified, and 227 hospital workers were quarantined. The index patient and neighboring patients were isolated. A chest physician team reviewed medical charts and chest radiographs and monitored the development of SARS in patients staying in the ward. The presence of underlying lung disease and immunocompromise in some patients made the diagnosis of SARS difficult. Some cases of SARS were diagnosed after the patients had died. Medical personnel were infected only if they cared for patients with unrecognized SARS, and caretakers played important roles in transmission of SARS to family members. As the number of cases of nosocomial SARS increased, the hospital closed the affected ward and expedited construction of negative-pressure rooms on other vacated floors for patient cohorting, and the last case in the hospital was identified 1 week later. CONCLUSIONS: Timely recognition of SARS is extremely important. However, given the limitations of SARS testing, possible loss of epidemic links, and the nonspecific clinical presentations in hospitalized patients, it is very important to establish cohorts of persons with low, medium, and high likelihoods of SARS acquisition. Rapid closure of affected wards may minimize the impact on hospital operations. Establishment of hospitals dedicated to appropriate treatment of patients with SARS might minimize the impact of the disease in future epidemics. PMID- 16671028 TI - Cluster of cases of severe acute respiratory syndrome among Toronto healthcare workers after implementation of infection control precautions: a case series. AB - OBJECTIVE: To review the severe acute respiratory syndrome (SARS) infection control practices, the types of exposure to patients with SARS, and the activities associated with treatment of such patients among healthcare workers (HCWs) who developed SARS in Toronto, Canada, after SARS-specific infection control precautions had been implemented. METHODS: A retrospective review of work logs and patient assignments, detailed review of medical records of patients with SARS, and comprehensive telephone-based interviews of HCWs who met the case definition for SARS after implementation of infection control precautions. RESULTS: Seventeen HCWs from 6 hospitals developed disease that met the case definition for SARS after implementation of infection control precautions. These HCWs had a mean age (+/-SD) of 39+/-2.3 years. Two HCWs were not interviewed because of illness. Of the remaining 15, only 9 (60%) reported that they had received formal infection control training. Thirteen HCWs (87%) were unsure of proper order in which personal protective equipment should be donned and doffed. Six HCWs (40%) reused items (eg, stethoscopes, goggles, and cleaning equipment) elsewhere on the ward after initial use in a room in which a patient with SARS was staying. Use of masks, gowns, gloves, and eyewear was inconsistent among HCWs. Eight (54%) reported that they were aware of a breach in infection control precautions. HCWs reported fatigue due to an increased number and length of shifts; participants worked a median of 10 shifts during the 10 days before onset of symptoms. Seven HCWs were involved in the intubation of a patient with SARS. One HCW died, and the remaining 16 recovered. CONCLUSION: Multiple factors were likely responsible for SARS in these HCWs, including the performance of high-risk patient care procedures, inconsistent use of personal protective equipment, fatigue, and lack of adequate infection control training. PMID- 16671029 TI - Lack of association between the increased incidence of Clostridium difficile associated disease and the increasing use of alcohol-based hand rubs. AB - OBJECTIVE: To determine whether there is an association between the increasing use of alcohol-based hand rubs (ABHRs) and the increased incidence of Clostridium difficile-associated disease (CDAD). SETTING: A 500-bed university-affiliated community teaching hospital. METHODS: Use of ABHRs during the period 2000-2003 was expressed as the number of liters of ABHR used per 1000 patient-days. The proportion of hand hygiene episodes performed by using an ABHR was determined by periodic observational surveys. CDAD was defined as a physician-ordered stool assay positive for C. difficile toxin A or A/B. The incidence of CDAD was expressed as the number of unique patients who had 1 or more positive CDAD test results per 1,000 patient-days. RESULTS: During 2000-2003, the use of ABHR increased 10-fold, from 3 to greater than 30 L/1,000 patient-days (P<.001). The proportion of hand hygiene episodes performed using an ABHR increased from 10% to 85% (P<.001). The incidence of CDAD in 2000, 2001, 2002, and 2003 was 1.74, 2.33, 1.14, and 1.18 cases/1,000 patient-days, respectively. CONCLUSION: Despite a significant and progressive increase in the use of ABHRs in our facility during a 3-year period, there was no evidence that the incidence of CDAD increased. These findings suggest that factors other than the increased use of ABHRs are responsible for the increasing incidence of CDAD noted since 2000 in other facilities. PMID- 16671030 TI - Why healthcare workers don't wash their hands: a behavioral explanation. AB - OBJECTIVE: To elucidate behavioral determinants of handwashing among nurses. DESIGN: Statistical modeling using the Theory of Planned Behavior and relevant components to handwashing behavior by nurses that were derived from focus-group discussions and literature review. SETTING: The community and 3 tertiary care hospitals. PARTICIPANTS: Children aged 9-10 years, mothers, and nurses. RESULTS: Responses from 754 nurses were analyzed using backward linear regression for handwashing intention. We reasoned that handwashing results in 2 distinct behavioral practices--inherent handwashing and elective handwashing--with our model explaining 64% and 76%, respectively, of the variance in behavioral intention. Translation of community handwashing behavior to healthcare settings is the predominant driver of all handwashing, both inherent (weighted beta =2.92) and elective (weighted beta =4.1). Intended elective in-hospital handwashing behavior is further significantly predicted by nurses' beliefs in the benefits of the activity (weighted beta =3.12), peer pressure of senior physicians (weighted beta =3.0) and administrators (weighted beta =2.2), and role modeling (weighted beta =3.0) but only to a minimal extent by reduction in effort (weighted beta =1.13). Inherent community behavior (weighted beta =2.92), attitudes (weighted beta =0.84), and peer behavior (weighted beta =1.08) were strongly predictive of inherent handwashing intent. CONCLUSIONS: A small increase in handwashing adherence may be seen after implementing the use of alcoholic hand rubs, to decrease the effort required to wash hands. However, the facilitation of compliance is not simply related to effort but is highly dependent on altering behavioral perceptions. Thus, introduction of hand rub alone without an associated behavioral modification program is unlikely to induce a sustained increase in hand hygiene compliance. PMID- 16671031 TI - Use of multistate models to assess prolongation of intensive care unit stay due to nosocomial infection. AB - BACKGROUND: Reliable data on the costs attributable to nosocomial infection (NI) are crucial to demonstrating the real cost-effectiveness of infection control measures. Several studies investigating this issue with regard to intensive care unit (ICU) patients have probably overestimated, as a result of inappropriate study methods, the part played by NIs in prolonging the length of stay. METHODS: Data from a prospective study of the incidence of NI in 5 ICUs over a period of 18 months formed the basis of this analysis. For describing the temporal dynamics of the data, a multistate model was used. Thus, ICU patients were counted as case patients as soon as an NI was ascertained on any particular day. All patients were then regarded as control subjects as long as they remained free of NI (time to-event data analysis technique). RESULTS: Admitted patients (n=1,876) were observed for the development of NI over a period of 28,498 patient-days. In total, 431 NIs were ascertained during the study period (incidence density, 15.1 NIs per 1,000 patient-days). The influence of NI as a time-dependent covariate in a proportional hazards model was highly significant (P< .0001, Wald test). NI significantly reduced the discharge hazard (hazard ratio, 0.72 [95% confidence interval, 0.63-0.82])--that is, it prolonged the ICU stay. The mean prolongation of ICU length of stay due to NI (+/- standard error) was estimated to be 5.3+/ 1.6 days. CONCLUSIONS: Further studies are required to enable comparison of data on prolongation of ICU length of stay with the results of various study methods. PMID- 16671032 TI - Automatic detection of patients with nosocomial infection by a computer-based surveillance system: a validation study in a general hospital. AB - OBJECTIVE: To validate an automated system for the detection of patients with nosocomial infection (NI) in an intensive care unit (ICU). DESIGN: Retrospective analysis of data from the hospital information system. We applied 3 different NI suspicion criteria (positive microbiology reports, antibiotic administration, and diagnosis of clinical infection) and compared the results to those of a prospective NI incidence study done in the ICU during the same period (1999 2002). SETTING: A 250-bed general hospital in Barcelona, Spain. PATIENTS: From April 15, 1999, through June 30, 2002, 1380 patients were admitted to the ICU. Of these, 1043 had an ICU stay of more than 24 hours and were included in the study. RESULTS: At least one NI suspicion criterion was present for 242 patients (23.2%); 2 criteria were present for 184 patients (17.6%); and all 3 criteria were present for 112 (11.7%). Comparison of hospital information system data to the results of the prospective study indicated that the combination of 2 criteria demonstrated the most satisfactory sensitivity (94.3%; 95% confidence interval [CI], 79.5%-99.0%) and specificity (83.6%; 95% CI, 76.8%-88.9%). The positive predictive value was 55.9% (95% CI, 42.5%-68.6%); the negative predictive value was 98.5% (95% CI, 94.2%-99.7%). The system could assign a site of infection for 90.4% of the NIs detected. CONCLUSION: The hospital information system was a useful tool for retrospectively detecting patients with an NI during the ICU stay. Given its high sensitivity, it may be useful as an alert for the NI team. PMID- 16671033 TI - Detection and quantification of dental unit water line contamination by oral streptococci. AB - OBJECTIVE: (1) To investigate the prevalence of oral streptococci (OS) and biological indicators of water contamination by oral fluids in water from dental unit water lines (DUWs) by detection and quantification and of saprophytes indigenous to the oral cavity. (2) To test whether measurement of the total cultivable mesophilic flora (TCF), the parameter commonly used to monitor water quality in DUWs, is an effective predictor for OS contamination. DESIGN: Survey of 21 dental units equipped with antiretraction devices. Water samples were collected from air-water syringes, cup fillers, tap water, and before and during the working day. SETTING: Units were from 7 public dental offices selected for convenience from among those in proximity of the microbiological laboratory. METHODS: For detection of OS, samples were plated on an enriched medium, to revitalize the organisms. Colonies were subcultured on a selective medium and biochemically identified (lower detection limit, 1 cfu/mL). For measurement of the TCF, samples were plated on a nutrient-poor medium. Cultures with colony counts greater than 200 cfu/mL were considered to be TCF positive. The sensitivity and specificity of TCF positivity in predicting OS detection was calculated. RESULTS: Prevalence rates for OS contamination and for TCF positivity were, respectively, 34.4% (11 of 32 samples) and 25.0% (8 of 32 samples) for syringes, 27.8% (10 of 36 samples) and 8.3% (3 of 36 samples) for cup fillers, and 0.0% (0 of 7 samples) for tap water. OS contamination levels ranged from 1 to 6 cfu/mL. No statistically significant differences were found between samples obtained before and during the working day. TCF positivity did not predict OS contamination effectively, because of low sensitivity. CONCLUSIONS: Given the absence of OS in tap water, the reported prevalence of OS contamination suggests that oral fluids are aspirated during dental therapy with relatively high frequency and that DUWs can potentially expose successive patients to bloodborne cross-infections. PMID- 16671034 TI - Screening of hospital workers for pulmonary tuberculosis in a medical center in Taiwan. AB - At a medical center in Taiwan, all workers were examined by chest radiography, to determine the prevalence of pulmonary tuberculosis. The prevalence of tuberculosis among all hospital workers was 0.12%, that among nurses was 0.35%, and that among externally contracted cleaners was 0.57%. All of the Mycobacterium tuberculosis isolates recovered from 2 nurses and from a patient with pulmonary tuberculosis were the Beijing strain, but the strains had different serotypes. PMID- 16671035 TI - The importance of two-step tuberculin skin testing for newly employed healthcare workers. AB - At the time of hire, 4059 of 6522 healthcare workers required a 2-step tuberculin skin test; 114 workers (2.8%) demonstrated a boosted reaction after the second step. Boosted reactions were significantly associated with male sex and older age. A verbal history of previous tuberculin skin test results was not a reliable indicator of baseline tuberculin skin test status at the time of hire. PMID- 16671036 TI - Discontinuation of respiratory isolation for possible tuberculosis: do two negative sputum smear results suffice? AB - Analysis of acid-fast bacillus smear results at a hospital with a moderate incidence of tuberculosis confirms recent recommendations that 2 negative smear results suffice for discontinuation of respiratory isolation. Use of polymerase chain reaction analysis further increases the confidence with which the diagnosis of active tuberculosis likely to be transmitted to others can be excluded. PMID- 16671037 TI - An outbreak of scabies in a long-term care facility: the role of misdiagnosis and the costs associated with control. AB - In August 2003, an outbreak of scabies was detected in a Canadian long-term care facility. The outbreak was likely associated with 2 index cases, 1 involving classic scabies and the other involving Norwegian scabies. The scabies control costs totaled CDN $200,000, and the facility received negative publicity for a short period after the outbreak. PMID- 16671038 TI - Hospital waste generation during an outbreak of severe acute respiratory syndrome in Taiwan. AB - During the SARS outbreak in Taiwan, the number of ambulatory patients and inpatients treated at one medical center decreased by 40%-70% because of the increasing number of SARS patients. At the peak of the epidemic, the amount of hospital infectious waste had increased from a norm of 0.85 kg per patient-day to 2.7 kg per patient-day. However, the hospital was able to return the generation of waste to normal levels within 10 days. PMID- 16671039 TI - Airborne severe acute respiratory syndrome coronavirus concentrations in a negative-pressure isolation room. AB - This study used a sensitive polymerase chain reaction method coupled with filter sampling to detect the presence of airborne severe acute respiratory syndrome (SARS) coronavirus in an isolation patient room with a patient with severe acute respiratory syndrome receiving mechanical ventilatory support. Polymerase chain reaction results were negative for SARS coronavirus in room air both before and after patient extubation. PMID- 16671040 TI - Prospective surveillance effectively reduced rates of surgical site infection associated with elective colorectal surgery at a university hospital in Japan. AB - At a university hospital in Japan, the introduction of prospective surveillance and subsequent interventions was effective in reducing the rate of surgical site infection associated with elective colorectal surgery from 27.5% to 17.8% of surgeries. Japan should both recognize the importance of broader surveillance for surgical site infection and establish its own nationwide surveillance database. PMID- 16671041 TI - Influenza vaccination: impact of an intervention campaign targeting hospital staff. PMID- 16671042 TI - On the need for a separate standard for performance testing of negative-pressure isolation rooms. PMID- 16671043 TI - Colonization of a water system by Legionella organisms and nosocomial legionellosis: a 5-year report from a large Italian hospital. PMID- 16671044 TI - Bacteremia caused by Stenotrophomonas maltophilia in a dialysis patient with a long-term central venous catheter. PMID- 16671046 TI - Interactions of neutral and cationic transition metals with the redox system of hydroquinone and quinone: theoretical characterization of the binding topologies, and implications for the formation of nanomaterials. AB - To understand the self-assembly process of the transition metal (TM) nanoclusters and nanowires self-synthesized by hydroquinone (HQ) and calix[4]hydroquinone (CHQ) by electrochemical redox processes, we have investigated the binding sites of HQ for the transition-metal cations TM(n+)=Ag(+), Au(+), Pd(2+), Pt(2+), and Hg(2+) and those of quinone (Q) for the reduced neutral metals TM(0), using ab initio calculations. For comparison, TM(0)-HQ and TM(n+)-Q interactions, as well as the cases for Na(+) and Cu(+) (which do not take part in self-synthesis by CHQ) are also included. In general, TM-ligand coordination is controlled by symmetry constraints imposed on the respective orbital interactions. Calculations predict that, due to synergetic interactions, silver and gold are very efficient metals for one-dimensional (1D) nanowire formation in the self-assembly process, platinum and mercury favor both nanowire/nanorod and thin film formation, while palladium favors two-dimensional (2D) thin film formation. PMID- 16671045 TI - Palladium-catalyzed cross-coupling reactions of silanolates: a paradigm shift in silicon-based cross-coupling reactions. AB - This paper chronicles the conceptual development, proof of principle experiments, and recent advances in the palladium-catalyzed cross-coupling reactions of the conjugate bases of organosilanols. The discovery that led to the design and refinement of this process represents a classical illustration of how mechanistic studies can provide a fertile ground for the invention of new reactions. On the basis of a working hypothesis (which ultimately proved to be incorrect) and the desire to effect silicon-based cross-coupling without the agency of fluoride activation, a mild and practical palladium-catalyzed cross-coupling of alkenyl-, aryl-, and heteroaryl silanolates has been developed. The mechanistic underpinnings, methodological extensions, and the successful applications of this technology to the synthesis of complex molecules are described. PMID- 16671047 TI - The prediction of the nuclear quadrupole splitting of 119Sn Mossbauer spectroscopy data by scalar relativistic DFT calculations. AB - The electric field gradient components for the tin nucleus of 34 tin compounds of experimentally known structures and (119)Sn Mossbauer spectroscopy parameters were computed at the scalar relativistic density functional theory level of approximation. The theoretical values of the electric field gradient components were used to determine a quantity, V, which is proportional to the nuclear quadrupole splitting parameter (DeltaE). In a subsequent linear regression analysis the effective nuclear quadrupole moment, Q, was evaluated. The value of (11.9+/-0.1) fm(2) is a significant improvement over the non-relativistic result of (15.2+/-4.4) fm(2) and is in agreement with the experimental value of (10.9+/ 0.8) fm(2). The average mean square error DeltaE(calcd)-DeltaE(exptl)=+/-0.3 mm s(-1) is a factor of two smaller than in the non-relativistic case. Thus, the approach has a quality which provides accurate support for the structure interpretation by (119)Sn spectroscopy. It was noted that geometry optimization at the relativistic level does not significantly increase the quality of the results compared with non-relativistic optimized structures. The accuracy in the approach called on us to consider the singlet-triplet state nature of the electronic structure of one of the investigated compounds. PMID- 16671048 TI - Conformational behavior of pyrazine-bridged and mixed-bridged cavitands: a general model for solvent effects on thermal "vase-kite" switching. AB - The controllable switching of suitably bridged resorcin[4]arene cavitands between a "vase" conformation, with a cavity capable of guest inclusion, and a "kite" conformation, featuring an extended flattened surface, provides the basis for ongoing developments of dynamic molecular receptors, sensors, and molecular machines. This paper describes the synthesis, X-ray crystallographic characterization, and NMR analysis of the "vase-kite" switching behavior of a fully pyrazine-bridged cavitand and five other mixed-bridged quinoxaline-bridged cavitands with one methylene, phosphonate, or phosphate bridge. The pyrazine bridged resorcin[4]arene cavitand displayed an unexpectedly high preference for the kite conformation in nonpolar solvents, relative to the quinoxaline-bridged analogue. This observation led to extensive solvent-dependent switching studies that provide a detailed picture of how solvent affects the thermal vase-kite equilibration. As for any thermodynamic process in the liquid phase, the conformational equilibrium is affected by how the solvent stabilizes the two individual states. Suitably sized solvents (benzene and derivatives) solvate the cavity of the vase form and reduce the propensity for the vase-to-kite transition. Correspondingly, the kite geometry becomes preferred in bulky solvents such as mesitylene, incapable of penetrating the vase cavity. As proposed earlier by Cram, the kite form is preferred at low temperatures due to the more favorable enthalpy of solvation of the enlarged surface. Furthermore, the kite conformation is more preferred in solvents with substantial hydrogen bonding acidity: weak hydrogen-bonding interactions between the mildly basic quinoxaline and pyrazine nitrogen atoms and solvent molecules are more efficient in the open kite than in the closed vase form. Vase-to-kite conversion is entirely absent in dipolar aprotic solvents lacking any H-bonding acidity. Thermal vase-kite switching requires fully quinoxaline- or pyrazine-bridged cavitands, whereas pH-controlled switching is also applicable to systems incorporating only two or three such bridges. PMID- 16671049 TI - Peculiarities of C60*- coordination to cobalt(II) octaethylporphyrin in ionic multicomponent complexes: Observation of the reversible formation of Co-C(C60-) coordination bonds. AB - Ionic multicomponent complexes containing the C60- anion, cobalt(II) octaethylporphyrin (OEP), and the noncoordinating tetramethylphosphonium cation (TMP+), [(TMP+){Co(II)OEP(C60-)}(C6H5CN)x(C6H4Cl2)(1-x)] (x congruent with 0.75) (1), or the coordinating cation of N-methyldiazabicyclooctane (MDABCO+), [{(MDABCO+)Co(II)OEP(C60-)}(C6H5CN)x(C6H4Cl2)(1-x)] (x congruent with 0.67) (2), were obtained. Diamagnetic sigma-bonded {Co(II)OEP(C60-)} units in 1 have the Co...C(C60-) distance of 2.268(1) A at 100 K and are stable up to 290 K. Both MDABCO+ and C60- coordinate to Co(II)OEP in 2. In this case, a noticeably longer Co...CC60-) distance of 2.508(4) A was observed at 100 K. As a result, the unprecedented reversible formation of the Co-C(C60-) coordination sigma bond is realized in 2 and is accompanied by a transition from a paramagnetic to a diamagnetic state in the 50-250 K range. It was shown, for the first time, that the Co...C distance of about 2.51 A is a boundary distance below which the Co C(C60-) coordination bond is formed. PMID- 16671050 TI - Noncovalent functionalization and solubilization of carbon nanotubes by using a conjugated Zn-porphyrin polymer. AB - A highly soluble, conjugated Zn-porphyrin polymer was synthesized and found to strongly interact with the surface of single-walled carbon nanotubes, producing a soluble polymer-nanotube complex. Successful complexation required the addition of trifluoroacetic acid to the solvent (THF). It was found that the complex remained soluble after excess free polymer was removed from solution, and could be centrifuged at high speed with no observable sedimentation. Furthermore, the polymer-nanotube assembly resulted in enhanced planarization and conjugation within the porphyrin polymer, which was manifested in a 127 nm bathochromic shift of the Q-band absorption. Control experiments with the Zn-porphyrin monomer indicated that homogeneous solutions could be prepared by means of sonication, but the monomer-nanotube interactions were significantly weaker, leading to nanotube precipitation within minutes. Atomic force microscopy (AFM) studies indicated that the polymer enables exfoliation of nanotube bundles and is able to "stitch" multiple nanotubes together into a series of long, interconnected strands. PMID- 16671051 TI - Drug release of pH/temperature-responsive calcium alginate/poly(N isopropylacrylamide) semi-IPN beads. AB - A series of semi-interpenetrating, polymer network (semi-IPN), hydrogel beads, composed of calcium alginate (Ca-alginate) and poly(N-isopropylacrylamide) (PNIPAAM), were prepared for a pH/temperature-sensitive drug delivery study. The equilibrium swelling showed the independent pH- and thermo- responsive nature of the developed materials. At pH=2.1, the release amount of indomethacin incorporated into these beads was about 10% within 400 min, while this value approached to 95% at pH=7.4. The release rate of the drug was higher at 37 degrees C than that at 25 degrees C and increased slightly with increasing PNIPAAM content. These results suggest that the Ca-alginate/PNIPAAM beads have the potential to be used as an effective pH/temperature sustainable delivery system of bioactive agents. [GRAPHS: SEE TEXT] A summary of the temperature- and pH-dependence on the release of the drug over a period of 450 min. The effect of the temperature on the swelling of the beads is shown in the inset. PMID- 16671052 TI - Cerebral plasticity in crossed C7 grafts of the brachial plexus: an fMRI study. AB - In order to rescue elbow flexion after complete accidental avulsion of one brachial plexus, seven patients underwent a neurotization of the biceps with fibers from the contralateral C7 root. The C7 fibers used for the graft belonged to the pyramidal pathway, which descends from the cerebral hemisphere ipsilateral to the damaged plexus, and which controls extension and abduction of the contralateral arm. After several months of reeducation, a functional magentic resonance imaging study was performed with a 1.5 tesla clinical magnetic resonance scan system, in order to investigate the central neural networks involved in the recovery of elbow flexion. Functional brain images were acquired under four conditions: flexion of each of the two elbows, and imagined flexion of each elbow. Results show that flexion of the neurotized arm is associated with a bilateral network activity. The contralateral cortex originally involved in control of the rescued arm still participates in the elaboration and control of the task through the bilateral premotor and primary motor cortex. The location of the ipsilateral clusters in the primary motor, premotor, supplementary motor area, and posterior parietal areas is similar among patients. The location of contralateral activations within the same areas differs across patients. PMID- 16671053 TI - Structure elucidation of a novel group of dithiocarbamate derivatives using 1D and 2D NMR spectroscopy. AB - Compounds 1-7 form a novel group of dithiocarbamates, first synthesized from the reaction of a series of primary amines with carbon disulfide and 3-bromo ethyl pyruvate in the presence of anhydrous potassium phosphate. Structure elucidation of this group of compounds was accomplished using extensive 1D and 2D NMR spectroscopic studies, including (1)H, (13)C, COSY, NOESY, HSQC, and gHMBC experiments. The distinction between the linear structures I, II and the cyclic structure III was made mainly on the basis of the analysis of the cross peak between H-2 and H-4a in the COSY spectra, in combination with the long-range correlation between H-2 and C-4, 6 in the gHMBC spectra. PMID- 16671054 TI - Computed coupling constants in X(CH3)nH(4-n) moieties where X = 13C and 15N+, and n = 0-4: comparisons with experimental data. AB - Seventy-three unique spin-spin coupling constants have been analyzed for the ten species in the two series X(CH3)nH(4-n), where the central atom X is 13C or 15N+. Thirty-seven experimental values have been obtained from the literature, and several new coupling constants have been measured for the methyl-substituted ammonium ions. Both DFT with the B3LYP functional and ab initio EOM-CCSD calculations have been carried out on these same systems. Coupling constants computed by these two methods are in agreement with experimental values. Some problems related to coupling constants for the cationic ammonium systems have been resolved when these were recomputed at EOM-CCSD for complexes in which NH4+ is hydrogen-bonded to H2O molecules. PMID- 16671055 TI - Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure. AB - Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of approximately = 90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance. PMID- 16671056 TI - Fluorescein-labeled stable neurotensin derivatives. AB - Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. PMID- 16671057 TI - Metabolism of Maillard reaction products by the human gut microbiota- implications for health. AB - The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e. g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed. PMID- 16671058 TI - Predicting heat inactivation of Staphylococcus aureus under nonisothermal treatments at different pH. AB - The aim was to assess whether heat resistance data obtained from isothermal treatments allow the estimation of survivors of Staphylococcus aureus under nonisothermal conditions and to find a model that accurately predicts its heat inactivation at constantly rising heating rates (0.5-9 degrees C/min) in media of different pH (4.0-7.4). S. aureus showed a higher heat resistance under isothermal treatments at pH 4.0 than at pH 5.5-7.4. However, under nonisothermal treatments S. aureus increased its heat resistance at pH 5.5-7.4 and became more thermotolerant than at pH 4.0. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments did not adequately fit experimental values. Whereas the number of survivors was much higher than estimated at pH 5.5-7.4, that obtained at the slower heating rates at pH 4.0 was lower. An equation based on the Weibullian-like distribution (log10 S(t) = (t/delta)p) accurately described survival curves obtained under nonisothermal conditions. A nonlinear relationship was observed among the scale parameter (delta) and the heating rate which allowed the development of two equations capable of predicting the inactivation rate of S. aureus under nonisothermal treatments. This study might contribute to prevent public health risks in foods requiring long heating lag phases during their processing. PMID- 16671059 TI - Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways: correlation with DNA-adduct formation. AB - Ochratoxin A (OTA), a nephrotoxic mycotoxin probably implicated in human Balkan endemic nephropathy and associated urothelial tumors, induces renal carcinomas in rodents and nephrotoxicity in pigs. OTA induces DNA-adduct formation, but the structure of the adducts and their role in nephrotoxicity and carcinogenicity have only partly been elucidated. In vivo, 2-mercaptoethane sulfonate (MESNA) protects rats against OTA-induced nephrotoxicity but not against carcinogenicity, indicating two different mechanisms leading to nephrotoxicity or carcinogenicity. To better understand how DNA-adduct could be generated, opossum kidney cells (OK) have been treated by OTA alone or in presence of several compounds such as MESNA or N-acetylcysteine (another agent that, like MESNA, reduces oxidative stress by increasing of free thiols in kidney), buthionine sulfoximine (BSO) (an inhibitor of glutathione-synthase), and alpha amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid (ACIVICIN) (an inhibitor of gamma glutamyl transpeptidase). Cytotoxicity of OTA on OK cells was evaluated by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. None of the listed agents diminished OTA cytotoxicity significantly; ACIVICIN even increases OTA cytotoxicity. In contrast, analysis of the HPLC profiles of OTA metabolites produced during these incubations indicated that the pattern, the quantity of metabolites, and the nature of the derivatives were modulated by these agents. Ochratoxin B (OTB), open-ring ochratoxin A (OP-OA), 4 hydroxylated OTA, 10 hydroxylated OTA, OTA without phenylalanine, OTB without phenylalanine, and a dechlorinated OTA metabolite could be identified by nano-ESI-IT-MS. PMID- 16671060 TI - Advantages and drawbacks of immunoaffinity columns in analysis of mycotoxins in food. AB - A number of countries are setting legislations on mycotoxins. In order to reduce dispute between importing and exporting countries, the analytical data should be as comparable as possible, especially when levels are close to the regulatory limits. The present trend in the analysis of mycotoxins is to use immunoaffinity column (IAC) as a clean-up and enrichment technique, and Association of Official Analytical Chemists and European Union have validated methods which address a few food commodities. This study describes our experience using both conventional and IAC approaches in the analysis of three mycotoxins. Aflatoxins (AFs): Aflatoxin G1 has been detected by liquid-liquid partitioning methods with HPLC detection as false-positive in some maize. On IACs, this compound behaves as an AF, lowering the amount of the AFs trapped. The problem was solved using either TLC or HPLC with detection in the Kobra cell. Depending on the additives to food during the processing and cooking, the AFs might appear as an opened ring not recognised by the antibody. Fumonisins (FB): Compounds interfering with the FB's antibodies were also observed while analysing breakfast cereals leading to underestimation of FB. Ochratoxin A (OTA): Depending on the food composition and extraction techniques, OTA is underestimated with IAC in some breakfast cereals and coffee. These data strengthen the necessity to validate methods using IAC for each complex matrix. PMID- 16671061 TI - Soil dissipation and biological activity of metolachlor and S-metolachlor in five soils. AB - The resolved isomer of metolachlor, S-metolachlor, was registered in 1997. New formulations based primarily on the S-metolachlor isomer are more active on a gram for gram metolachlor basis than formulations based on a racemic mixture of metolachlor containing a 50:50 ratio of the R and S isomers. The labelled use rates of S-metolachlor-based products were reduced by 35% to give equivalent weed control to metolachlor. However, several companies have recently registered new metolachlor formulations with the same recommended use rates for weed control as S-metolachlor. This research was done to compare the soil behaviour and the biological activity of metolachlor and S-metolachlor in different soils under greenhouse and field conditions. Although K(d) ranged from 1.6 to 6.9 across the five soils, there were no differences in the binding of metolachlor and S metolachlor to soil or in the rate of soil solution dissipation in a given soil. However, both greenhouse and field studies showed that S-metolachlor was 1.4-3 fold more active than metolachlor against Echinochloa crus-galli (L.) Beauv. in five different soils and that S-metolachlor was more active than metolachlor in three Colorado field locations. When the rates of metolachlor and S-metolachlor were adjusted for S isomer concentrations in the formulations, there were no differences between the formulations in field, greenhouse or bioassay studies. Thus herbicidal activity is due to the S isomers, with the R isomers being largely inactive. PMID- 16671062 TI - Association between early systemic inflammatory response, severity of multiorgan dysfunction and death in acute pancreatitis. AB - BACKGROUND: Mortality in patients with acute pancreatitis is associated with the number of failing organs and the severity and reversibility of organ dysfunction. The aim of this study was to assess the significance of early systemic inflammatory response syndrome (SIRS) in the development of multiorgan dysfunction syndrome (MODS) and death from acute pancreatitis. METHODS: Data for all patients with a diagnosis of acute pancreatitis between January 2000 and December 2004 were reviewed. Serum C-reactive protein (CRP), Acute Physiology And Chronic Health Evaluation (APACHE) II scores and presence of SIRS were recorded on admission and at 48 h. Marshall organ dysfunction scores were calculated during the first week of presentation. Presence of SIRS and raised serum CRP levels on admission and at 48 h were correlated with the cumulative organ dysfunction scores in the first week. RESULTS: A total of 759 patients with acute pancreatitis were identified, of whom 45 (5.9 per cent) died during the index admission. SIRS was identified in 162 patients on admission and was persistent in 138 at 48 h. The median (range) cumulative Marshall score in patients with persistent SIRS was significantly higher than that in patients in whom SIRS resolved and in those with no SIRS (4 (0-12), 3 (0-7) and 0 (0-9) respectively; P < 0.001). Thirty-five patients (25.4 per cent) with persistent SIRS died from acute pancreatitis, compared with six patients (8 per cent) with transient SIRS and four (0.7 per cent) without SIRS (P < 0.001). No correlation was observed between CRP level on admission and Marshall score (P = 0.810); however, there was a close correlation between CRP level at 48 h and Marshall score (P < 0.001). CONCLUSION: Persistent SIRS is associated with MODS and death in patients with acute pancreatitis and is an early indicator of the likely severity of acute pancreatitis. PMID- 16671063 TI - European experience with laparoscopic Roux-en-Y gastric bypass in 466 obese patients. AB - BACKGROUND: Roux-en-Y gastric bypass (RYGBP) is usually considered as the procedure of choice for morbid obesity, but its use has been limited in Europe. It is not known whether results with European patients match those from the USA. METHODS: A total of 466 patients were followed prospectively regarding weight loss, co-morbidities, quality of life and morbidity after primary laparoscopic RYGBP. Overall assessment was done using the bariatric analysis and reporting outcome system (BAROS). RESULTS: Conversion to open surgery was necessary in three patients. The overall early morbidity rate was 17.0 per cent and the rate of major complications was 4.7 per cent. The mortality rate was 0.2 per cent. Major morbidity decreased over time. Excess weight loss of over 50 per cent was maintained for up to 4 years in 71.4 per cent of the morbidly obese and 65.2 per cent of the super-obese patients. Co-morbidities resolved or improved in most patients and quality of life improved. At 3 years, the BAROS score was excellent or very good in 77.1 per cent and good in 22.8 per cent. Late complications leading to reoperation developed in 19 patients (4.1 per cent). CONCLUSION: These results are satisfactory and comparable to those reported from the USA. Owing to limitations associated with purely restrictive bariatric procedures, laparoscopic RYGBP is likely to become the procedure of choice for treatment of morbid obesity in Europe. PMID- 16671064 TI - Combined preoperative endoscopic and laparoscopic ultrasonography for prediction of R0 resection in upper gastrointestinal tract cancer. AB - BACKGROUND: This study evaluated the ability of combined endoscopic and laparoscopic ultrasonography to predict R0 resection and avoid unnecessary surgery in patients with upper gastrointestinal tract cancer (UGIC). METHODS: A total of 411 consecutive patients with UGIC (182 pancreatic cancers, 134 gastric cancers and 95 oesophageal cancers) treated between January 2002 and May 2004 were analysed prospectively. The allocation of patients into resectability groups by endoscopic ultrasonography (EUS) and laparoscopic ultrasonography (LUS) was compared with the treatment actually undertaken. RESULTS: The combination of EUS and LUS correctly predicted R0 resection in 90.6 per cent, R1-R2 in 91 per cent and irresectability in 91.4 per cent of patients. Ten patients (2.4 per cent) had explorative laparotomy only. There were no complications associated with the EUS and LUS procedures. CONCLUSION: The routine use of EUS and LUS before surgery predicted R0 resection in nine of ten patients and reduced the number of unnecessary laparotomies to less than 3 per cent. PMID- 16671065 TI - Topical local anaesthetic (tetracaine) reduces pain from botulinum toxin injections for axillary hyperhidrosis. PMID- 16671066 TI - Neoadjuvant chemotherapy and resection of advanced synchronous liver metastases before treatment of the colorectal primary. AB - BACKGROUND: In many patients with advanced synchronous liver metastases from colorectal tumours, the metastases progress during treatment of the primary, precluding curative treatment. The authors have investigated a management strategy that involves high-impact chemotherapy first, resection of liver metastases second and finally removal of the primary tumour in patients with adverse prognostic factors. METHODS: Twenty consecutive patients with non obstructive colonic (nine patients) or rectal (11 patients) cancer and advanced synchronous liver metastases were treated according to this strategy. Median age was 56 years. Patients received between two and six cycles of 5-fluorouracil, oxaliplatin and irinotecan-based chemotherapy. Data were collected prospectively. RESULTS: Overall survival rates at 1, 2, 3 and 4 years after the start of treatment were 85, 79, 71 and 56 per cent respectively, with a median survival of 46 months. Sixteen of the 20 patients had complete removal of liver metastases and colorectal tumours (resectability rate 80 per cent). CONCLUSION: This new strategy produced resectability and survival rates better than those expected from the published data on patients with disease of similar severity. It allows initial control and downstaging of liver metastases, and delivery of preoperative radiotherapy for rectal cancer without the fear that liver metastases will meanwhile progress beyond the possibility of cure. PMID- 16671067 TI - Ecotoxicity testing of chemicals with particular reference to pesticides. AB - Ecotoxicity tests are performed on vertebrates and invertebrates for the environmental risk assessment of pesticides and other chemicals and for a variety of ecotoxicological studies in the laboratory and in the field. Existing practices and strategies in ecotoxicity testing are reviewed, including an account of current requirements of the European Commission for the testing of pesticides and the recent REACH (Registration, Evaluation, Authorisation and Restrictions of Chemicals) proposals for industrial chemicals. Criticisms of existing practices have been made on both scientific and ethical grounds, and these are considered before dealing with the question of possible alternative methods and strategies both for environmental risk assessment and for ecotoxicological studies more generally. New approaches from an ecological point of view are compared with recent developments in laboratory-based methods such as toxicity tests, biomarker assays and bioassays. With regard to the development of new strategies for risk assessment, it is suggested that full consideration should be given to the findings of earlier long-term studies of pollution, which identified mechanisms of action by which environmental chemicals can cause natural populations to decline. Neurotoxicity and endocrine disruption are two cases in point, and biomarker assays for them could have an important role in testing new chemicals suspected of having these properties. In a concluding discussion, possible ways of improving testing protocols are discussed, having regard for current issues in the field of environmental risk assessment as exemplified by the debate over the REACH proposals. The importance of flexibility and the roles of ecologists and ecotoxicologists are stressed in the context of environmental risk assessment. PMID- 16671068 TI - Mutation of Phe102 to Ser in the carboxyl terminal helix of Escherichia coli thioredoxin affects the stability and processivity of T7 DNA polymerase. AB - Processivity of T7 DNA polymerase relies on the coupling of its cofactor Escherichia coli thioredoxin (Trx) to gene 5 protein (gp5) at 1:1 stoichiometry. We designed a coexpression system for gp5 and Trx that allows in vivo reconstitution of subunits into a functional enzyme. The properties of this enzyme were compared with the activity of commercial T7 DNA polymerase. Examination of purified enzymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the thioredoxin subunit of the two enzymes did not comigrate. To our surprise, we identified a mutation (Phe102 to Ser) in the Trx component from the commercial T7 DNA polymerase (gp5/TrxS102) that was not in the enzyme from the coexpression system (wild type gp5/Trx). A comparison of polymerase activity of the T7 DNA polymerases shows that both enzymes possessed similar specific activity but they were different in their residual activity at 37 degrees C. The half-life of gp5/TrxS102 was 7 min at 37 degrees C and 12 min for gp5/Trx. gp5/TrxS102 polymerase activity was reduced by fourfold with 3'-5' exonuclease activity as the prominent activity detected after 10 min of heat inactivation at 37 degrees C. Supplementation of reaction mixtures containing gp5/TrxS102 with exogenous nonmutant thioredoxin restored the enzyme activity levels. Pulse proteolysis was used to demonstrate that TrxS102 unfolded at lower urea concentrations than wild type thioredoxin. Thus, Ser substitution at position 102 affected the structural stability of thioredoxin resulting in a reduced binding affinity for gp5 and loss of processivity. PMID- 16671069 TI - Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury. AB - BACKGROUND: This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. METHODS: Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. RESULTS: The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp cmk. CONCLUSION: Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. PMID- 16671070 TI - Surgeon experience and trends in intraoperative complications in laparoscopic cholecystectomy. AB - BACKGROUND: Intraoperative complications, particularly bile duct injuries (BDIs), have increased since the introduction of laparoscopic cholecystectomy (LC). This excess risk is expected to decline as surgeon experience in laparoscopic surgery increases. METHODS: This was a population-based study of trends in intraoperative injuries in 33 309 cholecystectomies carried out in Western Australia between 1988 and 1998, based on hospital discharge abstracts. Endpoints were identified from diagnostic and procedure codes in index or postoperative readmissions, or a register of endoscopic retrograde cholangiopancreatography procedures, and validated using hospital records. Multivariate analysis was used to estimate the risk of complications associated with potential risk factors. RESULTS: Following the introduction of LC in 1991, the prevalence of all complications doubled by 1994 then stabilized, whereas that of BDI declined after 1994. The risk of complications increased with age, was higher in men, teaching and country hospitals, and was higher for LC and more complicated operations. It was lower when intraoperative cholangiography was performed and with increasing surgeon experience. Approximately 20 per cent of all complications and 30 per cent of BDIs were attributable to surgeons who had performed 200 or fewer cholecystectomies in the previous 5 years. CONCLUSION: The risk of intraoperative complications declined with increasing surgical experience and use of intraoperative cholangiography. PMID- 16671071 TI - Randomized clinical trial and follow-up study of cost-effectiveness of laparoscopic versus conventional Nissen fundoplication. AB - BACKGROUND: Laparoscopic Nissen fundoplication (LNF) has essentially replaced its conventional open counterpart (CNF). An economic evaluation of LNF compared with CNF based on prospective data with adequate follow-up is lacking. METHODS: Data from two consecutive studies (a randomized clinical trial (RCT) of 57 patients undergoing LNF and 46 undergoing CNF that was terminated prematurely, and a follow-up study of 121 consecutive patients with LNF) were combined to determine incremental cost-effectiveness 1 year after surgery. RESULTS: Mean operating time, reoperation rate and hospital costs of LNF were lower in the second series. The mean overall hospital cost per patient was euro 9126 for LNF and euro 6989 for CNF at 1 year in the initial RCT, and euro 7782 in the second LNF series. The success rate of both LNF and CNF at 1 year was 91 per cent in the RCT, and LNF was successful in 90.1 per cent in the second series. A cost reduction of euro 998 for LNF would cancel out the cost advantage of CNF. Similarly, if the reoperation rate after LNF decreased from 0.05 to below 0.008 and/or if the mean duration of sick leave after LNF was reduced from 67.2 to less than 61.1 days, the procedure would become less expensive than CNF. Complications, reoperation rate and quality of life after both operations were similar. CONCLUSION: Including reinterventions, the outcome at 1 year after LNF and CNF was similar. In a well organized setting with appropriate expertise, the cost advantage of CNF may be neutralized. PMID- 16671073 TI - Bilateral subthalamic nucleus deep brain stimulation improves certain aspects of postural control in Parkinson's disease, whereas medication does not. AB - Postural control requires precise integration of sensory inputs and motor output, but clinical assessments of postural control do not differentiate between these. Previously, we found that this differentiation is important in Parkinson's disease (PD) as there was a dissociated effect of medication versus pallidotomy on sensory aspects of postural instability. In this study, we address several questions that emerged from that work in 28 different patients with PD off and on medication, before and after bilateral subthalamic nucleus deep brain stimulation (B-STN DBS): (1) In a different cohort is there still an unusually large percentage of patients with postural instability in sensory-deprived conditions? (2) Are more specific measures of motor aspects of postural control using dynamic posturography (postural movement velocity [MV] and reaction time [RT]) abnormal in PD as seen clinically using the Postural Instability and Gait Disorder score of the Unified Parkinson's Disease Rating Scale? (3) What is the effect of B-STN DBS versus medication on sensory versus motor aspects of postural instability in PD? The results included (1) substantially more patients (39%) versus controls (5%) exhibited postural instability in conditions of limited sensory feedback; (2) postural MV and postural RT were abnormal off medication preoperatively (N(subset) = 23; P < 0.001 for both); (3) B-STN DBS improved abnormal sensory aspects of postural instability (P < 0.05) and postural MV (P = 0.005), whereas medication did not. Neither B-STN DBS nor medication improved postural RT. For the group as a whole, STN DBS plus medication was better therapy than medication preoperatively for sensory aspects of postural control (P = 0.003). PMID- 16671072 TI - A genome-wide screen for nicotine dependence susceptibility loci. AB - Genome-wide model free linkage analysis was conducted for nicotine dependence and tobacco use phenotypes in 607 members of 158 nuclear families consisting of at least two ever smokers (100 or more cigarettes smoked in lifetime). DNA from whole blood was genotyped for 739 autosomal microsatellite polymorphisms with an average inter-marker distance of 4.6 cM. A peak LOD score of 2.7 was observed on chromosome 6 for scores for the Fagerstrom Test for Nicotine Dependence. Exploratory analyses were conducted to determine whether sequence variation at other loci affected other measures of dependence or tobacco use. Four additional loci with LOD scores of 2.7 or more were associated with alternative measures of nicotine dependence, one with current frequency of use, and one with smoking cessation. Several of the corresponding support intervals were near putative loci reported previously (on chromosomes 6, 7, and 8) while others appear to be novel (on chromosomes 5, 16, and 19). PMID- 16671074 TI - Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations. AB - We describe the regulated transcriptome of CACNA1G, a human gene for T-type Ca(v)3.1 calcium channels that is subject to extensive alternative RNA splicing. Fifteen sites of transcript variation include 2 alternative 5'-UTR promoter sites, 2 alternative 3'-UTR polyadenylation sites, and 11 sites of alternative splicing within the open reading frame. A survey of 1580 fetal and adult human brain full-length complementary DNAs reveals a family of 30 distinct transcripts, including multiple functional forms that vary in expression with development. Statistical analyses of fetal and adult transcript populations reveal patterns of linkages among intramolecular splice site configurations that change dramatically with development. A shift from nearly independent, biased splicing in fetal transcripts to strongly concerted splicing in adult transcripts suggests progressive activation of multiple "programs" of splicing regulation that reorganize molecular structures in differentiating cells. Patch-clamp studies of nine selected variants help relate splicing regulation to permutations of the gating parameters most likely to modify T-channel physiology in expressing neurons. Gating behavior reflects combinatorial interactions between variable domains so that molecular phenotype depends on ensembles of coselected domains, consistent with the observed emergence of concerted splicing during development. We conclude that the structural gene and networks of splicing regulatory factors define an integrated system for the phenotypic variation of Ca(v)3.1 biophysics during nervous system development. PMID- 16671075 TI - Learning and retention of movement sequences in Parkinson's disease. AB - The purpose of this study was to examine motor learning and retention given extensive practice in two fundamentally different movement sequences. One sequence was a memory-driven task (performing a series of whole body positions from memory) and the other a context-driven task (buttoning). Practice took place over 3 weeks, with performance measured weekly; retention was measured weekly for 3 weeks after practice. There were 7 people with Parkinson's disease (PD) and 7 age-matched neurologically healthy people who participated in this study. Both groups improved performance on both tasks with practice, with the majority of the change for the PD group occurring between 1 and 2 weeks of practice. Although those with PD did not necessarily perform as well as age-matched controls, they learned both sequences in a manner similar to age-matched controls, and exhibited retention across the 3-week retention interval. If people with PD are given sufficient practice they can learn and retain both memory-based and context driven movement sequences as well as age-matched controls. The results provide support for maintaining physical activity and for intervention through movement therapy. PMID- 16671076 TI - Use of antiparkinsonian drugs in Denmark: results from a nationwide pharmacoepidemiological study. AB - The objective of the present study was to record the use of antiparkinsonian drugs (APD) in Denmark and discuss estimates of the incidence and prevalence rates of Parkinson's disease (PD). The main indication for treatment with APD is idiopathic PD. The use of APD is, therefore, an indicator of the epidemiology of PD and Parkinsonism. We used a drug tracer design, which previously has been found applicable in estimating the frequency of PD. From a national prescription database, all persons who purchased APD from 1995 to 2002 could be identified on an individual level. Results show an age-standardized prevalence rate for APD purchase of 164.0 persons per 100,000, and an incidence rate of 55.9 persons per 100,000. The total number of persons purchasing APD was 11,656 per year on average. Our results showed higher figures of persons purchasing APD than the estimated prevalence of idiopathic PD in Denmark, which is approximately 100 persons per 100,000, corresponding to 5,000 to 6,000 persons. The differences might in part be explained by other indications for APD prescription in addition to PD and in part by misdiagnosis. However, the possibility of somewhat higher incidence and prevalence rates of PD than hitherto estimated should be considered. PMID- 16671077 TI - Cardiac 123I-MIBG scintigraphy in patients with essential tremor. AB - In some cases, it is difficult to differentiate essential tremor (ET) from Parkinson's disease (PD), especially in the early stages of the disease. We investigated cardiac sympathetic dysfunction using (123)I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy in 22 patients with ET, in comparison with early PD and tremor-dominant PD (TDPD). The mean ratio of (123)I-MIBG uptake in the region of interest in the heart to that in the mediastinum (H/M ratio) was significantly greater in patients with ET (1.99 +/- 0.21) than in those with either TDPD (1.28 +/- 0.11) or early PD (1.28 +/- 0.17; each P < 0.001). The H/M ratio in all patients with ET was greater than two standard deviations above the range of the ratio in the patients with early PD or TDPD. PMID- 16671078 TI - Striatal dopamine transporter binding in Parkinson's disease associated with the LRRK2 Gly2019Ser mutation. AB - We measured striatal dopamine transporter binding using [(123)I]ioflupane and SPECT in patients with Parkinson's disease associated with the LRRK2 (PARK8) Gly2019Ser gene mutation (LRRK2-PD) and in gene-negative patients with idiopathic Parkinson's disease (IPD) of comparable disease duration and severity. The LRRK2 PD group consisted of a total of 10 patients (3 sporadic) with mean age 62 +/- 14 years, disease duration 9 +/- 3 years, and UPDRS III motor score 21.60 +/- 6.65. The control IPD group consisted of 15 patients with mean age 59 +/- 9 years, disease duration 9 +/- 5 years, and UPDRS III motor score 23.80 +/- 8.69. [(123)I]ioflupane-specific uptake ratios were calculated for caudate nucleus and putamen using the occipital cortex as reference region. We found no differences between the LRRK2-PD group and IPD in all items studied. In particular, putamen and caudate uptake values as well as side asymmetry indexes and putamen/caudate ratios all revealed comparable between-group values. We conclude that in these patients carrying the LRRK2 Gly2019Ser mutation, the neurodegenerative process results in a pattern of nigrostriatal dopaminergic dysfunction similar to that observed in IPD. PMID- 16671079 TI - Antiamphiphysin-positive stiff-person syndrome associated with small cell lung cancer. AB - The paraneoplastic amphiphysin(+) stiff-person syndrome (SPS) has so far only been described in women with breast adenocarcinoma. Here, we describe the rare case of a female patient with antiamphiphysin(+) SPS due to small cell cancer of the lung. PMID- 16671080 TI - Fractal dimension in human cortical surface: multiple regression analysis with cortical thickness, sulcal depth, and folding area. AB - Fractal dimension (FD) has been widely used to provide a quantitative description of structural complexity in the cerebral cortex. FD is an extremely compact measure of shape complexity, condensing all details into a single numeric value. We interpreted the variation of the FD in the cortical surface of normal controls through multiple regression analysis with cortical thickness, sulcal depth, and folding area related to cortical complexity. We used a cortical surface showing a reliable representation of folded gyri and manually parcellated it into frontal, parietal, temporal, and occipital regions for regional analysis. In both hemispheres the mean cortical thickness and folding area showed significant combination effects on cortical complexity and accounted for about 50% of its variance. The folding area was significant in accounting for the FD of the cortical surface, with positive coefficients in both hemispheres and several lobe regions, while sulcal depth was significant only in the left temporal region. The results may suggest that human cortex develops a complex structure through the thinning of cortical thickness and by increasing the frequency of folds and the convolution of gyral shape rather than by deepening sulcal regions. Through correlation analysis of FD with IQ and the number of years of education, the results showed that a complex shape of the cortical surface has a significant relationship with intelligence and education. Our findings may indicate the structural characteristics that are revealed in the cerebral cortex when the FD in human brain is increased, and provide important information about brain development. PMID- 16671081 TI - Calibration of BOLD fMRI using breath holding reduces group variance during a cognitive task. AB - The proportionality of blood oxygen level-dependent (BOLD) response during a cognitive task and that from a hypercapnic challenge was investigated in cortical structures involved in working memory (WM). Breath holding (BH) following inspiration was used to induce a BOLD response characteristic of regional vasomotor reactivity but devoid of metabolic changes. BOLD effects measured during BH were used to normalize individual subject activations during WM, which effectively reduced the confounding influence of individual- and region-specific differences in hemodynamic responsivity common to both tasks. In a study of seven subjects, the BH calibration reduced intersubject variability in WM effect amplitude by 24.8% (P < 0.03). Reduced intersubject variability resulted in a 23.7% increase in group WM activation voxel extent significant at P < 0.001, with further increases at more stringent thresholds. Because the BH task does not require CO(2) inhalation or other invasive manipulations and is broadly applicable across cortical regions, the proposed approach is simple to implement and may be beneficial for use not only in quantitative group fMRI analyses, but also for multicenter and longitudinal studies. PMID- 16671082 TI - Statistical neuroanatomy of the human inferior frontal gyrus and probabilistic atlas in a standard stereotaxic space. AB - We manually defined the inferior frontal gyrus (IFG) on high-resolution MRIs in native space in 30 healthy subjects (15 female, median age 31 years; 15 male, median age 30 years), resulting in 30 individual atlases. Using standard software (SPM99), these were spatially transformed to a widely used stereotaxic space (MNI/ICBM 152) to create probabilistic maps. In native space, the total IFG volume was on average 5%, and the gray matter (GM) portion 12% larger in women (not significant). Expressed as a percentage of ipsilateral frontal lobe volume (i.e., correcting for brain size), the IFG was an average of 20%, and the GM portion of the IFG 27%, larger in women (P < 0.005). Correcting for total lobar volume yielded the same result. No asymmetry was found in IFG volumes. There were significant positional differences between the right and left IFGs, with the right IFG being further lateral in both native and stereotaxic space. Variability was similar on the left and right, but more pronounced anteriorly and superiorly. We show differences in IFG volume, composition, and position between sexes and between hemispheres. Applications include probabilistic determination of location in group studies, automatic labeling of new scans, and detection of anatomical abnormalities in patients. PMID- 16671083 TI - Botulinum toxin as a treatment for tardive dyskinesia. AB - Two older female patients with a history of psychotic depression were treated with a combination of an antipsychotic and an antidepressant. Both developed persistent tardive dyskinesia with severe tongue protrusion. Botulinum toxin was injected into the genioglossal area and the embarrassing tongue protrusion practically disappeared. Both patients chose to continue taking botulinum toxin. PMID- 16671085 TI - Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8-associated polyclonal body cavity effusions that mimic primary effusion lymphomas. PMID- 16671086 TI - NNK activates ERK1/2 and CREB/ATF-1 via beta-1-AR and EGFR signaling in human lung adenocarcinoma and small airway epithelial cells. AB - We have shown that the tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) is an agonist for -adrenergic receptors (beta-ARs) and increased DNA synthesis of human lung adenocarcinoma cells with features of bronchiolar Clara cells by binding to these receptors. Using a cell line derived from a human pulmonary adenocarcinoma with Clara cell phenotype (PACC) and immortalized human small airway epithelial cells (HPLD1), the putative cells of origin of this cancer type, our current studies have analyzed signaling initiated by binding of NNK to the beta 1-AR. NNK upregulated ERK1/2 and CREB/ATF-1 phosphorylation in a PKA-dependent manner in both cell lines. This response was further increased by transient overexpression of the beta 1-AR. Pre-exposure of cells to the selective beta 1-AR antagonist, atenolol, attenuated the stimulatory effects of NNK, suggesting the latter upregulated ERK1/2 and CREB/ATF-1 via this receptor. In vivo labeling and immunoprecipitation assays revealed that NNK phosphorylated the epidermal growth factor receptor (EGFR) at tyrosine residues, 991, 1068 and 1173, an effect inhibited by atenolol. The inhibitor of EGFR-specific tyrosine kinases, AG1478, reduced NNK ability to stimulate ERK1/2 and CREB/ATF-1. Genomic analysis of the exons 18-21 of the EGFR genes showed that no mutations were present in either gene. Collectively, our data provide evidence, for the first time, that NNK targets ERK1/2 and CREB/ATF-1 proteins via dual signaling involving beta 1-AR and EGFR pathways in PACCs and their putative cells of origin. PMID- 16671087 TI - Host-bacterial interaction in the development of gastric precancerous lesions in a high risk population for gastric cancer in Venezuela. AB - Helicobacter pylori (HP) infection affects over 50% of the world's population. The prevalence is over 90% in populations at high risk for gastric cancer, but clinical outcomes of the infection are highly variable and thus host genetic factors have been suggested to play a role in its outcomes in addition to bacterial factors. In this study, we examined the effects of common functional genetic polymorphisms of several proinflammatory cytokines known to be overexpressed in HP-infected gastric mucosa on the risk of various stages of gastric premalignant lesions. The odds ratios (ORs) and 95% confidence intervals (CI) for atrophic gastritis, intestinal metaplasia and dysplasia were estimated by multinominal logistic regression analysis among 2,033 Venezuelan subjects. There was a significant effect of IL8 -251A allele on the prevalence of dysplasia (p = 0.021). The OR associated with the A-allele was 1.34 (95% CI: 0.82-2.18) for heterozygotes and 2.00 (95% CI: 1.13-3.56) for homozygotes, compared with the TT genotype. Furthermore, there was a statistically significant interaction between the number of A-alleles and HP cag A genotype (p = 0.009), suggesting that the A allele increased the risk of dysplasia only when cag A was present. The OR for the AA compared with TT genotype was 3.22 (95% CI: 1.60-6.52) in this group. There were no associations with other proinflammatory cytokines studied, i.e., IL1 beta, IL6, monocyte chemoattractant protein 1 (MCP1) and TNF alpha, or with other stages of premalignant lesions. The present study provides important evidence suggesting host-bacterial interactions in the development of gastric precancerous lesions. PMID- 16671088 TI - Analysis of genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine in gastric cancer cell lines. AB - In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC), cDNA microarray analysis (AceGene(R), containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real-time PCR, and the methylation status of 5'CpG islands was determined by bisulfite DNA sequencing or methylation-specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3-fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5'CpG islands of BCL2L10, DKK1 and DNAJD1. 5'CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5'CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth-inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis. PMID- 16671089 TI - The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma. AB - Vascular endothelial growth factor receptor 1 (VEGFR-1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR-1 on tumor cells. Here, we report that VEGFR-1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR-1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen activated protein kinase (MAPK) or Akt pathways. Ligand-stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti-VEGFR-1 neutralizing monoclonal antibody. Treatment with anti-VEGFR-1 mAb significantly suppressed the growth of DU4475, MCF-7, BT-474 and MDA-MB-231 breast xenografts in athymic mice. Histological examination of anti-VEGFR-1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR-1 on tumor cells and murine VEGFR-1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors. PMID- 16671090 TI - Survivin in esophageal cancer: An accurate prognostic marker for squamous cell carcinoma but not adenocarcinoma. AB - We quantified the expression of survivin, both as mRNA in real-time PCR and protein in immunohistochemistry, in tumor samples of 112 patients with esophageal cancer (56 squamous cell carcinomas and 56 adenocarcinomas). Overall survival of squamous cell carcinoma patients with high survivin mRNA levels was significantly less than that of patients with low survivin mRNA levels (p = 0.0033). Distribution pattern of survivin (nuclear vs. cytoplasmic or mixed) was not correlated to survival, while the extent of immunostaining was significantly correlated to survivin mRNA values (p = 0.016) and had prognostic relevance in univariate analysis (p = 0.0012). Cox's proportional-hazard regression model showed that tumor survivin expression in esophageal squamous cell carcinoma was the most important prognostic factor, independent of tumor stage and other histopathological factors, both as mRNA relative value (p = 0.0259) and protein immunostaining (p = 0.0147). In esophageal adenocarcinoma, survivin expression and pattern of distribution had no prognostic relevance. Thus, quantifying survivin expression provides a prognostic marker only for esophageal squamous tumors. PMID- 16671091 TI - Methotrexate resistance in vitro is achieved by a dynamic selectionprocess of tumor cell variants emerging during treatment. AB - Genetic instability leads to tumor heterogeneity, which in turn provides a source of cell variants responsible for drug resistance. However, the source of resistant cells during the process of acquired resistance is poorly understood. Our aim has been to characterize the mechanism by which acquired resistance to methotrexate emerges during the course of cancer cell treatment in vitro. We recently demonstrated that, in vitro, HT-29 colon cancer cells become transiently sensitive to methotrexate by depleting the extracellular milieu of survival factors; on the other hand, the cell population under treatment can reversibly adapt to grow below a critical cell density in the presence of the drug. Here, we show that this adapted cell population gives rise to permanent resistant populations through repeated cycles of cell death and growth. This increased cell turnover, but not merely cell proliferation, is required for the appearance of increasing degrees of stable resistance that are progressively selected by drug pressure. Such a process, taking place in multiple steps, is here designated "dynamic selection." The analysis of sensitive and resistant HT-29 cell populations revealed that methotrexate induces genomic instability--characterized by centrosome amplification and aberrant chromosome recombination--leading to a low-level amplification of the 5q chromosome arm as one of the earliest genetic events selected during treatment. Therefore, this model provides a mechanism by which a tumor cell population lacking resistant subpopulations before treatment is able to acquire the genetic changes required for stable drug resistance. PMID- 16671092 TI - Chemokines and cancer. AB - The chemokines and their receptors are a superfamily of small secreted molecules that control the migration of many cell types in the body. Several years ago it became clear that some chemokines and receptors regulate the migration of certain cells in the lymphoid system, and this raised the possibility that chemokines could also control the migration of tumor cells in the body. Breast cancer cells were found to express chemokine receptors in a nonrandom manner, and these observations pointed to several chemokine/receptor pairs that control tumor-cell migration. The most important ligand/receptors pairs in these phenomena include CXCL12/CXCR4 and CCL21/CCR7. Since then, there has been intense interest in this area and many studies have been published, especially on CXCR4. These studies point to the following conclusions: (i) Tumors express chemokine receptors in a nonrandom manner. (ii) CXCR4 is the most widely expressed chemokine receptor in many different cancers. (iii) CCR7 is also expressed by many cancers, and is likely to mediate metastasis to the lymph nodes in selected cancers. (iv) The effects of CXCL12 on CXCR4-bearing tumor cells likely include many other functions (growth, differentiation) besides migration. During normal development, the interaction CXCL12/CXCR4 is known to be involved in organogenesis. This process shares many characteristics with metastasis, and represents one of the key areas of future research. PMID- 16671093 TI - Restless legs syndrome in Parkinson's disease patients may improve with subthalamic stimulation. AB - We report on 6 advanced Parkinson's disease (PD) patients who underwent bilateral subthalamic nucleus (STN) deep brain stimulation (DBS) surgery whose restless legs syndrome (RLS) improved postoperatively. Despite a mean 56% decrease in their levodopa equivalents postoperatively, their RLS scores dropped by a mean of 84% (100% in three). Our findings suggest that bilateral STN DBS surgery can improve RLS in patients with advanced PD. PMID- 16671094 TI - Performing functional magnetic resonance imaging in patients with Parkinson's disease treated with deep brain stimulation. AB - Deep brain stimulation (DBS) is a relatively novel treatment in advanced Parkinson's disease (PD). Functional magnetic resonance imaging (fMRI) is a useful technique for examining the effects of DBS both within the basal ganglia and its cortical connectivity. There are technical difficulties in imaging patients with PD, and the DBS itself can generate image artifacts. We describe aspects related to optimizing the fMRI acquisition parameters in patients with DBS and the results of sensorimotor activation tasks performed by four PD patients during hand, foot, and tongue movements, both before and after DBS implant. Provided that all safety conditions are followed, it is possible to perform fMRI in patients with PD and DBS. The standard DBS surgical procedure has to be slightly modified in order to reduce image artifacts. The event-related design provided increased power to detect sensorimotor cortex and basal ganglia activation. PMID- 16671095 TI - A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson-Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS. AB - Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder normally caused by a spontaneous heterozygous mutation in the LMNA gene that codes for the nuclear lamina protein lamin A. Several enzymes are involved in the processing of its precursor, prelamin A, to the mature lamin A. A functional knockout of one of the enzymes involved in prelamin A processing, the zinc metalloprotease ZMPSTE24, causes an even more severe disorder with early neonatal death described as restrictive dermatopathy (RD). This work describes a HGPS patient with a combined defect of a homozygous loss-of-function mutation in the ZMPSTE24 gene and a heterozygous mutation in the LMNA gene that results in a C terminal elongation of the final lamin A. Whereas the loss of function mutation of ZMPSTE24 normally results in lethal RD, the truncation of LMNA seems to be a salvage alteration alleviating the clinical picture to the HGPS phenotype. The mutations of our patient indicate that farnesylated prelamin A is the deleterious agent leading to the HGPS phenotype, which gives further insights into the pathophysiology of the disorder. PMID- 16671096 TI - m.6267G>A: a recurrent mutation in the human mitochondrial DNA that reduces cytochrome c oxidase activity and is associated with tumors. AB - Complete sequencing of the mitochondrial genome of 13 cell lines derived from a variety of human cancers revealed nine novel mitochondrial DNA (mtDNA) variations. One of them, m.6267G>A, is a recurrent mutation that introduces the Ala122Thr substitution in the mitochondrially encoded cytochrome c oxidase I (MT CO1): p.MT-CO1: Ala122Thr (GenBank: NP_536845.1). Biochemical analysis of the original cell lines and the transmitochondrial cybrids generated by transferring mitochondrial DNAs to a common nuclear background, indicate that cytochrome c oxidase (COX) activity, respiration, and growth in galactose are impaired by the m.6267G>A mutation. This mutation, found twice in the cancer cell lines included in this study, has been also encountered in one out of 63 breast cancer samples, one out of 64 colon cancer samples, one out of 260 prostate cancer samples, and in one out of 15 pancreatic cancer cell lines. In all instances the m.6267G>A mutation was associated to different mtDNA haplogroups. These findings, contrast with the extremely low frequency of the m.6267G>A mutation in the normal population (1:2264) and its apparent absence in other pathologies, strongly suggesting that the m.6267G>A missense mutation is a recurrent mutation specifically associated with cancer. PMID- 16671097 TI - Genome-wide identification of pseudogenes capable of disease-causing gene conversion. AB - Pseudogenes are remnants of gene duplication (nonprocessed pseudogenes) and retrotransposition (processed pseudogenes) events. This study describes methods for identifying gene conversion candidates from predicted pseudogenes. Pseudogenes may accumulate and harbor sequence variations over time that become disease-causing mutations when transferred to genes by gene conversion. A total of 14,476 pseudogenes were identified, including 3,426 nonprocessed pseudogenes. In addition, 1,945 nonprocessed pseudogenes that are localized near their progenitor gene were evaluated for their possible role in gene conversion and disease. All 11 known, human cases of gene conversion (with deleterious effects) involving pseudogenes were successfully identified by these methods. Among the pseudogenes identified is a retinitis pigmentosa 9 (RP9) pseudogene that carries a c.509A>G mutation which produces a p.Asp170Gly substitution that is associated with the RP9 form of autosomal dominant retinitis pigmentosa (adRP). The c.509A>G mutation in RP9 is a previously unrecognized example of gene conversion between the progenitor gene and its pseudogene. Notably, two processed pseudogenes also contain mutations associated with diseases. An inosine monophosphate dehydrogenase 1 (IMPDH1) pseudogene carries a c.676G>A mutation that produces a p.Asp226Asn substitution that causes the retinitis pigmentosa 10 (RP10) form of adRP; and a phosphoglycerate kinase 1 (PGK1) pseudogene (PGK1P1) carries a c.837T>C mutation that produces a p.Ile252Thr substitution that is associated with a phosphoglycerate kinase deficiency. Ranking of nonprocessed pseudogenes as candidates for gene conversion was also performed based on the sequence characteristics of published cases of pseudogene-mediated gene conversion. All results and tools produced by this study are available for download at: http://genome.uiowa.edu/pseudogenes. PMID- 16671098 TI - Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores. AB - The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies. PMID- 16671099 TI - Regulation of lipocalin-2 gene by the cancer chemopreventive retinoid 4-HPR. AB - N-(4-Hydroxyphenyl)retinamide (4-HPR) is a nonclassical retinoid with cancer preventive effects in vivo and antiproliferative and apoptotic activities in vitro. Examining the transcriptional profile of human breast cancer cell lines, MCF7 and T47D, treated with 4-HPR, we identified the lipocalin member LCN2 (NGAL or 24p3) as a gene, markedly induced by the retinoid. Because of its presumed function in apoptosis, LCN2 was examined more thoroughly in response to 4-HPR. Like mRNA, the expression of LCN2 protein in MCF7 and T47D cells was highly induced in a time-dependent manner by 4-HPR, but not by its inactive metabolite 4 MPR and, to some extent, this event was linked to the free radicals normally generated by 4-HPR. All-trans retinoic acid also induced LCN2 protein, particularly in T47D cells. Ectopic LCN2 compromised cell viability, and the few MCF7 clones that survived LCN2 overexpression were less sensitive than do mock cells to 4HPR, indicating that selective pressure for survival to LCN2 confers cross-resistance to 4-HPR. Significantly, ablation of LCN2 induction by siRNA did not modify the response to 4-HPR, implying that LCN2 is not critical for apoptosis by 4-HPR. Our results indicate that 4-HPR markedly induces LCN2 expression, but this event may not represent an apoptotic response. PMID- 16671100 TI - Cancer survival is dependent on season of diagnosis and sunlight exposure. AB - Sunlight is essential for the production of vitamin D in the body. Evidence exists to suggest that vitamin D metabolites may have a role in tumor growth suppression. In this large study, involving over a million cancer patients from the United Kingdom, we have analyzed the role of season of diagnosis and sunlight exposure in cancer survival for cancers of the breast, colorectum, lung, prostate and at all sites combined. We used population-based data from the Thames Cancer Registry to analyze cancer survival in periods 0-1 and 0-5 years after diagnosis. The analysis was performed using Cox proportional regression analysis adjusting for age and period at diagnosis and including season of diagnosis and sunlight exposure in the preceding months as factors in the analysis. We found evidence of substantial seasonality in cancer survival, with diagnosis in summer and autumn associated with improved survival compared with that in winter, especially in female breast cancer patients and both male and female lung cancer patients (hazard ratios 0.86 [95% CI 0.83-0.89], 0.95 [95% CI 0.92-0.97] and 0.95 [95% CI 0.93-0.98] respectively). Cumulative sunlight exposure in the months preceding diagnosis was also a predictor of subsequent survival, although season of diagnosis was a stronger predictor than cumulative sunlight exposure. We found seasonality in cancer survival to be stronger in women than in men. Our results add to a growing body of evidence that vitamin D metabolites play an important role in cancer survival. PMID- 16671101 TI - Er:YAG laser cavity preparation and composite resin bonding with a single component adhesive system: relationship between shear bond strength and microleakage. AB - BACKGROUND AND OBJECTIVES: The purpose of this study was to assess the performances of a single-component adhesive system when an Er:YAG laser was used to prepare cavities. STUDY DESIGN/MATERIALS AND METHODS: Shear bond strength tests were performed on composite rods bonded with a single-component adhesive system to dentin surfaces prepared using either bur+acid, Er:YAG laser (fluence = 44 J/cm2), or Er:YAG laser+acid. Class V cavities were prepared differently according to the molar surface: buccal surfaces were prepared using a diamond bur, and lingual surfaces using the Er:YAG laser. In group 1, cavity walls were not acid treated after Er:YAG laser preparation. In group 2, only the enamel walls were acid treated. In group 3, all the enamel and dentin walls were acid treated. Microleakage was assessed using a methylene blue dye penetration method. RESULTS: Bond strength values did not differ significantly between the three groups (P = 0.162). Better marginal adaptation was obtained between the composite resin and cavity walls when Er:YAG laser preparation was followed by a total acid etching (P = 0.008). CONCLUSIONS: Our results supported the use of single component adhesive systems in cavities prepared using Er:YAG laser. The step of acid etching must be maintained for the whole cavity walls in regard of the microleakage values. PMID- 16671102 TI - Targeted photodynamic therapy. AB - BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is an emerging modality for the treatment of various neoplastic and non-neoplastic pathologies. STUDY DESIGN/MATERIALS AND METHODS: PDT usually occurs when reactive oxygen species (ROS) generated from light-activated chemicals (photosensitizer, PS) destroy the target. For non-dermatologic applications the PS are delivered systemically and accumulate, at different concentrations, in most organs. RESULTS AND CONCLUSION: Typically there is a modest enhanced accumulation of the PS in tumor tissues, providing a first level of selectivity. Additional selectivity is provided by the confined illumination of the target area with the appropriate wavelength of light. For the treatment of pathologies in complex anatomical sites, such as in the peritoneal cavity, where restricted illumination is difficult; improved targeting of the PS is necessary to prevent damage to the surrounding healthy tissue. This article will focus on targeted PDT. PMID- 16671103 TI - The application of photodynamic therapy in the treatment of metastatic endobronchial disease. AB - BACKGROUND AND OBJECTIVE: We utilized photodynamic therapy (PDT) for palliation of metastatic endobronchial tumors employing sensitization with synthetic porphyrin, application of non-thermal light, and endoscopic debridement of necrotic tumor. STUDY DESIGN/MATERIALS AND METHODS: Nine patients with symptomatic endobronchial metastasis from carcinomas of the colon(3), breast(3), kidney(2), and tongue(1) received PDT. RESULTS: After two PDT treatments, patients showed substantial response, which was complete in all but one. One patient had perioperative complications and expired 2 days after developing massive hemoptysis during tumor debridement. Patient survival was 6.38 months (mean) and 4.2 months (median). Most patients died from advanced metastatic disease. One patient with metastasis limited to the airway is still alive 24 months following endobronchial presentation. CONCLUSIONS: PDT ameliorates symptoms of metastatic airway obstruction, is easy to apply, has low morbidity and improves the quality of life. PMID- 16671104 TI - Genetic risk factors associated with lipid-lowering drug-induced myopathies. AB - Lipid-lowering drugs produce myopathic side effects in up to 7% of treated patients, with severe rhabdomyolysis occurring in as many as 0.5%. Underlying metabolic muscle diseases have not been evaluated extensively. In a cross sectional study of 136 patients with drug-induced myopathies, we report a higher prevalence of underlying metabolic muscle diseases than expected in the general population. Control groups included 116 patients on therapy with no myopathic symptoms, 100 asymptomatic individuals from the general population never exposed to statins, and 106 patients with non-statin-induced myopathies. Of 110 patients who underwent mutation testing, 10% were heterozygous or homozygous for mutations causing three metabolic myopathies, compared to 3% testing positive among asymptomatic patients on therapy (P = 0.04). The actual number of mutant alleles found in the test group patients was increased fourfold over the control group (P < 0.0001) due to an increased presence of mutation homozygotes. The number of carriers for carnitine palmitoyltransferase II deficiency and for McArdle disease was increased 13- and 20-fold, respectively, over expected general population frequencies. Homozygotes for myoadenylate deaminase deficiency were increased 3.25-fold with no increase in carrier status. In 52% of muscle biopsies from patients, significant biochemical abnormalities were found in mitochondrial or fatty acid metabolism, with 31% having multiple defects. Variable persistent symptoms occurred in 68% of patients despite cessation of therapy. The effect of statins on energy metabolism combined with a genetic susceptibility to triggering of muscle symptoms may account for myopathic outcomes in certain high-risk groups. PMID- 16671105 TI - Linkage analysis with gene-environment interaction: model illustration and performance of ordered subset analysis. AB - The ordered subset analysis (OSA) method allows for the incorporation of covariates into the linkage analysis of a dichotomous disease phenotype in order to reduce genetic heterogeneity. Complex human diseases may involve gene environment (G x E) interactions, which represent a special form of heterogeneity. Here, we present results of a simulation study to evaluate the performance of OSA when the disease-generating mechanism includes G x E interaction, in the absence of main effects of gene and environment. First, the complex simulation models are illustrated graphically. Second, we show that OSA is underpowered to detect small to moderate interaction effects, consistent with previous evaluations of other linkage analysis methods. When interaction effects are large enough to produce substantial marginal effects, standard linkage methods have sufficient power to detect significant baseline linkage evidence in the entire dataset. The power of OSA to improve upon a high baseline lod score is then strongly dependent on the underlying genetic model, especially the susceptibility allele frequency. If significant, OSA identifies family subsets that are more efficient for follow-up analysis than the entire dataset, in terms of the proportion of susceptible genotypes among generated marker genotypes. For example, when strong G x E interaction with RR(G x E) = 10 is operating in at least 70% of families in the dataset, OSA has at least 70% power to detect a subset of families with significantly greater linkage evidence, the majority of linked families are captured in the OSA subset, and the per-genotype efficiency in the subset is 20-30% greater than in the entire dataset. PMID- 16671106 TI - Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrafish wound inflammation. AB - The inflammatory response is one of the most dramatic examples of directed cell movement in nature. Inflammation is triggered at the site of injury and results in the migration of immune cells to the site to protect the host from infection. We have devised an in vivo inflammation assay using translucent zebrafish embryos, which allow live imaging and pharmacological manipulation of macrophage chemotaxis to wounds inflicted with a laser. Using this assay, we test the role of the microtubule cytoskeleton in macrophage chemotaxis in vivo using nocodazole to disrupt microtubule polymerization. We find that de-stabilisation of microtubules with nocodazole impairs macrophage recruitment to wounds, but that addition of the Rho kinase inhibitor Y-27632 suppresses these effects and restores the recruitment of macrophages to wounds. Taken together, these results suggest that destabilizing microtubules activates Rho kinase and that this increase in Rho kinase activity interferes with leukocyte recruitment in vivo. PMID- 16671107 TI - Nonparametric linkage analysis using person-specific covariates. AB - Linkage analysis provides an important tool for mapping genes for complex disease. However its usefulness has been limited by inadequate marker density, inadequate sample sizes and the possibility that different genes account for different subtypes of the disease (phenotypic heterogeneity). The first two limitations can be addressed by high-density single nucleotide polymorphism (SNP) genotyping and the pooling of large sets of multiple-case families. Phenotypic heterogeneity can be addressed by analyses that weigh the contributions of affected family members according to characteristics of their disease phenotypes. Here we introduce a method for including such person-specific weights in nonparametric linkage analysis. We show with simulations that such weighting can provide stronger linkage signals when a causal polymorphism affects some manifestations of the disease more than others. We applied the method to prostate cancer linkage data in a region on chromosome 19p, and obtained higher lod scores by assigning weights of one to men with early-onset aggressive cancers, weights of zero to those with late-onset nonaggressive cancers, and intermediate weights to all other affected men. We have developed a modified version of GENEHUNTER that allows inclusion of person-specific weights in the nonparametric analyses. This program is freely available at http://med.stanford.edu/epidemiology/statisticalSoftware/weightedKAC. PMID- 16671108 TI - Acute motor and sensory axonal neuropathy in Burkitt-like lymphoma. AB - Immune-mediated neuropathies associated with non-Hodgkin's lymphoma are rare and can be difficult to delineate from neuropathies of other etiologies. We report the clinical and pathological findings of a 36-year-old patient with fulminant quadriplegic neuropathy, left facial nerve palsy, and Burkitt-like lymphoma. Features of the neuropathy, which occurred during induction chemotherapy with a total cumulative dose of 4 mg vincristine, suggested axonal Guillain-Barre syndrome. There was no evidence of direct malignant infiltration of the peripheral nervous system. We hypothesize that immune mechanisms triggered by the lymphoma initiated damage to the peripheral nervous system and enhanced its vulnerability to the toxic effects of vincristine. PMID- 16671109 TI - Omnibus permutation tests of the association of an ensemble of genetic markers with disease in case-control studies. AB - An omnibus permutation test of the overall null hypothesis can be used to assess the association of an entire ensemble of genetic markers with disease in case control studies. In this approach, p-values for univariate marker-specific Armitage trend tests are combined to form a scalar statistic, which is then used in a permutation test to determine an overall p-value. Two previously described competing methods utilize either a standard two-sample Hotelling's T2 statistic or a global U statistic that is a weighted sum of univariate U statistics. In contrast to Hotelling's test, omnibus tests are much less sensitive to missing data, and utilize all available data. In contrast to the global U test, omnibus tests do not require that the direction of the effects of the individual markers on the risk of disease be correctly specified in advance; in fact, any combination of one- and two-sided univariate tests can be used. Simulations show that, even under circumstances favoring the competing tests (no missing data; direction of effects known), omnibus permutation tests based on Fisher's combining function or the Anderson-Darling statistic typically have power comparable to or greater than Hotelling's and the global U tests. PMID- 16671110 TI - Variance component models for X-linked QTLs. AB - This paper discusses the theory and implementation of a model for mapping X linked quantitative trait loci (QTL). As a result of X inactivation, a female's body is subdivided into a number of patches. In each patch one of her two X chromosomes is randomly switched off. This smooths the allelic contributions in a heterozygote and implies that females should show less trait variation than males for an X-linked trait. The latest version of the genetic analysis program Mendel incorporates a simple variance component version of this model. An application to head circumference in autistic children illustrates Mendel in action. PMID- 16671111 TI - FISH detection of t(14;18) in follicular lymphoma on Papanicolaou-stained archival cytology slides. AB - BACKGROUND: The t(14;18)(q32;q21) translocation is present in about 85% of follicular lymphomas (FL) and can be identified using fluorescence in situ hybridization (FISH). In the diagnostic laboratory setting, the cytologic archival material consists of stained slides, and only rarely is material saved for molecular testing. The authors proposed FISH for FL using Papanicolaou stained archival cytology material as a practical ancillary technique for diagnosing FL. METHODS: Cases included 35 FL, 6 small lymphocytic lymphomas/chronic lymphocytic leukemias (SLL/CLL), 4 mantle cell lymphomas (MCL), 4 marginal zone lymphomas (MZL), 1 lymphoplasmacytic lymphoma (LPL), and 10 reactive lymphoid tissues (RLT). FISH was performed on Papanicolaou-stained archival cytology slides using probes for immunoglobulin heavy chain (IGH) on chromosome 14 and BCL2 on chromosome 18. RESULTS: In all, 25 of 32 (81%) FL cases exhibited the t(14;18) translocation, whereas 7 of 32 (19%) lacked the translocation. No cases of non-FL were positive for t(14;18). This series shows a sensitivity of 81% and specificity of 100% for detecting the t(14;18) translocation as a diagnostic tool in FL. CONCLUSIONS: When performed on Papanicolaou-stained cytology slides, FISH for t(14;18) is relatively sensitive and quite specific for FL. These findings are similar to those reported on other specimens, such as paraffin-embedded tissue and unstained cytology slides. The authors proposed that their technique would allow the pathologist and clinician the flexibility to utilize previously stained fine-needle aspiration slides for FISH evaluation. PMID- 16671112 TI - Rapid prescreening of Papanicolaou smears: a practical and efficient quality control strategy. PMID- 16671114 TI - The preparation and characterisation of hetero- and homobimetallic complexes containing bridging naphthalene-1,8-dithiolato ligands. AB - Homo- and heterobimetallic complexes of the form [(PPh(3))(2)(mu(2)-1,8-S(2) nap){ML(n)}] (in which (1,8-S(2)-nap)=naphtho-1,8-dithiolate and {ML(n)}={PtCl(2)} (1), {PtClMe} (2), {PtClPh} (3), {PtMe(2)} (4), {PtIMe(3)} (5) and {Mo(CO)(4)} (6)) were obtained by the addition of [PtCl(2)(NCPh)(2)], [PtClMe(cod)] (cod=1,5-cyclooctadiene), [PtClPh(cod)], [PtMe(2)(cod)], [{PtIMe(3)}(4)] and [Mo(CO)(4)(nbd)] (nbd=norbornadiene), respectively, to [Pt(PPh(3))(2)(1,8-S(2)-nap)]. Synthesis of cationic complexes was achieved by the addition of one or two equivalents of a halide abstractor, Ag[BF(4)] or Ag[ClO(4)], to [{Pt(mu-Cl)(mu-eta(2):eta(1)-C(3)H(5))}(4)], [{Pd(mu-Cl)(eta(3) C(3)H(5))}(2)], [{IrCl(mu-Cl)(eta(5)-C(5)Me(5))}(2)] (in which C(5)Me(5)=Cp*=1,2,3,4,5-pentamethylcyclopentadienyl), [{RhCl(mu-Cl)(eta(5) C(5)Me(5))}(2)], [PtCl(2)(PMe(2)Ph)(2)] and [{Rh(mu-Cl)(cod)}(2)] to give the appropriate coordinatively unsaturated species that, upon treatment with [(PPh(3))(2)Pt(1,8-S(2)-nap)], gave complexes of the form [(PPh(3))(2)(mu(2)-1,8 S(2)-nap){ML(n)}][X] (in which {ML(n)}[X]={Pt(eta(3)-C(3)H(5))}[ClO(4)] (7), {Pd(eta(3)-C(3)H(5))}[ClO(4)] (8), {IrCl(eta(5)-C(5)Me(5))}[ClO(4)] (9), {RhCl(eta(5)-C(5)Me(5))}[BF(4)] (10), {Pt(PMe(2)Ph)(2)}[ClO(4)](2) (11), {Rh(cod)}[ClO(4)] (12); the carbonyl complex {Rh(CO)(2)}[ClO(4)] (13) was formed by bubbling gaseous CO through a solution of 12. In all cases the naphtho-1,8 dithiolate ligand acts as a bridge between two metal centres to give a four membered PtMS(2) ring (M=transition metal). All compounds were characterised spectroscopically. The X-ray structures of 5, 6, 7, 8, 10 and 12 reveal a binuclear PtMS(2) core with PtM distances ranging from 2.9630(8)-3.438(1) A for 8 and 5, respectively. The napS(2) mean plane is tilted with respect to the PtP(2)S(2) coordination plane, with dihedral angles in the range 49.7-76.1 degrees and the degree of tilting being related to the PtM distance and the coordination number of M. The sum of the Pt(1)coordination plane/napS(2) angle, a, and the Pt(1)coordination plane/M(2)coordination plane angle, b, a+b, is close to 120 degrees in nearly all cases. This suggests that electronic effects play a significant role in these binuclear systems. PMID- 16671115 TI - A new type of in situ single-molecule rectifier. PMID- 16671116 TI - Interaction of carboranes with biomolecules: formation of dihydrogen bonds. AB - Noncovalent interactions of the polyhedral carborane 1-carba-closo-dodecaborane (CB(11)H(12))(-) with building blocks of biomolecules, modelled by glycine (GLY), serine (SER), phenylalanine (PHE), glutamic acid (GLU), lysine (LYS) and arginine (ARG), were investigated in vacuo by molecular dynamics simulations with the UFF empirical potential. Selected structures were further studied by accurate ab initio quantum chemical procedures. Interactions with a peptide bond (GLY-SER dipeptide) and a nucleic acid building block (guanine) were also considered. The RESP and NPA charges of carboranes and small model systems are compared and their use is discussed. The dominant interaction between carboranes and biomolecules is the formation of unconventional proton-hydride hydrogen bonds (dihydrogen bonds) characterized by a short distance between hydrogen atoms (as close as 1.8 A) and an average strength in the range of 4.2-5.8 kcal mol(-1). The total stabilization energy of complexes investigated is rather large, and the largest value (approximately 15 kcal mol(-1)) was found for the carborane complexes with ARG and the GLY-SER dipeptide. These interactions are ubiquitous under geometrical constraints influencing the strength of the interaction. The carborane forms dihydrogen bonds with biomolecules preferably with the hydrogen atoms of its lower hemisphere (i.e. the part of the cage opposite to the carbon atom). These two geometrical factors can be used to explain the specificity of inhibition of HIV protease by carboranes. PMID- 16671118 TI - A hybrid water-chloride structure with discrete undecameric water moieties self assembled in a heptaprotonated octaamino cryptand. PMID- 16671119 TI - A highly enantioselective, Pd-TangPhos-catalyzed hydrogenation of N-tosylimines. PMID- 16671120 TI - Insertion of arynes into sigma bonds. PMID- 16671121 TI - The glycine-water complex. PMID- 16671122 TI - A quantum chemical study of the quintuple bond between two chromium centers in [PhCrCrPh]: trans-bent versus linear geometry. PMID- 16671123 TI - Biotin-functionalized cellulose-based monolayers as sensitive interfaces for the detection of single molecules. PMID- 16671124 TI - The molecular mechanism of bee venom phospholipase A2 inactivation by bolinaquinone. AB - The molecular basis of the inactivation of bee venom PLA2 by the marine natural product bolinaquinone (BLQ) was studied by several spectral techniques (CD, fluorescence, and NMR spectroscopy, mass spectrometry), biomimetic reactions, and molecular modeling. Our data suggest competitive inhibition based on a BLQ-PLA2 noncovalent molecular recognition. However, BLQ is also able to react selectively with Lys133 through conjugate addition followed by a beta elimination. The biological implications of both the covalent and noncovalent molecular events are discussed. PMID- 16671125 TI - A new supramolecular structure made of two different plasmids linked by a circular oligonucleotide. PMID- 16671126 TI - Replacement of natural cofactors by selected hydrogen peroxide donors or organic peroxides results in improved activity for CYP3A4 and CYP2D6. PMID- 16671127 TI - Detection of mRNA in mammalian cells with a split ribozyme reporter. PMID- 16671128 TI - Theoretical investigation of the decarbonylation of acetaldehyde by Fe+ and Cr+. AB - We report a comprehensive theoretical study on the decarbonylation of acetaldehyde by Fe+ and Cr+. Various intermediates, transition states, and products involved in the decarbonylation reactions are fully optimized at the B3LYP/6-311+G(2df,2pd) level of theory. The potential energy surfaces (PESs) corresponding to [M,O,C2,H4]+(M=Cr and Fe) are examined in detail using B3LYP and CCSD(T) methods, respectively. The validity of these theoretical methods is calibrated with respect to the available thermochemical data. Calculations suggest that the Cr+ mediated decarbonylation of acetaldehyde takes place in four steps on the sextet surface: encounter complexation, C-C activation, aldehyde H shift, and nonreactive dissociation, in good accordance with the Co+ mediated decarbonylation of acetaldehyde [Zhao, Zhang, Guo, Wu, Lu, Chem. Phys. Lett. 2005, 414, 28], while for the Fe+/acetaldehyde system decarbonylation can occur on both the quartet and the sextet PESs. The quartet pathway, which experiences spin-orbit coupling between the two surfaces, is energetically more favorable; whereas along the sextet decarbonylation coordinate several high-energy barriers are revealed. The theoretical results are compared with the experimental product kinetic energy and angular distributions of decarbonylation of acetaldehyde by Fe+ and Cr+ measured using a crossed-beam technique [Sonnenfroh, Farrar, J. Am. Chem. Soc. 1986, 108, 3521]. PMID- 16671130 TI - Oxygenated edge plane sites slow the electron transfer of the ferro-/ferricyanide redox couple at graphite electrodes. AB - The electron transfer kinetics of ferrocyanide, potassium hexachloroiridate(III), hexaammineruthenium(III) chloride, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) have been examined at basal plane and edge plane pyrolytic graphite electrodes which have been allowed to oxidise in air for various periods of time. It is demonstrated via voltammetric and X-ray photoelectron spectroscopy (XPS) analysis that oxygenated species formed at edge plane sites/defects decrease the electron transfer kinetics of ferrocyanide but that the rates for potassium hexachloroiridate(III), hexaammineruthenium(III) chloride and TMPD are insensitive to the oxygenated species. The behaviour of the ferro-/ferricyanide couple contrasts with that seen on single-walled carbon nanotubes where oxygenation of the tube ends is known to speed up the electron transfer kinetics (A. Chou, T. Bocking, N. K. Singh, J. J. Gooding, Chem. Commun. 2005, 842); the possible reasons for this contrasting behaviour are discussed. PMID- 16671129 TI - Details of the partial unfolding of T4 lysozyme on quartz using site-directed spin labeling. PMID- 16671131 TI - Chemical sensing with familiar devices. PMID- 16671132 TI - Construction of enantiomerically enriched tertiary alpha-hydroxycarboxylic acid derivatives by phase-transfer-catalyzed asymmetric alkylation of diaryloxazolidin 2,4-diones. PMID- 16671133 TI - Catalytic enantioselective and diastereoselective addition of aldehyde-derived enecarbamates to alpha-oxo aldehydes. PMID- 16671134 TI - A facile experimental method to determine the hydride affinity of polarized olefins in acetonitrile. PMID- 16671135 TI - Synthesis and characterization of [PhC(NtBu)2]SiCl: a stable monomeric chlorosilylene. PMID- 16671136 TI - Starbons: new starch-derived mesoporous carbonaceous materials with tunable properties. PMID- 16671137 TI - Ir- and Ru-catalyzed sequential reactions: asymmetric alpha-alkylative reduction of ketones with alcohols. PMID- 16671138 TI - A chemically switchable molecular pinwheel. PMID- 16671139 TI - Directed meta-metalation using alkali-metal-mediated zincation. PMID- 16671140 TI - With molecular-oxygen-activated Lewis acids: dinuclear molybdenum complexes for aza-Diels-Alder reactions of acyl hydrazones. PMID- 16671141 TI - The enantioselective allylation and crotylation of sterically hindered and functionalized aryl ketones: convenient access to unusual tertiary carbinol structures. PMID- 16671142 TI - Crystallographic characterization of a synthetic 1:1 end-on copper dioxygen adduct complex. PMID- 16671143 TI - Chirality-induced switch in hydrogen-bond topology: tetrameric methyl lactate clusters in the gas phase. PMID- 16671144 TI - In(OTf)3-catalyzed tandem nucleophilic addition and cyclization of ortho alkynylarylaldimines to 1,2-dihydroisoquinolines. PMID- 16671145 TI - A eta4-dithio-para-benzoquinone metal complex. PMID- 16671146 TI - Hermaphroditic chirality of a D2-symmetric saddle-shaped porphyrin in multicomponent spontaneous optical resolution: inclusion cocrystals with double helical porphyrin arrays. PMID- 16671147 TI - A carbocyclic carbene as an efficient catalyst ligand for C-C coupling reactions. PMID- 16671148 TI - Spectroscopic and theoretical studies of optically active porphyrin dimers: a system uninterpretable by exciton coupling theory. AB - The electronic excited states of a meso-meso beta-beta doubly linked bis porphyrin are comprehensively investigated by measuring its circular dichroism (CD) and magnetic circular dichroism (MCD) spectra. The observed spectroscopic properties are rationalized by DFT calculations. The frontier molecular orbitals (MOs) are constructed by the linear combinations of the constituent monomers' four MOs. Comparison of a theoretical CD spectrum based on time-dependent DFT (TDDFT) with the experimental spectra resulted in the assignment of the helical conformation of the dimer. This assignment is contrary to the previous assignment based on the point-dipole approximation (exciton coupling theory). PMID- 16671149 TI - Discovery of low-molecular-weight ligands for the AF6 PDZ domain. PMID- 16671150 TI - Vibrational frequencies of water adsorbed on (111) and (221) nickel surfaces from first principle calculations. PMID- 16671151 TI - Density functional study of methyl chemisorption on polycyclic aromatic hydrocarbons. AB - The reactions of methyl radicals with large (up to C(96)H(24)) polycyclic aromatic hydrocarbons (PAHs) are studied by density functional calculations to shed light on the experimentally observed deposition of carbon on highly oriented pyrolytic graphite (HOPG), which occurs when hot HOPG (decorated by nanometre sized defects) is exposed to methyl radicals. The equilibrium structures of the reaction products, together with transition structures for PAHs up to the size of phenanthroperylene, are determined using the density functionals B3LYP, TPSSh, BP86 and TPSS. The structures are analysed by computing the pi orbital axis vector (POAV) and the altitude of the reactive carbon above the molecular plane of the PAH. The strongest C-CH(3) bonds are found at the edges of the PAHs, where the s character of the C orbital involved in the bond is roughly 25 % (sp(3) hybrid orbital). Carbon atoms inside the PAH form bonds with the methyl radical through atomic orbitals with about 16 % s character in the POAV analysis. These bonds are much weaker than those at the edges of the PAH, while the reactive carbon has moved about 40 pm above the molecular plane. At the edges, the PAH carbon atoms do not leave the molecular plane to this extent. The computed barrier heights and geometrical parameters of the transition structures are in agreement with Hammond's postulate, and the relative energies of all of the equilibrium structures can be rationalized by Huckel molecular orbital (HMO) theory. PMID- 16671152 TI - Magic and antimagic protonated water clusters: exotic structures with unusual dynamic effects. PMID- 16671153 TI - The theory of non-Cottrellian diffusion on the surface of a sphere or truncated sphere. AB - A method is developed whereby spherical and other particles can be derivatised with electroactive species on their surface and then immobilised on the surface of an electrode. The chronoamperometric and voltammetric responses in the limit of reversible electrode kinetics are modelled using a theory of charge movement over the surface of the spheres where this movement is considered as a diffusional process. The model is extended to include different distributions of sphere radii and to model the scenario of truncated spheres resting on the electrode surface. It is found that a good estimation of the truncation angle can be found by fitting the experimental data with theoretical predictions. PMID- 16671155 TI - Hydrogen-bond-directed head-to-tail orientation of dipolar merocyanine dyes: a strategy for the design of electrooptical materials. PMID- 16671154 TI - Siphonazole, an unusual metabolite from Herpetosiphon sp. PMID- 16671157 TI - Kid-friendly food pyramid. PMID- 16671156 TI - Warning on Strattera for attention-deficit hyperactivity disorder. PMID- 16671158 TI - FDA proposes additional mad cow safeguards. PMID- 16671159 TI - [Free will and neurobiology: a methodological analysis]. AB - Whether or not the neurobiological basis of mental processes is compatible with the philosophical postulate of free will is a matter of committed debating in our days. What is the meaning of those frequently-quoted experiments concerning voluntary action? Both convictions, being autonomous subjects and exercising a strong influence on the world by applying sciences, have become most important for modern human self-conception. Now these two views are growing apart and appear contradictory because neurobiology tries to reveal the illusionary character of free will. In order to cope with this ostensible dichotomy it is recommended to return to the core of scientific thinking, i. e. to the reflection about truth and methods. The neurobiological standpoint referring to Libet as well as the philosophical approaches to free will must be analysed, considering pre-conceptions and context-conditions. Hence Libet's experiments can be criticised on different levels: methods, methodology and epistemology. Free will is a highly complex system, not a simple fact. Taking these very complicated details into account it is possible to define conditions of compatibility and to use the term free will still in a meaningful way, negotiating the obstacles called pure chance and determinism. PMID- 16671160 TI - [Misdemeanor in frontotemporal dementia]. AB - The nature and prevalence of misdemeanor in patients with dementia due to frontotemporal lobar degeneration has been described in a few case reports and in two small U.S. studies. Our clinical impression suggests that antisocial and aggressive behaviour are relatively frequent in this patient population. The objective of the present study was to verify this observation. For this purpose we developed a standardized questionnaire on misdemeanor in Frontotemporal Dementia. Using this instrument caregivers of 30 patients with Frontotemporal Dementia (FTD), 11 patients with Semantic dementia (SD) and 33 patients with Alzheimer-type dementia (AD) were interviewed. The interview included questions about theft, burglary, damaging other peoples' belongings, verbal or physical offence, bodily harm, drug abuse and use of weapons. Questions about the frequency of criminal behaviour, the amount of damages and consequences if applicable completed the questionnaire. Misdemeanor was found in half of the patients with FTD (15 out of 30) and in 7 out of 11 patients with SD, but only in one out of 33 patients with AD. The most frequent type of inappropriate behaviour was theft (13 patients), particularly shoplifting. 8 patients with FTD, 1 patient with SD and 1 patient with AD entered someone else's house without permission. 10 patients with FTD and 3 patients with SD but none of the patients with AD had physically threatened spouses, relatives or strangers. In one case another person was hurt. PMID- 16671161 TI - [A survey on trials focussing on the treatment of cannabis-dependence]. AB - Epidemiological studies show that the prevalence of cannabis use in Germany is approaching that of countries known for their high consumption rate, such as Australia and some parts of the USA. In these countries, the resulting cannabis related psychosocial damage has led to mainly psychotherapeutic treatment studies (conducted in the past ten years) that will be introduced here. According to these studies, the optimal treatment of cannabis-dependent adults would be a short intervention which consists of a combination of motivational-enhancement and cognitive-behavioural elements as well as individual case-counselling (c. f. "Marihuana Treatment Project"). In parallel to a clear consume-reduction over time, these interventions were associated with abstinence rates between 15 % and 22 % also in longer follow-ups (greater than 12 months). Additionally, an approximately 50 % reduction of cannabis-related problems was described. Without any specific treatment, however, abstinence rates were found to be only between 0 % and 9 %, also without any change in prominent social problems. A similar though less obvious trend occurred in a large multisite study regarding the specific treatment of young people, in which besides motivational and cognitive behavioural also milieu and family therapeutic interventions were tested (c. f. "Cannabis Youth Treatment"). Unfortunately, there exists no controlled treatment study up to now that considered a relevant psychiatric co-morbidity, which usually accompany cannabis-dependence. Whether the interventions above can be recommended and implemented under German conditions should be further investigated, especially considering the growing number of cannabis-consumers seeking treatment. PMID- 16671162 TI - Readers' responses to "are 80 hours a week enough to train a surgeon?". PMID- 16671163 TI - Readers' responses to "are 80 hours a week enough to train a surgeon?". PMID- 16671164 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671165 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671166 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671167 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671168 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671169 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671170 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671171 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671172 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671173 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671174 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671175 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671176 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671177 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671178 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671180 TI - Current awareness in prenatal diagnosis. PMID- 16671179 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671181 TI - Spotlight on Ingrid Oberhansli-Weiss, MD. PMID- 16671182 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671183 TI - Readers' responses to "it is time for marijuana to be reclassified as something other than a schedule I drug". PMID- 16671184 TI - Is C-reactive protein an innocent bystander or proatherogenic culprit? C-reactive protein promotes atherothrombosis. PMID- 16671185 TI - Readers' responses to "thought for food: 2400-year-old advice for today's worldwide weight crisis". PMID- 16671187 TI - Condoms still contentious in Uganda's struggle over AIDS. PMID- 16671186 TI - Japan moves to guard against future health-spending rises. PMID- 16671188 TI - LifePoint rethinks HCA deal. Renegotiations are under way for five hospitals. PMID- 16671189 TI - New immune globulin product. PMID- 16671190 TI - FDA: so-called 'Canadian' products really from other countries. PMID- 16671191 TI - New treatment for myelodysplastic syndrome. PMID- 16671193 TI - Safety alert on blood glucose meters. PMID- 16671192 TI - New warnings for two eczema drugs. PMID- 16671194 TI - The FDA speeds medical treatments for serious diseases. PMID- 16671195 TI - Proper use of fentanyl pain patches. PMID- 16671196 TI - Taking care of your feet. PMID- 16671197 TI - The FDA announces new prescription drug information format. AB - In January 2006, the Food and Drug Administration unveiled a major revision to the format of prescription drug information, commonly called the package insert. To manage the risks of medication use and to reduce medical errors, the newly designed package insert will provide the most up-to-date information in an easy to-read format that draws physician and patient attention to the most important pieces of drug information before a product is prescribed. The new format also will make prescription information more accessible for use with electronic prescribing tools and other electronic information resources. PMID- 16671199 TI - Managing migraines. PMID- 16671198 TI - First inhaled insulin product approved. AB - There is a new, potential alternative for many of the more than 5 million Americans who take insulin injections, with the Food and Drug Administration's approval of the first-ever inhaled insulin. Exubera, an inhaled powder form of recombinant human insulin (rDNA) for the treatment of adult patients with type 1 and type 2 diabetes, is the first new insulin delivery option introduced since the discovery of insulin in the 1920s. PMID- 16671200 TI - Food labels identify allergens more clearly. PMID- 16671202 TI - Symptoms you should know by heart. How to recognize "atypical" danger signs sooner to get help faster. PMID- 16671201 TI - Treating migraine: be proactive. PMID- 16671203 TI - What a new study on low-fat diets means for you. PMID- 16671204 TI - When heart threats gang up. Why cardiac care means treating multiple diseases. PMID- 16671205 TI - How to block medical mistakes. What to do about a major heart threat: errors of omission. PMID- 16671206 TI - Pacemakers: the new generation. Today's implantable units do far more than previous models. PMID- 16671207 TI - Ask the doctors. I've been blessed with good health, but have been seeing an internist for hypertension. I think my blood pressure is too low. I wonder if I should be under the care of a cardiologist to make sure I'm on the correct medication and to head off potential problems. PMID- 16671208 TI - [Serum amyloid A as an effective marker for the assessment of surgical trauma and risk of post-operative complications]. AB - OBJECTIVE: To analyze relations among acute phase reactants in a group of 40 women operated for uterine myom by laparoscopy and open surgery. DESIGN: Prospective study. METHODS: Plasma concentrations of C-reactive protein (CRP), serum amyloid A (SAA) and interleukin 6 (IL-6) were measured together with leukocytes in blood before operation, 24 and 72 hours post operation, respectively. RESULTS: Leukocytes and IL-6 displayed minimal response and decreased quickly after operation to preoperative levels. Concentrations of CRP and SAA remained increased after operation. There were no relationships between leukocytes and acute phase reactants. Normal leukocytes 72 hours post operation were found in 1/3 of women with increased at least one acute phase reactants and in 1/4 of women with increased at least two markers. Typ of surgery, surgical stress and length of surgery were related to the concentration of CRP, IL-6 and SAA. CONCLUSION: Changes in SAA 24 hours after operation are similar to CRP and IL-6. Surgical stress, length of operation and possible risk 72 hours after operation are best predicted by CRP and SAA (at that time IL-6 and leukocytes are practically normal). Maximal increase was found for SAA concentrations. Thus SAA seems to be suitable marker of early postoperative complications. PMID- 16671209 TI - Outbreak news. Avian influenza, Azerbaijan. PMID- 16671210 TI - Outbreak news. Avian influenza, Egypt. PMID- 16671211 TI - Prevention and control of avian influenza in humans in China: achieving the national objectives of the WHO Global Influenza Preparedness Plan. PMID- 16671212 TI - Performance of acute flaccid paralysis (AFP) surveillance and incidence of poliomyelitis, 2004-2005. (Data received in WHO headquarters as of 7 March 2006). PMID- 16671213 TI - Future directions for research on enterotoxigenic Escherichia coli vaccines for developing countries. PMID- 16671214 TI - Outbreak news. Cholera, southern Sudan. PMID- 16671215 TI - Progress in reducing global measles deaths: 1999-2004. PMID- 16671216 TI - Influenza in the world. PMID- 16671217 TI - Outbreak news. Avian influenza, Nigeria. PMID- 16671218 TI - Recommended composition of influenza virus vaccines for use in the 2006-2007 influenza season. PMID- 16671219 TI - The global plan to stop TB, 2006-2015: summary. PMID- 16671220 TI - Outbreak news. Avian influenza--spread of the virus to new countries. PMID- 16671221 TI - Outbreak news. Avian influenza, India. PMID- 16671222 TI - Outbreak news. Avian influenza, Iraq--update. PMID- 16671223 TI - Nocturnal lagophthalmos: an overview and classification. AB - Nocturnal lagophthalmos is the inability to close the eyelids during sleep. Lagophthalmos is associated with exposure keratopathy, poor sleep, and persistent exposure-related symptoms. There are a variety of causes of lagophthalmos, grouped as proptosis/eye exposure etiologies and palpebral insufficiency etiologies. Although obvious lagophthalmos is usually detected, it is sometimes difficult to recognize obscure lagophthalmos, due either to eyelash obstruction or overhang of the upper lid anterior and inferior to the most superior portion of the lower lid in a closed position. We present a novel classification system and illustrations of obvious and obscure lagophthalmos. A diagnosis can usually be made with a focused history and slit lamp examination. Treatment is multipronged and may include minor procedures or ocular surgery to correct the lid malposition; natural, topical or oral agents; and punctal plugs to manage ocular surface effects. Correct and timely diagnosis allows greater opportunity for relief of patient suffering and prevention of severe ocular surface pathology, as well as educated planning for future ocular surgical procedures. PMID- 16671224 TI - Outbreak news. Avian influenza, China--update. PMID- 16671225 TI - Outbreak news. Avian influenza, Indonesia--update. PMID- 16671226 TI - Resurgence of wild poliovirus type 1 transmission and effect of importation into polio-free countries, 2002-2005. PMID- 16671227 TI - Outbreak news. Avian influenza, Indonesia--update. PMID- 16671229 TI - Outbreak news. Meningococcal disease, Sudan. PMID- 16671228 TI - Outbreak news. Avian influenza, Iraq--update. PMID- 16671230 TI - Development and evaluation of new diagnostic tests for human African trypanosomiasis. PMID- 16671231 TI - Outbreak news. Avian influenza, China--update. PMID- 16671232 TI - Outbreak news. Avian influenza, Iraq. PMID- 16671233 TI - Outbreak news. Meningococcal disease, Uganda. PMID- 16671234 TI - Influenza. PMID- 16671235 TI - Outbreak news. Avian influenza, China--update. PMID- 16671236 TI - Confirmation of the elimination of neonatal tetanus in Togo by lot quality assurance-cluster sampling. PMID- 16671238 TI - Outbreak news. Avian influenza, Indonesia. PMID- 16671237 TI - UNAIDS Reference Group on estimates, modelling and projections--statement on the use of the BED assay for the estimation of HIV-1 incidence for surveillance or epidemic monitoring. PMID- 16671239 TI - Outbreak news. Avian influenza, Turkey--update. PMID- 16671240 TI - Diphtheria vaccine. PMID- 16671241 TI - Influenza. PMID- 16671242 TI - Outbreak news. Avian influenza, China. PMID- 16671243 TI - Global Advisory Committee on Vaccine Safety, 1-2 December 2005. PMID- 16671244 TI - New tuberculosis therapy offers potential shorter treatment. PMID- 16671245 TI - Outbreak news. Avian influenza, China--update. PMID- 16671246 TI - Outbreak news. Avian influenza, Indonesia--update. PMID- 16671247 TI - Black fluid from a traumatic wound. PMID- 16671248 TI - Fraudulent, unapproved influenza-related products. PMID- 16671249 TI - Historical background of stereotactic surgery: reflection on stereotactic surgery and the introduction of microelectrode recording in Montreal. PMID- 16671250 TI - Recombination nodule mapping and chiasma distribution in spermatocytes of the pigeon, Columba livia. AB - Pigeon spermatocytes were processed with a drying-down technique and their synaptonemal complex (SC) complements were analyzed by electron microscopy. The synaptonemal complex karyotype of the macrobivalents shows an excellent correspondence with the mitotic karyotype. The number and distribution of recombination nodules (RNs) were scored in complete nuclei stained with phosphotungstic acid. The average number of RNs per nucleus is 64.7. The number of nodules per bivalent shows a clear linear relationship with SC length in the 10 longest synaptonemal complexes, while the microbivalents usually bear a single RN. The location of RNs has a non-random distribution along the largest synaptonemal complexes, with lower frequencies near kinetochores and higher frequencies toward the telomeres. The ZZ bivalent is the fourth in size and shows free recombination, having on average 3.8 RNs. The mean number of nodules per cell and the mean number of nodules in the largest bivalents show very good agreement with the corresponding number of chiasmata scored in metaphase-I spermatocytes. It is concluded that the recombination nodules provide a good check for reciprocal exchanges in this and other species of birds. Additionally, a new morphology for the recombination nodules is presented, consisting of groups of electron-dense particles measuring 43 nm in diameter. PMID- 16671251 TI - [The social sciences' other health-care discourse]. AB - Since the late 1960s and early 1970s, concern over inadequate social conditions - which is what has truly hampered human development - has been focused on growth together with equity. If human development is a process which should steadily broaden human options, what place does this concept play in the discourse of the social sciences and politics as 'master' - whose major product today is greater poverty, overpopulation, and less options for people? How can this situation be reverted, so that people can enjoy a long, healthy life, receive an education, and have access to the resources that are needed if their life chances are to be greater than their death chances? What role does or should the social sciences play in devising an ethical discourse on health care? To what objectives will we need to return, if we are to refrain from using scientific knowledge to validate the unjust social inequalities espoused in the discourses of power and of the master? PMID- 16671252 TI - [Bleeders and healers in Brazil (1808-28)]. AB - Based on documentation from the institution known as Fisicatura-mor (1808-28), and mainly on proceedings involving its official 'accreditation' of medical activities, this analysis of popular therapists opens access to categories like bleeders and healers, who were persecuted for their activities down through the nineteenth century. The curing practices employed by these bleeders and curandeiros ranked low on the hierarchy of procedures acceptable by the Fisicatura-mor. Quantitative data analysis demonstrates a link between these practices and disadvantaged social positions. Also analyzed was information that popular therapists and their clientele provided on these practices and on how they are accepted. PMID- 16671253 TI - [Brazil within the concert of nations: the struggle against racism in the early days of Unesco]. AB - This article has the objective of approaching the political and scientific context that led Brazil to the position of Unesco paradigm for racial matters. I set out from the hypothesis that soon after the Holocaust, the positive image of race relations in Brazil was in greater evidence, becoming sort of anti-Nazi Germany. At this time, a number of unforeseen efforts took place in the sense of striving for an association between the search for understanding the German totalitarian phenomenon, the radical critique of the scientific standing of the concept of race, the evidencing of socio-economic demands of underdeveloped countries, and the choice of Brazil as a socio-anthropological laboratory. This combination of aims only became viable from the onset of a transnational political-academic alliance universalistic in nature. PMID- 16671254 TI - [Visual representation of biological structures in teaching material]. AB - Parameters must be defined for presenting and handling scientific information presented in the form of teaching materials. Through library research and consultations with specialists in the health sciences and in graphic arts and design, this study undertook a comparative description of the first examples of scientific illustrations of anatomy and the evolution of visual representations of knowledge on the cell. The study includes significant examples of illustrations which served as elements of analysis. PMID- 16671255 TI - [Delayed progress on the road to an impact factor]. PMID- 16671256 TI - [Difficulty of epidural puncture for obstetric analgesia: risk factors]. AB - INTRODUCTION: Predicting technical difficulties in performing an epidural block can affect the anesthesiologist's choice of technique or decisions about who should carry it out. Our aim was to determine patient characteristics associated with difficulty in performing an epidural block for obstetric analgesia. MATERIAL AND METHODS: We designed a prospective observational study in 540 pregnant women who requested epidural analgesia for obstetric labor. Patient characteristics (age, weight, height), anatomical features (body constitution, visible and/or palpable spinal apophyses, structural anomalies), technical features (number of attempts, repositioning of the needle), anesthesiologist's experience (resident or staff), block success, time taken to accomplish the procedure, and obstetrical variables (parity, gestational age, cervical dilatation). RESULTS: The factor that best correlates with puncture difficulty in the obstetric patient is the presence of visible or palpable spinal apophyses and palpation prior to carrying out the block. Factors like weight, body constitution, or anesthesiologist's experience seem to influence the rate of success or failure of the epidural block for obstetric analgesia, although to a lesser degree. CONCLUSIONS: The most important factor for predicting success of an epidural block for obstetric analgesia in our practice is the presence of palpable spinal apophyses. PMID- 16671257 TI - [Postoperative analgesia in cardiac surgery: spinal versus intravenous morphine]. AB - OBJECTIVE: To compare the effects of spinal and intravenous administration of morphine to supplement anesthesia with remifentanil in terms of analgesia during early postoperative recovery and considering time until extubation. MATERIAL AND METHODS: This prospective, randomized, blinded trial enrolled 59 patients scheduled for cardiac surgery. The patients were assigned to receive either a spinal infusion of morphine (15 microg x Kg(-1)) or an intravenous infusion (0.3 mg x Kg(-1)). Anesthesia was maintained with 0.15 to 0.50 microg x Kg(-1) x min( 1) of remifentanil and 2 to 4 mg x Kg(-1) x h(-1) of propofol in perfusion. After the period of extracorporeal circulation, all patients were given an intravenous infusion of 30 mg of ketorolac. Later intravenous ketorolac was ministered at a dose of 30 mg per 8 hours; intravenous morphine (bolus dose of 3 mg) was also administered until pain was relieved. RESULTS: The same quality of postoperative analgesia and anesthetic recovery was achieved with both spinal and intravenous administration. The incidence of side effects was also similar. Likewise, the extubation times were similar in the 2 groups (spinal infusion group: 294.5 [SD, 150.5] minutes; intravenous group: 325.0 [139.9] minutes; P>0.05). Less postoperative intravenous morphine was administered in the first 24 hours to patients in the spinal morphine group (P<0.05) and fewer patients in that group required intravenous morphine boluses (P<0.05). CONCLUSIONS: Our study suggests that spinal morphine does not offer advantages over intravenous morphine with regard to postoperative analgesia, hemodynamic stability and respiratory parameters, time until extubation, or adverse effects. PMID- 16671258 TI - [Interaction between mivacurium and succinylcholine from a different point of view]. AB - OBJECTIVES: Succinylcholine (SCH) may first be used and continue with mivacurium (MIV). MIV has been suggested as a pretreatment. Conflicting results arises from studies on SCH-MIV interaction. The following trial revisits this interaction. PATIENTS AND METHODS: The patients were intubated after randomized administration of 100 microg x Kg(-1) of mivacurium (group 1) or 1 mg x Kg(-1) of succinylcholine and, after 50% recovery, 100 microg x Kg(-1) of mivacurium (group 2). A third group received the same regimen as group 2, preceded by pretreatment with 10 microg x Kg(-1) of mivacurium. Maximum effect (MAX), onset time, the 10% 25% recovery index, and duration of effect of mivacurium were determined by electromyography. In groups 2 and 3, the corrected MAX was defined as the difference between the actual MAX effect and the residual block after administration of succinylcholine, and speed of action was defined as the ratio between MAX or corrected MAX and onset time. Data were subjected to analysis of variance and Student-Newman-Keuls and t tests for bivariate comparisons. A value of P less than 0.05 was considered significant. RESULTS: Groups 2 and 3 had significantly greater MAX effects (97% and 98%, respectively) in comparison with group 1 (93%), shorter onset times (135 and 158 seconds in groups 2 and 3 vs 279 seconds in group 1), and greater speed of action without changes in duration of effect. MAX was halved when corrected (to 47% and 49% in groups 2 and 3, respectively), and speed of action was significantly reduced (from 1.34 and 1.62 seconds/% in groups 2 and 3 respectively, to 2.69 and 3.36 seconds/%). Mivacurium pretreatment did not produce relevant clinical changes. CONCLUSIONS: When mivacurium is used before the effects of succinylcholine disappear, a residual effect is not usually taken into consideration. This study corrected MAX and calculated speed of action, demonstrating a reduction in net block and speed of action, consistent with an antagonistic action when the 2 blockers are administered sequentially. PMID- 16671259 TI - [Recombinant activated factor VII in the postanesthetic recovery unit: review of 16 cases]. AB - We describe a series of 16 cases in which recombinant activated factor VII (rFVIIa) was used in our postanesthetic recovery unit. The mean age of the patients was 53.5 years (range, 30-84 years). Eleven were men and 5 women. The mean dose of rFVIIa used was 75 microg x kg(-1) (range, 60-90 microg x kg(-1)) and 25% of the patients needed a second dose. All the patients had postoperative bleeding, 62.5% after general surgery, 25% after a liver transplant, and 12.5% after a lung transplant. rFVIIa therapy was effective in 66% of the patients and no adverse thrombotic events related to treatment were observed. rFVIIa can be an efficacious therapeutic option for bleeding and coagulation disorders that are refractory to conventional replacement therapy. Approval to use rFVIIa in this setting and the establishment of indications should be based on further research. PMID- 16671260 TI - [Perioperative management of cardiac arrhythmia: part II]. AB - Cardiac arrhythmias are an important cause of complications throughout the perioperative period. Although our understanding of arrhythmias has increased considerably in recent years, they remain a source of concern for anesthesiologists. Our objective was to review steps to take when diagnosing arrhythmia. Although treatment is still largely influenced by therapies used in nonsurgical patients, we will review the approaches that are most applicable to practice situations in which anesthesiologists must manage patients with arrhythmias or at high risk of developing them. PMID- 16671261 TI - [Emergency axillary brachial plexus block in a patient allergic to atropine]. AB - Peripheral nerve blocks can provide a valid alternative for anesthetizing patients with allergy to drugs such as atropine that are often used for general anesthesia. We report the case of a 7-year-old boy (physical status ASA II, weight 28 Kg). Relevant medical history included generalized skin rash and breathing difficulties due to bronchospasm after administration of atropine eyedrops. The boy had never undergone skin testing for allergy. With a diagnosis of distal metaphyseal fracture of the right radius, he was proposed for urgent closed reduction and osteosynthesis of the lesion. After premedication with midazolam, an axillary brachial plexus block with 0.4 mL x Kg(-1) of 1% mepivacaine was performed with the aid of a nerve stimulator. The intervention was uneventful and the patient was free of pain during and after the procedure. Axillary brachial plexus block anesthesia in this pediatric patient was easy to perform, safe, and effective. It provided satisfactory conditions for surgery and made it possible to avoid administering drugs with vagotonic effects that are often used for general anesthesia and require the use of atropine. PMID- 16671262 TI - [Prolonged coma due to fat embolism syndrome after fracture of the femur]. AB - A 24-year-old male came to the emergency department with a diaphyseal fracture of the femur resulting from a motorcycle accident. Neurological deterioration was progressive, although a computed tomography scan was normal. Endotracheal intubation for mechanical ventilation was necessary. His condition progressed to sepsis and multiorgan failure before resolving. Magnetic resonance images of the brain suggested a fat embolism. The presence of a patent foramen ovale was investigated. The patient remained in a state of coma vigil for 3 months after the accident. After ruling out other more likely causes of neurological deterioration after trauma with fractures, fat embolism should be suspected. The prognosis for the neurological manifestations of fat embolism syndrome are generally good. Severe cases suggest massive (paradoxical) embolization of the brain and are associated with a patent foramen ovale. Early diagnosis will identify the patient at high surgical risk. A favorable course and outcome have been reported with preoperative closure of the foramen ovale. PMID- 16671263 TI - [Cerebrospinal fluid drainage to treat delayed paraplegia after stent grafting of the descending thoracic aorta]. AB - Delayed onset of paraplegia secondary to spinal cord ischemia is a rare but serious complication that can appear after endovascular repair of an aneurysm in the descending thoracic aorta, although this complication is significantly less frequent after stent grafting than after conventional surgical repair. We report the case of a man who developed paraplegia 36 hours after insertion of 4 stents in the descending thoracic aorta. The paraplegia reversed after a spinal catheter was placed to monitor cerebrospinal fluid pressure and to provide drainage. PMID- 16671264 TI - [Aspiration pneumonia and pulmonary edema due to laryngeal spasm: differential diagnosis]. PMID- 16671265 TI - [Hemostatic drugs in a patient with antiphospholipid syndrome and clinically significant perioperative bleeding]. PMID- 16671266 TI - [Ventilation and airway seal failure with a ProSeal laryngeal mask secondary to arytenoid edema caused by a nasogastric tube]. PMID- 16671267 TI - [On the new guidelines for inhibitors of hemostasis and regional neuraxial anesthesia]. PMID- 16671268 TI - [Postpartum rupture of a healthy uterus]. PMID- 16671269 TI - [Distancing of a central venous catheter from the pericardium as a radiographic sign of pericardial effusion]. PMID- 16671270 TI - [Placebo surgery: surprises and ethical questions]. AB - Placebo has a well-known therapeutic potential and this explains why placebo controlled trials are considered necessary for showing the efficacy (and to a lesser extent the safety) of a new drug treatment. However, there are clear examples of a potent surgical placebo-effect in several pathological fields (cardiology, osteoarthropathy, slipped disk, post-traumatic lesions, abdominal adhesions, Parkinson's disease). The efficacy of the placebo surgery would suggest the advisability of placebo-controlled studies also for some surgical treatments. This type of trials, however, raises a number of reservations and ethical questions. In particular, it is necessary to consider the possible unethical nature of placebo surgery as against the unethical nature of surgical operations lacking any evidence of "specific" efficacy (i.e. being better than placebo). PMID- 16671271 TI - [Peer review about gastro-esophageal reflux disease in primary care: epidemiology, diagnostic and therapeutic management]. AB - Retrospective research carried out by 29 General Practitioners in their databases, in order to evaluate the prevalence of gastro-esophageal reflux disease in its different clinical outbreaks and the incidence of new diagnosis in the last quinquennium, the diagnostic approach through instrumental examinations (endoscopy) or empirical tests (PPI test), and the therapeutical aspects, in particular concerning the usage of PPI. The prevalence has been of 3.82%, while the data concerning the incidence have pointed out a progressive increase of the diagnosis in the last quinquennium, specially for the atypical outbreaks. Moreover, it has been noted a likely excessive use of endoscopy, in the follow up as well, while less used is the IPP test. Gastroesophageal reflux disease is the most important item in the expenditure for the usage of IPP. PMID- 16671272 TI - [Screening of adult patients with fever of unknown origin. A prospective study on the role of primary cytomegalovirus infection]. AB - PATIENTS AND METHODS: In a three-year prospective survey of 135 consecutive adult patients referred for fever of unknown origin often associated with a broad spectrum of constitutional signs and symptoms, 21 (15.5%) were found to have a primary Cytomegalovirus infection. RESULTS: In the majority of cases, this syndrome was consistently associated with altered white blood cell count, abnormal T-lymphocyte subsets and hepatosplenomegaly. On the other hand, altered white blood cell differential and serum hepatic enzymes, and constitutional signs and symptoms were absent with a rate ranging from 11.1% to 27.4% of cases, and an initial laboratory cross-reaction with anti-Epstein-Barr IgM antibodies was detected in 48.1% of episodes. Non-specific signs and symptoms were the only features in 27.4% of patients, thus confirming that this disorder may be still clinically underestimated in its real frequency, until virologic assays are carried out. An extensive and varied spectrum of subjective disturbances, sometimes of duration prolonged beyond six months involved nearly 30% of subjects, and lasted for 3-15 months after recovery of acute, primary Cytomegalovirus disease. CONCLUSIONS: In a multidisciplinary (clinical, laboratory, and instrumental) workup for a fever of unknown origin, a rapid recognition of a primary Cytomegalovirus disease is useful to exclude alternative diagnoses, avoid unnecessary exposure to antimicrobial agents, and reassure patients of the benign and self-limiting course of their illness. PMID- 16671273 TI - [An uncommon cause of gynecomastia: testicular Leydig cell tumor. Hormonal profile before and after orchiectomy]. AB - Leydig cell tumors (LTC) are uncommon neoplasms arising from gonadal stroma that account for 1-3% of all testicular tumors. We report a case of LCT in a 36 years old man who had been suffering from painful bilateral gynecomastia for one year. Endocrine function tests showed decreased gonadotropin concentrations, and reduction of testosterone/estradiol ratio. Ultrasonography revealed a 10-12 mm hypoechoic area in the right testis, not evident on physical examination. Right orchiectomy was performed and histological examination confirmed the supposed existence of a LCT. After surgery, the gynecomastia has completely disappeared and hormonal alterations returned to normal. PMID- 16671274 TI - [Experimental treatment of the hand affected by rheumatoid arthritis with the use of biometric equipment]. PMID- 16671275 TI - [Role of renal failure in the pathogenesis of adverse drug reactions in the elderly]. AB - The high prevalence of multiple chronic disorders and polypharmacotherapy expose elderly patient to an increased risk of adverse drug reactions. Aging is associated with relevant morphological and functional changes in the kidney, with consequent changes in the pharmacokinetics of hydrosoluble drugs. Changes in renal function are not always clinically evident, and a concealed renal failure can lead to an increased risk of adverse drug reactions to hydrosoluble drugs. Targeting these at-risk patients could contribute to reduce the risk of adverse drug reactions with relevant cost saving. PMID- 16671276 TI - [Pharmacologic therapy of depression during pregnancy]. AB - The pregnancy is considered to be relatively high risk period for depressive episodes in women, particularly for those with pre-existing affective disorders. Epidemiological studies indicate that between 10% to 16% of pregnant women fulfil the diagnostic criteria for major depression and on average 20% is affected by an anxiety disorder. Pharmacological treatment of depression during pregnancy, however, brings with it certainties and dilemmas. It has been reported that untreated depression is associated with impaired feto-placental function, premature delivery, miscarriage, low fetal growth and perinatal unwanted effects. On the other hand, the use of antidepressant drugs in pregnancy might be at risk of major malformations (teratogenesis), neonatal toxicity, especially withdrawal symptoms and neuropsychological-behavioural impairment. In addition, the abrupt discontinuation of antidepressants, because of fear for adverse fetal effects, exposes women to serious clinical problems, in particular the disease relapse. A number of reviews indicates that among antidepressant drugs, the older SSRIs (in particular fluoxetine, sertraline, citalopram) seem to be avoided of teratogenic risks; for these reasons such drugs are nowadays considered of choice for the treatment of depression during pregnancy. Less information is available for other drugs, including triciclycs, venlafaxine, mirtazapine, bupropion, escitalopram and duloxetine. Withdrawal symptoms have been reported for all antidepressants; these symptoms, however, were self-limiting in majority of cases and had a favourable outcome. Inconclusive findings emerge, so far, from the few longitudinal studies focusing on the long-term neurodevelopment outcome in children. PMID- 16671278 TI - Reflections on the social determinants of women's health. Exploring intersections: does racialization matter? PMID- 16671277 TI - [Anti-TNF (infliximab) treatment in Crohn disease: safety profile]. AB - Anti-tumor necrosis factor (anti-TNF) therapy is an important therapeutic addition in the treatment of active Crohn's disease. Although controlled trials have confirmed the efficacy of anti-TNF (infliximab) treatment, serious toxicities related to the therapies have emerged. The purpose of this article was to review the safety profile of infliximab, and in particular analyse the infectious complications, the autoimmune disorders and the theoretical risk of cancer and lymphoma. PMID- 16671279 TI - Gender-based challenges faced by older Sikh women as immigrants: recognizing and acting on the risk of coronary artery disease. AB - Gender and ethnocultural affiliation can influence people's health beliefs and their ability to make behavioural changes associated with risk reduction. The authors undertook a series of grounded theory studies aimed at describing and explaining how gender and ethnocultural affiliation influence the process that people undergo when faced with the need to make behavioural changes to reduce the risk of coronary artery disease (CAD). Here, they describe the gender-based influences associated with managing CAD risk in a small sample of older Sikh immigrants to Canada. Data were collected through semi-structured interviews, using an interpreter when necessary. Interviews were audiotaped to enable verification of interpretation and transcription. Data were analyzed using constant comparative methods. The core variable that emerged in the series of studies was "meeting the challenge." The process of managing CAD risk included pre-diagnosis or event, liminal or changing self, and living with CAD. Intra-, inter-, and extrapersonal factors as well as sociodemographic characteristics influenced the participants' ability to meet the challenge of managing CAD risk. Health-care providers and policy-makers have a responsibility to work with ethnocultural communities in order to (1) enhance the ability of health-care providers to provide ethnoculturally sensitive care, and (2) develop ethnoculturally relevant resources to enable health promotion and disease prevention. The ultimate aim is to improve health outcomes for Sikh immigrants as vulnerable members of society. PMID- 16671280 TI - Lesbian disclosure: disrupting the taken for granted. AB - The purpose of this interpretive inquiry was to generate understandings about the experience of lesbian disclosure. The inquiry relied on Gadamerian hermeneutic and feminist philosophical thought and was situated in women's health. In a feminist understanding of women's health, experiences of health are inseparable from the everyday experiences of an embodied life and are constituted within each woman's social, material, and discursive realities. The study was informed by conversations with 15 women who self-identified as lesbian for the purpose of the inquiry, accounts of women in the media, and the researcher's reflective journals. The findings move us towards new understandings about the multiple meanings of "lesbian." They challenge nurses to consider the binary categories of homosexual and heterosexual as inadequate signifiers for the reality of women's lives, to consider the particular arrangements of each woman's life, and to disrupt assumptions of heterosexism in order to reduce the negative impact of social exclusion, isolation, discrimination, and stigmatization as social determinants of health. PMID- 16671281 TI - Relationship among employment status, stressful life events, and depression in single mothers. AB - This purpose of this study was to extend our understanding of employment status as a social determinant of psychological distress among single mothers. A cross sectional survey assessing stressful life events and depression was completed with 96 single mothers (48 employed and 48 social assistance [SA] recipients) between November 2003 and March 2004. The prevalence of depressive symptoms was significantly higher for the SA recipients. Mild, moderate, and severe depressive symptoms were reported by 2%, 23%, and 67%, respectively, of SA recipients. Total stressful events were markedly greater for SA recipients. In addition, SA recipients reported larger numbers of housing, health, social, and financial stressors. Regression analysis indicated that 40.6% of the variation in depressive symptoms among single mothers was explained by their employment status and stressful events. The findings suggest that women's employment status significantly impacts on their psychological well-being. Implications for nursing practice, policy development, and future research are identified and discussed. PMID- 16671282 TI - The health-care practices of Vietnamese-Canadian women: cultural influences on breast and cervical cancer screening. AB - Breast and cervical cancer are major contributors to morbidity and mortality among Vietnamese-Canadian women. Vietnamese women are at risk due to their low participation rate in screening programs for these cancers. The purpose of this exploratory qualitative study, informed by Kleinman's Exploratory Model of Health and Illness, was to explore the participation of Vietnamese-Canadian women in screening for breast and cervical cancer; the appropriateness of current cancer prevention services for Vietnamese women; and the influence of social, cultural, political, historical, and economic factors, shaped by race, gender, and class, on the screening practices of Vietnamese-Canadian women. Fifteen Vietnamese Canadian women and 6 health-care providers were interviewed. Analysis revealed that several factors influenced the women's participation in cancer screening. This paper reports on the process by which cultural knowledge and beliefs contributed to the women's health-care practices. The study revealed that the following cultural factors influenced the women's level of participation in screening programs for breast and cervical cancer: cultural knowledge and values with regard to women's bodies, conceptualization of health and illness, and beliefs and values concerning the patient/health-care provider relationship. The author offers recommendations on the promotion of screening for breast and cervical cancer among Vietnamese-Canadian women. PMID- 16671283 TI - Beliefs and practices of First Nation women about weight gain during pregnancy and lactation: implications for women's health. AB - Many Cree women in northern Quebec experience excessive weight gain during pregnancy and retain the weight between pregnancies. This contributes to poor maternal-fetal outcomes and increased rates of obesity-related health problems. The purpose of this qualitative descriptive study was to explore (a) Cree women's perceptions of weight gain in pregnancy and weight loss following pregnancy, (b) the barriers that women face in maintaining a healthy body weight, and (c) the sociocultural context of health. Semi-structured interviews were conducted with 30 women who had given birth within the preceding 12 months. Many women spoke of the negative health consequences of excessive weight gain, yet they found it difficult to lose weight due to individual constraints such as lack of time, cultural beliefs about diet while breastfeeding, and community constraints including lack of child care and lack of community programs for mothers. PMID- 16671284 TI - Perspectives of women with dementia receiving care from their adult daughters. AB - The caregiving experience within Alzheimer disease is fairly well documented. However, little research has been conducted from the perspective of the person living with dementia. The purpose of this study, part of a larger qualitative investigation of mother-daughter relationships within the care process of dementia, was to elicit the perceptions and experiences of mothers receiving care from their adult daughters. Guided by feminist and life-course perspectives, the researchers conducted in-depth, semi-structured interviews with a diverse sample of 10 community-dwelling women with mild to moderate cognitive impairment. In general, the health perceptions and experiences of the women were shaped by gender and how its meaning is constructed. While mothers reported mostly positive relationships with their daughters, cultural ideologies of individualism and familism manifested in feelings of "grateful guilt." Participants managed their contradictory experiences of receiving care from their daughters by doing care, undemanding care, determining care, and accepting care. The authors recommend changes in practice, policy, and research, with the aim of addressing relevant social determinants of health such as gender and social support, thereby promoting the health and well-being of women with dementia. PMID- 16671285 TI - Developing an evidence-based health advocacy intervention for women who have left an abusive partner. PMID- 16671286 TI - Researching the social determinants of women's health. PMID- 16671287 TI - Optimization of requantization codebook for vector quantization. AB - Requantization is a key technology for reducing the bit rate of a previously compressed data. When recompression ratio is high, the requantizer may cause unacceptable quality degradation. To improve the quality of the requantized image, an optimization scheme for the requantization codebook has been proposed. The proposed scheme constructs an optimal requantization codebook in an iterative manner for a given original quantization codebook of transmitter. The construction of codebook is iteratively repeated until they reach a local optimum solution. Our approach can be applied not only to the scalar quantization, but to any method which employs vector quantization-based system. Simulation results show that the optimized system based on the proposed algorithm outperforms the conventional system which is made without consideration of requantization. The proposed algorithm enables a reliable image communication over heterogeneous networks. PMID- 16671288 TI - Multiple particle tracking in 3-D+t microscopy: method and application to the tracking of endocytosed quantum dots. AB - We propose a method to detect and track multiple moving biological spot-like particles showing different kinds of dynamics in image sequences acquired through multidimensional fluorescence microscopy. It enables the extraction and analysis of information such as number, position, speed, movement, and diffusion phases of, e.g., endosomal particles. The method consists of several stages. After a detection stage performed by a three-dimensional (3-D) undecimated wavelet transform, we compute, for each detected spot, several predictions of its future state in the next frame. This is accomplished thanks to an interacting multiple model (IMM) algorithm which includes several models corresponding to different biologically realistic movement types. Tracks are constructed, thereafter, by a data association algorithm based on the maximization of the likelihood of each IMM. The last stage consists of updating the IMM filters in order to compute final estimations for the present image and to improve predictions for the next image. The performances of the method are validated on synthetic image data and used to characterize the 3-D movement of endocytic vesicles containing quantum dots. PMID- 16671289 TI - Wavelet-domain approximation and compression of piecewise smooth images. AB - The wavelet transform provides a sparse representation for smooth images, enabling efficient approximation and compression using techniques such as zerotrees. Unfortunately, this sparsity does not extend to piecewise smooth images, where edge discontinuities separating smooth regions persist along smooth contours. This lack of sparsity hampers the efficiency of wavelet-based approximation and compression. On the class of images containing smooth C2 regions separated by edges along smooth C2 contours, for example, the asymptotic rate-distortion (R-D) performance of zerotree-based wavelet coding is limited to D(R) (< or = 1/R, well below the optimal rate of 1/R2. In this paper, we develop a geometric modeling framework for wavelets that addresses this shortcoming. The framework can be interpreted either as 1) an extension to the "zerotree model" for wavelet coefficients that explicitly accounts for edge structure at fine scales, or as 2) a new atomic representation that synthesizes images using a sparse combination of wavelets and wedgeprints--anisotropic atoms that are adapted to edge singularities. Our approach enables a new type of quadtree pruning for piecewise smooth images, using zerotrees in uniformly smooth regions and wedgeprints in regions containing geometry. Using this framework, we develop a prototype image coder that has near-optimal asymptotic R-D performance D(R) < or = (log R)2 /R2 for piecewise smooth C2/C2 images. In addition, we extend the algorithm to compress natural images, exploring the practical problems that arise and attaining promising results in terms of mean-square error and visual quality. PMID- 16671290 TI - Invariance properties of gabor filter-based features--overview and applications. AB - For almost three decades the use of features based on Gabor filters has been promoted for their useful properties in image processing. The most important properties are related to invariance to illumination, rotation, scale, and translation. These properties are based on the fact that they are all parameters of Gabor filters themselves. This is especially useful in feature extraction, where Gabor filters have succeeded in many applications, from texture analysis to iris and face recognition. This study provides an overview of Gabor filters in image processing, a short literature survey of the most significant results, and establishes invariance properties and restrictions to the use of Gabor filters in feature extraction. Results are demonstrated by application examples. PMID- 16671291 TI - Fingerprint registration by maximization of mutual information. AB - Fingerprint registration is a critical step in fingerprint matching. Although a variety of registration alignment algorithms have been proposed, accurate fingerprint registration remains an unresolved problem. We propose a new algorithm for fingerprint registration using orientation field. This algorithm finds the correct alignment by maximization of mutual information between features extracted from orientation fields of template and input fingerprint images. Orientation field, representing the flow of ridges, is a relatively stable global feature of fingerprint images. This method uses the statistics and distribution of global feature of fingerprint images so that it is robust to image quality and local changes in images. The primary characteristic of this method is that it uses this stable global feature to align fingerprints, and that its behavior may resemble the way humans compare fingerprints. Experimental results show that the occurrence of misalignment is dramatically reduced and that registration accuracy is greatly improved at the same time, leading to enhanced matching performance. PMID- 16671292 TI - Combining geodesic interpolating splines and affine transformations. AB - Geodesic spline interpolation is a simple and efficient approach for landmark matching by nonambiguous mappings (diffeomorphisms), combining classic spline interpolation and flows of diffeomorphisms. Here, we extend the method to incorporate the estimation of a affine transformation, yielding a consistent and numerically stable algorithm. A theoretical justification is provided by studying the existence of the global minimum of the energy. PMID- 16671293 TI - Fast image and video colorization using chrominance blending. AB - Colorization, the task of coloring a grayscale image or video, involves assigning from the single dimension of intensity or luminance a quantity that varies in three dimensions, such as red, green, and blue channels. Mapping between intensity and color is, therefore, not unique, and colorization is ambiguous in nature and requires some amount of human interaction or external information. A computationally simple, yet effective, approach of colorization is presented in this paper. The method is fast and it can be conveniently used "on the fly," permitting the user to interactively get the desired results promptly after providing a reduced set of chrominance scribbles. Based on the concepts of luminance-weighted chrominance blending and fast intrinsic distance computations, high-quality colorization results for still images and video are obtained at a fraction of the complexity and computational cost of previously reported techniques. Possible extensions of the algorithm introduced here included the capability of changing the colors of an existing color image or video, as well as changing the underlying luminance, and many other special effects demonstrated here. PMID- 16671294 TI - On global and local convergence of half-quadratic algorithms. AB - This paper provides original results on the global and local convergence properties of half-quadratic (HQ) algorithms resulting from the Geman and Yang (GY) and Geman and Reynolds (GR) primal-dual constructions. First, we show that the convergence domain of the GY algorithm can be extended with the benefit of an improved convergence rate. Second, we provide a precise comparison of the convergence rates for both algorithms. This analysis shows that the GR form does not benefit from a better convergence rate in general. Moreover, the GY iterates often take advantage of a low cost implementation. In this case, the GY form is usually faster than the GR form from the CPU time viewpoint. PMID- 16671295 TI - Exact histogram specification. AB - While in the continuous case, statistical models of histogram equalization/specification would yield exact results, their discrete counterparts fail. This is due to the fact that the cumulative distribution functions one deals with are not exactly invertible. Otherwise stated, exact histogram specification for discrete images is an ill-posed problem. Invertible cumulative distribution functions are obtained by translating the problem in a K-dimensional space and further inducing a strict ordering among image pixels. The proposed ordering refines the natural one. Experimental results and statistical models of the induced ordering are presented and several applications are discussed: image enhancement, normalization, watermarking, etc. PMID- 16671296 TI - A fuzzy impulse noise detection and reduction method. AB - Removing or reducing impulse noise is a very active research area in image processing. In this paper we describe a new algorithm that is especially developed for reducing all kinds of impulse noise: fuzzy impulse noise detection and reduction method (FIDRM). It can also be applied to images having a mixture of impulse noise and other types of noise. The result is an image quasi without (or with very little) impulse noise so that other filters can be used afterwards. This nonlinear filtering technique contains two separated steps: an impulse noise detection step and a reduction step that preserves edge sharpness. Based on the concept of fuzzy gradient values, our detection method constructs a fuzzy set impulse noise. This fuzzy set is represented by a membership function that will be used by the filtering method, which is a fuzzy averaging of neighboring pixels. Experimental results show that FIDRM provides a significant improvement on other existing filters. FIDRM is not only very fast, but also very effective for reducing little as well as very high impulse noise. PMID- 16671297 TI - PDE-based image restoration: a hybrid model and color image denoising. AB - The paper is concerned with PDE-based image restoration. A new model is introduced by hybridizing a nonconvex variant of the total variation minimization (TVM) and the motion by mean curvature (MMC) in order to deal with the mixture of the impulse and Gaussian noises reliably. We suggest the essentially nondissipative (ENoD) difference schemes for the MMC component to eliminate the impulse noise with a minimum (ideally no) introduction of dissipation. The MMC TVM hybrid model and the ENoD schemes are applied for both gray-scale and color images. For color image denoising, we consider the chromaticity-brightness decomposition with the chromaticity formulated in the angle domain. An incomplete Crank-Nicolson alternating direction implicit time-stepping procedure is adopted to solve those differential equations efficiently. Numerical experiments have shown that the new hybrid model and the numerical schemes can remove the mixture of the impulse and Gaussian noises, efficiently and reliably, preserving edges quite satisfactorily. PMID- 16671298 TI - A binary level set model and some applications to Mumford-Shah image segmentation. AB - In this paper, we propose a PDE-based level set method. Traditionally, interfaces are represented by the zero level set of continuous level set functions. Instead, we let the interfaces be represented by discontinuities of piecewise constant level set functions. Each level set function can at convergence only take two values, i.e., it can only be 1 or -1; thus, our method is related to phase-field methods. Some of the properties of standard level set methods are preserved in the proposed method, while others are not. Using this new method for interface problems, we need to minimize a smooth convex functional under a quadratic constraint. The level set functions are discontinuous at convergence, but the minimization functional is smooth. We show numerical results using the method for segmentation of digital images. PMID- 16671299 TI - A faster converging snake algorithm to locate object boundaries. AB - A different contour search algorithm is presented in this paper that provides a faster convergence to the object contours than both the greedy snake algorithm (GSA) and the fast greedy snake (FGSA) algorithm. This new algorithm performs the search in an alternate skipping way between the even and odd nodes (snaxels) of a snake with different step sizes such that the snake moves to a likely local minimum in a twisting way. The alternative step sizes are adjusted so that the snake is less likely to be trapped at a pseudo-local minimum. The iteration process is based on a coarse-to-fine approach to improve the convergence. The proposed algorithm is compared with the FGSA algorithm that employs two alternating search patterns without altering the search step size. The algorithm is also applied in conjunction with the subband decomposition to extract face profiles in a hierarchical way. PMID- 16671300 TI - Road extraction from aerial images using a region competition algorithm. AB - In this paper, we present a user-guided method based on the region competition algorithm to extract roads, and therefore we also provide some clues concerning the placement of the points required by the algorithm. The initial points are analyzed in order to find out whether it is necessary to add more initial points, and this process will be based on image information. Not only is the algorithm able to obtain the road centerline, but it also recovers the road sides. An initial simple model is deformed by using region growing techniques to obtain a rough road approximation. This model will be refined by region competition. The result of this approach is that it delivers the simplest output vector information, fully recovering the road details as they are on the image, without performing any kind of symbolization. Therefore, we tried to refine a general road model by using a reliable method to detect transitions between regions. This method is proposed in order to obtain information for feeding large-scale Geographic Information System. PMID- 16671301 TI - Performance characterization of the dynamic programming obstacle detection algorithm. AB - A computer vision-based system using images from an airborne aircraft can increase flight safety by aiding the pilot to detect obstacles in the flight path so as to avoid mid-air collisions. Such a system fits naturally with the development of an external vision system proposed by NASA for use in high-speed civil transport aircraft with limited cockpit visibility. The detection techniques should provide high detection probability for obstacles that can vary from subpixels to a few pixels in size, while maintaining a low false alarm probability in the presence of noise and severe background clutter. Furthermore, the detection algorithms must be able to report such obstacles in a timely fashion, imposing severe constraints on their execution time. For this purpose, we have implemented a number of algorithms to detect airborne obstacles using image sequences obtained from a camera mounted on an aircraft. This paper describes the methodology used for characterizing the performance of the dynamic programming obstacle detection algorithm and its special cases. The experimental results were obtained using several types of image sequences, with simulated and real backgrounds. The approximate performance of the algorithm is also theoretically derived using principles of statistical analysis in terms of the signal-to-noise ration (SNR) required for the probabilities of false alarms and misdetections to be lower than prespecified values. The theoretical and experimental performance are compared in terms of the required SNR. PMID- 16671302 TI - Robust ego-motion estimation and 3-D model refinement using surface parallax. AB - We present an iterative algorithm for robustly estimating the ego-motion and refining and updating a coarse depth map using parametric surface parallax models and brightness derivatives extracted from an image pair. Given a coarse depth map acquired by a range-finder or extracted from a digital elevation map (DEM), ego motion is estimated by combining a global ego-motion constraint and a local brightness constancy constraint. Using the estimated camera motion and the available depth estimate, motion of the three-dimensional (3-D) points is compensated. We utilize the fact that the resulting surface parallax field is an epipolar field, and knowing its direction from the previous motion estimates, estimate its magnitude and use it to refine the depth map estimate. The parallax magnitude is estimated using a constant parallax model (CPM) which assumes a smooth parallax field and a depth based parallax model (DBPM), which models the parallax magnitude using the given depth map. We obtain confidence measures for determining the accuracy of the estimated depth values which are used to remove regions with potentially incorrect depth estimates for robustly estimating ego motion in subsequent iterations. Experimental results using both synthetic and real data (both indoor and outdoor sequences) illustrate the effectiveness of the proposed algorithm. PMID- 16671303 TI - Adaptive MAP error concealment for dispersively packetized wavelet-coded images. AB - In this paper, we present an adaptive maximum a posteriori (MAP) error concealment algorithm for dispersively packetized wavelet-coded images. We model the subbands of a wavelet-coded image as Markov random fields, and use the edge characteristics in a particular subband, and regularity properties of subband/wavelet samples across scales, to adapt the potential functions locally. The resulting adaptive MAP estimation gives PSNR advantages of up to 0.7 dB compared to the competing algorithms. The advantage is most evident near the edges, which helps improve the visual quality of the reconstructed images. PMID- 16671304 TI - The multiscale hermite transform for local orientation analysis. AB - The efficient representation of local differential structure at various resolutions has been a matter of great interest for adaptive image processing and computer vision tasks. In this paper, we derive a multiscale model to represent natural images based on the scale-space representation: a model that has an inspiration in the human visual system. We first derive the one-dimensional case and then extend the results to two and three dimensions. The operators obtained for analysis and synthesis stages are derivatives of the Gaussian smoothing kernel, so that, for the two-dimensional case, we can represent them either in a rotated coordinate system or in terms of directional derivatives. The method to perform the rotation is efficient because it is implemented by means of the application of the so-called generalized binomial filters. Such a family of discrete sequences fulfills a number of properties that allows estimating the local orientation for several image structures. We also define the discrete counterpart in which the coordinate normalization of the continuous case is approximated as a subsampling of the discrete domain. PMID- 16671305 TI - Image denoising using a tight frame. AB - We present a general mathematical theory for lifting frames that allows us to modify existing filters to construct new ones that form Parseval frames. We apply our theory to design nonseparable Parseval frames from separable (tensor) products of a piecewise linear spline tight frame. These new frame systems incorporate the weighted average operator, the Sobel operator, and the Laplacian operator in directions that are integer multiples of 45 degrees. A new image denoising algorithm is then proposed, tailored to the specific properties of these new frame filters. We demonstrate the performance of our algorithm on a diverse set of images with very encouraging results. PMID- 16671306 TI - Acoustical diffraction tomography in a finite form based on the Rytov transform. AB - A new reconstruction algorithm in a finite form based on the Rytov transform is presented for acoustical diffraction tomography. Applying the Rytov transform to the governing differential wave equation necessarily introduces the so-called generalized scattering. Our analysis shows that the generalized scattered wave is asymptotically equivalent to the physically scattered wave, and also satisfies the Sommerfeld radiation condition in the far field. Using the method of formal parameter expansion, we further find that all other terms in the expansion of the object function vanish except the first- and second-order ones, and thus reach a finite form solution to the diffraction tomography. Our computer simulation confirms the effectiveness of the algorithm in the case of the scattering objects with cylindrical symmetry, also shows its limitations when it applies to the strong scattering. PMID- 16671307 TI - Blue-noise halftoning for hexagonal grids. AB - In this paper, we closely scrutinize the spatial and spectral properties of aperiodic halftoning schemes on rectangular and hexagonal sampling grids. Traditionally, hexagonal sampling grids have been shunned due to their inability to preserve the high-frequency components of blue-noise dither patterns at gray levels near one-half, but as will be shown, only through the introduction of diagonal correlations between dots can even rectangular sampling grids preserve these frequencies. And by allowing the sampling grid to constrain the placement of dots, a particular algorithm may introduce visual artifacts just as disturbing as excess energy below the principal frequency. If, instead, the algorithm maintains radial symmetry by introducing a minimum degree of clustering, then that algorithm can maintain its grid defiance illusion fundamental to the spirit of the blue-noise model. As such, this paper shows that hexagonal grids are preferrable because they can support gray-levels near one-half with less required clustering of minority pixels and a higher principal frequency. Along with a thorough Fourier analysis of blue-noise dither patterns on both rectangular and hexagonal sampling grids, this paper also demonstrates the construction of a blue noise dither array for hexagonal grids. PMID- 16671308 TI - A new framework for characterization of halftone textures. AB - Characterization of halftone texture is important for quantitative assessment of halftone quality. In this paper, we develop a new framework based on directional local sequency analysis and a filter bank structure. We decompose a halftone image into subband images, from which we can easily reconstruct the original halftone. Based on these subband images, we define the directional sequency spectrum which is analogous to the two-dimensional Fourier spectrum, and formulate several texture measures. Two test image sets are used to justify these measures. PMID- 16671309 TI - A new texture generation method based on pseudo-DCT coefficients. AB - In this paper, a new method for generating different texture images is presented. This method involves a simple transform from a certain one-dimensional (1-D) signal to an expected two-dimensional (2-D) image. Unlike traditional methods, the input signal is generated by a simple 1-D function in our work instead of a sample texture. We first transform the 1-D input signal into frequency domain using fast Fourier transform. Based on the sufficient analysis in 2-D discrete cosine transform (DCT) domain, where each of the coefficients expresses a texture feature in a certain direction, the 2-D pseudo-DCT coefficients are then constructed by appropriately rearranging the Fourier coefficients in terms of their frequency components. Finally, the corresponding texture image can be produced by 2-D inverse DCT algorithm. We applied the proposed method to generate several stochastic textures (i.e., cloud, illumination, and sand), and several structural texture images. Experimental results indicate the good performance of the proposed method. PMID- 16671310 TI - Prevention of mental illness in primary care. The IMHPA manual and the general practitioners' role. PMID- 16671311 TI - Do German general practitioners support euthanasia? Results of a nation-wide questionnaire survey. AB - OBJECTIVE: To learn more about the attitudes of German general practitioners (GPs) concerning euthanasia and the frequency of its performance in Germany. METHODS: 500 GPs from all parts of Germany were randomly selected from telephone listings, and were sent a postal questionnaire with anonymous return envelopes. Participants were asked to make decisions based on hypothetical scenarios involving terminally ill patients and were questioned about their attitudes towards active euthanasia or physician assisted suicide (PAS). RESULTS: The questionnaire was returned by 48% of all 481 eligible GPs (mean age 51 years, 68% male). Although the option of performing euthanasia was rarely chosen in hypothetical scenarios, its performance was considered acceptable by 34% (active euthanasia) and 80% (PAS). Seventy-seven percent of respondents believed that a comparison between euthanasia today and the atrocities committed during the 3rd Reich was not appropriate. Sixty-two percent of respondents had received requests for active euthanasia and 73% for PAS. Thirteen percent and 38% of respondents seem to have performed euthanasia themselves in the past. CONCLUSIONS: The majority of German GPs reject active euthanasia and physician-assisted suicide (PAS). Nonetheless, requests for and performance of euthanasia do not seem to be a rare occurrence. Only a small proportion of respondents are willing to perform euthanasia at a patient's request under the current legislation which make these acts illegal in Germany. German history seems to play only a minor role in shaping respondents' attitudes towards active euthanasia or PAS. PMID- 16671312 TI - Retrospective evaluation of palliative care in Romania: the perspective of the involved professionals. AB - OBJECTIVE: Palliative care at home is a new service provision in Romania. This study evaluated retrospectively, after the patient's death, the provision of care at home from the perspective of professionals directly involved in the care process. The evaluation included assessment of the role of the professional, the functioning of the team, communication with patients and family, and overall judgment. The study is part of the development of palliative care at home, by multidisciplinary teams in Romania. METHODS: Over a period of eighteen months five teams with a total of 19 professionals, provided palliative care for 103 patients with cancer in its terminal stage. During this period 80 patients died. A questionnaire was sent to the professionals involved. All responded resulting in 181 evaluations. These evaluations are the basis for analysis. The data were analyzed using SPSS. RESULTS: Of the 181 cases evaluated, 63 indicated an emotional burden on the professionals. GP's and nurses reported such a burden more frequently. A lack of knowledge of how to treat a special case was reported 56 times. This was especially so when a case was judged to be different from other cases, which often included (unexpected) complications. GP's reported cases with complications more frequently than oncologists and nurses. The multidisciplinary teams functioned very satisfactorily in treating patients. Although communication with patients/families was generally judged positively, communication with patients was sometimes viewed as problematic. The overall verdict was that the care delivered to the 80 patients was positive. Most professionals reported that they would deal with the cases in the same way again. CONCLUSIONS: The patients who received palliative care and died, were treated well according to the professionals who evaluated the care process. Improvement of palliative care services at home may be achieved by provision of additional information on (acute) complications. Since palliative care at home is a new phenomenon in Romania, professionals working closely with the patient have to learn to cope with the emotional burden certain cases may include. PMID- 16671313 TI - Cardiovascular outcome stratification using the ankle-brachial pressure index. AB - OBJECTIVES: To investigate, in patients with peripheral arterial disease (PAD), the association between the level of the Ankle-Brachial Pressure Index (ABPI) at time of diagnosis and clinical outcome. METHODS: The data set of the Limburg PAOD Longitudinal Study (n=3649, mean follow-up time 7.2 years) was analyzed for this purpose. The ABPI was measured by trained personnel using handheld Doppler devices. Subjects were classified into 6 baseline ABPI categories ranging from ABPI > or = 1.10 ('certainly normal') to ABPI<0.50 ('seriously reduced'). Outcome measures were progressive limb ischaemia, cardiovascular morbidity and cardiovascular mortality. Cox proportional hazard models were used to investigate the associations (including hazard ratio's, HR) between different ABPI categories and outcome events. RESULTS: As compared to persons with an ABPI > or = 1.10 ('certainly normal'), patients with a baseline ABPI <1.00 (categories 'probably normal' and worse) were at high risk to develop progressive limb ischaemia (HR 3.3 - 9.0). Additionally, patients with a baseline ABPI <0.90 (categories 'moderately reduced' and worse) were at higher risk to experience a non-fatal cardiovascular disease (HR 1.5 - 1.7). Moreover, patients with a baseline ABPI <0.70 ('clearly' and 'seriously' reduced ABPI) were at high risk to die from a cardiovascular or other cause (HR 2.3). CONCLUSION: The ABPI can be helpful in identifying subjects at high risk for a poor cardiovascular outcome, in whom strict cardiovascular risk management would be appropriate. PMID- 16671314 TI - General practitioners' management of lumbosacral radicular syndrome compared with a clinical guideline. AB - OBJECTIVE: To investigate the current treatment policy of general practitioners (GPs) in patients with a lumbosacral radicular syndrome (LRS) compared with their clinical guideline. DESIGN: A cross sectional survey. METHODS: Sixty-three GPs completed questionnaires about their treatment policy in individual LRS patients at baseline and at six months follow-up. Simultaneously, 136 LRS patients of these GPs were interviewed at baseline, and at three and six month's follow-up. RESULTS: Of the 12 recommendations in the guideline related to history taking, four were not adhered to by the GPs in about 25% of the patients. Of the ten recommended physical examinations, three are not frequently carried out by the GPs. Almost 40% of the patients were referred to physiotherapy and 27% received muscle relaxants. CONCLUSION: The majority of the GPs support the content of the LRS guideline. Overall, there was a good adherence with the guideline for history taking and physical examination, and a moderate adherence for treatment policy. PMID- 16671315 TI - 'Antidepressants unlimited'. Are antidepressants the best option for treating depression in general practice? Debating session WONCA 2004. AB - Antidepressants are among the worlds' most prescribed drugs. However, there are several controversies around their extensive use in primary care, and doubts about their effects, especially in children and older people. Psychological interventions are probably at least as effective as antidepressants, and are preferred by many patients. These issues were discussed at the debating sessions at WONCA 2004 in Amsterdam. PMID- 16671316 TI - General practice based teaching exchanges in Europe. Experiences from the EU Socrates programme 'primary health care'. AB - This paper reviews the experience of international exchange of medical students for general practice. The experience is based on the EU Socrates programme 'Primary Health Care' that offers, since 1992, clinical attachments and research electives in primary care. This programme involves 11 university departments of general practice/primary care in eight countries: Austria - Vienna; Belgium - Gent; Germany Dusseldorf; Italy - Monza, Udine; Netherlands Nijmegen; Slovenia - Ljubljana; Sweden - Goteborg; and the UK - Edinburgh, Imperial College London and Nottingham. More than 150 students have taken part in the programme, most in the last four years. For clinical attachment communication to patients is essential, and students should be able to speak the language of the host university. A research elective in primary care is less demanding and requires students' ability to communicate in English. Despite marked differences in health care structure in the countries involved, it is quite possible to provide a valuable teaching environment in general practice, and the experience gained by students in the exchanges more than equals that what they would gain at home. The added value is in experiencing the influence of another health care system and of working in another academic primary care centre. A substantial number of research electives have been published in international peer reviewed scientific journals with the student as first (occasionally second) author and staff members of the student's host and home university as co-authors. A further benefit of the exchange programme lies in the transfer teaching innovations between universities. PMID- 16671317 TI - Family medicine in South Africa: where are we now and where do we want to be? AB - OBJECTIVES: This paper outlines the development of family medicine in South Africa with special reference to the process leading to the recognition of this discipline as a medical specialty. It also examines the constraints under which the discipline has had to function, considers where the discipline should be, seeks to identify the barriers to its further development and suggests ways in which to overcome these. METHODS: A short review was carried out of the available South African literature to record the advancement of the discipline and the international literature was searched for articles supporting this direction. RESULTS: The situation in South Africa is complicated by the existence of many doctor groupings claiming to represent the generalist and the perception that family medicine only addresses the needs of middle-class citizens. A flawed consultative process leading up to the present stage has contributed to this perception. CONCLUSIONS: The available literature supports the establishment of family medicine as a speciality. Developments in South Africa, such as raising the status of Family Medicine and creating a compulsory rotation through family medicine as an extended internship and the creation of registrar posts in family medicine will advance the discipline in a positive manner, whilst possibly attempting to resolve the medical migration issues that are presently destroying Sub-Saharan Africa's health services. PMID- 16671319 TI - Electronic patient record and the family: a pilot European questionnaire. PMID- 16671318 TI - Greek-Turkish collaboration in general practice/family medicine: an example of country-to-country collaboration. PMID- 16671320 TI - General practice medical records: patients' views on storage, use and access. PMID- 16671321 TI - The investigation of acute gastroenteritis in general practice: a survey of general practitioners in Northern Ireland and Republic of Ireland. PMID- 16671322 TI - Variation in outpatient antibiotic use in three European countries: exploration of possible determinants. PMID- 16671323 TI - Nursing home medicine in The Netherlands. PMID- 16671324 TI - [Allergic asthma and intrinsic inhibitors of cytokine signaling]. AB - T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play an important role in allergic asthma. These cytokines transmit signals through the JAK/STAT and the Ras/ERK signaling pathways, and SOCS family proteins and SPRED family proteins regulate these pathways. SOCS3 controls IL-12-dependent STAT4 activation and Th2 differentiation process. SPRED-1 modulates IL-5-dependent ERK activation and eosinophilia. SOCS3 and SPRED-1 may be targets for therapeutic strategies in allergic asthma. PMID- 16671326 TI - [Clinical approach to hypereosinophilia-associated diseases]. PMID- 16671325 TI - [Prevention of onset and severity of adult asthma]. PMID- 16671327 TI - [Status of allergen immunotherapy in the treatment of allergic diseases]. PMID- 16671329 TI - [A case of drug eruption induced by hydroxyzine pamoate]. AB - A 62-year-old woman with rheumatoid arthritis, Basedow's disease and arrhythmia has been treated with antirheumatic, antiarrhythmic drugs and so on. She developed pruritic diffuse erythema with papules on the trunk and extremities 2 days after taking hydroxyzine pamoate for asteatotic eczema. Laboratory data showed increased levels of eosinophils. Histopathological examination revealed a infiltrate of inflammatory cells in the upper dermis. Patch tests with hydroxyzine pamoate and hydroxyzine hydrochloride were positive. From these findings, we diagnosed this case as drug eruption due to hydroxyzine. Her eruption subsided after she discontinued hydroxyzine pamoate and other drugs which were started within 5 days before the onset of the eruption and was treated with systemic steroid, systemic antiallergic drug and topical steroid. PMID- 16671328 TI - [Development of the simple method for measurement the content of Cry j 1 in the air by latex agglutination test]. AB - BACKGROUND: In order to develop an easy method for measure the airborne C. japonica pollen allergen (Cry j 1) without any utensils or a special apparatus, we examined latex agglutination test for the purpose. METHODS: The latex beads were reacted with the anti-Cry j 1 polyclonal antibody, and the beads were blocked with bovine serum albumin (sensitized beads). The measurement is as follows; two-fold diluted series of solutions of airborne samples are reacted with the sensitized beads, and the results are examined after 2 hrs. RESULTS: Agglutination was recognized with 5ng/ml Cry j 1 standard solution and 20pg/m3 of airborne samples. CONCLUSION: We have developed a simple method for measure the airborne Cry j 1 by use of latex agglutination test. The strong point of the method is 1) measurement is easy and does not affect the numbers of pollen, 2) do not need any utensils or a special apparatus, 3) measure the disease causative materials themselves. PMID- 16671330 TI - [Oral allergy syndrome due to cashew nuts in the patient without pollinosis]. AB - A 26-year-old woman felt tingling on her tongue and itching both in the throat and on the face immediately after she put a cashew nut on her tongue. She had a history of atopic dermatitis and bronchial asthma, but not of pollinosis. CAP FEIA and skin prick test (SPT) were positive for cashew nuts. The results showed negative for peanuts and other tree nuts than cashew nuts. Consequently, she was diagnosed with oral allergy syndrome due to cashew nuts. In addition, the result of skin prick test with cashew nuts normalized one year after she began avoiding cashew nuts, indicating that cashew nuts allergy would be due to sensitization by itself rather than to cross-reactivity between cashew nuts and pollens in this case. PMID- 16671331 TI - Selective angiotensin receptor antagonism with valsartan decreases arterial stiffness independently of blood pressure lowering in hypertensive patients. AB - Angiotensin II plays a key role in the development of vascular disease. We examined the long-term effects of selective angiotensin II receptor (ATR) blockade with valsartan on arterial wall stiffness. Brachial to ankle pulse wave velocity (baPWV) was measured in 28 women and 25 men with hypertension (mean age: 62+/-2 years). The measurements were repeated after 24 weeks of treatment with valsartan, 40 to 160 mg/day, with (n=10) or without (n=36) concomitant statin therapy. By multiple regression analysis, baseline baPWV was correlated with age (p<0.001), systolic blood pressure (SBP, p<0.0001), body mass index (p=0.018), and pulse pressure (p=0.005), but not with total cholesterol (p=0.446). Valsartan lowered mean SBP and diastolic blood pressure (DBP) from 155+/-3 to 140+/-3 mmHg and from 90+/-2 to 82+/-2 mmHg, respectively, and mean baPWV from 1,853+/-49 to 1,682+/-52 cm/s. Lowering of baPWV was not influenced by statin therapy. An overlap analysis was performed to separate the effect of angiotensin II receptor blockade from that of blood pressure (BP) lowering. The decrease in the baPWV value of 1,794+/-46 cm/s before valsartan (n=39) vs. 1,663+/-45 cm/s during valsartan (p=0.048, n=31) at a similar mean SBP level (149+/-2 vs. 146+/-3 mmHg, p=0.304) confirmed that ATR blockade had a beneficial effect independent of BP lowering. SBP strongly influences baPWV. However, the decrease in baPWV with valsartan was independent of BP lowering. Statins had no synergistic effect on baPWV. Lowering of baPWV may account for the therapeutic benefit conferred by valsartan independent of its BP-lowering effect. PMID- 16671332 TI - Insulin resistance is associated with arterial stiffness in nondiabetic hypertensives independent of metabolic status. AB - We sought to determine whether insulin resistance (IR) is related to arterial stiffness in nondiabetic hypertensive patients, independent of metabolic status and gender. IR has been associated with increased arterial stiffness in patients with diabetes. In nondiabetic hypertensive patients, the correlation between IR and arterial stiffness has yet to be investigated. We enrolled 284 nondiabetic patients who were being treated for hypertension. At the time of enrollment, the patients underwent a baseline laboratory assessment including homeostatic model assessment (HOMA) IR index and pulse wave velocity (PWV). The HOMA IR index is used as a marker of IR, and brachial to ankle PWV (baPWV) was used as a marker of arterial stiffness. Of the 284 study subjects, 121 were classified as having metabolic syndrome. The patients with metabolic syndrome were older than the non metabolic syndrome patients (55.4+/-10.7 vs. 52.1+/-11.6 years, p=0.013), but there was no gender difference between the two groups. The average baPWV was significantly higher in the patients with metabolic syndrome (1,506+/-235 vs. 1,435+/-211 cm/s, p=0.009). The HOMA index was independently associated with an increase in arterial stiffness (r=0.548, p<0.001) after controlling for age, systolic blood pressure (SBP), heart rate, medication and gender. The independent association of HOMA with arterial stiffness was demonstrated in subgroup analysis, regardless of the metabolic status and gender. In conclusion, increased IR was associated with arterial stiffness, independent of age, baseline SBP, gender and heart rate. This independent association of IR was demonstrated regardless of gender and metabolic status. PMID- 16671333 TI - Long-term compliance with salt restriction in Japanese hypertensive patients. AB - The purpose of the present study was to investigate the long-term compliance with salt restriction in Japanese hypertensive patients. Subjects included 389 patients, 230 women and 159 men, mean age 58+/-11 years, who underwent successful 24-h home urine collection more than three times over an interval of a year. Urinary salt, potassium, and creatinine were measured. Additionally, family history, habitual alcohol intake, smoking habit, physical activities, and job status were assessed by use of a questionnaire. During the follow-up period (average 3.5 years), participants underwent urine collection 4.6 times in average. Urinary salt excretion at the last visit was significantly lower than that at the first visit (8.7+/-3.4 vs. 9.6+/-4.1 g/day; p<0.01). Urinary potassium excretion also decreased significantly during this period (from 2.0+/ 0.7 to 1.9+/-0.7 g/day; p<0.05). Among the mean 4.6 urine collections, 45.2% (men 34.6%, women 52.6%) of the patients successfully achieved <6 g (100 mmol of sodium)/day of salt excretion on at least one occasion. The rate of achievement of averaged urinary salt excretion <6 g/day dropped to 10.3% (men 4.4%, women 14.3%). Only 2.3% (men 0.6%, women 3.5%) of the patients achieved <6 g/day on all occasions. There were no significant differences in age, habitual alcohol intake, smoking habit, physical activities, or job status between patients who complied with the salt-restricted diet and those who did not. Results suggest that long term compliance with salt restriction is poor in Japanese hypertensive patients. Since no specifically defining characteristics were found in the compliant patients, repeated measurements of urinary salt excretion seem to be important to encourage salt restriction. PMID- 16671334 TI - Long-term anti-hypertensive therapy with benidipine improves arterial stiffness over blood pressure lowering. AB - Pulse wave velocity (PWV) reflects arterial stiffness and is an independent predictor of cardiovascular mortality and morbidity. However, because it is closely related to blood pressure (BP), PWV is an imperfect measure for evaluating the effects of anti-hypertensive drugs on arterial wall properties. To clarify the effect of benidipine on arterial properties, we first derived the regression line between BP and PWV changes in a short-term experiment. Using this line, we evaluated the long-term effect of benidipine on PWV changes. In the short-term experiment, 29 participants were intravenously administered nicardipine for 90 min. Maximum decreases of brachial-ankle PWV (baPWV) were plotted against the corresponding decreases in BP. In the long-term experiment, 9 hypertensive patients were treated with benidipine for 1 year, during which BP and baPWV were monitored. After 1 year, benidipine was suspended for 2 weeks, and BP and baPWV were reevaluated. In the short-term experiment, PWV was dependent on BP only, and the equation of the regression line was deltaPWV (cm/s) =10.114 x deltaMBP (mmHg) (r=0.913) or deltaPWV (%) =0.719 x deltaMBP (%) (r=0.926). In the long-term therapy, benidipine treatment achieved stable BP control within 3 months; the real PWV decreases (r-PWV) were almost identical to the PWV decrease estimated (e-PWV) from BP lowering at 3 months. However, r-PWV exceeded e-PWV after 6 months. Relative BP and PWV improvements compared to the control were maintained 2 weeks after suspension of benidipine. In conclusion, long-term benidipine administration improves arterial wall properties beyond what can be accounted for by changes in BP. PMID- 16671335 TI - Relationships between echocardiographic findings, pulse wave velocity, and carotid atherosclerosis in type 2 diabetic patients. AB - The purpose of the present study was to analyze the relationships between echocardiographic findings, brachial-ankle pulse wave velocity, and carotid atherosclerosis in type 2 diabetic patients. In 70 type 2 diabetic patients without cardiovascular disease, pulse wave velocity was measured using an automatic waveform analyzer, and the carotid plaque score was obtained by carotid ultrasonography. The left ventricular wall thickness and the indexes of left ventricular diastolic function (the peak velocity of early rapid filling [E velocity], the peak velocity of atrial filling [A velocity], and the E/A ratio) were obtained by echocardiography. Brachial-ankle pulse wave velocity correlated significantly with the carotid plaque score, but the correlation was weak (r=0.37, p=0.001). The brachial-ankle pulse wave velocity demonstrated a strong correlation with the A velocity (r=0.73, p<0.001), the ratio of E to A (E/A) (r= 0.63, p<0.001), and the deceleration time of the E velocity (r=0.48, p<0.001). Stepwise regression analysis showed that the A velocity (beta coefficient=0.42, p<0.001) and ventricular septal thickness at the left ventricular outflow tract (beta coefficient=0.27, p=0.001) were independently associated with brachial ankle pulse wave velocity. Stepwise regression analysis indicated that ventricular septal thickness at the left ventricular outflow tract (beta coefficient=0.38, p=0.001) was independently associated with the plaque score. These results indicate that left ventricular diastolic dysfunction as revealed by increased peak velocity of atrial filling reflects arterial stiffening in type 2 diabetic patients. In addition, myocardial wall thickening at the left ventricular outflow tract reflects not only arterial stiffening but also carotid atherosclerosis. Therefore, these abnormal echocardiographic findings of left ventricular diastolic dysfunction and myocardial wall thickening may be useful markers of the presence of progressive arteriosclerosis in type 2 diabetic patients. PMID- 16671336 TI - Oxidation and carboxy methyl lysine-modification of albumin: possible involvement in the progression of oxidative stress in hemodialysis patients. AB - Hemodialysis (HD) patients are frequently in a state of increased oxidative stress, and hyperglycemia appears to be a major factor. We recently found that oxidized human serum albumin (HSA) is a reliable marker of oxidative stress in HD patients. However, the issue of whether oxidized HSA is associated with the progression of oxidative stress in HD patients with or without diabetes is not clear. In the present study, we examined the effect of a qualitative modification of HSA in HD patients with or without diabetes. Blood samples from 10 HD patients with diabetes, 7 HD patients without diabetes, and 10 healthy age-matched controls were examined. The increase in plasma protein carbonyl content and advanced glycation endproducts (AGEs) in HD patients was largely due to an increase in the levels of oxidized HSA. Furthermore, these increases were greatest in HD patients with diabetes. Purified HSA from HD patients (non-DM-HSA) was carbonylated and AGE-modified. The amount of modified HSA was the highest in HD patients with diabetes (DM-HSA). Carboxy methyl lysine (CML)-modified HSA triggered a neutrophil respiratory burst, and this activity was closely correlated with the increase in the CML/HSA ratio. These findings indicate that uremia plays an important role in the progression of oxidative stress in HD patients via an increase in CML-modified HSA. They also indicate that diabetic complications further exacerbate the progression of oxidative stress by further increasing the amount of these modified HSA molecules. PMID- 16671337 TI - T1198C polymorphism of the angiotensinogen gene and antihypertensive response to angiotensin-converting enzyme inhibitors. AB - This study examined the association between T1198C polymorphism of the angiotensinogen (AGT) gene and the blood pressure response to ACE inhibitors in a Chinese hypertensive cohort. After a 2-week single-blind placebo run-in period, benazepril (10-20 mg/day) or imidapril (5-10 mg/day) was administered for 6 weeks to 509 patients with mild-to-moderate essential hypertension. Polymerase chain reaction combined with restriction enzyme digestion was used to detect the polymorphism, and the patients were classified as having the TT, TC, or CC genotype. The achieved changes in systolic and diastolic blood pressure (SBP and DBP) were analyzed to determine their association with genotypes at the AGT gene locus. In the total 509 patients, the TT genotype was observed in 44 patients (8.7%), the TC genotype in 214 patients (42.0%), and the CC genotype in 251 patients (49.3%). The SBP reductions in patients with the TT genotype, TC genotype, and CC genotype were -15.3+/-12.7 mmHg, -14.0+/-12.7 mmHg, and -14.4+/ 12.4 mmHg, respectively (p=0.809). The DBP reductions in patients with the TT genotype, TC genotype, and CC genotype were -8.5+/-8.1 mmHg, -8.3+/-7.5 mmHg, and -8.9+/-6.6 mmHg, respectively (p=0.638). There were no significant differences in the changes in SBP or DBP after treatment among the three genotype groups. In conclusion, these results suggest that the AGT genotype does not predict the blood pressure-lowering response to antihypertensive treatment with ACE inhibitors in Chinese hypertensive patients. PMID- 16671338 TI - Effect of telmisartan on ambulatory blood pressure monitoring, plasma brain natriuretic peptide, and oxidative status of serum albumin in hemodialysis patients. AB - The effect of telmisartan on ambulatory blood pressure, plasma neurohormonal parameters, and oxidation of serum albumin has not been investigated in hemodialysis (HD) patients. Thirteen hypertensive HD patients were treated with 40 mg telmisartan once daily, and 24-h ambulatory blood pressure monitoring was performed after 0, 4, and 8 weeks of treatment. Plasma renin activity, plasma aldosterone concentration (PAC), brain natriuretic peptide (BNP) level, and serum oxidized albumin level were determined at the same time points. Serum telmisartan concentration was also measured at 4 and 8 weeks. Telmisartan significantly reduced systolic blood pressure and diastolic blood pressure (both awake and sleeping) after 4 weeks, and these pressures showed a further significant decrease after 8 weeks. Plasma levels of aldosterone, BNP, and serum oxidized albumin were markedly decreased after 4 weeks and these lower levels were maintained at 8 weeks. The trough serum telmisartan concentration was not significantly different at 8 weeks compared with 4 weeks. Throughout the treatment period, there were no significant adverse effects. Telmisartan effectively lowers blood pressure and reduces PAC, BNP, and oxidative stress and is safe and well-tolerated by HD patients. A long-term study in a large population is required to determine the influence of telmisartan therapy on cardiovascular mortality and morbidity in HD patients. PMID- 16671339 TI - Collagen metabolism in extracellular matrix may be involved in arterial stiffness in older hypertensive patients with left ventricular hypertrophy. AB - Collagen metabolism in the extracellular matrix (ECM) is related to the pathogenesis of cardiovascular stiffness and remodeling in hypertension. We evaluated the association between collagen metabolism markers and the newly developed parameter, brachial-ankle pulse wave velocity (baPWV), in older hypertensive patients with left ventricular hypertrophy (LVH). We performed echocardiography and baPWV measurement using a new device, form PWV/ABI (Colin Medical Technology, Komaki, Japan), and measured plasma levels of markers of collagen metabolism such as procollagen type I C-terminal propeptide (PICP: a marker of collagen synthesis), collagen type I pyridinoline cross-linked C terminal telopeptide (ICTP: a marker of collagen type I degradation), matrix metalloproteinase-1 (MMP-1: a marker of collagen degradation) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in 46 hypertensive patients with LVH. BaPWV was correlated with the plasma level of PICP (r=0.33, p=0.03) and ICTP (r=0.29, p=0.05) and the total TIMP-1/MMP-1 ratio (an index of collagen turnover; r=0.30, p=0.04). BaPWV was negatively correlated with the E/A ratio of left ventricular inflow (r=-0.36, p<0.05), while baPWV was not correlated with left ventricular mass index (LVMI; r=-0.175, p=0.25) or deceleration time of the mitral E wave (DCT; r=0.15, p=0.31). The measures of hypertensive heart disease, such as the E/A ratio, DCT or LVMI were not correlated with any collagen markers in this study. In multiple regression analysis adjusted for confounding factors such as age, sex, pulse pressure, mean blood pressure, pulse rate, LVMI, E/A ratio and DCT, the positive correlation between baPWV and total TIMP-1/MMP-1 ratio remained significant (p<0.05). In conclusion, arterial stiffness in high risk older hypertensive patients may involve ECM collagen metabolism. PMID- 16671340 TI - Comparison of the effects of cilnidipine and amlodipine on ambulatory blood pressure. AB - Cilnidipine is a novel and unique 1,4-dydropyridine derivative calcium antagonist that exerts potent inhibitory actions not only on L-type but also on N-type voltage-dependent calcium channels. Blockade of the neural N-type calcium channel inhibits the secretion of norepinephrine from peripheral neural terminals and depresses sympathetic nervous system activity. The purpose of this study was to assess the effect of cilnidipine and amlodipine on ambulatory blood pressure (BP) levels. We performed 24-h ambulatory BP monitoring before and after once-daily use of cilnidipine (n=55) and amlodipine (n=55) in 110 hypertensive patients. Both drugs significantly reduced clinic and 24-h systolic BP (SBP) and diastolic BP (DBP) (p < 0.005). However, the reductions of 24-h (-1.19+/-6.78 vs. 1.55+/ 6.13 bpm, p=0.03), daytime (-1.58+/-6.72 vs. 1.68+/-7.34 bpm, p=0.02) and nighttime (-1.19+/-5.72 vs. 1.89+/-6.56 bpm, p=0.01) pulse rate (PR) were significantly greater in the cilnidipine group than the amlodipine group. There was no correlation between the degree of daytime SBP change and that of daytime PR change after amlodipine treatment (r=-0.08, n.s.), but there was a significant negative correlation between the degree of daytime SBP change and that of day time PR change after cilnidipine treatment (r=-0.27, p<0.05). N-type calcium channel blockade by cilnidipine may not cause reflex tachycardia, and may be useful for hypertensive treatment. PMID- 16671341 TI - Renoprotective effect of pravastatin in salt-loaded Dahl salt-sensitive rats. AB - The pathophysiological features of nephrosclerosis may be analogous to those of atherosclerosis, which is intimately related to lipid metabolism. Thus, we examined whether a lipid-lowering agent, pravastatin, would ameliorate renal damage in hypertensive model animals. Salt-loaded Dahl salt-sensitive (S) rats were given pravastatin (2 mg/ml in drinking water) for 5 weeks. Pravastatin decreased systolic blood pressure. Although pravastatin did not influence the serum total, high-density, or low-density lipoprotein cholesterol, serum triglycerides were decreased. Pravastatin decreased urinary protein excretion and ameliorated histopathological damage in salt-loaded Dahl S rats. Increased urinary excretion of 8-iso-prostagaldin F2alpha and 8-hydroxy-2'-deoxyguanosine and renal superoxide overproduction and decreased reduced glutathione in the renal parenchyma were ameliorated with pravastatin in Dahl S rats fed a high salt diet. Therefore, pravastatin inhibited the progression of renal injury in salt loaded Dahl S rats, through its antioxidant as well as its depressor effects. PMID- 16671342 TI - Effects of a new calcium channel blocker, azelnidipine, on systemic hemodynamics and renal sympathetic nerve activity in spontaneously hypertensive rats. AB - Antihypertensive treatment with dihydropyridine calcium channel blockers elicits sympathetic nerve activation, which may contribute to cardiovascular events. However, recent clinical studies showed that treatment with azelnidipine, a new dihydropyridine calcium channel blocker, significantly reduced blood pressure in hypertensive patients while either maintaining or actually decreasing heart rate (HR). In this study, we examined the effects of azelnidipine and amlodipine on systemic hemodynamics and renal sympathetic nerve activity (RSNA) in anesthetized spontaneously hypertensive rats (SHR). We also examined the effects of these agents on baroreflex functions by infusing phenylephrine (30 microg/kg/min, i.v.) and sodium nitroprusside (10 microg/kg/min, i.v.) into azelnidipine- or amlodipine-treated SHR. Fifty min after administration of azelnidipine (10 microg/kg/min for 10 min, i.v.), mean arterial pressure (MAP) significantly decreased from 153+/-5 to 122+/-5 mmHg; however, HR and integrated RSNA did not change significantly (from 352+/-9 to 353+/-10 beats/ min and 115+/-5% of baseline, respectively). Infusion of amlodipine (50 microg/kg/min for 10 min) elicited similar effects on MAP (from 152+/-5 to 120+/-4 mmHg). However, amlodipine significantly increased HR (from 351+/-9 to 375+/-11 beats/min) and integrated RSNA (165+/-5% of baseline). Analyses of baroreflex function curves revealed that azelnidipine-treated rats showed a smaller baroreflex function than amlodipine-treated rats (p<0.05). These data suggest that azelnidipine possesses sympathoinhibitory effects, which may be one reason why it had less pronounced effects on HR in hypertensive patients. PMID- 16671343 TI - How do you cope with bundled claims? PMID- 16671344 TI - Reinventing family medicine. PMID- 16671345 TI - Should you cut out the middle plan? PMID- 16671346 TI - Should you cut out the middle plan? PMID- 16671347 TI - Level-IV visits. PMID- 16671348 TI - Creating a lean practice. PMID- 16671349 TI - Outpatient vs. inpatient treatment of community acquired pneumonia. PMID- 16671350 TI - Using a simple patient registry to improve your chronic disease care. PMID- 16671351 TI - Same-day E/M services: what to do when a health plan won't pay. PMID- 16671352 TI - Can your patients afford the medications you prescribe? PMID- 16671353 TI - Turning frustration into fulfillment. PMID- 16671354 TI - Ubiquitin wins Nobel. PMID- 16671355 TI - Ubiquitin-independent degradation: lessons from the p53 model. AB - Ubiquitin-proteasome degradation is a key cellular process involved in almost every aspect of cell life. According to the current concept, proteins are stable unless they are marked by poly-ubiquitination for degradation by the 26S proteasomes. A new twist in the concept became evident while studying the degradation of the tumor suppressor p53, a protein that appeared to satisfy this principle. We have discovered that native p53 is also prone to ubiquitin independent 20S proteasomal degradation, suggesting that certain proteins are inherently unstable. We further found that this process of degradation is mediated by 20S proteasomes and inhibited by NADH quinone oxidoreductase 1. Our recent findings together with previous observations of ubiquitin-independent degradation suggest the existence of ubiquitin-independent mechanisms for proteasomal protein degradation in the cells. PMID- 16671356 TI - Monoubiquitylation: a recurrent theme in membrane protein transport. AB - Polyubiquitylation of cellular proteins has long been recognized as a prelude to a degradative fate in proteasomes. In recent years, however, ubiquitin conjugation has emerged as a regulatory strategy of considerable versatility. Most notably, monoubiquitylation is attributed an intimate role in trafficking of membrane proteins between various cellular compartments. Diverse classes of transmembrane proteins from across the eukaryotic spectrum (e.g., epidermal growth factor-receptor and other receptor tyrosine kinases) become modified with monoubiquitin molecules. Monoubiquitylation of substrates, in turn, regulates both their endocytosis at the plasma membrane and sorting in endosomes for delivery to lysosomes or vacuoles. A mechanistic rationale lies in the identification of a growing list of ubiquitin-binding domains carried by a variety of endocytic adaptor proteins. Thus, ubiquitin-conjugated membrane proteins may form extensive contacts with the endocytic machinery. Further, ubiquitin-binding adaptors and other endocytic components are, likewise, often monoubiquitylated. In this case, ubiquitin conjugation may serve to enhance intermolecular avidity in cargo-bound endocytic complexes, or alternatively, to mediate timely inactivation of ubiquitin-binding adaptors. Interestingly, the ubiquitin/endocytosis interface is appropriated by pathogenic organisms, for instance, during budding of viruses from host-infected cells. Moreover, compromised ubiquitin-mediated transport of certain signaling receptors is associated with disease states, including oncogenic transformation. PMID- 16671357 TI - Quality control in the secretory pathway. PMID- 16671358 TI - The ubiquitin system and morphogenesis of fungal pathogens. AB - Distinct fungal species exhibit different cellular morphologies, such as yeast and filamentous (hyphal and pseudohyphal) forms, that are reflected in the macroscopic colony morphology. Dimorphic and multimorphic fungi can switch between these different morphologies, enabling the utilization of different food supplies in the case of saprophytes, and contributing to pathogenesis in the case of parasites. Cellular morphogenesis is often regulated by signal transduction pathways, and is intimately linked to the cell cycle machinery. Here we describe the role of ubiquitin-mediated degradation of cell cycle regulators and transcription factors involved in fungal morphogenesis. PMID- 16671359 TI - The F-box protein, Ufo1, maintains genome stability by recruiting the yeast mating switch endonuclease, Ho, for rapid proteasome degradation. AB - We describe a unique E3, the F-box protein, Ufo1, of yeast. Ufo1 recruits the mating switch endonuclease, Ho, to the SCF complex for ubiquitylation. In addition to the F-box and WD40 protein-protein interaction domains found in all F box proteins, Ufo1 has a unique domain comprising multiple copies of the ubiquitin-interacting motif. Ufo1 interacts with the UbL-UbA protein, Ddi1, via its UIMs, and this is required for turnover of SCFUfo1 complexes. This is a novel function for an UbL-UbA protein. Deletion of the genomic UFO1UIMs is lethal and our data indicate that Ufo1deltaUIM acts as a dominant negative leading to inhibition of the SCF pathway of substrate degradation and to cell cycle arrest. Furthermore, we found that Ddi1 is required for the final stages of degradation of Ho endonuclease. In the absence of Ddi1, Ho does not form a complex with the 19S RP and is stabilized. Stabilization of Ho leads to perturbation of the cell cycle and to the formation of multi-budded cells. Our experiments uncover a novel role for the ubiquitin-proteasome system in maintenance of genome stability. PMID- 16671360 TI - Ubiquitination of, and sumoylation by, the Arf tumor suppressor. AB - The Ink4a-Arf locus, which encodes two distinct tumor suppressor proteins, is inactivated in many cancers. Whereas p16Ink4a is an inhibitor of cyclin D dependent kinases, p19Arf (p14ARF in humans) antagonizes the E3 ubiquitin protein ligase activity of Mdm2 to activate p53. We now recognize that Arf functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals originating from proto-oncogene activation, but its p53-independent activities remain poorly understood. Arf proteins are highly basic (> 20% arginine content, pl > 12) and predominantly localize within nucleoli in physical association with an abundant acidic protein, nucleophosmin (NPM/B23). When bound to NPM, Arf proteins are relatively stable with half-lives of 6-8 hours. Although mouse p19Arf contains only a single lysine residue and human p14ARF has none, both proteins are N-terminally ubiquitinated and degraded in proteasomes. Through as yet uncharacterized mechanisms, p19Arf induces p53-independent sumoylation of a variety of cellular target proteins with which it interacts, including both Mdm2 and NPM. A naturally occurring NPM mutant (NPMc) expressed in myeloid leukemia cells redirects both wild-type NPM and p19Arf to the cytoplasm, inhibits Arf-induced sumoylation, and attenuates p53 activity. Thus, ubiquitination and sumoylation can each influence Arf tumor suppressor activity. PMID- 16671361 TI - Pulmonary arterial hypertension associated with autoimmune disease: a single medical center experience. AB - BACKGROUND: New drugs have significantly improved the prognosis and quality of life of patients with pulmonary arterial hypertension. However, PAH associated with autoimmune disease, particularly progressive sclerosis, remains a very serious problem. OBJECTIVES: To evaluate whether the course of the disease and survival is significantly different in patients with PAH related to autoimmune disease as compared to other patients with PAH and to determine the prognostic factors in these patients. METHODS: We retrospectively compared 24 patients with PAH associated with autoimmune disease to 42 patients with other causes of PAH. We focused on the clinical and hemodynamic parameters and on the outcome. RESULTS: The early mortality rate was slightly higher in patients with PAH associated with autoimmune disease (13% after the first year, 25% after the fifth year). The prognostic factor was a shorter distance on the 6 minute walking distance test (r = 0.2, P= 0.01). CONCLUSIONS: The early detection of PAH associated with autoimmune disease should encourage earlier and more aggressive treatment than in idiopathic PAH. PMID- 16671362 TI - The Israeli anesthesiology physician workforce. AB - BACKGROUND: Anesthesiology is a vital specialty that permits the safe and humane performance of painful procedures. Most Israeli anesthesiologists are immigrants, while only a minimal number of Israeli medical school graduates enter the specialty. Unfortunately, the supply of immigrant physicians is declining due to falling immigration rates. OBJECTIVES: To examine the current Israeli anesthesiology workforce and project future needs. METHODS: Demographic and professional information about Israeli hospital anesthesiologists was solicited from anesthesiology department heads. Data were also gathered about the past, present and projected future growth, age distribution and birth rate of the Israeli population. Needs and demand-based analyses were used to project future anesthesiology workforce requirements. RESULTS: Data on 711 anesthesiologists were obtained from 30 hospital anesthesiology department heads. Eighty-seven anesthesiologists (12.2%) graduated from Israeli medical schools and 459 (64.6%) graduated from medical schools in the former Soviet Union. Among the 154 anesthesiology residents < or = 40 years old, only 13 (8.4%) graduated from Israeli medical schools. There are approximately 10.8 anesthesiologists per 100,000 population. Projections for 2005-2015 revealed a need for 250-300 new anesthesiologists. CONCLUSIONS: The anesthesiology workforce is predominantly composed of immigrants. This has vast implications for the future viability of the specialty because of the continuing reduction in immigration, the lack of interest in the specialty by Israeli medical school graduates, and the projected need for many new anesthesiologists to replace retirees and to provide care to a growing and aging population. PMID- 16671363 TI - Virtual reality immersion method of distraction to control experimental ischemic pain. AB - BACKGROUND: Virtual reality immersion has been advocated as a new effective adjunct to drugs for pain control. The attenuation of pain perception and unpleasantness has been attributed to the patient's attention being diverted from the real, external environment through immersion in a virtual environment transmitted by an interactive 3-D software computer program via a VR helmet. OBJECTIVES: To investigate whether VR immersion can extend the amount of time subjects can tolerate ischemic tourniquet pain. METHODS: The study group comprised 20 healthy adult volunteers. The pain was induced by an inflated blood pressure cuff during two separate, counterbalanced, randomized experimental conditions for each subject: one with VR and the control without VR exposure. The VR equipment consisted of a standard computer, a lightweight helmet and an interactive software game. RESULTS: Tolerance time to ischemia was significantly longer for VR conditions than for those without (P < 0.001). Visual Analogue Scale (0-10) ratings were recorded for pain intensity, pain unpleasantness, and the time spent thinking about pain. Affective distress ratings of unpleasantness and of time spent thinking about pain were significantly lower during VR as compared with the control condition (P< 0.003 and 0.001 respectively). CONCLUSIONS: The VR method in pain control was shown to be beneficial. The relatively inexpensive equipment will facilitate the use of VR immersion in clinical situations. Future research is necessary to establish the optimal selection of clinical patients appropriate for VR pain therapy and the type of software required according to age, gender, personality, and cultural factors. PMID- 16671364 TI - Techniques for terminating patient-physician encounters in primary care settings. AB - BACKGROUND: Physicians in the community work on a tight and often pressured schedule; verbal and non-verbal techniques to terminate the patient-physician encounter are therefore necessary. OBJECTIVES: To characterize ways of terminating the encounter. METHODS: Using a structured questionnaire we observed seven family physicians and nine consultants and recorded patient-physician encounters to assess techniques for terminating the encounter. RESULTS: In all, 320 encounters were recorded, 179 (55.9%) by consultants and 141 (44.1%) by family physicians. The mean duration of the encounters was 9.02 +/- 5.34 minutes. The mean duration of encounters with family physicians was longer than with consultants (10.39 vs. 7.93 minutes, P< 0.001). In most cases the encounter ended with the patient receiving printed documentation from the physician (no difference between family physicians and with consultants). Consultants were more likely to end the encounter with a positive concluding remark such as "feel good" or "be well" (P < 0.01). There was no single occasion where termination of the encounter was initiated by the patient. CONCLUSIONS: Giving a printed document to the patient appears to be perceived by both patients and physicians as an accepted way to end an encounter. Another good way to end the encounter is a positive comment such as "feel good" or "be well." PMID- 16671365 TI - Public and private patients in Jerusalem hospitals: who operates on whom? AB - BACKGROUND: Debate continues in Israel as to whether to allow patients in public hospitals to choose their physician in return for an additional, out-of-pocket payment. One argument against this arrangement is that the most senior physicians will devote most of their time to private patients and not be sufficiently available to public patients with complex cases. OBJECTIVES: To analyze the patterns of surgical seniority in Jerusalem hospitals from a number of perspectives, including the extent to which: a) opting for private care increases the likelihood of being treated by a very senior surgeon; b) public patients undergoing complex operations are being treated by very senior surgeons, c) the most senior surgeons allocate a significant portion of their time to private patients. METHODS: Demographic and clinical data were retrieved from the operating room records of three of the public hospitals in Jerusalem for all 38,840 operations performed in 2001. Of them, roughly 6000 (16%) were performed privately. Operations were classified as "most complex," "moderately complex" and "least complex" by averaging the independent ratings of eight medical and surgical experts. The surgeon's seniority was graded as "tenured" (tenured board certified specialists, including department heads), "senior" (non-tenured board certified specialists), and "residents." For each operation, we considered the seniority of the lead surgeon and of the most senior surgeon on the surgical team. RESULTS: The lead surgeon was of tenured rank in 99% of the most complex private cases and 74% of the most complex public cases, in 93% of the moderately complex private and 35% of the moderately complex public cases, and in 92% of the least complex private and 32% of the least complex public cases. The surgical team included a tenured physician in 97%, 66%, and 53% of the most complex, moderately complex, and least complex public operations, respectively. In both private and public cases, a board-certified (tenured or senior) specialist was a member of the surgical team for almost all of the most complex and moderately complex operations. On average, over half of the operations in which the lead surgeon was a department head were performed on public patients. Among tenured surgeons, those who spent more hours than their colleagues leading private operations also tended, on average, to spend more hours leading public operations. CONCLUSIONS: Private patients have an advantage over public patients in terms of the seniority of the lead surgeon. However, there is also substantial involvement of very senior surgeons in the treatment of public patients, particularly in those cases that are most complex. PMID- 16671366 TI - Primary pulmonary arterial hypertension and the systemic autoimmune diseases. PMID- 16671367 TI - The impending crisis of anesthesiology in Israel. PMID- 16671368 TI - Myocardial regeneration by adult stem cells. AB - Ventricular remodeling and heart failure are the inevitable consequences of myocardial infarction. Current options to cure myocardial infarction and subsequent heart failure suffer from specific limitations. Thus, alternative, additional long-term therapeutic strategies are needed to cure this costly and deadly disease. Cardiac regeneration is a promising new therapeutic option. Through cellular and molecular therapies, the concept of in situ "growing" heart muscle, vascular tissue and manipulating the extracellular matrix environment promises to revolutionize the approach of treating heart disease. Recent studies have suggested that stem cells resident within the bone marrow or peripheral blood can be recruited to the injured heart. The regeneration of damaged heart tissue may include the mobilization of progenitor or stem cells to the damaged area or stimulation of a regenerative program within the organ. There is now evidence accumulating that the heart contains resident stem cells that can be induced to develop into cardiac muscle and vascular tissue. The present review aims to describe the potential, the current status and the future challenges of myocardial regeneration by adult stem cells. PMID- 16671369 TI - Desmoid tumor causing duodenal obstruction. PMID- 16671370 TI - Thoracic epidural for Nissen fundoplication in a child with familial dysautonomia. PMID- 16671371 TI - Sentinel lymph node biopsy in the diagnosis and treatment of multicentric malignant melanoma of the penis. PMID- 16671372 TI - Myositis in Behcet's disease after tonsillectomy. PMID- 16671373 TI - Multidetector-row CT enteroclysis: indications and clinical applications. AB - This paper examines the diagnostic potential of multislice computed tomography enteroclysis (MSCT-E) to detect and assess different diseases affecting the small bowel, emphasising the increasingly important role assumed by the technique in the study of this anatomical region. After a short summary of the technical aspects, we discuss the different findings that can be observed during an MSCT-E study and that enable detection of small-bowel disease and, if necessary, assessment of the extent and stage of disease. PMID- 16671374 TI - Multislice CT with single-phase technique in patients with suspected pancreatic cancer. AB - PURPOSE: The purpose of this study was to evaluate the role of multislice computed tomography (MSCT) with a single-phase technique in patients with suspected pancreatic cancer (PC). MATERIALS AND METHODS: Seventy-eight patients underwent MSCT with the following technical parameters: collimation: 4x1 mm; pitch 1; 120 kVp; 260 mAs. The pre-contrast scan was followed by a single acquisition phase in the caudocranial direction from the inferior hepatic margin to the diaphragm with a 60-s delay after IV administration of 150 ml of iodinated contrast material at a rate of 3 ml/s. Two radiologists assessed the single images independently. Receiver operating characteristics (ROC) curves were obtained for each of the two observers. RESULTS: The final diagnosis was pancreatic cancer in 46 cases and chronic pancreatitis in 32 cases. Areas under the curve (AZ) for diagnosis and evaluation of disease resectability were 0.97 and 0.93 for the first observer (p=ns), and 0.97 and 0.90 for the second observer (p=ns). The mean difference in tissue attenuation values between the cancer and normal pancreas was 72 +/- 3 Hounsfield units (HU). No statistically significant differences were observed in the degree of opacification between the peripancreatic arteries and veins. CONCLUSIONS: MSCT with a single-phase technique is an accurate and reproducible method for diagnosis and evaluation of disease resectability in patients with suspected PC, ensuring optimal tumour-to pancreas contrast and maximal opacification of the main peripancreatic arterial and venous structures. PMID- 16671375 TI - Ultrasonographic and spiral CT evaluation of simple and complicated acute cholecystitis: diagnostic protocol assessment based on personal experience and review of the literature. AB - PURPOSE: The aim of this study was to identify and classify the ultrasonographic and computed tomography (CT) signs of simple and complicated acute cholecystitis and to define the correct diagnostic protocol. MATERIALS AND METHODS: We retrospectively reviewed 35 patients (23 men and 12 women; mean age 66.6 years) presenting with acute cholecystitis who were assessed by emergency ultrasonography (US) (30/35 cases) and spiral CT (12/35 cases); all patients underwent emergency surgery. The US signs were analysed and classified as major criteria (wall thickening and stratification, distension, Murphy's sign), minor criteria (bile stones, sludge, and biliary tract dilatation), and complication signs (gas collections, aerobilia, fluid collection, difficult or missed identification of the gallbladder). Imaging results were compared with histological findings (gold standard), and accuracy, sensitivity, specificity, and positive and negative predictive values (PPVs and NPVs) were assessed for each modality. Concordance between the US and CT findings was also evaluated for cases undergoing both examinations; additional findings provided by one or the other modality were also assessed. RESULTS: US had an accuracy of 66.6%, a sensitivity of 37.5%, a specificity of 70%, a PPV of 100%, and an NPV of 58.3%. CT had 100% accuracy, sensitivity, and specificity. Concordance between US and CT was observed for diagnosis of complications, but CT provided additional findings in all cases. The majority of complicated cases undergoing US examination (68.7%) revealed more than two major criteria and one minor criterion or at least one sign of complication. CONCLUSIONS: If more than two major signs associated with one minor sign or at least one sign of complication are present at US, CT is mandatory to recognise and thoroughly evaluate the type of complication and indicate appropriate treatment. PMID- 16671376 TI - Multidetector CT with double arterial phase and high-iodine-concentration contrast agent in the detection of hepatocellular carcinoma. AB - PURPOSE: The purpose of this study was to determine whether the double arterial phase with multidetector computed tomography MDCT and high-iodine-concentration contrast material (CM) improves identification of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Fifty patients with 111 HCC foci (61 confirmed histologically, 46 confirmed by percutaneous interventional procedures, four confirmed by CT follow-up of at least 6 months) underwent MDCT with a double arterial phase and a portal venous phase after administration of contrast material with a high iodine concentration (400 mgI/ml, 2 ml/kg, 5 ml/s). Two radiologists independently evaluated the images in three distinct reading sessions (early arterial phase (EAP), late arterial phase (LAP) and double arterial phase) to determine presence, number and degree of suspicion of HCC. The sensitivity and the positive predictive value (PPV) were calculated for every reading session. The following statistical evaluations were used: k statistic and McNemar's test. RESULTS: Mean sensitivity and PPV in the detection of HCC were, respectively, 83.8% and 93.5% for EAP, 90.5% and 94.8% for LAP, and 94.1% and 95.1% for the double arterial phase. Sensitivity of the double arterial phase was statistically higher when compared with EAP alone but showed no statistically significant difference when compared with LAP. The k values ndicated moderate-to excellent interobserver agreement in all reading sessions. CONCLUSIONS: Sensitivity and PPV increase progressively when passing from EAP to LAP to double arterial-phase images obtained with contrast material with a high iodine concentration. However, the difference in sensitivity between LAP and the double arterial phase was not statistically significant. PMID- 16671377 TI - Pulmonary nodules in osteosarcoma patients: differential diagnosis of central venous catheter-related infections in the lungs. AB - PURPOSE: The purpose of this study was to evaluate the differential diagnosis of pulmonary nodules by conventional radiography and computed tomography (CT) in osteosarcoma patients with central venous catheter. MATERIALS AND METHODS: Between March 1997 and December 2001 at our Department of Musculoskeletal Oncology, 231 patients with peripheral osteosarcoma received a central venous catheter to allow infusion therapy and blood sampling. The mean age of these patients was 16 years (range 4-63), and 90 were aged 15 years or younger. All patients underwent radiological investigation for tumour staging and comparison with the following study in accordance with the protocol in place at our Department of Oncology and Division of Diagnostic Imaging. RESULTS: Of a total of 231 patients, 13 (5.6%) developed an infection of the central venous line, with fever that was very high in some cases. In ten cases (4.3%), radiology showed damage to the alveolar interstitium typical of inflammatory forms, whereas in the remaining three (1.3%) it depicted nodular opacities, which required differentiation between lung metastases and septic emboli. After appropriate antibiotic and replacement of the central venous line, CT demonstrated disappearance of the lung nodules in all three patients, enabling a diagnosis of nodular septic embolism. CONCLUSIONS: Placement of a central venous catheter for infusion therapy, chemotherapy and blood sampling has improved the quality of life of cancer patients. The most common complications of the use of central venous catheters include infection and venous thrombosis whereas pulmonary septic emboli are rare. PMID- 16671378 TI - Organizing pneumonia: typical and atypical HRCT patterns. AB - Organizing pneumonia (OP) is a clinical-pathological entity that, if idiopathic, makes part of the idiopathic interstitial pneumonias classification of the American Thoracic Society/European Respiratory Society (ATS/ERS) of 2002 (50% of the cases, called cryptogenic organizing pneumonia, or COP). In the remaining 50% it is secondary, representing one modality of response of the lung to various forms of injury. Typical computed tomography (CT) pattern has been widely described and consists of peripheral parenchymal consolidations with air bronchogram with or without surrounding ground-glass-like opacities. The purpose of this article is to describe the less frequent imaging pattern of this disease represented by single or multiple focal lesions (nodules or masses that place diagnostic problems with malignancy), bronchocentric pattern (parenchymal consolidations with peribronchovascular distribution), atoll sign (central area of ground-glass-like density and peripheral area of consolidation), nodular lesions (poorly defined micronodular pattern), linear and band-like opacities (subpleural linear opacities that can have disposition parallel or perpendicular in relation to the pleura), perilobular pattern (thickening of the interlobular septa with reticular pattern) and progressive fibrotic pattern (irregular thickening of the interlobular septa with associated ground-glass-like appearance and consolidations). PMID- 16671379 TI - Integrated multislice CT and Tc-99m Sestamibi SPECT-CT evaluation of solitary pulmonary nodules. AB - PURPOSE: The purpose of this study was to evaluate efficacy of multislice computed tomography (MSCT) and single photon emission computed tomography (SPECT) CT with Tc-99m Sestamibi in the assessment of solitary pulmonary nodules of uncertain significance. Scintigraphy was performed using a 'hybrid' g-camera that allows simultaneous acquisition of SPECT and CT images, with interesting results in diagnostic oncology. MATERIALS AND METHODS: Between September 2003 and August 2004, 23 patients with a solitary pulmonary nodule detected on CT underwent SPECT CT using Tc-99m Sestamibi as a radiotracer. Nodules with positive scintigraphy were immediately subjected to biopsy or surgical resection. Nodules with negative scintigraphy were followed up after 3-4 months by MSCT with automatic segmentation software (Advanced Lung Analysis, ALA) and histological characterisation. RESULTS: Of the 23 nodules (size range 0.8-2 cm) discovered with MSCT, 11 showed intense uptake of Tc-99m Sestamibi. Ten lesions were true positive: seven adenocarcinomas, one squamous cell carcinoma, one large cell carcinoma and one metastasis. The only false positive was histologically classified as a large cell granuloma. Twelve lesions had negative scintigraphy: five fibrous lesions, three hamartomas, three granulomas and one adenocarcinoma (false negative). Benign nodules without tracer uptake underwent another CT scan 3-4 months later, which confirmed stability of the nodule size. Correlation of Sestamibi SPECT with histology showed sensitivity (Se) of 90.9 %, specificity (Sp) of 91.6 %, diagnostic accuracy of 91.3 %, positive predictive value (PPV) of 90.9% and negative predictive value (NPV) of 91.6 %. CONCLUSIONS: The integrated use of MSCT and Tc-99m Sestamibi SPECT-CT could be very useful in the management of solitary pulmonary nodules (SPNs). In particular, in our preliminary study, scintigraphy provided significant diagnostic information to differentiate benign from suspicious pulmonary nodules. The use of scintigraphy could be helpful to anticipate histological assessment and surgical treatment of SPNs identified at CT. PMID- 16671380 TI - Peripheral arterial occlusive disease: role of MR angiography. AB - PURPOSE: Magnetic resonance angiography (MRA) has recently become instrumental in the diagnosis of arterial disease in various body districts and is gaining an increasingly important role in the study of peripheral vascularisation. The aim of our study was to evaluate the reliability of MRA using digital subtraction angiography (DSA) as the reference standard. MATERIALS AND METHODS: Between November 2003 and August 2004, 30 patients with known peripheral arterial disease were studied by MRA and DSA. MRA was performed with a Philips Intera 1.5 T, with acquisitions from the coeliac trunk to the feet. For acquisitions of the feet and ankles we used unenhanced time-of-flight (TOF) sequences with a head coil. The angiographic sequence was acquired in three volumes of 40-45 cm after administration of paramagnetic contrast material. RESULTS: In the patients with peripheral arterial disease, the technique provided a precise evaluation of the stenosis (mild, moderate, severe) or obstruction of the peripheral district as well as the detection of other diseases, such as stenosis of the renal arteries or aneurysms. CONCLUSIONS: Total-body three-dimensional (3D) MRA allows a fast, safe, and accurate assessment of the arterial system in patients with arteriosclerosis and can be considered an alternative to DSA in the management of patients with steno-obstructive disease of the peripheral arteries. PMID- 16671381 TI - Optimisation of multislice computed tomography protocols in angio-CT examinations. AB - PURPOSE: The aim of this paper is to explain a general procedure for the optimisation of multislice computed tomography (MSCT) protocols. MATERIALS AND METHODS: Four angio-CT protocols with a GE LightSpeed Plus 4-slice CT scanner were considered. Effective doses were computed for a sample of patients. First the dose was optimised for arterial-phase scans on a standard patient and adapted to the weight of individual patients with a scaling factor. RESULTS: The mean effective dose for an angio-CT examination ranged from 18.8 mSv to 28.8 mSv, depending on the protocol adopted. Following the optimisation procedure, we drew up a table indicating tube current values for each patient weight. Calculation of the effective dose before and after the optimisation procedure revealed a dose reduction of about 40%. CONCLUSIONS: Angio-CT examinations deliver high doses, but these doses can be reduced without affecting image quality. PMID- 16671382 TI - Emergency radiology today between philosophy of science and the reality of "emergency care". AB - In the past 20 years, emergency care concept has substantially changed on a cultural point of view, going well beyond the boundaries of medical science. It is now a general understanding that the real enemy of the critical patient is time; thus, functional organisation and collocation of human and technological resources in the emergency department (ED) can help avoid the loss of human lives. This "cultural revolution" led to the creation and development of structural and organisational models (layouts) of EDs. Now, emergency radiology has a central role in ED organisation, and the radiologist, providing 24-h coverage in the emergency room, is crucial for the correct diagnostic approach and rapid management of trauma. If this is the cultural background to the "emergency care" concept, an overview of such care in our country shows great differences from a structural, technological and organisational point of view. The presence of the radiologist providing 24-h coverage in the emergency room is still uncommon in many EDs The qualification of emergency care must be sought by studying the needs of the population and by seeking qualified personnel with high professional skill levels. All this must be understood and pursued by politicians and health care managers whose aim should be to coordinate and check the measures and human resources applied to the system. This process necessarily involves rewarding those health care professionals who prove to be up to the job. PMID- 16671383 TI - Systematic approach to human error in radiology. AB - We propose a systematic approach to human errors in radiology. We consider perceptual, cognitive and system errors. Practical aspects with psychological and clinical involvement are discussed with tree diagrams. PMID- 16671384 TI - Imaging nervous pathways with MR tractography. AB - This paper describes the state of the art of tractography, a technique which enables the virtual reconstruction of axon bundles of the central nervous system using diffusion-weighted magnetic resonance images. This technique has raised enthusiasm and expectations among specialists because it is the only non-invasive method for studying the three-dimensional architecture of axonal fibres in vivo. Tractography is a new technique used to assess the anatomy of the central nervous system, and it will be available for routine clinical use in the future. Understanding its potential applications and limitations is therefore important. PMID- 16671385 TI - Evaluation of basal ganglia haemodynamic changes with perfusion-weighted magnetic resonance imaging in patients with Parkinson's disease. AB - PURPOSE: The aim of our study was to assess the regional cerebral blood flow (rCBF) of basal ganglia and thalami in patients with Parkinson's disease (PD) using perfusion-weighted magnetic resonance imaging (PW-MRI). MATERIAL AND METHODS: Twenty subjects affected by idiopathic PD according to the United Kingdom Brain Bank criteria were enrolled in the study. Twenty normal subjects matched for age and gender were included as controls. After 20-day therapy withdrawal, the PD patients underwent PW-MRI. The rCBF was calculated both in patients and in controls. The regions of interest were manually positioned on rCBF maps over the caudate nucleus, the putamen, the external and internal globus pallidus, and over the ventrolateral nucleus of the thalamus. Data were normalised with those obtained from parieto-occipital white matter (POWM). Statistical analysis was performed using a parametric ANOVA test. RESULTS: Patients showed a significant (p<0.01) interhemispheric asymmetry; rCBF values were higher on the more severely affected side. Controls showed no interhemispheric asymmetry. CONCLUSION: Our study suggests that PW-MRI is a valuable tool for assessing haemodynamic changes in PD patients. Haemodynamic change pattern may be useful in the early diagnosis of PD. PMID- 16671386 TI - Non-ST-Segment elevation acute coronary syndrmes: management strategies for optimal outcomes. PMID- 16671387 TI - Nursing education: at the crossroads of change. PMID- 16671388 TI - The many benefits of certification. PMID- 16671389 TI - Alexander H. Leighton's and Jane Murphy's scientific contributions in psychiatric epidemiology: a personal appreciation. AB - This article introduces the special issue of Transcultural Psychiatry in honour of Alexander Leighton. A sketch of his research career is followed by a discussion of the work of his wife, Dr. Jane Murphy, first on St. Lawrence Island, near the Bering Strait, and later as a key figure in the Stirling County project. A brief conclusion highlights the main aspects of their joint legacy to cultural psychiatry and psychiatric epidemiology. PMID- 16671390 TI - Johns Hopkins legionary: Leighton's lineage and legacy. AB - What is the relationship between sociocultural environment and psychiatric disorder? In particular, what is 'social disintegration', its characteristics, dangers and possible remediation? Alexander Hamilton Leighton and Jane Murphy's interdisciplinary contributions derive from those consuming concerns. This article contextualizes the famous Stirling County Study by suggesting that a hitherto unrecognized shaping role was played by William Henry Welch of Johns Hopkins and The Rockefeller Foundation, especially so through the example of Hopkins's epidemiological 'demonstration areas'. The article then details the Stirling County Study itself, including its relations with Dalhousie University and Canadian psychiatry. The concluding section identifies and assesses a wider set of achievements and contributions forming Leighton and Murphy's legacy to our several but interdependent fields of cross-cultural endeavour. PMID- 16671391 TI - Alexander Leighton and the evolutionary perspective. AB - A comparison of the theoretical approach of Alexander H. Leighton with that of current work in evolutionary psychology and psychiatry reveals key similarities. For both, a core human nature/psychology is assumed to underlie human behavior. Individuals may vary with regard to the salience of components of that core, which in any event are differentially expressed in response to varying cultural/environmental factors. As with many of today's evolutionists, Leighton's research involved natural settings combined with the collection of rigorously analyzed quantitative data. Evolutionary psychiatrists would benefit greatly from studying Leighton's work, particularly in connection with the hypothesis that psychopathology is a product of our departure from the environments in which we evolved. PMID- 16671392 TI - Longitudinal research to promote effective refugee resettlement. AB - Canada's relative generosity in admitting refugees and fairness in considering refugee claims has earned this country an enviable reputation. However, having opened its doors to those selected, Canada's resettlement policies and programs fail to provide for their needs, and to promote their optimal adaptation. Based on a decade-long investigation of the resettlement of more than 1300 Southeast Asian refugee--'Boat People'--the current report examines how research concerning (a) the impact of pre-migration trauma; (b) the mental health impact of social resources such as the like-ethnic community, refugee sponsorship programs, and language training; and (c) individual coping strategies such as suppressing the past, can contribute both to theory and to improving policy and practice. The presentation acknowledges the contributions of Dr. Alexander H. Leighton by demonstrating the importance of his insistence on the need for a longitudinal perspective both for conducting research and for planning programs and services. PMID- 16671393 TI - The view from the Hogan: cultural epidemiology and the return to ethnography. AB - Alexander Leighton's seminal work has clearly demonstrated how ethnographic experience provides the rich cultural context in which epidemiological data are best interpreted. This article reviews recent trends in cultural epidemiology, and especially the emergence of the EMIC (Explanatory Model Interview Catalogue) as a quantitatively oriented tool designed to assess culture. It is suggested that such efforts do not reflect more recent trends in culture theory, and tend to view 'cultures' as easily bounded and largely homogenous units to facilitate the generation of quantitative data. It is argued that cultural epidemiologists should take a step back and ask, 'what is the culture in question here?' and 'how do I know if it is appropriate to place any given member of my sample into a specific cultural category?' before proceeding with any 'culturally appropriate' instrument. The answer to these questions begins with a return to ethnography as a means to elucidate and describe culture within the context in which it is being presented and studied. PMID- 16671394 TI - Environmental sensitivity: equivocal illness in the context of place. AB - This article presents a phenomenologically oriented description of the interaction of illness experience, social context, and place. This is used to explore an outbreak of environmental sensitivities in Nova Scotia, Canada. Environmental Sensitivity (ES) is a popular designation for bodily reactions to mundane environmental stimuli that are insignificant for most people. Mainstream medicine cannot support the popular models of this disease process and consequently illness experience is subject to ambiguity and contestation. As an 'equivocal illness', ES generates considerable social action around the nature, meaning and validity of suffering. Sense of place plays an important role in this process. In this case, the meanings that accrue to illness experience and that produce salient popular disease etiology are grounded in the experience and social construction of the Nova Scotian landscape over time. Shifting representations of place are reflected in illness experience and the meanings that arise around illness are emplaced in landscape. PMID- 16671395 TI - Speaking about culture in multiple voices. AB - As a mark of gratitude to Alexander Leighton, this article engages him in a dialogue, reopening several debates that were enriched by his research and reflections on ethics, the 'aesthetic dimension' of the research enterprise, the processes that mediate between collective and individual variables, and his strong distrust of theory. The authors discuss some of the features of Leighton's perspective that they have retained and transformed in their own work on community responses to chronic mental illness in rural Quebec, on the course of schizophrenia in India, and on culture and psychosis in clinical settings in Montreal. The challenge remains the one that Leighton identified: how can findings derived from different disciplines be made to co-ordinate? The authors argue that this question must be answered from within the centre of each discipline rather than from their frontiers or zones of interface. PMID- 16671396 TI - Beyond the 'new cross-cultural psychiatry': cultural biology, discursive psychology and the ironies of globalization. AB - The 'new cross-cultural psychiatry' heralded by Kleinman in 1977 promised a revitalized tradition that gave due respect to cultural difference and did not export psychiatric theories that were themselves culture bound. In the ensuing years, the view of culture within anthropology has continued to change, along with our understanding of the relationship of biological processes to cultural diversity, and the global political economic contexts in which mental health care is delivered. This article considers the implications of these new notions of culture, biology and the context of practice for theory in cultural psychiatry. The future of cultural psychiatry lies in advancing a broad perspective that: (a) is inherently multidisciplinary (involving psychiatric epidemiology, medical anthropology and sociology, cognitive science and social psychology), breaking down the nature/culture dichotomy with an integrative view of culture as a core feature of human biology, while remaining alert to cultural constructions of biological theory; (b) attends to psychological processes but understands these as not exclusively located within the individual but as including discursive processes that are fundamentally social; and (c) critically examines the interaction of both local and global systems of knowledge and power. Globalization has brought with it many ironies for cultural psychiatry: Transnational migrations have resulted in cultural hybridization at the same time as ethnicity has become more salient; the call for evidence-based medicine has been used to limit the impact of cultural research; and cultural psychiatry itself has been co-opted by pharmaceutical companies to inform marketing campaigns to promote conventional treatments for new populations. Cultural psychiatry must address these ironies to develop the self-critical awareness and flexibility needed to deliver humane care in shifting contexts. PMID- 16671397 TI - New strategies in drug discovery. AB - Gene identification followed by determination of the expression of genes in a given disease and understanding of the function of the gene products is central to the drug discovery process. The ability to associate a specific gene with a disease can be attributed primarily to the extraordinary progress that has been made in the areas of gene sequencing and information technologies. Selection and validation of novel molecular targets have become of great importance in light of the abundance of new potential therapeutic drug targets that have emerged from human gene sequencing. In response to this revolution within the pharmaceutical industry, the development of high-throughput methods in both biology and chemistry has been necessitated. Further, the successful translation of basic scientific discoveries into clinical experimental medicine and novel therapeutics is an increasing challenge. As such, a new paradigm for drug discovery has emerged. This process involves the integration of clinical, genetic, genomic, and molecular phenotype data partnered with cheminformatics. Central to this process, the data generated are managed, collated, and interpreted with the use of informatics. This review addresses the use of new technologies that have arisen to deal with this new paradigm. PMID- 16671398 TI - Basic microarray analysis: strategies for successful experiments. AB - Microarrays offer a powerful approach to the analysis of gene expression that can be used for a wide variety of experimental purposes. However, several types of microarray platforms are available. In addition, microarray experiments are expensive and generate complicated data sets that can be difficult to interpret. Success with microarray approaches requires a sound experimental design and a coordinated and appropriate use of statistical tools. Here, the advantages and pitfalls of utilizing microarrays are discussed, as are practical strategies to help novice users succeed with this method that can empower them with the ability to assay changes in gene expression at the whole-genome level. PMID- 16671399 TI - From microarray to biological networks: Analysis of gene expression profiles. AB - Powerful new methods, such as expression profiles using cDNA arrays, have been used to monitor changes in gene expression levels as a result of a variety of metabolic, xenobiotic, or pathogenic challenges. This potentially vast quantity of data enables, in principle, the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell. Here we present a general approach to developing dynamic models for analyzing time series of whole-genome expression. The parameters in the model show the influence of one gene expression level on another and are calculated using singular value decomposition as a means of inverting noisy and near-singular matrices. Correlative networks can then be generated based on these parameters with a simple threshold approach. We also demonstrate how dynamic models can be used in conjunction with cluster analysis to analyze microarray time series. Using the parameters from the dynamic model as a metric, two-way hierarchical clustering could be performed to visualize how influencing genes affect the expression levels of responding genes. Application of these approaches is demonstrated using gene expression data in yeast cell cycle. PMID- 16671400 TI - Microarray analysis in drug discovery and clinical applications. AB - DNA microarray analyzes genome-wide gene expression patterns and is used in many areas including drug discovery and clinical applications. This chapter summarizes some of these applications such as identification and validation of anti infective drug target, study mechanisms of drug action and drug metabolism, classification of different types of tumors, and use of molecular signatures for prediction of disease outcome. A step-by-step protocol is provided for sample preparation, sample labeling and purification, hybridization and washing, feature extraction, and data analysis. Important considerations for a successful experiment are also discussed with emphasis on drug discovery and clinical applications. Finally, a clinical study is presented as an example to illustrate how DNA microarray technology can be used to identify gene signatures, and to demonstrate the promise of DNA microarray as a clinical tool. PMID- 16671401 TI - Ontology-driven approaches to analyzing data in functional genomics. AB - Ontologies are fundamental knowledge representations that provide not only standards for annotating and indexing biological information, but also the basis for implementing functional classification and interpretation models. This chapter discusses the application of gene ontology (GO) for predictive tasks in functional genomics. It focuses on the problem of analyzing functional patterns associated with gene products. This chapter is divided into two main parts. The first part overviews GO and its applications for the development of functional classification models. The second part presents two methods for the characterization of genomic information using GO. It discusses methods for measuring functional similarity of gene products, and a tool for supporting gene expression clustering analysis and validation. PMID- 16671402 TI - Gene evolution and drug discovery. AB - Mutation and selection are the principle forces governing gene and protein sequence. Mutation is the major source of variation, and selection removes variation. Although many mutations are likely to be neutral with respect to natural selection, much of the extant sequence that is functionally important has experienced selective pressures in the past. By examining the history of DNA sequences, we can infer the functional importance of particular residues and the selective pressures that have influenced their evolution. In this chapter, we review the most interesting approaches for inferring the evolutionary history of DNA and protein sequences and indicate how these analyses can be useful in the drug discovery process. PMID- 16671403 TI - Standardization of microarray and pharmacogenomics data. AB - This chapter provides a bottom-up perspective on bioinformatics data standards, beginning with a historical perspective on biochemical nomenclature standards. Various file format standards were soon developed to convey increasingly complex and voluminous data that nomenclature alone could not effectively organize without additional structure and annotation. As areas of biochemistry and molecular biology have become more integral to the practice of modern medicine, broader data representation models have been created, from corepresentation of genomic and clinical data as a framework for drug research and discovery to the modeling of genotyping and pharmacogenomic therapy within the broader process of the delivery of health care. PMID- 16671404 TI - Clinical applications of bioinformatics, genomics, and pharmacogenomics. AB - Elucidation of the entire human genomic sequence is one of the greatest achievements of science. Understanding the functional role of 30,000 human genes and more than 2 million polymorphisms was possible through a multidisciplinary approach using micro-arrays and bioinformatics. Polymorphisms, variations in DNA sequences, occur in 1% of the population, and a vast majority of them are single nucleotide polymorphisms. Genotype analysis has identified genes important in thrombosis, cardiac defects, and risk of cardiac disease. Many of the genes show a significant correlation with polymorphisms and the incidence of coronary artery disease and heart failure. In this chapter, the application of current state-of the-art genomic analysis to a variety of these disorders is reviewed. PMID- 16671405 TI - Protein interactions probed with mass spectrometry. AB - Understanding the interactions of proteins with other proteins and/or with drug molecules is essential for understanding the progression of diseases. In this chapter, we present several methods utilizing mass spectrometry (MS) for the analysis of protein-protein, protein-drug, and protein-metal interactions. We describe the analysis of protein interactions with hydrogen exchange MS methods. Hydrogen exchange methods can be used to analyze conformational changes on binding, to estimate dissociation constants, and to locate the sites of interaction/binding between binding partners. We also discuss more direct MS methods, including the analysis of metal ion complexation with proteins. PMID- 16671406 TI - Discovering new drug-targeting sites on flexible multidomain protein kinases: Combining segmental isotopic and site-directed spin labeling for nuclear magnetic resonance detection of interfacial clefts. AB - A novel structure-based approach to study the structure and dynamics of flexible multi-domain monomeric protein kinases, which otherwise do not yield diffraction quality crystals, is described. A combination of segmental 15N-isotopic labeling of a regulatory domain with site-directed paramagnetic nitroxide spin labeling of the kinase domain is employed. Nuclear magnetic resonance studies of the enhancement of amide proton relaxation rates of the 15N-isotopically labeled regulatory domain caused by insertion of the paramagnetic nitroxide spin label on the kinase domain provide long-range distance restraints for determination of both the average positional structure and the relative flexibility exhibited between the two contiguous domains. Clefts and crevices detected around the dynamic domain-domain interface provide new targeting sites for tethered-based extension of current small-molecule lead compounds to produce more potent and selective pharmaceutical agents. PMID- 16671407 TI - Nuclear magnetic resonance-based screening methods for drug discovery. AB - Nuclear magnetic resonance (NMR) techniques are widely used in the drug discovery process. The primary feature exploited in these investigations is the large difference in mass between drugs and receptors (usually proteins) and the effect that this has on the rotational or translational correlation times for drugs bound to their targets. Many NMR parameters, such as the diffusion coefficient, spin diffusion, nuclear Overhauser enhancement, and transverse and longitudinal relaxation times, are strong functions of either the overall tumbling or translation of molecules in solution. This has led to the development of a wide variety of NMR techniques applicable to the elucidation of protein and nucleic acid structure in solution, the screening of drug candidates for binding to a target of choice, and the study of the conformational changes that occur in a target on drug binding. High-throughput screening by NMR methods has recently received a boost from the introduction of sophisticated computational techniques for reducing the time needed for the acquistion of the primary NMR data for multidimensional studies. PMID- 16671408 TI - Receptor-binding sites: bioinformatic approaches. AB - It is increasingly clear that both transient and long-lasting interactions between biomacromolecules and their molecular partners are the most fundamental of all biological mechanisms and lie at the conceptual heart of protein function. In particular, the protein-binding site is the most fascinating and important mechanistic arbiter of protein function. In this review, I examine the nature of protein-binding sites found in both ligand-binding receptors and substrate binding enzymes. I highlight two important concepts underlying the identification and analysis of binding sites. The first is based on knowledge: when one knows the location of a binding site in one protein, one can "inherit" the site from one protein to another. The second approach involves the a priori prediction of a binding site from a sequence or a structure. The full and complete analysis of binding sites will necessarily involve the full range of informatic techniques ranging from sequence-based bioinformatic analysis through structural bioinformatics to computational chemistry and molecular physics. Integration of both diverse experimental and diverse theoretical approaches is thus a mandatory requirement in the evaluation of binding sites and the binding events that occur within them. PMID- 16671409 TI - In silico protein design: fitting sequence onto structure. AB - In the last 10 yr, efforts have begun to combine the goals and approaches of computational molecular design and protein sequence analysis to provide tools for the rational mutagenesis and functional modification of proteins. These approaches use analysis of the three-dimensional structure of a protein to guide the selection of appropriate amino acid sequences to create desired properties or functions. The convergence of low-cost, high-speed computers, a tremendous increase in protein structure information, and a growing understanding of the forces that control protein structure has resulted in dramatic advances in the ability to control protein function and structure and to create the first truly artificial proteins. Various academic software packages have been developed for in silico protein design. The methods for selecting the protein structure, defining the portion to be designed, and choosing the input parameters for the software are described in this chapter. PMID- 16671410 TI - Chemical database preparation for compound acquisition or virtual screening. AB - Virtual and high-throughput screening are time-saving techniques that have been successfully applied to identify novel chemotypes in biologically active molecules. Both methods require the ability to aptly handle large numbers of chemicals prior to an experiment or acquisition. We describe a step-by-step preparation procedure for handling large collections of existing or virtual compounds prior to virtual screening or acquisition. PMID- 16671411 TI - Bioinformatics platform development: from gene to lead compound. AB - In the past 10 yr, the field of bioinformatics has been characterized by the mapping of many genomes. These efforts have stimulated explosive development of novel bioinformatics and experimental approaches to predict the functions and metabolic role of the new proteins. The main application of the work is to search, validate, and prioritize new targets for designing a new generation of drugs. Modern computer and experimental methods for discovery of new lead compounds have also expanded and integrated into the process referred to as rational drug design. They are directed to accelerate and optimize the drug discovery process using experimental and virtual (computer-aided drug discovery) methods. Recently, these methods and approaches have merged into a "from gene to lead" platform that includes the processes from new target discovery through obtaining highly effective lead compounds. This chapter describes the strategies as employed by the "From Gene to Lead" platform, including the major computer and experimental approaches and their interrelationship. The latter part of the chapter contains some examples of the steps required for implementing this platform. PMID- 16671412 TI - Surgical management of early massive edema caused by cerebral contusion in head trauma patients. AB - Early massive edema caused by severe cerebral contusion results in elevation of intracranial pressure (ICP) and clinical deterioration within 24-72 hours post trauma. Previous studies indicate that cells in the central area of the contusion undergo shrinkage, disintegration, and homogenization, whereas cellular swelling is predominant in the peripheral area, suggesting that early massive edema is attributable to high osmolality within necrotic brain tissue and may generate an osmotic potential across central and peripheral areas. We analyzed the effects of surgical excision of necrotic brain tissue in 182 patients with cerebral contusion registered with Japan Neurotrauma Data Bank; 121 patients (66%; Group I) were treated conservatively, and 61 (34%; Group II) were treated surgically. Most Group II cases (90%) underwent complete excision of necrotic brain tissue and evacuation of clots. Group I demonstrated higher mortality at 6 months post trauma compared to Group II (48%) vs. 23%; p = 0.0001; n = 182). Striking differences were observed in patients scoring 9 or more on Glasgow Coma Scale at admission (56% vs. 17%); p = 0.017; n = 45) and demonstrated "talk-and deteriorate" (64% vs. 22%: p = 0.026; n = 29), supporting our hypothesis that early massive edema is caused by cerebral contusion accompanied by necrotic brain tissue, indicating that surgical excision of necrotic brain tissue provides satisfactory control of progressive elevation in ICP and clinical deterioration in many cases. PMID- 16671413 TI - BrainIT: a trans-national head injury monitoring research network. AB - BACKGROUND: Studies of therapeutic interventions and management strategies on head injured patients are difficult to undertake. BrainIT provides validated data for analysis available to centers that contribute data to allow post-hoc analysis and hypothesis testing. METHODS: Both physiological and intensive care management data are collected. Patient identification is eliminated prior to transfer of data to a central database in Glasgow. Requests for missing/ ambiguous data are sent back to the local center. Country coordinating centers provide advice, training, and assistance to centers and manage the data validation process. RESULTS: Currently 30 centers participate in the group. Data collection started in January 2004 and 242 patients have been recruited. Data validation tools were developed to ensure data accuracy and all analysis must be undertaken on validated data. CONCLUSION: BrainIT is an open, collaborative network that has been established with primary objectives of i) creating a core data set of information, ii) standardizing the collection methodology, iii) providing data collection tools, iv) creating and populating a data base for future analysis, and v) establishing data validation methodologies. Improved standards for multi center data collection should permit the more accurate analysis of monitoring and management studies in head injured patients. PMID- 16671414 TI - Decompressive craniectomy in traumatic brain injury: outcome following protocol driven therapy. AB - Although decompressive craniectomy following traumatic brain injury is an option in patients with raised intracranial pressure (ICP) refractory to medical measures, its effect on clinical outcome remains unclear. The aim of this study was to evaluate the outcome of patients undergoing this procedure as part of protocol-driven therapy between 2000-2003. This was an observational study combining case note analysis and follow-up. Outcome was assessed at an interval of at least 6 months following injury using the Glasgow Outcome Scale (GOS) score and the SF-36 quality of life questionnaire. Forty-nine patients underwent decompressive craniectomy for raised and refractory ICP (41 [83.7%] bilateral craniectomy and 8 [16.3%] unilateral). Using the Glasgow Coma Scale (GCS), the presenting head injury grade was severe (GCS 3-8) in 40 (81.6%) patients, moderate (GCS 9-12) in 8 (16.3%) patients, and initially mild (GCS 13-15) in 1 (2.0%) patient. At follow-up, 30 (61.2%) patients had a favorable outcome (good recovery or moderate disability), 10 (20.48) remained severely disabled, and 9 (18.4%) died. No patients were left in a vegetative state. Overall the results demonstrated that decompressive craniectomy, when applied as part of protocol driven therapy, yields a satisfactory rate of favorable outcome. Formal prospective randomized studies of decompressive craniectomy are now indicated. PMID- 16671416 TI - Cerebral hemisphere asymmetry in cerebrovascular regulation in ventilated traumatic brain injury. AB - Disturbances in cerebrovascular regulation in the form of diminished cerebral vasoreactivity (CVR) to carbon dioxide and an altered pressure autoregulatory response (PAR) are common after traumatic brain injury (TBI) and correlate with clinical outcome. Daily assessment of the state of cerebrovascular regulation may assist in the clinical management of TBI patients. This study examined 20 ventilated TBI patients. We employed blood flow velocity (BFV) measurement using transcranial Doppler ultrasonography to assess the impact of injury type (focal and diffuse) on cerebral hemisphere asymmetry in cerebrovascular regulation and to examine whether impairment in CVR and PAR correlate with clinical outcomes. Significant hemisphere asymmetries were found in BFV and PAR. Impairment in CVR was associated with unfavorable outcomes and bilateral CVR impairment predicted mortality. PMID- 16671415 TI - Decompressive craniectomy in traumatic brain injury: the randomized multicenter RESCUEicp study (www.RESCUEicp.com). AB - The RESCUEicp (Randomized Evaluation of Surgery with Craniectomy for Uncontrollable Elevation of intracranial pressure) study has been established to determine whether decompressive craniectomy has a role in the management of patients with traumatic brain injury and raised intracranial pressure that does not respond to initial treatment measures. We describe the concept of decompressive craniectomy in traumatic brain injury and the rationale and protocol of the RESCUEicp study. PMID- 16671417 TI - Traumatic brain edema in diffuse and focal injury: cellular or vasogenic? AB - The objective of this study was to confirm the nature of the edema, cellular or vasogenic, in traumatic brain injury in head-injured patients using magnetic resonance imaging techniques. Diffusion-weighted imaging methods were quantified by calculating the apparent diffusion coefficients (ADC). Brain water and cerebral blood flow (CBF) were also measured using magnetic resonance and stable Xenon CT techniques. After obtaining informed consent, 45 severely injured patients rated 8 or less on Glasgow Coma Scale (32 diffuse injury, 13 focal injury) and 8 normal volunteers were entered into the study. We observed that in regions of edema, the ADC was reduced, signifying a predominantly cellular edema. The ADC values in diffuse injured patients without swelling were close to normal and averaged 0.89 +/- 0.08. This was not surprising, as ICP values for these patients were low. In contrast, in patients with significant brain swelling ADC values were reduced and averaged 0.74 +/- 0.05 (p < 0.0001), consistent with a predominantly cellular edema. We also found that the CBF in these regions was well above ischemic threshold at time of study. Taking these findings in concert, it is concluded that the predominant form of edema responsible for brain swelling and raised ICP is cellular in nature. PMID- 16671418 TI - CT prediction of contusion evolution after closed head injury: the role of pericontusional edema. AB - BACKGROUND: Cerebral contusions have a 51% incidence of evolution in the first hours after injury. Evolution is associated with clinical deterioration and is the reason for ICP monitoring or surgical intervention. We sought to define CT features that predict cerebral contusion evolution. METHODS: Patients treated for cerebral contusion who had 2 CT scans within 24 hours after injury were evaluated (n = 21). CT scans were analyzed for area of contusion, hemorrhagic components, and edema. Increase (%) in contusion size was recorded. Contusion evolution was defined as > 5% size increase. Ratios of hemorrhagic components to surrounding edema were calculated. RESULTS: Ten patients (47.6%) showed contusion evolution and 11 (52.4%) did not. Age, sex ratio, or injury severity between the 2 groups did not differ. Eight of 10 patients with evolving contusions had minimal or no perilesional edema on first CT; only 2 of 11 nonevolution patients had perilesional edema (p < 0.005). Mean ratio of area of surrounding edema to area of hemorrhagic products on first CT was 0.770 in evolution group versus 2.22 in non-evolution group (p = 0.055). CONCLUSIONS: A higher proportion of patients without contusion evolution had perilesional edema present on first CT scan. The absence of pericontusional edema on early CT may be a useful marker to predict contusion evolution. PMID- 16671419 TI - Organ dysfunction assessment score for severe head injury patients during brain hypothermia. AB - The purpose of this study was to evaluate the utility of a novel organ dysfunction assessment score developed for patients with severe traumatic brain injury during therapeutic brain hypothermia. The Brain Hypothermia Organ Dysfunction Assessment (BHODA) score is calculated through the combined assessment of 6 indices: central nervous system (CNS) function, respiratory function, cardiovascular function, hepatosplanchnic circulation, coagulation, and metabolism. The CNS, hepatosplanchnic circulation, and metabolic indices were based on measurements of cerebral perfusion pressure, gastric tonometry, and blood glucose, respectively. Thirty-nine patients with severe closed head injuries (scores of 3 to 8 on the Glasgow Coma Scale) were enrolled. Seven patients (18%) died during hospitalization. Outcome was favorable in 20 patients and unfavorable in 19. The BHODA score proved useful in describing sequences of complications during therapeutic brain hypothermia. A total maximum BHODA score of more than 13 points corresponded to a mortality of 70%. In a multivariate model, the total maximum BHODA score was independently associated with neurological outcome (odds ratio for unfavorable neurological outcome, 2.590: 95% confidence interval, 1.260, 5.327). In conclusion, the BHODA score can help assess multiple organ dysfunction/failure during therapeutic hypothermia and may be useful for predicting outcome. PMID- 16671420 TI - Importance of cerebral perfusion pressure management using cerebrospinal drainage in severe traumatic brain injury. AB - OBJECTIVE: To evaluate hemodynamics in patients with severe traumatic brain injury (TBI) after cerebral perfusion pressure (CPP) management using cerebrospinal fluid (CSF) drainage. METHODS: Twenty-six patients with TBI (Glasgow Coma Score = 8 or less) were investigated. Mean arterial blood pressure, CPP, cardiac index (CI), systemic vascular resistance index (SVRI), and central venous pressure were measured. The patients were divided into 2 groups after craniotomy: the intraparenchymal ICP (IP-ICP) monitoring group (n = 14) and ventricular ICP (V-ICP) monitoring group (n = 12). Patient hemodynamics were investigated on the second hospital day to identify differences. Measurements indicated a target CPP above 70 mmHg and a central venous pressure of 8 10 mmHg in both groups. Mannitol administration (IP-ICP group) or CSF drainage (V-ICP group) was performed whenever the CPP remained below 70 mmHg. RESULTS: High SVRI and low CI (p < 0.05) were observed in the IP-ICP group. The V-ICP group exhibited a reduction in the total fluid infusion volume of crystalloid (p < 0.01) and a reduction in the frequency of hypotensive episodes after the mannitol infusion. CONCLUSIONS: CPP management using CSF drainage decreases the total infusion volume of crystalloid and may reduce the risk of aggravated brain edema after excess fluid resuscitation. PMID- 16671421 TI - Acute hemispheric swelling associated with thin subdural hematomas: pathophysiology of repetitive head injury in sports. AB - INTRODUCTION: The most common head injury in sports is concussion, and repeated concussions occurring within a short period occasionally can be fatal. Acute subdural hematoma is the most common severe head injury and can be associated with severe neurologic disability and death in sports. We investigated severe brain damage resulting from repetitive head injury in sports, and evaluated the pathophysiology of sports-related repetitive injury. METHODS: We reviewed the literature containing detailed descriptions of repetitive severe sports-related head injury. In total, 18 cases were analyzed with regard to age, gender, type of sports, symptoms before second injury, and pathology of brain CT scans. RESULTS: The majority of cases involved young males aged 16 to 23 years old, who sustained a second head injury before symptoms from the first head injury had resolved. Ten of 15 cases did not suffer loss of consciousness at insult. Eight cases were confirmed on brain CT scans after the second injury, and all 8 cases revealed brain swelling associated with a thin subdural hematoma. CONCLUSIONS: Second impact syndrome is thought to occur because of loss of autoregulation of cerebral blood flow, leading to vascular engorgement, increased intracranial pressure, and eventual herniation. Our investigation suggests that the existence of subdural hematoma is a major cause of brain swelling following sports-related, repetitive head injury. PMID- 16671422 TI - Rewarming following accidental hypothermia in patients with acute subdural hematoma: case report. AB - A 57-year-old man was admitted to the Emergency and Critical Care Department with accidental hypothermia (31.5 degrees C) after resuscitation from cardiopulmonary arrest (CPA). Brain CT revealed an acute subdural hematoma. Active core rewarming to 33 degrees C was performed using an intravenous infusion of warm crystalloid. The patient underwent craniotomy soon after admission, with bladder temperature maintained at 33 to 34 degrees C throughout the surgery. Therapeutic hypothermia (34 degrees C) was continued for 2 days, followed by gradual rewarming. After rehabilitation, the patient was able to continue daily life with assistance. Traumatic brain injury (TBI) following CPA is associated with extremely unfavorable outcomes. Very few patients with acute subdural hematomas presenting with accidental hypothermia and CPA have been reported to recover. No suitable strategies have been clearly established for the rewarming performed following accidental hypothermia in patients with TBI. Our experience with this patient suggests that therapeutic hypothermia might improve the outcome in some patients with severe brain injury. It also appears that the method used for rewarming might play an important role in the therapy for TBI with accidental hypothermia. PMID- 16671423 TI - Clinical characteristics of postoperative contralateral intracranial hematoma after traumatic brain injury. AB - OBJECTIVES: To investigate the clinical characteristics of contralateral intracranial hematoma (ICH) after traumatic brain injury. METHODS: The subjects included 149 patients with traumatic ICH treated by hematoma evacuation. The patients were retrospectively divided into a bilateral ICH (B-ICH) group and unilateral ICH (U-ICH) group after craniotomy using brain CT scans for comparison of the following parameters: complicated expanded brain bulk from the cranial window, hypotension during craniotomy, and outcome. RESULTS: Post-craniotomy brain CT scans revealed U-ICH in 106 patients and B-ICH in 43 patients. Average Glasgow Coma Scale on arrival did not differ between the groups, but a higher proportion of patients in the B-ICH group deteriorated after admission (p = 0.02). The B-ICH patients also exhibited a significantly higher rate of expanded brain bulk from the cranial window (p < 0.05). No significant difference was observed between the groups with hypotension during craniotomy. The B-ICH group exhibited a lower rate of favorable outcome (p < 0.05) and higher mortality (p < 0.05). CONCLUSION: The B-ICH patients had a worse outcome than the U-ICH patients. Contralateral ICH was difficult to forecast based on pre- and intraoperative clinical conditions. Subdural hematoma or contusional ICH was frequently observed as a contralateral ICH. PMID- 16671424 TI - Diagnostic impact of the spectrum of ischemic cerebral blood flow thresholds in sedated subarachnoid hemorrhage patients. AB - OBJECTIVE: Ischemia is the main cause of secondary damage in subarachnoid hemorrhage (SAH). Cerebral blood flow (CBF) measurement is useful to detect critical values. We analyzed the diagnostic impact of CBF ischemic thresholds to predict a new low attenuation area on computed tomography (CT) due to failure of large vessel perfusion. METHODS: We analyzed 48 xenon CT (Xe-CT) studies from 10 patients with SAH. CBF measurements were obtained by means of Xe-CT and cortical regions of interest (ROls). The ROIs which appeared in a hypoattenuation area were recorded. Cortical CBF was tested for specificity and sensitivity as a predictor of hypoattenuation by means of a receiver operating characteristic curve. RESULTS: Mean age was 58 (SD +/- 12.4) years. The median Fisher score and Hunt and Hess scale were 2 and 3, respectively. The area under the receiver operating characteristic curve was 0.912 (CI 0.896 to 0.926). The cut-off value for best accuracy was 6 mL/ 100 g/min, with a likelihood ratio of 37. CONCLUSION: The present study suggests a threshold of 6 mL/100 g/ min as a predictor of a new low attenuation area. However, each clinician should choose the most useful threshold according to pre-test probability and the cost/effectiveness ratio of the applied therapies. PMID- 16671425 TI - Pharmacological brain cooling with indomethacin in acute hemorrhagic stroke: antiinflammatory cytokines and antioxidative effects. AB - We evaluated the effects of a novel pharmacological brain cooling (PBC) method with indomethacin (IND), a nonselective cyclooxygenase inhibitor, without the use of cooling blankets in patients with hemorrhagic stroke. Forty-six patients with hemorrhagic stroke (subarachnoid hemorrhage; n = 35, intracerebral hemorrhage; n = 11) were enrolled in this study. Brain temperature was measured directly with a temperature sensor. Patients were cooled by administering transrectal IND (100 mg) and a modified nasopharyngeal cooling method (positive selective brain cooling) initially. Brain temperature was controlled with IND 6 mg/kg/day for 14 days. Cerebrospinal fluid concentrations of interleukin-1beta (CSF IL-1beta) and serum bilirubin levels were measured at 1, 2, 4, and 7 days. The incidence of complicating symptomatic vasospasm after subarachnoid hemorrhage was lower than in non-PBC patients. CSF IL-1beta and serum bilirubin levels were suppressed in treated patients. IND has several beneficial effects on damaged brain tissues (anticytokine, free radical scavenger, antiprostaglandin effects, etc.) and prevents initial and secondary brain damage. PBC treatment for hemorrhagic stroke in patients appears to yield favorable results by acting as an antiinflammatory cytokine and reducing oxidative stress. PMID- 16671426 TI - The significance of crossovers after randomization in the STICH trial. AB - INTRODUCTION: Of all forms of stroke, spontaneous intracerebral haemorrhage (ICH) causes the highest morbidity and mortality. The Surgical Trial in Intracerebral Haemorrhage (STICH) found no difference in outcomes between patients randomized to surgical or conservative treatment. PATIENTS AND METHODS: Of 530 patients randomized to initial conservative treatment, 140 crossed over to surgery. This study examines the variables associated with crossover. RESULTS: Dominant features of the crossover group were: male, (p = 0.04), right-sided clot (p = 0.03), lobar clot (p = 0.003), clot volume (median 64 mL for crossovers vs. 38 mL for others, p < 0.00001), midline shift (median 6 mm for crossovers vs. 3 mm for others, p < 0.00001), superficial clot (median 1.3 mm for crossovers vs. 11.5 mm for others, p < 0.00001), and randomization within 12 hours of ictus (p < 0.0005). Thalamic location (p = 0.002) was under-represented. Intraventricular haemorrhage, hydrocephalus, and focal deficits were not associated with crossover. Craniotomy was the method of evacuation in 85% of crossover patients. CONCLUSIONS: Crossover to surgery was more likely when ICH had these features: Right side, lobar location, superficial, large volume, big shift, and early randomization. Crossovers formed a worse prognostic group compared to non crossovers. Surgery did not affect trial results, which were analyzed by intention-to-treat. PMID- 16671427 TI - Intraventricular hemorrhage and hydrocephalus after spontaneous intracerebral hemorrhage: results from the STICH trial. AB - INTRODUCTION: Intraventricular hemorrhage (IVH), either independent of or as an extension of intracranial bleed, is thought to carry a grave prognosis. Although the effect of IVH on outcome in patients with subarachnoid hemorrhage has been extensively reviewed in the literature, reports of spontaneous intracerebral hemorrhage (ICH) in similar situations have been infrequent. The association of hydrocephalus in such situations and its influence on outcome is also uncertain. PATIENTS AND METHODS: As a sub-analysis of data obtained through the international Surgical Trial in Intracerebral Hemorrhage (STICH), the impact of IVH, with or without the presence of hydrocephalus, on outcome in patients with spontaneous ICH was analyzed. CT scans of randomized patients were examined for IVH and/or hydrocephalus. Other characteristics of hematoma were evaluated to see if they influenced outcome, as defined by the STICH protocol. RESULTS: Favorable outcomes were more frequent when IVH was absent (31.4% vs. 15.1%; p < 0.00001). The presence of hydrocephalus lowered the likelihood of favorable outcome still further to 11.5% (p = 0.031). In patients with IVH, early surgical intervention had a more favorable outcome (17.8%) compared to initial conservative management (12.4%) (p = 0.141). CONCLUSION: The presence of IVH and hydrocephalus are independent predictors of poor outcome in spontaneous ICH. Early surgery is of some benefit in those with IVH. PMID- 16671428 TI - Changes in coagulative and fibrinolytic activities in patients with intracranial hemorrhage. AB - OBJECTIVE: To investigate whether any changes occur in the coagulative/fibrinolytic cascade in patients with subarachnoid hemorrhage (SAH) or hypertensive intracerebral hemorrhage (HICH). DESIGN AND METHODS: Subjects included 143 patients with intracranial hemorrhage (SAH, n = 50; HICH, n = 82; ROSC-SAH [return of spontaneous circulation after cardiopulmonary arrest due to SAH], n = 11). Coagulative and fibrinolytic factors were measured in blood samples taken on admission. RESULTS: The prothrombin fragment 1+2 level was significantly higher (p < 0.005) in SAH patients than in HICH patients. The fibrinolytic factors (plasmin alpha 2-plasmin inhibitor complex, D-dimer, or fibrinogen degradation products) in SAH and ROSC-SAH were both significantly higher than those in HICH, but the significance of difference was stronger in the case of ROSC-SAH (p < 0.05). DISCUSSION: Both coagulative and fibrinolytic activities were altered after the onset of SAH. These results demonstrate that the coagulative/fibrinolytic cascade might be activated via different mechanisms in different types of stroke. It remains unclear, however, whether a significant alteration of the fibrinolytic cascade in patients with ROSC-SAH might be a nonspecific phenomenon attributable to the reperfusion after collapse. PMID- 16671429 TI - The effect of hematoma removal for reducing the development of brain edema in cases of putaminal hemorrhage. AB - INTRODUCTION: Surgical intervention in putaminal hemorrhage has been a controversial issue. The aim of this research is to evaluate the benefits of surgery for reducing the development of brain edema. MATERIALS AND METHODS: Sixteen cases of putaminal hemorrhage were examined. Eight patients were treated conservatively (C group), and the other 8 patients were treated surgically (S group). Head CT scans were performed on the day of onset (day 0) in C group or performed just after surgery (day 0) in S group, and performed again once per period on days 1-7, 8-14, and 15-21. The volume of the mass including hematoma and edema (H + E) was measured using CT scans and the (H + E)/H0 ratios were calculated (H0; hematoma volume on day 0). The (H + E)/H0 ratios for each period were compared statistically between the 2 groups using a t-test. RESULTS: The mean values of(H + E)/H0 ratios at each period were 2.19, 2.63, 2.53 in C group, and 1.29, 1.29, 0.66 in S group. The values in S group were significantly lower as compared with C group in every period (p < 0.01, < 0.05, < 0.01). CONCLUSIONS: Hematoma volume reduction by surgery reduced the development of brain edema. PMID- 16671430 TI - Spontaneous intracerebral hemorrhage in humans: hematoma enlargement, clot lysis, and brain edema. AB - Early hematoma enlargement and delayed clot lysis contribute to brain injury after intracerebral hemorrhage (ICH). We investigated hematoma growth, clot lysis, and brain edema formation in patients with spontaneous ICH. A total of 17 spontaneous ICH patients who received regular medication were chosen for this study. All patients had their first CT scan within 5 hours of onset of symptoms (day 0). The patients then underwent second, third, and fourth CT scans at 1, 3, and 10 days later. Hematoma size and absolute and relative brain edema volumes were measured. Hematoma enlargement was defined as a > 33% increase in volume. Relative brain edema volume = absolute brain edema volume/hematoma size. Hematoma enlargement occurred in 4 of the 17 ICH patients (24%) within the first 24 hours. The hematoma sizes were reduced significantly at day 10 (p < 0.05) because of clot lysis. However, both absolute and relative brain edema increased gradually with time (p < 0.01). These results suggest that delayed brain edema following ICH may result from hematoma lysis. This study also shows that early hematoma enlargement occurs in Chinese patients with ICH. Reducing early hematoma growth and limiting clot lysis-induced brain toxicity could be potential therapies for ICH. PMID- 16671431 TI - Evaluation of acute perihematomal regional apparent diffusion coefficient abnormalities by diffusion-weighted imaging. AB - In this study, we investigated 40 patients (18 male, 22 female; mean age = 64.5 +/- 11.0; GCS = 9 to 14) with acute supratentorial spontaneous intracerebral hemorrhage (SICH) at admission by using a 1-tesla magnetic resonance imaging (MRI) unit equipped for single-shot echo-planar spin-echo isotropic diffusion weighted imaging (DWI) sequences. All DWI studies were obtained within 48 hours after symptom onset. Regional apparent diffusion coefficient (rADC) values were measured in 3 different regions of interest (ROIs) drawn freehand on the T2 weighted images at b 0 s/mm2 on every section in which hematoma was visible: 1) the perihematomal hyperintense area; 2) 1 cm of normal appearing brain tissue surrounding the perilesional hyperintense rim; 3) an area mirroring the region including the clot and perihematomal hyperintense area placed in the contralateral hemisphere. rADC mean values were higher in perihematomal hyperintense and in contralateral than in normal appearing areas (p < 0.001), with increased rADC mean levels in all regions examined. Our findings show that rADC values indicative of vasogenic edema were present in the perihematomal area and in normal appearing brain tissue located both ipsilateral and contralateral to the hematoma, with lower levels in non-injured areas located in the T2 hyperintense rim around the clot. PMID- 16671432 TI - Reperfusion of low attenuation areas complicating subarachnoid hemorrhage. AB - Hypoattenuation areas shown on brain CT scans after subarachnoid hemorrhage (SAH) are believed to be associated with persistent ischemia. The aim of this study was to evaluate regional cerebral blood flow (rCBF) in hypoattenuation areas and its evolution over time by means of Xenon CT (Xe-CT). We enrolled 16 patients with SAH who developed a hypoattenuation area in the middle cerebral artery territory. Patients were studied at time zero (the first Xe-CT), within 24 to 96 hours, and 96 hours after the initial Xe-CT. We analyzed 19 hypoattenuation areas caused by vascular distortion, vasospasm, or post-surgical embolization in 48 Xe-CT studies. Areas of hypoattenuation were divided in 2 groups according to initial rCBF. In the first group (n = 15), rCBF was initially above 6 mL/100 gr/min but only 2 were still ischemic (rCBF < 18 mL/ 100 gr/min) 96 hours after the first Xe CT, while 7 (58%) were hyperemic. Conversely, in the second group with severe ischemia (rCBF < 6 mL/100 gr/min; n = 4) mean rCBF increased (p = 0.08) but still remained below the ischemic threshold. In severely ischemic lesions, rCBF reperfusion occurs but is probably marginally relevant. Conversely, in lesions not initially severely ischemic, residual CBF gradually improved and frequently became hyperemic. The functional recovery of these zones remains to be evaluated. PMID- 16671433 TI - Stroke in the young: relationship of active cocaine use with stroke mechanism and outcome. AB - BACKGROUND: Cocaine and other vasoactive substances are known causes of cerebrovascular disease. Ictus during drug intake adversely affects outcome. MATERIALS AND METHODS: A retrospective review revealed 42 patients with cocaine abuse and stroke. Aneurysmal bleed occurred in 15 patients; the rest had stroke. The outcome of stroke because of cocaine intoxication was analyzed. RESULTS: Mean age for stroke was 38 (+/- 8.5 SD) years; males out-numbered females (20 : 7) similar to the pattern seen in subarachnoid hemorrhage (SAH) following aneurysm rupture. Nine had intracerebral hematomas, 6 had SAH with intracerebral hemorrhage (ICH)/ infarct, I had transverse myelopathy. Transient ischemic attack was identified in 4. Carotid occlusion was found in 2, and slow-flow in the vertebrobasilar system in 1. Fifteen were known hypertensives. Cocaine was the principal substance in all patients; 7 used other substances including marijuana and heroin. Three patients had HIV, 3 had hepatitis, 2 had syphilis, and 1 had tuberculosis. Urinalysis was positive for cocaine metabolites in 15; 2 had late analysis. Nine had ICH or SAH with poor neurological status at admission and died. Cocaine intoxication correlated with fatal cerebrovascular accident (CVA) (p < 0.001) and poor Glasgow Outcome Score (GOS) (p < 0.001). CONCLUSION: Stroke and cocaine use correlated with fatal CVA and poor outcome. Prompt diagnostic intervention may reveal the incidence of CNS injury with cocaine abuse. PMID- 16671434 TI - Brain oxygen metabolism may relate to the temperature gradient between the jugular vein and pulmonary artery after cardiopulmonary resuscitation. AB - OBJECTIVE: A gradient between the jugular vein temperature and core body temperature has been reported in animal and clinical studies; however, the pathophysiological meaning of this phenomenon remains unclear. This study was conducted to identify the temperature gradient between the jugular vein and pulmonary artery in comatose patients after cardiopulmonary resuscitation. MATERIALS AND METHODS: The temperatures of the jugular vein and pulmonary artery were measured in 19 patients at 6 and 24 hours after cardiopulmonary resuscitation. Jugular venous blood saturation (SjO2; %) was also measured concomitantly. The patients were divided into 2 groups: high SjO2 (SjO2 > 75%: H group; n = 10) and normal SjO2 (SjO2 < or = 75%: N-group; n = 9). The temperature gradient was calculated by subtracting the temperature of the pulmonary artery from that of the jugular vein (jugular - pulmonary = dT degrees C). Statistical significance was defined as p < 0.05. RESULTS: dT was significantly lower in the H-group than in the N-group at 6 hours (0.120 +/- 0.011: mean +/- SD vs. 0.389 +/ 0.036: p = 0.0012) and 24 hours (0.090 +/- 0.005 vs. 0.256 +/- 0.030: p = 0.0136) after cardiopulmonary resuscitation. CONCLUSION: The temperature gradient between the jugular vein and pulmonary artery was significantly lower in patients with high SjO2 after cardiopulmonary resuscitation. This temperature gradient may be reflected in brain oxygen metabolism. PMID- 16671435 TI - Intracranial pressure monitoring: modeling cerebrovascular pressure transmission. AB - OBJECTIVES: To examine changes in cerebrovascular pressure transmission derived from arterial blood pressure (ABP) and intracranial pressure (ICP) recordings by autoregressive moving average modeling technique. METHODS: Digitized ICP and ABP recordings were obtained from patients with brain injury. Two groups were defined: Group A with 4 patients who demonstrated plateau waves, and Group B with 4 intracranial hypertensive, hypoperfused patients. For each 16.5 s interval, mean values of ICP, ABP, cerebral perfusion pressure (CPP), and corresponding highest modal frequency (HMF) of cerebrovascular pressure transmission were computed. RESULTS: Mean values of CPP and HMF of 56.2 mmHg and 2.0 Hz for Group A were significantly higher (p < 0.005) than corresponding mean values of 31.9 mmHg and 0.744 Hz for Group B. The mean value of the slope of the regression line between HMF and CPP for group A of -0.034 Hz/mmHg was significantly different (p < 0.025) than the mean value of 0.0077 Hz/mmHg for Group B. Computations of HMF, pressure reactivity, and correlation pressure reactivity index on continuous pressure recordings are illustrated. CONCLUSIONS: Values of HMF of cerebrovascular pressure transmission are inversely related to CPP when pressure regulation is thought to be intact, and directly related when regulation is likely lost. PMID- 16671436 TI - Use of ICM+ software for on-line analysis of intracranial and arterial pressures in head-injured patients. AB - OBJECTIVE: To summarize our experience from the first 2 years of use of the ICM+ software in our Neurocritical Care Unit (NCCU). MATERIALS AND METHODS: Ninety five head-injured patients (74 males, 21 females), average age 36 years, were managed in the NCCU. Intracranial pressure (ICP) was monitored using Codman intraparenchymal probes and arterial blood pressure (ABP) was measured from the radial artery. Signals were monitored by ICM+ software calculating mean values of ICP, ABP, cerebral perfusion pressure (CPP) and various indices describing pressure reactivity, compensation and vascular waveforms of ICP (pulse amplitude, respiratory, and slow waves), etc. RESULTS: Mean ICP was 17 mmHg, mean CPP was 73 mmHg. Seven patients showed permanent disturbance of cerebral autoregulation (mean pressure reactivity index above 0.3). Pressure reactivity index demonstrated significant U-shape relationship with CPP, suggesting loss of pressure reactivity at too low (CPP < 55 mmHg) and too high CPPs (CPP > 95 mmHg). Mean ICP was inversely correlated with respiratory rate (R = 0.46; p < 0.0001; reciprocal model). CONCLUSION: The new version of ICM+ software proved to be useful clinically in the NCCU. It allows continuous monitoring of pressure reactivity and exploratory analysis of factors implicating intracranial hypertension. PMID- 16671437 TI - Monitoring and interpretation of intracranial pressure after head injury. AB - OBJECTIVE: To investigate the relationships between long-term computer-assisted monitoring of intracranial pressure (ICP) and indices derived from its waveform versus outcome, age, and sex. MATERIALS AND METHODS: From 1992 to 2002, 429 sedated and ventilated head-injured patients were continuously monitored. ICP and arterial blood pressure (ABP) were recorded directly and stored in bedside computers. Additional calculated variables included: 1) Cerebral perfusion pressure (CPP) = ABP - ICP; 2) a PRx calculated as a moving correlation coefficient between slow waves (of periods from 20 seconds to 3 minutes) of ICP and ABP. RESULTS: Fatal outcome was associated with higher ICP (p < 0.000002), worse PRx (p < 0.0006), and lower CPP (p < 0.001). None of these parameters differentiated severely disabled patients from patients with a favorable outcome. Higher average ICP, lower CPP, worse outcome, and worse pressure reactivity were observed in females than in males (age-matched). Worse outcome, lower mean ICP, worse PRx, and higher CPP were significantly associated with the older age of patients. CONCLUSION: High ICP and low PRx are strongly associated with fatal outcome. There is a considerable heterogeneity amongst patients; optimization of care depends upon observing the time-trends for the individual patient. PMID- 16671438 TI - The temporal profile of edema formation differs between male and female rats following diffuse traumatic brain injury. AB - Although female hormones are known to influence edema formation following traumatic brain injury (TBI), no studies have actually compared the temporal profile of edema formation in both male and female rats following diffuse TBI. In this study, male, female, and female ovariectomized rats were injured using the 2 m impact acceleration model of diffuse TBI. The temporal profile of brain water content was assessed over 1 week post-trauma. Male animals demonstrated increased (p < 0.05) edema at 5 hours, 24 hours, 3 days, 4 days, and 5 days after TBI with a peak at 5 hours post-injury. This time point was associated with increased blood-brain barrier (BBB) permeability. In contrast, intact females showed increased levels of edema (p < 0.05) at 5 hours, 24 hours, 3 days, and 4 days post-TBI, with a peak at 24 hours. No BBB opening was present in intact females at 5 hours. Female animals demonstrated more edema than male animals at 24 hours, but less at 5 hours, 3 days, and 5 days. Ovariectomy produced an edema profile that was similar to that observed in males. The temporal profile of edema formation after TBI seems to depend on endogenous hormone levels, a difference which may have an influence on clinical management. PMID- 16671439 TI - The effect of intravenous fluid replacement on the response to mannitol in experimental cerebral edema: an analysis of intracranial pressure, serum osmolality, serum electrolytes, and brain water content. AB - Albino rabbits that had undergone a cryogenic insult over the left parieto occipital cortex were analyzed for serum osmolality, serum electrolytes, brain water content, and intracranial pressure (ICP) following either a baseline infusion of intravenous (i.v.) fluid (45 mL total) for 3 hours or above maintenance isotonic saline (73.5 +/- 12 mL or 90.5 +/- 1.5 mL) and mannitol therapy. The subgroups were compared amongst themselves and to sham-operated controls. Serum osmolality was elevated in the higher-dose mannitol subgroup compared with maintenance i.v. fluids subgroup (1 g/kg/h vs 1 g/kg/3 h; p < 0.05), accompanied by an insignificant reduction of serum sodium. A significant reduction in brain water in the injured left hemisphere was seen following high dose mannitol in the subgroup that received less i.v. (maintenance) fluids than the group that received above-maintenance i.v. fluids (p < 0.025). No reduction in brain water was seen in the subgroup that received above-maintenance i.v. fluids (non-treated groups). Reduction of ICP was not found in the lower mannitol dose group. We conclude that the ability of mannitol to reduce cerebral edema is related to the total amount of i.v. fluid replacement. This implies that the amount of i.v. crystalloid fluid that is administered to patients with cerebral edema and raised ICP requiring mannitol for control needs to be carefully monitored. PMID- 16671440 TI - Matrix metalloproteinase-9 is associated with blood-brain barrier opening and brain edema formation after cortical contusion in rats. AB - Matrix metalloproteinases (MMPs) are associated with blood-brain opening and may be involved in the pathophysiology of acute brain injury. Previous research demonstrated that knockout mice deficient in MMP-9 subjected to transient focal cerebral ischemia had reduced blood-brain barrier (BBB) disruption and attenuated cerebral infarction. In this study, we examined MMP-9 up-regulation, BBB disruption, and brain edema formation after cortical impact injury in rats. Cortical contusion was induced by controlled cortical impact. Animals were sacrificed at intervals after injury. MMP up-regulation was assessed by gelatin zymography, and BBB integrity was evaluated using Evans blue dye with a spectrophotometric assay. Brain water content was measured by comparing wet and dry weights of each hemisphere as an indicator of brain edema. Zymograms showed elevated MMP-9 as early as at 3 hours after injury, reaching a maximum at 18 hours. Peak levels of BBB disruption occurred 6 hours after injury. Brain edema became progressively more severe, peaking 24 hours after injury. Compared to control group, treatment with MMP-inhibitor GM6001 significantly reduced BBB disruption 6 hours and brain water content (85.9 +/- 0.5% vs. 82.6 +/- 0.3%; p < 0.05) 24 hours after injury. These findings suggest that MMP-9 may contribute to BBB disturbance and subsequent brain edema after traumatic brain injury. PMID- 16671441 TI - Delayed precursor cell marker response in hippocampus following cold injury induced brain edema. AB - The purpose of this study was to examine the possibility of neuronal remodeling and repair after cold injury-induced brain edema using immunoassays of nestin, 3CB2, and TUC-4. Male ddN strain mice were subjected to cold-induced cortical injury. Animals were divided into the following 6 groups: 1) 1-day after injury, 2) 1-week after injury, 3) 2-weeks after injury, 4) 1-month after injury, 5) sham, and 6) normal controls. Brain water content measurement, Western blot analysis, histological examination, and neurobehavioral examination were performed. Brain water content was significantly increased in the ipsilateral cortex at 1-day after injury. At 1-day and 1-week after injury, immunoreactivity of nestin, 3CB2, and TUC-4 were absent. Nestin was expressed in 3CB2-positive astrocytes at 1-month after injury, and nestin expression with TUC-4 was present in the hippocampal cell layer. Neurobehavioral function of the 1-month after injury group was significantly improved compared with function 1-day after injury. These results suggest that delayed precursor cell marker expression in glia and neuron-like cells might be part of adaptation to the injury. Although brain injury causes brain edema and neuronal death, there is the possibility of remodeling. PMID- 16671442 TI - Granulocyte colony-stimulating factor does not affect contusion size, brain edema or cerebrospinal fluid glutamate concentrations in rats following controlled cortical impact. AB - INTRODUCTION: Granulocyte colony-stimulating factor (G-CSF) is an established treatment in the neutropenic host. Usage in head-injured patients at risk for infection may aggravate brain damage. In contrast, evidence of G-CSF neuroprotective effects has been reported in rodent models of focal cerebral ischemia. We investigated effects of G-CSF in acute focal traumatic brain injury (TBI) in rats. METHODS: Thirty-six male Sprague-Dawley rats were anesthetized with 1.2%) to 2.0% isoflurane and subjected to controlled cortical impact injury (CCII). Thirty minutes following CCII, either vehicle or G-CSF was administered intravenously. Animals were sacrificed 24 hours following CCII. Glutamate concentrations were determined in cisternal cerebrospinal fluid (CSF). Brain edema was assessed gravimetrically. Contusion size was estimated by 2,3,5 triphenyltetrazolium chloride staining and volumetric analysis. RESULTS: Dose dependent leukocytosis was induced by infusion of G-CSF. Physiological variables were unaffected. Water content of the traumatized hemisphere and CSF glutamate concentrations were unchanged by treatment. Contusion volume was similar in all groups. CONCLUSIONS: A single injection of G-CSF did not influence cortical contusion volume, brain edema, or glutamate concentrations in CSF determined 24 hours following CCII in rats. G-CSF, administered 30 minutes following experimental TBI, failed to exert neuroprotective effects. PMID- 16671443 TI - Unilateral spatial neglect and memory deficit associated with abnormal beta amyloid precursor protein accumulation after lateral fluid percussion injury in Mongolian gerbils. AB - The purpose of this study was to investigate cognitive/memory dysfunctions and the pathological process contributing to such dysfunction following moderate lateral fluid percussion injury (LFPI) in Mongolian gerbils. Mongolian gerbils were subjected to moderate LFPI (1.3 1.6 atm). During 7 days post-trauma, spatial cognitive and memory dysfunctions were evaluated by T-maze test (TMT). At 6 hours, 24 hours, and 7 days post-injury, animals were sacrificed and the brains were prepared for Kluver-Barrera staining and immunostaining of beta-amyloid precursor protein (APP). In LFPI animals, the spontaneous alternation rate in the TMT remained below the random alternation rate (< 50%) on all post-injury test days. These animals also showed a transient tendency to choose only the right arm (ipsilateral to the injury) in the TMT at 6 hours and 24 hours after injury. Significant accumulation of APP was found widespread in the ipsilateral hemisphere including directly injured cortex, subcortical white matter, and hippocampal formation at 6 hours and 24 hours post-injury, while on day 7, the increased immunoreactivity of APP subsided. These results suggest that the widespread axonal degeneration of the white matter might contribute to the unilateral spatial neglect and memory deficit in the acute stage after LFPI. PMID- 16671444 TI - Alteration of gap junction proteins (connexins) following lateral fluid percussion injury in rats. AB - Gap junctions are intercellular channels that mediate the cytoplasmic exchange of small hydrophilic molecules and are formed by a family of integral membrane proteins called connexins (Cxs). Cx43 is expressed predominantly in astrocytes, while Cx36 is expressed in neurons. In this study, we show alteration of Cx43 and Cx36 in the hippocampus after traumatic brain injury in rats. Adult male Sprague Dawley rats were subjected to lateral fluid percussion injury of moderate severity. Brain coronal sections were used for immunohistochemistry with Cx43 and Cx36 antibodies. Cx43 immunoreactivity was increased in reactive astrocytes in the damaged hippocampus 24 hours after injury, and persisted for 72 hours. On the other hand, Cx36 immunoreactivity increased in CA3 neurons 1 hour after injury, and decreased later. These results indicate that gap junctions might participate in the pathophysiological process after traumatic brain injury. PMID- 16671445 TI - Zinc protoporphyrin IX attenuates closed head injury-induced edema formation, blood-brain barrier disruption, and serotonin levels in the rat. AB - The role of heme oxygenase (HO) in closed head injury (CHI) was examined using a potent HO and guanylyl cyclase inhibitor, zinc protoporphyrin (Zn-PP) in the rat. Blood-brain barrier (BBB) permeability to Evans blue and radioiodine, edema formation, and plasma and brain levels of serotonin were measured in control, CHI, and Zn-PP-treated CHI rats. CHI was produced by an impact of 0.224 N on the right parietal bone by dropping 114.6 g weight from a height of 20 cm in anesthetized rats. This concussive injury resulted in edema formation and brain swelling 5 hours after insult that was most pronounced in the contralateral hemisphere. The whole brain was edematous and remained in a semi-fluid state. Microvascular permeability disturbances to protein tracers were prominent in both cerebral hemispheres and the underlying cerebral structures. Plasma and brain serotonin showed pronounced increases and correlated with edema formation. Pretreatment with Zn-PP (10 mg/ kg, i.p) 30 minutes before or after CHI attenuated edema formation, brain swelling, plasma and brain serotonin levels, and microvascular permeability at 5 hours. Brain edema, BBB permeability, and serotonin levels were not attenuated when the compound was administered 60 minutes post-CHI suggesting that HO is involved in cellular and molecular mechanisms of edema formation and BBB breakdown early after CHI. PMID- 16671446 TI - A novel neuroprotective compound FR901459 with dual inhibition of calcineurin and cyclophilins. AB - Brain ischemia leads to severe damage in the form of delayed neuronal cell death. In our study, we show that the marked neuroprotection of the new immunosuppressant FR901495 in forebrain ischemia is due not only to inhibition of calcineurin, but also to protection against mitochondrial damage caused by mitochondrial permeability transition pore formation through cyclophilin D, one of the prolyl cis/trans isomerase family members. These findings shed light on the clinical application and development of new drugs for the treatment of ischemic damage in the brain as well as in the heart and liver. PMID- 16671447 TI - Search for novel gene markers of traumatic brain injury by time differential microarray analysis. AB - Neuronal and glial cell death caused by axonal injury sometimes contributes to whole brain pathology after traumatic brain injury (TBI). We show that neuroprotection by 2 types of immunosuppressants, cyclosporin A (CsA) and tacrolimus (FK506), in a cryogenic brain injury model results from inhibition of calcineurin and protection from mitochondrial damage caused by formation of a mitochondrial permeability transition pore induced by cyclophilin D (CyPD), one of the prolyl cis/trans isomerase family members. We evaluated why CsA is neuroprotective by microarray analysis of gene expression in the cryogenic brain injury rat model. Analyses of expression patterns demonstrated that expression of over 14,000 genes changed between the groups with and without CsA treatment, and about 350 genes among them were extracted showing a significant difference. We learned that the differential expression of several gene targets showed specific patterns in a time-dependent manner. These results may help elucidate the mechanisms of neuronal cell death after TBI and the neuroprotective effects of CsA after TBI. PMID- 16671448 TI - Diffusion tensor feature in vasogenic brain edema in cats. AB - We investigated the correlation between the changes in diffusion tensor magnetic resonance imaging, regional water content, and tissue ultrastructure after vasogenic brain edema induced by cortical cold lesioning. In this cat model, E3 in the white matter was dominantly increased while fractional anisotropy (FA) was significantly decreased 8 hours after cortical cold lesioning. This finding indicates that water diffusion in the cortical white matter mainly increased perpendicularly rather than parallel to the direction of the nerve fibers. Additionally, in the area where edema is mild or moderate (tissues with water content of 65% to 75%), FA in the chronic phase was significantly lower than that in the acute phase. Histological examination demonstrated disordered arrangement of nerve fibers, highly dissociated neuronal fibers due to extracellular accumulation of protein rich-fluid, and enlarged interfiber spaces in the acute phase. PMID- 16671449 TI - Bolus tracer delivery measured by MRI confirms edema without blood-brain barrier permeability in diffuse traumatic brain injury. AB - INTRODUCTION: Previous studies have shown that edema formation after diffuse traumatic brain injury (TBI) with secondary insult is cytotoxic and not vasogenic. This assumption is based on observations of reduced apparent diffusion coefficient (ADC) and lack of significant accumulation of intravascular tracer in brain tissue. However, ADC reduction does not exclude vasogenic edema, and intravascular tracer can only accumulate when it reaches the tissue and is not perfusion limited. This study aims to confirm tissue delivery of intravascular tracer and lack of BBB opening during a phase of rapid brain swelling after diffuse TBI. METHODS: Rats were exposed to either TBI using the impact acceleration model combined with 30 minutes of hypoxia and hypotension, or sham injury. At 2 or 4 hours after injury, ADC and tissue water content were assessed using MRI. Gd-DTPA was given followed by a combination of rapid T2 imaging (60 seconds) and T1 imaging (30 minutes). Signal intensity changes were analyzed to determine a bolus effect (dynamic susceptibility contrast) and longer-term tissue accumulation of Gd-DTPA. RESULTS: Mean increase in cortical water content on the left was 0.8% at 2 hours, 2.1% at 4 hours; on the right it was 0.5% at 2 hours and 1.7% at 4 hours (p < 0.05). Mean ADC reduction over 4 hours was 0.04 x 10(-3) mm2/s on the left and 0.06 x 10(-3) mm2/s on the right. Kinetic analysis of signal intensity changes after Gd-DTPA showed no significant difference in inward transfer coefficient (BBB permeability) between sham injury and 2 or 4 hours post injury. T2 imaging showed consistent tissue delivery of a bolus of Gd-DTPA to the tissue at 2 and 4 hours post-injury, comparable to sham animals. CONCLUSIONS: Progressive cerebral edema formation after diffuse TBI occurred during ADC reduction and without continued BBB permeability. Tissue delivery of Gd-DTPA was confirmed, verifying that lack of tracer accumulation is due to an intact BBB and not to limited perfusion. PMID- 16671450 TI - Delayed profound local brain hypothermia markedly reduces interleukin-1beta gene expression and vasogenic edema development in a porcine model of intracerebral hemorrhage. AB - White matter (lobar) intracerebral hemorrhage (ICH) can cause edema-related deaths and life-long morbidity. In our porcine model, ICH induces oxidative stress, acute interstitial and delayed vasogenic edema, and up-regulates interleukin-1beta (IL-1beta), a proinflammatory cytokine-linked to blood-brain barrier (BBB) opening. In brain injury models, hypothermia reduces inflammatory cytokine production and protects the BBB. Clinically, however, hypothermia for stroke treatment using surface and systemic approaches can be challenging. We tested the hypothesis that an alternative approach, i.e., local brain cooling using the ChillerPad System, would reduce IL-1beta gene expression and vasogenic edema development even if initiated several hours after ICH. We infused autologous whole blood (3.0 mL) into the frontal hemispheric white matter of 20 kg pentobarbital-anesthetized pigs. At 3 hours post-ICH, we performed a craniotomy for epidural placement of the ChillerPad. Chilled saline was then circulated through the pad for 12 hours to induce profound local hypothermia (14 degrees C brain surface temperature). We froze brains in situ at 16 hours after ICH induction, sampled perihematomal white matter, extracted RNA, and performed real-time RT-PCR. Local brain cooling markedly reduced both IL-1beta RNA levels and vasogenic edema. These robust results support the potential for local brain cooling to protect the BBB and reduce injury after ICH. PMID- 16671451 TI - Alterations in intracerebral hemorrhage-induced brain injury in the iron deficient rat. AB - BACKGROUND: Iron contributes to brain edema and cellular toxicity after intracerebral hemorrhage (ICH). Knowledge regarding ICH in the context of iron deficiency anemia (IDA), a common nutritional disorder, is limited. OBJECTIVE: To determine the effect of IDA on brain and behavioral outcome after ICH in rats. METHODS: Six-week-old male rats (n = 75) were randomized to non-IDA or IDA groups. After 1 month of iron sufficient or deficient diets, 100 microl autologous blood was infused into the right basal ganglia (BG). Brains were assessed for iron concentration, regional water content, BG transferrin, and transferrin receptor concentrations after ICH. Recovery of upper extremity sensorimotor function was assessed. Brain and behavioral variables were compared by diet group. Significance was set at p < 0.05. RESULTS: Whole brain iron was decreased and water content was increased for IDA rats in injured cortex and BG at day 3 (p < 0.05) compared with non-IDA rats. Transferrin and transferrin receptor content were increased in injured BG for IDA compared to non-IDA in the first week after ICH (p < 0.05). IDA rats had greater left vibrissae-stimulated forelimb-placing deficits and forelimb-use asymmetry than non-IDA after ICH (p < 0.05). CONCLUSIONS: Brain iron status may be an important determinant of injury severity and recovery after ICH. PMID- 16671452 TI - Neuroprotective effect of hyperbaric oxygen in a rat model of subarachnoid hemorrhage. AB - Acute brain ischemia after subarachnoid hemorrhage (SAH) induces oxidative stress in brain tissues. Up-regulated NADPH oxidase (NOX), a major enzymatic source of superoxide anion in the brain, may contribute to early brain injury after SAH. We evaluated the effects of hyperbaric oxygen (HBO) on protein expression of gp91(phox) catalytic subunit of NOX, lipid peroxidation as a marker of oxidative stress, and on neurological and neuropathological outcomes after SAH. Twenty-nine male Sprague-Dawley rats (300 to 350 g) were randomly allocated to control (sham operation), SAH (endovascular perforation), and SAH treated with HBO groups (2.8 ATA for 2 hours, at 1 hour after SAH). Cerebral blood flow was measured using laser Doppler flowmetry. Rats were sacrificed after 24 hours and brain tissues collected for histology (Nissl staining and gp91 (phox) immunohistochemistry) and biochemistry. Mortality and neurological scores were evaluated. Neuronal injury associated with enhanced gp91 (phox) immunostaining was observed in the cerebral cortex after SAH. The lipid peroxidation product, malondialdehyde, accumulated in the ipsilateral cerebral cortex. HBO treatment reduced expression of NOX, diminished lipid peroxidation, and reduced neuronal damage. HBO caused a drop in mortality and ameliorated functional deficits. HBO-induced neuroprotection after SAH may involve down-regulation of NOX and a subsequent reduction in oxidative stress. PMID- 16671453 TI - Iron-induced oxidative brain injury after experimental intracerebral hemorrhage. AB - We investigated the occurrence of DNA damage in brain after intracerebral hemorrhage (ICH) and the role of iron in such injury. Male Sprague-Dawley rats received an infusion of 100 microL autologous whole blood or 30 microL FeCl2 into the right basal ganglia and were sacrificed 1, 3, or 7 days later. 8-hydroxyl-2' deoxyguanosine (8-OHdG) was analyzed by immunohistochemistry, while the number of apurinic/apyrimidinic abasic sites (AP sites) was also quantified. 8-OHdG and AP sites are two hallmarks of DNA oxidation. DNA damage was also examined using PANT and TUNEL labeling. Dinitrophenyl (DNP) was measured by Western blot to compare the time course of protein oxidative damage to that of DNA. DNA repair APE/Ref-1 and Ku-proteins were also measured by Western blot. Bipyridine, a ferrous iron chelator, was used to examine the role of iron in ICH-induced oxidative brain injury. An increase in 8-OHdG, AP sites, and DNP levels, and a decrease in APE/Ref-1 and Ku levels were observed. Abundant PANT-positive cells were also observed in the perihematomal area 3 days after ICH. Bipyridine attenuated ICH induced changes in PANT and DNP. These results suggest that iron-induced oxidation causes DNA damage in brain after ICH and that iron is a therapeutic target for ICH. PMID- 16671454 TI - Deferoxamine reduces CSF free iron levels following intracerebral hemorrhage. AB - Iron overload occurs in brain after intracerebral hemorrhage (ICH). Deferoxamine, an iron chelator, attenuates perihematomal edema and oxidative stress in brain after ICH. We investigated the effects of deferoxamine on cerebrospinal fluid (CSF) free iron and brain total iron following ICH. Rats received an infusion of 100-microL autologous whole blood into the right basal ganglia, then were treated with either deferoxamine (100 mg/kg, i.p., administered 2 hours after ICH and then at 12-hour intervals for up to 7 days) or vehicle. The rats were killed at different time points from 1 to 28 days for measurement of free and total iron. Behavioral tests were also performed. Free iron levels in normal rat CSF were very low (1.1 +/- 0.4 micromol). After ICH, CSF free iron levels were increased at all time points. Levels of brain total iron were also increased after ICH (p < 0.05). Deferoxamine given 2 hours after ICH reduced free iron in CSF at all time points. Deferoxamine also reduced ICH-induced neurological deficits (p < 0.05), but did not reduce total brain iron. In conclusion, CSF free iron levels increase after ICH and do not clear for at least 28 days. Deferoxamine reduces free iron levels and improves functional outcome in the rat, indicating that it may be a potential therapeutic agent for ICH patients. PMID- 16671455 TI - Up-regulation of brain ceruloplasmin in thrombin preconditioning. AB - Pretreatment with low-dose thrombin attenuates brain edema induced by iron or intracerebral hemorrhage (ICH). Ceruloplasmin is involved in iron metabolism by oxidizing ferrous iron to ferric iron. The present study examines whether thrombin modulates brain ceruloplasmin levels and whether exogenous ceruloplasmin reduces brain edema induced by ferrous iron in vivo. In the first set of experiments, rats received intracerebral infusion of saline or 1 U thrombin into the right basal ganglia. Rats were killed 1, 3, or 7 days later for Western blot analysis and RT-PCR analysis. In the second set of experiments, rats received either ferric iron, ferrous iron, or ferrous iron plus ceruloplasmin, then were killed 24 hours later for brain edema measurement. We found that ceruloplasmin protein levels in the ipsilateral basal ganglia increased on the first day after thrombin stimulation and peaked at day 3. Brain ceruloplasmin levels were higher after thrombin infusion than after saline injection. RT-PCR showed that brain ceruloplasmin mRNA levels were also up-regulated after thrombin injection (p < 0.05). We also found ipsilateral brain edema after intracerebral infusion of ferrous iron but not ferric iron at 24 hours. Co-injection of ferrous iron with ceruloplasmin reduced ferrous iron-induced brain edema (p < 0.05). Our results demonstrate that thrombin increases brain ceruloplasmin levels and exogenous ceruloplasmin reduces ferrous iron-induced brain edema, suggesting that ceruloplasmin up-regulation may contribute to thrombin-induced brain tolerance to ICH by limiting the injury caused by ferrous iron released from the hematoma. PMID- 16671456 TI - Hydrocephalus in a rat model of intraventricular hemorrhage. AB - The aims of the current study were 1) to establish an adult rat model of intraventricular hemorrhage (IVH) and post-hemorrhagic ventricular dilatation, and 2) to examine the role of alterations in cerebrospinal fluid (CSF) drainage and parenchymal injury in that dilatation. Rats underwent infusion of 200 microl of autologous blood over 15 minutes. The rats were used to measure hematoma mass, ventricular dilatation, and cortical mantle volume (with T2 imaging), resistance to CSF absorption, and brain edema (as a marker of brain injury). IVH resulted in ventricular dilatation peaking at day 2 but persisting for at least 8 weeks. Although there was an increased resistance to CSF absorption at 3 days, it returned to normal at day 7. Long-term ventricular dilatation was not associated with an alteration in cortical mantle volume, although there was evidence of cortical damage (edema). It is possible that initial ventricular distension (due to the hematoma and the impaired CSF drainage) in combination with periventricular white matter damage results in structural changes that prevent total recoil once the hematoma has resolved and CSF drainage is normalized, leading to long-term hydrocephalus. PMID- 16671457 TI - Early hemostatic therapy using recombinant factor VIIa in a collagenase-induced intracerebral hemorrhage model in rats. AB - Neurological deterioration during the first day after intracerebral hemorrhage (ICH) is associated with early hematoma growth in 18 to 38% of patients. While clinical studies continue to evaluate efficacy of activated recombinant factor VII (rFVlla) for reducing frequency of early hematoma growth, there have been no studies investigating the effect of rFVIIa on early hematoma growth. We used a collagenase-induced ICH model in the rat to evaluate the effects of rFVIIa on early hematoma growth. Two hours after injection of 0.14 U of type IV bacterial collagenase in 10 microL of saline into the basal ganglia, a small amount of blood collected in the striatum. The ICH gradually increased in size, extending posteriorly to the thalamus by 24 hours after injection. Intravenous administration of rFVIIa immediately after collagenase injection decreased average hematoma volume at 24 hours compared with vehicle-treated group (168.1 +/ 13.4 mm3 vs. 118.3 +/- 23.0 mm3, p < 0.01). There was also a decrease in total hemoglobin content in rats treated with rFVlla compared with vehicle-treated rats (optical density at 550 nm: 0.87 +/- 0.08 vs. 0.71 +/- 0.09, p < 0.05). There was no difference in cortical brain water content overlying the hematoma between the rFVlla- and vehicle-treated groups (81.4 +/- 0.7% vs. 81.7 +/- 0.4%). Our study indicates that treatment with rFVIla may be useful in reducing the frequency of early hematoma growth in ICH patients. PMID- 16671458 TI - Effects of endogenous and exogenous estrogen on intracerebral hemorrhage-induced brain damage in rats. AB - The present study examined differences in intracerebral hemorrhage (ICH)-induced brain injury in male and female rats, whether delayed administration of 17beta estradiol can reduce ICH-induced brain damage, and whether these effects are estrogen receptor (ER)-dependent. Male and female Sprague-Dawley rats received an infusion of 100-microL autologous whole blood into the right basal ganglia. The effects of 1beta-estradiol (5 mg/kg, i.p.) on ICH-induced brain injury were examined by measuring brain edema and neurological deficits 24 hours later. Heme oxygenase-1 (HO-1) was investigated by immuno-analysis. Brain edema was significantly less in female compared to male rats. The ER antagonist ICI182,780 exacerbated ICH-induced brain edema in female but not in male rats, suggesting that ER activation during ICH is protective in female rats. Administration of 17beta-estradiol to male (but not female) rats significantly reduced brain edema, neurological deficits, and ICH-induced increases in brain HO-1 levels when given 2 hours after ICH. This study showed that female rats have less ICH-induced injury than male rats. ER is involved in limiting ICH-induced injury in female rats. ICH-injury in male rats can be reduced by 17beta-estradiol. Since 17beta estradiol treatment was effective in male rats, it could be a potential therapeutic agent for ICH. PMID- 16671459 TI - Dopamine changes in a rat model of intracerebral hemorrhage. AB - Recent case reports suggest that dopamine (DA) replacement may reduce behavioral deficits resulting from hemorrhages along the nigrostriatal tract. In the rat model of intracerebral hemorrhage (ICH), behavioral deficits are first evident on day 1, with return to near control levels by day 28. The current study was conducted to determine if striatal dopamine alterations are correlated with behavioral deficits. Gamma-aminobutyric acid (GABA) levels were measured to determine selectivity. Striatal DA, DA metabolites, and GABA were determined at days 1, 3, 7, and 28 after ICH by high-pressure liquid chromatography with electrochemical detection. ICH resulted in significant increases above control in DA contralateral to the lesion (177 to 361% above control, days 1 to 28). There were also significant, but much less marked changes in GABA. In the ipsilateral striatum, significant DA increases also occurred (approximately 200%, at day 3 and approximately 275% day 28), while GABA alterations were not significant. These results indicate that the striatal DA system is selectively altered after ICH. Further studies will be needed to determine if regional dopamine alterations occur relative to the location of the hematoma. PMID- 16671460 TI - Intracerebral hemorrhage in complement C3-deficient mice. AB - The complement cascade is activated and contributes to brain damage after intracerebral hemorrhage (ICH). The present study investigated ICH-induced brain damage in complement C3-deficient mice. This study was divided into 2 parts. Male C3-deficient and C3-sufficient mice received an infusion of 30-microl autologous whole blood into the right basal ganglia. In the first part of our study, mice were killed 3 days later for brain water content measurement. Behavioral assessments including forelimb use asymmetry and corner turn tests were also preformed before and after ICH. In the second part of the study, brain heme oxygenase-1 (HO-1) was measured by Western blot analysis and immunohistochemistry 3 days after the infusion. We found that brain water content in the ipsilateral basal ganglia 3 days after ICH was less in C3-deficient mice compared to C3 sufficient mice (p < 0.05). The C3-deficient mice had reduced ICH-induced forelimb use asymmetry deficits compared with C3-sufficient mice (p < 0.05), although there was no significant difference in the corner turn test score. Western blot analysis showed that HO-1 contents were significantly lower in C3 deficient mice (day 3: 2024 +/- 560 vs. 5140 +/- 1151 pixels in the C3-sufficient mice, p < 0.05). We conclude that ICH causes less brain edema and behavioral deficits in complement C3-deficient mice. These results suggest that complement C3 is a key factor contributing to brain injury following ICH. PMID- 16671461 TI - Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage induced brain injury. AB - Hemoglobin degradation products result in brain injury after intracerebral hemorrhage (ICH). Recent studies found that intracerebral infusion of heme oxygenase inhibitors reduces hemoglobin- and ICH-induced brain edema in rats and pigs. The present study examined whether systemic use of zinc protoporphyrin (ZnPP), a heme oxygenase inhibitor, can attenuate brain edema, behavioral deficits, and brain atrophy following ICH. All rats had intracerebral infusion of 100-microL autologous blood. ZnPP (1 nmol/hour/rat) or vehicle was given immediately or 6 hours following ICH. ZnPP was delivered intraperitoneally up to 14 days through an osmotic mini-pump. Rats were killed at day 3 and day 28 after ICH for brain edema and brain atrophy measurements, respectively. Behavioral tests were performed. We found that ZnPP attenuated brain edema in animals sacrificed 3 days after ICH (p < 0.05). ZnPP also reduced ICH-induced caudate atrophy (p < 0.05) and ventricular enlargement (p < 0.05). In addition, ZnPP given immediately or 6 hours after ICH improved neurological deficits (p < 0.05). In conclusion, systemic zinc protoporphyrin treatment started at 0 or 6 hours after ICH reduced brain edema, neurological deficits, and brain atrophy after ICH. These results indicate that heme oxygenase may be a new target for ICH therapeutics. PMID- 16671462 TI - Restitution of ischemic injuries in penumbra of cerebral cortex after temporary ischemia. AB - We investigated, at both light and ultrastructural levels, the fate of swollen astrocytes and remodeling of neurites connected to disseminated, dying neurons in the ischemic neocortical penumbra. Specimens from left cerebral cortex were cut coronally at the infundibulum and observed by light and electron microscopy. We measured synapses and spines, and the thickness of neuritic trunks in the neuropil on electron microscopy photos. We also determined percent volume of axon terminals and spines by Weibel's point-counting method. Astrocytic swelling gradually subsided from day 4 after the ischemic insult, with increases in cytoplasmic glial fibrils and GFAP-positive astrocytes. Disseminated dying electron-dense neurons were fragmented by invading astrocytic cell processes and accumulated as granular pieces. The number of synapses and spines and total percent volume of axon terminals and spines decreased with an increasing sparsity of synaptic vesicles until day 4. One to 12 weeks after the ischemic insult, these values increased to or exceeded control values, and sprouting and increased synaptic vesicles were seen. Axons that had been attached to the dying neurons appeared to have shifted their connections to the spines and the neurites of the surviving neurons, increasing their thickness. Astrocytic restitution and neuronal remodeling processes started at 4 days continuing until 12 weeks after ischemic insult. PMID- 16671463 TI - Inhibition of Na+/H+ exchanger isoform 1 attenuates mitochondrial cytochrome C release in cortical neurons following in vitro ischemia. AB - Na+/H+ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism following intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to disruption of Na+ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Oxygen and glucose deprivation (OGD) for 3 hours followed by 21 hours of reoxygenation (REOX) led to 68 +/- 10% cell death. Inhibition of NHE1 with the potent inhibitor HOE 642 or genetic ablation of NHE1 reduced OGD-induced cell death by approximately 40% to 50% (p < 0.05). In NHE1 +/+ neurons, OGD/REOX triggered significant increases in Na+ and Ca(i)2+. Genetic ablation of NHE1 and HOE 642 treatment reduced the rise of Na(i)+ by approximately 40% to 50% and abolished the OGD/REOX-mediated Ca2+ accumulation. Moreover, mitochondrial cytochrome C release was significantly attenuated by inhibition of NHE1 activity. These results imply that NHE1 activity disrupts Na+ and Ca2+ homeostasis and contributes to ischemic neuronal damage. PMID- 16671464 TI - Controlled normothermia during ischemia is important for the induction of neuronal cell death after global ischemia in mouse. AB - A stable model of neuronal damage after ischemia is needed in mice to enable progression of transgenic strategies. We performed transient global ischemia induced by common carotid artery occlusions with and without maintaining normal rectal temperature (Trec) in order to determine the importance of body temperature control during ischemia. We measured brain temperature (Tb) during ischemia/reperfusion. Mice with normothermia (Trec within +/- 1 degrees C) had increased mortality and neuronal cell death in the CA1 region of hippocampus, which did not occur in hypothermic animals. If the Trec was kept within +/- 1 degrees C, the Tb decreased during ischemia. After reperfusion, Tb in the normothermia group developed hyperthermia, which reached > 40 degrees C and was > 2 degrees C higher than Trec. We suggest that tightly controlled normothermia and prevention of hypothermia (Trec) during ischemia are important factors in the development of a stable neuronal damage model in mice. PMID- 16671465 TI - Ex vivo measurement of brain tissue viscoelasticity in postischemic brain edema. AB - Knowledge of the biomechanical properties of postischemic brain tissue is important for understanding the mechanisms of postischemic secondary brain tissue injury. We describe the method and results of biomechanical property measurement in ex vivo postischemic brain tissue by applying an indentation method. Mongolian gerbils were subjected to a transient unilateral hemispheric ischemia. At day 1 after ischemia, multi-parametric MRI was performed, the brain was removed under anesthesia, sliced, and kept in a container with silicone oil for the measurement. A compression probe attached to a pressure transducer was inserted to a pre-determined depth at the regions of interest and maintained at a constant speed. A pressure relaxation curve was recorded for the calculation of elasticity modulus (E) and viscosity modulus (eta) according to Maxwell-Voigt's 3-element model. One day after ischemia, E and eta decreased to 78.7% and 73.1% of the control level, respectively. This decrease corresponded to a mild decrease in apparent diffusion coefficient (ADC) and magnetization transfer ratio, and an increase in T2 value. Tissue water content increased to 105.1% of control. Microvacuolation with demyelination and axonal disruption was evident in the postischemic brain tissue. PMID- 16671466 TI - Effect of dimethyl sulfoxide on blood-brain barrier integrity following middle cerebral artery occlusion in the rat. AB - Dimethyl sulfoxide (DMSO) is widely used as a solvent for other drugs, i.e., for the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) and the V1a receptor-antagonist SR49059, to reduce brain edema. We studied the effect of DMSO on blood-brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO) and the consequences on brain edema development. Male Sprague-Dawley rats were randomly assigned to sham procedure or infusion of 1% DMSO, PMA (230 microg/kg in 1% DMSO), or SR49059 (1 mg/kg in 1% DMSO) followed by MCAO (each group n = 10). After a 2-hour period of ischemia and 2 hours reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentration. BBB integrity was assessed by Evans blue extravasation. Statistical analysis was performed by ANOVA followed by a Tukey post hoc test. Low-dose DMSO treatment following MCAO significantly opened the BBB on the ischemic side (p < 0.037). PMA and SR49059 did not have any additional effect on BBB compromise compared to DMSO (p = 1.000, p < 0.957, respectively). We conclude that DMSO as a vehicle for drug administration may increase the drug concentration into the extracellular space, but since BBB permeability is increased, it may also provide an avenue for development of vasogenic edema. PMID- 16671467 TI - Increased substance P immunoreactivity and edema formation following reversible ischemic stroke. AB - Previous results from our laboratory have shown that neurogenic inflammation is associated with edema formation after traumatic brain injury (TBI). This neurogenic inflammation was characterized by increased substance P (SP) immunoreactivity and could be attenuated with administration of SP antagonists with a resultant decrease in edema formation. Few studies have examined whether neurogenic inflammation, as identified by increased SP immunoreactivity, occurs after stroke and its potential role in edema formation. The present study examines SP immunoreactivity and edema formation following stroke. Experimental stroke was induced in halothane anaesthetized male Sprague-Dawley rats using a reversible thread model of middle cerebral artery occlusion. Increased SP immunoreactivity at 24 hours relative to the non-infarcted hemisphere was observed in perivascular, neuronal, and glial tissue, and within the penumbra of the infarcted hemisphere. It was not as apparent in the infarct core. This increased SP immunoreactivity was associated with edema formation. We conclude that neurogenic inflammation, as reflected by increased SP immunoreactivity, occurs following experimental stroke, and that this may be associated with edema formation. As such, inhibition of neurogenic inflammation may represent a novel therapeutic target for the treatment of edema following reversible, ischemic stroke. PMID- 16671468 TI - Micro-blood-brain barrier openings and cytotoxic fragments of amyloid precursor protein accumulation in white matter after ischemic brain injury in long-lived rats. AB - Our study demonstrates that ischemia-reperfusion brain injury induces an increase in blood-brain barrier (BBB) permeability in the periventricular white matter. This chronic insufficiency of BBB may allow entry of neurotoxic fragments of amyloid precursor protein (APP) and other blood components such as platelets into the perineurovascular white matter tissue. These components may have secondary and chronic harmful effects on the ischemic myelin and axons and can intensify the phagocytic activity of microglial cells. Pathological accumulation of toxic fragments of APP in myelinated axons and oligodendrocytes appears after ischemic BBB injury and seem to be concomitant with, but independent of neuronal injury. It seems that ischemia-reperfusion disturbances may play important roles, both directly and indirectly, in the pathogenesis of white matter lesions. This pathology appears to have distribution similar to that of sporadic Alzheimer's disease. We noted micro-BBB openings in ischemic white matter lesions that probably would act as seeds of future Alzheimer's-type pathology. PMID- 16671469 TI - Time profile of eosinophilic neurons in the cortical layers and cortical atrophy. AB - Eosinophilic neurons (ENs) appear in the post-ischemic cortex; however, whether there are differences in the time profile for different cortical layers and the fate of the cortex with ENs is largely unknown. We examined the time profile of ENs in different cortical layers and evolution of cortical atrophy after transient cerebral ischemia in Mongolian gerbils. Unilateral forebrain ischemia was induced twice by 10-minute unilateral common carotid artery occlusions. Brains at 24 hours, 4 days, and 2, 4, and 16 weeks post-ischemia were prepared for morphometric analysis. Quantitative analysis of ENs in regions of interest in the rostral and caudal cortex showed the highest number of ENs at 4 days post ischemia in layers 3 and 6. Reduction in ENs after this peak was slower in layer 6 than in layer 3 in both rostral and caudal cortex, and this difference was significant in layer 6 of the caudal cortex. Infarcts with significant atrophy appeared in the rostral cortex. In the caudal cortex, only selective neuronal death with mild but distinct atrophy was observed. We observed a significant difference between cortical layers in the time profile of ENs in the post ischemic cortex. Selective neuronal death without infarction was sufficient to induce cortical atrophy after transient cerebral ischemia. PMID- 16671470 TI - Forebrain ischemia and the blood-cerebrospinal fluid barrier. AB - Although the effects of cerebral ischemia on the blood-brain barrier have been extensively studied, the effects on the blood-cerebrospinal fluid barrier (BCSFB) at the choroid plexuses have received much less attention. This paper reviews evidence on the effects of cerebral ischemia on the choroid plexus, particularly focusing on the degree of blood flow reduction required to damage the lateral ventricle choroid plexuses during transient forebrain ischemia, and whether disruption of the BCSFB might affect nearby tissues. Studies have shown that 2 common models of forebrain ischemia (4-vessel and 2-vessel with hypotension) cause damage to the lateral ventricle choroid plexus via necrosis and apoptosis. We have found that bilateral common carotid artery occlusion with hypotension causes an 87% reduction in lateral ventricle choroid plexus blood flow during ischemia and an approximate tripling of the permeability of the BCSFB to inulin after 6 hours of reperfusion. Interestingly, evidence suggests that this disruption of the BCSFB rather than disruption to the blood-brain barrier is the major cause of enhanced inulin entry into the hippocampus. The hippocampus undergoes selective delayed neuronal loss in that model of forebrain ischemia and the BCSFB disruption may participate in or modulate that delayed injury. PMID- 16671471 TI - Neurological dysfunctions versus apparent diffusion coefficient and T2 abnormality after transient focal cerebral ischemia in Mongolian gerbils. AB - We examined temporal profiles of neurological dysfunctions and compared them with apparent diffusion coefficient (ADC) and T2 changes in ischemic cortical regions after transient focal cerebral ischemia in Mongolian gerbils. Mongolian gerbils (n = 7) underwent right common carotid artery occlusion for 20 minutes. Asymmetric motor behavior and unilateral somatosensory dysfunction were quantified by the elevated body swing test and the bilateral asymmetry test at 0, 2, 3, and 8 days after ischemia. The results were compared to the ADC and T2 changes in the primary motor cortex and the somatosensory cortex. Transient motor dysfunction was observed at day 2 after ischemia. MRI revealed transient and mild ADC decrease without T2 increase at day 2 after ischemia in the primary motor cortex. Persistent somatosensory dysfunction was observed at 2, 3, and 8 days after ischemia, which corresponded to a moderate ADC decrease, and a mild T2 increase in the primary somatosensory cortex at days 2 and 3 after ischemia. Time profiles of neurological deficits concurred with ADC changes of the post-ischemic cortex responsible for the deficits. The post-ischemic lesions responsible for the neurological deficits were detectable by using ADC mapping in the acute phase after transient focal cerebral ischemia. PMID- 16671472 TI - Progressive expression of vascular endothelial growth factor (VEGF) and angiogenesis after chronic ischemic hypoperfusion in rat. AB - Cerebrovascular stenosis caused by arteriosclerosis induces failure of the cerebral circulation. Even if chronic cerebral hypoperfusion does not induce acute neuronal cell death, cerebral hypoperfusion may be a risk factor for neurodegenerative diseases. The purpose of this study was to determine if vasodilation, expression of VEGF, and neovascularization are homeostatic signs of cerebral circulation failure after permanent common carotid artery occlusion (CCAO) in the rat. Neuronal cell death in neocortex was observed 2 weeks after CCAO and gradually increased in a time-dependent manner. The diameter of capillaries and expression of VEGF also increased progressively after CCAO. Moreover, we observed unusual irregular angiogenic vasculature at 4 weeks. In conclusion, chronic hypoperfusion results in mechanisms to compensate for insufficiency in blood flow including vasodilation, VEGF expression, and neovascularization in the ischemic region. These results suggest that angiogenesis might be induced in adult brain through the support of growth factors and transplantation of vascular progenitor cells, and that neovascularization might be a therapeutic strategy for children and adults with diseases such as vascular dementia. PMID- 16671473 TI - Intracerebral administration of neuronal nitric oxide synthase antiserum attenuates traumatic brain injury-induced blood-brain barrier permeability, brain edema formation, and sensory motor disturbances in the rat. AB - The role of nitric oxide (NO) in traumatic brain injury (TBI)-induced sensory motor function and brain pathology was examined using intracerebral administration of neuronal nitric oxide synthase (nNOS) antiserum in a rat model. TBI was produced by a making a longitudinal incision into the right parietal cerebral cortex limited to the dorsal surface of the hippocampus. Focal TBI induces profound edematous swelling, extravasation of Evans blue dye, and up regulation of nNOS in the injured cerebral cortex and the underlying subcortical areas at 5 hours. The traumatized animals exhibited pronounced sensory motor deficit, as seen using Rota-Rod and grid-walking tests. Intracerebral administration of nNOS antiserum (1 : 20) 5 minutes and 1 hour after TBI significantly attenuated brain edema formation, Evans blue leakage, and nNOS expression in the injured cortex and the underlying subcortical regions. The nNOS antiserum-treated rats showed improved sensory motor functions. However, administration of nNOS antiserum 2 hours after TBI did not influence these parameters significantly. These novel observations suggest that NO participates in blood-brain barrier disruption, edema formation, and sensory motor disturbances in the early phase of TBI, and that nNOS antiserum has some potential therapeutic value requiring additional investigation. PMID- 16671474 TI - Effects of 2,4-dinitrophenol on ischemia-induced blood-brain barrier disruption. AB - This study examines the effect of 2,4-dinitrophenol (DNP), a mitochondrial uncoupling agent, during focal brain ischemia induced by middle cerebral artery (MCA) occlusion. Blood-brain barrier (BBB) disruption was assessed after 2 hours of occlusion with 2 hours of reperfusion or 4 hours of permanent occlusion by measurement of the influx rate constant (K(i)) for 3H-inulin in the MCA territory ipsi- and contralateral to the occlusion. Three experimental groups were examined: vehicle and 1 and 5 mg/kg DNP treated animals (given 30 minutes prior to occlusion). Four hours of permanent MCA occlusion only induced a modest increase in the K(i) for inulin in vehicle-treated animals (0.09 +/- 0.01 vs. 0.07 +/- 0.01 microL/g/min in contralateral tissue). Although 5 mg/kg DNP significantly increased this disruption (p < 0.01), this effect was relatively minor (0.14 +/- 0.02 microL/g/min). In contrast, DNP treatment in transient ischemia markedly increased barrier disruption. The ipsilateral K(i) for 3H inulin were 0.15 +/- 0.04, 0.37 +/- 0.06, and 0.79 +/- 0.17 microL/g/min in vehicle, 1 mg/kg DNP and 5 mg/kg DNP groups, respectively. DNP did not induce barrier disruption in the contralateral hemisphere. Thus, while there is evidence that DNP can be neuroprotective, it has adverse effects on the BBB during ischemia, particularly with reperfusion. Considering the importance of naturally- or therapeutically-induced reperfusion in limiting brain damage, this may limit the utility of DNP and mitochondrial uncouplers as therapeutic agents. PMID- 16671475 TI - Long-term cognitive and neuropsychological symptoms after global cerebral ischemia in Mongolian gerbils. AB - The objective of this study was to establish a rodent model of vascular dementia that showed long-term cognitive and neuropsychological deficits, and to correlate those behavioral deficits with the patterns of ischemic lesions, thus providing a platform for future testing of potential therapeutic agents. In Mongolian gerbils, either 5-minute single bilateral common carotid artery occlusion (SBCCAO) or repetitive bilateral common carotid artery occlusion (two 7-minute occlusions, RBCCAO) was induced, and the behavioral deficits were evaluated using 2 tests: a modified open-field test with an escape zone to evaluate changes in anxiety and locomotor activity, and a T-maze test to assess cognitive dysfunction. SBCCAO did not induce anxiety changes but caused transient locomotor hyperactivity and mild cognitive deficits. Only pyramidal neuronal death was found in the bilateral CA1 sector of the hippocampus following SBCCAO. In contrast, RBCCAO induced persistent locomotor hyperactivity, reduced anxiety, and caused severe cognitive deficits at 4 weeks post-ischemia. RBCCAO caused significant atrophy associated with diffuse selective neuronal death in the bilateral cerebral cortex and caudate nucleus, as well as the CA1 region. The repetitive ischemia model appears to be a potentially useful platform for the long-term analysis of cognitive and neuropsychological symptoms associated with vascular dementia. PMID- 16671476 TI - Protective effect of the V1a receptor antagonist SR49059 on brain edema formation following middle cerebral artery occlusion in the rat. AB - There exists no pharmacological treatment for fulminating brain edema. Since evidence indicates that brain aquaporin-4 (AQP4) water channels are modulated by vasopressin V1a receptors, we examined the edema-reducing properties of the selective V1a receptor antagonist, SR49059, following middle cerebral artery occlusion (MCAO). Male Sprague-Dawley rats were randomly assigned to sham procedure, vehicle, or SR49059 infusion at different dosages (each n = 6,480 microL/hr, 640 microL/hr, 720 microL/hr) and starting 60 minutes before or after MCAO. After a 2-hour period of ischemia and 2 hours of reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentration. Statistics were performed using an ANOVA followed by a Tukey post hoc analysis. SR049059 treatment reduced brain water content in the infarcted area given at 640 microL/hr (p = 0.036), 720 microL/hr 60 minutes before (p = 0.002) or 60 minutes after (p = 0.005) MCAO. The consecutive sodium shift into the brain was prevented (p = 0.001), while the potassium loss was inhibited only by pre-treatment (p = 0.003). These findings imply that in ischemia-induced brain edema, the selective V1a receptor-antagonist SR49059 inhibits brain edema and the subsequent sodium shift into brain. This substance offers a new avenue in brain edema treatment and prompts further study into AQP4 modulation. PMID- 16671477 TI - Topical application of dynorphin A (1-17) antibodies attenuates neuronal nitric oxide synthase up-regulation, edema formation, and cell injury following focal trauma to the rat spinal cord. AB - Previous investigations from our laboratory show that up-regulation of neuronal nitric oxide synthase (NOS) following spinal cord injury (SCI) is injurious to the cord. Antiserum to dynorphin A (1-17) induces marked neuroprotection in our model of SCI, indicating an interaction between dynorphin and NOS regulation. The present investigation was undertaken to find out whether topical application of dynorphin A (1-17) antiserum has some influence on neuronal NOS up-regulation in the traumatized spinal cord. SCI was produced in anesthetized animals by making a unilateral incision into the right dorsal horn of the T10-11 segments. The antiserum to dynorphin A (1-17) was applied (1 : 20, 20 microL in 10 seconds) 5 minutes after trauma over the injured spinal cord and the rats were allowed to survive 5 hours after SCI. Topical application of dynorphin A (1-17) antiserum significantly attenuated neuronal NOS up-regulation in the adjacent T9 and T12 segments. In the antiserum-treated group, spinal cord edema and cell injury were also less marked. These observations provide new evidence that the opioid active peptide dynorphin A may be involved in the mechanisms underlying NOS regulation in the spinal cord after injury, and confirms our hypothesis that up-regulation of neuronal NOS is injurious to the cord. PMID- 16671478 TI - Histamine receptors influence blood-spinal cord barrier permeability, edema formation, and spinal cord blood flow following trauma to the rat spinal cord. AB - The role of histamine in edema formation, blood-spinal cord barrier (BSCB) permeability, and spinal cord blood flow (SCBF) following spinal cord injury (SCI) was examined using modulation of histamine H1, H2, and H3 receptors in the rat. Focal trauma to the spinal cord at the T10-11 level significantly increased spinal cord edema formation, BSCB permeability to protein tracers and SCBF reduction in the T9 and T12 segments. Pretreatment with histamine H1 receptor antagonist mepyramine (1 mg, 5 mg, and 10 mg/kg, i.p.) did not attenuate spinal pathophysiology following SCI. Blockade of histamine H2 receptors with cimetidine or ranitidine (1 mg, 5 mg, or 10 mg/kg 30 minutes before injury) significantly reduced early pathophysiological events in a dose dependent manner. The effects of ranitidine were far superior to cimetidine in identical doses. Pretreatment with a histamine H3 receptor agonist alpha-methylhistamine (1 mg and 2 mg/kg/i.p.), that inhibits histamine synthesis and release in the CNS, thwarted edema formation, BSCB breakdown, and SCBF disturbances after SCI. The lowest dose of histamine H3 agonist was most effective. Blockade of histamine H3 receptors with thioperamide (1 mg, 5 mg/kg, i.p.) exacerbated spinal cord pathology. These observations suggest that stimulation of histamine H3 receptors and blockade of histamine H2 receptors is neuroprotective in SCI. PMID- 16671479 TI - Post-injury treatment with a new antioxidant compound H-290/51 attenuates spinal cord trauma-induced c-fos expression, motor dysfunction, edema formation, and cell injury in the rat. AB - The neuroprotective efficacy of post-injury treatment with the antioxidant compound H-290/51 (10, 30, and 60 minutes after trauma) on immediate early gene expression (c-fos), blood-spinal cord barrier (BSCB) permeability, edema formation, and motor dysfunction was examined in a rat model of spinal cord injury (SCI). SCI was produced by a longitudinal incision into the right dorsal horn of the T10-11 segment under Equithesin anesthesia. Focal SCI in control rats resulted in profound up-regulation of c-fos expression, BSCB dysfunction, edema formation, and cell damage in the adjacent T9 and T12 segments at 5 hours. Pronounced motor dysfunction was present at this time as assessed using the Tarlov scale and the inclined plane test. Treatment with H-290/51 (50 mg/kg, p.o.) 10 and 30 minutes after SCI (but not after 60 minutes) markedly attenuated c-fos expression and motor dysfunction. In these groups, BSCB permeability, edema formation, and cell injuries were mildly but significantly reduced. These observations suggest that (i) antioxidants are capable of attenuating cellular and molecular events following trauma, and (ii) have the capacity to induce neuroprotection and improve motor function if administered during the early phase of SCI, a novel finding. PMID- 16671481 TI - Chronic spinal nerve ligation induces microvascular permeability disturbances, astrocytic reaction, and structural changes in the rat spinal cord. AB - The possibility that a chronic nerve ligation impairs the spinal cord cellular microenvironment was examined using leakage of endogenous albumin, reaction of astrocytes, and structural changes in a rat model. Rats subjected to 8 weeks of unilateral L4/L5 nerve ligation (a model of neuropathic pain) showed leakage of albumin, up-regulation of glial fibrillary acidic protein (GFAP) immunoreaction, and abnormal cell reaction. Distortion and loss of nerve cells as well as general sponginess of the gray matter was clearly evident. Cell changes were present in both dorsal and ventral horns and were most marked on the ipsilateral side compared to the contralateral cord. Nerve cell and glial cell changes are normally present in the regions showing intense albumin immunoreactivity, indicating disruption of the blood-spinal cord barrier (BSCB). Our observations indicate that a chronic nerve lesion has the capacity to induce selective breakdown of the BSCB that could be responsible for activation of astrocytes and abnormal cell reaction. These findings enhance our understanding of the pathophysiology of neuropathic pain and/or other spinal cord disorders. PMID- 16671480 TI - Post-traumatic application of brain-derived neurotrophic factor and glia-derived neurotrophic factor on the rat spinal cord enhances neuroprotection and improves motor function. AB - We examined the potential efficacy of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) applied over traumatized spinal cord, alone or in combination, for attenuating motor dysfunction, blood-spinal cord barrier (BSCB) breakdown, edema formation, and cell injury in a rat model. Under Equithesin anesthesia, spinal cord injury (SCI) was performed by making a unilateral incision into the right dorsal horn of the T10-11 segment. The rats were allowed to survive 5 hours after trauma. The BDNF or GDNF was applied (0.1 to 1 microg/10 microl in phosphate buffer saline) 30, 60, or 90 minutes after SCI. Topical application of BDNF or GDNF 30 minutes after SCI in high concentration (0.5 microg and 1 microg) significantly improved motor function and reduced BSCB breakdown, edema formation, and cell injury at 5 hours. These beneficial effects of neurotrophins were markedly absent when administered separately either 60 or 90 minutes after injury. However, combined application of BDNF and GDNF at 60 or 90 minutes after SCI resulted in a significant reduction in motor dysfunction and spinal cord pathology. These novel observations suggest that neurotrophins in combination have potential therapeutic value for the treatment of SCI in clinical situations. PMID- 16671482 TI - Gravitational valves: relevant differences with different technical solutions to counteract hydrostatic pressure. AB - Two different technical principles of gravitational valves (G-valves) have been presented: counterbalancer and switcher G-valves. The objective of our prospective study was to look for clinically relevant differences between both. A total of 54 patients with normal-pressure hydrocephalus (NPH) were treated; 30 patients received an Aesculap-Miethke GA-Valve (GAV; counterbalancer), and in 24 patients an Aesculap-Miethke Dualswitch-Valve (DSV; switcher) was implanted. The opening pressure of the posture-independent valve was 5 cm H2O in both devices. The outcome was clearly better with the usage of the GAV than with the DSV. The frequency and severity of complications was pronounced in the DSV group. We recommend the Aesculap-Miethke-GAV valve with a low opening pressure in a posture independent valve for patients with NPH. PMID- 16671483 TI - Brain tissue water content in patients with idiopathic normal pressure hydrocephalus. AB - Relatively little is known regarding the water content of brain tissue in idiopathic normal-pressure hydrocephalus (NPH) patients. The objective of our study was to determine absolute water content non-invasively in hydrocephalic patients, particularly in the anterior and posterior ventricular horns and in the periventricular white matter. Ten patients who were diagnosed and treated for idiopathic NPH in our clinic were selected for study. Magnetic resonance imaging (MRI) techniques were used to obtain anatomical image slices for quantitative brain water measurements. Apparent diffusion coefficient measures were also extracted from regions of interest. To our knowledge, this is the first study to confirm that periventricular lucency seen on MRI represents increased water content in the extracellular space that is markedly elevated prior to shunting. PMID- 16671484 TI - Predictors of outcome in patients with normal-pressure hydrocephalus. AB - From 1982 until 2000 we examined 200 patients diagnosed with normal-pressure hydrocephalus (NPH) in a prospective study. From the patients who were surgically treated by a shunt implantation we could re-examine 155 (78%) at a mean time interval of 7 months after the operation. NPH differed in severity according to the results of the intrathecal infusion test in an early state NPH (without brain atrophy) and late state NPH (with brain atrophy). In our study, we focused on the possible predictors: patient age; length of disease; clinical signs including gait ataxia, dementia, and bladder incontinence; idiopathic vs. secondary origin; implanted valve type and the resistance of the valve to cerebrospinal fluid outflow. In 80 patients without cerebral atrophy and a short course of disease (< 1 year), a slight amount of dementia and an implanted Miethke Dualswitch-Valve were significant predictors for a positive postoperative outcome. The outflow resistance measured in the intrathecal infusion test showed only minimal relevance for outcome. Seventy-five patients with cerebral atrophy had a better outcome when dementia was not present, outflow resistance was above 20 mmHg/mL/min, the CSF tap-test was positive, and a Miethke Dualswitch-Valve was implanted. PMID- 16671485 TI - On the optimal opening pressure of hydrostatic valves in cases of idiopathic normal-pressure hydrocephalus: a prospective randomized study with 123 patients. AB - Does the opening pressure of hydrostatic shunts influence the clinical outcome for patients suffering from idiopathic normal-pressure hydrocephalus (NPH)? Between September 1997 and January 2003, 123 patients with idiopathic NPH were surgically treated by implanting a hydrostatic shunt at the Departments of Neurosurgery of the Unfallkrankenhaus Berlin and the University Homburg/Saar. As part of a prospective randomized study, all patients were examined preoperatively, postoperatively, and 1 year after the intervention. Forty-three percent of the patients showed a very good outcome, 25% good outcome, 20% fair outcome, and 12% poor outcome 1 year after the shunt implantation. Patients treated with an opening pressure rating of 50 mmH2O in the low-pressure stage of the gravitational valve showed a better outcome than those with an opening pressure of 100 or 130 mmH2O. According to present knowledge, hydrostatic shunts with an opening pressure of 50 mmH2O for the low-pressure stage are the best option for patients with idiopathic NPH. Due to the prompt switching function when the patient changes posture (lying down, standing, sitting, slanting etc.), the Miethke gravity-assisted valve (GAV) is more suitable in such cases than the Miethke Dual-Switch valve (DSV). PMID- 16671486 TI - Outcome predictors for normal-pressure hydrocephalus. AB - The objective of this prospective study was to find outcome predictors for better selection for treatment of normal-pressure hydrocephalus (NPH) patients. A total of 125 patients were evaluated and provided with a gravitational shunt. Cerebrospinal fluid hydrodynamics provided better predictive values if an algorithm to shunt all patients with a pressure/volume index of < 30 mL or resistance to outflow > 13 mmHg/mL x min was used. In general, outcome became worse with increasing anamnesis duration, worse preoperative clinical state, and increasing comorbidity. If one of these parameters was lower than a critical value, the shunt-responder rate was about 90% and the normally negative influence of older age was not seen. The well-known paradigm of a worse prognosis with NPH is not the result of the hydrocephalus etiology itself, but the consequence of a typical accumulation of negative outcome predictors as a consequence of the misinterpretation of normal aging and delayed adequate treatment. PMID- 16671487 TI - First clinical experiences in patients with idiopathic normal-pressure hydrocephalus with the adjustable gravity valve manufactured by Aesculap (proGAV(Aesculap)). AB - OBJECTIVE: Improved clinical outcomes after implantation of a low pressure valve in patients with idiopathic normal-pressure hydrocephalus is usually achieved at the expense of a higher overdrainage rate. Can an adjustable valve with a gravitational unit provide optimal results? METHOD: In a prospective clinical outcome study conducted in the Unfallkrankenhaus Berlin, 30 patients with idiopathic normal-pressure hydrocephalus were treated surgically between June 2004 and May 2005 with the valve combination described above, and reexamination 3 months or 6 months postoperatively. RESULTS: Clinical outcome correlates with opening pressure level of the valve. Controlled adjustment of the valve from 100 mmH2O to 70 mmH2O, and then to 50 mmH2O after 3 months, permits optimum adaptation of the brain to the implanted valve without overdrainage complications. CONCLUSIONS: Advantages of this programmable gravity valve include: 1) the absence of unintentional readjustment through external magnets, and 2) the possibility of controlling the valve setting using an accessory instrument without the need for x-ray monitoring. A significant disadvantage is adjusting the valve after implantation. From the clinical point of view, this new "proGAV(Aesculap)" valve is a necessary development in the right direction, but at the moment it is still beset with technical problems. PMID- 16671488 TI - Decompressive craniectomy for severe head injury in patients with major extracranial injuries. AB - Neurosurgical therapy aims to minimize secondary brain damage after a severe head injury. This includes the evacuation of intracranial space-occupying hematomas, the reduction of intracranial volumes, external ventricular drainage, and aggressive therapy in order to influence increased intracranial pressure (ICP) and decreased P(ti)O2. When conservative treatment fails, a decompressive craniectomy might be successful in lowering ICP. From September 1997 until December 2004, we operated on 836 patients with severe head injuries, of whom 117 patients (14%) were treated by means of a decompressive craniectomy. The prognosis after decompression depends on the clinical signs and symptoms at admission, patient age, and the existence of major extracranial injuries. Our guidelines for decompressive craniectomy after failure of conservative interventions and evacuation of space-occupying hematomas include: patient age below 50 years without multiple trauma, patient age below 30 years in the presence of major extracranial injuries, severe brain swelling on CT scan, exclusion of a primary brainstem lesion or injury, and intervention before irreversible brain stem damage. PMID- 16671490 TI - Is it possible to optimize treatment of patients with idiopathic normal pressure hydrocephalus by implanting an adjustable Medos Hakim valve in combination with a Miethke shunt assistant? AB - A better course of the disease after implantation of a low-pressure valve in patients with idiopathic normal pressure hydrocephalus normally comes at the cost of a distinctly higher rate of overdrainage. Can combining an adjustable valve with a gravity unit produce optimization of treatment results? In a prospective observation of the course of the disease, 18 patients with idiopathic normal pressure hydrocephalus were surgically treated with the aforementioned valve combination during the period January to June 2004 at the Unfallkrankenhaus Berlin and examined after 6 and 12 months. The course of the disease correlates with the opening pressure level of the valve. The controlled setting of the valve from 100 mmH2O to 70 mmH2O, then to 50 mmH2O after 3 months permits the brain to adapt optimally to the implanted valve without complications from overdrainage. In our view, combining an adjustable differential pressure valve with a gravity unit currently represents the optimal treatment variant for patients with idiopathic normal pressure hydrocephalus. In the future, the gravity valve should also be adjustable. PMID- 16671489 TI - Clinical outcome of patients with idiopathic normal pressure hydrocephalus three years after shunt implantation. AB - OBJECTIVE: To investigate the outcomes of surgical treatment of idiopathic normal pressure hydrocephalus (iNPH). PATIENTS AND METHODS: We prospectively investigated 51 patients treated for iNPH by insertion of a ventriculoperitoneal shunt with gravitational valve. RESULTS: The proportion of excellent, good, and satisfactory outcomes immediately following surgery was 80%; the same clinical outcome was later verified in 67% of patients on average of 34 months postoperatively. These results are similar to those reported in the literature. Ventricle volume decreased minimally during the course of treatment using Evans' index, which was concordant with recent literature and with current understanding of hydrocephalus treatment. CONCLUSIONS: A gravitational valve for treatment of iNPH is the logical implementation of current knowledge of the pathophysiology of this illness enabling us to solve the problem of cerebrospinal fluid drainage with the lowest possible opening pressure while simultaneously protecting from overdrainage. The use of programmable valves, in combination with a gravitational component, is the next evolutionary stage, potentially making revision operations unnecessary. PMID- 16671491 TI - Increased seizure duration in mice lacking aquaporin-4 water channels. AB - Aquaporins are intrinsic membrane proteins involved in water transport in fluid transporting tissues. In the brain, aquaporin-4 (AQP4) is expressed widely by glial cells, but its function is unclear. Extensive basic and clinical studies indicate that osmolarity affects seizure susceptibility, and in our previous studies we found that AQP4 -/- mice have an elevated seizure threshold in response to the chemoconvulsant pentylenetetrazol. In this study, we examined the seizure phenotype of AQP4 -/- mice in greater detail using in vivo electroencephalographic recording. AQP4 -/- mice were found to have dramatically longer stimulation-evoked seizures following hippocampal stimulation as well as a higher seizure threshold. These results implicate AQP4 in water and potassium regulation associated with neuronal activity and seizures. PMID- 16671492 TI - Modulation of AQP4 expression by the protein kinase C activator, phorbol myristate acetate, decreases ischemia-induced brain edema. AB - The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), is known to interact with aquaporin-4 (AQP4), a water-selective transporting protein abundant in astrocytes and ependymal cells, that has been found to decrease osmotically-induced swelling. The purpose of this study was to examine whether PMA given at different time points following focal ischemia induced by middle cerebral artery occlusion (MCAO) reduces brain edema by AQP4 modulation. Male Sprague-Dawley rats were randomly assigned to sham procedure, vehicle, or PMA infusion (230 microg/kg), starting either 60 minutes before, or 30 or 60 minutes after MCAO (each group n = 12). After a 2-hour period of ischemia and 2 hours of reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentrations. AQP4 expression was assessed by immunoblotting. Statistical analysis was performed by ANOVA followed by Tukey's post hoc test. PMA treatment significantly reduced brain water content concentration in the infarcted area when started before or 30 minutes post occlusion (p < 0.001, p = 0.022) and prevented the subsequent sodium shift (p < 0.05). Furthermore, PMA reduced ischemia-induced AQP4 up-regulation (p < 0.05). Attenuation of the ischemia-induced AQP4 up-regulation by PMA suggests that the reduction in brain edema formation following PMA treatment was at least in part mediated by AQP4 modulation. PMID- 16671493 TI - Astrocytes co-express aquaporin-1, -4, and vascular endothelial growth factor in brain edema tissue associated with brain contusion. AB - INTRODUCTION: Brain edema may be life threatening. The mechanisms underlying the development of traumatic brain edema are still unclear; however, mixed mechanisms including vasogenic, ischemic, and neurotoxic types of edema may be contributors. Recent studies indicate that astrocytes, aquaporins (AQPs; a protein family of water channels), and vascular endothelial growth factor (VEGF) may have important roles in the formation and resolution of brain edema. We studied the expression of AQPs and VEGF in the edematous brain. METHODS: We investigated the expression of AQP1, AQP4, and vascular endothelial growth factor (VEGF) in contusional brain tissue surgically obtained from 6 patients. Glial fibrillary acidic protein (GFAP) was also stained to detect astrocytes and to clarify the location of those proteins. The specimens received immunohistological staining and 3-color immunofluorescent staining, and were observed using confocal laser scanning microscopy. RESULTS: AQP1, AQP4, and VEGF were co-expressed in GFAP-positive astrocytes. AQP1 and AQP4 were expressed strongly in astrocytic end-feet. The astrocytes were located in the edematous tissue, and some cells surrounded cerebral capillaries. CONCLUSION: Our results suggest that AQP1, AQP4, and VEGF are induced in astrocytes located in and surrounding edematous tissue. Those astrocytes may regulate the water in- and out-flow in the injured tissue. PMID- 16671494 TI - Magnesium restores altered aquaporin-4 immunoreactivity following traumatic brain injury to a pre-injury state. AB - Magnesium reduces edema following traumatic brain injury (TBI), although the associated mechanisms are unknown. Recent studies suggest that edema formation after TBI may be related to alterations in aquaporin-4 (AQP4) channels. In this study, we characterize the effects of magnesium administration on AQP4 immunoreactivity following TBI. Male Sprague-Dawley rats were injured by impact acceleration diffuse TBI and a subgroup was administered 30 mg/kg magnesium sulphate 30 minutes after injury. Animals were fixed by perfusion 5 hours later, which corresponded to the time of maximum edema formation according to previous studies. One half of the brain was cut using a Vibratome and the other half blocked in paraffin wax. Wax and Vibratome sections were immunostained for detection of AQP4 by light and electron microscopy, respectively. In untreated animals, AQP4 immunoreactivity was increased in the subependymal inner glia limitans and the subpial outer glia limitans, and decreased in perivascular astrocytic processes in the cerebrum and brain stem. In contrast, animals treated with magnesium sulphate had AQP4 profiles similar to normal and sham control animals. We conclude that magnesium decreases brain edema formation after TBI, possibly by restoring the polarized state of astrocytes and by down-regulation of AQP4 channels in astrocytes. PMID- 16671495 TI - Positive selective brain cooling method: a novel, simple, and selective nasopharyngeal brain cooling method. AB - Brain damage is worsened by hyperthermia and prevented by hypothermia. Conventional hypothermia is a non-selective brain cooling method that employs cooling blankets to achieve surface cooling. This complicated method sometimes induces unfavorable systemic complications. We have developed a positive selective brain cooling (PSBC) method to control brain temperature quickly and safely following brain injury. Brain temperature was measured in patients with a ventriculostomy CAMINO catheter. A Foley balloon catheter was inserted to direct chilled air (8 to 12 L/min) into each side of the nasal cavity. The chilled air was exhaled through the oral cavity. In most patients, PSBC maintained normal brain temperature. This new technique provides quick induction of brain temperature control and does not require special facilities. PMID- 16671496 TI - Mechanism of neuroprotective effect induced by QingKaiLing as an adjuvant drug in rabbits with E. coli bacterial meningitis. AB - OBJECTIVE: To explore the neuroprotective effects and underlying mechanism of QingKaiLing (QKL) as an adjuvant treatment for bacterial meningitis. METHOD: E. coli bacterial meningitis rabbits were treated with antibiotics (ampicillin) alone or in combination with QKL. The number of leukocytes and the concentration of protein in the cerebrospinal fluid (CSF) of rabbits were determined at 0, 16, and 26 hours after treatment. Brain water, sodium, potassium, and calcium contents were determined at the 26-hour time point. The level of matrix metalloproteinase-9 in the brain was also determined by Western blot. RESULT: The average number of leukocytes and the concentration of protein in CSF of the QKL adjuvant treatment group were reduced compared with the ampicillin alone group. Brain water, sodium, and calcium contents were reduced in the QKL adjuvant treatment group. The level of MMP-9 in brain tissue was also reduced in the QKL adjuvant treatment group. CONCLUSION: QKL adjuvant treatment alleviates the aggravated inflammatory reaction and partially protects brain tissue from antibiotic-induced injury. The mechanism of this neuroprotective effect of QKL may be due to decreased levels of Ca2+ and MMP-9 in the brain. PMID- 16671497 TI - Acceleration of chemokine production from endothelial cells in response to lipopolysaccharide in hyperglycemic condition. AB - Chronic hyperglycemia is an established risk factor for endothelial damage. It remains unclear, however, whether brief hyperglycemic episodes after acute stress alter the function of vascular endothelial cells in response to endotoxin. We hypothesize that brief hyperglycemic episodes enhance the production of interleukin-8 (IL-8) after lipopolysaccharide (LPS) stimulation. METHODS: Human umbilical vein endothelial cells (HUVECs; 1 x 10(5) cells/mL, cells from subcultures 2-5, n = 6) were cultivated in various concentrations of glucose (200, 300, 400, and 500 mg/dL) with or without LPS stimulation (1 microg/mL) for 24 hours. After culture, IL-8 levels in the supernatant were measured using ELISA. RESULTS: HUVECs cultured at glucose concentrations of 300 and 400 mg/dL produced more (p < 0.01) IL-8 than control cells (200 mg/dL). HUVECs cultured at glucose concentrations of 300 and 400 mg/dL also produced more (p < 0.01) IL-8 than those cultured in the absence of LPS. CONCLUSIONS: Hyperglycemic conditions enhance IL-8 production by vascular endothelial cells, and this response is augmented by LPS. Infections may foster neutrophil accumulation at injury sites. These results suggest that it is important to manage even short-term increases in blood glucose after acute stress. PMID- 16671498 TI - Photodynamic therapy increases brain edema and intracranial pressure in a rabbit brain tumor model. AB - The objective of this study was to evaluate the effect of a single photodynamic therapy (PDT) on brain edema and intracranial pressure (ICP) in a rabbit model of brain tumor. A total of 57 adult New Zealand rabbits were assigned to 3 groups: the PDT group, the tumor group, and the tumor plus PDT group. Rabbits in the PDT group (n = 9) received PDT but no tumor implantation; rabbits in the tumor group (n = 18) received VX2 carcinoma implantation but no PDT; rabbits in the tumor plus PDT group (n = 30) received tumor implantation with subsequent PDT 16 days later. Brain edema and ICP levels were then evaluated. We found that ICP in the PDT group was 7.43 +/- 0.50 mmHg. After tumor implantation, ICP increased rapidly (18.43 +/- 1.10 mmHg, 21 days later). PDT alone did not increase ICP, but compared with that in the tumor group, ICP increased significantly in the tumor plus PDT group (9.55 +/- 1.32 vs. 13.31 +/- 1.13 mmHg, p < 0.01) 24 hours after treatment. Brain water content in the tumor group increased rapidly after tumor implantation. PDT again increased perineoplastic brain edema 24 hours after treatment (81.09 +/- 0.97% vs. 78.32 +/- 0.49%, p < 0.01). It should be noted that PDT alone did not induce brain edema. In conclusion, PDT causes transient brain edema and increases ICP in a rabbit brain tumor model. PMID- 16671499 TI - Whole-body hyperthermia in the rat disrupts the blood-cerebrospinal fluid barrier and induces brain edema. AB - The present investigation was undertaken to find out whether whole-body hyperthermia (WBH) alters blood-cerebrospinal fluid barrier (BCSFB) permeability to exogenously-administered tracers and whether choroid plexus and ependymal cells exhibit morphological alterations in hyperthermia. Rats subjected to 4 hours of heat stress at 38 degrees C in a biological oxygen demand (BOD) incubator exhibited a profound increase in the BCSFB to Evans blue and radioiodine. Blue staining of the dorsal surface of the hippocampus and caudate nucleus and a significant increase in Evans blue and [131]Iodine in cisternal cerebrospinal fluid were seen following 4-hour heat stress compared to control. Degeneration of choroidal epithelial cells and underlying ependyma, a dilated ventricular space, and degenerative changes in the underlying neuropil were frequent. Hippocampus, caudate nucleus, thalamus, and hypothalamus exhibited profound increases in water content after 4 hours of heat stress. These observations suggest that hyperthermia induced by WBH is capable of breaking down the BCSFB and contributing to cell and tissue injury in the central nervous system. PMID- 16671500 TI - Dynamics of cerebral venous and intracranial pressures. AB - Traumatic brain injury and stroke are both characterized by an ischemic core surrounded by a penumbra of low to hyperemic flows. The underperfused ischemic core is the focus of edema development, but the source of the edema fluid is not known. We hypothesized that flow of edema fluid into the tissue is derived from cerebral venous circulation pressure, which always exceeds intracranial pressure (ICP). As a first step toward testing this hypothesis, the aim of the current study was to determine whether cerebral venous pressure in the normal brain is always equal to or higher than ICP. In studies on 2 pigs, cerebral cortical venous, intracranial (subarachnoid), sagittal sinus, and central venous pressures were monitored with manipulation of ICP by raising and lowering a reservoir above and below the external auditory meatus zero point. The results show that cerebral venous pressure is always higher than or equal to ICP at pressures of up to 60 mmHg. On the basis of these observations, we hypothesize that increased cerebral venous pressure initiated after traumatic brain injury and stroke drives edema fluid into the tissue, which thereby increases ICP and a further increase in cerebral venous pressure in a vicious cycle of brain edema. PMID- 16671501 TI - Effects of angiopoietin-1 on vascular endothelial growth factor-induced angiogenesis in the mouse brain. AB - A better understanding of angiogenic factors and their effects on angiogenesis in brain is necessary to treat cerebral vascular disorders such as ischemic brain injury. Vascular endothelial growth factor (VEGF) induces angiogenesis and increases blood-brain barrier (BBB) permeability in adult mouse brain. The effect of angiopoietin-1 on BBB leakage during the angiogenesis process is unclear. We sought to identify the effects of combining VEGF with angiopoietin-1 on cerebral angiogenesis and BBB. Adult male CD-1 mice underwent AdFc (adenoviral vector control), AdAng-1, VEGF protein, VEGF protein plus AdAng-1, or saline (negative control) injection. Brain microvessels were counted using lectin staining on tissue sections after 2 weeks of adenoviral gene transfer. The presence of zonula occludens-1 (ZO-1) was determined by Western blot analysis and immunohistochemistry. Microvessel count and augmented capillary diameter increased in mice treated with either VEGF protein or AdAng-1 plus VEGF protein compared to saline, AdFc, or AdAng-1 alone (p < 0.05). Double-labeled immunostaining demonstrated that ZO-1-positive staining was more complete on the microvessel wall in the AdAng-1 and AdAng-1 plus VEGF protein treated group compared to VEGF protein group. The results of ZO-1 expression from Western blot analysis paralleled that from immunohistochemistry (p < 0.05). We conclude that focal VEGF and angiopoietin-1 hyperstimulation in mouse brain increases microvessel density while maintaining ZO-1 protein expression, suggesting that angiopoietin-1 plays a role in synergistically inducing angiogenesis and BBB integrity. PMID- 16671502 TI - Inflammation and brain edema: new insights into the role of chemokines and their receptors. AB - Brain edema is associated with a variety of neuropathological conditions such as brain trauma, ischemic and hypoxic brain injury, central nervous system infection, acute attacks of multiple sclerosis, and brain tumors. A common finding is an inflammatory response, which may have a significant impact on brain edema formation. One critical event in the development of brain edema is blood brain barrier (BBB) breakdown, which may be initiated and regulated by several proinflammatory mediators (oxidative mediators, adhesion molecules, cytokines, chemokines). These mediators not only regulate the magnitude of leukocyte extravasation into brain parenchyma, but also act directly on brain endothelial cells causing the loosening of junction complexes between endothelial cells, increasing brain endothelial barrier permeability, and causing vasogenic edema. Here we review junction structure at the BBB, the effects of pro-inflammatory mediators on that structure, and focus on the effects of chemokines at the BBB. New evidence indicates that chemokines (chemoattractant cytokines) do not merely direct leukocytes to areas of injury. They also have direct and indirect effects on the BBB leading to BBB disruption, facilitating entry of leukocytes into brain, and inducing vasogenic brain edema formation. Chemokine inhibition may be a new therapeutic target to reduce vasogenic brain edema. PMID- 16671503 TI - Atrial natriuretic peptide: its putative role in modulating the choroid plexus CSF system for intracranial pressure regulation. AB - Evidence continues to build for the role of atrial natriuretic peptide (ANP) in reducing cerebrospinal fluid (CSF) formation rate, and thus, intracranial pressure. ANP binds to choroid plexus (CP) epithelial cells. This generates cGMP, which leads to altered ion transport and the slowing of CSF production. Binding sites for ANP in CP are plentiful and demonstrate plasticity in fluid imbalance disorders; however, specific ANP receptors in epithelial cells need confirmation. Using antibodies directed against NPR-A and NPR-B, we now demonstrate immunostaining not only in the choroidal epithelium (including cytoplasm), but also in the ependyma and some endothelial cells of cerebral microvessels in adult rats (Sprague-Dawley). The choroidal and ependymal cells stained almost universally, thus substantiating the initial autoradiographic binding studies with 125I-ANP. Because ANP titers in human CSF have previously been shown to increase proportionally to increments in ICP, we propose a compensatory ANP modulation of CP function to down-regulate ICP in hydrocephalus. Further evidence for this notion comes from the current finding of increased frequency of "dark" epithelial cells in CP of hydrocephalic (HTx) rats, which fits our earlier observation that the "dark" choroidal cells, associated with states of reduced CSF formation, are increased by elevated ANP in CSF. Altogether, ANP neuroendocrine-like regulation at CSF transport interfaces and blood-brain barrier impacts brain fluid homeostasis. PMID- 16671504 TI - [EBM in pediatric surgery. Critical reading of articles. Treatment (II)]. PMID- 16671505 TI - [Morpho-functional study of electrostimulated latissimus dorsi muscle flap for diaphragm substitution]. AB - Based on practical applications of the muscle latissimus dorsi (MLD) as much in reconstruction thoracic-abdominal defects as functionally in cardiomyoplasty, and in our experience in the electrostimulation with diaphragm pacing, we elaborated the primary objective of this work consisting of the creation of functional muscle plasty of MLD for substitution of the diaphragm. In two groups of cats formed by 6 animals each one, with section of the corresponding phrenic nerve, MLD plasty has been done to replace hemidiaphragm, conserving the thoracodorsal pedicle, by thoracic route in a group (intrathoracic plasty), and in the other by abdominal route (intraabdominal plasty). In a second intervention the stimulador electrode is implanted on thoracodorsal pedicle and the rest of components of the diaphragmatic pacing in a subcutaneous pocket. During a month progressive electroestimulation of MLD plasty is made, carrying out radiological and spirometric evaluation, that compares with evaluation control made before making plasty. After the sacrifice of the animals planimetric and histologic evaluation of the extirpated diaphragm were performed. Morphologic and functional results obtained allow us to affirm that functional plasty of MLD is an effective diaphragmatic substitute, although with a smaller degree of function than the original diaphragm, and functionally intraabdominal plasty has been superior to intratoracica plasty. PMID- 16671506 TI - [Endoscopic treatment of ureterocele in children: is a long term effective technique?]. AB - INTRODUCTION: The endoscopic treatment of the ureterocele is a less invasive procedure. Currently, there are only a few published articles regarding the long term outcomes of this treatment. MATERIAL AND METHODS: We have introduced this technique in our practice since 1995, and have been tracking 15 pediatric patients for long-term evaluation. RESULTS: The average operative age for endoscopic treatment is 19.40 months (1 week-8 years). In 53% of the cases, the diagnosis was made during prenatal screening. In 27% of the cases, the ureterocele was bilateral, and 47% of the patients have a double system. The indication for endoscopic treatment is 66% ureteric obstruction and 34% vesicoureteric reflux. We perform "uncapping" and incision in the two first cases and puncture in the subsequent procedure. The patients were followed for 4 years (7 months-7 years). During this time, two ureteroceles needed a second perforation, out of the seven that continued having reflux, two were treated with quimioprofilaxis and two were treated endoscopically for anti-reflux. Two patients continued to have reflux, and two were diagnosed with renal atrophy and hemiatrophy. DISCUSSION: The endoscopic treatment avoided an open surgical procedure in almost all cases. The need for anti-reflux puncture occurred in 33% of the cases. PMID- 16671507 TI - [Sigmoid colon vaginoplasty: experience with five cases]. AB - Vaginal atresia compounds a rare congenital anomaly and results from anomalies in the development of Muller ducts as happens in the Mayer-Rokitansky syndrome or in other cases of intersexual states. Some different tissues have been issued for the creation of a neovagina, but none of them has been accepted as ideal. We have chosen sigmoid colon in order to create a new vagina functionally appropriate. We present five cases affected of vaginal atresia. Two of them were Mayer-Rokitansky syndromes and the other were diagnosed during the study for primary amenorrhea as a result of an intersexual state. In all cases a vaginoplasty was performed according to Baldwin's technique in the ages between 15 and 20 year-old. The follow up has been from 1 to 16 years. No postoperative complications have been observed. The external aspect of the genitalia is normal and a proper sized with length and lubrificated vagina was attained in all cases without any retraction in any patient. Two of the patients maintain sexual relations without problems. In our experience, the cosmetic and functional result of sigmoid vaginoplasty were excellent. PMID- 16671508 TI - [Platelet-rich plasma (platelet gel) in secondary alveoloplasty in cleft patients]. AB - INTRODUCTION: The use of Platelet-rich plasma (platelet gel--PRP--) was introduced in the oral and maxilofacial surgery 10 years ago. Its good results are due to the quickly generation of new bone and the acceleration of the period of surgical scar formation. Its employment in the alveolar reconstruction of the cleft patient is not still consistent and the works published in the literature are infrequent. The objective of this preliminary study is presenting our experience with the use of PRP in the reconstruction of the alveolar congenital defects of cleft patients. PATIENTS AND METHODS: Between July 2002 and January 2004 were operated 14 patients with congenital alveolar cleft carrying out an standard secondary alveoloplasty. In 12 cases was employed cancellous bone of iliac crest and in two cases the donor area was tibial. The laboratory of Hematology of our Hospital prepared the plasma gel rich in platelets by means of a double centrifuge of autologous serum of the patient. After adding calcium a rich gel in platelets in approximate quantities of 1-2 ml was obtained. In this preliminary report we have studied the aspect of the surgical injury the 3er postoperative day, and the first and second weeks after intervention. These results were compared carried out previously with the Standard alveoloplasty without use of the PRP. Likewise we evaluated the bony density by means of intraoral Rx at 3 and 6 months postoperative. RESULTS: The injury of the alveoloplasty healed more quickly in the patients in which plasma enriched gel was employed. The patients referred less pain and edema in the first days of the postoperative period. The alveolar bony regeneration was faster to the 3 months, though the result was similar in the intraoral Rx to the 6 months. The necessary quantities of cancellous bone were smaller in all cases in which the plasma enriched gel was used (30% less). CONCLUSIONS: The use of Platelet-rich plasma (PRP) is a valid protocol for the reconstruction in patients with congenital alveolar clefts. Its low price and morbidity do it recommendable for its employment in cleft patients. PMID- 16671509 TI - [VOT analysis in cleft palate patients after surgery]. AB - OBJECTIVES: To determine the factors that modify the measures of voice onset time (VOT) as acoustic parameter in operated cleft palate children. The voice of cleft palate patient is defined by the generation of an abnormal intraoral pression. This doesn't allow the normal generation of voiceless plosives consonants and produces an increased VOT. Acoustic analysis of voice quantifies these parameters. In this study we observed an increased VOT if the patient hadn't got a normal teeth occlusion and a normal soft palate function. Also VOT measures were influenced by cleft lip and delay in beginning of ortodoncy. Hearing loss is associated with increased VOT. Our results are based in a correct sample and statistical analysis by multiple lineal regression. PMID- 16671510 TI - [Choledocal cyst: analysis of 29 cases and review or the literature]. AB - The aim of the present study has been to systematize the clinical presentation of the entity named choledochal cyst, in relation with its probable etiopathology and the intraoperative findings as well as its evolution after surgery, based on the revision of the literature and of our experience in 29 cases. MATERIAL AND METHODS: 29 cases of cystic dilatation of the biliar duct extra and/or intrahepatic are analyzed. In 4 cases the diagnosis was prenatal and two were excluded of the study after it has been confirmed they suffered biliary atresia type I. In the left 27 cases, 19 variables are analyzed retrospectively, like age, sex, weight, symptoms, ultrasonographic images, etc. Subsequently, intra operative cholangiographic findings were correlated with the clinic presentation and the evolution of the patients after surgery. RESULTS: Of the 27 cases analyzed 16 (59,25%) were cystic dilatations from which 14 had a neonatal or early clinic presentation (before 2 years), however the fusiform dilatations were presented later on. From the analyzed symptoms, in relation with the age only the pain and the jaundice showed significant differences, being the pain most frequent in later presentation ande the jaundice in the early form. The two cases of type 3 of Todani or choledochocele were of later presentation. An anomaly in the bilionpacreatric junction was detected in 15 patients; the majority had a later presentation, associated to pancreatitis in 4 cases. Primary cyst excision and biliary Roux-en-Y reconstruction was the treatment of election in the majority of cases. In 3 cases we used the appendix to replace the choledocus, but all three cases were reconverted two years later because of permanent elevation of ALT and GGT. CONCLUSIONS: In favour of the literature and of our experience nowadays it would be possible to systematize this malformation and make a division in two groups, depending on the cholangiographic findings and clinical presentation: 1. Cystic dilatations with a clinical neonatal presentation or beneath 2 years. 2. Fusiform dilatations with a later clinical predominance and associated frequently to pancreatitis and anomalous pancreatobiliary junction. Choledochocele is an entity that must be considered not only for its etiology but for its clinical presentation and treatment. Primary cyst excision and biliary Roux-en-Y reconstruction is the treatment of election. Regular long-term review of these patients is mandatory in the surveillance of sub-clinic cholangitis and the risk of possible long-term malignance of this entity. PMID- 16671511 TI - [Esophageal atresia type I. Is impossible possible?]. AB - INTRODUCTION: Treatment of esophageal atresia with "long gap" remains difficult and controversial. According to the idea that esophageal anastomosis is imposible in most cases, several esophageal substitution methods have been proposed, as esophagocoloplasty, gastric transposition or reversed gastric tube. Nevertheless reconstruction of native esophagus is accepted as the best option if posible. "Long gap" definition is imprecise, expressed by variability in percent of these cases in total esophageal atresias reported in different series in literature. We report our experience in seven cases type I esophageal atresia with long gap and the different therapeutic options used, with attention to delayed or early esophageal anastomosis feasibility and outcome. MATERIAL AND METHODS: We have treated 121 patients with esophageal atresia from whom we analized 7 cases with pure esophageal atresia with "long gap" (5.8%). Six patients underwent gastrostomy and two gastrostomy and esophagostomy. Five patient underwent primary repair with esophageal anastomosis, delayed between 14 days and 4 months in 4 cases. One patient underwent esophageal anastomosis in the first day without gastrostomy. Retroesternal esophagocoloplasty was performed in 2 patients about their first year of life. Esophagogram was done in first month after surgery and pH monitoring of gastroesophageal reflux. Follow-up ranged from 6 months to 28 years. RESULTS: Esophageal anastomosis was feasible in all 5 patients in whom it was tried. Stricture occurred in two patients, one patient underwent anastomotic resection and new esophageal anastomosis. Esophageal reflux was present in two patients, one of them required funduplication. One patient was dead by complications of cardiac malformation. Remaining patients have normal swallowing and are in normal growth curves. Patients with esophagocoloplasty had not relevant early or late complications. CONCLUSIONS: In most pure esophageal atresia, delayed or even early esophageal anastomosis is feasible, making use of surgical and anesthesic sources that favour it. Esophageal substitution techniques can be reserved if this approach fails. PMID- 16671512 TI - [The vacuum chest wall lifter in the treatment of pectus excavatum]. AB - Since 2002, the Eckart Klobe vacuum has been used in our Service as an innovate and non-invasive procedure in patients with pectus excavatum. This vacuum method contributes not only to hold promise as a valuable adjunct in minimally invasive surgery, but also as an effective method to lift and fix the funnel for repairing pectus excavatum in some patients. Our experience is limited to the use of this device in 10 patients, during the Nuss technique. This procedure has been useful for safer passage of the introducer, and as a definitive treatment in a 10 years old girl with pectus excavatum and a Haller index of 5.1 with successful result after one year of treatment. The aim of this study is to report the safety and efficacy of this procedure in appropriate patients. Long-term results will be necessary to confirm this method as an alternative to more invasive techniques. PMID- 16671514 TI - [Surgery strategy on obstructive sleep apnea hypopnea syndrome]. PMID- 16671513 TI - [Meckel's diverticulum as a cause of massive rectal bleeding]. AB - A fit three months old male baby was admitted in our hospital with a history of massive painless fresh rectal bleeding. Blood transfusion, hemodynamic stabilization and emergency laparotomy were necessary because of the high suspect of Meckel's diverticulum as the cause of bleeding. It is an uncommon case due to the low age of the patient and the severity of clinical presentation. PMID- 16671515 TI - [Noninvasive ventilation for obstructive sleep apnea hypopnea syndrome in children]. AB - OBJECTIVE: To study the efficacy and safety of noninvasive ventilation (NV) for treating children with obstructive sleep apnea. METHODS: Twenty-one children confirmed obstructive sleep apnea hypopnea syndrome (OSAHS) with full-night polysomnography (PSG) or ambulatory screening device were enrolled in the study. The NV treatment was carried out successfully for all cases. Nasal continuous positive airway pressure (nCPAP) compliance data were gathered via clinical follow-up examination, telephone interview, or mailed questionnaire. The statistical analysis was performed with SPSS 11.0 statistical software. Pre- and on-nCPAP parameters were compared with paired t-test. Twenty-one OSAHS children (17 boys, 4 girls) were enrolled into the study. The mean age of the children was 4. 5 years; ranging from 40 days to 11 years. RESULTS: nCPAP pressure was increased from 4 cm H2O by (1 cm H2O = 0.098 kPa) 0.2 cm H2O each time to the treating pressure which was between 4.8 and 16 cm H2O. Before nCPAP treatment, apnea hypopnea index (AHI) was (80.8 +/- 45.1)/h, the lowest pulse oxygen saturation (SPO2) 0.557 +/- 0.135 and SPO2 <0.90 time during sleep (42.9 +/- 31.9) %, which were much worse compared to that with nCPAP treatment, the above parameters decreased to (6.7 +/- 12.4)/h, 0.862 +/- 0.082, (1.1 +/- 2.5) % respectively(P <0.01). CONCLUSIONS: Noninvasive ventilation is a safe and effective treatment for OSAHS children. It is possible to use nCPAP as a short term treatment or as a long-term treatment at home. PMID- 16671516 TI - [Effectiveness of combined surgery for treating severe obstructive sleep apnea hypopnea syndrome]. AB - OBJECTIVE: To explore the efficiency of a comprehensive surgical approach of genioglossus advancement and hyoid suspension (GAHM) plus uvulopalatopharyngoplasty (UPPP) for treating severe obstructive sleep apnea hypopnea syndrome (OSAHS) and to evaluate related factors on surgery outcomes. METHODS: Eighteen patients with severe OSAHS (apnea hypopnea index, AHI > 40/h) confirmed with polysomnography received genioglossus advancement and hyoid suspension plus uvulopalatopharyngoplasty. The obstruction in both the oropharynx and the hypopharynx were evaluated by preoperative physical examination, fiberoptic pharyngolaryngoscopy, cephalometry, and computed tomography of the upper airway. The follow up was at least 6 months postoperatively. The Wilcoxon signed rank test was used to compare the preoperative and postoperative results by SPSS 11.0 for windows. The Mann-Whitney test was used to analyze the difference between responders and nonresponders. RESULTS: The follow up time ranges from 6 to 24 months, there were statistically significance in all but body mass index (BMI) between preoperative and postoperative measurements. Mean AHI was reduced from preoperative (x +/- s, 63.8 +/- 16.3)/h to postoperative (23.6 +/- 19.5)/h, lowest mean oxygen saturation increased from 0.72 +/- 0.07 to 0.81 +/- 0.13(x +/- s). According to criterion at home, the 6-month rate of responder is 83%, if AHI <20/h and decreased by at least 50% as success, the rate of success is 67%. The age, posterior airway space (PAS) and percentage of time with oxyhemoglobin saturation below 0.90 (CT90) were (39.1 +/- 7.4) years, (8.3 +/- 0.9) mm, (18.5 +/- 10.9)% in responder, while (52.5 +/- 9.4) years, (6.8 +/- 1.3) mm, (37.7 +/- 23.6) % in nonresponder, and there are statistically significant between responder and nonresponder. CONCLUSIONS: GAHM plus UPPP is effective surgical approach for patients with severe OSAHS who suffer from oropharyngeal and hypopharyngeal obstruction. Age, PAS and CT90 were possible affective factors on surgical outcomes. PMID- 16671517 TI - [Clinical application of uvulopalatopharyngoplasty with uvula preservation and tongue base radiofrequency reduction]. AB - OBJECTIVE: To explore the effect of uvulopalatopharyngoplasty with uvula preservation and radiofrequency tongue base reduction for obstructive sleep apnea hypopnea syndrome (OSAHS). METHODS: Forty six patients with OSAHS were enrolled. One group (n = 22) of patients only received uvulopalatopharyngoplasty with uvula preservation, while the other group (n = 24) had both uvulopalatopharyngoplasty with uvula preservation and radiofrequency tongue base reduction. Polysomnography and distance between anterior pillars (DBAP), distance between posterior pillars (DBPP), length of roft palate, distance between tongue base and posterior pharyngeal wall (DBTP) were measured before and 6 months after surgery. RESULTS: The pre-operation apnea hypopnea index (AHI), x +/- s, lowest SaO2 (LSaO2) of first group were (56. 5 +/- 6. 0)/h, and 0.626 +/- 0.060 respectively, and 6 months after surgery, AHI was (23.7 +/- 2.7)/h, LSaO2 was 0.797 +/- 0.053. The pre-operation AHI, LSaO2 of second group were (58.4 +/- 5.1)/h, and 0.650 +/- 0.057 respectively, and 6 months after surgery, AHI was (15.5 +/- 3.2)/h, LSaO2 was 0.864 +/- 0.064. After surgery AHI and LSaO2 have changed in both groups (P<0.001). Six months after operation, DBAP and DBPP became withy, length of soft palate became short (P<0. 001). In one group the validity ratio is 72.7% (16/22), the other group the validity ratio is 87.5% (21/24) (P< 0.05), and pharyngeal posterior airway width (PPAW) became withy (P <0.001). CONCLUSIONS: For OSAHS patients, the obstructive regions should be evaluated. The combined surgery of uvulopalatopharyngoplasty with uvula preservation and radiofrequency tongue base reduction could have a better result. PMID- 16671518 TI - [Perioperative management of modified uvulopalatopharyngoplasty]. AB - OBJECTIVE: Experiences and lessons of uvulopalatopharyngoplasty (UPPP ) perioperative management, especially causes of postoperative tracheotomy, were analyzed, and related strategy was raised to have a better perioperative management and to avoid tracheotomy. METHODS: Two hundred and fifty eight cases of obstructive sleep apnea hypopnea syndromes (OSAHS) diagnosed with polysomnography (PSG) were treated with modified uvulopalatopharyngoplasty (UPPP). The perioperative management was summarized. Patients were divided into two groups according to the perioperative management: without or with perioperative comprehensive management. In group A, there were 32 patients, without comprehensive management, and in group B there were 226 cases with comprehensive management. Sixty eight cases in group B whose apnea hypopnea index over 50 times per hour and the lowest arterial oxygen saturation was less than 0.5 were treated with continuous positive airway pressure (CPAP) for 1 to 3 weeks. For all the 258 cases, perioperative management includes treatment of medical complications, treatment with antibiotics 2 or 3 days before the operation. None of these cases had tracheotomy before surgery. RESULTS: In group A, three of 32 patients had postoperative tracheotomy, two because of bleeding, and another one because of laryngeal spasm. In group B, none of 226 patients underwent tracheotomy, which owing to modified operative apparatus and effective perioperative and postoperative treatment (chi2 = 21.35, P < 0.001). In group A, 5 of 32 patients had oral pharynx bleeding after 24 hours of the operation. While 26 of 226 patients in group B did so (chi2 = 0.15, P > 0.05). CONCLUSION: Comprehensive perioperative management can effectively lower down the complication rate for patients receiving uvulopalatopharyngoplasty. PMID- 16671519 TI - [Infants hearing screening in the coastal villages of Shandong province]. AB - OBJECTIVE: To study how to perform the hearing screening on the infants in the rural area. METHODS: Three thousand nine hundreds and twenty-two infants, about 84% of them from rural, were born in the People Hospital of LaiZhou City from January to December in 2004. The infants were performed fast hearing screening by transient evoked otoacoustic emission (TEOAE) after the birth in 2-7 days. The fail cases were checked again after 4-6 weeks, and then were diagnosed if they still failed after following-up. RESULTS: The infants (3612/3922, 92.1%) have been checked by TEOAE, and the examination was free in the poverty cases. The rate passed on the first check was 69.96% (2527/3922), but 1085 infants failed (30.4%), while 310 infants have not been checked (7.9%). In the 1085 cases that should be rechecked, there was only 633 cases (58.34%) accepted the check on time, while 452 cases (41.66%) missed. In the 163 cases with high-risk infants in 2004, 114 infants (69.96%) were checked, but 49 infants (29.04%) were not checked. Fourteen cases failed in the recheck, and 11 of them were checked by ABR. Two cases were found to be moderate and severe hearing loss in binaural respectively and 4 cases with mild hearing loss in monaural while 3 cases were normal. CONCLUSIONS: It is necessary and viable for the infants on hearing screening in the rural area It should be set up and perfected the model for infants on hearing screening in rural area as soon as possible; it should be free for the poor infants to make sure everyone enjoy the health care. PMID- 16671520 TI - [Case control study on risk factors of congenital microtia]. AB - OBJECTIVE: To identify the risk factors associated with congenital microtia. METHODS: A case-control, retrospective study was performed. A total of 207 cases of congenital microtia and 209 age-frequently-matched controls were interviewed individually according to the uniform questionnaire. The questionnaire included: (1) Essential information about patients and their parents. (2) First trimester diseases and medication of mother. (3) Past gestation history of mother. (4) Family history and life style of the parent. The available data were analyzed by chi-square test and multivariate Logistic regression model. RESULTS: The factors, such as gender (OR = 5.893), first trimester disease of mother (OR = 34.49) and medication (OR = 4.299), excessive drinking and smoking of father (OR = 4. 347, OR = 4.304), non-primiparous parity (OR = 9.524), abortion (spontaneous and induced, OR = 1.723), low-education of mother (OR = 2.275) were risk factors contributing to microtia in multivariate study. The factors, such as tea (OR = 0.179) were protective factors in multivariate study. CONCLUSIONS: Multi-risks factors are contributing to microtia. Preventive measures according to risk factors should be put forward. PMID- 16671522 TI - [Transseptal endoscopic excision of nasal dermoid sinus cyst]. AB - OBJECTIVE: Surgery is the curative treatment for nasal dermoid cysts. Many different surgical incisions have been advocated. All approaches described have the advantage of either improved accessibility or improved patients'cosmetic satisfaction, but none is optimal. We designed a new transseptal endoscopic approach for the treatment of nasal dermoid sinus cyst and reported the result of a special case. METHODS: The diagnostic studies, the operative technique, and the result of 18 month follow-up were presented. RESULTS: The patient, a 16 years old girl, remains free of disease 18 months after surgical treatment, most importantly without facial scar. CONCLUSIONS: Transseptal endoscopic approach is an alternative surgical technique for selected nasal dermoid cysts. So far as we know, the operative method presented in the current report is the first surgical approach reported in the world literature. PMID- 16671521 TI - [Auditory capability evaluation for children after cochlear implantation using meaningful auditory integration scale]. AB - OBJECTIVE: To analyze the auditory capability of preschool children before and after cochlear implantation using meaningful auditory integration scale (MAIS) questionnaire. METHODS: Eighty-two prelingually deaf patients participated in this study. They received a cochlear implant at the age of 3 to 6 years and 11 months. The audiologists who were trained for the research used the MAIS questionnaire. Audiologists asked for the parents' answers and recorded all the information about the device using (Q1,2) and the patient's spontaneous auditory behavioural responses including spontaneous alerting to sound Q3 approximately 6 and deriving meaning from sound (Q7 approximately 10). The evaluation was performed before operation and 1 , 3, 6 months, 1, 1.5, 2 years after switch-on. RESULTS: The scores of question 1a and 1b were not significantly different among the different periods after switch-on. The scores of question 2 to 10 were significantly different among the different periods after switch-on. CONCLUSIONS: Considerable variability across subjects' auditory ability after cochlear implantation was noted. Most of the patients showed no consistent response to sound in everyday life before implantation. After cochlear implantation, a significant increase in auditory capability occurred. The children demonstrated faster development of device using relative to spontaneous alerting to sound and deriving meaning from sound. PMID- 16671523 TI - [Diagnosis and treatment of laryngeal web in infants]. AB - OBJECTIVE: To summarize the clinical manifestation, operative method and therapeutic effect of various type of laryngeal web in infants. METHODS: The clinical data of 12 cases were analyzed, 5 cases of which were congenital laryngeal web (4 cases, glottic type; 1 case, subglottic type), 7 cases of which were secondary laryngeal web (1 case, tuberculous laryngeal web; 6 cases, traumatic laryngeal web). Diagnosis was mainly depended on history and clinical manifestation. Final diagnosis was depended on fibrolaryngoscope and pathological report. Microlaryngoscopic surgery was the main operative method. However, specific infection should be cured before operation. RESULTS: During 3-18 months follow-up, 4 glottic laryngeal webs were cured. One subglottic laryngeal web case well recovered and secondary surgery is not needed at least recently. One tuberculous laryngeal web was followed up for 6 months, no vocal adhesion was observed. During 3-6 months follow-up, 1 traumatic laryngeal web was cred, while the other 6 cases need secondary surgery. CONCLUSIONS: Final diagnosis of congenital laryngeal web is mainly depended on fibrolaryngoscope. And prognosis of it is well. Laryngeal web induced by specific infection should be cured specific infection before operation. The prevention is the key for traumatic laryngeal web because the surgery outcome is not satisfactory. PMID- 16671524 TI - [Comprehensive treatment of advanced pyriform sinus cancer]. AB - OBJECTIVE: To evaluate the treatment results of advanced pyriform sinus cancer according to therapy modalities. METHODS: Two hundred and eighty seven patients with locally advanced pyriform sinus cancer were included in this retrospective analysis. One hundred and thirty four patients were treated with radiotherapy alone (Ra group) with 68.8 Gy. One hundred and twenty five patients were treated with preoperation radiotherapy with 45.5 Gy followed by surgery including 32 laryngeal function sparing surgery (R + S group). Thirteen patients were treated with surgery alone (Sa group). Fifteen patients were treated with surgery followed by post-operation radiotherapy with 56.3 Gy (S + R group) . There was no laryngeal function sparing surgery in the last two groups. RESULTS: Survival rate and laryngeal sparing rate were calculated using Kaplan-Meier method. The overall 5-year survival rates were 23. 13% for Ra group, 46.51% for R + S group,18. 33% for Sa group and 44. 44% for S + R group, 46. 2% for combined therapy group ( R + S group and S + R group) and 22. 60% for single therapy group (Sa group and Ra group). There was statistical difference between combined therapy group and single therapy group (P = 0. 000 ) . Laryngeal sparing rate were 14. 08% ,13.61% , 0 and 0,respectively. R + S group showed superiority over Ra group, Sa group and S + R group in teams of both overall survival rate and laryngeal sparing rate. Laryngeal function spare was analyzed using Cox model. Therapy mode is the most important factor that contribute to laryngeal sparing. No significant differences were found in complication rate among the groups. CONCLUSION: Preoperation radiotherapy conduce to preserve laryngeal function of patients with locally advanced pyriform sinus carcinoma. PMID- 16671525 TI - [Clinical anatomy measurement of accessory nerve in neck dissection]. AB - OBJECTIVE: To investigate the relations between accessory nerve and its surrounding structures. METHODS: One hundred and thirty six patients were divided into two groups: has or has no neck surgical history. Neck dissection were performed and the four distance were measured simultaneously. The distance of accessory nerve and the great auricular nerve going out the posterior edge of sternocleidomastoid muscle; the distance of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to clavicular midpoint; the distance of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to sternoclavicular articulation; the distance of the point accessory nerve enter trapezius muscle to clavicular midpoint. RESULTS: In no neck dissection group, the point accessory nerve going out sternomastoid muscle were supra the point of great auricular nerve going out the sternomastoid muscle, the average length of two points is (0. 61 +/- 0. 35) cm , the significance has not observed between genders (P > 0.05), however, there has significant difference between two groups of has or has no neck surgical history (P < 0.05). 88.2% (112/127) accessory nerve going out supra the great auricular within 1.0 cm, 11.8% (15/127) within 1.0 approximately 2.0 cm. 67.7% (86/127) accessory nerve adopt branch from cervical plexus before entering trapezius. The distances of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to clavicular midpoint and to sternoclavicular articulation were significant relative not with before neck surgical history but gender. The distance of the point accessory nerve enter trapezius muscle to clavicular midpoint is (4.96 +/- 0.78) cm, it has no difference both before neck surgical history and gender (P > 0.05). CONCLUSION: In no neck surgical history group,both of the distance that accessory nerve and the great auricular nerve going out the posterior edge of sternocleidomastoid muscle and the point accessory nerve enter trapezius muscle to clavicular midpoint were helpful for search accessory nerve in surgery. But in patients who have neck surgical history or great auricular have been injured, accessory nerve could be looked for associating with the distances of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to clavicular midpoint and to sternoclavicular articulation; the distance of the point accessory nerve enter trapezius muscle to clavicular midpoint. PMID- 16671526 TI - [Observation of auditory brainstem response and distortion product otoacoustic emission on the animal model of autoimmune auditory neuropathy]. AB - OBJECTIVE: To set up an animal model of autoimmune auditory neuropathy and to observe the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) in guinea pigs. METHODS: The spiral ganglion and the cochlear nerve were obtained and purified by electrophoresis from 250 normal guinea pigs. The purified cochlear nerve antigen was mixed with an equal volume of complete Freunds adjuvant for immunization. Seventy guinea pigs were divided into three groups: experiment group (50 guinea pigs), control group (10 guinea pigs), normal group (10 guinea pigs). ABR, DPOAE, serum IgG levels, and morphological changes of spiral ganglion cells and the cochlear nucleus were observed. The protein expressions of the antigen were examined by immunohistochemistry and the super structure of the auditory nerve were observed. RESULTS: The threshold of ABR response increased ranged from 10 to 25 dB in 32% (32/100 ears) of the guinea pigs. The peak latencies of waves I , III and the interpeak latency I approximately III were prolonged in the hearing loss group of guinea pigs. Prolonged peak latency of wave III was noted in hearing loss group at 2 and 3 weeks post immunization and slowly decreased to normal peak latency. The amplitude of DPOAE was no difference in the guinea pigs. The levels of serum IgG increased significantly compared with those of the control group. Inflammatory cell infiltration was observed in the cochlear nerve and the number of spiral ganglion cells detected. On the contrary, inflammatory cell infiltration was not observed in the cochlear nucleus. The cell densities and the across-sectional areas of neurons in anteroventral cochlear nucleus and posteroventral cochlear nucleus were no difference in the guinea pigs. The antigen protein distributed strictly in cochlear nerve and the spiral ganglion. Some demyelinated areas in cochlear nerve was observed in this group. The threshold of ABR response in 68% guinea pigs (68/100 ears) did not increase. The data of DPOAE and the serum IgG levels show no difference compared with the control group. There were not pathological observation in spiral ganglion cells, cochlear nucleus and cochlear nerve. CONCLUSION: An animal model of autoimmune auditory neuropathy has been set up successfully and the character of the ABR and DPOAE was observed. PMID- 16671527 TI - [Adenoid remnant investigation after traditional adenoidectomy]. PMID- 16671528 TI - [Immortalization of human laryngeal epithelial cells induced by human papillomavirus type 16 DNA]. PMID- 16671530 TI - [Sedation technique for postoperative nasal endoscopic debridement]. PMID- 16671529 TI - [Fungal sinusitis: a case report and clinical analysis]. PMID- 16671531 TI - [Endoscopic surgery for nasal meningoencephalocele: a case report]. PMID- 16671532 TI - [A case of caseous maxillary sinusitis with nasal polyp]. PMID- 16671533 TI - [A case of giant neurofibroma in nasal cavity and ethmoid sinus]. PMID- 16671534 TI - [Otorhinolaryngology and obstructive sleep apnea hypopnea syndrome]. PMID- 16671535 TI - New attention to the IUD: expanding women's contraceptive options to meet their needs. AB - Modern intrauterine devices (IUDs) are safe, effective, and quickly reversible long-term contraceptives that require little attention after insertion. Yet safety concerns and programmatic challenges have held back IUD services in many countries. New assessment of research findings, recently translated into guidance by the World Health Organization, should help reassure providers that most women can use IUDs safely. PMID- 16671536 TI - [Significant role of intestinal transporters in drug absorption]. PMID- 16671537 TI - [Advances in studies on inhibitor of apoptosis proteins and cancer therapy]. PMID- 16671538 TI - [Synthesis of phenyloxyisobutyric acid derivatives and their antidiabetic activity in vitro]. AB - AIM: To design and synthesize new phenyloxyisobutyric acid analogues as antidiabetic compounds. METHODS: Eight new target compounds were synthesized by combination of lipophilic moieties and acidic moiety with nucleophilic replacement or Mitsunobu condensation. The eight compounds were confirmed by 1H NMR, 13C NMR, IR and MS. RESULTS: In vitro insulin-sensitizing activity (3T3-L1 adipocyte) demonstrated, that the cultured glucose concentration of up-clear solution detected with GOD-POD assay were 5.942, 6.339, 6.226 and 6.512 mmol x L( 1), respectively, when rosiglitazone, pioglitazone, compounds A and B were added to the insulin-resistant system. CONCLUSION: In vitro insulin-sensitizing activity of target compound A is in between that of rosiglitazone and pioglitazone, and activity of target compound B is slightly less than that of pioglitazone. PMID- 16671539 TI - [Synthesis and AchE inhibitory activity of 2-phenoxy-indan-1-one derivatives]. AB - AIM: To design and synthesize novel AchE inhibitors. METHODS: The condensation of 2-bromo-5, 6-dimethoxy-indan-1-one with various aminoalkyl phenols in the presence of K2CO3 and acetonitrile gave the corresponding title compounds, and the in vitro AchE and BchE inhibitory activities were evaluated by the modified Ellman method. RESULTS: Sixteen novel target compounds 8a - p were synthesized, their structures were confirmed by 1H NMR, MS, IR and elemental analysis. Preliminary pharmacological test demonstrated that most of these compounds displayed high AchE inhibitory activities, the IC50 of the most potent inhibitor 8h was 50.0 nmol x L(-1), similar to that of Huperzine A (IC50 = 53.0 nmol x L( 1)), while all the compounds were almost inactive against BchE. CONCLUSION: 2 Phenoxy-indan-1-one derivatives exhibit high activities of AchE inhibition and are worthy of further investigation. PMID- 16671540 TI - [Structural modification of cyclovirobuxine D and their inhibition activity on lipid peroxidation]. AB - AIM: To search for compounds through structural modification of cyclovirobuxine D, using 20 or 21-aminosteroids as lead compound for the treatment of cerebrovascular diseases with better lipid peroxidation inhibitory activity. METHODS: According to rational drug design principle, a series of cyclovirobuxine D analogues were prepared, and their bioactivities were tested. RESULTS: Four new compounds were obtained and confirmed by spectra. CONCLUSION: Lipid peroxidation inhibitory effects of cyclovirobuxine D analogues were tested by using TBA method. Some compounds showed better activity than that of cyclovirobuxine D. PMID- 16671541 TI - [The chemical constituents of Breynia rostrata]. AB - AIM: To study the chemical constituents of Breynia rostrata Merr. METHODS: Chromatography was used to isolate and purify the chemical constituents, their structures were identified by spectral analysis. RESULTS: Four glycosides were identified as 6-O-methylpropanoyl-alpha-D-glucopyranoside (1), 4"-phenolic-6-O methylpropanoyl-beta-D-glucopyranoside (2), 1-O-galloyl-beta-D-glucopyranoside (3), arbutin (4). CONCLUSION: Compounds 1 and 2 are new compounds; 3 and 4 were isolated from Breynia rostrata Merr. for the first time. PMID- 16671542 TI - [Isolation and identification of hetisine-type alkaloids from Aconitum coreanum by high speed countercurrent chromatography]. AB - AIM: To search for more bioactive compounds from the roots of Aconitum coreanum (Levl.) Rapaics. METHODS: High speed countercurrent chromatography was successfully applied to the separation of alkaloids from Aconitum coreanum. The structures were elucidated by their physicochemical properties and spectroscopic analysis. RESULTS: Two-phase solvent system composed of CHCl3-CH3OH-0.2 mol x L( 1) HCl (10:3:3, volume ratio) was used in this experiment, eight alkaloids were obtained from the roots of Aconitum coreanum, which were identified as: 2alpha propionyl-11alpha,13beta-diacetyl-14-hydroxyhetisine (I), Guanfu base P (II), Guanfu base G (III), Guanfu base F (IV), Guanfu base Z (V), Guanfu base O (VI), Guanfu base A (VII), Guanfu base B (VIII). CONCLUSION: Compound I is a new alkaloid, named Guanfu base R. PMID- 16671543 TI - [Immunogenicity of single-dose HBsAg-PLGA controlled release microspheres in mice]. AB - AIM: To investigate the level of immune response and the immune mechanism of the single-dose hepatitis B surface antigen (HBsAg)-poly (d, l)-lactide-co-glicolide acid (PLGA) microspheres in BALB/c mice. METHODS: Three kind of HBsAg-PLGA microspheres, HBsAg-PLGA50/50-COOH microspheres, HBsAg-PLGA75/25 microspheres and HBsAg-PLGA50/50 microspheres, were prepared by double emulsion microencapsulation technique used three kinds of PLGA with different L/G ratio. The single-dose of HBsAg-PLGA microspheres was subcutaneously injected into BALB/c mice at the dose of 7.5 microg HBsAg per mouse. The conventional aluminum-adjuvant vaccine was subcutaneously injected at 0, 1 and 2 month as positive control. In certain time interval, the induced immune level of total antibody was detected by enzyme linked immunosorbent assay (ELISA). For subclass of IgG antibody and cytokines studies, the dose of HBsAg was 2.5 microg per mouse. RESULTS: The HBsAg-PLGA microspheres could successfully induce a humoral immune response in BALB/c mice. Compared with the conventional aluminum-adjuvant vaccine, the antibody response of the HBsAg-PLGA50/50-COOH microspheres was significantly lower than the group received three injections of aluminum-adjuvant vaccine (P < 0.01) except for a higher priming response during the early 6 weeks. The results were ascribed to the relatively rapid degradation charactics of PLGA50/50-COOH polymer. The immune response for the HBsAg-PLGA50/50 microspheres and HBsAg-PLGA75/25 microspheres were comparable to the group administered with aluminum-adjuvant vaccine (P > 0.05) which was due to the sustained degradation of PLGA50/50 and PLGA75/25 polymer. CONCLUSION: The HBsAg-PLGA microsphere is a promising candidate for the controlled delivery of a vaccine which does not require multiple injections. PMID- 16671544 TI - [The absorption characteristics of silybin in small intestine of rat]. AB - AIM: To investigate the intestinal absorption of silybin (SLB) in male rats. METHODS: Single-pass intestinal perfusion (SPIP) technique was performed in each isolated region of the small intestine at a flow rate of 0.1 mL x min(-1). The samples of perfusate and portal plasma were collected at the designated periods of time after rat intestinal perfusion and analyzed for drug by HPLC. RESULTS: The absorption rate constant (k(a)) and the effective permeability (P(eff)) of SLB at 190 microg x mL(-1) were determined for each segment. These data indicated the absorption rate were duodenum > jejunum > ileum > colon. SPIP was also performed in duodenum with three concentrations of SLB (80, 190 and 300 microg x mL(-1)). The concentration dependent changes of k(a) and P(eff) were evident in the duodenum perfusion of silybin. At silybin perfusate concentrations of 80 microg x mL(-1), k(a) and P(eff) were different from the values at either of the two higher concentrations (190 and 300 microg x mL(-1), P < 0.05). However there was no difference between 190 microg x mL(-1) and 300 microg x mL(-1) groups. The drug mass appearing in the plasma further indicated the absorption were duodenum > jejunum > ileum > colon. CONCLUSION: SLB can be absorbed in whole intestinal sections. When the concentration raises to a certain level, the uptake of SLB will not increase. PMID- 16671545 TI - [Cell penetrating peptides enhance intracellular translocation and function of siRNA encapsulated in Pegylated liposomes]. AB - AIM: To prepare the PEGylated liposomes modified with cell penetrating peptides, which protect siRNA from nuclease degradation and deliver efficiently siRNA into cells to facilitate silencing of target gene. METHODS: The purity of R8-PEG-PE and pNP-PEG-PE was detected by HPLC; the quantity of R8, PEG-DPPE modified R8, and R8 attached to the out membrane surface of the liposomal siRNA by transfer from R8-PEG-DPPE micelles to the liposomes was tested by fluorescence; Size and size distribution of siRNA loaded liposomes with and without attached R8 were determined by Zetasizer 5000; A comparison of mediated siRNA transfection efficiency between R8-liposomes and lipofectamine 2000 was examined by individual inside cell fluorescence intensity; The growth inhibition of small cell lung carcinoma NCI-H446 cells treated with R8-liposomal hdm2-siRNA or lipofectamine 2000-hdm2-siRNA complex was tested by MTT assay. RESULTS: The retention times of PEG-DPPE and R8-PEG-DPPE were 9.0 min and 7.8 min, respectively. Fluorescence scanning indicated that lipids composed of liposomes and siRNAs didn't interfere to the determination of R8 when it was attached to the liposomal siRNA. The cells treated with R8-liposomal hdm2-siRNA significantly enhanced the cellular uptake of hdm2-siRNA and facilitated the functions of hdm2-siRNA through silencing of target gene which, in turn, inhibited tumor cell growth, compared with lipofectamine 2000. CONCLUSION: The R8 attached liposomes are shown to be powerful carriers for delivery siRNAs into cell to silence targeted gene. PMID- 16671546 TI - [Effect of absorption enhancers on nasal ginsenoside Rg1 delivery and its nasal ciliotoxicity]. AB - AIM: The enhancing activity and safety of several absorption enhancers were evaluated as potential nasal absorption enhancers to increase intranasal absorption of ginsenoside Rg1. METHODS: Nasal circulatory perfusion test in vivo had been employed to investigate the effect of absorption enhancers for nasal mucosa absorption of ginsenoside Rgl in rats. The safety of the absorption enhancers were evaluated by testing cilia movement of the in situ toad palate model, the hemolysis of erythrocyte membrane of the rabbit, leaching of protein and LDH from the mice nasal mucosa and the effect on cilia structural and specific cellular changes of nasal mucosa. RESULTS: Absorption enhancers were necessary to facilitate ginsenoside Rg1 absorption by nasal mucosa. Among the absorption enhancers 1% sodium deoxycholate had great effect to facilite ginsenoside Rgl absorption by nasal mucosa; 1% dipotassium glycyrrhizinate and 1% azone had moderate effect to facilitate ginsenoside Rg1 absorption by nasal mucosa; 1% Tween-80, 2% beta-cyclodextrin, 0.5% borneol (dissolved in paraffin liquid), 0.5% chitosan, 5% hydroxypropyl-beta-cyclodextrin and 0.1% EDTA had low effect to facilitate ginsenoside Rgl absorption by nasal mucosa. 1% sodium deoxycholate, 1% azone and 1% dipotassium glycyrrhizinate had serious nasal toxicity; 1% Tween-80, 2% beta-cyclodextrin, 5% hydroxypropyl-beta-cyclodextrin had moderate nasal toxicity; 0.5% borneol (dissolved in paraffin liquid), 0.5% chitosan and 0.1% EDTA have little nasal toxicity. CONCLUSION: 0.5% borneol and 0.5% chitosan were the promising candidates having a good balance between enhancing activity and safety for nasal ginsenoside Rg1 delivery. PMID- 16671547 TI - Effect of ginkgolide B on the platelet-activating factor induced changes of chemotaxis and cytoskeleton of macrophages. AB - AIM: To study the inhibitory effect of ginkgolide B (BN52021) on the PAF induced changes of chemotaxis of murine peritoneal macrophages and the related polymerization of F-actin. METHODS: Chemotaxis assays were performed using a modified 48-well Boyden chamber. Actin polymerization of murine peritoneal macrophages was analyzed by flow cytometry using a specific fluorescent stain. RESULTS: Peritoneal macrophages significantly migrated toward platelet-activating factor (PAF) through a micropore filter; however, in the presence of PAF receptor antagonist BN52021 (0.01 nmol x L(-1) -0.1 micromol x L(-1)), the migration was significantly inhibited. Moreover, BN52021 inhibited the actin polymerization of murine peritoneal macrophages induced by PAF in the presence of Ca2+, but not in Ca2+ -free medium. CONCLUSION: The results suggested that preventing polymerization of F-actin may be a pathway by BN52021 to inhibit the chemotaxis of macrophages, and this effect seems to be Ca2+ dependent. The data further indicated that inhibition of PAF induced macrophage chemotaxis is an important mechanism underlying the anti-inflammatory action of BN52021. PMID- 16671548 TI - Inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated proliferation of rat smooth muscle cells. AB - AIM: To investigate the bio-affinities of ligustilide and butylidenephthalide to rat aortic smooth muscle cells and the inhibitory effects of them on bFGF stimulated proliferation of rat vascular smooth muscle cell (VSMC). METHODS: VSMCs were cultured from rat aorta pectoralis and identified by an immunohistochemical method. The bio-affinities between solute (ligustilide or butylidenephthalide) and cell membrane were measured by rat aortic cell membrane chromatography (CMC). The inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated VSMC proliferation were evaluated by MIT colorimetric method. RESULTS: Both ligustilide and butylidenephthalide had selective affinities to rat aortic smooth muscle cell as the same as verapamil, one of the calcium ion antagonists. They could potently inhibit the bFGF stimulated VSMC proliferation at the concentrations of 5.5 and 11.1 micromol x L( 1), separately (P < 0.05), but had no effects on the normal VSMC growth. CONCLUSION: Both ligustilide and butylidenephthalide can inhibit the abnormal proliferation of VSMC induced by bFGF. PMID- 16671549 TI - [Effect and mechanism of action of total glucosides of paeony on synoviocytes from rats with collagen-induced arthritis]. AB - AIM: To study the effect and mechanism of action of total glucosides of paeony (TGP) on synoviocytes from rats with collagen-induced arthritis (CIA). METHODS: Chicken type II collagen was used to induce CIA in rats. Synoviocytes were separated by incubation with collagenase and trypsin, and its ultrastructural changes were observed under transmission electron microscope. Synoviocyte proliferation was determined by 3-(4,5-dimethylthiazal-2yl) 2,5- diphenyltetrazoliumbromide (MTT) assay, and IL-1 activity in synoviocytes supernatant was measured by thymocyte proliferation assay. TNFa and PGE, produced by synoviocytes were determined by radioimmunoassay. RESULTS: TGP was shown to protect CIA rats against the ultrastructural damages of synoviocytes. Meanwhile, TGP also suppressed the excessive synoviocyte proliferation and over-production of IL-1, TNFalpha and PGE2. CONCLUSION: TGP has inhibitory effect on hyperfunctional synoviocytes of CIA rats and its mechanism of action may be related with the inhibition of abnormal proliferation and secretion of synoviocytes. PMID- 16671550 TI - [Protective effects of hydroxyethylpuerarin against brain astrocytes injury induced by hydrogen peroxide]. AB - AIM: To study the protective effects of hydroxyethylpuerarin against the injury of astrocytes induced by hydrogen peroxide (H2O2). METHODS: Experiments were performed with cells from passage 4. Plasma membrane integrity was measured by lactate dehydrogenase (LDH) release. The occurrence of apoptosis was measured by flow cytometry. The glutamate uptake of astrocytes was studied with [3H] glutamate incorporation. Intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were assessed by automatic biochemistry analyzer. RESULTS: Compared with H2O2 injured group, the occurrence of apoptosis, levels of LDH release and intracellular MDA of astrocytes reduced in hydroxyethylpuerarin pre-treated groups, but the glutamate uptake and intracellular SOD activity of astrocytes increased. CONCLUSION: Hydroxyethylpuerarin could reduce the occurrence of apoptosis and improve neurotrophic function of astrocytes, which may be related with its antioxidant effects during oxidative stress. PMID- 16671551 TI - [Spectroscopic studies on the binding of sibutramine hydrochloride and bovine serum albumin]. AB - AIM: To study the binding of sibutramine hydrochloride (SH) and bovine serum albumin (BSA) in physiological condition by spectroscopic method. METHODS: The quenching mechanism of the fluorescence of bovine serum albumin by sibutramine hydrochloride was studied with the fluorescence and the absorption spectroscopy. The binding constants K and the number of binding sites were determined at different temperatures according to Scatchard equation and the main binding force was discussed by thermodynamic equations. The effect of the drug on bovine serum albumin conformation was also studied by using synchronous fluorescence spectroscopy. RESULTS: The quenching mechanism of sibutramine hydrochloride to bovine serum albumin was static quenching. The binding constants K at 8 degrees C, 25 degrees C, 37 degrees C were 1.21 x 10(5), 8.31 x 10(4), 6.97 x 10(4) L x mol(-1) with one binding site, respectively. The thermodynamic parameters of the reaction were deltaH = -9.70 kJ x mol(-1), deltaS = 56.41 J x mol(-1) x K(-1). CONCLUSION: The binding force is electrostatic interaction. Sibutramine hydrochloride can be deposited and transported by serum protein in vivo. Sibutramine hydrochloride has nearly no effect on the serum protein conformation. PMID- 16671552 TI - [Fingerprint of Polygala tenuifolia by high performance liquid chromatography]. AB - AIM: To study and establish the fingerprint of Polygala tenuifolia by RP-HPLC. METHODS: The fingerprint of P. tenuifolia was built by using Kromasil C18 as column and acetonitrile-0.05% phosphoric acid aqueous in gradient as mobile phase. The flow rate was 1.0 mL x min(-1), and the detecting wavelength was set at 318 nm. Total 14 batches of P. tenuifolia from different habitats were detected, and different commodities and preparative methods were compared. RESULTS: Samples from different habitats were of high similarities, and the qualities of "yuanzhitong" and "yuanzhirou" were better than that of "yuanzhigun", while the fingerprints of P. tenuifolia with different preparative methods have not obvious differences. Moreover, the standard fingerprint of P. tenuifolia was originated from the "Computer Aided Similarity Evaluation" software, and 29 common peaks existed in the fingerprint. Each peak in the fingerprint was separated very well under the above chromatographic condition, with good accuracy, stability and repeatability, satisfied to the requests of fingerprint. CONCLUSION: The RP-HPLC fingerprint method can be used in the quality control of P. tenuifolia. PMID- 16671553 TI - Effect of dipfluzine on delayed afterdepolarizations and triggered activity induced by isoprenaline in human atrial fibers. PMID- 16671554 TI - [Simultaneous determination of ephedrine and chlorpheniramine in human plasma by a highly sensitive liquid chromatography-tandem mass spectrometric method]. AB - AIM: To develop and validate a liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantification of ephedrine and chlorpheniramine in human plasma after oral administration of a compound preparation. METHODS: The analytes and the internal standard, diphenhydramine, were isolated from plasma by protein precipitation with methanol, then chromatographied on a Zorbax SB-C18 column (150 mm x 4.6 mm ID) using a mobile phase consisted of methanol-water-formic acid (80: 20: 0.5, v/v), at a flow rate of 0.5 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor to produce ion combinations of m/z 166-->115, m/z 275-->230 and m/z 256-->167 were used to quantify ephedrine, chlorpheniramine and the internal standard, respectively. Results The linear concentration ranges of the calibration curves for ephedrine and chlorpheniramine were 0.50 - 200 microg x L(-1) and 0.050 - 20.0 microg x L( 1), respectively. The lower limits of quantification were 0. 50 microg x L(-1) for ephedrine and 0.050 microg x L(-1) for chlorpheniramine, individually. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 9.3% for both ephedrine and chlorpheniramine. The inter-day accuracy (RE) was within +/- 3.4% for the analytes. Each sample was chromatographied within 3.3 min. The method was successfully used in pharmacokinetics study of ephedrine and chlorpheniramine in human plasma after oral administration of a compound preparation containing 5 mg ephedrine hydrochloride, 1 mg chlorpheniramine maleate, 50 mg phenytoin, 12.5 mg theophylline, 12.5 mg theobromine and 7.5 mg caffeine. No interaction among the six components was observed on their pharmacokinetic parameters. CONCLUSION: The method was proved to be highly sensitive, selective, and suitable for pharmacokinetics investigations of different compound preparations containing low dosage of both ephedrine and chlorpheniramine. PMID- 16671555 TI - Structural analysis of heart rhythm spectrogram in narcotized cats. AB - Eight types of peaks were revealed in the cardiac rhythm spectrum during acute experiments on vagotomized cats. Some peaks had no physiological nature and resulted from specificity of ECG processing by Fourier analysis, while others reflected myogenic reaction of the sinoatrial node (length-dependence of automaticity) to changes in venous return caused by respiratory-induced and other variations of the blood flow in the cardiovascular system. PMID- 16671556 TI - Estradiol-dependent effect of nitric oxide on meiotic maturation of mouse oocytes. AB - Estradiol dipropionate decreased the number of oocytes in mouse ovary and suppressed meiotic maturation of these cells. Administration of NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine increased the ability of ovarian oocytes to complete meiotic maturation and counteracted the effect of estradiol dipropionate. Our results suggest that estradiol is involved in the regulation of production and release of ovarian NO. PMID- 16671557 TI - Temperature dependence of H+ transport across erythrocyte membrane of Rana temporaria grass frog in media containing Cl- and SO4(2-). AB - H+ transport across the erythrocyte membrane was studied in Rana temporaria grass frog. The temperature coefficients and activation energy of H+ transport were calculated in media containing Cl- and SO4(2-). Our results show that kinetic characteristics of H+ transport depend on function of band 3 protein in the erythrocyte membrane. PMID- 16671558 TI - Spectral and coherent characteristics of EEG in women during various phases of menstrual cycle. AB - EEG recording was performed in healthy women during various phases of menstrual cycle. Comparative study showed that the power spectrum of EEG a-waves in central and medio-temporal areas significantly decreases during ovulation. In the menstrual phase coherent characteristics of EEG alpha-waves increased in symmetrical occipital and intrahemispheric parieto-occipital areas. PMID- 16671559 TI - Change in resonance properties of the central nervous system in 6-11 year-old children. AB - Basic and reactive characteristics of EEG in children of 6-11 years were studied under conditions rhythmic photostimulation. We showed that the efficiency of photostimulated entrainment reaction determines functional maturity of the corresponding brain oscillators. PMID- 16671560 TI - Activity of angiotensin-converting enzyme in hereditary stress-induced arterial hypertension. AB - We measured activity of angiotensin-converting enzyme in plasma and tissue of NISAG and normotensive WAG rats. In different organs of NISAG rats, activity of this enzyme did not differ from the corresponding values of WAG rats, although enzyme activity in the plasma of NISAG rats was significantly lower than that of WAG rats. Since NISAG rats are characterized by low activity of renin in the renal cortex, it is hypothesized that NISAG rats simulate the low-renin hypertension, in which inhibition of activity of the angiotensin-production system results from elevation of arterial pressure of central origin. PMID- 16671561 TI - Activity of nitric oxide synthase and concentration of nitric oxide end metabolites in the gingiva under experimental pathological conditions. AB - Parameters of NO metabolism in the gingiva were studied during experimental periodontitis accompanied by alloxan diabetes and exogenous hypercholesterolemia. We measured activities of inducible and constitutive NO synthase and concentrations of stable NO end metabolites in rat gingival tissue (total contents of nitrite and nitrate). Under pathological conditions NO-metabolism significantly differed from the control. Treatment with mexidol for 14 days significantly decreased activity of inducible NO synthase in the gingiva of experimental animals. PMID- 16671562 TI - Diurnal variations in qualitative composition of breast milk in women with iron deficiency. AB - Biorhythms of iron metabolism in healthy women and patients with iron deficiency were studied during lactation. Healthy nursing women were characterized by circadian variations in the concentrations of iron, alpha-tocopherol, and malonic dialdehyde in breast milk. Diurnal variations in iron concentration, antioxidant potential, and lipid peroxidation in breast milk depended on iron metabolism in nursing women. Iron deficiency was accompanied by a decrease in the concentrations of alpha-tocopherol and iron, increase in malonic dialdehyde content, and suppression of circadian variations in these parameters in breast milk. PMID- 16671563 TI - Metabolic alterations in rat myocardium in experimental acute atrial fibrillation. AB - Experimental atrial fibrillation in intact rats significantly decreased the content of catecholamines in atrial adrenergic fibers and phosphorylase activity, which attests to enhanced glycogen consumption in the heart. These changes were specific of the fibrillating myocardium and atria, but were absent in the ventricles. Induced atrial fibrillation did not modify activities of SDH and monoamine oxidase in cardiac subdivisions. It was hypothesized that increased energy requirements in the atria during myocardial fibrillation led to activation of anaerobic metabolism. PMID- 16671564 TI - Effect of ketorolac tromethamine on lymph circulation, contractile activity of the lymphangion and lymphatic microvessels, and cellular composition and toxicity of the lymph during fever. AB - Single parenteral administration of ketorolac tromethamine produced a lymphotropic effect, which was manifested in acceleration of lymph flow in the thoracic duct and increase in contractile activity of the wall and valves in mesenteric lymphatic microvessels of rats with fever. These changes improved lymph circulation. PMID- 16671565 TI - Lanthanum potentiates GABA-activated currents in rat pyramidal neurons of CA1 hippocampal field. AB - In CA1 hippocampal pyramidal neurons, lanthanum ions increased the amplitude of GABA-activated currents and shifted the dose-dependence curve to the left, which attests to increased affinity of GABAA-receptors to GABA. The data made it possible to compare the sensitivity GABAA-receptors of pyramidal neurons and similar receptors of other cells to GABA and lanthanum. PMID- 16671566 TI - Apolipoprotein A-I possessing activity of defensin proteins modifies structure of plasma membranes. AB - We studied the in vitro effect of apolipoprotein A-I possessing activity of defensin proteins on the structure of plasma membranes in donor erythrocytes and lymphocytes. Incubation of erythrocyte membranes with apolipoprotein A-I was accompanied by significant changes in biophysical characteristics of a fluorescent probe pyrene in the hydrophobic membrane region and a decrease in Na+/K(+)-ATPase activity. Microviscosity of annular lipids in the plasma membrane of lymphocytes increased during incubation with apolipoprotein A-I. Our results suggest that membranotropic activity of apolipoprotein A-I is associated with the presence of amphipathic alpha-helix regions. PMID- 16671567 TI - Relationship between blood cortisol level and blood parameters in animals with experimental bile peritonitis. AB - We studied the relationship between blood cortisol level and hemogram parameters in animals with bile peritonitis. A strong correlation was revealed between variations in cortisol concentration and leukocyte count. We found differences in the hormonal regulation of individual cell populations in the peripheral blood. PMID- 16671568 TI - In vitro effect of fucose-specific lectins on rat erythrocyte membranes under normal conditions and during toxic stress. AB - We studied the effect of various fucose-specific lectins on lipid peroxidation in rat erythrocyte membranes under normal conditions and during exposure to the toxic agent. Under normal conditions parameters of lipid peroxidation in erythrocyte membranes increased after treatment with lectins or cadmium nitrate. Lectins possess biological activity modulated lipid peroxidation in membranes of erythrocytes from experimental animals, which depended on the time of adaptation. Differences were revealed in the effect of bacterial and plant lectins on lipid peroxidation in erythrocytes treated with the test xenobiotic. PMID- 16671569 TI - Free radical oxidation in rat brain during chronic stress and pharmacological regulation of this process. AB - We studied free radical oxidation in the brain and blood serum from experimental animals exposed to chronic stress and receiving psychotropic drugs (phenazepam, Atarax, Fluanxol, and valerian). Chronic stress was accompanied by activation of free radical oxidation, which could be modulated by psychotropic drugs. PMID- 16671570 TI - Phospholipid hydrolysis with phospholipases A2 and C impairs apolipoprotein B-100 conformation on the surface of low density lipoproteins by reducing their association resistance. AB - Modification of apolipoprotein B-100 conformation on the surface of LDL isolated from human blood was demonstrated by enzyme immunoassay with a panel of monoclonal antibodies to this protein. The study by the light transmission fluctuation method showed that incubation of LDL with phospholipases A2 or C led to association of LDL particles. This lipolytic modification seems to impair LDL surface properties inducing association of these particles, which can play an important role in lipid accumulation in the vascular wall and at early stages promote the development of atherosclerosis. PMID- 16671571 TI - Effect of nifedipine on cerebral saturation parameters. AB - The effect of 10 mg nifedipine on cerebral oxygenation was studied in healthy individuals. The duration of investigation was 30 min or 3 h. Nifedipine appreciably increased cerebral oxygenation (by 5.9%, p<0.01) against the background of significant increase in heart rate and negligible decrease in systolic arterial pressure. Drowsiness was the most serious side effect of nifedipine (observed in 52.8% examined subjects). PMID- 16671572 TI - Effect of zosterin on protein-synthesizing activity of hepatocytes. AB - The effect of zosterin (pectin polysaccharide) on the protein-synthesizing function of mouse liver cells was studied after its intragastric administration of 1% gel zosterin. The drug modified the morphology and function of the nucleolar apparatus by changing the number and summary area of the nucleoli, content of Ag proteins, nucleolar/nuclear ratio, and increased protein content in hepatocyte cytoplasm. PMID- 16671573 TI - Radiobiological effects of olipifat during radiation exposure. AB - Preventive injections of olipifat to rats (intramuscularly in a single dose of 250 mg/kg 24 h before gamma-exposure in a dose of 7.6 Gy) decrease animal mortality from 70 to 50%, the mean life span of dead rats remained unchanged. Injection of olipifat before exposure in a dose of 5.2 Gy did not change animal mortality in comparison with irradiated controls, but stimulated postradiation recovery of leukocyte count (mainly the granulocytic component and less so the increase in lymphocyte count). No appreciable effects of olipifat on the postradiation changes in individual fractions of blood leukocytes were observed in animals exposed in a dose of 7.6 Gy. PMID- 16671574 TI - Lymphotropic effect of mexidol in reactive fever. AB - Single injection of mexidol (drug with antioxidant and membranotropic effects) to animals with reactive fever produces a multicomponent effect on the lymph circulation. The drug increased the number of functioning lymph capillaries and contractile activity of wall and valvular leaflets in rat small intestinal mesenteric lymphangion, accelerated lymph drainage, thus stimulating lymph formation and lymph flow. PMID- 16671575 TI - Protective effect of reamberin on functional activity of mitochondria during skin ischemia. AB - Reamberin in a dose of 25 mg/kg (succinate concentration) was injected intravenously for 3 days starting from the 1st hour after skin ischemia modeling. This treatment decreased activities of lactate dehydrogenase, aspartate transaminase, and creatine phosphokinase in skin homogenates by 1.6 times, 19%, and 51.3%, respectively. The index of cytolysis decreased by 18%. Reamberin had an energotropic effect, which manifested in an increase in the total ATP content and concentration of creatine phosphate (by 16 and 10%, respectively). After administration of Reamberin, activity of the succinate-ubiquinone reductase system increased by 17%. Under these conditions succinate dehydrogenase activity exceeded the normal by 21%. Reamberin had no effect on the mitochondrial NADH ubiquinone reductase system in dermal cells during skin ischemia. Superoxide dismutase activity in the area of necrosis increased to the control level on day 3 of treatment with Reamberin. Activities of catalase and glutathione peroxidase increased by 13 and 19%, respectively. Our results indicate that the course of intravenous treatment with Reamberin for 3 days contributes to an increase in reserve capacities of the antioxidant protection system and produces a protective effect during skin ischemia. PMID- 16671576 TI - Preventive and therapeutic effects of alpha-acid glycoprotein in mice infected with B. anthracis. AB - We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha. PMID- 16671577 TI - Lectin binding to mouse blood lymphocytes during tumor growth. AB - Binding of FITC-labeled lectins to lymphocytes from intact mice and mice with transplanted Ehrlich carcinoma and CaO-1 ovarian carcinoma was studied by flow cytofluorometry. Specific binding of lectins by mannose and N-acetylgalactosamine was demonstrated. Lectin binding to lymphocytes from animals with tumors decreased by more than 50% in comparison with intact animals. Changed protein glycosylation during tumor growth is specific and differs for tumors of different origin. PMID- 16671578 TI - Stimulation of macrophages increases, while suppression of these cells inhibits metastatic dissemination of two transplantable mouse tumors in the liver and lungs. AB - Stimulation of mouse tissue macrophages with carboxymethylated beta-(1-->43)-D glycan 1 day before intravenous injection of tumor cells increased the number and weight of implants (experimental metastases) of mouse hepatocarcinoma and adenocarcinoma in the liver and lungs, respectively. Suppression of liver macrophages with gadolinium chloride or sequestration of cells during intraperitoneal administration of macrophage attractants inhibited metastatic dissemination of hepatocarcinoma and adenocarcinoma in the liver and lungs, respectively. In the latter case animal lifespan increased. Our results indicate that at certain stages of metastatic dissemination, activation of mononuclear phagocytes can stimulate the formation and growth of metastases. PMID- 16671579 TI - Effect of tetrapeptide on insulin biosynthesis in rats with alloxan-induced diabetes. AB - Analysis of amino acid sequences of insulinotropic polypeptides revealed a common short fragment consisting of four amino acid residues. We synthesized KEDWa tetrapeptide, analog of this fragment protected from the effects of gastrointestinal proteinases. This tetrapeptide partially restored insulin synthesis in rats with alloxan-induced diabetes. The slope of the sugar curve in this case was similar to that in normal animals. Presumably, this tetrapeptide activates the preproinsulin gene promotor site via complementary interactions with the ggcagg and cctgcc nucleotide sequences of the leading strand of double stranded DNA. PMID- 16671580 TI - Correlation of intracellular Ca(2+)-activated proteinase activity and cholesterol content in white sea mussel (Mytilus edulis) membranes at different water saltiness. AB - Correlation between changes in activity of intracellular Ca(2+)-activated proteinases and cholesterol content in mussels in response to changes in habitat saltiness was detected in mollusks from littoral and sublittoral White sea zones. Calpain activity in mussels decreased in low water saltiness and increased in high saltiness in parallel with decrease in cholesterol content, which attests to decreased microviscosity and modification of permeability of biomembranes. A complex pattern of interactions between metabolic routes in mussels under conditions of different habitat saltiness was detected. PMID- 16671581 TI - Variants of cardiomyopathic heart pathomorphogenesis. comparison of echocardiographic and endomyocardial biopsy findings. AB - The main variants of cardiomyopathies (undifferentiated, dilatational, hypertrophic, and restrictive) were distinguished using a complex pathomorphological analysis of 600 cardiomyopathic hearts detected in 5000 autopsies after cardiovascular death. The main pathomorphological diagnostic criteria for each variant were defined. High diagnostic value of lifetime echocardiographic diagnosis in comparison with myocardial biopsy was shown. The informative value of endomyocardial biopsy in cardiomyopathic heart is higher, if the method is combined with clinical examinations, noninvasive and invasive studies. PMID- 16671582 TI - Morphogenesis of adaptation and compensatory reactions in mouse adrenals during restitution after thermal exposure. AB - Single hyperthermia session causes an appreciable decrease in adrenocorticocyte count in the adrenal cortex in mice on day 3 of postthermal restitution. Morphological changes in the adrenals under conditions of developing stress reaction manifested in increased structural and functional heterogeneity of adrenocorticocytes in all cortical layers, exhaustion of lipid incorporations in the zona fasciculata cells, pronounced shrinkage of zona reticularis, developing against the background of hemodynamic disorders. Recovery of the total count of adrenocorticocyte population was associated with the appearance of small accumulations of immature cells in the subcapsular area of zona glomerulosa. Despite the absence of pronounced changes in the architectonics of all adrenocortical zones during postthermal restitution, recovery of zona reticularis was incomplete, which manifested in its shrinkage and persistence of degenerative changes in adrenocorticocytes. PMID- 16671583 TI - Topography of 3H-DHA, 3H-QNB, 3H-dopamine, and 3H-DAGO binding sites distribution in the central part of the sinoatrial node in rat heart. AB - The topography of distribution of 3H-dihydroalprenolol, 3H-quinucledinyl benzilate, 3H-dopamine, and 3H-DAGO binding sites in the central part of the sinoatrial node in rat heart was studied by autoradiography after electrophysiological identification of the dominant pacemaker region location. Receptor asymmetry between the lateral and median regions of the central part of the sinoatrial node was shown. The dominant pacemaker region lay in the lateral area of the sinoatrial node; the number of binding sites for all four ligands was minimum in it. The number of binding sites gradually increased in the cranial and caudal directions from the dominant pacemaker region along the sinoatrial node artery (more smoothly in the caudal direction). The relative densities of bindings sites for 3H-dihydroalprenolol and 3H-dopamine were higher in the lateral region compared to the perinodal working myocardium, while the densities for 3H-quinucledinyl benzilate and 3H-DAGO were virtually the same. The distribution of binding sites along the artery in the median region of the sinoatrial node was even for 3H-quinucledinyl benzilate and 3H-DAGO. For 3H-DAGO these parameters were close to those in the perinodal atrial myocardium, for 3H quinucledinyl benzilate somewhat lower. Curves presenting the distribution of binding site densities for 3H-dihydroalprenolol and 3H-dopamine in the median region of the sinoatrial node were similar, with a pronounced peak in the region contralateral to the dominant pacemaker region, and significantly higher binding parameters compared to those for the perinodal atrial myocardium. The difference consisted in higher density of 3H-dopamine binding sites in the median region of the sinoatrial node in comparison with the lateral region. Binding activity was maximum in the wall of the sinoatrial node artery. The distribution of binding sites for ligands to the main autonomic nervous system neurotransmitters in the rat heart sinoatrial node is heterogeneous. PMID- 16671584 TI - Differential diagnosis of various forms of myasthenia and endocrine ophthalmopathy by immunoblotting. AB - Immunoblotting can be used for screening a population of antibodies to acetylcholine receptor subunits circulating in the blot of patients with myasthenia. Torpedo Californica acetylcholine receptor served as the antigen. We found that in generalized myasthenia autoantibodies bind to alpha1- or alpha1- and gamma-subunits, while in ophthalmic form they bind only gamma-subunit of acetylcholine receptor. No antibodies to any of the acetylcholine receptor subunits were detected in patients with endocrine ophthalmopathy and in healthy volunteers. This method can be used for differential diagnosis of ophthalmic myasthenia and endocrine ophthalmopathy and for predicting generalization of the pathological process in patients with myasthenia. PMID- 16671585 TI - Memory lymphocytes of young and old C57BL/6 mice express high levels of class I major histocompatibility complex (H-2 Kb) protein. AB - Memory lymphocytes play a central role in the secondary immune response. The concentration of memory lymphocytes increases with age. A high level of cell surface CD44 is a marker of memory lymphocytes compared to naive lymphocytes which express a low level of CD44. Major histocompatibility complex (MHC) class I protein expression also increases with age. To explore a possible correlation between the expression of CD44 and MHC class I protein (Kb), peripheral blood lymphocytes from 27 C57BL/6 mice ranging in age from 3 months to 33 months were isolated by Ficoll-Hypaque gradient centrifugation. Single and double indirect immunofluorescence assays were then performed with rat IgG anti-CD44 and/or mouse IgG anti-Kb as first antibodies, and phycoerythrin (PE) labeled goat anti-rat IgG and/or fluorescein (FITC) labeled goat anti-mouse IgG as second antibodies. Cells were then analyzed by using a FACScan flow cytometer. As expected, the percentage of lymphocytes expressing high levels of CD44 (memory cells) increased significantly with age and the expression of Kb increased significantly with age. Interestingly, the expression of Kb in lymphocytes expressing high levels of CD44 (memory cells) was 72% more than in cells expressing low levels of CD44 (naive cells) regardless of age. PMID- 16671586 TI - Distribution of components of the insulin-like growth factor system in the temporomandibular joint of the aging mouse. AB - The temporomandibular joint (TMJ) is a exceptional joint involved in growth as well as mastication. In adult mice, it provides a model for age related natural osteoarthritis (OA). The insulin-like growth factor (IGF-) system plays was tested because it plays important roles in cartilage biology and OA pathogenesis. Decalcified and paraffin embedded TMJs of 48 NMRI mice sacrificed in groups of three male and females each at the ages of 3, 4, 5, 7, 9, 10, 12, and 18 months were prepared for histopathology and immunohistochemistry for IGF-I and -II, IGF1 receptor and IGF binding proteins -1, -2, -3, -4, -5, and -6. Histopathological signs of OA were obvious in the TMJ of all animals older than 5 months, but did not show a clear age-related staging. Immunoreactivity for all IGF components was found in unchanged anterior and posterior regions of the condyle and in regions of advanced OA lesions. Receptor immunostaining was obvious in all ages. Most IGFBPs showed immunostaining patterns similar to IGFs. While the anterior and posterior zones of the condylar cartilage appear to be specialized judging by structure and IGF immunostaining pattern, probably due to metabolic or biomechanical peculiarities, the central portion undergoes early degeneration. In advanced OA stages, the IGF system seems to be upregulated to induce repair processes. According to their mainly inhibiting functions, IGFBPs may suppress anabolic IGF activities. PMID- 16671587 TI - Evidence from catch-up growth and hoarding behavior of rats that exposure to hypobaric air lowers the body-mass set point. AB - The depression of body growth rate and the reduction of body mass for chronological age and gender in growing experimental animals exposed to hypobaric air (simulated high altitude = SHA) have been associated with hypophagia because of reduced appetite. Catch-up growth during protein recovery after a short period of protein restriction only occurs if food intake becomes super-normal, which should not be possible under hypoxic conditions if the set-point for appetite is adjusted by the level of SHA. The present investigation was designed to test the hypothesis that growth retardation during exposure to SHA is due to an alteration of the neural mechanism for setting body mass size rather than a primary alteration of the central set-point for appetite. One group of female rats aged 35 d were exposed to SHA (5460m) in a SHA chamber for 27 d (HX rats). Other group was maintained under local barometric pressure conditions (NX rats). One half of both NX and HX rats were fed a protein-free diet for the initial 9 d of the experimental period. From this time on, they were fed a diet containing 20% protein, as were the remaining rats of both groups during the entire experimental period. The growth rates of both mass and length of the body were significantly depressed in well-nourished rats exposed to SHA during the entire observation period when compared to normoxic ones. At its end, body mass and body length were 24% and 21% less in HX than in NX rats. Growth rates were negatively affected by protein restriction in both NX and HX rats. During protein recovery, they reached supernormal values in response to supernormal levels of energy intake that allowed a complete catch-up of both body mass and length. The finding that energy intake during the period of protein rehabilitation in HX rats previously stunted by protein restriction was markedly higher than in HX control ones at equal levels of hypoxia demonstrates that the degree of hypoxia does not determine directly the degree of appetite and energy intake. Furthermore, the finding that catch-up growth in the stunted HX rats returns the animal only to the stunted size appropriate for the hypoxic animal supports the hypothesis that hypoxia lowers the set-point for body mass size, which is reached by inhibition of appetite. Confirmation of the hypothesis was done by assessment of the set-point of body mass by the behavioral method of the weight threshold to hoard food. It was lowered by 17.0% in HX rats. PMID- 16671588 TI - Catch-up in body weight of intrauterine growth retarded (IUGR) rats castrated and treated with growth hormone. AB - The aim of the present study was to assess body weight recovery in rats with intrauterine growth retardation (IUGR) castrated and treated with growth hormone (GH). Wistar albino rats were divided into the following experimental groups: control (C), sham-operated (SH), IUGR, IUGR castrated (IUGR+C), and IUGR castrated and injected with GH (IUGR+C+GH). IUGR was induced by partial bending of uterine vessels at day 14 of pregnancy. GH (Genotropin 3.0 mg/kg/day) was administered from weaning (21 days old) to 60 days of age. SH rats were injected only with diluent. Castration was performed at weaning. Body weight, body weight velocity and relative food intake were registered weekly. Data were analyzed by ANOVA and LSD post hoc test. The between-subjects analysis showed significant differences for sex and treatment factors. Differences in body weight were significant among treatments, being SH > IUGR > IUGR+ C+GH > IUGR+C in males, and SH > IUGR+C > IUGR+C+GH > IUGR in females. Differences in relative food intake were IUGR+C > IUGR+C+GH > IUGR > SH in males, and IUGR+C+GH > IUGR+C > IUGR > SH in females. These results indicate that nutritional rehabilitation impairs the effects of IUGR. While the absence of testosterone inhibited body weight gain, the absence of estrogens promoted catch-up growth. Castrated animals with or without GH treatment ate relatively more, suggesting the absence of an anabolic effect of gonadal steroids. PMID- 16671589 TI - Aging-associated loci in Canis familiaris. AB - Although recent endeavors to discover the mechanisms of the aging process have been numerous and successful, there is still much to be learned. Genes implicated in the aging process were mapped to the canine genome and will serve as additional framework markers for the assignment of contiguous segments from the canine genome sequence to chromosomes. The 54 genes were selected because of their demonstrated contribution to longevity in other organisms or based upon their proximity to a marker, D4S1564, on human chromosome 4 (Puca et al., 2001). This effort lays the necessary groundwork for our utilization of the domestic dog as a model organism to define the genes that govern aging and longevity. Within the species, naturally diverse life expectancies and highly homogeneous populations create an ideal population structure for studying the genetic components of aging (Patronek et al., 1997). PMID- 16671590 TI - Advantage of dichromats over trichromats in discrimination of color-camouflaged stimuli in humans. AB - This study investigated whether 12 participants with color-vision deficiency had superior visual discrimination of color-camouflaged stimuli shown on a computer screen compared with 12 participants with normal trichromatic vision. Participants were asked to distinguish a circular pattern from other patterns in which textural elements differed from the background in orientation and thickness. In one condition, stimuli were single-colored, green or red; in the other condition, stimuli were color camouflaged with a green and red mosaic overlaid onto the pattern. Color-vision deficient participants selected the correct stimuli in the color-camouflaged condition as quickly as they did in the single-colored condition. However, normal color-vision participants took longer to select the correct choice in the color-camouflaged condition than in the single-colored condition. These results suggest that participants with color vision deficiency may have a superior visual ability to discriminate the color camouflaged stimuli. PMID- 16671591 TI - Multiple microswitches for persons with multiple disabilities: a basic procedure to examine the persons' choice behavior. AB - This study examined a basic choice-analysis procedure to clarify choice between two responses and related stimuli by five persons with multiple disabilities ages 17.1 to 50.2 yr. The procedure was based on reversing the links between responses and stimuli to assess whether the stimuli accounted for differences in response levels. When this was not the case, the procedure added extra stimuli for the less preferred response to judge whether such an increase would compensate for the apparent disadvantage of that response, e.g., its higher physical cost. Analysis showed that the choice of three of the five participants seemed motivated by the stimuli following the responses. The choice of the other two persons seemed related to a preference for one of the responses which was partially or largely modified when extra stimuli were added. PMID- 16671592 TI - Dream recall and the full moon. AB - There is ongoing debate on whether the full moon is associated with sleep and dreaming. The analysis of diaries kept by the participants (N = 196) over 28 to 111 nights showed no association of a full moon and dream recall. Psychological factors might explain why some persons associate a full moon with increased dream recall. PMID- 16671593 TI - Transfer of training for two ball-throwing tasks. AB - The effects of training overhand ball throws to enhance underhand ball-throwing accuracy were studied with 921 Italian high school pupils (575 girls, M age = 16.5 yr., SD = 1.3; 346 boys, M = 16.5 yr., SD = 1.2) ages 14 to 18 years. A standardized test (10 underhand tennis-ball throws into the floor area delimited by an 80-cm radius wooden hoop lying on the floor 9 m from the standing pupil) was performed and scored. An Experimental group (435 girls, M age = 16.5 yr., SD = 1.3; 204 boys, M age = 16.5 yr., SD = 1.2) was randomly selected to participate in 10 weekly training periods. The set included 10 overhand throws of a rubber bouncing ball (14.5 cm diameter, weight 240 g) into a basketball hoop from 5 standardized positions (in front of the basketball hoop; at 45 degrees, 90 degrees on the right and on the left of the basketball hoop) at a distance of 4.40 m. The underhand test was repeated for both groups. On the average, boys obtained higher test scores than girls of the same age. Older adolescents had higher mean scores than younger adolescents. After 10 weeks, boys and girls of all ages improved. Mean differences between sessions were significant for the Experimental group (Student t test, p < .01). In Session 2, consistent differences between Experimental and Control groups were also found (Student t test, p < .05). PMID- 16671594 TI - Hand preference consistency and eye-hand coordination in young children during a motor task. AB - Indications of earlier research are that individuals exhibiting a consistent hand preference are better coordinated on selected motor tasks than peers with inconsistent hand preference. The present study examined eye-hand coordination via inter- and intramodal matching behavior by handedness groups for 55 5- and 6 yr.-olds using the Pinboard Test. Analysis indicated the Consistent group scored better, leading to the speculation that children with consistent laterality may possess an advantage in interhemispheric communication, especially when the task requires coordination of both limbs. PMID- 16671595 TI - Polarization of perceived Procedural Justice. AB - This study examined polarization of perceptions of Procedural Justice. Two polarization mechanisms are examined, Persuasive Arguments and Social Comparisons. Participants were students enrolled in a first-year introductory business class. There were 216 participants in the Persuasive Arguments study, 429 in the Social Comparisons study. The average age of all participants was 22.3 yr. (SD = 2.1); 56% were women. Fields of study represented were business, engineering, information technology, and sports. Analysis showed under conditions of low Procedural Justice, polarization effects were only found with the Persuasive Arguments mechanism. Under conditions of high Procedural Justice, polarization effects were only found with Social Comparisons. Implications for group polarization and Procedural Justice theories are considered. PMID- 16671596 TI - Teaching 'Yes' and 'No' responses to children with multiple disabilities through a program including microswitches linked to a vocal output device. AB - The present study introduced two children (a boy and a girl of 9.2 and 12.3 yr. of age, respectively, with multiple disabilities) to an intervention program for establishing Yes and No responses in relation to preferred and nonpreferred stimuli and assessed the acquisition and generalization of those responses. The program involved the use of microswitches, which monitored the nonverbal Yes and No responses performed by the children and ensured that these responses produced the matching verbal utterances via a vocal output device. Analysis showed children acquired Yes and No responses in relation to the stimuli used for the intervention and, to a comparable degree, to the stimuli used for generalization checks as well. An interview of 30 educational staff of a center for persons with multiple disabilities indicated that they considered the presence of Yes and No verbal utterances preferable to the absence of such utterances, and thought that the utterances could be helpful for the children's response acquisition and would be useful and pleasing for staff and parents. General implications of the findings were discussed. PMID- 16671597 TI - Modeling sport services in Greece: a re-evaluation of outcome dimension. AB - The Service Quality Scale, developed to evaluate perceived service quality in sport services in Greece, was administrated to 389 participants, 208 men and 181 women. Their mean age was 36.4 yr. (SD = 8.7). A confirmatory factor analysis did not support the prior five-factor solution. When the outcome dimension was removed, a four-factor model appeared an adequate fit. Practical implications are discussed. PMID- 16671598 TI - Construct validity of the Bruininks-Oseretsky Test of Motor Proficiency-short form for a sample of Greek preschool and primary school children. AB - The construct validity of the short form of the Bruininks-Oseretsky Test of Motor Proficiency for the assessment of gross and fine motor skills was assessed in 377 nondisabled Greek preschool and primary school children (age range 5 yr. to 8:3 mo.) from urban areas of northern Greece. Analysis showed the three factors accounted for 54.1% of the total score variance, agreeing with the earlier findings. Moreover, the item scores had statistically significant relationships with the total short-form score, except for that of copying a circle with the preferred hand. This latter item was also the only one with a small effect size. Age confirmed a statistically significant effect on the scores of the half items of the test battery, also an earlier finding. This test seemed to be a valid test of motor proficiency in normal Greek preschool and primary school children. PMID- 16671599 TI - Buying books online: a replication. AB - In a replication study, purchasing books online by 137 state college students was predicted by computer/Internet skills. PMID- 16671600 TI - Comment on IQ, sustained attention, and visual processing speed in children and adolescents with schizophrenia spectrum disorders. AB - This paper is intended to lend support to the 2005 findings of McCarthy, Kraseski, Schvartz, Mercado, Daisy, Tobing, and Ryan in relation to the WISC-III performance of children and adolescents with Schizophrenia Spectrum Disorders. Relevant evidence from the literature and recent studies conducted by our group at the Institute of Psychiatry, UK, are discussed. PMID- 16671601 TI - Relation of attention control and school performance in normal children. AB - This study examined the relation of attention control and school performance in a sample of 113 school children (51 boys, 62 girls), ages 9 to 12 years (M = 10.86, SD = 0.91). Children and parents completed the Attention Control Scale for Children, which measures children's ability to focus and shift attention, and also a scale for assessing symptoms of Attention-Deficit Hyperactivity Disorder. Teachers provided a global rating of children's school performance. Analysis indicated that attention control correlated positively with school performance. PMID- 16671602 TI - Interval endurance capacity of talented youth soccer players. AB - The purpose of this study, in which 113 talented youth soccer players (M = 16.0 yr., SD = 1.5), selected by their age and level of performance participated, was to investigate interval endurance capacity needed to play at the highest level of competition in the age-category 12 through 18. Multivariate analyses of covariance with factors of level of performance and age category showed that players ages 16 through 18 years outscored the players ages 12 through 15 years on the interval endurance capacity (p < .05). In both age categories, elite players outscored less skilled players (p < .05). In the age category 12-15 years, the elite players participated more in soccer training than less skilled players (p < .05), whereas less skilled players participated more in additional training than the elite players (p < .05). No differences were found for total training (p > .05). In conclusion, development of the interval endurance capacity seems crucial for a talented youth soccer player to be successful. PMID- 16671603 TI - Goal orientation, motivational climate, and dispositional flow of high school students engaged in extracurricular physical activity. AB - The purpose of this study was to examine the role of goal orientation, motivational climate, and dispositional flow in physical education lessons on extracurricular involvement in physical activity. Questionnaires were administered to 1,103 (792 athletes; 311 nonathletes) secondary school students (M age = 14.3 yr., SD = 0.7). Analysis showed significant mean differences between groups on goal orientation and dispositional flow in physical education lessons, but none for perception of motivational climate. These findings suggest that dispositional variables are related to extracurricular involvement in physical activity. PMID- 16671604 TI - Mode of grasp, materials, and grooved chopstick tip on gripping performance and evaluation. AB - This study evaluated the effects of the mode of grasping, chopstick materials, and the groove of the chopstick-tip on gripping performance of 32 participants, among whom 16 were accustomed to using chopsticks with a pliers-grasping mode and the other 16 with a scissors-grasping mode. Based on a random sequence, 8 kinds of chopsticks with different combinations (materials: bamboo, wood, plastic, and stainless steel, with and without grooves in the chopstick-tip) were used to carry out the action of gripping two different objects (peanuts and longan). The time to finish gripping 50 pieces of peanut and longan, the number which fell on the table when gripping, the subjective effort and the subjective preference were measures of efficiency of pinching for 8 kinds of chopsticks. Analysis showed that chopstick materials significantly affect time of gripping. The large object (such as longan) could be gripped more easily with the pliers-grasping mode or gripped with the grooved chopstick-tip. However, for small objects (such as peanuts), there was no significant difference in performance whether the chopstick-tip was grooved or not. A combination of bamboo chopsticks with grooved tips used with a pliers-grasping seem preferrable for more favorable gripping performance. PMID- 16671605 TI - Suicide attacks in Israel and suicide rates. AB - In Israel from 1983 to 1999, the frequency and lethality of suicide attacks was negatively associated with the suicide rate. PMID- 16671606 TI - Geographical and political predictors of emotion in the sounds of favorite baby names. AB - The top 5 favorite boys' and girls' names from each state of the USA in 2000 and 2003 were analyzed in terms of the emotional associations of their component sounds and sound pronounceability. These were significantly and variously correlated with a historical factor (year), geographic factors (compass directions), and a political factor (percentage of the popular vote cast for President Bush in 2004). The expected stereotypical sex differences were observed: girls' names were longer, more pleasant, less active, and easier to pronounce (p < .01). It was possible to predict emotional associations and pronounceability (R2 = .27-.48, p < .01) on the basis of historical, geographical, and political variables. PMID- 16671607 TI - Comprehensive analysis of golf performance on the PGA Tour: 1990-2004. AB - Researchers have investigated the relationship between different shot-making measures and performance on the PGA Tour. Prior studies have typically focused on a short period of time or used a restricted sample so long-term trends were not discernible. To remedy this situation, the present study looked at the longitudinal performance of professional golfers from 1990-2004. The findings indicated a remarkable stability in terms of the relative importance of Greens In Regulation and Putting Average in explaining the variability in Scoring Average. The findings also indicated a declining importance of driving in recent years due, in part, to a strengthening of the negative relationship between Driving Distance and Driving Accuracy. PMID- 16671608 TI - Comment on evaluating cognitive demand. AB - In 2005 Shieh and Chen found differences in EEG responses when using computer- and paper-based display media, which they attributed to different cognitive demands. This paper supports this interpretation by reporting an unpublished study in which cognitive demand was measured using a self-report workload measure. PMID- 16671609 TI - Emotion in the sounds of pets' names. AB - Several thousand cats' and dogs' names were compared with each other and with several thousand men's and women's names in terms of their use of various sounds and the emotional associations of these sounds. Emotional associations were scored according to the system developed by Whissell in 2000. In general, cats' names stood in comparison to dogs' names as women's names stood in comparison to men's names. Names from the first group in each pairing included more pleasant and soft phonemes and fewer unpleasant and sad ones than those in the second group (one-way analyses of variance with post hoc LSD tests, p < .0001). As well, pets' names were longer and more easily pronounced by children than the human names (p < .0001). PMID- 16671610 TI - Directional uncertainty in visually guided pointing. AB - Studies of the relationship between stimulus-response uncertainty and reaction times indicate three qualitatively different functions: Hick's law, simple-choice step function, or flat curve (no effect at all). The extent of stimulus-response S-R) compatibility appears to interact with the effects of uncertainty on response times. One possible hypothesis regarding these various S-R uncertainty functions is that uncertainty will have an effect whenever the stimuli and their associated responses are not within the same egocentric spatial coordinates. We tested this hypothesis in 5 undergraduate participants (2 men, M age 18.7 yr., range 18-20) by investigating the time-course of pointing to peripherally located visual targets under four different levels of uncertainty (1, 2, 4, or 8 possible locations). Surprisingly, the resulting response function does not match any of those previously reported. Visually guided pointing produced a quadratic reaction time function as S-R uncertainty increases in log2 steps from 1 to 8. PMID- 16671611 TI - Construct validity of the Bender-Gestalt II: comparison with Wechsler Intelligence Scale for Children-III. AB - This study investigated the relationships of visuomotor constructs as measured by the Bender-Gestalt II with the subtests of the WISC-III. A factor analysis included the standardized Copy and Recall scores of the Bender-Gestalt II and subtest scores of the WISC-III. The Copy score was predicted to load on measures of visual and spatial thinking and the Recall score on a short-term memory factor. The results of a principal components analysis suggest a four-factor solution with the Bender-Gestalt II Copy score loading on a visual and spatial thinking factor primarily with WISC-III Perceptual Organization subtests. The Recall score loaded on a visual and spatial thinking factor as well as a short term memory factor with the WISC-III Digit Span subtest. The results suggest the Bender-Gestalt II Copy subtest shared commonality with the visual and spatial tasks given the similarity in the visuomotor demands of each test and was less influenced by processing speed or cultural knowledge than other tests on the visual and spatial thinking factor. Also, results suggest Bender-Gestalt II Recall measures elements of both visual and spatial thinking as well as aspects of short-term memory and memory retrieval. Clinical guidelines for co administration and underlying processing demands are discussed. PMID- 16671612 TI - Sex differences in the Poggendorff illusion: identifying the locus of the effect. AB - Extensive research has identified individual differences associated with sex in a range of visual task performances, including susceptibility to visual illusions. The aim of this study was to identify the locus of sex differences within the context of the Poggendorf illusion. 79 women and 79 men participated within a mixed factorial design. Analyses indicated that sex differences were only present in the stimulus context with the full inducing element present. This finding replicates recent research and provides qualifying evidence as to the locus of the effect. The findings are discussed within the functional framework of perceptual processes involved in extrapolating 3-dimensional characteristics from 2-dimensional visual stimuli. PMID- 16671613 TI - Visual behaviors of soccer players while kicking with the inside of the foot. AB - This study analyzed the visual behaviors of soccer players while they kicked with the inside of the foot, which involved near and far aiming skills. Participants (N = 8) were required to step forward and kick a ball to hit a target. The top three scorers were defined as the High-score group, and the three low scorers were defined as the Low-score group. Analysis indicated that the High-score group was characterized by longer quiet eye durations, which were defined as the final fixation durations on the target prior to the initiation of a kicking movement, than the Low-score group in the preparation phase. The High-score group also set their visual pivot on the frontal space between the target and the ball in the kicking phase. These two visual behaviors of the High-score group are important for soccer players to kick a ball successfully with the inside of the foot. PMID- 16671614 TI - Relation of game location and experience on mood states. AB - The study investigated relationships between game location, performers' experience, and mood states. 31 experienced collegiate soccer players completed the Brunel Mood Scale to assess anger, calmness, confusion, depression, fatigue, happiness, tension, and vigor before eight competitive games (four home and four away). Participants were categorized into Experienced and Less Experienced groups, based on the level of performance at which they played. Repeated-measures multivariate analyses of variance compared mean mood scores across location and experience, and follow-up univariate analyses suggested the increase in mood scores on Tension and decrease in scores on Calmness, Happiness, and Vigor between playing away and at home were significantly greater for Less experienced soccer players than Experienced players. Implications of these findings for the applied practitioner are discussed. PMID- 16671615 TI - Evaluation of some tasks used for specifying handedness and footedness. AB - Healthy men (n = 42) and women (n = 45) who were right-handed and men (n = 21) and women (n = 20) who were left-handed were studied. Men's mean age was 21.1 +/- 3.5 yr. and women's 20.7 +/- 3.1 yr. These students in various faculties reported they were right- or left-handed. Then their hand and foot preferences (handedness and footedness) were ascertained by asking each of the subjects to perform 11 tasks for handedness and 9 tasks for footedness. A discriminate function analysis test showed that each of the 11 tasks used for assessing their self-reported handedness was significant, but, of the 9 tasks used for assessing self-reported footedness, only 7 were significant. Strength of the hand or foot played no role in reports of handedness or footedness. A combination of four tasks, such as pulling a door, pushing a door, holding an object, and hammering a nail, on which the maximum number of subjects performed with the right or left hand, depending upon their self-reported handedness, would be ideal for ascertaining handedness. A combination of three tasks, namely, kicking a football, pushing an object with the foot, and stamping on the ground, would be ideal for ascertaining footedness. PMID- 16671616 TI - Saccadic latency as a function of target duration in a spatial localization task. AB - This study examined a potential confound in the inverse relationship between target duration and saccadic latency reported by Adam, Ketelaars, Kingma, and Hoek in 1993. Eight participants located a briefly flashed target by moving the eyes and the cursor toward its position in a (backward) mask condition and in a no-mask condition. Analysis showed similar saccadic latencies in both conditions, thereby refuting the potentially confounding role of the backward masking procedure. It is tenatively suggested that the longer saccadic latencies noted for shorter target durations may be associated with delayed accumulation of evidence for the detection of the target. PMID- 16671617 TI - Collegiate year of eligibility, sex, and sports achievement motivation. AB - The relations among year of eligibility, sex, and achievement motivation were investigated with 88 athletes representing three levels of collegiate eligibility (Year 1, Year 2/3, and Year 4) by sex. Participants were given Achievement Motivation subscales for Sporting Environments (approach-success practice, approach-success competition, failure-avoidance practice, failure-avoidance competition). A 3 x 2 analysis of variance showed significant main effects for year of eligibility and sex, and an interaction for age x sex for the failure avoidance competition scores. Mean approach-success and failure-avoidance practice scores were not significantly different. Fear of failure may be less for male athletes in Year 4 of eligibility. PMID- 16671618 TI - Perceptuomotor functioning in preschool children with symptoms of attention deficit hyperactivity disorder. AB - Given limited research on perceptual-motor functioning of preschool children with symptoms of ADHD, the purpose of this study was to compare the fine motor, gross motor, visuomotor, and oral-motor functioning of 49 4- to 6-yr.-old children with symptoms of Attention Deficit Hyperactivity Disorder and 48 typically developing children. Analysis showed scores of the ADHD group were significantly lower than those of the control group on all perceptuomotor measures. In addition, scores on all measures yielded significant correlations with scores on the Hyperactive Inattentive scale of the Preschool Behavior Questionnaire. Regression analyses indicated that the scores of gross motor and visuomotor functioning were significant predictors of group classification (with and without ADHD). These findings extend the well documented findings of perceptuomotor deficits among school-age children with ADHD into the preschool period and emphasize the importance of early assessment and treatment of these deficits in young children with symptoms of ADHD. PMID- 16671619 TI - Irrational beliefs in major depression and generalized anxiety disorders in an Iranian sample: a preliminary study. AB - Previous research on Rational Emotive Theory using western samples has shown that irrational belief systems are associated with emotional disorders such as anxiety and depression. In a replication of this work the present study was conducted to compare the scores of 29 clinically depressed, 31 clinically anxious, and 34 normal controls on the Irrational Beliefs Test in an Iranian sample. Consistent with Ellis's theory, depressed and anxious patients scored higher than the controls on scores for the full scale and all 10 subscales of the Irrational Beliefs Test. Also, the two patient groups differed significantly in their endorsement of 2 out of 10 irrational belief subscales but not in overall score on irrational beliefs. Results were interpreted in light of previous literature. PMID- 16671620 TI - Event-related potential as a measure of effects of report order and training on identification of multidimensional stimuli. AB - This experiment investigated effects of report order and training on multidimensional stimulus identification. Subjects were required to identify each two-dimensional symbol. There were two orders of reporting the two dimensions of a stimulus. Subjects performed the task for three practice sessions. Both behavioral data and event-related potentials were recorded. Analysis showed order of report had no significant effect on behavior data. But when participants responded in Order Shape/ Part, they showed more N2 inhibition for Order Part/Shape. The P3 mean amplitude was marginally significantly greater for Order Part/Shape than Order Shape/Part. This indicated Order Part/Shape, according to natural language habits, was more appropriate and less cognitively demanding than Order Shape/Part. For the two report orders, along with reduction of reaction time, amplitude of N1 and N2 increased with practice. Amplitude of P2 decreased with practice, and no P3 habituation was found. ERP data indicated no habituation effect. Results suggested neural activity depended not only on perceptual mechanism but also on extent of learning. PMID- 16671621 TI - WAIS-III task perception and performance as a function of stopwatch display. AB - Examiner's instructions for the Wechsler Adult Intelligence Scale-Third Edition indicate that stopwatch display is optional. Since verbal instructions do not consistently inform examinees whether a subtest is timed, examinees may rely upon the visual cue of the stopwatch. Furthermore, examinees' understanding of the actual subtest demands may affect their performances. In the present study, 58 volunteers were randomly assigned to two groups, one in which the examiner displayed the stopwatch and one in which the stopwatch was concealed. Participants were administered the first five subtests. Predictions were that (1) participants to whom the stopwatch was displayed would more accurately perceive whether the subtests were timed, and (2) participants who accurately understood the demands of subtests would score higher than those who did not. Results supported the first hypothesis for three subtests out of five, one remaining inconclusive due to a ceiling effect. The second hypothesis was not supported. Both the implications and limitations of the present study are discussed. PMID- 16671622 TI - Lack of impairment in patients with Parkinson's disease on an object-based negative priming task. AB - 12 nondemented patients with Parkinson's disease (M age = 67.3) and 12 normal control participants were administered an object-based attention task that enabled examination of both negative and positive priming. Unlike previous studies in which spatial-based attention tasks were used, results of the present study indicated that the patients displayed negative and positive priming not different from those shown by controls. These results suggest that certain object based attentional processes may not be impaired in patients with Parkinson's disease. PMID- 16671623 TI - Influence of target location on coincident timing performance. AB - This study examined the influence of two stimulus-presentation methods on coincident timing performance. 50 participants performed a button press and striking task in coincidence with a target stimulus. Participants were randomly divided into either the Terminating Group, whose target light was the last in the series, or the Apparent Motion Group, whose target was not the final light in the series and allowed the apparent motion of the stimulus to continue. 20 trials at each of three velocities, 4, 8, and 12 mph, were randomly performed for each task. The analysis yielded no significant differences between groups, indicating that coincident timing performance was not dependent on the method of stimulus presentation. PMID- 16671625 TI - Age and education in moral judgment of participants in team sports. AB - The present aim was to investigate the effect of age and education on the moral reasoning of the same 535 individuals in sports for whom nature of sport experience was reported. All 535 participants (M age = 24.9 yr., SD = 8.3) were involved in sports at the time of the study as athletes (n = 342), referees (n = 145), or coaches (n = 48), and had a wide range of education. Analysis of variance of scores on the Defining Issues Test of Rest showed moral judgment in sports differs significantly amongst different age groups (F5.510 = 5.37, p < .001) and amounts of education (F4.511 = 6.24, p < .001). Generally, with more education, higher moral judgment can be expected. It is apparent that moral development in sport is related to age and education, as also holds for a wider social setting. PMID- 16671624 TI - Laboratory strength measures of talented and less talented Greek children ages 9 to 10 years. AB - The purpose was twofold: (1) to assess whether laboratory tests can distinguish talented groups and less talented groups of children identified by their total scores in a battery of field tests and (2) to evaluate whether scores in field and laboratory tests are correlated. 36 children (ages 9 to 10 years) were divided into groups of higher and lower total scores on standing long jump, medicine ball throw, and sprint. Both groups were tested for maximum isometric force, force produced at the initial 100 msec., reactive force index, drop jump height, squat jump height, and maximum pedaling rate. Analysis showed the talented group had significantly (p < or = .05) higher scores on maximum isometric force, force produced in the initial 100 msec., and drop jump height than less talented children. Moderate to high correlations were found among scores for field performance tests with maximum pedaling rate. In conclusion, the laboratory tests distinguished the talented and less talented children in maximal isometric force, the force produced at the initial 100 msec., the reactive force index, and the drop jump height. PMID- 16671626 TI - Relations of body mass index and coronary risk as estimated by the Framingham Risk Score. AB - The Framingham Risk Score is considered to predict 10-yr. risk of developing coronary heart disease. Other risk factors, such as a family history of coronary heart disease, sedentary lifestyle, and obesity should also be considered when estimating the risk of development of coronary heart disease. The last two factors can be modified by therapeutic lifestyle alterations. This cross sectional coronary risk assessment of 16,871 Japanese subjects was estimated by the Framingham Risk Score. Sex and age were included in the calculation of the Framingham Risk Score. When multiple regression analysis was conducted controlling for age, regular physical exercise, smoking, and drinking, the body mass index was significantly associated with the Framingham Risk Score. The standardized regression coefficients for body mass index were .271 in men (p < .001) and .211 in women (p < .001), respectively. The significant association of body mass index with coronary heart disease risk, as estimated by the Framingham Risk Score, confirmed prior work. PMID- 16671627 TI - Effects of chromaticities and occlusion of shaft on magnitude of the Muller-Lyer illusion. AB - The magnitude of the Muller-Lyer illusion was investigated using the Brentano figure by varying the chromaticities of the shaft and the fins along the cone excitation axes and by varying the occlusion status of the shaft by the fins. 72 college students participated using the staircase method. The stimuli whose shaft and the fin chromaticities were the same produced larger illusions than stimuli with different chromaticities regardless of the luminance contrast between the figure and the surround. When the shaft appeared in front of the fins, the illusion effect was stronger than when the shaft was occluded by the fins. PMID- 16671628 TI - An examination of practice and laterality effects on the Purdue Pegboard and Moving Beans with Tweezers. AB - The purpose of this study was to examine the relation of practice or learning and laterality on the Purdue Pegboard and the Moving Beans with Tweezers test. The subjects were 30 right-handed, healthy young male adults (age: M = 21.1, SD = 1.9 yr.). The subjects performed both tests five times with each hand. A two-way analysis of variance (hand x trial) for scores on the Purdue Pegboard showed that Trial 1 had a significantly lower mean than Trials 4 and 5 with the dominant hand, and scores on Trials 1 and 3 were lower than that on Trial 5 for the nondominant hand. For the Moving Beans with Tweezers test, Trial 1 had a significantly lower score than Trial 5 with the nondominant hand. The dominant hand produced significantly higher values on all trials of both tests. Intraclass correlation coefficients of Trials 2 and 3 were high, and means were significantly higher for the dominant hand. A correlation between other tests was not high (r = .37-.46) for both hands. All correlations among five trials on both tests were significant and high (Purdue Pegboard-Dominant: average r =.72, [r] = .60-.80, Purdue Pegboard-Nondominant: average r = .59, [r] = .43-.76), (Moving Beans-Dominant: average r = .74, [r] = .57-.81, Moving Beans-Nondominant: average r =.71, [r] = .55-.87). Correlations among five trials for the ratio of dominant:nondominant hand on both tests were significant and moderate (Moving Beans: average r = .28, [r] = .08-.57, Purdue Pegboard: average r = .41, [r] = .19.56). Performance with the dominant hand is superior to that of the nondominant hand on both tests. Practice effects are found for both tests across five trials. The Moving Beans with Tweezers test also showed laterality of practice effects. The two tests may measure different abilities. PMID- 16671629 TI - Three feedback methods in acquisition and retention of badminton skills. AB - Comparison of three different feedback methods using information about the correct execution of the technique, errors of execution, and a combination of these on the acquisition and retention of two badminton skills of different difficulty were examined. Participants were 48 young male athletes, 10 to 14 years of age, with 2 to 4 years of training. They were divided into three equal groups and instructed on the long forehand serve, a more difficult serve than the short backhand. The first group received instructions for correct execution, the second group received instructional cues on errors of execution, and the third group received instructions on errors and how to correct them. The training program lasted 12 practice units. There was a pretest, a posttest after the end of the 10-wk. training program, and 2 wk. later, a retention test. A three-way (2 difficulty x 3 groups x 3 measures) analysis of variance with repeated measures on the last factor was applied to analyze scores with the three practice methods of corrective feedback for the two badminton skills. Analysis showed the group receiving correct technique information had increased scores on both skills. The group receiving information on errors increased their scores on the less difficult skill. The group receiving information on errors and correct technique increased their scores on the more difficult skill. Physical education teachers or coaches teaching skills to young participants should consider difficulty of skills and use appropriate corrective feedback for better acquisition and retention of sport skills. PMID- 16671630 TI - Response to Taguchi and Noma on "relationship between directionality and orientation in drawings by young children and adults. AB - When assessing the relationship between direction and orientation in drawings by young children and adults, Taguchi and Noma used a fish-drawing task. However, the fish is not convenient enough as an object for such a task so it is highly preferable to use, instead of a single object, a set of several objects to assess directionality quantitatively. These authors' conclusions do not acknowledge alternative explanations. PMID- 16671632 TI - Privacy and security in an electronic age. PMID- 16671631 TI - Validity and reliability of the Test of Early Reading Ability-Second Edition with preschool age children. AB - Criterion validity and test-retest reliability across the two forms of the Test of Early Reading Ability-Second Edition were examined. 18 preschool age children, 4 boys and 14 girls, between the ages of 37 and 59 mo. (M = 48.5, SD = 7.6) were randomly administered Forms A and B as well as the sound-blending, letter-word identification, spelling, and sound-awareness reading subtests from the 2001 Woodcock Johnson-III. Correlations were moderate between the children's performance on the Test of Early Reading Ability-2 Forms A and B and their reading subtest scores from the Woodcock Johnson-III and also between Forms A and B of the Test of Early Reading Ability-2. However, Forms A and B of the Test of Early Reading Ability-2 were not clinically equivalent. PMID- 16671633 TI - Speaking of EHRs: parsing EHR systems and the start of IT projects. PMID- 16671634 TI - CIO calling. How much IT does an IT project require? PMID- 16671635 TI - The language of health data exchange. PMID- 16671636 TI - Word power. A glossary of software licensing terms. PMID- 16671637 TI - Lessons from Katrina. PMID- 16671638 TI - Running out of room for data. HIPAA requires healthcare organizations to re assess data storage. PMID- 16671639 TI - HIM in corrections. PMID- 16671640 TI - Wanted: user-friendly system. Usability testing evaluates software through the eyes of the user. PMID- 16671641 TI - The current state of speech recognition. Despite high accuracy and growing interest, behavioral barriers remain. PMID- 16671642 TI - Using HL7 standards to evaluate an EHR. PMID- 16671643 TI - Coding technology today. AHIMA survey sheds light on coding's progress toward automation. PMID- 16671644 TI - Characteristics and challenges of long-term acute care hospitals. PMID- 16671645 TI - Mixing knowledge and data management. Using health IT skills to leverage technology. PMID- 16671647 TI - Medicare and long term care. PMID- 16671646 TI - The future of home health PPS. PMID- 16671648 TI - Home health and Medicare privatization: the managed care challenge. PMID- 16671649 TI - Quality improvement organizations: transforming the future of home health. AB - Every QIO is individually contracted by CMS to improve the quality of care to Medicare beneficiaries, to safeguard the Medicare Trust Fund, to protect beneficiaries by expeditiously addressing individual complaints, notices and appeals, and to ensure that Medicare beneficiaries receive the highest possible quality of health care. PMID- 16671650 TI - The medicare home health wage index. Is it time for change? AB - Of all of the elements and factors that make up the calculation of the payment rate under the Medicare home health benefit, the wage index may be the most important, yet overlooked, component. PMID- 16671651 TI - Medicare Part D and your agency. PMID- 16671652 TI - Medicare Hospice Benefit needs to be updated...not cut. PMID- 16671653 TI - National Association for Home Care & Hospice: key legislative issues January 2006. PMID- 16671654 TI - Pay for perfornmance: what, how, and when? PMID- 16671655 TI - Pandemic influenza planning: a guide for individuals and families. PMID- 16671656 TI - The "state" of home care & hospice. AB - There is at least one home care and hospice industry association in each of our 50 United States and Puerto Rico. These associations represent tens of thousands of home care and hospice providers. They provide state and local leadership, professional and para-professional training and other services designed to help home care and hospice providers care for our nation's sick, disabled and elderly. These are their stories. PMID- 16671657 TI - Pay-for-performance programs in home health care. PMID- 16671658 TI - The importance of best practices. PMID- 16671659 TI - Are you getting outsold? PMID- 16671660 TI - Hatathli: portrait of a healer. PMID- 16671661 TI - Caring thoughts. I am proud to have played a role in the enactment of Medicare in 1965, some 41 years ago. PMID- 16671662 TI - Electron transfer reactivity in matrix-assisted laser desorption/ionization (MALDI): ionization energy, electron affinity and performance of the DCTB matrix within the thermochemical framework. AB - DCTB [(H(3)C)(3)C-p-Ph-CH=C(CH(3))-trans-CH=C(CN)(2)] has recently advanced to the most promising matrix material for matrix-assisted laser desorption/ionization (MALDI) within material sciences. However, data that would allow the evaluation of the electron-transfer reactivity within a thermochemical framework are sparse. The present study reports the first-time determination of the ionization energy (IE) of DCTB applying photoelectron (PE) spectroscopy. The experimental IE (8.54 +/- 0.05 eV) is in excellent agreement with the theoretical value of 8.47 eV, obtained by AM1 calculations. The same level of theory determines the electron affinity (EA) as 2.31 eV. Model analytes of known thermochemistry (phenanthrene [C(14)H(10)], anthracene [C(14)H(10)] and fluorofullerene [C(60)F(46/48)]) are used to bracket the electron-transfer reactivity within DCTB-MALDI. The formation of molecular ions of these analytes either is expected or is beyond the thermochemical accessibility of the DCTB matrix. PMID- 16671664 TI - Time-dependent wave packet studies on the Cl + HCl hydrogen exchange reaction. AB - The initiation of the hydrogen exchange reaction Cl((2)P)+HCl --> ClH+Cl((2)P) by excitation of the HCl molecular stretch to v=2 is studied for total angular momentum quantum number J=(1)/(2) and both even and odd parity. The calculations were performed using a time-dependent propagation from an initial quasi-bound state and employed all three relevant potential energy surfaces and the nonadiabatic couplings between them. Coriolis and spin-orbit coupling were also taken into account. The electronic and HCl rotational distributions of the products in both dissociation channels are analyzed, and the results are interpreted using features of the potential energy surfaces. PMID- 16671663 TI - Resonance Raman scattering of rhodamine 6G as calculated using time-dependent density functional theory. AB - In this work, we present the first calculation of the resonance Raman scattering (RRS) spectrum of rhodamine 6G (R6G) which is a prototype molecule in surface enhanced Raman scattering (SERS). The calculation is done using a recently developed time-dependent density functional theory (TDDFT) method, which uses a short-time approximation to evaluate the Raman scattering cross section. The normal Raman spectrum calculated with this method is in good agreement with experimental results. The calculated RRS spectrum shows qualitative agreement with SERS results at a wavelength that corresponds to excitation of the S(1) state, but there are significant differences with the measured RRS spectrum at wavelengths that correspond to excitation of the vibronic sideband of S(1). Although the agreement with the experiments is not perfect, the results provide insight into the RRS spectrum of R6G at wavelengths close to the absorption maximum where experiments are hindered due to strong fluorescence. The calculated resonance enhancements are found to be on the order of 10(5). This indicates that a surface enhancement factor of about 10(10) would be required in SERS in order to achieve single-molecule detection of R6G. PMID- 16671665 TI - Empirical description of chiral autocatalysis. AB - The only known example of chiral autocatalysis is the alkylation of N heterocyclic aldehydes with iPr(2)Zn (Soai reaction). The mechanism and some details of this reaction are not yet clear. An empirical formula is proposed here for the description of this chiral autocatalytic reaction. This formula allows the calculation of some very informative parameters. PMID- 16671666 TI - Competing isomerizations: a combined experimental/theoretical study of phenylpentenone isomerism. AB - The possible competition of Z/E versus hydrogen-shift isomerization in (E)-5 phenyl-3-penten-2-one (E-1) and (E)-5-phenyl-4-penten-2-one (E-2) was studied, both experimentally and theoretically. Iodine-catalyzed isomerization experiments and computational modeling studies show that the equilibrated system consists predominantly of E-1 and E-2, with E-2 in moderate excess, and with no detectable amounts of the Z (cis) diastereoisomers. Density functional theory (DFT) calculations corroborated the free energy difference (Delta(r) and Delta(r) were 0.7 and -1.1 kcal mol(-1), respectively), and computations of Boltzmann-weighted (1)H NMR spectra were found to be useful in confirming the assignment of the isomers. The relevance of this equilibrium to earlier work on double-bond stabilization is discussed. PMID- 16671667 TI - The effect of solvent polarity on the balance between charge transfer and non charge transfer pathways in the sensitization of singlet oxygen by pipi triplet states. AB - A large set of literature kinetic data on triplet (T(1)) sensitization of singlet oxygen by two series of biphenyl and naphthalene sensitizers in solvents of strongly different polarity has been analyzed. The rate constants and the efficiencies of singlet oxygen formation are quantitatively reproduced by a model that assumes the competition of a non-charge transfer (nCT) and a CT deactivation channel. nCT deactivation occurs from a fully established spin-statistical equilibrium of (1)(T(1)(3)Sigma) and (3)(T(1)(3)Sigma) encounter complexes by internal conversion (IC) to lower excited complexes that dissociate to yield O(2)((1)Sigma(g)(+)), O(2)((1)Delta(g)), and O(2)((3)Sigma(g)(-)). IC of (1,3)(T(1)(3)Sigma) encounter complexes is controlled by an energy gap law that is generally valid for the transfer of electronic energy to and from O(2). (1,3)(T(1)(3)Sigma) nCT complexes form in competition to IC (1)(T(1)(3)Sigma) and (3)(T(1)(3)Sigma) exciplexes if CT interactions between T(1) and O(2) are important. The rate constants of exciplex formation depend via a Marcus type parabolic model on the corresponding free energy change DeltaG(CT), which varies with sensitizer triplet energy, oxidation potential, and solvent polarity. O(2)((1)Sigma(g)(+)), O(2)((1)Delta(g)), and O(2)((3)Sigma(g)(-)) are formed in the product ratio (1/6):(1/12):(3/4) in the CT deactivation channel. The balance between nCT and CT deactivation is described by the relative contribution p(CT) of CT induced deactivation calculated for a sensitizer of known triplet energy from its quenching rate constant. It is shown how the change of p(CT) influences the quenching rate constant and the efficiency of singlet oxygen formation in both series of sensitizers. p(CT) is sensitive to differences of solvent polarity and varies for the biphenyls and the naphthalenes as sigmoidal with DeltaG(CT). This quantitative model represents a realistic and general mechanism for the quenching of pipi triplet states by O(2), surpassing previous advanced models. PMID- 16671668 TI - Solvent and pH dependent fluorescent properties of a dimethylaminostyryl borondipyrromethene dye in solution. AB - Steady-state and time-resolved fluorescence techniques have been used to study the photophysical properties of the fluorescent BODIPY-derived dye 3-{2-[4 (dimethylamino)phenyl]ethenyl}-4,4-difluoro-8-(4-methoxyphenyl)-1,5,7-trimethyl 3a,4a-diaza-4-bora-s-indacene. This compound has been synthesized via a microwave assisted condensation of p-N,N-dimethylaminobenzaldehyde with the appropriate 1,3,5,7-tetramethyl substituted borondipyrromethene unit. The fluorescence properties of the dye are strongly solvent dependent: increasing the solvent polarity leads to lower fluorescence quantum yields and lifetimes, and the wavelength of maximum fluorescence emission shifts to the red. The Catalan solvent scales are found to be the most suitable for describing the solvatochromic shifts of the fluorescence emission. These are dominated by polarity/polarizability effects, as confirmed by quantum-chemical calculations performed in the dielectric continuum approximation. Fluorescence decay profiles of the dye can be described by a single-exponential fit in most solvents investigated, while two decay times are found in alcohols. The dye undergoes a reversible protonation-deprotonation reaction in the acidic pH range with a pK(a) of 2.25 in acetonitrile solution. Fluorimetric titrations as a function of pH produce fluorescence emission enhancements at lower pH. The fluorescence excitation spectra show a hypsochromic shift from 600 nm for the neutral amine to 553 nm for the ammonium form, so that ratiometric measurements can be used to determine pK(a). PMID- 16671669 TI - ZEKE-PFI spectroscopy of benzocaine. AB - The adiabatic ionization threshold (AIT) of trans- and gauche-benzocaine has been measured by zero electron kinetic energy-pulsed field ionization (ZEKE-PFI) spectroscopy to be 7.8412+/-0.0008 eV (lasers at 34,134.4 and 29,109.3 cm(-1)) and 7.8421+/-0.0004 eV (34,144.8+29105.7 cm(-1)), respectively. AITs computed at the B3LYP/AUG-cc-p-VDZ level for the two conformers are some approximately 2,500 cm(-1) lower than the experimental; in contrast their energy difference is very close. The trans-benzocaine cation ZEKE spectra has been recorded taking a number of S(1) intermediate vibronic states. The spectra provide an energy threshold for the appearance of intramolecular vibrational redistribution (IVR) around approximately 540 cm(-1) in the S(1) state. PMID- 16671670 TI - Photoinduced amino-imino tautomerism: an infrared study of 2-amino-5 methylpyridine in a low-temperature argon matrix. AB - The photoreaction of 2-amino-5-methylpyridine was investigated by matrix isolation infrared spectroscopy and DFT calculation. Photoinduced reversible amino (N=C-NH(2))-imino (NH-C=NH) tautomerism was found between 2-amino-5 methylpyridine and 5-methyl-2(1H)-pyridinimine; the amino tautomer changes to the imino tautomer by UV irradiation (340>lambda>or= 300 nm) and the reverse change occurs by longer-wavelength light irradiation (420>lambda>or= 340 nm). The results of the CASSCF calculation revealed that the amino-imino tautomerism proceeds in vibrational relaxation process from electronic excited state to the ground state. The IR spectra of 2-amino-5-methylpyridine in the T(1) state and 5 methyl-2-pyridinamino radical were also obtained by UV irradiation (lambda>or= 300 nm). PMID- 16671672 TI - Theoretical study of mixed silicon-lithium clusters Si(n)Li(p)(+) (n=1-6, p=1-2). AB - Theoretical study on the structures of neutral and singly charged Si(n)Li(p)((+)) (n=1-6, p=1-2) clusters have been carried out in the framework of the density functional theory (DFT) with the B3LYP functional. The structures of the neutral Si(n)Li(p) and cationic Si(n)Li(p)(+) clusters are found to keep the frame of the corresponding Si(n), Li species being adsorbed at the surface. The localization of the lithium cation is not the same one as that of the neutral atom. The Li(+) ion is preferentially located on a Si atom, while the Li atom is preferentially attached at a bridge site. A clear parallelism between the structures of Si(n)Na(p) and those of Si(n)Li(p) appears. The population analysis show that the electronic structure of Si(n)Li(p) can be described as Si(n)(p)(-)+pLi(+) for the small sizes considered. Vertical and adiabatic ionization potentials, adsorption energies, as well as electric dipole moments and static dipolar polarizabilities, are calculated for each considered isomer of neutral species. PMID- 16671671 TI - A combined electron paramagnetic resonance and fourier transform infrared study of the co(c(6)h(6))(1,2) complexes isolated in neat benzene or in cryogenic matrixes. AB - The products obtained in the reaction of cobalt atoms in neat benzene or in a benzene/argon mixture at low temperature have been reinvestigated. At least three cobalt-containing species were detected by IR, namely, Co(C(6)H(6)), Co(C(6)H(6))(2), and Co(x)(C(6)H(6)), x>1. The IR bands were assigned to these complexes by monitoring their behavior as a function of (a) Co and C(6)H(6) concentration, (b) isotopic substitution, and (c) photoirradiation. We were able to analyze the sample in neat benzene by both electron paramagnetic resonance (EPR) and IR spectroscopy and to determine the magnetic parameters (g tensor and Co hyperfine interaction) for the Co(C(6)H(6))(2) sandwich compound. The large number of fundamental bands observed in the IR spectrum of Co(C(6)H(6))(2), the absorption pattern observed in the Co-ring stretching region of the IR spectrum of the mixed complex, Co(C(6)H(6))(C(6)D(6)) and the orthorhombic g-values extracted from the EPR spectrum are most consistent with nonequivalent benzene ligands in Co(C(6)H(6))(2), i.e., C(s) symmetry. A bonding scheme consistent with both the EPR and IR data for Co(C(6)H(6))(2) is discussed. PMID- 16671673 TI - Coriolis-coupled wave packet dynamics of H + HLi reaction. AB - We investigated the effect of Coriolis coupling (CC) on the initial state selected dynamics of H+HLi reaction by a time-dependent wave packet (WP) approach. Exact quantum scattering calculations were obtained by a WP propagation method based on the Chebyshev polynomial scheme and ab initio potential energy surface of the reacting system. Partial wave contributions up to the total angular momentum J=30 were found to be necessary for the scattering of HLi in its vibrational and rotational ground state up to a collision energy approximately 0.75 eV. For each J value, the projection quantum number K was varied from 0 to min (J, K(max)), with K(max)=8 until J=20 and K(max)=4 for further higher J values. This is because further higher values of K do not have much effect on the dynamics and also because one wishes to maintain the large computational overhead for each calculation within the affordable limit. The initial state-selected integral reaction cross sections and thermal rate constants were calculated by summing up the contributions from all partial waves. These were compared with our previous results on the title system, obtained within the centrifugal sudden and J-shifting approximations, to demonstrate the impact of CC on the dynamics of this system. PMID- 16671674 TI - Electronic spectra of hydrogen-bonded 2-fluoropyridine clusters with water in a supersonic free jet. AB - Fluorescence excitation, multiphoton ionization, and dispersed fluorescence spectra of bare and hydrogen-bonded 2-fluoropyridine with water were measured in a supersonic free jet. For bare 2-fluoropyridine, fluorescence quantum yield decrease in the higher vibronic levels was observed even under collision-free conditions. The inter-system crossing channel was probed experimentally by two color R2PI and found to be negligible. The non-radiative relaxation process of 2 fluoropyridine is mainly governed by the relaxation to the electronic ground state. Electronic spectra of 2-fluoropyridine-(water)(n) (n=1 approximately 3) were also obtained. The hydrogen bond formation with water increases the quantum yield in the higher vibronic levels. Rather low frequency vibrations were observed in the hole burning spectrum of bare 2-fluoropyridine; however, these vibronic bands disappeared with the hydrogen bond formation with water. The appearance of low frequency vibronic bands observed for bare 2-fluoropyridine is ascribed to the existence of closely lying (n,pi) state. PMID- 16671675 TI - Structural and conformational properties and intramolecular hydrogen bonding of (methylenecyclopropyl)methanol, as studied by microwave spectroscopy and quantum chemical calculations. AB - The microwave spectra of (methylenecyclopropyl)methanol (H(2)C=C(3)H(3)CH(2)OH) and one deuterated species (H(2)C=C(3)H(3)CH(2)OD) have been investigated in the 20-80 GHz spectral range. Accurate spectral measurements have been performed in the 40-80 GHz spectral interval. The spectra of two rotameric forms, denoted conformer I and conformer IX, have been assigned. Both these rotamers are stabilized by intramolecular hydrogen bonds formed between the hydrogen atom of the hydroxyl group and the pseudo-pi electrons on the outside of the cyclopropyl ring, the so-called "banana bonds". The carbon-carbon bond lengths in the ring are rather different. The bonds adjacent to the methylene group (H(2)C=) are approximately 7 pm shorter that the carbon-carbon bond opposite to this group. It is found from relative intensity measurements of microwave transitions that conformer IX, in which the hydrogen bond is formed with the banana bonds of the long carbon-carbon bond, is 0.4(3) kJ/mol more stable than conformer I, where the hydrogen bond is formed with the pseudo-pi electrons belonging to the shortest carbon-carbon bond of the ring. The microwave study has been augmented by quantum chemical calculations at the MP2/6-311++G, G3 and B3LYP/6-311++G levels of theory. PMID- 16671676 TI - Atom and ion chemistry in low pressure hydrogen dc plasmas. AB - The chemical composition of a low-pressure hydrogen dc plasma produced in a hollow cathode discharge has been measured and modeled. The concentrations of H atoms and of H(+), H(2)(+) and H(3)(+) ions were determined with a combination of optical spectroscopic and mass spectrometric techniques, over the range of pressures (p approximately 0.008-0.2 m bar) investigated. The results were rationalized with the help of a zero-order kinetic model. A comparatively high fraction ( approximately 0.1+/-0.05) of H atoms, indicative of a relatively small wall recombination, was observed. Low ionization degrees (<10(-4)) were obtained in all cases. In general, the ionic composition of the plasma was found to be dominated by H(3)(+), except at the lowest pressures, where H(2)(+) was the major ion. The key physicochemical processes determining the plasma composition were identified from the comparison of experimental and model results, and are discussed in the paper. PMID- 16671677 TI - New experimental data and mechanistic studies on the bromate-dual substrate-dual catalyst batch oscillator. AB - The bromate-hypophosphite-acetone-Mn(II)-Ru(bpy)(3)(2+) batch oscillator was recently suggested for studying two-dimensional pattern formation. The system meets all major requirements that are needed for generation of good quality traveling waves in a thin solution layer. The serious drawback of using the system for studying temporal and spatial dynamical phenomena is its unknown chemical mechanism. In order to develop a mechanism that explains the observed long-lasting batch oscillations the bromate-hypophosphite-acetone-Mn(II) Ru(bpy)(3)(2+) oscillator was revisited. We studied the dynamics both in the total system and in some composite reactions, and kinetic measurements were carried out in three subsystems. From the new experimental results we concluded that the two oscillatory sequences observed in the full system are originated from two oscillatory subsystems, the Mn(II)-catalyzed bromate-hypophosphite acetone and the Ru(bpy)(3)(2+)-catalyzed bromate-bromoacetone reactions. Here we propose a mechanism which is capable of simulating the dynamical features that appeared in the complex system. PMID- 16671678 TI - Mechanistic study of the CH(3)O(2)(*) + HO(2)(*) --> CH(3)O(2)H + O(2) reaction in the gas phase. computational evidence for the formation of a hydrogen-bonded diradical complex. AB - In an attempt to understand the mechanism of the reaction of alkylperoxy radicals with hydroperoxy radical, a key reaction in both atmospheric and combustion chemistry, the singlet and triplet potential energy surfaces (PESs) for the gas phase reaction between CH(3)O(2)(*) and HO(2)(*) leading to the formation of CH(3)OOH and O(2) have been investigated by means of quantum-mechanical electronic structure methods (CASSCF and CASPT2). In addition, standard transition state theory calculations have been carried out with the main purpose of a qualitative description of the strong negative temperature dependence observed for this reaction. All the pathways on both the singlet and triplet PESs consist of a reversible first step involving the barrierless formation of a hydrogen-bonded pre-reactive complex, followed by the irreversible formation of products. This complex is a diradical species where the two unpaired electrons are not used for bonding and is lying about 5 kcal/mol below the energy of the reactants at 0 K. The lowest energy reaction pathway occurs on the triplet PES and involves the direct H-atom transfer from HO(2) to CH(3)O(2) in the diradical complex through a transition structure lying 3.8 kcal/mol below the energy of the reactants at 0 K. Contradicting the currently accepted interpretation of the reaction mechanism, the observed strong negative temperature dependence of the rate constant is due to the formation of the hydrogen-bonded diradical complex rather than a short-lived tetraoxide intermediate CH(3)OOOOH. PMID- 16671679 TI - Self-consistent combination of the three-dimensional RISM theory of molecular solvation with analytical gradients and the Amsterdam density functional package. AB - The three-dimensional reference interaction site model with the closure relation by Kovalenko and Hirata (3D-RISM-KH) in combination with the density functional theory (DFT) method has been implemented in the Amsterdam density functional (ADF) software package. The analytical first derivatives of the free energy with respect to displacements of the solute nuclear coordinates have also been developed. This enables study of chemical reactions, including reaction coordinates and transition state search, with the molecular solvation described from the first principles. The method yields all of the features available by using other solvation approaches, for instance infrared spectra of solvated molecules. To evaluate the accuracy of the present method, test calculations have been carried out for a number of small molecules, including four glycine conformers, a set of small organic compounds, and carbon nanotubes of various lengths in aqueous solution. Our predictions for the solvation free energy agree well with other approaches as well as experiment. This new development makes it possible to calculate at modest computational cost the electronic properties and molecular solvation structure of a solute molecule in a given molecular liquid or mixture from the first principles. PMID- 16671680 TI - Theoretical analysis of the structural and electronic properties of metalloporphyrin pi-cation radicals. AB - A method for analyzing the A(1u)/A(2u) contents of metalloporphyrin pi-cation radicals is developed and applied to a series of unsubstituted planar metalloporphines (MPs) (M=Cr, Mn, Fe, Co, Ni, Cu, and Zn). The structures and electronic properties of the MPs and their cation radicals were calculated by density functional theory (DFT) and subsequently analyzed. It was found that the MPs with small core sizes have a tendency to form A(1u)-type radicals, while the MPs with large core size have a preference for an A(2u)-type. Neither of these pure-state species, however, is stable under the D(4)(h) symmetry, and both radical cation types are subject to pseudo-Jahn-Teller (pJT) distortion. The pJT distortion leads to structures with lower symmetry and states that have mixed character with respect to the A(1u) and A(2u) components. The degree of mixing could be estimated by employing orbital projection technique or a complementary spin density decomposition. Both techniques produce very similar results, pointing out that the frontier orbital, which becomes empty upon electron removal, plays a critical role in determining electronic properties. PMID- 16671681 TI - Folded and unfolded conformations of the omega-3 polyunsaturated fatty acid family: ch(3)ch(2)[ch=chch(2)](b)[ch(2)](m)cooh: first principles study. AB - Polyunsaturated fatty acids (PUFA) like stearidonic acid (SDA;18:4 n-3) eicosapentaenoic acid (EPA; 20:5 n-3), and docosahexaenoic acid (DHA; 22:6 n-3) and its chain fragment models were studied at B3LYP/6-31G(d) levels of theory. Significant conformations for the cis and trans isomers were selected to obtained the thermodynamic functions (DeltaH, DeltaS, DeltaG) for the cis-trans isomerization and for folding using the B3LYP/6-311+G(2d,p)//B3LYP/6-31G(d) level of theory. The structural analysis shows that there are significant differences in thermodynamic function of the trans- and cis-PUFAs. The trans-cis isomerization energy values reinforce the consistency and the relative accuracy of theoretical model calculations. The observed flexibility of naturally cis PUFAs could be explained by a very special "smooth basin" PES of the motif of sp(2)-sp(3)-sp(2) hybrid states as reported previously (J. Phys. Chem. A 2005, 109, 520-533). We assumed that intrinsic thermodynamic functions may describe this flexible folding process. The folding enthalpy as well as the folding entropy suggests that there is a new role of the cis-PUFAs in membranes: these cis isomers may have a strong influence on membrane stability and permeability. The average length of the cis helix and beta PUFA was approximated. The difference between the lengths of these two structures is approximately 10 A. PMID- 16671682 TI - On the competition between scavenging and recombination in solutions of macromolecules. AB - This paper is concerned with the competition between recombination of a radical pair and radical attack on targets such as macromolecules or nanoparticles in solution, which are large on the molecular scale. The difference in scale between radicals and targets causes the kinetics to be transient over a long period. The specific novel feature of the analysis is the effect of the initial spatial correlation of the radicals on the kinetics of attack on the targets. The main results are (i) a simple modification of the Smoluchowski rate coefficient for scavenging and (ii) the probability of multiple hits on the same target. Both effects arise from the clustering of the radicals. The latter is of particular interest in radiation biology, because multiple hits result in complex damage. The analysis is validated against results from random flights computer simulation; excellent agreement is obtained. PMID- 16671683 TI - Structures of heterogeneous proton-bond dimers with a high dipole moment monomer: covalent vs electrostatic interactions. AB - A number of calculated structures of heterogeneous proton-bound dimers containing monomers such as acetonitrile, cyanamide, vinylene carbonate, and propiolactone, which have high dipole moments, are presented. These proton-bound dimers are predicted to have a structural anomaly pertaining to the bond distances between the central proton and the basic sites on each of the monomers. The monomers with the high dipole moments also have the larger proton affinity and, on the basis of difference in proton affinities, it would be expected that the proton would be closer to this monomer than the one with the lower proton affinity. However, the proton is found to lie substantially closer to the monomer with the lower proton affinity in most cases, unless the difference in proton affinity is too large. Simply stated, the difference in proton affinities is smaller than the difference in the affinity to form an ion-dipole complex for the two monomers and it is the larger affinity for the high dipole moment monomer (which also has the higher proton affinity) to form an ion-dipole complex that is responsible for the proton lying closer to the low proton affinity monomer. The bond distances between the central proton and the monomers are found to be related to the difference in proton affinity. It is found, though, that the proton-bound dimers can be grouped into two separate groups, one where the proton-bound dimer contains a high dipole moment monomer and one group where the proton-bound dimer does not contain a high dipole moment monomer. From these plots it has been determined that a high dipole moment monomer is one that has a dipole moment greater than 2.9 D. PMID- 16671684 TI - H-Atom abstraction from CH(3)NHNH(2) by NO(2): CCSD(T)/6-311++G(3df,2p)//MPWB1K/6 31+G(d,p) and CCSD(T)/6-311+G(2df,p)//CCSD/6-31+G(d,p) calculations. AB - Stationary points of paths for H atom abstraction from CH(3)NHNH(2) (monomethylhydrazine) by NO(2) were characterized via CCSD(T)/6 311++G(3df,2p)//MPWB1K/6-31+G(d,p) and CCSD(T)/6-311+G(2df,p)//CCSD/6-31+G(d,p) calculations. Five transition states connecting CH(3)NHNH(2)-NO(2) complexes to a manifold that includes CH(3)NHNH-HONO, CH(3)NNH(2)-HONO, CH(3)NNH(2)-HNO(2), and CH(3)NHNH-HNO(2) complexes were identified. Transition states that connect CH(3)NHNH-HONO, CH(3)NNH(2)-HONO, CH(3)NNH(2)-HNO(2), and CH(3)NHNH-HNO(2) complexes to each other via H atom exchange and/or hindered internal rotation were also identified. The high point in the minimum energy path from the CH(3)NHNH(2) + NO(2) reactant asymptote to the manifold of HONO-containing product states is a transition state 8.6 kcal/mol above the reactant asymptote. From a kinetics standpoint, this value is considerably higher than the 5.9 kcal/mol value that was estimated for it based on theoretical results for H atom abstraction from NH(3) by NO(2). PMID- 16671686 TI - Mechanisms of glycerol dehydration. AB - Dehydration of neutral and protonated glycerol was investigated using quantum mechanical calculations (CBS-QB3). Calculations on neutral glycerol show that there is a high barrier for simple 1,2-dehydration, E(a)=70.9 kcal mol(-1), which is lowered to 65.2 kcal mol(-1) for pericyclic 1,3-dehydration. In contrast, the barriers for dehydration of protonated glycerol are much lower. Dehydration mechanisms involving hydride transfer, pinacol rearrangement, or substitution reactions have barriers between 20 and 25 kcal mol(-1). Loss of water from glycerol via substitution results in either oxirane or oxetane intermediates, which can inter-convert over a low barrier. Subsequent decomposition of these intermediates proceeds via either a second dehydration step or loss of formaldehyde. The computed mechanisms for decomposition of protonated glycerol are supported by the gas-phase fragmentation of protonated glycerol observed using a triple--quadrupole mass spectrometer. PMID- 16671685 TI - Theoretical study of interaction of urate with li(+), na(+), k(+), be(2+), mg(2+), and ca(2+) metal cations. AB - The geometries and energetics of complexes of Li(+), Na(+), K(+), Be(2+), Mg(2+), and Ca(2+)metal cations with different possible uric acid anions (urate) were studied. The complexes were optimized at the B3LYP level and the 6-311++G(d,p) basis set. Complexes of urate with Mg(2+), and Ca(2+)metal cations were also optimized at the MP2/6-31+G(d) level. Single point energy calculations were performed at the MP2/6-311++G(d,p) level. The interactions of the metal cations at different nucleophilic sites of various possible urate were considered. It was revealed that metal cations would interact with urate in a bi-coordinate manner. In the gas phase, the most preferred position for the interaction of Li(+), Na(+), and K(+) cations is between the N(3) and O(2) sites, while all divalent cations Be(2+), Mg(2+), and Ca(2+) prefer binding between the N(7) and O(6) sites of the corresponding urate. The influence of aqueous solvent on the relative stability of different complexes has been examined using the Tomasi's polarized continuum model. The basis set superposition error (BSSE) corrected interaction energy was also computed for complexes. The AIM theory has been applied to analyze the properties of the bond critical points (electron densities and their Laplacians) involved in the coordination between urate and the metal cations. It was revealed that aqueous solvation would have significant effect on the relative stability of complexes obtained by the interaction of urate with Mg(2+) and Ca(2+)cations. Consequently, several complexes were found to exist in the water solution. The effect of metal cations on different NH and CO stretching vibrational modes of uric acid has also been discussed. PMID- 16671687 TI - The electronic spectrum of chloroformic acid in comparison to formic acid. AB - The electronic spectra of chloroformic acid ClCOOH and formic acid HCOOH are computed in large-scale multireference configuration interaction (MRD-CI) calculations. The computed spectrum of formic acid is in reasonable agreement with prior calculations and experimental data. The first electronic transition of ClCOOH is computed at 6.41 eV (193.4 nm), about 0.5 eV higher than in HCOOH. Together with five strong transitions calculated at 7.66 eV (161.9 nm; 2(1)A' <-- X(1)A'), 8.36 eV (148.3 nm; 3(1)A' <-- X(1)A'), 8.49 eV (146.0 nm; 4(1)A' <-- X(1)A'), 9.00 eV (137.8 nm; 5(1)A' <-- X(1)A'), and 9.44 eV (131.3 nm; 7(1)A' <-- X(1)A'), this can serve as a guideline for experimental search of ClCOOH. PMID- 16671688 TI - Raman spectroscopic study on solvation of diphenylcyclopropenone and phenol blue in room temperature ionic liquids. AB - We investigated the solvation of several room temperature ionic liquids by Raman spectroscopy using diphenylcyclopropenone (DPCP) and phenol blue (PB) as probe molecules. We estimated acceptor numbers (AN) of room temperature ionic liquids by an empirical equation associated with the Raman band of DPCP assigned as a C=C stretching mode involving a significant C=O stretching character. According to the dependence of AN on cation and anion species, the Lewis acidity of ionic liquids is considered to come mainly from the cation charge. The frequencies and bandwidths of the C=O and C=N stretching modes of phenol blue are found to be close to those in conventional polar solvents such as methanol and dimethyl sulfoxide. The frequencies of these vibrational modes show similar dependence upon the electronic absorption band center as is observed in conventional liquid solvents. However, peculiar behavior was found in the Raman bandwidths and the excitation wavelength dependence of the C=N stretching mode in room temperature ionic liquids. Both the bandwidth of the C=N stretching mode and the extent of the excitation wavelength dependence of the Raman shift of the C=N stretching mode tend to decrease as the absorption band center decreases, in contrast to the case of conventional solvents. This anomaly is discussed in terms of the properties of room temperature ionic liquids. PMID- 16671689 TI - On the electronic properties of dehydrogenated polycyclic aromatic hydrocarbons. AB - The electronic excited-state properties of a series of dehydrogenated polycyclic aromatic hydrocarbons from phenyl through to decacyl are reported. The radicals were investigated by use of time-dependent density functional theory in conjunction with the B3LYP functional. The pi and n orbitals were seen to converge in energy as the system increased in size, yet all radicals were found to have A' ground states. In addition to the study of the electronic state symmetries, the excited-state transitions and oscillator strengths were investigated with the resulting transitions found within the visible region of the spectrum, placing these radicals in the large group of candidate carriers of the diffuse interstellar bands. PMID- 16671690 TI - Density functional theory study of the properties of N-H...N, non cooperativities, and intermolecular interactions in linear trans-diazene clusters up to ten molecules. AB - We investigate aspects of N-H...N hydrogen bonding in the linear trans-diazene clusters (n=2-10) such as the N...H and N-H lengths, n(N) --> sigma(N-H) interactions, N...H strengths, and frequencies of the N-H stretching vibrations utilizing the DFT/B3LYP theory, the natural bond orbital (NBO) method, and the theory of atoms in molecules (AIM). Our calculations indicate that the structure and energetics are qualitatively different from the conventional H-bonded systems, which usually exhibit distinct cooperative effects, as cluster size increases. First, a shortening rather than lengthening of the N-H bond is found and thus a blue rather than red shift is predicted. Second, for the title clusters, any sizable cooperative changes in the N-H and N...H lengths, n(N) --> sigma(N-H) charge transfers, N...H strengths, and frequencies of the N-H stretching vibrations for the linear H-bonded trans-diazene clusters do not exist. Because the n(N) --> sigma(N-H) interaction hardly exhibits cooperative effects, the capability of the linear trans-diazene cluster to localize electrons at the N...H bond critical point is almost independent of cluster size and thereby leads to the noncooperative changes in the N...H lengths and strengths and the N-H stretching frequencies. Third, the dispersion energy is sizable and important; more than 30% of short-range dispersion energy not being reproduced by the DFT leads to the underestimation of the interaction energies by DFT/B3LYP. The calculated nonadditive interaction energies show that, unlike the conventional H-boned systems, the trans-diazene clusters indeed exhibit very weak nonadditive interactions. PMID- 16671691 TI - Contribution of omega-3 fatty acids to the thermodynamics of membrane protein solvation. AB - Recent NMR experiments and molecular dynamics simulations have indicated that rhodopsin is preferentially solvated by omega-3 fatty acids compared to saturated chains. However, to date no physical theory has been advanced to explain this phenomenon. The present work presents a novel thermodynamic explanation for this preferential solvation based on statistical analysis of 26,100 ns all-atom molecular dynamics simulations of rhodopsin in membranes rich in polyunsaturated chains. The results indicate that the preferential solvation by omega-3 chains is entropically driven; all chains experience an entropic penalty when associating with the protein, but the penalty is significantly larger for saturated chains. PMID- 16671692 TI - Sequence dependence of charge transport properties of DNA. AB - The electrical conduction through three short oligomers (26 base pairs, 8 nm long) with differing numbers of GC base pairs was measured. One strand is poly(A) poly(T), which is entirely devoid of GC base pairs. Of the two additional strands, one contains 8 and the other 14 GC base pairs. The oligomers were adsorbed on a gold substrate on one side and to a gold nanoparticle on the other side. Conducting atomic force microscope was used for obtaining the current versus voltage curves. We found that in all cases the DNA behaves as a wide band gap semiconductor, with width depending on the number of GC base pairs. As this number increases, the band-gap narrows. For applied voltages exceeding the band gap, the current density rises dramatically. The rise becomes sharper with increasing number of GC base pairs, reaching more than 1 nA/nm2 for the oligomer containing 14 GC pairs. PMID- 16671693 TI - Pulsed laser syntheses of layer-structured WS2 nanomaterials in water. AB - We used water as an environmental friendly medium for the synthesis of hexagonal WS2 nanoparticles by the pulsed laser method. The materials collected on substrates were oriented with the 2H-WS2 basal planes parallel to the surface. The use of water, UV lasers, and large WS2 targets prevented the nanoparticles from restructuring into inorganic fullerenes, which were observed in research using hydrocarbon solvents, longer wavelength lasers, and dispersed powder targets. Fairly good dispersion of nanoparticles suggests that large surface areas are available for chemical reactivity. PMID- 16671694 TI - Computational observation of enhanced solvation of the hydroxyl radical with increased NaCl concentration. AB - Classical molecular dynamics simulations with many-body potentials were carried out to quantitatively determine the effect of NaCl salt concentration on the aqueous solvation and surface concentration of hydroxyl radicals. The potential of mean force technique was used to track the incremental free energy of the hydroxyl radical from the vapor, crossing the air-water interface into the aqueous bulk. Results showed increased NaCl salt concentration significantly enhanced hydroxyl radical solvation, which should significantly increase its accommodation on water droplets. This has been experimentally observed for ozone aqueous accommodation with increased NaI concentration, but, to our knowledge, no experimental study has probed this for hydroxyl radicals. The origin for this effect was found to be very favorable hydroxyl radical-chloride ion interactions, being stronger than those for water-chloride. PMID- 16671695 TI - Solvent triggered change of the electron excitation route of KI in supercritical NH3. AB - The UV-spectroscopic behavior of KI contact ion pairs (CIPs) dissolved in supercritical NH3 was studied combining classical molecular dynamics simulations with electronic structure calculations, and the results show that an abrupt change of the photoexcitation route of KI CIPs occurs at very low solvent densities. Few NH3 solvating molecules are required to hamper the well-known photoinduced intramolecular electron (e-) transfer observed in isolated ion pairs of alkali metal halides in the vapor drawing the e- to solvent cavities leading to a charge-transfer-to-solvent process. PMID- 16671696 TI - Assembly of disperse red 1 molecules in the channels of AlPO4-5 single crystals for second-harmonic generation. AB - Disperse red 1 (DR1) molecules have been successfully incorporated into the one dimensional channels of AlPO4-5 single crystals by means of vapor-phase diffusion. Polarizing microscope and SHG results indicate that the DR1 molecules are well aligned in a preferred direction along the crystal channels. The p polarization (parallel to the c-axis of AlPO4-5 crystal) SH intensity (Ip-ex), and s-polarization (perpendicular to the c-axis of AlPO4-5 crystal) SH intensity (Is-ex) of DR1-loaded AlPO4-5 single crystals can be well fitted as a function of input polarization angle (alpha): Ip-ex = 0.69 cos4 alpha + 0.37 sin4 alpha - 0.17, Is-ex = 0.69 cos2 alpha sin2 alpha + 0.06, respectively. This polarization angle dependence can be well explained by three different SHG processes of Ip (0 degrees), Ip (90 degrees), and Is (45 degrees), originated from different combinations of two polarized photons. PMID- 16671697 TI - Quantum chemical study of the inhibitive properties of 2-pyridyl-azoles. AB - Four molecules that have been proven to act as corrosion inhibitors of mild steel in acidic media are studied. The inhibitive efficiency of these molecules is explained by means of electronic structure calculations of the protonated species that seem to represent better the actual situation of the experimental conditions. By assuming that the interaction between the inhibitor and the metallic surface occurs through donation and back-donation, it is shown, with a simple charge transfer model, that the interaction energy is favored when hardness increases, in agreement with the experimentally observed inhibition efficiencies. A local analysis with Hirshfeld condensed Fukui functions, and local Fukui functions, provides further support to the donation and back-donation mechanism. PMID- 16671698 TI - Changes in the fluorescence spectrum of individual single-wall carbon nanotubes induced by light-assisted oxidation with hydroperoxide. AB - Through fluorescence-spectrum measurements, we investigated the effects of light assisted oxidation with H2O2 (LAOx) on single-wall carbon nanotubes (SWNTs) that were individually dispersed in an aqueous solution of surfactant. The intensities of the fluorescence spectra were decreased remarkably by the LAOx when the light's wavelength was 400-500 nm and a little when 600-700 nm. The spectrum intensity did not recover even when the pH was restored to an original value of 6.5. The spectra changed little when the LAOx wavelength was 500-600 nm or the light was not irradiated. In addition, the effect of LAOx on SWNTs was related to the diameters of SWNTs. We inferred that these phenomena reflected that H2O2 was dissociated by absorbing the fluorescence light emitted from optically excited SWNTs, which, in turn, accelerated the burning out of SWNTs. PMID- 16671699 TI - Hydration of poly(ethylene oxide)s in aqueous solution as studied by dielectric relaxation measurements. AB - The hydration state of poly(ethylene oxide)s (PEOs) in aqueous solutions was investigated using dielectric relaxation measurements at 25 degrees C over a frequency range up to 20 GHz, which is the relaxation frequency of water molecules in a bulk state. The dielectric relaxation spectra obtained indicated decomposition into two major and one minor relaxation modes with relaxation times of 8.3, 22, and 250 ps, respectively. The two major modes were attributed to rotational relaxation of water molecules belonging to the bulk state and water molecules hydrogen bonded to ethylene oxide (EO) monomer units. The number of hydration water molecules per EO unit depended on the molar mass of PEO (M) and reached a constant value of 3.7 at M > 1500, which agrees with the value obtained by other experiments. PMID- 16671700 TI - Short- and medium-range order in sodium aluminophosphate glasses: new insights from high-resolution dipolar solid-state NMR spectroscopy. AB - The structures of sodium aluminophosphate glasses prepared by both sol-gel as well as melt-cooling routes have been extensively characterized by high resolution solid-state 23Na, 27Al, and 31P single and double-resonance NMR techniques, including quantitative connectivity studies by 27Al <--> 31P and 23Na <--> 31P rotational echo double-resonance (REDOR) methods. Studies along four compositional lines, I: (AlPO4)x -(NaPO3)1-x, II: (Na2O)x -(AlPO4)1-x, III: (NaAlO2)x -(NaPO3)1-x, and IV: (Al2O3)x (NaPO3)1-x, reveal that the network structures of those glasses that are accessible by either preparation method are essentially identical. However, the significantly extended glass-forming ranges available by the sol-gel route facilitate exploration of the structure/composition relationships in more detail, revealing a number of interesting universal features throughout the whole glass system. Both short- and medium-range order appear to be controlled strongly by the O/P ratio of the glasses studied: Up to an O/P ratio of 3.5 (pyrophosphate composition), aluminum is predominantly six-coordinated and fully connected to phosphorus (Al(OP)6 sites). In the region 3.5 < or = O/P < or = 4.0, a dramatic structural transformation takes place, leading to the appearance of additional four- and five-coordinated aluminum species whose second coordination spheres are also entirely dominated by phosphorus. The structure of glasses with an O/P ratio of precisely 4.0 (orthophosphate) is dominated by Al(OP)4 units. As the O/P ratio increases beyond 4.0, the average extent of Al-O-P connectivity is decreased significantly. Here, new types of five- and six-coordinated aluminum units, which are only weakly connected to phosphorus, are formed, while the network modifier is attracted mainly by the phosphate units. PMID- 16671701 TI - Electronic and geometric properties of ETS-10: QM/MM studies of cluster models. AB - Hybrid DFT/MM methods have been used to investigate the electronic and geometric properties of the microporous titanosilicate ETS-10. A comparison of finite length and periodic models demonstrates that band gap energies for ETS-10 can be well represented with relatively small cluster models. Optimization of finite clusters leads to different local geometries for bulk and end sites, where the local bulk TiO6 geometry is in good agreement with recent experimental results. Geometry optimizations reveal that any asymmetry within the axial O-Ti-O chain is negligible. The band gap in the optimized model corresponds to a O(2p) --> Tibulk(3d) transition. The results suggest that the three Ti atom, single chain, symmetric, finite cluster is an effective model for the geometric and electronic properties of bulk and end TiO6 groups in ETS-10. PMID- 16671702 TI - Electrodeposition of Bi(x)Fe(1-x) intermetallic compound nanowire arrays and their magnetic properties. AB - There have been few reports on Bi-Fe intermetallic compounds because Bi and Fe are immiscible in the equilibrium states and neither alloy nor intermetallic compound exists in the binary system. In this paper, we show that, by using the nanometer-scale templates based synthesis in conjunction with the electrochemical deposition, it is possible to mix in solid solution elements that are immiscible in traditional fabrication methods. The preparation of Bi-Fe intermetallic compound nanowire arrays was investigated via an electrodeposition route by using a polycarbonate (PC) membrane template. Cyclic voltammetry, potentiostatic transient, and potentiostatic stripping were used to study the formation of Bi(x)Fe(1-x) intermetallic compounds. The compositions of Bi(1-x)Fe(x) intermetallic compound nanowire arrays were sensitive to the bath compositions and the electrodeposition potentials, and the length could be easily adjusted by varying the electrodeposition time. The electrodeposited Bi(1-x)Fe(x) intermetallic compound nanowire arrays had a parallel-to-the-wire easy magnetization. Furthermore, the spin-glass such as behavior and an unusually large characteristic time, which was about 5.26 h, were found in Bi(1-x)Fe(x) intermetallic compound nanowire arrays at room temperature. PMID- 16671703 TI - Molecular dynamics simulations applied to electric field induced second harmonic generation in dipolar chromophore solutions. AB - Electric field induced second harmonic generation (EFISH) is an important experimental technique in extracting the first hyperpolarizability of an organic chromophore molecule. Such experiments are carried out in solutions with chromophore molecules dissolved in some common solvents. A known fact is that the first hyperpolarizabilities extracted from EFISH experiments are subject to the use of local field factors. In this work, we apply simulations to study the EFISH properties of chromophore solutions. By combining quantum chemistry calculations with the results derived from molecular dynamics simulations, we show how macroscopic EFISH properties can be modeled, using 4-(dimethylamino)-4' nitroazobenzene dissolved in chloroform as a demonstration case. The focus of the study is on deriving accurate local field factors. We find that the local field approach applies very well to dipolar solutions, such as the one studied here, but that the local field factors derived are much smaller than the commonly used Onsager or Lorentz local field factors. Our study indicates that many of the reported first hyperpolarizabilities for dipolar molecules from EFISH experiments are most probably underestimated because the Onsager/Lorentz approach, commonly used in extracting the molecular first hyperpolarizability, neglects the effects of the shapes of dipolar chromophore molecules on the local field factors. PMID- 16671704 TI - Biomolecule-assisted synthesis and electrochemical hydrogen storage of Bi2S3 flowerlike patterns with well-aligned nanorods. AB - Bi2S3 flowerlike patterns with well-aligned nanorods were synthesized using a facile solution-phase biomolecule-assisted approach in the presence of L-cysteine (an ordinary and cheap amino acid), which turned out to serve as both the S source and the directing molecule in the formation of bismuth sulfide nanostructures. Emphatically, no nauseous scent (H2S) appeared in our experiments, which could not be avoided in other previous reports. The morphology, structure, and phase composition of the as-prepared Bi2S3 products were characterized using various techniques (scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, transmission electron microscopy, selected area electron diffraction, and high-resolution transmission electron microscopy). The formation mechanism for the bismuth sulfide flowerlike assemblies with well-arranged nanorods was also discussed. In addition, other Bi2S3 homogeneous nanostructures (e.g., networklike nanoflakes, nanorod-based bundles, and nanoflakes) were obtained through varying the experimental parameters. Interestingly, we have found that these synthesized bismuth sulfide nanostructures using the biomoleucle-assisted approach could electrochemically charge and discharge with the capacity of 142 (mA h)/g (corresponding to 0.51 wt % hydrogen in single-walled carbon nanotubes) under normal atmosphere at room temperature. A novel two-plateau phenomenon was observed in the synthesized Bi2S3 nanostructures, suggesting that there were two independent steps in the charging process. It has been demonstrated that the bismuth sulfide's morphology and the constant charge-discharge current density had a noticeable influence on their capacity of electrochemical hydrogen storage. These differences in hydrogen storage capacity are likely due to the size and density of space/pores as well as the morphology of different Bi2S3 nanostructures. The novel Bi2S3 nanomaterials may find potential applications in hydrogen storage, high-energy batteries, luminescence, optoelectronic and catalytic fields, as well as in the studies of structure-property relationships. This facile, environmentally benign, and solution-phase biomolecule-assisted method can be potentially extended to the preparation of other metal chalcogenides including FeS, CuS, NiS, PbS, MnS, and CoS nanostructures. PMID- 16671706 TI - A synthetic route toward well-defined stoichiometric silica fullerene and nanotubes based on metastable four-membered rings. AB - On the basis of computational considerations, using metastable four-membered rings as building blocks, we propose novel synthetic routes toward well-defined stoichiometric silica nanofullerenes and nanotubes. The viability of the routes has been demonstrated by performing high-level density functional calculations, and the so-formed nanoarchitectures were proved to be energetically and structurally stable. Such nanostructures, if synthesized, are expected to have potential application in nanotechnology. PMID- 16671705 TI - Dye-labeled silver nanoshell-bright particle. AB - Silica beads with average diameters of 40-600 nm were prepared, and Ru(bpy)3(2+) complexes were incorporated into the beads. These beads were coated by silver layer by layer to generate porous but continuous metal nanoshells. The thicknesses of these metal shells were 5-50 nm. The emission band from the dyes in the silica cores was more narrow and the intensity was enhanced with growth of silver shell thickness due to coupling of the emission light from Ru(bpy)3(2+) in the cores with the metal plasmon from the silver shells. The enhancement of emission intensity was also dependent on the size of the silica core, showing that the enhancement efficiency decreased with an increase in the size of the silica beads. Lifetime measurements support the coupling mechanism between the dye and metal shell. This study can be used to develop novel dye-labeled metal particles with bright and narrow emission bands. PMID- 16671707 TI - Theoretical study of the electronic structure and stability of titanium dioxide clusters (TiO2)n with n = 1-9. AB - The electronic structure and the stability of both neutral and singly charged (TiO2)n clusters with n = 1-9 have been investigated using the density functional B3LYP/LANL2DZ method. The lowest-lying singlet clusters tend to form some compact structures with one or two terminal Ti-O bonds, which are about 1.4-2.5 eV more stable than the corresponding triplet structures. For the lowest-lying structures, strong infrared absorption lines at 988-1020 cm(-1) due to terminal Ti-O bonds and below 930 cm(-1) due to Ti-O-Ti bridging bonds may be observed, with some characteristic lines at 530-760 cm(-1) due to 3-fold coordinated O atoms that are comparable with the spectra of rutile and anatase bulk. The holes and excited electrons within triplet structures tend to be localized on the least coordinated O- and Ti-atoms, respectively, with some exceptions possibly due to the electron-hole interaction. The extra electrons within (TiO2)n- clusters and the holes within (TiO2)n+ clusters show a clearer preference of location on the least coordinated Ti- and O-atoms, respectively. For the lowest-lying (TiO2)n clusters, the cluster formation energy per TiO2 unit and the electron affinity tend to increase whereas the ionization potential tends to decrease with the cluster size n. On the other hand, the singlet-triplet and HOMO-LUMO gaps represent the lower and upper limits of the TiO2 bulk band gaps, respectively. The theoretical results agree well with the available experimental data and may be helpful for understanding the chemistry of small (TiO2)n clusters. PMID- 16671708 TI - Synthesis and photophysical properties of EuS nanoparticles from the thermal reduction of novel Eu(III) complex. AB - EuS nanoparticles were synthesized by the thermal reduction of single source precursor (SSP), (PPh4)[Eu(S2CNEt2)4].2H2O, under microwave irradiation. The average size of the EuS nanoparticles was found to be 8 nm (3-16 nm in size). The organic products on the EuS surface were observed by using FT-IR, NMR, and MS analyses. We have found that these are resulted from the chemical reactions of SSP and cover the nanocrystal surface. A thermal reaction of SSP gave EuS nanoparticles and the organic product (*SCN(Et)2). The organic product would make a dimmer, (Et)2NC(S)-(S)CN(Et)2, by the couping of the radicals formed in the thermal reaction and/or thiopolymer in the solution through the polymerization of the radicals. The effective surface modification by the organic products led to protection of the EuS surface, resulting in the formation of the strongly luminescent EuS nanoparticles at room temperature (emission peak = 350 nm, fwhm = 58 nm, emission quantum yield = 27 +/- 5%). PMID- 16671709 TI - Interfacial electron transfer between the photoexcited porphyrin molecule and TiO2 nanoparticles: effect of catecholate binding. AB - Interfacial electron transfer (ET) dynamics of 5,10,15-trisphenyl-20-(3,4 dihydroxybenzene) porphyrin (TPP-cat) adsorbed on TiO2 nanoparticles has been studied by femtosecond transient absorption spectroscopy in the visible and near IR region exciting at 400 and 800 nm. TPP-cat molecule forms a charge transfer (CT) complex with TiO2 nanoparticles through the catechol moiety with the formation of a five-membered ring. Optical absorption measurements have shown that the Q-band of TPP-cat interacts strongly with TiO2 due to chelation; however, the Soret band is affected very little. Optical absorption measurements indicate that the catechol moiety also interacts with TiO2 nanoparticles showing the characteristic band of pure catechol-TiO2 charge transfer (CT) in the visible region. Electron injection has been confirmed by monitoring the cation radical, instant bleach, and injected electron in the conduction band of TiO2 nanoparticles. Electron injection time has been measured to be < 100 fs and recombination kinetics has been best fitted with a multiexponential function, where the majority of the injected electrons come back to the parent cation radical with a time constant of approximately 800 fs for both excitation wavelengths. However, the reaction channel for the electron injection process has been found to be different for both wavelengths. Excitation at 800 nm, found to populate the CT state of the Q-band, and from the photoexcited CT state electron injection into the conduction band, takes place through diffusion. On the other hand, with excitation at 400 nm, a complicated reaction channel takes place. Excitation with 400 nm light excites both the CT band of Cat-TiO2 and also the Soret band of TPP-cat. We have discussed the reaction path in the TPP-cat/TiO2 system after exciting with both 400 and 800 nm laser light. We have also compared ET dynamics by exciting at both wavelengths. PMID- 16671710 TI - Nanostructure origins of C60 fluorescence in pyridine. AB - The nanostructure origins of three distinct and strong fluorescence bands of C60 in room-temperature pyridine are reported for the first time. Fluorescence study and in-situ TEM observation on C60-pyridine solutions with different setting times exhibit that the blue fluorescence peaks centered at 440 nm originate from C60 nanoparticles; the yellow-green fluorescence band located at 575 nm derives from C60 lace-like cluster; and the salmon pink fluorescence band around 700 nm arises from C60 microbulk in solution. The conclusions are supported further by investigation on the C60-toluene system as well. PMID- 16671711 TI - Morphological characterization of self-assembled peptide nucleic acid amphiphiles. AB - Peptide nucleic acid amphiphiles (PNAA) are a promising set of materials for sequence-specific separation of nucleic acids from complex mixtures. To implement PNAA in micellar separations, the morphology and size of PNAA micelles in the presence and absence of a sodium dodecyl sulfate (SDS) cosurfactant have been studied by small-angle X-ray scattering and dynamic light scattering. We find that a 6-mer PNAA with a 12-carbon n-alkane tail forms ellipsoidal micelles (a = 5.15 nm; b = 3.20 nm) above its critical micelle concentration (CMC) of 110.9 microM. On addition of a stoichiometric amount of complementary DNA, PNAA hybridizes to DNA, suppressing the formation of PNAA micelles. At a ratio of 19:1 SDS/PNAA (total concentration = 20 mM), spherical micelles are formed with outer radius Rs = 2.67 nm, slightly larger than spherical micelles of pure SDS. Capillary electrophoresis studies show that PNAA/DNA duplexes do not comicellize with SDS micelles. No such effects are observed using noncomplementary DNA. The shape and size of the PNAA micelles is also verified by dynamic light scattering (DLS) studies. These results provide an interesting case study with competing electrostatic, hydrophobic, and hydrogen-bonding interactions in micellar systems and make possible the use of PNAA in micellar separations of DNA oligomers. PMID- 16671712 TI - Microwave-assisted aqueous synthesis: a rapid approach to prepare highly luminescent ZnSe(S) alloyed quantum dots. AB - In this paper, we present a new procedure for the rapid synthesis of luminescent ZnSe nanocrystals in aqueous phase by microwave irradiation with controllable temperature. The effects of microwave irradiation and experimental conditions on the synthesis of nanocrystals were investigated systematically. It was found that there were significant effects of pH value of reaction solutions, molar ratio of precursors, and heating time of microwave irradiation on the optical properties of the ZnSe nanocrystals. A series of nanocrystals with different size was prepared in 1 h, and the photoluminescence quantum yield reached up to 17% at the optimal reaction condition. The results of HRTEM and XRD showed that the as prepared nanocrystals had high crystallinity. The characterizations of EDS spectra and elemental analysis showed that the sulfur content of nanocrystals increased with the growth of nanocrystals. We speculated that the structure of nanocrystals was an alloy ZnSe(S) shell on the surface of the ZnSe particles core. Furthermore, we found that the oxygen from air in the reaction vessel played an important role in the decomposition of the thiol group under microwave irradiation. PMID- 16671713 TI - Rapid, large-scale synthesis and electrochemical behavior of faceted single crystalline selenium nanotubes. AB - This article describes a rapid, solution-phase approach to the large-scale synthesis of faceted single-crystalline Se nanotubes, in the presence of cetyltrimethylammonium bromide. The products were characterized by scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, laser Raman spectrography, differential scanning calorimetry analysis, and thermogravimetric analysis. The growth mechanism of the Se nanotubes was investigated by a series of experiments, and the rationality of the faceted morphology model for the Se nanotubes was demonstrated from the energetics and geometry. Furthermore, the electrochemical behavior of the Se nanotubes was studied by voltammetric techniques. PMID- 16671714 TI - Kinetic modeling of the polymer-derived ceramics route: investigation of the thermal decomposition kinetics of poly[B-(methylamino)borazine] precursors into boron nitride. AB - A complete kinetic modeling of the polymer-derived ceramics (PDCs) route is achieved for the first time through the investigation of the solid-state decomposition of a typical melt-spinnable poly[B-(methylamino)borazine] into boron nitride fibers at various heating rates. Through the use of the Lorentz fitting approach, it is shown that the two-step weight loss associated with the polymer-to-ceramic conversion is governed by a complex interplay of five diffusion-type transport mechanisms that are independent of the applied heating schedule. The application of the Friedman method to dynamic thermogravimetry data yields Ea and ln A values that are seen to increase with the extent of the ceramic conversion from region one (Ea = 38.73 kJ mol(-1)) to region five (Ea = 146.64 kJ mol(-1)). This fact indicates that both the mechanisms within those regions are parallel routes to the formation of the final solid-state material and their complexity increases with the conversion progress. The cross-linking process (first weight loss) of the polymer is governed by three dependent poorly energetic mechanisms. The first weight loss is activated by ammonolysis reactions that provide a modified polymer capable of undergoing condensation reactions in regions two and three to yield a highly cross-linked polymer. A large evolution of methylamine is identified during this process. Mineralization (region four) and ceramization (region five) steps are represented by two highly energetic multistep mechanisms. The mineralization step is associated with a large evolution of methylamine and occurs during the transition between the cross linking and ceramization processes through the cleavage of the inter-ring B-N bonds. Ceramization represents the end of the polymer-to-ceramic conversion in which the planar consolidation of BN hexagons occurs through complex structural rearrangements of the borazine units (cleavage of the intraring B-N bonds) accompanied with an ammonia evolution. Finally, the simulation of the polymer-to ceramic conversion was demonstrated through a simplified model that appropriately predicted experimental data. PMID- 16671715 TI - Influence of stereoregularity of the polymer chain on interactions with surfactants: binding of cetylpyridinium chloride by isotactic and atactic poly(methacrylic acid). AB - Association of a cationic surfactant cetylpyridinium chloride, CPC, with isotactic and atactic poly(methacrylic acid), i-PMA and a-PMA, respectively, in aqueous 0.01 M NaCl solutions was studied by pH and fluorescence measurements in conjunction with potentiometric studies using a surfactant-sensitive membrane electrode. pH measurements have demonstrated that the presence of an oppositely charged surfactant increases ionization of carboxyl groups on PMA at low degrees of neutralization. The increase is more pronounced in the case of i-PMA. The isotactic form of PMA is not soluble in water at zero degrees of neutralization but can be rendered soluble by the addition of CPC at the surfactant to a polyion molar ratio of around 0.4. In the solubilized complex, the positive charge of the CPC molecule is facing the polar solvent, whereas surfactant tails are oriented toward the i-PMA compact coil. Binding isotherms and cooperativity parameters show that chain tacticity has an important influence on the interaction of cetylpyridinium cation with polymethacrylate anion. At the onset of cooperative binding, the association is stronger with i-PMA than with the atactic form, as demonstrated by lower CAC values and higher values of the cooperativity parameters. In contrast, more surfactant is bound by a-PMA in the region where polyion becomes saturated with surfactant ions. Results are interpreted by taking into account local chain conformations as obtained from quantum mechanical semiempirical molecular orbital calculations. Greater hydrophobicity and possibly higher charge density of i-PMA on one hand and more flexibility of the a-PMA chain on the other are held responsible for these observations. PMID- 16671716 TI - Thermal hysteresis and thickness dependence of the molecular orientation of poly(di-n-hexylsilane) in the film state. AB - Molecular orientation of poly(di-n-hexylsilane) adsorbed on poly(vinyl alcohol) film has been studied by making use of the stretching technique. Dichroic ratio, Rd, strongly depended on the thickness of poly(di-n-hexylsilane) thin film and the highest value ca. 19 was observed at the film thickness of 110 +/- 30 nm. The thermal hysteresis of the molecular orientation was observed in the heating cooling cycles. By studying the fluorescence spectrum it was confirmed that a portion of the poly(di-n-hexylsilane) molecules were in transoid conformation even at 320 K, although most of poly(di-n-hexylsilane) molecules were in disordered conformation (conformation D). This poly(di-n-hexylsilane) in transoid conformation is formed in the stretching process and may play a role of crystallization nucleus to induce the whole orientation of the poly(di-n hexylsilane) in the film state. PMID- 16671717 TI - Stability and phase behavior of acrylamide-based emulsions before and after polymerization. AB - The stability and phase behavior of acrylamide-based emulsions, prepared with surfactants consisting of lipophilic Span80 and hydrophilic OP10, before or after polymerization were investigated. The research results indicated that the phase separation behavior of the W/O-type emulsions is related to the toluene/water ratio. When the water volume fraction was larger, the phase separation mechanism was mainly a penetration of aqueous molecules from the dispersed-phase droplets. When the water volume fraction was smaller, the phase separation mechanism was mainly a sedimentation of the separated aqueous droplets. At a fixed toluene/water ratio, the emulsion stability and the emulsion type are related not only to the ratio of the two surfactants but also to the acrylamide concentration, and the effect of increasing acrylamide concentration on the character of the emulsions is similar to that of increasing OP10 mass fraction (increasing HLB value), which determines the corresponding relationship between acrylamide concentration and HLB value in the most stable emulsion system. To obtain the most stable emulsion at a fixed acrylamide concentration, the emulsion with higher acrylamide concentration needs a lower HLB value for the emulsion systems. PMID- 16671718 TI - How does bovine serum albumin prevent the formation of kidney stone? A kinetics study. AB - To attain a better understanding of the crystallization of calcium oxalate crystals under the influence of the protein bovine serum albumin, we examined not only the nucleation kinetics but also the structural synergy between the biomineral and the biosubstrate. It follows that during the crystallization process of calcium oxalate crystals bovine serum albumin inhibits the nucleation of calcium oxalate by increasing the kink kinetics barrier. The results of scanning electron microscopy and X-ray diffraction show, however, that bovine serum albumin promotes the formation of calcium oxalate dihydrate. Apart from this, bovine serum albumin facilitates the ordered calcium oxalate crystal assembly by suppressing the supersaturation-driven interfacial structure mismatch. The physics questions behind the mentioned effects have been addressed from the kinetics point of view. This may explain why bovine serum albumin plays an important role in suppressing urine stone formation. PMID- 16671719 TI - UV-induced nonlinear absorption in lanthanum calcium borate single crystals. AB - It has been revealed that lanthanum calcium borate (La2CaB10O19) crystals show two-photon absorption (TPA) induced by a UV laser field. UV-induced TPA measurements were performed in the spectral range of 475-1130 nm using as fundamental beam the third harmonics of the 28 ps Nd-YAG pulsed laser as a pumping beam for LiB3O5 optical parametrized generator using Z-scan method. Investigations performed by the Z-scan method were done during illumination by a Xe-F laser (lambda = 217 nm) as a photoinducing (pumping) beam. The pumping laser beam created a thin surface layer (about 80-90 nm) that was the source of the observed photoinduced TPA. The highest values of the TPA beta coefficients were achieved for polarization of the pumping light directed along the second-order crystallographic axis of the investigated crystals. The obtained values of the TPA coefficients were higher than those for the BiB3O6 crystals investigated earlier by us. PMID- 16671720 TI - Water-soluble multiwalled carbon nanotubes functionalized with sulfonated polyaniline. AB - Multiwalled carbon nanotubes (MWNTs) functionalized with a water-soluble conducting polymer, sulfonated polyaniline (SPAN), were prepared by in situ polymerization of aniline followed by sulfonation with chlorosulfonic acid in an inert solvent and by hydrolysis in water. Electron microscopy, laser Raman spectroscopy, X-ray photoelectron spectroscopy, and UV-vis absorption spectroscopy were employed to characterize the morphology and chemical structure of the resulting product. The results show that the quinonoid structure of SPAN preferentially interacts with the nanotubes and is stabilized by strong pi-pi interaction between two components. The structure of MWNTs was not perturbed by the incorporation of SPAN, since the pi-pi interaction between MWNTs and SPAN is much weaker in comparison to that of the carbon covalent bond. The SPAN functionalized MWNTs are highly dispersible in water, thus opening new possibilities for their prospective technological applications. PMID- 16671721 TI - Evidence of nanoconfinement effects of MCM-41 on propylene polymerization catalyzed by MCM-41 supported metallocene catalyst in the presence and absence of beta-cyclodextrin. AB - Propylene polymerization was carried out with MCM-41 supported rac-Et(Ind)2ZrCl2 catalysts, in the presence and absence of beta-cyclodextrin. The resultant PP was studied by X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, and differential scanning calorimetry. Through comparison of the results, it was found that the channels of MCM-41 could act as a nanoreactor of propylene polymerization and the polypropylene (PP) contained in the channels had noncrystal structure. However, the PP could grow out of the channels and form some crystals after the active sites on the surface of MCM-41 were destroyed. This showed that the channels of MCM-41 had great confinement effects on propylene polymerization. PMID- 16671722 TI - Fast H-vacancy dynamics during alanate decomposition by anelastic spectroscopy. proposition of a model for Ti-enhanced hydrogen transport. AB - A systematic study of the dehydrogenation process of undoped and of catalyzed NaAlH4 by means of anelastic spectroscopy is presented. Evidence is reported of the formation of a highly mobile species during decomposition, which has been identified in off-stoichiometric AlH6-x units, giving rise to fast H vacancy local dynamics. The formation of such stoichiometry defects starts at temperatures much lower in Ti doped than in undoped samples, and concomitantly with the decomposition reaction. The catalyst atoms decrease the energy barrier to be overcome by H to break the bond, thus enhancing the kinetics of the chemical reactions and decreasing the temperature at which the dehydrogenation processes take place. The experimental data show that not all the hydrogen released by the formula units during the evolution of decomposition evolves out of the sample, but part of it remains in the lattice and migrates on a long-range scale within the sample. We identify, in this H mobilized population, the species which induces the fast tetragonal to monoclinic phase transformation accompanying decomposition. A partial spontaneous thermally activated regression of decomposition has also been observed by aging experiments. A model is proposed which accounts for the action of the Ti catalyst and for the atomistic mechanism of decomposition. PMID- 16671723 TI - Aggregation of cesium perfluorooctanoate on poly(ethylene glycol) oligomers in water. AB - The interaction of cesium perfluorooctanoate (CsPFO) with poly(ethylene glycol) (PEG) of different molecular weight (300 < or = MW < or = 20000 Da) has been investigated at 298.15 K by isothermal titration calorimetry (ITC), density, viscosity, and conductivity measurements. Calorimetric titrations exhibited peculiar trends analogous to those already observed for sodium dodecyl sulfate (SDS). Micelles of the perfluorosurfactant, as compared to those of SDS, yield complexes with the polymer of similar thermodynamic stability but are able to interact with shorter PEG oligomers. The average number of surfactant molecules bonded per polymer chain at the saturation is about twice that observed for SDS. ITC data at 308.15 K indicate a larger thermodynamic stability of the aggregates but an almost constant stoichiometry. The peculiar thermal effects and the viscosity trend observed during the titration of an aqueous PEG solution with the surfactant appear consistent with a conformational change of the polymer. The PEG chain would evolve from a strained to an expanded conformation, induced by the growing of the surfactant micellar clusters bonded to the polymer, as suggested in a previous study of the PEG/SDS/H2O system. PMID- 16671724 TI - Crystalline-like molecularly ordered mesoporous aluminosilicates derived from aluminosilica-surfactant mesophases via benign template removal. AB - We report the preparation of mesoporous aluminosilicate materials that exhibit molecular-scale ordering in their pore wall framework. The materials were derived from mesoporous aluminosilica-surfactant mesophases via benign template removal methods, which allowed the retention of molecular ordering in surfactant-free materials. The molecularly ordered aluminosilica-surfactant mesophases were obtained from hydrothermal crystallization of cetyltrimethylammonium hydroxide/Al,Si/H2O systems at 135 degrees C for 12 days. Benign template removal via H2O2-mediated oxidation of the surfactant at room temperature was found to be the most effective method in generating surfactant-free materials with molecular ordering, high textural properties (depending on Al content), and high acidity. The Al in the resulting aluminosilicates was entirely incorporated in framework (tetrahedrally coordinated) sites. Template extraction in acidified ethanol also generated molecularly ordered materials but compromised the Al content and acidity. Template removal via conventional calcination generated porous materials with high textural properties but which exhibited only limited molecular ordering and had relatively low acidity and significant amounts of nonframework Al. This work demonstrates that molecular ordering in mesoporous silicate-surfactant mesophases is due to crystallographic ordering within inorganic frameworks rather than the arrangement/packing of surfactant molecules. PMID- 16671725 TI - Phase transition behavior of unimolecular micelles with thermoresponsive poly(N isopropylacrylamide) coronas. AB - This paper describes the double phase transition behavior of a thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) brush at the surface of a hydrophobic core. Reversible addition-fragmentation transfer (RAFT) polymerization of N isopropylacrylamide (NIPAM) was conducted by using a hyperbranched polyester (Boltorn H40) based macroRAFT agent. The resultant multiarm star block copolymer (H40-PNIPAM) exists as unimolecular micelles with hydrophobic H40 as the core, densely grafted PNIPAM brush as the shell. A combination of laser light scattering (LLS) and microdifferential scanning calorimetry (micro-DSC) studies of H40-PNIPAM in aqueous solution reveals double phase transitions of the PNIPAM corona, which is in contrast to the fact that free PNIPAM homopolymer in aqueous solution exhibits a lower critical solution temperature (LCST) at approximately 32 degrees C. The first phase transition takes place in the broad temperature range 20-30 degrees C, which can be tentatively ascribed to the n-cluster-induced collapse of the inner region of the PNIPAM brush close to the H40 core; the second phase transition occurs above 30 degrees C, which can be ascribed to the outer region of PNIPAM brush. Employing the RAFT chain extension technique, the inner and outer part of PNIPAM brush were then selectively labeled with pyrene derivatives, respectively; temperature-dependent excimer fluorescence measurements further support the conclusion that the inner part of PNIPAM brush collapses first at lower temperatures, followed by the collapse of the outer part at higher temperatures. PMID- 16671726 TI - Structural characterization and oxidative dehydrogenation activity of V2O5/Ce(x)Zr(1-x)O2/SiO2 catalysts. AB - The thermal stability of a nanosized Ce(x)Zr(1-x)O2 solid solution on a silica surface and the dispersion behavior of V2O5 over Ce(x)Zr(1-x)O2/SiO2 have been investigated using XRD, Raman spectroscopy, XPS, HREM, and BET surface area techniques. Oxidative dehydrogenation of ethylbenzene to styrene was performed as a test reaction to assess the usefulness of the VOx/Ce(x)Zr(1-x)O2/SiO2 catalyst. Ce(x)Zr(1-x)O2/SiO2 (1:1:2 mol ratio based on oxides) was synthesized through a soft-chemical route from ultrahigh dilute solutions by adopting a deposition coprecipitation technique. A theoretical monolayer equivalent to 10 wt % V2O5 was impregnated over the calcined Ce(x)Zr(1-x)O2/SiO2 sample (773 K) by an aqueous wet impregnation technique. The prepared V2O5/Ce(x)Zr(1-x)O2/SiO2 sample was subjected to thermal treatments from 773 to 1073 K. The XRD measurements indicate the presence of cubic Ce0.75Zr0.25O2 in the case of Ce(x)Zr(1-x)O2/SiO2, while cubic Ce0.5Zr0.5O2 and tetragonal Ce0.16Zr0.84O2 in the case of V2O5/Ce(x)Zr(1 x)O2/SiO2 when calcined at various temperatures. Dispersed vanadium oxide induces more incorporation of zirconium into the ceria lattice, thereby decreasing its lattice size and also accelerating the crystallization of Ce-Zr-O solid solutions at higher calcination temperatures. Further, it interacts selectively with the ceria portion of the composite oxide to form CeVO4. The RS measurements provide good evidence about the dispersed form of vanadium oxide and the CeVO4 compound. The HREM studies show the presence of small Ce-Zr-oxide particles of approximately 5 nm size over the surface of amorphous silica and corroborate with the results obtained from other techniques. The catalytic activity studies reveal the ability of vanadium oxide supported on Ce(x)Zr(1-x)O2/SiO2 to efficiently catalyze the ODH of ethylbenzene at normal atmospheric pressure. The remarkable ability of Ce(x)Zr(1-x)O2 to prevent the deactivation of supported vanadium oxide leading to stable activity in the time-on-stream experiments and high selectivity to styrene are other important observations. PMID- 16671727 TI - Hydrophobic contribution of amino acids in peptides measured by hydrophobic interaction chromatography. AB - The adsorption behaviors of amino acids in short chain peptides were examined. Each amino acid, aliphatic or charged, was inserted between the two tryptophans of a peptide, GWWG. The capacity factors of these peptides on an Ocytl-Sepharose column were measured. The adsorption enthalpies, entropies, and the number of repelled water molecules after adsorption were estimated to analyze the contribution of each different amino acid to its hydrophobic adsorption. The peptides inserted with aliphatic amino acids owned the highest capacity factors but released the least amount of adsorption heat among all the peptides under examination. It was found that the hydrophobic contribution of aliphatic amino acids was derived from the entropy gain by repelling the ordered water surrounding them. The insertion of negatively charged amino acids greatly reduced the capacity factors but still repelled a significant number of water molecules after adsorption. This indicated that the water molecules surrounding ionic amino acids were not orderly aligned. The dehydration cost energy but the water repelling did not offer enough entropy to drive the adsorption. Subsequently, lower retention was obtained from the peptides inserted with negatively charged ionic amino acids. The insertion of lysine increased the adsorption enthalpy but repelled no water molecules after adsorption. It was speculated that the inserted lysine still interacted with hydrophobic ligands but disturbed the interaction between ligands and adjacent tryptophans. Therefore, the adsorption enthalpy increased and the capacity factors decreased. Different amino acids contributed to hydrophobic interaction in different ways. The simultaneous analysis of capacity factor, adsorption enthalpy, adsorption entropy, and the number of repelled water molecules facilitated the understanding of the adsorption processes. PMID- 16671728 TI - Fischer-Tropsch synthesis on anchored Co/Nb2O5/Al2O3 catalysts: the nature of the surface and the effect on chain growth. AB - A series of Co/x%Nb2O5/Al2O3 catalysts were prepared by anchoring niobia on an Al2O3 support at different niobia concentrations. Characterization of the structure and nature of surface active sites was attempted in order to correlate the CO hydrogenation activity of these systems with those of the Co/Al2O3 and Co/Nb2O5 catalysts. The effect of the reduction temperature on the CO hydrogenation activity and selectivity was studied, showing that interaction of cobalt and niobia surface species favored the selectivity for hydrocarbon chain growth. However, this effect is less pronounced on the niobia-promoted Co/Al2O3 compared to Co/Nb2O5 catalysts. X-ray photoelectron spectroscopy (XPS) and diffuse reflectance spectroscopy (DRS) results on Co/x%Nb2O5/Al2O3 showed prevailing amounts of Co2+ and Co3+ after calcination and reduction at 573 K, while, after reduction at 773 K, besides metallic cobalt, the Co2+ species still remains in contact with alumina, even for higher niobia loading. It seems that during this process formation and destruction of new interfaces involving Co0 NbOx sites takes place. Results suggest that Co0, Co0-Co2+, and Co0-NbOx are the active sites at the surface. The relative abundance of Co2+ species affects greatly the performance of the catalysts. DRIFTS and selectivity results suggest that these sites might be responsible for the reaction chain growth and therefore for the drastic change in the selectivity of CH4 and C5+ hydrocarbons mainly on the Co/Nb2O5 catalyst. DRIFTS results on Co/Nb2O5/Al2O3 showed the formation of C=C- and -CH3- besides CHxO species. With increasing reduction temperature, the C=C- species disappear while -CH3- fragments increased markedly, suggesting the formation of increasing amounts of hydrocarbons with higher chain length. PMID- 16671729 TI - Single molecule spectroscopy studies of diffusion in mesoporous silica thin films. AB - Single molecule spectroscopy is applied in studies of diffusion and surface adsorption in sol-gel-derived mesoporous silica thin films. Mesoporous films are obtained by spin casting surfactant-templated sols onto glass substrates. Small angle X-ray diffraction results are consistent with hexagonally ordered mesophases in as-synthesized (i.e., surfactant-containing) films. Upon calcination, a 30% contraction and disordering of these structures occurs. Nile Red is used as a fluorescent probe of both the as-synthesized and calcined films. It is loaded into the samples at subnanomolar levels either prior to spin casting or after calcination. Fluorescence imaging and single-point fluorescence time transients show the dye molecules to be relatively mobile in the as-synthesized samples. In contrast, the molecules appear entrapped at fixed locations in dry calcined films. In calcined films rehydrated under high humidity conditions, the Nile Red molecules again become mobile. Time transients obtained from the as synthesized and rehydrated samples provide clear evidence for frequent reversible adsorption of the dye to the silica surfaces. Autocorrelations of the time transients provide quantitative data on the mean diffusion coefficients (D = 2.4 x 10(-10) and 2.6 x 10(-10) cm2/s) and mean desorption times (1/k = 25 and 40 s) for the as-synthesized and rehydrated films, respectively. The results prove both water and surfactant play important roles in governing matrix interactions and mass transport. PMID- 16671730 TI - Reversible activation of diblock copolymer monolayers at the interface by pH modulation, 1: Lateral chain density and conformation. AB - This study focuses on the design of chemically regulated surfaces that allow for reversible control of the interactions between biological matter (cells and proteins) and planar substrates. As a tunable interlayer, we use a monolayer of a near-monodisperse poly[2-(dimethylamino)ethyl methacrylate-block-methyl methacrylate] (PDMAEMA-PMMA) diblock copolymer. Owing to the relatively large fraction (50%) of the hydrophobic PMMA block, this copolymer forms a stable Langmuir monolayer at the air/water interface. Both in situ and ex situ film balance experiments suggest that the hydrophilic PDMAEMA block adsorbs to the air/water interface in its uncharged state (pH 8.5), but stretches into the subphase in its charged state (pH 5.5). Optimization of the preparation protocols enables us to fabricate stable, homogeneous diblock copolymer films on hydrophobized substrates via Langmuir-Schaefer transfer at well-defined lateral chain densities. Ellipsometry and X-ray reflectivity studies of the transferred films confirm that the film thickness can be systematically regulated by the lateral chain densities. The transferred copolymer films remain stable in water for about a week, suggesting that they are promising materials for the creation of pH-controlled solid substrates for the support of biological matter such as proteins and cells. PMID- 16671731 TI - Reversible activation of diblock copolymer monolayers at the interface by pH modulation, 2: Membrane interactions at the solid/liquid interface. AB - A monolayer of the pH-responsive poly[2-(dimethylamino)ethyl methacrylate-block methyl methacrylate] diblock copolymer [PDMAEMA-PMMA] was transferred from the air/water interface to a silicon substrate for evaluation as a tunable interlayer between biological material and solid substrates. Specular neutron reflectivity experiments revealed that the weak polyelectrolyte PDMAEMA chains at the solid/liquid interface can be reversibly activated by pH modulation. The thickness, scattering length density, and surface roughness of the polymer film can be systematically controlled by pH titration. As a simple model of plasma membranes, a lipid bilayer was deposited onto the polymer film. The membrane substrate interaction was characterized by neutron reflectivity experiments, demonstrating that the membrane-substrate distance could be reversibly regulated by pH titration. These results confirm the potential of stimuli-responsive polymers for precise control of cell-surface interactions. PMID- 16671732 TI - Investigation of the morphology of the mesoporous SBA-16 and SBA-15 materials. AB - Mesoporous SBA-16 and SBA-15 were studied in order to control their possible morphologies. SBA-16 is synthesized using a silicon source (tetraethoxysilane, TEOS) and a ternary system consisting of surfactant F127 (EO106PO70EO106), water, and butanol. The same ternary system, with higher butanol concentration, is used to form SBA-15 material as well. An increase of the TEOS concentration results in a morphology shift of SBA-16 from micron-sized spheres, over randomly shaped aggregated particles, to macrospheres with a size of 15 mm. An identical increase in TEOS concentration also results in the formation of SBA-15 macrospheres, which can be controlled in size. Micron-sized spheres of SBA-15 were formed using a quaternary system of surfactant P123 (EO20PO70EO20), cetyltrimethylammonium bromide (CTAB), ethanol, and water. All mesoporous silica materials were characterized using SEM, XRD, and N2 sorption techniques. PMID- 16671733 TI - Water molecule adsorption properties on the BiVO4 (100) surface. AB - The water absorption properties at the surface of BiVO4 are attracting a great deal of attention because the system is a promising candidate as a photocatalyst operating in the visible light range. This has motivated the present investigation via first principles molecular dynamics, which has revealed that a H2O molecule is adsorbed molecularly, instead of dissociatively, at the fivefold Bi site with an adsorption energy of approximately 0.58 eV/molecule. The band gap of the system shrinks slightly (by approximately 0.2 eV) upon water adsorption and it is likely that oxygen atoms belonging to the adsorbed water molecules to the Bi sites are oxidized, as inferred by the small Bi-Owater equilibrium distance (approximately 2.6-2.8 A) very close to the Bi-O bond in the bulk crystal. In the case of water adsorption at a Bi site, the distance between Hwater and V, which is a reduction site, is larger than in the case of adsorption at a V site, indicating that the proton reduction processes may be suppressed. PMID- 16671734 TI - Surface potential switching by metal ion complexation/decomplexation using bipyridinethiolate monolayers on gold. AB - Surface potential switching on gold(111) surfaces is induced by complexation/decomplexation reactions of a bipyridine (BP) derivative and palladium(II) chloride, as observed by Kelvin probe force microscopy (KFM). On the basis of the theoretical predictions, a 4-(5-phenylethynyl-2,2'-bipyridine-5' yl-ethynyl)benzenethiol (PhBP) derivative was synthesized and used as an active monolayer to catch transition metal ions. By using the microcontact printing (CP) technique, micron-size patterned PhBP monolayers, which act as effective hosts to coordinate palladium(II) chloride, were prepared on gold(111) surfaces. The KFM signal decreases by complexation of the Pd(II) chloride in PhBP monolayers and is recovered by removal of Pd ions using an ethylenediamine solution, as confirmed by X-ray photoelectron spectroscopy. This process is reversible, indicating that the surface potential switching is realized by complexation/decomplexation of Pd(II). A CP PhBP monolayer, when it detects the target palladium ion, shows sensitivity for the picomolar level detection judged from surface potential changes in KFM measurements. The dipole moment estimated by the surface potentials is much smaller than the calculated value, indicating that mechanisms for the reduction of the surface dipole moment exist in real monolayers prepared by the CP method. PMID- 16671735 TI - Spectroscopic measurement of diffusion kinetics through subnanometer and larger Al2O3 particles by a new method: the interaction of 2-chloroethylethyl sulfide with gamma-Al2O3. AB - A new method to study the diffusion properties of molecules into porous materials using transmission IR spectroscopy is employed. A measurement of the diffusion of the 2-chloroethylethyl sulfide (2-CEES) molecule into two types of gamma-Al2O3 powder is performed, showing that the diffusion rate into subnanometer crystallite particle size gamma-Al2O3 powders (subnano-Al2O3) is higher than that into the larger crystallite particle size powder. It is shown that a surface diffusion mechanism can be used to model the diffusion process giving good agreement with the experimental results, where Dsubnano-Al2O3 is approximately 5 times larger than Dmultinano-Al2O3 at 170 K for the 2-CEES molecule. PMID- 16671736 TI - On the nature and accessibility of the Bronsted-base sites in activated hydrotalcite catalysts. AB - Base catalysis is of importance for organic synthesis in general and fine chemicals manufacture in particular. Activated hydrotalcites have recently received a great deal of attention as solid base catalysts; however, no systematic work on the nature of their active sites has been published up till now. In this work two different methods have been applied to activate Mg-Al hydrotalcites to obtain Bronsted-base catalysts for liquid-phase condensation reactions. Activation via thermal treatment followed by rehydration (HT-reh) resulted in irregularly stacked platelets ( approximately 60 nm), whereas the sample activated via aqueous ion-exchange (HT-exc) preserved its original hexagonal hydrotalcite platelets ( approximately 100 nm). The specific activity for the self-condensation of acetone of HT-reh was over 10 times that of HT-exc. The enthalpy of CO2 adsorption on the activated hydrotalcites determined with calorimetry to gain insight into the strength of the basic sites showed very similar values. IR spectra of adsorbed CDCl3 as probe molecule on the differently activated samples revealed large differences in adsorbed amounts, but again the strength of the basic sites appeared to be the same. These results point to steric hindrance for the substrate molecules as the main factor determining differences in catalytic activity. The high accessibility of Bronsted-base sites in HT-reh is proposed to involve a distorted edge structure of the platelets. The edge structure of exchanged samples could be distorted too, either by exchange under reflux conditions or under ultrasonic treatment. In line with the proposed model, the distorted exchanged samples displayed a much higher catalytic activity than HT-exc. PMID- 16671737 TI - Surface charge density and electrokinetic potential of highly charged minerals: experiments and Monte Carlo simulations on calcium silicate hydrate. AB - In this paper, we are concerned with the charging and electrokinetic behavior of colloidal particles exhibiting a high surface charge in the alkaline pH range. For such particles, a theoretical approach has been developed in the framework of the primitive model. The charging and electrokinetic behavior of the particles are determined by the use of a Monte Carlo simulation in a grand canonical ensemble and compared with those obtained through the mean field theory. One of the most common colloidal particles has been chosen to test our theoretical approach. That is calcium silicate hydrate (C-S-H) which is the main component of hydrated cement and is known for being responsible for cement cohesion partly due to its unusually high surface charge density. Various experimental techniques have been used to determine its surface charge and electrokinetic potential. The experimental and simulated results are in excellent agreement over a wide range of electrostatic coupling, from a weakly charged surface in contact with a reservoir containing monovalent ions to a highly charged one in contact with a reservoir with divalent ions. The electrophoretic measurements show a charge reversal of the C-S-H particles at high pH and/or high calcium concentration in excellent agreement with simulation predictions. Finally, both simulation and experimental results clearly demonstrate that the mean field theory fails not only quantitatively but also qualitatively to describe a C-S-H dispersion under realistic conditions. PMID- 16671738 TI - Stability of an ultrathin plasma polymerized film in aqueous solution: in situ detection by surface plasmon resonance. AB - Recently, surface plasmon resonance (SPR) has been successfully implemented to characterize the film stability in real time: a radio frequency (RF) plasma polymerized film was monitored by swelling or extracting behaviors in aqueous solution. The high-resolution SPR revealed that film stability strongly depended on the monomers, plasma mode, and substrate locations as well as plasma polymerization conditions: incident power and working pressure. By Fourier transform infrared (FTIR), the possible reasons of film stabilization affected by plasma conditions and sample locations are explained. It is recommended that as an adhesive layer for future applications the plasma polymerized polymer shall be prepared in low working pressure pulsed plasma. PMID- 16671739 TI - Growth mechanism of textured MgO thin films via SSCVD. AB - Magnesium oxide thin films have been deposited with use of single source chemical vapor deposition (SSCVD). The resultant films were examined by using transmission electron microscopy, X-ray texture analysis, and pole figure analysis. Due to the nature of the chemical reactions occurring at the surface during SSCVD growth, which result in a high growth rate/low flux environment, films of (111) orientation have been achieved without an amorphous underlayer, an unusual result for films of this orientation. Moreover the films have a strong degree of biaxial texturing in the x-y plane as found with X-ray texture analysis. These findings have important implications for buffer layers in perovskite thin film devices. The mechanism producing these structures has been revealed by using TEM and is discussed here. PMID- 16671740 TI - Surface-enhanced Raman scattering investigation of the adsorption of 2 mercaptobenzoxazole on smooth copper surfaces doped with silver colloidal nanoparticles. AB - The adsorption of 2-mercaptobenzoxazole on copper has been investigated by means of surface-enhanced Raman scattering (SERS) by doping smooth copper surfaces with silver colloidal nanoparticles. The metal surfaces have been characterized by means of atomic force microscopy measurements. The compound adsorbs on the Cu/Ag surfaces in its ionized thiolic form, adopting a tilted orientation with respect to the metal surface. The anion is chemisorbed through the sulfur and nitrogen atoms on the smooth copper surface, and the silver colloidal nanoparticles only enhance the Raman signal due to the electromagnetic mechanism. SERS data have been interpreted with the help of DFT calculations on models of the ligand bound to copper adclusters. PMID- 16671741 TI - Ordering of TiO2-based nanostructures on SrTiO3(001) surfaces. AB - A class of nanostructured surface phases on SrTiO3(001) is reported and characterized through atomic-resolution scanning tunneling microscopy and Auger electron spectroscopy. These surface phases are created via argon ion sputtering and UHV annealing and form close-packed domains of highly ordered nanostructures. Depending on the type of nanostructures present, the domain ordering exhibit either (6 x 2), (9 x 2), (12 x 2), (6 x 8), or (7 x 4) surface patterning. The nanostructures are composed of TiO2-derived complexes surrounded by a TiO2 surface termination. Such surface ordering phenomena introduce another level of complexity in the chemistry of perovskite oxide surfaces and provide a basis from which potential photocatalytic and molecular-ordering applications may be developed. PMID- 16671742 TI - Perturbed chain-statistical associating fluid theory extended to dipolar and quadrupolar molecular fluids. AB - The perturbed chain statistical associating fluid theory (PC-SAFT) is extended to polar molecular fluids, namely dipolar and quadrupolar fluids. The extension is based on the perturbation theory for polar fluids by Stell and co-workers. Appropriate expressions are proposed for dipole-dipole, quadrupole-quadrupole, and dipole-quadrupole interactions. Furthermore, induced dipole interactions are calculated explicitly in the model. The new polar PC-SAFT model is relatively complex; for this purpose, a truncated polar PC-SAFT model is proposed using only the leading term in the polynomial expansion for polar interactions. The new model is used for the calculation of thermodynamic properties of various quadrupolar pure fluids. In all cases, the agreement between experimental data and model predictions is very good. PMID- 16671743 TI - Modeling of the carbon dioxide solubility in imidazolium-based ionic liquids with the tPC-PSAFT equation of state. AB - In this work, an equation of state (EoS) is developed to predict accurately the phase behavior of ionic liquid + CO2 systems based on the truncated perturbed chain polar statistical associating fluid theory (tPC-PSAFT) EoS. This EoS accounts explicitly for the dipolar interactions between ionic liquid molecules, the quadrupolar interactions between CO2 molecules, and the Lewis acid-base type of association between the ionic liquid and the CO2 molecules. Physically meaningful model pure-component parameters for ionic liquids are estimated based on literature data. All experimental vapor-liquid equilibrium data are correlated with a single linearly temperature-dependent binary interaction parameter. The ability of the model to describe accurately carbon dioxide solubility in various 1-alkyl-3-methylimidazolium-based ionic liquids with different alkyl chain lengths and different anions at pressures from 0 to 100 MPa and carbon dioxide fractions from 0 to 75 mol % is demonstrated. In all cases, good agreement with experimental data is obtained. PMID- 16671744 TI - Computational determination of aqueous pKa values of protonated benzimidazoles (part 1). AB - Benzimidazoles are the organic compounds investigated in this work. The experimental determination of the pKa values of protonated benzimidazoles in water is a challenge because of their low solubility. In addition, some derivatives are involved in tautomeric equilibria which increase the complexity of the theoretical pKa determinations. In the present study, different approaches are considered to develop a methodology for the accurate prediction of aqueous pKa values of protonated benzimidazoles at 298.15 K. We have considered different reaction schemes for approximating the acid dissociation equilibrium; two distinct equations are used for the calculation of pKa values, and a number of levels of theory and empirical corrections are applied in the process of working toward this aim. The best correlations between the experimental and calculated data are obtained at the B3LYP/6-31+G(d,p)-PCM(opt) level of theory. The predictive capabilities of the methodologies attempted are tested with two compounds that were not included in the set of benzimidazoles initially investigated. The direct calculations differ significantly from the expected values, but the pKa values calculated using the correlation equations are very similar and in reasonable agreement with the expected pKa values. PMID- 16671745 TI - Determination of the enthalpy of solute-solvent interaction from the enthalpy of solution: aqueous solutions of erythritol and L-threitol. AB - In this work the enthalpy of the solute-solvent interaction of erythritol and L threitol in aqueous solution was determined from the values obtained for the enthalpy of solvation. The values for this property were calculated from those determined for the enthalpies of solution and sublimation. To determine the values of the enthalpy of solute-solvent interaction, the solvation process is considered as taking place in three steps: opening a cavity in the solvent to hold the solute molecule, changing the solute conformation when it passes from the gas phase into solution, and interaction between the solute and the solvent molecules. The cavity enthalpy was calculated by the scaled particle theory and the conformational enthalpy change was estimated from the value of this function in the gas phase and in solution. Both terms were determined by DFT calculations. The solvent effect on the solute conformation in solution was estimated using the CPCM solvation model. The importance of the cavity and conformational terms in the interpretation of the enthalpy of solvation is noted. While the cavity term has been used by some authors, the conformational term is considered for the first time. The structural features in aqueous solution of erythritol and L threitol are discussed. PMID- 16671746 TI - Study of molecular behavior in a water nanocluster: size and temperature effect. AB - Temperature and size effects on the behavior of nanoscale water molecule clusters are investigated by molecular dynamics simulations. The flexible three-centered (F3C) water potential is used to model the inter- and intramolecular interactions of the water molecule. The differences between the structural properties for the surface region and those for the interior region of the cluster are also investigated. It is found that as the temperature rises, the average number of hydrogen bonds per water molecule decreases, but the ratio of surface water molecules increases. After comparing the water densities in interior regions and the average number of hydrogen bonds in those regions, we find there is no apparent size effect on water molecules in the interior region, whereas the size of the water cluster has a significant influence on the behavior of water molecules at the surface region. PMID- 16671747 TI - Vibrational spectroscopy at high pressure of the permanganate anion trapped in potassium bromide and potassium perchlorate matrices. AB - The effect of high pressure on the resonance Raman spectra of the permanganate ion isolated in potassium bromide and potassium perchlorate matrices has been investigated at room temperature for pressures up to 50 kbar. The pressure dependences of the anharmonicity constants and harmonic frequencies have been determined from the overtones of the totally symmetric nu1(A1) mode of the permanganate ion. For both matrices, as the pressure increases, the anharmonicity constants decrease slightly, while the harmonic frequencies increase steadily. The effect of the potassium bromide phase transition from a face-centered to a body-centered structure was observed on the permanganate ion Raman spectrum at approximately 24 kbar. The perchlorate matrix does not exhibit any phase transition under the experimental conditions used in this study. PMID- 16671748 TI - Quantitative description of the hydrophobic effect: the enthalpic contribution. AB - A new method of experimental determination of the hydrophobic effect enthalpy is proposed. The method is based on regarding the hydration enthalpy as the sum of the nonspecific hydration enthalpy, specific hydration enthalpy, and the hydrophobic effect enthalpy. The hydrophobic effect enthalpies of noble and simple substance gases, alkanes, arenes, and normal aliphatic alcohols are determined. For the noble gases and alkanes, the hydrophobic effect enthalpy is found to be negative and independent of the size of molecule. For aromatic hydrocarbons, it is positive and grows up with the size of the hydrocarbon. The hydrophobic effect enthalpies of normal aliphatic alcohols are determined by assuming that the specific interaction enthalpies of alcohols in water and in methanol are equal. The hydrophobic effect enthalpy values for the aliphatic alcohols (-10.0 +/- 0.9 kJ.mol(-1)) were found to be close to the alkanes hydrophobic effect enthalpies (-10.7 +/- 1.5 kJ.mol(-1)). PMID- 16671749 TI - Electrostatics of ligand binding: parametrization of the generalized Born model and comparison with the Poisson-Boltzmann approach. AB - An accurate and fast evaluation of the electrostatics in ligand-protein interactions is crucial for computer-aided drug design. The pairwise generalized Born (GB) model, a fast analytical method originally developed for studying the solvation of organic molecules, has been widely applied to macromolecular systems, including ligand-protein complexes. However, this model involves several empirical scaling parameters, which have been optimized for the solvation of organic molecules, peptides, and nucleic acids but not for energetics of ligand binding. Studies have shown that a good solvation energy does not guarantee a correct model of solvent-mediated interactions. Thus, in this study, we have used the Poisson-Boltzmann (PB) approach as a reference to optimize the GB model for studies of ligand-protein interactions. Specifically, we have employed the pairwise descreening approximation proposed by Hawkins et al.(1) for GB calculations and DelPhi for PB calculations. The AMBER all-atom force field parameters have been used in this work. Seventeen protein-ligand complexes have been used as a training database, and a set of atomic descreening parameters has been selected with which the pairwise GB model and the PB model yield comparable results on atomic Born radii, the electrostatic component of free energies of ligand binding, and desolvation energies of the ligands and proteins. The energetics of the 15 test complexes calculated with the GB model using this set of parameters also agrees well with the energetics calculated with the PB method. This is the first time that the GB model has been parametrized and thoroughly compared with the PB model for the electrostatics of ligand binding. PMID- 16671750 TI - The link between sequence and conformation in protein structures appears to be stereochemically established. AB - In search of the link between sequence and conformation in protein structures, we perform molecular dynamics analysis of the effect of stereochemical mutation in end-protected octa-alanine Ac-Ala8-NHMe from poly-L to an alternating-L,D structure. The mutation has a dramatic effect, transforming the peptide from a condition of extreme sensitivity to one of extreme insensitivity to solvent. Examining the molecular folds of poly-L and alternating-L,D structure in atomistic detail, we find them to differ in the relationship between peptide dipolar interactions at the local and nonlocal levels, either conflicting or harmonious depending upon the chain stereochemistry. The stereochemical transformation of interpeptide electrostatics from a condition of conflict to one of harmony explains the long-standing puzzle of why poly-L and alternating-L,D peptides strongly differ in properties such as "stiffness" and solvent sensitivity. Furthermore, it is possible that poly-L stereochemistry is also the fulcrum of protein sensitivity to the effects of amino acid side-chain structures via dielectric arbitrations in interpeptide electrostatics. Indeed the evidence is accumulating that the amino acid side chains differing in alpha-helix and beta sheet propensities also differ in their desolvating effects in the adjacent and nearest-neighbor peptides and thus possibly in the solvent screening of peptide dipolar interactions. PMID- 16671751 TI - Homonuclear and heteronuclear NMR studies of a statherin fragment bound to hydroxyapatite crystals. AB - Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond. PMID- 16671752 TI - Charge transfer through a protein-nano junction. AB - We address the problem of charge transfer (CT) between a nanosized inorganic system and a protein from a theoretical and numerical perspective. The CT process is described on an atomistic level by applying an electronic Hamiltonian that takes into account the chemical bond, vibronic coupling effects, and polarization degrees of freedom. As a structurally well-characterized example, we consider a complex of C60 and its antibody. For this system, we find a novel efficient protein CT mechanism; through-space superexchange is mediated by stacked pi orbital systems. The predicted rates are comparable to those obtained for short range electron tunneling through covalent bonds, the fastest ground-state CT process known for proteins. PMID- 16671753 TI - Orientational control of the physiological reaction of cytochrome c oxidase tethered to a gold electrode. AB - The physiological reaction of a membrane protein is reconstituted on a solid supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry. PMID- 16671754 TI - The chromophore of asFP595: a theoretical study. AB - We investigate the electronic and structural properties of the chromophore of the asCP/asFP595, a newly discovered protein of the (green) fluorescent protein family. The use of theoretical methods with different degrees of accuracy and efficiency (DFT, TDDFT, CASSCF and perturbative corrections) allows us to compare the properties of a large number of hypothetic molecular models for the chromophore. The models are sorted on the basis of the relative stability and through a comparison with the experimental values of the excitation energy. Our study indicates that the most probable structure of the photoactive moiety in the protein and in water is the one resulting from the GFP-like rather than the "alternative" cyclization scheme. PMID- 16671756 TI - Molecular motions in polymer films near the glass transition: a single molecule study of rotational dynamics. PMID- 16671755 TI - Liquid phase behavior of ionic liquids with alcohols: experimental studies and modeling. AB - Ionic liquids (ILs) have been suggested as potential "green" solvents to replace volatile organic solvents in reaction and separation processes due to their negligible vapor pressure. To develop ILs for these applications, it is important to gain a fundamental understanding of the factors that control the phase behavior of ionic liquids with other liquids. In this work, we continue our study of the effect of chemical and structural factors on the phase behavior of ionic liquids with alcohols, focusing on pyridinium ILs for comparison to imidazolium ILs from our previous studies. The impact of different alcohol and IL characteristics, including alcohol chain length, cation alkyl chain length, anion, different substituent groups on the pyridinium cation, and type of cation (pyridinium vs imidazolium) will be discussed. In general, the same type of behavior is observed for pyridinium and imidazolium ILs, with all systems studied exhibiting upper critical solution temperature behavior. The impacts of alcohol chain length, cation chain length, and anion, are the same for pyridinium ILs as those observed previously for imidazolium ILs. However, the effect of cation type on the phase behavior is dependent on the strength of the cation-anion interaction. Additionally, all systems from this study and our previous work for imidazolium ILs were modeled using the nonrandom two-liquid (NRTL) equation using two different approaches for determining the adjustable parameters. For all systems, the NRTL equation with binary interaction parameters with a linear temperature dependence provided a good fit of the experimental data. PMID- 16671757 TI - Chiral silanes via asymmetric hydrosilylation with catalytic CuH. AB - [reaction: see text] CuH-catalyzed asymmetric conjugate reduction of beta-silyl alpha,beta-unsaturated esters has been developed. Using PMHS as a stoichiometric source of hydride and in situ generated CuH ligated by Solvias' JOSIPHOS analogue PPF-P(t-Bu)(2) leads to highly enantioselective 1,4-reductions. PMID- 16671758 TI - Rhodium-catalyzed asymmetric cyclization/hydroboration of 1,6-enynes. AB - [reaction: see text] Reaction of enyne 1 with catecholborane catalyzed by a 1:1 mixture of [Rh(COD)(2)](+)SbF(6)(-) and (S)-BINAP (5 mol %) followed by Pd catalyzed arylation with p-IC(6)H(4)CF(3) gave benzylidenecyclopentane 5 in 65% yield with 88% ee. Rhodium-catalyzed asymmetric cyclization/hydroboration followed either by Pd-catalyzed arylation or by oxidation was applied to the synthesis of a number of chiral, nonracemic carbocycles and heterocycles. PMID- 16671759 TI - Simple protocol for NMR analysis of the enantiomeric purity of diols. AB - [reaction: see text] A practically simple three-component chiral derivatization protocol for determining the enantiopurity of chiral 1,2-, 1,3-, and 1,4-diols by (1)H NMR spectroscopic analysis is described. The method involves treatment with 2-formylphenylboronic acid and enantiopure alpha-methylbenzylamine to afford a mixture of diastereoisomeric iminoboronate esters whose ratio is an accurate reflection of the enantiopurity of the parent diol. PMID- 16671760 TI - Total synthesis of stephanotic acid methyl ester. AB - [structure: see text]The methyl ester of the naturally occurring macrocyclic pentapeptide stephanotic acid, containing an unusual beta-substituted alpha-amino acid with a tryptophan C-6 to leucine beta-carbon link, has been synthesized. The key steps include the formation of this amino acid through a thioxo-oxazolidine intermediate and a Horner-Wadsworth-Emmons reaction using a phosphonoglycine, derived by a dirhodium(II)-catalyzed N-H insertion reaction, to give a dehydroamino acid and subsequent rhodium(I)-catalyzed asymmetric hydrogenation to introduce the modified tryptophan residue. PMID- 16671761 TI - Catalytic C-H arylation of SEM-protected azoles with palladium complexes of NHCs and phosphines. AB - [reaction: see text] The synthesis and catalytic evaluation of palladium complexes containing imidazolyl carbene ligand of varying steric and electronic properties is reported. These complexes catalyze the efficient C-H arylation of SEM-protected azole heteroarenes and thus provide a good method for preparation of a wide range of arylated free (NH)-azoles including pyrroles, indoles, imidazoles, and imidazo[1,2-a]pyridines. The reaction is operationally simple; the complexes are insensitive to moisture. PMID- 16671762 TI - Enediynes from aza-enediynes: C,N-dialkynyl imines undergo both aza-Bergman rearrangement and conversion to enediynes and fumaronitriles. AB - [reaction; see text] Aza-enediynes (C,N-dialkynyl imines) undergo thermal aza Bergman rearrangement to beta-alkynyl acrylonitriles through 2,5 didehydropyridine (2,5-ddp) intermediates. Certain aza-enediynes also undergo an alternative process affording enediynes and fumaronitriles. Studies employing a specifically (l3)C-labeled aza-enediyne show that the conversion to enediyne is second order in aza-enediyne, proceeds by a "head-to-tail" coupling, and affords the (Z)-enediyne. PMID- 16671763 TI - Photodimerizable ditopic ligand. AB - [reaction: see text] The synthesis, photophysical properties, and structural characterization of a photodimerizable ditopic ligand are described. Upon irradiation at 366 nm, ligand 1 dimerizes to the head-to-tail tetra-bpy ligand 2. This thermally stable photodimer can be dissociated back to 1 using higher energy irradiation (254 nm). PMID- 16671764 TI - General approach to the coupling of organoindium reagents with imines via copper catalysis. AB - [reaction: see text] A copper-catalyzed three-component coupling of organoindium reagents with imines and acid chlorides is described. This mild carbon-carbon bond forming reaction requires only one-third of an equivalent of indium reagent to proceed in high yield, with the sole byproduct being indium trichloride. The reaction demonstrates broad generality, with aryl-, heteroaryl-, vinyl-, and alkylindiums, as well as functionalized imines and acid chlorides, all providing alpha-substituted amides or N-protected amines in a single step. PMID- 16671765 TI - Novel calixarene hemisphere synthesized via pinacol rearrangement of [2.1.2.1]metacyclophane. AB - [reaction: see text] Tetramethoxy[2.1.2.1]metacyclophane ([2.1.2.1]MCP) was prepared by the pinacol coupling reaction of diphenylmethane dialdehyde. The treatment of [2.1.2.1]MCP with trimethylsilyl chloride and sodium iodide yielded two unexpected calixarene derivatives, cone (hemisphere) and 1,2-alternate types, instead of octahydroxy[2.1.2.1]MCP. The X-ray structure of the cone-type derivative and its inclusion property with acetonitrile were also discussed. PMID- 16671766 TI - Encapsulation of neutral guests by tri(ethylene oxide)-pyrrole-terminated dendrimer hosts in water. AB - [reaction: see text] This work focuses on the synthesis and guest hosting capabilities of novel, water-soluble tri(ethylene oxide)-pyrrole-functionalized poly(propylene imine), PPI, dendrimers (DAB-TEOPy(n)). The high-generation DAB TEOPy(n) (n = 32 and 64) possesses the ability to encapsulate hydrophobic guest molecules, such as Nile Red, in aqueous media. PMID- 16671767 TI - Highly diastereoselective asymmetric Mannich reactions of 1,3-dicarbonyls with acyl imines. AB - [reaction: see text] The cinchona alkaloids catalyze direct asymmetric Mannich reactions of cyclic 1,3-dicarbonyl compounds with acyl imines to afford alpha quaternary carbon-bearing reaction products in yields of up to 98%, a diastereomeric excess of 90% or greater, and enantioselectivities up to 99% ee. A model is proposed that accounts for both the observed diastereoselectivities and the enantioselectivities for the reactions. PMID- 16671768 TI - Direct synthesis of bipyrroles using phenyliodine bis(trifluoroacetate) with bromotrimethylsilane. AB - [reaction: see text] The hypervalent iodine(III) reagent, phenyliodine bis(trifluoroacetate) (PIFA), mediates the unprecedented, oxidative coupling reaction of pyrroles to give alpha-linked bipyrroles selectively in the presence of bromotrimethylsilane. This straightforward synthesis could provide 2,3' bipyrrole by the choice of a N-substituent of pyrrole. Mechanistic consideration of the present reaction is also described. PMID- 16671769 TI - Highly regioselective transformation of alkenyl bromides into alpha bromoaziridines and alpha-bromohydrazones. AB - [reaction: see text] The synthetic utility of alpha-halohydrazones is an underexplored area due to the lack of chemo- and regioselective routes towards these molecules. Herein, we describe a general method for alpha-bromohydrazone synthesis via the rearrangement of alpha-bromoaziridines, which can be readily prepared for the first time from the corresponding alkenyl bromides. The rearrangement of alpha-bromoaziridines into alpha-bromohydrazones proceeds with high yields and with high selectivities. PMID- 16671770 TI - Solid-phase synthesis of cyclic PNA and PNA-DNA chimeras. AB - [structure: see text] A new and versatile on-line automated solid-phase approach to obtain cyclic PNA (I and III) and cyclic PNA-DNA chimeras (II) in highly pure form has been developed. Starting from a Tentagel matrix functionalized with a 3 chloro-4-hydroxyphenylacetic linker, the synthesis of representative, new cyclic molecules by standard peptide and phosphoramidite-based chemistry has been achieved. PMID- 16671771 TI - Regiospecific hydration of gamma-hydroxy-alpha,beta-acetylenic esters: a novel asymmetric synthesis of tetronic acids. AB - [reaction: see text] The optically active gamma-hydroxy-alpha,beta-acetylenic esters are obtained from the enantioselective reaction of methyl propiolate with both aliphatic and aromatic aldehydes. These compounds can undergo regiospecific hydration in the presence of Zeise's dimer, [PtCl(2)(C(2)H(4))](2), to generate the optically active tetronic acids. PMID- 16671772 TI - Fluorogenic phospholipid substrate to detect lysophospholipase D/autotaxin activity. AB - [reaction: see text] Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA). Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug target. PMID- 16671773 TI - Palladium asymmetric allylic alkylation of prochiral nucleophiles: horsfiline. AB - [reaction; see text] The asymmetric synthesis of the oxindole alkaloid horsfiline is described. A palladium-catalyzed asymmetric allylic alkylation (AAA) is used to set the spiro(pyrrolidine-oxindole) stereogenic center. PMID- 16671774 TI - Synthesis of functionalized organotrifluoroborates via halomethyltrifluoroborates. AB - [reaction: see text] Potassium bromo- and iodomethyltrifluoroborates have been prepared via in situ reaction of n-BuLi with dibromo- and diiodomethane, respectively, in the presence of trialkyl borates, followed by treatment with KHF(2). Moreover, a new synthetic method for the preparation of potassium organotrifluoroborates through nucleophilic substitution of the halide in these potassium halomethyltrifluoroborates is described. PMID- 16671775 TI - A concise, enantioselective approach to (-)-quinic acid. AB - [reaction: see text] An expedient, enantioselective synthesis of a key precursor to (-)-quinic acid has been achieved from an ephedrine-derived morpholine-dione. The salient features of this approach are a highly diastereoselective conversion of the dione to a dialkenyl morpholinone and a subsequent ring-closing metathesis reaction. Removal of the ephedrine portion generates an enantiomerically enriched hydroxycyclohexene carboxamide that is readily converted to the quinic acid precursor. PMID- 16671777 TI - Preparation of annulated nitrogen-containing heterocycles via a one-pot palladium catalyzed alkylation/direct arylation sequence. AB - [reaction: see text] A palladium-catalyzed/norbornene-mediated sequential coupling reaction involving an aromatic sp(2) C-H functionalization as the key step is described, in which an alkyl-aryl bond and an aryl-heteroaryl bond are formed in one pot. A variety of highly substituted six- and seven-membered annulated pyrroles and pyrazoles were synthesized in a one-step process in good yields from readily accessible N-bromoalkyl pyrroles or pyrazoles and aryl iodides. PMID- 16671776 TI - Synthetic studies on (+)-ophiobolin A: asymmetric synthesis of the spirocyclic CD ring moiety. AB - [reaction; see text] Asymmetric synthesis of the spirocyclic CD-ring moiety of (+)-ophiobolin A is described. Fragment A, which was prepared via pig liver esterase (PLE)-mediated kinetic resolution, and fragment B, which was prepared via diastereoselective allylation and subsequent kinetic iodolactonization, were coupled to afford the allylsilane 2, which was successfully cyclized to the desired spirocyclic CD-ring moiety 1a in the presence of a Lewis acid. PMID- 16671778 TI - Regioselective [5,5]-sigmatropic rearrangement reactions of aryl hydrazides. AB - [reaction: see text] N,N'-Aryl hydrazides with substituents at the ortho or meta positions undergo highly regioselective [5,5]-sigmatropic rearrangement reactions to furnish benzidines in good to excellent isolated yields. The presence of single substituent at either the ortho or meta position provides sufficient bias, effectively suppressing the formation of diphenylene, the major byproduct of the conventional benzidine rearrangement reaction. PMID- 16671779 TI - Synthesis of C-aryldeoxyribosides by [2 + 2 + 2]-cyclotrimerization catalyzed by Rh, Ni, Co, and Ru complexes. AB - [reaction: see text] A novel approach to the synthesis of functionalized C nucleosides was developed. Cyclotrimerization of C-alkynyldeoxyriboside with a variety of substituted 1,6-heptadiynes to the corresponding C-aryldeoxyribosides was catalyzed by various transition metal complexes (Rh, Ir, Co, Ru, and Ni). The most general catalyst proved to be RhCl(PPh(3))(3), which could catalyze most of the cyclotrimerizations in high yields (52-95%). PMID- 16671780 TI - Computational study and analysis of the kinetic isotope effects of the rearrangement of cis-bicyclo[4.2.0]oct-7-ene to cis,cis-cycloocta-1,3-diene. AB - [reaction: see text] On the basis of KIE experiments, the ring opening of cis bicyclo[4.2.0.]oct-7-ene has been suggested as an anti-Woodward-Hoffmann reaction candidate. We hereby report the results of a high-level computational study of the alternate reaction pathways which proves that the energy profiles show a clear preference for the conrotatory (W-H allowed) ring opening followed by double-bond isomerization. Computed KIE values for the aforementioned mechanism are in good agreement with the experimental values. PMID- 16671781 TI - spiro-Mamakone A: a unique relative of the spirobisnaphthalene class of compounds. AB - [structure: see text] A spirobisnaphthalene derivative with a new spiro-nonadiene skeleton, spiro-mamakone A (1), has been isolated from the extract of a cultured nonsporulating fungal endophyte derived from the New Zealand native tree Knightia excelsa (rewarewa). The carbon skeleton of spiro-mamakone A represents a new structural entity and an intriguing addition to the structurally diverse spirobisnaphthalene group of compounds. spiro-Mamakone A is potently cytotoxic and is also antimicrobial. PMID- 16671782 TI - Macrocycloadditions leading to conformationally restricted small molecules. AB - [reaction: see text] The Cu(I)-catalyzed cycloaddition of alkynes and azides (click reaction) provides a robust method for the construction of macrocyclic small molecules via an intramolecular macrocycloaddition. A three-subunit system has been used to explore the tolerance of this macrocycloaddition to variations of stereochemistries and substituents. PMID- 16671783 TI - Unusual temperature dependence of enantioselectivity in asymmetric reductions by chiral NADH models. AB - [reaction: see text] Unusual stereoselectivity changes, i.e., enhancement and inversion of enantioselectivity with increasing temperature, were observed in the asymmetric reduction of methyl benzoylformate with chiral 1,4-dihydropyridines possessing amino acid residues as ligating chiral auxiliaries. The differential activation parameters, DeltaDeltaH(S-R) and DeltaDeltaS(S-R), obtained from the Eyring plots demonstrate that the entropy term controls the enantiodifferentiating step, accounting for the observed unique temperature dependencies. PMID- 16671784 TI - Asymmetric, anti-selective scandium-catalyzed Sakurai additions to glyoxyamide. Applications to the syntheses of N-boc D-alloisoleucine and D-isoleucine. AB - [reaction: see text] An enantio- and diastereoselective Sakurai-Hosomi reaction, catalyzed by chiral scandium pyridyl-bis(oxazoline) (pybox) complexes, has been developed. Both alkyl- and aryl-substituted allylsilanes are effective coupling partners with N-phenylglyoxamide. Applications of this reaction to the asymmetric syntheses of N-Boc D-alloisoleucine and D-isoleucine are described. PMID- 16671786 TI - Highly efficient one-pot three-component Mannich reaction in water catalyzed by heteropoly acids. AB - [reaction: see text] Heteropoly acids efficiently catalyzed the one-pot, three component Mannich reaction of ketones with aromatic aldehydes and different amines in water at ambient temperature and afforded the corresponding beta-amino carbonyl compounds in good to excellent yields and with moderate diastereoselectivity. This method provides a novel and improved modification of the three-component Mannich reaction in terms of mild reaction conditions and clean reaction profiles, using very a small quantity of catalyst and a simple workup procedure. PMID- 16671785 TI - One-flask synthesis of dinucleoside tetra- and pentaphosphates. AB - [reaction: see text] We report a one-flask route for the synthesis of dinucleoside tetra- and pentaphosphates, in isolated yields of 50-85%. This route relies on a mixture of P(III) and P(V) chemistries, using phosphitylation of a protected nucleoside with 2-chloro-4H-l,3,2-benzo-dioxaphosphorin-4-one (salicylchlorophosphite), followed by sequential reaction with inorganic pyrophosphate and a nucleoside 5' mono- or diphosphate. PMID- 16671787 TI - Palladium-catalyzed synthesis of 2-(aminomethyl)indoles from ethyl 3-(o trifluoroacetamidophenyl)-1-propargyl carbonate. AB - [reaction: see text] The palladium-catalyzed reaction of ethyl 3-(o trifluoroacetamidophenyl)-1-propargyl carbonate with piperazines in the presence of Pd(PPh(3))(4) in THF at 80 degrees C affords 2-(piperazin-1-ylmethyl)indoles in excellent yields. Good to excellent yields are also obtained with other secondary amines. PMID- 16671788 TI - 2-Phenylthio-3-bromopropene, a valuable synthon, easily prepared by a simple rearrangement. AB - [reaction: see text] Treatment of allyl phenyl sulfide with bromine, followed by aqueous sodium hydroxide, provides a good yield of 2-phenylthio-3-bromopropene 2 via a mechanism that is elucidated by isolation of the 1,3-dibromo-2 (phenylthio)propene intermediate 7. Three uses of 2 as an annulating agent for cycloheptanone are illustrated, demonstrating that this reagent is a synthetic equivalent of acetonyl halide and an alpha-halo vinyllithium. It is also readily converted to the useful synthons 2,3-bis(phenylthio)propene 16 and 2-phenylthio-3 (phenylsulfonyl)propene 17. PMID- 16671789 TI - Rearrangement of homoallylic alcohols induced by DAST. AB - [reaction: see text] Treatment of beta,gamma-unsaturated monoprotected 1,2-diols with diethylaminosulfur trifluoride (DAST) allows the stereoselective formation of beta,gamma-unsaturated aldehydes in good yields and with a good transfer of chirality. PMID- 16671790 TI - 1,2-Bis(diphenylphosphino)carborane as a dual mode ligand for both the Sonogashira coupling and hydride-transfer steps in palladium-catalyzed one-pot synthesis of allenes from aryl iodides. AB - [reaction: see text] One-pot allene synthesis from aryl iodides 1 and propargyldicyclohexylamine 2 proceeded in the presence of Pd(2)(dba)(3).CHCl(3) catalyst (2.5 mol %), 1,2-bis(diphenylphosphino)carborane 5 (10 mol %), CuI (15 mol %), and Et(3)N (150 mol %) to give the corresponding allenes 4 in good to high yields. Electron-deficient bidentate phosphines, such as 1,2 bis(diphenylphosphino)carborane 5 and (C(6)F(5))(2)PC(2)H(4)P(C(6)F(5))(2), play the role of a dual mode ligand for both the Sonogashira coupling and hydride transfer reactions. PMID- 16671791 TI - Palladium-catalyzed asymmetric hydrophosphorylation of norbornenes. AB - [reaction: see text] By using Josiphos ligands, palladium-catalyzed hydrophosphorylation of norbornenes with hydrogen phosphonates proceeded efficiently to give the corresponding phosphonates in high enantioselectivities. PMID- 16671792 TI - Dipeptide analogues containing 4-ethoxy-3-pyrrolin-2-ones. AB - [reaction: see text] Pyrrolidine-2,4-diones (1) are naturally occurring analogues of amino acids. We herein present a facile synthesis of N-acylated, O-alkylated pyrrolin-2-ones (2) in high yield and excellent enantiopurity. Molecular mechanics calculations suggest that the resulting dipeptide analogues adopt a linear, extended conformation. PMID- 16671793 TI - A bis-guanidine-based multisignaling sensor molecule that displays redox ratiometric behavior or fluorescence enhancement in the presence of anions and cations. AB - [structure: see text]The guanidine bridge in the new structural motif [3,3]ferrocenophane acts as a dual binding site for anions and metal ions. Sensing of anions (Cl(-), F(-), NO(3)(-), HSO(4)(-), and H(2)PO(4)(-)) takes place by redox-ratiometric measurements, whereas metal ion (Zn(2+), Ni(2+), and Cd(2+)) recognition is achieved by fluorescence measurements. PMID- 16671794 TI - A new approach to the furan degradation problem involving ozonolysis of the trans enedione and its use in a cost-effective synthesis of eplerenone. AB - [reaction: see text] Whereas ozonization of furan 3a affords little or no carboxylic acid 5, ozonization of the corresponding trans-enedione 6 afforded carboxylic acid 5 in 82.4% yield (cryst., overall from furan, 100 g scale; after workup with dimethyl sulfide, followed by mildly basic hydrogen peroxide). This new approach to furan degradation is showcased in a cost-effective synthesis of eplerenone, an important new medicine for cardiovascular indications. PMID- 16671795 TI - Enantioselective Friedel-Crafts alkylation of indoles with nitroalkenes catalyzed by bifunctional tridentate bis(oxazoline)-Zn(II) complex. AB - [reaction: see text] A more practical and efficient catalytic asymmetric Friedel Crafts alkylation of indoles with nitroalkenes using bifunctional tridentate bis(oxazoline)-Zn(OTf)(2) as catalyst has been developed. Various types of the nitroalkylated indoles were obtained in excellent yields (85-99%) and high enantioselectivities (up to 98% ee). PMID- 16671796 TI - Path selection for conformational interconversions in [2]catenanes. AB - [strucure: see text]The conformational interconversions of several [2]catenanes containing a dibenzo-34-crown-10 ether (BPP34C10) interlocked with rings containing two 4,4'-dipyridyls tethered by different aryl spacers have been studied. Blocking groups on the tethers enabled the two pathways for circumrotation of the BPP34C10 to be open or blocked. The activation barrier for migration along the open tethers varied from 11 to 13 kcal/mol. This study demonstrates an ability to select the pathway for conformational interconversions in [2]catenanes. PMID- 16671797 TI - Synthesis of ent-thallusin. AB - [reaction: see text] A three-step route from sclareol oxide (6) to bromo ester 4 in 53% overall yield was achieved using the efficient oxidation of an allylic bromide to an enal with bis(2,4,6-trimethylpyridine)silver(I) hexafluorophosphate in DMSO. Stille coupling of bromo ester 4 with stannylpyridine 5 gave the trimethyl ester of ent-thallusin in 54-92% yield by the stoichiometric conversion of 4 to a vinyl palladium intermediate prior to the addition of 5 to the reaction. PMID- 16671798 TI - Stereoselective synthesis of highly substituted gamma-lactams by the [3+2] annulation of alpha-siloxy allylic silanes with chlorosulfonyl isocyanate. AB - [reaction: see text] A stereoselective synthesis of gamma-lactams by the [3+2] annulation of alpha-siloxy allylic silanes with N-chlorosulfonyl isocyanate (ClSO(2)NCO) was developed. The use of these allylic silanes allowed for further diastereoselective substitution of the resultant N,O-acetal to give highly substituted gamma-lactams. Oxidation of the silyl group afforded access to complex beta-hydroxy-gamma-lactams. PMID- 16671799 TI - Total synthesis and stereochemical reassignment of (+)-dolastatin 19. AB - [reaction: see text] A revised configurational assignment for the cytotoxic marine macrolide dolastatin 19 is proposed and validated by total synthesis. Key features of the route include an asymmetric vinylogous aldol reaction to install the isolated C13 stereocenter and (E)-trisubstituted alkene, two sequential 1,4 syn boron-mediated aldol reactions, and a Mukaiyama glycosylation to append the l rhamnose-derived pyranoside. PMID- 16671800 TI - New additivity schemes for hydrocarbon energies. AB - [structure: see text] A new isodesmic additivity scheme based on the energetic relationships among the simplest hydrocarbon molecules reproduces the experimental heats of formation for a broad range of unstrained hydrocarbons with remarkable accuracy. The stabilizations of radicals, double, and triple CC bonds by alkyl substituents (hyperconjugation), as well as the stabilization by 1,3 alkyl group interactions at the same carbon (branching), support conventional interpretations. Statistical data fitting can also be achieved by using only four adjustable parameters. PMID- 16671801 TI - Cross metathesis with strained exocyclic enones: synthesis of 3-alkylideneoxetan 2-ones from 3-methyleneoxetan-2-ones. AB - [reaction: see text] 3-Alkylideneoxetan-2-ones have been prepared in good to excellent yields with high Z-selectivity by olefin cross metathesis with 3 methyleneoxetan-2-ones in the presence of second generation metathesis catalysts 1 or 2. PMID- 16671802 TI - New protocols for the assembly of the tetracyclic framework associated with the aromatic erythrina alkaloids. AB - [reaction: see text] Treatment of the anion derived from the ring-fused gem dichlorocyclopropane 4c with silver tetrafluoroborate afforded the spirocyclic compound 17 in 74% yield. Product 17 was readily converted, over three steps, into the beta-iodoethyl derivative 20 and treatment of this latter compound with n-Bu(3)SnH then afforded, in 93% yield and via a radical addition/elimination sequence, compound 2 incorporating the ABCD framework of the aromatic erythrina alkaloids. PMID- 16671803 TI - 1,2-Bisanionic coupling approach to 2,3-disubstituted cyclopentenols and cyclopentenones. AB - [reaction: see text] We describe a new approach to 2,3-disubstituted cyclopentenols and cyclopentenones through two consecutive regioselective additions of equal or different electrophiles to a cyclopentene bisanionic synthon. Indeed, on exposure to BuLi, 3-bromo-2-iodocyclopent-2-enol O-TBS ether undergoes iodine-lithium permutation with complete regioselectivity. Successive reaction of the monolithium anion with different C(sp(2))- and C(sp(3)) electrophiles affords the corresponding 2-substituted-3-bromocyclopentenol derivative. Subsequent bromo-lithium exchange with t-BuLi, followed by reaction with an equal or different electrophile, affords the desired 2,3-disubstituted cyclopentenol. PMID- 16671804 TI - Synthesis of highly substituted pyrroles via a multimetal-catalyzed rearrangement condensation-cyclization domino approach. AB - [reaction: see text] In a convenient one-pot process, easily accessed propargyl vinyl ethers and aromatic amines are effectively converted into tetra- and pentasubstituted 5-methylpyrroles which can further be transformed into 5 formylpyrroles via IBX-mediated oxidation. The cascade reaction proceeds through a silver(I)-catalyzed propargyl-Claisen rearrangement, an amine condensation, and a gold(I)-catalyzed 5-exo-dig heterocyclization. PMID- 16671805 TI - Skeletal reorganization of enynes catalyzed by InCl3. AB - [reaction: see text] The skeletal reorganization of enynes is achieved by the presence of InCl(3) as the catalyst. The reaction of enynes having a terminal acetylenic moiety proceeds in a stereospecific manner to give 1 vinylcycloalkenes. The reaction of enynes containing an alkyl group on the acetylenic terminal carbon resulted in a new type of skeletal reorganization to give 1-allylcycloalkenes, formation of which involves a double cleavage of the C C double bond and the triple bond. PMID- 16671806 TI - Kinetically controlled self-assembly of pseudorotaxanes on crystallization. AB - [reaction: see text] Mixing of equimolar amounts of cyclobis(paraquat-p phenylene) (CBPQT(4+)) with a bis-4-methylphenyl ether (MPE twice) of a 1,5 dioxynaphthalene (DNP) derivative in MeCN/CH(2)Cl(2) (3:1) results in the formation of a [2]pseudorotaxane which, on crystallization, yields a [4]pseudorotaxane in the solid state that is stabilized by multiple [C-H...F] interactions: a mixture of the same components in a 1:3 ratio affords a crystalline [2]pseudorotaxane after vapor diffusion of methyl-tert-butyl ether into a solution of these components in MeCN/CH(2)Cl(2) (3:1). PMID- 16671807 TI - Shape-selective fluorescent sensing ensemble using a tweezer-type metalloreceptor. AB - [reaction: see text] A fluorescent sensing ensemble for pyridine-derived compounds is described. The receptor portion of the ensemble is prepared from a bisimidazole pyridine which coordinates copper to form a well-defined cavity. Small heteroaromatic guests such as adenine bind strongly in the cavity. The fluorescent response is provided by a dye which is coordinated to the receptor and quenched by the metal ion. The dye is released upon guest binding providing up to 25-fold fluorescence increases. PMID- 16671808 TI - A new modular indole synthesis. Construction of the highly strained CDEF parent tetracycle of nodulisporic acids A and B. AB - [reaction: see text] Construction of the highly strained CDEF parent tetracycle, a structural motif found only in the potent ectoparasiticidal agents (+) nodulisporic acids A and B and related congeners, has been achieved via a new modular indole synthesis, exploiting a sequential Stille cross-coupling/Buchwald Hartwig union/cyclization tactic. The new indole synthesis holds the promise of rapid assembly of diverse, highly substituted indoles possessing uncommon substitution patterns. PMID- 16671809 TI - Chemical transformation of prostaglandin-A2: a novel series of C-10 halogenated, C-12 hydroxylated prostaglandin-A2 analogues. AB - [reaction: see text] Synthesis of a novel class of C-10 halogenated and C-12 oxygenated prostaglandin-A(2) derivatives (6a-6c) has been accomplished. (15S) Prostaglandin-A(2) (1), from the gorgonian Plexaura homomalla, served as the starting material for the synthesis. The absolute configuration was determined using NMR. PMID- 16671811 TI - Two novel tetrairidoid glucosides from Dipsacus asper. AB - [reaction: see text] Two new iridoid glucoside tetramers, dipsanosides A (1) and B (2), the first-reported iridoid tetramers with four glucosides, were isolated from Dipsacus asper. Their structures were determined by analysis of 1D and 2D NMR data as well as by comparison with model compounds. Their cytotoxicities were tested, but neither of them showed obvious activity. PMID- 16671810 TI - Solvolysis of a tetrahydropyranyl mesylate: mechanistic implications for the Prins cyclization, 2-oxonia-cope rearrangement, and Grob fragmentation. AB - [reaction: see text] A solvolysis reaction is used to demonstrate that a tetrahydropyranyl cation is a common intermediate for Prins cyclizations, 2 oxonia-Cope rearrangements, and Grob fragmentations of tetrahydropyran rings. PMID- 16671812 TI - Aziridinium from N,N-dibenzyl serine methyl ester: synthesis of enantiomerically pure beta-amino and alpha,beta-diamino esters. AB - [reaction: see text] Reaction of N,N-dibenzyl-O-methylsulfonyl serine methyl ester with a variety of heteronucleophiles (sodium azide, sodium phthalimide, amines, thiols) and carbanions (sodium malonate) gave, via an aziridinium intermediate, the corresponding beta-amino or alpha,beta-diamino ester in good to excellent yield. A short synthesis of orthogonally protected and enantiomerically pure 2,3-diamino propionate (Dap) is described. PMID- 16671813 TI - Biomimetic approach to perophoramidine and communesin via an intramolecular cyclopropanation reaction. AB - [reaction: see text] Starting from tryptamine 4 and isatin 5, a biomimetic approach to the pentacyclic substructure 1 of perophoramidine and communesin was developed. The key steps were to create a stable three/six bicyclic system 2 on the 2,3-double bond of an indole derivative 3 by an intramolecular cyclopropanation, followed by ring opening of the resulting cyclopropane ring with the in situ generated amine group of an aniline. PMID- 16671814 TI - Formal synthesis of 6-deoxyerythronolide B. AB - [reaction: see text] The enantioselective synthesis of the carbon skeleton of 6 deoxyerythronolide B has been achieved in 23 linear steps from propionaldehyde. The synthesis relies on an iterative approach employing an asymmetric acyl thiazolidinethione propionate aldol reaction to establish eight of nine stereogenic centers. The remaining stereogenic center at C6 was set through a Myers alkylation employing a complex alkyl iodide. PMID- 16671815 TI - Regioselective carbene insertion on polysubstituted dihydroaromatic compounds. AB - [reaction: see text] The rhodium-catalyzed CH-insertion reaction of diazo ester derivatives into dihydroaromatic compounds was expanded to the application of various polysubstituted and functionalized 1,4-dienes generated from a cobalt catalyzed Diels-Alder reaction. The highly regioselective CH-insertion process produces after DDQ oxidation polysubstituted, polyfunctionalized aromatic compounds in good overall yields. PMID- 16671816 TI - Hydrophobic effects are dominant over secondary orbital interactions for a simple Diels-Alder reaction in salt solutions. AB - [reaction: see text] The stereoselectivity ratios for a Diels-Alder reaction between cyclopentadiene with methyl trans-crotonate carried out in salt solutions demonstrate the dominance of hydrophobic effects over secondary orbital interactions. PMID- 16671819 TI - Nanofunctionalisation for the treatment of peripheral nervous system injuries. AB - A construct based on the electrostatic layer-by-layer self assembly technique has been fabricated, to be used as a tailored device to encourage nerve regeneration. A multilayered nanocoating composed of three precursor bilayers of cationic poly(dimethyldiallylammonium) chloride (PDDA) and anionic poly(styrenesulfonate) (PSS), followed by bilayers of poly-D-lysine (PDL) and antibody specific to transforming growth factor 1 (anti-TGF-1), has been deposited on HYAFF 11. The assembly process has been monitored by quartz crystal microbalance (QCM) for its characterisation and then it has been used on HYAFF 11. Structural studies of the resulting multilayers confirmed stepwise deposition of anti-TGF-1, with an average layer thickness of 2.2+/-0.2 nm and an average surface density of 0.36+/ 0.03 mug cm(-2). Scanning electron microscopy has been used to characterise multilayer uniformity. Finally, the immunological activity of the multilayered structure has been assessed. The results show that anti-TGF-1 can be included in its active form in a predetermined multilayered structure onto HYAFF 11 with quantitative control of layer thickness and weight, providing a high tool with great potential in tissue engineering. PMID- 16671820 TI - Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin ergosterol modulated vesicle fusion. AB - The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render protein-carrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatin-ergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the -amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems. PMID- 16671821 TI - Sensors made of RNA: tailored ribozymes for detection of small organic molecules, metals, nucleic acids and proteins. AB - Nucleic acids are well suited to serve as biosensors for the fast and reliable detection of small organic molecules, such as a number of metabolites or antibiotics, specific nucleic acid sequences, peptides, proteins or metal ions. One of the main advantages of using nucleic acids as biosensors is that they can be modulated to respond allosterically to specific effectors. Thus molecular recognition is transformed directly into a catalytic process with observable results. In particular, catalytic RNA structures, such as the hammerhead and hairpin ribozymes, have been used for biosensor engineering. The review reports on the function mode of nucleic acid biosensors and introduces recent developments and applications in the field. PMID- 16671835 TI - The role of music in deaf culture: deaf students' perception of emotion in music. AB - Although emotional interpretation of music is an individual and variable experience, researchers have found that typical listeners are quite consistent in associating basic or primary emotions such as happiness, sadness, fear, and anger to musical compositions. It has been suggested that an individual with a sensorineural hearing loss, or any lesion in auditory perceptors in the brain may have trouble perceiving music emotionally. The purpose of the present study was to investigate whether students with a hearing loss who associate with the deaf culture, assign the same emotions to music as students without a hearing loss. Sixty-two elementary and junior high students at a Midwestern state school for the deaf and students at neighboring elementary and junior high schools served as participants. Participants at the state school for the deaf had hearing losses ranging from moderate to severe. Twelve film score excerpts, composed to depict the primary emotions-happiness, sadness, and fear, were used as the musical stimuli. Participants were asked to assign an emotion to each excerpt. Results indicated a significant difference between the Deaf and typical hearing participants' responses, with hearing participants' responses more in agreement with the composers' intent. No significant differences were found for age or gender. Analyses of the Deaf participants' responses indicate that timbre, texture, and rhythm are perhaps the musical elements most influential in transmitting emotion to persons with a hearing loss. Adaptive strategies are suggested for assisting children who are deaf in accessing the elements of music intended to portray emotion. PMID- 16671833 TI - Linkage disequilibrium mapping of CHEK2: common variation and breast cancer risk. AB - BACKGROUND: Checkpoint kinase 2 (CHEK2) averts cancer development by promoting cell cycle arrest and activating DNA repair in genetically damaged cells. Previous investigation has established a role for the CHEK2 gene in breast cancer aetiology, but studies have largely been limited to the rare 1100delC mutation. Whether common polymorphisms in this gene influence breast cancer risk remains unknown. In this study, we aimed to assess the importance of common CHEK2 variants on population risk for breast cancer by capturing the majority of diversity in the gene using haplotype tagging single nucleotide polymorphisms (tagSNPs). METHODS AND FINDINGS: We analyzed 14 common SNPs spanning 52 kilobases (kb) of the CHEK2 gene in 92 Swedish women. Coverage evaluation indicated that these typed SNPs would efficiently convey association signal also from untyped SNPs in the same region. Six of the 14 SNPs predicted well both the haplotypic and single SNP variations within CHEK2. We genotyped these six tagSNPs in 1,577 postmenopausal breast cancer cases and 1,513 population controls, but found no convincing association between any common CHEK2 haplotype and breast cancer risk. The 1100delC mutation was rare in our Swedish population--0.7% in cases and 0.4% in controls--with a corresponding odds ratio for carriers versus noncarriers of 2.26 (95% confidence interval, 0.99-5.15). Estimates of the population frequency and the odds ratio of 1100delC indicate that our sample is representative of a Northern European population. CONCLUSIONS: Notwithstanding the involvement of the CHEK2 gene in breast cancer aetiology, we show that common polymorphisms do not influence postmenopausal breast cancer risk. PMID- 16671834 TI - ApoE plasma levels and risk of cardiovascular mortality in old age. AB - BACKGROUND: The epsilon2, epsilon3, and epsilon4 alleles of the apolipoprotein E gene (APOE) encode three isoforms, apoE2, E3, and E4, respectively. The apoE isoforms circulate in different plasma concentrations, but plasma concentrations of the same isoform also differ between individuals. Whereas the isoforms have been associated with cardiovascular disease, the relation between plasma apoE levels and cardiovascular disease is unknown. METHODS AND FINDINGS: We assessed APOE genotypes, plasma levels of apoE, cardiovascular risk factors, and mortality in a population-based sample of 546 individuals aged 85 y who participated in the Leiden 85-plus Study and were prospectively followed for specific causes of death for 5 y. Participants in the highest tertile of apoE levels suffered a twofold increased risk of cardiovascular mortality (hazard ratio compared to lowest tertile, 2.08; 95% confidence interval [CI], 1.30 to 3.33). Among the 324 participants with the epsilon3epsilon3 genotype, the hazard from cardiovascular disease was threefold increased (highest versus lowest tertile 3.01; 95% CI 1.60 to 5.66), with similar estimates for men and women. Other causes of death were not increased significantly. Plasma levels of apoE in epsilon3epsilon3 participants were positively correlated with total cholesterol (p < 0.001), low density lipoprotein cholesterol (p < 0.001) and triglycerides (p < 0.001) and negatively with high-density lipoprotein cholesterol levels (p = 0.010). Adjustment for plasma lipids did not change the hazard ratios, whereas interaction was absent. The risk associated with high levels of apoE, however, was strongest in participants from the lowest tertile of C-reactive protein (CRP) levels and absent in those from the highest tertile (p(interaction) < 0.001). Among participants from the lowest tertile of CRP levels, those with a high apoE levels had a significantly steeper increase in CRP than those with low apoE levels (p = 0.020). Similar cardiovascular mortality risks as in epsilon3epsilon3 participants were found in epsilon2 and epsilon4 carriers. CONCLUSIONS: In old age, high plasma apoE levels precede an increase of circulating CRP and strongly associates with cardiovascular mortality, independent of APOE genotype and plasma lipids. PMID- 16671836 TI - The effect of music versus nonmusic on behavioral signs of distress and self report of pain in pediatric injection patients. AB - Receiving vaccinations is a part of growing up; however, as necessary as vaccinations are, many children find them to be frightening and painful. Music has been examined as a potential distraction during pediatric medical procedures, but research findings have been mixed, due, in part, to the fact that children were primarily instructed to merely "listen to the music." The present study sought to determine if a focus of attention activity involving music would affect levels of distress and perceptions of pain in pediatric injection patients. Sixty four 4- to 6(1/2) -year old children receiving routine immunizations were randomly assigned to one of three conditions: musical story, spoken story, or standard care/control. Children in the two treatment conditions listened to a recorded story and pointed at corresponding pictures throughout the injection process. Observational data on distress and pain were collected, in addition to the child's self-rating of pain. Participants in the musical story condition tended to be less distressed and report less pain than participants in the other two conditions, although these differences were not statistically significant. Subsequent analysis indicated that children who received more injections tended to benefit more from the music intervention, in terms of their perceived pain. PMID- 16671838 TI - The administration of music therapy training clinics: a descriptive study. AB - A two-part study was conducted to describe issues and administrative practices related to university and college affiliated music therapy training clinics. First, all 72 AMTA academic directors were surveyed in order to discover (a) which programs had a clinic, and (b) the reasons why other directors did not operate a clinic. Second, 12 survey respondents, who reported that they were involved with a training clinic, participated in in-depth interviews discussing: (a) their motivations for establishing a clinic, (b) the possible effects of a clinic on the community, (c) the individuals and groups involved in clinic operations, d) clinic space and equipment, (e) policy and procedure topics, (f) specific administrative practices related to clients and students, (g) finances, (h) research (i) quality assurance, (j) dual roles, and (k) liability issues. The administrative practices described by the interviewees varied greatly across clinics and provided a wealth of information that could be considered both useful and thought provoking for those interested in operating a music therapy clinic. PMID- 16671837 TI - Instructional and improvisational models of music therapy with adolescents who have attention deficit hyperactivity disorder (ADHD): a comparison of the effects on motor impulsivity. AB - This study compared the impact of instructional and improvisational music therapy approaches on the level of motor impulsivity displayed by adolescent boys (n = 13) who have Attention Deficit Hyperactivity Disorder (ADHD). A combination of a multiple contrasting treatment and an experimental control group design was used. No statistical difference was found between the impact of the contrasting approaches as measured by a Synchronised Tapping Task (STT) (Humphrey, 2003) and the parent and teacher versions of Conners' Rating Scales (Conners, 1997) Restless-Impulsive (R-I) and Hyperactive-Impulsive (H-I) subscales. However, while no firm conclusions can be drawn, there are indications that the instructional approach may have contributed to a reduction of impulsive and restless behaviors in the classroom. Further, over the period of the study, both music therapy treatment groups significantly improved accuracy on the STT, and teachers reported a significant reduction in Conners' DSM-IV Total and Global Index subscale scores. These findings tentatively suggest that music therapy may contribute to a reduction in a range of ADHD symptoms in the classroom, and that increasing accuracy on the STT could be related to improvement in a range of developmental areas-not specifically motor impulsivity. PMID- 16671839 TI - Using of osmotic fragility (OF) and dichlorophenol-indolephenol (DCIP) tests screening for antenatal clinic: appraisal of usefulness of the program in rural Thai communities. PMID- 16671840 TI - Nonword repetition and sentence repetition as clinical markers of specific language impairment: the case of Cantonese. AB - PURPOSE: Recent research suggests that nonword repetition (NWR) and sentence repetition (SR) tasks can be used to discriminate between children with SLI and their typically developing age-matched (TDAM) and younger (TDY) peers. METHOD: Fourteen Cantonese-speaking children with SLI and 30 of their TDAM and TDY peers were compared on NWR and SR tasks. NWR of IN nonwords (CV combinations attested in the language) and OUT nonwords (CV combinations unattested in the language) were compared. SR performance was compared using 4 different scoring methods. RESULTS: The SLI group did not score significantly lower than the TDAM group on the test of NWR (overall results were TDAM = SLI > TDY). There were nonsignificant group differences on IN syllables but not on OUT syllables. The results do not suggest a limitation in phonological working memory in Cantonese speaking children with SLI. The SR task discriminated between children and their TDAM peers but not between children with SLI and their TDY peers matched for mean length of utterance. CONCLUSIONS: SR but not NWR discriminates between children with SLI and their TDAM peers. Poorer NWR for English-speaking children with SLI might be attributable to weaker use of the redintegration strategy in word repetition. Further cross-linguistic investigations of processing strategies are required. PMID- 16671841 TI - Can severely language delayed 3-year-olds be identified at 18 months? Evaluation of a screening version of the MacArthur-Bates Communicative Development Inventories. AB - PURPOSE: To evaluate the effectiveness of a screening instrument (the Swedish Communication Screening at 18 months of age; SCS18), derived from the Swedish MacArthur-Bates Communicative Development Inventory, in identification of 18 month-old children who will be severely language disabled by 3 years of age, the authors (a) analyzed which SCS18's component yielded the best prediction, (b) compared the productivity figures of the SCS18 with those of the traditional method of identification, and (c) tried different cutoff criteria of the SCS18. METHOD: Half of the child health care (CHC) centers in a Swedish county were randomly selected to use the SCS18 (e.g., a checklist supporting parents in assessing their child's word production, word comprehension, and communicative gestures). Remaining CHC centers used an informal assessment. Expressive and receptive language was subsequently judged with an observation for 3-year-olds that is routine in the county. An unselected population of 2,080 children participated at 18 months of age and again at 3 years of age. RESULT: Number of spoken words yielded the best prediction, and SCS18 was superior to the traditional method. A sensitivity of 50%, however, was not enough, and a stricter criterion resulted in too many false positives to be acceptable as routine. CONCLUSION: Although the SCS18 has strength, the age of 18 months seems to be too early for identification of severe language disability. PMID- 16671842 TI - The impact of augmentative and alternative communication intervention on the speech production of individuals with developmental disabilities: a research review. AB - PURPOSE: This article presents the results of a meta-analysis to determine the effect of augmentative and alternative communication (AAC) on the speech production of individuals with developmental disabilities. METHOD: A comprehensive search of the literature published between 1975 and 2003, which included data on speech production before, during, and after AAC intervention, was conducted using a combination of electronic and hand searches. RESULTS: The review identified 23 studies, involving 67 individuals. Seventeen of these studies did not establish experimental control, thereby limiting the certainty of evidence about speech outcomes. The remaining 6 studies, involving 27 cases, had sufficient methodological rigor for the "best evidence analysis" (cf. >R. E. Slavin, 1986). Most of the participants (aged 2-60 years) had mental retardation or autism; the AAC interventions involved instruction in manual signs or nonelectronic aided systems. None of the 27 cases demonstrated decreases in speech production as a result of AAC intervention, 11% showed no change, and the majority (89%) demonstrated gains in speech. For the most part, the gains observed were modest, but these data may underestimate the effect of AAC intervention on speech production because there were ceiling effects. CONCLUSIONS: Future research is needed to better delineate the relationship between AAC intervention and speech production across a wider range of participants and AAC interventions. PMID- 16671843 TI - Visuospatial immediate memory in specific language impairment. AB - PURPOSE: Investigations of the cognitive processes underlying specific language impairment (SLI) have implicated deficits in verbal short-term and working memory and in particular the storage and processing of phonological information. This study investigated short-term and working memory for visuospatial material for a group of children with SLI, to test whether the verbal memory impairments already established extend to the visuospatial domain. METHOD: Fifteen children with SLI and control groups of children matched on chronological age and language age completed tests of visuospatial short-term and working memory. RESULTS: The SLI group performed comparably with age-matched control children on all measures and at a higher level than the language-age control group on several measures. CONCLUSIONS: The visuospatial short-term and working memory abilities were at age appropriate levels in this SLI group. This contrasts markedly with their impairments on tests of verbal short-term and working memory. PMID- 16671844 TI - Language deficits in poor comprehenders: a case for the simple view of reading. AB - PURPOSE: To examine concurrently and retrospectively the language abilities of children with specific reading comprehension deficits ("poor comprehenders") and compare them to typical readers and children with specific decoding deficits ("poor decoders"). METHOD: In Study 1, the authors identified 57 poor comprehenders, 27 poor decoders, and 98 typical readers on the basis of 8th-grade reading achievement. These subgroups' performances on 8th-grade measures of language comprehension and phonological processing were investigated. In Study 2, the authors examined retrospectively subgroups' performances on measures of language comprehension and phonological processing in kindergarten, 2nd, and 4th grades. Word recognition and reading comprehension in 2nd and 4th grades were also considered. RESULTS: Study 1 showed that poor comprehenders had concurrent deficits in language comprehension but normal abilities in phonological processing. Poor decoders were characterized by the opposite pattern of language abilities. Study 2 results showed that subgroups had language (and word recognition) profiles in the earlier grades that were consistent with those observed in 8th grade. Subgroup differences in reading comprehension were inconsistent across grades but reflective of the changes in the components of reading comprehension over time. CONCLUSIONS: The results support the simple view of reading and the phonological deficit hypothesis. Furthermore, the findings indicate that a classification system that is based on the simple view has advantages over standard systems that focus only on word recognition and/or reading comprehension. PMID- 16671845 TI - School-aged children's phonological production of derived English words. AB - PURPOSE: Little is known about the phonological aspects of derivational processes. Neutral suffixes (e.g., -ness) that do not change stress and rhythmic or nonneutral suffixes (e.g., -ity) that alter stem stress were used in a production task that explored developmental changes in phonological accuracy of derived English words. METHOD: Three groups of typically achieving children, aged 7 (n = 19), 8 (n = 18), and 9 (n = 15) years, produced derived words in isolation (12 words with rhythmic suffixes and 10 with neutral suffixes). Productions were transcribed from audio-recordings. RESULTS: Stress accuracy was at ceiling levels for neutral derived words but steadily improved in words with rhythmic suffixes. The predominant stress error was maintaining stem stress in the derived form. Children also made syllabification and consonant errors (in isolation and overlapping with stem stress errors). More errors occurred on derived words with vowel changes than without. CONCLUSIONS: Morphophonological knowledge for words with rhythmic suffixes undergoes development in early school-aged children. The number or degree of phonological changes between the stem and derived word appears to be an important variable in accurate production. PMID- 16671846 TI - Children's naming and word-finding difficulties: descriptions and explanations. AB - PURPOSE: There is a substantial minority of children for whom lexical retrieval problems impede the normal pattern of language development and use. These problems include accurately producing the correct word even when the word's meaning is understood. Such problems are often referred to as word-finding difficulties (WFDs). This article examines the nature of naming and lexical retrieval difficulties in these and other groups of children. METHOD: A review of the relevant literature on lexical access difficulties in children with word finding difficulties was conducted. Studies were examined in the terms of population parameters and comparison groups included in the study. RESULTS AND CONCLUSIONS: Most discussions of the cognitive processes causing lexical retrieval difficulties refer to semantics, phonology, and processing speed. The authors propose that understanding of these topics will be further advanced by the use of appropriate methodology to test developmental models. In this way it will be possible to identify the processes that contribute to successful lexical retrieval and the processes that result in retrieval difficulties. PMID- 16671847 TI - Sampling context affects MLU in the language of adolescents with Down syndrome. AB - PURPOSE: The authors describe the procedures used to explain an unexpected finding that adolescents with Down syndrome (DS) had a lower mean length of utterance (MLU) than typically developing (TD) children in interviews without picture support, but not in narratives supported by wordless picture books. They hypothesize that the picture support of the narrative context increased the MLU for the group with DS alone. METHOD: Adolescents with DS (n = 14) and TD children (n = 14) matched for receptive syntax narrated picture storybooks and participated in interviews. Transcription reliability, intelligibility/fluency, grammatical errors, discourse and sampling contexts, and discourse characteristics were examined for their effects on MLU. RESULTS: The DS group showed a greater responsiveness to adult questions than the TD group; an alternate MLU without yes/no responses showed the same interaction of group and context as the original finding. An additional comparison of MLUs, obtained from narratives present in the interview and narratives elicited using picture books, showed that picture support in narrative increased MLUs only for the group with DS. CONCLUSION: Picture support, rather than narrative context alone, increased MLUs for the group with DS. Clinical use of narratives and picture support in assessment and intervention with individuals with DS is discussed. PMID- 16671848 TI - Speech perception for adults who use hearing aids in conjunction with cochlear implants in opposite ears. AB - This study aimed to (a) investigate the effect of using a hearing aid in conjunction with a cochlear implant in opposite ears on speech perception in quiet and in noise, (b) identify the speech information obtained from a hearing aid that is additive to the information obtained from a cochlear implant, and (c) explore the relationship between aided thresholds in the nonimplanted ear and speech perception benefit from wearing a hearing aid in conjunction with a cochlear implant in opposite ears. Fourteen adults who used the Nucleus 24 cochlear implant system in 1 ear participated in the study. All participants had either used a hearing aid in the nonimplanted ear for at least 75% of waking hours after cochlear implantation, and/or, hearing loss less than 90 dB HL in the low frequencies in the nonimplanted ear. Speech perception was evaluated in 3 conditions: cochlear implant alone (CI), hearing aid alone (HA), and cochlear implant in conjunction with hearing aid in opposite ears (CIHA). Three speech perception tests were used: consonant-vowel nucleus-consonant (CNC) words in quiet, City University of New York style (CUNY) sentences in coincident signal and noise, and spondees in coincidental and spatially separated signal and noise. Information transmission analyses were performed on the CNC responses. Of the 14 participants tested, 6 showed significant bimodal benefit on open-set speech perception measures and 5 showed benefit on close-set spondees. However, 2 participants showed poorer speech perception with CIHA than CI in at least 1 of the speech perception tests. Results of information transmission analyses showed that bimodal benefit (performance with CIHA minus that with CI) in quiet arises from improved perception of the low frequency components in speech. Results showed that participants with poorer aided thresholds in the mid-to-high frequencies demonstrated greater bimodal benefit. It is possible that the mid-to high frequency information provided by the hearing aids may be conflicting with the cochlear implants. PMID- 16671849 TI - Development of auditory saltation and its relationship to reading and phonological processing. AB - PURPOSE: The temporal offset of auditory saltation is thought to be reflective of the limits of temporal resolution, and has recently been used to compare dyslexic and control adults and children, with mixed results (R. Hari & P. Kiesila, 1996; M. Kronbichler, F. Hutzler, & H. Wimmer, 2002). This study sought to document and understand normative changes in saltation thresholds as a function of age, and examine the relationship of saltation thresholds to reading and phonological processing across development. METHOD: This study used a cross-sectional developmental design. Groups of 7-8-, 9-10-, and 11-13-year-old children, and adults, undertook a 2-alternative forced-choice saltation task, along with standard tests of reading and phonological processing. RESULTS: Significantly higher and more variable saltation thresholds were evident in the 7-8-year-old group. Group distributions were skewed: Only a few young children obtained poor thresholds while most showed adult-like performance. Saltation thresholds were not related to reading or phonological processing skills at any stage of development. CONCLUSIONS: The temporal offset of saltation is unlikely to reflect the true limits of temporal resolution in young children, but rather the conflation of perceptual and nonperceptual factors (such as poor short-term memory, inattention, and confusion) to task performance. Effort should be made to minimize such nonperceptual factors, particularly when using saltation as a measure of temporal resolution in participants with dyslexia. PMID- 16671850 TI - Speech perception in individuals with auditory neuropathy. AB - PURPOSE: Speech perception in participants with auditory neuropathy (AN) was systematically studied to answer the following 2 questions: Does noise present a particular problem for people with AN? Can clear speech and cochlear implants alleviate this problem? METHOD: The researchers evaluated the advantage in intelligibility of clear speech over conversational speech in 13 participants with AN. Of these participants, 7 had received a cochlear implant. Eight sentence recognition experiments were conducted to examine the clear speech advantage in 2 listening conditions (quiet and noise) using 4 stimulation modes (monaural acoustic, diotic acoustic, monaural electric, and binaurally combined acoustic and electric stimulation). RESULTS: Participants with AN performed more poorly in speech recognition in noise than did the normal-hearing, cochlear-impaired, and cochlear implant controls. A significant clear speech advantage was observed, ranging from 9 to 23 percentage points in intelligibility for all listening conditions and stimulation modes. Electric stimulation via a cochlear implant produced significantly higher intelligibility than acoustic stimulation in both quiet and in noise. Binaural hearing with either diotic acoustic stimulation or combined acoustic and electric stimulation produced significantly higher intelligibility than monaural stimulation in quiet but not in noise. CONCLUSIONS: Participants with AN most likely derive the clear speech advantage from enhanced temporal properties in clear speech and improved neural synchrony with electric stimulation. Although the present result supports cochlear implantation as one treatment choice for people with AN, it suggests that the use of innovative hearing aids may be another viable option to improve speech perception in noise. PMID- 16671851 TI - Using preference-based measures to assess quality of life in stuttering. AB - PURPOSE: The purpose of this study was to determine whether standard pharmaco economic preference methods can be used to assess perceived quality of life in stuttering. METHOD: Seventy-five nonstuttering adults completed a standardized face-to-face interview that included a rating scale, standard gamble, and time trade-off preference measures for 4 health states (your health and mild, moderate, and severe stuttering) in the context of 2 anchor states (perfect health and death). RESULTS: Results showed mean utility values between .443 for severe stuttering estimated using the rating scale technique and .982 for respondents' own current health estimated using a standard gamble technique. A two-way repeated measures analysis of variance and post hoc tests showed significant effects for method, health state, and the interaction. CONCLUSIONS: These results confirm that utility estimates can differentiate between stuttering severity levels and that utility scores for stuttering conform to the known properties of data obtained using these standard measurement techniques. These techniques, therefore, can and should be further investigated as potential contributors to complete measurement protocols for the study and treatment of stuttering. PMID- 16671852 TI - Parametric quantitative acoustic analysis of conversation produced by speakers with dysarthria and healthy speakers. AB - PURPOSE: This study's main purpose was to (a) identify acoustic signatures of hypokinetic dysarthria (HKD) that are robust to phonetic variation in conversational speech and (b) determine specific characteristics of the variability associated with HKD. METHOD: Twenty healthy control (HC) participants and 20 participants with HKD associated with idiopathic Parkinson's disease (PD) repeated 3 isolated sentences (controlled phonetic content) and 2 min of conversational speech (phonetic content treated as a random variable). A MATLAB based program automatically calculated measures of contrastivity: speech-pause ratio, intensity variation, median and maximum formant slope, formant range, change in the upper and lower spectral envelope, and range of the spectral envelope. t tests were used to identify which measures were sensitive to HKD and which measures differed by task. Discriminant analysis was used to identify the combination of measures that best predicted HKD, and this analysis was then used as a general measure of contrastivity (Contrastivity Index). Differential effects of HKD on maximum and typical contrastivity levels were tested with interaction of maximum, minimum, and median observations of individual speakers and with pairwise comparisons of skewness and kurtosis of the contrastivity index distributions. RESULTS: Group differences were detected with pairwise comparisons with t tests in 8 of the 9 measures. Percentage pause time and spectral range were identified as the most specific (95%) and accurate (95%) differentiators of HKD and HC conversational speech. Sentence repetition elicited significantly higher levels of contrastivity than conversational speech in both HC and HKD speakers. Maximum and minimum contrastivities were significantly lower in HKD speech, but there was no evidence that HKD affects maximum contrastivity levels more than median contrastivity levels. The HKD speakers' contrastivity distributions were significantly more skewed to lower levels of production. CONCLUSION: HKD can be consistently distinguished from HC speech in both sentence repetition and conversational speech on the basis of intensity variation and spectral range. Although speakers with HKD were effectively able to produce higher contrastivity levels in sentence repetition tasks, they habitually performed closer to the lower end of their production ranges. PMID- 16671853 TI - Effect of facemask use on respiratory patterns of women in speech and singing. AB - PURPOSE: Research into respiratory behavior during singing and speech makes extensive use of standard respiratory and vented pneumotachograph facemasks. This study investigated whether the use of such facemasks would affect respiratory behavior in terms of lung volume (excursion, at initiation and at termination) or duration (of inspiration and of expiration) during speech or singing. METHOD: The respiratory patterns of 6 females were recorded using uniaxial surface magnetometry during 4 tasks: quiet breathing, a /pa/ syllabic train, reading ("The Rainbow Passage"), and singing a Christmas carol ("Silent night"). Each task was performed in 4 facemask conditions: wearing no facemask, wearing a facemask rim only, wearing a standard respiratory facemask, and wearing a vented pneumotachograph facemask. RESULTS: No significant effect was found for any of the facemask conditions on lung volume or duration measures during any tasks. CONCLUSION: The results confirm earlier studies that the vented pneumotachograph facemask does not affect breathing behavior in speech research studies and extends the finding to the study of breathing behavior in singing and to the use of a standard respiratory facemask. PMID- 16671854 TI - Analysis of high-frequency electroencephalographic-electromyographic coherence elicited by speech and oral nonspeech tasks in Parkinson's disease. AB - PURPOSE: Corticomuscular electroencephalographic-electromyographic (EEG-EMG) coherence elicited by speech and nonspeech oromotor tasks in healthy participants and those with Parkinson's disease (PD) was examined. Hypotheses were the following: (a) corticomuscular coherence is demonstrable between orbicularis oris (OO) muscles' EMG and scalp EEG recording; (b) the presence, location, and magnitude of coherence is task specific; (c) differences in corticomuscular coherence patterns exist between healthy and PD participants; and (d) differences will manifest as either increased or decreased coherence values in certain frequency bands, with EEG localization at primary sensorimotor cortex and/or supplementary motor area (SMA). METHOD: Simultaneous EEG, EMG (OO), and speech samples were recorded on 20 healthy and 20 PD participants during speech and nonspeech tasks. Fast Fourier transform and coherence analysis was performed with Neuroscan software on 1,000 randomly generated epochs per task per group. Corticomuscular coherence was analyzed between each EEG electrode and right and left superior and inferior OO muscles up to 200 Hz. Significant coherence peaks exceeded 95% confidence limits (.003). RESULTS: Corticomuscular coherence existed for both groups and for all tasks, but to varying degrees in primary sensorimotor cortex and SMA. CONCLUSIONS: Results support task specificity for both groups and, in PD, a diminished modulation flexibility linked to the sensorimotor area and reduced corticomuscular coherence at the SMA. PMID- 16671855 TI - Theoretical analysis of maximum flow declination rate versus maximum area declination rate in phonation. AB - PURPOSE: Maximum flow declination rate (MFDR) in the glottis is known to correlate strongly with vocal intensity in voicing. This declination, or negative slope on the glottal airflow waveform, is in part attributable to the maximum area declination rate (MADR) and in part to the overall inertia of the air column of the vocal tract (lungs to lips). The purpose of this theoretical study was to show the possible contributions of air inertance and MADR to MFDR. METHOD: A simplified computational model of the kinematics of vocal fold movement was utilized to compute a glottal area function. The glottal flow was computed interactively with lumped vocal tract parameters in the form of resistance and inertive reactance. RESULTS: It was shown that MADR depends almost entirely on the ratio of vibrational amplitudes of the lower to upper margins of the vocal fold tissue. Adduction, vertical phase difference, and prephonatory convergence of the glottis have a lesser effect on MADR. A relatively simple rule was developed that relates MFDR to a vibrational amplitude ratio and vocal tract inertance. CONCLUSION: It was concluded that speakers and singers have multiple options for control of intensity, some of which involve more source-filter interaction than others. PMID- 16671856 TI - Voice training and therapy with a semi-occluded vocal tract: rationale and scientific underpinnings. AB - PURPOSE: Voice therapy with a semi-occluded vocal tract has a long history. The use of lip trills, tongue trills, bilabial fricatives, humming, and phonation into tubes or straws has been hailed by clinicians, singing teachers, and voice coaches as efficacious for training and rehabilitation. Little has been done, however, to provide the scientific underpinnings. The purpose of the study was to investigate the underlying physical principles behind the training and therapy approaches that use semi-occluded vocal tract shapes. METHOD: Computer simulation, with a self-oscillating vocal fold model and a 44 section vocal tract, was used to elucidate source-filter interactions for lip and epilarynx tube semi-occlusions. RESULTS: A semi-occlusion in the front of the vocal tract (at the lips) heightens source-tract interaction by raising the mean supraglottal and intraglottal pressures. Impedance matching by vocal fold adduction and epilarynx tube narrowing can then make the voice more efficient and more economic (in terms of tissue collision). CONCLUSION: The efficacious effects of a lip semi occlusion can also be realized for nonoccluded vocal tracts by a combination of vocal fold adduction and epilarynx tube adjustments. It is reasoned that therapy approaches are designed to match the glottal impedance to the input impedance of the vocal tract. PMID- 16671858 TI - Orientation coding: a specific deficit in Williams syndrome? AB - Williams syndrome (WS) is a rare genetic disorder with a unique cognitive profile in which verbal abilities are markedly stronger than visuospatial abilities. This study investigated the claim that orientation coding is a specific deficit within the visuospatial domain in WS. Experiment 1 employed a simplified version of the Benton Judgement of Line Orientation task and a control, length-matching task. Results demonstrated comparable levels of orientation matching performance in the group with WS and a group of typically developing (TD) controls matched by nonverbal ability, although it is possible that floor effects masked group differences. A group difference was observed in the length-matching task due to stronger performance from the control group. Experiment 2 employed an orientation discrimination task and a length-discrimination task. Contrary to previous reports, the results showed that individuals with WS were able to code by orientation to a comparable level as that of their matched controls. This demonstrates that, although some impairment is apparent, orientation coding does not represent a specific deficit in WS. Comparison between Experiments 1 and 2 suggests that orientation coding is vulnerable to task complexity. However, once again, this vulnerability does not appear to be specific to the population with WS, as it was also apparent in the TD controls. PMID- 16671859 TI - Developmental changes in unimanual and bimanual aiming movements. AB - The aim of this study was twofold: (a) analyze the development of reaction time (RT) and movement time (MT) for bimanual and unimanual movements and (b) investigate the interaction of age and sex on the changes in RT and MT. Participants (5-, 8-, and 11-year-olds) were asked to aim at target buttons under three conditions of movement: unimanual, bimanual symmetrical, and bimanual nonsymmetrical. As expected, RTs for bimanual symmetrical movements were shorter than RTs for unimanual and bimanual nonsymmetrical movements in the 5-year-olds. By the age of 8, bimanual nonsymmetrical movements still yielded longer RTs than unimanual and bimanual symmetrical movements, which no longer differed from each other. Regarding MT, in the 2 younger groups there was an advantage of unimanual over bimanual symmetrical movements. The latter were executed faster than nonsymmetrical movements at all ages. These results suggest that the evolution of RT and MT with age reflects development of interhemispheric transfer of information. It appears that the functional improvement of such transfer, which depends on the corpus callosum, progressively enables contralateral motor inhibition and the coordination of complex bilateral movements. The exchange of movement feedback information could mature more slowly than that of feed-forward information, explaining the extended time course of MT evolution. PMID- 16671860 TI - Error-related electrocortical responses are enhanced in children with obsessive compulsive behaviors. AB - The error-related negativity (ERN or Ne) and positivity (Pe) are event-related potential components elicited during simple discrimination tasks after an error response. The ERN and Pe have a fronto-central scalp distribution and may be an indirect measure of anterior cingulate (AC) activity as it relates to performance monitoring. Brain imaging studies suggest that obsessive-compulsive disorder (OCD) is associated with exaggerated activity of the AC while electrophysiological studies have found an association between OCD and pronounced ERNs in adults. The present study explored the relation between obsessive compulsive behaviors, the ERN, and the Pe in a sample of nonclinical 10-year-old children. It was found that more parent-reported obsessive-compulsive behaviors were associated with larger ERN and Pe components in the children. Results suggest unique contributions of the ERN and Pe in predicting obsessive-compulsive behaviors. PMID- 16671861 TI - Revisiting inhibitory control across the life span: insights from the ex-Gaussian distribution. AB - Changes in inhibitory control occur across the life span and have been associated with alterations in prefrontal function. In this study, ex-Gaussian analysis was used to reexamine data from an inhibitory control task. Participants (ages 6 to 82 years) composed three groups: children, young adults, and older adults. In fitting the ex-Gaussian distribution to reaction time data, estimates of three parameters were obtained: mu (mu), reflecting average performance; sigma (sigma), reflecting variability in performance; and tau (tau), reflecting extremes in performance. Older adults differed from young adults in terms of mu, sigma, and tau. For children, mu and tau values were comparable to those of young adults; sigma, however, was different. Thus, inhibitory changes in older adults were due to slower, more variable, and more extreme responding. Inhibitory changes in children were due only to more variable responding. These findings suggest that different mechanisms underlie age-related changes in inhibitory control during different epochs of the life span. This study demonstrates that the ex-Gaussian approach provides a finer level of analysis than data analytic approaches typically used in neuropsychological research. PMID- 16671862 TI - Executive functions and performance on high-stakes testing in children from urban schools. AB - High-stakes achievement testing is a centerpiece of education reform. Children from socially disadvantaged backgrounds typically perform more poorly than their more advantaged peers. The authors evaluated 91 fifth-grade children from low income urban schools using clinical neuropsychological tests and behavioral questionnaires and obtained fourth-grade scores on state mandated standards-based testing. Goals were to determine whether executive functions are selectively diminished in children from poor urban environments and to evaluate to what extent integrity of executive functions is associated with test scores. Neuropsychological variables (particularly executive functions) accounted for 40% of the variance in English scores and 30% in mathematics. Efforts to improve children's academic achievement should consider developmental factors as well as curricular content. PMID- 16671863 TI - Cognitive reserve, age, and neuropsychological performance in healthy participants. AB - The first aim of this study was to explore the relation between cognitive reserve, age, and neuropsychological functioning in a healthy sample; and second, to determine the risk of showing cognitive deficits as a function of cognitive reserve. One hundred forty-six healthy participants between the ages of 20 and 79 were submitted to neuropsychological assessment, focusing on attention, memory, visuo-construction, conceptualization and reasoning. Premorbid IQ as measured with the Wechsler Adult Intelligence Scale Vocabulary subtest was used as a proxy of cognitive reserve. Multivariate regression analysis with age and premorbid IQ as explanatory factors revealed a significant effect in all neuropsychological tests. Logistic regression revealed that participants with low cognitive reserve were more likely to obtain deficient scores (< or =1.5 SD below the mean) in the cognitive domains of attention (odds ratio [OR], 3.13; 95% confidence interval [CI], 1.059.29), memory (OR, 6.17; 95% CI, 1.69-22.61) and global functioning (OR, 6.44; 95% CI, 2.56-16.22) than participants with high cognitive reserve. Results suggest that cognitive reserve acts as a protective factor against the expression of cognitive decline related to age in healthy individuals. PMID- 16671864 TI - The development of naming and word fluency: evidence from Hebrew-speaking children between ages 8 and 17. AB - Naming and word fluency tests are commonly used in neuropsychological evaluations of both children and adults. The current work examines at which age performance on these tests reaches adult level. One hundred fifty children, 30 in each of 5 age groups (8-9, 10-11, 12-13, 14-15, 16-17), and 30 adults ages 18 to 29, participated in the study. Participants completed a Hebrew naming test, a three letter phonemic fluency task, and a three-category semantic fluency task (animals, fruits and vegetables, and vehicles). Results show that all measures increase steadily from age 8 to age 17. No difference between the 16- to 17-year old adolescents and the adults was found on the naming test and on the phonemic fluency task, but such a difference was documented for semantic fluency. The relative contribution of the maturation of vocabulary and the development of efficient retrieval processes to performance on naming and fluency tasks is discussed. PMID- 16671865 TI - Neuropsychological function in adults with von Recklinghausen's neurofibromatosis. AB - The cognitive function of adults with von Recklinghausen's neurofibromatosis (NF 1) was examined. This study sought to replicate in an adult sample the findings of decreased visuospatial and attention abilities reported for many children with NF-1. Specifically, it was anticipated that adults with NF-1 would be classified separately from unaffected controls according to visual and attention-executive function skills. Second, this study examined whether language skills discriminated between adults with NF-1 and unaffected controls. The sample included 20 participants with NF-1 and 25 control participants. All participants were administered a battery of neuropsychological tests (Developmental Test of Visual-Motor Integration (VMI), Judgment of Line Orientation (JLO), Visual Form Discrimination, Booklet Category Test, Figure Cancellation, Peabody Picture Vocabulary Test-Revised (PPVT-R), Sentence Repetition, Controlled Oral Word Association). The results of a discriminant function analysis partially supported the hypothesis: Two of the tests of visual-spatial skill (VMI, JLO) and one of the language tests (PPVT-R) were found to be the best predictors for group membership. The discriminant function accounted for 45% of the variance between the groups and correctly classified 15 of the NF-1 participants and 21 of the control participants. Post hoc exploratory analyses revealed that the VMI was the most important test in discriminating between the groups. It is suggested that patients with NF-1 will tend to have sparing of basic cognitive functions but will have greater impairment on tests that use multiple cognitive skills. PMID- 16671866 TI - Hereditary gingival fibromatosis: a systematic review. AB - Generalized gingival enlargement can be caused by a variety of etiological factors. It can be inherited (hereditary gingival fibromatosis [HGF]); associated with other diseases characterizing a syndrome; or induced as a side effect of systemic drugs, such as phenytoin, cyclosporin, or nifedipine. HGF, previously known as elephantiasis gingivae, hereditary gingival hyperplasia, and hypertrophic gingiva, is a genetic disorder characterized by a progressive enlargement of the gingiva. This review will focus on diagnosis, treatment, and control of HGF. The pattern of inheritance, the histopathologic characteristics, and the known biologic and genetic features associated with HGF are also emphasized. PMID- 16671867 TI - The effect of varying the particle size of beta tricalcium phosphate carrier of recombinant human bone morphogenetic protein-4 on bone formation in rat calvarial defects. AB - BACKGROUND: Beta tricalcium phosphate (beta-TCP) has been developed as one of the carriers of recombinant human bone morphogenetic protein (rhBMP). However, it is not known whether the particle size of beta-TCP is related to its resorption rate and the degree of bone formation. The purpose of this study was to evaluate the effect of using beta-TCP with different particle sizes on the ability of rhBMP-4 to enhance bone formation in the rat calvarial defect model. METHODS: Calvarial, 8-mm-diameter, critical-size defects were created in 100 male Sprague-Dawley rats. Five groups of 20 animals each received either rhBMP-4 (2.5 microg) using beta-TCP with a particle size of 50 to 150 microm, rhBMP-4 (2.5 microg) using beta-TCP with a particle size of 150 to 500 microm, a beta-TCP control with a particle size of 50 to 150 microm, a beta-TCP control with a particle size of 150 to 500 microm, or a sham-surgery control, respectively, and were evaluated by measuring their histologic and histometric parameters following a 2- and 8-week healing interval. RESULTS: There were no significant differences in the defect closure, new bone area, or augmented area between either the two rhBMP-4/beta-TCP groups or between the two beta-TCP control groups at 2 and 8 weeks. CONCLUSIONS: rhBMP-4 combined with either small- or large-particle beta-TCP had a significant effect on the induction of bone formation compared to either a small- or large particle beta-TCP control or a sham-surgery control. Within the parameters of this study, varying the particle size of beta-TCP did not seem to have a significant effect on bone formation. PMID- 16671868 TI - Longitudinal evaluation of loss of attachment in HIV-infected women compared to HIV-uninfected women. AB - BACKGROUND: The Women's Interagency HIV Study (WIHS) is the largest, most detailed, controlled longitudinal collection of data to evaluate the influence of human immunodeficiency virus (HIV) disease and its therapies on the periodontium. METHODS: This report evaluates periodontal probing depth (PD), attachment loss (AL), and tooth loss from 584 HIV-seropositive and 151 HIV-seronegative women, recorded at 6-month intervals from 1995 to 2002. Using the random split-mouth method, PD and AL were recorded from four sites per tooth: mesial-buccal, buccal, distal-buccal, and lingual. Influence of viral load, CD4 count, race, smoking, drug use, low income, and level of education were evaluated. RESULTS: At baseline, AL was 1.6 versus 1.1 mm (P = 0.003) and PD was marginally deeper (2.1 versus 2.0 mm; P = 0.02) in HIV-seropositive versus HIV-seronegative women. Adjusted longitudinal analysis showed that HIV infection did not increase the mean PD (rate ratio [RR], 1.00; 95% confidence interval [CI], 0.96 to 1.04), worst PD (RR, 1.03; 95% CI, 0.98 to 1.09), mean AL (RR, 0.97; 95% CI, 0.96 to 1.02), worst AL (RR, 1.01; 95% CI, 0.94 to 1.07), or tooth loss (RR, 1.02; 95% CI, 1.0 to 1.05). CONCLUSIONS: CD4 count and viral load had no consistent effects on PD or AL. Among HIV-infected women, a 10-fold increase in viral load was associated with a marginal increase in tooth loss. The progression of periodontal disease measured by PD and AL did not significantly differ between HIV-infected and HIV-uninfected women. The HIV-seropositive women lost more teeth. Race, smoking, drug use, income, and education level did not influence the results for either group. PMID- 16671869 TI - Autogenous bone graft with or without a calcium sulfate barrier in the treatment of Class II furcation defects: a histologic and histometric study in dogs. AB - BACKGROUND: The purpose of this study was to histologically evaluate the healing of surgically created Class II furcation defects treated using an autogenous bone (AB) graft with or without a calcium sulfate (CS) barrier. METHODS: The second, third, and fourth mandibular premolars (P2, P3, and P4) of six mongrel dogs were used in this study. Class II furcation defects (5 mm in height x 2 mm in depth) were surgically created and immediately treated. Teeth were randomly divided into three groups: group C (control), in which the defect was filled with blood clot; group AB, in which the defect was filled with AB graft; and group AB/CS, in which the defect was filled with AB graft and covered by a CS barrier. Flaps were repositioned to cover all defects. The animals were euthanized 90 days post surgery. Mesio-distal serial sections were obtained and stained with either hematoxylin and eosin or Masson's trichrome. Histometric, using image-analysis software, and histologic analyses were performed. Linear and area measurements of periodontal healing were evaluated and calculated as a percentage of the original defect. Percentage data were transformed into arccosine for statistical analysis (analysis of variance; P <0.05). RESULTS: Periodontal regeneration in the three groups was similar. Regeneration of bone and connective tissue in the furcation defects was incomplete in most of the specimens. Statistically significant differences were not found in any of the evaluated parameters among the groups. CONCLUSION: Periodontal healing was similar using surgical debridement alone, AB graft, or AB graft with a CS barrier in the treatment of Class II furcation defects. PMID- 16671870 TI - Application of periodontal tissue engineering using enamel matrix derivative and a human fibroblast-derived dermal substitute to stimulate periodontal wound healing in Class III furcation defects. AB - BACKGROUND: Enamel matrix derivative (EMD) has been shown to promote several aspects of periodontal regeneration in vitro and in vivo. Recently, a bioengineered tissue (DG) was developed to promote wound healing of chronic skin ulcers. This pilot study sought to assess the effects of EMD and DG, alone or in combination, on periodontal wound healing in surgically created Class III furcation defects. METHODS: Six female baboons received bilateral ostectomy of approximately 10 mm around the first and second mandibular molars to achieve Class III, subclass C furcation defects. Wire ligatures and cotton pellets were left in place for 2 months to maintain the depth of the defects and promote plaque accumulation. Each furcally involved molar was then assigned to one of four treatments: open flap debridement (OFD), OFD plus EMD, OFD plus DG, or OFD plus DG and EMD. This resulted in six total sites per treatment group. Seven months after defect creation and 5 months after treatment, and after no oral hygiene, tissue blocks of the mandible were taken for blinded histometric analysis to assess parameters of periodontal regeneration adjacent to furcal root surfaces and from the mid-furcal aspect (i.e., new bone, new connective tissue attachment, new epithelial attachment, and new cementum formation). RESULTS: Histometric analysis demonstrated differential regenerative responses with respect to treatment within each animal. However, statistically significant differences between treatments from all six animals were not observed (P >0.20, mixed-model analysis of variance). EMD-treated sites presented mildly positive regenerative results and no negative responses. Both DG only and combination therapy demonstrated similar or less than positive responses relative to OFD controls. CONCLUSION: The descriptive analysis may suggest a positive effect of enamel matrix proteins and a negative effect of DG used alone or in combination with enamel matrix proteins on the regeneration of Class III furcation defects in baboons. PMID- 16671871 TI - Promotion of functioning of human periodontal ligament cells and human endothelial cells by nerve growth factor. AB - BACKGROUND: We have previously shown that cultured human periodontal ligament (HPL) cells produce nerve growth factor (NGF) and express mRNA of tyrosine kinase receptor (trkA), a high-affinity receptor of NGF. These findings suggest that NGF modulates the differentiation and proliferation of the periodontal ligament cells by paracrine and autocrine functions in vivo. Endothelial cells also express NGF and trkA. Therefore, NGF may regulate functions of periodontal ligament cells and endothelial cells during periodontal tissue regeneration. METHODS: Effects of NGF on expressions of bone/cementum-related proteins (osteocalcin [OC], bone sialoprotein [BSP], bone morphogenetic protein [BMP-7], core binding factor alpha [Cbfa-1], and type I collagen), calcification in HPL cells, and proliferation and mRNA expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen, in human microvascular endothelial cells (HMVECs) were examined. RESULTS: NGF elevated mRNA levels of OC, BSP, BMP-7, Cbfa-1, and type I collagen and enhanced mineral deposition in cultures of HPL cells. Furthermore, NGF stimulated mRNA expressions of VEGF-A and VEGF-B and cell proliferation in HMVEC. CONCLUSION: These findings suggest that the functional regulation of periodontal ligament cells and endothelial cells by NGF might result in the acceleration of periodontal tissue regeneration in vivo. PMID- 16671872 TI - Supragingival plaque may modify the effects of subgingival bacteria on attachment loss. AB - BACKGROUND: Bacterial interactions in response to local and systemic factors may explain important features of the pathogenesis of periodontal disease. This study evaluates the role of supragingival plaque level on the relationship between subgingival microorganisms and the risk of attachment loss. METHODS: The study population consisted of 386 white women aged 45 years and older who had at least seven teeth. Subgingival plaque samples were taken from the mesio-buccal surface of six maxillary and six mandibular teeth using paper point technique. The samples were pooled, and immunofluorescence microscopy was performed to identify the following target microorganisms: Actinobacillus actinomycetemcomitans (Aa), Tannerella forsythensis (Tf), Campylobacter rectus (Cr), Prevotella intermedia (Pi), Capnocytophaga species (Cs), Porphyromonas gingivalis (Pg), Eubacterium saburreum (Es), and Fusobacterium nucleatum (Fn). The presence or absence of supragingival plaque and clinical attachment loss (CAL) were assessed at the same 12 sites where subgingival plaque samples were taken. The association of each microorganism with CAL was tested using multiple logistic regressions controlling for age, smoking status, and diabetes. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated. RESULTS: At low supragingival plaque levels, only the presence of Pg was significantly associated with CAL (OR: 6.41, 95% CI: 1.30 to 31.70); all remaining microorganisms were also associated with increased but non-significant risk of CAL. At high supragingival plaque levels, the presence of Tf (OR: 2.40, 95% CI: 1.42 to 4.04) and Pg (OR: 3.71, 95% CI: 1.63 to 8.42) was significantly associated with increased risk of attachment loss. By contrast, the presence of Cs (OR: 0.55, 95% CI: 0.30 to 1.01) and Es (OR: 0.35, 95% CI: 0.15 to 0.82) was associated with decreased risk of attachment loss. Fn, Pi, and Cr were not significantly associated with CAL at either low or high supragingival plaque levels. CONCLUSIONS: This study suggests that the association of certain subgingival microorganisms with CAL changes in relation to supragingival plaque levels in older adult women. It also suggests that the overall effect of dental plaque is a function of the balance between pathogenic and other microorganisms that colonize this environment. Therefore, all microorganisms in the biofilm, including those with no apparent association with overt disease and those with negative associations, may play roles in the pathogenesis of periodontal diseases. PMID- 16671873 TI - Dietary supplementation of omega-3 fatty acid and circulating levels of interleukin-1beta, osteocalcin, and C-reactive protein in rats. AB - BACKGROUND: In this study, we evaluated the effects of two different regimes of dietary supplementation of omega-3 fatty acid on serum levels of interleukin-1 beta (IL-1beta), osteocalcin (OC), and C-reactive protein (CRP) in experimental periodontitis. METHODS: Experimental periodontitis was induced by repeated injections of Escherichia coli lipopolysaccharide (LPS). Thirty-nine adult male Sprague-Dawley rats were divided into four study groups as follows: an LPS positive control group; a saline (negative) control group; and two different groups with omega-3 fatty acid dietary supplementation, one in which we gave the supplement subsequent to disease induction (TO3) and the other in which the agent was started prior to and continued subsequent to LPS injections (P + TO3). In the TO3 group, omega-3 fatty acid administration was performed for 14 days following induction of experimental periodontitis. In the P + TO3 group, omega-3 fatty acid was given for 14 days prior to the start of LPS injections and was continued for another 14 days subsequent to the induction of experimental periodontitis. On day 15 of the first LPS injection, serum samples were obtained and rats were sacrificed. Serum samples were analyzed for IL-1beta, OC, and CRP concentrations by enzyme-linked immunosorbent assay. Defleshed jaws were analyzed morphometrically for alveolar bone loss. Data were evaluated statistically by non parametric tests. RESULTS: LPS injection resulted in statistically significantly more bone loss compared to the saline control group (P <0.05). None of the omega 3 fatty acid administration groups showed evidence that this fatty acid was effective in preventing LPS-induced alveolar bone loss. TO3 and P + TO3 groups revealed significantly higher IL-1beta and OC levels than the LPS group (P <0.05). The study groups exhibited no significant differences in the serum CRP levels. CONCLUSIONS: Omega-3 fatty acid administration does not seem to influence circulating levels of CRP. The significantly increased serum OC level observed in both omega-3 fatty acid regimes is curious and could have an effect on bone turnover, as could the further significant increase in serum IL-1beta, which could counteract any osteoblastic induction by OC through promotion of osteoclast activity. The lack of a therapeutic benefit of omega-3 fatty acid in this study, despite the effects on OC and IL-1beta, is difficult to explain, and further studies are required to more fully assess the potential role of this fatty acid in periodontal treatment. PMID- 16671874 TI - Periodontitis and plasma C-reactive protein during pregnancy. AB - BACKGROUND: Periodontitis has been associated with increased risk of adverse pregnancy outcomes and elevated C-reactive protein (CRP) concentrations in non pregnant adults. We examined the relationship between periodontitis and CRP among women who provided dental radiographs and had blood collected during early pregnancy, excluding smokers and diabetic patients. METHODS: From Project Viva, an ongoing cohort study, we measured plasma CRP in 35 subjects with periodontitis (i.e., at least one site with > or =3 mm of alveolar bone loss) and a random sample of 66 periodontally healthy subjects matched on age and race/ethnicity. We performed linear regression analysis with log-transformed CRP levels as the outcome. RESULTS: The mean (+/- SE) CRP level was 65% higher (95% confidence interval: -2%, 180%; P = 0.06) in women with periodontitis (2.46 +/- 0.52 mg/l) than in controls (1.49 +/- 0.22 mg/l), adjusting for factors related to CRP levels, including age, race/ethnicity, pre-pregnancy body mass index, alcohol intake, education, income, and gestational age at blood collection. CONCLUSIONS: These findings suggest that periodontitis may increase CRP levels in pregnancy. CRP could potentially mediate the association of periodontitis with adverse pregnancy outcomes. PMID- 16671875 TI - Topical application of drugs influencing cytoskeleton and cell contractility affects alveolar bone loss in rats. AB - BACKGROUND: Several studies have shown that sectioning bundles of collagen fibers in the marginal gingiva during surgical procedures in animals is a distinct stimulus for alveolar bone resorption. Normally, gingival and periodontal fibroblasts, which reside on these collagen fibers, create physiological traction forces generated by the cytoskeleton. By splitting the fibers, traction forces are released, inducing changes in the cytoskeleton and cell shape. In this study, four drugs were selected, including cytochalasin D, EDTA, sodium orthovanadate, and H-7, all influencing the cytoskeleton-integrin-extracellular matrix (ECM) pathway, for their ability to reduce alveolar bone loss by local application. METHODS: The drugs were applied locally only once at the site of mucoperiosteal flap surgery in a rat model. Cytochalasin D (1 microl/microl), EDTA (0.24 mg/microl), sodium orthovanadate (0.02 mg/microl), and H-7 (0.10 microl/microl), each separately, were carried by a protective paste and placed immediately after elevating the flap. The analysis of alveolar bone loss was performed 3 weeks after surgery by scanning the microradiographic films of the mandible cross sections. The percentages of cross sections with no, moderate, or severe bone loss in treated in comparison to non-treated rats are presented. RESULTS: EDTA, sodium orthovanadate, and H-7 were significantly effective in reducing alveolar bone loss. They were effective in reducing the amount of severe bone loss by 53%, 20%, and 58% while increasing the number of sections with no bone loss by 25%, 23%, and 35%, respectively. Cytochalasin D reduced alveolar bone loss insignificantly. CONCLUSION: EDTA, sodium orthovanadate, and H-7 are effective in reducing alveolar bone loss in rats following mucoperiosteum surgery. PMID- 16671876 TI - Cyclosporin-induced downregulation of the expression of E-cadherin during proliferation of edentulous gingival epithelium in rats. AB - BACKGROUND: To examine the role of E-cadherin in epithelial hyperplasia of cyclosporin A (CsA)-induced gingival enlargement, mRNA and protein levels of E cadherin, beta-catenin, proliferating cell nuclear antigen (PCNA), and Cyclin D1 were examined in the edentulous gingiva of rats following CsA treatment. METHODS: Three weeks after the extraction of all maxillary molars, 20 male Sprague-Dawley rats were assigned to a CsA-fed group (30 mg/kg daily) or a control group. Five rats per group were sacrificed at weeks 1 and 4. Edentulous ridge specimens were taken, and the expression levels of E-cadherin, beta-catenin, Cyclin D1, and PCNA mRNAs were estimated by reverse transcription-polymerase chain reaction (RT-PCR). Tissue specimens of the week 4 groups were examined using immunohistochemical (IHC) staining for proteins. RESULTS: The mRNA expression of E-cadherin was significantly weaker in the CsA-treated group than the control group at both times. Using IHC staining, a weaker level of membrane-bonded E-cadherin was also observed in the gingival epithelial cells in the CsA group than in controls. By contrast, significantly stronger beta-catenin and Cyclin D1 mRNA expressions and protein levels were found in CsA-treated rats than controls by RT-PCR and immunohistochemistry at week 4, whereas PCNA production was stronger at both times. CONCLUSIONS: CsA treatment reduced the production of E-cadherin but increased the production of beta-catenin, Cyclin D1, and PCNA. Thus, CsA may downregulate E-cadherin gene expression, leading to the epithelial cell proliferation of gingival overgrowth. PMID- 16671877 TI - Junctional epithelium in rats is characterized by slow cell proliferation. AB - BACKGROUND: The integrity of junctional epithelium (JE) and a firm epithelial adhesion to the tooth surface are maintained by the balance between cell proliferation and cell death. Maintaining the JE structure is essential for the protection of periodontal connective tissues against oral microbes. In this study, the proliferative activity and the expression of caspase 3, a cysteine protease associated with cell death, were studied in rat JE and other epithelial structures during molar tooth development. METHODS: Fourteen rats aged 10 to 70 days were injected with bromodeoxyuridine (BrdU). Samples of first and second molars were selected for immunohistochemical staining. BrdU incorporation was studied in oral epithelium (OE) covering the erupting tooth, reduced enamel epithelium (REE), and gingival epithelium (GE), sulcular epithelium (SE), and JE. Samples were also subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA) and caspase 3. RESULTS: The basal cells of the GE were actively proliferating, but in the JE, only a few cells were positive for BrdU or PCNA immunostaining. Some outer REE cells were proliferating during tooth eruption. Caspase 3 expression was in specific areas of REE after completion of amelogenesis. CONCLUSIONS: Results showed slow proliferative activity in the rat JE. However, specific studies on cellular turnover and cell migration are needed to understand tissue homeostasis in this area. PMID- 16671878 TI - A model of spontaneous periodontitis in the miniature goat. AB - BACKGROUND: The pathogenesis and progression of periodontal disease have been extensively studied through the use of animal models. However, no animal model has yet been established that is precisely similar to periodontitis in humans. In the present study, we examined the use of Shiba goats as a model for spontaneous periodontitis. METHODS: Thirty-four Shiba goats (seven males and 27 females, aged 10 to 98 months) were used. We examined periodontitis in Shiba goats clinically, histopathologically, and microbiologically. RESULTS: The mean probing depth (PD) of the 68 teeth examined was 2.7 +/- 0.8 mm. The incidences of PD > or =3 and 4 mm were 46.4% and 22.1%, respectively. The incidence of bleeding on probing in 68 sites and in 34 animals was 60.7% and 73.5%, respectively. The formation of vertical alveolar bone defects and downgrowth of gingival epithelial cells were found in the areas of periodontitis. The prevalence of Tannerella forsythensis, Campylobacter rectus, Fusobacterium necrophorum, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans in subgingival plaque by polymerase chain reaction was 46.4%, 28.5%, 28.5%, 17.8%, and 3.5%, respectively. These percentages were increased in subgingival plaque from PD > or =3 mm. CONCLUSIONS: The clinical, histopathological, and microbiological features of spontaneous periodontitis in Shiba goats were somewhat similar to those in human periodontitis. Moreover, there are some advantages of using the Shiba goat; the size of the oral cavity is suitable for periodontal treatment, and handling and housing are relatively easy. Therefore, these results suggest that the Shiba goat is a useful animal model for human periodontitis. PMID- 16671879 TI - Profiling the cytokines in gingival crevicular fluid using a cytokine antibody array. AB - BACKGROUND: Various compounds have been detected in gingival crevicular fluid (GCF) as indicators of periodontal disease activity. Therefore, the analysis of GCF may be especially beneficial for diagnosing current periodontal status and addressing the effects of treatment. Moreover, the identification of new markers in GCF may also contribute to elucidating novel mechanisms involved in periodontal disease. This study sought novel marker proteins specific to chronic periodontitis by profiling cytokines in GCF using a cytokine antibody array system. METHODS: Human cytokine array V, which detects 79 cytokines on one membrane, was used to determine the profile of cytokines in GCF from seven subjects with chronic periodontitis and seven subjects with healthy periodontia. The profile was exposed to x-ray film and quantified using image analysis software. Healthy and diseased sites were compared statistically. RESULTS: We detected 10 cytokines in periodontally healthy sites and 36 cytokines in periodontally diseased sites. Interleukin-8 (IL-8) and transforming growth factor beta 2 (TGF-beta2) were detected at high levels in healthy and diseased subjects. There were significant differences between healthy and diseased subjects in the levels of tissue inhibitor of metalloproteinases-2 (TIMP-2), tumor necrosis factor-beta (TNF-beta), growth-related oncogene (GRO), interferon-inducible protein-10 (IP-10), angiogenin (Ang), vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-3 (IGFBP-3), osteoprotegerin (OPG), epidermal growth factor (EGF), glial-derived neurotrophic factor (GDNF), pulmonary and activation-regulated chemokine (PARC), oncostatin M (OSM), fibroblast growth factor-4 (FGF-4), IL-16, homologous to lymphotoxins (LIGHT), and placenta growth factor (PlGF). Of these, the newly detected cytokines were GRO, Ang, IGFBP-3, GDNF, PARC, OSM, FGF-4, IL-16, LIGHT, and PlGF. CONCLUSIONS: In this study, we detected several cytokines in GCF using a cytokine antibody array system, including both inflammatory cytokines and various growth factors. Therefore, periodontal disease may participate in the wound healing process and in tissue destruction via the inflammatory process. Our results suggest that the quantification of these cytokines in GCF provides useful information for the diagnosis of periodontal disease status. PMID- 16671880 TI - The relationship of transforming growth factor-beta1 gene polymorphism, its plasma level, and gingival overgrowth in renal transplant recipients receiving different immunosuppressive regimens. AB - BACKGROUND: Cyclosporin A (CsA) induces gingival overgrowth (GO) in patients who seem to be prone to this disorder. It is still impossible to determine which patients will develop GO. Patients treated with the new immunosuppressive drug tacrolimus seem not to have GO. The aims of this study were to investigate transforming growth factor-beta1 (TGF-beta1) gene polymorphisms in renal transplant recipients treated with CsA or tacrolimus and to establish an association between these polymorphisms and TGF-beta1 plasma concentration and the incidence of GO. METHODS: The examined group consisted of 134 renal transplant recipients. Ninety-two underwent CsA treatment (50 with and 42 without GO), and 42 underwent tacrolimus treatment. Age, gender, time after transplantation, calcineurin inhibitor total dosage, number of teeth, and sulcus bleeding index were analyzed. TGF-beta1 plasma levels were estimated in 60 CsA- and 30 tacrolimus-treated patients. Two biallelic polymorphisms of the TGF-beta1 gene were studied at codon 10 (at position +869) and at codon 25 (at position +915) in patients from the examined group and in 108 healthy volunteers (the control group). RESULTS: The distribution of the high, intermediate, and low TGF beta1 producer phenotypes was comparable in all the studied groups and in the healthy controls. The high producer phenotype was more frequent in patients with GO. TGF-beta1 levels in the CsA group showed correlation with the phenotypes. The lowest incidence of GO was observed in the 10C/C genotype, whereas the highest was observed in the 10T/C genotype. CONCLUSION: High and intermediate TGF-beta1 producer phenotypes and heterozygous genotype 10T/C might be considered risk factors for GO in patients treated with CsA. PMID- 16671881 TI - Effect of flapless implant surgery on soft tissue profile: a randomized controlled clinical trial. AB - BACKGROUND: Flapless implant surgery has been suggested as one possible treatment option for enhancement of implant esthetics. METHODS: Twenty-four patients with a missing tooth in the premaxillary region were randomly assigned to one of two groups (12 each): immediate loading (IL) or delayed loading (DL) (loading after 4 months). An endosseous implant was placed in each patient via a flapless surgery. Clinical measurements including the papillary index (PPI) (0, no papilla; 1, less than half; 2, more than half but not complete fill; 3, complete fill; and 4, overfill), marginal levels of the soft tissue (ML), probing depths (PDs), modified bleeding index (mBI), modified plaque index (mPI), and the width of the keratinized mucosa (WKM) were performed at baseline (at the time of loading) and at 2, 4, and 6 months. RESULTS: The soft tissue profile remained stable up to 6 months, without significant differences between the two groups (mean PPI and ML at 6 months, 2.16 and 0.30 mm, respectively). Mean PPI in the IL group significantly increased from 1.50 at baseline to 2.09 at 2 months, and the significance remained up to 6 months (2.30 at 6 months) (P <0.05), whereas in the DL group, no significant changes were found from baseline to 6 months in mean PPI (2.06 at both time points). Mean PPI increased over time when two treatment groups were combined; however, no statistical significance was found. In ML, the difference at baseline between the two groups (-0.28 mm for DL versus 0.17 mm for IL; P <0.05) was no longer significant at 2 months (0 versus 0.08 mm for DL and IL, respectively) and thereafter (P >0.05). No significant differences were detected between groups at each time and over time in the other clinical parameters, PD, mBI, mPI, and WKM (P >0.05). CONCLUSIONS: The results of this study indicate that creeping attachment (i.e., soft tissue recovery) might occur within 2 months after IL. The study suggests that flapless implant surgery provides esthetic soft tissue results in single-tooth implants either immediately or delayed loaded. Other long-term randomized controlled clinical trials with a large sample size and comparison group (i.e., implant surgery with flap) are recommended to verify the conclusions drawn in this preliminary study. PMID- 16671882 TI - Inhibitory effects of tumor necrosis factor-alpha on migration of human periodontal ligament cells. AB - BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is associated with chronic gingival inflammation and is suspected to influence periodontal destruction. However, the exact roles of TNF-alpha in wound healing and periodontal tissue regeneration are largely unknown. In the present study, we examined the effects of TNF-alpha on migration and proliferation of human periodontal ligament (PDL) cells. METHODS: PDL cells were cultured in the presence of TNF-alpha to determine its effects on cellular migration and proliferation. The protein expression profiles of alpha5 and beta1 integrin subunits and their related molecules, paxillin and focal adhesion kinases (FAK), were investigated. Gene expression of fibronectin also was assayed. Further, the activation of Rho-family small guanosine triphosphate (GTP)-binding protein (RhoA) was evaluated using a GTP loading pull-down assay, and focal adhesion formation by PDL cells after transfection with the expression vector of paxillin-fused green fluorescent protein (GFP) also was observed with confocal microscopy. RESULTS: Cellular migration was impaired by TNF-alpha and recovered following the addition of anti TNF-alpha antibodies. In contrast, PDL cell proliferation was not affected by TNF alpha. TNF-alpha upregulated the expression of the alpha5 and beta1 integrin subunits, whereas fibronectin was not overexpressed. Phosphorylation of paxillin and FAK by PDL cells was induced, and RhoA activation also was induced. Confocal microscopic analysis revealed that TNF-alpha induced focal adhesion and stress fiber formation in all parts of the cells. CONCLUSION: Our results suggested that TNF-alpha impairs cellular migration by enhancing cellular adhesive ability following significant focal adhesion and stress fiber formation. PMID- 16671883 TI - Effects of enamel matrix derivative on bone-related mRNA expression in human periodontal ligament cells in vitro. AB - BACKGROUND: Enamel matrix derivative (EMD) has demonstrated the potential to stimulate periodontal regeneration with mineralized tissue formation. Molecular regulators of bone metabolism include osteoprotegrin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), cyclooxygenase 2 (COX2), and core binding factor alpha 1 (Cbfa1). The role of these regulatory molecules within the context of EMD stimulation of mineralized tissue formation is unknown. Therefore, the purpose of this investigation was to explore the effects of EMD on these bone related molecules in human periodontal ligament (PDL) cells. METHODS: Human PDL cell cultures were treated with EMD (5 to 100 microg/ml) for 24 hours. Total RNA was isolated using phenolchloroform, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers specific for OPG, RANKL, COX2, Cbfa1, and aldolase, with amplification in the exponential range for each molecule studied. RESULTS: The results of this study show that there is a significant (P <0.05) increase in COX2 mRNA levels with EMD treatment, and no effects were noted on mRNA levels for Cbfa1. RANKL mRNA levels were significantly decreased (P <0.01) up to 50% with EMD treatment > or =25 microg/ml. OPG levels showed minimal effects with EMD treatment. However, the RANKL/OPG ratio showed a 40% to 55% reduction with EMD >or =25 microg/ml. CONCLUSION: This study supports a role for EMD stimulation of mineralized tissue formation consistent with periodontal regeneration by modulating regulatory molecules critical to bone metabolism at the RNA level. PMID- 16671884 TI - Connective tissue graft for gingival recession treatment: assessment of the maximum graft dimensions at the palatal vault as a donor site. AB - BACKGROUND: The palatal masticatory mucosa is widely used as a connective tissue donor site in gingival recession treatment. However, concern has been raised regarding the potential risk of damaging the greater palatine artery (GPA) due to anatomical variations in the palatal vault. The anatomy of the palatal vault in terms of size and shape may affect the maximum dimensions of the graft that can be safely taken from the palatal vault. In a cohort of patients free of periodontal disease, the purpose of this study was to assess the maximum dimensions of the graft, particularly the height and length, that could be safely taken from the palatal vault. METHODS: Plaster impressions were made from 198 patients free of periodontal disease. Because the connective tissue graft is usually taken from an area extending from the mid-palatal aspect of the canine to the mid-palatal aspect of the second molar, this interval was measured and represented the maximum length dimension. The emergence of the GPA was assumed to be localized at the junction of the vertical and horizontal palatal walls of vault, and its course was marked on the plaster casts. The maximum height of the graft corresponded to the distances measured from the gingival margin to the marked course of the GPA of each tooth at its interproximal and mid-palatal aspects. RESULTS: The length of the maximum available tissue graft was 31.7 +/- 4.0 mm. The distance extending from the gingival margin to the greater palatine artery ranged from 12.07 +/- 2.9 mm at the canine level to 14.7 +/- 2.9 mm at the mid-palatal aspect of the second molar level. Therefore, in the premolar area, it was possible to harvest a connective tissue graft measuring 5 mm in height in all cases and 8 mm in height in 93% of cases. CONCLUSION: Our findings suggest that the maximum available tissue graft as measured in the palatal vault was large enough to allow a safe withdrawal from this donor site in a high percentage of our patient population free of periodontal disease. PMID- 16671885 TI - Maxillary sinus septa: prevalence, height, location, and morphology. A reformatted computed tomography scan analysis. AB - BACKGROUND: The sinus lift technique may be difficult to perform if an aberrant sinus anatomy is encountered during surgical exposure, such as when a septum is present on the sinus floor. The objective of this study was to determine the prevalence, size, location, and morphology of maxillary sinus septa in the atrophic/edentulous and non-atrophic/dentate maxillary segments. METHODS: The sample population consisted of 100 patients (41 women and 59 men, with a mean age of 50 years, ranging between 19 and 87 years) for whom treatment was being planned for implant-supported restorations. Reformatted computerized tomograms (CT) from 200 sinuses were analyzed using imaging software. RESULTS: The prevalence of one or more septa per sinus was found to be 26.5% (53/200), 31.76% (27/85), and 22.61% (26/115) in the overall study population and the atrophic/edentulous and the non-atrophic/dentate maxillary segments, respectively. In the analysis of the anatomic location of the septa within the sinus, it was revealed that 15 (25.4%) were located in the anterior region, 30 (50.8%) in the middle region, and 14 (23.7%) in the posterior region. The measured heights of the septa varied among the different areas. The mean heights of the septa were 1.63 +/- 2.44, 3.55 +/- 2.58, and 5.46 +/- 3.09 mm in the lateral, middle, and medial areas, respectively. CONCLUSIONS: It can be inferred that there is a wide anatomical variation in the prevalence, size, location, and morphology of maxillary sinus septa, irrespective of the degree of atrophy. Therefore, to prevent the likelihood of complications arising during sinus augmentation procedures, a thorough and extensive understanding of the anatomic structures inherent to the maxillary sinus is indispensable. PMID- 16671886 TI - Subepithelial connective tissue grafts in the treatment of multiple recession type defects. AB - BACKGROUND: There is little documentation regarding the treatment of multiple recession-type defects. The objective of this study was to evaluate the results obtained with a connective tissue graft placed under a coronally advanced flap for the treatment of multiple gingival recessions. METHODS: Twenty-eight patients, with at least two recession defects affecting adjacent teeth, were treated with a combination of a connective tissue graft and a coronally advanced flap. At baseline, the following measurements were recorded: 1) recession depth; 2) probing depth; 3) clinical attachment level; and 4) width of keratinized tissue. At 6 months post-surgery, all clinical measurements were repeated. RESULTS: The mean root coverage from baseline to 6 months post-surgery results was 96%. Complete root coverage was achieved in 20 (71%) of the 28 defects. The mean recession depth decreased from 3.84 +/- 1.50 mm to 0.14 +/- 0.23 mm. Statistically significant improvements were found for all clinical parameters from baseline to 6 months (P <0.01). Patients with maxillary recessions recorded statistically superior outcomes than patients with mandibular recessions. CONCLUSIONS: The results of the present study demonstrated that the connective tissue graft associated with a coronally advance flap is an effective procedure to cover multiple gingival recessions, especially in defects localized in the maxillary arch. However, further investigation focusing on the effects of this root coverage procedure in mandibular sites is necessary. PMID- 16671887 TI - Eosinophilic granuloma masquerading as aggressive periodontitis. AB - BACKGROUND: Eosinophilic granuloma (EG) is the mildest and mainly localized form of the clinicopathologic spectrum of Langerhans' cell histiocytosis. It is a destructive osseous lesion characterized by a vast number of eosinophils and histiocytes. The etiology remains unknown. In this paper, a case of EG is presented that was initially diagnosed and treated as aggressive periodontitis (AP). METHODS: Despite treatment procedures, the EG continued to expand very quickly, destroying the lingual cortical bone and the neighboring soft tissues and exhibiting periosteal reaction. Diagnosis of EG was established on the basis of histopathologic and immunohistochemical evaluation. Moreover, certain manifestations in the skeletal and respiratory system were observed. RESULTS: Surgical curettage of the lesions was effective; however, corticosteroids and low dose radiation were used as adjunctive therapy. CONCLUSION: The rapid progress of eosinophilic granuloma, the diagnostic problems, and the consequences of late diagnosis and treatment are discussed. PMID- 16671888 TI - Periodontal plastic surgery associated with treatment for the removal of gingival overgrowth. AB - BACKGROUND: Excisional biopsies of gingival overgrowths, performed with safety margins, frequently result in mucogingival defects. These defects may produce esthetic problems and increase the chances of dentin hyperesthesia and its possibility of hindering oral hygiene. METHODS: Two clinical cases are reported in which gingival overgrowths were removed by excisional biopsy, resulting in unsightly defects. The first clinical case presents an invasive approach for the treatment of a recurrent pyogenic granuloma in the anterior maxilla, and the second depicts a complete removal of a peripheral odontogenic fibroma in the posterior maxilla. In both situations, the soft-tissue defects were repaired by periodontal plastic surgery, including a laterally positioned flap and a coronally positioned flap, respectively. RESULTS: Periodontal plastic surgery successfully restored the defects that resulted from biopsies, and no recurrence has been noticed in the 5-year postoperative follow-up period. CONCLUSIONS: The combination of biopsy and periodontal plastic surgery in a one-step procedure seems to be suitable to remove gingival overgrowths in most areas of the mouth, regardless of esthetic significance. Such procedures seem to restore gingival health, encourage healing, and create both esthetics and function in the excised area. PMID- 16671889 TI - Re: Tobacco smoking and periodontal health in a Saudi Arabian population. Natto S, Baljoon M, Bergstrom J (2005;76:1919-1926). PMID- 16671891 TI - Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols. AB - Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease. PMID- 16671892 TI - Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking. AB - Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis. PMID- 16671893 TI - NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes. AB - Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post translational modification enzyme that converts protein-bound arginine residues into citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) bind to this region in vitro and in vivo. Moreover, mutation of the Sp1- or NF-Y-binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region. PMID- 16671894 TI - dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX. AB - The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, we used full-length dDYRK2 (Drosophila DYRK2) as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102 in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling. PMID- 16671895 TI - Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. AB - The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single strand break was determined. Between 4 and 37 degrees C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50 1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 degrees C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'. PMID- 16671896 TI - Dirty little secrets. PMID- 16671898 TI - 2005 Gregg Lecture: Congenital cataract--from rubella to genetics. AB - On the 65th anniversary of Gregg's observation "Congenital cataract following German measles in the mother", rubella has retired as the leading cause of congenital cataract, from 87% of Gregg's cohort to less than 3% over the last 25 years and almost zero now in Australia and other developed countries. However, people must keep vigilance in maintaining immunization rates and encourage immunization in developing countries. At least one-fifth of congenital cataract is familial. Understanding the genetics of familial cataract will lead to better treatment of congenital as well as age-related cataract. PMID- 16671897 TI - Mitomycin C in the treatment of pterygium. PMID- 16671899 TI - Glaucoma management trends in Australia and New Zealand. AB - BACKGROUND: To describe self-reported patterns of care for glaucoma of ophthalmologists in Australia and New Zealand and summarize current practice styles and patterns associated with glaucoma management. METHODS: A questionnaire of glaucoma management practices was mailed to all ophthalmologists registered with the Royal Australian and New Zealand College of Ophthalmologists in June 2003. The questionnaire assessed practice preferences for medical management, examination techniques and indications for surgery. The results were cross tabulated by age, country and subspecialty training in glaucoma. RESULTS: Fifty one per cent of 761 surveys were returned, 14% being from glaucoma specialists. New Zealand ophthalmologists proceeded to surgical management of glaucoma earlier than did their Australian colleagues. Australian ophthalmologists tended to use argon laser trabeculoplasty more frequently. Ninety-six per cent of ophthalmologists routinely use gonioscopy in diagnosing glaucoma. Disc drawings and recording cup:disc ratios were the most commonly used methods of documenting disc morphology; glaucoma specialists were more likely to use imaging technologies. SITA-Standard 24-2 was the most commonly used modality of perimetry, and was favoured by glaucoma specialists. CONCLUSIONS: This survey represents the first Australian and New Zealand effort to identify glaucoma management practices. Although a substantial consensus was found in most areas of treatment, a few areas showed diversity. The information gathered will enable ophthalmologists to compare their own practices with those of their colleagues. In addition, this survey provides a baseline allowing future trends in management to be determined. PMID- 16671900 TI - Glaucoma prescribing trends in Australia and New Zealand. AB - PURPOSE: To summarize current practice styles and patterns associated with glaucoma management in ophthalmologists of Australia and New Zealand as derived from a survey. METHOD: A questionnaire was sent to all Australian and New Zealand ophthalmologists, which anonymously assessed demographic characteristics and prescribing patterns for each major class of glaucoma medication. RESULTS: A total of 761 questionnaires were sent with a response rate of 51%. Of respondents 14% were glaucoma subspecialists. In 69%, the first-line drug-class of choice was a prostaglandin analogue. New Zealand ophthalmologists favoured beta-blockers as their first-line agent because of cost, government restrictions and familiarity. Most respondents stated "hypotensive efficacy" as the most important factor in class choice. Alpha-2-agonists, carbonic anhydrase inhibitors and miotics were considered second-line agents, because of side-effects and lack of hypotensive potency. CONCLUSIONS: The choice of first-line agent for the treatment of glaucoma differed between Australian and New Zealand ophthalmologists, in part as the result of government restriction of prostaglandin-class drugs. Practice patterns seen in Australasia parallel the current evidence base reported in peer reviewed literature. PMID- 16671901 TI - Combined "symmetrical conjunctival flap transposition" and intraoperative low dose mitomycin C in the treatment of primary pterygium. AB - PURPOSE: To introduce a new technique of conjunctival reconstruction after primary pterygium excision, and to evaluate the efficacy of combined "symmetrical conjunctival flap transposition" and intraoperative low-dose mitomycin C application in preventing recurrence of primary pterygium. METHODS: In a prospective, non-comparative case series, 43 eyes of 41 consecutive patients with primary pterygium were studied. In all patient eyes, after excision of pterygia, 0.02% mitomycin C was applied topically for 2 min over the exposed scleral surface and "symmetrical conjunctival flap transposition" was performed to reconstruct the conjunctival defect. The main outcome measures were pterygium recurrence or any complications related with surgery or mitomycin C. RESULTS: The mean age of the patients was 47.5 +/- 12.4 years (range 30-70 years). Among the 38 patients who were not lost to follow up, three patient eyes (7.5%) had grade 1, 24 eyes (60.0%) had grade 2 and 13 eyes (32.5%) had grade 3 pterygium. The mean follow up was 12.2 +/- 5.7 months (range 7-28 months). No recurrence or sight-threatening complications were encountered in any patient eye throughout the follow-up period. CONCLUSION: Combined "symmetrical conjunctival flap transposition" and intraoperative low-dose mitomycin C application may be an effective surgical alternative in preventing recurrence of primary pterygium. Although the procedure seems to be free from severe complications, surgeons and patients should be well aware of the risk of late radiomimetic complications of mitomycin C. Comparative, randomized trials with more number of patients and longer follow up are required to further establish the safety and efficacy of this treatment strategy. PMID- 16671902 TI - Visual function following transpupillary thermotherapy with adjusted laser parameters for the treatment of exudative age-related macular degeneration: a pilot study. AB - PURPOSE: To evaluate the efficacy of transpupillary thermotherapy (TTT) using adjusted laser parameters for the treatment of choroidal neovascularization (CNV) secondary to age-related macular degeneration. METHODS: TTT was performed on patients with CNV using a diode laser (810 nm) for 60 s in a subthreshold manner. Power settings were varied between 460 and 1200 mW, depending on lesion size, presence of pigment epithelial detachment and the amount of fundal pigmentation and subretinal fluid. LogMAR visual acuity, contrast sensitivity (Pelli-Robson) and metamorphopsia (Amsler chart) were assessed prior to and 6 months following treatment. Subjects also self-administered the National Eye Institute 25-Item Visual Function Questionnaire. RESULTS: Thirty occult/minimally classic and eight predominantly classic membranes were treated with TTT. At 6 months, absence or significant reduction of fluorescein leakage was observed in 20 (53%) patients. Stabilization of vision (loss of less than 15 letters) was observed in 25/30 (83%) eyes with occult/minimally classic CNV and 5/8 (63%) eyes with predominantly classic CNV. Improvement of contrast sensitivity was noted in 15 (35%) eyes, in 10 (26%) eyes it remained unchanged and in 13 (34%) eyes it deteriorated. There was no statistically significant effect of TTT on the National Eye Institute 25-Item Visual Function Questionnaire composite or subscale scores. CONCLUSIONS: TTT using adjusted parameters depending on fundal characteristics appears to be effective in stabilizing subjective and objective visual ability in a considerable number of patients with subfoveal CNV due to age related macular degeneration. Larger-scale studies are required to confirm the benefit of this technique as opposed to the natural history of occult CNV. PMID- 16671903 TI - Variable phenotypes in patients diagnosed with idiopathic multifocal choroiditis. AB - PURPOSE: To report investigations in a case series of patients diagnosed with idiopathic multifocal choroiditis (MFC) in order to determine whether or not a new clinical phenotype could be identified and to determine the long-term visual prognosis. METHODS: Twenty-eight patients were identified from the clinic database of approximately 1200 and subsequently reviewed. After ocular examination, which included fluorescein angiography, 20 of these patients were determined to have idiopathic MFC. Visual outcome, patient demographics, presenting symptoms, the distribution of inflammatory cells, the pattern of chorioretinal lesions and the development of peripapillary atrophy, choroidal new vessels or cystoid macular oedema were compared. RESULTS: The range of follow up was 1-27 years (mean 10 years). A Kaplan-Meier plot showed that approximately 60% of patients maintained their best-corrected visual acuity for 10 years after diagnosis. Poor vision at final review was associated with eyes having choroidal new vessels or cystoid macular oedema. Oral steroids were used to treat 11 patients, of which 10 responded with a visual improvement. The one non-responsive patient had foveal ischaemia angiographically. Generally the idiopathic phenotype was variable and only four patients showed some overlap with known subgroups of MFC. Unusual vascular features were observed in two patients. CONCLUSIONS: No specific phenotype was seen to emerge from this cohort of patients diagnosed with idiopathic MFC, although most were significantly different from the retinal white dot syndromes currently described. The visual prognosis was relatively good and most cases responded to oral steroids. PMID- 16671904 TI - Pathological evaluation of orbital tumours in Japan: analysis of a large case series and 1379 cases reported in the Japanese literature. AB - PURPOSE: To review epidemiological features of orbital tumours in Japan. METHODS: Retrospective, observational case series and systematic review. A total of 104 patients with orbital tumours collected at the authors' institution during 1983 2002 were assessed. In addition, 1379 cases from a large series of orbital tumours diagnosed by histopathological analysis that were previously published in Japanese ophthalmological journals from 1980 to 2004 were analysed. RESULTS: After combining the current data with the previously published cases, there were a total of 1483 lesions with 47% classified as primary, 30% as secondary and 22% as inflammatory. The most common primary tumour was malignant lymphoma (12%) followed by pleomorphic adenoma (7%). Carcinomas from the lung, breast and thyroid were found to predominate among orbital metastases. Inflammatory pseudotumour had the highest lesion frequency (18%). CONCLUSIONS: Malignant lymphoma by far was found to have the most dramatic increases within the recent reported series. Pleomorphic adenomas in Japan were found to be much more common compared with that reported for American and European studies. PMID- 16671906 TI - Epidemiology of open- and closed-globe trauma presenting to Cairns Base Hospital, Queensland. AB - PURPOSE: To review the epidemiology of serious ocular trauma presenting to Cairns Base Hospital, from the far north Queensland health districts. METHODS: A retrospective study of cases from January 1995 to November 2002 inclusive. Cases were analysed with respect to demographics, cause and nature of injury, method of transport and time to and type of ophthalmic treatment, and visual outcomes. RESULTS: There were 226 cases identified, including 71 open-globe and 155 closed globe injuries. The annual rate of injury was 3.7 per 100 000 for open-globe and 11.8 per 100 000 in total. The Aboriginal and Torres Strait Islander population from the far north Queensland districts showed a disproportionate incidence, with 38% of the total number of injuries, despite representing only 12.3% of the population. Assault in the Aboriginal and Torres Strait Islander population resulted in 69.6% of injuries in men and 75.8% of injuries in women. Of all assaults 76.2% were alcohol-related. The majority (71.5%) of injuries in the Caucasian population were due to accidental blunt and sharp trauma. In total, 77.4% of injuries occurred in men, with an average age of 31 years. Of all open and closed injuries in the study, a final visual acuity of 6/12 or better was achieved in 47.8% of eyes and a final visual acuity of 6/60 or less occurred in 17.7% of patients, 20.8% patients were lost to follow up. In total, 14.1% of open injuries required enucleation/evisceration. CONCLUSIONS: The incidence of ocular trauma in far north Queensland is equal to other Australian populations. However, there is a disproportionately high incidence in the Aboriginal and Torres Strait Islander population. Alcohol-related assault is a significant cause of visual loss in the Aboriginal and Torres Strait Islander population. Closed-globe injuries are more common than open globe; however, the latter have poorer visual prognosis. Initial visual acuity of all injuries correlated with final visual acuity. PMID- 16671905 TI - Intermittent exotropia: comparison of treatments. AB - PURPOSE: To identify the best treatment option for intermittent exotropia. METHODS: A retrospective analysis of the progress of 150 treated intermittent exotropia patients was performed. Treatment forms considered are: (i) surgery combined with orthoptic/occlusion therapy; (ii) surgery; (iii) orthoptic/occlusion therapy; and (iv) observation. Pearson's chi(2)-analysis of association of therapy form with success was performed. Reduction of exodeviation in prism dioptres between groups and subgroups were compared at 6 months, 1, 2 and 5 years follow up and the "within group" variations were compared. Exodeviation reduction in prism dioptres per millimetre of horizontal rectus surgery performed in the "surgery with orthoptic/occlusion therapy" and "surgery only" groups were compared. RESULTS: Chi(2)-analysis revealed a significantly highest (P < 0.001) association with success in the "surgery with orthoptic/occlusion therapy" group at follow up. ANOVA analysis revealed that surgery with orthoptic/occlusion therapy resulted significantly (P < 0.001) in the highest reduction of exodeviation as compared with the other three treatment modalities at each follow up. Reduction of exodeviation in prism dioptres per millimetre of horizontal rectus surgery performed was significantly higher (P < 0.05) in the surgery with orthoptic/occlusion therapy group as compared with surgery only at all follow ups. Chi(2)-test revealed no significant association of success with the magnitude of initial exodeviation (P > 0.05). CONCLUSION: Surgery with preoperative orthoptic/occlusion therapy had the highest success rates. Surgery with orthoptic/occlusion therapy was more effective in reducing exodeviation (prism dioptres per millimetre of horizontal rectus surgery), compared with surgery only. PMID- 16671907 TI - Aureobasidium pullulans keratitis. AB - BACKGROUND: Corneal ulcer caused by Aureobasidium pullulans is considered to be a rare entity. So far very few reports have appeared in the world literature and the authors' hospital is the first to report from Nepal. Although A. pullulans is regarded as a contaminant, it should be considered as a pathogen if isolated from corneal ulcer specimen with clinical signs of infection and with growth of the organism on two or more culture media or growth in one medium with consistent direct microscopy findings or growth of the same organism on repeated corneal scrapings. In the present study, a series of proven cases of A. pullulans corneal ulcers at a tertiary eye care centre of Eastern Nepal is reported. METHODS: A retrospective analysis of stored data of microbiological and clinical cases of corneal ulcer was carried out. All consecutive patients (447 patients) with presumed microbial keratitis from 1 August 1998 to 31 July 2001 were evaluated with regards to clinical details, microbiological examination and management. RESULTS: Of 200 fungal organisms isolated from the cultures, 25 were identified as A. pullulans. These ulcers showed negligible improvement to topical natamycin and required either topical fluconazole or topical itraconazole in all along with systemic intravenous fluconazole in eight patients. Of 25 eyes, 22 responded well to antifungal therapy and 2 required therapeutic penetrating keratoplasty. One patient was lost to follow up for 3 months and revealed phthisis bulbi on subsequent examination. CONCLUSIONS: Aureobasidium pullulans corneal infection should be considered as a cause of keratomycosis. PMID- 16671908 TI - Inhibitory effects of Triptolide on interferon-gamma-induced human leucocyte antigen-DR, intercellular adhesion molecule-1, CD40 expression on retro-ocular fibroblasts derived from patients with Graves' ophthalmopathy. AB - PURPOSE: To explore the effects of Triptolide, the principal active diterpenoid from the Chinese Medicinal Herb Tripterygium Wilfordii Hook F that has immunosuppressive and anti-inflammatory properties, on cell proliferation, hyaluronic acid (HA) synthesis, and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) and CD40 on cultured retro ocular fibroblasts (RFs) from patients with Graves' ophthalmopathy. METHODS: After two to five passages, cultured RFs were incubated for 48 h within a medium alone or in the presence of recombinant human interferon-gamma (IFN-gamma) and various concentrations of Triptolide. Cell viability was assessed by MTT (3-[4.5 dimethylahiazol-2-yl]-2,5-diphenyltetrazolium Bromide). RFs proliferation was assessed by [(3)H]-thymidine incorporation assay. Flow cytometry was used to investigate the amount of HLA-DR, ICAM-1 and CD40. HA synthesis was measured by radioimmunoassay. RESULTS: Cell viability was not detrimentally affected when incubated with Triptolide from 0.01 microg/L to 10 microg/L for 48 h, and decreased with 20 microg/L Triptolide. The incorporation of [(3)H]-thymidine of RFs was 55 476 +/- 15 842 cpm incubated with medium alone or 18 352 +/- 3568 cpm with 10 microg/L Triptolide (t = 5.600, P < 0.01). Initially, the percentage of positive cells of HLA-DR, ICAM-1 and CD40 on RFs were 4.75 +/- 2.13%, 17.53 +/- 10.12% and 6.38 +/- 2.23%, respectively, and the synthesis of HA was 100 +/- 12%. Compared with basal values, 48-h incubation with IFN-gamma (100 U/mL) significantly enhanced the amount of HLA-DR, ICAM-1 and CD40, and HA synthesis. The values were 60.58 +/- 10.12% (t = 13.224, P < 0.01), 62.66 +/- 18.17% (t = 5.315, P < 0.01), 57.67 +/- 13.61% (t = 9.110, P < 0.01) and 164 +/- 22% (t = 9.238, P < 0.01), respectively. Triptolide 0.01 microg/L had little effect on IFN gamma-induced HLA-DR, ICAM-1 and CD40 amounts, as well as HA synthesis. When the concentration ranged from 0.1 microg/L to 10 microg/L, Triptolide inhibited IFN gamma-induced RFs activation in a dose-dependent manner. It was also found that Triptolide had the same inhibiting effects on IFN-gamma-induced RFs and skin fibroblasts from patients with normal individual conditions. CONCLUSIONS: Triptolide could inhibit IFN-gamma-induced activation of RFs derived from patients with Graves' ophthalmopathy. PMID- 16671909 TI - Clinicopathological features of severe corneal blood staining associated with proliferative diabetic retinopathy. AB - A 54-year-old man with a history of severe proliferative diabetic retinopathy in both eyes and profound visual impairment presented with severe corneal blood staining in the left eye secondary to a "spontaneous" total hyphaema and raised intraocular pressure in an eye with iris neovascularization. Despite anterior chamber washout, the cornea remained virtually opaque and thickened. The subject subsequently underwent pars plana vitrectomy with endolaser using a temporary keratoprosthesis, insertion of a Morcher iris-surround intraocular lens and penetrating keratoplasty. Histopathology of the excised corneal button revealed fine eosinophilic granules composed of aggregations of haemoglobin and its breakdown products dispersed throughout the stroma, with occasional foci of weakly positive Perl staining for intracellular haemosiderin. Fluorescence confocal microscopy revealed a marked increase in fluorescence throughout the corneal stroma and the basal epithelial layer. This case highlights the microstructural features and aspects of the surgical management of severe corneal blood staining. PMID- 16671910 TI - Malignant fibrous histiocytoma of the conjunctiva. AB - Fibrous histiocytomas are mesenchymal tumours composed of cells with fibroblastic to histiocytic differentiation. They can occur in any part of the body including the orbital tissues. To date, there are 18 cases of fibrous histiocytoma arising from the corneoscleral limbus reported in the literature. Eleven of these were classified as benign, and the rest were malignant fibrous histiocytomas. Benign fibrous histiocytomas have been reported in the orbit, eyelid, episclera and conjunctiva. Malignant fibrous histiocytoma has been well described in the orbit, but rarely as a primary conjunctival tumour. The rarity of the tumour makes its diagnosis and management a challenge. Herein, the clinicopathological features of a case of malignant fibrous histiocytoma are presented and its management with wide excision and cryotherapy followed by ocular reconstruction with amniotic membrane transplant is discussed. PMID- 16671911 TI - Spontaneous resolution of corneal blood staining. AB - Herein, a case of a 66-year-old man who developed corneal blood staining secondary to hyphaema after trabeculectomy is reported. No significant increase in intraocular pressure was recorded. The corneal staining spontaneously cleared from the periphery and resolved fully over the following 2 years. PMID- 16671912 TI - Systemic non-Hodgkin's lymphoma masquerading as Vogt-Koyanagi-Harada disease in an HIV-positive patient. AB - A 29-year-old man presented with decreased visual acuity in both eyes secondary to exudative retinal detachment resembling Vogt-Koyanagi-Harada disease. Although fluorescein angiographic pictures supported the clinical findings, there was no choroidal thickening evident with ultrasonography. In 3 days he developed increased disc oedema with peripapillary haemorrhages in both eyes. Further evaluation revealed HIV-positive status and a systemic non-Hodgkin's lymphoma. The patient responded favourably to the treatment for systemic non-Hodgkin's lymphoma confirming our diagnosis of intraocular metastasis. In bilateral exudative detachment, an absence of choroidal thickening on ultrasonography and the presence of peripapillary haemorrhages should prompt a systemic evaluation for causes other than Vogt-Koyanagi-Harada disease, especially in HIV-positive patients. PMID- 16671913 TI - Correlation study between optical coherence tomography and fluorescein angiography in post-membrane peeling recalcitrant macular oedema. AB - Correlation between optical coherence tomography and fluorescein angiography in a post-membrane peeling patient indicated a surgically disturbed retinal vessel as the cause of recalcitrant macular oedema. PMID- 16671914 TI - Medial canthal tendon release and lower eyelid retractor plication for Centurion syndrome. AB - Centurion syndrome is an uncommon, idiopathic medial canthal anomaly that causes epiphora due to the forward displacement of the lacrimal punctum out of the tear lake associated with the abnormal anterior insertion of the medial canthal tendon and enophthalmos. A case of Centurion syndrome is presented together with the description of a previously unreported surgical treatment: the combined surgical techniques of medial canthal tendon release and lower eyelid retractor plication. PMID- 16671915 TI - Marked discordance for myopia in female monozygotic twins. AB - Female monozygotic twins aged 54 years discordant for myopia are reported. One twin presented with bilateral high myopia (right eye = -6.00/+0.50 x 5 degrees , left eye = -6.00/+0.50 x 45 degrees ) and her identical twin had no significant refractive error (right eye = -0.50/plano, left eye = -0.50/+0.75 x 40 degrees ). An explanation for the striking refractive discordance seen in this case report is yet to be determined. PMID- 16671916 TI - Tools for cup:disc ratio measurement. PMID- 16671918 TI - Doctor of nursing practice: forward direction or digression for child psychiatric nursing? PMID- 16671919 TI - Differences in trait anger among children with varying levels of anger expression patterns. AB - PROBLEM: Little research has been done with children to determine effects of using various patterns of anger expression on trait anger. The purpose was to examine differences in trait anger of children who indicated high, moderate, or low use of three patterns of anger expression. METHODS: A convenience sample of 1,060 third through sixth graders completed trait anger and patterns of expressing anger instruments. FINDINGS: High users of anger-out (anger expressed outwardly) had the highest trait anger for every grade while high users of anger reflection/control had the lowest. CONCLUSIONS: Anger-reflection/control may be more effective than anger-out in reducing trait anger in school-age children. PMID- 16671920 TI - Integrating nursing care into systems of care for children with emotional and behavioral disorders. AB - PROBLEM: Recent developments in providing care to children with emotional and behavioral disorders, especially those with serious emotional disturbance, have included the establishment of systems of care. Guided by a set of principles and values, these systems of care have organized and delivered services to children and families with complex needs. To date, nurses have not had a salient role in systems of care. RESULTS: It is estimated that 20% of American children and adolescents have an emotional or mental disorder. As many as two thirds of these children are not receiving services. Systems of care have been funded to provide services for these children, particularly for the most severely affected. To date, nursing has not had a prominent role in these systems of care. CONCLUSIONS: Based on their knowledge, skills, and holistic approach to care, nurses could better integrate nursing care into systems of care. Possible roles as case managers, primary therapists, in-home interventionists, and in educational programs are suggested. PMID- 16671921 TI - Aromatherapy, used as an integrative tool for crisis management by adolescents in a residential treatment center. AB - PROBLEM: Little is known about the acceptance and effectiveness of aromatherapy as a complementary support for agitated and aggressive adolescents. METHODS: Chi square test was applied to satisfaction surveys, administration records of p.r.n. medication, aromatherapy, and seclusion/ restraint data. FINDINGS: Seventy-seven percent of the population accepted and used aromatherapy for crisis management. A trend indicates this population uses less p.r.n. medication for crisis management. CONCLUSIONS: This adolescent population was receptive to using less invasive tools for crisis management. A longitudinal study is needed to determine the long-term efficacy of this treatment modality. PMID- 16671922 TI - Understanding runaway teens. AB - PROBLEM: Large numbers of teenagers become "runaways" and put themselves at risk for physical and emotional harm. During the year 2002, an estimated 1.6 million U.S. youth aged 12 to 17 had run away from home and slept on the streets. METHODS: A qualitative exploratory method was used to study a sample of primarily ethnic minority youth found in a Midwestern inner city detention center. This study examined run behavior from the teenager's own perspective to identify why they ran, how they kept themselves safe, and what factors contributed to ending a run. FINDINGS: Teens ran to gain control of their lives by changing their situation. They developed new affiliations to meet safety and sustenance needs and learned from experience that one cannot run from your problems. With experience, running became habitual. CONCLUSIONS: Safer alternatives are needed for youth who cannot live at home. PMID- 16671923 TI - The quality of mental healthcare chasm. AB - The Institute of Medicine (IOM) released the latest in the Quality Chasm Series. PMID- 16671925 TI - Complementary theoretical and empirical approaches to studying the role of attention in the development of psychopathology. PMID- 16671926 TI - Annotation: the therapeutic alliance--a significant but neglected variable in child mental health treatment studies. AB - BACKGROUND: There has been relatively little research into therapeutic alliance in child and adolescent mental health and virtually no incorporation of alliance measures as a variable in treatment trials in Child and Adolescent Mental Health Services (CAMHS). METHOD: A selective literature review on studies in therapeutic alliance in adulthood and childhood along with a theoretical formulation of possible mechanisms of alliance. RESULTS: Therapeutic alliance is reliably measurable both by observation and questionnaire methods at all points in the treatment cycle. In both adult and child studies it shows a consistent, albeit modest, association with treatment outcome. In specific adult studies it has shown a high predictive validity in relation to outcome compared to other variables. In child studies alliance is particularly salient in externalising disorder and predicts outcome of inpatient treatment. Child alliance and parental alliance are independent factors. Theoretical models of alliance outlined in this paper suggest testable hypotheses regarding predictors for positive and negative alliance. CONCLUSIONS: Therapeutic alliance in CAMHS is measurable and worth measuring. It is likely to be an important variable for treatment outcome studies and should be included in future trial designs. PMID- 16671927 TI - Child-therapist and parent-therapist alliance and therapeutic change in the treatment of children referred for oppositional, aggressive, and antisocial behavior. AB - BACKGROUND: We examined the therapeutic alliance in evidence-based treatment for children (N = 77, 19 girls, 58 boys, ages 6-14) referred clinically for oppositional, aggressive, and antisocial behavior. METHOD: Different alliances (child-therapist, parent-therapist) were assessed from each participant's perspective at two points over the course of treatment. Both the quality of the child-therapist and the parent-therapist alliance predicted therapeutic changes in the children; the parent-therapist alliance also predicted improvements in parenting practices in the home. RESULTS: The findings could not easily be attributed to the influence of other domains (socioeconomic disadvantage, parent psychopathology and stress, and severity of child dysfunction) known to predict therapeutic change or to rater effects (common rater variance) in the predictors and criteria. CONCLUSION: The therapeutic alliance warrants increased attention to understand the precise role in treatment and whether or how the alliance can be mobilized to enhance change. PMID- 16671928 TI - Assessing medication effects in the MTA study using neuropsychological outcomes. AB - BACKGROUND: While studies have increasingly investigated deficits in reaction time (RT) and RT variability in children with attention deficit/hyperactivity disorder (ADHD), few studies have examined the effects of stimulant medication on these important neuropsychological outcome measures. METHODS: 316 children who participated in the Multimodal Treatment Study of Children with ADHD (MTA) completed the Conners' Continuous Performance Test (CPT) at the 24-month assessment point. Outcome measures included standard CPT outcomes (e.g., errors of commission, mean hit reaction time (RT)) and RT indicators derived from an Ex Gaussian distributional model (i.e., mu, sigma, and tau). RESULTS: Analyses revealed significant effects of medication across all neuropsychological outcome measures. Results on the Ex-Gaussian outcome measures revealed that stimulant medication slows RT and reduces RT variability. CONCLUSIONS: This demonstrates the importance of including analytic strategies that can accurately model the actual distributional pattern, including the positive skew. Further, the results of the study relate to several theoretical models of ADHD. PMID- 16671929 TI - Infrequent, but not frequent, reinforcers produce more variable responding and deficient sustained attention in young children with attention deficit/hyperactivity disorder (ADHD). AB - BACKGROUND: The underlying behavioral/psychological processes of attention deficit/hyperactivity disorder are unclear. Motivational factors, related to dopamine dysfunction, may play an important role in the development of the behavioral symptoms. Particularly, infrequent, but not frequent, reinforcers have been suggested to be associated with altered responding and deviant behavior in children with ADHD. The present study was designed to analyze the influence of reinforcement frequency on operationalized measures of hyperactivity, impulsiveness, sustained attention, and response variability. METHODS: Fifty-six boys, half of whom were diagnosed with ADHD, completed a computerized task presented as a game with two squares on the screen. One square was associated with reinforcement. The task required responses by the computer mouse under contingencies alternating between variable interval schedules of short (mean 2 s) and long (mean 20 s) durations. Reinforcers were cartoon pictures and small trinkets. RESULTS: Overall, there was no difference between children with ADHD and comparison children when reinforcers were given frequently. Statistically significant differences on measures of sustained attention and variability, but not hyperactivity and impulsiveness, were found during infrequent reinforcement only. Age effects interacted with group effects on sustained attention, showing that group differences were found in the young children only. Surprisingly, older comparison children showed increased response variability and no learning. CONCLUSIONS: When reinforcers are infrequent, children with ADHD show deficient sustained attention and increased variability in responding. Computer experience may have interfered with measures of hyperactivity and impulsiveness. The unexpectedly poor performance of the older comparison children may have been due to inefficient reinforcers or to verbally governed behavior overruling reinforcer effects. Reinforcer characteristics and experimental procedures are important factors influencing findings in studies investigating motivational factors. The study provides some support for the dynamic developmental theory of ADHD predicting relationships between neurobiological deficits, altered reinforcement mechanisms, and treatment recommendations. PMID- 16671930 TI - Predictive validity of ICD-10 hyperkinetic disorder relative to DSM-IV attention deficit/hyperactivity disorder among younger children. AB - BACKGROUND: Little is known about the predictive validity of hyperkinetic disorder (HKD) as defined by the Diagnostic Criteria for Research for mental and behavioral disorders of the tenth edition of the International Classification of Diseases (ICD-10; World Health Organization, 1993), particularly when the diagnosis is given to younger children. METHODS: The predictive validity of HKD was evaluated over a 6-year period and compared to the predictive validity of DSM IV attention-deficit/hyperactivity disorder (ADHD) in 95 4-6-year-old children who met full criteria for at least ADHD and 122 demographically-matched nonreferred comparison children. Diagnoses were based on structured assessments of both parents and teachers. RESULTS: All children who met full criteria for HKD also met full DSM-IV criteria for ADHD, but only 26% of ADHD children met criteria for HKD. Children who met criteria for HKD (N = 24), children who would have met criteria for HKD but were excluded from the diagnosis because they concurrently met criteria for an anxiety disorder or depression (N = 16), and the remaining children who met DSM-IV criteria for ADHD (N = 55) all exhibited significantly more symptoms of ADHD and greater social and academic impairment during years 2-7 than nonreferred comparison children. Unlike the two other diagnostic groups, however, children who met strict criteria for HKD were not more likely than comparison children to be injured unintentionally or to be placed in special education. CONCLUSIONS: Both ICD-10 HKD and DSM-IV ADHD exhibit predictive validity over 6 years, but ICD-10 HKD appears to under-identify children with persistent ADHD symptoms and related impairment. Children who met criteria for DSM-IV ADHD but not HKD exhibited at least as much functional impairment over time as hyperkinetic children. PMID- 16671931 TI - Institutional care: associations between inattention and early reading performance. AB - BACKGROUND: Recent government papers have expressed concern about the poor educational attainment of 'looked after' children. Early reading development has been found to be significant in their subsequent academic achievement. The possibility that biosocial factors extraneous to their experiences in public care may underpin their low attainment has not been investigated to date. METHODS: The reading ability of 19 primary school children, who had been raised in institutional care from an early age, were compared with 19 children, matched for age and sex, who were comparable in biological background and who had experienced uninterrupted family foster care. Both groups were compared with classroom controls using teacher questionnaires, interviews, systematic observations and cognitive testing. RESULTS: Reading delay was more prevalent in the institutional group and as a group they had lower reading scores than the children reared in family foster care. Variation in IQ accounted for the lower reading scores of the family foster care group compared with their matched classroom controls. Inattention, found in a previous study to be much more evident in the institutional group, partially accounted for the group difference in reading scores, and was situationally specific to formal teacher-directed tasks. Differential effects of caregivers' interest in terms of help with homework were also found. CONCLUSIONS: Early reading performance was associated with the experience of being raised 'in care' but was not an inevitable outcome. It was concluded that the type of substitute caregiving experienced affected reading performance. Institutional upbringing affected reading performance both directly and indirectly through the heightened levels of inattention associated with institutional care. PMID- 16671932 TI - Behavioural problems in 2-year-olds: links with individual differences in theory of mind, executive function and harsh parenting. AB - BACKGROUND: Cognitive and family factors are implicated in the origins of behavioural problems, but little is known about their independence or interplay. METHODS: We present data from 127 two-year-olds from predominantly disadvantaged families who completed tests of 'theory of mind' (ToM), executive function (EF) and verbal ability. Researchers' home-visit ratings and detailed video-based coding of mother-child interactions were combined to give an aggregate measure of harsh parenting, while behavioural problems were indexed by a multi-informant, multi-setting, multi-measure aggregate. RESULTS: Harsh parenting and deficits in ToM and verbal ability each predicted unique variance in behavioural problems; independent effects of EF were only marginally significant. Harsh parenting and ToM interacted significantly in their effects on behavioural problems. CONCLUSIONS: Child and family influences on behavioural problems should be considered in tandem, as they show significant interplay; in particular, advanced ToM skills appear to buffer young children against effects of harsh parenting. PMID- 16671933 TI - Predictors of between-family and within-family variation in parent-child relationships. AB - BACKGROUND: Previous studies have found that multiple factors are associated with parent-child relationship quality, but have not distinguished potential sources of between-family and within-family variation in parent-child relationship quality. METHODS: Approximately equal numbers of biological (non-stepfamilies), single-mother, stepfather, and complex/stepmother stepfamilies were selected from a large community study in England. The sample comprised 404 children in 171 families. Parent-child warmth/support and conflict/negativity were assessed using questionnaire and interview methods; family socio-demographic data were collected from parent report. RESULTS: Multilevel model analyses indicated substantial within-family variation in parent-child relationship quality, and greater within family variation in complex/stepmother families compared to other family types. Within-family variation was largely accounted for by differences in the siblings' biological relatedness to the mother and father and to child-specific factors, notably aggressive behavior. CONCLUSIONS: The findings illustrate how research using multilevel model designs and analytic strategies may enhance our understanding of family process. PMID- 16671934 TI - The development of siblings of children with autism at 4 and 14 months: social engagement, communication, and cognition. AB - AIMS: To compare siblings of children with autism (SIBS-A) and siblings of children with typical development (SIBS-TD) at 4 and 14 months of age. METHODS: At 4 months, mother-infant interactional synchrony during free play, infant gaze and affect during the still-face paradigm, and infant responsiveness to a name calling paradigm were examined (n = 21 in each group). At 14 months, verbal and nonverbal communication skills were examined as well as cognition (30 SIBS-A and 31 SIBS-TD). RESULTS: Most SIBS-A were functioning as well as the SIBS-TD at 4 and 14 months of age. However, some differences in early social engagement and later communicative and cognitive skills emerged. Synchrony was weaker in the SIBS-A dyads, but only for infant-led interactions. Infant SIBS-A revealed more neutral affect during the still-face procedure and were less upset by it than was true for the SIBS-TD. A surprising result was that significantly more SIBS-A responded to their name being called by their mothers compared to SIBS-TD. At 14 months, SIBS-A made fewer nonverbal requesting gestures and achieved lower language scores on the Bayley Scale. Six SIBS-A revealed a language delay of 5 months and were responsible for some of the significant differences between SIBS A and SIBS-TD. Furthermore, infant SIBS-A who showed more neutral affect to the still face and were less able to respond to their name being called by their mothers initiated fewer nonverbal joint attention and requesting behaviors at 14 months, respectively. DISCUSSION: Focused on the genetic liability for the broad phenotype of autism as well as the possible influence of having a sibling with autism. PMID- 16671935 TI - Physical and emotional development, appetite and body image in adolescents who failed to thrive as infants. AB - BACKGROUND: Previous studies suggest that failure to thrive in infancy may be associated with adverse sequelae in childhood. Although cognitive abilities have been extensively investigated, little systematic research is available on other aspects of development. METHODS: Eighty-nine children who failed to thrive as infants and 91 controls were followed up when twelve years old and examined using anthropometric measurement, self-ratings of appetite and body image, the Dutch Eating Behaviour Questionnaire, the Self-perception Profile for Children, The Revised Children's Manifest Anxiety Scale, the parent and child form of the Mood and Feelings Questionnaire and the parent and teacher's form of the Child Behavior Checklist. RESULTS: The children who failed to thrive were significantly shorter and lighter at twelve and had significantly lower BMIs, but they did not go into puberty any later. They were more likely to rate their appetite as lower than their best friend's, were generally more satisfied with their body shape, and had significantly lower restraint score on the Dutch Eating Behaviour Questionnaire. They were not significantly different from controls on any of the measures reflecting anxiety, depression or low self-esteem. CONCLUSIONS: Failure to thrive in infancy is not associated with adverse emotional development in childhood. PMID- 16671941 TI - Cysteine cathepsins in the immune response. AB - Antigen (Ag) processing by major histocompatibility complex class II (MHC) class II molecules is tightly linked with the proteases of the endosomal/lysosomal system. Cysteine (Cys) cathepsins, which constitute a major portion of this proteolytic system, have been found to have essential roles in both Ag processing and maturation of the MHC class II molecules. In this review, we will cover some specific functions of individual Cys cathepsins and particularly those most relevant to the immune system. PMID- 16671942 TI - Distribution of killer-cell immunoglobulin-like receptor (KIR) in Comoros and Southeast France. AB - Killer-cell immunoglobulin-like receptors (KIRs) expressed by natural killer cells are cell surface molecules able to recognize groups of HLA class I alleles. The number and distribution of KIR genes vary among individuals and populations. The aim of this study is to analyse the KIR gene content in a Comorian population in order to investigate genetic relationships with other populations and to reconstruct past migration events. The Comorian population consisted of 54 unrelated immigrants living in France and a control population consisted of 38 individuals from Southeast France. We investigated the presence or absence of 15 KIR genes, two pseudogenes expressed and non-expressed forms of KIR2DL5 and the two major subtype full-length and deleted forms of KIR2DS4. All individuals were typed positive for the framework genes, i.e. KIR2DL4, KIR3DL2 and KIR3DL3, and the two pseudogenes KIR3DP1 and KIR2DP1. The frequencies of full-length KIR2DS4 (*00101/00102/002) were lower in the French population (F = 29%) than in the Comorian population (F = 72%) (P(c) < 0.05). No significant differences were found for other KIR genes. A total of 11 genotypes were identified in the Southeast French population and 22 genotypes in the Comorian population. The most common genotype (2DL1, 2DL3, 2DL4, 3DL1, 3DL2, 3DL3 and 2DS4) accounted for 41% in the Comorian population and 34% in the Southeast French population. Principal component analysis using KIR gene data from 20 populations was performed to determine genetic differences and relations between populations. The Comorian population exhibited closest kinship with Africans and Asians. As KIR gene content is heterogeneous among ethnic groups, it can probably be used to assess the genetic relationships among populations from different geographic areas. PMID- 16671943 TI - Fusion of CpG-ODN-stimulating dendritic cells with Lewis lung cancer cells can enhance anti-tumor immune responses. AB - Immunogenicity of tumor cells is generally weak. Therefore, dendritic cells (DCs) have been used to boost anti-tumor responses of DC-based vaccines. DC function is highly dependent on its subsets and the level of its maturation. Nowadays, DC/tumor cell fusion vaccines are already used in clinical trials, and there are numerous studies discussing the effects of cytidine-phosphate-guanosine containing oligonucleotides (CpG-ODN) on various cell types including DC. CpG-ODN a powerful immuno-stimulant can drive DCs fully mature, thus improve the efficacy of vaccine therapy. There are two simple ways to help load tumor antigens onto DCs by direct contact with cells themselves: fusion or co-culture of DCs with whole tumor cells. In this study, we combined these two approaches to improve the efficacy of DC/tumor cell-based vaccine. Mature DCs are adept at presenting processed Ag to T cells with loss of its capacity to capture Ag, while immature DCs are on the contrary. Our results emphasize the necessity of considering the stage of DC maturation and corresponding choice of tumor antigen delivery when designing approaches for prophylaxis or therapy of tumors using DC-based immunization protocols. We used CpG-ODN-1826-stimulated mature DCs and non-CpG ODN-stimulating DCs as sources of tumor antigen carriers to investigate the appropriate Ag-loading ways between fusion and co-culture. Our results displayed that DC/tumor vaccine using CpG-ODN-stimulating mature DCs fused, not co cultured, with tumor cells can generate a consistent and highly effective anti tumor immune responses in vivo. PMID- 16671944 TI - TNF-alpha SNP haplotype frequencies in equidae. AB - Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine that plays a crucial role in the regulation of inflammatory and immune responses. In all vertebrate species the genes encoding TNF-alpha are located within the major histocompatability complex. In the horse TNF-alpha has been ascribed a role in a variety of important disease processes. Previously two single nucleotide polymorphisms (SNPs) have been reported within the 5' un-translated region of the equine TNF-alpha gene. We have examined the equine TNF-alpha promoter region further for additional SNPs by analysing DNA from 131 horses (Equus caballus), 19 donkeys (E. asinus), 2 Grant's zebras (E. burchellii boehmi) and one onager (E. hemionus). Two further SNPs were identified at nucleotide positions 24 (T/G) and 452 (T/C) relative to the first nucleotide of the 522 bp polymerase chain reaction product. A sequence variant at position 51 was observed between equidae. SNaPSHOT genotyping assays for these and the two previously reported SNPs were performed on 457 horses comprising seven different breeds and 23 donkeys to determine the gene frequencies. SNP frequencies varied considerably between different horse breeds and also between the equine species. In total, nine different TNF-alpha promoter SNP haplotypes and their frequencies were established amongst the various equidae examined, with some haplotypes being found only in horses and others only in donkeys or zebras. The haplotype frequencies observed varied greatly between different horse breeds. Such haplotypes may relate to levels of TNF-alpha production and disease susceptibility and further investigation is required to identify associations between particular haplotypes and altered risk of disease. PMID- 16671945 TI - Promoter region -318 C/ T and -1661 A/G CTLA-4 single nucleotide polymorphisms and type 1 diabetes in North Indians. AB - Single nucleotide polymorphisms (SNPs) of the CTLA-4 gene have been associated with manifestation of type 1 diabetes in several populations. We assessed the association of five SNPs present in the CTLA-4 gene [-318C/T, -1661A/G and 1722C/T in the promoter region, +49A/G in exon 1 and CT60 in the 3' untranslated region (UTR) region] with type 1 diabetes in North Indian subjects. Genotyping was performed in the patients (n = 130) and the healthy control (n = 180) subjects by polymerase chain reaction-fragment length polymorphism analysis using MseI, BbvI, BstEII and NcoI restriction endonucleases for the -318, -1661, -1722, +49 and CT60 SNPs, respectively. The frequency of G alleles at -1661 locus was significantly higher in the patient group compared with the control subjects. Although the frequency of T alleles at -318 SNP was significantly higher in patients with type 1 diabetes compared with the controls, it did not remain significant after Bonferroni correction for the number of alleles tested. The frequencies of C/T alleles and genotypes at -1722C/T and G allele at +49A/G and CT60 SNPs were not significantly different between the patient and the control groups. Of the various possible haplotypes constructed using the five genetic loci tested (-318, -1661, -1722, +49, CT60), the frequency of 'TGTAG' haplotype was significantly higher in the patients when compared with the controls. The results of the present study indicate that the presence of G allele at -1661 locus at the CTLA-4 gene (IDDM12 locus) is associated with increased susceptibility to type 1 diabetes in North Indians, whereas A allele is protective. PMID- 16671946 TI - Four-digit allele genotyping of the HLA-A and HLA-B genes in Japanese patients with Behcet's disease by a PCR-SSOP-Luminex method. AB - The present study represents the first four-digit allele genotyping of HLA-A and B in Japanese Behcet's disease (BD) patients and controls using a new genotyping method (named the PCR-SSOP-Luminex method) to determine the association of certain HLA-A or -B alleles with BD. Peripheral blood lymphocytes were collected from 180 Japanese BD patients and 170 healthy controls. The genotype frequency of HLA-B*5101 was significantly increased in the patients (61.7%) as compared with the controls (15.9%) (Pc = 1 x 10(-16), OR = 8.5). When we recalculated the phenotype frequencies after excluding the HLA-B*51-positive patients and controls to account for the effects of the linkage disequilibrium and the abundance of the HLA-B*51 allele, the frequencies of HLA-A*2602 and HLA-B*3901 had a weak association in the patient group without HLA-B*51 as compared with the control group without HLA-B*51 (A*2602; Pc = 0.130, OR = 4.3, B*3901; Pc = 0.099, OR = 3.5). This study confirmed on the basis of using a new and more accurate genotyping method that Japanese BD patients have a strong primary association with HLA-B*5101. The significant increase of HLA-A*2602 and B*3901 in the patient group without HLA-B*51 suggests that these two alleles might also have some secondary influence on the onset of BD. PMID- 16671947 TI - The dbMHC microsatellite portal: a public resource for the storage and display of MHC microsatellite information. AB - Major histocompatibility complex (MHC) region Microsatellites (Msat) have been extensively used in various applications, such as disease mapping, forensics, and population genetics. A comprehensive review of HLA Msat primers has been previously published based on literature and sequence analysis, but electronic tools are lacking to make it easily accessible and actually used by the community. We have integrated data from this review, with an overlapping set of 31 Msat markers used in the 13th International Histocompatibility and Immunogenetics Workshop (IHIWS) to create a public archive that will synchronize published descriptions to a common framework. http://www.ncbi.nlm.nih.gov/projects/mhc. Currently, the dbMHC contains 389 primer pairs across the extended MHC targeting 281 distinct repeat regions (approximately 1/45 kb). Literature review and analysis of the primers reveal that over 200 synonymous names have been published for these markers. Users may view or download specific Msat data sets using the portal. Query options include name or partial name, primer sequence, neighboring genes, and/or position. Query results include locus name(s), a graphic showing of the relative location of the marker in relation to the classical HLA genes, a listing of the constituent primer pairs and name, a link to UniSTS, aliases, allele range (bp), overlapping single nucleotide polymorphisms, a link to e-polymerase chain reaction, and physical mapping information. To increase the utility of this resource, researchers using Msat markers in the HLA region are encouraged by the authors to submit new primers to the dbMHC. The minimal Msat submission consists of primers sequences, a submitter's name and contact information. Additional information recommended but not required is the laboratory protocol(s), known allele size range (bp), known aliases, and an exemplar sequence. Assigned UniSTS numbers can be used for primer pair standard identification. PMID- 16671948 TI - Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations. AB - The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters. PMID- 16671949 TI - Clinical behavior of multiple sclerosis is modulated by the MHC class I-chain related gene A. AB - It is well known that certain HLA class II alleles confer an increased risk for developing multiple sclerosis (MS). Recent studies have suggested HLA class I as a region that may also contribute to the development of MS. In this study, we investigated the association between HLA-DR, HLA-B alleles, and major histocompatibility complex (MHC) class I-chain-related gene A (MICA) transmembrane (MICA-TM) polymorphisms and disease progression in 104 MS patients and 116 healthy controls. DR1 was found to be decreased in patients when compared with controls (p(c) = 0.012). Neither HLA-B nor HLA-DR alleles were found to be associated with MS susceptibility. Furthermore, the prevalence of MICA-A5 in patients with relapsing MS was 9% while the prevalence in progressive forms was 42% (p(c) = 0.0015). The extended haplotypes related to MICA-TM5 that were found in our population were DR7-MICA5-B64 (EH 64.1, delta(s) = 0.38), DR4-MICA5-B62 (EH 62.1, delta(s) = 0.28), and DR11-MICA5-B35 (EH35.1, delta(s) = 0.10), but none of them were found to be associated to MS susceptibility or disease progression. Our data could indicate a possible role of MICA-TM in MS prognosis. PMID- 16671950 TI - Interleukin-18 promoter polymorphism in patients with rheumatoid arthritis. AB - Rheumatoid arthritis (RA) is a chronic inflammatory disease in which interleukin (IL)-18 plays an important role. However, there are controversial reports on IL 18 promoter polymorphism as an independent marker of RA susceptibility. The aim of present study was to examine the IL-18 promoter polymorphism in patients with RA, and its association with disease susceptibility, activity and severity. We examined 309 patients with RA from a Polish population diagnosed according to the criteria of American College of Rheumatology. An allele-specific polymerase chain reaction was used for analysis of the polymorphisms in positions - 137 and - 607 in promoter region of IL-18 gene. A significantly decreased number of subjects with AC/AC and AG/AG diplotypes was observed among RA patients as compared with healthy controls (OR - 0.51, 95%CI 0.28-0.95, P = 0.045) and (OR - 0.12, 95% CI 0.02-0.97, P = 0.042), respectively. Nevertheless, there was no significant association with disease activity, joint erosions, extra-articular manifestations, rheumatoid factor. Above results suggest that IL-18-137 and - 607 promoter polymorphisms are not the significant factors influencing RA course and severity in a Polish population. PMID- 16671951 TI - Molecular cloning and characterization of pig, cow and sheep MAdCAM-1 cDNA and the demonstration of cross-reactive epitopes amongst mammalian homologues. AB - Full-length cDNA clones for the pig, cow and sheep mucosal addressin cellular adhesion molecule (MAdCAM)-1 homologues were isolated from Peyer's patches by a combination of reverse transcription (RT)-polymerase chain reaction and 5' and 3' RACE strategies. Degenerate primers based on conserved amino acid (aa) sequences within the N-terminal immunoglobulin (Ig)-like domains of the human and rodent MAdCAM-1 molecules were used for initial sequencing of the Ig-like domains. MAdCAM-1 transcripts of 1425 bp, 1525 bp and 1510 bp obtained for the pig, cow and sheep contained an open-reading frame for proteins of 390, 424 and 418 aa, respectively. The pig and ruminant MAdCAM-1 had two N-terminal Ig-like domains, a mucin-like region and a third Ig-like domain found in rodent but not human MAdCAM 1. Antibodies raised against bacterially expressed N-terminal Ig-like domains of pig, human and sheep MAdCAM-1 demonstrated the existence of cross-reactive epitopes, raising the possibility of producing monoclonal antibodies which can be used as multi-species MAdCAM-1-targeting reagent for the development of mucosal vaccines. PMID- 16671952 TI - Identification of a new allele, HLA-DRB5*0113, through three different molecular biology techniques. AB - A new HLA-DRB5 allele, HLA-DRB5*0113, has been identified in an Italian patient during routine HLA typing in order to activate a bone marrow donor search. HLA typing was performed by different molecular biology techniques, and the results showed that the HLA-DRB5*0113 allele differs from HLA-DRB5*010101 allele for three nucleotide substitutions at codons 57 (GAC-->GAT; Asp) and 58 (GCT-->GAG; Ala-->Glu) of exon 2. PMID- 16671953 TI - The 1858T PTPN22 gene variant contributes to a genetic risk of type 1 diabetes in a Ukrainian population. AB - The 1858T variant of the protein tyrosine phosphatase gene, PTPN22, is associated with an increased risk of several autoimmune diseases. The aim of this study has been to investigate the possible association of 1858C-->T PTPN22 polymorphism and type 1 diabetes (T1D) in Caucasians from Ukraine. Overall, the distribution of 1858 PTPN22 genotypes differed significantly between the T1D patient group (n = 296) and the control group (n = 242) (P = 0.0036). When both groups were classified according to sex, the TT genotype and T allele showed a statistically significant higher frequency in T1D female patients (5.9 and 22.8%, respectively) in comparison with the female controls (0 and 11.9%) (P = 0.008 for both analyses). The patients with the TT genotype were significantly younger at the onset of T1D compared with those with genotypes TC and CC (P = 0.035 and 0.019, respectively). In our Ukrainian Caucasian cohort, we confirmed the association between T1D and the PTPN22,1858T allele. PMID- 16671954 TI - The PTPN22 1858T variant is not associated with primary biliary cirrhosis. AB - The minor allele of a single nucleotide polymorphism (SNP) in the PTPN22 gene (1858T) encoding the Lyp-tyrosine phosphatase has been recently associated with multiple autoimmune disorders, raising the possibility that this variant may also represent a risk allele for primary biliary cirrhosis (PBC). We therefore investigated the possible association of the PTPN22(1858T) variant with PBC in a Canadian population. We studied 160 Caucasian patients with biopsy and antimitochondrial antibodies (AMA)-proven PBC who were genotyped for the PTPN22(C1858T) SNP using a single-base primer extension assay and mass spectrometry. The frequency of the PTPN22(1858T) allele was then compared between the patients and 290 healthy controls. No association was detected between the PTPN22(1858T) allele and PBC, the frequency of this variant being similar in patients with PBC (7.5%) and controls (8.4%). Restricting the analysis to patients with PBC with any second autoimmune condition or specifically with sicca syndrome or autoimmune thyroid disease also revealed no association with this variant. Thus the PTPN22(1858T) variant is not associated with PBC or with the combination of PBC and a second autoimmune disease. These data suggest that this variant does not confer risk for PBC and does not account for the frequent presence of other autoimmune diseases in patients with PBC. PMID- 16671955 TI - Nomenclature for factors of the HLA system, update January 2006. PMID- 16671956 TI - Nomenclature for factors of the HLA system, update February 2006. PMID- 16671957 TI - Benzodiazepines prescription in Dakar: a study about prescribing habits and knowledge in general practitioners, neurologists and psychiatrists. AB - Benzodiazepines are relatively well-tolerated medicines but can induce serious problems of addiction and that is why their use is regulated. However, in developing countries like Senegal, these products are used without clear indications on their prescription, their dispensation or their use. This work focuses on the prescription of these medicines with a view to make recommendations for their rational use. Benzodiazepine prescription was studied with psychiatrists or neurologists and generalists in 2003. Specialist doctors work in two Dakar university hospitals and generalists in the 11 health centres in Dakar. We did a survey by direct interview with 29 of 35 specialists and 23 of 25 generalists. All doctors were interviewed in their office. The questionnaire focused on benzodiazepine indications, their pharmacological properties, benzodiazepines prescribed in first intention against a given disease and the level of training in benzodiazepines by doctors. Comparisons between specialists and generalists were made by chi-square test. Benzodiazepines were essentially used for anxiety, insomnia and epilepsy. With these diseases, the most benzodiazepines prescribed are prazepam against anxiety and insomnia and diazepam against epilepsy. About 10% of doctors do not know that there is a limitation for the period of benzodiazepine use. The principal reasons of drugs choice are knowledge of the drugs, habit and low side effects of drugs. All generalists (100%) said that their training on benzodiazepines is poor vs. 62.1% of specialists, and doctors suggest seminars, journals adhesions and conferences to complete their training in this field. There are not many differences between specialists and generalists except the fact that specialists prefer prazepam in first intention in the insomnia treatment where generalists choose bromazepam. In addition, our survey showed that specialists' training in benzodiazepines is better than that of generalists. Overall, benzodiazepine prescription poses problems particularly in training, and national authorities must take urgent measures for rational use of these drugs. PMID- 16671958 TI - Effect of acute inhibition of nitric oxide synthesis by L-NAME on cardiovascular responses following peripheral autonomic blockade in rabbits. AB - The pressor and chronotropic responses to acute inhibition of nitric oxide synthase enzyme by N(G)-nitro-L-arginine methyl ester (L-NAME) were studied in anaesthetized rabbits with intact autonomic nervous system (ANS) activity. Also, they were investigated when administration of L-NAME was preceded by peripheral autonomic blockade. Autonomic blockade had different forms: ganglionic (hexamethonium-induced), post-ganglionic beta-adrenergic blockade (propranolol induced), parasympathetic blockade (atropine induced), and complete autonomic blockade by coadministration of hexamethonium and atropine simultaneously. L-NAME injected intravenously (10 mg/kg) in animals with intact and blocked autonomic activity induced a pressor response. This pressor response was accompanied by bradycardia in rabbits with either intact autonomic activity or hexamethonium induced ganglionic blockade. L-NAME exerted no effect on heart rate in animals with beta-adrenergic blockade or parasympathetic blockade. In rabbits with complete autonomic blockade, L-NAME evoked tachycardia. These experiments indicate that L-NAME-induced hypertension is not relying only on ANS. Also, L NAME-induced tachycardia in rabbits treated with atropine plus hexamethonium suggests other humoral mechanisms that may be involved in the L-NAME induced chronotropic response. PMID- 16671959 TI - Modulation of pentylenetetrazole-induced seizures and oxidative stress parameters by sodium valproate in the absence and presence of N-acetylcysteine. AB - In view of a role of oxidative stress in epilepsy and the evidence for the involvement of peroxidative injury in sodium valproate (SVP)-induced adverse effects on liver and kidneys, we investigated whether the combination of SVP with N-acetylcysteine (NAC), an antioxidant, may help us to achieve maximal efficacy in terms of seizure control, with minimal toxicity on liver and kidneys. Pentylenetetrazole (PTZ)-induced seizures were used to evaluate the anticonvulsant effect of drugs. Biochemical estimations included the determination of oxidative stress markers like thiobarbituric acid-reactive substances in brain tissue and glutathione (GSH) levels in liver and kidney tissues. Aspartate aminotransferase and alanine aminotransferase concentrations in the serum were also determined to assess liver function. In our study, NAC exhibited a nondose-dependent anticonvulsant effect. The concurrent administration of NAC with SVP significantly prolonged the latency to jerks, myoclonus and clonic generalized seizures. No significant oxidative stress was evident in brain tissue following PTZ-induced seizures, though an elevation of serum transaminase enzymes was seen. SVP at the dose studied did not produce any significant oxidative stress on the liver and kidneys, while treatment with NAC elevated liver and kidney GSH levels. The concurrent administration of NAC with SVP had beneficial effects on liver and kidney cells. PMID- 16671960 TI - Effect of rofecoxib, a cyclo-oxygenase-2 inhibitor, on various biochemical parameters of brain associated with pentylenetetrazol-induced chemical kindling in mice. AB - Cyclo-oxygenase (COX) has been reported to play a significant role in neurodegeneration and other brain-related disorders. Recent studies have reported that COX plays a significant role in the pathophysiology of brain-related disorders and COX-2 inhibitors could be useful drug therapy in neurodegenerative disorders. The aim of the present study was to explore the possible role of COX and the effect of COX-2 inhibitor, rofecoxib in epilepsy. In the present study, kindling was induced in mice by chronic administration of a subconvulsive dose of pentylenetetrazol (PTZ, 40 mg/kg, i.p.) on every other day for a period of 15 days. Rofecoxib was administered orally daily 45 min before either PTZ or vehicle. The kindling score was recorded after PTZ administration. Seizure severity was measured according to a prevalidated scoring scale. Biochemical estimations were performed on the day 16 of PTZ treatment (24 h after the last dose of PTZ). Chronic treatment with selective COX-2 inhibitor, rofecoxib (2.0 and 5.0 mg/kg, p.o.) for 15 days showed significant decrease in PTZ-induced kindling score. Biochemical analysis showed that chronic treatment with PTZ significantly increased lipid peroxidation, nitrite levels (NO levels), and myeloperoxidase levels and decreased the reduced glutathione levels in brain homogenate. Chronic treatment with rofecoxib, a selective COX-2 inhibitor, significantly reversed the PTZ-induced kindling score as well as various biochemical alterations suggesting the use of COX-2 inhibitor rofecoxib in epilepsy. In conclusion, results of the present study suggested that COX-2 plays an important role in the pathophysiology of PTZ-induced kindling in mice and rofecoxib is protective against various biochemical alterations against PTZ induced kindling in mice. PMID- 16671961 TI - Modulation of gentamicin-induced renal dysfunction and injury by the phenolic extract of soybean (Glycine max). AB - Gentamicin (GM) is one of the most important of the aminoglycoside antibiotics used widely for the treatment of serious and life-threatening infections and whose clinical use is limited by its nephrotoxicity. As the pathogenesis of GM induced renal dysfunction and injury involves reactive oxygen species, the polyphenolic constituents of soybean with antioxidant property may protect against GM-induced renal toxicity. We therefore tested this hypothesis using phenolic extract of soybean (PESB) on GM-induced nephrotoxicity rat model. Administration of GM (80 mg/kg, s.c.) for 12 days to rats induced marked renal failure, characterized by a significantly increased plasma creatinine, urea and Na(+) ions levels, with K(+) depletion. This was also associated with decreases in the activity of the renal antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST)] measured and depletion of both blood and renal reduced glutathione (GSH) levels. The activities of membrane bound glucose-6-phosphatase (G6Pase) and 5(1)-nucleotidase (5(1)-NTD) enzymes as well as gamma-glutamyltransferase (gamma-GT) and aspartate aminotransferase (AST) (enzymes that are located in the proximal tubule) were decreased. Renal histology examination further confirmed the damage to the kidney as it reveals severe necrosis of the proximal renal tubules with deposition of colloid casts. These alterations were ameliorated in rats pretreated with PESB. The decrease in the activities of SOD, CAT, GST as well as GSH depletion observed in GM-treated rats was prevented in the rats pretreated with PESB. The activities of gamma-GT, AST and G6Pase were also increased in the kidney. These protective effects were dose dependent except for G6Pase activity and GSH levels that were preserved only at 500 mg/kg dose of PESB, and 5'-NTD activity that was dose dependently decreased. Furthermore, the extent of tubular damage induced by GM was reduced in rats that also received PESB. The lower dose (500 mg/kg) of the extract, however, appeared to provide better histological protection. These results suggest that the PESB has protective effects on GM-mediated nephropathy and this may be related to the action of the antioxidant polyphenolic content of the soybean. PMID- 16671962 TI - Characterization of beta-adrenoceptor antagonists as substrates and inhibitors of the drug transporter P-glycoprotein. AB - Transporter proteins such as P-glycoprotein are major determinants of intracellular drug concentrations. Moreover, inhibition or induction of transporters is an important mechanism underlying drug interactions in humans. However, very little is known whether beta-adrenoceptor antagonists are substrates and/or inhibitors of P-glycoprotein. Therefore, we investigated the P glycoprotein-mediated transport of propranolol, metoprolol, bisoprolol, carvedilol and sotalol in P-glycoprotein-expressing Caco-2 monolayers and inhibition of P-glycoprotein-mediated digoxin transport by the beta-adrenoceptor antagonists. A significant inhibition of polarized, basal to apical drug transport by the P-glycoprotein inhibitor PSC-833 was observed for bisoprolol (0.5 and 5 microm) and carvedilol (0.5 microm). Moreover, propranolol and carvedilol inhibited P-glycoprotein-mediated digoxin transport with IC(50) values of 24.8 and 0.16 microm, respectively, whereas metoprolol and sotalol had no effect. Bisoprolol significantly inhibited directional digoxin transport at 50 and 250 microm by 31% and 44%, respectively. Taken together, P-glycoprotein is likely to be one determinant of bisoprolol and carvedilol disposition in humans. In addition, the beta-adrenoceptor antagonists propranolol and carvedilol significantly inhibit P-glycoprotein function thereby possibly contributing to drug interactions in humans (e.g. digoxin-carvedilol and cyclosporine carvedilol). PMID- 16671963 TI - Protective role of caffeic acid phenethyl ester in the liver of rats exposed to cold stress. AB - Cold exposure can induce a form of environmental stress. Cold stress (CS) alters homeostasis, results in the creation of reactive oxygen species and leads to alterations in the antioxidant defense system. The caffeic acid phenethyl ester (CAPE), an active component of propolis, has an antioxidant capacity. We investigated the effect of CS on oxidative stress and antioxidant defense system and the possible protective effect of CAPE in rat liver tissue. Twenty-four female Wistar Albino rats were divided into four groups: Control, CAPE-treated, CS, and CAPE-treated CS (CS + CAPE) group. Catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities and total glutathione (GSH) and malondialdehyde (MDA) levels were measured. In addition, histological changes in liver tissue were examined by light microscopy. SOD, CAT and GSH-Px activities and total GSH level were significantly declined in the CS group. In the CS + CAPE group, the activities of these three enzymes and GSH level significantly raised with regard to the CS group. MDA levels increased in the CS group and decreased in the CS + CAPE group. The tissues of the CS group showed some histopathological changes such as necrosis, hepatocyte degeneration, sinusoidal dilatation, hemorrhage and vascular congestion and dilatation. In the CS + CAPE group, the histopathological evidence of hepatic damage was markedly reduced. Histological parameters were consistent with biochemical parameters. In this study, CS increased oxidative stress in liver tissue. CAPE regulated antioxidant enzymes, inhibited lipid peroxidation and reduced hepatic damage. PMID- 16671964 TI - Cisatracurium: myographical and electrophysiological studies in the isolated rat muscle. AB - Myographical and electrophysiological studies of cisatracurium were performed, in vitro, in the isolated sciatic nerve-extensor digitorum longus muscle preparation of the rat. Indirect twitches were generated at 0.1 Hz and tetanic contractions at 50 Hz. endplate potentials (epps) were generated in trains of 50 Hz. The electrophysiological variables used in the analysis of the epps were: amplitude of the first epp in the train, average amplitude of the 30 degrees to the 59 degrees epp in the train (epps-plateau), tetanic rundown (percent loss in amplitude of epps-plateau relative to the first epp in the train), quantal size and quantal content. The myographical results showed that the inhibitory concentration 50% (IC(50)) of cisatracurium for the blockade of twitches (0.48 microm) is 12 times its IC(50) for the induction of tetanic fade (0.04 microm). The electrophysiological results showed a concentration dependent decrease in the amplitudes of first epps in the trains and of epps-plateau in the two used concentrations (0.13 microm and 0.38 microm). The tetanic rundown was intensified only in the presence of the higher (0.38 microm) concentration of cisatracurium. In cisatracurium 0.13 microm (a concentration which affects only tetanic contractions, inducing their fade, while leaving the twitch unaffected) there was a decrease in the quantal content of the first epp and of epps-plateau in the train. In cisatracurium (0.38 microm), a concentration, which affects the twitch, there was a decrease of the quantal size and of quantal content of epps-plateau, but not of the quantal content of the first epp in the train. The results indicate that the fade of the tetanic contraction induced by cisatracurium at the concentration of 0.13 microm is entirely because of a pre-synaptic blocking effect while the decrease in the twitch induced by cisatracurium at the concentration of 0.38 microm is due to a post-synaptic blocking effect. PMID- 16671965 TI - Pongamia pinnata modulates the oxidant-antioxidant imbalance in ammonium chloride induced hyperammonemic rats. AB - The effect of Pongamia pinnata (an indigenous plant used in Ayurvedic Medicine in India) leaf extract (PPEt) on circulatory lipid peroxidation and antioxidant status was evaluated in ammonium chloride-induced hyperammonemic rats. Enhanced lipid peroxidation in the circulation of ammonium chloride-treated rats was accompanied by a significant decrease in the levels of vitamin A, vitamin C, vitamin E, reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). PPEt-administered rats experienced a significant reduction in lipid peroxidation with a simultaneous elevation in antioxidant levels. Our results indicate that PPEt modulates these changes by reversing the oxidant-antioxidant imbalance during ammonium chloride-induced hyperammonemia and this could be due to its (i) antihyperammonemic effect by means of detoxifying excess ammonia, urea and creatinine and (ii) antioxidant property. The exact mechanism has to be still investigated and isolation of active constituents is required. PMID- 16671966 TI - Effect of inhaled corticosteroids on risk of development of cataract: a meta analysis. AB - The objective of this study was to quantify the risk of cataract among users of inhaled corticosteroids (ICS). Studies that examined the association of ICS use with risk of cataract were identified through computerized (MEDLINE, EMBASE), manual searches using Index Medicus, and checking cross-references to recover all published articles and scientific session abstracts. Pooled odds ratio (OR) with 95% confidence intervals (CI) were estimated using two methods, fixed effects Mantel-Haenszel model and random effects DerSimonian-Laird model. Four studies satisfied all the inclusion/exclusion criteria and were included in the analysis. The pooled OR (95% CI) by the Mantel-Haenszel method was 1.48 (1.39-1.57) and by the DerSimonian-Laird method was 1.48 (1.30-1.68). The test for heterogeneity was not significant. A total number of nine negative studies would be required to make the results of our meta-analysis non-significant. Number needed to harm is 16 with 95% CI of 13-19. Contrary to popular belief, inhaled ICS may be associated with systemic side effects like cataract as shown by this meta analysis. The risk of increased cataract shown in our analysis needs to be weighted against the benefits of ICS. Further evaluations are required to clarify the causal association between the dosage and duration of drug use. PMID- 16671967 TI - Clinical methods for the evaluation of endothelial function-- a focus on resistance arteries. AB - Endothelial dysfunction is a key event in the pathophysiology of cardiovascular diseases and appears as a strong independent predictor of cardiovascular events. In this context, biological evaluation of endothelial circulating markers can be helpful. However, functional tests using pharmacological stimuli appear more specific for the study of resistance arteries. These methods consist in the evaluation of the endothelium-dependent changes in regional vascular flow in response to local infusion of substances that act through endothelial receptors without modification of systemic arterial pressure and in comparison with a non endothelium-dependent relaxation. Flow is measured by Doppler and intravascular ultrasound in coronary circulation, laser Doppler in skin and by venous occlusion plethysmography in peripheral muscular arteries. Similar studies can be performed ex vivo using isolated resistance arteries obtained from fat subcutaneous biopsies. In addition, other information can be obtained from reactive hyperemia and the study of the flow-mediated dilatation of conduit arteries to enable a selective and comprehensive approach of the heterogeneity of endothelial function in pathophysiology. PMID- 16671968 TI - Use of an indirect effect model to describe the LDL cholesterol-lowering effect by statins in hypercholesterolaemic patients. AB - Statins are the most commonly prescribed agents for the treatment of hypercholesterolaemia. This is due to their efficacy in reducing low-density lipoprotein cholesterol (LDL) level which is the primary goal of the treatment especially for patients with multiple risk factors or with established coronary heart diseases. The purpose of this study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model that describes the LDL-lowering process in patients with hypercholesterolaemia treated with atorvastatin, fluvastatin or simvastatin. A total of 100 patients were studied retrospectively. They received atorvastatin (n = 57), fluvastatin (n = 26) or simvastatin (n = 17). As no pharmacokinetic data were available, the absorption rate was fixed to 1/h and atorvastatin, simvastatin and fluvastatin elimination half-lives were fixed to 14, 2 and 2.5 h respectively. A total of 309 LDL levels were measured and the data were analysed by nonmem v. The time course of the LDL-lowering effect of statins was described by an indirect-response model with precursor (LDL synthesis, input rate K(in)) and response (circulating LDL, input and output rates K) compartments. The following parameters were estimated: LDL input rate (K(in)) 0.14 +/- 0.015 g/L/day (mean +/- SD); inhibition fraction of K(in) (INH) 0.21 +/- 0.017; and dose producing 50% increase of LDL removal (D50), 26 +/- 7.8, 1.3 +/- 0.48 and 15 +/- 5.25 mg for atorvastatin, simvastatin and fluvastatin, respectively. Gender, bodyweight, age, calories/day, sugar/day, lipids/day, hyperlipidaemia types and waist/hip circumference, renal and hepatic functions had no effect on the pharmacodynamic parameters. The pharmacodynamic parameters for the three statins were accurately estimated. The PK/PD model developed successfully predicted the time course of the LDL-lowering effect of statins. PMID- 16671970 TI - The experience of surviving traumatic brain injury. AB - AIM: This paper reports a study describing the experience of surviving traumatic brain injury as narrated by individuals 1 year after injury. BACKGROUND: The experience of surviving traumatic brain injury is an individual and invisible experience. Healing and resolution of grief is still problematic for many years after injury. This experience includes those with severe injury. There is limited literature focusing on the survivor. METHODOLOGY: Sixty people with hospital admission diagnoses of traumatic brain injury were interviewed 1 year after injury using the Extended Glasgow Outcome Score questionnaire. Survivors then described their experiences of recovery. Data were collected through unstructured in-depth interviews, transcribed from audio-tapes and analysed into references. Qualitative content analysis was used to compile their frequencies from recurring themes. FINDINGS: Sixty survivors with a median age of 35 years were enrolled in the study, with 100% follow-up. Twelve per cent returned to full preinjury functioning, 35% had residual psychosocial and physical sequelae, 38% had significant restrictions in lifestyle and work capacity, and 15% were unable to care for themselves throughout the 24-hour period. The common narratives were classified into five categories: 'regret and grief within self'; 'insensitivity of health professionals'; 'invisibility of self'; 'stranded self'; and 'recovery in self'. These self-narratives reflected renewed ways to view the self, which were conceptualized to be intact 'in spite of' or to be worthwhile 'because of' the traumatic brain injury. CONCLUSIONS: Self-narratives could be a worthwhile focus of psychological intervention by substituting positive narratives for negative ones, demonstrating hope and a positive outlook on life in order to enhance self-reflection and improve mental health. They can assist people to minimize, avoid or overcome devaluation and, as such, provide a basis for considerable nursing rehabilitation practice, even in those with severe injury. PMID- 16671971 TI - Conceptions of pain among Somali women. AB - AIM: This paper reports a study of a group of Somali mothers' views on pain, the causes of pain, pain behaviour and pain treatment concerning themselves and their family members. BACKGROUND: Both the meaning of pain and pain behaviour are associated with values in a patient's social and cultural context. Culture also provides models for how to treat and relieve pain. Several studies report a lack of cultural sensitivity and competence among healthcare professionals, resulting in lower quality of and less access to health care and pain treatment for minority groups. However, the majority of the scientific literature on pain and culture concerns adults in the United States of America. METHOD: Focused conversational interviews were carried out with a convenience sample of nine Somalia women living in Sweden in order to describe and explore their conceptions of pain. Qualitative content analysis was conducted through meaning condensation. The data were collected in 2002-2003. FINDINGS: The women expressed a number of different ideas about definitions and causes of pain. Somalis, especially men, are expected to be stoic about pain. The women had different strategies for communicating about and relieving pain. Children from the ages of 6-8 years upwards were expected to control their pain expression. Respondents used both formal and informal care to relieve pain. For some of the women, consulting a psychologist was not a culturally acceptable way of seeking pain relief. CONCLUSIONS: Nurses must strive for increased cultural competence and explore ways to make healthcare services sensitive to culturally diverse groups. Nurses have an educational role in educating parents and children about pain and the importance of sufficient pain relief. All healthcare providers should be aware of their own cultural values and the risk of stereotyping people. PMID- 16671972 TI - Effectiveness of nurse-led brief alcohol intervention: a cluster randomized controlled trial. AB - AIM: This paper reports an evaluation of the effectiveness and cost-effectiveness of nurse-led screening and brief intervention in reducing excessive alcohol consumption among patients in primary health care. BACKGROUND: Excessive alcohol consumption is a major source of social, economic and health problems. However, such consumption is responsive to brief alcohol intervention. To date, brief intervention research in primary health care has focused on general practitioner led interventions, and there is only circumstantial evidence of effectiveness in nurse-led interventions. However, nurses are increasingly taking a lead in health promotion work in primary care. METHODS: A pragmatic cluster-randomized controlled trial was carried out between August 2000 and June 2003 to evaluate the effects of a brief intervention compared with standard advice (control condition). A total of 40 general practice clusters (intervention = 21 and control = 19) recruited 127 patients (intervention = 67 and control = 60) to the trial. Excessive consumption was identified opportunistically via the Alcohol Use Disorders Identification Test. After baseline assessment, patients received either a 5-10 minutes brief intervention using the 'Drink-Less' protocol or standard advice (control condition). Follow-up occurred at 6 and 12 months postintervention. RESULTS: Analysis of variance weighted for cluster size revealed no statistically significant differences between intervention and control patients at follow up. A majority of patients in both conditions reduced their alcohol consumption between assessment and subsequent measurement. Economic analysis suggested that the brief intervention led to no statistically significant changes in subsequent health service resource use relative to standard treatment. CONCLUSION: The brief intervention evaluated in this trial had no effect over standard advice delivered by nurses in primary health care. However, there was a reduction in excessive drinking across both arms of the trial over time. Due to nurse drop-out, this trial was significantly underpowered. Future research should explore barriers to nurses' involvement in research trials, particularly with an alcohol focus. A larger trial is required to evaluate the effectiveness of nurse-led screening and brief alcohol intervention in primary care. PMID- 16671973 TI - Protecting sick children from exposure to passive smoking through mothers' actions: a randomized controlled trial of a nursing intervention. AB - AIMS: The aim of this study was to evaluate the effectiveness of a nursing educational intervention with mothers of sick children to decrease passive smoking exposure. BACKGROUND: Passive smoking represents a serious health hazard and is a substantial threat to child health causing major risk factors for acute respiratory illness in children. Nurses are in a vital position to conduct health education to improve children's health, which is a legitimate activity in a pediatric ward. METHODS: A randomized controlled trial was conducted in the general paediatric wards of four major hospitals in Hong Kong. The participants were non-smoking mothers of sick children admitted to the paediatric ward and with smoking husbands living in the same household. FINDINGS: A total of 1483 women were randomized into the intervention (n = 752) and control (n = 731) group. The intervention group received from the nurses (1) standardized health advice; (2) two purpose-designed booklets about preventing exposure to passive smoking and helping fathers quit; (3) a no smoking sticker; and (4) a telephone reminder 1 week later. No intervention was given to the controls. Baseline comparison showed no significant differences between the two groups in the mothers' actions to protect the children from passive smoking exposure. More mothers in the intervention group than the control group had always moved the children away when they were exposed to the fathers' smoke at home at 3-month follow up (78.4% vs. 71.1%; P = 0.01) but became non-significant at 6 and 12 months. CONCLUSIONS: A simple health education intervention provided by nurses to the mothers in a busy clinical setting can be effective in the short-term to motivate the mothers to take actions to protect the children from exposure to passive smoking produced by the fathers. PMID- 16671974 TI - Being reborn: the recovery process of postpartum depression in Taiwanese women. AB - AIM: This paper reports a qualitative study describing the process of recovery from postpartum depression in Taiwanese women. METHODS: We carried out a study using grounded theory with a purposive sample of 23 postnatally depressed women in 2001-2002. The Beck Depression Inventory was used to screen for severe depression. Women with scores higher than 16 at 6 weeks after childbirth were categorized as having postpartum depression. Only two out of the 23 mothers were being treated for depression at the time. The data were analysed by constant comparative method. FINDINGS: 'Being reborn' was the core concept that emerged from the data on the mothers' experience of going through postnatal depression, which was a process of descent into near-death insanity and eventual rebirth. Such postnatally depressed mothers often underwent four stages of coping with the loss of self or loss of former identity and attachment to their new lives as mothers. The four stages were: (1) shattered role identity, (2) feeling trapped and breaking down, (3) struggling for self-integrity and (4) regaining vitality. CONCLUSION: The experience of postpartum depression should be examined within the social and cultural contexts in which it occurs. These Taiwanese data can be used to develop culturally-sensitive health care. The nursing role is primarily that of reflective listening to help the women adjust to the process of being reborn- an internal process of painful growth represented by motherhood and striving to protect the real self in order to maintain emotional health while negotiating a developmental transition. Preventive interventions might include providing guidance for parenting, counselling of individual mothers, and facilitating the development of support groups. PMID- 16671975 TI - Functioning and subjective health among stroke survivors after discharge from hospital. AB - AIM: This paper reports a study exploring functioning (functions, activities and participation) and subjective health among stroke survivors 6 months after discharge from hospital. A further aim was to investigate patterns of functions and activities, and associations between functioning and subjective health. BACKGROUND: Nurses tend to emphasize functional and activity outcomes and rarely look to people's participation in their social world. By integrating individual and social contextual factors in their assessments, nurses will have a powerful tool to broaden their outlook. The International Classification of Functioning, Disability and Health can be used as a framework for understanding the consequences of stroke. Studies are needed, however to make the International Classification of Functioning operational for practical use. METHODS: A cross sectional and explorative design was used using structured interviews and assessments with a number of scales with 89 stroke survivors 6 months after their hospital stay. The measures used were Personal Activities of Daily Living, including cognitive ability, transfer, dressing, bathing, energy to eat, ingestion, swallowing, going to the toilet, faecal continence, and urinary continence; and Instrumental Activities of Daily Living, including washing clothes, cooking, cleaning, transport and shopping. Factor analysis was performed for functions and activities and the final solution had an explained variance of 70%. The data were collected during autumn 2003 and spring 2004. FINDINGS: Close associations (r(s) > 0.5) were found between Activities of Daily Living in relation to participation and subjective physical health. CONCLUSION: Existing Activities of Daily Living scales were expanded and found to relate to a participation scale and a subjective health scale. Using these scales in nursing care has the potential to correct current bias towards functions and activities by broadening the focus to include the social as well as the physical. PMID- 16671976 TI - Relational conversation as method for creating partnerships: pilot study. AB - AIM: This paper reports a pilot study testing the intervention, Self-Care TALK, whose aim was to describe communication skills used by an advanced practice nurse to create partnerships with caregivers. The communication process and examples of skills used in creating partnerships are described. BACKGROUND: Decades of exploring nurse-client relationships provide a knowledge base for describing a structure and process for building partnerships. Self-Care TALK functions as a vehicle for an education and support partnership. Self-Care TALK, a theory derived intervention, is based on the Self-Care for Health Promotion in Aging model. Implementation of the intervention was evaluated in a pilot study in 2003, with older spouse caregivers of persons with early dementia. METHODS: Pilot testing of Self-Care TALK followed Lichstein's criteria of tracking three phases of intervention: delivery, receipt and enactment. Henson's communication elements for establishing partnership relationships provided a guide for advanced practice nurse communication. Self-Care TALK was delivered during six phone conversations with caregivers for a total of 36 phone conversations. All phone conversations were audiotape recorded. Field notes were taken during each conversation. Data analysis followed a qualitative descriptive process of comparative analysis in which communication skills were compared across conversations. Categories of skills were named based on communication elements that dominated the category. Communication skills were compared to literature addressing communication and nurse-client partnerships. FINDINGS: Communication examples described the structure for interaction (Self-Care TALK protocol), process of interaction (relational conversation) and caregiver identified outcomes (intentions to enact self-care). CONCLUSION: Conversations to create partnerships depended on one theme: language that supported relational conversation. Four communication skills illustrated the how of relational conversation: listening with intent, affirming emotions, creating relational images, and planning enactment. PMID- 16671977 TI - Nutritional screening and assessment tools for older adults: literature review. AB - AIM: This paper reports a literature review to examine the range of published tools available for use by nurses to screen or assess nutritional status of older adults, and the extent to which validity, reliability, sensitivity, specificity and acceptability of the tools has been addressed. BACKGROUND: The incidence of malnutrition in older adults is high. One method by which malnutrition or risk of malnutrition can be detected is by the use of nutritional screening or assessment tools. METHODS: A comprehensive literature review methodology was employed. A variety of electronic databases were searched for the period 1982-2002. Search terms incorporating nutrition, screening, validity, reliability and sensitivity and specificity were combined to retrieve relevant literature. In addition, manual searches were conducted and articles retrieved from those listed in key papers. In this paper, nutritional screening or assessment tools are described as tools which use a questionnaire-type format containing more than one risk factor for malnutrition, and give a quantitative or categorical assessment of risk. RESULTS: Seventy-one nutritional tools were located, 21 of which were identified as designated for use with an older population. A wide variety of risk factors for malnutrition are used with the tools, ranging from objective measurements to subjective assessment. Some tools identify an action plan based on the score obtained. Many tools appear not to have been subjected to validity and/or reliability testing but are used clinically. CONCLUSIONS: As malnutrition is present in the older adult population, nutritional assessment and screening tools can be useful to highlight those in need of a nutritional care plan. However, many have not been subjected to evaluation and consequently may not demonstrate sensitivity and/or specificity in clinical use. The decision to use a particular tool should therefore be considered carefully. PMID- 16671978 TI - Effectiveness of acupuncture for migraine: critical literature review. AB - AIM: The aim of this paper is to evaluate previous research studies on acupuncture for migraine with reference to the Standards for Reporting Interventions in Controlled Trials of Acupuncture guidelines. BACKGROUND: It is estimated that around 2-15% of the world's population are affected by migraine headaches. Thirteen per cent of adults in the United Kingdom suffer with chronic pain, migraine headaches accounting for 7% of cases. Migraine pain relief is grounded in pharmacology. Acupuncture for migraine has been widely researched. However, inconsistent and low quality results have been produced. Recently, published Standards for Reporting Interventions in Controlled Randomized Trials of Acupuncture guidelines recommend important information that must be included in research in order to be valid and reliable. METHODS: Searches were conducted between September 2003 and May 2004 using the Ovid Medline 1966-2004, British Medical Journal, Blackwell Synergy, Science Direct, The Lancet and Cochrane Library Issue 1 databases. Searches were limited to the previous 20 years and to publications in the English language only. FINDINGS: Thirteen randomized controlled trials met the inclusion criteria and were critically reviewed for methodological quality, reporting of acupuncture needling details, practitioner background, control interventions and use of a diagnostic criterion. Findings agreed with previous literature reviews that the majority of studies of acupuncture for migraine research are of poor quality, with conflicting results. Few studies met the criteria of the Standards for Reporting Interventions in Controlled Trials of Acupuncture recommendations. Overall, the quality of research in this area must be questioned. CONCLUSIONS: In the light of these findings, practitioners may face a dilemma when considering the use of acupuncture for migraine. Therefore, large, high quality randomized controlled trials of acupuncture for migraine are needed. Until better quality research is published, with verification of the benefits of acupuncture for migraine, provision of this alternative therapy should not be expanded or withdrawn. PMID- 16671979 TI - Is the humanistic view of the nurse role still alive--in spite of an academic education? AB - AIM: This paper is a report of a study exploring what 'being a good nurse' means by following nursing students during their pre-registration education and for some years after graduation. BACKGROUND: There have been few studies focusing on the nurse role in the transition from student to experienced nurse. Studies with a qualitative design, in particular, are limited in number. METHODS: A longitudinal survey design was adopted, using an open-ended questionnaire at the beginning of education (n = 164), just before graduation (n = 123), and 3-5 years after graduation (n = 77). The participants were students in the nursing programme at a Swedish university. The data were collected during the period 1993 2002. Latent and manifest content analyses were used. FINDINGS: Four categories were identified in the data. 'To do good for others', with the sub-category 'to care for others', was most frequent over time and quite stable. The category 'to be competent and skilled' was frequent and increased over time. 'To have professional courage and pride' and 'to seek professional development' were mentioned to lesser extent and showed a slight increase over time. The meaning of being a good nurse grew in complexity over time and informants' professional awareness seemed to increase, especially concerning 'to be competent and skilled'. CONCLUSION: Attention needs to be paid both to nursing education and practice. Clinical supervision given by nurses with Master's degrees is suggested in order to convey positive attitudes towards nursing development and research into practice. Further studies are needed to compare what 'a good nurse' means to graduate nurses and how they actually behave when performing good nursing care. PMID- 16671980 TI - Factors influencing use of home care and nursing homes. AB - AIM: This paper reports a study comparing the characteristics of patients who use home care services and those who are cared for in nursing homes, and identifying the factors that influence the use of these care settings. BACKGROUND: The increase in the functionally dependent older population has led to an increase in the number of nursing homes and home care agencies. It has become clear that, rather than disputing which is the better of these options, it would be better to determine the characteristics of patients who use the two long-term care services. Gaining an understanding of the unique characteristics of patients who are cared for by home care agencies and those who are cared for in nursing homes will be imperative for reforming and developing long-term care systems. METHOD: The research model was based on the Anderson Model of Health Services Utilization. Interviews were conducted with 99 stroke survivors from two home care agencies and four nursing homes, and their family members, between May and December 2001. RESULTS: The patient characteristics that predicted greater use of home care rather than nursing home services were: being married, poor physical function, impaired cognitive function, higher rates of comorbidity, various medical complications, and/or number of catheters (e.g. urinary catheter, naso gastric tube). CONCLUSION: Contrary to the findings of previous studies conducted in countries with ageing populations, our findings indicate that in South Korea home care agencies, rather than nursing homes, provide care for severely impaired patients. This may be due to differences between countries in their long-term care systems and cultural attitudes toward end-of-life care. Our results will contribute to the development or reformation of long-term care systems in countries with ageing populations, and to the development of strategies for increasing access to these services. PMID- 16671981 TI - The safety of immunization delivery improves, but challenges remain. PMID- 16671984 TI - Clinical factors affecting the outcome of occlusal splint therapy of temporomandibular joint disorders. AB - The purpose of this study was to test the hypothesis that (1) the presence of a clinical temporomandibular joint-related disorder has effects on short-term changes in temporomandibular joint pain, and that (2) clinical variables of time since pain onset predict treatment outcomes of occlusal splint therapy. The study comprised 76 patients with unilateral temporomandibular joint pain. The clinical disorder subgroup included 47 patients with a clinical pain side-related diagnosis of internal derangement type I (n = 16), internal derangement type III (n = 19), and degenerative joint disease (n = 12). The clinical non-disorder subgroup consisted of 29 patients without a temporomandibular joint disorder. A logistic regression analysis was used to compute the odds ratio for the clinical variables of time since pain onset, adjusted for age, gender, pretreatment pain level, and clinical subgroup. For the temporomandibular joint pain measurements there was no significant 'session'/'clinical subgroup' interaction (P = 0.470). Significant increase in benefit of a successful outcome of 'pain reduction >70%' occurred with a time since pain onset of 2 years might belong to the unsuccessful treatment group of 'pain reduction <30%' was strong (6.0) and significant (P = 0.026). Diagnosis of temporomandibular joint disorder proved not to be linked to changes in therapeutic outcome measures of temporomandibular joint pain. Time since pain onset was an important prognostic determinant of successful occlusal splint therapy. PMID- 16671985 TI - Effect of reclining and chin-tuck position on the coordination between respiration and swallowing. AB - Chin-tuck position and reclining posture have been used in dysphagia patients to prevent aspiration during swallowing. However, both behavioural treatments may affect respiratory function. This study was carried out to test the hypothesis that if chin-tuck posture and body reclining affected respiratory function, this would be associated with altered coordination between respiration and swallowing. To investigate this hypothesis, respiratory parameters and manometry were used in each of four combinations of reclining posture and chin-tuck position. In the 60 degrees reclining with 60 degrees chin-tuck position, duration of swallowing apnea (0.89 s.d. 0.17 s) and submental electromyography burst (2.34 s.d. 0.84 s) were significantly longer when compared to both upright sitting and 30 degrees reclining positions. We conclude that 60 degrees reclining from vertical with 60 degrees chin-tuck may affect oral processing stages which delay and reduce a variety of oropharyngeal movements. These in turn significantly influence the coordination between respiration and swallowing. PMID- 16671986 TI - Effect of adding impression material to mandibular denture space in Piezography. AB - The purpose of the study was to examine the effect of adding impression material on denture space using a piezographical record. Subjects were ten voluntary edentulous patients, aged from 61 to 84 years old. A maxillary trial denture with anterior artificial teeth and a mandibular base plate with a keel were inserted into the oral cavity. Three ml of tissue-conditioning materials was injected on the base plate for each trial. Afterwards, the patients were instructed to pronounce various phonemes, so that tongue, cheeks and lips conformed to the denture space. The impression complexes were cut at the level of the estimated occlusal plane. Occlusal analogues were made by duplicating the impression complexes. Measurements were performed for five analogues from the first to fifth additions for each subject. The data were compared using analysis of variance (ANOVA), and a Friedman's test followed by a Bonferroni test for multiple comparisons with a level of significance at 5%. At the molar and premolar positions, the bucco-lingual widths of the occlusal table increased significantly at incremental injection of impression materials from P1 to P4. The midpoints of the analogues were located at a distance of 1.5 mm buccally at the molar position and at a distance of 1.9 mm buccally at the premolar position from the top of the alveolar crest, independent of the addition of impression material. It was concluded that denture space was regulated by volume of material and was located slightly on the buccal side from the crest of the residual alveolar ridge. PMID- 16671987 TI - Evaluation of transitional implant stabilized overdentures: a case series report. AB - Transitional implants (TI) can immediately improve the stability and retention of existing unstable mandibular complete dentures. This study evaluated the improvement of mandibular complete denture stability and retention with the use of TI. Three TIs were placed in the intraforaminal region of the edentulous mandible of seven patients (three men, four women; mean age 69.7 years). The patients' existing mandibular complete dentures were immediately modified to TI stabilized overdentures. Their masticatory movements (mandibular movements during mastication) were measured using a commercially available tracking device (BioPACK, Bioresearch, Japan), both before TI placement and about 1 month after delivery of the TI-stabilized overdentures. The mean time of each chewing phase (opening, closing, and occluding) and coefficients of variation were calculated. The patients also completed a questionnaire about the foods they could chew and indicated on a 100 mm Visual Analog Scale (VAS) their personal levels of comfort, ease of chewing, speech, and stability. For masticatory movements, there were no significant differences (P > 0.05) between the TI-stabilized overdentures and existing complete dentures. However, the number of foods that could be chewed increased, and the stability and comfort were reported as improved with the TI stabilized overdentures. Using TIs, the stability and comfort of the existing mandibular complete dentures studied in this report could be immediately improved. PMID- 16671988 TI - Deviation from the Broadrick occlusal curve following posterior tooth loss. AB - Production of an appropriate occlusal curve for dentitions which have become deranged because of tooth loss, overeruption, tipping and drifting can present challenges for the dental technician. An earlier paper (J Oral Rehabil, 2005 32: 895-900.) demonstrates that the use of the Broadrick flag method for producing the occlusal curve is relatively accurate for most intact arches. This study demonstrates that when a posterior tooth has remained unopposed for 5 years or more positional changes, which cause deviation from the Broadrick curve, occur. The extent of the deviation may be extreme, potentially leading to difficulties in restoring a harmonious occlusal scheme. This study also demonstrates that the Broadrick curve may provide an accurate reproduction of the occlusal curve, even when the tooth forming the posterior determinant of the curve is tipped. A moderate degree of tipping of this tooth has little effect on the radius of the Broadrick curve. PMID- 16671989 TI - Aesthetic effect of minor changes in incisor angulation: an internet evaluation. AB - The aim of this study was to evaluate the aesthetic assessment of different tiltings of the upper incisors with surrounding tissues. A picture of a smiling mouth revealing the upper anterior teeth from 15 to 25 was digitally transformed producing different tiltings of the incisors. These seven versions presented in a web-based survey were evaluated concerning their attractiveness. Overall 439 judges joined the evaluation. However, 249 judges were included in analysis, because only judges with normal wellbeings and completed questionnaires were accepted. These judges consisted of 38 dentists, 26 dental and 24 other students, 45 academics and 89 non-academics, while 27 gave no data about their profession. The results [median (first; third quartile)] showed significant that symmetrical incisors [67.5 (47.5; 85.0)] and minor changes in the angulation of one [65.0 (42.5; 80.0)] or both lateral incisors [65.0 (42.5; 80.0)], respectively, are aesthetically more attractive than the angulation of one [45.0 (27.5; 62.5)] or both central incisors [27.5 (16.3; 45.0)], or the combination of one tilted lateral and central incisor [37.5 (25.0; 60.0)]. These results confirm the results of a previous study where similar image versions without surrounding tissues were assessed concerning their attractiveness on printed photos. PMID- 16671991 TI - Nd:YAG laser penetration into cast titanium and gold alloy with different surface preparations. AB - This study investigated the effect of surface preparation on the Nd:YAG laser penetration into cast titanium and gold alloy. Cast blocks of each metal were given four different surface preparations: (i) coloured with black marker; (ii) air-abraded with 50 microm Al2O3; (iii) ground with SiC points and (iv) polished with 1 microm Al2O3 (mirror-polished). Two blocks with each of the surface preparations were abutted and laser-welded at their interface using the voltages of 210-260 V in increments of 10 V. After the welded blocks were mechanically separated, the laser penetration was measured using computer graphics. Regardless of the surface preparation, an increase in voltage increased the laser penetration for both metals. The laser penetration into titanium prepared with black marker and air-abrasion was significantly deeper than into the titanium ground with SiC points and mirror-polished. Although there were no statistical differences in penetration among the surface preparations for the gold alloy, the penetration in the mirror-polished specimens was shallower than any of the other preparation methods at higher voltages of 240-260 V. The results obtained in this study suggested that broken metal frameworks with finished surfaces should be painted with black marker or air-abraded before laser welding. PMID- 16671990 TI - In vivo and in vitro evaluation of marginal fit of class II ceromer inlays. AB - The aim of this study was to evaluate the marginal adaptation of class II ceromer (Targis, Vivadent) indirect inlay restorations under both in vivo and in vitro conditions. Twenty Targis inlays were produced for class II inlay cavities (13 mandibular and seven maxillary teeth) in 20 patients. The in vivo adaptation of the inlay to the tooth cavity was evaluated by means of silicone replica technique. For in vitro evaluation, 20 mesio-occluso-distal (MOD) Targis inlays were made in extracted upper molars. Half of the inlays were cemented with Variolink high-viscosity resin cement while the other half was cemented with Variolink Ultra. The replica specimens and in vitro samples were sectioned buccolingually and mesiodistally, and marginal adaptation was evaluated at both proximal and occlusal margins at 200x magnification under a light microscope. The data was analyzed with anova (P < 0.05). The in vivo mean film thickness values for occlusal and proximal locations were recorded as 73 and 132 microm respectively. In vitro mean marginal fit values were observed as 48 and 67 microm for occlusal and proximal margins of inlays luted with Variolink II high viscosity. The marginal fit values recorded under in vivo conditions were higher in magnitude than the measurements obtained under in vitro conditions. The use of a highly filled resin luting agent with an ultrasonic insertion technique did not cause an increase in marginal gap width of the inlay. PMID- 16671992 TI - The influence of electron beam irradiation on fibre-reinforced composite specimens. AB - This study investigated whether glass fibre-reinforced composite (FRC) specimens can benefit from post-curing using electron beam irradiation. Twenty-four frameworks of the Vectris and 24 of the Stick glass fibre-reinforced system were veneered with their correspondent veneer materials. Eight specimens of both systems were post-cured using electron beam irradiation (3 x 33 kGy, 10 MeV). The specimens were fixed in a restrained-end apparatus and inserted in an artificial mouth. With the exception of controls (n = 8 each) all other groups were thermally cycled and mechanically loaded (TCML). Finally, all samples were loaded to fracture using a universal testing machine. In two of eight non-irradiated Vectris/Targis specimens facing fracture occurred during TCML. Irradiation avoided these failures. No Stick/Sinfony facing fractured. However, Stick frameworks showed considerable torsions. Post-curing with electron beam irradiation made Stick frameworks stiffer. The fracture load of irradiated Stick/Sinfony specimens reached 520 +/- 31 N; control (without TCML and irradiation) 396 +/- 14 N, TCML-group without irradiation 362 +/- 41 N. Irradiated Vectris/Targis had a fracture resistance of 575 +/- 57 N; the control 556 +/- 36 N and the TCML group without irradiation 383 +/- 51 N. This investigation demonstrated that different types of FRC systems could considerably benefit from electron beam irradiation. The reconstructions became stiffer and resisted higher load. PMID- 16671993 TI - Reactionary dentinogenesis after applying restorative materials and bioactive dentin matrix molecules as liners in deep cavities prepared in nonhuman primate teeth. AB - OBJECTIVE: The aim of this in vivo study was to evaluate the response of the pulp dentin complex following application of resin-modified glass-ionomer cement, calcium hydroxide hard-setting cement and EDTA-soluble preparation of dentine matrix proteins (ESDP) in deep cavities prepared in non-human primate teeth. METHODS: Eighteen deep Class V buccal cavities were prepared in premolars of four capuccin monkeys. In Groups 1 and 2, the cavity floor was lined with ESDP or a resin-modified glass-ionomer cement (Vitrebond - 3M ESPE), respectively. In Group 3 (control), the cavity was lined with a hard setting calcium hydroxide cement (Dycal - Dentsply). The cavities were subsequently filled with amalgam. After 6 months, the animals were sacrificed and the teeth were prepared for microscopic assessment. Six-micron thick serial sections were stained with H/E, Masson's trichrome and Brown & Brenn techniques. RESULTS: No inflammatory pulpal response was observed for all experimental and control Groups. However, the amount of reactionary dentin deposition differed between groups in the rank order ESDP (Group 1) > calcium hydroxide (Group 3) > resin-modified glass-ionomer (Group 2). These differences were statistically significant. CONCLUSIONS: All materials were biocompatible when applied in deep cavities. ESDP stimulated higher deposition of reactionary dentin matrix than Vitrebond and Dycal. PMID- 16671994 TI - Genotoxicity evaluation of five different dentin bonding agents by chromosomal aberration analysis. AB - Dentin bonding agents became unavoidable in today's aesthetic restorative dentistry. Nevertheless, more and more evidences on their possible cytotoxicity and/or genotoxicity emerge. Still, only limited number of studies has been published on that issue. In our work we evaluated possible genotoxicity of five different adhesives: Adper Single Bond, Adper Single Bond 2 with nanofiller, Excite, OptiBond Solo Plus and Prompt L-pop. Genotoxicity assessment was carried out on human lymphocytes in vitro, using chromosomal aberration analysis. Polymerized adhesives were tested at three different dilutions of the 0.5 g mL( 1) eluate stock (2.5 x 1:10(6), 1:10(6) and 1:10(5)) after 1 h, 24 h and 5 days of elution. Slight but significant increase in the number of chromatid breaks was observed after 24-h elution period, for adhesives Adper Single Bond 2, Excite, and OptiBond Solo Plus at dilutions of 1:10(6) and 1:10(5), and for other two only at dilution of 1:10(5). First three adhesives also appeared to be slightly genotoxic after 1 h of elution but only at 1:10(5). As a bonding agent remains in close contact with living dental tissue over a long period of time, information on their possible genotoxicity and carcinogenicity should be more clearly clarified in the near future. PMID- 16671995 TI - Crypt dynamics and colorectal cancer: advances in mathematical modelling. AB - Mathematical modelling forms a key component of systems biology, offering insights that complement and stimulate experimental studies. In this review, we illustrate the role of theoretical models in elucidating the mechanisms involved in normal intestinal crypt dynamics and colorectal cancer. We discuss a range of modelling approaches, including models that describe cell proliferation, migration, differentiation, crypt fission, genetic instability, APC inactivation and tumour heterogeneity. We focus on the model assumptions, limitations and applications, rather than on the technical details. We also present a new stochastic model for stem-cell dynamics, which predicts that, on average, APC inactivation occurs more quickly in the stem-cell pool in the absence of symmetric cell division. This suggests that natural niche succession may protect stem cells against malignant transformation in the gut. Finally, we explain how we aim to gain further understanding of the crypt system and of colorectal carcinogenesis with the aid of multiscale models that cover all levels of organization from the molecular to the whole organ. PMID- 16671996 TI - Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes. AB - The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high affinity EGF receptors. PMID- 16671997 TI - Effect of transfection with a gene coding for the fibronectin FNIII/10 fragment upon contact inhibition of C3H10T1/2 fibroblasts. AB - The density-dependent growth inhibition of non-transformed cells may be associated with inefficient transduction of the proliferative signal from cell adhesion molecules. To verify this concept, the C3H10T1/2 fibroblasts were stably transfected with the gene coding for the fibronectin fragment III/10 (FNIII/10). This resulted in differences in gene's expression between original C3H10T1/2 cells and their FNIII/10 transfectants. No significant differences in growth properties were observed in the original or in the transfected cells. C3H10T1/2 cells and their transfectants, when co-cultured, displayed more cells at confluence than the cells cultured alone. Moreover, co-cultured C3H10T1/2 cells and their transfectants showed elevated levels of phospho-ERK1/2 compared to homogenous cultures. Results obtained indicate that cellular homogeneity is responsible for density-dependent growth inhibition. PMID- 16671998 TI - Spontaneous senescence in the MDA-MB-231 cell line. AB - Normal human somatic cells have a limited division potential when they grow in vitro. It is believed that shortening of telomeres, specialized structures at the ends of chromosomes, controls cell growth. When one telomere achieves a critical minimal length, the cell cycle control mechanism recognizes it as DNA damage and causes the cell's exit from the cycle in G1-phase. Because it is not possible to extend telomeres in normal cells, this non-dividing state is prolonged indefinitely, and is known as cellular senescence. The immortal cell line MDA-MB 231 has active telomerase, which prevents telomere shortening and allows cells' permanent divisions. However, there is a fraction of cells that do not divide over several days in culture as documented for some other tumour cell lines. Combination of methods has made it possible to isolate these non-growing cells and compare them with the fraction of fast-growing cells from the same culture. Although the non-growing fraction contains a significant percentage of typical senescent cells, both fractions have equal telomerase activity and telomere length. In this paper we discuss possible mechanisms that cause the appearance of this non-growing fraction of cells in cultures of MDA-MB-231, which indicate stress and genome instability rather than variation in telomerase activity or telomere shortening to affect individual cells. PMID- 16671999 TI - Primate embryonic stem cells create their own niche while differentiating in three-dimensional culture systems. AB - Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells. PMID- 16672000 TI - Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage. AB - Several methods to synchronize cultured cells in the cell cycle are based on temporary inhibition of DNA replication. Previously it has been reported that cells synchronized this way exhibited significant growth imbalance and unscheduled expression of cyclins A and B1. We have now observed that HL-60 cells exposed to inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea), at concentrations commonly used to synchronize cell populations, had histone H2AX phosphorylated on Ser-139. This modification of H2AX, a marker of DNA damage (induction of DNA double-strand breaks; DSBs), was most pronounced in S-phase cells, and led to their apoptosis. Thus, to a large extent, synchronization was caused by selective kill of DNA replicating cells through induction of replication stress. In fact, similar synchronization has been achieved by exposure of cells to the DNA topoisomerase I inhibitor camptothecin, a cytotoxic drug known to target S-phase cells. A large proportion of the surviving cells 'synchronized' by DNA replication inhibitors at the G1/S boundary had phosphorylated histone H2AX. Inhibitors of DNA replication, thus, not only selectively kill DNA replicating cells, induce growth imbalance and alter the machinery regulating progression through the cycle, but they also cause DNA damage involving formation of DSBs in the surviving ('synchronized') cells. The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication. PMID- 16672001 TI - Global Allergy and Asthma European Network (GA2LEN)--a focus for paediatric allergy research. PMID- 16672002 TI - Cytokine production, activation marker, and skin homing receptor in children with atopic dermatitis and bronchial asthma. AB - T cells are known to develop a critical role in the pathogenesis of atopic dermatitis (AD) and bronchial asthma. T cells involved in AD express the skin homing receptor CLA, but no lung homing receptor has been identified in bronchial asthma. We compared different cell markers and the cytokine production in T cells from children with AD or bronchial asthma. We studied the involvement of CLA+ and CLA- T-cell subpopulations in these diseases. We studied 20 children with acute AD lesions, 15 with mild persistent asthma, and 15 non-atopic controls. All patients were sensitized to house dust mite (DP) and evaluated during the acute phase. Total and specific IgE were measured by immunoassay and the expression of different cell markers and the cytokine production was analyzed by flow cytometry in peripheral blood mononuclear cells. Total IgE was significantly higher in AD children and IgE to DP in the asthmatic children. There was a significant increase in CD25+ CD4+ cells in asthmatic children and in HLA-DR+ CD4+ and HLA DR+ CD8+ cells in AD. In the CD4+ subsets, there was an increase in IL-13, IL-5 and TNF-alpha in AD compared to controls, a decrease in IFN-gamma in asthmatic children compared to controls, and an increase in IL-13, IL5, IL2, TNF-alpha, and IFN-gamma in the AD compared to asthmatic children. Changes in cytokine production were mainly detected in CLA+ cells in AD and in CLA- cells in asthma. Differences exist in total and specific IgE, activation markers, and cytokine patterns between AD children and children with asthma, with the former expressing a Th2 pattern whereas in asthmatic children we only detected a decrease in IFN gamma. Moreover, the subpopulations (CLA+ vs. CLA-) expressing these changes were different, indicating that the underlying mechanisms in the two diseases are not exactly the same. PMID- 16672003 TI - Cytokine levels in healthy and allergic mothers and their children during the first year of life. AB - To assess the regulatory changes of immune system in children genetically pre disposed to allergic diseases and in their mothers, we tested cytokines IL-4, IL 5, IL-6, IL-10, IL-13, IFN-gamma and TGF-beta in 21 healthy and 21 allergic mothers (serum at the time of delivery, colostrum and milk throughout the suckling period) and their children (cord blood, venous blood and stool filtrates) up to 1 yr of age. Samples were taken at the time of delivery, 4 days post-partum and then after 3, 6 and 12 months. Significant differences between the healthy and the allergic group were found in the levels of IL-4, IL-10, IL-13 and IFN-gamma. The levels of IL-4 in the allergic group were generally higher; the levels in the sera of children of allergic mothers during the post-natal life decreased, reaching levels typical for the healthy group at 1 yr of age. Allergic mothers exhibited markedly higher IL-10 levels in the serum at the time of delivery and in milk 3 months after delivery than healthy mothers while after 6 months the IL-10 levels in all samples from the allergic group were very low. Children from allergic group had lower intestinal content of IL-13 in comparison with the healthy counterparts. At 1 yr of age, the levels of IFN-gamma in sera and stool of children from the allergic group sharply increased. TGF-beta levels in the sera of both groups were high, while in the milk they were relatively low and substantially lower that in the children's stool. TGF-beta of mammary secretions is therefore unlikely to exert a decisive regulatory influence on the children's immunity. Long-term clinical monitoring of the children will be performed to evaluate the potential prognostic significance of these changes for the future development of allergies. PMID- 16672004 TI - Atopic wheezing and early life antibiotic exposure: a nested case-control study. AB - Several factors including early-life antibiotic usage have been implicated in the rising prevalence of allergic sensitization and asthma. A nested case-control study comparing antibiotic exposure of 37 sensitized children with recurrent wheeze (age 3-5 yr) and 37 non-sensitized children who had never wheezed was carried out within a population-based birth cohort (matching for age, sex, parental atopy, allergen exposure, and pet ownership). We collected data on antibiotic prescriptions during first 3 yr of life (timing, type, indication) from the primary care medical records. Significantly, more cases than controls received one or more antibiotic courses during the first year of life (92% vs. 70%, p = 0.04). The median time to first antibiotic course was shorter for the cases than the controls (6 vs. 8 months, p = 0.03). The total number of antibiotic receipts was greater amongst cases in each of the first 3 yr of life, but this reached significance only when the whole three-year period was considered (249 vs. 182 courses, p = 0.05). The increased ratio of antibiotic receipt in cases over controls was highest in the first year of life (1.32, 95% CI 0.99-1.78). Significantly more cases than controls were prescribed antibiotics for lower respiratory tract infection during the first 3 yr (p = 0.007), but not during the first year of life (p = 0.52). Antibiotics use by class was similar in the two groups. Our data support the hypothesis that early life exposure to broad spectrum antibiotics may have a causative role in sensitisation and the expression of wheeze. PMID- 16672005 TI - Daily ambulatory exhaled nitric oxide measurements in asthma. AB - Exhaled NO (FENO) is a non-invasive, validated marker for asthmatic airway inflammation. Recently, a new hand-held NO-analyzer has been developed which makes it possible to monitor FENO at home. We assessed feasibility and analyzed variability of daily FENO home measurements. Twenty-one asthmatics (mean age 14.5 yr; range 8-25 yr) participated. Nineteen used a stable dose of inhaled corticosteroids and all of them were in a stable clinical condition. FENO was measured twice daily for 14 consecutive days. Measurements and symptom scores were recorded on a smart card in the analyzer. Symptom score items included well being, wheeze, activity, and nocturnal symptoms. Measurements showed a success rate of 93%. We found a significant diurnal variation in FENO with geometric mean morning levels 14% higher than evening levels (95% CI: 4%-25%; p = 0.013). Individual subjects showed marked fluctuation of FENO. The mean intrasubject coefficient of variation of FENO was 40% for morning and 36% for evening values. FENO and cumulative symptom scores did not correlate. Home FENO measurements are feasible, and offer the possibility to asses airway inflammation on a daily basis. Further study is needed to interpret and evaluate possible benefits of FENO home monitoring. PMID- 16672006 TI - Increased glycosaminoglycans in the urine of asthmatic children on inhaled corticosteroids. AB - Increased extracellular matrix (ECM) deposition in the airway wall contributes to the airway remodeling in asthmatics. Glycosaminoglycans (GAGs) are polysaccharides attached to a protein core in order to form proteoglycans, a component of the ECM. In this study, we investigated the possible influence of long-term treatment with inhaled corticosteroids (ICS) on urinary GAGs levels of asthmatic children. Seventy asthmatic children (41 boys), aged 6.8-12.5 yr, participated in the study. About 44 were treated with inhaled budesonide via turbuhaler for 2-35 months (median 12 months) and 26 were on relief medications. About 30 healthy controls were also studied. GAGs were precipitated from early morning urine samples, collected, isolated and quantified using uronic acid carbazole reaction and expressed as uronic acid (UA) in microg/g/Cr(u)/m2. Urinary GAGs values did not differ significantly between controls and asthmatics but significant differences were found between children on ICS and asthmatics on relief medications (p < 0.001). There was a positive correlation between the daily dose of inhaled budesonide and the urinary GAGs values (r = 0.32, p = 0.037) whereas a threshold distinguishing 'low' vs. 'high' doses of ICS was found to be at 300 microg/m2 per day with a significant difference in urinary GAGs secretion (p = 0.006). Our data show that urinary GAGs secretion is reduced in asthmatic children that used only relief medication but it is increased in those on long-term treatment with ICS. A dose dependent effect of ICS was also detected. PMID- 16672007 TI - The role of computerized order sets in pediatric inpatient asthma treatment. AB - Condition-specific order sets within computerized physician order entry (CPOE) systems are designed to decrease unnecessary practice variation and to promote evidence-based practice. This study quantitatively assessed the relationship between use of a computerized order set and processes of care in inpatient pediatric asthma treatment, and qualitatively assessed user attitudes toward order set use. The study population included 790 patients with a primary diagnosis of asthma admitted to Columbus Children's Hospital between November 1, 2001 and November 30, 2003. Rates of systemic corticosteroid (SCS) use, metered dose inhaler use, and pulse oximetry (PulseOx) were calculated for three patient groups: those admitted prior to order set implementation ('pre-set'); those admitted after implementation but without the order set used ('no set'); and those admitted after implementation with the order set used ('set'). Financial measures of length of stay, total charges, and pharmacy charges were also calculated. Focus groups exploring attitudes about order sets were held with physician users of order sets. Order set patients were significantly more likely to receive SCS and PulseOx than 'pre-set' and 'no set' patients. 'No set' patients did not differ significantly from 'pre-set' patients. No significant differences were found in financial measures. Results from focus groups suggested that order set use would be optimized by promoting order set awareness and maximizing order set quality. These results give further credence to policy makers' calls for expanded use of CPOE systems with condition-specific order sets to facilitate provision of evidence-based care. PMID- 16672008 TI - Pediatric Asthma Quality of Life Questionnaire (PAQLQ): validation among asthmatic children in Thailand. AB - Quality of life (QoL) is an important consideration among asthma sufferers. The Pediatric Asthma Quality of Life Questionnaire (PAQLQ) is one of the most widely used instruments for measuring health-related QoL in children with asthma. The standardized version of PAQLQ contains 23 questions in three domains, i.e., activity limitation, symptoms and emotional function. The objective of this study was to validate the Thai-translated version of the PAQLQ. The study design consisted of a five-week single cohort study. Patients recorded symptoms, and peak expiratory flow rate (PEFR) each morning and evening during the first and fifth week of the study in asthma diary. At each clinic visit, a trained interviewer administered the PAQLQ and performed spirometric measurements. Fifty one children, ages between 7 and 17 yr participated in the study. Scores from the asthma diary were used to classify patients into stable vs. unstable groups. The construct validity of the questionnaire was confirmed in both cross-sectional and longitudinal studies by demonstrating correlations between various PAQLQ domains with clinical asthma parameters (asthma diary, beta-agonist use and PEFR). There was high internal consistency for scores of the three domains (Cronbach's alpha coefficient = 0.83-0.95). For those with stable asthma, the reliability of PAQLQ was good for the rating scale (intra-class correlation coefficient--ICC = 0.84) and for total score (alpha = 0.97) indicating high reproducibility of the PAQLQ. The significant difference of changes QoL scores between stable and unstable groups was observed in all domains. We conclude that the Thai version of PAQLQ is valid and reliable for implementing in Thai children with asthma. PMID- 16672009 TI - Proposal for a standardized interpretation of the atopy patch test in children with atopic dermatitis and suspected food allergy. AB - The interpretation of the atopy patch test (APT) to foods is not standardized. This study aimed to validate the reading of the APT in terms of the diagnostic accuracy of individual skin signs. Eighty-seven children (mean age 2.4 +/- 2.5 yr, range 0.5-13.5; 57 male) with atopic dermatitis (AD) and suspected food allergies underwent APT to cow's milk, hen's egg, wheat and soy. Twelve millimetre Finn chambers were applied for 48 h, and results were read after 48 and 72 h. Skin changes were graded for erythema, induration, papule formation and 'crescendo' phenomenon (increase of skin sign severity from 48 to 72 h). Food allergy was assessed by double blind, placebo-controlled food challenges (DBPCFC). Sensitivity, specificity and predictive values were calculated for each skin signs in relation to challenge outcome. Of 165 DBPCFC children, 75 (45%) were positive. The combination of any skin induration plus papules (seven or more), or of moderate erythema plus any induration plus seven or more papules had a positive predictive value (PPV) and specificity for the challenge outcome of 100%; however, the sensitivity was low (8% and 15%). The best diagnostic accuracy for single signs was found for induration beyond the Finn chamber margin (PPV 88%, specificity 99%, sensitivity 9%) and presence of at least seven papules (PPV 80%, specificity 96% sensitivity 21%). Presence of both induration and of at least seven papules at 72 h were the APT skin signs with the greatest diagnostic accuracy for food allergy in children with AD. PMID- 16672010 TI - Hypomethylation of DNA and resistance to apoptosis in tonsillar hypertrophy in children. AB - We analyzed the hypomethylation of DNA and the sensitivity to apoptosis of tonsillar cells and peripheral blood lymphocytes (PBL) in twenty children with either recurrent tonsillitis (RT) or tonsillar hypertrophy (TH). We found no significant differences in DNA methylation of PBL obtained from RT and TH groups. Hypomethylation of DNA extracted from tonsillar tissue was higher in TH than in RT and was associated with lower spontaneous and thapsigargin-induced apoptosis. By contrast, RT showed a low level of DNA hypomethylation and was associated with high sensitivity to spontaneous and thapsigargin-induced apoptosis. PMID- 16672011 TI - Parental knowledge and use of epinephrine auto-injector for children with food allergy. AB - Epinephrine should be prescribed for patients at risk of anaphylaxis. Our purpose was to determine the use of Anapen prescribed for food-allergic children, to assess parental knowledge regarding Anapen, and to evaluate the arrangements for emergency kits and personalized care projects in everyday life. A prospective study was performed with a questionnaire sent to families with a food-allergic child previously prescribed Anapen. One hundred and fifty two families were contacted and finally 111 children included (median age 6.5 yrs). Main food allergens were peanuts (n = 89), egg (n = 39) and cow's milk (n = 10). The use of Anapen had been demonstrated to 90% of parents (by prescribing physician, 69%; pharmacist, 25%; general practitioner, 5%; nurse 1%), with a training device (76%) and/or written instructions (49%). When asked to list symptoms requiring injection, 48% of parents cited more than one response: breathing difficulties only (23%), or with angio-edema (41%), collapse or faintness (38%), anaphylactic shock (48%). Of 107 children attending school, 54% had a personalized care project, 72% an Anapen device, and 60% a complete emergency kit (epinephrine, inhaled beta-agonist, corticosteroid, anti-H1 drug). Beta-agonists were forgotten at school by 34 children (13 asthmatics). Anapen was used in one child for angio edema and dyspnea after inadvertent ingestion of egg at home. In our population, epinephrine auto-injectors and emergency kits were insufficiently available at schools and in daily life. The use of auto-injectors was not adequately demonstrated. The prescription of epinephrine for food-allergic children at risk of anaphylaxis requires accurate diagnosis, educational programs, information, and follow up. PMID- 16672012 TI - Factors determining the ability of parents to effectively administer intramuscular adrenaline to food allergic children. AB - Intramuscular adrenaline is the treatment of choice for food-related anaphylactic reactions. Although auto-injectable adrenaline devices are routinely prescribed for patients at risk of serious reactions, previous studies have shown that only one-third to one and a half of patients or their carers are able to properly use these devices. The aim of this study was to determine which factors are most strongly associated with the effective use of these devices. A 122 children with food allergies who had previously been prescribed EpiPens and were attending a single specialist pediatric allergy center in the UK. were studied prospectively. A 69% of parents were unable to use the EpiPen, did not have it available, or did not know when it should be administered. A prior practical demonstration was associated with a 4-5 fold greater chance that parents would be able to use the device (p < 0.005). Prior consultation with an allergy specialist rather than a general physician, and parents who independently sought additional information from the national self-help allergy organization were also four to six times more likely to be competent with these devices (p < 0.005). The study clearly shows that for EpiPens to be used safely and effectively it is essential to educate the carer at the time the device is prescribed. PMID- 16672013 TI - Acute allergic reaction to an iron compound in a milk-allergic patient. AB - A child allergic to cow's milk developed a mild systemic allergic reaction after the first dose of Ferplex 40 (iron proteinsuccinylate). Skin tests and in vitro studies were performed in the child, in three cow's milk-allergic controls and in a non-allergic control. Milk, casein and iron proteinsuccinylate (Ferplex 40) were used for skin tests, specific immunoglobulin E (IgE) determination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and enzyme allergo sorbent test (EAST) inhibition. A review of the drug information sheet and contact with the manufacturer were also performed. Although proteinsuccinylate is indeed a succinylated casein (each dose containing about 575 mg of casein) there was no indication of the milk protein content in the prescribing information provided by the manufacturer. Skin tests and specific IgE were positive in the case and in all allergic controls, except for EAST to iron proteinsuccinylate in one control. In EAST, iron proteinsuccinylate in solid phase was 100% inhibited by casein and casein in solid phase was inhibited 74% by iron proteinsuccinylate. SDS-PAGE of iron proteinsuccinylate showed a broad 46 kDa band and a blur of aggregated material. On immunoblot, the patient's IgE reacted to this heavily aggregated material and in the native casein extract recognized a 35-kDa band. The allergenicity of succinylated casein (proteinsuccinylate) among milk-allergic children is demonstrated. The protein source used in drug-protein conjugates should always be indicated by the manufacturer (as it should be in foods) to avoid potential risks to allergic patients. PMID- 16672014 TI - Childhood immunization and atopic disease. PMID- 16672016 TI - Beta-lactam allergy in children. PMID- 16672017 TI - Bone healing and graft resorption of autograft, anorganic bovine bone and beta tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs. AB - OBJECTIVE: The purpose was to qualitatively and quantitatively compare the bone formation and graft resorption of two different bone substitutes used in both orthopedic and oral surgery, with autogenous bone as a positive control. MATERIALS AND METHODS: Three standardized bone defects were prepared in both mandibular angles of 12 adult minipigs. The defects were grafted with either autograft, anorganic bovine bone (ABB), or synthetic beta-tricalcium phosphate (beta-TCP). Sacrifice was performed after 1, 2, 4, and 8 weeks for histologic and histomorphometric analysis. RESULTS: At 2 weeks, more new bone formation was seen in defects filled with autograft than with ABB (P approximately 0.0005) and beta TCP (P approximately 0.002). After 4 weeks, there was no significant difference between beta-TCP and the two other materials. Defects grafted with ABB still exhibited less bone formation as compared with autograft (P approximately 0.004). At 8 weeks, more bone formation was observed in defects grafted with autograft (P approximately 0.003) and beta-TCP (P approximately 0.00004) than with ABB. No difference could be demonstrated between beta-TCP and autograft. beta-TCP resorbed almost completely over 8 weeks, whereas ABB remained stable. CONCLUSION: Both bone substitutes seemed to decelerate bone regeneration in the early healing phase as compared with autograft. All defects ultimately regenerated with newly formed bone and a developing bone marrow. The grafting materials showed complete osseous integration. Both bone substitutes may have a place in reconstructive surgery where different clinical indications require differences in biodegradability. PMID- 16672018 TI - Significance of primary stability for osseointegration of dental implants. AB - AIM: To investigate the significance of the initial stability of dental implants for the establishment of osseointegration in an experimental capsule model for bone augmentation. MATERIAL AND METHODS: Sixteen male rats were used in the study. In each rat, muscle-periosteal flaps were elevated on the lateral aspect of the mandibular ramus on both sides, resulting in exposure of the bone surface. Small perforations were then produced in the ramus. A rigid, hemispherical Teflon capsule with a diameter of 6 mm and a height of 4 mm and with a hole in its middle portion, prepared to fit the circumference of an ITI HC titanium implant of 2.8 mm in diameter, was fixed to the ramus using 4 mini-screws. On one side of the jaw, the implant was placed through the hole in such a way that its apex did not make contact with the mandibular ramus (test). This placement of the implant did not ensure primary stability. On the other side of the jaw, a similar implant was placed through the hole of the capsule in such a way that contact was made between the implant and the surface of the ramus (control). This provided primary stability of the implant. After placement of the implants, the soft tissues were repositioned over the capsules and sutured. After 1, 3, 6 and 9 months, four animals were sacrificed and subjected to histometric analysis. RESULTS: The mean height of direct bone-to-implant contact of implants with primary stability was 38.8%, 52.9%, 64.6% and 81.3% of the implant length at 1, 3, 6 and 9 months, respectively. Of the bone adjacent to the implant surface, 28.1%, 28.9%, 52.6% and 69.6%, respectively, consisted of mineralized bone. At the test implants, no bone-to-implant contact was observed at any observation time or in any of these non-stabilized specimens. CONCLUSION: The findings of the present study indicate that primary implant stability is a prerequisite for successful osseointegration, and that implant instability results in fibrous encapsulation, thus confirming previously made clinical observations. PMID- 16672019 TI - Enhanced bone apposition around biofunctionalized sandblasted and acid-etched titanium implant surfaces. A histomorphometric study in miniature pigs. AB - Microrough titanium (Ti) surfaces of dental implants have demonstrated more rapid and greater bone apposition when compared with machined Ti surfaces. However, further enhancement of osteoblastic activity and bone apposition by bio functionalizing the implant surface with a monomolecular adsorbed layer of a co polymer - i.e., poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its derivatives (PLL-g-PEG/PEG-peptide) - has never been investigated. The aim of the present study was to examine early bone apposition to a modified sandblasted and acid-etched (SLA) surface coated with an Arg-Gly-Asp (RGD)-peptide-modified polymer (PLL-g-PEG/PEG-RGD) in the maxillae of miniature pigs, and to compare it with the standard SLA surface. Test and control implants had the same microrough topography (SLA), but differed in their surface chemistry (polymer coatings). The following surfaces were examined histomorphometrically: (i) control - SLA without coating; (ii) (PLL-g-PEG); (iii) (PLL-g-PEG/PEG-RDG) (RDG, Arg-Asp-Gly); and (iv) (PLL-g-PEG/PEG-RGD). At 2 weeks, RGD-coated implants demonstrated significantly higher percentages of bone-to-implant contact as compared with controls (61.68% vs. 43.62%; P < 0.001). It can be concluded that the (PLL-g-PEG/PEG-RGD) coatings may promote enhanced bone apposition during the early stages of bone regeneration. PMID- 16672020 TI - Osteoblast-like cells are sensitive to submicron-scale surface structure. AB - OBJECTIVES: Studies showing that osteoblasts exhibit a more differentiated phenotype on rough titanium (Ti) surfaces and osteoclast-resorbed bone surfaces used materials characterized by average peak to valley distance (Ra). Other surface features impacting the cells include distance between peaks, curvature of the valleys, and relative distribution of flat and smooth regions. We used novel Ti surfaces prepared by electrochemical micromachining as models to examine specific contributions of individual design features to osteoblast response. Results show that micron-scale topography modulates cell number, cell morphology and prostaglandin E2 (PGE2). In the presence of the appropriate microtopography, submicron-scale rugosity modulates differentiation and transforming growth factor beta1 (TGF-beta1) levels. In this study, we examined the role of different types of submicron-scale structures. MATERIAL AND METHODS: Thirty micrometer diameter craters on Ti disks were produced by photolithography resulting in an electropolished smooth surface, and arranged so that inside crater area vs. outside flat area was 6 (30/6). Submicron-scale structures were superposed by acid etching and porous anodization. Ra's were 700, 400, 60 nm on acid-etched, porous anodized and smooth 30/6 surfaces, respectively. RESULTS: MG63 osteoblast like cells were sensitive to submicron-scale architecture. Cell morphology on anodized surfaces was similar to morphology on smooth surfaces, whereas on etched surfaces, cells had a more elongated differentiated shape. Cell number was greatest on smooth surfaces > anodized > etched. Osteocalcin and PGE2 were affected in a reverse manner. Active TGF-beta1 was greatest on etched 30/6 surfaces > anodized > smooth; latent TGF-beta1 was elevated on all rough surfaces. CONCLUSIONS: These results support our previous observations that submicron-scale structures modulate osteoblastic phenotype and show that the physical properties of the submicron-scale structures are important variables in determining osteoblast response to substrate topography. PMID- 16672021 TI - Dental implants placed in expanded narrow edentulous ridges with the Extension Crest device. A 1-3-year multicenter follow-up study. AB - OBJECTIVE: This study has been designed to evaluate the capability of a new surgical device (Extension Crest) to widen narrow edentulous alveolar ridges and to allow a correct placement of endosseous implants in horizontally atrophied sites. MATERIAL AND METHODS: Forty-five patients, 20 males and 25 females, aged 20-66 years, affected by edentulism associated to horizontal resorption of the ridges, were treated by means of a sagittal osteotomy and expansion of the ridge with a new surgical device (Extension Crest) to obtain a wider bony base for ideal implant placement. In the same procedure in 33 patients, and 1 week afterwards in 12 patients, 110 endosseous titanium implants (ITI TE) were placed. Three to four months later, the patients were rehabilitated with implant supported prostheses. RESULTS: The success rate of the expansion technique was 97.8%. A total of 110 implants were inserted in the expanded ridges. The mean follow-up after the start of prosthetic loading was 20.4 months. Three implants were removed before the start of prosthetic loading, because of non-integration, while no other implants failed after the completion of the prosthetic rehabilitation. Three implants, although integrated and in function, did not fulfill success criteria: cumulative success and survival rates at the end of the observation period were 95.4% and 97.3%, respectively. CONCLUSION: Within the limits of this study, this technique appeared to be reliable and simple, with reduction of morbidity and times of dental rehabilitation as compared with other techniques such as autogenous bone grafts and guided bone regeneration. Survival and success rates of implants placed in the treated areas are consistent with those placed in native bone. PMID- 16672022 TI - Lethal photosensitization and guided bone regeneration in treatment of peri implantitis: an experimental study in dogs. AB - The purpose of this study was to evaluate the effect of lethal photosensitization and guided bone regeneration (GBR) on the treatment of ligature-induced peri implantitis in different implant surfaces. The treatment outcome was evaluated by clinical and histometric methods. A total of 40 dental implants with four different surface coatings (10 commercially pure titanium surface (cpTi); 10 titanium plasma-sprayed (TPS); 10 acid-etched surface; 10 surface-oxide sandblasted) were inserted into five mongrel dogs. After 3 months, the animals with ligature-induced peri-implantitis were subjected to surgical treatment using a split-mouth design. The controls were treated by debridment and GBR, while the test side received an additional therapy with photosensitization, using a GaAlAs diode laser, with a wavelength of 830 nm and a power output of 50 mW for 80 s (4 J/cm2), and sensitized toluidine blue O (100 microg/ml). The animals were sacrificed 5 months after therapy. The control sites presented an earlier exposition of the membranes on all coating surfaces, while the test group presented a higher bone height gain. Re-osseointegration ranged between 41.9% for the cpTi surface and 31.19% for the TPS surface in the test sites; however differences were not achieved between the surfaces. The lethal photosensitization associated with GBR allowed for better re-osseointegration at the area adjacent to the peri-implant defect regardless of the implant surface. PMID- 16672023 TI - Performance of six bone collectors designed for dental implant surgery. AB - OBJECTIVES: The aim of this study was to perform an in vitro comparison of six bone collectors for harvesting of particulate bone. MATERIAL AND METHODS: Four commercially available bone collectors (Frios, Osseous Coagulum Trap, ACE Autografter, Bone Trap) and two custom-designed models were tested. Three different in vitro tests were performed to determine the harvesting capabilities of the collectors. In test I, a bovine mandible was drilled and the bone collectors were used to collect bone chips. The harvested bone volumes and dry weights were measured after harvesting. In test II, three dental implant sites were prepared in a bovine mandible. The bones from the implant osteotomies were collected, and bone volumes and dry weights were measured. In test III, 1 ml of bone chips was mixed with water, and suctioned through the bone collectors. The volumes of the bone chips retained were measured to determine the efficiency of each collector. RESULTS: The Osseous Coagulum Trap and the custom-made collectors were the most effective instruments in test I. The mean volumes ranged from 0.17 to 0.38 ml. In test II, the difference between the collectors was small and the bone volume ranged from 0.28 to 0.37 ml. In test III, the Bone Trap became blocked before the other collectors, and its bone procurement was therefore limited. CONCLUSION: Comparison of six different bone collectors in this in vitro study showed that all collectors are usable in clinical situations but their effectiveness varies. PMID- 16672024 TI - Resorbable screws for fixation of autologous bone grafts. AB - The aim of this study was to evaluate the suitability of resorbable screws made of poly (D.L-lactide) acid (PDLLA) for fixation of autologous bone grafts related to graft regeneration and osseointegration of dental implants. In eight edentulous patients suffering from insufficient retention of their upper denture related to a severely resorbed maxilla, the floor of both maxillary sinus and width of the alveolar crest were augmented with an autologous bone graft from the iliac crest. Randomly, the bone graft used to augment the alveolar crest was fixed with two titanium screws on one side and two resorbable screws on the other side (split-mouth design). Three months after the reconstruction, a bone biopsy was taken with a trephine including one resorbable screw (N = 8). Subsequently, six implants were placed in the left and right posterior maxilla. Six months later, at the abutment connection, a bone biopsy was taken including the other resorbable screw (N = 8). The biopsies were processed for light microscopic examination. In addition, clinical parameters were scored. Wound healing was uneventful. Clinically no difference in wound healing was observed between sides treated with either a resorbable or titanium screw. No implants were lost. Six months after implantation, implant retained overdentures could be fabricated in all patients. All patients functioned well with their overdentures (follow-up 22.2 +/- 4.3 months). Three as well as 9 months after insertion (remnants of), the resorbable screws were still visible after reflecting the mucoperiosteum. Histological examination confirmed that a considerable quantity of remnants of the resorbable screws was still present, although areas with some fragmentation of the PDLLA were also observed. The screws were separated by a fibrous tissue layer containing many giant cells from the bone. Particles of PDLLA were observed within these giant cells. This study revealed that resorbable screws made of PDLLA can be used for fixation of bone grafts. The bulk of the PDLLA material is still present after 9 months. PMID- 16672025 TI - Effect of mechanical and antiseptic therapy on peri-implant mucositis: an experimental study in monkeys. AB - OBJECTIVES: This experiment was performed to evaluate clinically and histologically the effect of mechanical therapy with or without antiseptic therapy on peri-implant mucositis lesions in nine cynomolgus monkeys. MATERIAL AND METHODS: Two ITI titanium implants were inserted into each side of the mandibles. After 90 days of plaque control and soft tissue healing, a baseline clinical examination was completed. Peri-implant lesions were induced by placing silk ligatures and allowing plaque to accumulate for 6 weeks. The clinical examination was then repeated, and the monkeys were randomly assigned to three treatment groups: group A, mechanical cleansing only; group B, mechanical cleansing and local irrigation with 0.12% chlorhexidine (CHX) and application of 0.2% CHX gel; and group C, control, no treatment. The implants in treatment groups A and B were treated and maintained according to the assigned treatment for two additional months. At the end of the maintenance period, a final clinical examination was performed and the animals were sacrificed for biopsies. RESULTS: The mean probing depths (PD) values at mucositis were: 3.5, 3.7, and 3.4 mm, and clinical attachment level (CAL) = 3.8, 4.1, and 3.9 mm for treatment groups A, B and C, respectively. The corresponding values after treatment were: PD = 1.7, 2.1, and 2.5 mm, and CAL=2.6, 2.6, and 3.1 mm. ANOVA of mean changes (Delta) in PD and CAL after treatment showed no statistical difference between the treatment groups. Comparison of the mean changes in PD and CAL after treatment yielded statistical differences between the control and treatment groups P < 0.01. According to the t-test, no statistical difference was found between treatment groups A and B for the PD reduction but there was a significant difference for the CAL change, P < 0.03. Group A had significantly more recession and less CAL gain than group B. Non-parametric tests yielded no significant differences in modified plaque index (mPlI) and gingival index (GI) after treatment between both treatment groups. Frequencies and percent distributions of the mPlI and GI scores changed considerably for both treatment groups when compared with the changes in the control group after treatment. With regard to the histological evaluation, no statistical differences existed between the treatments for any linear measurement. The proportion of inflammation found in the mucosal tissues of the control implants was greater than the one found for both treatment groups, P < 0.01. More importantly, both treatment groups showed a similar low proportion of inflammation after 2 months of treatment. CONCLUSIONS: Within the limitations of this experiment, and considering the supportive plaque control rendered, it can be concluded that for pockets of 3-4 mm: (1) mechanical therapy alone or combined with CHX results in the clinical resolution of peri-implant mucositis lesions, (2) histologically, both treatments result in minimal inflammation compatible with health, and (3) the mechanical effect alone is sufficient to achieve clinical and histologic resolution of mucositis lesions. PMID- 16672026 TI - The bone regenerative effect of platelet-rich plasma in combination with an osteoconductive material in rat cranial defects. AB - The effect of platelet-rich plasma (PRP) on bone regeneration, in combination with an osteoconductive material, was evaluated in a rat model. Cranial defects, 6.2 mm in diameter, were filled with HA/beta-TCP particles, HA/beta-TCP particles combined with PRP and HA/beta-TCP particles combined with PRP gel, where some were left empty as a control. After 4 weeks of implantation histological, histomorphometrical and micro-computed tomography analyses revealed no difference in new bone formation among the groups. Further, no additional effect of PRP gel in comparison with PRP liquid was detected, except for the increased handling capacity of the graft. These findings suggest that PRP had no positive effect on bone formation in addition to an osteoconductive material after an implantation period of 4 weeks. Also, no negative effect was seen, and neither PRP nor HA/beta TCP hampered bone ingrowth into the defects. PMID- 16672027 TI - Influence of platelet-rich plasma on a bioglass and autogenous bone in sinus augmentation. An explorative study. AB - Platelet-rich plasma (PRP) has been introduced to the field of oral and maxillofacial surgery for a decade, but its beneficial effects on maxillary sinus augmentation remain unclear. The aim of this study was to evaluate the short- and long-term effects of PRP on osseointegration following single-stage sinus augmentation in a randomized prospective animal study. The maxillary premolars of 24 minipigs were extracted bilaterally and allowed to heal for 2 months. Consecutively all animals underwent bilateral sinus floor elevation using autogenous bone, Biogran as well as a combination of the materials with PRP. Three dental implants (Ankylos, Dentsply Co., Mannheim, Germany) were installed in each sinus simultaneously. Four animals were sacrificed at each period of observation (1, 2, 8 and 12 months). Microradiographic images of the specimens were made for quantitative evaluation of the bone-implant contact (BIC) and light microscopic images were made for qualitative analysis. An increment of the BIC during the observation time could be seen over the observation time in all groups. Autogenous bone exhibited a level of BIC from 25.1 +/- 9.96% at 1 month to 55.1 +/- 13.10% at 12 months; on adding PRP, the BIC ranged from 28.4 +/- 4.64% to 52.5 +/- 17.06%. Biogran with and without PRP led to BIC levels from 16.3 +/- 4.64% to 37.6 +/- 16.40% and 21.7 +/- 4.33% to 46.6 +/- 19.37%, respectively. The results of this study did not show a significantly positive effect of PRP on the BIC following sinus augmentation in both groups. PMID- 16672028 TI - Platelet releasate increases the proliferation and migration of bone marrow derived cells cultured under osteogenic conditions. AB - Concentrated platelets and their products are currently being used as a clinical tool to accelerate endosseous wound healing. However, there is little understanding regarding the actions of platelets and platelet-released products on osteogenic cells. We show, herein, that releasate from thrombin-activated platelets increases the migration and proliferation of osteogenic cultures of bone marrow cells. Using a scratch wound assay, we demonstrated that platelet releasate (PR) stimulated up to a 2.4 +/- 0.5-fold increase in wound closure in serum-free medium, relative to a control containing thrombin. In the presence of serum, the addition of PR resulted in a 1.45 +/- 0.13-fold increase in scratch closure. To isolate cell migration from the effects of cell proliferation, cell monolayers were pre-incubated with 5, 10 and 20 microg/ml of Mitomycin C (MMC), which is a potent inhibitor of cell proliferation. This resulted in a large decrease in the leading front of scratch closure, which indicates that PR stimulated cell mitogenesis. However, irrespective of MMC pre-treatment, PR stimulated a motogenic response. These results provide evidence of possible mechanisms by which platelets could influence bone regeneration. PMID- 16672029 TI - Oral myofunctional and vocal characteristics in subjects subjected to oral rehabilitation with osseointegrated implants. AB - PURPOSE: The aim of this study was to evaluate the effect of oral rehabilitation using immediately loaded fixed implant-supported mandibular prostheses, considering chewing, swallowing, speech and voice aspects. MATERIAL AND METHODS: Fourteen patients subjected to this treatment were evaluated by a clinical examination of the stomatognathic system and perceptual evaluation of voice quality before oral rehabilitation, as well as after 1 and 4 months. RESULTS: After oral rehabilitation, the results showed that there were no significant modifications in the aspects investigated. CONCLUSION: Thus, implant-supported prosthetic rehabilitation, despite providing a more satisfactory occlusal standard, did not yield changes in the functional patterns of individuals in this study. PMID- 16672030 TI - Implant-tooth-supported fixed partial prostheses: correlations between in vivo occlusal bite forces and marginal bone reactions. AB - PURPOSE: To evaluate maximal occlusal bite forces (MOF) and marginal bone level (MBL) changes in patients with implant-tooth-supported fixed partial prostheses (FPP). MATERIAL AND METHODS: Twenty nine partially edentulous patients consecutively who received 34 three-occlusal unit FPP with terminal implant and tooth support were subjected to quantification of MOFs using a sub-miniature load cell connected to a data acquisition system and measurement of the MBL changes around implants in digitalized periapical radiographs obtained at prostheses delivery and 24-month follow-up. RESULTS: MOFs for implant support (mean: 353.61 N) significantly differed from tooth support (mean: 275.48 N) (P < 0.05), while gender did not influence MOFs (P > 0.05). MBL changes at mesial and distal sites of the implants at 24 months of functional loading were 0.28 and 0.097 mm respectively. CONCLUSION: Although MOFs under functional loading might indicate an increase in load participation for supporting implant, the rigid connection between implant and natural tooth via three-occlusal unit FPP does not jeopardize the time-dependent MBL stability of the implant under functional loads. PMID- 16672031 TI - Implant survival in mandibles of irradiated oral cancer patients. AB - OBJECTIVE: The aim of this study was to analyze long-term implant survival in the mandible after radiotherapy and radical surgery in oral cancer patients. STUDY DESIGN: Between 1990 and 2003, 71 patients (15 females, 56 males; average age 57.8 years, range 16-84.1 years) were treated with dental implants after radiochemotherapy and ablative surgery of oral cancer. Radiation therapy was delivered in daily fractions of 2 Gy given on 25 days (total dose of 50 Gy). Oral defects were reconstructed microsurgically with jejunal, iliac crest or radial forearm grafts. Thereafter 316 dental implants were placed in the non-irradiated residual bone (84; 27%), irradiated residual bone (154; 49%) or grafted bone (78; 25%) at various intervals (mean interval 1.41 (+/- 1.01) years, range 0.34-6.35 years). RESULTS: The mean follow-up time after implant insertion was 5.42 (+/- 3.21) years (range 0.3-13.61 years). The overall 2-, 3-, 5-, and 8-year survival rates of all implants were 95%, 94%, 91% and 75%. Forty-four implants were lost in 21 patients during the observation period. Irradiation of the mandibular bone showed significantly (P = 0.0028) lower implant survival compared with non irradiated mandibular bone. The 8-year survival rate in the non-irradiated residual bone (two loss), irradiated residual bone (29 loss) or grafted bone (13 loss) were 95%, 72% and 54%, respectively. Time of implantation after irradiation showed no statistically significant influence. Implant brand, length or diameter or the incidence of resective surgery on the mandible and gender of patients had no statistically significant influence on implant survival. CONCLUSION: Radiation therapy with 50 Gy was significantly related to shorter implant survival in mandibular bone. Survival was lowest in grafted bone. Time of implant placement had no statistically significant influence on survival under the conditions of this study. Although implant survival is lower in irradiated mandibles, implants significantly facilitate prosthodontic treatment and enhance outcome of oral rehabilitation in cancer patients. PMID- 16672032 TI - Loading of bone surrounding implants through three-unit fixed partial denture fixation: a finite-element analysis based on in vitro and in vivo strain measurements. AB - Implant-borne fixed partial dentures (FPDs), whether cementable or screwable superstructures, ought to display a true passive fit. The objective of this in vivo-based finite-element analysis is, therefore, to quantify the degree of stress which occurs in the bone around the implants as a result of the fixation of cemented and screw-retained FPDs. On the basis of a simulated patient situation with two implants, six groups of implant-supported superstructures containing 10 samples each were fabricated. Strain gauges which were mounted on the pontics of the restorations were subsequently used to take in vivo measurements (Ethics Commission Approval No. 2315). Taking the values obtained as a basis, the von Mises equivalent stress was chosen to illustrate bone loading in three-dimensional finite-element models. Superstructure fixation caused residual interface stress as high as 30 MPa. Similar stress magnitudes can be observed for axial implant loading of 200 N. Assuming that the axial loading of a single implant with 200 N is within the bone's physiological range, it can be concluded that the degree of stress resulting from the fixation of superstructures alone does not constitute a risk. PMID- 16672033 TI - Behavioural phenotypes in disability research: historical perspectives. AB - Western medicine has a long history of accounting for behaviour by reducing the body to ultimate explanatory entities. In pre-modern medicine these were invisible "animal spirits" circulating the body. In modern medicine, they are "genes". Both raise questions. The psychological phenotype is defined by human consensus, varying according to time and place, while the genotype's DNA exists in a realm of material reality. There are deep philosophical and methodological problems in linking one realm to the other. Nyhan's original application of the phenotype-genotype pairing merely claimed that the two realms could be matched because of their common susceptibility to statistical treatment. His behavioural example was "stereotypy". It has since extended to include such things as "social cognition" in Turner's syndrome (Skuse), thus revealing increasingly clearly that the two realms are fundamentally and ontologically separate. The problems are not merely epistemological but ethical, since the looseness of psychological categories involves a blurring of the boundaries between behavioural phenotype and social stereotype. The latter may then be underwritten as "real" by being associated, spuriously, with the empirically demonstrable reality of genetic material. PMID- 16672034 TI - The Assessment of Basic Learning Abilities (ABLA) test predicts the relative efficacy of task preferences for persons with developmental disabilities. AB - BACKGROUND: Prior studies have showed that presentation methods could affect the accuracy of a choice assessment. METHODS: In the current study, high- and low preferred work tasks were identified in nine adults with developmental disabilities. Both tasks were then introduced in pairs within a choice assessment using the actual tasks, pictures of the tasks and spoken descriptions of the tasks. Participants were also given the Assessment of Basic Learning Abilities (ABLA) test that evaluated their discrimination skills. RESULTS: For five of the participants, their consistent choice of preferred task was predicted by their discrimination skills as assessed by the ABLA. CONCLUSIONS: These findings extend that of Conyers et al. whereby the systematic assessment of discrimination skills could predict the effectiveness of different presentation methods in this population. PMID- 16672035 TI - Assessing the reading comprehension of adults with learning disabilities. AB - BACKGROUND: This study's aim was to begin the process of measuring the reading comprehension of adults with mild and borderline learning disabilities, in order to generate information to help clinicians and other professionals to make written material for adults with learning disabilities more comprehensible. METHODS: The Test for the Reception of Grammar (TROG), with items presented visually rather than orally, and the Reading Comprehension sub-test of the Wechsler Objective Reading Dimensions (WORD) battery were given to 24 service users of a metropolitan community learning disability team who had an estimated IQ in the range 50-79. RESULTS & CONCLUSIONS: These tests were demonstrated to have satisfactory split-half reliability and convergent validity with this population, supporting both their use in this study and in clinical work. Data are presented concerning the distribution across the sample of reading-ages and the comprehension of written grammatical constructions. These data should be useful to those who are preparing written material for adults with learning disabilities. PMID- 16672036 TI - Community and cluster centre residential services for adults with intellectual disability: long-term results from an Australian-matched sample. AB - BACKGROUND: Changes in residential accommodation models for adults with intellectual disability (ID) over the last 20 years in Australia, the United Kingdom and the United States have involved relocation from institutions primarily into dispersed homes in the community. But an evolving alternative service style is the cluster centre. METHODS: This paper reports on the relocation of a matched group of 30 pairs of adults with moderate and severe IDs and challenging behaviour who were relocated from an institution into either dispersed housing in the community or cluster centres but under the same residential service philosophy. Adaptive and maladaptive behaviour, choice-making and objective life quality were assessed prior to leaving the institution and then after 12 and 24 months of living in the new residential model. RESULTS: Adaptive behaviour, choice-making and life quality increased for both groups and there was no change in level of maladaptive behaviour compared with levels exhibited in the institution. However, there were some significant differences between the community and cluster centre group as the community group increased some adaptive skills, choice-making and objective life quality to a greater extent than the cluster centre group. CONCLUSIONS: Both cluster centre and dispersed community living offer lifestyle and skill development advantages compared with opportunities available in large residential institutions. Dispersed community houses, however, offer increased opportunities for choice making, acquisition of adaptive behaviours and improved life quality for long term institutionalized adults with IDs. PMID- 16672037 TI - Young people with intellectual disabilities attending mainstream and segregated schooling: perceived stigma, social comparison and future aspirations. AB - BACKGROUND: Mainstream schooling is a key policy in the promotion of social inclusion of young people with learning disabilities. Yet there is limited evidence about the school experience of young people about to leave mainstream as compared with segregated education, and how it impacts on their relative view of self and future aspirations. METHODS: Sixty young people with mild to moderate intellectual disabilities in their final year of secondary school participated in this study. Twenty-eight individuals came from mainstream schools and 32 attended segregated school. They completed a series of self-report measures on perceptions of stigma, social comparison to a more disabled and non-disabled peer and the likelihood involved in attaining their future goals. RESULTS: The majority of participants from both groups reported experiencing stigmatized treatment in the local area where they lived. The mainstream group reported significant additional stigma at school. In terms of social comparisons, both groups compared themselves positively with a more disabled peer and with a non-disabled peer. While the mainstream pupils had more ambitious work-related aspirations, both groups felt it equally likely that they would attain their future goals. Although the participants from segregated schools came from significantly more deprived areas and had lower scores on tests of cognitive functioning, neither of these factors appeared to have an impact on their experience of stigma, social comparisons or future aspirations. CONCLUSIONS: Irrespective of schooling environment, the young people appeared to be able to cope with the threats to their identities and retained a sense of optimism about their future. Nevertheless, negative treatment reported by the children was a serious source of concern and there is a need for schools to promote the emotional well-being of pupils with intellectual disabilities. PMID- 16672038 TI - Temperament and social behaviour at home and school among typically developing children and children with an intellectually disability. AB - BACKGROUND: There has been limited research on differences in temperament between typically developing children and children with an intellectual disability (ID). Individual differences have generally been neglected in previous investigations of children with an ID. The present research investigated differences in temperament and social behaviour between typically developing children and children with an ID, in both home and school settings. METHODS: Participants were 100 children (M = 10.7 years, SD = 0.88) from both regular and special education schools. Temperament was measured using the Emotionality, Activity, Sociability (EAS) Temperament Survey for Children (parental and teacher ratings), while social behaviour was measured using the School Social Behaviour Scales, Second Edition (SSBS-2) and the Home and Community Social Behaviour Scales (HCSBS). RESULTS: There were minimal significant differences in temperament between the regular and special education groups. Children who attended regular schools had a significantly higher level of social competence and significantly lower level of antisocial behaviour both at home and school, when compared with children who attended special schools. CONCLUSIONS: The difference in social behaviour between children attending regular and special schools was attributed to the difference in intellectual ability of the two groups, as well as contextual influences of regular or special school environments. PMID- 16672039 TI - A multiple case study of verbal short-term memory in velo-cardio-facial syndrome. AB - BACKGROUND: Velo-cardio-facial syndrome (VCFS, 22q11.2 deletion) is characterized by severely delayed language development. The current study explored the integrity of verbal short-term memory (STM), a cognitive function critically involved in language development, in eight children with VCFS. METHODS: Using a multiple case study design, we presented a series of STM tasks exploring immediate serial recall for word and non-word lists to eight children with VCFS (aged 8-12 years) and to chronological-age-matched control groups. A first task assessed the integrity of phonological coding in verbal STM by comparing recall for phonologically similar and dissimilar words. Subsequently, the interaction between verbal knowledge and STM capacity was investigated by comparing recall for high- and low-imageability words, for high- and low-frequency words, and for words and non-words. A final task assessed short-term serial order recognition for digit sequences. RESULTS: When computing the number of items recalled in the word recall tasks, independently of their serial position, only one child presented consistent difficulties. Short-term recall of non-words was normal in each child. Phonological similarity and verbal knowledge influenced STM performance to a similar extent in children with VCFS and controls. On the other hand, when applying a strict serial recall criterion, difficulties with the word and non-word recall tasks were observed in most children. Half of the patients were also impaired in the serial order recognition task. CONCLUSIONS: Despite mild intellectual disability, it is possible for short-term retention capacities for verbal item information to be at an age-appropriate level in VCFS. However, STM for serial order information could be impaired more specifically. PMID- 16672040 TI - Visual impairments in people with severe and profound multiple disabilities: an inventory of visual functioning. AB - BACKGROUND: The prevalence of visual impairments in people with severe and profound multiple disabilities (SPMD) is the subject of considerable debate and is difficult to assess. METHODS: In a typical Dutch care organization, all clients with SPMD (n = 76) participated in the study and specific instruments adapted to these clients (requiring a minimum of cooperation) were used to measure visual acuity, the visual field, binocular vision, contrast sensitivity, refractive errors and visual functioning behaviour. RESULTS: We found an unexpected 92% of clients with SPMD to have visual impairments. Previously, only 30% were known to have visual problems. None of the persons observed had normal visual acuity. Subnormal visual acuity was the best result. The severity of the visual impairment was related to the severity of the intellectual disability. In addition to the problem of acuity, impairments in the visual field, impaired contrast sensibility and impaired binocular functioning were found, as well as impaired visual attention, fixation and following. In 22% of the clients observed, refractive errors were found and glasses were advised. CONCLUSIONS: Consequences for caregiving and for modifications of the environment were discussed. PMID- 16672041 TI - Time trend and age-period-cohort effect on kidney cancer mortality in Europe, 1981-2000. AB - BACKGROUND: The incorporation of diagnostic and therapeutic improvements, as well as the different smoking patterns, may have had an influence on the observed variability in renal cancer mortality across Europe. This study examined time trends in kidney cancer mortality in fourteen European countries during the last two decades of the 20th century. METHODS: Kidney cancer deaths and population estimates for each country during the period 1981-2000 were drawn from the World Health Organization Mortality Database. Age- and period-adjusted mortality rates, as well as annual percentage changes in age-adjusted mortality rates, were calculated for each country and geographical region. Log-linear Poisson models were also fitted to study the effect of age, death period, and birth cohort on kidney cancer mortality rates within each country. RESULTS: For men, the overall standardized kidney cancer mortality rates in the eastern, western, and northern European countries were 20, 25, and 53% higher than those for the southern European countries, respectively. However, age-adjusted mortality rates showed a significant annual decrease of -0.7% in the north of Europe, a moderate rise of 0.7% in the west, and substantial increases of 1.4% in the south and 2.0% in the east. This trend was similar among women, but with lower mortality rates. Age period-cohort models showed three different birth-cohort patterns for both men and women: a decrease in mortality trend for those generations born after 1920 in the Nordic countries, a similar but lagged decline for cohorts born after 1930 in western and southern European countries, and a continuous increase throughout all birth cohorts in eastern Europe. Similar but more heterogeneous regional patterns were observed for period effects. CONCLUSION: Kidney cancer mortality trends in Europe showed a clear north-south pattern, with high rates on a downward trend in the north, intermediate rates on a more marked rising trend in the east than in the west, and low rates on an upward trend in the south. The downward pattern observed for cohorts born after 1920-1930 in northern, western, and southern regions suggests more favourable trends in coming years, in contrast to the eastern countries where birth-cohort pattern remains upward. PMID- 16672042 TI - A hidden Markov model approach for determining expression from genomic tiling micro arrays. AB - BACKGROUND: Genomic tiling micro arrays have great potential for identifying previously undiscovered coding as well as non-coding transcription. To-date, however, analyses of these data have been performed in an ad hoc fashion. RESULTS: We present a probabilistic procedure, ExpressHMM, that adaptively models tiling data prior to predicting expression on genomic sequence. A hidden Markov model (HMM) is used to model the distributions of tiling array probe scores in expressed and non-expressed regions. The HMM is trained on sets of probes mapped to regions of annotated expression and non-expression. Subsequently, prediction of transcribed fragments is made on tiled genomic sequence. The prediction is accompanied by an expression probability curve for visual inspection of the supporting evidence. We test ExpressHMM on data from the Cheng et al. (2005) tiling array experiments on ten Human chromosomes. Results can be downloaded and viewed from our web site. CONCLUSION: The value of adaptive modelling of fluorescence scores prior to categorisation into expressed and non-expressed probes is demonstrated. Our results indicate that our adaptive approach is superior to the previous analysis in terms of nucleotide sensitivity and transfrag specificity. PMID- 16672044 TI - Coping and back problems: analysis of multiple data sources on an entire cross sectional cohort of Swedish military recruits. AB - BACKGROUND: As the literature now stands, a bewildering number and variety of biological, psychological and social factors are, apparently, implicated in back problems. However, if and how these have a direct influence on back problems is not clear. Obesity, for example, has in many studies been shown to be associated with back problems but there is no evidence for a causal link. This could be explained by a dearth of suitably designed studies but also because obesity may be but a proxy for some other, truly explanatory variable. Coping has been linked with, particularly, persistent back problems as well as with health in general. The question is, whether coping could be the explanatory link between, for example, these two variables. A cross-sectional study was undertaken using data from the Swedish Army, consisting of the entire cohort of males (N = 48,502) summoned in 1998 to serve in the military. The purpose of the study was to investigate the relation between five independent variables and two dependent variables ("outcome variables"). The independent variables were two anthropomorphic variables (height and body mass index), two psychological variables (intellectual capacity and coping in relation to stress), and one social variable (type of education). The two outcome variables were back problems and ill health. In particular, we wanted to determine whether controlling for coping would affect the associations between the other four independent variables and the two outcome variables. METHODS: Data for the analysis come from a battery of standardized examinations, including medical examinations, a test of intellectual capacity, and a test of coping in relation to stress. Each of these examinations was conducted independently of the others. Unadjusted and adjusted odds ratios were calculated for the outcome variables of back problems and ill health. RESULTS: The associations between height, body mass index, intellectual capacity, type of education and the two outcome variables (back problems and ill health) were weak to moderate. Additionally, there were strong associations between coping and the two outcome variables and when controlling for coping the previously noted associations diminished or disappeared, whereas none of the other variables had a large effect on the association between coping and the two outcome variables. CONCLUSION: Coping emerged as strongly associated with both back problem and ill health and coping had a leveling effect on the associations between the other independent variables and the two outcome variables. This study is noteworthy particularly because the association with coping is so robust. It is a retrospective, cross-sectional study, however, and, as such it raises questions of causality; which - if any - came first, inability to cope or back pain? The results of this study call attention to the need for a prospective study, in which coping is clearly defined. Such a study has been undertaken and will be presented separately. Index terms: back pain, coping, education, height, BMI, intellectual capacity, bio-psycho-social model, epidemiology, cohort, cross sectional study. PMID- 16672043 TI - Estimating and projecting HIV prevalence and AIDS deaths in Tanzania using antenatal surveillance data. AB - BACKGROUND: The Estimations and Projections Package (EPP 2005) for HIV/AIDS estimates and projects HIV prevalence, number of people living with HIV and new HIV infections and AIDS cases using antenatal clinic (ANC) surveillance data. The prevalence projection produced by EPP can be transferred to SPECTRUM, a demographic projection model, to calculate the number of AIDS deaths. This paper presents estimates and projections of HIV prevalence, new cases of HIV infections and AIDS deaths in Tanzania between 2001 and 2010 using the EPP 2005 and SPECTRUM soft-wares on ANC data. METHODS: For this study we used; the 1985-2004 ANC data set, the 2005 UN population estimates for urban and rural adults, which is based on the 2002 population census, and results of the 2003 Tanzania HIV Indicator Survey. The ANC surveillance sites were categorized into urban and rural areas on the basis of the standard national definitions of urban and rural areas, which led to 40 urban and 35 rural clinic sites. The rural and urban epidemics were run independently by fitting the model to all data and on level fits. RESULTS: The national HIV prevalence increased from 0% in 1981 to a peak of 8.1% in 1995, and gradually decreased to 6.5% in 2004 which stabilized until 2010. The urban HIV epidemic increased from 0% in 1981 peaking at 12.6% in 1992 and leveled to between 10.9% and 11.8% from 2003 to 2010. The rural epidemic peaked in 1995 at 7.0% and gradually declined to 5.2% in 2004, and then stabilized at between 5.1% and 5.3% from 2005 to 2010. New infections are projected to rise steadily, resulting in 250,000 new cases in 2010. Deaths due to AIDS started in 1985 and rose steadily to reach 120,000 deaths in 2010, with more females dying than men. CONCLUSION: The fact that the number of new infections is projected to increase steadily to reach 250,000 per year in 2010 calls for more concerted efforts to combat the spread of HIV infection particularly in the rural areas where the infrastructure needed for prevention programmes such as counseling and testing, condom accessibility and AIDS information is less developed. PMID- 16672045 TI - How artificial intelligence tools can be used to assess individual patient risk in cardiovascular disease: problems with the current methods. AB - BACKGROUND: In recent years a number of algorithms for cardiovascular risk assessment has been proposed to the medical community. These algorithms consider a number of variables and express their results as the percentage risk of developing a major fatal or non-fatal cardiovascular event in the following 10 to 20 years DISCUSSION: The author has identified three major pitfalls of these algorithms, linked to the limitation of the classical statistical approach in dealing with this kind of non linear and complex information. The pitfalls are the inability to capture the disease complexity, the inability to capture process dynamics, and the wide confidence interval of individual risk assessment. Artificial Intelligence tools can provide potential advantage in trying to overcome these limitations. The theoretical background and some application examples related to artificial neural networks and fuzzy logic have been reviewed and discussed. SUMMARY: The use of predictive algorithms to assess individual absolute risk of cardiovascular future events is currently hampered by methodological and mathematical flaws. The use of newer approaches, such as fuzzy logic and artificial neural networks, linked to artificial intelligence, seems to better address both the challenge of increasing complexity resulting from a correlation between predisposing factors, data on the occurrence of cardiovascular events, and the prediction of future events on an individual level. PMID- 16672047 TI - Calcium adsorption and displacement: characterization of lipid monolayers and their interaction with membrane-active peptides/proteins. AB - BACKGROUND: The first target of antimicrobial peptides (AMPs) is the bacterial membrane. In the case of Gram-negative bacteria this is the outer membrane (OM), the lipid composition of which is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is made up solely from lipopolysaccharides (LPSs). LPS, therefore, represents the first target of AMPs. The binding and intercalation of polycationic AMPs is driven by the number and position of negatively charged groups of the LPS. Also, proteins other than cationic AMPs can interact with LPS, e.g. leading eventually to a neutralization of the endotoxic effects of LPS. We compared different biophysical techniques to gain insight into the properties of the electrical surface potentials of lipid monolayers and aggregates composed of LPSs and various phospholipids and their interaction with peptides and proteins. RESULTS: The net negative charge calculated from the chemical structure of the phospholipid and LPS molecules is linearly correlated with the adsorption of calcium to two-dimensional lipid monolayers composed of the respective lipids. However, the zeta-potentials determined by the electrophoretic mobility of LPS aggregates can only be interpreted by assuming a dependence of the plane of shear on the number of saccharides and charged groups. Various peptides and proteins were able to displace calcium adsorbed to monolayers. CONCLUSION: To characterize the electrical properties of negatively charged phospholipids and LPSs and their electrostatic interaction with various polycationic peptides/proteins, the adsorption of calcium to and displacement from lipid monolayers is a suitable parameter. Using the calcium displacement method, the binding of peptides to monolayers can be determined even if they do not intercalate. The interpretation of zeta-potential data is difficulty for LPS aggregates, because of the complex three-dimensional structure of the LPS molecules. However, the influence of peptides/proteins on the zeta-potential can be used to characterize the underlying interaction mechanisms. PMID- 16672046 TI - Comparison of exercise, dobutamine-atropine and dipyridamole-atropine stress echocardiography in detecting coronary artery disease. AB - BACKGROUND: Dipyridamole and dobutamine stress echocardiography testing are most widely utilized, but their sensitivity remained suboptimal in comparison to routine exercise stress echocardiography. The aim of our study is to compare, head-to-head, exercise, dobutamine and dipyridamole stress echocardiography tests, performed with state-of-the-art protocols in a large scale prospective group of patients. METHODS: Dipyridamole-atropine (Dipatro: 0.84 mg/kg over 10 min i.v. dipyridamole with addition of up to 1 mg of atropine), dobutamine atropine (Dobatro: up to 40 mcg/kg/min i.v. dobutamine with addition of up to 1 mg of atropine) and exercise (Ex, Bruce) were performed in 166 pts. Of them, 117 pts without resting wall motion abnormalities were enrolled in study (91 male; mean age 54 +/- 10 years; previous non-transmural myocardial infarction in 32 pts, angina pectoris in 69 pts and atypical chest pain in 16 pts). Tests were performed in random sequence, in 3 different days, within 5 day period under identical therapy. All patients underwent coronary angiography. RESULTS: Significant coronary artery disease (CAD; > or =50% diameter stenosis) was present in 69 pts (57 pts 1-vessel CAD, 12 multivessel CAD) and absent in 48 pts. Sensitivity (Sn) was 96%, 93% and 90%, whereas specificity (Sp) was 92%, 92% and 87% for Dobatro, Dipatro and Ex, respectively (p = ns). Concomitant beta blocker therapy did not influence peak rate-pressure product and Sn of Dobatro and Dipatro (p = ns). CONCLUSION: When state-of-the-art protocols are used, dipyridamole and dobutamine stress echocardiography have comparable and high diagnostic accuracy, similar to maximal post-exercise treadmill stress echocardiography. PMID- 16672048 TI - Genome-wide linkage analysis of inguinal hernia in pigs using affected sib pairs. AB - BACKGROUND: Inguinal and scrotal hernias are of great concern to pig producers, and lead to poor animal welfare and severe economic loss. Selection against these conditions is highly preferable, but at this time no gene, Quantitative Trait Loci (QTL), or mode of inheritance has been identified in pigs or in any other species. Therefore, a complete genome scan was performed in order to identify genomic regions affecting inguinal and scrotal hernias in pigs. Records from seedstock breeding farms were collected. No clinical examinations were executed on the pigs and there was therefore no distinction between inguinal and scrotal hernias. The genome scan utilised affected sib pairs (ASP), and the data was analysed using both an ASP test based on Non-parametric Linkage (NPL) analysis, and a Transmission Disequilibrium Test (TDT). RESULTS: Significant QTLs (p < 0.01) were detected on 8 out of 19 porcine chromosomes. The most promising QTLs, however, were detected in SSC1, SSC2, SSC5, SSC6, SSC15, SSC17 and SSCX; all of these regions showed either statistical significance with both statistical methods, or convincing significance with one of the methods. Haplotypes from these suggestive QTL regions were constructed and analysed with TDT. Of these, six different haplotypes were found to be differently transmitted (p < 0.01) to healthy and affected pigs. The most interesting result was one haplotype on SSC5 that was found to be transmitted to hernia pigs with four times higher frequency than to healthy pigs (p < 0.00005). CONCLUSION: For the first time in any species, a genome scan has revealed suggestive QTLs for inguinal and scrotal hernias. While this study permitted the detection of chromosomal regions only, it is interesting to note that several promising candidate genes, including INSL3, MIS, and CGRP, are located within the highly significant QTL regions. Further studies are required in order to narrow down the suggestive QTL regions, investigate the candidate genes, and to confirm the suggestive QTLs in other populations. The haplotype associated with inguinal and scrotal hernias may help in achieving selection against the disorder. PMID- 16672049 TI - Four genomic islands that mark post-1995 pandemic Vibrio parahaemolyticus isolates. AB - BACKGROUND: Vibrio parahaemolyticus is an aquatic, halophilic, Gram-negative bacterium, first discovered in 1950 in Japan during a food-poisoning outbreak. Infections resulting from consumption of V. parahaemolyticus have increased globally in the last 10 years leading to the bacterium's classification as a newly emerging pathogen. In 1996 the first appearance of a pandemic V. parahaemolyticus clone occurred, a new O3:K6 serotype strain that has now been identified worldwide as a major cause of seafood-borne gastroenteritis. RESULTS: We examined the sequenced genome of V. parahaemolyticus RIMD2210633, an O3:K6 serotype strain isolated in Japan in 1996, by bioinformatic analyses to uncover genomic islands (GIs) that may play a role in the emergence and pathogenesis of pandemic strains. We identified 7 regions ranging in size from 10 kb to 81 kb that had the characteristics of GIs such as aberrant base composition compared to the core genome, presence of phage-like integrases, flanked by direct repeats and the absence of these regions from closely related species. Molecular analysis of worldwide clinical isolates of V. parahaemolyticus recovered over the last 33 years demonstrated that a 24 kb region named V. parahaemolyticus island-1 (VPaI 1) encompassing ORFs VP0380 to VP0403 is only present in new O3:K6 and related strains recovered after 1995. We investigated the presence of 3 additional regions, VPaI-4 (VP2131 to VP2144), VPaI-5 (VP2900 to VP2910) and VPaI-6 (VPA1254 to VPA1270) by PCR assays and Southern blot analyses among the same set of V. parahaemolyticus isolates. These 3 VPaI regions also gave similar distribution patterns amongst the 41 strains examined. CONCLUSION: The 4 VPaI regions examined may represent DNA acquired by the pandemic group of V. parahaemolyticus isolates that increased their fitness either in the aquatic environment or in their ability to infect humans. PMID- 16672050 TI - PPARdelta status and mismatch repair mediated neoplasia in the mouse intestine. AB - BACKGROUND: Therapeutic regulation of PPARdelta activity using selective agonists has been proposed for various disorders. However, the consequences of altered peroxisome proliferator-activated receptor delta (PPARdelta) activity in the context of intestinal tumourigenesis remain somewhat unclear. Contradictory evidence suggesting PPARdelta either attenuates or potentiates intestinal neoplasia. To further investigate the PPARdelta dependency of intestinal tumourigenesis, we have analysed the consequences of PPARdelta deficiency upon intestinal neoplasia occurring in mice with impaired mismatch DNA repair. METHODS: Mice deficient for both PPARdelta and the mismatch repair gene Mlh1 were produced and the incidence and severity of intestinal neoplasia recorded. RESULTS: No significant differences between the control genotypes and the double mutant genotypes were recorded indicating that deficiency of PPARdelta does not modify impaired mismatch repair induced neoplasia. CONCLUSION: In contrast with the previously observed acceleration of intestinal neoplasia in the context of the ApcMin/+ mouse, PPARdelta deficiency does not alter the phenotype of mismatch repair deficiency. This data supports the notion that PPARdelta is not required for adenoma formation and indicate that any pro-tumourigenic effect of PPARdelta inactivation may be highly context dependent. PMID- 16672051 TI - The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction. AB - BACKGROUND: In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. METHODS: To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm2 for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (microPIV) experiments. RESULTS: Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within +/ 10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. CONCLUSION: The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable. PMID- 16672052 TI - Compensation by tumor suppressor genes during retinal development in mice and humans. AB - BACKGROUND: The RB1 gene was the first tumor suppressor gene cloned from humans by studying genetic lesions in families with retinoblastoma. Children who inherit one defective copy of the RB1 gene have an increased susceptibility to retinoblastoma. Several years after the identification of the human RB1 gene, a targeted deletion of Rb was generated in mice. Mice with one defective copy of the Rb gene do not develop retinoblastoma. In this manuscript, we explore the different roles of the Rb family in human and mouse retinal development in order to better understand the species-specific difference in retinoblastoma susceptibility. RESULTS: We found that the Rb family of proteins (Rb, p107 and p130) are expressed in a dynamic manner during mouse retinal development. The primary Rb family member expressed in proliferating embryonic retinal progenitor cells in mice is p107, which is required for appropriate cell cycle exit during retinogenesis. The primary Rb family member expressed in proliferating postnatal retinal progenitor cells is Rb. p130 protein is expressed redundantly with Rb in postmitotic cells of the inner nuclear layer and the ganglion cell layer of the mouse retina. When Rb is inactivated in an acute or chronic manner during mouse retinal development, p107 is upregulated in a compensatory manner. Similarly, when p107 is inactivated in the mouse retina, Rb is upregulated. No changes in p130 expression were seen when p107, Rb or both were inactivated in the developing mouse retina. In the human retina, RB1 was the primary family member expressed throughout development. There was very little if any p107 expressed in the developing human retina. In contrast to the developing mouse retina, when RB1 was acutely inactivated in the developing human fetal retina, p107 was not upregulated in a compensatory manner. CONCLUSION: We propose that intrinsic genetic compensation between Rb and p107 prevents retinoblastoma in Rb- or p107 deficient mice, but this compensation does not occur in humans. Together, these data suggest a model that explains why humans are susceptible to retinoblastoma following RB1 loss, but mice require both Rb and p107 gene inactivation. PMID- 16672053 TI - Chronic renal insufficiency among Asian Indians with type 2 diabetes: I. Role of RAAS gene polymorphisms. AB - BACKGROUND: Renal failure in diabetes is mediated by multiple pathways. Experimental and clinical evidences suggest that renin-angiotensin-aldosterone system (RAAS) has a crucial role in diabetic kidney disease. A relationship between the RAAS genotypes and chronic renal insufficiency (CRI) among type 2 diabetes subjects has therefore been speculated. We investigated the contribution of selected RAAS gene polymorphisms to CRI among type 2 diabetic Asian Indian subjects. METHODS: Twelve single nucleotide polymorphisms (SNPs) from six genes namely-renin (REN), angiotensinogen (ATG), angiotensin converting enzyme I (ACE), angiotensin II type 1 receptor (AT1) and aldosterone synthase (CYP11B2) gene from the RAAS pathway and one from chymase pathway were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and tested for their association with diabetic CRI using a case-control approach. Successive cases presenting to study centres with type 2 diabetes of > or =2 years duration and moderate CRI diagnosed by serum creatinine > or =3 mg/dl after exclusion of non-diabetic causes of CRI (n = 196) were compared with diabetes subjects with no evidence of renal disease (n = 225). Logistic regression analysis was carried out to correlate various clinical parameters with genotypes, and to study pair wise interactions between SNPs of different genes. RESULTS: Of the 12 SNPs genotyped, Glu53Stop in AGT and A>T (-777) in AT1 genes, were monomorphic and not included for further analysis. We observed a highly significant association of Met235Thr SNP in angiotensinogen gene with CRI (O.R. 2.68, 95%CI: 2.01-3.57 for Thr allele, O.R. 2.94, 95%CI: 1.88-4.59 for Thr/Thr genotype and O.R. 2.68, 95%CI: 1.97-3.64 for ACC haplotype). A significant allelic and genotypic association of T>C (-344) SNP in aldosterone synthase gene (O.R. 1.57, 95%CI: 1.16-2.14 and O.R. 1.81, 95%CI: 1.21-2.71 respectively), and genotypic association of GA genotype of G>A (-1903) in chymase gene (O.R. 2.06, 95%CI: 1.34-3.17) were also observed. CONCLUSION: SNPs Met235Thr in angiotensinogen, T>C (-344) in aldosterone synthase, and G>A (-1903) in chymase genes are significantly associated with diabetic chronic renal insufficiency in Indian patients and warrant replication in larger sample sets. Use of such markers for prediction of susceptibility to diabetes specific renal disease in the ethnically Indian population appears promising. PMID- 16672054 TI - Direct and heterologous approaches to identify the LET-756/FGF interactome. AB - BACKGROUND: Fibroblast growth factors (FGFs) are multifunctional proteins that play important roles in cell communication, proliferation and differentiation. However, many aspects of their activities are not well defined. LET-756, one of the two C. elegans FGFs, is expressed throughout development and is essential for worm development. It is both expressed in the nucleus and secreted. RESULTS: To identify nuclear factors associated with LET-756, we used three approaches. First, we screened a two-hybrid cDNA library derived from mixed stages worms and from a normalized library, using LET-756 as bait. This direct approach allowed the identification of several binding partners that play various roles in the nucleus/nucleolus, such as PAL-1, a transcription regulator, or RPS-16, a component of the small ribosomal subunit. The interactions were validated by co immunoprecipitation and determination of their site of occurrence in mammalian cells. Second, because patterns of protein interactions may be conserved throughout species, we searched for orthologs of known mammalian interactors and measured binary interaction with these predicted candidates. We found KIN-3 and KIN-10, the orthologs of CK2alpha and CK2beta, as new partners of LET-756. Third, following the assumption that recognition motifs mediating protein interaction may be conserved between species, we screened a two-hybrid cDNA human library using LET-756 as bait. Among the few FGF partners detected was 14-3-3beta. In support of this interaction we showed that the two 14-3-3beta orthologous proteins, FTT-1 and FTT-2/PAR-5, interacted with LET-756. CONCLUSION: We have conducted the first extensive search for LET-756 interactors using a multi directional approach and established the first interaction map of LET-756/FGF with other FGF binding proteins from other species. The interactors identified play various roles in developmental process or basic biochemical events such as ribosome biogenesis. PMID- 16672055 TI - Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study. AB - BACKGROUND: Hereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted by iron-overload. Two popular mutations in HFE, p.C282Y and p.H63D, have been discovered and found to associate with HH in different ethnic backgrounds. p.C282Y and p.H63D diagnosis is usually made by restriction enzyme analysis. However, the use of this technique is largely limited to research laboratories because they are relatively expensive, time-consuming, and difficult to transform into a high throughput format. METHODS: Single nucleotide variations in target DNA sequences can be readily identified using molecular beacon fluorescent probes. These are quenched probes with loop and hairpin structure, and they become fluorescent upon specific target recognition. We developed high throughput homogeneous real-time PCR assays using molecular beacon technology, to genotype p.C282Y and p.H63D variants. Representative samples of different genotypes for these variants were assayed by restriction enzyme analysis and direct sequencing as bench mark methods for comparison with the newly developed molecular beacon-based real-time PCR assay. RESULTS: Complete concordance was achieved by all three assay formats. Homozygotes (mutant and wildtype) and heterozygotes were readily differentiated by the allele specific molecular beacons as reported by the associated fluorophore in the real-time assay developed in this study. Additionally, these assays were used in a high throughput format to establish the allele frequency of C282Y and H63D in Saudis for the first time. CONCLUSION: These assays may be reliably applied as a diagnostic test or large scale method for population screening. PMID- 16672056 TI - Intra-cluster correlation coefficients in adults with diabetes in primary care practices: the Vermont Diabetes Information System field survey. AB - BACKGROUND: Proper estimation of sample size requirements for cluster-based studies requires estimates of the intra-cluster correlation coefficient (ICC) for the variables of interest. METHODS: We calculated the ICC for 112 variables measured as part of the Vermont Diabetes Information System, a cluster-randomized study of adults with diabetes from 73 primary care practices (the clusters) in Vermont and surrounding areas. RESULTS: ICCs varied widely around a median value of 0.0185 (Inter-quartile range: 0.006, 0.037). Some characteristics (such as the proportion having a recent creatinine measurement) were highly associated with the practice (ICC = 0.288), while others (prevalence of some comorbidities and complications and certain aspects of quality of life) varied much more across patients with only small correlation within practices (ICC<0.001). CONCLUSION: The ICC values reported here may be useful in designing future studies that use clustered sampling from primary care practices. PMID- 16672057 TI - Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey. AB - BACKGROUND: Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. AIMS: In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. RESULTS: The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. CONCLUSION: We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome. PMID- 16672058 TI - Self-reported drug utilization, health, and lifestyle factors among 70-74 year old community dwelling individuals in Western Norway. The Hordaland Health Study (HUSK). AB - BACKGROUND: To examine the level and patterns of self-reported medication use (prescription and non-prescription drugs) among 70-74 year old individuals living in the community, and to explore self-reported indications for use, and factors possibly predictive of drug use. METHODS: A health survey carried out in 1997-99 in the county of Hordaland (Western Norway) in the setting of a population study. A self-administered questionnaire was mailed to 4338 persons born in 1925-27, and a health check-up was offered. Drug use the previous day was reported (point prevalence). 3341 (77.0%) persons who responded, comprise the material for the analyses. RESULTS: Between one third (males) and one quarter (females) did not take any drug the previous day. Mean number of drugs among users was 2.8 (men and women). 32% used three or more drugs and 11.5% five or more. Hypertension and other cardiovascular problems were by far the most common reasons for drug use, followed by respiratory, musculoskeletal and mental health problems. Self reported poor health, a high Body Mass Index (BMI), and being an ex-smoker (but not currently a smoker) correlated with increasing number of drugs taken. CONCLUSION: Among 70-74-year old individuals living in the community no use of medication was more common than major polypharmacy (5+ drugs). Persons who had fallen ill and were put on regular medication, probably tended to quit smoking, while those who remained healthy, continued to smoke. PMID- 16672059 TI - Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis. AB - BACKGROUND: Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. RESULTS: Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10 phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood brain barrier perturbations. CONCLUSION: Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae. PMID- 16672060 TI - Thyroid papillary carcinoma arising in ectopic thyroid tissue within a neck branchial cyst. AB - BACKGROUND: Thyroid gland derives from one median anlage at the base of the tongue, and from the two fourth branchial pouches. A number of anomalies may occur during their migration. These can be in form of ectopic tissues, which are frequently found along the course of thyroglossal duct and rarely in other sites, many of these may develop same diseases as the thyroid gland. CASE PRESENTATION: A 36-years-old female presented with a 3 month history of left side neck mass. The mass disappeared following aspiration of brown colored fluid, which on cytological examination showed cells with nuclear irregularities that warranted the resection of the lesion. The histology demonstrated a thyroid papillary carcinoma arising within the branchial cyst. Thereafter, the patient underwent a total thyroidectomy with central lymph nodes dissection. Histology showed a multifocal papillary carcinoma with central lymph nodes metastases. Only four cases of primary thyroid carcinomas in neck branchial cyst have been described so far. CONCLUSION: In a lateral cystic neck mass, although rare, occurrence of ectopic thyroid tissue and presence of a papillary thyroid carcinoma should be kept in mind. PMID- 16672061 TI - Electrical impedance tomography system: an open access circuit design. AB - BACKGROUND: This paper reports a simple 2-D system for electrical impedance tomography EIT, which works efficiently and is low cost. The system has been developed in the Sharif University of Technology Tehran-Iran (for the author's MSc Project). METHODS: The EIT system consists of a PC in which an I/O card is installed with an external current generator, a multiplexer, a power supply and a phantom with an array of electrodes. The measurement system provides 12-bit accuracy and hence, suitable data acquisition software has been prepared accordingly. The synchronous phase detection method has been implemented for voltage measurement. Different methods of image reconstruction have been used with this instrument to generate electrical conductivity images. RESULTS: The results of simulation and real measurement of the system are presented. The reconstruction programs were written in MATLAB and the data acquisition software in C++. The system has been tested with both static and dynamic mode in a 2-D domain. Better results have been produced in the dynamic mode of operation, due to the cancellation of errors. CONCLUSION: In the spirit of open access publication the design details of this simple EIT system are made available here. PMID- 16672062 TI - Waterborne microbial risk assessment: a population-based dose-response function for Giardia spp. (E.MI.R.A study). AB - BACKGROUND: Dose-response parameters based on clinical challenges are frequently used to assess the health impact of protozoa in drinking water. We compare the risk estimates associated with Giardia in drinking water derived from the dose response parameter published in the literature and the incidence of acute digestive conditions (ADC) measured in the framework of an epidemiological study in a general population. METHODS: The study combined a daily follow-up of digestive morbidity among a panel of 544 volunteers and a microbiological surveillance of tap water. The relationship between incidence of ADC and concentrations of Giardia cysts was modeled with Generalized Estimating Equations, adjusting on community, age, tap water intake, presence of bacterial indicators, and genetic markers of viruses. The quantitative estimate of Giardia dose was the product of the declared amount of drinking water intake (in L) by the logarithm of cysts concentrations. RESULTS: The Odds Ratio for one unit of dose [OR = 1.76 (95% CI: 1.21, 2.55)] showed a very good consistency with the risk assessment estimate computed after the literature dose-response, provided application of a 20 % abatement factor to the cysts counts that were measured in the epidemiological study. Doing so, a daily water intake of 2 L and a Giardia concentration of 10 cysts/100 L, would yield an estimated relative excess risk of 12 % according to the Rendtorff model, against 11 % when multiplying the baseline rate of ADC by the corresponding OR. This abatement parameter encompasses uncertainties associated with germ viability, infectivity and virulence in natural settings. CONCLUSION: The dose-response function for waterborne Giardia risk derived from clinical experiments is consistent with epidemiological data. However, much remains to be learned about key characteristics that may heavily influence quantitative risk assessment results. PMID- 16672065 TI - Acupuncture is a feasible treatment for post-thoracotomy pain: results of a prospective pilot trial. AB - BACKGROUND: Thoracotomy is associated with severe pain that may persist for years. Acupuncture is a complementary therapy with a proven role in pain control. A randomized trial showed that acupuncture was effective in controlling pain after abdominal surgery, but the efficacy of this technique for the treatment of thoracotomy pain has not been established. We developed a novel technique for convenient application of acupuncture to patients undergoing thoracotomy, and in a Phase II trial evaluated the safety of this intervention and the feasibility of doing a randomized trial. METHODS: Adult patients scheduled for unilateral thoracotomy with preoperative epidural catheter placement received acupuncture immediately prior to surgery. Eighteen semi-permanent intradermal needles were inserted on either side of the spine, and four were inserted in the legs and auricles. Needles were removed after four weeks. Using a numerical rating scale, pain was measured on the first five postoperative days. After discharge, pain was assessed using the Brief Pain Inventory at 7, 30, 60 and 90 days. RESULTS: Thirty six patients were treated with acupuncture. Of these, 25, 23, and 22 patients provided data at 30, 60, and 90 days, respectively. The intervention was well tolerated by patients with only one minor and transient adverse event of skin ulceration. CONCLUSION: The rate of data completion met our predefined criterion for determining a randomized trial to be feasible (at least 75% of patients tolerated the intervention and provided evaluable data). This novel intervention is acceptable to patients undergoing thoracotomy and does not interfere with standard preoperative care. There was no evidence of important adverse events. We are now testing the hypothesis that acupuncture significantly adds to standard perioperative pain management in a randomized trial. PMID- 16672064 TI - How effective is tetracaine 4% gel, before a peripherally inserted central catheter, in reducing procedural pain in infants: a randomized double-blind placebo controlled trial [ISRCTN75884221]. AB - BACKGROUND: Procedural pain relief is sub-optimal in infants, especially small and vulnerable ones. Tetracaine gel 4% (Ametop, Smith-Nephew) provides pain relief in children and larger infants, but its efficacy in smaller infants and for peripherally inserted central catheters (PICC) remains uncertain. The objective of this trial was to assess the safety and efficacy of tetracaine gel on the pain response of very low birth weight (VLBW) infants during insertion of a PICC. METHODS: Medically stable infants greater than or equal to 24 weeks gestation, requiring a non-urgent PICC, were included. Following randomization and double blinding, 1.1 g of tetracaine or placebo was applied to the skin for 30 minutes. The PICC was inserted according to a standard protocol. Pain was assessed using the Premature Infant Pain Profile (PIPP). A 3-point change in the pain score was considered clinically significant, leading to a sample size of 54 infants, with 90% statistical power. Local skin reactions and immediate adverse cardiorespiratory events were noted. The primary outcome, PIPP score at 1 minute, was analysed using an independent Student's t-test. RESULTS: Fifty-four infants were included, 27 +/- 2 weeks gestation, 916 +/- 292 grams and 6.5 +/- 3.2 days of age. Baseline characteristics were similar between groups. The mean PIPP score in the first minute was 10.88 in the treatment group as compared to 11.74 in the placebo group (difference 0.86, 95% CI -1.86, 3.58). Median duration of crying in non-intubated infants was 181 seconds in the tetracaine group compared to 68 seconds in the placebo group (difference -78, 95% CI -539, 117). Local skin erythema was observed transiently in 4 infants (3 in the treatment and 1 in the placebo group). No serious harms were observed. CONCLUSION: Tetracaine 4% when applied for 30 minutes was not beneficial in decreasing procedural pain associated with a PICC in very small infants. PMID- 16672067 TI - Assessing effects of a media campaign on HIV/AIDS awareness and prevention in Nigeria: results from the VISION Project. AB - BACKGROUND: In response to the growing HIV epidemic in Nigeria, the U.S. Agency for International Development (USAID) initiated the VISION Project, which aimed to increase use of family planning, child survival, and HIV/AIDS services. The VISION Project used a mass-media campaign that focused on reproductive health and HIV/AIDS prevention. This paper assesses to what extent program exposure translates into increased awareness and prevention of HIV/AIDS. METHODS: This analysis is based on data from the 2002 and 2004 Nigeria (Bauchi, Enugu, and Oyo) Family Planning and Reproductive Health Surveys, which were conducted among adults living in the VISION Project areas. To correct for endogeneity, two-stage logistic regression is used to investigate the effect of program exposure on 1) discussion of HIV/AIDS with a partner, 2) awareness that consistent condom use reduces HIV risk, and 3) condom use at last intercourse. RESULTS: Exposure to the VISION mass media campaign was high: 59%, 47%, and 24% were exposed to at least 1 VISION radio, printed advertisement, or TV program about reproductive health, respectively. The differences in outcome variables between 2002 baseline data and the 2004 follow-up data were small. However, those with high program exposure were almost one and a half (Odds Ratio [O.R.] = 1.47, 95% Confidence Interval [C.I.] 1.01-2.16) times more likely than those with no exposure to have discussed HIV/AIDS with a partner. Those with high program exposure were over twice (O.R. = 2.20, C.I. 1.49-3.25) as likely as those with low exposure to know that condom use can reduce risk of HIV infection. Program exposure had no effect on condom use at last sex. CONCLUSION: The VISION Project reached a large portion of the population and exposure to mass media programs about reproductive health and HIV prevention topics can help increase HIV/AIDS awareness. Programs that target rural populations, females, and unmarried individuals, and disseminate information on where to obtain condoms, are needed to reduce barriers to condom use. Improvements in HIV/AIDS prevention behaviour are likely to require that these programmatic efforts be continued, scaled up, done in conjunction with other interventions, and targeted towards individuals with specific socio demographic characteristics. PMID- 16672066 TI - SNP-SNP interactions in breast cancer susceptibility. AB - BACKGROUND: Breast cancer predisposition genes identified to date (e.g., BRCA1 and BRCA2) are responsible for less than 5% of all breast cancer cases. Many studies have shown that the cancer risks associated with individual commonly occurring single nucleotide polymorphisms (SNPs) are incremental. However, polygenic models suggest that multiple commonly occurring low to modestly penetrant SNPs of cancer related genes might have a greater effect on a disease when considered in combination. METHODS: In an attempt to identify the breast cancer risk conferred by SNP interactions, we have studied 19 SNPs from genes involved in major cancer related pathways. All SNPs were genotyped by TaqMan 5'nuclease assay. The association between the case-control status and each individual SNP, measured by the odds ratio and its corresponding 95% confidence interval, was estimated using unconditional logistic regression models. At the second stage, two-way interactions were investigated using multivariate logistic models. The robustness of the interactions, which were observed among SNPs with stronger functional evidence, was assessed using a bootstrap approach, and correction for multiple testing based on the false discovery rate (FDR) principle. RESULTS: None of these SNPs contributed to breast cancer risk individually. However, we have demonstrated evidence for gene-gene (SNP-SNP) interaction among these SNPs, which were associated with increased breast cancer risk. Our study suggests cross talk between the SNPs of the DNA repair and immune system (XPD-[Lys751Gln] and IL10-[G(-1082)A]), cell cycle and estrogen metabolism (CCND1-[Pro241Pro] and COMT-[Met108/158Val]), cell cycle and DNA repair (BARD1 [Pro24Ser] and XPD-[Lys751Gln]), and within carcinogen metabolism (GSTP1 [Ile105Val] and COMT-[Met108/158Val]) pathways. CONCLUSION: The importance of these pathways and their communication in breast cancer predisposition has been emphasized previously, but their biological interactions through SNPs have not been described. The strategy used here has the potential to identify complex biological links among breast cancer genes and processes. This will provide novel biological information, which will ultimately improve breast cancer risk management. PMID- 16672068 TI - A new water-based topical carrier with polar skin-lipids. AB - A new water-based topical formulation is presented that aims at providing good penetration properties for both lipophilic and hydrophilic drugs with as small a disturbance of the skin barrier function as possible. The formulation contains dispersed lipids in a ratio resembling that of human skin. The capacity to deliver is addressed in this first study while the mild effect on skin will be presented later. Three variations of the lipid formulation were investigated by use of pigskin in vitro diffusion cell. The hydrophilic 5(6)-carboxyfluorescein (CF) and the lipophilic acridine orange 10-nonyl bromide (AO) were used as model drug substances. The results showed that the delivery properties of the new formulation exceeded that of the references (vaseline and xanthan gum gel). The effect was largest for lipophilic AO where all lipid matrix formulations were superior in amount detected in the skin. The results for the hydrophilic CF were also promising. Especially efficient was the lipid formulation containing the non ionic adjuvants tetra ethylene glycol monododecyl ether and polyoxyethylene 23 dodecyl ether. The additional in vivo study suggests that the used in vitro model has qualitative bearing on relevant in vivo situations. PMID- 16672069 TI - Validation of a patient satisfaction questionnaire for anemia treatment, the PSQ An. AB - BACKGROUND: Treating anemia associated with chemotherapy and many cancers is often necessary. However, patient satisfaction with anemia treatment is limited by the lack of validated instruments. We developed and validated a new treatment specific patient satisfaction instrument: the Patient Satisfaction Questionnaire for Anemia Treatment (PSQ-An). Treatment burden and overall satisfaction scales were designed for ease of use in clinical practice. METHODS: 312 cancer patients (141 breast, 69 gynecological, and 102 non-small cell lung) were targeted to complete the PSQ-An at 4 week intervals. Data from weeks 5 and 9 were analyzed. Patients also completed the MOS SF-36 Global Health assessment and questions concerning resources devoted to anemia treatment. Item reduction used endorsement rates, floor/ceiling effects, and item-item correlations. Factor analysis identified meaningful subscales. Test-retest reliability was assessed. Construct validity was tested, using Pearson's correlations, by comparing subscale scores to Global Health, hemoglobin levels, and resources devoted to anemia treatment. RESULTS: The overall response rate was 92.9% (264/284) at week 5. Most (84.2%) of the patients were female, and the mean (SD) age was 60.2 (+/- 11.8) years. Two distinct subscales were identified measuring treatment burden (7 items) and overall satisfaction (2 items). Test-retest reliability was examined (ICC: 0.45 0.67); both were internally consistent (alpha = 0.83). Both subscales exhibited convergent and divergent validity with independent measures of health. ANOVA results indicated that the PSQ-An Satisfaction subscale discriminated between 5 levels of MOS SF-36 Global Health (P = 0.006). CONCLUSION: The PSQ-An is a validated, treatment-specific instrument for measuring satisfaction with anemia treatment for cancer patients. PSQ-An subscales reflect the burden of injection anemia treatment on cancer patients and their assessment of the overall treatment value. PMID- 16672070 TI - Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines. AB - BACKGROUND: In order to compare the gene expression profiles of human embryonic stem cell (hESC) lines and their differentiated progeny and to monitor feeder contaminations, we have examined gene expression in seven hESC lines and human fibroblast feeder cells using Illumina bead arrays that contain probes for 24,131 transcript probes. RESULTS: A total of 48 different samples (including duplicates) grown in multiple laboratories under different conditions were analyzed and pairwise comparisons were performed in all groups. Hierarchical clustering showed that blinded duplicates were correctly identified as the closest related samples. hESC lines clustered together irrespective of the laboratory in which they were maintained. hESCs could be readily distinguished from embryoid bodies (EB) differentiated from them and the karyotypically abnormal hESC line BG01V. The embryonal carcinoma (EC) line NTera2 is a useful model for evaluating characteristics of hESCs. Expression of subsets of individual genes was validated by comparing with published databases, MPSS (Massively Parallel Signature Sequencing) libraries, and parallel analysis by microarray and RT-PCR. CONCLUSION: we show that Illumina's bead array platform is a reliable, reproducible and robust method for developing base global profiles of cells and identifying similarities and differences in large number of samples. PMID- 16672071 TI - Coexistence of K-ras mutations and HPV infection in colon cancer. AB - BACKGROUND: Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. METHODS: K ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Statistical analyses were performed by the chi-square and Fisher's exact tests RESULTS: HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. CONCLUSION: Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype. PMID- 16672072 TI - The interferon gamma gene polymorphism +874 A/T is associated with severe acute respiratory syndrome. AB - BACKGROUND: Cytokines play important roles in antiviral action. We examined whether polymorphisms of IFN-gamma,TNF-alpha and IL-10 affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). METHODS: A case control study was carried out in 476 Chinese SARS patients and 449 healthy controls. We tested the polymorphisms of IFN-gamma,TNF-alpha and IL-10 for their associations with SARS. RESULTS: IFN-gamma +874A allele was associated with susceptibility to SARS in a dose-dependent manner (P < 0.001). Individuals with IFN-gamma +874 AA and AT genotype had a 5.19-fold (95% Confidence Interval [CI], 2.78-9.68) and 2.57-fold (95% CI, 1.35-4.88) increased risk of developing SARS respectively. The polymorphisms of IL-10 and TNF-alpha were not associated with SARS susceptibility. CONCLUSION: IFN-gamma +874A allele was shown to be a risk factor in SARS susceptibility. PMID- 16672073 TI - Patterns of ambulatory care utilization in Taiwan. AB - BACKGROUND: We used the insurance claims of a representative cohort to quantify the patterns of ambulatory care visits, especially the doctor-shopping phenomenon, in Taiwan. METHODS: The ambulatory visit files of the 200,000-person cohort datasets from the National Health Insurance Research Database in 2002 were analyzed. Only a visit with physician consultation would be considered. We computed the visit patterns both by visit count and by patient count. RESULTS: In 2002, there were 182,474 eligible people with 2,443,003 physician consultations. During the year, 87.4% of the cohort had visited physician clinics and 57.5% had visited hospital-based outpatient or emergency departments. On average, a person had 13.4 physician consultations and consulted 3.4 specialties, 5.2 physicians, and 3.9 healthcare facilities in a year. In 2002, 17.3% of the cohort had ever visited different healthcare facilities on the same day; 23.5% had ever visited physicians of the same specialty at different healthcare facilities within 7 days and the percentage of second visits was 3.8% of all visits. Besides, 7.6% of the cohort had visited two or more specialties at the same facility on the same day, and such visits make up 2.5% of all visits. CONCLUSION: The people in Taiwan did visit the physicians and outpatient departments frequently. Many patients not only consulted several physicians of different specialties and at different healthcare facilities during the year, but also switched the physicians and facilities quickly. An effective referral system with efficient data exchange between facilities might be the solution. PMID- 16672074 TI - Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package. AB - BACKGROUND: Availability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA) for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment. RESULTS: Three-dimensional structure of rRNA is visualized in OpenGL 3D environment with the abilities to change the display and overlay information onto the molecule, dynamically. Phylogenetic information derived from the multiple sequence alignments can be overlaid onto the molecule structure in a real time. Superimposition of both statistical and non-statistical sequence associated information onto the rRNA 3D structure can be done using customizable color scheme, which is also applied to a textual sequence alignment for reference. Oligonucleotide probes designed by ARB probe design tools can be mapped onto the 3D structure along with the probe accessibility models for evaluation with respect to secondary and tertiary structural conformations of rRNA. CONCLUSION: Visualization of three-dimensional structure of rRNA in an intuitive display provides the biologists with the greater possibilities to carry out structure based phylogenetic analysis. Coupled with secondary structure models of rRNA, RNA3D program aids in validating the sequence alignments of rRNA genes and evaluating probe target sites. Superimposition of the information derived from the multiple sequence alignment onto the molecule dynamically allows the researchers to observe any sequence inherited characteristics (phylogenetic information) in real-time environment. The extended ARB software package is made freely available for the scientific community via http://www.arb-home.de. PMID- 16672075 TI - Herbal medicine: the science of the art. AB - In the last 50 years science has provided new perspectives on the ancient art of herbal medicine. The present article discusses ways in which the evidence base for the professional use of 'Western' herbal medicine, as therapy to treat disease, known as phytotherapy, can be strengthened and developed. The evidence base for phytotherapy is small and lags behind that for the nutritional sciences, mainly because phytochemicals are ingested as complex mixtures that are incompletely characterised and have only relatively recently been subject to scientific scrutiny. While some methodologies developed for the nutritional sciences can inform phytotherapy research, opportunities for observational studies are more limited, although greater use could be made of patient case notes. Randomised clinical trials of single-herb interventions are relatively easy to undertake and increasing numbers of such studies are being published. Indeed, enough data are available on three herbs (ginkgo (Ginkgo biloba), St John's wort (Hypericum perforatum) and saw palmetto (Serenoa repens)) for meta analyses to have been undertaken. However, phytotherapy is holistic therapy, using lifestyle advice, nutrition and individually-prescribed mixtures of herbs aimed at reinstating homeostasis. While clinical experience shows that this approach is applicable to a wide range of conditions, including chronic disease, evidence of its efficacy is scarce. Strategies for investigating the full holistic approach of phytotherapy and its main elements are discussed and illustrated through the author's studies at the University of Reading. PMID- 16672076 TI - Biofortification of essential nutritional compounds and trace elements in rice and cassava. AB - Plant biotechnology can make important contributions to food security and nutritional improvement. For example, the development of 'Golden Rice' by Professor Ingo Potrykus was a milestone in the application of gene technology to deliver both increased nutritional qualities and health improvement to wide sections of the human population. Mineral nutrient and protein deficiency as well as food security remain the most important challenges for developing countries. Current projects are addressing these issues in two major staple crops, cassava (Manihot esculenta Crantz) and rice. The tropical root crop cassava is a major source of food for approximately 600 million of the population worldwide. In sub Saharan Africa >200 million of the population rely on cassava as their major source of dietary energy. The nutritional quality of the cassava root is not sufficient to meet all dietary needs. Rice is the staple food for half the world population, providing approximately 20% of the per capita energy and 13% of the protein for human consumption worldwide. In many developing countries the dietary contributions of rice are substantially greater (29.3% dietary energy and 29.1% dietary protein). The current six most popular 'mega' rice varieties (in terms of popularity and acreage), including Chinese hybrid rice, have an incomplete amino acid profile and contain limited amounts of essential micronutrients. Rice lines with improved Fe contents have been developed using genes that have functions in Fe absorption, translocation and accumulation in the plant, as well as improved Fe bioavailability in the human intestine. Current developments in biotechnology assisted plant improvement are reviewed and the potential of the technology in addressing human nutrition and health are discussed. PMID- 16672077 TI - Improving the bioavailability of nutrients in plant foods at the household level. AB - Plant foods are the major staples of diets in developing countries, in which the consumption of animal-source foods is often low because of economic and/or religious concerns. However, such plant-based diets are often associated with micronutrient deficits, exacerbated in part by poor micronutrient bioavailability. Diet-related factors in plant foods that affect bioavailability include: the chemical form of the nutrient in food and/or nature of the food matrix; interactions between nutrients and other organic components (e.g. phytate, polyphenols, dietary fibre, oxalic acid, protein, fat, ascorbic acid); pretreatment of food as a result of processing and/or preparation practices. Consequently, household strategies that reduce the content or counteract the inhibiting effects of these factors on micronutrient bioavailability are urgently needed in developing-country settings. Examples of such strategies include: germination, microbial fermentation or soaking to reduce the phytate and polyphenol content of unrefined cereal porridges used for young child feeding; addition of ascorbic acid-containing fruits to enhance non-haem-Fe absorption; heating to destroy heat-labile anti-nutritional factors (e.g. goitrogens, thiaminases) or disrupt carotenoid-protein complexes. Such strategies have been employed in both experimental isotope-absorption and community-based studies. Increases in Fe, Zn and Ca absorption have been reported in adults fed dephytinized cereals compared with cereals containing their native phytate. In community-based studies in rural Malawi improvements in dietary quality and arm muscle area and reductions in the incidence of anaemia and common infections in young children have been observed. PMID- 16672078 TI - Biofortification of UK food crops with selenium. AB - Se is an essential element for animals. In man low dietary Se intakes are associated with health disorders including oxidative stress-related conditions, reduced fertility and immune functions and an increased risk of cancers. Although the reference nutrient intakes for adult females and males in the UK are 60 and 75 microg Se/d respectively, dietary Se intakes in the UK have declined from >60 microg Se/d in the 1970s to 35 microg Se/d in the 1990s, with a concomitant decline in human Se status. This decline in Se intake and status has been attributed primarily to the replacement of milling wheat having high levels of grain Se and grown on high-Se soils in North America with UK-sourced wheat having low levels of grain Se and grown on low-Se soils. An immediate solution to low dietary Se intake and status is to enrich UK-grown food crops using Se fertilisers (agronomic biofortification). Such a strategy has been adopted with success in Finland. It may also be possible to enrich food crops in the longer term by selecting or breeding crop varieties with enhanced Se-accumulation characteristics (genetic biofortification). The present paper will review the potential for biofortification of UK food crops with Se. PMID- 16672079 TI - Linking biodiversity, diet and health in policy and practice. AB - Simplification of human diets associated with increased accessibility of inexpensive agricultural commodities and erosion of agrobiodiversity leads to nutrient deficiencies and excess energy consumption. Non-communicable diseases are growing causes of death and disability worldwide. Successful food systems in transition effectively draw on locally-available foods, food variety and traditional food cultures. In practice this process involves empirical research, public policy, promotion and applied action in support of multi-sectoral, community-based strategies linking rural producers and urban consumers, subsistence and market economies, and traditional and modern food systems. Implementation of the International Plant Genetic Resources Institute's Global Nutrition Strategy in Sub-Saharan Africa offers a useful case study. Relevant policy platforms, in which biodiversity conservation and nutrition are and should be linked, include the Millennium Development Goals, Millennium Ecosystem Assessment, Convention on Biological Diversity, Global Strategy on Diet, Physical Activity and Health, Food-Based Dietary Guidelines, Right to Adequate Food and UN Human Rights Commission's Permanent Forum on Indigenous Issues. The largely unexplored health benefits of cultivated and wild plants include micronutrient intake and functions related to energy density, glycaemic control, oxidative stress and immuno-stimulation. Research on the properties of neglected and underutilized species and local varieties deserves higher priority. In tests of the hypothesis that biodiversity is essential for dietary diversity and health, quantitative indicators of dietary and biological diversity can be combined with nutrition and health outcomes at the population level. That traditional systems once lost are hard to recreate underlines the imperative for timely documentation, compilation and dissemination of eroding knowledge of biodiversity and the use of food culture for promoting positive behaviours. PMID- 16672080 TI - Wild-gathered fungi for health and rural livelihoods. AB - Fungi are a good source of digestible proteins and fibre, are low in fat and energy and make a useful contribution to vitamin and mineral intake. In terms of current dietary advice, 80 g fungi represent one portion of vegetables. Dried fungi and concentrated extracts are also used as medicines and dietary supplements. Some species show strong anti-tumour and antioxidant activity by enhancing various immune system functions and lowering cholesterol levels. Nevertheless, there are also some safety concerns. Edible species might be mistaken for poisonous ones, high heavy-metal concentrations in wild edible fungi (WEF) are a known source of chronic poisoning and the consumption of WEF can contribute markedly to the radiocaesium intake of human subjects. Some regions of Europe have a strong WEF tradition, especially eastern Europe. In the UK the consumption of wild fungi is considered of minor importance. Only one-third of adults consume fungi (cultivated species and WEF) throughout the UK; the average intake of fungi in the UK is estimated to be 0.12 kg fresh weight per capita per year. At least eighty-two species of wild fungi are recorded as being consumed in the UK, although certain species (e.g. chanterelle (Cantharellus cibarius), cep (Boletus edulis), oyster mushroom (Pleurotus ostreatus)) are favoured over others. Although WEF are not essential components in the daily diet, they are a nutritionally-valuable addition to the range of vegetables consumed, and their role in helping to avert food shortages in less-favoured areas should be definitely considered. PMID- 16672081 TI - Plant foods for human health: research challenges. AB - Plants provide the major part of human food intake. Whilst advances in agronomic characteristics (improved yield and better pest and disease resistance) continue to be a very high priority, there is increasing opportunity to enhance the nutritional value of plant based diets by improving the nutritional quality of staple foods. We now have proof of principle that genetic engineering can be used to produce plant-derived human vaccines. In relation to plant foods for human health, the research challenges include understanding: (1) why certain foods cause adverse reactions in some individuals but not in others; (2) the mechanisms of action of apparently 'protective' foods such as fruits and vegetables. There is also a need to develop much more informative and robust methods for measuring dietary exposure to specific plant foods or food constituents, including both recent exposure, for which a metabolomics approach may be particularly helpful, and long-term exposure. PMID- 16672082 TI - Genetic variation in genes of folate metabolism and neural-tube defect risk. AB - Neural-tube defects (NTD) are common congenital malformations that can lead to severe disability or even death. Periconceptional supplementation with the B vitamin folic acid has been demonstrated to prevent 50-70% of NTD cases. Since the identification of the first genetic risk factor of NTD, the C677T single nucleotide polymorphism (SNP) in the methylenetetrahydrofolate reductase (MTHFR) gene, and the observation that elevated plasma homocysteine levels are associated with NTD, research has focused on genetic variation in genes encoding for enzymes of folate metabolism and the closely-related homocysteine metabolism. In the present review relevant SNP in genes that code for enzymes involved in folate transport and uptake, the folate cycles and homocysteine metabolism are summarised and the importance of these SNP discussed in relation to NTD risk. PMID- 16672084 TI - Recurrent outbreaks of cryptosporidiosis associated with calves among students at an educational farm programme, Minnesota, 2003. AB - Enteric illness outbreaks among middle-/high-school students in consecutive semesters of an educational farm programme were investigated with retrospective cohort studies. During the first outbreak, 31/92 (34%) interviewed students were ill. Risk factors included participating in animal science class (RR 8.1, 95% CI 1.2-55.2) and contact with calves (RR 4.2, 95% CI 1.1-16.2). Stool samples from seven students and two calves yielded Cryptosporidium parvum. Students cared for animals in street clothes and practised poor hand washing. During the second outbreak, 37/81 (46%) interviewed animal science students were ill. Risk factors included having visible manure on hands, and wearing coveralls and boots. Stool samples from seven students and eight calves yielded C. parvum. Student hand washing was still inadequate. Coveralls/boots were cleaned infrequently and removed after hand washing. These outbreaks of cryptosporidiosis resulted from calf contact and inadequate hygiene practices. The failure to adequately implement recommended interventions contributed to the second outbreak. PMID- 16672085 TI - The Larynxane ST intralaryngeal endoprosthesis for laryngotracheal pathologies. AB - OBJECTIVES: The authors present their experience of a new intralaryngeal silicone prosthesis used to manage laryngotracheal pathologies associated with severe deglutition and respiratory disorders. STUDY DESIGN: This retrospective study, conducted in the head and neck surgery department of the Strasbourg University Hospital, France, included 10 patients (12 prostheses inserted) and was conducted from November 2000 to November 2003. METHODS: A pre-operative clinical examination and a computed tomography and/or magnetic resonance scan assessed patients' laryngeal function. Two different semirigid laryngotracheal prostheses were used, inserted under general anaesthetic into the region from the supraglottic area to the first tracheal cartilages including the vocal folds and the ventricles. In group one (n = 5), patients presented with laryngeal stenosis with preserved deglutition function, and patent prostheses were used to restore laryngeal patency. In group two (n = 5), patients suffered from severe deglutition disorders and were in poor general condition and so required a cuffed tracheostomy tube, therefore obstructed prostheses were inserted to stop aspirations and to suppress the cuff of the tracheostomy tube. The study was performed under the authorization of the Biomedical Research Patients' Protection Committee of Alsace I, France. RESULTS: The mean implantation duration was six months. The prosthesis used in the first group restored laryngeal patency without further complications such as aspiration or prosthesis (mucous) obstruction. The prosthesis used in group two prevented aspiration and allowed to change a cuff by an uncuffed tracheostomy tube. CONCLUSIONS: The intralaryngeal prosthesis used in group one constituted a reversible and effective method of treating laryngotracheal stenoses. The intralaryngeal prosthesis used in group two constituted an alternative to classical larynx exclusion techniques. The insertion is performed in few minutes under general anaesthetic through an endoscopic procedure. This reversible technique aimed to treat patients rapidly to reduce complications and post-operative morbidity. PMID- 16672086 TI - Assessment of expressive vocabulary outcomes in hearing-impaired children with hearing aids: do bilaterally hearing-impaired children catch up? AB - OBJECTIVE: To evaluate expressive vocabulary growth in hearing-impaired preschool children wearing hearing aids. DESIGN: Prospective analysis of the outcomes of children included in the 1994 German 'Goettinger Hoer-Sprachregister' (GHR) series, using a repeated-measures paradigm in six- to nine-month intervals (t1 t3). SUBJECTS: Twenty-seven children (aged 2.0-4.4 years) with bilateral sensorineural hearing loss (with averages at frequencies of 0.5, 1, 2 and 4 kHz of > 20 to > 90 dB in the better ear) from the 1994 GHR series. The children were diagnosed at a mean age of 31.4 months (standard deviation (SD) 10.6 months) and fitted with a binaural hearing aid at a mean age of 32.3 months (SD 10.5 months). Nonverbal intelligence was average (five missing data entries). Standardized, age appropriate picture naming tests (the 'Sprachentwicklungstest fur 2-jahrige Kinder', the Kaufman Assessment Battery for Children subtest vocabulary, and the 'Aktiver Wortschatztest fur drei- bis sechsjahrige Kinder') were carried out at three time points and results compared with data from children with normal hearing. The test raw scores were converted to T scores (mean = 50; SD = 10). RESULTS: On average, the children scored far below the normative population at t1 (mean = 28.9; SD = 11.3) and slowly improved as they got older (at t3, mean = 34.1; SD = 16.1; p = 0.010). Children with mild or moderate hearing loss improved most notably (mean difference t1-t3; p = 0.001), except for one child of deaf parents. Two of the five mildly hearing-impaired children and two of the eleven moderately hearing-impaired children caught up with their normal hearing peers with regards to expressive vocabulary. Such expressive vocabulary achievements were not seen in any children with > 70 dB hearing loss or in six of the eleven children (55 per cent) with a 40-70 dB hearing loss, despite receiving adequate personal amplification. CONCLUSION: Testing expressive vocabulary size is a useful clinical tool in assessing linguistic lexical outcome. PMID- 16672087 TI - Covering ENT out of hours: how confident are senior house officers? AB - BACKGROUND: The implementation of the European working time directive has led to an increase in cross-speciality out-of-hours cover. This survey illustrates ENT out-of-hours cover arrangements and assesses the implications for senior house officers (SHOs) responsible for managing emergencies. METHODS: A telephone survey of 100 ENT departments was conducted, asking the on-call SHO about departmental structure, on-call rota design, their previous ENT experience, access to SHO training and their confidence in managing emergencies. RESULTS: 44 per cent of departments used only ENT SHOs on the on-call rota. 73 per cent always had an ENT middle grade on call. In 60 per cent of hospitals, the ENT consultant was sometimes on call with only a non-ENT SHO. At the time of the study, 5 per cent of SHOs had no ENT experience, no access to training, were not confident in managing simple emergencies and were on-call without middle-grade cover. CONCLUSION: The current junior on-call structure for ENT has implications for patient management. PMID- 16672088 TI - Optimizing training opportunities for otolaryngology senior house officers: an audit. AB - Implementation of the European Working Time Directive and the Modernising Medical Careers initiative will mean junior surgeons must be trained in fewer hours over a shorter period. For this reason, junior surgeon training opportunities must be optimized. We undertook a departmental audit to identify where opportunities to train senior house officers (SHOs) in theatre were being lost, so that appropriate timetable changes could be made in order to optimize exposure to suitable surgical cases. During the first audit cycle, the SHOs followed their existing timetable and theatre attendance was monitored prospectively over a two week period. Only 30 per cent of theatre sessions were attended and case participation was only 27 per cent. Simple timetable changes were made to maximize SHO theatre attendance, and a second prospective two-week audit was undertaken. The new rota yielded 46 per cent theatre attendance and 48 per cent case participation. PMID- 16672089 TI - Sinonasal inverted papilloma with malignant transformation in the middle ear: a multicentric origin? AB - Inverted papilloma is a rare, benign tumour representing only 0.5 to 4 per cent of all sinonasal neoplasms; its involvement of the middle ear is extremely rare. We present a case of multicentric inverted papilloma in the sinonasal region and middle ear in a 54-year-old man. The patient later developed neck metastasis secondary to malignant transformation of the inverted papilloma in the middle ear. PMID- 16672090 TI - Bone-anchored hearing aids: results of the first eight years of a programme in a district general hospital, assessed by the Glasgow benefit inventory. AB - OBJECTIVES: To report the clinical results of a bone-anchored hearing aid (BAHA) programme in a district general hospital, compared with those in an established, large, university teaching hospital centre. DESIGN: A retrospective postal questionnaire sent to BAHA patients, with two month waiting time and one reminder, combined with case note analysis. Results compared by appropriate statistical tests with published outcomes data from the largest UK series. SETTING: Public sector (National Health Service) district general hospital, England. Catchment population: 300,000 (mixed rural and small towns). PARTICIPANTS: Case note analysis of sixty-three patients implanted between 1994 and 2003 (age range, six to 88 years). The commonest indication was chronic otitis media, with inability to wear a conventional hearing aid. Otitis externa, otosclerosis and sensorineural hearing loss were other indications. The questionnaire was sent to 59 patients who had worn their aid for at least six months; it was returned by 41 (69 per cent). MAIN OUTCOME MEASURES: Glasgow benefit inventory (GBI, change in health status following otolaryngological intervention); incidence of complications. RESULTS: Bone-anchored hearing aid implantation significantly improved quality of life as measured by the GBI (p<0.001). The degree of improvement was similar to that achieved in Birmingham by Proops et al. (p>0.05, chi-squared test). Minor temporary skin infection was common (33 per cent). Thickening of the skin around the implant occurred in 17 per cent. One implant (2 per cent) failed. There were no serious complications. CONCLUSION: The BAHA is a safe, reliable and effective treatment for selected patients. A successful BAHA programme can be run in a district general hospital. PMID- 16672091 TI - Changes in expression of p53, proliferating cell nuclear antigen and bcl-2 in recurrent laryngeal cancer after radiotherapy. AB - The biological changes in recurrent laryngeal cancer following radiotherapy are not fully understood. The authors investigated differences in the expression of p53, proliferating cell nuclear antigen (PCNA) and bcl-2 in laryngeal cancer specimens before radiotherapy and in recurrent laryngeal cancer specimens following radiotherapy in the same patients. The authors investigated the expression of p53, PCNA and bcl-2 by immunohistochemical stain in 30 specimens from 15 patients with primary laryngeal cancer and recurrent laryngeal cancer after radiotherapy. The expression of p53 protein was significantly different in laryngeal cancer before radiotherapy (4/15, 26.7 per cent) compared with recurrent laryngeal cancer after radiotherapy (8/15, 53.3 per cent) (p<0.05). The PCNA index was also significantly different in laryngeal cancer specimens before radiotherapy (mean, 11.9 per cent) compared with recurrent laryngeal cancer after radiotherapy (mean, 18.0 per cent) (p<0.05). However, there was no statistically significant alteration of bcl-2 expression in primary compared with recurrent laryngeal cancer. The expression of p53 and PCNA increased in recurrent laryngeal cancers after radiotherapy, compared with that in laryngeal cancers before radiotherapy. Recurrent laryngeal cancers arising following radiotherapy became biologically aggressive. PMID- 16672092 TI - Feasibility of closed-loop titration of propofol guided by the Bispectral Index for general anaesthesia induction: a prospective randomized study. AB - BACKGROUND: This study was designed to evaluate the feasibility of propofol infusion by a closed-loop system for the titration of anaesthetic induction guided by Bispectral Index. METHODS: Forty patients were prospectively and randomly allocated into two groups: the target control infusion (TCI) group, where propofol titration was performed manually guided by the Bispectral Index using a commercial pharmacokinetic model (Diprifusor device) and the closed-loop group where titration was performed using a proportional differential algorithm. For both groups, the objective was to achieve a Bispectral Index of 50. Remifentanil TCI was infused at a target of 2 ng mL-1 and was maintained constant throughout the study. Feasibility of automatic induction was evaluated with performance error and haemodynamic data. RESULTS: Bispectral Index overshoot (-9 +/- 13% vs. -16 +/- 20%, P = 0.035) and mean duration of induction (381 +/- 106 s vs. 490 +/- 131 s, P = 0.004) were lower in the closed-loop group than in the TCI group. Haemodynamic data were similar between groups with a similar use of ephedrine bolus. CONCLUSION: The system was able to allow induction clinically for all patients. Automated titration guided by Bispectral Index for propofol infusion was feasible without increase in haemodynamic adverse effects. PMID- 16672093 TI - The effect of adding two target-controlled concentrations (1-3 ng mL -1 ) of remifentanil on MAC BAR of desflurane. AB - BACKGROUND AND OBJECTIVES: The aim of this prospective, randomized, double-blind study was to determine the effects of adding two different target-controlled concentrations of remifentanil (1 and 3 ng mL(-1)) on the desflurane requirement for blunting sympathetic responses after surgical incision (minimum anaesthetic concentration (MAC(BAR)). METHODS: 67 patients, aged 20-50 yr, ASA I, undergoing general anaesthesia for elective abdominal surgery were enrolled and randomly allocated to receive no remifentanil infusion (n = 21) or a target-controlled effect-site concentration of 1 ng mL(-1) (n = 24) or 3 ng mL(-1) remifentanil (n = 22). All patients were anaesthetized with propofol, cisatracurium and desflurane with a mixture of 60% nitrous oxide in oxygen. Sympathetic responses to surgical incision were determined after a 20-min period of stable end-tidal desflurane and target-controlled remifentanil concentrations. Predetermined end tidal desflurane concentrations and the MAC(BAR) for each group were determined using an up-and-down sequential-allocation technique. RESULTS: The MAC(BAR) of desflurane was higher in the group receiving no remifentanil (6.25% [95% confidence interval: 5.9-6.5%]) as compared with patients of the groups receiving 1 ng mL(-1) (2.7% [2.6-2.8%]; P < 0.001) and 3 ng mL(-1) remifentanil (2% [1.9 2.2%]; P < 0.01). When considering a MAC value in this age population and the contribution of 60% nitrous oxide (0.55 MAC), the combined MAC(BAR) values, expressed as multiples of the MAC, were 1.9, 0.8 and 0.6 MAC, in the three groups, respectively. CONCLUSION: A target-controlled concentration of 1 ng mL( 1) remifentanil results in a 57% decrease in the MAC(BAR) of desflurane combined with 60% nitrous oxide. Increasing the target concentration of remifentanil to 3 ng mL(-1) produces a further 26% decrease in the MAC(BAR) values of desflurane. PMID- 16672094 TI - An evaluation of the GlideScope, a new video laryngoscope for difficult airways: a manikin study. AB - BACKGROUND AND OBJECTIVES: The GlideScope is a new video laryngoscope. The aim of our study was to assess its use compared to a Macintosh blade in airway scenarios on the Airman airway simulator. The scenarios were: 'normal' or resting state of the manikin, pharyngeal obstruction, cervical rigidity and tongue oedema. METHODS: Thirty anaesthetists from the Christchurch Anaesthetic Department attempted to pass a bougie or stylet through the vocal cords of the manikin with a size 3 Macintosh blade, then the GlideScope. View at laryngoscopy, ease of 'intubation' and whether they thought the GlideScope would be useful in clinical practice were recorded. RESULTS: Forty three percent (P = 0.02) found an improved view with the GlideScope in the pharyngeal obstruction scenario. In the other scenarios there was no significant difference in view. Reportage of ease of intubation showed no statistical difference in any scenarios. However, 93% of anaesthetists considered having the GlideScope would be useful if faced clinically with one or more of the studied scenarios. CONCLUSIONS: The GlideScope improved the view in one of three difficult airway situations when used by anaesthetists with no formal training in its use. No single airway device offers a solution to all scenarios, however, we consider that the GlideScope is a useful addition to the range of difficult airway devices available. PMID- 16672095 TI - Accessory myocardial pathway mimicking an inferior myocardial infarction after major vascular surgery. PMID- 16672097 TI - Significance of cocaine abuse in the past. PMID- 16672098 TI - Isoflurane damage to a Draeger Primus water trap. PMID- 16672104 TI - Onset properties of mivacurium measured by mechanomyography depend on the twitch height of the adductor pollicis muscle. AB - INTRODUCTION: The influence of the twitch height of the adductor pollicis muscle during baseline measurements on the pharmacodynamic parameters of mivacurium was prospectively evaluated. PATIENTS AND METHODS: Fifty adult patients were anaesthetized with propofol and alfentanil. Neuromuscular function was monitored mechanomyographically by measuring the force of the adductor pollicis muscle following stimulation of the ulnar nerve. Following a stabilization period of 20 min, the individual twitch height of the adductor pollicis muscle was determined before a single bolus of mivacurium (75 microg kg-1) was administered. Patients were divided into two groups. The data of patients whose thumb adduction force was below the median value of all patients were the 'low force' group (9.1 +/- 1.4 N) and the data of all other patients were the 'high force' group (13.7 +/- 1.8 N). RESULTS: In the 'high force' group, maximum neuromuscular blockade of mivacurium was deeper (0.97 +/- 0.05 vs. 0.93 +/- 0.06; P < 0.05) and onset faster (2.9 +/- 1.1 min vs. 4.0 +/- 1.2 min; P < 0.05). Neuromuscular recovery did not differ between the groups. CONCLUSION: The different onset speeds reflect either different sensitivity to neuromuscular blocking agents with respect to patients' muscle power or a problem of the mechanomyographic measuring technique. PMID- 16672099 TI - Lidocaine vs. mepivacaine for peribulbar anaesthesia in cataract surgery: a randomized double-blind study. PMID- 16672105 TI - The origin of human pathogens: evaluating the role of agriculture and domestic animals in the evolution of human disease. AB - Many significant diseases of human civilization are thought to have arisen concurrently with the advent of agriculture in human society. It has been hypothesised that the food produced by farming increased population sizes to allow the maintenance of virulent pathogens, i.e. civilization pathogens, while domestic animals provided sources of disease to humans. To determine the relationship between pathogens in humans and domestic animals, I examined phylogenetic data for several human pathogens that are commonly evolutionarily linked to domestic animals: measles, pertussis, smallpox, tuberculosis, taenid worms, and falciparal malaria. The majority are civilization pathogens, although I have included others whose evolutionary origins have traditionally been ascribed to domestic animals. The strongest evidence for a domestic-animal origin exists for measles and pertussis, although the data do not exclude a non-domestic origin. As for the other pathogens, the evidence currently available makes it difficult to determine if the domestic-origin hypothesis is supported or refuted; in fact, intriguing data for tuberculosis and taenid worms suggests that transmission may occur as easily from humans to domestic animals. These findings do not abrogate the importance of agriculture in disease transmission; rather, if anything, they suggest an alternative, more complex series of effects than previously elucidated. Rather than domestication, the broader force for human pathogen evolution could be ecological change, namely anthropogenic modification of the environment. This is supported by evidence that many current emerging infectious diseases are associated with human modification of the environment. Agriculture may have changed the transmission ecology of pre-existing human pathogens, increased the success of pre-existing pathogen vectors, resulted in novel interactions between humans and wildlife, and, through the domestication of animals, provided a stable conduit for human infection by wildlife diseases. PMID- 16672106 TI - Regulation of mouse brain glycogen synthase kinase-3 by atypical antipsychotics. AB - Glycogen synthase kinase-3 (GSK3) has been recognized as an important enzyme that modulates many aspects of neuronal function. Accumulating evidence implicates abnormal activity of GSK3 in mood disorders and schizophrenia, and GSK3 is a potential protein kinase target for psychotropics used in these disorders. We previously reported that serotonin, a major neurotransmitter involved in mood disorders, regulates GSK3 by acutely increasing its N-terminal serine phosphorylation. The present study was undertaken to further determine if atypical antipsychotics, which have therapeutic effects in both mood disorders and schizophrenia, can regulate phospho-Ser-GSK3 and inhibit its activity. The results showed that acute treatment of mice with risperidone rapidly increased the level of brain phospho-Ser-GSK3 in the cortex, hippocampus, striatum, and cerebellum in a dose-dependent manner. Regulation of phospho-Ser-GSK3 was a shared effect among several atypical antipsychotics, including olanzapine, clozapine, quetiapine, and ziprasidone. In addition, combination treatment of mice with risperidone and a monoamine reuptake inhibitor antidepressant imipramine or fluoxetine elicited larger increases in brain phospho-Ser-GSK3 than each agent alone. Taken together, these results provide new information suggesting that atypical antipsychotics, in addition to mood stabilizers and antidepressants, can inhibit the activity of GSK3. These findings may support the pharmacological mechanisms of atypical antipsychotics in the treatment of mood disorders. PMID- 16672107 TI - Salmonella Mississippi infections in Tasmania: the role of native Australian animals and untreated drinking water. AB - Salmonella Mississippi infections are very common in Australia's island state - Tasmania - with an annual rate of 17 cases/100000 population. A case-control study conducted during 2001-2002 found single variable associations with indirect exposure to many native animal species, untreated drinking water, travelling within the state, hand-mouth behaviours and contact with pet faeces. No associations were detected with farm animal or pet species or with any food. Indirect contact with native birds, untreated drinking water and travel within the state remained significant predictors of infection in the final model with population attributable fractions of 0.57 and 0.54 for native animals and untreated drinking water respectively. In Tasmania, Australian wildlife species are the likely reservoir for S. Mississippi, contaminating land and water environments. To decrease infection rates requires treatment of water supplies, particularly private rainwater collection systems and advising people to wash their hands after being outdoors and prior to eating. PMID- 16672108 TI - Risk factors for encephalitis and death from West Nile virus infection. AB - We conducted a nested case-control study to determine potential risk factors for developing encephalitis from West Nile virus (WNV) infection. Retrospective medical chart reviews were completed for 172 confirmed WNV cases hospitalized in Houston between 2002 and 2004. Of these cases, 113 had encephalitis, including 17 deaths, 47 had meningitis, and 12 were fever cases; 67% were male. Homeless patients were more likely to be hospitalized from WNV compared to the general population. A multiple logistic regression model identified age [odds ratio (OR) 1.1, P<0.001], history of hypertension, including those cases taking hypertension inducing drugs (OR 2.9, P=0.012), and history of cardiovascular disease (OR 3.5, P=0.061) as independent risk factors for developing encephalitis from WNV infection. After adjusting for age, race/ethnicity (being black) (OR 12.0, P<0.001), chronic renal disease (OR 10.6, P<0.001), hepatitis C virus (OR 23.1, P=0.0013), and immunosuppression (OR 3.9, P=0.033) were identified as risk factors for death from WNV infection. PMID- 16672110 TI - Impact of fibrin glue and urinary bladder cell spraying on the in-vivo acellular matrix cellularization: a porcine pilot study. AB - PURPOSE: Urinary bladder tissue engineering utilizing autologous cell-seeded scaffolds requires enough bladder cells to populate a large surface area which may be difficult to obtain from abnormal bladders. We evaluated whether a fibrin glue spray technique enhances cell seeded acellular matrix (ACM) repopulation in a porcine bladder model. MATERIALS AND METHODS: Porcine urothelial and smooth muscle cells cultured from open bladder biopsy were sprayed with or without fibrin glue onto porcine bladder ACM. After 10 days in vitro, constructs were implanted onto porcine bladders (4/group) and harvested after 1 or 6 weeks for H&E and immunohistochemical staining. RESULTS: In vitro, fibrin glue was associated with more continuous cell growth and enhanced cellular organization, maintained particularly in the periphery in vivo, where both groups demonstrated central fibrosis. CONCLUSIONS: While fibrin glue enhanced cellular organization on ACM in vitro, central fibrosis in vivo suggests that factors supporting seeded cell survival are lacking. PMID- 16672111 TI - Pelvic chemoradiotherapy after chemotherapy for metastatic bladder cancer. AB - OBJECTIVE: Consolidative radiotherapy has improved local control in other tumors with high local recurrence rates but has not been well studied in urothelial cancer. We hypothesized that pelvic chemoradiotherapy (PCRT) given after systemic chemotherapy for metastatic bladder cancer (MTCC) might alter the pattern of disease recurrence, and reduce the complications and morbidity of intrapelvic disease relapse. A 74% locoregional relapse rate has been observed in MTCC patients with intrapelvic nodal disease after response to chemotherapy. To explore this hypothesis further, we performed a retrospective analysis and report the efficacy, toxicity and pattern of failure with this approach. MATERIALS AND METHODS: Patients treated for MTCC who received consolidative PCRT following at least a partial response to systemic chemotherapy were identified and their charts reviewed for pelvic relapse, disease progression, survival, and toxicity. RESULTS: Twelve patients were identified and median follow-up was 15.6 months. Nine patients developed progressive disease and died, and median survival was 15.6 months. Three patients had pelvic progression (pelvic failure rate 25%). Median time to pelvic failure was 12.8 months. At last follow-up, three patients were alive and disease-free. No life-threatening toxicities were observed. The most common acute non-hematological toxicities were diarrhea and nausea. CONCLUSIONS: These data support a hypothesis that consolidative PCRT following chemotherapy in MTCC patients with systemic disease control may be feasible and efficacious for improving pelvic disease control. This intervention should be considered for further study in prospective controlled clinical trials. PMID- 16672112 TI - The Erectile Function Visual Analog Scale (EF-VAS): a disease-specific utility instrument for the assessment of erectile function. AB - This article presents the responsiveness results of the Erectile Function Visual Analog Scale (EF-VAS) and reports, for the first time, utilities associated with erectile dysfunction (ED), as calculated by a disease-specific utility assessment. The EF-VAS is a new quality of life (QoL) instrument specific to ED that combines the strengths of the disease-specific approach to measuring QoL (greater disease relevance and responsiveness, with relevance to clinicians and patients) with those of preference-based assessments (generalizability and relevance to decision makers). The EF-VAS has demonstrated feasibility, reliability, and validity as reported in a recent publication. METHODS: Standard instrument development methodology was utilized and the finalized content was integrated into a preference based scoring instrument comprised of two visual analogue scales (VAS). The EF-VAS was implemented in a clinical trial and data from the trial was subjected to validation analysis. Three methods were used to evaluate the responsiveness of the EF-VAS: Spearman correlations, effect size and standardized response means. VAS scores were converted to von Neumann-Morgenstern (vNM) utilities through a conversion curve. RESULTS: The EF-VAS was established to be responsive to changes in disease state within and between patients with ED. The EF-VAS allowed the calculation of vNM utility values and a significant increase in utility was observed in the sildenafil group compared to placebo at study end. CONCLUSION: The EF-VAS represents an important advance in the understanding of the impact of ED on patients' QoL and in providing a mechanism to allow the quantification of the health status that patients associate with ED. Based on its responsiveness, the EF-VAS will provide an important clinical tool to assess and contribute to the understanding of the impact of treatment for ED. The EF-VAS represents a major advance in the science of health-related quality of life (HRQol) assessment, as it is the first validated ED-specific utility assessment reported in the literature. PMID- 16672114 TI - Injection of botulinum toxin type A in the urethral sphincter to treat lower urinary tract dysfunction: a review of indications, techniques and results. AB - The first application of botulinum toxin type A (BTA) in urology was its injection into the urinary sphincter to treat neurogenic detrusor-sphincter dyssynergia (DSD) in quadriplegic men. Since that first report in the eighties, the results of focal BTA injections into the sphincter, the bladder wall and lately into the prostate, have raised the interest of the urology community in this promising new therapeutic modality. An evidence-based review is presented of current indications, techniques and outcome of BTA injections into the urethral sphincter. PMID- 16672115 TI - Non-palpable testicular lesion: the case for testicular preservation. AB - OBJECTIVES: To report our experience in the management of patients with non palpable lesions of the testis with a review of published studies. Even though radical orchiectomy remains the golden standard treatment for solid testicular masses there is an increasing role for testes-sparing surgery in a select group of patients with non-palpable tumors. PATIENTS AND METHODS: Between April 2000 and October 2004, a non-palpable testicular lesion was discovered on ultrasonography in six patients. Ultrasonography was performed for infertility in two patients, for testicular pain in three patients, and in one case for previous history of seminoma in the contra-lateral testis. RESULTS: Radical orchiectomy was performed in two patients, testis-sparing surgery with microsurgical excision and frozen-section examination (FSE) in two patients, and a conservative watchful waiting attitude was adopted in two patients. CONCLUSIONS: In a select group of patients, testis-sparing surgery with microsurgical dissection and FSE or a watchful waiting attitude can be safely adopted as an alternative to radical orchiectomy in the presence of incidentally discovered non-palpable testicular lesion. PMID- 16672116 TI - Emphysematous pyelonephritis: a case report and review of the literature. AB - We present a case of fulminant emphysematous pyelonephritis in a 44-year-old diabetic woman culminating in emergent nephrectomy. Current management strategies and their outcomes are reviewed. PMID- 16672117 TI - Tuberculosis of the bladder without previous renal infection. AB - Tuberculous (TB) infections are usually limited to the pulmonary system but the hematogenous spread of TB can result in secondary infections in any part of the body. Genitourinary TB is uncommon and follows hematogenous spread from a primary pulmonary infection to the kidneys. A rare case of a TB infection of the bladder without renal involvement is described. PMID- 16672118 TI - The laparoscopic experience of recently trained Canadian urologists. AB - OBJECTIVE: While laparoscopy represents an increasingly important aspect of operative urology, the experience of Canadian urology trainees is poorly defined. The purpose of this study was to determine the level of laparoscopic training of Canadian urology trainees during residency as well as their comfort level with various laparoscopic techniques. METHODS: An e-mail-and web-based questionnaire was administered to the two most recent cohorts of Canadian-trained urologists (residencies completed in 2003 or 2004). A total of 50 questionnaires were sent. Overall laparoscopic experience and experience with specific laparoscopic tasks (e.g. colonic mobilization) were assessed. Subjects also reported on their relative comfort level (Likert scale of 1 to 5) with various laparoscopic procedures and tasks. Finally, attitudes towards the future role of laparoscopy in the practice of urology were determined. RESULTS: Thirty-six individuals (72%) responded to the questionnaire. Of the respondents, 28 (78%) were performing fellowships; of those pursuing fellowship training, 13 (36%) involved laparoscopy. Thirty-five respondents (97%) had received some laparoscopic exposure during residency; 29 (81%) had mobilized the colon, spleen or liver and 27 (75%) had dissected the renal vessels. Only 7 out of 33 respondents (21%) felt that their residency adequately prepared them to perform laparoscopy independently. There were no significant differences between the responses of the two cohorts. CONCLUSION: Most Canadian urology trainees are being exposed to laparoscopy, but more exposure is required in order to perform laparoscopy in practice. Many residents still require fellowship training to become facile in laparoscopy. PMID- 16672119 TI - Primary malignant melanoma of the prostate: case report and review of the literature. AB - Primary malignant melanoma of the prostate is very rare. Most cases attributed to the prostate actually originate from the prostatic urethra. Due to its infrequency, primary malignant melanoma of the genitourinary tract presents a difficult diagnostic and management challenge. We report a case of primary malignant melanoma of the prostate found incidentally following transurethral resection of the prostate (TURP). PMID- 16672120 TI - Inguinal herniation of the bladder in an infant. AB - Inguinal herniation of the bladder is an uncommon finding with fewer than 200 cases reported in the literature. It is found most commonly in older, obese men with lower urinary tract symptoms. We report a case of inguinal herniation of the bladder in a premature infant. PMID- 16672122 TI - Nomogram prediction for prostate cancer and aggressive prostate cancer at time of biopsy: utilizing all risk factors and tumor markers for prostate cancer. AB - BACKGROUND: There is a large amount of confusion in interpreting prostate specific antigen (PSA) values for prostate cancer. More precise risk assessments for prostate cancer detection are needed for men faced with an abnormal PSA. METHODS: We studied a sample of 2,637 men who underwent a prostate biopsy for an abnormal digital rectal exam (DRE) or PSA. Using factors including age, ethnicity, family history of prostate cancer, previous negative biopsy, presence of voiding symptoms, prostate volume, DRE and PSA, we constructed nomograms to predict the probability of prostate cancer at biopsy. RESULTS: Of the 2,637 men, 1,282 men (48.6%) had prostate cancer detected. Age, ethnicity, family history of prostate cancer, a previous negative biopsy, prostate volume, DRE and PSA were all significant predictors of prostate cancer. Nomograms were constructed based on these factors to predict the risk of prostate cancer and of aggressive prostate cancer (defined as a Gleason Score 7 or more). The positive predictive value varied from 5% to 95% based on the nomograms. The nomograms were validated using bootstrapping methods and the expected and observed proportions were found to be highly concordant. CONCLUSIONS: For men with an abnormal PSA or DRE, the risk for prostate cancer can be accurately estimated using a nomogram based on age, ethnicity, family history of prostate cancer, previous negative biopsy, presence of voiding symptoms, prostate volume, DRE and PSA. This tool will aid physicians and patients in determining the need for prostate biopsy. PMID- 16672123 TI - Obesity and prostate cancer. AB - The relationship between obesity and prostate cancer is currently a hotly debated topic, but despite the number of publications devoted to the topic, the actual nature of the relationship remains uncertain. Obesity has been shown to have a direct relationship with the incidence of prostate cancer in a number of studies but an equal number of studies have shown no association. The relationship is further obscured with recent findings that obesity in younger obese men may actually be protective against prostate cancer. Confounding factors include the lack of correlation of body mass index (BMI) as a measure of central obesity and the lack of consistency in timing of BMI measurements, i.e. before or after diagnosis and in young or advanced adulthood. Evidence for increased BMI as a risk factor for prostate cancer is unclear, but less ambiguous is the mounting substantiation that obesity is associated with prognostically worse disease, poorer post-surgical outcomes and increased prostate cancer mortality, irregardless of margin status. From a biologic perspective, one can put forth a number of potential mechanisms by which obesity might promote prostate cancer and/or prostate cancer progression including; low levels of testosterone, increased levels of estrogen, co-existing diabetes or metabolic syndrome, increased circulating insulin-growth factor-one (IGF-1), increased levels of leptin, decreased levels of adiponectin and increased dietary saturated fats. Evidence for the association of these factors with prostate cancer are examined herein. The timing of serum measurements is crucial in elucidating whether these factors have causative influence on prostate cancer or rather are produced by the prostate cancer cells and are better understood as markers of disease. The interaction between obesity and prostate cancer is important to clarify because it will have impact on the prevention, prognostication and treatment of prostate cancer. Future study with careful attention to avoid the methodological pitfalls of the past need be accomplished to bear out the nature of the interaction of obesity and prostate cancer. PMID- 16672124 TI - Prostate cancer nomograms are superior to neural networks. AB - INTRODUCTION: Several nomograms have been developed to predict PCa related outcomes. Neural networks represent an alternative. METHODS: We provide a descriptive and an analytic comparison of nomograms and neural networks, with focus on PCa detection. RESULTS: Our results indicate that nomograms have several advantages that distinguish them from neural networks. These are both quantitative and qualitative. CONCLUSION: In the field of PCa detection, nomograms appear to outweigh the benefits of neural networks. However, the neural network methodology represents a valid alternative, which should not be underestimated. PMID- 16672125 TI - Changing management of localized prostate cancer: a comparison survey of Ontario radiation oncologists. AB - BACKGROUND AND PURPOSE: Annual genitourinary radiation oncology meetings aim to assist in the dissemination of knowledge that may affect current practice. We aim to measure changes in practice approaches that have occurred while these meetings have been conducted. MATERIALS AND METHODS: A previously published survey from 2002 was sent to all genitourinary radiation oncologists in Ontario. Six prostate cancer patient scenarios were used: three definitive (low risk, intermediate risk, high risk), and three post-operative (extracapsular extension, margin positive, slowly rising PSA). There were 21 responders from seven cancer centers. RESULTS: Using biological equivalent dose (BED), there is significant dose escalation in 2005, particularly for intermediate risk patients (mean BED 73.0 Gy2 in 2002 versus 76.1 Gy2 in 2005, p=0.0003). There has been a corresponding move away from the use of neoadjuvant hormones in these patients (2002: 62% versus 2005: 24%, p=0.0097). More accurate prostate localization using fiducials is more common, leading to less use of rectal barium and urethrograms in the simulation process. In the definitive settings there is more utilization of rigid immobilization and more complex treatment delivery including intensity modulated radiotherapy. There is also greater use of multileaf collimation, electronic portal imaging and dose volume histograms in 2005 compared with 2002. CONCLUSIONS: There have been significant changes in the way that prostate cancer is managed with radiotherapy in Ontario between 2002 and 2005. Dose escalation and more complex treatment planning is widely evident. PMID- 16672126 TI - High-precision radiotherapy: where are we going and how do we get there? AB - In January 2006, physicians with an interest in urologic cancers met to discuss patient care at the 4th Annual Current Problems in Urology Conference. A portion of the meeting was focused on technical issues in prostate cancer radiotherapy. This portion of the meeting sought to answer the questions: where are we going? And how can we get there? Work performed at the Princess Margaret Hospital (PMH) and the London Regional Cancer Program (LRCP) served as the basis for discussion and to present examples of options for implementation of new techniques. The response to the first question reviewed the issue of improved outcomes with dose escalation and the preliminary implementation of hypofractionated treatment. The impact on toxicity was reviewed in detail. The response to the second question revolved around the options available to ensure adequate tumor localization. As dose is increased, the need to localize the prostate accurately has become more important in order to ensure tumor control and to avoid toxicity. Selection of appropriate margins around the prostate is determined by a center's ability to localize the target. Options to localize organs including three-dimensional ultrasound, fiducial markers, megavoltage CT and cone beam CT were discussed. The basic research to enable selection and implementation of these options were presented. PMID- 16672127 TI - Early detection of prostate cancer. What do we tell our patients? AB - INTRODUCTION: Early detection of prostate cancer is possible; overdetection of early disease that may never surface clinically during a lifetime is likely. On the other hand, early detection measures will detect life-threatening disease of which some maybe amenable to cure, while otherwise it would kill the patient. Proof of effectiveness of early detection is unavailable. Those who decide to be screened and those who decide not to be screened take risks that are difficult to balance against each other. How do we deal with this situation? What do we tell our patients? DISCUSSION: Ongoing randomized studies are likely to produce definitive answers on the question of whether screening will save men from prostate cancer deaths within a few years. The trials will also provide answers to the optimal way of screening and to the question of how to avoid overdiagnosis and treatment. In the meantime, however, men who are considering to undergo testing have a difficult decision to make. Our profession is obliged to provide assistance. The information provided unfortunately at this time cannot be the simple message: if you undergo testing your cancer will be detected early and be curable, so you will resolve the problem. It is necessary to stress the potential benefits and also the downside of testing. Elements of such information are provided in this article. Benefits may include the reassurance resulting from a normal test result, the early diagnosis of aggressive and still-curable cancer, and the avoidance of the consequences of advanced prostate cancer such as the occurrence of metastatic disease. The downside includes the possibility of missing prostate cancer and providing a false reassurance, the fact that screening may lead to unnecessary anxiety and medical tests when no cancer is present, and the fact that it may detect slow-growing cancer that may never cause any symptoms or shorten lifespan. All treatments have side effects to which men will be exposed, even those who do not have life-threatening cancer. In addition, there is no certainty that treatment will be successful. CONCLUSION: In the present situation, it is important to emphasize to patients that capabilities are being developed to identify those prostate cancers that may not cause any harm and to exclude them from immediate treatment. This recent development is a very important aspect for those who consider to be tested outside ongoing trials. PMID- 16672128 TI - PSA recurrence: definitions, PSA kinetics, and identifying patients at risk. AB - Uncertainty exists for clinicians and patients with respect to choosing the optimal therapy for patients with PSA recurrence. There is no consensus as to what the PSA cutpoint should be to define PSA failure after radical prostatectomy (RP) or radiation therapy (XRT). We do, however, have validated nomograms which allow the stratification of patients according to their risk of disease progression and cancer specific death. This is based in large part on PSA kinetics. A short PSA doubling time (PSA-DT) is associated with a marked increase in the risk of prostate cancer death in the 5-10 year time frame. PSA DT can also be used to identify patients most likely to respond to local salvage therapy. PMID- 16672129 TI - Treatment of radiation failure in prostate cancer. AB - Radiotherapy, both external beam and more recently, interstitial, have been therapeutic options for localized prostate cancer. Management of patients who have failed of local radiotherapy remains a challenge. Herein the current therapeutic options are reviewed. PMID- 16672130 TI - Therapeutic approach to hormone-refractory prostate cancer. AB - Over 60 years ago, Huggins and Hodges discovered androgen deprivation as an effective first-line therapy for metastatic prostate cancer. This leads to significant cancer control but in almost all men prostate cancer ultimately progresses to a hormone-refractory (HRPC) state resulting in significant morbidity and eventual death. In 2004, two landmark studies using docetaxel based chemotherapy demonstrated, for the first time, a survival advantage in HRPC. This has set a new standard of care for this disease. In addition, treatment with the bisphosphonate zoledronic acid has been shown to significantly reduce bone complications in metastatic HRPC. Building on these advances, several new docetaxel/zoledronic acid based combinations as well as new targeted therapies are under development. Introducing these effective therapies earlier in high risk patients is also under investigation to further improve outcomes. PMID- 16672131 TI - The role of adjuvant therapy in non-metastatic RCC. AB - Renal cell carcinoma (RCC) presents as localized disease in 54% of the cases. For these patients, surgery is the primary curative treatment. Unfortunately, up to 65% of all patients show recurrent disease. For metastatic RCC non-specific immunotherapy is currently the treatment of choice. Nevertheless, several new modalities, e.g. WX-G250, oncophage and anti-angiogenic compounds like sunitinib and sorafenib are being explored with favorable results. Still, their place in the primary treatment of advanced RCC has yet to be determined. Because of the high percentage of recurrent disease, there is a need to identify these patients with conventional and molecular risk factors. Furthermore, adjuvant therapy to reduce risk of recurrence of RCC following nephrectomy is of clinical relevance. A review of recent literature was performed on the topics prognostic models, risk factors and adjuvant treatment for non-metastasized RCC. Combining classical risk factors for progression of RCC has shown to be effective for stratifying patients into risk groups. The UCLA integrated staging system (UISS) is the currently the only validated prognostic model. Whether molecular markers are able to better identify high-risk patients is still under investigation. Adjuvant therapy has been explored in the treatment for RCC and the use of non-specific cytokine regimens has so far not shown to be effective in the adjuvant setting. More specific therapies, e.g. WX-G250, oncophage and anti-angiogenic drugs are clinically active in patients with advanced RCC. Large randomized clinical trials with these drugs are currently ongoing to evaluate their effect in patients with localized RCC. PMID- 16672132 TI - Low and intermediate risk prostate cancer-- role of hormonal therapy with external beam radiation therapy. AB - Risk categorization based on pre-treatment PSA, clinical stage and Gleason score is now widely used in the management of patients with localized prostate cancer. In patients with low-risk disease (cT1-T2a, PSA < 10 ng/ml and Gleason score < 6) there is no role for the routine use of adjunctive hormonal therapy. In intermediate-risk patients (T1-T2, PSA < 20 ng/ml and Gleason 3microgL(-1) to less than 0.5microgL(-1) when prochloraz was added, could be problematic as these concentrations are environmentally realistic. Furthermore, insecticides and ergostrol-biosynthesis-inhibitors (EBI-fungicides) are often applied together, and are therefore likely to co-exist in surface waters, enhancing the problem of the already very potent insecticides. PMID- 16672167 TI - Waterborne and dietary hexavalent chromium exposure causes DNA-protein crosslink (DPX) formation in erythrocytes of largemouth bass (Micropterus salmoides). AB - Formation of DNA-protein crosslinks (DPXs) was demonstrated in erythrocytes from fathead minnows (Pimephales promelas) and largemouth bass (Micropterus salmoides) exposed to hexavalent chromium [Cr(VI)], a known carcinogenic and mutagenic metal contaminant of many industrial waterways. Tank water exposure of 2-3 in. fathead minnows to 2 ppm Cr(VI) led to significant DPX formation in erythrocytes, with over 140-200% elevations above background levels at 3-4 days, respectively. Largemouth bass exposed similarly were found to have 62% elevation of DPX levels after 4 days. When largemouth bass were fed a diet of minnows injected with 20 microg Cr(VI) for 5 days, a significant (p<0.01) increase of DPXs in erythrocytes was observed, with 80% elevation above erythrocytes from bass fed minnows injected only with saline. However, when largemouth bass were fed a diet exclusively of minnows exposed to 2 ppm Cr(VI) for 21 days, there was no significant difference in DPX levels compared to bass fed control (unexposed) minnows. This study provides evidence that DPX formation occurs in erythrocytes of fathead minnows exposed under controlled conditions to low ppm Cr(VI) concentrations, which is at or below concentrations previously assigned no observable effect levels. Furthermore, it appears that both waterborne and high dose dietary exposure to Cr(VI) can lead to DPX formation in erythrocytes of predatory fish species such as bass. However, it is unlikely that a bioconcentration of chromium in the food chain would be a major concern at these low ppm levels of exposure. Further, it may be difficult to achieve dietary Cr(VI) levels high enough to elicit DPXs in predatory fish under most environmental exposure scenarios. PMID- 16672168 TI - Oral cobalamin remains medicine's best kept secret. AB - A cross-sectional survey was conducted in order to describe the use of oral cobalamin among geriatricians, hematologists, and general practitioners, and to explore factors related to its use. The study population consisted of all geriatricians (n = 138) and hematologists (n = 317) listed in the Canadian Medical Directory plus a random sample of 307 general practitioners. The overall response rate was 40%. Intramuscular and oral cobalamin was prescribed by 76 and 32% of the respondents, respectively. Twenty seven percent reported using both oral and intramuscular cobalamin and 6% reported using only oral cobalamin. Only 25% of respondents indicated they were aware of a RCT demonstrating the efficacy of oral cobalamin prior to reading a synopsis of the study in the survey. After multivariate adjustment, only the belief that oral cobalamin was effective and certainty about who carried oral preparations remained independently associated with oral cobalamin use. Oral cobalamin has been shown to be an efficacious, cost efficient and safe method of treating cobalamin deficiency. Nonetheless, it is not used by the majority of physicians treating this condition. Strategies to promote the use of oral cobalamin should be directed at educating physicians of its efficacy and providing them with prescribing information on where it can be purchased. PMID- 16672169 TI - Effects of chronic ethanol administration on brain interstitial fluid levels of Methionine-enkephalin as measured by microdialysis in vivo. AB - The level of Met-enkephalin in the brain is inversely correlated with ethanol consumption and is controlled partially through efflux activity of peptide transport system-1 (PTS-1) at the blood-brain barrier (BBB). Prolonged alcohol drinking can perturb aspects of this system, including a loss of control of Met enkephalin levels at the transcriptional and translational levels, and impaired release of Met-enkephalin from tissue sources. Met-enkephalin levels in whole brain homogenates often first paradoxically increase after a few days of ethanol drinking and then decrease with the development of physical dependence. Which of those various changes drives the others is unclear. To clarify these interactions, we here determined the levels of Met-enkephalin in striatal interstitial fluid (ISF) by microdialysis, striatal tissue homogenates, and serum after chronic ethanol treatment and alcohol withdrawal. Mice received ethanol (5%) in liquid diet for 7 days (ethanol-treated) and others withdrawn for a day following 7-day treatment (withdrawal). There was a significant (P<0.05) difference in the levels of Met-enkephalin in striatal microdialysate between the control (79.1+/-5.9 pg/ml) and ethanol-treated group (94.9+/-4.3 pg/ml), which was lost by withdrawing ethanol (83.9+/-3.8 pg/ml). In contrast, ethanol treatment did not affect Met-enkephalin levels in the striatal tissue. In the ethanol-treated group, there was a significant (P<0.05) reduction of the levels of Met-enkephalin in serum to 70.5% of control levels. This decrease was restored to the level of control by withdrawing ethanol. These reversible changes in ISF and serum are readily explained by the known changes in the efflux activity of PTS-1 at the BBB. PMID- 16672170 TI - Decoy mRNAs reduce beta-amyloid precursor protein mRNA in neuronal cells. AB - Overproduction of amyloid precursor protein (APP) and beta-amyloid likely contribute to neurodegeneration in Alzheimer's disease (AD). In an effort to understand neuronal APP gene regulation, we identified a 52 base element (52sce) immediately downstream from the stop codon that stabilizes APP mRNA. Deletion of this domain drastically destabilized APP mRNAs and reduced APP synthesis in vitro. Chimeric globin-APP mRNAs containing the globin coding sequence fused to the entire APP 3'-UTR, showed regulation similar to full-length APP mRNA. A variety of cytoplasmic lysates contain 52sce RNA binding activity, suggesting cis trans interactions regulate the element's functionality. Finally, the overexpression of chimeric mRNAs, containing the GFP coding sequence and APP 3' UTR, dramatically reduced endogenous APP steady-state levels in SH-SY5Y neuroblastoma cells and suggests a novel approach to reduce the amyloid burden in AD patients. PMID- 16672171 TI - Sperm thermotaxis. AB - Thermotaxis--movement directed by a temperature gradient--is a prevalent process, found from bacteria to human cells. In the case of mammalian sperm, thermotaxis appears to be an essential mechanism guiding spermatozoa, released from the cooler reservoir site, towards the warmer fertilization site. Only capacitated spermatozoa are thermotactically responsive. Thermotaxis appears to be a long range guidance mechanism, additional to chemotaxis, which seems to be short-range and likely occurs at close proximity to the oocyte and within the cumulus mass. Both mechanisms probably have a similar function--to guide capacitated, ready-to fertilize spermatozoa towards the oocyte. The temperature difference between the site of the sperm reservoir and the fertilization site is generated at ovulation by a temperature drop at the former. The molecular mechanism of sperm thermotaxis waits to be revealed. PMID- 16672172 TI - MAP-quest: could we produce constitutively active variants of MAP kinases? AB - Constitutively active mutants that acquired intrinsic activity and escaped regulation, serve as powerful tools for revealing the biochemical, biological and pathological functions of proteins. Such mutants are not available for mitogen activated protein kinases (MAPKs). It is not known how to mimic the unusual mode of MAPK activation and to enforce, by mutations, their active conformation. In this review we describe the strategies employed in attempts to overcome this obstacle. We focus on a recent breakthrough with the p38 family that suggests that active variants of all MAPKs will soon be available. PMID- 16672173 TI - Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland. AB - Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland. PMID- 16672174 TI - Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat. AB - We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20 h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred at 17 h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16 h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9 h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9h followed by a second rise at 22 h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns. PMID- 16672175 TI - Lack of evidence for functional TRPV1 vanilloid receptors in rat hippocampal nerve terminals. AB - Although TRPV(1) vanilloid receptors (TRPV(1)Rs) have been assumed to be present in the brain, their role is not well-defined. Here, we tested the widely used TRPV(1)R agonists (E)-capsaicin (0.1-100 microM) and resiniferatoxin (RTX, 0.1 microM) on resting and K(+)-evoked Ca(2+) entry and radiolabelled GABA release in rat hippocampal nerve terminals. (E)-capsaicin and RTX failed to evoke Ca(2+) transients or to trigger [(3)H]GABA outflow. Both (E)-capsaicin (EC(50), 40.4 microM) and its enantiomer (Z)-capsaicin (EC(50), 22.9 microM), which is inactive at the TRPV(1)R, inhibited the K(+)-evoked Ca(2+) entry, and to similar extent, the Ca(2+)-dependent K(+)-evoked [(3)H]GABA release. The TRPV(1)R enhancer/partial agonist 2-aminoethoxydiphenyl borate (1-300 microM) induced rapid Ca(2+) entry. None of the above-mentioned findings proved to be sensitive to the TRPV(1)R antagonists iodoresiniferatoxin (I-RTX; 3 microM) and SB366791 (3 microM). The CB(1) cannabinoid receptor antagonist AM251 (EC(50), 1.1 microM) and I-RTX (EC(50), 4.6 microM) also diminished the K(+)-evoked Ca(2+) entry per se. We observed competitive antagonism between I-RTX and AM251, indicating that the two molecules may act at the same site. In conclusion, there is a need to examine the discrepancy between ex vivo and in vitro data to understand the neurochemical and physiological functions of brain TRPV(1)Rs. PMID- 16672177 TI - Characterization of Toxoplasma gondii isolates in free-range chickens from Chile, South America. AB - The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 85 free-range chickens (Gallus domesticus) from Chile was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 47 of 85 (55.3.9%) chickens with titers of 1:5 in six, 1:10 in four, 1:20 in four 1: 40 in three, 1: 80 in nine, 1: 160 in four 1:320 in nine, and 1: 640 or higher in eight. Hearts and brains of 47 chickens with titers of 1:5 or higher were pooled for each chicken and bioassayed in mice. Tissues from 16 seronegative (MAT<1:5) chickens were pooled and fed to one T. gondii-free cat. Feces of the cat were examined for oocysts but none was found based on bioassay of fecal floats in mice. Hearts and brains from seven seronegative (<1:5) were pooled and bioassayed in mice; T. gondii was not isolated. T. gondii was isolated by bioassay in mice from 22 chickens with MAT titers of 1:20 or higher. Genotyping of these 22 isolates using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed three genotypes. Seventeen isolates had type II alleles and four isolates had type III alleles at all loci. One isolate contained the combination of type I and III alleles. This is the first report of genetic characterization of T. gondii isolates from Chile, South America. PMID- 16672178 TI - The geographic distribution of the putative agent of epizootic bovine abortion in the tick vector, Ornithodoros coriaceus. AB - Epizootic bovine abortion (EBA), also known as "foothill abortion", is a vector borne disease of beef cattle that graze in the mountainous regions of California, southern Oregon and western Nevada transmitted by the argasid tick Ornithodoros coriaceus. Recently, the putative agent of EBA was identified as a novel Deltaproteobacter in the order Myxococcales. In this study, a TaqMan real-time PCR (TM-PCR) protocol specific to the putative EBA agent was developed. The new real-time TM-PCR assay functioned sensitively and specifically to detect pathogen DNA in field-collected O. coriaceus ticks. The assay had an analytical sensitivity of a single plasmid copy and, when evaluated with a collection of tick-borne pathogens, yielded a positive PCR-result only for the agent of EBA. Use of the TM-PCR represents an effective tool for rapid and highly sensitive assessment of environmental risk and spatial and statistical analysis to highlight areas where there may be increased risk for EBA in susceptible cattle. PMID- 16672179 TI - Analysis of the IgG antibody response against Paramphistomidae trematoda in naturally infected cattle. Application to serological surveys. AB - The IgG antibody response to Calicophoron daubneyi (Digenea: Paramphistomidae) excretory/secretory antigens was evaluated in naturally infected cattle from Lugo (Galicia, NW Spain) by using an ELISA procedure. Two studies were conducted, first a survey in 524 cattle separated into three groups according to age, G-1 (0 2 years old), G-2 (3-5 years old) and G-3 (> 6 years old). In the second study, three groups of cattle were employed: G-I, naturally infected; G-T, naturally infected and treated with oxyclozanide plus levamisole (Nilzan Plus); G-C, cattle maintained in a farm where C. daubneyi has never diagnosed. Variations on egg output and haematic parameters (erythrocytes, haematocrite, leukocytes and lymphocytes) were also analyzed. The ELISA procedure showed that 61.2% of the cattle in the first study had been exposed to the trematode, but only 10.1% passed eggs in the feces. Age-association with egg-output was shown but not with the IgG values. In the second experiment, the administration of the anthelmintic reduced significantly the IgG kinetic levels and the C. daubneyi-egg-output was suppressed during 12 weeks in the G-T group. The values of red cells, haematocrite, leukocytes and lymphocytes increased significantly in the treated cattle 5 weeks after chemotherapy; however, new reduction after week 5 was recorded, as results of the challenge of these cattle. This is the first investigation in which evaluation of the IgG humoral response against C. daubneyi in cattle has been carried out. We proved that a notable IgG response in naturally infected cattle is induced, and can be detected by using an ELISA procedure. The IgG antibodies did not increase after challenge infection. Our results proved an important percentage of cattle were exposed to this trematode in the area of study and suitable measures for preventing this relationship must be considered. PMID- 16672180 TI - Performance characteristics and optimisation of cut-off values of two enzyme linked immunosorbent assays for the detection of antibodies to Neospora caninum in the serum of cattle. AB - AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis. PMID- 16672181 TI - Characterization of selected genes upregulated in non-tuberculous European wild boar as possible correlates of resistance to Mycobacterium bovis infection. AB - Bovine tuberculosis (bTB), caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle and wildlife worldwide. These animal hosts can serve as reservoirs of infection, thus increasing the risk of human exposure and infection. In this study we quantified by RNA macroarray fluorescent hybridization and real-time RT-PCR the mRNA levels of genes differentially expressed in oropharyngeal tonsils and mandibular lymph nodes of three and seven individual non-tuberculous and tuberculous wild boars naturally exposed to M. bovis, respectively. These results demonstrated upregulation of two genes, complement component 3 (C3) and methylmalonyl-CoA mutase (MUT), in the non-tuberculous wild boars. These upregulated genes may contribute to resistance of wild boars to bTB by modifying the innate immunity, which limits the ability of the mycobacterium to infect and persist within macrophages. The C3 and MUT genes, therefore, are likely to be good candidates to study as markers of bTB resistance using functional genomics in animal model systems. Identification of genes upregulated in wild animals resistant to bTB contributes to our understanding of the mechanisms of protective immunity and resistance to mycobacterial organisms. PMID- 16672182 TI - Infection of pigs in Ireland with lymphotropic gamma-herpesviruses and relationship to postweaning multisystemic wasting syndrome. AB - Three species of porcine lymphotropic herpesviruses (PLHVs) have been described but there are few reports on the distribution and prevalence of these viruses in domestic pigs. We aimed to determine the PLHV status of Irish commercial pig herds, and to this end spleens taken from 110 healthy adult pigs sourced from 22 geographically distributed farms in Ireland were analysed for PLHV DNA using novel species-specific polymerase chain reaction assays. We now report that PLHV infection is widespread in the Irish domestic pig population and that PLHV-1 infections are most common (74% of all animals tested), followed by PLHV-3 and PLHV-2 (45% and 21%, respectively) and that infections with multiple PLHV species were frequently detected. As the PLHVs are lymphotrophic agents, we also investigated if co-infection with PLHVs was linked to the development of porcine circovirus-2 (PCV2)-associated postweaning mutlisystemic wasting syndrome (PMWS), a disease characterised in part by histopathological lesions in lymphoid tissues. We examined the PLHV infection status of young animals on two farms that were experiencing outbreaks of PMWS. Overall the findings are further evidence of the widespread prevalence of PLHVs in domestic pigs and are a first indication that co-infection with PCV2 and PLHVs does not lead to the development of PMWS in the absence of other cofactors. PMID- 16672183 TI - A validated chiral CE method for Frovatriptan, using cyclodextrin as chiral selector. AB - A cyclodextrin modified capillary zone electrophoretic method has been developed for the evaluation of chiral purity of Frovatriptan using sulfobutyl ether beta cyclodextrin (SB-beta-CD) as the chiral selector. The method is highly specific, accurate and reproducible. The method was optimized with a systematic method development approach by optimizing the pH of electrolyte, attempting the separation in different classes of chiral selectors and modifying parameters such as cyclodextrin concentration and the organic modifier type and concentration. The optimized method was validated for specificity, precision, linearity, accuracy and stability in solution using Imidazole as the internal standard. The limit of detection (LOD) and limit of quantification (LOQ) were 1.0 microg/mL and 5.0 microg/mL respectively for each isomer. The method was applied for estimating the chiral purity of various batches of Frovatriptan. PMID- 16672184 TI - Salicylic acid determination in cow urine and drugs using a bienzymatic sensor. AB - An enzymatic biosensor was developed for salicylic acid (salicylate ion) determined using a Clark type gas diffusion electrode and two enzymes (tyrosinase and salicylate hydroxylase) entrapped in a cellulose triacetate membrane. After optimization, the method was applied to the determination of salicylic acid in cow urine. Relatively good recoveries were achieved, between about 83% and 109%, using the calibration curve, and acceptable precision (R.S.D. about 8%). The method is now being tested for the determination of salicylic acid contained in commercially available drug specialities or galenic products. So far agreement with nominal values has been found to be between 75% and 110% with a R.S.D. of less than 8%. PMID- 16672185 TI - Sentinel node biopsy for clear cell sarcoma. AB - Clear cell sarcoma (CCS), also known as clear cell sarcoma of tendons and aponeuroses or malignant melanoma of soft tissue, is a rare malignant tumor and is histogenitically related to melanoma. The aim of this study was to describe our experience with the sentinel node (SN) procedure for CCS patients and to discuss the potential value of this technique for CCS patients. Five patients with a subcutaneous CCS, who underwent an SN procedure, are described. Two patients had positive SNs, with additional tumor positive nodes in both lymph node dissection specimens. Only the patients with tumor positive SNs developed recurrent disease during an average follow-up of 33 months. None of the negative SN patients developed recurrent disease and all were alive after an average follow-up of 39 months. SN status seems to predict additional nodal involvement and recurrent disease as well as survival. The SN procedure might be a useful and accurate staging procedure in CCS patients, comparable to the situation in melanoma. PMID- 16672186 TI - Safety considerations for operating room personnel during hyperthermic intraoperative intraperitoneal chemotherapy perfusion. AB - The new treatment strategy for Peritoneal Surface Malignancy combines a cytoreductive surgery and perioperative intraperitoneal chemotherapy. Cytoreduction removes all macroscopic tumor. Intraperitoneal chemotherapy avoids implantation of microscopic residual tumor cells on intra-abdominal surfaces when it is administered intraoperatively and/or early in the postoperative period. Delivering cytotoxic drugs directly into the peritoneal cavity maximizes dose intensity and minimizes systemic toxicity. Hyperthermia is selectively cytotoxic for malignant cells and potentiates the effect of chemotherapy. Implementation of this procedure makes the perioperative personnel to face a risk of exposure to cytotoxic agents. Furthermore, peritonectomies and electro-evaporation of tumor nodules are performed with high voltage electrocautery, generating a large amount of surgical smoke during several hours. Inhalation of these fumes may be also a risk for healthcare workers. In this article, we analyse in depth these new risks of the operating room personnel, we review the literature, and we give guidelines for secure performance of cytoreductive surgery and hyperthermic intraoperative intraperitoneal chemotherapy, as well as for early postoperative intraperitoneal chemotherapy administration. These new procedures are safe techniques for patients and healthcare workers provided adequate policies are adopted to avoid occupational exposure. PMID- 16672187 TI - How Tai Chi improves balance: biomechanics of recovery to a walking slip in impaired seniors. AB - BACKGROUND AND AIMS: This study examined the effect of Tai Chi (TC) training on biomechanical responses to large, fast walking perturbations in balance-impaired seniors. METHODS: Twenty-two seniors (age 68-92, BERG 44 or less) with surgical interventions to knees, hips, and back were randomly divided into control or TC groups. Groups trained 1.5h/day, 5 days/week for 3 weeks. Controls received TC training after post-control testing. Subjects walked across a force plate triggered to move forward 15cm at 40cm/s at right heel strike (RHS). Kinematics, center of pressure (COP) and center of mass (COM) responses were measured. RESULTS: TC but not control training significantly reduced tripping (p25 kg/m2 (WHO, 2000). Children were considered overweight when their BMI was >95th percentile (CDC, 2000). Prevalence of overweight was calculated and logistic regressions were performed. The sensitivity and specificity of parental overweight were calculated. A total of 26% (805/3059) parents were overweight. Of the families with one overweight parent, 15% (N = 109) had an overweight child. When both parents were overweight, 17% (N = 9) had an overweight child. After adjusting for child's age and gender, parental education, family income, and spouse's BMI as required, the odds of having an overweight child were 2.5 [1.8, 3.3] for one overweight parent, and 3.2 [1.4, 7.1] for both overweight parents. While the sensitivity of one overweight parent to identify families with overweight school age children was 44%, specificity was 75%. The presence of both overweight parents provided a 3% sensitivity and 98% specificity for the identification of an overweight school age child. Although parental overweight was obtained from self-reported weight and height in Busan (Korea), it is a practical indicator to identify families with an overweight school age child, it has poor sensitivity. PMID- 16672204 TI - Body image differences among Malay, Samoan, and Australian women. AB - Comparisons of body attitudes and associated behaviours were undertaken using Malay, Samoan, and Australian female students. The general goal of the research was to determine the degree to which the observed pattern of attitudes and behaviours was attributable to culture. The specific analyses comprised an examination of group differences using standard measures that included the Body Attitudes Questionnaire, the Three-Factor Eating Questionnaire and detailed questions concerning the use of diet and exercise as weight control strategies. The main findings concerned a number of cultural differences, particularly in relation to diet and exercise, that were evident even with the effect of body mass index held constant. These results are interpreted in terms of the efficacy of entrenched cultural beliefs in protecting against introduced, more dominant, cultural values. The Australian sample exhibited the most negative body image, although there was some evidence that Malays and Samoans were influenced by Western ideals of weight and shape. It is proposed that to fully understand the differential meaning of negative body image across cultures and the potential impact of westernisation, both within-group and between-group differences in body size need to be acknowledged. PMID- 16672205 TI - Differences in nutrient intakes and physical activity levels of Japanese and Australian Caucasian males living in Australia and Japanese males living in Japan. AB - The aim of the study was to determine the nutritional status and nutrient intakes of young Japanese males living in Australia and compared with Japanese males living in Japan and Australian Caucasian males. Four-day dietary records were obtained from 65 Japanese living in Australia (JA), 81 Japanese living in Japan (JJ), and 70 Australian Caucasian males (AA) aged 18-30 years old, together with body composition and physical activity level assessments using anthropometry and the questionnaire. Australian males were significantly taller and heavier than the Japanese counterparts and also showed a greater percent body fat (%BF) and height-corrected sum of skinfolds compared with Japanese males living in Japan (%BF: JJ = 16.6 +/- 5.2, AA = 18.7 +/- 5.6; height corrected sum of skinfolds: JJ = 78.8 +/- 37.3, AA = 96.0 +/- 39.5) (P<0.05). A greater proportion of Australian Caucasian males (98.6%) were involved in vigorous physical exercise than Japanese males (JA = 72.3%; JJ = 85.2%). The JA group consumed a greater amount of energy from protein and fat sources as well as greater calcium, iron, dietary fibre and niacin equivalents intakes than the JJ group (P<0.05). The results suggest that Japanese males living in Australia consumed more energy-dense westernised diet than Japanese males living in Japan. Because of lower physical activity level than Australian males, consumption of energy-dense diet may increase the risk of weight gain among Japanese males who stay in Australia for a long-term. PMID- 16672206 TI - Physical activity and nutritional status of children of low socioeconomic status. Two interrelated problems: undernutrition and overweight. AB - The aim of this study was to analyse the level of habitual physical activity (HPA) and nutritional status of children attending selected public schools in Brazil. The sample comprised 1719 participants who ranged in age from 11 to 14 yrs with 861 females (F) aged 12.7+/-1.0 yr and 858 males (M) aged 12.7+/-1.0 yr. The short form of the International Physical Activity Questionnaire (IPAQ) was used to measure HPA. Nutritional status was assessed using the body mass index (BMI). Statistical analyses included ANOVA followed by the post-hoc Tukey-test (P<0.05) which showed a statistically significant difference between M and F in time spent in moderate intensity physical activity. F completed more moderate intensity physical activity than M whereas time in vigorous physical activities was higher in M than F. Analyses of BMI and IPAQ categorical score using Pearson product moment correlations with subsequent Fisher Z transformation showed values of Z = 0.49 for females and Z = 0.44 for males, indicating a low relationship between these variables. HPA levels showed 93.7% of the M and 91.1% of the F were at least minimally active. According to BMI values, 7.3% of the cohort was underweight; 83.0% normal weight; 8.3% overweight and 1.3% obese. These results demand attention, particularly for children at the lower end of nutritional status and HPA levels due to the potential negative impact on their growth and development. At the other end of the nutritional spectrum, one needs to be concerned regarding the levels of excess body weight, mainly considering the poor region in which the children live. PMID- 16672207 TI - Diet composition and body mass index in Tehranian adults. AB - Human studies investigating the relationship between macronutrients intake and obesity, have failed to achieve consistent findings. This study was undertaken to assess the relationship between macronutrients intake and body mass index in a group of Tehranians. From 15,005 participants of the Tehran Lipid and Glucose Study, 1290 subjects aged over 10 years (565 males and 725 females) were selected randomly for dietary survey. Anthropometric indices were measured according to standard protocols and BMI was calculated. Dietary data were collected by trained interviewers using two non-consecutive 24-hour dietary recalls. Data on smoking habits, educational level and physical activity were compiled. Under- and over reporting of energy intake were defined as EI: BMR < 1.35 and > or = 2.4, respectively. Calorie-adjusted amounts of macronutrients were calculated by the residual method, following which energy intakes from all calorie-adjusted macronutrients were simultaneously included in the multiple regression models controlling for age, physical activity, educational level and smoking and mutual effects of macronutrients. Total energy intake was not included to avoid collinearity. BMI increased with age in either gender. Controlling for confounding variables, energy intake from fat was positively associated with BMI in males in the 10-18, 19-24, 25-50 and 51+ year age categories (beta = 0.06, 0.13, 0.33, 0.48, P<0.05 for all, respectively) and females in the 19-24, 25-50 and 51+ age categories (beta = 0.17, 0.43, 0.52, P<0.05 for all, respectively). This relationship remained after excluding misreporters (beta = 0.06, 0.15, 0.36, 0.50 for males and beta = 0.21, 0.46, 0.54 for females in the corresponding age categories, respectively). The correlation of fat intake to BMI was not significant in younger females (10-18 year). No association was seen between energy intake from protein and carbohydrate with BMI in subjects before and after exclusion of misreporters. In conclusion, energy from fat was found to be independently and positively associated with obesity in adults. No other association was observed between energy from protein and carbohydrate with BMI. PMID- 16672208 TI - Skinfold thickness, body fat percentage and body mass index in obese and non obese Indian boys. AB - Childhood obesity is presently increasing worldwide and has created enormous concern for researchers working in the field of obesity related diseases with special interest in child health and development. Selected anthropometric measurements including stature, body mass, and skinfolds are globally accepted sensitive indicators of growth patterns and health status of a child. The present study was therefore aimed not only at evaluating the body mass index (BMI), skinfolds, body fat percentage (%fat) in obese school going boys of West Bengal, India, but also aimed to compare these data with their non-obese counterparts. Ten to sixteen year old obese boys (N = 158) were separated from their non-obese counterparts using the age-wise international cut-off points of BMI. Skinfolds were measured using skinfold calipers, BMI and %fat were calculated from standard equations. Body mass, BMI, skinfolds and %fat were significantly (P<0.001) higher for the sample of obese boys when compared to their non-obese counterparts. The obese group also showed progressive age-wise increments in all recorded anthropometric parameters. Stature (cm) showed no significant inter-group variation except in the 10 year age group (P<0.001). All data for the non-obese group were comparable with other national and international studies, but those collected for the obese group could not feasibly be compared because the availability of data on obese children is limited. Current data and prediction equations will not only serve as a reference standard, but also be of vital clinical importance in order to identify or categorize obese boys, and to take preventative steps to minimise serious health problems that appear during the later part of life. PMID- 16672209 TI - Prevalence of obesity, overweight and underweight in a Hong Kong community: the United Christian Nethersole Community Health Service (UCNCHS) primary health care program 1996-1997. AB - The aim of this cross-sectional observation study was to assess thoroughly the body mass index (BMI) profile in Hong Kong Chinese and report all categories of BMI cutoff points as suggested by the World Health Organization (WHO). A cohort of 17,242 subjects (4822 men and 12420 women) aged 15 or above from the community of Hong Kong presenting themselves voluntarily from April 1996 to August 1997 for primary health assessment at the Three Health Centers of the United Christian Nethersole Community Health Service (UCNCHS). The mean age (+/- SD) was 51.0 +/- 16.2 years (range 15-96 years, median 49.6 years). The mean BMI of the 17,242 subjects was 23.5 +/- 3.3 kg/m2 in men and 23.0 +/- 3.7 kg/m2 in women. The BMI peaked at age 30 to 50 years in men and 50 to 70 years in women. The age standardized prevalence of BMI > or = 30 kg/m2 or > or = 25 kg/m2 in Hong Kong Chinese was 3.0% in men and 3.2% in women, and 29.1% in men and 21.3% in women, respectively. For underweight, 35.9% and 27.8% of women and 27.5% and 10.3% of men aged 15-20 and 20-30 years, respectively, had BMI < 18.5 kg/m2. In conclusion, a significant proportion of Hong Kong Chinese had a BMI > or = 25 kg/m2. Among Hong Kong Chinese aged < 30 years, the prevalence of underweight was also high. PMID- 16672210 TI - Are energy drinks contributing to the obesity epidemic? AB - The consumption of energy drinks containing sucrose and caffeine is increasing worldwide. Ten healthy women aged 18 to 22 years and fasted overnight were randomly allocated to a standardised dose of sucrose either as an "energy" drink (containing sucrose and caffeine) or lemonade on the first day and then crossed over to the alternative drink on a second day. For thirty minutes before and thirty minutes after drinking oxygen consumption and carbon dioxide production were measured in the resting subject breath-by-breath by indirect calorimetry and the rates of carbohydrate and fat oxidation calculated. Energy drink consumption apparently caused increased carbohydrate oxidation (P = 0.004) and reduced lipid oxidation (P = 0.004) compared to lemonade. The longer term effects of combined caffeine and sucrose intake, particularly in sedentary individuals, on metabolism and body fatness needs further examination. PMID- 16672211 TI - Fruits and vegetables, 5+ a day: are we getting the message across? AB - Fruit and vegetables have important health promoting properties. The 5+ a day programme aims to promote awareness of the need to eat more of these foods. This paper presents and discusses the results of two surveys designed to determine the success of the 5+ a day programme across New Zealand. Household surveys were carried out by a marketing research company in 1999 and 2000. The 1999 questionnaire focused on awareness and understanding of the 5+ a day campaign. The 2000 questionnaire focused on attitudes to health and on intakes of fruits and vegetables. Data were collected from households nationwide (1999 survey N = 200, 2000 survey N = 520). Spontaneous consumer awareness of messages promoting the need to eat more fruit and vegetables was high. Seventy-one percent of all respondents identified the 5 servings a day message from the 5+ a day logo regardless of whether they had seen it before. The meaning of the hand in the logo was less clear with only 2.5% identifying the 'serving size' element of the logo. Fruit and vegetable intakes of respondents were influenced by demographic factors: gender, ethnicity, education and occupation (all P < or = 0.05). Positive attitude towards the relationship between fruit, vegetables and health was influenced by similar factors and in turn affected fruit and vegetable intakes. The 5+ a day message is well recognised and understood. Portion size is less well understood. The 5+ a day message promotes positive attitudes towards healthy eating which are associated with healthier eating habits, but some groups within society may need further attention. PMID- 16672212 TI - Drinking tea is associated with lower plasma total homocysteine in older women. AB - Dietary polyphenols are suggested to elevate plasma total homocysteine concentration (tHcy). Although tea is rich in polyphenols, it has been associated with lower tHcy, which may be due to its folate content. Our aims were to investigate relationships of tea intake and 4-O-methylgallic acid (4OMGA)--a biomarker of exposure to tea-derived polyphenols--with tHcy in older women. In a cross-sectional study of 232 women over 70 years of age, we measured tHcy, tea intake, 24 h urinary excretion of 4OMGA, and red cell folate. Tea intake and 4OMGA excretion were inversely related to tHcy. Tea intake (>2 cups) and 4OMGA excretion above the median were associated with lower tHcy by approximately 1 mmol/L (P <0.01). Red cell folate was not associated with tea intake or 4OMGA excretion. The observed lower tHcy in women with higher tea intake is consistent in direction and magnitude with previous epidemiological studies, but any mechanisms remain unclear. PMID- 16672213 TI - Concordance of diets and eating practices in a rural Guatemalan setting with the cancer prevention recommendations of the World Cancer Research Fund: estimates from existing dietary intake. AB - To evaluate concordance of eating practices in a rural Guatemalan setting with the 14-point cancer prevention recommendations of the World Cancer Research Fund/American Institute for Cancer Research. Two-hundred sixty nine food frequency questionnaires based on the Willett model were analysed with respect to concordance with the recommendations, aimed at constraining intakes of: fat, sugar, salt, red meat and alcoholic beverages within specific ranges; restricting nutritional supplements' use for the express purpose of avoiding cancer; maximizing the consumption of: edible plants, especially fruits, vegetables, cereals, tubers and legumes; achieving dietary variety and nutrient adequacy; avoiding high-temperature cooking, and exposure to food additives, pesticides and residues; adequately preserving perishable and fungal-prone foods; maintaining normal body composition and regular levels of physical activity. The study was conducted in the county seat and three hamlets from the rural province of Santa Rosa. 214 females and 55 males were evaluated. Daily food servings of plant origin varied from 18.2 to 99.1% (74.6 +/- 10.7%). The guidelines' criteria were met by more than 80% for total fat, red meat, ethanol, fruits and vegetables, cereals-tubers-legumes, sugar and nutritional adequacy, whereas 80% concordance was not met for nutritional supplements, and dietary variety. The other variables could not be evaluated. Concordance with national standards for micronutrient intake was not achieved most frequently for calcium, riboflavin, and iron. Guatemalan cuisine and diet in this rural setting has features of a cancer protective diet, but complementary actions must be taken, within the social and economic realities of the region. PMID- 16672214 TI - Lactagogue effects of Torbangun, a Bataknese traditional cuisine. AB - Coleus amboinicus Lour (CA) has been used as a breast milk stimulant (a lactagogue) by Bataknese people in Indonesia for hundreds of years. However, the traditional use of CA is not well documented, and scientific evidence is limited to establish CA as a lactagogue. This investigation was conducted to elucidate the effect of traditional use of CA during the first month of lactation on quantity and quality of the breast milk. The results collected from the study show that CA supplementation increased breast milk production without compromising the nutritional quality of the breast milk. Lactating women receiving CA supplementation had a 65% increase in milk volume during the last two weeks of supplementation (from Day 14 to Day 28). This increase was greater than that of lactating women receiving Molocco+B12 tablets (10%) or Fenugreek seeds (20%). The residual effects of CA supplementation were seen even after the supplementation had ended for one month. Results of the present study confirmed the belief and the practice amongst the Bataknese people that CA can be used as a lactagogue in humans, and the use of CA might be suitable for lactating women in general. PMID- 16672215 TI - Phytochemical intakes of the Fijian population. AB - The dietary intakes of major phytochemicals in Fijian population were estimated from the consumption of 90 plant foods reported in five major surveys conducted in Fiji from 1952 to 2001. These surveys included the Naduri Longitudinal study, for which food intake data were collected on four occasions in 1952, 1953, 1963 and 1994), the 1982 and 1993 National Nutritional Surveys, the 1996 Suva-Nausori Corridor cross-sectional study, the 1999 Verata cross-sectional study, and the 2001 Fiji Food Choice study. It was found that the Fijian population generally had low intakes of total phenols (275 mg/day), and total flavonoids (17.5 mg/day), but high intake of total carotenoids (20 mg/day), in comparisons with the intakes of other populations reported in literature. It has been speculated that the change of eating patterns resulting in the low intakes of phytochemicals may have partly contributed to the increase in the nutritionally chronic disease morbidity and mortality among the Fijians. It is further recommended that the traditional Fijian food patterns with high fruits and vegetables should be revived, and the consumption of sweet potato leaves and drumstick leaves, both of which were rich in phytochemicals, should be promoted. PMID- 16672217 TI - The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter. AB - Protein S (PS) is a vitamin K-dependent plasma protein that inhibits blood coagulation by serving as a nonenzymatic cofactor for activated protein C in the protein C anticoagulant pathway. Low PS levels are a risk factor for the development of deep venous thrombosis. The regulation of PS levels through transcriptional regulation of the PS gene was investigated in this report. A minimal PS gene promoter 370 bp upstream from the translational initiation codon was sufficient for maximal promoter activity in transient transfections regardless of the cell type. A pivotal role for Sp1 in the constitutive expression of the PS gene was demonstrated through electrophoretic mobility shift assay experiments, transient expression of mutant PS promoter-reporter gene constructs, and chromatin immunoprecipitations in HepG2 cells. At least four Sp binding sites were identified. The two sites most proximal to the translational start codon were found to be indispensable for PS promoter activity, whereas mutation of the two most distal Sp-binding sites had a negligible influence on basal promoter activity. In addition, all other major promoter-binding proteins that were found by electrophoretic mobility shift assay could be positively identified in supershift assays. We identified binding sites for the hepatocyte specific forkhead transcription factor FOXA2, nuclear factor Y, and the cAMP response element-binding protein/activating transcription factor family of transcription factors. Their relevance was investigated using site-directed mutagenesis. PMID- 16672216 TI - The alternative stimulatory G protein alpha-subunit XLalphas is a critical regulator of energy and glucose metabolism and sympathetic nerve activity in adult mice. AB - The complex imprinted Gnas locus encodes several gene products including G(s)alpha, the ubiquitously expressed G protein alpha-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific G(s)alpha isoform XLalphas. XLalphas is only expressed from the paternal allele, whereas G(s)alpha is biallelically expressed in most tissues. XLalphas knock-out mice (Gnasxl(m+/p-)) have poor suckling and perinatal lethality, implicating XLalphas as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxl(m+/p-) mice. Gnasxl(m+/p-) mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLalphas is not normally expressed in adult adipose tissue, and Gnasxl(m+/p-) mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a beta3-adrenergic agonist. Gnasxl(m+/p-) mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLalphas deficiency (or deficiency of the related alternative truncated protein XLN1). XLalphas (or XLN1) is a negative regulator of sympathetic nervous system activity in mice. PMID- 16672218 TI - Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX. AB - LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function. PMID- 16672219 TI - Avian and canine aldehyde oxidases. Novel insights into the biology and evolution of molybdo-flavoenzymes. AB - Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity. PMID- 16672220 TI - Identification of far upstream element-binding protein-1 as an authentic Parkin substrate. AB - Aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 was recently identified as an authentic substrate of the ubiquitin E3 ligase, parkin, a gene associated with autosomal recessive juvenile parkinsonism. Far upstream element binding protein 1 is known to be degraded in an aminoacyl-tRNA synthetase interacting multifunctional protein type 2 dependent manner, which is crucial for lung cell maturation in early development. Therefore, we wondered whether far upstream element-binding protein 1 levels are altered in the absence of Parkin and in Parkinson disease. We herein report that far upstream element-binding protein 1 accumulates in Parkin knock-out mice, patients with autosomal recessive juvenile parkinsonism, sporadic Parkinson disease, and diffuse Lewy Body disease as well as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. Moreover, Parkin interacts with and ubiquitinates far upstream element-binding protein 1 facilitating its degradation through the ubiquitin proteasome system. Taken together, these results suggest that far upstream element-binding protein 1 is an authentic substrate of Parkin and that far upstream element-binding protein 1 might play an important role in development of Parkinson disease pathology along with aminoacyl-tRNA synthetase interacting multifunctional protein type 2. PMID- 16672221 TI - Identification of tyrosine residues critical for the function of an ion-coupled multidrug transporter. AB - Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site directed mutagenesis. Two of these residues, Tyr-40 and Tyr-60, can be partially replaced with hydroxyamino acids, but in the case of Tyr-40, replacement with either Ser or Thr generates a protein with modified substrate specificity. Replacement of Tyr-4 with either Trp or Phe generates a functional transporter. A Cys replacement at this position generates an uncoupled protein; it binds substrate and protons and transports the substrate downhill but is impaired in uphill substrate transport in the presence of a proton gradient. The role of these residues is discussed in the context of the published structures of EmrE. PMID- 16672222 TI - Structure of pyrimidine 5'-nucleotidase type 1. Insight into mechanism of action and inhibition during lead poisoning. AB - Eukaryotic pyrimidine 5'-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5'-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35 A resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25 A, 2) phosphoenzyme intermediate analog at 2.30 A, 3) product-transition complex analog at 2.35 A, and 4) product complex at 2.1A resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35 A) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides. PMID- 16672223 TI - Enhanced expression of multidrug resistance-associated protein 2 and reduced expression of aquaglyceroporin 3 in an arsenic-resistant human cell line. AB - Arsenic-resistant cells (R15), derived from a human lung adenocarcinoma cell line (CL3), were 10-fold more resistant to sodium arsenite (As(III)). Because R15 cells accumulated less arsenic than parental CL3 cells, this arsenic resistance may be due to higher efflux and/or lower uptake of As(III). We therefore compared expression of the multidrug resistance-associated proteins MRP1, MRP2, and MRP3 in these two cell lines. MRP2 expression was 5-fold higher in R15 cells than in CL3 cells, whereas MRP1 and MRP3 expression levels were similar. Furthermore, verapamil and cyclosporin A, inhibitors of multidrug resistance transporters, significantly reduced the efflux of arsenic from R15. Thus, increased arsenic extrusion by MRP2 may contribute to arsenic resistance in R15 cells. We also examined the expression of several aquaglyceroporins (AQPs), which mediate As(III) uptake by cells. Little AQP7 or AQP9 mRNA was detected by reverse transcription-PCR in either cell line, whereas AQP3 mRNA expression was 2-fold lower in R15 cells than in CL3 cells. When AQP3 expression in CL3 cells was knocked down by RNA interference, CL3 cells accumulated less arsenic and became more resistant to As(III). Conversely, overexpression of AQP3 in human embryonic kidney 293T cells increased arsenic accumulation, and the cells were more susceptible to As(III) than 293T cells transfected with vector alone. These results suggest that AQP3 is involved in As(III) accumulation. Taken together, our results suggest that enhanced expression of MRP2 and lower expression of AQP3 are responsible for lower arsenic accumulation in arsenic-resistant R15 cells. PMID- 16672224 TI - Casein kinase 2 Is activated and essential for Wnt/beta-catenin signaling. AB - Wnt/beta-catenin signaling is essential to early development. Activation of Frizzled-1 by Wnts induces nuclear accumulation of beta-catenin and activation of Lef/Tcf-dependent gene expression. Casein kinase 2 has been shown to affect Wnt/beta-catenin signaling. How casein kinase 2 exerts an influence in Wnt signaling is not clear; casein kinase 2 has been reported to be constitutively active (i.e. not regulated). Herein we show to the contrary that casein kinase 2 activity is rapidly and transiently increased in response to Wnt3a stimulation and is essential for Wnt/beta-catenin signaling. Chemical inhibition of casein kinase 2 or suppression of its expression blocks Frizzled-1 activation of Lef/Tcf sensitive gene expression. Treatment with pertussis toxin or knock down of Galpha(q) or Galpha(o) blocks Wnt stimulation of casein kinase 2 activation, as does suppression of the phosphoprotein Dishevelled, demonstrating that casein kinase 2 is downstream of heterotrimeric G proteins and Dishevelled. Expression of a constitutively active mutant of either Galpha(q) or Galpha(o) stimulates casein kinase 2 activation and Lef/Tcf-sensitive gene expression. Thus, casein kinase 2 is shown to be regulated by Wnt3a and essential to stimulation of the Frizzled-1/beta-catenin/Lef-Tcf pathway. PMID- 16672225 TI - Syntaxin-1A actions on sulfonylurea receptor 2A can block acidic pH-induced cardiac K(ATP) channel activation. AB - During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of K(ATP) channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125-47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac K(ATP) channels. K(ATP) channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-deltaC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn 1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the K(ATP) channels, which may serve as a "brake" to temper the fluctuation of low pH-induced K(ATP) channel opening that could induce fatal reentrant arrhythmias. PMID- 16672226 TI - Anti-hemostatic effects of a serpin from the saliva of the tick Ixodes ricinus. AB - Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response. PMID- 16672227 TI - Uncoupling the enzymatic and autoprocessing activities of Helicobacter pylori gamma-glutamyltranspeptidase. AB - Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions. PMID- 16672228 TI - Crystal structure of the Orf virus NZ2 variant of vascular endothelial growth factor-E. Implications for receptor specificity. AB - Mammalian vascular endothelial growth factors constitute a family of polypeptides, vascular endothelial growth factor (VEGF)-A, -B, -C, -D and placenta growth factor (PlGF), that regulate blood and lymphatic vessel development. VEGFs bind to three types of receptor tyrosine kinases, VEGF receptors 1, 2, and 3, that are predominantly expressed on endothelial and some hematopoietic cells. Pox viruses of the Orf family encode highly related proteins called VEGF-E that show only 25-35% amino acid identity with VEGF-A but bind with comparable affinity to VEGFR-2. The crystal structure of VEGF-E NZ2 described here reveals high similarity to the known structural homologs VEGF-A, PlGF, and the snake venoms Vammin and VR-1, which are all homodimers and contain the characteristic cysteine knot motif. Distinct conformational differences are observed in loop L1 and particularly in L3, which contains a highly flexible GS rich motif that differs from all other structural homologs. Based on our structure, we created chimeric proteins by exchanging selected segments in L1 and L3 with the corresponding sequences from PlGF. Single loop mutants did not bind to either receptor, whereas a VEGF-E mutant in which both L1 and L3 were replaced gained affinity for VEGFR-1, illustrating the possibility to engineer receptor specific chimeric VEGF molecules. In addition, changing arginine 46 to isoleucine in L1 significantly increased the affinity of VEGF-E for both VEGF receptors. PMID- 16672229 TI - Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking. AB - The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655 1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface exposed side chains in this interaction. Mutations causing delta deltaG > or = 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR x uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction. PMID- 16672230 TI - The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9 bioavailability via inhibition and binding to cargo receptors. AB - Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of approximately 64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor related protein-1 (LRP-1). Furthermore, megalin/LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs. PMID- 16672231 TI - The biochemical characterization of ferret carotene-9',10'-monooxygenase catalyzing cleavage of carotenoids in vitro and in vivo. AB - Previous studies have shown that beta-carotene 15,15'-monooxygenase catalyzes the cleavage of beta-carotene at the central carbon 15,15'-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9',10' monooxygenase (CMO2) in beta-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10' carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9',10'-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of beta-carotene/lycopene cleavage into beta-apo 10'-carotenal/apo-10'-lycopenal in vitro and the formation of apo-10'-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-beta-carotene and the 5-cis- and 13-cis isomers of lycopene at the 9',10'-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with beta-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10' lycopenol, as well as up-regulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10'-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10' lycopenoic acid and apo-10'-lycopenol, respectively, depending upon the presence of NAD+ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10'-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo lycopenoids may have specific biological activities related to human health. PMID- 16672232 TI - Protein SRP68 of human signal recognition particle: identification of the RNA and SRP72 binding domains. AB - The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68-SRP72 interaction remained stable at elevated salt concentrations and engaged approximately 150 amino acids from the N terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68. PMID- 16672234 TI - Glu257 in GroEL is a sensor involved in coupling polypeptide substrate binding to stimulation of ATP hydrolysis. AB - The ATPase activity of many types of molecular chaperones is stimulated by polypeptide substrate binding via molecular mechanisms that are, for the most part, unknown. Here, we report that such stimulation of the ATPase activity of GroEL is abolished when its conserved apical domain residue Glu257 is replaced by alanine. This mutation is also found to convert the ATPase profile of GroEL, a group I chaperonin, into one that is characteristic of group II chaperonins. Steady-state and transient kinetic analysis indicate that both effects are due, at least in part, to a reduction of the affinity of GroEL for ADP. This finding indicates that nonfolded proteins stimulate ATP hydrolysis by accelerating the off-rate of the ADP formed, thereby allowing more rapid cycles of ATP binding and hydrolysis. PMID- 16672233 TI - Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC. AB - The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin. PMID- 16672235 TI - The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1. AB - The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure. PMID- 16672236 TI - Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1. AB - The "mu loop," an 11-residue loop spanning amino acid residues 33-43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35-44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the "Deletion Enzyme." Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4 degrees C and 10 degrees C, but it rapidly loses activity at 25 degrees C, unlike the wild-type enzyme. In the temperature range of 4 degrees C through 25 degrees C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1 1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates. PMID- 16672237 TI - Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain. AB - We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family. PMID- 16672238 TI - Structural characterization of GntR/HutC family signaling domain. AB - The crystal structure of Escherichia coli PhnF C-terminal domain (C-PhnF) was solved at 1.7 A resolution by the single wavelength anomalous dispersion (SAD) method. The PhnF protein belongs to the HutC subfamily of the large GntR transcriptional regulator family. Members of this family share similar N-terminal DNA-binding domains, but are divided into four subfamilies according to their heterogenic C-terminal domains, which are involved in effector binding and oligomerization. The C-PhnF structure provides for the first time the scaffold of this domain for the HutC subfamily, which covers about 31% of GntR-like regulators. The structure represents a mixture of alpha-helices and beta-strands, with a six-stranded antiparallel beta-sheet at the core. C-PhnF monomers form a dimer by establishing interdomain eight-strand beta-sheets that include core antiparallel and N-terminal two-strand parallel beta-sheets from each monomer. C PhnF shares strong structural similarity with the chorismate lyase fold, which features a buried active site locked behind two helix-turn-helix loops. The structural comparison of the C-PhnF and UbiC proteins allows us to propose that a similar site in the PhnF structure is adapted for effector binding. PMID- 16672239 TI - Recurrent use of evolutionary importance for functional annotation of proteins based on local structural similarity. AB - The annotation of protein function has not kept pace with the exponential growth of raw sequence and structure data. An emerging solution to this problem is to identify 3D motifs or templates in protein structures that are necessary and sufficient determinants of function. Here, we demonstrate the recurrent use of evolutionary trace information to construct such 3D templates for enzymes, search for them in other structures, and distinguish true from spurious matches. Serine protease templates built from evolutionarily important residues distinguish between proteases and other proteins nearly as well as the classic Ser-His-Asp catalytic triad. In 53 enzymes spanning 33 distinct functions, an automated pipeline identifies functionally related proteins with an average positive predictive power of 62%, including correct matches to proteins with the same function but with low sequence identity (the average identity for some templates is only 17%). Although these template building, searching, and match classification strategies are not yet optimized, their sequential implementation demonstrates a functional annotation pipeline which does not require experimental information, but only local molecular mimicry among a small number of evolutionarily important residues. PMID- 16672240 TI - Enhanced automated function prediction using distantly related sequences and contextual association by PFP. AB - The impetus for the recent development and emergence of automated function prediction methods is an exponentially growing flood of new experimental data, the interpretation of which is hindered by a shortage of reliable annotations for proteins that lack experimental characterization or significant homologs in current databases. Here we introduce PFP, an automated function prediction server that provides the most probable annotations for a query sequence in each of the three branches of the Gene Ontology: biological process, molecular function, and cellular component. Rather than utilizing precise pattern matching to identify functional motifs in the sequences and structures of these proteins, we designed PFP to increase the coverage of function annotation by lowering resolution of predictions when a detailed function is not predictable. To do this we extend a traditional PSI-BLAST search by extracting and scoring annotations (GO terms) individually, including annotations from distantly related sequences, and applying a novel data mining tool, the Function Association Matrix, to score strongly associated pairs of annotations. We show that PFP can correctly assign function using only weakly similar sequences with a significantly better accuracy and coverage than a standard PSI-BLAST search, improving it more than fivefold. The most descriptive annotations predicted by PFP (GO depth > or = 8) can identify a significant subgraph in the GO with > 60% accuracy and approximately 100% coverage for our benchmark set. We also provide examples of the superb performance of PFP in an assessment of automated function prediction servers at the Automated Function Prediction Special Interest Group meeting at ISMB 2005 (AFP-SIG '05). PMID- 16672242 TI - Conversion of trypsin to a functional threonine protease. AB - The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10(2) -10(4) -fold less active than wild type in kcat/KM but >10(6)-fold more active than the Ser 195 --> Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'- P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195. PMID- 16672241 TI - The crystal structure of Mycobacterium tuberculosis adenylate kinase in complex with two molecules of ADP and Mg2+ supports an associative mechanism for phosphoryl transfer. AB - The crystal structure of Mycobacterium tuberculosis adenylate kinase (MtAK) in complex with two ADP molecules and Mg2+ has been determined at 1.9 A resolution. Comparison with the solution structure of the enzyme, obtained in the absence of substrates, shows significant conformational changes of the LID and NMP-binding domains upon substrate binding. The ternary complex represents the state of the enzyme at the start of the backward reaction (ATP synthesis). The structure is consistent with a direct nucleophilic attack of a terminal oxygen from the acceptor ADP molecule on the beta-phosphate from the donor substrate, and both the geometry and the distribution of positive charge in the active site support the hypothesis of an associative mechanism for phosphoryl transfer. PMID- 16672243 TI - A categorization approach to automated ontological function annotation. AB - Automated function prediction (AFP) methods increasingly use knowledge discovery algorithms to map sequence, structure, literature, and/or pathway information about proteins whose functions are unknown into functional ontologies, typically (a portion of) the Gene Ontology (GO). While there are a growing number of methods within this paradigm, the general problem of assessing the accuracy of such prediction algorithms has not been seriously addressed. We present first an application for function prediction from protein sequences using the POSet Ontology Categorizer (POSOC) to produce new annotations by analyzing collections of GO nodes derived from annotations of protein BLAST neighborhoods. We then also present hierarchical precision and hierarchical recall as new evaluation metrics for assessing the accuracy of any predictions in hierarchical ontologies, and discuss results on a test set of protein sequences. We show that our method provides substantially improved hierarchical precision (measure of predictions made that are correct) when applied to the nearest BLAST neighbors of target proteins, as compared with simply imputing that neighborhood's annotations to the target. Moreover, when our method is applied to a broader BLAST neighborhood, hierarchical precision is enhanced even further. In all cases, such increased hierarchical precision performance is purchased at a modest expense of hierarchical recall (measure of all annotations that get predicted at all). PMID- 16672244 TI - Functional annotation prediction: all for one and one for all. AB - In an era of rapid genome sequencing and high-throughput technology, automatic function prediction for a novel sequence is of utter importance in bioinformatics. While automatic annotation methods based on local alignment searches can be simple and straightforward, they suffer from several drawbacks, including relatively low sensitivity and assignment of incorrect annotations that are not associated with the region of similarity. ProtoNet is a hierarchical organization of the protein sequences in the UniProt database. Although the hierarchy is constructed in an unsupervised automatic manner, it has been shown to be coherent with several biological data sources. We extend the ProtoNet system in order to assign functional annotations automatically. By leveraging on the scaffold of the hierarchical classification, the method is able to overcome some frequent annotation pitfalls. PMID- 16672245 TI - An update on antenatal screening for Down's syndrome and specific implications for assisted reproduction pregnancies. AB - Since the introduction of antenatal serum screening for Down's syndrome (DS) more than two decades ago, several screening approaches have been utilized in routine clinical practice. The current DS screening strategies involve mid-trimester serum biochemistry tests, first trimester tests combining sonographic markers and serum biochemistry and integration of first and second trimester markers. In this review, we evaluate the performance of DS screening strategies according to the Serum, Urine and Ultrasound Screening Study (SURUSS), the First and Second Trimester Evaluation of Risks (FASTER) Trial and the Serum Biochemistry and Fetal Nuchal Translucency Screening (BUN) Study. We also evaluate the performance of first trimester screening in studies and meta-analyses by other groups. Specific issues related to assisted reproduction technology (ART) pregnancies are also addressed in this review. PMID- 16672246 TI - Ovarian feedback, mechanism of action and possible clinical implications. AB - The secretion of gonadotrophins from the pituitary in women is under ovarian control via negative and positive feedback mechanisms. Steroidal and non steroidal substances mediate the ovarian effects on the hypothalamic-pituitary system. During the follicular phase of the cycle, estradiol (E(2)) plays a key role, while circulating progesterone (at low concentrations) and inhibin B contribute to the control of LH and FSH secretion respectively. During the luteal phase, both E(2) and progesterone regulate secretion of the two gonadotrophins, while inhibin A plays a role in FSH secretion. The intercycle rise of FSH is related to changes in the levels of the steroidal and non-steroidal substances during the luteal-follicular transition. In terms of the positive feedback mechanism, E(2) is the main component sensitizing the pituitary to GnRH. Activity of a non-steroidal ovarian substance, named gonadotrophin surge-attenuating factor (GnSAF), has been detected after ovarian stimulation. It is hypothesized that GnSAF, by antagonizing the sensitizing effect of E(2) on the pituitary, regulates the amplitude of the endogenous LH surge at midcycle. Disturbances in the feedback mechanisms can occur in various abnormal conditions or after treatment with pharmaceutical compounds that interfere with the production or the action of endogenous hormones. PMID- 16672247 TI - The sensitivity of the child to sex steroids: possible impact of exogenous estrogens. AB - The current trends of increasing incidences of testis, breast and prostate cancers are poorly understood, although it is assumed that sex hormones play a role. Disrupted sex hormone action is also believed to be involved in the increased occurrence of genital abnormalities among newborn boys and precocious puberty in girls. In this article, recent literature on sex steroid levels and their physiological roles during childhood is reviewed. It is concluded that (i) circulating levels of estradiol in prepubertal children are lower than originally claimed; (ii) children are extremely sensitive to estradiol and may respond with increased growth and/or breast development even at serum levels below the current detection limits; (iii) no threshold has been established, below which no hormonal effects can be seen in children exposed to exogenous steroids or endocrine disruptors; (iv) changes in hormone levels during fetal and prepubertal development may have severe effects in adult life and (v) the daily production rates of sex steroids in children estimated by the Food and Drug Administration in 1999 and still used in risk assessments are highly overestimated and should be revised. Because no lower threshold for estrogenic action has been established, caution should be taken to avoid unnecessary exposure of fetuses and children to exogenous sex steroids and endocrine disruptors, even at very low levels. PMID- 16672248 TI - Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression. AB - The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells. PMID- 16672249 TI - Burden of overweight in Germany: prevalence differences between former East and West German children. AB - BACKGROUND: Given the increasing prevalence of childhood overweight, we aimed to quantify the population burden and evaluate potential regional differences in anthropometric characteristics and prevalence of overweight in fourth graders in two German cities. METHODS: Data were analysed from a cross-sectional school based study conducted in 1995-96 in Dresden (former East Germany) and Munich (former West Germany) as part of the International Study of Asthma and Allergies in Childhood. Height and weight of the children were measured, and the parents completed a questionnaire. 2474 children age 9-10 years provided anthropometric data. Overweight was defined based on the age-specific and gender-specific international cut-off values for body mass index. RESULTS: Dresden children were on average 1.2 kg lighter and >1 cm taller than their Munich peers. The prevalence of overweight in Dresden was 15.2% in girls and 14.2% in boys compared with 24 and 22.2%, respectively, in Munich. Differences were observed between Dresden and Munich with respect to the proportion of children of non-German nationality, household smoking, breastfeeding practices, and individual dietary behaviours. Even in combination these factors were not able to explain entirely the between-city overweight differences. CONCLUSIONS: A substantial proportion of pre-adolescent children in Germany is now considered overweight with marked regional differences in prevalence. Comparison of population-level factors indicate that other unmeasured determinants of overweight may be responsible for the marked differences in the prevalence of overweight. PMID- 16672250 TI - Attitudes towards smoking policies and tobacco control measures in relation to smoking status and smoking behaviour. AB - BACKGROUND: To examine support for various smoking policies and tobacco control measures among lifetime smokers in a country with weak anti-smoking legislation and an underdeveloped anti-smoking climate. METHODS: Current (n = 624) and former smokers (n = 131) from a general population survey filled in the 30-item Smoking Policy Index (SPI). Structural equation modelling was used to confirm the SPI factorial structure and to test whether smoking status and smoking behaviour variables were related to the six dimensions of the SPI. RESULTS: The dimension with the highest support was penalties for sales to minors. Sanctions against smokers had the lowest support. Current smokers compared with former smokers showed lower support on the taxes/fees, public education, and environmental restrictions dimensions while controlling for gender, age, and social status. Within current smokers, unfavourable smoking behaviours were associated with lower support. CONCLUSION: Even in a country with poor tobacco control conditions, lifetime smokers including smokers with highly unfavourable smoking behaviours strongly support smoking policies and tobacco control measures concerning penalties and advertising/promotion. These measures should be used to promote anti-smoking legislation, and strict law enforcement of these measures is expected to be accepted by all smokers. For measures that are not supported by all lifetime smokers, interventions may be useful to increase acceptability. A limitation of the present study is the absence of never-smokers as a comparison group. PMID- 16672251 TI - Use of health care services by Afghan, Iranian, and Somali refugees and asylum seekers living in The Netherlands. AB - BACKGROUND: Although asylum seekers have been coming to The Netherlands since the 1980s, very few epidemiological studies have focused on this group of inhabitants, or on the refugees who have resettled in this country. The objective of this study is to estimate the use of health care services by refugees and asylum seekers and to identify determinants for this utilisation. METHODS: A population-based study was conducted in The Netherlands from June 2003 to April 2004 among adult refugees and asylum seekers from Afghanistan, Iran, and Somalia. A total of 178 refugees and 232 asylum seekers, living in 3 municipalities and 14 reception centres, participated. RESULTS: This study showed that there are no differences between refugees and asylum seekers in the self-reported use of health care services. Respondents from Somalia reported less contacts with a general practitioner, less use of mental health services, and less medication use than respondents from Afghanistan and Iran. Both female gender and older age were related to more contacts with a general practitioner and a medical specialist, and with higher medication use. Poor general health was related to more contacts with a medical specialist and mental health services, and with higher medication use. CONCLUSION: Asylum seekers and refugees seem to have equal access to the Dutch health care system in general. However, there are differences in the self reported use of health care services by the different ethnic groups. PMID- 16672252 TI - Disentangling gender and age effects on risky single occasion drinking during adolescence. AB - OBJECTIVES: To determine the association between adolescent risky single occasion drinking (RSOD) and gender, age, and the relative age position of students within their class. METHOD: A cross-sectional national representative sample of 7088 from 8th to 10th graders in Switzerland (mean age 14.6; SD = 0.94) as part of the ESPAD international study was analysed using hierarchical linear modelling. RESULTS: Being male, older than the class average and a member of older classes (on average) was associated with a higher RSOD frequency. Additionally, interactions between student and class level were found. The higher the mean class age the greater the difference in RSOD between boys and girls, and the lower the impact on RSOD of the relative age position in a class. CONCLUSIONS: In early and mid-adolescence, prevention efforts should try to impede the modelling of alcohol use of older classmates, whereas in late adolescence gender-specific motives for RSOD should be taken into account. PMID- 16672253 TI - The incidence of spina bifida in Sweden 1973-2003: the effect of prenatal diagnosis. AB - BACKGROUND: Many studies have been conducted on the accuracy of prenatal ultrasound diagnosis of foetal CNS-malformations. These studies were mostly hospital-based or, sometimes, multicentre studies. We present here a population based study of the prenatal diagnosis of spina bifida in Sweden over a period of 31 years. METHODS: We compared the number of newborns with spina bifida and the elective terminations because of the prenatal diagnosis of spina bifida for different periods. RESULTS: The rate of spina bifida among newborns diminished gradually from 0.55 per 1000 to 0.29 per 1000 during the study period. In M county the rate of spina bifida at birth decreased very rapidly and from 1993 onwards was about half of that in the rest of the country. CONCLUSION: There has been a decline in the rate of spina bifida at birth. This decline can be seen earlier in the southern part of the country, M county. The decline is probably, to a great extent, a consequence of prenatal ultrasound screening. PMID- 16672255 TI - An individual-based framework for the study of medical error. AB - BACKGROUND: In the late 1990s, medical error came into focus as a problem to be explicitly acknowledged and addressed. Research on this topic is amassing in the epidemiology of medical error and the system and human factors that contribute to error. In addition, however, an understanding of medical errors in terms of the underlying decision process is needed. OBJECTIVE: To present an individual-based framework for the study of medical errors in the context of the decision maker. RESULTS: A framework is developed in terms of four state spaces: the decision environment, problem, goal, and action spaces. The role of information uncertainty is discussed. The framework is purposefully simple to provide flexibility and options for research-specific extensions, but sufficient structure is imposed to guide understanding and investigation. CONCLUSION: Understanding medical error in terms of the proposed framework can guide research and subsequent interventions by illuminating where in the decision process such errors are generated. PMID- 16672254 TI - Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 Cells. AB - Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (GFP)-fimbrin (BY GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)-fimbrin in tobacco BY-2 cells stably expressing GFP AtVam3p (BY-GV7) and BY-2 cells stably expressing gamma-tonoplast intrinsic protein (gamma-TIP)-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating region. Three dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3 butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for the acto myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis. PMID- 16672256 TI - BIOCHAM: an environment for modeling biological systems and formalizing experimental knowledge. AB - BIOCHAM (the BIOCHemical Abstract Machine) is a software environment for modeling biochemical systems. It is based on two aspects: (1) the analysis and simulation of boolean, kinetic and stochastic models and (2) the formalization of biological properties in temporal logic. BIOCHAM provides tools and languages for describing protein networks with a simple and straightforward syntax, and for integrating biological properties into the model. It then becomes possible to analyze, query, verify and maintain the model with respect to those properties. For kinetic models, BIOCHAM can search for appropriate parameter values in order to reproduce a specific behavior observed in experiments and formalized in temporal logic. Coupled with other methods such as bifurcation diagrams, this search assists the modeler/biologist in the modeling process. AVAILABILITY: BIOCHAM (v. 2.5) is a free software available for download, with example models, at http://contraintes.inria.fr/BIOCHAM/. PMID- 16672257 TI - SHARP2: protein-protein interaction predictions using patch analysis. AB - SHARP2 is a flexible web-based bioinformatics tool for predicting potential protein-protein interaction sites on protein structures. It implements a predictive algorithm that calculates multiple parameters for overlapping patches of residues on the surface of a protein. Six parameters are calculated: solvation potential, hydrophobicity, accessible surface area, residue interface propensity, planarity and protrusion (SHARP2). Parameter scores for each patch are combined, and the patch with the highest combined score is predicted as a potential interaction site. SHARP2 enables users to upload 3D protein structure files in PDB format, to obtain information on potential interaction sites as downloadable HTML tables and to view the location of the sites on the 3D structure using Jmol. The server allows for the input of multiple structures and multiple combinations of parameters. Therefore predictions can be made for complete datasets, as well as individual structures. AVAILABILITY: http://www.bioinformatics.sussex.ac.uk/SHARP2. PMID- 16672258 TI - Ensemble classifier for protein fold pattern recognition. AB - MOTIVATION: Prediction of protein folding patterns is one level deeper than that of protein structural classes, and hence is much more complicated and difficult. To deal with such a challenging problem, the ensemble classifier was introduced. It was formed by a set of basic classifiers, with each trained in different parameter systems, such as predicted secondary structure, hydrophobicity, van der Waals volume, polarity, polarizability, as well as different dimensions of pseudo amino acid composition, which were extracted from a training dataset. The operation engine for the constituent individual classifiers was OET-KNN (optimized evidence-theoretic k-nearest neighbors) rule. Their outcomes were combined through a weighted voting to give a final determination for classifying a query protein. The recognition was to find the true fold among the 27 possible patterns. RESULTS: The overall success rate thus obtained was 62% for a testing dataset where most of the proteins have <25% sequence identity with the proteins used in training the classifier. Such a rate is 6-21% higher than the corresponding rates obtained by various existing NN (neural networks) and SVM (support vector machines) approaches, implying that the ensemble classifier is very promising and might become a useful vehicle in protein science, as well as proteomics and bioinformatics. AVAILABILITY: The ensemble classifier, called PFP Pred, is available as a web-server at http://202.120.37.186/bioinf/fold/PFP Pred.htm for public usage. PMID- 16672259 TI - TopoICE-R: 3D visualization modeling the topology of DNA recombination. AB - TopoICE-R is a three-dimensional visualization and manipulation software for solving 2-string tangle equations and can be used to model the topology of DNA bound by proteins such as recombinases and topoisomerases. AVAILABILITY: This software, manual and example files are available at www.knotplot.com/download for Linux, Windows and Mac. PMID- 16672260 TI - Integrative Array Analyzer: a software package for analysis of cross-platform and cross-species microarray data. AB - The rapid accumulation of microarray data translates into an urgent need for tools to perform integrative microarray analysis. Integrative Array Analyzer is a comprehensive analysis and visualization software toolkit, which aims to facilitate the reuse of the large amount of cross-platform and cross-species microarray data. It is composed of the data preprocess module, the co-expression analysis module, the differential expression analysis module, the functional and transcriptional annotation module and the graph visualization module. PMID- 16672261 TI - Query Chem: a Google-powered web search combining text and chemical structures. AB - Query Chem (www.QueryChem.com) is a Web program that integrates chemical structure and text-based searching using publicly available chemical databases and Google's Web Application Program Interface (API). Query Chem makes it possible to search the Web for information about chemical structures without knowing their common names or identifiers. Furthermore, a structure can be combined with textual query terms to further restrict searches. Query Chem's search results can retrieve many interesting structure-property relationships of biomolecules on the Web. PMID- 16672262 TI - Molecular mechanism for pterin-mediated inactivation of tyrosine hydroxylase: formation of insoluble aggregates of tyrosine hydroxylase. AB - Tyrosine hydroxylase (TH), an iron-containing enzyme, catalyzes the first and rate-limiting step of catecholamine biosynthesis, and requires tetrahydrobiopterin (BH4) as a cofactor. We found that preincubation of recombinant human TH with BH4 results in the irreversible inactivation of the enzyme at a concentration far less than the Km value toward BH4 in spite of its cofactor role, whereas oxidized biopterin, which has no cofactor activity, does not affect the enzyme activity. We show that TH is inactivated by BH4 in competition with the binding of dopamine. The sequential addition of BH4 to TH results in a gradual decrease in the intensity of the fluorescence and CD spectra without changing their overall profiles. Sedimentation velocity analysis demonstrated an association of TH molecules with each other in the presence of BH4, and studies using gel-permeation chromatography, turbidity measurements, and transmission electron microscopy demonstrated the formation of amorphous aggregates with large molecular weights following the association of the TH proteins. These results suggest that BH4 not only acts as a cofactor, but also accelerates the aggregation of TH. We propose a novel mechanism for regulating the amount of TH protein, and discuss its physiological significance. PMID- 16672263 TI - Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry. AB - Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry. PMID- 16672264 TI - Preparation and characterization of a novel rice plant-specific kinesin. AB - Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. The kinesin family is classified into subfamilies. Kinesin species derived from vertebrates have been well characterized. In contrast, plant kinesins have yet to be adequately characterized. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, K16, in Escherichia coli, and then determined its enzymatic characteristics and compared them with those of kinesin 1. Our findings demonstrated that the rice kinesin motor domain has different enzymatic properties from those of well known kinesin 1. PMID- 16672265 TI - The signal transducer and activator of transcription 1alpha and interferon regulatory factor 1 are not essential for the induction of indoleamine 2,3 dioxygenase by lipopolysaccharide: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappaB pathways, and synergistic effect of several proinflammatory cytokines. AB - Indoleamine 2,3-dioxygenase (IDO) is induced by interferon (IFN)-gamma-mediated effects of the signal transducer and activator of transcription 1alpha (STAT1alpha) and interferon regulatory factor (IRF)-1. The induction of IDO can also be mediated through an IFN-gamma-independent mechanism, although the mechanism of induction has not been identified. In this study, we explored whether lipopolysaccharide (LPS) or several proinflammatory cytokines can induce IDO via an IFN-gamma-independent mechanism, and whether IDO induction by LPS requires the STAT1alpha and IRF-1 signaling pathways. IDO was induced by LPS or IFN-gamma in peripheral blood mononuclear cells and THP-1 cells, and a synergistic IDO induction occurred when THP-1 cells were cultured in the presence of a combination of tumor necrosis factor-alpha, interleukin-6 or interleukin 1beta. An electrophoretic mobility shift assay using STAT1alpha and IRF-1 consensus oligonucleotide probes showed no STAT1alpha or IRF-1 binding activities in LPS-stimulated THP-1 cells. Further, the LPS-induced IDO activity was inhibited by both p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) inhibitors. These findings suggest that the induction of IDO by LPS in THP-1 cells is not regulated by IFN-gamma via recruitment of STAT1alpha or IRF-1 to the intracellular signaling pathway, and may be related to the activity of the p38 MAPK pathway and NF-kappaB. PMID- 16672266 TI - Thin layer chromatography-blotting, a novel method for the detection of phosphoinositides. AB - Phosphoinositides are believed to be involved in fundamental cellular events such as signal transduction and vesicular trafficking. Aberrant metabolisms of this lipid, caused by mutations in phosphoinositide kinases, phosphatases and lipases are known to be related to variety of human disorders such as diabetes and cancer. While the majority of such information is obtained by analyzing genetic and biochemical properties of phosphoinositide-metabolic enzymes, direct measurement of cellular content of the lipid is hindered by the lack of a simple method that is sensitive enough to measure phosphoinositides present in trace amounts in vivo. Here, we describe a novel, thin layer chromatography (TLC)-based method by which cellular phosphoinositides are separated, transferred and detected by specific phosphoinositide-binding domains. This method was applied to follow the generation of minor phosphoinositides, such as PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to insulin and to compare PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels in several cancer cell lines. The method has potential application not only in investigating the physiological roles of phosphoinositides, but also in diagnosing metabolic disease and cancer by directly assessing phosphoinositide levels in samples obtained from patients. PMID- 16672267 TI - Enhancement by cigarette smoke extract of the radical formation in a reaction mixture of 13-hydroperoxide octadecadienoic acid and ferric ions. AB - The effects of cigarette smoke extract on radical formation were examined in reaction mixtures containing 13-hydroperoxide octadecadienoic acid (13-HPODE), FeCl3, cigarette smoke extract, ethylenediaminetetraacetic acid (EDTA), alpha-(4 pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and phosphate buffer (pH 7.4). Cigarette smoke extract enhanced the formation of both 7-carboxyheptyl and pentyl radicals in the reaction. Ferric ions were reduced in the reaction mixture, suggesting that cigarette smoke extract enhances the formation of 7-carboxyheptyl and pentyl radicals by reducing ferric irons. Although there is a large body of evidence supporting the involvement of radicals such as the semiquinone radical, hydroxyl radical, superoxide radical, nitric oxide radicals in smoking-related diseases, the enhancement by cigarette smoke of lipid-derived radical formation, which we first report here, may be one of the other causes of smoking-related diseases. PMID- 16672268 TI - A disulfide bridge mediated by cysteine 574 is formed in the dimer of the 70-kDa heat shock protein. AB - The 70-kDa heat shock protein (Hsp70) is predominantly present intracellularly as a monomer, but a small population is converted to dimers and oligomers under certain conditions. In the present study, we investigated the dimeric structure of human inducible Hsp70. As reported earlier, the C-terminal client-binding domain (amino acids 382-641) was required for the dimerization. A 40-amino acid deletion in the client-binding domain from either the N-terminus or C-terminus greatly enhanced the dimerization potential of Hsp70. Limited proteolysis indicated that the dimer formed through truncation from the C-terminus had a conformation similar to that of the non-truncated form. Truncation experiments demonstrated that the client-binding sub-domain (amino acids 382-520) with its adjacent region up to amino acid 541 was not sufficient for the dimerization but that the region up to amino acid 561 was sufficient. Interestingly, the dimer formed through truncation from the C-terminus acquired a homomeric disulfide bridge at Cys574. PMID- 16672269 TI - Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence. AB - Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo. PMID- 16672270 TI - Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification. AB - We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis. PMID- 16672271 TI - Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells. AB - Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene. PMID- 16672272 TI - Binding investigation of human 5-lipoxygenase with its inhibitors by SPR technology correlating with molecular docking simulation. AB - The binding features of a series of 5-lipoxygenase (5-LOX) inhibitors (caffeic acid, NDGA, AA-861, CDC, esculetin, gossypol and phenidone) to human 5-LOX have been studied by using surface plasmon resonance biosensor (SPR) technology based Biacore 3000 and molecular docking simulation analyses. The SPR results showed that the equilibrium dissociation constant (KD) values evaluated by Biacore 3000 for the inhibitors showed a good correlation with its reported IC50, suggesting that SPR technology might be applicable as a direct assay method in screening new 5-LOX inhibitors at an early stage. In addition, the 3D structural model of 5-LOX was generated according to the crystal structure of rabbit reticulocyte 15 lipoxygenase, and the molecular docking simulation analyses revealed that the predicted binding free energies for the inhibitors correlated well with the KD values measured by SPR assay, which implies the correctness of the constructed 3D structural model of 5-LOX. This current work has potential for application in structure-based 5-LOX inhibitor discovery. PMID- 16672273 TI - Two novel proteins bind specifically to trichosanthin on choriocarcinoma cell membrane. AB - Trichosanthin is the active protein component in the Chinese herb Trichosanthes kirilowi, which has distinct pharmacological properties. The cytotoxicity of trichosanthin was demonstrated by its selective inhibition of various choriocarcinoma cells. When Jar cells were treated with trichosanthin, the influx of calcium into the cells was observed by confocal laser scanning microscopy. When the distribution of trichosanthin-binding proteins on Jar cells was studied, two classes of binding sites for trichosanthin were shown by radioligand binding assay. Furthermore, the cytoplasmic membrane of Jar cells was biotinylated and the trichosanthin-binding proteins were isolated with trichosanthin-coupled Sepharose beads. Two protein bands with molecular masses of about 50 kDa and 60 kDa were revealed, further characterization of which should shed light on the mechanism of the selective cytotoxicity of trichosanthin to Jar cells. PMID- 16672274 TI - The effect of Abeta conformation on the metal affinity and aggregation mechanism studied by circular dichroism spectroscopy. AB - The conformational change and associated aggregation of beta amyloid (Abeta) with or without metals is the main cause of Alzheimer's disease (AD). In order to further understand the effects of Abeta and its associated metals on the aggregation mechanism, the influence of Abeta conformation on the metal affinity and aggregation was investigated using circular dichroism (CD) spectroscopy. The Abeta conformation is dependent on pH and trifluoroethanol (TFE). The binding of metals to Abeta was found to be dependent on the Abeta conformation. The aggregation induced by Abeta itself or its associated metals is completely diminished for Abeta in 40% TFE. Only in 5% and 25% TFE can Abeta undergo an alpha-helix to beta-sheet aggregation, which involve a three-state mechanism for the metal-free state, and a two-state transition for the metal-bound state, respectively. The aggregation-inducing activity of metals is in the order, Cu2+ > Fe3+ > or = Al3+ > Zn2+. PMID- 16672275 TI - Novel regulatory properties of Saccharomyces cerevisiae Arp4. AB - ARP4, an essential gene of Saccharomyces cerevisiae, codes for a nuclear actin related protein. Arp4 is a subunit of several chromatin-modifying complexes and is known to be involved in the transcriptional regulation in yeast. We used a mutant strain with a single amino acid substitution (G161D) in the conserved actin fold domain to investigate the influence of Arp4 on stress and nitrogen catabolite repression genes. The deficiency of functional Arp4 caused a highly increased sensitivity towards nitrogen starvation and to the macrolide antibiotic rapamycin. We show the changes of mRNA levels of selected genes under these conditions. The upregulation of stress genes as a consequence of treatment with rapamycin was largely Msn2p/Msn4p-dependent. The sensitivity towards rapamycin indicates a participation of Arp4 in the regulation of the TOR pathway. Consistently, arp4G161D cells exhibited an affected cell cycle. Long-term cultivation, which leads to a G1 arrest in wild-type cells, provoked arrest in G2/M (more than 60%) in the mutant strain. The same effect was observed upon treatment with rapamycin, indicating an unexpected relationship of Arp4 to TOR mediated cell cycle arrest. PMID- 16672276 TI - Structural analysis of a mutant of the HIV-1 integrase zinc finger domain that forms a single conformation. AB - HIV-1 integrase consists of three functional domains, an N-terminal zinc finger domain, a catalytic core domain and a C-terminal DNA binding domain. NMR analysis of an isolated N-terminal domain (IN(1-55)) has shown that IN(1-55) exists in two conformational states [E and D forms; Cai et al. (1997) Nat. Struct. Biol. 4, 567 577]. The two forms differ in the coordination of the zinc ion by two histidine residues. In the present study, structural analysis of a mutant of IN(1-55), Y15A, by NMR spectroscopy indicated that the mutant protein folds correctly but takes only the E form. Since the Y15A mutation abrogates the HIV-1 infectivity, Y15 might have some important role in the full-length integrase activity during the virus infection cycle. Our results suggest a possible role of Y15 in structural transition between the E and D forms of HIV-1 integrase to allow the optimal tetramerization. PMID- 16672277 TI - Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding. AB - The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14 3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites. PMID- 16672278 TI - Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow. AB - Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis. PMID- 16672279 TI - Kinetic mechanism of quinol oxidation by cytochrome bd studied with ubiquinone-2 analogs. AB - Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897-29902]. Kinetic analysis of the oxidation of the ubiquinol 2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)S(n) on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3 methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry. PMID- 16672280 TI - Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen. AB - The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained. PMID- 16672281 TI - Activation of the HOG pathway upon cold stress in Saccharomyces cerevisiae. AB - When Saccharomyces cerevisiae cells are exposed to hyper-osmotic stress, the high osmolarity glycerol response (HOG) pathway is activated to induce osmotic responses. The HOG pathway consists of two upstream osmosensing branches, the SLN1 and SHO1 branches, and a downstream MAP kinase cascade. Although the mechanisms by which these upstream branches transmit signals to the MAP kinase cascade are well understood, the mechanisms by which they sense and respond to osmotic changes are elusive. Here we show that the HOG pathway is activated in an SLN1 branch-dependent manner when cells are exposed to cold stress (0 degrees C treatment). Dimethyl sulfoxide (DMSO) treatment, which rigidifies the cell membrane, also activates the HOG pathway in both SLN1 branch- and SHO1 branch dependent manners. Moreover, cold stress, as well as hyper-osmotic stress, exhibits a synergistic effect with DMSO treatment on HOG pathway activation. On the other hand, ethanol treatment, which fluidizes the cell membrane, partially represses the cold stress-induced HOG pathway activation. Our results suggest that both osmosensing branches respond to the rigidification of the cell membrane to activate the HOG pathway. PMID- 16672282 TI - Characterization of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum. AB - The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids that are involved in its flocculation. These were found to be produced concomitantly with cell growth. The RNA fraction of these extracellular nucleic acids was subjected to cDNA analysis by applying a micro RNA cloning method and found to contain mainly fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Analyses of modified bases and genes of the RNAs revealed no structural difference between the intracellular and extracellular RNAs. This is the first report of structural analyses of bacterial extracellular RNAs. PMID- 16672284 TI - An improved statistical method for detecting heterotachy in nucleotide sequences. AB - The principle of heterotachy states that the substitution rate of sites in a gene can change through time. In this article, we propose a powerful statistical test to detect sites that evolve according to the process of heterotachy. We apply this test to an alignment of 1289 eukaryotic rRNA molecules to 1) determine how widespread the phenomenon of heterotachy is in ribosomal RNA, 2) to test whether these heterotachous sites are nonrandomly distributed, that is, linked to secondary structure features of ribosomal RNA, and 3) to determine the impact of heterotachous sites on the bootstrap support of monophyletic groupings. Our study revealed that with 21 monophyletic taxa, approximately two-thirds of the sites in the considered set of sequences is heterotachous. Although the detected heterotachous sites do not appear bound to specific structural features of the small subunit rRNA, their presence is shown to have a large beneficial influence on the bootstrap support of monophyletic groups. Using extensive testing, we show that this may not be due to heterotachy itself but merely due to the increased substitution rate at the detected heterotachous sites. PMID- 16672283 TI - Genetic signatures of a Mediterranean influence in Iberian Peninsula sheep husbandry. AB - Highly adaptable and versatile populations of domestic sheep, the result of millennia of intense husbandry, are found in almost every corner of the world. Here we describe a genetic survey of sheep from the western fringe of its European distribution. We studied the mitochondrial DNA control region sequences from 161 individuals belonging to 7 Portuguese sheep breeds. Our study revealed a high level of genetic diversity, with an average breed haplotype diversity of 0.983, substantially above that observed in central European breeds, as well as the presence of maternal lineages until now only found in the Middle East and Asia. A broad north-south pattern describes the most important trend in the Portuguese sheep population with a southern population clearly distinct from most other breeds. A recurrent influx of new genetic diversity, probably via the Mediterranean Sea, may explain these patterns and appears to corroborate the importance of this maritime route in the history of both mankind and livestock. Zooarchaeological studies of sheep bones from southern Portugal indicate a marked size increase during the Moslem period that may reflect an improvement of this animal--perhaps part of the well known "Arab agricultural revolution" in Andalusia. This could have been a time when the gene pool of Iberian sheep was substantially enriched and may help to explain the history of modern sheep breeds in this peninsula. PMID- 16672285 TI - Prediction of function divergence in protein families using the substitution rate variation parameter alpha. AB - Protein families typically embody a range of related functions and may thus be decomposed into subfamilies with, for example, distinct substrate specificities. Detection of functionally divergent subfamilies is possible by methods for recognizing branches of adaptive evolution in a gene tree. As the number of genome sequences is growing rapidly, it is highly desirable to automatically detect subfamily function divergence. To this end, we here introduce a method for large-scale prediction of function divergence within protein families. It is called the alpha shift measure (ASM) as it is based on detecting a shift in the shape parameter (alpha [alpha]) of the substitution rate gamma distribution. Four different methods for estimating alpha were investigated. We benchmarked the accuracy of ASM using function annotation from Enzyme Commission numbers within Pfam protein families divided into subfamilies by the automatic tree-based method BETE. In a test using 563 subfamily pairs in 162 families, ASM outperformed functional site-based methods using rate or conservation shifting (rate shift measure [RSM] and conservation shift measure [CSM]). The best results were obtained using the "GZ-Gamma" method for estimating alpha. By combining ASM with RSM and CSM using linear discriminant analysis, the prediction accuracy was further improved. PMID- 16672286 TI - Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces. AB - DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed. PMID- 16672287 TI - Stability in a mathematical model of neurite elongation. AB - We have developed a continuum partial differential equation model of tubulin driven neurite elongation and solved the steady problem. For non-zero values of the decay coefficient, the authors identified three different regimes of steady neurite growth, small, moderate and large, dependent on the strength of the tubulin flux into the neurite at the soma. Solution of the fully time-dependent moving boundary problem is, however, hampered by its analytical intractibility. A linear instability analysis, novel to moving boundary problems in this context, is possible and reduces to finding the zeros of an eigen-condition function. One of the system parameters is small and this permits solutions to the eigen condition equation in terms of asymptotic series in each growth regime. Linear instability is demonstrated to be absent from the neurite growth model and a Newton-Raphson root-finding algorithm is then shown to corroborate the asymptotic results for some selected examples. By numerically integrating the fully non linear time-dependent system, we show how the steady solutions are non-linearly stable in each of the three growth regimes with decay and oscillatory behaviour being as predicted by the linear eigenvalue analysis. PMID- 16672288 TI - Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. AB - A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6 antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation. PMID- 16672289 TI - A new autosomal recessive spastic ataxia associated with frequent white matter changes maps to 2q33-34. AB - Recessive ataxias are a heterogeneous group of diseases. We identified a group of 23 French-Canadian cases belonging to 17 families affected by an autosomal recessive spastic ataxia associated with frequent white matter changes. The fact that 59% of these families have a genealogical relationship to the Portneuf County of Quebec suggests that this is a new form of ataxia with a regional founder effect. All cases present with cerebellar ataxia and spasticity. There is great intrafamilial and interfamilial variability, as illustrated by the spectrum of age of diagnosis (range: 2-59 years, mean: 15.0) and the presence of white matter changes on MRI in 52.4% of cases. The more severe cases have spasticity from birth, scoliosis, dystonia and cognitive impairment and were considered cases of cerebral palsy. Brain MRI constantly shows cerebellar atrophy, which in some cases may be associated with cortical atrophy, leucoencephalopathy and corpus callosum thinning. A genome wide scan uncovered linkage of three families to marker D2S2321 localized on chromosome 2q33-34. Linkage analysis confirmed that all families are linked to the same region [multipoint log of the odds (LOD) score of 5.95]. Haplotype analysis and allele sharing suggest that one common mutation may account for 97% of carrier chromosomes in Quebec. The uncovering of the mutated gene may point to a common pathway for pyramidal and cerebellar degeneration as both are often observed in recessive ataxias and complicated paraplegias. PMID- 16672290 TI - Probabilistic diffusion tractography: a potential tool to assess the rate of disease progression in amyotrophic lateral sclerosis. AB - The goal of probabilistic tractography is to obtain a connectivity index along a white matter pathway that reflects fibre organization and is sensitive to pathological abnormalities contributing to disability. Here, we present the development of voxel-based connectivity measures along the tractography-derived corticospinal tract (CST). We investigated whether these connectivity measures are different in patients with amyotrophic lateral sclerosis (ALS) and correlate with the rate of disease progression. We also investigated whether fractional anisotropy (FA), which reflects directional coherence of fibre tracts, is reduced in the CST of ALS patients and relates to disease progression rate. Thirteen patients with probable or definite ALS and 19 healthy subjects were studied. The probabilistic tractography algorithm segmented the bilateral CST, along which FA and connectivity values were obtained. To take into account the asymmetric distribution of connectivity values, two summary statistic measures that focused on voxels with higher connectivity values were selected and then used in the analysis, together with the mean connectivity and the mean FA. To complete the analysis, the same summary measures for FA were included. Differences in all these indices between patients with moderate or rapid disease progression rate and controls were investigated using linear regression, adjusted for age and white matter fraction. The association between FA or connectivity in the CST and the disease progression rate was assessed using linear regression. Patients with a rapid disease progression rate had significantly lower summary connectivity measures than controls in the left CST, but there was only a borderline statistical difference in mean connectivity. Patients with rapid progression had a significantly lower mean FA, and any other FA measure, in both CSTs than controls. When only patients were considered, strong associations between the rate of disease progression and all the connectivity measures in the left CST were found (P-values between P < 0.001 and P = 0.002, partial correlation coefficients between -0.90 and -0.82). However, there was no evidence of an association between disease progression rate and any of the FA measures in the bilateral CST. Our findings suggest that FA and connectivity provide complementary information, since FA is sensitive to the detection of all the group differences, whereas the summary connectivity measures correlate with disease progression rate. The development of such connectivity measures raises their potential as markers of disease progression in ALS, and provides guidance for their use in other neurological diseases. PMID- 16672291 TI - MRI and clinical studies of facial and bulbar muscle involvement in MuSK antibody associated myasthenia gravis. AB - A proportion of patients with myasthenia gravis (MG) without acetylcholine receptor (AChR) antibodies have antibodies to muscle-specific kinase (MuSK). MG with MuSK antibodies (MuSK-MG) is often associated with persistent bulbar involvement, including marked facial weakness and tongue muscle wasting. The extent of muscle wasting in MuSK-MG, and whether it is also found in the few acetylcholine receptor (AChR-MG) patients who have persistent bulbar involvement, is not clear. We studied 12 MuSK-MG patients and recruited 14 AChR-MG patients matched broadly for age, sex ratio, duration of disease and degree of ocular, bulbar and facial weakness. We used coronal and sagittal T1-weighted (T1W) and T2 weighted (T2W) magnetic resonance imaging (MRI) to assess muscle wasting in facial and tongue muscles. Hyperintense signal on T1W MRI and comparison of axial T1W sequences with cUTE sequences were used to assess fibrous/fatty tissue in the tongue. We compared the results with those of four patients with myotonic dystrophy and 12 healthy individuals. We correlated the changes with clinical and treatment histories, and established a new ocular-bulbar-facial-respiratory (OBFR) score. At the time of study, none of the clinical measures, including the OBFR score, differed between the two MG groups. MRI demonstrated thinning of the buccinator, orbicularis oris (O.oris) and orbicularis oculi (O.oculi) muscles in MuSK-MG patients compared with healthy controls, whereas thinning of these muscles was not significant in AChR-MG. Tongue areas with T1W high signal were increased in MuSK-MG patients and the intensity of the signal on axial T1W sequences was greater in MuSK-MG than in controls. To look for possible correlations between imaging and clinical findings, we pooled results from all MG patients. The duration of treatment with prednisolone at >40 mg on alternate days (AD) correlated positively with the percentage of tongue area with high signal (P = 0.006) and negatively with MRI measurements of individual muscles and with the mean muscle dimensions (P = 0.001). The new OBFR score correlated positively with current Myasthenia Gravis Foundation of America grades and with the percentage of high signal (P = 0.004) and negatively with the mean muscle dimensions (P < 0.001). The results show that bulbar and facial muscle weakness and wasting are associated with significant muscle atrophy and fatty replacement in MuSK-MG, which was not found in the AChR-MG patients. MuSK antibodies per se may predispose to muscle thinning, but the difficulties in obtaining clinical remission under steroid therapy in some patients, resulting in long duration of treatment with higher doses (>40 mg AD), may be an additional factor. PMID- 16672292 TI - Plasticity in the human central nervous system. AB - Long-term potentiation (LTP) is a well-characterized form of synaptic plasticity that fulfils many of the criteria for a neural correlate of memory. LTP has been studied in a variety of animal models and, in rodents in particular, there is now a strong body of evidence demonstrating common underlying molecular mechanisms in LTP and memory. Results are beginning to emerge from studies of neural plasticity in humans. This review will summarize findings demonstrating that synaptic LTP can be induced in human CNS tissue and that rodent and human LTP probably share similar molecular mechanisms. We will also discuss the application of non invasive stimulation techniques to awake human subjects to induce LTP-like long lasting changes in localized neural activity. These techniques have potential therapeutic application in manipulating neural plasticity to treat a variety of conditions, including depression, Parkinson's disease, epilepsy and neuropathic pain. PMID- 16672293 TI - The cost-effectiveness of a child nutrition education programme in Peru. AB - This article reports impact and cost results from a health facility-based nutrition education programme targeting children less than 2 years of age in Trujillo, Peru. Key elements of the programme included participative complementary feeding demonstrations, growth monitoring sessions and an accreditation process. Data were collected from six intervention and six control health facilities to measure utilization and costs associated with the intervention. To calculate the unit costs of services, these costs are allocated using activity-based costing. To measure the effects of the intervention, 338 children were followed through household surveys at regular intervals from birth until the age of 18 months. The intervention had a clear positive impact both on the use of nutrition-related services and on children's growth outcomes. Children in the intervention areas made 17.6 visits to health facilities in the first 18 months of life, compared with 14.1 visits for children in the control areas (P < 0.001). This pattern holds true for all socioeconomic groups. The intervention prevented 11.1 cases of stunting per 100 children. In multivariate logistic regression analysis, children in the intervention were 0.33 times as likely to be stunted as the controls (P = 0.002). The marginal cost of the intervention - including external costs, training, health education materials and extra travel and equipment - is 6.12 US dollars per child reached and 55.16 US dollars per case of stunting prevented. The estimated marginal cost of the intervention per death averted is 1952 US dollars. PMID- 16672294 TI - Strengthening health systems to meet MDGs. PMID- 16672295 TI - Surveillance imaging during remission identifies a group of patients with more favorable aggressive NHL at time of relapse: a retrospective analysis of a uniformly-treated patient population. AB - BACKGROUND: Approximately one-third of the patients with relapsed aggressive non Hodgkin's lymphoma (NHL) are cured by second-line chemotherapy followed by high dose consolidation. The age-adjusted international prognostic index determined at the time of relapse (sAAIPI) predicts outcome in relapsed diffuse large B-cell lymphoma, suggesting that the success of salvage therapy could be enhanced by early relapse detection. This study evaluated the role of surveillance imaging in detection of relapsed disease and its impact on outcome of salvage treatment. PATIENTS AND METHODS: One hundred and eight patients with relapsed aggressive NHL were treated with ICE-based second-line chemotherapy. Relapses were categorized as detected by imaging, examination, or patient-reported symptoms. RESULTS: Twenty per cent of relapses were detected by routine imaging while 80% were identified by reported symptoms or abnormalities on exam. Patients were 4.1 times (95% CI: 1.7-10.2) more likely to have low risk disease if relapse was diagnosed by routine imaging (group 1) compared with those diagnosed by reported symptoms or physical findings (group 2). Median overall 5-year survival for group 1 versus group 2 was 54% and 43% respectively (P = 0.13). CONCLUSION: These results suggest that routine surveillance imaging can identify a population of patients with a more favorable outcome based on the sAAIPI. PMID- 16672296 TI - Combined auditory and visual stimuli facilitate head saccades in the barn owl (Tyto alba). AB - The barn owl naturally responds to an auditory or visual stimulus in its environment with a quick head turn toward the source. We measured these head saccades evoked by auditory, visual, and simultaneous, co-localized audiovisual stimuli to quantify multisensory interactions in the barn owl. Stimulus levels ranged from near to well above saccadic threshold. In accordance with previous human psychophysical findings, the owl's saccade reaction times (SRTs) and errors to unisensory stimuli were inversely related to stimulus strength. Auditory saccades characteristically had shorter reaction times but were less accurate than visual saccades. Audiovisual trials, over a large range of tested stimulus combinations, had auditory-like SRTs and visual-like errors, suggesting that barn owls are able to use both auditory and visual cues to produce saccades with the shortest possible SRT and greatest accuracy. These results support a model of sensory integration in which the faster modality initiates the saccade and the slower modality remains available to refine saccade trajectory. PMID- 16672297 TI - Saccade-related spread of activity across superior colliculus may arise from asymmetry of internal connections. AB - The superior colliculus (SC) receives a retinotopic projection of the contralateral visual field in which the representation of the central field is expanded with respect to the peripheral field. The visual projection forms a nonlinear, approximately logarithmic, map on the SC. Models of the SC commonly assume that the function defining the strength of neuronal connections within this map (the kernel) depends only on the distance between two neurons, and is thus isotropic and homogeneous. However, if the connection strength is based on the distance between two stimuli in sensory space, the kernel will be asymmetric because of the nonlinear projection onto the brain map. We show, using a model of the SC, that one consequence of these asymmetric intrinsic connections is that activity initiated at one point spreads across the map. We compare this simulated spread with the spread observed experimentally around the time of saccadic eye movements with respect to direction of spread, differing effects of local and global inhibition, and the consequences of localized inactivation on the SC map. Early studies suggested that the SC spread was caused by feedback of eye displacement during a saccade, but subsequent studies were inconsistent with this feedback hypothesis. In our new model, the spread is autonomous, resulting from intrinsic connections within the SC, and thus does not depend on eye movement feedback. Other sensory maps in the brain (e.g., visual cortex) are also nonlinear and our analysis suggests that the consequences of asymmetric connections in those areas should be considered. PMID- 16672298 TI - Two types of TMS-induced movement variability after stimulation of the primary motor cortex. AB - Using transcranial magnetic stimulation, we studied the role of the primary motor cortex (M1) in repetitive movements, examining whether the functional contribution of this region is associated with controlling response timing, response implementation, or both. In two experiments, participants performed a rhythmic tapping task, attempting to produce isochronous intervals (range of 350 550 ms) while stimulation was applied over M1 or a control site. M1 stimulation was associated with increased variability of the inter-tap intervals (ITI), and, by manipulating stimulation intensity, we identified two distinct changes in performance: a generalized increase in ITI variability and a delay in the subsequent response when the pulse fell within a restricted window prior to movement onset. Using a series of simulations, we demonstrate that the general increase in variability and the temporally specific delay reflect disruption of response implementation processes rather than an increase in noise associated with response timing. PMID- 16672299 TI - Distinct short-term and long-term adaptation to reduce saccade size in monkey. AB - In monkeys, saccades that repeatedly overshoot their targets adapt to become smaller by the time the monkey has made 1,000-2,000 saccades. In life, adaptation must keep movements accurate for long periods of time. Previous work describes only saccade adaptation that occurs within a few hours. Here we describe long term saccade adaptation elicited in three monkeys by 19 days of training. Each day a monkey made saccades to track 16 degrees leftward and rightward target movements. During saccades, the target stepped back toward its starting position 6.4 degrees (40%) in two monkeys or 8 degrees (50%) in the third. After each day's adaptation, we blindfolded the monkey with goggles and returned it to its cage overnight. We found that adapting saccades for 19 days elicited significantly larger, long-lasting reduction in saccade size than did adapting for only 1 day. Further, after 19 days of adaptation we could elicit additional, apparently normal, short-term reduction in saccade size by increasing the size of the intra-saccade target movement. In contrast, we could elicit only small additional size reduction after only 1 day of adaptation. A simple model using separate short- and long-term adaptation mechanisms can reproduce many of the features of saccade gain exhibited by monkeys during a 19-day adaptation. We conclude that there is a long-term saccade-adaptation mechanism that is distinct from the well-characterized short-term system and that this newly recognized system is responsible for long-term maintenance of saccade accuracy. PMID- 16672300 TI - Mathematical models of proprioceptors. II. Structure and function of the Golgi tendon organ. AB - We developed a physiologically realistic mathematical model of the Golgi tendon organ (GTO) whose elements correspond to anatomical features of the biological receptor. The mechanical interactions of these elements enable it to capture all salient aspects of GTO afferent behavior reported in the literature. The model accurately describes the GTO's static and dynamic responses to activation of single motor units whose muscle fibers insert into the GTO, including the different static and dynamic sensitivities that exist for different types of muscle fibers (S, FR, and FF). Furthermore, it captures the phenomena of self- and cross-adaptation wherein the GTO dynamic response during motor unit activation is reduced by prior activation of the same or a different motor unit, respectively. The model demonstrates various degrees of nonlinear summation of GTO responses resulting from simultaneous activation of multiple motor units. Similarly to the biological GTO, the model suggests that the activation of every additional motor unit to already active motor units that influence the receptor will have a progressively weaker incremental effect on the GTO afferent activity. Finally, the proportional relationship between the cross-adaptation and summation recorded for various pairs of motor units was captured by the model, but only by incorporating a particular type of occlusion between multiple transduction regions that were previously suggested. This occlusion mechanism is consistent with the anatomy of the afferent innervation and its arrangement with respect to the collagen strands inserting into the GTO. PMID- 16672301 TI - Mathematical models of proprioceptors. I. Control and transduction in the muscle spindle. AB - We constructed a physiologically realistic model of a lower-limb, mammalian muscle spindle composed of mathematical elements closely related to the anatomical components found in the biological spindle. The spindle model incorporates three nonlinear intrafusal fiber models (bag(1), bag(2), and chain) that contribute variously to action potential generation of primary and secondary afferents. A single set of model parameters was optimized on a number of data sets collected from feline soleus muscle, accounting accurately for afferent activity during a variety of ramp, triangular, and sinusoidal stretches. We also incorporated the different temporal properties of fusimotor activation as observed in the twitchlike chain fibers versus the toniclike bag fibers. The model captures the spindle's behavior both in the absence of fusimotor stimulation and during activation of static or dynamic fusimotor efferents. In the case of simultaneous static and dynamic fusimotor efferent stimulation, we demonstrated the importance of including the experimentally observed effect of partial occlusion. The model was validated against data that originated from the cat's medial gastrocnemius muscle and were different from the data used for the parameter determination purposes. The validation record included recently published experiments in which fusimotor efferent and spindle afferent activities were recorded simultaneously during decerebrate locomotion in the cat. This model will be useful in understanding the role of the muscle spindle and its fusimotor control during both natural and pathological motor behavior. PMID- 16672302 TI - Changes in mIPSCs and sIPSCs after kainate treatment: status epilepticus-induced neuronal loss or direct activation of kainate receptors? PMID- 16672303 TI - Fabrication and use of high-speed, concentric h+- and Ca2+-selective microelectrodes suitable for in vitro extracellular recording. AB - Ion-selective microelectrodes (ISMs) have been used extensively in neurophysiological studies. ISMs selective for H(+) and Ca(2+) are notable for their sensitivity and selectivity, but suffer from a slow response time, and susceptibility to noise because of the high electrical resistance of the respective ion exchange cocktails. These drawbacks can be overcome by using a "coaxial" or "concentric" inner micropipette to shunt the bulk of the ion exchanger resistance. This approach was used decades ago to record extracellular [Ca(2+)] transients in cat cortex, but has not been subsequently used. Here, we describe a method for the rapid fabrication of concentric pH- and Ca(2+) selective microelectrodes useful for extracellular studies in brain slices or other work in vitro. Construction was simplified compared with previous implementations, by using commercially available, thin-walled borosilicate glass, drawing an outer barrel with a rapid taper (similar to a patch pipette), and by use of a quick and reliable silanization procedure. Using a piezoelectric stepper to effect a rapid solution change, the response time constants of the concentric pH and Ca(2+)-electrodes were 14.9 +/- 1.3 and 5.3 +/- 0.90 ms, respectively. Use of these concentric ISMs is demonstrated in rat hippocampal slices. Activity dependent, extracellular pH, and [Ca(2+)] transients are shown to arise two- to threefold faster, and attain amplitudes two- to fourfold greater, when recorded by concentric versus conventional ISMs. The advantage of concentric ISMs for studies of ion transport and ion diffusion is discussed. PMID- 16672304 TI - Smooth pursuit of nonvisual motion. AB - Unlike saccades, smooth pursuit eye movements (SPEMs) are not under voluntary control and their initiation generally requires a moving visual target. However, there are various reports of limited smooth pursuit of the motion of a subject's own finger in total darkness (pursuit based on proprioceptive feedback) and to the combination of proprioception and tactile motion as an unseen finger was moved voluntarily over a smooth surface. In contrast, SPEMs to auditory motion are not distinguishable from pursuit of imagined motion. These reports of smooth pursuit of nonvisual motion cues used a variety of paradigms and different stimuli. In addition, the results have often relied primarily on qualitative descriptions of the smooth pursuit. Here, we directly compare measurements of smooth pursuit gain (eye velocity/stimulus velocity) to visual, auditory, proprioceptive, tactile, and combined tactile + proprioceptive motion stimuli. The results demonstrate high gains for visual pursuit, low gains for auditory pursuit, and intermediate, statistically indistinguishable gains for tactile, proprioceptive, and proprioceptive + tactile pursuit. PMID- 16672305 TI - Kv1.1-containing channels are critical for temporal precision during spike initiation. AB - Low threshold, voltage-gated potassium currents (Ikl) are widely expressed in auditory neurons that can fire temporally precise action potentials (APs). In the medial nucleus of the trapezoid body (MNTB), channels containing the Kv1.1 subunit (encoded by the Kcna1 gene) underlie Ikl. Using pharmacology, genetics and whole cell patch-clamp recordings in mouse brain slices, we tested the role of Ikl in limiting AP latency-variability (jitter) in response to trains of single inputs at moderate to high stimulation rates. With dendrotoxin-K (DTX-K, a selective blocker of Kv1.1-containing channels), we blocked Ikl maximally (approximately 80% with 100 nM DTX-K) or partially (approximately 50% with 1-h incubation in 3 nM DTX-K). Ikl was similar in 3 nM DTX-K-treated cells and cells from Kcna1(-/-) mice, allowing a comparison of these two different methods of Ikl reduction. In response to current injection, Ikl reduction increased the temporal window for AP initiation and increased jitter in response to the smallest currents that were able to drive APs. While 100 nM DTX-K caused the largest increases, latency and jitter in Kcna1(-/-) cells and in 3 nM DTX-K-treated cells were similar to each other but increased compared with +/+. The near-phenocopy of the Kcna1(-/-) cells with 3 nM DTX-K shows that acute blockade of a subset of the Kv1.1-containing channels is functionally similar to the chronic elimination of all Kv1.1 subunits. During rapid stimulation (100-500 Hz), Ikl reduction increased jitter in response to both large and small inputs. These data show that Ikl is critical for maintaining AP temporal precision at physiologically relevant firing rates. PMID- 16672306 TI - Seeing through the glass darkly? A qualitative exploration of GPs' drinking and their alcohol intervention practices. AB - BACKGROUND: Brief alcohol intervention is influenced by patients' personal characteristics as well as their clinical risk. Risk-drinkers from higher social status groups are less likely to receive brief intervention from GPs than those from lower social-status groups. Thus GPs' perception of social similarity or distance may influence brief intervention. OBJECTIVE: To explore the role that GPs' drinking behaviour plays in their recognition of alcohol-related risk in patients. METHOD: A qualitative interview study with 29 GPs recruited according to maximum variation sampling. All interviews were audio-recorded and transcribed verbatim. Analysis was inductive with constant comparison within and between themes plus deviant case analysis. Analysis developed until category saturation was reached. RESULTS: GPs described a range of personal drinking practices that broadly mirrored population drinking patterns. Many saw themselves as part of mainstream society, sharing in culturally sanctioned behaviour. For some GPs, shared drinking practices could increase empathy for patients who drank, and facilitate discussion about alcohol. However, several GPs regarded themselves as distinct from 'others', separating their own drinking from that of patients. Several GPs described a form of bench-marking, wherein only patients who drank more, or differently, to themselves were felt to be 'at risk'. CONCLUSION: Alcohol is clearly a complex and emotive health and social issue and GPs are not immune to its effects. For some GPs' shared drinking behaviour can act as a window of opportunity enabling insight on alcohol issues and facilitating discussion. However, other GPs may see through the glass more darkly and selectively recognize risk only in those patients who are least like them. PMID- 16672308 TI - Commentary: the epidemiology of neurodevelopmental disorders in Sub-Saharan Africa--moving forward to understand the health and psychosocial needs of children, families, and communities. PMID- 16672310 TI - Commentary: malaria death rates remain highly pertinent. PMID- 16672309 TI - A comparison of two dietary instruments for evaluating the fat-breast cancer relationship. AB - BACKGROUND: Previous research suggests food diaries may be more efficient than food frequency questionnaires (FFQ) in detecting a dietary fat-breast cancer relationship. We assessed this further using 4 day food records (FRs) and FFQs in a large sample. METHODS: Participants were from the non-intervention group of the dietary modification component of the Women's Health Initiative Clinical Trial: 603 breast cancer cases and 1206 controls matched on age, clinic, and length of follow-up. Relative risks (RRs) were estimated using unconditional logistic regression, adjusted for confounders and for the selection into the trial of women with an FFQ report exceeding 32% calories from fat. Direct comparison of the statistical power of the two instruments used the standardized log RR. An alternative analysis after removing subjects with missing covariate data was also conducted. RESULTS: The RR estimate for breast cancer in the top quintile of total fat intake, adjusted for confounders and total energy, was 1.82 (P for trend 0.02) for the FR but 0.67 for the FFQ (P for trend 0.24). Following adjustment for selection, estimates were 2.09 (P for trend 0.008) for the FR (alternative: 2.54, P for trend 0.006) and 1.71 (P for trend 0.18) for the FFQ (alternative: 1.24, P for trend 0.41). Similar results were seen for fat subtypes, particularly unsaturated fats. Comparisons showed higher statistical power for the FR than the FFQ (e.g. total fat, P = 0.08: alternative P = 0.01). CONCLUSIONS: Alternative instruments, such as FRs, may be preferable to FFQs for evaluating diet-disease relationships in cohort studies. The results support a positive association between dietary fat and breast cancer. PMID- 16672311 TI - Commentary: statistical analysis or biological analysis as tools for understanding biological causes. PMID- 16672312 TI - Prospective study of alcohol drinking patterns and coronary heart disease in women and men. AB - OBJECTIVE: To determine the association between alcohol drinking patterns and risk of coronary heart disease in women and men. DESIGN: Population based cohort study. SETTING: Denmark, 1993-2002. PARTICIPANTS: 28 448 women and 25 052 men aged 50-65 years, who were free of cardiovascular disease at entry to the study. MAIN OUTCOME MEASURES: Incidence of coronary heart disease occurring during a median follow-up period of 5.7 years. RESULTS: 749 and 1283 coronary heart disease events occurred among women and men. Women who drank alcohol on at least one day a week had a lower risk of coronary heart disease than women who drank alcohol on less than one day a week. Little difference was found, however, between drinking frequency: one day a week (hazard ratio 0.64, 95% confidence interval 0.51 to 0.81), 2-4 days a week (0.63, 0.52 to 0.77), five or six days a week (0.79, 0.61 to 1.03), and seven days a week (0.65, 0.51 to 0.84). For men an inverse association was found between drinking frequency and risk of coronary heart disease across the entire range of drinking frequencies. The lowest risk was observed among men who drank daily (0.59, 0.48 to 0.71) compared with men who drank alcohol on less than one day a week. CONCLUSIONS: Among women alcohol intake may be the primary determinant of the inverse association between drinking alcohol and risk of coronary heart disease whereas among men, drinking frequency, not alcohol intake, seems more important. PMID- 16672313 TI - Impact of renin angiotensin system modulation on the hyperfiltration state in type 1 diabetes. AB - The initial stages of diabetic nephropathy are characterized by glomerular hyperfiltration and hypertension, processes that have been linked to initiation and progression of renal disease. Renin angiotensin system (RAS) blockade is commonly used to modify the hyperfiltration state and delay progression of renal disease. Despite this therapy, many patients progress to ESRD, suggesting heterogeneity in the response to RAS modulation. The role of the RAS in the hyperfiltration state in adolescents with uncomplicated type 1 diabetes was examined, segregated on the basis of the presence of hyperfiltration. Baseline renal hemodynamic function was characterized in 22 patients. Eleven patients exhibited glomerular hyperfiltration (GFR>or=135 ml/min), and in the remaining 11 patients, the GFR was <130 ml/min. Renal hemodynamic function was assessed in response to a graded angiotensin II (AngII) infusion during euglycemic conditions and again after 21 d of angiotensin-converting enzyme (ACE) inhibition with enalapril. AngII infusion under euglycemic conditions resulted in a significant decline in GFR and renal plasma flow in the hyperfiltration group but not in the normofiltration group. After ACE inhibition, GFR fell but did not normalize in the hyperfiltration group; the normofiltration group showed no change. These data show significant differences in renal hemodynamic function between hyperfiltering and normofiltering adolescents with type 1 diabetes at baseline, after AngII infusion and ACE inhibition. The response to ACE inhibition and AngII in hyperfiltering patients suggests that vasodilation may complement RAS activation in causing the hyperfiltration state. The interaction between glomerular vasoconstrictors and vasodilators requires examination in future studies. PMID- 16672307 TI - Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling. AB - Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization. PMID- 16672314 TI - Glial cell line-derived neurotrophic factor and its receptor ret is a novel ligand-receptor complex critical for survival response during podocyte injury. AB - Glomerulosclerosis correlates with a reduction in podocyte number that occurs through mechanisms that include apoptosis. Whether glial cell line-derived neurotrophic factor (GDNF), a growth factor that is critical for neural and renal development, is a survival factor for injured podocytes was investigated. Ret, the GDNF receptor tyrosine kinase, was upregulated in podocytes in the passive Heymann nephritis and puromycin aminonucleoside (PA) nephrosis rat models of podocyte injury. In addition, Ret mRNA and protein were upregulated in mouse podocytes in vitro after injury that was induced by sublytic C5b-9 and PA. GDNF, which also was induced during podocyte injury, inhibited significantly the apoptosis of podocytes that was induced by ultraviolet C irradiation. Knockdown of Ret expression by small interference RNA in podocytes exacerbated apoptosis that was induced by both ultraviolet C and PA. Ret knockdown, upon injury, decreased AKT phosphorylation, suggesting that the phosphoinositol-3 kinase/AKT pathway mediated the survival effect of GDNF on podocytes. Consistent with this hypothesis, the selective phosphoinositol-3 kinase inhibitor LY294002 blocked the survival-promoting effects of GDNF. In conclusion, GDNF is a novel podocyte survival factor. Furthermore, Ret is highly upregulated during podocyte injury in vitro and in vivo, suggesting that Ret activation is a critical adaptive response for podocyte remodeling and repair. PMID- 16672315 TI - Role of parathyroid hormone-related protein in tubulointerstitial apoptosis and fibrosis after folic acid-induced nephrotoxicity. AB - Parathyroid hormone-related protein (PTHrP) is shortly upregulated in acute renal injury, but its pathophysiologic role is unclear. Investigated was whether PTHrP might act as a profibrogenic factor in mice that do or do not overexpress PTHrP in the proximal tubule after folic acid (FA) nephrotoxicity, a model of acute renal damage followed by partial regeneration and patchy tubulointerstitial fibrosis. It was found that constitutive PTHrP overexpression in these animals conveyed a significant increase in tubulointerstitial fibrosis, associated with both fibroblast activation (as alpha-smooth muscle actin staining) and macrophage influx, compared with control littermates at 2 to 3 wk after FA damage. Cell proliferation and survival was higher (P<0.01) in the renal interstitium of PTHrP overexpressing mice than in control littermates within this period after injury. Moreover, the former mice had a constitutive Bcl-XL protein overexpression. In vitro studies in renal tubulointerstitial and fibroblastic cells strongly suggest that PTHrP (1-36) (100 nM) reduced FA-induced apoptosis through a dual mechanism involving Bcl-XL upregulation and Akt and Bad phosphorylation. PTHrP (1-36) also stimulated monocyte chemoattractant protein-1 expression in tubuloepithelial cells, as well as type-1 procollagen gene expression and fibronectin (mRNA levels and protein secretion) in these cells and renal fibroblastic cells. Our findings indicate that this peptide, by interaction with the PTH1 receptor, can increase tubulointerstitial cell survival and seems to act as a proinflammatory and profibrogenic factor in the FA-damaged kidney. PMID- 16672316 TI - Ethnic disparities in cardiovascular risk factors and coronary disease prevalence among individuals with chronic kidney disease: findings from the Third National Health and Nutrition Examination Survey. AB - Differences in coronary disease have been reported among ethnic minorities in the US population. Whether these persist in patients with chronic kidney disease is unknown. The prevalence of myocardial infarction (MI) and angina was compared by race and GFR in the Third National Health and Nutrition Examination Survey using the Modification of Diet in Renal Disease Study equation. Age-gender standardized estimates were computed for each GFR category (>or=90, 60 to 89, and <60 ml/min per 1.73 m2), and odds ratios were compared using weighted multivariable logistic regression for each race. The age-gender standardized prevalence of MI was 3.0, 3.1, and 4.9% in white individuals; 2.8, 3.8, and 9.9% in black individuals; and 1.9, 2.9, and 3.8% in Mexican-American individuals in each category: >or=90, 60 to 89, and <60 ml/min, respectively. Compared with the referent (Mexican American; GFR>or=90 ml/min; odds ratio 1.00), Mexican-American individuals with GFR of 60 to 89 and <60 ml/min had more than four and nine times the odds for MI; black individuals at successively lower GFR levels had 1.6, 6.1, and 16.3 times the odds for MI, whereas white individuals had 1.9, 4.7, and 20.2 times that of the referent, respectively. After adjustment for traditional risk factors, the inverse association of GFR with MI was substantially attenuated in black and white individuals and completely abolished in Mexican-American individuals. The burden of coronary disease is lower in Mexican-American than in white or black individuals with reduced kidney function even accounting for differences in traditional risk factors. PMID- 16672317 TI - The enlarging body of evidence: obesity and chronic kidney disease. PMID- 16672318 TI - Hyperosmolality in vivo upregulates aquaporin 2 water channel and Na-K-2Cl co transporter in Brattleboro rats. AB - There are considerable experimental results that indicate that arginine vasopressin (AVP)-independent factors are involved in urinary concentration. This study examined the role of hyperosmolality in vivo to modulate aquaporin 2 (AQP2) and Na-K-2Cl co-transporter (NKCC2), pivotal factors in urinary concentration, in AVP-deficient Brattleboro (BB) rats. Hyperglycemia with associated hyperosmolality occurred in diabetic BB rats (BBDM). Protein abundance of AQP2 increased and was reversed by insulin in the inner medulla (IM; control 100+/-5%; BBDM 146+/-8%; BBDM+Ins 122+/-9%; P<0.001) and inner stripe of outer medulla (ISOM; control 100+/-4%; BBDM 123+/-8%; BBDM+Ins 93+/-6%; P<0.05). These results were confirmed by immunohistochemistry studies. NKCC2 rose in the ISOM but was not reversed with insulin treatment. For investigation of the role of hyperosmolality in the absence of hyperglycemia on the regulation of the expression of renal AQP and NKCC2, studies were performed with hyperosmolality that was induced by 0.5% NaCl in drinking water in BB rats. Hyperosmolality that was induced by NaCl increased significantly the protein abundance of IM AQP2 (121+/-2 versus 100+/-5%; P<0.01), ISOM AQP2 (135+/-6 versus 100+/-5%; P<0.001), cortex plus outer stripe of outer medulla AQP2 (121+/-4 versus 100+/-1%; P<0.001), ISOM NKCC2 (133+/-1 versus 100+/-4%; P<0.05), and cortex plus outer stripe of outer medulla NKCC2 (142+/-16 versus 100+/-9%; P<0.05). In conclusion, hyperosmolality, secondary to either glucose or NaCl, upregulated renal AQP2 and NKCC2 in vivo in BB rats. PMID- 16672319 TI - IGF-binding protein-3 modulates TGF-beta/BMP-signaling in glomerular podocytes. AB - Podocyte apoptosis initiates progressive glomerulosclerosis in TGF-beta1 transgenic and CD2AP-knockout (CD2AP-/-) mice. It was previously shown that in both mouse models, activation of the TGF-beta pathway is the key event during development of podocyte apoptosis. Furthermore, CD2AP is an important modifier of TGF-beta-induced survival signaling via activation of the phosphoinositol 3 kinase/AKT signaling pathway. This article presents IGF-binding protein-3 (IGFBP 3) as a new modulator of apoptosis and survival signaling in glomerular podocytes. High expression of IGFBP-3 protein in the urine of diseased CD2AP-/- mice was discovered, and IGFBP-3 expression in glomerular podocytes and parietal cells was detected. IGFBP-3 can induce changes in podocyte actin cytoskeleton, leads to apoptosis in cultured murine podocytes, and can enhance TGF-beta1 induced apoptosis in vitro. For studying this process on a molecular level, proapoptotic p38 mitogen-activated protein kinase pathways and antiapoptotic phosphoinositol 3-kinase/AKT pathways were examined in cultured murine podocytes. It was found that IGFBP-3 increments the level of TGF-beta1-induced phosphorylated p38 mitogen-activated protein kinase and decreases the phosphorylation of antiapoptotic AKT. This effect is specific for the co stimulation of IGFBP-3 with TGF-beta1 because a combination of IGFBP-3 with bone morphogenic protein-7 (BMP-7), another member of the TGF-beta superfamily, results in apoptosis opposing signaling effects with a strong increase of phosphorylated AKT and subsequent functional effects. These results demonstrate that the IGF/IGFBP axis plays an important role in the development of podocyte apoptosis by modulation of TGF-beta and BMP-7-induced pro- and antiapoptotic signals. PMID- 16672320 TI - Suppression of ureteric bud apoptosis rescues nephron endowment and adult renal function in Pax2 mutant mice. AB - The molecular mechanisms that set congenital nephron number are unknown. However, humans with modest suboptimal nephron number may be at increased risk for essential hypertension, and those with more severe nephron deficits at birth may develop progressive renal insufficiency. A model of branching morphogenesis during fetal kidney development in which the extent of ureteric bud arborization is dependent on suppression of programmed cell death has been proposed. This study shows that the increased apoptosis and reduced ureteric bud branching of heterozygous Pax2 mutant mice is associated with 40% decrease in nephron number at birth. This leads to postnatal glomerular hypertrophy and long-term renal insufficiency in the absence of glomerulosclerosis. To determine whether restoration of antiapoptotic factors alone is sufficient to rescue the nephron deficit in these mice, a BCL2 transgene that is under the control of the PAX2 promoter was targeted to the ureteric bud. The transgene suppressed programmed cell death in the ureteric bud lineage, increased nephron number to 90% of that of wild-type littermates at birth, and normalized renal function at 1 yr. These observations lend strong support to the hypothesis that factors that control ureteric bud apoptosis are powerful determinants of congenital nephron endowment. PMID- 16672321 TI - Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure. AB - The goal of this research was to develop a strategy to couple stem cell and gene therapy for in vivo delivery of erythropoietin (Epo) for treatment of anemia of ESRD. It was shown previously that autologous bone marrow stromal cells (MSCs) can be genetically engineered to secrete pharmacologic amounts of Epo in normal mice. Therefore, whether anemia in mice with mild to moderate chronic renal failure (CRF) can be improved with Epo gene-modified MSCs (Epo+MSCs) within a subcutaneous implant was examined. A cohort of C57BL/6 mice were rendered anemic by right kidney electrocoagulation and left nephrectomy. In these CRF mice, the hematocrit (Hct) dropped from a prenephrectomy baseline of approximately 55% to 40% after induction of renal failure. MSCs from C57BL/6 donor mice were genetically engineered to secrete murine Epo at a rate of 3 to 4 units of Epo/10(6) cells per 24 h, embedded in a collagen-based matrix, and implanted subcutaneously in anemic CRF mice. It was observed that Hct increased after administration of Epo+MSCs, according to cell dose. Implants of 3 million Epo+MSCs per mouse had no effect on Hct, whereas 10 million led to a supraphysiologic effect. The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCs rose to a peak 54+/-4.0 or 63+/-5.5%, respectively, at 3 wk after implantation and remained above 48 or 54% for >19 wk. Moreover, mice that had CRF and received Epo+MSCs showed significantly greater swimming exercise capacity. In conclusion, these results demonstrate that subcutaneous implantation of Epo secreting genetically engineered MSCs can correct anemia that occurs in a murine model of CRF. PMID- 16672322 TI - Copper-induced stimulation of extracellular signal-regulated kinase in trout hepatocytes: the role of reactive oxygen species, Ca2+, and cell energetics and the impact of extracellular signal-regulated kinase signaling on apoptosis and necrosis. AB - The present study investigated if copper (Cu) exposure of trout hepatocytes, which stimulates formation of reactive oxygen species (ROS) and increases intracellular free Ca(2+) (Ca(2+)i), leads to an activation of extracellular signal-regulated kinase (ERK), the mechanisms underlying this activation, and the role of ERK signaling in cell death. Cu stimulated a time- and dose-dependent increase of phosphorylated extracellular signal-regulated kinase (pERK), and preventing the associated Ca(2+) influx or radical formation diminished or inhibited ERK activation, respectively. Furthermore, Cu enhanced caspase 3/7 activity and necrosis, and both effects were inhibited by treatments diminishing radical production and by chelating extracellular Ca(2+). In addition, ERK activity, and to a lesser extent caspase activity, was reduced by inhibiting mitochondrial ATP production, suggesting ATP dependence of the process. Inhibition of the ERK activator MEK, as well as of p38, significantly reduced caspase activation and necrosis, whereas c-Jun N-terminal kinase (JNK) inhibition diminished only caspase activity. Likewise, inhibition of MEK and p38, but not of JNK, prevented Cu-induced ROS production. In summary, we found that stimulation of ERK by Cu exposure of trout hepatocytes is dependent on radical formation and ATP, whereas Ca(2+) only modulates ERK activity. At the same time, activated ERK, as well as p38, contributes to enhanced ROS formation, whereas JNK did not. All three mitogen-activated protein kinases appear to promote apoptotic cell death upon Cu exposure, and ERK and p38 also stimulate necrosis. PMID- 16672323 TI - Activation of multiple mitogen-activated protein kinases in pro/pre-B cells by GW7845, a peroxisome proliferator-activated receptor gamma agonist, and their contribution to GW7845-induced apoptosis. AB - There is growing interest in using peroxisome proliferator-activated receptor (PPAR) gamma agonists as chemotherapeutic agents in hematologic malignancies. PPARgamma agonists of diverse chemical structure induce apoptosis in several malignant B cell lines. However, PPARgamma agonists also induce apoptosis in normal B cells. One such agonist, GW7845, rapidly induces apoptosis in early B cells. Understanding the mechanisms of PPARgamma agonist-induced death is essential to minimizing loss of normal cells during chemotherapy. PPARgamma agonists influence mitogen-activated protein kinase (MAPK) cascades in other systems, and MAPKs can be associated with apoptosis. Therefore, we investigated the activation of MAPKs in primary pro-B cells and cultured pro/pre-B cells and their role in GW7845-induced apoptosis. Treatment of a nontransformed murine pro/pre-B-cell line with GW7845 transiently induced the phosphorylation of extracellular signal-related protein kinase (ERK) 1/2, but strongly and persistently induced the activation of p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). In primary pro-B-cells, p38 MAPK and JNK were activated following treatment with GW7845. Phosphorylation of activating transcription factor-2 (ATF 2) was induced strongly in both B-cell types. In pro/pre-B cells, pretreatment with the p38 MAPK/JNK inhibitor PD169316 potently suppressed multiple facets of GW7845-induced apoptosis signaling. However, when a series of p38 MAPK and JNK inhibitors were used, only SB202190, also a dual inhibitor, completely suppressed GW7845-induced apoptosis. Inhibitors specific for p38 MAPK and JNK were only partially effective, suggesting that suppression of a single MAPK is not sufficient to inhibit death. The results support the hypothesis that GW7845 initiates an apoptotic pathway in early B cells through the activation of a kinase cascade that includes at least p38 MAPK and JNK. PMID- 16672324 TI - Intraocular pressure following phacoemulsification in patients with and without exfoliation syndrome: a 2 year prospective study. AB - AIM: To determine the long term intraocular pressure (IOP) response to phacoemulsification in patients with and without exfoliation syndrome (XFS). METHODS: Prospective, multicentre, cohort study with the following inclusion criteria: age over 50 years, open iridocorneal angle, and cataract. Two groups were enrolled: those with XFS and those without. The main outcome was mean IOP reduction 2 years after phacoemulsification cataract extraction (PCE). Univariate and multivariate analyses were performed. RESULTS: 183 patients were enrolled, 71 with and 112 without XFS. There were 29 patients with glaucoma in both groups. Mean baseline IOP was higher in XFS compared to control eyes (17.60 (SD 3.23) mm Hg v 16.08 (3.18) mm Hg, p = 0.002). Overall IOP reduction was significantly greater in the XFS group at the 2 year time point (-1.85 mm Hg v -0.62 mm Hg in the controls (p = 0.0037)). Multivariate analysis demonstrated that the IOP lowering effect in the XFS group may be related to irrigation volume at the time of surgery. In the subgroup analyses IOP lowering was significantly greater in the XFS and XFG patients than in controls without glaucoma, and POAG controls, respectively. The percentage of patients with a postoperative IOP spike was similar and relatively high in both XFS and control groups (34% v 25%; p = 0.54). CONCLUSION: IOP decreases more in patients with XFS following PCE compared to control eyes without XFS. This effect is more pronounced in glaucoma patients and persists for at least 2 years. PMID- 16672325 TI - Complications of mitomycin C therapy in 100 eyes with ocular surface neoplasia. AB - AIM: To determine the complications associated with mitomycin C (MMC) in the treatment of ocular surface neoplasia. METHODS: A retrospective and consecutive study of 100 eyes in 91 patients with ocular surface neoplasia treated with MMC in a single centre between November 1998 and January 2005. Outcome measures included complications of MMC and the treatment required for these complications. RESULTS: One to three 7 day cycles of topical MMC 0.04% four times a day were given to 59 eyes with localised corneal-conjunctival intraepithelial neoplasia (CIN), 19 eyes with diffuse CIN, six eyes with recurrent CIN, one eye with ocular surface squamous cell carcinoma, three eyes with primary acquired melanosis (PAM) with atypia, nine eyes with conjunctival malignant melanoma (MM), two eyes with sebaceous carcinoma with pagetoid spread, and one eye with recurrent atypical fibroxanthoma. Nine patients had bilateral CIN. 31 (34%) cases developed an allergic reaction to MMC and 14 (14%) eyes had epiphora secondary to punctal stenosis at a mean follow up period of 26.5 months. CONCLUSION: In the largest study looking at complications of topical MMC in the treatment of ocular surface neoplasia, allergic reaction and punctal stenosis are relatively common. Serious complications were not observed suggesting the safe use of MMC in mid-term follow up. PMID- 16672327 TI - Long term ultrasonic follow up of choroidal naevi and their transformation to melanomas. AB - AIMS: To compare ultrasonographic (US) predicting factors for conversion of choroidal naevi into melanomas. METHODS: 659 consecutive eyes with choroidal naevi were examined between 1984 and 2004. 165 clinically suspicious naevi were followed clinically and ultrasonographically (thickness, base diameters, internal reflectivity and location in the eye) for 5.08 (SE 0.24) years. RESULTS: 17 naevi (2.6% of all naevi, 10.3% of suspicious naevi) converted to small choroidal melanomas. The thickness of benign and premalignant naevi differed significant only after 1.5 years of follow up. The mean initial thickness of benign and premalignant naevi was significantly different (p = 0.001), as was mean initial internal reflectivity (p = 0.002) and mean initial largest base diameter (LBD, p = 0.05). Posterior pole and nasally located naevi were more likely to become malignant. A thickness of > or = 2 mm and a LBD > or = 7 mm were most predictive of conversion to melanoma, as was a combined K(I) index of > or = 14.5 (KI = LBD + 4 x thickness + 1 (for nasal location) + 1 (for posterior pole location)). An artificial neural network did not have a better forecasting accuracy than the KI index. Logistic regression found the only significant parameters to influence the risk of conversion to melanoma to be the KI value and the initial tumour thickness. CONCLUSIONS: A follow up of at least 1.5 years is necessary to detect conversion of naevi to choroidal melanomas. The thickness and LBD of the lesion can be used for predicting the risk. PMID- 16672326 TI - Depth perception deficits in glaucoma suspects. AB - AIM: To investigate depth perception in glaucoma suspects compared to glaucoma patients and controls. METHODS: Glaucoma suspects (n = 16), patients (n = 18), and normal age matched controls (n = 19) aged 40-65 years were prospectively evaluated for depth perception deficits using the Frisby test. Stereoacuity was measured by stereothreshold in seconds of arc for each group. RESULTS: Glaucoma suspects showed significantly increased mean stereothreshold compared to age matched normals (144.1 (SE 35.2) v 26.6 (3.7) seconds of arc; p = 0.0004). The mean stereothreshold in glaucoma patients was also increased compared to age matched normals 148.1 (33.8) v 26.6 (3.7) seconds of arc; p = 0.0004). CONCLUSIONS: Glaucoma suspects show depth perception deficits. The impaired stereovision in glaucoma suspects suggests that binocular interactions can be disrupted in the presence of normal visual fields by standard achromatic automated perimetry. PMID- 16672328 TI - Down's syndrome and early cataract. AB - AIMS: To estimate the occurrence of early cataract among patients with Down's syndrome and to evaluate the clinical characteristics of the cases. METHODS: Cases with Down's syndrome were ascertained from a cohort of all Danish children between 0 and 17 years of age, who were diagnosed with cataract during the period 1977-2001 (n = 1027). Information on the patients was obtained from the medical records. RESULTS: Of the total of 1027 cases with non-traumatic, non-acquired cataract there were 29 cases (13 males, 16 females) with Down's syndrome (2.8%). This corresponds to an occurrence of early cataract among patients with Down's syndrome of 1.4%; 27 had bilateral cataract and two had unilateral cataract. Half of the patients (n = 14) underwent cataract surgery, of whom two had bilateral primary lens implantation. 10 patients had bilateral cataract observed soon after birth, and five of these underwent cataract surgery within the first 6 months of life. CONCLUSION: The frequency of early cataract among children with Down's syndrome is estimated to be 1.4%, with cataracts requiring surgery during childhood being even rarer. In one third of the 29 cases, bilateral cataract was detected in the neonatal period. PMID- 16672330 TI - Comparison of rebound tonometry with Goldmann applanation tonometry and correlation with central corneal thickness. AB - BACKGROUND/AIMS: Rebound tonometry (RT) is performed without anaesthesia with a hand held device. The primary aim was to compare RT with Goldmann applanation tonometry (GAT) and to correlate with central corneal thickness (CCT). The secondary aim was to prove tolerability and practicability of RT under "study conditions" and "routine practice conditions." METHODS: In group 1 (52 eyes/28 patients), all measurements were taken by the same physician, in the same room and order: non-contact optical pachymetry, RT, slit lamp inspection, GAT. Patients were questioned about discomfort or pain. In group 2 (49 eyes/27 patients), tonometry was performed by three other physicians during routine examinations. RESULTS: RT was well tolerated and safe. Intraocular pressure (IOP) ranged between 6 mm Hg and 48 mm Hg. No different trends were found between the groups. RT tended to give slightly higher readings: n = 101, mean difference 1.0 (SD 2.17) mm Hg; 84.1% of RT readings within plus or minus 3 mm Hg of GAT; 95% confidence interval in the Bland-Altman analysis -3.2 mm Hg to +5.2 mm Hg. Both RT and GAT showed a weak positive correlation with CCT (r2 0.028 and 0.025, respectively). CONCLUSIONS: RT can be considered a reliable alternative for clinical screening and in cases where positioning of the head at the slit lamp is impossible or topical preparations are to be avoided. PMID- 16672329 TI - Primary lymphoma of the lacrimal sac: an EORTC ophthalmic oncology task force study. AB - AIM: To define the clinical and histopathological characteristics of primary lacrimal sac lymphoma in a predominantly white population. METHODS: Specimens of lacrimal sac lymphoma and follow up data were solicited from members of the Ophthalmic Oncology Task Force of the European Organization for Research and Treatment of Cancer (EORTC) and the European Ophthalmic Pathology Society (EOPS). Specimens were stained with haematoxylin and eosin and an immunohistochemical panel against leucocyte antigens was applied. Diagnosis was reached by consensus of five experienced pathologists according to the World Health Organization classification system. The histopathological findings were correlated with the clinical data. RESULTS: Of 15 primary lacrimal sac lymphomas, five (33%) were diffuse large B cell lymphoma (DLBCL), five (33%) were extranodal marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma), three were classified as "transitional MALT lymphoma," being in transition from MALT lymphoma to DLBCL, and two were unclassified B cell lymphomas. Nine of the patients were female, and the median age at the time of diagnosis was 71 years (range 45-95 years). The most frequent presenting symptoms were epiphora (85%), swelling in the region of the lacrimal sac (79%), and dacryocystitis (21%). All but one patient presented in stage I. Systemic spread occurred in three of nine patients (33%). The 5 year overall survival was 65%. CONCLUSIONS: DLBCL and MALT lymphoma are equally common in the lacrimal sac in contrast with the remaining periorbital and/or orbital region where MALT lymphoma predominates. PMID- 16672331 TI - Chromatin and epigenetics in development: blending cellular memory with cell fate plasticity. AB - The epigenetic regulation of chromatin structure and composition has often been studied molecularly in the context of specific DNA-dependent processes. However, epigenetics also play important global roles in shaping and maintaining cell identity, and in patterning the body plan during normal development. Moreover, alterations in epigenetic regulation are involved in many diseases, including cancer. The advances in our understanding of the impact of epigenetics in development and disease were discussed at a recent Keystone symposium. PMID- 16672332 TI - GABA induces terminal differentiation of Dictyostelium through a GABAB receptor. AB - When prespore cells approach the top of the stalk in a Dictyostelium fruiting body, they rapidly encapsulate in response to the signalling peptide SDF-2. Glutamate decarboxylase, the product of the gadA gene, generates GABA from glutamate. gadA is expressed exclusively in prespore cells late in development. We have found that GABA induces the release of the precursor of SDF-2, AcbA, from prespore cells. GABA also induces exposure of the protease domain of TagC on the surface of prestalk cells where it can convert AcbA to SDF-2. The receptor for GABA in Dictyostelium, GrlE, is a seven-transmembrane G-protein-coupled receptor that is most similar to GABA(B) receptors. The signal transduction pathway from GABA/GrlE appears to be mediated by PI3 kinase and the PKB-related protein kinase PkbR1. Glutamate acts as a competitive inhibitor of GABA functions in Dictyostelium and is also able to inhibit induction of sporulation by SDF-2. The signal transduction pathway from SDF-2 is independent of the GABA/glutamate signal transduction pathway, but the two appear to converge to control release of AcbA and exposure of TagC protease. These results indicate that GABA is not only a neurotransmitter but also an ancient intercellular signal. PMID- 16672333 TI - Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression. AB - Vertebrate limb development occurs along three cardinal axes-proximodistal, anteroposterior and dorsoventral-that are established via the organization of signaling centers, such as the zone of polarizing activity (ZPA). Distal limb development, in turn, requires a molecular feedback loop between the ZPA expression of sonic hedgehog (Shh) and the apical ectodermal ridge. The TALE homeoprotein Pbx1 has been shown to be essential for proximal limb development. In this study, we first uncover that Pbx1 and Pbx2 are co-expressed in the lateral plate and early limb field mesoderm. Later, Pbx2 is expressed throughout the limb, unlike Pbx1, which is expressed only in the proximal bud. By exploiting a Pbx1/Pbx2 loss-of-function mouse model, we demonstrate that, despite the lack of limb abnormalities in Pbx2-deficient (Pbx2(-/-)) embryos, compound Pbx1(-/-); Pbx2(+/-) mutants, in addition to their exacerbated proximal limb defects, exhibit novel and severe distal abnormalities. Additionally, we reveal that Pbx1( /-); Pbx2(-/-) embryos lack limbs altogether. Furthermore, we establish that, unlike in flies, where the leg develops independently of Hox and where the Pbx ortholog Exd is required for specification of proximal (but not distal) limbs, in vertebrates, distal limb patterning is Pbx1/Pbx2 dependent. Indeed, we demonstrate that Pbx genetic requirement is mediated, at least in part, through their hierarchical control of Hox spatial distribution and Shh expression. Overall, we establish that, by controlling the spatial expression of Hox genes in the posterior limb and regulating ZPA function, Pbx1/Pbx2 exert a primary hierarchical function on Hox genes, rather than behaving merely as Hox ancillary factors. PMID- 16672334 TI - Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans. AB - C. elegans develops through four larval stages (L1 to L4) separated by molts. The identity of larval stages is mostly determined by stage-specific expression of heterochronic genes, which constitute an intrinsic genetic timer. However, extrinsic cues such as food availability or population density also modulate the developmental timing of C. elegans by mechanisms that remain largely unknown. To investigate a potential role of the nervous system in the temporal regulation of C. elegans development, we pharmacologically manipulated nicotinic neurotransmission, which represents a prominent signaling component in C. elegans nervous system. Exposure to the nicotinic agonist DMPP during post-embryonic development is lethal at the L2/L3 molt. Specifically, it delays cell divisions and differentiation during the L2 stage but does not affect the timing of the molt cycle, hence causing exposure of a defective L3 cuticle to the environment after the L2/L3 molt. Forcing development through a previously uncharacterized L2 diapause resynchronizes these events and suppresses DMPP-induced lethality. Nicotinic acetylcholine receptors (nAChRs) containing the UNC-63 subunit are required, probably in neurons, to trigger the action of DMPP. Using a forward genetic screen, we further demonstrated that the nuclear hormone receptor (NHR) DAF-12 is necessary to implement the developmental effects of DMPP. Therefore, a novel neuroendocrine pathway involving nAChRs and the NHR DAF-12 can control the speed of stage-specific developmental events in C. elegans. Activation of DMPP sensitive nAChRs during the second larval stage uncouples a molting timer and a developmental timer, thus causing a heterochronic phenotype that is lethal at the subsequent molt. PMID- 16672335 TI - Sustained Bmp signaling is essential for cloaca development in zebrafish. AB - Bone morphogenetic protein (Bmp) signaling has long been known to be important for the early development of the ventral mesoderm, including blood, vasculature and kidney cells. Although Bmp genes are continually expressed in the ventral cells throughout gastrulation and somitogenesis, previous studies in zebrafish have not addressed how the role of Bmp signaling changes over time to regulate ventral mesoderm development. Here, we describe the use of a transgenic inducible dominant-negative Bmp receptor line to examine the temporal roles of Bmp signaling in ventral mesoderm patterning. Surprisingly, we find that Bmp signaling from the mid-gastrula stage through early somitogenesis is important for excluding blood and vascular precursors from the extreme ventral mesoderm, and we show that this domain is normally required for development of the cloaca (the common gut and urogenital opening). Using a novel assay for cloacal function, we find that larvae with reduced mid-gastrula Bmp signaling cannot properly excrete waste. We show that the cloacal defects result from alterations in the morphogenesis of the cloaca and from changes in the expression of genes marking the excretory system. Finally, we show that HrT, a T-box transcription factor, is a Bmp-regulated gene that has an essential function in cloacal development. We conclude that sustained Bmp signaling plays an important role in specification of the zebrafish cloaca by maintaining the fate of extreme ventral cells during the course of gastrulation and early somitogenesis. Furthermore, our data suggest that alterations in Bmp signaling are one possible cause of anorectal malformations during human embryogenesis. PMID- 16672336 TI - Zebrafish endoderm formation is regulated by combinatorial Nodal, FGF and BMP signalling. AB - In the zebrafish embryo, the mesoderm and endoderm originate from common precursors and segregate during gastrulation by mechanisms that are largely unknown. Understanding how the signalling pathways that regulate endoderm and mesoderm formation interact is crucial to understanding how the germ layers are established. Here, we have analysed how the FGF and BMP pathways interact with Nodal signalling during the process of endoderm formation. We found that activation of the FGF/ERK pathway disrupts endoderm formation in the embryo and antagonizes the ability of an activated form of Tar/Acvr1b to induce endoderm at the animal pole. By contrast, inhibition of FGF signalling increases the number of endodermal precursors and potentiates the ability of Tar*/Acvr1b to induce endoderm at the animal pole. Using a pharmacological inhibitor of the FGF receptor, we show that reducing FGF signalling partially rescues the deficit of endoderm precursors in bon mutant embryos. Furthermore, we found that overexpression of BMPs compromises endoderm formation, suggesting that formation of endoderm precursors is negatively regulated by BMPs on the ventral side. We show that simultaneous inhibition of the FGF/Ras and BMP pathways results in a dramatic increase in the number of endoderm precursors. Taken together, these data strongly suggest that BMP and FGF-ERK pathways cooperate to restrict the number of endodermal progenitors induced in response to Nodal signalling. Finally, we investigated the molecular basis for the FGF-MAPK-dependent repression of endoderm formation. We found that FGF/ERK signalling causes phosphorylation of Casanova/Sox32, an important regulator of endoderm determination, and provide evidence that this phosphorylation attenuates its ability to induce sox17. These results identify a molecular mechanism whereby FGF attenuates Nodal-induced endodermal transcription factors and highlight a potential mechanism whereby mesoderm and endoderm fates could segregate from each other. PMID- 16672337 TI - BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis. AB - The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development. PMID- 16672338 TI - VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation. AB - During vertebrate neurogenesis, multiple extracellular signals influence progenitor cell fate choices. The process by which uncommitted progenitor cells interpret and integrate signals is not well understood. We demonstrate here that in the avascular chicken retina, vascular endothelial growth factor (VEGF) secreted by postmitotic neurons acts through the FLK1 receptor present on progenitor cells to influence cell proliferation and commitment. Augmenting VEGF signals increases progenitor cell proliferation and decreases retinal ganglion cell genesis. Conversely, absorbing endogenous VEGF ligand or disrupting FLK1 activity attenuates cell proliferation and enhances retinal ganglion cell production. In addition, we provide evidence that VEGF signals transmitted by the FLK1 receptor activate divergent intracellular signaling components, which regulate different responses of progenitor cells. VEGF-induced proliferation is influenced by the MEK-ERK pathway, as well as by the basic helix-loop-helix factor HES1. By contrast, VEGF-dependent ganglion cell suppression does not require MEK-ERK activation, but instead relies on VEGF-stimulated HES1 activity, which is independent of NOTCH signaling. Moreover, elevated HES1 expression promotes progenitor cell proliferation and prevents overproduction of retinal ganglion cells owing to the loss of VEGF or sonic hedgehog (SHH), another signal that suppresses ganglion cell development. Based on previous and current findings, we propose that HES1 serves as a convergent signaling node within early retinal progenitor cells to integrate various cell-extrinsic cues, including VEGF and SHH, in order to control cell proliferation and neuronal specification. PMID- 16672339 TI - The left-right axis in the mouse: from origin to morphology. AB - The past decade or so has seen rapid progress in our understanding of how left right (LR) asymmetry is generated in vertebrate embryos. However, many important questions about this process remain unanswered. Although a leftward flow of extra embryonic fluid in the node cavity (nodal flow) is likely to be the symmetry breaking event, at least in the mouse embryo, it is not yet known how this flow functions or how the asymmetric signal generated in the node is transferred to the lateral plate. The final step in left-right patterning - translation of the asymmetric signal into morphology - is also little understood. PMID- 16672340 TI - Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex. AB - Most GABAergic interneurons originate from the basal forebrain and migrate tangentially into the cortex. The migratory pathways and mode of interneuron migration within the developing cerebral cortex, however, previously was largely unknown. Time-lapse imaging and in vivo labelling with glutamate decarboxylase (GAD)67-green fluorescence protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)ergic neurons indicated that multidirectional tangential (MDT) migration of interneurons takes place in both the marginal zone (MZ) and the ventricular zone (VZ) of the cortex. Quantitative analysis of migrating interneurons showed that rostrocaudally migrating neurons outnumber those migrating mediolaterally in both of these zones. In vivo labelling with a lipophilic dye showed that the MDT migration in the MZ occurs throughout the cortex over distances of up to 3 mm during a period of a few days. These results indicate that MZ cortical interneurons undergo a second phase of tangential migration in all directions and over long distances, after reaching the cortex by dorsomedial tangential migration. The MDT migration in the MZ may disperse and intermix interneurons within the cortex, resulting in a balanced distribution of interneuron subtypes. PMID- 16672341 TI - Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium. AB - The Dkk family of secreted cysteine-rich proteins regulates Wnt/beta-catenin signaling by interacting with the Wnt co-receptor Lrp5/6. Here, we show that Dkk2 mediated repression of the Wnt/beta-catenin pathway is essential to promote differentiation of the corneal epithelial progenitor cells into a non keratinizing stratified epithelium. Complete transformation of the corneal epithelium into a stratified epithelium that expresses epidermal-specific differentiation markers and develops appendages such as hair follicles is achieved in the absence of the Dkk2 gene function. We show that Dkk2 is a key regulator of the corneal versus epidermal fate of the ocular surface epithelium. PMID- 16672342 TI - Drosophila Plexin B is a Sema-2a receptor required for axon guidance. AB - Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how PlexB functions during neural development and which ligands serve to activate this receptor. Here, we show that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. We observe a physical association between these two plexin receptors in vivo and find that they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema 2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance. PMID- 16672343 TI - A beta1,4-galactosyltransferase is required for Bmp2-dependent patterning of the dorsoventral axis during zebrafish embryogenesis. AB - Complex carbohydrates are highly polymorphic macromolecules that are involved in diverse biological processes; however, a detailed understanding of their function remains obscure. To better define the roles of complex carbohydrates during vertebrate embryogenesis, we have initiated an analysis of glycosyltransferase function using the zebrafish system. In this study, we report the characterization of a zebrafish beta1,4-galactosyltransferase (GalT), which has substantial homology with mammalian beta4GalT5 and is expressed zygotically throughout the zebrafish embryo. Downregulating the expression of beta4GalT5 by injection of specific morpholino oligonucleotides results in dorsalized zebrafish embryos, suggesting a role of beta4GalT5 in Bmp2-mediated specification of the dorsoventral axis. Consistent with this, morpholino-injected embryos have ventrally expanded chordin expression and reduced activation of the Bmp-dependent transcription factors Smad1/5/8. Because other growth factors, such as Egf and Fgf, require binding to extracellular proteoglycans for delivery and/or binding to their cognate receptors, we examined whether proteoglycans isolated from control and morpholino-injected embryos show differential binding affinities for Bmp2. In this regard, proteoglycans isolated from beta4GalT5 morphant embryos are underglycosylated and are unable to bind recombinant Bmp2 as efficiently as proteoglycans from control-injected embryos, whereas the binding of Bmp7 is relatively unaffected. These results suggest that beta4GalT5 is a previously unidentified zebrafish galactosyltransferase that is essential for proper patterning of the dorsoventral axis by regulating Bmp2 signaling. Furthermore, this work demonstrates that a relatively simple carbohydrate modification to endogenous proteoglycans can modulate the specificity of cytokine signaling. PMID- 16672344 TI - The GATA2 transcription factor negatively regulates the proliferation of neuronal progenitors. AB - Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion that requires proper spatial and temporal coordination of proliferation, fate determination, differentiation and morphogenesis. This probably relies on complex interplay between mechanisms that control cell cycle, specification and differentiation. In this respect, we have studied the possible implication of GATA2, a transcription factor that is involved in several neuronal specification pathways, in the control of the proliferation of neural progenitors in the embryonic spinal cord. Using gain- and loss-of-function manipulations, we have shown that Gata2 can drive neural progenitors out of the cycle and, to some extent, into differentiation. This correlates with the control of cyclin D1 transcription and of the expression of the p27/Kip1 protein. Interestingly, this functional aspect is not only associated with silencing of the Notch pathway but also appears to be independent of proneural function. Consistently, GATA2 also controls the proliferation capacity of mouse embryonic neuroepithelial cells in culture. Indeed, Gata2 inactivation enhances the proliferation rate in these cells. By contrast, GATA2 overexpression is sufficient to force such cells and neuroblastoma cells to stop dividing but not to drive either type of cell into differentiation. Furthermore, a non-cell autonomous effect of Gata2 expression was observed in vivo as well as in vitro. Hence, our data have provided evidence for the ability of Gata2 to inhibit the proliferation of neural progenitors, and they further suggest that, in this regard, Gata2 can operate independently of neuronal differentiation. PMID- 16672345 TI - Programmed cell death mechanisms of identifiable peptidergic neurons in Drosophila melanogaster. AB - The molecular basis of programmed cell death (PCD) of neurons during early metamorphic development of the central nervous system (CNS) in Drosophila melanogaster are largely unknown, in part owing to the lack of appropriate model systems. Here, we provide evidence showing that a group of neurons (vCrz) that express neuropeptide Corazonin (Crz) gene in the ventral nerve cord of the larval CNS undergo programmed death within 6 hours of the onset of metamorphosis. The death was prevented by targeted expression of caspase inhibitor p35, suggesting that these larval neurons are eliminated via a caspase-dependent pathway. Genetic and transgenic disruptions of ecdysone signal transduction involving ecdysone receptor-B (EcR-B) isoforms suppressed vCrz death, whereas transgenic re introduction of either EcR-B1 or EcR-B2 isoform into the EcR-B-null mutant resumed normal death. Expression of reaper in vCrz neurons and suppression of vCrz-cell death in a reaper-null mutant suggest that reaper functions are required for the death, while no apparent role was found for hid or grim as a death promoter. Our data further suggest that diap1 does not play a role as a central regulator of the PCD of vCrz neurons. Significant delay of vCrz-cell death was observed in mutants that lack dronc or dark functions, indicating that formation of an apoptosome is necessary, but not sufficient, for timely execution of the death. These results suggest that activated ecdysone signaling determines precise developmental timing of the neuronal degeneration during early metamorphosis, and that subsequent reaper-mediated caspase activation occurs through a novel DIAP1-independent pathway. PMID- 16672346 TI - Capicua regulates follicle cell fate in the Drosophila ovary through repression of mirror. AB - The dorsoventral axis of the Drosophila egg is established by dorsally localized activation of the epidermal growth factor receptor (Egfr) in the ovarian follicular epithelium. Subsequent positive- and negative-feedback regulation generates two dorsolateral follicle cell primordia that will produce the eggshell appendages. A dorsal midline domain of low Egfr activity between the appendage primordia defines their dorsal boundary, but little is known about the mechanisms that establish their ventral limit. We demonstrate that the transcriptional repressor Capicua is required cell autonomously in ventral and lateral follicle cells to repress dorsal fates, and functions in this process through the repression of mirror. Interestingly, ectopic expression of mirror in the absence of capicua is observed only in the anterior half of the epithelium. We propose that Capicua regulates the pattern of follicle cell fates along the dorsoventral axis by blocking the induction of appendage determinants, such as mirror, by anterior positional cues. PMID- 16672347 TI - Islet1 and Islet2 have equivalent abilities to promote motoneuron formation and to specify motoneuron subtype identity. AB - The expression of LIM homeobox genes islet1 and islet2 is tightly regulated during development of zebrafish primary motoneurons. All primary motoneurons express islet1 around the time they exit the cell cycle. By the time primary motoneurons undergo axogenesis, specific subtypes express islet1, whereas other subtypes express islet2, suggesting that these two genes have different functions. Here, we show that Islet1 is required for formation of zebrafish primary motoneurons; in the absence of Islet1, primary motoneurons are missing and there is an apparent increase in some types of ventral interneurons. We also provide evidence that Islet2 can substitute for Islet1 during primary motoneuron formation. Surprisingly, our results demonstrate that despite the motoneuron subtype-specific expression patterns of Islet1 and Islet2, the differences between the Islet1 and Islet2 proteins are not important for specification of the different primary motoneuron subtypes. Thus, primary motoneuron subtypes are likely to be specified by factors that act in parallel to or upstream of islet1 and islet2. PMID- 16672348 TI - Glucose utilization and the PI3-K pathway: mechanisms for cell survival in preimplantation embryos. AB - The maintenance of optimal glucose utilization during the preimplantation period is critical for embryo survival. A decrease in glucose transport during preimplantation development has been linked to the early steps of programmed cell death in these embryos. Decreased glucose transport is not thought to be simply a consequence of cell death, rather it is thought to be a trigger that can initiate the apoptotic cascade. Extensive apoptosis during the preimplantation period may manifest later in pregnancy as a malformation-- or miscarriage, if cell loss is excessive. Phosphatidylinositol 3-kinase (PI3-K) is a known regulator of a number of physiologic responses including cellular proliferation, growth, and survival as well as glucose metabolism. Studies performed in other cell systems have demonstrated that the PI3-K pathway plays a critical role in maintaining glucose transport and metabolism. This review will present the current evidence that suggests that PI3-K is vital for preimplantation embryo survival and development. In addition, data demonstrating that PI3-K activity is important for glucose metabolism during this early developmental period will be discussed. PMID- 16672349 TI - Gene and protein expression in the myometrium in pregnancy and labor. AB - Microarray technologies widen our comprehension of the major structural and metabolic transformations which affect the myometrium from the very beginning of pregnancy until parturition. The results are coherent with the mass of information which was accumulated previously, primarily on the basis of studies of selected critical factors. They highlight the activation of precise signaling pathways, some of which may have been previously under evaluated. The remodelling and maturation processes that the myometrium undergoes in pregnancy appear clearly as phenomena which last during the full course of gestation. Comparatively, the onset of labor is perhaps the phenomenon which remains the least well described by these methods of analysis. Nevertheless, genomic studies constitute a necessary first step of orientation and help establishing new links between the generic signaling pathways that are activated during the normal or pathological gestation. These studies also represent an indicative step that will have to be paralleled, in the future, with the results of the systematic proteomic analysis of the myometrium. PMID- 16672350 TI - Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts. AB - Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured before in vitro fertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts. PMID- 16672351 TI - Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA. AB - RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis. PMID- 16672352 TI - Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa. AB - Recent experimental data show that incubating bovine sperm with cholesterol loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L(pg)) and activation energy (E(Lp))). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 degrees C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 microm and radius 0.4 microm, with osmotically inactive cell volume (V(b)) of 0.61 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best fit water transport parameters (L(pg) and E(Lp)) were determined. The predicted best-fit permeability parameters ranged from L(pg) = 0.02 to 0.036 microm/min-atm and E(Lp) = 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 degrees C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa. PMID- 16672353 TI - Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. AB - The response of sperm to cryopreservation and the fertility of frozen-thawed semen varies between species. Besides species differences in sperm physiology, structure and biochemistry, factors such as sperm transport and female reproductive tract anatomy will affect fertility of frozen-thawed semen. Therefore, studying differences in sperm cryotolerance between breeds and individuals instead of between species may reveal sources of variability in sperm cryotolerance. In the present study, the effect of cooling, re-warming and freezing and thawing on plasma membrane and acrosome integrity of sperm within and between Norwegian Landrace and Duroc breeds was studied. Furthermore, the relation between post-thaw survival rate and fatty acid composition of the sperm plasma membranes was investigated. Flow cytometry assessments of plasma membrane and acrosome integrity revealed no significant differences between breeds; however there were significant male-to-male variations within breeds in post-thaw percentages of live sperm (plasma membrane intact). The most abundant fatty acids in the plasma membranes from both breeds were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, n-9), docosapentaenoic acid (22:5, n-6) and docosahexaenoic acid (22:6, n-3). The ratio of sigma operator 22:5, n-6 and 22:6, n-3/ sigma operator all other membrane fatty acids was significantly related to survival rate (plasma membrane integrity) of sperm for both Norwegian Landrace (correlation coefficient (r(s)) = 0.64, P < 0.05) and Duroc (r(s) = 0.67, P < 0.05) boars. In conclusion, male-to-male differences in sperm survival rate after freezing and thawing may be partly related to the amount of long-chain polyunsaturated fatty acids in the sperm plasma membranes. PMID- 16672354 TI - Developmental and molecular correlates of bovine preimplantation embryos. AB - Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were cultured in vitro in three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR. In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P < 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P < 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P < 0.001). Expression of interferon tau (IF-tau) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P < 0.001). Gene expression did not differ between in vivo-derived blastocysts and their in vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression. PMID- 16672355 TI - Murine homologues of the Drosophila gustavus gene are expressed in ovarian granulosa cells. AB - Mammalian homologues of genes that control oogenesis in other organisms may play similar roles in mammalian ovarian development. In Drosophila melanogaster, GUSTAVUS (GUS) protein physically interacts with and is necessary for the proper posterior localization of VASA protein, and thus is required for specification of germ cells. We identified two mouse genes, SSB-1 and SSB-4 (SPRY domain SOCS box protein), whose protein products share 75% identity and are each approximately 70% identical to Drosophila GUS. Both SSB-1 and SSB-4 mRNA were detectable in mouse ovaries by Northern blotting of total and poly(A) + RNA, but were expressed in few other tissues. SSB-1 was detectable in testes, although the 3' untranslated region of the mRNA was considerably shorter than the ovarian mRNA. In situ hybridization and RT-PCR analysis of ovaries revealed that both genes were expressed in granulosa cells at all stages of follicular development. In contrast, expression was barely detectable in in oocytes. Immunoblotting analysis revealed that SSB-1 protein was present in follicles at different stages of growth, and immunocytochemistry confirmed that SSB-1 and SSB-4 were detectable in granulosa cells of primary and subsequent stage follicles and that they were present in both mural and cumulus granulosa cells of antral follicles. These results establish that GUS-related proteins, which in Drosophila are restricted to the germ cells, are in the mouse instead expressed in the granulosa cells and are present throughout folliculogenesis. Based on their tissue-restricted pattern of expression and apparent abundance in granulosa cells, we propose that SSB-1 and SSB-4 play key roles in regulating granulosa cell physiology. PMID- 16672356 TI - Developmental regulation of prostacyclin synthase and prostacyclin receptors in the ovine uterus and conceptus during the peri-implantation period. AB - This study documents the expression of prostacyclin (PGI2) synthase (PTGIS) and PGI2 receptors in the trophoblast and uterus of the ewe at the time of maternal recognition of pregnancy (i.e. days 7, 9, 12, 14 and 17). The membrane receptor for PGI2 (PTGIR) and the nuclear receptors, i.e. peroxisome proliferator activated receptors (PPAR) and their heterodimer partners the retinoid X receptors (RXR), were analysed. In the endometrium, PTGIS transcript and protein were expressed at day 9 of pregnancy and levels declined from days 12 to 17. Immunohistochemistry and in situ hybridization indicated that PTGIS was mainly located in the luminal epithelium of the endometrium. Endometrial PTGIR, PPARA, PPARG and RXRG expression was regulated during the peri-implantation period whereas PPARD, RXRA and RXRB were consistently expressed. In the trophoblast, PTGIS transcript levels rose as development progressed and peaked at day 17. PTGIR and PPARA transcripts peaked before day 12 and then declined and became nearly undetectable by day 17, whereas PPARD and PPARG transcript levels rose steadily from days 12 to 17. Because the PPARs and the RXRs display different expression profiles, we suggest that different heterodimers may form and support distinct functions as development proceeds. Our results also underline the importance of PTGIS and PPARD in the trophoblast and PTGIR in the uterus, suggesting that PGI2 is of both uterine and trophoblastic origin and is involved in a complex signalling pathway at around the time of implantation in the ewe. PMID- 16672357 TI - Modulation of peroxisome proliferator-activated receptor delta and gamma transcripts in swine endometrial tissue during early gestation. AB - Recent evidence points to a role for peroxisome proliferator-activated receptors (PPARs) delta and gamma in embryo implantation and survival. In this study, we report the porcine PPARdelta complete coding sequence and mRNA abundance of PPARdelta, PPARgamma1 and gamma2, angiopoietin-like protein 4 (ANGPTL4) and adipocyte determination and differentiation-dependent factor 1 (ADD1) genes in the pregnant sow endometrium. Real-time PCR analysis was used to study the effect of parity (Yorkshire-Landrace multiparous (YL) and nulliparous (YLn)), site of endometrial tissue sampling (between and at embryo attachment sites) in crossbred DurocxYorkshire-Landrace (DYL) sows and stages of pregnancy (non-pregnant, day 15 and day 25 after mating) in Meishan-Landrace (ML) on mRNA levels. Parity effects were observed for PPARdelta, ANGPTL4, and ADD1, with higher mRNA levels in YL than YLn sows. In DYL sows, lower mRNA levels were present at attachment sites compared to between attachment sites for PPARdelta, PPARgamma1, and ANGPTL4. Finally, day 15 pregnant ML sows had lower PPARdelta mRNA levels compared to day 15 cycling ML sows. A significant increase of PPARgamma1 mRNA levels was found on day 25 pregnant ML and DYL sows relative to day 15 ML or DYL pregnant sows. PPARdelta and gamma immunostaining was detected in endometrial tissue of day 15 cycling sows, day 15 and 25 pregnant sows and epithelial cells of day 25 embryos. Collectively, our results suggest a role for PPARdelta, PPARgamma1, and ANGPTL4, but not PPARgamma2, during the peri-implantation period in pregnant sows. PMID- 16672358 TI - Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs. AB - While uterotrophic effects of relaxin are well documented, the mechanism through which relaxin promotes uterine growth is incompletely understood. Studies in rats suggest that relaxin-stimulated uterine edema depends on estrogen receptor (ER) activation. Here, neonatal pigs were used to investigate the interaction between relaxin and ER signaling pathways. Gilts were treated either at birth (postnatal day (PND) 0) (study 1) before the onset of endometrial ERalpha expression, or on PND 12 (study 2) after the onset of ERalpha expression. In study 1, gilts were treated with estradiol-17beta or porcine relaxin for two days and uteri were collected on PND 2. In study 2, PND 12 gilts were treated with a single injection of the ER antagonist ICI 182,780 (ICI) or vehicle. Two hours later, gilts were given either estradiol-17beta or porcine relaxin for two days. When administered for two days from birth (study 1), neither estradiol-17beta nor relaxin affected uterine weight or protein content. However, uterine luminal epithelial height was greater in relaxin- than in vehicle-treated gilts. In contrast, in study 2, both estradiol and relaxin increased uterine weight, protein content and uterine luminal epithelial height on PND 14. These effects were inhibited by pre treatment with ICI in both estradiol- and relaxin-treated gilts. The results indicate that uterotrophic effects of relaxin in the neonatal pig are related to age and to both the relative presence and state of activation of the ER system in developing uterine tissues between birth and PND 14. PMID- 16672359 TI - Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction. AB - Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using alpha(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non essential plus 0.5 x essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved. PMID- 16672360 TI - Regulatory roles for mitochondria in the peri-implantation mouse blastocyst: possible origins and developmental significance of differential DeltaPsim. AB - Studies of mitochondria in mouse and human oocytes and preimplantation stage embryos have focused primarily on their metabolic capacity to generate ATP. However, it is becoming increasingly apparent that mitochondria are also regulatory agents in other processes involved in the establishment of developmental competence, including calcium homeostasis and apoptosis. The magnitude of the inner mitochondrial membrane potential, or its polarity (DeltaPsim), is a physiochemical property of mitochondria related to levels of organelle activity, and differences in the magnitude and spatial distribution of high- and low-polarized mitochondria have been suggested to influence oocyte and early embryo competence. Here, we investigated mitochondrial polarity in normal and diapausing peri-implantation-stage mouse blastocysts, and their corresponding outgrowths, for indications of cell-type-specific regulatory functions or activities in which these organelles may be engaged. The results demonstrate that cell-type- and location-specific domains of differential DeltaPsim exist in the peri-implantation blastocyst and remain unchanged during blastocyst outgrowth and during delayed implantation, which for the latter, is accompanied by the suppression of mitochondrial oxidative phosphorylation. Our findings demonstrate that cell-type-specific DeltaPsim in the peri-implantation blastocyst is not an intrinsic property of the corresponding mitochondria but one that can be mediated by the dynamics of intercellular contact. Cells with high- or low-polarized mitochondria are differentially affected by photosensitization, with developmental consequences related to embryo behavior and outgrowth performance. Differences in polarity are discussed with respect to the participation of mitochondria in regulatory and morphogenetic processes in the normal peri implantation embryo. The persistence of high DeltaPsim in the diapausing embryo is suggested to be associated with the regulation of levels of cytoplasmic free calcium and the ability of the embryo to reactivate development when delayed implantation terminates. PMID- 16672361 TI - Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax. AB - Prevention of inhalational anthrax after Bacillus anthracis spore exposure requires a prolonged course of antibiotic prophylaxis. In response to the 2001 anthrax attack in the United States, approximately 10,000 people were offered 60 days of antibiotic prophylaxis to prevent inhalational anthrax, but adherence to this regimen was poor. We sought to determine whether a short course of antibiotic prophylaxis after exposure could protect non-human primates from a high-dose spore challenge if vaccination was combined with antibiotics. Two groups of 10 rhesus macaques were exposed to approximately 1,600 LD50 of spores by aerosol. Both groups were given ciprofloxacin by orogastric tube twice daily for 14 days, beginning 1-2 h after exposure. One group also received three doses of the licensed human anthrax vaccine (anthrax vaccine adsorbed) after exposure. In the ciprofloxacin-only group, four of nine monkeys (44%) survived the challenge. In contrast, all 10 monkeys that received 14 days of antibiotic plus anthrax vaccine adsorbed survived (P = 0.011). Thus postexposure vaccination enhanced the protection afforded by 14 days of antibiotic prophylaxis alone and completely protected animals against inhalational anthrax. These data provide evidence that postexposure vaccination can shorten the duration of antibiotic prophylaxis required to protect against inhalational anthrax and may impact public health management of a bioterrorism event. PMID- 16672362 TI - Ordered assembly of the duplicating Golgi in Trypanosoma brucei. AB - The new Golgi in the protozoan parasite Trypanosoma brucei grows near to the old and adjacent to the growing new endoplasmic reticulum exit site. Growth is now shown to be at least a two-stage process, in which a representative matrix marker (GRASP) and enzyme (GntB) are delivered to the site of assembly, followed approximately 10 min later by a COPI component (epsilon-COP) and a trans-Golgi network (TGN) marker (GRIP70). A secretory cargo marker (signal sequence-YFP) appeared early near the new endoplasmic reticulum exit site but did not enter the Golgi until the second stage. Together these data suggest that structural and enzymatic components of the new Golgi stack are laid down first, followed by those needed to move and sort the cargo passing through it. PMID- 16672363 TI - Structure of the ternary signaling complex of a TGF-beta superfamily member. AB - The crystal structure of the complete signaling complex formed between bone morphogenetic protein 2 (BMP-2) and the extracellular domains (ECDs) of its type I receptor [bone morphogenetic protein receptor type Ia (BMPR-Ia)-ECD] and its type II receptor [activin receptor type II (ActRII)-ECD] shows two fundamental structural constraints for receptor assembly. First, the homodimeric BMP-2 ligand assembles two pairs of each receptor symmetrically, where each of the receptor ECDs does not make physical contact. Therefore, conformational communication between receptor ECDs, if any, should be propagated through the central ligand. Second, the type I and II receptor interfaces of the complex, when compared with those of binary complexes such as BMP-2/BMPR Ia-ECD, BMP-7/ActRII-ECD, and activin/ActRIIb-ECD, respectively, show there are common sets of positions repeatedly used by both ligands and receptors. Therefore, specificity-determining amino acid differences at the receptor interfaces should also account for the disparity in affinity of individual receptors for different ligand subunits. We find that a specific mutation to BMP-2 increases its affinity to ActRII-ECD by 5 fold. These results together establish that the specific signaling output is largely determined by two variables, the ligand-receptor pair identity and the mode of cooperative assembly of relevant receptors governed by the ligand flexibility in a membrane-restricted manner. PMID- 16672364 TI - Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias. AB - Catecholaminergic polymorphic ventricular tachycardia is a form of exercise induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias. PMID- 16672365 TI - Influence of FHIT on benzo[a]pyrene-induced tumors and alopecia in mice: chemoprevention by budesonide and N-acetylcysteine. AB - The FHIT gene has many hallmarks of a tumor-suppressor gene and is involved in a large variety of cancers. We treated A/J mice and (C57BL/6J x 129/SvJ)F1 (B6/129 F1) mice, either wild-type or FHIT+/-, with multiple doses of benzo[a]pyrene (B[a]P) by gavage. B[a]P caused a time-related increase of micronuclei in peripheral blood erythrocytes. Both A/J and B6/129 F1 mice, irrespective of their FHIT status, were sensitive to induction of forestomach tumors, whereas B[a]P induced glandular stomach hyperplasia and a high multiplicity of lung tumors in A/J mice only. Preneoplastic lesions of the uterus were more frequent in FHIT+/- mice. B6/129 F1 mice underwent spontaneous alopecia areata and hair bulb cell apoptosis, which were greatly accelerated either by FHIT heterozygosity or by B[a]P treatment, thus suggesting that FHIT plays a role in the pathogenesis of alopecia areata. The oral administration of either budesonide or N-acetyl-L cysteine (NAC) inhibited the occurrence of this inflammatory skin disease. In addition, these agents prevented B[a]P-induced glandular stomach hyperplasia and decreased the size of both forestomach tumors and lung tumors in A/J mice. Budesonide also attenuated lung tumor multiplicity. In B6/129 F1 mice, NAC significantly decreased the proliferating cell nuclear antigen in lung tumors. Both budesonide and NAC inhibited B[a]P-induced forestomach tumors and preneoplastic lesions of the respiratory tract in B6/129 F1 mice. In conclusion, heterozygosity for FHIT affects susceptibility of mice to spontaneous alopecia areata and B[a]P-induced preneoplastic lesions of the uterus and does not alter responsiveness to budesonide and NAC. PMID- 16672366 TI - Birth of a chimeric primate gene by capture of the transposase gene from a mobile element. AB - The emergence of new genes and functions is of central importance to the evolution of species. The contribution of various types of duplications to genetic innovation has been extensively investigated. Less understood is the creation of new genes by recycling of coding material from selfish mobile genetic elements. To investigate this process, we reconstructed the evolutionary history of SETMAR, a new primate chimeric gene resulting from fusion of a SET histone methyltransferase gene to the transposase gene of a mobile element. We show that the transposase gene was recruited as part of SETMAR 40-58 million years ago, after the insertion of an Hsmar1 transposon downstream of a preexisting SET gene, followed by the de novo exonization of previously noncoding sequence and the creation of a new intron. The original structure of the fusion gene is conserved in all anthropoid lineages, but only the N-terminal half of the transposase is evolving under strong purifying selection. In vitro assays show that this region contains a DNA-binding domain that has preserved its ancestral binding specificity for a 19-bp motif located within the terminal-inverted repeats of Hsmar1 transposons and their derivatives. The presence of these transposons in the human genome constitutes a potential reservoir of approximately 1,500 perfect or nearly perfect SETMAR-binding sites. Our results not only provide insight into the conditions required for a successful gene fusion, but they also suggest a mechanism by which the circuitry underlying complex regulatory networks may be rapidly established. PMID- 16672367 TI - Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. AB - The plant-derived cannabinoids delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) both have immunosuppressive effects; although some effects of THC are mediated by the CB2 receptor, CB2 binds CBD weakly. In examining the effects of THC and CBD on microglial proliferation, we found that these compounds potently inhibit [3H]thymidine incorporation into a murine microglial cell line with no effect on cell cycle. Treatment with THC and CBD decreased [3H]thymidine uptake into microglia, with IC50 values that match inhibition of [3H]thymidine incorporation into DNA. CBD and, less potently, THC decreased uptake of [3H]adenosine to a similar extent as [3H]thymidine in both murine microglia and RAW264.7 macrophages. Binding studies confirm that CBD binds to the equilibrative nucleoside transporter 1 with a Ki < 250 nM. Because adenosine agonists have antiinflammatory effects, and because uptake of adenosine is a primary mechanism of terminating adenosine signaling, we tested the hypothesis that CBD is immunosuppressive because it enhances endogenous adenosine signaling. In vivo treatment with a low dose of CBD decreases TNFalpha production in lipopolysaccharide-treated mice; this effect is reversed with an A2A adenosine receptor antagonist and abolished in A2A receptor knockout mice. These studies demonstrate that CBD has the ability to enhance adenosine signaling through inhibition of uptake and provide a non-cannabinoid receptor mechanism by which CBD can decrease inflammation. PMID- 16672368 TI - Protease specificity determination by using cellular libraries of peptide substrates (CLiPS). AB - We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics. PMID- 16672369 TI - The sulfur acclimation SAC3 kinase is required for chloroplast transcriptional repression under sulfur limitation in Chlamydomonas reinhardtii. AB - Sulfur (S) deprivation responses have been studied extensively in algae and land plants; however, little is known of the signals that link perception of S status to chloroplast gene expression. Here, we have compared the chloroplast S limitation response in WT vs. sac1 and sac3 sulfur acclimation mutants of the green alga Chlamydomonas reinhardtii. We provide evidence that in the WT, chloroplast transcriptional activity rapidly decreases after removal of S from the medium, leading to reduced transcript accumulation. This decrease correlates with reduced abundance of a sigma70-like factor, Sig1, which is most likely the unique chloroplast transcription specificity factor. We further show that reduced transcription activity and diminished Sig1 accumulation are mediated by the SAC3 gene product, a putative Snf1-type Ser/Thr kinase previously shown to have both positive and negative effects on nuclear gene expression. Inclusion of the protein kinase inhibitor 6-dimethylaminopurine during S limitation yielded a pattern of expression that was largely similar to that seen in the sac3 mutant, lending support to the hypothesis that Sac3 kinase activation leads to transcriptional repression and Sig1 proteolysis. The finding that Sac3 regulates chloroplast gene expression suggests that it has a previously unknown role in integrating the S limitation response in multiple subcellular compartments. PMID- 16672370 TI - Elimination of damaged proteins during differentiation of embryonic stem cells. AB - During mammalian aging, cellular proteins become increasingly damaged: for example, by carbonylation and formation of advanced glycation end products (AGEs). The means to ensure that offspring are born without such damage are unknown. Unexpectedly, we found that undifferentiated mouse ES cells contain high levels of both carbonyls and AGEs. The damaged proteins, identified as chaperones and proteins of the cytoskeleton, are the main targets for protein oxidation in aged tissues. However, the mouse ES cells rid themselves of such damage upon differentiation in vitro. This elimination of damaged proteins coincides with a considerably elevated activity of the 20S proteasome. Moreover, damaged proteins were primarily observed in the inner cell mass of blastocysts, whereas the cells that had embarked on differentiation into the trophectoderm displayed drastically reduced levels of protein damage. Thus, the elimination of protein damage occurs also during normal embryonic development in vivo. This clear-out of damaged proteins may be a part of a previously unknown rejuvenation process at the protein level that occurs at a distinct stage during early embryonic development. PMID- 16672371 TI - The road from Santa Rosalia: a faster tempo of evolution in tropical climates. AB - Using an appropriately designed and replicated study of a latitudinal influence on rates of evolution, we test the prediction by K. Rohde [(1992) Oikos 65, 514 527] that the tempo of molecular evolution in the tropics is greater than at higher latitudes. Consistent with this prediction we found tropical plant species had more than twice the rate of molecular evolution as closely related temperate congeners. Rohde's climate-speciation hypothesis constitutes one explanation for the cause of that relationship. This hypothesis suggests that mutagenesis occurs more frequently as productivity and metabolic rates increase toward the equator. More rapid mutagenesis was then proposed as the mechanism that increases evolutionary tempo and rates of speciation. A second possible explanation is that faster rates of molecular evolution result from higher tropical speciation rates [e.g., Bromham, L. & Cardillo, M. (2003) J. Evol. Biol. 16, 200-207]. However, we found the relationship continued to hold for genera with the same number of, or more, species in temperate latitudes. This finding suggests that greater rates of speciation in the tropics do not cause higher rates of molecular evolution. A third explanation is that more rapid genetic drift might have occurred in smaller tropical species populations [Stevens, G. C. (1989) Am. Nat. 133, 240-256]. However, we targeted common species to limit the influence of genetic drift, and many of the tropical species we used, despite occurring in abundant populations, had much higher rates of molecular evolution. Nonetheless, this issue is not completely resolved by that precaution and requires further examination. PMID- 16672372 TI - Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors. AB - The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI 272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation. PMID- 16672373 TI - Langerhans cells cross-present antigen derived from skin. AB - Dendritic cells (DC) efficiently cross-present exogenous antigen on MHC class I molecules to CD8+ T cells. However, little is known about cross-presentation by Langerhans cells (LC), the DCs of the epidermis. Therefore, we investigated this issue in detail. Isolated murine LCs were able to cross-present soluble ovalbumin protein on MHC-class I molecules to antigen-specific CD8+ T cells, albeit less potently than the CD8+ DC subsets from spleen. Furthermore, LCs cross-presented cell-associated ovalbumin peptide and protein expressed by neighboring keratinocytes. Use of transporter associated with antigen processing (TAP-1) deficient mice suggested a TAP-dependent pathway. Similar observations were made with migratory LC. Antigen expressed in the epidermis was ingested by LCs during migration from the epidermis and presented to antigen-specific T cells in vitro. Cross-presentation of ovalbumin protein by LCs induced IFN-gamma production and cytotoxicity in antigen-specific CD8+ T cells. Additionally, epicutaneous application of ovalbumin protein induced in vivo proliferation of OT-I T cells in the draining lymph nodes; this was markedly enhanced when antigen was applied to inflamed, barrier-disrupted skin. Thus, LCs cross-present exogenous antigen to CD8+ T cells and induce effector functions, like cytokine production and cytotoxicity, and may thereby critically contribute in epicutaneous vaccination approaches. PMID- 16672374 TI - Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans. AB - Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1 GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1 GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization. PMID- 16672375 TI - Cellular analyses of the mitotic region in the Caenorhabditis elegans adult germ line. AB - The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions. First, all germ cells within the mitotic region are actively cycling, as visualized by bromodeoxyuridine (BrdU) labeling. No quiescent cells were found. Second, germ cells in the mitotic region lose BrdU label uniformly, either by movement of labeled cells into the meiotic region or by dilution, probably due to replication. No label-retaining cells were found in the mitotic region. Third, the distal tip cell niche extends processes that nearly encircle adjacent germ cells, a phenomenon that is likely to anchor the distal-most germ cells within the niche. Fourth, germline mitoses are not oriented reproducibly, even within the immediate confines of the niche. We propose that germ cells in the distal-most rows of the mitotic region serve as stem cells and more proximal germ cells embark on the path to differentiation. We also propose that C. elegans adult germline stem cells are maintained by proximity to the niche rather than by programmed asymmetric divisions. PMID- 16672376 TI - Substrate-dependent contribution of double-stranded RNA-binding motifs to ADAR2 function. AB - ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine (A-to-I). ADAR2 contains two tandem double-stranded RNA-binding motifs (dsRBMs) that are not only important for efficient editing of RNA substrates but also necessary for localizing ADAR2 to nucleoli. The sequence and structural similarity of these motifs have raised questions regarding the role(s) that each dsRBM plays in ADAR2 function. Here, we demonstrate that the dsRBMs of ADAR2 differ in both their ability to modulate subnuclear localization as well as to promote site-selective A-to-I conversion. Surprisingly, dsRBM1 contributes to editing activity in a substrate-dependent manner, indicating that dsRBMs recognize distinct structural determinants in each RNA substrate. Although dsRBM2 is essential for the editing of all substrates examined, a point mutation in this motif affects editing for only a subset of RNAs, suggesting that dsRBM2 uses unique sets of amino acid(s) for functional interactions with different RNA targets. The dsRBMs of ADAR2 are interchangeable for subnuclear targeting, yet such motif alterations do not support site-selective editing, indicating that the unique binding preferences of each dsRBM differentially contribute to their pleiotropic function. PMID- 16672377 TI - A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans. AB - The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2 Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen. PMID- 16672378 TI - Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress. AB - The unfolded protein response (UPR) regulates the protein-folding capacity of the endoplasmic reticulum (ER) according to cellular demand. In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, initiate distinct UPR signaling branches. We show that these UPR components display distinct sensitivities toward different forms of ER stress. ER stress induced by ER Ca2+ release in particular revealed fundamental differences in the properties of UPR signaling branches. Compared with the rapid response of both IRE1 and PERK to ER stress induced by thapsigargin, an ER Ca2+ ATPase inhibitor, the response of ATF6 was markedly delayed. These studies are the first side-by-side comparisons of UPR signaling branch activation and reveal intrinsic features of UPR stress sensor activation in response to alternate forms of ER stress. As such, they provide initial groundwork toward understanding how ER stress sensors can confer different responses and how optimal UPR responses are achieved in physiological settings. PMID- 16672379 TI - CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25. AB - SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway. PMID- 16672380 TI - Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis. AB - Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites. PMID- 16672381 TI - The stress-activated mitogen-activated protein kinase signaling cascade promotes exit from mitosis. AB - In budding yeast, a signaling network known as the mitotic exit network (MEN) triggers exit from mitosis. We find that hypertonic stress allows MEN mutants to exit from mitosis in a manner dependent on the high osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase cascade. The HOG pathway drives exit from mitosis in MEN mutants by promoting the activation of the MEN effector, the protein phosphatase Cdc14. Activation of Cdc14 depends on the Cdc14 early anaphase release network, a group of proteins that functions in parallel to the MEN to promote Cdc14 function. Notably, exit from mitosis is promoted by the signaling branch defined by the Sho1 osmosensing system, but not by the Sln1 osmosensor of the HOG pathway. Our results suggest that the stress MAP kinase pathway mobilizes programs to promote completion of the cell cycle and entry into G1 under unfavorable conditions. PMID- 16672382 TI - Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements. AB - Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin independent endocytosis. PMID- 16672383 TI - Role of cell cycle-regulated expression in the localized incorporation of cell wall proteins in yeast. AB - The yeast cell wall is an essential organelle that protects the cell from mechanical damage and antimicrobial peptides, participates in cell recognition and adhesion, and is important for the generation and maintenance of normal cell shape. We studied the localization of three covalently bound cell wall proteins in Saccharomyces cerevisiae. Tip1p was found only in mother cells, whereas Cwp2p was incorporated in small-to-medium-sized buds. When the promoter regions of TIP1 and CWP2 (responsible for transcription in early G1 and S/G2 phases, respectively) were exchanged, the localization patterns of Tip1p and Cwp2p were reversed, indicating that the localization of cell wall proteins can be completely determined by the timing of transcription during the cell cycle. The third protein, Cwp1p, was incorporated into the birth scar, where it remained for several generations. However, we could not detect any role of Cwp1p in strengthening the birth scar wall or any functional interaction with the proteins that mark the birth scar pole as a potential future budding site. Promoter exchange experiments showed that expression in S/G2 phase is necessary but not sufficient for the normal localization of Cwp1p. Studies of mutants in which septum formation is perturbed indicate that the normal asymmetric localization of Cwp1p also depends on the normal timing of septum formation, composition of the septum, or both. PMID- 16672384 TI - Comparison of a conventional antimicrobial susceptibility assay to an oligonucleotide chip system for detection of drug resistance in Mycobacterium tuberculosis isolates. AB - An oligonucleotide chip (Combichip Mycobacteria chip) detecting specific mutations in the rpoB, katG, and inhA genes of Mycobacterium tuberculosis was compared with conventional antimicrobial susceptibility results. The probes detecting drug resistance were as follows: 7 wild-type and 13 mutant probes for rifampin and 2 wild-type and 3 mutant probes for isoniazid. Target DNA of M. tuberculosis was amplified by PCR, followed by hybridization and scanning. Direct sequencing was performed to verify the results of the oligonucleotide chip. One hundred seven of 115 rifampin-resistant strains (93%) had mutations in the rpoB gene. Eighty-five of 119 isoniazid-resistant strains (71%) had mutations in the katG gene or inhA gene. The diagnostic oligonucleotide chip with mutation specific probes is a reliable and useful tool for the rapid and accurate diagnosis of resistance against rifampin and isoniazid in M. tuberculosis isolates. PMID- 16672385 TI - Emergence of an invasive clone of nontoxigenic Corynebacterium diphtheriae in the urban poor population of Vancouver, Canada. AB - Invasive disease due to Corynebacterium diphtheriae is rare in North America. Here we describe the emergence of a predominant clone of a nontoxigenic strain of C. diphtheriae in the impoverished population of Vancouver's downtown core. This clone has caused significant morbidity and contributed to at least two deaths. Over a 5-year period, seven cases of bacteremia due to C. diphtheriae were detected in patients admitted to Vancouver hospitals. Injection drug use, diabetes mellitus, skin colonization/infection with C. diphtheriae, and homelessness all appeared to be related to the development of bacteremia with the organism. Ribotyping of isolates recovered from blood culture revealed a predominant ribotype pattern that has not previously been reported in North America. PMID- 16672386 TI - Longitudinal farm study of extended-spectrum beta-lactamase-mediated resistance. AB - Extended-spectrum beta-lactamase (ESBL)-mediated resistance is of considerable importance in human medicine. Recently, such enzymes have been reported in bacteria from animals. We describe a longitudinal study of a dairy farm suffering calf scour with high mortality rates. In November 2004, two Escherichia coli isolates with resistance to a wide range of beta-lactams (including amoxicillin clavulanate and cefotaxime) were isolated from scouring calves. Testing by PCR and sequence analysis confirmed the isolates as being both bla(CTX-M14/17) and bla(TEM-35) ((IRT-4)) positive. They had indistinguishable plasmid and pulsed field gel electrophoresis (PFGE) profiles. Transferability studies demonstrated that bla(CTX-M) was located on a conjugative 65-MDa IncK plasmid. Following a farm visit in December 2004, 31/48 calves and 2/60 cows were positive for E. coli with bla(CTX-M). Also, 5/48 calf and 28/60 cow samples yielded bla(CTX)- and bla(TEM)-negative E. coli isolates that were resistant to cefotaxime, and sequence analysis confirmed that these presented mutations in the promoter region of the chromosomal ampC gene. Fingerprinting showed 11 different PFGE types (seven in bla(CTX-M)-positive isolates). Six different PFGE clones conjugated the same bla(CTX-M)-positive IncK plasmid. One clone carried a different-sized, bla(CTX-M)-positive, transformable plasmid. This is the first report of bla(CTX M) from livestock in the United Kingdom, and this report demonstrates the complexity of ESBL epidemiology. Results indicate that horizontal plasmid transfer between strains as well as horizontal gene transfer between plasmids have contributed to the spread of resistance. We have also shown that some clones can persist for months, suggesting that clonal spread also contributes to the perpetuation of resistance. PMID- 16672387 TI - Comparison of DNA fingerprinting methods for use in investigation of type E botulism outbreaks in the Canadian Arctic. AB - Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, and automated ribotyping were compared for epidemiological typing of Clostridium botulinum type E using clinical and food isolates associated with four botulism outbreaks occurring in the Canadian Arctic. All type E strains previously untypeable by PFGE, even with the use of a formaldehyde fixation step, could be typed by the addition of 50 microM thiourea to the electrophoresis running buffer. Digestion with SmaI or XhoI followed by PFGE was used to link food and clinical isolates from four different type E botulism outbreaks and differentiate them from among 39 group II strains. Strain differentiation was unsuccessful with the automated ribotyping system, producing a single characteristic EcoRI fingerprint common to all group II strains. RAPD analysis of C. botulinum group II strains was not consistently reproducible with primer OPJ-6 or OPJ-13, apparently discriminating between epidemiologically related strains. A modified PFGE protocol was judged to be the most useful method for typing epidemiologically related C. botulinum type E strains, based on its ability to type all strains reproducibly and with an adequate level of discrimination. PMID- 16672388 TI - Molecular epidemiology of Haemophilus influenzae type b causing vaccine failures in the United Kingdom. AB - Haemophilus influenzae type b (Hib) was a major cause of pediatric disease in the United Kingdom prior to the introduction of routine Hib immunization in 1992. An unexpected resurgence of cases of vaccine failure was observed with fully vaccinated children from 1999 onward. We investigated whether Hib isolates causing vaccine failures in the United Kingdom could have undergone a change in their population structure to elude the protective effect of Hib vaccine. Molecular epidemiology studies were carried out with 376 isolates from invasive infections (164 vaccine failures and 212 controls). Genetic variability was higher in controls than in vaccine failures. Of the four major clusters obtained, cluster I comprised 92.2% of the total isolates: 156 vaccine failures (95%) and 193 (91%) controls. Cluster IV was specific for vaccine failures but included only four isolates. The increased number of cases of invasive Hib in fully vaccinated children in the United Kingdom was caused by isolates belonging not to a particular or new genotype but to genotypes already circulating in the prevaccination era, before conjugate Hib vaccines were available. PMID- 16672389 TI - Nutritional requirements and antibiotic resistance patterns of Helicobacter species in chemically defined media. AB - The growth of the gastric pathogen Helicobacter pylori in the absence of serum remains challenging, and nutritional requirements have only partially been defined, while almost nothing is known about nutritional requirements of other Helicobacter spp. Although previous data showed that H. pylori grows in the chemically defined medium F-12, but not in other tissue culture media examined, the specific components responsible for growth were not entirely understood. Here we describe the optimization of amino acids, metals, and sodium chloride for H. pylori. Iron, zinc, and magnesium were critical for growth; copper was not required. Optimization of sodium chloride was further beneficial. Nutritional requirements and antibiotic resistance patterns of several other Helicobacter spp. revealed that all except H. felis grew in serum-free, unsupplemented F-12. All Helicobacter spp. were resistant to at least six antimicrobial agents when cultured in the presence of serum. However, in the absence of serum, H. pylori, H. mustelae, and H. muridarum became sensitive to polymyxin B and/or trimethoprim. Much of the data were obtained using a convenient ATP assay to quantify growth. H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter spp. are not as fastidious as previously thought. The data also suggest that chemically defined media described herein could yield the growth of a wide range of Helicobacter spp., allowing a more detailed characterization of Helicobacter physiology and interactions with host cells. PMID- 16672390 TI - Trends in production of extended-spectrum beta-lactamases among enterobacteria of medical interest: report of the second Italian nationwide survey. AB - Results of a 2003 survey carried out in Italy to evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing enterobacteria are presented. Eleven Italian Microbiology Laboratories investigated 9,076 consecutive nonreplicate isolates (inpatients, 6,850; outpatients, 2,226). ESBL screening was performed by MIC data analysis. Confirmation was obtained using the double-disk synergy test and the combination disk test based on CLSI methodology. ESBL determinants were investigated by colony blot hybridization and confirmed by sequencing. Results were compared to those of the 1999 Italian survey (8,015 isolates). The prevalence of ESBL producers was 7.4% among isolates from inpatients (in 1999, 6.3%) and 3.5% among outpatients (no data were available for 1999). Among hospitalized patients, the most prevalent ESBL-positive species was Escherichia coli (Klebsiella pneumoniae in 1999). Proteus mirabilis was the most prevalent ESBL-positive species among outpatients. In both groups, most ESBL positive pathogens were obtained from urinary tract infections. TEM-type ESBLs were the most prevalent enzymes (45.4%). Non-TEM, non-SHV determinants emerged: CTX-M-type in E. coli and K. pneumoniae, and PER-type in P. mirabilis, Providencia spp., and E. coli. With the exception of 3/163 P. mirabilis isolates and 1/44 Providencia stuartii isolate (all of which were intermediate for imipenem), carbapenems were active against all ESBL-positive enterobacteria. Susceptibility to other drugs was as follows: 84.7% for amikacin, 84.4% for piperacillin-tazobactam, 48.0% for gentamicin, and 32.8% for ciprofloxacin. Carbapenems appear to be the drug of choice. Amikacin and beta-lactam/beta lactamase inhibitor combinations represent an alternative in non-life-threatening infections. The appearance of ESBL-positive enterobacteria in the community makes it mandatory that family physicians learn how to treat these pathogens. PMID- 16672391 TI - Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies. AB - Antibodies that inhibit replication of Plasmodium falciparum in erythrocytes are thought to be important both in acquired immunity to malaria and as mediators of immunity generated by candidate blood-stage vaccines. However, several constraints have limited the study of these functional antibodies in population studies and vaccine trials. We report the development and optimization of high throughput growth inhibition assays with improved sensitivity that use minimal volumes of test serum. The major inhibitory activity of serum from exposed donors was antibody mediated, but nonspecific inhibitory factors were found in untreated serum. Culture volumes could be effectively reduced to 25 microl to limit amounts of test serum or inhibitors used in assays. Performing inhibition assays over two cycles of parasite replication gave greater sensitivity than single-cycle assays, and a simple two-cycle inhibition assay was developed that yielded highly reproducible results. Determination of parasite growth by flow cytometry was most suitable for high-throughput assays using small culture volumes and was more sensitive than parasite lactate dehydrogenase assays and less prone to error and variation than microscopy. We evaluated and optimized methods to remove antimalarials and nonspecific inhibitory factors from serum that are suitable for use with small volumes of samples that are typically obtained from clinical studies. Both microdialysis and immunoglobulin purification by ammonium sulfate precipitation were effective and practical. These methods should facilitate evaluation of vaccine trials and clinical studies of immunity and are also suitable for testing drugs and other compounds for antimalarial activity. PMID- 16672392 TI - Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis. AB - The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis. PMID- 16672393 TI - Epidemiology, risk factors, and prognosis of Candida parapsilosis bloodstream infections: case-control population-based surveillance study of patients in Barcelona, Spain, from 2002 to 2003. AB - Candida parapsilosis has emerged as an important yeast species causing fungemia. We describe the incidence and epidemiology of C. parapsilosis fungemia. Data from active population-based surveillance in Barcelona, Spain, from January 2002 to December 2003 were analyzed. We focused on 78 episodes of C. parapsilosis fungemia, and we compared them with 175 Candida albicans controls. C. parapsilosis accounted for 23% of all fungemias. The annual incidences were 1 episode per 10(5) patients, 1.2 episodes per 10(4) discharges, and 1.7 episodes per 10(5) patient days. All isolates but one (99%) were fluconazole susceptible. Seventy-two isolates (92%) were inpatient candidemias. Forty-two episodes (51%) were considered catheter-related fungemia, 35 (45%) were considered primary fungemia, and 3 (4%) were considered secondary fungemia. Risk factors for candidemia were vascular catheterization (97%), prior antibiotic therapy (91%), parenteral nutrition (54%), prior surgery (46%), prior immunosuppressive therapy (38%), malignancy (27%), prior antifungal infection (26%), transplant recipient (16%), neutropenia (12%), and prior colonization (11%). Multivariate analysis of the differential characteristics showed that the factors that independently predicted the presence of C. parapsilosis fungemia were neonate patients (odds ratio [OR], 7.5; 95% confidence interval [CI], 2.1 to 26.8; P = 0.002), transplant recipients (OR, 9.2; 95% CI, 1.9 to 43.3; P = 0.005), patients with a history of prior antifungal therapy (OR, 5.4; 95% CI, 1.8 to 15.9; P = 0.002), and patients who received parenteral nutrition (OR, 2.2; 95% CI, 1.09 to 4.6; P = 0.028). The overall mortality rate was lower than that associated with C. albicans candidemia (23% versus 43%; P < 0.01). In summary, C. parapsilosis was responsible for 23% of all candidemias and was more frequent in neonates, in transplant recipients, and in patients who received parenteral nutrition or previous antifungal therapy, mainly fluconazole. The mortality rate was lower than that associated with C. albicans fungemia. PMID- 16672394 TI - Clonal composition of Staphylococcus aureus isolates at a Brazilian university hospital: identification of international circulating lineages. AB - In only a few instances has the clonal composition of Staphylococcus aureus collections that include methicillin-susceptible S. aureus (MSSA) been extensively characterized. In order to investigate the clonal composition of MSSA and methicillin-resistant S. aureus (MRSA) and examine whether the infections diagnosed at our hospital were related to internationally distributed S. aureus lineages, we collected 89 clinical S. aureus isolates from patients at a public university hospital in Rio de Janeiro, Brazil, from September 1999 to June 2000. All S. aureus isolates were genotyped by pulsed-field gel electrophoresis and multilocus restriction fragment typing (MLRFT), and a subset (n = 17) was further characterized by multilocus sequence typing (MLST). The 34 MRSA isolates were additionally characterized by SCCmec typing. The MSSA population (n = 55) was grouped into 18 restriction fragment types (RFTs); of these, five RFTs accounted for 67% (37) of the MSSA isolates. MRSA isolates were clustered into only three RFTs (P = 0.02). The majority of MSSA RFTs were related to sequence type 30 (ST30) (12 isolates, 22%), ST1, ST188, and ST432 (6 isolates, 11% each). The predominant MRSA RFT comprised 31 (91%) of 34 isolates; four randomly selected isolates of this RFT were ST239, the previously described widely disseminated Brazilian clone. However, a fifth isolate belonging to this RFT was the ST644, a new single locus variant of ST239. By applying MLRFT and MLST, we found evidence for a clonal structure in MSSA isolates and detected the dissemination of MSSA clonal complexes 1, 5, 8, 30, and 45. PMID- 16672395 TI - Incidence of Helicobacter felis and the effect of coinfection with Helicobacter pylori on the gastric mucosa in the African population. AB - Helicobacter pylori and Helicobacter felis are two of the Helicobacter spp. that infect humans. H. pylori has been linked to significant gastric pathology. Coinfection with Helicobacter spp. may influence infectious burden, pathogenesis, and antibiotic resistance; however, this has not been studied. The aims of this study were to identify the incidence of H. felis and to analyze the effects of coinfection with both organisms on gastric pathology in a well-characterized South African population. Biopsy samples from the gastric corpora and antra of volunteers (n = 90) were subjected to histological examination and PCR for the identification of H. pylori and H. felis. We further investigated the effect of global strain type on the occurrence of precursor lesions by assigning nucleotide sequences derived from PCR amplification of three genes to global groupings (ancestral Africa1, ancestral Africa2, ancestral Europe, ancestral Asia, and mixed). H. pylori was detected in 75 (83.3%), H. felis in 23 (25.6%), and coinfection in 21 (23.3%) of the volunteers by PCR. H. felis was randomly distributed among adults and children but clustered within families, suggesting intrafamilial transmission. Analysis of histopathology scores revealed no differences in atrophy, activity, and helicobacter density between H. felis positive and H. felis-negative volunteers. H. pylori substrains common to southern Africa showed no differences in inflammation or atrophy scores. The incidences of H. felis and coinfection with H. pylori in the African population are high. H. felis infection, however, does not influence specific gastric pathology in this population. PMID- 16672396 TI - Molecular diversity of rabies viruses associated with bats in Mexico and other countries of the Americas. AB - Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus, Histiotus montanus, and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus. Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species. PMID- 16672397 TI - In vitro release by Aspergillus fumigatus of galactofuranose antigens, 1,3-beta-D glucan, and DNA, surrogate markers used for diagnosis of invasive aspergillosis. AB - Aspergillus markers are becoming increasingly important for the early diagnosis of invasive aspergillosis. The kinetics of release of these surrogate markers, however, is largely unknown. We investigated the release of beta-(1-5) galactofuranosyl (galf) antigens (Platelia Aspergillus), 1,3-beta-D-glucan (BG) (Fungitell), and DNA (PCR) in an in vitro model of Aspergillus fumigatus. The results showed that release is correlated to the growth phase of the fungus, which depends on available nutrients. Whereas galf antigens and BG are released during logarithmic growth, DNA is released only after mycelium breakdown. During early logarithmic growth, galf antigens seem to be released somewhat earlier than BG. Furthermore, galf antigen concentrations of more than 120,000 times the serum cutoff value (0.5 ng/ml) can be measured, while BG concentrations reach a value only 978 times the serum cutoff value (60 pg/ml). During lytical growth, release of galf antigens further increased to a maximum level, which depended on pH. After that, the concentration of galf antigens stayed high (pH 7.4) or decreased to zero within 4 days (pH 5.0). In contrast to galf antigens, BG concentration decreased after 1 day of growth. The decrease of galf components seems to be due to the enzyme beta-galactofuranosidase, which is able to destroy galf epitopes and whose activity fluctuates in the culture filtrates in parallel with galf antigen concentration. Fungal DNA seems to be released only due to autolysis caused by nutrient limitation. In conclusion, several factors clearly influence the release of surrogate markers in vitro. These same factors might also play a role at the infection site of Aspergillus disease in humans. PMID- 16672398 TI - Viable bacteria present within oral squamous cell carcinoma tissue. AB - Despite increasing interest in the possible relationships between bacteria and the different stages of cancer development, the association of bacteria with cancer of the oral cavity has yet to be adequately examined. With that in mind, the primary objective of this study was to identify any bacterial species within oral squamous cell carcinoma tissue using a standard microbiological culture approach. At the time of surgery, a 1-cm3 portion of tissue was harvested from deep within the tumor mass using a fresh blade for each cut. Whenever possible, "superficial" portions from the mucosa overlying the tumor and nontumorous control specimens from at least 5 cm away from the primary tumor site were also obtained. Surface contamination was eliminated by immersion in Betadine and washing with phosphate-buffered saline. Each specimen was aseptically macerated and cultured on nonselective media under both aerobic and anaerobic conditions. Isolates were identified by 16S rRNA gene sequencing. Twenty deep-tissue specimens, 19 with corresponding superficial tissues and 12 with control tissues, were successfully processed. A diversity of bacterial taxa were isolated and identified, including several putatively novel species. Most isolates were found to be saccharolytic and acid-tolerant species. Notably, some species were isolated only from either the tumorous or nontumorous tissue type, indicating a degree of restriction. Successful surface decontamination of the specimens indicates that the bacteria detected were from within the tissue. A diversity of bacterial groups have been isolated from within oral squamous cell carcinoma tissue. The significance of these bacteria within the tumor warrants further study. PMID- 16672399 TI - Multilaboratory comparison of hepatitis C virus viral load assays. AB - We report a multilaboratory evaluation of hepatitis C virus (HCV) viral load assays to determine their linear range, reproducibility, subtype detection, and agreement. A panel of HCV RNA samples ranging in nominal concentration from 1.0 to 7.0 log10 IU/ml was constructed by diluting a clinical specimen (genotype 1b). Replicates of the panel were tested in multiple laboratories using the Abbott TaqMan analyte-specific reagent (Abbott reverse transcription-PCR [RT-PCR]), Roche TaqMan RUO (Roche RT-PCR), Roche Amplicor Monitor HCV 2.0 (Roche Monitor), and Bayer VERSANT HCV RNA 3.0 (Bayer bDNA) assays. Bayer bDNA-negative specimens were tested reflexively using the Bayer VERSANT HCV RNA qualitative assay (Bayer TMA). Abbott RT-PCR and Roche RT-PCR detected all 28 replicates with a concentration of 1.0 log10 IU/ml and were linear to 7.0 log10 IU/ml. Roche Monitor and Bayer bDNA detected 27 out of 28 and 13 out of 28 replicates, respectively, of 3.0 log10 IU/ml. Bayer TMA detected all seven replicates with 1.0 log10 IU/ml. Bayer bDNA was the most reproducible of the four assays. The mean viral load values for panel members in the linear ranges of the assays were within 0.5 log10 for the different tests. Eighty-nine clinical specimens of various genotypes (1 through 4) were tested in the Bayer bDNA, Abbott RT-PCR, and Roche RT-PCR assays. For Abbott RT-PCR, mean viral load values were 0.61 to 0.96 log10 greater than the values for Bayer bDNA assay for samples with genotype 1, 2, or 3 samples and 0.08 log10 greater for genotype 4 specimens. The Roche RT-PCR assay gave mean viral load values that were 0.28 to 0.82 log10 greater than those obtained with the Bayer bDNA assay for genotype 1, 2, and 3 samples. However, for genotype 4 samples the mean viral load value obtained with the Roche RT-PCR assay was, on average, 0.15 log10 lower than that of the Bayer bDNA. Based on these data, we conclude that the sensitivity and linear range of the Abbott and Roche RT-PCR assays enable them to be used for HCV diagnostics and therapeutic monitoring. However, the differences in the viral load values obtained with the different assays underscore the importance of using one assay when monitoring response to therapy. PMID- 16672400 TI - Comparison between the Hybrid Capture II Test and an SPF1/GP6+ PCR-based assay for detection of human papillomavirus DNA in cervical swab samples. AB - We compared the efficacy of human papillomavirus (HPV) DNA detection between a PCR-based genechip (Easychip HPV Blot [hereafter referred to as HPV Blot]; King Car, Taiwan) method and Hybrid Capture II (HCII; Digene, Gaithersburg, MD) in women with previous normal (n = 146) or abnormal (> or =atypical squamous cells of undetermined significance [ASCUS] [n = 208]) cytology. A total of 354 cervical swab samples were collected for HPV DNA assay by both HCII and SPF1/GP6+ PCR followed by HPV Blot tests. Colposcopy-directed biopsy was performed if clinically indicated. Of the 354 samples, HPV-positive rates by these two methods (HCII and HPV Blot) were 12.6% and 18.2% in 143 normal samples, 36.2% and 45.7% in 105 ASCUS samples, 57.4% and 57.4% in 94 low-grade squamous intraepithelial lesion samples, and 83.3% and 75.0% in 12 high-grade squamous intraepithelial lesion samples, respectively. The concordance of HPV Blot and HCII was 80.8% (286/354), and the agreement between the methods (kappa value, 0.68) was substantial. Discrepancies were further investigated by at least one of the following three methods: direct sequencing, type-specific PCR, and HPV Blot genotyping of cervical biopsy tissue. In the 15 HCII-positive samples, HPV Blot detected only non-HCII HPV genotypes; results of further verification methods were consistent with the latter test in the 15 samples. Of the 20 samples with HCII-negative and HPV Blot-positive results, 18 were found to contain the 13 HCII high-risk genotypes by verification methods. In only 16.7% (3/18) of the HCII positive but HPV Blot-negative samples, further studies detected the 13 HCII genotypes. We conclude that HPV Blot seemed comparable to HCII for detection of HPV DNA in cervical swab samples. PMID- 16672401 TI - Clinical significance of azole antifungal drug cross-resistance in Candida glabrata. AB - Candida glabrata, which can become resistant to fluconazole, is a common cause of bloodstream infection. This study was performed to determine the significance of cross-resistance to new azole drugs among C. glabrata isolates recovered as a cause of infection in azole-treated hematopoietic stem cell transplant (HSCT) recipients. Seven cases of invasive candidiasis caused by C. glabrata occurred in HSCT recipients who were receiving azole therapy between January 2000 and December 2004 in our institution. Case characteristics were ascertained. Sequential colonizing and invasive isolates were examined to determine susceptibilities to fluconazole, itraconazole, and voriconazole, and molecular relatedness by restriction fragment length polymorphism (RFLP) analysis. Twenty three C. glabrata isolates were recovered from 4 patients who developed candidemia while receiving fluconazole and three patients who developed candidemia while receiving voriconazole. The mode MICs of fluconazole, itraconazole, and voriconazole for these isolates were > or =64 microg/ml (range, 4 to > or =64 microg/ml), 2 microg/ml (range, 0.25 to > or =16 microg/ml), and 1 microg/ml (range, 0.03 to > or =16 microg/ml), respectively. Kendall tau b correlation coefficients demonstrated significant associations between the MICs of voriconazole with fluconazole (P = 0.005) and itraconazole (P = 0.008). Colonizing and invasive isolates exhibiting variable susceptibilities had similar RFLP patterns. These observations suggest that C. glabrata exhibits considerable clinically significant cross-resistance between older azole drugs (fluconazole and itraconazole) and voriconazole. Caution is advised when considering voriconazole therapy for C. glabrata candidemia that occurs in patients with extensive prior azole drug exposure. PMID- 16672402 TI - Multilaboratory evaluation of disk diffusion antimicrobial susceptibility testing of Neisseria meningitidis isolates. AB - In 2005, the Clinical and Laboratory Standards Institute published MIC interpretive criteria for 13 antimicrobial agents used for either therapy or prophylaxis of Neisseria meningitidis infections. The MIC method includes the use of lysed horse blood-supplemented Mueller-Hinton broth with incubation in 5% CO2 for 20 to 24 h. Since some clinical laboratories might prefer the option of disk diffusion testing for infrequently encountered isolates a multicenter collaborative study was conducted to evaluate the reproducibility of a disk diffusion method for testing isolates of N. meningitidis. Interpretive criteria were developed for 12 antimicrobial agents. Four laboratories tested a common collection of 50 meningococcal strains and then tested 25 unique isolates per laboratory. Isolates were tested using Mueller-Hinton sheep blood agar plates incubated for 20 to 24 h in 5% CO2; they were also tested by the reference broth microdilution method in parallel. Pooling of the MIC and disk diffusion data from the common and unique isolates provided a sufficient sample size to develop susceptible, intermediate, and resistant zone diameter interpretive criteria using the error rate-bounded method for the following agents: chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and rifampin. Due to the lack of resistant strains at the present time, "susceptible only" interpretive criteria were proposed for cefotaxime, ceftriaxone, meropenem, azithromycin, and minocycline. The numbers of minor interpretive errors with penicillin and ampicillin disk tests were unacceptably high and precluded recommended testing of those agents by the disk method. However, amdinocillin, an agent that preferentially binds to the altered penicillin binding protein responsible for diminished penicillin susceptibility, has potential utility as a surrogate screening reagent for ampicillin resistance. A disk diffusion breakpoint was derived for nalidixic acid to serve as a surrogate marker for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Disk diffusion testing with meningococci can be performed in a reproducible manner with several antimicrobial agents and represents a practical and cost-effective option for testing sporadic clinical isolates or for surveillance purposes by resource limited laboratories. PMID- 16672403 TI - Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer. AB - Integration of human papillomavirus type 16 (HPV-16) into the host DNA has been proposed as a potential marker of cervical neoplastic progression. In this study, a quantitative real-time PCR (qRT-PCR) was used to examine the physical status of HPV-16 in 126 cervical carcinoma in situ and 92 invasive cervical cancers. Based on criteria applied to results from this qRT-PCR assay, HPV-16 was characterized in carcinoma in situ cases as episomal (61.9%), mixed (i.e., episomal and integrated; 29.4%), and integrated (8.7%) forms. In invasive cervical cancer samples, HPV-16 was similarly characterized as episomal (39.1%), mixed (45.7%), and integrated (15.2%) forms. The difference in the frequency of integrated or episomal status estimated for carcinoma in situ and invasive cervical cancer cases was statistically significant (P = 0.003). Extensive mapping analysis of HPV-16 E1 and E2 genes in 37 selected tumors demonstrated deletions in both E1 and E2 genes with the maximum number of losses (78.4%) observed within the HPV-16 E2 hinge region. Specifically, deletions within the E2 hinge region were detected most often between nucleotides (nt) 3243 and 3539. The capacity to detect low frequency HPV-16 integration events was highly limited due to the common presence and abundance of HPV episomal forms. HPV-16 E2 expressed from intact episomes may act in trans to regulate integrated genome expression of E6 and E7. PMID- 16672404 TI - Spoligotyping of Mycobacterium tuberculosis isolates from Pakistan reveals predominance of Central Asian Strain 1 and Beijing isolates. AB - The estimated incidence of tuberculosis in Pakistan is 181 per 100,000; however, there is limited information on Mycobacterium tuberculosis genotypes circulating in the country. We studied 314 M. tuberculosis clinical isolates; of these, 197 (63%) isolates grouped into 22 different clusters, while 119 (37%) had unique spoligotypes. Eighty-nine percent of the isolates were pulmonary (Pul), and 11% were extrapulmonary (E-Pul). We identified Central Asian Strain (CAS), Beijing, T1, Latin American-Mediterranean, and East African-Indian genogroups. Beijing strains, reportedly the most prevalent spoligotype worldwide, constituted 6% of our strain population. The CAS1 strain comprised 121 (39%) of the study isolates. No difference was observed between clustered isolates from cases of Pul and E-Pul tuberculosis. However, E-Pul isolates included a greater number of unique spoligotypes than Pul isolates (P = 0.005). The overall percentage of drug resistance was 54%, and that of MDR strains was 40%. While CAS1 strains were not associated with drug resistance, the relative risk of MDR was significant in Beijing strains compared to the non-Beijing groups (95% confidence interval, 1.2 to 8.9). The fact that the predominant strain, CAS1, is not associated with drug resistance is encouraging and suggests that an effective tuberculosis control program should be able to limit the high incidence of disease in this region. PMID- 16672405 TI - Evaluation of the GenoType Mycobacterium Assay for identification of mycobacterial species from cultures. AB - A new commercially available DNA strip assay (GenoType Mycobacterium CM/AS; Hain Lifescience, Nehren, Germany) was evaluated for the ability to differentiate mycobacterial species. The test is based on a PCR technique targeting a 23S rRNA gene region, followed by reverse hybridization and line probe technology. The GenoType CM is capable of identifying 23, the GenoType AS a further 14, species either alone or in combination with one or more species. Both tests were evaluated with 156 mycobacterial strains composed of 61 validly published species including different subspecies, 6 not validly published species, and 3 strains other than mycobacterial species. All strains were precharacterized by sequencing of the 5' region of the 16S rRNA gene and biochemical tests. In total, results for 151 strains were interpretable. Concordant results were obtained for 137 (92.6%) of 148 mycobacterial strains with the CM assay and 133 (89.9%) of 148 mycobacterial strains with the AS assay, and all three non-Mycobacterium species were identified. PMID- 16672407 TI - International surveillance of Candida spp. and Aspergillus spp.: report from the SENTRY Antimicrobial Surveillance Program (2003). AB - During 2003, a total of 1,397 Candida isolates, 73 Aspergillus isolates, 53 Cryptococcus neoformans isolates, and 25 other fungal isolates from infected, normally sterile, body sites in patients hospitalized in North America, Europe, and Latin America were studied as a component of the longitudinal SENTRY Antimicrobial Surveillance Program. The MICs for seven antifungal agents were determined in a central laboratory (JMI Laboratories, North Liberty, IA) using testing methods promulgated by the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards). The rank order of Candida spp. occurrence was as follows: C. albicans (48.7%), C. parapsilosis (17.3%), C. glabrata (17.2%), C. tropicalis (10.9%), C. krusei (1.9%), and other Candida spp. (4.0%). C. albicans accounted for 51.5, 47.8, and 36.5% of candidal infections in North America, Europe, and Latin America, respectively. Ravuconazole, voriconazole, and fluconazole were highly active against C. albicans, C. parapsilosis, and C. tropicalis, with both former agents being more potent (MIC at which 90% of the isolates tested are inhibited [MIC90] of < or =0.008 to 0.12 microg/ml) than fluconazole (MIC90 of 0.5 to 2 microg/ml). C. glabrata isolates were less susceptible to these agents, with MIC90s of 1, 1, and 64 microg/ml, respectively. Ravuconazole and voriconazole were the most active agents tested against C. krusei (MIC90 of 0.5 microg/ml). Among Aspergillus spp., A. fumigatus was the most commonly (71.2% of isolates) recovered species; 96.2, 96.2, 84.6, and 11.5% of strains were inhibited by < or =1 microg/ml of ravuconazole, voriconazole, itraconazole, and amphotericin B, respectively. Of the antifungal agents tested, ravuconazole and voriconazole displayed the greatest spectrum of activity against pathogenic Candida and Aspergillus spp., regardless of geographic origin. These results extend upon previous findings from SENTRY Program reports (1997 to 2000), further characterizing species composition as seen in local clinical practice and demonstrating the potent activity of selected, newer triazole antifungal agents. PMID- 16672406 TI - Pulsed-field gel electrophoresis typing of Escherichia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women. AB - The primary infecting Escherichia coli strains from 156 women with community acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE). In the pivmecillinam treatment group PFGE showed that among patients having a negative urine culture at the first follow-up 77% (46/60) had a relapse with the primary infecting E. coli strain and 23% (14/60) had reinfection with a new E. coli strain at the second follow-up. Among patients having E. coli at the first follow-up PFGE showed that 80% (32/40) had persistence with the primary infecting E. coli strain, 15% (6/40) had reinfection with a new E. coli strain, and 5% (2/40) had different E. coli strains at the two follow-up visits (one had reinfection followed by relapse, and the other had persistence followed by reinfection). In the placebo group the majority had E. coli at the first follow-up. PFGE showed that among these patients 96% (50/52) had persistence with the primary infecting E. coli strain and 4% (2/50) had different E. coli strains at the two follow-up visits (both had persistence followed by reinfection). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment, constituting a reservoir for recurrent UTI. PMID- 16672408 TI - Comparative evaluation of the performance of the Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for measurement of HIV-1 plasma viral load following automated specimen preparation. AB - The performance of the new Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for HIV-1 RNA load determination in plasma was compared to that of the Abbott LCx HIV-1 RNA quantitative assay following automated RNA isolation by the Abbott m1000 extractor. The measured viral loads of 89 clinical specimens differed by mean 0.19 log10 copies/ml (95% confidence interval, 0.12 to 0.26 log10 copies/ml). Although the difference in viral load determinations was positively skewed in favor of the LCx assay, it did not reach statistical significance (P = 0.42). Results were linearly associated (R2 = 0.94) and strongly correlated (R = 0.96). Good performance was observed with HIV-1 subtypes other than B and circulating recombinant forms, although results obtained with two subtype G specimens and one H specimen showed a more substantial difference. PMID- 16672409 TI - Evaluation of a novel highly sensitive, broad-spectrum PCR-reverse hybridization assay for detection and identification of beta-papillomavirus DNA. AB - Human papillomavirus can be detected by amplification of viral DNA. A novel one step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material. PMID- 16672410 TI - G8 rotavirus strains isolated in the Democratic Republic of Congo belong to the DS-1-like genogroup. AB - Several G8P[6] and G8P[8] rotavirus strains were isolated from hospitalized patients in the Democratic Republic of Congo in 2003. To investigate their overall genomic relatedness and to determine to which genogroup they belonged, the complete genomes of strains DRC88 (G8P[8]) and DRC86 (G8P[6]) were determined. Genomic comparison of these two African G8 strains revealed that 10 out of their 11 gene segments, except for VP4, were nearly identical (>98.9% identical at the nucleotide level), suggesting that this rare G8P[8] rotavirus strain originated recently from a reassortment between a common G8P[6] strain and a strain with a P[8] specificity. A very close evolutionary relationship between 9 out of the 11 gene segments of DRC88 and DRC86 and rotavirus strains belonging to the DS-1-like (G2P[4]) "genogroup" was found, and several possible reassortment events preceding the occurrence of G8P[8] and G8P[6] human rotaviruses were hypothesized. Since the genes of G2P[4] rotavirus strains are very well adapted to infect humans, the acquirement of a new VP7 (G8) gene, and especially the replacement of P[6] (believed to be of animal origin) by P[8] (most common in human rotaviruses), might make DRC88-like rotaviruses very well equipped to become a predominant human rotavirus strain and an important pathogen on the African continent and the rest of the world. These findings have important implications for rotavirus vaccine development and highlight that typing of new rotavirus strains by merely sequencing their VP7 and VP4 genes provides us with only the tip of the iceberg regarding rotavirus diversity. PMID- 16672412 TI - Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens. AB - Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numbers of tdh-positive V. parahaemolyticus organisms, and their enrichment cultures were tested with TDH-ICA. The results of detection of TDH in the enrichment cultures by TDH-ICA were in accord with the results of recovery of the spiked V. parahaemolyticus organisms from the enrichment cultures by plating onto thiosulfate-citrate-bile salts-sucrose agar. When enrichment cultures of 217 stool specimens from patients with diarrhea were tested with TDH ICA, the TDH-ICA results showed 100% sensitivity and specificity compared to the results of isolation of V. parahaemolyticus from the stool specimens by a conventional bacterial culture test. Since TDH-ICA was able to detect TDH in a fecal enrichment culture within 10 min, TDH-ICA testing of a fecal enrichment culture could be completed rapidly and easily within approximately 16 h, including incubation time for the fecal enrichment culture. These results indicate that TDH-ICA is a rapid, simple, and sensitive TDH detection method and that TDH-ICA testing of a fecal enrichment culture is useful as an adjunct to facilitate the early diagnosis of V. parahaemolyticus diarrhea. PMID- 16672413 TI - Rapid detection of Brucella spp. in blood cultures by fluorescence in situ hybridization. AB - Brucellosis is a severe systemic disease in humans. We describe a new 16S rRNA based fluorescence in situ hybridization assay that facilitates rapid and specific detection of all human pathogenic species of Brucella and that can be applied directly to positive blood cultures. PMID- 16672411 TI - Multilocus sequence typing reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract. AB - Candida albicans is a human commensal that is also responsible for superficial and systemic infections. Little is known about the carriage of C. albicans in the digestive tract and the genome dynamics that occur during commensalisms of this diploid species. The aim of this study was to evaluate the prevalence, diversity, and genetic relationships among C. albicans isolates recovered during natural colonization of the digestive tract of humans, with emphasis on Crohn's disease patients who produce anti-yeast antibodies and may have altered Candida sp. carriage. Candida sp. isolates were recovered from 234 subjects within 25 families with multiple cases of Crohn's disease and 10 control families, sampled at the oral and fecal sites. Prevalences of Candida sp. and C. albicans carriage were 53.4% and 46.5%, respectively, indicating frequent commensal carriage. No differences in prevalence of carriage could be observed between Crohn's disease patients and healthy subjects. Multilocus sequence typing (MLST) of C. albicans isolates revealed frequent colonization of a subject or several members of the same family by genetically indistinguishable or genetically close isolates. These latter isolates differed by loss-of-heterozygosity events at one or several of the MLST loci. These loss-of-heterozygosity events could be due to either chromosome loss followed by duplication or large mitotic recombination events between complementary chromosomes. This study was the first to jointly assess commensal carriage of C. albicans, intrafamilial transmission, and microevolution. The high frequency of each of these events suggests that the digestive tract provides an important and natural niche for microevolutions of diploid C. albicans through the loss of heterozygosity. PMID- 16672414 TI - Sensitivity of the Multispot HIV-1/HIV-2 rapid test using samples from human immunodeficiency virus type 1-positive individuals with various levels of exposure to highly active antiretroviral therapy. AB - The Multispot HIV-1/HIV-2 rapid test detects human immunodeficiency virus type 1 (HIV-1) gp41 antibodies, which can wane over time in some HIV-1-infected populations, resulting in false-negative screening results. Multispot sensitivity was 100% using 248 sera from one such population, and it correctly identified serostatus in individuals who previously tested false negative with rapid testing. PMID- 16672415 TI - Prospective study of the value of quantitative culture of organisms from blood collected through central venous catheters in differentiating between contamination and bloodstream infection. AB - Collection of blood through a central venous catheter for the diagnosis of bacteremia is a debated topic. Quantitative cultures of organisms from blood collected through central venous catheters were found to be highly sensitive, specific, and predictive of bacteremia, especially when a cutoff point of 15 colonies of skin organisms was used. PMID- 16672416 TI - Rapid differentiation between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis within beta-hemolytic group C and G streptococci by PCR. AB - Based on a pair of primers developed initially for differentiating the anginosus group from other viridans streptococci, the PCR reported here can also differentiate between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis among beta-hemolytic group C and G streptococci. The resulting 742-bp PCR product was specific for members of the anginosus group, although a smaller, nonspecific product (361 bp) was generated from S. dysgalactiae subsp. equisimilis. Restriction digestion of the amplicon with XbaI and BsmI further differentiated Streptococcus anginosus from Streptococcus constellatus within the anginosus group. PMID- 16672417 TI - Polymorphism of the cell wall-anchoring domain of the autolysin-adhesin AtlE and its relationship to sequence type, as revealed by multilocus sequence typing of invasive and commensal Staphylococcus epidermidis strains. AB - We sequenced the adhesin-cell wall-anchoring domain of the atlE gene of 49 invasive and commensal Staphylococcus epidermidis strains. We identified 22 alleles, which could be separated into two main groups: group 1 (alleles 1 and 6 to 16, 32/49 strains) and group 2 (alleles 2 to 5 and 17 to 22, 17/49 strains). Allele 1 (the type strain sequence) was by far the most prevalent (21 of 49 strains). Multilocus sequence typing showed a clear relationship between the atlE allele and the sequence type (ST), with the "nosocomial" ST27 clone and closely related STs expressing group 1 alleles. PMID- 16672418 TI - Cytolethal distending toxin in Escherichia coli O157:H7: spectrum of conservation, structure, and endothelial toxicity. AB - We identified the cytolethal distending toxin V (CDT-V) gene cluster in 19 (4.9%) of 391 enterohemorrhagic Escherichia coli O157:H7. cdt-V+ strains belonged to five phage types (PTs) and were most frequent within PTs 14 and 34. CDT-V was expressed in all but two cdt-V+ strains and was lethal to cultured endothelial cells. Subtyping schemes should include cdt-V as a marker to differentiate E. coli O157:H7 even within the same phage type. PMID- 16672419 TI - Polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation. AB - We analyzed 90 polymorphisms in 17 genes related to immune function for association with human cytomegalovirus (HCMV) reactivation and disease in patients after allogeneic stem cell transplantation. We found relevant markers (i) in CCR5 and IL-10 genes conferring a higher risk for the development of HCMV disease and (ii) in the MCP1 gene associated with HCMV reactivation. Testing of high-risk patients for the presence of these single-nucleotide polymorphisms might be useful for individualizing antiviral prophylaxis. PMID- 16672420 TI - Emergence of Cryptococcus gattii in a novel environment provides clues to its incubation period. AB - Cryptococcus gattii emerged in 1999 in a distinct ecologic environment on Vancouver Island, Canada. Estimates of incubation period were derived from an analysis of travelers with discreet exposures to this region. Individual case incubation periods ranged from 2 to 11 months, with a median of 6 to 7 months. PMID- 16672421 TI - Evaluation of a commercial latex agglutination assay for serological diagnosis of leptospirosis. AB - Leptospirosis is a febrile zoonosis of worldwide distribution. A latex agglutination assay was evaluated in two studies, the first using a panel of well characterized sera from patients with leptospirosis and from patients with other disease states and the second, a prospective hospital-based study, evaluating sera from 186 consecutive patients admitted to hospital with acute febrile illness. The confirmed leptospirosis serum panel included paired acute- and convalescent-phase specimens from 40 cases, of which 34 gave positive latex tests (case sensitivity, 85%; 95% confidence interval [95% CI], 70 to 94%). The other diseases represented in the panel of 112 specimens from nonleptospirosis patients included autoimmune diseases, brucellosis, dengue, melioidosis, malaria, syphilis, toxoplasmosis, viral hepatitis, and a number of other viral infections. The specificity of latex agglutination using this panel was 81% (95% CI, 73 to 87%). Among the patients with acute febrile illness, there were 25 cases of leptospirosis and 161 patients with other diagnoses. The sensitivity and specificity of latex agglutination in this group were 88% (95% CI, 72 to 97%) and 98% (95% CI, 95 to 100%), respectively. In this evaluation, the two distinct groups of specimens gave similar results for sensitivity, but specificity was different in each study. The sensitivity and specificity observed for the hospital study were similar to those obtained in evaluations of other rapid tests in the same population. The results of this study suggest that multiple evaluations of new diagnostic assays should be performed, because performance characteristics may vary in different populations. PMID- 16672422 TI - Use of an acute seroconversion panel to evaluate a third-generation enzyme-linked immunoassay for detection of human immunodeficiency virus-specific antibodies relative to multiple other assays. AB - A human immunodeficiency virus type 1 (HIV-1)/HIV-2 antibody screening assay, the Genetic Systems HIV-1/HIV-2 PLUS O EIA, was compared to several established screening or confirmatory tests using an acute HIV seroconversion panel. The HIV 1/HIV-2 PLUS O EIA showed an improved sensitivity over all tested antibody screening methods, and detected antibody in 7 of 19 specimens found to be negative by a first-generation EIA but positive for the presence of HIV RNA. PMID- 16672423 TI - Novel approach to designing primers for identification and distinction of the human pathogenic fungi Coccidioides immitis and Coccidioides posadasii by PCR amplification. AB - We developed a pair of primers that specifically identifies Coccidioides species, etiologic agents of the human fungal disease coccidioidomycosis. These primers could be used for distinguishing Coccidioides immitis and Coccidioides posadasii by simply comparing the amplicon sizes on an agarose gel. PMID- 16672424 TI - Isolation, characterization, and epidemiological assessment of shiga toxin producing Escherichia coli O84 isolates from New Zealand. AB - Shiga toxin-producing Escherichia coli O84 isolates (n = 22) were examined using culture- and molecularly based methods in order to compare their phenotypic and genotypic characteristics. These analyses directly linked Shiga toxin-producing Escherichia coli O84 isolates from cattle and sheep with human isolates indicating that New Zealand livestock may be a reservoir of infection. PMID- 16672425 TI - Lack of association between the presence of the pVir plasmid and bloody diarrhea in Campylobacter jejuni enteritis. AB - The main mechanisms by which Campylobacter jejuni causes diarrhea are unknown. In contrast to a recent communication, we report here the absence of an association with the plasmid pVir in patients infected with C. jejuni who developed bloody diarrhea in The Netherlands, and we suggest a role for other virulence determinants. PMID- 16672426 TI - Evaluation of the Q score and Q234 systems for cost-effective and clinically relevant interpretation of wound cultures. AB - The Q score and Q234 systems were compared to our current protocol for interpreting wound cultures. The Q score and Q234 systems were more cost effective than our current method, with the Q234 system being considered the most useful protocol for implementation by both the laboratory and our clinicians. PMID- 16672427 TI - Increasing incidence of severe Epstein-Barr virus-related infectious mononucleosis: surveillance study. AB - Older patients are more susceptible to severe Epstein-Barr virus (EBV)-related infectious mononucleosis (IM). This condition may increase in industrialized countries where primary EBV infection occurs later in life. Between 1990 and 2004, 38 patients were admitted to our department with EBV-related IM. Two patients died. The annual incidence increased significantly (r = 0.623; P = 0.013). PMID- 16672428 TI - Presence of a novel DNA methylation enzyme in methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis. AB - Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in the genus Staphylococcus with the recognition sequence CCNGG. PMID- 16672429 TI - Plasma and liver hepatitis C virus variability in patients coinfected with human immunodeficiency virus. AB - Liver and plasma hepatitis C virus (HCV) variability was compared by E2 cloning and sequencing in three patients coinfected with HCV and human immunodeficiency virus (HIV) before and after interferon treatment and in three patients solely infected with HCV. The plasma and liver samples contained unique sequences. In the patients coinfected with HIV, accumulated random mutations produced mostly nonsynonymous substitutions in contrast to the reduced HCV genetic variability seen after treatment. PMID- 16672430 TI - Occurrence of the enterotoxin gene cluster and the toxic shock syndrome toxin 1 gene among clinical isolates of methicillin-resistant Staphylococcus aureus is related to clonal type and agr group. AB - Analysis of 177 methicillin-resistant Staphylococcus aureus (MRSA) strains for the presence of egc and tst revealed that 60 strains carried at least one of the tested genes. MRSA strains were classified by pulsed-field gel electrophoresis into four clones belonging to agr groups I and III. Toxin genotypes were related by clonal type and agr group. PMID- 16672431 TI - PCR amplification of the IS6110 insertion element of Mycobacterium tuberculosis in fecal samples from patients with intestinal tuberculosis. AB - PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis in fecal samples was evaluated in the diagnosis of intestinal tuberculosis (ITB). The numbers of samples that tested positive by PCR with SalI digestion were 16/18 untreated-ITB samples, 0/8 treated-ITB samples, 12/14 smear-positive pulmonary tuberculosis samples, and 0/30 control samples. The sensitivity, specificity, positive predictive value, and negative predictive value of fecal PCR were 88.8%, 100%, 100%, and 93.7%, respectively. PMID- 16672432 TI - Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes. AB - We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well characterized isolates, representing all 90 serotypes, and 268 "unknowns." The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera. PMID- 16672433 TI - Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay. AB - The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID- 16672434 TI - Application of minimal sequence quality values prevents misidentification of the blaSHV type in single bacterial isolates carrying different SHV extended-spectrum beta-lactamase genes. AB - Nucleotide sequencing is the standard molecular method for determination of the beta-lactamase gene present in an isolate. Using minimal sequence quality values prevents misidentification of bla(SHV) genes, as illustrated by three strains of three different species that each contained two different bla(SHV) alleles, SHV-2 and SHV-12. PMID- 16672435 TI - Development and evaluation of a loop-mediated isothermal amplification method for rapid diagnosis of Bordetella pertussis infection. AB - We developed a loop-mediated isothermal amplification (LAMP) method to detect Bordetella pertussis infection. This LAMP assay detected B. pertussis with high sensitivity, but not other Bordetella species. Among nasopharyngeal swab samples from subjects with suspected pertussis, LAMP results showed a high level of agreement with results of conventional PCR. This method is a rapid, sensitive, and specific method for diagnosis of B. pertussis infection even in clinical laboratories with no specific equipment. PMID- 16672436 TI - Early diagnosis of Acanthamoeba infection during routine cytological examination of cerebrospinal fluid. AB - Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection. PMID- 16672437 TI - Native valve endocarditis due to Gordonia polyisoprenivorans: case report and review of literature of bloodstream infections caused by Gordonia species. AB - We report the first case of endocarditis caused by Gordonia polyisoprenivorans and concisely review the English literature regarding bloodstream infections caused by Gordonia species. PMID- 16672438 TI - Campylobacter curvus-associated hepatic abscesses: a case report. AB - Campylobacter curvus was isolated from blood cultures of a patient with liver abscesses. Bacterial identification involved Gram staining, biochemical analysis, gas-liquid chromatography, and 16S rRNA sequencing. The difficulty in isolation, identification, and growth of the species confirms previous work that these organisms may be overlooked by conventional detection methods. PMID- 16672439 TI - Spondylodiscitis caused by Candida krusei: case report and susceptibility patterns. AB - A 62-year-old man with amphotericin B-resistant Candida krusei spondylodiscitis, following an episode of candidemia caused by the same strain, was successfully treated with caspofungin plus voriconazole. Amphotericin B fungicidal concentrations were better predictors of the clinical outcome than were MICs. This is the first case of C. krusei spondylodiscitis reported in the literature. PMID- 16672440 TI - Central venous catheter colonization by linezolid-resistant, vancomycin susceptible Enterococcus faecalis. AB - Resistance to linezolid is rare in clinical isolates of Enterococcus faecalis. A strain resistant to this antimicrobial but susceptible to vancomycin was found to cause central venous catheter colonization in a patient who never received linezolid. PMID- 16672441 TI - Listeriosis due to infection with a catalase-negative strain of Listeria monocytogenes. AB - A strain of Listeria monocytogenes recovered from blood and cerebrospinal fluid had no detectable catalase activity, a characteristic used for primary identification. The sporadic occurrence of pathogenic catalase-negative strains highlights the need for a reconsideration of diagnostic criteria and questions the role of catalase in the pathogenesis of listeria infection. PMID- 16672442 TI - Detection of CTX-M-1, CTX-M-15, and CTX-M-2 in clinical isolates of Enterobacteriaceae in Bogota, Colombia. PMID- 16672443 TI - Verification of antimicrobial susceptibility testing of Mycobacterium tuberculosis. PMID- 16672444 TI - High prevalence of teicoplanin resistance among Staphylococcus epidermidis strains in a 5-year retrospective study. PMID- 16672446 TI - Elimination of Escherichia coli O157:H7 from fermented dry sausages at an organoleptically acceptable level of microencapsulated allyl isothiocyanate. AB - Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log(10) CFU/g Pediococcus pentosaceus and 6 log(10) CFU/g Staphylococcus carnosus) and a five strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log(10) CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at < or =26 degrees C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13 degrees C for at least 25 days. The water activity (a(w)), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a(w) from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log(10) CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log(10) CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists. PMID- 16672445 TI - Diversity and distribution of marine microbial eukaryotes in the Arctic Ocean and adjacent seas. AB - We analyzed microbial eukaryote diversity in perennially cold arctic marine waters by using 18S rRNA gene clone libraries. Samples were collected during concurrent oceanographic missions to opposite sides of the Arctic Ocean Basin and encompassed five distinct water masses. Two deep water Arctic Ocean sites and the convergence of the Greenland, Norwegian, and Barents Seas were sampled from 28 August to 2 September 2002. An additional sample was obtained from the Beaufort Sea (Canada) in early October 2002. The ribotypes were diverse, with different communities among sites and between the upper mixed layer and just below the halocline. Eukaryotes from the remote Canada Basin contained new phylotypes belonging to the radiolarian orders Acantharea, Polycystinea, and Taxopodida. A novel group within the photosynthetic stramenopiles was also identified. One sample closest to the interior of the Canada Basin yielded only four major taxa, and all but two of the sequences recovered belonged to the polar diatom Fragilariopsis and a radiolarian. Overall, 42% of the sequences were <98% similar to any sequences in GenBank. Moreover, 15% of these were <95% similar to previously recovered sequences, which is indicative of endemic or undersampled taxa in the North Polar environment. The cold, stable Arctic Ocean is a threatened environment, and climate change could result in significant loss of global microbial biodiversity. PMID- 16672447 TI - Diazotrophic burkholderia species associated with field-grown maize and sugarcane. AB - Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N(2)-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N(2)-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N(2)-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed. PMID- 16672448 TI - Horizontal gene transfer of PIB-type ATPases among bacteria isolated from radionuclide- and metal-contaminated subsurface soils. AB - Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with co-occurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G+C- and low-G+C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb resistant (Pb(r)) isolates was amplified with PCR primers specific for P(IB)-type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P(IB)-type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb(r) P(IB)-type ATPase positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO(2)(2+)) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P(IB)-type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC. PMID- 16672449 TI - Pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas stutzeri KC reduces and precipitates selenium and tellurium oxyanions. AB - The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H(2)S, forming zero valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids. PMID- 16672450 TI - Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei. AB - Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae, respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli. PMID- 16672451 TI - Genetic and biochemical evidence for the involvement of a molybdenum-dependent enzyme in one of the selenite reduction pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106. AB - Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity. PMID- 16672452 TI - Isolation and sequencing of a temperate transducing phage for Pasteurella multocida. AB - A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae, presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA(Leu). By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation. PMID- 16672453 TI - Wolbachia infections in the Cimicidae: museum specimens as an untapped resource for endosymbiont surveys. AB - Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality. PMID- 16672454 TI - Bacillus anthracis multiplication, persistence, and genetic exchange in the rhizosphere of grass plants. AB - Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat resistant spores. Co-inoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species. PMID- 16672455 TI - Effect of nutrient periodicity on microbial community dynamics. AB - When microbes are subjected to temporal changes in nutrient availability, growth rate and substrate affinity can contribute to competitive fitness and thereby affect microbial community structure. This hypothesis was tested using planktonic bacterial communities exposed to nutrient additions at 1-, 3-, 7-, or 14-day intervals. Growth rates after nutrient addition were inversely proportional to the pulse interval and declined from 0.5 h(-1) to 0.15 h(-1) as the pulse interval increased from 1 to 14 days. The dynamics of community structure were monitored by 16S rRNA gene PCR-denaturing gradient gel electrophoresis. At pulse intervals of more than 1 day, the community composition continued to change over 130 days. Although replicate systems exposed to the same pulse interval were physiologically similar, their community compositions could exhibit as much dissimilarity (Dice similarity coefficients of <0.5) as did systems operated at different intervals. Bacteria were cultivated from the systems to determine if the physiological characteristics of individual members were consistent with the measured performance of the systems. The isolates fell into three bacterial divisions, Bacteroidetes, Proteobacteria, and Actinobacteria. In agreement with community results, bacteria isolated from systems pulsed every day with nutrients had higher growth rates and ectoaminopeptidase specific activities than isolates from systems pulsed every 14 days. However, the latter isolates did not survive starvation longer than those provided with nutrients every day. The present study demonstrates the dynamic nature of microbial communities exposed to even simple and regular environmental discontinuities when a substantial pool of species that can catabolize the limiting substrate is present. PMID- 16672456 TI - Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes. AB - Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor. PMID- 16672457 TI - Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models. AB - The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30 degrees C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls. PMID- 16672458 TI - Plasmid pCAR3 contains multiple gene sets involved in the conversion of carbazole to anthranilate. AB - The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed. PMID- 16672460 TI - Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp. AB - A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N(2) (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N(2) fixation when combined nitrogen (NO(3)(-), NH(4)(+)) is available and the increased rate of N(2) fixation when combined nitrogen is insufficient to cover the demand. The daily N(2) fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics. PMID- 16672459 TI - Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation. AB - Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 x 10(-1) and 3 x 10(-3) per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid. PMID- 16672461 TI - Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid. AB - Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+) lactic acid at 50 degrees C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [(13)C]lactate accounted for more than 90% of the total (13)C-labeled products from [(13)C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals. PMID- 16672462 TI - Metal reduction and iron biomineralization by a psychrotolerant Fe(III)-reducing bacterium, Shewanella sp. strain PV-4. AB - A marine psychrotolerant, dissimilatory Fe(III)-reducing bacterium, Shewanella sp. strain PV-4, from the microbial mat at a hydrothermal vent of Loihi Seamount in the Pacific Ocean has been further characterized, with emphases on metal reduction and iron biomineralization. The strain is able to reduce metals such as Fe(III), Co(III), Cr(VI), Mn(IV), and U(VI) as electron acceptors while using lactate, formate, pyruvate, or hydrogen as an electron donor. Growth during iron reduction occurred over the pH range of 7.0 to 8.9, a sodium chloride range of 0.05 to 5%, and a temperature range of 0 to 37 degrees C, with an optimum growth temperature of 18 degrees C. Unlike mesophilic dissimilatory Fe(III)-reducing bacteria, which produce mostly superparamagnetic magnetite (<35 nm), this psychrotolerant bacterium produces well-formed single-domain magnetite (>35 nm) at temperatures from 18 to 37 degrees C. The genome size of this strain is about 4.5 Mb. Strain PV-4 is sensitive to a variety of commonly used antibiotics except ampicillin and can acquire exogenous DNA (plasmid pCM157) through conjugation. PMID- 16672463 TI - Characterization of Shiga toxin gene (stx)-positive and intimin gene (eae) positive Escherichia coli isolates from wastewater of slaughterhouses in France. AB - Wastewater samples from 12 slaughterhouses located in different regions in France were tested for the presence of stx-positive and eae-positive Escherichia coli isolates, and characteristics of the isolates obtained were determined. A total of 224 wastewater samples were collected in wastewater treatment plants at different stages of wastewater processing. Altogether, 5,001 E. coli isolates were obtained by colony counting and screened for the presence of stx and eae genes by multiplex PCR. stx-positive and eae-positive E. coli isolates were detected in 25% of the samples collected; they were found in 13% and 3% of the samples obtained from treated effluent and sludge, respectively, suggesting that they could be spread into the environment. Screening of the samples collected by immunomagnetic separation allowed us to isolate 31 additional E. coli serogroup O157 isolates. Four of these isolates harbored stx and eae genes. All stx positive and eae-positive E. coli isolates were analyzed for eae and stx genetic variants, as well as for additional virulence factors and serotypes. Our results suggest that the majority of the stx- and eae-positive E. coli isolates from wastewater have low virulence for humans. However, the diversity of the enterohemorrhagic E. coli-associated virulence factors in the strains indicates that the environment may play an important role in the emergence of new pathogenic enterohemorrhagic E. coli strains. PMID- 16672464 TI - Determination of cyanobacterial diversity during algal blooms in Daechung Reservoir, Korea, on the basis of cpcBA intergenic spacer region analysis. AB - The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria) like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes. PMID- 16672465 TI - Glycosylphosphatidylinositol-anchored Ecm33p influences conidial cell wall biosynthesis in Aspergillus fumigatus. AB - ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils. PMID- 16672466 TI - Inactivation of enterohemorrhagic Escherichia coli in rumen content- or feces contaminated drinking water for cattle. AB - Cattle drinking water is a source of on-farm Escherichia coli O157:H7 transmission. The antimicrobial activities of disinfectants to control E. coli O157:H7 in on-farm drinking water are frequently neutralized by the presence of rumen content and manure that generally contaminate the drinking water. Different chemical treatments, including lactic acid, acidic calcium sulfate, chlorine, chlorine dioxide, hydrogen peroxide, caprylic acid, ozone, butyric acid, sodium benzoate, and competing E. coli, were tested individually or in combination for inactivation of E. coli O157:H7 in the presence of rumen content. Chlorine (5 ppm), ozone (22 to 24 ppm at 5 degrees C), and competing E. coli treatment of water had minimal effects (<1 log CFU/ml reduction) on killing E. coli O157:H7 in the presence of rumen content at water-to-rumen content ratios of 50:1 (vol/wt) and lower. Four chemical-treatment combinations, including (i) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.05% caprylic acid (treatment A); (ii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.1% sodium benzoate (treatment B); (iii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.5% butyric acid (treatment C); and (iv) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 100 ppm chlorine dioxide (treatment D); were highly effective (>3 log CFU/ml reduction) at 21 degrees C in killing E. coli O157:H7, O26:H11, and O111:NM in water heavily contaminated with rumen content (10:1 water/rumen content ratio [vol/wt]) or feces (20:1 water/feces ratio [vol/wt]). Among them, treatments A, B, and C killed >5 log CFU E. coli O157:H7, O26:H11, and O111:NM/ml within 30 min in water containing rumen content or feces, whereas treatment D inactivated approximately 3 to 4 log CFU/ml under the same conditions. Cattle given water containing treatment A or C or untreated water (control) ad libitum for two 7-day periods drank 15.2, 13.8, and 30.3 liters/day, respectively, and cattle given water containing 0.1% lactic acid plus 0.9% acidic calcium sulfate (pH 2.1) drank 18.6 liters/day. The amounts of water consumed for all water treatments were significantly different from that for the control, but there were no significant differences among the water treatments. Such treatments may best be applied periodically to drinking water troughs and then flushed, rather than being added continuously, to avoid reduced water consumption by cattle. PMID- 16672468 TI - Quantitative microbial risk assessment models for consumption of raw vegetables irrigated with reclaimed water. AB - Quantitative microbial risk assessment models for estimating the annual risk of enteric virus infection associated with consuming raw vegetables that have been overhead irrigated with nondisinfected secondary treated reclaimed water were constructed. We ran models for several different scenarios of crop type, viral concentration in effluent, and time since last irrigation event. The mean annual risk of infection was always less for cucumber than for broccoli, cabbage, or lettuce. Across the various crops, effluent qualities, and viral decay rates considered, the annual risk of infection ranged from 10(-3) to 10(-1) when reclaimed-water irrigation ceased 1 day before harvest and from 10(-9) to 10(-3) when it ceased 2 weeks before harvest. Two previously published decay coefficients were used to describe the die-off of viruses in the environment. For all combinations of crop type and effluent quality, application of the more aggressive decay coefficient led to annual risks of infection that satisfied the commonly propounded benchmark of < or =10(-4), i.e., one infection or less per 10,000 people per year, providing that 14 days had elapsed since irrigation with reclaimed water. Conversely, this benchmark was not attained for any combination of crop and water quality when this withholding period was 1 day. The lower decay rate conferred markedly less protection, with broccoli and cucumber being the only crops satisfying the 10(-4) standard for all water qualities after a 14-day withholding period. Sensitivity analyses on the models revealed that in nearly all cases, variation in the amount of produce consumed had the most significant effect on the total uncertainty surrounding the estimate of annual infection risk. The models presented cover what would generally be considered to be worst case scenarios: overhead irrigation and consumption of vegetables raw. Practices such as subsurface, furrow, or drip irrigation and postharvest washing/disinfection and food preparation could substantially lower risks and need to be considered in future models, particularly for developed nations where these extra risk reduction measures are more common. PMID- 16672467 TI - Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity. AB - Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase. PMID- 16672470 TI - Inhibition of plant-pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides. AB - Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH(2)). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED(50)) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation were also determined. Pep3 and several analogues inhibited growth of the three pathogens and had a bactericidal effect at low micromolar concentrations (ED(50) of 1.3 to 7.3 microM). One of the analogues consisting of a replacement of both Trp and Val with Lys and Phe, respectively, resulted in a peptide with improved bactericidal activity and minimized cytotoxicity and susceptibility to protease degradation compared to Pep3. The best analogues can be considered as potential lead compounds for the development of new antimicrobial agents for use in plant protection either as components of pesticides or expressed in transgenic plants. PMID- 16672471 TI - Genetic diversity and quinolone resistance in Campylobacter jejuni isolates from poultry in Senegal. AB - We used the multilocus sequence typing (MLST) method to evaluate the genetic diversity of 46 Campylobacter jejuni isolates from chickens and to determine the link between quinolone resistance and sequence type (ST). There were a total of 16 ST genotypes, and the majority of them belonged to seven clonal complexes previously identified by using isolates from human disease. The ST-353 complex was the most common complex, whereas the ST-21, ST-42, ST-52, and ST-257 complexes were less well represented. The resistance phenotype varied for each ST, and the Thr-86-Ile substitution in the GyrA protein was the predominant mechanism of resistance to quinolone. Nine of the 14 isolates having the Thr-86 Ile substitution belonged to the ST-353 complex. MLST showed that the emergence of quinolone resistance is not related to the diffusion of a unique clone and that there is no link between ST genotype and quinolone resistance. Based on silent mutations, different variants of the gyrA gene were shown to exist for the same ST. These data provide useful information for understanding the epidemiology of C. jejuni in Senegal. PMID- 16672472 TI - Control of Listeria spp. by competitive-exclusion bacteria in floor drains of a poultry processing plant. AB - In previous studies workers determined that two lactic acid bacterium isolates, Lactococcus lactis subsp. lactis C-1-92 and Enterococcus durans 152 (competitive exclusion bacteria [CE]), which were originally obtained from biofilms in floor drains, are bactericidal to Listeria monocytogenes or inhibit the growth of L. monocytogenes both in vitro and in biofilms at 4 to 37 degrees C. We evaluated the efficacy of these isolates for reducing Listeria spp. contamination of floor drains of a plant in which fresh poultry is processed. Baseline assays revealed that the mean numbers of Listeria sp. cells in floor drains sampled on six different dates (at approximately biweekly intervals) were 7.5 log(10) CFU/100 cm(2) for drain 8, 4.9 log(10) CFU/100 cm(2) for drain 3, 4.4 log(10) CFU/100 cm(2) for drain 2, 4.1 log(10) CFU/100 cm(2) for drain 4, 3.7 log(10) CFU/100 cm(2) for drain 1, and 3.6 log(10) CFU/100 cm(2) for drain 6. The drains were then treated with 10(7) CE/ml in an enzyme-foam-based cleaning agent four times in 1 week and twice a week for the following 3 weeks. In samples collected 1 week after CE treatments were applied Listeria sp. cells were not detectable (samples were negative as determined by selective enrichment culture) for drains 4 and 6 (reductions of 4.1 and 3.6 log(10) CFU/100 cm(2), respectively), and the mean numbers of Listeria sp. cells were 3.7 log(10) CFU/100 cm(2) for drain 8 (a reduction of 3.8 log(10) CFU/100 cm(2)), <1.7 log(10) CFU/100 cm(2) for drain 1 (detectable only by selective enrichment culture; a reduction of 3.3 log(10) CFU/100 cm(2)), and 2.6 log(10) CFU/100 cm(2) for drain 3 (a reduction of 2.3 log(10) CFU/100 cm(2)). However, the aerobic plate counts for samples collected from floor drains before, during, and after CE treatment remained approximately the same. The results indicate that application of the two CE can greatly reduce the number of Listeria sp. cells in floor drains at 3 to 26 degrees C in a facility in which fresh poultry is processed. PMID- 16672473 TI - Characterization of novel carbazole catabolism genes from gram-positive carbazole degrader Nocardioides aromaticivorans IC177. AB - Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta 2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl. PMID- 16672474 TI - L-methionine degradation pathway in Kluyveromyces lactis: identification and functional analysis of the genes encoding L-methionine aminotransferase. AB - Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced. PMID- 16672469 TI - Environmental whole-genome amplification to access microbial populations in contaminated sediments. AB - Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173. PMID- 16672475 TI - Development of a biofilm production-deficient Escherichia coli strain as a host for biotechnological applications. AB - Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Delta(csgG-csgC); colanic acid biosynthesis and assembly, Delta(wcaL-wza); and type I pilus biosynthesis, Delta(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were approximately 16% and approximately 25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena. PMID- 16672476 TI - Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid. AB - Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules. PMID- 16672477 TI - Levels of polyamines and kinetic characterization of their uptake in the soybean pathogen Phytophthora sojae. AB - Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-(14)C]putrescine and [1,4-(14)C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection. PMID- 16672478 TI - Diversity, composition, and geographical distribution of microbial communities in California salt marsh sediments. AB - The Pacific Estuarine Ecosystem Indicators Research Consortium seeks to develop bioindicators of toxicant-induced stress and bioavailability for wetland biota. Within this framework, the effects of environmental and pollutant variables on microbial communities were studied at different spatial scales over a 2-year period. Six salt marshes along the California coastline were characterized using phospholipid fatty acid (PLFA) analysis and terminal restriction fragment length polymorphism (TRFLP) analysis. Additionally, 27 metals, six currently used pesticides, total polychlorinated biphenyls and polycyclic aromatic hydrocarbons, chlordanes, nonachlors, dichlorodiphenyldichloroethane, and dichlorodiphenyldichloroethylene were analyzed. Sampling was performed over large (between salt marshes), medium (stations within a marsh), and small (different channel depths) spatial scales. Regression and ordination analysis suggested that the spatial variation in microbial communities exceeded the variation attributable to pollutants. PLFA analysis and TRFLP canonical correspondence analysis (CCA) explained 74 and 43% of the variation, respectively, and both methods attributed 34% of the variation to tidal cycles, marsh, year, and latitude. After accounting for spatial variation using partial CCA, we found that metals had a greater effect on microbial community composition than organic pollutants had. Organic carbon and nitrogen contents were positively correlated with PLFA biomass, whereas total metal concentrations were positively correlated with biomass and diversity. Higher concentrations of heavy metals were negatively correlated with branched PLFAs and positively correlated with methyl- and cyclo substituted PLFAs. The strong relationships observed between pollutant concentrations and some of the microbial indicators indicated the potential for using microbial community analyses in assessments of the ecosystem health of salt marshes. PMID- 16672479 TI - Modeling for gellan gum production by Sphingomonas paucimobilis ATCC 31461 in a simplified medium. AB - Gellan gum production was carried out by Sphingomonas paucimobilis ATCC 31461 in a simplified medium with a short incubation time, and a kinetic model for understanding, controlling, and optimizing the fermentation process was proposed. The results revealed that glucose was the best carbon source and that the optimal concentration was 30 g liter(-1). As for the fermenting parameters, considerably large amounts of gellan gum were yielded by an 8-h-old culture and a 4% inoculum at 200 rpm on a rotary shaker. Under the optimized conditions, the maximum level of gellan gum (14.75 g liter(-1)) and the highest conversion efficiency (49.17%) were obtained in a 30-liter fermentor in batch fermentation. Logistic and Luedeking-Piret models were confirmed to provide a good description of gellan gum fermentation, which gave some support for the study of gellan gum fermentation kinetics. Additionally, this study is the first demonstration that gellan gum production is largely growth associated by analysis of kinetics in its batch fermentation process. Based on model prediction, higher gellan gum production (17.71 g liter(-1)) and higher conversion efficiency (57.12%) were obtained in fed-batch fermentation at the same total glucose concentration (30 g liter(-1)). PMID- 16672480 TI - Identification of poly(cis-1,4-Isoprene) degradation intermediates during growth of moderately thermophilic actinomycetes on rubber and cloning of a functional lcp homologue from Nocardia farcinica strain E1. AB - The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis 1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50 degrees C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4 isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis 1,4-isoprene), confirming its involvement in initial polymer cleavage. PMID- 16672481 TI - Lactococcin Q, a novel two-peptide bacteriocin produced by Lactococcus lactis QU 4. AB - A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, alpha and beta, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qalpha plus Qbeta) and lactococcin G (Galpha plus Gbeta) clarified that hybrid combinations (Qalpha plus Gbeta and Galpha plus Qbeta) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qalpha plus Gbeta was 32 times higher than that of Qalpha plus Qbeta, suggesting that the difference in beta peptides was important for the intensity of antibacterial activity. PMID- 16672482 TI - Recovery, bioaccumulation, and inactivation of human waterborne pathogens by the Chesapeake Bay nonnative oyster, Crassostrea ariakensis. AB - The introduction of nonnative oysters (i.e., Crassostrea ariakensis) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8 ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 x 10(5) transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters. PMID- 16672483 TI - Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40. AB - Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50 dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha neoagarobiose hydrolase to release galactose from agarose. PMID- 16672484 TI - Interaction of Escherichia coli and soil particles in runoff. AB - A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (> 45 microm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (< 2 microm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix. PMID- 16672485 TI - High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and Synechocystis sp. strain PCC6803. AB - A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium Synechocystis sp. strain PCC6803 and the eubacterium Escherichia coli to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In Synechocystis, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In E. coli, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high throughput evaluation of combinatorial libraries of Synechocystis and E. coli using fluorescence-activated cell sorting to identify mutants with increased PHB accumulating properties. PMID- 16672486 TI - Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. AB - The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertes, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics. PMID- 16672487 TI - Rhizosphere selection of highly motile phenotypic variants of Pseudomonas fluorescens with enhanced competitive colonization ability. AB - Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks. PMID- 16672488 TI - Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables. AB - A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic. PMID- 16672489 TI - Dissemination of fluoroquinolone-resistant Campylobacter spp. within an integrated commercial poultry production system. AB - While characterizing the intestinal bacterial community of broiler chickens, we detected epsilon-proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks. PMID- 16672490 TI - Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions. AB - Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as alpha-amylase (TAA) and beta glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture. PMID- 16672492 TI - Classification tree method for bacterial source tracking with antibiotic resistance analysis data. AB - Various statistical classification methods, including discriminant analysis, logistic regression, and cluster analysis, have been used with antibiotic resistance analysis (ARA) data to construct models for bacterial source tracking (BST). We applied the statistical method known as classification trees to build a model for BST for the Anacostia Watershed in Maryland. Classification trees have more flexibility than other statistical classification approaches based on standard statistical methods to accommodate complex interactions among ARA variables. This article describes the use of classification trees for BST and includes discussion of its principal parameters and features. Anacostia Watershed ARA data are used to illustrate the application of classification trees, and we report the BST results for the watershed. PMID- 16672491 TI - Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis. AB - Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment. PMID- 16672493 TI - High-pressure-mediated survival of Clostridium botulinum and Bacillus amyloliquefaciens endospores at high temperature. AB - Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120 degrees C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110 degrees C) accelerated the inactivation of C. botulinum spores. However, incubation at 100 degrees C, 110 degrees C, or 120 degrees C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120 degrees C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120 degrees C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment. PMID- 16672494 TI - Survival strategy of Erwinia amylovora against copper: induction of the viable but-nonculturable state. AB - Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu(2+) was inoculated with 10(7) CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper. PMID- 16672495 TI - Diversity and seasonal dynamics of Actinobacteria populations in four lakes in northeastern Germany. AB - The phylogenetic diversity and seasonal dynamics of freshwater Actinobacteria populations in four limnologically different lakes of the Mecklenburg-Brandenburg Lake District (northeastern Germany) were investigated. Fluorescence in situ hybridization was used to determine the seasonal abundances and dynamics of total Actinobacteria (probe HGC69a) and the three actinobacterial subclusters acI, acI A, and acI-B (probes AcI-852, AcI-840-1, and AcI-840-2). Seasonal means of total Actinobacteria abundances in the epilimnia of the lakes varied from 13 to 36%, with maximum values of 30 to 58%, of all DAPI (4',6'-diamidino-2-phenylindole) stained cells. Around 80% of total Actinobacteria belonged to the acI cluster. The two subclusters acI-A and acI-B accounted for 60 to 91% of the acI cluster and showed seasonal means of 49% (acI-B) and 23% (acI-A) in relation to the acI cluster. Total Actinobacteria and members of the clusters acI and acI-B showed distinct seasonal changes in their absolute abundances, with maxima in late spring and fall/winter. In eight clone libraries constructed from the lakes, a total of 76 actinobacterial 16S rRNA gene sequences were identified from a total of 177 clones. The majority of the Actinobacteria sequences belonged to the acI and acIV cluster. Several new clusters and subclusters were found (acSTL, scB1-4, and acIVA-D). The majority of all obtained 16S rRNA gene sequences are distinct from those of already-cultured freshwater Actinobacteria. PMID- 16672496 TI - Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride. AB - In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC. PMID- 16672497 TI - Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and genes similar to those encoding isopropylbenzene dioxygenases. AB - The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic alpha subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant alpha subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase alpha subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an alpha subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket. PMID- 16672498 TI - Characterization of an exo-beta-1,3-galactanase from Clostridium thermocellum. AB - A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3 galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase treated AGP. PMID- 16672499 TI - A single monooxygenase, ese, is involved in the metabolism of the organochlorides endosulfan and endosulfate in an Arthrobacter sp. AB - In this paper we describe isolation of a bacterium capable of degrading both isomers of the organochloride insecticide endosulfan and its toxic metabolite, endosulfate. The bacterium was isolated from a soil microbial population that was enriched with continuous pressure to use endosulfate as the sole source of sulfur. Analysis of the 16S rRNA sequence of the bacterium indicated that it was an Arthrobacter species. The organochloride-degrading activity was not observed in the presence of sodium sulfite as an alternative sulfur source, suggesting that the activity was part of the sulfur starvation response of the strain. A gene, ese, encoding an enzyme capable of degrading both isomers of endosulfan and endosulfate was isolated from this bacterium. The enzyme belongs to the two component flavin-dependent monooxygenase family whose members require reduced flavin for activity. Nuclear magnetic resonance analyses identified the metabolite of endosulfan as endosulfan monoalcohol and the metabolite of endosulfate as endosulfan hemisulfate. The ese gene was located in a cluster of 10 open reading frames encoding proteins with low levels of sulfur-containing amino acids. These open reading frames were organized into two apparent divergently orientated operons and a gene encoding a putative LysR-type transcriptional regulator. The operon not containing ese did contain a homologue whose product exhibited 62% amino acid identity to the ese-encoded protein. PMID- 16672501 TI - Coupled photochemical and enzymatic Mn(II) oxidation pathways of a planktonic Roseobacter-Like bacterium. AB - Bacteria belonging to the Roseobacter clade of the alpha-Proteobacteria occupy a wide range of environmental niches and are numerically abundant in coastal waters. Here we reveal that Roseobacter-like bacteria may play a previously unrecognized role in the oxidation and cycling of manganese (Mn) in coastal waters. A diverse array of Mn(II)-oxidizing Roseobacter-like species were isolated from Elkhorn Slough, a coastal estuary adjacent to Monterey Bay in California. One isolate (designated AzwK-3b), in particular, rapidly oxidizes Mn(II) to insoluble Mn(III, IV) oxides. Interestingly, AzwK-3b is 100% identical (at the 16S rRNA gene level) to a previously described Pfiesteria-associated Roseobacter-like bacterium, which is not able to oxidize Mn(II). The rates of manganese(II) oxidation by live cultures and cell-free filtrates are substantially higher when the preparations are incubated in the presence of light. The rates of oxidation by washed cell extracts, however, are light independent. Thus, AzwK-3b invokes two Mn(II) oxidation mechanisms when it is incubated in the presence of light, in contrast to the predominantly direct enzymatic oxidation in the dark. In the presence of light, production of photochemically active metabolites is coupled with initial direct enzymatic Mn(II) oxidation, resulting in higher Mn(II) oxidation rates. Thus, Roseobacter like bacteria may not only play a previously unrecognized role in Mn(II) oxidation and cycling in coastal surface waters but also induce a novel photooxidation pathway that provides an alternative means of Mn(II) oxidation in the photic zone. PMID- 16672500 TI - Population dynamics within a microbial consortium during growth on diesel fuel in saline environments. AB - The diversity and dynamics of a bacterial community extracted from an exploited oil field with high natural soil salinity near Comodoro Rivadavia in Patagonia (Argentina) were investigated. Community shifts during long-term incubation with diesel fuel at four salinities between 0 and 20% NaCl were monitored by single strand conformation polymorphism community fingerprinting of the PCR-amplified V4 V5 region of the 16S rRNA genes. Information obtained by this qualitative approach was extended by flow cytometric analysis to follow quantitatively the dynamics of community structures at different salinities. Dominant and newly developing clusters of individuals visualized via their DNA patterns versus cell sizes were used to identify the subcommunities primarily involved in the degradation process. To determine the most active species, subcommunities were separated physically by high-resolution cell sorting and subsequent phylogenetic identification by 16S rRNA gene sequencing. Reduced salinity favored the dominance of Sphingomonas spp., whereas at elevated salinities, Ralstonia spp. and a number of halophilic genera, including Halomonas, Dietzia, and Alcanivorax, were identified. The combination of cytometric sorting with molecular characterization allowed us to monitor community adaptation and to identify active and proliferating subcommunities. PMID- 16672502 TI - Auxofuran, a novel metabolite that stimulates the growth of fly agaric, is produced by the mycorrhiza helper bacterium Streptomyces strain AcH 505. AB - The mycorrhiza helper bacterium Streptomyces strain AcH 505 improves mycelial growth of ectomycorrhizal fungi and formation of ectomycorrhizas between Amanita muscaria and spruce but suppresses the growth of plant-pathogenic fungi, suggesting that it produces both fungal growth-stimulating and -suppressing compounds. The dominant fungal-growth-promoting substance produced by strain AcH 505, auxofuran, was isolated, and its effect on the levels of gene expression of A. muscaria was investigated. Auxofuran and its synthetic analogue 7-dehydroxy auxofuran were most effective at a concentration of 15 microM, and application of these compounds led to increased lipid metabolism-related gene expression. Cocultivation of strain AcH 505 and A. muscaria stimulated auxofuran production by the streptomycete. The antifungal substances produced by strain AcH 505 were identified as the antibiotics WS-5995 B and C. WS-5995 B completely blocked mycelial growth at a concentration of 60 microM and caused a cell stress-related gene expression response in A. muscaria. Characterization of these compounds provides the foundation for molecular analysis of the fungus-bacterium interaction in the ectomycorrhizal symbiosis between fly agaric and spruce. PMID- 16672503 TI - The general secretory pathway of Burkholderia gladioli pv. agaricicola BG164R is necessary for cavity disease in white button mushrooms. AB - Cavity disease in white button mushrooms is caused by Burkholderia gladioli pv. agaricicola. We describe the isolation and characterization of six mutants of the strain BG164R that no longer cause this disease on mushrooms. The mutations were mapped to genes of the general secretory pathway (GSP). This is the first report of the association of the type II secretion pathway with a disease in mushrooms. Phenotypes of the six avirulent mutants were the following: an inability to degrade mushroom tissue, a highly reduced capacity to secrete chitinase and protease, and a reduced number of flagella. Using these mutants, we also made the novel observation that the factors causing mushroom tissue degradation, thereby leading to the expression of cavity disease, can be separated from mycelium inhibition because avirulent mutants continued to inhibit the growth of actively growing mushroom mycelia. The GSP locus of B. gladioli was subsequently cloned and mapped and compared to the same locus in closely related species, establishing that the genetic organization of the gsp operon of B. gladioli pv. agaricicola is consistent with that of other species of the genus. We also identify the most common indigenous bacterial population present in the mushroom fruit bodies from a New Zealand farm, one of which, Ewingella americana, was found to be an apparent antagonist of B. gladioli pv. agaricicola. While other investigators have reported enhanced disease symptoms due to interactions between endogenous and disease-causing bacteria in other mushroom diseases, to the best of our knowledge this is the first report of an antagonistic effect. PMID- 16672504 TI - Short-term metabolome dynamics and carbon, electron, and ATP balances in chemostat-grown Saccharomyces cerevisiae CEN.PK 113-7D following a glucose pulse. AB - The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O(2) uptake and CO(2) evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO(2) evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses. PMID- 16672505 TI - Dynamics of a pig slurry microbial community during anaerobic storage and management. AB - The microbial community of a pig slurry on a farm was monitored for 6 months using both molecular and cultural approaches. Sampling was carried out at all the different stages of effluent handling, from the rearing build-up to slurry spreading. Total DNA of each sample was extracted and analyzed by PCR-single strand conformation polymorphism (SSCP) analysis using primers targeting the 16S rRNA genes from the archaeal and bacterial domains and also the Eubacterium Clostridium, Bacillus-Streptococcus-Lactobacillus, and Bacteroides-Prevotella groups. A comparison of the SSCP profiles showed that there were rapid changes in the dominant bacterial community during the first 2 weeks of anaerobic storage and that the community was relatively stable thereafter. Several bacterial populations, identified as populations closely related to uncultured Clostridium and Porphyromonas and to Lactobacillus and Streptococcus cultured species commonly isolated from pig feces, remained present and dominant from the rearing build-up to the time of spreading. Enumeration of fecal indicators (enterococci and Escherichia coli) performed in parallel using cultural methods revealed the same trends. On the other hand, the archaeal community adapted slowly during pig slurry storage, and its diversity increased. A shift between two hydrogenotrophic methanogenic Methanobrevibacter populations from the storage pit to the pond was observed. Microorganisms present in pig slurry at the time of spreading could not be detected in soil after spreading by either molecular or cultural techniques, probably because of the detection limit inherent in the two techniques. PMID- 16672506 TI - RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater. AB - Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [(13)C(6)]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the (13)C RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [(14)C]benzene showed that the labeled carbon was mostly incorporated into (14)CO(2) within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function. PMID- 16672507 TI - Two routes of metabolic cross-feeding between Bifidobacterium adolescentis and butyrate-producing anaerobes from the human gut. AB - Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [(13)C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross feeding of partial breakdown products from complex substrates. PMID- 16672508 TI - Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry. AB - Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms. PMID- 16672509 TI - Uranium(VI) reduction by Anaeromyxobacter dehalogenans strain 2CP-C. AB - Previous studies demonstrated growth of Anaeromyxobacter dehalogenans strain 2CP C with acetate or hydrogen as the electron donor and Fe(III), nitrate, nitrite, fumarate, oxygen, or ortho-substituted halophenols as electron acceptors. In this study, we explored and characterized U(VI) reduction by strain 2CP-C. Cell suspensions of fumarate-grown 2CP-C cells reduced U(VI) to U(IV). More-detailed growth studies demonstrated that hydrogen was the required electron donor for U(VI) reduction and could not be replaced by acetate. The addition of nitrate to U(VI)-reducing cultures resulted in a transitory increase in U(VI) concentration, apparently caused by the reoxidation of reduced U(IV), but U(VI) reduction resumed following the consumption of N-oxyanions. Inhibition of U(VI) reduction occurred in cultures amended with Fe(III) citrate, or citrate. In the presence of amorphous Fe(III) oxide, U(VI) reduction proceeded to completion but the U(VI) reduction rates decreased threefold compared to control cultures. Fumarate and 2 chlorophenol had no inhibitory effects on U(VI) reduction, and both electron acceptors were consumed concomitantly with U(VI). Since cocontaminants (e.g., nitrate, halogenated compounds) and bioavailable ferric iron are often encountered at uranium-impacted sites, the metabolic versatility makes Anaeromyxobacter dehalogenans a promising model organism for studying the complex interaction of multiple electron acceptors in U(VI) reduction and immobilization. PMID- 16672511 TI - Microeukaryote community patterns along an O2/H2S gradient in a supersulfidic anoxic fjord (Framvaren, Norway). AB - To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O(2)/H(2)S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O(2)/H(2)S gradient. PMID- 16672510 TI - Genetic population structure of the soil bacterium Myxococcus xanthus at the centimeter scale. AB - Myxococcus xanthus is a gram-negative soil bacterium best known for its remarkable life history of social swarming, social predation, and multicellular fruiting body formation. Very little is known about genetic diversity within this species or how social strategies might vary among neighboring strains at small spatial scales. To investigate the small-scale population structure of M. xanthus, 78 clones were isolated from a patch of soil (16 by 16 cm) in Tubingen, Germany. Among these isolates, 21 genotypes could be distinguished from a concatemer of three gene fragments: csgA (developmental C signal), fibA (extracellular matrix-associated zinc metalloprotease), and pilA (the pilin subunit of type IV pili). Accumulation curves showed that most of the diversity present at this scale was sampled. The pilA gene contains both conserved and highly variable regions, and two frequency-distribution tests provide evidence for balancing selection on this gene. The functional domains in the csgA gene were found to be conserved. Three instances of lateral gene transfer could be inferred from a comparison of individual gene phylogenies, but no evidence was found for linkage equilibrium, supporting the view that M. xanthus evolution is largely clonal. This study shows that M. xanthus is surrounded by a variety of distinct conspecifics in its natural soil habitat at a spatial scale at which encounters among genotypes are likely. PMID- 16672512 TI - Isolation and biochemical characterization of two novel metagenome-derived esterases. AB - The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to beta lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50 degrees C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47 degrees C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications. PMID- 16672513 TI - Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae). AB - Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor "Candidatus Portiera aleyrodidarum," an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others. PMID- 16672514 TI - Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio. AB - Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses. PMID- 16672515 TI - Sequence analysis of the 144-kilobase accessory plasmid pSmeSM11a, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment. AB - The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules. PMID- 16672516 TI - Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses. AB - Two quaternary ammonium silanes (QAS) were used to coat silicone rubber tracheoesophageal shunt prostheses, yielding a positively charged surface. One QAS coating [(trimethoxysilyl)-propyldimethyloctadecylammonium chloride] was applied through chemical bonding, while the other coating, Biocidal ZF, was sprayed onto the silicone rubber surface. The sprayed coating lost its stability within an hour, while the chemically bonded coating appeared stable. Upon incubation in an artificial throat model, allowing simultaneous adhesion and growth of yeast and bacteria, all coated prostheses showed significant reductions in the numbers of viable yeast (to 12% to 16%) and bacteria (to 27% to 36%) compared with those for silicone rubber controls, as confirmed using confocal laser scanning microscopy after live/dead staining of the biofilms. In situ hybridization with fluorescently labeled oligonucleotide probes showed that yeasts expressed hyphae on the untreated and Biocidal ZF-coated prostheses but not on the QAS-coated prostheses. Whether this is a result of the positive QAS coating or is due to the reduced number of bacteria is currently unknown. In summary, this is the first report on the inhibitory effects of positively charged coatings on the viability of yeasts and bacteria in mixed biofilms. Although the study initially aimed at reducing voice prosthetic biofilms, its relevance extends to all biomedical and environmental surfaces where mixed biofilms develop and present a problem. PMID- 16672517 TI - Modeling shifts in microbial populations associated with health or disease. AB - Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents. PMID- 16672518 TI - Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat. AB - We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (> or =97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O(2) and H(2)S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity. The sequences determined in this study have been submitted to the GenBank database and assigned accession numbers DQ 329539 to DQ 331020, and DQ 397339 to DQ 397511. PMID- 16672519 TI - Identifying functionally important mutations from phenotypically diverse sequence data. AB - Here we present a simple statistical method to determine the phenotypic contribution of a single mutation from libraries of mutants with diverse phenotypes in which each mutant contains a multitude of mutations. The central premise of this method is that, given M phenotypic classes, mutations that do not affect the phenotype should partition among the M classes according to a multinomial distribution. Deviations from this distribution are indicative of a link between specific mutations and phenotypes. We suggest that this method will aid the engineering of functional nucleic acids, proteins, and other biomolecules by uncovering target sites for rational mutagenesis. As a proof of the principle, we show how the method can be used to deduce the individual effects of mutations in a set of 69 P(L)-lambda promoter variants. Each of these promoters was generated by error-prone PCR and incorporated numerous mutations. The activity of the promoters was assayed using flow cytometry to measure the fluorescence of a green fluorescent protein reporter gene. Our analysis of the sequences of these mutants revealed seven positions having a statistically significant correlation with promoter activity. Using site-directed mutagenesis, we constructed point mutations for several sites, both statistically significant and insignificant, and combinations of these sites. Our results show that the statistical method correctly elucidated the phenotypic manifestations of these mutations. We suggest that this method may be useful for expediting directed evolution experiments by allowing both desired and undesired mutations to be identified and incorporated between rounds of mutagenesis. PMID- 16672520 TI - Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor. AB - A methanogenic bioreactor that utilized wastepaper was developed and operated at 55 degrees C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium. PMID- 16672521 TI - Use of fluorescence resonance energy transfer for rapid detection of enteroviral infection in vivo. AB - Enteroviruses can be easily transmitted through the fecal-oral route and cause a diverse array of clinical manifestations. Recent outbreaks associated with enteroviral contamination in aquatic environments have called for the development of a more efficient and accurate virus monitoring system. To develop a simple, rapid, and direct method for identifying enteroviral infections, we generated a fluorescent reporter system in which genetically engineered cells express a hybrid fluorescent indicator composed of a linker peptide, which is exclusively cleaved by the 2A protease (2A(pro)), flanked with a cyan fluorescent protein (CFP) and a yellow fluorescent protein undergoing fluorescence resonance energy transfer. The covalent linkage between two fluorophores is disrupted due to 2A(pro) activity upon viral infection, which results in an increase in CFP intensity. This allows the rapid (within 7.5 h) detection of very low numbers (10 PFU or fewer) of infectious enteroviruses. PMID- 16672522 TI - Extracellular production of neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae. AB - Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, alpha-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2 like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests. PMID- 16672523 TI - Characterization of a culturable alphaproteobacterial symbiont common to many marine sponges and evidence for vertical transmission via sponge larvae. AB - A closely related group of alphaproteobacteria were found to be present in seven genera of marine sponges from several locations and were shown to be transferred between sponge generations through the larvae in one of these sponges. Isolates of the alphaproteobacterium were cultured from the sponges Axinella corrugata, Mycale laxissima, Monanchora unguifera, and Niphates digitalis from Key Largo, Florida; Didiscus oxeata and Monanchora unguifera from Discovery Bay, Jamaica; an Acanthostronglyophora sp. from Manado, Indonesia; and Microciona prolifera from the Cheasapeake Bay in Maryland. Isolates were very similar to each other on the basis of 16S rRNA gene sequence (>99% identity) and are closely related to Pseudovibrio denitrificans. The bacterium was never isolated from surrounding water samples and was cultured from larvae of M. laxissima, indicating that it is a vertically transmitted symbiont in this sponge. Denaturing gradient gel electrophoresis, 16S rRNA gene clone library analysis, and fluorescent in situ hybridization with probes specific to the alphaproteobacterium confirmed the presence of this bacterium in the M. laxissima larvae. The alphaproteobacterium was densely associated with the larvae rather than being evenly distributed throughout the mesohyl. This is the first report of the successful culture of a bacterial symbiont of a sponge that is transferred through the gametes. PMID- 16672524 TI - Improved methodology for bioremoval of black crusts on historical stone artworks by use of sulfate-reducing bacteria. AB - An improved methodology to remove black crusts from stone by using Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, a sulfate-reducing bacterium, is presented. The strain removed 98% of the sulfates of the crust in a 45-h treatment. Precipitation of black iron sulfide was avoided using filtration of a medium devoid of iron. Among three cell carriers, Carbogel proved to be superior to both sepiolite and Hydrobiogel-97, as it allowed an easy application of the bacteria, kept the system in a state where microbial activity was maintained, and allowed easy removal of the cells after the treatment. PMID- 16672525 TI - arsRBOCT arsenic resistance system encoded by linear plasmid pHZ227 in Streptomyces sp. strain FR-008. AB - In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1 arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs. PMID- 16672526 TI - Multiniche screening reveals the clinically relevant metallo-beta-lactamase VIM-2 in Pseudomonas aeruginosa far from the hospital setting: an ongoing dispersion process? AB - A screening study of the presence of metallo-beta-lactamases (IMP and VIM types and SPM-1) in isolates from different nonhospital sources was conducted, and it revealed the presence of bla(VIM-2), associated with the In58 class 1 integron, in two unrelated Pseudomonas aeruginosa strains from aquatic habitats. The results suggest that the hospital setting was the possible origin of these bla(VIM-2)-carrying strains. PMID- 16672527 TI - Influence of sporulation medium composition on transcription of ger operons and the germination response of spores of Bacillus cereus ATCC 14579. AB - Bacillus cereus ATCC 14579 endospores were produced in Y1 medium, a nutrient rich, chemically defined sporulation medium, and in modified G medium, containing low amounts of nutrients. The average transcription level of the seven ger operons per cell was 3.5 times higher in Y1 medium, and the spores grown in this medium showed an enhanced germination response. PMID- 16672528 TI - High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library. AB - A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms. PMID- 16672529 TI - Involvement of coenzyme M during aerobic biodegradation of vinyl chloride and ethene by Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD. AB - The involvement of coenzyme M in aerobic biodegradation of vinyl chloride and ethene in Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD was demonstrated using PCR, hybridization, and enzyme assays. The results of this study extend the range of eubacteria known to use epoxyalkane:coenzyme M transferase. PMID- 16672531 TI - Assessment of the effectiveness of low-pressure UV light for inactivation of Helicobacter pylori. AB - Three strains of Helicobacter pylori were exposed to UV light from a low-pressure source to determine log inactivation versus applied fluence. Results indicate that H. pylori is readily inactivated at UV fluences typically used in water treatment regimens. Greater than 4-log(10) inactivation was demonstrated on all three strains at fluences of less than 8 mJ cm(-2). PMID- 16672530 TI - Novel thermophilic sulfate-reducing bacteria from a geothermally active underground mine in Japan. AB - Thermophilic sulfate-reducing bacteria were enriched from samples obtained from a geothermal underground mine in Japan. The enrichment cultures contained bacteria affiliated with the genera Desulfotomaculum, Thermanaeromonas, Thermincola, Thermovenabulum, Moorella, "Natronoanaerobium," and Clostridium. Two novel thermophilic sulfate-reducing strains, RL50JIII and RL80JIV, affiliated with the genera Desulfotomaculum and Thermanaeromonas, respectively, were isolated. PMID- 16672532 TI - Comparison of cultures from rectoanal-junction mucosal swabs and feces for detection of Escherichia coli O157 in dairy heifers. AB - Fecal culture for Escherichia coli O157:H7 was compared to rectoanal mucosal swab (RAMS) culture in dairy heifers over a 1-year period. RAMS enrichment culture was as sensitive as fecal culture using immunomagnetic separation (IMS) (P = 0.98, as determined by a chi-square test). RAMS culture is less costly than fecal IMS culture and can yield quantitative data. PMID- 16672533 TI - Salmonella serovars in the herpetofauna of Indiana County, Pennsylvania. AB - Herpetofaunal Salmonella enterica serovars have not been fully examined in any U.S. region. Thirty-three Salmonella serovars were isolated from 156 samples from 34 species, all within Indiana County, Pennsylvania. Results suggest that herpetofaunas could potentially pose a threat to humans. Further understanding of Salmonella in herpetofaunas may prevent future human cases. PMID- 16672534 TI - Effect of biofilm growth on expression of surface proteins of Actinomyces naeslundii genospecies 2. AB - The predominant surface proteins of biofilm and planktonic Actinomyces naeslundii, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly up-regulated relative to planktonic cells. PMID- 16672535 TI - Development and validation of an oligonucleotide microarray for detection of multiple virulence and antimicrobial resistance genes in Escherichia coli. AB - An oligonucleotide microarray detecting 189 Escherichia coli virulence genes or markers and 30 antimicrobial resistance genes was designed and validated using DNA from known reference strains. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging E. coli pathotypes and antimicrobial resistance, as well as for environmental, epidemiological, and phylogenetic studies including the evaluation of genome plasticity. PMID- 16672536 TI - Single-nucleotide-polymorphism-specific PCR for quantification and discrimination of Chlamydia pneumoniae genotypes by use of a "locked" nucleic acid. AB - Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP. PMID- 16672537 TI - Fecal bacterial diversity in a wild gorilla. AB - We describe the bacterial diversity in fecal samples of a wild gorilla by use of a 16S rRNA gene clone library and terminal-restriction fragment length polymorphism (T-RFLP). Clones were classified as Firmicutes, Verrucomicrobia, Actinobacteria, Lentisphaerae, Bacteroidetes, Spirochetes, and Planctomycetes. Our data suggest that fecal populations did not change temporally, as determined by T-RFLP. PMID- 16672538 TI - Combination of multiplex PCR and PCR-denaturing gradient gel electrophoresis for monitoring common sourdough-associated Lactobacillus species. AB - A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs. PMID- 16672539 TI - Improvement of an unusual twin-arginine transporter leader peptide by a codon based randomization approach. AB - Secretion of Escherichia coli penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with respect to the wild-type value). PMID- 16672541 TI - The Phuket Declaration. PMID- 16672542 TI - Overview and research agenda arising from the 5th World Workshop on Oral Health and Disease in AIDS. PMID- 16672543 TI - HIV pathogenesis: knowledge gained after two decades of research. AB - Great progress has been made in our understanding of HIV since its initial discovery about 20 years ago. The ability of HIV to infect CD4+ lymphocytes and a wide variety of other cells in the body is appreciated, as is its role in immunologic, gastrointestinal, and brain disorders. HIV enters cells via the CD4 molecule, chemokine co-receptors (CXCR4, CCR5), and other cell-surface proteins. Several accessory virus-associated genes (e.g., Rev, Tat, Nef) have uncovered unique pathways that can also be observed in normal cells. Recently, the discovery of natural cellular resistant factors (APOBEC3G and TRIM5a) has provided avenues for novel antiviral therapies. Studies of long-term survivors have given insight into immune responses that control HIV and can prevent infection. Neutralizing antibodies and CD8+ cell cytotoxic responses, as well as plasmacytoid dendritic cells and CD8+ cell non-cytotoxic antiviral responses, are adaptive and innate immune activities mediating this anti-HIV effect. HIV vaccine studies have indicated that conventional approaches do not work against this integrated intracellular parasite. While much has been learned about HIV, more details are needed about its infection cycle and its pathologic effects in the body. The past 20 years have yielded important information on HIV/AIDS that should lead to effective anti-HIV therapies and a vaccine. PMID- 16672544 TI - Policy for prevention of oral manifestations in HIV/AIDS: the approach of the WHO Global Oral Health Program. AB - The HIV/AIDS pandemic has become a human and social disaster, particularly affecting the developing countries of Africa, Southeast Asia, and Latin America. By the end of 2004, about 40 million people were estimated to be infected by HIV globally. The health sectors in many affected countries are facing severe shortages of human and financial resources, and are struggling to cope with the growing impact of HIV/AIDS. In most developed countries, the availability of antiretroviral treatment has resulted in a dramatic reduction in HIV/AIDS-related mortality and morbidity. In contrast, in the developing countries, there is little access to treatment, and access to HIV-prevention services is poor. The '3 by 5' initiative was launched by the WHO and UNAIDS in 2003 with the aim of providing antiretrovirals to three million people in developing countries by the year 2005. HIV infection has a significant negative impact on oral health, with approximately 40-50% of HIV-positive persons developing oral fungal, bacterial, or viral infections early in the course of the disease. Oral health services and professionals can contribute effectively to the control of HIV/AIDS through health education and health promotion, patient care, effective infection control, and surveillance. The WHO Global Oral Health Program has strengthened its work for prevention of HIV/AIDS-related oral disease. The WHO co-sponsored conference, Oral Health and Disease in AIDS, held in Phuket, Thailand (2004), issued a declaration calling for action by national and international health authorities. The aim is to strengthen oral health promotion and the care of HIV-infected persons, and to encourage research on the impact that HIV/AIDS, public health initiatives, and surveillance have on oral health. PMID- 16672545 TI - Innate and adaptive mucosal immunity in protection against HIV infection. AB - The appalling toll on the populations of developing countries as a result of the HIV epidemic shows no signs of abatement. While costly drug therapies are effective in developed nations, the sheer scale of the epidemic elsewhere makes the need for a vaccine an ever more urgent goal. The prevalent DNA prime-viral boost strategy aims to elicit cytotoxic lymphocytes (CTL) against HIV, but this approach is undermined by the rapid mutation of HIV, which thereby escapes CTL control. Alloimmunity has been found to be protective in vertical transmission from infected mothers to their babies, in alloimmunization of women with their partners' mononuclear cells, and in monkeys immunized with SIV grown in human T cells. Vaginal mucosal immunization, as a result of unprotected sex with a regular partner, induced in vitro protection against HIV infection, and this was confirmed in macaques. The second type of natural protection is found in persons with the homozygous 32 CCR5 mutation, a 32-base-pair deletion of the CCR5 gene, which results in a lack of cell-surface expression of CCR5, which is associated with an increase in CC chemokines and the development of CCR5 antibodies. These two 'experiments of nature' have been used to develop vaccine strategies--first, in vaginal immunization of macaques with CCR5 peptides, in addition to HIV envelope (env) and SIV core (gag) antigens, all of which were linked to the 70-kD heat-shock protein (HSP70); and second, in mucosal allo-immunization of macaques, which also gave rise to in vitro protection from infection. Immunization with this vaccine elicited serum and vaginal IgG and IgA antibodies, IFNgamma- and IL 12-producing cells, and increased concentrations of CCL-3 and CCL-4. Vaginal challenge with a simian immunodeficiency virus engineered to carry a human envelope protein (SHIV 89.6) showed significant clearance of SHIV in the immunized macaques. This platform strategy will now be developed to activate the co-stimulatory pathways with the aim of enhancing the primary allogeneic and CCR5 directed responses which are involved in natural protection against HIV infection. PMID- 16672546 TI - The effects of HIV infection on oral mucosal immunity. AB - Oral mucosal infections, especially candidiasis, are a feature of HIV disease, suggesting that compromised mucosal immunity within the oral cavity is a consequence of the viral infection. However, how this mucosal immunity is compromised and at what stage of HIV infection this occurs are unclear. Better understanding of the protection of the oral cavity against infection has allowed us to gain some insight into the local consequences of HIV infection. From a humoral perpective, IgA2 subclasses are reduced in HIV infection in saliva, and total secretory IgA levels are reduced in later disease. Similarly, mucosal antibody responses appear near normal in early HIV infection but reduced in AIDS. There is now convincing evidence that salivary IgA can be neutralizing to HIV 1 and HIV 2, as well as block epithelial transmigration. Oral cellular immunity is also affected by HIV infection. Transmission of HIV from one oral cell type to another appears to be confirmed by work showing that HIV can bind to or infect epithelial cells, Langerhans cells, and other mucosal cells. CXCR4 tropic (via GalCer and CXCR4) and dual tropic HIV strains have been shown to be able to infect normal human oral keratinocytes (NHOKs), and infectious HIV virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection. There is evidence of up-regulation of various receptors, including HIV receptors, on the surface of oral epithelium, and the epithelium may become more permeable. HIV may exploit this antigen uptake mechanism to cross epithelial barriers during co-infection with damage-inducing pathogens such as Candida. Immune responsiveness to many of the co-pathogens associated with HIV has been demonstrated to depend on a family of innate recognition molecules, known as Toll like receptors (TLR), and recognition of a single pathogen can involve activation of multiple TLRs. Consequently, TLR-pathogen interactions could play an indirect but major role in regulating HIV-associated disease in the oral cavity. Thus, HIV infection appears to have both direct and indirect effects on oral mucosal immunity, affecting both cellular and humoral immunity as well as both specific and innate immunity. PMID- 16672547 TI - Dendritic cells and HIV infection: activating dendritic cells to boost immunity. AB - Dendritic cells (DCs) are white blood cells that coordinate innate and adaptive immunity. They are distributed within epithelia and mucosal-associated lymphoid tissues, positioned to entrap incoming pathogens or vaccines. Human immunodeficiency virus (HIV) and the non-human primate equivalent (SIV) exploit DCs to amplify infection, underscoring the need to harness strategies that promote presentation of virus by DCs to stimulate potent anti-viral immunity instead of virus transmission. Two main subsets of DCs need to be considered: myeloid (MDC) and plasmacytoid (PDC) subsets. Using the SIV-macaque system to advance oral vaccine research, we examined macaque PDC and MDC biology, identifying ways to activate DCs and boost antiviral immunity. Immunostimulatory oligodeoxyribonucleotides (ISS-ODNs) stimulated PDC/MDC mixtures to up-regulate co-stimulatory molecule expression and to secrete both IFN-alpha and IL-12. Additionally, ISS-ODNs augmented SIV-specific IFN-gamma responses induced by virus-bearing DCs. ISS-ODN-driven DC activation is being pursued to improve oral/nasopharyngeal mucosal vaccines and therapies against HIV. PMID- 16672548 TI - Role of human beta-defensins in HIV infection. AB - Mechanisms of resistance to HIV-1 infection in the human oral cavity are incompletely understood. While salivary components have been implicated in protection, there is growing evidence that human beta-defensins (hBDs), originating in oral epithelial cells, may be playing an important role in the prevention of HIV infection. New antiviral, chemotactic, and immunosurveillance properties are being attributed to hBDs, which are small cationic antimicrobial innate response molecules expressed in mucosal epithelium. Inducible hBDs are always expressed in normal oral epithelium, a property not shared by other mucosal barriers. Data reviewed in this paper demonstrate that: (1) HIV-1 X4 and R5 phenotypes induce hBD-2 and -3 mRNA in normal human oral epithelial cells; (2) hBD-2 and -3 inhibit HIV-1 infection by both viral strains, with greater activity against X4 viruses; and (3) this inhibition is due to a direct interaction with virions and through modulation of the CXCR4 co-receptor. These properties may be exploited as strategies for mucosal protection against HIV-1 transmission. PMID- 16672549 TI - Oral mucosal expression of HIV-1 receptors, co-receptors, and alpha-defensins: tableau of resistance or susceptibility to HIV infection? AB - The basic premise of whether transmission of HIV-1 through the oral mucosa actually occurs, and through what route, is a topic of intense interest. Our work has focused on HIV-1 receptors/co-receptors and alpha-defensin-1 in situ in human gingiva. Regardless of HIV-1 infection, the role that C-type lectin receptors might play in periodontal pathogenesis is of great interest. We have shown that the gingival lamina propria, when inflamed, becomes increasingly infiltrated with DC-SIGN+MR+ dermal dendritic cells (DDCs), while the inflamed epithelium shows a decrease in Langerin+ Langerhans cells (LCs). Moreover, DDCs and LCs contribute to the mature CD83+ DC pool in situ, and form immune conjugates with CD4+ T-cells in the lamina propria (Jotwani and Cutler, 2003). This raises the intriguing possibility that oral mucosal DCs may be involved in HIV-1 transfer to T-cells in situ. However, this possibility is tendered by the challenges faced by the virus in gaining access to oral mucosal immune cells, including their ability to survive the salivary defenses, cross the mucosal barrier, resist inactivation by alpha-defensins, and overcome the paucity of co-receptor CCR5 in (healthy) oral mucosa (i.e., required for productive infection [Jotwani et al., 2004]). To date, there is little evidence of direct infection by HIV-1 of oral mucosal DCs/T cells and other cells in situ. Abbreviations used in this paper: CP, chronic periodontitis; CCR5, chemokine receptor 5; CXCR4, C-X-C receptor 4; DCs, dendritic cells; DC-SIGN, DC-specific ICAM-3 grabbing non-integrin; DDC, dermal dendritic cells; LCs, Langerhans cells; LP, lamina propria; MR, mannose receptor. PMID- 16672550 TI - Differential mucosal susceptibility in HIV-1 transmission and infection. AB - HIV infection occurs primarily through mucosal surfaces, indicating that protection at mucosal sites may be crucial in prevention and treatment. The host innate and adaptive immune elements provide a level of protection, which differs between mucosal compartments, and appears to be most successful in the oral environment, where transmission is rare. In addition to the distinct oral mucosal architecture and cellular constituents, oral fluids, unlike other mucosal secretions, are rarely a vehicle for HIV infection. Multiple soluble factors may contribute to this antiviral activity, including neutralizing antibodies, secretory leukocyte protease inhibitor (SLPI), antiviral peptides such as defensins and cystatins, glycoproteins including thrombospondin and lactoferrin, and complement components. Understanding the antiviral activities of these and other potential resistance factors is becoming increasingly important in attempts to design treatments in the era of HAART resistance. In this regard, the mechanism of anti-HIV action of SLPI has recently been further elucidated by the discovery of its binding protein/receptor, which plays a key role in the infection of macrophages and may consequently be a novel therapeutic target. Continued elucidation of the unique features of mucosal HIV immunology is essential for understanding HIV pathogenesis and for developing effective vaccines and therapeutics. PMID- 16672551 TI - Oral lesions of HIV disease and HAART in industrialized countries. AB - The epidemiology of HIV-related oral disease in industrialized nations has evolved following the initial manifestations described in 1982. Studies from both the Americas and Europe report a decreased frequency of HIV-related oral manifestations of 10-50% following the introduction of HAART (highly active antiretroviral therapy). Evidence suggests that HAART plays an important role in controlling the occurrence of oral candidosis. The effect of HAART on reducing the incidence of oral lesions, other than oral candidosis, does not appear as significant, possibly as a result of low lesion prevalence in industrialized countries. In contrast to other oral manifestations of HIV, an increased prevalence of oral warts in patients on HAART has been reported from the USA and the UK. HIV-related salivary gland disease may show a trend of rising prevalence in the USA and Europe. The re-emergence of HIV-related oral disease may be indicative of failing therapy. A range of orofacial iatrogenic consequences of HAART has been reported, and it is often difficult to distinguish between true HIV-related oral disease manifestations and the adverse effects of HAART. A possible association between an increased risk of oral squamous cell carcinoma and HIV infection has been suggested by at least three epidemiological studies, with reference to the lip and tongue. These substantial and intensive research efforts directed toward enhancing knowledge regarding the orofacial consequences of HIV infection in the industrialized nations require dissemination in the wider health care environment. PMID- 16672552 TI - Oral lesions in HIV infection in developing countries: an overview. AB - HIV infection is a major global health problem affecting developing and developed countries alike. Oral lesions that are associated with this disease are important, since they affect the quality of life of the patient and are useful markers of disease progression and immunosuppression. Oral lesions in HIV infection have been well-documented in developed countries, but there are fewer reports on oral lesions from developing countries. Oral candidiasis is the most common opportunistic infection seen in all continents. Kaposi's sarcoma has been reported only from Africa and Latin America, while histoplasmosis and penicilliosis were reported in patients with advanced disease from Thailand. HIV associated salivary gland disease has a high prevalence in Africa and Latin America, especially in the pediatric group. It is clear that there are considerable regional variations in the oral manifestations of HIV infection, depending both on the populations studied and on the clinical expertise available, among other factors. Well-designed and -documented studies are necessary for the correct assessment of the nature and magnitude of the problem in developing countries, if oral health measures are to be effectively formulated for the HIV-infected. PMID- 16672553 TI - Skin lesions: mirror images of oral lesion infections. AB - Skin lesions can be the presenting signs for HIV disease and are among the most prevalent manifestations throughout the course of HIV disease. Correlation of skin diseases and HIV disease staging has long been recognized and used to guide medical management in resource-limited settings. The purpose of this paper is to give a review of common skin infections presented in HIV-infected patients. Common skin infections presenting in HIV-infected patients include viral, fungal, mycobacterial, and bacterial infections, along with skin infestation. Key diagnostic points correlate with certain HIV disease staging for many skin diseases. These can help facilitate appropriate diagnosis and referral by health care personnel when treating HIV-infected patients who have skin lesions. Knowledge of common skin manifestations found in HIV-infected patients is essential for all health care personnel who work in the HIV field. Most skin infections presenting in HIV-infected patients can be treated effectively if the correct diagnosis and appropriate referral are made promptly. PMID- 16672554 TI - Implications of HIV disease for oral health services. AB - This paper, by means of a quality framework, reviews health services research in relation to people with HIV infection. The relevance of oral health care services to people's needs is considered in terms of the goal of health services to reduce the burden of disease on the everyday life of the population. Dental services may therefore have a role in primary prevention in the HIV epidemic, passing on information about HIV and promoting health through the early diagnosis and treatment of oral disease. Effectiveness research of oral health care in HIV assesses the usefulness of oral diagnosis, whether care is safe, and whether treatment is clinically effective. Few data are available on the efficiency of services. People with HIV still have problems accessing dental care, due to the volume of care available in relation to their need and acceptability of care. Access problems in the US are compounded by social inequality. Health services research data are particularly sparse in resource-poor countries, and there is a need to translate the available information into treatment guidelines appropriate to these settings. The research community and funding agencies should place greater emphasis on the quality of oral health services for people with HIV. PMID- 16672555 TI - Candida-host interactions in HIV disease: relationships in oropharyngeal candidiasis. AB - Oropharyngeal candidiasis (OPC) caused by the commensal organism, Candida albicans, is the most common oral infection in HIV disease. Although cell mediated immunity (CMI) by Th1-type CD4+ T-cells is considered the predominant host defense mechanism against OPC, other systemic or local immune mechanisms are critical when blood CD4+ T-cells are reduced below a protective threshold. For example, the Th cytokine profile in saliva may influence resistance or susceptibility to OPC. In OPC lesions, CD8+ T-cells become accumulated at the lamina propria-epithelium interface, suggesting some role for CD8+ T-cells against OPC. However, the absence of CD8+ T-cells close to Candida at the outer epithelium indicates that susceptibility to OPC involves a dysfunction in the CD8+ T-cells or in the micro-environment. Further evaluation of the buccal mucosa lesion showed that CD8 T-cell-associated cytokine and chemokine mRNA is increased compared with buccal mucosa from lesion-negative matched controls. The majority of CD8+ T-cells present possess the alphabeta T-cell receptor and several homing receptors (i.e., 4beta7, 4beta1, ebeta7). While several adhesion molecules are similar in OPC+ vs. OPC- persons, E-cadherin is reduced in the tissue of OPC+ persons. These results support evidence for a role for CD8+ T-cells against OPC, but suggest that a putative dysfunction in mucosal T-cell trafficking may be associated with susceptibility to infection. Similar levels of Candida-specific antibodies in persons with and without OPC confirmed a limited role for humoral immunity. Finally, oral epithelial cells inhibit the growth of Candida in vitro in a static rather than a cidal manner. Clinically, oral epithelial cell anti Candida activity is reduced in HIV+ persons with OPC, compared with controls. The mechanism of action includes a strict requirement for cell contact by an acid labile moiety on intact, but not necessarily live, epithelial cells, with no role for soluble factors. Taken together, host defense against OPC involves several levels of activity. The status and efficiency of local host defenses when blood CD4+ T-cells are not available appear to play a role in protection against or susceptibility to OPC. PMID- 16672556 TI - Conceptual emergence of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) as an oral herpesvirus. AB - Recognition of the various clinico-epidemiologic forms of Kaposi's sarcoma, a disease putatively caused by an infectious agent, did not provide ready clues as to how that agent might be transmitted, although fecal and sexual routes were implicated. Application of serologic and genome-detection assays, and cell culture studies following the identification of human herpesvirus 8 as the causative agent now implicate that virus as one that is orally shed. While oral transmission of the virus might account for the viral endemicity in Africa and Mediterranean countries, why it is particularly prevalent among male homosexuals in the West remains more difficult to explain. Such explanation may be sought from behavioral studies into the role saliva plays in sexual interactions. PMID- 16672557 TI - Oral EBV and KSHV infection in HIV. AB - The gamma herpesviruses, Kaposi's-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are tightly associated with the development of AIDS associated oral disease and malignancy during immune suppression. The objective of this investigation was to characterize oral infection and pathogenesis in healthy and immune-suppressed individuals. To characterize oral EBV and KSHV infection, we examined throat washings and oral epithelial cells from HIV positive and HIV-negative individuals. Quantitative/real-time polymerase-chain reaction (PCR) assays, transmission electronmicroscopy, immunostaining, and sequence analysis were used to identify viral infection. Virus was isolated from throat-wash samples and was used to infect epithelial and lymphoid cell lines. We detected EBV and KSHV in the oral cavity in healthy and immune-suppressed individuals. Viral strain analysis of KSHV K1 in multiple clones from the oral cavities of healthy persons and immunosuppressed patients detected several strains previously detected in KS lesions, with minor strain variation within individuals. Immunoelectron microscopy for multiple viral antigens detected consistent expression of viral proteins and oral epithelial specimens. In oral epithelial cells infected with wild-type KSHV in vitro, the K8.1 glycoprotein associated with lytic KSHV infection was detected in both primary and telomerase immortalized oral epithelial cultures by 24 hours post-infection. Virions were detected, subsequent to infection, by scanning electron microscopy. Oral epithelial cells were also infected in vitro with wild-type EBV originating from throat washes. Analysis of these data suggests that, like EBV, KSHV infection is present in the oropharynx of healthy individuals, is transmissible in vitro, and may be transmitted by saliva. PMID- 16672558 TI - Cytomegalovirus co-infection in AIDS-associated oral Kaposi's sarcoma. AB - The increasing appearance of AIDS-associated oral Kaposi's sarcoma (KS) in South Africa may be ascribed to the later start of the HIV epidemic, more patients reaching stages III and IV, and the inaccessibility of most patients to anti retroviral therapy. The objective of this study was to demonstrate cytomegalovirus (CMV) co-infection in oral KS and to consider its possible significance. We reviewed 20 cases of oral KS in known HIV-positive patients without active CMV disease. HHV8 PCR and CMV immunohistochemistry were performed. HHV8 DNA was present in all cases. CMV inclusions were detected in five cases. The significance of CMV co-infection in oral KS is unclear. The inclusions suggest active infection, although there is no evidence to support CMV in the pathogenesis of KS. Nonetheless, it is vital that physicians be alerted to active CMV infection, so that timely intervention and careful observation can be instituted, ensuring early diagnosis and treatment. PMID- 16672559 TI - Biology of HPV in HIV infection. AB - HIV-positive men and women are at increased risk of anogenital and oral HPV infection. The risks for HPV-associated high-grade intra-epithelial neoplasia (IN) and cancer are also increased. The prevalence of oral, anal, and cervical HPV infection in HIV-positive individuals compared with HIV-negative individuals increases with progressively lower CD4+ levels, as does incident high-grade IN. In contrast to IN, development of cancer is not related to lower CD4+ level. With increasing grades of IN and cancer, the proportion of tissues with copy-number abnormalities (CNA) increases, with one of the most common genetic changes being amplification of chromosome 3q. The presence of CNA is associated with the integration of HPV DNA into the host genome, with loss of HPV E2 and/or E2 rearrangement. This suggests a link between CNA and increased HPV-induced chromosomal instability mediated through de-repressed E6 and E7 expression consequent to loss of functional E2 protein. In addition, epigenetic changes occur with increasing frequency in high-grade IN and cancer, such as hypermethylation leading to down-regulation of potential tumor suppressor genes. Analysis of these data together suggests that immune suppression plays a more prominent role in the earlier stages of HPV-associated disease, up to and including incident high-grade IN. Persistent high-grade IN and development of cancer may be more strongly related to the cumulative effect of HPV-associated genetic instability and the resulting host genetic changes. There are few data to suggest a direct role for HIV in the pathogenesis of HPV-associated neoplasia, but HIV-associated attenuation of HPV-specific immune responses may allow for persistence of high-grade IN and sufficient time for accumulation of genetic changes that are important in progression to cancer. PMID- 16672560 TI - (A1) Identification of oral health care needs in children and adults, management of oral diseases. AB - The workshop considered five questions reviewing the identification of international oral health care needs of children and adults, and the management of oral diseases in resource-poor countries: (1) What is the role of the dental profession in the management of the HIV-infected individual? (2) Identifying health care needs-What are the epidemiology and disparities of HIV-associated oral lesions in children from different continents? (3) How effective is HIV treatment in controlling oral diseases? (4) Could we develop basic inexpensive oral and dental care protocols for economically deprived HIV-infected patients? and (5) What is the best method of arranging resources to meet the oral health care needs of people with HIV disease? The consensus of the workshop participants was that there is a need to re-target research efforts to non-established market economy countries and prioritize research in these regions to children with HIV disease. It will be important to assess commonalities and variations in oral health needs across geographical and cultural boundaries, and research efforts should be centralized in resource-poor countries to support multi-center longitudinal standardized studies. It is essential that oral health research be integrated into other health care research programs, to make these research priorities and public health initiatives feasible. PMID- 16672561 TI - (A2) Oral health and general health. AB - The interactions between oral and systemic health are bi-directional and complex, involving many pathways. Regarding health as not merely the absence of disease, but as a state of total well-being, these interactions profoundly influence the progress of many diseases, and the quality of life and economic performance of HIV-infected individuals and populations. The evidence base for specific interactions is currently weak, partly because few good-quality studies have been published, partly because of the naivete of the instruments currently available for recording these interactions and their inherent complexity. Recording quality of life should be a fundamental aspect of all future studies. The most significant conclusion of this Workshop is the need for all involved in oral health research and oral health care to be seen as, and to act as, essential partners in comprehensive care for whole patients and communities. PMID- 16672562 TI - (A3) HIV Phenotypes, oral lesions, and management of HIV-related disease. AB - Workshop participants discussed: the role of HIV subtypes in disease; the treatment of oral candidiasis; the relationship between and among viral load, CD4+ counts, oral candidiasis and oral hairy leukoplakia, pigmentation; and the development of a reliable oral index to predict disease progression. Regarding HIV, the literature revealed that Type I (HIV-I), in particular group M, is involved in the majority (90%) of documented infections, and groups N and O to a lesser extent. Viral envelope diversity led to the subclassification of the virus into nine subtypes, or clades-A-D, F-H, J, and K-each dominating in different geographical areas. HIV-2, currently occurring mostly in West Africa, appears to be less virulent. No evidence could be produced of any direct impact of type, subtype, or clade on oral lesions, and participants believed that further research is not feasible. Oral candidiasis in patients from resource-poor countries should be prevented. When the condition does occur, it should be treated until all clinical symptoms disappear. Oral rinsing with an antimicrobial agent was suggested to prevent recurrence of the condition, to reduce cost, and to prevent the development of antifungal resistance. Lawsone methyl ether, isolated from a plant (Rhinacanthus nasutus leaves) in Thailand, is a cost effective mouthrinse with potent antifungal activity. Evidence from a carefully designed prospective longitudinal study on a Mexican cohort of HIV/AIDS patients, not receiving anti-retroviral treatment, revealed that the onset of oral candidiasis and oral hairy leukoplakia was heralded by a sustained reduction of CD4+, with an associated sharp increase in viral load. Analysis of the data obtained from a large cohort of HIV/AIDS patients in India could not establish a systemic or local cause of oral melanin pigmentation. A possible explanation was a dysfunctional immune system that increased melanin production. However, longitudinal studies may contribute to a better understanding of this phenomenon. Finally, a development plan was presented that could provide a reliable prediction of disease progression. To be useful in developing countries, the index should be independent of costly blood counts and viral load. PMID- 16672563 TI - (B1) Candida and mycotic infections. AB - Oral candidiasis (OC) is the most common mucosal manifestation of HIV infection. This workshop examined OC and other mycoses associated with HIV infection. Historically, blood CD4 cell numbers were the primary prognosticator for the development of OC. However, a study that statistically evaluated the predictive role of HIV viral load vs. CD4 cell counts revealed viral load to be a stronger predictor for OC. The role of biofilms and antifungal resistance in recalcitrant OC is unclear at present. In general, micro-organisms including yeasts in biofilms are more resistant to antifungals than their planktonic counterparts. When the remaining organisms are eliminated, the few resistant organisms may not be problematic, because they are present in low numbers. Unusual exotic mycoses in HIV-infected patients are more common in patients from the developing than the developed world. These infections may be recurrent and recalcitrant to therapy, be present in multiple and uncommon sites, increase with the progression of HIV disease, and may play a role similar to that of the more common mycoses. Typing and subtyping of yeasts are probably not critical to the clinical management of candidiasis caused by Candida albicans and non-albicans strains, including C. dubliniensis, because it is responsive to antifungal therapy. C. glabrata is probably the only exception. The presence of oral thrush in infants younger than 6 months of age is associated with an increased post-natal transmission risk of HIV infection. Thus, perinatal retroviral therapy should be combined with the treatment of oral thrush to prevent the post-natal acquisition of HIV. PMID- 16672564 TI - (B2) Periodontal diseases and other bacterial infections. AB - The workshop addressed the following questions with respect to periodontal diseases and bacterial infections seen in HIV infection: (1) What is linear gingival erythema? Is it prevalent only in HIV disease? A crude Delphi technique was used to ascertain whether LGE existed, but a consensus could not be reached. It was agreed that a diagnosis of LGE should be considered only if the lesion persists after removal of plaque in the initial visit. (2) Do periodontal pockets contribute to viremia in HIV infection? At present, the data are not available to answer this question. (3) Do anti-viral drugs reach the sulcular fluid in significant concentrations? No one at the workshop was aware of data that could answer this question. (4) Does concurrent tuberculosis infection modify the oral manifestations of HIV infection? Though analysis of data from the developing countries does suggest an association between tuberculosis and oral candidiasis, more data and multivariate analysis considering immunosuppression as a confounding factor are necessary, for any conclusions to be derived. (5) What pathogens are involved in periodontal diseases in HIV infection? Periodontal disease may be initiated by conventional periodontal pathogens. But the progression and tissue destruction depend upon the presence of typical and atypical micro-organisms, including viruses, their by-products, increased secretion of potentially destructive inflammatory mediators, and overwhelming host response. (6) How can we diagnose the diseases seen in HIV infection? The answer can be obtained only with data from controlled and blinded studies. It is necessary to design collaborative multi-center longitudinal studies. The results obtained from such large sample sizes can contribute eventually to interpretation of the outcome. PMID- 16672565 TI - (B3) Markers of immunodeficiency and mechanisms of HAART therapy on oral lesions. AB - Highly active anti-retroviral therapy (HAART) has revolutionized the treatment and prognosis of HIV disease and AIDS in those who can take advantage of the treatment. There are currently 20 different anti-retroviral drugs in 4 different classes that are used in specific combinations. Suppression of HIV replication and immune reconstitution are goals of therapy. Since the prevalence of some easily detectable oral manifestations of HIV/AIDS (OMHIV/AIDS) decreases with HAART, it has been suggested that they might be clinically useful surrogate markers of HAART efficacy and immune status. This might be particularly useful if their recurrence presaged or accompanied HAART failure. To date, there has been little work in this area, but its potential value to the clinical management of HIV/AIDS is apparent, especially if frequent measures of viral load and CD4 cell counts are not readily available. However, the usefulness of OMHIV/AIDS as signals for HAART failure is complicated by three phenomena: the immune reconstitution syndrome, the similarity of some adverse reactions of HAART to OMHIV/AIDS, and the direct inhibitory effect of HAART medications on some OMHIV/AIDS (e.g., inhibition of oral candidosis by protease inhibitors). This workshop considered the current evidence and proposed pertinent research questions. PMID- 16672566 TI - (C2) Saliva, breast milk, and mucosal fluids in HIV transmission. AB - The oral environment has received various amounts of attention in association with HIV infection and pathogenesis. Since HIV infection occurs through mucosal tissue, oral factors-including tissue, fluids, and compartments-are of interest in furthering our understanding of the diagnosis, infectivity, transmission, and pathogenesis of disease. This report reviews: (1) HIV testing and diagnoses with oral fluids; (2) post-natal acquisition of HIV in association with breast-feeding from HIV-positive mothers; and (3) oral sex and HIV transmission. In the first, we examine how oral fluids are used to detect HIV infection and review current consensus on the role of salivary molecules as markers for immunosuppression. Second, lactation-associated HIV acquisition is reviewed, with special consideration of emerging issues associated with the impact of anti-retroviral therapies. Last, we consider current data on the risk of HIV infection in association with oral sex. Investigation of these diverse topics has a common goal: understanding how HIV presents in the oral environment, with an aim to rapid and accessible HIV diagnosis, and improved prevention and treatment of infection. PMID- 16672567 TI - (C3) The oral epithelial cell and first encounters with HIV-1. AB - The oral epithelium is the site of first exposure of HIV-1 to host tissues during oral sex with an infected partner or through breast-feeding by an infected mother. Although the oral epithelium is distinguishable by its apparent resistance, the mucosal surfaces represent a primary target of HIV-1. After oral exposure and swallowing, infection is detected prominently in the gastrointestinal tract, which becomes depleted of CD4+ T-cells. The oral cavity and palatine tonsils appear to resist infection and transfer to susceptible lymphoid cells in the lamina propria by local anti-HIV-1 mechanisms. In some cases, expression of these antiviral mechanisms increases after exposure to HIV 1. During primary exposure and before seroconversion, based on limited in vitro and primate data, a window of opportunity for capture of HIV-1 by the oral epithelium may exist. After seroconversion, the risk of infectious HIV-1 appearing in saliva is negligible. This report considers evidence that oral epithelium has the potential both to enable and to resist infection by HIV-1. PMID- 16672570 TI - The comparative pathology of Clostridium difficile-associated disease. AB - Clostridium difficile is a confirmed pathogen in a wide variety of mammals, but the incidence of disease varies greatly in relation to host species, age, environmental density of spores, administration of antibiotics, and possibly, other factors. Lesions vary as well, in severity and distribution within individuals, and in some instances, age groups, of a given species. The cecum and colon are principally affected in most species, but foals and rabbits develop severe jejunal lesions. Explanations for variable susceptibility of species, and age groups within a species, are largely speculative. Differences in colonization rates and toxin-receptor densities have been proposed. Clostridium difficile associated disease is most commonly diagnosed in Syrian hamsters, horses, and neonatal pigs, but it is reported sporadically in many other species. The essential virulence factors of C. difficile are large exotoxins, toxin A (TcdA) and toxin B (TcdB). Receptor-mediated endocytosis of the toxins is followed by endosomal acidification, a necessary step for conversion of the toxin to its active form in the cytosol. Cell-surface receptors have been characterized for TcdA, but remain to be identified for TcdB. Both TcdA and TcdB disrupt the actin cytoskeleton by disrupting Rho-subtype, intracellular signaling molecules. Disruption of the actin cytoskeleton is catastrophic for cellular function, but inflammation and neurogenic stimuli are also involved in the pathogenesis of the disease. PMID- 16672569 TI - Antinociceptive and hypothermic effects of Salvinorin A are abolished in a novel strain of kappa-opioid receptor-1 knockout mice. AB - Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the kappa-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as kappa-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 microg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of kappa-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional kappa-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for kappa 1- but not kappa 2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for kappa(1)-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the kappa-opioid receptor. PMID- 16672571 TI - Canine indolent nodular lymphoma. AB - Sixty-six cases of indolent canine lymphoid proliferation were reviewed. Age ranged from 1.5 to 16 years (median 9.0 years). Dogs of 26 breeds, plus 13 of mixed breeding or unknown lineage, were represented. B-Cell lymphomas (CD79a+) predominated. Marginal zone lymphoma (MZL), the largest group, involved lymph node (33 cases) and spleen (13 cases), with both tissues involved in five of these cases. Follicular lymphoma (FL) involved lymph nodes (five cases), and mantle cell lymphoma (MCL) occurred as solitary splenic masses (three cases). Nodal CD3+ T-zone lymphomas (TZL) (10 cases), were included since they resembled late-stage MZL at the architectural level. Two cases of marginal zone hyperplasia (MZH) were included to aid in differentiation of early MZL. Clonality status was determined in 54 cases by analysis of immunoglobulin heavy chain (IGH) and T-cell antigen receptor gamma (TCRG) gene rearrangement. Clonal rearrangement of IGH was detected in 28 of 35 MZL cases (80%), four of four FL cases (100%) and three of three MCL cases (100%). Concurrent cross lineage rearrangement of TCRG was detected in six MZL and two FL cases. Clonal rearrangement of TCRG was documented in five of eight TZL cases (63%). Limited survival data obtained for 18 dogs indicated that the B-cell lymphomas (MZL, MCL, and FL) and the T-cell lymphoma (TZL) were associated with indolent behavior and long survival. Although to the authors' knowledge, the true incidence of canine indolent lymphomas is unknown, the tumors are not rare and may have been underrecognized. Recognition of their architectural features, routine application of immunophenotyping, and molecular clonality assessment should alleviate this. PMID- 16672572 TI - Comparison of histopathologic criteria and skeletal muscle fixation techniques for the diagnosis of polysaccharide storage myopathy in horses. AB - The purpose of the study reported here was to determine the effect of three methods of fixation of skeletal muscle biopsy specimens on the histopathologic appearance of muscle sections and to determine criteria that were most consistently associated with a diagnosis of polysaccharide storage myopathy (PSSM) in horses. Surgically excised semimembranosus muscle biopsy specimens were obtained from nine horses previously diagnosed with PSSM and from 15 control horses. Portions of each specimen were fixed in formalin, frozen immediately, and chilled for 24 hours prior to freezing. Sections stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and amylase-PAS were scored for histopathologic criteria by three investigators blinded to the sample origin. The presence of amylase-resistant, abnormal polysaccharide was found to be the most sensitive and specific diagnostic indicator for PSSM, and was readily detected regardless of the fixation technique or investigator. Other less-specific features associated with PSSM included atrophy and cytoplasmic and subsarcolemmal vacuoles; however, their histologic scores varied among fixation technique and investigators. Scores for subsarcolemmal and cytoplasmic amylase-sensitive glycogen in horses with PSSM were similar to those for control horses and varied among fixation techniques. In conclusion, PSSM is most accurately diagnosed in muscle biopsy specimens on the basis of appearance of amylase-resistant, abnormal polysaccharide, not amylase-sensitive glycogen, regardless of fixation technique. In general, frozen sections appeared to be better suited for studying myopathies because many histopathologic features of skeletal muscle were obscured by formalin fixation. PMID- 16672573 TI - Ubiquitin expression in muscle from horses with polysaccharide storage myopathy. AB - Serial sections of formalin-fixed, paraffin-embedded muscle biopsy specimens from 28 Quarter Horse, Paint, and draft-related breeds, aged 0.5-23 years, were treated with periodic acid-Schiff (PAS) stain for glycogen and were immunostained to detect ubiquitin expression. On the basis of findings in PAS-stained sections, a diagnosis of equine polysaccharide storage myopathy (EPSSM) was made in 22 horses aged 2-23 years (mean, 9.4 years); samples from 6 horses aged 0.5-15 years (mean, 7.3 years) had a normal PAS staining pattern, with no relevant lesions. Ubiquitin expression was detected in all but a 2-year-old EPSSM-affected horse and was not detected in the non-EPSSM-affected horses. Ubiquitin expression was greater than the degree of PAS-positive, amylase-resistant material, and ubiquitin was detected in aggregates of amylase-sensitive glycogen as well as in aggregates of amylase-resistant material. Results suggest that glycogen aggregates develop and are ubiquitinated prior to development of amylase resistant inclusions. Ubiquitin immunostaining may be most useful for confirming the diagnosis of EPSSM in horses with only amylase-sensitive glycogen aggregates and in horses with early amylase-resistant inclusions. However, ubiquitin immunostaining is no more sensitive than is PAS staining for diagnosis of EPSSM. PMID- 16672574 TI - Renal tubular-cell neoplasms in black-footed ferrets (Mustela nigripes)--38 cases. AB - Thirty-eight cases of renal tubular cell neoplasms were diagnosed in 184 captive, adult (>1-year-old), black-footed ferrets (Mustela nigripes) examined from 1985 to 1996. This prevalence (20.7%) is one of the highest reported for this neoplasm in a population of animals. These tumors rarely metastasized (1/38), and usually were incidental postmortem findings, associated clinical disease being present in only 3 (8%) of the 38 cases. The prevalence of renal tubular cell neoplasms found at postmortem examination increased linearly with age, up to 67% in ferrets >8 years old. Both males (prevalence = 19%) and females (prevalence = 24%) were affected. Multiple renal tumors were common, and seven ferrets (18.4% of affected animals) had bilateral tumors. The cause of this neoplastic syndrome could not be determined. Since most of the animals affected by this condition were in their postreproductive years of life, the impact of this neoplastic syndrome on the captive propagation of this species is negligible. PMID- 16672575 TI - Evidence of breed-dependent differences in susceptibility to porcine circovirus type-2-associated disease and lesions. AB - Porcine circovirus type 2 (PCV2) has been confirmed as the primary cause of postweaning multisystemic wasting syndrome (PMWS). However, in the field, PMWS is seen only in a small percentage of pigs infected with PCV2. The overall objective of the study reported here was to determine whether host genetic differences in the susceptibility to PCV2-associated disease exist among selected breeds of pigs. This study included Duroc (n = 23), Landrace (n = 19), and Large White (n = 21) pigs. The pigs were infected intranasally and intramuscularly at 5-7 weeks of age with PCV2. A portion of the pigs (31/63; 30.2%) had low passively acquired PCV2 antibodies at the time of infection. There were no differences in mean weight gain, rectal temperature, or respiratory score. Clinical disease compatible with PMWS was observed only in the Landrace pigs. Most of the PCV2 infected pigs had enlarged lymph nodes, and individual Duroc and Landrace pigs had mottled tan lungs. PCV2-associated lymphoid depletion and granulomatous inflammation were observed in pigs of all breeds. Three of 19 Landrace pigs and none of the Duroc or Large White pigs developed severe lymphoid lesions associated with large amounts of intralesional PCV2 antigen typical of PMWS. Compared with seronegative Landrace pigs, Landrace pigs that had low maternal antibodies at the time of PCV2 inoculation had significantly (P < 0.05) less severe PCV2-associated lesions. The results suggest a predisposition of the Landrace pigs of this study to PCV2-induced disease and lesions, and that low levels of passively acquired antibodies are protective. PMID- 16672576 TI - Cerebellar hypoplasia associated with an avian leukosis virus inducing fowl glioma. AB - Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), shows tumorigenicity and pathogenicity, mainly in the nervous system, and causes astrocytoma and perineurioma. Apart from these neoplasms, cerebellar anomaly was found in chickens infected with FGV in ovo. The study reported here describes the morphologic characteristics of the affected cerebellum. Specific-pathogen-free chickens (C/O) were inoculated with FGV through the yolk sac on the 7th day of incubation. The cerebellar anomaly included diffuse depletion of granular cells of the internal granular layer (IGL), remnants of the external granular layer (EGL), and disorganization of the Purkinje cell layer. These cerebellar changes were observed in all birds except one. In the infected embryos, the EGL was thicker and had an irregular arrangement with a thin molecular layer (ML) and IGL, compared with the control. The granular cells were immunohistochemically positive for ALV common antigen. Immunohistochemical analysis for vimentin revealed disarrangement and decreased number of Bergmann's fibers. Use of the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling method and electron microscopy indicated that apoptotic granular cells were frequently observed in the EGL and ML. These results suggested that the cerebellar anomaly was hypoplasia, principally resulting from the apoptosis of granular cells in the EGL and ML caused by FGV infection and that the cell loss induced obstruction of granular cell migration and disarrangement of Bergmann's fibers in the ML. PMID- 16672577 TI - Massive mortality of common carp (Cyprinus carpio carpio) in the St. Lawrence River in 2001: diagnostic investigation and experimental induction of lymphocytic encephalitis. AB - A massive fish kill affecting exclusively common carp (Cyprinus carpio carpio) in the St. Lawrence River, Quebec, Canada, during the summer of 2001 was investigated by use of laboratory diagnostic methods and by an attempt to experimentally induce the disease. The ultimate causes of mortality were opportunistic bacterial infections with Aeromonas hydrophila and Flavobacterium sp. secondary to immunosuppression induced by physiologic (i.e., spawning) and environmental (i.e., high temperatures and low water levels) stressors, and possibly enhanced by an infection causing lymphocytic encephalitis observed in 9 of 18 (50%) fish examined. Experimental induction of disease was attempted in captured wild carp by administration of crude and filtered (particulate <0.22 microm) inocula prepared from a homogenate of tissues from carp affected by the natural outbreak. Although significant clinical disease or mortality was not induced by experimental challenge, lymphocytic encephalitis similar to the one observed in naturally affected carp was induced in four of seven (57%) fish administered crude inoculum and four of seven (57%) fish administered filtered inoculum. None of the control fish inoculated with sterile phosphate-buffered saline (n = 6) were affected by encephalitis. The cause of the encephalitis observed in carp from the natural outbreak and in experimentally inoculated fish could not be determined by use of virus isolation and transmission electron microscopy. PMID- 16672578 TI - Intranuclear coccidiosis in tortoises: nine cases. AB - Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. Intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. Intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises. PMID- 16672579 TI - The 2000 canine distemper epidemic in Caspian seals (Phoca caspica): pathology and analysis of contributory factors. AB - More than 10,000 Caspian seals (Phoca caspica) were reported dead in the Caspian Sea during spring and summer 2000. We performed necropsies and extensive laboratory analyses on 18 seals, as well as examination of the pattern of strandings and variation in weather in recent years, to identify the cause of mortality and potential contributory factors. The monthly stranding rate in 2000 was up to 2.8 times the historic mean. It was preceded by an unusually mild winter, as observed before in mass mortality events of pinnipeds. The primary diagnosis in 11 of 13 seals was canine distemper, characterized by broncho interstitial pneumonia, lymphocytic necrosis and depletion in lymphoid organs, and the presence of typical intracytoplasmic inclusion bodies in multiple epithelia. Canine distemper virus infection was confirmed by phylogenetic analysis of reverse transcriptase-polymerase chain reaction products. Organochlorine and zinc concentrations in tissues of seals with canine distemper were comparable to those of Caspian seals in previous years. Concurrent bacterial infections that may have contributed to the mortality of the seals included Bordetella bronchiseptica (4/8 seals), Streptococcus phocae (3/8), Salmonella dublin (1/8), and S. choleraesuis (1/8). A newly identified bacterium, Corynebacterium caspium, was associated with balanoposthitis in one seal. Several infectious and parasitic organisms, including poxvirus, Atopobacter phocae, Eimeria- and Sarcocystis-like organisms, and Halarachne sp. were identified in Caspian seals for the first time. PMID- 16672580 TI - T-cell lymphoma with eosinophilic infiltration involving the intestinal tract in 11 dogs. AB - Among the intestinal tumors of hematopoietic cell origin, lymphoma is the most common in the dog. Herein, we characterized the clinical and pathologic features of 11 dogs (average age, 10.6 +/- 2.5 years) with T-cell lymphoma of the intestinal tract with eosinophil infiltrates. No sex predominance was apparent. All had localized tumor masses in the small intestine. Grossly, the intestinal wall was thickened, and the lumen of the affected intestine was usually narrowed. Microscopically, we observed transmural diffuse invasion of round to pleomorphic tumor cells. Tumor cells showed varying morphology, from scanty to abundant cytoplasm, and round to ovoid nuclei with scattered to dense chromatin. In seven of the dogs, tumor cells had infiltrated into the epithelium. All showed infiltration of eosinophils and all 11 tumors had a T-cell phenotype (CD3+, CD79 ). Only one tumor stained positive for the mast cell marker c-kit and none was positive for mast cell tryptase. We did not observe ultrastructurally apparent granules in any of the tumor cells. These results suggest that, in dogs, T-cell lymphomas of intestinal origin resemble mast cell tumors of intestinal origin with respect to cell structure and eosinophil infiltration. Therefore, in the absence of epitheliotropism, it is difficult to confirm the differential diagnosis without immunostaining for mast cell and lymphocyte markers, including mast cell tryptase, c-kit, CD3, and CD79. PMID- 16672581 TI - Multiple endocrine neoplasia type-I-like syndrome in two cats. AB - Multiple endocrine neoplasia (MEN) embodies a group of diseases in human patients and domestic animals that are characterized by hyperplasia or neoplasia, or both, of two or more endocrine tissues. The MEN-1 syndrome is associated with menin gene mutations that induce various combinations of parathyroid, pituitary, and pancreatic endocrine tumors in humans. Two male, Domestic Shorthair cats developed symmetric alopecia, insulin-resistant diabetes mellitus, and pituitary dependent hyperadrenocorticism at 12 and 13 years of age. Examination of skin biopsy specimens revealed atrophic dermatosis associated with hyperadrenocorticism. In one cat, cutaneous lesions consistent with paraneoplastic alopecia associated with pancreatic adenocarcinoma also were evident. Multiple invasive pancreatic beta cell carcinomas, pituitary corticotroph adenomas, and thyroid C-cell and parathyroid chief cell hyperplasia were diagnosed on the basis of results of gross, histologic, and immunohistochemical findings in both cats. Pancreatic exocrine adenocarcinoma was diagnosed in both cats. One cat also had hepatocellular carcinoma. Exons 1-8 of the feline menin gene were sequenced and were found to bear 93% homology with the human gene sequence, and the corresponding amino acid sequences shared 98% homology. Purification of total RNA and amplification of cDNA from lesional tissues to document mutations in the feline menin gene sequence were unsuccessful. The combination of lesions observed was consistent with the diagnosis of MEN-1-like syndrome in both cats. PMID- 16672582 TI - Renal adenocarcinoma in a ferret. AB - A spontaneous case of renal tumor was observed in a 7-year-old ovariectomized female pet ferret (Mustela putorius furo). Clinical signs included exhaustion, emaciation, anorexia, and stooping position. At necropsy, a solid and cystic mass replaced the left kidney and adrenal gland. The tumor was composed of pleomorphic epithelial cells with a large number of giant cells. Metastases were recognized in the lung, liver, greater omentum, right renal pelvis, and systemic lymph nodes. Immunohistochemical stains revealed that the tumor cells were positive for CD10, cytokeratin (CAM 5.2), and Ki-67 (MIB-1). On the basis of morphologic and immunohistochemical features, the tumor was diagnosed as a pleomorphic renal adenocarcinoma. This type of neoplasm is very rare in all species and has never been reported in a ferret. PMID- 16672583 TI - Intestinal choristoma in the subcutis of a dog. AB - A 2-year-old, spayed female, Labrador Retriever-cross presented with a subcutaneous mass of several weeks' duration in the right flank region. Surgical excision and histologic examination were performed. The 1.0-cm-diameter mass was circumscribed, unencapsulated, and cystic with a bilayer wall. The inner layer resembled intestinal mucosa, including a tall columnar lining epithelium, crypt like glands containing scattered neuroendocrine cells that were strongly immunopositive for synaptophysin, and a supporting lamina propria-like fibrovascular tissue that contained lymphocytes and plasma cells. The outer layer was 1- to 2-mm thick and was composed of intersecting and blending bundles of smooth muscle and collagen. Given the presence of organized intestinal tissues in the subcutis, the lesion was consistent with intestinal choristoma. PMID- 16672584 TI - Gastric adenocarcinoma and chronic gastritis in two related Persian cats. AB - Two 12.5-year-old castrated male Persian cats from the same household, whose dams were littermates, presented simultaneously with gastric adenocarcinoma associated with proliferative and fibrosing gastritis. Intralesional adult Ollulanus tricuspis nematodes and rare surface-associated spiral-shaped bacteria were identified in one cat. No etiologic agents were identified in tissues from the second cat; however, gastric mucosa was examined following anthelmintic treatment. Clinical signs in each cat had commenced 2 months apart and included vomiting, hematemesis, intermittent melena, and weight loss. This is the first report of gastric adenocarcinoma occurring in housemate cats or cats of common descent. Carcinogenesis may have been influenced by shared undetermined genetic and environmental factors, possibly including Ollulanus tricuspis, spiral-shaped bacteria, or other etiologies for chronic gastritis that remain unidentified. PMID- 16672586 TI - Abnormal prion protein in ectopic lymphoid tissue in a kidney of an asymptomatic white-tailed deer experimentally inoculated with the agent of chronic wasting disease. AB - Chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) of deer and elk, is one of a group of fatal, neurologic diseases that affect several mammalian species, including human beings. Infection by the causative agent induces accumulations of an abnormal form of prion protein (PrPres) in nervous and lymphoid tissues. This report documents the presence of PrPres within ectopic lymphoid follicles in a kidney of a white-tailed deer that had been experimentally inoculated by the intracerebral route with CWD 10 months previously. The deer was nonclinical, but spongiform lesions characteristic of TSE were detected in tissues of the central nervous system (CNS) and PrPres was seen in CNS and in lymphoid tissues by immunohistochemistry. The demonstration of PrPres in lymphoid tissue in the kidney of this deer corroborates a recently published finding of PrPres in lymphoid follicles of organs other than CNS and lymphoid tissues in laboratory animals with TSE (scrapie). PMID- 16672585 TI - Immunophenotypic characterization of peripheral blast cells in a leukemic miniature pig. AB - The health status of a 4-year-old female, dd-haplotype miniature pig deteriorated rapidly, so the animal finally had to be euthanized because of poor clinical condition. Necropsy revealed a massive leukocytic infiltration in the parenchymatous organs of the abdominal cavity. On hematologic cell counting, severe leukocytosis (69.3 x 10(9) cells/liter) and high-grade basophilia (6.9 x 10(9) cells/liter) were evident. Cytologic examination, as well as analysis of expression of leukocyte differentiation antigens by means of flow cytometry, classified blasts, which accounted for about 22% of leukocytes, as biphenotypic cells co-expressing the myeloid marker SWC3 (CD172a) and the lymphoid markers CD5 and CD25. Hematologic features resembled those seen in humans with chronic myeloid leukemia at blast phase. PMID- 16672587 TI - Sudden death associated with Clostridium sordellii in captive lions (Panthera leo). AB - In the spring of 2003, a series of sudden deaths in a group of adult lions (Panthera leo) with a previous history of depression, inanition, and lethargy, was investigated. Five animals died within 24 to 36 hours after onset of signs of disease. Serologic screening for viral disease detection was negative, evidence of parasites was not detected, and results of a complete blood count and serum biochemical analysis were within reference intervals in all lions. The most relevant lesions observed were multiple areas of necrosis and hemorrhage in the intestinal outer muscular layer, and cellulitis with an intense bloody edema in the mesenteric and the pericardial fat tissue. On the basis of the fulminant course of the disease, the gross and histologic findings, and the isolation and identification of Clostridium sordellii, a diagnosis of infectious myositis and cellulitis associated with acute clostridiosis was made. To the authors' knowledge, this is the first report of sudden death associated with C. sordellii in felines. PMID- 16672588 TI - Carcinoma ex pleomorphic adenoma with sebaceous differentiation in the mandibular salivary gland of a dog. AB - A carcinoma ex pleomorphic adenoma was diagnosed in the left mandibular salivary gland of an 8-year-old female spayed dog. The animal presented with a large nonpainful swelling in the left submandibular region. A computed tomography scan detected an irregularly enhancing soft tissue mass that was closely associated with the left external ear canal and extended to the left wing of the atlas. On surgical exploration, the mass was intimately associated with the left mandibular salivary gland. Both the mass and the adjacent gland were removed, and the diagnosis was determined by histopathology. The tumor was comprised of basaloid and low columnar epithelial cells, many glandular units formed by well differentiated sebocytes, and multifocal regions of necrosis, mineralization, and hemorrhage. Salivary gland tumors with sebaceous differentiation are very rare in animals, with one previously reported case in a cat. PMID- 16672589 TI - Spontaneous deciduosarcoma in a domestic rabbit (Oryctolagus cuniculus). AB - Deciduosarcoma is a rare, hormonally dependent neoplasm with features of malignancy, previously reported only in rabbits enrolled in chronic toxicology studies involving estrogens with or without progestins. An exploratory laparotomy was performed on a 6-year-old pet Dutch dwarf rabbit following palpation of a 6 cm-diameter abdominal mass. Grossly, the mass was fleshy and nodular, adhered to but not appearing to originate from the small intestine, with a smaller mass of similar appearance involving the uterus, and an effaced mesenteric lymph node. Histologically, the mass was characterized by spindloid cells and large epithelioid cells with abundant pale eosinophilic vacuolated cytoplasm and an infiltrative pattern of growth. Giant cells with large, bizarre, hyperchromatic nuclei were common. Cells were positive by immunohistochemistry for vimentin and progesterone and estrogen receptors and negative for pancytokeratin (AE1/AE3), cytokeratin 18, desmin, alpha-smooth muscle actin (SMA), and CD10. Based on histologic and immunohistochemical findings, a diagnosis of deciduosarcoma was made. PMID- 16672590 TI - Listeric meningoencephalomyelitis in a cougar (Felis concolor): characterization by histopathologic, immunohistochemical, and molecular methods. AB - Listeria monocytogenes has been recognized as an important food-borne pathogen in animals. Records of the disease caused by this bacterium in large felids are, however, rare. The nervous form of listeriosis was diagnosed in a 12-year-old male cougar (Felis concolor) with a several-day history of neurologic disease characterized by excess salivation, head pressing, and circling that progressed to recumbency and death. Microscopically, the main alteration in the brain and spinal cord was a variably severe meningoencephalomyelitis composed mainly of mononuclear cell aggregates with fewer neutrophils. L. monocytogenes was isolated from the brain by microbiological culture, and L. monocytogenes antigen was detected in formalin-fixed, paraffin-embedded sections of brain and spinal cord by immunohistochemical analysis. On the basis of the nucleotide sequence of the 16S rRNA gene, the isolated strain was determined to be serotype 1/2a. Food-borne transmission of the bacterium was suspected, but food was not available for testing. PMID- 16672591 TI - Canine cardiac mesothelioma with granular cell morphology. AB - Cardiac mesothelioma with granular cell features was diagnosed in a 10-year-old Golden Retriever presenting with pericardial and abdominal effusions. The diagnosis was based on gross, morphologic, and immunohistochemical features. The immunohistochemical profile of the neoplasm was pancytokeratin positive, vimentin positive, and S-100 negative; most gross and morphologic features were consistent with both mesothelioma and granular cell tumor. To the authors' knowledge, the prognosis for either primary cardiac mesothelioma or granular cell tumor in the dog is unknown. At 4 months after thoracotomy, pericardectomy, and mass excision, this dog was alive and without clinical evidence of pericardial or abdominal effusions. We describe a granular morphologic variant of cardiac mesothelioma in a dog. PMID- 16672592 TI - Multicentric physeal dysplasia in two cats. AB - Feline physeal dysplasia typically presents as unilateral or bilateral, atraumatic, slipped capital femoral epiphysis. The femoral physeal lesion consists of retention of a cartilaginous physis beyond the expected age of closure, with disorganization of the chondrocytes and subsequent slippage. In this article, we describe two cats with feline physeal dysplasia and slipped capital femoral epiphysis that died of unrelated causes (cardiomyopathy and lymphosarcoma). At necropsy, additional sites were found to have retained physes with similar abnormal arrangement of chondrocytes. This confirms that physeal dysplasia in cats is a widespread multicentric disorder of chondrocytes that precedes the development of slipped capital femoral epiphysis. PMID- 16672593 TI - Systemic granulomatous disease and sialometaplasia in a dog with Bartonella infection. AB - Systemic granulomatous disease involving the spleen, heart, lymph nodes, omentum, liver, kidney, lung, mediastinum, and salivary glands developed in an 8-year-old Rottweiler. The dog also had sialometaplasia of both submandibular salivary glands. Bartonella henselae and B. vinsonii subsp. berkhoffii DNA was amplified from the salivary gland by polymerase chain reaction analysis. Bartonellae may be the cause of this systemic disease, but to the authors' knowledge, involvement of omentum, mediastinum, and salivary glands has not previously been reported in association with Bartonella infection. Bartonellae should be considered potential causes of sialometaplasia. PMID- 16672595 TI - The difficult road from sequence to function. PMID- 16672596 TI - The International Bacillus anthracis, B. cereus, and B. thuringiensis Conference, "Bacillus-ACT05". PMID- 16672597 TI - Diffusion of green fluorescent protein in three cell environments in Escherichia coli. AB - Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments. PMID- 16672598 TI - Characterization of the ArsRS regulon of Helicobacter pylori, involved in acid adaptation. AB - The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR approximately P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR approximately P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the -10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR approximately P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene. PMID- 16672599 TI - Ecf, an alternative sigma factor from Neisseria gonorrhoeae, controls expression of msrAB, which encodes methionine sulfoxide reductase. AB - A DNA microarray was used to identify genes transcribed in Neisseria gonorrhoeae using Ecf, an alternative sigma factor. No differences between the transcriptional profiles of strain FA1090 and a mutant where ecf had been inactivated could be detected when both were grown in vitro. We therefore constructed a gonococcal strain in which Ecf can be overexpressed. Some differentially expressed genes are clustered with ecf on the genome and appear to form a single transcriptional unit. Expression of the gene encoding MsrAB, which possesses methionine sulfoxide reductase activity, was also dependent on Ecf, suggesting that the regulon responds to oxidative damage. Western blotting confirmed that the increased level of MsrAB protein is dependent on the presence of Ecf. PMID- 16672600 TI - Localization of the Escherichia coli RNA polymerase beta' subunit residue phosphorylated by bacteriophage T7 kinase Gp0.7. AB - During bacteriophage T7 infection, the Escherichia coli RNA polymerase beta' subunit is phosphorylated by the phage-encoded kinase Gp0.7. Here, we used proteolytic degradation and mutational analysis to localize the phosphorylation site to a single amino acid, Thr(1068), in the evolutionarily hypervariable segment of beta'. Using a phosphomimetic substitution of Thr(1068), we show that phosphorylation of beta' leads to increased rho-dependent transcription termination, which may help to switch from host to viral RNA polymerase transcription during phage development. PMID- 16672601 TI - Loss- and gain-of-function mutations in the F1-HAMP region of the Escherichia coli aerotaxis transducer Aer. AB - The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants. PMID- 16672602 TI - Signaling interactions between the aerotaxis transducer Aer and heterologous chemoreceptors in Escherichia coli. AB - Aer, a low-abundance signal transducer in Escherichia coli, mediates robust aerotactic behavior, possibly through interactions with methyl-accepting chemotaxis proteins (MCP). We obtained evidence for interactions between Aer and the high-abundance aspartate (Tar) and serine (Tsr) receptors. Aer molecules bearing a cysteine reporter diagnostic for trimer-of-dimer formation yielded cross-linking products upon treatment with a trifunctional maleimide reagent. Aer also formed mixed cross-linking products with a similarly marked Tar reporter. An Aer trimer contact mutation known to abolish trimer formation by MCPs eliminated Aer trimer and mixed trimer formation. Trimer contact alterations known to cause epistatic behavior in MCPs also produced epistatic properties in Aer. Amino acid replacements in the Tar trimer contact region suppressed an epistatic Aer signaling defect, consistent with compensatory conformational changes between directly interacting proteins. In cells lacking MCPs, Aer function required high level expression, comparable to the aggregate number of receptors in a wild-type cell. Aer proteins with clockwise (CW)-biased signal output cannot function under these conditions but do so in the presence of MCPs, presumably through formation of mixed signaling teams. The Tar signaling domain was sufficient for functional rescue. Moreover, CW-biased lesions did not impair aerotactic signaling in a hybrid Aer-Tar transducer capable of adjusting its steady-state signal output via methylation-dependent sensory adaptation. Thus, MCPs most likely assist mutant Aer proteins to signal productively by forming collaborative signaling teams. Aer evidently evolved to operate collaboratively with high-abundance receptors but can also function without MCP assistance, provided that it can establish a suitable prestimulus swimming pattern. PMID- 16672603 TI - Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. AB - Bacterial populations contain persisters, cells which survive exposure to bactericidal antibiotics and other lethal factors. Persisters do not have a genetic resistance mechanism, and their means to tolerate killing remain unknown. In exponentially growing populations of Escherichia coli the frequency of persister formation usually is 10(-7) to 10(-5). It has been shown that cells overexpressing either of the toxic proteins HipA and RelE, both members of the bacterial toxin-antitoxin (TA) modules, have the ability to form more persisters, suggesting a specific role for these toxins in the mechanism of persistence. However, here we show that cells expressing proteins that are unrelated to TA modules but which become toxic when ectopically expressed, chaperone DnaJ and protein PmrC of Salmonella enterica, also form 100- to 1,000-fold more persisters. Thus, persistence is linked not only to toxicity caused by expression of HipA or dedicated toxins but also to expression of other unrelated proteins. PMID- 16672604 TI - WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. AB - WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM and the K(m)(benzoquinone) was 5.8 +/- 0.12 microM. For A. fulgidus WrbA, the K(m)(NADH) was 19 +/- 1.7 microM and the K(m)(benzoquinone) was 37 +/- 3.6 microM. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress. PMID- 16672605 TI - Multiple groESL operons are not key targets of RpoH1 and RpoH2 in Sinorhizobium meliloti. AB - Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets. PMID- 16672606 TI - Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae. AB - Chimeras created by fusing the monomeric red fluorescent protein (RFP) to a bacterial lipoprotein signal peptide (lipoRFPs) were visualized in the cell envelope by epifluorescence microscopy. Plasmolysis of the bacteria separated the inner and outer membranes, allowing the specific subcellular localization of lipoRFPs to be determined in situ. When equipped with the canonical inner membrane lipoprotein retention signal CDSR, lipoRFP was located in the inner membrane in Escherichia coli, whereas the outer membrane sorting signal CSSR caused lipoRFP to localize to the outer membrane. CFSR-RFP was also routed to the outer membrane, but CFNSR-RFP was located in the inner membrane, consistent with previous data showing that this sequence functions as an inner membrane retention signal. These four lipoproteins exhibited identical localization patterns in a panel of members of the family Enterobacteriaceae, showing that the lipoprotein sorting rules are conserved in these bacteria and validating the use of E. coli as a model system. Although most predicted inner membrane lipoproteins in these bacteria have an aspartate residue after the fatty acylated N-terminal cysteine residue, alternative signals such as CFN can and probably do function in parallel, as indicated by the existence of putative inner membrane lipoproteins with this sequence at their N termini. PMID- 16672608 TI - Purification and characterization of a hemolysin-like protein, Sll1951, a nontoxic member of the RTX protein family from the Cyanobacterium Synechocystis sp. strain PCC 6803. AB - The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca(2+) binding, was purified from the glucose tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca(2+) was not chelated with EGTA; however, it was released after being heated at 100 degrees C for 1 min, and it rebound to the Ca(2+)-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca(2+), respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca(2+). Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca(2+)-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature. PMID- 16672607 TI - Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL. AB - Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components. PMID- 16672609 TI - Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1. AB - Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Go1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes. PMID- 16672611 TI - In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors. AB - Our understanding of Escherichia coli biofilm formation in vitro is based on studies of laboratory K-12 strains grown in standard media. However, pathogenic E. coli isolates differ substantially in their genetic repertoire from E. coli K 12 and are subject to heterogeneous environmental conditions. In this study, in vitro biofilm formation of 331 nondomesticated E. coli strains isolated from healthy (n = 105) and diarrhea-afflicted children (n = 68), bacteremia patients (n = 90), and male patients with urinary tract infections (n = 68) was monitored using a variety of growth conditions and compared to in vitro biofilm formation of prototypic pathogenic and laboratory strains. Our results revealed remarkable variation among the capacities of diverse E. coli isolates to form biofilms in vitro. Notably, we could not identify an association of increased biofilm formation in vitro with a specific strain collection that represented pathogenic E. coli strains. Instead, analysis of biofilm data revealed a significant dependence on growth medium composition (P < 0.05). Poor correlation between biofilm formation in the various media suggests that diverse E. coli isolates respond very differently to changing environmental conditions. The data demonstrate that prevalence and expression of three factors known to strongly promote biofilm formation in E. coli K-12 (F-like conjugative pili, aggregative adherence fimbriae, and curli) cannot adequately account for the increased biofilm formation of nondomesticated E. coli isolates in vitro. This study highlights the complexity of genetic and environmental effectors of the biofilm phenotype within the species E. coli. PMID- 16672610 TI - Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities. AB - The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO(2) tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO(2), the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO(2)-responsive chromosome- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities. PMID- 16672612 TI - Synergistic effects in mixed Escherichia coli biofilms: conjugative plasmid transfer drives biofilm expansion. AB - Bacterial biofilms, often composed of multiple species and genetically distinct strains, develop under complex influences of cell-cell interactions. Although detailed knowledge about the mechanisms underlying formation of single-species laboratory biofilms has emerged, little is known about the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from the cocultivation of 403 undomesticated E. coli strains with a characterized E. coli K-12 strain was detected at a significant frequency. The survey suggests that different mechanisms underlie the observed stimulation, yet synergistic development of biofilm within the subset of E. coli isolates (n = 56) exhibiting the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes present initially in the isolate, and (iii) was inhibited through the presence in the cocultured K-12 strain of a related conjugative plasmid, presumably due to surface exclusion functions. Synergistic effects of cocultivation of pairs of natural isolates were also observed, demonstrating that biofilm promotion in this system is not dependent on the laboratory strain and that the described model system could provide relevant insights on mechanisms of biofilm development in natural E. coli populations. PMID- 16672613 TI - Structural and functional analysis of Rv3214 from Mycobacterium tuberculosis, a protein with conflicting functional annotations, leads to its characterization as a phosphatase. AB - The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4'-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Angstroms resolution (R = 0.229; R(free) = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM alpha/beta structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4' phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function. PMID- 16672614 TI - Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di GMP level. AB - Vibrio cholerae, the causative agent of cholera, is a facultative human pathogen with intestinal and aquatic life cycles. The capacity of V. cholerae to recognize and respond to fluctuating parameters in its environment is critical to its survival. In many microorganisms, the second messenger, 3',5'-cyclic diguanylic acid (c-di-GMP), is believed to be important for integrating environmental stimuli that affect cell physiology. Sequence analysis of the V. cholerae genome has revealed an abundance of genes encoding proteins with either GGDEF domains, EAL domains, or both, which are predicted to modulate cellular c-di-GMP concentrations. To elucidate the cellular processes controlled by c-di-GMP, whole genome transcriptome responses of the El Tor and classical V. cholerae biotypes to increased c-di-GMP concentrations were determined. The results suggest that V. cholerae responds to an elevated level of c-di-GMP by increasing the transcription of the vps, eps, and msh genes and decreasing that of flagellar genes. The functions of other c-di-GMP-regulated genes in V. cholerae are yet to be identified. PMID- 16672615 TI - Role of conserved amino acids in the catalytic activity of Escherichia coli primase. AB - The role of conserved amino acid residues in the polymerase domain of Escherichia coli primase has been studied by mutagenesis. We demonstrate that each of the conserved amino acids Arg146, Arg221, Tyr230, Gly266, and Asp311 is involved in the process of catalysis. Residues Glu265 and Asp309 are also critical because a substitution of each amino acid irreversibly destroys the catalytic activity. Two K229A and M268A mutant primase proteins synthesize only 2-nucleotide products in de novo synthesis reactions under standard conditions. Y267A mutant primase protein synthesizes both full-size and 2-nucleotide RNA, but with no intermediate size products. From these data we discuss the significant step of the 2 nucleotide primer RNA synthesis by E. coli primase and the role of amino acids Lys229, Tyr267, and Met268 in primase complex stability. PMID- 16672616 TI - Cation-induced transcriptional regulation of the dlt operon of Staphylococcus aureus. AB - Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with D-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. D-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating D-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na(+) and moderate concentrations of Mg(2+) and Ca(2+) but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg(2+)-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg(2+)-induced repression of the dlt operon in S. aureus. PMID- 16672617 TI - The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is a part of the Fur regulon. AB - Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS, located upstream from orbI. The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) sigma factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron regulated sigma(70)-dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF sigma factor gene orbS. A mechanism is also proposed for the biosynthesis of ornibactin. PMID- 16672618 TI - Application of comparative phylogenomics to study the evolution of Yersinia enterocolitica and to identify genetic differences relating to pathogenicity. AB - Yersinia enterocolitica, an important cause of human gastroenteritis generally caused by the consumption of livestock, has traditionally been categorized into three groups with respect to pathogenicity, i.e., nonpathogenic (biotype 1A), low pathogenicity (biotypes 2 to 5), and highly pathogenic (biotype 1B). However, genetic differences that explain variation in pathogenesis and whether different biotypes are associated with specific nonhuman hosts are largely unknown. In this study, we applied comparative phylogenomics (whole-genome comparisons of microbes with DNA microarrays combined with Bayesian phylogenies) to investigate a diverse collection of 94 strains of Y. enterocolitica consisting of 35 human, 35 pig, 15 sheep, and 9 cattle isolates from nonpathogenic, low-pathogenicity, and highly pathogenic biotypes. Analysis confirmed three distinct statistically supported clusters composed of a nonpathogenic clade, a low-pathogenicity clade, and a highly pathogenic clade. Genetic differences revealed 125 predicted coding sequences (CDSs) present in all highly pathogenic strains but absent from the other clades. These included several previously uncharacterized CDSs that may encode novel virulence determinants including a hemolysin, a metalloprotease, and a type III secretion effector protein. Additionally, 27 CDSs were identified which were present in all 47 low-pathogenicity strains and Y. enterocolitica 8081 but absent from all nonpathogenic 1A isolates. Analysis of the core gene set for Y. enterocolitica revealed that 20.8% of the genes were shared by all of the strains, confirming this species as highly heterogeneous, adding to the case for the existence of three subspecies of Y. enterocolitica. Further analysis revealed that Y. enterocolitica does not cluster according to source (host). PMID- 16672619 TI - Flavonoid-inducible modifications to rhamnan O antigens are necessary for Rhizobium sp. strain NGR234-legume symbioses. AB - Rhizobium sp. strain NGR234 produces a flavonoid-inducible rhamnose-rich lipopolysaccharide (LPS) that is important for the nodulation of legumes. Many of the genes encoding the rhamnan part of the molecule lie between 87 degrees and 110 degrees of pNGR234a, the symbiotic plasmid of NGR234. Computational methods suggest that 5 of the 12 open reading frames (ORFs) within this arc are involved in synthesis (and subsequent polymerization) of L-rhamnose. Two others probably play roles in the transport of carbohydrates. To evaluate the function of these ORFs, we mutated a number of them and tested the ability of the mutants to nodulate a variety of legumes. At the same time, changes in the production of surface polysaccharides (particularly the rhamnan O antigen) were examined. Deletion of rmlB to wbgA and mutation in fixF abolished rhamnan synthesis. Mutation of y4gM (a member of the ATP-binding cassette transporter family) did not abolish production of the rhamnose-rich LPS but, unexpectedly, the mutant displayed a symbiotic phenotype very similar to that of strains unable to produce the rhamnan O antigen (NGRDeltarmlB-wbgA and NGROmegafixF). At least two flavonoid-inducible regulatory pathways are involved in synthesis of the rhamnan O antigen. Mutation of either pathway reduces rhamnan production. Coordination of rhamnan synthesis with rhizobial release from infection threads is thus part of the symbiotic interaction. PMID- 16672620 TI - Role of the Fur regulon in iron transport in Bacillus subtilis. AB - The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT. PMID- 16672621 TI - Identification of a mutation in the Bacillus subtilis S-adenosylmethionine synthetase gene that results in derepression of S-box gene expression. AB - Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5' region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors. PMID- 16672623 TI - Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum. AB - Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen. PMID- 16672622 TI - Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. AB - Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M. PMID- 16672624 TI - D-alanyl ester depletion of teichoic acids in Lactobacillus plantarum results in a major modification of lipoteichoic acid composition and cell wall perforations at the septum mediated by the Acm2 autolysin. AB - The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase. PMID- 16672625 TI - Evidence that Clostridium difficile TcdC is a membrane-associated protein. AB - Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile. PMID- 16672626 TI - Variable expression patterns of Mycobacterium tuberculosis PE_PGRS genes: evidence that PE_PGRS16 and PE_PGRS26 are inversely regulated in vivo. AB - Evaluation of expression of 16 PE_PGRS genes present in Mycobacterium tuberculosis under various growth conditions demonstrated constitutive expression of 7 genes, variable expression of 7 genes, and no expression of 2 genes. An inverse expression profile for genes PE_PGRS16 and PE_PGRS26 was observed to occur in macrophages and in mice infected with M. tuberculosis. Variable expression of PE_PGRS proteins could have implications for their role in the immunopathogenesis of tuberculosis. PMID- 16672628 TI - Innovative model for building academic nursing. PMID- 16672627 TI - Helicobacter pylori initiates the stringent response upon nutrient and pH downshift. AB - Helicobacter pylori was previously reported to lack a stringent response. In contrast, we show that after nutrient downshift, H. pylori produced abundant ppGpp and less total RNA. pH downshift also caused (p)ppGpp accumulation. Our observations indicate that nutrient deprivation and acid shock activate the stringent response in H. pylori. PMID- 16672629 TI - Stigma associated with Ghanaian caregivers of AIDS patients. AB - This study explores the experiences of informal caregivers of AIDS patients in Accra, the capital city of Ghana. Fifteen interviews were completed in 2002 with 11 informal caregivers, including wives, mothers, boyfriends, daughters, sons and brothers of AIDS patients. Three major themes emerge in the analysis of the interviews with caregivers: stigma, caregiver burden, and caregiver commitment. In this article, the authors focus on the theme of stigma by documenting its presence and highlighting its impact on caregiving activities. Caregivers go to great effort to not only "hide" their patients but also their care giving activities, resulting in the social isolation of both patients and their caregivers. Many caregivers live in secrecy, not sharing their family member's diagnosis with extended family members. As a result, they receive limited support from the extended family. Stigma results in negative attitudes of neighbors, relatives, and health care workers toward caregivers and their patients. PMID- 16672630 TI - Keeping nursing home residents hydrated. AB - The purpose of this research is to ascertain nursing home (NH) staffs' perspectives of the problem of dehydration in NHs (skilled nursing facilities) and have them identify clinically practical interventions to ensure that NH residents consistently take adequate fluids. Four focus groups with 28 NH staff members in attendance are held in three NHs in metropolitan Los Angeles. The majority of participants are certified nursing assistants; however, licensed vocational nurses, registered nurses, and other NH staff also attend the groups. Content analytic techniques are used to analyze the data. Three themes emerge focusing on residents' reasons for not drinking, signs and symptoms of dehydration, and strategies for improving hydration in NH residents. NH staff members identify the complexity of providing adequate hydration that was influenced by the resident's relationship with family and NH staff and communication between staff members. PMID- 16672631 TI - Moral reckoning in nursing. AB - Analysis of qualitative data resulted in an original substantive grounded theory of moral reckoning in nursing, a three-stage process. After a novice period, the nurse experiences a stage of ease in which there is comfort in the workplace and congruence of internal and external values. Unexpectedly, a situational bind occurs in which the nurse's core beliefs come into irreconcilable conflict with external forces. This compels the nurse into the stage of resolution, in which he or she either gives up or makes a stand. The nurse then moves into the stage of reflection in which he or she lives with the consequences and iteratively examines beliefs, values, and actions. The nurse tries to make sense of experiences through remembering, telling the story, and examining conflicts. This study sets the stage for further investigation of moral distress. The theory of moral reckoning challenges nurses to tell their stories, examine conflicts, and participate as partners in moral decision making. PMID- 16672632 TI - Determinants of older rural women's activity and eating. AB - This research examined the relationship of cognitive-perceptual determinants from the Health Promotion Model to physical activity and healthy eating at baseline of a clinical trial among rural women aged 50 to 69. Two multivariate regression analyses with canonical correlation (N = 179) each yielded one interpretable canonical variate that explained similar amounts of variance (21.7% and 22.5%) in sets of activity and eating variables. In both analyses, the determinants set is a linear combination of all four cognitive-perceptual constructs from the model (perceived self-efficacy, benefits, barriers, interpersonal influences). The activity behavior set included caloric expenditure, VO2max, and stretching and strengthening activity, whereas the eating behavior set included daily percentage of calories from fat and servings of fruits and vegetables, meat, and whole grains. As predicted by theory, greater self-efficacy, benefits and interpersonal support, and fewer barriers are associated with desirable healthy lifestyle behaviors. PMID- 16672633 TI - Hepatitis C virus infection among homeless men referred from a community clinic. AB - In this study of factors related to Hepatitis C virus (HCV) infection in 104 homeless HCV-infected men and 94 uninfected homeless men, findings reveal that homeless men with HCV infection are older and more likely to be cocaine injectors than those not infected. Moreover, the sharing of needles, use of injected cocaine during the past 6 months, previous incarceration, veteran status, fair or poor health, and having multiple tattoos are also associated with HCV infection. Reports of having completed the HBV vaccination series, weekly marijuana use, and snorting cocaine or methamphetamine are negatively associated with HCV infection. Among men not reporting lifetime injection drug use, factors such as sharing toothbrushes, having multiple tattoos, being in fair or poor health, and past incarceration are associated with HCV infection. These findings may need to be considered when making screening decisions and counseling homeless male patients about HCV. PMID- 16672634 TI - Adipose-specific effect of rosiglitazone on vascular permeability and protein kinase C activation: novel mechanism for PPARgamma agonist's effects on edema and weight gain. AB - PPARgamma agonists, thiazolidinediones, cause fluid retention and edema due to unknown mechanisms. We characterized the effect of rosiglitazone (RSG), a thiazolidinedione, to induce vascular permeability, vascular endothelial growth factor (VEGF) expression, and protein kinase C (PKC) activation with edema and wt gain. In lean, fatty and diabetic Zucker rats, and endothelial insulin receptor knockout mice, RSG increased wt and vascular permeability, selectively in fat and retina, but not in heart or skeletal muscle. H2O content and wt of epididymal fat were increased by RSG and correlated to increases in capillary permeability in fat and body wt. RSG induced VEGF mRNA expression and PKC activation in fat and retina up to 2.5-fold. Ruboxistaurin, a PKCbeta isoform inhibitor, in the latter 2 wk of a 4-wk study, normalized vascular permeability in fat and decreased total wt gain, H2O content, and wt of fat vs. RSG alone but did not decrease VEGF expression, basal permeability, or food intake. Finally, RSG did not increase wt or vascular permeability in PKCbeta knockout vs. control mice. Thus, thiazolidinedione's effects on edema and wt are partially due to an adipose tissue-selective activation of PKC and vascular permeability that may be prevented by PKCbeta inhibition. PMID- 16672635 TI - HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism. AB - This study investigated whether inducible heme oxygenase-1[corrected] (HO-1) [corrected] is targeted to mitochondria and its putative effects on oxidative metabolism in rat liver. Western blot and immune-electron microscopy in whole purified and fractionated organelles showed basal expression of HO-1 protein in both microsomes and mitochondria (inner membrane), accompanied by a parallel HO activity. Inducers of HO-1 increased HO-1 targeting to the inner mitochondrial membrane, which also contained biliverdin reductase, supporting that both enzymes are in the same compartmentalization. Induction of mitochondrial HO-1 was associated with a decrease of mitochondrial heme content and selective reduction of protein expression of cytochrome oxidase (COX) subunit I, which is coded by the mitochondrial genome and synthesized in the mitochondria depending on heme availability; these changes resulted in decreased COX spectrum and activity. Mitochondrial HO-1 induction was also associated with down-regulation of mitochondrial-targeted NO synthase expression and activity, resulting in a reduction of NO-dependent mitochondrial oxidant yield; inhibition of HO-1 activity reverted these effects. In conclusion, we demonstrated for the first time localization of HO-1 protein in mitochondria. It is surmised that mitochondrial HO-1 has important biological roles in regulating mitochondrial heme protein turnover and in protecting against conditions such as hypoxia, neurodegenerative diseases, or sepsis, in which substantially increased mitochondrial NO and oxidant production have been implicated. PMID- 16672637 TI - Catastrophic antiphospholipid syndrome. AB - Catastrophic antiphospholipid syndrome is a rapidly progressive life-threatening disease that causes multiple organ thromboses and dysfunction in the presence of antiphospholipid antibodies. A high index of clinical suspicion and careful investigation are required to make an early diagnosis so that treatment with anticoagulation and corticosteroids can be initiated; plasma exchange and/or intravenous immunoglobulins can be added if the life-threatening condition persists. Despite aggressive treatment and intensive care unit management, patients with catastrophic antiphospholipid syndrome have a 48% mortality rate, primarily attributable to cardiopulmonary failure. This article reviews the current information on the etiopathogenesis, clinical manifestations, diagnosis, management, and prognosis of catastrophic antiphospholipid syndrome. PMID- 16672638 TI - Immunologic responses to critical injury and sepsis. AB - Almost 2 million patients are admitted to hospitals in the United States each year for treatment of traumatic injuries, and these patients are at increased risk of late infections and complications of systemic inflammation as a result of injury. Host response to injury involves a general activation of multiple systems in defending the organism from hemorrhagic or infectious death. Clinicians have the capability to support the critically injured through their traumatic insult with surgery and improved critical care, but the inflammatory response generated by such injuries creates new challenges in the management of these patients. It has long been known that local tissue injury induces systemic changes in the traumatized patient that are often maladaptive. This article reviews the effects of injury on the function of immune system cells and highlights some of the clinical sequelae of this deranged inflammatory-immune interaction. PMID- 16672639 TI - The changing epidemiology of mechanical ventilation: a population-based study. AB - The number of critical care beds in the United States has been increasing considerably, but it is unclear how these additional beds have been used. Mechanical ventilation for acute respiratory failure almost always demands ICU care and is likely to be a reliable indicator of critical care resource requirements on a population level. The objective of this study was to measure changes in the yearly incidence of mechanical ventilation in a statewide population. The North Carolina Hospital Discharge Database contains data on all discharges from nonfederal, nonpsychiatric hospitals in North Carolina. Authors extracted data on adult patients with International Classification of Diseases, 9th Revision, Clinical Modification procedure codes for mechanical ventilation from 1996 to 2002. The incidence of mechanical ventilation for adults grew from 284/100,000 population in 1996 to 314/100,000 in 2002, an increase of 11% (P < .05). While patients aged >64 had the highest age-specific incidence of mechanical ventilation each year, the greatest increase in incidence occurred in younger age groups (19% increase for age 18-64 vs 4% increase for age >64). The mean Charlson score increased from 1.76 +/- 1.73 to 1.89 +/- 1.86 (P < .001). Renal disease became more prevalent among patients requiring mechanical ventilation (17% of patients in 1996 vs 24% in 2002). Hospital charges adjusted for the medical consumer price index increased by 12%. The proportion of patients discharged to home declined from 45.4% to 34.4%, and discharges to nursing homes grew from 7.3% to 10.7%. The incidence of mechanical ventilation is increasing, and the increase is associated with a higher burden of comorbidities and fewer discharges to home. PMID- 16672636 TI - Pharmacotherapy of acute lung injury and the acute respiratory distress syndrome. AB - Acute lung injury and the acute respiratory distress syndrome are common syndromes with a high mortality rate that affect both medical and surgical patients. Better understanding of the pathophysiology of acute lung injury and the acute respiratory distress syndrome and advances in supportive care and mechanical ventilation have led to improved clinical outcomes since the syndrome was first described in 1967. Although several promising pharmacological therapies, including surfactant, nitric oxide, glucocorticoids and lysofylline, have been studied in patients with acute lung injury and the acute respiratory distress syndrome, none of these pharmacological treatments reduced mortality. This article provides an overview of pharmacological therapies of acute lung injury and the acute respiratory distress syndrome tested in clinical trials and current recommendations for their use as well as a discussion of potential future pharmacological therapies including beta(2)-adrenergic agonist therapy, keratinocyte growth factor, and activated protein C. PMID- 16672640 TI - Levosimendan for low cardiac output: a pediatric experience. AB - This was a retrospective observational study in a pediatric intensive care unit, in which 19 patients received levosimendan. There were no adverse events attributable to levosimendan and no instances where the clinical condition worsened after administration. Arterial lactate levels decreased significantly following levosimendan administration during cardiopulmonary bypass for anticipated low cardiac output. In those with established low cardiac output, trends toward improved hemodynamics were seen, with heart rate reduction, an increase in mean blood pressure, a reduction in arterial lactate, and reduced conventional inotrope use. Levosimendan was safely used in a small number of pediatric patients with established low cardiac output state who demonstrated improved hemodynamics and tissue perfusion, with a tendency to reduced conventional inotrope usage, and this warrants its evaluation as an inotrope in the pediatric population. PMID- 16672641 TI - Adding up the zeros. PMID- 16672642 TI - Utilization of critical care resources is increasing--are we ready? PMID- 16672643 TI - The three-substituted indolinone cyclin-dependent kinase 2 inhibitor 3-[1-(3H imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) kills human leukemia cells via down-regulation of Mcl-1 through a transcriptional mechanism. AB - Mechanisms of lethality of the three-substituted indolinone and putatively selective cyclin-dependent kinase (CDK)2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth (Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations > or =5 microM rapidly (i.e., within 4 h) induced cytochrome c release, Bax mitochondrial translocation, and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase (Pol) II on serine 2 but not serine 5. Reverse transcription-polymerase chain reaction analysis revealed pronounced down regulation of Mcl-1 mRNA levels in SU9516-treated cells. Similar results were obtained in Jurkat and HL-60 leukemia cells. Furthermore, cotreatment with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked SU9516-mediated Mcl-1 down-regulation, implicating proteasomal degradation in diminished expression of this protein. Ectopic expression of Mcl-1 largely blocked SU9516-induced cytochrome c release, Bax translocation, and apoptosis, whereas knockdown of Mcl-1 by small interfering RNA potentiated SU9516 lethality, confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. It is noteworthy that SU9516 treatment resulted in a marked increase in reactive oxygen species production, which was diminished, along with cell death, by the free radical scavenger N-acetylcysteine (NAC). We were surprised to find that NAC blocked SU9516-mediated inhibition of RNA Pol II CTD phosphorylation on serine 2, reductions in Mcl-1 mRNA levels, and Mcl-1 down regulation. Together, these findings suggest that SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation in association with oxidative damage and down-regulation of Mcl-1 at the transcriptional level, culminating in mitochondrial injury and cell death. PMID- 16672644 TI - Short amyloid-beta (Abeta) immunogens reduce cerebral Abeta load and learning deficits in an Alzheimer's disease mouse model in the absence of an Abeta specific cellular immune response. AB - Amyloid-beta (Abeta) immunotherapy lowers cerebral Abeta and improves cognition in mouse models of Alzheimer's disease (AD). A clinical trial using active immunization with Abeta1-42 was suspended after approximately 6% of patients developed meningoencephalitis, possibly because of a T-cell reaction against Abeta. Nevertheless, beneficial effects were reported in antibody responders. Consequently, alternatives are required for a safer vaccine. The Abeta1-15 sequence contains the antibody epitope(s) but lacks the T-cell reactive sites of full-length Abeta1-42. Therefore, we tested four alternative peptide immunogens encompassing either a tandem repeat of two lysine-linked Abeta1-15 sequences (2xAbeta1-15) or the Abeta1-15 sequence synthesized to a cross-species active T1 T-helper-cell epitope (T1-Abeta1-15) and each with the addition of a three-amino acid RGD (Arg-Gly-Asp) motif (R-2xAbeta1-15; T1-R-Abeta1-15). High anti-Abeta antibody titers were observed in wild-type mice after intranasal immunization with R-2xAbeta1-15 or 2xAbeta1-15 plus mutant Escherichia coli heat-labile enterotoxin LT(R192G) adjuvant. Moderate antibody levels were induced after immunization with T1-R-Abeta1-15 or T1-Abeta1-15 plus LT(R192G). Restimulation of splenocytes with the corresponding immunogens resulted in moderate proliferative responses, whereas proliferation was absent after restimulation with full-length Abeta or Abeta1-15. Immunization of human amyloid precursor protein, familial AD (hAPP(FAD)) mice with R-2xAbeta1-15 or 2xAbeta1-15 resulted in high anti-Abeta titers of noninflammatory T-helper 2 isotypes (IgG1 and IgG2b), a lack of splenocyte proliferation against full-length Abeta, significantly reduced Abeta plaque load, and lower cerebral Abeta levels. In addition, 2xAbeta1-15-immunized hAPP(FAD) animals showed improved acquisition of memory compared with vehicle controls in a reference-memory Morris water-maze behavior test that approximately correlated with anti-Abeta titers. Thus, our novel immunogens show promise for future AD vaccines. PMID- 16672645 TI - Antibodies against the NG2 proteoglycan promote the regeneration of sensory axons within the dorsal columns of the spinal cord. AB - The NG2 chondroitin sulfate proteoglycan inhibits axon growth in vitro. Levels of NG2 increase rapidly in the glial scars that form at sites of CNS injury, suggesting that NG2 may inhibit axon regeneration. To determine the functions of NG2, we infused mixtures of neutralizing or non-neutralizing anti-NG2 monoclonal antibodies into the dorsally transected adult rat spinal cord and analyzed the regeneration of ascending mechanosensory axons anatomically. At 1 week after injury, ascending sensory axons in control animals terminated caudal to the lesion within an area containing dense deposits of NG2 immunoreactivity. In animals treated with the neutralizing anti-NG2 antibodies, labeled axons penetrated the caudal border of the lesion and grew into and beyond the lesion center. The low intrinsic growth capacity of adult neurons may also limit the ability of damaged axons to regenerate. To enhance growth, we combined antibody treatment with a peripheral nerve conditioning lesion. After a conditioning lesion and treatment with control, non-neutralizing antibodies, many sensory axons grew into the lesion core. These axons did not grow past the rostral border of the lesion; rather, they grew along the dorsal surface of the spinal cord and within any remaining pieces of the dorsal roots. In contrast, combining a peripheral nerve conditioning lesion with neutralizing anti-NG2 antibodies resulted in sensory axon regeneration past the glial scar and into the white matter rostral to the injury site. The combinatorial approach used here that neutralizes extrinsic inhibition and increases intrinsic growth results in anatomically correct axon regeneration, a prerequisite for functional recovery. PMID- 16672646 TI - Localization of diacylglycerol lipase-alpha around postsynaptic spine suggests close proximity between production site of an endocannabinoid, 2-arachidonoyl glycerol, and presynaptic cannabinoid CB1 receptor. AB - 2-arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-alpha (DAGLalpha), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGLalpha was predominantly expressed in Purkinje cells. DAGLalpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to proximal gradient from spiny branchlets toward somata. DAGLalpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGLalpha was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLalpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLalpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLalpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types. PMID- 16672647 TI - The time of prenatal immune challenge determines the specificity of inflammation mediated brain and behavioral pathology. AB - Disturbance to early brain development is implicated in several neuropsychiatric disorders including autism, schizophrenia, and mental retardation. Epidemiological studies have indicated that the risk of developing these disorders is enhanced by prenatal maternal infection, presumably as a result of neurodevelopmental defects triggered by cytokine-related inflammatory events. Here, we demonstrate that the effects of maternal immune challenge between middle and late gestation periods in mice are dissociable in terms of fetal brain cytokine responses to maternal inflammation and the pathological consequences in brain and behavior. Specifically, the relative expression of pro- and anti inflammatory cytokines in the fetal brains in response to maternal immune challenge may be an important determinant among other developmental factors for the precise pathological profile emerging in later life. Thus, the middle and late gestation periods correspond to two windows with differing vulnerability to adult behavioral dysfunction, brain neuropathology in early adolescence, and of the acute cytokine responses in the fetal brain. PMID- 16672648 TI - An in vitro study of horizontal connections in the intermediate layer of the superior colliculus. AB - Some models propose that the spatial and temporal distributions of premotor activity in the intermediate layer of the superior colliculus are shaped by neuronal ensembles that give rise to local excitatory and distant inhibitory connections. One function proposed for these connections is to mediate a "winner take-all" network; the short-range excitatory connections build up the activity of neighboring cells that command orienting movements in one direction, whereas the wide-ranging inhibitory projections attenuate the activity of remote cells that command incompatible movements. We used in vitro photostimulation and whole cell patch-clamp recording to test these models by measuring the spatial extent of synaptic interactions within the rat intermediate layer. Uncaging glutamate over whole-cell patch-clamped cells in the intermediate layer elicited long lasting inward currents, resulting from direct activation of glutamate receptors expressed by the cells, and brief synaptic currents evoked by activation of presynaptic neurons. The synaptic responses comprised clusters of excitatory and inhibitory currents. The size of these responses depended on the location of the stimulus with respect to the clamped cell. Large responses were commonly evoked by stimuli within 200 microm of the soma in the intermediate layer; smaller responses could occasionally be evoked from sites as distant as 500 microm. Responses evoked by stimulation beyond this distance were rare. Although the results demonstrated powerful local excitatory and inhibitory connections, they did not support the pattern of short-range excitation and widespread inhibition predicted by the winner-take-all hypothesis. PMID- 16672649 TI - The anterior cingulate and error avoidance. AB - The precise role of the anterior cingulate cortex in monitoring, evaluating, and correcting behavior remains unclear despite numerous theories and much empirical data implicating it in cognitive control. The present event-related functional magnetic resonance imaging study was able to separate monitoring from error specific functions by allowing subjects to reject a trial so as to avoid errors. Cingulate and left dorsolateral prefrontal activity was greatest on rejection trials but comparable for correct and error trials, whereas an error-specific response was observed in bilateral insula. A dissociation was also observed between the cingulate and the nucleus accumbens with the latter more active for error than reject trials. These results reveal that the functional role of the cingulate is not particular to errors but instead is related to an evaluative function concerned with on-line behavioral adjustment in the service of avoiding losses. PMID- 16672650 TI - Oxidative stress and mitogen-activated protein kinase phosphorylation mediate ammonia-induced cell swelling and glutamate uptake inhibition in cultured astrocytes. AB - Hepatic encephalopathy (HE) is a major neurological complication in patients with severe liver failure. Elevated levels of ammonia have been strongly implicated as a factor in HE, and astrocytes appear to be the primary target of its neurotoxicity. Mechanisms mediating key aspects of ammonia-induced astrocyte dysfunction such as cell swelling and inhibition of glutamate uptake are not clear. We demonstrated previously that cultured astrocytes exposed to ammonia increase free radical production. We now show that treatment with antioxidants significantly prevents ammonia-induced astrocyte swelling as well as glutamate uptake inhibition. Because one consequence of oxidative stress is the phosphorylation of mitogen-activated protein kinases (MAPKs), we investigated whether phosphorylation of MAPKs may mediate astrocyte dysfunction. Primary cultured astrocytes exposed to 5 mm NH4Cl for different time periods (1-72 h) significantly increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38(MAPK), and c-Jun N-terminal kinase (JNK) 1/2/3, which was inhibited by appropriate MAPK inhibitors 1, 4-diamino-2, 3-dicyano-1, 4-bis (2 aminophenylthio) butadiene (UO126; for ERK1/2), trans-1-(4-hydroxyclyclohexyl)-4 (4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole (SB 239063; for p38(MAPK)), and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125; for JNK1/2/3), as well as by antioxidants. Kinase inhibitors partially or completely prevented astrocyte swelling. Although SB239063 and SP600125 significantly reversed glutamate uptake inhibition and ammonia-induced decline in glutamate-aspartate transporter protein levels, UO126 did not, indicating a differential effect of these kinases in ammonia-induced astrocyte swelling and glutamate transport impairment. These studies strongly suggest the involvement of oxidative stress and phosphorylation of MAPKs in the mechanism of ammonia-induced astrocyte dysfunction associated with ammonia neurotoxicity. PMID- 16672651 TI - Plasticity of temporal pattern codes for vocalization stimuli in primary auditory cortex. AB - It has been suggested that "call-selective" neurons may play an important role in the encoding of vocalizations in primary auditory cortex (A1). For example, marmoset A1 neurons often respond more vigorously to natural than to time reversed twitter calls, although the spectral energy distribution in the natural and time-reversed signals is the same. Neurons recorded in cat A1, in contrast, showed no such selectivity for natural marmoset calls. To investigate whether call selectivity in A1 can arise purely as a result of auditory experience, we recorded responses to marmoset calls in A1 of naive ferrets, as well as in ferrets that had been trained to recognize these natural marmoset calls. We found that training did not induce call selectivity for the trained vocalizations in A1. However, although ferret A1 neurons were not call selective, they efficiently represented the vocalizations through temporal pattern codes, and trained animals recognized marmoset twitters with a high degree of accuracy. These temporal patterns needed to be analyzed at timescales of 10-50 ms to ensure efficient decoding. Training led to a substantial increase in the amount of information transmitted by these temporal discharge patterns, but the fundamental nature of the temporal pattern code remained unaltered. These results emphasize the importance of temporal discharge patterns and cast doubt on the functional significance of call-selective neurons in the processing of animal communication sounds at the level of A1. PMID- 16672652 TI - Fatigue-sensitive afferents inhibit extensor but not flexor motoneurons in humans. AB - The role of group III and IV muscle afferents in controlling the output from human muscles is poorly understood. We investigated the effects of these afferents from homonymous or antagonist muscles on motoneuron pools innervating extensor and flexor muscles of the elbow. In study 1, subjects (n = 8) performed brief maximal voluntary contractions (MVCs) of elbow extensors before and after a 2 min MVC of the extensors. During MVCs, electromyographic responses from triceps were evoked by stimulation of the corticospinal tracts [cervicomedullary motor evoked potentials (CMEPs)]. The same subjects repeated the protocol, but input from fatigue-sensitive afferents was prolonged after the fatiguing contraction by maintained muscle ischemia. In study 2, CMEPs were evoked in triceps during brief extensor MVCs before and after a 2 min sustained flexor MVC (n = 7) or in biceps during brief flexor MVCs before and after a sustained extensor MVC (n = 7). Again, ischemia was maintained after the sustained contractions. During sustained MVCs of the extensors, CMEPs in triceps decreased by approximately 35%. Without muscle ischemia, CMEPs recovered within 15 s, but with maintained ischemia, they remained depressed (by approximately 28%; p < 0.001). CMEPs in triceps were also depressed (by approximately 20%; p < 0.001) after fatiguing flexor contractions, whereas CMEPs in biceps were facilitated (by approximately 25%; p < 0.001) after fatiguing extensor contractions. During fatigue, inputs from group III and IV muscle afferents from homonymous or antagonist muscles depress extensor motoneurons but facilitate flexor motoneurons. The more pronounced inhibitory influence of these afferents on extensors suggests that these muscles may require greater cortical drive to generate force during fatigue. PMID- 16672653 TI - The presence of background dopamine signal converts long-term synaptic depression to potentiation in rat prefrontal cortex. AB - Executive functions of the brain are believed to require tonic dopamine inputs to the prefrontal cortex (PFC). It is unclear, however, how this background dopamine activity controls synaptic plasticity in the PFC, a possible underlying mechanism of executive functions. Using PFC slices, we show that pairing of dopamine with weak tetanic stimulation, a maneuver that otherwise induces NMDA receptor independent long-term depression (LTD), induces long-term potentiation (LTP) when "primed" with dopamine. This "priming" occurs through the combined activation of D1 and D2 receptors and requires 12-40 min to develop. Moreover, concurrent synaptic activation of NMDA receptors during priming is necessary for this novel form of LTP. We suggest that a role of background dopamine signals in the PFC is to prevent high-frequency synaptic inputs from abnormally inducing LTD and to secure the induction of LTP. PMID- 16672654 TI - ARF6 and EFA6A regulate the development and maintenance of dendritic spines. AB - The cellular and molecular mechanisms underlying the development and maintenance of dendritic spines are not fully understood. ADP-ribosylation factor 6 (ARF6) is a small GTPase known to regulate actin remodeling and membrane traffic. Here, we report involvement of ARF6 and exchange factor for ARF6 (EFA6A) in the regulation of spine development and maintenance. An active form of ARF6 promotes the formation of dendritic spines at the expense of filopodia. EFA6A promotes spine formation in an ARF6 activation-dependent manner. Knockdown of ARF6 and EFA6A by small interfering RNA decreases spine formation. Live imaging indicates that ARF6 knockdown decreases the conversion of filopodia to spines and the stability of early spines. The spine-promoting effect of ARF6 is partially blocked by Rac1. ARF6 and EFA6A protect mature spines from inactivity-induced destabilization. These results suggest that ARF6 and EFA6A may regulate the conversion of filopodia to spines and the stability of both early and mature spines. PMID- 16672655 TI - Involvement of basal protein kinase C and extracellular signal-regulated kinase 1/2 activities in constitutive internalization of AMPA receptors in cerebellar Purkinje cells. AB - AMPA receptor (AMPAR) internalization provides a mechanism for long-term depression (LTD) in both hippocampal pyramidal neurons and cerebellar Purkinje cells (PCs). Cerebellar LTD at the parallel fiber (PF)-PC synapse is the underlying basis of motor learning and requires AMPAR activation, a large Ca2+ influx, and protein kinase C (PKC) activation. However, whether these requirements affect the constitutive AMPAR internalization in PF-PC synapses remains unclarified. Tetanus toxin (TeTx) infusion into PCs decreased PF-EPSC amplitude to 60% within 20-30 min (TeTx rundown), without change in paired-pulse facilitation ratio or receptor kinetics. Immunocytochemically measured glutamate receptor 2 (GluR2) internalization ratio decreased at the steady state of TeTx rundown. TeTx rundown did not require AMPAR activity nor an increase in intracellular Ca2+ concentration. TeTx rundown was suppressed partially by the inhibition of either conventional PKC or mitogen-activated protein kinase kinase (MEK) and completely by the inhibition of both kinases. The background PKC activity was shown to be sufficient, because a PKC activator did not facilitate TeTx rundown. The inhibition of protein phosphatase 1/2A (PP1/2A) enhanced TeTx rundown slightly, and both inhibition of PP1/2A and activation of PKC maximized it, but one-half of AMPARs at PF-PC synapses remained in the TeTx-resistant pool. The inhibition of actin depolymerization suppressed TeTx rundown and decreased the GluR2 internalization ratio. In contrast, the inhibition of actin polymerization enhanced TeTx rundown and increased the GluR2 internalization ratio. We suggest that the regulation of actin polymerization is involved in the surface expression of AMPARs and the surface expressing AMPARs are constitutively internalized through both basal PKC and MEK-ERK1/2 (extracellular signal regulated kinase 1/2) activities at PF-PC synapses. PMID- 16672656 TI - Dynamics of suppression in macaque primary visual cortex. AB - The response of a neuron in primary visual cortex (V1) to an optimal stimulus in its classical receptive field (CRF) can be reduced by the presence of an orthogonal mask, a phenomenon known as cross-orientation suppression. The presence of a parallel stimulus outside the CRF can have a similar effect, in this case known as surround suppression. We used a novel stimulus to probe the time course of cross-orientation suppression and found that it is very fast, starting even before the response to optimal excitatory stimuli. However, it occurs with some delay after the offset response, considered to be a measure of the earliest excitatory signals that reach the CRF. We also examined the time course of response to a stimulus presented outside the CRF and found that cross orientation suppression begins substantially earlier than surround suppression measured in the same cells. Together, these findings suggest that cross orientation suppression is attributable to either direct feedforward signal paths to V1 neurons or a circuit involving fast local interneurons within V1. Feedback from higher cortical areas is implicated in surround suppression, but our results make this an implausible mechanism for cross-orientation suppression. We conclude that suppression from inside and outside the CRF occur through different mechanisms. PMID- 16672657 TI - A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels. AB - Temperature transduction in mammals is possible because of the presence of a set of temperature-dependent transient receptor potential (TRP) channels in dorsal root ganglia neurons and skin cells. Six thermo-TRP channels, all characterized by their unusually high temperature sensitivity (Q10 > 10), have been cloned: TRPV1-4 are heat activated, whereas TRPM8 and TRPA1 are activated by cold. Because of the lack of structural information, the molecular basis for regulation by temperature remains unknown. In this study, we assessed the role of the C terminal domain of thermo-TRPs and its involvement in thermal activation by using chimeras between the heat receptor TRPV1 and the cold receptor TRPM8, in which the entire C-terminal domain was switched. Here, we demonstrate that the C terminal domain is modular and confers the channel phenotype regarding temperature sensitivity, channel gating kinetics, and PIP2 (phosphatidylinositol 4,5-bisphophate) modulation. Thus, thermo-TRP channels contain an interchangeable specific region, different from the voltage sensor, which allows them to sense temperature stimuli. PMID- 16672658 TI - Lipid rafts serve as a signaling platform for nicotinic acetylcholine receptor clustering. AB - Agrin, a motoneuron-derived factor, and the muscle-specific receptor tyrosine kinase (MuSK) are essential for the acetylcholine receptor (AChR) clustering at the postjunctional membrane. However, the underlying signaling mechanisms remain poorly defined. We show that agrin stimulates a dynamic translocation of the AChR into lipid rafts-cholesterol and sphingolipid-rich microdomains in the plasma membrane. This follows MuSK partition into lipid rafts and requires its activation. Disruption of lipid rafts inhibits MuSK activation and downstream signaling and AChR clustering in response to agrin. Rapsyn, an intracellular protein necessary for AChR clustering, is located constitutively in lipid rafts, but its interaction with the AChR is inhibited when lipid rafts are perturbed. These results reveal that lipid rafts may regulate AChR clustering by facilitating the agrin/MuSK signaling and the interaction between the receptor and rapsyn, both necessary for AChR clustering and maintenance. These results provide insight into mechanisms of AChR cluster formation. PMID- 16672659 TI - Dynamics of hippocampal and cortical activation during consolidation of a nonspatial memory. AB - Observations of temporally graded retrograde amnesia after hippocampal damage suggest that the hippocampal region plays a critical, time-limited role in memory consolidation. However, these observations do not indicate where permanent memory is stored, nor do they clarify whether the hippocampus normally remains involved in a nonessential way. Evidence from multiple neural imaging studies indicate the time-limited role of the hippocampus and suggest that the anterior cingulate cortex is a critical storage site of different types of long-term memory. However, each of the previous studies examined spatial memory, leaving open the question of whether different cortical areas support long-term memory for other types of material. We characterized the course of involvement of cortical and hippocampal areas in animals trained in an explicitly nonspatial task. First, we confirmed previous findings that hippocampal damage produces temporally graded retrograde amnesia for the social transmission of a food preference (STFP) within our experimental protocol. Damage to the hippocampal region 1 d, but not 21 d, after training impaired subsequent recall of STFP. Then, we characterized the anatomical patterns of activation of the immediate early gene c-fos during retrieval of STFP immediately and 1, 2, and 21 d after training. The ventral subiculum was activated during retrieval shortly after learning, but the level of activation declined at successive times. In contrast, olfactory recipient regions including piriform, entorhinal, and orbitofrontal cortex showed the opposite pattern, increasingly greater activation in successively later retrieval tests. These findings support the view that different cortical networks support long term memory for different types of information. PMID- 16672660 TI - A mechanism for vocal-respiratory coupling in the mammalian parabrachial nucleus. AB - Mammalian vocalizations require the precise coordination of separate laryngeal and respiratory motor pathways. Precisely how and where in the brain vocal motor patterns interact with respiratory rhythm control is unknown. The parabrachial nucleus (PB) is known to mediate key respiratory reflexes and is also considered a principle component of the mammalian vocal motor pathway, making it a likely site for vocal-respiratory interactions, yet a specific role for the PB in vocalizing has yet to be demonstrated. To investigate the role of the PB in vocal respiratory coordination, we pharmacologically manipulated synaptic activity in the PB while spontaneously vocalizing horseshoe bats were provoked to emit either short, single syllable or long, multisyllabic vocal motor patterns. Iontophoresis of the GABAA agonist muscimol (MUS) into the lateral PB extended expiratory durations surrounding all vocalizations and increased mean call durations. Alternatively, application of the GABAA antagonist bicuculline methiodide (BIC) shortened expirations and call durations. In addition, BIC eliminated the occurrence of multisyllabic vocalizations. BIC caused a mild increase in quiet breathing rates, whereas MUS tended to slow quiet breathing. The results indicate that GABAA receptor-mediated inhibition in the lateral PB modulates the time course of respiratory phase switching during vocalizing, and is needed for proper coordination of calling and breathing in mammals. We hypothesize that vocal respiratory rhythm entrainment is achieved at least in part via mechanisms similar to other forms of locomotor-respiratory coupling, namely somatosensory feedback influences on respiratory phase-switching in the lateral PB. PMID- 16672661 TI - Regulation of nuclear factor kappaB in the hippocampus by group I metabotropic glutamate receptors. AB - An increasing amount of evidence suggests that the family of nuclear factor kappaB (NF-kappaB) transcription factors plays an important role in synaptic plasticity and long-term memory formation. The present study investigated the regulation of NF-kappaB family members p50, p65/RelA, and c-Rel in the hippocampus in response to metabotropic glutamate receptor (mGluR) signaling. Activation of group I metabotropic glutamate receptors (GpI-mGluRs) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) resulted in a time-dependent increase in DNA binding activity of p50, p65, and c-Rel in area CA1 of the hippocampus. An antagonist of mGluR5, 2-Methyl-6-(phenylethynyl)pyridine, inhibited the DHPG-induced activation of NF-kappaB, whereas an antagonist of mGluR1, (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid, did not. Using a series of inhibitors, we investigated the signaling pathways necessary for DHPG induced activation of NF-kappaB and found that they included the phosphatidyl inositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and p38-mitogen-activated protein kinase pathways. To determine the functional significance of mGluR-induced regulation of NF-kappaB, we measured long-term depression (LTD) of Schaffer-collateral synapses in the hippocampus of c-Rel knock-out mice. Early phase LTD was normal in c-rel(-/-) mice. However, late phase LTD (>90 min) was impaired in c-rel(-/-) mice. The observations of this deficit in hippocampal synaptic plasticity prompted us to further investigate long-term memory formation in c-rel(-/-) mice. c-rel(-/-) mice exhibited impaired performance in a long-term passive avoidance task, providing additional evidence for c-Rel in long-term memory formation. These results demonstrate that the NF kappaB transcription factor family is regulated by GpI-mGluRs in the hippocampus and that the c-Rel transcription factor is necessary for long-term maintenance of LTD and formation of long-term memory. PMID- 16672662 TI - Distinct physiological mechanisms underlie altered glycinergic synaptic transmission in the murine mutants spastic, spasmodic, and oscillator. AB - Spastic (spa), spasmodic (spd), and oscillator (ot) mice have naturally occurring glycine receptor (GlyR) mutations, which manifest as motor deficits and an exaggerated "startle response." Using whole-cell recording in hypoglossal motoneurons, we compared the physiological mechanisms by which each mutation alters GlyR function. Mean glycinergic miniature IPSC (mIPSC) amplitude and frequency were dramatically reduced (>50%) compared with controls for each mutant. mIPSC decay times were unchanged in spa/spa (4.5 +/- 0.3 vs 4.7 +/- 0.2 ms), reduced in spd/spd (2.7 +/- 0.2 vs 4.7 +/- 0.2 ms), and increased in ot/ot (12.3 +/- 1.2 vs 4.8 +/- 0.2 ms). Thus, in spastic, GlyRs are functionally normal but reduced in number, whereas in spasmodic, GlyR kinetics is faster. The oscillator mutation results in complete absence of alpha1-containing GlyRs; however, some non-alpha1-containing GlyRs persist at synapses. Fluctuation analysis of membrane current, induced by glycine application to outside-out patches, showed that mean single-channel conductance was increased in spa/spa (64.2 +/- 4.9 vs 36.1 +/- 1.4 pS), but unchanged in spd/spd (32.4 +/- 2.1 vs 35.3 +/- 2.1 pS). GlyR-mediated whole-cell currents in spa/spa exhibited increased picrotoxin sensitivity (27 vs 71% block for 100 microM), indicating alpha1 homomeric GlyR expression. The picrotoxin sensitivity of evoked glycinergic IPSCs and conductance of synaptic GlyRs, as determined by nonstationary variance analysis, were identical for spa/spa and controls. Together, these findings show the three mutations disrupt GlyR-mediated inhibition via different physiological mechanisms, and the spastic mutation results in "compensatory" alpha1 homomeric GlyRs at extrasynaptic loci. PMID- 16672663 TI - Enhanced excitatory synaptic connectivity in layer v pyramidal neurons of chronically injured epileptogenic neocortex in rats. AB - Formation of new recurrent excitatory circuits after brain injuries has been hypothesized as a major factor contributing to epileptogenesis. Increases in total axonal length and the density of synaptic boutons are present in layer V pyramidal neurons of chronic partial isolations of rat neocortex, a model of posttraumatic epileptogenesis. To explore the functional consequences of these changes, we used laser-scanning photostimulation combined with whole-cell patch clamp recording from neurons in layer V of somatosensory cortex to map changes in excitatory synaptic connectivity after injury. Coronal slices were submerged in artificial CSF (23 degrees C) containing 100 microM caged glutamate, APV (2-amino 5-phosphonovaleric acid), and high divalent cation concentration to block polysynaptic responses. Focal uncaging of glutamate, accomplished by switching a pulsed UV laser to give a 200-400 micros light stimulus, evoked single- or multiple-component composite EPSCs. In neurons of the partially isolated cortex, there were significant increases in the fraction of uncaging sites from which EPSCs could be evoked ("hot spots") and a decrease in the mean amplitude of individual elements in the composite EPSC. When plotted along the cortical depth, the changes in EPSCs took place mainly between 150 and 200 microm above and below the somata, suggesting a specific enhancement of recurrent excitatory connectivity among layer V pyramidal neurons of the undercut neocortex. These changes may shift the balance within cortical circuits toward increased synaptic excitation and contribute to epileptogenesis. PMID- 16672664 TI - Two brain sites for cannabinoid reward. AB - The recent findings that Delta9tetrahydrocannabinol (Delta9THC), the active agent in marijuana and hashish, (1) is self-administered intravenously, (2) potentiates the rewarding effects of electrical brain stimulation, and (3) can establish conditioned place preferences in laboratory animals, suggest that these drugs activate biologically primitive brain reward mechanisms. Here, we identify two chemical trigger zones for stimulant and rewarding actions of Delta9THC. Microinjections of Delta9THC into the posterior ventral tegmental area (VTA) or into the shell of the nucleus accumbens (NAS) increased locomotion, and rats learned to lever-press for injections of Delta9THC into each of these regions. Substitution of vehicle for drug or treatment with a cannabinoid CB1 receptor antagonist caused response cessation. Microinjections of Delta9THC into the posterior VTA and into the posterior shell of NAS established conditioned place preferences. Injections into the core of the NAS, the anterior VTA, or dorsal to the VTA were ineffective. These findings link the sites of rewarding action of Delta9THC to brain regions where such drugs as amphetamines, cocaine, heroin, and nicotine are also thought to have their sites of rewarding action. PMID- 16672665 TI - Estrogen receptors alpha and beta differentially regulate the transcriptional activity of the Urocortin gene. AB - Urocortin (Ucn), a highly conserved metazoan gene, is related to stress and feeding, behaviors with significant gender differences. We investigated whether estrogens regulate the expression of the Ucn gene using transient transfection in PC12 cells with the human Ucn (hUcn) promoter coupled to luciferase and either alpha or beta estrogen receptors (ERalpha or ERbeta, respectively). The results demonstrate that estradiol (E2) increases the activity of the hUcn promoter via ERalpha, and decreases hUcn promoter activity through ERbeta. Deletions of the hUcn promoter show that the increase in promoter activity mediated by E2-ERalpha depends on a promoter region containing a half-estrogen response element and an Sp1 site, and the decrease mediated by E2-ERbeta depends on a proximal promoter region containing a cAMP response element. Ucn and ERs coexist in neurons of rat hypothalamic nuclei, giving anatomical support for a direct effect of estrogen receptors on the Ucn gene. By in situ hybridization, we observed that cycling female rats have a higher number of cells expressing Ucn mRNA than males in the paraventricular nucleus of the hypothalamus (PVN) and the septum. Both of these brain nuclei are related to stress behaviors and express moderate levels of Ucn. Furthermore, Ucn mRNA was significantly decreased in the PVN and increased in the septum 30 d after ovariectomy. Acute E2 administration to ovariectomized rats significantly increased Ucn mRNA expression in the PVN and septum. In conclusion, our in vitro and in vivo evidence suggests that estrogens exert a direct and differential transcriptional regulation of the Ucn gene. PMID- 16672666 TI - Perceptual knowledge retrieval activates sensory brain regions. AB - Although knowledge indexes our experiences of the world, the neural basis of this relationship remains to be determined. Previous neuroimaging research, especially involving knowledge biased to visual and functional information, suggests that semantic representations depend on modality-specific brain mechanisms. However, it is unclear whether sensory cortical regions, in general, support retrieval of perceptual knowledge. Using neuroimaging methods, we show that semantic decisions that index tactile, gustatory, auditory, and visual knowledge specifically activate brain regions associated with encoding these sensory experiences. Retrieval of tactile knowledge was specifically associated with increased activation in somatosensory, motor, and premotor cortical regions. In contrast, decisions involving flavor knowledge increased activation in an orbitofrontal region previously implicated in processing semantic comparisons among edible items. Perceptual knowledge retrieval that references visual and auditory experiences was associated with increased activity in distinct temporal brain regions involved in the respective sensory processing. These results indicate that retrieval of perceptual knowledge relies on brain regions used to mediate sensory experiences with the referenced objects. PMID- 16672667 TI - Premotor correlates of integrated feedback control for eye-head gaze shifts. AB - Simple activities like picking up the morning newspaper or catching a ball require finely coordinated movements of multiple body segments. How our brain readily achieves such kinematically complex yet remarkably precise multijoint movements remains a fundamental and unresolved question in neuroscience. Many prevailing theoretical frameworks ensure multijoint coordination by means of integrative feedback control. However, to date, it has proven both technically and conceptually difficult to determine whether the activity of motor circuits is consistent with integrated feedback coding. Here, we tested this proposal using coordinated eye-head gaze shifts as an example behavior. Individual neurons in the premotor network that command saccadic eye movements were recorded in monkeys trained to make voluntary eye-head gaze shifts. Head-movement feedback was experimentally controlled by unexpectedly and transiently altering the head trajectory midflight during a subset of movements. We found that the duration and dynamics of neuronal responses were appropriately updated following head perturbations to preserve global movement accuracy. Perturbation-induced increases in gaze shift durations were accompanied by equivalent changes in response durations so that neuronal activity remained tightly synchronized to gaze shift offset. In addition, the saccadic command signal was updated on-line in response to head perturbations applied during gaze shifts. Nearly instantaneous updating of responses, coupled with longer latency changes in overall discharge durations, indicated the convergence of at least two levels of feedback. We propose that this strategy is likely to have analogs in other motor systems and provides the flexibility required for fine-tuning goal-directed movements. PMID- 16672669 TI - Synchronization between background activity and visually evoked potential is not mirrored by focal hyperoxygenation: implications for the interpretation of vascular brain imaging. AB - We performed an electroencephalography and optical topography study simultaneously exploring electrophysiological and vascular response magnitude as a function of stimulus frequency. To elicit a response in the visual cortex, subjects were exposed to flicker frequencies varying from 1 to 25 Hz (1 Hz steps, eyes closed). Extending the standard view to compare magnitudes of the evoked neuronal to the evoked vascular response, we additionally investigated modulations of alpha-power, a marker of "background" EEG activity. The results show two discrepancies between the electrophysiological and vascular response: (1) VEP and alpha-power exhibit a discontinuous peak when stimulating at the individual alpha-frequency (IAF) (approximately 10-11 Hz), indicating resonance between background oscillations and evoked response; this is not mirrored by the vascular response. (2) The vascular response, in contrast, steadily increases up to a maximum at 7-8 Hz and slightly decreases with higher frequencies. This continuous frequency dependence is partly reflected by the decrease in alpha power up to frequencies of 8-9 Hz and a slight increase in alpha-power beyond the IAF resonance. Although indicating an inverse relationship between alpha-power and vascular response, the frequency dependence of the evoked response does not show such a correlation. Thus, electrophysiological resonance between an individual's alpha-frequency and isofrequent stimulation is not mirrored by the vascular response. Also, spontaneous background EEG activity is an important modulator of the vascular response magnitude. We discuss these deviations from a simple one-to-one translation between evoked potential and vascular response amplitude in the light of questions concerning synchronization, attenuation, and induction of background oscillations such as the alpha-rhythm. PMID- 16672670 TI - Involvement of protein synthesis and degradation in long-term potentiation of Schaffer collateral CA1 synapses. AB - Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system. PMID- 16672668 TI - Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide. AB - Increased expression of glial fibrillary acidic protein (GFAP) represents astroglial activation and gliosis during neurodegeneration. However, the molecular mechanism behind increased expression of GFAP in astrocytes is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of GFAP. Bacterial lipopolysachharides (LPSs) induced the production of NO and the expression of GFAP in mouse primary astrocytes. Either a scavenger of NO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (PTIO)] or an inhibitor of inducible nitric oxide synthase [l-N6-(I iminoethyl)-lysine hydrochloride] blocked this induction of GFAP expression. Similarly, other inducers of NO production such as interferon-gamma, interleukin 1beta, human immunodeficiency virus type 1 gp120, fibrillar amyloid beta peptides, and double-stranded RNA (polyinosinic-polycytidilic acid) also induced the expression of GFAP through NO. The role of NO in the expression of GFAP was supported further by increased expression of GFAP by S-nitroso glutathione (GSNO), an NO donor. Interestingly, inhibition of nuclear factor kappaB (NF kappaB) suppressed LPS- but not GSNO-induced expression of GFAP, suggesting that NO does not require NF-kappaB to induce GFAP and that NF-kappaB functions upstream of NO production. However, inhibition of LPS- and GSNO-induced expression of GFAP either by NS-2028 [a specific inhibitor of guanylate cyclase (GC)] or by KT5823 [a specific inhibitor of cGMP-activated protein kinase (PKG)], and induction of GFAP expression by either 8-Br cGMP (a cell-permeable cGMP analog) or MY-5445 (a specific inhibitor of cGMP phosphodiesterase) suggests that NO induces GFAP via GC-cGMP-PKG. This study illustrates a novel biological role of NO in regulating the expression of GFAP in astrocytes through the GC-cGMP-PKG pathway that may participate in the pathogenesis of neurodegenerative disorders. PMID- 16672671 TI - Plasticity-associated gene Krox24/Zif268 is required for long-lasting behavioral effects of cocaine. AB - The extracellular signal-regulated kinases (ERKs) 1/2 pathway is stimulated by drugs of abuse in striatal neurons through coincident activation of dopamine D1 and glutamate NMDA receptors and is critical for long-lasting behavioral effects of these drugs. Although regulation of transcription is a major target of ERK, the precise mechanisms by which it contributes to behavioral alterations is not known. We examined the role of Zif268, an immediate-early gene induced by drugs of abuse under the control of ERK, in behavioral responses to cocaine using knock in mutant mice in which Zif268 was replaced by LacZ. No biochemical or behavioral differences between mutant and wild-type mice were observed in basal conditions or in acute responses to cocaine injection. In contrast, locomotor sensitization to single or repeated cocaine injections was dramatically diminished in both heterozygous and homozygous Zif268 mutant mice. Conditioned place preference in response to cocaine was prevented in Zif268-deficient mice. This effect was not attributable to a general learning deficit because the mutant mice displayed normal conditioned place preference when food was used as reward. Our results provide direct genetic evidence for the requirement of Zif268 for long-lasting association of environmental context with specific behavioral responses after short exposures to cocaine. They also underline the common molecular machinery involved in long-lasting drug-induced behavioral alterations and the formation of other types of memory. PMID- 16672673 TI - Perceptual learning directs auditory cortical map reorganization through top-down influences. AB - The primary sensory cortex is positioned at a confluence of bottom-up dedicated sensory inputs and top-down inputs related to higher-order sensory features, attentional state, and behavioral reinforcement. We tested whether topographic map plasticity in the adult primary auditory cortex and a secondary auditory area, the suprarhinal auditory field, was controlled by the statistics of bottom up sensory inputs or by top-down task-dependent influences. Rats were trained to attend to independent parameters, either frequency or intensity, within an identical set of auditory stimuli, allowing us to vary task demands while holding the bottom-up sensory inputs constant. We observed a clear double-dissociation in map plasticity in both cortical fields. Rats trained to attend to frequency cues exhibited an expanded representation of the target frequency range within the tonotopic map but no change in sound intensity encoding compared with controls. Rats trained to attend to intensity cues expressed an increased proportion of nonmonotonic intensity response profiles preferentially tuned to the target intensity range but no change in tonotopic map organization relative to controls. The degree of topographic map plasticity within the task-relevant stimulus dimension was correlated with the degree of perceptual learning for rats in both tasks. These data suggest that enduring receptive field plasticity in the adult auditory cortex may be shaped by task-specific top-down inputs that interact with bottom-up sensory inputs and reinforcement-based neuromodulator release. Top-down inputs might confer the selectivity necessary to modify a single feature representation without affecting other spatially organized feature representations embedded within the same neural circuitry. PMID- 16672672 TI - RanBPM contributes to Semaphorin3A signaling through plexin-A receptors. AB - Secreted Semaphorin3A (Sema3A) proteins are known to act as diffusible and repellant axonal guidance cues during nervous system development. A receptor complex consisting of a Neuropilin and a Plexin-A mediates their effects. Plexin A signal transduction has remained poorly defined despite the documented involvement of collapsin response mediator protein and molecule interacting with CasL proteins (MICALs) as mediators of Plexin-A activation. Here, we defined a domain of Plexin-A1 required for Sema3A signaling in a reconstituted environment and then searched for proteins interacting with this domain. RanBPM is shown to physically interact with Plexin-A1, and the RanBPM/Plexin complex is regulated by MICAL expression. Overexpression of RanBPM cooperates with PlexinA1 to reduce non neuronal cell spreading and strongly inhibit axonal outgrowth in vitro and in vivo. A truncated RanBPM protein blocks Sema3A responsiveness in non-neuronal and neuronal cells. Suppression of RanBPM expression reduces Sema3A responsiveness. Thus, RanBPM is a mediator of Sema3A signaling through Plexin-A. RanBPM has the potential to link Plexin-A receptors to retrograde transport and microtubule function in axonal guidance. PMID- 16672674 TI - Behavior-change action plans in primary care: a feasibility study of clinicians. AB - PURPOSE: Collaborative goal-setting--with clinician and patient together deciding on concrete behavior-change goals-may be more effective in encouraging healthy behaviors than traditional clinician-directed advice. This study explores whether it is feasible for clinicians to engage patients with coronary heart disease (CHD) risk factors in collaborative goal-setting and concrete action planning during the primary care visit. METHODS: Primary care clinicians were trained in goal-setting and action planning techniques and asked to conduct action plan discussions with study patients during medical visits. Clinicians' experiences were documented through post-visit surveys and with questionnaires and semistructured interviews at the end of the study. RESULTS: Forty-three clinicians and 274 patients with CHD risk factors participated in the study; 83% of the patient encounters resulted in a behavior-change action plan. Goal-setting discussions lasted an average of 6.9 minutes. Clinicians rated 75% of the discussions as equally or more satisfying than previous behavior-change discussions, and identified time constraints as the most important barrier to adopting the goal-setting process. CONCLUSIONS: Collaborative goal-setting between clinicians and patients for improved health behaviors is viewed favorably by clinicians in primary care. Time constraints could be addressed by delegating goal-setting to other caregivers. PMID- 16672675 TI - Using action plans to help primary care patients adopt healthy behaviors: a descriptive study. AB - PURPOSE: An action plan is an agreement between clinician and patient that the patient will make a specific behavior change. The goals of this study are to: determine whether it is feasible for patients to make action plans in the primary care visit; determine whether patients report carrying out their action plans; and describe the action plans patients choose. METHODS: Forty-three clinicians in 8 primary care sites were recruited to hold action-plan discussions with patients. Research assistants contacted patients by telephone 3 weeks later to assess whether patients had conducted their action plans. RESULTS: Eighty-three percent of enrolled patients (228) made an action plan during a primary care visit. Of the 79% who recalled making the action plan when interviewed by telephone 3 weeks later, 56% recalled the details of their action plan, and an additional 33% recalled the general nature of the action plan. At least 53% of patients making an action plan reported making a behavior change consistent with that action plan. CONCLUSIONS: Most patients reported making a behavior change based on an action plan, suggesting that action plans may be a useful strategy to encourage behavior change for patients seen in primary care. PMID- 16672676 TI - Clinical decision-making in blood pressure management of patients with diabetes mellitus: an Oklahoma Physicians Resource/Research Network (OKPRN) Study. AB - BACKGROUND: Outcomes can be improved when the blood pressure (BP) is kept below 130/80 in patients with diabetes mellitus. However, physicians and patients achieve this target less than 50% of the time. The purpose of this study was to determine the reasons for this apparent quality deficit from the perspective of a small random sample of family physicians. METHODS: Nine family physicians completed interviews about encounters with diabetic patients with BP >or=130/80 for whom no change in management was recorded. Four investigators analyzed the transcribed interviews to identify and categorize reasons for failure to intervene. RESULTS: Ninety-eight (62%) of 159 patients had BP >or=130/80 at the index visit. No change in management was recorded in 73 (74%). Physicians gave 175 reasons for nonintervention in 3 broad categories, physician-related, patient related, and information/measurement-related reasons, and 10 subcategories. In most cases they gave more than one reason (mean 2.4) per case. The most frequent subcategories were limited treatment options (47/73; 64%), inadequate information on which to intervene (43/73; 59%), and patient nonadherence (27/73; 37%). Competing demands were mentioned in 10 cases (13.7%). Physicians differed with regard to the kinds of reasons given. CONCLUSIONS: Physicians have a variety of clinical reasons for not responding to elevated BP in diabetic patients. Some might be addressed with better technology (eg, more reliable BP measurements) or health care system reforms (eg, less expensive medications). Others (eg, patient nonadherence) are more challenging. Methods for measuring quality must be robust enough to account for legitimate clinical reasons for not achieving BP targets. Physician-based interventions will need to take into account different physician personalities and practice styles. PMID- 16672677 TI - Further investigation of the negative association between hypertension and peripheral neuropathy in the elderly: an Oklahoma Physicians Resource/Research Network (OKPRN) Study. AB - BACKGROUND: In a previous cross-sectional study of age-associated peripheral neuropathy (AAPN), we found that a history of hypertension was protective. The purpose of this study, conducted in the same cohort, was to investigate further this association in the same subjects and in a comparison group of older subjects with diabetes mellitus. METHODS: Baseline data from 584 subjects involved in a longitudinal study of primary care patients 65 years of age and older, with no history of 10 medical conditions known to cause peripheral neuropathy, were analyzed for associations between peripheral neuropathy by examination and history of hypertension, number of antihypertensive medications, systolic blood pressure, diastolic blood pressure, pulse pressure, and orthostatic hypotension. In addition, we examined associations between neuropathy at baseline and use of specific classes of antihypertensive medications and non-steroidal anti inflammatory agents (NSAIDs) in year 3. The analyses were repeated in 110 subjects with diabetes mellitus. RESULTS: History of hypertension, but not the other hypertension-related variables, was negatively associated (OR, 0.60; 95% CI: 0.40 to 0.90) with AAPN after controlling for age and body mass index (BMI). In 287 subjects evaluated in the 3rd year of the study, beta-blocking agents (OR, 3.56; 95% CI: 1.58 to 8.03) and NSAIDs (OR, 2.65; 95% CI: 1.37 to 5.10) were positively associated with AAPN. In subjects with diabetes mellitus, a history of hypertension was again protective, but current pulse pressure (OR, 1.03; 95% CI: 1.00 to 1.05) was a positive predictor of peripheral neuropathy. There were interesting interactions between pulse pressure and military service and pulse pressure and BMI in diabetic patients. CONCLUSIONS: The negative association between hypertension and AAPN remains unexplained. The positive association between pulse pressure and neuropathy in diabetic subjects supports findings from previous studies and suggests that AAPN and the neuropathy of diabetes may be distinct entities. The incidental finding of an association between NSAIDs and AAPN is concerning and should be investigated further. PMID- 16672678 TI - Physician-patient interaction for smoking cessation medications: a dance of mutual accommodation? AB - OBJECTIVE: To study physician-smoker interactions around the receipt of smoking cessation medication prescriptions to better understand the low rates of reported assistance and follow-up. METHODS: A stratified random sample of smokers recently filling prescriptions for cessation medications was identified for a phone interview about the quitting experience. The transcriptions of those portions of 50 interviews that addressed cessation contacts with clinicians were reviewed by the co-authors and analyzed for quantifiable data, observations, and themes. RESULTS: Although there were low levels of reported physician adherence to the Assist and Arrange recommendations of the Public Health Service 5As clinical guideline for smoking cessation, 27 (55%) of these smokers were quit at 1 to 3 months after the medication fill. Smoker descriptions of the contacts with their physicians about smoking cessation suggested nonconfrontational, collaborative, and satisfying interactions that were flexibly dominated by either party. Physician assistance predominantly concerned use of the medication (66%). CONCLUSIONS: These physician-smoker interactions seemed to be mutually accommodative. Given the apparent high quit rates and limited evidence of smoker interest in other forms of assistance, perhaps a physician-dominant encounter is not as common or as necessary as has been thought. PMID- 16672679 TI - Factors that influence the receipt of well baby care in the first 2 weeks of life. AB - PURPOSE: To determine the percentage of babies born at a community hospital who received follow-up visits at home or clinic by 2 weeks of age. Also to describe the characteristics of the mothers whose newborns received visits within 2 weeks of birth. METHODS: A convenience sample of 335 mothers giving birth at a 430-bed community hospital in St. Paul, MN from September 2003 to September 2004 were surveyed by telephone when their infants were approximately 3- to 4-weeks old. RESULTS: 84% of the infants had a home or clinic visit within 2 weeks of birth. In bivariate analyses, the likelihood of having a visit within 2 weeks was significantly lower for mothers having more children (P = .002), lower maternal education level (P = .002), lower income (P = .02), mothers' lack of knowledge of baby's insurance (P = .02), mothers of nonwhite race (P = .03), and mothers' having no medical insurance (P = .04). The likelihood of a visit was not significantly related to whether English was spoken in the home or marital status. In logistic regression analyses, lower maternal education, and more children were significant predictors of the lack of visits. CONCLUSIONS: Infants whose mothers had little education and had other children were a high-risk group that was less likely to receive care. When discharging newborns belonging to this group, extra effort should be made to ensure that appropriate postdischarge follow-up occurs. PMID- 16672680 TI - Characteristics of frequent attenders at a community health center. AB - BACKGROUND: As high utilizers of health care resources, frequent attenders to medical practices pose a significant issue for primary health care. Studies documenting content of visits and characteristics of frequent attenders have paid limited attention to community health center populations. This study profiles these high utilizers comparing them to non-frequent attenders. METHODS: Through medical record abstraction, retrospective and longitudinal patient data were obtained for a 30-month time period for 382 established patients. RESULTS: Older patients 45 to 64 years (OR = 1.85; 95% CI: 0.92, 3.70) and Medicaid recipients (OR = 3.22; 95% CI: 1.54, 6.72) were more likely to be frequent attenders. Patients residing furthest away from the CHC were also the most frequent of attenders (OR = 3.68; 95% CI: 1.22, 11.13), as were those with a diagnosis of diabetes (OR = 5.03; 95% CI: 2.17, 11.70) or depression (OR = 1.85; 95% CI: 0.96, 3.56). Patients who used the Emergency Department more (OR = 1.54; 95% CI: 1.23,1.91) had the highest number of visits, although those patients who missed more appointments as a proportion of the number of scheduled appointments were less likely to be frequent attenders (OR = 0.49; 95% CI: 0.36, 0.66). CONCLUSIONS: Through developing interventions such as customized social report cards and applying elements of the Chronic Care Model, these results can help administrators and policy makers ensure that frequent attenders are cared for adequately and that safety net providers' resources are appropriate to the tasks demanded of them. PMID- 16672681 TI - Group visits: a qualitative review of current research. AB - PURPOSE: The group visit model has emerged as one possible solution to problems posed by the limitations of current structures of care and the demands of a growing chronic illness load. In this article, we summarize current group visit research and develop suggestions for furthering this care model. METHODS: An electronic review of all group visit articles published from the years 1974 to 2004 was conducted via the PubMed and MedLine databases. Reference sections of articles thus obtained were mined for additional citations. Articles were excluded if: (1) they were not research studies (ie, purely descriptive, with no evaluative component); or (2) the group visit intervention was subsumed under larger primary or hospital-based interventions. RESULTS: Although the heterogeneity of the studies presented renders the assessment of this care model problematic, there is sufficient data to support the effectiveness of group visits in improving patient and physician satisfaction, quality of care, quality of life, and in decreasing emergency department and specialist visits. CONCLUSION: Group visits are a promising approach to chronic care management for the motivated patient. Future research may benefit, however, from abandoning old nomenclatures and clearly defining the structure, processes of care, content of visits, and appropriate outcome measures. PMID- 16672682 TI - A systematic review of studies comparing myocardial infarction mortality for generalists and specialists: lessons for research and health policy. AB - BACKGROUND: Much of the research comparing specialists and generalists is from studies of patients who had a myocardial infarction. The present study systematically examined this research. METHODS: Medline was used to search for all articles published from 1990 to 2003 that compared cardiologists and generalists for adjusted mortality rates of patients with myocardial infarction. From each article identified, information was abstracted on factors that could have influenced the comparisons. RESULTS: The studies consistently found that patients of generalists were at greater risk of mortality from both cardiac and noncardiac risk factors and had higher unadjusted mortality rates. Adjusting for risk factors decreased the differences between cardiologists and generalists. Studies that seemed to do the best job taking into account patient differences had similar adjusted-mortality rates for the cardiologists and generalists. No studies adequately took into account reasons the patient did not have care by a cardiologist, eg, patient preferences, severity of comorbid disease, general health status, or resource availability. CONCLUSIONS: Generalists and cardiologists differ substantially with respect to their patients and practice environments. Results comparing patient outcomes by specialty are often influenced by important patient or resource characteristics that were not taken into account. PMID- 16672683 TI - Reorganizing medicare for older adults with chronic illness. AB - A major challenge for the US health care system during coming years will be the financial viability and reorganization of Medicare--a program in which over 90% of family physicians participate. Although chronic illness constitutes a trajectory characterized by long-term patterns of health and functional states, fee-for-service Medicare is largely directed to the treatment of acute, episodic illness. Beyond the prescription drug benefit, there were several provisions in the Medicare Prescription Drug Improvement and Modernization Act of 2003 that were designed to improve the quality of care and reduce costs for chronically ill beneficiaries, an important first step in the reorientation of Medicare to chronic illness care. Quality is the foundation of Medicare's movement to a chronic care program and paying physicians for quality care is on the horizon. Family physicians will need to be actively engaged in Medicare's reorientation by articulating and promoting a quality of care that effectively integrates evidence based medicine with a person-centered focus. PMID- 16672684 TI - Hypertriglyceridemia. AB - Hypertriglyceridemia is a commonly encountered lipid abnormality frequently associated with other lipid and metabolic derangements. The National Cholesterol Education Program recommends obtaining a fasting lipid panel in adults over the age of 20. The discovery of hypertriglyceridemia should prompt an investigation for secondary causes such as high fat diet, excessive alcohol intake, certain medications, and medical conditions (eg, diabetes mellitus, hypothyroidism). In addition, patients should be evaluated for other components of the metabolic syndrome. These include abdominal obesity, insulin resistance, low high-density lipoprotein (HDL), high triglyceride, and hypertension. Hypertriglyceridemia is classified as primary hypertriglyceridemia when there are no secondary causes identified. Primary hypertriglyceridemia is the result of various genetic defects leading to disordered triglyceride metabolism. It is important to treat hypertriglyceridemia to prevent pancreatitis by reducing triglyceride levels to <500 mg/dL. Furthermore, lowering triglycerides while treating other dyslipidemias and components of the metabolic syndrome will reduce coronary events. However, it is controversial how much isolated hypertriglyceridemia correlates directly with coronary artery disease and further studies are needed to clarify whether treatment for this condition leads to meaningful clinical outcomes. Therapeutic lifestyle changes (TLC) are the first line of treatment for hypertriglyceridemia. These changes include a low saturated fat, carbohydrate controlled diet, combined with alcohol reduction, smoking cessation, and regular aerobic exercise. High doses of omega-3 fatty acids from fish and fish oil supplements will lower triglyceride levels significantly. When patients do not reach their goals by TLC, drug therapy should be started. In cases of isolated hypertriglyceridemia, fibrates are initially considered. When elevated low density lipoprotein levels accompany hypertriglyceridemia, 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors are preferred. In patients with low HDL levels and hypertriglyceridemia, extended release niacin can be considered. A combination of the medicines may be necessary in recalcitrant cases. PMID- 16672685 TI - Parvovirus B19 presenting with severe sepsis in a previously healthy 25-year-old female. PMID- 16672686 TI - Chronic addiction to dextromethorphan cough syrup: a case report. AB - BACKGROUND: Serious drug abuse and addiction related to dextromethorphan containing cough preparations has been a problem in the United States since the 1950s, but few physicians are aware of it. Physicians must be alert to the type of substances and quantities used and misused by patients in obtaining a thorough routine history of over-the-counter medication use. METHODS: We describe the case of a 66-year-old clerical worker who ingested 4 to 16 oz of dextromethorphan on a regular basis over an 8-year period. We consulted with our local Poison Control Center and undertook a literature search to research previous reports of similar cases to identify the features that would aid physicians in recognition and management of this problem. RESULTS AND CONCLUSION: Despite the availability of a substantial number of case reports in specialty journals, there are almost no reports in the primary care literature of chronic dextromethorphan addiction. Our case highlights the difficulties in making an appropriate diagnosis and in obtaining effective help for the patient. PMID- 16672687 TI - Action planning: a call to action. PMID- 16672689 TI - Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism. AB - Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties and inhibits factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The mechanism and molecular target for intrinsic tenase inhibition were determined and compared with inhibition by low-molecular-weight heparin (LMWH). DHG inhibited factor X activation in a noncompetitive manner (reduced V(max(app))), with 50-fold higher apparent affinity than LMWH. DHG did not affect factor VIIIa half-life or chromogenic substrate cleavage by factor IXa phospholipid but reduced the affinity of factor IXa for factor VIIIa. DHG competed factor IXa binding to immobilized LMWH with an EC(50) 35-fold lower than soluble LWMH. Analysis of intrinsic tenase inhibition, employing factor IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (K(i)) for DHG was as follows: wild type > K241A > H92A > R170A > > R233A, with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity and the potency of LMWH for intrinsic tenase. DHG also accelerated decay of the intact intrinsic tenase complex. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interactions. PMID- 16672690 TI - Rat glomerular mesangial cells require laminin-9 to migrate in response to insulin-like growth factor binding protein-5. AB - Temporal and spatial differences in extracellular matrix play critical roles in cell proliferation, differentiation and migration. Different migratory stimuli use different substrates and receptors to achieve cell migration. To understand the mechanism of insulin-like growth factor binding protein-5 (IGFBP-5)-induced migration in mesangial cells, the roles of integrins and substrates were examined. IGFBP-5 induced an increase in mRNA expression for laminin (LN) chains lama4, lamb2, and lamc1, suggesting that LN-9 might be required for migration. Antibodies to the LNalpha(4) and LNbeta(2) chains, but not LNbeta(1), blocked IGFBP-5-induced migration. Anti-sense morpholino oligonucleotide inhibition of expression of LNalpha(4) substantially reduced expression of LN-8/9 (alpha(4)beta(1)gamma(1)/alpha(4)beta(2)gamma(1), 411/421) and prevented IGFBP-5 induced migration. Anti-sense inhibition of lamb2 reduced expression of LN-9. Absence of LN-9 prevented IGFBP-5-induced migration, which was not preserved by continued expression of LN-8. The requirement for LN-9 was further supported by studies of T98G cells, which express predominantly LN-8. IGFBP-5 had little effect on migration in these cells, but increased migration when T98G cells were plated on LN-8/9. IGFBP-5-mediated mesangial cell migration was inhibited by antibodies that block attachment to alpha(6)beta(1)-integrins but was unaffected by antibodies and disintegrins that block binding to other integrins. Furthermore, in cells with anti-sense inhibited expression of LN-9, integrin alpha(6)beta(1) was no longer detected on the cell surface. These studies suggest the specificity of mechanisms of migration induced by specific stimuli and for the first time demonstrate a unique function for LN-9 in mediating IGFBP-5 induced migration. PMID- 16672691 TI - JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells. AB - Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1 MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP. PMID- 16672692 TI - Palytoxin-induced cell death cascade in bovine aortic endothelial cells. AB - The plasmalemmal Na(+)-K(+)-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca(2+). However, the ability of PTX to directly increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via Na(+) pump channels and to initiate Ca(2+) overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca(2+)](i) and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca(2+)](i) that was dependent on extracellular Ca(2+). The increase in [Ca(2+)](i) initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC(50) < 1 microM. The elevation in [Ca(2+)](i) could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca(2+)](i) occurs via the Na(+) pump. Subsequent to the rise in [Ca(2+)](i), PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca(2+) or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca(2+) overload. PMID- 16672693 TI - Muscle cell survival mediated by the transcriptional coactivators p300 and PCAF displays different requirements for acetyltransferase activity. AB - Normal skeletal muscle development requires the proper orchestration of genetic programs by myogenic regulatory factors (MRFs). The actions of the MRF protein MyoD are enhanced by the transcriptional coactivators p300 and the p300/CBP associated factor (PCAF). We previously described C2 skeletal myoblasts lacking expression of insulin-like growth factor-II (IGF-II) that underwent progressive apoptotic death when incubated in differentiation-promoting medium. Viability of these cells was sustained by addition of IGF analogs or unrelated peptide growth factors. We now show that p300 or PCAF maintains myoblast viability as effectively as added growth factors through mechanisms requiring the acetyltransferase activity of PCAF but not of p300. The actions of p300 to promote cell survival were not secondary to increased expression of known MyoD targets, as evidenced by results of gene microarray experiments, but rather appeared to be mediated by induction of other genes, including fibroblast growth factor-1 (FGF-1). Conditioned culture medium from cells expressing p300 increased myoblast viability, and this was blocked by pharmacological inhibition of FGF receptors. Our results define a role for p300 in promoting cell survival, which is independent of its acetyltransferase activity and acts at least in part through FGF-1. PMID- 16672694 TI - Mechanism of inhibition of TREK-2 (K2P10.1) by the Gq-coupled M3 muscarinic receptor. AB - TREK-2 is a member of the two-pore domain K(+) channel family and provides part of the background K(+) current in many types of cells. Neurotransmitters that act on receptors coupled to G(q) strongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and M(3) receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5'-O-(3 thiotriphosphate) (10 microM), Ca(2+) (5 microM), or diacylglycerol (DAG; 10 microM) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 microM wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 microM) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist induced inhibition of TREK-2 via M(3) receptor occurs primarily via PKC-mediated phosphorylation. PMID- 16672695 TI - Images in clinical medicine. Scrofula. PMID- 16672696 TI - Injury to research volunteers--the clinical-research nightmare. PMID- 16672697 TI - Compensation for injured research subjects. PMID- 16672698 TI - Disabled receptor signaling and new primary immunodeficiency disorders. PMID- 16672699 TI - Balloon angioplasty versus implantation of nitinol stents in the superficial femoral artery. AB - BACKGROUND: Because stent implantation for disease of the superficial femoral artery has been associated with high rates of late clinical failure, percutaneous transluminal angioplasty is preferred for endovascular treatment, and stenting is recommended only in the event of suboptimal technical results. We evaluated whether primary implantation of a self-expanding nitinol (nickel-titanium) stent yielded anatomical and clinical benefits superior to those afforded by percutaneous transluminal angioplasty with optional secondary stenting. METHODS: We randomly assigned 104 patients who had severe claudication or chronic limb ischemia due to stenosis or occlusion of the superficial femoral artery to undergo primary stent implantation (51 patients) or angioplasty (53 patients). Restenosis and clinical outcomes were assessed at 6 and 12 months. RESULTS: The mean (+/-SD) length of the treated segment was 132+/-71 mm in the stent group and 127+/-55 mm in the angioplasty group. Secondary stenting was performed in 17 of 53 patients (32 percent) in the angioplasty group, in most cases because of a suboptimal result after angioplasty. At 6 months, the rate of restenosis on angiography was 24 percent in the stent group and 43 percent in the angioplasty group (P=0.05); at 12 months the rates on duplex ultrasonography were 37 percent and 63 percent, respectively (P=0.01). Patients in the stent group were able to walk significantly farther on a treadmill at 6 and 12 months than those in the angioplasty group. CONCLUSIONS: In the intermediate term, treatment of superficial-femoral-artery disease by primary implantation of a self-expanding nitinol stent yielded results that were superior to those with the currently recommended approach of balloon angioplasty with optional secondary stenting. (ClinicalTrials.gov number, NCT00281060.). PMID- 16672700 TI - Outcomes among newborns with total serum bilirubin levels of 25 mg per deciliter or more. AB - BACKGROUND: The neurodevelopmental risks associated with high total serum bilirubin levels in newborns are not well defined. METHODS: We identified 140 infants with neonatal total serum bilirubin levels of at least 25 mg per deciliter (428 micromol per liter) and 419 randomly selected controls from a cohort of 106,627 term and near-term infants born from 1995 through 1998 in Kaiser Permanente hospitals in northern California. Data on outcomes were obtained from electronic records, interviews, responses to questionnaires, and neurodevelopmental evaluations that had been performed in a blinded fashion. RESULTS: Peak bilirubin levels were between 25 and 29.9 mg per deciliter (511 micromol per liter) in 130 of the newborns with hyperbilirubinemia and 30 mg per deciliter (513 micromol per liter) or more in 10 newborns; treatment involved phototherapy in 136 cases and exchange transfusion in 5. Follow-up data to the age of at least two years were available for 132 of 140 children with a history of hyperbilirubinemia (94 percent) and 372 of 419 controls (89 percent) and included formal evaluation at a mean (+/-SD) age of 5.1+/-0.12 years for 82 children (59 percent) and 168 children (40 percent), respectively. There were no cases of kernicterus. Neither crude nor adjusted scores on cognitive tests differed significantly between the two groups; on most tests, 95 percent confidence intervals excluded a 3-point (0.2 SD) decrease in adjusted scores in the hyperbilirubinemia group. There was no significant difference between groups in the proportion of children with abnormal neurologic findings on physical examination or with documented diagnoses of neurologic abnormalities. Fourteen of the children with hyperbilirubinemia (17 percent) had "questionable" or abnormal findings on neurologic examination, as compared with 48 controls (29 percent; P=0.05; adjusted odds ratio, 0.47; 95 percent confidence interval, 0.23 to 0.98; P=0.04). The frequencies of parental concern and reported behavioral problems also were not significantly different between the two groups. Within the hyperbilirubinemia group, those with positive direct antiglobulin tests had lower scores on cognitive testing but not more neurologic or behavioral problems. CONCLUSIONS: When treated with phototherapy or exchange transfusion, total serum bilirubin levels in the range included in this study were not associated with adverse neurodevelopmental outcomes in infants born at or near term. PMID- 16672701 TI - An antibody-deficiency syndrome due to mutations in the CD19 gene. AB - BACKGROUND: The CD19 protein forms a complex with CD21, CD81, and CD225 in the membrane of mature B cells. Together with the B-cell antigen receptor, this complex signals the B cell to decrease its threshold for activation by the antigen. METHODS: We evaluated four patients from two unrelated families who had increased susceptibility to infection, hypogammaglobulinemia, and normal numbers of mature B cells in blood. We found a mutation in the CD19 gene in all four patients. The CD19 gene in the patients and their first-degree relatives was sequenced, and flow-cytometric immunophenotyping of B cells, immunohistochemical staining of lymphoid tissues, and DNA and messenger RNA analysis were performed. B-cell responses on the triggering of the B-cell receptor were investigated by in vitro stimulation; the antibody response after vaccination with rabies vaccine was also studied. RESULTS: All four patients had homozygous mutations in the CD19 gene. Levels of CD19 were undetectable in one patient and substantially decreased in the other three. Levels of CD21 were decreased, whereas levels of CD81 and CD225 were normal, in all four patients. The composition of the precursor B-cell compartment in bone marrow and the total numbers of B cells in blood were normal. However, the numbers of CD27+ memory B cells and CD5+ B cells were decreased. Secondary follicles in lymphoid tissues were small to normal in size and had a normal cellular composition. The few B cells that showed molecular signs of switching from one immunoglobulin class to another contained V(H)-C(alpha) and V(H)-C(gamma) transcripts with somatic mutations. The response of the patients' B cells to in vitro stimulation through the B-cell receptor was impaired, and in all four patients, the antibody response to rabies vaccination was poor. CONCLUSIONS: Mutation of the CD19 gene causes a type of hypogammaglobulinemia in which the response of mature B cells to antigenic stimulation is defective. PMID- 16672702 TI - Inherited and somatic CD3zeta mutations in a patient with T-cell deficiency. AB - A four-month-old boy with primary immunodeficiency was found to have a homozygous germ-line mutation of the gene encoding the CD3zeta subunit of the T-cell receptor-CD3 complex. CD3zeta is necessary for the development and function of T cells. Some of the patient's T cells had low levels of the T-cell receptor-CD3 complex and carried the Q70X mutation in both alleles of CD3zeta, whereas other T cells had normal levels of the complex and bore the Q70X mutation on only one allele of CD3zeta, plus one of three heterozygous somatic mutations of CD3zeta on the other allele, allowing expression of poorly functional T-cell receptor-CD3 complexes. PMID- 16672703 TI - Shattuck Lecture. The family business--to educate. PMID- 16672705 TI - Images in clinical medicine. Spurious platelet count. PMID- 16672704 TI - Clinical practice. Thrombotic thrombocytopenic purpura. PMID- 16672706 TI - Clinical problem-solving. Ring around the diagnosis. PMID- 16672707 TI - Treatment of peripheral arterial disease--extending "intervention" to "therapeutic choice". PMID- 16672708 TI - Neonatal hyperbilirubinemia--what are the risks? PMID- 16672709 TI - Saw palmetto for benign prostatic hyperplasia. PMID- 16672710 TI - Racial differences in lung cancer. PMID- 16672711 TI - Aprotinin in cardiac surgery. PMID- 16672712 TI - Positron-emission tomography in cancer therapy. PMID- 16672713 TI - Chronic daily headache. PMID- 16672714 TI - Medicare drug benefit. PMID- 16672715 TI - Resolution of recurrent focal segmental glomerulosclerosis proteinuria after rituximab treatment. PMID- 16672716 TI - A delayed complication after injury in World War II. PMID- 16672717 TI - The estrogen early response gene FOS is altered in a baboon model of endometriosis. AB - Endometriosis, the presence of a functional endometrium outside of the uterine cavity, is associated with infertility. In our simulated model of pregnancy in baboons with experimental endometriosis, hCG infusion fails to induce expression of the immunoregulatory protein glycodelin. To test the hypothesis that the development of endometriosis is associated with an aberrant endometrial immunological environment, we examined the expression of a series of immunoregulatory genes in endometrium from baboons with and without endometriosis. Six months following intraperitoneal inoculation with menstrual endometrium, eutopic endometrium was surgically collected between Days 9 and 11 postovulation. Control endometrium was similarly collected from disease-free animals. Total RNA was extracted, and biotinylated cDNA probes were hybridized to the SuperArray GEArray Q series Th1/Th2/Th3 cDNA array, representing 96 genes. Gene expression levels were determined using ScanAlyze and GEArray Analyzer software. Seven genes were upregulated, including JUND, FOS, CCL11, NFKB1 and others, in the endometrium from baboons with endometriosis compared with the endometrium from disease-free animals; one gene, IL1R1, was downregulated. Quantitative RT-PCR confirmed upregulation of FOS and CCL11 in endometriotic eutopic endometrium. Immunohistochemical analysis revealed altered levels and distribution of FOS protein in the eutopic endometrium of baboons with induced endometriosis. These data suggest that in an induced model of endometriosis an aberrant eutopic immunological environment results in a decreased apoptotic potential and in rapid alterations in endometrial gene expression. We propose that the reduced fecundity associated with endometriosis has a multifold etiology in spontaneous and induced disease. PMID- 16672718 TI - Cloned transgenic swine via in vitro production and cryopreservation. AB - It has been notoriously difficult to successfully cryopreserve swine embryos, a task that has been even more difficult for in vitro-produced embryos. The first reproducible method of cryopreserving in vivo-produced swine embryos was after centrifugation and removal of the lipids. Here we report the adaptation of a similar process that permits the cryopreservation of in vitro-produced somatic cell nuclear transfer (SCNT) swine embryos. These embryos develop to the blastocyst stage and survive cryopreservation. Transfer of 163 cryopreserved SCNT embryos to two surrogates produced 10 piglets. Application of this technique may permit national and international movement of cloned transgenic swine embryos, storage until a suitable surrogate is available, or the long-term frozen storage of valuable genetics. PMID- 16672719 TI - Analysis of transcription factor AP-2 expression and function during mouse preimplantation development. AB - The activating protein 2 (AP-2) transcription factor family is required for multiple aspects of mouse postimplantation development, but much less is known about the expression and possible function of these genes during the preimplantation period. In the present study, we have examined the expression of all five members of the mouse AP-2 gene family in the unfertilized oocyte and from zygote formation to the blastocyst stage of development. Four AP-2 genes are differentially expressed during the preimplantation period,Tcfap2a, Tcfap2b, Tcfap2c, and Tcfap2e. Furthermore, with the exception of Tcfap2a, these genes are also expressed in unfertilized oocytes, indicating that they may be important for oogenesis, maternal-effect functions, or both. Given these findings, we have initiated studies to assess how various combinations of maternal and zygotic AP-2 gene expression might function together to regulate pre- and peri-implantation development. The present study focuses on the interplay between the expression of zygotic Tcfap2aand maternal and zygoticTcfap2c. These studies indicate that zygotic, but not maternal, Tcfap2cexpression is required for normal embryogenesis. In addition, the combined loss of both Tcfap2a and Tcfap2caccelerates embryonic lethality compared to the loss of either gene alone, demonstrating that genetic redundancy exists between these two AP-2 family members during the peri-implantation period of embryogenesis. PMID- 16672720 TI - Adrenomedullin peptide: gene expression of adrenomedullin, its receptors and receptor activity modifying proteins, and receptor binding in rat testis--actions on testosterone secretion. AB - Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 +/- 0.42 fmol of immunoreactive ADM per milligram of protein and 84 +/- 8 fg of ADM mRNA per picogram of Actb (beta-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22-52) and by human calcitonin gene-related peptide (8-37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22-52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis. PMID- 16672721 TI - Grafting period and donor age affect the potential for spermatogenesis in bovine ectopic testis xenografts. AB - Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life. The objective of this research was to xenograft testis tissue from multiple ages of bull calves for 24 or 36 wk in order to gain a better understanding of early bovine testis development. Testis tissue from 1-, 2-, 4-, and 8-wk-old calves was grafted onto the backs of castrated immunodeficient mice. Testis tissue from all donor ages grew, differentiated, and produced testosterone and elongating spermatids. Testis tissue grafts from 1- and 8-wk-old calves had elongating spermatids in greater than 5.5% of seminiferous tubule cross sections at the time of graft removal regardless of grafting period. Four-week-old donor tissue never had more than 5.2% of seminiferous tubule cross sections with elongating spermatids. Extending the grafting period from 24 to 36 wk resulted in an increase in the percentage of seminiferous tubule cross sections with elongating spermatids from 2% to 10% in 2 wk donor tissue. These data demonstrate that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting. PMID- 16672723 TI - In praise of insulin resistance. PMID- 16672724 TI - What good is growing old? PMID- 16672725 TI - A shared splice site? PMID- 16672722 TI - Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat. AB - Morphological male sex determination is dependent on migration of endothelial and preperitubular cells from the adjacent mesonephros into the developing testis. Our hypothesis is that VEGFA and its receptor KDR are necessary for both testicular cord formation and neovascularization. The Vegfa gene has 8 exons with many splice variants. Vegfa120, Vegfa164, and Vegfa188 mRNA isoforms were detected on Embryonic Day (E) 13.5 (plug date=E0) in the rat. Vegfa120, Vegfa144, Vegfa164, Vegfa188, and Vegfa205 mRNA were detected at E18 and Postnatal Day 3 (P3). Kdr mRNA was present on E13.5, whereas Fms-like tyrosine kinase 1 receptor (Flt1) mRNA was not detected until E18. VEGFA protein was localized to Sertoli cells at cord formation and KDR to germ and interstitial cells. The VEGFA signaling inhibitors SU1498 (40 microM) and VEGFR-TKI (8 microM) inhibited cord formation in E13 testis cultures with 90% reduced vascular density (P<0.01) in VEGFR-TKI-treated organs. Furthermore, Je-11 (10 microM), an antagonist to VEGFA, also perturbed cord formation and inhibited vascular density by more than 50% (P<0.01). To determine signal transduction pathways involved in VEGFA's regulation of testis morphogenesis, E13 testis were treated with LY 294002 (15 microM), a phosphoinositide 3-kinase (PI3K) pathway inhibitor, resulting in inhibition of both vascular density (46%) and cord formation. Thus, we support our hypothesis and conclude that VEGFA, secreted by the Sertoli cell, is involved in both neovascularization and cord formation and potentially acts through the PI3K pathway during testis morphogenesis to elicit its effects. PMID- 16672726 TI - Neuropathology in Alzheimer's disease: awaking from a hundred-year-old dream. AB - For one hundred years after Alois Alzheimer's first report of Alzheimer's disease (AD) in 1906, the pathological hallmarks of the disease, senile plaques and neurofibrillary tangles (NFTs), have been attractive targets for researchers. Therefore, not surprisingly, efforts to understand disease mechanisms have concentrated on the cell biology of amyloid-beta (Abeta) deposition as senile plaques or on the phosphorylation and aggregation of tau as NFTs. However, it now appears that this focus on pathology as a central contributor to disease may be misguided. Indeed, neurons associated with Abeta and NFTs in AD brain show a decrease in oxidative damage relative to those in vulnerable but morphologically intact areas of the brain, suggesting that neurodegenerative lesions are compensatory phenomena, and thus manifestations of cellular adaptation. That Abeta and tau accumulations indicate an age-related physiological reaction to chronic stress calls into question the rationale of current therapeutic efforts targeted toward lesion removal. Moreover, if this concept holds true for pathology in other neurodegenerative diseases, we may need to restructure our thinking and undergo a paradigm shift before substantial progress can be made in therapeutic intervention. PMID- 16672727 TI - Toward a better understanding of Klotho. AB - klotho mutant mice were originally described as a short-lived mouse model with premature aging-like disorders. The klotho gene responsible for these phenotypes encodes a type I membrane protein with a considerable similarity to beta glycosidase. klotho is predominantly expressed in tissues functioning in the regulation of calcium homeostasis. Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1; and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety. How can we reconcile these pleiotropic functions of Klotho? Is there any common mechanism? Further in vivo studies, and biochemical as well as physiological analyses, are required for a better understanding of the molecular aspects of Klotho. PMID- 16672728 TI - Psychometric properties of an abbreviated instrument of the five-factor model. AB - Brief measures of the five-factor model (FFM) have been developed but none include an assessment of facets within each domain. The purpose of this study was to examine the validity of a simple, one-page, facet-level description of the FFM. Five data collections were completed to assess the reliability and the convergent and discriminant validity of the rating form with other measures of the FFM and to replicate correlations with measures of maladaptive personality functioning that have been obtained with more extensive measures. Results appeared to support the validity of the FFM rating form (FFMRF) because it obtained relatively good internal consistency, convergent validity, and discriminant validity. In addition, self-descriptions of persons in terms of the FFMRF related to maladaptive personality traits in a manner that was consistent with theoretical expectations. Negative findings and limitations of the rating form also are discussed. PMID- 16672729 TI - Conceptions of perfectionism and interpersonal problems: evaluating groups using the structural summary method for circumplex data. AB - This study examined two conceptions of perfectionism in relation to interpersonal problems. Two hundred and seventy-nine undergraduate participants completed the Almost Perfect Scale-Revised (APS-R) and the Dyadic Almost Perfect Scale (DAPS). The authors used empirically derived discriminant functions (APS-R) and cluster analysis (DAPS) to identify three groups for both measures: adaptive and maladaptive perfectionists and nonperfectionists. Analyses of group profiles were performed on the Inventory of Interpersonal Problems Circumplex (IIP-C) scales using the structural summary method for circumplex data. APS-R and DAPS groups were compared on interpersonal problems endorsement and IIP-C profile characteristics. For both measures, results supported the hypotheses that maladaptive perfectionists would exhibit elevated profiles reflecting hostile dominant and friendly-submissive interpersonal problems, whereas the adaptive perfectionists would exhibit low profile elevation indicative of interpersonal adjustment. Overall, results supported Slaney and colleagues' (2001) model of perfectionism and provided evidence for the validity of the APS-R and DAPS. PMID- 16672730 TI - Evaluation of the Computerized Assessment System for Psychotherapy Evaluation and Research (CASPER) as a measure of treatment effectiveness with psychiatric inpatients. AB - Assessment procedures to evaluate inpatient treatment effectiveness can provide information to inform clinical practice. The Computerized Assessment System for Psychotherapy Evaluation and Research (CASPER) represents a standardized approach to assess patients' target problems that combines elements of individualized and nomothetic approaches. Although previous evaluations of CASPER with other patient populations have been encouraging, its use as a measure of change with an inpatient population has not been examined. In this study, 75 psychiatric inpatients completed CASPER and the Behavior and Symptom Identification Scale-32 (BASIS-32) at hospital admission and discharge. Results supported the concurrent validity of CASPER as a measure of pre- and posttreatment functioning and suggested that CASPER may provide a sensitive measure of change during inpatient treatment. Findings also supported the notion that patients' ratings of the extent to which their treatment focused on the problems they identified as a high priority were related to their overall treatment satisfaction. PMID- 16672731 TI - Depression as measured by the Beck Depression Inventory-II among injecting drug users. AB - This study conducts a confirmatory factor analysis of the Beck Depression Inventory-II (BDI-II) with a sample of 598 individuals who reported recent injecting drug use. Findings indicate that out of four models tested, the best model for this sample is a three-factor solution (somatic, affective, and cognitive) previously reported by Buckley, Parker, and Heggie. The findings that nearly 50% of participants provided BDI-II scores indicating significant depressive symptomatology reveals that these individuals are in need of treatment for their psychiatric symptoms as well as substance use. Somatic symptoms are endorsed more strongly than affective or cognitive symptoms of depression, suggesting a possible, but yet poorly defined, relationship between depressive symptomatology and drug use that centers on shared somatic symptomatology. PMID- 16672732 TI - Incremental validity of the MMPI-2 PSY-5 scales in assessing self-reported personality disorder criteria. AB - The Minnesota Multiphasic Personality Inventory-2 (MMPI-2) Personality Psychopathology-Five (PSY-5) scales were developed to measure abnormal personality symptomatology. The present study examines the incremental validity of the PSY-5 scales beyond the clinical and content scales in assessing criteria associated with personality disorders. The current sample includes 240 male and 407 female clients from private practice settings who completed the MMPI-2 and the Multiaxial Diagnostic Inventory (MDI), a self-report checklist of Diagnostic and Statistical Manual of Mental Disorders (3rd ed., revised) symptoms. Six of the MDI personality disorder scales, conceptually related to the PSY-5 scales, are used as criteria. Hierarchical regression analyses determine the incremental validity of each PSY-5 scale. In most analyses, PSY-5 scales add a significant increment of variance to the clinical and content scales. Implications of the results are discussed. PMID- 16672733 TI - Agreement among adolescents, parents, and teachers on adolescent personality. AB - Agreement between adolescents, mothers, fathers, and teachers on adolescents' personality traits was investigated in a longitudinal study. The targets for personality ratings were the adolescents who participated in the European Youth Heart Study in Estonia. There were 593 participants in the first wave and 480 participants in the follow-up study 3 years later. Adolescents' self-reports as well as father, mother, and teacher ratings were collected using questionnaires to measure the five-factor model of personality. In both waves, inter-rater agreement was highest between mothers and fathers, was low to moderate for parent self ratings, and was lowest for ratings between self and teacher, mother and teacher, and father and teacher. Test-retest correlations were moderate for parent and self-ratings but failed to reach statistical significance for three of the five teacher-rated traits, suggesting lower reliability of teacher ratings. Possible explanations for the low agreement between teachers and other judges are discussed. PMID- 16672734 TI - The Self-Report Psychopathy Scale and passive avoidance learning: a validation study of race and gender effects. AB - The reliability and validity of the Self-Report Psychopathy Scale (SRPS) was examined in a noninstitutionalized offender sample of mixed gender and race. Adequate alpha coefficients were obtained for the total sample and across gender and race. The SRPS was compared to measures of trait anxiety and passive avoidance errors. SRPS total, primary, and secondary scores were positively and significantly correlated with trait anxiety and passive avoidance (commission) errors, but not omission errors. Employing hierarchical regression models, no anxiety, gender, or ethnic effects were found. Intelligence confounded the relationship between psychopathic traits and passive avoidance errors. Findings provide tentative support of the SRPS as a valid measure of psychopathy. PMID- 16672735 TI - Two-, three-, and four-factor PCL-R models in applied sex offender risk assessments. AB - The authors compared 2-, 3-, 4-factor, and 2-factor/4-facet Psychopathy Checklist Revised (PCL-R) models in a previously unpublished sample of 1,566 adult male sex offenders assessed under applied clinical conditions as part of a comprehensive state-mandated community notification risk assessment procedure. "Testlets" significantly improved the performance of all models. The 3-factor model provided the best fit to the current data, followed by the 2-factor/4-facet model. The 2 factor model was not supported. PMID- 16672736 TI - Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. AB - Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways. PMID- 16672737 TI - Recidivism among spousal abusers: predictions and program evaluation. AB - The relative effectiveness of two interventions for dealing with 200 court referred spousal abusers is examined. The overall failure rate is 17.5%, with most recidivism occurring during the first 6 months after treatment. Offenders who completed a 14-week group treatment program called SAFE manifest significantly lower rates of recidivism (10.6%) than do offenders who did not complete the mandated treatment (38.8%). Some high-risk clients are referred to a cognitive restructuring treatment program called R&R, and those completing both programs (despite their high-risk status) have a recidivism rate of only 23.5%. Prediction of recidivism is difficult, with the LSI-R scores correctly predicting only 66% of the outcomes, using a cut score of 11.5. The exploration of other predictors is encouraged. PMID- 16672738 TI - Dating violence among college men and women: evaluation of a theoretical model. AB - This study empirically evaluates the Riggs and O'Leary (1989) model of dating violence. A sample of 200 college students completes assessments concerning the occurrence of violence in their dating relationships. The incidence of self reported partner violence is 25% for women and 10% for men. Multivariate logistic regression analyses are performed to determine the most salient predictors of dating violence for each gender. Findings reveal that the model is more accurate in predicting female, as compared with male perpetration of dating violence. The model accurately classifies 83% of violent women and only 30% of violent men. This study has several implications for the field of dating violence. Results indicate that although there is some degree of overlap, variant constructs predict violence for each gender. Identifying these constructs will guide prevention efforts in more effectively decreasing the occurrence of dating violence. PMID- 16672739 TI - A prospective analysis of the relationship between childhood sexual victimization and perpetration of dating violence and sexual assault in adulthood. AB - The majority of studies evaluating the relationship between childhood sexual assault and subsequent sexual assault perpetration by men have been conducted retrospectively and with incarcerated populations. The present study seeks to improve on previous research by prospectively investigating the relationship between childhood sexual assault and subsequent perpetration of dating violence in adulthood in men. Although there is a significant relationship between childhood sexual abuse and history of sexual assault perpetration at baseline, prospective analyses indicate that childhood sexual assault is not predictive of perpetration during the follow-up period. The role of family factors, including parental conflict resolution, is implicated in subsequent sexual aggression. These results are supportive of the idea that the effects of childhood sexual abuse may be mediated by a variety of factors. PMID- 16672740 TI - Urban adolescent mothers exposed to community, family, and partner violence: is cumulative violence exposure a barrier to school performance and participation? AB - Using a risk and resilience perspective, the authors assessed urban adolescent mothers' exposure to community, family, and partner violence and analyzed the relationships between cumulative violence exposure and multiple school outcomes, within the context of welfare reforms. Positive attitude toward school and social support were examined as moderators of violence exposure on school outcomes. The authors pilot tested the questionnaire with 10 participants, then surveyed 120 adolescent mothers regarding their violence exposure, school performance and participation, positive attitude toward school, and social support. Results indicate very high rates of lifetime exposure to violence; intercorrelations and regression analyses indicate that as violence exposure increases, school outcomes tend to worsen, with positive attitude toward school found to be a significant moderator of the effects of exposure to community violence on behavior problems in school. Implications for researchers, practitioners, school policies and programs, and welfare policies and programs conclude the article. PMID- 16672741 TI - Persistence of intimate partner violence among families referred to child welfare. AB - The purpose of this study is to examine the longitudinal course of intimate partner violence (IPV) among female caregivers of children receiving child welfare services. Data are derived from the National Survey of Child and Adolescent Well-Being, a national probability study of children investigated for child abuse and neglect in the United States. Caregivers (n = 861) are interviewed about demographic characteristics, mental health, substance use, and physical violence by a partner at the close of the investigation and at an 18 month follow-up. Polychotomous logistic regression examines the associations of severe and minor IPV controlling for caregiver and environmental characteristics. The results suggest that factors related to initial risk for IPV do not affect the continuation of IPV and that patterns of IPV differ for racial and ethnic groups. PMID- 16672742 TI - The role of victim-offender relationship in women's sexual assault experiences. AB - This study's goal is to identify differences in background, assault, and postassault factors according to the victim-offender relationship. A mail survey is conducted with more than 1,000 female sexual assault survivors (response rate 90%) recruited from college, community, and mental health agency sources. Stranger assailants are associated with a greater victim perceived life threat, more severe sexual assaults, and ethnic minority victims. Positive social reactions do not vary according to the victim-offender relationship, but stranger victims report more negative social reactions from others than do victims of acquaintances or romantic partners. Assaults by strangers and relatives are associated with more posttraumatic stress disorder (PTSD) symptoms than assaults by acquaintances and romantic partners. As expected, survivors' social cognitive responses to rape and social reactions from support providers are stronger correlates of PTSD symptoms than demographic or assault characteristics in general, but correlates vary across victim-offender relationship groups. PMID- 16672743 TI - Toward a more comprehensive understanding of violence against impoverished women. AB - Research and knowledge of violence against impoverished women continues to be limited. To achieve a more comprehensive understanding of violence against impoverished women and therefore inform prevention and intervention efforts for this population, the authors report on recent (past 6 months) physical, sexual, and psychological violence among 898 women who were randomly sampled from temporary shelter settings (n = 460) and low-income housing (n = 438) in Los Angeles County. Women experienced notable rates of violence during the past 6 months (e.g., 23% of sheltered women and 9% of housed women reported physical violence). Perpetrators were diverse, particularly for the sheltered women, including sexual partners, family, and strangers. These findings, and others suggesting that the different types of violence are distinct and severe, may call for more comprehensive screening and intervention efforts to enhance the safety of impoverished women. PMID- 16672745 TI - 'First do no harm'--a clear line in law and medical ethics. PMID- 16672747 TI - Medical experts, the law and professional regulation. PMID- 16672746 TI - Self-image in obesity: clinical and public health implications. PMID- 16672748 TI - Localization in clinical neurology. PMID- 16672749 TI - Localization in clinical neurology. PMID- 16672750 TI - Public health in the UK: success or failure? PMID- 16672751 TI - Inadvertent toxic drug reaction in the management of atrial fibrillation. PMID- 16672752 TI - Depression: epidemic or pseudo-epidemic? PMID- 16672753 TI - Contribution of general practice to NHS patients. PMID- 16672754 TI - A pandemic on the horizon. AB - A pandemic of human influenza is on the horizon: the question is not whether it will arrive but when. Knowing that a pandemic is coming provides an opportunity to plan ahead to mitigate against its impact. However, there are inevitably many uncertainties and managing these, under a constant media spotlight, is an additional and novel challenge. PMID- 16672755 TI - Payment by results or payment by outcome? The history of measuring medicine. PMID- 16672756 TI - Research misconduct: the poisoning of the well. PMID- 16672757 TI - Surgical insights into Parkinson's disease. AB - Surgery for Parkinson's disease was popularized in the mid-twentieth century before the advent of effective medical therapies. Early lesioning treatments contributed to our understanding of the functional anatomy of Parkinson's disease. Observations of the limitations and long-term complications of established pharmacological therapies for Parkinson's disease, together with major contributions from animal research to elucidate the roles of the basal ganglia in movement disorders, inspired a recent renaissance in neurosurgical interventions for Parkinson's disease including deep brain stimulation; this continues to yield much neurophysiological information. The development of potentially restorative treatment modalities, such as gene therapy, neural transplantation and nanotechnology, hold much promise for surgery, both therapeutically and in revealing further insights into Parkinson's disease pathophysiology. PMID- 16672758 TI - Adrenomyeloneuropathy in patients with 'Addison's disease': genetic case analysis. AB - OBJECTIVE: To review the clinical presentations and diagnostic issues in adrenomyeloneuropathy and adrenoleukodystrophy, which are different presentations of the same single gene disorder. DESIGN: Observational study. PARTICIPANTS: Three generations of an affected kindred. INTERVENTION: None. MAIN OUTCOME MEASURES: Neurological features suggestive of adrenoleukodystrophy or adrenomyeloneuropathy. Measurement of very long chain fatty acids. Molecular analysis of the adrenoleukodystrophy gene. RESULTS: Three adults presented with adrenomyeloneuropathy and two children with adrenoleukodystrophy. Circulating concentrations of long chain fatty acids were raised consistent with clinical features. A mutation in exon 6 of the adrenoleukodystrophy gene (P543L) was identified. This had not previously been identified but has subsequently been reported by other groups. CONCLUSIONS: Adrenomyeloneuropathy should be considered in the differential diagnosis in male patients presenting with adrenal failure. Early diagnosis allows genetic counselling in such families and may become more important as treatment strategies evolve. PMID- 16672759 TI - Trends in national and state-level obesity in the USA after correction for self report bias: analysis of health surveys. AB - OBJECTIVES: To quantify population-level bias in self-reported weight and height as a function of age, sex, and the mode of self-report, and to estimate unbiased trends in national and state level obesity in the USA. DESIGN: Statistical analysis of repeated cross-sectional health examination surveys (the National Health and Nutrition Examination Survey [NHANES]) and health surveys (the Behavioral Risk Factor Surveillance System [BRFSS]) in the USA. SETTING: The 50 states of the USA and the District of Columbia. RESULTS: In the USA, on average, women underreported their weight, but men did not. Young and middle-aged (<65 years) adult men over-reported their height more than women of the same age. In older age groups, over-reporting of height was similar in men and women. Population-level bias in self-reported weight was larger in telephone interviews (BRFSS) than in-person interviews (NHANES). Except in older adults, height was over-reported more often in telephone interviews than in-person interviews. Using corrected weight and height in the year 2000, Mississippi (30%) and Texas (31%) [corrected] had the highest prevalence of obesity for men; Texas (37%), Louisiana (37%), Mississippi (37%), District of Columbia (37%), Alabama (37%), and South Carolina (36%) for women. CONCLUSIONS: Population-level bias in self-reported weight and height is larger in telephone interviews than in-person interviews. Telephone interviews are a low-cost method for regular, nationally- and sub nationally representative monitoring of obesity. It is possible to obtain corrected estimates of trends and geographical distributions of obesity from telephone interviews by using systematic analysis which measure weight and height from an independent sample of the same population. PMID- 16672760 TI - Erectile incontinence post radical prostatectomy. PMID- 16672761 TI - Simultaneous presentation of myasthenia gravis and mesothelioma. PMID- 16672762 TI - Quantitative evidence for judgments on the efficacy of inoculation for the prevention of smallpox: England and New England in the 1700s. PMID- 16672763 TI - From anecdote to EBM. PMID- 16672764 TI - The effects of low dose leukotriene receptor antagonist therapy on airway remodeling and cysteinyl leukotriene expression in a mouse asthma model. AB - Airway structural changes that occur in patients with asthma in response to persistent inflammation are termed airway remodeling. The cysteinyl leukotrienes (LTC(4), D(4) and E(4)) are known to play important roles in the pathobiology of asthma. To evaluate the effect of low dose montelukast (MK) on the development of airway remodeling using a chronic murine model of allergic airway inflammation with subepithelial fibrosis, BALB/c mice, after intraperitoneal ovalbumin (OVA) sensitization on days 0 and 14, received intranasal OVA periodically on days 14 75. MK treated mice received montelukast sodium intraperitoneally on days 26-75. The OVA sensitized/challenged mice developed an extensive eosinophil cell inflammatory response, goblet cell hyperplasia, mucus occlusion, and smooth muscle hypertrophy of the airways. In addition, in OVA sensitized/challenged mice, dense collagen deposition/fibrosis was seen throughout the lung interstitium surrounding the airways, blood vessels, and alveolar septae. The cysteinyl leukotriene 1 (CysLT1) receptor antagonist, MK significantly reduced the airway eosinophil infiltration, goblet cell hyperplasia, mucus occlusion, and lung fibrosis except airway smooth muscle hypertrophy in the OVA sensitized/challenged mice. The OVA sensitized/challenged mice had significantly increased epithelial desquamation compared with control mice. MK markedly reduced epithelial desquamation of airways in OVA/MK treated animals compared with OVA sensitized/challenged mice. MK treatment did not affect the levels of CysLT in lung tissue. Our results show that the important role of cysteinyl leukotrienes in the pathogenesis of asthma. Lower dose of CysLT1 receptor antagonism has a significant anti-inflammatory effect on allergen-induced lung inflammation and fibrosis but not airway smooth muscle hypertrophy in an animal model of asthma. PMID- 16672765 TI - Diagnostic mutational analysis of MECP2 in Korean patients with Rett syndrome. AB - Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000-15,000 female births worldwide. The disease-causing gene has been identified as MECP2 (methyl-CpG-binding protein 2). In this study, we performed diagnostic mutational analysis of the MECP2 gene in RTT patients. Four exons and a putative promoter of the MECP2 gene were analyzed from the peripheral blood of 43 Korean patients with Rett syndrome by PCR-RFLP and direct sequencing. Mutations were detected in the MECP2 gene in approximately 60.5% of patients (26 cases/43 cases). The mutations consisted of 14 different types, including 9 missense mutations, 4 nonsense mutations and 1 frameshift mutation. Of these, three mutations (G161E, T311M, p385fsX409) were newly identified and were determined to be disease-causing mutations by PCR- RFLP and direct sequencing analysis. Most of the mutations were located within MBD (42.3%) and TRD (50%). T158M, R270X, and R306C mutations were identified at a high frequency. Additionally, an intronic SNP (IVS3+23C>G) was newly identified in three of the patients. IVS3+23C>G may be a disease-related and Korea-specific SNP for RTT. L100V and A201V are apparently disease-causing mutations in Korean RTT, contrary to previous studies. Disease-causing mutations and polymorphisms are important tools for diagnosing RTT in Koreans. The experimental procedures used in this study should be considered for clinical molecular biologic diagnosis. PMID- 16672766 TI - Caveolin-1 upregulation in senescent neurons alters amyloid precursor protein processing. AB - Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin 1 was co-localized with APP in caveolae-like structures. In DIG fractions, beta APP secretion was up-regulated by caveolin-1 over- expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by beta-secretase via PKC downregulation. PMID- 16672767 TI - Downstream molecular events in the altered profiles of lysophosphatidic acid induced cAMP in senescent human diploid fibroblasts. AB - Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G protein-coupled receptors. Previously, we demonstrated an altered profile of LPA dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ialpha. Senescence associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes. PMID- 16672768 TI - Protein kinase A-dependent phosphorylation of B/K protein. AB - We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolation and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H 89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed. PMID- 16672769 TI - betaig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through alphavbeta5 integrin. AB - Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis. PMID- 16672770 TI - Association of anti-obesity activity of N-acetylcysteine with metallothionein-II down-regulation. AB - People with upper body or visceral obesity have a much higher risk of morbidity and mortality from obesity-related metabolic disorders than those with lower body obesity. In an attempt to develop therapeutic strategies targeting visceral obesity, depot- specific differences in the expression of genes in omental and subcutaneous adipose tissues were investigated by DNA array technology, and their roles in adipocyte differentiation were further examined. We found that levels of metallothionein-II (MT-II) mRNA and protein expression were higher in omental than in subcutaneous adipose tissues. The study demonstrates that MT-II may play an important role in adipocyte differentiation of 3T3L1 preadipocytes, and that N acetylcysteine (NAC) inhibits the adipocyte differentiation of 3T3L1 cells by repressing MT-II in a time- and dose-dependent manner. Furthermore, the intraperitoneal administration of NAC to rats and mice resulted in a reduction of body weights, and a marked reduction in visceral fat tissues. These results suggest that MT-II plays important roles in adipogenesis, and that NAC may be useful as an anti-obesity drug or supplement. PMID- 16672771 TI - Inhibition of corneal neovascularization by rapamycin. AB - The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent. PMID- 16672772 TI - Implication of phosphorylation of the myosin II regulatory light chain in insulin stimulated GLUT4 translocation in 3T3-F442A adipocytes. AB - In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca(2+)/calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca(2+) in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3-F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca(2+) dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin-stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca(2+)/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes. PMID- 16672774 TI - Cardiocerebral resuscitation should replace cardiopulmonary resuscitation for out of-hospital cardiac arrest. PMID- 16672775 TI - Are the new resuscitation guidelines optimal? AB - PURPOSE OF REVIEW: The purpose of this review is to evaluate the 2005 guidelines on cardiopulmonary resuscitation. RECENT FINDINGS: International guidelines are based ideally on results from robust clinical trials. They are necessarily constrained in how far they can draw conclusions from experimental data, and have to pay regard to perceived safety and educational issues. Informed opinion can be more radical in drawing from compelling recent experimental findings, particularly when supported by unreplicated or indirect clinical evidence. Those already available cover a range of issues relevant to the guidelines; the most important ones are reviewed here. SUMMARY: The 2005 guidelines represent a major advance on those previously in use, but on the evidence already available they cannot be considered optimal. Deviations based on good evidence should not be discouraged provided they are approved and preferably monitored by authoritative bodies that should see this as a legitimate role in developing the science of resuscitation medicine. Guidelines for the most pressing of medical emergencies should not be set and inflexible over several years whilst the science behind them continues to advance. PMID- 16672777 TI - Incidence and significance of gasping or agonal respirations in cardiac arrest patients. AB - PURPOSE OF REVIEW: This review examines the clinical significance of agonal respirations associated with cardiac arrest. RECENT FINDINGS: Observational data indicate that agonal respirations are frequent (55% of witnessed cardiac arrests and probably higher) and that they are associated with successful resuscitation. They also are found more commonly in ventricular fibrillation compared with other rhythms. Agonal respirations pose the greatest challenge to bystanders at the scene and to emergency dispatchers. Bystanders are often lulled into thinking the person is still breathing thus identification of cardiac arrest may be missed by the dispatcher. In a study from King County, Washington, cardiopulmonary resuscitation instructions were not provided by emergency dispatchers in 20% of cardiac arrest cases because the caller reported signs of life - typically abnormal breathing. SUMMARY: Agonal respirations occur frequently in cardiac arrest. Emergency dispatchers and the general public must be more aware of their presence and significance. PMID- 16672776 TI - Haemodynamics of cardiac arrest and resuscitation. AB - PURPOSE OF REVIEW: This review will summarize the available data regarding the haemodynamic changes occurring following cardiac arrest in humans and animal models. RECENT FINDINGS: Following cardiac arrest due to ventricular fibrillation without cardiopulmonary resuscitation, blood flow exponentially falls but continues for approximately 5 min until the pressure gradient between the aorta and the right heart is completely dissipated. During cardiopulmonary resuscitation forward flow occurs into the aorta during the compression phase. Coronary blood flow is retrograde during the compression phase and antegrade during the decompression phase. Carotid blood flow takes over a minute to reach plateau levels following the initiation of chest compressions, and even brief interruptions of compressions result in a dramatic reduction in carotid blood flow which takes a minute or so to recover to plateau levels when compressions are reinstituted. Coronary perfusion pressure during the release phase of cardiopulmonary resuscitation has been shown to be a powerful predictor of the likelihood of recovery of spontaneous circulation following restoration of electrical activity. SUMMARY: Recent studies have provided important insights into the haemodynamics of cardiac arrest and of cardiopulmonary resuscitation which may inform more effective strategies for the management of cardiac arrest in the future. PMID- 16672778 TI - The problem with and benefit of ventilations: should our approach be the same in cardiac and respiratory arrest? AB - PURPOSE OF REVIEW: Recent advances in cardiopulmonary resuscitation have led to greater understanding of cardio-cerebral-pulmonary interactions during the process. The purpose of this discussion is to update the physiologic understanding of these interactions during cardiopulmonary resuscitation, review the detrimental and beneficial effects of ventilation, and identify implications for clinical practice. RECENT FINDINGS: There is an inversely proportional relationship between mean intrathoracic pressure, coronary perfusion pressure, and survival from cardiac arrest. Increased ventilation rates and increased ventilation duration impede venous blood return to the heart, decreasing hemodynamics and coronary perfusion pressure during cardiopulmonary resuscitation. It has also been shown that there is a direct and immediate transfer of the increase in intrathoracic pressure to the cranial cavity with each positive pressure ventilation, also reducing cerebral perfusion pressure. The reduced amount of blood flowing through the pulmonary bed during cardiopulmonary resuscitation tends to be overventilated, compromising hemodynamics to both the heart and brain and resulting in ventilation/perfusion mismatch. SUMMARY: The fundamental hemodynamic principle of intrathoracic pressure defines cardio-cerebral-pulmonary interactions during cardiopulmonary resuscitation. Further research is essential to optimize these interactions during treatment of profound shock. PMID- 16672779 TI - Therapeutic hypothermia after cardiac arrest. AB - PURPOSE OF REVIEW: Patients who are successfully resuscitated following cardiac arrest often have a significant medical condition termed postresuscitation disease. This includes myocardial stunning, metabolic abnormalities and neurologic injury from global ischemia. There are no clinical signs or diagnostic tests for 24-72 h to distinguish patients who will and will not recover neurologic function. RECENT FINDINGS: Therapeutic hypothermia had been advocated for decades as a treatment to improve neurologic outcome after cardiac arrest. The early studies focused on moderate hypothermia, which was associated with complications and was not clearly beneficial. Over the past decade, studies have focused on mild hypothermia with target temperatures of 32-34 degrees C. Two recent multicentered, randomized, controlled trials have demonstrated improved neurologic outcome with mild therapeutic hypothermia applied to comatose survivors after cardiac arrest compared with a normothermic control group. SUMMARY: As a result of these studies the International Liaison Committee on Resuscitation recommends that 'Unconscious adult patients with spontaneous circulation after out-of-hospital cardiac arrest should be cooled to 32 degrees C to 34 degrees C for 12 to 24 hours when the initial rhythm was ventricular fibrillation'. Mild therapeutic hypothermia should also be considered for patients with in-hospital arrest and asystole and pulseless electrical activity who are comatose after return of spontaneous circulation. PMID- 16672781 TI - Central venous pressure monitoring. AB - PURPOSE OF REVIEW: The proper use of central venous pressure requires a good understanding of basic measurement techniques and features of the waveform. RECENT FINDINGS: If attention is not paid to proper leveling of the transducer and consideration of transmural pressure then major errors are made in the use of central venous pressure. Besides the information gained from the relationship of changes in central venous pressure to changes in cardiac output, there is also much information to be obtained by examining the waveforms of the central venous pressure tracing. Examples are given of rhythm disorders, tricuspid regurgitation, cardiac tamponade, cardiac restriction, and decreased thoracic compliance. SUMMARY: There is much more to the measurement of central venous pressure than the simple digital value on the monitor and the actual waveform should always be examined. PMID- 16672782 TI - Pulmonary arterial hypertension. AB - PURPOSE OF REVIEW: The prevalence of pulmonary arterial hypertension is rising worldwide. Significant progress in our understanding of the pathobiology of pulmonary arterial hypertension has resulted in a shift from vasodilator therapy to the development of specific drugs targeting seminal molecular derangements of this disorder. This review highlights the recent advances in treatment and provides directions for the future care. RECENT FINDINGS: Induction of endothelin 1 and decreased transcription of nitric oxide and prostacyclin leads to pulmonary vasoconstriction that triggers several downstream molecular events which result in pulmonary vascular remodeling. Treatment with endothelin receptor antagonists, prostanoids and phosphodiesterase-5 inhibitors that prevent breakdown of cGMP have all demonstrated benefits in prospective, randomized, controlled trials of pulmonary arterial hypertension patients. Future strategies will combine these therapies to explore whether targeting more than one pathway provides synergistic long-term benefit. SUMMARY: Treatment guidelines developed by the American College of Chest Physicians aim to ensure that evidence-based approaches are practiced. Because of genetic heterogeneity in treatment effects and outcomes among patients, pharmacogenetics which will study polymorphisms that modulate the response to treatment will enable physicians to deliver cost-effective, tailored treatments for all pulmonary arterial hypertension patients in the future. PMID- 16672783 TI - Invasive measures of left ventricular preload. AB - PURPOSE OF REVIEW: Cardiac preload is frequently altered during hemodynamic failure and is a major focus of therapeutic management. The aim of this review was to summarize the invasive indicators of preload and the invasive predictors of preload responsiveness. RECENT FINDINGS: The static assessment of preload is based on the measurement of pulmonary artery occlusion pressure, which is still considered a gold standard. The reliability of the transpulmonary dilution method for bedside monitoring of cardiac volumes and preload has been clearly documented. Nonetheless, a number of recent studies have emphasized the poor value of static markers of preload for predicting a positive response to fluid therapy in comparison to 'dynamic' or 'functional' indices. Among them, the respiratory variation of arterial pulse pressure has been confirmed by numerous studies as an excellent indicator of volume responsiveness. The limitations for using these dynamic parameters have recently been emphasized so that alternative methods, such as passive leg raising or the respiratory systolic variation test, have been developed. SUMMARY: The best prediction of the hemodynamic response to fluid therapy is afforded by functional evaluation of preload responsiveness rather than by static markers of preload. PMID- 16672784 TI - Echocardiographic measurement of ventricular function. AB - PURPOSE OF REVIEW: We review new findings concerning ventricular function in patients in intensive care units with shock or unexplained respiratory distress syndrome analyzed using echocardiography. RECENT FINDINGS: Bedside echocardiography is not only an imaging technique but should be considered as a hemodynamic method. Left-ventricular systolic function can be assessed in daily clinical practice by measuring shortening fraction, fraction area change and ejection fraction. But these indices are dependent on load conditions. Index of myocardial performance can be also used. Rate of left-ventricular pressure increase may be measured from mitral regurgitation. Other indices such a maximal elastance and preload-adjusted maximal power were developed to evaluate myocardial systolic function but are not still used in clinical practice in patients in intensive care. Cardiac output measurement can be calculated easily from aortic annulus diameter and the velocity time integral of aortic blood flow. To complete the assessment of ventricular function, left-ventricular diastolic function and pressure as well as right ventricular size, septal movement and right pressures should be analyzed. SUMMARY: Using echocardiography the intensivist can examine both the mechanism and the cause of shock or pulmonary edema. It is time to increase the use of this technique in intensive care units. PMID- 16672785 TI - Echocardiographic measurement of fluid responsiveness. AB - PURPOSE OF REVIEW: Fluid responsiveness is a relatively new concept. It enables the efficacy of volume expansion to be predicted before use, rather than assessed afterwards, thus avoiding inappropriate fluid infusion. Echocardiography is a fantastic noninvasive tool which can directly visualize the heart and assess cardiac function. Its use was long limited by the absence of accurate indices to diagnose hypovolemia and predict the effect of volume expansion. In the last few years, several French teams have used echocardiography to develop new parameters of fluid responsiveness, taking advantage of its ability to monitor cardiac function beat by beat during the respiratory cycle. RECENT FINDINGS: In mechanically ventilated patients perfectly adapted to the respirator, respiratory variations in superior and inferior vena cava diameters and in left ventricular stroke volume have been validated as parameters of fluid responsiveness. In our opinion, the collapsibility index of the superior vena cava is the most reliable of these parameters, but does require transesophageal echocardiography. SUMMARY: Echocardiography has been widely demonstrated to predict fluid responsiveness accurately. This is now a complete and noninvasive tool able to accurately determine hemodynamic status in circulatory failure. PMID- 16672786 TI - Radiographic measures of intravascular volume status: the role of vascular pedicle width. AB - PURPOSE OF REVIEW: A valid, low-cost, high-yield instrument to assess intravascular volume status in critically ill patients does not exist. The portable chest X-ray is a common part of any intensivist's or chest clinician's daily rounds. RECENT FINDINGS: A simple, objective, valid measure of intravascular volume status, the vascular pedicle width, remains underappreciated in the medical literature. While more invasive, more expensive, and less common technologies are looked upon to assist in the clinical evaluation of volume status among critically ill patients, the vascular pedicle width stands alone in its low-cost, nearly risk-free potential to impact clinical practice. Even as the daily chest X-ray has become less common in practice, the role of measuring vascular pedicle width is potentially significant, particularly among mechanically ventilated patients. A standardized approach to reading the portable chest X-ray (supine or erect) is needed to facilitate interpretation of complex medical problems among the critically ill. Prospective evaluation of its appropriate use, particularly as compared with other, typically more invasive measures of intravascular volume, is warranted. SUMMARY: Vascular pedicle width measurement using a standardized approach to daily chest X-ray interpretation represents untapped potential for improving the non-invasive assessment of volume status in critically ill patients. PMID- 16672787 TI - Venous oximetry. AB - PURPOSE OF REVIEW: Mixed venous and central venous oxygen saturations reflect the balance between oxygen requirement and oxygen delivery, and thus may be used to assess the adequacy of tissue oxygenation. This review discusses recent data on the impact of using venous oximetry by obtaining mixed venous oxygen saturation or central venous oxygen saturation as useful monitoring parameters in critically ill patients. RECENT FINDINGS: Monitoring of changes in the central venous oxygen saturation after major surgery has been shown to be associated with outcome. Furthermore, early goal-directed therapy for patients with severe sepsis or septic shock, which includes treatment goals for mean arterial pressure, central venous pressure, and central venous oxygen saturation, was able to increase survival in these patients. In contrast, pulmonary artery catheter usage does not affect outcome in critically ill patients. SUMMARY: Early goal-directed therapy should be implemented in the initial resuscitation of septic patients. Central venous saturation may have prognostic significance following major surgery. Further evaluation of peri-operative trends in central venous saturation is required. Measurement of central venous oxygen saturation can easily be applied in intensive care unit patients and offers a useful, indirect indicator for the adequacy of tissue oxygenation. PMID- 16672788 TI - Pulse oximetry. AB - PURPOSE OF REVIEW: Pulse oximetry is now a ubiquitous and essential tool of modern medicine, and while it is a relatively recent invention, the technology has rapidly matured since the first commercially available oximeters were introduced in the 1970s. This review seeks to provide an overview of the basic physical operation of the probe and discuss its limitations, sources of error and some current advances in the use of multi-wavelength probes. RECENT FINDINGS: New multi-wavelength oximeters and plethysmographic waveform analysis may expand the information that we can collect and use non-invasively. This includes distinguishing between haemoglobinopathies, monitoring volume status and volume loss, and potentially monitoring cardiac output non-invasively. SUMMARY: The pulse oximeter, like any basic tool, must be used properly. There is considerable misunderstanding and lack of education among junior clinicians as to the use and interpretation of pulse oximeters. The introduction of the pulse oximeter has demonstrated a cost saving, although the cost-benefit of new multi-wavelength probes remains unproven. PMID- 16672789 TI - Gastric and sublingual capnometry. AB - PURPOSE OF REVIEW: Tissue hypoperfusion is a common pathophysiologic process leading to multiple organ dysfunction and death. Increases in tissue PCO2 can reflect an abnormal oxygen supply to the cells, so that monitoring tissue PCO2 by the use of gastric or sublingual capnometry may help identify circulatory abnormalities and guide their correction. This review provides an update on these technologies. RECENT FINDINGS: Gastric tonometry aims at monitoring PCO2 in the stomach, an organ that becomes ischemic quite early when the circulatory status is jeopardized. Despite substantial initial enthusiasm, this technique has never been widely implemented due to methodological problems. The measurement of sublingual mucosal PCO2 (PslCO2) by sublingual capnometry is technically simple and noninvasive. Experimental studies have suggested that PslCO2 is a reliable marker of tissue perfusion. Clinical studies have demonstrated that high PslCO2 values are associated with impaired microcirculatory blood flow and a worse prognosis in critically ill patients. SUMMARY: Gastric tonometry was proposed for regional PCO2 monitoring, but it is prone to a number of technical limitations. Sublingual capnometry could offer a valuable alternative for tissue PCO2 monitoring in clinical practice, representing a simple, noninvasive method to monitor tissue perfusion and titrate therapeutic interventions in critically ill patients. PMID- 16672790 TI - Bibliography. Current world literature. Cardiovascular resuscitation. PMID- 16672791 TI - Multiple auditory steady-state response thresholds to bone-conduction stimuli in young infants with normal hearing. AB - OBJECTIVE: Multiple auditory steady-state responses (ASSRs) probably will be incorporated into the diagnostic test battery for estimating hearing thresholds in young infants in the near future. Limiting this, however, is the fact that there are no published bone-conduction ASSR threshold data for infants with normal or impaired hearing. The objective of this study was to investigate bone conduction ASSR thresholds in infants from a Neonatal Intensive Care Unit (NICU) and in young infants with normal hearing and to compare these with adult ASSR thresholds. DESIGN: ASSR thresholds to multiple bone-conduction stimuli (carrier frequencies: 500 to 4000 Hz; 77 to 101-Hz modulation rates; amplitude/frequency modulated; single-polarity stimulus) were obtained in two infant groups [N = 29 preterm (32 to 43 wk PCA), tested in NICU; N = 14 postterm (0 to 8 mo), tested in sound booth]. All infants had passed a hearing screening test. ASSR thresholds, amplitudes, and phase delays for preterm and postterm infants were compared with previously collected adult data. RESULTS: Mean (+/-1 SD) ASSR thresholds were 16 (11), 16 (10), 37 (10), and 33 (13) dB HL for the preterm infants and 14 (13), 2 (7), 26 (6), and 22 (8) dB HL for the postterm infants at 500, 1000, 2000, and 4000 Hz, respectively. Both infant groups had significantly better thresholds for 500 and 1000 Hz compared with 2000 and 4000 Hz, in contrast to adults who have similar thresholds across frequency (22, 26, 18, and 18 dB HL). When 500- and 1000-Hz thresholds were pooled, pre- and postterm infants had better low frequency thresholds than adults. When 2000- and 4000-Hz thresholds were pooled, pre- and postterm infants had poorer thresholds than adults. ASSR amplitudes were significantly larger for low frequencies compared with high frequencies for both infant groups, in contrast to adults, who show little difference across frequency. ASSR phase delays were later for lower frequencies compared with higher frequencies for infants and adults, except for 500 Hz in the preterm group. ASSR phase delays were later for infants compared with adults across frequency. CONCLUSIONS: Infant bone-conduction ASSR thresholds are very different from those of adults. Overall, these results indicate that low-frequency bone conduction thresholds worsen and high-frequency bone-conduction thresholds improve with maturation. Bone-conduction ASSR threshold differences between the postterm infants and adults probably are due to skull maturation. Differences between preterm and older infants may be explained both by skull changes and a masking effect of high ambient noise levels in the NICU (and possibly to other issues due to prematurity). PMID- 16672792 TI - The Performance-Perceptual Test (PPT) and its relationship to aided reported handicap and hearing aid satisfaction. AB - OBJECTIVE: Results of objective clinical tests (e.g., measures of speech understanding in noise) often conflict with subjective reports of hearing aid benefit and satisfaction. The Performance-Perceptual Test (PPT) is an outcome measure in which objective and subjective evaluations are made by using the same test materials, testing format, and unit of measurement (signal-to-noise ratio, S/N), permitting a direct comparison between measured and perceived ability to hear. Two variables are measured: a Performance Speech Reception Threshold in Noise (SRTN) for 50% correct performance and a Perceptual SRTN, which is the S/N at which listeners perceive that they can understand the speech material. A third variable is computed: the Performance-Perceptual Discrepancy (PPDIS); it is the difference between the Performance and Perceptual SRTNs and measures the extent to which listeners "misjudge" their hearing ability. Saunders et al. in 2004 examined the relation between PPT scores and unaided hearing handicap. In this publication, the relations between the PPT, residual aided handicap, and hearing aid satisfaction are described. DESIGN: Ninety-four individuals between the ages of 47 and 86 yr participated. All had symmetrical sensorineural hearing loss and had worn binaural hearing aids for at least 6 wk before participating. All subjects underwent routine audiological examination and completed the PPT, the Hearing Handicap Inventory for the Elderly/Adults (HHIE/A), and the Satisfaction for Amplification in Daily Life questionnaire. Sixty-five subjects attended one research visit for participation in this study, and 29 attended a second visit to complete the PPT a second time. RESULTS: Performance and Perceptual SRTN and PPDIS scores were normally distributed and showed excellent test-retest reliability. Aided SRTNs were significantly better than unaided SRTNs; aided and unaided PPDIS values did not differ. Stepwise multiple linear regression showed that the PPDIS, the Performance SRTN, and age were significant predictors of scores on the HHIE/A such that greater reported handicap is associated with underestimating hearing ability, poorer aided ability to understand speech in noise, and being younger. Scores on the Satisfaction with Amplification in Daily Life were not well explained by the PPT, age, or audiometric thresholds. When individuals were grouped by their HHIE/A scores, it was seen that individuals who report more handicap than expected based on their audiometric thresholds, have a more negative PPDIS, i.e., underestimate their hearing ability, relative to individuals who report expected handicap, who in turn have a more negative PPDIS than individuals who report less handicap than expected. No such patterns were apparent for the Performance SRTN. CONCLUSIONS: The study showed the PPT to be a reliable outcome measure that can provide more information than a performance measure and/or a questionnaire measure alone, in that the PPDIS can provide the clinician with an explanation for discrepant objective and subjective reports of hearing difficulties. The finding that self-reported handicap is affected independently by both actual ability to hear and the (mis)perception of ability to hear underscores the difficulty clinicians encounter when trying to interpret outcomes questionnaires. We suggest that this variable should be measured and taken into account when interpreting questionnaires and counseling patients. PMID- 16672793 TI - Acoustic and perceptual study of Cantonese tones produced by profoundly hearing impaired adolescents. AB - OBJECTIVE: This study investigated five acoustic parameters of fundamental frequency (f0), one related to average f0 level and four related to change of f0 (f0 change) over four consecutive quarters of the vocalic segment, for their roles as acoustic correlates to the perception of Cantonese lexical tones produced by profoundly hearing-impaired speakers. DESIGN: Speakers were 20 Cantonese adolescents, 10 of whom had normal-hearing (mean age = 13;05); the other 10 were profoundly hearing-impaired (mean age = 13;05). The hearing impaired speakers were selected on the basis of the criterion of being prelingually deaf; they showed pure-tone average thresholds of 90 dB HL or more in the better ear at 0.5, 1.0, and 2.0 kHz. Twenty-four consonant-vowel Cantonese words made up four sets of words that minimally contrasted in the six contrastive Cantonese tones. The words were read by the speakers once in random order. Listeners were 10 final-year speech therapy students who were asked to identify the Cantonese tones. The f0 of each syllable (tone) was measured at five consecutive positions of the vocalic segment (initial, 25%, 50%, 75%, final). Discriminant analysis was used to determine how average f0 and f0 change accounted for both the production of intended tones and the perception of correctly identified tones. RESULTS: Average f0 and f0 change over the second half of the vocalic segment were reliable acoustic and perceptual correlates of tones produced by control speakers. By contrast, for hearing-impaired speakers, production of intended tones were not reliably distinguished by average f0 or f0 change. Furthermore, listeners mainly relied on f0 change over the second half of the vocalic segment for the distinction between tone 25 and level tones (55, 33, and 22) produced by hearing-impaired speakers. CONCLUSIONS: Average f0 and f0 change over the second half of the vocalic segment are important cues for accurate identification of Cantonese tones produced by speakers with normal hearing. By contrast, these cues are less reliable for perceiving tones produced by profoundly hearing-impaired speakers. PMID- 16672794 TI - Neonatal hearing screening: evaluation of tone-burst and click-evoked otoacoustic emission test criteria. AB - OBJECTIVE: Click-evoked otoacoustic emissions (CEOAEs) are widely used in universal neonatal hearing screening programs. A common finding in many such programs is a relatively high false-positive rate. This is often due to infant physiological noise adversely affecting the emission recording, leading to a "refer" screening outcome. In an attempt to reduce false-positive screening outcomes related to the effects of noise on otoacoustic emission response detection, tone-burst-evoked otoacoustic emissions (TBOAEs) were included in a neonatal hearing screening program because TBOAEs may elicit a greater signal-to noise ratio than CEOAEs. The research project compared the pass/refer rate for a CEOAE-based test using established pass/refer criteria with the pass/refer rate for screening criteria that were based on TBOAE results alone or on combined CEOAE and TEOAE results. DESIGN: Neonates were recruited at the Hong Kong Adventist Hospital, and both CEOAEs and TBOAEs were performed. Six passing criteria were used in this study, based on CEOAEs only; CEOAEs plus 1 kHz TBOAEs; CEOAEs plus 2 kHz TBOAEs; CEOAEs plus 3 kHz TBOAEs; CEOAEs plus 1, 2, and 3 kHz TBOAEs; and TBOAEs only. RESULTS: Data from 298 neonates (546 ears) were obtained. Criteria set 1, using CEOAEs only, demonstrated a pass rate of 79.1%, and 114 ears were referred. Criteria set 2, using CEOAEs together with TBOAEs recorded at 1 kHz, passed 39 more ears than Protocol 1, and the pass rate was 86.3%. Hence, the overall referral rate for total number of screened ears decreased by 7.2 percentage points. Criteria set 3, using CEOAEs together with TBOAEs recorded at 2 kHz, and Criteria set 4, using CEOAEs in conjunction with TBOAEs recorded at 3 kHz, gave pass rates similar to Criteria set 1. Criteria set 5, using TBOAE information at frequencies where CEOAEs were not rated as "pass," raised the pass rate from 79.1 to 87.6%, reducing the overall referral rate by 8.5 percentage points. Criteria set 6, in which neonates were screened with TBOAEs recorded at 1, 2, and 3 kHz, gave a pass rate of 78.4%, similar to results for the CEOAE-only procedure. CONCLUSIONS: Both Criteria sets 2 and 5, which combined CEOAE and TBOAE recordings, gave significantly higher pass rates than Criteria sets 1, 3, 4, and 6. The results suggest that the introduction of combined CEOAE and TBOAE protocols may assist in the reduction of refer outcomes, and hence the false-positive rates, of neonatal hearing screening programs. PMID- 16672795 TI - Effect of training on word-recognition performance in noise for young normal hearing and older hearing-impaired listeners. AB - OBJECTIVE: The objective of this study was to evaluate the effectiveness of a training program for hearing-impaired listeners to improve their speech recognition performance within a background noise when listening to amplified speech. Both noise-masked young normal-hearing listeners, used to model the performance of elderly hearing-impaired listeners, and a group of elderly hearing impaired listeners participated in the study. Of particular interest was whether training on an isolated word list presented by a standardized talker can generalize to everyday speech communication across novel talkers. DESIGN: Word recognition performance was measured for both young normal-hearing (n = 16) and older hearing-impaired (n = 7) adults. Listeners were trained on a set of 75 monosyllabic words spoken by a single female talker over a 9- to 14-day period. Performance for the familiar (trained) talker was measured before and after training in both open-set and closed-set response conditions. Performance on the trained words of the familiar talker were then compared with those same words spoken by three novel talkers and to performance on a second set of untrained words presented by both the familiar and unfamiliar talkers. The hearing-impaired listeners returned 6 mo after their initial training to examine retention of the trained words as well as their ability to transfer any knowledge gained from word training to sentences containing both trained and untrained words. RESULTS: Both young normal-hearing and older hearing-impaired listeners performed significantly better on the word list in which they were trained versus a second untrained list presented by the same talker. Improvements on the untrained words were small but significant, indicating some generalization to novel words. The large increase in performance on the trained words, however, was maintained across novel talkers, pointing to the listener's greater focus on lexical memorization of the words rather than a focus on talker-specific acoustic characteristics. On return in 6 mo, listeners performed significantly better on the trained words relative to their initial baseline performance. Although the listeners performed significantly better on trained versus untrained words in isolation, once the trained words were embedded in sentences, no improvement in recognition over untrained words within the same sentences was shown. CONCLUSIONS: Older hearing impaired listeners were able to significantly improve their word-recognition abilities through training with one talker and to the same degree as young normal hearing listeners. The improved performance was maintained across talkers and across time. This might imply that training a listener using a standardized list and talker may still provide benefit when these same words are presented by novel talkers outside the clinic. However, training on isolated words was not sufficient to transfer to fluent speech for the specific sentence materials used within this study. Further investigation is needed regarding approaches to improve a hearing aid user's speech understanding in everyday communication situations. PMID- 16672796 TI - Influence of dynamic compression on directional hearing in the horizontal plane. AB - OBJECTIVE: The purpose of this study was to assess the influence of dynamic compression on directional hearing in the frontal horizontal plane. DESIGN: Compression schemes with various compression ratios and attack times were created by using a digital signal processor. The influence of compression on isolated interaural level differences (ILDs) and interaural time differences (ITDs) was examined in discrimination experiments. The combination of ILDs and ITDs was investigated with measurements of localization based on head-related transfer functions. Five normal listeners and seven hearing-impaired subjects participated in the study. RESULTS: Dynamic compression revealed substantial effects on discrimination of ILDs but not on ITDs. With higher compression ratios and shorter attack times, just-noticeable differences for ILDs increased. Lateralization via head-related transfer functions was predominantly affected by compression in the higher frequency range because for lower frequencies, interaural time cues dominate over interaural level cues. The effect of compression was similar for normal and hearing-impaired listeners, but the latter performed worse on almost all tasks, especially with ITD discrimination at 4000 Hz. CONCLUSIONS: The influence of compression ratio and attack time could be shown for ILDs, but the effect was rather small, at least for the low compression ratios commonly used in hearing aids. The influence further decreased when attack time was prolonged. The dramatic impairment of the discrimination of ITDs with the hearing-impaired subjects in the high-frequency range suggests that they primarily rely on ILD cues. PMID- 16672798 TI - Peripheral audition of children on the autistic spectrum. AB - OBJECTIVE: To determine whether the peripheral audition of a group of children comprehensively diagnosed as being on the autistic spectrum was quantitatively different from that of a matched group of typically developing children. METHODS: Thirty-seven children with autism and an equal number of control participants matched for chronological age within 6 mo were examined by means of behavioral and physiologic measures of auditory function. All participants had normal hearing (thresholds <15 dB HL; 0.25 to 8 kHz) and normal middle ear function by quantitative tympanometry. Conventional behavioral audiometry, a computer assisted threshold assessment procedure, acoustic middle ear muscle reflex thresholds, and evoked otoacoustic emissions (both transient and distortion product) tests were administered to both groups. RESULTS: No significant differences between children with autism and those developing typically on any behavioral or physiologic measure of peripheral auditory function were found. CONCLUSIONS: There was no evidence of intrinsic differences in the peripheral auditory mechanism of children with autism that would account for the auditory processing disorders and sound sensitivity that are commonly reported in this population. PMID- 16672797 TI - Effects of early auditory experience on the spoken language of deaf children at 3 years of age. AB - OBJECTIVE: By age 3, typically developing children have achieved extensive vocabulary and syntax skills that facilitate both cognitive and social development. Substantial delays in spoken language acquisition have been documented for children with severe to profound deafness, even those with auditory oral training and early hearing aid use. This study documents the spoken language skills achieved by orally educated 3-yr-olds whose profound hearing loss was identified and hearing aids fitted between 1 and 30 mo of age and who received a cochlear implant between 12 and 38 mo of age. The purpose of the analysis was to examine the effects of age, duration, and type of early auditory experience on spoken language competence at age 3.5 yr. DESIGN: The spoken language skills of 76 children who had used a cochlear implant for at least 7 mo were evaluated via standardized 30-minute language sample analysis, a parent completed vocabulary checklist, and a teacher language-rating scale. The children were recruited from and enrolled in oral education programs or therapy practices across the United States. Inclusion criteria included presumed deaf since birth, English the primary language of the home, no other known conditions that interfere with speech/language development, enrolled in programs using oral education methods, and no known problems with the cochlear implant lasting more than 30 days. RESULTS: Strong correlations were obtained among all language measures. Therefore, principal components analysis was used to derive a single Language Factor score for each child. A number of possible predictors of language outcome were examined, including age at identification and intervention with a hearing aid, duration of use of a hearing aid, pre-implant pure-tone average (PTA) threshold with a hearing aid, PTA threshold with a cochlear implant, and duration of use of a cochlear implant/age at implantation (the last two variables were practically identical because all children were tested between 40 and 44 mo of age). Examination of the independent influence of these predictors through multiple regression analysis revealed that pre-implant-aided PTA threshold and duration of cochlear implant use (i.e., age at implant) accounted for 58% of the variance in Language Factor scores. A significant negative coefficient associated with pre-implant-aided threshold indicated that children with poorer hearing before implantation exhibited poorer language skills at age 3.5 yr. Likewise, a strong positive coefficient associated with duration of implant use indicated that children who had used their implant for a longer period of time (i.e., who were implanted at an earlier age) exhibited better language at age 3.5 yr. Age at identification and amplification was unrelated to language outcome, as was aided threshold with the cochlear implant. A significant quadratic trend in the relation between duration of implant use and language score revealed a steady increase in language skill (at age 3.5 yr) for each additional month of use of a cochlear implant after the first 12 mo of implant use. The advantage to language of longer implant use became more pronounced over time. CONCLUSIONS: Longer use of a cochlear implant in infancy and very early childhood dramatically affects the amount of spoken language exhibited by 3-yr-old, profoundly deaf children. In this sample, the amount of pre-implant intervention with a hearing aid was not related to language outcome at 3.5 yr of age. Rather, it was cochlear implantation at a younger age that served to promote spoken language competence. The previously identified language-facilitating factors of early identification of hearing impairment and early educational intervention may not be sufficient for optimizing spoken language of profoundly deaf children unless it leads to early cochlear implantation. PMID- 16672799 TI - Reflections on the Institute of Medicine Report: arriving December 2005. PMID- 16672800 TI - The giant muscle protein titin is an adjustable molecular spring. AB - When muscles are stretched, the giant protein titin develops passive force. Titin's force performs important functions that include maintaining the structural integrity of the sarcomere, and triggering signal transduction pathways. We propose that the mechanical properties of titin can be tuned according to the mechanical demands places on muscle, using mechanisms that include alternative splicing and posttranslational modifications. PMID- 16672801 TI - Changes in alpha-motoneuron properties with altered physical activity levels. AB - In this article I summarize recent results indicating that several basic electrophysiological properties of alpha-motoneurons change with both increases and decreases in neuromuscular activity. The properties that change are important in determining how easily these cells are recruited and their firing behaviors once recruited. PMID- 16672802 TI - Mechanomyography for studying force fluctuations and muscle fatigue. AB - Measurement of the transverse displacement of the skin over a contracting muscle is known as the surface mechanomyogram. With the concurrent measurement of the electromyogram and twitch force, the mechanomyogram can provide supplementary information about muscle activity and motor performance during such tasks as fatiguing contractions and attempts to perform steady contractions. PMID- 16672803 TI - Magnitude and time course of arterial vascular adaptations to inactivity in humans. AB - We demonstrate that extensive arterial vascular adaptations occur within 3-8 wk of inactivity in humans. We put forth the hypothesis that the diameter decrease represents an adaptation to a lack of variation in peak shear stress. Furthermore, an enhanced flow-mediated dilation in deconditioned arteries implies that functional vascular adaptations to inactivity are not simply the inverse of adaptations to exercise. PMID- 16672804 TI - Metabolic consequences of exercise-associated autonomic failure. AB - Type I diabetes mellitus patients who tightly control blood glucose levels toward normal have increased frequency of hypoglycemia, a major barrier to physical activity. This article presents the hypothesis that dysfunctional autonomic regulation of metabolism after recent bouts of exercise or hypoglycemia contributes to exercise-induced hypoglycemia in these patients. PMID- 16672805 TI - Attentional and biomechanical deficits interact after mild traumatic brain injury. AB - Even a mild traumatic brain injury leads to deficits in the ability to maintain gait stability that is exacerbated when a secondary cognitive task is performed at the same time. This implies that executive function is disrupted after concussion-a supposition that is confirmed when tested directly. PMID- 16672806 TI - The built-in environment: the role of personality and physical activity. AB - Personality trait psychology has seen a reemergence in recent years, and researchers are beginning to apply personality models to understand health behavior habits. This review features recent research on personality correlates of physical activity (PA) and the integration of personality with PA-related social cognition. The current definition and status of general personality work is introduced followed by a review and discussion of the evidence for personality and PA relations. The review concludes with recommendations for personality matched PA marketing and experimental and longitudinal designs. PMID- 16672807 TI - Health beliefs: how they shape how we live. PMID- 16672809 TI - Psychological and physical etiologies of heart palpitations. PMID- 16672811 TI - Examining spirituality among women with breast cancer. AB - A diagnosis of cancer can conjure up a whole host of emotions. Many women with breast cancer will try to deal with the disease by ascribing meaning for its occurrence and searching for mechanisms by which to cope with its sequelae. Research supports that women who have been diagnosed with breast cancer are employing complementary and alternative medicine use as a means of gaining a sense of control over the disease as well as reducing stress and achieving inner peace. Spirituality is one such mechanism that women with breast cancer utilize in order to achieve that goal; an underlying premise being that taking care of the soul is a necessary element in healing the body. PMID- 16672812 TI - Development of the healing touch comfort questionnaire. AB - This study provided preliminary evidence for internal consistency reliability (Cronbach alpha = .94) of the newly developed Healing Touch Comfort Questionnaire. Fifty-six Healing Touch (HT) recipients (51 women and 5 men with a mean age of 51) completed the questionnaire. Participants with more than 4 HT treatments had higher comfort levels than those with fewer than 4. PMID- 16672813 TI - What tools help make "whole-of-patient" practices happen?: a Tasmanian falls prevention project. AB - The development of "whole-of-patient" approaches is explored using transcripts of 10 interviews with registered nurses implementing the Quickscreen Clinical Falls Risk Assessment Tool in general practices in northern Tasmania. These data suggest that while the tool helped develop holistic nursing practices, the development of multidisciplinary practices requires different tools and strategies. PMID- 16672814 TI - Stroke patients in the acute care phase: role of hope in self-healing. AB - Hope affects stroke survival positively and should therefore be facilitated via holistic nursing care. This qualitative study explored the perceptions of hope of 8 stroke patients during the acute care phase. Data generated 5 themes: storytelling, vision of hope, worries and concerns for the future, type of hope, and self-healing. Two types of hope were identified; with a "passive type of hope," participants retained disturbed feelings preventing progression; with an "active type of hope," participants expressed progression from suffering to a healing phase. Knowledge of this progression of phases of hope type can inform on training for effective nursing care to facilitate self-healing. PMID- 16672815 TI - Mind-body intelligence: a new perspective integrating Eastern and Western healing traditions. AB - The purpose of this article is to introduce a new approach that integrates mindfulness meditation practices and Western psychotherapeutic approaches into a holistic program. PMID- 16672816 TI - An evidence-based conceptual analysis of presence. AB - Presence is recognized as a quality and an intervention of nursing care. As technology replaces human touch, economic constraints decrease time spent with the patient and the demand for holistic care increases, the act and quality of presence gains significance. Presence is described as transformative to the patient care experience, having the potential to hinder or facilitate the healing process. This conceptual analysis utilizes an evidence-based approach of presence for use in theory development and concept measurement. PMID- 16672818 TI - Healthier aging: reaching and teaching older adults. PMID- 16672830 TI - Continuous thermoregulatory responses to mass-participation distance running in heat. AB - PURPOSE: To continuously measure core temperature (T(c)) and heart rate(HR), and quantify fluid balance during a 21-km mass-participation road racein warm, humid environmental conditions. METHODS: Eighteen heat-acclimatized male soldiers ingested a telemetric Tc sensor on the evening prior to the race and wore an ambulatory T(c) data recorder and HR monitor during the race. Pre- to postrace changes in nude body mass quantified fluid balance. RESULTS: Environmental wet bulb globe temperature averaged 26.5 degrees C. All runners finished the race asymptomatic of heat illness in a mean +/- SD (range) time of 118 +/- 13 (105 146) min, corresponding to an average running speed of 10.8 +/- 1.1 (8.6-12.0) km.h(-1). All runners recorded peak T(c) > 39 degrees C; 56% (N = 10) > 40 degrees C; and 11% (N = 2) > 41 degrees C. Peak T(c) was 40.1 +/- 0.7 (39.3-41.7) degrees C at 86 +/- 36 (13-130) min, with T(c) 39.9 +/- 0.8 (38.3-41.7) degrees C at race finish. The magnitude of T(c) response was unrelated (P > 0.05) to running time or fluid balance (e.g., fluid intake, % dehydration). Cumulative heat strain index was 2790 +/- 1112 (1046-5144) units at race finish. CONCLUSION: Ingestible telemetric temperature sensors demonstrated utility for continuous measurement of T(c) during mass-participation running. Successful application of this technology has highlighted the magnitude and duration of T(c) elevation that runners will voluntarily achieve during mass-participation distance races in heat and high humidity without medical consequence. PMID- 16672831 TI - Injuries among disabled athletes during the 2002 Winter Paralympic Games. AB - PURPOSE: This prospective injury surveillance study was conducted to better understand the types of and risk factors of injuries sustained by athletes with disabilities competing in adapted winter sports at the elite level. METHODS: Detailed information was collected on all injuries evaluated by polyclinic or venue medical personnel during the operational 20-d period of the 2002 Winter Paralympics. RESULTS: A total of 39 injuries involving 9% of the Paralympic athletes were recorded in the injury registry. Most of these injuries were of acute, traumatic onset and involved the disciplines of alpine skiing and sledge hockey. Sprains (32%), fractures (21%), and strains and lacerations (14% each) represented the most common diagnoses. Of the recorded injuries, eight (21%) resulted in time lost from training or competition. CONCLUSIONS: The injury patterns observed among winter Paralympians in this study are not appreciably different from able-bodied athletes competing in similar disciplines, although in many instances the risk factors for sport-specific injury appear to be unique to disabled or adapted competition. Our preliminary observations suggest that several of the more severe injuries were potentially preventable. Ongoing data collection by the International Paralympic Committee should enable feasible injury prevention strategies to be designed and implemented. PMID- 16672832 TI - The congenital long QT syndrome and implications for young athletes. AB - The congenital long QT syndrome (LQTS) is caused by cardiac ion channel mutations, which predispose young individuals to sudden cardiac death often related to exercise. The issue of LQTS and sports participation has received significant publicity due to reports of sudden death in young competitive athletes. This article reviews the pathophysiology, clinical characteristics, and management of LQTS in the physically active and athletic population. PMID- 16672833 TI - Physical activity in individuals at risk for diabetes: Diabetes Prevention Program. AB - PURPOSE AND METHODS: Leisure physical activity was assessed using questionnaires with different time frames in the Diabetes Prevention Program (DPP) cohort of 3234 overweight individuals aged > 25 yr with impaired glucose tolerance (IGT) from 27 centers across the United States. The three questionnaires were the Modifiable Activity Questionnaire (MAQ; past year), the Low-Level Physical Activity Recall (LOPAR; past 7 d), and the Third National Health and Nutrition Examination Survey (NHANES III; past month). This provided the opportunity to examine the relationship between the three activity measures and to compare activity levels of the DPP sample with that of a national sample with IGT. RESULTS: Leisure activity determined by the three questionnaires significantly correlated with each other, although the correlations between MAQ and NHANES III were stronger (men: rho = 0.52; women: rho = 0.49; P < 0.01) than between LOPAR and either measure (men: rho = 0.20 for MAQ, 0.24 for NHANES; women: rho = 0.10 for MAQ, 0.13 for NHANES). In the DPP, measures of obesity and glucose tolerance were significantly correlated with activity levels determined by MAQ and NHANES, but not LOPAR. Activity levels in DPP participants determined by the NHANES III questionnaire were generally higher than those reported by individuals meeting DPP eligibility criteria who were part of the NHANES cohort for similar age, body mass index, and race or ethnicity. CONCLUSION: If the DPP participants were more active than a national sample of individuals with IGT, this would have implications for translation when using the DPP lifestyle intervention in less active or less motivated populations. Finally, the weak relationship between activity levels obtained with MAQ and LOPAR may result from the fact that they encompass different time frames and different components of leisure activity. PMID- 16672834 TI - Effects of cyclosporine-A on rat soleus muscle fiber size and phenotype. AB - PURPOSE: Organ transplant patients treated with cyclosporine-A (CsA) often exhibit weight loss and muscle weakness. The cellular target of CsA, calcineurin, has been implicated in maintenance of muscle fiber size and in expression of the type I skeletal muscle phenotype. We hypothesized that CsA treatment would cause fiber atrophy, as well as increase type IIa myosin heavy chain (MHC) content and oxidative enzyme activities in the soleus muscle. METHODS: Rats were treated with CsA for 21 d (20 mg.kg(-1).d(-1); N = 16) and compared with control rats given olive oil vehicle (Veh; N = 16). Soleus muscles were excised bilaterally. MHC content was determined by gel electrophoresis, oxidative enzyme activities by spectrophotometric methods, and fiber type and size by histochemistry. RESULTS: Lymphocyte count was depressed in CsA rats (P < 0.05), indicating treatment efficacy. Type IIa MHC content was increased in the soleus muscle with CsA (Veh, 10.4 +/- 1.7%; CsA, 15.1 +/- 2.0; P < 0.05) at the expense of type I MHC. Soleus muscle oxidative enzyme activities were also increased with CsA treatment (P < 0.05). Soleus muscle atrophy occurred, reflected by a 22% decrease in fiber cross sectional area (Veh, 3255 +/- 105 microm(2); CsA, 2533 +/- 125; P < 0.05). CONCLUSION: These findings indicate that CsA treatment is associated with changes in skeletal muscle fiber size and phenotype. The former may underlie clinical symptoms of transplant patients treated with CsA. PMID- 16672835 TI - Ibuprofen inhibits skeletal muscle hypertrophy in rats. AB - PURPOSE: We sought to determine whether cyclooxygenase (COX) activity is necessary for overload-induced growth of adult rat skeletal muscle, and whether nitric oxide synthase (NOS) activity is involved in upregulation of COX messenger RNA (mRNA) expression in skeletal muscle. METHODS: Unilateral surgical removal of the gastrocnemius and soleus was performed on the right hindlimb of 16 female Sprague-Dawley rats (approximately 230 g) to induce chronic overload (OL) of the plantaris for 14 d, with sham surgeries performed on the contralateral leg as a normally loaded (NL) control. Half of the rats were treated with the nonspecific COX inhibitor, ibuprofen (0.2 mg.mL(-1) in drinking water; approximately 20 mg.kg(-1).d(-1)). In a second experiment, the plantaris was unilaterally overloaded for 5 or 14 d in male rats (approximately 350 g; N = 16 rats per time point) and half of the animals were treated with the NOS inhibitor, L-NAME (0.75 mg.mL(-1) in drinking water; approximately 90 mg.kg(-1).d(-1)). RESULTS: Ibuprofen treatment inhibited plantaris hypertrophy by approximately 50% (P < 0.05) following 14 d of OL, as did L-NAME treatment (P < 0.05). COX-1 and COX-2 mRNA did not differ between any groups at 5 d. At 14 d, however, L-NAME caused a 30-fold increase in plantaris COX-1 mRNA expression independent of loading condition. Additionally, OL induced a 20-fold increase in COX-2 mRNA expression compared with NL (P < 0.05) at 14 d, without affecting COX-1 mRNA level. L-NAME treatment significantly inhibited OL-induced expression of COX-2 mRNA. CONCLUSION: COX activity is important for in vivo muscle hypertrophy, and plantaris overload is associated with NOS activity-dependent COX-2 expression. PMID- 16672836 TI - Enhanced acidotic myocardial Ca2+ responsiveness with training in hypertension. AB - PURPOSE: We tested how hypertension-induced compensated hypertrophy, both alone and coupled with exercise training, affects left ventricular (LV) Ca(2+) responsiveness during acidosis. METHODS: Four-month-old female, spontaneously hypertensive rats (SHR) (N = 23) were assigned to a sedentary (SHR-SED) or treadmill-trained (SHR-TRD) group (60% VO(2peak), 5 d.wk(-1), 6 months), while Wistar-Kyoto rats (WKY) (N = 12) served as normotensive controls. LV performance was established in response to supraphysiologic Ca(2+) infusion (4 mmol.L(-1)) alone and concomitant with isoproterenol (ISO) (1 x 10 mol.L(-1)) at pH 7.4 and 6.8. RESULTS: HR, rate-pressure product (RPP), and blood pressure were greater in SHR than in WKY (P < 0.05). HR and RPP were attenuated with training. Heart weight and LV anterior wall thickness (diastole) were increased in SHR relative to WKY (P < 0.05) and augmented with training. ISO + 4 mmol.L(-1) [Ca]o resulted in similar LV performance at pH 7.4. At pH 6.8, LV developed pressure was greater in both SHR groups (P < 0.05) versus WKY rats and a twofold increase in the [Ca(2+)]o rescued LV performance to the greatest extent in SHR-TRD. During acidosis, the added stimulus of ISO coupled with elevated [Ca(2+)](o) improved WKY LV performance to near baseline (P < 0.05). Neither elevated [Ca(2+)](o) nor ISO was effective in rescuing LV performance in SHR-SED during acidosis. Phospholamban phosphorylation at Ser(16) and Thr(17) residues were positively correlated with LV functional recovery. Regulatory proteins such as the Na(+)/H(+) exchanger, Na(+)/Ca(2+) exchanger, and the L-type Ca(+) channel were not correlated with LV function. CONCLUSION: Myocardial tolerance to acidosis is improved during the adaptive phase of compensatory hypertrophy. Furthermore, exercise training in SHR induced a myocardial phenotype that preserved Ca(2+) responsiveness during acidosis. PMID- 16672837 TI - Low-intensity exercise training improves survival in Dahl salt hypertension. AB - PURPOSE: The present study examined whether exercise training could increase survival in a rodent model of salt-sensitive hypertension. METHODS: Male, inbred Dahl salt-sensitive rats arriving at 8 wk of age were randomly divided into a sedentary control group (N = 5) or an exercise-trained group (N = 8). Exercise training consisted of running 20 m.min(-1), 0% incline, 60 min.d(-1), 5 d.wk(-1) on a motorized driven treadmill. On arrival, animals were fed a low-salt diet (0.12% NaCl) during a 1-wk acclimatization period. At the end of this period, all rats were then fed a high-salt diet (7.8% NaCl) for the remainder of the study. Arterial systolic blood pressure (SBP) was measured via the tail-cuff method. RESULTS: Systolic blood pressure (SBP) measured on the low salt diet was similar between groups. After 2 wk of a high-salt diet, SBP was similarly significantly elevated in both control and exercise groups relative to the low salt diet. Kaplan-Meier analysis showed that exercise training increased survival (P < 0.02) with an approximate 30% increase in the mean days survived with exercise training (P < 0.02). CONCLUSION: These data suggest that exercise training is an important intervention for salt-sensitive hypertension and that the enhanced survival observed with exercise training appears to be independent of training-induced SBP lowering effects. PMID- 16672838 TI - Gender differences in macrophage antiviral function following exercise stress. AB - INTRODUCTION: In male mice, exhaustive exercise increases susceptibility to respiratory infection following intranasal inoculation with herpes simplex virus 1 (HSV-1), whereas moderate exercise decreases the risk of infection. These responses have been linked with altered macrophage antiviral resistance, among other immune mechanisms. Female mice appear to be better protected from death than male mice following HSV-1 infection, although their response to exercise stress is similar. The possible immune mechanisms, however, have not been explored. PURPOSE: This study was conducted to examine gender differences in macrophage antiviral resistance following repeated moderate and exhaustive treadmill exercise. METHODS: Male (M, N = 36) and female (F, N = 36) CD-1 mice were randomly assigned to moderate exercise (Mod), exhaustive exercise (Exh), or control (C) groups. Exercise was done daily for 3 d; moderate exercise consisted of treadmill running for 90 min, whereas exhaustive exercise consisted of running to volitional fatigue (approximately 50 min). RESULTS: Females had greater macrophage antiviral resistance to HSV-1 than males in C and Mod (P < 0.05), but not Exh; Mod increased resistance, whereas Exh decreased resistance similarly in both genders (P < 0.001). CONCLUSIONS: These data suggest that altered macrophage antiviral resistance to HSV-1 may contribute to gender differences in in vivo resistance to HSV-1 respiratory infection at rest, as well as following moderate and exhaustive exercise. PMID- 16672839 TI - Puberty effects on NK cell responses to exercise and carbohydrate intake in boys. AB - Previous research has demonstrated that younger versus older animals and humans experience smaller perturbations in natural killer (NK) cells in response to physiological stress. PURPOSE: To determine whether the smaller perturbations in NK cells induced by strenuous exercise and carbohydrate (CHO) intake, previously reported in children, are influenced by puberty. METHODS: Twenty 12-yr-old boys, distinguished as prepubertal (Tanner (T) 1, N = 7), early pubertal (T2, N = 7), or pubertal (T3-5, N = 6), cycled for 60 min at 70% VO(2max) while drinking 6% CHO (CT) or flavored water (WT). Blood was collected at rest and during (30 and 60 min) and following (30 and 60 min) exercise to identify NK cells as CD3( )CD56(dim) or CD3(-)CD56(dim). CD69 expression on CD3(-)CD56(+) cells was also determined. RESULTS: A puberty x CHO x exercise interaction was found for the proportion, but not number, of CD56(dim) cells (P = 0.06). CD56(dim) cell counts were lower in CT versus WT (P < 0.001). Responses of CD56(bright) proportions (P = 0.007) and counts (P = 0.03) depended on pubertal status, but not CHO. The CD56(bright):CD56(dim) ratio remained stable during exercise, but during recovery was higher in T1 and T3-5 versus T2 (P = 0.08) and in CT versus WT (P = 0.04). During recovery, CD3(-)CD56(+) cells expressed higher levels of CD69 (P = 0.01), with no change in the proportion of CD69(+) cells. CONCLUSION: These results confirm the influence of puberty on the distribution of NK cell subsets in response to exercise and CHO intake. Increased CD69 expression suggests that NK cells increase activation status during recovery from physiological stress. PMID- 16672840 TI - Heart rate variability during recovery from a Wingate test in adolescent males. AB - PURPOSE: To evaluate the effect of maturity status on the autonomic nervous system at rest and recovery after short-term, high-intensity exercise in adolescents. METHODS: A biological maturity age was estimated in 27 males by calculating the years from peak height velocity (PHV) using a multiple regression equation. Subjects were divided into two groups: pre-PHV (years from PHV < 0.49), N = 14, mean age = 12.29 +/- 0.91 yr; post-PHV (years from PHV > 0.5, N = 13, mean age = 15.12 +/-0.76 yr). HR variability was used to evaluate autonomic function. ECG tracings were collected during 5 min at rest and recovery after a Wingate test and were analyzed in the frequency domain (low-frequency (LF), high frequency (HF), LF/HF, total power (TP)). Data are presented as natural logarithms (LN). RESULTS: Changes in HR from HR(peak) during exercise to HR measured at minute 4 after exercise ([DELTA]HR4) were significantly greater in the pre-PHV group (84.31 +/-17.58 bpm) compared with the post-PHV group (69.42 +/ 17.63 bpm). There were no significant differences in resting HR variability between pre- and post-PHV groups (P > 0.05). Significant group x time interactions were found for LF(LN) (ms(2)) and TP(LN) (ms(2)) measured during recovery (P < 0.05). Post hoc tests showed that the pre-PHV group had significantly higher postexercise LF(LN) (5.02 +/- 0.97 vs 4.19 +/- 0.79) and TP(LN) (6.36 +/- 1.02 vs 5.62 +/- 0.65) compared with the post-PHV group. When postexercise LF(LN) (ms(2)) was normalized for TP(LN) (ms(2)), there were no significant differences between groups (P > 0.05). CONCLUSION: The pre-PHV group had higher total HR variability than the post-PHV group after a Wingate test, suggesting that maturity status significantly affects total HR variability during recovery after high-intensity exercise. PMID- 16672841 TI - Genotype related differences in beta2 adrenergic receptor density and cardiac function. AB - INTRODUCTION: Several common polymorphisms of the beta2 adrenergic (ADRB2) have been described including a Glycine (Gly) for arginine (Arg) substitution at amino acid 16. In vivo studies have attributed phenotypic differences in the Arg16Gly polymorphism of the ADRB2 to differences in agonist-mediated desensitization. Some studies, however, have shown differences between genotype groups under non agonist-mediated conditions suggesting baseline differences in receptor function or in receptor density. We sought to determine whether genetic variation of the ADRB2 influenced ADRB2 density and, consequently, resting cardiovascular function. METHODS: We measured ADRB2 density on isolated lymphocytes in 30 healthy subjects (15 homozygous for Arg, Arg16, and 15 homozygous for Gly, Gly16) matched for age, cardiovascular fitness, BMI, and gender. In addition, we measured cardiac output (Q), heart rate (HR), and stroke volume (SV) after 5 min of quiet rest in these same subjects. RESULTS: Arg16 subjects had lower receptor density (1220 +/- 78 vs 1574 +/- 110, mean +/- SE, P < 0.01) as well as lower resting cardiac output due to a reduced stroke volume, but a higher HR when compared with the Gly16 subjects (Q = 4.3 +/- 0.2 vs 5.0 +/- 0.3 L.min(-1), SV = 65 +/- 6 vs 86 +/- 7 mL.beat(-1), HR = 70 +/- 4 vs 60 +/- 3 beats.min(-1), for the Arg16 and Gly16 groups, respectively, P < 0.01). In addition, ADRB2 density for all subjects was positively associated with cardiac output (r = 0.428, P = 0.009) and stroke volume (r = 0.407, P = 0.001). CONCLUSIONS: These data suggest that the Arg16Gly polymorphism of the ADRB2 influences receptor density, which, in turn, contributes to resting differences in cardiac output and stroke volume. PMID- 16672842 TI - Validity of the polar S810 heart rate monitor to measure R-R intervals at rest. AB - PURPOSE: This study was conducted to compare R-R intervals and the subsequent analysis of heart rate variability (HRV) obtained from the Polar S810 heart rate monitor (HRM) (Polar Electro Oy) with an electrocardiogram (ECG) (Physiotrace, Estaris, Lille, France) during an orthostatic test. METHODS: A total of 18 healthy men (age: 27.1 +/- 1.9 yr; height: 1.82 +/- 0.06 m; mass 77.1 +/- 7.7 kg) performed an active orthostatic test during which R-R intervals were simultaneously recorded with the HRM and the ECG recorder The two signals were synchronized and corrected before a time domain analysis, the fast Fourier transform (FFT) and a Poincare plot analysis. Bias and limits of agreement (LoA), effect size (ES), and correlation coefficients were calculated. RESULTS: R-R intervals were significantly different in the supine and standing position between the ECG and the HRM uncorrected and corrected signal (P < 0.05, ES = 0.000 and 0.006, respectively). The bias +/- LoA, however, were 0.9 +/- 12 ms. HRV parameters derived from both signals in both positions were not different (P > 0.05) and well correlated (r > 0.97, P < 0.05), except root mean square of difference (RMSSD) and SD1 in standing position (P < 0.05, ES = 0.052 and 0.057; r = 0.99 and 0.98, respectively). CONCLUSION: Narrow LoA, good correlations, and small effect sizes support the validity of the Polar S810 HRM to measure R-R intervals and make the subsequent HRV analysis in supine position. Caution must be taken in standing position for the parameters sensitive to the short-term variability (i.e., RMSSD and SD1). PMID- 16672843 TI - Energy expenditure in men and women during 54 h of exercise and caloric deprivation. AB - Fifty U.S. Marine recruits (30 men, 20 women) were studied during a physically intense, energy intake-restricted, winter-time 54-h field training exercise (FEX) at Parris Island Marine Corps Recruit Depot. Men and women completed the same physical tasks. PURPOSE: To characterize and compare the total energy expenditure (TEE) and core temperature responses in men and women working almost continuously for 2.25 d in an outdoor environment while developing a substantial energy deficit. METHODS: TEE was measured using doubly labeled water (D(2)O(18)). Energy intake was estimated using beverage diaries and collecting ration wrappers saved by each volunteer and adding the known caloric value of each food item consumed. Core temperature was measured using an ingested thermometer pill. Physical activity level (PAL) was calculated by dividing TEE by the calculated basal metabolic rate. RESULTS: TEE was higher (P < 0.001) for the men (25.7 MJ.d(-1)) than women (19.8 MJ.d(-1)), but there were no differences between men and women in TEE normalized to body mass (men, 0.35 +/- 0.05 MJ.d(-1).kg(-1); women, 0.34 +/- 0.06 MJ.d(-).kg(-1)), corrected body mass (men, 0.29 +/- 0.04 MJ.d(-1).kg(-1) corrected body mass; women, 0.27 +/- 0.04 MJ.d(-1).kg(-1) corrected body mass), fat-free mass (men, 0.41 +/- 0.07 MJ.d(-1).kg(-1) FFM; women, 0.46 +/- 0.07 MJ.d( 1).kg(-1) FFM), or corrected fat-free mass (men, 0.30 +/- 0.05 MJ.d(-1).kg corrected body mass; women, 0.30 +/- 0.04 0.30 +/- 0.05 MJ.d(-1).kg(-1) corrected body mass). PAL was the same for men (3.4 +/- 0.5) and women (3.3 +/- 0.4). Energy intakes were higher (P < 0.05) in men (6.0 +/- 2.0 MJ.d(-1)) than women (4.8 +/- 1.8 MJ.d(-1)). The average minimum core temperature was 36.0 +/- 0.4 degrees C, and the mean maximum core temperature was 38.5 +/- 0.3 degrees C. CONCLUSIONS: For both men and women, total energy expenditures were among the highest observed for a military FEX. TEE, when normalized or corrected to body mass and fat-free mass, and PAL were the same for men and women. PMID- 16672844 TI - Fluid ingestion attenuates the decline in VO2peak associated with cardiovascular drift. AB - INTRODUCTION/PURPOSE: This study investigated whether manipulation of cardiovascular drift (CV drift) by changing exercise duration or by fluid ingestion is associated with altered peak oxygen uptake VO(2peak). METHODS: VO(2peak) was measured in 11 trained men immediately after they cycled at 60% control VO(2max) in 30 degrees C, 40% relative humidity for 15, 60, and 120 min with no fluid (15 NF, 60 NF, 120 NF) or 120 min with fluid (120 F). Stroke volume (SV), heart rate (HR), and related measures were measured in 120 NF and 120 F at 15, 60, and 120 min. RESULTS: Body mass decreased 0.7, 2.3, and 3.7% in 120 F, 60 NF, and 120 NF. SV at the end of submaximal exercise and VO(2peak) measured immediately thereafter were reduced significantly (P < 0.05) from 15-min values in 120 NF (13.8 and 8.7%) but not in 60 NF (4.6 and 1.2%) or 120 F (2.1 and 1.9%). CONCLUSIONS: The progressive decline in SV during prolonged, constant-rate submaximal exercise in a warm environment, reflective of increased cardiovascular strain associated with hyperthermia, dehydration, and other changes that occur over time, reduces VO(2peak). Fluid ingestion improves performance in prolonged exercise, in part, by mitigating the decline in SV and its determinants, and preserving VO(2peak). PMID- 16672845 TI - Family structure and children's television viewing and physical activity. AB - OBJECTIVES: This study aimed to examine how physical activity (PA) and television (TV) viewing time of children varied according to family structure. METHODS: In 2001, 5- to 6-yr-old (N = 296) and 10- to 12-yr-old (N = 919) children and their parents were recruited from 19 state elementary schools in Melbourne, Australia. Children's PA was objectively assessed using accelerometers worn for 8 d. Sociodemographic and family structure information and time spent watching TV was collected via questionnaire completed by parents. RESULTS: ANCOVA revealed that, after controlling for socioeconomic status and age of child, boys without any siblings spent more minutes per day watching TV (153.2 +/- 71.3) compared with those who have siblings (129.0 +/- 64.4, P < 0.05). There were also significant differences in TV viewing time between boys with one sibling (125.5 +/- 59.9), two siblings (141.9 +/- 70.1), or three or more siblings (111.6 +/- 62.6, P < 0.001). Girls from single-parent families (145.7 +/- 85.1) spent significantly more minutes per day watching TV compared with girls from two-parent families (125.1 +/- 67.7, P < 0.05). Girls with siblings spent more minutes per day in PA (148.3 +/- 67.7) compared with those who were an only child (131.0 +/- 58.9, P < 0.05). There were significant interactions between parental status and having a sibling, with PA, and also with TV viewing for girls and between parental status and having a brother with PA for boys. There were also significant interactions between having an older sibling and child's sex with PA and between number of siblings and sex with TV viewing. CONCLUSIONS: Family structure may be an important source of influence on children's PA and TV viewing time. Aspects of family structure interact differently with PA and TV viewing, suggesting interventions may need to be tailored with consideration of the family structure of children. PMID- 16672846 TI - Risk of obesity in relation to physical activity tracking from youth to adulthood. AB - PURPOSE: Maintaining a high level of physical activity throughout one's lifetime may decrease the risk of obesity. We evaluated how physical activity patterns from youths (9-18 yr) to adulthood are associated with body mass index (BMI) and waist circumference (WC) in a population of young adults. METHODS: As part of the longitudinal Cardiovascular Risk in Young Finns Study, we assessed physical activity over a 21-yr follow-up in a cohort of 1319 subjects. Physical activity was measured using a questionnaire completed in conjunction with a medical examination. RESULTS: During the follow-up, 33.1% of men and 32.0% of women were classified as persistently active, and 11.5% of men and 7.4% of women as persistently inactive. Both decreasingly active and persistently inactive subjects were more likely to be obese as adults compared with persistently active subjects. In women, being decreasingly active from youth to adulthood compared with being persistently active was independently associated with the risk of being overweight (BMI = 25.0-29.9 kg.m(-2), odds ratio (OR) = 2.35, confidence interval (CI) = 1.16-4.78), obese (BMI > or = 30.0 kg.m(-2), OR = 2.72, CI = 1.04 7.09), mildly abdominally obese (WC = 800-879 mm, OR = 2.21, CI = 1.01-4.84), and severely abdominally obese (WC > or = 880 mm, OR = 2.19, CI = 1.03-4.67), after adjustment for several variables including childhood fatness. In men, decreasing physical activity during their lifetime was associated with mild (WC = 940-1019 mm, odds ratio (OR) = 1.78, CI = 1.00-3.19) and severe (WC > or = 1020 mm, OR = 2.47, CI = 1.27-4.78) abdominal obesity in unadjusted analyses, but these two associations disappeared after adjustment for confounding variables (OR = 1.51, CI = 0.72-3.17 and OR = 1.62, CI 0.66-4.02, respectively). In men, changes in physical activity were not associated with obesity or overweight as defined by cut-points of BMI. CONCLUSIONS: Maintaining a high level of physical activity from youth to adulthood is independently associated with lower risk of abdominal obesity in among women, but not men. These findings suggest that changes in physical activity patterns during the lifetime may contribute to the development of abdominal obesity in women. PMID- 16672847 TI - School-level intraclass correlation for physical activity in sixth grade girls. AB - PURPOSE: The Trial for Activity in Adolescent Girls (TAAG) is a group-randomized trial (GRT) to reduce the usual decline in moderate to vigorous physical activity (MVPA) among middle school girls. We report the school-level intraclass correlation (ICC) for MVPA from the TAAG baseline survey of sixth grade girls and describe the relationship between the schedule of data collection and the ICC. METHODS: Each of six sites recruited six schools and randomly selected 60 sixth grade girls from each school; 74.2% participated. Girls were grouped in waves defined by the date measurements began and asked to wear an Actigraph accelerometer for 6 d. Occasional missing data were replaced by imputation, and counts above 1500 per 30 s were treated as MVPA, converted into metabolic equivalents (METs), and summed over 6 a.m.-midnight to provide MET-minutes per 18 h day. Mixed-model regression was used to estimate ICC. RESULTS: The school-level ICC were higher when estimated from a single wave compared with three waves (e.g., 0.057 vs 0.022) and across weekdays compared with weekend days (e.g., 0.024 vs 0.012). Power in a new trial would be greater with some schedules (e.g., 88% given three waves and 6 d) than with others (e.g., 23% given one wave and Tuesday only). CONCLUSIONS: The schedule of data collection can have a dramatic effect on the ICC for MVPA. In turn, this can have a dramatic effect on the standard error for an intervention effect and on power. Investigators will need to consider the expected magnitude of the ICC and the validity of the MVPA estimates associated with their data collection schedule in planning a new study. PMID- 16672848 TI - Neural activation after maximal isometric contractions at different muscle lengths. AB - PURPOSE: To investigate i) whether neural activation dependence on muscle length is preserved with neuromuscular fatigue and ii) whether fatigue induced by a maximal isometric exercise is muscle length dependent. METHODS: Twelve male subjects performed two fatiguing quadriceps muscle exercises: FS is the fatigue carried out at short muscle length (S) (S = 40 degrees of knee flexion) and FL is the fatigue at long muscle length (L) (L = 100 degrees). Before and after each fatiguing exercise (i.e., three maximal isometric contractions maintained until 80, 60, and 40% of the initial maximal torque, respectively), activation level (AL, assessed by means of twitch interpolation technique), EMG activity (RMS), and peak doublet torque (Pd) were measured at the two lengths (S and L). RESULTS: First, AL was greater (P < 0.05) at L compared with S before and after both exercises. Second, despite a similar decrease in maximal voluntary torque (approximately 21% of the initial value) after the two exercises, AL and RMS were significantly reduced after FS (P < 0.05) but remained unchanged after FL, whereas the Pd decrease was more pronounced after FL than FS (P < 0.05). Nevertheless, after a given fatiguing exercise (i.e., FS or FL), AL, RMS, and Pd changes were similar at both postexercise test lengths (S and L). CONCLUSION: These results clearly demonstrate that i) the neural activation dependence on quadriceps muscle length is maintained with fatigue, and ii) neuromuscular fatigue after maximal isometric contractions is dependent on the muscle length at which the exercise is performed: short length preferentially induces neural activation impairment, whereas long length leads to higher contractile failure. PMID- 16672849 TI - Core strength and lower extremity alignment during single leg squats. AB - INTRODUCTION/PURPOSE: Muscles of the trunk, hip, and knee influence the orientation of the lower extremity during weight bearing activities. The purpose of this study was threefold: first, to compare the orientation of the lower extremity during a single leg (SL) squat among male and female athletes; second, to compare the strength of muscle groups in the trunk, hips, and knees between these individuals; and third, to evaluate the association between trunk, hip, and knee strength and the orientation of the knee joint during this activity. METHODS: Twenty-four male and 22 female athletes participated in this study. Peak isometric torque was determined for the following muscle actions: trunk flexion, extension, and lateral flexion, hip abduction and external rotation, and knee flexion and extension. The frontal plane projection angle (FPPA) of the knee during a 45 degrees SL squat was determined using photo editing software. RESULTS: Males and females moved in opposite directions during the SL squat test (F(1,42) = 5.05, P = 0.03). Females typically moved toward more extreme FPPA during SL squats (P = 0.056), while males tended to move toward more neutral alignment (P = 0.066). Females also generated less torque in all muscle groups, with the exception of trunk extension. The projection angle of the knee during the SL squat test was most closely associated with hip external rotation strength. CONCLUSION: Using instruments suitable for a clinical setting, females were found to have greater FPPA and generally decreased trunk, hip, and knee isometric torque. Hip external rotation strength was most closely associated with the frontal plane projection angle. PMID- 16672850 TI - Performance at high pedaling cadences in well-trained cyclists. AB - PURPOSE: This study was conducted to determine the effect of high pedaling cadences on maximal cycling power output (W(max)). METHODS: Nine well-trained cyclists performed a continuous, incremental cycle-ergometer test to exhaustion (25 W increases every 3 min) either at 80, 100, or 120 rpm on three different occasions. RESULTS: W(max) was approximately 9% lower during 120 rpm in comparison with 80 and 100 rpm (335 +/- 9, 363 +/- 7, and 370 +/- 12 W, respectively; P < 0.05). During 120 rpm, ventilation rate (V(E)) increased above the increases in expired CO(2), which reduced the power output (PO) at the ventilatory anaerobic threshold (VT(2)) by 11% (P < 0.05). Gross efficiency (GE) did not differ among trials. At 120 rpm, capillary blood lactate concentration ([Lac]) increased above the 80-rpm trial (5.3 +/- 1.2 vs 3.0 +/- 0.7 mM at 300 W; P < 0.05), although pH was not reduced. At 120 rpm, expired CO(2) increased and reduced blood bicarbonate concentration ([HCO(3)(-)]) was reduced, maintaining blood pH similar to the other trials. CONCLUSION: A high pedaling cadence (i.e., 120 rpm) reduces performance (i.e., W(max)) and anaerobic threshold during an incremental test in well-trained cyclists. The data suggest that ventilatory anaerobic threshold (VT(2)) is a sensitive predictor of optimal pedaling cadence for performance, whereas blood pH or efficiency is not. PMID- 16672851 TI - Exercise responses and adaptations in rowers and spinal cord injury individuals. AB - Elite rowers (ROWERS) and those who have had a spinal cord injury (SCI) are different physically in many realms. Both have physical activity histories that affect their lower-extremity extensor muscles in a dramatically different fashion. ROWERS can sustain a 500-W power output during their 5- to 6-min race. After a complete SCI, a 75-W power output might be achieved during a VO(2peak) test. Elite SCI wheelchair racers can achieve a higher value that is similar to that of a sedentary able-bodied person. ROWERS can attain a VO(2 max) of more than 7.5 L.min(-1) and can tolerate a blood lactate of 30 mmol.L(-1). After a complete SCI in which muscles become markedly atrophied, a peak VO(2) of 2 L.min( 1) and a blood lactate of 10 mmol.L(-1) might be achieved. ROWERS rely on the 75% slow-twitch fiber composition of their trained thigh muscles to train and race. Such activity modestly increases fiber size and markedly increases mitochondrial content. After a complete SCI, affected muscle fibers markedly atrophy, maintain most of their mitochondrial content, and become fast-twitch. These data suggest remarkable plasticity of physical function to the extreme that a marked increase in energy demanding, rather continuous physical activity can make a muscle more "slow-twitch"; so it will demand less energy when contracted. In contrast, SCI eventually causes muscle to be composed of more fast-twitch fibers. Molecular biologists may explain why fast-twitch fibers, which appear ideal for some athletes because of their high power output, are abundant in muscles that are seldom recruited. Until then, our results indicate that the fiber type composition of muscle in humans is stable unless extreme alterations in physical activity are endured. PMID- 16672852 TI - NCAA rule change improves weight loss among national championship wrestlers. AB - PURPOSE: The present investigation was initiated to examine the weight management practices among wrestlers participating in the NCAA Division I, II, and III wrestling tournaments. Part 1 examined the efficacy of body composition assessment between preseason (PRE) and postseason (POST). Part 2 examined rapid weight loss (RWL) during the 20 h preceding the weigh-in and the rapid weight gained (RWG) during the first day's competition. METHODS: Subjects include 811 competitors from Divisions I, II, and III participating in the NCAA national championship tournaments between 1999 and 2004. Measurements included relative body fatness (% BF) and weight (WT) on the day preceding the tournament and the evening of the first day. Minimal weight (MW) was computed with 5% BF. Retrospectively, MW, % BF, and WT from the previous fall were obtained for comparisons from NCAA records. RESULTS: Part 1: WT and % BF decreased significantly PRE (WT 74.0 +/- 11.1 kg; % BF 12.3 +/- 3.4%) to POST (WT 71.5 +/- 10.4 kg; % BF 9.5 +/- 1.8%), but MW (PRE MW 68.0 +/- 9.2 kg, POST MW 67.9 +/- 9.1 kg) remained unchanged. Heavier wrestlers and Division I and II wrestlers showed the greatest changes in WT and % BF. Part 2: RWL averaged (+/- SD) 1.2 +/- 0.9 kg and relative to weight 1.7 +/- 1.2%. Division I and lighter wrestlers showed the greatest change. RWG averaged 0.9 +/- 0.8 kg, or 1.3 +/- 1.2%. RWG was greater among lighter and Division I and II wrestlers. CONCLUSIONS: Minimal weight estimates PRE appear valid compared with POST. RWL and RWG are reduced significantly over previous investigations with only mat-side weigh-ins. The NCAA weight management program appears effective in reducing unhealthy weight cutting behaviors and promoting competitive equity. Efforts to institute similar programs among younger wrestlers seem warranted. PMID- 16672853 TI - Psychosocial factors related to physical activity and weight loss in overweight women. AB - PURPOSE: This study examined whether psychosocial factors related to physical activity in overweight, previously sedentary women were affected by a 6-month behavioral weight loss program. In addition, these psychosocial factors were examined across levels of weight loss and self-reported physical activity in response to a weight loss intervention. METHODS: Data from 165 overweight (body mass index (BMI) = 32.7 +/- 4.2 kg.m(-2)) women (age = 37.6 +/- 5.5 yr) who participated in a comprehensive behavioral weight loss program that included behavioral education, moderate caloric restriction, and progressive home-based exercise were examined. Body weight was assessed at 0 and 6 months. Perceived benefits and barriers, physical activity self-efficacy, and physical activity processes of change were assessed at 0 and 6 months. Physical activity (minutes per week of at least moderate-intensity activity) was assessed using the 7-Day Physical Activity Recall at 0 and 6 months. RESULTS: The intervention resulted in increases in physical activity self-efficacy, behavioral processes of change, and several cognitive processes of change(P < 0.05). There was a reduction in expected barriers for physical activity (P < 0.05). Individuals with > or = 10% weight loss reported higher levels of physical activity self-efficacy, greater use of behavioral strategies to elicit social support, and fewer barriers to physical activity than those with lower levels of physical activity and less weight loss (P < 0.05). Individuals reporting higher levels of exercise also reported higher levels of physical activity self-efficacy, greater use of behavioral strategies, and fewer barriers to physical activity than those individuals with lower levels of physical activity (P < 0.05). CONCLUSION: Targeting self-efficacy, behavioral strategies, and barriers in weight management programs may improve physical activity, which may result in improved weight loss in overweight adults. PMID- 16672854 TI - The influence of fitness and body weight on preferred exercise intensity. AB - PURPOSE: The purpose of this investigation was to determine the individual and combined effects of aerobic fitness and body weight on physiological responses, perceived exertion, and speed variables during self-selected steady-state treadmill (TM) walking in 60 healthy college-age women. METHODS: The women were placed into one of four categories based on body mass index (BMI) and fitness level, assessed by a graded TM test. Subjects walked continuously on a TM at a self-selected pace for 15 min at a 2.5% grade. The dependent variables were oxygen uptake (VO(2)), HR, percentage of maximal oxygen uptake (VO(2max)), percentage of HRmax (%HRmax), RPE for the overall body, TM belt speed, and total energy expenditure (EE). RESULTS: There were no significant interactions or body weight main effects for any of the dependent variables. However, lower-fitness subjects walked at a TM speed that resulted in a higher (P < 0.0005) VO(2max) (52.4 vs 39.56) than the higher-fitness subjects. CONCLUSION: These findings suggest that fitness, and not body weight, influences preferred exercise intensity as measured by VO(2max) during TM walking in college-age women. The self-selected walking speed did not result in an intensity, as determined by VO(2max), that is consistent with the enhancement of cardiorespiratory fitness for higher-fitness women regardless of body weight. PMID- 16672855 TI - Impact of physical activity during pregnancy and postpartum on chronic disease risk. AB - Research over the past 20 years has focused on the safety of physical activity during pregnancy. Guidelines for health care providers and pregnant/postpartum women have been developed from the results of these studies. The overwhelming results of most studies have shown few negative effects on the pregnancy of a healthy gravida, but rather, be beneficial to the maternal-fetal unit. Recently, researchers have begun to consider the role of maternal physical activity in a more traditional chronic disease prevention model, for both mother and offspring. To address the key issues related to the role of physical activity during pregnancy and postpartum on chronic disease risk, the American College of Sports Medicine convened a Scientific Roundtable at Michigan State University in East Lansing, MI. Topics included preeclampsia, gestational diabetes, breastfeeding and weight loss, musculoskeletal disorders, mental health, and offspring health and development. PMID- 16672856 TI - A simplified strategy for the estimation of the exercise ventilatory thresholds. AB - PURPOSE: To analyze the limits of agreement between exercise ventilatory threshold values (VT1 and VT2) estimated from a combination of pulmonary gas exchange and ventilatory variables (cardiopulmonary exercise testing) and those derived from an alternative approach based on the ventilatory response only (V(E), ventilometry). METHODS: Forty-two nontrained subjects (24 males, aged 18 48, peak VO(2) = 33.1 +/- 8.6 mL.min(-1).kg(-1)) performed a maximum incremental cardiopulmonary exercise testing on an electromagnetically braked cycle ergometer. The participants breathed through a Pitot tube (Cardio2 System, MGC) and a fixed-resistance ventilometer (Micromed, Brazil), which were connected in series. HR values at the estimated VT (VTHR1 and VTHR2) were obtained by the conventional method (ventilatory equivalents, end-expiratory pressures for O(2) and CO(2), and the V-slope procedure) and an experimental approach (V(E) vs time, V(E)/time vs time, and breathing frequency vs time). RESULTS: There were no significant between-method differences on VT(HR1), VT(HR2), VT(VE1), VT(VE2), and peak V(E) (P > 0.05). After certification of data normality, a Bland-Altman analysis revealed that the mean bias +/- 95% confidence interval of the between method differences were lower for VT(HR2) than VT(HR1) (2 +/- 9 and 0 +/- 17 bpm, respectively). VT(HR2) according to ventilometry differed more than 10 bpm from the standard procedure in 3 out of 42 subjects (9%). Between-method differences were independent of the level of fitness, as estimated from peak VO(2) (P > 0.05). CONCLUSIONS: : A simplified approach, based on the ventilatory response as a function of time, can provide acceptable estimates of the exercise ventilatory thresholds--especially VT2--during ramp-incremental cycle ergometry. This new strategy might prove to be useful for exercise training prescription in nontrained adults. PMID- 16672857 TI - Validity of PCERT and OMNI walk/run ratings of perceived exertion. AB - PURPOSE: This study was conducted to test the validity of the Pictorial Children's Effort Rating Table (PCERT) and OMNI walk/run scales. METHODS: Children (26 boys age 11.2 yr +/- 1.6 and 25 girls age 11.1 yr +/- 1.4) performed a five-stage incremental exertion treadmill test. The undifferentiated perceived exertion from the PCERT and OMNI scales was assessed for construct validity using Pearson correlations with VO(2) and heart rate as criteria and concurrent validity by correlating PCERT and OMNI scores. RESULTS: Increases in PCERT and OMNI scale scores were correlated with increases in VO(2) (r = 0.90 and 0.92) and heart rate (r = 0.89 and 0.92). No difference was found in slope of the PCERT and OMNI scores when regressed against heart rate or VO(2) and the slopes were invariant across sexes. To test concurrent validity of the PCERT and OMNI scales, subject scores at each stage were converted to a percentage of the maximal scale. No effect of sex was noted on perceived exertion (P = 0.32), and the percentage of the maximal PCERT and OMNI scales was almost identical at each stage (P = 0.73). CONCLUSIONS: Validity of both the PCERT and OMNI scales was established for submaximal exercise. PCERT and OMNI scores are not interchangeable because they have different scales, but at a given exercise intensity youth assign similar percentages of the maximal scores. PMID- 16672858 TI - Methodological problems in studying the female athlete triad. PMID- 16672859 TI - Female athlete triad errors and misunderstandings. PMID- 16672861 TI - Revision total knee arthroplasty: editorial comment. PMID- 16672862 TI - Bone graft for tibial defects in total knee arthroplasty. 1986. AB - Twenty-four knees with bone grafts for tibial defects at the time of either primary or revision total knee arthroplasty were followed for three to six years. With 22 of 24 bone grafts, union and revascularization were seen and no clinical collapse was present. In two, nonunion occurred, accompanied by collapse in one. Failure was attributed to varus alignment of the leg in one (a medial condylar graft) and to insufficient preparation of the bony bed in the second (bleeding bone was not exposed). Evidence for incorporation of the grafts was obtained by tomogram, bone scan, and bone biopsy. Incorporation was present by six months, but the time to complete remodeling was not determined. A bone graft is recommended for tibial defect involving 50% or more of the bony support of either tibial plateau. A bone graft is indicated whenever a cement column under the prosthesis would measure more than 5 mm in height. PMID- 16672864 TI - Polycentric knee arthroplasty: prosthetic simulation of normal knee movement. 1971. PMID- 16672865 TI - A critical view of total joint replacements. 1977. PMID- 16672866 TI - Socioeconomic issues and demographics of total knee arthroplasty revision. AB - Despite rising numbers of total knee arthroplasty revision (TKAR) procedures there remains a paucity of information regarding the relationships between total knee arthroplasty failure and socioeconomic and educational status, demographics, general health and functional disability. We performed a multicenter prospective study of 290 consecutive TKAR patients in order to determine whether they differed from the population they were drawn from in terms of socioeconomic or educational status, race or gender. Secondary aims were to establish the relative comorbid status of this population, social supports and their general health status compared to national norms and their modes of failure. Our cohort consisted of 137 males and 153 females with a mean age of 68.6 years (range, 34 85 years), substantial overall functional disability according to the SF-36 and a large average number of comorbidities at baseline. We found a relative overrepresentation of patients of comparatively low socioeconomic and educational status and also of Caucasian patients in the TKAR population. This large prospective investigation demonstrates demographic features associated with TKA failure and provides a platform for further investigations on the effect demographic characteristics have on the outcomes of TKAR. LEVEL OF EVIDENCE: Prognostic Study, Level II (Lesser quality prospective study). See Guidelines for Authors for a complete description of the Levels of Evidence. PMID- 16672867 TI - Economic burden of revision hip and knee arthroplasty in Medicare enrollees. AB - The economic burden to Medicare due to revision arthroplasty procedures has not yet been studied systematically. The economic burden of revisions was calculated as annual reimbursements for revision arthroplasties relative to the sum total reimbursements of primary and revision arthroplasties. We evaluated this revision burden for total hip and knee arthroplasties through investigation of trends in charges and reimbursements in the Medicare population (Parts A and B claims from 1997-2003), while taking into account age and gender effects. Mean annual economic revision burdens were 18.8% (range, 17.4-20.2%) and 8.2% (range, 7.5 9.2%) for total hip arthroplasties and total knee arthroplasties, respectively. Procedural charges increased while reimbursements decreased over the study period, with higher charges observed for revisions than primary arthroplasties. Reimbursements per procedure were 62% to 68% less than associated charges for primary and revision total hip and knee arthroplasties. The effect of age and gender on reimbursements varied by procedure type. Unless some limiting mechanism is implemented to reduce the incidence of revision surgeries, the diverging trends in reimbursements and charges for total hip and knee arthroplasties indicate that the economic impact to the Medicare population and healthcare system will continue to increase. LEVEL OF EVIDENCE: Prognostic study, level II-1 (retrospective study). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672868 TI - Cost effectiveness of revision total knee arthroplasty. AB - Total knee arthroplasty now exceeds total hip arthroplasty as the most commonly performed joint replacement. Projections suggest the need for revision knee arthroplasty in the future will produce an immense economic burden. The excellent cost effectiveness of primary knee arthroplasty has been well established. This article explores the cost effectiveness of revision knee arthroplasty, and makes a comparison of costs between different international health care systems. While revision knee arthroplasty is more costly, technically difficult, and complicated than primary knee arthroplasty, it is still a cost effective means of improving function, pain relief, and quality of life. The role of national arthroplasty registries will be important in guiding decision making toward reducing the requirements for revision surgery. LEVEL OF EVIDENCE: Prognostic study, level II 1 (prospective study). See Guidelines for Authors for complete description of levels of evidence. PMID- 16672869 TI - Agreement about indications for total knee arthroplasty. AB - Total knee arthroplasty is an effective and cost efficient procedure that improves the quality of life for patients with end stage knee arthritis. With the prevalence of arthritis expected to increase with the aging of the population, the demand for total knee arthroplasty will increase substantially. We examined current indications for total knee arthroplasty by reviewing articles extracted from a comprehensive Med-line search. We studied the indications for proceeding with a total knee arthroplasty, the indications for referring a patient for a total knee arthroplasty, and the contraindications among orthopaedic surgeons, rheumatologists, and primary care providers. We evaluated 27-42 different patient factors. "Pain not responsive to drug therapy" was the only factor with consensus for total knee arthroplasty. "Major psychiatric disorder, including dementia" was the only contraindication to total knee arthroplasty that had a consensus. The lack of consensus within orthopaedics and across specialties likely represents a limitation in empirical data associated with total knee arthroplasty outcomes. Achieving better consensus will require conducting and disseminating better research on patient characteristics that predict the success of total knee arthroplasty. LEVEL OF EVIDENCE: Economic and decision analyses, level IV (analyses with no sensitivity analyses). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672870 TI - Preoperative evaluations in revision total knee arthroplasty. AB - There are many causes of total knee arthroplasty failure, and an accurate preoperative diagnosis is essential to optimize the results of revision surgery. We discuss our standard pre-operative evaluation routine and we retrospectively reviewed the last 295 patients who underwent revision total knee arthroplasty to establish the clinical value of the most commonly performed investigations used to diagnose sepsis. Routinely performed preoperative investigations include erythrocyte sedimentation rate, C-reactive protein, microbiology, bacteriology cultures of preoperative knee aspirations, and intraoperative tissue bacteriology cultures. Each investigation was compared with the reason for revision (eg, septic or aseptic) to establish the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of each. 79 cases (26.8%) were revised for infection. Of the investigations, the ESR had a sensitivity of 0.63, a specificity of 0.55, a positive predictive value of 0.39, a negative predictive value of 0.77, and an accuracy of 0.57. The respective values for C-reactive protein were 0.6, 0.63, 0.45, 0.76, and 0.62, and 0.53, 0.94, 0.75, 0.85, and 0.83 for intraoperative tissue culture. There was no preoperative investigation accurate enough to be solely relied on for diagnosing infection. We believe that clinical findings and the routine use of simple tests such as C-reactive protein, ESR, and knee aspiration yield predictable results. LEVEL OF EVIDENCE: Prognostic study, level II-1 (retrospective study). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672871 TI - Current etiologies and modes of failure in total knee arthroplasty revision. AB - Although total knee arthroplasty is a very effective intervention and increasing in prevalence, failures do occur. We studied patients presenting for total knee arthroplasty revision to determine any modifiable causes of failure, both short and long term, and where future efforts should be directed to reduce the incidence of failure. A multicenter prospective observational cohort study of 318 consecutive patients, with minimum 1 year follow-up, undergoing total knee arthroplasty revision was performed. Associations between modes of failure were also assessed. The mean time from primary procedure to total knee arthroplasty revision was 7.9 years. Many patients (64.4%) had more than one cause of failure. Thirty-one percent of patients were early (< 2 years) failures at a mean of 11 months. These had a higher prevalence of infection, perioperative factors and comorbidities. Late failures occurred at a mean of 119.2 months. Other major causes of failure included instability (28.9%), wear (24.5%) and component loosening suggesting the importance of modifications in technique, implants and other areas. Application of these findings will ultimately reduce revision numbers through continued refinement of total knee arthroplasty practice and through further specific investigation of these modes of failure. LEVEL OF EVIDENCE: Therapeutic study, level II-2 (prospective study). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672872 TI - Radiographic prediction of intraoperative bone loss in knee arthroplasty revision. AB - A key challenge for orthopaedic surgeons performing revision total knee arthroplasty is the management of bone loss. The goal of our study was to test the validity of predicting bone loss from preoperative radiographs using two commonly utilized bone loss assessments: the Anderson Orthopaedic Research Institute and University of Pennsylvania systems and secondarily to assess the frequency and severity of bone loss in a prospective study of total knee arthroplasty revisions. Ninety-eight total knee arthroplasty revision patients were assessed and bone loss was detected in 76 (77.6%) patients preoperatively and intraoperatively. The validity of both systems was established. Agreement between preoperative and intraoperative Anderson Orthopaedic Research Institute classification was fair for the femur and good for the tibia. All University of Pennsylvania preoperative measures were correlated with intraoperative measures. Establishing valid and reliable preoperative systems of measuring bone loss facilitates planning of total knee arthroplasty revision and rehabilitation and meaningful comparisons between different series of patients and treatment protocols. LEVEL OF EVIDENCE: Diagnostic study, level I (prospective testing of previously developed diagnostic criteria on consecutive patients [with universally applied reference "gold" standard]). See Author Guidelines for a complete description of levels of evidence. PMID- 16672873 TI - Surgical exposures in revision total knee arthroplasty. AB - Safely obtaining adequate exposure at the time of revision total knee arthroplasty is an integral step in successfully performing the procedure. A medial capsular approach combined with an extensive intraarticular synovectomy provides adequate exposure for most patients. If further exposure is required, a quadriceps snip can be used to free the proximal extensor mechanism. The benefits of this approach include its technically simple nature and an unaltered postoperative rehabilitation regimen. We report a series of 126 consecutive revision knee procedures in which a medial capsular approach was adequate in 111 cases, representing 92% of the patients with an intact extensor mechanism. A quadriceps snip was required in nine cases. If more extensive exposure is required for an excessively stiff or difficult to expose knee, a tibial tubercle osteotomy or V-Y quadricepsplasty provides wider exposure. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672874 TI - Minimally invasive revision total knee arthroplasty. AB - Exposure in revision total knee arthroplasty can be difficult. For example, eversion of the patella may require a number of procedures including vastus snips, V-Y turndowns, and tibial tubercle osteotomies. A minimally invasive approach further adds to exposure difficulties. We report a new approach for exposure during difficult revision total knee arthroplasties. A soft tissue envelope technique allows retraction of the patella and avoids eversion. This technique reduces the size of the incision, reduces quadriceps muscle damage, and enhances exposure. It is essential that appropriate instrumentation is used along with down sized femoral and tibial cutting blocks. We report all 17 revision knee arthroplasties using this technique including unicompartmental revisions, tibial component revisions, and femoral component revisions. Followup averaged 29 months (range, 24-39 months). We had one case of posterior retained cement after a full revision with no subsequent complications. One patient required an arthroscopic lateral release for patellar maltracking. Preliminary results are encouraging using improved instrumentation we find essential for enhanced exposure. We believe this a reasonable approach for selected total knee arthroplasty revisions. LEVEL OF EVIDENCE: Therapeutic study, level IV (retrospective case series). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672875 TI - Removing well-fixed total knee arthroplasty implants. AB - Revision total knee arthroplasty requires the removal of one or all of the prior knee components. Thoughtful and careful removal of well-fixed total knee components can preserve host bone stock and facilitate subsequent reconstructive efforts. The tools and techniques for knee component removal include simple osteotomes, saws, extraction devices, ultrasonic devices, and frequently a combination of some or all of the above. It is the unique nature of each revision arthroplasty, which presents the surgeon with such challenge. Each of the techniques discussed in this review reflects the anecdotal experience acquired in over 800 revision total knee arthroplasties performed at our center. We review our empiric bias regarding the individual applicability of these techniques. LEVEL OF EVIDENCE: Therapeutic studies, Level V (expert opinion). See Guidelines for Authors for a complete description of Levels of Evidence. PMID- 16672876 TI - Management of bone loss: augments, cones, offset stems. AB - Trabecular metal augmentation has added new treatment options for severe proximal tibial bone defects in revision knee arthroplasty. Porous tantalum tibial cones provide mechanical support for the tibial component and have the potential for long-term biologic fixation. These cones facilitate restoration of the proximal tibia metaphysis in Type 2 and 3 defects. Ten tantalum tibial cones were press fit into the prepared cavitary defect of a series of revision knee arthroplasties. Voids between the cone and host bone were filled with morselized grafting material. The core tibial component was cemented into the implanted tibial cone; fixation was enhanced with stem extensions, which were press-fit in four knees and cemented in six knees. Extensions ranged from 75-200 mm with length dependent upon the residual bone quality. Offset stems were used in 3 tibias. At follow-up (average 10 months), radiographic evaluation revealed no evidence of loosening or change in position. Strength, range of motion, and stability were comparable to previously reported series of revision arthroplasties. Trabecular metal cones can help reconstruct large cavitary defects and, along with stem extensions and offset stems, may eliminate the need for extensive bone grafting or structural allograft in revision knee arthroplasty. LEVEL OF EVIDENCE: Therapeutic study, level V (expert opinion). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672877 TI - Modular femoral offset stems facilitate joint line restoration in revision knee arthroplasty. AB - Restoration of proper joint line position in revision total knee arthroplasty is essential in promoting recovery of function. We retrospectively analyzed joint line restoration and clinical outcomes in 22 consecutive femur revision cases using modular offsets of variable length and direction between the intramedullary fixation rod and the femoral component. Flexion and extension gap balancing techniques and medial epicondylar referencing was used to achieve proper position of the joint line. Position of the reconstructed joint line from postoperative radiographs was compared to the baseline position of intended anatomic placement determined from pre-operative planning radiographs. Postoperative joint line height averaged 1.6 mm distal to baseline (range, 5 mm distal to 2.5 proximal). Joint line was restored to within 2 mm of anatomic position in 12 of the 22 knees. Sixteen patients received conventional, minimally constrained tibial inserts, and joint stability was achieved in all cases. LEVEL OF EVIDENCE: Therapeutic study, Level IV (case series). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672878 TI - Impaction grafting for bone defects in revision total knee arthroplasty. AB - Bone loss in revision total knee arthroplasty is a complex and challenging problem. Frequently, the defects encountered are irregular in size and shape. Since 1998, we have been using impaction bone allografting for constrained and unconstrained defects in revision total knee arthroplasty. We prospectively studied the mid-term results of 48 consecutive revision total knee arthroplasties with substantial bone loss treated with impaction allograft. Average followup was 3.8 years. Knee Society scores improved from a preoperative average of 57.0 to a postoperative average of 89.8 points (p < 0.001). Knee Society functional scores improved from a pre-operative average of 52.3 to a postoperative average of 80.3 points (p < 0.001). There have been no mechanical failures of the revisions and all radiographs have shown incorporation and remodeling of the bone graft. There were six complications out of the 42 revisions available for followup (14%); two periprosthetic fractures, one early infection salvaged with irrigation and antibiotics, one late infection resulting in fusion, and two patellar clunk syndromes. Though time consuming and technically demanding, impaction grafting for bone loss in revision total knee arthroplasty has excellent durability and versatility. It has become our preferred technique for the management of substantial bone loss in revision total knee arthroplasty. LEVEL OF EVIDENCE: Therapeutic study, Level IV (case series). PMID- 16672879 TI - Management of bone loss: structural grafts in revision total knee arthroplasty. AB - Massive bone defects are challenging problems in revision knee surgery. When defects are large and uncontained (without a cortical rim), structural allografts may be used to provide support for femoral and tibial components. This study reviewed 68 structural allografts at a mean of 5.4 years for clinical and radiographic outcomes. Indications for grafts included periprosthetic fracture in 19 knees, aseptic loosening in 29, infection in 11 and instability in 2. Seven knees had both femoral and tibial allografts. Multiple implant designs were used including 7 hinged prostheses. Thirteen knees (13/61) failed due to graft related complications including one graft nonunion, three aseptic loosenings, three periprosthetic fractures, four infections, and two for instability. The case of graft nonunion was successfully treated with revision fixation and autologous bone graft. There were three cases of graft resorption, two graded as severe and one as moderate. These results are satisfactory given the nature and complexity of the problem, however, reconstructive procedures require careful preoperative preparation and extensive experience in complex knee arthroplasty. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672880 TI - Distal femur replacement is useful in complex total knee arthroplasty revisions. AB - Revision total knee arthroplasty can be considerably more difficult in cases involving severe bone loss, complete absence of collateral ligaments, and persistent periprosthetic supracondylar femoral fractures. Modular segmental endoprosthetic distal femur replacement is a limb salvage option when other surgical options are unfeasible. The clinical performance of rotating hinge knee prostheses has greatly improved with the evolution of second and third generation designs. The increased freedom of rotation decreases the prosthetic bone stresses and the longevity. While designs have improved, the prostheses still do not match the function and longevity of condylar components with approximately a 90% survivorship at 20 years. The modular endoprosthesis rotating hinge knee prosthesis is useful for total knee arthroplasty revision in patients with inadequate bone stock, ligamentous instability, or difficult supracondylar femur fractures, especially in low demand elderly patients. LEVEL OF EVIDENCE: Therapeutic study, level V (expert opinion). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672881 TI - Flexion and extension gap balancing in revision total knee arthroplasty. AB - Revision total knee arthroplasty presents a unique set of problems when attempting to balance flexion and extension gaps. Loss of soft tissue support and established deformity can make balancing difficult. One needs to balance the flexion and extension gap heights as well as medial and lateral symmetry, which may not always be attainable. We used a set of stepwise techniques to reestablish the joint line in extension using femoral augments, and then balanced the flexion gap using different sized femoral components. We retrospectively analyzed 45 patients who had revision total knee replacement with an average of 4 years followup. These patients had a mean flexion of 105 degrees and none had signs of instability in flexion or extension or on clinical exam. Despite the complex nature of revision knee arthroplasty, cases utilizing an algorithm to balance the extension and flexion gaps, with increased implant constraint when necessary, can aid in obtaining a good outcome. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672882 TI - Knee arthrodesis with the Wichita Fusion Nail: an outcome comparison. AB - The Wichita Fusion Nail (WFN) is a knee arthrodesis stabilization system that employs compression via an intramedullary rod. It was designed for use in the salvage of the irretrievably failed total knee arthroplasty and other severe knee pathologies. Questionnaires covering the fusion success rate, fusion time, and complication rate were obtained from 33 surgeons who were among the first to use the device. Data from these questionnaires were analyzed to determine if the rate of successful fusion was close to 100%, which was the primary hypothesis of this study. The average time required to achieve fusion and the rate of complications were also calculated and compared to similar results available in the literature. The results for 44 selected patients were included and it was determined that all achieved fusion for a success rate of 100%. This compared favorably with reported success rates in the range of 54% to 96%. The average fusion time was 15.5 weeks. Complications included: six delayed unions, three deep infections, and two periimplant fractures for a major complications rate of 20.4%. Both the fusion times and complication rate compared favorably with other reported results. Surgeons using the device for the first time had outcomes equal to those of more experienced users. Our results demonstrated that a rate of successful arthrodesis close to 100% could be consistently achieved with the WFN. Overall, the WFN facilitated an improved outcome for a previously difficult procedure. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series). See the Guidelines for Authors for a complete description of level of evidence. PMID- 16672883 TI - Cementless fixation in revision total knee arthroplasty. AB - A surgical approach to revision total knee arthroplasty that includes minimal bone resection, minimal soft-tissue stripping, cementless fixation of the femoral and tibial components, and morselized allografting of defects was used and evaluated in 105 patients (110 knees) with severe bone loss. The patients were followed for 60 to 127 months postoperatively. Fixation included a tightly-fit fluted titanium stem in the femoral and tibial canals and rim contact on the peripheral rim of the tibia. One tibia loosened and one knee failed because of infection. Ligamentous stability and pain relief were consistent through the followup period. At 10 years the mean valgus laxity was 4 degrees +/- 2.5 degrees, and the mean varus laxity was 5.2 degrees +/- 3.3 degrees. Mean Knee Society pain score was 47 +/- 2.1. Pain was mild in 28 knees, moderate in eight knees, and severe in two knees. Evidence of bone healing occurred in the bone defects that could be seen on radiographs. Increase in radiodensity always was found at postoperative intervals greater than one year. An approach to revision total knee arthroplasty that maintains bone and soft tissue about the knee establishes an effective and durable construct. LEVEL OF EVIDENCE: Therapeutic study, level II (prospective study). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672884 TI - Patellofemoral instability after total knee arthroplasty. AB - Despite advances in surgical technique and implant design, complications involving the extensor mechanism and patellofemoral joint after total knee arthroplasty (TKA) continue to be the most common cause of pain and the most commonly cited reason for revision TKA surgery. A thorough understanding of the etiologies of patellofemoral instability, careful preoperative planning, and meticulous surgical techniques will optimize clinical outcome. Evaluation of patellofemoral stability should begin in the operating room. Postoperatively, thorough history, physical examination, and dedicated radiographic studies should be obtained. Computed tomography scan is the most accurate and reliable way to assess component positioning. Treatment of patellofemoral instability is directed by its etiology. Revision of one or both components is indicated if malpositioning is present. If the components are determined to be in satisfactory positions, soft tissue procedures can be pursued. Future advancements in prosthetic design and the routine use of computer-assisted navigation systems will minimize patellofemoral instability. LEVEL OF EVIDENCE: Therapeutic study, Level V (expert opinion). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672885 TI - Periprosthetic patellar fractures. AB - Patellar fracture after total knee arthroplasty is a rare yet challenging complication. Patellar fracture can occur as a result of trauma or it may be atraumatic. A multitude of factors can lead to periprosthetic patellar fracture including patient related factors, surgical technique related factors, and implant specific factors. Understanding the etiologic factors leading to atraumatic patellar fractures could result in minimizing complications. We present the results of peri-prosthetic patellar fractures in 12 patients. All type I non-displaced fractures (7 cases) were treated nonoperatively. Surgical treatment was selected for the remaining 5 cases which included resection arthroplasty combined with open reduction and internal fixation of the fracture (3 knees), partial patellectomy (1 knee), and total patellectomy (1 knee). The outcome was excellent in 1 knee, good in 8 knees, and fair in the remaining 3 knees at the latest follow-up. There were 2 reoperations; 1 for disruption of the extensor mechanism and 1 for refracture. One patient developed a superficial wound infection. We reviewed the available literature regarding the etiology, surgical strategies, and outcomes for periprosthetic patellar fracture. LEVEL OF EVIDENCE: Therapeutic studies, level IV (case series). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672886 TI - Extensor mechanism disruption after total knee arthroplasty. AB - Extensor mechanism disruption after total knee arthroplasty is a challenging complication for orthopaedic surgeons. The treatment options for repair include observation, direct primary repair, direct primary repair with synthetic ligament or autogenous tissue augmentation, or reconstruction with allograft tissue. A computerized systemic review and literature search was performed to identify the relevant literature on extensor mechanism disruptions associated with total knee arthroplasty. A comprehensive review of the literature and description of relevant treatment options and outcomes were performed using the information gained with the literature review. A multi-center prospective study on a consecutive series of patients recruited from the North American Knee Arthroplasty Revision (NAKAR) study was performed and data collected pre operatively, intra-operatively, and post-operatively on patients that had a failed total knee arthroplasty using validated health related quality of life measures was analyzed. Six out of 290 patients in the study had extensor mechanism disruption and this group of patients had overall worse functional outcomes. The results of the study have solidified our knowledge that patients with extensor mechanism disruptions have worse functional outcomes and will need intensive management and rehabilitation. LEVEL OF EVIDENCE: Economic and decision analyses, level III (systematic review of level III studies). See Guide for Authors for a complete description of levels of evidence. PMID- 16672887 TI - Medial gastrocnemius flap coverage for treatment of skin necrosis after total knee arthroplasty. AB - Skin necrosis after total knee arthroplasty is a rare complication that can rapidly lead to deep infection of the prosthetic components. The medial gastrocnemius transposition flap usually provides adequate soft tissue coverage to salvage the total knee arthroplasty. However, variations in defect location and excursion of the muscle flap can affect results. Twelve patients were treated with a medial gastrocnemius transposition flap after total knee arthroplasty. The skin defect that required flap coverage was located over the tibial tubercle or patellar tendon in eight patients (Group 1). The defect extended proximally to the patella or quadriceps tendon in four patients (Group 2). A functioning total knee arthroplasty was salvaged in 11 patients (92%). The medial gastrocnemius flap healed primarily in all patients in Group 1. Three patients in Group 2 required additional fasciocutaneous, lateral gastrocnemius, or free flap coverage, and one patient underwent above knee amputation. The medial gastrocnemius flap is most effective for coverage of distal defects over the tibial tubercle or patellar tendon. Defects that extend more proximally over the patella or quadriceps tendon are more likely to require additional procedures to achieve adequate soft tissue coverage. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series). See Author Guidelines for a complete description of levels of evidence. PMID- 16672889 TI - Revision total knee arthroplasty does not increase PACU utilization. AB - The hospital records of 232 consecutive cases of patients undergoing primary and revision total knee arthroplasty were analyzed to determine differences in operating room time, postanesthesia care unit time, operating room narcotic usage, and postanesthesia care unit narcotic usage between the two groups. The average operating room time for a surgeon performing revision total knee arthroplasty on a patient was greater than that for a primary total knee arthroplasty. However, there was no difference in average postanesthesia care unit time nor operating room and postanesthesia care unit narcotic usage. When stratified to anesthetic type and perioperative pain intervention, there was no difference in any of the measured parameters between the primary and revision groups. Thus, even with longer operating times, a patient undergoing revision total knee arthroplasty did not utilize more postanesthesia care unit time, nor more perioperative narcotics, than a patient undergoing primary total knee arthroplasty. LEVEL OF EVIDENCE: Therapeutic study, Level III (retrospective comparative study). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672888 TI - Inpatient rehabilitation outcomes in primary and revision total knee arthroplasty patients. AB - Inpatient rehabilitation is an effective means of improving physical function and independence following total knee arthroplasty (TKA). Revision TKA (TKAR) is becoming increasingly more prevalent and it is unclear if revision TKAR patients attain similar improvements following inpatient rehabilitation compared to primary TKA (TKAP) patients. This investigation compared functional outcomes following interdisciplinary inpatient rehabilitation in 286 TKAP and 138 TKAR consecutive male and female patients. Functional Independence Measure (FIM) scores improved from admission to discharge for the TKAP (81.6 to 110.5) and TKAR (74.4 to 101.4) groups (p = 0.015). TKAP patients had shorter length of stay (LOS) compared to TKAR (9.2 and 11.3). FIM efficiency (FIM/LOS) was greater for the TKAP compared to TKAR (3.6 and 2.6). Total hospital charges were 11,399 dollars and 13,407 dollars for the TKAP and TKAR groups, respectively. TKAP patients were more likely to be discharged home compared to the TKAR patients (97.6 vs. 78.3%). Both TKAP and TKAR patients demonstrate gains in FIM scores during inpatient rehabilitation. However, the amount of FIM efficiency is lower, and LOS and hospital charges are greater when comparing TKAR and TKAP. In addition, discharge disposition may be influenced by the type of TKA, primary and revision. LEVEL OF EVIDENCE: Prognostic study, level II-1 (retrospective study). See Guidelines for Authors for a complete description of levels of evidence. PMID- 16672890 TI - Conversion of failed unicompartmental knee arthroplasty to TKA. AB - As the number of unicompartmental knee arthroplasties performed continues to rise, so too will the number of failures. In order to justify its continued use, conversion to total knee arthroplasty must be evaluated. From 1993-2004, 22 consecutive knees from 18 patients with a failed unicondylar knee arthroplasty underwent conversion to total knee arthroplasty. The most common modes of failure were polyethylene wear (12 patients), loosening of the femoral (4 patients) or tibial component (3 patients), and osteoarthritis progression (3 patients). All patients were converted to primary cruciate retaining components. Twenty-seven percent of patients had contained defects on the femoral condyle that required bone graft. No femoral stems or metal augmentation were required. Forty-five percent of patients had contained defects on the tibia that required bone graft. Metal wedge augmentation was required in five knees (23%), and stems were used in two patients. Sixteen of 22 knees (73%) were followed for an average of 64.5 months. Knee Society knee scores and functional scores at latest followup were 93 and 78, respectively. Conversion of a failed unicondylar knee arthroplasty to a total knee arthroplasty is technically demanding, but may be done successfully with careful preoperative planning and possible need for revision techniques. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series). See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672891 TI - The increasing financial burden of knee revision surgery in the United States. AB - The popularity of total knee arthroplasty combined with the aging US population indicates a dramatic increase in revision TKA procedures. Our objective was to project revision surgery costs in the United States, and to estimate the financial burden for hospitals historically under-reimbursed for these complex surgical procedures. Inflation adjusted charge data derived from a series of knee revision surgeries performed by a single surgeon practice (CJL) (n = 100) were applied to population projections of the number of revision surgeries expected for the Medicare population from 2005-2030. The average charge of TKA revision surgery was 73,696 dollars, (Cost was 36,848 dollars) with substantially higher costs for patients undergoing surgery because of deep joint infection, patients receiving a three component exchange, and patients receiving hinged or constrained condylar knee implants. The number of revision procedures is expected to increase from 37,544 in 2005 to 56,918 in 2030. Projected hospital costs for these procedures may exceed 2 billion dollars by 2030. The number of revision knee surgeries may increase by 66% in the next 25 years. Reimbursement rates will not cover hospital costs for this procedure despite recent increases in Medicare payments for revision arthroplasty. LEVEL OF EVIDENCE: Economic analysis study, level III. See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672892 TI - Early internal fracture fixation prevents bacterial translocation. AB - The aim of our study was to determine whether early internal fixation of major bone fractures helps prevent bacterial translocation in patients with multitrauma. Thirty-seven Sprague-Dawley rats were divided into three groups: (1) anesthesia only (n = 12); (2) the trauma group: tibia and femur fractures and moderate head trauma under anesthesia (n = 14); and (3) the fixation group: fixation of tibia and femur fractures and moderate head trauma under anesthesia (n = 11). After 24 hours, mesenteric lymph nodes, liver, spleen, and systemic blood samples were quantitatively cultured. The terminal ileum was assessed histopathologically. The incidence of bacterial translocation was less in the anesthesia group (two of 12 rats) and the fixation group (two of 11 rats) than in the trauma group (10 of 14 rats). The number of organs containing viable bacteria was significantly lower in the fixation group than in the trauma group. Histopathologically, villous architecture was preserved mostly in the fixation group; however, marked mucosal damage was detected in the trauma group. Our data suggest early internal fixation of long bone fractures in polytraumatized experimental animals with head injury results in preservation of the intestinal mucosal barrier and decreased bacterial translocation from the gut. PMID- 16672893 TI - Buttock mass in a 37-year-old woman. PMID- 16672895 TI - Recurrent instability after total hip arthroplasty: beware of subtle component malpositioning. AB - Most patients exhibiting instability after total hip arthroplasty can be treated nonoperatively. However, instability may become recurrent and require surgical intervention. Abductor insufficiency and component malpositioning constitute two of the most important causes of recurrent instability, although the exact cause may not be identifiable in some patients. There is relative scarcity of reports in the literature regarding the outcome of surgical intervention for recurrent instability; however, it is known that surgical intervention is likely to have a better outcome in patients for whom the cause of recurrent instability can be identified. We hypothesized that component malpositioning, which may be subtle in some cases, is the cause of recurrent instability for many patients. The outcomes of revision arthroplasty in 93 patients who were treated at our institution for recurrent instability were reviewed. Component malpositioning was found to be the major cause of recurrent instability in this successfully treated cohort. PMID- 16672896 TI - Revision hip arthroplasty for late instability secondary to polyethylene wear. AB - We evaluated the outcome of revision arthroplasty for polyethylene wear presenting as late dislocation. The computerized databases at two institutions were reviewed to identify all patients presenting with first time dislocation five or more years after total hip arthroplasty. Records and radiographs were then evaluated, and patients whose late dislocation occurred in the presence of greater than two millimeters of polyethylene liner wear with no other etiology for dislocation were identified. There were 22 patients with a mean age of 57.8 years at primary procedure. The average time from initial arthroplasty to dislocation was 9.0 years. Revision surgery to address polyethylene wear and instability was performed at a mean of 11.1 years (range 5.8 to 23 years). Revision surgery restored stability to eighteen patients (eighty-two percent). Polyethylene wear can and is associated with late dislocation after hip arthroplasty. Exchange of polyethylene lining of a metal backed implant or revision of the all polyethylene acetabular component can successfully address late instability in the majority of patients. PMID- 16672897 TI - Epidemiology of dislocation after total hip arthroplasty. AB - Instability after total hip arthroplasty is an important complication. It usually occurs in the immediate postoperative period, but the risk also increases with time. There are numerous surgical treatment options, but they have relatively unpredictable outcomes. Numerous factors are associated with dislocation, but research has mainly focused on surgical factors. Epidemiological factors remain the subject of much debate. We aimed to establish the most significant epidemiological factors in Scotland and in particular the dislocation rate in neuromuscular conditions. The Scottish National arthroplasty nonvoluntary registry is based on SMR01 records (Scottish Morbidity Record) data. We analyzed the Scottish National Arthroplasty Project to find patients' dislocation rates up to 1 year postoperatively for surgeon volume, age, gender, previous surgery, diagnosis, and followup duration. There were 14,314 total hip arthroplasties performed from April 1996 to March 2004 with an annual incidence of dislocation of 1.9%. We found an association between rate of dislocation with age, surgical volume, and previous fracture. However, there was no increase in the rate of dislocation associated with gender or with diagnoses of stroke or Parkinson's disease. Our prognostic assessment of dislocation risk allows assessment for methods of reducing dislocation in high risk patients. PMID- 16672898 TI - Increased surgical volume is associated with lower THA dislocation rates. AB - The presumed correlation between an increasing volume of health care procedures and an improvement in outcomes is sometimes referred to as the practice-makes perfect effect. Growing interest in outcomes-based research has led to numerous papers examining this relationship for various surgical procedures, including total hip arthroplasty. The results of these studies have important implications for consumers, providers, and healthcare financers. Accordingly, we review the literature to date examining surgeon and hospital volume effects on hip arthroplasty outcomes, with a specific focus on the effects of volume on dislocation. A systemic review of the literature demonstrates a substantial positive association between surgical volumes and improvement in most THA outcomes, including dislocation; that is, increasing surgical volume is associated with lower dislocation rates. This correlation appears to be stronger and is more clearly established for surgeon volumes than it is for hospital volumes. PMID- 16672899 TI - Comparison of capacitive versus resistive joint contact stress sensors. AB - Cartilage contact stress elevations might be associated with pain or other symptoms after malunited, incongruous intraarticular fractures. Studies identifying fractures with patterns of elevated contact stresses would help to ensure more appropriate choices of treatment. However, appropriate instrumentation for such studies is crucial. We tested two such systems, one capacitive and one resistive, under identical loading conditions presumed to occur in the ankle. We used a materials testing machine and customized-loading fixtures to measure force detection error, contact area error, repeatability, homogeneity, creep, and one-axis and two-axis bending artifacts. The loading regimen caused pressures up to 2.5 MPa. An error in force detection between -3% and +5% was observed with the capacitive sensor whereas an error between -12% and +20% was observed with the resistive sensor. Repeatability and homogeneity were greater for the capacitive sensor. Errors in contact area measurement were less than 2% for the resistive sensor and less than 6% for the capacitive sensor. The resistive sensor could not conform to spherical surfaces without crinkling. Creep artifact was observed with both sensors. We concluded that the capacitive sensor had superior performance even though its thickness and high compliance may be disadvantageous in intraarticular measurements. The resistive sensor is required for use where higher pressures are expected despite its inferior accuracy. PMID- 16672900 TI - Ulnar coronoid process anatomy: possible implications for elbow instability. AB - Ulnar coronoid process fractures are relatively uncommon injuries usually occurring with elbow dislocations and contributing to elbow instability. Recent evidence suggests coronoid tip fractures have a role in the instability. We sought to quantify the capsular and brachialis attachments of the ulnar coronoid process to better understand why instability occurs. We prepared eight fresh frozen cadaveric specimens to ascertain the specific attachment locations. After dissection, we isolated and resected the proximal ulna, including the coronoid process and its soft tissue attachments. We then embedded, sectioned, and stained the specimens. The average distance from the tip of the coronoid to the proximal capsule was 2.36 +/- 0.39 mm. The average distance from the tip of the coronoid to the proximal brachialis insertion was 10.13 +/- 1.6 mm. Most coronoid tip fractures included disruption of the anterior capsule, which potentially explains why instability can be associated with these fractures. PMID- 16672901 TI - Qualitative and quantitative accuracy of CAOS in a standardized in vitro spine model. AB - Pedicle breach with screw implantation is relatively common. For clinical application of computer-assisted orthopaedic surgery, it is important to quantitatively know the accuracy and localization of any guidance modality. We ascertained the accuracy of computed tomography and C-arm-based navigated drilling versus conventional fluoroscopy using an artificial thoracic and lumbar spine model. The 3.2-mm diameter transpedicle drilling target was the center of a 4-mm steel ball fixed in the anterior left pedicle axis. After drilling, we used computed tomography to verify the position of the steel ball and the canal and visually explored for cortex perforation. Quantitative vector calculation showed computed tomography-based navigation had the greatest accuracy (median, d(thoracic) = 1.4 mm; median, d(lumbar) = 1.8 mm) followed by C-arm navigation (median, d(thoracic) = 2.6 mm; median, d(lumbar) = 2 mm) and the conventional procedure (median, d(thoracic) = 2.2 mm; median, d(lumbar) = 2.7 mm). Visual examination showed a decreased perforation rate in navigated drillings. We found no correlation between pedicle breaches and inaccurate drilling. The data suggest computer-assisted orthopaedic surgery cannot provide sub-millimeter accuracy, and complete prevention of pedicle perforation is not realistic. PMID- 16672902 TI - Necrotizing soft tissue infections of the extremities and back. AB - Necrotizing soft tissue infections are potentially fatal infections that often involve extremities. Studies of mixed anatomic sites suggest several factors increase mortality (eg, age, medical comorbidities, laboratory values, treatment timing). We hypothesized that patients with necrotizing soft tissue infections of the extremities would have similar factors associated with mortality. We retrospectively reviewed 150 patients with necrotizing soft tissue infections of the extremities treated at San Francisco General Hospital from 1993-1997. We recorded cofactors, treatment, physical findings, radio- graphs, and laboratory findings at presentation. No cofactor or examination finding was associated with increased mortality. Compared with survivors, nonsurvivors had a higher leukocyte count, blood urea nitrogen, creatinine, potassium, partial thromboplastin time, and aspartate aminotransferase, but had lower pH and bicarbonate. Nonsurvivors did not have delays in treatment relative to survivors. Univariate analysis showed an increased risk of mortality in patients with hypotension, hypothermia, Clostridium species in the wound culture, low leukocyte count and bicarbonate levels, and elevated blood urea nitrogen, aspartate aminotransferase, creatinine, and potassium levels. Several signs of shock and organ dysfunction were associated with mortality in patients with necrotizing soft tissue infections of the extremities. The overall mortality rate (9.3%) was lower than in some other reports. PMID- 16672903 TI - Scapulopexy of winged scapula secondary to facioscapulohumeral muscular dystrophy. AB - Facioscapulohumeral muscular dystrophy is an hereditary disease that causes weakness of the scapulothoracic muscles and leads to winged scapula. Patients with facioscapulohumeral muscular dystrophy are unable to sustain shoulder abduction or flexion and are limited in daily activities. We retrospectively reviewed nine patients (18 procedures) who had scapulothoracic fixation without arthrodesis (scapulopexy). The technique consists of repositioning the scapula over the rib cage and fixation to four ribs with metal wires. We assessed improvement in range of motion of the shoulder, maintenance of the correction with time, and cosmetic and functional results. The average age of the patients at surgery was 25.2 years (range, 15-35 years), and there were no major complications. The average followup was 9.9 years (range, 3-16 years). All patients had complete resolution of the winged scapula and improved range of motion. Arm abduction increased from an average of 68.3 degrees (range, 45 degrees-90 degrees) preoperatively to 96.1 degrees (range, 60 degrees-120 degrees) postoperatively. Arm flexion increased from an average of 57.2 degrees (range, 45 degrees-90 degrees) preoperatively to 116.1 degrees (range, 80 degrees 180 degrees) postoperatively. The position of the scapula obtained by surgery was maintained with time, and the patients had satisfactory cosmetic results. LEVEL OF EVIDENCE: Therapeutic study, Level IV. See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672904 TI - Misuse of baseline comparison tests and subgroup analyses in surgical trials. AB - It is unclear whether the misuse of statistical tests that compare patients' baseline characteristics and subgroup analyses in randomized controlled trials can be extrapolated to the surgical literature. We did an observational study evaluating the current use of baseline comparability tests and subgroup analyses in surgical randomized controlled trials. Published surgical randomized controlled trials in four medical journals were identified. We also identified randomized controlled trials in the Journal of Bone and Joint Surgery (American and British volumes). We identified 72 randomized controlled trials, with a mean of 10 +/- 8 baseline variables. Of 166 significance tests, 17 (10%) were significant. Twenty-seven (38%) trials included 54 subgroup analyses with a minimum of one and maximum of 23 subgroup analyses per study. Inappropriate emphasis on subgroup analyses occurred frequently. Forty-nine (91%) analyses were performed post hoc without prior hypotheses. Investigators reported differences between subgroups in 31 (57%) of the analyses, all of which were featured in the summary or conclusion. These inferences may be misleading, making their application to clinical practice unwarranted. PMID- 16672905 TI - Two-year outcomes of computed tomography-based and computed tomography free navigation for total knee arthroplasties. AB - Optimal component position in all planes and well-balanced soft tissues facilitate a good clinical outcome and long-term survival after total knee arthroplasties. We investigated the accuracy of implantation of navigated total knee arthroplasties at 3 months followup and the influence on the clinical outcome at 2 years followup. Forty-four patients (44 procedures) were enrolled in our prospective study. One half of the surgeries were performed using a computed tomography-based navigation system, and half were performed with imageless navigation. Outcomes were based on the Insall knee score parameters, anterior knee pain, patient satisfaction, feeling of instability, and step test. The radiographic parameters were the mechanical axis, tibial slope, lateral distal femoral angle, and medial proximal tibial angle. The radiographic measurements were similar in both groups (patients within +/- 3 degrees inaccuracy range in computed tomography-based/imageless groups; mechanical axis 86%/81%, tibial slope 95%/91%, lateral distal femoral angle 95%/91%, medial proximal tibial angle 91%/95%). The imageless system provided equal radiographic results, but we found improved ligament balancing in the computed tomography free group. The computed tomography-based approach has a good pre-operative planning procedure, but is more expensive and time consuming. LEVEL OF EVIDENCE: Therapeutic Study, Level II. See the Guidelines for Authors for a complete description of levels of evidence. PMID- 16672906 TI - Angiotensin-(1-7) inhibits angiotensin II-stimulated phosphorylation of MAP kinases in proximal tubular cells. AB - Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE, which is not blocked by ACE inhibitors. High amounts of ACE2 are present in the proximal tubule, and ACE2 catalyzes generation of angiotensin 1-7 (Ang-(1-7)) by this segment. Ang-(1 7) binds to a receptor distinct from the AT1 or AT2 Ang II receptor, identified as the mas receptor. We studied the effects of Ang-(1-7) on Ang II-mediated cell signaling pathways in proximal tubule. In primary cultures of rat proximal tubular cells, activation of mitogen-activated protein kinases (MAPK) was detected by immunoblotting, in the presence or absence of agonists/antagonists. Transforming growth factor-beta1 (TGF-beta1) was measured by enzyme-linked immunosorbent assay. Ang II (5 min, 10(-7) M) stimulated phosphorylation of the three MAPK (p38, extracellular signal-related kinase (ERK 1/2), and c-Jun N terminal kinase (JNK)). While incubation of proximal tubular cells with Ang-(1-7) alone did not significantly affect MAPK phosphorylation, Ang-(1-7) (10(-7) M) completely inhibited Ang II-stimulated phosphorylation of p38, ERK 1/2, and JNK. This inhibitory effect was reversed by the Ang-(1-7) receptor antagonist, D-Ala7 Ang-(1-7). Ang II significantly increased production of TGF-beta1 in proximal tubular cells, an effect that was partly inhibited by Ang-(1-7). Ang-(1-7) had no significant effect on cyclic 3',5'-adenosine monophosphate production in these cells. In summary, Ang-(1-7) inhibits Ang II-stimulated MAPK phosphorylation in proximal tubular cells. Generation of Ang-(1-7) by proximal tubular ACE2 could thereby serve a protective role by counteracting the effects of locally generated Ang II. PMID- 16672907 TI - Method for detecting the disconnection of an extracorporeal device using a patient's endogenous electrical voltages. AB - Tubing (especially venous) disconnections using pumped devices cause significant hemorrhage, and current monitoring techniques are imperfect because they rely on intraluminal pressure changes. We devised a passive detection method based on a patient's electrical voltages being transmitted via blood tubing to our alarm circuit. As the arterial and venous access sites are in close proximity, the signals are nearly identical during connection, and markedly different with disconnection. We built a prototype and tested it in vitro with saline and during hemodialysis treatments (n=7). The connection status is determined by examining the difference between endogenous voltages in the blood tubing from and to the patient, and when it exceeds a threshold an alarm condition is triggered. We tested for possible confounding by an electrical shunt through the dialyzer and determined that pathway had an impedance approximately three times (>350 kOmega) that of the tubing to the patient. As the roller blood-pump periodically occluded the tubing, the resultant intermittent very high impedance prevented that potential shunting problem and improved the sensitivity of our device. Disconnections were detectable at various bloodline sites (needles, sampling ports, drip chambers). Thus, the circuit's sensors can be placed remotely at the dialysis machine, with electrical continuity to blood made by inexpensive conductive elements at the tubing wall or drip chambers. Appropriate threshold and noise-eliminating circuitry, as well as alarm states that alert the staff and stop the blood pump, make our prototype a promising low-cost safety enhancement. PMID- 16672908 TI - Characteristics of sudden death in hemodialysis patients. AB - Hemodialysis (HD) is an intermittent procedure during which large fluid and electrolyte shifts occur. We hypothesized that sudden death occurrences in HD patients are related to the timing of HD, and that they occur more frequently in the 12 h period starting with dialysis and in the 12 h period at the end of the dialysis-free weekend interval. In a retrospective study, 228 patient deaths were screened to determine if they met the criteria for sudden death. Information was obtained from clinic charts, dialysis center records, and interview of witnesses of the death event. There were 80 HD patients who met the criteria for sudden death. A bimodal distribution of death occurrences was present, with a 1.7-fold increased death risk occurring in the 12 h period starting with the dialysis procedure and a threefold increased risk of death in the 12 h before HD at the end of the weekend interval (P=0.011). Patients with sudden death had a high prevalence of congestive heart failure and coronary artery disease. Only 40% of patients experiencing sudden death were receiving beta-blockers, and the prior monthly serum potassium value was less than 4 mEq/l in 25%. Sudden death is temporally related to the HD procedure. Every other day HD could be beneficial in preventing sudden death. Careful attention to the usage of beta-blockers and to the maintenance of normal serum potassium values is indicated in HD patients at risk for sudden death. PMID- 16672909 TI - Kidney-specific upregulation of vitamin D3 target genes in ClC-5 KO mice. AB - Mutations in ClC-5 cause Dent's disease, a disorder associated with low molecular weight proteinuria, hyperphosphaturia, and kidney stones. ClC-5 is a Cl(-)/H(+) exchanger predominantly expressed in the kidney, where it facilitates the acidification of proximal tubular endosomes. The reduction in proximal tubular endocytosis resulting from a lack of ClC-5 raises the luminal concentration of filtered proteins and peptides like parathyroid hormone (PTH). The increase in PTH may explain the hyperphosphaturia observed in Dent's disease. Expression profiling, quantitative reverse transcriptase-polymerase chain reaction (qRT PCR), and hormone measurements were used to investigate whether the disruption of ClC-5 affects other signalling pathways. Although the upregulation of 25(OH)(2) vitamin D(3) 1alpha-hydroxylase and downregulation of vitamin D(3) 24-hydroxylase suggested an increased formation of 1,25(OH)(2)-vitamin D(3), the concentration of this active metabolite was reduced in the serum of ClC-5 knockout (KO) mice. However, target genes of 1,25(OH)(2)-vitamin D(3) were upregulated in KO kidneys. Expression analysis of intestine and bone revealed that the upregulation of 1,25(OH)(2)-vitamin D(3) target genes was kidney intrinsic and not systemic. In spite of reduced serum levels of 1,25(OH)(2)-vitamin D(3) in ClC-5 KO mice, 1,25(OH)(2)-vitamin D(3) is increased in later nephron segments as a consequence of impaired proximal tubular endocytosis. This leads to a kidney-specific stimulation of 1,25(OH)(2)-vitamin D(3) target genes that may contribute to the pathogenesis of Dent's disease. The activation of genes in distal nephron segments by hormones that are normally endocytosed in the proximal tubule may extend to other pathways like those activated by retinoic acid. PMID- 16672910 TI - Metabolomic study of cisplatin-induced nephrotoxicity. AB - We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARalpha ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARalpha ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARalpha ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity. PMID- 16672911 TI - Vasopressin receptor antagonists. AB - The first non-peptide vasopressin receptor antagonist (VRA) was recently approved by the United States Food and Drug Administration, and several others are now in late stages of clinical development. Phase 3 trials indicate that these agents predictably reduce urine osmolality, increase electrolyte-free water excretion, and raise serum sodium concentration. They are likely to become a mainstay of treatment of euvolemic and hypervolemic hyponatremia. Although tachyphylaxis to the hydro-osmotic effect of these agents does not appear to occur, their use is accompanied by an increase in thirst, and they do not always eliminate altogether the need for water restriction during treatment of hyponatremia. Experience with use of these agents for treatment of acute, severe, life-threatening hyponatremia as well as chronic hyponatremia is limited. Further studies are needed to determine how they are best used in these situations, but the risk of overly rapid correction of hyponatremia seems low. Results of long-term trials to determine the ability of VRAs to reduce morbidity or mortality in congestive heart failure or to slow the progression of polycystic kidney disease are awaited with great interest. PMID- 16672912 TI - Efficacy of local dipyridamole therapy in a porcine model of arteriovenous graft stenosis. AB - Perivascular delivery of antiproliferative drugs has been proposed as an approach to prevent neointimal hyperplasia associated with hemodialysis polytetrafluoroethylene (PTFE) grafts. We examined this approach to deliver dipyridamole in a porcine graft model. PTFE grafts were implanted between the carotid artery and external jugular vein bilaterally in pigs. During the surgery or 1 week post-graft placement, dipyridamole (0.26-52 mg) alone or incorporated in microspheres was mixed with an injectable polymeric gel and applied to the graft-arterial and graft-venous anastomoses on one side, whereas the contralateral control graft received no treatment. Three or four weeks after operation, the grafts and adjacent vessels were explanted en bloc and cross sections of the anastomoses were examined histologically. The degree of neointimal hyperplasia was quantified by planimetry. In separate experiments, dipyridamole was extracted from the explanted tissues and assayed by spectrofluorometry. The normalized median hyperplasia areas of the treated and control graft-venous anastomoses were 0.45 (25th-75th percentile, 0.30-0.86) and 0.24 (0.21-0.30), respectively (N=7; P=0.08). The median hyperplasia areas of the treated and control graft-arterial anastomoses were 0.12 (0.07-0.39) and 0.11 (0.09-0.13), respectively (N=7; P=0.31). The dipyridamole levels in the vascular walls around the anastomoses were at or above the in vitro inhibitory concentrations for approximately 3 weeks. These results suggest that the local perivascular sustained delivery of dipyridamole, even at high dosages, was ineffective in inhibiting neointimal hyperplasia associated with PTFE grafts in a porcine model. PMID- 16672913 TI - Minimal change nephrotic syndrome and classical Hodgkin's lymphoma: report of 21 cases and review of the literature. AB - Minimal change nephrotic syndrome (MCNS) is described as a paraneoplastic manifestation of classical Hodgkin's lymphoma (cHL). We reassessed the pathophysiological and clinical significance of this association. A retrospective study was performed to evaluate a cohort of adult patients who developed MCNS and cHL. Twenty-one patients recruited in 15 French centers were analyzed. cHL was associated with inflammatory and general symptoms in most cases. The morphological subtype was predominantly nodular sclerosis (71.4%). MCNS appeared before the diagnosis of lymphoma in eight patients (38.1%) and in this case, it was characterized by a nephrotic syndrome (NS) frequently resistant (50%) or dependent (12.5%) to steroid treatment. Interestingly, diagnosis (3-120 months after MCNS) and effective treatment of the hemopathy were associated with the disappearance of the MCNS. cHL was diagnosed before MCNS in nine patients (42.9%), and in this case, glomerulopathy was associated with cHL relapse in 55.5% of cases. In four patients (19%), the two diseases occurred simultaneously. Extensive immunohistochemical study of lymph nodes was performed in eight patients and did not reveal particular features. In conclusion, MCNS associated with cHL is frequently dependent or resistant to steroid regimen, but remission of NS is obtained with the cure of lymphoma. PMID- 16672914 TI - A cross-sectional study of HIV-seropositive patients with varying degrees of proteinuria in South Africa. AB - Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is the most common finding on renal biopsy in HIV-infected black patients and is also the commonest cause of end-stage renal disease in these patients. Early detection of HIVAN may be beneficial in evaluating early treatment. This study examined the pattern of renal diseases in HIV-infected South Africans and also attempted to diagnose HIVAN at an early stage. In this single-center cross-sectional study, 615 HIV-infected patients were screened for proteinuria. Thirty patients with varying degrees of proteinuria underwent renal biopsy. Patients with diabetes mellitus, uncontrolled hypertension, known causes of chronic kidney disease, and serum creatinine above 250 mumol/l were excluded. Patients in this study were not on antiretroviral therapy. HIVAN was found in 25 (83%) patients. Six of them (24%) had microalbuminuria. Altogether, seven patients with persistent microalbuminuria were biopsied and six (86%) showed HIVAN. Other biopsy findings included membranoproliferative nephropathy in two (7%) and interstitial nephritis in three (10%). Four patients with HIVAN had associated membranous nephropathy. HIVAN is the commonest biopsy finding among our study patients with HIV infection who present with varying degrees of proteinuria. Microalbuminuria is a manifestation of HIVAN in our study patients. Therefore, microalbuminuria may be an early marker of HIVAN, and screening for its presence may be beneficial. Renal biopsy may be considered in seropositive patients who present with persistent microalbuminuria, especially with low CD4 counts irrespective of good renal function. This will allow diagnosis and treatment of HIVAN at an early stage and may prevent further disease progression. PMID- 16672915 TI - Determinants of baroreflex function in juvenile end-stage renal disease. AB - Arterial baroreflex sensitivity (BRS) is markedly reduced in middle-aged patients with end-stage renal disease (ESRD), due to the combined effects of aging, arterial stiffening, and autonomic neuropathy. Much less is known about the effects of ESRD on arterial baroreflex in juvenile patients. Therefore, we investigated baroreflex function and its relation to carotid artery elasticity and heart rate variability in children and young adults with ESRD. We studied 42 subjects (9-30 years): 14 patients on maintenance hemodialysis (HD), 14 renal transplant recipients (RT), and 14 healthy control subjects (C). Baroreflex function was determined by pharmacological (BRS) and spontaneous (sequence and spectral indices) techniques. Carotid artery elasticity was characterized by stiffness index beta. Heart rate variability was assessed using time and frequency domain measures. Data are expressed as mean+/-s.d. BRS was markedly reduced in HD as compared to C (10.0+/-4.2 vs 25.7+/-5.9 ms/mm Hg); spontaneous indices were reduced to similar extent. Carotid artery stiffness was approximately 50% higher in HD than in C and was inversely related to BRS. Heart rate variability was also compromised in HD, and was directly related to spontaneous indices. No significant differences existed in any of these variables between RT and C. Decreased baroreflex function in juvenile HD is partly due to loss of carotid artery elasticity and partly due to impaired heart rate variability. Renal transplantation may partly prevent impairment or improve compromised baroreflex function in young patients with ESRD. PMID- 16672917 TI - The PPARalpha ligand fenofibrate: meeting multiple targets in diabetic nephropathy. AB - Fibrate peroxisome proliferator-activated receptor (PPAR)-alpha ligands are mainly used as hypolipidemic drugs. But this commentary highlights their potential in treating insulin resistance, dyslipidemia, and hypertension and in preventing diabetic nephropathy, inflammation, and cardiovascular disease. Because diabetes is a major contributor to chronic kidney disease and cardiovascular disease, PPAR-alpha agonists may provide greater opportunities for hitting multiple targets in this complex metabolic disease. PMID- 16672918 TI - Water-only pores and peritoneal dialysis. AB - The article by Ni et al. solidifies the important role of aquaporin-1 in the process of fluid removal from anephric patients treated with peritoneal dialysis. The presence of the water-only channel in the subperitoneal endothelia provides the mechanism for solute-free ultrafiltrate observed early in dialysis and accounts for approximately half of all the filtration observed in dialysis. PMID- 16672919 TI - Relaxin in cardiovascular and renal disease. AB - Fibrosis (organ scarring) is a hallmark of many forms of cardiovascular and renal disease, and causes organ dysfunction and structural changes when normal tissue is replaced with scar tissue; the accumulation of scar tissue being a leading cause of death around the world. Despite deep organ scarring potentially existing in many forms (including myocardial and vascular sclerosis, renal interstitial fibrosis, and glomerulosclerosis), current therapies have only had limited success in delaying end-stage disease. The peptide hormone relaxin is emerging as a potent antifibrotic therapy with rapid-occurring efficacy. Recent studies have demonstrated the antifibrotic actions of relaxin in experimental models of cardiac and renal disease in vivo, and the various levels at which relaxin acts to inhibit fibroblast-induced collagen overproduction leading to fibrosis, in vitro. Separate studies using relaxin gene-knockout mice have demonstrated the significance of endogenous relaxin as a naturally occurring and protective moderator of collagen turnover, while the therapeutic potential of relaxin has been enhanced by its ability to promote vasodilation and renal hyperfiltration. This review will summarize these coherent findings as a means of highlighting the clinical potential of relaxin in cardiovascular and renal disease. PMID- 16672920 TI - Renin/prorenin receptors. AB - The existence of a tissue renin-angiotensin (RAS) system independent of the circulating RAS has prompted the search for cellular binding sites for angiotensinogen and for renin in order to explain their tissue uptake. Two receptors that bind with similar affinity mature renin and prorenin were identified, the mannose-6-phosphate receptor (M6P-R) and a specific receptor. The M6P-R is a clearance receptor that binds exclusively the glycosylated forms of renin and prorenin. Binding of renin and prorenin to the M6P-R is followed by internalization and degradation, and the intracellular proteolysis of prorenin in mature renin did not provoke any generation of intracellular angiotensins. In contrast to the M6P-R, (pro)renin bound to the specific receptor was not degraded. Instead, receptor-bound renin showed increased catalytic activity, and receptor-bound prorenin exhibited full catalytic activity. This 'gain of activity' was explained by a conformational change of the (pro)renin molecule upon binding. Furthermore, (pro)renin binding provoked a rapid activation of the mitogen-activated protein kinases p44/p42, indicating that the receptor has mediated specific, angiotensin II-independent effects of (pro)renin. This receptor represents an elegant concept to explain the existence of active prorenin in vivo, and it provides a pathological role for prorenin in situations with paradoxical low renin and high prorenin concentrations such as in diabetes. Experimental models of rats overexpressing the receptor either in vascular smooth muscle cells and developing high blood pressure or with ubiquitous expression associated with glomerulosclerosis and proteinuria confirm a role for the receptor in cardiovascular and renal diseases. PMID- 16672922 TI - Reactive oxygen species deglycosilate glomerular alpha-dystroglycan. AB - In the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG. By using specific antibodies directed against the core protein or glyco epitopes of alpha-DG, this was studied in a solid-phase assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes. PMID- 16672921 TI - PPARalpha agonist fenofibrate improves diabetic nephropathy in db/db mice. AB - Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARalpha by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARalpha, decreased type I collagen production. Taken together, the PPARalpha agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARalpha by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy. PMID- 16672923 TI - Cross-sectional validity of a modified Edmonton symptom assessment system in dialysis patients: a simple assessment of symptom burden. AB - Subjective symptom assessment should be a fundamental component of health-related quality of life (HRQL) assessment in end-stage renal disease (ESRD). Unfortunately, no symptom checklist has established reliability or validity in ESRD. We report the validation of a modified Edmonton Symptom Assessment System (ESAS) in 507 dialysis patients who concurrently completed the Kidney Dialysis Quality of Life-Short Form (KDQOL-SF) questionnaire. The ESAS demonstrated a mean of 7.5+/-2.5 symptoms. The symptoms reported as most severe were tiredness, well being, appetite, and pain. The overall symptom distress score was strongly correlated with the KDQOL-SF subscales symptom/problem list (r=-0.69, P<0.01), effects of kidney disease (r=-0.52, P<0.01), and burden of kidney disease (r= 0.50, P<0.01), as well as lower RAND-12 physical health composite (PHC) (r=-0.54, P<0.01) and lower RAND-12 mental health composite (MHC) (r=-0.62, P<0.001). In the multivariate regression analysis, after controlling for potential confounding variables including comorbidity using the modified Charlson Comorbidity Index, the ESAS symptom distress score remained strongly associated with the MHC (slope= 0.82+/-0.07, P<0.01) and PHC (slope=-0.48+/-0.07, P<0.01). The ESAS symptom distress score accounted for 29% of the impairment in PHC and 39% of the impairment in MHC. The intraclass correlation coefficient for the total symptom distress score in a 1-week test-retest was 0.70, P<0.01. Symptom burden is high and adversely affects HRQL in dialysis patients. The modified ESAS is a reliable, valid, simple, and useful method for regular symptom assessment in this patient population. PMID- 16672924 TI - Etiology and prognostic significance of severe uremic pruritus in chronic hemodialysis patients. AB - Although uremia is well known as the most common cause of pruritus, the mechanisms of pruritus in chronic hemodialysis patients remain unclear. The purpose was to characterize uremic pruritus in more detail and to investigate whether severe pruritus is a marker for poor prognosis. A total of 1773 adult hemodialysis patients were studied. A questionnaire was given to each patient to assess the intensity and frequency, as well as pruritus-related sleep disturbance. We analyzed the relationship between clinical and laboratory data and the severity of pruritus in hemodialysis patients and followed them for 24 months prospectively. In total, 453 patients had severe pruritus with a visual analogue scale (VAS) score more than or equal to 7.0. Among them, more than 70% complained of sleep disturbance, whereas the majority of patients with a VAS score of less than 7.0 had no sleep disturbance. Male gender, high levels of blood urea nitrogen, beta2-microglobulin (beta2MG), hypercalcemia, and hyperphosphatemia were identified as independent risk factors for the development of severe pruritus, whereas a low level of calcium and intact-parathyroid hormone were associated with reduced risk. During the follow-up, 171 (9.64%) patients died. The prognosis of patients with severe pruritus was significantly worse than the others. Moreover, severe pruritus was independently associated with death even after adjusting for other clinical factors including diabetes mellitus, age, beta2MG, and albumin. Severe uremic pruritus caused by multiple factors, not only affects the quality of life but may also be associated with poor outcome in chronic hemodialysis patients. PMID- 16672925 TI - Finding function in novel targets: C. elegans as a model organism. AB - Despite its apparent simplicity, the nematode worm Caenorhabditis elegans has developed into an important model for biomedical research, particularly in the functional characterization of novel drug targets that have been identified using genomics technologies. The cellular complexity and the conservation of disease pathways between C. elegans and higher organisms, together with the simplicity and cost-effectiveness of cultivation, make for an effective in vivo model that is amenable to whole-organism high-throughput compound screens and large-scale target validation. This review describes how C. elegans models can be used to advance our understanding of the molecular mechanisms of drug action and disease pathogenesis. PMID- 16672926 TI - Therapeutic implications of the gender-specific aspects of cardiovascular disease. AB - The manifestations of cardiovascular diseases differ between men and women, as do outcomes after therapeutic interventions. It is important that those involved in drug discovery and development, as well as disease treatment, are aware of these differences because such variations are likely to have an increasing role in therapeutic decisions in the future. Here, I review gender differences in the most frequent cardiovascular diseases and their underlying sex-dependent molecular pathophysiology, and discuss gender-specific effects of current cardiovascular drugs and the implications for novel strategies for drug development. PMID- 16672928 TI - Theoretical biology: comparing models of species abundance. AB - Ecologists are struggling to explain how so many tropical tree species can coexist in tropical forests, and several empirical studies have demonstrated that negative density dependence is an important mechanism of tree-species coexistence. Volkov et al. compare a model incorporating negative density dependence with a dispersal-limited neutral model and claim that each predicts six empirical species-abundance distributions of tropical-tree communities equally well. However, we show here that their main conclusion is premature: when the two models are compared in an improved analysis, we find that the dispersal limited model outcompetes the density-dependent model in all six cases. Hence, although density dependence is certainly an important diversity-maintaining mechanism, our improved approach indicates that the dispersal-limited model provides a more parsimonious explanation of empirical species-abundance distributions. PMID- 16672932 TI - Painful transition at the NIH. PMID- 16672933 TI - Amateur night. PMID- 16672934 TI - Small first step. PMID- 16672937 TI - US posts sensitive climate report for public comment. PMID- 16672940 TI - Tempers flare at hurricane meeting. PMID- 16672939 TI - Will medics' qualms kill the death penalty? PMID- 16672941 TI - Chemists get out begging bowl to avert closure. PMID- 16672943 TI - Biomedical research: facing the opposition. PMID- 16672944 TI - It's a postdoc's life. PMID- 16672945 TI - The quiet rise of the clinical contractor. PMID- 16672948 TI - Colossus was the first electronic digital computer. PMID- 16672949 TI - A logical alternative for biological computing. PMID- 16672950 TI - Computer 'recycling' builds garbage dumps overseas. PMID- 16672957 TI - Particle Physics: the first axion? PMID- 16672951 TI - Computing: report leaps geographical barriers but stumbles over gender. PMID- 16672959 TI - Planetary Science: a new spin on Saturn. PMID- 16672958 TI - Cell biology: divining cancer cell weaknesses. PMID- 16672960 TI - Molecular biology: chromosome guardians on duty. PMID- 16672961 TI - Materials science: polymers show they're metal. PMID- 16672962 TI - Animal communication: complex call production in the tungara frog. AB - Animals' sound-producing organs often act as an integrated whole--particular vocal structure are not directly associated with the creation of discrete syllables. But here we show that the 'chuck' of the 'whine-chuck' mating call of the tungara frog, Physalaemus pustulosus, is caused by a fibrous mass attached to the vocal folds; the chuck is eliminated by removal of this structure, although the frog still tries to produce the sound. Sexual selection affects the acoustic complexity of the frog's call, so evolution may have shaped this unusual vocalization, which is akin to the two-voiced song of songbirds. PMID- 16672963 TI - The search for signs of recovery of the ozone layer. AB - Evidence of mid-latitude ozone depletion and proof that the Antarctic ozone hole was caused by humans spurred policy makers from the late 1980s onwards to ratify the Montreal Protocol and subsequent treaties, legislating for reduced production of ozone-depleting substances. The case of anthropogenic ozone loss has often been cited since as a success story of international agreements in the regulation of environmental pollution. Although recent data suggest that total column ozone abundances have at least not decreased over the past eight years for most of the world, it is still uncertain whether this improvement is actually attributable to the observed decline in the amount of ozone-depleting substances in the Earth's atmosphere. The high natural variability in ozone abundances, due in part to the solar cycle as well as changes in transport and temperature, could override the relatively small changes expected from the recent decrease in ozone-depleting substances. Whatever the benefits of the Montreal agreement, recovery of ozone is likely to occur in a different atmospheric environment, with changes expected in atmospheric transport, temperature and important trace gases. It is therefore unlikely that ozone will stabilize at levels observed before 1980, when a decline in ozone concentrations was first observed. PMID- 16672964 TI - A regular period for Saturn's magnetic field that may track its internal rotation. AB - The rotation rate of a planet is one of its fundamental properties. Saturn's rotation, however, is difficult to determine because there is no solid surface from which to time it, and the alternative 'clock'--the magnetic field--is nearly symmetrically aligned with the rotation axis. Radio emissions, thought to provide a proxy measure of the rotation of the magnetic field, have yielded estimates of the rotation period between 10 h 39 min 22 s and 10 h 45 min 45 s (refs 8-10). Because the period determined from radio measurements exhibits large time variations, even on timescales of months, it has been uncertain whether the radio emission periodicity coincides with the inner rotation rate of the planet. Here we report magnetic field measurements that revealed a time-stationary magnetic signal with a period of 10 h 47 min 6 s +/- 40 s. The signal appears to be stable in period, amplitude and phase over 14 months of observations, pointing to a close connection with the conductive region inside the planet, although its interpretation as the 'true' inner rotation period is still uncertain. PMID- 16672965 TI - Metallic transport in polyaniline. AB - Despite nearly three decades of materials development, the transport properties in the 'metallic state' of the so-called conducting polymers are still not typical of conventional metals. The hallmark of metallic resistivity--a monotonic decrease in resistivity with temperature--has not been obtained at temperatures over the full range below room temperature; and a frequency dependent conductivity, sigma(omega), typical of metals has also not been observed. In contrast, the low-temperature behaviour of 'metallic' polymers has, in all previous cases, exhibited an increase in resistivity as temperature is further decreased, as a result of disorder-induced localization of the charge carriers. This disorder-induced localization also changes the infrared response such that sigma(omega) deviates from the prediction of Drude theory. Here we report classic metallic transport data obtained from truly metallic polymers. With polyaniline samples prepared using self-stabilized dispersion polymerization, we find that for samples having room-temperature conductivities in excess of 1,000 S cm(-1), the resistivity decreases monotonically as the temperature is lowered down to 5 K, and that the infrared spectra are characteristic of the conventional Drude model even at the lowest frequencies measured. PMID- 16672966 TI - Towards molecular electronics with large-area molecular junctions. AB - Electronic transport through single molecules has been studied extensively by academic and industrial research groups. Discrete tunnel junctions, or molecular diodes, have been reported using scanning probes, break junctions, metallic crossbars and nanopores. For technological applications, molecular tunnel junctions must be reliable, stable and reproducible. The conductance per molecule, however, typically varies by many orders of magnitude. Self-assembled monolayers (SAMs) may offer a promising route to the fabrication of reliable devices, and charge transport through SAMs of alkanethiols within nanopores is well understood, with non-resonant tunnelling dominating the transport mechanism. Unfortunately, electrical shorts in SAMs are often formed upon vapour deposition of the top electrode, which limits the diameter of the nanopore diodes to about 45 nm. Here we demonstrate a method to manufacture molecular junctions with diameters up to 100 microm with high yields (> 95 per cent). The junctions show excellent stability and reproducibility, and the conductance per unit area is similar to that obtained for benchmark nanopore diodes. Our technique involves processing the molecular junctions in the holes of a lithographically patterned photoresist, and then inserting a conducting polymer interlayer between the SAM and the metal top electrode. This simple approach is potentially low-cost and could pave the way for practical molecular electronics. PMID- 16672967 TI - Weakening of tropical Pacific atmospheric circulation due to anthropogenic forcing. AB - Since the mid-nineteenth century the Earth's surface has warmed, and models indicate that human activities have caused part of the warming by altering the radiative balance of the atmosphere. Simple theories suggest that global warming will reduce the strength of the mean tropical atmospheric circulation. An important aspect of this tropical circulation is a large-scale zonal (east-west) overturning of air across the equatorial Pacific Ocean--driven by convection to the west and subsidence to the east--known as the Walker circulation. Here we explore changes in tropical Pacific circulation since the mid-nineteenth century using observations and a suite of global climate model experiments. Observed Indo Pacific sea level pressure reveals a weakening of the Walker circulation. The size of this trend is consistent with theoretical predictions, is accurately reproduced by climate model simulations and, within the climate models, is largely due to anthropogenic forcing. The climate model indicates that the weakened surface winds have altered the thermal structure and circulation of the tropical Pacific Ocean. These results support model projections of further weakening of tropical atmospheric circulation during the twenty-first century. PMID- 16672968 TI - A primitive fish provides key characters bearing on deep osteichthyan phylogeny. AB - Osteichthyans, or bony vertebrates, include actinopterygians (teleosts and their relatives) and sarcopterygians (coelacanths, lungfishes and tetrapods). Despite features found in basal actinopterygians (for example, Dialipina and Ligulalepis) and basal sarcopterygians (for example, Psarolepis and Achoania), the morphological gap between the two lineages remains wide and how sarcopterygians developed a dermal surface covering known as cosmine (composed of a pore-canal network and a single layer of odontodes and enamel) is still poorly known. Here we describe a primitive fossil fish, Meemannia eos gen. et sp. nov., that possesses an actinopterygian-like skull roof and a cosmine-like dermal surface combining a pore-canal network (found in various fossil sarcopterygians) with superimposed layers of odontodes and enamel (previously known in actinopterygians and some acanthodians). This 405-million-year-old fish from the Lower Devonian of Yunnan (China) demonstrates that cosmine in many fossil sarcopterygians arose step by step through the acquisition of a pore-canal network followed by the subsequently developed ability to resorb previous generations of odontodes and enamel. Meemannia provides key characters for studying deep osteichthyan phylogeny and indicates a possible morphotype for the common ancestor of actinopterygians and sarcopterygians. PMID- 16672969 TI - Climate change and population declines in a long-distance migratory bird. AB - Phenological responses to climate change differ across trophic levels, which may lead to birds failing to breed at the time of maximal food abundance. Here we investigate the population consequences of such mistiming in the migratory pied flycatcher, Ficedula hypoleuca. In a comparison of nine Dutch populations, we find that populations have declined by about 90% over the past two decades in areas where the food for provisioning nestlings peaks early in the season and the birds are currently mistimed. In areas with a late food peak, early-breeding birds still breed at the right time, and there is, at most, a weak population decline. If food phenology advances further, we also predict population declines in areas with a late food peak, as in these areas adjustment to an advanced food peak is insufficient. Mistiming as a result of climate change is probably a widespread phenomenon, and here we provide evidence that it can lead to population declines. PMID- 16672970 TI - Testing the genetics underlying the co-evolution of mate choice and ornament in the wild. AB - One of the most debated questions in evolutionary biology is whether female choice of males with exaggerated sexual displays can evolve as a correlated response to selection acting on genes coding for male attractiveness or high overall viability. To date, empirical studies have provided support for parts of this scenario, but evidence for all key genetic components in a natural population is lacking. Here we use animal-model quantitative genetic analysis on data from over 8,500 collared flycatchers (Ficedula albicollis) followed for 24 years to quantify all of the key genetic requirements of both fisherian and 'good genes' models on sexual selection in the wild. We found significant additive genetic variances of all the main components: male ornament (forehead patch size), female mate choice for this ornament, male fitness and female fitness. However, when the necessary genetic correlations between these components were taken into account, the estimated strength of indirect sexual selection on female mate choice was negligible. Our results show that the combined effect of environmental influences on several components reduces the potential for indirect sexual selection in the wild. This study provides insight into the field of sexual selection by showing that genes coding for mate choice for an ornament probably evolve by their own pathways instead of 'hitchhiking' with genes coding for the ornament. PMID- 16672971 TI - A small-molecule screen in C. elegans yields a new calcium channel antagonist. AB - Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4 dihydropyridines (DHPs) that antagonize the alpha1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel alpha1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets. PMID- 16672972 TI - Downstream nuclear events in brassinosteroid signalling. AB - Brassinosteroids (BRs) are steroid hormones that control many aspects of plant growth and development. BRs bind to the plasma membrane receptor kinase BRI1, and act through a signalling pathway that involves a glycogen synthase kinase-3-like kinase (BIN2) and a serine/threonine phosphatase (BSU1). Previous models proposed that BIN2 negatively regulates BR signalling by controlling the stability and subcellular localization of the related transcription factors BES1 and BZR1 by phosphorylation, in a manner reminiscent of the canonical Wnt signalling pathway of metazoans. Here we present strong evidence for a different mode of regulation of BR signalling. We show that BES1 is localized constitutively to the nucleus, where its activity is modulated by nuclear-localized BIN2 kinase. BIN2-mediated phosphorylation of BES1 inhibits its DNA-binding activity on BR-responsive target promoters and its transcriptional activity through impaired multimerization. Our observations demonstrate that phosphorylation-dependent inhibition of DNA binding and trans-activation is the key primary mechanism of BES1 regulation. PMID- 16672973 TI - The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. AB - The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family. These proteins are crucial for the control of microtubule length in cell division, neuronal development and interphase microtubule dynamics. The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme-substrate kinetics. To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family. Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK-microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 microm2 s(-1). Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a "reduction in dimensionality" that has been proposed as the search strategy by which DNA enzymes find specific binding sites. We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution. PMID- 16672979 TI - Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis. AB - During meiosis, cohesins--protein complexes that hold sister chromatids together- are lost from chromosomes in a step-wise manner. Loss of cohesins from chromosome arms is necessary for homologous chromosomes to segregate during meiosis I. Retention of cohesins around centromeres until meiosis II is required for the accurate segregation of sister chromatids. Here we show that phosphorylation of the cohesin subunit Rec8 contributes to step-wise cohesin removal. Our data further implicate two other key regulators of meiotic chromosome segregation, the cohesin protector Sgo1 and meiotic recombination in bringing about the step-wise loss of cohesins and thus the establishment of the meiotic chromosome segregation pattern. Understanding the interplay between these processes should provide insight into the events underlying meiotic chromosome mis-segregation, the leading cause of miscarriages and mental retardation in humans. PMID- 16672980 TI - Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. AB - Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson's disease characterized by motor disturbances and dopaminergic neurodegeneration. To address its underlying molecular pathogenesis, we generated and characterized loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINK1), a novel AR-JP-linked gene. Here, we show that PINK1 mutants exhibit indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINK1 mutants. Notably, we also found that PINK1 mutants share marked phenotypic similarities with parkin mutants. Transgenic expression of Parkin markedly ameliorated all PINK1 loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINK1. Taken together, our genetic evidence clearly establishes that Parkin and PINK1 act in a common pathway in maintaining mitochondrial integrity and function in both muscles and dopaminergic neurons. PMID- 16672981 TI - Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. AB - Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis. PMID- 16672982 TI - Phi c31 integrase induces chromosomal aberrations in primary human fibroblasts. AB - Phi c31 integrase is investigated as a novel tool for nonviral gene therapy as the enzyme can direct site-specific integration into a host chromosome. In order to investigate effects of phi c31 integrase expression in normal human cells, we have generated stably transfected primary human fibroblasts expressing the enzyme. All control cells were cytogenetically normal, but in cells expressing phi c31 integrase, numerous chromosomal abnormalities including various translocations were found, suggesting that the enzyme itself acts as a mutagen. PMID- 16672983 TI - Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia. AB - The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD Ia, including hypoglycemia, hyperlipidemia and growth failure. PMID- 16672984 TI - Combination of MIG (CXCL9) chemokine gene therapy with low-dose cisplatin improves therapeutic efficacy against murine carcinoma. AB - MIG (monokine induced by interferon-gamma) is a CXC chemokine ligand (CXCL9) that can potently inhibit angiogenesis, and displays thymus-dependent antitumor effects. The effectiveness of a treatment combining gene therapy with plasmid borne MIG (pORF-MIG) and low-dose cisplatin chemotherapy was determined using colon carcinoma (CT26) and Lewis lung carcinoma (LL/2c) murine models. The program was carried out via intramuscular delivery of pORF-MIG at 100 mug/mouse twice a week for 4 weeks, and/or intraperitoneal delivery of cisplatin at 0.6 mg/kg/mouse every 3 days for 48 days. Tumor volume and survival time were evaluated after treatment. CD31 immunohistochemical staining in tumor tissues and alginate capsule models in vivo was used to evaluate angiogenesis. Induction of apoptosis and cytotoxic T-lymphocyte (CTL) activity were also assessed. The combination of pORF-MIG and low-dose cisplatin produced significant antitumor activity, with complete tumor regression in 4/10 of CT26 colon carcinomas and 3/10 of LL/2c lung carcinomas, low vascularity, in alginate capsules, apparently degraded tumor microvessel density, and increased induction of apoptotic and CTL activities compared with either treatment alone. This study suggests that the combination of pORF-MIG plus cisplatin augments the inhibition of angiogenesis and the induction of apoptosis or CTL activity, all of which enhance antitumor activity. These findings may prove useful in further explorations of the application of combinatorial approaches to the treatment of solid tumors. PMID- 16672986 TI - Is it possible to survive pancreatic cancer? PMID- 16672987 TI - The tragedy of (unpublished) errors. PMID- 16673000 TI - Transanal excision or radical surgery--which is the best treatment for T1 rectal cancer? PMID- 16673001 TI - Is high-dose ursodeoxycholic acid effective for the treatment of patients with primary sclerosing cholangitis? PMID- 16673002 TI - Long-term efficacy of percutaneous thermal ablation for patients with recurrent hepatocellular carcinoma. PMID- 16673003 TI - What is the clinical impact of double-balloon enteroscopy? PMID- 16673004 TI - Success of colonoscopy in children. PMID- 16673005 TI - Therapy insight: drugs for gastrointestinal disorders in pregnant women. AB - The management and treatment of gastrointestinal ailments in pregnant women requires special attention and expertise, since the safety of the mother, fetus and neonate remains the primary focus. Nausea and vomiting during pregnancy is common, as is symptomatic gastroesophageal reflux disease. Peptic ulcer disease occurs less frequently and with fewer complications. Gastroenterologists and obstetricians should be familiar with safe treatment options for these conditions, because they can profoundly impair the quality of life of pregnant women. During pregnancy, constipation can develop de novo, or chronic constipation can increase in severity. Given the array of therapies for constipation, physicians must apprise themselves of drugs that are safe for both mother and fetus. Management of acute, self-limited diarrhea should focus on supportive therapy, dietary changes and maintenance of hydration. Treatment of chronic diarrhea should be considered in the context of therapy for the underlying disorder. Inflammatory bowel disease and irritable bowel syndrome present a unique therapeutic challenge--to control the disease while minimizing toxicity to the fetus and mother. Initiation and alteration of medical therapy for gastrointestinal disorders during pregnancy must be undertaken after discussion with the patient's obstetrician. PMID- 16673006 TI - Mechanisms of disease: from stem cells to colorectal cancer. AB - Over the past decade, the advances in our understanding of stem cell biology and the role of stem cells in diseases, such as colorectal cancer, have been remarkable. In particular, discoveries related to the control of stem cell proliferation and how dysregulation of proliferation leads to oncogenesis have been foremost. For intestinal stem cells, the WNT family of growth factors, and events such as the regulation of the nuclear localization of beta-catenin, seem to be central to normal homeostasis, and mutations in the components of these pathways seem to lead to the development of colorectal cancer. A paradigm of abnormal stem cell biology is illustrated by patients with familial adenomatous polyposis, who have mutations in the adenomatous polyposis coli gene. The wild type protein encoded by this gene is important for the prevention of mass beta catenin accumulation in the nucleus and the subsequent overtranscription of cell cycle proteins. This review discusses the basic mechanisms behind stem cell regulation in the gut and follows their role in the natural history of tumor progression. PMID- 16673007 TI - Mechanisms of disease: the hygiene hypothesis revisited. AB - In industrialized countries the incidence of diseases caused by immune dysregulation has risen. Epidemiologic studies initially suggested this was connected to a reduction in the incidence of infectious diseases; however, an association with defects in immunoregulation is now being recognized. Effector T(H)1 and T(H)2 cells are controlled by specialized subsets of regulatory T cells. Some pathogens can induce regulatory cells to evade immune elimination, but regulatory pathways are homeostatic and mainly triggered by harmless microorganisms. Helminths, saprophytic mycobacteria, bifidobacteria and lactobacilli, which induce immunoregulatory mechanisms in the host, ameliorate aberrant immune responses in the setting of allergy and inflammatory bowel disease. These organisms cause little, if any, harm, and have been part of human microecology for millennia; however, they are now less frequent or even absent in the human environment of westernized societies. Deficient exposure to these 'old friends' might explain the increase in immunodysregulatory disorders. The use of probiotics, prebiotics, helminths or microbe-derived immunoregulatory vaccines might, therefore, become a valuable approach to disease prevention. PMID- 16673008 TI - Eosinophilic esophagitis: an overlooked entity in chronic dysphagia. AB - BACKGROUND: A 40-year-old white male with atopy presented to our department in March 2004 with a history of chronic heartburn and solid-food dysphagia since 1994. The patient was taking on-demand salbutamol for asthma and ranitidine for mild heartburn, occurring less than once per week. Eight years previously, he had undergone esophageal dilatation for a Schatzki's ring. INVESTIGATIONS: Physical examination, laboratory investigations, video esophagram, upper endoscopy with mid-esophageal biopsies, and skin testing for a number of food and environmental allergens. Diagnosis Eosinophilic esophagitis. MANAGEMENT: Topical steroids with a fluticasone 220 microg multiple-dose inhaler, four puffs swallowed twice a day for 6 weeks. PMID- 16673009 TI - Combined oral and topical mesalazine treatment for extensive ulcerative colitis. AB - BACKGROUND: A 32-year-old white Jewish woman was referred to our clinic in October 2002 for a second opinion on the management of her active extensive ulcerative colitis that did not respond to oral mesalazine (mesalamine) 3.6 g/day. The severity of symptoms had affected her plans to have children. INVESTIGATIONS: Laboratory investigations, including perinuclear antineutrophil cytoplasmic antibodies and antibodies to Saccharomyces cerevisiae, stool cultures, and sigmoidoscopy to 40 cm from the rectum, with biopsies. DIAGNOSIS: Moderate-to-severe active extensive ulcerative colitis, unresponsive to mesalazine at 3.6 g/day. MANAGEMENT: Oral mesalazine 4.8 g/day and 4 g mesalazine enemas nightly. PMID- 16673010 TI - Beta-receptor blockers in primary prevention for cardiovascular disease: forgotten benefits? PMID- 16673011 TI - Blood pressure response to calcium supplementation: a meta-analysis of randomized controlled trials. AB - Calcium plays a role in blood pressure (BP) regulation, but the importance of supplemental calcium intake for the prevention of hypertension is still debated. We conducted a meta-analysis of randomized controlled trials to determine the effect of calcium supplementation on BP. A systematic search for randomized trials of calcium supplementation and BP in non-pregnant subjects was performed in Medline from 1966 to June 2003. Seventy-one trials were identified, 40 of which met the criteria for meta-analysis (total of 2492 subjects). Two persons independently extracted data from original publications on changes in calcium intake and BP. In addition, data were collected on subjects' characteristics, that is, age, gender, initial BP and initial calcium intake. A random effects model was used to obtain the effect of calcium supplementation on BP, overall and in predefined population subgroups. Calcium supplementation (mean daily dose: 1200 mg) reduced systolic BP by -1.86 mm Hg (95% confidence interval: -2.91 to 0.81) and diastolic BP by -0.99 mm Hg (-1.61 to -0.37). In people with a relatively low calcium intake (< or =800 mg per day) somewhat larger BP estimates were obtained, that is, -2.63 (-4.03 to -1.24) for systolic BP and -1.30 (-2.13 to -0.47) for diastolic BP. Our study suggests that an adequate intake of calcium should be recommended for the prevention of hypertension. More research on BP in people with calcium-deficient diets is warranted. PMID- 16673012 TI - Fatality of acute coronary events in relation to hypertension and low-grade inflammation: a population-based cohort study. AB - Hypertension has been associated with increased case-fatality rates among individuals who subsequently suffer from acute coronary events. It is unknown whether inflammation modifies this relationship. This population-based study explored the effects of inflammation and hypertension on incidence of coronary event, and on the fatality of the future events. Blood pressure (BP) and five inflammation-sensitive plasma proteins (ISPs, fibrinogen, orosomucoid, alpha 1 antitrypsin, haptoglobin and ceruloplasmin) were determined in 6071 healthy men. During the mean follow-up of 19 years, 679 men had a first coronary event (non fatal myocardial infarction or death from coronary heart disease). Of them, 197 (29%) were fatal cases (death during the first day). As expected, hypertension was associated with increased incidence of coronary events and increased proportion of fatal cases. At all levels of BP, high ISPs (> or =2 ISPs in top quartile) significantly added to the incidence of events. Men with high ISPs had the highest case-fatality rates. The difference in case-fatality rate between men with and without high ISPs was, however, significant only in men with normal BP (<130/85 mm Hg) (33 vs 19%, P < 0.05), and not in men with moderate or severe hypertension (> or =160/100 mm Hg) (40 vs 35%, P = 0.32). High ISPs add to the incidence of coronary events at all levels of BP. Hypertension and inflammation are both independently associated with increased case-fatality in subjects who later have an acute coronary event. The influence of ISPs on the case-fatality rate seems to be most important in men with normal BP. PMID- 16673013 TI - Statins and hypertension. PMID- 16673015 TI - Aristotle: the first student of angiogenesis. PMID- 16673014 TI - Aggressive antihypertensive strategies based on hydrochlorothiazide, candesartan or lisinopril decrease left ventricular mass and improve arterial compliance in patients with type II diabetes mellitus and hypertension. AB - We investigated the effects of aggressive antihypertensive therapy based on hydrochlorothiazide, candesartan or lisinopril on left ventricular mass (LVM) index and arterial stiffness in hypertensive type II diabetic individuals. Seventy hypertensive type II diabetic individuals were treated with three antihypertensive strategies in a randomized, double-blind, double-dummy design. Blood pressure was titrated to levels below 130/85 mm Hg or a decrease in systolic pressure of 10% with a diastolic pressure below 85 mm Hg. After titration, patients were treated for 12 months. Mean blood pressures were 157/93, 151/94 and 149/93 mm Hg at baseline in the hydrochlorothiazide (n = 24), candesartan (n = 24) and lisinopril (n = 22) groups, and 135/80, 135/82 and 131/80 mm Hg after titration. About 70% reached target blood pressures, with the median use of three antihypertensive drugs. Left ventricular mass index and all estimates of arterial stiffness showed significant improvement after 12 months: that is, LVM index (-11 g/m(2); -8%); carotid distensibility coefficient (DC; +2.8 x 10(-3) kPa(-1); +27%), compliance coefficient (CC; +0.13 mm2/kPa; +21%) and elastic modulus (-0.19 kPa; -16%); femoral DC (+1.6 x 10(-3) kPa(-1); +50%) and CC (+0.08 mm2/kPa; +26%); brachial DC (+2.1 x 10(-3) kPa(-1); +39%) and CC (+0.03 mm2/kPa; +27%) and total systemic arterial compliance (+0.29 ml/mm Hg; +16%). No differences in outcome variables between treatment groups were observed. Aggressive antihypertensive treatment, although difficult to achieve, resulted in substantial reductions of LVM index and arterial stiffness in relatively uncomplicated hypertensive type II diabetic individuals. Strategies based on renin-angiotensin system inhibitors were not clearly superior to conventional (i.e. diuretic-based) strategies. PMID- 16673016 TI - MTB, the murine homolog of condensin II subunit CAP-G2, represses transcription and promotes erythroid cell differentiation. AB - Chromosome condensation is essential for proper segregation of duplicated sister chromatids in mitosis. Mammalian erythroid maturation is also associated with gradual nuclear condensation. However, few proteins that are directly involved in chromosome condensation during erythropoiesis have been identified. In this report, we show that MTB (more than blood), which was initially isolated in a yeast two-hybrid screen for proteins that interact with the basic helix-loop helix (bHLH) protein stem cell leukemia (SCL), and later identified as the murine homolog of the condensin II subunit CAP-G2, participates in erythroid cell development. MTB interacts with SCL and another hematopoietic bHLH protein, E12, and is recruited to the nucleus by SCL and E12. In addition, MTB can repress SCL/E12-mediated transcriptional activation. Consistent with the model that MTB may function together with SCL/E12 heterodimer during erythroid cell development, MTB is highly expressed in the erythroid lineage and is upregulated upon erythroid differentiation. Moreover, overexpression of MTB promotes the terminal differentiation of the murine erythroleukemia erythroid cell line. Together, these findings demonstrate that the condensin II subunit MTB/mCAP-G2 plays a novel function during erythropoiesis and suggest that key hematopoietic transcription factors such as SCL and E12 may regulate the terminal differentiation of hematopoietic cells through the interaction with condensin complexes. PMID- 16673017 TI - Human telomerase reverse transcriptase protects hematopoietic progenitor TF-1 cells from death and quiescence induced by cytokine withdrawal. AB - Telomerase is a complex ribonucleoprotein enzyme that exhibits elevated activity in the majority of cases of human leukemia. We have previously shown that retroviral expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), in human cord blood CD34+ cells leads to an enhanced survival of mature hematopoietic cells. The mechanism for this pro survival effect is not known. Here, we show that telomerase may play a role in leukemogenesis as a survival factor, independent of its role in maintaining telomere length. Retroviral expression of hTERT in the cytokine-dependent, human hematopoietic progenitor cell line, TF-1, resulted in the survival of cells following the withdrawal of cytokine, with protection from apoptosis, but did not promote unlimited replicative potential. This hTERT-mediated effect on cell survival does not involve Bcl-2 family members, results in accumulation of cells in G1 and appears to operate via autocrine expression of IL-3 and activation of the p53/p21 pathway. Survival in the absence of cytokine stimulation was also observed following retroviral expression of hTERT in normal cord blood CD34+ cells. This study demonstrates a novel pro-survival role for hTERT and may have important implications for the role of hTERT in the pathogenesis of leukemia and drug resistance. PMID- 16673018 TI - A novel TEL-AML1 fusion transcript involving the pro-apoptotic gene BCL-G in pediatric precursor B acute lymphoblastic leukemia. PMID- 16673019 TI - A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia. AB - Over the last decade, genetic characterization of T-cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities. In this study, we used array-comparative genome hybridization (array-CGH) and identified a novel recurrent 9q34 amplification in 33% (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed among patients, but the critical region encloses approximately 4 Mb and includes NOTCH1. The 9q34 amplification may lead to elevated expression of various genes, and MRLP41, SSNA1 and PHPT1 were found significantly expressed at higher levels. Fluorescence in situ hybridization (FISH) analysis revealed that this 9q34 amplification was in fact a 9q34 duplication on one chromosome and could be identified in 17-39 percent of leukemic cells at diagnosis. Although this leukemic subclone did not predict for poor outcome, leukemic cells carrying this duplication were still present at relapse, indicating that these cells survived chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our patient cohort. We showed that both of these genetic abnormalities occur independently from this newly identified 9q34 duplication. PMID- 16673020 TI - Genetic rearrangement of FOXP1 is predominantly detected in a subset of diffuse large B-cell lymphomas with extranodal presentation. PMID- 16673021 TI - Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCRbeta locus rearrangements and putative new T-cell oncogenes. AB - Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRalphadelta (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRalphadelta (14q11), TCRbeta (7q34) and TCRgamma (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRbeta locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRalphadelta locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRalphadelta/TCRbeta chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRbeta and TCRalphadelta loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRbeta locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis. PMID- 16673022 TI - Presence of JAK2 V617F tyrosine kinase mutation as a myeloid-lineage-specific mutation in chronic neutrophilic leukaemia. PMID- 16673024 TI - Modular corneal surgery. PMID- 16673025 TI - Patient pathways for macular disease: what will the new optometrist with special interest achieve? PMID- 16673028 TI - A niobaziridine hydride system for white phosphorus or dinitrogen activation and N- or P-atom transfer. AB - This short review describes a breakthrough embodied by the synthesis of a niobaziridine hydride complex. This reactive entity reacts directly with white phosphorus to provide a bridging diphosphorus diniobium complex that upon reduction splits to afford a terminal niobium phosphide anion, isolated as its sodium salt. Reactions of the latter with acid chlorides constitute a new synthesis of phosphaalkynes, while treatment with chlorodiorganophosphanes leads to complexed 1,1-diorganophosphanylphosphinidene systems. Additionally, reactions of the sodium salt of the niobium phosphide anion with divalent main group element salts (E = Ge, Sn, or Pb) provide complexed triatomic EP2 triangles. Dinitrogen cleavage was realized via reduction of a heterodinuclear niobium/molybdenum dinitrogen complex, and this provided an entry to a nitrogen 15 labeled terminal nitride anion of niobium as its sodium salt. In a fashion analogous to the aforementioned phosphaalkyne synthesis, acid chlorides are transformed upon reaction with the niobium nitride anion into corresponding nitrogen-15 labeled organic nitriles. Complete synthetic cycles are achieved in both the phosphaalkyne and the organic nitrile syntheses, as the oxoniobium(v) byproduct can be recycled in high yield to the title niobaziridine hydride complex. PMID- 16673029 TI - A trinuclear complex containing MnII MnIII MnIV, radicals, quinone and chloride ligands potentially relevant to PS II. AB - Reaction of MnCl2 with a non-innocent ligand H3L results in an unprecedented mixed-valence trinuclear complex [MnII MnIII MnIV (L)(L*)2(L(IQ))Cl] whose structural and magnetic properties are described. PMID- 16673030 TI - A cross-coupling strategy for the synthesis of dimetallic assemblies containing mixed bipyridine-terpyridine bridging ligands: luminescence and energy transfer properties. AB - Tris-bidentate complexes of the form [Ir(N[symbol: see text]C)2(N[symbol: see text]N)]+ and [Ru(N[symbol: see text]N)3]2+ incorporating a boronic acid substituent (N[symbol: see text]C=2-phenylpyridyl, N[symbol: see text]N=a 2,2' bipyridyl ligand), are cross-coupled with bromo-substituted bis-terpyridyl ruthenium and iridium complexes to generate heterometallic assemblies comprising bpy-phi(n)-tpy bridges; they display efficient energy transfer from Ir(III) to Ru(II). PMID- 16673031 TI - Preparation and structures of 1,2-dihydro-1,2-diphosphaacenaphthylenes and rigid backbone stabilized triphosphenium cation. AB - The effect of the special peri-geometry of rigid naphthalene-1,8-diyl backbone in phosphenium formation reaction was investigated. 1,8-Bis(diphenylphosphino) naphthalene and P2I4 afforded triphosphenium iodide in a clean reaction. The reaction of 1,8-bis(dimethylaminophosphino) naphthalene with P2I4 is complex, it afforded four products, all containing the 1,2-dihydro-1,2 diphosphaacenaphthylene motif and heterophosphonium functionalities. Two examples of the rare structural motif of two acenaphthylene units connected head to head and thus a contiguous chain of four phosphorus atoms were also isolated, one compound showing diastereomerization in the solution. All new compounds were fully characterised including single crystal X-ray diffraction. PMID- 16673032 TI - Influence of anions on the dimensionality of extended networks based on Cu I cations and 1,4,5,8,9,12-hexaazatriphenylene (HAT) ligands. AB - Reactions of Cu I salts with 1,4,5,8,9,12-hexaazatriphenylene (HAT) afford three types of cationic coordination polymers depending on the anion present in the reaction solution. In the crystal structure of {[Cu(HAT)][BF4]x1/3(C6H6)}infinity, (1), Cu ions and HAT molecules form extended layers that are best described as strongly distorted honeycomb nets. The space between the layers is occupied by [BF4]- anions and solvent molecules. {[Cu(HAT)][PF6]}infinity, (2), crystallizes as a chiral (10,3)-a net with [PF6]- anions residing in the cavities of the three-dimensional metal-organic framework. The crystal structure of {[Cu4(HAT)3][SbF6]4x3C6H6}infinity, (3), is based on unique extended [Cu4(HAT)3]infinity "nanotubules" filled with solvent molecules and [SbF6]- anions. PMID- 16673033 TI - Dynamics of an [Fe4S4(SPh)4]2- cluster explored via IR, Raman, and nuclear resonance vibrational spectroscopy (NRVS)-analysis using 36S substitution, DFT calculations, and empirical force fields. AB - We have used four vibrational spectroscopies--FT-IR, FT-Raman, resonance Raman, and 57Fe nuclear resonance vibrational spectroscopy (NRVS)--to study the normal modes of the Fe-S cluster in [(n-Bu)4N]2[Fe4S4(SPh)4]. This [Fe4S4(SR)4]2- complex serves as a model for the clusters in 4Fe ferredoxins and high-potential iron proteins (HiPIPs). The IR spectra exhibited differences above and below the 243 K phase transition. Significant shifts with 36S substitution into the bridging S positions were also observed. The NRVS results were in good agreement with the low temperature data from the conventional spectroscopies. The NRVS spectra were interpreted by normal mode analysis using optimized Urey-Bradley force fields (UBFF) as well as from DFT theory. For the UBFF calculations, the parameters were refined by comparing calculated and observed NRVS frequencies and intensities. The frequency shifts after 36S substitution were used as an additional constraint. A D 2d symmetry Fe4S4S'4 model could explain most of the observed frequencies, but a better match to the observed intensities was obtained when the ligand aromatic rings were included for a D 2d Fe4S4(SPh)4 model. The best results were obtained using the low temperature structure without symmetry constraints. In addition to stretching and bending vibrations, low frequency modes between approximately 50 and 100 cm(-1) were observed. These modes, which have not been seen before, are interpreted as twisting motions with opposing sides of the cube rotating in opposite directions. In contrast with a recent paper on a related Fe4S4 cluster, we find no need to assign a large fraction of the low frequency NRVS intensity to 'rotational lattice modes'. We also reassign the 430 cm(-1) band as primarily an elongation of the thiophenolate ring, with approximately 10% terminal Fe-S stretch character. This study illustrates the benefits of combining NRVS with conventional Raman and IR analysis for characterization of Fe-S centers. DFT theory is shown to provide remarkable agreement with the experimental NRVS data. These results provide a reference point for the analysis of more complex Fe-S clusters in proteins. PMID- 16673034 TI - Synthesis of diazahexathia-24-crown-8 derivatives and structures of Ag+ complexes. AB - Diazahexathia-24-crown-8 (2) has been isolated from the reaction mixture during the preparation of monoazatrithia-12-crown-4 (1). When N-Boc protected bis(2 chloroethyl)amine was employed as a starting material, N-Boc protected monoazatrithia-12-crown-4 (3) and N-Boc protected diazahexathia-24-crown-4 (4) were separated easily. Double-armed diazahexathia-24-crown-8 having two 3',5' dichlorobenzyl groups (5a) or two 2-phenylethyl groups (5b) were also prepared using reductive amination. The stoichiometry and detailed structures of the Ag+ complexes with and were investigated by 1H NMR titration experiments and X-ray crystallography. PMID- 16673035 TI - Highly conjugated multiporphyrins: synthesis, spectroscopic and electrochemical properties. AB - A series of meso-to-meso ethynyl-bridged multiporphyrin arrays have been synthesized using Sonogoshira palladium-catalyzed cross-coupling reactions involving the appropriate ethynylporphyrin and iodoporphyrin precursors. The absorption spectra of these multiporphyrins show splitting of the Soret bands and significant red shifts of the Q bands as compared to the combination of the corresponding components. These conjugated multiporphyrins also show red shifts in their emission spectra as the pi-conjugation is expanded. In the electrochemical measurements, the porphyrins dimer 7 shows two 1 - e- oxidations at E(1/2) = +0.63 and +0.76 V for the first electron abstraction from the two porphyrin rings, indicating electronic communication between the two porphyrin units. The porphyrin trimer 4 exhibits the first and second 1 - e- oxidations at E(1/2) = +0.68 and +0.77 V, respectively, which correspond to the two outer porphyrins. The cyclic voltammogram of pentamer 5 shows two overlapping 1 - e- couples at E(1/2) = +0.56 and +0.66 V, and one 2 - e- couple at E(1/2) = +0.86 V, for the four outer porphyrin units. These results demonstrate that in the porphyrin trimer and pentamer the individual peripheral porphyrin units are electrochemically coupled via a central porphyrin core. The UV-Vis-NIR spectra of the oxidized species of these multiporphyrins exhibit a broad intervalence charge transfer (IVCT) band in the region from 1200 to 3000 nm. The present work shows that a central porphyrin unit appended with ethynyl bridges affords strong electronic interactions between the peripheral porphyrin rings over a distance of about 15 A. PMID- 16673036 TI - P-Heterocyclic carbenes as potential ligands in the design of new metathesis catalysts. A computational study. AB - Density functional calculations are reported concerning the olefin metathesis characteristics of a variety of P-heterocyclic carbene (PHC) complexes. The calculations employ model catalysts of the type (PMe3)(PHC)Cl2Ru=CH2, the PHC ligands being 1,3-dihydro-1,3-diphosphol-2-ylidene PH, 1,3-diphenyl-1,3 diphosphol-2-ylidene PPH, and 1,4-dihydro-1,4-diphosphol-2-azol-5-ylidene PNH. Complexes with N-heterocyclic carbenes (NHC) are included for comparison. Associative and dissociative reaction pathways are considered, the latter ones representing the favored reaction mechanisms. Calculations show that the rate determining step is ring opening of a ruthena-cyclobutane intermediate. In comparison with NHC model catalysts, the PHC compounds have lower phosphine dissociation energies, and also form weaker pi-complexes with an olefinic substrate. Compared to the initially formed pi-complexes, the ruthena-cyclobutane is more stable for PHC- than for NHC-catalysts. The catalytic activity of model PHC-compounds in comparison with NHC-compounds is discussed on the basis of the calculated reaction profiles. In this context, different models for enhanced reactivity of NHC-based catalysts that have been proposed in the literature are considered as well. It is demonstrated that the nature of the substituent of the carbene phosphorus not only exhibits a steric influence on the course of the reaction, but a significant stereoelectronic effect as well. Further, agostic interactions in ruthena-cyclobutane intermediates are investigated. PMID- 16673037 TI - Derivatives of dipyrido[3,2-a:2',3'-c]phenazine and its ruthenium complexes, influence of arylic substitution on photophysical properties. AB - The synthesis and photophysical properties of a series of substituted dipyridophenazine (dppz) ruthenium complexes of the type [(tbbpy)2Ru(dppz-R2)]2+ (where tbbpy = 4,4-tert-butyl-2,2-bipyridine and dppz = dipyrido[3,2-a:2',3' c]phenazine and R represents substitution at the 11 and 12 position with: Br, phenyl, 4-tert-butyl-phenyl and para-biphenyl) are described. The ligands could be obtained in high yields using Suzuki-type coupling reactions, an approach which also has been successfully applied to the analogous dppz-Br2 ruthenium complex. All compounds are fully characterised by NMR, MS and UV-vis spectroscopy. The solid state structures of dppz-bi-para-biphenyl and the ruthenium complex [(tbbpy)2Ru(dppz-Br2)]2+ are also reported. The investigation of the free ligands reveals a pronounced effect of the arylic substitution on absorption and emission properties. These properties are mirrored in the corresponding complexes, which possess emission lifetimes of up to 900 ns. The resonance Raman investigation of the complex [(tbbpy)2Ru(dppz-Br2)]2+ supports the assumption that the excited state properties of the substituted complexes are related to the parent [(bpy)2Ru(dppz)]2+ compound, but that important differences may be expected based on the differences observed in the lowest energy absorption band. PMID- 16673038 TI - Synthesis, structure and dioxygen reactivity of a bis(micro-iodo)dicopper(I) complex supported by the [N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N,N-di-(2 pyridylmethyl)]amine ligand. AB - The air-sensitive bis(micro-iodo)dicopper(I) complex 1 supported by [N-(3,5-di tert-butyl-2-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine (L) has been prepared by treating copper(I) iodide with L in anhydrous THF. Compound 1 crystallizes as a dimer in space group C2/c. Each copper(I) center has distorted tetrahedral N2I2 coordination geometry with Cu-N(pyridyl) distances 2.061(3) and 2.063(3) A, Cu-I distances 2.6162(5) and 2.7817(5) and a Cu...Cu distance of 2.9086(8) A. Complex 1 is rapidly oxidized by dioxygen in CH2Cl2 with a 1 : 1 stoichiometry giving the bis(micro-iodo)peroxodicopper(II) complex [Cu(L)(micro-I)]2O2 (2). The reaction of 1 with dioxygen has been characterized by UV-vis, mass spectrometry, EPR and Cu K-edge X-ray absorption spectroscopy at low temperature (193 K) and above. The mass spectrometry and low temperature EPR measurements suggested an equilibrium between the bis(micro-iodo)peroxodicopper(II) complex 2 and its dimer, namely, the tetranuclear (peroxodicopper(II))2 complex [Cu(L)(micro-I)]4O4 (2'). Complex 2 undergoes an effective oxo-transfer reaction converting PPh3 into O=PPh3 under anaerobic conditions. At sufficiently high concentration of PPh3, the oxygen atom transfer from 2 to PPh3 was followed by the formation of [Cu(PPh3)3I]. The dioxygen reactivity of 1 was compared with that known for other halo(amine)copper(I) dimers. PMID- 16673039 TI - A convenient route to silyl linked bis(amidinate) ligands and synthesis of group(I) metal derivatives. AB - A novel type of linked bis(amidinate) ligands (D) were developed successfully. Their lithium derivatives 1-4 were synthesized by treating the silyl-bridged diamines I-IV with two equivalents of LiBu(n) and PhCN in sequence, which underwent a silyl-bridge migration process. In addition, the linked bis(amidinate) configuration proved to be the thermodynamically stable form rather than the mono(amidinate) type by contrasting reaction of I with one equivalent of LiBu(n) and PhCN in sequence. PMID- 16673040 TI - Structure and reactivity of new phosphine ligands containing the hemi-labile sulfone moiety. AB - New heterofunctional phosphine ligands have been synthesised, incorporating the substitutionally labile sulfone and sulfonamide moieties as chelating groups, which display activity in the palladium-catalysed Suzuki and amination cross coupling reactions. Single-crystal X-ray diffraction studies of the complexes formed with [Pd(mu-Cl)(dmba)] (dmba-H = N,N-dimethylbenzylamine) highlight the coordinating nature of these ligands; showing the formation of a bis chelate complex through a six-membered Pd-P-C-C-S-O ring with the sulfonamide class of ligands. PMID- 16673041 TI - The Postpartum Bonding Questionnaire: a validation. AB - This is a validation of a self-rating questionnaire designed to detect disorders of the mother-infant relationship. 125 subjects filled in the questionnaire, and were also interviewed using the 5(th) Edition of the Birmingham Interview for Maternal Mental Health. On the basis of these interviews and the case records, we made consensus diagnoses of various forms and degrees of mother infant relationship disorder, according to criteria published in this paper. We calculated specificity, sensitivity and positive predictive value of the four scale scores generated by the questionnaire. Scale 1 (a general factor) had a sensitivity of 0.82 for all mother-infant relationship disorders. Scale 2 (rejection and pathological anger) had a sensitivity of 0.88 for rejection of the infant, but only 0.67 for severe anger. The performance of scale 3 (infant focused anxiety) was unsatisfactory. Scale 4 (incipient abuse) selected only a few mothers, but was of some value in identifying those at high risk of child abuse. Revision of the thresholds can improve sensitivity, especially of scale 2, where a cut-off point of 12 = normal, 13 = high better identifies mothers with threatened rejection. These new cut-off points would need validation in another sample. PMID- 16673042 TI - Identifying the behavioural phenotype in Fetal Alcohol Spectrum Disorder: sensitivity, specificity and screening potential. AB - BACKGROUND: In most cases of Fetal Alcohol Spectrum Disorder (FASD), the pathognomonic facial features are absent making diagnosis challenging, if not impossible, particularly when no history of maternal drinking is available. Also because FASD is often comorbid with Attention Deficit Hyperactivity Disorder (ADHD), children with FASD are frequently improperly diagnosed and receive the wrong treatment. Since access to psychological testing is typically limited or non-existent in remote areas, other diagnostic methods are needed to provide necessary interventions. OBJECTIVES: To determine if a characteristic behavioural phenotype distinguishes children with FASD from typically developing children and children with ADHD and use this information to create a screening tool for FASD diagnosis. METHODS: Parents and caregivers completed the Child Behavior Checklist (CBCL), a well-established standardized tool for evaluating children's behavioural problems. Results from 30 children with Fetal Alcohol Syndrome or Alcohol-Related Neurodevelopmental Disability, 30 children with ADHD, and 30 typically developing healthy children matched for age and socioeconomic status with FASD were analyzed. Based on our previous work, 12 CBCL items that significantly differentiated FASD and control groups were selected for further analyses. Stepwise discriminant function analysis identified behavioural characteristics most strongly differentiating groups and Receiver Operating Characteristics (ROC) curve analyses determined sensitivity and specificity of different item combinations. RESULTS: Seven items reflecting hyperactivity, inattention, lying and cheating, lack of guilt, and disobedience significantly differentiated children with FASD from controls. ROC analyses showed scores of 6 or higher on these items differentiated groups with a sensitivity of 86%, specificity of 82%. For FASD and ADHD, two combinations of items significantly differentiated groups with high sensitivity and specificity (i) no guilt, cruelty, and acts young (sensitivity = 70%; specificity = 80% (ii) acts young, cruelty, no guilt, lying or cheating, steals from home, and steals outside (sensitivity = 81%; specificity = 72%). These items were used to construct a potential FASD screening tool. CONCLUSIONS: Our findings identifying the behavioural characteristics differentiating children with FASD from typically developing children or children with ADHD have the potential for development of an empirically derived tool for FASD tool to be used in remote areas where psychological services are not readily available. This technique may speed up diagnosis and intervention for children without ready access to formal assessments. PMID- 16673043 TI - The activation of lytic replication of Epstein-Barr virus by baculovirus-mediated gene transduction. AB - A baculoviral mammalian-cell vector was constructed to express Rta, a protein of Epstein-Barr virus (EBV) responsible for the transition from latent infection to lytic replication. EBV lytic replication and cell-growth inhibition was observed in infected D98/HR1 cells. The baculovirus caused little cytotoxicity in the non targeted HeLa cells, compared to an adenovirus vector. It is concluded that recombinant baculovirus might have the potential as a vector for the therapy of EBV-related cancer. PMID- 16673044 TI - Cytochrome P4503A: evidence for mRNA expression and catalytic activity in rat brain. AB - Studies initiated to investigate the presence of cytochrome P4503A (CYP3A) isoenzymes in brain revealed constitutive mRNA and protein expression of CYP3A1 in rat brain. Western blotting studies showed that pretreatment with CYP3A inducer such as pregnenolone-16alpha -carbonitrile (PCN) significantly increased the cross reactivity comigrating with hepatic CYP3A1 and CYP3A2 in rat brain microsomes. RT-PCR studies have also shown increase in mRNA expression of CYP3A1 following pretreatment of rats with PCN. The ability of rat brain microsomes to catalyze the demethylation of erythromycin, known to be mediated by CYP3A isoenzymes in liver and significant increase in the activity of erythromycin demethylase (EMD) following pretreatment with dexamethasone or PCN have indicated that CYP3A isoenzymes expressed in brain are functionally active. Kinetic studies revealed that increase in the enzyme activity following pretreatment with PCN resulted in increase in the apparent affinity (Km) and Vmax of the reaction. Similarities in the inhibition of the constitutive and inducible brain and liver EMD activity following in vitro addition of ketoconazole, a inhibitor specific for CYP3A catalysed reactions and anti-CYP3A have further indicated that like in liver, CYP3A isoenzymes catalyse the activity of EMD in rat brain. Data also revealed regional differences in the activity of EMD in the brain. Relatively higher constitutive as well as inducible mRNA expression of CYP3A1 in hypothalamus and hippocampus, the brain regions responsive to steroid hormones have suggested that CYP3A isoenzymes may not only be involved in the process of detoxication mechanism but also in the metabolism of endogenous substrates in brain. PMID- 16673050 TI - Urethrovaginal fistula--a rare complication after the placement of a suburethral sling (IVS). AB - A sixty-year-old woman with stress urinary incontinence had undergone a placement of an IVS (Tyco Health Care UK) in another hospital in February 2003. Seventeen months after the procedure, she complained about a suppurative discharge from the vagina and a recurrent severe stress urinary incontinence. The gynecological examination revealed an erosion of the sling into the vagina and a large urethrovaginal fistula bordered by granuloma. After removal of the sling, the urethrovaginal fistula was closed using a vaginal flap. A subsequent conservative treatment regime with duloxetine and pelvic floor training improved the stress urinary incontinence to the patient's satisfaction. PMID- 16673051 TI - Is there a long-lasting effect of pelvic floor muscle training in women with urinary incontinence after ischemic stroke? A 6-month follow-up study. AB - The aim of this study was to evaluate the long-lasting effect of pelvic floor muscle training (PFMT) in women with urinary incontinence after stroke measured by quality of life parameters. Twenty-four (24/24) women with urinary incontinence after stroke, who had completed a prospective, randomised controlled and single-blinded trial evaluating the effect of 12 weeks PFMT, were included in this follow-up study. The follow-up assessments were done by telephone interview 6 months after the intervention. The effect was evaluated by The Short Form 36 (SF-36) Health Survey Questionnaire and Incontinence Impact Questionnaire (IIQ). Twenty-four subjects completed the study. In the treatment group, the SF-36 showed a trend to a long-lasting effect in one of the eight domains and the IIQ showed a tendency to decreased impact of UI in two sub-scales compared to the control group. Our data indicated that PFMT may have a long-lasting effect measured by quality of life parameters. PMID- 16673052 TI - Prophylactic antibiotics after urodynamics in women: a decision analysis. AB - The objective of this paper is to compare the risks and benefits of antibiotics to prevent urinary tract infection (UTI) after urodynamics. We developed a decision analytic model to compare the use of prophylactic antibiotics with no antibiotic use after urodynamics to prevent UTI. Risks and benefits were estimated from the literature and by consensus. The main outcome measure was the occurrence of UTI. Secondary outcomes were the development of adverse events and possible sequelae. One-way sensitivity analyses estimated the effect of varying each characteristic through its range while all other characteristics were fixed at their baseline values. The effectiveness of prophylaxis after urodynamics was a reduction of UTI from 9.2 (no antibiotics) to 5.3% (antibiotics). One-way sensitivity analysis of the probability of UTI without prophylaxis yielded a threshold infection rate of 10%, below which, the strategy of no prophylaxis was favored. In this model, prophylactic antibiotics after urodynamics are not beneficial until the rate of UTI without antibiotics exceeds 10%. PMID- 16673054 TI - Low-level laser therapy in management of postmastectomy lymphedema. AB - The aim of this paper was to study the effects of low-level laser therapy (LLLT) in the treatment of postmastectomy lymphedema. Eleven women with unilateral postmastectomy lymphedema were enrolled in a double-blind controlled trial. Patients were randomly assigned to laser and sham groups and received laser or placebo irradiation (Ga-As laser device with a wavelength of 890 nm and fluence of 1.5 J/cm2) over the arm and axillary areas. Changes in patients' limb circumference, pain score, range of motion, heaviness of the affected limb, and desire to continue the treatment were measured before the treatment and at follow up sessions (weeks 3, 9, 12, 18, and 22) and were compared to pretreatment values. Results showed that of the 11 enrolled patients, eight completed the treatment sessions. Reduction in limb circumference was detected in both groups, although it was more pronounced in the laser group up to the end of 22nd week. Desire to continue treatment at each session and baseline score in the laser group was greater than in the sham group in all sessions. Pain reduction in the laser group was more than in the sham group except for the weeks 3 and 9. No substantial differences were seen in other two parameters between the two treatment groups. In conclusion, despite our encouraging results, further studies of the effects of LLLT in management of postmastectomy lymphedema should be undertaken to determine the optimal physiological and physical parameters to obtain the most effective clinical response. PMID- 16673053 TI - A novel modelling and experimental technique to predict and measure tissue temperature during CO2 laser stimuli for human pain studies. AB - Laser nerve stimulation is now accepted as one of the preferred methods for applying painful stimuli to human skin during pain studies. One of the main concerns, however, is thermal damage to the skin. We present recent work based on using a CO2 laser with a remote infrared (IR) temperature sensor as a feedback system. A model for predicting the subcutaneous skin temperature derived from the signal from the IR detector allows us to accurately predict the laser parameters, thus maintaining an optimum pain stimulus whilst avoiding dangerous temperature levels, which could result in thermal damage. Another aim is to relate the modelling of the CO2 fibre laser interaction to the pain response and compare these results with practical measurements of the pain threshold for various stimulus parameters. The system will also allow us to maintain a constant skin temperature during the stimulus. Another aim of the experiments underway is to review the psychophysics for pain in human subjects, permitting an investigation of the relationship between temperature and perceived pain. PMID- 16673055 TI - Hyperplasia suppression by Ho:YAG laser intravascular irradiation in rabbit. AB - The proliferation of smooth muscle cells (SMCs) was suppressed in denudated rabbit aorta by holmium-yttrium-aluminum-garnet (Ho:YAG) laser intravascular irradiation. This study was dedicated to determine the applicability of the Ho:YAG laser irradiation on chronic restenosis after balloon angioplasty. The proliferation of SMCs in denudated rabbit aortas was suppressed in vivo 6 weeks after the laser irradiation of 20 pulses with 60 mJ per pulse. To investigate the mechanisms of this in vivo effect, the death of SMCs by the Ho:YAG laser-induced bubble collapse pressure was studied in vitro. No significant cell death attributed to this pressure was found. We conclude that the suppression of the proliferation of SMCs in vivo might not be caused by a reduction in density of SMCs induced by the collapse in pressure. We submit that the suppression of SMC proliferation in vivo could be caused by the bubble expansion pressure and/or heat induced by the laser irradiation. PMID- 16673057 TI - MG132 induced apoptosis is associated with p53-independent induction of pro apoptotic Noxa and transcriptional activity of beta-catenin. AB - Noxa is a pro-apoptotic BH3-only member of the Bcl-2 family of proteins that is up-regulated at a transcriptional level by the nuclear protein p53 in response to cellular stresses such as DNA damage or growth factor deprivation. Noxa is able to interact with anti-apoptotic members of the Bcl-2 family and causes release of cytochrome c into the cytosol, leading to the activation of caspases and induction of apoptosis. Here we demonstrate that MG132, a proteasomal inhibitor, rapidly induces Noxa mRNA and protein in two human cell lines, T/C28a and Saos2. The induction of Noxa is associated with a significant reduction in the number of metabolically active cells over the first 24 h of exposure to MG132 and progressive activation of caspase-3, a hallmark of caspase-dependent apoptosis. Partial rescue of the phenotype is observed when cells are transfected with Noxa siRNA prior to treatment with MG132, indicating functional significance of the induction of Noxa. p53 has previously been shown to be non-functional in the T/C28a cell line and is absent by Western blotting in Saos2 cells, suggesting that the induction of Noxa is through a p53 independent mechanism. Western blotting and confocal microscopy showed that total beta-catenin protein is increased in both cell lines at the time of Noxa induction, with the bulk of the beta-catenin present in the nucleus. Transfection with the Tcf reporter vector pTOPFLASH confirms that treatment with MG132 leads to early increased transcriptional activity of beta-catenin in both T/C28a and Saos2 cells. However, although over-expression of transcriptionally active beta-catenin in T/C28a cells also induced apoptosis through a p53-independent mechanism, the levels of Noxa protein were unchanged, suggesting that beta-catenin mediated signaling and Noxa may play independent roles in MG132 induced apoptosis. In summary, our results demonstrate that MG132 induces the pro-apoptotic protein Noxa via a p53 independent mechanism that leads to caspase-dependent apoptosis. This is the first report showing that treatment with MG132 induces Noxa. This study also provides further evidence for a link between beta-catenin mediated signaling and the induction of apoptosis. PMID- 16673058 TI - [Perspectives of radiation therapy in benign diseases]. AB - PURPOSE: The numbers of patients with nonmalignant diseases referred for radiation therapy had to be evaluated for the last 4 years. PATIENTS AND METHODS: In the years 2002, 2004, and 2005 radiation therapy was performed in 61, 40, and 26 patients, respectively. Regularly, more women than men were treated, median age annually was 57, 54, and 55 years, respectively (Table 1). The radiotherapy scheme was not modified within the evaluated period. RESULTS: The proportion of nonmalignant diseases among all patients treated decreased from 4.7% in 2002 to 3.3% in 2004 and 2.2% in 2005, respectively. A shift was noticed toward the treatment of four main diseases (endocrine orbitopathy, prevention of heterotopic ossification, meningeoma, tendinitis, Table 2). The number of referring physicians decreased from 19 to six. CONCLUSION: Due to administrative restrictions for treatment in hospitals, budget restrictions in private practices and lasting, insufficient revenues for radiotherapy in nonmalignant diseases, radiation therapy for the entire group of benign diseases is endangered. PMID- 16673056 TI - Measuring the quality of colorectal cancer screening: the importance of follow up. AB - PURPOSE: As evidence mounts for effectiveness, an increasing proportion of the United States population undergoes colorectal cancer screening. However, relatively little is known about rates of follow-up after abnormal results from initial screening tests. This study examines patterns of colorectal cancer screening and follow-up within the nation's largest integrated health care system: the Veterans Health Administration. METHODS: We obtained information about patients who received colorectal cancer screening in the Veterans Health Administration from an existing quality improvement program and from the Veterans Health Administration's electronic medical record. Linking these data, we analyzed receipt of screening and follow-up testing after a positive fecal occult blood test. RESULTS: A total of 39,870 patients met criteria for colorectal cancer screening; of these 61 percent were screened. Screening was more likely in patients aged 70 to 80 years than in those younger or older. Female gender (relative risk, 0.92; 95 percent confidence interval, 0.9-0.95), Black race (relative risk, 0.92; 95 percent confidence interval, 0.89-0.96), lower income, and infrequent primary care visits were associated with lower likelihood of screening. Of those patients with a positive fecal occult blood test (n = 313), 59 percent received a follow-up barium enema or colonoscopy. Patient-level factors did not predict receipt of a follow-up test. CONCLUSIONS: The Veterans Health Administration rates for colorectal cancer screening are significantly higher than the national average. However, 41 percent of patients with positive fecal occult blood tests failed to receive follow-up testing. Efforts to measure the quality of colorectal cancer screening programs should focus on the entire diagnostic process. PMID- 16673059 TI - Gemcitabine concurrent with thoracic radiotherapy after induction chemotherapy with gemcitabine/vinorelbine in locally advanced non-small cell lung cancer: a phase I study. AB - PURPOSE: To determine the maximum tolerated dose (MTD) of gemcitabine every 2 weeks to a concurrent radiotherapy administered during an aggressive program of sequential and simultaneous radio-/chemotherapy for locally advanced, unresectable non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Ten patients with histologically confirmed NSCLC were observed and treated in accordance with a combined radio-/chemotherapy protocol. This included two cycles of induction chemotherapy with gemcitabine (1,200 mg/m(2)) and vinorelbine (30 mg/m(2)) at days 1, 8 and 22, 29, followed by concurrent radiotherapy including [(18)F] fluorodeoxyglucose positron emission tomography-(FDG-PET-)based target volume definition (2.0 Gy/d; total dose 66.0 Gy) and chemotherapy with gemcitabine every 2 weeks at days 43, 57, and 71. The initial dose was 300 mg/m(2). The dose of gemcitabine was increased by 100 mg/m(2) until the MTD was realized. Three patients were enrolled for each dose level. RESULTS: Dose limiting toxicity (DLT) was identified for the patient group receiving gemcitabine 500 mg/m(2), due to grade 2 esophagitis (next to grade 3) in all patients. 6 weeks after the completion of radio-/chemotherapy, most patients still presented treatment-induced esophagitis. In accordance with expected complications, such as esophagitis, dysphagia and odynophagia, the MTD was defined at this dose level, although no DLT grade 3 was reached. CONCLUSION: After induction chemotherapy, the MTD and frequency of gemcitabine in locally advanced NSCLC is 500 mg/m(2) every 2 weeks during a maximum of 7 weeks of thoracic radiotherapy. PMID- 16673060 TI - Religious belief as a coping strategy: an explorative trial in patients irradiated for head-and-neck cancer. AB - PURPOSE: To explore the role of religious belief in coping with disease symptoms and treatment-related side effects in patients with head-and-neck cancer under radiotherapy. PATIENTS AND METHODS: Prospectively collected data were used with a cohort of head-and-neck cancer patients treated by radiotherapy and epoetin beta or placebo within a double-blind multicenter trial. All patients were divided into believers and nonbelievers. Answers to a quality of life questionnaire at four points in time during radiotherapy were analyzed according to both groups. Clinical parameters and therapy side effects were controlled regularly. RESULTS: 62.1% of the patients (66/105) sent back a baseline questionnaire discriminating between believers and nonbelievers. For 34.2% (40/105) data of all four measures could be obtained. On average, believers felt better in all categories of side effects at all points of time before, during and directly after therapy. CONCLUSION: Religious faith seems to play an important role in coping strategies of radiotherapy patients. More research in this area would be worthwhile. PMID- 16673061 TI - Influence of organ at risk definition on rectal dose-volume histograms in patients with prostate cancer undergoing external-beam radiotherapy. AB - PURPOSE: To evaluate rectal dose-volume relations during three-dimensional conformal radiotherapy of patients with prostate cancer by means of different rectal volume contours. PATIENTS AND METHODS: 55 patients with prostate cancer underwent three-dimensional conformal external-beam radiotherapy. Rectal dose volume histograms were calculated for four separately contoured rectal volumes in all patients resulting in four groups. In group 1 the outer rectal wall was contoured two CT slices above and below the planning target volume. The rectal contour of group 2 was drawn from the anal verge up to the sigmoid. Furthermore, the posterior half of the rectum was contoured for both volumes mentioned above (groups 1a and 2a). Statistical analysis was then performed using nonparametric Wilcoxon tests. RESULTS: The mean target dose was 72.9 Gy (standard deviation [SD] +/- 2.1 Gy). The minimum target dose was 70.2 Gy. Mean rectum dose (+/- SD) over all patients was 50.7 Gy (+/- 4.6 Gy), 45.2 Gy (+/- 5.4 Gy), 43.2 Gy (+/- 4.2 Gy), and 38.7 Gy (+/- 5.5 Gy) for group 1, 2, 1a, and 2a, respectively. The corresponding volumes receiving > or = 70 Gy for groups 1 and 2 were 14.0% (+/- 5.3%) and 11.9% (+/- 4.5%). These differences were statistically significant. Comparison of minimum and mean rectal dose also revealed a statistically significant difference toward higher doses in groups 1 and 1a (p < 0.001). Maximum rectal doses for groups 1 and 2 as well as for groups 1a and 2a revealed no statistically significant difference (p = 1.0). CONCLUSION: Data from the literature on normal-tissue complication probability (rectal bleeding) refer to different rectal contours. When applying dose restrictions to the rectum, contouring becomes a significant factor that determines the risk of rectal toxicity. The results of this study show that different ways of rectal contouring significantly influence doses to the rectum. The influence of organ at risk contouring should be considered thoroughly in conformal radiotherapy of prostate cancer patients, especially in dose escalation studies. It is recommended to calculate the doses for absolute rectal volumes and correlate these data with toxicity in order to be able to achieve comparable results among different institutions. PMID- 16673062 TI - Osteoradionecrosis of the mandible: minimized risk profile following intensity modulated radiation therapy (IMRT). AB - BACKGROUND AND PURPOSE: Osteoradionecrosis (ON) of the mandible is a serious late complication of high-dose radiation therapy for tumors of the oropharynx and oral cavity. After doses between 60 and 72 Gy using standard fractionation, an incidence of ON between 5% and 15% is reported in a review from 1989, whereas in more recent publications using moderately accelerated or hyperfractionated irradiation and doses between 69 and 81 Gy, the incidence of ON is between < 1% and ~ 6%. Intensity-modulated radiation therapy (IMRT) is expected to translate into a further important reduction of ON. The aim of this descriptive study was to assess absolute and relative bone volumes exposed to high IMRT doses, related to observed bone tolerance. PATIENTS AND METHODS: Between December 2001 and November 2004, 73 of 123 patients treated with IMRT were identified as subgroup "at risk" for ON (> 60 Gy for oropharyngeal or oral cavity cancer). 21/73 patients were treated in a postoperative setting, 52 patients underwent primary definitive irradiation. In 56 patients concomitant cisplatin-based chemotherapy was applied. Mean follow-up time was 22 months (12-46 months). Oral cavity including the mandible bone outside the planning target volume was contoured and dose-volume constraints were defined in order to spare bone tissue. Dose-volume histograms were obtained from contoured mandible in each patient and were analyzed and related to clinical mandible bone tolerance. RESULTS: Using IMRT with doses between 60 and 75 Gy (mean 67 Gy), on average 7.8, 4.8, 0.9, and 0.3 cm(3) were exposed to doses > 60, 65, 70, and 75 Gy, respectively. These values are substantially lower than when using three-dimensional conformal radiotherapy. The difference has been approximately quantified by comparison with a historic series. Additional ON risk factors of the patients were also analyzed. Only one grade 3 ON of the lingual horizontal branch, treated with lingual decortication, was observed. CONCLUSION: Using IMRT, only very small partial volumes of the mandibular bone are exposed to high radiation doses. This is expected to translate into a further reduction of ON and improved osseointegration of dental implants. PMID- 16673063 TI - Contralateral testicular cancer in spite of TIN-negative double biopsies and interval cisplatin chemotherapy. AB - BACKGROUND: Current models of tumorigenesis postulate that testicular germ cell cancer uniformly develops through a preinvasive lesion termed testicular intraepithelial neoplasia (TIN). An open testicular biopsy is a simple and highly sensitive method to diagnose TIN, and this procedure constitutes the basis for curative treatment of TIN. Patients with testis cancer carry a significantly increased risk of developing contralateral testicular tumors. Therefore, a contralateral biopsy has been recommended in these patients. A negative biopsy was assumed to exclude the risk of a subsequent germ cell cancer in the testis due to the high sensitivity of the method. Reports on false-negative biopsies gave rise to the idea that TIN is not uniformly distributed throughout the testis. Consequently, double biopsies are thought to increase the diagnostic sensitivity. CASE REPORT: A 24-year-old patient with nonseminomatous testis cancer is reported. The patient had TIN-negative double biopsies in the contralateral testis. He received three cycles of standard PEB (cisplatin, etoposide, bleomycin) chemotherapy for visceral metastasis. 1 year after treatment the patient developed a nonseminomatous contralateral testis cancer which was treated by partial orchiectomy and subsequent local radiotherapy with 20 Gy. CONCLUSION: The case presented here highlights some clinically important aspects: a) even double biopsies of the testis may fail to detect TIN. b) Systemic cisplatin-based chemotherapy may fail to prevent contralateral testicular germ cell cancer. c) A metachronous contralateral testis cancer may-in contrast to common clinical perception-develop even soon after the diagnosis of the first testis tumor. Furthermore, the case could foster the hypothesis that testicular germ cell tumors may in some cases develop without a preceding stage of TIN. PMID- 16673064 TI - [Letter of U.D. Braumann [corrected] and A. Pfafflin regarding the comment by T. Herrmann: ASARA or the art to avoid waiting list: in Strahlenther Onkol 2005;181:808-9 (Nr. 12)]. PMID- 16673065 TI - Global motion mechanisms compensate local motion deficits in a patient with a bilateral occipital lobe lesion. AB - Successive stages of cortical processing encode increasingly more complex types of information. In the visual motion system this increasing complexity, complemented by an increase in spatial summation, has proven effective in characterizing the mechanisms mediating visual perception. Here we report psychophysical results from a motion-impaired stroke patient, WB, whose pattern of deficits over time reveals a systematic shift in spatial scale for processing speed. We show that following loss in sensitivity to low-level motion direction WB's representation of speed shifts to larger spatial scales, consistent with recruitment of intact high-level mechanisms. With the recovery of low-level motion processing WB's representation of speed shifts back to small spatial scales. These results support the recruitment of high-level visual mechanisms in cases where lower-level function is impaired and suggest that, as an experimental paradigm, spatial summation may provide an important avenue for investigating functional recovery in patients following damage to visually responsive cortex. PMID- 16673069 TI - [Results from biomedical aging research. Trends and current examples from immunology]. AB - The public health of our society is challenged by a continuous increase in life expectancy. Hence, biomedical aging research is enjoying a steadily increasing popularity but also enlightens our understanding of age-related diseases by a number of striking results from basic research. One of the most striking changes that occurs during normal human aging is an overall diminution of immune functions, a phenomenon often termed immunosenescence. Starting from some highly exciting examples from basic immunological research, this article sheds light on which impact normal human aging has on several immune defence mechanisms. In addition, clinical consequences in view of Alzheimer's disease, immunogenicity of vaccines and autoimmune diseases are discussed. PMID- 16673070 TI - [Activity, physical and psychological mobility in old age]. AB - Old age and aging are accompanied by a number of physical and mental changes. However, these so-called age-dependent processes are not exclusively genetically determined or irreversible but can be partially delayed, prevented, or compensated and some can even be reversed. The goal of this article is to highlight the plasticity, or the "mobility", of physical and mental aging. We will point out in what respects an appropriate lifestyle or purposeful interventions can positively influence the reserve capacity of aging human beings and the aging process. Using the example of physical activity, we will illustrate how we can influence physiological development, cognitive performance, longevity, as well as the development and the occurrence of chronic diseases. Additionally, it is shown that cognitive development is malleable as well. It is facilitated or debilitated by behavior and activity-this covers not only cognitive but also physical activity. It is our particular concern to demonstrate the close interconnectedness of body and mind. PMID- 16673071 TI - [Morbidity and health in old age. A challenge for prevention and health care]. AB - Due to chronic illness and multimorbidity, the physical and psychosocial impairments increase significantly in the second half of life. Even though the health condition of the elderly has improved in recent years, the potential for prevention in older people is still undervalued. On the basis of surveys, data from the Association of CHI Physicians and health insurance data, this article provides a review of health and health behaviour of the elderly in Germany. It aims to point out areas of particular relevance for prevention and preventive approaches. Strategies that consider mental and social aspects of aging beside the physical ones are necessary. Systematic, personal and professional factors still inhibit the realisation of these strategies. In this context, doctors and nurses are particularly important as primary professional contact persons for the target group. However, prevention and health promotion in that age group are social tasks, which need the appropriate involvement of further professionals and an explicit political agenda setting. PMID- 16673072 TI - [Living conditions of aging and old people in Germany]. AB - This contribution aims to convey a general overview of the living conditions of aging and old people in Germany. It introduces a series of contributions devoted to the topic "health in old age" and focuses on older people as a very heterogeneous group in society. Who exactly are these older people? We start by discussing the definition of "old age" as a stage of life and the distinction between a "third" and "fourth" age. This is followed by a presentation of some facts describing demographic change at the population level. The main body of the contribution looks at households and housing, family relations and social integration, income, life satisfaction and the health of older people. The two waves of the German Aging Survey, a representative study of persons in the second half of life undertaken in 1996 and 2000, provide the empirical base for the paper. PMID- 16673073 TI - The effect of intravenous gadolinium-DTPA on diffusion-weighted imaging. AB - INTRODUCTION: Diffusion-weighted imaging (DWI) is usually performed before injection of intravenous paramagnetic contrast medium. Occasionally, it may be necessary to perform or to repeat DWI after such administration. Our purpose was to evaluate the effect of intravenous gadodiamide (Gd [DTPA-BMA]) on DWI. METHODS: DWI was performed on 88 brain lesions immediately before, immediately after, and 5-10 min following the end of 0.1 mmol/kg Gd [DTPA-BMA] administration. Signal-to-noise ratios (SNRs) and contrast-to-noise ratios (CNRs) of the lesions, and the SNRs of normal brain tissue were calculated on b=0 s/mm(2) and b=1,000 s/mm(2) DW images. Apparent diffusion coefficient (ADC) values of the lesions were measured on ADC maps. A paired t-test was used to determine the significance of differences between the values before and after administration of contrast medium. RESULTS: The lesions consisted of 23 intraaxial and 11 extraaxial masses, 19 ischemic strokes, 15 intracranial hemorrhages and 20 demyelinating lesions. Images before and after contrast administration were not significantly different regarding SNRs and CNRs on DWI. This statement was also true for strongly enhanced lesions. However, ADC values significantly decreased after contrast medium injection on early post-contrast DWI in normal brain tissue (1%, P<0.049) and (3%, P<0.008) in lesions. By contrast, on late images, ADC values were normalized. CONCLUSION: Contrast medium injection had significant and time-dependent effects on ADC values. Therefore, only pre-contrast and late DW images should be used in quantitative ADC studies. PMID- 16673074 TI - Transient splenium lesions in presurgical epilepsy patients: incidence and pathogenesis. AB - INTRODUCTION: Transient splenium corporis callosi (SCC) lesions are related to rapid reduction of antiepileptic drugs (AEDs). The range of substances with predilection for SCC changes, their pathophysiology and their occurrence are still unknown. METHODS: In a prospective 2-year study an epilepsy-dedicated MRI protocol supplemented by DWI and ADC maps was performed after AED withdrawal for diagnostic seizure provocation in all patients with pharmacoresistant seizures locally admitted to the Department of Epileptology. RESULTS: Of 891 presurgical epilepsy patients, 6 (0.7%) had SCC lesions with cytotoxic edema on DWI. Carbamazepine combined with other AEDs was administered in five of those patients. In the study period we observed identical lesions in a schizophrenic patient treated with olanzapine and citalopram, in a patient with oropharyngeal carcinoma treated with alkylating agents, and in a hypernatremic patient following neurohypophyseal granular cell tumor surgery. CONCLUSION: Transient SCC lesions are related to rapid AED reduction but may occur in similar conditions with fluid balance alterations. We contribute further clinical data in this field to better classify the pharmaceuticals that are prone to the described cerebral cytotoxic side effects in the SCC and to clarify their incidence among presurgical epilepsy patients. PMID- 16673075 TI - Determinants of household health expenditure on western institutional health care. AB - We try to identify determinants of illness reporting, provider choice and resulting expenditure with different econometric models using data from a representative household panel survey of 800 households in Nouna health district, Burkina Faso, during 2000-2001. The factors "being an adult", "married", "illness occurred in rainy season" and "severe illness" significantly increased the magnitude of health expenditure. Compared to malaria, individuals spent more on other infectious diseases, injury and the other disease category. In contrast, people were less likely to spend on chronic illness. An individual who belonged to a household headed by a female, a literate household head and with a higher household expenditure had a significantly positive association with the magnitude of expenditure. Findings from this study can be used for policy implication to improve health system performance in Burkina Faso through enhancing health care utilization. PMID- 16673076 TI - [123I-Metaiodobenzylguanidine- (MIBG-) scintigraphy: paradoxical positivity in an oncocytic adrenocortical carcinoma]. AB - (123)I-metaiodobenzylguanidine (MIBG), a radio-labeled catecholamine analogue, is used for the imaging of pheochromocytoma based on the selective uptake of MIBG by chromaffin tissues. MIBG scintigraphy displays high sensitivity (90%) and specificity (close to 100%). In contrast, the false-positive uptake of MIBG by adrenal cortical carcinoma is rare. Here, we report a metastatic oncocytic adrenal cortical carcinoma with MIBG uptake used for therapeutic purposes. PMID- 16673077 TI - A quasi-Laue neutron crystallographic study of D-xylose isomerase. AB - The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464 6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase. PMID- 16673078 TI - X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases. AB - Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing non hydrolysable analogues of the scissile peptide bond. Until recent years, the positions of protons on the catalytic aspartates and the ligand in these complexes had not been determined with certainty due to the inadequate resolution of these analyses. There has been much interest in locating the catalytic protons at the active site of aspartic proteinases since this has major implications for detailed understanding of the mechanism of action and the design of improved transition state mimics for therapeutic applications. In this review we discuss the results of studies which have shed light on the locations of protons at the catalytic centre. The first direct determination of the proton positions stemmed from neutron diffraction data collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex at a resolution of 2.1 A provided evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. Atomic resolution X-ray studies of inhibitor complexes have corroborated this finding. A similar study of the native enzyme established that it, unexpectedly, has a dipeptide bound at the catalytic site which is consistent with classical reports of inhibition by short peptides and the ability of pepsins to catalyse transpeptidation reactions. Studies by NMR have confirmed the findings of low-barrier and single-well hydrogen bonds in the complexes with transition state analogues. PMID- 16673079 TI - Huge retropharyngeal lipoma causing obstructive sleep apnea: A case report. AB - Lipoma of the retropharyngeal space is a very rare benign tumor often causing unspecific clinical symptoms. The most common symptoms are dysphagia and/or respiratory disturbances. The clinical diagnosis may be difficult. The radiological imaging techniques (CT and MRI) can provide adequate information with regard to the composition and extension of the tumor, although final histological confirmation is essential. Surgery is the treatment of choice. We present a case of 40-year-old male patient complaining of obstructive sleep apnea symptoms (respiratory disturbances, excessive daytime somnolence, morning headache). The radiological examination (CT) showed a huge (11.7 x 7.2 cm) lipoma of the retropharyngeal space extending from the nasopharynx to the superior mediastinum. The tumor was removed via transcervical approach with complete amelioration of symptoms. PMID- 16673080 TI - Optimal site for throat culture: tonsillar surface versus posterior pharyngeal wall. AB - To determine the optimal site of throat culture for the detection of potential pathogens by comparing culture results from the tonsillar surface and the posterior pharyngeal wall in children selected for adenotonsillectomy and in children without upper respiratory disease. Cotton culture swabs were taken from the tonsillar surface and the posterior pharyngeal wall of 50 children selected for adenotonsillectomy for symptoms of recurrent tonsillitis and/or adenotonsillar hypertrophy and of 50 children without upper respiratory disease. Potential respiratory pathogens were identified. In the overall group (n = 100), positive culture results were found in 67 posterior pharyngeal wall samples and 47 tonsillar surface samples (P = 0.001). Haemophilus influenzae was the most frequently isolated micro-organism both in the posterior pharyngeal wall and the tonsillar surface samples; 55 and 35%, respectively (P = 0.001). Group A beta haemolytic streptococci were found in the samples of the posterior pharyngeal wall and the tonsillar surface in 17 and 13%, respectively (P = 0.2). When dealing with patients with sore throat, sampling both tonsillar surfaces is enough for the detection of group A beta-haemolytic streptococci. When detection of other bacteria is also important, such as for research purposes, the posterior pharyngeal wall should be sampled as well. PMID- 16673083 TI - A fast surface-matching procedure for protein-ligand docking. AB - A very simple, fast, and efficient scheme is proposed for performing preliminary protein-ligand docking as the first step of intensive high-throughput virtual screening. The procedure acts as a surface-complementarity filter that first calculates the 2D-contour maps of both the protein cavity and of the ligands using a spherical harmonics description of the associated molecular surfaces. Next, the obtained 2D-fingerprint images are compared to detect their complementarity. This scheme was tested on three typical cases of protein cavities, namely, a well-closed pocket, a small open pocket, and a large open one. For that purpose, for each case, a sample of 101 ligand conformers was generated (the X-ray one and 100 different conformers generated using simulated annealing), and these conformational samples were ranked according to the complementarity with the protein cavity surface. Compared to traditional docking procedures such as FRED (considered as typical of a very fast rigid body docking algorithms) and GOLD (considered as typical of the more accurate flexible docking algorithms), our procedure was much faster and more successful in detecting the right X-ray conformation. We did, however, identify a certain weakness in the case of the very large pocket where results were not as expected. In general, our method could be used for incorporating indirectly flexibility in protein-ligand docking calculations as such a scheme can easily handle several conformational states of both the protein and the ligand. PMID- 16673085 TI - SPME in environmental analysis. AB - Recent advances in the use of solid-phase microextraction (SPME) in environmental analysis, including fiber coatings, derivatization techniques, and in-tube SPME, are reviewed in this article. Several calibration methods for SPME, including traditional calibration methods, the equilibrium extraction method, the exhaustive extraction method, and several diffusion-based calibration methods, are presented. Recent developed SPME devices for on-site sampling and several applications of SPME in environmental analysis are also introduced. PMID- 16673084 TI - FlgM anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure function relationships. AB - FlgM proteins, also known as Anti-sigma-28 factor (sigma28), are negative regulators of flagellin synthesis. Recently, a three-dimensional structure of the Aquifex aeolicus sigma28/FlgM complex (PDB code: 1rp3) was determined by X-ray crystallography at 2.3 A resolution. Furthermore, experimental data on bacterial FlgM, including site-directed mutagenesis and structural characterization by NMR are also available. However, an interpretation of the sequence-structure-function relationships combining X-ray and NMR data with the evolutionary information extracted from the increasing number of FlgM-related sequences annotated in databases is not available. In the present study, we combined database sequence searches and sequence-analysis tools to update the multiple sequence alignment of a previously characterized cluster of orthologs (COG2747) and the PFAM classification of protein domains (PF04316) for the FlgM family. A phylogenetic analysis of 77 protein sequences revealed the presence of at least three major sequence clades within the FlgM family. Besides, we predicted functional residues using a SequenceSpace method. We also generated homology models for Bacillus subtilis and Salmonella typhimurium FlgM proteins, for which sequence-structure function relationship data are available, and used the docking program ClusPro to hypothesize about the dimer association between FlgM proteins. In conclusion, the analysis presented in this work will be useful in designing new experiments to understand better protein-protein interactions between FglM, sigma factors, and putative molecules from the flagellar export apparatus. Electronic Supplementary Material is available in the online version of this article at http://link.springer.de/ PMID- 16673086 TI - Chemometrical investigation of the presence and distribution of organochlorine and polyaromatic compounds in sediments of the Ebro River Basin. AB - A multivariate statistical data analysis, using principal component analysis, of historical data from 1996 to 2003 concerning the concentration of different polycyclic aromatic hydrocarbons and organochlorine compounds in sediment samples from different sampling sites of the Ebro River Basin was performed under the UE funded project AQUATERRA. Three major contamination sources were identified and their composition and distribution profiles were resolved. The first contamination profile was mostly loaded by polycyclic aromatic hydrocarbons, the second contamination profile was loaded by some organochlorine compounds and the third contamination profile was more specifically loaded by naphthalene. Samples from the different geographical regions of the Ebro River Basin were grouped according to the contamination described by these three major profiles. PMID- 16673089 TI - Bienzyme sensors based on novel polymethylferrocenyl dendrimers. AB - Amperometric bienzyme electrodes with horseradish peroxidase (HRP) and glucose oxidase (GOx) co-immobilized on polymethylferrocenyl dendrimers deposited onto platinum electrodes have been used for determination of the hydrogen peroxide produced by the oxidase during the enzymatic reaction. The redox dendrimers consist of flexible poly(propylenimine) dendrimer cores functionalised with octamethylferrocenyl units. The effects of dendrimer generation, the thickness of the dendrimer layer, substrate concentration, interferences, and reproducibility on the response of the sensors were investigated. The new bienzyme biosensors respond to substrate at work potential values between 200 and 50 mV (vs. SCE), have good sensitivity, and are resistant to interferences. Figure. PMID- 16673090 TI - Case report: a gossypiboma in the shoulder. AB - We present the case of a 15-year-old boy who underwent shoulder surgery for repair of a Bankart lesion after dislocation of his right shoulder. A compress was left in the surgical wound. This case is presented to highlight an important pitfall in the diagnosis of gossypiboma (foreign body reaction): when the wires of a compress are visualized on X-ray, beware of the fact that it is possibly located inside the body. The diagnosis of an abscess was made by ultrasound. The compress wires were visualized on radiographs. PMID- 16673091 TI - Odontoid osteomyelitis masquerading as a C2 fracture in an 18-month-old male with torticollis: CT and MRI features. AB - Odontoid osteomyelitis is a rare entity and can be confused with other disease processes, requiring imaging to clarify the diagnosis. The following describes a pediatric case and the associated MR and CT findings. PMID- 16673092 TI - Contrast-enhanced 3D MRI of lung perfusion in children with cystic fibrosis- initial results. AB - This paper is a feasibility study of magnetic resonance imaging (MRI) of lung perfusion in children with cystic fibrosis (CF) using contrast-enhanced 3D MRI. Correlation assessment of perfusion changes with structural abnormalities. Eleven CF patients (9 f, 2 m; median age 16 years) were examined at 1.5 T. Morphology: HASTE coronal, transversal (TR/TE/alpha/ST: 600 ms/28 ms/180 degrees /6 mm), breath-hold 18 s. Perfusion: Time-resolved 3D GRE pulse sequence (FLASH, TE/TR/alpha: 0.8/1.9 ms/40 degrees ), parallel imaging (GRAPPA, PAT 2). Twenty five data sets were acquired after intravenous injection of 0.1 mmol/kg body weight of gadodiamide, 3-5 ml/s. A total of 198 lung segments were analyzed by two radiologists in consensus and scored for morphological and perfusion changes. Statistical analysis was performed by Mantel-Haenszel chi-square test. Results showed that perfusion defects were observed in all patients and present in 80% of upper, and 39% of lower lobes. Normal lung parenchyma showed homogeneous perfusion (86%, P<0.0001). Severe morphological changes led to perfusion defects (97%, P<0.0001). Segments with moderate morphological changes showed normal (53%) or impaired perfusion (47%). In conclusion, pulmonary perfusion is easy to judge in segments with normal parenchyma or severe changes. In moderately damaged segments, MRI of lung perfusion may help to better assess actual functional impairment. Contrast-enhanced 3D MRI of lung perfusion has the potential for early vascular functional assessment and therapy control in CF patients. PMID- 16673093 TI - Imaging of small intestinal Crohn's disease: comparison between MR enteroclysis and conventional enteroclysis. AB - The purpose of this study was to compare MR enteroclysis (MRE) with conventional enteroclysis (CE) in patients with small intestinal Crohn's disease. Fifty-two consecutive patients with known or suspected Crohn's disease underwent MR and conventional enteroclysis, which was considered the gold standard. Eleven imaging features, classified in three groups, mucosal, transmural and extraintestinal, were subjectively evaluated by two experienced radiologists. MRE and CE were in full agreement in revealing, localizing and estimating the length of all involved segments of the small bowel. The sensitivity of MRE for the detection of superficial ulcers, fold distortion and fold thickening was 40, 30 and 62.5%, respectively. The sensitivity of MRE for the detection of deep ulcers, cobble stoning pattern, stenosis and prestenostic dilatation was 89.5, 92.3, 100 and 100%, respectively. Additional findings demonstrated on MRE images included fibrofatty proliferation in 15 cases and mesenteric lymphadenopathy in 19 cases. MRE strongly correlates with CE in the detection of individual lesions expressing small intestinal Crohn's disease. It provides additional information from the mesenteries; however, its capability to detect subtle lesions is still inferior to conventional enteroclysis. PMID- 16673094 TI - Contrast-medium-induced nephropathy: is there a new consensus? A review of published guidelines. AB - The interest in contrast-medium-induced nephropathy has increased considerably during the last few years. Various guidelines regarding identifying patients at risk and measures to reduce the incidence of this complication have been proposed. The aim of this review was to analyse whether there is some consistency amongst these guidelines. A Medline search for the keyword "contrast medium induced nephropathy" during the period from the beginning of 2003 through the end of September 2005 was carried out. Only papers in English were reviewed. Thirteen guidelines were identified. Inconsistency was observed regarding advise on the prophylactic use of drugs and the isoosmolar dimer to reduce the incidence of contrast-medium-induced nephropathy. Consistency was found in relation to the importance of hydration, cessation of intake of nephrotoxic drugs and administration of the lowest possible dose of contrast medium. No new consensus has been observed in comparison to the European Society for Urogenital Radiology (ESUR) guidelines, which were published in 1999. PMID- 16673096 TI - Immunohistochemical localization of sodium-dependent L-ascorbic acid transporter 1 protein in rat kidney. AB - Recently, two L-ascorbic acid transporters were identified; sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2. The previous study suggested that SVCT protein might be present on the apical membrane in the straight segment (S3) of proximal tubule. In the present study, SVCT1 immunoreactivity (IR) was observed in the brush border of proximal straight tubules in the medullary ray of renal cortex and the outer stripe of outer medulla, while SVCT2 IR was not localized in any region of the kidney. Since the mechanism of VC reabsorption in the kidney has not been fully elucidated up to the present time, it is meaningful to demonstrate the exact cellular distribution of SVCT protein in the kidney. PMID- 16673095 TI - Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers. AB - We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 x 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman's layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics. PMID- 16673097 TI - Toxic osteoblastoma of the scapula. AB - Osteoblastoma rarely occurs in the scapula, and toxic osteoblastoma is a very rare subtype of this bone-forming tumour. This report details the clinical, radiological and pathological features of a toxic osteoblastoma of the scapula; it is the first reported case to be diagnosed correctly pre-operatively and treated appropriately by excision. PMID- 16673101 TI - Associations between socio-economic factors and the use of prescription medication during pregnancy: a population-based study among 19,874 Danish women. AB - OBJECTIVE: To examine the association between socio-economic factors and use of prescription medication during pregnancy in a population of Danish women. METHODS: This was a population-based cohort study. Using record linkage from public administrative registries, we described the use of prescription medication during pregnancy and the financial and educational resources for each pregnant woman in the cohort. RESULTS: The analyses included all 19,874 primiparous women delivering singletons in North Jutland county, Denmark, in 1991-1998. We identified 24,243 prescriptions filled by the women during their pregnancies. The highest overall prescription medication use was among women with basic schooling (OR 1.3; 95% CI 1.2-1.4), and lowest among women with the highest education (OR: 0.8; 95% CI 0.7-0.9) compared with women who had vocational education. Stratified analysis of therapeutic subgroups revealed that socio-economic factors were associated with the use of anti-infective and anti-asthmatic medications during pregnancy. CONCLUSION: Maternal educational level, and to a lesser degree household income, paternal educational level and cohabitation status, was associated with the use of prescription medication during pregnancy. However, the analyses did not take into account important clinical variables such as maternal illness, and the results could be affected by differential misclassification of exposure information, by confounding or chance. PMID- 16673102 TI - Sacroiliac joint tuberculosis. AB - Infections of the sacroiliac joint are uncommon and the diagnosis is usually delayed. In a retrospective study, 17 patients who had been treated for tuberculosis sacroiliitis between 1994 and 2004 were reviewed. Two patients were excluded due to a short follow-up (less than 2 years). Low back pain and difficulty in walking were the most common presenting features. Two patients presented with a buttock abscess and spondylitis of the lumbar spine was noted in two patients. The Gaenslen's and FABER (flexion, abduction and external rotation) tests were positive in all patients. Radiological changes included loss of cortical margins with erosion of the joints. An open biopsy and curettage was performed in all patients; histology revealed chronic infection and acid-fast bacilli were isolated in nine patients. Antituberculous (TB) medication was administered for 18 months and the follow-up ranged from 3 to 10 years (mean: 5 years). The sacroiliac joint fused spontaneously within 2 years. Although all patients had mild discomfort in the lower back following treatment they had no difficulty in walking. Sacroiliac joint infection must be included in the differential diagnosis of lower back pain and meticulous history and clinical evaluation of the joint are essential. PMID- 16673103 TI - Staged arthroplasty as salvage procedure for deep hip infection following intertrochanteric fracture. AB - Deep hip infection is a rare complication of intertrochanteric fractures and an optimal treatment has not yet been reported. Twenty-seven patients who contracted deep hip sepsis following the failed primary treatment of an intertrochanteric fracture were treated with two-stage hip arthroplasty. Antibiotic-impregnated cement beads were implanted following resection arthroplasty in the first 15 patients, and a temporary cement spacer prosthesis was used in the other 12 hips. There was only one recurrence of infection at an average follow-up of 4.8 years. Complications included non-union of the greater trochanter in four patients, intraoperative femoral fracture in two and fracture of the cement prosthesis in one. The use of an interim spacer was associated with better hip function in the interim period; a decreased operative time and less blood loss at the time of arthroplasty; and a higher hip score at final follow-up. Staged arthroplasty is an effective salvage procedure for deep hip infection after the failed treatment of an intertrochanteric fracture. The use of a temporary spacer maintains hip function between stages, makes arthroplasty less complicated, and improves the clinical outcome. PMID- 16673104 TI - Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells. AB - Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer's patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2+/-3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens. PMID- 16673107 TI - [Sonography of enlarged lymph nodes: Pathogenetic categorization using contrast enhanced power Doppler sonography]. AB - BACKGROUND: The purpose of this study was to categorize enlarged superficial lymph nodes as benign or malignant using sonomorphologic features and vascularization pattern. PATIENTS AND METHODS: Enlarged superficial lymph nodes in 57 patients were assessed with B-mode and contrast-enhanced power Doppler sonography. Morphology and vascularization were evaluated. The lymph nodes were categorized as benign or malignant. Correlation was made with histology and follow-up results. RESULTS: In 55 patients, 40 lymph nodes were correctly categorized as benign and 15 lymph nodes correctly as malignant. The most reliable criteria were shape and vascularization pattern. Intact hilar vessels and branching indicated benign enlargement, destruction of the hilum with vessels running peripherally along the capsule indicated metastatic destruction. Two benign lymph nodes were considered malignant (false positive). CONCLUSION: B-mode ultrasound along with contrast-enhanced power Doppler ultrasound is an easy, cost effective, and reliable tool for differentiation and categorization of enlarged superficial lymph nodes. PMID- 16673108 TI - Community structure and function in a H(2)-based membrane biofilm reactor capable of bioreduction of selenate and chromate. AB - Two different H(2)-based, denitrifying membrane-biofilm reactors (MBfRs) initially reduced Se(VI) or Cr(VI) stably to Se(0) or Cr(III). When the oxidized contaminants in the influent were switched, each new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been in its original MBfR. The precipitation of reduced selenium and chromium in the biofilm was verified by scanning electron microscopy and energy dispersive X-ray analysis. These results on selenate and chromate reduction are consistent with the interpretation that the H(2)-based biofilm community had a high level of functional diversity. The communities' structures were assessed by cloning analysis. Dechloromonas spp., a known perchlorate-reducing bacteria, dominated the clones from both reactors during selenate and chromate reductions, which suggests that it may have functional diversity capable of reducing selenate and chromate as secondary and dissimilatory acceptors. PMID- 16673109 TI - Mutation on N-terminus of polyhydroxybutyrate synthase of Ralstonia eutropha enhanced PHB accumulation. AB - Polyhydroxyalkanoate (PHA) synthase is the central enzyme involved in the biosynthesis of PHA, a family of bacterial biodegradable polyesters. Due to its high variability, the N-terminal fragment of this enzyme was previously considered as unnecessary for a functionally active enzyme. In this study, polyhydroxybutyrate synthase from Ralstonia eutropha (PhbC(Re)) with a deletion on N-terminal 88 amino acid residues showed a significant reduced activity, as reflected by only 1.5% PHB accumulation compared with the wild type which produced 58.4% PHB of the cell dry weight. Whilst several site-specific mutagenesis results revealed the amphiphilic alpha-helix assembled by the amino acid region, D70-E88 played an important role in both maintaining the PHB synthase activity and regulating molecular weight and polydispersity of accumulated PHB homopolymer. PMID- 16673110 TI - Direct electrochemical immunoassay based on a silica nanoparticles/sol-gel composite architecture for encapsulation of immunoconjugate. AB - A highly hydrophobic and non-toxic colloidal silica nanoparticle/polyvinyl butyral sol-gel composite membrane was prepared on a platinum wire electrode. With diphtheria-toxoid (D-Ag) as a model antigen and encapsulation of diphtheria antibody (D-Ab) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. It displayed a porous and homogeneous composite architecture without the aggregation of the immobilized protein molecules. The formation of immunoconjugate by a simple one-step immunoreaction between D-Ag in sample solution and the immobilized D-Ab introduced the change in the potential. Under optimal conditions, the D-Ag analyte could be determined in the linear ranges from 10 to 800 ng ml(-1) with a relatively low detection limit of 2.3 ng ml(-1) at 3delta. The D-Ag immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy, and reproducibility. This composite membrane could be used efficiently for the entrapment of different biomarkers and clinical applications. PMID- 16673111 TI - Aspergillus oryzae strains with a large deletion of the aflatoxin biosynthetic homologous gene cluster differentiated by chromosomal breakage. AB - Recently we divided Aspergillus oryzae RIB strains into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-1, aflR, norA, avnA, verB, and vbs), and group 2, having three homologues (avnA, verB, and vbs). Here, partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8 kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups' strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage. PMID- 16673115 TI - An ultrastructural analysis of cellular death in the CA1 field in the rat hippocampus after transient forebrain ischemia followed by 2, 4 and 10 days of reperfusion. AB - An ultrastructural study was performed to investigate the type of cellular death that occurs in hippocampal CA1 field pyramidal neurons after 10 and 20 min of transient cerebral ischemia in the male adult Wistar rats, followed by 2, 4 and 10 days of reperfusion. The four-vessel occlusion method was used to induce ischemic insult for either 10 or 20 min, following which the animals were submitted to either 2, 4 or 10 days of reperfusion. The animals were then anaesthetised, and their brains removed, dehydrated, embedded, sectioned and examined under a transmission electron microscope. After ischemic insult, neurons from the CA1 field presented alterations, corresponding to the initial, intermediate and final stages of the degenerative process. The only difference observed between the 10 and 20 min ischemic groups was the degree of damage; the reaction was stronger in 20 min groups than in the 10 min groups. While neurons were found in the different stages of oncotic necrosis in all groups, differences were found between the groups in relation to prevalent stages. In both ischemic groups, after 2 days of reperfusion, the initial stage of oncotic necrosis was prevalent and large numbers of neurons appeared normal. In both groups, after 4 days of reperfusion, most of the neurons showed more advanced alterations, typical of an intermediate stage. In both groups, after 10 days of reperfusion, alterations corresponding to the intermediate and final stages of oncotic necrosis were also predominant. However, few intact neurons were identified and the neuropile appeared more organised, with numerous glial cells. In summary, the pyramidal neurons of the CA1 field displayed selective vulnerability and exhibited a morphological death pattern corresponding exclusively to an oncotic necrotic pathway. PMID- 16673116 TI - C4d deposits mark sites of meniscal tissue disintegration. AB - Although the frequent occurrence of meniscal degeneration is a well-known fact and its consequences include rupture and even loss of the meniscus, the pathogenetic factors are established insufficiently. Because complement factors and leukocytes are present in synovial fluid, we tried to detect complement deposits and macrophages in menisci, which displayed degenerative changes. We therefore performed a retrospective analysis by immunohistochemical staining of C4d and CD68 in meniscal tissue derived from patients (n=15) who underwent meniscectomy because of meniscal tears and from three autopsy cases (n=3). In this study, focal C4d deposits in the meniscal extracellular matrix in areas of mucoid degeneration or fibrillation were demonstrated for the first time, while no C4d deposits in the avascular zone of menisci without signs of degeneration or injury could be detected. In addition, colocalization of C4d and CD68+ cells was found at sites of meniscal tissue disintegration in five cases. These results represent the first evidence for an involvement of complement and macrophages in meniscal tissue disintegration, indicating a complement mediated reaction at the site of tissue alteration. PMID- 16673117 TI - Atypical thymoma (WHO B3) with neuroendocrine differentiation: report of a case. AB - We report a case of type-B3 thymoma manifesting neuroendocrine differentiation. The patient was a 42-year-old woman who complained of shoulder pain but had no symptoms of myasthenia gravis or anemia. The tumor was located in the anterior mediastinum and had directly invaded the pericardium and left lung. Histological examination revealed that the tumor was lobulated by bands of fibrous tissue, perivascular spaces were scattered throughout the tumor, and there were a few intraepithelial lymphocytes. The vast majority of lymphocytes in the perivascular spaces and in the lobulated tumor were immunohistochemically positive for TdT, MIC2, and CD1a. The majority of tumor cells were polygonal and medium or large in size. The tumor cells were weakly positive for synaptophysin, chromogranin A, CD56, and NSE. Small nests of small, relatively uniform polygonal cells were observed facing the fibrous bands. These cells resembled the cells of carcinoid tumors and were strongly positive for NSE, synaptophysin, chromogranin A, and CD56. Ultrastructurally, sparse dense-core granules were observed in the cytoplasm of a few tumor cells. This is a unique case of thymoma with neuroendocrine differentiation, and to the best of our knowledge this is the first such case ever reported. PMID- 16673118 TI - CD10 expression by fusiform stromal cells in nasopharyngeal carcinoma correlates with tumor progression. AB - CD10 is a cell surface zinc metalloprotease expressed through a variety of normal cell types, including lymphoid precursor cells, germinal center B lymphocytes, and some epithelial cells. Many studies showed that CD10 expression is associated with the tumor progression of a large variety of cancers, such as breast and colorectal carcinomas. The aim of this study was to investigate the expression of CD10 in nasopharyngeal carcinoma (NPC). The expression of CD10 was immunohistochemically examined in 47 paraffin embedded NPC biopsies from Tunisian patients compared with 16 reactional nasopharyngeal mucosas. A significant expression of CD10 was observed in stromal fusiform cells in 46.8% of NPC cases but was not in malignant and normal epithelial cells. There was no significant expression of CD10 in control group. The stromal expression of CD10 was more frequently detected in advanced clinical stage than early stage (56 vs 23%; p=0.04) and in patients older than 25 years than in patients under 25 years (56.2 vs 26.5%; p=0.05). Our study is the first in investigating CD10 expression in nasopharyngeal carcinoma and showed that CD10 expression by stromal cells in this malignancy play an important role in tumor progression, particularly in older patients. PMID- 16673119 TI - An ultrastructural and immunohistochemical study of a combined submucosal granular cell tumor and lipoma of the colon showing a unique nodule-in-nodule structure: putative implication of CD34 or prominin-2-positive stromal cells in its histopathogenesis. PMID- 16673120 TI - Accessible calculation of multirater kappa statistics for pathologists. PMID- 16673121 TI - Preinvasive intraductal neoplasia in salivary adenocarcinoma, not otherwise specified. AB - Preinvasive intraductal neoplasia of the salivary glands has only been identified in the rare salivary-duct carcinoma, whereas, it is an established feature of carcinomas of other glands. A fortuitous observation of what appeared to be intraductal tumor in a salivary adenocarcinoma, not otherwise specified, led to the present investigation to determine whether intraductal neoplasia is a significant feature of this carcinoma. Intraductal tumor confined by normal CK14 positive, actin-negative ductal basal cells was identified in 15 of 22 cases (68%). The degree of cellular atypia and the pattern of growth of intraductal tumor was similar to that of the invasive tumor. Cases with intraductal tumor devoid of invasive tumor were not found. Intraductal tumor is identified as the pre-invasive precursor of adenocarcinoma, not otherwise specified, and apparently develops in excretory ducts. The findings support the possibility that different salivary tumors arise from different types of parenchymal cell. Possibly intraductal neoplasia is a universal feature of many types of salivary tumor, but has been overlooked because of the need to use immunohistology to demonstrate it and because it may no longer be present as such when the tumor presents as a clinical lesion. PMID- 16673124 TI - [Rectal perforation as a late complication of ProACT implantation]. AB - Operative approaches to solve severe urinary incontinence following radical prostatectomy include artificial urinary sphincters, bulking agents, urethral sling procedures, and the ProACT device (Uromedica Inc.). Previously reported complications of the ProACT procedure comprise ruptures and dislocations of balloons as well as erosions and perforations of the bladder or urethra. We report the case of a rectal perforation as a late complication of the ProACT procedure after prostatectomy and adjuvant radiotherapy. To the best of our knowledge this complication has not been described before. PMID- 16673125 TI - [Coincidence of testicular germ cell tumor and sarcoidosis: A diagnostic challenge]. AB - Several case reports and small case series have described a total of 66 patients with sarcoidosis and testicular cancer so far. This report describes three additional cases. We highlight the association of sarcoidosis and testicular cancer and comment on the potential impact of this connection on the interpretation of the radiological and pathological findings in suspected cancer relapse. Sarcoidosis, a condition that can be combined with testicular cancer, should always be considered in the differential diagnosis. PMID- 16673126 TI - Unusual radiological findings in a case of myelofibrosis secondary to polycythemia vera. PMID- 16673127 TI - Clinical role of Bcl-2, Bax, or p53 overexpression in peripheral T-cell lymphomas. AB - Apoptosis pathways are known to be involved in the pathogenesis of peripheral T cell lymphomas (PTCLs). As such, the current study attempted to investigate the overexpression of Bcl-2, Bax, or p53 with respect to the progression of PTCL. Paraffin-embedded specimens from 74 patients were analyzed immunohistochemically for Bcl-2, Bax, or p53 overexpression including PTCL-unspecified (n=45), extranodal natural killer cell/T-cell lymphoma (n=10), angioimmunoblastic T-cell lymphoma (n=7), anaplastic large cell lymphoma (n=7), and cutaneous T-cell lymphoma (n=5). The Bcl-2 overexpression was exhibited in 33 (45%), Bax, 17 (23%), and p53, 33 patients (45%). Bcl-2 overexpression was strongly associated with advanced stage (p=0.021) and higher international prognostic indices (IPI) (p=0.038). Bcl-2(+)/p53(+) group was found to be associated with advanced stage (p=0.008) and higher IPI (p=0.001), compared with the other groups. The independent expression of Bcl-2 or p53 was not correlated with survival. Meanwhile, when confined to Bcl-2 overexpressing groups, p53 overexpression was significantly associated with poor survival (p=0.05), as the 3-year OS rate was 82.5% for Bcl-2(+)/p53- cases, yet only 32.9% for Bcl-2(+)/p53(+) cases. Multivariate analyses for OS found the Bcl-2/p53 co-expression (p=0.004) as independent prognostic factor, together with advanced stage (p<0.001) and higher prognostic index for PTCL (p=0.008). Bcl-2 overexpression seemed to correlate with the progression of PTCL interacting with a p53-dependent pathway. PMID- 16673129 TI - Pure esophageal atresia with no gap and associated intra-abdominal calcification. AB - Pure esophageal atresia (PEA) is characterized by a long gap in between the two pouches. Primary anastomosis is usually not feasible and various techniques have been described in the literature to bridge this gap. We describe a case of PEA where there was no gap externally in between the two pouches and associated intra abdominal calcification. PMID- 16673131 TI - Anemia in rheumatoid arthritis: high prevalence of iron-deficiency anemia in Indian patients. AB - Anemia in rheumatoid arthritis (RA) is multi-factorial. We studied the prevalence and type of anemia and its correlation with disease variables in RA patients. Among patients with RA anemia was defined as hemoglobin A (W1326X) and 9557C>G (S3186X), were observed in five Pakistani families. Four novel nonsynonymous sequence variants, 9118insCATT, 9238A>T (L3080X), 9539A>C (Q3180P), and 1260delTCAAGTC, were found to segregate within four families, but were not observed in 200 Pakistani control chromosomes. One of the variants, 9539A>C (Q3180P), occurred in the IQ 79 domain, but its functional significance awaits definition. PMID- 16673150 TI - Insulin inhibits phagocytosis in normal human neutrophils via PKCalpha/beta dependent priming of F-actin assembly. AB - OBJECTIVE: This study investigated the effects of insulin on the phagocytosis of C3bi - and IgG-opsonized yeast particles in normal human neutrophils. METHODS: Neutrophils were incubated in different insulin concentrations for 30 minutes and stimulated by C3bi - or IgG-opsonized yeast particles. Phagocytosis was quantified by both light microscopy and FACscan flow cytometry. Laser confocal microscopy was used for quantification of F-actin levels. RESULTS: Elevated insulin concentrations decreased neutrophil phagocytosis of both types of targets. This defect was shown to be in part due to a delayed phagocytosis in the presence of insulin. Following a 30 minute incubation, insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The specific PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. CONCLUSION: Hyperinsulinemia in vitro can inhibit phagocytosis of opsonized targets in normal human neutrophils. This effect of insulin is dependent on activation of PKCalpha and/or PKCbeta, and these insulin signals may interfere with the dynamic assembly/disassembly and/or distribution of F-actin, which is required for the phagocytosis process. PMID- 16673151 TI - Anti-CD45 antibody enhances lipoxygenase pathway of human naive mononuclear cells and cyclooxygenase pathway of neutrophils. AB - OBJECTIVE AND DESIGN: Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human naive MNC and PMN. MATERIALS AND METHODS: Human naive MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab')(2) antibody or non-specific IgG F(ab')(2) for 30 min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5 lipoxygenase (5-LOX) and leukotriene A(4) hydrolase (LTA(4) hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE(2) and LTB(4) in cultured supernatants was measured by EIA kits. RESULTS: Anti-CD45 IgG F(ab')(2) up-regulated LTA(4) hydrolase mRNA expression and LTB(4) production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of naive MNC compared to non-specific IgG F(ab')(2). In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway. CONCLUSIONS: A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA(4) hydrolase in MNC were enhanced. PMID- 16673152 TI - Inflammation-induced modulation of cellular galectin-1 and -3 expression in a model of rat peritonitis. AB - OBJECTIVE AND DESIGN: To investigate the effect of galectin-1 (Gal-1) and -3 (Gal 3) on leukocyte migration and analyze the expression of both galectins in inflammatory cells using a model of rat peritonitis. MATERIAL OR SUBJECTS: Sprague-Dawley rats (n = 4 per group). TREATMENT: Peritonitis was induced in animals through intraperitoneal injection of carrageenin (1.5 mg/kg) and rat mesenteries were analyzed at different time points (0, 4, 24 and 48h). For pharmacological treatment, rats received intravenous injection of Gal-1 or -3 (3microg/kg) followed by carrageenin. METHODS: Western blotting and immunoelectron microscopy analysis. Statistical analysis was performed using ANOVA followed by Bonferroni test. RESULTS: Pharmacological treatment with Gal-1, but not Gal-3, inhibited (approximately 50 %) leukocyte recruitment into the peritoneal cavity at 4 h time-point. In this early phase, immunogold staining of mesenteries showed a diminished Gal-3 expression in degranulated mast cells and Gal-1 in transmigrated neutrophils (approximately 20 % reduction compared to intravascular cells). In the later phases (24 and 48h), leukocyte turnover was associated with augmented Gal-1 expression in neutrophils and macrophages and Gal 3 in mast cells and macrophages. CONCLUSIONS: These results point to a balanced expression of cell-associated-Gal-1/Gal-3 and might impact on the development of new therapeutic strategies for inflammatory diseases. PMID- 16673153 TI - Verapamil modulates LPS-induced cytokine production via inhibition of NF-kappa B activation in the liver. AB - OBJECTIVE: To investigate the effect of verapamil on Lipopolysaccharide (LPS) induced cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-10 (IL-10)] and nuclear factor kappa B (NF-kappa B) in the liver. METHODS AND MATERIALS: Adult male Sprague-Dawley rats were randomly divided into seven groups of eight rats each: control rats treated with saline (0.9 % NaCl); rats treated with saline and then challenged intraperitoneally with LPS (10 mg/kg); rats treated intraperitoneally with different levels of verapamil (1, 2.5, 5, 10 mg/kg) and then challenged with LPS (10 mg/kg); and rats treated only with verapamil (10 mg/kg). TNF-alpha, IL-6, IL-10 and NF-kappa B in the liver tissues were investigated as well as the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) one hour after LPS injection. RESULTS: LPS alone stimulated production of TNF-alpha, IL-6 and IL-10, and activated NF kappa B in the liver. Pretreatment with verapamil before LPS challenge reduced acute liver injury, down-regulated production of LPS-induced pro-inflammatory cytokines (TNF-alpha and IL-6), up-regulated production of anti-inflammatory cytokines (IL-10) and inhibited NF-kappa B activation in the liver in a dose dependent manner. CONCLUSION: Verapamil can attenuate acute liver injury by down regulating the production of TNF-alpha and IL-6 and up-regulating IL-10 in the liver, possibly via inhibition of NF-kappa B. PMID- 16673154 TI - Establishment of a cell-based drug screening system for identifying selective down-regulators of mPGES-1. AB - OBJECTIVE: To establish a drug screening system based on transcriptional regulation of microsomal PGE(2) synthase 1 (mPGES-1), cyclooxygenase 1 (COX-1) and 2 (COX-2) for discovering selective down-regulators of mPGES-1. METHODS: The upstream regulatory sequences of mPGES-1, COX-1, COX-2 were respectively cloned into pGL3B-neo vector containing luciferase gene and neomycin resistance gene (the pGL3B-neo vector had been previously constructed by cloning the neomycin resistance gene into the Sal I site of the pGL3-Basic vector). After that, the recombinant reporter gene vectors pGL3B-neo/mPGES-1, pGL3B-neo/COX-1, pGL3B neo/COX-2 were respectively transfected into A549 cells and therefore stable cell lines, namely M 1, M 2 and M 3, were obtained. Samples were detected then by testing luciferase activity of M 1, M 2 and M 3 cells in microtiter wells to identify compounds that can selectively down-regulate mPGES-1 expression. Through luciferase activity testing, the compounds which had more than 40 % inhibition ratio on M 1 and less than 20 % inhibition ratio on M 2 and M 3 cells could be regarded as hits. RESULTS: Using the cell-based reporter gene assay, we screened compounds for selectively down-regulation of mPGES-1 expression and several compounds were discovered. CONCLUSION: A cell-based drug screening system was established to screen selective down-regulators of mPGES-1 expression, and compound CM188 was identified, which might become a lead compound for novel anti arthritic drugs. PMID- 16673155 TI - Mechanisms of cell death induced by the neutrophil antimicrobial peptides alpha defensins and LL-37. AB - OBJECTIVE: The aim of this study was to investigate the mechanisms of cell death mediated by the antimicrobial peptides neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) and LL-37. MATERIALS AND METHODS: HNP1-3- and LL-37 mediated cell death was assessed in human lung epithelial cells and Jurkat T cells in serum-free culture media. RESULTS: Both HNP1-3 and LL-37 induced cell death in Jurkat T-cells and A549 cells. HNP1-3 but not LL-37 induced caspase-3/-7 activity and caused cleavage of [ADP-ribose] polymerase (PARP) in Jurkat cells, while in A549 cells neither peptides induced caspase-3/-7 activation. Furthermore, both peptides increased mitochondrial cytochrome c release in A549 and Jurkat cells. Our observation that over-expression of the anti-apoptotic protein Bcl-2 in Jurkat cells did not affect HNP1-3- or LL-37-induced cell death indicates that antimicrobial peptide-induced cytochrome c release is not involved in peptide-induced cell death. Finally, in A549 cells and in primary bronchial epithelial cells, both HNP1-3 and LL-37 induced DNA breaks as demonstrated by increased TUNEL labelling. CONCLUSIONS: The results from this study suggest that the antimicrobial peptides HNP1-3 and LL-37 induce cell death, which is associated with mitochondrial injury and mediated via different intracellular pathways. PMID- 16673157 TI - Risky sexual behavior among women with protective orders against violent male partners. AB - The purpose of this study was to describe risky sexual behaviors among women with protective orders against violent male partners (N=673), as well as to examine associations of relationship factors, psychological abuse, severity of partner violence, sexual violence, and women's substance abuse with risky sexual behavior. An HIV sexual risk index was computed based on the participant's self reported risky sexual behavior, the participants' estimations of the abusive partner's extra dyadic sexual behavior and the abusive partner's illicit drug use. The majority of women engaged in risky sexual practices and had partners who engaged in risky sexual practices. Results of OLS regression analysis showed that the participant's age, length of the participant's relationship with the abusive partner, severity of physical violence, substance abuse/dependence (alcohol and illicit drug) were significantly associated with risky sexual behavior. Implications for future research and HIV prevention interventions with partner violence victims are discussed. PMID- 16673158 TI - Examining the role of partner status in an HIV prevention trial targeting HIV positive gay and bisexual men: the seropositive urban men's intervention trial (SUMIT). AB - The goal of this analysis was to examine whether partner status impacted behavioral outcomes from the Seropositive Urban Men's Intervention Trial (SUMIT). We contrasted sample characteristics of HIV-positive gay and bisexual men with and without main partners and then examined differences in sexual behaviors among men participating in an enhanced HIV prevention intervention compared to those in a standard condition. Logistic regression analyses were performed to differentiate characteristics of men with and without main partners and mixed model logistic regression analyses were performed to test for intervention effects. Men with main partners showed a greater likelihood of having unprotected anal sex with any partner, although the number of male sexual partners was higher for men without a main partner. SUMIT generally produced few differences in sexual risk behaviors and did not affect several potential mediator variables of the relationship between the intervention and sexual behaviors with main and nonmain partners. PMID- 16673160 TI - DNA damage in male gonad cells of Green mussel (Perna viridis) upon exposure to tobacco products. AB - DNA damage (determined by the Comet Assay) and the occurrence of deformed nuclei were measured as endpoints of genotoxicity in male gonad cells of the marine mussel (Perna viridis). Upon exposure of the organism to varying concentrations of extracts of smoked and non-smoked cigar tobacco over a period of 16 days, DNA damage was found to be highest in marine mussels exposed to extracts of smoked cigar tobacco. Conversely, more deformed nuclei were detected in marine mussels exposed to extracts of non-smoked cigar tobacco. The level of DNA damage and the number of deformed nuclei reach a maximum at day 12 of exposure to both extracts but decrease thereafter. This phenomenon is attributed to the organism's capacity to maintain the integrity of its genetic material upon exposure to potential genotoxicants present in the tobacco extracts. A dose response in DNA damage and deformed nuclei was also detected in isolated gonad cells upon in vitro exposure to hydrogen peroxide a known DNA strand breaking agent. The results of this study indicate that the DNA in male gonad cells of the marine mussel is damaged upon exposure to genotoxicants, and suggests the suitability of the organism for future investigations into the effect of such agents on its reproductive capacities. PMID- 16673161 TI - Genotoxicity of cadmium in marine diatom Chaetoceros tenuissimus using the alkaline Comet assay. AB - Genotoxic effects of cadmium on phytoplankton Chaetoceros tenuissimus have been evaluated using DNA damage by Comet assay. Cadmium concentrations ranging from 2.4 to 10 mg/l were used to evaluate the effects. Results showed that as the concentration of Cd increased growth of the diatom decreased. Alkaline single cell gel electrophoresis (Comet assay) method, which is highly sensitive in detection of DNA damage in eukaryotic cells, was used to observe genomic changes in marine diatom cells. DNA damage was measured as percent number of comets and normal cells. 65% cells were found to be damaged at 10 mg/l concentration of Cd as compared to 23% in 2.4 mg/l and only 5% in controls. More than 50% apoptotic cells were observed on 8th day at 10 mg/l and 12th day at 7.5 mg/l concentrations. At lower Cd concentrations (4.5 mg/l and below) the damage was below 30% till the last day. This suggested that higher Cd levels have early damaging effects on cell nuclear material and that % injury increases with advancement of exposure period. One advantage of use of C. tenuissimus is the ease with which it can be cultured in a defined medium. C. tenuissimus diatom can be used as an in vivo model for ecogenotoxicity assessment using the Comet assay. PMID- 16673162 TI - Subcellular/tissue distribution and responses to oil exposure of the cytochrome P450-dependent monooxygenase system and glutathione S-transferase in freshwater prawns (Macrobrachium malcolmsonii, M. lamarrei lamarrei). AB - Subcellular fractions (mitochondrial, cytosolic and microsomal) prepared from the tissues (hepatopancreas, muscle and gill) of freshwater prawns Macrobrachium malcolmsonii and Macrobrachium lamarrei lamarrei were scrutinized to investigate the presence of mixed function oxygenase (MFO) and conjugating enzymes (glutathione-S-transferase, GST). Cytochrome P450 (CYP) and other components (cytochrome b(5); NADPH-cytochrome c (CYP) reductase and NADH-cytochrome c reductase activities) of the MFO system were predominantly present in the hepatic microsomal fraction of M. malcolmsonii and M. lamarrei lamarrei. The results are in agreement with the notion that monooxygenase system is mainly membrane bound in the endoplasmic reticulum, and that the hepatopancreas is the major metabolic tissue for production of biotransformation enzymes in crustaceans. Further, the prawns were exposed to two sublethal (0.9 ppt (parts per thousand) and 2.3 ppt) concentrations of oil effluent. At the end of 30th day, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. The accumulations of hydrocarbon in the tissues gradually increased when exposed to sublethal concentrations of oil effluent and were associated with significantly enhanced levels of cytochrome P450 (180.6+/-6.34 pmol mg(-1) protein (P<0.05 versus control, 136.5+/-7.1 pmol mg(-1) protein) for 2.3 ppt and 305.6+/-8.5 pmol mg(-1) protein (P<0.001 versus control, 132.3+/-6.8 pmol mg(-1) protein] for 0.9 ppt of oil exposed M. malcolmsonii; 150+/-6.5 pmol mg(-1 )protein (P<0.01 versus control, 84.6+/-5.2 pmol mg(-1) protein) for 2.3 ppt and 175+/-5.5 pmol mg(-1) protein (P<0.01 versus control, 87.6+/-5.4 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei), NADPH cytochrome c-reductase activity (14.7+/ 0.6 nmol min(-1 )mg(-1) protein (P<0.05 versus control, 6.8+/-0.55 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 12.1+/-0.45 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 6.9+/-0.42 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. malcolmsonii; 12.5+/-0.31 nmol min(-1 )mg(-1) protein (P<0.001 versus control, 4.6+/-0.45 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 9.6+/-0.32 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 4.9+/-0.41 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei) and cytochrome b(5 )(124.8+/-3.73 pmol mg(-1) protein (P<0.01 versus control, 76.8+/-4.2 pmol mg(-1) protein) for 2.3 ppt and 115.3+/-3.86 pmol mg(-1) protein (P<0.01 versus control, 76.4+/-4.25 pmol mg(-1 )protein) for 0.9 ppt of oil exposed M. malcolmsonii and 110+/-3.11 pmol mg(-1) protein (P<0.01 versus control, 63.7+/-3.24 pmol mg(-1 )protein) for 2.3 ppt and 95.3+/-2.63 pmol mg(-1) protein (P<0.01 versus control, 61.4+/-2.82 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei). The enhanced levels of biotransformation enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in crustaceans, and the response offers the possibility of use as a biomarker for the early detection of oil pollution. PMID- 16673163 TI - Biomarkers of marine pollution and bioremediation. PMID- 16673164 TI - Tribute to a frontline scientist in marine pollution research. PMID- 16673172 TI - The birth of neurochemical maps. PMID- 16673165 TI - Bioremediation of toxic substances by mercury resistant marine bacteria. AB - Bioremediation of toxic substances includes microbe-mediated enzymatic transformation of toxicants to non-toxic, often assimilable, forms. Mercury resistant marine bacteria are found to be very promising in dealing with mercury, and a host of other highly toxic heavy metals and xenobiotics. In the present studies we have shown that the Pseudomonas aeruginosa CH07 (NRRL B-30604) has been able to degrade a variety of PCB congeners including a complete degradation of CB-126 and CB-181. The culture was able to remove over 70% Cd from growth medium when supplemented with 100 ppm Cd. The same bacterium rapidly biotransformed/removed toxic mercury from wastewater in a bioreactor system. PMID- 16673173 TI - Lessons learned from Miki. PMID- 16673174 TI - Synaptic plasticity in the hippocampus: effects of estrogen from the gonads or hippocampus? AB - Different effects of estrogen on synaptic plasticity have [corrected] been reported. Here, we summarise effects of low, gonad-derived serum estrogen concentrations, of intermediate concentrations, provided by hippocampal cells, and of pharmacological doses of estrogen on synapses and spines and on the expression of synaptic proteins. No effects of low concentrations were found. To study the effects of hippocampus-derived estradiol, we inhibited hippocampal estrogen synthesis by treatment of hippocampal cell cultures with letrozole, an aromatase inhibitor. Alternatively, we used siRNA against Steroidogenic acute regulatory protein (StAR). Spines, synapses, and synaptic proteins were significantly down regulated in response to letrozole and in siRNA-StAR transfected cells. Application of high pharmacological doses of estradiol promoted only synaptophysin expression, a presynaptic protein, but did not increase the number of boutons. Our results point to an essential role of endogenous hippocampal estrogen in hippocampal synaptic plasticity rather than to a direct influence of estrogens derived from peripheral sources, such as the gonads. PMID- 16673175 TI - Continuous monitoring of post mortem temperature changes in the human brain. AB - Recent developments in neurochemistry research on the post mortem human brain require a detailed understanding of the post mortem changes in the human brain, including the correlation between time related temperature changes and alterations in biochemical parameters. As an initial step towards our deeper insight into the intricate relationships between post mortem time, temperature and neurochemical processes, in the present study we set out to monitor continuously temperature changes in the post mortem human brain in eight cadavers for a period of up to 24 h after death under 'standard' clinical conditions at a neurosurgery clinic. A main objective of the study was to find a simple and reliable mathematical formula, requiring only time and an easily obtainable body temperature measurement parameter, with the help of which the superficial and deep brain temperatures can be obtained without invasive interactions. With a portable thermoprobe data logger system superficial (4 cm from skull surface) and deep (8 cm) brain temperatures, the temperature of the liver and that of the forehead skin, as well as the ambient temperature of the room were measured at regular time intervals (every 1 or 5 min). Various mathematical models were fitted to the data in order to create a simple model capable to predict brain temperatures from easily accessible measurements, such as that of the forehead skin. On the basis of the tested models we propose that with simple polynomial equations the deep and superficial brain temperatures can be described reliably as T (br4) ( degrees C)=T (fh)-0.001t (3)+0.0541t (2)-1.0622t+7.5933 and T (br8) ( degrees C)=T (fh)-0.0003t (3)+0.0201t (2)-0.619t+7.9036, respectively, where T (br4) is the superficial (4 cm) brain temperature, T (br8) is the deep (8 cm) brain temperature, T (fh) is the forehead temperature and t is the time from death. These measurements can, in combination with further neurochemical studies, contribute to our better understanding of the human brain's time- and temperature related post mortem biochemical changes. PMID- 16673176 TI - Regional distribution and relative abundance of serotonin(2c) receptors in human brain: effect of suicide. AB - Abnormalities in serotonin receptor subtypes have been observed in the postmortem brain of suicide victims. We examined the regional distribution of serotonin (5HT)(2C) receptor mRNA in several areas of the human brain and also compared its protein and mRNA expression in the prefrontal cortex (PFC), hippocampus, and choroid plexus between suicide victims and normal control subjects. 5HT(2C) receptors were found to be distributed in several areas of the human brain (in order of abundance): highly concentrated and richest in choroid plexus; hypothalamus; nucleus accumbens; with the lowest abundance in PFC and cerebellum. Comparison of 5HT(2C) receptors between suicide victims and control subjects showed higher protein levels in the PFC but not the hippocampus or choroid plexus of suicide victims. However, there were no significant differences in mRNA levels between suicide victims and control subjects in these brain areas. These results suggest that 5HT(2C) receptors are richly distributed throughout the brain with the highest level in the choroid plexus and that abnormalities in protein expression of 5HT(2C) receptors in the PFC may be associated with suicide. PMID- 16673178 TI - Engaging diversity's underbelly: a story from an immigrant parish community. AB - : This story explores an intervention conducted in a Catholic parish community in New York City. The intervention, conducted by the author and a Jesuit priest, focused on issues of unity and diversity among the various Chinese immigrant subgroups in the parish (primarily Cantonese- and Mandarin-speakers). Issues of class, power, and a history of colonialism in the Catholic Church are explored as central to the relations among culturally diverse Chinese American community members and between the members and the practitioners and the church authority. The author especially focuses on how the dynamics that played out in the intervention reflected wider issues of economics, labor practices, and political elitism in the wider Chinatown community. A central part of the author's argument is about power relationships between this parish community and Chinatown and how these power relationships are embedded within broader racial and economic oppression within the United States. PMID- 16673179 TI - Maternal body mass index and self-reported postpartum depressive symptoms. AB - OBJECTIVES: The association between extremes of body mass index (BMI) and depression in women has been documented, yet little is known about the relationship between obesity and postpartum depression (PPD). This study seeks to characterize the association between BMI and PPD. METHODS: The 2000-2001 Utah data from Pregnancy Risk Assessment Monitoring System (PRAMS) were used to determine the proportion of women, stratified by prepregnancy body mass index, reporting postpartum depressed mood and stressors during pregnancy. RESULTS: The prevalence of self-reported moderate or greater depressive symptoms was 27.7% (S.E. +/-2.2) in underweight, 22.8% (+/-1.2) in normal weight, 24.8% (+/-2.9) in overweight and 30.8% (+/-2.5) in obese women. After controlling for marital status and income, normal BMI (19.8-25.9) was associated with the lowest rate of self-reported postpartum depressive symptoms. There was a two-fold increase in self-reported depressive symptoms requiring assistance among overweight and obese women compared to normal weight women (1.53% normal, 2.99% overweight, and 3.10% obese [p < 0.001]). Obese women were significantly more likely to report emotional and traumatic stressors during pregnancy than normal weight women. CONCLUSION: This population-based survey suggests a potential association between prepregnancy body mass index and self-reported postpartum depressive symptoms. Prospective studies of association between obesity and PPD, with improved diagnostic precision are warranted. PMID- 16673183 TI - Reader response to colonoscopy versus sigmoidoscopy. PMID- 16673181 TI - Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle. AB - PURPOSE: The circadian rhythm of endogenous corticosterone (CS) may produce fluctuations of downstream gene expression in normal rats. This study examined changes in glucocorticoid receptor (GR) and glutamine synthetase (GS) expression in rat skeletal muscle in relation to plasma CS over a 24-h period. METHODS: Fifty-four normal male Wistar rats were sacrificed at 18 time points (n = 3) over 24 h. Plasma CS concentrations and gastrocnemius muscle GR and GS mRNA and GS activity were measured. RESULTS: The circadian rhythm of plasma CS was captured by a two-harmonic function. The expression of GR and GS mRNA and GS activity follow a circadian rhythm in normal rat skeletal muscle. GR mRNA reaches a trough at 4 h after the peak of plasma CS and it fluctuates between 0.55 and 0.9 fmol g tissue(-1). GS mRNA and activity reach peaks at 6 and 12 h after the endogenous CS peak. GS mRNA oscillates between 3 and 6 fmol g tissue(-1), whereas GS activity fluctuates between 17 and 23 micromol min(-1) g protein(-1). Mechanistic receptor/gene-mediated pharmacodynamic models were applied to describe the temporal patterns of GR mRNA, GS mRNA, and GS activity within the circadian cycle. CONCLUSIONS: The integrated models were able to capture the circadian expression patterns of plasma CS, and GR and GS in normal rat skeletal muscle showing a dependence of tissue gene expression on plasma CS. PMID- 16673184 TI - Reader response to colonoscopy versus sigmoidoscopy. PMID- 16673187 TI - A look forward: arrows in our quiver. PMID- 16673185 TI - Reader response to colonoscopy versus sigmoidoscopy. PMID- 16673188 TI - Teaching the ESSEnCE of office-based prescribing. PMID- 16673192 TI - The wizard of Osler: a brief educational intervention combining film and medical readers' theater to teach about power in medicine. PMID- 16673193 TI - Trained standardized patients can train their peers to provide well-rated, cost effective physical exam skills training to first-year medical students. AB - BACKGROUND AND OBJECTIVES: Teaching physical examination skills effectively, consistently, and cost-effectively is challenging. Faculty time is the most expensive resource. One solution is to train medical students using lay physical examination teaching associates. In this study, we investigated the feasibility, acceptability, and cost-effectiveness of training medical students using teaching associates trained by a lay expert instead of a clinician. METHODS: We used teaching associates to instruct students about techniques of physical examination. We measured students' satisfaction with this teaching approach. We also monitored the financial cost of this approach compared to the previously used approach in which faculty physicians taught physical examination skills. RESULTS: Our program proved practical to accomplish and acceptable to students. Students rated the program highly, and we saved approximately $9,100, compared with our previous faculty-intensive teaching program. CONCLUSIONS: We believe that our program is popular with students, cost-effective, and generalizable to other institutions. PMID- 16673194 TI - Initiating abortion training in residency programs: issues and obstacles. AB - OBJECTIVES: Early abortion is a common outpatient procedure, but few family medicine residencies provide abortion training. We wished to assess experiences and obstacles among residency programs that have worked to establish early abortion services. METHODS: From 2001-2004, 14 faculty participated in a collaborative program to initiate abortion training at seven family medicine residencies. Ten focus groups with all trainees were followed by individual semi structured interviews with a smaller group (n=9) that explored the progress and obstacles they experienced. Individual interviews were recorded and analyzed to identify major themes and sub-themes related to initiating abortion training. RESULTS: Five of seven sites established abortion training. Five major themes were identified: (1) establishing support, (2) administration, (3) finance, (4) legal matters, and (5) security/demonstrators. Faculty from sites where training was ultimately established rated the sub-themes of billing/reimbursement, obtaining staff support, and state/hospital regulations as most difficult. Gaining support from within the department and institution was most difficult for the two sites that could not establish training. None experienced difficulty with security/demonstrators. CONCLUSIONS: Developing the clinical and administrative capacity to provide early abortion services in family medicine residency programs is feasible. Support from leadership within departments and from the wider institution is important for implementation. PMID- 16673195 TI - Attending physicians' and residents' attitudes and beliefs about prescribing buprenorphine at an urban teaching hospital. AB - BACKGROUND AND OBJECTIVES: Opioid abuse and dependence are increasing. Pharmacotherapy with an opioid agonist reduces adverse consequences of opioid dependency. Physicians can now prescribe buprenorphine for opioid dependency in the primary care setting. This study assessed primary care providers' attitudes and beliefs about opioid addiction treatment with buprenorphine. METHODS: Ninety nine resident and attending physicians from six ambulatory clinics associated with a university hospital were interviewed with an adapted questionnaire eliciting attitudes and beliefs about opioid addiction treatment options, including buprenorphine. RESULTS: While only 37.8% of respondents believed primary care providers should prescribe buprenorphine, and 35.7% reported interest in prescribing buprenorphine, 72.1% were willing to prescribe it with training and support. Common training/support needs were buprenorphine education/training (83.8%), available consultation (19.2%), and on-site counselors (18.2%). The most frequent reasons for not prescribing buprenorphine were lack of knowledge or training (47.5%) and lack of time (25.3%). Physicians involved in primary care-oriented programs (versus non-primary care programs) were more likely to have positive attitudes regarding buprenorphine. CONCLUSIONS: Most physicians would be willing to prescribe buprenorphine with proper training and support. Barriers and training/support needs must be addressed to develop effective opioid addiction treatment programs in the primary care setting. PMID- 16673196 TI - Research participation, protected time, and research output by family physicians in family medicine residencies. AB - BACKGROUND AND OBJECTIVES: The Future of Family Medicine project concluded that research must become a greater part of the culture of the specialty. We examined the participation of family physician residency faculty in research, their protected time, and their research output and how these varied by program type. METHODS: This was a cross-sectional survey of all family medicine residency programs in the United States. The response rate was 66% (298/453). RESULTS: The majority of programs reported at least one family physician who participates in research, though the medical school-based (MSB) programs reported a higher total number of faculty than the community-based, medical school affiliated (MSA) programs (9.53 versus 2.72) and percentage of faculty (56% versus 37%). Substantially more MSB programs reported that they had at least one family physician with significant protected time for research (48% versus 7% for > 25% protected time) or any protected time (69% for MSB versus 45% for MSA). MSB programs and MSA programs reported similar success at producing at least one poster or paper for national meetings within the last 3 years (63% versus 41%) but not for published papers (86% versus 43%). CONCLUSIONS: We found that only about half of the family medicine residencies produced any nationally recognized research over a 3-year period and that this represents only a small improvement over the last 10 years. Our findings suggest that more support is needed if research is to become an integral part of the culture of family medicine. PMID- 16673197 TI - Use of antibiotics for adult upper respiratory infections in outpatient settings: a national ambulatory network study. AB - BACKGROUND AND OBJECTIVES: Studies have suggested that antibiotics are often inappropriately used in the treatment of acute upper respiratory tract infections (URIs). This study examined antibiotic use for adult URIs in a large national network of offices that use electronic health records (EHRs). METHODS: Using the Medical Quality Improvement Consortium (MQIC) database, URI episodes were identified for patients ages 18 to 65 years from January 1, 1998, to March 31, 2003. The percentage of episodes for which antibiotics were prescribed and the proportion of antibiotics that were broad spectrum were calculated. RESULTS: Of the 52,135 URI episodes identified, 65% received antibiotics. Antibiotics were prescribed for 78% of acute bronchitis episodes, 65% of acute pharyngitis episodes, 81% of acute sinusitis episodes, and 33% of nonspecific URI episodes. The proportion of antibiotics that were broad spectrum was 56% for all URI episodes, 68% for acute bronchitis, 55% for acute sinusitis and nonspecific URI, and 40% for pharyngitis. CONCLUSIONS: This study demonstrates overuse of antibiotics for adult URIs in a large national ambulatory care network, with particular overuse of broad-spectrum antibiotics. The study also illustrates the enormous potential of EHR data for conducting practice-based research across large national office networks. PMID- 16673198 TI - Is family medicine ready to move toward having professional researchers? PMID- 16673199 TI - Family medicine needs a generation of dreamers. PMID- 16673200 TI - United Kingdom: the role of nurse practitioners in acute care. PMID- 16673201 TI - Sweden: one way to build academic family medicine. PMID- 16673202 TI - Norway: can general practitioners ever be satisfied with their careers? PMID- 16673203 TI - Successful uterus cryopreservation in an animal model. AB - Cryopreservation of whole organs would not only be advantageous for experimental biology but also for transplantation surgery. The objective of this study was to evaluate the feasibility of cryopreserving swine uterus as an experimental model of cryopreservation affecting a whole visceral muscle organ. Organs were frozen slowly (0.2 degrees C/min) after arterial perfusion with 10% dimethyl sulfoxide solution for 10 min and equilibration in this solution for 30 min. Viability of the organs was tested by biochemical parameters, LIVE/DEAD fluorescent staining and capability for contraction in a perfusion system. Ten fresh and ten cryopreserved uteri showed living cells in the LIVE/DEAD assay, and were viable for at least 7 h as shown by their ability to produce rhythmic contractions after oxytocin administration. This is the first report known to us on successful cryopreservation of a whole swine uterus to demonstrate that perfusion with a cryoprotectant and slow freezing provides a promising method for cryopreserving whole organs, and may encourage further studies with other cryoprotective agents and freezing protocols as well as other organs. PMID- 16673204 TI - Morphological and histopathological changes in tongues of experimentally developed acromegaly-like rats. AB - An acromegaly-like rat model recently developed by exogenous administration of insulin-like growth factor I (IGF-I) was used to investigate morphological and histopathological tongue changes and clarify whether the changes were reversible. Human recombinant IGF-I (640 microg/day) was continuously subcutaneously infused into ten-week-old male rats for four weeks (IGF-I group; n = 6). Control sham operated animals were injected saline alone (control group; n = 6). Rats were sacrificed immediately on ending administration at the age of fourteen weeks. Another 12 rats (6 from each group) were housed for an additional four weeks after administration ended. Total IGF-I (human + rat) increased significantly during administration, returning to control levels afterwards. Tongue weights significantly increased with histopathological changes present (increases in the muscle-bundle width, spaces between muscle-bundles and epithelium thickness) in the IGF-I group compared to control rats. Tongue size returned to control levels after discontinuation of IGF-I administration. These findings suggest that the characteristic tongue enlargement was developed experimentally in our acromegaly like rat model, and that such morphological and histopathological tongue changes are reversible on normalization of circulating IGF-I levels. PMID- 16673205 TI - Minodronate suppresses prostaglandin F2alpha-induced vascular endothelial growth factor synthesis in osteoblasts. AB - In our previous study, we showed that prostaglandin F2alpha (PGF2alpha) stimulates vascular endothelial growth factor (VEGF) synthesis via activation of p44/p42 mitogen-activated protein (MAP) kinase via protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells. In addition, we demonstrated that incadronate amplified, and tiludronate suppressed PGF2alpha-induced VEGF synthesis among bisphosphonates, while alendronate or etidronate had no effect. In the present study, we investigated the effects of minodronate, a newly developed bisphosphonate, on PGF (2alpha)-induced VEGF synthesis in MC3T3-E1 cells. Minodronate significantly reduced VEGF synthesis induced by PGF2alpha dose dependently at levels between 3 and 100 microM. PGF2alpha-stimulated phosphorylation of Raf-1, MEK1/2 and p44/p42 MAP kinase were suppressed by minodronate. 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator VEGF synthesis induced by PKC, was inhibited by minodronate. Minodronate inhibited Raf 1, MEK1/2 and p44/p42 MAP kinase phosphorylation induced by TPA. Mevalonate failed to affect the suppressive effect of minodronate on PGF2alpha-induced VEGF synthesis. Taken together, these results indicate that minodronate suppresses PGF2alpha-stimulated VEGF synthesis at the point between PKC and Raf-1 in osteoblasts. PMID- 16673206 TI - Restoration of transforming growth factor-beta type II receptor reduces tumorigenicity in the human adrenocortical carcinoma SW-13 cell line. AB - Transforming growth factor-beta (TGF-beta) is a potent growth suppressor. Acquisition of TGF-beta resistance has been reported in many tumors, and has been associated with reduced TGF-beta receptor expression. In this study, we examined TGF-beta 1, TGF-beta type I receptor (TbetaRI) and TGF-beta type II receptor (TbetaRII) expression in SW-13 adrenocortical carcinoma cells by Northern and Western blot analysis. SW-13 cells did not express TbetaRII mRNA or protein. We have investigated the role of TbetaRII in modulating tumorigenic potential using stably transfected SW-13 cells with TbetaRII expression plasmid. TbetaRII positive SW-13 cell growth was inhibited by exogenous human TGF-beta1 (hTGF beta1) in a dose-dependent manner. In contrast, SW-13 cells and control clones transfected with empty vector remained hTGF-beta1-insensitive. Xenograft examination in athymic nude mice demonstrated that TbetaRII-positive SW-13 cells reduced tumor-forming activity. Reconstructing the TbetaRII can lead to reversion of the malignant phenotype of TbetaRII-negative human adrenocortical carcinoma, which contains SW-13 cells. Reduced TbetaRII expression may play a critical role in determining the malignant phenotype of human adrenocortical carcinoma. PMID- 16673207 TI - Transient chemokine receptor blockade does not prevent, but may accelerate type 1 diabetes in prediabetic NOD mice. AB - The influx of autoreactive lymphocytes into the site of an autoimmune inflammation is mediated by certain chemokines. Autoimmune insulitis in type 1 diabetes is viewed as the result of destructive Th-1-cells and their corresponding antigen-presenting cells infiltrating the pancreatic islets. Blocking the chemokine receptors that mediate a Th-1-reaction has been shown to reduce autoimmunity in other experimental autoimmune disorders. We used the NOD mouse model to investigate the potency of anti-CCR2 and anti-CCR5 antibodies to inhibit the influx of Th-1-cells into the pancreatic islets, thus preventing diabetes onset. Eleven-week-old female NOD mice were treated with 500 microg of a monoclonal anti-CCR5 or anti-CCR2 or an isotype control antibody every third day over two weeks. We did not observe any preventive effect in either treatment group, but accelerated diabetes onset in the anti-CCR5 treated group. The number of autoantigen-specific Th-1-cells detected in the two treated groups was not reduced, but increased in the anti-CCR5 group. Redundancy within the chemokine system may account for this lack of prevention, or the intervention may have come too late in the disease process. Furthermore, blocking Th-1 chemokine receptors in the late autoimmune process may also inhibit regulatory T-cells, thus accelerating rather than preventing the disease. PMID- 16673208 TI - Once-daily insulin glargine administration in the morning compared to bedtime in combination with morning glimepiride in patients with type 2 diabetes: an assessment of treatment flexibility. AB - AIMS: To compare the incidence of nocturnal hypoglycemia and glycemic control following bedtime or morning insulin glargine (LANTUS; glargine) plus glimepiride. METHODS: In this 24-week, multinational, open, randomized study, 624 patients with type 2 diabetes poorly controlled on oral therapy received morning or bedtime glargine plus morning glimepiride (2, 3 or 4 mg) titrated to a target fasting blood glucose level < or = 5.5 mmol/l. RESULTS: The incidence of nocturnal hypoglycemia was equivalent between the two groups, with morning glargine non-inferior to bedtime (13.0 VS. 14.9 % of patients; between-treatment difference -1.9 %; one-sided 95 % confidence interval -100 %; 2.84 %). At endpoint, similar improvements in glycemic control were observed with morning compared to bedtime glargine: HbA1c: -1.65 +/- 1.21 VS. -1.57 +/- 1.16 %; p = 0.42; fasting blood glucose: -4.25 +/- 2.82 VS. -4.48 +/- 2.75 mmol/l; p = 0.08. The endpoint mean daily glargine dose was comparable (34.7 +/- 17.4 VS. 32.4 +/- 17.0 IU; p = 0.15), and there was no significant between-treatment difference in the change in body weight (2.1 VS. 1.8 kg; p = 0.39). CONCLUSIONS: Once-daily glargine can be administered in a flexible morning or bedtime regimen (plus morning glimepiride) to achieve good glycemic control without any difference in hypoglycemia. PMID- 16673209 TI - Beta cell function, insulin resistance and plasma adiponectin concentrations are predictors for the change of postprandial glucose in non-diabetic subjects at risk for type 2 diabetes. AB - Insulin resistance, impaired insulin secretion, and low adiponectin levels have been shown to be predictors for type 2 diabetes. However, it is not yet clear whether these associations (1) are independent of changes in body weight, or (2) are valid for changes in glucose tolerance in the prediabetic state. Sixty-two non-diabetics (50 with normal glucose tolerance) aged 41 +/- 11 years, BMI 30.5 +/- 5.3 kg/m2 (mean +/- SD) were studied twice with a standard oral glucose tolerance test (oGTT, mean follow-up time 3.0 +/- 1.8 years (mean +/- SD) [range 0.5-6.5 years]). Insulin sensitivity and insulin secretion were estimated from oGTT using validated indices. Two-hour blood glucose during oGTT deteriorated over time (baseline 2 h glucose 6.32 +/- 0.21 VS. follow-up 2 h glucose 7.14 +/- 0.22 mM, p < 0.001) while the percentage body fat did not change (32.7 +/- 1.2 VS. 32.6 +/- 1.2%, p = 0.46). Follow-up 2 h blood glucose was predicted by adiponectin (p = 0.01), baseline insulin sensitivity (p = 0.02) and baseline insulin secretion relative to insulin sensitivity (p = 0.03) independent of sex, age, baseline 2 h blood glucose or change in percentage body fat. Our results suggest that low adiponectin levels, insulin resistance and low beta cell function predict the continuous deterioration of glucose tolerance in early prediabetic states, independent of changes in adiposity. Therefore, the early influence of these parameters should be the subject of future prevention programs to prevent deterioration of glucose tolerance. PMID- 16673210 TI - Effects on the male endocrine system of long-term treatment with gonadotropin releasing hormone agonists and estrogens in male-to-female transsexuals. AB - We studied hormonal changes resulting from long-term treatment with gonadotropin releasing hormone agonist and 17beta estradiol valerate in 40 healthy middle-aged male-to-female transsexuals over a period of two years. All of the participants received injections of 3.8 mg goserelin acetate every four weeks in combination with 6 mg oral 17beta estradiol valerate per day for cross-sex hormone treatment for male-to-female transsexuals. There was a significant reduction in the levels of serum luteinizing hormone and follicle-stimulating hormone to the hypogonadal stage. Mean testosterone levels decreased by 97% to 0.52 and 0.59 nmol/l after 12 months and 24 months, respectively. There was a significant reduction in dehydroepiandrosterone sulfate by 37% after 12 months and 43% after 24 months, and androstendione by 29% after 12 months and 27% after 24 months, respectively. Cortisol levels were reduced by 43% and 50%, respectively. Estrogen levels were significantly increased from 77.51 to 677 after 12 months and 661 pmol/l after 24 months. Sex hormone-binding globulin and corticoid-binding globulin levels were significantly increased after 12 and 24 months. There was a significant decrease in all measured androgen fractions and cortisol during long-term treatment with gonadotropin-releasing hormone agonist and 17beta estradiol valerate. Apart from suppression of testicular hormone production, one possible interpretation is that treatment with long-term gonadotropin-releasing hormone agonist and 17beta estradiol valerate influences adrenal hormone levels in healthy middle-aged male to-female transsexuals. Cortisol serum levels may be decreased due to estrogen induced increase in corticoid-binding globulin. PMID- 16673211 TI - Effects of allopurinol on beer-induced increases in plasma concentrations and urinary excretion of purine bases (uric acid, hypoxanthine, and xanthine). AB - To determine the effects of allopurinol on beer-induced increases in plasma and urinary excretion of purine bases (hypoxanthine, xanthine, and uric acid), we performed three experiments on five healthy study participants. In the first experiment (combination study), the participants ingested beer (10 ml/kg body weight) eleven hours after taking allopurinol (300 mg). In the second experiment (beer-only study), the same participants ingested beer (10 ml/kg body weight) alone, while in the third experiment (allopurinol-only study), they took allopurinol (300 mg) alone. There was a two-week interval between each of the studies. Beer-induced increases in plasma concentration and urinary excretion of hypoxanthine in the combination study were markedly higher than those in the beer only study. On the other hand, the sum of increases in plasma concentrations of purine bases in the beer-only study was greater than in the combination study, whereas the increase in plasma uridine concentration in the combination study did not differ from the beer-only study. In addition, allopurinol administration inhibited the beer-induced increase in plasma concentration of uric acid. These results suggest that abrupt adenine nucleotide degradation may increase plasma concentration and urinary excretion of hypoxanthine under conditions of low xanthine dehydrogenase activity, which is mostly ascribable to allopurinol. Further, the difference in the sum of increases in plasma concentrations of purine bases between the combination study and beer-only study was largely ascribable to a greater increase in urinary excretion of hypoxanthine in the combination study. In addition, allopurinol intake seems to be effective in controlling the rapid increase in plasma uric acid caused by ingestion of alcoholic beverages. PMID- 16673212 TI - Serum levels of macrophage colony-stimulating factor in singleton and twin pregnancy. AB - Macrophage colony-stimulating factor (M-CSF) is present in the placenta and has been implicated in placental growth and development. M-CSF levels in peripheral blood increase significantly with progression of pregnancy in uncomplicated singleton pregnancy. This study investigated whether serum M-CSF levels in singleton pregnancy differed from those in twin pregnancy. A hundred and four pregnant women, of whom fifty-nine were women carrying single fetuses and forty five were women with twin fetuses, participated in the study. Their average gestational age at entry was 10, 20, 30, and 38 weeks of gestation in singleton pregnancy and 10, 20, 30, and 35 weeks in twin pregnancy. Peripheral blood was collected and serum was separated after centrifugation and stored at - 20 degrees C. M-CSF levels were determined by ELISA. In both groups, the serum levels of M CSF increased significantly as pregnancy progressed. M-CSF levels were relatively higher in twin pregnancy than in singleton pregnancy at 10, 20, and 30 weeks. At 35 and 38 weeks, the M-CSF levels were significantly higher in twin pregnancy than in singleton pregnancy. Elevation of serum M-CSF supports M-CSF production in the placenta. This elevation in twin pregnancy may be related to increased demand of M-CSF in twin pregnancy. PMID- 16673214 TI - Gout--new insights into a forgotten disease. PMID- 16673213 TI - Acute vascular events and electrolytes variations in elderly patients. AB - Alterations in electrolyte balance have been claimed to play a role in the pathophysiology of coronary heart disease; however, the relationship between the electrolyte pattern and other clinical variables immediately after an acute vascular event is unclear. The aim of the present study was to test whether electrolyte and microelement changes characterize the acute phase in patients with different degrees of glucose tolerance admitted to the hospital shortly after an acute cardiovascular or cerebrovascular ischemic event. Two hundred consecutive patients with either myocardial infarction or stroke (SP group), stratified by degree of glucose tolerance, were studied within six hours of admission, and compared against 125 patients admitted for conditions other than acute vascular ischemia (CP). Routine laboratory parameters and serum Na, K, Cl, Mg and Ca concentrations were determined in all patients and compared to those recorded within six months before the admission. Relative to CP and independently of confounding factors including glucose tolerance status, the SP group showed significantly higher plasma glucose and insulin concentrations, higher creatinine and a modified serum electrolyte pattern characterized by significantly lower potassium and magnesium levels and by hypercalcemia and hyperphosphatemia. Irrespective of glucose tolerance, the first hours following an acute vascular event are characterized by marked insulin resistance with a consistent shift in the serum electrolyte pattern. This pattern is the physiological consequence of the attendant compensatory hyperinsulinemia. Its significance for the evolution of ischemic damage remains to be established. PMID- 16673215 TI - [Surgery -- interface in medicine]. PMID- 16673216 TI - [Clinical pathways -- first results of a systematic IT-supported application at a surgical department of a university hospital]. AB - BACKGROUND AND OBJECTIVE: In analogy to industrial process management, clinical pathways have been introduced for operational process organisation in orthopedic and interventional procedures as well as for conservative treatments. Within this study, introduction and continuous, systematic application of clinical pathways at a surgical department of a university hospital was investigated. METHODS: Analysis was performed by IT-based (SAP/i. s. h.med) evaluation of the registered data regarding pathway violations. Additionally, patients' records were matched with the computerized data for quality assurance of pathway documentation. RESULTS: 501 patients were entered in 16 clinical pathways (31 +/- 39 patients per pathway). The mean period from the introduction of a distinct pathway to the data acquisition of the present study ranged from 2 to 14 months (mean 7+/-7.5 months). Pathway violations correlated with the complexity, but not with the period since the pathway was introduced. Organisational reasons next to medical reasons (i. e. delayed gastric function or wound healing) were the most frequent causes for pathway violations. CONCLUSION: Clinical pathways may not only be realised for operative procedures or conservative treatments, but represent a clinical and administrative management instrument at a surgical department of a university hospital for daily work routine. They are suitable as an instrument for clinical quality and risk management, as an economic control instrument in reorganisation phases as well as in the routine of surgical clinics. PMID- 16673217 TI - [Results of the certification of vascular centers by the German Society for Vascular Surgery]. AB - INTRODUCTION: Advances in conventional vascular surgery, endovascular procedures and conservative treatment require close interdisciplinary cooperation. The term Vascular Centre indicates to patients and referring doctors optimal professional and organisational competence. METHODS: 73 applications by vascular centres for certification were made between June 2002 and December 2005, of which after audit 54 were successful for three years, including one centre each in Austria and Switzerland. 13 centres were not accepted and six are still under consideration. This analysis is based on the data of 52 certified centres in the Federal Republic of Germany. RESULTS: A mean of 1149 patients were hospitalized in these centres per year (12 % emergency admissions) and 2159 patients per year were treated as outpatients, 60 % with arterial vascular disease. All centres have vascular surgery and radiology department, 10 of the 52 centres cooperate with specialists in private practice to provide an angiology service. A mean of 460 arterial vascular operations and 239 arterial interventional procedures were performed at each centre annually. In addition there were operations for varicose veins (n = 217), shunt operations (n = 58) and major amputations (n = 42). Inpatient or outpatient conservative treatment (for peripheral vascular disease, diabetic foot syndrome, phlebothrombosis or chronic venous insufficiency) was given to a mean of 338 patients annually. CONCLUSIONS: Certification of interdisciplinary vascular centres provides structural and medical data and the beginnings of comparing such centres. A future task will be standardization of documentation and continuing assessment of the quality of results. PMID- 16673218 TI - [A rare cause of a central bile duct stricture]. AB - HISTORY AND CLINICAL FINDINGS: A 55 year old female patient was diagnosed because of painless jaundice and weight loss. She had undergone conventional cholecystectomy 15 years before. INVESTIGATIONS: Bilirubin level before ERCP and stent implantation was 287 micromol/l at maximum. A subtotal stenosis was found in the proximal common hepatic duct. Endosonographically, a mass of 14 mm diameter was seen. She was suspected of having a Klatskin tumour Bismuth type II. Tumour markers (CEA, CA19-9) were within normal ranges. TREATMENT AND COURSE: One month after stent implantation open surgery was performed suspecting a malignant tumour of the bile duct. A solid tumour at the hepatic bifurcation was resected completely. Reconstruction was performed by hepatico-jejunostomy was formed and the blind end of the jejunum was anastomosed to the gastric antrum to form an "access loop". Upon histological analysis no malignancy was found. However, a distinct fibrosis within the suspicious area was seen, formed around braided suture material. CONCLUSION: Preoperative distinction between benign and malignant bile duct stenosis can be difficult, even impossible. Thus, every resectable bile duct tumour should undergo surgery even and especially if a histological diagnosis cannot be reached preoperatively and the condition of the patient is adequate. PMID- 16673220 TI - [Tetanus following an abrasion injury]. AB - HISTORY AND CLINICAL FINDINGS: A 80 year-old woman contracted an abrasion of her right forearm while gardening. 5 days later, dysphagia, trismus and a cramp of the right hand developed. On admission (8 days after the injury) cramps of the platysma and pharyngeal muscles were observed. A tetanus vaccination had not been carried out for years. INVESTIGATIONS: Electrophysiologically a pathological masseter inhibitory reflex with a missing "silent period" was observed. The level of serum anti-tetanus-toxoid IgG antibodies was 0.03 IU/ml (definitively protective above 0.1 IU/ml). Free tetanus toxin was not detectable in the serum upon mouse challenge. The diagnosis of tetanus was made based on the typical symptoms and the serological susceptibility. TREATMENT AND COURSE: On admission the wound was antiseptically cleaned and a bandage was applied. On the next day a debridement was performed. After taking a serum sample the patient was immunized against tetanus (active and passive). On the following two days the patient received a further dose of tetanus-antitoxin. The patient was treated with metronidazole for 8 and with penicillin G for 10 days. Within the first days after hospital admission laryngospasms, tachycardia and tachypnoe occurred. From the 7 (th) day in hospital the trismus and the cramp of the hand improved. 4 weeks after admission the patient was discharged; the trismus had completely disappeared, but a slight cramp of the hand was still present. CONCLUSION: Minor superficial injuries can lead to acute tetanus, if the level of anti-tetanus toxin-antibodies is non-protective and a disinfective wound cleaning and a postexpositional vaccination are not performed. PMID- 16673221 TI - [Diagnostic and therapeutic management of patients receiving antithrombotic drugs: what to heed?]. PMID- 16673222 TI - [Clinical structures of the future]. PMID- 16673223 TI - [Developing model of university medicine in Dresden]. PMID- 16673224 TI - [Progression of late metastases of renal cell carcinoma]. PMID- 16673225 TI - [Colorectal carcinoma: postoperative chemotherapy is not applied sufficiently]. PMID- 16673226 TI - [Drugs during pregnancy and breastfeeding: new risk categories--antibiotics as a model]. AB - BACKGROUND AND OBJECTIVE: In selecting drugs for treatment during pregnancy and breastfeeding it is essential to be certain of their safety. But categories are lacking that systematically relate the current state of knowledge of individual substances--rather than just drug classes--to each trimester of pregnancy. As such a stratification of risk would be helpful, it was the aim of this study to propose a new and more differentiated classification. METHODS: Internationally available literature and electronic data bases providing details on adverse effects of individual drugs during pregnancy and breastfeeding as well as summaries of product characteristics of the drugs served as comprehensive sources of information. Account was taken of compound-specific data on toxicity during the reproductive and developmental stages, genotoxicity and carcinogenicity (in animals and in-vitro experiments) and drug-specific experience as documented in women during pregnancy and breastfeeding. RESULTS: A new risk classification and appropriate recommendations for clinical management were developed to ensure the safety of drugs given during pregnancy and breastfeeding, taking into account the varying risks during the three trimesters of pregnancy and the perinatal period. Antibiotics were selected as a model for drugs in general and classified according to the new system. CONCLUSION: The proposed new classification of risk makes it possible to select safe agents in the treatment of pregnant and/or breastfeeding women. It is based on the current state of knowledge about a particular substance, also in relationship to the developmental phase of the breastfed child. PMID- 16673227 TI - [Wound botulism in heroin addicts in Germany]. AB - HISTORY AND ADMISSION FINDINGS: 5 heroin addicts (aged 31-44 years; 1 female, 4 men) presented with a history of blurred vision and diplopia followed by dysarthria. 3 of the patients also developed respiratory failure requiring long term ventilatory support. Physical examination revealed cranial nerve deficits and abscesses at injection sites in 3 of them. DIAGNOSIS: In 4 patients wound botulism was diagnosed on the basis of symptoms, course of the illness and response to specific treatment. Clostridium botulinum was grown from wound swab in one patient. TREATMENT AND COURSE: Two of the patients, having been injected with antitoxin immediately after admission, were discharged almost symptom-free after only a few days. Adjuvant antibiotics and, in 3 patients, surgical debridement of the abscesses were needed. CONCLUSIONS: Progressive cranial nerve pareses in addicts who inject drugs intravenously or intramuscularly should raise the suspicion of wound botulism and require hospitalization. While indirect demonstration of toxin supports the diagnosis, false-negative results are common. PMID- 16673229 TI - [Bosentan: a new pillar in the treatment of pulmonary hypertension?]. PMID- 16673230 TI - [Duplexsonography of the liver: state-of-the-art and perspectives]. PMID- 16673231 TI - [Syncope of uncertain cause and sudden cardiac death in young patients]. PMID- 16673232 TI - [Informed consent in complementary and alternative medicine]. PMID- 16673234 TI - [Nephropathy, clinical aspects and prevention]. PMID- 16673235 TI - [Laboratory diagnosis of Pneumocystis pneumonia -- new developments]. PMID- 16673237 TI - Low-grade gliomas -- current concepts. AB - Diffuse astrocytomas, oligodendrogliomas, and oligoastrocytomas (mixed gliomas) WHO grade II, pleomorphic xanthoastrocytomas (PXAs), pilocytic astrocytomas, and subependymal giant cell astrocytomas (SEGAs) are often referred to as low-grade gliomas. WHO grade II astrocytomas, oligodendrogliomas, and mixed gliomas are characterized by their infiltrative growth, frequent tumor recurrence and a more than 50 % risk for malignant progression. In contrast, pilocytic astrocytomas and SEGAs are circumscribed tumors amenable to a (radio)surgical cure. There are few universally accepted guidelines for the treatment of low-grade gliomas. In this review, three neurosurgeons, a neurologist, a neuropathologist, and a radiation oncologist discuss some of the difficult issues surrounding the diagnosis and treatment of low-grade gliomas from their individual points of view (i. e., classification and neuropathology, MR imaging, stereotactic biopsy, microsurgery, interstitial radiotherapy/brachytherapy, radiotherapy, wait and see strategy). PMID- 16673238 TI - Interdisciplinary pain therapy: an innovative therapeutic but pre-DRG Economical Center of Medical Excellence. AB - OBJECTIVE: After the implementation of the G-DRG system in Germany, doubts arose whether and how interdisciplinary pain therapy centers should be restructured to remain profitable and maintain medical excellence for patients with a long ordeal of malaise. METHODS: To reveal structural deficits, we performed a detailed economic analysis of all patients treated at an interdisciplinary pain therapy center of a German University hospital in 2004. RESULTS: 3,672 patients were treated: 2,163 outpatients, 753 at the daycare clinic, 619 as consults and 132 inpatients. The costs for personnel were euro 736,645, consumables euro 105,061, and infrastructure euro 277,762. We calculated fixed costs of euro 236, and consumables of euro 24 per patient. The costs for surgery were euro 1,595, and for a neuroradiological examination euro 245 per patient. Overall treatment costs were euro 319 per patient. We calculated an overall loss of euro 476,752 or euro 109.19 per patient. Outpatients caused a total loss of euro 456,665.83 or euro 211 per patient, consults a total loss of euro 161 683.16 or euro 261.20 per patient, daycare patients a slight profit of euro 30,370 or euro 40 per patient and inpatients a total profit of euro 111,225 or euro 135 per day. CONCLUSION: Managerial optimization can yield considerable cost reductions in the G-DRG coding system, without any change in treatment strategies, selection of profitable patients or dismissal of personnel. Inversely, additional personnel are needed to accomplish the implementation process. Board certification was unveiled to constitute the key structural implementation that ensures the economic survival of the department and continuing medical excellence for the patients. PMID- 16673239 TI - Traumatic delayed epidural hematoma. AB - OBJECT: Traumatic delayed epidural hematoma (DEH) can be defined as a hematoma that is insignificant or not present on the initial computerized tomography (CT) scan made after trauma but subsequent CT scan shows sizeable epidural bleeding. During a 3-year period we have treated a total of 96 epidural hematomas, eight (8.3 %) of which had a delayed onset. CASE REPORT: We present here an analysis of the eight patients with traumatic DEH which had a significant mass effect in all patients and required surgical evacuation. In three patients with mild head injury (GCS > 12) neurological deterioration indicated the necessity of repeating the CT scan and preceded the detection of DEH. In only one case of the five patients with moderate (8 < GCS < 13) and severe head injury (GCS < 9) was neurological deterioration the precursor of the DEH. All patients were immediately operated on after diagnosis. Postoperative outcome was favorable in all cases. CONCLUSIONS: DEHs are highly unpredictable and continue to cause diagnostic difficulty. Close observation for signs of clinical deterioration and repeat CT scan are the most important factors for early detection of DEH. Early diagnosis and prompt operation offers excellent results for DEHs. PMID- 16673240 TI - Dynamic cerebral autoregulation in patients with ruptured and unruptured aneurysms after induction of general anesthesia. AB - INTRODUCTION: Blood pressure management in patients undergoing surgery for clipping of aneurysms is demanding. More information about the ability of cerebral vessels to normally regulate cerebral blood flow may have a direct influence on the intraoperative management. In patients with subarachnoid hemorrhage (SAH) a disturbance of cerebral autoregulation has been reported and it correlated with the severity of the bleeding in these studies. The impairment of autoregulation was demonstrated using static measurements of cerebral pressure autoregulation. However, the dynamic component of the autoregulatory capacity seems to be of importance in the acute setting after SAH. The aim of this study was to evaluate dynamic pressure autoregulation in patients undergoing surgery for intracranial aneurysms. PATIENTS/MATERIAL AND METHODS: 36 patients with a mean age of 45 years were evaluated, 26 patients with acute SAH, 10 patients with unruptured aneurysms. Cerebral autoregulation in normocapnia was tested using thigh cuffs to alter arterial blood pressure and continuous registration of the blood flow velocities with transcranial Doppler sonography. After the induction of general anesthesia under normocapnia the autoregulatory index (ARI) was calculated (values between 0-9). Patient groups were compared using Wilcoxon- and Spearman's rank test. RESULTS: The two patient groups were comparable with regard to gender, age, PaCO(2), blood flow velocities and blood pressure. In patients with SAH mean ARI was 3.1/3.3 (right/left side) compared to 4.7/4.6 (right/left side) in patients without SAH. The difference was statistically significant (Wilcoxon p = 0.0399). The degree of impairment of the autoregulatory capacity increased significantly (p = 0.006) with the severity of the SAH (Hunt&Hess and Fisher scale). CONCLUSION: Dynamic pressure autoregulation is impaired in patients after SAH compared to patients without SAH and correlates with the severity of the SAH. We propose that autoregulation should be measured in all patients with SAH or that an impaired autoregulation should be taken into account in patients with SAH undergoing surgery in the acute phase. PMID- 16673241 TI - Erich Fischer-brugge (28.12.1904-4.2.1951) -- founder of neurosurgery at the university of Munster. AB - In 1936, Erich Fischer-Brugge, who worked as a surgeon at the University Clinic of Munster, Westfalia, Germany, visited Wilhelm Tonnis in Wurzburg, to receive neurosurgical training. He commenced his work in the field of neurosurgery in Munster from 1937. In 1938 he published a new classification of the anterior circulation of cerebral arteries in states of tumorous mass lesions. From 1939 through to the end of WW II, Tonnis and Fischer-Brugge worked closely together, mainly in the field of war surgery. After WW II, in 1949, Fischer-Brugge published another relevant clinical contribution on the "Clivuskantensyndom". He recognised the ipsilateral osseous compression of the oculomotor nerve at the sphenoidal ridge in raised intracranial pressure. Paul Sunder-Plassmann, successor of Hermann Coenen as chief of surgery at the University Clinic in 1946, inhibited Fischer-Brugge's neurosurgical work massively. After numerous unsuccessful applications for newly installed neurosurgical units, Fischer-Brugge died at the age of only 46 years. PMID- 16673242 TI - Delayed diagnosis of spinal dural arteriovenous fistula in the absence of pathological vessels on MRI. AB - The authors report on a 69-year-old man presenting with progressive leg weakness and gait ataxia over two years. A central intramedullary cord lesion ranging from T8-12 on MR imaging was misdiagnosed as a low-grade glioma and a biopsy was attempted followed by temporary clinical deterioration. Selective spinal angiography revealed a spinal dural arteriovenous (AV) fistula on the left L3 nerve root sheath despite the absence of pathological vessels on MR imaging. The fistula was successfully treated by microsurgical interruption of the arterialized intradural vein. The present case should remind us to include selective spinal angiography in our diagnostic work-up in patients predisposed for spinal dural AV fistula by male sex, advanced age and clinical presentation of slowly progressive sensorimotor symptoms with myelopathy on MR imaging, even in the absence of any pathological vascular structures. PMID- 16673243 TI - ["Baby cradle coop slots" for abandoned (anonymous) babies: legal problems]. AB - It is illegal to propagate or offer anonymous births or to "cradle" abandoned babies in anonymous coop slots ("Babyklappen" in German). Such offers must be rescinded. Offers concerning anonymity of births are ill-suited, since they do not save life. They are unnecessary, because women and children can be assisted even if they are in dire straits, by utilising available help as offered by statutory German child and adolescent aid institutions. Those illegal offers do not solve any problems but create new difficulties and result in lifelong misery for children and parents. Offers for anonymousness create a previously non existent demand and discredit legal help facilities. Persons assisting anonymous deliveries or recovering "baby cradle" ("Babyklappen") children are obliged to report this to the local registrar's office (in hospitals by the hospital director). Anonymous babies must be immediately reported by hospitals or institutions to local police authorities, and they should be reported to the municipal Youth Office. PMID- 16673244 TI - [Legal limitations of freedom of medical practice]. PMID- 16673245 TI - [Albert and Gustav Doderlein -- a critical view to the biographies of two German professors]. AB - Albert Doderlein and his son Gustav had as professors in Munich and Jena a formative influence on the German gynecology in the first half of the 20(th) century. Albert Doderlein discovered the lactobacillus named after him and its function, he was one of the first who employed radiotherapeutics in cancer therapy and introduced the rubber glove in obstetrics and gynecology. But he also wrote the gynecologic text to the "Bill for prevention of progeny suffering from an inherited disease dated 14(th) June 1933". After his time at the university hospital Charite, between 1936 and 1945 he was medical superintendent of the department of gynecology and obstetrics at the Police Hospital in Berlin. He can be regarded as the founder of antenatal care in Germany. Both scientific and historic aspects of the biographies of these two professors are critically looked right past. PMID- 16673246 TI - [Magnetic resonance imaging (MRI) of the pelvis in the diagnosis of cervical carcinoma -- review]. AB - Magnetic resonance imaging (MRI) is an accepted imaging method for diagnosing cervical cancer staging before therapy and for recurrent cervical carcinoma. This review article describes the usefulness and the limits of MRI in the diagnosis of cervical cancer. Typical manifestations are illustrated with sample images. PMID- 16673247 TI - [Paraurethral abscess developing after mid-urethral Zuidex-injection in women with stress urinary incontinence -- management of complications and retrospective comparison with bladder neck located injection technique]. AB - PURPOSE: To evaluate the cause and management of paraurethral abscess developing after injection of a mixture of hyaluronic acid and dextranomer (Zuidex) for treating stress urinary incontinence. PATIENTS AND METHOD: A total of 127 women having undergone midurethral Zuidex injection and 34 women after endoscopically guided Deflux injection into the tissue around the bladder neck were followed up 1 to 24 months after the intervention. At follow-up, the paraurethral tissue was evaluated clinically by gynecologic examination and by introital ultrasound. RESULTS: Thirteen of 127 women (10 %) having undergone midurethral Zuidex injection had a sterile paraurethral abscess that was treated by transvaginal puncture (1 to 3 punctures with removal of 10-60 ml of fluid per patient). No case of postoperative paraurethral abscess formation was demonstrated in the control group treated by Deflux injection into the area around the bladder neck. CONCLUSIONS: Paraurethral abscess must be excluded in the postinterventional follow-up of patients after Zuidex injection. Patients in whom an abscess is demonstrated can be treated by transvaginal puncture. Abscess formation can be avoided by injecting dextranomer/hyaluronic acid into the area around the bladder neck. PMID- 16673248 TI - [Stereotactic body radiotherapy (SBRT) -- an overview under special consideration of SBRT and LITT in the therapy of liver metastases]. AB - Stereotactic body radiotherapy (SBRT) is a high dose radiotherapy, the radiation dose is applicated one-time or in a few fractions exactly to the tumor or metastases under maximal separation from the normal tissue. Because of this a major expenditure of medical and technical efforts are necessary, a detailed description of the proceedings is following in the methodic part of this article. Indications of SBRT are especially medical irresectable lung tumors of early stages, primary liver/biliary tumors and pulmonary or liver metastases if there is an oncological benefit for the patients. The side effects are moderate, the local tumor control rate is between 78 to 100 %. Depending on the primary tumor the overall survival is prolonged statistically significant. In the Klinikum Krefeld indications of SBRT vs chemotherapy, LITT or resection are discussed by an interdisciplinary conference of gastroenterologists/oncologists, radiologists, radiooncologists and surgeons. An example of LITT will be shown also. PMID- 16673249 TI - Gene expression profiles of breast cancer obtained from core cut biopsies before neoadjuvant docetaxel, adriamycin, and cyclophoshamide chemotherapy correlate with routine prognostic markers and could be used to identify predictive signatures. AB - BACKGROUND: Neoadjuvant administration of chemotherapy provides a unique opportunity to monitor response to treatment in breast cancer and assesses response exactly. Global gene expression profiling by microarrays has been used as a valuable tool for the identification of prognostic and predictive marker genes. Even though this technology is now wide spread and relatively standardized, there are only few data available which compare established parameters with expression values to determine reliability of this method. Therefore we analyzed gene expression data of pretreatment biopsies of breast cancer patients and compared them with the results of the immunohistochemical receptor expression for ER/ PR and Her-2, as well as FISH testing for HER-2 amplification. We analyzed the change of expression of these markers before and after neoadjuvant chemotherapy. Furthermore we evaluated the predictive significance of prognostic gene signatures as described by Sorlie, van't Veer and Ahr for response to neoadjuvant chemotherapy. METHODS: Pretherapeutic core biopsies were obtained from 70 patients undergoing neoadjuvant TAC chemotherapy within the GEPARTRIO-trial. Samples were characterized according to standard pathology including ER, PR and HER2 IHC and amount of cancer cells. Only biopsies with more than 80 % tumor cells were considered for further examination. RNA was isolated and expression profiling performed using Affymetrix Hg U133 Arrays (22 500 genes). GeneData's Expressionist software was used for bioinformatic analyses. RESULTS: More than two thirds of the biopsies yielded sufficient amounts (> 5 microg) of RNA for expression profiling and high quality data were obtained for 50 samples. Unsupervised clustering broadly revealed a correlation with hormone receptor status. When ER-alpha, PR and HER2 as analyzed by immunohistochemistry were compared to the corresponding mRNA data from gene chips more than 90 % concordance was observed. We could observe a switch of receptor expression for ER, PR or HER-2 from positive to negative and vice versa in 16/35 cases (45.7 %) and 5/22 cases (22.7 %) respectively. The prognostic marker sets of Sorlie, van't Veer and Ahr could not discriminate responders from non responders in our patient group. CONCLUSIONS: Our results demonstrate that reliable expression profiles can be achieved by using limited amounts of tissue obtained during neoadjuvant chemotherapy. Microarray data capture conventional prognostic markers but might contain additional informative gene sets correlated with treatment outcome. Prognostic marker sets are not suitable to predict tumor response in the neoadjuvant setting, suggesting the necessity of class prediction methods to identify marker sets predictive for the type of therapy used. PMID- 16673250 TI - Prevention of mother-to-child transmission of HIV in Cameroon: experiences from the University Teaching Hospital in Yaounde (Cameroon). AB - OBJECTIVE: The aim of this study was to assess the effectiveness of nevirapine (NVP) in the prevention of mother-to-child transmission (MTVT) of HIV-1 in children in the Yaounde University Teaching Hospital, Cameroon. METHODS: This was a prospective and descriptive hospital-based study in which pre-counselled HIV infected pregnant women who gave their consent were included. At the onset of labour a single dose of 200 mg oral NVP was administered to each woman. Their newborns were also given 2 mg/kg of oral NVP syrup within 72 hours of their birth. These NVP-treated babies were assessed at 6 weeks, 6 months and between 15 and 18 months for viral load levels, using a branched DNA technology. Viral load levels were classified as undetectable (< 50 copies/ml); intermediate (50-3 500 cp/ml) or high (> 3 500 cp/ml). RESULTS: Of the 350 women screened for HIV 22 (6.3 %) were positive for HIV-1, but only 18 children were duly tested until 18 months. Viral load levels were undetectable at 6 weeks in 61.1 % of the children; intermediate in 38.9 % and no child had a high VL (> 3 500 cp/ml). At 6 months 5 of 7 children with intermediate levels had dropped to undetectable levels. By 15 18 months, a total of 16 children (88.9 %) had undetectable levels while 2 children (11.1 %) with detectable levels were confirmed to be HIV positive using ELISA test and immunoblot analysis. CONCLUSIONS: We conclude that the rate of HIV 1 MTCT with NVP is about 11 % in CHU Yaounde. PMID- 16673251 TI - [Clinical management of an androgen insensitivity syndrome]. AB - The seldom occurring androgen insensitivity syndrome is characterised by male karyotype in a phenotypical women. We report on a complete androgen insensitivity syndrome with the rare coincidence of bilateral Fallopian tubes and a testicular intraepithelial neoplasia. Additionally we discuss aspects of incidence, molecular background, characteristics, diagnostic pitfalls and therapy against the background of the current literature. PMID- 16673255 TI - Clinical services and the regulatory environment: an overview. AB - Most clinicians have difficulty participating in the political process. Patients/clients come first, and there is often little time to pursue information about the political and regulatory processes affecting clinical practice. Yet, such activity is imperative if speech-language pathologists and other practitioners want to achieve individual and collective professional goals and ensure sufficient funding for services to consumers. This article is an overview of politics, health-care policy, and regulation. Definitions are presented, and concepts regarding how politics become policy are cast in the familiar structure process-outcome triad. Four types of regulation are discussed, and current regulatory initiatives are explained from the perspective of policy-makers. The discussion concludes by applying these concepts to issues and behaviors of specific interest to speech-language pathologists and sets the stage for the other articles in this publication. PMID- 16673252 TI - [Mediastinal metastasis of a tubal choriocarcinoma following ectopic pregnancy as a rare cause of thoracic pain]. AB - We report on a 26-year old female patient with thoracic pain and dyspnea, in whom a large tumorous mass in the anterior mediastinum with a pleural effusion was diagnosed by computed tomography and magnetic resonance imaging. After rapid progression of tumor growth and detection of malignant cells within the pleural effusion operative intervention including resection of the tumor was performed. Histologic examination of the tumor revealed the typical morphology of a large mediastinal choriocarcinoma. The excessively high hCG-levels returned to normal values post-operatively. A thorough history making revealed an ectopic pregnancy which had made unilateral salpingectomy necessary. Although primary histologic examination of the tubarian tissue had shown no malignancy, a secondary look revealed a choriocarcinoma with identical histological features compared to the mediastinal tumor. Thus, final diagnosis of a mediastinal metastasis of a tubarian choriocarcinoma in ectopic pregnancy was made. We discuss this extremely rare disease and provide a short overview of the literature. PMID- 16673256 TI - Health care quality and safety issues. AB - Our health-care system is burdened with high costs, health-care disparities, overtreatment, undertreatment, high error rates, and fraud and abuse. At the same time, the United States has achieved spectacular medical advances using the latest technology. As a result, health-care quality measurement, publicly reported patient safety and quality indicators, and evaluation of patients' experience of care are watchwords of a new era of accountability for health-care professionals and organizations. The health-care industry is subject to increasing regulation, private sector challenges, and public demand to make significant improvements in all three components of the quality triad: structure, process, and outcome. This article examines regulatory initiatives and industry trends pertaining to patient safety and quality measurement and concludes with specific suggestions for the professions of speech-language pathology and audiology. PMID- 16673257 TI - HIPAA privacy regulations. AB - The Health Insurance Portability and Accountability Act of 1996 (HIPAA) impacts all health-care professionals, including speech-language pathologists (SLPs). The administration simplification section of HIPAA includes specific regulations designed to protect the privacy of an individual's health records. These privacy regulations are not designed to impede upon essential health-care practices, but do require that covered providers take reasonable steps to limit the use and disclosure of protected health information. Important aspects of the privacy regulations include the development of facility- or practice-specific privacy policies, the development of a privacy manual, staff training on privacy policies and procedures, and the drafting and posting of a notice of privacy practices. In addition, authorization forms are mandated for instances of use and disclosure of public health information not related to treatment, payment, or health-care operations. PMID- 16673258 TI - Clinical documentation, coding, and billing. AB - Practitioners must be aware of ever-changing requirements for documentation and correct diagnostic and procedural coding for billing. These requirements are often dependent on the setting, and clinicians who practice in more than one setting have to understand multiple regulations and guidelines. Some practices are dictated by the payer, such as precertification or using a specific form (e.g., HCFA 1500) to submit the charges. Documentation of services provided encompasses evaluation reports, treatment plans, recertifications, and discharge summaries. Different forms and formats are required in different settings and by different payers. The clinician must document in a clear and concise format that can be understood by others. When documentation is completed accurately and well, reimbursement for services is more likely. PMID- 16673259 TI - The regulatory context for public education. AB - The provision of special education and related services in public schools involves the intertwining of federal, state, and local laws, regulations, other levels of policy, and practices. Such a multilayered system can obviously lead to misunderstanding and misinformation regarding school-based services, creating challenges in terms of implementation and advocacy. However, by determining the source of the requirements and the intent of the laws, it is possible not only to remain in compliance with mandates but also to influence implementation decisions and improvements that will ultimately be in the best interest of students. Educators must identify and analyze the source and intentions of requirements, understand the responsibilities of states and localities, and when necessary, identify vehicles for advocating for change. This article provides information regarding major federal mandates that affect public education, with comments specific to speech-language services in the schools. PMID- 16673260 TI - Challenges for professional practice in the next decade. AB - This article is presented in a question and answer format and shares the thoughts of the 2006 President of the American Speech-Language-Hearing Association (ASHA), Alex Johnson, and current Executive Director of ASHA, Arlene Pietranton, regarding several regulatory issues that the professions of audiology and/or speech-language pathology are likely to have to address over the next decade. Specific topics mentioned include: trends affecting our professions; anticipated service delivery changes; public (i.e., Medicaid and Medicare) and private (i.e., health plans, consumer-driven plans) reimbursement; competing for funding dollars; pay-for-performance; the Individuals with Disabilities Education Act; No Child Left Behind; and ASHA's work with various national organizations and federal agencies in the regulatory arena. Several ASHA resources for further information are listed. PMID- 16673261 TI - Thrombin biology in the 21st century: implications in hemostasis, inflammation, coagulopathies, and cancer. Proceedings of a seminar. September, 2005. PMID- 16673262 TI - An overview of the structure and function of thrombin. AB - The fundamental importance of thrombin in biology and medicine has made it one of the most extensively studied of all proteases. Thrombin performs essential functions in vertebrate biology as the central enzyme involved in blood coagulation and platelet aggregation, and as a mitogen and secretagogue for a variety of cell types. Thrombin is synthesized in the liver and secreted into the general circulation in an inactive zymogen form (prothrombin), a complex multidomain glycoprotein that is activated to yield thrombin at sites of vascular injury by limited proteolysis following upstream activation of the coagulation cascade. Thrombin shares its general architecture and catalytic mechanism with those of pancreatic trypsin, the prototypical digestive serine protease. However, the specificity of thrombin toward substrates and cofactors, as well as its spatiotemporal regulation by effectors and inhibitors, is directed by features of the molecule that distinguish it from relatively nonspecific serine proteases like trypsin. Structural and functional studies have demonstrated the presence of surface loops that partially occlude the active site and make specific contacts with residues adjacent to the scissile bond of substrates. Specificity toward macromolecular substrates and cofactors is additionally enhanced by anion-binding exosites that are spatially distinct from the active site. More than five decades of multidisciplinary research on thrombin have produced an abundance of functional and structural information and provided a robust framework for understanding the role of thrombin in vertebrate biology. PMID- 16673263 TI - The structure of thrombin: a janus-headed proteinase. AB - Through a series of successive, cascade-like proteinase activation and amplification steps, any vascular injury triggers a rapid burst of alpha thrombin, a trypsin-like serine proteinase. Thrombin, the main executioner of the coagulation cascade, has procoagulant as well as anticoagulant and antifibrinolytic properties. It exhibits quite diverse physiological functions, but also gives rise to several thrombotic disorders, such as thromboembolism, myocardial infarction, and stroke, thus making it an attractive target for antithrombotic agents. Thrombin interacts specifically with several protein substrates, receptors, cofactors, inhibitors, carbohydrates, and modulators. It cleaves fibrinogen, factors XI (FXI) and FXIII, cofactors V and VIII, and the thrombin receptors; uses thrombomodulin to activate protein C and thrombin activatable-fibrinolysis inhibitor; is inhibited by heparin cofactor II and antithrombin III with the help of acidic carbohydrates; and its activity/specificity is modulated by sodium ions. A large number of crystal structures of alpha-thrombin in complexes with synthetic polypeptides and protein inhibitors, substrate fragments, cofactors, and carbohydrates have displayed extended recognition sites on the thrombin surface, reflecting the versatility and multifunctional specificity of this remarkable proteinase. These structures essentially show that the thrombin surface can be subdivided into several functional regions, which recognize different chemical moieties. By using different combinations of these surface elements, thrombin can interact with a variety of molecules with high specificity, accounting for its multifunctional properties. PMID- 16673264 TI - A cell-based model of thrombin generation. AB - We have developed a cell-based model of thrombin generation using activated monocytes as a source of tissue factor (TF) and platelets serving as a surface for thrombin generation. Monocytes are activated by lipopolysaccharide and express cell-bound TF. To these are added physiologic (plasma) concentrations of all the plasma procoagulants as well as TF pathway inhibitor, antithrombin, and C1-esterase inhibitor. Coagulation takes place in microtiter wells and is initiated by factor VIIa (FVIIa) and calcium. At time intervals, aliquots are removed, platelet activation is measured by the expression of P-selectin, and thrombin generation is measured by chromogenic assay. In addition, one can measure the activation of FIX, FX, FVIII, FV, and FXI. Initial results reveal that the FVIIa-TF interaction results in the activation of FX to FXa and FIX to FIXa. FXa stays in the vicinity of the TF-bearing cell and, in the presence of FVa, converts a small amount of prothrombin to thrombin on the surface of the TF cell. This small amount of thrombin is not sufficient to clot fibrinogen, but is sufficient to activate platelets and FVIII, FV, and FXI. Following platelet activation, FVIIIa, FVa, and FXa occupy sites on the activated platelet surface. FIXa, activated by TF-FVIIa, does not remain on the TF cell, but converts FX to FXa on the platelet surface. FXIa acts to boost FIXa generation on the activated platelet, increasing FXa and subsequent thrombin generation. We have also shown that activated protein C does not inactivate Va on the platelet surface but rather on endothelial cell surfaces. PMID- 16673265 TI - Protease-activated receptors: how proteases signal to cells to cause inflammation and pain. AB - Certain serine proteases that originate from the circulation (coagulation factors), inflammatory cells (mast cell tryptase, neutrophil granzyme A, and proteinase 3), and epithelial and neuronal tissues (trypsins) can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors. Proteases cleave PARs on multiple cell types to reveal tethered ligand domains that bind to and activate the cleaved receptors. The proteases that activate PARs are often generated and secreted during injury and inflammation, and PARs orchestrate tissue responses to these insults, including hemostasis, inflammation, nociception, and repair mechanisms. Agonists of PARs, notably PAR2, induce inflammation in many tissues that is characterized by hyperemia, extravasation of plasma proteins, granulocyte infiltration, and alterations in epithelial permeability. These effects are mediated in part by the release of neuropeptides substance P and calcitonin gene-related peptide from sensory nerve fibers in peripheral tissues. Proteases that activate PAR2 also induce the release of neuropeptides from the central projections of these nerves in the dorsal horn of the spinal cord, where they participate in pain transmission. Accumulating evidence from PAR-deficient mice indicates that these mechanisms may contribute to experimental models of disease and raise the possibility that protease inhibitors and PAR antagonists may be useful therapies for a variety of inflammatory and painful conditions. PMID- 16673266 TI - Inflammation and the activated protein C anticoagulant pathway. AB - After a coagulation stimulus, the blood clotting cascade amplifies largely unchecked until very high levels of thrombin are generated. Natural anticoagulant mechanisms (for example, the protein C anticoagulant pathway) are amplified to prevent excessive thrombin generation. Thrombin binds to thrombomodulin (TM) and this complex and then activates protein C approximately 1000 times faster than free thrombin. Protein C activation is enhanced approximately 20-fold further by the endothelial cell protein C receptor (EPCR). Activated protein C proteolytically inactivates factor Va (FVa) and FVIIIa, thereby blocking the amplification of the coagulation system, a process that is accelerated by protein S. TM not only accelerates protein C activation, but also decreases endothelial cell activation by blocking high-mobility group protein-B1 inflammatory functions and suppressing both nuclear factor-kappa B nuclear translocation and the mitogen activated protein kinase pathways. The thrombin-TM complex also activates thrombin-activatable fibrinolysis inhibitor, a procarboxypeptidase that renders fibrin resistant to clot lysis and neutralizes vasoactive molecules such as complement C5a. Activated protein C has a variety of antiinflammatory activities. It suppresses inflammatory cytokine elevation in animal models of severe sepsis, inhibits leukocyte adhesion, decreases leukocyte chemotaxis, reduces endothelial cell apoptosis, helps maintain endothelial cell barrier function through activation of the sphingosine-1 phosphate receptor, and minimizes the decrease in blood pressure associated with severe sepsis. Most of these functions are dependent on binding to EPCR. Overall this pathway is critical to both regulation of the blood coagulation process, and control of the innate inflammatory response and some of its associated downstream pathologies. PMID- 16673267 TI - Thrombin generation and the pathogenesis of cancer. AB - Advanced cancer is associated with a hypercoagulable state that is triggered by tissue factor (TF). TF-initiated thrombin generation is crucial for metastasis through fibrin and platelet deposition, as well as thrombin-dependent protease activated receptor (PAR) 1 signaling. Surprisingly, PAR2, which is not cleaved by thrombin, appears to cosignal with PAR1 to elicit thrombin effects in metastatic tumor cells. In contrast to TF-driven thrombin pathways in metastasis, direct TF signaling plays a role in angiogenesis-dependent tumor growth. In TF cytoplasmic domain-deleted mice, PAR2-dependent angiogenesis and tumor growth is enhanced, demonstrating a role for host cell TF signaling. In tumor cells, TF-factor VIIa (FVIIa) activates PAR2 and thereby regulates proangiogenic growth factor expression as well as integrins involving crosstalk with the TF cytoplasmic domain. In addition to thrombin-PAR signaling in metastasis and TF-FVIIa-PAR2 signaling in tumor growth, it is likely that additional protease pathways will prove to be crucial activators of PARs in cancer. Transmembrane serine proteases as well as matrix metalloproteinase are prime candidates for accessory pathways to regulate metastasis, tumor expansion, and angiogenesis dependent on specific features of the local tumor microenvironment. PMID- 16673268 TI - Activation of microglial cells by thrombin: past, present, and future. AB - In addition to its role in the coagulation cascade, the serine proteinase thrombin (factor IIa) activates cell surface proteinase-activated receptors (PARs) both within and outside the vascular system. PARs are expressed in the central nervous system and mediate thrombin-induced cellular responses in a variety of neural cell types, including microglial cells. Microglial activation by thrombin was reported to induce proliferation, cytokine release, and intracellular calcium signaling. Recently, additional experiments questioned whether these effects are mediated either by thrombin's proteolytic activity or by thrombin itself. Analysis of commercially available plasma-derived thrombin frequently used in the earlier studies showed that cyto/chemokine release activating properties were not residing with thrombin but were with high molecular weight contaminant(s). In the absence of such contamination, no microglial activation was seen. We compared commercial-grade plasma-derived thrombin to pharmaceutical-grade recombinant thrombin devoid of any measurable contamination. The pharmaceutical-grade thrombin displayed a much more limited profile of microglia-activating properties, triggering only intracellular calcium signals and small changes in surface antigen expression. The signals induced by the pharmaceutical-grade thrombin were completely abolished by proteolytic inhibition, indicating that they are proteolysis-dependent, are most likely PAR mediated, and reflect thrombin's true microglia-activating potential. Prior reports using nonpharmaceutical-grade thrombin need to be reinterpreted critically given these new findings. PMID- 16673269 TI - Mechanism of action of factor VIIa in the treatment of coagulopathies. AB - Recombinant factor VIIa (rFVIIa) has been developed for treatment of bleeding in patients with hemophilia who have inhibitors against factor VIII (FVIII) or FIX, and has been found to induce hemostasis during major orthopedic surgery. The use of rFVIIa treatment for hemophilia is a new concept and is based on the low affinity binding of FVIIa to the surface of thrombin-activated platelets. Administration of pharmacologic doses of exogenous rFVIIa enhances thrombin generation on the platelet surface at the site of injury independently of the presence of FVIII or FIX. Pharmacologic doses of rFVIIa induce hemostasis not only in hemophilia patients, but also in patients with thrombocytopenia, functional platelet defects, and with profuse bleeding triggered by extensive surgery or trauma. The general mechanism of action of rFVIIa to induce hemostasis under these conditions may be its capacity to generate a tight fibrin hemostatic plug through increased thrombin generation. A tight fibrin plug will aid in resisting the overwhelming local release of fibrinolytic activity triggered by vast tissue damage occurring in extensive trauma. Local fibrinolytic activity also occurs in the gastrointestinal tract as well as during profuse postpartum bleeding. Pharmacologic doses of rFVIIa induce hemostasis in these cases also. PMID- 16673270 TI - Comparison of recombinant human thrombin and plasma-derived human alpha-thrombin. AB - Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery. PMID- 16673271 TI - The clinical use and immunologic impact of thrombin in surgery. AB - Thrombin is a naturally derived enzyme that has been widely characterized for its roles in hemostasis, inflammation, and cell signaling. Thrombin has been purified from numerous sources and used as a clinical aid for topical hemostasis for more than 60 years. Due to both its ease of use and apparent effectiveness, thrombin has become used routinely as an aid for topical hemostasis in nearly all types of surgical procedures, including but not limited to cardiovascular, orthopedic, neurologic, general, gynecologic, and dental procedures. Due to the widespread acceptance of thrombin in the surgical setting, it is conservatively estimated that at least 1 million patients in the United States are treated with topical applications of thrombin each year. Although the U.S. Food and Drug Administration (FDA) has approved a wide array of topical and biologic products to stop surgical bleeding, the only thrombin that is currently FDA approved as a stand-alone hemostatic product in the United States is derived from bovine sources. Bovine-derived thrombin has potent biologic activity in its ability to convert fibrinogen to fibrin, activate platelets, and induce vascular contraction. However, it has also been shown to induce a robust immune response following human exposure. Numerous reports have documented an array of clinical events that follow bovine thrombin exposure, which include the development of antibodies against thrombin, prothrombin, factor V, and cardiolipin. In some well described cases, these antibodies have led to clinical syndromes that range from severe postoperative bleeding to high rates of vascular bypass graft thrombosis. Furthermore, experimental applications of bovine thrombin to various strains of mice have induced a postexposure autoimmune syndrome that was pathologically identical to lupus. Thrombin-derived products are well accepted by the surgical community for use as an aid for hemostasis, but the bovine-derived products have an unacceptably high and unnecessary association with immunologic side effects. If a nonimmunologic and effective thrombin were developed, one would expect it to be rapidly adopted by the clinical community. PMID- 16673273 TI - Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia. AB - The annual incidence of polycythemia vera (PV) and essential thrombocythemia (ET) is approximately 2 and 1.5 per 100,000 inhabitants, respectively, if based on large population surveys and adjusted to a standard population. Survival for ET patients does not differ from that of the general population. However, it appears that PV patients have a significantly higher mortality, at least for patients diagnosed before 1990. PMID- 16673274 TI - Clinical and laboratory features, pathobiology of platelet-mediated thrombosis and bleeding complications, and the molecular etiology of essential thrombocythemia and polycythemia vera: therapeutic implications. AB - Microvascular disturbances in essential thrombocythemia (ET) and polycythemia vera (PV), including erythromelalgia, and atypical and typical transient cerebral, ocular, and coronary ischemic attacks, are caused by platelet-mediated transient and occlusive thrombosis in the end-arterial circulation. ET patients with microvascular disturbances have shortened platelet survival, increased beta thromboglobulin (beta-TG), platelet factor 4 (PF4), and thrombomodulin (TM) levels, and increased urinary thromboxane B2 (TXB2) excretion, indicating platelet-mediated thrombotic processes. Inhibition of platelet cyclooxygenase-1 by aspirin is followed by relief of microvascular disturbances; correction of shortened platelet survival; correction of increased plasma beta-TG, PF4, and TM levels; and correction of increased TXB2 excretion to normal. In PV associated with thrombocythemia, increased hematocrit and whole blood viscosity aggravate the platelet-mediated microvascular syndrome of thrombocythemia to produce major arterial and venous thrombotic complications. Correction of hematocrit to normal by phlebotomy will reduce the major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated microvascular circulation disturbances in PV patients because thrombocythemia persists. Complete relief and prevention of microvascular and major thrombosis in ET and PV patients, in addition to phlebotomy, are obtained by treatment with aspirin and not with coumarin. The discovery of JAK2 V617F gain of function mutation in patients with myeloproliferative disorders (MPDs) expands our insights into the molecular etiology and biological features of ET, PV, and chronic idiopathic myelofibrosis (CIMF). The current concept is that heterozygous JAK2 V617F mutation with increased kinase activity is enough for megakaryocyte proliferation and increased hypersensitive platelets with no or slightly increased erythropoiesis in ET and in early PV mimicking ET. Homozygous JAK2 mutation with pronounced kinase activity is associated with trilinear megakaryocyte, erythroid, and granulocytic myeloproliferation, myeloid metaplasia, and secondary myelofibrosis (MF), with the most frequent clinical picture of classical PV complicated by major thrombosis in addition to the platelet-mediated microvascular thrombotic syndrome of thrombocythemia. The positive predictive value of a JAK2 V617F polymerase chain reaction test for the diagnosis of MPDs is high (near to 100%), but only half of ET and MF (sensitivity 50%) and the majority of PV (sensitivity 85 to 97%) are JAK2 V617F positive. Bone marrow histopathology, when used in combination with specific markers such as serum erythropoietin, PRV-1, endogenous erythroid colony formation, peripheral blood parameters and red cell mass, has a high sensitivity and specificity (near 100%) to detect the early and overt stages of the MPDs and to differentiate between ET, PV, and CIMF in both JAK2 V617F positive and -negative MPDs. PMID- 16673275 TI - Budd-Chiari syndrome and portal vein thrombosis associated with myeloproliferative disorders: diagnosis and management. AB - The improvement observed in the prognosis of Budd-Chiari syndrome (BCS) since 1985 may be related to a better identification of underlying prothrombotic states. In a recent investigation of the currently identified prothrombotic disorders, one or several prothrombotic disorders were evidenced in 72% of patients with portal vein thrombosis (PVT) and 87% of patients with hepatic vein thrombosis. According to the different criteria used for their diagnosis, myeloproliferative disorders (MPDs) were reported in 25 to 65% of patients with splanchnic vein thrombosis (STV). In a single-center retrospective study of 128 patients with STV, clusters of abnormal megakaryocytes in bone marrow biopsy combined with endogenous erythrocyte colony formation were used as reference standards for a diagnosis of MPD. These two criteria allowed the diagnosis of MPDs at risk of aggravation in 25% (26 of 103) of patients with STV (41% in BCS; 23% in PVT). After a mean follow-up of 6.09 +/- 6.6 years, hematologic progression was confirmed in 23% of patients with the two criteria both positive, whereas neither progression nor features suggestive of MPD were observed in patients with the same concordant negative criteria. The combination of marked splenomegaly and platelet count > 200 x 10 (9)/L that was never observed in patients without MPD, constitutes a simple index for the suspicion of an underlying MPD, but the absence of an obvious splenic enlargement and normal or decreased platelet numbers may even be observed in patients with MPD-associated STV. In the absence of reliable indicators of the exact expansion of the myeloproliferative clone, the therapeutic management of STV-associated MPD is difficult to standardize. Owing to the suspected incidence on the prognosis of BCS of an associated MPD, the present watchful-waiting attitude proposed in MPD without obvious hematologic expression may be challenged. PMID- 16673276 TI - Clinicopathological criteria for differential diagnosis of thrombocythemias in various myeloproliferative disorders. AB - Thrombocythemias with the presenting or developing complications of thromboembolic and hemorrhagic episodes may be encountered at strikingly different incidences in each subtype of chronic myeloproliferative disorders. A critical reappraisal of the Polycythemia Vera Study Group (PVSG) criteria for essential thrombocythemia (ET) reveals that differentiation is explicitly focused on the exclusion of chronic myeloid leukemia and polycythemia vera (PV), but not on prodromal stages of chronic idiopathic myelofibrosis (CIMF) or latent (initial) PV. Consequently, it may be assumed that most series of patients with so-called ET include a considerable fraction of patients with the latter entities. The diagnostic impact of bone marrow (BM) histopathology was recognized by the World Health Organization classification, which emphasizes for the first time positive criteria for ET. The need of a more accurate ET diagnosis is obvious, in particular regarding therapeutic strategies and outcome (i.e., progression into myelofibrosis and blastic crisis). Conversely, early CIMF with accompanying thrombocythemia mimicking (true) ET is characterized by a higher rate of evolution into myelofibrosis and fatal complications. A scrutinized discrimination of thrombocythemias resulting in a clear-cut diagnosis of true versus false ET is warranted by a professional evaluation of BM biopsies in ongoing and prospective clinical trials. PMID- 16673277 TI - Analysis of risk factors: the rationale of the guidelines of the Czech Hematological Society for diagnosis and treatment of chronic myeloproliferative disorders with thrombocythemia. AB - The rationale of the Czech Hematological Society guidelines for diagnosis and treatment of Philadelphia chromosome-negative myeloproliferative disorders with thrombocythemia (MPD-T) is reviewed. For diagnosis of MPD-T, the classification according to the World Health Organization or to the Rotterdam criteria is preferred because they distinguish true essential thrombocythemia from prefibrotic or early fibrotic idiopathic myelofibrosis and prepolycythemic polycythemia vera. The histopathology-based nosological distinction provided by these classifications yields valuable information on prognosis (including the risks of transition into secondary acute myeloid leukemia and myelofibrosis). Another serious complication in MPD-T is thrombosis (arterial or venous), the main risk factors of which are age, previous thrombosis, platelet counts 350 to 2,200 x 10 (9)/L (peak at approximately 900 x 10 (9)/L) and the presence of additional thrombophilic risk factors (hereditary thrombophilia, any hypercoagulable state, cardiovascular disease). The hemorrhagic risk starts increasing progressively at platelet counts > 1,000 x 10 (9)/L. Treatment should be stratified with respect to the thrombotic and hemorrhagic risks. In high-risk patients, thromboreductive therapy is warranted. All of the cytostatic drugs, including hydroxyurea, may be leukemogenic and should be given only to patients > 60 years old, whereas anagrelide or interferon alpha are preferred in younger individuals. In low-risk patients, antiaggregation therapy is sufficient, unless the platelet count exceeds 1,000 x 10 (9)/L, which is another indication for thromboreduction. Thrombopheresis is indicated in thrombocythemia > 2,000 x 10 (9)/L. PMID- 16673278 TI - Influence of the assays of endogenous colony formation and serum erythropoietin on the diagnosis of polycythemia vera and essential thrombocythemia. AB - Formation of endogenous erythroid colonies (EECs) or endogenous megakaryocytic colonies (EMCs) is a hallmark of myeloproliferative disorders (MPDs). The diagnostic value of EEC for polycythemia vera (PV) using standardized media has been demonstrated, and has led clinicians to consider EEC as a major diagnostic criterion in the WHO classification. The interest of EEC currently needs to be considered taking into account recent data about V617F JAK2 mutation in MPD. In particular, EECs and EMCs should be helpful for the diagnostic and the vascular risk evaluation of essential thrombocythemia (ET) and for mutation negative patients. A low serum erythropoietin (EPO) level is a consistent finding in PV. Recent studies have shown that commercial serum EPO assays provided a reliable, accurate, and low-cost criterion for the diagnosis of a significant proportion of PV. It suggests that diagnostic algorithms integrating serum EPO level could be elaborated. The diagnostic value of EPO assay for thrombocytosis has not been proved, but some data suggest a predictive value of low EPO levels for thrombosis in ET. PMID- 16673279 TI - Aspirin for the control of platelet activation and prevention of thrombosis in essential thrombocythemia and polycythemia vera: current insights and rationale for future studies. AB - Polycythemia vera and essential thrombocythemia are chronic myeloproliferative disorders characterized by a relatively benign clinical course that may be complicated by arterial and venous thromboses. A thrombotic diathesis often manifests at diagnosis or in the preclinical phase of the myeloproliferative disease. Peculiar microcirculatory disturbances such as erythromelalgia and visual and hearing symptoms also commonly occur in these patients, and are highly responsive to aspirin. In a placebo-controlled trial in relatively low-risk polycythemic subjects, low-dose aspirin recently was shown to reduce the incidence of both arterial and venous thrombosis with a limited increase of the hemorrhagic risk. Due to its favorable benefit/risk profile, low-dose aspirin should be prescribed to all patients with polycythemia vera who have no contraindication to this treatment. Future studies should assess primarily the efficacy and safety of aspirin in essential thrombocythemia, and test the possible use of more aggressive antithrombotic strategies in high-risk polycythemic patients. PMID- 16673280 TI - Anagrelide treatment in myeloproliferative disorders. AB - Platelet-lowering therapy in myeloproliferative disorders includes cytostatic drugs, mainly hydroxyurea, interferon alpha, and anagrelide. Anagrelide is the latest addition to the therapeutic arsenal, and the basis for its use is reviewed. The platelet-lowering efficacy is 70 to 80% in essential thrombocythemia, and the response is rapid; most of the patients reach the treatment goal within a few weeks. Side effects are common, mainly caused by the vascular effects, and include palpitation, headache, loose stools/diarrhea, and edema. Some side effects are time-limited, but late dropout from therapy is not uncommon. The total dropout rate in prospective studies is 30 to 50%. Pharmacologic treatment of side effects is often helpful. Cardiac insufficiency may be worsened in patients with previous heart failure, and special caution is warranted in such patients. Anagrelide has recently been registered in Europe as a second-line therapy in ET but is often used as first-line therapy in the United States, especially in younger patients, due to the concern about increased leukemia risk with cytostatic treatment. The first randomized anagrelide study, with its limitations, gives support for the second-line registration. Given that dose escalation is a problem in some patients with all therapeutic agents used, combination of two drugs in lower doses is a practical option already used by many clinicians without basis in any published study. PMID- 16673281 TI - Diagnosis and therapy of polycythemia vera. AB - Polycythemia vera (PV) is a hematopoietic stem cell disorder characterized by a predominant proliferation of the erythroid cell line. The diagnosis is commonly based on the WHO criteria. The acquired V617F mutation in the tyrosine kinase gene JAK2 represents a new molecular marker proving clonality in PV and other chronic myeloproliferative disorders. Phlebotomy is still the treatment of choice to reduce the red cell mass. Low-dose acetylsalicylic acid is successful in the primary prophylaxis of vascular complications. However, the majority of patients require myelosuppressive therapy during the course of their disease due to progressive myeloproliferation. Hydroxyurea still plays a role in patients of all age groups. Interferon alpha represents an alternative, particularly for younger patients. Apart from sporadic cases of bone marrow transplantation, there is no known curative treatment in PV. To date, the diagnosis of PV was based mainly on clinical criteria. The identification of the JAK2 mutation enables new approaches to the diagnosis, classification, and treatment of PV and of the other myeloproliferative disorders. PMID- 16673282 TI - Risk stratification, staging, and treatment of patients with polycythemia vera: Italian and European collaboration on low-dose aspirin in polycythemia data. AB - The clinical course of polycythemia vera (PV) is marked by a high incidence of thrombotic complications, which represent the main cause of morbidity and mortality. Major predictors of vascular events are increasing age and previous thrombosis. Myelosuppressive drugs can reduce the rate of thrombosis, but there is concern that their use increases the risk of transformation into acute leukemia. To tackle this dilemma, a risk-oriented management strategy is recommended. Low-risk patients should be treated with phlebotomy and low-dose aspirin based on the results of the European Collaboration on Low-Dose Aspirin in Polycythemia study. Cytotoxic therapy is indicated in high-risk patients, and the drug of choice is hydroxyurea because its leukemogenicity is low. New therapeutic options, that theoretically are devoid of leukemic risk (such as interferon alpha and imatinib), should be reserved for selected patients and require additional clinical experience. PMID- 16673283 TI - Management of essential thrombocythemia: implications of the medical research council primary thrombocythemia 1 trial. AB - There has been a dearth of prospective randomized studies in essential (or primary) thrombocythemia (ET). To date, only two reports have been published. Cortelazzo et al reported the first study in 1995. The second is the Medical Research Council Primary Thrombocythemia 1 trial (MRC PT-1), the results and implications of which are the subject of this article. PMID- 16673284 TI - Vascular complications in Chuvash polycythemia. AB - Chuvash polycythemia is characterized by a homozygous 598C> T germline mutation in the von Hippel-Lindau gene ( VHL), upregulation of hypoxia-inducible factor 1alpha during normoxia, and resulting augmentation of erythropoietin and several other hypoxia-controlled genes. Although endemic to the Chuvash population of Russia, this mutation occurs worldwide and usually originates from a single ancient event. Matched-cohort and case-control analyses have shown that VHL 598C> T homozygosity is associated with lower peripheral blood pressures, varicose veins, vertebral hemangiomas, lower white blood cell and platelet counts, and elevated serum concentrations of vascular endothelial growth factor and plasminogen activator inhibitor-1. These studies have also shown associations with arterial and venous thrombosis, major bleeding episodes, cerebral vascular events, and premature mortality. Spinocerebellar hemangioblastomas, renal carcinomas, and pheochromocytomas typical of classical VHL tumor predisposition syndrome have not been found, and no increased risk of cancer has been demonstrated. Retrospective analyses among patients with Chuvash polycythemia have not shown benefit for therapy with phlebotomy or aspirin, but these and other modes of therapy should be studied prospectively. Further investigation of the vascular complications of Chuvash polycythemia may increase our fundamental knowledge of thrombophilia, bleeding diatheses, and protection from cancer. PMID- 16673285 TI - Thrombopoietin and platelet function. AB - In hematopoietic stem cells and megakaryocytes, the thrombopoietin (TPO) receptor signals to control proliferation, maturation, and antiapoptosis. In the anucleated platelet, much of this signaling appears out of place. Nevertheless, platelets possess TPO receptors and the cascades for transduction of TPO signals, but the final effect has shifted from DNA regulation to control of platelet aggregation and secretion. Although at physiological concentrations, TPO is not a sufficiently strong stimulator to trigger platelet functions in the absence of a second stimulator, TPO increases the sensitivity of platelets to agonist stimulation, leading to better functions at lower stimulation. PMID- 16673287 TI - Hepatitis B virus: molecular virology and common mutants. AB - A high rate of viral turnover, combined with an error-prone polymerase, results in a very high frequency of mutational events during hepatitis B virus replication. Mutants may accumulate, particularly in individuals at advanced stages of persistent infection and with antibody- and cell-mediated immune responses to the virus. Specific mutants may be selected from these populations, especially by antibody and cytotoxic T lymphocyte responses and by antiviral therapy. In some cases, the mutants may be associated with especially severe acute hepatitis (including acute liver failure) and the development of sequelae such as cirrhosis and hepatocellular carcinoma. Antiviral resistance poses a challenge to therapy that might be overcome by the use of combinations of antiviral drugs. PMID- 16673288 TI - Serological and molecular diagnosis. AB - The development of serological assays to detect hepatitis B surface antigen (HBsAg) has played a major role in the diagnosis of hepatitis B virus (HBV) infection. With other hepatitis B serological assays, a diagnosis of acute or chronic HBV infection, past infection, or successful vaccination can be determined. However, serological profiles can at times be atypical or ambiguous. Some of these difficulties may be overcome by HBV DNA testing, which may elucidate an individual's true hepatitis B status. In addition, quantification of HBV DNA provides a means of monitoring the effectiveness of antiviral therapy and detecting the early development of antiviral drug resistance. Molecular diagnostics are also being applied to HBV-infected liver tissue. The use of molecular techniques to quantify intrahepatic HBV DNA and other key HBV replicative intermediates may provide additional options for monitoring and predicting treatment efficacy, but such assays require further validation before they can be used routinely. PMID- 16673289 TI - Immunopathogenesis: role of innate and adaptive immune responses. AB - Hepatitis B virus (HBV) infection in immunocompetent adults usually results in a self-limited, transient liver disease and viral clearance, with only a small percentage (5 to 10%) developing chronic hepatitis associated with viral persistence. In contrast, when neonates are infected, more than 90% become persistently infected, suffering differing degrees of chronic liver disease. Activation of immunity plays a central role in host-virus interactions, greatly influencing viral replication and the clinical outcome of infection. Although all of the specific mechanisms and consequences of this interaction have not been elucidated, the purpose of this article is to describe the basic arms of the immune system as they interact with the HBV and describe the present state of knowledge in this area. These arms may be divided broadly into innate and specific immune responses, and they have different roles and responses in acute and chronic infection. PMID- 16673290 TI - Hepatitis B e antigen-positive chronic hepatitis B: natural history and treatment. AB - The natural history of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B is very heterogeneous. Age at acquisition is a major factor in determining the natural history of chronic infection. The vigor of the host immune response to the virus, viral factors (genotype, core promoter mutations, and duration of viral replication) as well as exogenous factors (alcohol, immune suppression) all influence the severity of disease. The goal of antiviral therapy is HBeAg seroconversion, and preferably HB surface Ag seroconversion as this latter end point is associated with sustained immune control and the halting of disease progression. Although peginterferon is now considered as the first line of therapy for HBeAg-positive chronic hepatitis B, in most cases there are circumstances where nucleos(t)ide analogues are indicated (e.g., decompensated liver disease) for those requiring cancer chemotherapy/other immunosuppressive agents and for those with contraindications to interferon. The major challenge for the clinician using these agents is the emergence of antiviral drug resistance. Long-term immune control of viral replication is key to improving patient outcome. PMID- 16673291 TI - Hepatitis B e antigen-negative chronic hepatitis B: natural history and treatment. AB - Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B evolves in the natural history of chronic hepatitis B virus (HBV) infection linked with selection of nonproducing HBeAg but replication-competent HBV mutants, and may have a potentially severe and progressive course. Effective suppression of HBV replication is the main therapeutic target. Sustained off-therapy responses are rare with treatment of finite duration, except perhaps for interferon-based therapies, which induce such responses in a sizeable, yet small proportion of patients. Eventually, the majority of patients will be treated with long-term oral antiviral therapy, which improves patients' outcome but is associated with progressively increasing rates of viral resistance. The long-term resistance profile of adefovir is significantly better than that of lamivudine (LMV), whereas data for entecavir currently are limited to 2 years, with resistance developing in LMV-resistant but not in treatment-naive patients. Combination therapy with adefovir added to LMV in LMV-resistant patients is extremely effective; cases of adefovir-resistance have not been reported to date. PMID- 16673292 TI - Hepatitis B virus-related cirrhosis: natural history and treatment. AB - In patients with compensated hepatitis B virus (HBV) cirrhosis, active viral replication correlates significantly with the risk of hepatic flare, decompensation, and the development of hepatocellular carcinoma (HCC). The 5-year survival of patients with compensated cirrhosis was reported to be 80 to 85%, and is significantly lower in patients with replicative HBV. Both interferon and maintenance lamivudine therapy have been shown to reduce the risk of decompensation or HCC and prolong survival in responders. A finite course of interferon is recommended as the first-line agent. For patients who had a contraindication for or who have failed interferon therapy, direct antiviral(s) can be considered for long-term treatment. Once decompensation occurs, mortality increases remarkably. Early treatment with nucleoside analogues should be instituted. Lamivudine therapy is associated with rapid viral suppression, improvement in Child-Pugh scores, and improved survival, but drug resistance is a major problem and is associated directly with a poor clinical outcome. Adefovir or entecavir is preferred in patients with decompensated cirrhosis who require long duration of treatment, due to the lower rate of development of resistance. PMID- 16673293 TI - Hepatocellular carcinoma and hepatitis B virus. AB - Chronic hepatitis B is the most common cause of hepatocellular carcinoma (HCC) in Asia. Integration of hepatitis B virus (HBV) genome is likely an early event of carcinogenesis. The integrated HBV genome may activate neighboring cellular genes directly to offer a selective growth advantage to the liver cells. Production of hepatitis B X protein can act as a transactivator on various cellular genes for tumor development. Hepatic inflammation and cirrhosis also favors the process of carcinogenesis. Various viral factors associated with hepatocellular carcinoma development include HBV genotype, basal core promoter mutations, and high viral load. Polymorphisms at the androgen receptor-regulating genes and cytokine genes are possible host factors associated with HCC. This review article summarizes the pathogenesis of HBV-related carcinogenesis and the viral and host factors that may increase the risk of HCC development. PMID- 16673294 TI - Antiviral drug resistance: clinical consequences and molecular aspects. AB - Antiviral drug resistance now poses a major problem for the management of patients with chronic hepatitis B. In theory, resistance may be prevented if a sufficiently potent antiviral drug, or combination of antiviral agents, is used that prevents viral replication and thereby the ongoing selection of hepatitis B virus quasispecies. Emergence of drug resistance in patients with hepatitis B generally results in progression of liver disease and in some cases, significant clinical deterioration if hepatic reserve is compromised. Currently, there are two major patterns of resistance mutations found in the viral reverse transcriptase (rt) that can be selected during monotherapy: (1) those that include the codon rtM204, which is part of the catalytic domain (YMDD) of the enzyme; (2) and those that do not include the codon. The rtM204I/V is selected by lamivudine and L-nucleosides. It is also part of the entecavir resistance profile and the tenofovir-lamivudine combination resistance profile. In contrast, resistance to adefovir is associated with mutations at rtN236T +/- rtA181V. A reasonable clinical goal is to develop an overall strategy that prevents the selection of resistance. These strategies have yet to be optimized for hepatitis B, but may include multiple therapies such as immune-based therapies in combination with one or more nucleoside analogue treatments. Future treatment protocols can be modeled on the use of multiple agents comprising highly active anti-retroviral therapy regimens that have been developed for the successful management of patients infected with human immunodeficiency virus. PMID- 16673295 TI - In vitro models for studying hepatitis B virus drug resistance. AB - Hepatitis B virus resistance to antivirals is conferred by the selection of polymerase gene mutants with reduced susceptibility to nucleoside analogues. Because of the increasing number of resistant mutants to the different available drugs, several phenotypic assays have been developed to provide cross-resistance data to guide clinicians in the management of therapy. Their possible use in the routine follow-up of antiviral treatment is also discussed. The different in vitro models for the study of HBV resistance include enzymatic and cell-based assays. Their relevance for the analysis of viral fitness, drug susceptibility, and cross-resistance is also reviewed. PMID- 16673296 TI - Small animal model systems for studying hepatitis B virus replication and pathogenesis. AB - The lack of reliable in vitro infection systems and convenient animal models has hindered progress in hepatitis B virus (HBV) research and the development of new treatment options. Due to various restraints encountered using chimpanzees and other models of HBV infection that are based on HBV-related viruses, and due to the necessity to work with small and well-characterized animal systems, it is not surprising that most recent developments have focused on mice. Various strains of transgenic mice harboring the entire HBV genome, or selected viral genes, were developed and proved to be very useful to elucidate mechanisms of HBV replication and pathogenesis. Furthermore, novel, exciting alternative mouse models have been developed recently, which allow not only studies on viral replication, but also the investigation of mechanisms of viral entry and clearance. This article provides an overview and discussion of the different mouse models of HBV replication and infection that are currently available. PMID- 16673297 TI - Current issues and future directions in treatment. AB - The clinical sequelae of chronic hepatitis B (CHB), such as liver cirrhosis and hepatocellular carcinoma, are determined mainly by the host immune response to the hepatitis B virus-encoded antigens. Disease remission in CHB has been associated with sustained viral suppression < 10 (4) to 10 (5) copies/mL. This can be achieved by either restoring the host immune control on the virus with immunomodulatory therapy, such as conventional or pegylated interferon-alfa therapy, or by continuous suppression of the viral replication by nucleos(t)ide therapy, such as lamivudine, adefovir dipivoxil, or entecavir treatment. Given that not all patients can tolerate or will respond to interferon-alfa-based therapy, maintenance therapy with nucleos(t)ide therapy is the alternative. However, this latter approach can lead to development of viral resistance and long-term safety concerns. Further improvement in CHB therapy is limited by a lack of understanding of the host immune characteristics associated with sustained disease remission. PMID- 16673299 TI - [Long-term experiences in the therapy of esthesioneuroblastoma]. AB - BACKGROUND: Esthesioneuroblastoma is a rare and clinically variable tumor of nasal sinus and skull base and challenging for modern multidisciplinary therapy. There are no generally known prognostic factors or generally accepted standard therapy regimens. PATIENTS AND METHODS: Between 1975 and 2001 26 patients were treated after the diagnosis of esthesioneuroblastoma was established. The cases were evaluated retrospectively. According to the classification of Kadish 1 patient (4 %) had stage A, 16 patients (53 %) stage B and 11 cases (43 %) had stage C tumors. Hyams grading could be obtained in 22 cases (81 %). Tumors were in 12 patients (52 %) graded I or II and in 10 cases tumors had grade III or IV (48 %). Operative therapy was performed in 23 patients (88.5 %), being in 5 patients a single mode therapy. In 18 cases combined therapy was performed (surgery and radiotherapy or radiotherapy and chemotherapy). RESULTS: Actuarial survival was 61.5 % (16/26). Disease specific 10- and 15-year-survival according to the estimation of Kaplan-Meier was 76.2 %. Patients with small tumors (Kadish A/B) had a 15-year-survival of 86.7 % compared to 63.6 % in cases with advanced tumors (Kadish C). In 7 cases (26.9 %) recurrences developed. Salvage therapy was performed in 5 cases (71.4 %) with a success rate and a 15-year survival each of 60 %. CONCLUSIONS: Therapy of esthesioneuroblastoma is challenging because of rarity and biologic variability of the tumor and and because of lack of a standard therapy. An interdisciplinary multimodal therapeutic approach is necessary especially in case of advanced tumors with promising results. Histopathological grading according to Hyams and tumor stage are important factors for survival and prognosis. Although recurrence can occur with high frequency even after prolonged time interval, long time survival can be improved after aggressive salvage therapy. Therefore longterm follow up is mandatory. PMID- 16673300 TI - [Long term change in quality of life after adenotonsillectomy for pediatric obstructive sleep disorders]. AB - BACKGROUND: To study short-term and long-term changes in quality of life (QOL) in children before and after adenotonsillectomy (T and A) for obstructive sleep disorders (OSDs). MATERIALS AND METHODS: Prospective study of 20 children underwent T and A for OSDs at the University hospital of Ulm/Germany. Caregivers were asked to complete the Brouillette-Score (BS) and OSA-18 survey, validated instruments for detecting symptoms and QOL change in children with OSDs, at the initial office visit prior to surgery (BS/1 and OSA-18/1) within 7.5 +/- 1.14 weeks after surgery (BS/2 and OSA-18/2), and 14.8 +/- 1.98 months after surgery (BS/3 and OSA-18/3). The BS comprises 3 items including: difficulty breathing during sleep, apnea observed by the caregivers and snoring. The OSA-18 survey comprises 18 items in 5 domains of sleep disturbance, physical suffering, emotional distress, daytime problems, and caregiver concerns. Scores from the preoperative and postoperative surveys were compared using the paired T-test and SPEARMAN-RANK test. RESULTS: 13 children were male (65 %). The mean age at the time of inclusion in the study was 6.2 +/- 1.63 years, mean preoperative body mass index (BMI) was 17.4 +/- 1.24 kg/m(2). Brouillette-Score: The mean Brouillette-Score before T and A (2.72) was significantly higher ( P = or < .001) than in the short-term (- 2.19) and in the long-term. (- 3.54). OSA-18 SURVEY: The mean total score for OSA-18/1 (83.4) was significantly higher ( P = or < .001) than the mean total score for OSA-18/2 (29.8) and OSA-18/3 (25.6). The overall correlation between BS and OSA-18 was R = .887 ( P = or < .001). CONCLUSION: Children with suspicious OSDs demonstrate significant short term and long-term improvement in the Brouillette-Score and OSA-18 survey. Caregivers perceive a long-term improvement for a minimum of one year in QOL after T and A for OSDs, although these improvements are not uniform across all domains of the OSA-18 survey. Use of the Brouillette-Score and the OSA-18 survey should decrease the need for polysomnographic monitoring and facilitate selection of children for T and A, whereas the OSA-18 questionnaire permits the more differentiated finding. PMID- 16673301 TI - Massive subcutaneous emphysema after attempted endoscopic retrograde cholangiopancreatography in a patient with a history of bariatric gastric bypass surgery. PMID- 16673302 TI - Ligation of a bleeding colonic varix using an upper gastrointestinal endoscope. PMID- 16673303 TI - Deliberate puncture of the bile duct and duodenal wall for placement of a biliary endoprosthesis in a rendezvous procedure. PMID- 16673304 TI - Hyponatremic convulsion after oral sodium phosphate for bowel preparation in a patient with previous gastrectomy. PMID- 16673305 TI - Short-segment jejunal carcinoma mimicking multiple lesions of the small bowel at capsule endoscopy. PMID- 16673306 TI - A novel endoscopic marker: safety experiments in the rat stomach. AB - BACKGROUND AND STUDY AIMS: The study aimed to assess a newly developed endoscopic marker designed to cause only minor inflammatory reactions. MATERIALS AND METHODS: Chitosan and carbon powder were used in the marker substance. The product was a viscoelastic solution, which was injected into the submucosa in rat stomach walls. The tissue reactions were then examined histopathologically. The structure of the injected marker substance was examined with electron microscopy into rat stomach walls. India ink, which is currently used as an endoscopic marker, served as the control. RESULTS: Histopathological examination showed that inflammatory reactions with the novel agent were remarkably mild in the rat organs, while submucosally applied india ink caused severe inflammation in situ. The electron-microscopic findings showed that the carbon particles used were completely spherical in shape and that the carbon in the marker substance was entrapped in the chitosan networks. The india ink was shown to consist of a mixture of fine carbon particles and adhesive additives. CONCLUSIONS: The chitosan-carbon solution appears to be a promising endoscopic marker substance, causing significantly reduced inflammation. PMID- 16673307 TI - Improved colonoscopy success rate with a distally attached mucosectomy cap. AB - BACKGROUND AND STUDY AIMS: Although colonoscopy is a well-established procedure, various technical difficulties may cause failure. This report describes experience with cap-assisted colonoscopy (CAC), a technique in which a mucosectomy cap is attached to the end of colonoscope in order to improve the success rate. PATIENTS AND METHODS: From January 2003 to May 2005, CAC was used in consecutive patients with difficult colonoscopy procedures, which were defined firstly as failure to pass through the sigmoid colon after 20 min of examination, or secondly as failure to reach the cecum at the end of the procedure. Patients with prior colonic surgery, poor bowel preparation, distal colonic stricture, or obstructing tumors were excluded. The results with the CAC method were reviewed retrospectively. RESULTS: A total of 100 patients met the criteria for difficult colonoscopy during the study period. When CAC was used to repeat the procedure, cecal and terminal ileal intubation could then be achieved in 94 and 70 patients, respectively. In the remaining six patients in whom cecal intubation failed, one failure was due to a tumor obstruction at the splenic flexure. CONCLUSIONS: CAC can be used as a rescue method to improve the success rate of colonoscopy when failure is encountered. PMID- 16673308 TI - A new technique for endoscopic submucosal dissection for early gastric cancer using an external grasping forceps. AB - BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) has improved the success rate of en-bloc resection. We report here on a new technique using an external grasping forceps. PATIENTS AND METHODS: A total of 25 patients with suitable EGCs over 10 mm in diameter located in the gastric body were enrolled. After submucosal injection followed by circumcision of the lesion with a needle-knife, an external grasping forceps was introduced with the help of a second grasping forceps and anchored at the distal margin of the lesion. With gentle oral traction applied with this forceps, the lesion was dissected endoscopically in retroversion from the aboral side. RESULTS: The mean lesion size was 15.0 mm (range 10 - 25 mm). Using the technique described, all lesions could be resected en bloc with free margins. The mean procedure time was 45 min (range 30 - 80 minutes). No significant bleeding requiring blood transfusion or perforation occurred. CONCLUSIONS: This technical modification may simplify and shorten the gastric ESD procedure, except for lesions in distal locations, without compromising the efficacy. PMID- 16673309 TI - Detection of human aspartyl (asparaginyl) beta-hydroxylase and homeobox B7 mRNA in brush cytology specimens from patients with bile duct cancer. AB - BACKGROUND AND STUDY AIMS: The diagnosis of bile duct cancer is hampered by the low sensitivity of intraductal brush cytology and forceps biopsy. In the present study real-time reverse transcription polymerase chain reaction (RT-PCR) assays for the detection of human aspartyl (asparaginyl) beta-hydroxylase (HAAH) and homeobox B7 (HoxB7) mRNA from intraductal brush cytology specimens were established. Both markers are overexpressed in biliary cancer cell lines and possibly involved in the pathogenesis of bile duct cancer. PATIENTS AND METHODS: RT-PCR assays were validated for detection limit, in-assay variability, and inter assay variability. Target gene expression was determined in brush cytology specimens from 16 patients with biliary strictures (11 with histologically proven cholangiocarcinomas and five with benign biliary strictures). RESULTS: The assay was quick (about 3 h), highly sensitive (with detection limits between 3 and 106 molecules), and reproducible (maximum in-assay variability 10.3 %, maximum inter assay variability 11.8 %). The sensitivity of routine brush cytology alone was 36 % (four of 11 cases), with 100 % specificity. A combination with detection of HoxB7 and HAAH mRNA increased the overall diagnostic sensitivity to 82 %. CONCLUSIONS: Detection of these markers using the RT-PCR assays from brush cytology specimens described here may prove to be a useful additional tool for the diagnosis of bile duct carcinoma. PMID- 16673310 TI - Cholangioscopic characterization of dominant bile duct stenoses in patients with primary sclerosing cholangitis. AB - BACKGROUND AND STUDY AIMS: Primary sclerosing cholangitis (PSC) is associated with the development of cholangiocarcinoma in up to 10 % of patients. Cholangiography or endoscopic tissue sampling does not reliably distinguish between cholangiocarcinoma and a benign dominant bile duct stenosis. The aim of the present study was to assess the value of cholangioscopy for distinguishing between benign and malignant dominant stenoses in PSC patients. PATIENTS AND METHODS: Fifty-three PSC patients with dominant bile duct stenoses were prospectively studied. Transpapillary cholangioscopy and endoscopic tissue sampling were carried out in addition to endoscopic retrograde cholangiography (ERC). The cholangiography and cholangioscopic findings were classified as malignant or benign by the investigators. A final diagnosis of malignant stenosis was based on positive histology and/or cytology, whereas a benign condition was assumed in cases of negative tissue sampling and uneventful extended clinical follow-up. RESULTS: Twelve PSC patients (23 %) had dominant bile duct stenoses caused by cholangiocarcinoma, whereas 41 of the 53 patients (77 %) had benign dominant bile duct stenoses. Cholangioscopy was significantly superior to ERC for detecting malignancy in terms of its sensitivity (92 % vs. 66 %; P = 0.25), specificity (93 % vs. 51 %; P < 0.001), accuracy (93 % vs. 55 %; P < 0.001), positive predictive value (79 % vs. 29 %; P < 0.001), and negative predictive value (97 % vs. 84 %; P < 0.001). Transpapillary cholangioscopy is more sensitive and specific for characterizing malignant bile duct stenosis in comparison with endoscopic brush cytology. CONCLUSIONS: Transpapillary cholangioscopy significantly increases the ability to distinguish between malignant and benign dominant bile duct stenoses in patients with PSC. PMID- 16673311 TI - The outcome of suspected upper gastrointestinal bleeding with 24-hour access to upper gastrointestinal endoscopy: a prospective cohort study. AB - BACKGROUND AND STUDY AIM: The aim was to evaluate the 30-day mortality after endoscopy for suspected upper gastrointestinal bleed, following the implementation of national audit guidelines at our hospital. PATIENTS AND METHODS: All patients with suspected upper gastrointestinal bleeding, referred for endoscopy to our teaching hospital between October 2001 and December 2003, were included in a prospective cohort study. RESULTS: A total of 716 patients with suspected upper gastrointestinal tract haemorrhage were referred for urgent endoscopy. The median age was 69 years (interquartile range 51 - 80 years). Bleeding from peptic ulcer remained the single most common endoscopic diagnosis (40 %). The overall re-bleeding rate for all patients with a gastrointestinal haemorrhage was 10 %. The overall 30-day mortality rate was 14.6 %. This was not significantly different from the mortality rate in 1995 of 10.5 % ( P = 0.11). Patients who died were significantly older (78 vs. 67 years, 95 %CI of the difference 5 to 12, P < 0.001). However, in only 29 % (30/105) was gastrointestinal haemorrhage stated in the death certificate as a factor which contributed to their death. CONCLUSIONS: Our results show that implementing the good practice guideline has a limited impact on overall mortality because of contributing factors that are beyond the control of clinicians. PMID- 16673312 TI - Analysis of colonoscopic findings in the differential diagnosis between intestinal tuberculosis and Crohn's disease. AB - BACKGROUND AND STUDY AIMS: Intestinal tuberculosis and Crohn's disease are chronic inflammatory bowel disorders that are difficult to differentiate from one another. This study aimed to evaluate the diagnostic value of various colonoscopic findings in the differential diagnosis between intestinal tuberculosis and Crohn's disease. PATIENTS AND METHODS: Colonoscopic findings on initial work-up were prospectively recorded in patients with an initial diagnosis of either intestinal tuberculosis or Crohn's disease. These findings were analyzed after a final diagnosis of intestinal tuberculosis (n = 44) or Crohn's disease (n = 44) had been made after follow-up. RESULTS: Four parameters (anorectal lesions, longitudinal ulcers, aphthous ulcers, and cobblestone appearance) were significantly more common in patients with Crohn's disease than in patients with intestinal tuberculosis. Four other parameters (involvement of fewer than four segments, a patulous ileocecal valve, transverse ulcers, and scars or pseudopolyps) were observed more frequently in patients with intestinal tuberculosis than in patients with Crohn's disease. We hypothesized that a diagnosis of Crohn's disease could be made when the number of parameters characteristic of Crohn's disease was higher than the number of parameters characteristic of intestinal tuberculosis, and vice versa. Making these assumptions, we calculated that the diagnosis of either intestinal tuberculosis or Crohn's disease would have been made made correctly in 77 of our 88 patients (87.5 %), incorrectly in seven patients (8.0 %), and would not have been made in four patients (4.5 %). CONCLUSIONS: A systematic analysis of colonoscopic findings is very useful in the differential diagnosis between intestinal tuberculosis and Crohn's disease. PMID- 16673313 TI - Pneumatosis coli. PMID- 16673314 TI - Acute gastric ulcer associated with gamma knife treatment (conformal stereotactic radiotherapy) of recurrent hepatocellular carcinoma. PMID- 16673315 TI - Isolated vasculitis of the stomach: a novel or rare disease with a difficult differential diagnosis. AB - In up to 80 % of patients with vasculitis, signs of the disease are also seen in the gastrointestinal tract. However, no cases of exclusively gastric vasculitis have previously been reported. We report here the case of a 45-year-old woman with upper abdominal discomfort (no arthropathy), with gastroscopic and endoscopic ultrasound (EUS) findings that mimicked scirrhous gastric carcinoma. Gastroscopy revealed giant gastric folds and a suspicious antral ulcer (with histological findings suggesting chronic active pangastritis). EUS showed a concentric, thickened gastric wall (8 mm) with "pseudolamellation" and more than five enlarged lymph nodes in the paragastric region (lesser curvature). On the basis of suspected scirrhous gastric carcinoma, the patient underwent a four fifths gastric resection of the altered parts of the gastric tissue. The postoperative course was uneventful. Histological examination of the specimen revealed severe obliterative panvasculitis of the stomach. During a 22-month follow-up period, no signs or symptoms of systemic primary or secondary vasculitis were found in the patient's medical history, symptoms, laboratory parameters, or imaging. This case shows for the first time that a specific gastric panvasculitis can occur, either as a preliminary stage of the condition or as a distinct manifestation of vasculitis associated with the stomach alone. Gastric resection appears to be indicated in patients with isolated obliterative gastric vasculitis, since it avoids the side effects of long-term immunosuppressive therapy and provides prognostic information that takes account of the differential diagnosis of scirrhous gastric carcinoma. PMID- 16673319 TI - Valerian extract Ze 911 inhibits postsynaptic potentials by activation of adenosine A1 receptors in rat cortical neurons. AB - In this study we evaluated the adenosine A1 receptor-mediated effect of valerian extract (Ze 911) on postsynaptic potentials (PSPs) in pyramidal cells of the rat cingulate cortex in a slice preparation. We first observed that N6 cyclopentyladenosine (CPA, 0.01 - 10 microM), an adenosine A1 receptor agonist, inhibited PSPs in a concentration-dependent manner. The CPA (10 microM)-induced inhibition was antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM), an adenosine A1 receptor antagonist. Ze 911 concentration dependently (0.1 - 15 mg/mL) inhibited PSPs in the presence of the adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC, 0.2 microM) and adenosine deaminase (1 U/mL). The maximal inhibition induced by 10 mg/mL was completely antagonised by DPCPX (0.1 microM), an A1 receptor blocker. The data suggest that activation of adenosine A1 receptors is involved in the pharmacological effects of the valerian extract Ze 911. PMID- 16673329 TI - Ginsenosides Rg3 and Rh2 inhibit the activation of AP-1 and protein kinase A pathway in lipopolysaccharide/interferon-gamma-stimulated BV-2 microglial cells. AB - The anti-inflammatory effect of ginsenosides Rg3 and Rh2, which improves ischemic brain injury induced by middle cerebral artery occlusion, was investigated in lipopolysaccharide (LPS) and IFN-gamma-induced murine BV-2 microglial cells. Ginsenoside Rh2 inhibited the production of NO, with an IC50 value of 17 microM. The inhibitory effect of Rh2 on NO correlates with the decreased protein and mRNA expression of an inducible NO synthase (iNOS) gene. Additionally, ginsenoside Rh2 inhibited the expression of COX-2, pro-inflammatory TNF-alpha and IL-1beta in BV 2 cells induced by LPS/IFN-gamma, while it increased the expression of the anti inflammatory cytokine IL-10. Electrophoretic mobility shift assays revealed that ginsenoside Rh2 significantly inhibited the LPS/IFN-gamma-induced AP-1 DNA binding activity, while it enhanced the protein binding to CRE sequences. However, it did not affect NF-kappaB binding activity. Thus, the anti inflammatory effect of Rh2 appears to depend on the AP-1 and protein kinase A (PKA) pathway. The anti-inflammatory effect of ginsenoside Rg3 against LPS/IFN gamma-activated BV-2 cells was less potent than that of ginsenoside Rh2. These findings suggest that the in vivo anti-ischemic effect of ginsenoside Rg3 may originate from ginsenoside Rh2, which is a main metabolite of ginsenoside Rg3 by intestinal microflora, and that of ginsenoside Rh2 may be due to its anti inflammatory effect in brain microglia. PMID- 16673331 TI - Two new phthalides from Ligusticum chuanxiong. AB - Two new phthalide dimers, namely chuanxiongnolide A (1) and chuanxiongnolide B (2), along with eleven known phthalides and derivatives ( 3 - 13), were isolated from the roots of Ligusticum chuanxiong Hort. (Umbelliferae). The chemical structures of the new compounds were established by HR-ESI-MS, 1D- and 2D-NMR spectroscopic analysis, whilst the relative stereochemistry of 1 was confirmed by X-ray crystallographic data. PMID- 16673333 TI - Aeruginascin, a trimethylammonium analogue of psilocybin from the hallucinogenic mushroom Inocybe aeruginascens. AB - The hallucinogenic mushroom Inocybe aeruginascens contains several typical Psilocybe alkaloids including psilocybin. We have now elucidated the structure of a further indole derivative named aeruginascin as the quaternary ammonium compound N, N, N-trimethyl-4-phosphoryloxytryptamine. Aeruginascin is closely related to the frog skin toxin bufotenidine (5-HTQ), a potent 5-HT3 receptor agonist, and has been found exclusively in Inocybe aeruginascens so far. PMID- 16673335 TI - Molecular identification of Sinopodophyllum hexandrum and Dysosma species using cpDNA sequences and PCR-RFLP markers. AB - Rhizomes of Sinopodophyllum hexandrum and Dysosma species, which have long been used in the traditional Chinese herbal medicine, have similar morphology and chemical composition. However, the podophyllotoxin content is higher in the rhizomes of S. hexandrum than in those of Dysosma species. The PCR-amplified fragments of trnT -trnL showed length variation between S. hexandrum and Dysosma species, and sequence comparison indicated that the length variation resulted from differential indels. There were species-specific PCR-RFLP markers of the chloroplast trnD -trnT region. Our results suggest that both chloroplast intergenic regions can be used for the identification of S. hexandrum and Dysosma Rhizoma medicines on the market. PMID- 16673337 TI - [The symptom checklist-27 in Germany]. AB - The symptom-checklist SCL-27 is a short, multidimensional screening instrument for mental health problems. It contains six subscales, i. e. depressive, dysthymic, vegetative, agoraphobic, sociophobic symptoms, symptoms of mistrust and a global severity index (GSI-27). A survey is presented in two representative samples with overall more than 4500 subjects. The scales of the SCL-27 show satisfying reliability (Cronbach's alpha > 0.70 for all subscales and alpha > 0.90 for global severity) throughout, as well as a largely congruent six factor structure (Comparative Fit Index CFI = 0.91, Standardisized Root Mean square Residual SRMR = 0.04). While women presented with higher scores in the year 1996 on almost all subscales, this was no longer observed in 2003. Additionally, age effects were less pronounced in 2003 than in 1996. PMID- 16673338 TI - Migration and the genetic covariance between habitat preference and performance. AB - Studies of the genetic covariance between habitat preference and performance have reported conflicting outcomes ranging from no covariance to strong covariance. The causes of this variability remain unclear. Here we show that variation in the magnitude of genetic covariance can result from variability in migration regimes. Using data from walking stick insects and a mathematical model, we find that genetic covariance within populations between host plant preference and a trait affecting performance on different hosts (cryptic color pattern) varies in magnitude predictably among populations according to migration regimes. Specifically, genetic covariance within populations is high in heterogeneous habitats where migration between populations locally adapted to different host plants generates nonrandom associations (i.e., linkage disequilibrium) between alleles at color pattern and host preference loci. Conversely, genetic covariance is low in homogeneous habitats where a single host exists and migration between hosts does not occur. Our results show that habitat structure and patterns of migration can strongly affect the evolution and variability of genetic covariance within populations. PMID- 16673339 TI - A probabilistic analysis of fire-induced tree-grass coexistence in savannas. AB - Fires play an important role in determining the composition and structure of vegetation in semiarid ecosystems. The study of the interactions between fire and vegetation requires a stochastic approach because of the random and unpredictable nature of fire occurrences. To this end, this article develops a minimalist probabilistic framework to investigate the impact of intermittent fire occurrences on the temporal dynamics of vegetation. This framework is used to analyze the emergence of statistically stable conditions favorable to tree-grass coexistence in savannas. It is found that these conditions can be induced and stabilized by the stochastic fire regime. A decrease in fire frequency leads to bush encroachment, while more frequent and intense fires favor savanna-to grassland conversions. The positive feedback between fires and vegetation can convert states of tree-grass coexistence in semiarid savannas into bistable conditions, with both woodland and grassland as possible, though mutually exclusive, stable states of the system. PMID- 16673340 TI - Is it what we know or who we know? Choice of organism and robustness of inference in ecology and evolutionary biology: (American Society of Naturalists Presidential Address). PMID- 16673341 TI - The consequences of direct versus indirect species interactions to selection on traits: pollination and nectar robbing in Ipomopsis aggregata. AB - Organisms experience a complex suite of species interactions. Although the ecological consequences of direct versus indirect species interactions have received attention, their evolutionary implications are not well understood. I examined selection on floral traits through direct versus indirect pathways of species interactions using the plant Ipomopsis aggregata and its pollinators and nectar robber. Using path analysis and structural equation modeling, I tested competing hypotheses comparing the relative importance of direct (pollinator mediated) versus indirect (robber-mediated) interactions to trait selection through female plant function in 2 years. The hypothesis that provided the best fit to the observed data included robbing and pollination, suggesting that both interactors are important in driving selection on some traits; however, the direction and intensity of selection through robbing versus pollination varied between years. I then increased my scope of inference by assessing traits and species interactions across more years. I found that the potential for temporal variation in the direction and intensity of selection was pronounced. Taken together, results suggest that assessing the broader context in which organisms evolve, including both direct and indirect interactions and across multiple years, can provide increased mechanistic understanding of the diversity of ways that animals shape floral and plant evolution. PMID- 16673342 TI - The prerequisites for and likelihood of generalist-specialist coexistence. AB - Mathematical models of three-consumer-two-resource systems are used to explore the possibility of coexistence when one consumer is a generalist utilizing both resources, and the other two are specialists utilizing only one. Such coexistence requires strongly saturating functional or numerical responses in at least one consumer and the presence of sustained asynchronous variation in resource abundances. Given these conditions, the effects of three dichotomous factors on the range of parameters allowing coexistence are examined: flexible versus inflexible resource choice by the generalist, endogenous or exogenous cause of resource cycles, and location of the two resources in a single habitat versus two habitats. Coexistence of all three species is found to be possible for all combinations of these factors except for inflexible choice in a two-habitat environment. Generalists experience frequency-dependent fitness because, when they are abundant, they synchronize resource cycles and/or reduce their amplitude. When the generalist can adaptively adjust its relative foraging on the two resources, coexistence conditions are broadened considerably, and coexistence commonly occurs readily with exogenous variation in resource growth and with resources located in distinct habitats. Adaptive behavior increases the generalist's ability to both synchronize and dampen resource cycles. PMID- 16673343 TI - Evolution of clutch size in cavity-excavating birds: the nest site limitation hypothesis revisited. AB - There are two major competing hypotheses for variation in clutch size among cavity-nesting species. The nest site limitation hypothesis postulates that nesting opportunities are more limited for weak excavators, which consequently invest more in each breeding attempt by laying larger clutches. Alternatively, clutch size may be determined by diet; the clutch sizes of strong excavators may be smaller because they are able to specialize on a more seasonally stable prey. We built a conceptual model that integrated hypotheses for interspecific variation in clutch size and tested it with comparative data on life-history traits of woodpeckers (Picidae) and nuthatches (Sittidae). In most analyses, diet explained more variation in clutch size among species than did propensity to excavate. Migratory status was positively associated with clutch size but was difficult to distinguish from diet since resident species consumed more bark beetles (a prey available in winter) and had smaller clutches than migratory species. The literature suggests that cavities are not limited in natural, old growth forests. Although our data do not rule out nest site limitation, we conclude that annual stability of food resources has a larger impact on the evolution of clutch sizes in excavators than does limitation of nest sites. PMID- 16673344 TI - Sexual systems and population genetic structure in an annual plant: testing the metapopulation model. AB - The need for reproductive assurance during dispersal, along with the pressure of local mate competition, means that the importance of frequent or repeated colonization is implicit in the literature on sexual system evolution. However, there have been few empirical tests of the association between colonization history and sexual system in plants, and none within a single species. Here we use patterns of genetic diversity to provide such a test in the Mercurialis annua species complex, which spans the range of systems from self-compatible monoecy through androdioecy to dioecy. This variation has been hypothesized to result from differing patterns of metapopulation turnover and recolonization. Because monoecy should be favored during colonization, androdioecy and dioecy will be maintained only in regions with low rates of local extinction and recolonization, and these differences should also be reflected in patterns of neutral genetic diversity. We show that monoecious populations of M. annua display lower within population genetic diversity than androdioecious populations and higher genetic differentiation than dioecious and androdioecious populations, as predicted by metapopulation models. In contrast, regional diversity in M. annua appears to be primarily a product of postglacial range expansion from two refugia in the eastern and western Mediterranean Basin. PMID- 16673345 TI - A new perspective on developmental plasticity and the principles of adaptive morph determination. AB - Organisms can have divergent paths of development leading to alternative phenotypes, or morphs. The choice of developmental path may be set by environmental cues, the individual's genotype, or a combination of the two. Using individual-based simulation and analytical investigation, we explore the idea that from the viewpoint of a developmental switch, genetic morph determination can sometimes be regarded as adaptive developmental plasticity. We compare the possibilities for the evolution of environmental and genetic morph determination and combinations of the two in situations with spatial variation in conditions. We find that the accuracy of environmental cues in predicting coming selective conditions is important for environmental morph determination, in accordance with previous results, and that genetic morph determination is favored in a similar way by the accuracy of genetic cues, in the form of selectively maintained gene frequency differences between local populations. Restricted gene flow and strong selection acting on the phenotypic alternatives produce clearer gene frequency differences and lead to greater accuracy of genetic cues. For combined environmental and genetic morph determination, we show that the developmental machinery can evolve toward efficiently combining information in environmental and genetic cues for the purpose of predicting coming selective conditions. PMID- 16673346 TI - Context-dependent discrimination and the evolution of mimicry. AB - Many mimetic organisms have evolved a close resemblance to their models, making it difficult to discriminate between them on the basis of appearance alone. However, if mimics and models differ slightly in their activity patterns, behavior, or use of microhabitats, the exact circumstances under which a signaler is encountered may provide additional clues to its identity. We employ an optimality model of mimetic discrimination in which signal receivers obtain information about the relative risk of encountering mimics and models by observing an external background cue and flexibly adjust their response thresholds. Although such flexibility on the part of signal receivers has been predicted by theory and is supported by empirical evidence in a range of biological settings, little is known about the effects it has on signalers. We show that the presence of external cues that partly reveal signaler identity may benefit models and harm mimics, harm both, or even benefit both, depending on ecological circumstances. Moreover, if mimetic traits are costly to express, or mimics are related to their neighbors, context-dependent discrimination can dramatically alter the outcome of mimetic evolution. We discuss context-dependent discrimination among signal receivers in relation to small-scale synchrony in model and mimic activity patterns. PMID- 16673347 TI - The evolution of worker caste diversity in social insects. AB - Morphological diversification of workers is predicted to improve the division of labor within social insect colonies, yet many species have monomorphic workers. Individual-level selection on the reproductive capacities of workers may counter colony-level selection for diversification, and life-history differences between species (timing of caste determination, colony size, genetic variation available) may mediate the strength of this selection. We tested this through phylogenetically independent contrast analyses on a new data set for 35 ant species. Evidence was found that early divergence of queen-worker developmental pathways may facilitate the evolution of worker diversity because queen-worker dimorphism was strongly positively associated with diversity. By contrast, risks for colonies that invest in specialized workers and colony size effects on costs of worker reproduction seem unlikely to strongly affect the evolution of worker diversity because there was no significant association between colony size and diversity when controlling statistically for queen-worker dimorphism. Finally, worker diversity was greater in species with multiple lineages per colony, and it was negatively associated with relatedness in monogynous species. This could be due to high intracolonial genetic variance favoring the expression and evolution of great worker diversity or to diversity evolving more easily when there is selection for repression of worker reproduction (worker policing). PMID- 16673348 TI - Nonrandom mating preserves intrasexual polymorphism and stops population differentiation in sexual conflict. AB - Evolutionary conflict between the sexes is predicted to lead to sexual arms races in which male adaptations for acquiring mates ("offense" traits) are met by female counteradaptations--for example, to reduce mating rate ("defense" traits). Such coevolutionary chases may be perpetual. However, we show here that the coevolutionary process may also lead to a stable state in which multiple offense defense trait pairs are maintained. This type of polymorphism below the species level is a result of sexual conflict in combination with nonrandom mating. Our results show that if nonrandom mating occurs with respect to male and female conflict traits, genetic correlations will act to stabilize the trait frequencies so that all morphs are maintained. We discuss the results in special relation to the evolution of female polymorphism in diving beetles and argue that the process we describe may be a general force that maintains polymorphism in other taxa as well. PMID- 16673349 TI - Integral projection models for species with complex demography. AB - Matrix projection models occupy a central role in population and conservation biology. Matrix models divide a population into discrete classes, even if the structuring trait exhibits continuous variation (e.g., body size). The integral projection model (IPM) avoids discrete classes and potential artifacts from arbitrary class divisions, facilitates parsimonious modeling based on smooth relationships between individual state and demographic performance, and can be implemented with standard matrix software. Here, we extend the IPM to species with complex demographic attributes, including dormant and active life stages, cross-classification by several attributes (e.g., size, age, and condition), and changes between discrete and continuous structure over the life cycle. We present a general model encompassing these cases, numerical methods, and theoretical results, including stable population growth and sensitivity/elasticity analysis for density-independent models, local stability analysis in density-dependent models, and optimal/evolutionarily stable strategy life-history analysis. Our presentation centers on an IPM for the thistle Onopordum illyricum based on a 6 year field study. Flowering and death probabilities are size and age dependent, and individuals also vary in a latent attribute affecting survival, but a predictively accurate IPM is completely parameterized by fitting a few regression equations. The online edition of the American Naturalist includes a zip archive of R scripts illustrating our suggested methods.A zip archive of R scripts illustrating our suggested methods is also provided. PMID- 16673350 TI - Viral ecology and the maintenance of novel host use. AB - Viruses can occasionally emerge by infecting new host species. However, the early phases of emergence can hinge upon ecological sustainability of the virus population, which is a product of both within-host population growth and between host transmission. Insufficient growth or transmission can force virus extinction before the latter phases of emergence, where genetic adaptations that improve host use may occur. We examined the early phase of emergence by studying the population dynamics of RNA phages in replicated laboratory environments containing native and novel host bacteria. To predict the breadth of transmission rates allowing viral persistence on each species, we developed a simple model based on in vitro data for phage growth rate over a range of initial population densities on both hosts. Validation of these predictions using serial passage experiments revealed a range of transmission rates for which the native host was a source and the novel host was a sink. In this critical range of transmission rates, periodic exposure to the native host was sufficient for the maintenance of the viral population on the novel host. We argue that this effect should facilitate adaptation by the virus to utilize the novel host--often crucial in subsequent phases of emergence. PMID- 16673351 TI - Predation risk influences adaptive morphological variation in fish populations. AB - Predators can cause a shift in both density and frequency of a prey phenotype that may lead to phenotypic divergence through natural selection. What is less investigated is that predators have a variety of indirect effects on prey that could potentially have large evolutionary responses. We conducted a pond experiment to test whether differences in predation risk in different habitats caused shifts in behavior of prey that, in turn, would affect their morphology. We also tested whether the experimental data could explain the morphological variation of perch in the natural environment. In the experiment, predators caused the prey fish to shift to the habitat with the lower predation risk. The prey specialized on habitat-specific resources, and there was a strong correlation between diet of the prey fish and morphological variation, suggesting that resource specialization ultimately affected the morphology. The lack of differences in competition and mortality suggest that the morphological variation among prey was induced by differences in predation risk among habitats. The field study demonstrated that there are differences in growth related to morphology of perch in two different habitats. Thus, a trade-off between foraging and predator avoidance could be responsible for adaptive morphological variation of young perch. PMID- 16673352 TI - Evolutionary response to selection on clutch size in a long-term study of the mute swan. AB - Life-history traits in wild populations are often regarded as being subject to directional selection, and the existence of substantial variation and microevolutionary stasis of these characters is therefore a problem in need of explanation. Avian clutch size is an archetypal life-history trait in this context, and many studies have sought to test explanations for stasis in clutch size. Surprisingly, there are many fewer studies that used long-term data to ask how selection acts on clutch size, particularly in a multivariate framework. In this article, we report selection, inheritance, and evolution of clutch size over 25 years in a colony of mute swans using a multivariate quantitative genetic framework to control for correlations with breeding time. We show that clutch size is influenced by both additive genetic and permanent environmental effects and that selection acts on clutch size in combination with breeding time. Natural selection on clutch size is strongly directional, favoring larger clutches, and we observe an increase in clutch size of 0.35 standard deviations, consistent with the expected response based on selection and inheritance of clutch size. We hypothesize that these changes result from recent relaxation of food constraints and predation risks experienced by this colony. PMID- 16673353 TI - Endovenous laser treatment for long saphenous vein incompetence. AB - BACKGROUND: Endovenous laser treatment is a percutaneous technique used for the treatment of long saphenous vein (LSV) incompetence. This paper presents the results of an uncontrolled case series undertaken to assess the feasibility, safety and efficacy of this technique. METHODS: Some 145 incompetent LSVs in 136 patients with saphenofemoral reflux were treated with endovenous laser. The data were evaluated prospectively. Assessment was carried out at 1 week, 3 and 12 months for LSV occlusion and symptomatic relief. RESULTS: Primary procedural success was achieved in 124 (85.5 per cent) of 145 LSVs. Reasons for primary failure included failed cannulation, failure to pass the guidewire and patient discomfort. At 3 months' follow-up, 105 (89.7 per cent) of 117 veins were totally and nine (7.7 per cent) were partially occluded. At 12 months, 63 (76 per cent) of 83 veins were totally and 15 (18 per cent) were partially occluded. At this stage 73 (88 per cent) of 83 patients remained satisfied, but 26 (31 per cent) had residual or recurrent varicosities. Of these, only five required further treatment. Complications included saphenous nerve injury in one patient and superficial skin burns in a second. CONCLUSION: Endovenous laser treatment for LSV reflux is safe and can be carried out under local anaesthesia in an outpatient setting with good patient satisfaction and low complication rates. PMID- 16673354 TI - Dynamic contrast-enhanced magnetic resonance imaging is a poor measure of rectal cancer angiogenesis. AB - BACKGROUND: The aim of this study was to investigate the use of magnetic resonance imaging (MRI) for non-invasive measurement of rectal cancer angiogenesis and hypoxia. METHODS: Fifteen patients with rectal adenocarcinoma underwent preoperative dynamic contrast-enhanced (DCE) and blood oxygenation level-dependent (BOLD) MRI. Microvessel density (CD31 level), and expression of vascular endothelial growth factor (VEGF) and carbonic anhydrase (CA) 9 were measured immunohistochemically in histological tumour sections from 12 patients. Serum VEGF levels were also measured in 14 patients. Correlations between quantitative imaging indices and immunohistochemical variables were examined. RESULTS: There was good correlation between circulating VEGF and CD31 expression (r(S) = 0.88, P < 0.001). CD31 expression did not correlate with any dynamic MRI parameter, except transfer constant, with which it correlated inversely (r(S) = 0.65, P = 0.022). Tissue and circulating VEGF levels did not correlate, and neither correlated with any tumour DCE MRI parameter. No relationship was seen between BOLD MRI and CA-9 expression. CONCLUSION: The negative correlation between transfer constant (reflecting tumour blood flow and microvessel permeability) with CD31 expression is paradoxical. DCE MRI methods for assessing tissue vascularity correlate poorly with histological markers of angiogenesis and hypoxia, suggesting that DCE MRI does not simply reflect static histological vascular properties in patients with rectal cancer. PMID- 16673355 TI - Trends in paediatric circumcision and its complications in England between 1997 and 2003. AB - BACKGROUND: It has been suggested that too many English boys undergo circumcision. This report describes how circumcision rates have changed in England between 1997 and 2003, including data on complication rates and on how age, medical indication and surgical specialty affect postoperative haemorrhage rates. METHODS: Data were extracted from the Hospital Episode Statistics database of admissions to National Health Service hospitals in England. Patients were included in the study if an Office of Population Censuses and Surveys version 4 code for circumcision was present in any of the operative procedure fields of the database; 75 868 boys below 15 years of age were included in the study. RESULTS: Circumcision rates declined by about 20 per cent, from 2.6 per 1000 boys per year in 1997 to 2.1 in 2003. Between 2000 and 2003, circumcision rates remained static at 2.1 per 1000 boys per year. Circumcision rates fell by 31.2 per cent for boys aged 0-4 years, 9.3 per cent for boys aged 5-9 years and increased by 7.7 per cent in boys aged 10-14 years; 90.2 per cent of circumcisions were done for phimosis and 1.2 per cent of boys experienced a complication. CONCLUSION: Circumcision rates in England continued to fall up until 2000, particularly in those aged under 5 years, in whom pathological phimosis is rare. The circumcision rate remains five times higher than the reported incidence of Phimosis. PMID- 16673356 TI - Effects of arterial pressure in an experimental isolated haemoperfused porcine kidney preservation system. AB - BACKGROUND: Normothermic preservation provides metabolic support to an ischaemically damaged organ before use as a kidney transplant. Optimal conditions for ex vivo preservation have not yet been established. This study examined the effects of arterial pressure on renal preservation using isolated haemoperfused kidneys. METHODS: An isolated organ preservation system, developed using cardiopulmonary bypass technology, was used to perfuse porcine kidneys with normothermic oxygenated blood. Groups of kidneys (n = 6) were perfused at a mean arterial pressure of 95, 75 or 55 mmHg. RESULTS: Kidneys perfused at the higher mean arterial pressures of 95 and 75 mmHg demonstrated improved renal function: mean(s.d.) area under the curve (AUC) for creatinine clearance 71(19) and 55(30) respectively versus 14(12) in the 55-mmHg group, P = 0.002; AUC for serum creatinine 938(140) and 1290(394) versus 2404(595), P = 0.003. The higher perfusion pressures were also associated with better acid-base homeostasis and improved renal haemodynamics. CONCLUSION: Mean arterial pressures of either 95 or 75 mmHg were capable of sustaining physiological renal function, but kidneys in the 95-mmHg group demonstrated superior renal function overall. PMID- 16673357 TI - Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging. AB - Ferumoxides-protamine sulfate (FE-Pro) complexes are used for intracellular magnetic labeling of cells to non-invasively monitor cell trafficking by in vivo MRI. FE-Pro labeling is non-toxic to cells; however, the effects of FE-Pro labeling on cellular expression of transferrin receptor (TfR-1) and ferritin, proteins involved in iron transport and storage, has not been reported. FE-Pro labeled human mesenchymal stem cells (MSCs), HeLa cells and primary macrophages were cultured from 1 week to 2 months and evaluated for TfR-1 and ferritin gene expression by RT-PCR and protein levels were determined using Western blots. MTT (proliferation assay) and reactive oxygen species (ROS) analysis were performed. FE-Pro labeling of HeLa and MSCs resulted in a transient decrease in TfR-1 mRNA and protein levels. In contrast, Fe-Pro labeling of primary macrophages resulted in an increase in TfR-1 mRNA but not in TfR-1 protein levels. Ferritin mRNA and protein levels increased transiently in labeled HeLa and macrophages but were sustained in MSCs. No changes in MTT and ROS analysis were noted. In conclusion, FE-Pro labeling elicited physiological changes of iron metabolism or storage, validating the safety of this procedure for cellular tracking by MRI. PMID- 16673359 TI - Clinical and epidemiological findings in patients with central ray deficiency: split hand foot malformation (SHFM) in Manitoba, Canada. AB - We conducted a clinical population study to examine the incidence and epidemiology of split hand foot-malformation (SHFM) in Manitoba from 1957 to 2003. The total number of births during this period was 850,742. Forty-three patients with SHFM were identified, resulting in an incidence of 1 in 19,784 births. Most patients were ascertained through referrals to the Section of Genetics and Metabolism at the Children's Hospital, Winnipeg, Manitoba. Overall, 22 (51.2%) of affected individuals were females and 21 (48.8%) were male. The left upper limb (LUL) was the most frequently affected, (in 46.5% of patients). The right hand was involved in 39.5%. In 4 patients (9.3%) all four limbs were affected. SHFM is classified as a failure of formation of parts according to the International Federation of Surgical Societies of the Hand (IFSSH) and has also been categorized as Typical or Atypical. Individuals in the Manitoba cohort were classified into two main categories: Typical (29 cases) and Atypical (3 cases). However, 11 patients were not easily placed into either group and comprised a distinct category termed "difficult to classify." Patients in the three groups were then further subdivided depending on whether or not they had additional congenital anomalies. These complex patients included those with single gene disorders in which SHFM has been reported (e.g., ectodermal dysplasia Ectrodactyly Clefting (EEC), tibial aplasia with SHFM, fibular aplasia with SHFM), as well as those with other recognized or unknown patterns of anomalies. Two had deletions involving 9q and 5p respectively. Unlike some other studies, we did not find an excess of males or right-sided defects and only two of the cases- two sisters--were related. PMID- 16673360 TI - Magnetic resonance imaging with T1 dispersion contrast. AB - Prepolarized MRI uses pulsed magnetic fields to produce MR images by polarizing the sample at one field strength (approximately 0.5 T) before imaging at a much lower field (approximately 50 mT). Contrast reflecting the T(1) of the sample at an intermediate field strength is achieved by polarizing the sample and then allowing the magnetization to decay at a chosen "evolution" field before imaging. For tissues whose T(1) varies with field strength (T(1) dispersion), the difference between two images collected with different evolution fields yields an image with contrast reflecting the slope of the T(1) dispersion curve between those fields. Tissues with high protein content, such as muscle, exhibit rapid changes in their T(1) dispersion curves at 49 and 65 mT due to cross-relaxation with nitrogen nuclei in protein backbones. Tissues without protein, such as fat, have fairly constant T(1) over this range; subtracting images with two different evolution fields eliminates signal from flat T(1) dispersion species. T(1) dispersion protein-content images of the human wrist and foot are presented, showing clear differentiation between muscle and fat. This technique may prove useful for delineating regions of muscle tissue in the extremities of patients with diseases affecting muscle viability, such as diabetic neuropathy, and for visualizing the protein content of tissues in vivo. PMID- 16673358 TI - The spectrum of WRN mutations in Werner syndrome patients. AB - The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46-48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators. PMID- 16673361 TI - In vivo estimation of the flow-driven adiabatic inversion efficiency for continuous arterial spin labeling: a method using phase contrast magnetic resonance angiography. AB - The accurate quantification of perfusion with arterial spin labeling (ASL) requires consideration of a number of factors, including the efficiency of the inversion and control pulses used for spin labeling. In this study the effects of spin velocity on continuous ASL efficiency when using the amplitude modulated control strategy were investigated using simulations of the Bloch equations. The inversion efficiency was determined in vivo by combining the simulations with phase-contrast velocity mapping data acquired at the level of the tagging plane. Using this novel method, an average inversion efficiency of 69% was calculated for a group of 28 subjects, in good agreement with experimental data reported previously. There was, however, a large range in inversion efficiency measured across the subject group (50-76%), indicating that the velocity dependence of the amplitude modulated control efficiency may introduce additional variability into the perfusion calculations if not properly taken into account. PMID- 16673362 TI - Enlarged lymph nodes in porta hepatis: sonographic sign of chronic hepatitis B and C infections. AB - PURPOSE: Enlarged lymph nodes in the hepatoduodenal ligament are prevalent in chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV); however, the clinical significance of this sonographic finding in an endemic area is unknown. METHODS: Six hundred outpatients were categorized into 4 groups (nonviral, HBV, HCV, and HBV and HCV) using viral markers. The prevalence and size of enlarged lymph nodes were compared. Correlation between clinical parameters and nodal size was evaluated. RESULTS: The incidence of detectable nodes in both the HBV group and the HCV group was significantly increased (56.9% and 69.4%, respectively; both p < 0.001) compared with the nonviral group; this rate was independent of aminotransferase levels. Nodal width was the only significant parameter when viral and nonviral groups were compared (p < 0.05). If a width of more than 5 mm was used to predict HBV or HCV infection, the positive predictive rate was 88% and the specificity was 89%. CONCLUSIONS: Lymph nodes in the hepatoduodenal ligament, especially those wider than 5 mm, suggest chronic HBV or HCV infection instead of only chronic hepatitis, especially in an endemic area such as Taiwan. PMID- 16673363 TI - Doppler ultrasound assessment of posterior tibial artery size in humans. AB - PURPOSE: The difference between structural remodeling and changes in tone of peripheral arteries in the lower extremities has not been evaluated. The purpose of this study was to (1) evaluate the day-to-day reproducibility and interobserver reliability (IOR) of posterior tibial artery (PTA) diameter measurements and (2) evaluate the effect of posture on PTA diameter at rest (Drest), during 10 minutes of proximal cuff occlusion (Dmin), and after the release of cuff occlusion (Dmax), as well as range (Dmax - Dmin) and constriction [(Dmax - Drest)/(Dmax - Dmin) x 100] in vivo. METHODS: We used B-mode sonography to image the PTA during each condition. RESULTS: Day-to-day reliability was good for Drest (intraclass correlation coefficient [ICC] 0.95; mean difference 4.2%), Dmin (ICC 0.93; mean difference 5.4%), and Dmax (ICC 0.99; mean difference 2.2%). The coefficient of repeatability for IOR was 70.5 microm, with a mean interobserver error of 4.7 microm. The seated position decreased Drest (2.6 +/- 0.2 to 2.4 +/- 0.3 mm; p = 0.002), increased Dmin (2.1 +/- 0.2 to 2.4 +/- 0.2 mm; p = 0.001), and decreased Dmax (3.1 +/- 0.4 to 2.8 +/- 0.3 mm; p < 0.001) compared with the supine position. The seated position also decreased arterial range (Dmax - Dmin) from 0.9 +/- 0.2 to 0.5 +/- 0.1 mm (p = 0.003) and increased basal arterial constriction from 57 +/- 19% to 105 +/- 27% (p = 0.007). CONCLUSIONS: The system employed for measuring PTA diameter yields unbiased and consistent estimates. Furthermore, lower extremity arterial constriction and range change with posture in a manner consistent with known changes in autonomic activity. PMID- 16673364 TI - Ceftriaxone-associated biliary pseudolithiasis in children. AB - PURPOSE: Ceftriaxone is known to induce reversible precipitations, known as pseudolithiasis, in the gallbladder and urinary tract. The aim of this study was to investigate the incidence and predisposing factors that contribute to this side effect. METHODS: A prospective study was conducted in 156 children admitted for the treatment of various infections with different daily ceftriaxone doses (50 mg/kg, 75 mg/kg, and 100 mg/kg). Sonographic examinations of the gallbladder and urinary tract were performed before treatment on the third and seventh day of therapy, and at the first and second month after the end of treatment. Patients with positive findings were followed with weekly sonographic examinations until the abnormality resolved. RESULTS: Abnormal gallbladder sonograms were demonstrated in 27 children (17%); 16 of them (10%) had gallbladder lithiasis, 11 had gallbladder sludge (7%) (n = 4 on the third day, n = 23 on the seventh day), and 1 developed urolithiasis (0.6%). Five children (19%) were symptomatic. The abnormalities resolved after a mean of 16 days (range 10-30 days). Patients with pseudolithiasis were older and treated with higher drug doses than those with normal sonographic findings (P < 0.01 and P < 0.05, respectively). CONCLUSIONS: Biliary pseudolithiasis (and infrequently nephrolithiasis) usually occurs in children receiving high doses of ceftriaxone. It is generally asymptomatic. When this reversible complication becomes symptomatic, unnecessary cholecystectomy should be avoided. PMID- 16673365 TI - Heel pad thickness and athletic activity in healthy young adults: a sonographic study. AB - PURPOSE: The aim of this study was to investigate the sonographic changes of heel fat pad thickness and compressibility index in healthy young adults in relation to level of athletic activity. MATERIALS AND METHODS: One hundred ten young adults (55 women and 55 men) with a body mass index between 18.5 and 24.9 were divided into 3 groups according to their athletic activity level: group 1, sedentary (n = 50); group 2, athletic activity <7 hours/week (n = 30); and group 3, athletic activity >or=7 hours/week (n = 30). The loaded heel pad thickness (LHPT) and unloaded heel pad thickness (ULHPT) were measured via sonography, and the heel pad compressibility index (HPCI) was calculated. RESULTS: The mean values of ULHPT, LHPT, and HPCI in group 1 were similar to those of group 2 (p > 0.05) and group 3 (p > 0.05). The mean values of ULHPT, LHPT, and HPCI were similar in the left and right feet in the 3 groups (p > 0.05). There was no correlation between level of athletic activity and ULHPT, LHPT, or HPCI. CONCLUSIONS: The heel pad thickness and HPCI of individuals engaging in athletic activity up to an average of 11 hours/week were similar to those of sedentary individuals. PMID- 16673366 TI - Sonographic diagnosis of a toothpick traversing the duodenum and penetrating into the liver. AB - Ingested foreign bodies rarely cause gastrointestinal perforation, because the majority are passed out uneventfully in the feces. However, long, sharp, slender, hard, indigestible objects such as toothpicks are dangerous and may lead to potentially life-threatening complications. We report a case of duodenal perforation caused by a toothpick and complicated by liver abscess and methicillin-resistant Staphylococcus aureus sepsis. Although laparotomy was not performed because of the patient's refusal to undergo surgery, the liver abscess and sepsis were controlled successfully with antibiotics. We also conducted a literature search for reports on injuries caused by ingested toothpicks. PMID- 16673367 TI - Retroperitoneal neurilemoma diagnosed by endosonographically guided fine needle aspiration. AB - Retroperitoneal neurilemoma is an often asymptomatic benign tumor that is usually discovered incidentally. We report a case of retroperitoneal neurilemoma diagnosed via endosonographically guided fine needle aspiration and emphasize the importance of obtaining a definite diagnosis before surgical treatment. PMID- 16673368 TI - Anterior sacral meningocele simulating ovarian cyst. AB - Anterior sacral meningocele is a rare condition characterized by the herniation of meningeal membranes and cerebrospinal fluid through a defect in the anterior aspect of the sacrum. We report a case of an anterior sacral meningocele that was mimicking an ovarian cyst. PMID- 16673369 TI - Intraoperative sonographically guided resection of hemangioblastoma in the cerebellum. AB - We report a case of hemangioblastoma in a 37-year-old woman. Intraoperative sonography was used to significantly reduce the duration of the intervention and ensure thorough resection of the tumor. We recommend real-time intraoperative sonography as a routine procedure in the surgical treatment of hemangioblastoma. PMID- 16673370 TI - Transcranial Doppler monitoring after embolization of an unruptured left paraclinoid aneurysm led to detection of subarachnoid hemorrhage. AB - A 46-year-old man who presented with an unruptured left paraclinoid aneurysm was treated via endovascular embolization using Guglielmi detachable coils, obtaining its complete exclusion. Within 5 hours, the patient developed a transient mild headache and moderate speech difficulty. CT scans revealed a left temporal ischemic area. Continuous transcranial Doppler monitoring was initiated. Eighteen hours after embolization, the patient developed a mild headache associated with a transient decrease in consciousness, while the diastolic blood flow velocity decreased and the pulsatility index increased in the left middle cerebral artery. These changes prompted us to perform CT, which revealed a subarachnoid hemorrhage. Angiograms demonstrated partial revascularization of the newly embolized aneurysm. The patient underwent a second embolization procedure with additional coils for complete exclusion of the aneurysm. His postoperative course was uneventful, with no additional neurological deficits. Although TCD monitoring is not recommended as a routine procedure in such cases, and experimental studies are needed to evaluate the possible risk of rebleeding in this specific setting, it could be used to detect the hemodynamic consequences of an acute increase in intracranial pressure, as in patients at risk of subarachnoid hemorrhage after endovascular treatment. PMID- 16673371 TI - Tuberculous abscess of the thyroid gland: a report of two cases. AB - Tuberculosis of the thyroid gland is very rare, with an acute abscess formation being the least common form of presentation. We report the sono-graphic features of two cases of tuberculous thyroid abscess that were confirmed via ultrasound guided fine needle aspiration biopsy. PMID- 16673372 TI - Complete epididymal separation presenting as polyorchidism. AB - We describe an unusual case of a 21-month-old boy with complete separation of the testis and epididymis. Two homogeneous structures were observed in the scrotum of a newborn boy in addition to a third structure. Polyorchidism was suspected, but follow-up sonographic studies showed a decrease in the echogenicity of the left scrotal structure. Surgical exploration revealed the testis and epididymis to be completely separated, with no duplicated testis. Orchidopexy was then performed. PMID- 16673373 TI - Glutamate leakage from a compartmentalized intracellular metabolic pool and activation of the lipoxygenase pathway mediate oxidative astrocyte death by reversed glutamate transport. AB - Astrocytes have essential roles for neuron survival and function, so that their demise in neurodegenerative insults, such as ischemia, deserves attention. A major event of the cell death cascade in ischemia is the reversed operation of excitatory amino acid transporters (EAAT), releasing glutamate. Cytotoxicity is conventionally attributed to extracellular glutamate accumulation. We previously reported that mimicking such dysfunction by EAAT substrate inhibitors, whose uptake induces glutamate release by heteroexchange, triggers glutathione (GSH) depletion and oxidative death of differentiated astrocytes in culture. Here we demonstrate that astrocyte death, although correlated with glutamate release, is not resulting from high extracellular glutamate-mediated toxicity. L-glutamate per se was gliotoxic only at concentrations much higher than the maximum reached with the potent EAAT substrate inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), and toxicity was lower. Moreover, high glutamate concentrations offered protection against PDC. Protection was also provided by L-aspartate, which is both transported by EAAT and metabolized into glutamate, and by inhibiting glutamine synthetase, which uses transported glutamate to synthesize glutamine. Neither D-aspartate, a metabolically inert EAAT substrate, nor compounds that can provide glutamate intracellularly but are not EAAT substrates offered protection. Interestingly, only the compounds providing protection prevented PDC-induced GSH depletion. These data strongly suggest that reversed uptake-mediated astrocyte death results from the leakage of glutamate from a compartmentalized intracellular metabolic pool specifically fuelled by EAAT, crucial for preserving GSH contents. In addition, we provide evidence for a minor contribution of the cystine-glutamate antiporter x(c) (-) but a major role of the 5-lipoxygenase pathway in this death mechanism. PMID- 16673374 TI - Transcription factor co-expression patterns indicate heterogeneity of oligodendroglial subpopulations in adult spinal cord. AB - Oligodendrocytes and their precursors serve critical roles in the maintenance of neurological function. Although activity of the transcription factors (TFs) Olig1, Olig2, Sox10, and Nkx2.2 is required during early oligodendrocyte development, their later expression in adult central nervous system is rather poorly characterized. Here we have analyzed co-expression patterns of these transcriptional proteins in the mouse cervical spinal cord. Our findings indicate that TF co-expression patterns describe heterogeneity in adult oligodendroglial populations (1) in distinct sub-regions of grey and white matter and (2) with respect to level of maturation from proliferating precursors to myelinating oligodendrocytes. Our findings suggest that TF co-expression patterns identify and might regulate distinct functional classes of grey and white matter oligodendroglia. PMID- 16673375 TI - Analysis of single nucleotide polymorphisms in genes in the chromosome 12Q24.31 region points to P2RX7 as a susceptibility gene to bipolar affective disorder. AB - Previous results from our genetic analyses using pedigrees from a French Canadian population suggested that the interval delimited by markers on chromosome 12, D12S86 and D12S378, was the most probable genomic region to contain a susceptibility gene for affective disorders. Association studies with microsatellite markers using a case/control sample from the same population (n = 427) revealed significant allelic associations between the bipolar phenotype and marker NBG6. Since this marker is located in intron 9 of the P2RX7 gene, we analyzed the surrounding genomic region for the presence of polymorphisms in regulatory, coding and intron/exon junction sequences. Twenty four (24) SNPs were genotyped in a case/control sample and 12 SNPs in all pedigrees used for linkage analysis. Allelic, genotypic or family-based association studies suggest the presence of two susceptibility loci, the P2RX7 and CaMKK2 genes. The strongest association was observed in bipolar families at the non-synonymous SNP P2RX7-E13A (rs2230912, P-value = 0.000708), which results from an over-transmission of the mutant G-allele to affected offspring. This Gln460Arg polymorphism occurs at an amino acid that is conserved between humans and rodents and is located in the C terminal domain of the P2X7 receptor, known to be essential for normal P2RX7 function. PMID- 16673377 TI - Improvement of bladder storage function by alpha1-blocker depends on the suppression of C-fiber afferent activity in rats. AB - AIMS: Alpha1-blockers improve voiding symptoms through the reduction of prostatic and urethral smooth muscle tone; however, the underlying mechanism of improvement of storage symptoms is not known. Using a rat model of detrusor overactivity caused by cerebral infarction (CI), we undertook the present study to determine whether the effect of an alpha1-blocker, naftopidil, is dependent on the suppression of C-fiber afferents. METHODS: To induce desensitization of C-fiber bladder afferents, we injected resiniferatoxin (0.3 mg/kg, RTX) sub-cutaneously to female Sprague-Dawley rats 2 days prior to left middle cerebral artery occlusion (MCAO) (RTX-CI rats). As controls we used rats without RTX treatment (CI rats). MCAO and insertion of a polyethylene catheter through the bladder dome were performed under halothane anesthesia. We investigated the effects on cystometrography (CMG) of intravenous (i.v.), intracerebroventricular (i.c.v.), or intrathecal (i.t.) administration of naftopidil in conscious CI rats. RESULTS: Bladder capacity (BC) was markedly reduced after MCAO in both RTX-CI and CI rats. I.v. administration of naftopidil significantly increased BC in CI rats without an increase in residual volume, but it had no effects on BC in RTX-CI rats. I.t. administration of naftopidil significantly increased BC in CI but not in RTX-CI rats. CONCLUSIONS: These results suggest that naftopidil has an inhibitory effect on C-fiber afferents in the lumbosacral spinal cord, improving BC during the storage phase. PMID- 16673378 TI - Effect of partial bladder outlet obstruction on nitrotyrosine levels and their correlation with contractile function. AB - AIMS: It has been demonstrated that partial bladder outlet obstruction (PBOO) causes free radical generation that, in turn, results in cellular and subcellular damage. We tested the hypothesis that nitration of proteins is associated with contractile dysfunctions in obstructive bladder disease. METHODS: Thirty rabbits were subjected to 1-28 days of partial outlet obstruction. Sham operated rabbits served as controls. Western blotting was used to determine the amount of nitrotyrosine level at the protein level. At each time point, isolated strips of bladder body were mounted in individual baths and the contractile response to field stimulation (FS), carbachol, and KCl determined. RESULTS: Bladder weight increased rapidly during the first 7 days and then increased slowly thereafter. There was a fourfold increase in the amount of nitrotyrosine in the 7 day obstructed groups when compared to sham controls and the levels remain elevated at 14 and 28 days of obstruction. Contractile dysfunction in response to FS (8 and 32 Hz) was noted as early as 1 day after obstruction and increased progressively over the study period. The decrease in response to carbachol and KCl was significant only after 3 days of obstruction and the progressive increase in dysfunction was slower than with FS. CONCLUSIONS: PBOO is accompanied by an increase in nitrotyrosine, a marker of free radical damage. Simultaneously there was a progressive decrease in contractility of detrusor smooth muscles (DSMs). Nitrotyrosine may be usable as a marker of free radical damage and reperfusion injury. PMID- 16673379 TI - Transurethral radiofrequency energy collagen micro-remodeling for the treatment of female stress urinary incontinence. AB - AIMS: This prospective, randomized, controlled clinical trial was performed to demonstrate the 12 months safety and efficacy of transurethral radiofrequency energy (RF) collagen micro-remodeling in women with stress urinary incontinence (SUI). MATERIALS AND METHODS: Women with SUI, bladder outlet hypermobility, and leak point pressure (LPP) > or =60 cmH(2)O were randomized to RF micro-remodeling or "sham treatment." Adverse events (AEs) were recorded. Incidence of > or =10 point incontinence quality of life (I-QOL) score improvement, a magnitude of improvement with a demonstrated responsiveness to patient satisfaction with treatment and to > or =25% reduction in both incontinence episode frequency and stress pad weight, served as a subjective outcome measurement. Change in mean LPP served as an objective outcome measurement. RESULTS: 110 women underwent RF micro remodeling and 63 underwent virtually identical "sham treatment" (with the exception of RF delivery). The 12 months RF micro-remodeling safety profile was statistically no different than that of sham treatment (a brief bladder catheterization). Seventy-four percent of women with moderate to severe baseline SUI experienced > or =10 point I-QOL score improvement at 12 months (P = 0.04). Women who underwent RF micro-remodeling demonstrated LPP elevation at 12 months, while sham treated women demonstrated LPP reduction (P = 0.02). CONCLUSIONS: Non surgical, transurethral RF micro-remodeling is a safe treatment for women with SUI. In women with moderate to severe SUI, this novel therapy resulted in statistically significant improvement in quality of life of a magnitude associated with patient satisfaction with the treatment. Women who underwent RF micro-remodeling demonstrated a statistically significant elevation in mean LPP at 12 months. PMID- 16673380 TI - Event driven electrical stimulation of the dorsal penile/clitoral nerve for management of neurogenic detrusor overactivity in multiple sclerosis. AB - AIMS: The aim of this study was to evaluate the effect of automatic event driven electrical stimulation on the dorsal penile/clitoral nerve for management of neurogenic detrusor overactivity in patients suffering from Multiple Sclerosis. METHODS: A total of 10 patients participated in the study. Detrusor pressure was recorded during physiological filling of the bladder and electrical stimulation was applied with surface electrodes whenever the detrusor pressure exceeded 10 cm H(2)O. RESULTS: In seven of the eight patients, where neurogenic detrusor overactivity was observed an average of 12 detrusor contractions could be inhibited by stimulation. In one patient, however, stimulation failed to inhibit the detrusor contractions. The average increase in bladder volume from first suppressed detrusor contraction until leakage was 94% (range: 22-366%). On average, the time from first suppressed contraction until leakage was 15 min and 50 sec (range: 4 min 58 sec-32 min 5 sec) with an average physiological filling rate of 8 ml/min. Urgency was effectively suppressed at the onset of stimulation. CONCLUSIONS: The results indicate that involuntary detrusor contractions in patients with multiple sclerosis can effectively be inhibited with event driven stimulation, hereby improving bladder capacity and reducing the number of incontinence episodes. However, the used method for detecting detrusor contractions is not suitable in a chronic setting and alternative techniques needs to be investigated if stimulation should be applied automatically. PMID- 16673381 TI - Role of TGFbeta in skin inflammation and carcinogenesis. AB - The functions of transforming growth factor beta-1(TGFbeta1) are cell-context specific. We have found that TGFbeta1 expression in human skin squamous cell carcinoma (SCC) samples has two distinct distribution patterns: (1) either predominantly in suprabasal layers or (2) throughout tumor epithelia including basal proliferative cells. To understand whether the spatial TGFbeta1 expression patterns affect its functions, we have generated several keratinocyte-specific transgenic mouse models in which TGFbeta1 overexpression can be induced either predominantly in the suprabasal epidermis or in the basal layer of the epidermis and hair follicles. Suprabasal TGFbeta1 overexpression inhibits keratinocyte proliferation, suppresses skin carcinogenesis at early stages, but promotes tumor invasion at later stages. In contrast, TGFbeta1 overexpression in the basal layer of the epidermis and hair follicles causes a severe inflammatory skin disorder and epidermal hyperproliferation. Given the importance of inflammation in cancer development, our data suggest that TGFbeta1-induced skin inflammation may override its tumor suppressive effect at early stages during skin carcinogenesis. This hypothesis is further suggested by our recent study that Smad3 knockout mice are resistant to skin chemical carcinogenesis at least in part via abrogation of endogenous TGFbeta1-induced inflammation. This review intends to summarize current insights into the role of TGFbeta1 in skin inflammation and carcinogenesis. PMID- 16673382 TI - NF-kappaB and cancer: mechanisms and targets. AB - In addition to being a central coordinator of immune responses, NF-kappaB signaling also plays a critical role in cancer development and progression and it may determine the response to therapy. NF-kappaB activation was shown to provide a critical mechanistic link between inflammation and cancer and is a major factor that controls the ability of both preneoplastic and malignant cells to resist apoptosis-based tumor surveillance mechanisms. NF-kappaB may also be involved in regulation of tumor angiogenesis and invasiveness. Importantly, NF-kappaB and the signaling pathways that mediate its activation have become attractive targets for development of new chemopreventive and chemotherapeutic approaches. PMID- 16673383 TI - Proceedings--targeting carcinogenesis: transduction, transcription, translation. PMID- 16673384 TI - Transcriptional regulation via cysteine thiol modification: a novel molecular strategy for chemoprevention and cytoprotection. AB - Chemoprevention refers to the use of defined nontoxic chemical regimens to inhibit, reverse, or retard the process of multistage carcinogenesis that involves multiple signal transduction events. Identification of signaling molecules associated with carcinogenesis as prime targets of chemopreventive agents has become an area of great interest. Recent studies have implicated cysteine thiols present in various transcription factors, such as NF-kappaB, AP 1, and p53 as redox sensors in transcriptional regulation of many genes essential for maintaining cellular homeostasis. Some chemopreventive and cytoprotective agents have been found to target cysteine thiols present in key transcription factors or their regulators, thereby suppressing aberrant over-activation of carcinogenic signal transduction or restoring/normalizing or even potentiating cellular defense signaling. The focus of this review is the oxidation or covalent modification of thiol groups present in key representative redox-sensitive transcription factors and their regulating molecules as a unique strategy for molecular target-based chemoprevention and cytoprotection. PMID- 16673385 TI - RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor. AB - Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA-kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP-actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F-actin patches, the latter being an effect attributable to ROCK-mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F-actin polymerization underlying protrusive activity in the distal axon. PMID- 16673386 TI - Differential roles of two major brain structures, mushroom bodies and central complex, for Drosophila male courtship behavior. AB - Drosophila male courtship is a complex and robust behavior, the potential for which is genetically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions involved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibire(ts1), here we demonstrate the significance of two major brain structures, the mushroom bodies and the central complex, in experience independent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treatment affects neither initiation nor maintenance of courtship toward young males that release courtship-stimulating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity toward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mushroom bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in maintenance of male courtship regardless of the nature of courtship-stimulating cues. PMID- 16673387 TI - Site-specific interactions of neurotrophin-3 and fibroblast growth factor (FGF2) in the embryonic development of the mouse cochlear nucleus. AB - Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well-defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13-E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time-lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes. PMID- 16673388 TI - Covalent linkages between cellulose and lignin in cell walls of coniferous and nonconiferous woods. AB - Covalent linkages between wall polysaccharides and lignin, especially linkage between cellulose and lignin were discussed by carboxymethylation technique of whole cell walls of coniferous and nonconiferous woods. Hydroxyl groups of plant cell walls polysaccharides were highly substituted, but not those of lignin by carboxymethyl groups under the used conditions, and separated into water-soluble and insoluble fractions by water extraction. Carboxymethylated wall polysaccharides linked covalently with lignin were distributed into the water insoluble fractions. Composition of carboxymethylated sugar residues in the both fractions was analyzed quantitatively by 1H NMR spectroscopy after hydrolyzation with D2SO4 in D2O. More than half of cellulose linked covalently with lignin in coniferous wood, but only one-sixth of cellulose was involved in the linkage in nonconiferous wood. The major noncellulosic wall polysaccharides of coniferous wood also linked significantly with lignin. On the other hand, noncellulosic wall polysaccharides of nonconiferous wood were involved slightly in the covalent linkage with lignin. The situation of linkage between wall polysaccharides containing cellulose and lignin was visualized by scanning electron micrographs. PMID- 16673389 TI - Controlled release of fibroblast growth factor-2 from an injectable 6-O desulfated heparin hydrogel and subsequent effect on in vivo vascularization. AB - We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding growth factors, such as fibroblast growth factor (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 microg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about 15-20 days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS Hep hydrogel and the subsequent induction of neovascularization in vivo. PMID- 16673390 TI - Preparation and characterization of novel hybrid of chitosan-g-lactic acid and montmorillonite. AB - The utilization of biopolymers and the development of organic-inorganic hybrids are ever increasing interest of material science researchers around the globe for various applications. The present attempt is intended to prepare nanocomposites of lactic acid grafted chitosan and layered silicates. Nanocomposites were prepared by dissolving chitosan and dispersing sodium montmorillonite in aqueous solution of L-lactic acid with subsequent heating and film casting. They were characterized by conventional techniques such as Fourier transform infrared spectroscopy, X-ray diffractometry, thermogravimetric analysis, energy dispersive X-ray spectroscopy, and elemental analysis. The results from polar optical and transmission electron microscopic measurements are also discussed. Sorption behavior of samples has been followed by measuring swelling degree and contact angle. The films have shown enhanced hydrophilicity when compared with polylactic acid (PLA). Issues on the interactions of polycationic chitosan with clay are also discussed. It is observed that nanocomposites are exhibiting better thermal and physical properties than neat chitosan-g-LA and PLA. PMID- 16673391 TI - Modulation of neurosteroid production in human neuroblastoma cells by Alzheimer's disease key proteins. AB - Studies performed with animals suggest neurosteroid involvement in neuroprotection. However in humans, the role of neurosteroidogenesis in the regulation of degenerative processes is unknown. To determine whether cellular factors intervening in degenerative mechanisms may interfere with the process of neurosteroidogenesis in humans, we combined pulse-chase experiments with HPLC and continuous flow scintillation detection to compare neurosteroid production in normal and transfected SH-SY5Y cells with key proteins involved in Alzheimer's disease (AD). Microscope analyses revealed that cell morphology was unchanged in stably transfected SH-SY5Y cells overexpressing human native tau (hTau40), mutant tau (P301L), and wild-type amyloid precursor protein (APPwt) compared to controls. Biochemical investigations showed that hTau40 enhanced progesterone (PROG), 17OHPROG, testosterone, and 3alpha-androstanediol neosynthesis from pregnenolone. In contrast, tau with the pathogenic P301L mutation was devoid of action on neurosteroidogenesis. Overexpression of APPwt inhibited PROG formation, did not affect 17OHPROG and testosterone, but increased 3alpha-androstanediol and estradiol synthesis. Extracellular treatment of control cells with aggregated amyloid peptide mimicked the action of APPwt expression on PROG but not on 3alpha androstanediol and estradiol production. Moreover, PROG biosynthesis in APPwt cells was up-regulated in the presence of a gamma-secretase inhibitor. Our results provide the first evidence for the regulation of neurosteroid biosynthesis by key proteins involved in the etiology of AD. The data suggest that pathogenic factors may induce neurodegeneration in humans through the reduction of the synthesis of endogenous neuroprotective neurosteroids in nerve cells. PMID- 16673392 TI - Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers. AB - In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth. PMID- 16673393 TI - Evaluating therapeutic targets for reperfusion-related brain hemorrhage. AB - OBJECTIVE: Early reperfusion after an ischemic stroke can cause blood-brain barrier injury with subsequent cerebral edema and devastating brain hemorrhage. These complications of early reperfusion, which result from excess production of reactive oxygen species, significantly limit the benefits of stroke therapies. In this article, we use a novel animal model that facilitates identification of specific components of the reperfusion injury process, including vascular injury and secondary brain damage, and allows assessment of therapeutic interventions. METHODS: Knock-out (KO) mice containing 50% manganese-superoxide dismutase activity (SOD2-KO) and transgenic mice overexpressing SOD2 undergo transient focal ischemia and reperfusion followed by assessment of infarct, edema, hemorrhage rates, metalloproteinase activation, and microvascular injury. RESULTS: SOD2-KO mice demonstrate delayed (>24h) blood-brain barrier breakdown associated with activation of matrix metalloproteinases, inflammation, and high brain hemorrhage rates. These adverse consequences are absent in wild-type littermates and minocycline-treated SOD2-KO animals. Increased hemorrhage rates also are absent in SOD2 overexpressors, which have reduced vascular endothelial cell death. Finally, we show that the tight junction membrane protein, occludin, is an early and specific target in oxidative stress-induced microvascular injury. INTERPRETATION: This model is ideal for studying ischemia/reperfusion-induced vascular injury and secondary brain hemorrhage and offers a unique opportunity to evaluate antioxidant-based neurovascular protective strategies as potential adjunct treatments to currently approved stroke therapies such as thrombolysis and endovascular clot retrieval. PMID- 16673394 TI - Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host. AB - The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. PMID- 16673396 TI - Axis is a feature of handwritten spirals in essential tremor. PMID- 16673395 TI - alpha-Tocopherol affects neuronal plasticity in adult rat dentate gyrus: the possible role of PKCdelta. AB - Hippocampus dentate gyrus (DG) is characterized by neuronal plasticity processes in adulthood, and polysialylation of NCAM promotes neuronal plasticity. In previous investigations we found that alpha-tocopherol increased the PSA-NCAM positive granule cell number in adult rat DG, suggesting that alpha-tocopherol may enhance neuronal plasticity. To verify this hypothesis, in the present study, structural remodeling in adult rat DG was investigated under alpha-tocopherol supplementation conditions. PSA-NCAM expression was evaluated by Western blotting, evaluation of PSA-NCAM-positive granule cell density, and morphometric analysis of PSA-NCAM-positive processes. In addition, the optical density of synaptophysin immunoreactivity and the synaptic profile density, examined by electron microscopy, were evaluated. Moreover, considering that PSA-NCAM expression has been found to be related to PKCdelta activity and alpha-tocopherol has been shown to inhibit PKC activity in vitro, Western blotting and immunohistochemistry followed by densitometry were used to analyze PKC. Our results demonstrated that an increase in PSA-NCAM expression and optical density of DG molecular layer synaptophysin immunoreactivity occurred in alpha-tocopherol treated rats. Electron microscopy analysis showed that the increase in synaptophysin expression was related to an increase in synaptic profile density. In addition, Western blotting revealed a decrease in phospho-PKC Pan and phospho PKCdelta, demonstrating that alpha-tocopherol is also able to inhibit PKC activity in vivo. Likewise, immunoreactivity for the active form of PKCdelta was lower in alpha-tocopherol-treated rats than in controls, while no changes were found in PKCdelta expression. These results demonstrate that alpha-tocopherol is an exogenous factor affecting neuronal plasticity in adult rat DG, possibly through PKCdelta inhibition. PMID- 16673397 TI - Metric attributes of the unified Parkinson's disease rating scale 3.0 battery: Part I, feasibility, scaling assumptions, reliability, and precision. AB - This article sought to assess the feasibility, scaling assumptions, reliability, precision, and factor analysis of the three most widely used rating scales in Parkinson's disease (PD): modified Hoehn and Yahr (HY), Schwab and England (SE), and Unified Parkinson's Disease Rating Scale (UPDRS). A multicenter sample of 1,136 PD patients was assessed. The percentage of missing data was 8% for SE, HY, and UPDRS mentation, and almost negligible (<2%) for the other UPDRS subscales. A high floor effect was found for UPDRS mentation (23%) and complications scales (36%). Item content validity, measured by multitrait scaling analysis, was adequate for all UPDRS subscales (scaling successes > 90%). Internal consistency coefficients for the UPDRS scales ranged from 0.79 (mentation) to 0.92 (activities of daily living and motor). Factor structure of the UPDRS mentation, activities of daily living, and complications subscales was replicated. As a whole, the HY, SE, and UPDRS are acceptable, consistent, and potentially sensitive rating scales. PMID- 16673398 TI - Galpha(s) sensitizes human SH-SY5Y cells to apoptosis independently of the protein kinase A pathway. AB - Disturbances in Galpha(s-L) levels and function have been implicated in the pathophysiology of bipolar disorder, but the role of these changes in the development of the illness is not clear. In view of the critical role of Galpha(s)-mediated cAMP signaling in regulating cell survival, we investigated the potential role of Galpha(s-L) in modulating susceptibility to cellular stressors in human SH-SY5Y neuroblastoma cells. Overexpression of Galpha(s-L) to a level twice that of the vector-transfected cells did not directly affect cell viability but significantly increased the sensitivity to induction of cell death by serum deprivation and other apoptotic stimuli, including staurosporine, H(2)O(2), and tunicamycin. This enhanced sensitivity was associated with increased caspase-3 activation and appearance of fragmented nuclei (Hoechst 33342 staining). The broad-spectrum caspase inhibitor z-VAD-fmk completely suppressed cell death evoked by these apoptotic insults in both vector-transfected and Galpha(s-L)-overexpressing cells. The increased vulnerability conferred by increased Galpha(s-L) expression was neither mimicked by cAMP analogs 8-Br-cAMP, 8-CPT-cAMP, and 8-CPT-2Me-cAMP nor attenuated by PKA inhibitors Rp-cAMPS and KT5720. These data indicate that Galpha(s-L) may modulate apoptotic processes in a caspase-dependent manner through a signaling cascade that is independent of the cAMP/PKA or cAMP/Epac pathway. These results suggest that enhanced Galpha(s-L) expression, as was observed in post-mortem brain of bipolar patients, may impair cellular resilience in response to intracellular stress signals resulting from mitochondrial and/or endoplasmic reticulum dysfunction implicated in this disorder. PMID- 16673399 TI - Maturation-dependent oligodendrocyte apoptosis caused by hyperoxia. AB - In the immature human brain, periventricular leukomalacia (PVL) is the predominant white matter injury underlying the development of cerebral palsy. PVL has its peak incidence during a well-defined period in human brain development (23-32 weeks postconceptional age) characterized by extensive oligodendrocyte migration and maturation. We hypothesized that the dramatic rise of oxygen tissue tension associated with mammalian birth and additional oxygen exposure of the preterm infant during intensive care may be harmful to immature oligodendrocytes (OLs). We therefore investigated the effects of hyperoxia on rat oligodendroglia cells in vitro and in vivo. Immature OLs (OLN-93), their progenitors [preoligodendrocytes (pre-OL)], and mature OLs were subjected to 80% hyperoxia (24-96 hr). Flow cytometry was used to assess cell death. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT). In addition, 6-day-old rat pups were subjected to 80% oxygen (24 hr) and then sacrificed, and their brains were processed for immunfluorescence staining. Apoptosis was detected at various stages (annexin-V, activated caspase-3) after 24-48 hr of incubation in 80% oxygen in pre- and immature OLs. Mature OLs were resistant to oxygen exposure. These results were confirmed by MTT assay. This cell death was blocked by administration of the pan-caspase inhibitor zVAD-fmk. Degeneration of OLs was confirmed in 7-day-old rat brains by positive staining for activated caspase-3. Hyperoxia triggers maturation-dependent apoptosis in immature and pre-OLs and involves caspase activation. This mechanism may be relevant to the white matter injury observed in infants born preterm. PMID- 16673400 TI - Melanized nigral neuronal numbers in Nigerian and British individuals. AB - The role of genetic and environmental factors in etiopathogenesis of Parkinson's disease (PD) is debated. The prevalence of PD is higher among white than nonwhite populations, yet it is five times higher in nonwhites living in the United States than in Nigeria. We compare counts of melanized nigral neurons between neurologically normal Nigerians and British brains. Neuronal counts were estimated in an age-matched sample of 23 Nigerian and 7 British brains from neurologically normal individuals who had no Lewy bodies and Lewy neurites on alpha-synuclein immunostaining. Two investigators blind to age and ethnicity performed counts of melanized neurons in a single 7-mum hemisections showing the substantia nigra pars compacta. No significant difference exits in the number of neurons between the Nigerian and the British subjects (P = 0.1, NS). Differences in melanized nigral neuronal numbers may not explain differences in the prevalence of PD between white and nonwhite populations, suggesting factors other than neuronal numbers contribute to differential susceptibility of black vs. white races to PD. PMID- 16673401 TI - Head tremor in Parkinson's disease. AB - Head tremor is a typical feature of essential tremor. Patients with sporadic Parkinson's disease can have tremor of the tongue, lip, or chin, but classically do not have head tremor. We describe five patients with Parkinson's disease and head tremor in whom clinical and neurophysiological findings suggested that head tremor was a manifestation of Parkinson's disease. PMID- 16673402 TI - Engineering aromatic L-amino acid transaminase for the asymmetric synthesis of constrained analogs of L-phenylalanine. AB - An enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L-diphenylalanine using the rationally engineered aromatic L-amino acid transaminase (eAroATEs) obtained from Enterobacter sp. BK2K-1. To rationally redesign the enzyme, structural model was constructed by the homology modeling. The structural model was experimentally validated by the site-directed mutagenesis of the predicted pyridoxal-5'-phosphate (PLP) binding site and the substrate-recognition region, and the cell-free protein synthesis of mutated enzymes. It was suggested that Arg281 and Arg375 were the key residues to recognize the distal carboxylate and alpha-carboxylate group of the substrates, respectively. The model also predicted that Tyr66 forms hydrogen bond with the phosphate moiety of PLP and interacts with the side chain attached to beta-carbon of the amino acid substrate. Among the various site-directed mutants, Y66L variant was able to synthesize L-diphenylalanine with 23% conversion yield for 10 h, whereas the wild-type AroATEs was inactive for the transamination between diphenylpyruvate and L-phenylalanine as amino acceptor and amino donor, respectively. PMID- 16673403 TI - Metabolic GHB precursor succinate binds to gamma-hydroxybutyrate receptors: characterization of human basal ganglia areas nucleus accumbens and globus pallidus. AB - Binding of the metabolic gamma-hydroxybutyrate (GHB) precursor succinate to NCS 382-sensitive [3H]GHB-labeled sites in crude synaptosomal or purified synaptic membrane fractions prepared from the human nucleus accumbens (NA), globus pallidus (GP) and rat forebrain has been shown. This site can be characterized by binding of ethyl hemisuccinate and gap-junction blockers, including carbenoxolone hemisuccinate and beta-GRA. There was no significant binding interaction between GABAB receptor ligands (CGP 55845, (R)-baclofen) and these [3H]GHB-labeled sites. GHB, NCS-382 and succinate binding profile of [3H]GHB-labeled sites in rat forebrain, human NA or GP synaptic membranes were similar. The synaptic fraction isolated from the rat forebrain was characterized by GHB binding inhibition constants: Ki,NCS-382 = 1.2 +/- 0.2 microM, Ki,GHB = 1.6 +/- 0.3 microM and Ki,SUCCINATE = 212 +/- 66 microM. In crude membranes containing mainly extrasynaptic membranes, distinct GHB and GABAB receptor sites were found in the NA. By contrast, extrasynaptic GABAB receptor sites of rat forebrain and GP were GHB- and succinate-sensitive, respectively. The heterogeneity of GABAB sites found in native membranes indicates GABAB receptor-dependent differences in GHB action. Based on these findings, we suggest that succinate (and possibly drugs available as succinate salt derivatives) can mimic some of the actions of GHB. PMID- 16673404 TI - Relative risk of spread of symptoms among the focal onset primary dystonias. AB - Adult-onset primary torsion dystonia (PTD) may spread to multiple body parts, but the relative risk of spread by site of onset of dystonia has not been well characterized. We retrospectively identified 602 patients with PTD out of 1,500 dystonia patients in our electronic database and extracted age at onset, site of onset, family history, and spread. Survival analyses were performed for groups based on site of onset, and hazard ratios compared relative risk of spread across groups. Patients with adult-onset blepharospasm were more likely to spread (31% past the head) than those with dystonia starting in the neck (9%), larynx (12%), or upper extremities (16%). Hazard ratios proved that the blepharospasm group had the greatest relative risk of spread. The rate of spread after onset varied significantly between the different groups. Most spread occurred in the first 1 to 2 years after onset of blepharospasm, whereas the risk of spread was relatively constant over time in cervical and laryngeal dystonia. Different sites of onset of PTD confer different risks of spread, important for clinical prognosis. Different risks of spread may provide clues about underlying pathogenesis of adult-onset primary dystonias. PMID- 16673405 TI - Glutathione peroxidase 4 protects cortical neurons from oxidative injury and amyloid toxicity. AB - Polyunsaturated fatty acids (PUFA) in membrane lipids are prone to attack by reactive oxygen species (ROS), and the resulting lipid peroxidation can cause injury and death of cells. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme that can directly detoxify lipid hydroperoxides generated by ROS. Overexpression of Gpx4 has been shown to be protective against oxidative damage in several cell lines. We examined in this study the stress response of neurons with increased expression of Gpx4, because neurons are especially vulnerable to oxidative injury as a result of their high content of PUFA. Our results show that primary culture cortical neurons derived from Gpx4 transgenic mice, which had increased expression of Gpx4, had increased cell survival and reduced level of apoptosis after exposure to t-butyl hydroperoxide and hydrogen peroxide. We also studied the protective role of Gpx4 against beta-amyloid toxicity, because beta amyloid-induced neural toxicity is believed to be mediated through lipid peroxidation. Primary culture cortical neurons from Gpx4 transgenic mice had significantly less cell toxicity than their wild-type counterparts after exposure to Abeta25-35 and Abeta1-40 peptides, and apoptosis induced by Abeta25-35 was attenuated in neurons from Gpx4 transgenic mice. Our data demonstrate that overexpression of Gpx4 protects neurons against oxidative injury and beta-amyloid induced cytotoxicity. PMID- 16673406 TI - Subthalamic deep brain stimulation in patients with a previous pallidotomy. AB - The safety and efficacy of subthalamic nucleus (STN) deep brain stimulation (DBS) in patients who have had a previous unilateral pallidotomy is not clear. We identified 10 patients (9 male) at the Baylor College of Medicine Parkinson's Disease Center who underwent STN DBS after prior unilateral pallidotomy. Demographics, efficacy as determined by off Unified Parkinson's Disease Rating Scale (UPDRS) part III scores, and levodopa equivalent dosing were analyzed. We then compared these to an age- and sex-matched group of 25 DBS patients who had no prior pallidotomy. After their initial pallidotomy (mean age, 51.8 +/- 10.8 years), the mean UPDRS motor off medicine scores improved from 51.3 +/- 14.3 to 34.9 +/- 12.8, and the UPDRS dyskinesia score improved from 1.8 +/- 1.0 to 0.8 +/ 0.7. Their STN DBS off UPDRS motor scores (mean age, 56.0 +/- 10.2 years) improved by 16.0% from 53.1 +/- 9.7 (range, 42-68) to 44.6 +/- 11.1 (range, 25 67). In contrast, the UPDRS off motor scores in a control group of 25 DBS patients improved by 49.9%, from 49.7 +/- 11.1 to 25.7 +/- 18.9, (16.0% vs. 49.9%; P < 0.001). Changes in UPDRS dyskinesia scores were similar in both groups. AE thought to be related to the STN DBS following pallidotomy included worse dysarthria (three) and worse balance (two). STN DBS patients with prior pallidotomy had less improvement in UPDRS off motor score compared to other STN DBS patients, despite relatively good outcomes immediately after their pallidotomy. This may be partially due to a selection bias, but it may also indicate that prior pallidotomy is a negative predictor of outcome of STN DBS and should be considered in patient selection. PMID- 16673407 TI - In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion. AB - Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo. PMID- 16673408 TI - Mapping ribosomal RNA transcription activity in the mouse eye. AB - All cells must make ribosomes, in which rRNA transcription is the rate-limiting step; however, some cells may require more ribosomes than others. Cell-type specific regulation of rRNA synthesis has been largely ignored in the past, because of the inability to measure rRNA transcription rate in situ. Here we map rRNA transcription activity in individual cells in mouse ocular tissues detected by a novel in situ hybridization technique, which detects the full-length transcripts (47S pre-rRNA) as well as various rRNA processing intermediates. In the adult mouse eye, the corneal and lens epithelia and some retinal neurons contain a higher level of 47S pre-rRNA and rRNA processing intermediates, which are regulated developmentally in neonates prior to eye opening. In the cornea and lens epithelia, the higher rRNA level of 47S rRNA correlates with cell proliferation, which is consistent with the notion that dividing cells require more protein synthesis. Interestingly, in some retinal neurons, the high level of 47S pre-rRNA does not correlate with mature rRNA accumulation or protein synthesis, suggesting the existence of unappreciated biochemical needs of these cells. PMID- 16673409 TI - Defining a minimal clinically relevant difference for the unified Parkinson's rating scale: an important but still unmet need. PMID- 16673410 TI - Minimal clinically important change on the unified Parkinson's disease rating scale. AB - The Unified Parkinson's Disease Rating Scale (UPDRS) is the main outcome measure in clinical trials of Parkinson's disease (PD). The minimal change that represents a clinically meaningful improvement is unknown. The objective of this study was to determine the minimal change on the UPDRS that represents a clinically meaningful improvement in early PD after 6 months of treatment. Data from two independent randomized treatment trials over 6 months involving 603 patients with de novo PD were analyzed to determine the minimal clinically important change (MCIC), referred to the status before treatment, for the UPDRS motor, activities of daily living (ADL), and total scores. An anchor-based method using ratings on a seven-point global clinical improvement was used. A change of five points on the UPDRS motor part was found to be the most appropriate cutoff score for all Hoehn and Yahr stages I to III, and a change of eight points for the UDPRS total score. For the UDPRS ADL score, an MCIC of two points for Hoehn and Yahr stages I/I.5 and II and of three points for Hoehn and Yahr stage II.5/III was the most appropriate cutoff score. These data give the first estimate for cutoffs defining clinically important changes in UPDRS ADL and motor scores. Further studies using larger databases from more diverse study populations are encouraged to better define and solidify the MCIC for the UPDRS. PMID- 16673411 TI - DNA damage, oxidative mutagen sensitivity, and repair of oxidative DNA damage in nonmelanoma skin cancer patients. AB - Nonmelanoma skin cancer (NMSC) is the most frequent type of cancer in humans. Exposure to UV radiation is a major risk factor for NMSC, and oxidative DNA damage, caused either by UV radiation itself or by other agents, may be involved in its induction. Increased sensitivity to oxidative damage and an altered DNA repair capacity (DRC) increase the risk of many types of cancer; however, sensitivity to oxidizing agents has not been evaluated for NMSC, and results regarding DRC in NMSC are inconclusive. In the present study, we evaluated DNA damage and repair in leukocytes from 41 NMSC patients and 45 controls. The Comet assay was used to measure basal and H(2)O(2)-induced DNA damage, as well as the DRC, while the cytokinesis-block micronucleus assay was used to measure the basal level of chromosome damage. Although basal DNA damage was higher for the controls than for the patients, this finding was mainly due to sampling more controls in the summer, which was associated with longer comet tails. In contrast, H(2)O(2) induced DNA damage was significantly higher in cases than in controls, and this parameter was not influenced by the season of the year. The DRC for the H(2)O(2) induced damage was similar for cases and controls and unrelated to seasonality. Finally, the frequency of binucleated lymphocytes with micronuclei was similar for cases and controls. The results of this study indicate that NMSC patients are distinguished from controls by an increased sensitivity to oxidative DNA damage. PMID- 16673412 TI - DNA damage and repair measurements from cryopreserved lymphocytes without cell culture--a reproducible assay for intervention studies. AB - Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When gamma-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after gamma irradiation exposure, and DRC--measured as tail moment--were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H(2)O(2) was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37 degrees C (CVs. ranging from 8 to 35%) than for the more standard 4 degrees C protocol. Analyzing moment arm- the average distance of DNA migration within the tail--yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. PMID- 16673413 TI - Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention. AB - Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it. PMID- 16673414 TI - Second malignant neoplasia in early (TIS-T1) glottic carcinoma. AB - BACKGROUND: We performed a population-based study to determine the incidence and patterns of second malignant neoplasia (SMN) in early glottic carcinoma. METHODS: All patients diagnosed with Tis-T1 glottic carcinoma in the southwest of the Netherlands between 1982 and 1993 (359) were included. Sources of the data were patient charts and the regional cancer registry. RESULTS: SMN incidence was 27.7% (median follow-up, 89 months). Observed-to-expected ratios were increased for lung, bladder, urinary tract, pancreatic, colorectal, and head and neck cancers. The incidence of head and neck and esophageal cancer was surprisingly low. CONCLUSIONS: Patients with early glottic carcinoma are at a reliably increased risk of the development of tumors not only in the areas of the upper aerodigestive tract, but also in the bladder, pancreas, and colorectum. The low incidence of head and neck and esophageal tumors does not appear to support routine panendoscopy in this patient population. PMID- 16673415 TI - RNA interference of sialidase improves glycoprotein sialic acid content consistency. AB - An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN gamma produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-gamma, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process. PMID- 16673416 TI - Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes. AB - The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2. PMID- 16673417 TI - Donor site morbidity after harvesting of proximal tibia bone. AB - BACKGROUND: Bone-grafting procedures are common in head and neck surgery. Donor site morbidity is an important factor in deciding the site for harvest of cancellous bone. The tibia has been recommended as a harvest site. Use of the proximal tibia as a donor site is associated with few complications. Our present study used proximal tibia bone grafts to reconstruct maxillofacial defects and augment bone volume for implantation. METHODS: A retrospective study was undertaken to analyze 40 proximal tibia bone grafts in maxillofacial reconstruction. Minimal follow-up was 6 months. RESULTS: There were no major complications during the follow-up period. Early minor complications (15%) included temporary sensory loss and ecchymosis. Late minor complication (2.5%) was gait disturbance for 2 months. Long-term minor complication (2.5%) was an unsightly scar. CONCLUSION: The procedure for proximal tibia bone graft is easy, has less operative risk, and results in a lower postoperative morbidity rate. Based on our findings, we believe the proximal tibia offers a reliable site for harvest of sufficient quantities of good-quality cancellous bone. PMID- 16673418 TI - Effects of growth rate on cell extract performance in cell-free protein synthesis. AB - Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity. PMID- 16673419 TI - BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. AB - Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis. PMID- 16673420 TI - Sequencing batch membrane biofilm reactor for simultaneous nitrogen and phosphorus removal: novel application of membrane-aerated biofilm. AB - A sequencing batch membrane biofilm reactor (SBMBfR) was developed for simultaneous carbon, nitrogen, and phosphorus removal from wastewater. This reactor was composed of two functional parts: (1) a gas-permeable membrane on which a nitrifying biofilm formed and (2) a bulk solution in which bacteria, mainly denitrifying polyphosphate-accumulating organisms (DNPAOs), were suspended. The reactor was operated sequentially under anaerobic condition and then under membrane aeration condition in one cycle. During the anaerobic period, organic carbon was consumed by DNPAOs; this was accompanied by phosphate release. During the subsequent membrane aeration period, nitrifying bacteria utilized oxygen supplied directly to them from the inside of the membrane. Consequently, the nitrite and nitrate products diffused into the bulk solution, where they were used by DNPAOs as electron acceptors for phosphate uptake. In a long-term sequencing batch operation, the mean removal efficiencies of total organic carbon (TOC), total nitrogen (T-N), and total phosphorus (T-P) under steady-state condition were 99%, 96%, and 90%, respectively. In addition, fluorescence in situ hybridization (FISH) clearly demonstrated the difference in bacterial community structure between the membrane biofilm and the suspended sludge: ammonia oxidizing bacteria belonging to the Nitrosomonas group were dominant in the region adjacent to the membrane throughout the operation, and the occupation ratio of the well-known polyphosphate-accumulating organism (PAO) Candidatus "Accumulibacter phosphates" in the suspended sludge gradually increased to a maximum of 37%. PMID- 16673421 TI - Construction and evaluation of novel fusion proteins for targeted delivery of micro particles to cellulose surfaces. AB - The use of IgG antibodies and fragments has been limited to specific sectors of the biotechnology industry due to the high cost of producing large batches of product necessary for alternative applications. A novel class of Camelid antibodies, known as V(HH) offer a more economical opportunity to meet a wider application in industry. In this study, we report the evaluation of four llama V(HH)-cellulose binding domain fusion proteins displaying varying formats of V(HH) and CBD domains. Proteins were characterized in a targeted particle delivery system as a method of delivering agents such as perfume to laundry in the wash cycle. Fusion proteins were shown to be stable at high pH and in the presence of a detergent base. They were also shown to bind effectively to both the designated antigen, the azo-dye reactive-red 6 (either conjugated to BSA or attached to coacervate microparticles), and cellulose. Binding strength differences were observed between the different fusion protein formats using surface plasmon resonance. The effect of key laundry ingredients was also studied. Combining the fusion proteins and particles into a delivery and deposition study generated clear microscopy evidence for bifunctionality. Confirmation of this was validated by GC-MS analysis of retained fragrance. This research, reporting the construction and characterization of a variety of fusion proteins, illustrates that the single multidomain fusion protein route offers a new technology for successful targeted delivery of encapsulated benefit agents. Furthermore, the potential to modify or select for proteins to recognize a wide range of surfaces is also possible. PMID- 16673422 TI - Development and preliminary validation of a new measure to assess satisfaction with information among head and neck cancer patients: the satisfaction with cancer information profile (SCIP). AB - BACKGROUND: Our objective was to develop and examine the psychometric properties of the Satisfaction With Cancer Information Profile (SCIP), a new measure designed to assess the extent to which patients with head and neck cancer (HNC) are satisfied with information received about their treatment. METHODS: Patients with head and neck cancer (HNC) from 4 hospitals in the UK completed the SCIP before treatment and again 1 month after treatment. Psychometric properties of the measure were evaluated: internal consistency, subscale intercorrelations, discriminant validity, concurrent validity, and predictive validity. The responsiveness of the measure to change and ease of completion was also assessed. RESULTS: The SCIP performed well on all the psychometric tests; level of satisfaction with information was related to a number of key outcomes and patient beliefs, demonstrating criterion-related validity. CONCLUSIONS: The SCIP was developed to assess HNC patient satisfaction with treatment information and has demonstrated good reliability and validity. This measure may prove appropriate for use with patients with different types of cancer due to its applicability with a variety of treatment modalities. Preliminary psychometric testing has demonstrated its potential for use in routine clinical settings and research settings as a predictive tool or for audit, in addition to assessing individual patient satisfaction with information. PMID- 16673423 TI - Beta-lactoglobulin as a potential modulator of intestinal activity and morphology in neonatal piglets. AB - Studies were conducted to determine the effects of the whey protein beta lactoglobulin (blg) on the intestinal development and intestinal morphology in neonatal piglets. Two trials (5 and 28 days) were conducted on piglets in three separate groups. One group served as a control group while the remaining two were experimental groups based on diets. The two experimental diets were a bovine colostrum powder, one with supplementation with blg and the other without. The control group remained suckling on a sow. All groups were weaned on day 20 and placed onto a solid commercial piglet diet. Enzymatic activity, total DNA synthesis, crypt depth, and villus height were all parameters used to evaluate the effect of blg. Piglets fed a diet supplement with blg had the greatest total DNA after 5 days. Sow-reared piglets had the greatest intestinal morphology development with regards to villus height. No significant differences were found in enzymatic activity between treatments. PMID- 16673424 TI - Heuristic problems in defining the three-dimensional arrangement of the ventricular myocytes. AB - There is lack of consensus concerning the three-dimensional arrangement of the myocytes within the ventricular muscle masses. Bioengineers are seeking to model the structure of the heart. Although the success of such models depends on the accuracy of the anatomic evidence, most of them have been based on concepts that are far from anatomical reality, which ignore many significant previous accounts of anatomy presented over the past 400 years. During the 19th century, Pettigrew emphasized that the heart was built on the basis of a modified blood vessel rather than in the form of skeletal muscles. This fact was reemphasized by Lev and Simkins as well as Grant in the 20th century, but the caveats listed by these authors have been ignored by proponents of two current concepts, which state either that the myocardium is arranged in the form of a "unique myocardial band," or that the walls of the ventricles are sequestrated in uniform fashion by laminar sheets of fibrous tissue extending from epicardium to endocardium. These two concepts are themselves incompatible and are further at variance with the majority of anatomic studies, which have emphasized the regional heterogeneity to be found in the three-dimensional packing of the myocytes within a supporting matrix of fibrous tissue. We reemphasize the significance of this three dimensional muscular mesh, showing how the presence of intruding aggregates of myocytes extending in oblique transmural fashion also contends against the notion that all myocytes are orientated with their long axes parallel to the epicardial and enodcardial surfaces. PMID- 16673425 TI - Localization of CD44 and hyaluronan in the synovial membrane of the rat temporomandibular joint. AB - Previous studies have pointed out a lack of adhesion structures in the synovial lining layer of the rat temporomandibular joint (TMJ) despite showing an epithelial arrangement. CD44, a major cell adhesion molecule, plays crucial roles as an anchor between cells and extracellular matrices by binding hyaluronan (HA) for the development of organs or the metastasis of tumors. The present study examined the localization of CD44 in the synovial membrane of the rat TMJ by immunocytochemistry for OX50, ED1, and Hsp25, which are markers for the rat CD44, macrophage-like type A, and fibroblast-like type B synoviocytes, respectively. Histochemistry for HA-binding protein (HABP) was also employed for the detection of HA. OX50 immunoreactions were found along the cell surface and, in particular, accumulated along the surface of the articular cavity. Observations by a double immunostaining and immunoelectron microscopy revealed that all the OX50 immunopositive cells were categorized as fibroblastic type B cells, which had many caveolae and a few vesicles reactive to intense OX50. However, the macrophage-like type A cells did not have any OX50 immunoreaction in the synovial lining layer. A strong HABP reaction was discernable in the extracellular matrix surrounding both OX50-positive and -negative cells in the synovial lining layers, exhibiting a meshwork distribution, but weak in its sublining layer. This localization pattern of CD44 and HABP might be involved in the formation of the epithelial arrangement of the synovial lining layer. Furthermore, OX50 immunonegativity in the type A cells suggests their low phagocytotic activity in the rat TMJ under normal conditions. PMID- 16673426 TI - Analysis of host-assisted guest protonation exemplified for p sulfonatocalix[4]arene--towards enzyme-mimetic pKa shifts. AB - The pD dependence of the complexation of p-sulfonatocalix[4]arene (CX4) with the azoalkanes 2,3-diazabicyclo[2.2.1]hept-2-ene (1), 2,3-diazabicyclo[2.2.2]oct-2 ene (2), 2,3-diazabicyclo[2.2.3]non-2-ene (3), and 1-methyl-4-isopropyl-2,3 diazabicyclo[2.2.2]oct-2-ene (4) in D(2)O has been studied. The pD-dependent binding constants, determined by (1)H NMR spectroscopy, were analyzed according to a seven-state model, which included the CX4 tetra- and penta-anions, the protonated and unprotonated forms of the azoalkanes, the corresponding complexes, as well as the complex formed between CX4 and the deuteriated hydronium ion. The variation of the UV absorption spectra, namely the hypsochromic shift in the near UV band of the azo chromophore upon protonation, was analyzed according to a four state model. Measurements by independent methods demonstrated that complexation by CX4 shifts the pK(a) values of the guest molecules by around 2 units, thereby establishing a case of host-assisted guest protonation. The pK(a) shift can be translated into improved binding (factor of 100) of the protonated guest relative to its unprotonated form as a result of the cation-receptor properties of CX4. The results are discussed in the context of supramolecular catalytic activity and the pK(a) shifts induced by different types of macrocyclic hosts are compared. PMID- 16673427 TI - A highly tunable family of chiral bisphospholanes for rh-catalyzed enantioselective hydrogenation reactions. AB - A set of 16 new and closely related bisphospholane ligands have been prepared by using a highly flexible and convergent approach. Each synthesis can be performed on an industrially relevant scale. The bisphosphines differ in the nature of the bridge connecting both phospholane units. Bridges are formed by three-, four-, five- and six-membered heterocyclic or alicyclic rings. Bisphospholanes and their Rh-precatalysts have been investigated by using results of theoretical calculations (DFT) and analytic measurements ((31)P and (103)Rh NMR spectroscopy, X-ray structure analysis). The studies showed that catalysts based on ligands with maleic anhydride or maleimide bridges give constantly superior enantioselectivities in methanol as the solvent. This may account for optimised steric and electronic effects. However, by changing the solvent catalysts with other backbones can give rise to excellent results. This gives proof that simple correlations between steric and electronic properties and results in the enantioselective hydrogenation frequently claimed in literature are not general. PMID- 16673428 TI - Absolute configuration of new cytotoxic and other bioactive trichothecene macrolides. AB - Three new cytotoxic 10,13-cyclotrichothecane-derived macrolides, myrothecines A-C (1-3), were characterized from the extracts of two Myrothecium roridum strains, IFB-E009 and IFB-E012, isolated as endophytic fungi found on the traditional Chinese medicinal plants Trachelospermum jasminoides and Artemisia annua, respectively. The absolute configuration of myrothecines A-C was elucidated by a combination of spectral techniques (UV, IR, MS, circular dichroism (CD), (1)H and (13)C NMR, DEPT, (1)H-(1)H COSY, NOESY, HMQC, and HMBC spectrascopic analyses), Mosher's ester analysis, and single-crystal X-ray diffraction. The absolute configuration of the reported bioactive analogue, mytoxin B was established by correlating its spectral data with that of known absolute configurational structures. Furthermore, the significance in endophytism (or symbiosis) and biocatabolism, highlighted by production of those macrolides by the endophytic strains, is discussed in brief. PMID- 16673429 TI - Intermolecular insertion of an N,N-heterocyclic carbene into a nonacidic C-H bond: Kinetics, mechanism and catalysis by (K-HMDS)2 (HMDS = Hexamethyldisilazide). AB - The reaction of 2-[13C]-1-ethyl-3-isopropyl-3,4,5,6-tetrahydropyrimidin-1-ium hexafluorophosphate ([13C1]-1-PF6) with a slight excess (1.03 equiv) of dimeric potassium hexamethyldisilazide ("(K-HMDS)2") in toluene generates 2-[13C]-3-ethyl 1-isopropyl-3,4,5,6-tetrahydropyrimid-2-ylidene ([13C1]-2). The hindered meta stable N,N-heterocyclic carbene [13C1]-2 thus generated undergoes a slow but quantitative reaction with toluene (the solvent) to generate the aminal 2-[13C]-2 benzyl-3-ethyl-1-isopropylhexahydropyrimidine ([13C1]-14) through formal C-H insertion of C2 (the "carbene carbon") at the toluene methyl group. Despite a significant pKa mismatch (Delta pKa 1+ and toluene estimated to be ca. 16 in DMSO) the reaction shows all the characteristics of a deprotonation mechanism, the reaction rate being strongly dependent on the toluene para substituent (rho = 4.8(+/-0.3)), and displaying substantial and rate-limiting primary (k(H)/k(D) = 4.2(+/-0.6)) and secondary (k(H)/k(D) = 1.18(+/-0.08)) kinetic isotope effects on the deuteration of the toluene methyl group. The reaction is catalysed by K-HMDS, but proceeds without cross over between toluene methyl protons and does not involve an HMDS anion acting as base to generate a benzyl anion. Detailed analysis of the reaction kinetics/kinetic isotope effects demonstrates that a pseudo-first-order decay in 2 arises from a first-order dependence on 2, a first order dependence on toluene (in large excess) and, in the catalytic manifold, a complex noninteger dependence on the K-HMDS dimer. The rate is not satisfactorily predicted by equations based on the Bronsted salt-effect catalysis law. However, the rate can be satisfactorily predicted by a mole-fraction-weighted net rate constant: -d[2]/dt = ({x2 k(uncat)} + {(1-x2) k(cat)})[2]1[toluene]1, in which x2 is determined by a standard bimolecular complexation equilibrium term. The association constant (Ka) for rapid equilibrium-complexation of 2 with (K-HMDS)2 to form [2(K-HMDS)2] is extracted by nonlinear regression of the 13C NMR shift of C2 in [13C1]-2 versus [(K-HMDS)2] yielding: Ka = 62(+/-7) M(-1); delta(C(2)) in 2=237.0 ppm; delta(C(2)) in [2(K-HMDS)2] = 226.8 ppm. It is thus concluded that there is discrete, albeit inefficient, molecular catalysis through the 1:1 carbene/(K-HMDS)2 complex [2(K-HMDS)2], which is found to react with toluene more rapidly than free 2 by a factor of 3.4 (=k(cat)/k(uncat)). The greater reactivity of the complex [2(K-HMDS)2] over the free carbene (2) may arise from local Bronsted salt-effect catalysis by the (K-HMDS)2 liberated in the solvent cage upon reaction with toluene. PMID- 16673430 TI - Mapping electrochemiluminescence as generated at double-band microelectrodes by confocal microscopy under steady state. AB - A confocal microspectrometer was used to collect spatially resolved electrochemiluminescence (ECL) spectra from the annihilation reaction between Ru(bpy)3+ and Ru(bpy)3(3+), each species being generated at a platinum band microelectrode and separated by a micrometric insulating gap. The use of a confocal microspectrometer allows in situ photon detection of the ECL reaction with micrometric spatial resolution and observes the concentration distribution of the photon source in the vicinity of the interelectrode gap. PMID- 16673431 TI - Influence of cluster size on the structures and stability of trimetallic nitride fullerenes M3N@C80. AB - To provide insight into the influence of encaged clusters on the structures and stability of trimetallic nitride fullerenes (TNFs), extensive density functional theory calculations were performed on Sc3N@C80, Y3N@C80, and La3N@C80 as well as their encaged clusters. The calculated results demonstrated that both Sc3N and Y3N units are planar, whereas La3N units are pyramidal inside C80-I(h), and that both of the Y3N@C80 and La3N@C80 cages deform considerably in the planes of Y3 and La3. The calculated results suggest that M-cage attraction/repulsion and M-M repulsion interactions determine the geometries of these three complex molecules and the dynamics of the corresponding encaged clusters. These calculated findings distinctly reveal the influence of the size of the encaged clusters on the structures and stability of TNFs and may rationalize their significant differences in yields and chemical reactivity. PMID- 16673432 TI - Raman spectroscopy of short-lived terthiophene radical cations generated by photochemical and chemical oxidation. AB - The Raman spectra of various terthiophene radical cations are investigated; namely those of unsubstituted terthiophene and two styryl-substituted terthiophenes. Transient pump-probe resonance Raman spectroscopy is used to measure the short-lived radical cation spectra of non-end-capped 2,2':5',2'' terthiophene (3T) and 3'-[(E)-2-(4-nitrophenyl)ethenyl]-2,2':5',2''-terthiophene (NO2-pe3T). For these two compounds, the radical cations are generated via either direct photogeneration or photochemically using the electron acceptor tetracyanoethylene. The radical cation of 5,5''-dimethyl-3'-[(E)-2-phenylethenyl] 2,2':5',2''-terthiophene (DM-pe3T) is stable for up to five minutes as a result of the two alpha end caps and continuous-wave resonance Raman spectroscopy and chemical oxidation is used to obtain the spectrum of this radical cation. The resonance Raman spectra of all three terthiophene radical cations are dominated by a group of very intense bands in the low-frequency region. These bands have been assigned, by density functional theory methods, to C-S stretching modes coupled to thiophene ring deformations. These modes are significantly less intense in the sigma-dimer of NO2-pe3T [i.e. the corresponding styryl sexithiophene (NO2-pe3T)2]. This observation is attributed to a smaller change in the C--S bond order in the sexithiophene compared to the analogous terthiophene. This bond order difference may be rationalised by consideration of the singly occupied molecular orbital and lowest unoccupied molecular orbital, which are involved in the electronic transition probed by the laser excitation wavelength. PMID- 16673433 TI - High-silica ferrierite zeolite self-transformed from aluminosilicate gel. PMID- 16673434 TI - High density amorphous ice from cubic ice. PMID- 16673435 TI - Detection of single oxygen molecules opens up new vistas for the investigation of molecular cooperativity in hemocyanins. PMID- 16673436 TI - Caenorhabditis elegans as a screening tool for the endothelial cell-derived putative aging-related proteins detected by proteomic analysis. AB - Endothelial cells go through progressive pathophysiologic modification as cellular senescence progresses. In vitro, endothelial cell senescence is accompanied by failure of proliferation and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in the organism in vivo and free radical theory is accepted to be the most plausible explanation for this process. We have screened proteins involved in both cellular senescence and reactive oxygen species induced condition using 2-D gel analysis and found that ubiquitin carboxyl terminal hydrolase L1, peroxyredoxin 2, peroxyredoxin 4, fatty acid binding proteins (FABPs), and 5'-AMP-activated protein kinase beta-1 subunit were candidate aging related proteins. To evaluate in vivo function of these proteins, Caenorhabditis elegans (C. elegans) knock-down system using RNA interference was applied. Aging specific expression of lipofucsin and the lifespan of knocked-down C. elegans were observed to assess the outcome. Interestingly, the inhibition of the genes led to short lifespan and earlier accumulation of lipofucsin with increasing age when compared with the wild type. These results suggest that the above genes may be related to cellular senescence process in determining the longevity in C. elegans and that gene inactivation renders animals susceptible to oxidative stress. PMID- 16673437 TI - Identification of a plasma proteomic signature to distinguish pediatric osteosarcoma from benign osteochondroma. AB - Osteosarcoma (OS) is the most common malignant bone tumor in children. To identify a plasma proteomic signature that can detect OS, we used SELDI MS to perform proteomic profiling on plasma specimens from 29 OS and 20 age-matched osteochondroma (OC) patients. Nineteen statistically significant ion peaks that were differentially expressed in OS when compared with OC patients were identified (p < 0.001 and false discovery rate < 10%). Using the proteomic profiles, we constructed a multivariate 3-nearest neighbors classifier to distinguish OS from OC patients with a sensitivity of 97% and a specificity of 80% based on external leave-one-out crossvalidation. Permutation test showed that the classification result was statistically significant (p < 0.00005). One of the proteins (m/z 11 704) in the proteomic signature was identified as serum amyloid protein A (SAA) by PMF. The higher plasma level of SAA in OS patients was further validated by Western blotting when compared to that of osteochrondroma patients and normal subjects as reference. The classifier based on this plasma proteomic signature may be useful to differentiate malignant bone cancer from benign bone tumors and for early detection of OS in high-risk individuals. PMID- 16673438 TI - Outer membrane vesicles of the VA-MENGOC-BC vaccine against serogroup B of Neisseria meningitidis: Analysis of protein components by two-dimensional gel electrophoresis and mass spectrometry. AB - Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains. PMID- 16673439 TI - Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map. AB - Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified. PMID- 16673440 TI - Proteomic trajectory mapping of biological transformation: Application to developmental mouse retina. AB - In this report we introduce a new concept "proteomic trajectory mapping" for the investigation of a complex phenomenon underlying biological transformation and transition. We define proteomic trajectory to be the kinetic trace of protein expression and present a successful proteomic trajectory mapping of complex molecular events underlying postnatal development of mouse retina. Cluster analysis of the trajectory data using a two-state model identified four proteomic trajectory types: two distinct trajectory types accounting for the decline or the rise of protein molecules actively expressed in the juvenile stage (J-type) or in the adult stage (A-type), a class of transient trajectories that mediate the transformation from the juvenile to the adult stage (T-type), and the steady trajectories throughout the entire process of transformation (C-type). The dominance of particular protein categories expressed in each trajectory characterizes the stage of retinal development. Proteomic trajectory mapping will be a powerful tool to study the systematic changes of protein expression caused by physiological, genetic, or pathological agents and the reverse of such changes to the norm by a treatment. The proteomic trajectory mapping is applicable to any biological transformation and, therefore, will be a powerful tool in biomedical sciences. PMID- 16673441 TI - The direct formation of glycosyl thiols from reducing sugars allows one-pot protein glycoconjugation. PMID- 16673442 TI - "Green" oxidation catalysis for rapid deactivation of bacterial spores. PMID- 16673443 TI - Crystallization in a mixture of solvents by using a crystal modifier: morphology control in the synthesis of highly monodisperse CaCO3 microspheres. PMID- 16673444 TI - From a lamellar to hexagonal self-assembly of bis(4,4'-(m,m' di(dodecyloxy)phenyl)-2,2'-difluoro-1,3,2-dioxaborin) molecules: a trans-to-cis isomerization-induced structural transition studied with STM. PMID- 16673445 TI - A noninterpenetrating three-dimensional 4(6)6(9) iron(II) coordination polymer built with a trigonal-antiprismatic iron(III) metalloligand. PMID- 16673446 TI - Impaired maturation and function of dendritic cells by mycobacteria through IL 1beta. AB - Dendritic cells (DC) are pivotal for initiation and regulation of innate and adaptive immune responses evoked by vaccination and natural infection. After infection, mycobacterial pathogens first encounter monocytes, which produce pro inflammatory cytokines, including IL-1beta, TNF-alpha and IL-6. The role of these cytokines in DC maturation remains incompletely understood. Here, we show that maturation of DC from monocytes was impaired by pretreatment of monocytes with low doses of IL-1beta. Under these conditions, Mycobacterium leprae-infected DC failed to stimulate antigen-specific T cell responses. Expression of CD86 and CD83 and production of IL-12 in response to lipopolysaccharide and peptidoglycan were diminished. In contrast, these DC functions were not impaired by pretreatment with TNF-alpha, IL-6 or IL-10. When monocytes were infected with M. bovis Bacillus Calmette-Guerin, and subsequently differentiated to DC, the activity of these DC was suppressed as well. Thus, IL-1beta acts at early stages of differentiation of DC and impairs biological functions of DC at later stages. Therefore, production of IL-1beta by mycobacteria-infected antigen-presenting cells counteracts effective stimulation of innate and adaptive immune responses. PMID- 16673447 TI - CD4 T cell help is required for primary CD8 T cell responses to vesicular antigen delivered to dendritic cells in vivo. AB - Insight into the mechanisms by which dendritic cells (DC) present exogenous antigen to T cells is of major importance in the design of vaccines. We examined the effectiveness of free antigen as well as antigen with lipopolysaccharide, emulsified in complete Freund's adjuvant, and antigen encapsulated in liposomes in activating adoptively transferred antigen-specific CD4 and CD8 T cells. When contained in liposomes, 100- to 1000-fold lower antigen amounts were as efficient in inducing proliferation and effector functions of CD4 and CD8 T cells in draining lymph nodes as other antigen forms. CD11c(+)/CD11b(+)/CD205(mod)/CD8alpha(-) DC that captured liposomes were activated and presented this form of antigen in an MHC class I- and class II restricted manner. CD4 T cells differentiated into Th1 and Th2 effector cells. Primary expansion and cytotoxic activity of CD8 T cells were CD4 T cell-dependent and required the transporter associated with antigen processing (TAP). Finally, adoptively transferred CD4 and CD8 T cells were not deleted after primary immunization and rapidly responded to a secondary immunization with antigen containing liposomes. In conclusion, encapsulation of antigen in liposomes is an efficient way of delivering antigen to DC for priming of both CD4 and CD8 T cell responses. Importantly, primary CD8 T cell responses were CD4 T cell-dependent. PMID- 16673448 TI - Adenosine A2A receptor-mediated cell death of mouse thymocytes involves adenylate cyclase and Bim and is negatively regulated by Nur77. AB - Adenosine is generated in the microenvironment of emerging thymocytes through normal mechanisms of lymphocyte selection. In a normal thymus, most of the adenosine is catabolized by adenosine deaminase; however, in an environment where up to 95% of the cells undergo programmed cell death, a sufficient amount of adenosine is accumulated to trigger cell surface adenosine receptors. Here we show that accumulated adenosine can induce apoptosis in immature mouse thymocytes, mostly via adenosine A(2A) receptors. The signaling pathway is coupled to adenylate cyclase activation, induction of the Nur77 transcription factor, Nur77-dependent genes, such as Fas ligand and TRAIL, and the pro apoptotic BH3-only protein Bim. We analyzed several knockout and transgenic mouse lines and found that adenosine-induced killing of mouse thymocytes requires Bim, occurs independently of "death receptor" signaling and is inhibited by Bcl-2 and Nur77. Collectively our data demonstrate that adenosine-induced cell death involves signaling pathways originally found in negative selection of thymocytes and suggest a determining role of Bim and a regulatory role for Nur77. PMID- 16673449 TI - Evaluation of antiinflammatory activity of the total flavonoids of Laggera pterodonta on acute and chronic inflammation models. AB - The antiinflammatory effect of the total flavonoids of Laggera pterodonta (TFLP) was evaluated with various in vivo models of both acute and chronic inflammation. In the acute inflammation tests, TFLP significantly inhibited xylene-induced mouse ear oedema, carrageenan-induced rat paw oedema and acetic acid-induced mouse vascular permeability. In the carrageenan-induced rat pleurisy model, TFLP efficiently suppressed inflammatory exudate and leukocyte migration, reduced the serum levels of lysozyme (LZM) and malondialdehyde (MDA), increased the activity of serum superoxide dismutase (SOD), and also decreased the contents of total protein, nitric oxide (NO) and prostaglandin E2 (PGE2) in the pleural exudates. No marked effect of TFLP on the activity of serum glutathione peroxidase (GSH-PX) was observed. In the chronic inflammation experiment, TFLP inhibited cotton pellet-induced rat granuloma. The antiinflammatory mechanisms of TFLP are probably associated with the inhibition of prostaglandin formation, influence on the antioxidant systems and the suppression of LZM release. The acute toxicity study revealed that TFLP was nontoxic in mice up to an oral dose of 7.5 g/kg body weight. PMID- 16673450 TI - Treatment for advanced kidney cancer. PMID- 16673451 TI - Paxil and the risk of birth defects. PMID- 16673452 TI - The FDA advances scientific research. PMID- 16673453 TI - To predict your odds of future heart disease, look back in time. PMID- 16673454 TI - Ask the doctors. A friend of mine in her 60s was told she had a 75 percent blockage in the bend of her aorta, and it was currently inoperable. Her doctor said that when and only when she had a stroke, he would perform a high-risk operation to try to clear the blockage. I don't feel I can tell her to get a second opinion. What's your opinion? PMID- 16673455 TI - Ask the doctors. A doctor told my son, "God has given you a left bundle branch block." The doctor said his heart was otherwise healthy, but this scares me because the husband of a friend died from bundle branch block. I've given my son a wrist band saying he has a heart condition. What else can he and I do? PMID- 16673456 TI - Outbreak news. Chikungunya and dengue, south-west Indian Ocean. PMID- 16673457 TI - Outbreak news. Meningococcal disease, Kenya. PMID- 16673459 TI - Human African trypanosomiasis (sleeping sickness): epidemiological update. PMID- 16673458 TI - Outbreak news. Avian influenza, Indonesia--update. PMID- 16673460 TI - Outbreak news. Meningococcal disease, Uganda--update. PMID- 16673461 TI - Future needs and directions for Shigella vaccines. PMID- 16673462 TI - Outbreak news. Avian influenza, Turkey--update. PMID- 16673463 TI - International standards for tuberculosis care. PMID- 16673464 TI - Outbreak news. Avian influenza, Indonesia--update. PMID- 16673465 TI - Outbreak news. Avian influenza, Turkey. PMID- 16673466 TI - Conclusions and recommendations from the Immunization Strategic Advisory Group. PMID- 16673467 TI - Positive seafood report. PMID- 16673468 TI - Standardisation of methods for assessing mould germination: a workshop report. AB - The first workshop on predictive mycology was held in Marseille, France, 2--4 February 2005 under the auspices of the French Microbiological society. The purpose of the workshop was to list the different techniques and definitions used by scientists for assessing mould germination and to evaluate the influence of the different techniques on the experimental results. Recommendations were made when a large consensus was obtained. In order to facilitate the study of germination, alternative methods to microscopic examination were examined. PMID- 16673469 TI - President's budget, FY2006 final appropriations paint dim picture. ADAP waiting list near 1,000. PMID- 16673470 TI - ADAP funding still fails to meet the growing need. Waiting list near 1,000. PMID- 16673471 TI - Acute infection patients have more partners. Higher chance of infection spreading in networks. PMID- 16673473 TI - FDA discusses studies to support OTC HIV test kits. PMID- 16673472 TI - No evidence of HIV-1 strain cross transmission. Superinfection is very rare occurrence. PMID- 16673474 TI - Tijuana's HIV prevalence rate has had an alarming increase in recent years. Border town's epidemic could be new trend. PMID- 16673475 TI - Global Partners Forum advocates for children. 18 million AIDS orphans by 2010. PMID- 16673476 TI - Study: HIV prevention requires counseling. PMID- 16673477 TI - North Carolina's HIV testing access inspires other states. PMID- 16673478 TI - Custody. Neglect charge against HIV-positive mother fails. PMID- 16673479 TI - NYC councilman advocates HIV-transmission prosecution. PMID- 16673480 TI - Immigration. Deportation order is stayed for HIV-positive Cuban. PMID- 16673481 TI - Transmission. Lab to go to trial over HIV, hepatitis C exposure case. PMID- 16673482 TI - Denial of HIV-positive man's SSI petition reversed. PMID- 16673483 TI - Activism. Ohio antigay group campaigns against AIDS organizations. PMID- 16673484 TI - Gay games. HIV-positive athletes allowed to enter U.S. PMID- 16673485 TI - Global Fund's top leader stepping down. PMID- 16673486 TI - HIV-positive student prevails in education suit. PMID- 16673488 TI - Teacher's retaliation complaint against DOE survives. PMID- 16673487 TI - Discrimination. Police sued for failing to help HIV-positive man. PMID- 16673489 TI - Discrimination. HIV-bias against carpet retailer fails. PMID- 16673490 TI - Missed appointments trip up man's SSI claim. PMID- 16673491 TI - Charges filed. HIV-positive man sent to jail after biting officer. PMID- 16673492 TI - Sealing the cracks. PMID- 16673494 TI - Sanctuary site for HCV in HIV-coinfected women. PMID- 16673493 TI - Windy city hosts IAPAC's fifth annual North American HIV conclave. IAPAC North American Sessions 2005, June 3-4, 2005, Chicago. PMID- 16673495 TI - Progressive realization, interrupted. PMID- 16673496 TI - Detectable viral loads and slow development of resistance. PMID- 16673497 TI - Crystal methamphetamine use and antiretroviral drug resistance: a pilot study of behavioral and clinical correlates. PMID- 16673498 TI - Epidemic (and lesser) lessons from Rio Centro. Part 1. The inversions. 3rd IAS Conference on HIV Pathogenesis and Treatment. July 24-27, 2005, Rio de Janeiro. PMID- 16673499 TI - Focus on hepatitis. Low rate of liver problems seen with LPV/r. PMID- 16673500 TI - Debate over warnings for ADHD stimulants. PMID- 16673501 TI - Asthma/allergies. EpiPen: accidental injection into a finger...and should allergic patients carry two-dose injectors? PMID- 16673502 TI - Immunizations. Can fever-reducers prevent redness from DTP shots? PMID- 16673503 TI - Dog and cat foods and treats: a new source of Salmonella infections? PMID- 16673504 TI - Playdough: 50 years' old, and still gooey, fun, and educational. PMID- 16673505 TI - Product recalls. Recalls: night light fire and burn hazards...and children's toy jewelry. PMID- 16673506 TI - Progress towards interruption of wild poliovirus transmission in 2005. PMID- 16673507 TI - Schistosomiasis and soil-transmitted helminth infections--preliminary estimates of the number of children treated with albendazole or mebendazole. PMID- 16673508 TI - Inactivated poliovirus vaccine following oral poliovirus vaccine cessation. PMID- 16673509 TI - Avian influenza fact sheet (April 2006). PMID- 16673510 TI - Outbreak news. Avian influenza, Cambodia. PMID- 16673511 TI - Outbreak news. Botulism, Thailand. PMID- 16673513 TI - Outbreak news. Poliomyelitis, Somalia. PMID- 16673512 TI - Outbreak news. Meningococcal disease, African meningitis belt, epidemic season 2006. PMID- 16673514 TI - Influenza. PMID- 16673515 TI - After the tsunami: mental health challenges to the community for today and tomorrow. Proceedings of a symposium. February 2-3, 2005. Bangkok, Thailand. PMID- 16673516 TI - Syndromic surveillance in bioterrorist attacks. PMID- 16673517 TI - Telithromycin-resistant Streptococcus pneumoniae. PMID- 16673518 TI - Integrated human-animal disease surveillance. PMID- 16673519 TI - VanB-VanC1 Enterococcus gallinarum, Italy. PMID- 16673520 TI - Empyema thoracic from Salmonella choleraesuis. PMID- 16673521 TI - Asymptomatic Yersinia pestis infection, China. PMID- 16673522 TI - Sporotrichosis, Plain of Jars, Lao People's Democratic Republic. PMID- 16673523 TI - West Nile virus antibodies in Colombian horses. PMID- 16673524 TI - Wild poliovirus type 1, Central African Republic. PMID- 16673525 TI - Oneness, complexity, and the distribution of disease. PMID- 16673526 TI - Unusual complication of coarctation of the aorta. AB - The majority of older patients with uncorrected coarctation of the aorta die before the age of 50 years from many complications. We report our experience in treating this type of congenital malformation in a 40-year-old man who developed aortic endarteritis as a rare complication. PMID- 16673527 TI - Saw palmetto for benign prostatic hyperplasia. PMID- 16673528 TI - Racial differences in lung cancer. PMID- 16673529 TI - Aprotinin in cardiac surgery. PMID- 16673530 TI - Aprotinin in cardiac surgery. PMID- 16673531 TI - Aprotinin in cardiac surgery. PMID- 16673532 TI - Positron-emission tomography in cancer therapy. PMID- 16673533 TI - Medicare drug benefit. PMID- 16673534 TI - Is there still a place for free nipple areolar grafting in breast reduction surgery? A review of cases over a three year period. AB - Breast reduction using the technique of free nipple areolar grafting still has a place in selected cases. The surgical technique used by the senior author is described together with an analysis of the 22 cases operated on including the results of a questionnaire from 20 of these patients. The results confirmed that the technique still has a use. Relief of pre-operative symptoms by the surgery was obtained in all and there was a high degree of satisfaction with both the cosmetic results and maintaining of shape. The main criticisms relate to scars and poor return of nipple sensation, appearance, and function. Overall this did not appear to be a major issue in this cohort of patients. PMID- 16673535 TI - Our algorithm for nasal reconstruction. AB - Nasal reconstruction is always challenging for plastic surgeons. Its midfacial localisation and the relationship between convexities and concavities of nasal subunits make impossible to hide any sort of deformity without a proper reconstruction. Nasal tissue defects can be caused by tumor removal, trauma or by any other insult to the nasal pyramid, like cocaine abuse, developing an irreversible sequela. Due to the special characteristics of the nasal pyramid surface, the removal of the lesion or the debridement must be performed according to nasal subunits as introduced by Burget. Afterwards, the reconstructive technique or a combination of them must be selected according to the size and the localisation of the defect created, and tissue availability to fulfil the procedure. An anatomical reconstruction must be completed as far as possible, trying to restore the nasal lining, the osteocartilaginous framework and the skin cover. In our department, 35 patients were operated on between 2000 and 2002: three bilobed flaps, five nasolabial flaps, two V-Y advancement flaps from the sidewall, three dorsonasal flaps modified by Ohsumi, 19 paramedian forehead flaps, three cheek advancement flaps, three costocondral grafts, two full thickness skin grafts and two auricular helix free flaps for alar reconstruction. All flaps but one free flap survived with no postoperative complications. After 12-24 months of follow-up, all reconstructions remained stable from cosmetic and functional point of view. Our aim is to present our choice for nasal reconstruction according to the size and localization of the defect, and donor tissue availability. PMID- 16673536 TI - A comparison of aesthetic proportions between the healthy Caucasian nose and the aesthetic ideal. AB - BACKGROUND AND OBJECTIVE: The aim of septorhinoplasty is to create a nose that is aesthetically pleasing to the patient and to maintain nasal function. Although a small number of population cohort studies have been performed on the ethnic nose, little is known of the aesthetics standards of nasal proportions in the general Caucasian population. The aim of this study was to establish parameters of the average nose in the healthy population and to compare them with those of the aesthetic ideals. MATERIAL AND METHODS: Ethical approval was obtained to recruit a cohort of staff and students from the medical school. Two photographs were taken: anterior and right lateral. The following measurements were made: intercanthal width, alar width, length of the nose (nasion-pronasion length), naso-labial angle, nasal tip projection, naso-facial angle and naso-frontal angle. These parameters were compared with published aesthetic ideals. RESULTS AND CONCLUSION: Aesthetic nasal proportions of 50 healthy Caucasians were examined from a cohort of 57 volunteers. Results showed that the average nose did not conform to neo-classical facial canons. The alar width (average 3.6 cm) was significantly wider than the intercanthal width (average 3.0 cm, p<0.05). The nasal width-length ratio was also greater suggesting that the cohort average was shorter and wider than the aesthetic ideal. The Baum ratio was 2.5:1, indicating that the average nasal tip was more projected that the aesthetic ideal (2.8:1). The naso-frontal and naso-facial angles are both more obtuse, and in profile the average nose overall appeared more prominent, as a result of the reduced forehead projection. The only parameter where there appeared to be consistency between the average and the ideal nose was the naso-labial angle. This study shows that many aesthetic parameters from a cohort of healthy subjects differ from the widely used aesthetic standards. Rhinoplasty surgeons should, therefore, give thought to the frame of reference used when discussing aesthetic objectives with their patients. PMID- 16673537 TI - Reconstruction of the nasal tip including the columella and soft triangle using a mastoid composite graft. AB - This paper describes the use of a composite graft from the mastoid area consisting of full-thickness skin peripherally and selectively localised fascia fat tissue underneath the skin centrally for immediate reconstruction of moderate defects of the nasal tip including the columella and soft triangle. Mastoid composite grafting is a simple and safe procedure that avoids partial graft loss and provides adequate augmentation of soft tissue, easy reshaping of the new nostril rim, minimal post-operative shrinkage, and no donor-site morbidity. Then, it results in a satisfactory nasal appearance with adequate tip projection and symmetry. This procedure may represent a preferred method of nasal tip reconstruction. PMID- 16673538 TI - Towards performance measurement in reconstructive surgery: a multicentre pilot study of free and pedicled flap procedures. AB - OBJECTIVES: To pilot the acceptability and feasibility of clinical audit in free and pedicled flap reconstruction. To establish a baseline flap failure rate in participating units, so that a sample size calculation could be performed for future national audit. METHODS: A proforma was piloted over a 3-month period in four participating units, during which time data on 93 reconstructive procedures involving free and pedicled flaps was collected. The patients included those where large transfers of tissue were required such as for coverage of grade IIIb compound tibial fractures and breast reconstruction after mastectomy, and also smaller flap transfers such as after skin cancer excision. RESULTS: The proforma was found to be acceptable to clinicians and the feasibility of the data collection process was established. Overall there was a total flap survival of 89% and secondary operations to the donor or recipient sites were required in 11% of patients. CONCLUSIONS: This study demonstrates the feasibility of comparative audit for free and pedicled flap procedures using the methods proposed. Based on the incidence of flap failure observed in this pilot study, at least 18 months of prospective data collection on consecutive patients is required to fulfil the statistical requirements of comparative audit. The establishment of a routinely collected minimum dataset is proposed as one means of meeting these requirements. PMID- 16673539 TI - Ocular contamination during lesional surgery-a hazard for the plastic surgeon. AB - Protective glasses reduce the risk of transconjunctival virus transmission although this has never been characterised for local anaesthetic skin lesion surgery. This study investigated the use of, and need for, protective eyewear during Plastic Surgery and the surgeons' attitude to risk. MATERIALS AND METHODS: Study 1. A multi-center questionnaire recorded the attitudes of Plastic Surgeons' regarding google use and ocular viral transmission. Study 2. A single surgeon performed local anaesthetic skin lesion surgery over 8 months whilst wearing clean goggles. After each procedure the goggles were inspected for macroscopic splashes ans a record made of whether the operator had been aware of the splash occurrence. RESULTS: Study 1. There were 36 respondents to the questionnaire (response rate of 84%). All grades recognised the risk of conjunctival contamination but most respondents did not wear goggles. Respondents changed their behavior, however, in 'high risk' cases. Study 2. 143 procedures resulted in 42 splashes. The surgeon was only aware of the splash in 6 cases. CONCLUSION: Despite widespread recognition of the importance of eye protection, goggle use is still infrequent. Our study stresses the frequency of potential splashes to the eye and need for routine use of goggles to prevent viral transmission. PMID- 16673540 TI - Psychiatric assessment of patients with self-inflicted lacerations to the wrist and forearm admitted to a nonpsychiatric ward: the experience of a regional plastic surgery unit. AB - In 1984, the Department of Health (DoH) recommended that all patients with deliberate self-harm (DSH) must have a mental assessment before discharge. DSH patients, especially those with lacerations to wrist and forearm, are a regular source of admission to plastic surgery units. In Northern Ireland, the regional plastic surgery service is provided at the Ulster Hospital, which does not have an on-site psychiatric department. Consequently, it was often difficult to arrange a psychiatrist assessment for these patients on the ward even when the assessment was required urgently. The objective of this study was to develop and validate a protocol that would ensure that these patients were assessed and followed up for their mental health as recommended by the DoH. In the absence of clear guidelines from the DoH and utilising the existing arrangement between Accident and Emergency (A&E) and the psychiatric services, all referring A&E departments were instructed at the time of referring patients with DSH to wrist and forearm to arrange a psychiatric examination before transferring the patient to the plastic surgery unit. Data were collected retrospectively (August 2002 October 2002) and prospectively (November 2002-October 2003) and comprised demographic features, previous history of self-harm, conduct of the patient in A&E and the ward, length of stay in the hospital and various aspects of psychiatric assessment and follow-up. In total, 42 referrals were made during the period studied. The average age was 28 years with a male to female ratio of 2:1. The average hospital stay was two days. Despite difficulties, our practice was found effective in ensuring psychiatry assessment and follow-up without risking the patient's physical health. The study also highlighted the need for collaboration between plastic surgeons and psychiatrists to improve services in regards to DSH patients. PMID- 16673541 TI - Psychological and social factors in reconstructive surgery for hemi-facial palsy. AB - This paper examines the psychological and social impact of reconstructive surgery for hemi-facial palsy and considers psychosocial factors which may be associated with patient satisfaction. It reports a retrospective study in which 106 adults were assessed using primarily qualitative methods. All participants had undergone two-stage reconstruction using vascularised free muscle grafts, with all procedures having been carried out by the same surgeon. The participants were all at least 12 months post-surgery. They were assessed using demographic questionnaires, the hospital anxiety and depression scale (HADS) and the facial paralysis evaluation measure (FPEM). In addition, all participants were interviewed using a semi-structured format, the interviews were recorded verbatim and the transcripts were analysed using thematic analysis. Of the total study group, 67% had acquired facial palsy. The mean age of the total group was 44.7 years and 67.9% were female. As a group they were rather less depressed than the normal population with similar levels of anxiety to population norms. The primary motivation for surgery was appearance rather than function. Using interview data in addition to the FPEM, satisfaction with the process and outcome of surgery was assessed. Thirty five percent were very satisfied with both process and outcome, 34% were satisfied with the outcome but found the treatment process stressful, 15.1% were not entirely satisfied with process or outcome but felt surgery had been worthwhile as there had been some improvement. The remainder were very dissatisfied with both process and outcome and regretted having undergone surgery. There was no significant association between dissatisfaction and anxiety, the cause of the acquired palsy, longevity prior to surgery, gender nor whether the condition was acquired or congenital. There was a significant relationship with depression, in that those who were suffering from depression were more likely to be dissatisfied with surgery. Participants were asked in interview about social pressures and comments or remarks made by others about their condition. The majority (89.6%) of the total study group reported intrusive questions by acquaintances and strangers, with more than half of these being distressed by such questions. Following surgery, there was a significant reduction in the incidence of these questions. There was no relationship between distress in response to these questions prior to surgery and dissatisfaction with surgery. However, 27.4% also reported aggressive hurtful comments before surgery with a minimal improvement in incidence following surgery. These participants also reported consistent patterns of social avoidance and social isolation before and after surgery, and were more likely to be depressed than the rest of the study group. They were significantly more likely to be dissatisfied with surgery (p=.016). It is recommended that patients are screened and counseled prior to surgery to identify such problems and referred for psychological treatment in order to ensure they gain maximum benefit from reconstructive surgery. PMID- 16673542 TI - Sensory reinnervation of a musculocutaneous flap: an experimental rabbit study. AB - Sensory neurotisation of a muscle (sensory nerve transfer to the motor nerve of a muscle) produces muscle sensibility, but not skin sensibility. How to achieve sensation of a musculocutaneous flap remains a challenge to reconstructive microsurgeons. The purpose of our study was to determine if multiple nerve grafts which were placed vertically between the neuromuscular entrance zone of a muscle and a target area of dermis on the overlying skin could improve sensation. Thirty six gracilis musculocutaneous flaps (18 rabbits) were raised and divided into three groups: group 1 consisted of 12 sensory neurotised gracilis musculocutaneous flaps with five nerve grafts each; group 2 consisted of another 12 sensory neurotised gracilis flaps with 10 nerve grafts each; and the control group consisted of 12 sensory neurotised gracilis musculocutaneous flaps without any nerve grafts. All nerve grafts spanned the distance between the neuromuscular entrance zone of the gracilis muscle and a specified 3 cm diameter area of the skin island. The saphenous nerve (sensory) was coapted to the obturator nerve (motor nerve of the gracilis) in an effort to achieve improved sensation of the skin island in the musculocutaneous flaps. After 6 months, the flaps were individually evaluated using cortical somatosensory evoked potentials (CSSEP) using normal, painful, cold and hot stimuli. One unoperated rabbit was studied as the baseline CSEEP for comparison. Retrograde horseradish peroxidase (HRP) labelling was then performed to evaluate the possibility of newly established neural pathways. Results of the CSSEP testing revealed that flaps possessing 10 nerve grafts (group 2) demonstrated better sensation when compared to flaps possessing five nerve grafts (group 1) or no nerve grafts (control group). Furthermore, retrograde HRP labelling proved that a new neural pathway had been established from the skin island to the dorsal root ganglia of S1 and S2 via the interposed nerve grafts and the sensory neurotised gracilis muscle in groups 1 and 2 rabbits. The control group did not display any sensory regeneration. PMID- 16673543 TI - In response to: anatomy of the proximal cutaneous perforator vessels of the gracilis muscle. PMID- 16673544 TI - Postoperative situation after axillary suction-curettage: an endoscopical view. PMID- 16673545 TI - Bilateral isolated orbital blowout fractures among terrorist bombing victims. A very rare entity. PMID- 16673546 TI - Sump drainage with an atraumatic diffusion sucker in DIEP flaps. PMID- 16673547 TI - 'Trilobed flap' and 'the seagull flap' for syndactyly. PMID- 16673548 TI - Facial herpes simplex infection from possible cross contamination through the laser hand-piece following cutaneous laser resurfacing. PMID- 16673549 TI - The effect of the new deal on the operative experience of plastic surgical SHOs. PMID- 16673551 TI - Multiagency effort to focus on PET as biomarker. PMID- 16673550 TI - Lymphoedema following sentinel node biopsy-a need for informed consent. PMID- 16673552 TI - A memoir of pediatric nuclear medicine: part 1. Pioneers and early advances. PMID- 16673553 TI - Q&A: perspective on pediatric nuclear medicine: an interview with Michael J. Gelfand. PMID- 16673554 TI - Evolution of the prevalence and characteristics of anemia in inflammatory bowel diseases between 1993 and 2003. AB - INTRODUCTION: Anemia has been considered as an overlooked complication of inflammatory bowel disease. Studies dating back to the 80ties and the 90ties have shown 30% of anemia among inflammatory bowel disease (IBD) patients. More recently, the broader use of immunosuppressive drug and infliximab allowing better mucosal healing as well as a more aggressive treatment of anemia, including the use of safer form of IV iron, may have influenced the prevalence of anemia among IBD patients. Our aim was to asses the prevalence and characteristics of anemia among two cohorts of IBD patients at 10 years interval and to look for associated clinical or demographic factors. METHODS: Using the IBD patients register of one senior gastroenterologist, we identified IBD patients he had consecutively seen and who had blood test at the outpatient clinic during the years 1993 and 2003. Demographic and clinical characteristics, treatment for Crohn's disease, blood test results and treatment of anemia were recorded and compared between these two cohorts. Anemia was defined as an hemoglobin level lower than the normal value of the laboratory of our hospital. RESULTS: 80 and 90 patients were identified in 1993 and 2003, respectively. There was no significant difference between the two cohorts, according to age, gender, disease type, duration or location. There were 27/80 (33.8%) and 15/90 (16.7%) anemic patients in 1993 and 2003, respectively (P = 0.013). The prevalence of severe anemia (hemoglobin level < 10.5 g/100 ml) was similar in the two cohorts (6.3% and 5.6%). Characteristics of the anemia were similar in the two cohorts with a majority of iron deficiency anemia and inflammatory anemia. Ferritin and CRP levels were not significantly different in the two cohorts. The only significant difference was a more frequent use of immunosuppressive treatment and infliximab in 2003 than in 1993 (33.3% vs. 13.8% ; P = 0.0038, RR: 0.41, 0.22 0.77) CONCLUSIONS: Prevalence of mild to moderate anemia has significantly decreased in our population over the last 10 years. The only difference detected between the two cohorts was the increased use of immunosuppressive drug (mainly azathioprine). PMID- 16673555 TI - Serum vitamin-E levels and its relation to clinical features in nonalcoholic fatty liver disease with elevated ALT levels. AB - BACKGROUND: Oxidative stress and free oxygen radicals play an important role in the progression from simple fatty liver to steatohepatitis. Deficiency of antioxidants like vitamin-E has been reported to trigger this progression. The main aims of our study were to measure plasma vitamin-E levels in nonalcoholic fatty liver disease (NAFLD), to explain its relationship with biochemical parameters and to examine the possible therapeutic and prophylactic role of vitamin-E. METHODS: 52 patients with NAFLD and elevated liver function tests were enrolled. After 6 months of follow-up with a standard low-fat, low-calorie diet, changes in liver enzymes were evaluated. RESULTS: Deficiency of vitamin-E was detected in 16 patients with NAFLD. Homogenous echo pattern of the liver and attenuation was found to be significantly higher in the low vitamin-E group (p = 0.03). The low vitamin-E group had significantly higher levels of triglyceride (p = 0.02). After 6 months, patients in the low vitamin-E group did not respond to the diet and no decrease in ALT levels was detected (p = 0.04). CONCLUSION: This is the first study measuring the serum vitamin-E levels in nonalcoholic fatty liver disease. A correlation was found between low vitamin-E levels, high triglyceride levels, as well as sonographic findings, both of which are negative prognostic factors causing progression of fatty liver to steatohepatitis. Patients with low vitamin-E levels did not respond to a classical diet for fatty liver disease. Based on the data, we suggest that diet alone is not adequate for patients with fatty liver, and vitamin-E supplementation should be added. PMID- 16673556 TI - (18)FDG-PET-scan in staging of primary malignant melanoma of the oesophagus: a case report. AB - Primary malignant melanoma of the oesophagus is a rare disease, only 262 cases being reported up to June 2005. In general, the prognosis is dismal because of its tendency to present as an advanced neoplasm with aggressive biological behaviour. (18)FDG-PET-scan is a useful tool for evaluation of metastatic disease and locoregional lymph node metastasis. We present herein a case of a young adult with primary malignant melanoma of the oesophagus, followed by a literature review on the subject. PMID- 16673557 TI - Diffuse idiopathic colonic varices presenting with lower gastrointestinal bleeding in an elderly patient: a case report and review of the literature. AB - We report a case of lower gastrointestinal bleeding caused by idiopathic colonic varices. A 74-year-old woman presented with rectal bleeding. Colonoscopy revealed numerous varices of the entire colon, which, after an extensive work-up, proved to be idiopathic. No specific therapy or transfusions were required and there has been no further bleeding to date (follow-up 10 months). Review of the literature demonstrates that diffuse idiopathic colonic varices are a rare cause of lower gastrointestinal bleeding, especially as the first presentation in an elderly patient. PMID- 16673558 TI - Severe thrombophilic diathesis starting with hepatic vein thrombosis (BUDD-CHIARI syndrome) in a family with a new Protein S gene mutation. AB - We report the case of a 26-year-old man with a chronic Budd-Chiari syndrome with ascites, caused by a hereditary Protein S deficiency, in a Turkish family with consanguinity. In this family, the father, the two sisters and the young brother suffered from severe venous thrombosis of the limbs, with pulmonary embolism in two of them. Those thrombotic events are caused by a hitherto not reported mutation in the PROS 1 gene on chromosome 3, resulting in a severe familial Protein S deficiency. No other thrombophilic defect was detected in the family, despite extensive investigation. Furthermore, we observe hereditary twenty-nail dystrophy in this family, the two genes probably segregating independently. Prophylaxis is discussed. PMID- 16673559 TI - GLEM/LOK report on proctology practice in Belgium. Results, comments and recommendations. AB - The article summarizes the response of eighty-eight Belgian gastroenterologists to a questionnaire on proctology practice, established by the common evaluation groups (GLEM/LOK) of medical practice. The results of the inquiry concerning haemorrhoids, anal fissure, abscess and fistula, and faecal incontinence are discussed separately. Each chapter is completed by specific therapeutic and/or diagnostic recommendations. These are based on the official statements of the American Gastroenterological Association and on our own clinical practice. There are still no official guidelines on proctology in Belgium and neighbouring countries. The diagnostic and therapeutic quality of the approach of our proctologic patients should improve by appropriate proctologic education and training in selected centres. PMID- 16673560 TI - Diagnosis of small bowel malabsorption syndromes in adults. AB - Patients with malabsorption represent a small proportion of presentations with chronic diarrhea. In spite of some progress, the last twenty years were not very innovating in malabsorption investigations. Supporting history may direct investigations toward either the small bowel or pancreas. Serological testing for celiac disease will determine most cases without invasive investigation, but individuals suspected to have small bowel malabsorption, despite negative celiac serology, should have endoscopic distal duodenal biopsies taken to exclude other rare forms of small bowel enteropathy. This strategy has largely supplanted many older tests of small bowel function. PMID- 16673561 TI - Food allergy: a challenge for the clinician. AB - Adverse reactions to food resulting in gastrointestinal symptoms and due to immunologic reactions (allergy) are discussed: their pathogenesis, the prevalence of food allergens and the clinical digestive expressions of food allergy in children and adults are reviewed. In IgE-mediated food allergy, the usefulness of the biological available tests is considered, mainly CAP tests, for proceeding to the diagnosis and the monitoring of the allergic disease. Finally, the best actual diagnostic tools in food allergy are considered (clinical history, skin tests, biological tests and food oral challenges), with their limitations and indications. PMID- 16673562 TI - Sensitivity and specificity of food specific IgE and IgG determinations for the diagnosis of food allergy. AB - Among the methods currently used to demonstrate a sensitization to foods, the measurement of food specific IgE antibodies (sIgE) is the most practical but not the most accurate. The "sensitivity" of food sIgE determinations is, for example, suboptimal with unstable allergens in fruits and vegetables that are involved in the (birch) pollen-related immediate oral allergy syndromes. In this particular syndrome the history is often conclusive and can be substantiated by skin prick tests with fresh foods. The "sensitivity" of sIgE tests is much better when sIgE are directed to stable plant or animal food allergens which often cause non immediate generalized reactions. Foods, usually, contain many different (glyco)proteinic allergens of which some are stable and others not. The "sensitivity" of the sIgE test with a particular food, therefore, varies according to the type of allergen that is recognized by the patient. The "specificity" of sIgE tests with foods is affected by the existence of homologous food allergens which induce cross-reactive IgE that may or may not be clinically relevant. While variable, clinical cross-reactivity is more common among botanically-related fruits, among different nuts, among mammalian foods and among seafood than among cereals, grains and legumes. The "specificity" of food sIgE tests is much better when sIgE are directed to unique non-cross-reactive food allergens. Unfortunately, neither the presence of food sIgE nor its level are predictive of clinical reactivity. The identification of individual allergens in foods and the characterization of the relevant IgE binding sites in these allergens might lead to the development of tests that only measure sIgE to clinical relevant food allergens. PMID- 16673563 TI - Role of the pathologist in the differential diagnosis of malabsorption. AB - Histological examination of small intestinal biopsies is an essential step in the assessment of malabsorption syndromes. Multiple biopsies should be taken in the proximal jejunum or distal duodenum and correctly oriented. Histological evaluation has to be systematic considering villous architecture, inflammation and specific diagnostic elements. In celiac disease, even with the development of serology tests, the biopsy remains useful to follow the patient and to detect potential complications. PMID- 16673564 TI - Clinical implication of immunohistochemical markers. AB - The role of the pathologist is to establish accurate diagnosis more and more with the help of complementary techniques. At present immunohistochemical expression of some diagnostic and prognostic factors may possibly predict the response to specific therapies. We focus here on the expression of three of these markers: epidermal growth factor receptor which overexpression is correlated with aggressive tumoral behaviour and with the possibility of a targeted therapy, cytokeratins 7 and 20 with their diagnostic implication in carcinomatous differentiation, and we close this review with the identification of markers related to hereditary non-polyposis colorectal cancer involving microsatellite instability. PMID- 16673565 TI - Hepatic intra-arterial injection of Yttrium-loaded microspheres for liver metastasis secondary to colorectal cancer: best soups are sometimes made from old recipies. AB - Colorectal cancer is a severe disease with a significant incidence in Western world. In the course of disease, about 40% of patients will eventually develop metastases to the liver. The majority of them will never be candidate for curative surgical management. For those patients, systemic or intra-hepatic chemotherapy is the treatment's cornerstone. Unfortunately, despite evident improvements and apparition of several active new agents, no hope of cure emerges on the agenda for now. Hepatic intra-arterial injections of radioactive devices have since a long time drawn interest from the medical community. An anti tumoural activity has been demonstrated with Yttrium-loaded microspheres injected in the hepatic artery for several liver neoplasms including metastases from colorectal cancer. We lack however the results of large randomized phase III trials to define clearly the place of those interventional therapies in the management of colorectal cancer metastatic to the liver. PMID- 16673566 TI - Half of London trusts on red alert over finance recovery plans. PMID- 16673567 TI - Logistics sell-off sparks worries on jobs and prices. PMID- 16673568 TI - Community hospitals face closure. PMID- 16673569 TI - Could contracts make secure foundations for early entrants? PMID- 16673570 TI - On the case for clarity. PMID- 16673571 TI - No more policies that fall short on implementation and cost'. Interview by Jennifer Trueland. PMID- 16673572 TI - Data briefing. Variation in primary care spend on HRG. PMID- 16673573 TI - Social enterprise. The co-op conundrum. AB - Social enterprise has opponents at both ends of the political spectrum, viewing it as either having an unfair advantage or the first stage of privatising the NHS. Supporters say there is less bureaucracy and it will focus commissioners on getting better services. In future providers will need to improve their business and financial skills. PMID- 16673574 TI - Finance. Why it pays to move. PMID- 16673575 TI - Midwives. Left holding the baby. PMID- 16673576 TI - Interim staff. Do drop in anytime. PMID- 16673577 TI - [Efflux systems of Pseudomonas aeruginosa to carbapenem antibiotics]. PMID- 16673578 TI - [The combination effects of antibacterial agents against clinical isolated multiple-drug resistant Pseudomonas aeruginosa]. AB - The effectiveness of antibacterial agents against 70 strains of clinically isolated multiple-drug resistant Pseudomonas aeruginosa (MDRP) was measured by the micro dilution method. Fifty of all strains (71%) produced metallo-beta lactamase and the IMP-1 gene was detected by polymerase chain reaction (PCR). The MIC90 (the minimum inhibitory concentration of an antibiotic necessary to inhibit the growth of 90% of bacterial strains) values of biapenem (BIPM), meropenem (MEPM), tazobactam/piperacillin (TAZ/PIPC), sulbactam/ cefoperazone (SBT/CPZ), cefepime (CFPM), ciprofloxacin (CPFX), pazufloxacin (PZFX), amikacin (AMK) and aztreonam (AZT) were found to be 265, 512, 256, 512, 512, 64, 128, 128 and 128 microg/mL, respectively. The in vitro combination effects of antibacterial agents were examined against 62 strains of MDRP and the synergy or additive effects were evaluated by fractional inhibitory concentration (FIC) index calculated by the checkerboard method. The combination of AMK and AZT showed synergy effects on 15/59 (25.4%) strains of MDRP. The synergy and additive effects on the MDRP strains were also found by the other antibacterial agents combination such as TAZ/PIPC and AMK, CFPM and AMK, and SBT/CPZ and AZT. These results suggested the necessity of further investigation of clinical usefulness. PMID- 16673579 TI - [Antimicrobial susceptibilities of organisms isolated from patients with urinary tract infections in 2002]. AB - Using the agar dilution method, the antibacterial activity of 18 antibiotics inclusive of 4 carbapenems were investigated against 101 strains of urinary pathogens isolated from patients with urinary tract infections who visited the Department of Urology at Kagoshima University Hospital, between January and December 2002. 4 strains of Staphylococcus aureus, 3 strains of Staphylococcus spp. (exclusive of S. aureus), 14 strains of Enterococcus faecalis, 3 strains of Enterococcus spp. (exclusive of E. faecalis), 41 strains of Escherichia coli, 21 strains of Enterobacteriaceae (exclusive of E. coli), 12 strains of Pseudomonas aeruginosa and 3 strains of glucose-nonfermentative Gram-negative rods (exclusive of P. aeruginosa) were examined. 1. Against clinical isolates of Gram-positive bacteria, vancomycin and teicoplanin were active. Additionally, arbekacin was active against S. aureus clinical isolates and ampicillin was active against E. faecalis clinical isolates. Carbapenems were active against clinical isolates of Gram-positive bacteria, except for multi-drug resistant strains of Gram-positive bacteria, such as methicillin-resistant S. aureus. 2. As for clinical isolates of Gram-negative bacteria, meropenem was most active against Enterobacteriaceae among 13 antibiotics tested. Against P. aeruginosa clinical isolates, MIC90 of meropenem was the lowest among 13 antibiotics tested. In addition, resistant rate of meropenem and biapenem against P. aeruginosa clinical isolates was lower than those of the other carbapenems tested. 3. As main urinary pathogens showed no remarkable increase in resistance to carbapenems, it can be stated that carbapenems retain their position as the drug of first choice for severe infection. PMID- 16673580 TI - [Clinical effect of ceftriaxone and cefditoren administration against oral and genital gonococcal infection]. AB - The clinical findings of genococcal infection (oral and genital) and the clinical effect of ceftiaxone (CTRX) and cefditoren (CDTR) administration were studied in Commercial Sex Workers (CSW). The gonococci were detected by DNA probe method (mouth), PCR method (genitals) and ELISA method (chlamydial antibody detection). 1) In the oral infection group (n = 20), chlamydial infection (65%), herpes infection (25%), and genital gonococcal infection (35%) were noted. Pharyngeal pain was observed in 9 out of 9 patients with tonsillitis and 4 out of 11 patients with pharyngitis. High fever and cervical lymphadenopathy were observed in 3 out of 9 patients with tonsillitis. 40% (8/20) of the partners had infections. Both CTRX administration (1 - 2 g/day x 3 days) (n = 11) and consecutive administration of CDTR (300 mg/day x 3 - 7 days) following CTRX administration (1 - 2 g/days x 1 - 3 days) (n = 9) were effective in all patients. 2) In the genital infection group (n = 35), chlamydial infection (65.7%), herpes infection (25.7%) and oral gonococcal infection (17.1%) were observed in 3 out of 6 patients with tonsillitis and 3 out of 6 patients with pharyngitis. The treatment was effective in all patients in the CTRX (1 - 2 g/day x 2 - 3 days) group (n = 14), CDTR (300 mg/day x 5 - 7 days) group (n = 5) and consecutive administration of CDTR (300 mg/day x 3 - 7 days) after CTRX (1 - 2 g/day x 1 - 3 days) and (n = 14). In pelvic peritonitis (n = 2), CTRX administration (2 - 4 g/day x 3 - 7 days) were effective. PMID- 16673581 TI - [Prevalence and treatment of pharyngeal Chlamydia trachomatis infections]. AB - In recent years, the number of the patients with pharyngeal infection caused by Chlamydia trachomatis is believed to be on the rise due to diversification in sexual behaviors. In addition, pharyngeal infection by C. trachomatis is often asymptomatic, and this is also believed to be a major factor for the increase of the disease. In this study, we conducted a survey among general females and commercial sex workers (CSWs) to study their sexual behavior and prevalence of chlamydial infections (in uterine cervix and pharynx). The results showed that orogenital contact has become a common act, even for general females. Chlamydial infections of uterine cervix were found in 33.3% and 7.9% of CSWs and general females, respectively. Chlamydial infections of pharynx were found in 22.5% and 5.2% of CSWs and general females, respectively. The evaluation of treatments of these infections with clarithromycin, levofloxacin, and azithromycin showed that 7, 10, and 14 days administrations of 400 mg clarithromycin, 7, 10, and 14 days administrations of 300 mg levofloxacin, and a single dose of 1000 mg of azithromycin, would eradicate 100% of C. trachomatis for infections of uterine cervix. For pharyngeal infections, 10 and 14 days administrations of clarithromycin and levofloxacin were shown to eradicate 100% of C. trachomatis. However, the eradication rates for 7 days administrations of clarithromycin and levofloxacin were 83.9% and 86.2%, respectively, and the rate for a single dose of azithromycin was 85.0%. From these results, it was thought that more than 10 days of administrations of clarithromycin or fluoroquinolone antibacterial agents such as levofloxacin are necessary to treat pharyngeal chlamydial infection. Clinical significance of pharyngeal chlamydial infection is still not clear; however, this study have shown the need for more detailed investigations using culture assay, in corporation with doctors in otolaryngology and internal medicine. PMID- 16673582 TI - [Mutations of DNA gyrase and topoisomerase IV in clinical isolates of fluoroquinolone-resistant Proteus mirabilis]. AB - The presence of fluoroquinolone resistance-associated mutations within the quinolone resistance-determining region of DNA gyrase and topoisomerase IV was investigated genetically in clinical isolates of Proteus mirabilis recovered from patients with urinay tract infections. Two isolates of fluoroquinolone-resistant P. mirabilis possessed the mutations in GyrA (Ser-83 --> Arg or Ile), GyrB (Ser 464 --> Tyr or Phe) and ParC (Ser-80 --> Ile). A novel mutation with Glu-87 --> Lys in GyrA, where suggested to be responsible for fluoroquinolone resistance, was identified. These results demonstrate that the presence of an additional mutation at Glu-87 in GyrA may contribute to high-level fluoroquinolone resistance, too. PMID- 16673583 TI - [Studies of anthelmintic and insecticide antibiotics]. PMID- 16673584 TI - [Structure-based drug design of the irreversible inhibitors of metallo-beta lactamases]. PMID- 16673585 TI - [Role of alveolar macrophages in the innate immunity in the lung]. PMID- 16673586 TI - Prostate cancer--management of disseminated disease (intermittent androgen suppression). PMID- 16673587 TI - Clinical correlation, ecographic and levels of prostate specific antigen in patients with prostate cancer. AB - PURPOSE: This report provides information about clinical correlation, ecographic and levels of prostate specific antigen in patients with prostate cancer in the Regional Hospital from Trujillo, Peru. MATERIALS AND METHODS: Retrospective, descriptive observation study of all the clinical histories from patients with histopathology diagnosis of prostate cancer during the period of January 2000 - December 2004. The sample was constituted by 60 patients with clinics histories, study by image based on the prostate ecography and the prostate specific antigen results. The average of age found was of 75.4 years old; the most frequent clinical case reported a 91.6% of patients with obstructive and irritative symptoms. In the levels of prostate antigen the PSA found values between 10-50 ng/ml, in 48.78% of the patients. In the ecography prostate study there where evidence of hypoecogenic images in 56%, hyperecogenics images 12%, heterogenics images 20% and 12% homogenics images. In the TNM classification the higher percentage is located in T stage with an 81.67%, and in smaller proportions in the N1 and M1 stages with 8.33% and 10.0% respectively. According to Gleason it was found levels from 2-4 with 36.67%, in the level 5-7 with 23.33% and in the level 8-10 with 40.0%. CONCLUSIONS: All the patients with prostate cancer showed a clinical symptoms about, obstructive and irritative and in other organs a less percentage of symptoms that shows metastases; in more than a half of the patients, the digital rectal examination showed nodules and irregularities in its surface; in the prostate ecography it is report a higher percentage of hypoecogenics images suggestive of neoplasia. The levels of specific prostate antigen in more than 70% of the patients where over of 10 ng/ml; in the totally of the patients the histological type most frequent is the Adenocarcinoma, with a high grade of percentage of patients who had been bad prognosticated according to the Gleason score. According the NTM, more of the two thirds parts of patients with prostate cancer are in T stage, with a high percentage extending on the prostatic capsule. PMID- 16673588 TI - Screening for prostate cancer--how to manage in 2006? AB - National Societies usually recommend screening for Prostate Cancer (PC) with Serum Prostate Specific Antigen (PSA) and digital rectal examination annually beginning at age 50. In high risk population including men with a family history of PC or African population screening should start at age of 45 years. PSA has been widely used to detect PC despite the fact that PSA is not specific for PC. Over the years serum PSA level of greater than 4.0 ng/ml was considered the treshold to perform prostate biopsy, searching for PC. In 2005 the Prostate Cancer Prevention Trial (PCPT) demonstrated that the cut-off of 4.0 ng/ml for PSA is not anymore adapted due to the fact that this survey found in 15% of men with PSA < or = 4.0 ng/ml a prostate cancer on sextant biopsies. Today the value of PSA and the cut-off for Prostate biopsy is questionned suggesting that PSA level higher than 2.6 ng/ml must be the case to propose Prostate Biopsy. Catalona confirms that approximately 25% to 30% of men with PSA 2.6 to 4.0 ng/ml have prostate cancer. Schroder and Gosselaar assert that screening for PC at low PSA levels (< 4.0 ng/ml) risks to detect clinically insignificant cancers which are no threat to man. So far in the year 2006 screening for PC demonstrates accumulating evidences of efficacy but persistent uncertainty. The major question for an urologist at work when facing a young men searching early diagnosis of PC is: at which level of PSA do we have to perform rectal biopsy? PMID- 16673589 TI - Ultrasound guided biopsy, a gold standard diagnostical test of the prostate cancer. AB - The author discusses preparations for ultrasound guided prostate biopsy, its technique conditions and the process of performing a biopsy. Every author proposes the use of preoperative antibiotics based prophylaxis. Differences may be found in the type, dosage and the time span of preoperative application. For anaesthesia mostly lidocaine was proposed, which may be a gel applied in the rectum or used in the form a prostate infiltrate. The widest debate goes on in respect of defining the number of biopsies needed. Recently 8 or rather 10 samples are proposed to be taken. Twelve biopsies do offer an advantage compared to 6 although in case of 8 this isn't so. According to the site of sample taking the apex, the base and the middle part are proposed. In case of a palpable nodule or any lesion, made visible by TRUS an additional, targeted, biopsy has to be performed. Certain new techniques like the 3D Doppler, contrast, intermittent and others shall also be presented. A repeated biopsy shall be necessary in case of PIN atypia, beyond that the author also discusses other indications for a repeated biopsy. We may expect the occurrence of direct postoperative complications and it is necessary to know how to treat these. PMID- 16673590 TI - Clinically significant and non significant prostate cancer an ongoing question. AB - One of the most important problems in urological practice is how to differentiate clinically significant and non significant prostate cancer (Pca) i.e. how to avoid over treatment of tumors with low malignant potential in one hand, and inappropriate less aggressive treatment of significant tumors, on the other hand. At the first place, one should estimate precise local clinical stage and the grade of the disease. Transrectal ultrasound--guided prostate biopsy id the golden standard, but there are few dilemmas concerning prostate biopsy: the number of biopsy cores, inter and intra-observer variations in the grading, the significance of PIN, multifocal character of Pca etc. Our opinion is that sextant or octan biopsy is quite sufficient for the exact detection of clinically insignificant cancers. An additional problem is the discrepancy in grade between biopsy and radical prosatectomy specimen. Second, the treatment should not be the same for every patient and it is guided by the age and general condition of the patient. The aggressive treatment is recommendable for younger patients, younger than 70-72 years, even for tiny area of cancer in one of the biopsy samples. On the other hand, it is an ethical question, should we insist on detection of small cancer foci at older patients, and make them anxious and unhappy in their last years of life. PMID- 16673591 TI - Combination therapy-permanent interstitial brachytherapy and external beam radiotherapy for patients with localized prostate cancer. AB - BACKGROUND: Combination of permanent interstitial brachytherapy based on radioactive iodine with external beam radiotherapy is an alternative to other treatment modalities, such as radical prostatectomy or external beam radiotherapy alone in patients with intermediate-risk localized prostate cancer. In this article we report our experience with this combination modality. METHODS: Among patients who were treated in our institute with brachytherapy, there were 64 patients who received combination therapy for the above indication. Combination therapy enables administration of 110 Gy as brachytherapy and thereafter 45 Gy as external beam radiation. All patients received adjuvant androgen deprivation therapy for 6 months. The prospective follow-up was done with the aid of validated evaluation instruments (questionnaires). RESULTS: Combination therapy was administered without additional urinary (IPSS-based) or sexual (IIEF-based) side effects above those with brachytherapy alone. No severe perianal and lower intestinal tract side effects were observed. Short-to-moderate-term results based on serum PSA levels are encouraging, and are not inferior to what is accepted by the literature for other radical therapies. CONCLUSION: Combination of permanent interstitial brachytherapy and external beam radiotherapy in the appropriate patients does not cause any additional morbidity, and its biochemical results justify its application. This modality should be offered as an accepted and good alternative to other radical treatment options, to men with prostate cancer with moderate risk factors. PMID- 16673592 TI - Radical retropubic prostatectomy for prostate carcinoma; first results of the Clinic of Urology in Skopje. AB - OBJECTIVES: The aim of this retrospective study is to present our experience and results in the management of prostate carcinoma, with radical retropubic prostatectomy, for a period of seven years. MATERIAL AND METHODS: From December 1997 to April 2005, 61 radical retropubic prostatectomies for prostate carcinoma were performed at the Clinic of Urology in Skopje. Mean age of the treated patients was 66.4 years. Mean serum PSA level was 32.75 ng/ml. None of the patients had distant or bone metastases. Mean operative time was 160 minutes and from 2 to 4 units of blood were transfused intra and postoperatively. Mean follow up time was 39 months. RESULTS: In all of 61 patients, the RRP was performed for adenocarcinoma of the prostate. The pathological findings postoperatively showed the following pTNM grade: pT2a in 8, pT2b in 10, pT3a in 10, pT3b in 27and pT4 in 6 patients. Positive lymph nodes were found in 14 cases. Intraoperative complications occurred in 6 patients. Early postoperative complications were seen in 12 patients. Urine leakage was seen in 2 patients, incontinence (day and night) in 8 and pulmonary embolia in 2 patients. Late postoperative complications occurred in 11 patients. Stenosis of the vesicourethral anasthomosis was seen in 3 patients and incontinence (during the night only) in 8 patients. The rate of potency was not evaluated but in the last 30 cases we insisted on preservation of the neurovascular bundles in the cases that it was possible. CONCLUSION: Radical retropubic prostatectomy is the method of choice and the golden standard for treatment of organ confined prostate carcinoma in patients with long life expectancy, no neither local nor distant metastases and good overall status. With this technique complication rates are minimal, the cure rate is very big and the patients have high quality of life. The experience of the surgeon is very important since the learning curve is crucial for diminishing operative time, postoperative complications and blood transfusions. PMID- 16673593 TI - Standardization of transrectal ultrasound-guided biopsy. AB - Ultrasound-guided biopsy is more sensitive to biopsy performed under the digital control, because 29% of prostatic cancers are not palpable. On the other hand, at least 30% of cancers are isoechogenic, so they cannot be viewed by transrectal ultrasound examination. It means that target biopsy is not sufficient for diagnosis of localized prostatic cancer, i.e., randomized samples are needed as well. More than ten years ago, the technique of sampling the six specimens became a standard procedure to which previously harvested target specimens from suspected growths were added. Today, the expansion of biopsy protocol is recommended, by obtaining the additional specimens from peripheral lateral area, four plus two samples if the prostate has volume over 50 ml. Larger number of biopsies requires anesthesia. In order to reduce complication, the cleaning of rectal ampulla and prophylactic use of quinolone are suggested. PMID- 16673594 TI - Correlation of high-molecular cytokeratin in tissue of prostatic cancer with Gleason score and PSA. AB - The absence of basal cell layer of prostatic acini containing high-molecular cytokeratin, which is immunohistochemically detected by monoclonal antibody 34betaE12, is an essential diagnostic characteristic of prostatic cancer. The absence of immunohistochemical reaction in 3 or more pseudoglandular structures of prostatic tissue indicates malignant process. The percentage of immunohistochemically completely negative glandular structures was determined by semiquantitative measurement in tissue specimens obtained by TRUS biopsy of the prostate, and it was correlated with serum PSA concentration and Gleason score. The increase of percentage of glandular prostatic formations completely negative to high-molecular cytokeratin detected by 34betaE12 led to simultaneous rise of mean value of Gleason prostatic cancer score (p < 0.001) as well as the average serum PSA concentration in subjects (p < 0.05). PMID- 16673596 TI - Radical retropubic prostatectomy--results in 127 surgically treated patients. AB - Radical prostatectomy represents an optimal therapeutic method in treatment of the localized prostatic carcinoma. It may be performed using retropubic, perineal, transcoccygeal or laparoscopic approach. In a multicenter study, the authors have analyzed 127 patients surgically treated in the period 1992 - 2003. All the patients were preoperatively diagnosed with the localized prostatic carcinoma. The patients were operated at the Clinic of Urology in Belgrade (92 patients) and other departments of urology in Serbia (35 patients). The youngest patient was 49 while the oldest one was 75 (mean age 64 years). The surgical procedure duration ranged between 60 and 120 minutes. Over the last five years, the need for blood transfusion was below 50%. All the patients underwent retropubic radical prostatectomy. PMID- 16673595 TI - An evaluation of high-doses ketoconazole with hydrocortisone substitution in hormone-refractory prostate cancer. AB - We investigated the efficacy of ketoconazole, an inhibitor of testicular and adrenal biosynthesis, for treating patients with progression of hormone refractory prostate cancer. The study comprised 35 patients with progressive disease despite salvage treatment with estramustine with or without vinblastine. Treatment consisted high-doses ketoconazole (400 mg three times daily) and hydrocortisone substitution. Patients were monitored clinically and with serial PSA measurements every 3 months. The principal endpoint of the study was PSA response to applied therapy. Of the 35 patients, 18 (51.4%) showed a decrease in PSA 50% with a median duration of 30 weeks (range 6-60 weeks). A PSA reduction 50% was seen in 15 of 31 patients (48.4%) with established metastasis. Twelve patients (34.2%), all of whom had metastasis, exhibited a PSA decrease 80% with median duration of 9 months (range 3-48 months). The median time to progression was 6.3 months (range 0-27 months) and the median survival time was 12.5 months (range 3-48 months). Twelve (34.3%) reported toxicity related to ketoconazole, whereas no patients required discontinuation of therapy. It is apparent from this study that a reasonable percentage of patients failing salvage chemotherapy (estramustine with or without vinblastine) respond favorably to high-dose ketoconazole and that toxicity is mild. In the absence of studies demonstrating better survival with chemotherapy, we believe that a trail of ketoconazole should be considered when progression of PSA occurs, following initial hormonal androgen deprivation. PMID- 16673597 TI - Determination of biochemical relapse after radical prostatectomy by biological detection limit (BDL) after cystoprostatectomy using two PSA assays. AB - The monitoring of PSA values following prostatectomy demands for the use of highly sensitive tests with low detection level. The possibilities to use the EIA Dialab test to monitor the PSA values after radical prostatectomy for early detection of persistent diseases were investigated by determining the biological detection limit (BDL) in serum of patients who unrewent cystoprostatectomy. The obtained values were compared with Abbott Imx test for PSA determination. A good correlation between the two studied methods was establisahed, r = 0.9827 with the regression curve Yx = 0.20463 + 0.96277. Test indicated that there was no significant difference (p < 0.001) between the investigated methods. PMID- 16673598 TI - The nerve sparing radical prostatectomy--a step to better quality of life surgical aspects. AB - Radical surgical procedure (Radical Retropubic Prostatectomy) is treatment of choice for patients with localized (T1, T2) prostatic cancer. By radiologists, radical radiotherapy (brachi and external beam or only brachi therapy) could, also, be rational radical option for T1, T2. But, long-term survival is better after surgical procedure which is confirmed by many authors. Nerve sparing radical prostatectomy should be considered as a step to better life in all cases where it could be done. With this presentation we would like to point out the critical moments (the places of our-surgical mistakes), where Neurovascular Bundle (NVB) could be injured during the procedure (RRP). If those injures could be avoided urologist would be able to enhance the quality of life of these patients. The possible injures of NVB may happen during: 1. Dissection of the posterior wall of urethra. 2. Separation-dissection of external urethral sphincter. 3. Putting of sutures on urethra. 4. Preparation of the posterior side of prostate. 5. Dissection of the seminal vesicles. CONCLUSION: Careful surgical work of experienced urologist concerning all surgical tips mentioned above will result in an excellent continence (100%),preserved potency (more than 70%), and better quality of life for patients with localized prostate cancer. PMID- 16673599 TI - Urinary prostate specific antigen: is the clinical use likely? AB - Prostate specific antigen (PSA) blood test represents the standard procedure in prostate cancer (CaP) diagnosis and follow-up. However, determination of PSA in the urine, where PSA is present in much higher concentrations than in the blood, still remains in the field of research. OBJECTIVES: To determine urinary concentrations of PSA (uPSA) in different groups of patients (pts.), and to estimate is it possible to differentiate benign and malignant prostate diseases and to follow-up the results of treatment. METHODS: Between January 2001. and November 2003., urinary concentrations of PSA were determined at 142 pts. divided in seven groups: 1. young and healthy volunteers, 2. "BPH-24": pts. with benign prostatic hyperplasia (BPH) who collected the sample of 24-hour voided urine, 3. "BPH-I": pts. with BPH who collected the first portion of first urinary voiding, 4. "TRUS-CaP": pts. with CaP which gave the first portion of urine just prior to transrectal ultrasound-guided prostate biopsy (TRUS- biopsy), 5. "TRUS-non-CaP": pts. who gave first portion of urine prior to TRUS-biopsy, but biopsy did not prove the presence of CaP, 6. "RRP": pts. who underwent radical retropubic prostatectomy (RRP), 7. "AAT": pts. who underwent androgen deprivation therapy. RESULTS: Average uPSA value in the group of young and healthy volunteers, was 13.8 +/- 19.6 ng/ml, in "BPH-24": 38.0 +/- 44.4 ng/ml, in "BPH-I": 140.8 +/- 140.9 ng/ml, in "TRUS-CaP": 234.8 +/- 277.7 ng/ml, in TRUS-non-CaP: 113.1 +/- 148.5 ng/ml, and in the group "RRP": 4.4 +/- 4.7 ng/ml. There was no statistically significant difference of average uPSA values between "BPH-I" and "TRUS-CaP" groups. The significant difference was found between the group of young volunteers and "BPH-I". In "TRUS-CaP" group, there was strong correlation between tumour size and aggressivenes and uPSA concentration. Finally, PSA and uPSA decline during androgen deprivation therapy, strongly correlated (up to r = 0.95). CONCLUSIONS: Determination of uPSA cannot differentiate BPH and CaP. However, in the group of pts. with proven localized CaP, uPSA can provide additional information concerning T-staging. Moreover, simultaneous monitoring of PSA and uPSA response on hormonal therapy, can provide an early recognition of androgen-indiferent CaP (AIPCA) and hormone-resistent CaP (HRPCA). PMID- 16673600 TI - Radical retropubic prostatectomy--report on 41 cases. AB - Anatomic radical retropubic prostatectomy is an effective method for long-term control of the prostatic carcinoma. In the current era of the anatomic prostatic surgery, blood loss is reduced, complication rate is low and control of the disease may be excellent. Some authors reported cancer-specific survival rate after the radical retropubic prostatectomy of 85-90% over the 10-year period and 82% over the 15-year period. The survival may be more favorable in the patients with lower Gleason scores. The former indicates that radical retropubic prostatectomy is an excellent therapeutic option for treatment of the localized prostatic carcinoma, while selection of patients is a crucial factor for true success of therapy based on this method. PMID- 16673601 TI - The possibilities of age reference values of PSA density in prostatic cancer screening. AB - INTRODUCTION: The possibilities of PSA (prostate specific antigen) test in screening of prostatic cancer may be evaluated by measuring its sensitivity, specificity and positive predictive value. The majority of reference articles in this field has evaluated the possibility of detection of prostatic cancer by radioimmunoassay (RIA). MATERIAL AND METHODS: Prospective study included 2000 male patients over 50 years of age. The objective of the study was to examine the possibility of enzyme PSA test for differentiation of benign prostatic hyperplasia (BPH) and localized prostatic cancer (PC). The possibility of enzyme PSA assay for detection of prostatic cancer was compared with RIA method, by digital rectal examination and echotomography. The effect of PSA density and age reference values for differentiation of PC from BPH was also examined. DISCUSSION: The results of enzyme immunoassay test (EIA) were compatible with radioimmunoassay results (RIA). Diagnostic algorithm of prostatic cancer detection should be corrected with PSA density values according to aged-specific groups (49 years - 0.09 ng/ml/cm3; 59 years - 0.13 ng/ml/cm3; 69 years - 0.17 ng/ml/cm3; 79 years - 0.19 ng/ml/cm3) in order to reduce the number of false positive results in PSAD grey zone (0.10 - 0.20). PMID- 16673602 TI - The predictive value of PSA in diagnosis of prostate cancer in non screened population. AB - INTRODUCTION: [corrected] PSA is the most important tumor marker in all solid tumor, indispensable in the management of prostate cancer. Screening for prostate cancer is still not recomended, although performed in many countries, which introduced questions about the usefulnes of PSA in detection of prostate cancer. The PSA treshold has also been changed, the value of PSA derivatives revised. Whether such changes are applicable in non scrrened population is questionable. Aim of this study was to evaluate the predictive value of PSA, free/ total PSA and PSA density in our non screened population. PATIENTS AND METHODS: TRUS guided prostate biopsy was performed in 579 patients. The number of cores was 6-12. Mean age of the patients was 67.5 years (30-90). PSA was ranging from 0.41 to 2250 (mean 38.6 ng/ml, median: 11.95, SD 140,45). Digitorectal examination was considered positive in 351 patients. Free PSA was measured in 352 patients with the index ranging from 0.02 to 0.88 (mean free/total PSA: 0.14, median: 0.13). The volume of the prostate was measured in all patients according the prostate ellipsoid model, and PSA density calculated according to the formula PSA/PV. Patients were stratified in 6 groups according to PSA value (I: PSA ng/ml, II: PSA 2.5-4, III: PSA 4-10, IV: PSA 10-20, V: PSA: 20 to 50, Group 6: PSA 50). RESULTS: Non homogenicity of the patients can be seen through the wide range of PSA which was from 0.4 to 2025). Prostate cancer was diagnosed in 233 pts (40.2%). As expected, the probability of detecting cancer was raised with PSA (p), and was extremely rare in pts with PSA below 4 ng/ml. PSA, free/total PSA, volume of the prostate and PSA density were significantly different according to the presence of cancer. Most of our patients had PSA between 4 and 20 ng/ml. Predictive value of PSA was 20.6% for pts with PSA from 4 to 10 and 32.7% for those with PSA from 10 to 20 ng/ml. Sensitivity, specificity, positive and negative predictive values for different cut off's of PSA (4, 10 and 20) was performed. The best results were obtained for PSA cut off of 10 ng/ml. In the group of patient with PSA, PSA density more reliable than free/total PSA index. CONCLUSION: PSA is still valuable marker for detection of prostate cancer in our non screened population. According to our results PSA treshold should not be lowered below 4 ng/ml. PSA density is a reliable PSA derivative, free/total PSA index having less importance in pts with PSA below 20 ng/ml. PMID- 16673603 TI - New generation urethral stents in treatment bladder outlet obstruction caused by prostate cancer. AB - Our clinical trial included until now, 22 patients in whom new generation urethral stent named Allium, were inserted due to bladder outlet obstruction caused in 7 patients (pt) with benign prostate hyperplasia, in 13 pt with bulbar urethral stricture of different ethiology and in 2 pt with prostate cancer. Allium prostatic stents, designed by Daniel Yachia differs in some crucial characteristics from previously used stents: they are covered for the first time in urethra stenting history, without relatively low radiation force and because of that nonirritative. The indications, contraindications and preliminary results in this study are discussed concerning the patients with cancer of the prostate. PMID- 16673604 TI - Radiotherapy in prostate cancer treatment. AB - Prostate cancer is a complex disease, with many controversial aspects of management in almost all stages of disease. The natural history of this tumor is variable and is influenced by multiple prognostic factors. Radical prostatectomy and radiotherapy are standard treatment options for disease limited to the prostate. The data in literature does not provide clear- cut evidence for the superiority of any treatment. Neo- adjuvant or adjuvant hormonal therapy improves local control and survival in locally advanced disease. The patients treated with radiotherapy would have a relatively long life expectancy, not great risk factors for radiation toxicity and a preference for radiotherapy. The advantages of radiotherapy are that it has a significant potential for cure, it is well tolerated in the majority of men especially when the modern techniques of conformal radiotherapy and intensity modulated therapy are used and it is non invasive therapeutic options with no anesthesia risk. Expected complications like radiation cystitis, impotence and proctitis are registered in about 1% of patients. PMID- 16673605 TI - Radical radiotherapy for localized prostate cancer in elderly. AB - BACKGROUND: Prostate cancer is an age related neoplasm, with high incidence in the group of elderly man. The correct management must to be balanced between the benefits of the treatment and its disadvantages. Radiotherapy as definitive treatment is commonly reserved for older patients and patients with high surgical risk and is widely used as a treatment of choice. AIM: The aim of this study was to determine the role of radical radiotherapy for localized prostate carcinoma in patients 70 years age or older concerning treatment morbidity, local control, disease free and overall survival. MATERIAL AND METHODS: A clinical prospective non-randomized study was performed including 103 elderly patients with an age 70 or above, between January 1991 and April 2005, at the Institute for Oncology and Radiology of Serbia. Median age of patients was 74,89 years (range 70- 80 years). Stage distribution was as follows: stage A--3 patients (2.9%), stage B--69 patients (67%) and stage C--31 patients (30.1%). Out of 103 patients, initial PSA value was noted in 87 patients. The mean value of initial PSA was 18,06 ng/ml. Radical radiotherapy was conducted on megavoltage linear accelerators with high energy photons (10, 18 MeV) and total tumor dose of 65 Gy. RESULTS: Low grade acute complications were registered in 70 patients (65%). Mean follow up time was 40, 13 months. The disease outcome at the last follow up show that 79 patients (76.7%) had no evidence of disease and 24 patients (23.3%) relapsed. Overall survival rates were 65, 29% and 44, 52% and disease free survival 66, 59% and 63, 26% at 5 and 10 years. Disease specific survival was at 5 and 10 years 73,32% and 65, 42% respectively. Late sequelas (gradus I and II) are registered in 22 patients (21.36%), out of 103. CONCLUSION: Radical radiotherapy for localized carcinoma of the prostate is effective treatment option in elderly patients with good local control, present treatment tolerance providing good quality of life and long-term cure. PMID- 16673606 TI - Histopathological evaluation of radical prostatectomy in the treatment of localized prostate cancer. AB - Incidence of prostate cancer has risen dramatically in the past decade. Radical prostatectomy is indicated in patients who have disease localized to the prostate. The aim of the study is to make histopathological evaluation of radical prostatectomy in the treatment local prostate cancer. Authors analyzed 49 cases of radical prostatectomy due to cancer localized to the prostate in period 1996 2000 in Clinic of Urology in Clinical Center of Serbia, Belgrade. The average age of the patients was 65, 6 years (range 44-76, pick 61-70). The most cases 25 (51%, p < 0.001) we found in pT2a N0M0, in pT2b N1M0 9 (18.36%), in pT3bN0M0 10 (20.4%), in pT3bN1M0 3 (6.12%), in pT4aN0M0 2 (4.08%). Nodal status positive was in 12 cases: 9 (18%) in pT2bN1M0- iliac 3 (right 2, left 1), obturatory 6 (right 1, left 5) and 3 cases in pT3bN1M0-iliac left 1 and obturatory 2 (1 right and 1 left). We found Gleason score 8 in 9 cases (18.36%) in pT2bN1M0 versus 7 cases (14.5%) without nodal metastases. Gleason score 9 we found in 3 cases (6.1%) in pT3bN1M0 versus one case without nodal metastases (difference is not significant). Gleason score 3 was in 6.1%, 4 in 12.2%, 5 in 8.1%, 6 in 16, 3%, 7 in 24.5%. Grade 1 of tumors we found in 9 cases (18%), grade 2 in 11 (22%), grade 3 in 29 (60%). HG PIN was in 18 cases (36.7%), LG PIN in 10 (20.4%). In all cases was elevated PSA: 4-10 mmol/L in 24 pts, 11-20 in 15 pts and > 20 in 10 pts. Radical prostatectomy is most adequate method in surgical treatment cancer localized in the prostate. Pelvic lymphadenectomy is necessary for staging purposes in adenocarcinoma of the prostate. Early detection adenocarcinoma of the prostate is important factor in decreasing rate of death. PMID- 16673607 TI - Anesthesia for radical prostatectomy. AB - Radical prostatectomy is one of most common treatment options currently recommended for clinically localized prostate cancer. Evaluation of intraoperative and postoperative complications is important in evaluation of relative morbidity of this treatment option. Furthermore, investigation of complications of surgical treatment in correlation with not only surgical technique, but comorbidity, ASA stage and anesthetic technique enables improvements in complete perioperative treatment and decrease of incidence of complications resulting from the procedure. Improvement of anesthetic techniques and use of new anesthetic agents contributes to better outcome of surgical treatment. For radical surgery, combined epidural analgesia and general anesthesia reduces postoperative complications and mortality. Benefits can be conferred most likely by altered coagulation activation in surgery, increased blood flow, reduction of operative stress response. Modalities for reduction of intraoperative blood loss during radical prostatectomy are normovolemic haemodilution, preoperative donation of blood for autologus transfusion and use of erythropoietin for increasing red cell mass. PMID- 16673608 TI - Zometa in the treatment of diseminated prostate cancer. AB - Zoledronic acid is the first bisphosphonat that confirms the a efficacy in the treatment of bone metastases in prostate carcinoma. That is the most potent bisphosphonat up to date which safe and simple administration. Zometa is strong inhibitor of osteoclast activity. PMID- 16673609 TI - The puzzle of chronic pain: will genetics force a major breakthrough in the pathophysiology and the treatment of chronic pain? PMID- 16673610 TI - Severity of liver echogenicity is correlated to serum c-peptide levels in type 2 diabetic patients. AB - AIM: To investigate the correlation between liver echogenicity and serum C peptide levels in type 2 diabetic patients treated with oral hypoglycaemic agents. PATIENTS AND METHODS: The study included 231 type 2 diabetic patients (114 men) with a mean age of 64.3 +/- 6.9 years and a mean diabetes duration of 8.5 +/- 3.2 years. Liver echogenicity was graded by ultrasound examination as follows: Grade 0 (normal liver texture), grade 1 (slight increase of liver echogenicity), grade 2 (moderate increase of liver echogenicity with impaired visualization of intrahepatic vessels and right hemi-diaphragm), grade 3 (marked increase of liver echogenicity with very poor visualization or non-visualization of intrahepatic vessels and right hemi-diaphragm). Serum C-peptide was measured both in fasting state (Fasting C-peptide, FCP) and after glucagon administration (Glucagon-stimulated C-peptide, GCP). RESULTS: FCP (median; interquartile range) showed a significant difference (p=0.041) between patients with grade 0 (1.9 ng/dl; 1.1-2.7 ng/dl),grade 1 (2.7 ng/dl; 1.9-3.7 ng/dl), grade 2 (4.1 ng/dl; 2.6 5.1 ng/dl) and grade 3 (6.2 ng/dl; 4.6-7.5 ng/dl) liver echogenicity. GCP (median; interquartile range) also differed significantly (p=0.04) between patients with grade 0 (2.6 ng/dl; 1.8-3.3 ng/dl), grade 1 (4.3 ng/dl; 3.3-5 ng/dl), grade 2 (5.8 ng/dl; 4.6-6.9 ng/dl) and grade 3 (8.3 ng/dl; 6.6-9.5 ng/dl) liver echogenicity. In multiple regression analysis, both FCP and GCP showed significant (p < 0.05) positive correlations with waist circumference, triglycerides, WHR and liver echogenicity. CONCLUSIONS: In type 2 diabetic patients treated with oral hypoglycaemic agents, liver echogenicity shows a significant positive correlation with serum C-peptide levels, both in fasting state and after glucagon administration. PMID- 16673611 TI - Recommendations for antibiotic prophylaxis before ERCP: can we come to workable conclusions after review of the literature? AB - Infectious complications after endoscopic retrograde cholangiopancreatography (ERCP) are rare and can mainly be divided into endocarditis and sepsis/cholangitis. There still remains uncertainty about the indications, antibiotic regimens, and timing for antibiotic prophylaxis. Several guidelines for antibiotic prophylaxis have been published. It is the purpose of the present paper to make an extensive review of the literature and to yield general recommendations on antibiotic prophylaxis before ERCP. Antibiotic prophylaxis is recommended for patients at high risk for endocarditis, as well as for patients with a vascular graft less than 1 year old. For sepsis/cholangitis, prophylaxis is mostly recommended for patients with a bile duct obstruction or with a pancreatic pseudocyst. For other conditions, a case-by-case evaluation by the physician/endoscopist is still inevitable. PMID- 16673612 TI - [Treatment of iatrogenic femoral pseudoaneurysms by ultrasound-guided percutaneous thrombin injection: effectiveness and complications]. AB - AIM: We retrospectively assessed the effectiveness of ultrasound-guided percutaneous thrombin injection in the treatment of iatrogenic femoral pseudoaneurysms and registered the occurrence of complications in the systemic circulation. METHODS: We performed ultrasound-guided thrombin injection in 26 iatrogenic femoral pseudoaneurysms: 24 were classified as single en 2 as complex. We registered the volume and the pseudoaneurysm neck measurements, as well as the complication rate. RESULTS: Ultrasound-guided percutaneous thrombin injection led to a successful obliteration of pseudoaneurysm in 25 out of 26 cases (96.2%). The thrombin amount varied between 250 and 1000 IU. A thrombosis of the common femoral artery after the thrombin injection occurred only in one patient. CONCLUSION: Ultrasound-guided percutaneous thrombin injection is effective and safe in the treatment of iatrogenic femoral pseudoaneurysms provided the exclusion criteria are respected. Complications are rare. PMID- 16673613 TI - Portal vein thrombosis in a patient with HIV treated with a protease inhibitor containing regimen. AB - We report a case of an HIV seropositive female patient treated with a protease inhibitor-containing regimen who developed recurrent severe life-threathening episodes of haematemesis over time, caused by ruptured oesophageal varices as a consequence of a portal vein thrombosis. Coagulation tests revealed a protein S deficiency, an elevated homocysteinemia and a constitutional elevated plasma factor VIII coagulant activity. These coagulopathies and the HIV infection are independent risk factors for developing venous thromboembolic events. The protease inhibitor treatment may have played a role in increasing the thromboembolic risk. The recurrent bleedings only stopped after invasive surgery. The invasive splenorenal shunt operation was in this case a life-saving procedure. PMID- 16673614 TI - Cancer presenting as fatal pulmonary tumour embolism. AB - Rapidly fatal pulmonary tumour embolism is a rare complication of malignancy, and often presents as progressive dyspnea without obvious cause. We describe two cases presenting with a dramatic clinical picture of lactic acidosis and cardiopulmonary arrest soon after admission on ICU. The first patient was a 29 year old woman with a breast cancer seeming in remission who was admitted with rapidly increasing dyspnea since two weeks. The second patient was a 46-year old woman with HIV and no history of malignancy, who developed dyspnea and lactic acidosis over the course of a few days while she was investigated for an occipital brain lesion. Both patients died soon after admission and massive tumour emboli were found on autopsy. Breast cancer was the origin of the emboli in both cases. Symptoms were out of proportion to the initial physical cardiopulmonary findings and radiographic features. Clinical signs of pulmonary tumour embolism are non-specific and subacute. Prognosis is poor and definite diagnosis is usually made post-mortem. Solid malignancies such as breast cancer account for most of the cases. Pulmonary tumour embolism should be considered in critically ill patients with unexplained hypoxemia and lactic acidosis, mild or no radiological abnormalities and fast clinical deterioration. It may occur in young patients and in patients without history of malignancy. PMID- 16673615 TI - Dysphagia as a primary manifestation of hyperthyroidism: a case report. AB - Myopathy effecting mainly skeletal muscles of the limbs are frequently seen in hyperthyroidism. Rarely bulbar muscles may also be involved, causing dysphagia, nasal speech, and aspiration. We report a 70-year-old woman with severe dysphagia and aspiration pneumonia. Clinical examination and laboratory tests showed an underlying Graves' disease. Her dysphagia improved dramatically by antithyroid therapy. Considering its excellent response to medical therapy, hyperthyroidism- being a very rare factor - is well-worth to remember for the unexplained dysphagia cases. PMID- 16673617 TI - Is there any relationship between sexual attraction and gender typology? AB - People can be classified as attracted to both sexes, to men, to women, or to neither sex, and also as instrumental-expressive, instrumental, expressive, or non-instrumental-expressive. The two hypotheses tested herein are, on the one hand, the relative independence between these two typologies and, on the other, the close relation between sexual dimorphism and sexual attraction, in contrast to the relative independence between sexual dimorphism and the instrumental and expressive domains. A total of 503 university students (284 women and 219 men) completed two assessment instruments: The Sexual Attraction Questionnaire (SAQ) and the Bem Sex Role Inventory (BSRI, 12 items). Analysis of contingency tables was performed. The results provide empirical support for the hypothesis of independence of the two typologies, solid support for the relation between sexual dimorphism and sexual attraction, and clear support for the independence between the gender domains and sexual dimorphism. The implications of these data for the different outlooks concerning the relations between sex and gender are established. PMID- 16673616 TI - Indoxyl sulphate and the purple urine bag syndrome: a case report. AB - A case of purple urine bag syndrome, a rare condition in which the urinary catheter bag of chronically catheterised patients develops a discolouration, is reported. The excretion of indoxyl sulphate, an intermediate in the causal mechanism of this unusual phenomenon, was measured using Ehrlich's reagent and found not to be elevated in this 77 year-old man, when compared to elderly male control subjects. PMID- 16673618 TI - Affective priming with pictures of emotional scenes: the role of perceptual similarity and category relatedness. AB - Prime pictures portraying pleasant or unpleasant scenes were briefly presented (150-ms display; SOAs of 300 or 800 ms), followed by probe pictures either congruent or incongruent in emotional valence. In an evaluative decision task, participants responded whether the probe was emotionally positive or negative. Affective priming was reflected in shorter response latencies for congruent than for incongruent prime-probe pairs. Although this effect was enhanced by perceptual similarity between the prime and the probe, it also occurred for probes that were physically different, and the effect generalized across semantic categories (animals vs. people). It is concluded that affective priming is a genuine phenomenon, in that it occurs as a function of stimulus emotional content, in the absence of both perceptual similarity and semantic category relatedness between the prime and the probe. PMID- 16673619 TI - Facial-expression affective attributes and their configural correlates: components and categories. AB - The present study investigates the perception of facial expressions of emotion, and explores the relation between the configural properties of expressions and their subjective attribution. Stimuli were a male and a female series of morphed facial expressions, interpolated between prototypes of seven emotions (happiness, sadness, fear, anger, surprise and disgust, and neutral) from Ekman and Friesen (1976). Topographical properties of the stimuli were quantified using the Facial Expression Measurement (FACEM) scheme. Perceived dissimilarities between the emotional expressions were elicited using a sorting procedure and processed with multidimensional scaling. Four dimensions were retained in the reconstructed facial-expression space, with positive and negative expressions opposed along D1, while the other three dimensions were interpreted as affective attributes distinguishing clusters of expressions categorized as "Surprise-Fear," "Anger," and "Disgust." Significant relationships were found between these affective attributes and objective facial measures of the stimuli. The findings support a componential explanatory scheme for expression processing, wherein each component of a facial stimulus conveys an affective value separable from its context, rather than a categorical-gestalt scheme. The findings further suggest that configural information is closely involved in the decoding of affective attributes of facial expressions. Configural measures are also suggested as a common ground for dimensional as well as categorical perception of emotional faces. PMID- 16673620 TI - Comparing speed estimations from a moving vehicle in different traffic scenarios: absence versus presence of traffic flow. AB - The aim of this research was to study the performance in a speed estimation task of a passenger travelling in a real car in different scenarios: a closed track used in previous experimental studies was compared with interurban traffic environment involving a secondary road and a highway. At the same time, the effect of sex and driving experience on speed estimation was analyzed. Thirty-six participants (18 male and 18 female, half of each group being drivers and half non-drivers) estimated the speed of the car in which they travelled as passengers. The actual speed values varied in the range of 40-100 km/h for the secondary road, 70-120 km/h for the highway condition, and 40-120 km/h for the track. The results obtained for the track in previous studies (Recarte and Nunes, 1996; Recarte, Conchillo, and Nunes, 2004, 2005) were replicated in the same condition and were also verified for the secondary road scenario. However, a different pattern of errors was found for the highway. From the viewpoint of psychophysics, the participants were more accurate on the without-traffic track than in real traffic conditions, considered as a whole. The differences found between road and highway are discussed. No effect was found for between- subject variables, sex, and driving experience. PMID- 16673621 TI - Personality dimensions and subjective well-being. AB - This work examines the association between personality dimensions (extraversion and neuroticism) and subjective well-being. Subjective well-being is associated both with extraversion and neuroticism, and currently, neuroticism is generally considered the more important. A total of 368 students from the University of Rovira i Virgili completed the Extraversion and Neuroticism subscales of the revised Eysenck Personality Questionnaire (Eysenck, Eysenck, and Barrett, 1985), the Satisfaction with Life Scale (SWLS; Diener, Emmons, Larsen, and Griffin, 1985), and the Positive and Negative Affect Scale (Watson, Clark, and Tellegen, 1988). Regression analyses revealed the personality variable of neuroticism as one of the most important correlates of subjective well-being. Regression analyses also showed that 44% of the variance of subjective well-being was accounted for by neuroticism, whereas extraversion only explained 8% of the variance. PMID- 16673622 TI - Emotional intelligence as predictor of mental, social, and physical health in university students. AB - This study examined the association between emotional intelligence (EI), anxiety, depression, and mental, social, and physical health in university students. The sample was made up of 184 university students (38 men and 146 women). El was evaluated by the Trait Meta-Mood Scale (Salovey, Mayer, Goldman, Turvey, and Palfai, 1995), which evaluates the three dimensions (Attention, Clarity, and Mood Repair). Anxiety was evaluated with the Trait Anxiety Questionnaire (Spielberger, Gorsuch, Lushene, Vagg, and Jacobs, 1983) and depression with the Beck Depression Inventory (Beck, Rush, Shaw, and Emery, 1979). Mental, social, and physical health were evaluated with the SF-12 Health Survey (Ware, Kosinski, and Keller, 1996). Results showed that high Emotional Attention was positively and significantly related to high anxiety, depression, and to low levels of Role Emotional, Social Functioning, and Mental Health. However, high levels of emotional Clarity and Mood Repair were related to low levels of anxiety and depression, high Role Physical, Social Functioning, Mental Health, Vitality, and General Health. This study confirmed the predictive value of Attention, Clarity and Mood Repair regarding the levels of anxiety, depression, and areas related to mental, social, and physical health in university students. PMID- 16673623 TI - Memories of the terrorist attacks of September 11, 2001: a study of the consistency and phenomenal characteristics of flashbulb memories. AB - In this study, I investigated students' memories of the terrorist attacks of September 11, 2001, carried out by Al Qaeda terrorists against the World Trade Center in New York and the Pentagon in Washington. Participants completed on two occasions (2 weeks and 8 months after the events took place) a memory questionnaire that included an assessment of the phenomenal richness of their memories. The results showed that the participants remembered very well the circumstances in which they first heard about the terrorist attacks, that they were very confident about this information, and that these memories were characterized by a high phenomenal richness. Over time, there was a decrease in all of these variables, but people's ratings of phenomenology and confidence were still very high. PMID- 16673624 TI - Post-traumatic stress reactions following the March 11, 2004 terrorist attacks in a Madrid community sample: a cautionary note about the measurement of psychological trauma. AB - Posttraumatic stress reactions related to the Madrid March 11, 2004, terrorist attacks were examined in a sample of Madrid residents (N = 503) 18-25 days after the attacks, using multiple diagnostic criteria and different cut-off scores. Based on the symptoms covered by the Posttraumatic Stress Disorder Checklist Civilian (PCL-C; Weathers, Litz, Herman, Huska, and Keane, 1993), rates of probable posttraumatic stress disorder (PTSD) ranged from 3.4% to 13.3%. Taking into account additional criteria from the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 200; i.e., the impact of initial reaction and problems in daily functioning as a consequence of the traumatic event), only 1.9% of respondents reported probable PTSD. These results suggest that inferences about the impact of traumatic events on the general population are strongly influenced by the definition of traumatic response. Our findings also revealed that the magnitude of posttraumatic reactions is associated with several risk factors, including living close to the attacked locations, physical proximity to the attacks when they occurred, perception of one's life being at risk, intensity of initial emotional reactions, and being a daily user of the attacked train lines. The use of different cut-off scores did not affect the pattern of risk to develop traumatic stress. The implications of these results for public health policies related to terrorist attacks are discussed. PMID- 16673625 TI - Diabetes mellitus as a model of psychosomatic and somatopsychic interrelationships. AB - The article reviews research on the problem of interrelationship between different physical and psychosocial factors in type 1 diabetes mellitus (DM1). The authors consider methodological principles of health-related quality of life (HRQoL) assessment in DM1 patients and stress the need for an integrated biopsychosocial approach to the management of the disease. DM1 is a chronic metabolic disease with an absolute requirement for insulin replacement therapy. The stress-inducing nature of DM1 is associated with its unexpected and dramatic manifestation in juvenile years, life-threatening nature of severe hypo /hyperglycaemias and long-term complications, with the burden of diabetes self management, threat of work disability, employment and career problems etc. These features of DM1 increase the likelihood of the development of anxiety and depressive disorders, which, in turn, may negatively influence the course of diabetes and in particular, diabetes self-care. This necessitates early diagnosis of emotional and behavioral disturbances in DM1 using self-report instruments as well as clinical assessment. Evidence suggests that active problem-focused coping behavior and adequate social support promote adherence to diabetes regimes and may act as a buffer against negative effects of the disease on HRQoL in DM1 patients. The core element in the HRQoL structure is personal disease picture (as opposed by objective clinical picture)--the cognitive-affective-behavioral complex reflecting the patient's personal perception of the disease. Examination of the personal disease picture and attitude towards the ailment in DM1 patients may help to improve understanding of the mechanisms of poor adjustment. Problems in disease adjustment can be detected also by diabetes-specific HRQoL assessment. The measures of HRQoL can be applied as screening instruments useful in increasing the effectiveness of patient-provider interactions and diabetes care. PMID- 16673626 TI - Psychometric properties of a European Spanish version of the Perceived Stress Scale (PSS). AB - This paper presents evidence from a heterogeneous sample of 440 Spanish adults, for the reliability and validity of a European Spanish version of the Perceived Stress Scale (PSS), designed to measure the degree to which situations in one's life are appraised as stressful. The European Spanish version PSS (14-item) demonstrated adequate reliability (internal consistency, alpha = .81, and test retest, r = .73), validity (concurrent), and sensitivity. Additional data indicate adequate reliability (alpha = .82, test-retest, r = .77), validity, and sensitivity of a 10-item short version of the PSS. PMID- 16673627 TI - The development of a Spanish language version of the Worn Out scale of the General Well-Being Questionnaire (GWBQ). AB - The paper describes the development of a short Spanish-language version of the General Well-Being Questionnaire (GWBQ; Cox and Gotts, 1987), based on the 12 items of its Worn Out scale. Research has shown the English-version Worn Out scale to be sensitive to aspects of the design and management of work. This study aimed to test its cross-cultural consistency in a Spanish-language workplace context. The data were collected from a sample of 229 workers in Valencia (Spain). Confirmatory Factor Analyses showed the factorial validity, reliability, and concurrent validity of the new Spanish version to be adequate. The sensitivity of the new measure to safety behavior and the reporting of accidents was also assessed and shown to be good. The new questionnaire extends the usefulness of the parent questionnaire to occupational health psychology research in the Spanish language by offering a short assessment tool appropriate for workplace studies. PMID- 16673629 TI - Russia, 1961. PMID- 16673628 TI - Nursing Motives for Helping Scale (N-MHS): reliability and validity. AB - This paper presents the Nursing Motives for Helping Scale (N-MHS), an instrument designed for the evaluation of three of the four motives for helping derived from Batson's helping pathway theory. Dimensionality was analyzed by means of principal component analysis (n = 113), followed by confirmatory factor analysis. A 3-factor structure (corresponding to Batson's differentiation among altruistic motivation, reward-seeking motivation, and punishment-avoidance motivation, respectively), with 9 items distributed in three latent variables, revealed an acceptable fit to the data. Alpha values (.60 - .74) showed that internal consistency was acceptable for a newly developed subscale with a small number of items. Convergence validity was evaluated with correlations between N-MHS subscales scores and scores on the Professional Expectations Scale (Garrosa, Moreno-Jimenez, Rodriguez-Carvajal, and Morante, 2005). The three resulting subscales are a promising instrument for the evaluation of three nursing motives for helping that can contribute to reduce the potential risks and to improve the potential benefits both for the nurse and the patient. PMID- 16673630 TI - Schiavo's lessons for health attorneys when good law is all you have: reflections of the special guardian ad litem to Theresa Marie Schiavo. AB - Rarely have all the branches of federal and state government converged upon a single issue, a single person as they did in the tragic and acrimonious case of Theresa Maria Schiavo. In late 2003, the Florida Legislature passed what become known as "Terri's Law" and in Spring of 2005, Congress and the President of the United States sought to directly intervene in the care of the severely brain damaged woman. During that period, the state and federal court systems, through the highest courts in both venues, ruled on Ms. Schiavo's life, resulting in the removal of an artificial feeding tube and her death during Easter week. The legal and medical issues in this complex, politically and emotionally charged case continue to raise important questions for health attorneys. In this Article, Professor Wolfson, who served as the legislatively mandated, court appointed special guardian ad litem for Theresa Schiavo in late 2003, provides a distinctive first-person overview of the Schiavo case. PMID- 16673631 TI - Living and dying in a post-Schiavo world. AB - The tragic circumstances surrounding the death of Theresa Marie Schiavo have reignited discussion regarding end-of-life decisionmaking. In this Article, the author examines the current legal and ethical environment surrounding the decision to end life-sustaining treatment. Starting with the New Jersey Supreme Court's decision in In re Quinlan, the author discusses how such important issues as who should be the surrogate decisionmaker, attitudes towards artificial nutrition and dehydration, and difficulties in defining medical futility. Looking through a post-Schiavo lens, the Article examines state law addressing these issues and how these choices may be reexamined in the coming years. PMID- 16673632 TI - Medicare reimbursement for clinical trial services: understanding Medicare coverage in establishing a clinical trial budget. AB - In designing and setting up a clinical trial, investigators and private sponsors must take into account what costs will or will not be covered by third-party insurers and government payment programs like Medicare and Medicaid. Failure to "cost out" the clinical trials accurately can yield one of two results: either third-party payors are billed improperly, or even illegally, for experimental care, or significant research-related care is not billed, with either the investigating institution, or the research subjects themselves, shouldering the cost. Unfortunately, because Medicare has established different coverage principles to be applied depending on the type of trial being conducted, costing out the trial is not an easy task. This Article looks at the various Medicare coverage principles as they apply to clinical trials, including the 2000 National Coverage Decision and the recent expansion in coverage for Class A Investigational Devices created by the Medicare Prescription Drug, Improvement, and Modernization Act of 2003. The Article then examines how the Medicare secondary payor rule, which states that providers may not bill Medicare for items or services when another party has primary responsibility for those services, relates to clinical trails in light of recent commentary. The Article concludes with the presentation of a general framework that investigators can use to establish a clinical trial budgeting and billing system. PMID- 16673633 TI - The debate over specialty hospitals: how physician-hospital relationships have reached a new fault line over these "focused factories". AB - Within the last 20 years, the fragile symbiotic relationship between physicians and hospitals has become increasingly strained. Physicians have created new healthcare delivery systems, including a host of for-profit, physician-owned specialty hospitals. Proponents of specialty hospitals argue that they provide high quality facilities and allow for innovative treatments. Opponents, however, contend that such hospitals "cherry pick" patients who otherwise would help to subsidize general hospitals, and point to the various fraud and abuse issues raised by physician-owned specialty hospitals. This Article examines the specialty hospital phenomenon and the arguments for and against such entities. It also analyzes whether an extension of the present federal moratorium on the construction of new physician-owned specialty hospitals is in the best interests of the country's healthcare system. The author concludes that the data are unclear as the overall impact of such hospitals and that they should not be further constrained until when and if the facts are clear. PMID- 16673634 TI - Understanding the physician-owned specialty hospital phenomenon: the confluence of DRG payment methodology and physician self-referral laws. AB - Physician-owned specialty hospitals have flourished in today's healthcare arena, but also have been the subject of a great deal of controversy. The author argues that the rise of specialty hospitals has been the result of a confluence of two healthcare policies: (1) skewed DRG payment methodologies and (2) the misapplication of exemptions to the Stark Law's ban on physician self-referral. This Article examines the aspects of these healthcare policies that have allowed for the explosion of specialty hospitals, as well as the arguments for and against the creation and need for specialty hospitals. The Article also analyzes the reform proposals to correct the Stark and DRG methodologies and how those proposals will affect both specialty and general hospitals. PMID- 16673635 TI - Physician contract checklist: recruitment, employment, and independent contractors. PMID- 16673636 TI - [The organizational culture of ten Dutch veterinary practices. Based on the model of competitive value]. AB - The aim of this study was to study the organizational culture of ten Dutch veterinary practices. In each practice, two veterinary nurses, two associates and two partners filled out the Organizational Culture Assessment Instrument for the current and preferred situation. Results showed that practice culture could be characterised best as the clan culture and, to a lesser extent, an adhocracy culture. The market culture was least prevalent. The family, adhocracy and hierarchy cultures were preferred above the market culture. The difference between current and preferred (i.e. the discrepancy) cultures was highest for the veterinary nurses, followed by the associates and smallest for the partners. Agreement between the three groups was stronger for the preferred situation than for the current situation. PMID- 16673637 TI - [A case of very serious Sarcoptes mange in alpacas (Lama pacos)]. AB - After the diagnosis sarcoptic mange in four alpaca's (Lama pacos) we have tried to control this infection. Despite three treatments with doramectin, three with ivermectin, four with amitraz and two with diazinon we were unable to get the animals free of Sarcoptes mites and their condition deteriorated. One animal died six month after the first treatment. The three remaining animals were euthanized one month thereafter. PMID- 16673638 TI - [Radiographic esophagus imaging: NEVER anesthesia!]. PMID- 16673639 TI - [Careful discharge]. PMID- 16673640 TI - [Also veterinarians have to (and should) make the VWA an authority]. PMID- 16673641 TI - [Forum Companion Animal Welfare presents action plan]. PMID- 16673642 TI - [Instruments, books and other objects memorable to (almost) forgotten opinions, therapies, buildings, et cetera. Sperm separation]. PMID- 16673643 TI - [The market parties determine the quality standards, indeed, but most likely without involvement of the veterinarian interest groups]. PMID- 16673644 TI - [Quality control in veterinary medicine]. PMID- 16673645 TI - The Portuguese Interventional Cardiology Registry from 1992 to 2003. AB - OBJECTIVE: The aim of the present paper was to report trends in Portuguese interventional cardiology from 1992 to 2003 and to compare these data with other European countries. METHODS: Based on questionnaires distributed to and completed by Portuguese interventional cardiology centers we give an overview of the development of coronary interventions since 1992, when data were first collected. In 2003, 24,834 diagnostic catheterization procedures were performed, representing an increase of 315% in comparison to 1992. In 2003 the population adjusted rate was 2483 coronary angiograms per million population. Coronary interventions increased by 1193% in comparison to 1992, with a total of 8465 procedures and a rate of coronary interventions of 848 per million population in 2003. Coronary stents were the most frequently used devices, with an increase from 53% in 1996 to 89% in 2003. The present rate of stent implantation in Portugal is similar to that in Europe and Spain, but the rate of use of drug eluting stents in Portugal is one of the highest in Europe (55%). In 2003, multivessel percutaneous coronary interventions were performed in 24% of cases, with no significant increase during the last 10 years, and ad hoc interventions were performed in the course of diagnostic coronary angiography in 73 % of patients; glycoprotein IIb-IIIa inhibitors were used in 30% of procedures; percutaneous coronary interventions in patients with acute myocardial infarction accounted for 16% of all procedures; of the noncoronary interventions recorded, 49 percutaneous mitral valvuloplasties, 37 atrial septal defect closures and 15 patent foramen ovale closures were reported. The PCI rate per million population in Portugal was lower than the mean European rate (848 vs. 1194). CONCLUSION: Interventional cardiology in Portugal and other European countries has been expanding since 1992. We would emphasize the high rate of reporting by laboratories, which enables the Registry to compile data that are highly representative of activity in Portugal since 1992. PMID- 16673646 TI - Metabolic score--a simple risk marker in non-ST elevation acute coronary syndromes. AB - BACKGROUND: Atherothrombotic coronary artery disease is increasingly recognized as part of a systemic metabolic disorder. However, little is known about the significance of metabolic dysfunction in the setting of acute coronary syndrome. OBJECTIVE: Our aim was to assess the prognostic implications of markers of metabolic dysfunction at hospital admission obesity (BMI > 30), previous history of hypertension, admission glucose > 128 mg/dl, triglycerides > 150 mg/dl, and HDL cholesterol < 40 mg/dl for men, or < 50 mg for women--in patients with non-ST elevation acute coronary syndromes (ACS). METHODS: A total of 303 consecutive patients admitted to the CCU with ACS were included in the study. Mean age was 63 +/- 10 years, and 86% were male. The primary end-point was a composite of death or non-fatal acute myocardial infarction (MI) at one-year follow-up. Each marker was assigned one point, and a metabolic score (MetScore) was calculated for each individual patient by adding together the number of markers present at hospital admission. Three groups were considered: group 1 (MetScore 0) with 0 markers (n = 30); group 2 (MetScore 1 to 3) with 1 to 3 markers (n = 222); and group 3 (MetScore 4 to 5) with 4 to 5 markers (n = 51). RESULTS: The cumulative incidence of death or MI was 14.5%. We found a statistically significant relation between MetScore and outcome at one-year follow-up. The event rate was 3.3% in the MetScore 0 group, 13.9% in the MetScore 1 to 3 group and 23.5% in the MetScore 4 to 5 group (p = 0.0114). MetScore was an independent predictor of death or MI at one year, with a 2.3-fold risk increase (95% CI: 1.32-4.01; p = 0.003) from one group to the next. Other variables identified as independent predictors of outcome were advanced age, Killip class, ST-segment depression and previous CABG. The incidence of the primary end-point in diabetic patients without significant metabolic dysfunction and non-diabetic patients with SMD was similar (21.2% vs. 22.7%; p = NS). CONCLUSION: Assessment of markers of metabolic dysfunction on admission in patients with non-ST elevation acute coronary syndromes, adds important prognostic information to conventional clinical, ECG and risk stratification markers and could prove useful in establishing secondary prevention strategies. PMID- 16673647 TI - Metabolic syndrome, atherosclerosis and thrombogenic risk. PMID- 16673648 TI - Troponin I as a marker of right ventricular dysfunction and severity of pulmonary embolism. AB - BACKGROUND: Cardiac troponin I (cTnI) is a specific marker which allows detection of minor myocardial cell damage. In patients with severe pulmonary embolism (PE), the rise in pulmonary artery pressure can lead to progressive right ventricular dysfunction (RVD), and clinical studies have demonstrated the presence of ischemia and even right ventricular infarction. Our aims were to determine the prevalence and diagnostic utility of cTnI in identifying patients with RVD and to ascertain whether it correlates with severity of PE. METHODS: We studied 77 patients with PE diagnosed by pulmonary angiography, ventilation-perfusion lung scan, spiral computed tomography scan or a combination of abnormal echocardiogram with clinical presentation suggestive of PE or with positive subsidiary exams (d dimers, venous Doppler of the lower limbs, ECG, blood gas analysis). We further classified the PE according to the European Society of Cardiology severity levels, the PE being: 1) massive, if there was shock and/or hypotension; 2) submassive, if we found right ventricular hypokinesis on the echocardiogram; and 3) non-massive, in the remaining cases. We considered the highest cTnI serum value from the admission to 24 hours and a normal value of < 0.10 ng/ml. RESULTS: Among the 60 patients with cTnI measurements, 42 had elevated values. Among those with RVD, 26 (81.3%) had increased cTnI levels and only 14 (35%) with elevated cTnI values did not have RVD, indicating that positive cTnI tests were significantly associated with RVD (p = 0.038). Patients with positive cTnI tests had earlier onset of symptoms (24.0 vs. 144.0 hours, p=0.02), higher prevalence of emboli in proximal vessels (pulmonary trunk and right or left main pulmonary arteries) (OR = 12, CI= 1.6-88.7), and received more thrombolytic therapy (OR = 5.4, CI = 1.1-26.8) than those with normal cTnI tests. cTnI levels were higher among patients with submassive PE (median: 0.77 ng/ml) and lower in those with non-massive PE (0.08 mg/ml, p < 0.05). CONCLUSIONS: Around 70% of patients with PE have elevated cTnI values and this test is significantly associated with RVD. cTnI measurements provide additional information in the evaluation of patients with PE by identifying more severe cases and those at increased risk of hemodynamic deterioration, who can benefit from more aggressive therapeutic strategies. PMID- 16673649 TI - Activity of two enzymes associated with apoptosis and cell aging in arterial hypertension. AB - OBJECTIVE: To study the activity of two enzymes, transmembrane reductase (TMR) and cytosolic low molecular weight protein tyrosine phosphatase (ACP1), in arterial hypertension (HT) in a sample of adults aged over 50 years. METHODS: A sample of 153 adults, 77 with hypertension and 76 normotensive (NT), aged between 50 and 90 years (mean: 71.87 +/- 11.59), of both sexes, was studied. TMR (mmol/cell/h) and ACP1 (micromol/gHb/h) activity in red blood cells was determined by spectrophotometry. Statistical analysis was carried out using the Student's t test and Pearson's correlation. RESULTS: We observed a significant difference in TMR activity between normotensive and hypertensive subjects, with lower levels in the hypertensives (NT = 4.762 +/- 2.595; HT = 3.878 +/- 1.748), p = 0.01. ACP1 activity, although higher in hypertensive patients, did not differ significantly from normotensive subjects (p = 0.08) (NT = 242.827 +/- 97.618; HT = 309.561 +/- 150.738). No correlation was observed between the activity of the two enzymes in either the hypertensive or the normotensive group. CONCLUSIONS: The lower level of activity of transmembrane reductase in arterial hypertension may be implicated in cardiovascular aging processes, reinforced by greater cytosolic acid phosphatase activity, with repercussions on cell proliferation and energy metabolism, leading to atherosclerosis. PMID- 16673650 TI - Exercise stress echocardiography for detection of pulmonary arterial hypertension in a patient with systemic sclerosis. AB - The authors report the case of a 57-year-old patient, diagnosed with systemic sclerosis 16 years ago. She had been followed in the rheumatology outpatient clinic since 2003 and complained of marked fatigue and dyspnea on exertion. In addition to full medical evaluation, she also underwent standard Doppler echocardiography at rest, which was unremarkable, and exercise stress echocardiography. In the latter examination, she was diagnosed with pulmonary arterial hypertension, later confirmed during right heart catheterization. The authors discuss the potential value of this methodology for the study of patients with systemic sclerosis. PMID- 16673651 TI - Intraventricular thrombi with systemic embolization: two clinical cases. AB - Ventricular thrombi are a frequent complication of dilated cardiomyopathy and acute myocardial infarction, with variable risk of embolization according to the clinical setting and thrombus morphology. The authors report two cases of patients admitted on the same day for acute myocardial infarction and dilated cardiomyopathy respectively, with high embolic risk left ventricular thrombi that embolized to the lower limbs after admission. Echocardiography, considered the gold standard diagnostic tool for intraventricular thrombi, is also the mainstay of embolic risk evaluation, for characterizing their size, mobility and echostructure. In the absence of evidence-based guidelines, management options- anticoagulation therapy, thrombolysis or thrombectomy--must be decided according to each specific case, taking account of embolic, bleeding and surgical risk. PMID- 16673652 TI - Analgesia, sedation and neuromuscular blockade in mechanically ventilated cardiac intensive care unit patients. Part II--sedation. AB - This article reviews relevant clinical issues regarding sedation, analgesia and neuromuscular blockade in the cardiac intensive care unit, including monitoring tools and available therapeutic options. The pathophysiologic implications of pain, agitation, anxiety and delirium in the ventilated patient are also discussed. Although guidelines for sedation, analgesia and neuromuscular blocking drugs in critical care have recently been published, there is great variability in clinical practice. The complexity of the environment and associated pathologies makes it difficult to implement universally applicable therapeutic regimens. Knowledge of pharmacologic mechanisms is an important tool in the development of dynamic protocols adapted to each unit. Strategies that include monitoring resources are essential for the optimization of sedation, analgesia and neuromuscular blockade. PMID- 16673653 TI - Types of clinical studies. Systematic reviews. AB - Systematic reviews are analytic instruments that summarize the best available scientific evidence in order to provide evidence-based answers to clinically relevant questions. Meta-analyses are systematic reviews with a quantitative analysis of results. The process of conducting a systematic review is a rigorous and standardized procedure that includes: 1) formulating a clinically relevant question; 2) explicit selection of studies (primary or secondary) to be included in the review; 3) critical appraisal of the relevant evidence; 4) summarizing the evidence; and 5) statistical analysis of the results. In this paper we describe the steps involved in conducting a systematic review of scientific evidence, the methodology, and problems. PMID- 16673654 TI - Eisenmenger syndrome secondary to patent ductus arterious. Contribution of three dimensional echocardiography. PMID- 16673655 TI - Cryothermal ablation of a para-Hisian accessory pathway--case report. PMID- 16673656 TI - [Evaluation of the practical efficacy of the FIGO Classification for Early Invasive Squamous Cervical Cancer]. AB - The aim of our investigation was to evaluate the efficacy of the FIGO Classification for Early Invasive Squamous Cervical Cancer. METHODS: A clinicopathological analysis was performed for 500 patients with invasive squamous cervical cancer with stromal invasion 5 mm or less. RESULTS: We found lymph node metastases in 2% (8 patients) with stromal invasion 3 mm or less in stage I A1. The lymph node involvment in patients with stromal invasion of 3-5 mm was 7.5% (3 patients). When enlarging the tumour width horizontaly from 4% (horizontal spread less than 7 mm) to 10% (horizontal spread more than 7 mm), had no patients with parametrial metastases. From 8 patients recurrences, 6 had horizontal spread more than 7 mm, the other 2 were in stage I A2. CONCLUSIONS: The definition of FIGO for the early squamous cervical cancer is of very good practical importance for the moment. There be a little bit paid more attention on the horizontal spread of 7 mm, the oncogynaecological surgeon must be more radical. PMID- 16673657 TI - [Hyperlipidemic pancreatitis during pregnancy--a case report]. AB - We present a case of acute pancreatitis during pregnancy, associated with hyperlipidemia. The patient, 23 years old in 36 g. w., was hospitalized at the Clinic of Obstetrics and Gynecology with nausea, multiple vomitting, persistent abdominal pain and febrility. Because of the clinical apperance of acute pancreatitis Ceaserean Section was performed with subsequent revision of the abdominal cavity, necrectomy of the pancreas, laparostoma, lavage, drainage, nutritional yienostoma. Intubation with artificial pulmonary ventilation and high volume continuous veno-venous haemofiltration (HV CVVH) was performed because of the development of polyorgan insufficiency and ARDS. After the procedure the patient condition became stable. After four months of hospitalisation, she was discharged from hospital with stable vital signs. PMID- 16673658 TI - [C-reactive protein in women with pre-eclampsia]. AB - Preeclampsia is a disorder associated with generalized dysfunction of endothelial cells probably as a result of systematic inflammatory maternal reaction. CRP is a positive marker of inflammation higher at overt preeclampsia than in normal pregnancy. The aim of the present study is to observe the variations of the acute phase protein CRP at women with preeclampsia and those with normal pregnancy in the third trimester. MATERIAL AND METHODS: Sixty women are included in the study, separated into two groups; group A (n = 30), consisted of women with overt preeclampsia and group B (n = 30)- women with normal pregnancy. Plasma levels of CRP are measured by using immunoturbidimetric method and the results are statistically calculated by variative analysis. RESULTS: We found statistically significant difference in plasma concentration of CRP between pregnant women with preeclampsia and those with normal pregnancy (t = 2.92, P < 0.01). CONCLUSION: In conclusion our data about the significantly higher values of CRP at women with preeclampsia are in agreement with the statement for presence of pronounced inflammation at preeclampsia compared with normal pregnancy. PMID- 16673659 TI - [The significance of kinesitherapy for prophylaxis of osteoporosis]. AB - Osteoporosis is a significant public health problem. It is associated with bone fragility resulting mostly from low bone mass and a concomitant increase in the risk for fracture. Physical exercises are commonly prescribed intervention for prophylaxis of osteoporosis. The purpose of the present study is to state the necessity of appliance kynesitherapy under different forms and methods at osteoporosis. PMID- 16673660 TI - Just compensation? GAO investigating executive pay at not-for-profits. PMID- 16673661 TI - Shareholder feud continues. LifePoint, accipiter take nomination fight to court. PMID- 16673662 TI - Richard, Richard! Despite a new president, the American Hospital Association hopes the secret to continued success is staying the same. AB - Richard Umbdenstock arrives at his new position at the American Hospital Association with a definite agenda: Stay the course. "Continuity is a priority of the first order," says Umbdenstock, left, who was the only candidate considered for the president's job. "One of the reasons he was selected was because we felt the AHA was on the right path and doing well and we wanted the course to stay the same," says AHA Chairman-elect Kevin Lofton. PMID- 16673664 TI - Proposed PPS favors rurals. Specialty hospitals expected to take hit in new system. PMID- 16673663 TI - Davidson set AHA on new path. Association became focused on lobbying Congress. PMID- 16673665 TI - More than just EHR chitchat? Private call to CCHIT from vendors raises concerns. PMID- 16673666 TI - Sentara enters software deal. PMID- 16673667 TI - Geisinger puts IT to work. Pa. system partners with Thomson to sell order sets. PMID- 16673668 TI - AHA creates ripple effect. Umbdenstock's new job will affect several groups. PMID- 16673669 TI - No competition. Umbdenstock was only one considered to lead AHA. PMID- 16673670 TI - Need not apply? Umbdenstock's AHA appointment raises questions about spirit of competition. PMID- 16673671 TI - The new CNO. Male nurses battle stereotypes, low numbers on the way to landing chief nursing officer positions. PMID- 16673672 TI - Acting neighborly. Rural hospitals share resources to advance use of IT. PMID- 16673673 TI - 'Patient-centered' missing from care. PMID- 16673674 TI - Equity-efficiency trade-offs in health technology assessment. AB - Health technology assessment (HTA) currently focuses on efficiency, rather than equity, on the basis that its primary objective is to maximize population health. Yet a strict cost-effectiveness approach sometimes conflicts with important equity concerns, such as the reduction of socioeconomic health inequalities. Managing such equity-efficiency trade-offs on the basis of intuition is unsatisfactory in a democracy, as it arouses suspicions of special pleading and favoritism toward vested interests. Over the next few decades, therefore, decision making may progress through up to three further stages of development observed historically in other areas of resource allocation. Stage two involves case law, limited to principles distilled from precedent. Stage three involves codification, seeking to generalize these principles without specifying their relative weights. Finally, at stage four, quantitative trade-offs are incorporated into a formula. At stage four, deliberation centers on adjustments to the formula, which would then be applied impartially, transparently, and fair mindedly to all future decisions. Methods already exist for valuing equity efficiency trade-offs, based on established methodological principles for valuing trade-offs between different dimensions of health. Early findings indicate that the general public thinks that social class inequalities are more inequitable than those by smoking status, with inequalities between the sexes somewhere in between. Relative weights can be calculated from these data, although the data are not yet comprehensive enough to do this credibly for current policy purposes. In the mean time, the equity-efficiency trade-offs suggested by current decisions can be estimated using standard cost-effectiveness analysis. This is because every departure from a strict cost-effectiveness approach has an opportunity cost. The size of that opportunity cost is a test of how much weight a particular equity concern is deemed to merit. PMID- 16673675 TI - Framework for describing and classifying decision-making systems using technology assessment to determine the reimbursement of health technologies (fourth hurdle systems). AB - OBJECTIVES: Australia, Canada, and many European countries now use various forms of health technology assessment (HTA) in decision making regarding the reimbursement of drugs and other health technologies. To achieve a better understanding of the potential for use of HTA in this context, an analytical framework was developed to describe and classify existing fourth hurdle systems. METHODS: Based on a review of published literature, and official documentation, the key aspects of a fourth hurdle system were identified at two levels: policy implementation and individual technology decision. Characteristics of the systems were grouped under four main headings: constitution and governance, objectives, use of evidence and decision processes, and accountability. The comprehensiveness and relevance of this framework was assessed by an independent group of experts in HTA. A pilot study was undertaken, using only published sources, to test the feasibility of obtaining the information needed to complete the framework. RESULTS: The framework was found to be sufficiently broad to encompass all the issues of interest regarding the systems, but the proportion of information available from published sources was variable between sections of the framework and between countries, with average availability of 45 percent. CONCLUSIONS: The analytical framework will help researchers and policy makers in individual countries to understand their own systems and will allow some preliminary sharing of experience between countries. More experience of its application is needed to judge whether it will provide the basis for more formal comparison of systems and whether it will determine their appropriateness for particular decision contexts. PMID- 16673676 TI - What influences the choice of assessment methods in health technology assessments? Statistical analysis of international health technology assessments from 1989 to 2002. AB - OBJECTIVES: Health technology assessment (HTA) has been used as input in decision making worldwide for more than 25 years. However, no uniform definition of HTA or agreement on assessment methods exists, leaving open the question of what influences the choice of assessment methods in HTAs. The objective of this study is to analyze statistically a possible relationship between methods of assessment used in practical HTAs, type of assessed technology, type of assessors, and year of publication. METHODS: A sample of 433 HTAs published by eleven leading institutions or agencies in nine countries was reviewed and analyzed by multiple logistic regression. RESULTS: The study shows that outsourcing of HTA reports to external partners is associated with a higher likelihood of using assessment methods, such as meta-analysis, surveys, economic evaluations, and randomized controlled trials; and with a lower likelihood of using assessment methods, such as literature reviews and "other methods". The year of publication was statistically related to the inclusion of economic evaluations and shows a decreasing likelihood during the year span. The type of assessed technology was related to economic evaluations with a decreasing likelihood, to surveys, and to "other methods" with a decreasing likelihood when pharmaceuticals were the assessed type of technology. CONCLUSIONS: During the period from 1989 to 2002, no major developments in assessment methods used in practical HTAs were shown statistically in a sample of 433 HTAs worldwide. Outsourcing to external assessors has a statistically significant influence on choice of assessment methods. PMID- 16673677 TI - Improving the process of developing technical reports for health care decision makers: using the theory of constraints in the evidence-based practice centers. AB - OBJECTIVES: To apply the Theory of Constraints (TOC) to the Evidence-based Practice Center (EPC) process. METHODS: Participants in the EPC process were interviewed, and the technical contracting literature was reviewed. Undesirable effects (UDEs) that might occur in the EPC process were identified and, in a TOC Current Reality Tree (CRT), a fundamental UDE was selected and tracked back to potential root causes, focusing on the core "constraint". The resulting draft CRT was presented at an EPC directors' meeting and finalized. From possible solutions obtained from interviews and literature, a Future Reality Tree (FRT) was constructed. RESULTS: Three UDEs were identified and that a report is not used was deemed fundamentally undesirable. Nine root causes were identified, and using the CRT, a core constraint emerged: EPC partner does not know how to conceptualize and articulate needs, objectives, and specifications. Two change strategies ("injections") appeared to address the core constraint: establish an ongoing relationship between partners, EPC, and Agency for Healthcare Research and Quality early in the process, and use a changes clause; in a FRT the injections lead to the positive outcome-the production of a successful EPC report that meets partner needs and, therefore, will be used. CONCLUSIONS: As with any complex enterprise, the EPC needs to engage in self-reflection and active improvement to maximize its value. The development of technical reports intended to inform decision making represents a complicated area, involving a network of interrelated processes. Using the TOC permitted us to understand process failures; results serve as a guide to improvement. PMID- 16673678 TI - Evidence-based practice center network and health technology assessment in the United States: bridging the cultural gap. AB - OBJECTIVES: The purpose of this study was to identify the Evidence-Based Practice Center (EPC) network participants' perceptions of the characteristics of the EPC process and the relationship of the process to the success of EPC reports. METHODS: Semistructured interviews were conducted with the three groups involved in the EPC: EPC staff, Agency for Healthcare Research and Quality (AHRQ) staff, and representatives of partner organizations. RESULTS: The analysis of the coded transcripts revealed three related major themes, which form the conceptual basis for the interpretation presented here: the definition of a successful report, the determinants of a successful report, and the role of AHRQ in the process. CONCLUSIONS: A successful report is a report that is used. The ultimate success of the core health technology assessment objective, moving from research to policy, depends on balancing two values: excellence and relevance. Our findings are consistent with the "two communities thesis," which postulates the existence of two camps that confer different values to excellence and relevance, with resulting tension. A promising model for approaching this tension is integration or collaboration, which requires linking researchers and policy makers, promoting productive dialogues about the formulation and timing of analysis, and early consideration of how the resulting analysis will be used. This effort suggests that actively blurring the frontiers between these two groups will enhance their interaction. Furthermore, enhancing the role of the AHRQ as scientific broker will maximize the potential of the EPC network. PMID- 16673679 TI - Health technology assessment in social care: a case study of randomized controlled trial retrieval. AB - OBJECTIVES: The aim of this study was to evaluate the success of search strategies in retrieving key documents for a technology assessment report (TAR) on a social care topic. METHODS: This study measured the differential yield of relevant studies from various information sources and evaluated strategies in different databases, with particular reference to capturing randomized controlled trials (RCTs) as a study design. RESULTS: A combination of four major databases would have found all thirty-two key references. One database alone would have found 78 percent, with another two each locating 59 percent. Sixteen percent of the trials were unique references. In non-health care databases, more sensitive search strategies would have resulted in a higher yield of relevant studies, in part due to inconsistent indexing and in part to attempts to restrict searches to RCTs. Although additional terms could be used to increase the sensitivity of the original strategies, this raises the question of trading off time against exhaustiveness, given the greater number of irrelevant references likely to be retrieved. CONCLUSIONS: A successful search for evidence on this social care topic would be possible using a combination of MEDLINE, EMBASE, the Cochrane Library and PsyclNFO, supplemented by only limited use of supplementary databases. In areas such as social care where evidence-based research is not yet well established, attempts to replicate searches based on study design do not seem to be advisable, although this may be an area for future research. PMID- 16673680 TI - Are we measuring what matters in health technology assessment of disease management? Systematic literature review. AB - OBJECTIVES: An overview was produced of indicators currently used to assess disease management programs and, based on these findings, provide a framework regarding sets of indicators that should be used when taking the aims and types of disease management programs into account. METHODS: A systematic literature review was performed. RESULTS: Thirty-six studies met the inclusion criteria. It appeared that a link between aims of disease management and evaluated structure, process, as well as outcome indicators does not exist in a substantial part of published studies on disease management of diabetes and asthma/chronic obstructive pulmonary disease, especially when efficiency of care is concerned. Furthermore, structure indicators are largely missing from the evaluations, although these are of major importance for the interpretation of outcomes for purposes of decision-making. Efficiency of disease management is mainly evaluated by means of process indicators; the use of outcome indicators is less common. Within a framework, structure, process, and outcome indicators for effectiveness and efficiency are recommended for each type of disease management program. CONCLUSIONS: The link between aims of disease management and evaluated structure, process, and outcome indicators does not exist in a substantial part of published studies on disease management. The added value of this study mainly lies in the development of a framework to guide the choice of indicators for health technology assessment of disease management. PMID- 16673681 TI - Clinical practice guidelines and organizational adaptation: a framework for analyzing economic effects. AB - OBJECTIVES: The overall objective of this article was to review the theoretical and conceptual dimensions of how the implementation of clinical practice guidelines (CPGs) is likely to affect treatment costs. METHODS: An important limitation of the extant literature on the cost effects of CPGs is that the main focus has been on clinical adaptation. We submit that the process innovation aspects of CPGs require changes in both clinical and organizational dimensions. We identify five organizational factors that are likely to affect the relationship between CPGs and total treatment costs: implementation, coordination, learning, human resources, and information. We review the literature supporting each of these factors. RESULTS: The net organizational effects of CPGs on costs depends on whether the cost-reducing properties of coordination, learning, and human resource management offset potential cost increases due to implementation and information management. CONCLUSIONS: Studies of the cost effects of clinical practice guidelines should attempt to measure, to the extent possible, the effects of each of these clinical and organizational factors. PMID- 16673682 TI - Randomized trial of a brief physiotherapy intervention compared with usual physiotherapy for neck pain patients: cost-effectiveness analysis. AB - OBJECTIVES: To assess the cost-effectiveness of brief physiotherapy intervention versus usual physiotherapy management in patients with neck pain of musculoskeletal origin in the community setting. METHODS: A cost-effectiveness analysis was conducted alongside a multicenter pragmatic randomized controlled clinical trial. Individuals 18 years of age and older with neck pain of more than 2 weeks were recruited from physiotherapy departments with referrals from general practitioners (GPs) in the East Yorkshire and North Lincolnshire regions in the United Kingdom. A total of 139 patients were allocated to the brief intervention, and 129 to the usual physiotherapy. Resource use data were prospectively collected on the number of physiotherapy sessions, hospital stay, specialist, and GP visits. Quality-adjusted life years (QALYs) were estimated using EQ-5D data collected at baseline, 3 and 12 months from the start of the treatment. The economic evaluation was conducted from the U.K. National Health System perspective. RESULTS: On average, brief intervention produced lower costs (pounds -68; 95 percent confidence interval [CI], pounds--103 to pounds--35) and marginally lower QALYs (-0.001; 95 percent CI, -0.030 to 0.028) compared with usual physiotherapy, resulting in an incremental cost per QALY of pounds 68,000 for usual physiotherapy. These results are sensitive to patients' treatment preferences. CONCLUSIONS: Usual physiotherapy may not be good value for money for the average individual in this trial but could be a cost-effective strategy for those who are indifferent toward which treatment they receive. PMID- 16673683 TI - Cost-effectiveness of a graded exercise therapy program for patients with chronic shoulder complaints. AB - OBJECTIVES: The present study evaluated the cost-effectiveness of a behavioral graded exercise therapy (GET) program compared with usual care (UC) in terms of the performance of daily activities by patients with chronic shoulder complaints in primary care. METHODS: A total of 176 patients were randomly assigned either to GET (n=87) or to UC (n=89). Clinical outcomes (main complaints, shoulder disability [SDQ] and generic health-related quality of life [EQ-5D], and costs [intervention costs, direct health care costs, direct non-health-related costs, and indirect costs]) were assessed during the 12-week treatment period and at 52 weeks of follow-up. RESULTS: Results showed that GET was more effective than UC in restoring daily activities as assessed by the main complaints instrument after the 12-week treatment period (p = .049; mean difference, 7.5; confidence interval [CI], 0.0-15.0). These effects lasted for at least 52 weeks (p = .025; mean difference 9.2; CI, 1.2-17.3). No statistically significant differences were found on the SDQ or EQ5D. GET significantly reduced direct health care costs (p = .000) and direct non-health care costs (p = .029). Nevertheless, total costs during the 1-year follow-up period were significantly higher (p = .001; GET = Euro 530 versus UC = Euro 377) due to the higher costs of the intervention. Incremental cost-effectiveness ratios for the main complaints (0-100), SDQ (0 100), and EQ-5D (-1.0-1.0) were Euro 7, Euro 74, and Euro 5278 per unit of improvement, respectively. CONCLUSIONS: GET proved to be more effective in the short- and long-term and reduces direct health care costs and direct non-health care costs but is associated with higher costs of the intervention itself. PMID- 16673684 TI - Cost-effectiveness of a supplementary class-based exercise program in the treatment of knee osteoarthritis. AB - OBJECTIVES: The aim of this study was to assess the cost-effectiveness of a class based exercise program supplementing a home-based program when compared with a home-based program alone. In addition, we estimated the probability that the supplementary class program is cost-effective over a range of values of a decision maker's willingness to pay for an additional quality-adjusted life-year (QALY). METHODS: The resource use and effectiveness data were collected as part of the clinical trial detailed elsewhere. Unit costs were estimated from published sources. The net benefit approach to cost-effectiveness analysis is used to estimate the probability of the intervention being cost-effective. RESULTS: The addition of a supplementary class-based group results in an increase in QALYs and lower costs. For all plausible values of a decision maker's willingness to pay for a QALY, the supplementary class group is likely to be cost effective. CONCLUSIONS: The addition of a class-based exercise program is likely to be cost-effective and, on current evidence, should be implemented. PMID- 16673685 TI - Evaluation of the cost-effectiveness of drotrecogin alfa (activated) for the treatment of severe sepsis in the United Kingdom. AB - OBJECTIVES: The aim of this study was to assess the cost-effectiveness of drotrecogin alfa (activated) compared with best supportive care in a UK cohort of adult intensive-care patients with severe sepsis. METHODS: A systematic review of evidence on the clinical- and cost-effectiveness of drotrecogin alfa (activated) was undertaken, and a decision-analytic model was developed to estimate the cost effectiveness of treatment in the United Kingdom. Trial data from the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study have been synthesized with other data, including UK data on severe sepsis, to estimate the costs and consequences of treatment over time. RESULTS: For patients with severe sepsis and multiple organ dysfunction, the estimates of cost per life year and cost per quality-adjusted life year (QALY) are pounds 4931 and pounds 8228, respectively. For patients with severe sepsis alone, the cost per life-year and cost per QALY are pouhds 5495 and pounds 9161, respectively. CONCLUSIONS: Whereas the therapeutic cost for drotrecogin alfa (activated) appears high (at around pounds 5000 per patient) and the potential impact on the provider budget is considerable, drotrecogin alfa (activated) is clinically effective, represents a cost-effective use of resources, and is a significant advance in the treatment of severe sepsis in patients requiring intensive care. PMID- 16673686 TI - Cost-effectiveness of drotrecogin alfa (activated) in the treatment of severe sepsis with multiple organ failure. AB - OBJECTIVES: The aim of this study was to estimate the expected cost and clinical benefits associated with the use of drotrecogin alfa (activated) (Xigris; Eli Lilly and Company; Indianapolis, IN) in the French hospital setting. METHODS: The recombinant human activated PROtein C Worldwide Evaluation in Severe Sepsis (PROWESS) study results (1271 patients with multiple organ failure) were adjusted to 9,948 hospital stays from a database of Parisian area intensive-care units (ICUs)-the CubRea (Intensive Care Database User Group) database. The analysis features a decision tree with a probabilistic sensitivity analysis. RESULTS: The cost per life year gained (LYG) of drotrecogin treatment for severe sepsis with multiple organ failure (European indication) was estimated to be dollars 11,812. At the hospital level, the drug is expected to induce an additional cost of dollars 7545 per treated patient. The incremental cost-effectiveness ratio ranges from dollars 7873 per LYG for patients receiving three organ supports during ICU stay to dollars 17,704 per LYG for patients receiving less than two organ supports. CONCLUSIONS: Drotrecogin alfa (activated) is cost-effective in the treatment of severe sepsis with multiple organ failure when added to best standard care. The cost-effectiveness of the drug increases with baseline disease severity, but it remains cost-effective for all patients when used in compliance with the European approved indication. PMID- 16673687 TI - Value of magnetic resonance cholangiopancreatography in the diagnosis of biliary abnormalities in postcholecystectomy patients: a probabilistic cost-effectiveness analysis of diagnostic strategies. AB - BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is considered the gold standard for imaging of the biliary tract but is associated with complications. Less invasive imaging techniques, such as magnetic resonance cholangiopancreatography (MRCP), have a much lower complication rate. The accuracy of MRCP is comparable to that of ERCP, and MRCP may be more effective and cost-effective, particularly in cases for which the suspected prevalence of disease is low and further intervention can be avoided. A model was constructed to compare the effectiveness and cost-effectiveness of MRCP and ERCP in patients with a previous history of cholecystectomy, presenting with abdominal pain and/or abnormal liver function tests. METHODS: Diagnostic accuracy estimates came from a systematic review of MRCP. A decision analytic model was constructed to represent the diagnostic and treatment pathway of this patient group. The model compared the following two diagnostic strategies: (i) MRCP followed with ERCP if positive, and then management based on ERCP; and (ii) ERCP only. Deterministic and probabilistic analyses were used to assess the likelihood of MRCP being cost effective. Sensitivity analyses examined the impact of prior probabilities of common bile duct stones (CBDS) and test performance characteristics. The outcomes considered were costs, quality-adjusted life years (QALYs), and cost per additional QALY. RESULTS: The deterministic analysis indicated that MRCP was dominant over ERCP. At prior probabilities of CBDS, less than 60 percent MRCP was the less costly initial diagnostic test; above this threshold, ERCP was less costly. Similarly, at probabilities of CBDS less than 68 percent, MRCP was also the more effective strategy (generated more QALYs). Above this threshold, ERCP became the more effective strategy. Probabilistic sensitivity analyses indicated that, in this patient group for which there is a low to moderate probability of CBDS, there was a 59 percent likelihood that MRCP was cost-saving, an 83 percent chance that MRCP was more effective with a higher quality adjusted survival, and an 83 percent chance that MRCP had a cost-effectiveness ratio more favorable than dollars 50,000 per QALY gained. CONCLUSIONS: Costs and cost-effectiveness are dependent upon the prior probability of CBDS. However, probabilistic analysis indicated that, with a high degree of certainty, MRCP was the more effective and cost-effective initial test in postcholecystectomy patients with a low to moderate probability of CBDS. PMID- 16673688 TI - Cerebral protection devices for use during carotid artery angioplasty with stenting: a health technology assessment. AB - OBJECTIVES: This study sought to examine the safety, efficacy, and economic implications of the use of cerebral protection devices during carotid artery angioplasty and stenting (CAS) in high-risk patients with severe carotid artery disease (CAD). METHODS: A comprehensive search for peer- and non-peer-reviewed studies that compared carotid endarterectomy (CEA) or CAS without cerebral protection to CAS with cerebral protection and appeared in the English language literature between January 1990 and January 2005 was completed. Information from studies identified was extracted using a common data abstraction form and then critically appraised against published quality assessment criteria. RESULTS: Of the eight studies found, six provided information on technical or procedural success rates, with values ranging from 95.6 percent to 100 percent. Three of the four studies comparing groups of patients who received CAS with cerebral protection with those who received only CAS reported a non-statistically significantly higher 30-day incidence of death and stroke (major or minor) in the latter group. None of the three studies comparing CAS with cerebral protection to CEA demonstrated a statistically significant difference in the 30-day incidence of death, major stroke, or myocardial infarction between treatment groups. No economic analyses were found. CONCLUSIONS: In high-risk patients with severe CAD, the evidence suggests that CAS with cerebral protection may offer a safe and efficacious alternative to CEA, reducing the risk of embolic peri-procedural complications associated with CAS to acceptable levels. PMID- 16673690 TI - Evaluating telemedicine: a focus on patient pathways. AB - Evaluations of telemedicine have sought to assess various measures of effectiveness (e.g., diagnostic accuracy), efficiency (e.g., cost), and engagement (e.g., patient satisfaction) to determine its success. Few studies, however, have looked at evaluating the organizational impact of telemedicine, which involves technology and process changes that affect the way that it is used and accepted by patients and clinicians alike. This study reviews and discusses the conceptual issues in telemedicine research and proposes a fresh approach for evaluating telemedicine. First, we advance a patient pathway perspective, as most of the existing studies view telemedicine as a support to a singular rather than multiple aspects of a health care process. Second, to conceptualize patient pathways and understand how telemedicine impacts upon them, we propose simulation as a tool to enhance understanding of the traditional and telemedicine patient pathway. PMID- 16673689 TI - Principles for the design of the economic evaluation of COLOR II: an international clinical trial in surgery comparing laparoscopic and open surgery in rectal cancer. AB - OBJECTIVES: The objective is to describe the principles for the design of the economic evaluation of COLOR II, a randomized, multi-country study comparing laparoscopic and open surgery for rectal cancer. METHODS: By using the experiences gained in a recent economic evaluation in colon cancer, where the same surgical techniques were compared, we could improve the method for identifying and measuring resource use items and also accommodate the use of data from the global study population. RESULTS: In the design of the study, the uncertainty in the resource-use variables was reduced by considering (i) what aspects drive each variable, (ii) what resource use is related to the intervention, (iii) how data from different countries affects the variable. CONCLUSIONS: The aim was to refine the data collection so that the economic research question could be answered in the best possible way, given the circumstances in the clinical study. Thus, (i) some variables were treated as stochastic variables and others as deterministic variables, (ii) aggregate key cost-driving resource items were developed that corresponded to clinical events, and (iii) a surrogate variable was selected, instead of the "obvious variable", to reduce the impact of confounding factors for one particular resource unit. PMID- 16673691 TI - [Bipolar disorder type I]. PMID- 16673692 TI - [Humor in bipolar disorder]. PMID- 16673693 TI - [Typical melancholia in bipolar disorder]. PMID- 16673694 TI - [Major depressive disorder in personality disorder]. PMID- 16673695 TI - [Love, humor and psychosis in an atypical depression]. PMID- 16673696 TI - [Masked depression]. PMID- 16673697 TI - [Depression and masochism]. PMID- 16673699 TI - [Prodromic status of psychotic disorders]. PMID- 16673698 TI - [Recurring depression]. PMID- 16673700 TI - [Relapse of acute delirium]. PMID- 16673701 TI - [Hebephrenic schizophrenia]. PMID- 16673702 TI - [Pseudo-psychopathic schizophrenia]. PMID- 16673703 TI - [Schizophrenia or obsessive-compulsive disorder?]. PMID- 16673704 TI - [Cannabis and psychosis]. PMID- 16673705 TI - [Paranoid delirium of cognition]. PMID- 16673706 TI - [Psychosomatic disorders: about observation of Crohn's disease]. PMID- 16673707 TI - [Alcohol dependence and depressive disorders in the course of opiate dependence]. PMID- 16673708 TI - [Cannabis dependence and social phobia]. PMID- 16673709 TI - [Maxime's social phobia]. PMID- 16673710 TI - [Obsessive-compulsive disorder in personality disorders]. PMID- 16673711 TI - [Dementia and/or depression]. PMID- 16673713 TI - [Chronic psychosis in an adult with antecedents of neonatal tuberculous meningitis]. PMID- 16673712 TI - [Preventing emergency? Preventing agitation?]. PMID- 16673714 TI - [Sex identity disorders]. PMID- 16673715 TI - [Homosexual pedophilia]. PMID- 16673716 TI - [Atypical hypersomnia]. PMID- 16673717 TI - [Borderline personality disorder]. PMID- 16673718 TI - [Minor hypochondriasis]. PMID- 16673719 TI - [Infectious disease: look over a trajectory]. PMID- 16673720 TI - [Severe sepsis and septic shock]. AB - Severe sepsis and septic shock are frequent pathologies accounting for approximately 11% of all admissions in intensive care units (ICU). In the United States, between 1979 and 2000 the incidence of sepsis increased by 8,7% annually and septic shock) remains the second most frequent cause of death in non-coronary ICU. Although our understanding of the host defense mechanisms against infections and of the pathogenesis of septic shock have progressed during the last decade, these progresses have not yet yielded the anticipated advantages. Recent new therapeutic approaches, especially early-goal directed therapy, activated protein C (drotrecogin alpha activated), moderate doses of corticosteroids and intensive insulin therapy have given encouraging results. PMID- 16673721 TI - [Infective endocarditis and outpatient practice]. AB - The incidence of infectious endocarditis is fairly stable over the past decades. It is estimated at roughly 3-4 case per patient-year. However, as a consequence of medical progress, Staphylococcus aureus endocarditis has become more prevalent. This is particularly true for health-care associated endocarditis, especially in iv-drug abusers or hemodialysis patients. Mortality (15-20% of patients in the last series) remains high. About 50% of patients undergo surgical treatment, whereas outpatient therapy is more and more frequent for highly selected subgroups of patients without complications and infected with low-risk organims. The present paper reviews in detail the epidemiology of infective endocarditis and discuss in detail the different out-patients therapies. PMID- 16673722 TI - [HIV-AIDS: answers to new questions]. AB - The introduction of highly active antiretroviral therapy 10 years ago has revolutionized the treatment of HIV infection in industrialized countries. HIV infection has now become a chronic condition, often treatable with low number of pills. Pregnancy and childbirth are possible with minimal vertical transmission risk. Organ transplantation is no longer contra-indicated solely on the basis of HIV infection. New management issues have emerged, including the long-term cardiovascular risk and metabolic toxicity associated with anti-HIV medications, the appearance of drug-resistant HIV strains, and the re-emergence of risky sexual behavior, particularly among men who have sex with men. This article provides an update for primary care physicians regarding important questions and controversies in the treatment of HIV-infected patients in 2006. PMID- 16673723 TI - [Lyme disease Part I: epidemiology and diagnosis]. PMID- 16673724 TI - [Lyme disease Part 2: clinic and treatment]. PMID- 16673725 TI - [Lyme disease Part 3: prevention, pregnancy, immunodeficient state, post-Lyme disease syndrome]. PMID- 16673726 TI - [When can the treatment of isolated systolic hypertension be avoided?]. AB - The treatment of isolated systolic hypertension (ISH) in the elderly reduces the cardiovascular (CV) risk, in particular in patients with diabetes or previous CV events. However, in the very old (> 80-85 years) the treatment of ISH may increase global mortality, although it still decreases the risk of stroke. The benefits of treatment on the risk of dementia remain uncertain. To verify the indication for therapy, the diagnosis of ISH should be confirmed by ambulatory blood pressure monitoring. Since the absolute benefit of treatment is related to its duration, a limited life expectancy may restrict the real impact of treatment. The advantages and limitations of anti-hypertensive therapy in the elderly should be discussed individually, respecting the patient's autonomy. PMID- 16673727 TI - [Legislation in matters of direct active euthanasia? Delicate matters and practical principles. Concerning the report of the Federal Office of Justice]. PMID- 16673728 TI - [Can one reasonably prescribe antidepressants in a child?]. PMID- 16673729 TI - [Does physiologic depression exist?]. PMID- 16673730 TI - [AIDS, transfusion and marriage: from Lisbon to Bujumbura]. PMID- 16673732 TI - [A historical manifestation]. PMID- 16673731 TI - [Mad cow, 10 years after the announcement of the apocalypse]. PMID- 16673733 TI - Regional detention facility weight fluctuations of detainees in Iraq. PMID- 16673734 TI - The history of U.S. Military contributions to the understanding, prevention, and treatment of infectious diseases. PMID- 16673735 TI - The Army forward surgical team: update and lessons learned, 1997-2004. AB - Although the U.S. Army stood up two prototype airborne forward surgical teams (FSTs) in the early 1990s, it officially fielded the first 20-man FST in March 1997. Since then, Army FSTs (of which there are now 14 active and 23 reserve) have deployed on numerous combat, peacekeeping, and disaster-relief missions throughout the world. Although initially designed primarily for forward resuscitative surgery in support of short-term, offensive combat operations, the FST has become one of the Army Medical Department's most frequently deployed "long-term" health care facilities, i.e., long-term for the assigned personnel. The purpose of this article was to review the history of the FST, define its mission, delineate pitfalls in its employment and to make personnel and material acquisition recommendations for the future. PMID- 16673736 TI - The seven P's in battalion level combat health support in the Military Operations in Urban Terrain environment: the Fallujah experience, summer 2003 to spring 2004. AB - Combat health support in the Military Operations in Urban Terrain (MOUT) environment represents a common challenge on today's battlefield. We identified seven key aspects of battalion level health support which required consideration before combat operations in this type of environment. We called these the "seven P's" of combat health support: prevention, proportion, preparation, portability, proximity, protection, and projection. We developed an easy to use framework for using these principles to quickly develop combat health support plans during periods of high operations tempo. PMID- 16673737 TI - Traumatic shock. AB - The association of shock with trauma has long been recognized. There are three types of shock, i.e., (1) hypovolemic or hemorrhagic shock, (2) traumatic shock, and (3) septic shock. The conditions and their treatments are described. PMID- 16673738 TI - Effect of night vision goggles on performance of advanced life support skills by emergency personnel. AB - Night vision goggles (NVGs) are used by military personnel operating in low-light environments. It is not known whether NVGs can be used by medical personnel to provide emergency care under such conditions. This was a randomized controlled study to determine the effect of NVGs on the performance of intravenous line insertion (IVI) and endotracheal intubation (El) on training manikins. Emergency physicians and paramedics were randomized to perform EI and IVI in ambient light or in total darkness using NVGs. Each skill was repeated three times, and averages were determined. The average times for EI in ambient light and with NVGs were 48.4 and 188.2 seconds, respectively (SE of 13.4 seconds for both; p < 0.0001). The average times for IVI in ambient light and with NVGs were 34.7 and 73.7 seconds, respectively (SE of 4.1 seconds for both; p < 0.0001). Emergency personnel were able to successfully perform these skills using NVGs, but their times were significantly longer than in ambient light. PMID- 16673739 TI - Functional outcomes of unilateral lower limb amputee soldiers in two districts of Sri Lanka. AB - The purpose of this study was to describe functional outcomes of unilateral lower limb amputee soldiers, in comparison to nonamputee male subjects, and to identify factors affecting functional outcomes. Selected lower limb amputee soldiers of the Sri Lanka Army (n = 461) and matched nonamputee male subjects (n = 461) were evaluated with the prevalidated self-administered version of Short-Form Health Survey-36. A majority were below-knee amputees (n = 413; 89.6%), and the mean age was 30.2 +/- 4.6 years. Short-Form Health Survey-36 health profile scores of amputees were significantly lower than those of nonamputees (p < 0.001). The differences in profiles were largest in scales sensitive to physical health. Higher levels of amputation and problems with the stump and sound leg were significantly associated with poor physical and mental health scores. Amputee soldiers' assessment of changes in health over the past year was significantly worse than that of nonamputees (p < 0.001). The study concluded that amputees have comparatively poor functional outcomes, which could be improved by modifying some associated factors. PMID- 16673740 TI - Estimation of stature from upper extremity. AB - With the increasing frequency of mass disasters, identification of an isolated upper extremity and determination of the stature of the person it belonged to have created problems for investigation of the identity of some victims. Despite a need for such a study, there is a lack of systematic studies to identify fragmented and dismembered human remains. The purpose of this study was to analyze anthropometric relationships between dimensions of the upper extremity and body height. Analyses were based on a sample of middle class male (n = 202) and female (n = 108) Turks residing in Istanbul, Turkey. Five variables were entered into the analyses. For male subjects, forearm length was selected as the first factor, followed by hand length and finally upper arm length. For female subjects, upper arm length was selected first, followed by forearm length and finally hand length. There were also individually calculated formulae for some of these measurements that provided smaller R2 values. The study suggested that estimation of a living height could be made possible by using various dimensions of the upper extremity. One must consider differences between populations to apply such functions to other populations. PMID- 16673741 TI - Sequential metatarsal fatigue fractures secondary to abnormal foot biomechanics. AB - Sequential fatigue fractures of the fourth, second, and third metatarsals in the same foot are reported for a military aviator in the absence of abnormal stresses or underlying bone disease. The likely etiological factor is altered foot biomechanics, as identified in pedobarographic assessment. We have reviewed the literature regarding multiple metatarsal stress fractures. PMID- 16673742 TI - Iron supplementation and the female soldier. AB - Twenty-two percent of women in the United States are iron deficient. Iron deficiency adversely affects immune function as well as physical and cognitive performance. Although the risk of developing iron deficiency is high for female soldiers, this risk can be minimized with proper nutritional guidance. Recommended dietary modifications include (1) heme iron consumption, (2) ingestion of vitamin C and protein with meals, and (3) discontinued tea and coffee consumption with meals. PMID- 16673743 TI - Prevalence of adverse behaviors among young military conscripts in Taiwan. AB - OBJECTIVES: To evaluate cigarette smoking prevalence and to examine the clustering of adverse behaviors among young military conscripts in Taiwan. METHODS: Cross-sectional screening was conducted among conscripts in southern and eastern sections of Taiwan from August 1 to December 31, 2001. A total of 7,931 conscripts who had > 1 month of service were included in this multistage sampling study. RESULTS: Adjusted prevalence rates of cigarette smoking before and during military service were 49.2% and 51.9%, respectively. The prevalence of smoking slightly increased during military service, and this was statistically significant. Adjusted prevalence was highest in the Navy (53.8%) and lowest in the Air Force (48.4%). Less education and longer duration of service were associated with increased prevalence of smoking. Conscripts with high school educations or below and those in service > 18 months had higher adjusted prevalence rates (65.6% and 55.2%, respectively). During military service, the adjusted prevalence rates of smoking among conscripts were similar in different geographic areas. There was significant aggregation of adverse behaviors, such as cigarette smoking, alcohol consumption, and betel nut chewing, among these young military conscripts in Taiwan. CONCLUSIONS: The prevalence of cigarette smoking was slightly increased during military service. Furthermore, the prevalence of cigarette smoking was still higher among military conscripts, compared with the general population in Taiwan, which suggests that military service is a potential factor associated with cigarette smoking for adolescents and young adults. This finding and the aggregation of adverse behaviors among conscripts indicate that more surveillance and intervention studies are needed to evaluate the best way to control and to stop smoking among military conscripts in Taiwan. PMID- 16673744 TI - Audibility of patient clinical alarms to hospital nursing personnel. AB - The audibility of patient clinical alarms to nursing personnel was tested during the first shift on a medical/surgical in-patient ward at William Beaumont Army Medical Center. This study was conducted during normal hospital operations, and we tested the Joint Commission on Accreditation of Healthcare Organizations 2004 National Patient Safety Goal, Section 6b to determine whether patient alarms could sufficiently compete against environmental background noises. Patient clinical alarms were audible at distances of > or = 95 feet with room doors open but were not sufficiently audible to hospital staff members when room doors were closed or during floor-buffing activities. This study suggests that, under these circumstances, hospitals may not meet Joint Commission on Accreditation of Healthcare Organizations 2004 National Patient Safety Goal, Section 6b. Because the audibility of patient clinical alarms directly affects patient safety, satisfaction, and quality of care, we provide recommendations for engineering controls and modifications to work routines. PMID- 16673745 TI - Multiple-role dilemmas for military mental health care providers. AB - Military psychologists and psychiatrists frequently face ethical quandaries involving boundary crossings, or extratherapy contact, and multiple relationships. A multiple relationship is defined as necessarily engaging psychotherapy patients in nonclinical roles, such as coworker, superior officer, neighbor, or friend. In contrast to their civilian counterparts, military mental health professionals must often engage patients in many different contexts and roles. In this article, we consider the distinctive features of mental health practice in the military and offer military providers several practice guidelines for avoiding harm to patients in military settings. This article is also designed to enhance sensitivity to multiple-role risks among nonpsychiatric providers. PMID- 16673746 TI - Treatment of psychiatric disorders onboard an aircraft carrier assisted with psychotropic medication: a retrospective review describing one aspect of Navy Force health protection. AB - Navy clinical psychologists, assigned to aircraft carriers, are playing an increasing role in not only implementing Navy force health protection, but also in further specializing the delivery of mental health evaluation, treatment, and disposition services at the "tip of the spear." An aircraft carrier's medical department, augmented with a clinical psychologist, is now better able to coordinate diagnostic, psychotropic, and psychotherapeutic treatments for both shipboard and air wing personnel. This retrospective review reports the outcomes of a 6-month treatment program for personnel, assigned to the USS Constellation (CV-64), who were prescribed a psychotropic medication while receiving psychotherapy. We concluded that psychotropic medications can be safely and effectively used onboard an aircraft carrier. Furthermore, personnel prescribed psychotropic medication successfully completed their assigned duties and obtained recommendations for advancement and retention. Lastly, our medical department proactively fulfilled the Navy force health protection tenet of preserving a healthy and fit force. PMID- 16673747 TI - Factors that influence the decision of military dependents to decline smallpox vaccination during an outbreak. AB - OBJECTIVE: To examine factors that influence the decision of military dependents to decline smallpox vaccination in the event of an outbreak. METHODS: A total of 373 military dependents, 18 to 65 years of age, were surveyed in December 2003, at Fort Hood, Texas, and Fort Sill, Oklahoma, concerning smallpox vaccine knowledge, adverse reactions, and willingness to receive the vaccine. RESULTS: A total of 89% of respondents were willing to receive the vaccine. Respondents were less willing if the vaccine was unsafe during pregnancy or caused blood clots and heart attacks. African American and Hispanic respondents were less willing to be vaccinated than were Caucasian respondents (p < 0.0079), as were respondents with lower vaccine knowledge scores (p < 0.0029). CONCLUSIONS: The majority of military dependents would want to receive the vaccine in the event of an outbreak. Vaccine safety in pregnancy and an unproven link to blood clots and heart attacks may influence vaccine refusal. African American respondents, Hispanic respondents, and respondents with lower vaccine knowledge scores were less willing to receive the vaccine. PMID- 16673748 TI - Knowledge and risks of human immunodeficiency virus transmission among veterans with severe mental illness. AB - This study is among the first to examine knowledge about human immunodeficiency virus (HIV) and behavioral risks for HIV transmission among veterans with severe mental illness (SMI), a group at high risk for HIV infection. This study examined associations between accuracy of HIV knowledge, risk behaviors, and clinical and demographic characteristics in a sample of male veteran psychiatric inpatients diagnosed with SMI (N = 353). Results showed high rates of inaccurate HIV knowledge, with > 40% of patients demonstrating some inaccuracies, particularly those related to the progression and symptoms of acquired immunodeficiency syndrome. Inaccurate HIV knowledge was associated with older age, minority status, education level, marital status, no homelessness within the previous 6 months, and no reported history of illicit intranasal drug use. There is a need for more effective HIV prevention interventions for persons with SMI. PMID- 16673749 TI - A case of eosinophilic gastroenteritis with severe peripheral eosinophilia. AB - Eosinophilic gastroenteritis is a rare heterogeneous disorder of undetermined etiology that is characterized by eosinophilic infiltration of the gastrointestinal tissues and various clinical manifestations. We report an uncommon case of eosinophilic gastroenteritis involving a patient with a short history of mild upper abdominal pain, severe peripheral eosinophilia (absolute eosinophil count of > 5,000 cells per microL), and ascites. The patient was treated successfully with a course of methylprednisolone. PMID- 16673750 TI - The great disease enemy, Kak'ke (beriberi) and the Imperial Japanese Army. AB - Although Japanese military officials had discovered that an improved diet could prevent beriberi by the late 19th century, their soldiers in the army suffered from beriberi during the Russo-Japanese War and World War II. A change in diet at the end of the Russo-Japanese War solved the problem and the army applied the lesson learned, along with postwar scientific discoveries about nutrition, toward the diet used during World War II. However, beriberi again plagued Japanese soldiers, this time due to poor logistics and unpalatable dietary supplements. PMID- 16673751 TI - A 43-year-old colonel with chills, diaphoresis, and headache. AB - The objectives were to illustrate the ease with which one might attribute concomitant or subsequent illness to an exposure such as the anthrax vaccine and to demonstrate an approach that keeps the significance of such exposures in appropriate perspective. A 43-year-old, active duty, Army officer presents with a variety of nonspecific common symptoms and raises concerns about the relationship of his symptoms to receipt of the anthrax vaccine. He is admitted for an evaluation that includes a series of diagnostic tests and consultations. The course of his illness and the corresponding evaluation are reviewed using a series of questions and accompanying discussions to highlight key points regarding diagnostic considerations, the anthrax vaccine, and the ultimate identification of the correct diagnosis. PMID- 16673752 TI - Is there an association between sweetened beverages and adiposity? AB - Four mechanisms were reviewed to explain the possible association between sweetened beverages and increased overweight or obesity: excess caloric intake, glycemic index and glycemic load, lack of effect of liquid calories on satiety, and displacement of milk. The findings were inconsistent across studies. The strongest support was for the excess caloric intake hypothesis, but the findings were not conclusive. Assigning possible links between sweetened beverage consumption and adiposity requires research that compares and contrasts specific mechanisms, especially in populations at risk for obesity, while controlling for likely confounding variables. PMID- 16673753 TI - Vegetarian diets and weight status. AB - The increasing global health problems of overweight and obesity are associated with coronary heart disease, hypertension, diabetes, osteoarthritis, and certain cancers, among other health concerns. Vegetarian diets are associated with reduced body weight, lower incidence of certain chronic disease, and lower medical costs compared with non-vegetarian diets. We reviewed the literature to ascertain the extent to which and by what mechanism(s) a plant-based diet may mediate body weight. PMID- 16673754 TI - A prebiotic substance persistently enhances intestinal calcium absorption and increases bone mineralization in young adolescents. AB - A number of short-term (9 days to 5 weeks) studies have reported that non digestible. oligosaccharides enhance intestinal calcium absorption. Recent interesting data from an intervention trial in adolescents (9-13 years of age) suggest that a non-digestible oligosaccharide can persistently stimulate calcium absorption over 12 months and can also enhance bone mineralization during pubertal growth. PMID- 16673756 TI - Calcium supplementation trials and bone mass development in children, adolescents, and young adults. AB - The development of bone mass during childhood through young adulthood is an important determinant of bone health later in life, and calcium is the major building block. Most randomized, double-blind, placebo-controlled trials of calcium supplementation have been done in girls; however, calcium supplementation in boys has been investigated in recent studies. Positive short-term effects on bone measures during growth has been shown in boys and girls, particularly in weight-bearing appendicular bone, although the lifelong effect is not certain. PMID- 16673757 TI - Stocking density and physiological adaptive responses of broilers. AB - Three trials were conducted to assess the effects of stocking density on physiological adaptive responses of broilers. Male broilers were reared in floor pens under conditions similar to those used commercially in the United States. Accepted indicators of adaptation to a stressor were measured on d 49 including plasma concentrations of corticosterone, glucose, cholesterol, and total nitrites as an indicator of nitric oxide, as well as heterophil:lymphocyte ratio. In trial 1, calculated stocking densities were 20, 25, 30, 35, 40, 45, 50, and 55 kg of BW/ m2 and in trials 2 and 3, stocking densities were 30, 35, 40, and 45 kg of BW/m2. Stocking densities were calculated based on a final BW of 3.3 kg. Linear trend analyses were used to assess the role of stocking density on each of the physiological parameters. Results indicate that stocking density did not cause physiological adaptive changes indicative of stress. PMID- 16673758 TI - Effects of S6-strain Mycoplasma gallisepticum inoculation at 10, 22, or 45 weeks of age on the digestive and reproductive organ characteristics of commercial egg laying hens. AB - Experimental inoculation of commercial laying hens with the S6-strain of Mycoplasma gallisepticum (S6MG) at 20 wk of age, while being maintained under ideal conditions, has previously been shown to affect the lengths and weights of various portions of the reproductive tract. Two trials were conducted in the current study to compare the effects of S6MG inoculation prior to lay at 10 wk of age, during onset of lay at 22 wk of age, and during lay at 45 wk of age on the digestive and reproductive organs of commercial layers similarly housed and maintained under ideal conditions. In each trial, liver weight, liver moisture and lipid concentration, incidence of fatty liver hemorrhagic syndrome, ovary weight, ovarian mature follicle numbers, weights and lengths of the oviduct and oviductal regions, and weights and lengths of the small intestine and small intestinal regions were examined at 60 wk of hen age. At 60 wk, liver lipid concentration was depressed, and isthmus weight, as a percentage of total oviduct weight, was increased in birds that had been inoculated with S6MG at 45 wk. Alterations in liver lipid content and weight of the isthmal portion of the oviduct may occur in response to S6MG inoculation during the later stages of production in layers housed under ideal conditions. PMID- 16673755 TI - Perinatal choline influences brain structure and function. AB - Choline is derived not only from the diet, but also from de novo synthesis. It is important for methyl-group metabolism, the formation of membranes, kidney function, and neurotransmission. When deprived of dietary choline, most adult men and postmenopausal women develop signs of organ dysfunction (fatty liver or muscle damage) and have a decreased capacity to convert homocysteine to methionine. Choline is critical during fetal development, when it influences stem cell proliferation and apoptosis, thereby altering brain structure and function (memory is permanently enhanced in rodents exposed to choline during the latter part of gestation). PMID- 16673759 TI - Effects of feed particle size and feed form on survival of Salmonella typhimurium in the alimentary tract and cecal S. typhimurium reduction in growing broilers. AB - An in vitro experiment and an in vivo experiment were conducted to investigate the effects of feed particle size (coarse and fine) and feed form (mash and pellet) on the survival of Salmonella typhimurium (ST) in the alimentary tract, and the reduction of cecal ST in growing broilers in a 2 x 2 factorial design. All diets in the 2 trials were corn and soybean meal-based diets that differed only in physical characteristics. Diets were provided for birds from d 1 to 22 of age. In the in vitro trial, the relative gizzard weight was lower in birds that were fed the pellet diet (P < 0.01), whereas the relative weight of the cecum was higher in these birds (P < 0.01). Broilers receiving the pellet diet had enhanced concentrations of volatile fatty acids in contents from both the gizzards and the ceca. In addition, there was an increase in gizzard pH (P < 0.01) with the pellet diet, but a reduction in cecal pH (P < 0.05). Pellet-fed birds showed a significantly decreased and increased in vitro death rate of ST in the contents from gizzards (P < 0.01) and ceca (P < 0.05), respectively. A higher in vitro ST death rate in the gizzard was observed in birds given the coarse mash diet compared with those given the fine mash diet. In the in vivo experiment, cecal volatile fatty acid concentrations were increased, whereas cecal pH was decreased significantly (P < 0.05) when birds were fed the pellet diet compared with the mash diet. Furthermore, cecal ST concentrations were higher (P < 0.05) in broilers fed the pellet diet than in those fed the mash diet. Results indicated that the pellet diet increases the incidence of ST in gizzards and ceca in growing broilers and provide evidence demonstrating that the gizzard may play a critical role in reducing ST contamination in growing broilers. PMID- 16673760 TI - Heritability and genetic correlation estimates for performance and carcass and body composition traits in a male broiler line. AB - The current research was conducted to estimate the heritability coefficients and the genetic correlations for performance and carcass and body composition traits in a single sire broiler line. The performance traits analyzed were BW at 38 d, ultrasound records of pectoral muscle depth, feed intake, feed conversion ratio, and BW at 42 d. The carcass traits analyzed were eviscerated BW, breast weight, and leg weight, and the body composition traits analyzed were abdominal fat content, heart weight, gizzard weight, liver weight, and intestine weight. The number of observations varied between 4,120 and 29,040 for each trait. The (co)variance components, heritability, and genetic correlation estimates were obtained by restricted maximum likelihood. The numerator relationship matrix had 42,912 animals. Based on the heritability estimates obtained, the analyzed traits seemed to be able to respond to selection, at variable intensities. The genetic correlation estimates between a great number of performance traits, as well as between a great number of carcass traits, were suggestive of a close genetic relationship between these traits. The genetic correlation estimates between body composition traits were variable. A large genetic association between a great number of performance and carcass traits seemed to exist. The genetic correlation estimates between performance and body composition traits were variable, and important associations between carcass and body composition traits did not seem to exist. PMID- 16673761 TI - Lymphoid organ size varies among inbred lines 6(3) and 7(2) and their thirteen recombinant congenic strains of chickens with the same major histocompatibility complex. AB - The objective was to evaluate lymphoid organ size in chickens from a series of 13 recombinant congenic strains (RCS) and their highly inbred parental lines (6(3) and 7(2)). The parental line 6(3) was selected for resistance to tumors induced by Marek's disease virus and avian leukosis viruses, whereas line 7(2) was selected for susceptibility to these tumors. Each RCS on the average contains a random one-eighth of genome from the donor line 7(2). Previous studies have shown that lines 6(3) and 7(2) differ in the size of primary lymphoid organs; i.e., the bursa of Fabricius (BF) and the lobes of the thymus (T) are smaller in line 6(3) than line 7(2). In the current study, the relative size of the T, BF, and spleen was first examined in about 15 males from each of 13 RCS and the 2 parental lines at 60 to 69 d of age. The differences of relative BF, T, and spleen size among the RCS and the parental lines 6(3) and 7(2) differed significantly (P < 0.001). Males and females from 4 RCS and the 2 parental lines were evaluated a second time, and differences in the relative sizes in lymphoid organs among the RCS and parental lines were consistent. In 2 RCS, the size of the T and BF was comparatively large as in line 7(2), leading to the conclusion that different allelic forms at 1 or more loci in these RCS regulate the size of both organs. In 2 other RCS, the BF was large compared with the T, suggesting that allelic forms at some loci in these RCS influence the BF independent of the T. The relative lymphoid organ size among the RCS appeared to cosegregate with the concentration of IgG in the plasma measured previously. The evaluation of genomic variability of these lines is underway, and the RCS are available for research on traits that differ between lines 6(3) and 7(2). PMID- 16673762 TI - Intestinal microbial ecology of broilers vaccinated and challenged with mixed Eimeria species, and supplemented with essential oil blends. AB - Intestinal microbiota is an important component in the development of defense mechanisms in the gut mucosa. This project determined the dynamics of intestinal microbial communities (MC) of broilers vaccinated at first day of age with live oocysts of Eimeria species and fed diets supplemented with 2 specific essential oil (EO) blends, Crina Poultry (CP) and Crina Alternate (CA). Five treatments were analyzed: 1) unmedicated-uninfected (UU) control; 2) unmedicated-infected (UI) control; 3) vaccinated with Advent cocci-vaccine and without feed additive (COV) supplements; 4) vaccinated with Advent and supplemented with CP; and 5) vaccinated with Advent and supplemented with CA. The EO blends were added at 100 ppm to the same basal diets. Chicks were gavage-infected at 19 d of age with Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Duodenal, ileal, and cecal samples were taken from 12 birds per treatment just before the infection and 7 d after the challenge, pooled in 6 samples, and frozen. Denaturing gradient gel electrophoresis was used to examine PCR-amplified fragments of the bacterial 16S ribosomal DNA variable region. Results are presented as percentages of similarity coefficients (SC). Dendrograms of amplicon patterns indicated MC differences due to intestinal location, feed additives, and cocci infection. The EO blends CP and CA did affect MC in all gut sections. The cocci-infection caused drastic MC population shifts in duodenal, ileal, and cecal sections (36.7, 55.4, and 36.2% SC, respectively). The CP-supplemented birds had higher SC between pre- and postchallenge MC in duodenal and ileal (73.3, 81.8%) than COV (66.4, 66.5%). However, COV broilers had the smallest changes in cecal MC after infection (79.5% SC). We concluded that cocci-vaccination causes small changes in intestinal MC, but challenge causes drastic shifts. The EO blend supplementation modulates MC in cocci-vaccinated broilers, avoiding drastic shifts after a mixed coccidia infection. Correlations between MC dynamics and host responses are discussed. PMID- 16673763 TI - Salmonella challenge affects the antibody isotype profile of bile in hens differing in metabolic efficiency. AB - Gel precipitation reactions determined antibody isotypes in bile from hens differing in dietary efficiency. Ouchterlony double diffusion employing alpha chain specific goat-anti-chicken IgA, rabbit anti-chicken IgG, goat anti-chicken IgM, black turtle bean (BTB), and Jacalin lectins as precipitating reagents detected bile IgA, IgG, and IgM from Salmonella exposed and nonexposed hens. The IgA was present in 1 of 3 forms designated by reagent and frequency: IgAB (precipitated by BTB lectin) 100%; IgAA (precipitated by anti-alpha chain antibody) 98%, and IgAJ (precipitated by Jacalin) 97%. That both BTB and Jacalin precipitates contain IgA was confirmed by immuno-dot blots using affinity purified alpha-chain specific antibody, establishing each as IgA glycoforms. Three measurements of Ouchterlony precipitates were made; d1 and d2 indicate diffusion from sample or reagent wells, lambda indicates arc length. Mean values for lambda, estimating quantity, were IgAA (11.3 mm) and IgAB (11.6 mm) and IgAJ (8.3 mm). The crescent shape IgAJ arc and its slower diffusion (d1) suggested its molecular weight is greater than either IgAA or IgAB. Arc lengths of individual samples were not significantly correlated suggesting that these are independent components of bile. Oral Salmonella enteritidis challenge resulted in a highly significant difference in bile IgA profiles. The IgAJ arc lengths (lambda) in R- hens increased by 20% over those in nonchallenged R- hens. Conversely S. enteritidis challenge was associated with a decrease of 10% in IgAJ arc lengths in nonefficient (R+) hens. Salmonella enteritidis challenge was not associated with arc length differences in either IgAA or IgAB. The IgG was present in all specimens, and in 9 of 59 (15%) 2 forms were detected. The IgG quantity was unaffected by either efficiency type or S. enteritidis challenge. The IgM was detected in only 2 of 59 (3.4%) specimens. Our observations suggest IgA of bile is composed of multiple forms influenced both by diet efficiency status and S. enteritidis exposure. It appears that the latter resulted in an increased quantity of IgAJ in R- hens, and suggests the existence of functional differences among the various IgA types. PMID- 16673764 TI - A method for estimating the relative degree of saponification of xanthophyll sources and feedstuffs. AB - Saponification of xanthophyll esters in various feed sources has been shown to improve pigmentation efficiency in broiler skin and egg yolks. Three trials were conducted to evaluate a rapid liquid chromatography procedure for estimating the relative degree of xanthophyll saponification using samples of yellow corn, corn gluten meal, alfalfa, and 6 commercially available marigold meal concentrates. In each trial, samples were extracted using a modification of the 1984 Association of Official Analytical Chemists hot saponification procedure with and without the addition of KOH. A comparison of the chromatography results was used to estimate percent saponification of the original sample by dividing the nonsaponified extraction values by the saponified extraction values. A comparison of the percent saponified xanthophylls for each product (mg/kg) was: yellow corn, 101; corn gluten meal, 78; alfalfa, 97.9; and marigold concentrates A through F, 99.8, 4.6, 99.0, 95.6, 96.8, and 6.6, respectively. These results indicate that a modification of the 1984 Association of Official Analytical Chemists procedure and liquid column chromatography can be used to quickly verify saponification and can be used to estimate the relative degree of saponification of an unknown xanthophyll source. PMID- 16673765 TI - Response of turkey poults to soybean lectin levels typically encountered in commercial diets. 2. Effect on intestinal development and lymphoid organs. AB - Lectins are capable of altering intestinal morphology by binding to and disrupting the intestinal brush border membrane. They are also known to alter the weight of lymphoid organs. Therefore, we evaluated the effect of soybean lectin (SBL) on intestinal morphology and lymphoid organ weights of poults fed diets containing SBL. Dietary treatments evaluated in this study included a cornstarch and casein-based control (lectin-free) semipurified diet (PD) and semipurified diets containing 0.024 or 0.048% SBL (PDL and PDH, respectively). Experimental diets were fed from hatch to 14 d. Morphological evaluation of the intestine involved measurement of the villi height and perimeter, crypt depth, villus:crypt, and thickness of the muscle layer in the jejunum. Intestinal physical characteristics were also determined by measuring intestinal weight, length, and volume. Results indicated that 0.048% SBL in PDH increased villus:crypt and reduced total intestinal length in turkey poults. In addition, both the 0.024 and 0.048% dietary SBL levels reduced thymus weights. It was concluded that dietary SBL up to 0.048% enhanced intestinal development by increasing villus:crypt, but might alter the structural integrity of lymphoid organs. PMID- 16673766 TI - Phytic acid and phytase: implications for protein utilization by poultry. AB - The effect of the ingestion of myo-inositol hexaphosphate (IP6) and phytase (EC 3.1.3.26) on the digestibility of casein was investigated using growing broiler chickens. A total of 64 female Ross broilers were used in a precision feeding study. One group of 8 birds was fed a solution of glucose to estimate endogenous losses. Seven groups, each of 8 birds, were fed either casein, casein + 1,000 units of phytase activity (FTU), casein + 2,000 FTU, casein + 0.5 g of IP6, casein + 0.5 g of IP6 + 1,000 FTU, casein + 1 g of IP6, or casein + 1 g of IP6 + 1,000 FTU. The excretion of DM, amino acids, nitrogen, minerals, and phytate phosphorus was determined over a 48-h period and nutrient digestibility coefficients were calculated. Casein was found to be highly digestible, with true coefficients of DM, N, and amino acid digestibility of between 0.85 and 1.0. However, the ingestion of IP6 reduced (P < 0.05) the digestibility coefficients of amino acids, N, and DM of casein compared with birds fed casein alone. Supplementation of the mixture of casein and IP6 with phytase improved (P < 0.05) the digestibility coefficients of amino acids compared with birds fed on casein and IP6 with no supplemental phytase. The excretion of endogenous minerals was increased (P < 0.05) by the ingestion of IP6 and reduced (P < 0.05) by the supplementation of IP6 with phytase. In the absence of exogenous phytase, the recovery of phytate-P in excreta was approximately 80%. However, the recovery of phytate-P was significantly reduced by the addition of exogenous phytase to the IP6/casein mixture. It can be concluded that the ingestion of IP6 reduces the digestibility coefficients of amino acids and the metabolizability of nitrogen of casein. This is likely to be mediated partially through increased endogenous losses. However, the addition of phytase can partially ameliorate the detrimental effects of IP6 on protein utilization. PMID- 16673768 TI - Semen characterization and sperm storage in Cabot's Tragopan. AB - The semen quality of Cabot's Tragopan, the dependence of sperm yields on frequency of semen collection, and the duration of sperm storage in females were investigated. The results are as follows: 1) The average duration of the period in which Cabot's Tragopan can produce an ejaculate was about 70 d. The ejaculate volume ranged from 15 to 100 microL. The average concentration of the ejaculate was 2.31 x 10(9) mL(-1). There were 11.69 (+/- 0.77)% abnormal spermatozoa per ejaculate. Three of 11 males yielded more than 50 microL of semen per collection most of the time. 2) The ejaculate volumes, the concentration, the total number of sperm per ejaculate, and the daily sperm output were all markedly affected by the frequency of semen collection (P < 0.01). However, no significant difference was detected in characters between the 2 groups with relatively low collection frequency (P > 0.05) except the daily sperm output (P < 0.01). The highest frequency of semen collection did not yield more sperm. 3) The average duration of the period in which the female laid fertilized eggs after single insemination was 19.85 +/- 3.08 d (range 9 to 32 d, n = 7). This value was affected by the rhythm of egg laying and varied among individuals. All of the results will facilitate design of the optimal artificial insemination strategy and help to achieve the ultimate aim of ex situ conservation. PMID- 16673767 TI - Performance and gastrointestinal tract metabolism of turkeys fed diets with different contents of fructooligosaccharides. AB - Different levels of dietary fructooligosaccharide (FOS) administered for 8 wk to commercial male turkeys were evaluated for their efficacy on performance and physiological response in the digestive tract. Special attention was paid to cecal metabolism. The following levels of FOS were used in a diet: 0.5, 1, and 2%. After 8 wk of experimental feeding, the diet intake, body weight, and feed efficiency ratio were similar in all groups examined. The pH of ileal and cecal contents were reduced by dietary treatments, especially when 2% of dietary FOS was used. The highest ammonia and Lowry's protein concentrations were associated with elevated amounts of FOS preparation in a diet (1 and 2%). Bacterial enzyme activity remained statistically unaffected by experimental treatments; however, a slight beneficial decrease in the activity of beta-glucosidase and beta glucuronidase was observed after ingestion of the FOS preparation. The addition of FOS to a diet did not affect short-chain fatty acid concentration but gave greater short-chain fatty acid pool in the ceca of turkeys, especially in the case of the highest dose of FOS. PMID- 16673769 TI - Pulmonary arteriole remodeling in hypoxic broilers expressing different amounts of endothelial nitric oxide synthase. AB - Lung samples from 30 pulmonary hypertensive chickens expressing low nitric oxide (NO) synthase activity in endothelial arteriolar cells were compared with samples taken from 30 nonhypertensive animals expressing high activity to investigate a possible relationship between the just-mentioned expressions and pulmonary vascular remodeling. The external diameter and media muscular thickness in 20 arterioles of 50- to 200-microm external diameter were measured in lung samples from 17-, 30-, and 42-d-old broilers to calculate %T (medial wall width divided by external vessel diameter x 100). The %T was higher in hypertensive chickens than in nonhypertensive (P < 0.01) chickens and was inversely related to NO synthase enzyme (P < 0.01). It can be inferred from this study that NO is involved in the remodeling process in broilers with hypoxic pulmonary hypertension syndrome. PMID- 16673770 TI - Meat quality evaluation of minimally aged broiler breast fillets from five commercial genetic strains. AB - A total of 1,040 birds from 5 common commercial genetic broiler strains were raised and processed to analyze the effect of strain and deboning time on meat quality. The birds were processed at either 6 or 7 wk of age in 4 replications each. Carcasses were deboned at either 2 or 4 h postmortem (PM; n = 52 birds per treatment). Carcass and breast weights were measured on each bird to calculate breast yield. Muscle pH was measured at time of deboning. Fillets deboned at 4 h PM were measured for length, width, and height to evaluate footprint analysis. At 24 h PM, fillets were weighed to calculate drip loss, and color (L*) was also measured. The fillets were then cooked to 76 degrees C, and cook loss was calculated. Fillets were then subjected to shear analysis using the Meullenet Owens razor shear method where shear energy (N x mm) was calculated to evaluate tenderness. The strains in this study were chosen for differences in yield; therefore, as expected, breast yield was significantly different among strains. Variation in meat quality attributes existed among strains deboned at 2 h PM, but there was no consistent relationship between meat quality and breast yield. However, at 4 h PM, fewer differences among strains existed in meat quality characteristics (tenderness, water holding capacity, and pH). As expected, deboning at 2 h PM resulted in higher shear energy, higher muscle pH, and lower L* value compared with deboning at 4 h PM in all but one strain. However, water holding capacity was not affected by deboning time at either age interval. Footprint analysis showed that most differences among strains were in heights measured at the fillet midpoint and caudal end. These results suggest that early deboning may affect meat quality of broiler strains differently, resulting in greater variation within the industry. PMID- 16673771 TI - Effect of different cleaning regimens on recovery of Clostridium perfringens on poultry live haul containers. AB - Clostridium perfringens is important to both poultry producers and humans. The excretion rate of pathogenic foodborne bacteria increases after live haul; however, the majority of research into flock cross-contamination has been performed on Salmonella and Campylobacter. Research into the sources of C. perfringens in poultry operations have implied that dirty transport containers do harbor this organism and, therefore, can potentially contaminate subsequent flocks. The objectives of this study were to examine both small plastic crates and large dump coops to determine which cleaning regimens were most effective in reducing C. perfringens contamination. Additionally, 2 different holding periods for small crates were compared to determine whether holding time influences C. perfringens recovery before and after cleaning. Two experiments were performed. One involved small plastic crates; the other involved large dump coops. Four small crate cleaning and disinfection treatments consisted of pressure washing, pressure washing and sun-drying, pressure washing with a (5%, vol/vol) sodium hypochlorite dip, and pressure washing with a quaternary ammonium dip. The second experiment involved dump coops. The 5 dump coop cleaning and disinfection treatments consisted of pressure washing, pressure washing with a (5%, vol/vol) sodium hypochlorite spray, pressure washing with a quaternary ammonium spray, 48 h drying after the sodium hypochlorite spray, and 48-h drying after the quaternary ammonium spray. The recovery of C. perfringens from small and large dirty transport containers averaged 1.94 and 4.43 log10 cfu/mL, respectively. There was no significant difference in C. perfringens recovery based on holding time for small crates. With small crates, pressure washing provided a significant decrease in the amount of C. perfringens recovered. The greatest bacterial reduction in dump coops, 2 to 3 log10 cfu/mL, was observed after 48 h of drying. This information provides solutions to poultry operations to reduce the cross contamination of this food safety pathogen via transport containers. PMID- 16673772 TI - Effects of intramuscular fat levels on sensory characteristics of duck breast meat. AB - We conducted a study to evaluate the effects of intramuscular fat levels on the sensory characteristics of duck breast meat. Combining duck genotypes (Muscovy, Pekin, and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 wk of age vs. ad libitum feeding) enabled us to obtain a wide range of lipid levels in breast muscle. The average values were between 2.55 and 6.40 g per 100 g of muscle. Breast muscle from overfed ducks showed higher lipid and lower water levels than breast muscle from ducks fed ad libitum. Muscle from the overfed ducks was also paler in color and exhibited greater yellowness and cooking loss values. Juiciness was judged lower and flavor more pronounced in overfed ducks. Muscovy ducks exhibited higher breast weight and lower lipid levels than the other genotypes. At the other extreme, Pekin ducks exhibited the highest lipid levels and the lowest breast weights; values for these criteria were intermediate in hinny and mule ducks. Breast muscle of Muscovy ducks was paler, less red, and more yellow than that of other genotypes. Breast muscle of Pekin ducks exhibited the lowest values for lightness, yellowness, and energy necessary to shear meat, as well as the highest cooking loss values, and was judged more tender, juicy and less stringy than that of other genotypes. In contrast, scores for breast muscle of Muscovy ducks were the lowest for tenderness, juiciness, and flavor, and the highest for stringiness. Breast muscle of hinny and mule ducks scored the highest values for redness. Hinny ducks also scored the highest values for flavor. Genotype exerted a higher effect on the sensory quality of breast muscle than did feeding levels. Finally, increasing lipid levels in breast muscle increased lightness, yellowness, cooking loss, tenderness, and flavor, with correlation coefficients of 0.49, 0.47, 0.54, 0.43, and 0.28, respectively. However, breast meat color and tenderness were mainly influenced by genotype. PMID- 16673773 TI - Short-term number of pecks and feed intake levels: a link with the physical characteristics of feed in four-week-old turkeys. AB - Short-term reactions of young turkeys to changes in the physical characteristics of their feed sometimes give rise to problems of adaptation in the field. Specific feeders were designed for this study to automatically count the number of pecks given at feed by 4-wk-old male turkeys during 95-min sessions. The lighting program was intermittent (0140L:0100D). In experiment 1, 4 series of 12 birds were tested 4 times/day from 28 to 31 d of age. Four control turkeys were consistently tested with 1 feed, and 8 experimental turkeys ate 1 of 32 commercial feeds (30 as pellets and 2 as crumbs) with a distinct feed for each test (16 replicates/experimental feed). Feed intake, total number of pecks, and pecking efficiency (mg/peck) were recorded for each test and normalized for each turkey. The effects of series, day, and test were not significant for controls. The number of pecks and pecking efficiency differed among feeds (P < 0.001) but not feed intake. Hierarchical Cluster Analysis for these variables distinguished 3 groups of feed: group 3 (6 crumb and small pellets) resulted in high pecking and low efficiency. However, group 1 (10 feeds) were pecked at less but more efficiently than group 2 (16 feeds) for similar feed intake, although the average sizes of the pellets were similar (6.23 and 6.16 mm, respectively), as were the average bulk density, hardness, resistance to abrasion, and color characteristics of the pellets for both groups. Two feeds of each of these 2 groups were compared in a second experiment for 12 turkeys receiving the 4 feeds successively each day over 4 d in varying order. The turkeys consistently pecked the feeds of group 1 less and less efficiently compared with group 2. The automated measurement of pecking, combined with a more detailed image analysis of feed particles, might enhance the evaluation of the effects of feed technology on the behavior of turkeys and thus become a useful tool for the feed industry. PMID- 16673774 TI - The effects of suboptimal eggshell temperature during incubation on broiler chick quality, live performance, and further processing yield. AB - Different incubation conditions can cause eggshell temperature (EST) to deviate from optimum. Two experiments were performed to determine the effect of low EST at the start of incubation and high EST at the end of incubation on hatchability, chick quality, 6-wk live performance, and breast meat yield of broiler chickens. In each experiment, 1,800 eggs from a single flock were divided and set into 2 setters. From 0 to 10 d of incubation, one setter was set to attain an EST of 36.6 degrees C (considered low), whereas the other was set to 37.8 degrees C (the control temperature). Using an infrared thermometer, EST was measured daily on a sample of eggs to ensure treatment intentions. On d 11 of incubation, the temperature of the low EST setter was increased to 37.8 degrees C in synchrony with the other setter until transfer. On d 18 of incubation, eggs from both setters were combined into 2 equal groups and transferred to hatchers. The EST in one hatcher was set to 37.8 degrees C (control) and in the other to 39.5 degrees C (considered high) until 21 d of incubation. Hatched males were placed in battery cages (Experiment 1) or floor pens (Experiment 2) and reared on common feeds to 1 or 6 wk of age, respectively. Low EST in the first 10 d of incubation reduced hatchability, increased BW and chick yield, and reduced 1-wk gain compared with the control EST. Throughout rearing, BW was reduced for low EST chicks compared with control EST chicks; consequently, carcass, fillet, and tender weights were also reduced. High EST in the hatcher increased hatchability, and reduced BW, chick yield, and 1-wk gain compared with control EST in the hatcher. By 3 wk of age, there was no difference in BW between chicks in high EST and control EST treatments. Subsequent carcass and processing yields were also similar. Incubation at the control EST of 37.8 degrees C, particularly from 0 to 10 d, resulted in the best performance overall. PMID- 16673775 TI - Poult performance as influenced by age of dam, genetic line, and dietary vitamin E. AB - An experiment was conducted to measure the effects of age of dam, genetic line, and dietary levels of vitamin E on growth and immunocompetence of poults. Age of dam was defined as younger (in early egg production) and older (past peak production); line consisted of a commercial sire and dam line; and dietary vitamin E was supplemented into the diet at 10 and 300 IU/kg of feed. Traits measured included body, liver, gizzard, and yolk sac weights at hatch; BW and feed conversion to 9, 28, and 42 d; response to SRBC, Phaseolus vulgaris agglutinin-P, and Escherichia coli administered at 28 d of age; and response to a cold stress on d 5 posthatch. Differences among genetic lines were evident with growth greater for poults from the sire than from the dam line. Performance of poults from older dams was generally superior to that of poults from younger dams. The higher level of vitamin E resulted in a greater than 7-fold increase in blood plasma vitamin E and reduced mortality. There were interactions among the main effects in which the fitness of poults from younger dams was enhanced by the higher level of vitamin E and the effect of breeder age differed among genetic lines. PMID- 16673776 TI - Modeling growth characteristics of meat-type guinea fowl. AB - This study was conducted to describe the growth pattern of the French guinea fowl, a meat-type variety. Using BW data from hatch to 9 wk, 2 nonlinear mathematical functions (Gompertz and logistic) were used to estimate growth patterns of the French guinea fowl. The French guinea fowl did not exhibit sexual dimorphism for growth characteristics. From the Gompertz model, the asymptotic BW, growth rate, and age at maximum growth were 2.05 kg, 0.25 kg/wk, and 5.74 wk in males, respectively, and 2.03 kg, 0.25 kg/wk, and 5.72 wk in females, respectively. The ages at maximum growth were 5.75 and 5.74 wk for males and females, respectively, using the logistic model. Differences in asymptotic BW between males and females were not significant in both Gompertz and logistic models. However, the average asymptotic BW of about 1.50 kg for both sexes predicted by the logistic model was below the average predicted BW from the Gompertz model (2.04 kg) at 9 wk. Also, the logistic model overestimated hatching weight (0.06 kg) more than the Gompertz model (0.03 kg), suggesting that the growth pattern of the French guinea fowl is Gompertz. The inverse relationship between the asymptotic weight and age at maximum growth of the French guinea fowl is similar to that of the pearl gray guinea fowl, chickens, quail, and ducks. Understanding the growth characteristics of French guinea fowl will contribute to the efforts of improving production efficiency of this least studied avian species. PMID- 16673777 TI - Appropriate statistical methods to compare dose responses of methionine sources. AB - Two sources of methionine (Met) activity are frequently used in commercial feed formulation: DL-2-hydroxy-4-(methylthio) butanoic acid (HMTBA), most commonly available as an 88% solution with 12% water; and DL-methionine (DLM, 99% powder). Despite the fact that both compounds have been in commercial use for over 50 yr, controversy and confusion remain with respect to their relative bioefficacy (RBE). This paper presents a review of the use of a nonlinear common plateau asymptotic regression technique (NLCPAR) that has been used to compare the 2 Met sources with particular emphasis on the validity of the basic assumptions of that model. The thesis of this paper is that the controversy is due, at least in part, to the misapplication of this regression technique to estimate the RBE of HMTBA and DLM. The NLCPAR model is a bioassay with the key dependent assumptions that HMTBA is a dilution of DLM, and that each follows dose-response curves of the same form and approach a common plateau. Because both provide Met activity, it may be considered reasonable to accept these assumptions; however, specifically testing them demonstrated that the assumption of a common dose-response is not supported by data. The common plateau assumption was tested with an alternative approach of fitting nonlinear separate plateaus asymptotic regression (NLSPAR) to a set of 13 published broiler studies in which the NLCPAR model had been used to estimate RBE of HMTBA and DLM. The hypothesis of a common plateau was rejected (P < 0.01), meaning that the conclusion that HMTBA had lower bioefficacy than DLM based on the NLCPAR methodology was not valid. An example using published data demonstrated that the NLSPAR model was a significantly better fit than the NLCPAR model, and showed that HMTBA and DLM followed different dose responses. Consequently, there was no single value for RBE for the entire dose range; rather, the RBE of the 2 compounds varied with use level. The evidence presented here indicates that separate plateau models should be used when comparing these 2 products. These more valid models can then be used for predictions of differences between HMTBA and DLM at levels of expected use. PMID- 16673778 TI - Tools for transforming language. PMID- 16673779 TI - What's ahead for Medicaid. PMID- 16673780 TI - Politicking for the substance abuse field. PMID- 16673781 TI - A troubled beginning for Part D. PMID- 16673782 TI - Seven governing realities of mental health policy. PMID- 16673783 TI - New initiatives in the EAP field. PMID- 16673784 TI - Placing the risk of diabetes in context. PMID- 16673785 TI - Reexamining the suicide connection. PMID- 16673787 TI - A soft landing after benefit cuts. PMID- 16673786 TI - Point-of-care tests in behavioral health. PMID- 16673788 TI - Preparing for a new era: EHR standards for behavioral healthcare. PMID- 16673789 TI - Pick the right team for an EMR home run. PMID- 16673790 TI - You have to run faster than the other guy. PMID- 16673791 TI - Viable spore counts in biological controls pre-sterilization. AB - The aim of the present study was to evaluate the total count of viable spores in standardized inoculated carriers pre-sterilization. Samples of "Bacterial Spore Sterilization Strip" (R Biological Laboratories) (well before their expiry date) were divided into Group A (B. subtilis) and Group B (B. stearothermophylus). Twenty-four strips were tested per group. The strips were minced in groups of three, placed in chilled sterile water and vortexed for 5 minutes to obtain a homogenous suspension. Ten ml of the homogenous suspension were transferred to two sterile jars, i.e. one jar per group. The samples were then heated in a water bath at 95 degrees C (Group A) or 80 degrees C (Group B) for 15 minutes and cooled rapidly in an ice bath at 0- 4 degrees C during 15 minutes. Successive dilutions were performed until a final aliquot of 30 to 300 colony-forming units (CFU) was obtained. The inoculums were placed in Petri dishes with culture medium (soy extract, casein agar adapted for spores, melted and cooled to 45-50 degrees C) and incubated at 55 degrees C or 37 degrees C. Statistical analysis of the data was performed. A larger number of spores were found at 48 hours than at 24 hours. However, this finding did not hold true for all the groups. The present results show that monitoring viable spores pre-sterilization would guarantee the accuracy of the data. Total spore counts must be within 50 and 300% of the number of spores indicated in the biological control. The procedure is essential to guarantee the efficacy of the biological control. PMID- 16673792 TI - Antimicrobial effect in vitro of chlorhexidine and calcium hydroxide impregnated gutta-percha points. AB - The aim of this study was to evaluate the antimicrobial activity of points containing antiseptics used for temporary obturation in endodontics. Points containing calcium hydroxide (Roeko and Hygienic), and chlorhexidine (Roeko) were tested and gutta-percha points served as control (Meta Dental Corp.). The following microorganisms were studied: Streptococcus mutans (Sm), Enterococcus faecalis (Ef), Staphylococcus aureus (Sta), Candida albicans (Ca), Fusobacterium nucleatum (Fn) ATCC 25586, Porphyromonas gingivalis (Pg) ATCC 33277, and Prevotella intermedia (Pi) ATCC 25611. The experiments were organized in three stages: 1st stage: one point of each kind was placed on agar plates previously seeded with microorganisms. 2nd stage: Another group of points was immersed in broth for 24 hours and placed on the seeded agar. Samples were then incubated at 37 degrees in the conditions of oxygen and for the time required by each microorganism. The zones of bacterial inhibition around each point were measured. The pH values of the broths were recorded. 3rd stage: the immersion broths were inoculated with suspensions of microorganisms, incubated, seeded on plates by dissemination and incubated. CFU counts were performed. RESULTS: points containing chlorhexidine showed inhibition zones with every microorganism in the 1st stage and, in the 2nd stage, with most of the microorganisms studied, except for Fn and Pi. Calcium hydroxide containing points did not inhibit any of the microorganisms assessed. Broth pH values did not exhibit any changes. CFU counts of the broths in which chlorhexidine points had been immersed showed total inhibition for all the microorganisms. The differences between materials were statistically significant (p<0.05) (ANOVA). In the conditions of this study, chlorhexidine-containing points proved to be effective against most of the tested strains. PMID- 16673793 TI - A scanning electron microscopy evaluation of different root canal irrigating solutions. AB - The efficacy of solutions to remove the smear layer from human teeth was evaluated. One and 2.5% sodium hypochlorite, 1 and 5% calcium hydroxide, 0.2% (solution) and 2% (gel) chlorhexidine gluconate, 1% tea and distilled water as control were assessed. Fifty-six freshly extracted permanent human incisors were selected. Access cavities, conductometric measurements and apical sealing with wax were performed. Root canals were instrumented up to a #40 file at the apical level and with the step-back technique up to #60 file and irrigated with 25 ml of each solution. Roots were longitudinally cut and cleaning of dentinal walls in the coronal, middle and apical thirds was evaluated by scanning electron microscopy. The ANOVA test showed no statistically significant differences between the thirds treated with the same solution (p = 0.562), but showed differences between solutions (p = 0). Although none of the solutions showed complete elimination of the smear layer, sodium hyplochlorite solutions were the most efficientfollowed by calcium hydroxide solutions and chlorhexidine. Tea and distilled water were less effective. PMID- 16673794 TI - An experimental model in calvaria to evaluate bone therapies. AB - The clinical therapeutic application of experimental strategies requires extensive preclinical experimentation in appropriate animal models. Thus, a valid model must be established. The aim of the present study was to determine the critical size defect (CSD) of rat calvaria that is unable to undergo spontaneous bone regeneration. Forty Sprague Dawley female rats (body weight (bw): 250 +/- 20 g) were distributed in two groups. Circular surgical defects, 3 mm (Group A) and 5 mm (Group B) in diameter, were produced in the parietal bones. The animals were left untreated and sacrificed 1, 2, 3 and 6 weeks after surgery. Group A showed bone formation at the experimental site, increasing from 1 week (4.5%) to 6 weeks (46%). However, Group B showed scarce bone formation (less than 10%) throughout the experimental period. We may conclude that a defect 5 mm in diameter is a critical size defect (CSD) because it is the minimum bone defect size that requires treatment to heal. Thus, a circular defect 5 mm in diameter in rat calvaria would be an appropriate experimental model to study bone therapies. PMID- 16673795 TI - Early acquisition of Streptococcus mutans for children. AB - Existing evidence reveals that in Early Oral Infection the main route of transmission of Streptococcus mutans is mother-child saliva contacts and that initial acquisition takes place during a specific period of time denominated "Window of Infectivity" that lapses between 6 and 30 months of the child's life, with a higher risk between 18 and 30 months of age. The aim of the present study was to analyze Early Oral Infection. The levels of Streptococcus mutans in saliva and bacterial plaque in the binomial mother-child 6 to 18 months after childbirth were evaluated. Twenty-four mother-child binomials that attended the University Hospital of Maternity and Neonatology of Cordoba participated in the study. Samples of saliva and dental plaque were taken from mother and child and seeded in selective Agar Mitis Salivarius Bacitracin medium (0.28 mg/ml) and cultured for 48 hours at 37 degrees C and in 5% CO2 to allow for the growth of Streptococcus mutans. The colonies were identified morphologically and biochemically. At 6 months after childbirth: 58.33% of the samples of the mother's saliva were positive, while the presence of the microorganism was not detected in 100% of the samples of the infants' saliva. At 18 months of after childbirth: 79.16% of the samples of saliva and 100% of the samples of the mothers' dental plaque were positive, whereas the samples of saliva and dental plaque of the infants were positive for Streptococcus mutans in 20.83% and 70.83% of the cases respectively. The initial acquisition of Streptococcus mutans would have occurred in 20.8% of the children at the age of 18 months. Eruption of primary molars occurs at this age. PMID- 16673796 TI - Efficiency of a schoolchildren program for oral care. AB - The aim of this investigation was to evaluate the efficiency of an oral school program, applied during 3 years. DMFT index, the component D and CTNI of a sample of 590 children (Group I, aged 12.0 +/- 1.1 years) which received regular oral treatment during 3 consecutive years were compared with the same indicators of 586 children (Group II, aged 12.6 +/- 1.0 years) without treatment. The cost benefit ratio of the current required treatments in both groups was calculated. Results revealed: (a) in Group I the component D of DMFT was 0.9 +/- SEM = 0.3, and in Group II, 5.5 +/- SEM = 1.5; and (b) the cost-benefit ratio of the required treatment for Group I ranged from 1.5 to 7.5 times lower than that required for cumulative pathology resolution in groups lacking preventive coverage. It seems advisable to introduce management strategies to change spontaneous demand into programmed care with a strong preventive component. The accomplishment of this regular school program appears to contribute to strengthen equity and could reduce the differences in oral health care among schoolchildren at different social risk. PMID- 16673797 TI - Metronidazole in the treatment of rosacea: do formulation, dosing, and concentration matter? AB - BACKGROUND: Topical metronidazole is commonly used in the management of rosacea. No consensus on the optimal formulation, concentration, or dosing regimen exists. PURPOSE: To assess the relative efficacy of metronidazole cream, gel, and lotion at concentrations of 0.75% and 1%, in dosing regimens of once and twice daily. METHODS: A meta-analysis of published metronidazole efficacy rates was performed. RESULTS: In non-weighted analysis, the mean efficacy was 28.2% (95% confidence interval [CI], 22.0%-34.4%) for the cream, 38.4% (95% CI, 18.4%-58.4%) for the gel, and 35% for the lotion. Confidence intervals for QD versus BID dosing and 0.75% versus 1% concentrations also overlapped. In weighted analysis, the mean reduction was 31.3% for the cream, 22.1% for the gel, and 35% for the lotion. CONCLUSIONS: Metronidazole cream, gel, and lotion vehicles have similar efficacies. There were no substantial differences between concentrations of 0.75% and 1%, or between once daily and twice daily regimens. PMID- 16673798 TI - Sun protection strength of a hydroquinone 4%/retinol 0.3% preparation containing sunscreens. AB - BACKGROUND: Dyschromias are common and significantly impact patients' quality of life. Formulas containing hydroquinone 4% are effective in these conditions. Since exposure to ultraviolet radiation (UVR) can worsen disease and complicate treatment, the incorporation of sunscreens can avert this problem. METHODS: We tested the sun protection factor of a hydroquinone formulation (Lustra-Ultra, TaroPharma, Hawthorne, NY) containing avobenzone 3%, and octinoxate 7.5% according to the FDA Sunscreen Monograph on 20 volunteer subjects. We also determined the UVR absorbance spectrum of the preparation. RESULTS: The mean sun protection factor (SPF) of 21.7 satisfied labeling requirements for SPF 20. The formulation exhibited strongest photoprotection near the wavelengths of peak sun burning effectiveness in the UVB region and maintains significant UVR absorbance through the entire UVA region. CONCLUSIONS: Avobenzone 3% and octinoxate 7.5% provide broad spectrum UV protection. Incorporating these sunscreens into a hydroquinone preparation simplifies the treatment regimen while providing significant photoprotection for patients being treated for dyschromia. PMID- 16673799 TI - Short-term safety assessment of clobetasol propionate 0.05% shampoo: hypothalamic pituitary-adrenal axis suppression, atrophogenicity, and ocular safety in subjects with scalp psoriasis. AB - Clobetasol propionate is known to be a very effective treatment for psoriasis; however, its use is limited by potent corticosteroid class related side effects such as hypothalamic-pituitary-adrenal (HPA) axis suppression and atrophogenicity. The aim of this single-center, parallel group, randomized study was to assess the HPA axis suppression potential, atrophogenicity, and ocular tolerability of clobetasol propionate shampoo in 26 patients with scalp psoriasis. Suitable subjects were treated once daily for 4 weeks with clobetasol propionate shampoo, to be rinsed off after 15 minutes or with a leave-on clobetasol propionate gel. The study demonstrated that clobetasol propionate shampoo did not lead to HPA axis suppression or to skin atrophy. Conversely, the gel led to HPA axis suppression and a decrease in skin thickness. Neither formulation had an impact on ocular safety. Despite the short contact application time, the clobetasol propionate shampoo provides similar efficacy results to the gel. PMID- 16673800 TI - Topical tacrolimus for the treatment of psoriasis on the face, genitalia, intertriginous areas and corporal plaques. AB - Tacrolimus is an immunosuppressive drug that has proved effective in the treatment of psoriasis when administered systemically. Topically, it seems only useful in thin psoriasis plaques located on the face, genitalia, and intertriginous areas. We present an open-label clinical trial to test the efficacy of 0.1% tacrolimus ointment in patients with psoriasis on the face, intertriginous areas, both, and in corporal plaques. Efficacy was assessed with the evaluation of erythema, desquamation, infiltration, reduction of the PASI, and reduction of itching. A total of 15 patients were enrolled in the study. In all the localizations evaluated, each of the signs (erythema, desquamation, and infiltration) showed a statistically significant improvement when compared to the baseline (p < .001). Itching also improved rapidly. PASI was also reduced from a mean of 12 at baseline to 2.2 at the end of the study. Of the 15 patients, only 2 experienced an adverse effect (13%), which was described as a warm sensation in facial lesions which was transient and self-limited. In conclusion, tacrolimus ointment may be an alternative to classical options for the treatment of psoriasis, not only for intertriginous, genital, and facial areas, but also for corporal plaques without occlusion, with good tolerance. PMID- 16673801 TI - Alefacept in the treatment of psoriatic nail disease: a proof of concept study. AB - Nail psoriasis can be debilitating and as therapeutic options are limited, it can be notoriously difficult to treat. As there are many new medications currently undergoing clinical trials in psoriasis, questions have arisen concerning the effectiveness of these new therapies with regard to psoriatic nail disease. We present the results of a prospective, open-label, proof of concept study to determine the efficacy and safety of alefacept in subjects with moderate to severe nail psoriasis. PMID- 16673802 TI - The utility of benzoyl peroxide in hydrophase base (Brevoxyl) in the treatment of acne vulgaris. AB - Available for more than 5 decades, benzoyl peroxide has been a "workhorse" of acne therapy. The benefits of this agent include reduction in Propionibacterium acnes (P. acnes) with decrease in inflammatory lesions, efficacy as both "leave on" and cleanser formulations and reduced emergence of antibiotic-resistant P. acnes strains. As the effect of benzoyl peroxide on P. acnes is a direct toxic effect rather than as a "true" antibiotic, resistance to benzoyl peroxide does not occur and has never been reported. Benzoyl peroxide in hydrophase base (Brevoxyl Creamy Washes and Gels) has shown significant efficacy in the treatment of acne, with lower irritancy than other benzoyl peroxide preparations. It is felt that the low irritancy of this product is related to a unique delivery vehicle containing dimethyl isosorbide, which dissolves benzoyl peroxide crystals on the skin. Clinical studies demonstrating the efficacy and safety of benzoyl peroxide in hydrophase base will be reviewed. PMID- 16673803 TI - Photodynamic therapy induces less pain in patients treated with methyl aminolevulinate compared to aminolevulinic acid. AB - Photodynamic therapy (PDT) with topical application of either aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) is increasingly used for the treatment of actinic keratoses (AKs). Although PDT is a well-tolerated treatment, pain is the most severe side effect. Sixty-nine patients (mean age 69 years, 61 male, 8 female) suffering from multiple AKs on the scalp (field cancerization) were included in the study. PDT treatment was performed, using red light (Waldmann PDT 1200) with a light dose of 100 J/cm2 delivered at a dose rate of 160 mW/cm2. Either ALA or MAL were used as photosensitizer. Patients were asked to rate their pain on a scale from 0 to 10 (0 = no pain, 10 = unbearable pain). Upon reaching a score of 10 treatment was discontinued. Comparison of ALA and MAL in patients with AK revealed that MAL induced less pain than ALA. While 14% of the patients treated with MAL discontinued treatment before reaching the required light dose of 100 J/cm2, the number of patients treated with ALA who discontinued treatment reached 54%. PDT using MAL appears to be a better tolerated treatment for multiple AKs on the scalp compared to ALA-PDT. Differences in selectivity for tumor cells and transport of ALA in peripheral neurons may play a role. PMID- 16673804 TI - An intra-individual randomized safety and efficacy comparison of clobetasol propionate 0.05% spray and its vehicle in the treatment of plaque psoriasis. AB - BACKGROUND: Plaque psoriasis affects about 2% of the US population. A new and unique spray formulation of clobetasol propionate (CP) 0.05% was developed to provide advantages over the currently available treatment formulations. OBJECTIVES: To evaluate the efficacy and safety of CP 0.05% spray compared to its vehicle in the treatment of moderate to severe plaque psoriasis. METHODS: A 4 week, single-center, randomized, double-blind, vehicle-controlled, intra individual study in subjects with plaque psoriasis. Each of 2 target lesions per subject were randomized to receive either CP 0.05% spray or its vehicle twice daily over 4 weeks. Efficacy parameters included overall target plaque severity score, scaling, erythema, and plaque elevation at all visits. Adverse events were reported throughout the study. RESULTS: A total of 27 subjects were enrolled in the study. At all visits there was a significant intra-individual treatment effect for the overall target plaque severity (P < .001) in favor of CP spray. Throughout the study, results for scaling, erythema, and plaque elevation were significantly (P < .001) in favor of CP spray. After 4 weeks of treatment, all intra-individual response measures, with the exception of erythema, showed an average difference in severity scores of more than 4 points (based on a 9-point scale) between the lesions treated with CP 0.05% spray and the lesions treated with vehicle. No serious adverse event occurred during the course of the study. One local adverse event at the application site (5%) was considered probably related to study medication. CONCLUSION: CP 0.05% spray was effective and safe in reducing overall plaque severity, scaling, erythema, and plaque elevation from the first week of treatment continuing throughout the trial. PMID- 16673805 TI - Uncontrollable prurigo nodularis effectively treated by roxithromycin and tranilast. AB - Treatment of prurigo nodularis (PN) is often very difficult even with strong corticosteroid dressing and other available means. Macrolide roxithromycin (RXM) is used in consideration of its immunosuppressive effects in treating several skin disorders. Tranilast (N-(3,4-dimethoxycinnamoyl) is useful for treating atopic disorders and hypertrophic scars as well, suggesting its capacity to inhibit fibroblast proliferation. More adequate and effective therapy for this disorder has been requested. We report 3 cases of uncontrollable PN treated with 300 mg/day roxithromycin and 200 mg/day tranilast. Complete and/or remarkable regression of PN was observed on treatment with roxithromycin and tranilast in combination within 4 to 6 months. The 2 agents in combination can be used effectively for the treatment of uncontrollable PN. PMID- 16673806 TI - Recurrent injection site reactions from interferon beta 1-b. AB - Recombinant human interferon beta-1b is an immune-modulatory drug used for a variety of conditions including multiple sclerosis (MS). Skin reactions to therapeutic use of injectable interferon beta-lb are relatively common, including injection site reactions and exacerbation of underlying skin disease. Injection site reactions are seen much more frequently in females. We discuss a case of prolonged susceptibility to injection site reaction with over a decade of use of interferon beta 1-b for MS. Given the prevalence of such reactions, the dermatologist should be aware of the phenomenon and that it frequently does not necessitate discontinuation of therapy. PMID- 16673807 TI - Epidermal growth factor receptor (EGFR) inhibitor associated skin eruption. AB - EGFR Inhibitors are used to treat Non-Small-Cell Lung Cancer (NSCLC) and colorectal cancer (CRC). A common side effect of EGFR Inhibitors is a follicular/pustular skin eruption. We report a case of gefitinib (Iressa) associated skin eruption. The treatment regimen consisted of triamcinolone 0.1% cream twice daily, clindamycin 1% lotion twice daily and sodium sulfacetamide lotion twice daily. The clinical presentation, etiology, and management options of EGFR Inhibitor associated skin eruptions are discussed. PMID- 16673808 TI - Non-sarcoidal, non-tuberculoid granuloma in common variable immunodeficiency. AB - Sarcoidal (non-caseating) or tuberculoid granulomas are cutaneous manifestations of common variable immunodeficiency (CVID). In this case report, we describe a patient with CVID but with non-sarcoidal, non-tuberculoid granuloma. The 29-year old Egyptian male patient presented with a vitiliginous patch on the chin of 1 year duration and multiple recurrent warts on the hands and feet of 8 years duration. He is a known case of CVID with chronic diarrhea, recurrent otitis media, pneumonia, purulent conjunctivitis, septic arthritis, hepato-splenomegaly, and generalized lymphadenopathy. In addition, he had evidence of multiple non tender subcutaneous nodules predominantly juxta-articular and recurrent rheumatoid-like arthritis. The skin overlying the nodules was either normal or slightly erythematous. Laboratory findings revealed markedly reduced serum immunoglobulins (IgG 3.4, n = 7.2-16.9 g/l; IgA 0.1, n = 0.69-3.82 g/l and IgM 0.1, n = 0.63-2.77 g/l) and deficient T cell function. Histopathologic examination of a skin nodule showed well demarcated areas of fibrinoid degeneration of collagen that stain homogeneously and are surrounded by histiocytes in a palisading arrangement, suggestive of granuloma annulare. No microorganisms could be detected. Serology for rheumatoid factor and HIV infection has been persistently negative. Although most infections, including common warts responded well to intravenous immunoglobulin replacement therapy (12 g/i.v., every 2 weeks) and oral broad spectrum antibiotic therapy, the subcutaneous nodules persisted. The vitiliginous patch responded favorably and disappeared within 24 local PUVA sessions. Since skin nodules are asymptomatic, no further treatment was given. PMID- 16673809 TI - Dapsone in the treatment of persistent erythema multiforme. AB - Erythema multiforme (EM) is usually an acute and self-limited inflammatory reaction of the skin and mucous membranes. Attacks may be sporadic or recurrent, and generally last for 1 to 3 weeks. Rarely, an episode of EM may fail to abate. This continuous and uninterrupted occurrence of typical and atypical lesions is known as persistent EM. We present a case responsive to dapsone. PMID- 16673810 TI - Nicolau syndrome: a report of 2 cases. AB - Nicolau syndrome or embolia cutis medicamentosa is a very rare, but well recognized complication of intramuscular injections which clinically presents with extensive necrosis of the injected skin area. Intra-arterial or para arterial injection after unintentional perforation of certain vessels could be the possible mechanisms. We describe 2 cases of Nicolau syndrome resulting from intramuscular injection of Diclofenac. Since irreversible tissue damage may occur within a short duration and, as the there is no definitive treatment for this condition, precautionary measures are emphasized. PMID- 16673811 TI - Report on childhood obesity in China (2). Verification of BMI classification reference for overweight and obesity in Chinese children and adolescents. AB - OBJECTIVE: To verify Working Group for Obesity in China (WGOC) recommended body mass index (BMI) classification reference for overweight and obesity in Chinese children and adolescents using the data of 2002 China Nationwide Nutrition and Health Survey. METHODS: Pediatric metabolic syndrome (MetS) and abnormality of each risk factor for MetS were defined using the criteria for US adolescents. Definition of hyper-TC, LDL, and dyslipidemia in adults was applied as well. The average level and abnormality rate of the metabolic indicators were described by BMI percentiles and compared with general linear model analysis. Receiver operating characteristic analysis was used to summarize the potential of BMI to discriminate between the presence and absence of the abnormality of these indicators. RESULTS: There was neither significantly increasing nor significantly decreasing trend of biochemical parameter levels in low BMI percentile range (<65th). Slight increasing trend from the 75th and a significant increase were found when BMI > or =85th percentile. In general, the prevalence of the examined risk factors varied slightly when BMI percentile<75th, and substantial increases were consistently seen when BMI percentile > or =75th. As an indicator of hyper TG, hypertension and MetS, the sensitivity and specificity were equal at the point of BMI<75th percentile, and the Youden's index of risk factors also reached peak point before 75th percentile except for MetS. When the BMI percentile was used as the screening indicator of MetS, Youden's index reached peak point at 85th percentile, just the point in the ROC graph that was nearest to the upper left corner. CONCLUSION: The BMI classification reference for overweight and obesity recommended by WGOC is rational to predict and prevent health risks in Chinese children and adolescents. Lower screening cut-off points, such as 83th percentile or 80th percentile, should not be excluded when they are considered as overweight criteria in future intervention or prevention studies. PMID- 16673812 TI - Metabolism of terephthalic acid and its effects on CYP4B1 induction. AB - OBJECTIVE: To investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L( 1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder. CONCLUSION: Lack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites. PMID- 16673813 TI - Fenvalerate-induced alterations in calcium homeostasis in rat ovary. AB - OBJECTIVE: To observe the effects of fenvalerate on calcium homeostasis in rat ovary. METHODS: Female Sprague-Dawley rats were orally given fenvalerate at daily doses of 0.00, 1.91, 9.55, and 31.80 mg/kg for four weeks. The ovary ultrastucture was observed by electron microscopy. Serum free calcium concentration was measured by atomic absorption spectrophotometry. The activities of phosphorylase a in rat ovary were evaluated by the chromatometry. The total content of calmodulin in ovary was estimated by ELISA at each stage of estrous cycle. Radioimmunoassay (RIA) was used to evaluate the level of serum progesterone. RESULTS: Histopathologically, damages of ovarian corpus luteum cells were observed. An increase in serum free calcium concentration was observed in rats treated with 31.80 mg/kg fenvalerate. The activities of phosphorylase a enhanced in all treated groups, and fenvalerate increased the total content of calmodulin significantly in estrus period. Serum progesterone levels declined in fenvalerate exposed rats in diestrus. CONCLUSION: Fenvalerate interferes with calcium homeostasis in rat ovary. Also, the inhibitory effects of fenvalerate on serum progesterone levels may be mediated partly through calcium signals. PMID- 16673814 TI - In vitro cytotoxicity of TCDD on SPC-A1 cells. AB - OBJECTIVE: The toxicology of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has been studied mainly with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of TCDD, this study was designed to investigate its effect on SPC-A1 cells. METHODS: SPC-A1 cells were cultured in 1640 medium and treated with 10 nmol/L, 0.1 micromol/L, 1 micromol/L TCDD for either 24 h or 96 h at each concentration. SPC-A1 cells were co-cultured with TCDD at different concentrations. Then the cell morphology, DNA fragment electrophoresis, and cell cycle were analyzed by flow cytometry, and enzyme assays were used to observe the effect of TCDD on the morphology, growth rate, and enxyme change of SPC-A1 cells. RESULTS: With the increasing concentrations of TCDD and prolongation of culture time, the morphology of SPC-A1 cells was changed from round shape to spindle, and the ability of SPC-A1 cells to adhere to wall was decreased. With debris emitted around the cells, the morphologic changes included reduction in cell volume. Nuclear chromatin condensation and PI were observed. With the increasing concentrations of TCDD, DNA ladder occurred. After treatment with TCDD, extraction of cancer cells exhibited typical DNA fragmentation, and flow cytometry analysis showed apoptosis in a dose-dependent manner. As the concentration of TCDD rose from 10 nmol/L to 1 micromol/L, the ratio of apoptotic cells increased from 10.76% to 21.82%. CONCLUSIONS: TCDD has in vitro cytotoxicity on SPC-A1 cells, and the cytotoxicity is positively related to its concentration and culture time. TCDD may inhibit the growth and proliferation of SPC-A1 cells through the pathway of apoptosis introduction. PMID- 16673815 TI - Tyrosine hydroxylase as a target for deltamethrin in the nigrostriatal dopaminergic pathway. AB - OBJECTIVE: To study the effects of deltamethrin on tyrosine hydroxylase in nigrostriatum of male rats. METHODS: Sprague-Dawley rats were daily treated with deltamethrin at 6.25 or 12.5 mg/kg body weight by gavage for 10 days. Then HPLC fluorescence detection was used to analyze the contents of dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC) and homoranillic acid (HVA) in substantial nigra and striatum. The activities of tyrosine hydroxylase (TH) were also detected by HPLC-fluorescence detection. TH mRNA or TH protein levels were measured by RT-PCR and immunohistochemistry method. RESULTS: The content of DA in striatum was significantly decreased by the treatments, suggesting an inhibition of DA synthesis by deltamethrin. The contents of DA metabolites DOPAC and HVA increased, indicating increased dopamine turnover. Furthermore, deltamethrin significantly decreased the activity, as well as the mRNA and protein levels of TH. CONCLUSIONS: These findings reveal a novel aspect of deltamethrin neurotoxicity and suggest tyrosine hydroxylase as a molecular target of deltamethin on dopamine metabolism in the nigrostriatal pathway. PMID- 16673816 TI - Inhibitory effect of isoflavones on prostate cancer cells and PTEN gene. AB - OBJECTIVE: To explore the mechanisms by which genistein and daidzein inhibit the growth of prostate cancer cells. METHODS: LNCaP and PC-3 cells were exposed to genistein and daidzein and cell viability was determined by MTT assay and cytotoxicity of the drugs by LDH test. Flow cytometry (FCM) was used to assess the cell cycle in LNCaP and PC-3 cells. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to examine the expression of PTEN gene (a tumor suppressor gene), estrogen receptor alpha gene (ERalpha), estrogen receptor beta gene (ERbeta), androgen receptor gene (AR) and vascular endothelial growth factor gene (VEGF). RESULTS: The viability of PC-3 and LNCaP cells decreased with increasing concentrations and exposure time of genistein and daidzein. Genistein increased G2/M phase cells in PC-3 cells while decreased S phase cells in LNCaP cells in a dose-dependent manner. Daidzein exerted no influence on the cell cycle of LNCaP and PC-3 cells, but the apoptosis percentage of LNCaP cells was elevated significantly by daidzein. Genistein induced the expression of PTEN gene in PC-3 and LNCaP cells. Daidzein induced the expression of PTEN gene in LNCaP but not in PC-3 cells. The expression of VEGF, ERalpha and ERbeta genes decreased and AR gene was not expressed after incubation with genistein and daidzein in PC-3 cells. In LNCaP cells, the expression of VEGF and AR gene decreased but there was no change in the expression of ERalpha and ERbeta gene after incubation with genistein and daidzein. Conclusion Genistein and daidzein exert a time- and dose dependent inhibitory effect on PC-3 and LNCaP cells. The down-regulation of ER gene by daidzein influences the growth of PC-3 cells directly. The inhibition of PC-3 cells by genistein and that of LNCaP cells by genistein and daidzein may be via Akt pathway that is repressed by PTEN gene, which subsequently down-regulates the expression of AR and VEGF genes. Our results suggest that the expression of PTEN gene plays a key role and several pathways may be involved in the suppression of prostate cancer cells by genistein and daidzein. PMID- 16673817 TI - Comparison of ileal digested production of parental rice and rice genetically modified with cowpeas trypsin inhibitor. AB - OBJECTIVE: To compare the ileal digestibility of protein and amino acids in parental rice and rice genetically modified with sck gene. METHODS: Six experimental swines were surgically fixed with a simple T-cannula at the terminal ileum and fed with parental rice and rice genetically modified with sck gene alternately. The ileum digesta were collected and analyzed for determination of apparent and true digestibility of protein and amino acids. RESULTS: The apparent and true digestibility of protein was similar in these two types of rice. Except for the apparent digestibility of lysine, there was no difference in the apparent and true digestibility of the other 17 amino acids. CONCLUSION: The digestibility of protein and amino acids is not changed by the insertion of foreign gene, so it can meet the request of "substantial equivalence" in digestibility of protein and amino acids. PMID- 16673818 TI - Experimental study on denitrification using coated electrode of immobilized denitrifying bacteria. AB - OBJECTIVE: To develop a coated electrode of immobilized denitrificants and to evaluate the performance of a bioelectrochemical reactor to enhance and control denitrification. METHODS: Denitrifying bacteria were developed by batch incubation and immobilized with polyvinyl alcohol (PVA) on the surface of activated carbon fiber (ACF) to make a coated electrode. Then the coated electrode (cathode) and graphite electrode (anode) were transferred to the reactor to reduce nitrate. RESULTS: After acclimated to the mixtrophic and autotrophic denitrification stages, the denitrifying bacteria could use hydrogen as an electron donor to reduce nitrate. When the initial nitrate concentration was 30.2 mg NO3- -N / L, the denitrification efficiency was 57.3% at an applied electric current of 15 mA and a hydraulic retention time (HRT) of 12 hours. Correspondingly, the current density was 0.083 mA/cm2. The nitrate removal rate of the reactor was 34.4 g NO3- -N/m3 x d, and the surface area loading was 1.34 g NO3- -N / m2 x d. CONCLUSION: The coated electrode may keep high quantity of biomass, thus achieving a high denitrification rate. Denitrification efficiencies are related to HRT, current density, oxidation reduction potential (ORP), dissolved oxygen (DO), pH value, and temperature. PMID- 16673819 TI - Abnormal expression of eukaryotic translation factors in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide. AB - OBJECTIVE: To study the oncogenic potential of mouse translation initiation factor 3 (TIF3) and elongation factor-1delta (TEF-1delta) in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide (NiS). METHODS: Abnormal expressions of human TIF3 and TEF-1delta genes in two kinds of NiS-transformed cells and NiS-tumorigenic cell lines were investigated and analyzed by the reverse transcript polymerase chain reaction (RT PCR) and fluorescent quantitative polymerase chain reaction (FQ-PCR), respectively. RESULTS: RT-PCR analysis primarily showed that both human TIF3 and TEF-1delta mRNA expressions in two kinds of NiS-transformed cells and NiS tumorigenic cell lines were increased as compared with controls. FQ-PCR assay showed that the levels of TIF3 expressions in the transformed cells and tumorigenic cells were 3 and 4 times higher respectively, and the elevated expressions of TEF-1delta cDNA copies were 2.7- to 3.5-fold in transformed cells and 4.1- to 5.2-fold in tumorigenic cells when compared with non-transformed cells, indicating that the over-expressions of human TIF3 and TEF-1delta genes were related to malignant degree of the cells induced by nickel. CONCLUSIONS: These findings demonstrate that there are markedly abnormal expressions of TIF3 and TEF-1delta genes during malignant transformation of human bronchial epithelial cell lines induced by crystalline NiS. They seem to be the molecular mechanisms potentially responsible for human carcinogensis due to nickel. PMID- 16673820 TI - Effect of alpha-ketoglutarate on cyanide-induced biochemical alterations in rat brain and liver. AB - OBJECTIVE: To investigate the biochemical changes in rat brain and liver following acute exposure to a lethal dose of cyanide, and its response to treatment of alpha-ketoglutarate (alpha-KG) in the absence or presence of sodium thiosulfate (STS). METHODS: Female rats were administered 2.0 LD50 potassium cyanide (KCN; oral) in the absence or presence of pre-treatment (-10 min), simultaneous treatment (0 min) or post-treatment (+2-3 min) of alpha-KG (2.0 g/kg, oral) and/or STS (1.0 g/kg, intraperitoneal, -15 min, 0 min or + 2-3 min). At the time of onset of signs and symptoms of KCN toxicity (2-4 min) and at the time of death (5-15 min), various parameters particularly akin to oxidative stress viz. cytochrome oxidase (CYTOX), superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and oxidized glutathione (GSSG) in brain, and CYTOX, sorbitol dehydrogenase (SDH), alkaline phosphatase (ALP), GSH and GSSG in liver homogenate were measured. RESULTS: At both time intervals brain CYTOX, SOD, GPx, and GSH significantly reduced (percent inhibition compared to control) to 24%, 56%, 77%, and 65%, and 44%, 46%, 78%, and 57%, respectively. At the corresponding time points liver CYTOX and GSH reduced to 74% and 63%, and 44% and 68%, respectively. The levels of GSSG in the brain and liver, and hepatic ALP and SDH were unchanged. Pre-treatment and simultaneous treatment of a-KG alone or with STS conferred significant protection on above variables. Post-treatment was effective in restoring the changes in liver but failed to normalize the changes in the brain. CONCLUSIONS: Oral treatment with alpha-KG alone or in combination with STS has protective effects on cyanide-induced biochemical alterations in rat brain and liver. PMID- 16673822 TI - Anaerobic degradation of tetrachloroethylene using different co-substrates as electron donors. AB - OBJECTIVE: To investigate the biodegradation of tetrachloroethylene (PCE) by acclimated anaerobic sludge using different co-substrates, i.e., glucose, acetate, and lactate as electron donors. METHODS: HP-6890 gas chromatograph (GC) in combination with auto-sampler was used to analyze the concentration of PCE and its intermediates. Results PCE could be degraded by reductive dechlorination and the degradation reaction conformed to the first-order kinetic equation. The rate constants are k(lactate) > k(glucose) > k(acetate). The PCE degradation rate was the highest in the presence of lactate as an electron donor. CONCLUSION: Lactate is the most suitable electron donor for PCE degradation and the electron donors supplied by co-metabolic substrates are not the limiting factors for PCE degradation. PMID- 16673821 TI - Expression of c-fos in rat brain as a prelude marker of central nervous system injury in response to methylmercury-stimulation. AB - OBJECTIVE: To probe into the prelude marker of central nervous system injury in response to methyl mercury chloride (MMC) stimulation and the signal transduction molecular mechanism of injury in rat brain induced by MMC. METHODS: The expression of c-fos mRNA in brain and the expression of c-FOS protein in cortex, hippocampus and ependyma were observed using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods. The control group was injected with physiological saline of 0.9%, while the concentrations for the exposure groups were 0.05 and 0.5, 5 mg/kg MMC respectively, and the sampling times points were 20, 60, 240, 1440 min. RESULTS: The expression of c-FOS protein in cortex and hippocampus increased significantly, the accumulation of mercury in the brain induced by 0.05 mg/Kg MMC for 20 min had no significant difference compared with the control group. The mean value was 0.0044 mg/Kg, while the protein c-FOS expression had significant difference compared with the control group (P < 0.01). More sensitive expression occurred in hippocampus and cortex, but not in ependyma. Conclusion The expression of c-FOS protein in cortex and hippocampus can predict the neurotoxicity of MMC in the early time, and immediately early gene (IEG) c-fos participates in the process of brain injury induced by MMC. PMID- 16673823 TI - Consumer brand choice: individual and group analyses of demand elasticity. AB - Following the behavior-analytic tradition of analyzing individual behavior, the present research investigated demand elasticity of individual consumers purchasing supermarket products, and compared individual and group analyses of elasticity. Panel data from 80 UK consumers purchasing 9 product categories (i.e., baked beans, biscuits, breakfast cereals, butter, cheese, fruit juice, instant coffee, margarine and tea) during a 16-week period were used. Elasticity coefficients were calculated for individual consumers with data from all or only 1 product category (intra-consumer elasticities), and for each product category using all data points from all consumers (overall product elasticity) or 1 average data point per consumer (interconsumer elasticity). In addition to this, split-sample elasticity coefficients were obtained for each individual with data from all product categories purchased during weeks 1 to 8 and 9 to 16. The results suggest that: 1) demand elasticity coefficients calculated for individual consumers purchasing supermarket food products are compatible with predictions from economic theory and behavioral economics; 2) overall product elasticities, typically employed in marketing and econometric research, include effects of interconsumer and intraconsumer elasticities; 3) when comparing demand elasticities of different product categories, group and individual analyses yield similar trends; and 4) individual differences in demand elasticity are relatively consistent across time, but do not seem to be consistent across products. These results demonstrate the theoretical, methodological, and managerial relevance of investigating the behavior of individual consumers. PMID- 16673824 TI - Matching and conditioned reinforcement rate. AB - Attempts to examine the effects of variations in relative conditioned reinforcement rate on choice have been confounded by changes in rates of primary reinforcement or changes in the value of the conditioned reinforcer. To avoid these problems, this experiment used concurrent observing responses to examine sensitivity of choice to relative conditioned reinforcement rate. In the absence of observing responses, unsignaled periods of food delivery on a variable interval 90-s schedule alternated with extinction on a center key (i.e., a mixed schedule was in effect). Two concurrently available observing responses produced 15-s access to a stimulus differentially associated with the schedule of food delivery (S+). The relative rate of S+ deliveries arranged by independent variable-interval schedules for the two observing responses varied across conditions. The relation between the ratio of observing responses and the ratio of S+ deliveries was well described by the generalized matching law, despite the absence of changes in the rate of food delivery. In addition, the value of the S+ deliveries likely remained constant across conditions because the ratio of S+ to mixed schedule food deliveries remained constant. Assuming that S+ deliveries serve as conditioned reinforcers, these findings are consistent with the functional similarity between primary and conditioned reinforcers suggested by general choice theories based on the concatenated matching law (e.g., contextual choice and hyperbolic value-added models). These findings are inconsistent with delay reduction theory, which has no terms for the effects of rate of conditioned reinforcement in the absence of changes in rate of primary reinforcement. PMID- 16673825 TI - Rapid acquisition in concurrent chains: evidence for a decision model. AB - Pigeons' choice in concurrent chains can adapt to rapidly changing contingencies. Grace, Bragason, and McLean (2003) found that relative initial-link response rate was sensitive to the immediacy ratio in the current session when one of the terminal-link fixed-interval schedules was changed daily according to a pseudorandom binary sequence (e.g., Schofield & Davison, 1997). The present experiment tested whether the degree of variation in delays across sessions had any effect on acquisition rate in Grace et al.'s (2003) rapid-acquisition procedure. In one condition ("minimal variation"), the left terminal link was always fixed-interval 8 s and the right terminal link was either fixed-interval 4 s or fixed-interval 16 s. In the other condition ("maximal variation"), a unique pair of fixed-interval values was used in each session. Responding was sensitive to the current-session immediacy ratio in both conditions, but across subjects there was no systematic difference in sensitivity. These results challenge the view that initial-link responding in the rapid-acquisition procedure is determined by changes in the learned value of the terminal-link stimuli, and suggests instead that a process resembling categorical discrimination may control performance. A decision model based on the assumption that delays are categorized as short or long relative to the history of delays provided a good account of the data and shows promise in being able to explain other choice phenomena. PMID- 16673826 TI - Identity matching-to-sample with olfactory stimuli in rats. AB - Identity matching-to-sample has been difficult to demonstrate in rats, but most studies have used visual stimuli. There is evidence that rats can acquire complex forms of olfactory stimulus control, and the present study explored the possibility that identity matching might be facilitated in rats if olfactory stimuli were used. Four rats were trained on an identity match-to-sample procedure with odorants mixed in cups of sand as stimuli. Digging in the sample cup produced two comparison cups, and digging in the comparison cup that contained the same scent as the sample was reinforced. When criterion accuracy levels were reached, novel stimuli were added to the baseline training regimen. All 4 rats reached terminal performance of above 90% correct matching with more than 20 different baseline stimuli and matched novel stimulus combinations with above-chance accuracy; 3 of the 4 rats matched novel stimuli at levels significantly above chance. Accurate matching performance was demonstrated both with 2- and 3-comparison procedures. These results suggest that generalized matching-to-sample can be observed in rats when olfactory stimuli are used and, furthermore, that multiple-exemplar training may be important for its emergence. PMID- 16673827 TI - A derived transfer of simple discrimination and self-reported arousal functions in spider fearful and non-spider-fearful participants. AB - Two experiments investigated the derived transfer of functions through equivalence relations established using a stimulus pairing observation procedure. In Experiment 1, participants were trained on a simple discrimination (A1+/A2-) and then a stimulus pairing observation procedure was used to establish 4 stimulus pairings (A1-B1, A2-B2, B1-C1, B2-C2). Subsequently, a transfer of the simple discrimination functions through equivalence relations was observed (e.g., C1+/C2-). These procedures were modified in Experiment 2, which demonstrated that spider-fearful and non-spider-fearful participants show differing levels of a transfer of self-reported arousal functions for stimuli used in equivalence relations with video-based material depicting scenes with spiders. The results demonstrate that the stimulus pairing observation procedure provides a viable alternative to matching-to-sample, and also offer tentative support for a derived relations model of the acquisition of anxiety responses in at least one sub clinical population. PMID- 16673828 TI - Naming and categorization in young children: IV: listener behavior training and transfer of function. AB - Following pretraining with everyday objects, 14 children aged from 1 to 4 years were trained, for each of three pairs of different arbitrary wooden shapes (Set 1), to select one stimulus in response to the spoken word /zog/, and the other to /vek/. When given a test for the corresponding tacts ("zog" and "vek"), 10 children passed, showing that they had learned common names for the stimuli, and 4 failed. All children were trained to clap to one stimulus of Pair 1 and wave to the other. All those who named showed either transfer of the novel functions to the remaining two pairs of stimuli in Test 1, or novel function comprehension for all three pairs in Test 2, or both. Three of these children next participated in, and passed, category match-to-sample tests. In contrast, all 4 children who had learned only listener behavior failed both the category transfer and category match-to-sample tests. When 3 of them were next trained to name the stimuli, they passed the category transfer and (for the 2 subjects tested) category match-to sample tests. Three children were next trained on the common listener relations with another set of arbitrary stimuli (Set 2); all succeeded on the tact and category tests with the Set 2 stimuli. Taken together with the findings from the other studies in the series, the present experiment shows that (a) common listener training also establishes the corresponding names in some but not all children, and (b) only children who learn common names categorize; all those who learn only listener behavior fail. This is good evidence in support of the naming account of categorization. PMID- 16673829 TI - Mathematical models and the experimental analysis of behavior. AB - The use of mathematical models in the experimental analysis of behavior has increased over the years, and they offer several advantages. Mathematical models require theorists to be precise and unambiguous, often allowing comparisons of competing theories that sound similar when stated in words. Sometimes different mathematical models may make equally accurate predictions for a large body of data. In such cases, it is important to find and investigate situations for which the competing models make different predictions because, unless two models are actually mathematically equivalent, they are based on different assumptions about the psychological processes that underlie an observed behavior. Mathematical models developed in basic behavioral research have been used to predict and control behavior in applied settings, and they have guided research in other areas of psychology. A good mathematical model can provide a common framework for understanding what might otherwise appear to be diverse and unrelated behavioral phenomena. Because psychologists vary in their quantitative skills and in their tolerance for mathematical equations, it is important for those who develop mathematical models of behavior to find ways (such as verbal analogies, pictorial representations, or concrete examples) to communicate the key premises of their models to nonspecialists. PMID- 16673830 TI - Microwave enhanced recovery of nickel-copper ore: communition and floatability aspects. AB - A study describing the effect of microwave radiation, at a frequency of 2450 MHz, on the processes of communication and flotation of a complex sulphide nickel copper ore is presented. Ore communication has been investigated under standard radiation-free conditions and after ore treatment in a radiated environment as a function of ore size, exposure time to radiation, and microwave power. The findings show that communication is tremendously improved by microwave radiation with values of the relative work index as low as 23% at a microwave power of 1.406 kW and after 10 s of exposure time. Communication is affected by exposure time and microwave power in a nontrivial manner. In terms of ore floatability, the experimental tests have been carried out on a sample of 75 microm in size under different exposure times. The results show that both ore concentrate recoveries and grades of nickel and copper are significantly enhanced after microwave treatment of the ore with relative increases in recovered concentrate, grade of nickel, and grade of copper of 26 wt%, 15 wt%, and 27%, respectively, at a microwave power of 1330 kW and after 30 s of exposure time. PMID- 16673831 TI - A finite difference thermal model of a cylindrical microwave heating applicator using locally conformal overlapping grids: part I--theoretical formulation. AB - In this paper, we present a versatile mathematical formulation of a newly developed 3-D locally conformal Finite Difference (FD) thermal algorithm developed specificallyfor coupled electromagnetic (EM) and heat diffusion simulations utilizing Overlapping Grids (OGFD) in the Cartesian and cylindrical coordinate systems. The motivation for this research arises from an attempt to characterize the dominant thermal transport phenomena typically encountered during the process cycle of a high-power, microwave-assisted material processing system employing a geometrically composite cylindrical multimode heating furnace. The cylindrical FD scheme is only applied to the outer shell of the housing cavity whereas the Cartesian FD scheme is used to advance the temperature elsewhere including top and bottom walls, and most of the inner region of the cavity volume. The temperature dependency of the EM constitutive and thermo physical parameters of the material being processed is readily accommodated into the OGFD update equations. The time increment, which satisfies the stability constraint of the explicit OGFD time-marching scheme, is derived. In a departure from prior work, the salient features of the proposed algorithm are first, the locally conformal discretization scheme accurately describes the diffusion of heat and second, significant heat-loss mechanisms usually encountered in microwave heating problems at the interfacial boundary temperature nodes have been considered. These include convection and radiation between the surface of the workload and air inside the cavity, heat convection and radiation between the inner cavity walls and interior cavity volume, and free cooling of the outermost cavity walls. PMID- 16673832 TI - Dielectric spectroscopy of fresh fruit and vegetable tissues from 10 to 1800 MHz. AB - Dielectric spectroscopy data from measurements on tissue samples of nine fresh fruits and vegetables were used to study their dielectric behavior over the frequency range from 10 MHz to 1.8 GHz at 5 to 65 degrees C. Dielectric constant and loss-factor data are presented graphically for apple, avocado, banana, cantaloupe, carrot, cucumber, grape, orange, and potato, showing dielectric constants ranging from values of several hundred at 10 MHz to less than 100 at 1.8 GHz and loss factors on the order of one thousand at 10 MHz to less than 20 at 1.8 GHz. The dielectric loss factor increased consistently with increasing temperature at frequencies below 1 GHz. The dielectric constant increased with temperature at lower frequencies, but it decreased with temperature at the higher frequencies. This reversal of the sign of the temperature coefficient occurred at some point in the frequency range between 20 and 120 MHz where the temperature dependence of the dielectric constant was zero. At frequencies below this point, ionic conduction dominates the dielectric behavior, but above that point dipolar relaxation appears to control the behavior. Multiple linear regression provided equations for calculation of the loss factor in the frequency range from 10 to 300 MHz at temperatures from 5 to 65 degrees C. The data provide new information useful in understanding dielectric heating behavior and evaluating dielectric properties of such agricultural products for quality sensing applications. PMID- 16673833 TI - Microwave radiometry for continuous non-contact temperature measurements during microwave heating. AB - Temperature measurement during microwave heating in industrial and commercial processes can improve quality, throughput, and energy conservation. Conventional ways of measuring temperature inside a microwave oven cavity are costly, inconvenient, or unsuitable for high-volume industrial applications. In this paper, we describe the theory of microwave radiometry as applied to the measurement of temperature during microwave heating. By extending the theory of radiative transfer to the case of thermal microwave radiation inside a cavity, we show that the same characteristics which make a microwave cavity suitable for heating materials also assist in obtaining meaningful temperature data with microwave radiometry. We present experimental data from the heating of liquid and solid materials which confirm the essential features of the theory, and show agreement between this method and more conventional methods of +/-4 degrees C. PMID- 16673834 TI - Oncologist Assisted Spiritual Intervention Study (OASIS): patient acceptability and initial evidence of effects. AB - PURPOSE: Individuals with serious illness often desire to discuss spiritual concerns with their physician, yet substantial barriers exist to doing so, including limited evidence of value. This study evaluated acceptability, impact on satisfaction with care and on quality of life (QOL) of a brief (5-7 minute) semi-structured exploration of spiritual/religious concerns. PATIENTS AND METHODS: 118 consecutive patients of four oncologist-hematologists (95% recruitment; 55.1% female, 91.5% Caucasian, 81.3% Christian) with mixed diagnoses, duration (51.7% diagnosed within 2 years) and prognosis (54.2% in active treatment) were alternately assigned to receive the intervention or usual care during an office visit. Assessment occurred just prior to the visit, immediately after, and after 3 weeks. Measures included the FACT-G QOL and FACIT Sp (Spiritual Well-Being) Scales; BSI Depression Scale; the PCAS Interpersonal and Communication scales; and ratings of acceptability. RESULTS: Oncologists rated themselves as comfortable during the inquiry with 85% of patients. Of patients, 76% felt the inquiry was "somewhat" to "very" useful. At 3 weeks, the intervention group had greater reductions in depressive symptoms (F= 7.57,p < .01), more improvement in QOL (F = 4.04, p < .05), and an improved sense of interpersonal caring from their physician (F = 4.79, p < .05) relative to control patients. Effects on QOL remained after adjusting for other variables, including relationship to physician. Improvement on Functional Well-being was accounted for primarily by patients lower on baseline spiritual well-being (beta = .293, p < .001). CONCLUSIONS: This study supports the acceptability of a semi-structured inquiry into spiritual concerns related to coping with cancer; furthermore, the inquiry appears to have a positive impact on perception of care and well-being. PMID- 16673835 TI - A randomized trial of the efficacy of group therapy in changing viral load and CD4 counts in individuals living with HIV infection. AB - OBJECTIVE: This randomized pilot study evaluates whether seropositive patients who are randomly assigned to receive a supportive-expressive group therapy plus education intervention show greater improvements in increased immune function and decreased viral load compared to those randomly assigned to an education-only intervention. METHOD: Fifty-nine individuals who had been HIV-seropositive for at least 6 months prior to inclusion in the study and had been receiving standard pharmacologic treatment were entered in a prospective randomized trial of the effects of weekly supportive-expressive group therapy on changes in immune status. Participants were matched for AIDS status and sex and randomized to receive weekly sessions of group psychotherapy plus educational materials on HIV/AIDS, or to receive the educational materials alone. Participants were assessed before treatment and then 12 weeks later. RESULTS: Individuals who were randomized to group therapy showed a statistically significant increase in CD4 count and decrease in HIV viral load. Among individuals randomized to the education only condition, no significant change occurred in CD4 count or viral load. CONCLUSIONS: These results provide preliminary data suggesting that HIV seropositive individuals who receive supportive-expressive group psychotherapy may experience concomitant improvements in CD4 cell count and viral load. Further research with a larger sample should examine the possible underlying mechanisms of such benefits. PMID- 16673836 TI - Effects of mood state and psychosocial functioning on plasma Interleukin-6 in adult patients before cardiac surgery. AB - OBJECTIVE: The purpose of this study was to examine the potential effect of mood states and psychosocial functioning during the waiting weeks prior to major cardiac surgery on the plasma Interleukin-6 (IL-6) levels in 236 patients immediately before their operation. METHOD: The sample was recruited from patients at the cardiac clinic of the University of Michigan Medical Center (Ann Arbor). Two weeks before cardiac surgery, trained research assistants conducted a face-to-face interview with these middle-aged and older patients on their preoperative physical examination date at the clinic. Standardized instruments were used to assess mood states and psychosocial functioning. The blood samples of 236 patients, obtained on the morning of the operation, were analyzed for plasma IL-6. RESULTS: In bivariate analysis, poor psychological functioning and anxiety, as well as bodily pain and body mass index (BMI), were correlated with plasma IL-6 (p < .05), but sociodemographics, chronic illness and use of psychotropic medications were not. When the effect of bodily pain and BMI were taken into account, partial correlation analysis showed that psychological functioning continued to be associated with plasma IL-6 (p < .05); the association of IL-6 with depression now became significant (p < .05), whereas that with anxiety became even more significant (p < .001). CONCLUSIONS: Preoperative psychological disturbances during the waiting weeks before cardiac surgery may influence the plasma levels of IL-6 immediately prior to the procedure. The clinical implications of these findings remain to be determined. PMID- 16673838 TI - Family attitudes toward transgendered people in Turkey: experience from a secular Islamic country. AB - OBJECTIVE: In Turkey, an individual with gender identity disorder is stigmatized and isolated from society. The family largely reflects and reinforces these negative views because gender crossing poses a threat to the normatively sanctioned gender classification. METHODS: We examined the acceptance of gender identity differences by the families in 47 relatives of 39 transgendered individuals who applied to a psychiatry clinic for sex reassignment. RESULTS: Half of the relatives who came to the interview were mothers. While 85.1% of the families considered themselves as secular muslims, 14.9% were very religious. They first noticed the gender identity disorder during puberty (70.2%) or prepuberty (17%). In 63.8% it was remarked that it was a shocking experience. One third of them felt responsible for it. While 65.9% tried to change the situation by coercion, only 27.7% adopted a supportive attitude. The majority of families tried to conceal the situation from their immediate environment and one-third did not even inform their closest relatives. For half of relatives the mass media was their only source of information whereas one-third received information from doctors. Most of the families were satisfied with the treatment. Family members also reported that the conformity of the transgendered relative within the family improved. Of the family members, 40.4% accepted the transgendered identity and approved the sex reassignment surgery as a final step. CONCLUSION: Involvement of family members in the process of change for the transgendered individual is important for both the family as well as the individual concerned. PMID- 16673837 TI - Changes in depressive symptoms and metabolic control over 3 years among African Americans with type 2 diabetes. AB - OBJECTIVE: It is established that individuals with diabetes have high rates of depression, but the longitudinal relationship between depression and glycemic control has not been well examined, particularly among African Americans. The objective of this study was to evaluate the longitudinal relationship between depressive symptoms and metabolic control. METHOD: We conducted an earlier cross sectional study that demonstrated marginal and significant associations between depressive symptoms (using the Center for Epidemiologic Studies Depression Scale [CES-D]) and metabolic control (HbA1c, lipids, blood pressure) among 183 African Americans with type 2 diabetes. In this report, we present data on these individuals, followed for three years, and examine the relationship between change in depressive symptoms and change in metabolic control over that time period. RESULTS: Results showed that that there were no statistically significant associations between baseline or change in depressive symptoms and metabolic control over three years. Limited statistical power may explain this negative finding. CONCLUSIONS: This study provides insight into the relationship between depression and metabolic control. Prospective observational studies are needed to further evaluate this relationship. PMID- 16673839 TI - Childhood trauma and employment disability. AB - INTRODUCTION: While the relationship between childhood trauma and employment disability has undergone very limited study, existing data suggest a possible correlation. METHOD: In this study of 91 outpatients in an internal medicine setting, we surveyed participants and inquired about their childhood histories of sexual, physical, and emotional abuse, of physical neglect, and of witnessing violence. We also asked whether participants had ever been on employment disability, either psychiatric or non-psychiatric, and the length of that disability. RESULTS: Being or having been on disability was significantly related to childhood histories of emotional abuse, physical neglect, and witnessing violence. Being or having been on psychiatric disability was significantly related to childhood emotional abuse and physical neglect while being on non psychiatric medical disability was significantly related to witnessing violence. The percent of one's lifetime on disability was significantly related to physical and emotional abuse as well as witnessing violence. CONCLUSIONS: Maltreatment in childhood appears to have a relationship to employment disability in adulthood. The authors discuss the implications of these findings. PMID- 16673840 TI - Burnout in psychiatrists: an etiological model. AB - OBJECTIVE: This article reviews burnout in psychiatrists. A model of burnout is presented which considers three factors: those which may predispose, precipitate, and perpetuate burnout. Lastly, recommendations are made for future research. METHODS: We conducted a selective literature review to expand on two recent systematic reviews to examine the etiology, prevalence, and consequences of burnout in psychiatrists. We distinguish the effects of burnout, depression, and work-related stress. RESULTS: An etiological model for the development of burnout in psychiatrists is described here in terms of an interaction between the predisposing, precipitating, and perpetuating factors. CONCLUSIONS: Research is needed on the long-term effects of work-place stress on psychiatrists, to identify possible protective factors, and to utilize this information to inform the design of interventions that protect or mitigate the effects of work-place stress on psychiatrists. PMID- 16673841 TI - Coprophagia in an elderly man: a case report and review of the literature. AB - OBJECTIVE: Coprophagia or the ingestion of feces has long been associated with psychiatric illness. It is considered to be a variant of pica. This behavior requires an extensive medical and psychiatric differential diagnosis. Medical disorders associated with coprophagia include seizure disorders, cerebral atrophy, and tumors. Psychiatric disorders associated with coprophagia include mental retardation, alcoholism, depression, obsessive compulsive disorder, schizophrenia, fetishes, delirium, and dementia. In animals, coprophagia is associated with boredom, thiamine deficiency, and lesions of the amygdala. METHODS: A case of coprophagia in an elderly man is reported here. A 77-year-old man with mild mental retardation was referred for urgent psychiatric evaluation due to coprophagia. The case is discussed and the literature reviewed. RESULTS: Psychiatric evaluation revealed cognitive dysfunction and depression. Physical examination and laboratory evaluation were noncontributory. He was started on sertraline 25 mg daily with resolution of his coprophagia. Coprophagia has been treated using behavioral interventions, supportive psychotherapy, elemental diets, tricyclic anti-depressants, carbamazepine, haloperidol, and electroconvulsive therapy. CONCLUSIONS: Use of Selective Serotonin Reuptake Inhibitors (SSRIs) may also be an effective treatment for coprophagia, particularly in the setting of depression or anxiety. PMID- 16673842 TI - Aripiprazole in the treatment of delirium. AB - OBJECTIVE: Delirium is a common condition frequently seen in consultation-liaison psychiatry. It is especially common among medically compromised patients, and is an indicator of the severity of the medical illness. In addition, it is associated with a higher morbidity, mortality, and longer hospitalization. Traditionally, haloperidol has been used to treat agitation as it may occur in delirium. However, atypical antipsychotics are being increasingly used to treat delirium. METHOD: In this article, we will describe two cases of delirium successfully treated with aripiprazole. RESULTS: Both patients had significant improvement in their delirium as measured by the delirium rating scale. CONCLUSION: Aripiprazole appears to be effective in reducing the symptoms of delirium. PMID- 16673843 TI - The review article by Coetzer on obsessive-compulsive disorder following brain injury. PMID- 16673844 TI - Effects of driver cell-phone use on driver aggression. AB - Using 2 field procedures, the authors assessed impacts of cell-phone use on mild forms of driver aggression. Participants were 135 drivers traveling within a city of approximately 17,000 people in an otherwise little-populated region of western North Dakota. The authors videotaped the participants while a confederate driver in a low-status vehicle frustrated them. In Experiment 1, the confederate was traveling well under the posted speed limit. In Experiment 2, the confederate remained motionless at a stoplight that had turned green. When the confederate visibly talked on a hand-held cell phone (n = 67), male drivers exhibited their frustration by honking their horn more quickly and frequently than did drivers in no-cell-phone trials, and female drivers were more angry according to blind judgments of videotaped facial expressions that were compared with those of drivers in no-cell-phone trials (n = 68). The present results suggested that driver cell-phone use contributes to the growing crisis of roadway aggression. PMID- 16673845 TI - How do we decide whom our friends are? Defining levels of friendship in Poland and the United States. AB - Participants were 57 U.S. college students and 56 Polish university students and copper mine workers who judged the intimacy of 9 hypothetical relationships and also rated the intensity of their relationships with a best friend, a friend, and an acquaintance on the Friendship Intensity Measurement Scale (FIMS; T. S. Arunkumar & B. Dharmangadan, 2001). The present results confirmed that people perceive (a) relationships with best friends as more intense and intimate than other friendships and (b) other friendships as more intense and intimate than acquaintanceships. The results also indicated that Americans perceive all of their relationships, ranging from mere acquaintanceships to intimate friendships, as more intense and intimate than do Poles. It was somewhat surprising that there were no sex differences in either country in the perception of relationships. The authors discussed the research in the context of the difficulty of defining what friendship is and how an individual's cultural background might interact with person variables such as age and sex. PMID- 16673846 TI - Attachment style and individual differences in leadership perceptions and emergence. AB - The authors examined whether individuals' approaches to relationships, expressed in terms of attachment styles, was related to how they viewed ideal leadership and to their degree of tendency to emerge as team leaders. Specifically, the authors hypothesized that attachment styles, or cognitive representations of orientation to others, would explain individual differences in leadership perceptions and emergence. Participants were 127 American students in college teams. The authors found that securely attached individuals (n = 81) perceived themselves as more effective team members than did insecurely attached individuals (n = 46) and that fellow team members saw securely attached team members as emerging team leaders significantly more often than they did insecurely attached team members. PMID- 16673847 TI - The dynamics and validity of the group selection interview. AB - The authors examined the validity of the group interview, in which several applicants are interviewed simultaneously, vs. the traditional 1-on-1 selection interview. The authors also investigated perceptions of fairness for each interview format and whether the order in which the applicant was asked to respond in the group interview affected the quality of his or her response. Participants were 91 undergraduates who participated in 1-on-1 or group interviews for a scholarship award. The present results supported the hypothesis that interviewers in the 1-on-1 format were significantly better at predicting the applicants' academic potential. Also, in the group interviews, the order in which the applicants were asked to respond affected the quality of their responses significantly. PMID- 16673848 TI - "Cultural fit": individual and societal discrepancies in values, beliefs, and subjective well-being. AB - The author examined the relationship between cultural values, beliefs, and subjective well-being (SWB) in the context of the "cultural fit" proposition with 3 diverse Chinese samples from Taiwan and Mainland China (N = 581). The author found that beliefs regarding the independent self, the interdependent self, active control, and relationship harmony as forming individual-level culture were consistently related to SWB. Furthermore, the author found that the magnitude of cultural fit was associated with SWB for certain groups of the Chinese people. It is most interesting that the direction of cultural fit regarding independent self was also important for SWB. Specifically, people who endorsed higher independent self but expected lower societal endorsement of such views were better off in SWB than those of the opposite combination. PMID- 16673849 TI - Reexamining personal, social, and cultural influences on compliance behavior in the United States, Poland, and Hong Kong. AB - Researchers have extended the literature on strategies of gaining compliance with a request to incorporate cultural variations into the analytic framework. In the present investigation, the authors sought to go beyond previous studies of the factors increasing compliance rates by reexamining how researchers conceptualize and measure personal, social, and cultural influences on compliance behavior in the United States, Poland, and Hong Kong. The authors found that different levels of compliance were affected by culture, principles of influence, and the individual's personal orientation of idiocentrism/allocentrism (I/A). In the present study, the authors extended previous cross-cultural work by decomposing the I/A into 2 separate individual difference variables: normative perceptions and evaluative perceptions. The interaction of person and situation on compliance showed the power of situational demands and the strength of different aspects of personal collectivism. Different patterns of compliance at the culture level revealed the importance of culture in shaping this behavioral tendency. Thus, the authors' integration of personal, social, and cultural influences provided an interactive model to help researchers explain compliance more comprehensively. PMID- 16673850 TI - Gender and consent to organ donation. PMID- 16673851 TI - The relation between self-esteem and sexual functioning in collegiate women. PMID- 16673852 TI - Implications of cognitive busyness for the perception of category conjunctions. PMID- 16673853 TI - [Clinical characteristics of coinfection with hepatitis C virus in HIV-positive patients]. AB - INTRODUCTION: Patients with HIV are commonly coinfected with hepatitis C virus (HCV) mostly due to similar routes of transmission, especially in areas with high prevalence of intravenous drug use. In the era of highly active antiretroviral therapy (HAART), life expectancy of these patients has increased and end-stage liver disease is a common cause of death. MATERIAL AND METHODS: This study included 387 HIV-positive patients treated at the Institute of Infectious and Tropical Diseases "Dr. Kosta Todorovic" in Belgrade. We investigated epidemiological and clinical features of patients who underwent biochemical liver tests, ultrasonography and some of them blind liver biopsies. RESULTS: The prevalence of HCV infection among these patients was 58.13% with 225/387 ELISA anti-HCV-antibody positive patients. HCV coinfection was statistically more significant among male patients aged between 30 and 34, and among intravenous drug users. None of the biochemical parameters were statistically significant among coinfected patients, except aminotraspherase levels which were significantly higher among patients with chronic hepatitis C and HIV. 44 patients underwent biopsy and pathohistological findings showed that every 4th patient (25%) had chronic hepatitis C infection. The average survival rate in coinfected patients in the pre HAART era was the same as in AIDS patients. CONCLUSIONS: Liver biopsy, elevated aminotraspherase and positive serology can help in making the diagnosis of chronic hepatitis C, and possible future treatment options. PMID- 16673854 TI - A histological study of cerebral aqueduct. AB - Cerebral (sylvian) aqueduct is a narrow channel in the mesencephalon. It lies between the tectum and the tegmentum of the mesencephalon and is surrounded by the periaqueductal gray matter. The aim of this study was to determine the shape of the aqueduct of sylvius and the structure of its walls in a series of transverse histological sections. Serial transverse sections of the mesencephalon were examined in twenty adult brains of both sexes. Six sections were stained by the hematoxylin-eosin method. The rostral part of the the aqueduct has a triangular shape with dorsal concavity caused by retrocommissural fossae. In the middle, its shape is oval to irregular, the rostral part has a T shape due to isthmic recess on the floor. Walls of the aqueduct are coated with a layer of prismatic cells. Determination of the morphological and histological features of the mesencephalic aqueduct is important for differentiation between physiological and pathological processes in this region. PMID- 16673855 TI - [Dobutamine stress echocardiography in distinguishing ischemic from nonischemic dilated cardiomyopathy]. AB - INTRODUCTION: The aim of this study was to evaluate the diagnostic accuracy of dobutamine stress echocardiography for detection of coronary artery disease in patients with dilated cardiomyopathy. Detection of regional wall motion abnormalities at rest does not reliably distinguish ischemic from nonischemic cardiomyopathy. MATERIAL AND METHODS: To distinguish between ischemic and nonischemic dilated cardiomyopathy (DCM), we studied 50 patients with left ventricular dysfunction (20 ischemic and 30 nonischemic, detected by coronary angiography) using dobutamine stress echocardiography. Echocardiographic images were obtained at baseline, low and paek dose of dobutamine. Rest and stress left ventricular wall motion scores were derived from analysis of regional wall motion. RESULTS: Dobutamine infusion was terminated after achievement of the target heart rate or maximal protocol dose in 16 (80%) patients with ischemic heart disease and in 23 (73.3%) patients with nonischemic heart disease. At rest, there were more normal segments (p<0.001) and a trend toward more akinetic segments (p, not significant) per ischemic than per nonischemic DCM patients. However, either at rest or with low-dose dobutamine, individual data largely overlapped. At peak dose, in ischemic DCM, regional contraction worsened in many normal or dyssinergic regions at rest (in some cases after inprovement with low dose dobutamine); in contrast, in nonischemic DCM, further mild impovement was observed in a variable number of left ventricular areas. Thus, with peak-dose dobutamine, more akinetic and less normal segments were present per ishemic than per nonischemic DCM patient (both, p<0.001). A value of six or more akinetic segments was 90% sensitive and 98% specific for ischemic DCM. CONCLUSIONS: Our data show that analysis of regional contraction by dobutamine stress echocardiography can distinguish between. PMID- 16673856 TI - [Accuracy of ultrasonic fetal weight estimation using head and abdominal circumference and femur length]. AB - INTRODUCTION: Former investigations have shown that the accuracy of fetal weight estimation is significantly higher if several ultrasonic fetal parameters are measured, because the total body mass depends on the size of fetal head, abdominal circumference and femur length. The aim of this investigation was to establish the best regression model, that is a number of combinations of fetal parameters providing the most accurate fetal weight estimation in utero in our population. MATERIAL AND METHODS: This prospective study was carried out at the Gynecology and Obstetrics Clinic of the Clinical Center Novi Sad. It included 270 pregnant women with singleton pregnancies within 72 hours of delivery who underwent ultrasound measurements of the biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL). RESULTS: In regard to fetal weight estimation formulas, the deviation was lowest using regression models that simultaneously analyzed four fetal parameters (0. 55%) with SD +/- 7.61%. In these models the estimates of fetal weights were within +/- 5% of actual birth weight in 48.89%, and within +/- 10% of actual birth weight in 81.48%. Good results were also obtained using AC, FL measurements (0.92% +/- 8.20) as well as using AC, HC, FL measurements (-1.45% +/- 7.81). In our sample the combination of AC and FL model gave better results in fetal weight estimation (0.92 +/- 8.20%) than the one using BPD and AC (2.97 +/- 8.83%). Furthermore, the model using parameters AC, HC and FL showed a lower error in accuracy (-1.45 +/- 7.81%) than the model using BPD, AC and FL (2.51 +/- 7.82%). CONCLUSION: This investigation has confirmed that the accuracy of fetal weight estimation increases with the number of measured ultrasonic fetal parameters. In our population the greatest accuracy was obtained using BPD, HC, AC and FL model. In cases when fast estimation of fetal weight is needed, AC, HC, FL model may be appropriate, but if fetal head circumference cannot be measured (amnion rupture and/or fetal head already in the pelvis) the AC, FL model should be used. PMID- 16673857 TI - [Kinesitherapy: one of the most important aspects of medical rehabilitation]. AB - INTRODUCTION: Kinesitherapy, as a part of physical therapy, represents one of the most important aspects of medical rehabilitation. It involves movement of various parts of the body, or of the whole body, using exercises in order to maintain, establish, develop and change functions of the locomotor apparatus and organs of locomotion. AIM: The aim of kinesitherapy is to use all potentials of the treated patients, to achieve optimal recovery of the damaged function of locomotion. CLASSIFICATION: Kinesitherapy includes active and passive exercises. Active exercises are divided into: active exercises with assistance, without assistance and active exercises with resistance. INDICATIONS AND CONTRAINDICATIONS: Kinesitherapy is applied in almost all medical branches in numerous pathological conditions, as well as a method of prevention. Practically, there are no absolute contraindications, and relative are extremely rare. PMID- 16673858 TI - [The growth hormone axis and insulin-like growth factors]. AB - INTRODUCTION: Growth is regulated by the interaction of environmental signals with endogenous neuroendocrine responses to the genetic programs that determine the body plan. The insulin-like growth factors (IGFs) are integral components of multiple systems controlling both growth and metabolism. THE IGF SYSTEM: The IGF system is thouht to be more complex than other endocrine systems, as genes for six IGF-binding proteins (IGFBPs) have been identified so far. The IGFs play a critical role in both cell cycle control and apoptosis, two functions involved in regulation of tumorigenesis. Insulin-like growth factor-I (IGF-I) is essential for normal growth. Confirmation of the significance of IGF-I in human physiology was obtained by the discovery of a patient with intrauterine growth retardation and postnatal growth failure associated with a mutation in the IGF-I gene. STAGES OF EVOLUTION OF THE SOMATOMEDIN HYPOTHESIS: The original somatomedin hypothesis postulated that somatic growth was regulated by growth hormone's (GH's) stimulation of hepatic IGF-I production, with IGF-I acting in an endocrine fashion to promote growth. The dual effector theory proposed an alternative view, involving direct effects by GH on peripheral tissues not mediated by IGF-I and GH stimulated local IGF-I production for autocrine/paracrine action. It is now clear that GH stimulates the formation of ternary IGF binding complex, which stabilizes IGF-I in the serum. PMID- 16673859 TI - [Treatment of neglected and abused children and their families in the context of complicated divorce]. AB - INTRODUCTION: Coceptualisation of complicated divorce, as an escalation of systemic dysfunctioning in a highly disturbed and unbalanced system, acquires attributes of trauma-organised-system, and yields itself for therapeutic interventions. COMPLICATED DIVORCE ISSUES: The authors comment on their experience while treating families sent by Court order in divorce custody disputes, with explicit aim of evaluating parental competence. This type of systemic dysfunction is severely affecting communication styles, family rules and norms, whereas parenting is frequently marked by tendency to repeat relational style experienced in marital, partners' conflicts. Feelings of discontent and resentment are projected onto children, and may take form of disqualifications, rejection, discrimination or else, those feelings stemming out of personal ambition such as favoring children. Parental rivalry worsens these processes, leading to attempts of establishing inappropriate level of control over children (instrumentalisation or favorisation). Therefore, care and protection of children is inadequate and children are emotionally neglected DISCUSSION AND CONCLUSION: Many of these highly disturbed families, especially those sent by Court order are engaged in a network of different social, legal and mental health institutions. Intervention plans are thus conceived and specifically tailored to suit individual families, confronting dysfunctional patterns and appealing to enhance parental responsibilities. Overcoming family crisis of complicated divorce where childresn's emotional needs are neglected is highly indicated for systemic intervening. The authors discuss their experience in treating such families and offer their questions, dilemmas and recommendations. PMID- 16673860 TI - [Possibilities of preventing traumatic dental injuries--a prospective study]. AB - INTRODUCTION: The aim of this study was to investigate the most important etiological factors related to traumatic dental injuries, type of required treatment, period from injury to initial treatment and frequency of recall appointments. MATERIAL AND METHODS: The sample consisted of 283 children with dental injuries managed at the Department of Dentistry of the Faculty of Medicine in Novi Sad, during the last 7 years. Retrospective data relied on trauma protocols of this department, whereas collected data referred to etiological factors, type of initial treatment, period from injury to initial treatment, recall appointments and complications of traumatic dental injuries. These results were compared with other similar national and international studies, in order to promote implementation of preventive strategies that would reduce the increasing frequency of dental trauma. RESULTS: Fall accidents were the most common cause of dental trauma. Injuries were most frequent in autumn, in the street, and in the afternoon hours. Single tooth injury was predominant, while the injured mostly denied previous injuries. Almost half of the injured patients did not seek professional help in the first 24 hours after the injury. 40% of treated patients missed their recall appointment. CONCLUSION: Knowledge of the etiology is important for planning preventive measures, but because of the complexity of etiological factors, it is difficult to prevent traumatic dental injuries. It is also important to underline the importance of immediate initial treatment of traumatized patients, and significance of their regular professional supervision. Generally speaking, almost all treatment procedures in management of traumatic dental injuries include preventive component. PMID- 16673861 TI - [Effect of glycemic control on microalbuminuria and arterial blood pressure in patients with type 1 diabetes]. AB - INTRODUCTION: Diabetic nephropathy is the leading cause of hypertension in type 1 diabetes. Microalbuminuria is usually the first manifestation of renal disease and antedate hypertension. The aim of this study was to investigate relationships between glycemic control, hypertension and microalbuminuria in type 1 diabetics. MATERIAL AND METHODS: We studied 27 type 1 diabetics, 18 male and 9 female, aged 18-50 years, with a duration of diabetes <20 years. Glycemic control was assessed using glycosylated hemoglobin (HbA1c) measurements, fructosamine and lipid analysis. 24-h urinary albumin excretion rate was evaluated by radioimmunoassay. Patients with persistent urinary albumin excretion rate 30-300 mg/24 h were defined as microalbuminuric (Group A--41% patients) and lower than that, as normoalbuminuric (Group B--59%). We examined them twice: first in poor glycemic control and then in good glycemic control. RESULTS: We found significant differences (Student's t-test) between groups in regard to microalbuminuria (p <0.01), diabetes duration (p=0.05), systolic blood pressure (BP) and diastolic BP (p<0.05). Systolic BP (p<0.01), diastolic BP (p<0.01) and microalbuminuria (p=0.05) positively correlated (Spearman's rho) with poor glycemic control in Group A. In both groups there was a significant improvement in glycemic control and regression in systolic and diastolic BP (p<0.01), but only Group B showed significant reduction in urinary albumnin excretion rate (p<0.01). DISCUSSION AND CONCLUSION: In this study, type 1 diabetics showed regression in systolic and diastolic hypertension with improvements of glycemic control regardless of presence of microalbuminuria, but only normoalbuminuric showed significant reduction in urinary albumin excretion rate. PMID- 16673862 TI - [Residual renal function and nutritional status in patients on continuous ambulatory peritoneal dialysis]. AB - INTRODUCTION: During the last years, an increasing number of patients with end stage renal failure caused by various underlying diseases, all over the world, is treated by renal replacement therapy. NUTRITIONAL STATUS: Malnutrition is often found in patients affected by renal failure; it is caused by reduced intake of nutritional substances due to anorexia and dietary restrictions hormonal and metabolic disorders, comorbid conditions and loss of proteins, amino-acids, and vitamins during the dialysis procedure itself. Nutritional status significantly affects the outcome of patients on chronic dialysis treatment. Recent epiodemiological trials have proved that survival on chronic continuous ambulatory peritoneal dialysis program depends more on residual renal function (RRF) than on peritoneal clearances of urea and creatinine. MATERIAL AND METHODS: The aim of the study was to analyze the influence of RRF on common biochemical and anthropometric markers of nutrition in 32 patients with end-stage renal failure with various underlying diseases during the first 6 months on continuous ambulatory peritoneal dialysis (CAPD). The mean residual creatinine clearance was 8,3 ml/min and the mean RRF was 16,24 l/week in our patients at the beginning of the chronic peritoneal dialysis treatment. RESULTS AND CONCLUSION: During the follow-up, the RRF slightly decreased, while the nutritional status of patients significantly improved. Gender and age, as well as the leading disease and peritonitis didn't influence the RRF during the first 6 months of CAPD treatment. We found several positive correlations between RRF and laboratory and anthropometric markers of nutrition during the follow-up, proving the positive influence of RRF on nutritional status of patients on chronic peritoneal dialysis. PMID- 16673863 TI - [Gene similarity between hepatitis C virus and human proteins--a blood transfusion problem]. AB - INTRODUCTION: Hepatitis C is a post-transfusion hepatitis which causes serious problems in blood transfusion. Blood testing requires highly sensitive and specific assays with high predictive value. GENOMIC CHARACTERISTICS OF HEPATITIS C VIRUS: According to recommendations of International Association for the study of Liver Diseases etiological diagnosis of hepatitis is based on highly sensitive third generation assays: epitopes in the NS5 region comprising noncoding sequence UTR with 324-341 well conserved pair of homologous basis in 92% HCV genomes, therefore appropriate for virus RNA detection. DEVELOPMENT OF ASSAYS FOR HEPATITIS VIRUS: The first generation of immunoenzyme tests (IET) were based on detection of antibodies on antigen c 100-3, which is a part of the NS4 region of HCV genome. The second generation of tests with two recombinant proteins--c22-3 and c200, achieved higher sensitivity of assays. The third generation included epitopes from NS5 region, and removed the antigen c100-3. DEVELOPMENT OF AUTOIMMUNITY: Autoimmunity is a pathophysiological mechanism that's leads to chronic inflammatory diseases. Autoimunity is characterized by loss of tolerance towards self-antigens. Viral hepatitis C is associated with development of autoimmune phenomena. MOLECULAR MIMICRY: Molecular mimicry, as a mechanism of autoimmunity, was investigated to establish cross-Reactive immune reactions between HCV antigen and human nitrogen-oxide synthase, Tyrosine kinase Lck and hepatic growth factor activator. CROSS REACTIVITY BETWEEN HCV PROTEINS AND HUMAN PROTEINS: HCV capsid proteins initiate the autoimmune process in the liver because of cross reaction of antibodies with human Gor protein 19-27, which causes autoimmune chronic hepatitis. However, analysis of human protein from protein basis Swiss-prot shows homology between NS5 region and 3 human protein nitrogen oxide synthases, tyrosine kinase-Lck, proto-oncogene and hepatic growth factor activator. According to protein data analysis and competitive in vitro experiments, it was concluded that presence of auto-antibodies is probably the consequence of cross reactive immune response. CONCLUSION: Homology of amino acid sequences in the NS5 region of the HCV genome with nitrogen-oxide synthase, tyrosine kinase-Lck, and hepatic growth factor activator, causes auto-immune phenomena in HC, and can be a model for researching autoimmunity and human virus induced autoimmune diseases. PMID- 16673865 TI - [Ureteral triplication--a case report]. AB - INTRODUCTION: Of all children born with congenital anomalies, 30-40% have urogenital abnormalities. Most of them are asymptomatic, but if any symptoms occur, they are usually symptoms of urinary infections. CASE REPORT: A four-year old girl was admitted because of recurrent urinary infections and in order to perform a thorough clinical examination. Clinical examination showed an asymmetrical gluteus and presence of a fibroma in the same region. Detailed radiologic examinations (ultrasonography, plain X-ray, excretory urography, voiding urethrocystography) revealed a triplicate ureter on the right side with a vesicoureteral reflux of the fifth grade on the same side. Intraoperatively, type III ureteric triplication according to Smith's classification was established. Ureteral remodeling and reimplantation was done. The postoperative course was good, and follow-up voiding urethrocystography showed absence of reflux. DISCUSSION: Ureteral triplication is a consequence of either development of three buds on the mesonephric duct, or development of two buds, one of which further divided into two parts. Most common anomalies associated with ureteral triplication are: ureteral duplication on the other side (37%), ectopy of the ureteric orifice (28%) and kidney dysplasia (8%). Vesicoureteral reflux is very common on affected or on both sides. Symptomatology is the same as in ureteral duplication--dominated by signs of urinary infections. CONCLUSION: Diagnosis of ureteral duplication requires detailed imaging. Treatment of symptomatic abnormalities should be individual, regardless of the applied treatment (conservative or operative). PMID- 16673864 TI - [Agenesis of the corpus callosum in a premature infant associated with Langdon Down syndrome]. AB - INTRODUCTION: Agenesis of the corpus callosum is an abnormality of the part of the brain connecting the two cerebral hemispheres. It can be partial, complete or atypical. The fibers from the cerebral cortex project towards the homotypical region of the contra-lateral cortex passing through the corpus callosum, and crossing the middle line. The absence of corpus callosum causes failure of information transfer from one hemisphere to the other. Children with this anomaly present with learning disabilities and trouble with memorizing facts. Agenesis of corpus callosum may be of syndromic or non-syndromic type. The more common form is the one not associated with any syndrome. The agenesis of corpus callosum is more frequent in male children. CASE REPORT: This paper presents a child from a twin pregnancy with partial absence of corpus callosum. The pregnancy was not controlled. It ended in premature birth. The afflicted twin is a boy, second in birth order. Apart from agenesis of corpus callosum, he also suffers from Down syndrome. The first twin is healthy, with corpus callosum and with normal karyotype. There was no consanquinity. In the 6th month of pregnancy the mother suffered from infection of the upper respiratory tract that might be the etiological factor of this anomaly. The child was born with hypotrophy, and all anthropometric parameters were below the third percentile. In the neonatal period, the agenesis of corpus callosum was diagnosed by ultrasonic examination and confirmed by CT and MR examinations. The child is now three and a half month old. Active monitoring of the psychical and motor development will show whether there will be any retardation in the psycho-motor development and later deficiency of the higher corticalfunctions and intelligence. DISCUSSION AND CONCLUSION: The clinical characteristics of this anomaly are numerous. They range from asymptomatic cases, with normal intellectual capacity, to severe mental retardation. Radiological and genetic markers cannot make a difference between the asymptomatic and symptomatic characteristics of the disease. Therefore, it is very difficult to give genetic advice if the diagnosis is made prenatally. This part of the central nervous system is very well visualized by ultrasonic examination, thus in the absence of corpus callosum a special attention must be paid to other parts of the central nervous system, for diagnosing associated anomalies. If the diagnosis of agenesis of corpus callosum is made prenatally by ultrasound, a MR examination is advised for diagnosing other anomalies, especially in the central nervous system like lissencephalia, schizocephalia or heterotopia of the gray matter, where MR examination is superior to others. PMID- 16673866 TI - Scottish Women's Hospitals--the 90th anniversary of their work in Serbia. AB - The Scottish Women's Hospitals (SWH), a unique health institution in the history of medicine, staffed entirely by women, was founded soon after the outbreak of the First World War, August 12, 1914 in Edinburgh, by the National Union of Women's Suffrage Societies. The founder and the main driving force behind this organisation was Dr. Elsie Inglis (1864-1917). Although her proposition to the British War Office had been rejected, she offered her services to the Allies (France, Belgium, Russia and Serbia). The first 200 bed SWH unit was sent to France in November 1914, and soon after followed other units, so at the end there were 13 very well equipped SWH units working in the various theatres of war in Belgium, Serbia, Russia, Rumania and Greece. The first unit of SWH came to Serbia in early January 1915, and was located at Kragujevac. Soon after, three other SWH units arrived to Serbia and were stationed at Mladenovac, Valjevo and Lazarevac. It was an enormous help to Serbia, full of wounded and sick people, due to the dreadful typhus epidemic which was devastating the country. A large SWH unit, attached to the Southern Slav Volunteer Division, had worked on the Dobrudja front, and there were three hospitals and a special transport unit on the Salonika Front, which were all engaged in the treatment of Serbian wounded soldiers until the end of the First World War. Two other SWH units, located in France, were treating the Serbian refugees. Serving bravely and honorably on the various theatres of war, the legendary Scottish Women's Hospitals made enormous contributions to the allied war efforts, and helped Serbian people a great deal. PMID- 16673867 TI - [Suppression of tumor immunity by electromagnetic fields and glucocorticoids in mice with implanted Ehrlich carcinoma]. AB - INTRODUCTION: The immune system plays a major role in the origin, growth and evolution of tumors; factors that decrease the immune response in any way can cause higher tumor incidence and its faster or uncontrolled growth and evolution. MATERIAL AND METHODS: The research included 18 healthy male Han: NMRI mice, weighing between 25 and 30g, with ten-day-old tumor deposits, divided into three groups consisting of six mice each. The first group was continuously exposed to extremely low frequency electromagnetic fields (intensity 70-320 microT). The second group was treated with high doses of corticosteroids (dexamethasone). The control group was not treated with corticosteroids, nor was exposed to extremely low frequency electromagnetic fields. The exposure period lasted for ten days. The criteria used to evaluate tumor immunity were: histological findings of leukocyte infiltration around the tumor cells and white blood cell count. RESULTS: The control group presented with excellent immune response to tumor cells. Lymphoplasmacytic infiltrates widely surrounded the tumor. Numerous tumor cells showed signs of cell death. The results showed that exposure of animals to high doses of glucocorticoids resulted in extremely decreased leukocyte infiltration in the tumor tissue (single lymphocytes), while exposure to extremely low frequency electromagnetic fields significantly decreased leukocyte infiltration in comparison to the control group. Comparison of white blood cell count in treated groups revealed that the white blood cell count in both treated groups was decreased, compared with the control group. CONCLUSION: Extremely low frequency electromagnetic fields significantly suppress the immune response to tumor cells. Dexamethasone treatment resulted in almost complete absence of immune response to tumor cells. Electromagnetic fields and dexamethasone both decrease the white blood cell count. PMID- 16673868 TI - [Phoniatry at the end of its first years]. PMID- 16673869 TI - Isolation of mutagen-sensitive Chinese hamster cell lines by replica plating. AB - Mutant rodent cell lines hypersensitive to DNA-damaging agents have provided a useful tool for the characterization of DNA repair pathways and have contributed to a better understanding of the mechanisms involved in the cellular responses to mutagenic treatment. Here we present a detailed description of how to isolate mutagen-sensitive mutants from hamster "wild-type" cell lines. First, cells are treated with ethyl nitrosourea, and then the mutagenized cell populations are screened for cells with an increased sensitivity to various mutagens using a replica-plating method. Mutagen-sensitive clones are identified and then characterized by assessing their stability, degree of sensitivity to various mutagens, and by genetic complementation analysis. PMID- 16673870 TI - Complementation assays adapted for DNA repair-deficient keratinocytes. AB - Genetic alterations affecting nucleotide excision repair, the most versatile DNA repair mechanism responsible for removal of bulky DNA adducts including ultraviolet (UV) light-induced DNA lesions, may result in the rare, recessively inherited autosomal syndromes xeroderma pigmentosum (XP), Cockayne syndrome (CS), or trichothiodystrophy (TTD). Classical approaches such as somatic cell fusions or microinjection assays have formalized the genetic complexity of these related but clinically distinct syndromes, and contributed to the determination of seven, five, and three complementation groups for XP, CS, and TTD, respectively. XP patients are highly susceptible to photoinduced cutaneous cancers of epidermal origin. To better study the responses to UV irradiation of XP keratinocytes, and to objectively determine the extent to which cutaneous gene therapy may be realized, we set up experimental procedures adapted to ex vivo genetic complementation of keratinocytes from XP patients. We provide here detailed rationales and procedures for these approaches. PMID- 16673871 TI - Cytogenetic challenge assays for assessment of DNA repair capacities. AB - Different challenge assays have been used to investigate cellular responses following exposure to DNA damaging agents. Our protocol uses X- or gamma-rays or ultraviolet light to challenge cells to repair the induced damage, and chromosome aberrations as a biomarker to indicate DNA repair proficiency. The assay was used successfully to demonstrate base- and nucleotide-excision repair deficiency in certain polymorphic DNA repair genes, namely XRCC1 751Gln and XPD 312Asn, respectively. In addition, populations with elevated exposure to certain environmental mutagenic agents-cigarette smokers, pesticide sprayers, and residents who lived near uranium mining and milling sites-showed DNA repair deficiency. Because expression of chromosome aberrations is associated with a significantly increased incidence of both cancer morbidity and mortality, the challenge assay may be useful in predicting cancer risk. The protocol for the assay is straightforward and the data have practical applications. PMID- 16673872 TI - Evaluating the delayed effects of cellular exposure to ionizing radiation. AB - A number of ongoing delayed effects have now been described in the progeny of an irradiated cell. These are grouped under the rubric of radiation induced genomic instability. Perhaps the best characterized is the dynamic production of chromosomal rearrangements in some clonally expanded cells surviving irradiation. In this chapter we provide the protocols for irradiation, cell culture, chromosome analysis, and characterization of the status of genomic stability in the context of delayed radiation effects. PMID- 16673873 TI - Inhibition of DNA synthesis by ionizing radiation: a marker for an S-phase checkpoint. AB - Inhibition of replicative DNA synthesis by ionizing radiation is partly caused by an active, signal-mediated response termed the "S-phase checkpoint." Defects in this checkpoint were first discovered in the human inherited disorder ataxia telangiectasia (AT). gamma-Irradiated cells from AT patients consistently display a diminished inhibition of DNA synthesis, a feature called "radioresistant DNA synthesis" (RDS). RDS has been widely used as a diagnostic marker for AT, in postnatal as well as prenatal material. The regulation and control of the S-phase checkpoint is complex and multifaceted; it is not restricted to ionizing radiation, but can occur after many genotoxic stressors. Defects in both upstream control functions, such as ATM, NBS1, and MRE11, as well as downstream modulators can provoke an RDS phenotype. Here a simple, accurate and highly reproducible experimental protocol is presented for the generation of DNA synthesis inhibition curves from cells in culture. PMID- 16673874 TI - Analysis of inhibition of DNA replication in irradiated cells using the SV40 based in vitro assay of DNA replication. AB - The mechanisms of inhibition of DNA replication after DNA damage through the activation of the S-phase checkpoint have been the focus of several investigations over the last 40 yr. Recent studies have identified several components of this checkpoint response and there is strong interest in its biochemical characterization. Helpful for the delineation of the mechanism of the S-phase checkpoint is the observation that factors inhibiting DNA replication in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested using the simian virus 40 (SV40) assay for in vitro DNA replication. In this assay, replication of plasmids carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein. Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after exposure to DNA damage. The procedures include the preparation of components of the in vitro DNA replication reaction including cytoplasmic extracts from cells that have sustained DNA damage. The assay is powerful but is limited by the fact that initiation steps carried out by the TAg in vitro may have different cellular determinants. PMID- 16673875 TI - Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage. AB - DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as gammaH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of gammaH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of gammaH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase. PMID- 16673876 TI - Detection of DNA strand breaks by flow and laser scanning cytometry in studies of apoptosis and cell proliferation (DNA replication). AB - Extensive fragmentation of nuclear DNA occurs during apoptosis, and the presence of DNA strand breaks is considered to be a marker of the apoptotic mode of cell death. This chapter describes methods to label in situ DNA strand breaks with fluorochromes for detection by flow or laser scanning cytometry. By staining DNA with a fluorochrome of another color, cellular DNA content is measured concurrently and the bivariate analysis of such a data reveals DNA ploidy and cell-cycle phase position of apoptotic cells. The DNA strand break-labeling methodology is also used for detecting the incorporation of halogenated DNA precursors in studies of the cell cycle, proliferation, and DNA replication. In this application, termed "strand breaks induced by photolysis" (SBIP), the cells are incubated with 5-bromo-2'-deoxyuridine (BrdU) to incorporate it into DNA and sensitize the DNA to ultraviolet (UV) light. DNA strand breaks are then photolytically generated by exposing the cells to UV light. The DNA strand breaks resulting from UV-photolysis are subsequently fluorochrome-labeled as for labeling apoptotic-DNA breaks. Because SBIP, unlike the alternative method of detection of BrdU incorporation, does not require subjecting cells to harsh conditions (strong acid or heat) of DNA denaturation, it is compatible with concurrent detection of intracellular or cell surface antigens by immunocytochemical means. PMID- 16673877 TI - In vitro rejoining of double-strand breaks in genomic DNA. AB - Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end-joining (NHEJ) in higher eukaryotes, and have helped to characterize several components including DNA-PKcs, Ku, DNA ligase IV, and XRCC4. There is evidence, however, that additional factors involved in NHEJ remain to be characterized. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end-joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of double strand breaks (DSBs) induced in plasmid vs genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: "naked" DNA and DNA organized in chromatin. Here, we describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for preparation of cell extract and the preparation of the substrate DNA ("naked" DNA or nuclei). PMID- 16673878 TI - Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR. AB - Current techniques for examining the global creation and repair of DNA double strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location. PMID- 16673879 TI - Plasmid-based assays for DNA end-joining in vitro. AB - Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characterization of NHEJ has significantly benefited from in vitro plasmid end-joining assays that can complement and extend information obtained from genetic studies. There is evidence that several factors involved in DNA-PK-dependent NHEJ remain to be identified. In addition, under certain circumstances, cells utilize backup pathways of NHEJ that depend on unknown factors to remove DSBs. Characterization of these putative factors will benefit from plasmid-based assays of DNA end-joining. Here, we describe a protocol for in vitro end-joining using plasmid DNA as substrate. The required procedures include: (1) preparation of HeLa-cell nuclear extract; (2) preparation of plasmid substrate DNA; (3) assembly of in vitro DNA repair reactions; and (4) product analysis by gel electrophoresis. The assay is powerful and easy to perform, but one should be aware that it represents an oversimplification, as it does not consider the in vivo organization of DNA into chromatin. PMID- 16673880 TI - Use of gene targeting to study recombination in mammalian cell DNA repair mutants. AB - Gene targeting by homologous recombination in mammalian cells is an important tool for generating genetically modified mice used for modeling human diseases. Gene targeting approaches are also useful for studying the mechanisms of homologous recombination. We have developed gene targeting methods that we have specifically used to investigate the mechanisms of recombination in cultured mammalian cells. In this chapter, we describe the generation of Chinese hamster ovary (CHO) cell gene disruption ("knockout") mutants in the repair/recombination gene ERCC1. Using this approach, we have constructed pairs of isogenic ERCC1 proficient and -deficient (null) CHO cell lines and used them as recipients for gene targeting assays in which a hemizygous mutant hamster adenine phosphoribosyltransferase (APRT) locus is corrected by homologous recombination with plasmid vectors containing hamster APRT DNA sequence homologous to the target gene in each cell line. The configuration of the targeting vector leads to experimental outcomes in which certain classes of APRTrecombinants are over- or under-represented depending on the repair gene status of the transfection recipient. We describe methods both for targeted gene knockout of ERCC1, and for APRT targeted gene correction by homologous recombination, and some of our experimental results using these approaches. PMID- 16673881 TI - Gene-specific and mitochondrial repair of oxidative DNA damage. AB - The Southern blot gene-specific DNA damage and repair assay is a robust and flexible method for quantifying many kinds of induced damage and repair with high reproducibility. Specific nicking and loss of a restricted DNA fragment at the site of induced damage is visualized by Southern blot and quantified against a control; since the blot is gene specific, only the damage of interest is measured. Here we show how the assay may be adapted to assess mitochondrial DNA (mtDNA) damage. In the mitochondrion, 8-oxoguanine is a significant oxidative lesion; in the laboratory, photoactivated methylene blue may be used to introduce this lesion into cells. Other lesions may also be studied by using different DNA damaging agents. We find that damage induction by methylene blue is consistently far greater in the mitochondrion than the nucleus. Thus advantageously, mitochondrial 8-oxoguanine repair may be studied without mtDNA isolation or preparation, which are processes known to induce DNA damage and skew measurements. This chapter gives detailed instructions for using methylene blue and the gene-specific repair assay to accurately measure mitochondrial oxidative damage and repair rates. PMID- 16673882 TI - Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. AB - In this chapter, we describe a gene-specific quantitative polymerase chain reaction (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been extensively used to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins, and has proved particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA (mtDNA) damage. QPCR can be used to quantify the formation of DNA damage, as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria, or a separate step of mtDNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol for the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory. PMID- 16673883 TI - Measuring the formation and repair of DNA damage by ligation-mediated PCR. AB - There is a need to analyze the formation of DNA lesions in specific sequence contexts. The formation and repair of DNA damage at specific locations in the genome is modulated by the DNA sequence, by DNA methylation patterns, by the transcriptional status of the locus, and by chromatin proteins associated with the DNA. The only method currently available to allow a precise sequence mapping of DNA lesions in mammalian cells is the ligation-mediated polymerase chain reaction (LM-PCR). I describe the technical details of LM-PCR as exemplified by the mapping of DNA damage products in ultraviolet (UV) light-irradiated cells. PMID- 16673884 TI - Immunochemical detection of UV-induced DNA damage and repair. AB - Because of a substantial rise in the incidence of skin cancer in the United Kingdom and elsewhere a greater awareness of the role of sun-induced cutaneous genetic damage has developed. This, in turn, has increased interest in the cellular mechanisms responsible for tumorigenesis, and the need to develop experimental methodologies to investigate these mechanisms. DNA represents a most important cellular target for ultraviolet radiation (UVR), leading to the formation of various DNA damage products. A number of these products, such as the cyclobutane pyrimidine dimer, have been implicated in the pathogenesis of various UVR-related conditions. In this chapter we detail a number of methods for assessing UVR-induced DNA damage using two antisera which recognize cyclobutane thymine dimers (T-T). Immuno-approaches have a number of benefits over chromatographic techniques, and have been applied herein to quantitatively and qualitatively assess the presence of T-T in cultured keratinocytes, human skin, and urine, providing information about lesion induction and repair. PMID- 16673885 TI - A dot-blot immunoassay for measuring repair of ultraviolet photoproducts. AB - The method described here makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UV C)--pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot-blot and quantified using an enzyme-conjugated secondary antibody and a color assay. Though the polyclonal antiserum contains antibodies to both cyclobutane pyrimidine dimers and (64) photoproducts, repair of these can be measured separately by differential destruction of one or other photoproduct. The method is useful for measuring repair in total genomic DNA. It is more sensitive than most other methods and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UV-C in DNA from mammalian cells. PMID- 16673886 TI - Quantification of photoproducts in mammalian cell DNA using radioimmunoassay. AB - Over the past 20 yr, the use of polyclonal and monoclonal antibodies to quantify damage in DNA has burgeoned. Immunoassays offer distinct advantages over other anaytical procedures currently used to measure DNA damage including adaptability, sensitivity and selectivity. This combination of attributes allows for the development of powerful analytical techniques to visualize and quantify specific types of DNA damage in cells and organisms exposed to subtoxic levels of xenobiotics with distinct advantages over the other procedures in the analysis of DNA damage in human and environmental samples. Radioimmunoassay (RIA) is readily applied to a variety of biological materials and has typically been used to measure DNA damage in cell and organ cultures, tissue sections and biopsies, buccal cells, bone marrow aspirates, peripheral blood lymphocytes, and urine. Here we describe the use of a very sensitive RIA for the specific quantitation of cyclobutane dimers and (6-4) photoproducts in DNA extracted from mammalian cells and tissues. PMID- 16673888 TI - The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair. AB - The comet assay (single-cell gel electrophoresis) is a simple and sensitive method for studying DNA damage and repair. In this microgel electrophoresis technique, a small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus toward the anode, which resembles the shape of a comet. The assay has manifold applications in fundamental research for DNA damage and repair, in genotoxicity testing of novel chemicals and pharmaceuticals, environmental biomonitoring, and human population monitoring. This chapter describes a standard protocol of the alkaline comet assay and points to some useful modifications. PMID- 16673887 TI - DNA damage quantitation by alkaline gel electrophoresis. AB - Quantifying DNA lesions provides a powerful way to assess the level of endogenous damage or the damage level induced by radiation, chemical or other agents, as well as the ability of cells to repair such damages. Quantitative gel electrophoresis of experimental DNAs along with DNA length standards, imaging the resulting dispersed DNA and calculating the population average length allows accurate measurement of lesion frequencies. Number average length analysis provides high sensitivity and does not require any specific distribution of lesions within the DNA molecules. These methods are readily applicable to strand breaks and ultraviolet radiation induced pyrimidine dimers, but can also be used with appropriate modifications-for ionizing radiation-induced lesions such as oxidized bases and abasic sites. PMID- 16673889 TI - Fast micromethod DNA single-strand-break assay. AB - The Fast Micromethod is a convenient and quick fluorimetric microplate assay for the assessment of DNA single-strand breaks and their repair. This method measures the rate of unwinding of cellular DNA on exposure to alkaline conditions using a fluorescent dye which preferentially binds to double-stranded DNA, but not to single-stranded DNA or protein. The advantages of this method are that it requires only minute amounts of material (30 ng of DNA or about 3000 cells per single well), it allows simultaneous measurements of multiple samples, and it can be performed within 3 h or less (for one 96-well microplate). The Fast Micromethod can be used for the routine determination of DNA damage in cells and tissue samples after irradiation, exposure to mutagenic and carcinogenic agents, or chemotherapy. PMID- 16673890 TI - 32P-postlabeling DNA damage assays: PAGE, TLC, and HPLC. AB - 32P-Postlabeling analysis is a powerful technique for detecting, identifying, and quantifying DNA adducts induced by mutagens or carcinogens. The method involves enzymatic digestion of the DNA sample to nucleoside 3'-monophosphates, and partial purification of the adducted nucleotides followed by their 5'-labeling with 32P. For analysis of DNA adducts, polyethyleneimine-cellulose thin-layer chromatography (TLC) plates have traditionally been used to resolve 32P-labeled DNA adducts (32P-postlabeling/ TLC analysis). However, the TLC procedure is time consuming and labor intensive. To expedite analyses, we recently devised a 32P postlabeling protocol that utilizes nondenaturing polyacrylamide gel electrophoresis (PAGE) and permits multiple DNA samples to be run on a single gel (32P-postlabeling/PAGE analysis). Using this method, the detection limit for 5 microg of DNA is approx 7 adducts/10(9) nucleotides, similar to that for 32P postlabeling/TLC. For still higher sensitivity and resolution, high-performance liquid chromatography (HPLC) combined with a radioisotope detector system (32P postlabeling/HPLC analysis) can be used to increase the detection limit to approx 3 adducts/10(10) nucleotides. Here we describe all three 32P-postlabeling techniques. PMID- 16673891 TI - Electrophoretic mobility shift assays to study protein binding to damaged DNA. AB - The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes. PMID- 16673892 TI - Construction of MMR plasmid substrates and analysis of MMR error correction and excision. AB - We describe simple and efficient construction of mismatch repair (MMR) substrates, by generation of gapped plasmids using one sequence-specific nicking endonuclease (N.BstNBI), ligation of synthetic oligomers into the gaps, and introduction of defined single nicks for initiation of MMR excision using a second such endonuclease (N.AlwI). We further describe measurement of completed mismatch correction and a sensitive quantitative assay for MMR excision intermediates. These methods can be easily adapted for construction of substrates containing defined DNA lesions, for analysis of MMR responses to DNA damage and for studies of other DNA repair pathways. PMID- 16673893 TI - Characterization of enzymes that initiate base excision repair at abasic sites. AB - Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a source of mutations. Organisms have developed an enzymatic means of repairing abasic sites in DNA that generally involves a DNA repair pathway that is initiated by a repair protein creating a phosphodiester break ("nick") adjacent to the site of base loss. Here we describe a method for analyzing the manner in which repair endonucleases differ in the way they create nicks in DNA and how to distinguish between them using cellular crude extracts. PMID- 16673894 TI - Base excision repair in mammalian cells. AB - A rapid, convenient and safe in vitro assay system for base excision repair is described. Whole cell extracts are prepared by detergent-based cell lysis and provide a vigorous activity of AP site repair. A circular DNA substrate is used for detection of both DNA polymerase beta-dependent and proliferating cell nuclear antigen (PCNA)-dependent pathways. Repaired and unrepaired DNA substrates are separated by agarose gel electrophoresis as a linear DNA molecule and a nicked circular molecule, respectively, and detected by staining with SYBR Green I. This assay system does not require radioactive substrates or nucleotides, and provides a sensitivity in which 10 ng of a DNA substrate per reaction is sufficient for quantitative repair analysis. PMID- 16673895 TI - In vitro base excision repair assay using mammalian cell extracts. AB - Base excision repair (BER) is the main pathway for removal of endogenous DNA damage. This repair mechanism is initiated by a specific DNA glycosylase that recognizes and removes the damaged base through N-glycosylic bond hydrolysis. The generated apurinic/apyrimidinic (AP) site can be repaired in mammalian cells by two alternative pathways which involve either the replacement of one (short patch BER) or more nucleotides (long patch BER) at the lesion site. This chapter describes a repair replication assay for measuring BER efficiency and mode in mammalian cell extracts. The DNA substrate used in the assay is either a randomly depurinated plasmid DNA or a plasmid containing a single lesion that is processed via BER (for example a single AP site or uracil residue). The construction of a single lesion at a defined site of the plasmid genome makes the substrate amenable to fine mapping of the repair patches, thus allowing discrimination between the two BER pathways. PMID- 16673896 TI - Biochemical assays for the characterization of DNA helicases. AB - Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5'-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombination, and DNA repair. Furthermore, an increasing number of helicases have been linked to genomic instability and human disease. With the growing interest in helicase mechanism and function, we have set out to describe some basic protocols for biochemical characterization of DNA helicases. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding activity of DNA helicases are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of a given helicase, which would serve as a platform for further investigation of its molecular and cellular functions. PMID- 16673897 TI - Repair synthesis assay for nucleotide excision repair activity using fractionated cell extracts and UV-damaged plasmid DNA. AB - Methods are described for measuring nucleotide excision repair (NER) of damaged plasmid DNA using fractionated mammalian cell extracts. NER creates a single stranded gap of approx 25-30 nt. Filling of this gap by repair synthesis can be monitored by the incorporation of radioactive nucleotides. We first describe the preparation of ultraviolet light (UV)-damaged and control plasmid DNA substrates and purification of their closed-circular forms. To increase the specificity for NER, plasmid molecules containing pyrimidine hydrates and other lesions sensitive to Escherichia coli Nth protein are eliminated. The preparation of whole cell extracts active in NER is described, both for cells grown as attached cultures and those grown in suspension. Cell extracts are partially purified on phosphocellulose to produce a fraction that can carry out the full NER reaction when combined with purified RPA and PCNA proteins. This enables NER to be quantified in an assay with exceptionally low background in nondamaged DNA. PMID- 16673898 TI - Assaying for the dual incisions of nucleotide excision repair using DNA with a lesion at a specific site. AB - Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily specifically measured by detecting the 24-32 nucleotide products of the dual-incision reaction. This chapter describes several methods for detection of repair of a specific lesion in closed-circular DNA. As a model lesion, we use the well-repaired 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Three methods are given for analysis of repair. One is to incorporate a radioactive label internally near the lesion and measure excision by detecting radioactive excised oligomers. Two other methods use DNA that is not internally labeled so that it can be stored and used when convenient. The first method for detection of repair of such unlabeled DNA is to detect excision products with a labeled complementary oligonucleotide by Southern blot hybridization. The second method is to 3'- end-label the excised oligonucleotide directly with radiolabeled dNTP and a DNA polymerase, using a complementary oligonucleotide with a 5' overhang that serves as a template. This protocol is fast and sensitive, but relies on accurate foreknowledge of the site of 3'-incision for the particular lesion being used. PMID- 16673899 TI - Analysis of proliferating cell nuclear antigen (PCNA) associated with DNA. AB - Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein adopting a ring structure that may encircle DNA. In this form, PCNA functions as a sliding platform to which different types of catalytic and regulatory proteins are tethered to perform DNA transactions such as replication and repair synthesis, methylation, chromatin assembly and remodeling, as well as sister chromatid cohesion. In addition, PCNA coordinates DNA metabolism with cell cycle progression by interacting with cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors. PCNA participates in different pathways of DNA repair, including nucleotide and base excision repair, as well as mismatch repair, by interacting with proteins involved in these processes. A fundamental step in DNA repair involves the transition of PCNA from a freely soluble nucleoplasmic form, to a chromatin-bound form associated with repair sites. This chapter describes biochemical and immunofluorescence methods for detection of the chromatin-bound form of PCNA involved in DNA repair. Cellular fractionation and nuclear extraction procedures are provided for Western blot analysis, as well as for protein-protein interaction studies. An in situ extraction protocol is described for immunostaining, fluorescence microscopy and flow cytometric analyses of nuclear localization, and cell cycle distribution of PCNA associated with DNA repair sites. PMID- 16673900 TI - Analysis of DNA repair and chromatin assembly in vitro using immobilized damaged DNA substrates. AB - Significant advances have been made in identifying a complex network of proteins that could play a role in the repair of DNA damage in the context of chromatin. Insights into this process have been obtained by combining damaged DNA substrates with mammalian cell-free systems that contain both DNA repair and chromatin assembly activities. The methods described in this chapter provide a powerful approach for the detection of proteins recruited during the recognition and repair of DNA lesions, including repair proteins and chromatin associated factors. Substrates for the recruitment assay consist of DNA containing damage that is immobilized on magnetic beads. A human cell-free system that supports both DNA repair and chromatin assembly is incubated with the immobilized DNA damaged substrates, and proteins associated with the DNA are then isolated and subjected to analysis. We present here protocols for preparing bead-linked DNA substrates containing different types of lesions, for the reaction of the damaged DNA with cell-free systems, and for the subsequent analysis of proteins that are recruited to the immobilized damaged DNA substrates. PMID- 16673901 TI - An introduction to plant cell culture: Back to the future. AB - Plant cell, tissue, and organ culture is a set of techniques designed for the growth and multiplication of cells and tissues using nutrient solutions in an aseptic and controlled environment. This technology explores conditions that promote cell division and genetic reprogramming in in vitro conditions. Mainly developed in the early 1960s, plant tissue culture has turned into a standard procedure for modem biotechnology and today one can recognize five major areas where in vitro cell cultures are currently applied: large-scale propagation of elite materials, generation of genetic modified fertile individuals, as a model system for fundamental plant cell physiology aspects, preservation of endangered species, and metabolic engineering of fine chemicals. This chapter reviews the recent advances in such areas. PMID- 16673902 TI - History of plant tissue culture. AB - Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the beginning of the 20th century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. The historical development of these in vitro technologies and their applications are the focus of this chapter. PMID- 16673903 TI - Pathogen and biological contamination management: the road ahead. AB - Multiplication of certified pathogen-free stock plants in vitro makes an important contribution to the production of disease-free planting material for vegetatively propagated crops. Meristem culture is extensively used to eliminate pathogens and contaminants from microbially contaminated plants prior to micropropagation. The approach to pathogen and contamination management differs. It is essential to avoid the release of pathogen-contaminated microplants and to ensure this the plant pathogen-testing guidelines and protocols issued by the Food and Agriculture Organisation (FAO) and its regional representative organizations should be followed. Where in vitro methods are used to eliminate pathogens, the progeny plants should be established in vivo under quarantine conditions and tested under FAO guidelines before being used as stock plants for in vitro multiplication. At establishment of microplants in vitro (stage 1), cultures should be culture-indexed for the presence of microbial contaminants. If pathogen-and contaminant-free cultures are established, then the risk is that of managing laboratory contamination by common environmental microorganisms based on Hazard Analysis Critical Control Points (HACCP) principles. International plant health certification organizations are conservative and rely on established pathogen indexing protocols. They are reluctant to accept DNA-based tests and do not accept testing of in vitro cultures. Given that in time both of these restrictions may be relaxed, micropropagators may look forward to availing more of diagnostic service providers using polymerase chain reaction-based multiplex assays for pathogen-indexing and advances in diagnostic kits for environmental microorganisms in support of laboratory contamination management; with the caveat that molecular tests for pathogens may continue to require confirmation by inoculation of indicator plant species. PMID- 16673904 TI - Growth measurements: estimation of cell division and cell expansion. AB - The main parameters for the estimation of growth within in vitro cultures are reviewed. Procedures to measure these parameters are described, emphasizing in each case their convenience of use, depending on the features of the culture to evaluate. PMID- 16673905 TI - Callus and suspension culture induction, maintenance, and characterization. AB - Callus and cell suspension can be used for long-term cell cultures maintenance. This chapter describes procedures for the induction of somatic embryos of garlic, keeping a regeneration capacity for more than 5 yr, as well as the maintenance of a tobacco suspension culture (NT-1 cells), for more than 10 yr. Methods for plant regeneration and growth kinetics of garlic cultures are described, as well as for cell viability of NT-1 cells stained with 2,3,5 triphenyltetrazolium chloride. The packed cell volume determination as a parameter of growth is detailed. PMID- 16673906 TI - Measurement of cell viability in in vitro cultures. AB - An overview of the methods for assessing cell viability in in vitro cultures is presented. The protocols of four of the most commonly used assays are described in detail, so the readers may be able to determine which assay is suitable for their own projects using plant cell cultures. PMID- 16673907 TI - Cryopreservation of embryogenic cell suspensions by encapsulation-vitrification. AB - Encapsulation-vitrification, which is a combination of encapsulation-dehydration and vitrification procedures, is a newly developed technique for cryopreservation of plant germoplasm. Here, we describe the protocol of this methodology, using grapevine (Vitis) as a model plant. Cell suspensions at the exponential growth stage were encapsulated with 2.5% sodium alginate solution in 0.1 M calcium chloride solution for 20 min to form beads of about 4 mm in diameter containing 25% cells. The beads were stepwise precultured in increasing sucrose concentrations of 0.25, 0.5, and 0.75 M for 3 yr, with 1 d for each step. Precultured beads were treated with a loading solution for 60 min at room temperature and then dehydrated with PVS2 at 0 degrees C for 270 min, followed by direct immersion in liquid nitrogen for 1 h. The beads were rapidly rewarmed at 40 degrees C in a water bath for 3 min and then diluted with 1 M sucorse solution at room temperature for 30 min. Rewarmed, washed beads were post-cultured on a recovery medium for 3 d at 25 degrees C in the dark for survival. Surviving cells were transferred to a regrowth medium to induce cell proliferation. Embryogenic cell suspensions were re-established by suspending the cells in a cell suspension maintenance medium maintained on a gyratory shaker at 25 degrees C in the dark. For plant regeneration, surviving cells were transferred from the recovery medium to an embryo maturation medium and maintained at 25 degrees C under light conditions. Embryos at the torpedo stage were cultured on rooting medium until whole plantlet was developed. PMID- 16673908 TI - Somatic embryogenesis in Picea suspension cultures. AB - Generation of somatic embryos in spruce is achieved through the execution of five steps designated as: (1) induction of embryogenic tissue, (2) maintenance of embryogenic tissue, (3) embryo development, (4) embryo maturation, and (5) conversion into plants. Depending on species and genotypes within the same species, each step must be optimized for obtaining maximum results. In general, embryogenic tissue is generated from immature and mature zygotic embryos and maintained in either liquid or solid conditions in the presence of plant growth regulators auxin and cytokinin. Initiation of embryo development in suspension cultured is induced by removal of plant growth regulators, whereas continuation of development and completion of maturation require applications of abscisic acid and imposition of a desiccation period. Both treatments are needed for conferring morphological and physiological maturation to the embryos. Mature somatic embryos are germinated in the absence of plant regulators and embryo conversion (i.e., formation of a functional shoot and root, occurs after a few weeks in culture). PMID- 16673909 TI - Indirect somatic embryogenesis in cassava for genetic modification purposes. AB - In cassava both direct and indirect somatic embryogenesis is described. Direct somatic embryogenesis starts with the culture of leaf explants on Murashige and Skoog (MS) medium supplemented with auxins. Somatic embryos undergo secondary somatic embryogenesis when cultured on the same medium. Indirect somatic embryogenesis is initiated by subculture of directly induced embryogenic tissue on auxin-supplemented medium with Gresshoff and Doy salts and vitamins. A very fine friable embryogenic callus (FEC) is formed after a few rounds of subculture and stringent selection. This FEC is maintained by subculture on auxin supplemented medium. Lowering of the auxin concentration allows the FEC to form mature somatic embryos that develop into plants when transferred to a cytokinin supplemented medium. PMID- 16673910 TI - Direct somatic embryogenesis in Coffea canephora. AB - Somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several model systems have been reported for in vitro SE induction. SE for coffee was first reported in Coffea canephora. Several systems have been developed since then, including SE from callus cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with growth regulators. This is a simplified method to obtain a faster and more efficient protocol to produce direct somatic embryos in C. canephora. PMID- 16673911 TI - A new temporary immersion bioreactor system for micropropagation. AB - A new type of bioreactor system for plant micropropagation is described that incorporates a number of features specifically designed to simplify its operation and reduce production costs. The BioMINT unit is a mid-sized (1.2 L) reactor that operates on the principle of temporary immersion. It is built of polypropylene and is translucent, autoclavable, and reusable. It consists of two vessels, one for the plant tissues and the other one for the liquid culture media coupled together through a perforated adaptor piece that permits the flow of the liquid media from one vessel to the other. This flux is driven by gravity through a see saw movement provided by equipment (SyB) consisting of electric motor powered platforms that change position. The structural simplicity and the modular and independent nature of the bioreactors simplify their operation and reduce the amount of hand labor required for transfers, thereby reducing the cost of the whole micropropagation process. PMID- 16673912 TI - Protocol to achieve photoautotrophic coconut plants cultured in vitro with improved performance ex vitro. AB - This chapter presents a protocol that will show ways to obtain photoautotrophic coconut in vitro plants and outlines protocol for improving photosynthesis and field performance. This protocol involves reducing sucrose concentration from the growing medium while simultaneously increasing light intensity and enriching the CO2 concentration of growth rooms. PMID- 16673913 TI - Use of statistics in plant biotechnology. AB - Statistics and experimental design are important tools for the plant biotechnologist and should be used when planning and conducting experiments as well as during the analysis and interpretation of results. This chapter provides some basic concepts important to the statistical analysis of data obtained from plant tissue culture or biotechnological experiments, and illustrates the application of common statistical procedures to analyze binomial, count, and continuous data for experiments with different treatment factors as well as identifying trends of dosage treatment factors. PMID- 16673914 TI - An efficient method for the micropropagation of Agave species. AB - Despite their economic importance, the Agave spp. have not been genetically improved. This is probably owing to the fact that they have very long life cycles and many of them have an inefficient sexual reproduction mechanism. Micropropagation offers an alternative to this problem through the efficient cloning of selected high-yielding "elite" plants. We report here an efficient method to micropropagate agaves and a strategy for the management of large scale production that has been successfully applied to several Agave spp. PMID- 16673915 TI - Micropropagation of endangered plant species. AB - This chapter describes the multiple-shoot-based methods of micropropagation for endangered plant species. Taxus and aloe are used here as examples. For Taxus, the process of micropropagation includes initiating multiple shoots, elongating shoots, rooting shoots, and transplanting plantlets. For aloe, the process of micropropagation includes initiating multiple shoots, rooting shoots, and transplanting plantlets. PMID- 16673916 TI - Clonal propagation of softwoods. AB - Softwoods or gymnosperms, which make up 60% of the forested areas of the world, are economically important as a source of lumber, pulp, and paper. Reforestation is a major activity worldwide and the potential benefits of using clonal planting stock have long been recognized. Tissue culture clonal methods or micropropagation is a newer approach that can be achieved by enhancing axillary bud breaking, production of adventitious buds (organogenesis), and somatic embryogenesis. Plantlet production via organogenesis requires at least four stages: (1) bud induction on the explant, (2) shoot development and multiplication, (3) rooting of developed shoots, and (4) hardening of plantlets. Similarly, the production of plantlets via somatic embryogenesis, which has the potential to produce a larger number of plantlets, and in a shorter period of time, also requires several stages. These include (1) induction, maintenance, and proliferation of embryogenic tissue; (2) maturation (both morphological and physiological) of somatic embryos; and (3) germination and conversion of the somatic embryos. In this chapter, plantlet production via organogenesis from seedling and adolescent/mature explants and somatic embryogenesis from immature and mature seeds of white spruce (Picea glauca) are outlined. PMID- 16673917 TI - Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora. AB - The family Passifloraceae contains many species exploited in the food, pharmaceutical, and ornamental plant industries. The routine culture of isolated protoplasts (naked cells) followed by reproducible plant regeneration, is crucial to the genetic improvement of Passiflora spp. by somatic cell technologies. Such procedures include somatic hybridization by protoplast fusion to generate novel hybrid plants, and gene introduction by transformation. Seedling leaves are a convenient source of totipotent protoplasts. The protoplast-to-plant system developed for Passiflora edulis fv. flavicarpa is summarized in this chapter. The procedure involves enzymatic degradation of leaf tissue using commercially available Macerozyme R10, Cellulase R10, and Driselase. Isolated protoplasts are cultured in Kao and Michayluk medium, semi-solidified with agarose. The medium containing the suspended protoplasts is dispensed as droplets or thin layers and bathed in liquid medium of the same composition. Shoot regeneration involves transfer of protoplast-derived tissues to Murashige and Skoog-based medium. The protocols developed for P. edulis are applicable to other Passiflora spp. and will underpin the future biotechnological exploitation of a range of species in this important plant family. PMID- 16673918 TI - Isolation, culture, and plant regeneration from Echinacea purpurea protoplasts. AB - A plant regeneration system from the isolated protoplasts of Echinacea purpurea L. using an alginate solid/liquid culture is described in the chapter. Viable protoplasts were isolated rom 100 mg of young leaves of 4-wk-old seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase, and 0.3 mol/L mannitol. After isolation and purification, the mesophyll protoplasts were embedded into 0.6% Na-alginate at the density 1 x 10(-5) mL and cultured in modified Murashige and Skoog (MS) culture medium supplemented with 0.3 mol/L sucrose, 2.5 micromol/L benzylaminopurine (BA), and 5.0 micromol/L 2,4 dichlorophenoxyacetic acid (2,4-D). The visible colonies were present after 4 wk of culture. The protoplast-derived clones were transferred onto gellan gum solidified basal medium supplemented with 1.0 micromol/L BA and 2.0 micromol/L indole-3-butyric acid (IBA) and formed compact and green calli. Shoot development was achieved by subculturing the calli onto the same basal medium supplemented with 5.0 micromol/L BA and 2.0 micromol/L IBA. Further subculture onto basal medium resulted in the regeneration of complete plantlets. PMID- 16673919 TI - Production of cybrids in Brassicaceae species. AB - This chapter describes a method of cytoplasm transfer within the brassicaceae family through Ca-PEG-mediated protoplast fusion. The method includes a protocol of nonmutagenic albinism induction based on spectinomycin-induced plastid ribosome deficiency (PRD). The proposed application of spectinomycin-mediated albinism allows speeding up creation of albino lines, as well as of hybrid production with substituted cytoplasm. According to described method cybrids between Orychophragmus violaceus and Brassica napus, O. violaceus and Lesquerella fendleri have been produced. Methods of further molecular analysis are also presented. The time-scale and reliability of described methods are indicated. PMID- 16673920 TI - Guard cell protoplasts: isolation, culture, and regeneration of plants. AB - Guard cell protoplasts have been used extensively in short-term experiments designed to elucidate the signal transduction mechanisms that regulate stomatal movements. The utility of uard cell protoplasts for other types of longer-term signal transduction experiments is just now being realized. Because highly purified, primary isolates of guard cell protoplasts are synchronous initially, they are uniform in their responses to changes in culture conditions. Such isolates have demonstrated potential to reveal mechanisms that underlie hormonal signalling for plant cell survival, cell cycle re-entry, reprogramming of genes during dedifferentiation to an embryogenic state, and plant cell thermotolerance. Plants have been regenerated from cultured guard cell protoplasts of two species: Nicotiana glauca (Graham), tree tobacco, and Beta vulgaris, sugar beet. Plants genetically engineered for herbicide tolerance have been regenerated from cultured guard cell protoplasts of B. vulgaris. The method for isolating, culturing, and regenerating plants from guard cell protoplasts of N. glauca is described here. A recently developed procedure for large-scale isolation of these cells from as many as nine leaves per experiment is described. Using this protocol, yields of 1.5-2 x 10(7) per isolate may be obtained. Such yields are sufficient for standard methods of molecular, biochemical, and proteomic analysis. PMID- 16673921 TI - Production of interspecific hybrid plants in Primula. AB - The methods of production of inter-specific hybrids in Primula are categorized into four steps: (1) emasculation, (2) pollination, (3) rescue culture of immature embryo, and (4) confiration of hybridity and ploidy level of the regenerated plants. Although most of the Primula pecies have a heteromorphic self incompatibility system, an emasculation step is usually needed to avoid self pollination since self-incompatibility is not always complete. At the resue culture step, addition of plant hormones (e.g., auxin, cytokinin, and gibberellin) to the culture medium is proved to be effective. The hybridity of the plants is efficiently confirmed at seedling stage by DNA analysis in addition to the comparison of morphological characters. The analysis of relative DNA contents by flow cytometry is easy and rapid technique to confirm hybridity and to estimate ploidy level and genomic combination. PMID- 16673922 TI - Agrobacterium-mediated transformation of Petunia leaf discs. AB - Many dicotyledonous and also several monocotyledonous plant species are susceptible to Agrobacterium-mediated transformation. This current and well established method has been used successfully with a large number of plant species to mediate gene transfer. This chapter describes an Agrobacterium mediated transformation method of Petunia hybrida leaf discs, one of the first species that was routinely transformed using this method. PMID- 16673923 TI - Transformation of wheat via particle bombardment. AB - The protocol described in this chapter was successfully used to produce the first transgenic plants of wheat. It involves high velocity bombardment of explants with DNA-coated microprojectiles (the biolistics procedure). It highlights the importance of selecting the right explants (immature embryos and/or embryogenic callus tissues), pre- and post-osmotic treatment, obtaining high levels of gene expression with the maize ubiquitin promoter, and stringent selection (with bar) during culture to obtain stably transformed and normal fertile plants. The protocol is used widely for genetic transformation of wheat. PMID- 16673924 TI - Chloroplast transformation. AB - In this chapter we briefly review the developmental history and current research status of chloroplast transformation and introduce the merits of chloroplast transformation as compared with the nuclear genome transformation. Furthermore, according to the chloroplast transformation achieved in oilseed rape (Brassica napus), we introduce the preparation of explants, transformation methods, system selection, identification methods of the transplastomic plants, and experimental results. The technical points, the bottleneck, and the further research directions of the chloroplast transformation are discussed in the notes. PMID- 16673925 TI - The biochemical basis for the resistance to aluminum and their potential as selection markers. AB - Aluminum (Al) toxicity is one of the most widespread agronomic problems in world agriculture. The cellular mechanisms that some plant species use to tolerate Al are still not understood today. This knowledge is essential in order to develop crop species that can be cultivated on acid soils. Plant cell culture has been used for the investigation of both: Al-toxicity and -tolerance. The method used to obtain Al-tolerant cell line will be shown; as well as a protocol to measure Al-free concentration in the culture medium by using Morin (a fluorescent histochemical indicator for Al). Because Al affects different processes involved in the signal transduction pathway such as phospholipase C (PLC) activity, which could serve as selection marker, the PLC activity determination will be introduced. PMID- 16673926 TI - Transformation of maize via Agrobacterium tumefaciens using a binary co-integrate vector system. AB - This chapter describes a stepwise protocol to achieve success in genetic transformation of maize using Agrobacterium tumefaciens as a DNA delivery system. Researchers will be able to effectively transform immature embryos of Hi-II and related genotypes with this protocol. The outcome of the transformation process will be transgenic embryogenic callus tissue, transgenic plants, and transgenic progeny seeds. Recommendations for molecular confirmation and evaluation of transgenic tissue/plants are also provided. PMID- 16673927 TI - Capsaicin accumulation in Capsicum spp. suspension cultures. AB - Fruits of chili peppers (Capsicum spp.) specifically synthesize and accumulate a group of analogs known as capsaicinoids in the placenta tissues. These secondary metabolites are responsible for the hot taste of chili pepper fruits. Capsaicinoids are of economic importance because of their use in the food, cosmetic, military, and pharmaceutical industry. Several efforts have been focused to investigate the biosynthetic capacity of in vitro chili pepper cells and tissue cultures in order to determine the production feasibility of these compounds at the industrial level under controlled conditions. A description of techniques for the establishment of in vitro cultures of chili pepper, the addition of precursors and intermediates to the culture medium, and the selection of cell lines as a means to increase the production of capsaicinoids as well as the extraction, separation, and quantification of capsaicinoids from chili pepper cell cultures is reported in this chapter. PMID- 16673928 TI - Isolation and purification of ribosome-inactivating proteins. AB - Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in plants, fungi, and bacteria. RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively cleaving a specific adenine residue from a highly conserved, surface-exposed, stem-loop (S/R loop) structure in the 28S rRNA of ribosomes. Some RIPs also exhibit a number of other enzymatic activities such as RNase, DNase, phospholipase, and superoxide dismutase (SOD). RIPs are considered to be plant defense-related proteins as they are able to inhibit the multiplication and growth of several pathogenic virus, fungi, and bacteria either alone or in conjugation with other defense-related proteins. The mechanism of inhibitory activity of RIPs against fungal pathogens seems to be by directly inhibiting fungal growth rather than depurinating host plant ribosomes and causing cell death as previously envisaged. This chapter describes the protocol used to isolate and purify RIPs from plant tissues. PMID- 16673929 TI - Catharanthus roseus shoot cultures for the production of monoterpenoid indole alkaloids. AB - A protocol for the establishment of in vitro shoot cultures of Catharanthus roseus is described. Shoots can be maintained for more than 1 yr without evidence of tissue vitrification, disaggregation, or callus formation. Vindoline was the main alkaloid accumulated, reaching values similar to those found in leaves from field-grown plants, after a long period of culture. An induction methodology to reduce such waiting time is also presented. PMID- 16673930 TI - Methods for regeneration and transformation in Eschscholzia californica: A model plant to investigate alkaloid biosynthesis. AB - Eschscholzia californica Cham. (California poppy) is a plant species that accumulates pharmacologically active alkaloids biosynthetically related to the morphinan alkaloids of Papaver somniferum. This, in combination with the relative ease with which it is propagated in vitro, makes it a key model for benzylisoquinoline biosynthesis. Transformation techniques are an important tool for these studies and for metabolic engineering attempts. Agrobacterium mediated transformation techniques for this model species have been developed in our lab and used for modulation of transcript levels relevant to the biosynthesis of these alkaloids. Here we describe the techniques used in our lab for production of transgenic callus, hairy root cultures, and whole plants. PMID- 16673932 TI - Plant biotechnology and tissue culture resources on the internet. PMID- 16673931 TI - The components of the culture media. PMID- 16673933 TI - A root-knot nematode secretory peptide functions as a ligand for a plant transcription factor. AB - Parasitism genes expressed in the esophageal gland cells of root-knot nematodes encode proteins that are secreted into host root cells to transform the recipient cells into enlarged multinucleate feeding cells called giant-cells. Expression of a root-knot nematode parasitism gene which encodes a novel 13-amino-acid secretory peptide in plant tissues stimulated root growth. Two SCARECROW-like transcription factors of the GRAS protein family were identified as the putative targets for this bioactive nematode peptide in yeast two-hybrid analyses and confirmed by in vitro and in vivo coimmunoprecipitations. This discovery is the first demonstration of a direct interaction of a nematode-secreted parasitism peptide with a plant-regulatory protein, which may represent an early signaling event in the root-knot nematode-host interaction. PMID- 16673934 TI - The Rhizobium leguminosarum biovar trifolii ANU794 induces novel developmental responses on the subterranean clover cultivar Woogenellup. AB - The clover-nodulating Rhizobium leguminosarum bv. trifolii ANU794 initiates normal root-nodule development with abnormally low efficiency on the Trifolium subterraneum cv. Woogenellup. The cellular and developmental responses of Woogenellup roots to the site- and dose-defined inoculation of green fluorescent protein (gfp)-labeled cells of ANU843 (nodulation proficient) and ANU794 was investigated using light, fluorescence, and confocal microscopy. Strain ANU794 gfp induced three primordia types and four developmental responses at the inoculation site: true or aberrant nodules (on 5 and 25% of plants, respectively), hybrid structures (20% of plants), or lateral roots (50% of plants). The novel hybrid structures possessed nodule and lateral root-like features and unusual vascular patterning. Strain ANU794-gfp induces lateral root formation by stimulating pericycle cell divisions at all nearby protoxylem poles. Only true nodules induced by ANU794-gfp contained intracellular bacteria. In contrast, strain ANU843-gfp induced nodules only and lateral root formation was suppressed at spot inoculation sites. Primordium types were distinguishable by the emission spectrum characteristics of phenolic UV-absorbing and fluorescent compounds that accumulate in primordium cells. Hybrid primordia contained (at least) two fluorescent cell populations, suggesting that they are chimeric. The results suggest that ANU794 may produce both nodule- and lateral root-generating signals simultaneously. PMID- 16673935 TI - Transcriptome analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease development. AB - The clubroot disease of the family Brassicaceae is caused by the obligate biotrophic protist Plasmodiophora brassicae. Infected roots undergo a developmental switch that results in the formation of aberrant roots (clubs). To investigate host gene expression during the development of the disease, we have used the Arabidopsis ATH1 genome array. Two timepoints were chosen, an early timepoint at which the pathogen has colonized the root but has induced only very limited change of host cell and root morphology and a later timepoint at which more than 60% of the host root cells were colonized and root morphology was drastically altered. At both timepoints, more than 1,000 genes were differentially expressed in infected versus control roots. These included genes associated with growth and cell cycle, sugar phosphate metabolism, and defense. The involvement of plant hormones in club development was further supported; genes involved in auxin homeostasis, such as nitrilases and members of the GH3 family, were upregulated, whereas genes involved in cytokinin homeostasis (cytokinin synthases and cytokinin oxidases/dehydrogenases) were already strongly downregulated at the early timepoint. Cytokinin oxidase/dehydrogenase overexpressing lines were disease resistant, clearly indicating the importance of cytokinin as a key factor in clubroot disease development. PMID- 16673936 TI - A rice calcium- and calmodulin-dependent protein kinase restores nodulation to a legume mutant. AB - The Medicago truncatula DMI3 gene encodes a calcium- and calmodulin-dependent protein kinase (CCaMK) that is necessary for the establishment of both rhizobial and mycorrhizal symbioses. The two symbiotic signaling pathways diverge downstream of DMI3; therefore, it has been proposed that legumes have evolved a particular form of CCaMK, acting like a switch able both to discriminate between rhizobial and mycorrhizal calcium signatures and to trigger the appropriate downstream signaling pathway. To test this hypothesis, we examined whether a CCaMK gene from a nonlegume species was able to restore the rhizobial symbiotic properties of a M. truncatula dmi3 mutant. Our results show that a CCaMK gene from rice can restore nodule formation, indicating that CCaMKs from nonlegumes can interpret the calcium signature elicited by rhizobial Nod factors and activate the appropriate downstream target. The nodules did not contain bacteria, which suggests that DMI3 is also involved in the control of the infection process. PMID- 16673937 TI - Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp. AB - The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1 carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important. PMID- 16673938 TI - Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana, but the activity differed significantly among their molecular species. AB - Phytopathogenic fungi such as Fusarium spp. synthesize trichothecene family phytotoxins. Although the type B trichothecene, deoxynivalenol (DON), is thought to be a virulence factor allowing infection of plants by their trichothecene producing Fusarium spp., little is known about effects of trichothecenes on the defense response in host plants. Therefore, in this article, we investigated these effects of various trichothecenes in Fusarium-susceptible Arabidopsis thaliana. Necrotic lesions were observed in Arabidopsis leaves infiltrated by 1 microM type A trichothecenes such as T-2 toxin. Trichothecene-induced lesions exhibited dead cells, callose deposition, generation of hydrogen peroxide, and accumulation of salicylic acids. Moreover, infiltration by trichothecenes caused rapid and prolonged activation of two mitogen-activated protein kinases and induced expression of both PR-1 and PDF1.2 genes. Thus, type A trichothecenes trigger the cell death by activation of an elicitor-like signaling pathway in Arabidopsis. Although DON did not have such an activity even at 10 microM, translational inhibition by DON was observed at concentrations above 5 microM. These results suggested that DON is capable of inhibiting translation in Arabidopsis cells without induction of the elicitor-like signaling pathway. PMID- 16673939 TI - A symbiont-independent endo-1,4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita. AB - Substituted xylan polymers constitute a major part of the hemicellulose fraction of plant cell walls, especially in monocotyledons. Endo-1,4-beta-xylanases (EC 3.2.1.8) are capable of hydrolyzing substituted xylan polymers into fragments of random size. Many herbivorous animals have evolved intimate relationships with endosymbionts to exploit their enzyme complexes for the degradation of xylan. Here, we report the first finding of a functional endo-1,4-beta-xylanase gene from an animal. The gene (Mi-xyl1) was found in the obligate plant-parasitic root knot nematode Meloidogyne incognita, and encodes a protein that is classified as a member of glycosyl hydrolase family 5. The expression of Mi-xyl1 is localized in the subventral esophageal gland cells of the nematode. Previous studies have shown that M. incognita has the ability to degrade cellulose and pectic polysaccharides in plant cell walls independent of endosymbionts. Including our current data on Mi-xyl1, we show that the endogenous enzyme complex in root-knot nematode secretions targets essentially all major cell wall carbohydrates to facilitate a stealthy intercellular migration in the host plant. PMID- 16673940 TI - Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection. AB - Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. In Arabidopsis, 20 MAPKs have been identified and divided into four major groups. In rice, a monocot model and economically important cereal crop, only five MAPKs were characterized, including three related to the host defense response. In this study, we have identified 17 members of the rice MAPK gene (OsMPK) family through an in silico search of rice genome databases. Based on the phylogenetic analysis and pairwise comparison of Arabidopsis and rice MAPKs, we propose that MAPKs can be divided into six groups. Interestingly, the rice genome contains many more MAPKs with the TDY phosphorylation site (11 members) than with the TEY motif (six members). In contrast, the Arabidopsis genome contains more MAPKs with the TEY motif (12 members) than with the TDY motif (eight members). Upon inoculation with the blast fungus (Magnaporthe grisea), nine of 17 OsMPK genes were found to be induced at the mRNA level during either early, late, or both stages of infection. Four of the M. grisea-induced OsMPK genes were associated with host-cell death in the lesion-mimic rice mutant, and eight of them were differentially induced in response to defense signal molecules such as jasmonic acid, salicylic acid, abscisic acid, and ethylene. The genome-wide expression analysis suggests that about half of the rice MAPK genes are associated with pathogen infection and host defense response. PMID- 16673941 TI - Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana. AB - Twelve Arabidopsis accessions were challenged with Plum pox potyvirus (PPV) isolates representative of the four PPV strains. Each accession supported local and systemic infection by at least some of the PPV isolates, but high variability was observed in the behavior of the five PPV isolates or the 12 Arabidopsis accessions. Resistance to local infection or long-distance movement occurred in about 40% of all the accession-isolate combinations analyzed. Except for Nd-1, all accessions showed resistance to local infection by PPV-SoC; in the Landsberg erecta (Ler) accession, this resistance was compromised by sgt1 and rar1 mutations, suggesting that it could be controlled by an R gene-mediated resistance pathway. While most of the susceptible accessions were symptomless, PPV induced severe symptoms on inflorescences in C24, Ler, and Bay-0 as early as 15 days after inoculation. Genetic analyses indicated that these interaction phenotypes are controlled by different genetic systems. The restriction of long distance movement of PPV-El Amar and of another member of genus Potyvirus, Lettuce mosaic virus, in Col-0 requires the RTM genes, indicating for the first time that the RTM system may provide a broad range, potyvirus-specific protection against systemic infection. The restriction to PPV-PS long-distance movement in Cvi-1 is controlled by a single recessive gene, designated rpv1, which was mapped to chromosome 1. The nuclear inclusion polymerase b-capsid protein region of the viral genome appears to be responsible for the ability of PPV-R to overcome rpv1 mediated resistance. PMID- 16673942 TI - A putative role for fusaric acid in biocontrol of the parasitic angiosperm Orobanche ramosa. AB - Fusarium spp. are ubiquitous fungi found in soil worldwide as both pathogenic and nonpathogenic strains. The signals leading to disease or the absence of disease are poorly understood. We recently showed that fusaric acid (FA), a nonspecific toxin produced by most Fusarium spp., could elicit various plant defense responses at 100 nM without toxic effect. In this study, we checked for the effect of FA on root and root hairs, probable first site of contact between the fungi and the host. Large FA concentrations reduce root and root-hair growth and induce a rapid transient membrane hyperpolarization, followed by a large depolarization, due to the inhibition of H(+)-ATPase currents. Nanomolar concentrations of FA induced only an early transient membrane hyperpolarization of root hairs compatible with the induction of a signal transduction pathway. FA at 10(-7) M failed to induce salicylic acid- and jasmonic acid/ethylene-dependent defense-related genes but inhibited the germination of the angiosperm parasite Orobanche ramosa in contact of FA-pretreated Arabidopsis thaliana seedlings. These data suggest that FA at nontoxic concentrations could activate signal transduction components necessary for plant-defense responses that could contribute to biocontrol activity of Fusarium spp. PMID- 16673943 TI - Different mutations in the genome-linked protein VPg of potato virus Y confer virulence on the pvr2(3) resistance in pepper. AB - Five different amino acid substitutions in the VPg of Potato virus Y were shown to be independently responsible for virulence toward pvr2(3) resistance gene of pepper. A consequence of these multiple mutations toward virulence involving single nucleotide substitutions is a particularly high frequency of resistance breaking (37% of inoculated plants from the first inoculation) and suggests a potentially low durability of pvr2(3) resistance. These five mutants were observed with significantly different frequencies, one of them being overrepresented. Genetic drift alone could not explain the observed distribution of virulent mutants. More plausible scenarios were obtained by taking into account either the relative substitution rates, the relative fitness of the mutants in pvr2(3) pepper plants, or both. PMID- 16673944 TI - Sources of motivation, interpersonal conflict management styles, and leadership effectiveness: a structural model. AB - 126 leaders and 624 employees were sampled to test the relationship between sources of motivation and conflict management styles of leaders and how these variables influence effectiveness of leadership. Five sources of motivation measured by the Motivation Sources Inventory were tested-intrinsic process, instrumental, self-concept external, self-concept internal, and goal internalization. These sources of work motivation were associated with Rahim's modes of interpersonal conflict management-dominating, avoiding, obliging, complying, and integrating-and to perceived leadership effectiveness. A structural equation model tested leaders' conflict management styles and leadership effectiveness based upon different sources of work motivation. The model explained variance for obliging (65%), dominating (79%), avoiding (76%), and compromising (68%), but explained little variance for integrating (7%). The model explained only 28% of the variance in leader effectiveness. PMID- 16673945 TI - Neurotransmitter-related personality traits and money attitudes: a study in neuroeconomics. AB - In a sample of 82 undergraduates, the personality dimension of novelty seeking, which has been hypothesized to be influenced by levels of dopamine in the central nervous system, was associated with attitudes regarding money. PMID- 16673946 TI - Temperament, anxiety, and attentional control. AB - To regulate attention has been a significant component of individual differences in temperament, and other research supported a relation between measures of attentional control and anxiety, i.e., high scores on trait anxiety were correlated with inability to regulate attention in response to the environment, so enhanced attentional control may interact with dimensions of temperament to modify the effects of generalized anxiety. To test this hypothesis, 69 undergraduates completed the Revised Dimensions of Temperament Survey, the Burns Anxiety Scale, and the Derryberry and Reed measure of Attentional Control. Analysis suggested attentional control may be a significant factor in modifying reported anxiety. Further research is warranted, given the present small sample tested with numerous measures. PMID- 16673947 TI - Psychological processes and family variables as prosocial behavior predictors in a sample of Spanish adolescents. AB - A review of personal and family variables that regulate prosocial behavior is based on a sample of 1,433 Spanish adolescents (ages 9 to 13; 743 boys and 690 girls) who were assessed on the Prosocial Reasoning Objective Measure, the Physical and Verbal Aggression Scale, the Emotional Instability Scale, the State Trait Anger Inventory, the Child's Report of Parental Behavior Inventory, the Prosocial Behavior Scale, and the Index of Empathy for Children and Adolescents. Discriminant analyses showed that empathy, self-control in anger-provoking situations, and affection in family relationships are the best predictors of prosocial behavior. PMID- 16673948 TI - Prefrontal cortex dysfunction and income: a study in neuroeconomics. AB - In four samples of adults, individual income was negatively associated with a measure of prefrontal cortex dysfunction even after implementing controls for age, sex, and education. The small association was stronger for men than for women. These results provide support for a neuroeconomic approach to the study of micro-economic variables. PMID- 16673949 TI - Interrater reliability of scores derived from two methods for scoring the clock drawing test. AB - Scores on the Clock Drawing Test have long been considered a useful screening tool for neuropsychological dysfunction, and a number of scoring methods have been developed to evaluate various aspects of performance. This study compared quantitative and qualitative scoring by briefly trained students on 145 clock drawings produced by patients in a geriatric psychiatry outpatient clinic to estimate the interrater reliability of the methods, user's acceptance of the methods, and whether the methods provide differential diagnosis. Both systems showed acceptable interrater reliability. Using the quantitative method, raters scored drawings by patients with organic mental disease as more impaired than those patients diagnosed as depressed or schizophrenic. Results suggest that the Clock Drawing Test is a reliable screening tool for cognitive impairment in a geropsychiatric population, but the scoring methods examined do not yet appear psychometrically sound enough to provide a differential diagnosis. PMID- 16673950 TI - Religious identity among Christian Korean-American adolescents. AB - This exploratory study examined religious identity among Christian Korean American adolescents. Marcia's 1966 model of identity formation, which conceptualizes identity along dimensions of exploration and commitment, provided the framework for the current study. 49 participants, between the ages of 14 and 19 years, completed the Duke Religion Index, the religion items from the Extended Objective Measure of Ego Identity Status, the Pan-denominational Measure of Personal Devotion and Personal Conservatism, and six questions assessing perceived parental involvement. Analysis indicated that high scores on Organizational and Intrinsic Religiosity were positively correlated with the commitment statuses of Achievement and Foreclosure and negatively correlated with the noncommitment statuses of Moratorium and Diffusion. Also high scores on perceived parental involvement were positively correlated with the nonexploration status of Foreclosure and negatively correlated with the exploration status of Moratorium. Overall, the study provided a general portrait and initial examination of the formation of religious identity in a sample of Christian Korean-American adolescents. PMID- 16673951 TI - Comparison of the books of the New Testament (English Translation) in terms of emotion and word use. AB - The 27 books of the New Testament (English Translation) were scored using the Dictionary of Affect in Language. Books were compared with one another in terms of Activation, Pleasantness, and Imagery scores, and in terms of word length, use of the word "love," and mentions of Jesus. Significant differences among books were evident for all variables. A table of means and standard errors is provided. Measures of the books were related to one another, e.g., Pleasantness score and the use of the word "love" (p=.80) and to descriptors of the books, e.g., longer books tended to score lower on Pleasantness (p = -.79). PMID- 16673952 TI - A revised Emotional Intelligence Scale: factor re-evaluation and item reduction. AB - The original Emotional Intelligence Scale of Fukunishi utilized 65 items measuring 3 basic dimensions: intrapersonal, interpersonal, and situational. 170 psychiatric outpatients (84 men and 86 women) completed the inventory. The mean age of the sample was 41.1 yr. (SD= 12.4). Compared with the 65-item scale, factor re-analysis yielded a new 34-item solution with significant correlations for the Intrapsychic (.97) and Interpersonal dimensions (.96) of the larger scale, but not with the Situational dimension (.43). The correlation between the total inventory scores was also significant (.94). PMID- 16673953 TI - Coefficient of variation calculated from the range for skewed distributions. AB - In this research a coefficient of variation (CVS(high.low)) is developed that is calculated from the highest and lowest values in a set of data for samples from skewed distributions. A correction factor is determined such that CVS(high-low) is a dose estimate of the population coefficient of variation when sampling from three skewed chi-squared distributions and three skewed empirical distributions. The empirical distributions are from "real-world" data sets in psychology and education. PMID- 16673954 TI - Adolescents' communication with parents on sexual topics: a study of young people in Taiwan. AB - Communication between parents and adolescents about adolescents' sexual behavior has been studied. However, the types of issues on which adolescents are willing to communicate with their parents have been scarcely discussed. A survey was conducted among a random sample of 1,204 junior high school students in Taipei. The survey probed into adolescents' communication with their parents as related to sex, including physical development, sexuality, choice of partner, frequency of dating, whom to date, and intimacy during dating. Analysis showed that physical development was the topic most often discussed. Talking about dating was discussed less than the other three topics. Moreover, girls were more likely to communicate with their parents than boys about all six issues. PMID- 16673955 TI - Exploring ease in thinking aloud. AB - Thinking aloud seems unnatural and difficult to most subjects. The present study explored the relation between Ease in Thinking Aloud, verbal fluency, Self-talk, and Big Five personality traits for a heterogeneous sample of 101 German-speaking adults (50 women and 51 men). Ease in Thinking Aloud positively correlated with scores on verbal fluency (r =.31) and Conscientiousness (r =.38). The discussion considers implications for further research on ease in thinking aloud. PMID- 16673956 TI - Relationship between self-reported intensity of headache and magnitude of surface EMG. AB - The study was designed to examine the relationship between self-reported intensity of headache and surface EMG. 98 patients, diagnosed by their neurologists with "muscle-contraction headaches" (tension-type headaches) were referred to evaluate their suitability for biofeedback therapy. At the time of examination, they were asked to rate their average headache intensity on a 10 point scale. Surface EMG data were collected to assess actual muscle contraction. Analysis indicated that among patients diagnosed with muscle contraction headache, there is a positive significant correlation between self-reported intensity of headache and actual muscle-contraction. The current data lend support to the hypothesis that the tension in the headaches currently described as "tension-type" may in fact refer to actual muscular tension or contraction. PMID- 16673957 TI - Sex differences in correlations between personal incentives and self-motivation, and occurrence of perceived positive changes in feeling states after exercise. AB - Personal incentives for exercising of weight management and appearance improvement were significantly correlated with percentage of positive exercise induced feeling changes in women (n=23) and men (n=20) initiating 12-wk. exercise programs. No significant differences were found by sex. Self-motivation was also significantly related to occurrence of positive feeling changes for men. PMID- 16673958 TI - Evoking shame and guilt: a comparison of two theories. AB - Self-discrepancy theory associates shame with deviations from ideals that significant others hold for us and guilt with deviations from one's own moral guides. By contrast, Tangney posits that, although transgressions may engender both of these emotions, the nature of focus determines the specific reaction. Focusing upon the action committed engenders guilt, whereas focusing on the self who committed the action engenders shame. Participants recounted one of four types of discrepant actions and responded to measures of shame, guilt, anxiety, and depression. Consistent with Tangney's theorizing, across all types of discrepant actions, guilt was rated significantly higher than shame. PMID- 16673959 TI - Self-rated emotional health in adults with and without HIV. AB - Affect is an important indicator of quality of life; unfortunately, many people with Human Immunodeficiency Virus (HIV) disease are vulnerable to depression and negative affect. In a secondary data analysis, 50 HIV-positive and 50 HIV negative adults between 30 and 65 years old completed several affective measures and rated their emotional health. The HIV-positive adults reported more negative affect than the HIV-negative adults. Older age, loneliness, and HIV stigma were associated with such negative affect. PMID- 16673960 TI - Do relations between judgment and epistemological beliefs differentiate between mere acceptance and critical evaluation? Comment on Latifian & Bashash (2004). AB - In 2004 Latifian and Bashash presented Iranian students with questionnaires assessing (a) their epistemological beliefs regarding religion and science as sources of value and truth and (b) their acceptance of a scenario arguing for relative ethics. Acceptance was related to epistemological beliefs in a condition where the scenario was attributed to Freud but not in a condition where it was attributed to Tabatabaee, a highly authoritative theologian. This commentary questions the authors' explanation, particularly their assumption that the participants' reliance on epistemological beliefs would indicate a more critical attitude towards the scenario in the Freud condition than in the Tabatabaee condition. PMID- 16673961 TI - Selected organizational outcome correlates of spirituality in the workplace. AB - This study addressed the feasibility, practicality, and effects of a management style defined as "Management-by-Virtues," a management philosophy and practice based on virtues derived from religious beliefs. The study focused on Management by-Virtues as practiced in Christian firms and assessed the effect that Management-by-Virtues could have on such organizational outcomes as employees' Organizational Commitment, Job Satisfaction, and propensity to go beyond the call of duty in carrying out organizational tasks, i.e., Organizational Citizenship Behavior. The study, based on survey responses from 328 employees of 26 business firms, compared overall scores on Organizational Commitment, Job Satisfaction, and Organizational Citizenship Behavior in 10 businesses, described by owner/managers as being managed according to Christian beliefs and practices, with the same outcomes in 16 "control" firms that did not explicitly espouse such an orientation. The Management-by-Virtues firms were characterized by more committed, more satisfied employees, and employees' reports of higher incidence of Organizational Citizenship Behavior than the comparison firms. However, a measure of this construct based on employees' perceptions did not sharply differentiate the two groups of firms. PMID- 16673962 TI - Internet chat room use, satisfaction with life, and loneliness. AB - In this study, 50 chat room users responded online to the Satisfaction With Life Scale, the Revised UCLA Loneliness Scale, and demographic questions which included time spent per week in Internet chat rooms. Time spent in chat rooms was positively correlated with loneliness but only weakly and negatively related to satisfaction with life. PMID- 16673963 TI - Reliability and validity of the Spanish Version of the Crisis in Family Systems Revised. AB - Increasing the representation of Spanish-speaking study participants requires development and dissemination of reliable and valid translated scales. In the urrent study the construct validity was assessed of the Spanish version of the Crisis n Family Systems-Revised, a measure of contemporary life stressors, with a convenience sample of 377 parents interviewed in a study of childhood asthma, although over half of the respondents did not have children with asthma. Most respondents were foreign-born women between 20 to 60 years old (M=35, SD=7). 52% had not completed high school or its equivalent, and 55% reported a household income of dollar 15,000 or less. For a subsample of 25 respondents test-retest reliability was .86 over 2 wk. Reporting more life stressors was associated with greater depressive symptomatology, poorer physical and mental health function, and lower household income. These relationships support the construct validity of the test in Spanish. This study provided strong evidence that this version is a valid and reliable measure of life stressors for a Spanish-speaking population living in the United States. PMID- 16673964 TI - Fulfillment of promise: 40-year follow-up of creative adolescent girls. AB - A 40-yr. follow-up study of 7 women from among 10 identified as exceptionally creative during adolescence indicated that all had achieved moderate success in one or more artistic domains. A self-report measure of creative achievement was used, and influences which facilitated and inhibited creativity were described by the women. A continuing creative self-concept and social support were the most commonly cited facilitative factors, while financial concerns were listed as one of the primary inhibitors of further achievement. PMID- 16673965 TI - Attitude toward Christianity and premarital sex. AB - A Pearson correlation of .55 was found for a sample of 243 female undergraduates in Wales (M age=20.9 yr., SD=4.6) between high scores on the Francis Scale of Attitude toward Christianity and rejection of premarital sex. PMID- 16673966 TI - Validity and reliability of Spanish versions of the Ruminative Responses Scale Short Form and the Distraction Responses Scale in a sample of Spanish high school and college students. AB - This study investigated the construct validity and reliability of the Spanish Ruminative Responses Scale-Short From, and the Distraction Responses Scale of the Response Styles Questionnaire for a sample of 727 Spanish high school and college students who responded anonymously and voluntarily to a questionnaire (293 men, 434 women; ages 16 to 29 years, M=18.8, SD=3.0). In addition to the above scales, the questionnaire included the Spanish forms of the Beck Depression Inventory, the Trait Anxiety Scale from the State-Trait Anxiety Scale, the Satisfaction with Life Scale, and the Subjective Happiness Scale. The internal consistency of the scales was satisfactory (Cronbach alpha=.86 for the Ruminative Responses Scale and .78 for the Distraction Responses Scale). As expected, scores on the Spanish Ruminative Responses Scale showed positive correlations with those on the Beck Depression Inventory and the Trait Anxiety Scale and negative associations with the Satisfaction with Life Scale and the Subjective Happiness Scale. Conversely, the Spanish Distraction Responses Scale was negatively correlated with the Beck Depression Inventory and positively associated with the Satisfaction with Life Scale and the Subjective Happiness Scale. These results provide evidence of appropriate reliability for research purposes. Furthermore, the correlational analysis supported prior findings that ruminative response and distraction response styles are differentially associated with reported depressed and positive moods. PMID- 16673967 TI - Current issues in South African career counselling and availability of teachers in science and technology. AB - Employability seems to be a major consideration for prospective students' career choices. To facilitate accountable career counseling understanding of students' needs and choices of careers through research is essential. Programs for increased specific training are needed as well as improvement in basic training to meet a growing national labor crisis in South Africa. PMID- 16673968 TI - Test-retest reliability of subliminal facial affective priming. AB - Since the seminal 1993 demonstrations o f Murphy an d Zajonc, researchers have replicated and extended findings concerning subliminal affective priming. So far, however, no data on test-retest reliability of affective priming effects are available. A subliminal facial affective priming task was administered to 22 healthy individuals (15 women and 7 men) twice about 7 wk. apart. Happy and sad facial expressions were used as affective primes and neutral Chinese ideographs served as target masks, which had to be evaluated. Neutral facial primes and a no face condition served as baselines. All participants reported not having seen any of the prime faces at either testing session. Priming scores for affective faces compared to the baselines were computed. Acceptable test-retest correlations (rs) of up to .74 were found for the affective priming scores. Although measured almost 2 mo. apart, subliminal affective priming seems to be a temporally stable effect. PMID- 16673969 TI - Coping strategies and cognitive functioning in elderly people from a rural community in Italy. AB - Stressful situations may contribute to pathological states in elderly people facing harmful events. This study investigated the relationships between adopting appropriate coping strategies, transforming circumstances to personal preferences or adjusting personal preferences to situational constraints, and the efficiency of cognitive functioning in an elderly sample of 121 residents drawn from a rural community in southern Italy. They were administered the Mini-Mental State Examination, the Tenacious Goal Pursuit, Flexible Goal Adjustment, and Coping Inventory for Stressful Situations. Intercorrelations of scores suggested that facing stressful situations is somewhat associated with efficient cognitive functioning and coping strategies that ensure a wide range of solutions. PMID- 16673970 TI - Publication of nonsignificant results: a survey of psychologists' opinions. AB - A total of 236 Ph.D. and 1 M.A. faculty completed an e-mail survey regarding their opinions about publication of nonsignificant results. Tenured faculty were significantly less likely than nontenured faculty to write manuscripts for studies with nonsignificant results. Reasons for not writing a manuscript included a perceived inability to publish the manuscript, time concerns, flawed methods or design, inability to interpret results, and the belief that the results are unimportant. The presence of the file drawer problem is supported, and possible solutions are discussed. PMID- 16673971 TI - Acculturation scale for Korean-American college students. AB - Two-sample t test on the mean scores of the Suinn-Lew Asian Self-identity Acculturation Scale showed significant differences between 1.5-generation Korean American college students (n=71, M=2.4, SD=0.4) and second generation Korean American college students (n=85, M=3.3, SD=0.4). A two-sample t test on the mean scores by men and women was not significant (women: M=3.0, SD=.6; men: M=2.8, SD= 0.6). PMID- 16673972 TI - Note on social behavior of long-term captive female orangutans (Pongo pygmaeus abellii). AB - Some observations of social behavior of two aging female orangutans with a younger male son of one were made in an outdoor and in an indoor enclosure during the day. These included social distance, grooming, and locomotion plus wrestling and genital stimulation. As noted 15 years earlier, the two old females were never in contact with each other. Each showed contact and noncontact with the younger male. No clear social relation between the females was evident. Careful study of aging zoo populations could be useful. PMID- 16673973 TI - Patterns of adaptation to conflict and schizoid personality scale scores. AB - A previous investigation gave no evidence of a significant relationship of patterns of adaptation to conflict, as measured with the Serial Color-Word Test, with the Schizoid Personality Scale of the Coolidge Axis II Inventory. As a new scoring algorithm has subsequently been proposed for the latter scale, a replication was done with the modified schizoid scale. A group of 75 consecutive nonpsychotic women outpatients was given the Serial Color-Word Test and Coolidge's inventory. Both multiple and logistic regressions selected two significant predictors of schizoid personality, corresponding to high values of linear change in reading times during Trials 3 and 5 of the Serial Color-Word Test, i.e., to an increasingly rigid and inflexible style of the adaptive process. A multivariate analysis of variance yielded an effect size of .22 (partial eta2). PMID- 16673974 TI - Substituing supplementary subtests for core subtests on reliability of WISC-IV Indexes and Full Scale IQ. AB - The effects of replacing core subtests with supplementary subtests on composite score reliabilities were evaluated for the WISC-IV Indexes and Full Scale IQ. When Wechsler's guidelines are followed, i.e., only one substitution for each Index; no more than two substitutions from different Indexes when assessing the Full Scale IQ, summary score reliabilities remain high, and measurement error, as defined by confidence intervals around obtained scores, never increases by more than 1 index score point. In three instances, substitution of a supplementary subtest for a core subtest actually increased the reliabilities and decreased the amount of associated measurement error. PMID- 16673975 TI - Asian values scale: comparisons of Korean and Korean-American high school students. AB - This study was intended to examine mean differences on the Asian Values Scale between Korean and Korean-American high school students. For 199 Korean and 217 second generation Korean-American high school students, means on emotional self control and collectivism were significantly different. A two-sample t test on the mean scores of Korean high school students (M=4.5, SD=0.6) and second-generation Korean-American high school students (M=3.9, SD=0.5) indicated significant differences (p<.001). This present study contrasts directly with Kim, Atkinson, and Yang's past findings of no differences between first- and second-generation Asian college students. PMID- 16673976 TI - Relationship of personal authoritarianism with parenting styles. AB - This research investigated the relationship between the personality construct of right-wing authoritarianism and Baumrind's 1971 proposed parenting styles of authoritarian, authoritative, and permissive parenting. 68 youth ages 12-18 along with one of their parents participated. The children rated both parents on Buri's 1991 Parental Authority Questionnaire. One of the parents responded to Altemeyer's Right-Wing Authoritarian Scale. People with higher scores on Altemeyer's scale were more likely to prefer the authoritarian parenting style as their offspring reported (r = .33). Permissive parenting correlated negatively with the measure of authoritarianism as a personality variable (r = -.56). PMID- 16673977 TI - Shyness and informal help-seeking behavior. AB - The present study examined whether shy individuals are more reluctant to seek help than less shy individuals. A sample of 48 undergraduates (46% men, Mage=20.6 yr.) were selected from a pool of 72 students based on their relatively high or low scores on the Social Interaction Anxiety Scale. They were then exposed to a situation involving an impossible task, on which they had the option of seeking assistance. Analysis indicated that participants scoring high on Shyness took significantly more time to seek help than participants scoring low. This result suggests that even when help is readily available, shy individuals may refrain from seeking it. Presumably, shy people may be reluctant to seek help because that often requires them to initiate a social interaction. PMID- 16673978 TI - Item characteristics and answer-changing behaviors. AB - Difficulty and discrimination indices on 113 multiple-choice test items were compared with points gained or lost from students' changing answers. Difficulty of items was not significantly correlated with score gain from changing answers, a result consistent with previous research. Item discrimination had a low correlation with test score gain from changing answers, a result not consistent with previous research. PMID- 16673979 TI - Reflections on the meaning of a Semester at Sea voyage after twenty-two years. AB - 22 years after completion of a travel-study voyage around the world, a random sample of 29 participants was contacted by telephone and mail to reflect on the meaning of the experience. Respondents answered open-ended questions about their lives and outcomes of Semester at Sea, and they completed Crumbaugh's Purpose in Life Test based on Frankl's concepts. Participants maintained a global perspective and high test scores. Life events such as parenthood and major accomplishments were related to further discovery of meaning. Research earlier in the lives of participants suggested that Semester at Sea was a springboard for personal growth. Within a lifespan developmental model, as participants approach middle age, a more accurate metaphor for the meaning of the Semester at Sea experience may be an interwoven strand in a life composition. PMID- 16673980 TI - Sanctity of human life in war: ethics and post traumatic stress disorder. AB - This reflective paper focuses on the moral and ethical difficulties medical care providers meet in applying DSM-IV criteria of Post Traumatic Stress Disorder in therapy and in Compensation and Pension Examinations. Providers may be less interested in articulating a philosophy of life or their own ontology than about documenting care for the patient or providing effective management of symptoms of Post Traumatic Stress Disorder. The veteran comes for assessment or therapy after an embodiment of war conflicts. Examiners and therapists spend time in clinical judgments and ethical consideration. Ethical decisions involve the sanctity of human life and the distinction made between acceptable civil and war behavior. Insight into the conflicting values found in the veterans' struggle to cope with the stress of war is needed. The stress of sending young men into harm's way is an ethical issue for veterans and the society which sends them and thereby compromises the sanctity of human life. These ethical components require the doctor and the veteran to articulate their values within ontology or a personal philosophy of life. That exchange contributes to accurate assessment and adequate therapy. Not understanding the importance of personal ontologies in treatment may make the veteran's self-disclosure difficult. Self-disclosure of one's ontology is one side of coping with Post Traumatic Stress Disorder. The other side is for powerful decision-making groups to disclose to the young recruits the full and honest account of the human cost of war before sending them into harm's way. PMID- 16673981 TI - Androgenetic alopecia: stress of discovery. AB - The psychological problems of men in the initial stages of alopecia androgenetica (hereditary male hair loss) have seldom been studied. We evaluated two groups of 80 men with alopecia androgenetica in Stages II to IV, indicating the amount of hair loss (overall N=160; for Group I: M=48 yr., SD=18.2; for Group II: M=50 yr., SD=18.0) who visited a dermatology clinic for benign dermatological complaints but not for hair loss, by questionnaires and interview, retrospectively. As predicted, hair problems were reported to be significantly greater overall at the moment of discovery of hair loss than later. About half of the men reported feeling annoyed to very annoyed about the discovery of hair loss. For those patients, provision of information by internet might facilitate a visit to the dermatologist. PMID- 16673982 TI - Helping behavior in a rural and an urban setting: professional and casual attire. AB - This study assessed differences in helping behavior in a rural versus an urban location when directed toward either a professionally or a casually dressed woman. Convenience samples included 40 men and 40 women (10 people of each sex assigned to each condition: rural and professional, rural and casual, urban and professional, and urban and casual). A 21-yr.-old female confederate dropped an envelope near each target helper individually and recorded number of seconds for the target helper to retrieve or point out the dropped item. Analysis indicated significantly faster helping occurred in the rural than in the urban location and that men helped the confederate more often than women. No difference in frequencey of help was related to kind of attire. PMID- 16673983 TI - Criterion validity of Bermond-Vorst Alexithymia Questionnaire-20 Form B: a study of 63 alcoholic subjects. AB - The aim of the study was to set alexithymia cutoff scores on the 20-item Bermond Vorst Alexithymia Questionnaire-20 Form B. Alexithymia is a clinical construct describing individuals who have difficulty identifying and reporting their specific emotions. 63 subjects meeting the DSM-IV criteria for alcohol dependence completed the questionnaire and the 20-item Toronto Alexithymia Scale. Using cutoff scores for the latter, subjects were divided into alexithymic and nonalexithymic subgroups. Taking into account the sensitivity, specificity, and predictive positive value, several potential Bermond-Vorst questionnaire scores were tested. Using the Younden formula (Sensitivity+ Specificity-1), score of 52 was the most appropriate cutoff score for the Bermond-Vorst-20 to indicate the presence of alexithymia in these male alcoholics. PMID- 16673984 TI - Conditional standard errors of measurement for composite scores on the Wechsler Preschool and Primary Scale of Intelligence-Third Edition. AB - A specific recommendation of the 1999 Standards for Educational and Psychological Testing by the American Educational Research Association, the American Psychological Association, and the National Council on Measurement in Education is that test publishers report estimates of the conditional standard error of measurement (SEM). Procedures for calculating the conditional (score-level) SEM based on raw scores are well documented; however, few procedures have been developed for estimating the conditional SEM of subtest or composite scale scores resulting from a nonlinear transformation. Item response theory provided the psychometric foundation to derive the conditional standard errors of measurement and confidence intervals for composite scores on the Wechsler Preschool and Primary Scale of Intelligence-Third Edition. PMID- 16673985 TI - Relationships of Experiential and Reflective Self-Knowledge with Trait Meta-Mood Scale, constructive thinking inventory, and the Five Factors in Iranian managers. AB - The purpose of this study was to examine the validity and the incremental validity of recently developed Reflective and Experiential Self-knowledge Scales. Along with measures of the Five Factors and of psychological adjustment, 201 male Iranian managers responded to the Self-knowledge Scales along with tests relevant to emotional intelligence, including the Trait Meta-mood Scale and the Constructive Thinking Inventory. As hypothesized, Self-knowledge Scales predicted greater self-reported emotional intelligence. Multiple regression also confirmed the incremental validity of these scales, showed each explained a separate source of variance, and supported the presumed temporal dynamics that theoretically underlie these constructs. PMID- 16673986 TI - Further exploration of the Vedic Personality Inventory: validity, reliability and generalizability. AB - The purpose of this study was to explore the validity, reliability, and generalizability of the Vedic Personality Inventory which consists of constructs derived from the Vedic literature of India. There were 57 participants (20 men, 37 women). The mean age was 44.3 yr. (SD= 15.1). The sample was predominantly older, well-educated women who volunteered to participate as respondees to invitations to participate posted in supermarkets, churches, colleges, and on the internet. Analysis yielded statistically significant correlations for scores on the Vedic Personality Inventory and on the Daily Spiritual Experiences Scale and the Brief Symptom Inventory, each of which measures domains of interest addressed by the first inventory. PMID- 16673987 TI - A discriminant analysis of Whissell's New Testament data: on the statistical trail of the author of Hebrews. AB - Discriminant analyses of data on 6 dimensions from Whissell's analyses of 27 books of an English translation of the New Testament seem to support Clement of Alexandria's contention that Luke was involved in the authorship, but it is difficult after centuries to rule out definitively a few other contenders. PMID- 16673988 TI - Typicality Index of the Strong Interest Inventory: a binomial analysis. AB - The Typicality Index of the 2004 Strong Interest Inventory was developed by Donnay, Morris, Schaubhut, and Thompson to identify inconsistent or other atypical responding. Analysis by the binomial distribution shows that the index, with the recommended cut score, correctly classifies 95.1% of randomly completed inventories. PMID- 16673989 TI - Additional validation of the attitudes toward Seeking Professional Psychological Help Scale. AB - To provide information on the construct validity of the Attitudes Toward Seeking Professional Psychological Help scale, relationships between prior treatment experiences and scores on the scale were evaluated. Participants were 270 students recruited from undergraduate psychology courses at a medium-sized university in the midwestern USA. 79% were women: their ages ranged from 18 to 53 years (M= 19.0, SD=4.4). In addition to completing the scale, the subset of 76 participants who reported prior mental health care treatment also evaluated these experiences using a rating scale anchored by 0: very dissatisfied and 4: very satisfied. For the total sample, prior experience with the mental health care delivery system was associated with higher scores on the scale, denoting more positive attitudes. In the subgroup with prior treatment experience, evaluation ratings were also associated with treatment attitudes, accounting for 17% of the variance in their scale score. These results provide additional support for the construct validity of this scale. PMID- 16673990 TI - Sex differences in stipulated preferences and mate search by clients of a French marriage bureau. AB - Using data from the file of a French matchmaking agency, this study confirmed some previous results on traditional male-female differences in stipulations by placers of advertisements: women seek features influencing financial resources, while men offer these characteristics and seek younger women. An interesting and original result is that the sampled women are older than the men, which partially explains why they spend relatively more money in professional matchmaking services as they age. PMID- 16673991 TI - Reforms soak up vital cash. PMID- 16673992 TI - A private war in the NHS. PMID- 16673993 TI - Prescription for change. AB - With a shortage of doctors to mentor nurse prescribing students, a call to allow experienced nurses to take on the role is the subject of an RCN congress resolution. But not everyone is convinced. From May 1, nurses will be able to prescribe any licensed product from the British National Formulary, apart from controlled drugs. Physiotherapists, optometrists and radiographers are among other groups with prescribing powers. PMID- 16673994 TI - 'I know why I am here'. AB - As an African-American, Beverly Malone's path to RCN general secretary has not been easy. Brought up in the deep South, she has faced racism. PMID- 16673995 TI - The price of the game. AB - The health needs of people who engage in prostitution were debated at last year's RCN congress. Members considered the evidence of a Home Office report into the issues surrounding prostitution. PMID- 16673996 TI - Stunted growth. PMID- 16673997 TI - Learning by rote. PMID- 16673998 TI - Walking through the jungle. PMID- 16673999 TI - 'No tolerance' posters pander to the values of rude minority. PMID- 16674000 TI - The role of a telephone helpline in provision of patient information. AB - AIM: To explore why individuals contacted a telephone helpline specialising in neurological conditions and their level of satisfaction with the service. METHOD: Callers were asked to complete a confidential postal questionnaire. RESULTS: The majority of callers found the helpline useful and all respondents would use the service again. The main reasons for contacting the helpline were to obtain medical information or request an information booklet. Health professionals and the internet were the other main sources of information and support. CONCLUSION: A telephone helpline is important in the provision of information and support on neurological conditions. Through close collaboration, the NHS and voluntary organisations can fulfil many of the needs of patients with complex medical conditions. PMID- 16674002 TI - Infection risk of urinary catheters. PMID- 16674001 TI - Manual handling risk management: critical care beds and support systems. AB - This article examines the reduction of manual handling risk to nurses through discussion of the technical and ergonomic features of critical care beds and support systems. It describes the features of different types of manual handling equipment, in particular trolleys, beds and patient platform support surfaces. PMID- 16674003 TI - Understanding foot ulceration in patients with diabetes. AB - Foot ulceration is a complication that affects a significant proportion of the ever-increasing number of people with diabetes in the UK. Nurses caring for patients with diabetes are ideally placed to contribute to diabetic foot management through assessment, education and appropriate timely referral. This article examines how pathophysiological changes associated with diabetes and the demanding functional role of the foot predispose individuals to foot ulceration, how to assess and classify the diabetic foot and the principles of preventive and curative management. PMID- 16674004 TI - An overview of compression therapy in leg ulceration. AB - This article provides an overview of the current options for the application of compression therapy and a rationale for appropriate selection. PMID- 16674005 TI - Power failure. PMID- 16674006 TI - You can go your own way. PMID- 16674007 TI - Nursing healthy profits. PMID- 16674009 TI - Fertility and a woman's age. AB - Women show a progressive decline in fecundity as they pass through the reproductive years. The decline in fecundity can be attributed to numerous potential causes, including changes in oocyte quality, the frequency and efficiency of ovulation, sexual function, the health of the uterus and the risk of pregnancy complications. Screening women for their ovarian reserve uses a number of biochemical and imaging strategies that have a limited ability to predict the likelihood of pregnancy with or without fertility treatment. Consideration of the decline infertility as a woman ages is an important and integral part of health care for women. Physicians and other caregivers must continue to counsel women about their potential fertility as they age, whether or not they are actively seeking pregnancy. PMID- 16674008 TI - Male infertility. AB - With infertile couples, one third of cases can be due to a male factor. It is important to have a comprehensive yet efficient approach to identifying potential causes for appropriate counseling and treatment. We combined the experience of a urologist and a gynecologist, both fertility subspecialists, and reviewed current literature on the investigation of male infertility. A history and physical examination supplemented by relevant investigations will help unravel any significant diseases that can be associated with male subfertility or any conditions that may be transmitted to future offspring. Semen analysis is a common, convenient measure of assessing the male. It should precede any invasive tests of the female. While reference values are important in standardization, the current trend in using sperm morphology alone in predicting male fertility remains problematic. Overreliance on this reference can lead to misdiagnosis and unnecessary invasive treatment with intracytoplasmic sperm injection (ICSI). In those who have no identifiable or correctable causes, ICSI provides new hope for couples with male infertility who, in the past, could only choose among therapeutic donor insemination, adoption or voluntary childlessness. With increasing application of these assisted reproductive technologies, it is important to rule out genetic causes, such as cystic fibrosis, and to provide appropriate genetic counseling before embarking on these invasive and costly procedures. PMID- 16674010 TI - Endometriosis and infertility: a review. AB - The objective of this article is to review the current understanding of the relationship between endometriosis and infertility and the recommended management based on the latest evidence. The article is based on a MEDLINE search and bibliography review of the relevant literature from 1960 to 2003. The exact nature of the relationship between endometriosis and infertility remains uncertain. Surgical treatment of moderate and severe endometriosis improves the monthly fecundity rate. Surgical ablation of minimal and mild endometriosis seems superior to expectant management. Infertile patients with minimal and mild endometriosis can benefit from using clomiphene citrate and intrauterine insemination (IUI) or gonadotropins and IUI. Ovarian suppression should not be used in asymptomatic patients wishing to become pregnant except in the setting of in vitro fertilization and embryo transfer (IVF/ET). Prolonged suppression with gonadotropic-releasing hormone analogues should be considered for endometriosis patients about to undergo an IVF/ET cycle. IVF seems to be effective in endometriosis patients. PMID- 16674011 TI - Uterine factors and infertility. AB - A literature review was performed to explore the available information regarding the association of uterine factors--intrauterine adhesions, uterine septa, uterine myomas and endometrial polyps--with infertility and reproductive loss. The literature was reviewed also to ascertain evidence that treatment of these abnormalities improves fertility. A MEDLINE search was performed to identify the relevant publications in the English-language literature. There is minimal published evidence to demonstrate that intrauterine adhesions lead to infertility or pregnancy loss, but the literature does contain several observational series that demonstrate successful fertility, with term pregnancy rates ranging from 32% to 87% following hysteroscopic division of intrauterine adhesions. The evidence supporting a direct link between a septate uterus and reproductive loss/infertility is derived from the results of metroplasty. Several case series demonstrated a reduction in the spontaneous abortion rate, from 91% to 17%, on average, after hysteroscopic metroplasty. Furthermore, following metroplasty, the mean pregnancy rate in previously infertile patients is 47%. Little has been written regarding the association of endometrial polyps and infertility. One study did demonstrate a pregnancy rate of 78% after hysteroscopic polypectomy as compared to 42% in infertile patients with normal endometrial cavities. The literature that associates myomas with infertility/reproductive loss is more extensive but quite controversial. Evidence from the in vitro fertilization literature suggests that only those myomas that distort the endometrial cavity impair fertility. Pregnancy rates approximating 50% are achieved with myomectomy by laparotomy, laparoscopy or hysteroscopy. PMID- 16674012 TI - Microsurgery for tubal infertility. AB - In vitro fertilization/embryo transfer (IVF/ART) results have shown significant improvements during the last decade. In the United States the rate of live births per cycle improved gradually to become 27% in 2001. Assisted reproductive techniques (ARTs) are increasingly being used for the treatment of tubal factor infertility. In this review the data are derived largely from our department, where we have treated sufficient numbers of patients and have maintained substantial consistency in our surgical techniques. This 3-part review demonstrates a high success rate of intrauterine pregnancy (IUP) after anastomosis for sterilization reversal. This rate, for those who are < 35 years of age at the time of reversal, is >70%, with most pregnancies occurring within 18 months after surgery. Those who are 35 years of age or more will have a 55% rate of IUP. We note, too, the satisfactory IUP rate (50%) after tubocornual anastomosis for proximal tubal disease. We document the beneficial role of laparoscopic salpingoovariolysis, fimbrioplasty and salpingostomy performed during the initial diagnostic laparoscopy. The IUP rates after salpingoovariolysis and fimbrioplasty are 60% and 50%, respectively. The rates of IUP for salpingostomy are modest in comparison, yet they are 25% for liberal use of salpingostomy during the preliminary laparoscopy. Salpingostomy also provides a beneficial effect upon embryo implantation in both in vivo and in vitro attempts at conception. This stresses the need for an appropriate preliminary investigation and for the subsequent diagnostic laparoscopy to be performed at a center able to perform these procedures. The evidence suggests that surgery should retain its place in the treatment of tubal infertility. Surgery and ART are complementary approaches that can be used singly or in combination to improve the outcome for couples with tubal infertility. PMID- 16674013 TI - Prevalence of pelvic musculoskeletal disorders in a female chronic pelvic pain clinic. AB - OBJECTIVE: To determine the prevalence of 2 musculoskeletal pain disorders among women presenting to a referral chronic pelvic pain clinic. STUDY DESIGN: This was a retrospective, cross-sectional study of 987 women (aged 14-79) presenting for evaluation from 1993 to 2000 at a university-based gynecologic chronic pelvic pain clinic. RESULTS: At the initial visit, all women completed standardized interviews and underwent a pelvic examination. Single-digit palpation of the levator ani and piriformis muscles was performed intravaginally. Among these women, 212 of 955 (22%) had tenderness of the levator ani muscles, while 128 of 943 (14%) had tenderness of the piriformis muscle (pain score > 3 of 10 on a visual analogue scale). Both levator ani tenderness and piriformis tenderness were associated with a higher total number of pain sites, previous surgery for pelvic pain, Beck Depression Inventory score, McGill Pain Inventory score and pain worsened with bowel movements (p < 0.05). CONCLUSION: Piriformis and levator ani pain are present in a significant proportion of female chronic pelvic pain patients. Further research into the natural course, diagnosis and treatment of pelvic musculoskeletal pain is needed to determine its true contribution to chronic pain. PMID- 16674014 TI - Episiotomy rates in private vs. resident service deliveries: a comparison. AB - OBJECTIVE: To compare the use of episiotomy by private practitioners vs. resident staff and to determine if number of years in practice influences episiotomy use. STUDY DESIGN: A retrospective chart review of vaginal deliveries at 35 weeks or greater between January 2001 and June 2001. The number of years in practice by each private physician was documented. Independent sample t tests and chi2 tests were used to analyze data. RESULTS: In 995 deliveries, episiotomies were performed in 6% of low-risk resident deliveries vs. 26% of low-risk private deliveries (p<0.001). Physicians in practice > or = 15 years performed episiotomies in 32% of low-risk births. Physicians in practice <15 years performed episiotomies in 22% of low-risk births (p = 0.027). CONCLUSION: Deliveries performed by private practitioners are associated with a higher rate of episiotomy than those by resident staff. The number of episiotomies appears to increase by number of years in practice. PMID- 16674016 TI - Syndrome of inappropriate antidiuretic hormone in a healthy woman after diagnostic laparoscopy and hysteroscopy: a case report. AB - BACKGROUND: Hyponatremia is common after major surgery. It is now recognized as a potential complication after minor surgery. Premenopausal women are more likely to suffer permanent brain damage or death from hyponatremic encephalopathy than are men or postmenopausal women. CASE: A 36-year-old woman, gravida 2, para 0, undergoing diagnostic laparoscopy and hysteroscopy for secondary infertility and an abnormal hysterosalpingogram, experienced mental status changes and hyponatremia 10 hours after otherwise-uncomplicated surgery. Early recognition and treatment led to a complete recovery. CONCLUSION: Symptomatic hyponatremia is a potential complication after minor surgery and must be recognized and treated to avoid a catastrophic outcome. PMID- 16674015 TI - Effect of physiologic levels of glutamine on ICAM-1 expression in endothelial cells activated by preeclamptic plasma. AB - OBJECTIVE: To investigate whether the plasma glutamine (GLN) concentration would be depleted in pregnant women with preeclampsia and whether administering GLN comparable to physiologic levels would decrease cellular adhesion molecule expression in human umbilical vein endothelial cells (HUVECs) induced by plasma in preeclamptic women. STUDY DESIGN: We assessed plasma GLN levels from blood samples collected from 20 women with preeclampsia and 10 normal pregnant women. HUVECs were cultured in medium-199, containing fetal calf serum, antibiotics and growth factor, at different concentrations (0, 300, 500 microM) of GLN for 24 hours. We stimulated those cells for 1.5-6.0 hours with sera from patients with preeclampsia and then determined the expression of intercellular cell adhesion molecules (ICAM)-1 and vascular cell adhesion molecules (VCAM)-1 on endothelial cells by flow cytometry. RESULTS: Women with preeclampsia had significantly lower plasma GLN concentrations as compared with normal pregnant women. There were no differences in VCAM-1 expression in HUVECs among various GLN concentrations at each time point. However, ICAM-1 expression in HUVECs was significantly lower in the 500-microM GLN group than in the 0- and 300-microM groups at 3, 4.5 and 6 hours. CONCLUSION: This study showed that plasma from women with preeclampsia had significantly lower GLN levels than that from normal pregnant women and that administering GLN at physiologic levels reduces HUVEC ICAM-1 expression induced by preeclamptic plasma. PMID- 16674017 TI - Acute urinary retention caused by a large peritoneal inclusion cyst: a case report. AB - BACKGROUND: Pelvic masses have been known to cause bladder symptoms and compression. This is the first documented case of a large peritoneal inclusion cyst causing acute urinary retention from bladder outlet obstruction. CASE: A 36 year-old woman, gravida 2, para 2, presented to the gynecology clinic with an indwelling Foley catheter that was placed at an outside hospital secondary to acute urinary retention. Computed tomography, performed several days earlier for complaints of progressively worsening lower abdominal and pelvic pain, revealed a 10-cm, complex, cystic mass within the pelvis between the rectum and sigmoid colon, with anterior displacement of the bladder. The patient's past surgical history included a total abdominal hysterectomy as well as separate exploratory laparotomy for resection of a 20-cm peritoneal inclusion cyst and a prophylactic bilateral salpingo-oophorectomy. Due to the acute urinary retention and worsening pain, the decision was made to proceed with laparoscopic removal and drainage of the mass, which turned out to be consistent with a recurrent peritoneal inclusion cyst. Symptom relief was immediate. CONCLUSION: A large, recurrent, peritoneal inclusion cyst obstructed the bladder neck and presented as acute urinary retention. Laparoscopy relieved the symptoms. PMID- 16674018 TI - Rupture of the preterm uterus in the nonlaboring woman: a report of 3 cases. AB - BACKGROUND: Uterine rupture in a laboring patient is a well-known pregnancy complication. There is a paucity of information regarding uterine rupture at a preterm gestational age in a nonlaboring patient. CASE: Three women experienced spontaneous uterine rupture at a preterm gestational age prior to the onset of labor. All women presented with a primary complaint of abdominal pain that was nonfocal and had been noted over hours to days. In no case was a common risk factor for uterine rupture present. On initial evaluation, no woman appeared hemodynamically unstable, and all fetuses had a reassuring status. In all cases, rapid deterioration of maternal and/or fetal status resulted in emergency delivery. CONCLUSION: Uterine rupture at a preterm gestational age in a nonlaboring woman may present with nonspecific findings and be associated with rapid maternal and fetal decompensation. Awareness of this complication is necessary if maternal and fetal outcomes are to be optimized. PMID- 16674019 TI - Trisomy 20 mosaicism and nonmosaic trisomy 20: a report of 2 cases. AB - BACKGROUND: Together with chromosome 19, chromosome 20 belongs to group F, the group of small metacentric chromosomes. Trisomy 20 mosaicism is one of the most frequent chromosomal mosaicisms, representing approximately 16% of prenatally diagnosed cases. In nonmosaic trisomy 20, the usual findings are severe and manifold. Only 3 cases in the literature involved fetuses surviving past the first trimester. CASES: In case 1, a 42-year-old woman presented in her sixth pregnancy; she had had 4 vaginal deliveries of term infants and a miscarriage. Both her familial and personal genetic histories were unremarkable. Genetic amniocentesis was performed in the 18th gestational week for advanced maternal age. Sample analysis revealed a normal, male karyotype in 27 mitoses, while 4 were trisomy 20 (46,XY [27]/47,XY, +20 [4]). In the 37th gestational week a live, immature, male infant weighing 1,730 g was delivered. Chromosomal investigation of the newborn's blood sample did not reveal trisomy 20 but a normal male karyotype. In case 2, a healthy 37-year-old nullipara underwent amniocentesis at the 18th week of pregnancy for advanced maternal age. Amniotic fluid cell karyotype revealed trisomy 20 (47,XX, +20). Ultrasonography performed simultaneously with genetic amniocentesis showed slightly shortened fetal long bones, detectable narrowing of the cranium in the region of the frontal bone, lateral ventricles of 10 mm in width bilaterally, echogenic bowel and polyhydramnios. Abortion was induced in the 23rd week of pregnancy, and a 490-g female fetus was delivered. CONCLUSION: Based on these 2 well-documented, prenatally diagnosed cases, as far as genetic counseling is concerned, nonmosaic trisomy 20 is much less challenging than its mosaic form since the prognosis is uniformly poor in the former. PMID- 16674020 TI - Successful dizygotic twin pregnancy after recryopreservation by vitrification of human expanded blastocysts developed from frozen cleaved embryos on day 6: a case report. AB - BACKGROUND: Vitrification has evolved into an established technique for cryopreservation of human blastocysts. However, it is still unclear whether the blastocysts developed from frozen embryos can be cryopreserved a second time by vitrification for further embryo transfer. CASE: A 31-year-old woman underwent a long-treatment protocol for ovarian stimulation. Twenty-seven mature oocytes were obtained, and 21 were fertilized with intracytoplasmic sperm injection. On day 3, 2 cleaved embryos were transferred, but no implantation occurred. The remaining 19 embryos were cryopreserved with the slow freezing method. Three months after oocyte retrieval, 5 frozen day 3 embryos were thawed, the surviving 2 were transferred, but no implantation occurred. Six months after oocyte retrieval, the remaining 14 frozen day 3 embryos were thawed and the surviving 12 cultured. On day 5, 2 embryos reached the expanded blastocyst stage and were transferred, but no implantation occurred. On day 6, 5 of the nontransferred embryos became expanded blastocysts and were cryopreserved again by vitrification. Eight months after oocyte retrieval, 2 recryopreserved day 6 blastocysts were warmed and transferred. Implantation resulted in a dizygotic twin pregnancy. The pregnancy resulted in delivery of normal, healthy male and female infants weighing 2,155 and 2,590 g at birth, at 36 weeks of gestation. CONCLUSION: The blastocysts developed from frozen embryos on day 6 can be recryopreserved by vitrification and have pregnancy potential after warming. PMID- 16674021 TI - Selecting an appropriate hCG test for managing gestational trophoblastic disease and cancer. PMID- 16674022 TI - Vesicovaginal fistula after sterilization. PMID- 16674023 TI - Rotation abnormality in septate uterus with a double cervix. PMID- 16674024 TI - Working together to fulfill the AORN dream. PMID- 16674025 TI - The value of knowing the patient. PMID- 16674026 TI - Finding and refining article topic. PMID- 16674027 TI - Bloodborne pathogen exposure in the OR--what research has taught us and where we need to go. AB - Contracting a disease from bloodborne pathogens has been identified as an occupational hazard for perioperative personnel for more than two decades. Perioperative staff members are particularly vulnerable to percutaneous exposure. Despite known hazards, research has shown that perioperative staff members continue to take risks by not consistently complying with standard precautions and not reporting all percutaneous injuries. Health care workers (HCWs) and their employers need to work together to ensure that workplaces are safe. This article discusses mechanisms of bloodborne pathogen transmission, compliance with standard guidelines, and the social and economic costs of contracting a bloodborne illness. Steps to ensure that HCWs are protected also are outlined. PMID- 16674028 TI - Demystifying the Occupational Safety and Health Administration inspection process. AB - Being prepared for an Occupational Safety and Health Administration (OSHA) inspection can save a facility money, as well as potentially protect employees from serious illness or injury. This article explains the OSHA inspection process, types of violations that may be cited and the appeals process for employers and employees. Actual citations given in four recent OSHA health care facility inspections are discussed and general recommendations to prepare for an OSHA site visit are given. PMID- 16674029 TI - Perioperative patient safety: a multisite qualitative analysis. AB - This study was undertaken to identify specific events that affected patient safety in perioperative service areas during a three-year period. The three most common types o reported incidents were incorrect counts, equipment malfunction, and medication errors. Data revealed significant disparities between health care facilities regarding what types of incidents were reported. Standardized language and rporting mechanisms would aid in the consistency and validity of current incident reporting and could help to improve perioperative patient safety. PMID- 16674030 TI - Determining patient discharge criteria in an outpatient surgery setting. AB - Discharge of an outpatient surgical patient to home implies that the patient is clinically stable and capable, to some degree, of self-care. Nurses currently have no formal evidence-based clinical criteria to assist them in determining outpatient surgical patients' readiness for discharge to home. Nurses often make discharge decisions based on their education, experience, and personal beliefs. The goal of this quality improvement project was to identify specific factors that outpatient surgery nurses consider in assessing readiness of patients for discharge. The results were used to develop orientation and educational programs for outpatient surgery staff members and were incorporated into a patient discharge assessment checklist. PMID- 16674031 TI - Using a monitored test to assess risk of aspiration in postoperative patients. AB - Pulmonary aspiration is associated with high morbidity and mortality rates. Older adult patients have been shown to be particularly at risk for aspiration. A randomized controlled trial was conducted to investigate the efficacy of a simple, noninvasive screening test-the monitored sip test-in identifying patients at risk for aspiration, including "silent aspirators." Based on clinical judgment using the study's outcome measures, incidence of aspiration-related lung injury was high (ie, 37.5%); however, no identification of patients at risk for aspiration occurred after either the use of the monitored sip test or routine cautious introduction of fluids during the data collection phase of the study. PMID- 16674032 TI - AORN guidance statement: The role of the health care industry representative in the perioperative setting. PMID- 16674033 TI - AORN guidance statement: creating a patient safety culture. AB - AORN believes all health care organizations must strive to create a culture of safety. Such a culture will provide an atmosphere where all members of the perioperative team can openly discuss errors, process improvements, or system issues without fear of reprisal. A culture of safety places an emphasis on flexibility and learning as a means of improving safety and reducing errors. Characteristics of a culture of safety include the following: communication is open and honest; the emphasis is on the team rather than the individual; standards and practices are developed in a multidisciplinary framework; staff members are helpful and supportive of each other; staff members trust each other; surgical team members have a friendly, open relationship emphasizing credibility and attentiveness; the environment is resilient, encourages creativity, and is patient outcomes-driven; the focus is on work flow and process; and these attributes are supported by an informed culture that learns from incidents and near misses. A commitment to safety must be articulated at all levels of the organization. Safety must be valued as the top priority, even at the expense of efficiency. Health care organizations must allocate an appropriate amount of resources and provide the necessary incentives or rewards to promote a robust patient safety culture. AORN recognizes that most patient safety initiatives will fail in the absence of a viable safety culture. PMID- 16674034 TI - Revised National Patient Safety Goal on medication handling. PMID- 16674035 TI - A license without borders. PMID- 16674036 TI - Electrophilic intermediates and their reactions in superacids. AB - Acid-catalyzed reactions have played a major role in hydrocarbon chemistry involving electron-deficient intermediates such as carbocations, carbodications, onium ions, etc. The pioneering discovery of the use of superacids by George A. Olah, in the early 1960s, to characterize such intermediates under so-called long lived stable ion conditions led to the understanding of their structures and reactivity patterns much more clearly. Continuing studies in this area in the past 30 years have resulted in a paradigm shift in comprehending the stability and reactivity of electrophilic intermediates in superacid media. PMID- 16674037 TI - Combined experimental and computational study of the thermochemistry of methylpiperidines. AB - To understand the influence of the methyl group in the stability and conformational behavior of the piperidine ring, the standard (p0= 0.1 MPa) molar enthalpies of formation of 1-methylpiperidine, 3-methylpiperidine, 4 methylpiperidine, 2,6-dimethylpiperidine, and 3,5-dimethylpiperidine, both in the liquid and in the gaseous states, were determined at the temperature of 298.15 K. The numerical values of the enthalpies of formation in the liquid and in the gaseous state are, respectively, -(95.9 +/- 1.6) and -(59.1 +/- 1.7) kJ.mol(-1) for 1-methylpiperidine; -(123.6 +/- 1.4) and -(79.2 +/- 1.6) kJ.mol(-1) for 3 methylpiperidine; -(123.5 +/- 1.5) and -(82.9 +/- 1.7) kJ.mol(-1) for 4 methylpiperidine; -(153.6 +/- 2.1) and -(111.2 +/- 2.2) kJ.mol(-1) for 2,6 dimethylpiperidine; and -(155.0 +/- 1.7) and -(105.9 +/- 1.8) kJ.mol(-1) for 3,5 dimethylpiperidine. In addition, and to be compared with the experimental results, theoretical calculations were carried out considering different ab initio and density functional theory based methods. The standard molar enthalpies of formation of the four isomers of methylpiperidine and of the 12 isomers of dimethylpiperidine have been computed. The G3MP2B3-derived numbers are in excellent agreement with experimental data, except in the case of 2,6 dimethylpiperidine for which a deviation of 9 kJ.mol(-1) was found. Surprisingly, the DFT methods fail in the prediction of these properties with the exception of the most approximated SVWN functional. PMID- 16674038 TI - Hiscotropic rearrangements: hybrids of electrocyclic and sigmatropic reactions. AB - An unusual reaction manifold for cyclopropylcarbinyl cations, uncovered using B3LYP and MP2 calculations, is described. This reaction is a hybrid of a [1,2] sigmatropic hydrogen shift and a two-electron electrocyclic ring opening. These two processes occur through a single transition structure (i.e., they are concerted), although they are not synchronous. We call these reactions "hiscotropic" rearrangements. The potential energy surfaces for these reactions are often complicated, in some cases involving flat plateaus and bifurcations. Torquoselectivity occurs in some cases as a result of favorable orbital interactions between the breaking C-C and C-H bonds. PMID- 16674040 TI - Efficient synthesis of meso-substituted corroles in a H2O-MeOH mixture. AB - New and efficient conditions for the synthesis of meso-substituted corroles were developed. The first step, involving the reaction of aldehydes with pyrrole, was carried out in a water-methanol mixture in the presence of HCl. A relatively narrow distribution of aldehyde-pyrrole oligocondensates was obtained through careful control of their solubility in the reaction medium. After thorough optimization of various reaction parameters (cosolvent, reagent, and acid concentration), high yields of bilanes were obtained. Additionally, the bilane derived from 4-cyanobenzaldehyde was isolated, and the oxidative macrocyclization reaction was performed under various reaction conditions (different solvents, different concentrations, and various oxidants). As a result, triphenylcorrole was obtained in the highest yield (32%) reported to date. The scope and limitations studied showed that this method was particularly efficient for moderately reactive aldehydes and those bearing electron-donating groups (yields 14-27%). Using these conditions, corroles bearing strongly electron-donating groups were obtained for the first time. In addition, it was found that the reaction of unhindered dipyrromethanes with aldehydes under analogous conditions afforded trans-A2B-corroles in very high yields (45-56%; 8-fold higher than previously reported) without scrambling. The fact that scrambling was not observed in this reaction despite a very high HCl concentration (0.3 M) is unprecedented. Detailed studies on the oxidation of bilane, derived from sterically hindered dipyrromethane, allowed us to unequivocally establish that the yield of macrocyclization is insensitive to the concentration. It was found the 1H NMR spectra of corroles in deuterated TFA gave very sharp signals. PMID- 16674039 TI - Synthesis of fluorinated mucin core 2 branched oligosaccharides with the potential of novel substrates and enzyme inhibitors for glycosyltransferases and sulfotransferases. AB - Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C 3 or C-4 position of the pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases and 3-O-sulfotransferases involved in the biosynthesis of O-glycans. Our acceptor substrate specificity studies on three cloned sialyltransferases (Sia-Ts) revealed that a 3- or 4 fluoro substituent in beta1,4Gal resulted in poor acceptors for alpha2,6(N)Sia-T and alpha2,3(N)Sia-T, whereas 4-fluoro-Galbeta1,3GalNAcalpha was a good acceptor for alpha2,3(O)Sia-T. Uniquely, 4-F Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-OBn was an inhibitor of alpha2,6(N)Sia-T activity but not alpha2,3(N)Sia-T activity. Further we found that the activities of only Gal 3-O-sulfotransferases and not sialyltransferases were adversely affected by a C-3 fluoro substituent at the other Gal terminal of mucin core 2. The strategy of building branched mucin core 2 structures by three glycosidation sequence coupling three classes of glycosyl donors with the reactivity-matching acceptors proved to be successful in syntheses of modified mucin-type core structures of O-glycan. The relative poor yields of the glycosylations using fluorinated galactosyl donors indicated that the fluorine modification dramatically decreased the donor reactivity due to electron withdrawing effect. PMID- 16674041 TI - Hydrolysis mechanisms for indomethacin and acemethacin in perchloric acid. AB - The acid-catalyzed hydrolysis reactions of the antiinflammatory drugs indomethacin and acemethacin were investigated at 25.0 degrees C in a number of strongly concentrated perchloric acid media. The reaction rates were evaluated by UV measurements, and the intermediate species were detected by UV-vis, 1H NMR, 13C NMR, and mass spectroscopy measurements. A switchover from an A-2 to an A-1 mechanism as a function of the medium acidity is reported for the acid-catalyzed hydrolyses of the amide group of both indomethacin and acemethacin. In the A-2 hydrolysis, two water molecules are involved in the rate-determining step. An analysis of the kinetic data collected for acemethacin by the different techniques used reveals a complex mechanism, indomethacin being a metabolite intermediate species in the hydrolysis of acemethacin. The rate constants for the hydrolysis of the acemethacin ester group were considerably larger compared to those of the amide group. PMID- 16674042 TI - Tautomeric equilibria and pi electron delocalization for some monohydroxyarenes- quantum chemical studies. AB - Keto-enol tautomeric interconversions and variations of the pi-electron distribution were studied for 11 isolated monohydroxyarenes at the DFT(B3LYP)/6 311++G(2df,2p) level. For two monohydroxyarenes (phenol and 9-anthrol), the PCM model of solvation (water) was also applied to the DFT geometries. The geometry based HOMA index was applied to estimate pi-electron delocalization in the keto and enol tautomeric forms. Thermodynamic parameters of tautomeric interconversions (DeltaET, DeltaGT, TDeltaST, pKT) were calculated to estimate relative stabilities of individual tautomers and their percentage contents in the tautomeric mixtures. In almost all cases, the aromatic enol forms are strongly favored. An exception is 9-anthrol, which prefers its keto form. The resonance stabilization of this form comes from the central ring. Generally, aromaticity is the main factor that influences tautomeric equilibria in monohydroxyarenes. Hydration effect is considerably smaller and it does not change the tautomeric preference. PMID- 16674043 TI - Reaction of thianthrene and phenoxathiin cation radicals with 2,3-dimethyl-2 butene. Chemical and electrochemical studies. AB - Thianthrene cation radical tetrafluoroborate (Th*+ BF4-) has been found to add to 2,3-dimethyl-2-butene (DMB) at 0 degrees C and -15 degrees C. The adduct, 2,3 dimethyl-2,3-(5,10-thianthreniumdiyl)butane ditetrafluoroborate (12), was isolated at -15 degrees C, and its 1H NMR spectrum was recorded at that temperature. The adduct was stable in CD3CN solution at -15 degrees C but decomposed slowly at 0 degrees C and quickly at 23 degrees C, forming the salt of 2,4,4,5,5-pentamethyl-2-oxazoline (8) with loss of thianthrene (Th). These results explain why earlier attempts to prepare 12 and detect its formation at room temperature with NMR spectroscopy were not successful. Reaction of Th*+ with DMB was followed with cyclic voltammetry and was found to exhibit redox catalysis in which Th was regenerated. With the faster scanning techniques of cyclic voltammetry, the formation of 12 was detectable, with a reduction potential of about -1.0 V at 25 degrees C and 3 degrees C. The observed reduction potential was in harmony with reduction potentials of a number of other, stable monoadducts. Thus, the redox catalysis involved the rapid formation of 12 and its rapid decomposition into 8 and Th, the newly formed Th being responsible for the observed enhanced oxidation currents. In contrast, 8 appears to be formed directly by oxidation of DMB by PO*+PF6-. PMID- 16674044 TI - Stereoselective preparation of (E)- and (Z)-alpha-fluorostilbenes via palladium catalyzed cross-coupling reaction of high E/Z ratio and (Z)-1-bromo-1 fluoroalkenes. AB - A highly stereoselective method to prepare both (E)- and (Z)-alpha fluorostilbenes is described. 1-Bromo-1-fluoroalkenes (E/Z approximately 1:1), a readily available starting material, isomerizes to high E/Z ratios by storage at 20 degrees C or by photolysis at 254 nm. Stille coupling between these high E/Z 1 bromo-1-fluoroalkenes and aryl stannanes gave (Z)-alpha-fluorostilbenes in high stereoselectivity. (Z)-1-Bromo-1-fluoroalkenes, which were kinetically separated from 1-bromo-1-fluoroalkenes (E/Z approximately 1:1), can participate in Suzuki coupling reactions to give (E)-alpha-fluorostilbenes stereoselectively. PMID- 16674045 TI - Synthesis of indoles by intermolecular cyclization of unfunctionalized nitroarenes and alkynes, catalyzed by palladium-phenanthroline complexes. AB - Palladium-phenanthroline complexes efficiently catalyze the reaction of nitroarenes with arylalkynes and CO to give 3-arylindoles by an ortho-C-H functionalization of the nitroarene ring. Both electron-withdrawing and electron donating substituents are tolerated on the nitroarene, except for bromide and activated chloride. Nitroarenes bearing electron-withdrawing substituents react faster, but the selectivity of the reaction depends on both polar and radical stabilization effects. Among those tested, only arylalkynes afforded indoles under the investigated conditions. The reaction mechanism was partly investigated. The kinetics is first order in nitroarene concentration and the rate-determining step of the cycle is the initial nitroarene reduction. No primary isotope effect is observed on either rate or selectivity, implying that the cyclization step is fast. PMID- 16674046 TI - Halogenated 2'-chlorobithiazoles via Pd-catalyzed cross-coupling reactions. AB - Halogenated bithiazoles allow facile further functionalization and are, therefore, suitable intermediates for the synthesis of compounds with interesting biological activity or material science properties. The applicability of three coupling methods (Negishi, Suzuki, and Stille) for the synthesis of the title compounds was compared. The Negishi method proved to be troublesome, and side reactions were predominant. The synthesis of the first thiazoleboronic acid ester offered a new method for the formation of bithiazoles, not generally applicable so far. The lower toxicity compared to that of tin organyls make this method an approach with interesting perspectives. The Stille coupling proved to be superior to the other methods and enabled the synthesis of the title compounds with diverse connectivity. PMID- 16674047 TI - Indium-mediated diastereoselective allylation of D- and L-glyceraldimines with 4 bromo-1,1,1-trifluoro-2-butene: highly stereoselective synthesis of 4,4,4 trifluoroisoleucines and 4,4,4-trifluorovaline. AB - A practical and efficient route for the stereoselective synthesis of (2R,3S)- and (2S,3R)-4,4,4-trifluoroisoleucines and (2R,3S)-4,4,4-trifluorovaline was developed. Indium-mediated allylation of (R)-N-benzyl-2,3-O isopropylideneglyceraldimine 7 with 4-bromo-1,1,1-trifluoro-2-butene 4 gave the desired homoallylic amine 8 in high diastereoselectivity (>95% de) with moderate yield. The Cbz-protected (2R,3S)-4,4,4-trifluoroisoleucine 14 and Boc-protected (2R,3S)-4,4,4-trifluorovaline 21 were then readily prepared from 8. In addition, following the same procedure, Cbz-protected (2S,3R)-4,4,4-trifluoroisoleucine 28, the enantiomer of 14, was prepared starting from (S)-N-benzyl-2,3-O isopropylideneglyceraldimine 24. PMID- 16674048 TI - Syntheses of vinca alkaloids and related compounds. 104. A concise synthesis of ( )-vincapusine. AB - beta-Iodo-enamines with an eburnane skeleton (5a and 5c) were obtained with the aid of iodine from compounds 2a and 2c and were then transformed into hydroxyl lactams (6a and 6c) with CuSO4.5H2O in a mixture of DMF and water. Lactams (6a and 6c) were reduced selectively with BH3.SMe2 to result in the first synthesis of (-)-vincapusine (4a) as well as its natural 14-decarbomethoxy analogue (4c). PMID- 16674049 TI - Synthesis and characterization of a persistent paramagnetic rotaxane based on alpha-cyclodextrin. AB - The synthesis and spectroscopic properties of a novel paramagnetic [2]rotaxane is described. This rotaxane is made from molecules having an alkyl chain flanked by 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) groups. Complexation of sebacoyl chloride by alpha-cyclodextrin followed by reaction with a bulky aminonitroxide resulted in the trapping of the cyclodextrin, threaded by the alkyl chain, thus generating the rotaxane structure. The structure of the paramagnetic [2]rotaxane was fully characterized by ESI-MS, 1D and 2D NMR and ESR spectroscopy. PMID- 16674050 TI - 4'-substituted-4-biphenylyloxenium ions: reactivity and selectivity in aqueous solution. AB - Azide trapping shows that the 4'-substituted-4-biphenylyloxenium ions 1b-d are generated during hydrolysis of 4-aryl-4-acetoxy-2,5-cyclohexadienones, 2c and 2d, and O-(4-aryl)phenyl-N-methanesulfonylhydroxylamines, 3b and 3c. In addition, the 4'-bromo-substituted ester, 2d, undergoes a kinetically second-order reaction with N3- that accounts for a fraction of the azide adduct, 5d. Since both first order and second-order azide trapping occurs simultaneously in 2d, the second order reaction is not enforced by the short lifetime of 1d, which has similar azide/solvent selectivity to the unsubstituted ion, 1a. In contrast the 4'-CN and 4'-NO2 ions 1e and 1f cannot be detected by azide trapping during the hydrolysis of the dichloroacetic acid esters 2e' and 2f' even though 18O labeling experiments show that a fraction of the hydrolysis of both esters occurs through C(alkyl)-O bond cleavage. These esters exhibit only second-order trapping by azide. Correlations of the azide/solvent selectivities of 1a-d with the calculated relative driving force for hydration of the ions (DeltaE of eq 4) determined at the pBP/DN//HF/6-31G and BP/6-31G//HF/6-31G levels of theory suggest that 1e and 1f have lifetimes in the 1-100 ps range. Ions with these short lifetimes are not in diffusional equilibrium with nonsolvent nucleophiles, and must be trapped by such nucleophiles via a preassociation mechanism. The second-order trapping that is observed in these two cases is enforced by the short lifetime of the cations, and may occur by a concerted S(N)2' mechanism or by internal azide trapping of an ion sandwich produced by azide-assisted ionization. Comparison of azide/solvent selectivities of the oxenium ions 1a-c with the corresponding biphenylylnitrenium ions 8a-c shows that 4'-substituent effects on reactivity in both sets of ions are similar in magnitude, although the nitrenium ions are ca. 30-fold more stable in an aqueous environment than the corresponding oxenium ions. The magnitude of the 4'-substituent effects for electron-donating substituents suggest that both sets of ions are more accurately described as 4-aryl-1-imino-2,5-cyclohexadienyl or 4-aryl-1-oxo-2,5 cyclohexadienyl carbocations. Calculated structures of the oxenium ions are also consistent with this interpretation. PMID- 16674051 TI - Palladium-catalyzed Suzuki cross-coupling of 2-haloselenophenes: synthesis of 2 arylselenophenes, 2,5-diarylselenophenes, and 2-arylselenophenyl ketones. AB - We present herein our results on the Suzuki coupling reaction of 2 haloselenophenes with boronic acids catalyzed by palladium salt and describe a new route established to prepare 2-arylselenophenes and 2,5-diarylselenophenes in good yields. The reaction proceeded cleanly under mild conditions and was performed with aryl boronic acids bearing electron-withdrawing, electron donating, and neutral substituents, in the presence of Pd(OAc)2, K2CO3/H2O in DME. In addition, by this protocol unsymmetrical aryl ketones were also obtained from 2-iodoselenophene and boronic acids via a carbonylative process. PMID- 16674052 TI - Remote substituent effects in ruthenium-catalyzed [2+2] cycloadditions: an experimental and theoretical study. AB - The effects of a remote substituent on the regioselectivity of ruthenium catalyzed [2+2] cycloadditions of 2-substituted norbornenes with alkynes have been investigated experimentally and theoretically using density functional theory. Most of the cycloadditions occurred smoothly at room temperature, giving the exo cycloadducts in excellent yields. Regioselectivities of 1.2:1 to 15:1 were observed with various substituents on the C-2 position of the norbornenes. Exo-C-2-substituents usually showed greater remote substituent effects on the regioselectivities of the cycloadditions than the corresponding endo-C-2 substituents. The regioselectivity of the cycloadditions with C-2 substituents containing an exocyclic double bond (sp2 hybridized carbon at C-2) are much higher than the cycloadditions with the exo and endo 2-substituted norbornenes. Theoretical studies predicted the same trends as experiment and matched the experimental product ratios well. The nature of the regioselectivity in this reaction is discussed. Different strengths of the pi(C5-C6)-->pi(C2-Y) or pi(C5 C6)-->sigma(C2-Y) orbital interactions in 2-substituted norbornenes result in different degrees of C5-C6 double bond polarization. Stronger C5-C6 polarization will increase the difference in the activation energies between the major and minor pathways and thus lead to greater regioselectivities. PMID- 16674053 TI - Alkylation of phenylglycinol-derived oxazolopiperidone lactams. Enantioselective synthesis of beta-substituted piperidines. AB - The stereochemical outcome of the alkylation of a variety of phenylglycinol derived oxazolopiperidone lactams is studied. The influence of the configuration of the C-8a stereocenter and the effect of the substituents at the C-8 and C-8a positions on the stereoselectivity of the reaction are discussed. The synthetic utility of these alkylation reactions is illustrated with the synthesis of cis and trans 3,5-disubstituted, 2,5-disubstituted, and 2,3,5-trisubstituted enantiopure piperidines, and the indole alkaloids 20R- and 20S-dihydrocleavamine. PMID- 16674054 TI - (IPr)Pd(acac)Cl: an easily synthesized, efficient, and versatile precatalyst for C-N and C-C bond formation. AB - A very straightforward synthesis of (IPr)Pd(acac)Cl from two commercially available starting materials, Pd(acac)2 and IPr.HCl [acac = acetylacetonate; IPr = N,N'-bis(2,6-diisopropylphenyl)imidazol-2-ylidene], has been developed. The resulting complex, (IPr)Pd(acac)Cl (1), has proven to be a highly active PdII precatalyst in the Buchwald-Hartwig and the alpha-ketone arylation reactions. A wide range of substrates has been screened, including unactivated, sterically hindered, and heterocyclic aryl chlorides. PMID- 16674055 TI - Dihydroxyacetone variants in the organocatalytic construction of carbohydrates: mimicking tagatose and fuculose aldolases. AB - Dihydroxyacetone variants have been explored as donors in organocatalytic aldol reactions with various aldehyde and ketone acceptors. The protected form of dihydroxyacetone that was chosen for in-depth study was 2,2-dimethyl-1,3-dioxan-5 one, 1. Among the catalysts surveyed here, proline proved to be superior in terms of yield and stereoselectivities in the construction of various carbohydrate scaffolds. In a fashion analogous to aldolase enzymes, the de novo preparation of L-ribulose, L-lyxose, D-ribose, D-tagatose, 1-amino-1-deoxy-D-lyxitol, and other carbohydrates was accomplished via the use of 1 and proline. In reactions using 2,2-dimethyl-1,3-dioxan-5-one 1 as a donor, (S)-proline can be used as a functional mimic of tagatose aldolase, whereas (R)-proline can be regarded as an organocatalytic mimic of fuculose aldolase. PMID- 16674056 TI - Multiple isotope effect study of the acid-catalyzed hydrolysis of formamide. AB - Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 degrees C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one tetrahedral intermediate, which does not appreciably exchange its carbonyl oxygen with the solvent (kh/kex = 55). The pKa for formamide was determined by 15N NMR and found to be about -2.0. The formyl hydrogen kinetic isotope effect (KIE) is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl-carbon KIE (13kobs = 1.031) is in agreement with this conclusion. The small leaving-nitrogen KIE (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl-oxygen KIE (18kobs = 0.996) points to attack of water as the rate determining step. On the basis of these results, a mechanism is proposed in which attachment of the nucleophile to a protonated formamide molecule is rate determining. PMID- 16674057 TI - Diastereoselective and enantiospecific synthesis of gamma-substituted alpha,beta unsaturated nitriles from O-protected allylic cyanohydrins. AB - gamma-Functionalized alpha,beta-unsaturated nitriles are prepared diastereoselectively and enantiospecifically from enantioenriched cyanohydrin-O phosphates and carbonates derived from alpha,beta-unsaturated aldehydes, either by palladium or iridium-catalyzed nucleophilic allylic substitution reactions with different nucleophiles. Appropriate reaction conditions for dibenzylamine, benzylamine, sodium azide, NaOAc, tetra-n-butylammonium acetate (TBAA), the corresponding sodium salts of phenol and N-hydroxysuccinimide and the carbonucleophile sodium dimethyl malonate are described. Different substituted O protected cyanohydrins, such as carbonates and phosphates, derived from crotonaldehyde, (E)-hex-2-enal, oct-2-enal, 2-methylbut-2-enal, and cinnamaldehyde are used as allylic substrates. The substitution takes place with total retention of the configuration for the (E)-gamma-functionalized nitriles and with inversion of the configuration for the Z-isomers. In general, cyanohydrin-O-phosphates are the materials of choice to get the highest E diastereoselectivity. Dibenzylamine is the best nucleophile for the synthesis of gamma-nitrogenated alpha,beta-unsaturated nitriles in the presence of either palladium or iridium catalysts when aliphatic compounds and cinnamaldehyde derivative are used (up to 98% dr). For the synthesis of gamma-oxygenated alpha,beta-unsaturated nitriles sodium or TBAA the reagents are selected to avoid epimerizations in up to 76% dr. Finally, the Tsuji-Trost reaction with sodium malonate works only under palladium catalysis in up to 70% dr. PMID- 16674058 TI - Efficient and selective sulfoxidation by hydrogen peroxide, using a recyclable flavin--[BMIm]PF6 catalytic system. AB - A new flavin catalyst 2 immobilized in an ionic liquid ([BMIm]PF6) was used for the highly selective oxidation of sulfides to sulfoxides by hydrogen peroxide. The sulfoxides were obtained in good to high yields and high selectivity without any detectable overoxidation to sulfone. The catalyst in the ionic liquid was recycled up to seven times without loss of activity or selectivity. PMID- 16674059 TI - Palladium(II)-catalyzed asymmetric synthesis of (Z)-alpha-alkylidene-gamma butyrolactams from (Z)-N-allylic 2-Alkynamides. Total synthesis of (-) isocynometrine. AB - Pd(OAc)2 combined with nitrogen-containing ligands catalyzed the cyclization of (Z)-N-allylic 2-alkynamides in acetic acid to afford the alpha-(Z) acetoxyalkylidene-gamma-butyrolactams in high yield and high stereoselectivity. When chiral nitrogen-containing ligands were used, the catalytic asymmetric protocol was achieved with moderate enantioselectivity (up to 80 degrees C). The utility of this new methodology was exemplified by the total synthesis of (-) isocynometrine. PMID- 16674060 TI - A facile, general synthesis of 3,4-difluoro-6-substituted-2-pyrones. AB - Reaction of (2E)-2,3-difluoro-3-iodoacrylic acid with a variety of terminal acetylenes under cocatalysis of PdCl2(PPh3)2 and CuI gave difluorinated 2-pyrones as the sole product in good yields. PMID- 16674061 TI - Synthesis of N3,N'3-polymethylene-bis-hydantoins and their macrocyclic derivatives. AB - An efficient and straightforward two-step approach toward N3,N'3-polymethylene bis-hydantoins was developed. As a first step, a pyroglutamate is reacted with a diisocyanate to produce a bis-carbamoyllactam. The second step is a double-ring transformation by treatment of this bis-carbamoyllactam with KOtBu in ethanol. In this fashion N3,N'3-polymethylene-bis-hydantoins are produced in two quantitative steps and under very mild conditions. When properly derivatized, these compounds can be converted to their macrocyclic derivatives upon treatment with 5 mol % of second-generation Grubbs' catalyst. These macrocyclic derivatives are so far not described in the literature. It was proven that exclusively (E)-isomers are formed. PMID- 16674062 TI - Quantification of the push-pull effect in tolanes and a revaluation of the factors affecting the 13C chemical shifts of the carbon atoms of the CC triple bond. AB - Variously substituted tolanes were employed to show that the push-pull effect is also active in CC triple bonds by the successful correlation of the occupation quotient pi/pi of the pi orbital in resonance with the substituted phenyl moieties of tolanes versus the bond length of the CC triple bond. In addition, the influences of the ortho phenyl ring substituents on the 13C chemical shifts of the triple bond carbon atoms, which were estimated by Rubin et al.(4) to be "inapplicable for describing triple bond polarization", were re-evaluated, leading to the conclusion that, while anisotropic effects of ortho substituents are negligible, the steric ortho-substituent effects do in fact dominate the deviations obtained. A detailed theoretical NBO/NCS study has been employed to illuminate the facts of this case. PMID- 16674063 TI - Strain energy release and intrinsic barriers in internal nucleophilic reactions. AB - This paper reports computational data for the energetics of internal attacks, both in ring-opening reactions (eq 3) where strain energy is released and in model, strain-free systems (eq 4). A comparison is drawn with the corresponding bimolecular processes. The exothermicity of three-membered ring-opening reactions is significantly larger than that of the four-membered ring systems. However, using the Marcus equation, it is shown that the higher reactivity of the three membered rings is intrinsic to the system and does not stem only from a higher thermodynamic driving force. The intrinsic barriers for the strain-free reactions are shown to be dominated by the position of the nucleophilic and nucleofugic atoms in the periodic table, as in the bimolecular SN2 reactions, although a pi rather than a sigma bond is formed in these reactions. PMID- 16674064 TI - Remarkable effect of water on functionalization of the phenyl ring in methyl substituted benzene derivatives with F-TEDA-BF4. AB - Various N-F reagents reacted with hexamethylbenzene (1) forming side chain substituted alkoxides or esters in protic solvents, Ritter type side chain functionalization was observed in acetonitrile in the presence of trifluoroacetic acid, while in aqueous acetonitrile solution phenyl ring transformation took place, starting with ipso attack of water and further rearrangement of the methyl group as the main process. Rearranged 2,3,4,5,6,6-hexamethylcyclohexa-2,4-dienone (7) was transformed to 5-fluoro-2,3,5,6,6-pentamethyl-4-methylenecyclohex-2-en-1 one (8) or 5-hydroxy-2,3,5,6,6-pentamethyl-4-methylenecyclohex-2-en-1-one (9). 1,2,3,4,5,6-Hexamethyl-bicyclo[2.2.0]hexa-2,5-diene reacted with F-TEDA-BF4 in the presence of water and 7 was formed in high yield. Durene (12) followed similar ipso attack of water as 1, but on the other hand 1,2,3,4 tetramethylbenzene displayed different regioselectivity and 2,3,4,5 tetramethylphenol was formed, further transforming to 4-fluoro-2,3,4,5 tetramethylcyclohexa-2,5-dienone. The functionalizations of methylbenzenes obeyed a second-order rate equation v = d[N-F]/dt = k2[N-F][substrate], and DeltaG# values between 77 and 94 kJ/mol were determined. The presence of water did not significantly influence DeltaG# but considerably affected DeltaS# and positive values were found where methyl group migration was the dominant process (9.1 J/(mol K) for 1 and 0.5 J/(mol K) for 12). A higher reactivity of durene than mesitylene (k2(MES)/k2(DUR) = 0.23) was found, supporting the assumption that single electron transfer (SET) is the dominant process in the functionalizations of methyl-substituted benzene derivatives with F-TEDA-BF4. PMID- 16674065 TI - Photogenerated quinone methides as useful intermediates in the synthesis of chiral BINOL ligands. AB - The photoinduced synthesis of chiral 3,3'-CH2X-disubstituted BINOL ligands (X = NR2, SR, OH) has been achieved with excellent ee by UV-visible activation of BINOLAMs bearing L-proline ester arms. Quinone methides, detected by laser flash photolysis, are the key intermediates involved in such a synthetic protocol, which undergo reversible nucleophilic conjugate additions by a great variety of nitrogen nucleophiles (amines and alpha-amino acid derivatives) with complete configuration retention of the BINOL moiety. PMID- 16674066 TI - Selective terminal heck arylation of vinyl ethers with aryl chlorides: a combined experimental-computational approach including synthesis of betaxolol. AB - Reaction conditions have been developed for palladium-catalyzed terminal (beta-) arylation of acyclic vinyl ethers with high regioselectivity using inexpensive aryl chlorides as starting materials and the P(t-Bu)3 releasing preligand [(t Bu3)PH]BF4 as the key additive. This swift and straightforward protocol exploits non-inert conditions and controlled microwave heating to minimize handling and processing times and uses aqueous DMF or environmentally friendly PEG-200 as the reaction medium. The selectivity for linear beta-product in PEG-200 is slightly higher than in aqueous DMF. DFT calculations support a ligand-driven selectivity rationale, where the electronic and steric influence of bulky P(t-Bu)3 ligand provides improved beta-selectivity in the essential insertion step also with electron-rich aryl chlorides. A tentative computational rationalization of the improved selectivity in non-methylated PEG is discussed. Finally the synthetic methodology was used to provide efficient access to linear p-[2 (cyclopropylmethoxy)ethyl] phenol from p-nitrophenyl chloride, a key intermediate in the synthesis of the beta-adrenergic blocking agent Betaxolol. PMID- 16674067 TI - Chiral phosphoramide-catalyzed aldol additions of ketone trichlorosilyl enolates. Mechanistic aspects. AB - The mechanism of the catalytic, enantioselective addition of trichlorosilyl enolates to aldehydes has been investigated. Kinetic studies using ReactIR and rapid injection NMR (RINMR) spectroscopy have confirmed the simultaneous operation of dual mechanistic pathways involving either one or two phosphoramides bound to a siliconium ion organizational center. This mechanistic dichotomy was initially postulated on the basis of catalyst loading studies and nonlinear effects studies. This duality explains the difference in reactivity and stereoselectivity of various classes of phosphoramides. Determination of Arrhenius activation parameters revealed that aldol addition occurs through the reversible albeit unfavorable formation of an activated complex, and natural abundance 13C NMR kinetic isotope effect (KIE) studies have determined that the turnover limiting step is the aldol addition. A thorough examination of a range of phosphoramides has established empirical structure-activity selectivity relationships. In addition, the effects of catalyst loading, rate of addition, solvents, and additives have been studied and together allow the formulation of a unified mechanistic picture for the aldol addition. PMID- 16674068 TI - Mix-and-heat benzylation of alcohols using a bench-stable pyridinium salt. AB - 2-Benzyloxy-1-methylpyridinium triflate (1) is a stable, neutral organic salt that converts alcohols into benzyl ethers upon warming. The synthesis and reactivity of 1 are described herein. Benzylation of a wide range of alcohols occurs in good to excellent yield. PMID- 16674069 TI - Triazole-based monophosphine ligands for palladium-catalyzed cross-coupling reactions of aryl chlorides. AB - A variety of triazole-based monophosphines (ClickPhos) have been prepared via efficient 1,3-dipolar cycloaddition of readily available azides and acetylenes. Their palladium complexes provided excellent yields in the amination reactions and Suzuki-Miyaura coupling reactions of unactivated aryl chlorides. Ligand 7i, which has a 2,6-dimethoxybenzene moiety, provided good results in Suzuki-Miyaura reaction to form hindered biaryls. A CAChe model for the Pd/7i complex shows that the likelihood of a Pd-arene interaction might be a rationale for its high catalytic reactivity. PMID- 16674070 TI - Enantio- and diastereocontrolled total synthesis of (+)-boronolide. AB - An efficient stereoselective total synthesis of (+)-boronolide from valeraldehyde is described. The key steps include a Sharpless asymmetric hydroxylation, a chelation-controlled vinyl Grignard reaction followed by a Sharpless asymmetric epoxidation, hydrolytic kinetic resolution, and a ring-closing metathesis. PMID- 16674071 TI - Stereoselective synthesis of 3,6-disubstituted-3,6-dihydropyridin-2-ones as potential diketopiperazine mimetics using organocopper-mediated anti-SN2' reactions and their use in the preparation of low-molecule CXCR4 antagonists. AB - Organocopper-mediated anti-SN2' reactions of gamma-phosphoryloxy-alpha,beta unsaturated-delta-lactams were used to prepare highly functionalized diketopiperazine mimetics. The substrate phosphates 24, 32, and 47 were prepared from alpha-amino acid-derived allylic alcohols 10 by a sequence of reactions that included ring-closing metathesis. In the reactions of phosphates with organocopper reagents, the addition of LiCl dramatically improved anti-SN2' selectivity, indicating that an organocopper cluster containing lithium chloride plays an important role in the determination of regioselectivity. This reaction system was applied to the preparation of novel low molecular weight CXCR4 chemokine receptor antagonists. PMID- 16674072 TI - BF3.2CF3CH2OH (BF3.2TFE), an efficient superacidic catalyst for some organic synthetic transformations. AB - BF3.2CF3CH2OH complex was found to be a very effective superacidic catalyst comparable in acid strength to at least that of 100% anhydrous sulfuric acid for various acid-catalyzed organic transformations such as isomerizations, rearrangements, ionic hydrogenation of various ketones, and aromatics with triethylsilane and nitration of aromatics with metal nitrate. Studies of the pivalaldehyde-methyl isopropyl ketone rearrangement and the benzopinacol to phenanthrene transformation suggest that the complex has an acidity comparable to that of 100% anhydrous sulfuric acid. The structure and properties of the 1:2 boron trifluoride-trifluoroethanol complex have been further studied using NMR (1H, 13C, 19F, 11B) and DFT calculations at the B3LYP/6-311++G//B3LYP/6-31G level. PMID- 16674073 TI - Microwave-assisted preparation of fused bicyclic heteroaryl boronates: application in one-pot Suzuki couplings. AB - The rapid and efficient synthesis of various disubstituted 5,6-fused heterocycles using a microwave-assisted one-pot cyclization-Suzuki coupling approach is described. This work highlights the tolerance of the boronic ester functional group to a variety of reaction conditions and the utility of functionalized boronates as penultimate intermediates in the synthesis of diverse compound libraries. PMID- 16674074 TI - Synthesis and receptor binding evaluation of clavizepine analogues with no ring D substituents. AB - Assembly of the azepine ring of xantheno[9,1-cd]azepines by electrophilic cyclization of sulfonamide acetals provides access to clavizepine analogues in the form of 2,12b-dihydro- or 4-hydroxy-2,3,4,12b-tetrahydro-1H-xantheno[9,1 cd]azepines, in the latter case producing the trans derivative stereoselectively. Binding assays for clavizepine and analogues at adrenergic, dopaminergic, and serotonergic receptors are reported. PMID- 16674075 TI - Direct C-allylation of aryl-alkyl/glycosyl carbinols: facile synthesis of C linked carbo-beta2-/gamma2-/delta2-amino acids. AB - A facile ZrCl4-catalyzed direct allylation of the p-methoxyphenyl-alkyl/glycosyl carbinols at room temperature, and the conversion of the derived aryl-glycosyl alkenes into hitherto unknown C-linked carbo-beta2-/gamma2-/delta2-amino acids is reported. PMID- 16674076 TI - A new tetrathiafulvalene-anthracence dyad fusion with the crown ether group: fluorescence modulation with Na+ and C60, mimicking the performance of an "AND" logic gate. AB - In this Note, we describe a new TTF-anthracene dyad fusion with the crown ether unit. It is interesting to find that the fluorescence of this new dyad can be modulated with Na+ and C60, and its fluorescence intensity can be largely enhanced only in the presence of both Na+ and C60. Such fluorescence modulation behavior mimics the performance of a two-input "AND" logic gate. PMID- 16674078 TI - Regioselectivity in the formation of small- and medium-sized cyclic ethers by diene-ene ring-closing metathesis. AB - In the formation of medium-sized ethers by diene-ene ring-closing metathesis, the formation of cyclic allyl ethers with a smaller ring size and of pentadienyl ethers with a larger ring size compete with each other. In the competition between six- and eight-membered and seven- and nine-membered ring formation, the smaller rings are formed exclusively, whereas in the competition between the five and seven-membered rings, both products are formed in comparable amounts. PMID- 16674077 TI - Studies of substituent effect on asymmetric epoxidation of chromenes by chiral dioxirane. AB - A series of 6- and 8-substituted chromenes has been investigated for asymmetric epoxidation using chiral ketone catalysts. Up to 93% ee was achieved. Higher ee's are obtained when substrates are substituted at the 6-position. The enhanced enantioselectivity is likely due to the beneficial interaction between the 6 substituent of the substrate and the N-aryl or alkyl group of the ketone catalyst. PMID- 16674079 TI - Novel and efficient method for the allylation of carbonyl compounds and imines using triallylaluminum. AB - This is the first report of the use of triallylaluminum as a reagent for the allylation of carbonyl compounds and imines. The allylation of ketimines without additional metal catalyst is known so far only in the case of the Grignard reagent. Triallylaluminum is a useful alternative to provide the homoallylic amines in excellent yield upon addition to aldimines and ketimines. The significant reactivity of this reagent was confirmed by its reaction with a sterically rigid ketone such as adamantanone to provide 1-adamantyl-3-buten-1-ol in 98% yield. The chemoselectivity of triallylaluminum was demonstrated by using different ketoesters. It is noteworthy that triallylaluminum is prepared from allyl bromide and aluminum metal, and not from a Grignard reagent, and that the procedure is operationally simple, leading to good to excellent product yields. PMID- 16674080 TI - Synthesis of 1,3-dihydrobenzo[c]furans from ortho-lithiated aryloxiranes. AB - A general method for the synthesis of hydroxyalkyl 1,3-dihydrobenzo[c]furans from ortho-lithiated aryloxiranes and carbonyl compounds is described. PMID- 16674081 TI - Stereoselective hydroxycarbonylation of vinyl bromides to alpha,beta-unsaturated carboxylic acids in the ionic liquid [BMIM]PF6. AB - (E/Z)-isomers containing vinyl bromides were stereoselectively carbonylated to the corresponding (E)-alpha,beta-ethylenic carboxylic acids in the ionic liquid [BMIM]PF6. Vinyl dibromides also underwent hydroxycarbonylation to give monoacids. The products are pure by proton NMR spectroscopic determination without purification by silica gel column chromatography or recrystallization. PMID- 16674082 TI - Basic ionic liquid as catalysis and reaction medium: a novel and green protocol for the Markovnikov addition of N-heterocycles to vinyl esters, using a task specific ionic liquid, [bmIm]OH. AB - A basic ionic liquid, 1-methyl-3-butylimidazolium hydroxide ([bmIm]OH), has been introduced as a catalyst and reaction medium for the Markovnikov addition of N heterocycles to vinyl esters under mild conditions. The evidence for the role of this basic ionic liquid [bmIm]OH in promoting the Markovnikov addition has been given. On the basis of the evidence, a mechanism was postulated. PMID- 16674083 TI - Synthesis of biaryls and polyaryls by ligand-free Suzuki reaction in aqueous phase. AB - A highly efficient palladium acetate-catalyzed ligand-free Suzuki reaction in aqueous phase was developed in short reaction times (0.5-1 h) at 35 degrees C in air. The key for such a successful catalytic system was the use of a suitable amount of cosolvents in the aqueous phase. The method could be extended to the consecutive multi-Suzuki coupling, and polyaryls were prepared in a single one pot step in high selectivity and excellent yield under mild reaction conditions (60 degrees C). PMID- 16674084 TI - Asymmetric reduction of oxime ethers promoted by chiral spiroborate esters with an O3BN framework. AB - Enatioselective reduction of oxime ethers promoted by chiral spiroborate esters with an O3BN framework is reported for the first time. In the presence of (R,S) 1, 11 aralkyloxime ethers are reduced by borane-THF at 0-5 degrees C to give (S) 1-aralkylamine in high yield and excellent enatiomeric excess (up to 98% ee). Influence of reaction conditions on the enantioselectivity of the reduction is investigated, and a possible mechanism of the catalytic reduction is suggested. PMID- 16674085 TI - Lewis base catalyzed addition of trimethylsilyl cyanide to aldehydes. AB - A variety of achiral Lewis bases were found to catalyze the addition of TMSCN to the aldehydes. Among them, phosphines and amines were the most efficient catalysts. In addition, several chiral amines and phosphines were examined in a catalytic, asymmetric addition of TMSCN to benzaldehyde albeit with low enantioselectivity. A mechanistic study revealed that the reaction was first order in aldehyde, first order in Lewis base, and zeroth order in TMSCN, suggesting the complex formation of TMSCN and Lewis base formation of complex i. However, there are at least two possible scenarios for this catalytic process, and in view of the low selectivities observed, it is not clear which mechanism is operative. PMID- 16674086 TI - Synthesis of 2-phosphonopyrroles via a one-pot RCM/oxidation sequence. AB - A four-step synthesis of 2-phosphonopyrroles is presented starting from suitable aldehydes. The key step in the synthesis involves a one-pot ring-closing metathesis/oxidation sequence of a functionalized alpha-aminoalkenyl phosphonate. Notwithstanding the presence of a nucleophilic nitrogen atom and high substitution patterns in the substrate, the results of the RCM reaction are excellent using mild reaction conditions. Furthermore, a synergism is observed between the RCM catalyst and the oxidizing agent, causing higher oxidation rates and allowing reaction for substrates that normally fail to ring close under standard RCM conditions. PMID- 16674087 TI - Synthesis and biological evaluation of new bicyclic fluorinated uracils through ring-closing metathesis. AB - Two families of bicyclic fluorinated uracils have been prepared starting from a gem-difluorinated unsaturated nitrile, by means of a ring-closing metathesis reaction to form the new ring, which is fused at the C-5/C-6 or N-1/C-6 positions of the uracil moiety. The selective formation of olefin regioisomers in the metathesis process can be controlled according to the reaction conditions (catalyst, solvent, and temperature). The acaricidal activities of the resulting compounds have also been investigated. PMID- 16674088 TI - Synthesis and characterization of new triazenide salts. AB - Stable triethylammonium triazenide salts were obtained on treatment of the appropriate triazenes with triethylamine. Those salts are described and fully characterized for the first time and are used for the preparation of alkoxycarbonylvinyltriethylammonium triazenides, which are prone to transesterification. PMID- 16674089 TI - Efficient synthesis of 2'-C-beta-methylguanosine. AB - 2'-beta-Methyl nucleosides have potential value as therapeutic agents and as nucleoside analogues for exploring RNA biology. Here we develop a strategy for efficient synthesis for 2'-C-beta-methylguanosine (3). Starting from 1,2,3,5 tetra-O-benzoyl-2-C-beta-methyl-d-ribofuranose (1) and N2-acetylguanine, we obtained the title compound in two steps (78% overall yield) with high stereoselectivity (beta/alpha > 99:1) and high regioselectivity (N9/N7 > 99:1). Extension of this strategy to the classic synthesis of guanosine also resulted in high stereoselectivity (beta/alpha = 99:1) and improved regioselectivity (N9/N7 = 97:3). PMID- 16674090 TI - A practical synthesis of 2-((1H-pyrrolo[2,3-b]pyridine-4-yl)methylamino)-5- fluoronicotinic acid. AB - A practical synthesis of a key pharmaceutical intermediate, 2-[(1H-pyrrolo[2,3 b]pyridine-4-yl)methylamino]-5-fluoronicotinic acid (1), is described. To introduce the aminomethyl moiety of 2 via a palladium-catalyzed cyanation/reduction sequence, a regioselective chlorination of 7-azaindole via the N-oxide was developed. A highly selective monodechlorination of 2,6-dichloro 5-fluoronicotinic acid was discovered to afford the nicotinic acid 3. The two building blocks 2 and 3 were then coupled to complete the preparation of 1. PMID- 16674091 TI - New synthesis of 2-aryl-3-substituted benzo[b]furans from benzyl 2-halophenyl ethers. AB - Treatment of benzyl 2-halophenyl ethers with 3 equiv of t-BuLi results in Li halogen exchange and lithiation at benzylic methylene simultaneously. These dianions do not undergo Wittig rearrangement and can be trapped with electrophiles. Their reactions with carboxylic esters afford the corresponding 2 aryl-3-hydroxy-2,3-dihydrobenzo[b]furans as a mixture of diastereoisomers. Subsequent acid-catalyzed or mediated dehydration gives moderate to good overall yield of a variety of 2-aryl-3-substituted benzo[b]furans. PMID- 16674092 TI - Oxaziridine-mediated amination of branched allylic sulfides: stereospecific formation of allylic amine derivatives via [2,3]-sigmatropic rearrangement. AB - Reaction of branched allylic sulfides with the N-Boc-oxaziridine 1 results in [2,3]-sigmatropic rearrangement of the intermediate allylic N-Boc-sulfimides with a high level of chirality transfer. The first example of formation of a quaternary stereocenter using this transformation is reported. PMID- 16674093 TI - Array of informatics: Applications in modern research. AB - The advent of microarray technology in the past decade has greatly enhanced gene expression studies and allowed for the acquisition of a vast amount of information simultaneously. Microarrays have been used in numerous scientific fields to identify new genes, to determine the transcriptional activity of cells, and to discover downstream targets of different loci. Recently, DNA microarrays have also been utilized in disease studies to determine outcomes at many levels including diagnosis, prognosis, and drug therapy. The promise of protein microarrays is to allow us to study the molecular interactions of protein, lipids, small molecules, and carbohydrates. They can be exploited to analyze a single protein pair interaction, to address changes in multiple protein levels as a response to treatment (i.e., drug or radiation), or in a pathological condition. Tissue microarrays allow the analysis of numerous tumor samples simultaneously. Finally, live cell-based microarrays provide an opportunity to study the function of the entire proteome en masse within living cells. However, these exciting new areas still have to overcome many inherent problems. In this review, we discuss novel microarray-based approaches that are in development and that have potential in applications for medicine, biotechnology, and basic research. PMID- 16674094 TI - Better understanding of organ dysfunction requires proteomic involvement. AB - Organ dysfunction is defined as a systemic consequence of acute and chronic diseases, a critical and important phase of disease development. The mortality of patients with severe illness is highly correlated with the number and duration of dysfunctional organs. There is still not an efficient and specific therapy to improve the prognosis of patients with organ dysfunction, due to the complexity and severity of the disease. There is a great need to understand molecular mechanisms of the disease, identify disease-related biomarkers, and validate therapeutic effects. Thus, it is important to have a special attention from proteomic scientists to explore the combination between advanced proteomic biotechnology, clinical proteomics, tissue imaging and profiling, and organ dysfunction score systems, to improve the clinical outcomes of these patients. PMID- 16674095 TI - Predicting the solvent accessibility of transmembrane residues from protein sequence. AB - In this study, we propose a novel method to predict the solvent accessible surface areas of transmembrane residues. For both transmembrane alpha-helix and beta-barrel residues, the correlation coefficients between the predicted and observed accessible surface areas are around 0.65. On the basis of predicted accessible surface areas, residues exposed to the lipid environment or buried inside a protein can be identified by using certain cutoff thresholds. We have extensively examined our approach based on different definitions of accessible surface areas and a variety of sets of control parameters. Given that experimentally determining the structures of membrane proteins is very difficult and membrane proteins are actually abundant in nature, our approach is useful for theoretically modeling membrane protein tertiary structures, particularly for modeling the assembly of transmembrane domains. This approach can be used to annotate the membrane proteins in proteomes to provide extra structural and functional information. PMID- 16674096 TI - Miniaturized solid-phase extraction and sample preparation for MALDI MS using a microfabricated integrated selective enrichment target. AB - A microfabricated proteomic sample preparation and sample presentation device, Integrated Selective Enrichment Target, (ISET), comprising an array of 96 perforated nanovials is described. Each perforated nanovial can be filled with solid-phase extraction media for purification and concentration of peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The validity of the ISET sample preparation is shown by analysis of low nM-pM standard samples, as well as biological samples. The ISET solid-phase extraction sample preparation was compared to ZipTip and MassPREP PROtarget sample preparation, demonstrating a superior performance with respect to number of detected peptides and signal intensity of detected peptides. PMID- 16674097 TI - Comparative proteomics of ovarian epithelial tumors. AB - We analyzed 12 ovarian epithelial tumors using 2D PAGE-based comparative proteomics to construct intra- and inter-tumoral distance map trees and to discover surrogate biomarkers indicative of an ovarian tumor. The analysis was performed after laser microdissection of 12 fresh-frozen tissue samples, including 4 serous, 5 mucinous, and 3 endometrioid tumors, with correlation with their histopathological characteristics. Ovarian epithelial tumors and normal tissues showed an apparent separation on the distance map tree. Mucinous carcinomas were closest to the normal group, whereas serous carcinomas were located furthest from the normal group. All mucinous tumors with aggressive histology were separated from the low malignant potential (LMP) group. The benign looking cysts adjacent to the intraepithelial carcinoma (IEC) showed an expression pattern identical to that of the IEC area. The extent of change on the lineages leading to the mucinous and serous carcinoma was 1.98-fold different. The overall gene expression profiles of serous or endometrioid carcinomas appeared to be less affected by grade or stage than by histologic type. The potential candidate biomarkers screened in ovarian tumors and found to be significantly up-regulated in comparison to normal tissues were as follows: NM23, annexin-1, protein phosphatase-1, ferritin light chain, proteasome alpha-6, and NAGK (N-acetyl glucosamine kinase). In conclusion, ovarian mucinous tumors are distinct from other ovarian epithelial tumors. LMP mucinous tumors showing histologically aggressive features belong to mucinous carcinoma on the proteomic basis. PMID- 16674098 TI - More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry. AB - The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves approximately inversely with liquid flow rate (for the columns having the same separation efficiency, linear velocity, and porosity), making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 microm i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. A 50-microm-i.d. micro solid phase extraction precolumn was used for ease of sample injection and cleanup prior to the reversed-phase LC separation, enabling the sample volume loading speed to be increased by approximately 50-fold. Greater than 10-fold improvement in sensitivity was obtained compared to analyses using more conventional capillary LC, enabling e.g. the identification of >5000 different peptides by MS/MS from 100-ng of a Shewanella oneidensis tryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform responses for different peptides, and provided improved quantitative measurements compared to conventional separation systems without the use of internal standards or isotopic labeling. The improved sensitivity allowed LC-MS measurements of immunopurified protein phosphatase 5 that were in good agreement with quantitative Western blot analyses. PMID- 16674099 TI - 2-D DIGE analysis of butyrate-treated HCT-116 cells after enrichment with heparin affinity chromatography. AB - Butyrate, a 4-carbon short chain fatty acid, is responsible for the protective effects of fiber in colorectal cancer prevention. To better understand the 'blueprint' of butyrate's chemopreventive role in this disease, we performed 2 dimensional difference gel electrophoresis (2-D DIGE) of butyrate-treated HCT-116 colorectal cancer cells after pre-fractionation using heparin affinity chromatography. A combination of this enrichment step with overlapping narrow range IPGs (pH 4-7 and pH 6-11) in 2-D DIGE resulted in the detection of 46 differentially expressed spots. Twenty-four of these were identified by MS analyses, and 5 spots were found to be heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Three isoforms of 38 kDa were down-regulated while two with Mr approximately 26 kDa were up-regulated. These represent phosphorylated isoforms of hnRNP A1 as verified by immunoblotting with anti-phosphotyrosine and anti phosphoserine antibodies. Using 2-DE, subcellular fractionation and western blot analysis, we further showed that full-length hnRNP A1 underwent down-regulation, cleavage and cytoplasmic retention upon butyrate treatment. These indicate that modulations of hnRNP A1 may play a significant role in the mediation of growth arrest and apoptosis by butyrate. PMID- 16674100 TI - Direct protein detection from biological media through electrospray-assisted laser desorption ionization/mass spectrometry. AB - We report here using a novel technology-electrospray-assisted laser desorption ionization (ELDI)/mass spectrometry-for the rapid and sensitive detection of the major proteins that exist in dried biological fluids (e.g., blood, tears, saliva, serum), bacterial cultures, and tissues (e.g., porcine liver and heart) under ambient conditions. This technique required essentially no sample pretreatment. The proteins in the samples were desorbed using a pulsed nitrogen laser without the assistance of an organic matrix. The desorbed protein molecules were then post-ionized through their fusion into the charged solvent droplets produced from the electrospray of an acidic methanol solution; electrospray ionization (ESI) proceeded from the newly formed droplets to generate the ESI-like protein ions. This new ionization approach combines some of the features of electrospray ionization with those of matrix-assisted laser desorption ionization (MALDI), that is, sampling of a solid surface with spatial resolution, generating ESI-like mass spectra of the desorbed proteins, and operating under ambient conditions. PMID- 16674101 TI - Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE. AB - In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified. PMID- 16674102 TI - Complex graph matrix representations and characterizations of proteomic maps and chemically induced changes to proteomes. AB - We have presented a complex graph matrix representation to characterize proteomics maps obtained from 2D-gel electrophoresis. In this method, each bubble in a 2D-gel proteomics map is represented by a complex number with components which are charge and mass. Then, a graph with complex weights is constructed by connecting the vertices in the relative order of abundance. This yields adjacency matrices and distance matrices of the proteomics graph with complex weights. We have computed the spectra, eigenvectors, and other properties of complex graphs and the Euclidian/graph distance obtained from the complex graphs. The leading eigenvalues and eigenvectors and, likewise, the smallest eigenvalues and eigenvectors, and the entire graph spectral patterns of the complex matrices derived from them yield novel weighted biodescriptors that characterize proteomics maps with information of charge and masses of proteins. We have also applied these eigenvector and eigenvalue maps to contrast the normal cells and cells exposed to four peroxisome proliferators, namely, clofibrate, diethylhexyl phthalate (DEHP), perfluorodecanoic acid (PFDA), and perfluoroctanoic acid (PFOA). Our complex eigenspectra show that the proteomic response induced by DEHP differs from the corresponding responses of other three chemicals consistent with their chemical structures and properties. PMID- 16674103 TI - Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype. AB - Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-beta-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-beta1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype. PMID- 16674104 TI - Proteomic analysis of extracellular proteins from Escherichia coli W3110. AB - While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth. PMID- 16674105 TI - Bridging neuropeptidomics and genomics with bioinformatics: Prediction of mammalian neuropeptide prohormone processing. AB - Neuropeptides are an important class of cell to cell signaling molecules that are difficult to predict from genetic information because of their large number of post-translational modifications. The transition from prohormone genetic sequence information to the determination of the biologically active neuropeptides requires the identification of the cleaved basic sites, among the many possible cleavage sites, that exist in the prohormone. We report a binary logistic regression model trained on mammalian prohormones that is more sensitive than existing methods in predicting these processing sites, and demonstrate the application of this method to mammalian neuropeptidomic studies. By comparing the predictive abilities of a binary logistic model trained on molluscan prohormone cleavages with the reported model, we establish the need for phyla-specific models. PMID- 16674106 TI - Aqueous two-phase partitioning for proteomic monitoring of cell surface biomarkers in human peripheral blood mononuclear cells. AB - For proteomic monitoring of processes such as allergy or inflammation an efficient pre-fractionation strategy is required. We isolated plasma membranes from human peripheral blood mononuclear (PBM) cells by aqueous two-phase partitioning. After 1DE combined with LC-MS/MS, several cell surface marker proteins and in total 60 different plasma membrane proteins (out of 84 identified proteins, i.e., 72%) were detected. Plasma membranes obtained were from only one human donor, the procedure is therefore applicable for individual patient screening. PMID- 16674107 TI - Proteomic analysis of MCF-7 cell lines expressing the zinc-finger or the proline rich domain of retinoblastoma-interacting-zinc-finger protein. AB - To identify a growth-promoting activity related to retinoblastoma-interacting zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. PMID- 16674108 TI - Activity-based matrix metallo-protease enrichment using automated, inhibitor affinity extractions. AB - An automated inhibitor affinity extraction method for the activity-based enrichment of matrix metallo-proteases (MMPs) is presented. Samples containing purified MMP-12 were first extracted at different flow rates in a syringe pump setup, using cartridges packed with an MMP inhibitor affinity sorbent based on an immobilized hydroxamic acid containing peptide (PLG-NHOH) with mumol/L MMP affinity. Faster extractions, a reduced number of manual manipulations, and higher extraction yields (98.9%-99.3%) were obtained over the whole flow rate range compared to batch extractions. Application of the method to synovial fluid from a rheumatoid arthritis patient followed by gelatin-zymography revealed a strong enrichment of distinct MMPs from this biological sample that were not clearly visible in the original sample. The use of an auto-sampler and a solid phase extraction (SPE) workstation allowed full automation of the extraction procedure with the potential for on-line coupling to further sample preparation and analytical steps. MMP-12 extractions were optimized showing that ligand density is an important factor with a clear extraction yield optimum around 5 to 7.5 mmol/L. Conditioning of the stationary phase for 1 week prior to use resulted in a further slight increase in extraction yield. Under optimal conditions, an extraction yield of 99.5% was reached with a cartridge contact time of only 13 s for MMP-12. The efficacy of the extraction method for activity-based MMP profiling was further improved by the use of a broad-spectrum MMP inhibitor with nmol/L affinity (TAPI-2). This resulted in an increased extraction yield for all tested MMPs. For MMP-1, -7, -8, -10, -12, and -13 extraction yields of at least 98.8% were obtained, while for MMP-9 (full length and catalytic domain) an extraction yield of at least 96.1% was reached. PMID- 16674109 TI - Enhanced peptide mass fingerprinting through high mass accuracy: Exclusion of non peptide signals based on residual mass. AB - Peptide mass fingerprinting (PMF) is among the principle methods of contemporary proteomic analysis. While PMF is routinely practiced in many laboratories, the complexity of protein tryptic digests is such that PMF based on unrefined mass spectrometric peak lists is often inconclusive. A number of data processing strategies have thus been designed to improve the quality of PMF peak lists, and the development of increasingly elaborate tools for PMF data reduction remains an active area of research. In this report, a novel and direct means of PMF peak list enhancement is suggested. Since the monoisotopic mass of a peptide must fall within a predictable range of residual values, PMF peak lists can in principle be relieved of many non-peptide signals solely on the basis of accurately determined monoisotopic mass. The calculations involved are relatively simple, making implementation of this scheme computationally facile. When this procedure for peak list processing was used, the large number of unassigned masses typical of PMF peak lists was considerably attenuated. As a result, protein identifications could be made with greater confidence and improved discrimination as compared to PMF queries submitted with raw peak lists. Importantly, this scheme for removal of non-peptide masses was found to conserve peptides bearing various post translational and artificial modifications. All PMF experiments discussed here were performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), which provided the high mass resolution and high mass accuracy essential for this application. Previously reported equations relating the nominal peptide mass to the permissible range of fractional peptide masses were slightly modified for this application, and these adjustments have been illustrated in detail. The role of mass accuracy in application of this scheme has also been explored. PMID- 16674110 TI - Selective isolation of lysine-free tryptic peptides delimited by arginine residues: A new tool for proteome analysis. AB - Tryptic digestion of biotinylated Lys-C peptides followed by affinity chromatography allows the selective isolation of lysine-free tryptic peptides delimited by arginine residues (RRnK peptides). In silico analysis revealed that RRnK peptides represent 87% of the whole proteomes and their specific isolation simplifies the complex peptide mixture (5 peptides per protein). The good recoveries and high selectivity obtained in the isolation of RRnK peptides anticipate the applicability of this method in 2DE-free quantitative proteome analyses. PMID- 16674112 TI - Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation. AB - The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent. PMID- 16674111 TI - Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes. AB - A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers. PMID- 16674113 TI - Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility: Implication of multiple injections. AB - We analyzed 10 isobaric tags for relative and absolute quantitation (iTRAQ) experiments using three different model organisms across the domains of life: Saccharomyces cerevisiae KAY446, Sulfolobussolfataricus P2, and Synechocystis sp. PCC6803. A double database search strategy was employed to minimize the rate of false positives to less than 3% for all organisms. The reliability of proteins with single-peptide identification was also assessed using the search strategy, coupled with multiple analyses of samples into LC-MS/MS. The outcomes of the three LC-MS/MS analyses provided higher proteome coverage with an average increment in total proteins identified of 6%, 33%, and 50% found in S. cerevisiae, S. solfataricus, and Synechocystis sp., respectively. The iTRAQ quantification values were found to be highly reproducible across the injections, with an average coefficient of variation (CV) of 0.09 (scattering from 0.14 to 0.04) calculated based on log mean average ratio for all three organisms. Hence, we recommend multiple analyses of iTRAQ samples for greater proteome coverage and precise quantification. PMID- 16674114 TI - Glutamine-binding protein from Escherichia coli specifically binds a wheat gliadin peptide allowing the design of a new porous silicon-based optical biosensor. AB - In this work, the binding of the recombinant glutamine-binding protein (GlnBP) from Escherichia coli to gliadin peptides, toxic for celiac patients, was investigated by mass spectrometry experiments and optical techniques. Mass spectrometry experiments demonstrated that GlnBP binds the following amino acid sequence: XXQPQPQQQQQQQQQQQQL, present only into the toxic prolamines. The binding of GlnBP to gliadin suggested us to design a new optical biosensor based on nanostructured porous silicon (PSi) for the detection of trace amounts of gliadin in food. The GlnBP, which acts as a molecular probe for the gliadin, was covalently linked to the surface of the PSi wafer by a proper passivation process. The GlnBP-gliadin interaction was revealed as a shift in wavelength of the fringes in the reflectivity spectrum of the PSi layer. The GlnBP, covalently bonded to the PSi chip, selectively recognized the toxic peptide. Finally, the sensor response to the protein concentration was measured in the range 2.0-40.0 microg/L and the sensitivity of the sensor was determined. PMID- 16674115 TI - Capillary isoelectric focusing coupled with dynamic imaging detection: A one dimensional separation for two-dimensional protein characterization. AB - Here, we devised a novel approach for two-dimensional (2D) protein characterization using a single one-dimensional separation followed by a second characterization in the same instrument. The approach combines capillary isoelectric focusing (CIEF), which separates proteins according to pI, with dynamic imaging detection, which permits monitoring of protein diffusion in real time and thereby allows estimation of molecular weight from diffusion coefficient. Compared with classical 2D gel electrophoresis, the approach provided several significant advantages including speed and ease in operation and automation, while yielding comparable accuracy. The approach was applicable for protein samples of low to moderate complexity. PMID- 16674116 TI - Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation. AB - A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes. PMID- 16674117 TI - Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma. AB - Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces. PMID- 16674118 TI - Use of solution-IEF-fractionation leads to separation of 2673 mouse brain proteins including 255 hydrophobic structures. AB - Analyzing complex protein mixtures on a single gel does not allow separation of many extracted proteins. Herein, we tried a prefractionation approach and mouse brain proteins were separated on a narrow pH range ZOOM-IEF Fractionator (MicroSol-IEF device) and run on two-dimensional gel electrophoresis. A total number of 2673 protein spots including 255 hydrophobic structures were successfully analyzed by mass spectrometry. This nonsophisticated approach to increase protein identification of a brain protein extract is a step forward in neurochemistry. PMID- 16674119 TI - Nanobacteria-like particles in human arthritic synovial fluids. AB - We investigated the existence of nanosize particles in synovial fluids of rheumatoid arthritis and osteoarthritis patients. These specimens were cultured under mammalian cell culture conditions (37 degrees C; 5% CO2/95% air) for a long period. After about 2 months, many nanoparticles appeared and they gradually increased in number and in size. The nanobacteria-like particles exist in synovial fluids of arthritis patients. The possibility of their existence and pathogenesis in various diseases should be verified cautiously. PMID- 16674121 TI - Molecular dynamics simulations studies of nanoparticles in an isotropic liquid crystal matrix: Single particle behavior and pairwise interactions. AB - We report results of molecular dynamics simulation studies of the behavior of spherical nanoparticles (NPs) in a dense isotropic nematogen matrix comprised of soft spherocylinders (SSCs). The SSCs exhibit a tendency for frustrated planar anchoring at the NP surface that results in a long-range (compared to the size of the NPs and SSCs) reduction in local orientational ordering and increased fluctuations in local orientational ordering compared to the pure isotropic phase of the SSCs. The potential of mean force between two nanoparticles exhibits a novel long-range repulsive tail separated from short-range molecular packing peaks by a shallow local minimum in free energy. The long-range repulsion is caused by NP-induced ordering fluctuations while the shallow minimum results from increased local ordering within the confinement region in between two NPs. The influence of the NPs on local orientational order in the nematogen matrix and the nematogen-induced interaction between NPs are found to depend strongly on the size of the NPs. PMID- 16674122 TI - The fragile-to-strong dynamic crossover transition in confined water: nuclear magnetic resonance results. AB - By means of a nuclear magnetic resonance experiment, we give evidence of the existence of a fragile-to-strong dynamic crossover transition (FST) in confined water at a temperature T(L)=223+/-2 K. We have studied the dynamics of water contained in 1D cylindrical nanoporous matrices (MCM-41-S) in the temperature range 190-280 K, where experiments on bulk water were so far hampered by crystallization. The FST is clearly inferred from the T dependence of the inverse of the self-diffusion coefficient of water (1D) as a crossover point from a non Arrhenius to an Arrhenius behavior. The combination of the measured self diffusion coefficient D and the average translational relaxation time tau(T), as measured by neutron scattering, shows the predicted breakdown of Stokes-Einstein relation in deeply supercooled water. PMID- 16674123 TI - On the electron leak problem in orbital-free embedding calculations. AB - Computer simulation methods using orbital level of description only for a selected part of the larger systems are prone to the artificial charge leak to the parts which are described without orbitals. The absence of orbitals in one of the subsystems makes it impossible to impose explicitly the orthogonality condition. Using the subsystem formulation of density functional theory, it is shown that the absence of explicit condition of orthogonality between orbitals belonging to different subsystems, does not cause any breakdown of this type of description for the chosen intermolecular complexes (F(-)H(2)O and Li(+)H(2)O), for which a significant charge-leak problem could be a priori expected. PMID- 16674124 TI - Transition rate prefactors for systems of many degrees of freedom. AB - When a minimum on the potential energy surface is surrounded by multiple saddle points with similar energy barriers, the transition pathways with greater prefactors are more important than those that have similar energy barriers but smaller prefactors. In this paper, we present a theoretical formulation for the prefactors, computing the probabilities for transition paths from a minimum to its surrounding saddle points. We apply this formulation to a system of 2 degrees of freedom and a system of 14 degrees of freedom. The first is Brownian motion in a two-dimensional potential whose global anharmonicities play a dominant role in determining the transition rates. The second is a Lennard-Jones (LJ) cluster of seven particles in two dimensions. Low lying transition states of the LJ cluster, which can be reached directly from a minimum without passing through another minimum, are identified without any presumption of their characteristics nor of the product states they lead to. The probabilities are computed for paths going from an equilibrium ensemble of states near a given minimum to the surrounding transition states. These probabilities are directly related to the prefactors in the rate formula. This determination of the rate prefactors includes all anharmonicities, near or far from transition states, which are pertinent in the very sophisticated energy landscape of LJ clusters and in many other complex systems. PMID- 16674125 TI - Ultrasoft pseudopotentials for lanthanide solvation complexes: core or valence character of the 4f electrons. AB - The 4f electrons of lanthanides, because of their strong localization in the region around the nucleus, are traditionally included in a pseudopotential core. This approximation is scrutinized by optimizing the structures and calculating the interaction energies of Gd(3+)(H(2)O) and Gd(3+)(NH(3)) microsolvation complexes within plane wave Perdew-Burke-Ernzerhof calculations using ultrasoft pseudopotentials where the 4f electrons are included either in the core or in the valence space. Upon comparison to quantum chemical MP2 and CCSD(T) reference calculations it is found that the explicit treatment of the 4f electrons in the valence shell yields quite accurate results including the required small spin polarization due to ligand charge transfer with only modest computational overhead. PMID- 16674126 TI - Optimal transition paths of stochastic chemical kinetic systems. AB - We present a new framework for finding the optimal transition paths of metastable stochastic chemical kinetic systems with large system sizes. The optimal transition paths are identified, in terms of reaction advancement coordinates, to be the most probable paths according to large deviation theory for the limiting dynamics governed by stochastic differential equations. Dynamical equations for the optimal transition paths are obtained using the variational principle. A multiscale minimum action method is proposed as a numerical scheme to solve the optimal transition paths. Applications to the toggle switch model are presented. PMID- 16674127 TI - Binding energies in benzene dimers: Nonlocal density functional calculations. AB - The interaction energy and minimum energy structure for different geometries of the benzene dimer have been calculated using the recently developed nonlocal correlation energy functional for calculating dispersion interactions. The comparison of this straightforward and relatively quick density functional based method with recent calculations provides a promising first step to elucidate how the former, quicker method might be exploited in larger more complicated biological, organic, aromatic, and even infinite systems such as molecules physisorbed on surfaces and van der Waals crystals. PMID- 16674128 TI - Interaction energies of monosubstituted benzene dimers via nonlocal density functional theory. AB - We present density functional calculations for the interaction energy of monosubstituted benzene dimers. Our approach utilizes a recently developed fully nonlocal correlation energy functional, which has been applied to the pure benzene dimer and several other systems with promising results. The interaction energy as a function of monomer distance was calculated for four different substituents in a sandwich and two T-shaped configurations. In addition, we considered two methods for dealing with exchange, namely, using the revPBE generalized gradient functional as well as full Hartree-Fock. Our results are compared with other methods, such as Moller-Plesset and coupled-cluster calculations, thereby suggesting the usefulness of our approach. Since our density functional based method is considerably faster than other standard methods, it provides a computationally inexpensive alternative, which is of particular interest for larger systems where standard calculations are too expensive or infeasible. PMID- 16674129 TI - Vibrational structure and methyl C-H dynamics in propyne. AB - Our previous study [J. Chem. Phys. 122, 224316 (2005)] presented the photoacoustic and action spectra of the V=2, 3, 4, and 5 manifolds of the C-H methyl stretching vibrations of propyne and their analysis in terms of a simplified joint local mode/normal mode model. In the current paper the C-H transition intensities were calculated using B3LYP6-311++G(d,p) level of theory to obtain the dipole moment functions. The diagonalization of the vibrational Hamiltonian revealed new model parameters obtained by least square fitting of the eigenvalues to the action spectra band origins, while examining the correspondence between the calculated intensities and simulated band areas. The newly derived parameters predict well the band positions and the observed intensities, allowing new assignment of the features. The derived Hamiltonian was also used to obtain the overall temporal behavior of the C-H stretches as a result of the Fermi couplings and interactions with the bath states. These results indicate that any specificity attained by suitable excitation of the methyl C-H stretches is lost on picosecond time scale, primarily due to strong interactions with doorway states in the lower overtone and coupling with bath states in the region of the higher ones. PMID- 16674130 TI - Vibrational quenching of CO2(010) by collisions with O(3P) at thermal energies: a quantum-mechanical study. AB - The CO(2)(010)-O((3)P) vibrational energy transfer (VET) efficiency is a key input to aeronomical models of the energy budget of the upper atmospheres of Earth, Venus, and Mars. This work addresses the physical mechanisms responsible for the high efficiency of the VET process at the thermal energies existing in the terrestrial upper atmosphere (150 KH2+CH3 reaction. AB - Calculations for the cumulative reaction probability N(E) (for J=0) and the thermal rate constant k(T) of the H+CH(4)-->H(2)+CH(3) reaction are presented. Accurate electronic structure calculations and a converged Shepard-interpolation approach are used to construct a potential energy surface which is specifically designed to allow the precise calculation of k(T) and N(E). Accurate quantum dynamics calculations employing flux correlation functions and multiconfigurational time-dependent Hartree wave packet propagation compute N(E) and k(T) based on this potential energy surface. The present work describes in detail the various convergence test performed to investigate the accuracy of the calculations at each step. These tests demonstrate the predictive power of the present calculations. In addition, approximate approaches for reaction rate calculations are discussed. A quite accurate approximation can be obtained from a potential energy surface which includes only interpolation points on the minimum energy path. PMID- 16674137 TI - Structure and stability of xenon insertion compounds of hypohalous acids, HXeOX [X=F, Cl, and Br]: an ab initio investigation. AB - The structure and stability of xenon-inserted hypohalous acids HXeOX (X=F, Cl, and Br) have been investigated theoretically using ab initio molecular orbital calculations. All these molecules are found to consist of a nearly linear HXeO moiety and a bend XeOX fragment. Geometrical parameters of HXeOX are comparable with that of experimentally observed HXeOH species. The dissociation energies corresponding to the lowest-energy fragmentation products, HOX+Xe have been computed to be -398.1, -385.5, and -386.7 kJmol for HXeOF, HXeOCl, and HXeOBr, respectively, at the MP2 level of theory. The respective barrier heights corresponding to the bent transition states (H-Xe-O bending mode) have been calculated to be 138.1, 138.4, and 138.2 kJmol with respect to HXeOX minimum. These species are found to be metastable in their respective potential-energy surface, and the dissociation energies corresponding to the H+Xe+OX products are found to be 56.8, 66.0, and 80.8 kJmol for HXeOF, HXeOCl, and HXeOBr, respectively. The energies corresponding to the H+Xe+O+X dissociation channel have been computed to be 272.0, 309.3, and 299.7 kJmol for HXeOF, HXeOCl, and HXeOBr, respectively, at the same level of theory. Energetics as well as geometrical considerations suggests that it may be possible to prepare these species experimentally similar to that of HXeOH species at low-temperature laser photolysis experiments. PMID- 16674138 TI - Reactions associated with ionization in water: a direct ab initio dynamics study of ionization in (H2O)17. AB - Quasiclassical ab initio simulations of the ionization dynamics in a (H(2)O)(17) cluster, the first water cluster that includes a fourfold coordinated (internally solvated) water molecule, have been carried out to obtain a detailed picture of the elementary processes and energy redistribution induced by ionization in a model of aqueous water. General features observable from the simulations are the following: (i) well within 100 fs following the ionization, one or more proton transfers are seen to take place from the "ionized molecule" to neighboring molecules and beyond, forming a hydronium ion and a hydroxyl radical; (ii) two water molecules close to the ionized water molecule play an important role in the reaction, in what we term a "reactive trimer." The reaction time is gated by the encounter of the ionized water molecule with these two neighboring molecules, and this occurs anytime between 10 and 50 fs after the ionization. The distances of approach between the ionized molecule and the neighboring molecules indeed display best the time characteristics of the transfer of a proton, and thus of the formation of a hydronium ion and a OH radical. These findings are consistent with those for smaller cyclic clusters, albeit the dynamics of the proton transfer displays more varieties in the larger cluster than in the small cyclic clusters. We used a partitioning scheme for the kinetic energy in the (H(2)O)(17) system that distinguishes between the reactive trimer and the surrounding "medium." The analysis of the simulations indicates that the kinetic energy of the surrounding medium increases markedly right after the event of ionization, a manifestation of the local heating of the medium. The increase in kinetic energy is consistent with a reorganization of the surrounding medium, electrostatically forced in a very short time by the water cation and in a longer time by the formation of the hydronium ion. PMID- 16674139 TI - Structures and relative stability of medium-sized silicon clusters. V. Low-lying endohedral fullerenelike clusters Si31-Si40 and Si45. AB - We have performed unconstrained search for low-lying structures of medium-sized silicon clusters Si(31)-Si(40) and Si(45), by means of the minimum-hopping global optimization method coupled with a density-functional based tight-binding model of silicon. Subsequent geometric optimization by using density-functional theory with the PBE, BLYP, and B3LYP functionals was carried out to determine the relative stability of various candidate low-lying silicon clusters obtained from the unconstrained search. The low-lying characteristics of these clusters can be affirmed by comparing the binding energies per atom of these clusters with previously determined lowest-energy clusters(Si(n)) in the size range of 2114 eV. Si and SiCl were detected via nonresonant ionization of SiCl(x) precursors that are produced via the same states and mechanisms that yield Cl. The Si retains the kinetic energy profile of the desorbed precursors. PMID- 16674153 TI - The role of fluid wall association on adsorption of chain molecules at functionalized surfaces: a density functional approach. AB - We present a density functional theory to describe adsorption in systems where selected segments of chain molecules of fluids can bond (or associate) with functional groups attached to the surfaces. Association of active segments with the surface is modeled within the framework of the first-order thermodynamic perturbation theory. We discuss the influence of several parameters such as the density of surface active sites, the energy of association, the chain length, and the number of the active segment in the chain molecule on the structure of the fluid adjacent to the wall. The proposed model can be considered as a first step towards developing a density functional theory of molecular brushes chemically bonded to solid surfaces. PMID- 16674154 TI - X-ray study of the electric double layer at the n-hexane/nanocolloidal silica interface. AB - The spatial structure of the transition region between an insulator and an electrolyte solution was studied with x-ray scattering. The electron-density profile across the n-hexane/silica sol interface (solutions with 5, 7, and 12 nm colloidal particles) agrees with the theory of the electrical double layer and shows separation of positive and negative charges. The interface consists of three layers, i.e., a compact layer of Na(+), a loose monolayer of nanocolloidal particles as part of a thick diffuse layer, and a low-density layer sandwiched between them. Its structure is described by a model in which the potential gradient at the interface reflects the difference in the potentials of "image forces" between the cationic Na(+) and anionic nanoparticles and the specific adsorption of surface charge. The density of water in the large electric field (approximately 10(9)-10(10) Vm) of the transition region and the layering of silica in the diffuse layer is discussed. PMID- 16674155 TI - Steady Marangoni flow traveling with chemical fronts. AB - When autocatalytic chemical fronts propagate in thin layers of solution in contact with air, they can induce capillary flows due to surface tension gradients across the front (Marangoni flows). We investigate here such an interplay between autocatalytic reactions, diffusion, and Marangoni effects with a theoretical model coupling the incompressible Navier-Stokes equations to a conservation equation for the autocatalytic product concentration in the absence of gravity and for isothermal conditions. The boundary condition at the open liquid/air interface takes the surface activity of this product into account and introduces the solutal Marangoni number M representing the intensity of the coupling between hydrodynamics and reaction-diffusion processes. Positive and negative Marangoni numbers correspond, respectively, to the cases where the product decreases or increases surface tension behind the front. We show that, in both cases, such coupled systems reach an asymptotic dynamics characterized by a steady fluid vortex traveling at a constant speed with the front and deforming it, with, however, an asymmetry between the results for positive and negative M. A parametric study shows that increased propagation speed, front deformation, and possible transient oscillating dynamics occur when the absolute value of M is increased. PMID- 16674156 TI - Stability of the polar {111} NaCl crystal face. AB - We present a surface x-ray diffraction determination of the {111} NaCl-liquid interface structure. Using ultrathin water or formamide liquid layers we ascertained that the crystal surface is smooth at an atomic level and is not reconstructed. Our results reveal surprisingly small differences in surface structure between the two cases, which nevertheless lead to dramatic differences in crystal morphology. We determined that the rocksalt {111} surface is Na(+) terminated for both environmental conditions. A quarter to half a monolayer of laterally disordered Cl(-) ions is located on top of a fully ordered Na(+) crystal surface with occupancy 0.75-1.0. This means that the polar surface is stabilized through the formation of an electrochemical double layer. PMID- 16674157 TI - C60 thin film growth on graphite: Coexistence of spherical and fractal-dendritic islands. AB - The initial growth stage of C(60) thin film on graphite substrate has been investigated by scanning tunneling microscopy in ultrahigh vacuum at room temperature. The C(60) layer grows in a quasi-layer-by-layer mode and forms round, monolayer high islands on the graphite surface. The islands are confined by terraces on the graphite surface and the mobility of C(60) fullerenes across steps is low in all layers. The second and all subsequent layers adopt a fractal dendritic shape, which was confirmed by calculating the fractal dimension (D=1.74 prior to island coalescence) and is in agreement with a diffusion limited aggregation. The profound differences between the growth of C(60) layers on graphite (first layer) and on C(60) surfaces (second and higher layers) are caused by the restriction of the C(60) mobility on the highly corrugated fullerene surfaces. The orientation of the fractal islands follows the hexagonal symmetry of the densely packed (111) surface of the fullerene lattice, which introduces a bias in the direction of molecule movement. The differences in surface topography on the nanoscale determine the mode of film growth in this van der Waals bonded system. PMID- 16674158 TI - Equilibrium adsorption at crystal-melt interfaces in Lennard-Jones alloys. AB - Although the properties of crystal-melt interfaces have been extensively studied in pure materials, effects of alloying on the interfacial free energy remain relatively poorly understood. In this work we make use of Monte Carlo computer simulations for model binary Lennard-Jones alloys to explore the effects which variations in atomic-size mismatch and the chemical contributions to mixing energies have upon density and composition profiles, as well as the resulting magnitudes of equilibrium adsorption coefficients in concentrated alloys. We study four different model systems covering a range of chemical and size mismatch, finding relatively small adsorption values which are nevertheless statistically different from zero. PMID- 16674159 TI - Growth of Co clusters on thin films Al2O3NiAl(100). AB - We present a scanning tunnel microscopy study of Co clusters grown through vapor deposition on Al(2)O(3) thin films over NiAl(100) at different coverages and temperatures. Formation of Co clusters was observed at 90, 300, 450, and 570 K. At the three lower temperatures, we find narrow cluster size distributions and the mean sizes (with a diameter of 2.6 nm and a height of 0.7 nm) do not change significantly with the coverage and temperature, until the clusters start to coalesce. Even on 3-4-nm-wide crystalline Al(2)O(3) strips where the deposited Co atoms are confined, the same features sustain. Only at 570 K the normal growth mode where the cluster size increases with the deposition coverage is observed, although the data are less conclusive. A simple modeling of kinetic surface processes on a strip confirms the normal growth mode, but fails to show a favored size unless additional energetic constraints are applied on the cluster sizes. Increasing Co coverages to cluster coalescence, a larger preferable size (mean diameter of 3.5 nm and height of 1.4 nm) appears for growth at 450 K. These two sizes are corroborated by morphology evolution of high Co coverages deposited at 300 K and annealed to 750 K, in which the coalescence is eliminated and the two preferable geometries appear and coexist. PMID- 16674160 TI - Homogeneous nucleation and droplet growth in supersaturated argon vapor: the cryogenic nucleation pulse chamber. AB - We built a cryogenic nucleation pulse chamber for measuring homogeneous nucleation rates of argon. First measurements show that the growth rate of argon droplets at nucleation conditions is rather high so that nucleation and growth could not yet be decoupled. Nevertheless, the experiments permit an estimate of the onset of nucleation corresponding to a nucleation rate of J=10(7(+/-2)) cm( 3) s(-1) at temperatures 5285%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability. PMID- 16674180 TI - Reflection spectroscopy of atherosclerotic plaque. AB - Heart disease is the primary cause of death in the western world. Many of these deaths are caused by the rupture of vulnerable plaque. Vulnerable plaques are characterized by a large lipid core covered by a thin fibrous cap. One method for detecting these plaques is reflection spectroscopy. Several studies have investigated this method using statistical methods. A more analytic and quantitative study might yield more insight into the sensitivity of this detection modality. This is the approach taken in this work. Reflectance spectra in the spectral region from 400 to 1700 nm are collected from 77 measurement points from 23 human aortas. A measure of lipid content in a plaque based on reflection spectra is presented. The measure of lipid content is compared with the thickness of the lipid core, determined from histology. Defining vulnerable plaque as having a lipid core >500 microm and fibrous cap <500 microm, vulnerable plaques are detected with a sensitivity of 88% and a specificity of 94%. Although the method can detect lipid content, it is not very sensitive to the thickness of the fibrous cap. Another detection modality is necessary to detect this feature. PMID- 16674181 TI - Measurement of fibrous cap thickness in atherosclerotic plaques by spatiotemporal analysis of laser speckle images. AB - Necrotic-core fibroatheromas (NCFA) with thin, mechanically weak fibrous caps overlying lipid cores comprise the majority of plaques that rupture and cause acute myocardial infarction. Laser speckle imaging (LSI) has been recently demonstrated to enable atherosclerotic plaque characterization with high accuracy. We investigate spatio-temporal analysis of LSI data, in conjunction with diffusion theory and Monte Carlo modeling of light transport, to estimate fibrous cap thickness in NCFAs. Time-varying laser speckle images of 20 NCFAs are selected for analysis. Spatio-temporal intensity fluctuations are analyzed by exponential fitting of the windowed normalized cross-correlation of sequential laser speckle patterns to obtain the speckle decorrelation time constant, tau(rho), as a function of distance rho from the source entry location. The distance, rho', at which tau(rho) dropped to 65% of its maximum value is recorded. Diffusion theory and Monte Carlo models are utilized to estimate the maximum photon penetration depth, zmax(rho'), for a distance equal to rho', measured from LSI. Measurements of zmax(rho') correlate well with histological measurements of fibrous cap thickness (R=0.78, p<0.0001), and paired t-tests show no significant difference between the groups (p=0.4). These results demonstrate that spatio-temporal LSI may allow the estimation of fibrous cap thickness in NCFAs, which is an important predictor of plaque stability. PMID- 16674182 TI - Detection of morphological markers of vulnerable atherosclerotic plaque using multimodal spectroscopy. AB - Vulnerable plaques, which are responsible for most acute ischemic events, are presently invisible to x-ray angiography. Their primary morphological features include a thin or ulcerated fibrous cap, a large necrotic core, superficial foam cells, and intraplaque hemorrhage. We present evidence that multimodal spectroscopy (MMS), a novel method that combines diffuse reflectance spectroscopy (DRS), intrinsic fluorescence spectroscopy (IFS), and Raman spectroscopy (RS), can detect these markers of plaque vulnerability. To test this concept, we perform an MMS feasibility study on 17 human carotid artery specimens. Following the acquisition of spectra, each specimen is histologically evaluated. Two parameters from DRS, hemoglobin concentration and a scattering parameter, are used to detect intraplaque hemorrhage and foam cells; an IFS parameter that relates to the amount of collagen in the topmost layers of the tissue is used to detect the presence of a thin fibrous cap; and an RS parameter related to the amount of cholesterol and necrotic material is used to detect necrotic core. Taken together, these spectral parameters can generally identify the vulnerable plaques. The results indicate that MMS provides depth-sensitive and complementary morphological information about plaque composition. A prospective in vivo study will be conducted to validate these findings. PMID- 16674183 TI - Photosensitizer delivery to vulnerable atherosclerotic plaque: comparison of macrophage-targeted conjugate versus free chlorin(e6). AB - We have previously shown that a conjugate (MA-ce6) between maleylated serum albumin and the photosensitizer chlorin(e6) (ce6) is targeted in vitro to macrophages via class A scavenger receptors. We now report on the ability of this conjugate to localize in macrophage-rich atherosclerotic plaques in vivo. Both the conjugate and the free photosensitizer ce6 are studied after injection into New Zealand White rabbits that are rendered atherosclerotic by a combination of aortic endothelial injury and cholesterol feeding into normal rabbits. Rabbits are sacrificed at 6 and 24 h after injection and intravascular fluorescence spectroscopy is carried out by fiber-based fluorimetry in intact blood-filled arteries. Surface spectrofluorimetry of numbered excised aortic segments together with injured and normal iliac arteries is carried out, and quantified ce6 content by subsequent extraction and quantitative fluorescence determination of the arterial segments and also of nontarget organs. There is good agreement between the various techniques for quantifying ce6 localization, and high contrast between arteries from atherosclerotic and normal rabbits is obtained. Fluorescence correlates with the highest burden of plaque in the aorta and the injured iliac artery. The highest accumulation in plaques is obtained using MA ce6 at 24 h. Free ce6 gives better accumulation at 6 h compared to 24 h. The liver, spleen, lung, and gall bladder have the highest uptake in nontarget organs. Macrophage-targeted photosensitizer conjugates may have applications in both detecting and treating inflamed vulnerable plaque. PMID- 16674184 TI - Detection of macrophage activity in atherosclerosis in vivo using multichannel, high-resolution laser scanning fluorescence microscopy. AB - Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9% saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5x5-microm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9+/-1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques. PMID- 16674185 TI - Bioenzymatic detection of troponin C using micro-opto-electro-mechanical systems. AB - Diagnosis and monitoring of critical diseases such as acute myocardial infarction (AMI) require a quantitative analysis of biological molecules. A high-throughput identification of these biological molecules can be generated by using micro electro-mechanical systems (MEMS) structures like simple cantilever beams, which respond to the intermolecular forces resulting from binding these molecules. Biochemical markers like troponin C are considered the primary markers for myocardial injury and have generated considerable interest. A 26-residue lytic membrane protein of bee venom melittin (ME) is chosen to interact with rabbit skeletal muscle troponin C (TnC) on the surface of the cantilever beams. An optical beam deflection method is employed to identify the enzymatic reaction on the surface of the cantilever due to these proteins. Identification of these proteins is also done using fluorescence spectroscopy (FS) to compliment the optical monitored deflection method. A second set of proteins like horse radish peroxide (HRP) and hydrogen peroxide (H2O2) are applied to atomic force microscopy (AFM) cantilever beams to study their behavior under the enzymatic reactions of proteins. Identification of these proteins is done using Fourier transform infrared spectroscopy (FTIR). An analytical model of the cantilever beam is developed, and its mode shapes are studied by employing orthogonal polynomials in the classic Rayleigh-Ritz method. The surface stress caused by the enzymatic reaction of the proteins that leads to pure bending on the top surface of the cantilever is evaluated. The information provided by the experimental and analytical modeling reported in this work will be useful in the development of a portable biosensor for the detection of AMI. PMID- 16674186 TI - Fluorophore-mediated, fiber-optic, multi-analyte, immunosensing system for rapid diagnosis and prognosis of cardiovascular diseases. AB - A prototype of a fiber-optic, multi-analyte, immunobiosensing system was developed to simultaneously quantify disease-representing biomarkers in blood plasma. This system was for simultaneous quantification of two different groups of multi-biomarkers related to cardiovascular diseases (CVD): anticoagulants (protein C, protein S, antithrombin III, and plasminogen) for deficiency diagnosis; and cardiac markers (B-type natriuretic peptide, cardiac troponin I, myoglobin, and C-reactive protein) for coronary heart disease diagnosis. As an initial effort towards the development of a disposable and easy-to-use sensing cartridge as a rapid diagnostic tool for CVD related diseases, a prototype of a flow control system was also developed to automatically perform simultaneous four analyte quantification. Currently, the system is capable of quantifying the multiple anticoagulants in their clinically significant sensing ranges within 5 minutes, at an average signal-to-noise (S/N) ratio of 25. A simultaneous assay of the four cardiac markers can be performed within 10 min, at an average S/N ratio of 20. When this highly portable multi-analyte sensing system is completed and successfully tested for CVD patient's plasma, it can provide rapid (<10 min) and reliable diagnostic and prognostic information at a patient's bedside. PMID- 16674187 TI - Optical mapping of myocardial reactive oxygen species production throughout the reperfusion of global ischemia. AB - Reactive oxygen species (ROS) are short-lived, highly reactive chemical entities that play significant roles in all levels of biology. However, their measurement requires destructive preparation, thereby limiting the continuous measurement of ROS in a living tissue. We develop an optical mapping system to visualize ROS production in an isolated and perfused rat heart. By staining the heart with dihydroethidium (DHE), a 532-nm laser beam is directed to the epicardial surface, where we collect the red fluorescence (>600 nm) for semiquantitative analysis. With this system, ROS production as well as ventricular pressure and ECG in isolated perfused rat hearts are monitored throughout the reperfusion of global ischemia. Ischemia would decrease myocardial ROS production, while reperfusion would immediately result in sustained ROS overproduction. Optical mapping would provide information regarding the spatial distribution and temporal evolution of myocardial ROS production, which would enhance knowledge of the role of free radicals in cardiovascular biology. PMID- 16674188 TI - Optical recording-guided pacing to create functional line of block during ventricular fibrillation. AB - Low-energy defibrillation is very desirable in cardiac rhythm management. We previously reported that ventricular fibrillation (VF) can be synchronized with a novel synchronized pacing technique (SyncP) using low-energy pacing pulses. This study sought to create a line of block during VF using SyncP. SyncP was performed in six isolated rabbit hearts during VF using optical recording to control the delivery of pacing pulses in real time. Four pacing electrodes with interelectrode distances of 5 mm were configured in a line along and across the myocardial fiber direction. The electrodes were controlled independently (independent mode) or fired together (simultaneous mode). Significant wavefront synchronization was observed along the electrode line as indicated by a decrease in variance. With the independent SyncP protocol, the decrease in the variance was 19.3 and 13.7% (P<0.001) for the along-, and across-fiber configurations, respectively. With the simultaneous SyncP protocol, the variance was reduced by 24.2 and 10.7% (P<0.001) in the along- and across-fiber configurations. The effect of synchronization dropped off with distance from the line of pacing. We conclude that SyncP can effectively create a line of functional block that isolates regions of VF propagation. Further optimization of this technique may prove useful for low-energy ventricular defibrillation. PMID- 16674189 TI - Three-dimensional optical coherence tomography of the embryonic murine cardiovascular system. AB - Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology that has potential as a novel investigational tool in developmental biology and functional genomics. In this study, murine embryos and embryonic hearts are visualized with an OCT system capable of 2-microm axial and 15-microm lateral resolution and with real-time acquisition rates. We present, to our knowledge, the first sets of high-resolution 2- and 3-D OCT images that reveal the internal structures of the mammalian (murine) embryo (E10.5) and embryonic (E14.5 and E17.5) cardiovascular system. Strong correlations are observed between OCT images and corresponding hematoxylin- and eosin-stained histological sections. Real-time in vivo embryonic (E10.5) heart activity is captured by spectral-domain optical coherence tomography, processed, and displayed at a continuous rate of five frames per second. With the ability to obtain not only high-resolution anatomical data but also functional information during cardiovascular development, the OCT technology has the potential to visualize and quantify changes in murine development and in congenital and induced heart disease, as well as enable a wide range of basic in vitro and in vivo research studies in functional genomics. PMID- 16674190 TI - Control of cardiomyocyte orientation on a microscaffold fabricated by photopolymerization with laser beam interference. AB - We fabricate scaffolds with a fine linear grating (periodicity of 1 to 8 microm and height of 1 microm) on a glass substrate for controlling cardiomyocyte orientation. The fabrication is done by the solidification of a liquid photopolymerizable material using two laser beam interference. As the photopolymerizable material, we use acrylated trimethylene-carbonate-based oligomers initiated with trimethylolpropane (T/TMP), followed by acrylation at terminal ends. Rat cardiomyocytes cultured on the fabricated scaffolds exhibit cell elongation, orientation, and contraction along the scaffold grating. Fluorescence observation of bundles of actin filaments of the cultured cells show that the cytoskeleton of the cells is also generated and oriented parallel to the grating. With a change in grating periodicity from 8 to 1 microm, the percentage of the cells that show orientation along the grating increase from approximately 40 to 70%. The cell orientation along the grating is observed 18 h after seeding the cells on the scaffold. This result implies that the attachment between a cell and a well-defined microarchitectural substrate at an early stage of culture is a significant determinant of cell morphology. PMID- 16674191 TI - Visible lesion thresholds with pulse duration, spot size dependency, and model predictions for 1.54-microm, near-infrared laser pulses penetrating porcine skin. AB - Er:glass lasers have been in operation with both long pulses (hundreds of microseconds) and Q-switched pulses (50 to 100 ns) for more than 35 yr. The ocular hazards of this laser were reported early, and it was determined that damage to the eye from the 1.54-microm wavelength occurred mainly in the cornea where light from this wavelength is highly absorbed. Research on skin hazards has been reported only in the past few years because of limited pulse energies from these lasers. Currently, however, with pulse energies in the hundreds of joules, these lasers may be hazardous to the skin in addition to being eye hazards. We report our minimum visible lesion (MVL) threshold measurements for two different pulse durations and three different spot sizes for the 1.54-microm wavelength using porcine skin as an in vivo model. We also compare our measurements to results from our model, based on the heat transfer equation and the rate process equation. Our MVL-ED50 thresholds for the long pulse (600 micros) at 24 h postexposure were measured to be 20, 8.1, and 7.4 J cm(-2) for spot diameters of 0.7, 1.0, and 5 mm, respectively. Q-switched laser pulses of 31 ns had lower ED50 (estimated dose for a 50% probability of laser-induced damage) thresholds of 6.1 J cm(-2) for a 5-mm-diam, top-hat spatial profile laser pulse. PMID- 16674192 TI - Collagen birefringence in skin repair in response to red polarized-laser therapy. AB - We use the optical path difference (OPD) technique to quantify the organization of collagen fibers during skin repair of full-thickness burns following low intensity polarized laser therapy with two different polarization incidence vectors. Three burns are cryogenerated on the back of rats. Lesion L(parallel) is irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction. Lesion L(perpendicular) is irradiated using the electric field vector of the polarized laser radiation aligned perpendicularly to the aforementioned orientation. Lesion C is untreated. A healthy area labeled H is also evaluated. The tissue samples are collected and processed for polarized light microscopy. The overall finding is that the OPD for collagen fibers depends on the electric field vector of the incident polarized laser radiation. No significant differences in OPDs are observed between L(parallel) and H in the center, sides, and edges of the lesion. Lesions irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction show higher birefringence, indicating that collagen bundles in these lesions are more organized. PMID- 16674193 TI - Raman microspectrometry of laser-reshaped rabbit auricular cartilage: preliminary study on laser-induced cartilage mineralization. AB - Laser-assisted cartilage reshaping (LACR) is a relatively novel technique designed to noninvasively and permanently restructure cartilaginous tissue. It is believed that heat-induced stress relaxation, in which a temperature-mediated disruption of H2O binding is associated with conformational alterations in the proteoglycan and collagen-rich matrix, constitutes the underlying mechanism of LACR. Several reports have suggested that laser-mediated cartilage mineralization may contribute to the permanent shape change of laser-reshaped cartilage. In an effort to validate these results in the context of Er:glass LACR, we performed a preliminary Raman microspectrometric study to characterize the crystal deposits in laser-irradiated chondrocytes and extracellular matrix. For the first time, we identified intracellular calcium sulfate deposits and extracellular calcium phosphate (apatite) crystals in laser-reshaped rabbit auricular cartilage. Calcium carbonate deposits are localized in both irradiated and nonirradiated samples, suggesting that this mineral plays no role in conformational retention. In our discussion, we elaborate on the possible molecular and cellular mechanisms responsible for intra- and extracellular crystallization, and propose a novel hypothesis on the formation of apatite, inasmuch as the biological function of this mineral (providing structure and rigidity in bones and dental enamel) may be extrapolated to the permanent shape change of laser-irradiated cartilage. PMID- 16674194 TI - Use of surface-enhanced Raman spectroscopy for the detection of human integrins. AB - Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, alphaVbeta3 and alpha5beta1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance. PMID- 16674195 TI - Micro-Raman imaging analysis of monomer/mineral distribution in intertubular region of adhesive/dentin interfaces. AB - It is generally proposed that bonding of resins to dentin results from infiltration of the adhesive monomers into the superficially demineralized dentin. However, it is still not clear how well the mineral phase of dentin is removed and how far each monomer penetrates into the thin zone of "wet" demineralized dentin. The quality and molecular structure of adhesive/dentin interfaces formed under "wet" bonding conditions are studied using 2-D Raman microspectroscopic mapping/imaging techniques. Micro-Raman imaging analysis of the adhesive/dentin interface provides a reliable and powerful means of identifying the degree and depth of dentin demineralization, adhesive monomer distribution, and flaws or defects in the pattern of adhesive penetration. The image of mineral reveals a partially demineralized layer on the top of dentin substrate. Adhesive monomers readily penetrate into dentin tubules and spread into intertubular region through open tubules. The extent of adhesive monomer penetration is higher in the intertubular regions close to tubules as compared to the middle regions between the tubules. The diffusion of resin monomers differs substantially. In a comparison with a hydrophilic monomer, the hydrophobic monomer resists diffusion into the demineralized intertubular dentin area. PMID- 16674196 TI - Simple device for the direct visualization of oral-cavity tissue fluorescence. AB - Early identification of high-risk disease could greatly reduce both mortality and morbidity due to oral cancer. We describe a simple handheld device that facilitates the direct visualization of oral-cavity fluorescence for the detection of high-risk precancerous and early cancerous lesions. Blue excitation light (400 to 460 nm) is employed to excite green-red fluorescence from fluorophores in the oral tissues. Tissue fluorescence is viewed directly along an optical axis collinear with the axis of excitation to reduce inter- and intraoperator variability. This robust, field-of-view device enables the direct visualization of fluorescence in the context of surrounding normal tissue. Results from a pilot study of 44 patients are presented. Using histology as the gold standard, the device achieves a sensitivity of 98% and specificity of 100% when discriminating normal mucosa from severe dysplasia/carcinoma in situ (CIS) or invasive carcinoma. We envisage this device as a suitable adjunct for oral cancer screening, biopsy guidance, and margin delineation. PMID- 16674197 TI - Ability of laser fluorescence device associated with fluorescent dyes in detecting and quantifying early smooth surface caries lesions. AB - A laser fluorescence (LF) device is a portable tool, but it does not measure minor mineral changes. Our in vitro study aim is to propose the association of an LF with two fluorescent dyes and to evaluate the performance in detecting and quantifying early demineralization. Artificial caries lesions are created in 40 primary canine teeth using a demineralizing solution (pH=4.8) for 12, 24, 48, and 96 h. LF measurements are performed with DIAGNOdent after demineralization in these samples and in 20 sound primary teeth. Measurements with LF with 0.2-mM tetrakis(N-methylpyridyl)porphyrin (LF TMPyP) and with 4-mM protoporphyrin IX (LF PPIX) are made. The amount of calcium loss is determined by atomic emission spectrometry. A correlation between LF and LF with dyes and mineral loss and receiver operating characteristics analysis are performed, as well as comparisons of sensitivity, specificity, and accuracy values. Significant correlation is obtained with LF TMPyP and mineral loss of lesions demineralized for 24, 48, and 96 h. Better performance is achieved with LF TMPyP for all parameters than with LF alone. LF PPIX does not present good results. In conclusion, LF TMPyP provides good performance in detecting and quantifying very early enamel caries lesions. PMID- 16674198 TI - Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia. AB - We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation. PMID- 16674199 TI - Fluorescence spectroscopic analysis of surface and subsurface residual stress fields in alumina hip joints. AB - We aim to establish a confocal spectroscopic technique able to study the features of fluorescence spectra arising from native Cr3+ impurity in polycrystalline alumina (Al2O3) as a biomaterial and to use their emission lines as microscopic probes for the characterization of residual stress fields stored in artificial hip prostheses during their implantation in vivo. As an application of the technique, we report for the first time concerning the evolution of microscopic (residual) stress fields stored on the surface and in the subsurface of N=7 retrieved Al2O3 hip joints after exposure in the human body from a few months to 19 yr. The micrometric diameter of the laser beam waist impinging on the joint surface (typically about 1 microm in lateral resolution) enables us to estimate the patterns and magnitude of residual stress with high spatial resolution, at least comparable with the grain size of the material. In addition, a selected confocal configuration for the optical probe enables minimization of the probe size along the in-depth direction. According to a statistical collection of data on the microscopic level for retrieved femoral heads in toto, a residual stress field arising from loading history in vivo during the lifetime of the Al2O3 femoral head can be revealed. Finally, an interpretation is given of microscopic wear mechanisms in Al2O3 artificial hip joints consistent with the observed evolution of surface residual stress fields on elapsed time in vivo. PMID- 16674200 TI - Fluorescence microscopy imaging of electroperturbation in mammalian cells. AB - We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation--the effects of nanosecond, megavolt per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes. PMID- 16674201 TI - Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis. AB - Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3. PMID- 16674202 TI - Accurate single-pair Forster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy. AB - Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively. PMID- 16674203 TI - Simulation study of second-harmonic microscopic imaging signals through tissue like turbid media. AB - We establish, for the first time, a simulation model for dealing with the second harmonic signals under a microscope through a tissue-like turbid medium, based on the Monte Carlo method. With this model, the angle-resolved distribution and the signal level eta of second-harmonic light through a slab of the turbid medium are demonstrated and the effects of the thickness (d) of the turbid medium, the numerical aperture (NA) of the objective as well as the size (rho) of the scatterers forming the turbid medium are explored. Simulation results reveal that the use of a small objective NA results in a narrow angle distribution but strong second-harmonic signals. A turbid medium consisting of large scattering particles has a strong influence on the angle distribution and the signal level eta, which results in a low penetration limit for second-harmonic signals made up of ballistic photons. It is approximately 30 microm in our situation. PMID- 16674204 TI - Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy. AB - Spectral domain phase microscopy (SDPM) is a function extension of spectral domain optical coherence tomography. SDPM achieves exquisite levels of phase stability by employing common-path interferometry. We discuss the theory and limitations of Doppler flow imaging using SDPM, demonstrate monitoring the thermal contraction of a glass sample with nanometer per second velocity sensitivity, and apply this technique to measurement of cytoplasmic streaming in an Amoeba proteus pseudopod. We observe reversal of cytoplasmic flow induced by extracellular CaCl2, and report results that suggest parabolic flow of cytoplasm in the A. proteus pseudopod. PMID- 16674205 TI - Noninvasive imaging of hemoglobin concentration and oxygenation in the rat brain using high-resolution photoacoustic tomography. AB - Simultaneous transcranial imaging of two functional parameters, the total concentration of hemoglobin and the hemoglobin oxygen saturation, in the rat brain in vivo is realized noninvasively using laser-based photoacoustic tomography (PAT). As in optical diffusion spectroscopy, PAT can assess the optical absorption of endogenous chromophores, e.g., oxygenated and deoxygenated hemoglobins, at multiple optical wavelengths. However, PAT can provide high spatial resolution because its resolution is diffraction-limited by photoacoustic signals rather than by optical diffusion. Laser pulses at two wavelengths are used sequentially to acquire photoacoustic images of the vasculature in the cerebral cortex of a rat brain through the intact skin and skull. The distributions of blood volume and blood oxygenation in the cerebral cortical venous vessels, altered by systemic physiological modulations including hyperoxia, normoxia, and hypoxia, are visualized successfully with satisfactory spatial resolution. This technique, with its prominent sensitivity to endogenous contrast, can potentially contribute to the understanding of the interrelationship between neural, hemodynamic, and metabolic activities in the brain. PMID- 16674206 TI - Monitoring cerebral oxygen saturation during cardiopulmonary bypass using near infrared spectroscopy: the relationships with body temperature and perfusion rate. AB - During cardiopulmonary bypass (CPB) because of weak arterial pulsation, near-IR spectroscopy (NIRS) is almost the only available method to monitor cerebral oxygenation noninvasively. Our group develops a NIRS oximeter to monitor regional cerebral oxygenation especially its oxygen saturation (rScO2). To achieve optimal coupling between the sensor and human brain, the distances between the light source and the detectors on it are properly chosen. The oximeter is calibrated by blood gas analysis, and the results indicate that its algorithm is little influenced by either background absorption or overlying tissue. We used it to measure the rScO2 of 15 patients during CPB. It is shown that rScO2 is negatively correlated with body temperature and positively with perfusion rate. There are two critical stages during CPB when rScO2 might be relatively low: one is the low perfusion-rate stage, the other is the early rewarming stage. During cooling, the changes of total hemoglobin concentration (C(tHb)) compared with its original value is also monitored. It is shown that C(tHb) decreases to a small extent, which may mainly reflect cerebral vasoconstriction induced by cooling. All these results indicate that NIRS can be used to monitor cerebral oxygenation to protect cerebral tissue during CPB. PMID- 16674207 TI - Automatic identification of biological microorganisms using three-dimensional complex morphology. AB - We propose automated identification of microorganisms using three-dimensional (3 D) complex morphology. This 3-D complex morphology pattern includes the complex amplitude (magnitude and phase) of computationally reconstructed holographic images at arbitrary depths. Microscope-based single-exposure on-line (SEOL) digital holography records and reconstructs holographic images of the biological microorganisms. The 3-D automatic recognition is processed by segmentation, feature extraction by Gabor-based wavelets, automatic feature vector selection by graph matching, training rules, and a decision process. Graph matching combined with Gabor feature vectors measures the similarity of complex geometrical shapes between a reference microorganism and unknown biological samples. Automatic selection of the training data is proposed to achieve a fully automatic recognition system. Preliminary experimental results are presented for 3-D image recognition of Sphacelaria alga and Tribonema aequale alga. PMID- 16674208 TI - Phase function measurements on nonspherical scatterers using a two-axis goniometer. AB - We present a two-axis goniometer for measuring the phase function of scattering media with an angular resolution of about 0.2 deg having 12 decades of dynamic range and covering almost the full solid angle. The setup is evaluated with polystyrene spheres and with perpendicularly and obliquely illuminated thin glass cylinders. The scattering pattern and its intensity distribution are in excellent agreement with analytical theory. A multiple scattering configuration composed of two parallel cylinders is also examined. Finally, the phase function of dentin slabs is measured and its dependence on the dental microstructure is discussed. PMID- 16674209 TI - Use of light absorbers to alter optical interrogation with epi-illumination and transillumination in three-dimensional cardiac models. AB - Cardiac optical mapping currently provides 2-D maps of transmembrane voltage sensitive fluorescence localized near the tissue surface. Methods for interrogation at different depths are required for studies of arrhythmias and the effects of defibrillation shocks in 3-D cardiac tissue. We model the effects of coloading with a dye that absorbs excitation or fluorescence light on the radius and depth of the interrogated region with specific illumination and collection techniques. Results indicate radii and depths of interrogation are larger for transillumination versus epi-illumination, an effect that is more pronounced for broad-field excitation versus laser scanner. Coloading with a fluorescence absorber lessens interrogated depth for epi-illumination and increases it for transillumination, which is confirmed with measurements using transillumination of heart tissue slices. Coloading with an absorber of excitation light consistently decreases the interrogated depths. Transillumination and coloading also decrease the intensities of collected fluorescence. Thus, localization can be modified with wavelength-specific absorbers at the expense of a reduction in fluorescence intensity. PMID- 16674210 TI - Classifying structural alterations of the cytoskeleton by spectrum enhancement and descriptor fusion. AB - A classifier capable of ranking structural alterations of the cytoskeleton is developed. Images of cytoskeletal microtubules obtained from the epifluorescence microscopy of primary culture rat hepatocytes are analyzed. Morphological descriptors are extracted by contour and mass fractal analysis, direct methods, and spectrum enhancement. All methods are designed and tuned to make the extracted morphological descriptors insensitive to absolute fluorescence intensities. Spectrum enhancement is a nonlinear filter that involves spatial differentiation of the gray-scale image followed by conversion of power spectral density to the logarithmic scale and averaging over arcs in the reciprocal domain. Enhanced spectra exhibit local maxima that correspond to the structured microtubule bundles of a normal cytoskeleton. Descriptor fusion for classification is achieved by means of multivariate analysis. The classifier is trained by image sets representing normal ("negative control") microtubules and those altered by exposure to a fungicide at the highest dose of the experiment design. Some sensitivity and validation tests, including discriminant functions analysis, are applied to the classifier. The latter is applied to recognize images of microtubules not used in the training stage and comes from treatments at lower concentrations and shorter times. As a result, structural alterations are ranked and structural recovery after treatment is quantified. The method has potential use in quantitative, morphology-based tests on the cytoskeleton treated either by anticancer drugs or by cytotoxic agents. PMID- 16674211 TI - Signatures of non locality for short-ranged wetting at curved substrates. AB - The binding potential for wetting near planes, spheres, and cylinders in systems with short-ranged forces is shown to have a universal geometrical structure. This arises from the nonlocal nature of the interfacial interactions and is exactly described by a recently proposed binding potential functional, which provides a systematic framework for studying wetting at arbitrarily shaped substrates. The corrections to the equilibrium wetting layer thickness induced by nonlocality are comparable to those arising from a Tolman length and lead to diverging terms in the total mass adsorption. PMID- 16674212 TI - Correlated wave functions for the ground and some excited states of the iron atom. AB - We study the states arising from the [Ar]4s(2)3d6 and [Ar]4s(1)3d7 configurations of iron atom with explicitly correlated wave functions. The variational wave function is the product of the Jastrow correlation factor times a model function obtained within the parametrized optimized effective potential framework. A systematic analysis of the dependence of both the effective potential and the correlation factor on the configuration and on the term is carried out. The ground state of both, the cation, Fe+, and anion, Fe-, are calculated with correlated wave functions and the ionization potential and the electron affinity are obtained. PMID- 16674213 TI - A fragment energy assembler method for Hartree-Fock calculations of large molecules. AB - We present a fragment energy assembler approach for approximate Hartree-Fock (HF) calculations of macromolecules. In this method, a macromolecule is divided into small fragments with appropriate size, and then each fragment is capped by its neighboring fragments to form a subsystem. The total energy of the target system is evaluated as the sum of the fragment energies of all fragments, which are available from conventional HF calculations on all subsystems. By applying the method to a broad range of molecules, we demonstrate that the present approach could yield satisfactory HF energies for all studied systems. PMID- 16674214 TI - A comparative study of imaginary time path integral based methods for quantum dynamics. AB - The recently introduced approximate many-body quantum simulation method, ring polymer molecular dynamics (RPMD), is compared to the centroid molecular dynamics method (CMD). Comparisons of simulation results for liquid para-hydrogen at two state points and liquid ortho-deuterium at one state point are presented. The calculated quantum correlation functions for the two methods are shown to be in good agreement with one another for a large portion of the time spectrum. However, as the quantum mechanical nature of the system increases, RPMD is less accurate in predicting the kinetic energy of the system than is CMD. A simplified and highly efficient algorithm is proposed which largely corrects this deficiency. PMID- 16674215 TI - Growing multiconfigurational potential energy surfaces with applications to X + H2 (X = C,N,O) reactions. AB - A previously developed method, based on a Shepard interpolation procedure to automatically construct a quantum mechanical potential energy surface (PES), is extended to the construction of multiple potential energy surfaces using multiconfigurational wave functions. These calculations are accomplished with the interface of the PES-building program, GROW, and the GAMESS suite of electronic structure programs. The efficient computation of multiconfigurational self consistent field surfaces is illustrated with the C + H2, N + H2, and O + H2 reactions. PMID- 16674216 TI - Cooperating or fighting with decoherence in the optimal control of quantum dynamics. AB - This paper explores the use of laboratory closed-loop learning control to either fight or cooperate with decoherence in the optimal manipulation of quantum dynamics. Simulations of the processes are performed in a Lindblad formulation on multilevel quantum systems strongly interacting with the environment without spontaneous emission. When seeking a high control yield it is possible to find fields that successfully fight with decoherence while attaining a good quality yield. When seeking modest control yields, fields can be found which are optimally shaped to cooperate with decoherence and thereby drive the dynamics more efficiently. In the latter regime when the control field and the decoherence strength are both weak, a theoretical foundation is established to describe how they cooperate with each other. In general, the results indicate that the population transfer objectives can be effectively met by appropriately either fighting or cooperating with decoherence. PMID- 16674217 TI - Multigraining: an algorithm for simultaneous fine-grained and coarse-grained simulation of molecular systems. AB - A method to combine fine-grained and coarse-grained simulations is presented. The coarse-grained particles are described as virtual particles defined by the underlying fine-grained particles are described as virtual particles defined by the underlying fine-grained particles. The contribution of the two grain levels to the interaction between particles is specified by a grain-level parameter lambda. Setting lambda = 0 results in a completely fine-grained simulation, whereas lambda = 1 yields a simulation governed by the coarse-grained potential energy surface with small contributions to keep the fine-grained covalently bound particles together. Simulations at different lambda values may be coupled using the replica-exchange molecular dynamics method to achieve enhanced sampling at the fine-grained level. PMID- 16674218 TI - Improved Wang-Landau sampling through the use of smoothed potential-energy surfaces. AB - A method is presented to improve the speed of convergence of Wang-Landau simulations as used to calculate the density of states of continuous systems. The density of states is first crudely estimated with calculations employing a smoothed potential-energy surface. This estimate is then used as a seed for subsequent Wang-Landau simulations using the original potential. The performance of the method is demonstrated by employing several simple models, including an analytically solvable harmonic system as well as a Go model of a protein. For all systems considered, the seeded simulations were found to converge significantly faster and with higher accuracy than the standard Wang-Landau simulations. PMID- 16674219 TI - Density functionals from many-body perturbation theory: the band gap for semiconductors and insulators. AB - Theoretically the Kohn-Sham band gap differs from the exact quasiparticle energy gap by the derivative discontinuity of the exchange-correlation functional. In practice for semiconductors and insulators the band gap calculated within any local or semilocal density approximations underestimates severely the experimental energy gap. On the other hand, calculations with an "exact" exchange potential derived from many-body perturbation theory via the optimized effective potential suggest that improving the exchange-correlation potential approximation can yield a reasonable agreement between the Kohn-Sham band gap and the experimental gap. The results in this work show that this is not the case. In fact, we add to the exact exchange the correlation that corresponds to the dynamical (random phase approximation) screening in the GW approximation. This accurate exchange-correlation potential provides band structures similar to the local density approximation with the corresponding derivative discontinuity that contributes 30%-50% to the energy gap. Our self-consistent results confirm substantially the results for Si and other semiconductors obtained perturbatively [R. W. Godby et al., Phys. Rev. B 36, 6497 (1987)] and extend the conclusion to LiF and Ar, a wide-gap insulator and a noble-gas solid. PMID- 16674220 TI - Relativistic configuration interaction study of the electronic spectrum of SnTe and SnTe+. AB - Ab initio based relativistic configuration interaction calculations have been performed to study the electronic spectrum of the heaviest tin chalcogenide and its monopositive ion. Potential energy curves and spectroscopic constants of low lying states of both species within 7 eV are reported. The ground-state dissociation energies of SnTe and SnTe+ are computed to be 3.48 and 2.50 eV, respectively. The spin-orbit splitting between the two components of the X 2Pi state of SnTe+ is about 3030 cm(-1). Effects of the strong spin-orbit coupling on the potential curves and spectroscopic properties of both the species are investigated in detail. The electric dipole moments of some of the low-lying states of SnTe and SnTe+ are reported. Transition moments of some important spin allowed and spin-forbidden transitions are calculated from the configuration interaction wave functions. The radiative lifetime of the excited E 1sigma0+(+) state of SnTe is about 39 ns. The X2-X1 transition in SnTe+ is found to be more probable than the similar transition in the lighter ions. The vertical ionization energy of SnTe in the ground state is estimated to be 8.22 eV. PMID- 16674221 TI - Time-resolved photoelectron spectroscopy of proton transfer in the ground state of chloromalonaldehyde: wave-packet dynamics on effective potential surfaces of reduced dimensionality. AB - We report on a simple but widely useful method for obtaining time-independent potential surfaces of reduced dimensionality wherein the coupling between reaction and substrate modes is embedded by averaging over an ensemble of classical trajectories. While these classically averaged potentials with their reduced dimensionality should be useful whenever a separation between reaction and substrate modes is meaningful, their use brings about significant simplification in studies of time-resolved photoelectron spectra in polyatomic systems where full-dimensional studies of skeletal and photoelectron dynamics can be prohibitive. Here we report on the use of these effective potentials in the studies of dump-probe photoelectron spectra of intramolecular proton transfer in chloromalonaldehyde. In these applications the effective potentials should provide a more realistic description of proton-substrate couplings than the sudden or adiabatic approximations commonly employed in studies of proton transfer. The resulting time-dependent photoelectron signals, obtained here assuming a constant value of the photoelectron matrix element for ionization of the wave packet, are seen to track the proton transfer. PMID- 16674222 TI - Vibronic coupling in benzene cation and anion: vibronic coupling and frontier electron density in Jahn-Teller molecules. AB - Vibronic coupling constants of Jahn-Teller molecules, benzene radical cation and anion, are computed as matrix elements of the electronic part of the vibronic coupling operator using the electronic wave functions calculated by generalized restricted Hartree-Fock and state-averaged complete active space self-consistent field methods. The calculated vibronic coupling constants for benzene cation agree well with the experimental and theoretical values. Vibronic coupling density analysis, which illustrates the local properties of the coupling, is performed in order to explain the order of magnitude of the coupling constant from view of the electronic and vibrational structures. This analysis reveals that the couplings of the e2g2 and e2g3 modes in which the large displacements locate on C-C bonds are strong in the cation. On the other hand, they are greatly weakened in the anion because of the decrease of electron density in the region of the C-C bonds, which originates from the antibonding nature of the singly occupied molecular orbital of the anion. However, the difference of the electronic structure has a little influence on the vibronic coupling of the e2g4 mode. These results indicate that the vibronic coupling depends not only on the direction of the nuclear displacement but also on the frontier electron density. PMID- 16674223 TI - Reactive collisions between CH+ and O-. AB - Integral cross sections were measured for two reactions occurring in CH+ + O- collisions: the formation of the carbon monoxide cation CO+ via a reactive ionization process and the formation of the (iso)formyl cation HCO+ (HOC+) via the associative ionization process. Both carbon monoxide and formyl cations are present in the interstellar medium, the latter one being quite abundant in dense clouds. Provided the oxygen anion would also be present in the interstellar environment, the large efficiency of the two reactive processes reported here would justify their inclusion in astrochemical models. The whole set of data was obtained by means of a merged-beam setup operating with keV beams. PMID- 16674224 TI - Interatomic Coulombic decay in a heteroatomic rare gas cluster. AB - Interatomic decay in a heteroatomic rare gas cluster (NeAr) is studied in detail using ab initio electronic structure description and nuclear dynamics simulations. Decay widths of all possible interatomic decay processes are calculated by the recently developed method based on Green's function formalism. Kinetic energy spectra of the electrons emitted in the course of interatomic Coulombic decay (ICD) are simulated for a series of initial vibrational states of the neutral cluster. The effect of the nuclear dynamics on the ICD electron spectra is discussed. PMID- 16674225 TI - Effects of ionization on N-glycylglycine peptide: influence of intramolecular hydrogen bonds. AB - The ionization effects on 28 conformations of N-glycylglycine are analyzed by means of the hybrid B3LYP and the hybrid meta-MPWB1K density functionals and by single-point calculations at the CCSD(T) level of theory. The most favorable process observed corresponds to the ionization of the only neutral conformation that presents a OH...NH2 intramolecular hydrogen bond, which leads to CO2 elimination after a spontaneous proton transfer from -COOH to NH2. The remaining neutral structures evolve to 20 different conformations of N-glycylglycine radical cation, which lie about 25-40 kcal/mol higher than the decarboxylated [NH3CH2CONHCH2]+*...[CO2] complex. Structural changes induced by ionization depend on the intramolecular hydrogen bonds of the initial conformation, since they determine the nature of the electron hole formed. In most cases, ionization takes place at the terminal -NH2 and -CO of the amide bond, which produces a strengthening of the peptide bond and the formation of new -NH2...OC(amide) and NH2...OCOH hydrogen bonds. However, if -NH2 and -CO(amide) simultaneously act as proton acceptor in the neutral conformation, ionization is mainly localized at the carboxylic group, which produces a strengthening of the -COOH...OC(amide) bond. Both functionals lead to similar trends and compare well with CCSD(T) results except for a few cases for which B3LYP provides a too delocalized picture of the electron hole and consequently leads to artificial geometry reorganization. PMID- 16674226 TI - Bonding analysis using localized relativistic orbitals: water, the ultrarelativistic case and the heavy homologues H2X (X = Te, Po, eka-Po). AB - We report the implementation of Pipek-Mezey [J. Chem. Phys. 90, 4916 (1989)] localization of molecular orbitals in the framework of a four-component relativistic molecular electronic structure theory. We have used an exponential parametrization of orbital rotations which allows the use of unconstrained optimization techniques. We demonstrate the strong basis set dependence of the Pipek-Mezey localization criterion and how it can be eliminated. We have employed localization in conjunction with projection analysis to study the bonding in the water molecule and its heavy homologues. We demonstrate that in localized orbitals the repulsion between hydrogens in the water molecule is dominated by electrostatic rather than exchange interactions and that freezing the oxygen 2s orbital blocks polarization of this orbital rather than hybridization. We also point out that the bond angle of the water molecule cannot be rationalized from the potential energy alone due to the force term of the molecular virial theorem that comes into play at nonequilibrium geometries and which turns out to be crucial in order to correctly reproduce the minimum of the total energy surface. In order to rapidly assess the possible relativistic effects we have carried out the geometry optimizations of the water molecule at various reduced speed of light with and without spin-orbit interaction. At intermediate speeds, the bond angle is reduced to around 90 degrees , as is known experimentally for H(2)S and heavier homologues, although our model of ultrarelativistic water by construction does not allow any contribution from d orbitals to bonding. At low speeds of light the water molecule becomes linear which is in apparent agreement with the valence shell electron pair repulsion (VSEPR) model since the oxygen 2s12 and 2p12 orbitals both become chemically inert. However, we show that linearity is brought about by the relativistic stabilization of the (n + 1)s orbital, the same mechanism that leads to an electron affinity for eka-radon. Actual calculations on the series H2X (X = Te, Po, eka-Po) show the spin-orbit effects for the heavier species that can be rationalized by the interplay between SO-induced bond lengthening and charge transfer. Finally, we demonstrate that although both the VSEPR and the more recent ligand close packing model are presented as orbital free models, they are sensitive to orbital input. For the series H2X (X = O, S, Se, Te) the ligand radius of the hydrogen can be obtained from the covalent radius of the central atom by the simple relation r(lig)(H) = 0.67r(cov)(X) + 27 (in picometers). PMID- 16674227 TI - Ab initio study of the electronic structure of manganese carbide. AB - We report electronic structure calculations on 13 states of the experimentally unknown manganese carbide (MnC) using standard multireference configuration interaction (MRCI) methods coupled with high quality basis sets. For all states considered we have constructed full potential energy curves and calculated zero point energies. The X state, correlating to ground state atoms, is of 4sigma- symmetry featuring three bonds, with a recommended dissociation energy of D0 = 70.0 kcal/mol and r(e) = 1.640 angstroms. The first and second excited states, which also correlate to ground state atoms, are of 6sigma- and 8sigma- symmetry, respectively, and lie 17.7 and 28.2 kcal/mol above the X state at the MRCI level of theory. PMID- 16674228 TI - Interaction-induced dipole moment of the Ar-H2 dimer: dependence on the H2 bond length. AB - We present ab initio calculations of the interaction-induced dipole moment of the Ar-H2 van der Waals dimer. The primary focus of our calculations is on the H2 bond length dependence of the dipole moment, which determines the intensities of both the collision-induced H2 upsilon = 1 <-- 0 fundamental band in gaseous Ar-H2 mixtures and the dopant-induced H2 upsilon = 1 <-- 0 absorption feature in Ar doped solid H2 matrices. Our calculations employ large atom-centered basis sets, diffuse bond functions positioned between the two monomers, and a coupled cluster treatment of valence electron correlation; core-valence correlation effects appear to make negligible contributions to the interaction-induced dipole moment for the Ar-H2 configurations considered here. PMID- 16674229 TI - Relative stability of planar versus double-ring tubular isomers of neutral and anionic boron cluster B20 and B20-. AB - High-level ab initio molecular-orbital methods have been employed to determine the relative stability among four neutral and anionic B20 isomers, particularly the double-ring tubular isomer versus three low-lying planar isomers. Calculations with the fourth-order Moller-Plessset perturbation theory [MP4(SDQ)] and Dunning's correlation consistent polarized valence triple zeta basis set as well as with the coupled-cluster method including single, double, and noniteratively perturbative triple excitations and the 6-311G(d) basis set show that the double-ring tubular isomer is appreciably lower in energy than the three planar isomers and is thus likely the global minimum of neutral B20 cluster. In contrast, calculations with the MP4(SDQ) level of theory and 6-311+G(d) basis set show that the double-ring anion isomer is appreciably higher in energy than two of the three planar isomers. In addition, the temperature effects on the relative stability of both 10B20- and 11B20- anion isomers are examined using the density functional theory. It is found that the three planar anion isomers become increasingly more stable than the double-ring isomer with increasing the temperature. These results are consistent with the previous conclusion based on a joint experimental/simulated anion photoelectron spectroscopy study [B. Kiran et al., Proc. Natl. Acad. Sci. U.S.A. 102, 961 (2005)], that is, the double-ring anion isomer is notably absent from the experimental spectra. The high stability of the double-ring neutral isomer of B20 can be attributed in part to the strong aromaticity as characterized by its large negative nucleus-independent chemical shift. The high-level ab initio calculations suggest that the planar-to-tubular structural transition starts at B20 for neutral clusters but should occur beyond the size of B20- for the anion clusters. PMID- 16674230 TI - Structural, electronic, and chemical properties of multiply iodized aluminum clusters. AB - The electronic structure, stability, and reactivity of iodized aluminum clusters, which have been investigated via reactivity studies, are examined by first principles gradient corrected density functional calculations. The observed behavior of Al13I(x)- and Al14I(x)- clusters is shown to indicate that for x < or = 8, they consist of compact Al13- and Al14++ cores, respectively, demonstrating that they behave as halogen- or alkaline earth-like superatoms. For x > 8, the Al cores assume a cagelike structure associated with the charging of the cores. The observed mass spectra of the reacted clusters reveal that Al13I(x)- species are more stable for even x while Al14I(x)- exhibit enhanced stability for odd x(x > or = 3). It is shown that these observations are linked to the formation and filling of "active sites," demonstrating a novel chemistry of superatoms. PMID- 16674231 TI - Comprehensive theoretical studies on the low-lying electronic states of NiF, NiCl, NiBr, and NiI. AB - The low-lying electronic states of the nickel monohalides, i.e., NiF, NiCl, NiBr, and NiI, are investigated by using multireference second-order perturbation theory with relativistic effects taken into account. For the energetically lowest 11 lambda-S states and 26 omega states there into, the potential energy curves and corresponding spectroscopic constants (vertical and adiabatic excitation energies, equilibrium bond lengths, vibrational frequencies, and rotational constants) are reported. The calculated results are grossly in very good agreement with those solid experimental data. In particular, the ground state of NiI is shown to be different from those of NiF, NiCl, and NiBr, being in line with the recent experimental observation. Detailed analyses are provided on those states that either have not been assigned or have been incorrectly assigned by previous experiments. PMID- 16674232 TI - Special stability of cationic MPb12+ clusters and superalkali character of neutral MPb12 clusters (M = B, Al, Ga, In, and Tl). AB - The electronic structures and stabilities of cationic MPb12+ clusters (M = B, Al, Ga, In, and Tl) with 50 valence electrons are investigated within density functional theory. It is shown that, at the B3LYP/cc-pVDZ(-PP) and BPW91/cc-pVDZ( PP) levels of theory, the structures of MPb12+ with icosahedra (I(h)) symmetry are energetically favorable, and their high stabilities may arise from the closed shell nature of the pi subsystems which are subject to the 2(N(pi + 1)2 rule with N(pi = 1). In addition, the possessing of large nucleus-independent chemical shifts of the five kinds of clusters reflects the common aromatic character of these clusters. From the comparison of our studies on the binding energies and the highest occupied molecular orbital and the lowest unoccupied molecular orbital energy gaps, the cluster AlPb12+ has higher stability than the others and this is consistent with the recent mass-spectrometric discovery of Al-doped Pb(n)+ clusters, in which AlPb12+ is highly abundant. The same methods are used to search for the structures of the neutral MPb12 clusters. The calculations reveal that the most stable geometries of the BPb12 and GaPb12 clusters have I(h) symmetry, the AlPb12 and InPb12 clusters have T(h) symmetry, and the TlPb12 cluster has C5v symmetry. Furthermore, the vertical ionization potentials of the neutral MPb12 clusters are smaller than that of some alkali atoms, indicating that the neutral MPb12 clusters possess superalkali character. PMID- 16674233 TI - Quantum mechanical and quasiclassical trajectory scattering calculations for the C(1D) + H2 reaction on the second excited 1 1A" potential energy surface. AB - Time-independent quantum mechanical (QM) and quasiclassical trajectory (QCT) scattering calculations have been carried out for the C(1D) + H2 --> CH + H reaction at a collision energy of 80 meV on a newly developed ab initio potential energy surface [B. Bussery-Honvault et al., Phys. Chem. Chem. Phys. 7, 1476 (2005)] of 1 1A" symmetry, corresponding to the second singlet state 1 1B1 of CH2. A general good agreement has been found between the QM and QCT rotational distributions and differential cross sections (DCSs). In both cases, DCSs are strongly peaked in the forward direction with a small contribution in the backward direction in contrast with those obtained on the 1 1A' surface, which are nearly symmetric. Rotational distributions obtained on the 1 1A" surface are somewhat colder than those calculated on the 1 1A' surface. The specific dynamics and the contribution of the 1 1A" surface to the overall reactivity of this system are discussed. PMID- 16674234 TI - Ab initio study on deactivation pathways of excited 9H-guanine. AB - The complete active space with second-order perturbation theory/complete active space self-consistent-field method was used to explore the nonradiative decay mechanism for excited 9H-guanine. On the 1pipi* (1L(a)) surface we determined a conical intersection (CI), labeled (S0pipi*)(CI), between the 1pipi* (1L(a)) excited state and the ground state, and a minimum, labeled (pipi*)min. For the 1pipi* (1L(a)) state, its probable deactivation path is to undergo a spontaneous relaxation to (pipi*)min first and then decay to the ground state through (S0pipi*)(CI), during which a small activation energy is required. On the 1n(N)pi* surface a CI between the 1n(N)pi* and 1pipi* (1L(a)) states was located, which suggests that the 1n(N)pi* excited state could transform to the 1pipi* (1L(a)) excited state first and then follow the deactivation path of the 1pipi* (1L(a)) state. This CI was also possibly involved in the nonradiative decay path of the second lowest 1pipi* (1L(b)) state. On the 1n(O)pi* surface a minimum was determined. The deactivation of the 1n(O)pi* state to the ground state was estimated to be energetically unfavorable. On the 1pisigma* surface, the dissociation of the N-H bond of the six-membered ring is difficult to occur due to a significant barrier. PMID- 16674235 TI - The dependence of low-energy electron attachment to CF3Br on electron and vibrational energy. AB - In a joint experimental and theoretical effort, we have studied dissociative electron attachment (DEA) to the CF3Br molecule at electron energies below 2 eV. Using two variants of the laser photoelectron attachment method with a thermal gas target (T(G) = 300 K), we measured the energy dependent yield for Br- formation over the range E = 3-1200 meV with resolutions of about 3 meV (E < 200 meV) and 35 meV. At the onsets for excitation of one and two quanta for the C-Br stretching mode nu3, downward cusps are detected. With reference to the recommended thermal (300 K) attachment rate coefficient k(A)(CF3Br) = 1.4 x 10( 8) cm3 s(-1), absolute cross sections have been determined for Br- formation. In addition, we studied Br- and (CF3Br)Br- formations with a seeded supersonic target beam (10% CF3Br in helium carrier gas, with a stagnation pressure of 1-4 bars and nozzle temperatures of 300 and 600 K) and found prominent structure in the anion yields due to cluster formation. Using the microwave pulse radiolysis swarm technique, allowing for controlled variation of the electron temperature by microwave heating, we studied the dependence of the absolute DEA rate coefficient on the mean electron energy E over the range of 0.04-2 eV at gas temperatures T(G) ranging from 173 to 600 K. For comparison with the experimental results, semiempirical resonance R-matrix calculations have been carried out. The input for the theory includes the known energetic and structural parameters of the neutral molecule and its anion; the parameters of the resonant anion curves are chosen with reference to the known thermal rate coefficient for the DEA process. For the gas temperature T(G) = 300 K, good overall agreement of the theoretical DEA cross section with the experimental results is observed; moreover, rate coefficients for Br- formation due to Rydberg electron transfer, calculated with both the experimental and the theoretical DEA cross sections, are found to agree with the previously reported absolute experimental values. At T(G) = 300 K, satisfactory agreement is also found between the calculated and experimental attachment rate coefficients for mean electron energies E = 0.04-2 eV. The strong increase of the measured rate coefficients with rising gas temperature, however, could be only partially recovered by the R-matrix results. The differences may result from the influence of thermal excitations of other vibrational modes not included in the theory. PMID- 16674236 TI - The transient dynamics leading to spin turbulence in high-field solution magnetic resonance: a numerical study. AB - The dynamics under the joint action of radiation damping and the distant dipolar field in high-field solution magnetic resonance are investigated. Different dynamical regimes during the evolution are identified and their individual features are discussed. In the steady state, the dynamics can be associated with a strange attractor in phase space on which the motion is chaotic. The possibility of the observed chaotic motion being spatiotemporal is examined. PMID- 16674237 TI - Excluded volume in the generic van der Waals equation of state and the self diffusion coefficient of the Lennard-Jones fluid. AB - In the previous papers applying the generic van der Waals equation of state the mean excluded volume was defined with the contact diameter of particles at which the potential energy is equal to zero-the size parameter in the case of the Lennard-Jones potential. This parameter appears as the upper limit of the integral for the generic van der Waals parameter B (mean excluded volume divided by the density) in the generic van der Waals equation of state. Since the choice is not unique, in this paper we reexamine the manner of defining the upper limit and propose another choice for the upper limit. We also propose an interpretation of the free volume overlap factor alpha appearing in the free volume theory of diffusion and a method of estimating it in terms of the intermolecular potential energy only. It is shown that with the so-estimated free volume overlap factor and the new choice of the upper limit of the integral for B the self-diffusion coefficient in the modified free volume theory of diffusion not only acquires a better accuracy than before, but also becomes calculable in terms of only the intermolecular interaction potential without an adjustable parameter. We also assess some of effective diameters of molecules proposed in the literature for their ability to predict the self-diffusion coefficient within the framework of the modified free volume theory of diffusion. PMID- 16674238 TI - Fluid-phase behavior of binary mixtures in which one component can have two critical points. AB - We investigate theoretically the binary fluid-phase behavior of mixtures in which one water-like component can have two critical points. We consider three equal sized nonpolar solutes that differ in the strength of their dispersive interactions (a1 < a2 < a3, where a denotes the van der Waals attractive parameter). In each case, we compare the phase behavior predicted using two sets of parameters for water: one giving rise to a pure component low-temperature liquid-liquid transition terminating at a critical point (two-critical-point parameter set), and one in which no such second critical point exists (singularity-free parameter set). Regardless of the parameter values used, we find five mixture critical lines. Using the two-critical-point parameter set, we find that a critical line originates at water's second critical point for aqueous mixtures involving solutes 1, 2, or 3. For mixtures involving solutes 1 or 2, this line extends towards low pressures and high temperatures as the solute mole fraction increases, and is closely related to the critical line originating at water's ordinary vapor-liquid critical point: these two critical lines are loci of upper and lower consolute points corresponding to the same liquid-liquid transition. In mixtures involving solute 2, the critical locus emanating from water's second critical point is shifted to higher temperatures compared to mixtures involving solute 1, and extends up to T approximately 310 K at moderate pressures (ca. 200 bars). This suggests the possibility of an experimentally accessible manifestation of the existence of a second critical point in water. For binary mixtures involving solutes 1 or 2, changing the water parameters from the two critical points to the singularity-free case causes the disappearance of a lower consolute point at moderate pressures. For binary mixtures involving solute 3, the differences between two-critical-point and singularity-free behaviors occur only in the experimentally difficult-to-probe low-temperature and high-pressure region. PMID- 16674239 TI - Molecular dynamics simulations of melting and the glass transition of nitromethane. AB - Molecular dynamics simulations have been used to investigate the thermodynamic melting point of the crystalline nitromethane, the melting mechanism of superheated crystalline nitromethane, and the physical properties of crystalline and glassy nitromethane. The maximum superheating and glass transition temperatures of nitromethane are calculated to be 316 and 160 K, respectively, for heating and cooling rates of 8.9 x 10(9) Ks. Using the hysteresis method [Luo et al., J. Chem. Phys. 120, 11640 (2004)] and by taking the glass transition temperature as the supercooling temperature, we calculate a value of 251.1 K for the thermodynamic melting point, which is in excellent agreement with the two phase result [Agrawal et al., J. Chem. Phys. 119, 9617 (2003)] of 255.5 K and measured value of 244.73 K. In the melting process, the nitromethane molecules begin to rotate about their lattice positions in the crystal, followed by translational freedom of the molecules. A nucleation mechanism for the melting is illustrated by the distribution of the local translational order parameter. The critical values of the Lindemann index for the C and N atoms immediately prior to melting (the Lindemann criterion) are found to be around 0.155 at 1 atm. The intramolecular motions and molecular structure of nitromethane undergo no abrupt changes upon melting, indicating that the intramolecular degrees of freedom have little effect on the melting. The thermal expansion coefficient and bulk modulus are predicted to be about two or three times larger in crystalline nitromethane than in glassy nitromethane. The vibrational density of states is almost identical in both phases. PMID- 16674240 TI - A general perturbation approach for equation of state development: applications to simple fluids, ab initio potentials, and fullerenes. AB - A new perturbation scheme based on the Barker-Henderson perturbation theory [J. Chem. Phys. 47, 4714 (1967)] is proposed to predict the thermodynamic properties of spherical molecules. Accurate predictions of second virial coefficients and vapor-liquid coexistence properties are obtained for a large variety of potential functions (square well, Yukawa, Sutherland, Lennard-Jones, Buckingham, Girifalco). New Gibbs ensemble Monte Carlo simulations of the generalized exp-m Buckingham potential are reported. An extension of the perturbation approach to mixtures is proposed, and excellent predictions of vapor-liquid equilibria are obtained for Lennard-Jones mixtures. The perturbation scheme can be applied to complex potential functions fitted to ab initio data to predict the properties of real molecules such as neon. The new approach can also be used as an auxiliary tool in molecular simulation studies, to efficiently optimize an intermolecular potential on macroscopic properties or match force fields based on different potential functions. PMID- 16674241 TI - A new generalization of the Carnahan-Starling equation of state to additive mixtures of hard spheres. AB - We introduce an expansion of the equation of state for additive hard-sphere mixtures in powers of the total packing fraction with coefficients which depend on a set of weighted densities used in scaled particle theory and fundamental measure theory. We demand that the mixture equation of state recovers the quasiexact Carnahan-Starling [J. Chem. Phys. 51, 635 (1969)] result in the case of a one-component fluid and show from thermodynamic considerations and consistency with an exact scaled particle relation that the first and second orders of the expansion lead unambiguously to the Boublik-Mansoori-Carnahan Starling-Leland [J. Chem. Phys. 53, 471 (1970); J. Chem. Phys. 54, 1523 (1971)] equation and the extended Carnahan-Starling equation introduced by Santos et al. [Mol. Phys. 96, 1 (1999)]. In the third order of the expansion, our approach allows us to define a new equation of state for hard-sphere mixtures which we find to be more accurate than the former equations when compared to available computer simulation data for binary and ternary mixtures. Using the new mixture equation of state, we calculate expressions for the surface tension and excess adsorption of the one-component fluid at a planar hard wall and compare its predictions to available simulation data. PMID- 16674242 TI - Heat capacity of tetrahydrofuran clathrate hydrate and of its components, and the clathrate formation from supercooled melt. AB - We report a thermodynamic study of the formation of tetrahydrofuran clathrate hydrate by explosive crystallization of water-deficient, near stoichiometric, and water-rich solutions, as well as of the heat capacity, C(p), of (i) supercooled tetrahydrofuran-H2O solutions and of the clathrate hydrate, (ii) tetrathydrofuran (THF) liquid, and (iii) supercooled water and the ice formed on its explosive crystallization. In explosive freezing of supercooled solutions at a temperature below 257 K, THF clathrate hydrate formed first. The nucleation temperature depends on the cooling rate, and excess water freezes on further cooling. The clathrate hydrate melts reversibly at 277 K and C(p) increases by 770 J/mol K on melting. The enthalpy of melting is 99.5 kJ/mol and entropy is 358 J/mol K. Molar C(p) of the empty host lattice is less than that of the ice, which is inconsistent with the known lower phonon frequency of H2O in the clathrate lattice. Analysis shows that C(p) of THF and ice are not additive in the clathrate. C(p) of the supercooled THF-H2O solutions is the same as that of water at 247 K, but less at lower temperatures and more at higher temperatures. The difference tends to become constant at 283 K. The results are discussed in terms of the hydrogen-bonding changes between THF and H2O. PMID- 16674243 TI - Adding salt to an aqueous solution of t-butanol: is hydrophobic association enhanced or reduced? AB - Recent neutron scattering experiments on aqueous salt solutions of amphiphilic t butanol by Bowron and Finney [Phys. Rev. Lett. 89, 215508 (2002); J. Chem. Phys. 118, 8357 (2003)] suggest the formation of t-butanol pairs, bridged by a chloride ion via O-H...Cl- hydrogen bonds, leading to a reduced number of intermolecular hydrophobic butanol-butanol contacts. Here we present a joint experimental/theoretical study on the same system, using a combination of molecular dynamics (MD) simulations and nuclear magnetic relaxation measurements. Both MD simulation and experiment clearly support the more classical scenario of an enhanced number of hydrophobic contacts in the presence of salt, as it would be expected for purely hydrophobic solutes. [T. Ghosh et al., J. Phys. Chem. B 107, 612 (2003)]. Although our conclusions arrive at a structurally completely distinct scenario, the molecular dynamics simulation results are within the experimental error bars of the Bowron and Finney data. PMID- 16674244 TI - Ion association in aqueous LiCl solutions at high concentration: predicted results via molecular simulation. AB - We perform molecular dynamics simulations to study the ionic solvation and association behavior in concentrated aqueous LiCl solutions at ambient conditions, including consideration of expected signatures of ion pairing that might be found in neutron diffraction experiments with isotopic substitution. The ten possible pair radial distribution functions that define the microstructure of the systems are determined and used to assess the first-order difference of the neutron-weighted correlation functions for these solutions in heavy and null water. Then, both sets of correlation functions are applied to the interpretation of the ion's local environment in terms of the location of the relevant peaks and the penetration of ions into the counterion solvation shells as a signature of ion-pair formation. Finally, we illustrate how first-order difference experiments involving null and heavy water might be used to assess the magnitude of the M(v+) - X(v-) ion-pair formation for a salt M(v+)X(n) v- in an aqueous solution, provided the significant experimental challenges in these studies could be overcome. PMID- 16674245 TI - Ion dynamics in compacted clays: derivation of a two-state diffusion-reaction scheme from the lattice Fokker-Planck equation. AB - We show how a two-state diffusion-reaction description of the mobility of ions confined within compacted clays can be constructed from the microscopic dynamics of ions in an external field. The diffusion-reaction picture provides the usual interpretation of the reduced ionic mobility in clays, but the required partitioning coefficient K(d) between trapped and mobile ions is generally an empirical parameter. We demonstrate that it is possible to obtain K(d) from the microscopic dynamics of ions interacting with the clay surfaces by evaluating the ionic mobility using a novel lattice implementation of the Fokker-Planck equation. The resulting K(d) allows a clear-cut characterization of the trapping sites on the clay surfaces and determines the adsorption/desorption rates. The results highlight the limitations of standard approximation schemes and pinpoint the crossover from jump to Brownian diffusion regimes. PMID- 16674246 TI - Adsorption of pentacene on filled d-band metal surfaces: long-range ordering and adsorption energy. AB - The growth of pentacene on suitable metallic templates is studied by means of low energy electron diffraction and ultraviolet photoelectron spectroscopy. Highly ordered pentacene single layers can be prepared by deposition on filled d-band metal templates kept at 370 K. The presence of the steps for the Cu(119) vicinal surface and of the Au troughs for the Au(110)-(1 x 2) surface allows the formation of commensurate long-range ordered structures with (3 x 7) and (3 x 6) periodicities, respectively. A detailed analysis of the molecular induced electronic states evolution is performed for different growth morphologies. The adsorption energy of the ordered molecular single layers on the Au(110) surface is lower (1.90 eV) than on the Cu vicinal surface (2.36 eV), where the steps enhance the molecule adsorption energy. PMID- 16674247 TI - Exact phase boundary of an amphiphile-rich phase in a ternary solution on a Bethe lattice. AB - A model ternary solution is considered in which the bonds of a three-coordinate Bethe lattice are covered by rodlike molecules of types AA, BB, and AB, the AB type molecule representing an amphiphile. The phase boundary in temperature composition space of an ordered amphiphile-rich phase is calculated exactly. PMID- 16674248 TI - Electrical transport measurements on self-assembled organic molecular wires. AB - The electrical properties of supermolecular assemblies of oligo(p-phenylene vinylene) were studied. These materials self-assemble into well-defined cylindrical structures in solution with lengths in the range of 100 nm-10 microm and diameters between 5 and 200 nm. Atomic force microscopy showed that by adjusting the concentration, either individual molecular wires or a dense film could be deposited. The molecular wires showed poor electrical conduction. Several tests were performed that show that it was the molecular wires themselves, not the contacts, that limit the conductivity. PMID- 16674249 TI - Atomistic lattice-gas modeling of CO oxidation on Pd(100): temperature-programmed spectroscopy and steady-state behavior. AB - We have developed an atomistic lattice-gas model for the catalytic oxidation of CO on single-crystal Pd(100) surfaces under ultrahigh vacuum conditions. This model necessarily incorporates an detailed description of adlayer ordering and adsorption-desorption kinetics both for CO on Pd(100), and for oxygen on Pd(100). Relevant energetic parameters are determined by comparing model predictions with experiment, together with some guidance from density functional theory calculations. The latter also facilitates description of the interaction and reaction of adsorbed CO and oxygen. Kinetic Monte Carlo simulations of this reaction model are performed to predict temperature-programmed reaction spectra, as well as steady-state bifurcation behavior. PMID- 16674250 TI - A novel algorithm to model the influence of host lattice flexibility in molecular dynamics simulations: loading dependence of self-diffusion in carbon nanotubes. AB - We describe a novel algorithm that includes the effect of host lattice flexibility into molecular dynamics simulations that use rigid lattices. It uses a Lowe-Andersen thermostat for interface-fluid collisions to take the most important aspects of flexibility into account. The same diffusivities and other properties of the flexible framework system are reproduced at a small fraction of the computational cost of an explicit simulation. We study the influence of flexibility on the self-diffusion of simple gases inside single walled carbon nanotubes. Results are shown for different guest molecules (methane, helium, and sulfur hexafluoride), temperatures, and types of carbon nanotubes. We show, surprisingly, that at low loadings flexibility is always relevant. Notably, it has a crucial influence on the diffusive dynamics of the guest molecules. PMID- 16674251 TI - A QUICKSTEP-based quantum mechanics/molecular mechanics approach for silica. AB - Quantum mechanics/molecular mechanics (QM/MM) approaches are currently used to describe several properties of silica-based systems, which are local in nature and require a quantum description of only a small number of atoms around the site of interest, e.g., local chemical reactivity or spectroscopic properties of point defects. We present a QM/MM scheme for silica suitable to be implemented in the general QM/MM framework recently developed for large scale molecular dynamics simulations, within the QUICKSTEP approach to the description of the quantum region. Our scheme has been validated by computing the structural and dynamical properties of an oxygen vacancy in alpha-quartz, a prototypical defect in silica. We have found that good convergence in the Si-Si bond length and formation energy is achieved by using a quantum cluster of only eight atoms in size. We check the suitability of the method for molecular dynamics and evaluate the Si-Si bond frequency from the velocity-velocity correlation function. PMID- 16674252 TI - Finite-size effects in the microscopic structure of a hard-sphere fluid in a narrow cylindrical pore. AB - We examine the microscopic structure of a hard-sphere fluid confined to a small cylindrical pore by means of Monte Carlo simulation. In order to analyze finite size effects, the simulations are carried out in the framework of different statistical mechanics ensembles. We find that the size effects are specially relevant in the canonical ensemble where noticeable differences are found with the results in the grand canonical ensemble (GCE) and the isothermal isobaric ensemble (IIE) which, in most situations, remain very close to the infinite system results. A customary series expansion in terms of fluctuations of either the number of particles (GCE) or the inverse volume (IIE) allows us to connect with the results of the canonical ensemble. PMID- 16674253 TI - Screened hybrid density functionals applied to solids. AB - Hybrid Fock exchange/density functional theory functionals have shown to be very successful in describing a wide range of molecular properties. For periodic systems, however, the long-range nature of the Fock exchange interaction and the resultant large computational requirements present a major drawback. This is especially true for metallic systems, which require a dense Brillouin zone sampling. Recently, a new hybrid functional [HSE03, J. Heyd, G. E. Scuseria, and M. Ernzerhof, J. Chem. Phys. 118, 8207 (2003)] that addresses this problem within the context of methods that evaluate the Fock exchange in real space was introduced. We discuss the advantages the HSE03 functional brings to methods that rely on a reciprocal space description of the Fock exchange interaction, e.g., all methods that use plane wave basis sets. Furthermore, we present a detailed comparison of the performance of the HSE03 and PBE0 functionals for a set of archetypical solid state systems by calculating lattice parameters, bulk moduli, heats of formation, and band gaps. The results indicate that the hybrid functionals indeed often improve the description of these properties, but in several cases the results are not yet on par with standard gradient corrected functionals. This concerns in particular metallic systems for which the bandwidth and exchange splitting are seriously overestimated. PMID- 16674254 TI - Diffusion in multilayer media: transient behavior of the lateral diffusion coefficient. AB - A general formalism for treating lateral diffusion in a multilayer medium is developed. The formalism is based on the relation between the lateral diffusion and the distribution of the cumulative residence time, which the diffusing particle spends in different layers. We exploit this fact to derive general expressions which give the global and local time-dependent diffusion coefficients in terms of the average cumulative times spent by the particle in different layers and the probabilities of finding the particle in different layers, respectively. These expressions are used to generalize two recently obtained results: (a) A solution for the short-time behavior of the lateral diffusion coefficient in two layers separated by a permeable membrane obtained by a perturbation theory is extended to the entire range of time. (b) A solution for the time-dependent diffusion coefficient of a ligand, which repeatedly dissociates and rebinds to sites on a planar surface, obtained under the assumption that the medium above the surface is infinite, is generalized to allow for the medium layer of finite thickness. For the latter problem we derive an expression for the Fourier-Laplace transform of the propagator in terms of the double Laplace transform of the probability density of the cumulative residence time spent by the ligand in the medium layer. PMID- 16674255 TI - Selectivity principle of the ligand escape process from a two-gate tunnel in myoglobin: molecular dynamics simulation. AB - We proposed a selectivity principle for the ligand escape process from two fluctuating bottlenecks in a cavity with a multigate inside a myoglobin pocket. Our previous analytical theory proposed a fluctuating bottleneck model for a Brownian particle passing through two gates on a cavity surface of an enzyme protein and has determined the escape rate in terms of the time-dependent gate function and the competition effect. It illustrated that with two (or more than two) gates on a cavity surface the gate modulation, which is controlled by protein fluctuation, dominates the ligand escape pathway. We have performed a molecular dynamics simulation to investigate the selectivity principle of the ligand escape process from two-gate tunnel in myoglobin. The simulation results confirm our theoretical conjecture. It indicates that the escape process is actually entropy driven, and the ligand escape pathway is chosen via the gate modulation. This suggests an interesting intrinsic property, that is, the oxymyoglobin tertiary structure is favorable to the departure of the ligand from one direction rather than through a biased random walk. PMID- 16674256 TI - Theory of the statistics of kinetic transitions with application to single molecule enzyme catalysis. AB - Single-molecule spectroscopy can monitor transitions between two microscopic states when these transitions are associated with the emission of photons. A general formalism is developed for obtaining the statistics of such transitions from a microscopic model when the dynamics is described by master or rate equations or their continuum analog, multidimensional reaction-diffusion equations. The focus is on the distribution of the number of transitions during a fixed observation time, the distribution of times between transitions, and the corresponding correlation functions. It is shown how these quantities are related to each other and how they can be explicitly calculated in a straightforward way for both immobile and diffusing molecules. Our formalism reduces to renewal theory when the monitored transitions either go to or originate from a single state. The influence of dynamics slow compared with the time between monitored transitions is treated in a simple way, and the probability distributions are expressed in terms of Mandel-type formulas. The formalism is illustrated by a detailed analysis of the statistics of catalytic turnovers of enzymes. When the rates of conformational changes are slower than the catalytic rates which are in turn slower than the binding relaxation rate, (1) the mean number of turnovers is shown to have the classical Michaelis-Menten form, (2) the correlation function of the number of turnovers is a direct measure of the time scale of catalytic rate fluctuations, and (3) the distribution of the time between consecutive turnovers is determined by the steady-state distribution. PMID- 16674257 TI - Void-induced dissolution in molecular dynamics simulations of NaCl and water. AB - To gain a better understanding of the interaction of water and NaCl at the surface during dissolution, we have used molecular dynamics to simulate the interface with two equal-sized slabs of solid NaCl and liquid water in contact. The introduction of voids in the bulk of the salt, as well as steps or pits on the surface of the NaCl slab results in a qualitative change of system structure, as defined by radial distribution functions (RDFs). As an example, the characteristic Na-Na RDF for the system changes from regularly spaced narrow peaks (corresponding to an ordered crystalline structure), to a broad primary and smaller secondary peak (corresponding to a disordered structure). The change is observed at computationally short time scales of 100 ps, in contrast with a much longer time scale of 1 mus expected for complete mixing in the absence of defects. The void fraction (which combines both bulk and surface defects) required to trigger dissolution varies between 15%-20% at 300 K and 1 atm, and has distinct characteristics for the physical breakdown of the crystal lattice. The void fraction required decreases with temperature. Sensitivity studies show a strong dependence of the critical void fraction on the quantity and distribution of voids on the surface, with systems containing a balanced number of surface defects and a rough surface showing a maximum tendency to dissolve. There is a moderate dependence on temperature, with a 5% decrease in required void fraction with a 100 K increase in temperature, and a weak dependence on water potential model used, with the SPC, SPC/E, TIP4P, and RPOL models giving qualitatively identical results. The results were insensitive to the total quantity of water available for dissolution and the duration of the simulation. PMID- 16674258 TI - Surface tension in bilayer membranes with fixed projected area. AB - We study the elastic response of bilayer membranes with fixed projected area to both the stretching and shape deformations. A surface tension is associated to each of these deformations. By using model amphiphilic membranes and computer simulations, we are able to observe both the types of deformation, and thus, both the surface tensions, related to each type of deformation, are measured for the same system. These surface tensions are found to assume different values in the same bilayer membrane, in particular, they vanish for different values of the projected area. We introduce a simple theory which relates the two quantities and successfully apply it to the data obtained with computer simulations. PMID- 16674259 TI - Dynamic bond constraints in protein Langevin dynamics. AB - Bond constraint algorithms for molecular dynamics typically take, as the target constraint lengths, the values of the equilibrium bond lengths defined in the potential. In Langevin form, the equations of motion are temperature dependent, which gives the average value for the individual bond lengths a temperature dependence. In addition to this, locally constant force fields can shift the local equilibrium bond lengths. To restore the average bond lengths in constrained integration to their unconstrained values, we suggest changing the constraint length used by popular constraint methods such as RATTLE [H. C. Andersen, J. Comput. Phys. 52, 23 (1983)] at each step. This allows us to more accurately capture the equilibrium bond length changes (with respect to the potential) due to the local equilibration and temperature effects. In addition, the approximations to the unconstrained nonbonded energies are closer using the dynamic constraint method than a traditional fixed constraint algorithm. The mechanism for finding the new constrained lengths involves one extra calculation of the bonded components of the force, and therefore adds O(N) time to the constraint algorithm. Since most molecular dynamics calculations are dominated by the O(N2) nonbonded forces, this new method does not take significantly more time than a fixed constraint algorithm. PMID- 16674260 TI - Entropy and enthalpy of polyelectrolyte complexation: Langevin dynamics simulations. AB - We report a systematic study by Langevin dynamics simulation on the energetics of complexation between two oppositely charged polyelectrolytes of same charge density in dilute solutions of a good solvent with counterions and salt ions explicitly included. The enthalpy of polyelectrolyte complexation is quantified by comparisons of the Coulomb energy before and after complexation. The entropy of polyelectrolyte complexation is determined directly from simulations and compared with that from a mean-field lattice model explicitly accounting for counterion adsorption. At weak Coulomb interaction strengths, e.g., in solvents of high dielectric constant or with weakly charged polyelectrolytes, complexation is driven by a negative enthalpy due to electrostatic attraction between two oppositely charged chains, with counterion release entropy playing only a subsidiary role. In the strong interaction regime, complexation is driven by a large counterion release entropy and opposed by a positive enthalpy change. The addition of salt reduces the enthalpy of polyelectrolyte complexation by screening electrostatic interaction at all Coulomb interaction strengths. The counterion release entropy also decreases in the presence of salt, but the reduction only becomes significant at higher Coulomb interaction strengths. More significantly, in the range of Coulomb interaction strengths appropriate for highly charged polymers in aqueous solutions, complexation enthalpy depends weakly on salt concentration and counterion release entropy exhibits a large variation as a function of salt concentration. Our study quantitatively establishes that polyelectrolyte complexation in highly charged Coulomb systems is of entropic origin. PMID- 16674261 TI - Folding thermodynamics of pseudoknotted chain conformations. AB - We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. PMID- 16674262 TI - Describing the component dynamics in miscible polymer blends: towards a fully predictive model. AB - We have recently proposed [D. Cangialosi et al., J. Chem. Phys. 123, 144908 (2005)] an extension of the Adam-Gibbs [J. Chem. Phys. 43, 139 (1965)] theory, combined with the concept of self-concentration, to describe the temperature dependence of the relaxation time for the component segmental dynamics in miscible polymer blends. Thus, we were able to obtain the dynamics of each component in the blend starting from the knowledge of the dynamic and thermodynamic data of the pure polymers, with a single fitting parameter (alpha) which had to be obtained from the fitting of the experimental data. In the present work we demonstrate that this model is also suitable to describe the polymer segmental dynamics in concentrated polymer solutions. From this result we have developed a new route for determining the value of the alpha parameter associated with any given polymer. Once this value is known for the two components of a possible polymer blend, our model for polymer blends dynamics becomes fully predictive. PMID- 16674264 TI - Evolution of vibrational properties during a macromolecule's growth. AB - The elastic constants and vibrational contributions to thermal properties of three polymerizing liquids were investigated by using the available hypersonic velocity measured by Brillouin light scattering in real time. During the addition polymerization to a molecular network structure, Poisson's ratio upsilon(Poisson) decreases approximately according to exp[-(kt(polym))]n, where both k and n are composition dependent. The Debye frequency increases and the corresponding heat capacity, energy, and entropy approaching a limiting value. upsilon(Poisson) of the vitrified polymer continues to decrease but much more slowly, indicating its continued slow polymerization and structural relaxation with time. In the potential energy landscape interpretation, a polymerizing liquid's state point continuously shifts to another landscape's more curved, deeper minima. PMID- 16674263 TI - Free energy of a trans-membrane pore calculated from atomistic molecular dynamics simulations. AB - Atomistic molecular dynamics simulations of a lipid bilayer were performed to calculate the free energy of a trans-membrane pore as a function of its radius. The free energy was calculated as a function of a reaction coordinate using a potential of mean constraint force. The pore radius was then calculated from the reaction coordinate using Monte Carlo particle insertions. The main characteristics of the free energy that comes out of the simulations are a quadratic shape for a radius less than about 0.3 nm, a linear shape for larger radii than this, and a rather abrupt change without local minima or maxima between the two regions. In the outer region, a line tension can be calculated, which is consistent with the experimentally measured values. Further, this line tension can be rationalized and understood in terms of the energetic cost for deforming a part of the lipid bilayer into a hydrophilic pore. The region with small radii can be described and understood in terms of statistical mechanics of density fluctuations. In the region of crossover between a quadratic and linear free energy there was some hysteresis associated with filling and evacuation of the pore with water. The metastable prepore state hypothesized to interpret the experiments was not observed in this region. PMID- 16674265 TI - Microscopic dynamics of thin hard rods. AB - We analyze the microscopic dynamics and transport properties of a gas of thin hard rods. Based on the collision rules for hard needles we derive a hydrodynamic equation that determines the coupled translational and rotational dynamics of a tagged thin rod in an ensemble of identical rods. Specifically, based on a pseudo Liouville operator for binary collisions between rods, the Mori-Zwanzig projection formalism is used to derive a continued fraction representation for the correlation function of the tagged particle's density, specifying its position and orientation. Truncation of the continued fraction gives rise to a generalized Enskog equation, which can be compared to the phenomenological Perrin equation for anisotropic diffusion. Only for sufficiently large density do we observe anisotropic diffusion, as indicated by an anisotropic mean-square displacement, growing linearly with time. For lower densities, the Perrin equation is shown to be an insufficient hydrodynamic description for hard needles interacting via binary collisions. We compare our results to simulations and find excellent quantitative agreement for low densities and qualitative agreement for higher densities. PMID- 16674266 TI - Free-energy analysis of solubilization in micelle. AB - A statistical-mechanical treatment of the solubilization in micelle is presented in combination with molecular simulation. The micellar solution is viewed as an inhomogeneous and partially finite, mixed solvent system, and the method of energy representation is employed to evaluate the free-energy change for insertion of a solute into the micelle inside with a realistic set of potential functions. Methane, benzene, and ethylbenzene are adopted as model hydrophobic solutes to analyze the solubilization in sodium dodecyl sulfate micelle. It is shown that these solutes are more favorably located within the micelle than in bulk water and that the affinity to the micelle inside is stronger for benzene and ethylbenzene than for methane. The micellar system is then divided into the hydrophobic core, the head-group region in contact with water, and the aqueous region outside the micelle to assess the relative importance of each region in the solubilization. In support of the pseudophase model, the aqueous region is found to be unimportant to determine the extent of solubilization. The contribution from the hydrophobic-core region is shown to be dominant for benzene and ethylbenzene, while an appreciable contribution from the head-group region is observed for methane. The methodology presented is not restricted to the binding of a molecule to micelle, and will be useful in treating the binding to such nanoscale structures as protein and membrane. PMID- 16674267 TI - Mechanical unfolding revisited through a simple but realistic model. AB - Single-molecule experiments and their application to probe the mechanical resistance and related properties of proteins provide a new dimension in our knowledge of these important and complex biological molecules. Single-molecule techniques may not have yet overridden solution experiments as a method of choice to characterize biophysical and biological properties of proteins, but have stimulated a debate and contributed considerably to bridge theory and experiment. Here we demonstrate this latter contribution by illustrating the reach of some theoretical findings using a solvable but nontrivial molecular model whose properties are analogous to those of the corresponding experimental systems. In particular, we show the relationship between the thermodynamic and the mechanical properties of a protein. The simulations presented here also illustrate how forced and spontaneous unfolding occur through different pathways and that folding and unfolding rates at equilibrium cannot in general be obtained from forced unfolding experiments or simulations. We also study the relationship between the energy surface and the mechanical resistance of a protein and show how a simple analysis of the native state can predict much of the mechanical properties of a protein. PMID- 16674268 TI - Isotropic-nematic phase transition of nonaqueous suspensions of natural clay rods. AB - A novel model system for studying the behavior of hard colloidal rods is presented, consisting of sterically stabilized particles of natural sepiolite clay. Electron microscopy and scattering results confirmed that the organophilic clay particles were individual, rigid rods when dispersed in organic solvents. With a length-to-diameter ratio of approximately 27, the particles showed nematic ordering for volume fractions phi > 0.06. Polarizing microscopy revealed that the phase separation process involved nucleation, growth, and coalescence of nematic domains. The phase volumes and particle concentrations in the coexisting phases were determined. The dependence of these quantities on the total concentration of the suspension agrees well with Onsager's [Ann. N. Y. Acad. Sci. 51, 627 (1949)] isotropic-nematic phase transition theory extended to bidisperse and polydisperse rod systems, and with previous experimental results for rigid rodlike particles. Particle size distributions were obtained by analyzing transmission electron microscopy images. A significant fractionation with respect to rod length (but not diameter) was observed in the coexisting isotropic and nematic phases. The relative polydispersity of both daughter phases was distinctly smaller than that of the parent suspension. The phase behavior of these daughter fractions agrees well with the predictions for hard spherocylinders of corresponding aspect ratios. An isotropic-nematic-nematic phase equilibrium was seen to develop in phase separated samples after 1 month standing and is ascribed to the effect of polydispersity and possibly gravity. The second nematic phase appearing is dominated by very long rods. PMID- 16674269 TI - Rotational l-type resonance in BeH2, BeD2, and MgH2. PMID- 16674271 TI - Stereoselectivity in ene reactions with 1O2: matrix effects in polymer supports, photo-oxygenation of organic salts and asymmetric synthesis. AB - The ene reaction of chiral allylic alcohols is applied as a tool for the investigation of intrapolymer effects by means of the stereoselectivity of the singlet-oxygen addition. The diastereo selectivity strongly depends on the structure of the polymer, the substrate loading degree and also on the degree of conversion demonstrating additional supramolecular effects evolving during the reaction. The efficiency and the stability of polymer-bound sensitizers were evaluated by the ene reaction of singlet oxygen with citronellol. The ene reaction with chiral ammonium salts of tiglic acid was conducted under solution phase conditions or in polystyrene beads under chiral contact ion-pair conditions. The products thus obtained precipitate during the photoreaction as ammonium salts. Moderate asymmetric induction was observed for this procedure for the first time. PMID- 16674272 TI - A call to action. AB - OBJECTIVE: To summarize the challenges facing the U.S. health care system between now and 2013. SUMMARY: Between now and 2013, the American health care system will need to make major changes to successfully address Medicare's funding challenges. Making the patient a partner in care, developing systems that promote and reward prevention efforts, and ensuring that health care purchasers understand and pursue value will be key. The growing prevalence of diabetes and cardiometabolic conditions is one area in which concerted efforts are needed. PMID- 16674273 TI - Contemporary strategies for managing cardiometabolic risk factors. AB - OBJECTIVE: To review the metabolic syndrome as defined by the 2001 Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (ATP III) and its modifiable risk factors and to review currently available treatment modalities. SUMMARY: Metabolic syndrome, although still controversial, is a growing concern. Most definitions include elevated blood pressure, smoking, inflammation, insulin resistance, abdominal adiposity, elevated blood glucose, and atherogenic dyslipidemia as modifiable risk factors. Some researchers believe that additional signs of inflammation such as plasminogen activator inhibitor-1 and C-reactive protein should be added to this list. Current treatment options include lifestyle changes (diet and exercise), pharmacotherapy, and bariatric surgery. The endocannabinoid system appears to play a key role in metabolism and weight gain. The investigational agent rimonabant is a cannabinoid receptor type 1 blocker that has been employed in numerous trials involving more than 6,500 patients. It has led to significant weight loss, reduced central fat, and improved glycemic and lipid profiles. PMID- 16674274 TI - Patient-centric care management. AB - OBJECTIVE: To review managed care's current cost management trends and the consumerism movement; to elucidate the pros and cons of key issues; and to describe the philosophy of focusing on the patient, also called patient-centric care, while improving the patient's care through value-based purchasing and plan design. SUMMARY: Managed care is sometimes practiced using a silo approach with little concern for the consumer. In this model, medical and pharmaceutical issues are addressed in silos, and value is narrowly defined. Increasingly, cost and responsibility is shared with or shifted to the patients. Patients may be unable or unwilling to assume these costs or responsibilities. Several studies have demonstrated that they may react with noncompliance. Managed care's definition of value must expand and integrate across silos to consider the needs and interests of the patient's overall care, in particular, addressing key cost drivers in terms of diseases that cause recurring costs. Using predictive modeling can result in cost savings. A case study (Pitney Bowes) is included in this article. PMID- 16674278 TI - Review: bioprinting: a beginning. AB - An increasing demand for directed assembly of biologically relevant materials, with prescribed three-dimensional hierarchical organizations, is stimulating technology developments with the ultimate goal of re-creating multicellular tissues and organs de novo. Existing techniques, mostly adapted from other applications or fields of research, are capable of independently meeting partial requirements for engineering biological or biomimetic structures, but their integration toward organ engineering is proving difficult. Inspired by recent developments in material transfer processes operating at all relevant length scales--from nano to macro--which are amenable to biological elements, a new research field of bioprinting and biopatterning has emerged. Here we present a short review regarding the framework, state of the art, and perspectives of this new field, based on the findings presented at a recent international workshop. PMID- 16674279 TI - Review: tissue engineering of the urinary bladder: considering structure-function relationships and the role of mechanotransduction. AB - A variety of conditions encountered in urology result in bladder dysfunction and the need for bioengineered tissue substitutes. Traditionally, a number of synthetic materials and natural matrices have been used in experimental and clinical settings. However, the production of functional bladder tissue replacements remains elusive. The urinary bladder sustains considerable structural deformation during its normal function and represents an ideal model tissue in which to study the effects of biomechanical simulation on tissue morphogenesis, differentiation, and function. However, the actual role of mechanical forces within the bladder has received little attention. A strategy in which in vitro-generated tissue constructs are conditioned by exposure to the same mechanical forces as they would encounter in vivo could potentially be used both in the development of functional tissue replacements and to further study the role of biomechanical signalling. The purpose of this review is to examine the role and structure-function relationship of the urinary bladder and, through consultation of the literature available on mechanotransduction and tissue engineering of alternative tissues, to determine the factors that need to be considered when biomechanically engineering a functional bladder. PMID- 16674280 TI - Culture of human keratinocytes on polypyrrole-based conducting polymers. AB - Variously loaded polypyrrole films, including those containing proteins and polysaccharides, were prepared on gold-coated polycarbonate coverslips. The characteristics of human keratinocytes were studied on these films by microscopy, biochemical assays, and immunocytochemistry. We found keratinocyte viability to be load dependent. For chloride, polyvinyl sulphate, dermatan sulphate, and collagen-loaded polypyrrole films, keratinocyte viability as assessed by the AlamarBlue assay was respectively 47.22, 60.43, 87.71, and 22.65% of tissue culture polystyrene controls after 5 days. This was found to require a previously unreported polymer washing step prior to cell seeding due to the observed toxicity of untreated films. In the case of bare polycarbonate and gold substrates, viability was respectively 75.44 and 61.04% of tissue culture polystyrene controls after 5 days. Keratinocytes stained positive for PCNA (proliferation), K10 (suprabasal differentiation), and K16 (hyperproliferation) markers although cell morphology was poor for organotypical cultures on dermatan- loaded polypyrrole compared with de-epidermalized dermis. From our studies, we concluded that optimized polypyrrole films adequately support keratinocyte growth in submerged cultures with some improvements needed for organotypical cultures. Polypyrrole composites are attractive candidates for tissue-engineering applications since they may incorporate biomolecules and are electrically addressable with the potential to both direct and report on cell activity. PMID- 16674281 TI - Functional characterization of embryonic stem cell-derived cardiomyocytes using scanning ion conductance microscopy. AB - We report here the novel use of scanning ion conductance microscopy (SICM) to produce surface images of embryonic stem cell-derived cardiomyocytes (ESCM) to identify individual contracting cardiomyocytes among different cell types. By measuring amplitude and rhythm we can quantitate contraction of ESCM. This method gives, within the same experiment, an assessment of the number and position of ESCM within the layer of mixed cell types, as well as an accurate measure of the response of individual ESCM. Using different modulators of contraction as examples we showed how SICM could be used for recording their responses. We subsequently demonstrated that this model can be used to investigate the protective effect of antiarrhythmogenic drugs. PMID- 16674282 TI - Generation and differentiation of human embryonic stem cell-derived keratinocyte precursors. AB - Human embryonic stem cells (hESC) hold tremendous potential in the future of tissue engineering, offering promise as a source of virtually unlimited quantities of desired cell and tissue types. We have identified soluble chemical and extracellular matrix factors that permit isolation of keratinocyte precursors from hESCs. Culturing embryoid bodies (EB) formed from hESCs in a defined serum free keratinocyte growth medium on a gelatin matrix generated keratin 14 (K14) expressing cells with an epithelial morphology. These K14 expressing cells could be subcultured in medium supplemented with hydrocortisone and induced to stratify and terminally differentiate by addition of calcium. Optimum times for obtaining K14 expressing cells were found for EB formation and for differentiation and growth of cultures after EB plating. EB formation was not necessary to generate keratinocyte precursors; direct transfer of hESC colonies to keratinocyte growth medium permitted differentiation into the keratinocyte lineage. With further studies to optimize generation and purification of hESC-derived keratinocyte precursors, these cells could provide a source of epidermal cells for skin tissue engineering applications in vitro or in vivo. PMID- 16674283 TI - Effects of cell-to-collagen ratio in stem cell-seeded constructs for Achilles tendon repair. AB - The objective of the present study was to test the hypotheses that implantation of cell-seeded constructs in a rabbit Achilles tendon defect model would 1) improve repair biomechanics and matrix organization and 2) result in higher failure forces than measured in vivo forces in normal rabbit Achilles tendon (AT) during an inclined hopping activity. Autogenous tissue-engineered constructs were fabricated in culture between posts in the wells of silicone dishes at four cell to-collagen ratios by seeding mesenchymal stem cells (MSC) from 18 adult rabbits at each of two seeding densities (0.1 x 10(6) and 1 x 10(6) cell/mL) in each of two collagen concentrations (1.3 and 2.6 mg/mL). After 5 days of contraction, constructs having the two highest ratios (0.4 and 0.8 M/mg) were damaged by excessive cell traction forces and could not be used in subsequent in vivo studies. Constructs at the lower ratios (0.04 and 0.08 M/mg) were implanted in bilateral, 2 cm long gap defects in the rabbit's lateral Achilles tendon. At 12 weeks after surgery, both repair tissues were isolated and either failed in tension (n = 13) to determine their biomechanical properties or submitted for histological analysis (n = 5). No significant differences were observed in any structural or mechanical properties or in histological appearance between the two repair conditions. However, the average maximum force and maximum stress of these repairs achieved 50 and 85% of corresponding values for the normal AT and exceeded the largest peak in vivo forces (19% of failure) previously recorded in the rabbit AT. Average stiffness and modulus were 60 and 85% of normal values, respectively. New constructs with lower cell densities and higher scaffold stiffness that do not excessively contract and tear in culture and that further improve the repair stiffness needed to withstand various levels of expected in vivo loading are currently being investigated. PMID- 16674284 TI - Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. AB - This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model. PMID- 16674285 TI - Analysis of cell growth in three-dimensional scaffolds. AB - The in vitro growth of pre-osteoblasts in multi-layer, three-dimensional scaffolds was determined from experimental measurements and was compared to a mathematical model. Immediately following cell seeding, the initial cell density was uniform throughout the scaffold. After 10 days, the cell density increased from 2.1 x 10(5) cells/cm(3) to 1.3 x 10(7) cells/cm(3) at the fluid-scaffold interface. The increase in cell density was largely confined to the outermost 200 microm from the fluid-scaffold interface. The cell density profile was in good agreement with a mathematical model that simulated the cell growth based on the local oxygen tension. The improved understanding derived from this mathematical model may be useful in the design of three-dimensional scaffolds that can support more uniform growth of cells. PMID- 16674286 TI - Engineering three-dimensional pulmonary tissue constructs. AB - In this paper, we report on engineering 3-D pulmonary tissue constructs in vitro. Primary isolates of murine embryonic day 18 fetal pulmonary cells (FPC) were comprised of a mixed population of epithelial, mesenchymal, and endothelial cells as assessed by immunohistochemistry and RT-PCR of 2-D cultures. The alveolar type II (AE2) cell phenotype in 2-D and 3-D cultures was confirmed by detection of SpC gene expression and presence of the gene product prosurfactant protein C. Three dimensional constructs of FPC were generated utilizing Matrigel hydrogel and synthetic polymer scaffolds of poly-lactic-co-glycolic acid (PLGA) and poly-L lactic-acid (PLLA) fabricated into porous foams and nanofibrous matrices, respectively. Three-dimensional Matrigel constructs contained alveolar forming units (AFU) comprised of cells displaying AE2 cellular ultrastructure while expressing the SpC gene and gene product. The addition of tissue-specific growth factors induced formation of branching, sacculated epithelial structures reminiscent of the distal lung architecture. Importantly, 3-D culture was necessary for inducing expression of the morphogenesis-associated distal epithelial gene fibroblast growth factor receptor 2 (FGFr2). PLGA foams and PLLA nanofiber scaffolds facilitated ingrowth of FPC, as evidenced by histology. However, these matrices did not support the survival of distal lung epithelial cells, despite the presence of tissue-specific growth factors. Our results may provide the first step on the long road toward engineering distal pulmonary tissue for augmenting and/or replacing dysfunctional native lung in diseases, such as neonatal pulmonary hypoplasia. PMID- 16674287 TI - Biofabricated marine hydrozoan: a bioactive crystalline material promoting ossification of mesenchymal stem cells. AB - This study introduces a novel three-dimensional biomatrix obtained from the marine hydrocoral Millepora dichotoma as a scaffold for hard tissue engineering. Millepora dichotoma was biofabricated under field and laboratory conditions. Three-dimensional biomatrices were made in order to convert mesenchymal stem cells (MSCs) to exemplify osteoblastic phenotype. We investigated the effect of the biomatrices on MSCs proliferation and differentiation at 2, 3, 4, 7, 10, 14, 21, 28, and 42 days. Different analyses were made: light microscopy, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregate formation (oil red O), collagen type I immunofluorescence, DNA concentrations, alkaline phosphatase (ALP) activity, and osteocalcin concentrations. MSCs seeded on Millepora dichotoma biomatrices showed higher levels of calcium and phosphate incorporation and higher type I collagen levels than did control Porites lutea biomatrices. ALP activity revealed that MSCs seeded on M. dichotoma biomatrices are highly osteogenic compared to those on control biomatrices. The osteocalcin content of MSCs seeded on M. dichotoma remained constant up to 2 weeks before rising to surpass that of seeded P. lutea biomatrices after 28 days. Our study thus showed that M. dichotoma biomatrices enhance the differentiation of MSCs into osteoblast and hence have excellent potential as bioscaffold for hard tissue engineering. PMID- 16674288 TI - Regulative mechanisms of chondrocyte adhesion. AB - Interaction between chondrocytes and extracellular matrix is considered a key factor in the generation of grafts for matrix-associated chondrocyte transplantation. Therefore, our objective was to study the influence of differentiation status on cellular attachment. Adhesion of chondrocytes to collagen type II increased after removal from native cartilage up to the third day in monolayer in a dose-dependent manner. Following dedifferentiation after the second passage, adhesion to collagen types I (-84%) and II (-46%) decreased, whereas adhesion to fibrinogen (+59%) and fibronectin (+43%) increased. A cartilage construct was developed based on a clinically established collagen type I scaffold. In this matrix, more than 80% of the cells could be immobilized by mechanisms of adhesion, filtration, and cell entrapment. Confocal laser microscopy revealed focal adhesion sites as points of cell-matrix interaction, as well as collagen type II expression in the cartilage graft after two weeks of in vitro cultivation. Basic fibroblast growth factor (bFGF) treated chondrocytes showed increased adhesion to collagen types I and II, fibronectin, and fibrinogen. Attachment to these investigated proteins significantly enhanced cell proliferation. Matrix design in cartilage engineering must meet the biological demands of amplified cells, because adhesion of chondrocytes depends on their differentiation status and is regulated by bFGF. PMID- 16674289 TI - Hepatocyte and kupffer cells co-cultured on micropatterned surfaces to optimize hepatocyte function. AB - One strategy for temporarily extending the lives of patients with liver failure is the use of bioartificial liver (BAL) support devices. The functional components of BALs are the parenchymal liver cells known as hepatocytes. One design option for further improving current BAL performance levels is to include the non-parenchymal cells of the liver (e.g., Kupffer cells) in the design. In the current study, the effect of Kupffer cells on hepatocyte function was investigated using micropatterned co-cultures of these two liver cell populations. With traditional co-culture methods, the user is unable to control the relative proximity of one cell type to another. In this study, two different micropatterning techniques were used to engineer macro and fine micropatterned configurations for evaluating hepatocyte-Kupffer cell co-cultures. The ratio of one cell population to the other was also adjusted to evaluate the effects on hepatocyte function. The micropatterned co-cultures were maintained for ten days to evaluate for morphological and functional (e.g., albumin, urea) changes. The results illustrate that micropatterning hepatocytes, in the arrangements of this study, significantly improved hepatocyte function. PMID- 16674290 TI - Development and characterization of an acellular human pericardial matrix for tissue engineering. AB - This study aimed to produce an acellular human tissue scaffold with a view to recellularization with autologous cells to produce a tissue-engineered pericardium that can be used as a patch for cardiovascular repair. Human pericardia from cadaveric donors were treated sequentially with hypotonic buffer, SDS in hypotonic buffer, and a nuclease solution. Histological analysis of decellularized matrices showed that the human pericardial tissue retained its histioarchitecture and major structural proteins. There were no whole cells or cell fragments. There were no significant differences in the hydroxyproline (normal and denatured collagen) and glycosaminoglycan content of the tissue before and after decellularization (p > 0.05). There were no significant changes in the ultimate tensile strength after decellularization (p > 0.05). However, there was an increased extensibility when the tissue strips were cut parallel to the visualized collagen bundles (p = 0.005). No indication of contact or extract cytotoxicity was found when using human dermal fibroblasts and A549 cells. In summary, successful decellularization of the human pericardium was achieved producing a biocompatible matrix that retained the major structural components and strength of the native tissue. PMID- 16674291 TI - Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model. AB - Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering. PMID- 16674292 TI - Porous poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composite for reconstruction of bone defects. AB - Calcium phosphate (Ca-P) cements are injectable, self-setting ceramic pastes generally known for their favorable bone response. Ingrowth of bone and subsequent degradation rates can be enhanced by the inclusion of macropores. Initial porosity can be induced by CO(2) foaming during setting of the cement, whereas secondary porosity can develop after hydrolysis of incorporated poly(DL lactic- co-glycolic acid) (PLGA) microparticles. In this study, we focused on the biological response to porous PLGA/Ca-P cement composites. Pre-set composite discs of four formulations (4 wt% or 15 wt% PLGA microparticles and low or high CO(2) induced porosity) were implanted subcutaneously and in cranial defects in rats for 12 weeks. Histological analysis of the explanted composites revealed that bone and fibrous tissue ingrowth was facilitated by addition of PLGA microparticles (number average diameter of 66 +/- 25 microm). No adverse tissue reaction was observed in any of the composites. Significant increases in composite density due to bone ingrowth in cranial implants were found in all formulations. The results suggest that the PLGA pores are suitable for bone ingrowth and may be sufficient to enable complete tissue ingrowth without initial CO(2) induced porosity. Finally, bone-like mineralization in subcutaneous implants suggests that, under appropriate conditions and architecture, porous PLGA/Ca-P cement composites can exhibit osteoinductive properties. These PLGA/Ca P composites are a promising scaffolding material for bone regeneration and bone tissue engineering. PMID- 16674293 TI - Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor. AB - This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(epsilon-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities-- 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination with the culture conditions, may affect the functionality of tissues formed in vitro. PMID- 16674294 TI - In vitro evaluation of poly[bis(ethyl alanato)phosphazene] as a scaffold for bone tissue engineering. AB - Polyphosphazenes with amino acid ester as side groups are biocompatible polymers that could provide valid scaffolds for cell growth. In the present study we investigate the adhesion and growth of osteoblasts obtained from rat bone marrow on matrices composed of thin fibers of poly[bis(ethyl alanato)phosphazene] (PAlaP), poly(d,l-lactic acid) (PDLLA), or PAlaP/PDLLA blend. Our data show that scaffolds of PAlaP or PAlaP/PDLLA blend enhanced the cell adhesion and growth in comparison with that observed in cultures seeded on polystyrene tissue culture plates. Although collagenase-digestible protein synthesis remained unchanged, all scaffolds induced a decrease in alkaline phosphatase activity, suggesting that osteoblasts are in the proliferation phase. Both PAlaP and PAlaP blended with PDLLA may represent a new and interesting substrate for bone tissue engineering. PMID- 16674295 TI - Behavior of adult human mesenchymal stem cells entrapped in alginate-GRGDY beads. AB - This study demonstrates that adult human mesenchymal cells (MSC) can be encapsulated in alginate beads with a substantially retained viability (>80%) and that a Gly-Arg-Gly-Asp-Tyr (GRGDY) derivative encourages attachment and elongation to form a dense network of cells that is required for a tissue substitute. Because the availability of autologous human material is severely limited, we used and examined the beads in this study as a proxy for larger constructs. These bead constructs were assessed using phase contrast microscopy and standard histological preparations. In addition, we used a modified MTT (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to examine cell proliferation by dissociating the cell/alginate constructs using trisodium citrate and trypsin/EDTA. MSCs did not proliferate within the alginate-GRGDY matrix during the 2 weeks examined. These results were further substantiated by concurrent cell density measurements using a hemocytometer. In addition, the glucose consumption rate was measured and compared to that of MSCs grown in two dimensional culture vessels, indicating steady consumption albeit at a lower level in the entrapped MSCs. PMID- 16674296 TI - Production of extracellular matrix components in tissue-engineered blood vessels. AB - Morphology and compliance of tissue-engineered blood vessels (TEBV) are dependent on the culture period and production of extracellular matrix (ECM) components in order to increase the strength of the developing tissue. The aim of the present study was to evaluate the potential of TEBVs to produce an ECM similar to native arteries and veins. Human smooth muscle cells (SMC) were seeded onto the poly(glycolic acid) (PGA) scaffold and placed in bioreactors filled with DMEM supplemented with growth factors. After 6 weeks, the vessels were harvested from the bioreactors and seeded with human endothelial cells at the lumen for another 3 days. Then, the TEBVs were harvested for RNA and protein isolation for further RT-PCR and Western blot. TEBVs had a similar macroscopic appearance to that of native vessels with no visible evidence of the original PGA. Histological and immunohistochemical analyses indicated the presence of high cell density and development of a highly organized structure of ECM. After 6 weeks of culture, there were significantly lower gene expression of SMC-specific markers, such as alpha-actin, caldesmon, and vimentin, and proteoglycans, such as biglycan, decorin, and versican, and other ECM components, such as collagen I and elastin, in TEBVs, with and without pulsatile conditions, compared to that of native arteries. Gene expression of fibronectin was significantly lower in TEBVs grown during pulsatile conditions compared to that of native arteries. No difference was observed in TEBVs grown during non-pulsatile conditions. The presence of alpha-actin, collagen I, decorin, and fibronectin at protein level was demonstrated in TEBVs with and without pulsatile conditions after 6 weeks and in native veins and arteries as well. How this deviation translates into mechanical properties remains to be explored. PMID- 16674297 TI - MR assessment of osteogenic differentiation in tissue-engineered constructs. AB - Bone marrow stromal cells (MSC) are a promising source of osteoprogenitor cells for bone tissue engineering. However, the population of the osteoprogenitor cells and their differentiation potentials change with the gender, age, and health of the donor. Development of a noninvasive method to assess osteogenic progression is critical for successful bone tissue regeneration. High-resolution magnetic resonance imaging (MRI) (at 11.7 T, with spatial resolution of 62.5 x 62.5 microm in 500 microm slices) is used in the present study to monitor osteogenic differentiation of tissue-engineered constructs prepared by seeding human bone MSCs on gelatin sponge scaffolds. Quantitative measurements of the MR relaxation times (T1, T2) and the apparent diffusion coefficient (ADC) were performed for four successive weeks on control tissue constructs and constructs exposed to osteogenic differentiation medium. The T1 and T2 relaxation times and ADC were found to decrease as osteogenic progression proceeded in samples exposed to osteogenic differentiation medium. At week 4, the T1, T2, and ADC of TE constructs were 1.81 +/- 0.11 s, 19.5 +/- 11.02 ms, and 1.01 +/- 0.47 x 10(3) mm(2)/s, respectively, for osteogenic differentiated constructs, significantly different from control constructs 2.22 +/- 0.08 s, 50.39 +/- 5.57 ms, and 1.86 +/ 0.18 x 107(3) mm(2)/s (p < 0.05). The MR parameters were also highly correlated with the cell seeding densities and alkaline phosphatase (ALP) activities of the osteogenic constructs. In conclusion, periodic measurements of MR parameters (T1, T2, and ADC) provide a promising method for noninvasive monitoring of the status of tissue-engineered bone growth and differentiation. PMID- 16674298 TI - Embryonic myocardium shows increased longevity as a functional tissue when cultured in the presence of a noncardiac tissue layer. AB - A major aim of regenerative medicine is the construction of bioengineered organs and tissue for transplantation into human patients; yet living tissue is dynamic, and thus arranging cellular and extracellular constituents into an architecture resembling normal adult organs may not be sufficient to maintain tissue stability. In this study, we used cultures of embryonic chick heart tissue as a model to explore how newly formed cardiac tissue constructs can sustain their morphological structure and functional capabilities over extended periods. During the initial days of incubation, embryonic cardiac explants will thrive as beating three-dimensional tissue aggregates. However, within the first week of culture, cardiac aggregates lose their contractile function and flatten. After 2 weeks of incubation, the cardiac cells will have spread out into a homogeneous monolayer and dedifferentiated to a noncardiac phenotype. In contrast, when the embryonic heart tissue was co-cultured with a noncardiac cell layer obtained from adult bone marrow, the cardiac aggregates maintained their contractile function, three dimensional tissue morphology, and myocyte phenotype for a full month of incubation. The capacity of this noncardiac cell layer to sustain the phenotype and morphology of the cardiac explants was partially replicated by treatment of the heart tissue with conditioned media from bone marrow cells. These findings are discussed in regard to the importance of adjacent cell layers for facilitating organogenesis in the developing embryo and having potential utility in producing stable bioengineered tissue constructs. PMID- 16674299 TI - Derivation of distal airway epithelium from human embryonic stem cells. AB - The pluripotency of embryonic stem cells (ESC) is offering new opportunities in tissue engineering and cell therapy. We have shown previously that alveolar epithelial cells, specifically type II pneumocytes, can be derived from murine ESC and hypothesized that a similar protocol could be used successfully on human ESC. Undifferentiated human ESC were induced to form embryoid bodies that were transferred into adherent culture conditions and grown in a medium designed for the maintenance of mature small airway epithelium. On inverted microscopy, the generated cells showed the cobblestone-like morphology of epithelium. The presence of surfactant protein C, a specific marker of type II pneumocytes, and its corresponding RNA were demonstrated by immunostaining and reverse transcription polymerase chain reaction, respectively. Electron microscopy revealed frequent cells with the typical ultrastructure of type II pneumocytes. This study provides evidence for in vitro induction of the differentiation from human ESC of alveolar type II cells, which have the potential for therapeutic use or construction of an in vitro model of human lung. PMID- 16674300 TI - Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration. AB - There are several gene therapy approaches to tissue regeneration. Although usually efficient, virusbased approaches may elicit an immune response against the viral proteins. An alternative approach, nonviral transfer, is safer, and can be controlled and reproduced. We hypothesized that in vivo bone formation could be achieved using human mesenchymal stem cells (hMSCs) nonvirally transfected with the human bone morphogenetic protein-2 (hBMP-2) or -9 (hBMP-9) gene. Human MSCs were transfected using nucleofection, a unique electropermeabilization-based technique. Postnucleofection, cell viability was 53.6 +/- 2.5% and gene delivery efficiency was 51% to 88% (mean 68.2 +/- 4.1%), as demonstrated by flow cytometry in enhanced green fluorescent protein (EGFP)-nucleofected hMSCs. Transgene expression lasted longer than 14 days and was very low 21 days postnucleofection. Both hBMP-2- and hBMP-9-nucleofected hMSCs in culture demonstrated a significant increase in calcium deposition compared with EGFP-nucleofected hMSCs. Human BMP-2 and hBMP-9-nucleofected hMSCs transplanted in ectopic sites in NOD/SCID mice induced bone formation 4 weeks postinjection. We conclude that in vivo bone formation can be achieved by using nonvirally nucleofected hMSCs. This could lead to a breakthrough in the field of regenerative medicine, in which safer, nonviral therapeutic strategies present a very attractive alternative. PMID- 16674301 TI - Tissue-engineered valves with commissural alignment. AB - We present a novel approach to producing bioartificial valves using the tissue equivalent method of entrapping cells within a biopolymer gel and using a mold design that presents appropriate mechanical constraints to the cell-induced gel compaction to yield both the fibril alignment and the geometry of a native valve. Bileaflet valves were fabricated from bovine collagen and neonatal human dermal fibroblasts as proof of principle. The resultant valves possessed both commissure tocommissure alignment of collagen fibers in the leaflets and circumferential alignment in the root. While this alignment was manifested in planar biaxial tensile mechanical properties, histology of the leaflets revealed an aligned collagen matrix but lacking other extracellular matrix (ECM) components present in the native valve. The apparent lack of ECM production by the fibroblasts after contracting and aligning the collagen fibrils is consistent with peak loads during biaxial testing being only approximately 10% of native leaflet values and a 0:1 coupling index that was only approximately 50% of native leaflet values despite exhibiting comparable values for the anisotropy index. PMID- 16674302 TI - Valvular endothelial cells regulate the phenotype of interstitial cells in co culture: effects of steady shear stress. AB - Valvular endothelial cells interact with interstitial cells in a complex hemodynamic and mechanical environment to maintain leaflet tissue integrity. The precise roles of each cell type are difficult to ascertain in a controlled manner in vivo. The objective of this study was to develop a three-dimensional aortic valve leaflet model, comprised of valvular endothelium and interstitial cells, and determine the cellular responses to imposed lumenal fluid flow. Two leaflet models were created using type I collagen hydrogels. Model 1 contained 1 million/mL porcine aortic valve interstitial cells (PAVICs). Model 2 added a seeding of the lumenal surface of Model 1 with approximately 50,000/cm(2) porcine aortic valve endothelial cells (PAVECs). Both leaflet models were exposed to 20 dynes/cm(2) steady shear for up to 96 h, with static constructs serving as controls. Endothelial cell alignment, matrix production, and cell phenotype were monitored. The results indicate that PAVECs align perpendicularly to flow similar to 2D culture. We report that PAVICs in model 1 express vimentin strongly and alpha-smooth-muscle actin (SMA) to a lesser extent, but SMA expression is increased by shear stress, particularly near the lumenal surface. Model 1 constructs increase in cell number, maintain protein levels, but lose glycosaminoglycans in response to shear. Co-culture with PAVECs (Model 2) modulates these responses in both static and flow environments, resulting in PAVIC phenotype that is more similar to the native condition. PAVECs stimulated a decrease in PAVIC proliferation, an increase in protein synthesis with shear stress, and reduced the loss of glycosaminoglycans with flow. Additionally, PAVECs stimulated PAVIC differentiation to a more quiescent phenotype, defined by reduced expression of SMA. These results suggest that valvular endothelial cells are necessary to properly regulate interstitial cell phenotype and matrix synthesis. Additionally, we show that tissue-engineered models can be used to discover and understand complex biomechanical relationships between cells that interact in vivo. PMID- 16674303 TI - Macroporous elastomeric scaffolds with extensive micropores for soft tissue engineering. AB - Macroporous scaffolds are of great value in tissue engineering. We have developed a method to fabricate macroporous scaffolds from a biocompatible and biodegradable elastomer, poly(glycerol sebacate) (PGS). This method is potentially very useful for soft tissue engineering. Our fabrication method produced macroporous scaffolds with extensive micropores. We fabricated flat scaffolds and tubular scaffolds of uniform thickness. This fabrication method demonstrated good control of variables such as pore size, porosity, and pore interconnectivity. Sodium chloride (salt) crystals, which served as solid porogens, were packed into a mold and fused in a humid chamber. PGS was cured while dispersed throughout the fused salt template. Dissolution of the salt and subsequent lyophilization produced elastomer sponges with approximately 90% porosity, interconnected macropores (75-150 microm), and extensive micropores (5 20 microm). The macropores were generated by the salt particles, while the micropores were likely generated by glycerol vapor formed during PGS curing. Such numerous micropores could facilitate cell-cell interactions and mass transport. Fibroblasts adhered to and proliferated well within the PGS scaffolds and formed three-dimensional tissue-engineered constructs within 8 days. PMID- 16674304 TI - Bone-like tissue formation by three-dimensional culture of MG63 osteosarcoma cells in gelatin hydrogels using calcium-enriched medium. AB - The aim of this study was to investigate the effect of Ca(2+) concentration in culture medium on the promotion of osteogenesis by MG63 osteoblast-like cells and to prepare bone-like tissues by supplying Ca(2+)-enriched medium to MG63 cells immobilized in three-dimensional gelatin hydrogels. Human osteosarcoma MG63 cells were cultured on tissue culture dish under various Ca(2+) concentrations to evaluate the effect of Ca(2+) concentration on calcium deposition. When Ca(2+) concentration was 8 mM, the maximum calcium deposition was obtained at day 28. Then MG63 cells were entrapped in gelatin hydrogels cross-linked by transglutaminase and cultured for 28 days, either in a standard culture medium or in medium containing 8 mM Ca(2+). Effects of Ca(2+)-enriched medium on osteoblastic phenotype of MG63 cells in gelatin hydrogels were analyzed in terms of cell number, calcium deposition content, and alkaline phosphatase (ALP) activity. The characteristics of calcified gelatin hydrogels were evaluated by x ray diffraction (XRD), histological analysis, and scanning electron microscopy (SEM). After 28 days of culture, no significant difference in cell numbers was found between the different culture conditions. However, calcium content of gelatin hydrogels with cells cultured in Ca(2+)-enriched media was significantly higher than that of hydrogels with cells cultured in standard Ca(2+) concentration medium. After 14 days of culture, ALP activity of cells cultured in Ca(2+)-enriched media was down-regulated compared with that of cells cultured in standard Ca(2+) concentration media. XRD analysis indicated the formation of hydroxyapatite in gelatin hydrogels cultured in the Ca(2+)-enriched media at day 14, and the XRD pattern of the composite at day 21 was almost similar to that of mouse tibia. Moreover, histological analysis and SEM analysis revealed that cross sections of hydrogels cultured in Ca(2+)-enriched media had an organic/mineral layer structure analogous to that of mouse tibia. PMID- 16674305 TI - Induced cell clustering enhances islet beta cell formation from human cultures enriched for pancreatic ductal epithelial cells. AB - A better understanding of the culture conditions that stimulate in vitro beta cell differentiation from islet precursors would be useful for optimizing the production of tissue-engineered islets. In this study, high- and low-adherent substrates and high- and low-serum media were used to control the clustering of human pancreatic ductal epithelial cells and to determine its effect on their transdifferentiation to beta cells. While the initial epithelial cell cultures were devoid of any beta cells as assessed by dithizone staining, dithizone+ cells were generated during the next 3 weeks under all culture conditions. Although the rate of transdifferentiation was low, a approximately 4-fold greater number and percentage of dithizone+ cells were generated following 23-24 days of culture in the least adherent conditions (low-serum medium, low-adherent substrate), which stimulated cell clustering to the highest degree. Insulin immunohistochemistry data correlated well with the dithizone data (r(2) = 0.99), evidence that dithizone is a reliable measure of insulin+ cells. The preferential distribution of the dithizone+ cells to regions of cell aggregation and the increased efficiency of transdifferentiation in conditions that promote cell clustering suggest that cell-cell interactions and/or cell shape changes are important to the transdifferentiation of adult pancreatic ductal epithelial cells to beta cells in vitro. PMID- 16674306 TI - In vitro ossification and remodeling of mineralized collagen I scaffolds. AB - A promising strategy of bone tissue engineering is to repair bone defects by implanting biodegradable scaffolds that can undergo remodeling and be replaced completely by autologous bone tissue. For this purpose, it is necessary to create scaffolds that can be degraded by osteoclasts and enable osteoblasts to build new mineralized bone matrix. In order to achieve this goal a new porous material has been developed using biomimetically mineralized collagen I. These scaffolds were co-cultured with osteoclast-like cells and osteoblasts in order to characterize the capacity of these cells to remodel the material in vitro. It was possible to show the development of biologically active osteoclast- like cells that were able to invade and degrade the scaffold. They degraded the scaffold by internalizing it as intracellular vesicles, thereby making room for osteoblasts to invade and build new bone matrix. In addition, it could be shown that osteoblasts proliferated, differentiated, and produced new mineralized extracellular matrix. Hence, it could be shown that co-culture of osteoclastlike cells and osteoblasts on biomimetically mineralized collagen I is a promising approach for bone tissue engineering. In addition, it can be applied to study the process of bone remodeling in vitro. PMID- 16674307 TI - Localized angiogenesis induced by human vascular endothelial growth factor activated PLGA sponge. AB - The objective of this study was to assess the in vitro release kinetics and the in vivo angiogenic effect of human vascular endothelial growth factor (VEGF) activated poly(DL-lactide-co-glycolide) (PLGA) sponge. The highly porous sponges (each 3 x 4 x 4 mm(3)) were activated by soaking in a VEGF solution (2.5 or 5.0 microg) and then freeze-drying. In vitro release in PBS was investigated by a competitive enzyme immunoassay for up to 3 weeks. The burst-type initial release within the first 3 days followed a more controlled one lasting for >2 weeks. The angiogenic potential of the VEGF sponge was evaluated by subcutaneous implantation into the epigastric groin fascia of Wistar rats. Histomorphometry and SEM confirmed the formation of new capillaries infiltrating the sponge pores starting from the first week and the drastic anostomosis at weeks 2 and 3. However, the rats implanted with control sponges or receiving VEGF injection exhibited much lower or no angiogenic response, respectively. TEM revealed the neo-vessels had a single endothelial layer surrounded by the matrix inoculated with the rat circulation. The results indicate that VEGF-activated PLGA sponge can be considered as a tool to establish neovascularized subcutaneous transplantation sites for tissue-engineering applications. PMID- 16674308 TI - A self-assembling process in articular cartilage tissue engineering. AB - Current therapies for articular cartilage defects often result in fibrocartilaginous tissue. To achieve regeneration with hyaline articular cartilage, tissue-engineering approaches employing cell-seeded scaffolds have been investigated. However, limitations of scaffolds include phenotypic alteration of cells, stress-shielding, hindrance of neotissue organization, and degradation product toxicity. This study employs a self-assembling process to produce tissue-engineered constructs over agarose in vitro without using a scaffold. Compared to past studies using various meshes and gels as scaffolding materials, the self-assembly method yielded constructs with comparable GAG and collagen content. By 12 weeks, the self-assembling process resulted in tissue engineered constructs that were hyaline- like in appearance with histological, biochemical, and biomechanical properties approaching those of native articular cartilage. Overall, constructs contained two thirds more GAG per dry weight than calf articular cartilage. Collagen per dry weight reached more than one third the level of native tissue. IHC and gel electrophoresis showed collagen type II production and absence of collagen type I. More importantly, self-assembled constructs reached well over one third the stiffness of native tissue. PMID- 16674313 TI - Effect of the silk protein sericin on the production of adenovirus-based gene therapy vectors. AB - Adenoviral vectors are extensively used as gene-delivery vehicles in gene therapy. They are usually produced by HEK-293 cell (human embryonic kidney-293 cell) culture, which requires specially formulated serum-free medium, the cost of which is considerable or by supplementation with FBS (fetal bovine serum). The risk of infectious diseases such as BSE (bovine spongiform encephalopathy) and endogenous retrovirus derived from cattle is a serious concern. The present study reports the use of sericin protein derived from silkworm (Bombyx mori) as an effective supplement instead of FBS. Without FBS, HEK-293 cells significantly proliferated in the presence of 0.025-0.4% sericin, especially at 0.1%, but the effect was inferior to that of FBS. When a lower titre [MOI (multiplicity of infection) 0.03] of adenoviral vector pAxCAiLacZ was used as the inoculum, HEK 293 cells in the presence of 0.1% sericin produced a nearly 3-fold higher vector titre than culture in the presence of 5% (v/v) FBS. However, when a higher vector titre (MOI 3.7) was used as the inoculum, HEK-293 cells in the presence of sericin produced a slightly higher vector titre than in the presence of FBS, which might suggest that HEK-293 cells produce a maximum amount when a higher vector titre is used as the inoculum. These increases in vector production with sericin were confirmed by LacZ (beta-galactosidase reporter gene) activity assay. Supplementation with sericin decreased lactate dehydrogenase activity, an indicator of cell death, suggesting that sericin improved cell survival; hence, prolonging the culture period might be one of the reasons for increased vector production. On the basis of these results, sericin peptide seems to be a potent and effective alternative supplement for production of adenoviral vectors without such risks as BSE and retrovirus. PMID- 16674315 TI - Comparing the quality of care provided by health plans: are the data there? PMID- 16674314 TI - An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme. AB - The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle. PMID- 16674316 TI - Assessment of quality of care for managed care and fee-for-service patients based on analysis of avoidable hospitalizations. AB - As managed care has grown to dominate the US health care delivery system, questions have been raised about the impact on the quality of care provided to its enrollees. Two important aspects of health care quality are access to care and the appropriateness of care. This analysis evaluated the occurrence of preventable hospitalizations among managed care (MCO) versus fee for service (FFS) populations to compare access to and appropriateness of preventive, primary, and surgical health care services. Rates of preventable hospitalizations associated with ambulatory sensitive conditions (ASCs) were calculated based on all discharges from Massachusetts hospitals in 1995, and categorized by population characteristics including: age, sex, ethnicity, and insurance status. Multivariate logistic regression models were employed to explain the likelihood of having a preventable hospitalization. Rates of preventable hospitalizations for two of the conditions evaluated (perforated appendix and diabetes complications) were lower for MCO enrollees. For two additional indicators (immunization preventable pneumonia and low birth weight), MCO rates were no different from FFS rates. Results for pediatric asthma were inconclusive. For four out of five quality indicators evaluated, individuals in Massachusetts MCOs are doing better or no worse than their counterparts in FFS plans. Until population-based data on managed care enrollees becomes available, and until such data can be linked to utilization and health outcomes information, investigations into the quality of services provided by MCOs compared to FFS plans cannot be definitive. PMID- 16674317 TI - The role of SSRI antidepressants for treating depressed patients in the California Medicaid (Medi-Cal) program. AB - This research used paid claims data to investigate the likelihood that patients achieved an adequate course of antidepressant drug therapy and the impact of completed therapy on health care costs. Completed therapy was defined as six or more months of uninterrupted therapy at an adequate dose as determined by AHCPR treatment guidelines. Apparent average daily dose for each prescription filled was calculated from data on the prescription paid claim and allowances were made for titration of dose to therapeutic levels and changes in antidepressant therapy. A total of 1648 new episodes of antidepressant therapy were identified for analysis. The likelihood of achieving an adequate course of antidepressant therapy was 22%. Completion rates varied significantly across antidepressants with fluoxetine achieving the highest completion rate at nearly 51%. Total health care costs were significantly lower for patients who completed therapy (-dollar 1487; P = .0487) due primarily to lower ambulatory care costs (-dollar 1296; P = .0110). Fluoxetine was the only antidepressant therapy which exhibited significantly lower total health care cost per patient relative to the older tricyclic antidepressants (-dollar 3524; P = .0024). The total costs of treating depression in the ambulatory setting were found to vary widely across alternative antidepressants. Most of the cost-savings associated with fluoxetine use were associated with the increased likelihood of completed therapy. Further research is needed to verify if fluoxetine achieves better rates of completed therapy relative to other SSRI antidepressants using data from other settings. PMID- 16674318 TI - Towards a European consensus on conducting and reporting health economic evaluations--a report from the ISPOR Inaugural European Conference. AB - This report is a summary of key issues in consensus development regarding the conduct and reporting of health economic research in the European context, presented and discussed at the ISPOR Inaugural European Conference in Cologne, Germany, December 1998. Recommendations of the Harmonization by Consensus of the Methodology for Economic Evaluation of Health Care Technologies in the European Union (HARMET) project were presented, as well as two instruments under development: software for Reporting Economic Evaluation Results (REER) and software for collecting and managing cost data called the Health Cost Database Software (HCDS). Working independently, but interrelated with the objectives of the HARMET initiative, preliminary results from the ongoing European Network on Methodology and Application of Economic Evaluation Techniques (EUROMET) project were presented. Each presentation was followed by an expert discussion panel with audience participation. Issues raised included the development of standards and related topics such as usefulness to European decision-makers, and education and training in health economics in Europe. PMID- 16674319 TI - Cost-effectiveness of prolonged administration of a low molecular weight heparin for the prevention of deep venous thrombosis following total hip replacement. AB - Recently there has been discussion on the value of prolonged thromboprophylaxis after elective hip surgery to prevent thromboembolic complications up to one month. Continuing low molecular weight heparin for one month can significantly reduce the frequency of venographically-proven deep vein thrombosis (DVT). How this will influence health economics has hitherto not been evaluated. Data on costs and effect from a randomized comparison between 10 and 30 days of once daily low molecular weight heparin (enoxaparin) were used in a cost-effectiveness study. Data on treatment costs were based on patients actually treated for DVT within the randomized study. Various sensitivity analyses were performed. In the study both phlebographically detected and clinically manifested venous thromboembolism were registered. By using costs from the health care system in Sweden it could be shown that the cost was 6,075 Swedish Krona (SEK) per phlebographic DVT avoided and 13,184 Swedish Krona (SEK) per clinical DVT avoided. If more than 50% of the patients managed to self-administer the low molecular weight heparin injections, the use of prolonged prophylaxis is cost effective. Prolonged prophylaxis with the low molecular weight heparin enoxaparin after elective hip arthroplasty is cost-effective provided that at least 50% of the patients can administer the subcutaneous injections themselves. PMID- 16674320 TI - Postmarketing studies: benefits and risks. AB - OBJECTIVE: To consider the benefits and risks of large postmarketing outcomes studies, as demonstrated by studies of the statin drugs. METHODS: Literature review. RESULTS: The risks were that the statin studies had a strong coat-tail effect. Each new study was beneficial to all statins as well as the one studied. Economic analyses based on the results of the postmarketing studies concluded that the drugs were not cost-effective. Long-term postmarketing studies were slow to be put into perspective and did not immediately influence other researchers or clinicians. During that time, the sponsoring companies shouldered opportunity costs as well as the actual costs of the studies. The risk that one drug company would use another company's results instead of investing in their own research did not materialize. The benefits were that the studies definitively showed that the drugs and the lowering of lipids were safe and efficacious. The studies also expanded the indications for the drugs, generated goodwill in the medical and research communities for the sponsors, allowed sponsors to include specific claims in their advertisements, generated follow-up studies, spawned economic analyses that sparked interest in the medical and lay press, and had a major impact on clinicians' use of the drug. CONCLUSION: The risks and benefits of postmarketing studies may depend on the company's time perspective. In the short term, the risks may outweigh the benefits. Only companies that have a longer perspective may find it beneficial to undertake large postmarketing studies. PMID- 16674321 TI - An analysis of the impact of demographic, clinical, and social factors on health related quality of life. AB - This study investigated the impact of demographic, social, and clinical factors on cancer patients' self-ratings of health-related quality of life (HRQL). The sample consisted of 1342 ethnically diverse individuals in treatment at four member institutions of the Eastern Cooperative Oncology Group (ECOG). Multivariable regression analyses were employed to determine the relationship between demographic variables (age, gender, race/ethnicity, socio-economic status (SES), living arrangement), clinical factors (performance status rating (PSR), disease type, disease stage), and social characteristics (spiritual beliefs, religious affiliation, relationship with physician) and five outcome measures of HRQL. The dependent variables, four dimensions of HRQL and overall HRQL, were measured by the Functional Assessment of Cancer Therapy-General (FACT-G) Quality of Life Measurement System. The results indicated that the full set of predictor variables accounted for 45% of the variance in patients' reporting of overall HRQL, 25% of the variance in physical well-being, 27% of the variance in social well-being, 30% of the variance in emotional well-being, and 41% of the variance in the area of functional well-being. The findings suggest that there are multiple factors that influence an individual's assessment of their HRQL and that these factors need to be considered in the management and treatment of culturally diverse cancer patients. PMID- 16674322 TI - Pharmacoeconomics in European decision-making. PMID- 16674323 TI - Current trends in the use of pharmacoeconomics and outcomes research in europe. PMID- 16674324 TI - The influence of case mix, site selection, and methods biases on costs of hospitalization for acute exacerbations of chronic obstructive airways disease and lower respiratory tract infections. AB - OBJECTIVE: To compare costs of hospitalization for lower respiratory tract infection (LRTI) in patients who received antibiotics before admission to those who did not, and in patients with and without underlying chronic obstructive airways disease (COAD) or diabetes mellitus. METHODS: All hospitalizations in a population of 366,849 residents in Tayside Scotland from 1993 to 1994 were analyzed. Three groups of patients were identified by primary discharge diagnosis in 1993/94 and previous admissions from 1980 to 1992: 1) acute exacerbation of COAD; 2) LRTI plus a secondary diagnosis of COAD or previous admission with COAD; 3) LRTI but no secondary COAD or previous admission with COAD. Setting specific costs were applied (e.g., general medicine, intensive care, geriatrics). Dispensed antibiotic prescribing in the 28 days before admission was identified from all community pharmacies. Nonparametric statistical tests were used. RESULTS: Patients with COAD were more likely to have received antibiotics before admission: COAD (n = 893) 49%; COAD + LRTI (n = 316) 43%; LRTI only (n = 822) 33%. Patients who received antibiotics before admission had lower hospital costs than patients who did not. Mean total costs per admission: COAD pound1604 versus pound1625 (p = .5); COAD + LRTI pound2281 versus pound2297 (p = .5); LRTI only pound2365 versus pound3233; (p = .009). Increasing age and diabetes mellitus were associated with higher hospital costs in all three groups. CONCLUSION: Economic models of the value of preventing hospital admissions for COAD or LRTI will be subject to case mix bias unless they adjust for age, community antibiotic use, and comorbidity. PMID- 16674325 TI - The norwegian version of the psoriasis disability index--a validation and reliability study. AB - OBJECTIVE: The aim of this study was to translate the Psoriasis Disability Index (PDI) into Norwegian and validate it in a Norwegian setting. The PDI is a measure that was developed to assess the impact of psoriasis on the patient's life. METHODS: Two hundred and eighty-two patients with psoriasis were included in the study (80% outpatients, and 20% hospitalized). RESULTS: Face and content validity were assessed as satisfactory. The PDI seems to capture issues of importance to patients. The results indicate that the PDI does not capture a unidimensional concept. A factor analysis (principal component with orthogonal rotation) resulted in three factors (physical, social, and hygienic) that were substantially different. All three factors had satisfactory internal consistency. Altogether they explained 58% of the variance. In addition, there were differing patterns of correlations with external criteria, such as dimensions of SF-36, as well as with sex, age, and education. CONCLUSIONS: The PDI has been found to have acceptable reliability in this study. However, further validation is necessary to estimate the sensitivity to change. PMID- 16674327 TI - Health care costs and outcomes: how should we evaluate real world data? PMID- 16674328 TI - Stochastic cost-effectiveness analysis: a simultaneous marginal-effect approach. AB - OBJECTIVE: The purpose of this study is to develop a cost-effectiveness methodology in the context of a simultaneous modeling framework that provides consistent point and interval estimates. METHODS: A simultaneous model of cost and effectiveness functions was developed to measure the incremental cost effectiveness ratio for competing medical interventions. A feasible nonlinear least-squares method was suggested to estimate the simultaneous model. Using a series of hypothetical data, a simulation analysis was performed to show the superior performance of the proposed model, relative to the average-effect model, a widely used approach to cost-effectiveness estimation. RESULTS: The traditional average-effect approach has two shortcomings. First, it assumes two strong conditions: truly random distributions of all the significant nontreatment variables (both observed and unobserved) across study groups, and the independence of cost and effectiveness variables. Second, it does not give the confidence interval, an important measure to assess the stochastic nature and robustness of point estimates. In contrast, the simultaneous modeling approach provides marginal-effect estimates, imposing no restrictions on the random distributions of the individual characteristics across study groups. Furthermore, it takes into account the simultaneity of cost and effectiveness functions being estimated. The simulation analysis showed that the simultaneous modeling approach is significantly more unbiased and efficient in predicting the true cost effectiveness ratio. CONCLUSION: The simultaneous modeling approach is superior to the average-effect approach in the estimation of incremental cost effectiveness ratios using data with significant nontreatment confounding factors. The advantages of the simultaneous modeling approach are particularly appealing for evaluative studies dealing with large-scale retrospective data at the patient level. PMID- 16674329 TI - Estimating endogenous treatment effects in retrospective data analysis. AB - Treatment effect estimation is one of the mainstays of the field of outcomes research. It is, for example, a key component in analyzing the cost-effectiveness of a proposed qualitative intervention. Some outcomes researchers are hesitant to use retrospective data for treatment effect estimation because of the potential endogeneity of the treatment variable. This is unfortunate, given the abundance and other advantages of retrospective data. Others who have used retrospective data have ignored the endogeneity problem, or have not recognized its potential for causing bias in their estimates. In this paper, an econometric method that is unbiased in the presence of endogeneity and therefore broadens the potential for use of retrospective data in the estimation of treatment effects is proposed. This two-stage method is also designed to accommodate nonlinearity in the relationship between the treatment variable and the outcome. An easy to apply GAUSS implementation of the estimator is offered. PMID- 16674330 TI - Controlling for systematic selection in retrospective analyses: an application to fluoxetine and sertraline prescribing in the United Kingdom. AB - BACKGROUND: Criticism has been made of observational studies in clinical practice because of their failure to control for unobserved factors that correlate with both initial treatment selection and observed outcomes. METHOD: A two-stage statistical model was applied to data obtained from a large general practitioner medical records database (DIN-LINK) to estimate the effect of initial antidepressant selection on the duration of antidepressant therapy and on the likelihood of being prescribed an average daily dose above the minimum recommended dose. The statistical model controlled for unobserved factors correlated with initial treatment selection and the observed outcomes as well as for observed confounders. RESULTS: Unobserved factors correlated with treatment selection were not a statistically significant determinant of the number of days of antidepressant therapy. However, unobserved factors correlated with treatment selection were a statistically significant determinant of the likelihood of receiving an average dose during therapy greater than the minimum recommended. After controlling for relevant confounders, those patients who began treatment with sertraline as opposed to fluoxetine had fewer days of antidepressant therapy and were more likely to receive average doses greater than the minimum recommended during therapy. CONCLUSION: Unobserved factors correlated with treatment selection can impact outcomes in observational studies and should be tested and controlled for whenever possible. PMID- 16674331 TI - Noncompliance with drug therapy for chronic obstructive pulmonary disease: a risk factor for hospitalization? AB - OBJECTIVE: To determine if patients who were noncompliant with prescribed medications for chronic obstructive pulmonary disease (COPD) had higher rates of hospitalization. METHODS: A retrospective case-control study was performed in a tertiary-care university-affiliated Veterans Administration Health Care System setting. Subjects included 93 patients hospitalized for exacerbation of COPD and 93 controls with a diagnosis of COPD who did not require hospitalization. Utilizing pharmacy prescription fill records, medication noncompliance rates of patients who required hospitalization for exacerbation of COPD were compared with patients who did not require such hospitalization. RESULTS: The mean noncompliance ratio for the hospitalized patients was lower than the ratio for the controls (0.19 vs. 0.20) although the difference was not statistically significant (P = .95). There was no statistically significant difference between the demographics of the two groups. However, the patients who were hospitalized had a significantly greater number of COPD and nonCOPD medications (P < .0001, P < .0001) prescribed. They also had significantly more nonCOPD admissions and lengths of stay (P = .02, P = .01). CONCLUSION: At the levels of medication noncompliance observed in this population, there was no difference in rates of hospitalization. Hospitalization could be attributed to other causes such as severity of illness and existence of other comorbid conditions. The effects of environmental pollution and cigarette smoking were not studied. KEYWORDS: chronic obstructive pulmonary disease; hospitalization, noncompliance; risk factors. PMID- 16674334 TI - Pharmacoeconomic perspectives in Russia. PMID- 16674332 TI - Incontinence drug utilization patterns in Quebec, Canada. AB - OBJECTIVE: To assess prescription renewal and switch rates in an elderly population receiving oxybutynin or flavoxate for treatment of urinary incontinence in Quebec. METHODS: Sociodemographic, clinical, and drug claim data for patients > or = 65 years of age, with at least one claim for oxybutynin or flavoxate between January 1, 1994 and December 31, 1997, were randomly extracted from a database maintained by the Regie de l'assurance maladie du Quebec (RAMQ). Rates of renewal of the first drug claim and the number of patients switching from one incontinence drug to the other were determined. In addition, survival curves defining the time until cessation of initial treatment were constructed. RESULTS: The oxybutynin (n = 5718) and flavoxate (n = 972) treatment groups were similar in terms of gender (62.1% female) and age (mean age 77). For oxybutynin, 56.8% of the claims were written by general practitioners and 36.6% by urologists compared to 40.5% by general practitioners and 51.4% by urologists for flavoxate. Only 39.3% of the oxybutynin patients renewed their first claims, compared to 36.6% of the flavoxate group. Switch rates were higher for flavoxate patients with more than twice as many patients switching from this drug to oxybutynin than vice versa. Survival curves indicated that there was only an 11.4% probability of a patient taking oxybutynin for 6 months compared to 5.7% for flavoxate patients. CONCLUSIONS: Drug claim renewal rates were low for both oxybutynin and flavoxate, suggesting that money spent on these therapies provides an inadequate clinical return on investment since the majority of patients discontinue treatment prematurely. PMID- 16674335 TI - Pharmacoeconomics: identifying the issues overview and advisory panel report summary. PMID- 16674337 TI - Panel 2: methodological issues in conducting pharmacoeconomic evaluations- modeling studies. PMID- 16674336 TI - Panel 1: methodological issues in pharmacoeconomic evaluations--clinical studies. PMID- 16674338 TI - Panel 3: methodological issues in conducting pharmacoeconomic evaluations- retrospective and claims database studies. PMID- 16674339 TI - Panel 4: education and skills needed to conduct, interpret, and use economic evaluations in healthcare. PMID- 16674340 TI - Panel 5: application of healthcare intervention economic evaluations in healthcare decision-making. PMID- 16674341 TI - Panel 6: addressing questions of bias, credibility, and quality in health economic evaluations. PMID- 16674342 TI - Panel 7: communication and reporting health economic information. PMID- 16674343 TI - Recommendations for evaluating the validity of quality of life claims for labeling and promotion. AB - The pharmaceutical industry, the medical device industry, and national regulatory agencies such as the United States Food and Drug Administration (FDA) are faced with a number of difficult issues related to the development and evaluation of health-related quality of life (HRQL) claims for product labeling and promotion. This paper outlines some of the unique challenges of HRQL research and makes recommendations for assuring that claims are based on the results of rigorous studies designed and conducted according to accepted scientific principles and practices. Standards of evidence for HRQL are discussed in terms of research design and methodology, instrumentation, statistical analysis, and interpretation. Examples are provided to highlight important points. The paper concludes with a brief discussion of future trends in HRQL outcomes evaluation. PMID- 16674344 TI - A direction for Value in Health. PMID- 16674345 TI - The ISPOR Lipid Conference: pharmacoeconomics and outcomes modeling issues. PMID- 16674346 TI - The economics of hypercholesterolemia and lipid-lowering therapy: a brief historical tour. AB - The first formal economic evaluation of a lipid-lowering intervention was conducted almost 20 years ago. The field exploded in the mid-1980s following the publication of findings from the Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT), in which the bile-acid sequestrant, cholestyramine, was reported to reduce the incidence of coronary artery disease in adults with significant elevations in cholesterol. Almost all of the early pharmacoeconomic studies that followed focused on this agent. Later in the decade, the introduction of lovastatin, the first 3-hydroxy-3 methylglutaryl coenzyme A (HMG CoA) reductase inhibitor (or "statin"), revolutionized the treatment of hypercholesterolemia, as it was significantly more effective than earlier agents (as were the other statins that followed it). Pharmacoeconomic studies of the statins generally have reported that, despite their higher cost, they are significantly more cost-effective than bile acid sequestrants. Recent long-term clinical trials, such as the West of Scotland Coronary Prevention Study (WOSCOPS) and the Scandinavian Simvastatin Survival Study (4S), have provided firm evidence of the benefits of the statins in both the primary and secondary prevention of coronary artery disease. Formal economic evaluations were incorporated into most of these end-point studies-in contrast to morbidity and mortality trials of earlier lipid-lowering agents-and results from these evaluations are just now becoming available. The availability of primary economic data derived directly from large-scale, long-term clinical trials raises important questions about the future role of modeling in this area. PMID- 16674347 TI - Lipid-lowering pharmacoeconomic studies. Reactor panel and open forum. PMID- 16674348 TI - Compliance in the real world. AB - Until 1994, rates of noncompliance for lipid-lowering therapies were largely drawn from clinical trials and showed favorable risks for drug discontinuation, ranging from 4-15% for 1-year risk to 11-30% for 5-year risk. Although cross study comparisons are difficult to make because of variations in study design and measures collected, when evaluating compliance to antihyperlipidemic drugs in primary care settings, results in general show substantially higher rates of discontinuation than those reported from randomized clinical trials. Recent studies from the United States, Australia, and Canada support the conclusion that adherence to lipid-lowering drugs is very poor in primary care settings. PMID- 16674349 TI - Use of surrogate measures in cost-effectiveness analyses of lipid-lowering therapies. AB - Use of surrogate measures of effectiveness in cost-effectiveness analyses requires the assumption that a constant and monotonic relationship exists between the surrogate measure and the clinical outcome of interest. Results from epidemiologic studies and randomized controlled trials provide considerable support for the use of changes in lipids as a surrogate measure of effectiveness for changes in coronary heart disease risk and mortality in cost-effectiveness analyses of lipid-lowering therapies. Accordingly, the cost-effectiveness of lipid-lowering therapies is examined using efficiency-frontier analysis and a variety of surrogate measures, including the percent change in low density lipoprotein cholesterol (LDL-C) and the ratio of LDL-C to high density lipoprotein cholesterol (HDL-C), and the percentage of patients attaining goal LDL-C levels. These analyses suggest that niacin, fluvastatin (20 and 40 mg), simvastatin (5 mg), pravastatin (20 mg), and atorvastatin (10-80 mg) are cost effective therapies; simvastatin (10, 20, and 40 mg), pravastatin (10 and 40 mg), all dosages of lovastatin, and the bile acid sequestrants are not. Advantages and limitations of this methodology are discussed. PMID- 16674350 TI - Clinical trials and external validity. AB - Although a number of clinical trials are available estimating the benefits of lipid-lowering therapies that include economic end-points, development of modeling methodologies are essential to extend results of those trials over time and to other populations. We reviewed the key issues to be considered when extending trial data to real-world situations. The availability of recent randomized controlled trials of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in primary and secondary prevention has demonstrated the limitations of earlier modeling efforts to project benefits of lipid modification. The importance of risk stratification is demonstrated, particularly the importance of both LDL and HDL cholesterol either together or as a ratio measure. The selection of modeling methodology to extend benefits of a treatment beyond the end of a trial and over a lifetime is discussed. The relationship between benefits from lipid reduction and risk difference is described, demonstrating that for individuals with established coronary heart disease (CHD) and those older than age 58, benefits from lipid reduction are greater than those predicted from baseline lipid-related risk differences alone. The implications of these data for primary prevention of CHD in the elderly are discussed. PMID- 16674351 TI - Economic modeling and sensitivity analysis. AB - The field of pharmacoeconomics (PE) faces serious concerns of research credibility and bias. The failure of researchers to reproduce similar results in similar settings, the inappropriate use of clinical data in economic models, the lack of transparency, and the inability of readers to make meaningful comparisons across published studies have greatly contributed to skepticism about the validity, reliability, and relevance of these studies to healthcare decision makers. Using a case study in the field of lipid PE, two suggestions are presented for generally applicable reporting standards that will improve the credibility of PE. Health economists and researchers should be expected to provide either the software used to create their PE model or a multivariate sensitivity analysis of their PE model. Software distribution would allow other users to validate the assumptions and calculations of a particular model and apply it to their own circumstances. Multivariate sensitivity analysis can also be used to present results in a consistent and meaningful way that will facilitate comparisons across the PE literature. Using these methods, broader acceptance and application of PE results by policy-makers would become possible. To reduce the uncertainty about what is being accomplished with PE studies, it is recommended that these guidelines become requirements of both scientific journals and healthcare plan decision-makers. The standardization of economic modeling in this manner will increase the acceptability of pharmacoeconomics as a practical, real-world science. PMID- 16674352 TI - Methodology issues in lipid pharmacoeconomic investigations. Reactor panel and open forum. PMID- 16674353 TI - Introduction to the ISPOR Lipid Conference. PMID- 16674355 TI - Atherosclerosis, acute coronary events, and cholesterol-lowering: a new paradigm. AB - Our current understanding of some mechanisms involved with the occurrence of acute coronary events indicates that coronary atherosclerotic plaques need not develop to the point of stenosis before resulting in an acute event. Recent studies show that the majority of myocardial infarctions result from lesions that are 30-60% stenotic, many of which are not associated with any overt clinical symptoms. Considering that every year in the United States, approximately 200,000 persons with no previous history of coronary disease die from their first myocardial infarction, a major issue becomes identifying persons at risk for coronary disease. Risk assessment for coronary events is a critical area in terms of moving forward with decision-making for lipid-lowering therapies and developing strategies to reduce acute coronary events, and the morbidity and mortality related to them. PMID- 16674356 TI - Lipoproteins and cardiovascular disease: biological basis and epidemiological studies. AB - There is a long history to our understanding of the biological basis of lipoproteins in cardiovascular disease and to the key epidemiological studies in this field. Building on a wealth of laboratory evidence explaining the role of cholesterol and lipoproteins in the pathogenesis of atherosclerosis, epidemiological studies have characterized associations between serum lipid abnormalities and the risk of myocardial infarction. While elevation in total cholesterol has been associated with an increased risk of myocardial infarction, this information alone is not sufficient. To adequately predict cardiac risk, blood cholesterol must be further characterized by the high density lipoprotein (HDL) cholesterol present and the ratio of total cholesterol to HDL cholesterol. Several other factors, including such clinical syndromes as the "deadly quartet" of insulin resistance, central obesity, high triglycerides, and hypertension, are also associated with a markedly increased cardiac risk and should be identified; these may require unique therapeutic approaches. In addition to the costs of tertiary prevention and therapy after infarct, the impact of long-term morbidity and the economic consequences of this disease further emphasize the importance of optimizing current therapies and actual practice. PMID- 16674357 TI - Landmark trials in lipid reduction. AB - Since the first epidemiological evidence on the benefit of lipid reduction in coronary heart disease (CHD) was gathered, a number of questions have emerged in this field of research. Consistent findings across a number of landmark trials have shown that lipid-lowering therapies significantly reduced major subsequent cardiovascular events in coronary patients with high and moderate low density lipoprotein (LDL) blood cholesterol levels. Although there has been some confusion on the effect of lipid intervention on mortality, recently definite answers have come from several major trials that establish a clear decrease in coronary death for patients after reduction of plasma cholesterol levels. Primary prevention in individuals with high LDL cholesterol levels was shown to be beneficial in terms of cardiovascular event reduction; more data are needed to address this issue for subjects with moderate LDL cholesterol levels. Reduction of LDL cholesterol below normal levels appeared to be beneficial for coronary patients, but again, further research is needed to elucidate this point. To date, major trials have answered some of the many questions pertaining to lipid lowering therapies; further research will continue to provide information and allow patients to benefit maximally from lipid interventions. PMID- 16674358 TI - Prevention of heart disease: is LDL reduction the outcome of choice? Absolutely yes. AB - There is only one well-established relationship between blood cholesterol lipid fractions and coronary artery disease (CAD) that meets all the Heiss and Tyroler criteria of causality. While there are a number of blood lipid fractions, only LDL cholesterol satisfies these criteria. We review the effect of reducing low density lipo-protein (LDL) cholesterol levels on CAD in the landmark lipid intervention trials performed since 1972. Lowering of LDL cholesterol consistently and systematically resulted in a reduction of cardiovascular events, irrespective of the therapy applied or the changes in high density lipoprotein (HDL) cholesterol or triglyceride levels. Findings accumulated over many studies and many years provided sufficient evidence to satisfy all the criteria required to establish a causative relationship between LDL cholesterol and CAD. Consistent inter-ventional benefit demonstrated that reduction of LDL cholesterol is currently the treatment of choice in prevention of heart disease. PMID- 16674359 TI - Prevention of heart disease: is LDL reduction the outcome of choice? No, there is more. AB - The West of Scotland Coronary Prevention Study (WOSCOPS), a placebo-controlled 5 year cohort study, demonstrated that the use of pravastatin decreased low density lipoprotein (LDL) levels and associated risk of myocardial infarction. The rate of occurrence of coronary events, however, was similar across the four lowest quintiles of LDL reduction (23-41% reductions in mean LDL levels). The relationship between reduction of LDL and the reduction of risk was not linear. Further analysis indicated that even in overlap groups where patients exhibited equivalent mean LDL levels on treatment, pravastatin treatment was associated with less risk of occurrence of coronary events than placebo treatment. These results suggest that while LDL level does serve as a predictor of the risk of coronary events, other factors exist that should be considered and investigated further. PMID- 16674360 TI - Cardiovascular disease and lipoproteins: available evidence and remaining questions. Reactor panel and open forum. PMID- 16674361 TI - Health and economic outcomes modeling practices: a suggested framework. AB - In the field of outcomes research, modeling is increasingly being used to assess costs and outcomes associated with healthcare interventions. However, there is little standardization with regard to modeling practices, and the quality and usefulness of economic and health outcomes models may vary. We propose the following set of recommendations for good modeling practices to be used in both the development and review of models. These recommendations are divided into three sections: criteria to be addressed before model development or initial review; criteria used during model development or evaluation; and criteria used following model development. These recommendations also include examples regarding different modeling techniques and practices as well as a checklist (see appendix) to assess model correspondence with the recommendations. We hope that the use of good practice recommendations for modeling will not only improve the development and review of models but also lead to greater acceptance of this methodology. PMID- 16674538 TI - Willingness to pay versus willingness to buy: what defines value in healthcare? PMID- 16674539 TI - What is happening in the real world in lipid therapy and is it appropriate? The need for a paradigm shift. AB - Cardiovascular disease is now the leading cause of death and disability in our society. Current strategies in the United States have been very effective in treating the symptomatic manifestations of severe obstructions but have done little to alter the long-term outcome of cardiovascular disease. Although lipid lowering therapies have proven beneficial in secondary prevention for patients with cardiovascular disease, they are not as widely employed in contemporary practice as they should be. Better implementation of lipid-lowering therapies, including such major issues as clear treatment guidelines, physician and patient compliance, and delivery of healthcare and quality of care, must be addressed to shift thinking about the treatment of cardiovascular disease as we advance into the next century. PMID- 16674540 TI - Criteria for the economic evaluation of lipid studies in health systems. AB - From the perspective of a health system as a drug purchaser, it is becoming increasingly obvious that the traditional pharmacoeconomic cost-outcomes techniques of comparator drug evaluation are of limited application. The reason is that such approaches fail to address the information needs of drug purchasers. The key question that these evaluations should address is: If a new product is introduced to formulary, what will be the net impact on the costs of treatment of that disease or therapy area and on the outcomes profile of the treating population? It is on these criteria that claims made for lipid therapies should be judged. Issues that the traditional approach fails to address include: 1) the impact of budget constraints, 2) the determinants and impact of therapy switching, 3) the impact of poor compliance on claims for cost-effectiveness, and 4) the way in which more integrative approaches to treatment delivery might reinforce claims made for particular products. Guidelines are important in this process because they set out the informational requirements and analytical standards required of drug manufacturers making a submission to pharmacy and therapeutics committees. This paper identifies what may be considered to be the minimum informational requirements and suggests how a systems approach to drug evaluation could apply in the area of lipid therapy and the claims for competing products. PMID- 16674541 TI - Patient education and compliance: how to make it cost-effective. AB - The current level of noncompliance with prescription medications exerts a tremendous burden on the healthcare system in terms of both cost and poor health. The process of developing a program to improve compliance for lipid therapy is twofold. First, patients must be met at their specific level of need. This involves enabling, reinforcing, prompting, and problem solving. Second, the delivery of treatment must be addressed. Large centers and groups should refer lipid patients to a central team of professionals specializing in lipid therapy; or groups of physicians should contract extra patient support out to other groups, such as pharmacies. The implementation of a compliance program will not be immediately cost-effective but will have the long-term benefit of reduced costs through improved health. PMID- 16674542 TI - Commentary on Langley and McKenney Papers. PMID- 16674543 TI - A Canadian perspective. AB - Although the healthcare program in Canada is in many ways universal, there are several differences between the provinces. Two key federal bodies (the Health Protection Branch of Health Canada and the Patented Medicine Prices Review Board) are involved in the drug review process. In addition, the Canadian Coordinating Office for Health Technology Assessment (CCOHTA), an organization funded jointly by provinces and the federal government, reviews new or existing drugs at the request of provinces. Each province or territory and the federal government has its own drug plan or plans. The 10 Canadian provinces require a notice of compliance, information on pricing, and clinical and comparative information for submission of a new drug. Over the last 3 years, 8 of the 10 provinces have required pharmacoeconomic analyses to be added to the submissions. Four of the eight provinces requiring pharmacoeconomic evaluations have specified that these evaluations should be made according to the Canadian Coordinating Office for Health Technology Assessment guidelines. A review of the utilization patterns of lipid-lowering therapies is presented in this Canadian context. Statins comprised 80% of the lipid-lowering drug market in 1995. Prescribers use statins available in Canada interchangeably for all approved and nonapproved indications. The newest statin, atorvastatin, has attained a market share for new prescriptions equivalent to simvastatin and pravastatin, despite the lack of clinical outcome studies. CCOHTA reviewed the evidence of efficacy and effectiveness of statins, evaluated whether significant differences existed between statins, and analyzed published economic evaluations. It found that treatment of higher-risk patients with statins is more cost-effective than that of lower-risk subjects. The evaluation also concluded that the statins, as a class, probably act in reducing LDL and raising HDL, resulting in beneficial clinical outcomes. PMID- 16674544 TI - A UK Perspective. AB - Making decisions on lipid-lowering treatment varies across Europe. Legislation differs from country to country, so it is difficult to generalize about European policy. From the perspective of the United Kingdom, this paper addresses the impact of rationing, cost containment, information feedback, fund-holding and drug expenses. Primary versus secondary prevention for cardiovascular disease is also discussed, including current recommendations for prescribing statins and other interventions. PMID- 16674545 TI - A US military perspective. AB - This paper discusses lipid therapy decision-making at the population level within the United States (US) Military Health Services System. The US Military Health Services System serves approximately 8.2 million beneficiaries fairly representative of the general population. Of its $680 million annual pharmacy expenditure, $45-$50 million is spent on lipid-lowering drugs. The military Medical Treatment Facilities (MTF) carry a Tri-Service Drug Formulary list, which serves as a bare minimum to which individual facilities may add other agents. The Department of Defense Pharmacoeconomic Center (PEC) performs cost-effectiveness analyses, establishes the Tri-Service Drug Formulary list and the National Mail Order Pharmacy formulary list, and provides drug treatment guidelines. Hyperlipidemia treatment guidelines published by the Pharmacoeconomic Center in 1995, which recommend the use of niacin, colestipol, and pravastatin, are currently under revision to take into consideration new drugs and new information in the field of lipid-lowering therapies. Future changes to the MTF outpatient formulary management may include the introduction of a Basic Core List of essential agents and a Master Pharmaceutical Agent List comprising the only drugs from which facilities may choose to add to their Basic Core List. These changes are designed to standardize drug availability across military facilities to leverage market share for lower drug prices. PMID- 16674546 TI - A managed care perspective. AB - This paper highlights some of the problems associated with lipid therapy in the primary and secondary prevention of cardiovascular disorders and to make some potentially useful suggestions in the context of managed care. For managed care organizations, financial and logistical issues create obstacles to the provision of primary prevention of cardiovascular disease. These current obstacles necessitate the generation of external forces, perhaps regulatory or standards agencies, that may help increase accountability in managed care organizations for midterm and distant outcomes. In contrast, the provision of secondary prevention by managed care organizations has fewer limitations. One of the major challenges in secondary prevention, however, is the low rate of physician compliance with national treatment guidelines and standards. Among possible explanations for this observation are limitations in health data collection and integration. Improvements in data management are vital to the achievement of treatment goal optimization in secondary prevention. PMID- 16674547 TI - A pharmacy benefit manager perspective. AB - Lipid-lowering therapy can account for 7% of per patient per month drug costs. Because this can be a significant proportion of a payer's drug expenditure, this class of drugs attracts payers' attention and thus becomes a focus of efforts designed to control drug expenditures. Pharmacy benefit managers (PBMs) use several techniques and capabilities to affect the use of these drugs to improve overall medical care and to capture financial opportunities. There is a gap between the marketplace needs for lipid therapy value information and current pharmacoeconomic evaluations of lipid therapy. The measurement units that current pharmacoeconomic analyses tend to use are not necessarily intuitive and do not easily conform to the existing conceptual framework of policy makers. The successful evaluation of PBM activities will be contingent on a recognizable and widely accepted quantitative measurement framework. PMID- 16674548 TI - Employer and consumer perspectives. AB - This paper reviews various published reports from surveys on employer opinion, perception of needs, and trends with regard to healthcare benefits; the consumer perspective regarding healthcare is also discussed. Surveys indicate that businesses want continuous evidence that high-quality healthcare can positively impact company profits. Employers and labor unions are demanding more cost effective healthcare. At both employer and consumer levels, greater patient education is needed, as well as traditional educational media. Direct-to-consumer advertising and use of the World Wide Web are increasingly important in enabling consumers to participate more fully in their own lipid-related decision-making. Finally, the transition of lipid-lowering drugs to over-the-counter accessibility has great implications with respect to issues of patient preferences and willingness to pay in the evolving healthcare environment. Groups in the United States, such as the National Committee on Quality Assurance and the Foundation for Accountability, are setting standards and beginning to assess both process and outcomes in patient care. Further collaborative efforts are needed that raise standards of care and stimulate more cost-effective healthcare. The pharmacoeconomics and outcomes data gathered will, one hopes, also demonstrate to global businesses the positive financial impact of high-quality healthcare and appropriate lipid therapy. PMID- 16674549 TI - From research into practice: how should healthcare organizations/governments decide about lipid therapy and who will pay? Reactor panel and open forum. PMID- 16674550 TI - Prevalence-based economic evaluation. AB - OBJECTIVE: Researchers have often stated that economic evaluations of new drugs have rarely been used to inform healthcare decisions, despite the large volume of published studies. In this paper, a new categorization for economic evaluations of new drugs is proposed: incidence-based and prevalence-based. This categorization is designed to increase the likelihood that decision-makers are given more complete and usable economic information about new treatments. RESULTS: Incidence-based evaluations (such as cost-effectiveness analysis) focus on the impact of a new treatment on a health condition from onset until cure or death. Prevalence-based evaluations focus on the impact of a new treatment on a health condition during a 1-year period. An incidence-based evaluation may focus either on a representative individual or on a specific disease cohort. A prevalence-based evaluation generally focuses on a specific population. Incidence based evaluations measure the value of the new treatment compared to alternative treatments for the same health conditions and compared to commonly used treatments for other health conditions. Prevalence-based evaluations measure the impact of introducing the new treatment on annual healthcare budgets and population health. CONCLUSION: Both types of evaluation provide important information when a new treatment is introduced to a population. PMID- 16674551 TI - An analysis paradigm for investigating multi-locus effects in complex disease: examination of three GABA receptor subunit genes on 15q11-q13 as risk factors for autistic disorder. AB - Gene-gene interactions are likely involved in many complex genetic disorders and new statistical approaches for detecting such interactions are needed. We propose a multi-analytic paradigm, relying on convergence of evidence across multiple analysis tools. Our paradigm tests for main and interactive effects, through allele, genotype and haplotype association. We applied our paradigm to genotype data from three GABAA receptor subunit genes (GABRB3, GABRA5, and GABRG3) on chromosome 15 in 470 Caucasian autism families. Previously implicated in autism, we hypothesized these genes interact to contribute to risk. We detected no evidence of main effects by allelic (PDT, FBAT) or genotypic (genotype-PDT) association at individual markers. However, three two-marker haplotypes in GABRG3 were significant (HBAT). We detected no significant multi-locus associations using genotype-PDT analysis or the EMDR data reduction program. However, consistent with the haplotype findings, the best single locus EMDR model selected a GABRG3 marker. Further, the best pairwise genotype-PDT result involved GABRB3 and GABRG3, and all multi-locus EMDR models also selected GABRB3 and GABRG3 markers. GABA receptor subunit genes do not significantly interact to contribute to autism risk in our overall data set. However, the consistency of results across analyses suggests that we have defined a useful framework for evaluating gene-gene interactions. PMID- 16674552 TI - Obesity is associated with genetic variants that alter dopamine availability. AB - Human and animal studies have implicated dopamine in appetite regulation, and family studies have shown that BMI has a strong genetic component. Dopamine availability is controlled largely by three enzymes: COMT, MAOA and MAOB, and by the dopamine transporter SLC6A3, and each gene has a well-characterized functional variant. Here we look at these four functional polymorphisms together, to investigate how heritable variation in dopamine levels influences the risk of obesity in a cohort of 1150, including 240 defined as obese (BMI > or = 30). The COMT and SLC6A3 polymorphisms showed no association with either weight, BMI or obesity risk. We found, however, that both MAOA and MAOB show an excess of the low-activity genotypes in obese individuals (MAOA:chi2= 15.45, p = 0.004; MAOB:chi2= 8.05, p = 0.018). Additionally, the MAOA genotype was significantly associated with both weight (p = 0.0005) and BMI (p = 0.001). When considered together, the 'at risk genotype'--low activity genotypes at both the MAOA and MAOB loci--shows a relative risk for obesity of 5.01. These results have not been replicated and, given the experience of complex trait genetics, warrant caution in interpretation. In implicating both the MAOA and MOAB variants, however, this study provides the first indication that dopamine availability (as opposed to other effects of MAOA) is involved in human obesity. It is therefore a priority to assess the associations in replication datasets. PMID- 16674553 TI - A frequent partial AZFc deletion does not render an increased risk of spermatogenic impairment in East Asians. AB - The gene families in the AZFc region of the Y chromosome have been shown to be functionally important in human spermatogenesis. The gr/gr deletion, a partial AZFc deletion that reduces the copy numbers of all the AZFc gene families, was identified as a significant risk factor for spermatogenic impairment in Dutch, Spanish and Italians. However, the presence of this deletion in healthy French and Germans questioned its importance in male infertility. In this study, we have shown that the gr/gr deletion does not render an increased risk in Han Chinese. In fact, the gr/gr deletion is frequent (about 8%) in our survey of 886 East Asians from 8 ethnic groups. Furthermore, the DAZ1/DAZ2 deletion has been detected as the primary subtype of the gr/gr deletion in East Asians, though this doublet has been considered as crucial for normal spermatogenesis in Europeans. The different spermatogenic effects of various types of the partial AZFc deletion suggest that the functional difference between AZFc gene copies is a likely cause of inconsistent associations of the gr/gr deletion with spermatogenic impairment across populations. PMID- 16674554 TI - Quality assessment of DNA sequence data: autopsy of a mis-sequenced mtDNA population sample. AB - Published DNA data sets constitute a body of sequencing results resting in silico that are supposed to reflect the variation of (once) living cells. In cases where the DNA variation reported is suspected to be fraught with artefacts, an autopsy of the full body of data is needed to clarify the amount and causes of mis sequencing. In this paper we elaborate on strategies that allow a clear-cut identification of the problems in severely flawed mtDNA data. This approach is applied, by way of example, to a data set of HVS-I sequences from the Caucasus, published by Nasidze & Stoneking in 2001. These data bear numerous ambiguous nucleotide positions and suffer from an even higher number of phantom mutations, indicating that severe biochemical problems adversely influenced those sequencing results at the time. Furthermore, systematic omission of sequences with a long C stretch (incurred by a transition at position 16189) must have severely biased the data set. Since no complete correction of these data has appeared to date, this example of mis-sequencing necessitates circumstantial evidence that is bullet-proof. PMID- 16674555 TI - The patient is not dead yet: premature autopsy of a mtDNA data set. PMID- 16674556 TI - Design and analysis of genetic association studies to finely map a locus identified by linkage analysis: sample size and power calculations. AB - Association (e.g. case-control) studies are often used to finely map loci identified by linkage analysis. We investigated the influence of various parameters on power and sample size requirements for such a study. Calculations were performed for various values of a high-risk functional allele (fA), frequency of a marker allele associated with the high risk allele (f1), degree of linkage disquilibrium between functional and marker alleles (D') and trait heritability attributable to the functional locus (h2). The calculations show that if cases and controls are selected from equal but opposite extreme quantiles of a quantitative trait, the primary determinants of power are h2 and the specific quantiles selected. For a dichotomous trait, power also depends on population prevalence. Power is optimal if functional alleles are studied (fA= f1 and D'= 1.0) and can decrease substantially as D' diverges from 1.0 or as f(1) diverges from fA. These analyses suggest that association studies to finely map loci are most powerful if potential functional polymorphisms are identified a priori or if markers are typed to maximize haplotypic diversity. In the absence of such information, expected minimum power at a given location for a given sample size can be calculated by specifying a range of potential frequencies for fA (e.g. 0.1-0.9) and determining power for all markers within the region with specification of the expected D' between the markers and the functional locus. This method is illustrated for a fine-mapping project with 662 single nucleotide polymorphisms in 24 Mb. Regions differed by marker density and allele frequencies. Thus, in some, power was near its theoretical maximum and little additional information is expected from additional markers, while in others, additional markers appear to be necessary. These methods may be useful in the analysis and interpretation of fine-mapping studies. PMID- 16674557 TI - A comparison of individual genotyping and pooled DNA analysis for polymorphism validation prior to large-scale genetic studies. AB - Polymorphism validation is an important issue in genetic studies because only polymorphic markers provide useful information. We analyzed genetic data for 180 SNPs in the human major histocompatibility complex region in Caucasian and Taiwanese populations, and evaluated ethnic heterogeneity between these populations to illustrate the importance of polymorphism validation. An initial individual genotyping experiment (IGE) with 95 samples was compared with a DNA pooling allele-typing experiment (PAE) of 630 individuals for polymorphism validation based on authentic data sets. Afterwards, all samples were genotyped individually in a confirmation study. Under narrow (broad) polymorphism criteria, 24 (41) polymorphic SNPs in Caucasians could not be validated in the Taiwanese population, suggesting a 13% (23%) inconsistency rate and revealing a strong discrepancy between genetic backgrounds, probably due to ethnic heterogeneity. IGE yielded high sensitivity and specificity for polymorphism validation, but may be sensitive to sampling variation. PAE showed high sensitivity (97%) and specificity (100%) using a narrow polymorphism criterion, but reduced specificity (83%) using a broad criterion. Public domain polymorphism databases should therefore be used with caution and polymorphism validation should be performed routinely prior to conducting large-scale genetic studies. PAE is a cost-saving, reliable alternative to IGE for polymorphism validation, especially for a stringent polymorphism criterion. PMID- 16674558 TI - Appropriate use of information on family history of disease in recruitment for linkage analysis studies. AB - When conducting genetic studies for complex traits, large samples are commonly required to detect any of the number of genes with relatively low effect thought to underly such traits. This is because, in contrast to monogenic diseases, complex traits typically result from a number of different genetic pathways (genetic heterogeneity) and any sample is likely to contain a considerable fraction of sporadic cases (phenocopies). Such samples are time-consuming and costly to recruit and analyse. Methods which might be used to decrease sample size include attempting to select families, with the aim of reducing genetic heterogeneity or phenocopy rate within the sample. Selecting cases with positive family history of disease should reduce the phenocopy rate, and this strategy has been employed in linkage studies of complex disease, although evaluations of such a strategy have been equivocal. This paper shows how identity by descent (IBD) distributions may be calculated for affected relative pairs recruited conditional on the affection status of a third relative. These distributions are then used to calculate expected power in affected sib and half-sib linkage studies when recruitment is conditional on family history of disease. We consider the proxy conditions of recruitment conditional on disease in an affected parent or third sibling with single-locus and additive multilocus genetic models. We show that while such selection strategies can reduce power if disease risk alleles are common and environmental heterogeneity low, under models more likely to underly common complex diseases power will generally be increased, and that this effect is greater as more loci are involved. Though the proxy cases studied are more extreme than a general strategy of asking potential recruits whether they have any family history of disease, these results suggest that conditional recruitment is more generally useful than previous studies have suggested. PMID- 16674559 TI - Multiple imputation of missing genotype data for unrelated individuals. AB - The objective of this study was to investigate the performance of multiple imputation of missing genotype data for unrelated individuals using the polytomous logistic regression model, focusing on different missingness mechanisms, percentages of missing data, and imputation models. A complete dataset of 581 individuals, each analysed for eight biallelic polymorphisms and the quantitative phenotype HDL-C, was used. From this dataset one hundred replicates with missing data were created, in different ways for different scenarios. The performance was assessed by comparing the mean bias in parameter estimates, the root mean squared standard errors, and the genotype-imputation error rates. Overall, the mean bias was small in all scenarios, and in most scenarios the mean did not differ significantly from 'no bias'. Including polymorphisms that are highly correlated in the imputation model reduced the genotype-imputation error rate and increased precision of the parameter estimates. The method works well for data that are missing completely at random, and for data that are missing at random. In conclusion, our results indicate that multiple imputation with the polytomous logistic regression model can be used for association studies to deal with the problem of missing genotype data, when attention is paid to the imputation model and the percentage of missing data. PMID- 16674560 TI - Case-parent triads: estimating single- and double-dose effects of fetal and maternal disease gene haplotypes. AB - Case-parent triad data are considered a robust basis for studying association between variants of a gene and a disease. Methods evaluating statistical significance of association, like the TDT-test and its extensions, are frequently used. When there are prior hypotheses of a causal effect of the gene under study, however, methods measuring penetrance of alleles or haplotypes as relative risks will be more informative. Log-linear models have been proposed as a flexible tool for such relative risk estimation. We demonstrate an extension of the log-linear model to a natural framework for also estimating effects of multiple alleles or haplotypes, incorporating both single- and double-dose effects. The model also incorporates effects of single- and double-dose maternal haplotypes on a fetus during pregnancy. Unknown phase of haplotypes as well as missing parents are accounted for by the EM algorithm. A number of numerical improvements to maximum likelihood estimation are also implemented to facilitate a larger number of haplotypes. Software for these analyses, HAPLIN, is publicly available through our web site. As an illustration we have re-analyzed data on the MSX1 homeobox gene on chromosome 4 to show how haplotypes may influence the risk of oral clefts. PMID- 16674561 TI - Detecting linkage disequilibrium in the presence of locus heterogeneity. AB - Locus heterogeneity is a common phenomenon in complex diseases and is one of the most important factors that affect the power of either linkage or linkage disequilibrium (LD) analysis. In linkage analysis, the heterogeneity LOD score (HLOD) rather than LOD itself is often used. However, the existing methods for detecting linkage disequilibrium, such as the TDT and many of its variants, do not take into account locus heterogeneity. We propose two novel likelihood-based methods, an LD-Het likelihood and an LD-multinomial likelihood, to test linkage disequilibrium (LD) that explicitly incorporate locus heterogeneity in the analysis. The LD-Het is applicable to general nuclear family data but requires a working penetrance model. The LD-multinomial is only applicable to affected sib pair data but does not require specification of a trait model. For affected sib pair data, both methods have similar power to detect LD under the recessive model, but the LD-multinomial model has greater power when the underlying model is dominant or additive. PMID- 16674562 TI - Study of the PVRL1 gene in Italian nonsyndromic cleft lip patients with or without cleft palate. AB - Nonsyndromic cleft lip with or without cleft palate (CL/P) is a complex genetic trait and little is known about its aetiology. Recent investigations on rare clefting syndromes provided interesting clues about genes involved in face development. The PVRL1 gene encodes nectin1, a cell-to-cell adhesion molecule. Mutations in its sequence have been shown to cause the rare autosomal recessive syndrome CL/P-ectodermal dysplasia syndrome (CLPED1), while heterozygosity for the mutation W185X seemed to increase the risk of non syndromic CL/P in a population from northern Venezuela. In the present study, we screened 143 Italian CL/P patients for mutations in PVRL1. Three rare sequence variants in exon 3 that create amino-acid changes were detected in a total of 7 patients. Two of these mutations were not found in a panel of 292 unaffected controls, while the third was found in two controls. This study describes new mutations that may represent genetic risk factors for CL/P. Even though a study to look at the effects of the mutations on nectin1 function was not feasible, supporting evidence was reported, thus confirming the involvement of PVRL1 in the aetiology of non-syndromic CL/P malformation. PMID- 16674563 TI - Myotilin is not the causative gene for vocal cord and pharyngeal weakness with distal myopathy (VCPDM). AB - Myotilin (MYOT) is a promising candidate gene for Vocal Cord and Pharyngeal Weakness with Distal Myopathy (VCPDM, also known as MPD2). Located within the minimum VCPDM candidate interval, myotilin mutations also cause a similarly progressive and adult-onset muscle disease. We examined myotilin in VCPDM patients by sequence analysis, RT-PCR, Southern blotting, and western blotting. We detected no defects in the myotilin gene, transcript, or protein in VCPDM. We also report several useful SNPs and STRs for the analysis of myotilin in muscle diseases of suspected, yet unknown genetic origin. We conclude that MYOT mutations likely are not a cause of VCPDM. PMID- 16674564 TI - Sexually antagonistic coevolution in insects is associated with only limited morphological diversity. AB - Morphological traits involved in male-female sexual interactions, such as male genitalia, often show rapid divergent evolution. This widespread evolutionary pattern could result from sustained sexually antagonistic coevolution, or from other types of selection such as female choice or selection for species isolation. I reviewed the extensive but under-utilized taxonomic literature on a selected subset of insects, in which male-female conflict has apparently resulted in antagonistic coevolution in males and females. I checked the sexual morphology of groups comprising 500-1000 species in six orders for three evolutionary trends predicted by the sexually antagonistic coevolution hypothesis: males with species specific differences and elaborate morphology in structures that grasp or perforate females in sexual contexts; corresponding female structures with apparently coevolved species-specific morphology; and potentially defensive designs of female morphology. The expectation was that the predictions were especially likely to be fulfilled in these groups. A largely qualitative overview revealed several surprising patterns: sexually antagonistic coevolution is associated with frequent, relatively weak species-specific differences in males, but male designs are usually relatively simple and conservative (in contrast to the diverse and elaborate designs common in male structures specialized to contact and hold females in other species, and also in weapons such as horns and pincers used in intra-specific battles); coevolutionary divergence of females is not common; and defensive female divergence is very uncommon. No cases were found of female defensive devices that can be facultatively deployed. Coevolutionary morphological races may have occurred between males and females of some bugs with traumatic insemination, but apparently as a result of female attempts to control fertilization, rather than to reduce the physical damage and infections resulting from insertion of the male's hypodermic genitalia. In sum, the sexually antagonistic coevolution that probably occurs in these groups has generally not resulted in rapid, sustained evolutionary divergence in male and female external sexual morphology. Several limitations of this study, and directions for further analyses are discussed. PMID- 16674565 TI - Sociality, age at first reproduction and senescence: comparative analyses of birds. AB - Evolutionary theories of senescence suggest that aging evolves as a consequence of early reproduction imposing later viability costs, or as a consequence of weak selection against mutations that act late in life. In addition, highly social species that live in sites that are protected from extrinsic mortality due to predation should senesce at a slower rate than solitary species. Therefore, species that start reproducing late in life should senesce at a slower rate than species that start reproducing early. In addition, social species should senesce more slowly than solitary species. Here I investigate the rate of senescence using an extensive data set on longevity records under natural field conditions to test predictions about the evolution of senescence among 271 species of birds. Longevity records increased with sampling effort and body mass, but once these confounding variables were controlled statistically, there was a strongly positive relationship between relative longevity and relative adult survival rate. Relative longevity after controlling statistically for sampling effort, body mass and adult survival rate, increased with age at first reproduction, but not with degree of breeding sociality. These findings suggest that the evolution of senescence is related to timing of first reproduction, but that the evolution of breeding sociality has played a negligible role in the evolution of senescence. PMID- 16674566 TI - Ontogenic sources of variation in sexual size dimorphism in a viviparous lizard. AB - To elucidate the developmental aspects of the evolution of sexual size dimorphism (SSD), an understanding of the sex-specific ontogeny of body size is critical. Here, we evaluate the relative importance of genetic and environmental determinants of SSD in juvenile common lizards (Lacerta vivipara). We examined the prenatal and post-natal effects of population density and habitat humidity on SSD, as well as the maternal effects of food availability, corticosterone level, humidity and heat regime during gestation. Analyses indicated strong prenatal and post-natal plasticity in body size per se and yielded three main results with respect to SSD. First, SSD in juvenile common lizards matches qualitatively the SSD observed in adults. Secondly, SSD was influenced by none of the prenatal factors investigated here, suggesting poor sex-biased maternal effects on offspring size. Thirdly, SSD was sensitive to post-natal habitat humidity, which positively affected growth rate more strongly in females than in males. Thus, natural variation in SSD in juvenile common lizards appears to be primarily determined by a combination of sex-biased genetic factors and post-natal conditions. We discuss the possibility that viviparity may constrain the evolution of sex-biased maternal effects on offspring size. PMID- 16674567 TI - Distortion of symmetrical introgression in a hybrid zone: evidence for locus specific selection and uni-directional range expansion. AB - The fate of species integrity upon natural hybridization depends on the interaction between selection and dispersal. The relative significance of these processes may be studied in the initial phase of contact before selection and gene flow reach equilibrium. Here we study a hybrid zone of two salamander species, Lyciasalamandra antalyana and Lyciasalamandra billae, at the initial phase of hybridization. We quantify the degree and mode of introgression using nuclear and mtDNA markers. The hybrid zone can be characterized as an abrupt transition zone, the central hybrid zone being only c. 400 m, but introgressed genes were traced up to 3 km. Introgression was traced in both sexes but gene flow may be slightly male-biased. Indirect evidence suggests that hybrid males are less viable than females. Introgression occurred at two levels: (1) locus specific selection led to different allelic introgression patterns independent of species, while (2) asymmetrical species-level introgression occurred predominately from L. antalyana to L. billae due to range expansion of the former. This indicates that foreign genes can be incorporated into novel genomic environments, which in turn may contribute to the great diversity of morphological variants in Lyciasalamandra. PMID- 16674568 TI - Gis-based niche models reveal unifying climatic mechanisms that maintain the location of avian hybrid zones in a North American suture zone. AB - The existence of suture zones in North America has recently been verified, yet the environmental factors responsible for the maintenance of their structure and position have remained undetermined. The Great Plains suture zone in the US is perhaps the most significant and broadly studied in North America. Numerous avian hybrid zones that cluster in this region have been extensively studied over the last half-century. A primary result of this work is that exogenous factors present in this region have promoted and maintained avian species divergence. Yet, to date the variables most important in the generation and the clustered positioning of these hybrid zones in relation to one another have not been determined. The present study aims to advance our understanding of this North American suture zone by using niche modelling to quantify which environmental variables are most important in its formation and maintenance. Through the generation of fundamental niches for four hybrids and their parental species, similarities linking the hybrid zones were uncovered. The results show that temperature holds this suture zone in position and also suggest that temperature played a primary role in promoting niche differentiation among these sister species pairs during Pleistocene glacial cycles. PMID- 16674569 TI - A new route to the evolution of cooperation. AB - The Prisoner's Dilemma (PD) constitutes a widely used metaphor to investigate problems related to the evolution of cooperation. Whenever evolution takes place in well-mixed populations engaged in single rounds of the PD, cooperators cannot resist invasion by defectors, a feature, which is somewhat alleviated whenever populations are spatially distributed. In both cases the populations are characterized by a homogeneous pattern of connectivity, in which every individual is equivalent, sharing the same number of neighbours. Recently, compelling evidence has been accumulated on the strong heterogeneous nature of the network of contacts between individuals in populations. Here we describe the networks of contacts in terms of graphs and show that heterogeneity provides a new mechanism for cooperation to survive. Specifically, we show that cooperators are capable of exploring the heterogeneity of the population structure to become evolutionary competitive. As a result, cooperation becomes the dominating trait in scale-free networks of contacts in which the few highly connected individuals are directly inter-connected, in this way contributing to self-sustain cooperation. PMID- 16674570 TI - Supertree analyses of the roles of viviparity and habitat in the evolution of atherinomorph fishes. AB - Using supertree phylogenetic reconstructions, we investigate how livebearing and freshwater adaptations may have shaped evolutionary patterns in the Atherinomorpha, a large clade (approximately 1500 extant species) of ray-finned fishes. Based on maximum parsimony reconstructions, livebearing appears to have evolved at least four times independently in this group, and no reversions to the ancestral state of oviparity were evident. With respect to habitat, at least five evolutionary transitions apparently occurred from freshwater to marine environments, at least two transitions in the opposite direction, and no clear ancestral state was identifiable. All viviparous clades exhibited more extant species than their oviparous sister taxa, suggesting that transitions to viviparity may be associated with cladogenetic diversification. Transitions to freshwater were usually, but not invariably associated with increased species richness, but the trend was, overall, not significant among sister clades. Additionally, we investigated whether livebearing and freshwater adaptations are currently associated with elevated risks of extinction as implied by species' presence on the 2004 IUCN Red List. Despite being correlated with decreased brood size, livebearing has not significantly increased extinction risk in the Atherinomorpha. However, freshwater species were significantly more likely than marine species to be listed as endangered. PMID- 16674571 TI - Parallel evolution of the sexes? Effects of predation and habitat features on the size and shape of wild guppies. AB - Environmental gradients often lead to the parallel evolution of populations and species. To what extent do such gradients also lead to parallel evolution of the sexes? We used guppies (Poecilia reticulata) to examine the parallel and independent (sex-specific) aspects of population divergence in response to predation and habitat features. Geometric morphometrics was used to analyse size and shape variation for 1335 guppies from 27 to 31 sites sampled in each of 2 years. Body size showed strong parallel population divergence; both sexes were larger at sites with a more open canopy and with higher flow. Body shape showed a mixture of parallel and independent population divergence. The strongest and most consistent effects were (1) high-predation sites had males with smaller heads and deeper caudal peduncles, (2) open-canopy sites had females with smaller heads and more distended abdomens and (3) high-flow sites had males and females with smaller heads and deeper caudal peduncles. PMID- 16674572 TI - Phylogenetic relationships and evolution of cleaning behaviour in the family Labridae: importance of body colour pattern. AB - The family Labridae contains numerous fishes known to act as cleaners in the wild. Previous studies suggested that a small body size and specific colour patterns may be prerequisites for cleaning. We investigated whether cleaning behaviour is linked to particular fish phenotypes. We first present a phylogeny based on partial 12S rRNA gene sequences of 32 wrasses sampled from different localities in the Indo-Pacific and Atlantic oceans, and in the Mediterranean Sea. Secondly, descriptive data (fish body size, fish body shape and fish body colour patterns) were analyzed in a phylogenetic context using comparative methods. We found no relationship between fish cleaning behaviour and fish body size and shape, but instead a correlation between cleaning behaviour and the presence of a dark lateral stripe within wrasses. Our results suggest that the evolution of cleaning depends upon the presence of a dark median lateral stripe on the fish body surface. PMID- 16674573 TI - Response of fluctuating and directional asymmetry to selection on wing shape in Drosophila melanogaster. AB - We tested whether directional selection on an index-based wing character in Drosophila melanogaster affected developmental stability and patterns of directional asymmetry. We selected for both an increase (up selection) and a decrease (down selection) of the index value on the left wing and compared patterns of fluctuating and directional asymmetry in the selection index and other wing traits across selection lines. Changes in fluctuating asymmetry across selection lines were predominantly small, but we observed a tendency for fluctuating asymmetry to decrease in the up-selected lines in both replicates. Because changes in fluctuating asymmetry depended on the direction of selection, and were not related to changes in trait size, these results fail to support existing hypotheses linking directional selection and developmental stability. Selection also produced a pattern of directional asymmetry that was similar in all selected lines whatever the direction of selection. This result may be interpreted as a release of genetic variance in directional asymmetry under selection. PMID- 16674574 TI - No sympatric speciation here: multiple data sources show that the ant Myrmica microrubra is not a separate species but an alternate reproductive morph of Myrmica rubra. AB - No aspect of speciation is as controversial as the view that new species can evolve sympatrically, among populations in close physical contact. Social parasitism has been suggested to yield necessary disruptive selection for sympatric speciation. Recently, mitochondrial DNA phylogeography has shown that the ant Myrmica microrubra is closely related to its host, Myrmica rubra, leading to the suggestion that sympatric speciation has occurred. We investigated the relationships between the two ant forms using mitochondrial and nuclear DNA sequences, microsatellite genotyping and morphometrics. Molecular phylogenetic and population structure analyses showed that M. microrubra does not evolve separately to its host but rather shares a gene pool with it. Probability analysis showed that mitochondrial DNA data previously adduced in favour of sympatric speciation do not in fact do so. Morphometrically, M. microrubra is most readily interpreted as a miniature queen form of M. rubra, not a separate species. Myrmica microrubra is not an example of speciation. The large (typical M. rubra) and small (M. microrubra) queen forms are alternative reproductive strategies of the same species. Myrmica microrubraSeifert 1993 is consequently synonymized here with M. rubra Linnaeus, 1758. PMID- 16674575 TI - The costs and benefits in an unusual symbiosis: experimental evidence that bitterling fish (Rhodeus sericeus) are parasites of unionid mussels in Europe. AB - Interspecific symbiotic relationships involve a complex network of interactions, and understanding their outcome requires quantification of the costs and benefits to both partners. We experimentally investigated the costs and benefits in the relationship between European bitterling fish (Rhodeus sericeus) and freshwater mussels that are used by R. sericeus for oviposition. This relationship has hitherto been thought mutualistic, on the premise that R. sericeus use mussels as foster parents of their embryos while mussels use R. sericeus as hosts for their larvae. We demonstrate that R. sericeus is a parasite of European mussels, because it (i) avoids the cost of infection by mussel larvae and (ii) imposes a direct cost on mussels. Our experiments also indicate a potential coevolutionary arms race between bitterling fishes and their mussel hosts; the outcome of this relationship may differ between Asia, the centre of distribution of bitterling fishes, and Europe where they have recently invaded. PMID- 16674576 TI - Ornament evolution in dragon lizards: multiple gains and widespread losses reveal a complex history of evolutionary change. AB - The expression in females of ornaments thought to be the target of sexual selection in males is a long-standing puzzle. Two main hypotheses are proposed to account for the existence of conspicuous ornaments in both sexes (mutual ornamentation): genetic correlation between the sexes and sexual selection on females as well as males. We examined the pattern of ornament gains and losses in 240 species of dragon lizards (Agamidae) in order to elucidate the relative contribution of these two factors in the evolution of mutual ornamentation. In addition, we tested whether the type of shelter used by lizards to avoid predators predicts the evolutionary loss or constraint of ornament expression. We found evidence that the origin of female ornaments is broadly consistent with the predictions of the genetic correlation hypothesis. Ornaments appear congruently in both sexes with some lineages subsequently evolving male biased sexual dimorphism, apparently through the process of natural selection for reduced ornamentation in females. Nevertheless, ornaments have also frequently evolved in both sexes independently. This suggests that genetic correlations are potentially weak for several lineages and sexual selection on females is responsible for at least some evolutionary change in this group. Unexpectedly, we found that the evolutionary loss of some ornaments is concentrated more in males than females and this trend cannot be fully explained by our measures of natural selection. PMID- 16674577 TI - Temporal differentiation and spatial coexistence of sexual and facultative asexual lineages of an aphid species at mating sites. AB - Cases of coexisting sexual and asexual relatives are puzzling, as evolutionary theory predicts that competition for the same ecological niches should lead to the exclusion of one or the other population. In the cyclically parthenogenetic aphid, Rhopalosiphum padi, sexual and facultative asexual lineages are admixed in space at the time of sexual reproduction. We investigated how the interaction of reproductive mode and environment can lead to temporal niche differentiation. We demonstrated theoretically that differential sensitivity of sexual and facultatively asexual aphids to an environmental parameter (mating host suitability) shapes the two strategies: whereas the sexual lineages switch earlier to the production of sexual forms, the facultative asexual lineages delay and spread out their investment in sexual reproduction. This predicted pattern of niche specialization is in agreement with the temporal structure revealed in natura by demographic and genetic data. We propose that partial loss of sex by one pool of aphids and subsequent reduction in gene flow between lineages may favour temporal specialization through disruptive selection. PMID- 16674578 TI - Relative migration rates and local adaptation in a mosquito-protozoan interaction. AB - Theory predicts that the direction of local adaptation depends on the relative migration rates of hosts and parasites. Here we measured relative migration rates and tested for local adaptation in the interaction between a tree hole mosquito (Ochlerotatus sierrensis) and a protozoan parasite (Lambornella clarki). We found strong support for the hypothesis that the host migrates more than its parasite. Hosts colonized artificial tree holes in the field at a much higher rate than the parasite. Field releases of the parasite demonstrated that it colonizes and persists in natural tree holes where it was previously absent, suggesting that parasite distribution is limited by its migratory ability. Although the host migrates more than its parasite, we found no evidence for local adaptation by hosts and some evidence for local adaptation by parasites. Other life history traits of the host and parasite may also influence patterns in local adaptation, particularly parasite virulence and host dormancy. PMID- 16674579 TI - Population size, female fecundity, and sex ratio variation in gynodioecious Plantago maritima. AB - Theory predicts that the sex ratio of gynodioecious populations (in which hermaphrodites and females coexist) will be affected by the relative female fitness of females and hermaphrodites, and by founder events and genetic drift in small populations. We documented the sex ratio and size of 104 populations of the gynodioecious, perennial herb Plantago maritima in four archipelagos in eastern Sweden and western Finland (from latitude 53 to 64 degrees N). The sex ratio varied significantly both among and within archipelagos (range 0-70% females, median 6.3% females). The frequency of females was highest in the northernmost archipelago and lowest in the southernmost archipelago. As predicted, females were more frequently missing from small than from large populations, and the variance in sex ratio increased with decreasing population size. The relative fecundity of female plants (mean seed output per female/mean seed output per hermaphrodite) ranged from 0.43 to 2.16 (median 1.01, n = 12 populations). Among the 12 populations sampled for seed production (four in each of three archipelagos), the frequency of females was positively related to relative fecundity of females and negatively related to population size. The results suggest that the local sex ratio is influenced both by the relative fecundity of females and hermaphrodites and by stochastic processes in small populations. PMID- 16674580 TI - Analysis of the importance of genotypic variation, metabolic rate, morphology, sex and development time on immune function in the cricket, Gryllus firmus. AB - Immune defence is hypothesized to be a trait that bears significant fitness costs as well as benefits in that mounting a defence depreciates the value of other life-history traits. Thus the cost of mounting an immune response could affect the evolution of both the immune system and correlated life history traits. In this study we examined, by means of a diallel cross of four inbred lines, the genetic basis of two measures of immune function, metabolic rate and several traits in the sand cricket, Gryllus firmus. We specifically addressed the following questions: (1) is immune function determined primarily by genetic constitution or correlations with phenotypic traits that could reduce the effectiveness of the immune response; (2) do the two measures of immune function covary; (3) What are the contributions of additive, nonadditive and maternal effects to the immune function? As estimates of immune function, we used lytic activity and encapsulation rate. We found that inbred crickets were smaller than individuals from the crossed lines and took longer to develop. However, inbred lines did not differ from the crossed lines in immune function nor metabolic rates, suggesting that increased homozygosity has little or no effect on these traits in G. firmus. We found that both immune parameters showed significant genetic variation but no consistent relationships with the other phenotypic traits (metabolic rate, head width, body mass, development time and activity). There was significant additive genetic variation only in encapsulation rate, but, with the exception of the activity measure, significant nonadditive and reciprocal variances were found in all traits. Metabolic rate of crickets was heritable, but there was neither phenotypic nor genetic association between metabolic rate and the two parameters of immune function. Further, there was no correlation between these two measures. Females showed a higher encapsulation response than males, but there was no sex differences in lytic activity. Our study indicates that genetic variation in immune parameters can be a very significant contributor to phenotypic variation in immune function. PMID- 16674581 TI - Molecular evolution of the avian growth hormone gene and comparison with its mammalian counterpart. AB - The molecular evolution of all available avian growth hormone (GH) gene sequences was investigated using both maximum-likelihood and parsimony methods, and the patterns compared to those found in mammals. In contrast to the rapid bursts of evolution observed for mammalian GH, the evolutionary rate of the avian GH mature peptide appears to have been more constant. However several positively selected sites were identified at functionally important positions in the avian signal peptide by the site-specific likelihood method. This implies that sequence variation in the avian GH signal peptide may be adaptive, although more conservative parsimony methods failed to confirm this. Nevertheless, the differing patterns of avian and mammalian GH signal peptide molecular evolution are consistent with the apparently differing roles of GH in controlling growth in these taxonomic groups and support the hypothesis that signal peptide sequence variation may in fact be the basis for increased functional complexity. PMID- 16674582 TI - Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone. AB - Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada. PMID- 16674583 TI - Asymmetric gene flow and constraints on adaptation caused by sex ratio distorters. AB - Asymmetric gene flow is generally believed to oppose natural selection and potentially impede adaptation. Whilst the cause of asymmetric gene flow has been seen largely in terms of variation in population density over space, asymmetric gene flow can also result from varying sex ratios across subpopulations with similar population sizes. We model the process of adaptation in a scenario in which two adjacent subpopulations have different sex ratios, associated with different levels of infection with maternally inherited endosymbionts that selectively kill male hosts. Two models are analyzed in detail. First, we consider one host locus with two alleles, each of which possesses a selective advantage in one of the subpopulations. We found that local adaptation can strongly be impeded in the subpopulation with the more female biased population sex ratio. Second, we analyze host alleles that provide resistance against the male-killing (MK) endosymbionts and show that asymmetric gene flow can prevent the spread of such alleles under certain conditions. These results might have important implications for the coevolution of MK bacteria and their hosts. PMID- 16674584 TI - Sex ratio selection and multi-factorial sex determination in the housefly: a dynamic model. AB - Sex determining (SD) mechanisms are highly variable between different taxonomic groups and appear to change relatively quickly during evolution. Sex ratio selection could be a dominant force causing such changes. We investigate theoretically the effect of sex ratio selection on the dynamics of a multi factorial SD system. The system considered resembles the naturally occurring three-locus system of the housefly, which allows for male heterogamety, female heterogamety and a variety of other mechanisms. Sex ratio selection is modelled by assuming cost differences in the production of sons and daughters, a scenario leading to a strong sex ratio bias in the absence of constraints imposed by the mechanism of sex determination. We show that, despite of the presumed flexibility of the SD system considered, equilibrium sex ratios never deviate strongly from 1 : 1. Even if daughters are very costly, a male-biased sex ratio can never evolve. If sons are more costly, sex ratio can be slightly female biased but even in case of large cost differences the bias is very small (<10% from 1 : 1). Sex ratio selection can lead to a shift in the SD mechanism, but cannot be the sole cause of complete switches from one SD system to another. In fact, more than one locus remains polymorphic at equilibrium. We discuss our results in the context of evolution of the variable SD mechanism found in natural housefly populations. PMID- 16674585 TI - Fitness conflicts and the costs of sociality in communal egg layers: a theoretical model and empirical tests. AB - Individuals within complex social groups often experience reduced reproduction owing to coercive or suppressive actions of other group members. However, the nature of social and ecological environments that favour individual acceptance of such costs of sociality is not well understood. Taxa with short periods of direct social interaction, such as some communal egg layers, are interesting models for study of the cost of social interaction because opportunities to control reproduction of others are limited to brief periods of reproduction. To understand the conditions under which communal egg layers are in fitness conflict and thus likely to influence each other's reproduction, we develop an optimality model involving a brood guarding 'host' and a nonguarding disperser, or 'egg dumper'. The model shows that when, where intermediate-sized broods have highest survival, lifetime inclusive fitnesses of hosts and dumpers are often optimized with different numbers of dumped eggs. We hypothesize that resolution of this conflict may involve attempts by one party to manipulate the other's reproduction. To test model predictions we used a lace bug (Heteroptera: Tingidae) that shows both hosts and egg dumpers as well as increased offspring survival in response to communal egg laying. We found that egg-dumping lace bugs oviposit a number of eggs that very closely matches predicted fitness optimum for hosts rather than predicted optimum of dumpers. This result suggests that dumpers pay a social cost for communal egg laying, a cost that may occur through host suppression of dumper reproduction. Although dumper allocation of eggs is thus sub-optimal for dumpers, previous models show that the decision to egg dump is nevertheless evolutionarily stable, possibly because hosts permit just enough dumper oviposition to encourage commitment to the behaviour. PMID- 16674586 TI - Genotype by environment interactions in viability and developmental time in populations of cactophilic Drosophila. AB - The genetic and ecological basis of viability and developmental time differences between Drosophila buzzatii and D. koepferae were analysed using the isofemale line technique. Several isofemale lines were sampled from pairs of allopatric/sympatric populations of each species. Flies were reared in media prepared with decaying tissues of two of the main natural cactus hosts of each species. This experimental design enabled us to evaluate the relative contribution of phenotypic plasticity, genetic variation and genotype by environment interaction (G x E) to total phenotypic variation for two fitness traits, viability and developmental time. Our results revealed significant G x E in both traits, suggesting that the maintenance of genetic variation can be explained, at least in part, by diversifying selection in different patches of a heterogeneous environment in both species. However, the relative importance of the factors involved in the G x E varied between traits and populations within species. For viability, the G x E can be mainly attributed to changes in the rank order of lines across cacti. However, the pattern was different for developmental time. In D. buzzatii the G x E can be mainly accounted for by changes in among line variance across cacti, whereas changes in the rank order of lines across cacti was the main component in D. koepferae. These dissimilar patterns of variation between traits and species suggest that the evolutionary forces shaping genetic variation for developmental time and viability vary between populations within species and between species. PMID- 16674587 TI - Evolution of sex-biased maternal effects in birds: II. Contrasting sex-specific oocyte clustering in native and recently established populations. AB - In species that produce broods of multiple offspring, parents need to partition resources among simultaneously growing neonates that often differ in growth requirements. In birds, multiple ovarian follicles develop inside the female at the same time, resulting in a trade-off of resources among them and potentially limiting maternal ability for sex-specific allocation. We compared resource acquisition among oocytes in relation to their future sex and ovulation order in two populations of house finches with contrasting sex-biased maternal strategies. In a native Arizona population, where mothers do not bias offspring sex in relation to ovulation order, the male and female oocytes did not show sex specific trade-offs of resources during growth and there was no evidence for spatial or temporal segregation of male and female oocytes in the ovary. In contrast, in a recently established Montana population where mothers strongly bias offspring sex in relation to ovulation order, we found evidence for both intra-sexual trade-offs among male and female oocytes and sex-specific clustering of oocytes in the ovary. We discuss the importance of sex-specific resource competition among offspring for the evolution of sex-ratio adjustment and sex specific maternal resource allocation. PMID- 16674588 TI - Early maternal investment in mice: no evidence for compatible-genes sexual selection despite hybrid vigor. AB - Confronting a recently mated female with a strange male can induce a pregnancy block ('Bruce effect'). The physiology of this effect is well studied, but its functional significance is still not fully understood. The 'anticipated infanticide hypothesis' suggests that the pregnancy block serves to avoid the cost of embryogenesis and giving birth to offspring that are likely to be killed by a new territory holder. Some 'compatible-genes sexual selection hypotheses' suggest that the likelihood of a pregnancy block is also dependent on the female's perception of the stud's and the stimulus male's genetic quality. We used two inbred strains of mice (C57BL/6 and BALB/c) to test all possible combinations of female strain, stud strain, and stimulus strain under experimental conditions (N(total) = 241 mated females). As predicted from previous studies, we found increased rates of pregnancy blocks if stud and stimulus strains differed, and we found evidence for hybrid vigour in offspring of between-strain mating. Despite the observed heterosis, pregnancies of within strain matings were not more likely to be blocked than pregnancies of between strain matings. A power analysis revealed that if we missed an existing effect (type-II error), the effect must be very small. If a female gave birth, the number and weight of newborns were not significantly influenced by the stimulus males. In conclusion, we found no support for the 'compatible-genes sexual selection hypotheses'. PMID- 16674589 TI - The evolution of host preference in allopatric vs. parapatric populations of Timema cristinae walking-sticks. AB - Divergent habitat preferences can contribute to speciation, as has been observed for host-plant preferences in phytophagous insects. Geographic variation in host preference can provide insight into the causes of preference evolution. For example, selection against maladaptive host-switching occurs only when multiple hosts are available in the local environment and can result in greater divergence in regions with multiple vs. a single host. Conversely, costs of finding a suitable host can select for preference even in populations using a single host. Some populations of Timema cristinae occur in regions with only one host-plant species present (in allopatry, surrounded by unsuitable hosts) whereas others occur in regions with two host-plant species adjacent to one another (in parapatry). Here, we use host choice and reciprocal-rearing experiments to document genetic divergence in host preference among 33 populations of T. cristinae. Populations feeding on Ceanothus exhibited a stronger preference for Ceanothus than did populations feeding on Adenostoma. Both allopatric and parapatric pairs of populations using the different hosts exhibited divergent host preferences, but the degree of divergence tended to be greater between allopatric pairs. Thus, gene flow between parapatric populations apparently constrains divergence. Host preferences led to levels of premating isolation between populations using alternate hosts that were comparable in magnitude to previously documented premating isolation caused by natural and sexual selection against migrants between hosts. Our findings demonstrate how gene flow and different forms of selection interact to determine the magnitude of reproductive isolation observed in nature. PMID- 16674590 TI - Phylogeography and alpha taxonomy of the common dolphin (Delphinus sp.). AB - The resolution of taxonomic classifications for delphinid cetaceans has been problematic, especially for species in the genera Delphinus, Tursiops and Stenella. The frequent lack of correspondence between morphological and genetic differentiation in these species raises questions about the mechanisms responsible for their evolution. In this study we focus on the genus Delphinus, and use molecular markers to address questions about speciation and the evolution of population structure. Delphinus species have a worldwide distribution and show a high degree of morphological variation. Two distinct morphotypes, long-beaked and short-beaked, have been considered different species named D. capensis and D. delphis, respectively. However, genetic differentiation between these two forms has only been demonstrated in the Pacific. We analysed samples from eight different geographical regions, including two morphologically defined long-beaked form populations, and compared these with the eastern North Pacific populations. We found high differentiation among the populations described as long-beaked instead of the expected monophyly, suggesting that these populations may have evolved from independent events converging on the same morphotype. We observed low genetic differentiation among the short-beaked populations across a large geographical scale. We interpret these phylogeographical patterns in the context of life history and population structure in related species. PMID- 16674591 TI - Glacial refugia and the phylogeography of Steller's sea lion (Eumatopias jubatus) in the North Pacific. AB - Mitochondrial DNA sequence data were used to examine the phylogeographic history of Steller's sea lions (Eumetopias jubatus) in relation to the presence of Plio Pleistocene insular refugia. Cytochrome b and control region sequences from 336 Steller's sea lions reveal phylogenetic lineages associated with continental refugia south of the ice sheets in North America and Eurasia. Phylogenetic analysis suggests the genetic structure of E. jubatus is the result of Pleistocene glacial geology, which caused the elimination and subsequent reappearance of suitable rookery habitat during glacial and interglacial periods. The cyclic nature of geological change produced a series of independent population expansions, contractions and isolations that had analogous results on Steller's sea lions and other marine and terrestrial species. Our data show evidence of four glacial refugia in which populations of Steller's sea lions diverged. These events occurred from approximately 60,000 to 180,000 years BP and thus preceded the last glacial maximum. PMID- 16674592 TI - Common sex-linked deleterious alleles in a plant parasitic fungus alter infection success but show no pleiotropic advantage. AB - Microbotryum violaceum is a fungus that causes the sterilizing anther smut disease in Caryophyllaceae. Its diploid teliospores normally produce equal proportions of haploid sporidia of its two mating types. However natural populations contain high frequencies of individuals producing sporidia of only one mating type ('biased strains'). This mating type-ratio bias is caused by deleterious alleles at haploid phase ('haplo-lethals') linked to the mating type locus that can be transmitted only by intra-tetrad selfing. We used experimental inoculations to test some of the hypotheses proposed to explain the maintenance of haplo-lethals. We found a disadvantage of biased strains in infection ability and high intra-tetrad mating rates. Biased strains had no higher competitive ability nor shorter latency and their higher spore production per flower appeared insufficient to compensate their disadvantages. These findings were only consistent with the hypothesis that haplo-lethals are maintained under a metapopulation structure because of high intra-tetrad selfing rates, founder effects and selection at the population level. PMID- 16674593 TI - Differential effects of egg albumen content on barn swallow nestlings in relation to hatch order. AB - In diverse animal taxa, egg mass variation mediates maternal effects with long term consequences for offspring ontogeny and fitness. Patterns of egg mass variation with laying order differ considerably among birds, but no study has experimentally investigated the function of variation in albumen or yolk egg content in the wild. In barn swallows (Hirundo rustica), absolute and relative albumen mass increased with egg laying order. Experimental albumen removal delayed hatching, had larger negative effects on growth of late-hatched nestlings, and reduced nestling survival. Laying order positively predicted hatch order. Because nestling competitive ability depends on size, and albumen egg content influences hatchling size, present results suggest that by increasing albumen content of late eggs mothers reduce hatching asynchrony and enhance growth particularly of late-hatched nestlings. Thus, variation in albumen mass with laying order may function to mitigate the negative phenotypic consequences of hatching late in species that adopt a 'brood-survival' strategy. PMID- 16674594 TI - Data depth, data completeness, and their influence on quantitative genetic estimation in two contrasting bird populations. AB - Evolutionary biologists increasingly use pedigree-based quantitative genetic methods to address questions about the evolutionary dynamics of traits in wild populations. In many cases, phenotypic data may have been collected only for recent parts of the study. How does this influence the performance of the models used to analyse these data? Here we explore how data depth (number of years) and completeness (number of observations) influence estimates of genetic variance and covariance within the context of an existing pedigree. Using long-term data from the great tit Parus major and the mute swan Cygnus olor, species with different life-histories, we examined the effect of manipulating the amount of data included on quantitative genetic parameter estimates. Manipulating data depth and completeness had little influence on estimated genetic variances, heritabilities, or genetic correlations, but (as expected) did influence confidence in these estimates. Estimated breeding values in the great tit were not influenced by data depth but were in the mute swan, probably because of differences in pedigree structure. Our analyses suggest the 'rule of thumb' that data from 3 years and a minimum of 100 individuals per year are needed to estimate genetic parameters with acceptable confidence, and that using pedigree data is worthwhile, even if phenotypes are only available toward the tips of the pedigree. PMID- 16674598 TI - 11beta-Hydroxysteroid dehydrogenase activity in pregnancies complicated by hydatidiform mole. AB - PROBLEM: 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) plays an important role in regulating active glucocorticoid reaching the fetus. In normal pregnancy, placental 11beta-HSD functions primarily in oxidative direction. Placental tissue of patients with pregnancies complicated by pre-eclampsia exhibit significantly lower type 1 and 2 11beta-HSD activities and significantly high cortisol level in cord blood suggesting fetal exposure to higher level of active glucocorticoids. The activity of 11beta-HSD in gestational trophoblastic disease has not been determined. The objective of this study was to assess 11beta-HSD activity in tissue from normal second trimester and pregnancies complicated by hydatidiform mole. METHOD OF STUDY: Normal placental tissues were obtained from patients undergoing termination of pregnancy, and from patients undergoing uterine evacuation for hydatidiform mole. Both nicotinamide adenine dinucleotide (NAD)- and nicotinamide adenine dinucleotide phosphate (NADP)-dependent activities were assayed in central villous tissue. Comparison of groups was performed using Student's t-test. A P-value of 0.05 was considered significant. Data are presented as mean +/- S.D. RESULTS: Tissue obtained from five patients with pathology-proven hydatidiform mole demonstrated significantly lower 11beta-HSD activities compared with placental tissue obtained from normal pregnancies. The mean NAD-dependent 11beta-HSD activity in normal placentas was 386 +/- 109 pmol/min/g placenta and in hydatidiform mole was 74 +/- 54 pmol/min/g placenta (P < 0.01). The mean NADP-dependent 11beta-HSD activity in normal placentas was 370 +/- 120 pmol/min/g placenta and in trophoblastic disease was 68 +/- 69 pmol/min/g placenta (P < 0.01). CONCLUSION: Our data indicate significant impairment in the ability of hydatidiform mole tissue to inactivate glucocorticoids. PMID- 16674599 TI - Possible role of natural immune response against altered fibroblasts in the development of post-operative adhesions. AB - PROBLEM: Post-operative adhesion tissue fibroblasts (ATF) differ from normal peritoneal fibroblasts (NPF). Natural immune response participates in the elimination of altered cells. In this study, we investigated NPF and ATF expression patterns of immune response-related markers, and lymphokine-activated killer (LAK) cell-mediated fibroblast elimination in vitro. METHOD OF STUDY: Primary cell cultures of both NPF and ATF obtained from the same four patients were used in the experiments. The expression of CD54, CD40 and CD120b, and allogeneic LAK cell-mediated ATF and NPF elimination were studied by flow cytometry. RESULTS: Average expression of CD54 in ATF was greater by 12.3-fold compared with NPF (P = 0.021), with ratios of 2.4 and 1.9-fold for CD40 (P < 0.001) and CD120b (P = 0.013), respectively. Average LAK cell-mediated fibroblast killing was 1.8 +/- 0.8-fold greater in ATF over NPF (P = 0.008). Furthermore, LAK cell-mediated fibroblast elimination correlated significantly with the increased CD40, CD54 and CD120b expression (R > 0.956; P < 0.05 for each). CONCLUSIONS: These results demonstrate that ATF are more susceptible to lymphocyte-mediated elimination than NPF and the development of adhesions despite this could be explained by either impaired or overwhelmed autologous natural immune response against reactive fibroblasts. PMID- 16674600 TI - Increased IL-18 levels in seminal plasma of infertile men with genital tract infections. AB - PROBLEM: Interleukin (IL)-18 is a novel cytokine, previously known as interferon (IFN)-gamma inducing factor. We evaluated the levels of IL-18 and IFN-gamma in seminal plasma (SP) of fertile and infertile men. METHOD OF STUDY: Semen samples were obtained by masturbation from 80 men, and were examined for the levels of IL 18 and IFN-gamma by enzyme-linked immunosorbent assay. Seven groups were included: (i) fertile men (n = 18), (i) infertile men with genital tract infections (n = 17), (iii) with varicocele (n = 15), (iv) with Klinefelter syndrome (n = 6), (v) with cryptorchidism (n = 7), (vi) with mumps orchitis (n = 7), and (vii) with idiopathic testicular lesions (n = 10). RESULTS: Mean levels of IL-18 were higher in SP from infertile men with genital tract infections compared with SP from other groups except Klinefelter syndrome (P < 0.05). However, no significant differences could be detected for IFN-gamma. A significant positive correlations was found between IL-18 and IFN-gamma in total patient population (P < 0.001). Moreover, a negative correlation was observed between IL-18 and sperm concentrations, and motility (P < 0.01 and < 0.03, respectively). Furthermore, there was a positive and statistically significant association between IL-18 and IFN-gamma levels in SP of infertile men with genital tract infections (P < 0.0001). However, there was no relationship between IL-18 and IFN-gamma, and semen parameters in the same group. CONCLUSION: SP IL-18 levels were increased in men with urogenital infections. Thus, the elevated expression of IL-18 in SP may be used as a diagnostic marker in the male genital tract infections. PMID- 16674601 TI - Effect of toll-like receptor (TLR) agonists on TLR and microbicide expression in uterine and vaginal tissues of the mouse. AB - PROBLEM: Epithelial cells lining the uterine lumen are the first line of defense against pathogenic microbes. The objective of this study was to examine the expression of Toll-like receptors (TLRs), defensins and secretory leukocyte protease inhibitor (SLPI) in the mouse uterus and vagina and in primary uterine epithelial cells and to determine whether TLR agonists induce TLR and defensin expression. METHOD OF STUDY: The mRNA expression of alpha- and beta-defensins (AD1, 2 and 5 and BD1, 2 and 4) and SLPI was examined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) along with the secretion of macrophage chemotactic protein-1 (MCP-1), measured by enzyme-linked immunosorbent assay. RESULTS: Expression of TLR1-9 as well as beta-defensins 1, 2 and 4 and SLPI by uterine and vaginal tissues was demonstrated by RT-PCR. beta-Defensins and SLPI expression was greater in the vagina than in the uterus. Comparison of fresh and polarized uterine epithelial cells indicated that TLR2-6 expression was unaffected by culture. Incubation of polarized epithelial cells with TLR agonists [lipopolysaccharide (LPS), Pam3Cys, Poly (I:C) or PGN] induced TLR5 and TLR9 expression but had no effect on TLR4, defensins or SLPI. Furthermore, exposure to LPS, Pam3Cys, Poly (I:C) or PGN, induced MCP-1 secretion by polarized epithelial cells in culture. CONCLUSION: These results indicate that the uterus and vagina as well as uterine epithelial cells are responsive to bacterial and viral pathogens. Not only do epithelial cells respond to TLR agonists by releasing MCP 1, which mediates inflammatory responses, but they also influence the expression of selected TLR genes to further enhance innate immune protection. PMID- 16674602 TI - Benign course in multiple sclerosis: a review. AB - Since the 1950s, it has been recognized that a subgroup of multiple sclerosis (MS) patients exists that shows little or no progression in the severity of the disease over time. This group is referred to as 'benign' MS. Although a substantial amount of research in MS indicates a multifactorial background in disease severity, to date it is still difficult to predict whether the course will be benign at onset and it is difficult to find factors that influence the course of the disease over time. Maintaining or restoring neural conduction inside a central nervous system lesion seems to be the essence of staying 'benign'. PMID- 16674603 TI - Neural substrates, experimental evidences and functional hypothesis of acupuncture mechanisms. AB - OBJECTIVES: Although acupuncture therapy has demonstrated itself to be effective in several clinical areas, the underlying mechanisms of acupuncture in general and the analgesic effect in particular are, however, still not clearly delineated. We, therefore, have studied acupuncture analgesic effect through fMRI and proposed a hypothesis, based on the obtained result, which will enlighten the central role of the brain in acupuncture therapy. METHODS: The proposed model, termed as a broad sense hypothalamus-pituitary-adrenal (BS-HPA) axis, was based on our observed neuroimaging results. The model incorporates the stress-induced HPA axis model together with neuro-immune interaction including the cholinergic anti-inflammatory model. RESULTS: The obtained results coupled with accumulating evidence suggest that the central nervous system is essential for the processing of these effects via its modulation of the autonomic nervous system, neuroimmune system and hormonal regulation. CONCLUSIONS: Based on our fMRI study, it appears that understanding the effects of acupuncture within a neuroscience-based framework is vital. Further, we have proposed the broad sense-HPA axis hypothesis which incorporates the experimental results. PMID- 16674604 TI - Glatiramer acetate in treatment-naive and prior interferon-beta-1b-treated multiple sclerosis patients. AB - OBJECTIVE: This prospective, open-label study evaluated the efficacy, safety, and tolerability of glatiramer acetate (GA) in treatment-naive relapsing-remitting multiple sclerosis (RRMS) patients and in patients who had previously received interferon-beta (IFN-beta)-1b therapy. METHODS: Two treatment cohorts were defined based on prestudy IFN-beta-1b use. At entry, prior IFN-beta-1b patients (n = 247) were older, had longer disease duration, and had higher mean Expanded Disability Status Scale (EDSS) scores, relapse rates, and ambulation indexes than treatment-naive patients (n = 558). Safety was assessed every 3 months and EDSS every 6 months for up to 3.5 years. RESULTS: Overall, 247 treatment-naive and 107 prior IFN-beta-1b patients discontinued before study end. Median GA treatment durations were 36 and 24 months in treatment-naive and prior IFN-beta-1b patients, respectively. At last observation, annual relapse rates had declined by 75% in both cohorts (0.42 +/- 0.84 and 0.34 +/- 0.71 in treatment-naive and prior IFN-beta-1b groups, respectively, P = 0.1482). Mean changes in EDSS were less than 0.5 in both cohorts, regardless of entry EDSS, at 12 and 18 months and at last observation. CONCLUSIONS: Prior IFN-beta-1b treatment does not negatively influence the efficacy, safety, or tolerability of subsequent GA therapy. Switching to GA can benefit patients who discontinue IFN-beta therapy. PMID- 16674605 TI - Levetiracetam in a broad population of patients with refractory epilepsy: interim results of the international SKATE trial. AB - OBJECTIVE: To prospectively assess the safety and efficacy of levetiracetam in patients with uncontrolled focal epilepsy, in a common practice-based setting. PATIENTS AND METHODS: In this phase IV, open-label, 16-week community-based study, adult patients with focal seizures initially received levetiracetam 1,000 mg/day. Throughout the study, the dose was adjusted in increments of 1,000 mg (maximum 3,000 mg/day) to achieve seizure control and maintain tolerability. The outcome parameters were the percentage reduction in partial and total seizure frequency per week from historical baseline, global evaluation scale (GES), and adverse events (AE). RESULTS: Seven hundred and thirty-one patients were included in this analysis and 84.4% completed the study. The median percent reduction in all seizures was 47.8%, and 49.3% for all partial seizures. The 50% responder rate was 49%, and the seizure-free rate was 17.2% for all partial seizures. Approximately 60% of patients showed moderate to marked improvement on the GES. The majority of AE were of mild to moderate severity; the most commonly reported being asthenia, somnolence, headache, and dizziness. CONCLUSION: Levetiracetam is both efficacious and safe as an add-on therapy in patients with refractory epilepsy treated by clinicians in their daily practice. PMID- 16674606 TI - Oxcarbazepine in painful diabetic neuropathy: results of a dose-ranging study. AB - OBJECTIVES: To evaluate the efficacy and safety of oxcarbazepine in patients with diabetic neuropathy in a multicenter, double-blind, placebo-controlled, dose ranging 16-week study. METHODS: A total of 347 patients were randomized to oxcarbazepine 600 mg/day (n = 83), 1,200 mg/day (n = 87), 1,800 mg/day (n = 88), or placebo (n = 89). The primary efficacy variable was change in mean visual analog scale (VAS) score from baseline to the last week of the study. RESULTS: No difference between any oxcarbazepine group and the placebo group was noted for the primary efficacy variable. Both the 1,200- and 1,800-mg/day groups showed a trend toward statistical significance (P = 0.101, P = 0.096, respectively). Statistically significant differences were found between the oxcarbazepine 1,200 mg/day (P = 0.038) and 1,800-mg/day (P = 0.005) groups and placebo in the overall mean weekly VAS scores for the entire double-blind treatment phase. CONCLUSIONS: Although the primary efficacy variable did not reach statistical significance, patients taking oxcarbazepine 1,200 and 1,800 mg/day showed improvements in VAS scores compared with placebo. Oxcarbazepine may provide clinically meaningful pain relief in patients with painful diabetic neuropathy. PMID- 16674607 TI - Trends in utilization of antiepileptic drugs in Denmark. AB - OBJECTIVES: To describe utilization of antiepileptic drugs (AEDs) in Denmark during 1993-2002, with special emphasis on oxcarbazepine, and to assess probable indications for AED use. MATERIALS AND METHODS: We retrieved prescription data from Odense University Pharmacoepidemiological Database, in Funen County, Denmark (population in 2002: 472,869). Within each calendar year we estimated period prevalence, incidence rate and monotherapy rate. Based on co-medication we defined 'epilepsy' when only AEDs were prescribed, 'pain' with co-prescription of opioids, and 'mood disorder' with co-prescription of antipsychotics or antidepressants. RESULTS: We identified 15,604 AED users. The prevalence of using AED increased from 9.3 (95% CI, 9-9.5) to 12.1 (11.8-12.4)/1000 persons. The incidence rate increased from 1.4 (1.3-1.6) to 1.7 (1.6-1.9)/1000 personyears. The monotherapy rate was 79-82%. AED use for 'epilepsy' declined by 19.7%, whereas the proportion of 'pain' and 'mood disorder' treatment increased by 11.2% and 8.4% respectively. CONCLUSIONS: Antiepileptic drug utilization increased during the study period, the increase probably caused by expanding use in areas other than epilepsy. PMID- 16674608 TI - A prospective PET study of patients with glioblastoma multiforme. AB - OBJECTIVE: To study the post-surgical metabolic and structural cerebral changes in patients with glioblastoma multiforme (GBM). MATERIALS AND METHODS: We examined ten patients prospectively with newly diagnosed GBM. All patients were primarily treated with surgery, followed by chemotherapy (carmustine, cisplatine and etoposide) and radiotherapy. Positron emission tomography (PET) was used to measure tumor- and cerebral metabolism. CT or MRI was used to estimate tumor volume by measurements of tumor area. RESULTS: Tumor metabolism was not increased during chemotherapy (P = 0.71), but increased during radiotherapy (P = 0.01). CT/MRI showed similar results with no increase in tumor area during chemotherapy (P = 0.33) but increase during radiotherapy (P = 0.002). During the entire study, tumor metabolism and area increased evenly (P = 0.01). CONCLUSIONS: Our study did not show a gain of PET compared with structural imaging in the prospective evaluation of GBM. We found a difference in metabolic increase and tumor growth between the two treatment regimens, although this finding has limited relevance due to the design of the study. PMID- 16674609 TI - Polymorphism of PRNP codons in the normal Icelandic population. AB - OBJECTIVES: Polymorphisms in the prion protein gene in humans influence susceptibility to, and phenotype of, prion diseases. Methionine-methionine (MM) homozygosity at codon 129 is a risk factor for sporadic Creutzfeldt-Jakob disease (CJD). Polymorphism at codon 117 and changes in the octapeptide repeat region have been associated with genetic CJD. Knowledge of genetic background in normal populations may contribute to better understanding of prion diseases. MATERIALS AND METHODS: Polymorphism at codon 129, codon 117 and deletions of octapetide repeats were studied in 208 healthy blood donors of both genders and of different age. RESULTS: Polymorphism at codon 129 was: MM 46.6%, methionine-valine 44.7%, valine-valine 8.7%. Polymorphism at codon 117 was observed in 4.8%. Deletions of octapeptide repeats were not detected. There were no gender or age differences in the distribution of codon 129 polymorphism. The frequency of codon 129 polymorphisms was, with one exception, not significantly different from that observed elsewhere in Europe. PMID- 16674610 TI - Sensitivity of transcranial Doppler for confirming brain death: a prospective study of 270 cases. AB - OBJECTIVE: The reported sensitivity of transcranial Doppler ultrasonography (TCD) for confirming brain death (BD) ranges from 91% to 100%. We assessed the frequency and causes of false-negative results in TCD examination in a series of patients with BD and in the literature. METHODS: We carried out a prospective TCD examination of consecutive patients with the clinical diagnosis of BD. RESULTS: In 204 (75.5%) of 270 patients, TCD showed a pattern compatible with BD. The causes of the false-negative results were persistent flow in the intracranial arteries in 47 (17.4%) patients and a lack of signal in 19 (7%). Absence of sympathomimetic drug use [odds ratio (OR) 5.4, 95% confidence interval (CI) 1.8 16.0, P = 0.003) and female gender (OR 3.7, 95% CI 1.1-12.5, P = 0.03) were associated with false-negative results. A review of 16 studies showed a sensitivity of 88% and a specificity of 98% of TCD for confirming BD. CONCLUSIONS: The sensitivity of TCD for confirming BD may be lower than previously reported, but is probably similar to that of other non-invasive methods. The specificity of TCD is close to 100%. Uniform criteria are needed for the routine use of TCD as a confirmatory test for BD. PMID- 16674611 TI - Stroke after initiation of interferon-beta treatment for relapsing-remitting disseminated white matter disease. AB - BACKGROUND: Interferon-beta (INF-beta) is effective and used in reducing exacerbation frequency and disease progression in multiple sclerosis. In certain circumstances, INF-beta can lead to rare side effects. AIMS OF THE STUDY: We report the case of a 34-year-old female patient satisfying the McDonald criteria of multiple sclerosis without showing typical pathologic changes in cerebrospinal fluid (CSF). After introduction of INF-beta treatment, she quickly developed further progression of her disseminated neurological symptoms and finally an ischemic cerebral infarction. METHODS: Evaluation of the patient included arterial angiography, magnetic resonance and positron emission tomography, histopathological assessment as well as a broad spectrum of serum and CSF analysis. RESULTS: All diagnostic evaluations and the clinical course revealed evidences for a primary angiitis of the CNS. We discuss the possible worsening due to inappropriate INF-beta treatment in cerebral angiitis promoting severe cerebrovascular insufficiency. CONCLUSION: The authors suggest that all diagnostic multiple sclerosis criteria including typical CSF findings should be ascertained before INF-beta treatment is initiated. PMID- 16674612 TI - Extracting value from case reports: lessons from serotonin toxicity. PMID- 16674613 TI - Local clinical quality monitoring for detection of excess operative deaths. AB - A monitoring system for cardiac surgery has been in use at Papworth Hospital for 10 years. We wished to determine whether this system would have detected an increase in deaths associated with a single practitioner, whether a poorly performing doctor or a serial killer such as Dr Harold Shipman, whose activities went undetected in the absence of a monitoring system for nearly a quarter of a century. Random extra deaths were artificially introduced into the practice of a surgeon and an anaesthetist in a way that broadly reproduced Shipman's pattern. The standard monitoring system was then used to analyse the hypothetical data thus generated. Using the current standard monitoring, the excess deaths would have been detected in less than 10 months. Suspicions would have been raised even earlier. Robust local quality monitoring of risk-adjusted outcomes is possible and, in our opinion, essential. PMID- 16674614 TI - Nasopharyngeal oxygen insufflation following pre-oxygenation using the four deep breath technique. AB - This paper evaluates the effectiveness of nasopharyngeal oxygen insufflation following preoxygenation using the four deep breath technique within 30 s, on the onset of haemoglobin desaturation during the subsequent apnoea. Thirty ASA I or II patients were randomly allocated to one of two groups. In the study group (n = 15), pre-oxygenation was followed by insufflation of oxygen at a flow of 5 l.min( 1) via a nasopharyngeal catheter commenced at the onset of apnoea. In the control group, pre-oxygenation was not followed by nasopharyngeal oxygen insufflation (n = 15). In the control group, SpO2 fell to 95% within a mean (SD) apnoea time of 3.65 (1.15) min, whereas in the study group, SpO2 was maintained in all patients at 100% throughout the 6 min of apnoea, at which point apnoea was terminated and positive pressure ventilation commenced. We conclude that nasopharyngeal oxygen insufflation following pre-oxygenation using the four deep breath technique can delay the onset of haemoglobin desaturation for a significant period of time during the subsequent apnoea. PMID- 16674615 TI - Protein cross-contamination during batch cleaning and autoclaving of the ProSeal laryngeal mask airway. AB - We tested the hypothesis that protein cross-contamination occurs during batch cleaning and autoclaving of a reusable extraglottic airway device, the ProSeal laryngeal mask airway. At the end of each day for 10 days, nine laryngeal mask airways that had been used for non-intra-oral surgery were cleaned and autoclaved alongside a new unused laryngeal mask airway. In addition, a new unused laryngeal mask airway underwent the same cleaning and autoclaving procedures in isolation. Protein staining was more frequently detected on the unused laryngeal mask airways that were processed by batch rather than in isolation (p < 0.01). Protein staining was detected on all unused laryngeal mask airways that were processed by batch, but none on those processed in isolation. Protein staining was more severe with the used compared with the unused laryngeal mask airways (p < 0.001). We conclude that protein cross-contamination of the laryngeal mask airway occurs during batch cleaning and autoclaving and recommend that reusable airway devices are cleaned in isolation. PMID- 16674616 TI - The effect of fundal pressure at Caesarean section on maternal haemodynamics. AB - In 20 women undergoing Caesarean section, we determined brachial arterial blood pressure, heart rate and cardiac output (using an oesophageal Doppler probe) just after uterine incision and during the application of extra-abdominal uterine fundal pressure to facilitate delivery. Mean (SD) systolic aortic flow time (354.3 (35.4) ms), cardiac output (3.85 (0.68) l.min(-1)), heart rate (80.6 (14.2) beats.min(-1)) and blood pressure (84.7 (12.1) mmHg) decreased when fundal pressure was applied compared with after uterine incision (397.6 (31.6) ms, 4.69 (0.78) l.min(-1), 95.1 (14.0) beats.min(-1)and 100.3 (12.0) mmHg, respectively; p < 0.05). Peak velocity and stroke distance of the aortic flow when fundal pressure was applied was similar to after uterine incision. The mothers and babies were not observed to be clinically compromised. The results suggest that applying fundal pressure to facilitate Caesarean delivery produces significant haemodynamic effects but the impact of these effects may be clinically insignificant. PMID- 16674617 TI - The effects of the exaggerated lithotomy position for radical perineal prostatectomy on respiratory mechanics. AB - The exaggerated lithotomy position is used during radical perineal prostatectomy to increase perineal exposure. The aim of this study was to evaluate the effects of the exaggerated lithotomy position on respiratory mechanics and arterial blood gases. In the exaggerated lithotomy position, dynamic compliance and static compliance were found to be significantly decreased by 27.4% and 34.8%, respectively, whilst peak, plateau, and mean airway pressures increased significantly by 34.0%, 45.8% and 31.7%, respectively. The physiological dead space/tidal volume ratio and total inspiratory work of breathing increased significantly by 11.1% and 33.7%, respectively. Arterial oxygen tension was significantly decreased by 26.9%; however, no significant differences were seen in end-tidal or arterial carbon dioxide tension. These results indicate that the exaggerated lithotomy position under general anaesthesia can cause significant effects on respiratory system mechanics and arterial oxygenation and highlights the need for careful monitoring of patients placed in this position for surgery. PMID- 16674618 TI - Blood pressure and heart rate changes during intubation: a comparison of direct laryngoscopy and a fibreoptic method. AB - Blood pressure and heart rate changes during nasotracheal intubation under general anaesthesia were studied in 100 patients who were randomly allocated to fibreoptic bronchoscope or direct laryngoscopy intubation. Noninvasive blood pressure and heart rate were recorded before and immediately after anaesthesia induction, at anaesthesia intubation and every minute thereafter for 5 min. Nasotracheal intubation was accompanied by significant increases in blood pressure and heart rate compared to baseline values in both groups. Blood pressure and heart rate at intubation, and the maximum values of blood pressure during the observation were significantly higher in the fibreoptic bronchoscope group. However, the maximum values of heart rate were not significantly different between the two groups. Fibreoptic nasotracheal intubation may result in more severe pressor and tachycardiac responses than direct laryngoscopic nasotracheal intubation. PMID- 16674619 TI - Awake fibrecapnic intubation: a novel technique for intubation in head and neck cancer patients with a difficult airway. AB - Awake fibreoptic intubation is the gold standard for difficult airway management but failures are reported in the literature in up to 13% of cases. In case of failure, a tracheotomy is often indicated. We describe a novel technique for intubation in head and neck cancer patients with a difficult airway that we call awake fibrecapnic intubation. The aim of this study was to investigate the feasibility of this technique. We studied prospectively 15 consecutive intubations in head and neck cancer patients before diagnostic or therapeutic surgical procedures. After topical anaesthesia, a fibrescope was introduced into the pharynx. Spontaneous respiration was maintained in all patients. Through the suction channel of the fibrescope a special suction catheter was advanced into the airway for carbon dioxide measurements. When four capnograms were obtained, the fibrescope was railroaded over the catheter and after identification of tracheal rings, a tracheal tube was placed. Tracheal intubation was successful in all patients without bleeding or complications, with a median (range) time to intubation of 3 (2-15) min. Identification of the vocal cords and glottis was difficult in four patients due to extensive anatomical abnormalities or poor visibility; even in these patients, a capnogram was obtained within 4 s. PMID- 16674620 TI - Where is T5? A survey of anaesthetists. AB - The extent of a regional block for Caesarean section must be tested and documented before surgery commences. In recent years a block to 'touch' that includes T5 has increasingly been considered the best predictive test for a pain free Caesarean section. Our survey examines the consistency with which different anaesthetists identified the location of the T5 dermatome. Seventy-three anaesthetists were asked to mark a point on an anatomical picture to indicate where they would test for T5. Overall there was good agreement on the location of the T5 dermatome, but one in seven anaesthetists were inaccurate by two or more dermatomes. There were no statistically significant differences between the subgroups of senior house officer, specialist registrar and consultant anaesthetists. The knowledge of relevant dermatome levels should be an integral part of obstetric anaesthetic training. PMID- 16674621 TI - A national survey of epidural use and management in elderly patients undergoing elective and emergency laparotomy. AB - A postal questionnaire was sent to anaesthetic clinical tutors in the United Kingdom describing two hypothetical 75-year-old patients requiring abdominal surgery. Patient 1 (ASA 2) required elective anterior resection and patient 2 (ASA 3-4) required emergency laparotomy. There was a 65% response rate. For patient 1, 98.5% of respondents would insert an epidural, 93% inserting this awake and 50% placing it in the high-mid thoracic region. All respondents would use local anaesthesia (concentration varied four-fold) and 62% would use opioids. All respondents would place the epidural pre-operatively; although 36% would administer the epidural block pre-operatively and 3% postoperatively. For patient 2, 70% of respondents would insert an epidural (p < 0.0001), drug administration would be more frequently delayed until postoperatively (13%); p = 0.0005) and epidural opioid use decreased (57%); p = n/s. Epidural insertion influenced the postoperative destination in 42% of departments. Use of a critical care facility was anticipated for more than half of these patients; 60% reported difficulty accessing critical care beds. PMID- 16674622 TI - The mechanisms of carbon monoxide production by inhalational agents. AB - Carbon monoxide can be formed when volatile anaesthetic agents such as desflurane and sevoflurane are used with anaesthetic breathing systems containing carbon dioxide absorbents. This review describes the possible chemical processes involved and summarises the experimental and clinical evidence for the generation of carbon monoxide. We emphasise the different conditions that were used in the experimental work, and explain some of the features of the clinical reports. Finally, we provide guidelines for the prevention and detection of this complication. PMID- 16674623 TI - Evaluation of intubation using the Airtraq or Macintosh laryngoscope by anaesthetists in easy and simulated difficult laryngoscopy--a manikin study. AB - The Airtraq Laryngoscope is a novel intubation device which allows visualisation of the vocal cords without alignment of the oral, pharyngeal and tracheal axes. We compared the Airtraq with the Macintosh laryngoscope in simulated easy and difficult laryngoscopy. Twenty-five anaesthetists were allowed up to three attempts to intubate the trachea in each of three laryngoscopy scenarios using a Laerdal Intubation Trainer followed by five scenarios using a Laerdal SimMan Manikin. Each anaesthetist then performed tracheal intubation of the normal airway a second time to characterise the learning curve. In the simulated easy laryngoscopy scenarios, there was no difference between the Airtraq and the Macintosh in success of tracheal intubation. The time taken to intubate at the end of the protocol was significantly lower using the Airtraq (9.5 (6.7) vs. 14.2 (7.4) s), demonstrating a rapid acquisition of skills. In the simulated difficult laryngoscopy scenarios, the Airtraq was more successful in achieving tracheal intubation, required less time to intubate successfully, caused less dental trauma, and was considered by the anaesthetists to be easier to use. PMID- 16674624 TI - A comparison of the Seeing Optical Stylet and the gum elastic bougie in simulated difficult tracheal intubation: a manikin study. AB - Management of the difficult airway is one of the major challenges that anaesthetists face. The flexible fibreoptic scope is widely available but its use requires a level of skill, training and continued practice that is not universally found in all anaesthetists, particularly trainees. The Seeing Optical Stylet is a new, semirigid fibreoptic stylet 'scope. We compared the Seeing Optical Stylet with a gum elastic bougie in a simulated Cormack and Lehane Grade 3 laryngoscopy in a manikin. Forty-four anaesthetists were timed while intubating the manikin's trachea with both devices. The mean (SD) time taken with the Seeing Optical Stylet was 20.8 (9.3) s and with the bougie 30 (19.8) s (p = 0.001). Oesophageal intubation occurred six times with the bougie but did not occur with the Seeing Optical Stylet (p = 0.011). We conclude that the Seeing Optical Stylet may be superior to the bougie in difficult tracheal intubation. We feel that the results of this manikin trial are sufficiently encouraging to proceed to a clinical trial in patients. PMID- 16674625 TI - A randomised, crossover study of the Dorges, McCoy and Macintosh laryngoscope blades in a simulated difficult intubation scenario. AB - The use of alternative adult laryngoscope blades in an unexpected difficult intubation was evaluated with a human patient simulator manikin. Twenty anaesthetists of varying experience attempted to intubate the trachea of a high fidelity simulator in both its normal and difficult intubation settings using Macintosh, Dorges and McCoy laryngoscopes in a randomised order. The time taken to intubate, Cormack and Lehane scores, percentage of glottic opening visible, failure rate, number of attempts and subjective ease of use were recorded. The Dorges and McCoy blades did not perform any better than the standard Macintosh blade in either the easy or difficult tracheal intubation settings. Guidelines recommending the use of an alternative blade in an unexpected difficult intubation scenario have limited supporting evidence. This study does not support this recommendation. PMID- 16674626 TI - The reliability of laryngoscope lights. AB - The aim of this study is to compare the brightness of laryngoscope light between instruments, and the variability within the same instrument. A secondary aim is to compare the light from a reusable laryngoscope with a single use design. Testing equipment was constructed which provided a qualitative measure of light output from laryngoscopes. Anaesthetists assessed the minimum acceptable brightness for intubation using the intubating mannikin and a variable brightness laryngoscope. Laryngoscopes in anaesthetic rooms around the hospital were then tested repeatedly, and the results were compared to the anaesthetists' opinions. There was considerable intersubject and intrasubject variation in the reusable design, often failing to satisfy the minimum brightness. The single use blades were found to be brighter and more reliable than reusable blades. PMID- 16674627 TI - The pressure required to force liquid through breathing system filters. AB - Liquid in the form of sputum and condensation may be forced through a breathing system filter if sufficient pressure is applied to the filter layer, with the potential for cross-infection. Twenty-three different breathing system filters (12 adult, 11 paediatric) were assessed to determine the pressure causing liquid penetration. Five samples of each were tested by connecting the ventilator end of a breathing system filter to a water-filled U-tube and reservoir. The reservoir was positioned so that the water surface was level with the filter layer and raised until liquid appeared through the filter layer on the patient side and the water-column height recorded. There were substantial differences in performance between models. Liquid penetration occurred at lower pressures for adult electrostatic than adult pleated filters: median [IQR] 1.18 [1.08- 1.32] and 8.04 [4.27-12.80] kPa, respectively, p < 0.0001. Liquid penetration occurred at similar pressures in paediatric electrostatic filters (0.98 [0.74-1.32] kPa) compared to adult electrostatic filters. PMID- 16674628 TI - Severe Ecstasy poisoning in a toddler. AB - A 17-month-old toddler became critically ill after an accidental overdose with 'Ecstasy'. A single tablet was quickly retrieved intact from under her tongue, but within 5 min the child developed generalised tonic-clonic seizures requiring immediate transfer to hospital. She also had hyperthermia (38.5 degrees C), tachycardia (150 beats.min(-1)) and hypertension. Treatment to terminate the seizures necessitated intubation and ventilation, and cooling measures brought the temperature within normal limits. The child was admitted to the intensive care unit and made a rapid recovery. She was discharged to the ward 12 h later, and had no long-term sequelae. PMID- 16674629 TI - Intraperitoneal fluid therapy: an alternative to intravenous treatment in a patient with limited vascular access. AB - We describe a 58-year-old female with Crohn's disease and short bowel syndrome after repeated intestinal resections, with only 90 cm of small intestine left. She had been dependent on vascular access for total parenteral nutrition for 16 years. Due to intravascular complications after numerous long-term central venous catheters, her vascular accessibility became limited. During the course of a year she was fed enterally through a gastrostomy, but required supplementary fluid therapy through peripheral venous route. Because of extremely limited venous access, we decided to implant an intraperitoneal catheter for administration of crystalloid fluid. The first intraperitoneal catheter had to be removed because of a postoperative infection, but after antibiotic treatment, a second intraperitoneal catheter was implanted without complications, through which the patient is now fully provided with crystalloid fluid (Ringer's acetate). Abdominal ultrasound examination shows good absorption of the fluid, and for the first time in 16 years the patient does not need intravascular access. We suggest that intraperitoneal administration of fluid may be an alternative for patients with limited vascular access. PMID- 16674630 TI - Failed intubation in obstetric anaesthesia. PMID- 16674632 TI - Local anaesthetic toxicity: prevention or cure? PMID- 16674635 TI - Analgesia after total hip replacement. PMID- 16674636 TI - Platelet function and diet. PMID- 16674638 TI - Better pre-operative evaluation. PMID- 16674640 TI - A reservoir bag for the Triservice anaesthetic apparatus. PMID- 16674641 TI - Ultrasound for determining the cause of shock during pregnancy. PMID- 16674642 TI - Ultra-low dose combined spinal-epidural anaesthesia for Caesarean section in severe pre-eclampsia. PMID- 16674643 TI - Survival after marked hyperlactaemia. PMID- 16674644 TI - Awake intubation through the CTrach in the presence of an unstable cervical spine. PMID- 16674645 TI - Stridor: remember the oesophagus. PMID- 16674646 TI - Anaesthesia in patient with Hailey-Hailey disease. PMID- 16674647 TI - Retromolar intubation--a simple alternative to submental intubation. PMID- 16674648 TI - Mental nerve injury following facemask anaesthesia. PMID- 16674649 TI - Inaccurate tidal volume readings when checking Blease 7800 ventilator with a Siemens test lung. PMID- 16674650 TI - Ultrasound guided central venous catheter placement using Geliperm. PMID- 16674651 TI - Assessing the laryngeal view by conventional laryngoscopy after awake fibreoptic tracheal intubation. PMID- 16674653 TI - Proceedings of the Seventh International Symposium on the Composition, Properties and Fundamental Structure of Tooth Enamel. April 10-14, 2005. Brewster, Massachusetts, USA. PMID- 16674655 TI - Mutational analysis of candidate genes in 24 amelogenesis imperfecta families. AB - Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in dental enamel formation. The malformed enamel can be unusually thin, soft, rough and stained. The strict definition of AI includes only those cases where enamel defects occur in the absence of other symptoms. Currently, there are seven candidate genes for AI: amelogenin, enamelin, ameloblastin, tuftelin, distal-less homeobox 3, enamelysin, and kallikrein 4. To identify sequence variations in AI candidate genes in patients with isolated enamel defects, and to deduce the likely effect of each sequence variation on protein expression and structure, families with isolated enamel defects were recruited. The coding exons and nearby intron sequences were amplified for each of the AI candidate genes by using genomic DNA from the proband as template. The amplification products for the proband were sequenced. Then, other family members were tested to determine their genotype with respect to each sequence variation. All subjects received an oral examination, and intraoral photographs and dental radiographs were obtained. Out of 24 families with isolated enamel defects, only six disease-causing mutations were identified in the AI candidate genes. This finding suggests that many additional genes potentially contribute to the etiology of AI. PMID- 16674656 TI - Human enamel phenotype associated with amelogenesis imperfecta and a kallikrein-4 (g.2142G>A) proteinase mutation. AB - Kallikrein-4 is known to be highly expressed during the maturation stage of enamel formation and is thought to be critical for the final phase of crystallite growth. The purpose of this study was to evaluate the enamel phenotype in humans with a known KLK-4 mutation (g.2142G>A). Primary teeth from two individuals with a known KLK-4 mutation were evaluated using amino acid analysis and light and electron microscopy. Light microscopy showed the enamel was of normal thickness but opaque throughout its width compared with normal enamel. Electron microscopy showed enamel affected by the KLK-4 mutation had a normal prismatic structure and generally had a well-organized and discernable crystallite composition. In some areas, globular structures were present where crystallites were not discernable or appeared to have an altered morphology. The KLK-4 mutant enamel had an increased protein content compared with normal enamel. Human enamel formed with a lack of functioning KLK-4 proteinase is altered primarily in the completeness of crystallite growth, while enamel thickness and prism structure remains essentially normal. Collectively, these studies suggest that the KLK-4 proteinase is essential for the final crystallite growth of enamel but is not critical for crystallite orientation, prism formation or enamel thickness. PMID- 16674657 TI - A developmental comparison of matrix metalloproteinase-20 and amelogenin null mouse enamel. AB - Mutations in both the human amelogenin and human matrix metalloproteinase-20 (MMP20, enamelysin) genes cause amelogenesis imperfecta. Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP 20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern. PMID- 16674658 TI - Formation of the dentino-enamel interface in enamelysin (MMP-20)-deficient mouse incisors. AB - An anomalous dentino-enamel junction (DEJ), manifested by delamination of the enamel layer, was reported in enamelysin [matrix metalloproteinase-20 (MMP-20)] knockout (KO) mice. To better understand the possible role of MMP-20 in the formation of the DEJ, we performed transmission electron microscopy (TEM) studies of the DEJ at early stages of tooth morphogenesis in KO mice. Our TEM analysis revealed that in the incisors from KO mice the mantle dentin is hypomineralized at the onset of enamel mineralization. At this early stage, TEM revealed no apparent differences in nascent aprismatic enamel between the KO mice and the controls. Hypomineralized mantle dentin was also observed in the incisors from KO mice, as assessed by back-scattered SEM at the secretory and early maturation stages, but not in the late-maturation stage, suggesting that the mineralization of mantle dentin is not completely arrested, but rather postponed. Histological studies indicate that the organic content in the initial enamel layer remains very high throughout amelogenesis. These results imply that MMP-20 is involved in the regulation of mineralization in mantle dentin and demonstrate the complex nature of DEJ formation. They also suggest that the structural and functional properties of the DEJ are determined during the initial mineralization stages. PMID- 16674659 TI - Overexpression of transforming growth factor-beta1 in teeth results in detachment of ameloblasts and enamel defects. AB - Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF beta1 in teeth impacts the adhesion process of ameloblasts to dentin. PMID- 16674660 TI - The new Enamel Defects Index: testing and expansion. AB - The Enamel Defects Index (EDI) was created based on three innovative principles: (i) a basic level of the three major categories of defects; (ii) more detailed subcategories of each major category; and (iii) each category scored independently as present [1] or absent [0], simplifying decision making. The aim of this investigation was to further test the index in a number of applications and to expand it to record defect subtype and treatment need. Testing was undertaken by operators with different levels of clinical experience. A computer assisted learning (CAL) package was developed for operator training and calibration. The index was also used on clinical photographs and high-resolution digital images of exfoliated and extracted teeth. Scoring of photographs revealed substantial intra-operator agreement. Training using the CAL package resulted in significant improvement in index use. Intra-operator reproducibility was good to excellent, and interoperator reproducibility was good for buccal surfaces on digital images. Index expansion allowed information on defect subtype, location, and treatment need to be gathered readily. The EDI has high reproducibility and allows more rapid and accurate data collection from clinical and in vitro studies than the Federation Dentaire Internationale Developmental Defects of Enamel index. PMID- 16674662 TI - How do enamelysin and kallikrein 4 process the 32-kDa enamelin? AB - The activities of two proteases--enamelysin (MMP-20) and kallikrein 4 (KLK4)--are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion. PMID- 16674663 TI - Proteolysis on maturing enamel surface, as shown by gel-coating methods. AB - Degradation of enamel matrix proteins, and their removal during early maturation, is critical for the growth of large enamel crystals in the subsequent processes of enamel maturation. In this study, we sought to demonstrate, using in vivo zymography, the exact sites of proteolysis in maturing enamel and its relationship to the overlying ameloblasts. The maturing enamel surfaces of rat and bovine incisors were exposed and painted either with pre-exposed autoradiographic emulsion or with densely fluorescein-conjugated (DQ) gelatin. After a few hours, photographic development of the emulsion revealed alternate black and white banding patterns over the maturing enamel surface. DQ gelatin also revealed similar banding patterns of fluorescent and non-fluorescent regions. White, powdery areas of emulsion and fluorescent bands of DQ gelatin both corresponded to the areas of ruffle-ended ameloblasts, at least up to the mid stages of enamel maturation, implicating a predominant contribution of ruffle ended ameloblasts in the degradation of enamel matrix proteins. Powdery white bands in autoradiographic emulsion shifted from the areas of ruffle-ended to smooth-ended ameloblasts in late maturation in both bovine and rat incisors and were not influenced by proteinase inhibitors or heat inactivation, implicating non-enzymatic interactions. DQ gelatin, in fact, did not generate any fluorescence in such smooth-ended ameloblast regions. PMID- 16674664 TI - Assembly and processing of an engineered amelogenin proteolytic product (rP148). AB - The purpose of this study was to express, characterize, and investigate the self assembly of a recombinant porcine amelogenin lacking the hydrophilic 24 C terminal amino acids (rP148). To gain further insight into the function of amelogenin processing during enamel mineralization, this protein was also used as a substrate to examine the action of matrix metalloproteinase-20 (MMP-20). The assembly properties of rP148 were monitored by dynamic light scattering (DLS). In general, rP148 molecules assemble into monomers, dimers, oligomers, and some nanosphere-like particles. Depending on the solution conditions, large aggregates were also observed. Matrix metalloproteinase-20 cleaved the rP148 molecule at a few sites, creating a number of different products, including the tyrosine-rich amelogenin polypeptide (TRAP). Our data suggest that although rP148 self assembles into small particles, its assembly properties are different from those of the full-length rP172, indicating that the C-terminal 24 amino acids play a critical role in nanosphere assembly. We further demonstrate that MMP-20 digests rP148 in a manner that generates a similar proteolytic pattern, as would be expected to occur in vivo. PMID- 16674665 TI - The amelogenin story: origin and evolution. AB - Genome sequencing and gene mapping have permitted the identification of HEVIN (SPARC-Like1) as the probable ancestor of the enamel matrix proteins (EMPs), amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). We have undertaken a phylogenetic analysis to elucidate their relationships. AMEL genes available in databases, and new sequences obtained in blast searching genomes or expressed sequence tags, were compiled (22 full-length sequences), aligned, and the ancestral sequence calculated and used to search for similarities using psi blast. Hits were obtained with the N-terminal region of AMBN, ENAM, and HEVIN. We retrieved all available AMBN (n=8), ENAM (n=3), and HEVIN (n=4) sequences. The sequences of the four proteins were aligned and analyzed phylogenetically. AMEL and AMBN are sister genes, which diverged after duplication of a common ancestor issued from ENAM. The latter derived from a copy of HEVIN. Comparisons of gene organization, amino acid sequences and location of ENAM and AMBN, adjacent on the same chromosome, suggest that AMBN is closer to ENAM than AMEL. This supports AMEL as being derived from AMBN duplication. This duplication occurred long before tetrapod differentiation, probably in an ancestral osteichthyan. The story of AMEL origin is completed as follows: SPARC-->HEVIN-->ENAM-->AMBN-->AMEL. PMID- 16674666 TI - Porcine SPARC: isolation from dentin, cDNA sequence, and computer model. AB - Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain. PMID- 16674667 TI - Expression and characterization of a Rana pipiens amelogenin protein. AB - Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico-chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens. In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli. Our newly generated R. pipiens amelogenin-specific antisera resolved a major 19-kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue-specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence-based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41-nm radius subunits compared with 14-nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self-assembly in contrast with a homogeneous 'stippled' matrix in mouse amelogenin matrix self-assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico-chemical properties which may be related to specific modes of crystal formation in frog enamel. PMID- 16674669 TI - The effects of fluoride on the nanostructure and surface pK of enamel crystals: an atomic force microscopy study of human and rat enamel. AB - Atomic force microscopy (AFM) studies have revealed 30-40 nm-wide regular positively charged bands across maturation-stage rat enamel crystals. Low pH resolved these into positively charged spherical domains of approximately 30 nm diameter. Crystal surface pK values from adhesion force titrations were approximately 6.5. The effect of fluoride on this pK value and on the nanostructure of fluorosed human enamel crystals has not been reported. The nanostructure and surface chemistry (pK) of normal and fluorotic human and of fluoride-treated rat maturing enamel crystals was examined. Enamel was sectioned and polished, prior to examination, using AFM in height and friction modes. High resolution height images revealed 30 nm-diameter spherical domains within crystals, arranged as layers of hexagons or as a shallow spiral. Fluorotic enamel showed similar, but less well ordered, nanodomains. These could represent an arrangement of original initiation sites or binding sites for modulating matrix proteins. Surface pK was derived from adhesion-force measurements between functionalized tips (OH or COOH) and crystal surfaces between pH 2 and pH 10. pK values of approximately 6.5 for normal crystals were reduced to approximately 5.5 after fluoride treatment. Reduction in surface pK by fluoride might indicate lowered protonation with possible effects on matrix protein binding. PMID- 16674670 TI - Fluoride down-regulates the expression of matrix metalloproteinase-20 in human fetal tooth ameloblast-lineage cells in vitro. AB - Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10 microM fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10 microM NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10 microM NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel. PMID- 16674671 TI - Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization. AB - We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F-) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5-20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of >or=5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F- is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth. PMID- 16674672 TI - Effect of calcium, given before or after a fluoride insult, on hamster secretory amelogenesis in vitro. AB - We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [3H]proline and uptake of 45Ca and acid soluble 32PO4. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non mineralizing) enamel matrix, increased matrix secretion but could not prevent F induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front. PMID- 16674673 TI - Improving esthetically objectionable human enamel fluorosis with a simple microabrasion technique. AB - Mild-to-moderately severe enamel fluorosis (EF) is an unsightly maturation-phase dental disorder. Despite extensive epidemiological studies on EF, little is known about individual treatment options. This study was carried out to determine whether a simple microabrasion technique is effective in improving the esthetics of EF. Patients with a variety of severities were treated using a water-cooled fine diamond polishing bur at high speed to remove the surface enamel layers. Photographs of the affected teeth before and after treatment were shown by computer to a panel of three judges (two lay and one experienced), who rated the appearance of the teeth using a newly developed visual analog scale. The severity of EF was rated randomly and blind for 52 individual teeth (26 before and 26 after treatment). Reteated-measures analysis of variance was used to analyze the results. The lay judges rated the appearance of the teeth with EF as significantly more objectionable before treatment. All judges found a significant improvement in the severity of EF after treatment. Using a newly developed visual analog scale, our study indicates that EF of an objectionable nature can be significantly improved with a simple microabrasion technique, thus conserving tooth structure and minimizing the cost of treating EF. PMID- 16674675 TI - Imaging RNA polymerase-amelogenin gene complexes with single molecule resolution using atomic force microscopy. AB - The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel. PMID- 16674676 TI - Identification of secreted and membrane proteins in the rat incisor enamel organ using a signal-trap screening approach. AB - The secretome represents the subset of proteins that are targeted by signal peptides to the endoplasmic reticulum. Among those, secreted proteins play a pivotal role because they regulate determinant cell activities such as differentiation and intercellular communication. In calcified tissues, they also represent key players in extracellular mineralization. This study was carried out to establish a secretome profile of rat enamel organ (EO) cells. A functional genomic technology, based on the signal trap methodology, was applied, starting with a library of 5'-enriched cDNA fragments prepared from rat incisor EOs. A total of 2,592 clones were analyzed by means of macroarray hybridizations and DNA sequencing. Ninety-four unique clones encoding a signal peptide were retrieved. Among those were 84 matched known genes, many not previously reported to be expressed by the EO. Most importantly, 10 clones were classified as being novel, with EO-009 identified as the rat homolog of human APin protein. These data indicate that many secreted and membrane-embedded EO proteins still remain to be identified, some of which may play crucial roles in regulating processes that create an optimal environment for the formation and organization of apatite crystals into a complex three-dimensional calcified matrix. PMID- 16674677 TI - Evidence by signal peptide trap technology for the expression of carbonic anhydrase 6 in rat incisor enamel organs. AB - During screening of a rat incisor enamel organ cDNA library by signal peptide trap technology, we identified a DNA fragment matching a predicted translation sequence for rat carbonic anhydrase 6 (CA6). This result was unexpected because CA6, to date, has been associated primarily with secretions from glandular tissues. To further characterize this observation, reverse transcription polymerase chain reaction (RT-PCR) amplifications were carried out on total RNA extracted from freeze-dried secretory and maturation-stage rat incisor enamel organs. A cDNA fragment of the expected size was detected in control samples from rat salivary glands as well as within maturation-stage enamel organ samples. This CA6 RT-PCR fragment was further cloned and sequenced and found to match the nucleotide sequence 770-1079 from clone XM_216584 of GenBank. Northern blot analyses with the rat CA6 cDNA fragment confirmed its expression relative to maturation-stage enamel organ samples. It is at present unclear whether the CA6 expressed by enamel organ cells is secreted into the enamel layer or into the intercellular spaces of the enamel organ itself to assist in neutralizing excess protons arising from the growth of apatite crystals during the maturation stage of amelogenesis. PMID- 16674678 TI - Differentiation of human ameloblast-lineage cells in vitro. AB - Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5 15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering. PMID- 16674679 TI - Growth of ameloblast-lineage cells in a three-dimensional Matrigel environment. AB - Enamel organ epithelial cells grow in culture as two distinct cell populations- either stellate-shaped or polygonal-shaped cells. The polygonal cells have an ameloblast cell phenotype and are difficult to grow in culture beyond two passages. This study was designed to determine the effects of a Matrigel three dimensional (3D) environment on polygonal cells, as compared with stellate cells, derived from porcine tooth enamel organ. Enamel organs were dissected free from the unerupted molars of 30-kg pigs and then grown in LCH-8e media, either with or without serum. Cells grown in serum-free media were primarily polygonal shaped, whereas cells grown in media containing serum were stellate shaped. Both types of cells were grown in a 3D Matrigel matrix. In addition, polygonal-shaped cells were mixed with hydroxyapatite powder and transplanted subcutaneously into nude mice. Polygonal-shaped epithelial cells formed cell groups, similar to epithelial pearls, both in vitro and in vivo. The stellate-shaped cells, in contrast, did not form similar structures, but remained suspended in the Matrigel and gradually disappeared from the culture. These results suggest that a Matrigel environment, rich in basement membrane and matrix proteins, selects for polygonal-shaped ameloblast-lineage cells and induces the formation of epithelial pearls. PMID- 16674681 TI - Transcription factor sumoylation and factor YY1 serve to modulate mouse amelogenin gene expression. AB - Amelogenin proteins are essential in the control of enamel biomineralization and the amelogenin gene therefore is spatiotemporally regulated to ensure proper amelogenin protein expression. In this study, we examined the role of sumoylation to alter CCAAT/enhancer-binding protein alpha (C/EBPalpha) activity, and performed a search using a protein/DNA array system for other proteins that act co-operatively with C/EBPalpha to alter amelogenin expression. We observed that C/EBPalpha was modified by sumoylation, and that this modification played an indirect inhibitory role on the regulation of C/EBPalpha activity which appeared to act through other transcription factors. The protein/DNA array allowed us to single out the transcription factor, YY1, which acts in the absence of direct DNA binding to repress both the basal amelogenin promoter activity and C/EBPalpha mediated transactivation. Taken together, these pathways may account for part of the physiological modulation of the amelogenin gene expression in accordance with tooth developmental and enamel biomineralization requirements. PMID- 16674682 TI - Vitamin D and tissue non-specific alkaline phosphatase in dental cells. AB - Dental epithelium comprises different cell populations, including ameloblasts and stratum intermedium cells. Ameloblasts are vitamin D targets, and at least five proteins undergo specific modulation of their expression following the addition of 1alpha,25(OH)2 vitamin D3[1alpha,25(OH)2D3]. Stratum intermedium cells have not been studied in any great detail regarding vitamin D impact. Interestingly, in these cells, the tissue non-specific alkaline phosphatase (TNAP) is overexpressed. On the other hand, TNAP is a reliable bone marker of vitamin D action, similar to calbindins in kidney and intestine, previously used for studies of vitamin D activity in ameloblasts. Here, TNAP expression and activity were investigated in vivo in the microdissected epithelium and mesenchyme of mandible incisors. Physiological doses of 1alpha,25(OH)2D3 injected in control rats failed to modify TNAP activity in both dental epithelium and mesenchyme. No significant differences were observed in the steady-state levels of TNAP mRNAs of dental tissues from wild-type and vitamin D nuclear receptor (VDRnuc)-deficient mice of the same litters. These data suggest that, in contrast to ameloblasts, stratum intermedium cells are not sensitive to 1alpha,25(OH)2D3. An explanation for such a responsiveness of stratum intermedium cells to 1alpha,25(OH)2D3 is proposed based on the respective expressions of both vitamin D receptors (VDRnuc and 1,25D3-[MARRS]) and the Dlx2 homeobox gene. PMID- 16674683 TI - Amelogenin, a major structural protein in mineralizing enamel, is also expressed in soft tissues: brain and cells of the hematopoietic system. AB - The amelogenin protein is considered as the major molecular marker of developing and mineralizing ectodermal enamel. It regulates the shape, size, and direction of growth of the enamel mineral crystallite. Recent data suggest other roles for amelogenin beyond regulation of enamel mineral crystal growth. The present study describes our recent discovery of amelogenin expression in soft tissues: in brain and in cells of the hematopoietic system, such as macrophages, megakaryocytes and in some of the hematopoietic stem cells. Reverse transcription-polymerase chain reaction (RT-PCR) followed by cDNA sequencing revealed, in mouse brain, two amelogenin mRNA isoforms: the full-length amelogenin including exon 4, and the isoform lacking exon 4. Immunohistochemistry revealed amelogenin expression in brain glial cells. Mouse macrophages were found to express the full-length amelogenin sequence lacking exon 4. Confocal microscopy revealed colocalization of amelogenin and CD41 (a megakaryocyte marker), as well as amelogenin and CD34 (a hematopoietic stem cell marker) in some of the bone marrow cells. The expression of amelogenin, a major structural protein of the mineralizing extracellular enamel matrix, also in cells of non-mineralizing soft tissues, suggests that amelogenin is multifunctional. Several different potential functions of amelogenin are discussed. PMID- 16674684 TI - Comparison of body weight and gene expression in amelogenin null and wild-type mice. AB - Amelogenin (AmelX) null mice develop hypomineralized enamel lacking normal prism structure, but are healthy and fertile. Because these mice are smaller than wild type mice prior to weaning, we undertook a detailed analysis of the weight of mice and analyzed AmelX expression in non-dental tissues. Wild-type mice had a greater average weight each day within the 3-wk period. Using reverse transcription-polymerase chain reaction (RT-PCR), products of approximately 200 bp in size were generated from wild-type teeth, brain, eye, and calvariae. DNA sequence analysis of RT-PCR products from calvariae indicated that the small amelogenin leucine-rich amelogenin peptide (LRAP), both with and without exon 4, was expressed. No products were obtained from any of the samples from the AmelX null mice. We also isolated mRNAs that included AmelX exons 8 and 9, and identified a duplication within the murine AmelX gene with 91% homology. Our results add additional support to the hypothesis that amelogenins are multifunctional proteins, with potential roles in non-ameloblasts and in non mineralizing tissues during development. The smaller size of AmelX null mice could potentially be explained by the lack of LRAP expression in some of these tissues, leading to a delay in development. PMID- 16674685 TI - Identification of temporal and spatial expression patterns of amelogenin isoforms during mouse molar development. AB - Amelogenin synthesis is initiated in a restricted time frame during odontogenesis. Polypeptides translated from several alternatively spliced isoforms of amelogenin mRNA have been identified in ameloblasts and odontoblasts. Recent studies suggest that the isoforms deleting exons 6a, 6b, and 6c produce polypeptides that might exert regulatory functions governing the late stages of ameloblast and odontoblast differentiation. Herein, the spatial and temporal expression of mouse amelogenin mRNA isoforms M194, M180, M73, and M59 have been determined around the perinatal development period using splice form-specific probes. Expression levels and distribution patterns varied with developmental stage and cell location. Amelogenin mRNA expression was most prominent within the enamel organ at boundaries between cell layers, beginning at the newborn stage (PN0.5). Odontoblasts supported the expression of M73 and M59 mRNA from developmental stages PN0.5 to PN1.5 (1 d of age). In contrast, ameloblasts expressed predominantly the M180 mRNA isoform with full exon 6 but devoid of exon 4. In the enamel organ, the stratum intermediun cells supported expression of the full-length isoform, M194, including the full exon 6 and exon 4 sequences, and strikingly, expression of M180 message was inhibited. In conclusion, ameloblasts, odontoblasts, and stratum intermedium cells demonstrate selective alternative splicing patterns of the amelogenin pre-mRNA transcript. PMID- 16674687 TI - The effect of enamel matrix derivative on gene expression in osteoblasts. AB - Observations that amelogenins, in the form of enamel matrix derivative (EMD), have a stimulatory effect on mesenchymal cells and tissues, and on the regeneration of alveolar bone, justified investigations into the effect of EMD on bone-forming cells. The binding and uptake of EMD in primary osteoblastic cells was characterized, and the effect of EMD on osteoblast gene expression, protein secretion, and mineralization was compared with the effect of parathyroid hormone (PTH). Although no specific receptor(s) has yet been identified, EMD appeared to be taken up by osteoblasts through clathrin-coated pits via the interaction with clathrin adaptor protein complex AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells. EMD had a positive effect on factors involved in mineralization in vitro, causing an increased alkaline phosphatase (ALP) activity in the medium as well an as increased expression of osteocalcin and collagen type 1. Several hundred genes are regulated by EMD in primary human osteoblasts. There appear to be similarities between the effects of EMD and PTH on human osteoblasts. The expression pattern of several mRNAs and proteins upon EMD stimulation also indicates a secondary osteoclast stimulatory effect, suggesting that the osteogenic effect of EMD in vivo, at least partly, involves stimulation of bone remodelling. PMID- 16674688 TI - Porcine sheath proteins show periodontal ligament regeneration activity. AB - The purpose of this study was to identify the periodontal regeneration factors of enamel protein extracts that induce cementum and bone regeneration in vivo. Cementum regeneration, one aspect of periodontal ligament regeneration, was examined using a buccal dehiscence model of dogs. Enamel matrix protein fractions were prepared from developing porcine incisors. Cementum-regeneration activity was found to reside in a protein aggregate composed of amelogenins and sheath proteins extracted from newly formed secretory enamel. Cementum-regeneration activity was not observed in protein fractions containing only amelogenin or its derivatives. The sheath proteins were purified to homogeneity and tested for alkaline phosphatase (ALP)-inducing activity on human periodontal ligament (HPDL) cells. The induction of ALP was observed following application of the 17-kDa sheath protein but not of the lower-molecular-weight sheath proteins. Although transforming growth factor-beta1 also shows ALP-inducing activity, contamination with growth factors was excluded because synthetic peptides (based on the sheath protein's sequence) also showed ALP-inducing activity. The 17-kDa sheath protein showed both cytodifferentiation and cementum-regeneration activity, but it is unclear whether its cementum-regeneration activity is derived from its ALP inducing activity on HPDL cells. PMID- 16674689 TI - Evidence for direct amelogenin-target cell interactions using dynamic force spectroscopy. AB - Increasing evidence suggests that amelogenin, long held to be a structural protein of developing enamel matrix, may also have cell signaling functions. However, a mechanism for amelogenin cell signaling has yet to be described. The aim of the present study was to use dynamic chemical force spectroscopy to measure amelogenin interactions with possible target cells. Full-length amelogenin (rM179) was covalently attached to silicon nitride AFM tips. Synthetic RGD peptides and unmodified AFM tips were used as controls. Amelogenin-RGD cell binding force measurements were carried out using human periodontal ligament fibroblasts (HPDF) from primary explants and a commercially available osteoblast like human sarcoma cell line as the targets. Results indicated a linear logarithmic dependence between loading rate and unbinding force for amelogenin RGD target cells across the range of loading rates used. For RGD controls, binding events measured at 5.5 nN s-1 force loading rate resulted in a mean force of 60 pN. Values for amelogenin-fibroblast and amelogenin-osteoblast-like cell unbinding forces, measured at similar loading rates, were 50 and 55 pN, respectively. These data suggest that amelogenin interacts with potential target cells with forces characteristic of specific ligand-receptor binding, suggesting a direct effect for amelogenin at target cell membranes. PMID- 16674690 TI - Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins. AB - A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern. PMID- 16674691 TI - Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells. AB - Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A 4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4). PMID- 16674692 TI - Amelogenins regulate expression of genes associated with cementoblasts in vitro. AB - Amelogenins are major proteins expressed by ameloblasts during development of the crown (enamel and dentin). These matrix proteins guide crystal habits of the mineral phase of developing enamel and are possible regulators of other genes/proteins during development and maturation of crown and root (dentin and cementum). This study focused on defining the effect that a specific proteolytic cleavage product of amelogenin, tyrosine-rich amelogenin peptide (TRAP), has on cementoblast behavior. Immortalized cementoblasts (OCCM-30) were exposed to TRAP in vitro. Cells treated with TRAP were evaluated for cell proliferation, gene expression for osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP), and induction of mineral nodule formation. No significant difference in cell proliferation was found between vehicle-treated cells and those treated with TRAP for up to 9 d after treatment. Gene expression of OCN, OPN, and BSP in TRAP treated cementoblasts showed down-regulation, up-regulation, and no significant change, respectively, relative to vehicle control. A marked decrease in mineral nodule formation was found in cells treated with TRAP compared with the vehicle control, in a dose-dependent manner. These data, along with our previous results demonstrating similar activity with full-length amelogenin and leucine-rich amelogenin peptide (LRAP), suggest that amelogenin-like molecules regulate mesenchymal cell behavior. PMID- 16674693 TI - Amelogenin and ameloblastin show growth-factor like activity in periodontal ligament cells. AB - Enamel proteins, particularly amelogenin, have been associated with other functions in addition to regulating enamel biomineralization. Extracts of enamel proteins are currently being used to regenerate periodontal tissues, and new studies suggest that enamel proteins might have chondrogenic and osteogenic properties. In this study, we wanted to determine the effect, if any, of purified recombinant amelogenin and ameloblastin on the adhesion, proliferation, and differentiation of periodontal ligament cells in vitro. Immortomouse-derived periodontal ligament (PDL) cells were grown under permissive and differentiation conditions in the presence of different concentrations of mouse recombinant amelogenin, recombinant ameloblastin, or both. Cells were collected after 4 h to determine attachment, after 24 h to determine proliferation, and after 7, 14, 21 and 28 d to determine differentiation using reverse transcription-polymerase chain reaction (RT-PCR). Both amelogenin and ameloblastin had a small, but statistically significant, effect on increasing the cell attachment and proliferation of PDL cells. Both amelogenin and ameloblastin modulated bone morphogenetic protein (BMP) expression, down-regulated the expression of collagen type I, and induced the de novo expression of osteocalcin. Amelogenin also induced the expression of bone sialoprotein. These results suggest that amelogenin, as well as ameloblastin, might have some 'growth factor' activity during periodontium development and regeneration. PMID- 16674695 TI - Towards second-generation proteome analysis of murine enamel-forming cells. AB - Proteome analysis of rat enamel-forming cells, initiated over a decade ago, has provided valuable insights to enamel biology. In preparation for a more comprehensive, second-generation proteomic exploration, we evaluated an updated microsample-profiling strategy that comprises sequential extraction of enamel epithelium, parallel one- and two-dimensional gel electrophoresis, and mass spectrometric sequence analysis. The results indicated that several hundred proteins, representing various cellular compartments (including membranes), are amenable to identification with a starting tissue volume of <10 microl. With its increased proteomic depth and breadth, this straightforward approach constitutes a major advance from the first-generation work (10-fold increased proteome coverage), although care was needed to ensure a comparably high stringency of protein identification. Expression proteomics has an exciting potential to elucidate the inner workings of murine enamel epithelial cells, leading to an improved understanding of enamel in health and disease. PMID- 16674696 TI - Proteomic analysis of enamel matrix using a two-dimensional protein fractionation system. AB - Our objectives in this study were to perform separate proteomic analyses of porcine soft and hard enamel matrices, using the ProteomeLab PF-2D System, to compare the contents of the hard and soft enamel and to identify matrix constituents that are absent from the early maturation stage. Developing first permanent molars were dissected from 6-month-old pigs. Both immature and mature enamel samples were obtained by scraping the secretory-stage (soft) and maturation-stage (hard) enamel, respectively. Enamel matrix samples were sequentially extracted and fractionated with 50 mM phosphate buffer (pH 7.4) and then with 50 mM carbonate buffer (pH 10.8). The neutral enamel extract was separated into four fractions by successive ammonium sulfate precipitations. The alkaline enamel extract was separated into four fractions by ion-exchange chromatography. These eight extracts from both the soft and hard enamel were injected for chromatofocusing. Soft enamel fractions containing constituents absent from the hard enamel were further separated by reverse-phase high performance liquid chromatography. The major soft enamel constituents absent from the hard enamel were acidic glycoproteins, corresponding to the 32-kDa enamelin, and the 29-, 27-, 15-, 13-, 8- and 6-kDa C-terminal fragments of ameloblastin. Loss of these glycoproteins is associated with a post-transition increase in enamel mineralization. PMID- 16674697 TI - Comparison of three methods for enamel protein extraction in different developmental phases of rat lower incisors. AB - Protein extraction methods [urea, trichloroacetic acid (TCA), and acetic acid] were compared for protein recovery from rat incisor developing enamel in the S phase (intermediate/late secretion), M1 phase (early maturation), M2 phase (intermediate maturation), and M3 phase (final maturation). We compared the protein recoveries with the percentage of enamel matrix dry weight burnt off by incineration. Our results indicate that TCA and urea were equally efficient for the extraction of S-stage proteins (85% and 90% recovery, respectively), while urea was the best for M1-stage proteins (92% recovery), and TCA the best for M2 stage (99% recovery) and M3-stage (60% recovery) proteins. The other methods yielded less than 30% recovery in comparison to incineration for M2 and M3 stages. The fact that urea extraction works well in the S and M1 stages and not thereafter is probably related to the changes in the proteins during enamel development and the amount of mineral that needs to be dissolved. TCA is the single method that effectively recovered proteins from all developmental stages of the rat incisor enamel. PMID- 16674698 TI - Using the yeast two-hybrid assay to discover protein partners for the leucine rich amelogenin peptide and for tuftelin-interacting protein 11. AB - The established structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. Historically, tuftelin and tuftelin-interacting protein 11 (TFIP11) have also been discussed as possible enamel proteins. Protein complexes are achieved by protein-protein interactions, and it is protein complexes that control biomineralization. The purpose of our recent studies was to catalog protein partners for these proteins that are, or have been, implicated in tooth formation. We used the sensitive yeast two-hybrid assay to identify proteins that interact directly with amelogenin, ameloblastin, enamelin, the leucine-rich amelogenin peptide (LRAP) and TFIP11. In this manuscript we refer to, or document, potential protein partners for the proteins listed above. The yeast two-hybrid assay may ultimately prove to be a valuable proteomics methodology for using to decipher molecular events that ultimately result in enamel biomineralization. PMID- 16674699 TI - Intracellular nanosphere subunit assembly as revealed by amelogenin molecular cross-linking studies. AB - Enamel matrix comprises nanospheres predominantly composed of amelogenin. Studies have shown that recombinant amelogenin forms nanospheres similar to those formed in vivo, but it is unclear exactly how nanospheres assemble in vivo. Are amelogenin monomers secreted into the enamel matrix where they then self-assemble to form nanospheres, or does nanosphere assembly actually occur intracellularly? The aim of this study was to attempt to answer this question. Rat enamel organs were treated with the bifunctional cross-linker, dithio bis (succinimidyl propionate) (DSP), which cross-links primary amines lying in close molecular proximity. The key to this technique is the fact that DSP cross-links are later sensitive to reductive cleavage. The cross-linked proteins were first subjected to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) in the first dimension and then to reducing SDS-PAGE in the second dimension (so-called diagonal electrophoresis) followed by western blot probing with anti-amelogenin. The results indicated that intracellular amelogenin monomers are in close neighbor contact, forming complexes comprising up to six individual amelogenin monomers. We suggest that these initial complexes are prefabricated intracellularly before secretion. Once secreted, these prefabricated subunits assemble further to form the mature full-size nanospheres containing hundreds of individual amelogenins characteristic of enamel matrix. PMID- 16674701 TI - On the formation of amelogenin microribbons. AB - We recently reported the remarkable spontaneous self-assembly and hierarchical organization of amelogenin 'microribbons' and their ability to facilitate oriented growth of apatite crystals in vitro. In a letter of correction we communicated the finding that the X-ray diffraction pattern reported in our original report was that of cellulose contaminant and not amelogenin microribbon. We have re-evaluated our data and confirmed the protein nature of the microribbons using Fourier transform infrared and Raman microspectroscopy. Some microribbons were remarkably similar in their morphology to that of cellulose fibers. The size distribution of amelogenin microribbons was wider, particularly in width and length, and generally smaller than those originally reported. Here we present additional detailed information on the formation of a series of intermediate hierarchical structures of amelogenin assemblies prior to the formation of microribbon. The most significant finding was that full-length amelogenin nanospheres had a tendency to assemble into collinear arrays whose function is assumed to be critical at the initial stage of enamel mineral deposition. The present data gives an insight into the step-by-step assembly process of amelogenin from nanometer scale molecules to micrometer scale organized structures that can be used as templates for controlled and oriented growth of apatite mineralization in vitro. PMID- 16674702 TI - Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro. AB - An amorphous silica mineralization technique was used to produce inorganic/protein composites to elucidate the structure and mechanism of formation of amelogenin assemblies, which may play an important role in regulating enamel structure during the initial stages of amelogenesis. Full length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated along with key degradation products (rM166 and native P148) lacking the hydrophilic C terminus found in parent molecules. The resulting products were examined using transmission electron microscopy and/or small-angle X-ray scattering. Using protein concentrations of 0.1-3 mg ml-1, large monodisperse spheres of remarkably similar mean diameters were observed using rM179 (124+/-4 nm) and rP172 (126+/-7 nm). These spheres also exhibited 'internal structure', comprising nearly spherical monodisperse particles of approximately 20 nm in diameter. In the presence of rM166, P148, and bovine serum albumin (control), large unstructured and randomly shaped particles (250-1000 nm) were observed. Without added protein, large dense spherical particles of silica (mean approximately 500 nm) lacking internal structure were produced. These findings demonstrate that full-length amelogenins have the ability to form higher-order structures, whereas amelogenins that lack the hydrophilic C terminus do not. The results also suggest that full-length amelogenin can guide the formation of organized mineralized structures through co-operative interactions between assembling protein and forming mineral. PMID- 16674703 TI - Control of apatite crystal growth by the co-operative effect of a recombinant porcine amelogenin and fluoride. AB - Recently, we used native amelogenins extracted from developing pig enamel to examine the combined effect of fluoride and amelogenins on the growth of octacalcium phosphate (OCP) and apatite crystals. The purpose of the present study was to investigate this combined effect using a highly purified recombinant amelogenin. We applied porcine amelogenin (rP172) and fluoride in a dual-membrane system as a model for tooth enamel formation. The combination of rP172 and fluoride in this system resulted in the formation of rod-like apatite crystals. On the other hand, without fluoride, rod-like OCP crystals of a comparable size were formed, and rather large hexagonal prisms of mixed crystals of OCP and apatite grew without amelogenins. Thus, highly purified and homogeneous recombinant amelogenin, in co-operation with F, regulated the mineral phase, habit, and size of crystals in the same manner as the extracted heterogeneous porcine amelogenins. We suggest that in both cases the control over the crystal phase and morphology was a direct effect of amelogenin protein serving as a scaffold for apatite mineralization. PMID- 16674704 TI - Dynamic light scattering study of an amelogenin gel-like matrix in vitro. AB - Amelogenin self-assembly is critical for the structural organization of apatite crystals during enamel mineralization. The aim of the present study was to investigate the influence of temperature and protein concentration on the aggregation of amelogenin nanospheres at high protein concentrations (>4.4 mg ml 1) in order to obtain an insight into the mechanism of amelogenin self-assembly to form higher-order structures. Amelogenins were extracted from enamel scrapings of unerupted mandibular pig molars. The dynamics of protein solutions were measured using dynamic light scattering (DLS) as a function of temperature and at acidic pH. At pH 4-5.5, three kinds of particles were observed, ranging in size from 3 to 80 nm. At pH 6, heating the solution above approximately 30 degrees C resulted in a drastic change in the solution transparency, from clear to opaque. Low pH showed no aggregation effect, whilst solutions at a slightly acidic pH exhibited diffusion dynamics associated with the onset of aggregation. In addition, at the same temperature range, the hydrodynamic radii of the aggregates increased drastically, by almost one order of magnitude. These observations support the view that hydrophobic interactions are the primary driving force for the pH- and temperature-sensitive self-assembly of amelogenin particles in a 'gel like' matrix. The trend of self-assembly in a 'gel-like matrix' is similar to that in solution. PMID- 16674705 TI - Self-assembly and effect on crystal growth of the leucine-rich amelogenin peptide. AB - Amelogenins are a unique group of alternatively spliced proteins. While the full length amelogenin is known to assemble into nanospheres and alter apatite crystal growth and alignment, the function of the leucine-rich amelogenin peptide (LRAP) in biomineralization is not understood. This study tested the hypothesis that LRAP self-assembles into a supramolecular structure and guides crystal growth similarly to the full-length protein. Synthetic LRAP and recombinant full-length amelogenin (rH175) were used at different concentrations and either immobilized onto fluoroapatite substrates (FAP) or immersed into saturated calcium-phosphate solutions. The structure of the assembled protein and the height of apatite crystals formed on the FAP template were determined using atomic force microscopy. Both LRAP and rH175 assembled into nanospheres. LRAP self-assembly, however, was only observed at concentrations of >0.5 mg ml-1 and limited to sizes between 5 and 30 nm. Apatite crystal growth was not significantly affected by LRAP, while rH175 accelerated crystal growth by up to 50-fold. The increased growth rate was only observed when rH175 precipitated at concentrations of >0.8 mg ml-1. It was concluded that the ability of amelogenins to self-assemble into nanospheres and to bind to apatite in vitro is not inevitably an indication for the ability to control apatite crystal growth. PMID- 16674706 TI - Comparative calcium binding of leucine-rich amelogenin peptide and full-length amelogenin. AB - Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25 degrees C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca2+, pH 7.5 and 4.0, at 25 degrees C. Thermodynamic values showed that rLRAP had a Ca2+-binding affinity approximately 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca2+, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 (beta-spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization. PMID- 16674708 TI - Anisotropic properties of the enamel organic extracellular matrix. AB - Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel. PMID- 16674709 TI - SEM evidence that one ameloblast secretes one keyhole-shaped enamel rod in monkey teeth. AB - Primate enamel is subdivided into inner enamel, having Hunter-Schreger bands, and outer enamel with all rods parallel to each other. Outer inter-rod enamel may surround each rod, lie between rows of rods, or be absent, as in the 'keyhole pattern', which is composed entirely of rods. One theory on the formation of the 'keyhole' pattern overlays the hexagonal cross-sectional shape of four or more ameloblasts over the keyhole shape of the enamel rod. This ignores the likelihood that Tomes processes have a different shape from the cell body, and also ignores the observation that paths of enamel rods sometimes diverge. Scanning electron microscopy (SEM) revealed the keyhole shape of the forming face of monkey enamel. These forming rods were arranged in stepped rows with the head regions in each row separated by the tails of the preceding row. Consequently, each forming face of a rod was surrounded on three sides by previously formed enamel. The apical surface of the Tomes process was shaped exactly like the forming rod face, permitting direct apposition of one rod and one Tomes process. The conclusion was that, in the monkey, each rod of the keyhole enamel configuration is produced by one ameloblast. PMID- 16674710 TI - Developmental movements of the inner enamel epithelium as derived from micromorphological features. AB - It was the purpose of this article to analyze the (micro) morphological structure of enamel at different stages of development in order to deduce movement patterns of ameloblasts during formation of the human dental primordium. Developing enamel and overlying ameloblasts were dried and fractured for scanning electron microscopy (SEM) and sectioned for transmission electron microscopy (TEM). Specimens of human permanent enamel were either fractured and/or ground and etched to visualize the enamel rods. All specimens were viewed by SEM. Moreover, three-dimensional reconstructions were made from serial ground sections of enamel blocks to follow the enamel rods for a longer distance. In addition, the outline of the dentino-enamel junction was analyzed under the SEM after removal (using nitric acid) of the enamel cap, and in serial histological sections. Two basic movements of the inner enamel epithelium can be derived from the micromorphological features: (i) the scalloped dentino-enamel junction may be a consequence of a bulged inner enamel epithelium owing to initial spatial impediment; and (ii) the undulating path of the enamel rods may be a consequence of unequal growth of the cells in the cervical loop. PMID- 16674712 TI - Three-dimensional study of human dental fissure enamel by synchrotron X-ray microtomography. AB - The three-dimensional morphology of human tooth fissures and the quantification of mineral distribution in fissure enamel are pertinent to the development and diagnosis of caries. Synchrotron X-ray microtomography was used to measure linear attenuation coefficients (at 25 keV) at high spatial resolution with a volume imaging element (cubic voxel) of 4.9x4.9x4.9 microm3 in a block from a human premolar that included part of a stained fissure. From the linear attenuation coefficient, the mineral concentration, expressed as gHAp cm-3 (where HAp is stoichiometric hydroxyapatite), was calculated. The mean mineral concentration in bulk enamel was 2.84 gHAp cm-3. Well-defined regions (1.5-2.6 gHAp cm-3), extending up to approximately 130 microm from the base of some narrower lengths of the fissure and up to approximately 50 microm deep from the fissure surface, were attributed to hypomineralization. Other regions of low mineral concentration, some (1.4-2.3 gHAp cm-3) lying within the expected course of the fissure base and some (2.2-2.7 gHAp cm-3) deep to the pit, were also considered to be of developmental origin. However, a diffuse distribution of low mineral concentrations (2.2-2.7 gHAp cm-3) in the pit walls was attributed primarily to demineralization from caries. The fissure contained heterogeneous material (0.05) in flow (AH Plus: 38.57 mm; Epiphany: 35.74 mm) and film thickness (AH Plus: 10.6 microm; Epiphany: 20.1 microm). The solubility (AH Plus: 0.21%; Epiphany: 3.41%) and dimensional alterations following setting (AH Plus: expansion of 1.3%; Epiphany: expansion of 8.1%) were statistically different (P<0.05). The setting times of both sealants were in accordance with ANSI/ADA requirements. CONCLUSIONS: Setting time, flow, and film thickness tests for both cements conformed to ANSI/ADA standards. Dimensional alteration test for both cements were greater than values considered acceptable by ANSI/ADA. Epiphany values regarding solubility were also greater than values considered acceptable by ANSI/ADA. PMID- 16674742 TI - The influence of volume, type of irrigant and flushing method on removing artificially placed dentine debris from the apical root canal during passive ultrasonic irrigation. AB - AIM: To determine the influence of volume, irrigant and method of flushing on the removal of artificially-placed dentine debris from the apical part of root canals during passive ultrasonic irrigation. METHODOLOGY: Access cavities were prepared in 15 canine teeth and their root canals instrumented to size 20, 0.10 taper. Each root was split longitudinally, forming two halves. A groove was cut in the canal wall 2-6 mm from the apex in each half which was then filled with dentine debris prior to the roots being reassembled. All canals were ultrasonically irrigated, using a size 15, 0.02 taper smooth wire to a length of 21 mm that was placed in the canal to the apical foramen. In group 1 the canal was flushed with a continuous flow of 50 mL 2% sodium hypochlorite (NaOCl). In group 2 the continuous flow was not used but the canal was flushed with 12 mL 2% NaOCl, at a rate of 2 mL 30 s-1 using a syringe. Group 3 was treated in the same way as group 2 but the canal was flushed with 6 mL 2% NaOCl, at a rate of 2 mL min-1. Group 4 was treated in the same way as group 1 but water was used as the irrigant. Before and after irrigation, images of the grooves were captured and stored. The quantity of dentine debris in the groove was evaluated. The differences in debris scores between the experimental groups were analysed with the Kruskal-Wallis test and the Mann-Whitney U-test. The level of significance was set at P=0.05. RESULTS: The difference between all groups was statistically significant (K-W test P<0.001). Groups 1, 2 and 3 differed significantly from group 4 (P<0.001); there was no significant difference between groups 1, 2 and 3 (P=0.550). CONCLUSIONS: Syringe delivery of 2% NaOCl (6 and 12 mL) was as effective as a continuous flow of 2% NaOCl (50 mL). Water was not effective in removing dentine debris from grooves in the apical portion of root canals. PMID- 16674743 TI - Biodegradable porous calcium polyphosphate scaffolds for the three-dimensional culture of dental pulp cells. AB - AIM: To develop a three-dimensional culture model of human dental pulp cells (DPCs) with biodegradable porous calcium polyphosphate (CPP) scaffolds. METHODOLOGY: Human DPCs were isolated from three donors. The 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of CPP compared with hydroxyapatite (HA) and beta tricalcium phosphate (beta-TCP). Values were analysed using unpaired t-tests. Cells were seeded onto porous CPP scaffolds with pore sizes in the range of 200 300 microm. The nature of cellular adaptation in the three-dimensional culture model was then evaluated visually by scanning electronic microscopy (SEM) and confocal laser scanning microscopy (CLSM). The apoptotic property of cells on the scaffolds was also assessed by DNA staining with CLSM. RESULTS: The cytotoxicity assay indicated that there was no significant difference between CPP and HA for each donor's original cells (P>0.05). Calcium polyphosphate had no cytotoxic effect on DPCs, whilst SEMs showed that cells successfully adhered to CPP scaffolds and spread amongst pores. On the cell surface, fine processes and matrix secretory granules were found. Confocal laser scanning microscopy showed that cells took on a three-dimensional structure with signs of vitality. CONCLUSION: Porous CPP scaffolds are promising for the establishment of a three dimensional culture model of DPCs. PMID- 16674744 TI - In vivo evaluation of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue. AB - AIM: To determine in vivo, the degree of microbial reduction after chemo mechanical preparation of human root canals containing necrotic pulp tissue when using two endodontic irrigating reagents, sodium hypochlorite (NaOCl) or chlorhexidine gel (CHX). METHODOLOGY: Thirty-two single rooted teeth with necrotic pulp were divided into two groups. One group (n=16) was irrigated with 2.5% NaOCl, whilst the other group (n=16) was irrigated with 2% CHX gel. Assessment of the bacterial load was accomplished by use of real-time quantitative-polymerase chain reaction (RTQ-PCR) directed against the small subunit ribosomal DNA using the SYBRGreen and TaqMan formats. Statistical analysis was performed using the Mann-Whitney test. For contrast, bacterial load was also determined by traditional culture techniques. RESULTS: The bacterial load was reduced substantially in both groups (over 96%). However, using RTQ-PCR the bacterial load before and after chemo-mechanical preparation was greater when compared with evaluation using colony forming units (CFU). Furthermore, as measured by RTQ-PCR, the bacterial reduction in the NaOCl-group (SYBRGreen 99.99%; TaqMan: 99.63%) was significantly greater (P<0.01) than in the CHX-group (SYBRGreen 96.62%; TaqMan: 96.60%). According to culture technique 75% of cases were free of bacteria after chemo-mechanical preparation in the NaOCl-group, whilst 50% of cases were bacteria free in the CHX-group. CONCLUSION: NaOCl has not only a higher capacity to kill microorganisms but is also more able to remove cells from the root canal. PMID- 16674745 TI - Long-term assessment of the seal provided by root-end filling materials in large cavities through capillary flow porometry. AB - AIM: To evaluate the long-term sealing ability of a variety of materials when used as root-end fillings. METHODOLOGY: A total of 140 standardized horizontal bovine root sections (external diameter: 7 mm, height: 3 mm; internal diameter: 2.5 mm) were divided into seven groups, filled with either gutta-percha with AH26, Ketac Fil, Fuji IX, Tooth-Colored MTA, IRM, Ketac Fil with conditioner or Fuji IX with conditioner and submitted to capillary flow porometry at 1 and 6 months to assess minimum, mean flow and maximum pore diameters. Results of the different materials and results by material were analysed statistically using non parametric tests; the level of significance was set at 0.05. RESULTS: There were no significant differences between the minimum pore diameters associated with the materials at each time. At 1 month the mean flow pore diameters of Ketac Fil were significantly larger than those of gutta-percha, Ketac Fil with conditioner, Fuji IX with conditioner and IRM. There were significant differences between the maximum pore diameters at 1 month (all>IRM; Fuji IX>gutta-percha, Ketac Fil with conditioner, Fuji IX with conditioner) and 6 months (Fuji IX>gutta-percha, IRM; Ketac Fil>gutta-percha, IRM). There were significant differences in the minimum pore diameters between the different points in time for each material except IRM, in the mean flow pore diameters for each material and in the maximum pore diameters for each material except MTA. CONCLUSIONS: All materials were associated with capillary flow. IRM root-end fillings had through pores that were smaller than those associated with other materials. Conventionally setting glass ionomer cements had the largest pores, although dentine conditioning improved their performance. The seal of all materials improved after 6 months. PMID- 16674746 TI - Fracture of ProFile nickel-titanium rotary instruments: a laboratory simulation assessment. AB - AIM: To determine the incidence of ProFile nickel-titanium rotary instrument fracture in an endodontic laboratory simulation. METHODOLOGY: Three hundred and sixty dental students used 2880 ProFile nickel-titanium rotary instruments to prepare 1440 simulated root canals in 720 plastic teeth, and another 2880 ProFile nickel-titanium rotary instruments to prepare 1440 natural root canals in 720 extracted teeth. A standardized crown-down rotary instrumentation technique was used, which included measures to prevent fracture. Rotary instrument fracture was monitored during and after completion of the laboratory simulation exercises to determine the incidence of fracture. When fracture occurred, data were collected concerning the size of the instrument, the length of the instrument fragment, the location of the fragment in the canal and the curvature of the canal in which the instrument fractured. RESULTS: The incidence of instrument fracture was 0.41% in plastic simulated canals and 0.31% in natural root canals. The overall incidence of instrument fracture was 0.36%. Of the instruments that fractured 67% were size 25, 0.04 taper; and 81% of the fragments were located in the apical third of the canal. The mean, median and mode of the fragment lengths were all 3 mm. CONCLUSIONS: The low overall incidence of fracture in this study suggests that ProFile rotary instruments are safe for use by dental students in laboratory simulations and that if preventive measures are taken the incidence of instrument fracture can be minimized. PMID- 16674747 TI - Periapical central giant cell granuloma misdiagnosed as odontogenic cyst. AB - AIM: To present the clinicopathological features of a series of four periapically located central giant cell granulomas (CGCGs) that were misdiagnosed and treated as being of endodontic origin. SUMMARY: Four cases of periapical CGCGs were submitted with a clinical diagnosis of either radicular or residual cyst. In two cases, root canal treatment had been performed previously. The patients were two women and two men whose age ranged from 31 to 85 years. Two cases were located in the mandibular premolar-molar region, and two in the anterolateral region of the maxilla. Two lesions were submitted for histological examination with a diagnosis of radicular cyst whereas the remaining two were submitted with a diagnosis of residual cyst. KEY LEARNING POINTS: Periapical giant cell lesions may be unilocular and therefore misdiagnosed as an endodontic lesion because of their radiographic similarity to an inflammatory periradicular lesion, especially if the teeth have been root filled or if the vitality is negative or doubtful. It is important to follow up the healing process of a periapical radiolucency related to a root filled tooth and, in case of persistence, to perform surgery and to submit the specimen for histological examination. PMID- 16674748 TI - The concept of positive health: a review and commentary on its application in oral health research. AB - Although the concept of positive health has been around for more than 60 years, acceptable measures of this construct have yet to emerge. Potential explanations are that there is no consensus on how it is to be defined and its ambiguous status with respect to medical and socioenvironmental models of health. In this paper we review definitions of positive health, the origins of these definitions, the way the concept of positive outcomes has been used in research on the outcomes of oral and orofacial conditions and assess whether the concept of positive health has any merit in terms of applied oral health research. This literature reveals many competing and imprecise definitions, many of which are similar to other constructs, such as well-being. Most are lacking empirical referents or indicators. In examining the literature on oral health we found five distinct, although overlapping, ways in which the concept of positive health has been framed: (i) positive health as the absence of negative health states; (ii) positive health as positively worded items; (iii) the positive outcomes of oral health; (iv) positive oral health as a set of psychological and social attributes, and (v) the positive outcomes of chronic conditions such as oro- and craniofacial differences. Each of these ways can be challenged on conceptual or methodological grounds. For example, the states that comprise the upper end of the negative-positive health continuum have not been defined and health states and determinants of health are often confused. Moreover, the meaning of responses to health status questionnaires and the interpretation of accounts of the illness experience is often unclear. Nevertheless, the notion of positive health, irrespective of its merits and public policy implications, provides a context for methodological and theoretical debate that can only serve to enrich theory and practice with respect to measures of health and quality of life and therapeutic interventions at the individual and population. PMID- 16674749 TI - Pattern of dental caries experience on tooth surfaces in an adult population. AB - OBJECTIVES: To determine the pattern of caries experience across teeth and surfaces in an adult population depending on age and exposure to water fluoridation. METHODS: Between November 2002 and March 2003 a total of 973 subjects aged 17-51 years had a clinical examination using visual and tactile criteria. Subsequent to this examination, bitewing radiographs were taken and viewed separately. Approximal and occlusal surfaces of molars and premolars were examined on the radiographs. RESULTS: Caries experience was relatively low, with mean DMFS scores of 3.21, 5.12, 9.61, 13.04 and 24.35 for subjects aged 17-20, 21 25, 26-30, 31-35 and 36-51 years respectively. The first molar teeth had the greatest caries experience, and occlusal surfaces had more caries experience than approximal surfaces. Subjects with a lifetime exposure to fluoridated drinking water had significantly lower caries experience than those who had no exposure to fluoridated drinking water. CONCLUSION: This study showed that caries prevalence, although relatively low in the study population, was found predominantly in occlusal surfaces, with an increasing prevalence in approximal surfaces of posterior teeth in older subjects. Subjects with a lifetime exposure to fluoridated drinking water had a lower level of caries experience than those with no exposure to fluoridated drinking water, and this was more noticeable in approximal surfaces than occlusal surfaces. PMID- 16674750 TI - Social gradients in periodontal diseases among adolescents. AB - OBJECTIVE: To investigate the association between socioeconomic position and periodontal diseases among adolescents. METHODS: Data were obtained from 9203 Chilean high school students. Clinical examinations included direct recordings of clinical attachment level and the necrotizing ulcerative gingival lesions. Students answered a questionnaire on various dimensions of socioeconomic position. Seven periodontal outcomes were analyzed. Logistic regression analyses were used to identify socioeconomic variables associated with the periodontal outcomes. RESULTS: The occurrence of all periodontal outcomes investigated followed social gradients, and paternal income and parental education were the most influential variables. CONCLUSIONS: The study demonstrates the existence of significant social gradients in periodontal diseases already among adolescents. This is worrying, and indicates a new potential for further insight into the mechanisms of periodontal disease causation. PMID- 16674751 TI - Changes in dental fluorosis following the cessation of water fluoridation. AB - OBJECTIVES: To determine changes in the prevalence of dental fluorosis, and in perceptions of aesthetic concerns due to dental fluorosis after water fluoridation ceased. METHODS: Schoolchildren in second and third grades were examined in 1993-94, 1996-97 and 2002-03 to determine changes in the prevalence of dental fluorosis following fluoridation cessation of the public water supplies in 1992. The Thylstrup-Fejerskov Index (TFI) was used to quantify dental fluorosis. Perceptions of aesthetics were assessed by questionnaires which were sent home to parents. Residence and dental histories were confirmed on all children to determine the extent of exposure to all types of fluorides. Comparisons between the three surveys were used to establish the influence of fluoridated water and other fluoride sources on the occurrence and severity of dental fluorosis. Aesthetic ratings from parents were used to assess the aesthetic conditions of maxillary anterior teeth across the three surveys. RESULTS: When fluoride was removed from the water supply in 1992, the prevalence and severity of TFI scores decreased significantly from the 1993-94 survey cycle when compared with the 1996-97 and 2002-03 survey cycles. The use of fluoride supplements and fluoride dentifrice also decreased during this study period. Analyses were unable to determine the influence of these different fluoride exposures on the changes in TFI scores over time. Comparisons of aesthetic ratings from parents between survey cycles failed to show any significant differences. PMID- 16674752 TI - Can the development of new dental caries in Danish schoolchildren be predicted from surveillance data in the School Dental Service? AB - BACKGROUND: Dental screening programmes for Danish children generally target all children, irrespective of their individual caries risk. The standard screening interval is approximately 12 months. A valid systematic screening tool based on routine information sources is however indispensable, if more selective screening strategies should be developed to target the children at highest risk. OBJECTIVE: To estimate the precision with which Danish schoolchildren at high risk for developing dental caries within 1 year can be identified based on information from routine registers. METHODS: Based on data from the Danish National Board of Health's Recording System for the Danish Child Dental Services and from the Central Office of Civil Registration, 3705 schoolchildren aged 7-12 years were followed through 1994-1996. Dental health information as of 1994 and changes 1994 1995 were applied in multiple logistic regressions together with social data as of 1995 to estimate the individual 1-year (1995-1996) risk of developing caries. RESULTS: In 1995, 37.4% of the children had a DMFS index above 0, and during the following year 21.8% of all children developed new caries. The individual child's 1-year caries risk could be estimated relatively accurately at baseline as indicated by the area (76%) under the receiver operating characteristic curve. About 40% of children with an estimated risk of 20% and above developed new caries, whereas 90% of the rest of the children did not do so. CONCLUSION: Based on information from Danish routine registers children at low caries risk may be identified relatively precisely. This may form the basis for the continuous development and targeting of high-risk strategies, in which the screening for caries among children of estimated low risk may be postponed at least 1 year. PMID- 16674753 TI - Effect of different diagnostic thresholds on dental caries calibration - a 12 month evaluation. AB - OBJECTIVE: To analyze the reproducibility of a calibration trial, at different diagnostic thresholds of dental caries, in a 12-month evaluation. METHODS: A group of dental examiners (n = 11), who had previous experience in epidemiological surveys, participated in the study. An initial training phase (theoretical and clinical) and five calibration exercises (baseline, 3, 6, 9 and 12 months) were arranged. World Health Organization (WHO) criteria, including the active initial lesions (IL) were used. Six- to 7-year-old children took part in the study. They were selected according to past history and dental caries activity. The data were analyzed at WHO and WHO + IL diagnostic thresholds in accordance with tooth and dental surfaces. RESULTS: Excellent mean intra- and inter-examiner Kappa values were obtained for both diagnostic thresholds, in accordance with tooth and surface, during the calibration phases. However, the most relevant errors were related to the decayed component and to IL diagnosis. CONCLUSION: It was possible to use the methodology proposed in this study in epidemiological surveys when examining the mixed dentition, although new strategies to improve training in IL diagnosis and calibration are necessary. PMID- 16674754 TI - Challenges to dental access - England as a case study. AB - Access to dental services because of an insufficient workforce is a historic challenge faced by many developing countries. In recent years, however, it has become a major issue for many industrialized countries. The growing demand for cosmetic dentistry, an increase in patients' willingness to pay for dental treatment, and growing numbers of older dentate patients have all put pressure on dental systems. Ways of meeting these challenges and ensuring reasonable dental access will vary from country to country, but the solutions often lie in how the dental workforce is regulated. This case study of the dental reforms currently being implemented in England highlights progress at a particular point in time (Summer 2005). It is clear that it will take a number of years to find a new national dental payment system (the National Health Service) to replace the system which has changed little since 1948. However, the political pressure to address poor access to state-funded dental services calls for more immediate actions. The initial approach was to increase the dental workforce via international recruitment, and in the medium term to increase the number of dental students in training and to expand the numbers of other members of the dental team. An additional stratagem is to retain those already providing dental care under the National Health Service by the introduction of a new method of remuneration. England is trying to improve both access to care and the oral health of the population by creating a workforce more suitable to public demands and changing oral health needs. PMID- 16674755 TI - Access to oral health care - an Australian perspective. AB - The objectives of this paper are to give a brief description of the Australian context for its dental care services and to discuss some of the nationally recognized issues in access to dental care with special reference to the situation in the most populous state, New South Wales. Australia is the size of continental USA but with only around 21 million people, 85% of whom reside within 50 km of the coastline. Thus, access to health care has a strong urban-rural dimension. The universal healthcare coverage excludes dental care, 80-90% of which is delivered through traditional fee-for-service private dental care. A public dental care system exists with varying eligibility criteria from state to state, mostly directed at children, low-income individuals, pensioners, and defined disadvantaged groups. Thus, access to dental care also has a strong socioeconomic dimension with disadvantaged people having serious access problems and extensive waiting times. Government and other reports have documented considerable polarization issues both in oral health and in access to dental care. Suggested change strategies have ranged from broad political changes in the dental care system to local oral health promotion initiatives, but overall, dental care remains a pawn in state-commonwealth political squabbles. In response to strong public reactions documented shortcomings of the public dental care system the government of New South Wales has recently initiated a political inquiry into dental care. Unless new resources are injected and policy adjustments made, serious changes are unlikely. PMID- 16674756 TI - Access to care - what can the United States learn from other countries? AB - This paper briefly describes the US system for dental care services; asserts that there is much to be learned by considering the experience of other countries; identifies a few lessons that may be learned from comparisons with England, Australia, and other nations; and encourages the monitoring of outcomes associated with innovations in financing and delivery of services elsewhere. Oral health is affected by more factors than access to dental care. Because so many factors at the individual, environmental, and delivery system levels affect oral health, interpreting the findings from international studies is difficult. Furthermore, the findings of these international studies are confounded by significant intra-country variation in outcomes and expectations. While public funding and the public provision of services (such as programs in schools or community health centers) can be powerful instruments of change, they have their limitations. Examination of all types of public subsidization of dental care may reveal inadvertent distributions that may increase disparities. The discovery of best practices and lessons learned in the financing and organization of dental care may begin by comparing US experiences with those of other countries. PMID- 16674757 TI - Prophylactic treatment of migraine in children. Part 2. A systematic review of pharmacological trials. AB - The aim of this study was to assess the efficacy of pharmacological prophylactic treatments of migraine in children. Databases were searched from inception to June 2004 and references were checked. We selected controlled trials on the effects of pharmacological prophylactic treatments in children with migraine. We assessed trial quality using the Delphi list and extracted data. Analyses were carried out according to type of intervention. A total of 20 trials were included. Headache improvement was significantly higher for flunarizine compared with placebo (relative risk 4.00, 95% confidence interval 1.60, 9.97). There is conflicting evidence for the use of propranolol. Nimodipine, clonidine, L-5HTP, trazodone and papaverine showed no effect when compared with placebo. All medications were well tolerated and adverse events showed no significant differences. Flunarizine may be effective as prophylactic treatment for migraine in children. Because of the small number of studies and the methodological shortcomings, conclusions regarding effectiveness have to be drawn with caution. PMID- 16674758 TI - Prevalence of unexplained upper abdominal symptoms in patients with migraine. AB - Patients with functional gastrointestinal disorders frequently report migraine. We aimed to determine the prevalence of idiopathic upper abdominal symptoms in patients with migraine and compare it with a control population of healthy blood donors. We assessed abdominal symptoms using the Bowel Disease Questionnaire in a series of 488 consecutive blood donors without migraine and 99 patients with migraine. Upper abdominal symptoms were reported by 38%[95% confidence interval (CI) 32, 44] of blood donors compared with 81% (67, 91, P<0.001) of migraine patients. Of the blood donors, 23% (18, 28) reported frequent dyspepsia compared with 60% (44, 74, P<0.001) of the migraine patients. Migraine was associated with frequent upper abdominal symptoms (odds ratio 2.7, 95% CI 1.2, 6.1) after adjusting for age, gender, smoking and consumption of analgesics and alcohol. Upper abdominal symptoms are significantly more frequent in patients with migraine compared with healthy controls. The association between migraine and idiopathic upper abdominal symptoms may suggest common pathophysiological mechanisms. PMID- 16674759 TI - Brainstem dysfunction in alternating hemiplegia of childhood: a neurophysiological study. AB - Six patients with alternating hemiplegia of childhood (AHC) underwent motor evoked potentials (MEPs), somatosensory evoked potentials (SEPs) and blink reflex recording. No SEP abnormality was found. As for MEP recording, central conduction time did not differ between patients studied during either interictal or ictal phase, and normal subjects, suggesting that the pyramidal system function is spared. In blink reflex recording, the latencies of both the ipsilateral (iR2) and contralateral (cR2) R2 components were significantly longer (P<0.01) in patients during the interictal phase than in normal subjects. Moreover, the iR2 and cR2 areas were significantly reduced (P<0.01) in patients during the interictal phase, compared with normal subjects. During the ictal phase, the ipsilateral R2 latency was significantly decreased after stimulation of the hemiplegic side, compared with the interictal phase (P<0.05). The blink reflex abnormalities suggest a brainstem dysfunction, which may be linked to the pathophysiological mechanisms of the disease. PMID- 16674760 TI - Acupuncture in migraine prophylaxis: a randomized sham-controlled trial. AB - The purpose of the present trial was to evaluate semi-standardized acupuncture efficacy in migraine prophylaxis. Twenty-eight subjects with migraine were randomized to the real or sham acupuncture groups. Semi-standardized and standardized minimal acupuncture were used, respectively, in the two groups of patients. They were all treated with 16 acupuncture sessions in 12 weeks. Both groups exhibited similar reductions in: percentage of patients with reduction of migraine>or=40% and >or=50% regarding frequency of migraine attacks, days with migraine, frequency of migraine attacks, average duration of a migraine attack, rate of rescue medication used, average headache severity rate and other parameters compared with the baseline period. Associated symptoms, such as nausea and vomiting, also showed equal estimates in both groups. These findings showed that semi-standardized acupuncture shows no difference from sham acupuncture in preventing migraine attacks. PMID- 16674761 TI - Is vasospasm requisite for posterior leukoencephalopathy in patients with primary thunderclap headaches? AB - Primary thunderclap headache (TCH) is sometimes associated with cerebral vasospasm. However, the role of vasospasm in relation to the development of reversible or irreversible posterior leukoencephalopathy among patients with primary TCH has never been fully addressed. This paper includes a report on a 51 year-old woman with primary TCH complicated with posterior leukoencephalopathy and a literature review of 16 further patients with the same illness. Their magnetic resonance or conventional angiographic findings were clearly described. Our review found that all these 17 patients showed evidence of cerebral vasospasm. Eleven (65%) of them developed permanent ischaemic infarctions, almost exclusively located at the watershed zones. We suggest that the presence of vasospasm might be requisite for posterior leukoencephalopathy as well as for permanent infarctions in these patients. Therefore, searching for any clue of vasospasm is mandatory in treatment of patients with primary TCH. Absence of an accompanying vasospasm might predict a good outcome. PMID- 16674762 TI - Efficacy and tolerability of diclofenac potassium sachets in migraine: a randomized, double-blind, cross-over study in comparison with diclofenac potassium tablets and placebo. AB - A randomized, controlled, cross-over trial compared single doses of 50 mg diclofenac potassium sachets and tablets with placebo in 328 patients with migraine pain, treating 888 attacks. For the primary endpoint 24.7% of the patients were pain free at 2 h postdose with sachets, 18.5% for tablets and 11.7% for placebo. Treatment differences were significant for sachets vs. placebo (P<0.0001), tablets vs. placebo (P=0.0040) and for sachets vs. tablets (P=0.0035). The numbers needed to treat compared with placebo to achieve pain free at 2 h were 7.75 [95% confidence interval (CI) 5.46, 13.35] for sachets and 15.83 (95% CI 8.63, 96.20) for tablets. Sachets were also statistically superior to tablets for sustained headache response, sustained pain free and reduction in headache intensity within the first 2 h postdose measured on a visual analogue scale (P<0.05). Onset of analgesic effect was 15 min for sachets and 60 min for tablets. Fewer patients needed rescue medication, and there were marked improvements in accompanying symptoms and working ability with both sachets and tablets vs. placebo. No safety issues were identified. This study demonstrates that sachets offer patients suffering from migraine pain a more effective treatment with a faster onset of analgesia when compared with tablets. PMID- 16674763 TI - The relationship between migraine pain and other associated symptoms. AB - This study explores the relationship of the pain of the migraine headache and the associated features of migraine. Migraineurs (n=1025) (ICHD-2, 1.1-1.2 and 1.5.1) were evaluated retrospectively using a detailed database (daily unremitting excluded). Variables studied included headache intensity and duration, associated symptoms and pain characteristics. Non-parametric correlations were used to evaluate relationships among variables. Headache intensity correlated with nausea, vomiting, photophobia, phonophobia, dizziness (all P=0.000), running of the nose/tearing of the eyes (P=0.007), and osmophobia (P=0.044), but not with diarrhoea or taste abnormality. Headache duration correlated only with osmophobia (P=0.002) and taste abnormality (P=0.005). Throbbing, pressure and stabbing pain correlated with most of the associated symptoms. Aching correlated only with taste abnormality. This correlational study demonstrates that migraine pain is clearly related to nausea, but is also correlated with other associated migraine symptoms. Taste abnormality and osmophobia are better correlated with headache duration rather than headache intensity. PMID- 16674764 TI - Visual cortical inhibitory function in migraine is not generally impaired: evidence from a combined psychophysical test with an fMRI study. AB - A robust, visual masking test that was developed to be feasible with functional magnetic resonance imaging (fMRI) was used to examine the visual cortical inhibitory function in migraine patients with visual aura at both psychophysical and cortical levels. The study showed that the decreased visibility of a visual target was associated with a reduction in cortical activation in the primary visual cortex. The suppression of the transient on-response and after-discharge of neurons to the target was most likely to be responsible for reducing cortical activation, rendering the target less visible or invisible. The migraine patients were equally susceptible to visual masking and showed no difference in cortical activation when compared with age- and sex-matched non-headache controls, demonstrating that visual cortical inhibitory function was not impaired under the experimental conditions. Although these results are not in conflict with the general cortical hyperexcitability theory in migraine, they provide evidence to show the limitation to the theory. PMID- 16674765 TI - Deep brain stimulation for neuropathic cephalalgia. AB - The aim of this study was to determine the efficacy of deep brain stimulation (DBS) in the treatment of various types of intractable head and facial pains. Seven patients underwent the insertion of DBS electrodes into the periventricular/periaqueductal grey region and/or the ventroposteromedial nucleus of the thalamus. We have shown statistically significant improvement in pain scores (visual analogue and McGill's) as well as health-related quality of life (SF-36v2) following surgery. There is wide variability in patient outcomes but, overall, DBS can be an effective treatment. Our results are compared with the published literature and electrode position for effective analgesia is discussed. PMID- 16674766 TI - Patterns of experimentally induced pain in pericranial muscles. AB - Nociceptive mechanisms in the craniofacial muscle tissue are poorly understood. The pain pattern in individual pericranial muscles has not been described before. Experimental muscle pain was induced by standardized infusions of 0.2 ml 1 m hypertonic saline into six craniofacial muscles (masseter, anterior temporalis, posterior temporalis, trapezius, splenius capitis and sternocleidomastoid) in 20 healthy subjects. The pressure pain thresholds (PPTs) were determined before and after infusions. The subjects continuously reported intensity of saline-induced pain on an electronic visual analogue scale (VAS) and the perceived area of pain was drawn on anatomical maps. The pain areas were measured and the localization determined by a new centre-of-gravity method. The PPTs were lowest on the sternocleidomastoid muscle (anova: P<0.001), but the saline-evoked VAS pain scored highest following injection into the masseter muscle (anova: P<0.05). The centre-of-gravity measures demonstrated significantly different localization of the pain areas (anova: P<0.001). The trigeminally vs. the cervically innervated muscles had significantly different patterns of spread and referral of pain according to trigeminally vs. cervically innervated dermatomes (P<0.005). In conclusion, there appear to be characteristic pain patterns and pain sensitivity in different craniofacial muscles in healthy volunteers, which may be of importance for further research on different craniofacial pain conditions. PMID- 16674767 TI - Clinical features and pharmacological treatment of migraine patients referred to headache specialists in Canada. AB - We set out to examine selected clinical characteristics of migraine patients referred to neurologists specializing in headache in Canada, and to document their pharmacological therapy both before and after consultation with the neurologist. Demographic, clinical and pharmacotherapy data were collected at the time of consultation for 606 patients referred to five headache clinics and who were given a migraine diagnosis by the neurologist. Data were analysed as part of the Canadian Headache Outpatient Registry and Database (CHORD) Project. The mean age of the migraine patients was 39.7 years; and 82.5% were female. The majority of patients suffered severe impact from their headaches. Prior to consultation, 48.7% were taking a triptan; after consultation, 97.2% were on a triptan. Before consultation, 30.9% were on a prophylactic drug; after consultation, 70.4% were. 20.8% of patients were medication overusers. Of these medication overusers, 42.4% were overusing an opiate, usually in combination with other analgesics; 21.6% were overusing a triptan. Medication changes made by the neurologists at consultation included a large increase in the use of both triptans and prophylactic medications. Medication overuse, particularly opiate overuse, remains a significant problem in patients with migraine in Canada. PMID- 16674768 TI - Outcome of medication overuse headache after abrupt in-patient withdrawal. AB - One hundred and one patients suffering from chronic daily headache (CDH) and medication overuse were treated, in an in-patient setting, with abrupt discontinuation of the medication overused, intravenous hydrating, and intravenous administration of benzodiazepines and ademetionine. The mean time to CDH resolution was 8.8 days. The in-patient withdrawal protocol used was effective, safe and well tolerated. There was a trend for a shorter time to CDH resolution in patients who overused triptans (P=0.062). There was no correlation between time to CDH resolution and either the type of initial primary headache or duration of medication abuse, whereas time to CDH resolution was related to daily drug intake (P=0.01). In multiple regression analysis, daily drug intake, age and type of medication overused were independent predictors of time to CDH resolution. At 3-months' follow-up, no patient had relapsed and was again overusing symptomatic medications. PMID- 16674769 TI - Cognitive and behavioural effects of migraine in childhood and adolescence. AB - Since cognitive and behavioural characteristics of paediatric migraine sufferers have yet to be adequately defined, in this study we assessed the effect of migraine on the interictal functioning of children and adolescents by comparing the performance of two patient groups, 17 migraine sufferers with aura (MA) and 31 without aura (MoA) and by correlating the duration of the disorder, the frequency of attacks and interictal period with neuropsychological and behavioural findings. Both patient groups had cognitive performance within normal range except for a significant delay in the reaction time (RT) task. Both MA and MoA revealed a behavioural phenotype characterized by internalizing problems on Child Behaviour Check List (CBCL) scales. Slower RT to simple visual stimuli may be an early sign of a subclinical neuropsychological dysfunction, significantly correlated with the frequency of headache attacks and interictal period. The lack of a control group and other methodological limitations, such as patient selection bias and unadjusted P-value for multiple testing, make it difficult to give this finding a clearcut meaning. Further studies are needed on larger samples compared with a control group. PMID- 16674770 TI - Outcome of headache frequency in adolescence. AB - Only a few studies have been published of the outcome of adolescent headache (HA). The aim of this study was to examine the predictors of the outcome of headache frequency. A population-based sample of 13-year-olds with or without HA (N=228) was followed to the age of 16 years. HA was classified on the basis of a face-to-face interview and clinical examination. The outcomes of monthly HA (>1/month) and non-frequent HA (0-1/month) were studied. Frequent use of analgesics, female gender and multiple non-headache pain predicted the persistence of monthly HA (>1/month). Significant predictors for worsening non frequent HA (from 0 to 1/month to >1/month) were female gender, consistent migraine and high basic educational level of one parent. Adolescents frequently using analgesics constitute a risk group for a poor outcome of HA. Especially girls meeting this criterion should be considered a target group in the planning and implementing of preventive measures. PMID- 16674771 TI - Orthostatic hypacusis in a patient with CSF hypovolaemia. PMID- 16674772 TI - Cluster headache attack due to sildenafil intake. PMID- 16674773 TI - Secondary SUNCT syndrome to a variant of the vertebrobasilar vascular development. PMID- 16674774 TI - Predictors of hazardous alcohol consumption among patients with cluster headache. AB - The purpose of this study was to identify predictors of hazardous alcohol consumption in patients with cluster headache (CH). We investigated 246 German CH patients with the Alcohol Use Disorders Identification Test (AUDIT). The average daily alcohol consumption was 6.5 g. Predictors for hazardous drinking (AUDIT>or=5 points; 21.5% of patients) were male gender [odds ratio (OR) 4.15, 95% confidence interval (CI) 1.35, 12.71], episodic as opposed to chronic CH (OR 4.8, 95% CI 1.38, 16.67) and a low demanding job as opposed to a high demanding job (OR 2.28, 95% CI 1.15, 4.51). Our data indicate that CH patients drink less alcohol compared with the German population and that CH seems to protect against hazardous alcohol consumption. Moreover, predictors for hazardous alcohol consumption in CH patients are not different from the general population. PMID- 16674775 TI - Oral triptans vs. other classes of acute migraine medication. PMID- 16674776 TI - Migraine and silent infarcts. Are we making the proper decisions? PMID- 16674777 TI - Nurses, ageing and older people. PMID- 16674778 TI - Family caregiving of persons living with HIV/AIDS in Thailand: caregiver burden, an outcome measure. AB - The present paper provides an initial picture of HIV/AIDS-affected families. It is evident that families play a major role of support for HIV/AIDS patients in Thai society. Caregiver burden is one of the patient-related outcomes, which is the most common outcome measure in caregiver research. The demands on the family caregivers of these patients are enormous and need to be addressed. The determinants that are associated with caregiver burden such as caregiver characteristics, patient characteristics and social stigma are important for nurses to minimize the burden of care so that appropriate interventions can be developed for persons with HIV/AIDS and family members who share the work of managing their care at home. PMID- 16674779 TI - An exploration of the concept of patient education: implications for the development of educational programmes for relapsed post-bone marrow transplantation patients and their families in Korea. AB - Frequently, the nursing care of patients who undergo bone marrow transplantation (BMT) is focused on acute, curative care. However, given that recurrent disease following BMT has a poor prognosis, a focus on acute care alone appears inappropriate for those clients who experience relapse. Care for this group of patients should be more inclusive of principles that underpin palliative care. Patient education is an important element of palliative care. Therefore, patient education based on the principles of palliative care should play a significant part in the nursing care of BMT patients with recurrent disease. This paper proposes a framework for the development of a palliative educational programme for relapsed post-BMT patients and their families in Korea. The framework was developed through a concept analysis of patient education. PMID- 16674780 TI - Compassion fatigue and nursing work: can we accurately capture the consequences of caring work? AB - Health outcomes and, in particular, patient health outcomes have become a driving force within health-care delivery. Little emphasis has been placed on the potential health consequences for nurses providing care and caring within the health-care system. Compassion fatigue (or secondary traumatic stress) has emerged as a natural consequence of caring for clients who are in pain, suffering or traumatized. This paper sheds light on how nursing work might impact the health of nurses by exploring the concept of compassion fatigue. Limitations of current instruments to measure compassion fatigue are highlighted, and suggestions for future direction are presented. PMID- 16674781 TI - Self-reported subjective sleep quality and fatigue in patients with peritoneal dialysis treatment at home. AB - The aim of this study was to describe habitual sleep, daytime symptoms, sleep disturbing factors, current sleep during 1 week and fatigue in patients with peritoneal dialysis treatment at home and also discover predictions for sleep quality outcome. The knowledge should increase possibilities for supportive nursing health care. Fifty-five patients answered two mailed questionnaires and filled in a sleep diary. Of these, 60% had moderate, persistent sleep problems combined with daytime symptoms. Nocturnal awakenings with difficulties falling asleep again and a sleep duration predicted as 57% of sleep quality. Nocturnal pruritus and 'difficulties finding a comfortable sleeping position' were significant sleep-disturbing factors. Sleep quality predictors means progress in knowledge about the complexity of the situation for peritoneal dialysis patients. Poor sleep, daytime symptoms, sleep-disturbing factors and chronic fatigue need to be enlightened, especially for the nephrology nurses who are in a unique position to give supportive nursing health care. PMID- 16674782 TI - Evaluation of 'partnership care model' in the control of hypertension. AB - One of the shared common goals of World Hypertension League (WHL) and World Health Organization (WHO) is the control of hypertension. Despite many local and international interventions, the goal has not been achieved. This study evaluated an intervention based on the partnership care model to control hypertension in a rural population in the north of Iran. The results showed that the intervention was effective in decreasing systolic and diastolic blood pressure and in increasing the rate of controlled hypertensives (based on criteria of WHO/WHL). The intervention also had positive effects on health-related quality of life, body mass index, anxiety, high density lipoprotein level and compliance score. Based on these results, the partnership care model is effective in hypertension control and is recommended as a model to replace previous approaches in hypertension control. PMID- 16674783 TI - Hand dermatitis among Korean nursing students. AB - We used a previously validated questionnaire survey, which was translated into Korean and given to 270 students from a university-based nursing school in Seoul. A total of 202 questionnaires were received. The students' hand dermatitis prevalence ranged from 6.9% in the first year to 22.9% in the fourth year. Logistic regression indicated that students with systemic allergies were 4.2 times as likely to suffer from hand dermatitis. Year of study and living arrangements were also shown to have statistically significant correlations with hand dermatitis. Overall, this study suggests that hand dermatitis might be less prevalent among Korean nursing students when compared with their international counterparts. However, the identification of systemic allergies as a significant risk factor was consistent with previous reports from other countries. PMID- 16674784 TI - On paternalism, autonomy and best interests: telling the (competent) aged-care resident what they want to know. AB - This paper has been derived from a review of literature from a recent qualitative study that explored the meaning of truth-telling within the care provider-aged resident dyad in high-level (nursing home) care of elderly people. In the literature reviewed here, paternalism (as benevolent decision-making in another's best interests) is critically analysed. Autonomy is then described and its function in promoting health is underscored. Furthermore, this paper specifically challenges nurses to consider their capacity to really know another's best interests--particularly in the nursing home. Finally, a very simple recommendation is proposed for determining and managing the (competent) aged-care resident's informational disclosure preferences: ask them. PMID- 16674785 TI - The modified Morse Fall Scale. PMID- 16674786 TI - A modified method for purifying amelanotic melanocytes from human hair follicles. AB - We describe a modified method for establishing long-term pure cultures of amelanotic melanocytes (AMMC) derived from human hair follicles. Normal human corpse scalp (just after death, 1 h) was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. Hair follicle cell suspensions were prepared by 0.50% trypsin treatment for 30 min and cultured in an optimized melanoblast proliferation nature mitogen medium. Cells attached to the substratum were mostly amelanotic melanocytic in character with small, bipolar shapes in the early stage; only a few keratinocytes and rare fibroblasts were observed. Keratinocytes were easily removed by differential trypsinization. After the third passage, the proliferating cells were all amelanotic melanocytes as confirmed by immunostaining with polyclonal antibodies to alphaPEP7h, which recognized the tyrosinase protein located on melanosomes and NKI/beteb, which is a pre melanosomal antigen against synthetic peptides corresponding to the carboxyl termini of human melanosomal protein GP100. Cultured AMMC were highly positive to L-dopa reactivity after the addition of IBMX to the culture medium for 7 days. Many stage I and II melanosomes and occasional stage III melanosomes without stage IV melanosomes were found in the cytoplasm by transmission electron microscope. This modified technique is potentially more suitable for cultivating amelanotic melanocytes. The availability of pure cultures of hair-follicle amelanotic melanocytes will facilitate investigations of the roles of those cells in migration and differentiation during treatment of vitiligo. PMID- 16674787 TI - Syringocystadenoma papilliferum associated with apocrine poroma. AB - A 65-year-old Japanese man presented with a gradually enlarging mass on the right side of the abdomen, which he had first noticed about 4 years previously. He was otherwise asymptomatic. Histopathological examination of the mass revealed an aggregation of neoplastic cells (tumor cell nests) with cellular proliferation extending from the epidermis to the dermis. The tumor consisted of two histologically distinct parts. One part was composed of uniformly small cells with a cuboidal appearance. Some ductal structures were visualized, and some of the cells lining the ductal lumina contained decapitation secretions. These histological changes were consistent with the diagnosis of apocrine poroma. The remaining part of the tumor was composed of cystic invaginations with numerous projections oriented toward the lumen. There were two rows of cells in the projections; the cells on the luminal side were columnar, and those at the apical aspect were small cuboidal cells. These histological changes were characteristic of syringocystadenoma papilliferum (SCAP). Based on these findings, a diagnosis of SCAP associated with apocrine poroma was made. To the best of our knowledge, there have been no previous reports of such a case in the published work. PMID- 16674788 TI - Angioma serpiginosum: dermoscopy for diagnosis, pulsed dye laser for treatment. AB - Angioma serpiginosum is a rare benign vascular disorder, characterized clinically by multiple minute, red to purple, grouped macules in serpiginous and gyrate patterns and histopathologically by ectatic dilatation of capillaries. Patients can undergo unnecessary hematological tests, because the condition can be confused with chronic purpuric dermatoses. An 18-year-old man with angioma serpiginosum of his left arm was evaluated by dermoscopy and treated with pulsed dye laser. Numerous small, relatively well-demarcated, round to oval red lagoons were determined with dermoscopy, and approximately 75% of the area of his lesion disappeared after four sessions of pulsed dye laser. Our case supports the hypothesis that dermoscopy is beneficial in the diagnosis of angioma serpiginosum and that pulsed dye laser is effective in the treatment of this disorder. PMID- 16674789 TI - Non-anogenital (ectopic) hidradenoma papilliferum with sebaceous differentiation: a case report and review of reported cases. AB - Hidradenoma papilliferum is a benign tumor that usually occurs in the female anogenital region. We present a 52-year-old male with a non-anogenital (ectopic) hidradenoma papilliferum on his face. He had had a subcutaneous tumor on his right eyebrow for 30 years. The histological findings included variously shaped cystic and tubular structures in the tumor. The lumina were surrounded by a double layer of cells and showed active decapitation secretion. The lesion contained sebaceous differentiation, which has not been previously documented in the histopathology of hidradenoma papilliferum. We review the published work of non-anogenital (ectopic) hidradenoma papilliferum tumors on the head and neck. PMID- 16674790 TI - A case of juvenile hyaline fibromatosis. AB - Juvenile hyaline fibromatosis (JHF) is a rare, autosomally-recessive disease characterized by papulonodular skin lesions, soft tissue masses, joint contractures, gingival hypertrophy and osteolytic bone lesions. Its onset is in infancy or early childhood. The most commonly affected sites are the nose, chin, ears, scalp, back and knees. The accumulation of an amorphous, hyaline material is typical in the skin and the other organs. Herein, we report a 14-month-old boy who presented with confluent pink papules on the paranasal folds and the chin, and nodular lesions on the periauricular and perianal regions. He had gingival hypertrophy and contractures of the shoulders, knees and elbows. He also had third-degree consanguineous parents. Histopathological studies confirmed the diagnosis of JHF with the presence of increased numbers of fibroblasts embedded in a hyalinized connective tissue stroma. PMID- 16674791 TI - Successful steroid treatment of acquired idiopathic partial hypohidrosis. AB - The pathogenesis of idiopathic-acquired hypohidrosis remains unknown, and no specific causes have yet been established. We report a 34-year-old man with acquired idiopathic hypohidrosis successfully treated with prednisolone. The patient noticed heat intolerance and hypohidrosis of the pectoral and back during the summer. No systemic disease or neurological findings were identified. Eccrine sweat glands displayed infiltration by inflammatory cells, with immunoglobulin G and C3 deposition in the basement membrane. Steroid therapy improved the hypohidrosis. An immunological pathogenesis could be a major factor in idiopathic acquired hypohidrosis. PMID- 16674792 TI - Erythema induratum of Bazin in an infant after Bacille Calmette-Guerin vaccination. AB - We report the first case of erythema induratum of Bazin (EIB) after Bacille Calmette-Guerin (BCG) vaccination in an infant. The patient developed recurrent multiple erythematous and subcutaneous nodules on his legs 2 months after a BCG vaccination. He had no other symptoms or family history of tuberculosis (TB). Histopathological findings revealed a granulomatous lobular panniculitis. EIB often appears as a chronic, nodular eruptions that frequently occur on the lower legs of females with tuberculin hypersensitivity. To date, only a few cases of infants with EIB have been reported. There have been no clinical reports of EIB after BCG vaccination. Our case suggests that Mycobacterium bovis in the BCG vaccination may have caused EIB. PMID- 16674793 TI - A case of early onset confluent and reticulated papillomatosis with an unusual localization. AB - Confluent and reticulated papillomatosis is a relatively rare dermatosis of unknown origin. It is characterized by papules that become confluent in the center and reticulated at the periphery. The sites of predilection are the neck, interscapular region, inframammary area and abdomen. In a 15-year-old girl diagnosed with confluent and reticulated papillomatosis, the lesions first appeared on her knees and elbows when she was 4-years-old, and on the interscapular area when she was 13. Similar lesions arose on the left hand a few weeks previous to her visit. Two biopsy specimens were taken from the interscapular and elbow areas. The histopathological findings of this case fit the diagnosis of confluent and reticulated papillomatosis. We expect good results from azithromycin therapy. PMID- 16674794 TI - Two cases of perforating lichen nitidus. AB - Lichen nitidus is an unusual condition that usually affects children and young adults. Of its many variants, a perforating type has been rarely reported. We herein report two cases of lichen nitidus with perforation. Two young women in good health presented with multiple shiny papules that seems to be typical lichen nitidus clinically, but some of the papules were umbilicated. Histopathological examination revealed the typical findings of lichen nitidus with perforation in both cases. PMID- 16674795 TI - A case of Henoch-Schonlein purpura temporarily improved by hemodialysis. AB - We report a case of Henoch-Schonlein purpura that occurred in an adult patient with end-stage renal disease on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD). The patient's skin lesions clearly decreased in number during HD, but not during CAPD. This finding has not been previously reported. PMID- 16674796 TI - Pigmented coalescing papules on the dorsa of the hands: pigmented colloid milium associated with exogenous ochronosis. AB - Colloid milium is a rare cutaneous deposition disorder that frequently occurs in areas of chronic sun exposure such as the face, neck and backs of the hands and is characterized by multiple small, discrete, usually amber-colored, dome-shaped papules that cluster to form large plaques. A 50-year-old white woman with esophageal squamous cell carcinoma was referred to us with asymptomatic, slowly spreading lesions localized to the dorsa of her hands which had been present for 4 years. The condition was diagnosed histopathologically as pigmented colloid milium associated with exogenous ochronosis (EO). Colloid milium associated with EO is very rarely reported in the published work. We think that a possible interaction between sunlight and exposure to chemical fertilizers may have played a role in the pathogenesis of both of the disorders in our case. PMID- 16674797 TI - A case of the hyperkeratotic variant of porokeratosis Mibelli. AB - Herein, we report a case of the hyperkeratotic variant of porokeratosis Mibelli. A 58-year-old woman presented with two brownish verrucous plaques on the buttock for 20 years, and extremely hyperkeratotic verrucous nodules and brownish macules on the right foot for 1 year. Histopathological findings of the buttock were typical of porokeratosis. However, histopathological findings of the right foot were atypical for porokeratosis, including extreme hyperkeratosis and an increased number of and unusual sized cornoid lamellae. PMID- 16674798 TI - Sporothrix schenckii type 3D (mtDNA-RFLP): report of an osteoarticular case. AB - Sporotrichosis is a frequent subcutaneous mycosis in Mexico and lymphocutaneous cases are the most common type. Extracutaneous or disseminated forms are exceptional and are usually seen in immunosuppressed hosts. We report the case of a 74-year-old immunocompetent male with osteoarticular involvement. The isolated Sporothrix schenckii was classified as type 3D according to restriction fragment length polymorphism analysis of the mitochondrial DNA (mtDNA-RFLP). PMID- 16674799 TI - Serum levels of CCL17/TARC in various skin diseases. PMID- 16674800 TI - Syringocystadenoma papilliferum with extensive apocrine nevus. PMID- 16674801 TI - Chalazion masquerading as sporotrichosis. PMID- 16674802 TI - The oxanorbornene approach to 3-hydroxy, 3,4-dihydroxy and 3,4,5-trihydroxy derivatives of 2-aminocyclohexanecarboxylic acid. AB - The nitro oxanorbornene adduct derived from the Diels-Alder reaction of ethyl (E) 3-nitroacrylate and furan provides a versatile template for the stereoselective synthesis of hydroxylated derivatives of 2-aminocyclohexanecarboxylic acid (ACHC). PMID- 16674803 TI - From descriptive to predictive distribution models: a working example with Iberian amphibians and reptiles. AB - BACKGROUND: Aim of the study was to identify the conditions under which spatial environmental models can be used for the improved understanding of species distributions, under the explicit criterion of model predictive performance. I constructed distribution models for 17 amphibian and 21 reptile species in Portugal from atlas data and 13 selected ecological variables with stepwise logistic regression and a geographic information system. Models constructed for Portugal were extrapolated over Spain and tested against range maps and atlas data. RESULTS: Descriptive model precision ranged from 'fair' to 'very good' for 12 species showing a range border inside Portugal ('edge species', kappa (k) 0.35 0.89, average 0.57) and was at best 'moderate' for 26 species with a countrywide Portuguese distribution ('non-edge species', k = 0.03-0.54, average 0.29). The accuracy of the prediction for Spain was significantly related to the precision of the descriptive model for the group of edge species and not for the countrywide species. In the latter group data were consistently better captured with the single variable search-effort than by the panel of environmental data. CONCLUSION: Atlas data in presence-absence format are often inadequate to model the distribution of species if the considered area does not include part of the range border. Conversely, distribution models for edge-species, especially those displaying high precision, may help in the correct identification of parameters underlying the species range and assist with the informed choice of conservation measures. PMID- 16674805 TI - Combined expectancies: electrophysiological evidence for the adjustment of expectancy effects. AB - BACKGROUND: When subjects use cues to prepare for a likely stimulus or a likely response, reaction times are facilitated by valid cues but prolonged by invalid cues. In studies on combined expectancy effects, two cues can independently give information regarding two dimensions of the forthcoming task. In certain situations, cueing effects on one dimension are reduced when the cue on the other dimension is invalid. According to the Adjusted Expectancy Model, cues affect different processing levels and a mechanism is presumed which is sensitive to the validity of early level cues and leads to online adjustment of expectancy effects at later levels. To examine the predictions of this model cueing of stimulus modality was combined with response cueing. RESULTS: Behavioral measures showed the interaction of cueing effects. Electrophysiological measures of the lateralized readiness potential (LRP) and the N200 amplitude confirmed the predictions of the model. The LRP showed larger effects of response cues on response activation when modality cues were valid rather than invalid. N200 amplitude was largest with valid modality cues and invalid response cues, medium with invalid modality cues, and smallest with two valid cues. CONCLUSION: Findings support the view that the validity of early level expectancies modulates the effects of late level expectancies, which included response activation and response conflict in the present study. PMID- 16674806 TI - Effectiveness of a Minimal Intervention for Stress-related mental disorders with Sick leave (MISS); study protocol of a cluster randomised controlled trial in general practice [ISRCTN43779641]. AB - BACKGROUND: The main aims of this paper are to describe the setting and design of a Minimal Intervention in general practice for Stress-related mental disorders in patients on Sick leave (MISS), as well as to ascertain the study complies with the requirements for a cluster randomised controlled trial (RCT). The potential adverse consequences of sick leave due to Stress-related Mental Disorders (SMDs) are extensive, but often not recognised. Since most people having SMDs with sick leave consult their general practitioner (GP) at an early stage, a tailored intervention given by GPs is justified. We provide a detailed description of the MISS; that is more accurate assessment, education, advice and monitoring to treat SMDs in patients on sick leave. Our hypothesis is that the MISS will be more effective compared to the usual care, in reducing days of sick leave of these patients. METHODS: The design is a pragmatic RCT. Randomisation is at the level of GPs. They received the MISS-training versus no training, in order to compare the MISS vs. usual care at patient level. Enrollment of patients took place after screening in the source population, that comprised 20-60 year old primary care attendees. Inclusion criteria were: moderately elevated distress levels, having a paid job and sick leave for no longer than three months. There is a one year follow up. The primary outcome measure is lasting full return to work. Reduction of SMD- symptoms is one of the secondary outcome measures. Forty-six GPs and 433 patients agreed to participate. DISCUSSION: In our study design, attention is given to the practical application of the requirements for a pragmatic trial. The results of this cluster RCT will add to the evidence about treatment options in general practice for SMDs in patients on sick leave, and might contribute to a new and appropriate guideline. These results will be available at the end of 2006. PMID- 16674808 TI - The impact of imprecisely measured covariates on estimating gene-environment interactions. AB - BACKGROUND: The effects of measurement error in epidemiological exposures and confounders on estimated effects of exposure are well described, but the effects on estimates for gene-environment interactions has received rather less attention. In particular, the effects of confounder measurement error on gene environment interactions are unknown. METHODS: We investigate these effects using simulated data and illustrate our results with a practical example in nutrition epidemiology. RESULTS: We show that the interaction regression coefficient is unchanged by confounder measurement error under certain conditions, but biased by exposure measurement error. We also confirm that confounder measurement error can lead to estimated effects of exposure biased either towards or away from the null, depending on the correlation structure, with associated effects on type II errors. CONCLUSION: Whilst measurement error in confounders does not lead to bias in interaction coefficients, it may still lead to bias in the estimated effects of exposure. There may still be cost implications for epidemiological studies that need to calibrate all error-prone covariates against a valid reference, in addition to the exposure, to reduce the effects of confounder measurement error. PMID- 16674807 TI - Functional conservation of Pax6 regulatory elements in humans and mice demonstrated with a novel transgenic reporter mouse. AB - BACKGROUND: The Pax6 transcription factor is expressed during development in the eyes and in specific CNS regions, where it is essential for normal cell proliferation and differentiation. Mice lacking one or both copies of the Pax6 gene model closely humans with loss-of-function mutations in the PAX6 locus. The sequence of the Pax6/PAX6 protein is identical in mice and humans and previous studies have shown structural conservation of the gene's regulatory regions. RESULTS: We generated a transgenic mouse expressing green fluorescent protein (GFP) and neomycin resistance under the control of the entire complement of human PAX6 regulatory elements using a modified yeast artificial chromosome (YAC). Expression of GFP was studied in embryos from 9.5 days on and was confined to cells known to express Pax6. GFP expression was sufficiently strong that expressing cells could be distinguished from non-expressing cells using flow cytometry. CONCLUSION: This work demonstrates the functional conservation of the regulatory elements controlling Pax6/PAX6 expression in mice and humans. The transgene provides an excellent tool for studying the functions of different Pax6/PAX6 regulatory elements in controlling Pax6 expression in animals that are otherwise normal. It will allow the analysis and isolation of cells in which Pax6 is activated, irrespective of the status of the endogenous locus. PMID- 16674804 TI - Melatonin in Alzheimer's disease and other neurodegenerative disorders. AB - Increased oxidative stress and mitochondrial dysfunction have been identified as common pathophysiological phenomena associated with neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). As the age-related decline in the production of melatonin may contribute to increased levels of oxidative stress in the elderly, the role of this neuroprotective agent is attracting increasing attention. Melatonin has multiple actions as a regulator of antioxidant and prooxidant enzymes, radical scavenger and antagonist of mitochondrial radical formation. The ability of melatonin and its kynuramine metabolites to interact directly with the electron transport chain by increasing the electron flow and reducing electron leakage are unique features by which melatonin is able to increase the survival of neurons under enhanced oxidative stress. Moreover, antifibrillogenic actions have been demonstrated in vitro, also in the presence of profibrillogenic apoE4 or apoE3, and in vivo, in a transgenic mouse model. Amyloid-beta toxicity is antagonized by melatonin and one of its kynuramine metabolites. Cytoskeletal disorganization and protein hyperphosphorylation, as induced in several cell-line models, have been attenuated by melatonin, effects comprising stress kinase downregulation and extending to neurotrophin expression. Various experimental models of AD, PD and HD indicate the usefulness of melatonin in antagonizing disease progression and/or mitigating some of the symptoms. Melatonin secretion has been found to be altered in AD and PD. Attempts to compensate for age- and disease-dependent melatonin deficiency have shown that administration of this compound can improve sleep efficiency in AD and PD and, to some extent, cognitive function in AD patients. Exogenous melatonin has also been reported to alleviate behavioral symptoms such as sundowning. Taken together, these findings suggest that melatonin, its analogues and kynuric metabolites may have potential value in prevention and treatment of AD and other neurodegenerative disorders. PMID- 16674809 TI - No association between chronic musculoskeletal complaints and Val158Met polymorphism in the Catechol-O-methyltransferase gene. The HUNT study. AB - BACKGROUND: The Catechol-O-methyltransferase (COMT) gene contains a functional polymorphism, Val158Met, that has been found to influence human pain perception. In one study fibromyalgia was less likely among those with Val/Val genotype. METHODS: In the 1995-97 Nord-Trondelag Health Study (HUNT), the association between Val/Met polymorphism at the COMT gene and chronic musculoskeletal complaints (MSCs) was evaluated in a random sample of 3017 individuals. RESULTS: The distribution of the COMT Val158Met genotypes and alleles were similar between controls and the twelve different chronic MSCs groups. Even when the Met/Met and Val/Met genotypes were pooled, the distribution of the Val/Val genotype and other genotypes were similar between controls and the chronic MSCs groups. CONCLUSION: In this population-based study, no significant association was found between Val/Met polymorphism at the COMT gene and chronic MSCs. PMID- 16674810 TI - Combining evidence, biomedical literature and statistical dependence: new insights for functional annotation of gene sets. AB - BACKGROUND: Large-scale genomic studies based on transcriptome technologies provide clusters of genes that need to be functionally annotated. The Gene Ontology (GO) implements a controlled vocabulary organised into three hierarchies: cellular components, molecular functions and biological processes. This terminology allows a coherent and consistent description of the knowledge about gene functions. The GO terms related to genes come primarily from semi automatic annotations made by trained biologists (annotation based on evidence) or text-mining of the published scientific literature (literature profiling). RESULTS: We report an original functional annotation method based on a combination of evidence and literature that overcomes the weaknesses and the limitations of each approach. It relies on the Gene Ontology Annotation database (GOA Human) and the PubGene biomedical literature index. We support these annotations with statistically associated GO terms and retrieve associative relations across the three GO hierarchies to emphasise the major pathways involved by a gene cluster. Both annotation methods and associative relations were quantitatively evaluated with a reference set of 7397 genes and a multi cluster study of 14 clusters. We also validated the biological appropriateness of our hybrid method with the annotation of a single gene (cdc2) and that of a down regulated cluster of 37 genes identified by a transcriptome study of an in vitro enterocyte differentiation model (CaCo-2 cells). CONCLUSION: The combination of both approaches is more informative than either separate approach: literature mining can enrich an annotation based only on evidence. Text-mining of the literature can also find valuable associated MEDLINE references that confirm the relevance of the annotation. Eventually, GO terms networks can be built with associative relations in order to highlight cooperative and competitive pathways and their connected molecular functions. PMID- 16674811 TI - Alteration of gene expression profiles during mycoplasma-induced malignant cell transformation. AB - BACKGROUND: Mycoplasmas are the smallest microorganisms capable of self replication. Our previous studies show that some mycoplasmas are able to induce malignant transformation of host mammalian cells. This malignant transformation is a multistage process with the early infection, reversible and irreversible stages, and similar to human tumor development in nature. The purpose of this study is to explore mechanisms for this malignant transformation. METHODS: To better understand mechanisms for this unique process, we examined gene expression profiles of C3H cells at different stages of the mycoplasma-induced transformation using cDNA microarray technology. A total of 1185 genes involved in oncogenesis, apoptosis, cell growth, cell-cycle regulation, DNA repair, etc. were examined. Differences in the expression of these genes were compared and analyzed using the computer software AtlasImage. RESULTS: Among 1185 genes screened, 135 had aberrant expression at the early infection stage, 252 at the reversible stage and 184 at the irreversible stage. At the early infection stage, genes with increased expression (92 genes) were twice more than those with decreased expression (42 genes). The global gene expression at the reversible stage appeared to be more volatile than that at any other stages but still resembled the profile at the early infection stage. The expression profile at the irreversible stage shows a unique pattern of a wide range of expression levels and an increased number of expressing genes, especially the cancer-related genes. Oncogenes and tumor suppressors are a group of molecules that showed significant changes in expression during the transformation. The majority of these changes occurred in the reversible and irreversible stages. A prolonged infection by mycoplasmas lead to the expression of more cancer related genes at the irreversible stage. CONCLUSION: The results indicate that the expression profiles correspond with the phenotypic features of the cells in the mycoplasma induced transformation process. The early mycoplasma infection stage shares a common phenomenon with many other acute infections, genes with increased expression significantly outnumbering those with decreased expression. The reversible stage is a transition stage between benignancy and malignancy at the molecular level. Aberrant expression of oncogenes and tumor repressors plays a key role in mycoplasma-induced malignant transformation. PMID- 16674812 TI - Primary pancreatic lymphoma--pancreatic tumours that are potentially curable without resection, a retrospective review of four cases. AB - BACKGROUND: Primary pancreatic lymphomas (PPL) are rare tumours of the pancreas. Symptoms, imaging and tumour markers can mimic pancreatic adenocarcinoma, but they are much more amenable to treatment. Treatment for PPL remains controversial, particularly the role of surgical resection. METHODS: Four cases of primary pancreatic lymphoma were identified at Prince of Wales Hospital, Sydney, Australia. A literature review of cases of PPL reported between 1985 and 2005 was conducted, and outcomes were contrasted. RESULTS: All four patients presented with upper abdominal symptoms associated with weight loss. One case was diagnosed without surgery. No patients underwent pancreatectomy. All patients were treated with chemotherapy and radiotherapy, and two of four patients received rituximab. One patient died at 32 months. Three patients are disease free at 15, 25 and 64 months, one after successful retreatment. Literature review identified a further 103 patients in 11 case series. Outcomes in our series and other series of chemotherapy and radiotherapy compared favourably to surgical series. CONCLUSION: Biopsy of all pancreatic masses is essential, to exclude potentially curable conditions such as PPL, and can be performed without laparotomy. Combined multimodality treatment, utilising chemotherapy and radiotherapy, without surgical resection is advocated but a cooperative prospective study would lead to further improvement in treatment outcomes. PMID- 16674813 TI - Reduction/oxidation-phosphorylation control of DNA binding in the bZIP dimerization network. AB - BACKGROUND: bZIPs are transcription factors that are found throughout the eukarya from fungi to flowering plants and mammals. They contain highly conserved basic region (BR) and leucine zipper (LZ) domains and often function as environmental sensors. Specifically, bZIPs frequently have a role in mediating the response to oxidative stress, a crucial environmental signal that needs to be transduced to the gene regulatory network. RESULTS: Based on sequence comparisons and experimental data on a number of important bZIP transcription factors, we predict which bZIPs are under redox control and which are regulated via protein phosphorylation. By integrating genomic, phylogenetic and functional data from the literature, we then propose a link between oxidative stress and the choice of interaction partners for the bZIP proteins. CONCLUSION: This integration permits the bZIP dimerization network to be interpreted in functional terms, especially in the context of the role of bZIP proteins in the response to environmental stress. This analysis demonstrates the importance of abiotic factors in shaping regulatory networks. PMID- 16674814 TI - Out-of-hours primary care. Implications of organisation on costs. AB - BACKGROUND: To perform out-of-hours primary care, Dutch general practitioners (GPs) have organised themselves in large-scale GP cooperatives. Roughly, two models of out-of-hours care can be distinguished; GP cooperatives working separate from the hospital emergency department (ED) and GP cooperatives integrated with the hospital ED. Research has shown differences in care utilisation between these two models; a significant shift in the integrated model from utilisation of ED care to primary care. These differences may have implications on costs, however, until now this has not been investigated. This study was performed to provide insight in costs of these two different models of out-of-hours care. METHODS: Annual reports of two GP cooperatives (one separate from and one integrated with a hospital emergency department) in 2003 were analysed on costs and use of out-of-hours care. Costs were calculated per capita. Comparisons were made between the two cooperatives. In addition, a comparison was made between the costs of the hospital ED of the integrated model before and after the set up of the GP cooperative were analysed. RESULTS: Costs per capita of the GP cooperative in the integrated model were slightly higher than in the separate model (epsilon 11.47 and epsilon 10.54 respectively). Differences were mainly caused by personnel and other costs, including transportation, interest, cleaning, computers and overhead. Despite a significant reduction in patients utilising ED care as a result of the introduction of the GP cooperative integrated within the ED, the costs of the ED remained the same. CONCLUSION: The study results show that the costs of primary care appear to be more dependent on the size of the population the cooperative covers than on the way the GP cooperative is organised, i.e. separated versus integrated. In addition, despite the substantial reduction of patients, locating the GP cooperative at the same site as the ED was found to have little effect on costs of the ED. Sharing more facilities and personnel between the ED and the GP cooperative may improve cost efficiency. PMID- 16674815 TI - Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue. AB - BACKGROUND: Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. METHODS: Uterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. RESULTS: In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to non-pregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy. CONCLUSION: The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility. PMID- 16674816 TI - Validation of a microwave radar system for the monitoring of locomotor activity in mice. AB - BACKGROUND: The general or spontaneous motor activity of animals is a useful parameter in chronobiology. Modified motion detectors can be used to monitor locomotor activity rhythms. We modified a commercial microwave-based detection device and validated the device by recording circadian and ultradian rhythms. METHODS: Movements were detected by microwave radar based on the Doppler effect. The equipment was designed to detect and record simultaneously 12 animals in separate cages. Radars were positioned at the bottom of aluminium bulkheads. Animal cages were positioned above the bulkheads. The radars were connected to a computer through a digital I/O board. RESULTS: The apparatus was evaluated by several tests. The first test showed the ability of the apparatus to detect the exact frequency of the standard moving object. The second test demonstrated the stability over time of the sensitivity of the radars. The third was performed by simultaneous observations of video-recording of a mouse and radar signals. We found that the radars are particularly sensitive to activities that involve a displacement of the whole body, as compared to movement of only a part of the body. In the fourth test, we recorded the locomotor activity of Balb/c mice. The results were in agreement with published studies. CONCLUSION: Radar detectors can provide automatic monitoring of an animal's locomotor activity in its home cage without perturbing the pattern of its normal behaviour or initiating the spurt of exploration occasioned by transfer to a novel environment. Recording inside breeding cages enables long-term studies with uninterrupted monitoring. The use of electromagnetic waves allows contactless detection and freedom from interference of external stimuli. PMID- 16674818 TI - Effects of a carbohydrate-restricted diet on emerging plasma markers for cardiovascular disease. AB - BACKGROUND: Increasing evidence supports carbohydrate restricted diets (CRD) for weight loss and improvement in traditional markers for cardiovascular disease (CVD); less is known regarding emerging CVD risk factors. We previously reported that a weight loss intervention based on a CRD (% carbohydrate:fat:protein = 13:60:27) led to a mean weight loss of 7.5 kg and a 20% reduction of abdominal fat in 29 overweight men. This group showed reduction in plasma LDL-cholesterol and triglycerides and elevations in HDL-cholesterol as well as reductions in large and medium VLDL particles and increases in LDL particle size. In this study we report on the effect of this intervention with and without fiber supplementation on plasma homocysteine, lipoprotein (a) [Lp(a)], C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha). METHODS: Twenty nine overweight men [body mass index (BMI) 25-35 kg/m2] aged 20 69 years consumed an ad libitum CRD (% carbohydrate:fat:protein = 13:60:27) including a standard multivitamin every other day for 12 wk. Subjects were matched by age and BMI and randomly assigned to consume 3 g/d of either a soluble fiber supplement (n = 14) or placebo (n = 15). RESULTS: There were no group or interaction (fiber x time) main effects, but significant time effects were observed for several variables. Energy intake was spontaneously reduced (-30.5%). This was accompanied by an increase in protein intake (96.2 +/- 29.8 g/d to 107.3 +/- 29.7 g/d) and methionine intake (2.25 +/- 0.7 g/d, to 2.71 +/- 0.78 g/d; P < 0.001). Trans fatty acid intake was significantly reduced (-38.6%) while dietary folate was unchanged, as was plasma homocysteine. Bodyweight (-7.5 +/- 2.5 kg) was reduced as was plasma Lp(a) (-11.3%). Changes in plasma Lp(a) correlated with reductions in LDL-cholesterol (r = .436, P < 0.05) and fat loss (r = .385, P < 0,05). At wk 12, both CRP (-8.1%) and TNF-alpha (-9.3%) were reduced (P < 0.05) independently of weight loss. IL-6 concentrations were unchanged. CONCLUSION: A diet based on restricting carbohydrates leads to spontaneous caloric reduction and subsequent improvement in emerging markers of CVD in overweight/obese men who are otherwise healthy. PMID- 16674817 TI - Revascularization and cardioprotective drug treatment in myocardial infarction patients: how do they impact on patients' survival when delivered as usual care. AB - BACKGROUND: Randomized clinical trials showed the benefit of pharmacological and revascularization treatments in secondary prevention of myocardial infarction (MI), in selected population with highly controlled interventions. The objective of this study is to measure these treatments' impact on the cardiovascular (CV) mortality rate among patients receiving usual care in the province of Quebec. METHODS: The study population consisted of a "naturalistic" cohort of all patients > or = 65 years old living in the Quebec province, who survived a MI (ICD-9: 410) in 1998. The studied dependant variable was time to death from a CV disease. Independent variables were revascularization procedure and cardioprotective drugs. Death from a non CV disease was also studied for comparison. Revascularization procedure was defined as percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass graft (CABG). The exposure to cardioprotective drugs was defined as the number of cardioprotective drug classes (Acetylsalicylic Acid (ASA), Beta-Blockers, Angiotensin-Converting Enzyme (ACE) Inhibitors, Statins) claimed within the index period (first 30 days after the index hospitalization). Age, gender and a comorbidity index were used as covariates. Kaplan-Meier survival curves, Cox proportional hazard models, logistic regressions and regression trees were used. RESULTS: The study population totaled 5596 patients (3206 men; 2390 women). We observed 1128 deaths (20%) within two years following index hospitalization, of them 603 from CV disease. The CV survival rate at two years is much greater for patients with revascularization, regardless of pharmacological treatments. For patients without revascularization, the CV survival rate increases with the number of cardioprotective drug classes claimed. Finally, Cox proportional hazard models, regression tree and logistic regression analyses all revealed that the absence of revascularization and, to a lower extent, absence of cardioprotective drugs were major predictors for CV death, even after adjusting for age, gender and comorbidity. CONCLUSION: Considering usual care management of MI in the province of Quebec in 1998, CV survival is positively correlated to the presence of a revascularization procedure and to the intensity of cardioprotective pharmacological treatment. These results are coherent with data from randomized control trials. PMID- 16674819 TI - Using geographic information systems to identify prospective marketing areas for a special library. AB - BACKGROUND: The Center for Disability Resources (CDR) Library is the largest collection of its kind in the Southeastern United States, consisting of over 5,200 books, videos/DVDs, brochures, and audiotapes covering a variety of disability-related topics, from autism to transition resources. The purpose of the library is to support the information needs of families, faculty, students, staff, and other professionals in South Carolina working with individuals with disabilities. The CDR Library is funded on a yearly basis; therefore, maintaining high usage is crucial. A variety of promotional efforts have been used to attract new patrons to the library. Anyone in South Carolina can check out materials from the library, and most of the patrons use the library remotely by requesting materials, which are then mailed to them. The goal of this project was to identify areas of low geographic usage as a means of identifying locations for future library marketing efforts. METHODS: Nearly four years worth of library statistics were compiled in a spreadsheet that provided information per county on the number of checkouts, the number of renewals, and the population. Five maps were created using ArcView GIS software to create visual representations of patron checkout and renewal behavior per county. RESULTS: Out of the 46 counties in South Carolina, eight counties never checked out materials from the library. As expected urban areas and counties near the library's physical location have high usage totals. CONCLUSION: The visual representation of the data made identification of low usage regions easier than using a standalone database with no visual-spatial component. The low usage counties will be the focus of future Center for Disability Resources Library marketing efforts. Due to the impressive visual-spatial representations created with Geographic Information Systems, which more efficiently communicate information than stand-alone database information can, librarians may benefit from the software's use as a supplemental tool for tracking library usage and planning promotional efforts. PMID- 16674820 TI - The basic principles of migration health: population mobility and gaps in disease prevalence. AB - Currently, migrants and other mobile individuals, such as migrant workers and asylum seekers, are an expanding global population of growing social, demographic and political importance. Disparities often exist between a migrant population's place of origin and its destination, particularly with relation to health determinants. The effects of those disparities can be observed at both individual and population levels. Migration across health and disease disparities influences the epidemiology of certain diseases globally and in nations receiving migrants. While specific disease-based outcomes may vary between migrant group and location, general epidemiological principles may be applied to any situation where numbers of individuals move between differences in disease prevalence. Traditionally, migration health activities have been designed for national application and lack an integrated international perspective. Present and future health challenges related to migration may be more effectively addressed through collaborative global undertakings. This paper reviews the epidemiological relationships resulting from health disparities bridged by migration and describes the growing role of migration and population mobility in global disease epidemiology. The implications for national and international health policy and program planning are presented. PMID- 16674821 TI - Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages. AB - BACKGROUND: Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase A2 is a major down-stream target and this enzyme is regulated by both phosphorylation and an increase in intracellular Ca2+. Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cgamma2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk. METHODS: Arachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity (PP2 and the Src kinase inhibitor 1, SKI-1) were used as well as the Btk inhibitor LFM-A13. RESULTS: Arachidonate release initiated by either Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cgamma2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38) and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type. CONCLUSION: Arachidonate release induced by either Staphylococcus aureus or zymosan was found to depend on Src family kinases as well as Btk. While members of the Src kinase family were shown to act upstream of Btk and the MAP kinases, Btk plays another role independent of MAP kinases, but down-stream of the Src family kinases. PMID- 16674822 TI - Elicitation from virus-naive individuals of cytotoxic T lymphocytes directed against conserved HIV-1 epitopes. AB - Cytotoxic T lymphocytes (CTL) protect against viruses including HIV-1. To avoid viral escape mutants that thwart immunity, we chose 25 CTL epitopes defined in the context of natural infection with functional and/or structural constraints that maintain sequence conservation. By combining HLA binding predictions with knowledge concerning HLA allele frequencies, a metric estimating population protection coverage (PPC) was computed and epitope pools assembled. Strikingly, only a minority of immunocompetent HIV-1 infected individuals responds to pools with PPC >95%. In contrast, virus-naive individuals uniformly expand IFNgamma producing cells and mount anti-HIV-1 cytolytic activity. This disparity suggests a vaccine design paradigm shift from infected to normal subjects. PMID- 16674823 TI - Effect of step-synchronized vibration stimulation of soles on gait in Parkinson's disease: a pilot study. AB - BACKGROUND: Previous studies have suggested that impaired proprioceptive processing in the striatum may contribute to abnormal gait in Parkinson's disease (PD). METHODS: This pilot study assessed the effects of enhanced proprioceptive feedback using step-synchronized vibration stimulation of the soles (S-VS) on gait in PD. S-VS was used in 8 PD subjects (3 women and 5 men, age range 44-79 years, on medication) and 8 age-matched healthy subjects (5 women and 3 men). PD subjects had mild or moderate gait impairment associated with abnormal balance, but they did not have gait freezing. Three vibratory devices (VDs) were embedded in elastic insoles (one below the heel and two below the forefoot areas) inserted into the shoes. Each VD operates independently and has a pressure switch that activates the underlying vibratory actuator. The VD delivered the 70-Hz suprathreshold vibration pulse upon touch by the heel or forefoot, and the vibration pulse was deactivated upon respective push-offs. Six-minute hallway walking was studied with and without S-VS. Gait characteristics were measured using the force-sensitive foot switches. The primary outcome was the stride variability expressed as a coefficient of variation (CV), a measure of gait steadiness. Secondary outcome measures were walking distance and speed, stride length and duration, cadence, stance, swing and double support duration, and respective CVs (if applicable). RESULTS: The walking speed (p < 0.04) and the CV of the stride interval (p < 0.02) differed between the groups and S-VS conditions. In the PD group, S-VS decreased stride variability (p < 0.002), increased walking speed (p < 0.0001), stride duration (p < 0.01), stride length (p < 0.0002), and cadence (p < 0.03). In the control group, S-VS decreased stride variability (p < 0.006) and increased gait speed (p < 0.03), but other locomotion parameters were not significantly altered. CONCLUSION: Augmented sensory feedback improves parkinsonian gait steadiness in the short-term setting. Because the suprathreshold stimulation prevented blinding of subjects, the learning effect and increased attention can be a confounding factor underlying results. Long-term studies are needed to establish the clinical value of the S-VS. PMID- 16674824 TI - Transanal endoscopic micro-surgery (TEMS) for the management of large or sessile rectal adenomas: a review of the technique and indications. AB - In this review article the surgical technique of Transanal Endoscopic Microsurgery (TEMS) is examined. A number of techniques have been used to treat adenomas of the rectum. The treatment of large adenomas which occupy a large surface of the rectal lumen or adenomas which are flat and grow in a "carpet like" fashion is particularly challenging. Major rectal surgery carries a risk of morbidity and mortality, particularly in elderly and unfit patients. Although local excision with transanal resection (TAR) and the Kraske sacral operation have been used in the past, during the last twenty years TEMS has become the method of choice for those lesions. TEMS is efficient and minimally invasive. The technique allows the patient to recover rapidly and the incidence of complications is much lower than that of major surgery. In case of recurrence the option of repeat TEMS or major surgery remain available. TEMS has been slow to gain popularity mainly for reasons of cost and steep learning curve but it is now an established procedure and a valuable therapeutic option which is particularly useful for elderly and unfit patients. Gastroenterologists should be aware of the nature and indications of TEMS in order to advise and refer selected patients with rectal adenomas accordingly. PMID- 16674825 TI - Posttransplantation malignancy in a patient presenting with weight loss and changed bowel habits: a case report. AB - BACKGROUND: Advancements in immunosuppressive therapy have significantly improved patient and graft survival following renal transplantation. This is paralleled by an increasing occurrence of posttransplantation malignancy. CASE PRESENTATION: We report on a patient who presented with a history reminding of colon cancer seven years after receiving a kidney transplant. Initial diagnostic imaging seemed to confirm this diagnosis showing a constricting colonic lesion. To our surprise, colonoscopy findings were unremarkable. Review of the imaging studies revealed that the tumor-like picture was caused by the renal graft impressing the intestine. The following search for malignancy in other locations resulted in the diagnosis of glioblastoma multiforme of which the patient died several weeks later. CONCLUSION: Follow-up of renal transplant patients must include screening tests directed at tumor detection. Imaging studies and other tests in this patient group should be interpreted by physicians who are familiar with transplant related peculiarities. PMID- 16674826 TI - Sexually dimorphic gene expression that overlaps maturation of type II pneumonocytes in fetal mouse lungs. AB - BACKGROUND: In human, respiratory distress of the neonates, which occurs in prematurity, is prevalent in male. Late in gestation, maturation of type II pneumonocytes, and consequently the surge of surfactant synthesis are delayed in male fetuses compared with female fetuses. Although the presence of higher levels of androgens in male fetuses is thought to explain this sex difference, the identity of genes involved in lung maturation that are differentially modulated according to fetal sex is unknown. We have studied the sex difference in developing mouse lung by gene profiling during a three-day gestational window preceding and including the emergence of mature PTII cells (the surge of surfactant synthesis in the mouse occurs on GD 17.5). METHODS: Total RNA was extracted from lungs of male and female fetal mice (gestation days 15.5, 16.5, and 17.5), converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays (Affymetrix MOE430A). Analysis of data was performed using MAS5.0, LFCM and Genesis softwares. RESULTS: Many genes involved in lung maturation were expressed with no sex difference. Of the approximative 14,000 transcripts covered by the arrays, only 83 genes presented a sex difference at one or more time points between GDs 15.5 and 17.5. They include genes involved in hormone metabolism and regulation (i.e. steroidogenesis pathways), apoptosis, signal transduction, transcriptional regulation, and lipid metabolism with four apolipoprotein genes. Genes involved in immune functions and other metabolisms also displayed a sex difference. CONCLUSION: Among these sexually dimorphic genes, some may be candidates for a role in lung maturation. Indeed, on GD 17.5, the sex difference in surfactant lipids correlates with the sex difference in pulmonary expression of apolipoprotein genes, which are involved in lipid transport. This suggests a role for these genes in the surge of surfactant synthesis. Our results would help to identify novel genes involved in the physiopathology of the respiratory distress of the neonates. PMID- 16674827 TI - Direct hospital costs of chest pain patients attending the emergency department: a retrospective study. AB - BACKGROUND: Chest pain is one of the most common complaints in the Emergency Department (ED), but the cost of ED chest pain patients is unclear. The aim of this study was to describe the direct hospital costs for unselected chest pain patients attending the emergency department (ED). METHODS: 1,000 consecutive ED visits of patients with chest pain were retrospectively included. Costs directly following the ED visit were retrieved from the hospital economy system. RESULTS: The mean cost per patient visit was 26.8 thousand Swedish kronar (kSEK) (median 7.2 kSEK), with admission time accounting for 73% of all costs. Mean cost for patients discharged from the ED was 1.4 kSEK (median 1.3 kSEK), and for patients without ACS admitted 1 day or less 7.6 kSEK (median 6.9 kSEK). The practice in the present study to admit 67% of the patients, of whom only 31% proved to have ACS, was estimated to give a cost per additional life-year saved by hospital admission, compared to theoretical strategy of discharging all patients home, of about 350 kSEK (39 kEUR or 42 kUSD). CONCLUSION: Costs for chest pain patients are large and primarily due to admission time. The present admission practice seems to be cost-effective, but the substantial overadmission indicates that better ED diagnostics and triage could decrease costs considerably. PMID- 16674828 TI - Multipolar representation of protein structure. AB - BACKGROUND: That the structure determines the function of proteins is a central paradigm in biology. However, protein functions are more directly related to cooperative effects at the residue and multi-residue scales. As such, current representations based on atomic coordinates can be considered inadequate. Bridging the gap between atomic-level structure and overall protein-level functionality requires parameterizations of the protein structure (and other physicochemical properties) in a quasi-continuous range, from a simple collection of unrelated amino acids coordinates to the highly synergistic organization of the whole protein entity, from a microscopic view in which each atom is completely resolved to a "macroscopic" description such as the one encoded in the three-dimensional protein shape. RESULTS: Here we propose such a parameterization and study its relationship to the standard Euclidian description based on amino acid representative coordinates. The representation uses multipoles associated with residue Calpha coordinates as shape descriptors. We demonstrate that the multipoles can be used for the quantitative description of the protein shape and for the comparison of protein structures at various levels of detail. Specifically, we construct a (dis)similarity measure in multipolar configuration space, and show how such a function can be used for the comparison of a pair of proteins. We then test the parameterization on a benchmark set of the protein kinase-like superfamily. We prove that, when the biologically relevant portions of the proteins are retained, it can robustly discriminate between the various families in the set in a way not possible through sequence or conventional structural representations alone. We then compare our representation with the Cartesian coordinate description and show that, as expected, the correlation with that representation increases as the level of detail, measured by the highest rank of multipoles used in the representation, approaches the dimensionality of the fold space. CONCLUSION: The results described here demonstrate how a granular description of the protein structure can be achieved using multipolar coefficients. The description has the additional advantage of being immediately generalizable for any residue-specific property therefore providing a unitary framework for the study and comparison of the spatial profile of various protein properties. PMID- 16674829 TI - Percutaneous subclavian artery stent-graft placement following failed ultrasound guided subclavian venous access. AB - BACKGROUND: Ultrasound guidance for central and peripheral venous access has been proven to improve success rates and reduce complications of venous cannulation. Appropriately trained and experienced operators add significantly to diminished patient morbidity related to venous access procedures. We discuss a patient who required an arterial stent-graft to prevent arterial hemorrhage following inadvertent cannulation of the proximal, ventral, right subclavian artery related to unsuccessful ultrasound guided access of the subclavian vein. CASE PRESENTATION: During pre-operative preparation for aortic valve replacement and aorto-coronary bypass surgery an anesthetist attempted ultrasound guided venous access. The ultrasound guided attempt to access the right jugular vein failed and the ultrasound guided attempt at accessing the subclavian vein resulted in inappropriate placement of an 8.5 F sheath in the arterial system. Following angiographic imaging and specialist consultations, an arterial stent-graft was deployed in the right subclavian artery rather than perform an extensive anterior chest wall resection and dissection to extract the arterial sheath. The patient tolerated the procedure, without complication, despite occlusion of the right internal mammary artery and the right vertebral artery. There were no neurologic sequelae. There was no evidence of hemorrhage after subclavian artery sheath extraction and stent-graft implantation. CONCLUSION: The attempted ultrasound guided puncture of the subclavian vein resulted in placement of an 8.5 F subclavian artery catheter. Entry of the catheter into the proximal subclavian artery beneath the medial clavicle, the medial first rib and the manubrium suggests that the operator, most likely, did not directly visualize the puncture needle enter the vessel with the ultrasound. The bones of the anterior chest impede the ultrasound beam and the vessels in this area would not be visible to ultrasound imaging. Appropriate training and supervised experience in ultrasound guided venous access coupled with quality ultrasound equipment would most likely have significantly diminished the likelihood of this complication. The potential for significant patient morbidity, and possible mortality, was prevented by implantation of an arterial stent-graft. PMID- 16674830 TI - Plant use of the Maasai of Sekenani Valley, Maasai Mara, Kenya. AB - Traditional plant use is of tremendous importance in many societies, including most rural African communities. This knowledge is however, rapidly dwindling due to changes towards a more Western lifestyle, and the influence of modern tourism.In case of the Sekenani Maasai, the recent change from a nomadic to a more sedentary lifestyle has not, thus far lead to a dramatic loss of traditional plant knowledge, when compared to other Maasai communities. However, in Sekenani, plants are used much less frequently for manufacturing tools, and for veterinary purposes, than in more remote areas. While the knowledge is still present, overgrazing and over-exploitation of plant resources have already led to a decline of the plant material available.This paper examines the plant use of the Maasai in the Sekenani Valley, North of the Masaai Mara National Reserve. The Maasai pastoralists of Kenya and Tanzania use a large part of the plants in their environment for many uses in daily life. The plant use and knowledge of the Sekenani Maasai is of particular interest, as their clan, the "Il-Purko", was moved from Central Kenya to this region by the British Colonial Administration in 1904.The results of this study indicate that despite their relocation 100 years ago, the local population has an extensive knowledge of the plants in their surroundings, and they ascribe uses to a large percentage of the plants found. One-hundred-fifty-five plant species were collected, identified and their Maa names and traditional uses recorded. Although fifty-one species were reported as of "no use", only eighteen of these had no Maasai name. Thirty-three were recognized by a distinctive Maa name. Thirty-nine species had a medicinal use, and 30 species served as fodder for livestock. Six species could not be identified. Of these plants five were addressed by the Maasai with distinct names. This exemplifies the Sekenani Maasai's in-depth knowledge of the plant resources.Traditionally, the Maasai attribute most illnesses to the effect of pollutants that block or inhibit digestion. These pollutants can include "polluted" food, contact with sick people and witchcraft. In most cases the treatment of illness involves herbal purgatives to cleanse the patient. There are also frequent indications of plant use for common problems like wounds, parasites, body aches and burns. PMID- 16674831 TI - Air pollution and lung function among susceptible adult subjects: a panel study. AB - BACKGROUND: Adverse health effects at relatively low levels of ambient air pollution have consistently been reported in the last years. We conducted a time series panel study of subjects with chronic obstructive pulmonary disease (COPD), asthma, and ischemic heart disease (IHD) to evaluate whether daily levels of air pollutants have a measurable impact on the lung function of adult subjects with pre-existing lung or heart diseases. METHODS: Twenty-nine patients with COPD, asthma, or IHD underwent repeated lung function tests by supervised spirometry in two one-month surveys. Daily samples of coarse (PM10-2.5) and fine (PM2.5) particulate matter were collected by means of dichotomous samplers, and the dust was gravimetrically analyzed. The particulate content of selected metals (cadmium, chrome, iron, nickel, lead, platinum, vanadium, and zinc) was determined by atomic absorption spectrometry. Ambient concentrations of nitrogen dioxide (NO2), carbon monoxide (CO), ozone (O3), and sulphur dioxide (SO2) were obtained from the regional air-quality monitoring network. The relationships between concentrations of air pollutants and lung function parameters were analyzed by generalized estimating equations (GEE) for panel data. RESULTS: Decrements in lung function indices (FVC and/or FEV1) associated with increasing concentrations of PM2.5, NO2 and some metals (especially zinc and iron) were observed in COPD cases. Among the asthmatics, NO2 was associated with a decrease in FEV1. No association between average ambient concentrations of any air pollutant and lung function was observed among IHD cases. CONCLUSION: This study suggests that the short-term negative impact of exposure to air pollutants on respiratory volume and flow is limited to individuals with already impaired respiratory function. The fine fraction of ambient PM seems responsible for the observed effects among COPD cases, with zinc and iron having a potential role via oxidative stress. The respiratory function of the relatively young and mild asthmatics included in this study seems to worsen when ambient levels of NO2 increase. PMID- 16674833 TI - Brain SPECT study of common ground between hypothyroidism and depression. AB - Hypothyroidism and major depressive disorder (MDD) share neuropsychiatric features. Cerebral perfusion deficits are found in both disorders. We compared regional cerebral blood flow (rCBF) in hypothyroidism and MDD to determine if clinical similarities are mediated by common neurocircuitry. Ten hypothyroid and 10 depressed patients underwent 99mTc-HMPAO-SPECT and clinical evaluation before and after response to respective treatments. Ten healthy controls underwent a similar, single, evaluation. Before treatment, rCBF in hypothyroid and depressed patients was lower than in controls, in posterior and anterior aspects of the brain respectively. rCBF in hypothyroidism was lower than in MDD in right posterior cingulate and parieto/occipital regions, and higher in frontal, prefrontal and sub-genual regions. Reduced rCBF in pre- and post-central gyri was found in both groups. Following treatment, rCBF in depressed patients increased and normalized, but remained unchanged in hypothyroidism. Affective symptoms in hypothyroidism may be mediated by neurocircuitry different from that of major depression. PMID- 16674832 TI - Xenoestrogenic activity in blood of European and Inuit populations. AB - BACKGROUND: Human exposure to persistent organic pollutants (POPs) is ubiquitous and found in all individuals. Studies have documented endocrine disrupting effects and impact on reproduction. The aim of the present study was to compare the level of xenoestrogenic activity in serum of groups with varying POP exposure, and to evaluate correlations to the POP biomarkers, 2,2',4,4',5,5' hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE). METHODS: The study included 358 men: Greenlandic Inuit's, Swedish fishermen, and Warsaw (Poland) and Kharkiv (Ukraine) inhabitants. Xenoestrogenicity of serum extracts alone (XER) and XER competitive (XERcomp) effect on 17beta-estradiol induced estrogen receptor (ER) transactivity were assessed in the hormone free, lipophilic serum fraction containing the POPs using the MVLN human breast cancer cell line. RESULTS: No agonistic XER activity was exhibited for Inuit serum samples, while 12 - 24% of the European samples had detectable agonistic XER activity. On the contrary, 71% of Inuit serum samples antagonized XERcomp compared to 7 - 30 % in the other regions. XER and XERcomp were not or weakly correlated to the two POP markers. XER activity of Inuit samples was negatively associated to levels of CB-153 and p,p'-DDE. For the Warsaw group a positive and negative correlation between XER and p,p'-DDE and estradiol equivalence level and CB-153 levels was found. CONCLUSION: No strong consistent association between xenoestrogenic net activity and the two POP markers was found. The results showed that the selected POP markers alone can not predict the integrated xenoestrogenic serum activity. Correlations to the POP markers were found at the extreme edge; the Inuit's and Warsaw study groups eliciting high frequency of samples with ER antagonistic and agonistic activity, respectively. We suggest that the variation in xenoestrogenic serum activity reflects differences in POP exposure mixture, genetic factors and/or life style factors. PMID- 16674834 TI - Decreased circulatory levels of neuroactive steroids in behaviourally more extremely affected rats subsequent to exposure to a potentially traumatic experience. AB - This study examined the effects of stress exposure on plasma levels of corticosterone, dehydroepiandrosterone (DHEA) and its sulphate derivative DHEA-S in relation to behavioural responses. The magnitude of anxiety-like behaviours on the elevated plus-maze and of non-habituated exaggerated startle reactions were assessed in rats exposed to stress compared to controls. Individuals displaying extreme behavioural changes were termed extreme behavioural response (EBR), as opposed to minimal behavioural response (MBR) in both paradigms performed consecutively. Significantly increased circulating corticosterone levels and decreased DHEA levels were found 7 d post-exposure only in EBR individuals, not in their MBR counterparts. DHEA-S levels were reduced in both EBR and MBR stress exposed rats compared to controls. This suggests that concomitantly decreased circulatory levels of DHEA and elevated corticosterone levels may be associated with an extreme (pathological) response to stress, whilst maintenance of normal levels of both steroids may be associated with minimal response, denoting resilience. PMID- 16674835 TI - NMR structures of paramagnetic metalloproteins. AB - Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions. PMID- 16674837 TI - Secular trends in twinning rates. AB - Based on national data from civil birth registration systems, this paper provides an overview of trends in the twinning birth rates in fifteen developed countries. Patterns and differentials in such rates across populations are described, and trends over time are given. The evolution of the twinning rate in industrialized countries over the last century follows broadly the same pattern. One exception is the period around World War I, with a peak in the twinning rate being observed during the war, or just after it, although this was not registered in all countries. Since the mid-1970s twinning rates have increased in many developed countries in response to a growing use of fertility-stimulating treatments such as in vitro fertilization. PMID- 16674836 TI - Size of newborn and caesarean section deliveries among teenagers in sub-Saharan Africa: evidence from DHS. AB - This paper uses DHS data from 20 countries in sub-Saharan Africa, collected in the late 1990s and early 2000s, to examine perceived size of newborn and Caesarean section deliveries among teenagers in the region. A comparison between teenagers and older women, based on logistic regression analyses for individual countries, as well as multilevel logistic analyses applied to pooled data across countries, and controlling for the effects of important socioeconomic and demographic factors, shows that in general, births to teenagers are more likely to be small in size but are less likely to be delivered by Caesarean section compared with births among older women. An examination of the country-level variations shows significant differences in perceived size of newborn and Caesarean section deliveries between countries. However, the observed pattern by maternal age does not vary significantly between countries, suggesting that these patterns are generalizable for the region. For teenagers with characteristics associated with higher odds of Caesarean section, being in a country with an overall higher rate particularly amplifies their individual probability. PMID- 16674838 TI - The distribution of consecutive closed birth intervals in females in Uttar Pradesh. AB - Most studies of closed birth intervals are regarding their variation at specific orders among females. This paper attempts to study the nature of the distributions of consecutive closed birth intervals. Data from the Uttar Pradesh National Family Health Survey 1998-99 (NFHS-2) were analysed. It was found that, under certain assumptions, the postpartum amenorrhoea period and menstruating interval are negatively associated, indicating that socio-cultural factors are affecting the menstruating interval. PMID- 16674840 TI - Early PCR detection of tyramine-producing bacteria during cheese production. AB - Biogenic amines (BA) are toxic substances that appear in foods and beverages. Tyramine is the most abundant BA in cheeses. A PCR method was developed to detect the presence of tyramine-producing bacteria during cheese manufacture and ripening. Six different batches of a farmhouse blue cheese were analysed by PCR. Tyramine concentrations were also determined by HPLC. The PCR method was able to anticipate tyramine accumulation in the cheeses; the presence of tyramine producing microorganisms in the early stages of manufacture correlated well with a high concentration of BA in mature cheese samples. PMID- 16674839 TI - The complete set of Toxoplasma gondii ribosomal protein genes contains two conserved promoter elements. AB - Recently we showed that de novo ribosome biosynthesis is transcriptionally regulated in Coccidia, depending on their life-cycle stage. Since the expression of ribosomal protein genes is likely coordinated, the transcriptional control of all Toxoplasma gondii ribosomal protein (RP) genes was analysed. Therefore, the complete set of all cytoplasmic RPs was defined, containing 79 different RPs in T. gondii. RP genes were randomly distributed over the genome, each with a unique upstream region with the exception of 8 RP genes which were paired in a head-to head orientation. To study if the RP genes share conserved promoter elements, a database was made containing upstream sequences of all T. gondii RP genes. Promoter activity was confirmed for the upstream sequences of 8 RP genes, some of which are comparable in strength to the alpha-tubulin promoter. In the complete set of RP upstream sequences 2 novel and highly conserved elements were identified, named Toxoplasma Ribosomal Protein (TRP)-1 (consensus: TCGGCTTATATTCGG) and TRP-2 ([T/C]GCATGC[G/A]). TRP-1 and/or TRP-2 were present in 95% of all RP upstream sequences and moreover, were specifically localized in a small region near the presumptive transcriptional start site (10-330 bp upstream). Although TRP elements were mostly absent in known T. gondii promoters, they are present elsewhere in the T. gondii genome suggesting that they operate not only in RP genes but in a larger set of genes. The identification of TRP elements creates a basis to further study the underlying mechanism by which RP transcription is controlled in T. gondii. PMID- 16674841 TI - Influence of ethanol on the rennet-induced coagulation of milk. AB - The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10-20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of kappa-casein, was progressively slowed with increasing ethanol content (5-20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of kappa-casein-depleted micelles, was enhanced in the presence of 5-15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products. PMID- 16674842 TI - Disruption and reassociation of casein micelles under high pressure. PMID- 16674854 TI - [Administration in health services and quality of educational and research processes]. PMID- 16674855 TI - [The role of social networks in exclusive breastfeeding]. AB - OBJECTIVE: The purpose of the study was to explore the network that the social actors form around the mother with a neonate and the roles they play, either as support or as an obstacle of exclusive breast-feeding. MATERIAL AND METHODS: We applied a social network survey in two stages: in the first one, we asked mothers with babies under six months of age who had helped them breast-feed their babies and who had had a bearing on not letting them do it. Later, we asked other mothers who had told them to give formula, water, tea or solid food to the babies. With the answers we formed two "cases by actors" matrices. We computed the frequencies to find out those people that most influenced the mother for and against exclusive breast-feeding. We also graphed the results from the matrix to visualize the answers. RESULTS: The maternal grandmother of the child, the physician and the paternal grandmother appeared as those who most positively contributed with exclusive breast-feeding. Paradoxically, they are the same who most influenced its interruption. DISCUSSION: We were looking for a social network to support exclusive breast-feeding and another one to hinder it, but the two networks turned out to be very similar. The same characters who help a mother to breast-feed are the ones who make her stop. The implication for an intervention program is that it is necessary to strengthen some knowledge and inhibit some other within the same group of people. PMID- 16674856 TI - [The evolution of teaching education in the health area]. AB - OBJECTIVES: To evaluate the performance through time of the Centro de Investigacion Educativa y Formacion Docente (Educational Research and Teaching Education Center) at the Instituto Mexicano del Seguro Social (Mexican Institute of Social Security), Nuevo Leon Office using as an indicator the ability for critical reading of theoretical texts of education. MATERIAL AND METHODS: A comparative cross-sectional study was made among professors with and without teaching education, as well as among the several generations of professors that took teaching education courses at the Centro de Investigacion Educativa y Formacion Docente. A previously validated instrument was used to measure the ability for critical reading. It was applied to 358 professors, 107 with teaching education at the Centro de Investigacion Educativa y Formacion Docente in Monterrey and 251 without teaching education, at the IMSS Nuevo Leon Office. RESULTS: It was found that the previous teaching education constitutes a "positive risk" with an odds ratio of 17.66 (CI 9.02-34.12). Statistically significant differences were also found between not educated professors (median of 15) and educated professors (median of 35). In the scale, the grades at random and very low predominated in not educated professors, while the educated ones were located in the low and average. CONCLUSION: Professors at the Centro de Investigacion Educativa y Formacion Docente are improving their teaching labor using research as a means of self-criticism. PMID- 16674857 TI - [The meaning of type 2 diabetes mellitus from the patient's perspective]. AB - INTRODUCTION: Medical anthropology considers the sociocultural aspects of illnesses, from the biomedical definition of the experience of the one who suffers the illness. This is what makes the difference between a disease and an illness, in other words, an explanatory model of illness. OBJECTIVES: To show the cultural consensus elaborated and shared by a group of diabetics through personal experience, of the causes, symptoms, treatment and complications of type 2 diabetes mellitus; to highlight the importance this has in the daily medical practice, and to understand the meaning of type 2 diabetes from the patient's perspective. MATERIAL AND METHODS: 88 patients were studied in an interactive process, with an interview outline that was applied in an open form at the waiting rooms for outpatients at two family medicine units of the Mexican Institute of Social Security (IMSS) in Guadalajara, Jalisco. RESULTS: The way in which patients construct a definition and an explanation of their illness is a syncretism of lived experiences done in a procedural manner. In this reconciliation, patients with diabetes integrate biomedical elements, alternative medicines, deities and even sorcery. CONCLUSIONS: These previously mentioned factors should be considered when in contact with the patient if one thinks that the influence of a doctor or institutional health care provider has the objective of achieving a greater effectiveness in the long-term handling of these patients. PMID- 16674858 TI - [Platelet reference values in healthy children living in Mexico City]. AB - INTRODUCTION: There are few studies on normal values of platelet parameters (number of plate-lets, mean platelet volume [MPV], thrombocytocrit, and platelet distribution width [PDW]) in children. The objective of this study was to determine the normal values of these platelet indices in healthy children who live in Mexico City, located 2240 meters above sea level. MATERIAL AND METHODS: 642 healthy children, between 2 and 16 years old, who had normal growth and developmental features and no history of treatment with drugs that alter platelets, were selected. The children fasted for 12 hours before their blood was collected by venipuncture of the arm. Platelet values, determined in an automatic counter (Cell Dyn 3000), were used in the statistical analysis to calculate the mean, standard deviation, and the 3, 50, and 97 percentiles of each data distribution, stratified by age and sex. Correlations between platelet count, MPV, PcT, and PDW, according to age and sex, were evaluated with linear regression statistics. RESULTS: The number of platelets presented a negative correlation with age (r = -0.31, p = 0.001), whereas the mean platelet volume showed a positive correlation (r = 0.29, p = 0.001). The platelet distribution width correlated positively (r = 0.13, p = 0.001) and thrombocytocrit, negatively (r = -0.08, p = 0.05), with no evident variation in relation to age or sex. CONCLUSIONS: The decrease of platelet number with the subsequent increase of mean platelet volume in relation to age possibly resulted from the platelets having become more efficient in carrying out their haemostatic function. The values of platelet indices obtained in this study were similar to those of children living at sea level and, therefore, may be used as reference values in pediatric patients with clinical problems related to these cells. PMID- 16674859 TI - [Six-month's clinical course of patients with unstable angina admitted at the emergency room]. AB - OBJECTIVE: to know the mortality rate after six months in patients with unstable angina. MATERIAL AND METHODS: A representative sample of patients who were admitted at the emergency room with unstable angina diagnosis during a period of two years was selected. The presence of angina pectoris, heart attack and death was registered during their stay at the emergency room. A six-month follow-up took place to evaluate the frequency of angina, heart attack and death related to ischemic acute coronary syndrome, as well as the determination of the relative risk of factors associated to mortality. RESULTS: From the 110 admitted patients, we lost 4; 106 conformed the total of the cohort. There was a mortality rate within the emergency room of 1.8% and of 5.7% in six months. Heart attack was present in 6.6% and angina pectoris in 27.1% during their stay at the emergency room. None of the analyzed risk factors showed any relatively significant risk associated to mortality; the relative risk for age, sex, presence of angina during the stay at the emergency room, acute changes in EKG, history of diabetes mellitus, hypertension, current smoking and ischemic cardiac disease, analyzed with the Mantel-Haenszel test, did not show any significant differences by the Mann-Whitney U among the values of the laboratory markers, arterial tension when admitted, pain intensity, evolution time of diabetes, hypertension, hypercholesterolemia and previous ischemic heart disease. CONCLUSIONS: A low frequency of angina, heart attack and mortality was found during their stay at the emergency room, as well as a low mortality rate in six months; none of the risk factors was significantly associated to death. PMID- 16674860 TI - [Medical schools: opportunities to improve drug prescription]. AB - This paper reviews different strategies to improve medical prescription. Medical schools, responsible for the education and updating of physicians, should strengthen educative strategies, both curricular and extracurricular. They must also support managerial and regulatory strategies in collaboration with institutions that provide health services and governmental health ministries. Some characteristics of rational and irrational medicinal prescription are listed. Some educational strategies are described, such as a decrease in the number of drugs reviewed during pharmacology courses, the inclusion of the concept of essential medicines, the use of sources of information about drugs, and problem-based learning. Some managerial strategies are mentioned, such as a research on medicine use and the production of therapeutic guidelines that require the involvement of health services providers and that might be supported by medical schools. This paper also points out the need for continuing medical education and explains some related experiences that have been successful in improving drug prescription practices in other countries. PMID- 16674861 TI - [Human papilloma virus and its association with oral cancer]. AB - Oral cancer it a pathology of multifactorial etiology, where some factors such as age, sex, race, genetic predisposition, nutrition, and the use of tobacco and alcohol have a bearing on. In the last years, some authors showed the implication of the human papilloma virus (HPV) in the development of precarcinogenic lesions and of oral squamous cell carcinoma. The infection by HPV has been associated to hyperplastic epithelial lesions, papilloma and warty carcinoma in skin and in different types of mucosa, including the anus-genital, cervical, urethral, tracheobronchial, nasal, laryngeal and oral mucosa tracts. The viral high-risk geno-types (oncogenic) such as 16, 18, 31, 33 and 35 are frequently associated to leukoplakia and squamous carcinoma. An association of HPV with oral squamous carcinoma in patients that consume tobacco and alcohol has been fundamentally established. It is important to study and to frequently review the role that viral infections and cancer have, and maybe in the future, it would be possible to create a vaccine that diminishes the frequency of oncological problems. PMID- 16674862 TI - [Acromial osteophyte in shoulder impingement syndrome. Diagnosis and prevalence]. AB - INTRODUCTION: The shoulder impingement syndrome (SIS) is caused by the compression of the rotator cuff tendon tear and the subacromial bursa between the humeral head and the structures that conforms the subacromial arc. OBJECTIVE: To determine a proportion of the acromial spur in the SIS comparing it with the general population. A second one would be to evaluate the quality of the radiological technique. MATERIAL AND METHODS: A comparative cross-sectional study was made in 148 patients, divided in two groups: one conformed by 52 persons, for patients with SIS and the other, conformed by 96, for the general population of similar age. The technique used to detect the acromial spur was the anteroposterior shoulder plate with a ray at 30 degrees in caudal direction in a bilateral way that was blindly interpreted by an orthopedist in two occasions, obtaining a high concordance index of Kappa of 0.90. The criteria to consider positive spur was that the bone growth could exceed a prolongation of an imaginary line at the inferior edge of the clavicle. RESULTS: The average age in both groups was of 48.7 years +/- 14.5 years for the patients with SIS, and of 43.2 years +/- 12.9 for the patients of the control group. It was founded that 84.6% of the patients with SIS had acromial spur and in the ones of the control group, it was founded in a 36%, being this difference significant with OR of 9.3 times more frequent to have spur in patients with SIS. The quality of the technique was good in 92% of the Rx. CONCLUSION: The frequency of spur in SIS is high (84.6%), but it calls our attention that the frequency in general population was moderate (36%). PMID- 16674863 TI - [Transoperative tamponade due to perforation with a catheter for central blood pressure: report and case analysis]. AB - Pericardial tamponade due to myocardial perforation from central venous catheter is rather unusual, especially if it happens during a trans-operative period. When it comes up, it has a high mortality risk if the pericardial fluid is not drained at the proper time. At the Oncology Hospital of the National Medical Center, IMSS, Mexico City (Hospital de Oncologia del Centro Medico Nacional, Siglo XXI), at the Anesthesia Service, a case of tamponade in transoperative period came up, after a surgical procedure that lasted more than 6 hours, and that manifested itself with signs of cardiogenic shock that did not improve with treatment. The etiology was not diagnosed until a postmortem study. If it had been diagnosed on time, its clinical evolution would have been different with an opportune and correct treatment. PMID- 16674865 TI - [Biochemical factors associated with cardiovascular risk...]. PMID- 16674866 TI - [Teaching evaluation at Medical School, UNAM]. AB - The purpose of this article is to offer a synthesis of what has been done in the Teaching Evaluation Program at the Medical School of the Autonomous National University of Mexico (UNAM). The Program involves three questionnaires of the students' opinion that evaluate professors of the basic and sociomedical areas, microbiology and parasitology laboratory and surgery. Between 1994 and 2003, 134,811 questionnaires were answered to evaluate the teaching performance of 6262 professors of pregraduate students. Although the evaluation of teaching through a single way is insufficient, the results obtained allow us to affirm that the Medical School at UNAM has a good professor staff, as well as they are useful for the design of programs dedicated to the acknowledgment of excellence and the needs for teaching education. PMID- 16674867 TI - [Intervention of the family physician in alcoholism: a review of operative strategies]. AB - The actions of secondary prevention of alcohol abuse and alcoholism in the primary medical attention confront barriers that difficult their attention, beginning with those of the patient him or herself, those of the family physician and the multidisciplinary staff, and finally, those corresponding to the system of attention itself. The objective of this study is to show a review of international literature and experiences in Mexico, which present secondary prevention strategies that facilitate the family physician his or her performance in the containment of alcoholism through early detection and brief intervention. In this paper, we present an operative model that the World Health Organization made in 2001 as an advice of its experts on the problem, combining early detection through the application of the "Identification test of disorders due to alcohol addiction, abuse and/or dependence" (AUDIT) and based on the score obtained, it applies an intervention strategy that goes from the simple piece of advice to the brief therapy and if it is necessary, the derivation. PMID- 16674870 TI - [Clostridium difficile, an old acquaintance in new clothes]. PMID- 16674871 TI - [Clostridium difficile-associated diarrhoea--a changing disease?]. AB - In recent years an increase in the number of Clostridium difficile-associated diarrhoea cases has been reported worldwide. This is due mainly to the increasing number of elderly patients being admitted to hospitals, changes in hygiene standards and changes in the antibiotics used to combat other diseases. But changes in the virulence of C. difficile strains also seem to play a role. This review presents the current knowledge of C. difficile-associated diarrhoea in the light of these changes in incidence and virulence. PMID- 16674872 TI - [Antibiotic treatment of Clostridium difficile associated diarrhea in adults. A survey of a Cochrane review]. PMID- 16674873 TI - [Pseudomembraneous colitis caused by a toxin B-positive and a toxin A-negative strain of Clostridium difficile]. AB - We describe a case of pseudomembraneous colitis (PMC) caused by a toxin A- B+ strain of Clostridium difficile (CD). In Denmark only a few laboratories investigate for toxin production, and if they do, the toxin A enzyme immunoassay (EIA) is the test generally used when testing for CD. This toxin A negative but toxin B positive strain thus remains undetectable. If CD-associated diarrhea is clinically suspected and tests for toxin A are negative, infection with a toxin A B+ strain should be considered. Further diagnostic tests such as cellular cytotoxicity assays or toxin A/B EIA should be performed. The standard treatment of PMC in severely ill patients should be vancomycin, administered orally. PMID- 16674874 TI - [Reducing the use of addictive drugs in clinical practice]. AB - INTRODUCTION: The use of benzodiazepines (BDs) and cyclopyrrolones (CPs) has drawn a great deal of political attention over the past years. There are estimated to be approximately 100,000 BD addicts in Denmark. This article describes a simple but effective method of reducing the use of addictive drugs in clinical practice. MATERIALS AND METHODS: Two solo clinics in Thyboron-Harboore Community decided to work strictly according to directive CIR no. 12 of 13 January 2003. All BDs and CPs were prescribed for one month at a time and could be renewed only by the doctor after a personal consultation. This monthly requirement forced the doctor as well as the patient to evaluate whether the existing prescription pattern or a drug-reducing regime was indicated. The prescription pattern was monitored using Ordiprax, which showed the amount of prescription medicines sold by pharmacies. RESULTS: After 15 months, the patients" use of BD was reduced by 50% and their use of CP by 75%. The process of changing prescription habits was far easier than expected. An entire group of patients, previously invisible to the doctors, was exposed. During the first three months, only four to five additional consultations for every 1,000 assigned patients were required each week. There was practically no need of any assistance from our usual partners, such as psychiatrists, hospitals, special wards for addictive treatment or primary health care. CONCLUSION: We strongly recommend that this simple procedure be made a daily routine when prescribing either a BD or a CP. PMID- 16674875 TI - [Implementation of clinical guidelines in general practice. The effect of journal audit and continuing education for the treatment of cardiovascular risk factors in patients with and without type 2 diabetes]. AB - INTRODUCTION: The objectives of this study were, first, to describe the status of implementation of clinical guidelines for prevention of ischaemic heart disease in subjects with arterial hypertension, dyslipidemia and type 2 diabetes, but without known cardiovascular disease, and, second, to describe the changes in treatment following journal audit and education of GPs. MATERIAL AND METHODS: 230 general practitioners (GPs) were invited to include 20 consecutive at-risk patients. One year after the first data collection of 3,555 patients by 182 GPs, a new set of 3,023 patients was collected by 170 GPs from a new patient population. In the period between the two surveys, the GPs were educated through workshops and an international meeting focusing on cardiovascular risk factor treatment. RESULTS: The quality of the care being delivered was suboptimal. Approximately 15-40% of the patients had obtained the treatment goals for hypertension, dyslipidemia and type 2 diabetes. Some improvement was demonstrated following education and journal audit, but a gap still exists between the recommended treatment and the treatment patients actually receive. CONCLUSION: There is a need for improvement in the implementation of clinical guidelines focusing on diabetes and cardiovascular disease. PMID- 16674876 TI - [Self-assessed health status among young people from ethnic minorities living in Denmark]. AB - INTRODUCTION: Only a few studies on the health status of ethnic minorities in Denmark have been carried out. These studies have shown a higher mental and physical morbidity rate and an increased prevalence of social problems among adults. The purpose of this study was to compare the self-reported health status of ethnic Danish youths and young first- and second-generation immigrants from non-Western countries to establish whether there is an association between ethnicity and "belief in the ability to affect one's own health". MATERIALS AND METHODS: We surveyed a cohort of 6,203 ninth-grade students (aged 15 to 16 years), including 264 first-generation and 391 second-generation immigrants from non-Western countries. Self-reported health and self-efficacy, defined as the belief in the ability to affect one's own health, were included in the analyses, using SPSS cross-tabulations and logistical regression analysis. RESULTS: A direct association between ethnicity and self-reported health status was found. Both first- and second-generation immigrants rated their health worse than did ethnic Danish youths. The association was statistically significant for first generation immigrant girls when controlling for relevant risk factors for ill health. Significant associations between self-reported health status and belief in the ability to affect one's own health were found for both first- and second generation immigrants of both sexes, compared to ethnic Danish youths. CONCLUSION: Factors related to immigration and immigrants' social conditions may partially explain the ethnic differences in self-reported health status. However, cultural differences may influence the ethnic and gender differences in health status and self-efficacy, as ethnic minorities are less likely to believe in their ability to affect their own health. It is recommended that this knowledge be implemented when developing health promotion programs in the public schools. PMID- 16674877 TI - [Suicide and suicidal behavior among asylum seekers in Denmark during the period 2001-2003. A retrospective study]. AB - INTRODUCTION: Our aim was to examine suicidal behaviours among asylum seekers in Denmark. MATERIALS AND METHODS: A retrospective quantitative analysis of data from reports to the Danish Red Cross Asylum Department on suicidal behaviours among persons over 15 years of age in the period 2001-2003 and from 54 medical records of suicidal asylum seekers in Denmark in 2001 was carried out. RESULTS: The number of suicide attempts by asylum seekers in 2001 was 3.4 times higher than by Danish residents. Furthermore, the rate of suicide attempts by asylum seekers grew in the following two years. Suicidal behaviours are most frequent among asylum seekers between 30 and 39 years of age. There are national differences. The preferred method of suicide attempt is intake of medicine. Stress-related diagnoses constitute three fourths of all diagnoses. One analysis suggests that the long waiting time (average 20.8 months) faced by asylum seekers combined with rejection of asylum cases may trigger a rapid suicidal reaction. Other factors may also be active, as 44% of suicide attempts occur within six months after arrival in the country. DISCUSSION: The results are discussed in relation to other research on the vulnerability of refugees, immigrants and asylum seekers and also in relation to length of waiting time, growing mental morbidity and the increasing number of rejections of asylum applications during recent years, a period characterized by a reduction of staff at the asylum centers. It is recommended that prevention of suicidal behaviour shall be given higher legal and administrative priority. PMID- 16674878 TI - [High levels of industrially produced trans fatty acids in popular fast food - but not in Denmark--secondary publication]. AB - An intake of trans-fatty acids of 5 grams per day is associated with an increase of 25% in the risk of ischemic heart disease. In 2004 Denmark, as the first country in the world, introduced a limitation on the content of industrially produced trans-fatty acids in foods. The amount in a "high-trans menu" consisting of popular foods was, from 2001 to 2005, reduced in Denmark from 30 g to <1 g. The amount in the same menu bought in countries within and outside the European Union is 20-40 g. During a period of just a few years, Denmark has thus eliminated a risk factor for ischemic heart disease without noticeable side effects for consumers. This risk factor is, however, still present in many other countries. PMID- 16674879 TI - [Are invitations to mammography screening a reasonable basis for informed consent?-- Secondary publication]. AB - There is a delicate balance between the benefits and harms of mammography screening, and women should decide for themselves, on an informed basis. The invitation to have a mammography is the only source of information provided to all eligible women, and we explored whether it provides such a basis. We collected invitations from English-speaking and Scandinavian countries. The major benefit of screening, a reduction in breast cancer mortality, was mentioned in 30 of 31 invitations, whereas the most important harms, overdiagnosis and overtreatment, were not mentioned in any of the invitations. PMID- 16674880 TI - [Blood sugar regulation in critically ill patients]. PMID- 16674881 TI - [Picture of the month: buried bumper syndrome]. PMID- 16674887 TI - [Treatment of hypercholesterolemia--lower is not better]. PMID- 16674888 TI - [Try olive oil]. PMID- 16674890 TI - [Ergoline-induced retroperitoneal fibrosis]. PMID- 16674891 TI - [Back pain, radiology and end plate changes by means of Modic]. PMID- 16674892 TI - [Endovascular treatment of diseases of the descending thoracic aorta]. PMID- 16674895 TI - [Is Antabus therapy connected with reduced efficacy of nitroglycerin?]. PMID- 16674909 TI - Validation of the drop coating deposition Raman method for protein analysis. AB - Drop coating deposition Raman (DCDR) spectroscopy is critically evaluated to establish the limits to which it may be used to detect changes in protein conformation, binding, and purity. Difference spectroscopy is used to evaluate the reproducibility of the DCDR spectra under various experimental conditions. The results indicate (i) the absence of thermal/photochemical laser damage induced by the Raman excitation laser under typical DCDR data collection conditions, (ii) the reproducibility of DCDR spectra from samples with different volumes or concentrations, (iii) the water content of DCDR protein deposits and associated spectral signatures, and (iv) the degree of similarity between solution Raman spectra and DCDR spectra. PMID- 16674910 TI - A coupled spectrophotometric assay for l-cysteine:1-D-myo-inosityl 2-amino-2 deoxy-alpha-D-glucopyranoside ligase and its application for inhibitor screening. AB - Most actinomycetes, including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PP(i). MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2,024 compounds predicted to inhibit ATP dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC(50) approximately 100 microM. PMID- 16674911 TI - Masticatory muscle pain and disordered jaw motor behaviour: Literature review over the past decade. AB - The clinically important relationship between masticatory muscle pain (MMP) and disordered jaw motor behaviour is subject of this concise, evidence-based review of the literature that was published during the past 10 years. Mainly based on studies that used some sort of experimental MMP (e.g., the intramuscular injection of noxious substances like hypertonic saline), it was concluded that MMP has pronounced effects on jaw motor functions like maximal clenching and mastication. The pain-related modulation of oral reflexes further illustrated the effects of MMP on masticatory motor control. Protecting the painful muscle tissues against further damage and allowing for time to heal the damaged tissues by immobilization of the masticatory system seem to be the key outcomes of these effects. Further, MMP was shown to influence the cervical motor system, which may partly explain the mechanism behind the frequently observed co-occurrence of pain in the neck and the jaw. Finally, it was concluded that, even though the evidence is not yet conclusive, also remote pain (non-MMP) can modulate jaw motor behaviour, which indicates the involvement of central mechanisms in this modulation. PMID- 16674912 TI - Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position. AB - To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (> or =0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity. PMID- 16674913 TI - Sphingosine-1-phosphate receptor agonists suppress concanavalin A-induced hepatic injury in mice. AB - T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p<0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p<0.05) and successfully reduced serum transaminase elevation (p=0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury. PMID- 16674914 TI - Localization of the flagellum-specific secretion signal in Salmonella flagellin. AB - The flagellum-specific export system is a specialized type III export machinery. Terminally truncated fragments of flagellin (FliC) were used to identify the secretion signal in the main component of flagellar filaments. The first 13 residues were not essential for export, but removal of 29 or more residues destroyed export ability. When an 8kDa human protein domain was fused to various N-terminal fragments of FliC, the 26-47 sequence alone was sufficient to mediate secretion of this protein module through the flagellum specific export pathway. Neither half of this segment was enough to direct export of the attached protein domain. Our results demonstrate that the 22-residue long 26-47 segment within the disordered N-terminal region of Salmonella flagellin contains the recognition signal for the flagellar export machinery. PMID- 16674915 TI - Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel. AB - To examine conformational changes during slow inactivation involving domain 2 segment 6 (D2-S6) of human cardiac Na(+) channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na(+) currents were recorded using patch clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5 mM) applied in the closed state (-160 mV) has no significant effect on whole-cell Na(+) current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0 mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA. PMID- 16674916 TI - Ion dependence of ligand binding to metabotropic glutamate receptors. AB - The ionic requirements for ligand binding to metabotropic glutamate receptors were carried out on secreted truncated receptors containing only the extracellular ligand binding domains of the receptors. The influence of ions on agonist binding was examined in mGluR1, mGluR3, and mGluR4 representing Group I, II, and III metabotropic glutamate receptors, respectively. [(3)H]Quisqualic acid binding to mGluR1 required the presence of calcium (or magnesium) ions but not sodium or chloride ions while [(3)H]DCG-IV binding to mGluR3 was dependent upon both cations and anions. [(3)H]L-AP4 binding to mGluR4 required chloride ions but not monovalent or divalent cations. The EC(50) for chloride facilitation of L-AP4 binding to mGluR4 was 63mM; this value is approximately one-half of the normal resting extracellular chloride concentration. These results demonstrate that metabotropic glutamate receptor subtypes require different complements of ions for ligand binding and suggest that natural physiological fluctuations in synaptic ion concentrations may regulate receptor binding and activation. PMID- 16674917 TI - Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation. AB - Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P(2), S1P(3), S1P(4), but not S1P(1). When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P(1)- and S1P(4)-selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G(i) protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process. PMID- 16674918 TI - Influence of dendrimer's structure on its activity against amyloid fibril formation. AB - Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer's diseases. Highly branched, globular polymers-dendrimers-are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185-208 and the Alzheimer's peptide Abeta 1-28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer peptide interaction mechanism is presented based on the dendrimers' molecular structure. PMID- 16674919 TI - Proteasome inhibitor MG-132 enhances whole-body protein turnover in rat. AB - Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators. PMID- 16674920 TI - The human tumour suppressor LATS1 is activated by human MOB1 at the membrane. AB - Downregulation of the LATS1 tumour suppressor protein kinase contributes to tumour formation in mammals and flies. Strikingly, the tumour suppressor activity depends on the interaction with Dmob (Drosphila Mps1-One binder) in Drosophila melanogaster. Recently, human LATS1 was reported to interact with human MOB1 (hMOB1), but the activation of LATS1 was not addressed. Here, we identified a highly conserved hMOB1-binding motif within LATS1's primary structure. While co expression of LATS1 with hMOB1 did not elevate LATS1 kinase activity in mammalian cells, membrane-targeting of hMOB1 resulted in a significant increase of LATS1 activity. This stimulation was dependent on intact activation segment and hydrophobic motif phosphorylation sites, and was further found to occur a few minutes after membrane association. Therefore, we suggest a potential in vivo mechanism of LATS1 activation through rapid recruitment to the plasma membrane by hMOB1 followed by multi-site phosphorylation, thereby providing insight into the molecular regulation of the LATS tumour suppressor. PMID- 16674921 TI - RUNX1 suppression induces megakaryocytic differentiation of UT-7/GM cells. AB - The transcription factor RUNX1 plays a crucial role in hematopoiesis. RUNX1 regulates both differentiation and proliferation of hematopoietic cells. Several reports have shown that RUNX1 participates in megakaryopoiesis, which is a process that leads to formation of platelets. However, to date, the mechanisms by which this occurs have not been fully elucidated. In the present study, we investigated whether siRNA-mediated depletion of RUNX1 affected megakaryopoiesis of UT-7/GM cells. The depletion of RUNX1 in UT-7/GM cells resulted in up regulation of the expression of megakaryocytic markers and polyploidization, while cell proliferation was down-regulated. Furthermore, the overexpression of RUNX1 decreased the activity of megakaryocytic gene promoters. These results suggest that RUNX1 down-regulates terminal differentiation of megakaryocytes and promotes proliferation of megakaryocytic progenitors. PMID- 16674922 TI - Zinc-induced aggregation of Abeta (10-21) potentiates its action on voltage-gated potassium channel. AB - Zinc may play an important role in the pathogenesis of Alzheimer's disease (AD) through influencing the conformation and neurotoxicity of amyloid beta-proteins (Abeta). Zn(2+) induces rapid aggregation of synthetic or endogenous Abeta in a pH-dependent fashion. Here we show for the first time that Zn(2+)-induced aggregation of Abeta (10-21) potentiates its action on outward potassium currents in hippocampal CA1 pyramidal neurons. Using the whole-cell voltage-clamp technique, we showed that Abeta (10-21) blocked the fast-inactivating outward potassium current (I(A)) in a concentration- and aggregation-dependent manner, but with no effect on the delayed rectifier potassium current (I(K)). Both the unaggregated and aggregated forms of Abeta (10-21) significantly shifted the activation curve and the inactivation curve of I(A) to more negative potentials. But the aggregated form has more effects than the unaggregated form. These data indicated that aggregation of amyloid fragments by zinc ions is required in order to obtain full modulatory effects on potassium channel currents. PMID- 16674923 TI - A novel single step double positive double negative selection strategy for beta globin gene replacement. AB - beta-Thalassemias are a heterogeneous group of autosomal recessive disorders, characterized by reduced or absence of the beta-globin chain production by the affected alleles. Transplantation of genetically corrected autologous hematopoietic stem cell (HSC) is an attractive approach for treatment of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy due to its ability to target the mutant genes and restore their normal expression. In the present study, a specific gene construct for beta globin gene replacement was constructed consisting of: two homologous stems including, upstream and downstream regions of beta-globin gene, beta-globin gene lying between hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase expression cassettes at both termini as negative selection marker. All segments were subcloned into pBGGT vector. The final plasmid was checked by sequencing and named as pFBGGT. Mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing to confirm the occurrence of homologous recombination. In this novel strategy gene replacement was achieved in one step and by a single construct. PMID- 16674924 TI - Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A. AB - HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A(2) (PLA(2))/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca(2+)-mobilization and enhanced bradykinin-promoted Ca(2+)-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARgamma agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs. PMID- 16674925 TI - Effects of capsaicin on P-gp function and expression in Caco-2 cells. AB - Capsaicin is the pungent component of hot chilli, a popular spice in many populations. The aim of the present study was to evaluate the chronicity and reversibility of the modulating effect of capsaicin on both the P-gp expression and activity in the Caco-2 cell monolayers. Capsaicin at concentrations ranging from 10 to 100 microM, which were found to be non-cytotoxic towards the Caco-2 cells, were observed to inhibit P-gp mediated efflux transport of [3H]-digoxin in the cells. The acute inhibitory effect was dependent on the capsaicin concentration and duration of exposure, with abolishment of polarity of [3H] digoxin transport attained at 50 microM of capsaicin. In contrast, longer term (48 and 72 h) co-incubation of the Caco-2 cells with capsaicin (50 and 100 microM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by higher degree of [3H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. The induction of P-gp protein and mRNA levels was also influenced by capsaicin concentration and duration of exposure, with higher expression levels, in particular of the mRNA, seen at higher spice concentrations over prolonged period of incubation. Our data suggest that caution should be exercised when capsaicin is to be consumed with drugs that are P-gp substrates. In particular, the oral bioavailability of these drugs may be influenced by the P-gp status of populations that rely heavily on hot chilli in their diets. PMID- 16674926 TI - Acute and repeated cocaine induces alterations in FosB/DeltaFosB expression in the paraventricular nucleus of the hypothalamus. AB - Apart from activation of the brain reward system, cocaine administration influences the activity of the hypothalamo-pituitary-adrenal (HPA) axis by affecting CRH neurons in the paraventricular nucleus of the hypothalamus (PVN). In order to find a molecular mechanism of cocaine-evoked effects in the PVN, in the present study, we investigated the impact of cocaine on the expression of FosB/DeltaFosB transcription factors in the PVN. Using an immunohistochemical method, we found that acute cocaine treatment (25 mg/kg) induced a relatively long-lasting (at least 72 h) expression of FosB/DeltaFosB in the PVN, whereas repeated cocaine administration (25 mg/kg, once daily for 5 consecutive days) caused accumulation of FosB/DeltaFosB in the PVN. The latter observation was further confirmed by the Western blot technique which revealed that repeated exposure to cocaine specifically increased the expression of a stable isoform of DeltaFosB (35 kDa). Using a double-labeling immunofluorescent method, it was established that FosB/DeltaFosB proteins induced by repeated cocaine treatment were present in a small population of CRF-immunoreactive neurons of the PVN. Furthermore, it was found that pretreatment with the specific antagonist of dopamine D1-like receptors SCH 23390 (1 mg/kg) attenuated the expression and accumulation of FosB/DeltaFosB in the PVN, evoked by repeated cocaine administration. Although functional consequences of the above effects for the process of addiction remain to be established, the obtained results indicate that cocaine administration can produce relatively long-lasting changes in the expression of FosB/DeltaFosB transcription factors in PVN neurons (in some populations of CRF-immunoreactive neurons, among others) and that dopamine D1 like receptors are involved in the above effects. Finally, it is proposed that the long-lasting expression as well as the accumulation of DeltaFosB in the PVN may constitute a molecular basis underlying adaptive changes occurring in the HPA axis after relatively high doses of cocaine. PMID- 16674927 TI - Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis. AB - Our previous studies and the others have strongly suggested that JNK signaling pathway plays a critical role in ischemic brain injury. Here, we reported that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, SP600125 diminished the increased phosphorylation of c-Jun and the increased expression of FasL induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 and the release of Bax from Bcl-2/Bax dimers, SP600125 attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion (I/R). Furthermore, the activation of caspase-3 induced by ischemia/reperfusion was also significantly suppressed by preinfusion of SP600125. Importantly, the same neuropotective effect was showed by administration of SP600125 both before and after ischemia. Thus, our findings imply that SP600125 can inhibit the activation of JNK signaling pathway and induce neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via suppressing the extrinsic and intrinsic pathways of apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury. PMID- 16674928 TI - Increase in synaptic hippocampal zinc concentration following chronic but not acute zinc treatment in rats. AB - Electroconvulsive seizures (ECS), one of the most effective treatments of depression, induce mossy fiber sprouting (when assayed by means of synaptic zinc method), and this indicates an increase in the synaptic zinc level in the hippocampus following such therapy. The aim of the present study was to investigate the influence of acute and chronic zinc hydroaspartate administration on the synaptic and total zinc level in the rat hippocampus. We used two methods of zinc determination: (1) zinc-selenium method, which images the pool of synaptic zinc, and (2) flame atomic absorption spectrometry, which assays the total concentration of zinc. Our results indicate that chronic (14 x 65 mg/kg), but not acute, zinc hydroaspartate administration intraperitoneally (i.p.) increases the pool of synaptic zinc in the majority of rat hippocampal layers (by 72-190%), except for the stratum moleculare and stratum radiatum CA, and perforant path DG. On the other hand, no changes were found in total hippocampal zinc level, measured by flame atomic absorption spectrometry. These data suggest that chronic zinc treatment increases the pool of synaptic zinc in the hippocampus, and this effect is similar to that observed following chronic ECS treatment. The measurement of zinc concentration in the whole hippocampus by the flame atomic absorption spectrometry method is not sensitive enough to detect such subtle alteration. PMID- 16674929 TI - A new rat model of acute seizures induced by tutin. AB - Coriaria Lactone (CL) is a mixture that has been used to establish animal models of epilepsy. In this study, we focused on the epileptogenic action of tutin, a pure chemical component derived from CL. Rats were implanted with a guide cannula for microinjection of tutin into either of the lateral cerebral ventricles. Behavioral and electroencephalographic (EEG) changes were investigated for at least 2 h after tutin administration. Injected animals presented behavioral seizures: initially, facial and limbic clonus, and subsequently, tonic-clonic seizures that eventually progressed to status epilepticus. Accompanying the behavioral activities, a variety of EEG patterns were recorded. Spike-and-wave complexes occurred continuously at 3 Hz, with a mean amplitude of approximately 295 microV. Multiple spikes and slow waves occurred repetitively and became more frequent and intense. The amplitude of this EEG pattern was low (approximately 85 microV) at onset and gradually increased to approximately 200 microV. Spikes (8 Hz, approximately 555 microV) and slow waves (3 Hz, approximately 670 microV) occurred periodically at the onset of grand mal seizures. Behavioral and EEG changes induced in rats by tutin demonstrated that this is a potent convulsant, by which a new animal model of status epilepticus was established. This acute seizure model is productive and would be optional for investigation of seizures or status epilepticus. PMID- 16674930 TI - High fat/refined carbohydrate diet enhances the susceptibility to spatial learning deficits in rats exposed to intermittent hypoxia. AB - BACKGROUND: Intermittent hypoxia during sleep (IH), as occurs in sleep disordered breathing (SDB), induces spatial learning deficits associated with regulation of transcription factors associated with learning and memory in the hippocampal CA1 region in rats. high fat refined carbohydrate diet (HF/RC) can induce similar deficits and associated changes in signaling pathways under normoxic conditions. METHODS: Sprague-Dawley adult male rats were fed either with (HF/RC) or low fat/complex carbohydrate diet (LF/CC) starting at post-natal day 30 for 90 days, and were then exposed for 14 days during light phase (12 h/day) to either normoxia (RA) or IH (21% and 10% O2 alternations every 90 s). Place-training reference memory task deficits were assessed in the Morris water maze. Total and ser-133 phosphorylated CREB were assessed in different brain regions by Western blotting and immunostaining in rats exposed to normoxia or IH and to LF/CC or HF/RC. RESULTS: Substantial decreases in CREB phosphorylation occurred in CA1 but not in motor cortex following either IH, HF/RC, and HF/RC + IH. Place-training reference memory task deficits were observed in rats exposed to IH and to HF/RC, and to a much greater extent in rats exposed to HF/RC + IH. CONCLUSIONS: Nutritional factors alter recruitment of transcription factors, possibly via oxidative-related pathways, and modulate the vulnerability of the CA1 region of the hippocampus to the episodic hypoxia that characterizes SDB, thereby enhancing neurocognitive susceptibility in SDB patients. PMID- 16674931 TI - Maternal licking influences dendritic development of motoneurons in a sexually dimorphic neuromuscular system. AB - Maternal licking of pups' perineal regions affects the development of the spinal nucleus of the bulbocavernosus (SNB), a sexually dimorphic motor nucleus in the lumbar spinal cord that controls penile reflexes involved with copulation. Maternal licking influences SNB motoneuron number, with reductions in licking resulting in fewer motoneurons. Reduced maternal licking also has functional consequences in adulthood, resulting in increased latency to ejaculation and postejaculatory intromission and longer interintromission intervals. In this experiment, we assessed the potential effect of maternal licking on the development of SNB dendritic morphology. To reduce maternal licking, dams were treated with intranasal application of zinc sulfate during the first two postnatal weeks, which interferes with their ability to detect the pup odors that drive the licking behavior. At either postnatal day (P) 28 (when SNB dendritic length is normally maximal) or P49 (when SNB dendritic morphology is normally mature), SNB motoneurons were retrogradely labeled with cholera toxin-conjugated HRP, and dendritic arbor was reconstructed in three dimensions. At P28, the dendritic arbor of reduced maternal licking pups was not different from controls; however at P49, reduced licking pups showed a 23% reduction in dendritic arbor in the SNB, an effect that was especially pronounced in the rostral end of the nucleus, where reductions reached 48%. These results suggest that reductions in perineal stimulation provided by maternal licking could affect adult male copulatory behavior via alterations in SNB motoneuron morphology, and thus support maternal licking as an important factor in normal neural and behavioral development. PMID- 16674932 TI - Serotonin transporter promoter variants: Analysis in Indian autistic and control population. AB - Serotonin transporter (5-HTT) is a transmembrane protein belonging to Na+/Cl- dependent membrane transporter family and transports 5-HT across the membranes of presynaptic neurons. 5-HTT-linked polymorphic region (5-HTTLPR) gained much interest because of the differential regulation of expression and activity of 5 HTT by its various genotypes. A population-based study has been conducted on 5 HTTLPR with 358 individuals, which included 79 autistic probands, 136 parents, and 143 controls from two subpopulations of east and northeast regions of India. The genotypic frequencies of all the groups conform to Hardy-Weinberg equilibrium. With the finding of efficacy of serotonin reuptake inhibitors in ameliorating ritualistic behavior in autistic disorder, 5-HTT emerged as a putative candidate gene for autism and association studies have been carried out in different ethnic populations. But these studies were inconclusive due to conflicting results on association. Because such a study has never been performed in the Indian population, we have tested the possible involvement of 5-HTTLPR polymorphism with autism. The present study failed to establish any association or linkage of 5-HTTLPR with autism in the Indian population by case-control studies (chi2 = 1.314, P = 0.63) and family-based approaches (TDT chi2 = 0.22, P = 0.64 and HHRR-chi2 = 0.25, P = 0.61). However, when a meta-analysis of all the available TDT data, inclusive of the present study is carried out, we observed a significant preferential transmission of S-allele from parents to the affected offspring (chi2 = 7.51, P = 0.006) indicating an association of 5-HTTLPR with autism. PMID- 16674933 TI - High-voltage-activated Ca2+ currents show similar patterns of expression in stellate and pyramidal cells from rat entorhinal cortex layer II. AB - High-voltage-activated (HVA) Ca2+ currents were studied in acutely isolated neurons from rat entorhinal cortex (EC) layer II. Stellate and pyramidal cells, the two main neuronal types of this structure, were visually identified based on morphological criteria. HVA currents were recorded by applying the whole-cell, patch-clamp technique, using 5-mM Ba2+ as the charge carrier. In both neuronal types, the amplitude of total HVA Ba2+ currents (IBas) showed a significant tendency to increase with postnatal age in the time window considered [postnatal day 15 (P15) to P28-29]. At P20-P29, when IBa expression reached stable levels, IBa density per unit of membrane area was not different in stellate versus pyramidal cells. The same was also observed when Ca2+, instead of Ba2+, was used as the charge carrier. The pharmacological current subtypes composing total HVA currents were characterized using selective blockers. Again, no significant differences were found between stellate and pyramidal cells with respect to the total-current fractions attributable to specific pharmacological Ca2+ channel subtypes. In both cell types, approximately 52-55% of total IBas was abolished by the L-type channel blocker, nifedipine (10 microM), approximately 23-30% by the N type channel blocker, omega-conotoxin GVIA (1 microM), approximately 22-24% by the P/Q-type channel blocker, omega-agatoxin IVA (100 nM), and approximately 11 13% remained unblocked (R-type current) after simultaneous application of L-, N-, and P/Q-type channel blockers. The Cav 2.3 (alpha1E) channel blocker, SNX-482 (100 nM), abolished approximately 57-62% of total R-type current. We conclude that HVA Ca2+ currents are expressed according to similar patterns in the somata and proximal dendrites of stellate and pyramidal cells of rat EC layer II. PMID- 16674934 TI - Lysosomal storage diseases in non-immune hydrops fetalis pregnancies. AB - BACKGROUND: At least 20 inborn errors of metabolism may cause hydrops fetalis. Most of these are lysosomal storage diseases. The study proposes a diagnostic flowchart for prenatal diagnosis of non-immune hydrops fetalis. METHODS: This study contains a series of 75 non-immune hydrops fetalis pregnancies. Mucopolysaccharides, oligosaccharides, neuraminic acid and 21 lysosomal enzymes were measured in amniotic fluid and cultured amniotic cells. RESULTS: The study gives reference values for mucopolysaccharides and neuraminic acid at various stages of gestation. Four definite and two probable lysosomal diagnoses were found among the 75 investigated cases (=5.3-8%). Fetal death was found to cause false positive values for mucopolysaccharides in amniotic fluid. In the galactosialidosis case, two novel mutations were found in the cathepsin A gene. CONCLUSIONS: Reference values for mucopolysaccharides and neuraminic acid depend on gestational age. In a relatively high percentage of the hydrops foetalis pregnancies, a lysosomal aetiology is found. This study provides a strategy to diagnose lysosomal diseases in hydrops fetalis pregnancies. Awareness of lysosomal storage diseases causing hydrops fetalis is useful as it gives an opportunity for risk evaluation, genetic counseling to parents and targeted prenatal diagnostics for ensuing pregnancies. PMID- 16674935 TI - PCNS: a novel protocadherin required for cranial neural crest migration and somite morphogenesis in Xenopus. AB - Protocadherins (Pcdhs), a major subfamily of cadherins, play an important role in specific intercellular interactions in development. These molecules are characterized by their unique extracellular domain (EC) with more than 5 cadherin like repeats, a transmembrane domain (TM) and a variable cytoplasmic domain. PCNS (Protocadherin in Neural crest and Somites), a novel Pcdh in Xenopus, is initially expressed in the mesoderm during gastrulation, followed by expression in the cranial neural crest (CNC) and somites. PCNS has 65% amino acid identity to Xenopus paraxial protocadherin (PAPC) and 42-49% amino acid identity to Pcdh 8 in human, mouse, and zebrafish genomes. Overexpression of PCNS resulted in gastrulation failure but conferred little if any specific adhesion on ectodermal cells. Loss of function accomplished independently with two non-overlapping antisense morpholino oligonucleotides resulted in failure of CNC migration, leading to severe defects in the craniofacial skeleton. Somites and axial muscles also failed to undergo normal morphogenesis in these embryos. Thus, PCNS has essential functions in these two important developmental processes in Xenopus. PMID- 16674936 TI - The inhibitory profile of Ibudilast against the human phosphodiesterase enzyme family. AB - Ibudilast is widely used in Japan to treat ischemic stroke and bronchial asthma. Its mode of action is through the inhibition of cyclic nucleotide phosphodiesterases (PDEs). Growing evidence suggests this compound has utility in a range of neurological conditions linked to its ability to elevate cellular cyclic nucleotide concentrations, however limited data exists on Ibudilast's action on individual PDE families. We therefore used an extensive panel of human PDE enzymes to define the PDE inhibitory profile of this compound. Ibudilast preferentially inhibits PDE3A, PDE4, PDE10 and PDE11 with lesser inhibition of a number of other families. The significance of these findings is discussed in relation to Ibudilast's observed effects on certain disease states. PMID- 16674937 TI - The involvement of a cyclooxygenase 1 gene-derived protein in the antinociceptive action of paracetamol in mice. AB - Paracetamol is a widely used analgesic and antipyretic with weak anti inflammatory properties. Experimental evidence suggests that inhibition of prostaglandin biosynthesis contributes to its pharmacological actions. Three cyclooxygenase (COX) isoenzymes are involved in prostaglandin biosynthesis, COX 1, COX-2 and a recently discovered splice-variant of COX-1, COX-3. Our aim was to identify the relative roles for these enzymes in the antinociceptive action of paracetamol in mice. We compared the antinociceptive action of paracetamol with the non-selective non-steroid anti-inflammatory drug, diclofenac and studied paracetamol antinociception in COX-1 and COX-2 knockout mice. Paracetamol (100 400 mg/kg) inhibited both acetic acid- and iloprost-induced writhing responses. In contrast, diclofenac (10-100 mg/kg) inhibited only acetic acid-induced writhing. Only diclofenac reduced peripheral prostaglandin biosynthesis whereas both drugs reduced central prostaglandin production. Prostaglandin E(2) (PGE(2)) concentrations were reduced in different brain regions by administration of paracetamol. COX-1, COX-2 and COX-3 enzyme proteins were expressed in the same brain regions. The effects of paracetamol on writhing responses and on brain PGE(2) levels were reduced in COX-1, but not COX-2, knockout mice. The selective COX-3 inhibitors, aminopyrine and antipyrine also reduced writhing responses and brain PGE(2) biosynthesis. These results suggest that the antinociceptive action of paracetamol may be mediated by inhibition of COX-3. PMID- 16674938 TI - PPARgamma agonist induced cardiac enlargement is associated with reduced fatty acid and increased glucose utilization in myocardium of Wistar rats. AB - In toxicological studies, high doses of peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists cause cardiac enlargement. To investigate whether this could be explained by a large shift from free fatty acid to glucose utilization by the heart, Wistar rats were treated for 2-3 weeks with a potent, selective PPARgamma agonist (X334, 3 micromol/kg/d), or vehicle. X334 treatment increased body-weight gain and ventricular mass. Treatment lowered plasma triglycerides by 61%, free fatty acid levels by 72%, insulin levels by 45%, and reduced total plasma protein concentration by 7% (indicating plasma volume expansion) compared to vehicle animals. Fasting plasma glucose levels were unaltered. To assess cardiac free fatty acid and glucose utilization in vivo we used simultaneous infusions of non-beta-oxidizable free fatty acid analogue, [9,10-(3)H](R)-2-bromopalmitate and [U-(14)C]2-deoxy-d-glucose tracers, which yield indices of local free fatty acid and glucose utilization. In anesthetized, 7 h fasted animals, left ventricular glucose utilization was increased to 182% while free fatty acid utilization was reduced by 28% (P<0.05) compared to vehicle. In separate studies we attempted to prevent the X334-induced hypolipidemia. Various dietary fat supplements were unsuccessful. By contrast, restricting the time during which the treated animals had access to food (promoting endogenous lipolysis), restored plasma free fatty acid from 27% to 72% of vehicle control levels and prevented the cardiac enlargement. Body-weight gain in these treated-food restricted rats was not different from vehicle controls. In conclusion, the cardiac enlargement caused by intense PPARgamma activation in normal animals is associated with marked changes in free fatty acid/glucose utilization and the enlargement can be prevented by restoring free fatty acid availability. PMID- 16674939 TI - Dopamine uptake and cocaine binding mechanisms: the involvement of charged amino acids from the transmembrane domains of the human dopamine transporter. AB - The wild type human dopamine transporter (DAT) and five DAT mutants were transfected into COS-7 cells and their ability to uptake dopamine or to bind cocaine was examine three days later. In each mutant, a single charged amino acid, located in areas that initial hydrophobic analysis had indicated were DAT transmembrane domains was substituted by alanine. Mutants used in this study were lysines 257 and 525 (termed K257A and K525A), arginines 283 and 521 (termed R283A and R521A), and glutamate 491 (termed E491A). Dopamine affinity was significantly enhanced in the K257A and R283A mutants, and the IC(50) for displacement of the radioactive cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT) by cocaine was significantly elevated in the E491A mutant. All mutants displayed a reduction or complete loss of the maximal velocity (V(m)) of dopamine transport. PMID- 16674940 TI - 5-I A-85380 and TC-2559 differentially activate heterologously expressed alpha4beta2 nicotinic receptors. AB - The neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits expressed in heterologous expression systems assemble into at least two distinct subunit stoichiometries of alpha4beta2 receptor. The (alpha4)2(beta2)3 stoichiometry is about 100-fold more sensitive to acetylcholine than the (alpha4)3(beta2)2 stoichiometry. In order to investigate if agonists in general distinguish high- and low-affinity alpha4beta2 nicotinic acetylcholine receptors, we have expressed human alpha4 and beta2 nicotinic acetylcholine receptor subunits in two different expression systems. The relative amounts of alpha4beta2 nicotinic acetylcholine receptors with high- and low-affinity for acetylcholine were manipulated by (a) injecting the subunit cDNAs at different alpha:beta ratios into Xenopus oocytes and (b) by culturing HEK-293 cells stably expressing alpha4beta2 nicotinic acetylcholine receptors overnight at different temperatures. The sensitivities of the alpha4beta2 nicotinic acetylcholine receptors to the agonists acetylcholine, 5-I A-85380, and TC-2559 were investigated using the voltage-clamp technique on Xenopus oocytes and using a fluorescent imaging plate reader to measure calcium responses from HEK-293 cells. Like acetylcholine, 5-I A-85380 produced biphasic concentration-response curves and the high-affinity component became larger when the cells were manipulated to produce a greater proportion of (alpha4)2(beta2)3 nicotinic acetylcholine receptors. Interestingly, under all circumstances, TC 2559 produced monophasic concentration-response curves. In oocytes injected with alpha4 and beta2 subunits in the 1:1 ratio the maximum effect of TC-2559 was 28% of that of acetylcholine. The EC50 for TC-2559 was not changed when oocytes were manipulated to express exclusively (alpha4)2(beta2)3 nicotinic acetylcholine receptors, however, the maximum effect of TC-2559 was dramatically enhanced. These results suggest that TC-2559 is a selective agonist of the (alpha4)2(beta2)3 nicotinic acetylcholine receptor stoichiometry. PMID- 16674941 TI - Identification of protease-sensitive sites in Human Endothelial-Monocyte Activating Polypeptide II protein. AB - The cleaved approximately 22-kDa form of Endothelial-Monocyte Activating Polypeptide [mature (m)EMAP II] functions as a potent inhibitor of tumor growth. Although the anti-tumor effect of mEMAP II has been described, little is known regarding the cleavage of mEMAP II from its precursor form (pEMAP II). We determined that pEMAP II is expressed at the cell membrane surface and proteinases MMP-9, elastase, and cathepsin L release protein fragments consistent with mEMAP II molecular mass. MMP-9 and elastase generate a approximately 25-26 kDa spanning fragments, while cathepsin L generates a approximately 22 kDa fragment. Although several fragments are processed from pEMAP II within a 44 AA residue stretch, cathepsin L cleaves pEMAP II within 4 amino acids of the determined N-terminal sequence, suggesting that this region is sensitive to proteinases. PMID- 16674942 TI - Differential expression of retinal pigment epithelium (RPE) IP-10 and interleukin 8. AB - Interferon-gamma induced protein of 10 kDa (IP-10) is a C-X-C chemokine that attracts T lymphocytes and inhibits angiogenesis. In this study, we investigated the expression of IP-10 by human retinal pigment epithelial cells (HRPE) and compared IP-10 expression to that of interleukin-8 (IL-8), which is a leukocytic chemoattractant and pro-angiogenic factor. Cultured HRPE cells were incubated with either IL-1 beta (0.2-20 ng/ml) or tumor necrosis factor (TNF)-alpha (0.2-20 ng/ml) alone or in combination with interferon-gamma (IFN-gamma) (1000 U/ml). HRPE cells were also incubated with: (1) media conditioned by activated human T lymphocytes (CM), or (2) the same CM treated with neutralizing antibodies to IL 1, TNF, and/or IFN-gamma. IL-8 and IP-10 protein levels were measured by ELISA and mRNA levels by Northern blot analysis of HRPE cells. HRPE cells produced very high levels of IP-10 in response to either IL-1 beta/IFN-gamma, TNF-alpha/IFN gamma or CD3-activated T-lymphocyte CM. The levels of IP-10 were at least tenfold higher (p<.001) than IL-8 measured in the same samples. Neutralizing antibodies to TNF and IFN-gamma, but not to IL-1, abrogated the ability of the CD3-activated T lymphocytes CM to induce HRPE IP-10 (p<.001). HRPE cells produce differential levels of IP-10 and IL-8 in response to various combinations of recombinant and T lymphocyte-secreted pro-inflammatory cytokines. This may be important in evolving inflammatory and angiogenic ocular responses. PMID- 16674943 TI - T cell infiltration after chronic constriction injury of mouse sciatic nerve is associated with interleukin-17 expression. AB - Interleukin (IL)-17A, a recently described novel T cell cytokine, orchestrates inflammation in a variety of immune-mediated diseases. In the present investigation, we analyzed the temporal gene expression pattern of IL-17A and its main regulators IL-23 and IL-15 after chronic constriction injury (CCI) of the sciatic nerve, a lesion paradigm inducing neuropathic pain, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in mice. IL-17A displayed a monophasic expression in degenerating nerves at day 7 after CCI while transcripts for the IL-17A regulatory cytokines IL-23 and IL-15 peaked earlier. Accordingly, IL-17A positive T cells were detectable within the endoneurium of the injured nerves by immunocytochemistry. In support of a crucial role of T cell inflammation, RAG-1 knockout mice lacking functional T lymphocytes did not express IL-17A mRNA in distal nerve segments following CCI. Interestingly, T cell deficiency was associated with less thermal hyperalgesia and reduced mRNA levels for the macrophage marker molecule F4/80 and the chemokine macrophage chemoattractant protein-1 (MCP-1) after CCI. Our study supports the notion that T cells and T-cell-derived cytokines contribute to the inflammatory response after peripheral nerve injury. PMID- 16674944 TI - Cystoisospora belli: in vitro multiplication in mammalian cells. AB - Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells. PMID- 16674945 TI - Position-specific polymorphism of Plasmodium falciparum Stuttering motif in a PHISTc PFI1780w. AB - Several genes of Plasmodium falciparum are positively selected due to the pressure from the host immune system. This is a pattern completely opposite to that found in most housekeeping genes, which have few synonymous mutations. The discrepancy is an important topic in Plasmodium biology. We searched for unique polymorphism patterns in P. falciparum and identified a repetitive Stuttering motif in PFI1780w which was recently grouped as a gene in the PHIST family. The repeat has a position-specific polymorphism pattern in the otherwise highly conserved gene. Its mutations are limited to only one small region, and they are not consistent with replication slippage or gene conversion commonly found in low complexity regions. The repeat variation was analyzed in different strains of P. falciparum. The PFI1780w Stuttering motif can be a model to study gene diversification and used as a tool for strain typing. PMID- 16674946 TI - A novel retinoid binding property of human annexin A6. AB - Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A dependent tissue mineralization. PMID- 16674947 TI - The metabolic syndrome and adipocytokines. AB - Visceral fat accumulation has been shown to play crucial roles in the development of cardiovascular disease as well as the development of obesity-related disorders such as diabetes mellitus, hyperlipidemia and hypertension and the so-called metabolic syndrome. Given these clinical findings, adipocytes functions have been intensively investigated in the past 10 years, and have been revealed to act as endocrine cells that have been termed adipocytokines, which secrete various bioactive substances. Among adipocytokines, tumor necrosis factor-alpha, plasminogen activator inhibitor type 1 and heparin binding epidermal growth factor-like growth factor are produced in adipocytes as well as other organs, and may contribute to the development of vascular diseases. Visfatin has been identified as a visceral-fat-specific protein that might be involved in the development of obesity-related diseases, such as diabetes mellitus and cardiovascular disease. On the contrary to these adipocytokines, adiponectin, an adipose-tissue-specific, collagen-like protein, has been noted as an important antiatherogenic and antidiabetic protein, or as an anti-inflammatory protein. The functions of adipocytokine secretion might be regulated dynamically by nutritional state. Visceral fat accumulation causes dysregulation of adipocyte functions, including oversecretion of tumor necrosis factor-alpha, plasminogen activator inhibitor type 1 and heparin binding epidermal growth factor-like growth and hyposecretion of adiponectin, which results in the development of a variety of metabolic and circulatory diseases. In this review, the importance of adipocytokines, especially focusing on adiponectin is discussed with respect to cardiovascular diseases. PMID- 16674948 TI - The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria. AB - Mycobacterium tuberculosis PknB is an essential receptor-like protein kinase involved in cell growth control. Here, we demonstrate that mitoxantrone, an anthraquinone derivative used in cancer therapy, is a PknB inhibitor capable of preventing mycobacterial growth. The structure of the complex reveals that mitoxantrone partially occupies the adenine-binding pocket in PknB, providing a framework for the design of compounds with potential therapeutic applications. PknB crystallizes as a 'back-to-back' homodimer identical to those observed in other structures of PknB in complex with ATP analogs. This organization resembles that of the RNA-dependent protein kinase PKR, suggesting a mechanism for kinase activation in mycobacteria. PMID- 16674949 TI - Excess methionine suppresses the methylation cycle and inhibits neural tube closure in mouse embryos. AB - Suppression of one-carbon metabolism or insufficient methionine intake are suggested to increase risk of neural tube defects (NTD). Here, exogenous methionine unexpectedly caused frequent NTD in cultured mouse embryos. NTD were associated with reduced cranial mesenchyme cell density, which may result from a preceding reduction in proliferation. The abundance ratio of S-adenosylmethionine to S-adenosylhomocysteine was also decreased in treated embryos, suggesting methylation reactions may be suppressed. Such an effect is potentially causative as NTD were also observed when DNA methylation was specifically inhibited. Thus, reduced cranial mesenchyme density and impairment of critical methylation reactions may contribute to development of methionine-induced NTD. PMID- 16674950 TI - Integrated genome-wide gene expression map and high-resolution analysis of aberrant chromosomal regions in squamous cell lung cancer. AB - The recognition of recurrent aberrant regions in cancer is important to the discovery of candidate cancer related genes. Here we first constructed a genome wide gene expression map of squamous lung carcinoma from the Stanford Microarray Database. High-resolution detection of aberrant chromosomal regions was performed by using moving-median method. 84% (27 of 32) of our results were consistent with the previous studies of comparative genomic hybridization or loss of heterozygosity. One overrepresented region in Xq28 was newly discovered to be related to squamous cell lung carcinoma. These observations could be of great interest for further studies. PMID- 16674951 TI - Engrailed: complexity and economy of a multi-functional transcription factor. AB - Engrailed is a homeodomain-containing transcription factor with numerous, overlapping roles in neural development. Its multifunctional nature depends upon an extremely diverse group of molecular functions including the ability to regulate both transcription and translation, and to be released from and internalized by cells. Recent findings have shown how some of these functions relate to specific roles in development and disease. PMID- 16674952 TI - Pigment epithelium-derived factor (PEDF) inhibits advanced glycation end product (AGE)-induced C-reactive protein expression in hepatoma cells by suppressing Rac 1 activation. AB - Serum levels of advanced glycation end products (AGEs) are associated with an acute phase reactant, C-reactive proteins (CRP) in diabetic patients. However, whether AGEs could directly stimulate hepatic CRP production remains to be elucidated. We found here that AGEs upregulated CRP mRNA levels in cultured Hep3B cells via Rac-1 activation, which was blocked by pigment epithelium-derived factor (PEDF). Our present study suggests that AGEs are one of the potent inducers of CRP and that PEDF may work as an anti-inflammatory agent against AGEs in the liver. PMID- 16674953 TI - Acclimation to temperature and irradiance modulates PSII charge recombination. AB - Acclimation of wild type and the chlorina F2 mutant of barley to either high light or low temperature results in a 2- to 3-fold increase in non-photochemical quenching which occurred independently of either energy-dependent quenching (qE), xanthophyll cycle-mediated antenna quenching or state transitions. Results of in vivo thermoluminescence measurements used to address this conundrum indicated that excitation pressure regulates the temperature gap for S(2)Q(B)(-) and S(2)Q(A)(-) charge recombinations within photosystem II reaction centers. This is discussed in terms of photoprotection through non-radiative charge recombination. PMID- 16674954 TI - The search for factors in human feeders that support the derivation and propagation of human embryonic stem cells: preliminary studies using transcriptome profiling by serial analysis of gene expression. AB - Serial analysis of gene expression (SAGE) was used to obtain the transcriptome profiles of a supportive human fetal skin feeder (Detroit 551) and a nonsupportive human fetal lung feeder (MRC-5) for human embryonic stem cells. A pairwise comparison of the two SAGE profiles showed that fibroblast growth factor 2 (FGF2), a bone morphogenetic protein 4 pathway inhibitor, Gremlin 1, and several extracellular matrix proteins that could potentially aid human embryonic stem cell attachment and growth were highly expressed in Detroit 551 fibroblasts. PMID- 16674955 TI - Intrauterine adhesions after manual vacuum aspiration for early pregnancy failure. AB - OBJECTIVE: To describe the occurrence of intrauterine adhesions after manual vacuum aspiration for early pregnancy failure. DESIGN: Case series. SETTING: Tertiary care center. PATIENT(S): Three women with intrauterine adhesions after manual vacuum aspiration for the treatment of early pregnancy failure. INTERVENTION(S): Chart review. MAIN OUTCOME MEASURE(S): Hysteroscopic diagnosis of intrauterine adhesions after manual vacuum aspiration. RESULT(S): Three cases of symptomatic intrauterine adhesions after manual vacuum aspiration. CONCLUSION(S): Intrauterine adhesion formation may follow manual vacuum aspiration for early pregnancy loss. PMID- 16674956 TI - Matrix metalloproteinase-9 activity in the plasma of patients after surgical resection of endometriomas. AB - Matrix metalloproteinases (MMPs) may have a role in the pathogenesis of endometrioma. The level of MMP was lower compared to the level taken after resection of endometrioma. PMID- 16674957 TI - Molecular cloning of follicle-stimulating hormone (FSH)-beta subunit cDNA from duck pituitary. AB - We have cloned FSH-beta cDNA from duck pituitary gland by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned duck FSH-beta cDNA contains 1909-bp nucleotides including 396 bp of open-reading frame and 1491-bp of 3'-untranslational region. The open reading frame encodes a 131-amino acid protein with a putative 20-amino acid signal peptide and a putative 111-amino acid mature protein. The deduced amino acid sequence shows a remarkable similarity (94-98%) to those of other avian FSH beta subunits; while it exhibits lower similarities with those of turtles (82 84%), mammals (63-71%), and amphibians (53-57%). The structural model analysis of duck FSH suggests that the cysteine-knot and beta-strands for maintaining the specific structural frame, and the "seat-belt" loop for specific binding to FSH receptor have been conserved in tetrapodian FSH-betas. PMID- 16674958 TI - Divide and conquer: Cell addition and aggressive signaling in electric fish. PMID- 16674959 TI - White blood cell count is a marker of wave reflections and arterial stiffness in healthy individuals. PMID- 16674960 TI - Association of glucose metabolism, smoking and cardiovascular risk factors with incident peripheral arterial disease: the DESIR study. AB - AIMS: We determined the 6-year incidence of peripheral arterial disease (PAD) in a French population and assessed the association of glucose metabolism, smoking, cardiovascular risk factors and physical activity with incident PAD. METHODS: Participants from the French Data from a Epidemiological Study on the Insulin Resistance Syndrome (DESIR) were studied. Participants analysed were 30-65 years (at baseline) and had complete data (n=3805) after 6 years of follow-up. Diabetes was diagnosed according to the 1999 WHO criteria on the basis of fasting plasma glucose results or previous diagnosis of diabetes mellitus. The ankle brachial pressure index (ABPI) and a claudication question were used to classify PAD. RESULTS: The 6-year incidence of PAD (defined by ABPI<0.9 and or claudication present) among those with normal fasting glucose (NFG) and free of PAD at baseline was 5.1%. Among those with impaired fasting glucose (IFG) at baseline the incidence of PAD was 4.9% and among those with diabetes mellitus at baseline the incidence of PAD was 9.8%. The incidence of PAD among those who maintained NFG over 6 years was 4.7% and among those who progressed to diabetes over 6 years was 10.2%. Those who progressed from NFG or IFG to diabetes over 6 years were twice as likely to develop PAD compared to those who maintained NFG over 6 years, after adjustment for age and sex (OR (95% CI), 2.22 (1.12-4.42)). Independent risk factors for incident PAD using baseline population characteristics were diabetes (OR (95% CI) 2.11 (1.25-3.55)), systolic BP 122-135mmHg 1.06 (0.70 1.60), >135mmHg 1.54 (1.04-2.27) and current smoking 1.60 (1.10-2.34) after multivariate adjustment for age, sex, cholesterol, triglycerides and waist circumference. CONCLUSIONS: This French study shows that those who progress to diabetes are twice as likely to develop PAD, compared to those who maintain NFG. Peripheral arterial disease is a treatable condition and more aggressive management of atherosclerotic risk factors could reduce the numbers of people who develop PAD. PMID- 16674961 TI - The association between high-density lipoprotein cholesterol level and cholesteryl ester transfer protein TaqIB gene polymorphism is influenced by alcohol drinking in a population-based sample. AB - Cholesteryl ester transfer protein (CETP) is a key enzyme in high-density lipoprotein (HDL) cholesterol metabolism. We studied the association between CETP TaqIB polymorphism and the HDL cholesterol levels considering environmental factors in a population-based sample consisting of 1729 participants who did not use lipid-lowering agents (659 men and 1070 women). The CETP TaqIB genotypes were determined by PCR-RFLP analysis. The serum HDL cholesterol levels of female participants with the B2B2 genotype were significantly higher than those with other genotypes (p<0.001). Multiple regression analysis with covariates such as age, waist to hip (W/H) ratio, alcohol drinking, current smoking, non-HDL cholesterol, and logarithm of triglyceride revealed that the CETP TaqIB genotype was an independent determinant of HDL cholesterol levels in men (p=0.049) and women (p<0.001). Subgroup analysis revealed that an interaction was observed between the CETP TaqIB polymorphism and alcohol consumption in the regulation of HDL cholesterol levels in men (p=0.049) and women (p=0.022). No interactions were observed between the CETP TaqIB polymorphism and current smoking status, body mass index, or W/H ratio in the regulation of HDL cholesterol levels. The association between the CETP TaqIB polymorphism and HDL cholesterol levels was more evident in alcohol consumers than in non-drinkers. PMID- 16674962 TI - Stretch-induced nerve conduction deficits in guinea pig ex vivo nerve. AB - In the current communication, we characterized supraphysiologic elongations that elicited short-term nerve dysfunction. This was accomplished by assessing the electrophysiology of guinea pig tibial and peroneal nerves at predetermined elongation magnitudes. Results showed that a longitudinal supraphysiological stretch of lambda = 1.05 caused a 16% reduction in the mean compound action potential (CAP) amplitude. Upon relaxation to physiologic length, a full recovery in the CAP was observed. At lambda = 1.10, the CAP decreased by 50% with an 88% recovery after relaxation. For a supraphysiologic stretch of lambda = 1.20, severe conduction block with minimal acute recovery was observed. Latency also increased during periods of stretch and was proportional to the stretch magnitude. Additional studies showed some electrophysiological recovery during the sustained stretch phase. This attribute may be related to internal stress relaxation mechanisms. Since whole nerve elongations are averaged global deformations, we also used an incremental digital image correlation (DIC) technique to characterize the strain at the micro-tissue level. The DIC analysis revealed considerable heterogeneity in the planar strain field, with some regions exhibiting strains above the macroscale stretch. This non-uniformity in the strain map arises from structural inconsistencies of the nerve and we presume that zones of high local strain may translate into the observed conduction deficits. PMID- 16674964 TI - Improved endcapping method of monolithic silica columns. AB - Following a bonding reaction with octadecyldimethyl-N,N-diethylaminosilane (ODS DEA), a monolithic silica was subjected to consequential on-line endcapping reactions with hexamethyldisilazane (HMDS) or N-(trimethylsilyl)imidazole (TMSI) to compare the two reagents. Endcapping of monolithic silica C(18) columns prepared by a manufacturer was also studied. After endcapping with TMSI in acetonitrile (ACN), all columns provided better performance for hydrogen-bonding compounds and basic compounds than C(18) phases endcapped with HMDS. TMSI endcapping of commercial Chromolith Performance columns also gave improved performance. The results indicate that one can significantly improve the performance of monolithic silica C(18) columns for basic compounds by a simple on column reaction with TMSI in ACN at a relative low temperature. PMID- 16674963 TI - Ionic effect on the binding of bilirubin to the imprinted poly(methacrylic acid co-ethylene glycol dimethylacrylate). AB - A molecularly imprinted polymer (MIP) capable of detecting bilirubin was successfully synthesized. Bilirubin template was imprinted in poly(methacrylic acid-co-ethylene glycol dimethylacrylate) [poly(MAA-co-EGDMA)]. MAA and EGDMA were used as the monomer and the cross-linker, respectively. The optimal solvent conditions to maintain its stability were discussed. Solvent system based on ethylenediamine tetraacetic acid (EDTA) and ascorbic acid was compared with respect to the stability of bilirubin. pH and bilirubin concentration were both investigated for the bilirubin stability. Blue light as well as aeration was applied to inspect the regarding effects. The cross-linking effect was further confirmed by the thermogravimetric analysis (TGA). The effect of salts, such as NaCl and KCl on the binding capacity of the molecularly imprinted polymer was also discussed. Further, the rat serum and bile samples were applied and the binding of the MIPs for bilirubin was thus confirmed. PMID- 16674965 TI - A simple supported liquid hollow fiber membrane microextraction for sample preparation of trihalomethanes in water samples. AB - A simple and efficient liquid-phase microextraction (LPME) technique using a supported liquid hollow fiber membrane, in conjunction with gas chromatography electron capture detector has been developed for extraction and determination of trihalomethanes (THMs) in water samples. THMs were extracted from water samples through an organic extracting solvent impregnated in the pores and filled inside the porous hollow fiber membrane. Our simple conditions were conducted at 35 degrees C with no stirring and no salt addition in order to minimize sample preparation steps. Parameters such as types of hollow fiber membranes, extracting solvents and extraction time were studied and optimized. The method exhibited enrichment factors ranged from 28- to 62-fold within 30 min extraction time. The linearity of the method ranged from 0.2 to 100 microg l(-1). The limits of detection were in the low microg l(-1) level, ranging between 0.01 and 0.2 microg l(-1). The recoveries of spiked THMs at 5 microg l(-1) in water were between 98 and 105% with relative standard deviations (RSDs) less than 4%. Furthermore, the method was applied for determination of THMs in drinking water and tap water samples was reported. PMID- 16674966 TI - Influence of the spectrophotometric detection mode on the separation performance of systems with monolithic capillary columns: on-column and external-cell detection modes. AB - Poly(butyl methacrylate) monolithic columns were prepared by thermally initiated radical polymerization in fused-silica capillaries of 320 microm i.d. The prepared monolithic columns were tested by capillary liquid chromatography (CLC) combined with a UV-VIS spectrophotometric detector. The influence of the detection configuration (i.e., on-column and external-cell detection modes) on the performance of the chromatographic system was investigated. In the on-column detection mode within the monolith, the detection window was located inside the column section filled with the monolith. With the on-column detection configuration after the monolith, the detection window was positioned just behind the column section containing the monolith. Using the external-cell detection mode, an additional detection capillary, provided with a detection window defining the external-cell, was connected to the monolithic capillary column. These detection modes were critically compared in terms of the principal chromatographic parameters of the system involving the prepared monolithic capillary columns. PMID- 16674967 TI - Preparation of zinc oxide nanorods using pulsed laser ablation in water media at high temperature. AB - ZnO columnar single crystals were formed by pulsed laser ablation in deionized water and surfactant aqueous solutions of lauryl dimethylaminoacetic acid (LDA) and cetyltrimethylammonium bromide (CTAB) at 80 degrees C. ZnO particles produced by laser ablation were dissolved at a higher temperature than 60 degrees C, and then crystalline growth to columnar structure proceeded. While large ZnO columnar crystals were obtained in deionized water, the crystals prepared in surfactant solution were smaller than those in deionized water due to inhibition of crystalline growth by surfactant adsorption on ZnO surfaces. The size of ZnO nanorods depended on how surfactant molecules adsorb on ZnO surface. PMID- 16674968 TI - Rimes are not necessarily favored by prereaders: evidence from meta- and epilinguistic phonological tasks. AB - This article explores young children's facility in phonological awareness tasks requiring either the detection or the articulation of head, coda, onset, and rime subsyllabic units shared in word pairs. Data are reported from 70 nonreading children and 21 precocious readers attending preschools. Prereading children were able to articulate shared heads, codas, and onsets, although rimes rarely were articulated. Precocious readers were able to articulate shared rimes, but articulation performance was still most accurate for onsets and codas. Rimes and heads were equally accessible in the detection task and were identified more often than onsets and codas (nonreaders) and codas (readers). It is concluded that the articulation advantage for nonrime units cannot simply reflect early reading instruction. This disjoint pattern of phonological awareness in detection and production tasks does not support Goswami's phonological status hypothesis. Results may instead reflect quite distinct influences on epilinguistic and metalinguistic phonological development. PMID- 16674969 TI - Children's understanding of the arithmetic concepts of inversion and associativity. AB - Previous studies have shown that even preschoolers can solve inversion problems of the form a+b-b by using the knowledge that addition and subtraction are inverse operations. In this study, a new type of inversion problem of the form d x e/e was also examined. Grade 6 and 8 students solved inversion problems of both types as well as standard problems of the form a+b-c and d x e/f. Students in both grades used the inversion concept on both types of inversion problems, although older students used inversion more frequently and inversion was used most frequently on the addition/subtraction problems. No transfer effects were found from one type of inversion problem to the other. Students who used the concept of associativity on the addition/subtraction standard problems (e.g., a+b c=[b-c]+a) were more likely to use the concept of inversion on the inversion problems, although overall implementation of the associativity concept was infrequent. The findings suggest that further study of inversion and associativity is important for understanding conceptual development in arithmetic. PMID- 16674970 TI - A heart rate analysis of developmental change in feedback processing and rule shifting from childhood to early adulthood. AB - Over the course of development, the ability to switch between different tasks on the basis of feedback cues increases profoundly, but the role of performance monitoring remains unclear. Heart rate indexes can provide critical information about how individuals monitor feedback cues indicating that performance should be adjusted. In this study, children of three age groups (8-10, 12-14, and 16-18 years) performed a rule change task in which sorting rules needed to be detected following positive or negative feedback. The number of perseverative errors was lower for 16- to 18-year-olds than for 8- to 10-year-olds, and 12- to 14-year olds performed at an intermediate level. Consistent with previous findings, heart rate slowed following feedback indicating a rule change, and the magnitude of slowing was similar for all age groups. Thus, 8- to 10-year-olds are already able to analyze feedback cues. In contrast, 12- to 14-year-olds and 16- to 18-year olds, but not 8- to 10-year-olds, showed heart rate slowing following performance errors, suggesting that with age children are increasingly able to monitor their performance online. Performance monitoring may therefore be an important contributor to set-shifting ability. PMID- 16674971 TI - Floxed reporter genes: Flow-cytometric selection of clonable cells expressing high levels of a target gene after tamoxifen-regulated Cre-loxP recombination. AB - Tamoxifen treatment allows MerCreMer fusion recombinase to localize to the nucleus where MerCreMer can excise a floxed inhibitory DNA segment, thereby activating the expression of a downstream gene. This excision is irreversible, and it is therefore difficult to predict which non-activated clones will express the gene at high levels after recombination. We transfected a vector using HLA A2.1 as floxed inhibitory DNA element and its expression level as surrogate marker predicting future expression of the attenuated downstream target gene. The target gene encoded an EGFP-linked fusion protein. In the unsorted population, 6% of the cells expressed the transfected target gene after recombination and less than 10-fold higher than the population before recombination. However after flow cytometric selection for high HLA-A2.1 expression, 47% of the cells expressed the target gene after recombination and at levels 37-fold higher than the sorted population before recombination. 58% of the clones were capable of expressing the fusion protein and some over 200-fold above background of untransfected cells and greater than 20-fold higher levels of expression than before recombination. We describe an efficient method to select for clones expressing high levels of a target gene after tamoxifen regulated Cre-loxP recombination. Other floxed reporter genes should be equally useful. PMID- 16674972 TI - A fast mutagenesis procedure to recover soluble and functional scFvs containing amber stop codons from synthetic and semisynthetic antibody libraries. AB - The selection and production of scFvs from phage display synthetic antibody libraries are frequently delayed by the presence of amber (TAG) stop codons within the sequences corresponding to the variable CDRs. This is due to the use of randomised oligonucleotides for library design and amber mutations for joining the scFv to the phage protein pIII. The screening of such libraries may lead to the selection of scFvs containing stop codons. Then, multiple site-directed mutagenesis is required for their removal or, alternatively, the proteins must be expressed as scFv-pIII fusions, which are not suitable for many functional assays. We describe here an alternative procedure to express soluble scFvs, despite the presence of TAG stop codons, in the currently used Escherichia coli suppressor strain TG1. It is based on a simple mutagenesis protocol that replaces the amber codon between the scFv and the pIII gene by a different stop codon (TAA), functional in E. coli TG1. The expression of soluble scFvs in the suppressor strain TG1 permits their fully functional characterization including the determination of affinity constants, which are critical for selecting the right scFvs for further studies. PMID- 16674973 TI - Comparison of random and oriented immobilisation of antibody fragments on mixed self-assembled monolayers. AB - The sensitivity of immunosensors is strongly dependent on the amount of immobilised antibodies and their remaining antigen binding properties. The use of smaller and well-oriented antibody fragments as bioreceptor molecules influences the final immunosensor signal. The aim of this study was to compare the immunosensor responses of different immobilised antibody fragments, such as F(ab')2 and Fab', with their parental IgG. In addition, we evaluated the oriented versus the random covalent immobilisation method of the Fab' fragments. First, an optimisation of cleavage protocol to generate these F(ab')2 and Fab' fragments was performed. Subsequently, we pursued a study with limited denaturation effects during immobilisation of the bioreceptor molecules and with reduced steric hindrance during antigen binding using mixed self-assembled monolayers (SAM) of thiols as the chemical linking layer. The Surface Plasmon Resonance technique was used to evaluate the degree of immobilisation of the antibody fragments and their parental IgGs on the mixed SAMs and the binding signals of their specific antigens. In this study, we demonstrate that for a particular antibody/antigen system (anti-hIgG/hIgG), the optimised fragmentation protocol in combination with an oriented immobilisation of Fab' fragments on mixed SAMs leads to a >2-fold increase of the antigen binding signals compared to randomly covalent immobilised full-length antibodies. PMID- 16674974 TI - Inferring functional linkages between proteins from evolutionary scenarios. AB - Identifying potential protein interactions is of great importance in understanding the topologies of cellular networks, which is much needed and valued in current systematic biological studies. The development of our computational methods to predict protein-protein interactions have been spurred on by the massive sequencing efforts of the genomic revolution. Among these methods is phylogenetic profiling, which assumes that proteins under similar evolutionary pressures with similar phylogenetic profiles might be functionally related. Here, we introduce a method for inferring functional linkages between proteins from their evolutionary scenarios. The term evolutionary scenario refers to a series of events that occurred in speciation over time, which can be reconstructed given a phylogenetic profile and a species tree. Common evolutionary pressures on two proteins can then be inferred by comparing their evolutionary scenarios, which is a direct indication of their functional linkage. This scenario method has proven to have better performance compared with the classical phylogenetic profile method, when applied to the same test set. In addition, predicted results of the two methods are found to be fairly different, suggesting the possibility of merging them in order to achieve a better performance. We analyzed the influence of the topology of the phylogenetic tree on the performance of this method, and found it to be robust to perturbations in the topology of the tree. However, if a completely random tree is incorporated, performance will decline significantly. The evolutionary scenario method was used for inferring functional linkages in 67 species, and 40,006 linkages were predicted. We examine our prediction for budding yeast and find that almost all predicted linkages are supported by further evidence. PMID- 16674975 TI - Structure and reaction mechanism of L-rhamnulose kinase from Escherichia coli. AB - Bacterial L-rhamnulose kinase participates in the degradation of L-rhamnose, which is ubiquitous and particularly abundant in some plants. The enzyme catalyzes the transfer of the gamma-phosphate group from ATP to the 1-hydroxyl group of L-rhamnulose. We determined the crystal structures of the substrate-free kinase and of a complex between the enzyme, ADP and L-fructose, which besides rhamnulose is also processed. According to its chainfold, the kinase belongs to the hexokinase-hsp70-actin superfamily. The closest structurally known homologue is glycerol kinase. The reported structures reveal a large conformational change on substrate binding as well as the key residues involved in catalysis. The substrates ADP and beta-L-fructose are in an ideal position to define a direct in line phosphoryl transfer through a bipyramidal pentavalent intermediate. The enzyme contains one disulfide bridge at a position where two homologous glycerol kinases are regulated by phosphorylation and effector binding, respectively, and it has two more pairs of cysteine residues near the surface that are poised for bridging. However, identical catalytic rates were observed for the enzyme in reducing and oxidizing environments, suggesting that regulation by disulfide formation is unlikely. PMID- 16674977 TI - How mothers parent their children with behavior disorders: implications for unintentional injury risk. AB - INTRODUCTION: This study was designed to test the role of parental supervision in explaining why children with behavior disorders have increased risk of unintentional injury. METHOD: Children referred to a pediatric behavior disorders clinic and their mothers were unknowingly observed in a "hazard room" environment that housed several items that appeared dangerous but actually were altered to be safe. RESULTS: Mother and child behavior in the hazard room was correlated to parent-, teacher-, and observational-reports of children's externalizing behavior patterns, children's injury history, and mother's parenting styles. Maternal ignoring of children's dangerous behavior in the hazard room was the strongest correlate to children's injury history. CONCLUSIONS: Poor parental supervision might serve as a mechanism to explain why children with behavior disorders, and those with oppositional behavior patterns in particular, have increased risk of unintentional injury. PMID- 16674976 TI - Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation. AB - Chemotaxis, the environment-specific swimming behavior of a bacterial cell is controlled by flagellar rotation. The steady-state level of the phosphorylated or activated form of the response regulator CheY dictates the direction of flagellar rotation. CheY phosphorylation is regulated by a fine equilibrium of three phosphotransfer activities: phosphorylation by the kinase CheA, its auto dephosphorylation and dephosphorylation by its phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two spatially distinct protein-protein contacts: tethering of the two proteins to each other and formation of an active site for dephosphorylation. The former involves interaction of phosphorylated CheY with the small highly conserved C-terminal helix of CheZ (CheZ(C)), an indispensable structural component of the functional CheZ protein. To understand how the CheZ(C) helix, representing less than 10% of the full-length protein, ascertains molecular specificity of binding to CheY, we have determined crystal structures of CheY in complex with a synthetic peptide corresponding to 15 C terminal residues of CheZ (CheZ(200-214)) at resolutions ranging from 2.0 A to 2.3A. These structures provide a detailed view of the CheZ(C) peptide interaction both in the presence and absence of the phosphoryl analog, BeF3-. Our studies reveal that two different modes of binding the CheZ(200-214) peptide are dictated by the conformational state of CheY in the complex. Our structures suggest that the CheZ(C) helix binds to a "meta-active" conformation of inactive CheY and it does so in an orientation that is distinct from the one in which it binds activated CheY. Our dual binding mode hypothesis provides implications for reverse information flow in CheY and extends previous observations on inherent resilience in CheY-like signaling domains. PMID- 16674978 TI - Pseudobulbar affect in multiple sclerosis: toward the development of innovative therapeutic strategies. AB - Pseudobulbar affect (PBA), a condition involving involuntary and uncontrollable episodes of crying and/or laughing, occurs frequently in patients with a variety of neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury, dementia including Alzheimer's disease, and multiple sclerosis (MS). Although PBA results in considerable distress for patients and caretakers, it is underrecognized and undertreated. Agents used to treat psychiatric disorders--particularly tricyclic antidepressants and selective serotonin reuptake inhibitors--are useful in alleviating PBA, but act on diffuse neural networks rather than targeting those involved in emotional motor expression. As a result of their nonspecific activity, these agents are associated with a range of unwanted effects that preclude many patients from using them. Dextromethorphan, a common cough suppressant, specifically targets sigma(1) receptors concentrated in the brainstem and cerebellum, thus providing the possibility of targeting regions implicated in emotional expression. When administered in a fixed combination with quinidine, dextromethorphan is effective in treating PBA in patients with ALS, and preliminary results suggest that this therapy also is effective in treating MS-related PBA. PMID- 16674979 TI - Mutational analysis of the Cu/Zn superoxide dismutase gene in a Catalan ALS population: should all sporadic ALS cases also be screened for SOD1? AB - BACKGROUND: SOD1 gene mutations are the most common identified cause of ALS, accounting for approximately 20% of familial ALS cases and around 4% of sporadic ALS cases. However, the prevalence of SOD1 varies in different ethnic groups. No previous epidemiological studies have been carried out in Catalonia. OBJECTIVE: To determine the prevalence of SOD1 gene mutations in a Catalan ALS population, and to analyze the genotype-phenotype relationship. MATERIALS AND METHODS: 30 different FALS pedigrees and 94 sporadic ALS patients were screened for SOD1 mutations using direct sequence analysis. RESULTS: Five of the 30 FALS pedigrees (16.6%) carried a SOD1 mutant. The mutations identified in this group were G37R, D76V, S105L, I112M and N139H. Four SOD1 mutants (4.25%) were found in the sporadic ALS group (SALS). The overall frequency (FALS plus SALS) of SOD1 mutations in our series was 6.45%. In the SALS group, D90A was identified in a patient presenting the typical Scandinavian phenotype. A 53-year-old woman with no family history of ALS carried the N139H mutation. Two unrelated sporadic ALS cases carried the A140A SOD1 mutant. CONCLUSIONS: The prevalence of the SOD1 mutation in FALS in Catalonia is similar to levels in other Mediterranean countries, but lower than those in reports studying the Belgian, Japanese, and Scottish populations. The prevalence of the SOD1 mutation was 4.25% in patients with no family history of ALS. These results may have significant repercussions on genetic counseling, and screening for the SOD1 mutation in sporadic ALS cases must therefore be considered. PMID- 16674980 TI - Anticholinesterasics in the treatment of cognitive impairment in multiple sclerosis. AB - Neuropsychological impairment is a common manifestation in multiple sclerosis (MS) and is found in 40-60% of patients. The pattern of cognitive impairment in MS is characterized by difficulties in recent memory, sustained attention, executive functions and information processing speed. These cognitive deficits have a significant impact on the patients' daily activities. However, there is no specific treatment available at present for cognitive disorders in MS patients. Treatment with acetylcholinesterase inhibitors (AChEI) has shown a positive effect on cognitive functions of patients with Alzheimer's disease and other conditions such as Lewy Body dementia, subcortical vascular dementia and Parkinson's disease. In this paper we review the results from studies and clinical trials aiming to demonstrate that AChEI could be a potential treatment for cognitive disorders in MS patients. Finally, we discuss future issues to take into consideration for AChEI treatments in the context of MS. PMID- 16674981 TI - Advanced glycation end-products induce apoptosis involving the signaling pathways of oxidative stress in bovine retinal pericytes. AB - One of the histopathologic hallmarks of early diabetic retinopathy is the selective loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the effect of advanced glycation end products (AGEs) on apoptotic cell death in bovine retinal pericytes (BRPs). After incubation of BRPs with 0.47, 1.88, 7.5, 30 microM of AGE-bovine serum albumin (BSA) for 4 days, we assayed the pericytes apoptosis by FACS (fluorescence activated cell sorting), and further measured the signaling pathway involved. The results showed that AGE-BSA could induce significantly the apoptosis of BRPs in a dose-dependent manner compared with controls, associated with an increase in intracellular malondialdehyde level and caspase-3 activity; a decrease in intracellular catalase, SOD activities and Bcl-2/Bax ratio. SOD and selective caspase-3 inhibitor Z-DEVD-fmk can inhibit pericyte apoptosis induced by AGE-BSA. These data suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that elevated AGE observed in diabetes may cause apoptosis in BRPs through an oxidative stress mechanism. The decreased Bcl-2/Bax ratio and activation of caspase-3 are associated with apoptotic process. PMID- 16674982 TI - Exercise training promotes expression of apelin and APJ of cardiovascular tissues in spontaneously hypertensive rats. AB - Because apelin may play an important regulatory role in human cardiac dysfunction, we investigated alterations in cardiovascular content of apelin and its receptor, APJ, during hypertension and the effect of exercise training on the cardiovascular apelin/APJ system in hypertensive animals. Spontaneously hypertensive rats (SHRs) underwent swimming training consisting of 54 swimming sessions of 60 min each (6 days/week for 9 weeks). Systolic blood pressure (SBP) was verified weekly by tail-cuff plethysmography. Apelin levels in plasma and cardiovascular tissues were determined by radioimmunoassay. The level of apelin/APJ mRNA was determined by RT-PCR. SHRs showed severe hypertension and pathological cardiomegaly. The level of apelin immunoreactivity (apelin-ir) in plasma and ventricular and aortic tissues was lower, by 40%, 40% and 42% (all P<0.01), respectively, in SHRs than in control Wistar-Kyoto rats, and the mRNA level of apelin and APJ in myocardium and aorta was markedly decreased. Compared with sedentary SHRs, swimming-trained SHRs showed decreased SBP and elevated mRNA expression of apelin and APJ in cardiovascular tissues and elevated apelin-ir level in plasma, myocardium and aorta (all P<0.01). SBP and level of apelin-ir in plasma and cardiovascular tissues were negatively correlated. Long-term swimming training relieved the pathogenesis of hypertension and reversed the downregulation of the cardiovascular apelin/APJ system induced by hypertension, which suggests that the improving effect of exercise training on hypertension could be mediated by upregulating the cardiovascular apelin/APJ system. PMID- 16674983 TI - Learning sequence movements in a homogenous sample of patients with Parkinson's disease. AB - We investigated the acquisition of sequence movements in Parkinson's disease (PD) by means of the serial reaction time (SRT) task. To this end, we used a sample of PD patients that fell within the same stage of the disease. Sixteen PD patients and 16 age-, sex- and education-matched control subjects performed the SRT task with a first-order conditional (FOC) sequence and with a second-order conditional (SOC) sequence. The results showed that the group of PD patients could be divided into two distinct subgroups: a fast PD patient subgroup (n=11) and a slow PD patient subgroup (n=5). FOC and SOC sequence learning in faster PD patients proved to be highly comparable to the group of controls. In contrast, learning of FOC and SOC sequences was severely impaired in slower PD patients. Since slow PD patients also scored lower on measures of cognitive functioning than faster PD patients, we assume that the deficits in SRT learning of the former reflect some more general cognitive impairment. This indicates that SRT performance can provide additional information about the cognitive abilities of PD patients, and accordingly may contribute to disease screening. PMID- 16674984 TI - Competitive adsorption of phosphate and phosphonates onto goethite. AB - Phosphate and phosphonates are both strongly adsorbed onto mineral surfaces and their removal during wastewater treatment is mainly due to adsorptive processes. We have conducted experiments to study the mutual influence of phosphate and six different phosphonates on each other in buffered medium at pH 7.2. We have used phosphonates having one to five phosphonic acid groups (HMP, IDMP, HEDP, NTMP, EDTMP and DTPMP). The presence of phosphonates suppressed the adsorption of phosphate. The monophosphonate HMP had the smallest and the polyphosphonates the largest effect on phosphate adsorption. The presence of phosphate lowered phosphonate adsorption. The competition in the multicomponent system can reasonably well be predicted using a surface complexation model developed for single component systems. The competitive model only failed in systems containing the polyphosphonate DTPMP. With this approach we can predict the behavior of both compounds during wastewater treatment. The calculations show that phosphonates have a small effect on phosphate adsorption at the actual concentrations in observed wastewater. Adsorption of low concentrations of phosphonates was calculated to be significantly reduced by phosphate concentrations as observed in wastewater. PMID- 16674985 TI - Characterization of atmospheric particulates, particle-bound transition metals and polycyclic aromatic hydrocarbons of urban air in the centre of Athens (Greece). AB - The concentrations of trace metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed to total suspended particulate (TSP) and finer fractions of airborne particulate matter (PM) were determined from a site in the centre of Athens (Greece), which is characterized by heavy local traffic and is densely populated, during the winter and summer periods in 2003-2004. Also, we collected and analyzed samples of diesel and gasoline exhaust particles from local vehicles (buses, taxis and private cars) and from chimney exhaust of residential central heating appliances. A seasonal effect was observed for the size distribution of aerosol mass, with a shift to larger fine fractions in winter. The most commonly detected trace metals in the TSP and PM fractions were Fe, Pb, Zn, Cu, Cr, V, Ni and Cd and their concentrations were similar to levels observed in heavily polluted urban areas from local traffic and other anthropogenic emissions. Analysis of 16 PAHs bound to PM showed that they are mostly traffic related. In general, the fine particulate PAHs concentrations were higher than coarse particles. The most common PAHs in PM(10.2) and PM(2.1) were pyrene, phenanthrene, acenapthylene and fluoranthene, which are associated with diesel and gasoline exhaust particles. The results of this study underlined the importance of local emission sources, especially vehicular traffic, central heating and other local anthropogenic emissions. Compared with other big cities, Athens has much higher levels of airborne particles, especially of the finer fractions PM(10) and PM(2.5), correlated with traffic-related air pollution. PMID- 16674986 TI - Mercury toxicity induces oxidative stress in growing cucumber seedlings. AB - In this study, the effects of exogenous mercury (HgCl(2)) on time-dependent changes in the activities of antioxidant enzymes (catalase and ascorbate peroxidase), lipid peroxidation, chlorophyll content and protein oxidation in cucumber seedlings (Cucumis sativus L.) were investigated. Cucumber seedlings were exposed to from 0 to 500microM of HgCl(2) during 10 and 15 days. Hg was readily absorbed by growing seedlings, and its content was greater in the roots than the in shoot. Time and concentration-dependent reduction in root and shoot length was observed at all concentrations tested, equally in the roots and shoot, at both 10 and 15 days. At 50microM HgCl(2), root fresh weight of 15-day-old seedlings increased, and at other concentrations, it reduced. For 10-day-old seedlings, reduction in root and shoot fresh biomass was observed. At 15 days, only at 50microM HgCl(2) was there no observed reduction in shoot fresh biomass. Dry weight of roots increased at 500microM both at 10 and 15 days, though at 250microM HgCl(2) there was only an increase at 15 days. There was a significant effect on shoot dry weight at all concentrations tested. Hg-treated seedlings showed elevated levels of lipid peroxides with a concomitant increase in protein oxidation levels, and decreased chlorophyll content when exposed to between 250 and 500microM of HgCl(2). At 10 days, catalase activity increased in seedlings at a moderately toxic level of Hg, whereas at the higher concentration (500microM), there was a marked inhibition. Taken together, our results suggest that Hg induces oxidative stress in cucumber, resulting in plant injury. PMID- 16674987 TI - Transport of molybdenum in a biosolid-amended alkaline soil. AB - The transport of molybdenum (Mo) in a biosolid-amended, alkaline, agricultural soil was examined in the research reported herein. Batch-equilibrium and miscible displacement experiments were conducted to examine the transport of Mo in soil with and without 10% by weight biosolid amendment. The results of geochemical modeling, conducted using PHREEQC, indicated that no mineral dissolution or precipitation reactions were expected for the system under the prevailing conditions. Sorption equilibrium coefficients (Kd) obtained from moment analysis of the Mo breakthrough curves were similar to those calculated from the results of the batch-equilibrium experiments. Mo sorption was greater for the biosolid amended soil (Kd of 1.3 versus 0.35). Sorption of Mo was shown to be linear, rate limited, and reversible for both unamended and amended soil. The results suggest that Mo associated with biosolid-amended soils is relatively bioavailable and mobile. PMID- 16674988 TI - Source identifications of PM10 aerosols depending on hourly measurements of soluble components characterization among different events in Taipei Basin during spring season of 2004. AB - Continuous measurements of hourly PM10 soluble ions were performed by the in situ IC technology in order to assess the impact of Asian outflows on local air quality. The intensive aerosol observation was carried in Taipei from 11 February 22:00 to 7 April 19:00, 2004. Concentrations of the water-soluble ions (Cl(-), NO(-)(2), NO(-)(3), SO(2-)(4), Na(+), NH(+)(4), K(+), and Ca(2+)) were measured in a total of 3,300 samples. The characteristics of air pollutant events in Taipei Basin were classified as frontal dust, dust, northeast monsoon, south wind and sea/land breeze according to the hourly meteorology and air pollutant concentrations. Factor analysis was conducted based on hourly data for 13 variables to find the group of variables with similar behavior. According to the source characteristics of high loading species, the possible sources of PM10 aerosols in each group were identified. Three to four factors were identified for each event. The total variances of frontal dust, dust, northeast trade, south wind, and sea/land breeze events were explained about 85%, 86%, 76%, 77%, and 80%, respectively, indicating that the identified factors were satisfactory. PMID- 16674989 TI - Fluorescence-based monitoring of tracer and substrate distribution in an UASB reactor. AB - In this work, rhodamine-related fluorescence was measured on-line at four reactor heights in order to study hydrodynamics within an upflow anaerobic sludge bed reactor. A linear dependence of the dispersion coefficient (D) on the upflow velocity was observed, while the influence of the organic loading rate (OLR) was insignificant. Furthermore, the Bodenstein number of the reactor loaded with granulated sludge was found to be position-dependent with the largest values measured at the bottom of the sludge bed. This trend was not observed in the reactor without sludge. Chemical oxygen demand (COD) and volatile fatty acid (VFA) concentrations were measured at the same reactor heights as in rhodamine tests using conventional off-line analytical methods and on-line multiwavelength fluorometry. Significant spatial COD and VFA gradients were observed at organic loading rates above 6g COD l(R)(-1)d(-1) and linear upflow velocities below 0.8m h(-1). PMID- 16674990 TI - The phytotoxic effect of C(1)/C(2)-halocarbons and trichloroacetic acid on the steppe plant Artemisia lerchiana. AB - Artemisia lerchiana is a wormwood species of the Central Asian steppe regions, where it completely cover whole areas. For the first time it was possible to show through field experiments that C(1)/C(2) halocarbons (VCHCs), such as chloroform (CHL), tetrachloroethene (PER) and hexachloroethane (HEX), can be taken up by test plants of the species A. lerchiana via the soil/root pathway and metabolised inter alia into trichloroacetic acid (TCA) under semi-aride conditions. At the same time, chlorophyll a fluorescence measurements carried out on the test plants revealed a phytotoxic influence on plant vitality (max. decline in vitality of 52% with application of CHL) and less efficient energy flows in the photosynthesis mechanism of the A. lerchiana test plants. The authors examine possible links between the simultaneous appearance of VCHCs and additional drought stress in the acceleration of desertification processes. PMID- 16674991 TI - Glutathione levels and enzyme activity in the tissues of bank vole Clethrionomys glareolus chronically exposed to a mixture of metal contaminants. AB - The biochemical response to chronic heavy metal exposure was studied in tissues of bank voles Clethrionomys glareolus. Animals were collected from three sites located 4, 8 and 30km from a zinc-lead smelter, the area's main source of metal contamination. Concentrations of Cd, Pb, Zn and Fe were measured in the liver, kidneys and gonads to assess the level of metal intoxication. In response to intoxication, organisms activate detoxification mechanisms which can protect animals from metals' toxicity. Glutathione plays an important role in toxic substance detoxification. Total glutathione (tGSH) and glutathione disulfide (GSSG) were measured in the tissues. Also, the activity of glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione-S-transferase (GST) was measured in the studied tissues. Results indicate that levels of all studied parameters were tissue and site-dependent. Evidence indicates that the most sensitive parameter of metal toxicity for animals living in a chronically contaminated environment is the GSH/GSSG ratio. In our study, the GSH/GSSG ratio was decreased in the liver of animals with high Cd levels. However, the relationship between Pb and the GSH/GSSG ratio was positive in the gonads. Cadmium and lead negatively influenced GPX activity in the liver; this was probably connected with inhibition of the Se-dependent fraction. The relationship between iron and GR activity in the kidney was also negative, but other correlations for iron both in liver and kidney were not significant. Positive correlations between Zn levels and GST and GR activity were found in the gonads of bank voles. PMID- 16674992 TI - Ecological risk assessments of endocrine disrupting organotin compounds using marine neogastropods in Hong Kong. AB - As active ingredients of anti-fouling paints that are widely used on ship hulls, organotin compounds, in particular tributyltin (TBT), are well-known endocrine disruptors causing sex changes in marine organisms and widespread in coastal waters and sediments worldwide. In this study, a comprehensive ecological risk assessment (ERA) of organotins was conducted in Hong Kong waters through determining the imposex status, sex ratio and tissue burdens of these compounds in the neogastropods, Thais clavigera and Thais luteostoma collected from 29 coastal sites. We also investigated the historical trend of organotin effects on these gastropods, and performed a probabilistic ERA based on tissue burden of TBT in the animals. Our results demonstrated that imposex indices were positively correlated with the body burden of organotins in the gastropods. Across all sites, the sex ratio (female:male) decreased significantly with increasing imposex levels or tissue burden of organotins, implying that such pollutants can result in a male-biased population, potentially leading to local extinction in extreme cases. Based on the ERA, 5.4% of all populations of T. clavigera are at risk due to exposure to TBT; the risks include growth inhibition, impairment of immune functions and reduced fitness. Seriously impacted areas included Aberdeen, Repulse Bay, Butterfly Beach, Mui Wo and Ha Mei Wan. A comparison with historical data revealed that there had been some improvement in the areas with low marine traffic, and distant from the major harbour/port. This could partly be due to the restriction on the use of TBT on small vessels (<25m in length) since 1992. Nevertheless, the organotin contamination still remains severe in areas with high marine traffic or adjacent to large harbours/ports. In particular, the situation in the northeastern waters of Hong Kong has been getting worst since 1996 that is probably associated with the rapid development of the cargo container port at Yantian in China. PMID- 16674993 TI - Leaching of terbumeton and terbumeton-desethyl from mini-columns packed with soil aggregates in laboratory conditions. AB - Leaching of terbumeton (TER) and terbumeton-desethyl (TED) from mini-columns packed with natural soil aggregates was investigated. Five soil samples from the Champagne area (France) with different physicochemical parameters were used. The soil samples were hand-packed into a 50 mm column in laboratory conditions. An aqueous solution of TER or TED was percolated through the column and collected effluents were analyzed for TER or TED using HPLC-DAD. The leaching experiments showed that TER and TED were moderately mobile. TED was more mobile than TER, possibly because of its higher polarity. The proportion of organic matter affected the mobility of TER and TED through soil columns (r=0.971) and leaching was lowest for soil having the highest organic matter content (5.9%). TER and TED were not significantly influenced by leaching solution composition (deionized water or CaCl(2) solution), but were strongly affected by soil packing. Packing resulted in less rapid release of compounds suggesting that unpacking may have contributed to preferential pathways through the soil columns. Increasing contact time between TER and soils before leaching decreased the mobility of TER and increased its persistence in soils. Indeed, 76% of TER was released when leaching started after a 15 h contact time whereas it was down to 26% after an aging treatment of 360 h. A proportion of TER (from 8% to 32%) and TED (from 8% to 17%) remained in soil. Associated to its high stability in soils this could in part account for a very slow transfer over the years towards the groundwater. PMID- 16674994 TI - A morphological investigation of soot produced by the detonation of munitions. AB - The morphology of three different detonation soot samples along with other common soot materials such as carbon black, diesel soot and chimney soot was studied by elemental and proximate analysis, X-ray diffraction and electron microscopy. The goal of this study was to better define the morphology of the detonation soot in order to better assess the interactions of this type of soot with explosive residues. The detonation soot samples were obtained by the detonation of artillery 155mm projectiles filled with either pure TNT (2,4,6-trinitrotoluene) or composition B, a military explosive based on a mixture of TNT and RDX (trimethylentrinitramine). The carbon content of the soot samples varied considerably depending on the feedstock composition. Detonation soot contains less carbon and more nitrogen than the other carbonaceous samples studied, due to the molecular structure of the energetic materials detonated such as TNT and RDX. The ash concentration was higher for detonation soot samples due to the high metal content coming from the projectiles shell and to the soil contamination which occurred during the detonation. By X-ray diffraction, diamond and graphite were found to be the major crystalline carbon forms in the detonation soot. Two electron microscopy techniques were used in this study to visualise the primary particles and to try to explain the formation mechanism of detonation soot samples. PMID- 16674995 TI - Multivariate data analysis of organochlorines and brominated flame retardants in Baltic Sea guillemot (Uria aalge) egg and muscle. AB - Concentrations of organochlorines (OCs) and brominated flame-retardants (BFRs) were determined in guillemot (Uria aalge) eggs from the island of Stora Karlso in the Baltic Sea where 10 eggs/year were collected in the years 2000, 2001 and 2002. The dominating contaminant in egg was p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) with a geometric mean (GM) concentration of 18200 ng/g lipid weight (lw). The GM concentration in egg of hexabromocyclododecane (HBCD) of 140 ng/glw, was significantly higher than that of polybrominated diphenyl ether (summation operatorPBDE) of 77 ng/glw. For the evaluation of the data multivariate data analysis techniques namely principal components analysis (PCA), partial least squares regression (PLS), soft independent modelling of class analogy (SIMCA classification), and PLS discriminant analysis (PLS-DA), were used. We investigated whether the eggs' biological characteristics co-varied with egg concentrations of OCs and BFRs, and found e.g., significant negative correlations between egg weight and concentrations of HCB and p,p'-DDE. A PLS model with analyzed BFRs as the Y matrix and OCs as the X matrix could, with varying accuracy, calculate the concentrations of BFRs in the individual egg from their concentrations of OCs (e.g., R(2)Y of 0.89 for BDE47, and of 0.50 for HBCD). Lastly, we compared the contaminant concentrations in the eggs to those in previously analyzed pectoral muscles from adult guillemots from Stora Karlso, from the year 2000. A PLS-DA model, showed that some of the contaminants (e.g., HBCD and CB28) had significantly higher concentrations in egg than in muscle, although 7 of the 14 contaminants showed no difference in concentrations between the two matrices. PMID- 16674996 TI - Assessing the effects of the three herbicides acetochlor, 2,4,5 trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid on the compound action potential of the sciatic nerve of the frog (Rana ridibunda). AB - To assess the relative toxicity of the herbicides acetochlor and 2,4,5 trichlorophenoxyacetic acid (2,4,5-T) on the nervous system, the sciatic nerve of the frog (Rana ridibunda) nerve was incubated in saline inside a specially designed recording chamber. This chamber permits monitoring of the evoked compound action potential (CAP) of the nerve, a parameter that could be used to quantify the vitality of the nerve in normal conditions as well as when the nerve was exposed to the compounds under investigation. Thus, when the nerve was exposed to acetochlor, the EC(50) was estimated to be 0.22mM, while for 2,4,5-T the EC(50) was 0.90mM. Using the identical nerve preparation, the EC(50) of 2,4-D was estimated to be 3.80mM [Kouri, G., Theophilidis, G., 2002. The action of the herbicide 2,4-dichlorophenoxyacetic acid on the isolated sciatic nerve of the frog (Rana ridibunda). Neurotoxicol. Res. 4, 25-32]. The ratio of the relative toxicity for acetochlor, 2,4,5-T and 2,4-D was found to be 1:4:17.2. However, because it is well-known that the action of 2,4-D is dependent on the pH, the relative toxicity of the three compounds was tested at pH 3.3, since it has been found that the sciatic nerve of the frog is tolerant of such a low pH. Under these conditions, the EC(50) was 0.77mM (from 0.22mM at pH 7.2) for acetochlor, 0.20mM (from 0.90mM) for 2,4,5-T and 0.24mM (from 3.80mM at pH 7.2) for 2,4-D. Thus, the relative toxicity of the three compounds changed drastically to 1:0.25:0.31. This change in the relative toxicity is due not only to the increase in the toxicity of 2,4,5-T and 2,4-D at low pH levels, but also to the decrease in the toxicity of acetochlor at pH 3.3. PMID- 16674997 TI - Migration of fallout radiocaesium in a grassland soil from 1986 to 2001. Part I: activity-depth profiles of (134)Cs and (137)Cs. AB - The temporal changes of the vertical distribution of (134)Cs (deposited by the Chernobyl fallout in 1986) and (137)Cs (deposited by the Chernobyl and the global fallout) in the soil were investigated at an undisturbed Bavarian grassland site in Germany. At ten sampling dates between 1986 and 2001, the activity density of (134)Cs and (137)Cs was determined in various soil layers down to 80 cm depth. In 2001, the small-scale spatial variability of the radiocaesium activity was determined by sampling five plots within 10 m(2) (coefficient of variation about 20% for the upper soil layers). Between 1987 and 1990, substantial changes of the activity-depth profiles were observed. The percentage depth distributions of (134)Cs and (137)Cs were rather similar. The 50%-depth of the accumulated activity increased from 2.4 cm in 1988 to 5.3 cm in 2001 for (134)Cs and from 2.7 to 5.8 cm for (137)Cs. This indicates that at the study site the migration data of Chernobyl-derived (137)Cs can be estimated by those of total (137)Cs. In the second part of this study, the activity-depth profiles will be evaluated by the convection-dispersion model [Schimmack, W, Feria Marquez, F. Migration of fallout radiocaesium in a grassland soil from 1986 to 2001. Part II: Evaluation of the activity-depth profiles by transport models. Sci Total Environ 2006-this issue]. PMID- 16674998 TI - Influence of Asian dust storms on air quality in Taiwan. AB - In each year, dust storms triggered by cold air masses passing through northern China and Mongolia enhance the PM10 concentration over Taiwan region during winter and spring. On average, there are four to five dust events and 6.1 dust days in a year in Taiwan. Each event lasts for 1 day or even longer. A procedure to identify a dust event is rationalized and exercised on data collected during 1994-2005. Also, a ranking method named as the dust intensity rank (DIR) is developed to distinguish the intensity of each event affecting the local air quality. About 86% of dust days belong to ranks 1 and 2. In general, poorer air quality is associated with higher ranks. Ranks 4 and 5 correspond to a PSI (Pollution Standard Index) larger than 100. Linking DIR with the popular PSI is useful for both the public and the official forecasting system. It is also useful for inter-comparison between dust influences on air quality at different downstream regions in Taiwan. Composite analyses of the temporal and spatial variation of the hourly PM10 level indicate that dust particles usually arrive 12 h before the time of the peak PM10 concentration and last for 36 h at northern Taiwan, while the time of the peak concentration at eastern or western Taiwan, due to the evolution of the synoptic weather system, is about 3-12 h later. It is noted that the increase of PM10 level at the western side of Taiwan results from a mixture of upstream Asian dust inputs and local pollutants. PMID- 16674999 TI - What is the optimal pharmacological prophylaxis for the prevention of deep-vein thrombosis and pulmonary embolism in patients with acute ischemic stroke? AB - BACKGROUND: Pulmonary embolism after acute ischemic stroke (AIS) is associated with a high in-hospital mortality. The benefit from pharmacological prophylaxis for venous thromboembolism (VTE) is uncertain probably due to doubts about the optimal agent and dose. We evaluated the benefit/risk ratio of different anticoagulant regimens in the prevention of VTE in patients with AIS. METHODS: The MEDLINE, EMBASE, and Cochrane Library databases were searched up to January 2005. Randomized controlled trials (RCT) comparing early administration of either low-molecular-weight heparin (LMWH) or unfractionated heparin (UFH) with control were included. Endpoints were objectively diagnosed deep-vein thrombosis (DVT), pulmonary embolism, intracranial hemorrhage (ICH), and extracranial hemorrhage (ECH). Low-dose UFH was arbitrarily defined as < or =15,000 IU/day, low-dose LMWH as < or =6000 IU/day or weight-adjusted dose of < or =86 IU/kg/day. RESULTS: Sixteen trials involving 23,043 patients with AIS met the inclusion criteria. The number of events was small and different doses of anticoagulant treatment were used. Compared to control, high-dose UFH was associated with a reduction in pulmonary embolism (OR=0.49, 95% confidence interval (CI)=0.29-0.83), but also with an increased risk of ICH (OR=3.86, 95% CI=2.41-6.19) and ECH (OR=4.74, 95% CI=2.88-7.78). Low-dose UFH decreased the thrombosis risk (OR=0.17, 95% CI=0.11 0.26), but had no influence on pulmonary embolism (OR=0.83, 95% CI=0.53-1.31); the risk of ICH or ECH was not statistically significant increased (OR=1.67, 95% CI=0.97-2.87 for ICH; and OR=1.58, 95% CI=0.89-2.81 for ECH, respectively). High dose LMWH decreased both DVT (OR=0.07, 95% CI=0.02-0.29) and pulmonary embolism (0.44, 95% CI=0.18-1.11), but this benefit was offset by an increased risk for ICH (OR=2.01, 95% CI=1.02-3.96) and ECH (OR=1.78, 95% CI=0.99-3.17). Low-dose LMWH reduced the incidence of both DVT (OR=0.34, 95% CI=0.19-0.59) and pulmonary embolism (OR=0.36, 95% CI=0.15-0.87), without an increased risk of ICH (OR=1.39, 95% CI=0.53-3.67) or ECH (OR=1.44, 95% CI=0.13-16). For low-dose LMWH, the numbers needed to treat were 7 and 38 for DVT and pulmonary embolism, respectively. CONCLUSIONS: Indirect comparison of low and high doses of UFH and LMWH suggests that low-dose LMWH have the best benefit/risk ratio in patients with acute ischemic stroke by decreasing the risk of both DVT and pulmonary embolism, without a clear increase in ICH or ECH. PMID- 16675001 TI - DSP-4 prevents dopamine receptor priming by quinpirole. AB - Repeated treatments of rats with the dopamine (DA) D2 receptor agonist quinpirole, consistently produce long-lived DA D2 receptor supersensitization, by the process that has been termed priming. Rats so-primed in ontogeny behaviorally demonstrate adulthood enhancement of low-dose quinpirole-induced yawning. Because 1) dopaminergic neurons originate in midbrain nuclei (substantia nigra and ventral tegmental area), and 2) noradrenergic neurons originate in pontine (locus coeruleus) and medullary areas, it might be presumed that these two monoaminergic systems are independent, not interdependent. However, in the present study we demonstrate that there was an attenuation of quinpirole-enhanced yawning at 8 weeks in rats that were 1) primed by repeated neonatal quinpirole HCl treatments (50 microg/kg per day SC) during the first ten days of postnatal ontogeny, and 2) lesioned at 3 days after birth with DSP-4 (N-2-chloroethyl-N-ethyl-2 bromobenzylamine hydrochloride, 50 mg/kg SC). Dose-effect curves indicated a 23 45% reduction in yawning by DSP-4 treatment of quinpirole-primed rats, acutely treated as adults with quinpirole (25, 50, or 100 microg/kg). Effectiveness of DSP-4 is reflected by the 95% and 99% reductions in norepinephrine contents of frontal cortex and hippocampus, respectively (HPLC/ED method). The findings are supportive of a modulatory role of noradrenergic fibers on dopamine receptor priming (supersensitization) in rat brain. PMID- 16675000 TI - Antithrombotic effects of aspirin based on PLA1/A2 glycoprotein IIIa polymorphism in patients with coronary artery disease. AB - OBJECTIVE: The diallelic glycoprotein IIIa polymorphism P1A1/A2 was attributed to be an inherited risk factor for coronary events. Whether this polymorphism affects response to aspirin in patients with coronary artery disease is not known. METHODS: We assessed thrombin generation (prothrombin fragment F1+2) in consecutive blood samples collected from bleeding-time wounds in 28 men with coronary artery disease; P1A2 carriers, n=9; P1A1/A1, n=19. Thrombin generation and bleeding time were measured before and after 2 weeks of aspirin 300 mg/day. RESULTS: Aspirin-depressed thrombin generation in A1 homozygotes (p=0.04), but not in A2 carriers. Bleeding time after aspirin was also prolonged in A1 subjects only (p=0.02). CONCLUSION: Genotyping for glycoprotein IIIa polymorphism might be helpful in predicting antithrombotic action of aspirin in secondary prevention of coronary artery disease. PMID- 16675002 TI - Modulation of a (+)amphetamine discriminative stimulus in rats by 8-hydroxy-2 (N,N-di-n-propylamino)tetralin (8-OH DPAT). AB - It is well established that the discriminative stimulus (DS) effect of amphetamine involves a dopaminergic and/or noradrenergic mechanism. These catecholamines can be modulated by the 5-HT(1A) serotonin receptor agonist 8 hydroxy-2-(N,N-di-n-propylamino)tetralin (8-OH DPAT). The present study was conducted to determine whether 8-OH DPAT could influence the DS effects of (+)amphetamine. Administration of 8-OH DPAT doses to Sprague-Dawley rats trained to discriminate 1 mg/kg of (+)amphetamine (ED(50)=0.33 mg/kg) using a two-lever operant paradigm (VI-15 s schedule of reinforcement for appetitive reward) failed to result in stimulus generalization when administered alone, and failed to antagonize the stimulus effect when administered in combination with the training dose of (+)amphetamine. However, administration of 8-OH DPAT doses that produced saline-like responding (i.e., 0.01-0.1 mg/kg; <20% amphetamine-appropriate responding) in combination with the ED(50) dose of (+)amphetamine resulted in the animals' making a progressively greater number of responses on the drug appropriate lever such that a combination of 0.1 mg/kg of 8-OH DPAT plus (+)amphetamine (0.33 mg/kg) elicited 91% (+)amphetamine-appropriate responding. In a separate study, administration of (+)amphetamine doses in combination with fixed doses of 8-OH DPAT (either 0.01 or 0.1 mg/kg) resulted in an apparent leftward shift of the dose-response curve. The results indicate that (+)amphetamine can be more effective as a discriminative stimulus in the presence of 8-OH DPAT than in its absence. PMID- 16675003 TI - Waterpipe smoking among American military recruits. AB - BACKGROUND: Waterpipe smoking, a traditional Middle Eastern tobacco use method, has increased dramatically among Arab adolescents and young adults. Anecdotal evidence also suggests that usage is on the rise among young people in the U.S., but epidemiological data are lacking. METHODS: From self-report health surveys collected during 1999-2002, we examined the prevalence and predictors of waterpipe use among U.S. Air Force recruits (n = 20,673; mean age = 20.0 years; range = 17-35). RESULTS: Waterpipe use was reported by 0.3% (n = 59) of recruits and was unrelated to age, gender, ethnicity, or family income. Compared to non users, waterpipe users were more likely to plan to smoke cigarettes in the coming year (P value < 0.05) and to believe that switching from cigarettes to other tobacco products reduces smoking-related health risks (P values < 0.002). Multivariate (logistic regression) analyses revealed several factors that distinguished waterpipe users from non-users, including higher education level (odds ratio [OR] = 1.94), having experimented with cigarettes before Basic Military Training (BMT; OR = 1.99), and using cigarettes (OR = 2.17) and other tobacco products (OR = 13.81) at the time of entry into BMT. Compared to recruits who used cigarettes only, waterpipe smokers were more educated (OR = 1.83), more likely to have engaged in experimental (OR = 3.30) or regular (OR = 3.87) use of tobacco products other than cigarettes prior to BMT, and less likely to have been a current (OR = 0.10) or former (OR = 0.11) smoker at the time of entry into BMT. CONCLUSION: Despite concerns that waterpipe smoking is increasing among young people in the U.S., use was low among military recruits. PMID- 16675004 TI - Mammography screening rates decline: a person-time approach to evaluation. AB - OBJECTIVES: Early detection through mammography can reduce breast cancer mortality. This cohort study evaluated trends in mammography screening, demonstrating a person-time approach. METHODS: Included were women HMO members aged 50-69 from 1999 to 2002 who had not had breast cancer, dysplasia, fibrocystic disease, or implant. The amount of person-time covered by mammography as a percent of the time eligible for mammography screening (the prevention index (PI)) was calculated using electronic data. The denominator was the time during which the guidelines recommended that each participant should have been covered by a mammogram (every 24 months), excluding times when breast mass, abnormal mammogram, galactorrhea, or other breast disorders were under evaluation. The numerator was the time during which she was covered by a mammogram. RESULTS: The number of women who contributed person-time increased from 43,283 to 49,512 and the number of screening mammograms declined from 23,586 to 22,719. The overall PI for screening mammography declined from 67.0 (67% of eligible person-time was appropriately covered by a mammogram) to 62.5, and the proportion of women with no coverage during a given year increased 16%. CONCLUSIONS: This study shows a declining pattern of mammography screening using a person-time approach, a decline greater than that shown by methods that include diagnostic mammograms. The study highlights opportunities for use of the PI and quality improvement initiatives to improve breast cancer outcomes. PMID- 16675005 TI - "Smoke-free Class Competition": far-reaching conclusions based on weak data. PMID- 16675006 TI - Are private school students more likely to smoke than public school students in China? AB - BACKGROUND: Tobacco use is prevalent among adolescents in China in general, however, little is known about tobacco use among students in private schools with an enrollment of 1.5 million. METHOD: In 2001, cross-sectional survey data from 2725 students in grades seven and eight (1307 sampled from private schools and 1418 sampled from public schools) were included. Smoking measures (risk of susceptibility to smoking, smoking onset, ever smoking, smoking in the past 7 days and past 30 days, established smoking) were compared between private and public school students using chi-square test, logistic regression, and survival analysis. RESULTS: Cigarette smoking was more prevalent among private school students than among public school students (private vs. public: 21.9% vs. 12.3% for susceptibility to smoking, 53.9% vs. 38.2% for ever smoking, 22% vs. 12.0% for 30-day smoking, 15.9% vs. 4.0% for 7-day smoking, 4.5% vs. 1.2% for established smoking). Students in private schools are 3.4 to 3.8 times more likely to smoke than students in public schools after the adjustment of important covariates (gender, grade, peer smoking, parental smoking, and parents' occupation). Risk of smoking onset by age was also greater for private school students than for public school students. CONCLUSIONS: Being in private schools was associated with three to four times increases in the likelihood of current cigarette smoking and heightened risk of smoking onset by age. Findings from this study suggest the urgent need to collect additional data on risk and protective factors as well as the willingness to participate in effective tobacco use intervention prevention among private school students. PMID- 16675007 TI - Worksite interventions to increase stair climbing; reasons for caution. AB - Point-of-choice prompts to use the stairs rather than the escalator consistently increase physical activity at public access staircases such as those in shopping malls. More recently, exercise promoters have targeted stair climbing in the worksite. A review of interventions in worksites reveals little hard evidence of successful increases in stair climbing, though the increases in stair usage are encouraging. The contrast between the worksites and public access staircases, however, is not simply one of location. In a worksite, the choice is between the stairs and an elevator rather than an escalator. We reason that the availability of the elevator or the stairwell may be the major immediate determinant of stair climbing in worksites and dilute any possible effects of an intervention. PMID- 16675008 TI - Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer. AB - The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage. PMID- 16675009 TI - Shape memory properties of poly(D,L-lactide)/hydroxyapatite composites. AB - Poly(D,L-lactide) (PDLLA) and Hydroxyapatite (HA) are compounded, which possess biodegradation, biocompatibility and shape memory properties. In the paper, we prepared serial imposing shape memory composites with different shapes, composite ratios and sample thicknesses. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) were carried out to examine surface morphology, glass transition temperature (Tg), dynamic mechanical properties, and shape memory effect of PDLLA/HA composites, respectively. Moreover, some interesting shape memory behaviors were investigated. The results show that the better disperse morphology of HA grains using the experiment methods, and PDLLA/HA composites at a definite range of compound ratio have much better shape memory effect than pure PDLLA polymer. It indicates that HA particles can improve shape memory effect and PDLLA/HA composites are potential for biomedical applications. PMID- 16675010 TI - Hemocompatibility evaluation of poly(glycerol-sebacate) in vitro for vascular tissue engineering. AB - Poly(glycerol-sebacate) (PGS) is an elastomeric biodegradable polyester that could potentially be used to engineer blood vessels in vivo. However, its blood material interactions are unknown. The objectives of this study were to: (a) fabricate PGS-based biphasic tubular scaffolds and (b) assess the blood compatibility of PGS in vitro in order to get some insight into its potential use in vivo. PGS was incorporated into biphasic scaffolds by dip-coating glass rods with PGS pre-polymer. The thrombogenicity (platelet adhesion and aggregation) and inflammatory potential (IL-1beta and TNFalpha expression) of PGS were evaluated using fresh human blood and a human monocyte cell line (THP-1). The activation of the clotting system was assessed via measurement of tissue factor expression on THP-1 cells, plasma recalcification times, and whole blood clotting times. Glass, tissue culture plastic (TCP), poly(l-lactide-co-glycolide) (PLGA), and expanded polytetrafluorethylene (ePTFE) were used as reference materials. Biphasic scaffolds with PGS as the blood-contacting surface were successfully fabricated. Relative to glass (100%), platelet attachment on ePTFE, PLGA and PGS was 61%, 100%, and 28%, respectively. PGS elicited a significantly lower release of IL 1beta and TNFalpha from THP-1 cells than ePTFE and PLGA. Similarly, relative to all reference materials, tissue factor expression by THP-1 cells was decreased when exposed to PGS. Plasma recalcification and whole blood clotting profiles of PGS were comparable to or better than those of the reference polymers tested. PMID- 16675011 TI - Up-regulation of inositol 1,4,5-trisphosphate receptor type 1 is responsible for a decreased endoplasmic-reticulum Ca2+ content in presenilin double knock-out cells. AB - Presenilins (PS) are proteins involved in the pathogenesis of autosomal-dominant familial cases of Alzheimer's disease. Mutations in PS are known to induce specific alterations in cellular Ca2+ signaling which might be involved in the pathogenesis of neurodegenerative diseases. Mouse embryonic fibroblasts (MEF) deficient in PS1 and PS2 (PS DKO) as well as the latter rescued with PS1 (Rescue), were used to investigate the underlying mechanism of these alterations in Ca2+ signaling. PS DKO cells were characterized by a decrease in the [Ca2+]ER as measured by ER-targeted aequorin luminescence and an increased level of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The lower [Ca2+]ER was associated with an increase in a Ca2+ leak from the ER. The increased IP3R1 expression and the concomitant changes in ER Ca2+ handling were reversed in the Rescue cells. Moreover using RNA-interference mediated reduction of IP3R1 we could demonstrate that the up-regulation of this isoform was responsible for the increased Ca2+ leak and the lowered [Ca2+]ER PS DKO cells. Finally, we show that the decreased [Ca2+]ER in PS DKO cells was protective against apoptosis. PMID- 16675012 TI - Characterization of a rolling-circle replication plasmid from Thermus aquaticus NTU103. AB - The thermophilic bacterium Thermus aquaticus NTU103 harbors a 1,965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames (orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double stranded origin and single-stranded origin sites, indicating the importance of these cis elements in pTA103 replication. PMID- 16675013 TI - DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14. AB - A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses. PMID- 16675014 TI - Effects of methyl parathion on Chasmagnathus granulatus hepatopancreas: protective role of sesamol. AB - The protective role of sesamol, an inhibitor of the mixed function oxygenase (MFO) system, against histopathological effects of methyl parathion in the hepatopancreas of the estuarine crab, Chasmagnathus granulatus, was studied. Exposure (72 h) to a sublethal dose (0.05 mg/kg/day; 10% of 72 h-LD50) of injected methyl parathion increased the percentage of damaged hepatopancreatic tubules. Presence of melanin-like deposits in the connective tissue between hepatopancreatic tubules was also observed. Antioxidant enzyme activities (catalase and glutathione S-transferase) and lipid peroxidation (LPO) levels were also increased in hepatopancreas of crabs injected with methyl parathion. Pretreatment with sesamol (0.85 mg/kg/day) significantly protected against all these effects. These findings suggest that the hepatopancreatic damages induced by methyl parathion are due to LPO of hepatopancreatocytes membranes, as a consequence of the oxidative stress generated after methyl parathion oxidative biotransformation mediated by the MFO system. PMID- 16675015 TI - Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli. AB - Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis. PMID- 16675016 TI - Monomeric and polymeric IgA show a similar association with the myeloid FcalphaRI/CD89. AB - IgA is found in both mucosal secretions and serum and is the dominant immunoglobulin isotype produced in humans. It exists in different molecular forms, namely monomeric IgA, dimeric IgA, polymeric IgA and secretory IgA, all exhibiting interactions with FcalphaRI/CD89 to some extent. CD89 is an activating, gamma-chain associated, Fc receptor for IgA expressed on myeloid cells. Here, we investigated the interaction of monomeric and polymeric IgA purified from human serum with CD89 using surface plasmon resonance. The results demonstrate a similar association for monomeric and polymeric IgA with CD89. In contrast, monomeric IgA dissociated more rapidly from CD89 than polymeric IgA. Removal of N-glycans from mIgA resulted is an increased association with CD89, whereas the dissociation was more rapid, resulting in binding comparable to that of untreated monomeric IgA. We conclude that the initial interaction of monomeric and polymeric IgA with CD89 is similar, whereas monomeric IgA dissociates more rapidly from CD89. In view of the large excess of monomeric IgA in serum, monomeric IgA will compete for CD89 interaction with polymeric IgA, thereby preventing cell activation initiated by receptor aggregation contributing to the anti-inflammatory role of IgA. PMID- 16675017 TI - Lipopolysaccharide (LPS) regulates TLR4 signal transduction in nasopharynx epithelial cell line 5-8F via NFkappaB and MAPKs signaling pathways. AB - The lipopolysaccharide (LPS) of Gram-negative bacteria induces the expression of cytokines and proinflammatory genes via the TLR4 signaling pathway in diverse cell types. The purpose of the present study was to test the hypothesis that the nasopharynx epithelial cells (NECs) could recognize and respond to LPS. The underlying molecular mechanisms were further elucidated in the NEC line 5-8F for its ability to activate the NFkappaB and TNF-alpha reporter genes, in response to LPS. After LPS stimulation, the TNF-alpha promoter activity and the relevant production of TNF-alpha were significantly increased in 5-8F cells. Moreover, LPS activated NFkappaB p65, ERK1/2 and JNK1/2 and induced their translocation to the nucleus. Western blot analysis showed that the expression of NFkappaB p65, MEK1, ERK1/2, JNK1/2, phospho-ERK1/2 and phospho-JNK1/2 proteins also was increased in NEC 5-8F cells, following the LPS stimulation. Additionally, the expression of TLR1-6, MD2 and CD14 was examined by RT-PCR, and the CD14 expression was determined by flow cytometry analysis. We demonstrated that the expression of CD14, TLR4 and MD2 was crucial for the NEC responses to LPS. In conclusion, our results provide novel mechanisms for the response of nasopharnyx epithelial cells to LPS stimulation, through NFkappaB and MAPKs signaling pathways. PMID- 16675018 TI - Splicing of NOD2 (CARD15) RNA transcripts. AB - Mutated variants of NOD2, a cytosolic Toll-like receptor (TLR) that recognizes bacterial peptidoglycan, are responsible for increased susceptibility to Crohn's disease (CD). TLRs and their related plant counterparts, the disease-resistance R proteins, undergo alternative splicing as a means of controlling activity. Here we report that regions of NOD2 RNA transcripts that encode the N-terminal and leucine-rich repeat (LRR) domains are alternatively spliced, potentially creating at least eight putative NOD2 variants. The most common variant is a short truncated isoform designated NOD2-short which terminates at residue position 820 leaving three LRR domains. An N-terminally spliced variant designated NOD2-190 contains only CARD1 and a partial CARD2 domain. The expression of transcripts encoding full-length and alternatively spliced forms of NOD2 was altered in blood mononuclear cells and monocytic cell lines stimulated by bacterial products. NOD2 short and NOD2-190 were inactive and unresponsive to muramyl dipeptide (MDP), but did not antagonize the activity of wild-type NOD2. Alternative splicing of NOD2 transcripts represents a potential mechanism by which the intracellular bacterial sensing activity of NOD2 is altered or down-regulated. PMID- 16675019 TI - Factors associated with childhood strabismus: findings from a population-based study. AB - PURPOSE: To describe strabismus prevalence and associated factors in a representative sample of 6-year-old Australian children. DESIGN: Population-based cross-sectional study. PARTICIPANTS: One thousand seven hundred thirty-nine predominantly 6-year-old children resident in Sydney examined in 2003 and 2004. METHODS: Cover testing was performed at near and distance fixation, and with spectacles if worn. Logarithm of the minimum angle of resolution visual acuity was measured in both eyes before and after pinhole correction, after correcting any cylindrical refraction >0.50 diopters and with spectacles, if worn. Cycloplegic autorefraction (cyclopentolate) and detailed dilated fundus examination were performed. Each child's medical and perinatal histories were sought in a detailed parental questionnaire. MAIN OUTCOME MEASURES: Strabismus was defined as any heterotropia at near or distance fixation, or both, on cover testing. Microstrabismus was defined as a deviation of fewer than 10 prism diopters. RESULTS: Strabismus was diagnosed in 48 children (2.8% of the population), 5 of whom had previously undergone surgical correction; 26 children (54%) had esotropia, 14 (29%) had exotropia, 7 (15%) had microstrabismus, and 1 child had VIth cranial nerve palsy. Prematurity was associated with a 5-fold increase in the risk of esotropia (odds ratio, 5.0; 95% confidence interval, 1.8 14.1). Visual impairment (with presenting correction) was significantly more common in children with (22.9%) than without (1.3%) strabismus (P<0.0001). The presence of strabismus was significantly associated with hyperopia, astigmatism, anisometropia, and amblyopia (P<0.0001). CONCLUSIONS: This report documents the prevalence of strabismus and its relation to other ocular signs and visual impairment in a representative sample of Australian school children. Presence of strabismus was significantly associated with prematurity. PMID- 16675020 TI - Subphrenic abscess and recurring focal lesions due to tuberculosis in a patient with IgA deficiency. AB - Subdiaphragmatic abscess has not yet been reported as a manifestation of tuberculosis. We report an IgA deficient patient with recurrent episodes of unusual extrapulmonary manifestations of tuberculosis including subdiaphragmatic abscesses and metacarpophalangeal osteomyelitis that was improved each time with antituberculosis drugs. There was not any resistance to the drugs used against mycobacterium despite repeated courses of antituberculosis regimens. In such recurrent cases, one should rule out any immunodeficiency states. PMID- 16675021 TI - Cerebral cortex astroglia and the brain of a genius: a propos of A. Einstein's. AB - The glial fibrillary acidic protein immunoreactive astroglial layout of the cerebral cortex from Albert Einstein and other four age-matched human cases lacking any known neurological disease was analyzed using quantification of geometrical features mathematically defined. Several parameters (parallelism, relative depth, tortuosity) describing the primate-specific interlaminar glial processes did not show individually distinctive characteristics in any of the samples analyzed. However, A. Einstein's astrocytic processes showed larger sizes and higher numbers of interlaminar terminal masses, reaching sizes of 15 microm in diameter. These bulbous endings are of unknown significance and they have been described occurring in Alzheimer's disease. These observations are placed in the context of the general discussion regarding the proposal--by other authors--that structural, postmortem characteristics of the aged brain of Albert Einstein may serve as markers of his cognitive performance, a proposal to which the authors of this paper do not subscribe, and argue against. PMID- 16675022 TI - A reducing and denaturing step maximizes the immunoprecipitations of m-calpain and I-2(PP2A)/SET: an approach toward antibodies that do not work well in immunoprecipitation. AB - Immunoprecipitation is an elegant method to isolate a specific protein of interest from a complex protein mixture such as cell lysate. We tried to increase the efficiency of m-calpain immunoprecipitation with anti-m-calpain antibodies directed toward denatured antigens that only work for immunoblotting and immunohistochemistry. We found that a reducing and denaturing step prior to immunoprecipitation greatly potentiates the efficiency of the immunoreaction. This improved method is also applicable for the immunoprecipitation of oncoprotein I-2(PP2A)/SET with antibodies directed toward a synthetic peptide that only work for immunoblotting. Thus, our improved method provides a way to maximize immunoprecipitation when using antibodies that do not work well under conventional immunoprecipitation conditions. Furthermore, the improved method is also suitable for decreasing the contaminating proteins during immunoprecipitation. PMID- 16675023 TI - Continuous flow magnetic cell fractionation based on antigen expression level. AB - Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry. PMID- 16675024 TI - Detection of nonlinear event-related potentials. AB - The methods used to evaluate event-related potentials (ERPs) are generally insensitive to nonlinear responses. Our goal was to show that nonlinear ERPs could be detected using recurrence analysis (RA). When fixed-phase sine signals were added to baseline electroencephalograms (EEGs), the added linear determinism was detected by signal averaging, as expected, and by RA. However, when nonlinear determinism was simulated by adding either random-phase sine or Lorenz signals, the added signals were detected only by RA. Auditory evoked potentials (AEPs) were studied in five subjects using RA. We detected not only the characteristic linear effects caused by onset and offset of the sound, but also nonlinear AEPs not previously reported; they occurred at 473-661 ms after onset, and 282-602 ms after offset, depending on the subject. In five other subjects we found nonlinear magnetosensory evoked potentials; they occurred at 209-354 ms after field onset, depending on the subject. RA was less sensitive than time averaging for detecting linear ERPs, but had the advantage of being able to detect nonlinear ERPs. PMID- 16675025 TI - fMRI in alert, behaving monkeys: an adaptation of the human infant familiarization novelty preference procedure. AB - Functional magnetic resonance imaging (fMRI) is increasingly used in non-human primate research. In the present study, we adapt the familiarization-novelty preference (FNP) procedure used in human infant research to examine visual behavior in alert, unanaesthetized rhesus monkeys that were acclimated to the fMRI environment, but did not undergo behavioral training. In keeping with the typical FNP paradigm, we recorded eye movements (looking time and number of fixations) while monkeys viewed a series of four identical pictures (familiarization phase) followed by two different pictures (novelty phase). Number of fixations and looking time both increased during the novelty phase, thereby demonstrating visual discrimination of the new from the old picture. Importantly, discrimination did not occur on catch trials in which six identical pictures were presented. Moreover, brain activation in the amygdala was more strongly associated with the novelty phase than with the familiarization phase. In addition, magnitude of brain activation in the amygdala was correlated with the behavioral effect of visual discrimination. These findings demonstrate the feasibility of using eye movements as an index of visual discrimination in untrained monkeys during fMRI scanning. This methodological approach helps to extend the repertoire of research tools for fMRI in non-human primates. PMID- 16675027 TI - EEG analysis using wavelet-based information tools. AB - Wavelet-based informational tools for quantitative electroencephalogram (EEG) record analysis are reviewed. Relative wavelet energies, wavelet entropies and wavelet statistical complexities are used in the characterization of scalp EEG records corresponding to secondary generalized tonic-clonic epileptic seizures. In particular, we show that the epileptic recruitment rhythm observed during seizure development is well described in terms of the relative wavelet energies. In addition, during the concomitant time-period the entropy diminishes while complexity grows. This is construed as evidence supporting the conjecture that an epileptic focus, for this kind of seizures, triggers a self-organized brain state characterized by both order and maximal complexity. PMID- 16675026 TI - Functional cortical source imaging from simultaneously recorded ERP and fMRI. AB - Feasibility of continuously and simultaneously recording visual evoked potentials (VEPs) with fMRI was assessed by quantitatively comparing cortical source images by means of receiver operating characteristic (ROC) curve analysis. The averaged EEG source images coincided well with simultaneously acquired fMRI activations. Strong correlation was found between the cortical source images of VEPs recorded inside and outside the scanner. Application of fMRI prior information strengthened correlation between estimated source images as well as resulted in source estimates with higher spatial resolution. The present results demonstrate that reliable cortical source images can be acquired during simultaneous fMRI scanning and they may be used for multimodal functional source imaging studies. PMID- 16675028 TI - Toward a unitary perspective on the bipolar spectrum and substance abuse: opiate addiction as a paradigm. AB - Bipolar spectrum disorders and addiction often co-occur and constitute reciprocal risk factors that the authors believe are best considered under a unitary perspective. In particular, we submit that patients whose disorders fall under the bipolar spectrum - and its hyperthymic and cyclothymic temperamental substrates are at increased risk for substance use, possibly moving towards addiction through exposure to intrinsically dependence-producing substances. In our experience, the contribution of bipolar spectrum disorders to the addictive process is often clinically missed, because attenuated and subclinical expressions of such mood disorders as bipolar II and cyclothymia are not adequately appreciated by our current formal diagnostic system (e.g. DSM-IV, as well as research and clinical practice based on it). The use of agonist treatment in dual diagnosis heroin addicts has allowed us to gather valuable knowledge about the intrinsic, and historically and clinically documented mood-regulating effects of opiates. From the therapeutic point of view, the challenge of double diagnosis requires double competence from clinicians. The combination of opiate agonists and mood stabilizers often produces results difficult to obtain with the use of the two types of drugs separately. We therefore submit that the present conceptualization of the link between bipolar spectrum and addictive disorders has not only heuristic and scientific values, but also an important message for the clinician. PMID- 16675029 TI - Impact of the life goals group therapy program for bipolar patients: an open study. AB - BACKGROUND: To evaluate the level of satisfaction of bipolar patients participating in the structured group Life Goals Program (LGP), as well as the impact of this psychoeducative program on mood stability, treatment compliance and relapse prevention. METHODS: Forty-five patients (30 females and 15 males) with bipolar disorder were included. Their level of satisfaction was evaluated at the end of the 6-week phase 1 of LGP and after one-year participation in phase 2 (median duration 366 days, range 68-483). The impact of the LGP was also evaluated with respect to mood stability, compliance with pharmacological treatment and ability to prevent and cope with relapse. Participants' mood was rated at study entry, end of phase 1 and end of phase 2 with the Montgomery and Asberg Depression Rating Scale (MADRS) and Bech-Rafaelsen Mania Scale. RESULTS: Thirty-six participants (80.0%) completed phase 1 and 17 entered phase 2. A majority of participants (82.4%) were very satisfied with the information delivered during phase 1. After attending phase 2, participants reported a subjective improvement in mood stability, relapse prevention strategies as well as coping with relapse. MADRS score decreased significantly between the beginning of phase 1 and end of phase 2 (p=0.016). LIMITATIONS: This is an open study and results need to be replicated in a randomized controlled trial. CONCLUSIONS: Results of this open study suggest that the life goals psychoeducative program is well perceived by bipolar patients and has a positive impact on mood stability, relapse prevention and ability to go beyond symptoms in order to reach specific goals. PMID- 16675030 TI - Affective disorder in the parents of a clinic sample of children with anxiety disorders. AB - BACKGROUND: Family history studies in adults reveal strong familiality for the anxiety disorders with some specificity. The aim of the current study was to establish whether there was an elevated rate of anxiety disorders in the parents of children with anxiety disorders, and whether there was intergenerational specificity in the form of disorder. METHODS: The mental state of a clinic sample of 85 children with anxiety disorder and their parents was systematically assessed, together with a comparison sample of 45 children with no current disorder and their parents. RESULTS: Compared to the rate of anxiety disorder amongst parents of comparison children, the rate of current anxiety disorder in mothers of anxious children was significantly raised, as was the lifetime rate of anxiety disorder for both mothers and fathers. The mothers of children with generalised anxiety disorder, social phobia, specific phobia and separation anxiety disorder all had raised lifetime rates of the corresponding disorder, but also raised rates of others disorders. LIMITATIONS: Only 60% of the fathers of the anxious children were assessed. CONCLUSIONS: Strong familiality of anxiety disorders was confirmed, especially between child and maternal anxiety disorder. All child anxiety disorders were associated with several forms of anxiety disorder in the mother. Some specificity in the form of anxiety disorder in the child and the mother was apparent for social phobia and separation anxiety disorder. The findings have implications for the management of child anxiety. PMID- 16675031 TI - Chronic otitis media (COM) -- a personal philosophy. PMID- 16675032 TI - Detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay. AB - The recombinant antigen obtained by cloning and expressing two IBV nucleocapsid protein fragments (143-414 aa, 281-414 aa) in Escherichia coli was used for the detection of avian infectious bronchitis virus (IBV) specific antibodies in chicken sera by the indirect ELISA (rNpIBV-ELISA). As a result of testing 1524 serum samples the diagnostic sensitivity and specificity of rNpIBV-ELISA when comparing those of the routine whole IBV ELISA have been shown to be 93.81% and 87.36%, respectively. The agreement value was 91.5%. PMID- 16675033 TI - New mosaic subgenotype of varicella-zoster virus in the USA: VZV detection and genotyping by oligonucleotide-microarray. AB - A rapid and sensitive microarray-based method was used to distinguish the three major circulating genotypes of varicella-zoster virus (VZV). The method analyzes five variable positions located in a 447-nucleotide variable region 1 of open reading frame 22 (ORF 22r1); these single nucleotide polymorphisms (SNP) display in stably occurring patterns specific to each of the VZV genotypes established in previously published studies. Pairs of short oligonucleotide probes (oligoprobes) with sequences corresponding to all of the observed SNP were used to detect specific sequences. Fluorescently labeled ssRNA samples for hybridization with a chip were prepared by in vitro T7 polymerase driven transcription of the amplicons of ORF 22r1, followed by chemical labeling with Cy5 into RNA sample. Ratios between fluorescent hybridization signals from each pair of oligoprobes were used to assess the sequence at each SNP. We evaluated six reference VZV strains and 130 VZV clinical specimens to validate the method. The microarray method accurately identified strains isolated in the US in 2001-2002, representing all major genotypes as determined using more extensive sequence analysis, correctly assigning strains to genotypes E (81.5%), J (3%) and M (15.5%). In addition, a new M variant (M3) was identified. PMID- 16675034 TI - Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes. AB - Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples. PMID- 16675035 TI - Comparison of INNO-LiPA HPV Genotyping v2 with PCR product subcloning and sequencing for identification of genital human papillomavirus genotypes in African women. AB - The performance characteristics of the INNO-LiPA Genotyping v2 test for human papillomavirus (HPV) identification were assessed by comparing results with those obtained by PCR product sequencing after subcloning, in genital samples from 20 highly sexually exposed African women. The INNO-LiPA HPV Genotyping v2 test identified more HPV types than subcloning/sequencing (56 versus 37, respectively). Overall, 86.5% (32/37) of the HPV types identified by subcloning/sequencing were identified by the INNO-LiPA HPV Genotyping v2 test, whereas 57.1% (32/56) of the HPV types identified by the INNO-LiPA HPV Genotyping v2 test were identified by subcloning/sequencing. Of the 20 clinical samples tested, 7 had identical types detected under both methods and a further 11 had more types detected under INNO-LiPA HPV Genotyping v2 than subcloning/sequencing. Of the remaining two samples, the same number of types were detected under both methods, but different types were detected. INNO-LiPA HPV Genotyping v2 test appears as a valid method for identifying HPV subtypes in women with multiple HPV infection. PMID- 16675036 TI - Herpes simplex encephalitis: adolescents and adults. AB - Herpes simplex encephalitis (HSE) remains one of the most devastating infections of the central nervous system despite available antiviral therapy. Children and adolescents account for approximately one third of all cases of HSE. Clinical diagnosis is suggested in the encephalopathic, febrile patient with focal neurologic signs. However, these clinical findings are not pathognomonic because numerous other diseases in the central nervous system can mimic HSE. Neurodiagnostic evaluation can provide support for the diagnosis by the demonstration of temporal lobe edema/hemorrhage by magnetic resonance image scan and spike and slow-wave activity on electroencephalogram. In the current era, the diagnostic gold standard is the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid by polymerase chain reaction (PCR). Although PCR is an excellent test and preferable to brain biopsy, false negatives can occur early after disease onset. Acyclovir is the treatment of choice and is administered at 10 mg/kg every 8 h for 21 days. Even with early administration of therapy after the disease onset, nearly two thirds of survivors have significant residual neurologic deficits. Current investigative efforts are assessing the prognostic value of quantitative PCR detection of viral DNA at the onset of therapy as well as at the completion of therapy and the contribution of prolonged antiviral therapy to improved neurologic outcome. PMID- 16675037 TI - Rhinovirus chemotherapy. AB - Human rhinoviruses (HRV), members of the Picornaviridae family, are comprised of over 100 different virus serotypes. HRV represent the single most important etiological agents of the common cold [Arruda, E., Pitkaranta, A., Witek Jr., T.J., Doyle, C.A., Hayden, F.G., 1997. Frequency and natural history of rhinovirus infections in adults during autumn. J. Clin. Microbiol. 35, 2864-2868; Couch, R.B., 1990. Rhinoviruses. In: Fields, B.N., Knipe, D.M. (Eds.), Virology. Raven Press, New York, pp. 607-629; Turner, R.B., 2001. The treatment of rhinovirus infections: progress and potential. Antivir. Res. 49 (1), 1-14]. Although HRV-induced upper respiratory illness is often mild and self-limiting, the socioeconomic impact caused by missed school or work is enormous and the degree of inappropriate antibiotic use is significant. It has been estimated that upper respiratory disease accounts for at least 25 million absences from work and 23 million absences of school annually in the United States [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Rotbart, H.A., 2002. Treatment of picornavirus infections. Antivir. Res. 53, 83-98]. Increasing evidences also describe the link between HRV infection and more serious medical complications. HRV-induced colds are the important predisposing factors to acute otitis media, sinusitis, and are the major factors in the induction of exacerbations of asthma in adults and children. HRV infections are also associated with lower respiratory tract syndromes in individuals with cystic fibrosis, bronchitis, and other underlying respiratory disorders [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Gern, J.E., Busse, W.W., 1999. Association of rhinovirus infections with asthma. Clin. Microbiol. Rev. 12 (1), 9-18; Pitkaranta, A., Arruda, E., Malmberg, H., Hayden, F.G., 1997. Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-PCR. J. Clin. Microbiol. 35, 1791-1793; Pitkaranta, A., Virolainen, A., Jero, J., Arruda, E., Hayden, F.G., 1998. Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction. Pediatrics 102, 291-295; Rotbart, H.A., 2002. Treatment of picornavirus infections. Antivir. Res. 53, 83-98]. To date, no effective antiviral therapies have been approved for either the prevention or treatment of diseases caused by HRV infection. Thus, there still exists a significant unmet medical need to find agents that can shorten the duration of HRV-induced illness, lessen the severity of symptoms, minimize secondary bacterial infections and exacerbations of underlying disease and reduce virus transmission. Although effective over-the-counter products have been described that alleviate symptoms associated with the common cold [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Gwaltney, J.M., 2002a. Viral respiratory infection therapy: historical perspectives and current trials. Am. J. Med. 22 (112 Suppl. 6A), 33S-41S; Turner, R.B., 2001. The treatment of rhinovirus infections: progress and potential. Antivir. Res. 49 (1), 1-14; Sperber, S.J., Hayden, F.G., 1988. Chemotherapy of rhinovirus colds. Antimicrob. Agents Chemother. 32, 409-419], this review will primarily focus on the discovery and development of those agents that directly or indirectly impact virus replication specifically highlighting new advances and/or specific challenges with their development. PMID- 16675038 TI - Inhibition of feline (FIPV) and human (SARS) coronavirus by semisynthetic derivatives of glycopeptide antibiotics. AB - Various semisynthetic derivatives of glycopeptide antibiotics including vancomycin, eremomycin, teicoplanin, ristocetin A and DA-40926 have been evaluated for their inhibitory activity against feline infectious peritonitis virus (FIPV) and human (SARS-CoV, Frankfurt-1 strain) coronavirus in cell culture in comparison with their activity against human immunodeficiency virus (HIV). Several glycopeptide derivatives modified with hydrophobic substituents showed selective antiviral activity. For the most active compounds, the 50% effective concentrations (EC(50)) were in the lower micromolar range. In general, removal of the carbohydrate parts of the molecules did not affect the antiviral activity of the compounds. Some compounds showed inhibitory activity against both, whereas other compounds proved inhibitory to either, FIPV or SARS-CoV. There was no close correlation between the EC(50) values of the glycopeptide derivatives for FIPV or SARS-CoV. PMID- 16675039 TI - Differentiation between capsaicin-induced allodynia and hyperalgesia using a thermal operant assay. AB - Investigations of new analgesic treatments ideally are coupled with the use of compassionate methods for pain testing in animals. Recently, we described a novel operant thermal testing device that can be used to quantify orofacial pain. The objective of the current study was to differentiate thermal allodynia from hyperalgesia using this operant thermal assay. Rats were trained to complete a task whereby they had a conflict between a positive reward and tolerance for thermal nociceptive stimulation. They were subjected to cool to hot temperatures (24-45 degrees C) and evaluated under naive (untreated), capsaicin cream (0.075%), capsaicin/morphine, or morphine test conditions. The following outcome measures were evaluated: reward intake; licking contacts; facial contacts; time to complete 25, 50, and 75% of the events (licks and face contacts); facial contact duration; ratio of reward/stimulus contacts; and ratio of facial contact duration/event. Capsaicin produced an increase in mechanical sensitivity and a significant thermal allodynic effect at 42 degrees C and hyperalgesic effect at 45 degrees C. These effects were blocked with morphine pre-treatment. The temporal profile for completing the task was also significantly altered following capsaicin treatment. These data demonstrate that using the operant orofacial assay in conjunction with capsaicin cream can provide a reproducible, sensitive, minimally invasive, and powerful approach for quantifying and studying enhanced thermal pain within the trigeminal system. This technique provides an alternative to reflex tests of orofacial sensitivity, and it presents a pivotal link for translating basic pain research into clinic trial strategies. PMID- 16675040 TI - Influence of salinity and organic matter on silver accumulation in Gulf toadfish (Opsanus beta). AB - To help extend the freshwater based biotic ligand model for silver (Ag) into brackish and saltwater conditions, 50g Gulf toadfish (Opsanus beta) were acclimated to 2.5%, 5%, 10%, 20%, 40%, 80%, or 100% salt water and exposed for 6d to 1.0microM AgNO(3), with or without 10mg C/L organic matter. Suwannee River natural organic matter collected by reverse osmosis was used. Silver accumulation in toadfish gills and plasma decreased as salinity increased, indicating low bioavailability of AgCl complexes. Complexation of Ag by organic matter, normally important in freshwater conditions, was less important as salinity increased. Although relatively little intestinal Ag uptake was observed, both liver and bile accumulated Ag from water imbibed past the isosmotic salinity point ( approximately 1/3 salt water). Toadfish also produced intestinal carbonate pellets, minerals which did not influence Ag accumulation. Our results further stress the importance of Ag speciation, physiological mechanisms, and intestinal Ag uptake when modelling Ag uptake and toxicity beyond freshwater conditions. PMID- 16675042 TI - Biological activity and antidiabetic potential of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide, GIP(1-16) and (Pro3)GIP(1-16). AB - Glucose-dependent insulinotropic polypeptide (GIP) is a key physiological insulin releasing peptide and potential antidiabetic agent. The present study was undertaken in an attempt to develop small molecular weight GIP agonist and antagonist molecules. The bioactivity of two modified C-terminally truncated fragment GIP peptides, GIP(1-16) and (Pro3)GIP(1-16), was examined in terms of insulin secretion and glucose homeostasis using BRIN-BD11 cells and type 2 diabetic mice. In vitro insulin release studies demonstrated that GIP(1-16) and (Pro3)GIP(1-16) possessed weak GIP-receptor agonist and antagonistic properties, respectively. Intraperitoneal administration of GIP(1-16) in combination with glucose to obese diabetic (ob/ob) mice did not effect the glycaemic excursion and had a marginal effect on insulin release. GIP(1-16) was substantially less effective than the native GIP(1-42). (Pro3)GIP(1-16) administration significantly curtailed (P < 0.05) the insulinotropic and glucose lowering effects of native GIP, but was significantly less effective than (Pro3)GIP. Based on the established concept of a therapeutic benefit of GIP receptor antagonism in obesity-diabetes, ob/ob mice received once daily injection of (Pro3)GIP(1-16) for 14 days. No significant effects were observed on food intake, body weight, HbA1c, glucose tolerance, metabolic response to feeding and either insulin secretion or insulin sensitivity following prolonged (Pro3)GIP(1-16) treatment. These data demonstrate that C-terminal truncation of GIP or (Pro3)GIP yields small molecular weight GIP molecules with significantly reduced biological activity that precludes therapeutic utility. PMID- 16675041 TI - Characterization of the Babesia gibsoni P18 as a homologue of thrombospondin related adhesive protein. AB - Thrombospondin related adhesive proteins (TRAPs) are well conserved among several apicomplexans. In this study, we reported the identification of the Babesia gibsoni P18, designated by our group previously, as a homologue of TRAP and renamed the P18 as the B. gibsoni TRAP (BgTRAP). The amino acid sequence of BgTRAP consists of several typical regions, including a signal peptide, a vonWillebrand factor A domain, a thrombospondin type 1 domain, a transmembrane region, and a cytoplasmic C-terminus. The B. gibsoni infected dog serum recognized recombinant BgTRAP expressed in E. coli by Western blotting. The antiserum against recombinant BgTRAP recognized an 80kDa protein in the lysate of infected erythrocytes (RBCs), which was detectable in the micronemal area of the parasites by confocal microscopic observation. The BgTRAP showed a bivalent cation-independent binding to canine RBC, and the specific antiserum was found to inhibit the growth of B. gibsoni in the infected severe combined immune deficiency mice given canine RBC. These results suggest that the BgTRAP is a new member of TRAP family identified from the merozoites of B. gibsoni and functionally important in merozoite invasion; this protein may be useful as a vaccine candidate against canine B. gibsoni infection. PMID- 16675043 TI - Protein-DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera. AB - Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning -467 to -371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from -965 to -534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions -360 to -355 bp, a potential site for interaction with basic helix-loop-helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression. PMID- 16675044 TI - Transcription promoter activity of the human S100A7 gene in oral squamous cell carcinoma cell lines. AB - The S100A7 (psoriasin) gene has been shown to be markedly over-expressed in squamous cell carcinomas (SCCs) as well as in psoriasis. We herein examined the S100A7 gene promoter activity in human oral SCC cell lines to identify the putative SCC-specific regulatory regions for the S100A7 transcription. Functional deletion assays of 5'-flanking region demonstrated that the segments, (-1513 to 988), (-1954 to -1513) and (-3040 to -2578), play important roles in the transcription activity in the oral SCCs. The internal deletion of the short segments, (-1248 to -1110), (-1109 to -988) and (-1248 to -988), decreased this activity. These segments cloned upstream of the heterologous promoter increased the promoter activity in oral SCC cell line. Electrophoretic mobility shift assays, using the sequence segmental probes, (-1248 to -1110) and (-1109 to 988), showed different DNA-protein complex patterns depending on the types of used cell lines. One of the complexes was only observed in the oral SCCs. These data suggested that the segment from -1513 to -988 contains up-regulatory elements for the transcription activity of the S100A7 gene in oral SCCs. PMID- 16675045 TI - Zinc transporters and the cellular trafficking of zinc. AB - Zinc is an essential nutrient for all organisms because this metal serves as a catalytic or structural cofactor for many different proteins. Zinc-dependent proteins are found in the cytoplasm and within many organelles of the eukaryotic cell including the nucleus, the endoplasmic reticulum, Golgi, secretory vesicles, and mitochondria. Thus, cells require zinc transport mechanisms to allow cells to efficiently accumulate the metal ion and distribute it within the cell. Our current knowledge of these transport systems in eukaryotes is the focus of this review. PMID- 16675046 TI - Effect of postprandial insulinemia and insulin resistance on measurement of arterial stiffness (augmentation index). AB - BACKGROUND: Arterial stiffness, specifically augmentation index (AIx), is an independent predictor of cardiovascular risk. Previous studies suggest that insulin infusion decreases AIx and that this response is attenuated in insulin resistance. Whether physiological postprandial insulinemia similarly affects AIx measurements, and whether insulin resistance modifies this response, has not been studied. METHODS: Seven relatively insulin-resistant and seven insulin-sensitive postmenopausal women received low-carbohydrate and high-carbohydrate high-fat meals on separate days. Glucose and insulin levels were measured for 360-min following meal consumption. AIx was measured by radial artery applanation tonometry at regular intervals postprandially. RESULTS: Postprandial increases in glucose and insulin were greater following the high-carbohydrate high-fat meal in both insulin-sensitive and insulin-resistant subjects. AIx decreased in both groups following both meals. In insulin-sensitive subjects, the postprandial reduction (incremental area above the curve) in AIx was greater following the high-carbohydrate vs. low-carbohydrate high-fat meal (-6821+/-1089 vs. -3797+/ 1171% x min, respectively, P=0.009). In contrast, in insulin-resistant subjects, postprandial AIx responses were similar following the meals, suggesting that insulin resistance is associated with impaired postprandial arterial relaxation. CONCLUSIONS: This study demonstrates that the carbohydrate content of a meal, and, hence, the magnitude of the postprandial glucose and insulin responses it elicits, are important determinants of postprandial AIx measurements. The further observation that insulin resistance modified this effect raises the possibility that this phenomenon is a contributor to increased cardiovascular risk in insulin resistance. The results indicate that future studies of AIx need to control for the effects of these potentially confounding variables and that measurement of AIx should be standardized with respect to meals. PMID- 16675047 TI - Skeletal muscle cells expressing VEGF induce capillary formation and reduce cardiac injury in rats. AB - BACKGROUND: We tested a preemptive combined cell/gene therapy strategy of skeletal myoblasts transfected with Ad(5)RSVVEGF-165 in an ischemia/reperfusion rat model to increase collateral blood flow to nonischemic heart tissue. METHODS: Lewis rats were injected with placebo (Control), 10(6) skeletal myoblasts (SkM), or 10(6) skeletal myoblasts transfected with Ad(5)RSVVEGF-165 (SkM(+)) into the left ventricle 1week before ischemia. Left ventricle end-diastolic pressure, scar area, and capillary density were assessed 4weeks later. RESULTS: Local expression of human vascular endothelial growth factor was accompanied by an increase in capillary density in the SkM(+) group compared with that in the SkM and Control groups (700+/-40 vs. 289+/-18 and 318+/-59capillaries/mm(2), respectively; p<0.05). After 3weeks, the myocardial scar area was reduced in SkM(+) vs. Control (5.3+/-0.4% and 14.8+/-1.6%, p<0.05), while injected cells alone (SkM) did not cause improvement compared with Control (11.8+/-2.1% vs. 14.8+/-1.6%, p>0.05). The decrease in the scar area in SkM(+) was accompanied by an increase in the capillary density compared with that in SkM and Control 30days after cell injection (1005+/-108 vs. 524+/-16 and 528+/-26capillaries/mm(2), respectively; p<0.05). The scar areas were discrete (5.3-14.8%) and left ventricle end diastolic pressure in all groups were comparable (p>0.05). CONCLUSIONS: The combined cell/gene therapy strategy of genetically modified myoblast cells expressing angiogenic factors injected into the myocardium induced capillary formation and prevented the extension and development of cardiac damage associated with ischemia/reperfusion in rats. PMID- 16675048 TI - Early and late determinants of C-reactive protein release in patients with acute coronary syndrome. AB - Increased levels of C-reactive protein (CRP) could be detected in subjects with acute coronary syndrome (ACS). Several factors, atherosclerosis, coronary flow impairment, myocardial necrosis, each one acting during a different, earlier or later, phase of ACS, are supposed to be involved in CRP release in case of ACS. Role and relevance of each factor, not mutually exclusive, still need to be comparatively evaluated but a cooperative synergism could be presumably hypothesized. PMID- 16675049 TI - A case of PAGOD syndrome with hypoplastic left heart syndrome. AB - PAGOD syndrome is an extremely rare congenital malformation complex involving multiple organs. These include pulmonary artery and lung hypoplasia, diaphragm defects, omphalocele, sex reversal or ambiguous genitalia, and complex cardiac defects. Eight cases have been reported to date. We report a case of PAGOD syndrome that is manifested by right diaphragm eventration, horseshoe lung with right lung hypoplasia, hypoplastic left heart syndrome (mitral atresia, aortic atresia), scimitar syndrome, agonadism with ambiguous genitalia. A karyotype revealed normal 46-XY. This patient received a modified Norwood procedure for hypoplastic left heart syndrome as an initial palliation and bidirectional cavopulmonary anastomosis as a second stage of operation. The postoperative courses were uneventful. This patient is waiting for Fontan operation. PMID- 16675051 TI - Quantification and evaluation of antimicrobial drug use in group treatments for fattening pigs in Belgium. AB - To control the emergence of antimicrobial resistance, knowledge of antimicrobial drug consumption is essential. Because consumption data are not available in Belgium, a study was conducted between March and October 2003 to investigate the antimicrobial drug consumption in pigs, using the treatment incidence based on the animal daily dose pig (ADDpig), the treatment incidence based on the used daily dose pig (UDDpig) (number of ADDpig or UDDpig/1,000 pigs at risk/day), and the ratio UDDpig/ADDpig. The sampling frame consisted of 821 pig herds that (a) used a closed or semi-closed production system, (b) were located in the most dense pig areas of Belgium, and (c) had at least 150 sows and 600 fattening pigs each. Of 50 randomly selected herds, all group treatments with antimicrobial drugs, applied to fattening pigs that were within 2 weeks of slaughter (median age 187 days), were collected retrospectively. The treatment incidence based on ADDpig for all oral and injectable antimicrobial drugs was 178.1 per 1,000 pigs at risk per day. The treatment incidence based on UDDpig shows that in reality fewer pigs were treated, namely 170.3 per 1,000 pigs at risk per day. Proportionally, the most often applied oral antimicrobial drugs were: doxycycline, amoxicillin, combination trimethoprim-sulphonamides and polymyxin E. The most often applied injectable antimicrobial drugs were long-acting amoxicillin and ceftiofur. The distribution of the UDDpig/ADDpig ratio per antimicrobial drug shows that 50-75% of the oral formulations were underdosed. Injectable formulations were almost always overdosed (>90%). PMID- 16675052 TI - Access to primary health care and health outcomes: the relationships between GP characteristics and mortality rates. AB - This paper analyses the impact of economic conditions and access to primary health care on health outcomes in Norway. Total mortality rates, grouped into four causes of death, were used as proxies for health, and the number of general practitioners (GPs) at the municipality level was used as the proxy for access to primary health care. Dynamic panel data models that allow for time persistence in mortality rates, incorporate municipal fixed effects, and treat both the number and types of GPs in a district as endogenous were estimated using municipality data from 1986 to 2001. We reject the significant relationship between mortality and the number of GPs per capita found in most previous studies. However, there is a significant effect of the composition of GPs, where an increase in the number of contracted GPs reduces mortality rates when compared with GPs employed directly by the municipality. PMID- 16675053 TI - Partial volume rat lung irradiation: the protective/mitigating effects of Eukarion-189, a superoxide dismutase-catalase mimetic. AB - BACKGROUND AND PURPOSE: The purpose of the current study was to elucidate the protective/mitigating effects of a SOD-catalase mimetic, Eukarion-189 (EUK-189), on DNA damage in rat lung following irradiation. The particular focus of these studies was the efficacy of EUK-189 when given after irradiation (mitigation). PATIENTS AND METHODS: We exposed whole or lower lungs of female Sprague-Dawley rats to doses ranging from 10 to 20.5 Gray (Gy) of (60)Co gamma rays. Animals in the EUK-189 treated groups received 2 or 30 mg/kg intraperitoneally (i.p.) at various times postirradiation (PI). A micronucleus assay was used to examine DNA damage at various times up to 16 weeks PI. RESULTS: Our results indicated that EUK-189 administration after irradiation is effective at reducing micronucleus formation in lung fibroblasts at various times following radiation exposure. Treatment with EUK-189 in the first 3 days after thoracic irradiation did not, however, modify the dose required to cause severe morbidity at 2-3 months after irradiation. CONCLUSIONS: The protection produced when Eukarion-189 was given shortly after irradiation suggests that DNA damage observed in the lung may be caused by chronic production of ROS induced by a chronic inflammatory response initiated by the radiation treatment. We speculate that our failure to observe protection against severe morbidity at 2-3 months may be because our treatment regime only blocked the initial wave of ROS production and that treatment needs to be more prolonged to suppress the effects of a chronic inflammatory response. PMID- 16675055 TI - [Role of the Neurocom Balance Master in assessment of gait problems and risk of falling in elderly people]. AB - INTRODUCTION: Falls in elderly people is currently a health service problem because of the multiple consequences. Numerous teams have been interested in predicting the risk of falling with clinical and instrumental tests. Our study investigated instrumental evaluation by use of the Neurocom Balance Master in the global assessment of gait problems and risk of falling in elderly people. PATIENTS AND METHODS: Transverse study concerning 60 subjects older than 65 years distributed in 2 groups of 30 subjects each according to the existence or not of falling incidents during the past year. Evaluation by the Balance Master involved the following items: 1) the modified Clinical Test for the Sensory Interaction on Balance (CTSIB), which estimates balance by measuring the speed of oscillation of the center of pressure (CP) with open then closed eyes and firm then mossy ground; 2) support monopodal 5" to the left then to the right, eyes open then closed in moderated speeds of oscillation of the CP; 3) passage from standing to sitting, in moderated speeds of oscillation of the CP; 4) limits of stability: the possibilities of moving the CP towards a predetermined target without moving the feet in moderated time and speed; 5) study of the step: determine length and width of the step as well as speed; 6) most about-turn: measure of speed of oscillation of the CP during the right then left about-turn; 7) clearing: the force of the impact and the oscillations of the CP during the clearing of an obstacle 10 cm high to measure leverage. RESULTS: The oscillation speed of the CP in the 2 groups during modified CTSIB, support monopodal 5", passage from standing to sitting, about-turn and clearing were significantly improved the group of the patients with falls (P < 0.05). The step, length and speed of these patients were significantly reduced, with no difference in width of the step between the 2 groups. In the evaluation of the limits of stability, only time necessary to reach the target was significantly increased in the group with falls. Finally, the indication of leverage and the force of impact on the ground measured by the test of clearing were more important in the group of fallers than in non-fallers. CONCLUSION: The Neurocom Balance Master estimates not only postural balance, but also the vestibulary system and reproduces the physiological conditions of daily life. It has a certain role in the early assessment of gait problems and the risk of falling. This system also allows for rehabilitation of the impaired balance and offers a profit with the biofeedback. PMID- 16675054 TI - N-acetylcysteine selectively protects cerebellar granule cells from 4 hydroxynonenal-induced cell death. AB - 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, has been shown to induce neurotoxicity accompanied by multiple events. To clarify mechanisms of neuroprotective compounds on HNE-induced toxicity, the protective effects of N-acetylcysteine (NAC), alpha-tocopherol (TOC), ebselen and S-allyl-L cysteine (SAC) were compared in cerebellar granule neurons. The decrease in MTT reduction induced by HNE was significantly suppressed by pretreatment of the neurons with 1000 microM NAC or 10 and 100 microM TOC; however, lactate dehydrogenase (LDH) release and propidium iodide (PI) fluorescence studies revealed that neuronal death was suppressed by NAC but not by TOC. Treatment of these neurons with HNE resulted in a drastic reduction of mitochondrial membrane potential, and this reduction was also prevented by NAC but not by TOC. Ebselen and SAC, a garlic compound, were unable to protect these neurons against HNE induced toxicity. Pretreatment with NAC also prevented HNE-induced depletion of intracellular glutathione (GSH) levels in these neurons. These results suggest that NAC, but not other antioxidants such as TOC, SAC and ebselen, exerts significant protective effects against HNE-induced neuronal death in cerebellar granule neurons, and that this neuroprotective effect is due, at least in part, to preservation of mitochondrial membrane potential and intracellular GSH levels. PMID- 16675056 TI - [Radiculopathy following intrathecal baclofen pump implantation]. AB - INTRODUCTION: Intrathecally delivered baclofen has been used as a treatment for severe spasticity since 1984. After a successful intrathecal baclofen trial, a programmable drug delivery system was implanted. Few early complications such as infection or hematoma are observed after this surgery. OBJECTIVE: To describe an unusual and unknown complication of intrathecal baclofen therapy. METHOD: We report 2 cases of complications of intrathecal baclofen therapy, radiculalgy, that appeared early after pump implantation. The clinical symptoms and computed tomography (CT) results are described. RESULTS: The first patient described pain, which evoked left S1 radiculopathic features. The second had left L5 radiculopathic involvement. The mean pain level was estimated on a 10-point visual analog scale as 7.5 (range 4-9). Lumbar CT scan showed a conflict between the symptomatic root and the catheter and eliminated other causes of the symptoms. Treatment with analgesic drugs was successful in 1 patient. The other presented with proximal disconnection of the catheter, which led to surgical replacement of the catheter. The pain disappeared after this surgery. CONCLUSION: Intrathecal baclofen therapy with a subcutaneously implanted progammable pump can be complicated by radiculalgy secondary to a conflict between the catheter and symptomatic root. The diagnosis is made by CT lumbar scan. If medical treatment is not sufficient, surgery could be proposed to replace the catheter. PMID- 16675057 TI - [Transforaminal epidural steroid injection and paraplegia: case report and bibliographic review]. AB - OBJECTIVES: We report a patient in whom paraplegia developed following transforaminal epidural injection of a corticosteroid and discuss the physiopathology of this complication, based on a literature survey. CASE REPORT: A 40-year-old man presented with low-back pain and symptoms of L4 radiculopathy due to degenerative disc herniation resistant to conventional medical treatment. Computed tomography revealed posterolateral L4-L5 disc protrusion. A dosage of 125 mg of hydrocortisone was given by epidural transforaminal L4-L5 left injection under radioscopy guidance. Within minutes following the injection, intense pain developed in both legs, with T12 complete paraplegia. Emergency magnetic resonance imaging (MRI) 2 hours later did not reveal spinal cord abnormalities. The patient underwent immediate surgery consisting of excising the protruding disc and extensive L3-L5 laminectomy. MRI performed 3 months later did not reveal medullar abnormalities. Six months later, the patient continued to show slow neurologic improvement, permitting him to walk with crutches and to stop intermittent self-catheterisation. DISCUSSION: The occurrence of paraplegia following epidural transforaminal injection of corticosteroids is a rare complication. To our knowledge, only 5 similar cases have been described. Most of the authors proposed that the mechanism of this complication is ischemia of the terminal cone due to accidental suppression of medullary blood supply. Direct lesion of a medullar artery, arterial spasm, or corticosteroid-induced occlusion due to undetected intra-arterial injection could lead to this medullar infarction. Anatomical variations of the path followed by the Adamkievicz artery strongly support this hypothesis. PMID- 16675059 TI - Effects of snacks on energy intake: an evolutionary perspective. AB - The question addressed in this paper is whether the consumption of snacks contributes to higher energy intake and body weight in humans. Currently available snacks have a higher energy density than most of the foods that were available in Paleolithic diets. Humans have a weak defense against overeating, which is a functional trait from an evolutionary perspective. Various studies found that people do not compensate their energy intake after the consumption of snacks. This is particularly true for energy-containing drinks, which provide calories in liquid form. It is concluded that snack consumption may contribute to a positive energy balance. PMID- 16675058 TI - [Retraining between months 4 and 6 after anterior cruciate ligament reconstruction with hamstring graft: comparison between cycling and running with an untrained operated subject group]. AB - OBJECTIVE: To study the effect of cycling or running retraining between 4 and 6 months after patients underwent anterior cruciate ligament reconstruction with hamstring grafting (Semitendinosus-Gracilis) compared with that in patients who had the same surgery but were untrained. METHOD: Patients who had undergone surgery for an anterior cruciate ligament reconstruction by the same surgeon who used hamstring grafting were included if they were free of knee pain 4 months after the surgery. After giving consent, patients were randomized to receive controlled retraining (cycling or running 3 times a week) or not. The effect of retraining was measured by the evolution of the knee isokinetic peak torque at 60 degrees/s and 180 degrees/s 6 months after surgery. RESULTS: Fifteen patients were retrained with cycling (GI), 17 with running (GII) and 15 patients did not retrain (GIII). Before retraining, the 3 groups had the same peak torque deficit, measured at an angular speed of 60 degrees/s and 180 degrees/s, for knee extensors (GI: 33+/-11% and 27+/-8%; GII: 30+/-13% and 24+/-10%; GIII: 31+/-15% and 24+/-13%, respectively) and knee flexors (GI: 26+/-11% and 20+/-13%; GII: 20+/-14% and 17+/-13%; GIII: 19+/-15% and 14+/-15%, respectively). After retraining, progress measured at 60 degrees /s of knee extensors and flexors on the operated knees was 18+/-9% and 16+/-10% for GI, 16+/-9% and 11+/-11% for GII and 12+/-15% and 8+/-12 for GIII, respectively. Progress measured at 180 degrees /s followed the same evolution. After comparison of the 3 groups, any significant difference was put in relief according to the type of retraining. CONCLUSION: Retraining after anterior cruciate ligament reconstruction is necessary for patients to practice their previous sport. In our study, aerobic cycling or running between 4 and 6 months after surgery did not improve peak torque in the operated knee extensors and flexors. However, these 2 types of retraining are well-tolerated. PMID- 16675060 TI - Molecular cloning and sequence analyses of preprotemporin mRNAs containing premature stop codons from extradermal tissues of Rana tagoi. AB - The temporins are a family of hydrophobic, C-terminally alpha-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS(10)GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG(10)KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 microM) and producing hemolysis of human erythrocytes (LD50 = 50 microM). PMID- 16675061 TI - Apidaecin-type peptides: biodiversity, structure-function relationships and mode of action. AB - Apidaecins (apidaecin-type peptides) refer to a series of small, proline-rich (Pro-rich), 18- to 20-residue peptides produced by insects. They are the largest group of Pro-rich antimicrobial peptides (AMPs) known to date. Structurally, apidaecins consist of two regions, the conserved (constant) region, responsible for the general antibacterial capacity, and the variable region, responsible for the antibacterial spectrum. The small, gene-encoded and unmodified apidaecins are predominantly active against many gram-negative bacteria by special antibacterial mechanisms. The mechanism of action by which apidaecins kill bacteria involves an initial non-specific binding of the peptides to an outer membrane (OM) component. This binding is followed by invasion of the periplasmic space, and by a specific and essentially irreversible combination with a receptor/docking molecule that may be a component of a permease-type transporter system on inner membrane (IM). In the final step, the peptide is translocated into the interior of the cell where it meets its ultimate target. Evidence that apidaecins are non-toxic for human and animal cells is a prerequisite for using them as novel antibiotic drugs. This review presents the biodiversity, structure-function relationships, and mechanism of action of apidaecins. PMID- 16675062 TI - Influence of cholesterol and lovastatin on alpha-form of secreted amyloid precursor protein and expression of alpha7 nicotinic receptor on astrocytes. AB - The influence of cholesterol and the lovastatin (cholesterol-lowering drug) on secretion of alpha-secretase cleavage product of amyloid precursor protein (APP) and expression of nicotinic acetylcholine receptors (nAChRs) was investigated in human HTB-15 astrocytes. The results showed that exposure of cholesterol to astrocytes inhibited the secretion of alpha-form of secreted APP (alphaAPPs) and reduced cell viability, while lovastatin enhanced the alpha-secretase processing on astrocytes; cholesterol treatment decreased expression of alpha7 nAChR, whereas lovastatin induced an up-regulation of the receptor; the increase in alphaAPPs resulted from lovastatin was partially inhibited by the alpha7 nAChR antagonists, alpha-bungarotoxin or methyllycaconitine; cholesterol or lovastatin did not influence either whole APP level or expression of alpha4 nAChR. We suggest that high dose of cholesterol may inhibit both the activity of alpha secretase in APP metabolic processing and the expression of alpha7 nAChR, while lovastatin may stimulate alpha-secretase cleavage processing that might be regulated by alpha7 nAChR. PMID- 16675063 TI - Targeting soluble Abeta peptide with Tramiprosate for the treatment of brain amyloidosis. AB - Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in Alzheimer's disease (AD). Neurotoxicity results from the conformational transition of Abeta from random-coil to beta-sheet and its oligomerization. Among a series of ionic compounds able to interact with soluble Abeta, Tramiprosate (3 amino-1-propanesulfonic acid; 3APS; Alzhemedtrade mark) was found to maintain Abeta in a non-fibrillar form, to decrease Abeta(42)-induced cell death in neuronal cell cultures, and to inhibit amyloid deposition. Tramiprosate crosses the murine blood-brain barrier (BBB) to exert its activity. Treatment of TgCRND8 mice with Tramiprosate resulted in significant reduction (approximately 30%) in the brain amyloid plaque load and a significant decrease in the cerebral levels of soluble and insoluble Abeta(40) and Abeta(42) (approximately 20-30%). A dose dependent reduction (up to 60%) of plasma Abeta levels was also observed, suggesting that Tramiprosate influences the central pool of Abeta, changing either its efflux or its metabolism in the brain. We propose that Tramiprosate, which targets soluble Abeta, represents a new and promising therapeutic class of drugs for the treatment of AD. PMID- 16675064 TI - Weak independent association signals between IDE polymorphisms, Alzheimer's disease and cognitive measures. AB - Functional and genetic studies suggest that insulin-degrading enzyme (IDE) may be a strong functional and positional candidate. As there is a lack of consensus in regards to the level and location of IDE association signals we aimed to clarify these discrepancies through genotyping 28 SNPs in a large case-control collective together with quantitative measures of cognitive ability (MMSE). Four SNPs (rs11187007, rs2149632_ide12, rs11187033, rs11187040) were found to be associated with AD (nominal p<0.01). Tests with MMSE scores adjusted for disease duration identified associations, with the most significant result for rs1999763 (nominal p=0.008). Similarly, different reconstructed IDE haplotypes were associated with AD and higher MMSE scores. The association signals are only borderline significant after adjustment for multiple testing, but add further evidence to previous published results on the association between IDE and AD or MMSE. A subgroup analysis indicated more prominent associations with AD in younger, and with MMSE in older patients. There may be two independent effects mediated by IDE variants, risk for AD and modification of disease progression. PMID- 16675065 TI - Synthesis and antiproliferative activities of indolin-2-one derivatives bearing amino acid moieties. AB - A convenient synthesis of indolin-2-ones substituted in the 3 position by an aminomethylene group bearing different amino acid moieties is described. Their antiproliferative activities were evaluated toward a panel of human solid tumor cell lines (PC 3, DLD-1, MCF-7, M4 Beu, A549, PA 1) and healthy cell lines (a murine fibroblast L929 and a human fibroblast primary culture). PMID- 16675066 TI - Affinity of 1-aryl-1,2,3,4-tetrahydroisoquinoline derivatives to the ion channel binding site of the NMDA receptor complex. AB - A series of 1-aryl-1,2,3,4-tetrahydroisoquinoline and 8-methyl-1-aryl-1,2,3,4 tetrahydroisoquinoline derivatives was evaluated for affinity to the PCP binding site of the NMDA receptor complex. The (S)-configured tetrahydroisoquinoline derivative (S)-4 e x HCl bearing a 2-methylphenyl substituent in position 1 of the heterocyclic ring system and a methyl group in position 8 was found to exhibit the highest affinity among the derivatives with a K(i)-value of 0.0374 microM. In addition, this compound shows a remarkable enantioselectivity of binding by being almost 90 times more potent than the corresponding (R) enantiomer (R)-4 e x HCl. Additionally, a convenient and efficient synthetic approach to racemic 1-aryl-1,2,3,4-tetrahydroisoquinoline derivatives is described. PMID- 16675067 TI - Novel 5,7-disubstituted 6-amino-5H-pyrrolo[3,2-b]pyrazine-2,3-dicarbonitriles, the promising protein kinase inhibitors with antiproliferative activity. AB - New derivatives of pyrrolo[2,3-b]pyrazine were synthesized and tested on a panel of cultured human tumor cell lines. It was found that 6-amino-5-(3 chlorophenylamino)-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-5H-pyrrolo[3,2 b]pyrazine-2,3-dicarbonitrile (4j) exhibited a significant antiproliferative activity: GI50 for cell lines RXF 393 (renal cancer) and BT-549 (breast cancer) were 14 and 82 nM, respectively. To identify possible molecular targets, docking of the most active compounds into the active sites of cyclin-dependent kinases was performed. Molecular modeling of the inhibitor-enzyme complexes showed the differences in the binding poses of new pyrrolo[2,3-b]pyrazine derivatives in the kinase ATP-binding site compared with known pyrrolo[2,3-b]pyrazine inhibitors called aloisines. The patterns of drug kinase interactions correlated well with antiproliferative activities of novel derivatives. Key interactions and binding mode of docked compounds are discussed. PMID- 16675068 TI - [Changes in the arterial wall in aging]. PMID- 16675069 TI - Students' perception of the psycho-social clinical learning environment: an evaluation of placement models. AB - Nursing is a practice based discipline. A supportive environment has been identified as important for the transfer of learning in the clinical context. The aim of the paper was to assess undergraduate nurses' perceptions of the psychosocial characteristics of clinical learning environments within three different clinical placement models. Three hundred and eight-nine undergraduate nursing students rated their perceptions of the psycho-social learning environment using a Clinical Learning Environment Inventory. There were 16 respondents in the Preceptor model category, 269 respondents in the Facilitation model category and 114 respondents in the clinical education unit model across 25 different clinical areas in one tertiary facility. The most positive social climate was associated with the preceptor model. On all subscales the median score was rated higher than the two other models. When clinical education units were compared with the standard facilitation model the median score was rated higher in all of the subscales in the Clinical Learning Environment Inventory. These results suggest that while preceptoring is an effective clinical placement strategy that provides psycho-social support for students, clinical education units that are more sustainable through their placement of greater numbers of students, can provide greater psycho-social support for students than traditional models. PMID- 16675070 TI - Nutritional risk index predicts a high-risk population in patients with obstructive jaundice. AB - BACKGROUND: Malnutrition is common in obstructive jaundice but is difficult to define. The aim of this study was to compare definitions of malnutrition in patients with obstructive jaundice to identify correlation with mortality, complications and length of hospital stay after intervention. METHODS: Prospective case-control study comparing 39 inpatients with obstructive jaundice with 21 controls. Body mass index (BMI), skin-fold thickness (TSF), mid-arm muscle circumference (MAMC), percentage weight loss, nutritional risk index (NRI) and malnutrition universal screening tool (MUST) were measured and compared. Duration of admission, interventions, complications and outcome were recorded prospectively. RESULTS: Patients with obstructive jaundice were significantly malnourished compared to controls. Severe malnutrition was equally prevalent in benign and malignant disease. Malnourished patients had higher mortality and longer duration of stay after intervention compared to non-malnourished patients. NRI<83.5 was significantly associated with mortality and longer duration of hospital admission but not complication rate. CONCLUSION: NRI is simple to use and defines a high-risk sub-group of patients with obstructive jaundice. PMID- 16675071 TI - The TLR4 agonist, monophosphoryl lipid A, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease. AB - Formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) induces a poorly understood immunopathological response that leads to disease enhancement upon RSV infection of vaccinees. In the cotton rat model, inclusion of monophosphoryl lipid A (MPL) in the FI-RSV formulation was found to mitigate the lung pathology associated with vaccine-enhanced disease. Here we report that the protective effect of MPL on FI-RSV vaccine-enhanced disease is associated with a dramatic reduction in levels of Th1- and Th2-type cytokines and chemokines normally elicited in response to RSV challenge. Our data illustrate the complexity of proinflammatory response elicited by FI-RSV vaccination and RSV infection and the potential importance of MPL in modifying this response. PMID- 16675072 TI - Structure- and oil type-based efficacy of emulsion adjuvants. AB - Oil-based emulsions are well-known immunopotentiators for inactivated, "killed" vaccines. We addressed the relationship between emulsion structure and levels of in vivo antibody formation to inactivated New Castle Disease virus (NDV) and Infectious Bronchitis virus (IBV) as antigens in 3-week-old chickens. The use of a polymeric emulsifier allowed for direct comparison of three types of emulsions, water-in-oil (W/O), oil-in-water (O/W) and W/O-in-water (W/O/W), while maintaining an identical content of components for each vehicle. They were prepared with either non-metabolizable, mineral oil or metabolizable, Miglyol 840. In addition, we assessed the inherent release capacity of each emulsion variant in vitro. Remarkably, we noted that W/O-type emulsions induced the best immune responses, while they released no antigen during 3 weeks. In general, mineral oil vaccines showed superior efficacy compared to Miglyol 840-based vaccines. PMID- 16675073 TI - Helminth infection suppresses T-cell immune response to HIV-DNA-based vaccine in mice. AB - A number of HIV-1 vaccines are in various phases of clinical trials and many more are in the developmental pipeline. Vaccines are especially needed for developing countries where morbidity and mortality due to HIV/AIDS is most severe, the prevalence of HIV infection is highest, and its incidence is often still rising dramatically. Individuals living in these regions are often infected with one or more helminth parasites which systemically bias the immune system towards Th2 type as well as drive immune anergy. The goal of this study was to develop a multi-T-cell epitope DNA-based vaccine for HIV-1 subtype C and to determine the impact of helminth infection on the immune response to this vaccine. We found that vaccination of naive mice with the multi-epitope vaccine, designated TD158, induced a strong HIV-1C-specific T-cell immune response, and that the addition of the Igkappa leader sequence to the TD158 vaccine construct significantly increased the frequencies of IFN-gamma secreting CD8+ T cells. However, the TD158 vaccine specific response of mice infected with the human helminth Schistosoma mansoni was significantly suppressed. The impact of schistosome infection on suppressing the virus-specific immune response was the same whether mice were vaccinated with the TD158 vaccine or with the Igkappa enhanced TD158. The results of this study suggest that helminth infection may pose a serious problem for vaccination with the DNA-based HIV-1 vaccine in developing country populations, and that the prevalence of helminth infections in the vaccine cohorts should be taken into account for HIV-1 vaccine trial design. PMID- 16675074 TI - Eradication of established HPV 16-expressing tumors by a single administration of a vaccine composed of a liposome-encapsulated CTL-T helper fusion peptide in a water-in-oil emulsion. AB - Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide with half a million new cases per year. Despite the encouraging development of a preventive vaccine for HPV, a therapeutic vaccine for cervical cancer or pre-cancerous lesions remains a high priority. The preclinical study reported here used VacciMax((R)) (VM) to deliver a peptide based vaccine composed of an HPV 16 E7-derived cytotoxic T lymphocyte (CTL) epitope fused to the T helper epitope PADRE (FP) and combined with CpG or lipopeptide adjuvant. In the study, C57BL/6 mice received 0.5million HPV 16 expressing C3 tumor cells. Mice were inoculated post-tumor challenge with a single s.c. injection of FP-CpG-VM on either day 4, 5, 6, 9, or 14. All mice that received the FP-CpG-VM vaccine were tumor-free to day 130 when the experiment was terminated. In contrast, only a minority of mice that received a control vaccine were tumor-free on day 60. Cytotoxicity assays, ELISPOT and intracellular staining for interferon (IFN)-gamma showed the immune response was specific for the selected CTL epitope. All mice that received the FP-CpG-VM vaccine remained tumor-free when re-challenged with 6million C3 cells. Cytotoxicity assays 4 months post-challenge showed that only splenocytes from mice inoculated with the FP-CpG-VM vaccine had high lysis activity. These results indicate that VacciMax((R)) causes a rapid, robust, durable and therapeutic CTL response to HPV 16 E7 protein expressing tumors. PMID- 16675075 TI - Immunogenicity of CTLA4 fusion anti-caries DNA vaccine in rabbits and monkeys. AB - Enhancement of mucosal and systemic immune responses is still a challenge for the application of DNA vaccine. Here, we show anti-caries DNA vaccines, pGJA-P and pGJA-P/VAX, encoding Streptococcus mutans antigens fused to cytotoxic T lymphocyte antigen-4 (CTLA4), which binds to B7 molecule expressed on the surfaces of antigen-presenting cells. Rabbits and monkeys were immunized via intranasal or intramuscular routes. The fusion vaccine induced accelerated and increased specific antibody responses in serum and saliva compared with non fusion DNA vaccine in rabbits. Significant specific serum IgG and salivary IgA levels could be detected in fusion vaccine-immunized monkeys. Therefore, this study demonstrates that fusing antigens to CTLA4 results in enhancing immune efficacy and strongly suggests that it may represent a promising approach to prevent dental caries or other mucosal infectious diseases. These findings also suggest that CTLA4 fusion anti-caries DNA vaccine may be effective immunogen in primates. PMID- 16675076 TI - Effectiveness of a hepatitis A vaccination program for migrant children in Amsterdam, The Netherlands (1992-2004). AB - OBJECTIVE: To evaluate the impact and effectiveness of risk-group vaccination against hepatitis A targeted at migrant children living in a country with low endemicity of hepatitis A. METHODS: Retrospective population based data analysis. Routinely collected data on hepatitis A incidence in migrant children and other risk groups in Amsterdam from 1 January 1992 to 2004 were analyzed and related to exposure, immunity and vaccination coverage in migrant children. RESULTS: The overall hepatitis A incidence in Amsterdam declined after a pediatric vaccine was introduced in 1997. This decline was seen in migrant children traveling to hepatitis A-endemic countries, contacts with hepatitis A patients, primary school students, injecting drug users, and persons with unknown source of infection, but not in men who have sex with men (MSM) or in travelers to endemic countries other than migrant children. CONCLUSION: The hepatitis A vaccination campaigns are effective: they reduce both import and secondary HAV cases. The campaigns could be more efficient and cost-effective if the hepatitis B vaccinations currently given to these groups were replaced by a combined hepatitis A and B vaccine. This would increase the hepatitis A vaccination coverage considerably and further reduce the hepatitis A incidence. PMID- 16675077 TI - Protective immunity to tuberculosis with Ag85B-ESAT-6 in a synthetic cationic adjuvant system IC31. AB - In this study, we evaluated the potential of a novel synthetic adjuvant designated IC31 for the ability to augment the immune response and protective efficacy of the well-known mycobacterial vaccine antigen, Ag85B-ESAT-6. The IC31 adjuvant, consisting of a vehicle based on the cationic peptide KLKL(5)KLK and the immunostimulatory oligodeoxynucleotide ODN1a signalling through the TLR9 receptor, was found to promote highly efficient Th1 responses. The combination of Ag85B-ESAT-6 and IC31 exhibited significant levels of protection in the mouse aerosol challenge model of tuberculosis and a detailed analysis of the immune response generated revealed the induction of CD4 T cells giving rise to high levels of IFN-gamma secretion. Furthermore, the combination of Ag85B-ESAT-6/IC31 was found to confer efficient protection in the guinea pig aerosol model of tuberculosis infection and is at present moving towards clinical testing. PMID- 16675078 TI - Evaluation of adenyl cyclase toxin constructs from Bordetella pertussis as candidate vaccine components in an in vitro model of complement-dependent intraphagocytic killing. AB - Recombinant, genetically-detoxified adenylate cyclase toxin (CyaA) constructs from Bordetella pertussis have been developed as potential antigen delivery systems and as promising antigen candidates for inclusion in acellular pertussis vaccines. The major toxic effects of native CyaA are attributed to its enzymatic activity following delivery to cells of the innate immune system via the CD11b/CD18 (CR3) cell receptor. In view of the potential use of detoxified CyaA in vaccinology, a complement dependent in vitro model was used to investigate the potential effects of the interaction of detoxified CyaA with CD11b/CD18 (CR3) on phagocytic function. Interaction of CyaA with CD11b/CD18 (CR3) on human pro myelocytic NB-4 cells differentiated to a neutrophil-like phenotype was measured as inhibition of binding of a monoclonal antibody to the receptor. This interaction was dose-dependent and required acylation of CyaA. Treatment of the cells with either acylated or non-acylated detoxified CyaA constructs inhibited their phagocytic function. Washing the cells allowed recovery of phagocytic function after treatment with non-acylated toxin but not for cells treated with acylated CyaA constructs. However, availability of CD11b/CD18 receptors on acylated CyaA-treated cells was restored after washing and further incubation. The results suggest that the interaction of detoxified CyaA constructs to the CD11b/CD18 (CR3) receptor may temporarily influence the complement-dependent phagocytic function in neutrophil leukocytes. PMID- 16675079 TI - Thrombosis of cervical vascular malformation in neurofibromatosis type 1. PMID- 16675080 TI - Acute effects of static stretching on passive stiffness of the hamstring muscles calculated using different mathematical models. AB - BACKGROUND: The aim of this study was to assess the effects of static stretching on hamstring passive stiffness calculated using different data reduction methods. METHODS: Subjects performed a maximal range of motion test, five cyclic stretching repetitions and a static stretching intervention that involved five 30 s static stretches. A computerised dynamometer allowed the measurement of torque and range of motion during passive knee extension. Stiffness was then calculated as the slope of the torque-angle relationship fitted using a second-order polynomial, a fourth-order polynomial, and an exponential model. The second-order polynomial and exponential models allowed the calculation of stiffness indices normalized to knee angle and passive torque, respectively. FINDINGS: Prior to static stretching, stiffness levels were significantly different across the models. After stretching, while knee maximal joint range of motion increased, stiffness was shown to decrease. Stiffness decreased more at the extended knee joint angle, and the magnitude of change depended upon the model used. After stretching, the stiffness indices also varied according to the model used to fit data. Thus, the stiffness index normalized to knee angle was found to decrease whereas the stiffness index normalized to passive torque increased after static stretching. INTERPRETATION: Stretching has significant effects on stiffness, but the findings highlight the need to carefully assess the effect of different models when analyzing such data. PMID- 16675081 TI - Decline eccentric squats increases patellar tendon loading compared to standard eccentric squats. AB - BACKGROUND: Recent studies have shown excellent clinical results using eccentric squat training on a 25 degrees decline board to treat patellar tendinopathy. It remains unknown why therapeutic management of patellar tendinopathy using decline eccentric squats offer superior clinical efficacy compared to standard horizontal eccentric squats. This study aimed to compare electromyography activity, patellar tendon strain and joint angle kinematics during standard and decline eccentric squats. METHODS: Thirteen subjects performed unilateral eccentric squats on flat and a 25 degrees decline surface. During the squats, electromyography activity was obtained in eight representative muscles. Also, ankle, knee and hip joint goniometry was obtained. Additionally, patellar tendon strain was measured in vivo using ultrasonography as subjects maintained a unilateral isometric 90 degrees knee angle squat position on either flat or 25 degrees decline surface. FINDINGS: Patellar tendon strain was significantly greater (P<0.05) during the squat position on the decline surface compared to the standard surface. The stop angles of the ankle and hip joints were significantly smaller during the decline compared to the standard squats (P<0.001, P<0.05). Normalized mean electromyography amplitudes of the knee extensor muscles were significantly greater during the decline compared to the standard squats (P<0.05). Hamstring and calf muscle mean electromyography did not differ, respectively, between standard and decline squats. INTERPRETATION: The use of a 25 degrees decline board increases the load and the strain of the patellar tendon during unilateral eccentric squats. This finding likely explains previous reports of superior clinical efficacy of decline eccentric squats in the rehabilitative management of patellar tendinopathy. PMID- 16675082 TI - An identification technique for evaluating body segment parameters in the upper extremity from manipulator-hand contact forces and arm kinematics. AB - OBJECTIVE: To show that it is possible to determine segment masses and segment centers of mass by measuring manipulator-hand contact forces and joint angles during upper extremity movement. BACKGROUND: The method serves as a quick subject specific body segment parameter evaluation technique. Clinically we see this method as an alternative upper extremity body segment parameter evaluation study especially useful in rehabilitation treatment activities. METHODS: The experiment is based on coupling the human arm with a robotic manipulator which is then used for imposing a specified sagittal plane trajectory. Joint angles and forces in the contact point serve as input to the identification procedure. For verification purposes the proposed identification procedure was first performed on a mechanical arm. Afterwards a low velocity trajectory was imposed into all joints of the human upper extremity, with very small angular deviations. Within this small angular region the arm was assumed to be represented as a linear system. FINDINGS: The outcome of the identification procedure is an estimate of masses and center of mass coordinates for the lower arm and palm segments, their products for the upper arm and the passive moments around the measured angle of all joints in the sagittal plane. The results obtained for three particular human arms are eventually compared to the average population based literature. CONCLUSION: From the clinical point of view the study can become useful for biomechanical evaluation and for evaluating biomechanical properties of lower extremities or other body segments. This method may also provide a foundation to measuring body segment moments of inertia and joint viscoelastic parameters. PMID- 16675083 TI - The influence of experience on knee mechanics during side-step cutting in females. AB - BACKGROUND: It is thought that female athletes with limited experience in a sport perform athletic maneuvers differently than their more experienced counterparts, and that they do so in a manner that places them at greater risk for injury. The purpose of this study was to evaluate the influence of athletic experience on knee mechanics during the execution of a side-step cutting maneuver in young female athletes. METHODS: Three-dimensional kinematics, ground reaction forces and electromyographic activity (surface electrodes) were recorded during the early deceleration phase of side-step cutting in 30 high school females (15 experienced, 15 novice). Group differences in knee joint kinematics, peak moments, net joint moment impulse and average muscle activation were evaluated. FINDINGS: No significant group differences were found in knee kinematics. When compared to experienced females, novice females demonstrated significantly smaller flexor, adductor, and internal rotator peak moments and smaller net joint moment impulse in all three planes at the knee. No group differences were found for average EMG; however, novice athletes had significantly greater co contraction at the knee. INTERPRETATION: The finding of smaller knee moments and greater muscle co-contraction in the novice group suggests that these athletes may adopt a protective strategy in response to a relatively unfamiliar task. In addition, these results suggest that increased moments at the knee emerge with experience, indicating that more skilled athletes may be at greater risk for anterior cruciate ligament (ACL) injury. PMID- 16675084 TI - Acute and chronic activity of perchlorate and hexavalent chromium contamination on the survival and development of Culex quinquefasciatus Say (Diptera: Culicidae). AB - Effects of water contamination with perchlorate and hexavalent chromium [Cr (VI)] on the mosquito Culex quinquefasciatus were assessed. The chronic (10-day) LC50s values for perchlorate and chromium were 74+/-8.0 mg/L and 0.41+/-0.15 mg/L, respectively. Relative Growth Index, a measure of growth and mortality rates in a population, was significantly reduced within 5 days for levels of perchlorate as low as 25 mg/L and for levels of chromium as low as 0.16 mg/L. Neither compound altered wing length of surviving adults. In combination, contaminants were synergistic, causing 14% more mortality than predicted. Acute (24-h) LC50 values for perchlorate and Cr (VI) were 17,000+/-3200 and 38+/-1.3 mg/L, respectively. Effects on mosquito larvae in contaminated environments are likely to be observed for Cr (VI) but not for perchlorate, which generally does not occur at levels as high as those shown here to affect larval mosquitoes. PMID- 16675085 TI - Social welfare, genetic welfare? Boundary-work in the IVF/PGD clinic. AB - Through the lens of the 'welfare of the child' assessment, this paper explores how staff working in the area of in vitro fertilisation and preimplantation genetic diagnosis (IVF/PGD) balance reflexive relations of legitimacy and accountability between the public and private spheres, and between medicine, the citizen and the state. The wider research of which this analysis is a part uses multiple methods to study two National Health Service Assisted Conception Units in England. Research methods used included observation clinics and interviews with staff from a range of disciplines. We illustrate how the staff reveal tensions between their views that the welfare of the child assessment can be seen as intrusive and discriminatory, and on the other hand that medical intervention in reproduction should be socially and professionally accountable. These tensions can be understood sociologically in terms of a gradual movement from socially based solutions to fertility problems and disabilities, towards a biomedical, and arguably genetically oriented worldview of such problems. Rather than being viewed as discrete, these two orientations should be seen as indicating an emergent direction of travel along a continuum, with elements of both being present in the accounts. We argue that consideration of the welfare of the child involves staff in ethical boundary-work across the two orientations and between the accountabilities and responsibilities of healthcare professionals, individuals and the state. PMID- 16675086 TI - Neuroleptic malignant syndrome is malignant catatonia, warranting treatments efficacious for catatonia. PMID- 16675087 TI - Urinary concentrating mechanism and Aquaporin-2 abundance in rats chronically treated with aluminum lactate. AB - The aim of this work was to study the effects of chronic administration of aluminum (Al) on the urinary concentrating and diluting mechanisms in the distal tubules and collecting ducts. Male Wistar rats were chronically treated with aluminum lactate for 12 weeks (0.575 mg Al/100g of body weight, i.p., three times per week). After 12 weeks, renal function of control and Al-treated rats was evaluated by clearance techniques. To study urinary concentrating mechanisms, renal function was also measured in control and Al-treated rats deprived of water, after the administration of desmopressin (vasopressin agonist) and after the infusion of hypertonic saline at increasing infusion rates. Sodium and water balance were impaired. We found decreased urinary concentrating ability in situations in which endogenous (thirst or infusion of hypertonic saline) or exogenous plasma antidiuretic hormone was increased. Solute-free water formation, measured during the infusion of hypotonic saline showed normal transport in the thick ascending limb. Aquaporin-2 (AQP2) expression was measured by Western blot to evaluate water permeability in collecting ducts. We found that Al produced downregulation of AQP2 in plasma membranes and intracellular vesicles, that could account for the impaired water handling. Administration of desmopressin increased AQP2 in plasma membranes, suggesting that Al did not impair trafficking of this protein, but could interfere with AQP2 synthesis. PMID- 16675088 TI - Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions. AB - The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase. PMID- 16675089 TI - Feedback control of ribosome synthesis in Escherichia coli is dependent on eight critical amino acids. AB - When bacteria growing in minimal medium are supplied with exogenous amino acids, they respond by increasing the synthesis of ribosomes; this leads to more protein synthesis capacity and faster growth. To examine how amino acids control the synthesis of ribosomes, two strategies were used. First, single amino acids were added to bacteria growing in minimal medium and their effect on the relative strength of the rrnB P1 promoter was determined. The addition of any one of eight amino acids (alanine, glutamine, and glutamic acid, isoleucine, leucine, methionine, serine, valine) increased the strength of the P1 promoter by 1.25- to 2.0-fold with no appreciable effect on transcription from an isolated rrn P2 promoter or on the bacterial growth rate. The effects of adding combinations of these critical amino acids were partially additive. When any one of the other amino acids was added, no discernable stimulation in relative P1 expression or growth was observed. In the second strategy, all amino acids were present in the growth medium, but the carbon source was altered to change the growth rate. In this case the relative strength of the P1 promoter was always constant and maximal. We suggest that addition of any of the eight critical amino acids reduces the ppGpp synthesis activity of the spoT gene product; the lower ppGpp levels, in turn, increase the strength of the rrn P1 promoters. It is suggested that these amino acids are involved in a feedback chain of reactions that control the rate of ribosome function by adjusting the rate of ribosome synthesis. PMID- 16675090 TI - Reflections on the interaction of the mind and brain. AB - Problems associated with the topic of the mind-brain interaction are reviewed and analyzed. If there is an interaction, then the "mind" and "brain" are independent variables; the mind represents subjective experience and is therefore a non physical phenomenon. This fact led to the need for a field theory, termed here the "cerebral mental field" (CMF). By definition, the CMF is a system property produced by the appropriate activities of billions of neurons. An experimental test of this theory is possible and a test design is presented. The most direct experimental evidence has been obtained by use of intracranial stimulating and recording electrodes. Important information has also been developed, however, with extracranial imaging techniques. These can be very fast (in ms), but the cerebral neuronal events that produce changes in physiological properties require a time delay for their processing. A number of surprising time factors affecting the appearance of a subjective somatosensory experience are described, and their wider implications are discussed. Among these is a delay (up to 0.5 s) in the generation of a sensory awareness. Thus, unconscious cerebral processes precede a subjective sensory experience. If this can be generalized to all kinds of subjective experiences, it would mean that all mental events begin unconsciously and not just those that never become conscious. In spite of the delay for a sensory experience, subjectively there appears to be no delay. Evidence was developed to demonstrate that this phenomenon depends on an antedating of the delayed experience. There is a subjective referral backward in time to coincide with the time of the primary cortical response to the earliest arriving sensory signal. The subjective referral in time is analogous to the well-known subjective referral in space. In conclusion, features of the CMF can be correlated with brain events, even though the CMF is non-physical, by study of subjective reports from the human subject. PMID- 16675091 TI - The efficacy and safety of aceclofenac versus placebo and naproxen in women with primary dysmenorrhoea. AB - OBJECTIVE: To determine the analgesic efficacy and safety of a single oral dose of aceclofenac 100 mg and compare that with placebo and naproxen 500 mg in women with primary dysmenorrhoea. STUDY DESIGN: In this double-blind, prospective, multicentre, randomised, three-way, crossover study, women were randomly assigned to receive one of six treatment sequences, comprising single oral doses of aceclofenac 100 mg, naproxen 500 mg or placebo, when menstrual pain reached a predetermined level of severity. A single dose of the assigned study medication was taken on three menstrual periods; a different medication was taken on each treatment day. Analgesic efficacy was determined by self-reported analgesia scoring and participants' and investigators' global evaluation of treatment effectiveness. Measurements also included physical examination and adverse events. RESULTS: Total pain relief scores were not statistically significantly different for aceclofenac and naproxen, and both were statistically significantly more effective than placebo (p = 0.019 and 0.002, respectively). This finding was supported by secondary endpoints including sum of pain intensity differences (SPID/8), peak analgesia (peak pain intensity and peak pain relief), and participants' and investigators' overall evaluation of effectiveness. Both aceclofenac and naproxen were well tolerated. CONCLUSIONS: Aceclofenac (100 mg) and naproxen (500 mg) effectively treated the pain associated with primary dysmenorrhoea, and both were more effective than placebo at easing menstrual pain assessed by various pain relief criteria. PMID- 16675092 TI - The length of menstrual bleeding and the risk of urogenital infections in the context of the activity of hemoglobin-derived microbicidal peptides. AB - OBJECTIVE: Well-known natural unspecific antimicrobial factors acting in the vagina are additionally reinforced during menstrual bleeding by hemocidins--a recently discovered novel class of microbicidal peptides generated proteolytically from hemoglobin. The aim of the presented research was to investigate the relation between the average length of menstrual bleeding and the frequency of urogenital infections. We expected that the shorter menstrual bleeding might increase the risk of urogenital infections because is synonymous with the shorter period of exposition on bactericidal action of hemocidins. STUDY DESIGN: The study contains statistical analysis of an average declared length of menstrual bleeding in the group of 267 young, sexually active women with the symptoms of urogenital infections. The control group consisted of 300 young healthy women. RESULTS: The length of menstrual bleeding in the group of patients with urogenital infections (average 4.35 days) was statistically significantly shorter than in the control group (average 4.95 days). The average length of the menstrual cycle was equal and counted ca. 28 days in both groups. CONCLUSION: The length of menstrual bleeding seems to be a significant factor in the vaginal ecology maintenance. PMID- 16675093 TI - Distribution of contraceptive use in a Turkish population. AB - OBJECTIVE: This study is designed to estimate the distribution of contraceptive use among reproductive age women in a Turkish population (Turkey). STUDY DESIGN: We included 2365 women of reproductive age in this study. Women were given a questionnaire via a face-to-face interview. RESULTS: During the time period just after marriage, 1914 women (80.9%) declared that they did not use any type of contraception. Among 451 women who had used a contraceptive method just after marriage, the most commonly used method was condoms (34.2%), followed by oral contraceptives (31.2%) and coitus interruptus (25.9%). When their history of contraceptive use was asked, 1903 women (80.2%) stated that they used a method of contraception while 462 women (19.5%) had never used any type of contraceptive. Distribution of contraceptive methods among women was analyzed and intrauterine devices were detected to be the most commonly used method (n=1046, 55%). CONCLUSION: A point that should be emphasized is that there is a marked increase in contraceptive prevalence, and expansion of family planning activities in Turkey. This change, we believe, is due to improvements in health services of Turkey, realized in recent years. PMID- 16675094 TI - Postpartal deep-vein thrombosis revealing agenesis of the inferior vena cava. PMID- 16675095 TI - Isosorbide mononitrate induces increased cervical expression of cyclooxygenase-2, but not of cyclooxygenase-1, at term. AB - OBJECTIVE: Prostaglandin and nitric oxide (NO) are both known to be involved in cervical ripening at term. The aim of the study was to investigate if NO has an effect on cervical expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), the two main isoenzymes involved in prostaglandin synthesis, and to localize these enzymes within the cervix. STUDY DESIGN: Women with an unripe cervix scheduled for elective caesarean section at term were randomly selected to receive vaginally either the NO donor isosorbide mononitrate (IMN) or placebo 4h before surgery. At the operating theatre, cervical tissue specimens were obtained for immunoblotting and immunohistochemistry. RESULTS: Increased expression of COX 2 was found in specimens exposed to IMN compared to specimens obtained from women in the placebo group. There was no difference in the expression of COX-1. Immunohistochemistry revealed similar localization of the two enzymes in treated and untreated women. CONCLUSIONS: Vaginal administration of IMN induces increased cervical expression of COX-2, but not of COX-1. This pathway may be of importance in the process of cervical ripening at term. PMID- 16675096 TI - Therapeutic dendritic cell vaccination of patients with renal cell carcinoma. AB - OBJECTIVE: Dendritic cell (DC) vaccination against cancer is a new specific immunotherapeutic approach given with either therapeutic or adjuvant intent. We provide a review of DC vaccination as a treatment for metastatic renal cell carcinoma (RCC). METHOD: A total of 197 patients with metastatic RCC were treated with DC vaccination in 14 phase I/II clinical trials. Different vaccine preparations, administration routes, and treatment schedules have been tested in these trials. Clinical response and immune response were analysed. RESULTS: Seventy-three (37%) patients had clinical response with 4 complete responses, 8 partial responses and 61 disease stabilisations, whereas 4 patients had mixed response, but most of these responses have not been transformed into durable clinical effects. Immune responses were observed in a subset of the treated patients but were not always associated with a clinical response. Only mild toxicity was observed in these trials. CONCLUSION: DC vaccination therapy in patients with metastatic RCC is currently experimental but the results are encouraging with achievement of tumour regression and induction of antigen specific immune response combined with minimal toxicity in a subset of the treated patients. Future emphasis should be placed on therapy in the adjuvant setting because patients with minimal residual disease are more likely to benefit from the treatment. Combination approaches with DC vaccination and immune enhancing therapies or antiangiogenic therapy should be further investigated to develop new and more efficient treatment strategies for patients with RCC. PMID- 16675097 TI - Autologous fibrin glue using the Vivostat system for hemostasis in laparoscopic partial nephrectomy. AB - OBJECTIVES: Haemostasis remains the greatest challenge during laparoscopic partial nephrectomy. Use of fibrin sealant currently is increasing. We describe first a technique for achieving effective haemostasis during laparoscopic partial nephrectomy using the Vivostat system. METHODS: Ten patients underwent laparoscopic partial nephrectomy. Autologous fibrin sealant was prepared with the Vivostat system and applied to the resection bed. This system is an automated medical device for the preparation of an autologous fibrin sealant, generating up to 5 ml of sealant from 120 ml of the patient's blood. The concentration of fibrin and the volume of sealant are stable; the sealant may be kept at room temperature for up to 8 hours before application without a loss of properties and effectiveness. The patients were evaluated for acute and delayed bleeding. RESULTS: Mean patient's age was 54 years (range, 31-68). Haemostasis was immediate in all cases after application of the sealant for 1 to 2 minutes to the resection site; no additional haemostatic measures were required. Mean warm ischemia time was 23 minutes (range, 20-27); mean blood loss was 90 cc (range, 20 200). Pre-operative and post-operative serum haemoglobin did not differ significantly (mean, 14.9 vs 12.6g/dl) and creatinine values (mean, 0.91 vs 0.95 ng/ml). Mean operative time was 136 minutes (range, 60-180). No postoperative bleeding or other complications occurred. CONCLUSIONS: In this study, immediate haemostasis was achieved and maintained after the kidney was reperfused. Our initial experience with the Vivostat system in laparoscopic partial nephrectomy has been encouraging. PMID- 16675098 TI - Tissue sealants in laparoscopic conservative renal surgery. PMID- 16675099 TI - Role of cascades in converting oscillatory signals into stationary step-like responses. AB - In biological signal transduction pathways intermediates are often oscillatory and need to be converted into smooth output signals at the end. We show by mathematical modelling that protein kinase cascades enable converting oscillatory signals into sharp stationary step-like outputs. The importance of this result is demonstrated for the switch-like protein activation by calcium oscillations, which is of biological importance for regulating different cellular processes. In addition, we found that protein kinase cascades cause memory effects in the protein activation, which might be of a physiological advantage since a smaller amount of calcium transported in the cell is required for an effective activation of cellular processes. PMID- 16675100 TI - Sensitive dependence of the coefficient of variation of interspike intervals on the lower boundary of membrane potential for the leaky integrate-and-fire neuron model. AB - After the report of Softky and Koch [Softky, W.R., Koch, C., 1993. The highly irregular firing of cortical cells is inconsistent with temporal integration of random EPSPs. J. Neurosci. 13, 334-350], leaky integrate-and-fire models have been investigated to explain high coefficient of variation (CV) of interspike intervals (ISIs) at high firing rates observed in the cortex. The purpose of this paper is to study the effect of the position of a lower boundary of membrane potential on the possible value of CV of ISIs based on the diffusional leaky integrate-and-fire models with and without reversal potentials. Our result shows that the irregularity of ISIs for the diffusional leaky integrate-and-fire neuron significantly changes by imposing a lower boundary of membrane potential, which suggests the importance of the position of the lower boundary as well as that of the firing threshold when we study the statistical properties of leaky integrate and-fire neuron models. It is worth pointing out that the mean-CV plot of ISIs for the diffusional leaky integrate-and-fire neuron with reversal potentials shows a close similarity to the experimental result obtained in Softky and Koch [Softky, W.R., Koch, C., 1993. The highly irregular firing of cortical cells is inconsistent with temporal integration of random EPSPs. J. Neurosci. 13, 334 350]. PMID- 16675101 TI - A platform for evolving intelligently interactive adversaries. AB - Entertainment software developers face significant challenges in designing games with broad appeal. One of the challenges concerns creating nonplayer (computer controlled) characters that can adapt their behavior in light of the current and prospective situation, possibly emulating human behaviors. This adaptation should be inherently novel, unrepeatable, yet within the bounds of realism. Evolutionary algorithms provide a suitable method for generating such behaviors. This paper provides background on the entertainment software industry, and details a prior and current effort to create a platform for evolving nonplayer characters with genetic and behavioral traits within a World War I combat flight simulator. PMID- 16675102 TI - Genetically modified mouse models in studies of luteinising hormone action. AB - Numerous genetically modified mouse models have recently been developed for the study of the pituitary-gonadal interactions. They include spontaneous or engineered knockouts (KO) of the gonadotrophin-releasing hormone (GnRH) and its receptor, the gonadotrophin common-alpha(Calpha), luteinising hormone (LH) beta and follicle-stimulating hormone (FSH) beta subunits, and the two gonadotrophin receptors (R), LHR and FSHR. In addition, there are also transgenic (TG) mice overexpressing gonadotrophin subunits and producing supraphysiological levels of these hormones. These models have offered relevant phenocopies for similar mutations in humans and to a great extent expanded our knowledge on normal and pathological functions of the hypothalamic-pituitary-gonadal (HPG) axis. The purpose of this article is to review some of our recent findings on two such mouse models, the LHR KO mouse (LuRKO), and the hCG overexpressing TG mouse (hCG+). PMID- 16675103 TI - Changing cerebral blood flow velocity by transcranial Doppler during head up tilt in patients with diabetes mellitus. AB - OBJECTIVE: Diabetes mellitus is an independent risk factor for poor prognosis in patients with ischemic stroke. It is known that diabetes mellitus directly affects cerebral vasculature as a secondary, long-term complication of cerebral circulation, and causes cerebral blood flow abnormalities. The abnormalities of cerebral autoregulation also poorly affects the prognosis of ischemic stroke. In this study, we aimed to show the cerebral autoregulation with transcranial Doppler (TCD) ultrasound in diabetic patients with autonomic nervous system abnormalities, determined with electrophysiological studies. MATERIAL AND METHOD: Twenty healthy controls and 39 patients, who had at least 2 years of diabetes mellitus, were evaluated (age ranges: 42-75 years). The patients were divided into two groups according to sympathetic skin response and R--R interval variation studies: (1) patients with autonomic neuropathy; (2) patients without autonomic neuropathy. Blood flow velocities were measured during supine position and after the patients were raised upright position on head up tilt table. Arterial blood pressures and heart rates were also evaluated. RESULTS: Mean blood flow velocities of diabetic patients with autonomic neuropathy were found more decreased at 90s after the patients were raised upright position. DISCUSSION: Autonomic neuropathy due to diabetes mellitus affects cerebral autoregulation, and by this way cerebral perfusion loses protection against hemodynamical changes. PMID- 16675104 TI - Clinical implications of osteopontin in metastatic lesions of uterine cervical cancers. AB - Osteopontin (OPN) is a glycophosphoprotein that has variety of physiological functions. OPN is expressed in various human cancers and associated with tumor progression, invasion and metastasis in many manners. The purpose of this study is to investigate the clinical significance of OPN expression in metastatic lymph node of uterine cervical cancers, since the prognosis of the patients with lymph node metastasis is extremely poor. Immunohistochemical staining revealed OPN was distributed in the cytoplasm and nuclear compartments of the cancer and stromal cells within and around the tumor. In 25 of the 40 cases, stronger staining for OPN was found in the cancer cells or stromal cells of the metastatic lymph node lesion than in those of the primary tumor. The OPN level was significantly (P<0.05) increased in 25 of 40 metastatic lymph node lesions of uterine cervical cancers. The OPN increased cases identified by immunohistochemical staining were consistent with those identified by the sandwich immunoassay. The prognosis of the 25 patients with significant increase of OPN in uterine cervical cancers was extremely poor, whereas the 24-month survival rate of the 15 patients with no increase of OPN was 67%. This indicates that OPN may contribute to lymph node metastasis and its advancement, and that the OPN level in metastatic lesion may be a prognostic indicator in uterine cervical cancers. PMID- 16675105 TI - RNA regulation and cancer development. AB - Cancer is viewed as a genetic disease. According to the currently accepted model of carcinogenesis, several consequential mutations in oncogenes or tumor suppressor genes are necessary for cancer development. In this model, mutated DNA sequence is transcribed to mRNA that is finally translated into functionally aberrant protein. mRNA is viewed solely as an intermediate between DNA (with 'coding' potential) and protein (with 'executive' function). However, recent findings suggest that (m)RNA is actively regulated by a variety of processes including nonsense-mediated decay, alternative splicing, RNA editing or RNA interference. Moreover, RNA molecules can regulate a variety of cellular functions through interactions with RNA, DNA as well as protein molecules. Although, the precise contribution of RNA molecules by themselves and RNA regulated processes on cancer development is currently unknown, recent data suggest their important role in carcinogenesis. Here, we summarize recent knowledge on RNA-related processes and discuss their potential role in cancer development. PMID- 16675106 TI - CFD analysis of gas explosions vented through relief pipes. AB - Vent devices for gas and dust explosions are often ducted to safe locations by means of relief pipes. However, the presence of the duct increases the severity of explosion if compared to simply vented vessels (i.e. compared to cases where no duct is present). Besides, the identification of the key phenomena controlling the violence of explosion has not yet been gained. Multidimensional models coupling, mass, momentum and energy conservation equations can be valuable tools for the analysis of such complex explosion phenomena. In this work, gas explosions vented through ducts have been modelled by a two-dimensional (2D) axi symmetric computational fluid dynamic (CFD) model based on the unsteady Reynolds Averaged Navier Stokes (RANS) approach in which the laminar, flamelet and distributed combustion models have been implemented. Numerical test have been carried out by varying ignition position, duct diameter and length. Results have evidenced that the severity of ducted explosions is mainly driven by the vigorous secondary explosion occurring in the duct (burn-up) rather than by the duct flow resistance or acoustic enhancement. Moreover, it has been found out that the burn up affects explosion severity due to the reduction of venting rate rather than to the burning rate enhancement through turbulization. PMID- 16675108 TI - Barrier and operational risk analysis of hydrocarbon releases (BORA-Release). Part I. Method description. AB - Investigations of major accidents show that technical, human, operational, as well as organisational factors influence the accident sequences. In spite of these facts, quantitative risk analyses of offshore oil and gas production platforms have focused on technical safety systems. This paper presents a method (called BORA-Release) for qualitative and quantitative risk analysis of the platform specific hydrocarbon release frequency. By using BORA-Release it is possible to analyse the effect of safety barriers introduced to prevent hydrocarbon releases, and how platform specific conditions of technical, human, operational, and organisational risk influencing factors influence the barrier performance. BORA-Release comprises the following main steps: (1) development of a basic risk model including release scenarios, (2) modelling the performance of safety barriers, (3) assignment of industry average probabilities/frequencies and risk quantification based on these probabilities/frequencies, (4) development of risk influence diagrams, (5) scoring of risk influencing factors, (6) weighting of risk influencing factors, (7) adjustment of industry average probabilities/frequencies, and (8) recalculation of the risk in order to determine the platform specific risk related to hydrocarbon release. The various steps in BORA-Release are presented and discussed. Part II of the paper presents results from a case study where BORA-Release is applied. PMID- 16675107 TI - Liquid phase adsorption of Crystal violet onto activated carbons derived from male flowers of coconut tree. AB - Adsorption of Crystal violet, a basic dye onto phosphoric and sulphuric acid activated carbons (PAAC and SAAC), prepared from male flowers coconut tree has been investigated. Equilibrium data were successfully applied to study the kinetics and mechanism of adsorption of dye onto both the carbons. The kinetics of adsorption was found to be pseudo second order with regard to intraparticle diffusion. The pseudo second order is further supported by the Elovich model, which in turn intensifies the fact of chemisorption of dye onto both the carbons. Quantitative removal of dye at higher initial pH of dye solution reveals the basic nature of the Crystal violet and acidic nature of the activated carbons. Influence of temperature on the removal of dye from aqueous solution shows the feasibility of adsorption and its endothermic nature. Mass transfer studies were also carried out. The adsorption capacities of both the carbons were found to be 60.42 and 85.84 mg/g for PAAC and SAAC, respectively. Langmuir's isotherm data were used to design single-stage batch adsorption model. PMID- 16675109 TI - Organic carbon leaching behavior from incinerator bottom ash. AB - The understanding of the leaching behavior of organic carbon from incinerator bottom ash is an important aspect for the control of organic carbon emissions from landfills in order to minimize their potential risk to the environment. The leaching behavior of organic carbon from incinerator bottom ash samples, obtained from two different solid waste sources, as well as the effects of calcium (Ca) on organic carbon (DOC) leaching associated with pH were therefore investigated in this paper. The effect of pH on the leaching of DOC and other major elements from bottom ash was ascertained through pH-stat leaching experiments. Column leaching experiments were carried out to evaluate the dependence of the leached amount of DOC on Ca leaching. It was found that the bottom ash produced by incineration of municipal solid waste (MSW) was composed of two DOC fractions: DOC leached independent (fraction I) of Ca leaching, observed at alkaline-neutral pH, and DOC leached dependent (fraction II) on Ca leaching, observed at acid pH. However, the bottom ash produced by incineration of industrial solid waste (ISW) was composed of only DOC fraction I. The addition of calcium oxide during incineration and the differences in the paper to plastic ratio in the physical composition of the solid wastes incinerated would explain the distinct organic carbon leaching behaviors of bottom ash samples. PMID- 16675110 TI - Adsorptive removal of water poisons from contaminated water by adsorbents. AB - Adsorptive removal of water poisons such as Pb(II), Cu(II), Mn(II), Hg(II), CN( ), microbes, nerve and blister agents (concentration range from 100 to 1000 mg/L) were studied by using adsorbents such as active carbon, impregnated carbon and bentonite loaded fabric strip. Removal of water poisons (99.5%) could be achieved with an optimum stirring time of 5-15 min and weight of adsorbent of 0.8-8.0 g/100mL contaminated water, respectively. However, 85% bentonite loading was found to be most effective for Pb(II) removal. Effect of contaminants concentration was also studied. PMID- 16675111 TI - Dopaminergic D1 receptor agonist SKF 38393 induces GAP-43 expression and long term potentiation in hippocampus in vivo. AB - We evaluated whether activating dopamine D1 receptors (D1R) with an agonist will mimic the effects of long-term potentiation (LTP)-inducing electrical stimulation and trigger the expression of the presynaptic growth-associated protein 43 (GAP 43), a putative synaptic plasticity factor. Thus, we conducted GAP-43 protein analyses together with assessments of LTP across CA3/CA1 synapses in guinea pigs administered with SKF38393 (the D1R agonist) and/or SCH23390 (the D1R antagonist). Our results showed that guinea pigs treated with SKF38393 coupled with low-frequency stimulation gradually exhibited an LTP-like potentiation in correlation with increased GAP-43 protein expression. However, when SKF38393 treatment was preceded by administration of SCH23390, this antagonized the occurrence of both synaptic potentiation and GAP-43 up-regulation. By comparison, persistent LTP was readily expressed after brief high frequency tetanic stimulation in control guinea pigs, whereas animals injected with SCH23390 and tetanized only developed early-LTP but not late-LTP. Western blot analyses showed GAP-43 up-regulation in the tetanized control guinea pigs but not those injected with SCH23390. We conclude that direct D1R activations with an agonist can mimic LTP-inducing electrical stimulation to produce GAP-43 up-regulation and synaptic plasticity. PMID- 16675112 TI - Exaggeration of tissue trauma induces signs and symptoms of acute CRPS I, however displays distinct differences to experimental CRPS II. AB - As CRPS I frequently develops after tissue trauma, we proposed that an exaggerated inflammatory response to tissue trauma may underlie CRPS I. Therefore, we studied the vascular inflammatory, nociceptive and apoptotic sequelae of (i) soft tissue trauma and (ii) exaggerated soft tissue trauma in comparison to those of (iii) sciatic nerve chronic constriction injury, modeling CRPS II. Standardized soft tissue trauma (TR) was induced by means of a controlled impact injury technique in the hind limb of pentobarbital-anesthetized rats. Additional animals received soft tissue trauma and femoral arterial infusion of mediator-enriched supernatant achieved by homogenization and centrifugation of traumatized muscle tissue in order to provoke an exaggerated trauma response (ETR). Infusion of supernatant of non-traumatized muscle served as control intervention (STR, sham trauma response). Neuropathy was induced by chronic constriction injury of the sciatic nerve (CCI). Untreated animals served as controls (CO). Detailed nociceptive testing showed temporarily decreased mechanical pain thresholds in ETR animals that resolved within 14 days, while TR and STR animals, i.e. those with singular limb trauma, and controls remained free of pain. Neither cold- nor heat-evoked allodynia developed in post-traumatic animals, whereas CCI animals presented the well-known pattern of ongoing neuropathic pain. Using high-resolution in vivo multifluorescence microscopy, muscle tissue of traumatized animals revealed an enhanced inflammatory response that was found most pronounced in ETR animals. CCI of the sciatic nerve was not accompanied by tissue inflammation; however, induced myocyte apoptosis. Collectively, these data indicate that exaggeration of trauma response induces signs and symptoms of acute CRPS I. Pain perception displays differences to that in CRPS II. Apoptosis turns out to be a distinctive marker for CRPS, warranting further evaluation in clinical studies. PMID- 16675113 TI - Finding a solution to the problem of pain: conceptual formulation and the development of the Pain Solutions Questionnaire (PaSol). AB - We report the development of the Pain Solutions Questionnaire (PaSol), an instrument designed to measure assimilative (efforts at changing or solving pain) and accommodative (accepting that pain cannot be solved, and changing life goals) responses to the problems associated with pain. Data were collected from 476 adults suffering from chronic pain. Exploratory and confirmatory factor analyses resulted in a 14-item instrument with an adequate oblique 4-factor structure: (1) Solving Pain scale (4 items), (2) Meaningfulness of Life Despite Pain scale (5 items), (3) Acceptance of the Insolubility of Pain scale (3 items), and (4) Belief in a Solution scale (2 items). The validity of the PaSol was further tested by its value in explaining disability and affective distress after controlling for the effects of the demographic characteristics and pain severity. The Meaningfulness of Life Despite Pain scale was important in explaining disability and affective distress. The Solving Pain scale had a unique and independent contribution in explaining affective distress. Results are discussed in terms of how a persistence in assimilative coping, even though the pain problem is insoluble, may increase hypervigilance, catastrophizing, distress and disability. PMID- 16675114 TI - The role of tumor necrosis factor-alpha in the neuropathic pain induced by Lumbar 5 ventral root transection in rat. AB - Accumulating evidence has demonstrated that tumor necrosis factor-alpha (TNF alpha) plays an important role in neuropathic pain. Recently, it has been shown that Lumbar 5 ventral root transection (L5 VRT) induces persistent mechanical allodynia and thermal hyperalgesia in bilateral hind paws. In the present study, the role of TNF-alpha in the L5 VRT model was investigated. We found that immunoreactivity (IR) of TNF-alpha and TNF receptor 1 (TNFR1) in ipsilateral (but not in contralateral) L4 and L5 dorsal root ganglion (DRG) was increased following L5 VRT, started 1 day after the lesion and persisted for 2 weeks. Double immunofluorescence staining revealed that the increased TNF-alpha-IR in DRG was in satellite glial cells, immune cells and neuronal cells, while TNFR1-IR was almost restricted at DRG neuronal cells. L5 VRT increased TNF-alpha-IR and TNFR1-IR in bilateral L5 spinal dorsal horn, started 1 day after lesion and persisted for 2 weeks. The increased TNF-alpha-IR in spinal dorsal horn was observed in astrocytes, microglias and neurons, but the upregulation of TNFR1 was mainly in neurons. Intraperitoneal injection of thalidomide, an inhibitor of TNF alpha synthesis, started at 2h before surgery, blocked mechanical allodynia and thermal hyperalgesia. However, the drug failed to reverse the abnormal pain behaviors, when it was applied at day 7 after surgery. These data suggest that the upregulation of TNF-alpha and TNFR1 in DRG and spinal dorsal horn is essential for the initiation but not for maintenance of the neuropathic pain induced by L5 VRT. PMID- 16675115 TI - Surface characterization and adhesion and friction properties of hydrophobic leaf surfaces. AB - Super-hydrophobic surfaces as well as low adhesion and friction are desirable for various industrial applications. Certain plant leaves are known to be hydrophobic in nature. These leaves are hydrophobic due to the presence of microbumps and a thin wax film on the surface of the leaf. The purpose of this study is to fully characterize the leaf surface and to separate out the effects of the microbumps and the wax on the hydrophobicity. Furthermore, the adhesion and friction properties of the leaves, with and without wax, are studied. Using an optical profiler and an atomic/friction force microscope (AFM/FFM), measurements on the hydrophobic leaves, both with and without wax, were made to fully characterize the leaf surface. Using a model that predicts contact angle as a function of roughness, the roughness factor for the hydrophobic leaves has been calculated, which is used to calculate the contact angle for a flat leaf surface. It is shown that both the microbumps and the wax play an equally important role in the hydrophobic nature as well as adhesion and friction of the leaf. This study will be useful in developing super-hydrophobic surfaces. PMID- 16675116 TI - Investigation of scale effects and directionality dependence on friction and adhesion of human hair using AFM and macroscale friction test apparatus. AB - Macroscale testing of human hair tribological properties has been widely used to aid in the development of better shampoos and conditioners. Recently, literature has focused on using the atomic force microscope (AFM) to study surface roughness, coefficient of friction, adhesive force, and wear (tribological properties) on the nanoscale in order to increase understanding about how shampoos and conditioners interact with the hair cuticle. Since there are both similarities and differences when comparing the tribological trends at both scales, it is thus recognized that scale effects are an important aspect of studying the tribology of hair. However, no microscale tribological data for hair exists in literature. This is unfortunate because many interactions between hair skin, hair-comb, and hair-hair contact takes place at microasperities ranging from a few mum to hundreds of mum. Thus, to bridge the gap between the macro- and nanoscale data, as well as to gain a full understanding of the mechanisms behind the trends, it is now worthwhile to look at hair tribology on the microscale. Presented in this paper are coefficient of friction and adhesive force data on various scales for virgin and chemically damaged hair, both with and without conditioner treatment. Macroscale coefficient of friction was determined using a traditional friction test apparatus. Microscale and nanoscale tribological characterization was performed with AFM tips of various radii. The nano-, micro-, and macroscale trends are compared and the mechanisms behind the scale effects are discussed. Since the coefficient of friction changes drastically (on any scale) depending on whether the direction of motion is along or against the cuticle scales, the directionality dependence and responsible mechanisms are discussed. PMID- 16675117 TI - Effect of cholesterol on the physical properties of pulmonary surfactant films: atomic force measurements study. AB - Atomic force measurements were performed on supported pulmonary surfactant (PS) films to address the effect of cholesterol on the physical properties of lung surfactant films. We recently found that cholesterol in excess of a physiological proportion abolishes surfactant function, and is the reason that surfactant fails to lower the surface tension upon compression. In this study, we investigated how the loss of mechanical stability observed earlier is related to the local mechanical properties of the film by local force measurements. The presence of 20% of cholesterol in bovine lipid extract surfactant (BLES) resulted in a decrease of the observed adhesive interaction, and an increase in rigidity of the film. We discuss the implication the increased rigidity might have on the functional failure of PS. PMID- 16675118 TI - Dynamics of space and polarization charges of ferroelectric thin films measured by atomic force microscopy. AB - Retention behavior and local hysteresis characteristics in Pb(Zr(0.52)Ti(0.48))O(3) (PZT) thin films on Pt electrodes have been investigated by electrostatic force microscopy (EFM). A sol-gel method is used to synthesize PZT thin films and drying conditions are carefully explored over a wide range of temperature. Decay and retention mechanisms of single-poled and reverse-poled regions of the ferroelectric thin films are explained by space charge redistribution. Trapping behavior of space charges is dependent on the nature of interface between ferroelectric thin films and bottom electrodes. Local measurement of polarization-electric field curves by EFM shows inhomogeneous space charge entrapment. PMID- 16675119 TI - A femtogram resolution mass sensor platform, based on SOI electrostatically driven resonant cantilever. Part I: electromechanical model and parameter extraction. AB - A microcantilever based platform for mass detection in the femtogram range has been integrated in the doped top silicon layer of a SOI substrate. The on-plane fundamental resonance mode of the cantilever is excited electrostatically and detected capacitively by means of two parallel placed electrodes in a two port configuration. An electromechanical model of the cantilever-electrodes transducer and its implementation in a SPICE environment are presented. The model takes into account non-linearities from variable cantilever-electrode gap, fringing field contributions and real deflection shape of the cantilever for the calculation of the driving electrostatic force. A fitting of the model to the measured S(21) transmitted power frequency response is performed to extract the characteristic sensor parameters as Young modulus, Q factor, electrical parasitics and mass responsivity. PMID- 16675120 TI - Improving the contrast of topographical AFM images by a simple averaging filter. AB - New image-processing methods were applied to atomic force microscopy images in order to visualize small details on the surface of virus particles and living cells. Polynomial line flattening and plane fitting of topographical images were performed as first step of the image processing. In a second step, a sliding window approach was used for low-pass filtering and data smoothing. The size of the filtering window was adjusted to the size of the small details of interest. Subtraction of the smoothed data from the original data resulted in images with enhanced contrast. Topographical features which are usually not visible can be easily discerned in the processed images. The method developed in this study rendered possible the detection of small patterns on viral particles as well as thin cytoskeleton fibers of living cells. It is shown that the sliding window approach gives better results than Fourier-filtering. Our method can be generally applied to increase the contrast of topographical images, especially when small features are to be highlighted on relatively high objects. PMID- 16675121 TI - AFM studies of environmental effects on nanomechanical properties and cellular structure of human hair. AB - Characterization of cellular structure and physical and mechanical properties of hair are essential to develop better cosmetic products and advance biological and cosmetic science. Although the morphology of the cellular structure of human hair has been traditionally investigated using scanning electron microscopy and transmission electron microscopy, these techniques provide limited capability to in situ study of the physical and mechanical properties of human hair in various environments. Atomic force microscopy (AFM) overcomes these problems and can be used for characterization in ambient conditions without requiring specific sample preparations and surface treatment. In this study, film thickness, adhesive forces and effective Young's modulus of various hair surfaces were measured at different environments (humidity and temperature) using force calibration plot technique with an AFM. Torsional resonance mode phase contrast images were also taken in order to characterize the morphology and cellular structure changes of human hair at different humidity. The correlation between the nanomechanical properties and the cellular structure of hair is discussed. PMID- 16675122 TI - Proceedings of the 7th International Conference on Scanning Probe Microscopy, Sensors and Nanostructures, Cancun, Mexico, June 5-8, 2005. PMID- 16675123 TI - A new technical approach to quantify cell-cell adhesion forces by AFM. AB - Cell-cell adhesion is a complex process that is involved in the tethering of cells, cell-cell communication, tissue formation, cell migration and the development and metastasis of tumors. Given the heterogeneous and complex nature of cell surfaces it has previously proved difficult to characterize individual cell-cell adhesion events. Force spectroscopy, using an atomic force microscope, is capable of resolving such individual cell-cell binding events, but has previously been limited in its application due to insufficient effective pulling distances. Extended pulling range is critical in studying cell-cell interactions due to the potential for large cell deformations. Here we describe an approach to such experiments, where the sample stage can be moved 100 microm in the z direction, by closed loop, linearized piezo elements. Such an approach enables an increase in pulling distance sufficient for the observation of long-distance cell unbinding events without reducing the imaging capabilities of the atomic force microscope. The atomic force microscope head and the piezo-driven sample stage are installed on an inverted optical microscope fitted with a piezo-driven objective, to allow the monitoring of cell morphology by conventional light microscopy, concomitant with force spectroscopy measurements. We have used the example of the WM115 melanoma cell line binding to human umbilical vein endothelial cells to demonstrate the capabilities of this system and the necessity for such an extended pulling range when quantifying cell-cell adhesion events. PMID- 16675124 TI - Molecular fluorescence from H2TBP porphyrin film on Ag substrate excited by tunneling electrons. AB - Molecular fluorescence from H(2)TBP porphyrin (H(2)TBPP) films on Ag substrate is excited by a scanning tunneling microscope (STM) at ambient conditions. The molecular films are prepared by spin-casting method. The thickness of films can be controlled by the times of dropping H(2)TBPP solution. Molecular fluorescence of as-above prepared H(2)TBPP films with 4-14nm thickness at both polarities of applied bias voltage are well defined in good matching with its photoluminescence spectrum, which suggests the same decay channel associated with the HOMO-LUMO radiative transitions similar to H(2)TBPP/Au. The thickness dependence of light emission intensity of molecular fluorescence has been studied. The results show that for thicker film, molecular fluoresceence tends to be much stronger due to the effective decoupling of the emitter from the metal substrate. PMID- 16675125 TI - Ixodid ticks parasitizing Iberian red deer (Cervus elaphus hispanicus) and European wild boar (Sus scrofa) from Spain: geographical and temporal distribution. AB - Commercial hunting of Spanish wild ungulates has made them an important economic resource. Wild ungulates may have an important role in the maintenance of ixodid tick populations, and also as reservoirs of pathogens. We studied the ixodid ticks that parasitize Iberian red deer and European wild boar from Spain. Ixodid ticks (n=6,336) were collected from 431 Iberian red deer and 142 wild boar in different regions of Spain. We found 10 different ixodid tick species parasitizing Iberian red deer, mainly Hyalomma marginatum marginatum (63.7%), Rhipicephalus (Boophilus) annulatus (7.9%) and R. bursa (7.5%). R. (Boophilus) annulatus was only collected in the province of Cadiz (southern Spain). We found 8 ixodid tick species on the wild boar, mainly Hy. m. marginatum (68.7%), R. bursa (14.6%) and Dermacentor marginatus (9.3%). We found one adult Hy. marginatum rufipes and one adult Hy. anatolicum excavatum parasitizing wild boar from south-central Spain. Mean prevalence of ixodid ticks was 41.3+/-0.08% (n=475) and 31+/-0.09% (n=284) and intensity of parasitization was 13.9+/-0.2 (n=283) and 13.6+/-0.3 (n=130) ticks/animal for Iberian red deer and wild boar, respectively. Only 5 of the 13 ixodid tick species found were shared by Iberian red deer and wild boar. This finding could indicate a host preference when Iberian red deer and wild boar share common habitats. In both Iberian red deer and wild boar from south-central Spain the monthly relative frequencies of Hy. m. marginatum and R. bursa presented an inverse pattern. The highest Hy. m. marginatum relative frequencies coincided with the lowest R. bursa relative frequencies along the year. R. bursa and I. ricinus were present in areas from northern to southern Spain while Hyalomma sp. and D. marginatus were exclusively collected in the two southern thirds of Spain. Haemaphysalis sp. and D. reticulatus were collected in northern Spain. Hy. m. marginatum and R. bursa were present during the whole year in red deer and wild boar from south-central Spain, showing more than one life cycle per year. These results are important for understanding the role of wild ungulates in the maintenance of tick infestations and to improve tick control programmes. PMID- 16675126 TI - Small strongyle infection: consequences of larvicidal treatment of horses with fenbendazole and moxidectin. AB - The study was undertaken to evaluate adverse effects of larvicidal treatment in horses naturally infected with cyathostomins. Out of 24 ponies kept on pasture, four animals were housed in September and anthelmintically cured to serve as worm free controls (group C-0). The others were housed in December. Eight animals each were treated 8 weeks later with 5 x 7.5mg/kg fenbendazole (FBZ) or 1 x 0.4 mg/kg moxidectin (MOX). Four animals remained untreated (group C-i). Two, 4, 6 and 14 days after the end of treatment two animals of each of the treated groups were necropsied together with group C-0 and C-i animals. Infected animals before treatment showed weight loss, eosinophilia, increased plasma protein and globulin contents. Treatment was followed by weight gain and temporal plasma protein and globulin increase. Proportions of CD4+ and CD8+ T lymphocytes in the peripheral blood did not differ between the groups before treatment but dropped significantly temporally after FBZ treatment. Group C-0 was worm-free at necropsy. Group C-i animals contained variable numbers of luminal and tissue cyathostomins. Histological sections showed larval stages in the lamina propria und submucosa surrounded by macrophages. Either treatment was effective against luminal parasites and reduced the number of larvae in the bowel wall beginning 4 6 days after FBZ and 6-14 days after MOX treatment. Histologically, as a first reaction after FBZ application T lymphocytes accumulated around morphologically intact L4 in the submucosa. Subsequently T lymphocytes associated with eosinophils infiltrated the submucosa. Parasites became enclosed by granulomas with eosinophils adhering to and invading the larvae which started to disintegrate on day 4. Later on, particularly on day 14 inflammation extended into the mucosa and was frequently associated with ulcerations. Third stage larvae in general and L4 in the lamina propria, however, seemed not to be affected until day 14 and even then, parasites did usually not generate extensive inflammation. After MOX treatment severe morphologically detectable alterations of tissue larvae could not be observed earlier than day 14. Different from FBZ treatment, larvae disintegrated and were obviously resorbed without causing severe inflammation in the gut wall. In conclusion treatment with either drug was efficacious against tissue larvae of cyathostomins but there may be different clinical consequences: in contrast to MOX effects, killing of larvae due to FBZ was associated with severe tissue damage, which clinically may correspond to reactions caused by synchronous mass emergence of fourth stage larvae, i.e., may mimic larval cyathostominosis. PMID- 16675127 TI - A comparative longitudinal study of bovine trypanosomiasis in tsetse-free and tsetse-infested zones of the Amhara Region, northwest Ethiopia. AB - A study was conducted to determine the incidence of trypanosome infections in cattle in tsetse-free and tsetse-infested zones of the Amhara Region of northwest Ethiopia. A total of six sentinel herds were established and the cattle observed during a period of 8 consecutive months. The prevalence of seropositive cattle was high in both the tsetse-free and tsetse-infested zones. The average monthly incidence of trypanosome infection, determined using molecular diagnostic tools, was 20.9% and 25.7% in the tsetse-free and the tsetse-infested zones, respectively. In the tsetse-free, Trypanosoma vivax was responsible for 90.9% of the cattle trypanosome infections. In the tsetse-infested zone, Trypanosoma congolense and T. vivax contributed almost equally to the trypanosome infections in cattle. Trypanosome infection, regardless of species, resulted in anaemia as evidenced by a significant decrease in the packed cell volume of the infected animal. The outcome of this longitudinal study suggests that control of trypanosomiasis in the Amhara Region cannot be achieved by tsetse control alone. Supplemental measures to include drug therapy and biting fly control are discussed. PMID- 16675128 TI - From discovery to development: current industry perspectives for the development of novel methods of helminth control in livestock. AB - Despite the extraordinary success in the development of anthelmintics in the latter part of the last century, helminth parasites of domestic ruminants continue to pose the greatest infectious disease problem in grazing livestock systems worldwide. Newly emerged threats to continuing successful livestock production, particularly with small ruminants, are the failure of this chemotherapeutic arsenal due to the widespread development of anthelmintic resistance at a time when the likelihood of new products becoming commercially available seems more remote. Changing public attitudes with regards to animal welfare, food preferences and safety will also significantly impact on the ways in which livestock are managed and their parasites are controlled. Superimposed on this are changes in livestock demographics internationally, in response to evolving trade policies and demands for livestock products. In addition, is the apparently ever-diminishing numbers of veterinary parasitology researchers in both the public and private sectors. Industries, whether being the livestock industries, the public research industries, or the pharmaceutical industries that provide animal health products, must adapt to these changes. In the context of helminth control in ruminant livestock, the mind-set of 'suppression' needs to be replaced by 'management' of parasites to maintain long-term profitable livestock production. Existing effective chemical groups need to be carefully husbanded and non-chemotherapeutic methods of parasite control need to be further researched and adopted, if and when, they become commercially available. This will require veterinary parasitology researchers from both the public and private sectors to work in close co-operation to ensure 'sustainability' - not only of the livestock industries that they service - but also for their very own activities and enterprises. PMID- 16675129 TI - Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. AB - Exposure to estrogens is associated with increased risk of breast and other types of human cancer. Estrogens are converted to metabolites, particularly the catechol estrogen-3,4-quinones (CE-3,4-Q), that can react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone base excision repair of this damage may lead to the mutations that can initiate breast, prostate and other types of cancer. The reaction of CE 3,4-Q with DNA forms the depurinating adducts 4-hydroxyestrone(estradiol) [4 OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua. These two adducts constitute more than 99% of the total DNA adducts formed. Increased levels of these quinones and their reaction with DNA occur when estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of estrogen activating enzymes and/or deficient expression of the deactivating (protective) enzymes. This unbalanced metabolism has been observed in breast biopsy tissue from women with breast cancer, compared to control women. Recently, the depurinating adduct 4-OHE1(E2)-1-N3Ade has been detected in the urine of prostate cancer patients, but not in urine from healthy men. Mutagenesis by CE-3,4-Q has been approached from two different perspectives: one is mutagenic activity in the lacI reporter gene in Fisher 344 rats and the other is study of the reporter Harvey-ras gene in mouse skin and rat mammary gland. A-->G and G-->A mutations have been observed in the mammary tissue of rats implanted with the CE-3,4-Q precursor, 4-OHE2. Mutations have also been observed in the Harvey-ras gene in mouse skin and rat mammary gland within 6-12 h after treatment with E2-3,4-Q, suggesting that these mutations arise by error-prone base excision repair of the apurinic sites generated by the depurinating adducts. Treatment of MCF-10F cells, which are estrogen receptor-alpha-negative immortalized human breast epithelial cells, with E2, 4-OHE2 or 2-OHE2 induces their neoplastic transformation in vitro, even in the presence of the antiestrogen ICI-182,780. This suggests that transformation is independent of the estrogen receptor. The transformed cells exhibit specific mutations in several genes. Poorly differentiated adenocarcinomas develop when aggressively transformed MCF-10F cells are selected and injected into severe combined immune depressed (SCID) mice. These results represent the first in vitro/in vivo model of estrogen-induced carcinogenesis in human breast epithelial cells. In other studies, the development of mammary tumors in estrogen receptor-alpha knockout mice expressing the Wnt-1 oncogene (ERKO/Wnt-1) provides direct evidence that estrogens may cause breast cancer through a genotoxic, non-estrogen receptor alpha-mediated mechanism. In summary, this evidence strongly indicates that estrogens can become endogenous tumor initiators when CE-3,4-Q react with DNA to form specific depurinating adducts. Initiated cells may be promoted by a number of processes, including hormone receptor stimulated proliferation. These results lay the groundwork for assessing risk and preventing disease. PMID- 16675130 TI - Differential efficiency of the endocytic machinery in tonic and phasic synapses. AB - Efficient synaptic vesicle membrane recycling is one of the key factors required to sustain neurotransmission. We investigated potential differences in the compensatory endocytic machineries in two glutamatergic synapses with phasic and tonic patterns of activity in the lamprey spinal cord. Post-embedding immunocytochemistry demonstrated that proteins involved in synaptic vesicle recycling, including dynamin, intersectin, and synapsin, occur at higher levels (labeling per vesicle) in tonic dorsal column synapses than in phasic reticulospinal synapses. Synaptic vesicle protein 2 occurred at similar levels in the two types of synapse. After challenging the synapses with high potassium stimulation for 30 min the vesicle pool in the tonic synapse was maintained at a normal level, while that in the phasic synapse was partly depleted along with expansion of the plasma membrane and accumulation of clathrin-coated intermediates at the periactive zone. Thus, our results indicate that an increased efficiency of the endocytic machinery in a synapse may be one of the factors underlying the ability to sustain neurotransmission at high rates. PMID- 16675131 TI - Exercise enhances axonal growth and functional recovery in the regenerating spinal cord. AB - We investigated whether enhancing locomotory activity could accelerate the axonal growth underlying the significant recovery of function after a complete spinal transection in the eel, Anguilla. Eels with low spinal transections (at about 60% body length) were kept in holding tanks, where they were inactive, or made to swim continually against a water current at about one body length/s. Their locomotion was periodically assessed by measuring tail beat frequencies at different swimming speeds. Axonal growth was determined from anterograde labeling with 1,1'-diotadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, inserted postmortem into the spinal cord, just rostral to the transection. Twenty days after surgery, there were significantly more labeled growth cones more than 2 mm caudal from the transection in the exercised fish (74.6+/-2.3%; cf. 34.5+/-1.1%). This difference was still observed at 40 days (57.9+/-1.6% cf. 42.1+/-2% >2 mm), but the regenerated axons were of similar maximum lengths by 120 days (9.8+/-0.3 cf. 7.7+/-2.8 mm). After surgery, each eel undulated its whole body faster at any given swimming speed, thus changing the linear relationship between tail beat frequency and forward speed established before transection. The slope increased by up to 112.5+/-27.4% over the first 8 days post-surgery in inactive animals, while a smaller rise (45.6+/-10.5%) was observed in exercised fish during this period. Thereafter, the slope progressively declined to pre-surgery levels in both groups of animals, but the recovery occurred within 20+/-4 days in exercised eels, as opposed to 40+/-5 days in inactive fish. The locomotory performance of sham-operated fish was unaffected by 10 days of continual locomotion and remained similar to that of naive eels, pre-transection. These data show that elevated locomotory activity enhances axonal growth and accelerates recovery of locomotory function. PMID- 16675132 TI - Atp2b2, encoding plasma membrane Ca2+-ATPase type 2, (PMCA2) exhibits tissue specific first exon usage in hair cells, neurons, and mammary glands of mice. AB - Atp2b2 encodes the plasma membrane Ca(2+)-ATPase type 2 (PMCA2) expressed in various tissues, including stereocilia of cochlear and vestibular hair cells, cerebellar Purkinje cells, and lactating mammary epithelia. Mutations of the gene lead to deafness, ataxia, and reduced Ca(2+) levels in milk. Heterozygous mutants also have abnormal hearing, suggesting that precise regulation of Atp2b2 is required for normal function. In this study, we describe Atp2b2 5'-untranslated region genomic structure and transcript usage in mice. Using 5'-rapid amplification of cDNA ends, we observed four transcripts: types alpha, beta, mu and delta, each splicing into a common ATG-containing exon. Types alpha and beta correspond to previously published mammalian cDNA sequences. Types mu and delta constitute novel 5'-untranslated region sequences, and were observed at high levels only in lactating mammary gland. Using real-time reverse transcriptase polymerase chain reaction, we quantified relative transcript usage across several tissues. We show that alpha and beta are abundant throughout the CNS, as well as the cochlea. When we microdissected the cochlea into hair cell and spiral ganglion containing fractions, we found that cochlear hair cell expression is mediated through the type alpha transcript. In situ hybridization studies in cerebellum using exon-specific probes revealed that alpha dominates in Purkinje neurons, while beta is enriched in cerebellar granule neurons. We compared 5' untranslated region sequence across multiple species, and found high conservation around the first exons for alpha and beta in mammals, but not other species. The regions around the mu and delta first exons are highly conserved between rat and mouse, but less so with other species. Our results show that expression of Atp2b2 is highly regulated, using four different transcriptional start regions, two of which are differentially expressed in neuronal tissue. This suggests that unique regulatory mechanisms are used to control Atp2b2 expression in different types of cells. PMID- 16675133 TI - Spontaneous bursting and rhythmic activity in the cuneate nucleus of anaesthetized rats. AB - Spontaneous and rhythmic neuronal activity in dorsal column nuclei has long been identified in anesthetized cats. Here, we have studied the spontaneous behavior of cuneate cells in anesthetized rats through extracellular recording, showing that most cuneate neurones recorded (155 of 185) fired spontaneously. Overall, 74% of these spontaneously firing neurones were single-spiking and 26% were bursting. Cells were considered "bursting" when more than 50% of the spontaneous spikes belonged to bursts. Nevertheless, occasional bursts were seen in 33% of spontaneous cuneate cells which were classified as single-spiking. Rhythmic firing was observed in about 14% of both spontaneously bursting and single spiking cells, and these cells were located close to the obex (+/-0.5 mm). Although the spike-frequency was mostly in the range 0-15 spikes/s, spontaneous rhythmic activity was circumscribed mainly to the alpha/beta-like range, both in single-spiking (26.1+/-3.6 Hz, n=16) and bursting cells (19.5+/-4.1 Hz, n=6). Lemniscal stimulation often activated several antidromic units with the same latency. About 65% of cuneolemniscal cells were spontaneously active and of these, 83% were single-spiking and 11% rhythmic (all single-spiking). In cells that were not antidromically activated from the medial lemniscus, short latency orthodromic responses consistent with excitation by recurrent lemniscal collaterals were often observed following lemniscal activation. Interestingly, only cells completely unresponsive to lemniscal stimulation showed rhythmic bursting. Most spontaneous cells responded with a burst to natural receptive field stimulation, while rhythmic cells became temporally arrhythmic. These results demonstrate, for the first time, that rat cuneate neurones can fire bursts spontaneously. Besides, this bursting activity can be rhythmic. These two properties, and the fact that groups of cuneolemniscal cells share the same conduction velocity, probably imply the reinforcement of temporal and spatial summation at their targets when they are synchronously recruited by the stimulation of overlapping receptive fields. PMID- 16675134 TI - The left parietal cortex and motor intention: an event-related functional magnetic resonance imaging study. AB - Traditionally the posterior parietal cortex was believed to be a sensory structure. More recently, however, its important role in sensory-motor integration has been recognized. One of its functions suggested in this context is the forming of intentions, i.e. high-level cognitive plans for movements. The selection and planning of a specific movement defines motor intention. In this study we used rapid event-related functional magnetic resonance imaging of healthy human subjects to investigate the involvement of posterior parietal cortex in motor intention in response to valid imperative cues. Subjects were provided with either neutral, motor or spatial cues. Neutral cues simply alerted, motor cues indicated which hand to use for response, and spatial cues indicated on which side the target would appear. Importantly, identical targets and responses followed these cues. Therefore any differential neural effects observed are independent from the actual movement performed. Differential blood oxygen level dependent signal changes for motor vs. neutral as well as motor vs. spatial cue trials were found in the left supramarginal gyrus, as hypothesized. The results demonstrate that neural activity in the left supramarginal gyrus underlies motor plans independent from the execution of the movement and thus extend previous neuropsychological and functional imaging data on the role of the left supramarginal gyrus in higher motor cognition. PMID- 16675135 TI - gamma-Hydroxybutyrate induces cyclic AMP-responsive element-binding protein phosphorylation in mouse hippocampus: an involvement of GABA(B) receptors and cAMP-dependent protein kinase activation. AB - gamma-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate gamma-hydroxybutyrate's action. Acute administration of gamma-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP responsive element-binding protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABA(B) receptor antagonist [3-[1-(R)-[(3 cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of gamma-hydroxybutyrate, confirming a GABA(B) receptor-mediated mechanism. In addition, acute gamma-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent protein kinase activity in the hippocampus, and pretreatment with the cAMP-dependent protein kinase inhibitor H-89 could prevent the effect of gamma-hydroxybutyrate on cAMP-responsive element-binding protein phosphorylation, indicating a direct involvement of cAMP-dependent protein kinase in gamma-hydroxybutyrate-induced cAMP-responsive element-binding protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding protein was not observed in the hippocampus of mice subjected to repeated gamma hydroxybutyrate exposure, suggesting the development of a gamma-hydroxybutyrate induced desensitization of the signaling pathway leading to cAMP-responsive element-binding protein activation. Since cAMP-responsive element-binding protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying gamma-hydroxybutyrate-induced neuroadaptations. PMID- 16675136 TI - Development and pH sensitivity of the respiratory rhythm of fetal mice in vitro. AB - In newborn and adult mammals, chemosensory drive exerted by CO(2) and H(+) provides an essential tonic input: without it the rhythm of respiration is abolished. It is not known, however, whether this chemosensory drive and the respiratory rhythm appear simultaneously during development. In isolated brainstem-spinal cord preparations from fetal mice, we determined at what stage of fetal life the respiratory rhythm appeared in third to fifth cervical ventral roots (phrenic motoneurons) and whether this fetal rhythm was sensitive to chemosensory inputs. A respiratory-like rhythm consisting of short duration bursts of discharges recurring at 2-16 min(-1) was detected in two of nine embryonic day 13 fetuses; it was abolished by transection of the spinal cord between the first to second cervical segments and was phase-related to rhythmic activity from medullary units of the ventral respiratory group. At embryonic day 13, it coexisted with a slow rhythm (0.1-2.0 min(-1)) of long duration bursts of action potentials which was generated by the spinal cord. At later fetal stages, the respiratory-like rhythm became more robust and of higher frequency, while the spinal cord rhythm became less obvious. At all fetal stages, acidification of the superfusion medium from pH 7.5-7.2 or 7.4-7.3 or 7.4 to 7.2 increased the frequency of both the respiratory-like and the spinal cord rhythms. In addition, acidification reduced the amplitude of the integrated burst activity of the spinal cord rhythm of embryonic day 13-embryonic day 16 fetuses and the respiratory-like rhythm of embryonic day 17 and older fetuses. Our results indicate that the rhythms transmitted by phrenic motoneurons during fetal development are chemosensitive from early fetal stages. Through its effects on induction and patterning of the rhythm, chemosensory drive may play a role in activity-dependent formation of respiratory neural networks. PMID- 16675137 TI - Localization and ontogeny of damage-induced neuronal endopeptidase mRNA expressing neurons in the rat nervous system. AB - Neuropeptides are crucial mediators in nervous and endocrine systems. Processing and degradation, the major regulatory mechanisms, of enzymes are essential for the control of these peptidergic intercellular signaling systems. Damage-induced neuronal endopeptidase (or endothelin converting enzyme-like1), a member of the neprilysin family, has recently been identified as an M13 zinc metalloprotease. Damage-induced neuronal endopeptidase mRNA expression is strikingly restricted to neurons, and is remarkably induced in response to various types of neuronal injuries, although its function and substrate remain unknown. To clarify the role of damage-induced neuronal endopeptidase, we examined the localization and ontogeny of damage-induced neuronal endopeptidase mRNA expression in the rat nervous system using in situ hybridization. Damage-induced neuronal endopeptidase mRNA was detected at embryonic day 12, and its expression restricted to the ventral region of the neural tube. Subsequently, expression was also apparent in primordia of the striatum, hypothalamus, and cranial motor nuclei during neural development. This specific distribution was relatively maintained in the adult brain, although expression levels became weaker. Expression of damage-induced neuronal endopeptidase was absent in the cerebral cortex, hippocampus, and cerebellum. In addition to prominent expression in CNS, intestinal and sensory ganglia and retina demonstrated transient intense damage-induced neuronal endopeptidase mRNA expression during the embryonic period that then declined, and disappeared after birth. The results indicated that damage-induced neuronal endopeptidase might play an important role in embryonic neural development, in particular in peripheral ganglia derived from the neural crest, and in some neurons originating from the basal plate such as the hypothalamus and cranial motor neurons. PMID- 16675138 TI - Reduced expression and capacity of the striatal high-affinity choline transporter in hyperdopaminergic mice. AB - Behavioral and neuronal abnormalities observed in mice exhibiting a reduced expression of the dopamine transporter model important aspects of schizophrenia, addiction, and attentional disorders. As the consequences of a chronic hyperdopaminergic tone for striatal output regulation have remained poorly understood, the present experiments were designed to determine the status of striatal interneuronal cholinergic neurotransmission in dopamine transporter knockdown animals. The high-affinity choline transporter represents the rate limiting step of acetylcholine synthesis and release. Compared with wild type mice, striatal high-affinity choline transporter expression in dopamine transporter knockdown mice was significantly decreased. As in vivo basal striatal acetylcholine release did not differ between the strains, reduced high-affinity choline transporter expression in dopamine transporter knockdown mice was not due to reduced basal cholinergic activity. Furthermore, the proportion of high affinity choline transporters expressed in plasma membrane-enriched versus vesicular membrane-enriched fractions did not differ from wild type animals, suggesting that changes in intracellular high-affinity choline transporter trafficking were not associated with lower overall levels of striatal high affinity choline transporters. Synaptosomal choline uptake assays indicated a reduced capacity of striatal high-affinity choline transporters in dopamine transporter knockdown mice, and thus the functional significance of the reduced level of high-affinity choline transporter expression. Likewise, in vivo measures of the capacity of striatal high-affinity choline transporters to clear increases in extracellular choline concentrations, using choline-sensitive microelectrodes, revealed a 37-41% reduction in hemicholinium-sensitive clearance of exogenous choline in dopamine transporter knockdown mice. Furthermore, clearance of potassium-evoked choline signals was reduced in dopamine transporter knockdown mice (1.63+/-0.15 microM/s) compared with wild type animals (2.29+/-0.21 microM/s). Dysregulated striatal cholinergic neurotransmission is hypothesized to disrupt the integration of thalamic and cortical information at spiny projection neurons and thus to contribute to abnormal striatal information processing in dopamine transporter knockdown mice. PMID- 16675139 TI - Ischemia-induced disturbance of neuronal network function in the rat spinal cord analyzed by voltage-imaging. AB - Using a voltage-imaging technique, we analyzed the acute effect of ischemia, hypoxia and hypoglycemia on the neuronal network function of the rat spinal cord. Ischemic, hypoxic, or hypoglycemic stress was loaded to spinal cord slices with an oxygen- and glucose-free, oxygen-free, or glucose-free mock cerebrospinal fluid, respectively. Depolarizing signals in the dorsal horn, induced by dorsal root stimulation, consisted of fast (pre-synaptic) and slow (post-synaptic) components. The slow component was attenuated much more than the fast component under an ischemic condition (P<0.0002). Post-synaptic neuronal activities in lamina III-IV were suppressed earlier than those in lamina I-II. The nerve fiber was relatively resistant to ischemia. As long as the fast component was preserved in the dorsal horn, the suppression of the fast and slow components was reversible. There was a significant difference (P<0.05) in the recovered slow component sizes between the group in which the fast component was suppressed by more than 20% by ischemia and the group in which the suppression was less than 20%. Further prolonged stress irreversibly eliminated most of the slow component, and attenuated the fast component (to 59+/-8%) accompanied by cellular damage in histology. Suppression of neural activity by hypoxic or hypoglycemic stress was less prominent than that by ischemia. Prolonged ischemic stress suddenly and irreversibly eliminated depolarizing signals in the ventral horn accompanied by morphological damage of motoneurons. Immunohistochemical staining was negative for apoptosis. We have, for the first time, analyzed the processes of spinal cord disturbance induced by ischemia, hypoxia and hypoglycemia at the neuronal network level by directly observing the regional neuronal network activities within the spinal cord. We conclude that synaptic transmission in the dorsal horn, especially in deep regions, is vulnerable and first affected by these stresses. Severe ischemic stress induces irreversible dysfunction of neurons accompanied by eventual cell death in both dorsal and ventral horns. PMID- 16675140 TI - Current source density analysis of laser heat-evoked intra-cortical field potentials in the primary somatosensory cortex of rats. AB - The role of the primary somatosensory cortex in thermal pain perception has been established. However, the cortical circuitry that mediates the thermo-nociceptive information processing has not been elucidated. The aim of present study was to investigate the intracortical synaptic currents in primary somatosensory cortex evoked by short laser pulses and to determine their transmission pathway. Noxious CO2 laser pulse stimuli or innocuous electrical and mechanical stimuli were delivered to the hind paw of halothane-anesthetized rats. Multi-channel field potentials were recorded simultaneously in primary somatosensory cortex and laminar-specific transmembrane currents were analyzed using a current source density method. A distinct spatial-temporal pattern of intra-cortical sink source currents was evoked by laser pulse stimuli. The amplitude of the early component was graded by laser energy output and influenced by contralateral signals, whereas the late components were not intensity-dependent and exhibited bilateral excitation. Intra-cortical current flows revealed that synaptic activation occurred initially at layers IV and VI separately and then was relayed transynaptically to the more superficial and the deeper layers. Latency, amplitude and intracortical distributions of the activated intra-cortical currents evoked by noxious stimuli differed significantly from those evoked by innocuous stimuli. Conduction velocity data together with the results of tetrodotoxin, capsaicin and morphine treatments indicated that the early and late components were mediated separately by A-delta and C fibers. Our results suggest that large and small diameter thermal nociceptive afferents generated laminar specific intracortical synaptic currents in primary somatosensory cortex and that these excitatory synaptic currents were conveyed separately by lateral and medial thalamic nuclei. PMID- 16675141 TI - Absence of GABA type A signaling in adult medial habenular neurons. AB - Neural inhibition in the brain is mainly mediated by ionotropic GABA type A receptors. Apart from the GABA type A receptors, both K(+)-Cl(-) cotransporter isoform 2 and the GABA-synthesizing enzyme, glutamic acid decarboxylase, are essential determinants for GABA type A receptor-mediated inhibition. By using immunofluorescent staining, we observed that K(+)-Cl(-) cotransporter isoform 2, GABA type A receptor beta2/3 subunits and a presynaptically localized glutamic acid decarboxylase isoform, glutamic acid decarboxylase 65, were all absent in adult Sprague-Dawley rat medial habenular nucleus, while immunopositive staining for glutamic acid decarboxylase 67, GABA and GABA type B receptor type 2 subunit were present in the medial habenular nucleus. Consistent with the lack of GABA type A signaling as detected by immunohistochemistry, GABA (100 muM) evoked no measurable currents in the medial habenular nucleus but induced bicuculline sensitive currents in the lateral habenular nucleus and in the CA1 area of hippocampus. We also failed to record miniature inhibitory postsynaptic currents in medial habenular nucleus neurons. These results support the idea that GABAergic transmission in medial habenular nucleus is probably not mediated by any of the most common GABA type A receptor subtypes. Our data suggest that GABA type B receptor-mediated inhibition may play a role in balancing neuronal excitation in this special region. Further exploration for factors determining medial habenular nucleus neural inhibition will lead to a more complete understanding of control of synaptic balance in the CNS. PMID- 16675142 TI - Auditory inhibitory gating in medial prefrontal cortex: Single unit and local field potential analysis. AB - Medial prefrontal cortex is a crucial region involved in inhibitory processes. Damage to the medial prefrontal cortex can lead to loss of normal inhibitory control over motor, sensory, emotional and cognitive functions. The goal of the present study was to examine the basic properties of inhibitory gating in this brain region in rats. Inhibitory gating has recently been proposed as a neurophysiological assay for sensory filters in higher brain regions that potentially enable or disable information throughput. This perspective has important clinical relevance due to the findings that gating is dramatically impaired in individuals with emotional and cognitive impairments (i.e. schizophrenia). We used the standard inhibitory gating two-tone paradigm with a 500 ms interval between tones and a 10 s interval between tone pairs. We recorded both single unit and local field potentials from chronic microwire arrays implanted in the medial prefrontal cortex. We investigated short-term (within session) and long-term (between session) variability of auditory gating and additionally examined how altering the interval between the tones influenced the potency of the inhibition. The local field potentials displayed greater variability with a reduction in the amplitudes of the tone responses over both the short and long-term time windows. The decrease across sessions was most intense for the second tone response (test tone) leading to a more robust gating (lower T/C ratio). Surprisingly, single unit responses of different varieties retained similar levels of auditory responsiveness and inhibition in both the short and long-term analysis. Neural inhibition decreased monotonically related to the increase in intertone interval. This change in gating was most consistent in the local field potentials. Subsets of single unit responses did not show the lack of inhibition even for the longer intertone intervals tested (4 s interval). These findings support the idea that the medial prefrontal cortex is an important site where early inhibitory functions reside and potentially mediate psychological processes. PMID- 16675143 TI - The tetratricopeptide repeat domains of rapsyn bind directly to cytoplasmic sequences of the muscle-specific kinase. AB - Clustering of acetylcholine receptors at the developing vertebrate neuromuscular junction is initiated by neural agrin, which stimulates the activity of the muscle-specific kinase (MuSK). Acetylcholine receptor clustering is also dependent on the postsynaptic scaffolding protein, rapsyn, which binds to acetylcholine receptors. Here, we address the possibility that MuSK and rapsyn bind directly to each other by coexpressing sequences of the cytoplasmic domain of MuSK with rapsyn in COS-7 cells and assaying for codistribution and biochemical interaction. Sequences constituting the bulk of the kinase domain can interact with rapsyn. This interaction is mediated by the tetratricopeptide repeat domains, but not the coiled coil or zinc finger domains, of rapsyn. This interaction does not require tyrosine phosphorylation of the MuSK sequences. Binding is direct, as indicated by blot overlay and surface plasmon resonance experiments. The sequence of the cytoplasmic domain of MuSK that most effectively codistributes with rapsyn confers the ability of an otherwise inactive receptor tyrosine kinase, TrkA, to associate with rapsyn. Our results support a model in which the tetratricopeptide repeat domains of rapsyn bind directly to the cytoplasmic portion of MuSK, which could thereby serve as an initial scaffold for the clustering of acetylcholine receptors. PMID- 16675144 TI - Noxious cold stimulation induces mitogen-activated protein kinase activation in transient receptor potential (TRP) channels TRPA1- and TRPM8-containing small sensory neurons. AB - Two cold-sensitive transient receptor potential (TRP) channels, TRPA1 and TRPM8, have been identified and considered interesting because of their possible roles in thermosensation, nociception and other functions. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase and p38 mitogen-activated protein kinase occurred in primary afferent neurons in response to noxious heat stimulation of the peripheral tissue, i.e. activity-dependent activation of extracellular signal-regulated protein kinase and p38 in dorsal root ganglion neurons. In the present study, we investigated the phosphorylation of extracellular signal-regulated protein kinase, p38, and c-Jun N-terminal kinase in the rat dorsal root ganglion by cold stimulation using immunohistochemistry. Cold stimuli (28-4 degrees C) were applied by immersion of the hind paw into a water bath (six times of 10 s stimulation and 10 s interval, total 2 min). Noxious cold stimulation induced phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38, but not phosphorylated-c Jun N-terminal kinase, in small to medium diameter sensory neurons with a peak at 2 min after stimulation. We found that a cold stimulation at 4 degrees C showed a marked increase in the number of activated neurons. Furthermore, double staining for phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38 showed no colocalization in the dorsal root ganglion neurons. We then performed double-labeling experiments for TRPA1 and TRPM8 mRNA and phosphorylation of mitogen-activated protein kinase. The majority of phosphorylated-extracellular signal-regulated protein kinase-positive neurons also expressed TRPM8 mRNA, whereas phosphorylated-p38 heavily colocalized with TRPA1 mRNA after noxious cold stimulation. Our data suggest that the noxious, but not innocuous, cold stimulation in vivo induced differential activation of extracellular signal-regulated protein kinase and p38 pathways in each subpopulation containing TRPA1 or TRPM8 in dorsal root ganglion. PMID- 16675145 TI - Triggering receptor expressed in myeloid cells 2 (TREM2) trafficking in microglial cells: continuous shuttling to and from the plasma membrane regulated by cell stimulation. AB - Cell biology of triggering receptor expressed in myeloid cells 2, a receptor expressed in brain cells (microglia and possibly neurons and oligodendrocytes) which is responsible for a neurological and psychiatric genetic disease, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy otherwise called the Nasu-Hakola disease, is still largely unknown. Using immortalized mouse N9 microglial cells we demonstrate that triggering receptor expressed in myeloid cells 2 is mostly distributed intracellularly in two pools: a deposit in the Golgi complex and a population of exocytic vesicles, distinct from endosomes and lysosomes, which is continuously translocated to, and recycled from the cell surface. Results with ionomycin and gamma-interferon, showing rapid and slow increases, respectively, of triggering receptor expressed in myeloid cells 2 surface density, documented that the exocytosis of the receptor-rich vesicles is regulated. Pulse labeling in the cold of surface triggering receptor expressed in myeloid cells 2 with its antibody (or Fab fragment) followed by chase at 37 degrees C showed internalization, with recovery of the antibody in endosomes and lysosomes. However, part of the receptor/antibody complex, internalized for up to 30 min chase, was recycled to the cell surface within 2 min of ionomycin stimulation, together with a fraction of the total biotinylated surface protein chased in parallel. The internalized receptor appears therefore to get access to exocytic organelles distinct from lysosomes which may resemble the exocytic vesicles of resting cells. These results document that, in microglial cells, the surface density of the triggering receptor expressed in myeloid cells 2 and thus, presumably, the response to its activation, is continuously adapted and can be greatly increased, even at rapid rate, as a function of cell activation. PMID- 16675146 TI - Seasonality, waist-to-hip ratio, and salivary testosterone. AB - Patterns of seasonal variation in testosterone (T) and T-dependent measures are poorly understood in humans and particularly in women, despite their importance in other animals. We examined seasonal fluctuations in salivary T in women and men, and waist-to-hip ratio (WHR) in women. Participants were 220 women and 127 men from central and West Coast North America. Results showed that T was significantly highest in autumn for both women and men, and that WHR in women closely matched the seasonal variation in T, with high values in the fall and summer. This suggests that T does show a reliable fluctuation over the seasons, which may result in meaningful fluctuations in behavioral, cognitive, and somatic variables associated with T. PMID- 16675147 TI - Item response mixture modeling: application to tobacco dependence criteria. AB - This paper illustrates new hybrid latent variable models that are promising for phenotypical analyses. The hybrid models combine features of dimensional and categorical analyses seen in the conventional techniques of factor analysis and latent class analysis. The paper focuses on the analysis of categorical items, which presents especially challenging analyses with hybrid models and has recently been made practical in the Mplus program. The hybrid models are typically seen to fit data better than conventional models of factor analysis (IRT) and latent class analysis. An illustration is given in the form of analysis of tobacco dependence in a general population survey. PMID- 16675148 TI - Explicit and implicit effects of anti-marijuana and anti-tobacco TV advertisements. AB - Effects of anti-tobacco and anti-marijuana TV advertisements on explicit (i.e., semantic differential ratings) and implicit (i.e. Implicit Association Test, IAT) attitudes toward tobacco and marijuana were compared. Two hundred twenty nine, 18 to 19-year-old U.S. college students were randomly assigned to anti-tobacco or anti-marijuana PSA viewing conditions. Participants completed a short survey on attitudes to tobacco and marijuana. Afterwards they watched 15 PSAs embedded in a 15-min science program. At the end, all participants completed IAT for marijuana, IAT for tobacco and the assessment of explicit attitudes. Results of ANCOVA revealed a significant interaction between type of TV PSAs watched and implicit attitudes, F(1,223)=7.12, p<0.01 when controlling for preexisting attitudes to both substances; the implicit attitudes were more negative toward the substance that corresponded to the content of advertisements watched (i.e., anti-tobacco or anti-marijuana). However, analogical analysis on explicit measures showed that attitudes to marijuana became less negative among students that watched anti marijuana ads than the group with anti-tobacco ads, F(1,222)=5.79, p<0.02. The discussion focused on the practical and theoretical implications of the observed dissociation between implicit and explicit attitudes to marijuana after the exposure to anti-marijuana PSAs. PMID- 16675149 TI - Impulsivity and the reinforcing value of cigarette smoking. AB - The present study tested the hypothesis that impulsivity would predict perceptions of positive and negative reinforcement from smoking. The secondary hypothesis was that the relationship between impulsivity and smoking reinforcement expectations would be mediated by the character trait of self directedness. College students (n=202) who reported smoking cigarettes participated in the survey study. Hierarchical regression analyses confirmed that impulsivity predicted expectations about positive (beta=.22, p=.001) and negative (beta=.25, p=.001) reinforcement from smoking. These relationships were also mediated by self-directedness. Results suggest that impulsive smokers in the early stages of dependence may smoke because they expect smoking to be extremely pleasurable as well as to help dispel bouts with negative affect. Furthermore, their elevated expectations about smoking may be related to difficulties adapting to challenging environments and working toward long-term goals. PMID- 16675150 TI - Substance use pathways to methamphetamine use among treated users. AB - Considerable research has focused on patterns of substance use initiation among adolescents and for users of selected drugs; however, few data are available for methamphetamine (MA) users. This study describes substance initiation patterns for 352 MA users and assesses predictors of age of MA initiation and its sequencing. Subjects were randomly selected from treatment admissions in a large California county and interviewed using an extensive natural history protocol. Average age of MA initiation was 19 years. Nearly all (95%) had used alcohol, marijuana, and tobacco (average initiation age 13); inhalants, hallucinogens, and crack were also commonly used earlier in the drug sequence than MA. Earlier age of MA initiation was related to race/ethnicity (being non-African-American), younger age of first use of any substance, more types of early criminal behavior, and initiating MA use for sensation-seeking reasons. Following initiation of alcohol, marijuana, and/or tobacco, 27% initiated MA before other illicit drugs, 18% initiated another illicit drug before MA, and 56% initiated two or more other illicit drugs before MA. Later MA order in the initiation sequence was related to ethnicity (being African-American) and initiating MA to substitute for another drug. Results may support targeted prevention efforts and development of more effective interventions. PMID- 16675151 TI - Substance use disorder trajectory classes: diachronic integration of onset age, severity, and course. AB - BACKGROUND: Substance use disorders (SUDs) may be characterized by onset age, severity, substance type, course, and outcomes. SUD phenotypes in the literature typically consider each of these features in isolation. Conceptual frameworks and data collection procedures for assessing SUD phenotypes are increasingly "diachronic" in approach, providing for characterizations "throughout time". The recent availability of statistical procedures for the identification of latent classes offers the possibility of developing SUD phenotypes integrating these developmental features. This article illustrates the utilization of SAS-TRAJ mixture modeling to characterize variations in SUD symptom trajectories to define phenotypes. METHODS: The subjects were 332 adult males with SUDs. Their course of symptoms from early adolescence through middle adulthood was retrospectively determined. Symptom trajectories were defined by the number of DSM-IV SUD symptoms by year of age. SAS-TRAJ mixture models identified trajectory classes. Model development, evaluation, and selection using this approach are discussed. RESULTS: Among these men with SUDs, six trajectory classes were identified, including groups characterized by early-onset and severe SUD symptoms persisting into adulthood, an early-onset group similar in adolescence but improving in adulthood, and other groups with symptoms emerging later with varying degrees of severity and persistence. The SUD trajectory classes were significantly different on comorbid psychopathology, particularly childhood disruptive behavior disorders. CONCLUSION: The results present a new method for the comprehensive depiction of heterogeneity in SUD symptoms. Future studies may determine the extent to which SUDs phenotypes based on the course of symptom development inform etiology, prevention and treatment research. PMID- 16675152 TI - Genetic correlations between smoking initiation and smoking behaviors in a twin sample. AB - Early exposure to cigarettes is connected to increasing rate of regular use later in life. We examine genetic correlation between the age of first cigarette and later smoking related behaviors. The sample consisted of twin ages 25 to 75. Biometrical analysis was used to evaluate the heritabilities and a second analyses based on Cholesky decomposition determined the shared variance. The genetic variance ranged from 48% to 69%. Models comparing age of first cigarette to and smoking persistence, cessation, and number of cigarettes during year smoked most individually indicated a very small proportion of shared genetic variance (ranging from 0.004 to 0.056). These findings support separate genetic and unshared environmental processes for the age of first cigarette as compared to three other smoking related phenotypes. Research in substance abuse risk and prevention literature suggests that delaying initial exposure is a critical step reducing in the risk of later regular use. The interventional implications of the findings support the importance of a tailored approach to preventing smoking behavior and reducing harm in smokers. PMID- 16675153 TI - Comparisons of substance use disorders and correlates between aboriginal and non aboriginal adolescents living in a mountain region in southern Taiwan. AB - The purposes of this study were to examine the differences in prevalence of lifetime substance use disorders (SUDs), age at initial substance use, and knowledge and attitudes toward substance use between aboriginal and non aboriginal adolescents living in a mountain region of southern Taiwan, and to separately examine the correlates of SUDs among the two groups. A total of 251 aboriginal and 79 non-aboriginal adolescents were recruited into this study. The results revealed that although the prevalence of SUDs was high in both aboriginal and non-aboriginal adolescents, no difference in the prevalence of SUDs between the two groups was found. Attitudes toward substance use and several dimensions of peer influence were associated with SUDs in both aboriginal and non-aboriginal adolescents. However, there were different socio-demographic and family correlates with SUDs in aboriginal and non-aboriginal adolescents, and an association between characteristics of personality and SUDs was found only in aboriginal adolescents. Those who devise strategies to prevent adolescent substance use may consider the differences in the correlates of SUDs between aboriginal and non-aboriginal adolescents. PMID- 16675154 TI - Plaques of Alzheimer's disease originate from cysts of Borrelia burgdorferi, the Lyme disease spirochete. AB - Here is hypothesized a truly revolutionary notion that rounded cystic forms of Borrelia burgdorferi are the root cause of the rounded structures called plaques in the Alzheimer brain. Rounded "plaques' in high density in brain tissue are emblematic of Alzheimer's disease (AD). Plaques may be conceptualized as rounded "pock mark-like" areas of brain tissue injury. In this century, in brain tissue of AD, plaques are Amyloid Plaques according to the most up to date textbooks. In the last century, however, Dr. Alois Alzheimer did not require amyloid as the pathogenesis for either the disease or for the origin of its plaques. Surely, amyloid is an event in AD, but it may not be the primal cause of AD. Indeed in plaques, amyloid is regularly represented by the "congophilic core" structure which is so named because the waxy amyloid material binds the congo red stain and is congophilic. However an accepted subset of plaques in AD is devoid of a congophilic amyloid core region (these plaques "cotton wool" type plaques, lack a central congophilic core structure). Furthermore, there is "plaque diversity" in Alzheimer's; small, medium and large plaques parallel variable cystic diameters for Borrelia burgdorferi. Perturbations of AD plaque structure (i.e. young plaques devoid of a central core and older plaques with or without a central core structure) offer room for an alternate pathway for explanation of ontogeny of the plaque structures. If amyloid is not required to initiate all of the possible plaques in Alzheimer's, is it possible that amyloid just a by product of a more fundamental primal path to dementia? If a byproduct status is assigned to amyloid in the realm of plaque formation, then is amyloid also an epiphenomenon rather than a primary pathogenesis for Alzheimer's disease. In the "anatomy is destiny" model, cysts of borrelia are always round. Why then not accept roundness as a fundamental "structure determines function" argument for the answer to the mystery of why Alzheimer plaques are always round? Parataxis causality, a concept borrowed from philosophy, is the error that comes from linking two events, which occur contemporaneously or in close proximity to one another with a cause and effect relationship. Parataxis tells us that what appears to be cause and effect in the couplet "amyloid plaque" merely by a proximity relationship may be "spurious causality" which is a cognitive dead end. PMID- 16675155 TI - Dose effects in gene environment interaction: an enzyme kinetics based approach. AB - There have been many observations of different forms of the effect of dose in toxicological and pharmacological research into gene environment interactions. In one form, the effect of genetic variation is seen to be more potent at lower doses, while in the other, the genetic variant has a stronger effect at higher doses of exposure. The application of principles of classical enzyme kinetics to this problem has led to a hypothesis that for any gain of function polymorphism, a low exposure gene (LEG) or low dose effect will always be observed, while for any loss of function polymorphism, the high exposure gene (HEG) effect will always be seen. Furthermore the dose effect is found to be independent of any effects on Vmax, but is related to effects on Km of the gene product. The hypothesis is derived from the Michaelis-Menten equation, and if supported by experimental evidence, could lead to important implications for drug dosage and toxicological risk analysis in the context of individual differences in genetic profiles. PMID- 16675156 TI - Genetically engineered bacteria may be useful for delivery of mucolytics into cystic fibrosis lungs. PMID- 16675157 TI - Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer. AB - The INK4a/ARF locus (9p21) encodes two unique and unrelated cell cycle regulators, p16INK4a and p14ARF. This study was performed to evaluate the methylation status of p16INK4a and p14ARF genes, as well as its association with p16 and p53 expression, microsatellite instability (MI) status, and various clinicopathologic parameters in sporadic colorectal cancer. Sixty-five cases of primary colorectal adenocarcinoma with a series of clinicopathological parameters were obtained. We performed methylation-specific PCR of p16INK4a and p14ARF genes in colorectal cancer paraffin blocks with its paired normal samples, as well as immunohistochemical stainings for p16 and p53, and MI analysis. Aberrant methylations of p16INK4a and p14ARF gene were present in 21 (32.3%) and 33 (50.8%) out of 65 cases, respectively. p16INK4a aberrant methylation was correlated with p16 negativity (P=0.021) and p53 overexpression (P=0.007). p16INK4a aberrant methylation was more frequently present in poorly differentiated adenocarcinomas (P=0.002). Aberrant methylation of p14ARF gene occurred more frequently in patients under 50 years of age and in left-sided colon cancers, and was not statistically significant. Compared with the group with simultaneous absence of methylation in both promoters, the group showing concomitant alterations in both p16INK4a and p14ARF genes (n=10) more frequently presented lymph node metastasis (P=0.020) and higher tumor grade (P=0.014). There was no correlation between p16INK4a and p14ARF gene hypermethylation or MI status. This study suggests that simultaneous hypermethylation of both p16INK4a and p14ARF genes is greater prognostic value in sporadic human colorectal cancer. PMID- 16675158 TI - Individual and sex differences in learning abilities of ravens. AB - Behavioral and physiological characteristics of individuals within the same species have been found to be stable across time and contexts. In this study, we investigated individual differences in learning abilities and object and social manipulation to test for consistency within individuals across different tasks. Individual ravens (Corvus corax) were tested in simple color and position discrimination tasks to establish their learning abilities. We found that males were significantly better in the acquisition of the first discrimination task and the object manipulation task, but not in any of the other tasks. Furthermore, faster learners engaged less often in manipulations of conspecifics and exploration of objects to get access to food. No relationship between object and social manipulation and reversal training were found. Our results suggest that individual differences in regard to the acquisition of new tasks may be related to personalities or at least object manipulation in ravens. PMID- 16675159 TI - An assessment of context-specificity of the CS-preexposure effect in Pavlovian excitatory and inhibitory conditioning. AB - Non-reinforced preexposure to a to-be-conditioned stimulus (CS) results in retarded development of conditioned excitation and inhibition. In a magazine approach preparation in rats, we explored the role of background context on this CS-preexposure effect by changing contexts after the preexposure treatment. Experiment 1 demonstrated with a typical three-group design that changing background contexts attenuated the CS-preexposure effect in conditioned excitation. Experiment 2 employed the identical design except that conditioned inhibition was the target of study. Preexposure to stimulus X retarded subsequent differentiation of responding to reinforced A trials and non-reinforced AX trials, suggesting that CS-preexposure retarded development of inhibitory conditioning. However, changing contexts did not attenuate the preexposure effect. We discuss these results in the framework of the extended comparator hypothesis. PMID- 16675160 TI - Drug and alcohol-impaired driving among electronic music dance event attendees. AB - BACKGROUND: Drug-impaired driving has received increased attention resulting from development of rapid drug-screening procedures used by police and state laws establishing per se limits for drug levels in drivers. Venues that host electronic music dance events (EMDEs) provide a unique opportunity to assess drug impaired driving among a high proportion of young adult drug users. EMDEs are late-night dance parties marked by a substantial number of young adult attendees and elevated drug involvement. No studies to date have examined drug-impaired driving in a natural environment with active drug and alcohol users. METHODS: Six EMDEs were sampled in San Diego, California, and Baltimore, Maryland. A random sample of approximately 40 attendees per event were administered surveys about alcohol and other drug (AOD) use and driving status, given breath tests for alcohol, and asked to provide oral fluid samples to test for illicit drug use upon entering and exiting the events. RESULTS: Driving status reduced the level of alcohol use (including abstaining) but the impact on drug-taking was not significant. However, 62% of individuals who reported their intention to drive away from the events were positive for drugs or alcohol upon leaving. This suggests that these events and settings are appropriate ones for developing interventions for reducing risks for young adults. PMID- 16675161 TI - Tobacco smoking in relation to analgesic drug use in a national adult population sample. AB - BACKGROUND: Nicotine is known to produce pain-inhibitory effects. Here, we examine whether there is a relation between tobacco smoking and analgesic drug use (ADU). METHODS: A probability sample of the German national population aged 18-79 with 7124 participants (participation rate: 61.4%) was used. All individuals underwent a health examination and ADU was assessed as part of an interview conducted by a study physician. RESULTS: Current and former smokers had higher odds for ADU than never smokers. Current heavy smokers (>20 cigarettes per day) had an odds ratio (OR) of 1.8 (95% confidence interval, CI, 1.4-2.3) for ADU three times per month or less and an OR of 3.1 (CI 2.0-4.8) for ADU once a week to daily, with never smokers as the reference group. Former heavy smokers had an OR of 2.0 (CI 1.3-3.2) for ADU once a week to daily compared to never smokers. CONCLUSION: The findings suggest that current and former smokers, particularly current and former heavy smokers, are more likely to use analgesic drugs than never smokers. PMID- 16675162 TI - Concentration-dependent conditioned place preference to inhaled toluene vapors in rats. AB - OBJECTIVES: Toluene is present in many commercial products and is subject to abuse by inhalation. The goal of this study was to extend previous reports indicating that rats will exhibit a positive conditioned place preference to inhaled toluene vapors and to determine the dose-response relationship for inhaled toluene in terms of exposure concentration and number of exposures. For the conditioned place preference experiments rats were exposed to toluene vapors at concentrations of 800, 2000, 3000 or 5000 ppm in one compartment of a three compartment box. RESULTS: Following six conditioning sessions with toluene, a significant place preference was obtained at 2000 and 3000 ppm, but not at 800 or 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm concentration to 12 significantly enhanced the place preference compared to that at six pairings. CONCLUSIONS: These experiments extend our previous finding that rats will show a conditioned place preference to inhaled toluene, and indicate that a reinforcing "dose" of toluene depends on both the concentration and number of pairings. PMID- 16675163 TI - The effectiveness of outreach case management in re-enrolling discharged methadone patients. AB - BACKGROUND: Heroin dependence is a chronic relapsing disease often requiring multiple treatment experiences. Despite this knowledge, few methadone programs follow-up with discharged patients who frequently continue to engage in risky behaviors. The aim of this project was to evaluate the effectiveness of outreach case management for post-discharged methadone patients. METHODS: At 90 days post discharge 128 active out of treatment heroin users were randomly assigned to receive either a passive referral (PR) for drug treatment (n=52) or were provided with 6 weeks of outreach case management (OCM), an intervention designed to help motivate and coach patients to re-enter treatment (n=76). RESULTS: At 6 months post-baseline 29% of the OCM participants had successfully re-enrolled in drug treatment compared to 8% of the PR participants (chi(2)=7.6, d.f.=1, p=0.006). A logistic regression analysis showed that OCM participants were nearly six times more likely than PR participants to re-engage in MMT (OR=5.8, CI=1.6-20.8, p=0.008). Moreover, OCM subjects had fewer opiate and cocaine positive urines at the 6-month follow-up compared to PR subjects. CONCLUSIONS: The findings highlight the importance of engaging former patients in treatment and actively assisting in treatment re-entry. OCM is a simple approach to reduce the number of out-of-treatment drug users, although availability of treatment funding limits enrollment opportunities. PMID- 16675164 TI - Regulation of the PTEN phosphatase. AB - Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a phosphatidylinositol phosphate phosphatase and is frequently inactivated in human cancers. The balance between phosphoinositide 3-kinase (PI3K) and PTEN determines PI(3,4,5)P3 levels. PI3K is regulated by a variety of intracellular and extracellular signals, but little is known about the regulation of PTEN. In this article, we review control of PTEN function by phosphorylation as well as by binding of lipid and protein partners. PMID- 16675165 TI - Eye-hand co-ordination skills in very preterm infants <29 weeks gestation at 3 years: Effects of preterm birth and retinopathy of prematurity. AB - INTRODUCTION: Preterm infants are known to have low gross motor and fine motor skills. We questioned whether poor eye-hand coordination skills are associated with moderate to severe stages of Retinopathy of Prematurity (ROP). AIMS: The aim of this study was to examine development, with specific reference to eye-hand coordination skills, among preterm infants <29 weeks gestation with different stages of ROP at 3 years of age. METHODS AND MATERIALS: Fifteen preterm infants (<29 weeks gestation) who developed Stage 3 ROP were matched for gestation, birthweight and gender with infants who developed Stage 2 and Stage 1/no ROP. Developmental (Griffiths Mental Development Scales and Peabody Developmental Motor Scales) and ophthalmic assessments in the 3 matched groups of 15 were performed at 3 years of age. RESULTS: 1) Whilst the eye-hand coordination scores and Peabody fine motor scores were lower in the Stage 3 ROP group, they were not significantly lower than the other ROP groups. 2) Locomotor, Peabody gross motor skills and hearing and speech were significantly lower in the infants with Stage 3 ROP. The other developmental domains were not significantly different to the severe ROP group. 3) All 3 groups (of preterm infants) had lower eye-hand coordination and Peabody fine motor scores compared to test norms. 4) There were 8 of 15 infants with Stage 3 ROP who developed moderate visual problems by 3 years of age. CONCLUSION: In preterm infants, low eye-hand coordination/fine motor scores are likely to be due to their extreme prematurity. PMID- 16675166 TI - Preparation for parenting multiple birth children. AB - The experience of expecting and parenting multiples is decidedly different from that of a singleton pregnancy and parenthood. Multiple births are associated with substantial medical, health care, socio-emotional, developmental, educational and economic consequences for both families and society. This paper aims to advise health professionals on how best to help families prepare for and successfully respond to the demands of multiple pregnancy and the first 5 years of parenthood after the births of twins, triplets or more. Four inter-related principles of good practice are vital to the care of multiple birth families: the involvement of a range of disciplines, of the family and of the multiple birth community; the provision of specialised care; coordinated services; and the building of family competency including the capacity to make informed decisions. Preparation should include education on the special aspects of multiple pregnancy and parenting using multiples-focused resources, health promotion and risk modification strategies, infant care and feeding, child development and advice on securing help and support while ensuring family participation in all care decisions. PMID- 16675167 TI - Menopausal estrogen therapy predicts better nocturnal oxyhemoglobin saturation. AB - OBJECTIVES: The respiratory responses in the few previous studies evaluating the effects of short-term unopposed estrogen therapy on breathing in postmenopausal women have been inconsistent. We performed a study to investigate whether long term estrogen therapy would prevent age-related decline in nocturnal arterial oxyhemoglobin saturation and whether higher serum estradiol concentration is associated with better arterial oxyhemoglobin saturation. METHODS: Sixty-four healthy postmenopausal women were followed-up for 5 years in a 5-year prospective open follow-up study. The women were users or non-users of estrogen therapy according to their personal preference. RESULTS: Mean overnight arterial oxyhemoglobin saturation was similar at baseline (94.3 +/- 1.1%) and after follow up (94.5 +/- 1.6%). Present estrogen users had higher mean arterial oxyhemoglobin saturation (95.2 +/- 1.4%) than present non-users (94.0 +/- 1.5%), when adjusted for age and body mass index (p = 0.042). The change in mean arterial oxyhemoglobin saturation during follow-up was not associated with serum estradiol concentration at baseline but associated with estradiol at follow-up (p = 0.042), when adjusted for age and body mass index. At follow-up, women with higher serum estradiol concentration had also higher mean nocturnal arterial oxyhemoglobin saturation (Pearson r = 0.29, p = 0.019) and lower apnea-hypopnea index (Spearman r = -0.28, p = 0.031). The pooled current estrogen users spent proportionally less time with SaO(2) below 90% than non-users (ANCOVA adjusted for age and BMI, p = 0.017). CONCLUSIONS: Estrogen use and especially high serum estradiol concentration predict higher mean overnight arterial oxyhemoglobin saturation. The present data suggest that estrogen therapy has favorable respiratory effects. PMID- 16675168 TI - Profile of menopausal women in west Anatolian rural region sample. AB - OBJECTIVES: The aim of this study was to evaluate age at menopause, climacteric symptoms and related factors in women living in west Anatolian rural region of Turkey. METHOD: This study included 761 menopausal women living in three rural towns. Questionnaires regarding to 10 climacteric symptoms, menopausal status, sociodemographic characteristics, parity, breastfeeding, presence of chronic illness, direct sun exposure, smoking, caffeine use and hormone replacement therapy (HRT) were filled out by face-to-face interview. Logistic regression analysis was used to assess confounding factors on the age of menopause and menopausal status. RESULTS: Mean age at menopause was 44.38+/-5.30 years. Breastfeeding more than one year, low level of direct sun exposure and mother's early age at menopause were associated with early onset of menopause. The most prevalent climacteric symptoms were urine leakage, decreased libido, lack of energy and poor memory in post-menopausal and hot flushes in peri-menopausal women. Somatic and psychological symptoms were associated with hot flushes. HRT and osteoporosis treatment usage were higher in surgical menopause group. Osteoporosis and Type 2 Diabetes Mellitus (DT) were more common in natural menopause group. No association was found with onset of menopause and smoking, excess caffeine use, BMI, marital status and presence of chronic illness. Cardiovascular risk factors such as hypertension (HT), diabetes and obesity were common in post-menopausal women. CONCLUSION: Onset of menopause in Turkish women living in rural region is earlier on average women living in western or industrialized countries. Hot flushes in peri-menopausal, and urogenital, and psychological in post-menopausal women are the most prevalent symptoms. Primary care services in rural districts should focus on cardiovascular risks of menopausal women. PMID- 16675169 TI - Isoflavone therapy for menopausal flushes: a systematic review and meta-analysis. AB - OBJECTIVE: To perform a systematic review and meta-analysis of all randomized, controlled trials of isoflavone supplementation to determine the efficacy of isoflavone therapy in reducing the number of daily menopausal flushes. METHODS: A comprehensive search of published studies of isoflavone treatment and menopausal flushing was undertaken. Studies were selected if they were randomized, were placebo controlled, provided the number of baseline flushes, the variance in flushes and the reduction in flushes. Effects for isoflavone treatment compared to control were calculated and a meta-analysis was performed. Regression analysis, weighted for the size of the study was performed to investigate the relationship between the dose of isoflavone, or number of baseline flushes and the reduction in flushes achieved compared to control. RESULTS: Isoflavone supplementation was found to be associated with a significant reduction in flushes (effect size -0.28, 95% confidence intervals -0.39 to -0.18, P < 0.0001). Marked heterogeneity was found between the studies, but the effect remained significant when analyzed using a random effects model (delta = -0.49, 95% confidence intervals -0.81 to -0.17, P = 0.001). The percentage reduction in flushes was significantly related to the number of baseline flushes per day and the dose of isoflavone studied (beta = -0.49 and -0.26, respectively, both P < 0.0001). CONCLUSIONS: These results suggest that isoflavone supplementation may produce a slight to modest reduction the number of daily flushes in menopausal women and that the benefit may be more apparent in women experiencing a high number of flushes per day. PMID- 16675170 TI - Latin American position on the current status of hormone therapy during the menopausal transition and thereafter. AB - OBJECTIVE: Data from placebo-controlled, randomized clinical trials conducted during the past few years resulted in critical re-evaluation of the overall health benefits of hormone therapy (HT) in women during the menopausal transition and thereafter. These data stimulated vigorous debate among experts and produced several position papers by North American and European authorities providing guidance on the use of HT. It is well known that cultural, geographic and ethnic differences influence the acceptance and risk perception of HT. Therefore, it was considered essential to present a position specifically relevant to Latin American countries. METHODS: A Latin American Expert Panel, convening in Salvador, Bahia, Brazil, obtained consensus on recommendations for HT that incorporated the findings of the most recently published reports. The panelists' opinions were surveyed by means of the Likert scale along five categories ranging from complete agreement to complete disagreement. RESULTS: The Panel presented 13 recommendations and considered three additional issues relevant to HT use. There was consensus that HT during the perimenopause and thereafter is warranted in Latin American women in particular for the management of vasomotor symptoms. HT may also be an option for osteoporosis prevention in women at significant risk, after evaluation of risks/benefits and after consideration of alternative therapies. HT should be individualized and prescribed at the lowest effective dose. CONCLUSIONS: The Panel concluded that HT remains a safe and effective treatment option for peri- and postmenopausal Latin American women. PMID- 16675171 TI - Plasma concentrations of levonorgestrel in patients with an intrauterine progestogen delivery system: do they have any significance? PMID- 16675172 TI - Solid-state 13C NMR study of indomethacin polymorphism. AB - The purpose of this study was to analyze the difference in the molecular conformation packed in the crystal lattice between the meta-stable alpha-form and stable gamma-form of indomethacin on the basis of solid-state (13)C NMR spectral patterns. The chemical shifts of each resonance of the alpha-form were distinctly different from the gamma-form. Each carbon nucleus of the gamma-form showed a single signal with no splitting. In contrast, carbon nuclei of the alpha-form showed a complicated set of resonances for each carbon. For some carbons of the alpha-form, four signals assigned to one carbon were observed at 203 K. Two of these four signals were merged between the temperature range from 203 to 343 K without a transformation in the crystal structure. It was found that solid-state (13)C NMR can be a powerful tool to estimate the number of molecular conformations as well as configurational differences in the packing of molecules in a unit cell. PMID- 16675173 TI - Sigmoidal release of indomethacin from pectin matrix tablets: effect of in situ crosslinking by calcium cations. AB - Sigmoidal release pattern is therapeutically beneficial for timed release and colonic drug delivery, and is always observed in coated systems. In this study, sigmoidal release from pectin matrix tablets with indomethacin as a model drug was investigated. The underlying mechanisms are calcium cation-induced in situ crosslinking that retard the initial drug release to a limited percentage. Power law equation n values were estimated for sigmoidal release profiles. Results indicated that calcium chloride incorporated in pectin matrix functioned as retarding mechanisms on drug release. Larger amount of calcium chloride led to slower drug release and matrix erosion. Even at extremely high levels, retarding on drug release and matrix erosion rate was obvious, which highlighted the effect of calcium-induced in situ crosslinking as calcium chloride was a freely water soluble salt. The sigmoidal release profiles were characterized by power law equation with high correlation coefficients of about 0.99 or over. Power law n values increased up to as high as 1.20 when calcium chloride content kept increasing. Erosion correlated well with release in almost all pectin matrix tablets indicating erosion-controlled mechanisms. It is concluded that large amount of calcium induces in situ crosslinking of pectin matrix and leads to sigmoidal release of indomethacin, and power law n values, sometimes larger than 1.0, are suitable to be used to describe sigmoidal release profiles. PMID- 16675174 TI - Influence of disease stage on polyethylenimine-mediated plasmid DNA delivery in murine hepatitis. AB - In order to determine the influence of hepatic disease-stage on polyethylenimine mediated gene delivery, we investigated branched and linear polyethylenimine (B PEI, L-PEI)-mediated plasmid DNA delivery with time in murine hepatitis induced by a subcutaneous injection of tetrachloro carbon (CCl(4)). Plasmid DNA (pDNA) encoding firefly luciferase was used as the model reporter gene. We determined luciferase activity in various organs of CCl(4)-treated mice and control mice after an intravenous administration of B-PEI and L-PEI/pDNA complexes. Both B-PEI and L-PEI/pDNA complexes showed significantly lower gene expression in the liver, spleen, and lung at the stage of severe hepatitis (18 h after CCl(4) injection), whereas the complexes induced gene expression in the liver at the liver regeneration stage (48 h after CCl(4) injection). Significant differences in gene expressions between CCl(4)-treated mice and control mice vanished in most organs at the hepatitis subsidence stage (168 h after CCl(4) injection), indicating that the influence of hepatitis induced by CCl(4) was reversible with PEI-mediated gene delivery. Our findings demonstrated that murine hepatitis induced by CCl(4) could influence polyethylenimine-mediated plasmid DNA delivery according to the disease stage. These results indicate the necessity of considering the timing and dose of gene therapy according to the disease stage. PMID- 16675175 TI - A DSC and Raman spectroscopy study on the effect of PAMAM dendrimer on DPPC model lipid membranes. AB - The interaction between PAMAM (polyamidoamine) dendrimer generation 4 (G4) and 3,5 (G3,5) with model lipid membranes composed of dipalmytoylphosphatidylcholine (DPPC) has been investigated. Differential scanning calorimetry (DSC) and Raman spectroscopy were applied to assess the thermodynamic changes caused by PAMAM G4 and G3,5 and to specify the exact location of these dendrimers into the DPPC lipid bilayer. DSC thermograms indicated that the maximum percentages of PAMAM G4 and of G3,5 that can be incorporated in the DPPC membrane without deranging its integrity were 5% and 3%, respectively. The Raman intensity ratios I(2935/2880), I(2844/2880) and I(1090/1130) cm(-1) showed the degree of the fluidity of the lipid bilayer, while the absorption at 715 cm(-1) showed a strong interaction of PAMAM G4 and G3,5 with the polar head group of phospholipid. The results showed that the incorporation of the PAMAM G4 and G3,5 dendrimers in DPPC bilayers causes a concentration dependent increase of the membrane fluidity and that the bilayers interact strongly with both the lipophilic part and the polar head group of the phospholipids. Due to the current weak knowledge relating to the mechanism(s) under which dendrimers interact with lipidic membranes and transport through cells, these results may justify the tendency of dendrimers to disrupt biological membranes. The findings from this study could also prove helpful to rationally design new liposomal drug carriers for bioactive molecules by combining dendrimeric and liposomal technologies. PMID- 16675176 TI - Nanoparticles in inflammatory bowel disease: particle targeting versus pH sensitive delivery. AB - Tacrolimus proved its distinct mitigating potential in the treatment of inflammatory bowel disease (IBD). Due to the risk for severe adverse effects and to achieve increased efficiency and tolerability, a selective delivery to the site of inflammation is of interest. Tacrolimus nanoparticles (NP) were tested for their efficiency in local treatment of inflamed bowel tissue in IBD. Drug loaded NP were prepared from either biodegradable poly(lactide-co-glycolide) (PLGA) or pH-sensitive Eudragit P-4135F by using a simple oil/water emulsification method. Tests on the therapeutic effect were conducted using dextran sulfate model colitis in mice receiving tacrolimus formulations daily for 12 days. Clinical activity score and myeloperoxidase activity decreased while colon length increased significantly after administration of all tacrolimus containing formulations. Oral NP formulations were less efficient in mitigating the experimental colitis compared to subcutaneous drug solution (PLGA: 7.88 +/- 0.83; P-4135F: 7.48 +/- 0.42; subcutaneous: 5.27 +/- 0.68 U/mg) but superior to drug solution given by oral route (oral: 8.75 +/- 1.34; untreated colitis control: 9.95 +/- 0.92, all U/mg tissue). Tacrolimus solution groups (oral/subcutaneous) exhibited increased levels of adverse effects, whereas both NP groups demonstrated their potential to reduce nephrotoxicity. Both strategies showed similar mitigating effects while nephrotoxic adverse effects were slightly less expressed with pH-sensitive NP. PMID- 16675177 TI - Clinical impact of ectopic teeth in the maxillary sinus. AB - OBJECTIVE: To evaluate the clinical features, aetiologic factors, challenging properties (such as radio opacity in Water's view in a patient with no sinusitis), signs and symptoms of the ectopic teeth in the maxillary sinus. STUDY DESIGN: Clinical symptoms and radiographic findings of 14 patients with ectopic teeth in the maxillary sinus were evaluated. Computed tomography (CT) and conventional radiographic images of maxillary sinus and clinical findings were compared with each other with regard to the final diagnosis. RESULTS: Water's view is inadequate to diagnose ectopic tooth in the maxillary sinus in some cases. Panoramic radiographs may be preferred before CT to evaluate the ectopic tooth in the maxillary sinus as structure of a tooth can be clearly detected on panoramic radiographs. Crowding was the most common aetiologic factor among the 14 cases. CONCLUSION AND SIGNIFICANCE: The patients with ectopic tooth in the maxillary sinus should be evaluated thoroughly by complete otorhinolaryngologic, intraoral examinations and proper diagnostic imaging procedures in order to avoid misdiagnosis of maxillary sinusitis. As the opacity of the maxillary sinus in Water's view due to ectopic tooth can be misinterpreted as maxillary sinusitis, patients who have sinusitis-like complaints and opacity of maxillary sinus in Water's view who are resistant to medical treatment should be evaluated with respect to the presence of ectopic tooth. PMID- 16675178 TI - Mucocele in an Onodi cell with simultaneous bilateral visual disturbance. AB - The Onodi cell is a large pneumatized posterior ethmoid cell and closely related to optic nerve. We present an extremely rare case of retrobulbar optic neuropathy caused by mucocele in an Onodi cell. A 79-year-old man complained of headaches and simultaneous bilateral visual disturbance. A computed tomography (CT) scan demonstrated a mucocele in an Onodi cell, which involved bilateral optic nerves. The surgical treatment with a transnasal endoscopic approach was performed, resulting in the improving of visual acuity. The bilateral optic nerves were identified along each lateral wall into an Onodi cell accompanied with bone defect. In an Onodi cell, even if the lesion is isolated and/or small, it may be closely related to ocular symptoms. Imaging studies should be considered for the differential diagnosis because early diagnosis and prompt surgical treatment for mucocele are needed for recovery of visual impairment. PMID- 16675179 TI - Surgical intervention of intestinal malrotations in paediatric patients without other congenital anatomical abnormalities: overview from a single center. AB - The subject of malrotation in infants and children without other congenital anatomical abnormalities is reviewed from the perspective of experience with 97 patients operated in 11 years. Fifty-five patients were younger than 6 weeks at operation. They often presented with bilious vomiting, in contrast to older children who presented with non-bilious vomiting or feeding problems. Patients younger than 6 weeks were operated more often acutely than older patients. Volvulus was more common in infants younger than 6 weeks. Two patients with a resulting short bowel syndrome died. In 73 of the surviving 95 (76.8%) children their symptoms disappeared. In the children younger than 6 weeks persisting abdominal problems were significantly less frequent than in older children. In the children presenting with proven gastro-esophageal reflux disease before the malrotation operation, abdominal problems persisted significantly more often. Although there remains considerable controversy over how older children without signs of vascular problems should be managed, failure to respond to radiographic evidence of malrotation could be considered malpractice if volvulus was to occur subsequently. For this reason, every patient with a radiological proven malrotation merits diagnostic laparoscopy. PMID- 16675180 TI - Prevalence of "dural tail sign" in patients with different intracranial pathologies. AB - The purpose of this study is to clarify the prevalence, spectrum of associated tumors and the diagnostic value of the linear meningeal thickening and enhancement adjacent to a peripherally located cranial mass "dural tail sign"(DTS) in contrast enhanced magnetic resonance imaging (MRI). In this prospective descriptive cross-sectional study conducted from 2002 to 2005, 110 patients with imaging-proven intracranial lesions and no history of previous intracranial surgery were referred to the neurosurgery clinic of our hospital for surgical resection. All underwent imaging with a 1.5 T MR system with and without contrast injection. Twelve patients were excluded from our study and finally 98 patients were evaluated for the presence of "dural tail sign". Twenty-two of 98 patients (22.44%) with intracranial masses exhibited the "dural tail sign" (18 meningiomas, 2 pituitary adenomas, 1 primary cerebral lymphoma and 1 fungal brain abscess). Fifty-eight percent of the patients with biopsy-proven meningioma were observed to show "dural tail sign". In conclusion, we found the "dural tail sign" to have a sensitivity of 58.6% and specificity of 94.02% in diagnosis of meningioma. PMID- 16675181 TI - Enteric pathogens associated with diarrhea in children in Fayoum, Egypt. AB - In a cross-sectional study of children <60 months old from Fayoum, Egypt, presenting with diarrhea, 46% (162/356) had detectable enteric pathogens. Bacterial pathogens were identified in 25% (89/356), whereas rotavirus and Cryptosporidium were detected in 21% (54/253) and 15% (39/253), respectively. Cryptosporidium is an important pathogen in this region. PMID- 16675182 TI - [Maternal postures and epidural analgesia during labour]. AB - The evolution of birth is of interest for obstetricians and midwives. Postures with asymmetric stretching and balance, kneeling, or sitting have been claimed to be able to help foetal head rotation. Although walking during labour have no influence on the outcome of labour, hip-flexed postures enlarging the pelvic diameter are yet evaluated to improve the obstetric course of labour. In a prospective randomised study including 93 parturients, we compared the supine 30 degrees lateral tilt (control group) to three hip-flexed postures: sitting (S), right hip-flexed left lateral position (L) and left hip-flexed right lateral position (R). Epidural analgesia with 12 ml ropivacaine 0.1% and sufentanil 0.5 microg/ml was administered over a period of six minutes. The total epidural spread was 15+/-0.3 dermatomes and the upper level of thermo-analgesic blockade reached T7-T8 (T5 to T10) in each group. There were no differences between groups for the left and right total spread and upper level of epidural blockade, for the time to maximal block and pain relief. There was no motor block and no maternal or foetal side effects. We conclude that, for the three hip-flexed postures tested, position does not influence local anesthetic spread or symmetry of analgesia after induction of obstetric epidural anaesthesia. PMID- 16675183 TI - [Necrotizing cutaneous infections and Fournier's gangrene]. AB - Fournier's gangrene is a necrotizing infection of skin and soft tissue of the perineum. Several sources of contamination have been described: cutaneous, urological or anorectal. Anatomy of the perineum helps in the prediction of the extension of the infection. The bacterial flora (usually mixed aero-anaerobic flora) depends on the source of infection: gram-positive cocci (skin source) combined with gram-negative bacilli and anaerobes (urological or anorectal source). CT scan and echography are useful tools for the diagnosis without delaying surgery. In the diagnostic procedure and the management, proctological examination, retrograde uretrography, bowel and urine derivation should be discussed. PMID- 16675184 TI - [Anti-inflammatory modulators in traumatic brain injury]. AB - Traumatic brain injury leads to primary and secondary brain injuries. Primary brain injury results from mechanical forces applied to the head at the time of impact. Secondary brain injury occurs at some time after the primary impact. Numerous pathophysiological mechanisms have been postulated to explain the progressive tissue damage produced by secondary injuries. The endogenous neuroinflammatory response after traumatic brain injury contributes to the development of blood-brain barrier breakdown, cerebral oedema and neuronal cell death and this has led to various pharmacological therapies to try to limit this type of damage. Studies employing glutamate receptor antagonist for cerebral protection have yielded promising results in laboratory animals but failed to produce clinically significant improvements. The present review will summarize the mechanisms of post traumatic cerebral inflammation with a special focus on the anti-inflammatory drug targets. PMID- 16675185 TI - [Strategies of management of necrotizing soft tissue infections]. AB - Necrotizing soft tissue infections represent a group of rapidly progressive diseases requiring early and repeated debridement, associated with broad spectrum antibiotics. Delay in surgery or inadequate therapy are the main risk factors for death. Most patients need aggressive critical care management and intensive nutritional support. The management of these patients by experimented senior surgeons is mandatory. A plastic surgeon can help debridement in order to preserve possibilities of later myocutaneous or rotational skin flaps. Intravenous immunoglobulins are an efficacious adjunctive therapy for severe group A streptococcal infection. PMID- 16675186 TI - [Clinical study of the laryngeal mask in paediatric. Comparison of the LMA ProSeal and LMA-classic]. AB - OBJECTIVES: To compare the Classic laryngeal mask airway (MLC) and the ProSeal LMA (MLP), size 2 and 2.5 in terms of ease of insertion, leak pressure and side effects during insertion and extraction in the recovery room. STUDY TYPE: Randomised prospective. MATERIAL AND METHOD: All children between 10 and 30 kg scheduled for general anaesthesia with laryngeal mask (ML) were included. There was no imposed protocol for the anaesthesia. The ML size was determined according to the child's weight. The MLC was inserted using the standard technique. The MLP was inserted following the recommendations, with or without the handle according to the operator's choice. The data analysed were: insertion type, ease of insertion of the mask, of the nasogastric tube (SG), number of attempts of mask insertion, complications, gastric leaks. RESULTS: One hundred (and) twenty children were included. There was no statistical difference in terms of difficulty of insertion, number of failed attempts, leak pressure or side effects. The use of the handle did not make insertion easier. Insertion of a nasogastric tube was possible in 92% cases. CONCLUSION: MLP is as easy to use in children as the MLC. MLP has the advantage of allowing rapid access to the stomach. It seems that the MLP is safer since its correct position is confirmed by easy gastric tube insertion. PMID- 16675188 TI - [Therapeutic hypothermia]. AB - The benefit of therapeutic hypothermia after severe head injury is highly controversial. However, hypothermia is still used and studied in this context for multiple reasons. Efficacy of hypothermia is demonstrated after cerebral ischemia in numerous animal studies and after cardiac arrest in human studies. Hyperthermia is a major independent factor of outcome after cerebral ischemic or traumatic brain injury. Moreover, ICP is related to core temperature, and hypothermia may be used to decrease intracranial hypertension. However, many questions are still unresolved and can explain discrepancies between clinical studies: direct measurement of cerebral temperature, relationship between ICP, temperature and PaCO(2), level and duration of hypothermia and precise methods for cooling and particularly for rewarming. PMID- 16675189 TI - [Severe cervical skin and soft tissue infections and necrotizing fasciitis]. AB - Cervical severe skin and soft tissue infections and necrotizing fasciitis originate from dental or pharyngeal infections. When compared to other forms of skin and soft tissue infections, they are recognized late, usually after one week of evolution often in a patient receiving antibiotic treatments. Extensions toward adjacent anatomical structures including mediastinum lead to a life threatening prognosis. The cutaneous appearance of these severe infections is usually inflammatory cervical signs combined to facial oedema. These moderate clinical signs require immediate surgery after CT scan imaging. PMID- 16675187 TI - [How about blood pressure in brain injury?]. AB - One of the goals of the medical management of head injured patients is to get a cerebral perfusion pressure between 60 and 70 mmHg. To reach such a goal, catecholamines are used after fluid challenge. Systemic effects of catecholamines depend on their affinity for the receptors alpha and beta. The topical application of norepinephrine (alpha predominant) induced a vasoconstriction on large cerebral arteries only. Cerebral blood flow increased in the pericontusionnal area, suggesting a loss of autoregulation. The topical application of dopamine at low concentration relaxed large cerebral arteries. Dopamine increased cerebral blood flow in the pericontusional area but data suggest a possible raise in the volume of contusion. Four human comparative studies have been published. The first study, which was not randomized, showed an intracranial pressure increase associated with dopamine. Two randomized clinical trials, published by the same group, demonstrated a better predictability with norepinephrine. The fourth study did not find any difference regarding cerebral haemodynamics. In conclusion, the quality of data on the effects of catecholamines on cerebral haemodynamics of head injured patients do not make it possible to conclude about their use. PMID- 16675190 TI - [Decompressive craniectomy and intracranial hypertension]. AB - Decompressive craniectomy was purposed for the treatment of refractory intracranial hypertension after head injury. This review discusses results obtained by this surgery in severe head trauma. Several studies have confirmed a reduction in intracranial pressure secondary to decompressive craniectomy. Mortality decreased and the proportion of good outcome of the survivors increased. These results have not been confirmed prospectively, and indications have to be clarified. The positive effects of decompressive craniectomy compared to barbiturate or hypocapnia in the "second tier therapy" in refractory intracranial hypertension could be interesting to evaluate. PMID- 16675191 TI - [Peripheral polyneuropathy after heat stroke]. AB - The authors report the case of a woman who developed a peripheral polyneuropathy after heat stroke. All the classical aetiologies of neuropathy were excluded. The final diagnostic was residual peripheral neuropathy provoked by heat stroke. The sequella of heat stroke are dominated by cerebellar compromise, but this case shows that peripheral polyneuropathies exist even if they are rare. They raise the issue of care because of severe neurological sequella. PMID- 16675192 TI - [Necrotizing skin and soft tissue infections: definitions, clinical and microbiological features]. AB - Among the three types of soft tissue and skins infections (cellulitis, necrotising cellulitis and necrotising fasciitis) only the necrotising injuries are commonly managed in intensive care unit. Location, depth and severity of these potentially life threatening infections may vary and cannot be predicted on clinical grounds No microbiological deduction can be made from clinical signs. A mixed aerobic-anaerobic flora is cultured in 40 to 80% of the cases. Magnetic resonance imaging and CT-scan are the best techniques to assess the diagnosis and determine the extension of infection. However, these exams should not delay surgical treatment. Fifteen to 30% of the patients die. Three parameters play a key role in the prognosis: ability of the physician to recognise the disease and to evaluate the severity of infection, early onset of antimicrobial therapy, and most of all early surgical debridement. PMID- 16675193 TI - [Non pharmacological treatment of severe cutaneous infections: hyperbaric oxygen therapy, dressings and local treatments]. AB - Beside conventional therapy, the management of necrotizing cellulitis and fasciitis is based on non-pharmacological treatments. Hyperbaric oxygen therapy and dressings are the most frequently used techniques. The usefulness of hyperbaric oxygen therapy is clearly demonstrated in experimental studies while the efficacy of this technique is poorly assessed in clinical practice. The French consensus conference has concluded to an adjuvant role of hyperbaric oxygen therapy combined to intensive care management, surgery and antibiotic therapy. Occlusive conventional dressings using humid or vaseline gauze dressings are largely used. Calcium alginate or silver coated dressings might be useful. In addition, vacuum-assisted closure therapy could be proposed in replacement of conventional dressings. PMID- 16675194 TI - Prevention of the interaction between HVEM, herpes virus entry mediator, and gD, HSV envelope protein, by a Keggin polyoxotungstate, PM-19. AB - One of the Keggin-type heteropolyoxotungstates (K7[PTi2W10O40]6H2O:PM-19) is a potent inhibitor of the replication of herpes simplex virus (HSV) both standard strain 169 and the thymidine kinase-defective strain YS-4C-1 in vitro and in vivo. HSV envelope protein, gD, is necessary for virus entry into the cells. Some cellular molecules, such as HVEM, were reported to act as cofactors during the viral entry step. We determined whether PM-19 prevents these interactions between HSV-gD and HVEM. These activities were investigated using the Ciphergen and BIACORE system. Using a protein chip, many kinds of gD-specific binding proteins were captured, but these proteins could not be identified. Several proteins in these gD-binding proteins were inhibited its interaction with gD due to the presence of PM-19. Using the BIACORE system, the affinity of PM-19 to gD was low, because PM-19 has no direct inactivation activity against the virion. The specific binding of HVEM to the gD was shown as KD of 1.1e-9. The affinity of PM 19 for HVEM was high (KD:2e-9). To determine the competitive binding, the PM-19 (10 microg/ml) and several concentrations of HVEM solution mixtures were injected over the gD-fixed sensor surface. Each binding signal was stable in the range of approximately 270-300 RU. In the case of the addition of PM-19 to HVEM solution, the binding signals were elevated by PM-19 dose dependently. These results suggest that the bindings of PM-19 to gD are not disturbed by the presence of HVEM. PM-19 prevents the interaction between HVEM and gD. PMID- 16675195 TI - Design and development of liposomes incorporating a bioactive labdane-type diterpene. In vitro growth inhibiting and cytotoxic activity against human cancer cell lines. AB - The isolation and identification of bioactive compounds from natural sources has given insights in the discovery of new drugs against several diseases. Labdane type diterpenes isolated from nature, have been investigated in the past for their pharmaceutical activities. Labd-7,13-dien-15-ol (1), a major lipophilic constituent of the resin 'ladano' was found to exhibit cytotoxic and cytostatic activity against cell lines derived from solid tumors with a profound activity on small lung cancer cells, DMS114. The present study, based on liposomal technology, aimed to develop a suitable carrier of compound 1 to overcome its water insolubility that inhibits further in vivo administration. Liposomes have been proved adequate drug carriers which enhance the solubility of water insoluble drugs and reduce possible side effects. Dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (EPC) and dipalmitoyl phosphatidylglycerol (DPPG) were used to prepare liposomes incorporating compound 1 at various molar ratios. Their physicochemical characteristics were determined and liposomes composed of EPC:DPPG 9:0.1 (molar ratio) was chosen as the most suitable carrier concerning their physical characteristics, stability, incorporation efficiency as well as the ability to retain the capture of compound 1 in the presence of RPMI-1640 medium. The activity of the liposomal formulation of compound 1 against human cancer cell lines was compared to that of its free form. The results showed that EPC:DPPG:1 9:0.1:5 (molar ratio) liposomal formulation maintained the pharmacological activity of 1, improved its water solubility and was justified as suitable formulation for in vivo administration of compound 1. PMID- 16675196 TI - Social anxiety and fear of negative evaluation: construct validity of the BFNE II. AB - The Brief Fear of Negative Evaluation Scale [BFNE; Leary, M. R. (1983). A brief version of the Fear of Negative Evaluation Scale. Personality and Social Psychology Bulletin, 9, 371-375] is a self-report measure designed to assess fear of negative evaluation, a characteristic feature of social anxiety disorders [Rapee, R. M., & Heimberg, R. G. (1997). A cognitive-behavioral model of anxiety in social phobia. Behaviour Research and Therapy, 35, 741-756]. Recent psychometric assessments have suggested that a 2-factor model is most appropriate, with the first factor comprising the straightforwardly worded items and the second factor comprising the reverse-worded items [Carleton, R. N., McCreary, D., Norton, P. J., & Asmundson, G. J. G. (in press-a). The Brief Fear of Negative Evaluation Scale, Revised. Depression & Anxiety; Rodebaugh, T. L., Woods, C. M., Thissen, D. M., Heimberg, R. G., Chambless, D. L., & Rapee, R. M. (2004). More information from fewer questions: the factor structure and item properties of the original and brief fear of negative evaluation scale. Psychological Assessment, 2, 169-181; Weeks, J. W., Heimberg, R. G., Fresco, D. M., Hart, T. A., Turk, C. L., Schneier, F. R., et al. (2005). Empirical validation and psychometric evaluation of the Brief Fear of Negative Evaluation Scale in patients with social anxiety disorder. Psychological Assessment, 17, 179 190]. Some researchers recommend the reverse-worded items be removed from scoring [e.g., Rodebaugh, T. L., Woods, C. M., Thissen, D. M., Heimberg, R. G., Chambless, D. L., & Rapee, R. M. (2004). More information from fewer questions: the factor structure and item properties of the original and brief fear of negative evaluation scale. Psychological Assessment, 2, 169-181; Weeks, J. W., Heimberg, R. G., Fresco, D. M., Hart, T. A., Turk, C. L., Schneier, F. R., et al. (2005). Empirical validation and psychometric evaluation of the Brief Fear of Negative Evaluation Scale in patients with social anxiety disorder. Psychological Assessment, 17, 179-190]; however [Carleton, R. N., McCreary, D., Norton, P. J., & Asmundson, G. J. G. (in press-a). The Brief Fear of Negative Evaluation Scale, Revised. Depression & Anxiety; Collins, K. A., Westra, H. A., Dozois, D. J. A., & Stewart, S. H. (2005). The validity of the brief version of the fear of negative evaluation scale. Journal of Anxiety Disorders, 19, 345-359] recommend that these items be reworded to maintain scale sensitivity. The present study examined the reliability and validity of the BFNE-II, a version of the BFNE evaluating revisions of the reverse-worded items in a community sample. A unitary model of the BFNE-II resulted in excellent confirmatory factor analysis fit indices. Moderate convergent and discriminant validity were found when BFNE-II items were correlated with additional independent measures of social anxiety [i.e., Social Interaction Anxiety & Social Phobia Scales; Mattick, R. P., & Clarke, J. C. (1998). Development and validation of measures of social phobia scrutiny fear and social interaction anxiety. Behaviour Research and Therapy, 36, 455-470], and fear [i.e., Anxiety Sensitivity Index; Reiss, S., & McNally, R. J. (1985). The expectancy model of fear. In S. Reiss, R. R. Bootzin (Eds.), Theoretical issues in behaviour therapy (pp. 107--121). New York: Academic Press. and the Illness/Injury Sensitivity Index; Carleton, R. N., Park, I., & Asmundson, G. J. G. (in press-b). The Illness/Injury Sensitivity Index: an examination of construct validity. Depression & Anxiety). These findings support the utility of the revised items and the validity of the BFNE-II as a measure of the fear of negative evaluation. Implications and future research directions are discussed. PMID- 16675197 TI - Development of the fear survey for adults with mental retardation. AB - This paper describes the development of the fear survey for adults with mental retardation (FSAMR) and provides initial evidence of its psychometric properties. The FSAMR was designed to be sensitive to the assessment needs of individuals with mental retardation. The items were developed through open-ended interviews, a review of existing measures, expert input, and pilot testing. The sample consisted of 138 adults with mental retardation (73 from institutional settings and 65 from community settings). Cronbach's alpha internal consistency coefficients were 0.97 for the scale, and above the 0.60 threshold set for the study for all but the acquiescence response set (ARS) subscale. Significant concurrent validity coefficients with anxiety measures were in the moderate range (r=0.32 and 0.40) and are comparable to other similar studies. Implications for using the FSAMR are discussed. PMID- 16675198 TI - A pharmacogenetic exploration of vigabatrin-induced visual field constriction. AB - INTRODUCTION: Use of the antiepileptic drug (AED) vigabatrin is severely limited by irreversible visual field constriction, an adverse reaction to the drug reported in approximately 40% of patients. Given the evidence suggesting an idiosyncratic drug response, we set out to detect genetic variation of strong, clinically relevant effect that might guide clinicians in the safe, controlled prescribing of this otherwise usefuldrug. METHODS: Patients with a history of at least 1-year exposure to vigabatrin were enrolled at two independent referral centers. Using Goldmann perimetry, visual fields and the extent of constriction were calculated for each patient. We examined the correlation between the extent of vigabatrin induced visual field constriction and genetic variation across six candidate genes (SLC6A1, SLC6A13, SCL6A11, ABAT, GABRR1 and GABRR2). We availed of HapMap data and used a tagging SNP technique in an effort to efficiently capture all common variation within these genes. We attempted to replicate any positive associations before drawing conclusions from our results. RESULTS: The degree of visual field constriction correlated with three SNPs and one haplotype in a cohort of 73 patients. However we were unable to replicate these findings in a second independent cohort consisting of 58 patients, suggesting the initial results were possibly false positives, or variants of weak effect. CONCLUSION: Common variants of strong, clinically relevant effect do not appear to reside in the candidate genes studied here. This does not rule out the presence of genetic variants of weak effect in these genes, nor of variants of strong effect in other genes. PMID- 16675199 TI - Measuring the effects of antiepileptic medications on balance in older people. AB - BACKGROUND: Dizziness and ataxia are among the most common adverse events associated with antiepileptic medications. Despite this, few studies have attempted to quantitatively assess the effects of antiepileptic therapies on equilibrium. This study was undertaken to prospectively compare quantitative measures of balance in older people taking carbamazepine, gabapentin and lamotrigine. METHODS: Thirty patients on monotherapy for idiopathic partial or generalized epilepsy were enrolled after giving informed consent. Patients had to be at least 50 years old, able to give consent, and on a stable dose of carbamazepine, gabapentin or lamotrigine for at least 30 days. Since this was a study of asymptomatic patients, all patients had to be without complaint of dizziness or imbalance. Patients with a history of alcohol or drug abuse or any medical or neurological condition expected to adversely affect equilibrium were excluded. Each patient underwent a history and examination, computerized dynamic posturography, the activities-specific balance confidence (ABC) scale, Fregly ataxia battery, and the Berg balance scale. Serum drug levels of carbamazepine were obtained to eliminate patients with toxic levels upon enrollment. Two-tailed paired t-tests were used to determined statistical significance among those on each antiepileptic medication. RESULTS: Thirty patients were enrolled: 10 on gabapentin, 10 on lamotrigine and 10 on carbamazepine monotherapy for epilepsy. There were no differences in age or sex among those in each treatment group. The average dosages were 1,120 mg/day for those on gabapentin, 335 mg for lamotrigine, and 640 mg for carbamazepine. There were no differences in the activities-specific balance confidence (ABC) or the Berg balance scale scores. All patients had normal vestibular function by quantitative testing. Posturography showed no statistically significant differences. The Fregly ataxia battery includes the sum of timed trials in the sharpened Romberg (SR) position, standing on one leg with eyes closed (SOLEC), and when walking in tandem with eyes closed (WITEC). The patients on lamotrigine exhibited ability to maintain balance in these positions significantly longer than did those on carbamazepine: SR (P<0.05), SOLEC (P<0.05) and WITEC (P<0.05). CONCLUSIONS: The effects of antiepileptic medications on equilibrium in asymptomatic older people may require more dynamic and challenging measures of equilibrium than are commonly employed in physical therapy to monitor risk of falls. Although the sample size is small, this study suggests that lamotrigine may induce less disequilibrium than does carbamazepine in older people on monotherapy for epilepsy. Further study in this area is needed, particularly given the risks of falling from imbalance in the elderly. PMID- 16675200 TI - Galantamine added to antipsychotic treatment in chronic schizophrenia: cognitive improvement? PMID- 16675202 TI - Expression of SERCA2 (Darier's disease gene product) in acantholytic dermatoses. PMID- 16675201 TI - Impaired error monitoring contributes to face recognition deficit in schizophrenia patients. AB - BACKGROUND: It has been proposed that social and cognitive deficits in schizophrenia may result from impaired error monitoring. OBJECTIVE: We tested the hypothesis that among schizophrenia patients, impaired error monitoring contributes to poor face recognition, an important social skill. METHODS: 79 schizophrenia patients and 57 healthy individuals were administered a computerized face recognition test which allowed collection of accuracy and latency performance parameters. Error monitoring was assessed by analyzing reaction times for correct (RTC) and incorrect (RTI) responses. Tests of working memory (WM) and processing speed were also administered. RESULTS: RTI was longer than RTC in patients and controls and did not differ between the groups. RTC was significantly longer in patients than controls. Error monitoring effort, calculated by dividing the difference between RTI and RTC by the sum of RTC and RTI, was significantly smaller in patients than controls. A regression model with face recognition performance as dependent variable showed independent contributions of error monitoring effort, spatial working memory and group (patient/healthy) to test performance and explained 26.1% of the variance. CONCLUSION: Error monitoring function influences face recognition accuracy and is impaired in schizophrenia. Impairments in error monitoring, and spatial WM contribute to face recognition deficits in schizophrenia. PMID- 16675203 TI - Assessing aggressiveness quickly and efficiently: the Spanish adaptation of Aggression Questionnaire-refined version. AB - The assessment of aggressiveness and the prediction of aggression has become a relevant research and applied topic in Psychiatry and Psychology. There have been many attempts in order to get a fast and reliable tool to measure aggression. Buss and Durkee started the pathway, and recently Bryant and Smith developed a tool with an enormous potential, a fast-applicable, reliable and valid test. We herein report a Spanish adaptation of this test and we show that aggressiveness can be measured rapidly, and in a simple, valid and reliable way across different populations. We focus on the discriminant capacity of this test to detect aggressive individuals. PMID- 16675204 TI - APRAND programme: an intervention to prevent relapses of anxiety and depressive disorders. First results of a medical health promotion intervention in a population of employees. AB - OBJECTIVE: The study sought to determine whether an organized health promotion intervention during medical consultations improves the outcome for patients who meet the ICD10 criteria for anxiety or depressive disorders. METHODS: In 2001, physicians used the MINI to screen 9743 employees on sick leave and consulting physicians in 21 medical centres of a large company. A health promotion intervention was implemented in eight "active" centres. It took place immediately after diagnosis and consisted of explaining the disorders, delivering the test results and leaflets based on WHO recommendations, and advising patients to consult their personal physician, psychiatrist or occupational physician, if necessary. Its effectiveness, evaluated by symptom severity and outcome over 1 year, was assessed by comparing the eight active centres to 13 control centres (without the intervention). RESULTS: Disorders were detected among 10.6% of the subjects, 29.4% of whom had no previous diagnosis of anxiety or depressive disorder. The intervention was associated with a positive effect on the 6-week and 6-month HAD scores. Total absence of disorders at 1 year was associated with age, sex, and intervention, among all identified cases (OR=1.53), among those with at least one anxiety disorder (OR=1.45), and among those with at least one depressive disorder (OR=1.40). CONCLUSION: Combining detection with organized provision of information including printed material improves patients' outcome and physicians' diagnostic abilities. PMID- 16675205 TI - Does temperamental instability support a continuity between bipolar II disorder and major depressive disorder? AB - BACKGROUND: The current categorical split of mood disorders in bipolar disorders and depressive disorders has recently been questioned. Two highly unstable personality features, i.e. the cyclothymic temperament (CT) and borderline personality disorder (BPD), have been found to be more common in bipolar II (BP II) disorder than in major depressive disorder (MDD). According to Kraepelin, temperamental instability was the "foundation" of his unitary view of mood disorders. STUDY AIM: The aim was to assess the distributions of the number of CT and borderline personality items between BP-II and MDD. Finding no bi-modal distribution (a "zone of rarity") of these items would support a continuity between the two disorders. METHODS: STUDY SETTING: an outpatient psychiatry private practice. Interviewer: A senior clinical and mood disorder research psychiatrist. PATIENT POPULATION: A consecutive sample of 138 BP-II and 71 MDD remitted outpatients. Assessment instruments: The structured clinical interview for DSM-IV Axis I Disorders-Clinician Version (SCID-CV), the SCID-II Personality Questionnaire for self-assessing borderline personality traits (BPT) by patients, the TEMPS-A for self-assessing CT by patients. Interview methods: Patients were interviewed with the SCID-CV to diagnose BP-II and MDD, and then patients self assessed the questions of the Personality Questionnaire relative to borderline personality, and the questions of the TEMPS-A relative to CT. As clinically significant distress or impairment of functioning is not assessed by the SCID-II Personality Questionnaire, a diagnosis of BPD could not be made, but BPT could be assessed (i.e. all BPD items but not the impairment criterion). The distribution of the number of CT and BPT items was studied by Kernel density estimate. RESULTS: CT and BPT items were significantly more common in BP-II versus MDD. The Kernel density estimate distributions of the number of CT and BPT items in the entire sample had a normal-like shape (i.e. no bi-modality). CONCLUSIONS: The expected finding, on the basis of previous studies and of the present sample features, was a clustering of CT and BPT items on the BP-II side of the curves. Instead, no bi-modality was present in the distributions of the number of CT and BPT items in the entire sample, showing a normal-like shape. By using the bi modality approach, a continuity between BP-II and MDD seems supported, questioning the current categorical splitting of BP-II and MDD based on classic diagnostic validators. PMID- 16675206 TI - Country of birth and hospital admission rates for mental disorders: a cohort study of 4.5 million men and women in Sweden. AB - This study analysed the association between country of birth and psychotic, affective, and neurotic disorders in seven immigrant categories, after adjustment for demographic and socioeconomic factors. A 2-year national cohort study of 4.5 million individuals in the age group 25-64 years was performed. Swedish national registers including individual demographic and socioeconomic data were linked to the hospital discharge register. Cox regression was used in the analysis. Several groups of immigrants, both men and women, had risks of hospital admission for psychotic, affective, or neurotic disorders compared to the Swedish-born reference group. The impact of demographic and socioeconomic factors on these risks seemed to be larger for men than for women. For foreign-born men, several of the risks no longer remained significant after adjustment for income and marital status. In contrast, most of the risks for foreign-born women remained significant after adjustment for income and marital status. Low income and being single were associated with an increased risk of psychiatric hospital admission. These results represent important knowledge for clinicians and public health planners who are involved in treatment and prevention of mental disorders among certain groups of immigrants, and among low income men and women irrespective of immigrant status. PMID- 16675207 TI - Transgenic mouse models of amyotrophic lateral sclerosis. AB - The discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases was rapidly followed by the generation of transgenic mice expressing various forms of SOD1 mutants. The mice overexpressing high levels of mutant SOD1 mRNAs do develop motor neuron disease but unraveling the mechanisms of pathogenesis has been very challenging. Studies with mouse lines suggest that the toxicity of mutant SOD1 is unrelated to copper mediated catalysis but rather to propensity of a subfraction of mutant SOD1 proteins to form misfolded protein species and aggregates. However, the mechanism of toxicity of SOD1 mutants remains to be elucidated. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Here, we describe how transgenic mouse models have been used for understanding pathogenic pathways of motor neuron disease and for pre-clinical drug testing. PMID- 16675208 TI - [Urination difficulty in a 9-year-old boy]. PMID- 16675209 TI - Effect of hydrogen bonding interactions on the release mechanism of felodipine from nanodispersions with polyvinylpyrrolidone. AB - Solid dispersion systems are widely investigated for the dissolution enhancement of poorly water soluble drugs. Nevertheless, very limited commercial use has been achieved due to the poor predictability of such systems caused by the lack of a basic understanding of the dissolution optimization mechanism. In the present study an investigation of the release mechanism is performed for solid dispersion systems composed by polyvinylpyrrolidone (PVP) and felodipine (FEL), based on a correlation of their hydrophilicity with the intensity of interactions. The existing interactions were evaluated by using NMR and UV spectroscopy while molecular simulation techniques were also enabled. It was found that the interactions that take place correspond to the creation of hydrogen bonds. The correlation between the intensity of interactions and the concentration of PVP in the matrix showed a sigmoid function. The interactions are impressively increased for polymer concentration exceeding 75% (w/w). This phenomenon was well explained by using the molecular simulation technique. A similar sigmoid pattern was found for the function between dissolution profiles and polymer concentration in the matrix, indicating that the intensity of interactions promotes the dissolution enhancement. Investigation of the solubility and the particle size distribution of FEL in the binary system appeared to have similar behaviour indicating that the interactions affect the release profile through these two factors. The hydrophilicity of PVP does not significantly affect this enhancement as the contact angle was found to be linear to PVP concentration. Microscopic observation of the dissolution behaviour showed that FEL remains in fine dispersion in aqueous solution, verifying the release mechanism. PMID- 16675210 TI - Application of PVP/HPMC miscible blends with enhanced mucoadhesive properties for adjusting drug release in predictable pulsatile chronotherapeutics. AB - The aim of the present study was to prepare pulsatile release formulations consisting of two-layered tablets appropriate for preventing ischemic heart diseases. For this reason the active core was constituted by a FELO/PVP 10/90 w/w solid dispersion while for the adjustment of the drug release time the coating layer was composed of PVP/HPMC blends at different compositions, acting as a stimulus responsible layer. These blends as was found by DSC studies are miscible in the entire composition range, ensured by the interactions taking place between hydroxyl groups of HPMC and carbonyl groups of PVP. The miscibility of the system enhances the mucoadhesive properties of the blends, compared with those of pure HPMC, which is desired for such applications. The enhancement was attributed to the higher rate of wetting and flexibility of the new matrices due to the faster dissolution of the PVP macromolecules. Upon exposure of the prepared tablets to the release medium it was found that the coating layer disintegrates first, followed by the immediate release of FELO from the active core. The delaying time is based on a complicated mechanism, which is a combination of swelling and erosion of the PVP/HPMC polymer blends. Varying the PVP/HPMC blend ratios, the exact time that FELO is released during a daytime can be effectively adjusted and this ability is expressed mathematically by the equation t = 0.028 C1.5, where C is the concentration of HPMC in the blend. PMID- 16675211 TI - Integrity and stability studies of precipitated rhBMP-2 microparticles with a focus on ATR-FTIR measurements. AB - A major obstacle in the development of protein drug formulations is the need to maintain the native, active protein structure both during the formulation process and upon long time storage. Controlled precipitation was evaluated for its potential to supply stable microparticulate formulations of bone-regenerating recombinant human Bone Morphogenetic Protein-2 (rhBMP-2). Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) did provide insight into the protein formulation and stability. Temperature dependent ATR FTIR measurements and DSC measurements allow for the study of changes in the protein structure during melting. To address the question of isomerization, peptide mapping was performed, and protein aggregation was monitored by size exclusion chromatography (SEC). It could be demonstrated by ATR-FTIR that controlled precipitation did not harm the protein and the process is fully reversible. DSC measurements further confirmed these findings. No changes in the transition temperature and process were observed after precipitation and redissolution. Upon storage, isomerization and aggregation could be detected, but to a lower extent in the precipitated formulation as compared to a solution reference. Thus, controlled precipitation of rhBMP-2 is fully reversible and has the potential as alternative formulation tool for the generation of a microparticulate drug delivery system. PMID- 16675213 TI - Sarcoidosis after treatment with interferon-alpha: a case series and review of the literature. AB - Recombinant interferon-alpha (rINF-alpha) is an immunomodulator used in the treatment of various conditions, including viral infections and malignancies. The use of rINF-alpha has been associated with the development of sarcoidosis in recent case reports. In this series, we report the incidence of sarcoidosis in recipients of rINF-alpha for hepatitis C viral (HCV) infection at our institution. We also review the 57 additional cases of sarcoidosis associated with rINF-alpha described in the literature, including clinical presentation, radiographic findings, management, and outcomes, and discuss the potential mechanisms by which rINF-alpha may lead to the development of sarcoidosis. PMID- 16675212 TI - EXCEL: A randomised trial comparing salmeterol/fluticasone propionate and formoterol/budesonide combinations in adults with persistent asthma. AB - OBJECTIVES: This multicentre, parallel group, double-blind, double-dummy, randomised 24-week study was designed to compare the efficacy of salmeterol/fluticasone propionate combination (SFC) 50/250 microg one inhalation twice daily (bid) with formoterol/budesonide combination (FBC) 6/200 microg two inhalations bid in patients with persistent asthma, currently receiving 1000-2000 microg/day of inhaled corticosteroids. METHODS: The intent-to-treat population comprised 694 patients in the SFC group and 697 patients in the FBC group. RESULTS: The primary endpoint, mean rate of all exacerbations over 24 weeks, was similar in both treatment groups (SFC: 2.69; FBC: 2.79; SFC/FBC ratio 0.96; 95% CL 0.84, 1.10; P=0.571). A reduction in the rate of exacerbations over time was observed in both treatment groups. Overall, there was a 30% lower annual rate of moderate/severe exacerbations in the SFC group compared with the FBC group (95% CI 0-49%, 52% reduction vs. 1% increase; P=0.059). This effect increased with time: in weeks 17-24 the moderate/severe exacerbation rate was 57% lower in the SFC group compared with the FBC group (95% CI 21-77% reduction; P=0.006). Similar improvements in lung function, asthma symptoms and rescue medication usage were seen with both treatments and both were well tolerated. CONCLUSIONS: Twice-daily treatment with SFC and FBC over 6 months significantly improved asthma symptoms and lung function in patients with persistent asthma. The rate of exacerbations was significantly reduced over time on both treatments but SFC was found to be significantly superior to FBC in reducing the rate of moderate/severe exacerbations with sustained treatment. PMID- 16675214 TI - Determinants of respiratory symptom development in patients with chronic airflow obstruction. AB - BACKGROUND: This study was undertaken to identify the determinants of respiratory symptom development in patients with chronic airflow obstruction (CAO). METHODS: Categories of symptomatic and asymptomatic CAO were defined using questionnaire responses and spirometric results. We analyzed data obtained as part of the second South Korean National Health and Nutrition Examination Survey (Korean NHANES II). RESULTS: Among 187 patients with CAO, 69 had no respiratory symptoms. CAO patients with symptoms were significantly older than those without symptoms (P=0.026), and hypertension was more common among symptomatic CAO patients than among asymptomatic CAO patients (P=0.005). According to questionnaire responses, symptomatic CAO patients had more difficulty in walking or lifting (P<0.001), required more help with personal care (P=0.01), and had poorer general health than asymptomatic CAO patients (P=0.008). Symptomatic CAO patients had higher fasting blood glucose levels than asymptomatic CAO patients (P=0.028). Symptomatic CAO patients had significantly lower forced expiratory volume in 1s (FEV1) (P=0.001), forced vital capacity (FVC) (P=0.008), and a ratio of FEV1/FVC than asymptomatic CAO patients (P<0.001). Statistically significant predictors of symptom development were as follows: age (odds ratio (OR) 1.04, P=0.028), hypertension (OR 4.41, P=0.008), fasting blood glucose (OR 1.02, P=0.034), FEV1 (OR 0.07, P=0.002), FVC (OR 0.08, P=0.009), FEV1/FVC (OR 0.00, P=0.001). Multiple logistic regression analyses revealed two independent factors associated with symptom development: FEV1/FVC (OR 0.001, P=0.002) and hypertension (OR 5.95, P=0.005). CONCLUSIONS: In CAO, respiratory symptom development is significantly associated with low FEV1/FVC and the presence of hypertension. PMID- 16675215 TI - Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode. AB - A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication. PMID- 16675216 TI - Exocytosis unbound. AB - The accepted theory of vesicular release of neurotransmitter posits that only a single vesicle per synapse can fuse with the membrane following action potential invasion, and this exocytotic event is limited to the ultrastructurally defined presynaptic active zone. Neither of these dictums is universally true. At certain synapses, more than a single vesicle can be released per action potential, and there is growing evidence that neuronal exocytosis can occur from sites that are unremarkable in electron micrographs. The first discrepancy extends the dynamic range of synapses, whereas the second enables faster and more robust chemical transmission at sites distant from morphologically defined synapses. Taken together, these attributes expand the capabilities of cellular communication in the nervous system. PMID- 16675217 TI - Bivalent inhibitors of glutathione S-transferase: the effect of spacer length on isozyme selectivity. AB - Glutathione S-transferases (GSTs) are cytosolic enzymes that catalyze the conjugation of glutathione with a variety of exogenous and endogenous electrophiles. High affinity, isozyme-specific inhibitors of GST are required for use as pharmacological tools as well as potential therapeutics. The design of selective inhibitors is hindered due to the broad substrate binding capabilities of the GST enzymes. GSTs are dimeric enzymes, and therefore offer a unique discriminator for achieving inhibitor selectivity: the distance between binding sites on each monomer unit as a function of its quaternary organization. Bivalent analogs of the non-selective GST inhibitor ethacrynic acid were prepared, and selectivity for the GST A1-1 isozyme over GST P1-1 (IC50 values of 13.7 vs 1022 nM, respectively) was achieved through the optimization of the spacer length between the ethacrynic acid ligand domains. PMID- 16675218 TI - Cyclin D1 and breast cancer. AB - Cyclin D1 is one of the frequently overexpressed proteins and one of the commonly amplified genes in breast cancer. This article reviews the roles of cyclin D1 in cell-cycle regulation (normal and abnormal), mammary gland development and carcinogenesis and the relationship to oestrogen in breast tissues. It concludes by presenting the clinical, prognostic and therapeutic implications of our current knowledge of cyclin D1 in breast cancer. PMID- 16675219 TI - Wake up! Peptidoglycan lysis and bacterial non-growth states. AB - When stressed, bacteria can enter various non-dividing states, which are medically important. For example, dormancy is used by Mycobacterium tuberculosis to evade host responses. A major breakthrough has been the discovery of resuscitation-promoting factor (Rpf) from Micrococcus luteus, which is an extremely potent anti-dormancy factor. Mycobacteria have multiple proteins that contain this domain. Surprisingly, the highly conserved resuscitation-promoting factor domain has strong structural similarities to lysozyme and soluble lytic transglycosylases, and it has been demonstrated that resuscitation-promoting factors cleave peptidoglycan. This suggests that the activation of dormant cells requires peptidoglycan hydrolysis, which either alters the mechanical properties of the cell wall to facilitate cell division or releases lysis products that function as anti-dormancy signals. PMID- 16675220 TI - Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides. AB - N(1)-Phenyl-3,5-dinitro-N(4),N(4)-di-n-propylsulfanilamide (1) and N(1)-phenyl 3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (2) show potent in vitro antimitotic activity against kinetoplastid parasites but display poor in vivo activity. Seventeen new dinitroaniline sulfonamide and eleven new benzamide analogs of these leads are reported here. Nine of the sulfonamides display in vitro IC(50) values under 500 nM against African trypanosomes, and the most active antikinetoplastid compounds also inhibit the in vitro assembly of purified leishmanial tubulin with potencies similar to that of 2. While several of the potent compounds are rapidly degraded by rat liver S9 fractions in vitro, N(1)-(3 hydroxy)phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (21) displays an IC(50) value of 260 nM against African trypanosomes in vitro and is more stable than 2 in the in vitro metabolism assay. PMID- 16675221 TI - 5-Alkylated thiazolidinones as follicle-stimulating hormone (FSH) receptor agonists. AB - We prepared analogs of potent thiazolidinone-based follicle-stimulating hormone (FSH) agonists 1, that is, 3 that contained an additional 5-alkyl substituent. This extra substituent was added to reduce synthetic problems that arose during preparation of analogs of 1. These compounds (3) were evaluated in a Chinese hamster ovary (CHO) cell line that expressed recombinant human FSH receptor (FSHR) and a luciferase reporter gene regulated by a cAMP response element (CRE). Selected compounds were also tested on a CHO-cell line that over expressed the FSHR for the ability to induce cAMP production. When the 5-alkyl substituent was a methyl group as in analog 16a, similar FSH activity (i.e., EC(50) = 51 nM, 100% efficacy relative to hFSH) to the analogous 5-hydrogen series compound (e.g., 2) was observed; thus, proving that a small 5-alkyl substituent was well tolerated. New derivatives of 3, in which the potentially hydrolytically labile secondary amide function of 1 (-CONH-) was modified to other moieties (e.g., -CH(2)NH-, CH(2)S-, and -CH(2)OCONH-), were also prepared and evaluated. These congeners (namely 21, 22, and 24) also displayed good potency in the CRE-luciferase assay. PMID- 16675222 TI - Potent inhibitors of the hepatitis C virus NS3 protease: use of a novel P2 cyclopentane-derived template. AB - The HCV NS3 protease is essential for replication of the hepatitis C virus (HCV) and therefore constitutes a promising new drug target for anti-HCV therapy. Several potent and promising HCV NS3 protease inhibitors, some of which display low nanomolar activities, were identified from a series of novel inhibitors incorporating a trisubstituted cyclopentane dicarboxylic acid moiety as a surrogate for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. PMID- 16675223 TI - Which is more appropriate as a cerebral protection method--unilateral or bilateral perfusion? PMID- 16675224 TI - Demystifying the anatomic arrangement of the aortic valve. PMID- 16675225 TI - Primary cardiac sarcoma. AB - Primary malignant lesions of the heart are rare. Although myxomas have been extensively described, there is a paucity of large studies on non-myxomatous cardiac tumours. On the other hand, there are several case reports on specific histopathological variants, in small numbers. Consequently there exists no consensus on therapeutic modalities for cardiac sarcomas. The prognosis for these lesions remains dismal, despite the enhanced diagnostic ability of newer technology. The reasons for the dismal prognosis are (1) the advanced tumour stage at presentation, (2) non-specific symptomatolgy, (3) insufficient awareness of these lesions, due to their rarity, (4) delayed diagnosis and/or misdiagnosis, which leads to (5) advanced tumour stage at presentation. Thus a vicious cycle is created. This article addresses these issues, deals with the surgically relevant modes of presentation, rather than the histopathology, and reviews the diagnosis and management options for the various sarcomas, categorized by the site and extent of cardiac involvement. Clinicians should be familiar with the presentation of these tumours and have a high index of suspicion, since the potential for long-term survival following resection does exist. Wide surgical resection remains the cornerstone of sarcoma therapy. Complete characterization of tumour extent using echocardiography and CT/MRI is mandatory to achieve this goal. Radical resections such as 'bench surgery' and transplantation may reduce local recurrence, but the risk of metastatic disease remains. The clinical experience with such approaches is limited. The role of adjuvant therapy is not yet established. In no other field of cardiac surgery would a multidisciplinary approach be more useful, in achieving cure or long-term palliation. PMID- 16675226 TI - Pretreatment with recombined human erythropoietin attenuates ischemia-reperfusion induced lung injury in rats. AB - OBJECTIVE: Based on the findings that erythropoietin (EPO) has been proved to be a multiple functional cytokine to attenuate ischemia-reperfusion (I/R) injury in various organs such as brain, heart, and kidney in animals, this experiment was designed to evaluate the effect of pretreatment with recombined human erythropoietin (rhEPO) on I/R-induced lung injury. METHODS: Left lungs of rats underwent 90 min of ischemia and then were reperfused for up to 2 h. Animals were randomly divided into three experimental groups as sham group, I/R group, and rhEPO + I/R group (a single dose of rhEPO was injected intraperitoneally 3000 U/kg 24 h prior to operation). Lung injury was evaluated according to semi quantitative analysis of microscopic changes, tissue polymorphonuclear neutrophils (PMNs) accumulation (myeloperoxidase (MPO) activity), and pulmonary microvascular permeability (Evan's blue dying method). Peripheral arterial and venous blood samples were obtained for blood-gas analysis after 5 min occlusion of right lung hilus at the end of reperfusion. The serum concentration of tumor necrosis factor (TNF)-alpha was also measured by the method of enzyme-linked immunosorbent assay. RESULTS: Histological injury scoring revealed significantly lessened lung alveolus edema and neutrophils infiltration in the rhEPO pretreated group compared with I/R group (p < 0.05). The rhEPO pretreated animals exhibited markedly decreased lung microvascular permeability (p < 0.05) and myeloperoxidase activity (p < 0.05). Blood-gas analysis demonstrated that the pretreated animals had significantly ameliorated pulmonary oxygenation function (p < 0.05). The serum concentration of tumor necrosis factor-alpha in rhEPO pretreated group was markedly decreased compared with that of I/R group (p < 0.05). CONCLUSIONS: Pretreatment with rhEPO appears to attenuate I/R-induced lung injury. This function is partly related with the capacity that rhEPO inhibits the accumulation of polymorphonuclear neutrophils in lung tissue and decreases the systematic expression of tumor necrosis factor-alpha. PMID- 16675227 TI - The goal is performance evaluation not outcome prediction. PMID- 16675228 TI - Management of congenital tracheal stenosis in infancy. AB - OBJECTIVE: Congenital tracheal stenosis (CTS) is a very infrequent malformation. Till recently, the outlook for these patients was dismal because medical management was the only way of treatment. Surgical and endoscopical techniques developed in the last years have improved the prognosis. We review the short- and long-term outcomes of a single institution experience in the management of children with CTS, comparing different treatment modalities. METHODS: Between 1991 and 2004, 19 cases of CTS have been managed in our Unit. Respiratory symptoms varied from mild stridor on exertion to severe distress. Bronchoscopy was performed for diagnostic purposes in all cases; other imaging techniques (computed tomography (CT), magnetic resonance imaging (MRI), bronchography, angiography, doppler-ultrasound) were performed on an individual basis. According to clinical and endoscopical features, patients were classified into three groups. The following data have been studied in each case: sex, age at diagnosis and treatment, anatomical type, associated anomalies, treatment modality, complications, outcome and time of follow-up. RESULTS: Ten boys and nine girls have been included in this study. Age at diagnosis ranged from 3 days to 7 years (median, 4 months) and 84% of cases showed associated anomalies. Five patients presented mild or no symptoms and have been managed expectantly. The other 14 cases were operated on because of persistent or severe clinical symptoms. The following procedures were performed: slide tracheoplasty (n = 7), costal cartilage tracheoplasty (n = 5), tracheal resection and reconstruction (n = 3), endoscopical dilatation (n = 3), stent placement (n = 1), and laser resection (n = 1). Three patients required two or more procedures and surgical survival rate is 78%. Overall mortality in the series is 21% and all survivors (15 patients) are asymptomatic or show mild symptoms with respiratory infections only. Follow up is complete, ranging from 8 months to 12.3 years (mean, 5 years). CONCLUSIONS: Bronchoscopy is our preferred diagnostic tool. Selection of the type of treatment depends on the patient's clinical status and the anatomical pattern of the stenosis. In symptomatic cases with short-segment stenosis (<30% of total tracheal length), we prefer tracheal resection with end-to-end anastomosis; for long-segment stenosis (>30%), slide tracheoplasty is our procedure of choice. PMID- 16675229 TI - Assessment of left ventricular ejection fraction may invalidate the reliability of EuroSCORE. AB - OBJECTIVE: To evaluate the impact of different echocardiographic methods for assessment of left ventricular dysfunction on the predictive value of EuroSCORE and thus its reliability in daily practice and benchmarking. METHODS: Twenty-five recordings obtained by transthoracic echocardiography, optimal for study using four different echocardiographic methods, were examined and placed into relevant EuroSCORE classes. The four methods were eyeballing, Wall Motion Index, Simpson 2D volumetric method and Motion mode. RESULTS: Bland and Altman plots showed that the three specific methods were not fully interchangeable. Using eyeballing as the key reference, only 44% of patients were placed in the same EuroSCORE class with all four methods. The quantitative echocardiographic methods in general overestimate the ejection fraction compared to eyeballing. CONCLUSION: The left ventricular dysfunction factor in the EuroSCORE risk assessment system needs a more precise definition. PMID- 16675230 TI - Effect of risk-adjusted, non-dialysis-dependent renal dysfunction on mortality and morbidity following coronary artery bypass surgery: a multi-centre study. AB - OBJECTIVE: As little is known about the impact of non-dialysis-dependent renal dysfunction on short- and mid-term outcomes following coronary surgery we have conducted a large multi-centre study comparing patients with no history of renal dysfunction to those with preoperative renal dysfunction. METHODS: Data was prospectively collected on 19,625 consecutive patients undergoing isolated coronary surgery between 1997 and 2003 from four institutions. Sixty-seven patients had a history of dialysis support prior to coronary surgery, and were excluded from the main analysis of the study. The remaining 19,558 patients were divided into two groups based on preoperative serum creatinine level, patients with preoperative renal dysfunction with serum creatinine levels >200 micromol/L without dialysis support and control patients with preoperative serum creatinine levels <200 micromol/L. Case-mix was accounted for by developing a propensity score, which was the probability of belonging to the non-dialysis-dependent renal dysfunction group, and included in the multivariable analyses. RESULTS: There were 19,172 patients with preoperative serum creatinine levels <200 micromol/L and 386 patients with serum creatinine levels >200 micromol/L without dialysis support. The propensity score included sex, body mass index, co-morbidity factors (respiratory disease, diabetes, cerebrovascular disease, hypertension, and hypercholesterolemia), ejection fraction, left main stem stenosis, emergency status, prior cardiac surgery, off-pump surgery, and the logistic EuroSCORE. After adjusting for the propensity score, patients with preoperative non-dialysis dependent renal dysfunction had significantly higher in-hospital mortality (adjusted odds ratio 3.0, p < 0.001), stroke (adjusted odds ratio 2.0, p = 0.033), atrial arrhythmia (adjusted odds ratio 1.5, p = 0.003), prolonged ventilation (adjusted odds ratio 2.1, p < 0.001), and post-op stay > 6 days (adjusted odds ratio 2.6, p < 0.001). One thousand one hundred and eighty-three (6.1%) deaths occurred during 58,062 patient-years follow-up. After adjusting for the propensity score, the adjusted hazard ratio of mid-term mortality for non dialysis-dependent renal dysfunction was 2.7 (p < 0.001). CONCLUSIONS: Patients undergoing coronary surgery with non-dialysis-dependent renal dysfunction have significantly increased perioperative morbidity and mortality. Mid-term survival is also significantly reduced at 5-years. PMID- 16675233 TI - The upside-down technique. A novel method to correct posterior leaflet prolapse. AB - The upside-down technique is a method for 'in situ' secondary cordae transposition for posterior leaflet lesions. The segmental prolapse of the posterior leaflet is corrected by rotating the resected segment upside-down and reattaching it to the annulus and adjacent leaflet segments. As the procedure is completed, the original annular attachment becomes the new free edge. The secondary chords, originally positioned at the base of the segment, become primary chordae. It is indicated in all cases when quadrangular resection is not feasible such as in case of calcified annulus, posterior leaflet hypoplasia, or when the prolapsing portion is wide. PMID- 16675232 TI - Does perioperative high-dose prednisolone have clinical benefits for generalized myasthenia gravis? AB - OBJECTIVE: The purpose of this study was to clarify the clinical benefits of perioperative administration of high-dose prednisolone (PSL) combined with extended thymectomy on the long-term outcomes of 116 consecutive patients with generalized myasthenia gravis (MG). METHODS: A retrospective review was conducted on 116 patients diagnosed with generalized MG who received alternate-day oral administration of high-dose PSL (100 mg/alternate days) and had undergone transsternal extended thymectomy. Incidences of postoperative myasthenic crisis, adverse effects of steroid, long-term outcomes, such as complete stable remission (CSR), pharmacologic remission (PR) or improvement (Imp), and disease recurrence after CSR were evaluated. RESULTS: Six patients (5.2%) experienced post thymectomy myasthenic crisis. Crude cumulative CSR and PR + CSR rates were 44.8 and 62.7%, respectively. Life table analysis showed that 41.8, 52.8 and 63.4% of the patients were in CSR at 3, 5 and 10 years, respectively. Multivariate analysis revealed that age and pretreatment classification according to the Myasthenia Gravis Foundation of America (MGFA) criteria tended to be independent predictors of CSR. There were 6.9% with compressive vertebral fracture, 13.8% with cataract, and 5.2% with steroid-induced diabetes. Life table analysis revealed that recurrence rates after CSR were 36.8 and 46.0% at 3 and 5 years, respectively. Patients with thymoma had a significantly higher rate of recurrence than those without thymoma (p = 0.001). CONCLUSIONS: Alternate-day administration of high-dose prednisolone reduced the risk of post-thymectomy myasthenic crisis. Presence of thymoma was a risk factor for MG recurrence after CSR. PMID- 16675234 TI - Sympathetic stimulation increases the blood flow through the in situ right gastroepiploic artery graft after off-pump coronary artery bypass graft surgery. AB - OBJECTIVE: The right gastroepiploic artery is gaining popularity as an in situ arterial graft for coronary artery bypass surgery. Unlike the internal thoracic artery, the right gastroepiploic artery is a visceral artery and has a vasoconstrictive tendency in response to sympathetic stimulation. We hypothesized that blood flow through the in situ right gastroepiploic arterial graft might be compromised after sympathetic stimulation. METHODS: Thirty patients scheduled for off-pump coronary artery bypass surgery using the left internal thoracic artery and the right gastroepiploic artery as in situ arterial grafts were enrolled. Blood flow through both arteries was measured by transit time flow before (T1), during (T2), and after noradrenalinee infusion (T3). RESULTS: After sympathetic stimulation, blood flow of both the right gastroepiploic artery (30.1+/-13.9 mL/min at T1 vs 36.2+/-17.5 mL/min at T2; P = 0.001) and left internal thoracic artery grafts (37.3+/-19.1 mL/min at T1 vs 41.8+/-18.2 mL/min at T2; P = 0.01) was increased significantly. However, blood flow in proportion to cardiac output increased only in the right gastroepiploic artery graft (P = 0.01). CONCLUSIONS: Sympathetic stimulation increases, rather than compromises, blood flow through the right gastroepiploic artery graft after coronary revascularization. PMID- 16675235 TI - Intestinal ischaemia following cardiac surgery: a multivariate risk model. AB - OBJECTIVE: Intestinal ischaemia following cardiac surgery is a serious complication, which carries a high mortality rate. Several studies have examined pre-operative and intra-operative risk factors. We aimed to develop a multivariate risk model to identify those patients at highest risk of intestinal ischaemia. METHODS: Data was prospectively collected for 10,976 consecutive cardiac surgery patients from our institution between April 1997 and March 2004. Fifty (0.5%) patients developed post-operative intestinal ischaemia. A forward stepwise multivariate logistic regression analysis was undertaken to identify predictors of developing intestinal ischaemia. Intra-operative and post-operative variables were censored at the time of onset of intestinal ischaemia. RESULTS: The predictors of post-operative intestinal ischaemia were: post-op inotrope and dialysis support (OR 6.7; p < 0.001), post-op ventilation >48 h (OR 5.1; p < 0.001), age at operation (OR 1.06 [for each additional year]; p < 0.001), post-op atrial fibrillation (OR 2.3; p = 0.014) and blood loss in intensive care unit (ICU) >700 ml (OR 2.0; p = 0.037). The predictive ability of this model was very good with an area under the receiver operating characteristic curve of 0.93. In hospital mortality for the patients who developed intestinal ischaemia was 94% (47/50) compared to 3.6% (390/10,926) for the other patients (p < 0.001). CONCLUSIONS: Although the incidence of intestinal ischaemia following cardiac surgery is low, the prognosis for these patients is very poor. We have identified several risk factors, and developed a multivariate prediction tool, which may be useful in identifying patients at high-risk of developing intestinal ischaemia. PMID- 16675236 TI - The 'Valve Racket': a new and different concept of atrioventricular valve repair. AB - OBJECTIVE: Mitral regurgitation due to prolapse of the mitral leaflets frequently compromises annuloplasty repair procedures. We present a new annuloplasty ring that overcomes this difficulty, preventing displacement of the leaflets into the atrium. METHODS: The 'Valve Racket' is a prosthetic ring transformed into a racket by means of handmade mesh using expanded polytetrafluoroethylene (ePTFE Gore-Tex). After transection of the marginal chordae tendineae, five sheep had the new racket implanted in the mitral (n = 3) and in the tricuspid position (n = 2). The surviving sheep underwent postoperative evaluation. RESULTS: After six months of operation, a standard transthoracic study showed competent valves without significant gradients and without residual valve regurgitation. The ring appeared encapsulated by a uniform fibrous tissue but the threads showed a completely denuded surface except in the zone proximal to the ring. Thrombi or calcification deposits in the ring, racket's mesh, or cardiac chamber were not observed. CONCLUSION: This initial experience confirmed the efficacy and simplicity of the technique. PMID- 16675237 TI - Massive mediastinal chylothorax. PMID- 16675238 TI - Comparative hemodynamic effects of vasopressin and norepinephrine after milrinone induced hypotension in off-pump coronary artery bypass surgical patients. AB - OBJECTIVE: Phosphodiesterase inhibitor is essential to the pharmacologic management of decompensated heart failure because it increases contractility and decreases afterload of right ventricle. It also improves hemodynamics and increases blood flow of the grafted internal mammary arteries and middle cerebral arteries during coronary artery bypass surgery. However, it induces vasodilation and necessitates the use of vasoconstrictors, such as norepinephrine. We hypothesized that vasopressin could recover hypotension induced by milrinone with less effect on pulmonary vascular resistance (PVR) compared to norepinephrine. METHODS: Fifty patients, undergoing coronary artery bypass graft (CABG) surgery, were assigned randomly in a double-blind manner to receive either vasopressin or norepinephrine. After baseline hemodynamic measurements, a loading dose of milrinone 50 microg/kg was infused slowly for 20 min followed by continuous infusion of 0.5 microg/(kg min). Immediately after the loading dose of milrinone, hemodynamic variables were measured, and vasopressin (VP group) or norepinephrine (NE groups) was infused. After being titrated until the mean arterial pressure was increased by 20%, hemodynamic variables were measured again. RESULTS: Milrinone infusion reduced both systemic vascular resistance (SVR, 1218+/-299 dynes/cm5 vs 838+/-209 dynes/cm5, 1345+/-299 dynes/cm5 vs 1011+/-195 dynes/cm5) and PVR (95+/-34 dynes/cm5 vs 72+/-30 dynes/cm5, 119+/-85 dynes/cm5 vs 87+/-33 dynes/cm5) in the VP and NE groups, respectively. Vasopressin and norepinephrine infusion increased both SVR (838+/-209 dynes/cm5 vs 1100+/-244 dynes/cm5, 1011+/ 195 dynes/cm5 vs 1446+/-681 dynes/cm5, respectively) and PVR (72+/-30 dynes/cm5 vs 84+/-18 dynes/cm5, 87+/-33 dynes/cm5 vs 139+/-97 dynes/cm5, respectively). The PRV/SVR ratio was decreased after vasopressin infusion (0.10+/-0.03 vs 0.08+/ 0.03), while no changes were found after norepinephrine infusion (0.09+/-0.02 vs 0.09+/-0.02). CONCLUSIONS: In the patients undergoing CABG surgery, both norepinephrine and low dose vasopressin were effective in restoring milrinone induced decrease of SVR. However, only low-dose vasopressin decreased the PVR/SVR ratio that was increased by milrinone. Considering the importance of maintaining systemic perfusion pressure as well as reducing right heart afterload, milrinone vasopressin may provide better hemodynamics than milrinone-norephinephrine during the management of right heart failure. PMID- 16675239 TI - Long-term results after Heller-Dor operation for oesophageal achalasia. AB - OBJECTIVE: In the literature, reports on the definitive rate of cure of the surgical treatment of oesophageal achalasia are not numerous. The aim of this study is to assess the clinical-instrumental-based patient's outcome related to long-term follow-up. METHODS: One hundred and seventy-four patients (80 men, median age 57 years, range 7-83) consecutively submitted to first instance transabdominal Heller-Dor in the period 1978-2002 were considered. Follow-up consisted of clinical interview, endoscopy, barium-swallow and oesophageal manometry if required. Twenty-six cases (15%) were sigmoid achalasias. RESULTS: One patient died post-operatively (severe haemorrhage in a patient previously operated upon for a cardiovascular malformation and suffering for portal hypertension), 173 were followed-up (mean 109 months, range 12-288, median 93 months) of whom 68 for more than 15 years. On the whole 151 patients (87.3%) had satisfactory and 22 (12.7%) had poor long-term results. Seven out of 173 patients (4%), 6 of whom were pre-operatively classified as sigmoid achalasia, subsequently underwent oesophagectomy, 3 for epidermoid cancer, 1 for Barrett's adenocarcinoma, 2 for stasis oesophagitis and recurrent sepsis, 1 for severe dysphagia. Fifteen patients (8.7%) had an insufficient result due to reflux oesophagitis which appeared in 2 (one erosion) after 184 and 252 months. All 22 patients, whether surgically or medically retreated, achieved satisfactory control of dysphagia and reflux symptoms. CONCLUSIONS: In the long term, insufficient results strictly related to Heller-Dor failure, always due to reflux oesophagitis, were recorded in 15/173 patients (8.7%) although it is questionable whether reflux oesophagitis appearing after more than 15 years is due to the Dor incompetence or to ageing. In sigmoid achalasia, oesophagectomy rather than myotomy should be taken into consideration in the first instance. In the long term, surgery is the best definitive treatment for oesophageal achalasia. PMID- 16675240 TI - Axillary cerebral perfusion for arch surgery in acute type A dissection under moderate hypothermia. AB - BACKGROUND: Aortic arch surgery is still associated with increased mortality and morbidity especially in acute type A aortic dissection. Adequate brain protection is essential and commonly performed by either antegrade selective perfusion of the brachiocephalic arteries or an interval of profound hypothermic circulatory arrest. We present our experience for open aortic arch repair with continuous antegrade brain perfusion by means of direct cannulation of the right axillary artery, under moderate hypothermia in patients with acute type A aortic dissection. METHODS: In, 25 consecutive patients (17 men) with a mean age of 62.6+/-14.8 years, aortic repair extended to the arch, for acute type A aortic dissection, was performed through a midline sternotomy. The right axillary artery was used for arterial systemic and brain perfusion at a rectal temperature of 25 27 degrees C. RESULTS: Mean duration of CPB and aortic cross-clamping was 241+/ 55 and 155+/-72 min, respectively. The mean duration of circulatory arrest of the lower body and brain perfusion was 39.7 (range, 24-55 min). All the patients survived the procedure and all but one were discharged from hospital. One patient had left arm paralysis which he recovered the first postoperative month. There were no other transient or permanent neurologic deficits. A CT scan was performed at discharge for routine postoperative evaluation. There were no local neurovascular complications related to the cannulation site except for one local re-exploration for bleeding. CONCLUSIONS: The absence of any major permanent neurologic deficit or any visceral damages in our patients suggests that continuous moderate hypothermic cerebral perfusion, with an interval of circulatory arrest of the lower body, is adequate for acute type A aortic dissection surgery, allowing safe open repair of the distal aortic arch. PMID- 16675241 TI - Hemiarch replacement during pregnancy (19 weeks) utilizing normothermic selective cerebral perfusion. AB - A 20-year-old woman with Marfan syndrome in the 19 weeks of pregnancy was admitted for a dissecting aneurysm of the ascending aorta to the proximal arch. She wanted to continue with pregnancy. A cardiotocography and a trans-esophageal echo probe which allowed visualization of the fetal heart contraction were attached on the abdomen. A cardio-pulmonary bypass was established via the right axillar and femoral arteries with bicaval drainage. The aortic isthmus and arch branches were clamped. After opening the ascending aorta, selective cerebral perfusion was initiated by quick cannulation into the left carotid and left subclavian arteries. Peripheral pressure was maintained above 80 mmHg with the flow of the selective cerebral perfusion at 1.0 L/min and flow from the femoral artery at 3.5L/min. Hemi-arch replacement was performed using a Dacron graft. The body temperature passively descended to 35 degrees C. She normally delivered a healthy female baby after 37 weeks' of gestation. PMID- 16675243 TI - Do we know the ideal surgical treatment for primary spontaneous pneumothorax? PMID- 16675242 TI - The impact of mitral valve surgery combined with maze procedure. AB - OBJECTIVE: Recent studies indicated that successful maze procedure for atrial fibrillation (AF) adjunct to mitral valve surgery provided a lower incidence of stroke and recurrence of AF. The purpose of this study is to review the 13-year experience of these combined procedures and to identify the risk factors and late outcomes of successful maze procedures compared to failed maze procedures. METHODS: At a single institution, 521 consecutive patients underwent combined maze procedures with mitral valve replacements or valvuloplasties. Three kinds of maze techniques were primarily used: Cox-maze III, Kosakai maze, and cryo-maze procedure. Three months after the operation, 394 patients were in sinus rhythm (Group S) while the remaining 116 patients were in continuous or intermittent AF (Group F), excluding 11 early death patients. Risk factors for Group F were determined by the analysis of all patient demographics. Survival, freedom from stroke, cardiac events, and AF recurrence were analyzed. RESULTS: The proportion of the patients without any other simultaneous procedures was greater in Group S (41% vs 29%, P = 0.02). The distributions of mitral valve surgery and maze procedure techniques were similar in these two groups. A left atrium larger than 70 mm [hazard ratio (HR) = 2.6; 95% confidence interval range 1.04-6.3, P = 0.043], preoperative AF history longer than 10 years (HR = 8.2; 4.5-15.1, P < 0.001) and f-wave voltage in V1 smaller than 0.1 mV (HR = 6.2; 5.0-15.2, P < 0.001) were determined to be risk factors for unsuccessful maze procedures. All the results of Cox proportional hazards models showed superiority in Group S; actuarial survival rates (HR = 2.7; 1.04-7.0, P = 0.035), freedoms from stroke (HR = 3.0; 1.1-8.1, P = 0.003) and cardiac events (HR = 4.3; 2.9-6.1, P < 0.001). Freedom from AF recurrence rate was 98.4% at 5 years and 81.0% at 12 years in Group S, and 73.0% and 60.1% in overall patients. CONCLUSIONS: Patients with successful maze procedures resulted in higher survival rate, greater freedom from stroke and cardiac events. The large left atrium, small f-wave, and long AF duration were significant risk factors for failed maze procedures, suggesting that earlier surgical interventions would result in superior results in mitral valve surgery combined with maze procedure. PMID- 16675244 TI - Operative stabilization of flail chest using a prosthetic mesh and methylmethacrylate. AB - Surgical stabilization of flail chest is a controversial operation, but recent data has shown that selected patients benefit from it. We describe a simple and practical method of operative stabilization of flail chest using a prosthetic mesh and methylmethacrylate anchored to the ribs and sternum. The methylmethacrylate-mesh complex is inexpensive, can be extracted electively as soon as full thoracic stability is achieved, and can be used to stabilize extended chest wall injuries. PMID- 16675245 TI - Morbidity and mortality following acute conversion from off-pump to on-pump coronary surgery. AB - OBJECTIVE: Many studies have described reduced morbidity in hospital and equivalent midterm outcomes with off-pump coronary artery bypass (OPCAB) surgery compared to conventional CABG (CABG-CPB). However, OPCAB is sometimes converted acutely to CABG-CPB. We describe the risk of acute conversion and compare patients' outcomes for acutely converted OPCAB with unconverted OPCAB and CABG CPB. METHODS: Consecutive acute conversions, i.e. OPCAB patients in whom CPB was instituted urgently for hemodynamic or electrical instability, cardiac arrest or uncontrolled bleeding, were compared with propensity-matched unconverted OPCAB and CABG-CPB patients. Relative risks of death and complications in hospital, and subsequent survival were estimated. RESULTS: The risk of acute conversion between 1996 and 2004 was 1.1% (27/2492): 5.1% in the first 2 years, 2.2% in the third year and 0.8% subsequently. Odds ratios for death in hospital compared to unconverted OPCAB and CABG-CPB were 4.4 (95% confidence interval (CI) 0.67-29.1) and 4.7 (95% CI 1.03-21.1), respectively, and ranged from 0 to 4.5 for serious complications. Converted patients had an increased hazard of death for 3 years after surgery compared to unconverted OPCAB (hazard ratio 3.21, 95% CI 1.20-8.59) and CABG-CPB patients (hazard ratio 3.23, 95% CI 1.41-7.39). CONCLUSIONS: Experienced OPCAB surgeons have a low risk of acute conversion. Acutely converted patients have a moderately increased risk of death and serious complications in hospital. These risks are difficult to quantify precisely because conversion is rare. PMID- 16675246 TI - Needle tract implantation clearly visualized by computed tomography following needle biopsy of malignant mesothelioma. PMID- 16675247 TI - Unusual cause of aortic bioprosthesis dysfunction. PMID- 16675248 TI - AIRFIX: the first digital postoperative chest tube airflowmetry--a novel method to quantify air leakage after lung resection. AB - OBJECTIVE: Prolonged air leak after pulmonary resection is a common complication and a major limiting factor for early discharge from hospital. Currently there is little consensus on its management. The aim of this study was to develop and evaluate a measuring device which allows a simple digital bed-side quantification of air-leaks compatible to standard thoracic drainage systems. PATIENTS AND METHODS: The measuring device (AIRFIX) is based upon a 'mass airflow' sensor with a specially designed software package that is connected to a thoracic suction drainage system. Its efficacy in detecting pulmonary air-leaks was evaluated in a series of 204 patients; all postoperative measurements were done under standardized conditions; the patients were asked to cough, to take a deep breath, to breathe out against the resistance of a flutter valve, to keep breath and to breathe normally. As standard parameters, the leakage per breath or cough (ml/b) as well as the leakage per minute (ml/min) were displayed and recorded on the computer. RESULTS: Air-leaks within a range of 0.25-45 ml/b and 5-900 ml/min were found. Removal of the chest tubes was done when leakage volume on Heimlich valve was less than 1.0 ml/b or 20 ml/min. After drain removal based upon the data from chest tube airflowmetry none of the patients needed re-drainage due to pneumothorax. CONCLUSION: The AIRFIX device for bed-side quantification of air leaks has proved to be very simple and helpful in diagnosis and management of air leaks after lung surgery, permitting drain removal without tentative clamping. PMID- 16675249 TI - Assessment of pulmonary function after lobectomy for lung cancer--upper lobectomy might have the same effect as lung volume reduction surgery. AB - OBJECTIVE: Lung volume reduction surgery (LVRS) in well-selected patients with severe emphysema results in postoperative improvement in symptoms and pulmonary function. Experience with LVRS suggests that predicted postoperative FEV(1.0) may be underestimated after lobectomy in patients with lung cancer and emphysema. As most of the patients with lung cancer have more or less emphysematous changes in the lungs, we assumed that lobectomy would achieve the same effect as LVRS even in patients without chronic obstructive pulmonary disease on the pulmonary function test. We assessed changes in pulmonary function in terms of 'volume reduction effect' after lobectomy for lung cancer. METHODS: Forty-three patients underwent right upper lobectomy (RUL), 38 patients left upper lobectomy (LUL), 39 patients right lower lobectomy (RLL), and 38 patients left lower lobectomy (LLL). Pulmonary function tests were performed preoperatively and 6 months to 1 year after surgery. RESULTS: Percent change in FEV(1.0) after lobectomy was -6.9+/ 16.1% in RUL group, -11.2+/-16.9% in LUL group, -14.7+/-9.8% in RLL group, and 12.8+/-9.5% in LLL group. We evaluated the correlation between a preoperative FEV(1.0)% of predicted and percentage change in FEV(1.0) after lobectomy. There were no significant relationships between these variables in RLL or LLL group. In contrast, there were significant negative relationships between these variables in RUL and LUL groups. Correlation coefficients were r = -0.667, p < 0.0001 for RUL and r = -0.712, p < 0.0001 for LUL. In RUL and LUL groups, patients with a higher preoperative FEV(1.0)% of predicted had a more adverse percentage change in FEV(1.0) after surgery. In addition, all 13 patients with a preoperative FEV(1.0)% of predicted <60% in RUL and LUL groups had an increase in FEV(1.0) postoperatively. Patients with a lower preoperative FEV(1.0)% of predicted had a greater 'volume reduction effect' with an increase in FEV(1.0) after upper lobectomy. CONCLUSION: Upper lobectomy might have a volume reduction effect. PMID- 16675250 TI - Chronic dissection of the ascending aorta: surgical results during a 20-year period (previous surgery excluded). AB - OBJECTIVE: We study here the surgical results of chronic dissection involving the ascending aorta over the last 20 years. Patients with previous cardiac surgery, or proximal aortic repair, were excluded. The patients survived an acute dissection, undiagnosed as pauci- or asymptomatic. The aorta was normal or pathological (atheromatous aneurysm in 15 cases, Marfan's disease in 12 cases, and annuloectasic disease in 18 cases). Two patients had a bicuspid aortic valve. METHODS: Between January 1981 and December 2001, 77 patients (mean age 48+/-15) underwent surgery for chronic dissection of the ascending aorta; 60 patients had severe aortic regurgitation, 12 had Marfan syndrome, and 18 had annuloaortic ectasia. Only the ascending aorta was dissected in 37 patients, the ascending aorta and arch in 26, and the whole aorta in 14. Coronary artery disease occurred in five patients. Statistical analysis was performed using SAS software. Different surgical procedures were used. The aortic arch was repaired in 40 cases; selective antegrade cerebral perfusion and partial circulatory arrest were used. Total aortic replacement was performed on four patients. RESULTS: In hospital mortality was 10%. The only risk factor was the extent of the dissection. The rate of neurologic stroke was 2.5%. Late survival rate was 42+/ 7.5% at 12 years for all the patients; it was 71+/-10% when only the ascending aorta was dissected, 44+/-11% when the ascending aorta and arch were dissected, and 33+/-15% when the whole aorta was dissected (p = 0.0329). The extent of the dissection was the only risk factor for late mortality. Reoperation was required for one proximal and five distal problems. CONCLUSION: In chronic aortic dissection, in-hospital and late mortality were related to the extent of the dissection; in-hospital mortality remained unchanged during the operative period. PMID- 16675251 TI - Tricuspidisation of the aortic valve with creation of a crown-like annulus is able to restore a normal valve function in bicuspid aortic valves. AB - OBJECTIVE: To evaluate the early results of a new method to repair malfunctioning bicuspid aortic valves by creating a tricuspid valve with a crown-like (i.e. anatomic) annulus. MATERIAL AND METHODS: Twelve patients (ages from 10 to 27 years) with chronic regurgitation (and flow-dependent stenosis) of a bicuspid aortic valve underwent repair with the principle of creating a tricuspid valve and a crown-like annulus. The fused leaflets were trimmed and reinserted underneath the existing aortic annulus to create one new native cusp. The third leaflet was fashioned out of a xenopericard patch and was inserted underneath the existing annulus as well to restore the crown-like anatomy of a normal aortic annulus. A tricuspid aortic valve with a morphologically normal annulus was thus created, which resulted in improved coaptation of the leaflets. The repair was immediately assessed by transesophageal echocardiography (TEE) with the heart loaded at 50%. In two patients, a second run helped fine-tune the repair. Median cross-clamping time was 82 min. Follow-up ranged from 3 to 46 months (median 13 months). RESULTS: No significant complication occurred. The function of the aortic valve was excellent with trivial or mild regurgitation in 11 patients and moderate regurgitation in 1 patient. There was no stenosis across the valve. The repair remained stable over time. Remodelling of the left ventricle occurred as expected. CONCLUSIONS: Aortic valve repair is feasible in some dysfunctioning bicuspid aortic valves. Tricuspidisation of the valve can result in excellent systolic and diastolic functions. The creation of a crown-like annulus results in improved coaptation of the cusps and could lead to more reliable outcome. Although long-term results are needed, this anatomic correction seems to be a good alternative to valvular replacement in certain sub-groups of patients. PMID- 16675252 TI - The outcome of sternal wire removal on persistent anterior chest wall pain after median sternotomy. AB - OBJECTIVE: To evaluate the effect of wire removal on a consecutive series of patients with persistent anterior chest wall pain after median sternotomy. METHODS: Ninety-five patients receiving sternal wire removal during the period January 1994-October 2001 were included in a follow-up study. Preoperative data, data from the primary operation, the postoperative course, and the sternal wire removal were collected from patient histories. The patients were attempted contacted by telephone, and interviewed about the outcome of the wire removal. RESULTS: Wire removals were performed 2.7+/-3.3 (SD) years (40 days-20 years) after the primary procedure. Wire removal appeared to occur more frequently in patients with allergies, patients receiving valve-related procedures, and patients that had been reoperated for surgical complications within the first month after the primary operation. In 79 patients, the pain was unrelated to skin affection or infection. Full follow-up was available for 71 patients. For 24 patients only partial follow-up was possible since the patients were dead (n = 19) or unreachable (n = 5). Of the patients followed up, 86% reported complete or partial relief of symptoms, while 11% reported no change in symptoms, and 3% reported worsening of symptoms after wire removal. In patients (n = 23) where symptoms appeared to be related to specific wires, only these were removed, and this approach produced as good results as for patients where all wires were removed. CONCLUSIONS: The surgical results were generally good. Sternal wire removal should be offered to patients with persistent anterior chest wall pain after sternotomy, when other serious postoperative complications have been excluded. PMID- 16675253 TI - Turbulent stress measurements downstream of three bileaflet heart valve designs in pigs. AB - OBJECTIVE: Mechanical heart valves can cause thromboembolic complications, possibly due to abnormal flow patterns that produce turbulence downstream of the valve. The objective of this study was to investigate whether three different bileaflet valve designs would exhibit clinically relevant differences in downstream turbulent stresses. METHODS: Three bileaflet mechanical heart valves (Medtronic Advantage), CarboMedics Orbis Universal and St. Jude Medical Standard) were implanted into 19 female 90 kg pigs. Blood velocity was measured during open chest conditions in the cross sectional area downstream of the valves with 10 MHz ultrasonic probes connected to a modified Alfred Pulsed Doppler equipment. As a measure of turbulence, Reynolds normal stress (RNS) was calculated at three different cardiac output ranges (3-4, 4.5-5.5, 6-7 L/min). RESULTS: Data from 12 animals were obtained. RNS correlated with increasing cardiac outputs. The highest instantaneous RNS observed in these experiments was 47 N/m2, and the mean RNS taken spatially over the cross sectional area of the aorta during systole was between 3 N/m2 and 15 N/m2. In none of the cardiac output ranges RNS values exceeded the lower critical limit for erythrocyte or thrombocyte damage for any of the valve designs. CONCLUSIONS: Reynolds normal stress values were below 100 N/m2 for all three valve designs and the difference in design was not reflected in generation of turbulence. Hence, it is unlikely that any of the valve designs causes flow induced damage to platelets or erythrocytes. PMID- 16675254 TI - Direct imaging of bileaflet mechanical valve behavior in the tricuspid position. AB - OBJECTIVE: The optimal orientation of a bileaflet mechanical valve for tricuspid valve replacement (TVR) has not yet been determined. The aim of this study was to use fiberoptic cardioscopy to evaluate the effect of orientation of a mechanical valve implanted in the tricuspid position on bileaflet mechanical valve behavior. METHODS: Twelve pigs (50-59 kg) underwent TVR with a St. Jude Mechanical Heart Valve (25 mm standard cuff model) after cardioplegic arrest. The mechanical valve was implanted horizontally in six pigs (Group H), and vertically in another six pigs (Group V). The heart was perfused with pellucid Krebs-Henseleit solution in situ and the mechanical valve behavior was observed with a fiberoptic endoscope during different heart rates (HRs) induced by ventricular pacing (60, 90, 120, 150 min(-1)). All images were recorded on a high-speed video system every 4 ms. The closing time lag (CTL) between the valve leaflets was calculated and compared between the two groups. RESULTS: In Group H, the lower valve leaflet tended to open incompletely and close earlier than the upper leaflet. The calculated CTL was 303+/-60 ms, 65+/-48 ms, 40+/-9 ms, and 40+/-26 ms at pacing HRs of 60, 90, 120, and 150 min(-1), respectively. In contrast to Group H, there was little difference in CTL between the right and left leaflets in Group V. The calculated CTL was 9+/-12 ms, 11+/-10 ms, 1+/-3 ms, and 6+/-7 ms at pacing HRs of 60, 90, 120, and 150 min(-1), respectively. There were significant differences in CLT between the two groups at each ventricular pacing rate (P < 0.01). CONCLUSIONS: Orientation of an implanted bileaflet valve in the tricuspid position significantly influenced leaflet motion. In a horizontal orientation, the lower valve leaflet opened incompletely and closed earlier than the upper leaflet. These results suggest that the gravity might affect leaflet motion and that bileaflet mechanical valves should be implanted vertically in TVR to prevent abnormal leaflet motion and thrombus formation. PMID- 16675255 TI - Mitral valve repair by Alfieri's technique does not limit exercise tolerance more than Carpentier's correction. AB - OBJECTIVE: The main goal of this study was to evaluate if the edge-to-edge mitral repair could be a limiting factor for exercise tolerance and to compare these results to those of classical techniques. METHODS: Between 2000 and 2002, 54 consecutive patients were operated on for mitral valve regurgitation (MR). Twenty five patients were operated with Alfieri's technique (group A) and 29 patients with Carpentier's technique (group C). The mean age was 63.9 years in group A and 63.8 years in group C (p = 0.98). After a mean follow-up of 16.2+/-12 months, survivor patients were seen at the outpatient clinic, by the same physician for a clinical evaluation, an echocardiogram at rest and at peak exercise, and received a cardiorespiratory exercise testing with maximal oxygen uptake (VO2 max) recording. RESULTS: Clinical status improved with 0% of the patients in class NYHA III or IV in either group postoperatively versus 77% preoperatively. There was no significant MR in 80% of cases in group A versus 89.6% in group C (p = 0.54). The mean mitral valve area was 2.5 and 2.9 cm2 in groups A and C, respectively (p = 0.018). The mitral gradient at rest was 3.8 and 3.3 mmHg (p = 0.31) and the mitral gradient at peak exercise was 8.5 and 9.7 mmHg (p = 0.22) in groups A and C, respectively. Cardiorespiratory exercise testing showed a mean VO2 max of 73.7+/-15% of normal value in group A versus 79.6+/-13.1% in group C (p = 0.18). CONCLUSION: Alfieri's technique has the same efficiency on improvement of MR and clinical status than classical repair. Despite a higher restriction of mitral valve area at rest in group A, gradient and mean VO2 max at peak exercise were similar in both groups. PMID- 16675256 TI - Extensive and massive thrombosis of the thoracic aorta and pulmonary artery. PMID- 16675257 TI - A new bioabsorbable sleeve for lung staple-line reinforcement (FOREseal): report of a three-center phase II clinical trial. AB - OBJECTIVE: To investigate on the feasibility, safety, and effectiveness of a new bioabsorbable material for lung staple-line reinforcement. METHODS: This prospective open trial included 66 patients (mean age of 56+/-17 years) who underwent various types of lung resection using staplers with knitted calcium alginate sleeves for buttressing (FOREseal, Laboratoires Brothier, Nanterre, France) at three academic centers: 29 lobectomies, 22 emphysema surgeries, 15 wedge resections or lung biopsies. Intraoperative air leakage was assessed at a mean respiratory peak pressure of 30 cmH2O, and rated as grade 1, 2, or 3. Persistent air leakage in the postoperative course, as well as any relevant event, was assessed daily. The follow-up period was of 6 months. RESULTS: No technical problem linked to the device occurred. Hemostasis of the cutting edges was completed in all patients. Fifty-six percent of the patients had no intraoperative air leak and 27.3% had grade 1 leaks. Mean postoperative air leaks and thoracic drainage times were 1.9+/-2.3 days and 6+/-5.3 days, respectively. In-hospital mortality was nil. There was no empyema. Mean hospital stay was 9.1+/ 6.6 days. At follow-up, one patient underwent lung transplantation, and pathology of the explanted specimen showed the absence of device-related foreign-body inflammation. One patient complained from metalloptysis, and another one, with a metastatic invasive aspergillosis, developed an infectious recurrence that required reoperation. CONCLUSIONS: FOREseal is an ergonomic, safe, and promising new material instead of nonabsorbable materials and xenomaterials for staple-line reinforcement. A randomized comparative study is now in progress. PMID- 16675258 TI - Heparin resistance and increased platelet activation in coronary surgery patients treated with enoxaparin preoperatively. AB - OBJECTIVE: Patients with unstable coronary disease have changes in the hemostatic system. These patients are often treated with low molecular weight heparin. In patients who are accepted for coronary artery bypass grafting, treatment with low molecular weight heparin is frequently continued until surgery. We hypothesized that in coronary artery bypass grafting, the hypercoagulable state seen in unstable patients persists into the intra- and postoperative phase despite preoperative treatment with low molecular weight heparin. The aim of this study was to explore and describe the perioperative hemostatic process in patients with unstable coronary artery disease undergoing coronary artery bypass grafting. METHODS: Thirty-two patients with unstable coronary disease treated preoperatively with enoxaparin, and 32 stable control patients not treated with enoxaparin, were included. All patients were taking low dose aspirin until the day before surgery. Before cardiopulmonary bypass, all patients were given tranexamic acid as a bolus injection. Blood samples for analysis of platelet counts, international normalized ratio, activated partial thromboplastin time, fibrinogen, protein S, protein C, prothrombin fragment 1 + 2, thrombin antithrombin complex, antithrombin, plasmin-antiplasmin complex, D-dimer, neutrophil-activating peptide 2, platelet-monocyte complexes, and heparin concentrations were drawn preoperatively, after 30 min on cardiopulmonary bypass, and 30 min, 3 h, and 20 h postoperatively. Heparin was given during cardiopulmonary bypass to maintain an activated clotting time above 480 s. RESULTS: Patients in the enoxaparin group needed more heparin to maintain an activated clotting time above 480 s, and had higher heparin concentrations and lower antithrombin values compared with control patients. Neutrophil-activating peptide 2 concentrations were higher in the enoxaparin group. CONCLUSIONS: Patients treated with enoxaparin before coronary artery bypass grafting showed signs of heparin resistance intraoperatively. Enoxaparin-treated patients also had increased perioperative platelet activation. Reasons for the observed difference in platelet activation remain unclear. PMID- 16675259 TI - Postoperative complications after induction chemoradiotherapy in patients with non-small-cell lung cancer. AB - OBJECTIVE: This study evaluates the risks of postoperative complications in 124 patients with non-small-cell lung cancer who received pre-operative induction chemoradiotherapy and surgery. METHODS: All patients with non-small-cell lung cancer who underwent surgery after induction therapy between January 1990 and December 2003 were reviewed. We adopted univariate and multiple logistic regression models to identify predictors that increased the incidence of postoperative complications. RESULTS: Of 124 patients, 59 received carboplatin and docetaxel, 53 received cisplatin and etoposide, and 12 received other platinum-based combinations. Pre-operative thoracic radiotherapy was performed concurrently with chemotherapy. The median dose to the primary tumor was 40 Gy, and 29 patients (23.4%) received radiotherapy of more than 45 Gy before surgery. There were 25 pneumonectomies (20.2%). The overall postoperative mortality was 9 of 124 patients (7.3%), and complications developed in 54 patients (43.5%). Multivariate analysis demonstrated that only thoracic radiotherapy of more than 45 Gy predicted postoperative complications (P = 0.021; odds ratio, 3.620; 95% confidence interval, 1.214-10.797). CONCLUSIONS: Thoracic radiotherapy of more than 45 Gy, in combination with chemotherapy, was a significant risk factor for postoperative complications. PMID- 16675260 TI - Immunohistological findings for an extracardiac conduit in Fontan pathway constructed with pedicled autologous pericardium. AB - Although autologous pericardium has been used in pedicled fashion for various reconstructive procedures in congenital cardiovascular surgery with the expectation that it will be able to grow and remain viable after implantation, no clinical study has evaluated the histological characteristics of implanted pedicled pericardium long after previous implantation. We describe herein histological findings of pedicled pericardium, which had been used for three years as an extracardiac conduit in Fontan pathway. A four-year-old boy with a history of Fontan operation using pedicled autologous pericardial roll required conduit replacement three years after the previous operation, and resected pericardial tissue was examined immunohistologically. Staining for CD34 revealed abundant microvasculature, suggesting preservation of viability. Staining for factor VIII demonstrated the presence of endothelium on the luminal surface of the conduit. Elastica-van Gieson staining revealed a band of elastic tissue, which is generally found not in the native pericardium but in the vascular wall structures. These findings suggested that the pedicled pericardium thus appeared to have remained viable and might have differentiated to resemble tissue of the vascular wall after having been used in Fontan pathway. PMID- 16675261 TI - Diagnosis of inverted left-atrial appendage (LAA) with CT-chest. PMID- 16675262 TI - A study exploring the role of intercostal nerve damage in chronic pain after thoracic surgery. AB - OBJECTIVE: Our aim was to investigate the prevalence of intra-operative nerve damage and its association with chronic pain. METHODS: Our prospective study of 33 patients used nerve conduction studies to assess intercostal nerve function during elective thoracic surgical procedures. We used two methods to study nerve conduction: pre-operative magnetic stimulation (in 10 patients) and intra operative nerve conduction studies (in all patients) We correlated these findings with specific intra-operative parameters, pain and psychological questionnaires pre-op and 3 month post-op and altered cutaneous sensation. RESULTS: Magstim (magnetic stimulation) assessments were not reliable and were therefore abandoned. Intraoperative intercostal nerve studies revealed two distinct patterns of nerve injury and also that nerve injury was less in those cases where a rib was not resected. However, intercostal nerve damage detected at the time of operation is not associated with chronic pain or altered cutaneous sensation at 3 months post-op. CONCLUSIONS: The study findings suggest that either the amount of intra-operative intercostal nerve damage is not indicative of long-term nerve damage or that there is a more significant cause for chronic pain other than intercostal nerve injury. PMID- 16675263 TI - Expression of hypoxia-inducible factor-1 alpha and its prognostic significance in small-sized adenocarcinomas of the lung. AB - OBJECTIVE: To analyze the prognostic value of hypoxia-inducible factor-1 (HIF-1) alpha expression and its correlation with clinicopathologic variables and the expression of vascular endothelial growth factor-A and -C in patients with lung adenocarcinomas of small size. METHODS: The expression of hypoxia-inducible factor-1 alpha was immunohistochemically determined in 78 cases of small-sized adenocarcinoma (maximum dimension < or = 2 cm) using antibody against a recombinant protein corresponding to amino acids 575-780 of hypoxia-inducible factor-1 alpha. Data regarding patient survival, clinicopathologic factors, and immunohistochemical studies of vascular endothelial growth factors were also collected. RESULTS: Strong expression of hypoxia-inducible factor-1 alpha was observed in 23 of 78 cases; absent or minimal expression was found in the localized bronchioloalveolar carcinomas. Strong expression of hypoxia-inducible factor-1 alpha was significantly higher in cases with vascular invasion, lymph node involvement, and vascular endothelial growth factor-A expression. The 5-year survival rate was 63.2% if expression of hypoxia-inducible factor-1 alpha was strong and 85.1% if expression was weak (p < 0.05). CONCLUSION: Immunohistochemical staining of HIF-1 alpha, along with examination of metastatic potential via vascular pathways, may be valid defining a subpopulation of patients with small-sized adenocarcinoma of the lung whose tumors have aggressive angiogenesis potential. PMID- 16675264 TI - The involvement of LHCII-associated polyamines in the response of the photosynthetic apparatus to low temperature. AB - The influence of low temperature on the structure and function of the photosynthetic apparatus was investigated in Phaseolus vulgaris L. Ten-day-old plants (grown at 26 degrees C) have been exposed to low temperature (6 degrees C) for 52 h and, then, transferred to the initial temperature (26 degrees C) for additional 30 h. Biochemical and physico-chemical measurements performed in the low temperature-treated plants showed that the response of the photosynthetic apparatus to low temperature is affected by the changes occurring in the pattern of LHCII-associated putrescine (Put) and spermine (Spm) which adjust the size of LHCII. The decrease of Put/Spm ratio, mainly due to the reduction in the quantity of LHCII-associated Put led to an increase of the LHCII, especially of the oligomeric forms. These alterations in the structure of the photosynthetic apparatus combined with the reduction in the photosynthetic electron transfer rate resulted in the inactivation of active reaction centers and the increase of dissipated energy which diminished the photosynthetic efficiency and the maximal photosynthetic rate. The transfer of plants at 26 degrees C after the low temperature treatment showed that, structurally and functionally, the photosynthetic mechanism recovered quite fast to the initial condition. PMID- 16675265 TI - Properties of voltage-gated Na+ channels in the human rhabdomyosarcoma cell-line SJ-RH30: conventional and automated patch clamp analysis. AB - Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to -40 mV; in full current voltage curves maximum current occurred between -20 and -10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at -90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 microM. The NaV 1.4 selective toxin mu-CTx-GIIIB reversibly blocked the Na+ current approximately 60% at 10 microM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was -72+/-2 mV (slope factor 7.2+/-0.2) in automated patch clamp and -74+/-2 mV (slope factor 7.4+/-0.4) with conventional recording. The corresponding values for activation mid-point were -33.2+/-2.4 and -30.3+/-2.7 mV (slope 5.8+/-0.3 and 6.4+/-0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. Mu-CTx-GIIIB also inhibited the channel in a concentration dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels. PMID- 16675266 TI - NKG2D and cytotoxic effector function in tumor immune surveillance. AB - NKG2D is a type II transmembrane-anchored glycoprotein expressed as a disulfide linked homodimer on the surface of all mouse and human natural killer cells (NK cells). Stimulation of NK cells through NKG2D triggers cell-mediated cytotoxicity and in some cases induces the production of cytokines. NKG2D binds to family of ligands with structural homology to MHC class I, however, unlike conventional MHC class I molecules, NKG2D ligands often display up-regulated surface expression on stressed cells and are frequently over expressed by tumors. Recent evidence clearly implicates that NKG2D recognition plays an important role in tumor immune surveillance and that NKG2D primarily acts to trigger perforin-mediated apoptosis. The data begin to place the NKG2D pathway into the context of other recognition-effector systems used by NK cells. PMID- 16675267 TI - Mapping of magnesium and of different protein fragments in sea urchin teeth via secondary ion mass spectroscopy. AB - Mature portions of sea urchin are comprised of a complex array of reinforcing elements yet are single crystals of high and very high Mg calcite. How a relatively poor structural material (calcite) can produce mechanically competent structures is of great interest. In teeth of the sea urchin Lytechinus variegatus, we recorded high-resolution secondary ion mass spectrometry (SIMS) maps of Mg, Ca ,and specific amino acid fragments of mineral-related proteins including aspartic acid (Asp). SIMS revealed strong colocalization of Asp residues with very high Mg. Demineralized specimens showed serine localization on membranes between crystal elements and reduced Mg and aspartic acid signals, further emphasizing colocalization of very high Mg with ready soluble Asp-rich protein(s). The association of Asp with nonequilibrium, very high magnesium calcite provides insight to the makeup of the macromolecules involved in the growth of two different composition calcites and the fundamental process of biomineralization. PMID- 16675268 TI - Model structure of the prototypical non-fimbrial adhesin YadA of Yersinia enterocolitica. AB - Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-stalk-anchor architecture. Whereas head and stalk domains are diverse and appear (frequently repetitively) in different combinations, the anchor domains are homologous and display the properties of autotransporters. We have built a molecular model for the prototypical non-fimbrial adhesin, YadA, by combining the crystal structure of the head (PDB:1P9H) with theoretical models for the stalk and the anchor. The head domain is a single-stranded, left-handed beta-helix, connected to the stalk by a conserved trimerization element (the neck). The stalk consists of a right-handed coiled coil, containing ten 15 residue repeats with a C-terminal stutter (insertion of four residues). The stalk continues into the conserved anchor domain, which is formed by four heptads of a left-handed coiled coil, followed by four transmembrane beta-strands. Our model of the YadA coiled coil, generated with the program BeammotifCC, combines these periodicities into a structure that starts with a pronounced right-handed supercoil and ends with a canonical, left-handed conformation. The last two heptads of the coiled coil are located within a 12-stranded beta-barrel, formed by trimerization of the four transmembrane beta-strands in each monomer. We propose that this pore assembles in the outer membrane to form the opening through which the monomer chains exit the cell. After export is completed, the fiber folds and the pore is occluded by the coiled coil. Our model explains how these proteins can act as autotransporters in the absence of any homology to classical, single-chain autotransporters. PMID- 16675269 TI - Abnormal deep grey matter development following preterm birth detected using deformation-based morphometry. AB - Preterm birth is a leading risk factor for neurodevelopmental and cognitive impairment in childhood and adolescence. The most common known cerebral abnormality among preterm infants at term equivalent age is a diffuse white matter abnormality seen on magnetic resonance (MR) images. It occurs with a similar prevalence to subsequent impairment, but its effect on developing neural systems is unknown. MR images were obtained at term equivalent age from 62 infants born at 24-33 completed weeks gestation and 12 term born controls. Tissue damage was quantified using diffusion-weighted imaging, and deformation-based morphometry was used to make a non-subjective survey of the whole brain to identify significant cerebral morphological alterations associated with preterm birth and with diffuse white matter injury. Preterm infants at term equivalent age had reduced thalamic and lentiform volumes without evidence of acute injury in these regions (t = 5.81, P < 0.05), and these alterations were more marked with increasing prematurity (t = 7.13, P < 0.05 for infants born at less than 28 weeks) and in infants with diffuse white matter injury (t = 6.43, P < 0.05). The identification of deep grey matter growth failure in association with diffuse white matter injury suggests that white matter injury is not an isolated phenomenon, but rather, it is associated with the maldevelopment of remote structures. This could be mediated by a disturbance to corticothalamic connectivity during a critical period in cerebral development. Deformation-based morphometry is a powerful tool for modelling the developing brain in health and disease, and can be used to test putative aetiological factors for injury. PMID- 16675270 TI - Stimulus-driven modulation of motor-evoked potentials during observation of others' pain. AB - Empathy may allow interindividual sharing not only of emotions (e.g., joy, sadness, disgust) but also of sensations (e.g., touch, itching, pain). Although empathy for pain may rely upon both sensory and affective components of the pain experience, neuroimaging studies indicate that only the affective component of the pain matrix is involved in empathy for pain. By using transcranial magnetic stimulation (TMS), we highlighted the sensorimotor side of empathy for pain by showing a clear motor inhibition during the mere observation of needles penetrating body parts of a human model. Here, we explored stimulus-specific and instruction-specific influences on this inhibition by manipulating task instructions (request to adopt first- or third-person perspective vs. passive observation) and painfulness of the experimental stimuli (presentation of videos of needles deeply penetrating or simply pinpricking a hand). We found a significant reduction in amplitudes of motor-evoked potentials (MEPs) specific to the muscle the subjects observed being penetrated that correlated with the intensity of the pain attributed to the model. Crucially, this motor inhibition was present during observation of penetrating but not of pinpricking needles. Moreover, no MEPs modulation contingent upon different task instructions was found. Results suggest that the motor inhibition elicited by the observation of "flesh and bone" pain stimuli is more stimulus-driven than instruction-driven. PMID- 16675271 TI - In vivo micro-MRI of intracortical neurovasculature. AB - This work describes a methodology for in vivo MR imaging of arteries and veins within the visual cortex of the cat brain. Very high magnetic fields (9.4 T) and small field-of-view 3D acquisitions were used to image the neurovasculature at resolutions approaching the microscopic scale. A combination of time-of-flight MR angiography and T*(2)-weighted imaging, using both endogenous BOLD contrast and an exogenous iron-oxide contrast agent, provided high specificity for distinguishing between arteries and veins within the cortex. These acquisition techniques, combined with 3D image processing and display methods, were used to detect and visualize intracortical arteries and veins with diameters smaller than 100 microm. This methodology can be used for visualizing the neurovasculature or building models of the vascular network and may benefit a variety of research applications including fMRI, cerebrovascular disease and cancer angiogenesis. PMID- 16675272 TI - Cerebral atrophy measurements using Jacobian integration: comparison with the boundary shift integral. AB - We compared two methods of measuring cerebral atrophy in a cohort of 38 clinically probable Alzheimer's disease (AD) subjects and 22 age-matched normal controls, using metrics of zero atrophy, consistency, scaled atrophy and AD/control group separation. The two methods compared were the boundary shift integral (BSI) and a technique based on the integration of Jacobian determinants from non-rigid registration. For each subject, we used two volumetric magnetic resonance (MR) scans at baseline and a third obtained 1 year later. The case of zero atrophy was established by registering the same-day baseline scan pair, which should approximate zero change. Consistency was established by registering the 1-year follow-up scan to each of the baseline scans, giving two measurements of atrophy that should be very similar, while scaled atrophy was established by reducing one of the same-day scans by a fixed amount, and rigidly registering this to the other same-day scan. Group separation was ascertained by calculating atrophy rates over the two 1-year measures for the control and AD subjects. The results showed the Jacobian integration technique was significantly more accurate in calculating scaled atrophy (P < 0.001) and was able to distinguish between control and AD subjects more clearly (P < 0.01). PMID- 16675274 TI - The selective interactions and functions of regulators of G-protein signalling. AB - There is accumulating evidence that regulators of G-protein signalling (RGS) can have roles in signal transduction that are not related to GAP activity. Furthermore, RGSs have much more selective effects in vivo than might be anticipated from their behaviour in in vitro assays. I discuss the molecular mechanisms by which these phenomena might be explained including specific interactions between the RGS and G-protein coupled receptor, G-protein and effector. PMID- 16675273 TI - Cell-based assays for profiling activity and safety properties of cancer drugs. AB - INTRODUCTION: Our goal was to establish versatile and high capacity assays to characterize activity and toxicity of chemotherapeutics. The major anti-cancer activity indicators of these agents included inhibition of proliferation and induction of apoptosis to cancer cells. In addition, cytotoxicity and myelosuppressive activity to normal cells were parameters to evaluate toxicity of these drugs. METHODS: Using a panel of cell-based assay systems, we investigated activity and toxicity properties of selected cancer drugs. Drug effects on a number of normal and cancer cell types from human origin were evaluated. RESULTS: Topoisomerase inhibitors (camptothecin, doxorubicin and etoposide) and microtubule inhibitors (colchicine and paclitaxel) showed anti-proliferation activity and induced apoptosis in MDA-231 cancer cells. Except for doxorubicin, these drugs had relatively low toxicity to normal cells because the dosage required for cytotoxicity EC(50) was >200-folds higher than the dosage for anti proliferation EC(50) in cancer cells (MDA-231, HL60). However, these drugs were potent inducers of myelotoxicity in human bone marrow progenitor cells. In comparison, the DNA alkylating agents (cisplatin and carboplatin) were less potent proliferation inhibitors (EC(50)>10 microM) in MDA-231 cells and they were also less myelosuppressive. DISCUSSION: Using marketed drugs as examples, our study established a multiple assay platform for profiling in vitro properties of cancer drugs. In drug discovery, such a platform will help to expedite lead selection at early stage. PMID- 16675275 TI - The role of photoinduced electron transfer processes in photodegradation of the [Fe4(mu3-S)3 (NO)7]- cluster. AB - Spectroscopic and electrochemical study of the [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photochemical reaction and thermodynamic calculations of relevant systems demonstrate the redox character of this process. The photoinduced electron transfer between substrate clusters in excited and ground state (probably via exciplex formation) results in dismutation yielding unstable [Fe(4)(mu(3) S)(3)(NO)(7)](2-) and [Fe(4)(mu(3)-S)(3)(NO)(7)](0). Back electron transfer between the primary products is responsible for fast reversibility of the photochemical reaction in deoxygenated solutions. In the presence of an electron acceptor (such as O(2), MV(2+) or NO) an oxidative quenching of the (*)[Fe(4)(mu(3)-S)(3)(NO)(7)](-) is anticipated, although NO seems to participate as well in the reductive quenching. The electron acceptors can also regenerate the substrate from its reduced form ([Fe(4)(mu(3)-S)(3)(NO)(7)](2-)), whereas the other primary product ([Fe(4)(mu(3)-S)(3)(NO)(7)](0)) decomposes to the final products. The suggested mechanism fits well to all experimental observations and shows the thermodynamically favored pathways and explains formation of all major (Fe(2+), S(2-), NO) and minor products (N(2)O, Fe(3+)). The photodissociation of nitrosyl ligands suggested earlier as the primary photochemical step cannot be, however, definitely excluded and may constitute a parallel pathway of [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photolysis. PMID- 16675276 TI - Altered expression of key cellular gene products accompanies development of resistance to nitric oxide. AB - NALM-6 is a pre-B leukemia cell line sensitive to exogenous nitric oxide (NO), which enters into apoptosis during 24 h of exposure to low doses of the NO donors SNAP (100 microM) or DETA-NO (250 microM). By culturing NALM-6 with repeated and increasing concentrations of SNAP, we obtained a variant (NALM-6R) that retains >95% viability and does not enter into apoptosis during 24 h culture in the presence of up to 500 microM SNAP or 750 microM DETA-NO. A power blot screen performed with 277 antibodies on cell lysates from NALM-6 and NALM-6R cultured without NO donors served to determine the altered constitutive expression of 19 proteins in NALM-6R. Proteins affected in the less sensitive cell line NALM6-R are involved in the regulation of apoptosis, the cell cycle, cell interactions, signal transduction, cell morphology, and cell motility. This model shows that repeated exposure of tumor cells to NO may either select NO-resistant cells or contribute to NO-sensitive conversion into NO-resistant cells. The identification of the proteins that are affected during this transition may help us to define the mechanisms that are involved in cell resistance to NO-cytotoxicity which often accompany clinical progression. PMID- 16675277 TI - T1rho MRI contrast in the human brain: modulation of the longitudinal rotating frame relaxation shutter-speed during an adiabatic RF pulse. AB - Longitudinal relaxation in the rotating frame (T1rho) is the dominant mechanism during a train of adiabatic full passage (AFP) RF pulses with no interpulse intervals, placed prior to an excitation pulse. Asymptotic apparent time constants (T1rho') were measured for human occipital lobe 1H2O at 4T using brief imaging readouts following such pulse trains. Two members of the hyperbolic secant (HSn) AFP pulse family (n=1 or 4; i.e., arising from different amplitude- and frequency-modulation functions) were used. These produced two different non monoexponential signal decays during the pulse trains. Thus, there are differing contrasts in asymptotic T1rho' maps derived from these data. This behavior is quite different than that of 1H2O signals from an aqueous protein solution of roughly the same macromolecular volume fraction as tissue. The ROI-averaged decays from the two acquisitions can be simultaneously accommodated by a two-site exchange model for an equilibrium isochronous process whose exchange condition is modulated during the pulse. The model employs a two-spin description of dipolar interaction fluctuations in each site. The intrinsic site R1rho(identical with T1rho(-1)) value is sensitive to fluctuations at the effective Larmor frequency (omegaeff) in the rotating frame, and this is modulated differently during the two types of AFP pulses. Agreement with the data is quite good for site orientation correlation time constants characteristic of macromolecule interacting water (site A) and bulk-like water (site B). Since R1rhoA is significantly modulated while R1rhoB is not, the intrinsic relaxographic shutter speed for the process (identical with /R1rhoA-R1rhoB/), and thus the exchange condition, is modulated. However, the mean residence time (67 ms) and intrinsic population fraction (0.2) values found for site A are each rather larger than might be expected, suggesting a disproportionate role for the water molecules known to be "buried" within the large and concentrated macromolecules of in vivo tissue. PMID- 16675278 TI - [Smoking cessation in the pregnant woman: descriptive study in Limousin]. AB - OBJECTIVE: Among obstetricians and midwives, estimate the percentage of smokers, appraise the proportion of medical professionals who mention the subject of nicotine addiction to their pregnant patients, and assess the quality of the information delivered to patients as well as their own experience in the field of tobacology. PATIENTS AND METHODS: Between February and March 2002, 188 postal questionnaires were sent to obstetricians and gynecologists, midwives and midwife students of public hospitals in Limousin and private clinics in Limoges. RESULTS: The response rate was 75,5% (142 answers) and the median age was 37 years old (20 59). 43,6% of the respondents were current smokers. Patients' smoking habits would be asked for and the risks of tobacco smoking would be explained by the majority of praticians and midwives. Minimal counsel to be given was known and given by 34,4% of gynecologists an 26,5% of midwives. Smoking cessation counselling was never proposed by 62% of gynecologists and 79% of midwives. Seventy per cent of them did not have any specific training in the field of tobacology during their studies. DISCUSSION AND CONCLUSION: There seems to be a deficiency in smoking cessation help for pregnant women in Limousin. Specifics programs and trainings for obstetricians, gynecologists, as well as midwives should be organized. PMID- 16675279 TI - [What is the optimum rate of cesarean sections?]. PMID- 16675280 TI - [Human life, the fruit of a meeting -- in connection with couple infertility]. PMID- 16675281 TI - New flowmetric measurement methods of power dissipated by an ultrasonic generator in an aqueous medium. AB - Two new determination methods of the power dissipated in an aqueous medium by an ultrasound generator were developed. They are based on the use of a heat flow sensor inserted between a tank and a heat sink that allows to measure the power directly coming through the sensor. To be exploitable, the first method requires waiting for stationary flow. On the other hand, the second, extrapolated from the first one, makes it possible to determine the dissipated power in only five minutes. Finally, the results obtained with the flowmetric method are compared to the classical calorimetric ones. PMID- 16675282 TI - Implantable neural electrical stimulator for external control of gastrointestinal motility. AB - Functional electrical stimulation has been suggested as a possible avenue for treating a variety of gastrointestinal motility-related disorders such as gastroparesis, chronic constipation and morbid obesity. The aims of the present study were to design a radio-frequency controlled multi-channel implantable neural gastrointestinal electrical stimulator and test it in an acute canine model. The stimulation parameters can be reprogrammed after implantation, allowing the execution of parametric studies and the investigation of their efficacy in producing controlled gastrointestinal contractions. Bipolar pulse trains of 50Hz frequency, 8-16V(pp) amplitude, 10-100% duty cycle, 1-120s duration, and 2s to 1h pause between successive stimulation sessions were delivered to the stomachs of nine dogs. The resulting contractions were measured by force transducers and digitally recorded on a personal computer. The acute studies confirmed the effectiveness of electrical stimulation in producing invoked gastric contractile activity under the control of the implantable neurostimulator. PMID- 16675283 TI - Compression of surface EMG signals with algebraic code excited linear prediction. AB - Despite the interest in long timescale recordings of surface electromyographic (EMG) signals, only a few studies have focused on EMG compression. In this paper we investigate a lossy coding technique for surface EMG signals that is based on the algebraic code excited linear prediction (ACELP) paradigm, widely used for speech signal coding. The algorithm was adapted to the EMG characteristics and tested on both simulated and experimental signals. The coding parameters selected led to a compression ratio of 87.3%. For simulated signals, the mean square error in signal reconstruction and the percentage error in average rectified value after compression were 11.2% and 4.90%, respectively. For experimental signals, they were 6.74% and 3.11%. The mean power spectral frequency and third-order power spectral moment were estimated with relative errors smaller than 1.23% and 8.50% for simulated signals, and 3.74% and 5.95% for experimental signals. It was concluded that the proposed coding scheme could be effectively used for high rate and low distortion compression of surface EMG signals. Moreover, the method is characterized by moderate complexity (approximately 20 million instructions/s) and an algorithmic delay smaller than 160 samples (approximately 160ms). PMID- 16675284 TI - The keratocyte: corneal stromal cell with variable repair phenotypes. AB - Keratocytes, also known as fibroblasts, are mesencyhmal-derived cells of the corneal stroma. These cells are normally quiescent, but they can readily respond and transition into repair phenotypes following injury. Cytokines and other growth factors that provide autocrine signals for stimulating wound responses in resident cells are typically presented by platelets at the site of an injury. However, due to the avascular nature of the cornea many of the environmental cues are derived from the overlying epithelium. Corneal epithelial-keratocyte cell interactions have thus been extensively studied in numerous in vivo corneal wound healing settings, as well as in in vitro culture models. Exposure to the different epithelial-derived factors, as well as the integrity of the epithelial substratum, are factors known to impact the keratocyte response and determine whether corneal repair will be regenerative or fibrotic in nature. Finally, the recent identification of bone-marrow derived stem cells in the corneal stroma suggests a further complexity in the regulation of the keratocyte phenotype following injury. PMID- 16675285 TI - Genes, cognition and dyslexia: learning to read the genome. AB - Studies of dyslexia provide vital insights into the cognitive architecture underpinning both disordered and normal reading. It is well established that inherited factors contribute to dyslexia susceptibility, but only very recently has evidence emerged to implicate specific candidate genes. In this article, we provide an accessible overview of four prominent examples--DYX1C1, KIAA0319, DCDC2 and ROBO1--and discuss their relevance for cognition. In each case correlations have been found between genetic variation and reading impairments, but precise risk variants remain elusive. Although none of these genes is specific to reading-related neuronal circuits, or even to the human brain, they have intriguing roles in neuronal migration or connectivity. Dissection of cognitive mechanisms that subserve reading will ultimately depend on an integrated approach, uniting data from genetic investigations, behavioural studies and neuroimaging. PMID- 16675286 TI - Structure-based virtual screening of chemical libraries for drug discovery. AB - One of the main goals in drug discovery is to identify new chemical entities that have a high likelihood of binding to the target protein to elicit the desired biological response. To this end, virtual screening is being increasingly used as a complement to high-throughput screening to improve the speed and efficiency of the drug discovery and development process. The availability of inexpensive high performance computing platforms in recent years has transformed this field into one that is highly diverse and rapidly evolving, where large chemical databases have been successfully screened to identify hits for a wide range of targets such as Bcl-2 family proteins, G protein-coupled receptors, kinases, metalloproteins, nuclear hormone receptors, proteases and many more. PMID- 16675288 TI - Enzymatic tools for engineering natural product glycosylation. AB - Glycosylated natural products have served as reliable platforms for the development of many existing front-line drugs. In an effort to explore the contribution of the sugar constituents of these compounds, research groups have focused upon the development of chemical and enzymatic tools to diversify natural product glycosylation. Among the complementary routes available, in vivo pathway engineering, also referred to as 'combinatorial biosynthesis', is an emerging method that relies upon the co-expression of sugar biosynthetic gene cassettes and glycosyltransferases in a host organism to generate novel glycosylated natural products. An overview of recent progress in combinatorial biosynthesis is highlighted in this review, emphasizing the elucidation of nucleotide-sugar biosynthetic pathways and recent developments on glycosyltransferases. PMID- 16675289 TI - Discovery of novel metabolites from marine actinomycetes. AB - Recent findings from culture-dependent and culture-independent methods have demonstrated that indigenous marine actinomycetes exist in the oceans and are widely distributed in different marine ecosystems. There is tremendous diversity and novelty among the marine actinomycetes present in marine environments. Progress has been made to isolate novel actinomycetes from samples collected at different marine environments and habitats. These marine actinomycetes produce different types of new secondary metabolites. Many of these metabolites possess biological activities and have the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific but underexploited source for the discovery of novel secondary metabolites. PMID- 16675290 TI - Bacterial communities in industrial wastewater bioreactors. AB - Wastewater bioreactors have been used to treat domestic and industrial waste for nearly a century. Development of molecular tools such as PCR and DNA microarrays have enabled identification and characterization of some of the microbes in these bioreactors; however, molecular characterization of the microbes is still in its infancy, and only a few of the molecular tools have been applied to improving performance of wastewater bioreactors at the commercial level. Several new plasmids and enzymes have been isolated from wastewater bioreactors. There is enormous opportunity to use the microbes from wastewater for industrial bioprocesses. PMID- 16675291 TI - Gamma-butyrolactones: Streptomyces signalling molecules regulating antibiotic production and differentiation. AB - Small signalling molecules called gamma-butyrolactones are mainly produced by Streptomyces species in which they regulate antibiotic production and morphological differentiation. Their molecular mechanism of action has recently been unravelled in several streptomycetes, revealing a diverse and complex system. Gamma-butyrolactones and their receptors also occur in some other Actinobacteria, suggesting that this is a general regulatory system for antibiotic production. The gamma-butyrolactones bind to receptors, many of which are involved in regulation of specific antibiotic biosynthesis clusters. The importance of understanding how secondary metabolites are regulated and how environmental and physiological signals are sensed highlights the relevance of studying this system. PMID- 16675292 TI - Diversity and biogeography of marine actinobacteria. AB - The actinomycetes, although not all the Actinobacteria, are easy to isolate from the marine environment. However, their ecological role in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate strains for search and discovery of new drugs. However, the marine environment has become a prime resource in search and discovery for novel natural products and biological diversity, and marine actinomycetes turn out to be important contributors. Similarly, striking advances have been made in marine microbial ecology using molecular techniques and metagenomics, and actinobacteria emerge as an often significant, sometimes even dominant, environmental clade. Both approaches - cultivation methods and molecular techniques - are leading to new insights into marine actinobacterial biodiversity and biogeography. Very different views of actinobacterial diversity emerge from these, however, and the true extent and biogeography of this are still not clear. These are important for developing natural product search and discovery strategies, and biogeography is a hot topic for microbial ecologists. PMID- 16675294 TI - The importance of host ecology in thelastomatoid (Nematoda: Oxyurida) host specificity. AB - An experimental investigation of host specificity within the Thelastomatoidea is presented by means of a comparison of the thelastomatoids of two panesthiine cockroaches, Panesthia cribrata and P. tryoni tryoni, with those of other log dwelling arthropods and those of leaf litter dwelling arthropods found near by. 145 log-dwelling and leaf-litter dwelling arthropods, representing adjacent ecological niches, were collected from Lamington National Park, Queensland, Australia. A high degree of thelastomatoid species sharing (19 incidences from 26 specimens) occurs between log-dwelling arthropods and the two cockroach species. No overlap in thelastomatoid fauna was observed between the log dwelling and leaf litter dwelling groups. Our results suggest that host specificity of thelastomatoids is largely dictated by host ecology. PMID- 16675293 TI - Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds? AB - Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds. PMID- 16675295 TI - Organotin(IV) complexes of thiohydrazides and thiodiamines: synthesis, spectral and thermal studies. AB - Organotin(IV) complexes of tribenzyltin(IV) chloride and di(para chlorobenzyl)tin(IV) dichloride with thiohydrazides have been reported. The ligands synthesized were bidentate coordinating through sulphur and terminal nitrogen atoms. These form 1:1 metal-ligand complexes. The following organotin(IV) complexes have been synthesized: (C(6)H(5)CH(2))(3)Sn(L(1))Cl, (p ClC(6)H(4)CH(2))(2)Sn(L(1))Cl(2), (C(6)H(5)CH(2))(3)Sn(L(1))Cl, (p ClC(6)H(4)CH(2))(2)Sn(L(2))Cl(2), (C(6)H(5)CH(2))(3)Sn(L(3))Cl, (p ClC(6)H(4)CH(2))(2)Sn(L(3))Cl(2), where (L(1)): 2-phenylethyl N-thiohydrazide, (L(2)): N-(2-phenylethyl-N-thio)-1,3-propane diamine, (L(3)): N-(2-phenylethyl-N thio)-1,2-ethane diamine. The complexes were synthesized by directly mixing, refluxing and stirring the ligands with organotin(IV) chlorides in a suitable solvent. The complexes were found to be pure and were characterized by elemental analysis, electronic, infrared, (1)H and (13)C NMR spectroscopy. These complexes were also studied for their thermal decomposition by thermogravimetry (TG) and differential thermal analysis (DTA). Various kinetic and thermodynamic parameters, viz. activation energy (E(a)), order of reaction (n), apparent activation entropy (S(#)) and heat of reaction (DeltaH) have been determined by using Horowitz-Metzger method. It was observed that these complexes are highly stable and the thermal degradation of these complexes is a spontaneous process. The ligands and their tin complexes have also been screened for their fungitoxicity activity and found to be quite active in this respect. PMID- 16675296 TI - Anharmonic analysis of the vibrational spectra of some cyanides and related molecules of astrophysical importance. AB - A detailed analysis of the vibrational spectra of carbonyl cyanide, diethynyl ketone and acetyl cyanide has been conducted in harmonic and anharmonic approximations. RHF, MP2 and density functional theory (DFT) methods with 6 311++G(2df,2p) basis sets and B3LYP functionals have been employed. Spectroscopic constants such as anharmonicity constants, rotational and centrifugal distortion constants, rotation-vibration coupling constants and Coriolis coupling coefficients have been calculated for each molecule and compared with the experimental data, where available. A close agreement between the calculated and experimental values of the spectroscopic constants has been obtained. Complete assignments have been provided to the fundamental bands, overtones and combination tones of the molecules. Density functional theory based anharmonic frequencies compare well with the experimental frequencies within +/-18 cm(-1) on an average. RHF and MP2 methods, however, give much higher values for the frequencies that need scaling even in the anharmonic approximation. PMID- 16675297 TI - Viability study of a personalized and adaptive knowledge-generation telehealthcare system for nephrology (NEFROTEL). AB - OBJECTIVES: Several important problems in the majority of countries are challenging the centralized and overburdened current model of healthcare. Telehealthcare is presented as a new paradigm that offers high expectations to solve this picture. In this paper we present the major outcomes of the viability study of a novel personalized telehealthcare system for nephrology (NEFROTEL). METHODS: The study evaluates the accuracy and quality of the knowledge generated by two key processing layers, namely, sensor layer and patient physiological image (PPI) layer, in an independent way, thanks to its modular design. The first one was defined by a personalized falling detection monitor, on account of the consequences of falls in chronic renal patients. The second one was analyzed by means of a PPI's prototype based on a urea compartmental pharmacokinetic model. The experimental study of the falling detector monitor has been more extensive than the other because the latter has already been addressed in other works. RESULTS: The outcomes show, firstly, the capability of the PPIs to provide integrated and correlated physiological knowledge adapted to each patient, and secondly, demonstrate the reliability of the impact detection function of the adaptive human movement monitor compliant with the NEFROTEL paradigm. CONCLUSIONS: The study confirms that NEFROTEL is able to provide knowledge concerning a patient in a manner that cannot be accomplished by the ordinary healthcare model at the present time. PMID- 16675298 TI - Highly sensitive TaqMan RT-PCR assay for detection and quantification of both lineages of West Nile virus RNA. AB - BACKGROUND: Starting in 1999, the West Nile virus (WNV) epidemic represents the largest outbreak of arboviral encephalitis ever recorded in the U.S. The effective means to determine an infection are detection of viral nucleic acid and/or viral specific immunoglobulin, IgM and/or IgG. OBJECTIVE: To develop a highly sensitive and specific TaqMan RT-PCR assay for the detection and quantification of WNV RNA of lineage 1 and lineage 2. STUDY DESIGN: A TaqMan RT PCR primer-probe was designed to perfectly match target sequences of all sequenced WNV strains and isolates, which added a layer of protection against false-negative results due to strain variability. In addition, the inclusion of a low level RNA internal control (IC) in the assay increased the precision and accuracy of the assay. RESULTS: By optimizing the RNA preparation procedure for increased WNV RNA recovery, together with optimizing the primer-probe and TaqMan conditions for improved amplification efficiency, we developed a highly sensitive assay with the detection limit of 10 copies/mL. To evaluate the assay, we tested plasma samples from 12 transfusion-transmitted implicated cases in 2002 and from 68 positive blood donors in 2003. All tested specimens were WNV positive. The viral load of 68 positive blood donor samples collected in 2003 ranged from 10 copies/mL to 67,000 copies/mL with a mean of 8100 copies/mL. Furthermore, high sensitivity of the assay was achieved without compromising specificity. All 100 routine donor samples tested negative. CONCLUSIONS: The assay results demonstrate that our in-house TaqMan RT-PCR procedure can detect and quantify WNV RNA of lineage1 and lineage 2 in human plasma with high sensitivity and specificity. PMID- 16675299 TI - Cytomegalovirus disease in the era of highly active antiretroviral therapy (HAART). AB - Cytomegalovirus (CMV) infection was one of the most important opportunistic infections in HIV-infected patients before the introduction of highly active antiretroviral therapy (HAART), i.e. the combination of at least three antiretroviral drugs of different classes. Thereafter, life expectancy and quality of life increased dramatically with the persistent suppression of HIV viremia and a significant reduction in incidence of CMV disease. Nevertheless, evidence for a multitude of direct and indirect effects of CMV on HIV progression is accumulating. Even in the era of HAART, a considerable number of HIV-infected patients have a CD4 cell count below <100 mm(-3), which involves a high risk for CMV disease. The focus of the present review is on interpretation of test results, their predictive value for CMV disease, and guidance for the rational use of diagnostic assays in HIV-infected patients. Identification of patients at immediate risk for CMV disease may be accomplished by detection of CMV-DNA in leucocytes or plasma. Evidence is growing that CMV genotypes may be also relevant for the risk of CMV disease. Diagnosis of CMV disease requires in most instances demonstration of virus in biopsy specimen from the affected organ because presence of CMV in blood may not be causally related to symptoms observed. Clinical symptoms and patient characteristics are essential in the interpretation of laboratory test results and may guide the rational collection of clinical specimen and use of laboratory assays. As a consequence, a reliable diagnosis of CMV disease and early identification of patients at high risk for CMV disease requires an integrated interpretation of clinical and virological information. PMID- 16675300 TI - Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2alpha in response to phorbol 12-myristate 13 acetate and action of these prostanoids during life cycle of adipocytes. AB - Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF2alpha and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX 1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2alpha (cPLA2alpha) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF2alpha following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2alpha and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF2alpha, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF2alpha and the specific action of these prostanoids at different cycle stages of adipocytes. PMID- 16675301 TI - Attenuated secretion of very low density lipoproteins from McA-RH7777 cells treated with eicosapentaenoic acid is associated with impaired utilization of triacylglycerol synthesized via phospholipid remodeling. AB - In McA-RH7777 cells stably expressing human apolipoprotein (apo) B100, treatment with oleic acid (18:1(n-9)) promoted whereas treatment with eicosapentaenoic acid (EPA, 20:5(n-3)) attenuated assembly and secretion of VLDL. Under conditions where the cells were cultured in the presence of 20% serum, EPA (0.4 mM) had marginal effect on the secretion of total apoB100 (determined by pulse-chase analysis) but decreased (by 50%) secretion of triacylglycerol (TG), indicating that the inhibitory effect of EPA was exerted primarily on TG-rich VLDL. Analysis of phospholipid mass and species by tandem mass spectrometry showed increased phosphatidylethanolamine (PE) in EPA-treated cells, the increase was significant in the distal Golgi membranes (by 170%) and endoplasmic reticulum (by 116%). Lipid pulse-chase studies showed a major distinction between phospholipid species containing 20:5(n-3) and 18:1(n-9), which in turn was associated with distinct compartmentalization of TG containing 20:5(n-3) or 18:1(n-9) between cytosol and microsomes and their recruitment during VLDL assembly. Thus, 18:1-TG was secreted as VLDL but 20:5-TG was not. These results suggest that EPA attenuation of VLDL secretion is associated with impaired utilization of TG derived from phospholipid remodeling. PMID- 16675302 TI - The effect of match standard and referee experience on the objective and subjective match workload of English Premier League referees. AB - The aim of the present study was to examine the effect of match standard and referee experience on the objective and subjective workload of referees during English Premier League and Football League soccer matches. We also examined the relationship between heart rate (HR) and ratings of perceived exertion (RPE) for assessing match intensity in soccer referees. Heart rate responses were recorded using short-range telemetry and RPE scores were collected using a 10-point scale. Analysis revealed a significant relationship between mean match HR and match RPE scores (r=0.485, p<0.05, n=18). There were significant differences in match HR (Premier League 83.6+/-2.6% maximal HR (HRmax) versus Football League 81.5+/ 2.2%HRmax, p<0.05) and match RPE scores (Premier League 7.8+/-0.8 versus Football League 6.9+/-0.8, p<0.05) between standards of competition. Referee experience had no effect on match HR and RPE responses to Premier League and Football League matches. The results of the present study demonstrate the validity of using HR and RPE as a measure of global match intensity in soccer referees. Referee experience had no effect on the referees' objective and subjective match workload assessments, whereas match intensity was correlated to competition standard. These findings have implications for fitness preparation and evaluation in soccer referees. When progressing to a higher level of competition, referees should ensure that appropriate levels of fitness are developed in order to enable them to cope with an increase in physical match demands. PMID- 16675303 TI - Visualizing the dual space of biological molecules. AB - An important part of protein structure characterization is the determination of excluded space such as fissures in contact interfaces, pores, inaccessible cavities, and catalytic pockets. We introduce a general tessellation method for visualizing the dual space around, within, and between biological molecules. Using Delaunay triangulation, a three-dimensional graph is constructed to provide a displayable discretization of the continuous volume. This graph structure is also used to compare the dual space of a system in two different states. Tessellator, a cross-platform implementation of the algorithm, is used to analyze the cavities within myoglobin, the protein-RNA docking interface between aspartyl tRNA synthetase and tRNA(Asp), and the ammonia channel in the hisH-hisF complex of imidazole glycerol phosphate synthase. PMID- 16675305 TI - Laughter and the mesial and lateral premotor cortex. AB - We report the induction of laughter and smiling by cortical electrical stimulation of the frontal lobe of two patients: an 18-month-old boy with a left frontal cortical lesion extending to the vertex and the central gyrus, and a 35 year-old woman with a lesion in the right supplementary sensorimotor area (SSMA). The subjects underwent presurgical epilepsy evaluation with subdural grid electrodes to determine surgical candidacy. Stimulation of the prefrontal area reproducibly induced laughter. The adult patient reported absence of emotional content. Slowing of speech occurred under stimulation of electrodes in the upper and posterior vicinity. In this patient laughter was elicited in the anterior part of the SSMA. In the child, this response was induced by stimulation of the lateral prefrontal cortex near the midline. We conclude that the anterior portion of the SSMA/lateral premotor cortex is involved in generating the motor pattern of laughter. PMID- 16675304 TI - Role of indirect allo- and autoreactivity in anti-tumor responses induced by recipient leukocyte infusions (RLI) in mixed chimeras prepared with nonmyeloablative conditioning. AB - In mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism and anti-tumor responses against the A20 BALB/c B cell lymphoma. We also previously showed that RLI-mediated tumor rejection involved IFN-gamma-producing RLI-derived CD8+ cells and non-RLI, recipient-derived CD4 T cells, leading to the generation of anti-tumor cytotoxic cells. However, the mechanisms of such paradoxical anti-tumor responses remained to be clarified. In the present study, we further explored the cellular mechanisms of the anti-tumor effects of RLI in fully MHC-mismatched and haploidentical strain combinations. In both cases, we show that RLI breaks the tolerance of chimeric T cells toward donor antigens, in association with the in vivo expansion of recipient splenic T, B and CD4-CD8- cells and the production of IFN-gamma. RLI leads to the development of two types of tumor-specific responses. The first is mediated by indirect presentation of donor antigens and occurs independently of tumor injection. The second is observed only in recipients of RLI and tumor and may involve responses to self antigens. Anti-tumor cytotoxicity was mediated by CD8+ or CD4-CD8- effector cells. Thus, anti-tumor cytotoxic responses are generated following complex interactions between recipient APCs presenting donor and recipient antigens and host-type CD4+, CD8+ and CD4-CD8- cells. PMID- 16675306 TI - Intractable epilepsy: A survey of patients and caregivers. AB - The social and health consequences associated with epilepsy are often magnified among patients with refractory epilepsy. Despite recent advances in the treatment of seizure disorders, many people with epilepsy continue to suffer from uncontrolled seizures and adverse side effects from medical therapy. This survey is the first to focus solely on the experiences, attitudes, and quality of life of a refractory epilepsy population, both those with the condition and their caregivers. To participate in this survey, respondents had to currently be experiencing seizures or troubling treatment side effects and had to have tried at least two different epilepsy medications. These survey data represent three groups of participants (n = 903): those with epilepsy who self-reported on their condition (Group 1, n = 503), the caregivers of those with refractory epilepsy (Group 2, n = 200), and those with epilepsy who had their condition reported on by a caregiver (Group 3, n = 200). This survey revealed that the negative consequences associated with epilepsy tend to be greater among those experiencing treatment side effects and a greater number of seizures. Physicians must take into account medication side effects and quality-of-life issues when treating patients with epilepsy. PMID- 16675307 TI - Comparison of endoscopic ultrasound and computed tomography for the preoperative evaluation of pancreatic cancer: a systematic review. AB - BACKGROUND & AIMS: It is uncertain whether computed tomography (CT) or endoscopic ultrasound (EUS) is superior for the detection, staging, and resectability of pancreatic cancer. We therefore performed a systematic literature review to determine which test is more accurate. METHODS: We identified relevant studies from MEDLINE (1986-2004) and evaluated study quality, which was measured on the basis of guidelines for assessing studies of diagnostic tests. Quantitative outcomes data were abstracted from the studies. RESULTS: Eleven studies with 678 patients satisfied inclusion criteria. Nine studies assessed tumor detection, all of which concluded that the sensitivity of EUS was superior to CT. Four of 5 studies that assessed tumor staging accuracy and 5 of 8 that assessed nodal staging accuracy concluded that EUS was superior to CT. Among the 4 studies that assessed resectability, 2 showed no difference between EUS and CT, and 1 favored each modality. Three of 11 studies met all but one of the quality criteria. The most important and frequent study limitations were lack of a consecutive series of patients and biased patient selection for surgery. Quantitative comparisons among studies were precluded by differences in tumor staging classifications, surgical selection, CT and EUS techniques, and reporting of operating characteristics. CONCLUSIONS: The published literature comparing EUS and CT for preoperative assessment of pancreatic cancer is heterogeneous in study design, quality, and results. All studies have methodologic limitations that potentially affect validity. Prospective studies with state-of-the-art imaging are needed to further define the role of each test. PMID- 16675308 TI - Microsatellite typing reveals strong genetic structure of Schistosoma mansoni from localities in Kenya. AB - Genetic diversity and population structure of seven populations of Schistosoma mansoni sampled in Kenya were assessed using five microsatellite markers. The mean number of alleles per locus, expected heterozygosity in Hardy-Weinberg equilibrium and pairwise F(ST) values ranged from 5.2 to 10.7, 0.5-0.8 and 3.6 27.3%, respectively. These data reveal that S. mansoni populations in Kenyan have relatively high levels of genetic diversity and is significantly differentiated. Our data combined with information on biogeography support the hypothesis that the strong genetic structure in Kenyan schistosomes is as a result of limited gene flow and large population sizes. Resistance to anthelminthics has not been reported among the Kenyan schistosomes, we hypothesize that this is probably due to the very little gene flow among populations, thereby limiting opportunities for the spread of rare alleles that might confer resistance to the drugs. PMID- 16675309 TI - Oxidation chemistry of 2'-deoxyadenosine at pyrolytic graphite electrode. AB - The electrochemical oxidation of 2'-deoxyadenosine has been investigated in phosphate containing supporting electrolytes in pH range 2-10 at a pyrolytic graphite electrode by cyclic sweep voltammetry, spectral studies, controlled potential electrolysis and related techniques. The oxidation of 2'-deoxyadenosine occurred in a single well-defined oxidation peak (I(a)), over the entire pH range. The electrooxidation occurred by the loss of 6.0+/-0.5 e(-) per mole over the entire pH range. The kinetics of the decay of the UV-absorbing intermediates has been studied and found to follow pseudo first order kinetics having rate constant (k) in the range (5.7-7.7)x10(-4) s(-1). The major products of electrooxidation were separated by HPLC and characterized by GC-MS/MS, (1)H NMR and a tentative mechanism for electrooxidation of 2'-deoxyadenosine has been suggested. PMID- 16675310 TI - HPLC resolution of thioridazine enantiomers from pharmaceutical dosage form using cyclodextrin-based chiral stationary phase. AB - Resolution of racemic thioridazine obtained from Thioril tablets (Cipla Ltd., Goa, India) into its enantiomers has been achieved by HPLC using a beta cyclodextrin (CD)-bonded stationary phase. Thioridazine was isolated from commercial formulations and was purified using preparative TLC. The purity was ascertained by RP-HPLC. For the resolution of rac-thioridazine using cyclodextrin based CSP and mobile phase of 0.05 M phosphate buffer (pH 6.5)-acetonitrile (50:50) was found to be successful. The optimum conditions of resolution were established by systematically studying the effect of organic modifier, concentration of buffer, pH and flow rate of mobile phase. The detection limit was found to be 10 microg (5 microg of each enantiomer). The enantiomeric purity of each of the resolved isomers was verified by optical rotation. PMID- 16675312 TI - Determination of iohexol clearance by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). AB - We have developed a simple, rapid, and accurate HPLC-MS/MS method for the determination of iohexol in serum. The column used was a Zorbax Eclipse XDB-C8 (100 mm x 2.1 mm i.d., 3.5 microm). Mobile phases consisted of water containing 2mM ammonium acetate and 0.1% formic acid (A) and methanol containing 2 mM ammonium acetate and 0.1% formic acid (B). After simple protein precipitation with ZnSO4, serum samples were mixed with I.S. (bromperidol) and centrifuged for 3 min. The obtained extraction recovery at three levels was 94.6-107.4%. Quantitative analysis was performed in the multiple reaction-monitoring mode (m/z 822.0-->804.0 for iohexol, 420.1-->122.7 for I.S.) with the total running time of 3 min for each sample. The assay was linear between 0.5 and 1500 microg/mL (r2 > 0.997). The intra- and inter-assay coefficient of variations were 2.4-6.2% and 5.5-6.5%, respectively. Our method provided sufficient analytical range and specificity for the 210 clinical samples analyzed. PMID- 16675313 TI - In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin. AB - Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE 30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established. PMID- 16675314 TI - Identification of a novel mutation in the SRY gene in a 46, XY female patient. AB - BACKGROUND: The SRY gene encodes for a testis-specific transcription factor (TDF, testis determining factor) that plays a key role in sexual differentiation and development in males. Several SRY mutations have been described in patients with gonadal dysgenesis, accounting for 10-15% of the sex reversal cases. The reported mutations are both point mutations and deletions, mostly involving the high mobility group (HMG) box domain of SRY, which is a conserved region through the evolution, suggesting that SRY function strictly depends on the HMG box. CASE PRESENTATION: Here we describe the clinical, endocrinological and molecular data of a patient with complete 46, XY gonadal dysgenesis caused by SRY mutation located within the conserved HMG box. Using DNA direct sequencing of the SRY coding region, we identified a single nucleotide insertion at codon 89 with subsequent frameshift of the reading frame sequence, which results in a truncated protein as consequence of an introduction of a stop codon at the position 103. CONCLUSION: A novel SRY mutation has been described in a female with a gonadal dysgenesis associated with a 46, XY karyotype. The described case is of importance for genetic counseling. PMID- 16675316 TI - Clinical implications of nonopioid analgesia for relief of mild-to-moderate pain in patients with or at risk for cardiovascular disease. AB - Nonopioid analgesics, which include acetaminophen, aspirin, nonsteroidal anti inflammatory drugs (NSAIDs), and cyclooxygenase-2 (COX-2)-specific inhibitors (coxibs), are frequently used for the relief of mild-to-moderate pain. Although all of these agents are effective at controlling pain, inhibition of prostaglandins (PGs) by NSAIDs may result in untoward cardiorenal effects, including hypertension, fluid and electrolyte abnormalities, congestive heart failure, acute renal failure, and nephrotic syndrome. Individuals with an increased risk for cardiorenal effects from NSAIDs (eg, the elderly, and those with hypertension, cardiac disease, or gouty nephropathy) should be monitored for early onset of edema, destabilization of blood pressure control, and/or onset of congestive heart failure when started on NSAID therapy. Because acetaminophen has a different mechanism of action from the conventional NSAIDs, it does not inhibit peripheral PGs at recommended dosing and therefore appears to have a more favorable cardiovascular and gastrointestinal safety profile. This review discusses the effects of acetaminophen, traditional NSAIDs, and coxibs on fluid and electrolytes, blood pressure, congestive heart failure, and renal function, as well as their consequences in patients with or at risk for cardiovascular disease (CVD). It also summarizes information on the mechanisms by which NSAID induced cardiovascular adverse events develop, and it provides recommendations for the use of nonopioid analgesics for relief of mild-to-moderate pain in patients with or at risk for CVD. PMID- 16675317 TI - Nonnarcotic analgesics and hypertension. AB - In 2004, individuals in the United States spent >$2.5 billion on over-the-counter (OTC) nonsteroidal anti-inflammatory drugs (NSAIDs) and filled >100 million NSAID prescriptions. The most commonly used OTC analgesics include aspirin, acetaminophen, and nonaspirin NSAIDs. Nonnarcotic analgesics are generally considered safe when used as directed but do have the potential to increase blood pressure in patients with hypertension treated with antihypertensives. This is important because hypertension alone has been correlated with an increased risk of cardiovascular disease or stroke. Small increases in blood pressure in patients with hypertension also have been shown to increase cardiovascular morbidity and mortality. Therefore, when nonnarcotic analgesics are taken by patients with hypertension, there may be important implications. This review explores the potential connection among analgesic agents, blood pressure, and hypertension, and discusses possible mechanisms by which analgesics might cause increases in blood pressure. This is followed by a summary of data on the relation between analgesics and blood pressure from both observational and randomized trials. PMID- 16675318 TI - Gastrointestinal Considerations in Patients with Cardiovascular Disease Using Nonopioid Analgesics for Mild-to-Moderate Pain or Cardioprotection. AB - Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used classes of medications worldwide, available both through prescription and over the counter (OTC). Although these drugs are highly effective for pain, gastrointestinal (GI) complications may occur. Risk factors for GI complications from NSAIDs have been well studied, and the highest risk exists among the elderly and patients with a history of GI bleeding or complications. The increasingly widespread use of aspirin for both primary and secondary cardiovascular prophylaxis has also drawn attention to the potential increase in GI complications. Several strategies may minimize NSAID-mediated GI complications, including the use of drugs that do not injure the gut, such as acetaminophen or a low-dose opiate. The cyclooxygenase-2 (COX-2) inhibitors, which cause approximately 50% fewer GI complications than traditional NSAIDs, may also be used, although their cardiovascular safety has recently come into question. Antacid therapy with proton pump inhibitors (PPIs) may also be used to reduce NSAID-related dyspepsia and upper GI complications. Misoprostol is also effective in preventing NSAID-related complications, but is not as well tolerated. In any patient, the risk-benefit ratio must be assessed to determine the appropriate therapies to minimize GI complications resulting from daily aspirin therapy. PMID- 16675319 TI - Potential for drug-drug interactions in patients taking analgesics for mild-to moderate pain and low-dose aspirin for cardioprotection. AB - Millions of individuals in the United States take low-dose aspirin for cardioprotection. Physicians face a clinical dilemma when those same patients also have pain from arthritis or another condition. Nonsteroidal anti inflammatory drugs (NSAIDs) may increase the risk of gastrointestinal (GI) complications when used in conjunction with aspirin. In addition, NSAIDs, particularly ibuprofen, may interfere with the antithrombotic benefits of aspirin through competitive interaction with platelet cyclooxygenase-1 (COX-1). Evidence suggests that naproxen has antithrombotic effects; however, as with other NSAIDs, it poses a risk of gastrotoxicity. Selective COX-2 inhibitors reduce the risk of GI side effects, and although they inhibit platelet COX-1, it is to a far lesser extent than COX-2. However, it is unclear to what degree COX-2 inhibitors remain gastroprotective in the presence of aspirin. In addition, recent long-term trials have raised concerns about adverse cardiovascular events with prolonged use of both traditional and selective NSAIDs. Conversely, acetaminophen is well tolerated, has not been shown to contribute to gastrotoxicity when taken with aspirin, and has not been shown to interfere with the inhibition of platelet aggregation produced by aspirin. Acetaminophen is considered a first-line therapy for patients with mild-to-moderate joint pain. PMID- 16675321 TI - The many faces of the hygiene hypothesis. AB - About 15 years have gone by since Strachan first proposed the idea that infections and unhygienic contact might confer protection against the development of allergic illnesses. The so-called hygiene hypothesis has ever since undergone numerous more or less subtle modifications by various researchers in the fields of epidemiology, clinical science, and immunology. Three major tracts have developed exploring the role of overt viral and bacterial infections, the significance of environmental exposure to microbial compounds, and the effect of both on underlying responses of the innate and adaptive immunity. To date, a truly unifying concept has not yet emerged, but various pieces of a complex interplay between immune responses of the host, characteristics of the invading microorganism, the level and variety of the environmental exposure, and the interactions between a genetic background and a range of exposures becomes apparent. These influences are discussed as determinants for a number of complex allergic illnesses in this review, while we attempt to pay attention to the importance of different phenotypes, namely of the asthma syndrome. Even if today practical implications cannot directly be deduced from these findings, there is great potential for the development of novel preventive and therapeutic strategies in the future. PMID- 16675322 TI - Toll-like receptor function and signaling. AB - Mammals sense pathogen invasion through pattern-recognition receptors. A group of transmembrane proteins, Toll-like receptors (TLRs), play critical roles as pattern-recognition receptors. They are mainly expressed on antigen-presenting cells, such as macrophages or dendritic cells, and their signaling activates antigen-presenting cells to provoke innate immunity and to establish adaptive immunity. Each TLR has common effects, such as inflammatory cytokine induction or upregulation of costimulatory molecule expression, but also has its specific function, exemplified by type I IFN-inducing ability. These immunoadjuvant effects are not only critical in antimicrobial immunity but are also involved in manifestations of autoimmunity. Furthermore, some TLR agonists are now promising therapeutic tools for various immune disorders, including allergy. Therefore understanding molecular mechanisms on TLRs should be quite useful in the development of therapeutic maneuvers against allergy and autoimmune diseases. PMID- 16675323 TI - Eosinophilic bronchitis in asthma: a model for establishing dose-response and relative potency of inhaled corticosteroids. AB - BACKGROUND: Newer generations and formulations of inhaled corticosteroids have necessitated the development of a clinically relevant model to compare their clinical potency. OBJECTIVE: We evaluated whether sputum eosinophil counts could demonstrate a dose-response to inhaled corticosteroids, and compared the response with other inflammatory markers. METHODS: Fourteen steroid-naive patients with asthma with an initial sputum eosinophilia of > or = 2.5% entered a 6-week sequential, placebo-controlled, patient-blinded, cumulative dose-response study. After 7 days of placebo, they received incremental doses of fluticasone propionate (FP), 50, 100, 200, and 400 microg/d, each for 7 days. Measurements were made of sputum and blood eosinophils, exhaled nitric oxide, spirometry, airway responsiveness to methacholine (methacholine PC20), and symptom scores before and after each dose. RESULTS: Sputum eosinophils and exhaled nitric oxide were extremely sensitive to the effects of FP, and exhibited significant dose dependent reductions of 99.4% and 99.8 parts per billion, respectively, where each variable was expressed per 100 microg/d FP. This compared with a 0.5 doubling dose increase of airway responsiveness to methacholine and a 0.3 decrease in symptom scores. Airway responsiveness to methacholine was the only variable that increased throughout the study. CONCLUSION: These results suggest that the model of eosinophilic bronchitis could be used to compare the effect of cumulative doses of an inhaled corticosteroid delivered by different types of delivery systems or preparations using a relatively small number of patients. CLINICAL IMPLICATIONS: Future clinical studies based on this model will allow clinicians to make informed decisions regarding the relative potencies of different inhaled corticosteroids. PMID- 16675324 TI - Validation of a beta-agonist long-term asthma control scale derived from computerized pharmacy data. AB - BACKGROUND: Asthma control has been defined clinically by using validated tools, but an asthma control scale using administrative data has not been reported. OBJECTIVE: We sought to validate a beta-agonist asthma control scale derived from administrative data. METHODS: Surveys that included validated asthma symptom and control tools were completed by a random sample of 2250 health maintenance organization members aged 18 to 56 years with persistent asthma. Linked computerized pharmacy data provided beta-agonist canister and oral corticosteroid dispensings. The proposed 4-level asthma control scale was based on the number of short-acting beta-agonist canisters dispensed in 12 months. Construct validity and predictive validity were assessed. RESULTS: For construct validity, factor analysis showed significant loading of the beta-agonist scale on the symptom control factor, and the beta-agonist scale was significantly related to the validated asthma control and symptom scales (r = 0.31, P < .0001). For predictive validity, each progressive level of the proposed beta-agonist control scale was associated with an increased risk of subsequent asthma hospitalizations or emergency department visits and oral corticosteroid use, independent of prior use. CONCLUSION: A scale based on the number of beta-agonists dispensed in a 1 year period and derived from administrative data reflects asthma symptom control over that period of time. This scale can help identify patients who are at risk for future acute asthma health care use. CLINICAL IMPLICATIONS: This information can be used in population management and by clinicians to assess long-term asthma control and identify patients who need intervention to prevent future morbidity. PMID- 16675325 TI - Endogenous and exogenous sex steroid hormones and asthma and wheeze in young women. AB - BACKGROUND: Emerging evidence suggests that both endogenous and exogenous sex steroid hormones may influence the occurrence of asthma and wheeze among women. OBJECTIVE: We investigated the associations between exogenous sex hormone (oral contraceptive [OC]) use and wheezing in young women with and without asthma history. To investigate the role of endogenous sex hormones, we examined the association between age at menarche and the development of asthma after puberty. METHODS: We conducted a study among 905 women who had undergone menarche. Subjects were between 13 and 28 years of age and had participated in the Children's Health Study. RESULTS: In women without asthma, OC use was associated with higher risk of current wheeze (odds ratio [OR], 1.75; 95% CI, 1.15-2.65). In contrast, OC use was associated with a markedly reduced prevalence of current wheeze in women with a history of asthma (OR, 0.18; 95% CI, 0.06-0.56; P value for interaction = .003). These associations showed significant trends with duration of OC use. Age at menarche was associated with new-onset asthma after puberty. Compared with women who had menarche after age 12 years, women with menarche before age 12 years had a 2.08-fold (95% CI, 1.05-4.12) higher risk of asthma after puberty. CONCLUSION: Both endogenous and exogenous sex steroid hormones affect asthma and wheeze occurrences in young women. CLINICAL IMPLICATIONS: Because women have higher asthma risk after puberty, and OC use is common among young women, clinicians may inform women with asthma about the potential effects of OC on asthma-related respiratory symptoms. PMID- 16675326 TI - Effect of ciclesonide dose and duration of therapy on exercise-induced bronchoconstriction in patients with asthma. AB - BACKGROUND: Inhaled corticosteroid therapy improves exercise symptoms in asthmatic subjects. OBJECTIVE: We sought to evaluate exercise-induced bronchoconstriction (EIB) as a method of determining the dose and time responses of inhaled corticosteroid therapy. METHODS: In this double-blind, randomized, cross-over study with 2 parallel arms, 4 doses of inhaled ciclesonide (40 microg and 160 microg or 80 microg and 320 microg) were compared over 3 weeks of treatment. Twenty-six asthmatic subjects (age range, 14-27 years) with baseline FEV1 values of greater than 70% of predicted value were enrolled. The primary outcome was the maximum percentage decrease in FEV1 after standardized exercise challenge. RESULTS: After 1 week of therapy, the mean +/- SEM reduction in maximum decrease in FEV1 in the ciclesonide 40-microg/80-microg dose group was 9% +/- 2.6% (95% CI, 3.9% to 14%), with no additional reduction thereafter. In the ciclesonide 160-microg/320-microg dose group, there was an 8.7% +/- 2.5% (95% CI, 3.7% to 13.7%) reduction in maximum decrease in FEV1 after week 1, which continued in a linear fashion during subsequent weeks of treatment. No difference was found between the 2 treatment arms in the temporal response of EIB to ciclesonide treatment. The maximum percentage attenuation in EIB achieved was 51.1% +/- 7.9%, which was achieved by using the 320-microg dose after 3 weeks of treatment. CONCLUSIONS: A significant improvement in EIB was demonstrated for all doses of ciclesonide. Use of 160 microg/320 microg of ciclesonide resulted in a continuing improvement in FEV1 with time, and no plateau was seen in protective effect during 3 weeks of treatment. CLINICAL IMPLICATIONS: Attenuation in exercise-induced decrease can be seen as early as after 1 week of therapy with inhaled ciclesonide at doses greater than 40 microg. However, maximal attenuation in exercise response continues to increase at doses greater than or equal to 200 microg, even after 3 weeks of therapy. PMID- 16675327 TI - Socioeconomic status and inflammatory processes in childhood asthma: the role of psychological stress. AB - BACKGROUND: Although social environment variables such as socioeconomic status (SES) have been linked to childhood asthma, little is known about the psychobiological mechanisms underlying this relationship. OBJECTIVES: The goal of this study was to investigate relationships among SES, psychological stress, and immune processes implicated in asthma. METHODS: Thirty-seven children ages 9 to 18 years, physician-diagnosed with asthma, and 39 healthy children participated. Families were interviewed about chronic life stress, perceptions of threat, and SES. Blood samples were drawn from children to assess stimulated production of cytokines implicated in asthma (IL-4, IL-5, IL-13) and eosinophil counts. RESULTS: In children with asthma, lower SES was associated with heightened production of IL-5 and IL-13 and higher eosinophil counts (P values < .05). Lower SES also was associated with higher chronic stress and perceived threat (both groups: P values < .05). Higher levels of stress and threat perception were associated with heightened production of IL-5 and IL-13, and higher eosinophil counts in children with asthma (P values < .05). Statistical mediation tests revealed that chronic stress and threat perception represented statistically significant pathways between SES and immune processes in children with asthma (P values < .05). In healthy children, associations were in the opposite direction from the asthma group, though generally not significant. CONCLUSION: This is one of the first studies to document empirically a psychobiological explanation for the epidemiologic relationship between low SES and poor asthma outcomes. CLINICAL IMPLICATIONS: Associations among SES, psychological stress, and immune pathways suggest that the experience of stress, particularly among lower SES children, has implications for childhood asthma morbidity. PMID- 16675328 TI - Sublingual immunotherapy: a comprehensive review. AB - Sublingual immunotherapy (SLIT) has been used with increasing frequency in Europe and is viewed with increasing interest by allergists in the United States. To address this interest, a Joint Task Force of the American College of Allergy, Asthma and Immunology and the American Academy of Allergy, Asthma and Immunology's Immunotherapy and Allergy Diagnostic Committees reviewed the available literature on SLIT and prepared this report. The task force concluded that despite clear evidence that SLIT is an effective treatment, many questions remained unanswered, including effective dose, treatment schedules, and overall duration of treatment. Until these have been determined, an assessment of the cost/benefit ratio of the treatment cannot be made. SLIT does appear to be associated with few serious side effects, but it has not been administered in high-risk asthmatic patients, nor in the studies reviewed has it been administered as a mixture of non-cross-reacting allergens. Furthermore, there is currently no allergy extract approved for this use in the United States, nor is there a Current Procedural Terminology code for billing purposes. All of these factors should be given careful consideration by anyone contemplating initiating SLIT treatment for their allergic patients. PMID- 16675329 TI - Update on the role of prostaglandins in allergic lung inflammation: separating friends from foes, harder than you might think. AB - Prostaglandins (PGs), small lipid molecules derived from arachidonic acid by COX enzymes, are critical mediators of allergic inflammation. Our understanding of the role of PGs in allergic lung inflammation has been hampered by the very short biologic half-life of these mediators, which has made mechanistic studies difficult in human subjects. However, advances in molecular biology and pharmacology have given investigators the opportunity to examine the role of specific prostanoids in the development of allergic inflammation in animal models. Studies investigating specific PG receptors are also elucidating the mechanisms by which PGs regulate the pulmonary allergic phenotype. This review summarizes the current literature on the role of PGs and PG receptors in allergic lung inflammation. PMID- 16675331 TI - Advances in upper airway diseases and allergen immunotherapy. AB - The purpose of this review is to highlight important articles on upper airway disease and immunotherapy that appeared in the Journal of Allergy and Clinical Immunology and elsewhere during 2005. In recent studies of tissue from patients with chronic hypertrophic eosinophilic sinusitis, increased leukotriene C4 synthase and 5-lipoxygenase activity and increased levels of cysteinyl leukotriene production were demonstrated that correlated with disease severity but not with whether the patient was aspirin sensitive. However, the cysteinyl leukotriene 1 receptor was increased in leukocytes in the sinus tissue only in those patients with aspirin sensitivity. Major basic protein, released by eosinophils into the mucus in the paranasal sinus lumen, was found to reach concentrations capable of damaging the sinus epithelium, predisposing to bacterial infections. Testing the hypothesis that chronic hypertrophic eosinophilic sinusitis represents a reaction to common fungi, a double-blind trial of intranasal instillation of amphotericin B was conducted. There were marginal but significant differences in favor of amphotericin B treatment for sinus mucosal thickening on the basis of computed tomography and the evidence of eosinophilic inflammation in the sinus mucus. The effectiveness of topical nasal corticosteroids for treatment of nasal polyps was confirmed in 2 large studies. Improvement in sleep quality and daytime drowsiness in patients with allergic rhinitis treated with nasal corticosteroids was reported to correlate with reduction in nasal obstruction. The statistical analysis behind studies that reported a decrease in asthma exacerbations with nasal corticosteroids or oral antihistamines has been questioned. It appears that the results of at least one of these studies are indeed too good to be true. Although caution is still indicated in administering immunotherapy to patients receiving beta-adrenergic blocking agents, the prohibition might not be absolute. A study in patients with Hymenoptera sensitivity given venom immunotherapy revealed no increase in serious adverse reactions to venom injections and no greater incidence of reactions to insect stings in those taking beta-blocking agents. Sublingual immunotherapy for 8 to 12 weeks in patients with hazelnut sensitivity significantly increased their tolerance to hazelnut in double-blind, placebo-controlled challenges while inducing increased IgG4 and IL-10 levels, indicating induction of regulatory T cells. There were a number of articles in the Journal of Allergy and Clinical Immunology in 2005 that addressed the entity of chronic hypertrophic eosinophilic sinusitis. In addition, an update of the "Practice parameters on sinusitis" was published. The major focus in allergen immunotherapy continues to be sublingual administration. PMID- 16675330 TI - Role of CCL21 and CCL19 in allergic inflammation in the ovalbumin-specific murine asthmatic model. AB - BACKGROUND: Dendritic cells are the most powerful of the antigen-presenting cells and are known to play important roles in sensitization and inflammation in allergen-specific asthma. Various cytokines and chemokines are involved in the maturation and activation of dendritic cells. Among them is CC chemokine ligand (CCL)21, a key chemokine in the entry of naive T cells and antigen-stimulated dendritic cells into the T-cell zones of secondary lymphoid organs, which is a critical process in antigen-specific T-cell activation. OBJECTIVE: We studied the role of CCL21 in airway inflammation in asthma by using BALB/c-plt/plt (plt) mice, which possess genetic defects in expression of both CCL21 and CCL19. METHODS: Plt and control BALB/c mice were immunized with ovalbumin and alum 4 times and thereafter were subjected to a 2-week regimen of ovalbumin inhalation. RESULTS: In plt mice, ovalbumin-specific IgE response was delayed compared with control BALB/c mice, but they had the same level of response after final immunization. Although airway inflammation and response to acetylcholine were significantly reduced compared with BALB/c mice, significant eosinophilic inflammation and hyperresponsiveness were also observed in plt mice after 2 weeks of inhalation. Four weeks after cessation of inhalation, airway inflammation and hyperresponsiveness in plt mice were greater than in BALB/c mice. At the time of resolution of airway inflammation, IL-10 production was enhanced in BALB/c mice but not in plt mice. CONCLUSION: The chemokines CCL21 and CCL19 were critical for resolution of airway inflammation. CLINICAL IMPLICATIONS: The findings about the chemokines for induction and resolution of inflammation are key to establishing a new strategy for asthma immunotherapy. PMID- 16675332 TI - ARIA update: I--Systematic review of complementary and alternative medicine for rhinitis and asthma. AB - Complementary-alternative medicines are extensively used in the treatment of allergic rhinitis and asthma, but evidence-based recommendations are lacking. To provide evidence-based recommendations, the literature was searched by using MedLine and the Cochrane Library to March 2005 (Key words: Asthma [OR] Rhinitis, [AND] Complementary [OR] Alternative Medicine, [OR] Herbal, [OR] Acupuncture, [OR] Homeopathy, [OR] Alternative Treatment). Randomized trials, preferably double-blind and published in English, were selected. The articles were evaluated by a panel of experts. Quality of reporting was assessed by using the scale validated by Jadad. The methodology of clinical trials with complementary alternative medicine was frequently inadequate. Meta-analyses provided no clear evidence for the efficacy of acupuncture in rhinitis and asthma. Some positive results were described with homeopathy in good-quality trials in rhinitis, but a number of negative studies were also found. Therefore it is not possible to provide evidence-based recommendations for homeopathy in the treatment of allergic rhinitis, and further trials are needed. A limited number of studies of herbal remedies showed some efficacy in rhinitis and asthma, but the studies were too few to make recommendations. There are also unresolved safety concerns. Therapeutic efficacy of complementary-alternative treatments for rhinitis and asthma is not supported by currently available evidence. PMID- 16675333 TI - Renaissance of the hygiene hypothesis. PMID- 16675334 TI - Does early indoor microbial exposure reduce the risk of asthma? The Prevention and Incidence of Asthma and Mite Allergy birth cohort study. AB - BACKGROUND: Exposure to microbial agents might inhibit the development of atopy and asthma. OBJECTIVE: We measured the association between microbial exposure assessed at 3 months and the development of atopic sensitization and doctor diagnosed (DD) asthma and wheeze in the first 4 years in a birth cohort study of children with atopic mothers. METHODS: Endotoxin, fungal (1-->3)-beta-D-glucans, extracellular polysaccharides from the genera Penicillium and Aspergillus (EPS Pen/Asp), and dust on living room floors were measured at 3 months of age. Serum IgE levels against common allergens were determined at 1 and 4 years, and questionnaire information about respiratory morbidity was collected yearly. RESULTS: Microbial levels in mattresses were low and not associated with serum IgE levels, DD asthma, and wheeze. Floor levels of biocontaminants and dust, on the other hand, were inversely associated with DD asthma, being most pronounced for endotoxin (odds ratio [OR], 0.40; 95% CI, 0.21-0.77) and EPS-Pen/Asp (OR, 0.42; 95% CI, 0.18-0.99). Mutual adjustment for other exposures did not significantly alter the results for endotoxin and only moderately affected the results for EPS-Pen/Asp. Persistent wheeze was also consistently less common in the high-exposure group, being significant only for EPS-Pen/Asp (OR, 0.37; 95% CI, 0.15-0.96). Transient wheeze and wheeze in the past 12 months were also reduced, but effects were smaller and not significant. Relationships with serum specific IgE levels, which could only be assessed in 41% at age 4 years, were less pronounced and statistically significant only for EPS-Pen/Asp. CONCLUSIONS: Early exposure to common microbial contaminants, including fungal agents, might protect against asthma. CLINICAL IMPLICATIONS: Microbial exposure in early life might protect against asthma and might constitute a novel target for prevention. PMID- 16675335 TI - House dust extracts have both TH2 adjuvant and tolerogenic activities. AB - BACKGROUND: Although mechanisms remain a subject of controversy, there is general agreement that living environments influence allergic risk during the first years of life. We reasoned that sterile house dust extracts (HDEs) would have immunologic activities reflective of their environments of origin and therefore would be useful surrogates for investigations of how ambient exposures influence immune homeostasis. OBJECTIVE: These experiments determined how airway HDE exposures influence adaptive responses to a coadministered antigen and subsequent airway hypersensitivity responses to antigen challenge. METHODS: Mice received intranasal ovalbumin (OVA) vaccinations on a weekly basis. Select groups of mice also received intranasal HDE weekly with OVA; daily at one seventh the weekly dose, beginning 7 days before the first OVA sensitization; or both. RESULTS: Weekly intranasal vaccinations with OVA and HDE primed mice for the development of T(H)2-biased immune and airway hypersensitivity responses. In contrast, daily low-dose intranasal HDE exposures protected against the immunologic and pathologic outcomes associated with weekly intranasal OVA/HDE vaccinations. The T(H)2 adjuvant activities of HDEs were found to be dependant on MyD88, a molecule critical for signaling through a majority of Toll-like receptors. Moreover, the tolerogenic activity associated with daily intranasal HDE exposures could be replicated with LPS. CONCLUSION: These investigations demonstrate that in addition to allergens, living environments contain immunomodulatory materials with both T(H)2 adjuvant and tolerogenic activities. CLINICAL IMPLICATIONS: As the contents of HDEs are ubiquitous, these experiments might recapitulate and help explain clinically relevant immunologic events involved in the maintenance of aeroallergen tolerance and the dysregulated responses that lead to allergic respiratory diseases. PMID- 16675336 TI - Endotoxin in inner-city homes: associations with wheeze and eczema in early childhood. AB - BACKGROUND: An inverse association between domestic exposure to endotoxin and atopy in childhood has been observed. The relevance of this aspect of the hygiene hypothesis to US inner-city communities that have disproportionately high asthma prevalence has not been determined. OBJECTIVES: To measure endotoxin in the dust from inner-city homes, evaluate associations between endotoxin and housing/lifestyle characteristics, and determine whether endotoxin exposure predicted wheeze, allergic rhinitis, and eczema over the first 3 years of life. METHODS: As part of an ongoing prospective birth cohort study, children of Dominican and African-American mothers living in New York City underwent repeated questionnaire measures. Dust samples collected from bedroom floors at age 12 or 36 months were assayed for endotoxin. RESULTS: Among the samples collected from 301 participants' homes, the geometric mean endotoxin concentration (95% CI) was 75.9 EU/mg (66-87), and load was 3892 EU/m2 (3351-4522). Lower endotoxin concentrations were associated with wet mop cleaning and certain neighborhoods. Endotoxin concentration correlated weakly with cockroach (Bla g 2: r = 0.22, P < .001) and mouse (mouse urinary protein: r = 0.28; P < .001) allergens in the dust. Children in homes with higher endotoxin concentration were less likely to have eczema at age 1 year (odds ratio, 0.70 [0.53-0.93]) and more likely to wheeze at age 2 years (odds ratio, 1.34 [1.01-1.78]). These associations were stronger among children with a maternal history of asthma. CONCLUSION: Endotoxin levels in this inner-city community are similar to those in nonfarm homes elsewhere. In this community, domestic endotoxin exposure was inversely associated with eczema at age 1 year, but positively associated with wheeze at age 2 years. CLINICAL IMPLICATIONS: Endotoxin exposure in the inner-city community may be related to wheeze in the early life; however, given the inverse association seen with eczema, the long-term development of allergic disease is still in question. PMID- 16675337 TI - Validation of a mouse model of chemical-induced asthma using trimellitic anhydride, a respiratory sensitizer, and dinitrochlorobenzene, a dermal sensitizer. AB - BACKGROUND: Occupational asthma can be caused by chemicals. Previously, we established a murine model of immunologically mediated chemical-induced asthma using toluene diisocyanate. OBJECTIVE: We sought to verify this model using trimellitic anhydride (TMA), a respiratory sensitizer, and 1-chloro-2,4 dinitrobenzene (DNCB), a dermal sensitizer. METHODS: BALB/c mice received dermal applications (vehicle or chemical) on days 1 and 7. On day 10, they received an intranasal instillation (vehicle or chemical). Whole-body plethysmography (enhanced pause) was used to monitor changes in ventilatory function and methacholine reactivity. Pulmonary inflammation was assessed by using bronchoalveolar lavage (cells, TNF-alpha levels, and macrophage inflammatory protein 2 levels). Immunologic parameters included total serum IgE levels, lymphocyte distribution in auricular and cervical lymph nodes, and IL-4 and IFN gamma levels in supernatants of lymph node cells incubated with or without concanavalin A. RESULTS: Mice dermally treated and intranasally challenged with TMA experienced markedly increased enhanced pause immediately after intranasal challenge and increased methacholine reactivity (24 hours later). Mice similarly treated with DNCB did not show any ventilatory changes. Neutrophil influx and increased macrophage inflammatory protein 2 and TNF-alpha levels were found in bronchoalveolar lavage fluid in both TMA- and DNCB-treated mice. The proportion of CD19+ B cells was increased in auricular and cervical lymph nodes of TMA treated mice. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular and cervical lymph node cells of TMA- or DNCB treated mice; however, the relative proportions of IL-4 and IFN-gamma levels differed between TMA- and DNCB-treated mice. Serum total IgE levels were increased in TMA-treated mice only. CONCLUSION: Both compounds induce a mixed T(H)1-T(H)2 response, but only TMA induced ventilatory changes. CLINICAL IMPLICATIONS: In the workplace avoiding skin contact with chemical sensitizers might be advised to prevent chemical-induced asthma. PMID- 16675338 TI - Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis. AB - BACKGROUND: Several case reports show allergy and anaphylactic reactions to the fish parasite Anisakis in the domestic and occupational setting. Further research is needed on the prevalence and mechanisms of disease. OBJECTIVE: To determine the prevalence of Anisakis sensitization and related symptoms among workers in 2 fish-processing factories, and to use gene-deficient mice to determine the working mechanisms of Anisakis allergy. METHODS: A modified version of the European Community Respiratory Health Survey was used to interview 578 South African fish-processing workers. Sensitization to Anisakis, seafood, and common aeroallergens was determined by skin prick test. Lung function was measured by spirometry and methacholine challenge. Serum eicosapentaenoic acid levels were used as an index of seafood consumption. Sensitized wild-type, IL-4, or IL-4 receptor alpha-deficient mice were challenged orally with Anisakis extract. Allergic reactions, lung pathology, antibodies, cytokines, mast cell proteases, and histamine were evaluated. RESULTS: The prevalence of sensitization to Anisakis was higher than the prevalence of sensitization to fish (8% vs 6%). Anisakis-specific IgE reactivity was associated with bronchial hyperreactivity and dermatitis, and significantly increased with fish consumption. In mice, Anisakis infective larvae (L3) induced a striking T(H)2/type 2 response. Food allergic-type reactions induced by oral challenge with Anisakis extract were absent in IL-4 receptor alpha knockout mice. CONCLUSION: Anisakis sensitization in fish-processing workers is associated with allergic symptoms and correlates with high levels of fish consumption. Anisakis proteins induce allergic reactions in sensitized mice by IL-4/IL-13-mediated mechanisms. CLINICAL IMPLICATIONS: Anisakis allergy should be considered in fish-processing workers with allergic symptoms. PMID- 16675339 TI - Evidence for a reduced histamine degradation capacity in a subgroup of patients with atopic eczema. AB - BACKGROUND: A diminished histamine degradation based on a reduced diaminoxidase activity is suspected as a reason for non-IgE-mediated food intolerance caused by histamine. Atopic eczema (AE) is often complicated by relapses triggered by IgE mediated allergy to different kinds of food. However, in a subgroup of patients with AE, allergy testing proves negative, although these patients report a coherence of food intake and worsening of AE and describe symptoms that are very similar to histamine intolerance (HIT). OBJECTIVES: It was the aim of our study to evaluate symptoms of HIT in combination with diaminoxidase levels in a total of 360 individuals consisting of patients with AE (n = 162) in comparison with patients with HIT (n = 124) without AE and healthy control volunteers (n = 85). METHODS: Histamine plasma level was determined with an ELISA and diaminoxidase serum activity with the help of radio extraction assays using [3H]-labeled putrescine-dihydrochloride as a substrate. Detailed clinical evaluations of characteristic features of AE and HIT were performed. RESULTS: Reduced diaminoxidase serum levels leading to occurrence of HIT symptoms like chronic headache, dysmenorrhea, flushing, gastrointestinal symptoms, and intolerance of histamine-rich food and alcohol were significantly more common in patients with AE than in controls. Reduction of both symptoms of HIT and Severity Scoring of Atopic Dermatitis could be achieved by a histamine-free diet in the subgroup of patients with AE and low diaminoxidase serum levels. CONCLUSION: Higher histamine plasma levels combined with a reduced histamine degradation capacity might influence the clinical course of a subgroup of patients with AE. CLINICAL IMPLICATIONS: As HIT emerges in a subgroup of patients with AE, a detailed anamnestic evaluation of food intolerance and HIT symptoms complemented by an allergological screening for food allergy, a diet diary, and, in confirmed suspicion of HIT, measurement of diaminoxidase activity and a histamine-free diet should be undertaken. PMID- 16675340 TI - Plasma of patients with chronic urticaria shows signs of thrombin generation, and its intradermal injection causes wheal-and-flare reactions much more frequently than autologous serum. AB - BACKGROUND: Several aspects of the pathogenesis of chronic urticaria (CU) remain contradictory. Autologous serum skin tests (ASSTs) and in vitro histamine release assays seem to look into distinct aspects of the disease, and the specificity of ASST has been questioned. OBJECTIVE: We compared the autologous plasma skin test (APST) with ASST to detect autoreactivity in patients with CU. The clotting process was investigated as well by measuring in vivo thrombin generation. METHODS: A total of 96 adults with CU underwent ASST; 71 of them underwent APST with Na citrate-anticoagulated plasma. Prothrombin fragment 1+2 plasma levels were measured by a sandwich ELISA in Na citrate-anticoagulated plasmas from 28 patients and 27 controls. RESULTS: Fifty-one of 96 (53%) patients scored positive on ASST, whereas 61 of 71 (86%) patients scored positive on APST (21/30 [70%] ASST-negative and 40/41 [98%] ASST-positive). Plasma prothrombin fragment 1+2 was higher in patients than controls (3.06 [SD 3.36] vs 0.80 [0.34]; P < .001) and in ASST-positive/APST-positive than in ASST-negative/APST-positive patients (3.89 [SD 3.68] vs 1.33 [1.64]; P = 0.058) and was directly related to urticaria severity (r = 0.37; P < .05). CONCLUSION: Most patients with CU are positive on APST-Na citrate. CU is associated with the generation of thrombin, a serine protease able to activate mast cells and to cause relevant increase in permeability of endothelium. APST and ASST only partially depend on the presence of circulating antibodies to FcepsilonRI or to IgE. CLINICAL IMPLICATIONS: These findings provide new insights into the pathogenesis of CU and suggest new therapeutic opportunities for treating this disease. PMID- 16675341 TI - Incidence of parentally reported and clinically diagnosed food hypersensitivity in the first year of life. AB - BACKGROUND: There are very few population-based studies investigating the incidence of food hypersensitivity during the first year of life. OBJECTIVE: To determine the incidence of parentally reported food hypersensitivity and objectively diagnosed food hypersensitivity during the first year of life. METHODS: A birth cohort was recruited (n = 969). At 3, 6, 9, and 12 months, information regarding feeding practices and reported symptoms of atopy were obtained. At 1 year, infants underwent a medical examination and skin prick testing to a battery of allergens. Symptomatic infants underwent food challenges. RESULTS: Adverse reactions to foods were reported by 132 (14.2%) parents at 3, 83 (9.1%) at 6, 49 (5.5%) at 9, and 65 (7.2%) at 12 months. Of the subjects, 1.0% (8/763) were sensitized to aeroallergens and 2.2% (17/763) to food allergens. Between 6 and 9 months and 9 and 12 months, 1.4% (14/969) and 2.8% (27/969) infants were diagnosed with food hypersensitivity on the basis of open food challenges and 0.9% (9/969) and 2.5% (24/969) on the basis of double-blind, placebo-controlled food challenges. Cumulative incidence of food hypersensitivity by 12 months was 4% (39/969; 95% CI, 2.9% to 5.5%) on the basis of open food challenges and 3.2% (31/969; 95% CI, 2.2% to 4.5%) on the basis of double-blind, placebo-controlled food challenges. CONCLUSION: Between 2.2% and 5.5% of infants have food hypersensitivity in the first year of life. The rate of parental perception of food hypersensitivity is higher than the prevalence of atopic sensitization to main food allergens or objectively assessed food hypersensitivity. CLINICAL IMPLICATIONS: In the first year of life, the rate of parentally perceived food hypersensitivity is considerably higher than objectively assessed food hypersensitivity. PMID- 16675343 TI - Toll-like receptor ligands and atopy: a coin with at least two sides. AB - Allergic disease prevalence rates have increased dramatically in affluent countries over the last half century. One proposed explanation is that decreased exposures to microbes caused by modern public health practices has led to deficiencies in an important source of immune education and a consequent increase in the risk of pathogenic immune responses to environmental antigens. Recently, it has become clear that innate responses to microbes are mediated in large part by Toll-like receptors (TLRs), which recognize a diverse family of ligands produced by viruses, bacteria, and fungi. In this perspectives article we will review experimental evidence suggesting that TLRs also play a dominant role in innate responses to noninfectious immunostimulatory materials present in environments of daily living. We will further discuss how ligands for different TLRs can polarize the T(H) bias of adaptive responses in opposing directions. Finally, we will consider how TLRs might contribute to the genesis of atopy and the clinical potential of pharmacologic interventions that target TLRs for the prevention and treatment of allergic diseases. PMID- 16675342 TI - Food antigen causes TH2-dependent enteropathy followed by tissue repair in T-cell receptor transgenic mice. AB - BACKGROUND: Clarification of the mechanisms underlying the development of food sensitive intestinal inflammation will provide an important clue to combating food allergies. OBJECTIVE: To establish a model of intestinal inflammation caused by oral administration of antigen without additional treatments, we focused on the ovalbumin (OVA) 23-3 T-cell receptor transgenic mouse, which had been reported to have high serum antigen-specific IgE responses to the feeding of an egg white diet. METHODS: Changes in body weight of mice fed an egg white diet were monitored throughout the 28-day experimental period. After the 28-day feeding, intestinal tissues were harvested for histologic examination. Endogenous production of cytokines and histamine in the jejunum, and production of cytokines secreted by OVA-specific CD4+ T cells purified from mesenteric lymph nodes, were analyzed. RESULTS: Egg white diet-fed OVA23-3 mice developed weight loss and inflammation with villous atrophy and goblet cell hyperplasia, especially in the jejunum. A further characteristic feature was evidence of weight recovery and tissue repair. Jejunal inflammation was also observed in egg white diet-fed recombination activating gene (RAG)-2-deficient OVA23-3 mice. In addition, tissue sections revealed significant infiltration of specific IgE-positive cells and IgE positive degranulating mast cells. Higher levels of IL-4 and significant levels of histamine were detected in the tissues. In the supernatant of OVA-stimulated T cells, IL-10 levels were also markedly elevated. CONCLUSION: We report that high dose and continuous intake of primitive OVA alone induces enteropathy containing regions under repair in OVA23-3 mice. Antigen-specific T cells and inflammatory cells primed by T(H)2 responses play important roles in regulation of development and improvement of the disease. CLINICAL IMPLICATIONS: Long-term antigen intake causes T(H)2-dependent and food-sensitive enteropathy followed by tissue repair. PMID- 16675344 TI - Human dendritic cells conditioned with Staphylococcus aureus enterotoxin B promote TH2 cell polarization. AB - BACKGROUND: Immune surveillance against microbes at sites of interface with environment involves immediate recognition of pathogen-associated molecular patterns by dendritic cells (DCs). According to their first-line position, DCs are key parameters for the establishment of an appropriate innate and adaptive response against pathogens to avoid disease development. Even though their role in pathogenesis is well known, bacterial toxins have been less examined for their ability to drive DC activation and T-cell polarization. OBJECTIVE: We made the assumption that early conditioning of DCs with Staphylococcus aureus enterotoxins could take part in T-cell polarization. METHODS: Human monocyte-derived DCs were stimulated with S aureus enterotoxin B (SEB) and characterized with respect to secretion of inflammatory cytokines and their ability to drive polarization of naive allogenic T cells. RESULTS: We demonstrated that SEB induced maturation of DCs and that SEB-activated DCs secreted high levels of IL-2 but no IL-12p70, contrary to LPS-activated ones. Accordingly, we further showed that SEB-activated DCs were able to drive polarization of naive T cells into the T(H)2 subset. By using highly purified SEB and Toll-like receptor (TLR)-2 stably transfected Human Embryonic Kidney (HEK) 293 cells, we demonstrated for the first time the ability of SEB to induce TLR2 signaling. Furthermore, the involvement of SEB-TLR2 interaction in activation of dendritic cells was supported by neutralizing activity of anti-TLR2 antibodies. CONCLUSION: Altogether, our findings reinforce the notion that bacterial toxins may appear as new pathogen-associated molecular patterns, which could play a major role in inflammation and bacterial pathologies. PMID- 16675345 TI - Toll-like receptor 2 ligands inhibit TH2 responses to mite allergen. AB - BACKGROUND: There is intense interest in the interaction between microbial compounds and allergy. Although Toll-like receptor (TLR)-2 ligands derived from Gram-positive bacteria alter allergic sensitization in animal models, it is not clear what effect TLR2 ligands have on allergen-specific T-cell memory in human beings. OBJECTIVE: To determine whether in vitro exposure to TLR2 ligands modifies the immune response to house dust mite allergen (HDM). METHODS: Blood mononuclear cells were obtained from individuals both allergic (n = 23) and not allergic (n = 22) to HDM, and stimulated with HDM in the presence or absence of TLR2 ligands. RESULTS: In subjects allergic to HDM, IL-5 and IL-13 responses to HDM were inhibited by heat-killed Staphylococcus aureus, staphylococcal lipoteichoic acid, and the synthetic lipoprotein Pam3CSK4 (P < .005; all stimuli). Although the whole staphylococcal bacteria increased IFN-gamma responses, the purified TLR2 ligands lipoteichoic acid and Pam3CSK4 inhibited HDM specific IFN-gamma synthesis. In contrast, TLR2 ligands had minimal effects on responses to HDM in subjects without allergy. TLR2 ligands induced upregulation of HLA-DR expression but did not inhibit antigen uptake or processing by antigen presenting cells. CONCLUSION: Toll-like receptor 2 ligands inhibit allergen specific T(H)2 responses in sensitized individuals. This effect appears to be mediated by the actions of TLR2 ligands on antigen-presenting cells, and at least for the purified TLR2 ligands does not involve the induction of a strong T(H)1 immune response. CLINICAL IMPLICATIONS: These findings provide an impetus for further preclinical studies examining the potential use of TLR2 ligands in allergic disease. PMID- 16675346 TI - Toll-like receptor agonists differentially regulate cysteinyl-leukotriene receptor 1 expression and function in human dendritic cells. AB - BACKGROUND: Dendritic cells (DCs) acquire, during their maturation, the expression of the chemokine receptor CCR7 and the ability to migrate to lymph nodes in response to CC chemokine ligand 19 (CCL19). This migration is impaired in mice lacking the leukotriene (LT) C4 transporter and restored by addition of exogenous LTC4. OBJECTIVE: To define the role of LT in human DC function, we studied the expression and function of the cysteinyl-leukotriene (CysLT) receptors during DC differentiation from monocytes and subsequent maturation. METHODS: Receptor expression was measured by flow cytometry and real-time PCR. Responsiveness to LTD4 stimulation was assessed by calcium flux and chemotaxis. RESULTS: Maturation of DC with LPS, a classic Toll-like receptor 4 agonist, reduced CysLT receptor 1 (CysLT1) expression by 50%, whereas CysLT receptor 2 expression was increased. In contrast, the Toll-like receptor 3 agonist poly inosinic and cytidylic acid (polyI:C) had no effect on receptor expression. Downregulation of CysLT1 expression by LPS could not be mimicked by TNF-alpha alone or in combination with IL-1beta or IL-6. It was, however, prevented by inhibitors of COX and could be reproduced by a combination of TNF-alpha and prostaglandin E2. Immature DCs and DCs matured with polyI:C, but not with LPS, responded to LTD4 with a robust cytosolic calcium flux, which was prevented by the CysLT1 antagonist montelukast. LTD4 induced DC chemotaxis and enhanced DC migration in response to CCL19 in DCs matured with polyI:C, but only weakly in DCs matured with LPS. CONCLUSION: Our data suggest that human DCs may differentially respond to leukotriene, depending on their maturational stimuli. CLINICAL IMPLICATIONS: Our study demonstrates that some microbial agents can reduce the migration of dendritic cells in response to leukotrienes, with potential for differential involvement of these cells in allergic inflammation. PMID- 16675347 TI - Acute Pseudomonas challenge in cystic fibrosis mice causes prolonged nuclear factor-kappa B activation, cytokine secretion, and persistent lung inflammation. AB - BACKGROUND: Cystic fibrosis (CF) is characterized by an excessive and prolonged inflammatory response to Pseudomonas aeruginosa in the lung. There are high levels of cytokines and chemokines and an exaggerated PMN influx causing significant morbidity and mortality. OBJECTIVE: To compare the kinetics of the inflammatory response with the kinetics of clearance of acute bacterial challenge in the lungs of CF and wild-type (WT) mice. METHODS: We challenged CF knockout (KO) and WT mice intratracheally with P aeruginosa in suspension and evaluated bacteria counts, nuclear factor-kappaB (NF-kappaB), and inhibitor of NF-kappaB alpha protein (I-kappaBalpha) in lung tissue, cytokines, and PMN in bronchoalveolar lavage (BAL). RESULTS: Both groups of mice cleared the infection with the same kinetics. CF-KO mice had more PMN in BAL than WT mice. CF-KO mice had high concentrations of proinflammatory cytokines in BAL on days 2 and 4, whereas cytokines in BAL from WT mice were only slightly elevated. CF-KO mice failed to regenerate I-kappaBalpha once it was degraded, and consequently had prolonged and excessive activation of NF-kappaB for the entire 6-day duration of the study. In contrast, WT mice showed only slight NF-kappaB activation, which plateaued at day 4. CONCLUSION: These data suggest that NF-kappaB is dysregulated in CF lung infection and could be a good target for therapy. Prolonged responses to initial acute infections may contribute to the eventual establishment of chronic persistent inflammation. CLINICAL IMPLICATIONS: Dysregulation of the I kappaB/NF-kappaB pathway in cystic fibrosis leads to prolonged cytokine secretion and persistent inflammation in response to acute challenges and may be important in the development of chronic lung inflammation and infection. PMID- 16675348 TI - Low zone tolerance induced by systemic application of allergens inhibits Tc1 mediated skin inflammation. AB - BACKGROUND: The induction of tolerance may be a promising target of strategies aimed at preventing harmful allergic diseases. Low zone tolerance (LZT), induced by epicutaneous application of low doses of contact allergens, inhibits the development of T(C)1-mediated contact hypersensitivity (CHS). OBJECTIVE: We evaluated the effect of systemic (oral, intravenous) administration of low amounts of haptens on specific immune reactions and tolerance induction. METHODS: By using the mouse model of LZT, we analyzed immune reactions in vivo (skin inflammation) and T-cell responses in vitro after oral, intravenous, or epicutaneous application of low amounts of the contact allergen 2,4,6-trinitro-1 chlorobenzene (TNCB). RESULTS: Subimmunogenic doses of TNCB applied orally and intravenously induced a significant tolerance reaction in vivo comparable to epicutaneously tolerized mice, indicating that LZT is a systemically mediated tolerance reaction. In vitro analysis in all models of LZT revealed the generation of IL-10 secreting, regulatory CD4+ T cells that were absolutely required for the development of hapten-specific CD8+ T(C)2 cells. Adoptive transfer experiments identified CD8+ T(C)2 cells as effector T cells of LZT inhibiting the development of CHS-promoting T(C)1 cells and consequently the manifestation of CHS. These suppressor CD8+ T(C)2 cells were found as well in skin-draining as in mesenteric lymph nodes and in the spleen of tolerized animals independent of the route of tolerization. CONCLUSION: These data indicate that systemic uptake and presentation of small amounts of haptens (eg, contact allergens, drugs, metals) induce the development of LZT and thus prevent inappropriate activation of the immune system and protect from allergic diseases. CLINICAL IMPLICATIONS: These findings will be of particular importance because tolerance induction by protocols applying subimmunogenic, low amounts of haptens may be used as tools for immunotherapy in allergic and autoimmune diseases. PMID- 16675349 TI - Recognition of pollen-derived phosphatidyl-ethanolamine by human CD1d-restricted gamma delta T cells. AB - BACKGROUND: Evidences from mice and human beings indicate that gammadelta T cells could be relevant in recognition of stress-induced self and/or yet unidentified inhaled foreign antigens. Their specificity differs from classic MHC-restricted alphabeta T cells and involves the immunoglobulin-like structure of the gammadelta T-cell receptor with the recognition of small organic molecules, alkylamines, and self lipid compounds presented by CD1+ dendritic cells. OBJECTIVE: Because CD1 receptors are mainly devoted to lipid antigen presentation, we sought to determine whether exogenous pollen membrane lipids may act as allergens for CD1-restricted gammadelta T cells. METHODS: Peripheral blood and nasal mucosa-associated gammadelta T cells were cloned from normal controls and cypress-sensitive subjects and tested for their antigen specificity and CD1 restriction with phospholipids extracted from tree pollen grains, as well with other natural or synthetic compounds. Phospholipid reactivity of cloned gammadelta T cells was measured by mean of proliferative response and cytokine release as well as by testing their helper activity on IgE production in vitro and in vivo. RESULTS: Cloned gammadelta T lymphocytes from subjects with allergy, but not normal controls, were found to recognize pollen-derived phosphatidyl ethanolamine (PE) in a CD1d-restricted fashion. Only 16:0/18:2 and 18:2/18:2 PE were stimulatory, whereas no response was recorded for disaturated PE, phosphatidylcholine, neutral lipids, or protein extract. Proliferating clones secreted both T(H)1-type and T(H)2-type cytokines and drove IgE production in vitro and in vivo. CONCLUSION: CD1d-restricted gammadelta T cells specific for phospholipids can represent a key mucosal regulatory subset for the control of early host reactivity against tree pollens. CLINICAL IMPLICATIONS: By knowing how lipid allergen constituents interact with mucosal immune system, we can expand our possibilities in diagnostic and therapeutic interventions. PMID- 16675351 TI - Dominance of human innate immune responses in primary Francisella tularensis live vaccine strain vaccination. PMID- 16675350 TI - Inflammatory response of the airway to inhaled endotoxin correlates with body mass index in atopic patients with asthma but not in normal volunteers. PMID- 16675352 TI - Parecoxib tolerability in patients with hypersensitivity to nonsteroidal anti inflammatory drugs. PMID- 16675353 TI - The leukotriene C4 synthase (A-444C) promoter polymorphism is associated with the severity of exercise-induced asthma in Korean children. PMID- 16675354 TI - Chlamydia pneumoniae and airway remodeling. PMID- 16675356 TI - Ciclesonide inhalation aerosol for persistent asthma. PMID- 16675358 TI - Mycoplasma in severe asthma. PMID- 16675360 TI - Pebbles in a pond: NIMH grants stimulate improvements in primary care treatment of depression. PMID- 16675361 TI - Mental health and psychiatry training in primary care residency programs. Part I. Who teaches, where, when and how satisfied? AB - OBJECTIVE: Some 40% of patients treated by primary care physicians have significant mental health problems. Only about half eventually receive mental health care, usually by the primary care physicians, often inadequately. Recently, there has been an increased attempt to incorporate psychiatry in primary care training programs. The authors sought to assess the current status of psychiatry training in Internal Medicine (IM), Family Practice (FP), Pediatrics (Peds) and Obstetrics and Gynecology (Ob/Gyn) residency programs. METHOD: All 1365 directors of accredited residency training programs in IM, FP, Ob/Gyn and Peds received a 16-item anonymous questionnaire in 2001-2002, collecting descriptive data concerning their psychiatry training. RESULTS: A great majority of IM (71%), Ob/Gyn (92%) and Peds (85%) training directors felt that the training was minimal or suboptimal, as compared to 41% of FP training directors (P<.001). Sixty-four percent of FP program directors were satisfied with their training (P<.001). In contrast, 54% of other PC program directors were dissatisfied with their psychiatry training. All programs utilized ambulatory care setting extensively. Family Practice programs had more types of mental health teachers, teaching formats and teaching settings (P<.001). A majority of IM (57%) and Peds (70%) residencies desired more psychiatry training in their programs compared to only a third of FP and 40% of Ob/Gyn programs (P<.001). Teaching in clinical settings was preferred by all except Ob/Gyn programs (P<.001). Psychiatry departments contributed more to IM and Peds programs than others. CONCLUSION: A majority of primary care training programs are dissatisfied with the current status of their psychiatric training except for FP programs. Family Practice programs have the most variety in training formats, venues and teachers. There are some specialty-specific differences in perceived needs and desires in psychiatric training. PMID- 16675362 TI - Mental health and psychiatry training in primary care residency programs. Part II. What skills and diagnoses are taught, how adequate, and what affects training directors' satisfaction? AB - OBJECTIVE: The purpose of this study is to describe the psychiatric skills and diagnostic categories taught in primary care training programs, their adequacy, the perceived needs and desires for curriculum enhancement and the factors affecting training directors' satisfaction. METHOD: All 1365 directors of accredited residency training programs in Internal Medicine (IM), Family Practice (FP), Obstetrics and Gynecology (Ob/Gyn), Pediatrics (Peds) and psychiatry received a 16-item anonymous questionnaire about psychiatry training in their program. Responses to the questionnaire to items concerning the skills and diagnostic categories taught, assessment of adequacy of teaching and desires for curriculum enhancement for specific skills and diagnostic categories were analyzed. The factors affecting training directors' satisfaction were explored. RESULTS: Interviewing skills were taught by a majority of all training programs and were considered adequate by 81% of FP and 54% of IM programs, in contrast to less than a majority of Ob/Gyn and Peds programs (P<.001). A majority provided diagnostic interviewing and counseling training, but only FP considered it adequate. A majority taught psychopharmacology and various psychiatric diagnoses, but only in FP did a majority consider them adequate. Both Peds and FP programs teach child psychiatry; significantly, more Peds compared to FP consider their training to be adequate. A vast majority of IM, Ob/Gyn and Peds programs, and 50% of FP programs desired more training in interviewing techniques and diagnostic interview. A majority of all programs desired more counseling and psychopharmacology training and more training in disorders of childhood and adolescence. The overall satisfaction rate for psychiatric training across specialties was 46% (n=657). Sixty-four percent of FP programs were satisfied compared to 31% of non-FP programs. Satisfaction was associated with increased amount of psychiatric training, diversity of training formats, venues, faculty and settings, the amount of contribution to teaching by psychiatry departments and the presence of current teaching in interviewing skills. There were specialty specific differences in factors associated with satisfaction. In general, a smaller size of residency program was associated with satisfaction except in IM, where larger size was associated with satisfaction. Satisfaction was associated with the opinion that primary care physician should be ready and willing to treat more psychiatric conditions. CONCLUSION: Most primary care training programs currently offer training in most psychiatric skills and disorders, but a majority of training directors are dissatisfied with their psychiatry training. There is a difference in the estimation of adequacy concerning training between FP, which consistently rates their teaching to be adequate, and all other primary care programs, which consider their teaching inadequate. This difference may be partly due to actual differences in amount and diversity of training as well as differences in the threshold for satisfaction. A vast majority of primary care training programs desire more training in almost all aspects of psychiatry, and there may be specialty-specific needs and areas of curriculum enhancement. To enhance satisfaction, we should improve the quality as well as the quantity of training, as well as the diversity in training formats, venues and faculty. PMID- 16675363 TI - Watchful waiting for minor depression in primary care: remission rates and predictors of improvement. AB - OBJECTIVES: The objectives of this study were to determine remission rates and predictors of improvement for minor depression following a 1-month watchful waiting period in primary care and to describe the watchful waiting processes. METHODS: Prior to randomization into a clinical trial for minor depression, 111 participants were entered into a 1-month watchful waiting period. Depression severity and predictors of improvement were measured at the start of watchful waiting. At the end of watchful waiting, remission rates were calculated and predictor variables were analyzed for their contribution toward predicting improvement. RESULTS: Remission rates were low, ranging from 9% to 13%, depending on the measure. Avoidant coping style and frequency of engaging in active pleasant events at baseline accounted for the majority of change in depression. During watchful waiting, about one fifth of the sample (21%) had at least one contact with their physician and 27% reported using self-initiated treatments. CONCLUSIONS: There is a low likelihood of spontaneous remission for treatment seeking samples with minor depression in primary care. An avoidant coping style seriously interferes with remission, and engaging in regular active pleasant events confers an advantage. Feasible interventions for primary care that promote activity and decrease avoidant coping styles may improve outcomes. These findings may not generalize to community and non-treatment-seeking samples. PMID- 16675365 TI - Effectiveness of an intervention to reduce sickness absence in patients with emotional distress or minor mental disorders: a randomized controlled effectiveness trial. AB - OBJECTIVE: The purpose of this study was to evaluate the effectiveness of an activating intervention designed to reduce sick leave duration in patients with emotional distress or minor mental disorders. METHOD: In a 1.5-year randomized controlled trial, 194 patients with minor mental disorders received either an experimental intervention by social workers or general practitioners' usual care. The intervention focused on understanding causes, developing and implementing problem-solving strategies and promoting early work resumption. Outcome measures were sick leave duration, mental health and physical health (questionnaires included the Hospital Anxiety and Depression Scale, the Four-Dimensional Symptom Questionnaire and SF-36), all measured at baseline at and 3, 6 and 18 months later. Multilevel analyses were used to evaluate differences between groups. RESULTS: The groups did not differ significantly on any of the outcome measures, except that the experimental group reported higher satisfaction with treatment. CONCLUSION: Although the intervention has benefits, it was not successful at its primary goal (i.e., to reduce sick leave duration in patients with emotional distress or minor mental disorders). Programs aimed at the reduction of sick leave duration may yield better results if targeted at patients with more severe emotional problems than at those with exclusively emotional distress or minor mental disorders, or if delivered by caregivers who are closer to the work environment than are social workers, such as occupational physicians. PMID- 16675364 TI - Posttraumatic stress disorder in primary care one year after the 9/11 attacks. AB - OBJECTIVE: To screen for posttraumatic stress disorder (PTSD) in primary care patients 7-16 months after 9/11 attacks and to examine its comorbidity, clinical presentation and relationships with mental health treatment and service utilization. METHOD: A systematic sample (n=930) of adult primary care patients who were seeking primary care at an urban general medicine clinic were interviewed using the PTSD Checklist: the Primary Care Evaluation of Mental Disorders (PRIME-MD) Patient Health Questionnaire and the Medical Outcome Study 12-Item Short Form Health Survey (SF-12). Health care utilization data were obtained by a cross linkage to the administrative computerized database. RESULTS: Prevalence estimates of current 9/11-related probable PTSD ranged from 4.7% (based on a cutoff PCL-C score of 50 and over) to 10.2% (based on the DSM-IV criteria). A comorbid mental disorder was more common among patients with PTSD than patients without PTSD (80% vs. 30%). Patients with PTSD were more functionally impaired and reported increased use of mental health medication as compared to patients without PTSD (70% vs. 18%). Among patients with PTSD there was no increase in hospital and emergency room (ER) admissions or outpatient care during the first year after the attacks. CONCLUSIONS: In an urban general medicine setting, 1 year after 9/11, the frequency of probable PTSD appears to be common and clinically significant. These results suggest an unmet need for mental health care in this clinical population and are especially important in view of available treatments for PTSD. PMID- 16675366 TI - Review of evidence that posttransplantation psychiatric treatment commonly affects prolactin levels and thereby influences graft fate. AB - Delirium, depression and other psychiatric difficulties are commonly encountered by posttransplantation patients, and antipsychotic medicines are frequently used to treat these difficulties. This article reviews previous research data concerning the immunological effects of these medicines, with particular focus on the consequences of prolactin elevation. Unproven but of concern is that these effects may influence graft fate. Older antipsychotic medicines such as haloperidol and chlorpromazine have a high likelihood of elevating prolactin. Prolactin is an immunologically active molecule generally promoting bone marrow function. This may be of benefit post-stem-cell transplant, helping engraftment, but could further rejection of solid-organ transplants. Elevated prolactin is implicated in the facilitation of graft-versus-host disease. Aripiprazole is the antipsychotic medicine least likely to increase prolactin (and may actually decrease prolactin); risperidone, the most likely to increase prolactin. Olanzapine, quetiapine and ziprazadone are antipsychotic medicines with a lower likelihood of elevating prolactin. Older ("neuroleptic") antipsychotics, such as chlorpromazine, droperidol and haloperidol, perphenazine and many others, are likely to elevate serum prolactin. Among antidepressants, most serotonin reuptake inhibitors, with the exception of sertraline, can slightly elevate prolactin. The atypical (i.e., alone in their class) antidepressants bupropion and mirtazapine are prolactin neutral. The immunological consequences of psychiatric medicines should be considered when treating transplant patients for delirium, depression and thought disorders; in addition, if elevation of prolactin is thought to be of immunological importance during psychiatric treatment, then it should be monitored and treated. The dopamine agonists used to treat Parkinson's disease- bromocriptine, pergolide, pramipexole, ropinerole--usually reverse antipsychotic induced prolactin increases without compromising psychiatric effectiveness. PMID- 16675367 TI - Acute stress disorder and posttraumatic stress disorder symptoms among patients severely injured in motor vehicle accidents in Japan. AB - OBJECTIVE: The prevalence of acute stress disorder (ASD) and posttraumatic stress disorder (PTSD) in seriously injured survivors of motor vehicle accidents (MVAs) in Japan was investigated. Furthermore, predictive factors in the early stage for development of PTSD were evaluated. METHOD: Subjects were consecutive samples (N=100) of patients hospitalized with severe MVA injuries surveyed at two time points: within 1 month after the MVA and then 6 months later. In the first survey, we conducted the Acute Stress Disorder Interview and compiled results of a self-rating questionnaire; in the second survey, we conducted a structured clinical interview via telephone. RESULTS: The prevalence of ASD and PTSD were 9.0% and 8.5%, respectively. The shift from ASD to PTSD was more pronounced when we included partial diagnoses of ASD and PTSD. Three predictive factors for PTSD were identified through multiple logistic analysis: ASD-positive, presence of persistent physical disability and physical injury severity. CONCLUSIONS: Even among severely injured MVA survivors, most acute stress symptoms subside over time. However, having ASD or partial ASD in the early stage, and the presence of physical disability as an aftereffect are strong predictive factors for PTSD. These findings validate the importance of evidence-based intervention for ASD to forestall PTSD. PMID- 16675368 TI - Medical and psychiatric casualties caused by conventional and radiological (dirty) bombs. AB - Bombing is the preferred method of terrorist attack. Unfortunately, the frequency and prevalence of this form of terrorism/criminal act/means of extortion are increasing in the United States. These attacks result in specific physical and psychiatric trauma, and produce both acute and long-term psychiatric sequelae. It will become increasingly important for trauma surgeons, emergency physicians and psychiatrists to be familiar with the special needs and treatment of these patients. This paper reviews the physical and psychiatric consequences of high explosive and radiological (dirty) bombs, and makes recommendations for the psychiatric management of bombing victims. PMID- 16675369 TI - Access of behavioral health patients to prescribing professionals. AB - OBJECTIVE: Advanced practice nurses (APNs), including clinical nurse specialists and nurse practitioners, now have prescribing privileges in all states. This study examined the proportion of Department of Veteran Affairs (VA) mental health patients who were seen by any prescribing professional and specifically the proportion and characteristics of patients who were treated exclusively by APNs. METHOD: Logistic regression models were used to examine data on all patients who received care in VA specialty mental health clinics in 2002 (n=767,920). We first identified patient characteristics independently associated with prescriber contact and, secondly, among those with prescriber contact, exclusive contact with an APN. We also compared characteristics of patients seen exclusively by an APN with those who saw both a physician and an APN. RESULTS: The strongest predictors of both whether a veteran saw a prescriber (66.6%) and whether that prescriber was exclusively an APN (6.7%) were indicators of severity. Specifically, more severe diagnoses, such as schizophrenia or bipolar disorder, receipt of VA disability payments and greater service use increased the odds that a veteran would have had a prescriber contact, decreased the odds that they would see an APN exclusively and, among those who saw an APN, increased the odds that they would also see a physician. CONCLUSIONS: Patients who see physician prescribers have more severe mental health problems than those who see APNs. Our results indicate that APNs and physicians treat distinguishable patient populations, suggesting that APNs may not be substitutes for physicians. PMID- 16675370 TI - Respiratory depression from Symbyax overdose and binge drinking. PMID- 16675371 TI - Pornographic effect of olanzapine: induction of vivid intrusive sexual obsessional images. PMID- 16675372 TI - Psychogenic urinary retention. PMID- 16675373 TI - Mania secondary to right-sided stroke--responsive to olanzapine. PMID- 16675374 TI - Oxcarbazepine treatment for paroxetine-induced restless leg syndrome. PMID- 16675375 TI - Vacations: helping families travel safely. PMID- 16675376 TI - A developmental approach to understanding drawings and narratives from children displaced by Hurricane Katrina. AB - Using art as a process to help children externalize complex feelings can add another layer of assessment in the primary care setting. In the face of trauma, drawing may help children gain symbolic control over events that are confusing and frightening. Through examples of children who were affected by Hurricane Katrina, this article describes the use of drawings and narratives to understand children's experiences related to traumatic displacement. Recommendations include using a developmental lens to understanding children's art, asking children to talk about their drawings, and considering the significance of place for children who have been traumatically displaced. PMID- 16675377 TI - Epilepsy and migraine headache: is there a connection? AB - INTRODUCTION: This study investigated the prevalence of migraine headache in pediatric patients with epilepsy and the incidence of these two disorders in the family history. METHOD: A retrospective chart review was conducted on 475 patients with a primary diagnosis of epilepsy between January 2003 and June 2004. The patients were managed in a pediatric neurology outpatient clinic at a major teaching hospital. Cases were selected using the ICD-9-CM definition for epilepsy. Data collected included age, sex, headache, migraine, and family history. RESULTS: The study revealed a higher prevalence of migraine in epilepsy patients (14.7%) than in the general population (2.7% to 11%). Only 4.7% had a family history of migraine, but 20.6% had a positive family history of epilepsy, supporting current ideas of the genetic etiology of some epilepsies. DISCUSSION: This study specifically addressed epilepsy and migraine in children, but whenever a primary diagnosis is made, the potential for a coexisting disorder should be investigated. PMID- 16675378 TI - Mastering the preceptor role: challenges of clinical teaching. AB - This article aims to help both experienced and new preceptors become more effective teachers while maintaining their clinical workloads. A variety of strategies is essential to increase teaching effectiveness and decrease stress for the busy preceptor who juggles the roles of teacher and clinician. The article will begin with a review of role expectations and role strain factors for student, faculty, and preceptor. Principles of clinical teaching will be identified, followed by some strategies for teaching on busy days and concluding with suggestions for dealing with difficult students. PMID- 16675379 TI - Positive adolescent life skills training for high-risk teens: results of a group intervention study. AB - INTRODUCTION: This study tested the addition of a cognitive-behavioral skill building component called Positive Adolescent Life Skills (PALS) training to an existing intervention for urban adolescents to enhance resiliency. In previous pilot work with the existing intervention, called "Teen Club," it was found that participants in group meetings and intensive case management reported an enhanced ability to connect with positive resources. METHOD: Sixteen adolescents aged 12 to 16 years (10 boys and 6 girls) attending an urban secondary school were randomly assigned to Teen Club or Teen Club plus PALS. Boys and girls met separately in one of the two conditions for 30 weeks. The Problem-Oriented Screening Instrument for Teenagers (POSIT) subscale scores were measured at baseline and at the completion of the program. RESULTS: The sample consisted of 11 Black and five Hispanic teens. Between-group differences in the POSIT subscale scores were not significantly different in this small sample. Group interviews conducted at the conclusion of the intervention revealed that participants found the PALS intervention to be relevant and useful. DISCUSSION: Results suggest that the PALS component strengthened the existing intervention and lend preliminary support for the continuation of this combination of interventions. Future research with larger numbers is needed. PMID- 16675381 TI - Part II: Clinical Practice Guidelines for adolescents and young adults with Down Syndrome: 12 to 21 Years. PMID- 16675380 TI - Educational posters to reduce antibiotic use. AB - INTRODUCTION: Antibiotic overuse promotes resistant strains of bacteria and puts patients at risk for adverse reactions. Given the use of educational posters in government-sponsored public health campaigns, this study examined the effectiveness of a waiting room poster in reducing excessive antibiotic use in clinical practice. METHODS: Investigators conducted a 1-month trial of an educational poster with historical controls using three private pediatric group practices in Westchester County, New York. Children between the ages of 6 months and 10 years at the time of a visit to diagnose and treat symptoms of respiratory illness were enrolled as subjects. Antibiotic prescriptions for children with respiratory illnesses seen during the poster month were compared with prescriptions written during three 1-month historical control periods. The proportion of visits that resulted in a prescription for an antibiotic served as the outcome measure. RESULTS: Overall, 326 of the 720 patients (45.2%) enrolled in the study were treated with an antibiotic. Multiple logistic regression analysis revealed no statistically significant difference in the proportion of visits resulting in an antibiotic prescription among the 4 study months (P = .79), indicating that the educational poster had no effect on antibiotic use. DISCUSSION: Public education in the form of a waiting room poster was not sufficient to decrease antibiotic prescriptions. This finding has implications for current large-scale programs and for health care providers as they continue to attempt to educate patients on the appropriate use of antibiotics. PMID- 16675382 TI - The elusive stomachache. Celiac disease. PMID- 16675383 TI - Unhappy after an appendectomy. Gastric ulcer. PMID- 16675384 TI - Seven strategies for successful surveys. PMID- 16675385 TI - A guide to personal flotation devices and basic open water safety for pediatric health care practitioners. PMID- 16675386 TI - Pten and the brain: sizing up social interaction. AB - In a comprehensive set of experiments in this issue of Neuron, Kwon et al. demonstrate that conditional inactivation of Pten leads to behavioral abnormalities and neuropathological changes. Pten mutants displayed reduced social interaction and heightened responses to sensory stimuli. Additionally, neuronal hypertrophy, as well as macrocephaly was observed. Based on rare human mutations in PTEN and the PI3K pathway, the authors suggest they have produced a potential animal model of autism with macrocephaly. PMID- 16675387 TI - Presynaptic unsilencing: searching for a mechanism. AB - Nascent synaptic networks have a high incidence of silent synapses. In this issue of Neuron, Shen et al. show that a brief burst of action potentials rapidly awaken silent synapses by increasing the availability of synaptic vesicles for fusion through BDNF-triggered presynaptic actin remodeling mediated by the small GTPase Cdc42. PMID- 16675388 TI - Striatal D2 receptors and LTD: yes, but not where you thought they were. AB - D1 and D2 dopamine receptors are expressed in disjoint subsets of striatal projection neurons, the direct and indirect pathways, respectively. This differential distribution of receptors forms the basis for explanations of many aspects of basal ganglia function and dysfunction, but it seems incompatible with some other important properties of striatal neurons. In this issue of Neuron, Wang et al. discover the mechanism of D2 sensitivity of long term depression at synapses on the striatal projection neuron. They show that D2 dependence of LTD does not depend on dopamine receptors of on the projection cell but is mediated by dopamine-induced changes in release of acetylcholine by interneurons that contact projection cells of both types. PMID- 16675389 TI - Keeping time without a clock. AB - The accepted dogma in circadian biology is that the transcription factor CLOCK lies at the heart of the molecular clock that drives behavioral and molecular rhythms. In this issue of Neuron, the generation of CLOCK-deficient mice with only subtle clock defects by DeBruyne et al. shakes up this view of the mammalian clock. PMID- 16675390 TI - The secret life of memories. AB - Recent evidence has challenged the view that memories are made permanent by a consolidation process that happens just once and instead have suggested that memories are "re-consolidated" after reminders. The current findings of Morris et al. in this issue of Neuron suggest that reconsolidation may involve a complex interaction between synaptic and system processing of recent as well as remote experiences. PMID- 16675391 TI - New insights into prion structure and toxicity. AB - Prion diseases in humans and animals are due to conformational conversion of PrP(C), a cellular glycoprotein of unknown function, into PrP(Sc), an isoform that appears to be infectious in the absence of nucleic acids. Proteins that behave as prions are also found in yeast and filamentous fungi. Although there is now strong experimental support for the hypothesis that prions are infectious proteins, two subjects have remained poorly understood: the structure of prions, and the mechanisms by which they kill neurons. In this review, we will highlight recent studies that shed new light on these important issues. PMID- 16675392 TI - gamma-cleavage-independent functions of presenilin, nicastrin, and Aph-1 regulate cell-junction organization and prevent tau toxicity in vivo. AB - Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease. PMID- 16675394 TI - Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN. AB - Axonal growth is fundamental to the establishment of neuronal connectivity in the brain. However, the cell-intrinsic mechanisms that govern axonal morphogenesis remain to be elucidated. The ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) suppresses the growth of axons in postmitotic neurons. Here, we report that Cdh1-APC operates in the nucleus to inhibit axonal growth. We also identify the transcriptional corepressor SnoN as a key target of neuronal Cdh1-APC that promotes axonal growth. Cdh1 forms a physical complex with SnoN and stimulates the ubiquitin-dependent proteasomal degradation of SnoN in neurons. Knockdown of SnoN in neurons significantly reduces axonal growth and suppresses Cdh1 RNAi enhancement of axonal growth. In addition, SnoN knockdown in vivo suggests an essential function for SnoN in the development of granule neuron parallel fibers in the cerebellar cortex. These findings define Cdh1-APC and SnoN as components of a cell-intrinsic pathway that orchestrates axonal morphogenesis in a transcription-dependent manner in the mammalian brain. PMID- 16675393 TI - Pten regulates neuronal arborization and social interaction in mice. AB - CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD. PMID- 16675395 TI - Activity-induced rapid synaptic maturation mediated by presynaptic cdc42 signaling. AB - Maturation of presynaptic transmitter secretion machinery is a critical step in synaptogenesis. Here we report that a brief train of presynaptic action potentials rapidly converts early nonfunctional contacts between cultured hippocampal neurons into functional synapses by enhancing presynaptic glutamate release. The enhanced release was confirmed by a marked increase in the number of depolarization-induced FM4-64 puncta in the presynaptic axon. This rapid presynaptic maturation can be abolished by treatments that interfered with presynaptic BDNF and Cdc42 signaling or actin polymerization. Activation of Cdc42 by applying BDNF or bradykinin mimicked the effect of electrical activity in promoting synaptic maturation. Furthermore, activity-induced increase in presynaptic actin polymerization, as revealed by increased concentration of actin YFP at axon boutons, was abolished by inhibiting BDNF and Cdc42 signaling. Thus, rapid presynaptic maturation induced by neuronal activity is mediated by presynaptic activation of the Cdc42 signaling pathway. PMID- 16675396 TI - Functional maturation of CA1 synapses involves activity-dependent loss of tonic kainate receptor-mediated inhibition of glutamate release. AB - Early in development, excitatory synapses transmit with low efficacy, one mechanism for which is a low probability of transmitter release (Pr). However, little is known about the developmental mechanisms that control activity dependent maturation of the presynaptic release. Here, we show that during early development, transmission at CA3-CA1 synapses is regulated by a high-affinity, G protein-dependent kainate receptor (KAR), which is endogenously activated by ambient glutamate. By tonically depressing glutamate release, this mechanism sets the dynamic properties of neonatal inputs to favor transmission during high frequency bursts of activity, typical for developing neuronal networks. In response to induction of LTP, the tonic activation of KAR is rapidly down regulated, causing an increase in Pr and profoundly changing the dynamic properties of transmission. Early development of the glutamatergic connectivity thus involves an activity-dependent loss of presynaptic KAR function producing maturation in the mode of excitatory transmission from CA3 to CA1. PMID- 16675397 TI - Distance-dependent differences in synapse number and AMPA receptor expression in hippocampal CA1 pyramidal neurons. AB - The ability of synapses throughout the dendritic tree to influence neuronal output is crucial for information processing in the brain. Synaptic potentials attenuate dramatically, however, as they propagate along dendrites toward the soma. To examine whether excitatory axospinous synapses on CA1 pyramidal neurons compensate for their distance from the soma to counteract such dendritic filtering, we evaluated axospinous synapse number and receptor expression in three progressively distal regions: proximal and distal stratum radiatum (SR), and stratum lacunosum-moleculare (SLM). We found that the proportion of perforated synapses increases as a function of distance from the soma and that their AMPAR, but not NMDAR, expression is highest in distal SR and lowest in SLM. Computational models of pyramidal neurons derived from these results suggest that they arise from the compartment-specific use of conductance scaling in SR and dendritic spikes in SLM to minimize the influence of distance on synaptic efficacy. PMID- 16675398 TI - Dopaminergic control of corticostriatal long-term synaptic depression in medium spiny neurons is mediated by cholinergic interneurons. AB - Long-term depression (LTD) of the synapse formed between cortical pyramidal neurons and striatal medium spiny neurons is central to many theories of motor plasticity and associative learning. The induction of LTD at this synapse is thought to depend upon D(2) dopamine receptors localized in the postsynaptic membrane. If this were true, LTD should be inducible in neurons from only one of the two projection systems of the striatum. Using transgenic mice in which neurons that contribute to these two systems are labeled, we show that this is not the case. Rather, in both cell types, the D(2) receptor dependence of LTD induction reflects the need to lower M(1) muscarinic receptor activity-a goal accomplished by D(2) receptors on cholinergic interneurons. In addition to reconciling discordant tracts of the striatal literature, these findings point to cholinergic interneurons as key mediators of dopamine-dependent striatal plasticity and learning. PMID- 16675399 TI - Presynaptic mechanism for slow contrast adaptation in mammalian retinal ganglion cells. AB - Visual neurons, from retina to cortex, adapt slowly to stimulus contrast. Following a switch from high to low contrast, a neuron rapidly decreases its responsiveness and recovers over 5-20 s. Cortical adaptation arises from an intrinsic cellular mechanism: a sodium-dependent potassium conductance that causes prolonged hyperpolarization. Spiking can drive this mechanism, raising the possibility that the same mechanism exists in retinal ganglion cells. We found that adaptation in ganglion cells corresponds to a slowly recovering afterhyperpolarization (AHP), but, unlike in cortical cells, this AHP is not primarily driven by an intrinsic cellular property: spiking was not sufficient to generate adaptation. Adaptation was strongest following spatial stimuli tuned to presynaptic bipolar cells rather than the ganglion cell; it was driven by a reduced excitatory conductance, and it persisted while blocking GABA and glycine receptors, K((Ca)) channels, or mGluRs. Thus, slow adaptation arises from reduced glutamate release from presynaptic (nonspiking) bipolar cells. PMID- 16675400 TI - A clock shock: mouse CLOCK is not required for circadian oscillator function. AB - The circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function. PMID- 16675401 TI - Memory reconsolidation: sensitivity of spatial memory to inhibition of protein synthesis in dorsal hippocampus during encoding and retrieval. AB - Reconsolidation is a putative neuronal process in which the retrieval of a previously consolidated memory returns it to a labile state that is once again subject to stabilization. This study explored the idea that reconsolidation occurs in spatial memory when animals retrieve memory under circumstances in which new memory encoding is likely to occur. Control studies confirmed that intrahippocampal infusions of anisomycin inhibited protein synthesis locally and that the spatial training protocols we used are subject to overnight protein synthesis-dependent consolidation. We then compared the impact of anisomycin in two conditions: when memory retrieval occurred in a reference memory task after performance had reached asymptote over several days; and after a comparable extent of training of a delayed matching-to-place task in which new memory encoding was required each day. Sensitivity to intrahippocampal anisomycin was observed only in the protocol involving new memory encoding at the time of retrieval. PMID- 16675402 TI - Responses of collicular fixation neurons to gaze shift perturbations in head unrestrained monkey reveal gaze feedback control. AB - A prominent hypothesis in motor control is that endpoint errors are minimized because motor commands are updated in real time via internal feedback loops. We investigated in monkey whether orienting saccadic gaze shifts made in the dark with coordinated eye-head movements are controlled by feedback. We recorded from superior colliculus fixation neurons (SCFNs) that fired tonically during fixation and were silent during gaze shifts. When we briefly (0.1 mV, with odds ratios at 2-day lag of 1.53 [95% confidence interval (CI), 1.19-1.97] and 1.11 (95% CI, 1.02-1.20) per 1 microg/m3, respectively. In multipollutant models, where we used indicator elements for sources instead of source-specific PM2.5, only absorbance (elemental carbon), an indicator of local traffic and other combustion, was associated with ST segment depressions. Our results suggest that the PM fraction originating from combustion processes, notably traffic, exacerbates ischemic heart diseases associated with PM mass. PMID- 16675417 TI - Reproduction, embryonic development, and maternal transfer of contaminants in the amphibian Gastrophryne carolinensis. AB - Although many amphibian populations around the world are declining at alarming rates, the cause of most declines remains unknown. Environmental contamination is one of several factors implicated in declines and may have particularly important effects on sensitive developmental stages. Despite the severe effects of maternal transfer of contaminants on early development in other vertebrate lineages, no studies have examined the effects of maternal transfer of contaminants on reproduction or development in amphibians. We examined maternal transfer of contaminants in eastern narrow-mouth toads (Gastrophryne carolinensis) collected from a reference site and near a coal-burning power plant. Adult toads inhabiting the industrial area transferred significant quantities of selenium and strontium to their eggs, but Se concentrations were most notable (up to 100 microg/g dry mass). Compared with the reference site, hatching success was reduced by 11% in clutches from the contaminated site. In surviving larvae, the frequency of developmental abnormalities and abnormal swimming was 55-58% higher in the contaminated site relative to the reference site. Craniofacial abnormalities were nearly an order of magnitude more prevalent in hatchlings from the contaminated site. When all developmental criteria were considered collectively, offspring from the contaminated site experienced 19% lower viability. Although there was no statistical relationship between the concentration of Se or Sr transferred to eggs and any measure of offspring viability, our study demonstrates that maternal transfer may be an important route of contaminant exposure in amphibians that has been overlooked. PMID- 16675418 TI - Chlorpyrifos affects phenotypic outcomes in a model of mammalian neurodevelopment: critical stages targeting differentiation in PC12 cells. AB - The organophosphate insecticide chlorpyrifos (CPF) adversely affects mammalian brain development through multiple mechanisms. To determine if CPF directly affects neuronal cell replication and phenotypic fate, and to identify the vulnerable stages of differentiation, we exposed PC12 cells, a model for mammalian neurodevelopment, to CPF concentrations spanning the threshold for cholinesterase inhibition (5-50 microM) and conducted evaluations during mitosis and in early and mid-differentiation. In undifferentiated cells, exposure to 5 microM CPF for 1-3 days reduced DNA synthesis significantly without eliciting cytotoxicity. At the same time, CPF increased the expression of tyrosine hydroxylase (TH), the enzymatic marker for the catecholamine phenotype, without affecting choline acetyltransferase (ChAT), the corresponding marker for the cholinergic phenotype. Upon exposure to nerve growth factor (NGF), PC12 cells developed neuritic projections in association with vastly increased TH and ChAT expression accompanying differentiation into the two phenotypes. CPF exposure begun at the start of differentiation significantly reduced ChAT but not TH activity. In contrast, when CPF was added in mid-differentiation (4 days of NGF pretreatment), ChAT was unaffected and TH was increased slightly. Thus, CPF exerts stage-specific effects, reducing DNA synthesis in the undifferentiated state, impairing development of the cholinergic phenotype at the start of differentiation, and promoting expression of the catecholaminergic phenotype both in undifferentiated and differentiated cells. CPF administration in vivo produces deficits in the number of neurons and cholinergic function, and because we were able to reproduce these effects in vitro, our results suggest that CPF directly influences the phenotypic fate of neuronal precursors. PMID- 16675419 TI - Elevated levels of urinary 8-hydroxy-2 -deoxyguanosine, lymphocytic micronuclei, and serum glutathione S-transferase in workers exposed to coke oven emissions. AB - To investigate associations among occupational exposure to coke oven emissions (COEs), oxidative stress, cytogenotoxic effects, change in the metabolizing enzyme glutathione S-transferase (GST), and internal levels of polycyclic aromatic hydrocarbons (PAHs) in coke oven workers, we recruited 47 male coke oven workers and 31 male control subjects from a coke oven plant in northern China. We measured the levels of 1-hydroxypyrene (1-OHP) and 8-hydroxy-2 -deoxyguanosine (8 OHdG) in urine, micronucleated binucleated cells (BNMNs) in peripheral blood lymphocyte, and GST in serum. Our results showed that the group exposed to COEs had significantly increased levels of 1-OHP [median 5.7; interquartile range (IQR), 1.4-12.0 micromol/mol creatinine] compared with the control group (3; 0.5 6.4 micromol/mol creatinine). In addition, the median levels (IQR) of 8-OHdG, BNMNs, and GST were markedly increased in the exposed [1.9 (1.4-15.4) micromol/mol creatinine; 6 (2-8) per thousand ; 22.1 (14.9-31.2) U/L, respectively] compared with controls [1.3 (1.0-4.0) micromol/mol creatinine, 2 (0 4) per thousand; and 13.1 (9.5-16.7) U/L, respectively]. These results appeared to be modified by smoking. However, multivariate logistic regression analysis revealed that exposure to COEs had the highest odds ratio among variables analyzed and that smoking was not a significant confounder of the levels of studied biomarkers. Overall, the present findings suggest that COE exposure led to increased internal PAH burden, genetic damage, oxidative stress, and GST activity. The consequences of the changes in these biomarkers, such as risk of cancer, warrant further investigations. PMID- 16675420 TI - Weather variability, tides, and Barmah Forest virus disease in the Gladstone region, Australia. AB - In this study we examined the impact of weather variability and tides on the transmission of Barmah Forest virus (BFV) disease and developed a weather-based forecasting model for BFV disease in the Gladstone region, Australia. We used seasonal autoregressive integrated moving-average (SARIMA) models to determine the contribution of weather variables to BFV transmission after the time-series data of response and explanatory variables were made stationary through seasonal differencing. We obtained data on the monthly counts of BFV cases, weather variables (e.g., mean minimum and maximum temperature, total rainfall, and mean relative humidity), high and low tides, and the population size in the Gladstone region between January 1992 and December 2001 from the Queensland Department of Health, Australian Bureau of Meteorology, Queensland Department of Transport, and Australian Bureau of Statistics, respectively. The SARIMA model shows that the 5 month moving average of minimum temperature (b=0.15, p-value<0.001) was statistically significantly and positively associated with BFV disease, whereas high tide in the current month (b=-1.03, p-value=0.04) was statistically significantly and inversely associated with it. However, no significant association was found for other variables. These results may be applied to forecast the occurrence of BFV disease and to use public health resources in BFV control and prevention. PMID- 16675421 TI - Comparison of oxidative properties, light absorbance, total and elemental mass concentration of ambient PM2.5 collected at 20 European sites. AB - OBJECTIVE: It has been proposed that the redox activity of particles may represent a major determinant of their toxicity. We measured the in vitro ability of ambient fine particles [particulate matter with aerodynamic diameters64 years of age) admitted to the hospital because of respiratory diseases. Generalized linear models were adopted with natural cubic splines to control for season and linear terms to control for weather. Analyses were carried out for the entire period, as well as for burning and nonburning periods. Additional models were built using three factors obtained from factor analysis instead of particles or tracer elements. Increases of 10.2 microg/m3 in particles50% of the world's population. The global burden of disease attributed to indoor air pollution from biomass combustion accounts for approximately 3% of worldwide disability-adjusted life-years lost. This is due to pneumonia in children and chronic obstructive pulmonary disease and lung cancer in women. CASE PRESENTATION: A 53-year-old man from Mexico was referred to the pulmonary clinic for evaluation of chronic productive cough and pulmonary nodules. In his youth, he worked at a charcoal plant in Mexico, where he burned wood and was exposed to massive amounts of smoke. His evaluation revealed thickened bronchovascular bundles with nodules on thoracic computed tomography, dark black plaques in large airways on bronchoscopy, and carbon-laden macrophages and fibrotic scars on lung biopsy. DISCUSSION: The patient was diagnosed with "hut lung," a term that refers to the noninfectious, nonmalignant respiratory manifestations of chronic, high-level exposures to biomass smoke. This is the first reported case of hut lung associated with charcoal production. This case highlights that histopathologic abnormalities of the lung parenchyma may be present in patients with only mild symptoms and that clinical progression is likely a function of both the duration and intensity of exposure. RELEVANCE TO CLINICAL PRACTICE: As residents of lesser developed countries continue to be exposed to high levels of biomass smoke at work or at home and continue to immigrate to developed countries, it is important that health care providers in developed countries be aware of biomass-smoke related pulmonary disease. PMID- 16675434 TI - Case report: hexachloroethane smoke inhalation: a rare cause of severe hepatic injuries. AB - CONTEXT: We report on two patients, a 23-year-old man and a 24-year-old man, who had chemical pneumonitis and respiratory distress after inhaling hexachloroethane/zinc oxide (HC/ZnO) smoke during military training. CASE PRESENTATION: The patients had been healthy previously and denied any history of alcohol or drug abuse. Hematologic tests revealed leukocytosis with neutrophils predominant. The respiratory conditions of both patients improved after steroid therapy and oxygen support, but deterioration of liver function was found. The laboratory results showed that alanine aminotransferase (ALT) and gamma-glutamyl transpeptidase levels were elevated about 1.5-fold the normal limits and that aspartate aminotransferase (AST) levels were marginally elevated. The elevation of liver aminotransferase started from day 1 and day 2 and peaked from day 18 to day 22. ALT/AST levels then returned to normal in 6 weeks. Common viral hepatitis was ruled out after serologic tests. Abdominal sonography and physical examination failed to show any specific findings. DISCUSSION: The hepatotoxic effect was attributed to inhalation of high-concentration HC/ZnO smoke in an enclosed area, where several hepatotoxicants, including ZnCl2, HC, and chlorinated vapors, could have been generated and mixed in the smoke. RELEVANCE TO CLINICAL PRACTICE: These case reports elaborate the hepatic effects that may occur in addition to pulmonary effects of HC/ZnO smoke. PMID- 16675435 TI - Traffic, susceptibility, and childhood asthma. AB - Results from studies of traffic and childhood asthma have been inconsistent, but there has been little systematic evaluation of susceptible subgroups. In this study, we examined the relationship of local traffic-related exposure and asthma and wheeze in southern California school children (5-7 years of age). Lifetime history of doctor-diagnosed asthma and prevalent asthma and wheeze were evaluated by questionnaire. Parental history of asthma and child's history of allergic symptoms, sex, and early-life exposure (residence at the same home since 2 years of age) were examined as susceptibility factors. Residential exposure was assessed by proximity to a major road and by modeling exposure to local traffic related pollutants. Residence within 75 m of a major road was associated with an increased risk of lifetime asthma [odds ratio (OR)=1.29; 95% confidence interval (CI), 1.01-1.86], prevalent asthma (OR=1.50; 95% CI, 1.16-1.95), and wheeze (OR=1.40; 95% CI, 1.09-1.78). Susceptibility increased in long-term residents with no parental history of asthma for lifetime asthma (OR=1.85; 95% CI, 1.11 3.09), prevalent asthma (OR=2.46; 95% CI, 0.48-4.09), and recent wheeze (OR=2.74; 95% CI, 1.71-4.39). The higher risk of asthma near a major road decreased to background rates at 150-200 m from the road. In children with a parental history of asthma and in children moving to the residence after 2 years of age, there was no increased risk associated with exposure. Effect of residential proximity to roadways was also larger in girls. A similar pattern of effects was observed with traffic-modeled exposure. These results indicate that residence near a major road is associated with asthma. The reason for larger effects in those with no parental history of asthma merits further investigation. PMID- 16675436 TI - Effects of prenatal exposure to polychlorinated biphenyls and dioxins on mental and motor development in Japanese children at 6 months of age. AB - Several studies have shown that prenatal and/or postnatal background-level exposure to environmental chemicals, such as polychlorinated biphenyls (PCBs) and dioxins, induces adverse effects on the neurodevelopment of children. However, other studies have not detected any harmful influences on neurodevelopment. Furthermore, except in western countries, no developmental tests have been carried out in relation to detailed assessment of exposure to PCBs and dioxins. In this study (the Hokkaido Study on Environment and Children's Health), the effect of prenatal exposure to background levels of PCBs and dioxins on infant neurodevelopment in Japan/Sapporo was elucidated. The associations between the total or individual isomer level of PCBs and dioxins in 134 Japanese pregnant women's peripheral blood and the mental or motor development of their 6-month-old infants were evaluated using the second edition of the Bayley Scales of Infant Development. The mean level of total toxicity equivalency quantity (TEQ) was 18.8 (4.0-51.2) pg/g lipid in blood of 134 mothers. After adjustment for potential confounding variables, the total TEQ value was shown not to be significantly associated with mental developmental index (MDI) or psychomotor developmental index (PDI). However, the levels of one polychlorinated dibenzo-p-dioxin (PCDD) isomer, total PCDDs, and total PCDDs/polychlorinated dibenzofurans (PCDFs) were significantly negatively associated with MDI, and the levels of two PCDD isomers and three PCDF isomers were significantly negatively associated with the PDI. In conclusion, the background-level exposure of several isomers of dioxins during the prenatal period probably affects the motor development of 6-month-old infants more than it does their mental development. PMID- 16675437 TI - Prenatal exposure to polychlorinated biphenyls and postnatal growth: a structural analysis. AB - Normal endocrine function in utero and early in childhood influences later height and weight attainment. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants with suspected endocrine-disrupting properties. PCBs may mimic or inhibit hormone and endocrine processes based in part on their structural configuration, with non-ortho-substituted PCBs having a coplanar orientation and ortho-substituted PCBs becoming increasingly noncoplanar. Coplanar and noncoplanar PCBs have known differences in biologic effect. Animal studies link prenatal PCB exposure to adverse birth and early-life growth outcomes, but epidemiologic studies are conflicting. We examined whether prenatal exposure to PCBs, categorized by their degree of ortho-substitution, affected childhood height and weight attainment in 150 children (109 boys and 41 girls) with African-American mothers born at the Columbia-Presbyterian Hospital from 1959 through 1962. Stratifying by sex, we used regression models for repeated measures to investigate associations between maternal levels of PCBs and height and weight through 17 years of age. Maternal levels of ortho-substituted PCBs were associated with reduced weight through 17 years of age among girls but not among boys. Tri-ortho-substituted PCBs were marginally associated with increased height in boys. Although limited by sample size, our results suggest that prenatal exposure to PCBs may affect growth, especially in girls, and that ortho substitution is an important determinant of its effect on growth. PMID- 16675438 TI - Fine particulate matter (PM2.5) air pollution and selected causes of postneonatal infant mortality in California. AB - Studies suggest that airborne particulate matter (PM) may be associated with postneonatal infant mortality, particularly with respiratory causes and sudden infant death syndrome (SIDS). To further explore this issue, we examined the relationship between long-term exposure to fine PM air pollution and postneonatal infant mortality in California. We linked monitoring data for PM5 microg/L (n=50,326) to newborns from 287 communities with average concentrations5 microg/L were observed, with 20.4 expected [adjusted prevalence odds ratio (POR)=0.71; 95% confidence interval (CI), 0.40-1.19]. Although only 36% of all California newborns were screened before 24 hr of age in 1998, nearly 80% of newborns with high TSH were screened before 24 hr of age. Because of the physiologic postnatal surge of TSH, the results for newborns screened before 24 hr were uninformative for assessing an environmental impact. For newborns screened>or=24 hr, the adjusted POR for high TSH was 0.73 (95% CI, 0.40-1.23). All adjusted odds ratios (ORs) were controlled for sex, ethnicity, birth weight, and multiple birth status. Using an assessment of ClO4- in drinking water based on available data, we did not observe an association between estimated average ClO4- concentrations>5 microg/L in drinking water supplies and the prevalence of clinically diagnosed PCH or high TSH concentrations. PMID- 16675441 TI - Distant HNF1 site as a master control for the human class I alcohol dehydrogenase gene expression. AB - Gene duplication and divergence have contributed to the biochemical diversity of the alcohol dehydrogenase (ADH) family. Class I ADH is the major enzyme that catalyzes alcohol to acetaldehyde in the liver. To investigate the mechanism(s) controlling tissue-specific and temporal regulation of the three human class I ADH genes (ADH1A, ADH1B, and ADH1C), we compared genomic sequences for the human and mouse ADH loci and analyzed human ADH gene expression in BAC transgenic mice carrying different lengths of the upstream sequences of the class I ADH. A conserved noncoding sequence, located between the class I and class IV ADH (ADH7) genes, was found to be essential for directing class I ADH gene expression in fetal and adult livers. Within this region, a 275-bp fragment displaying liver specific DNase I hypersensitivity was bound by HNF1. The HNF1-containing upstream sequence enhanced all three class I ADH promoters in an orientation-dependent manner, and the transcriptional activation depended on binding to the HNF1 site. Deletion of the conserved HNF1 site in the BAC led to the shutdown of human class I ADH gene expression in the transgenic livers, leaving ADH1C gene expression in the stomach unchanged. Moreover, interaction between the upstream element and the class I ADH gene promoters was demonstrated by chromosome conformation capture, suggesting a DNA looping mechanism is involved in gene activation. Taken together, our data indicate that HNF1 binding, at approximately 51 kb upstream, plays a master role in controlling human class I ADH gene expression and may govern alcohol metabolism in the liver. PMID- 16675442 TI - Folding and fibril formation of the cell cycle protein Cks1. AB - The Saccharomyces cerevisiae Cks protein Cks1 has a COOH-terminal glutamine-rich sequence not present in other homologues. Cks proteins domain swap to form dimers but unique to Cks1 is the anti-parallel arrangement of protomers within the dimer. Despite the differences in Cks1 compared with other Cks proteins, we find the domain swapping properties are very similar. However, aggregation of Cks1 occurs by a route distinct from the other Cks proteins studied to date. Cks1 formed fibrillar aggregates at room temperature and neutral pH. During this process, Cks1 underwent proteolytic cleavage at a trypsin-like site into two fragments, the globular Cks domain and the glutamine-rich COOH terminus. At high protein concentrations, the rate of fibril formation was the same as the rate of proteolysis. The dominant species present within the fibrils was the glutamine rich sequence. Consistent with this result, fibril formation was enhanced by addition of trypsin. Moreover, a truncated variant lacking the glutamine-rich sequence did not form fibrils under the same conditions. A lag phase at low protein concentrations indicates that fibril formation occurs through a nucleation and growth mechanism. The aggregates appear to resemble amyloid fibrils, in that they show the typical cross-beta x-ray diffraction pattern. Moreover, infrared spectroscopy data indicate that the glutamine side chains are hydrogen-bonded along the axis of the fibril. Our results indicate that the proteolytic reaction is the crucial step initiating aggregation and demonstrate that Cks1 is a simple, tunable model system for exploring aggregation mechanisms associated with polyglutamine deposition diseases. PMID- 16675443 TI - Interleukin-4 induces 15-lipoxygenase-1 expression in human orbital fibroblasts from patients with Graves disease. Evidence for anatomic site-selective actions of Th2 cytokines. AB - Orbital fibroblasts orchestrate tissue remodeling in Graves disease, at least in part, because they exhibit exaggerated responses to proinflammatory cytokines. A hallmark of late stage orbital disease is vision-threatening fibrosis, the molecular basis of which remains uncertain. We report here that the Th2 cytokines, interleukin (IL)-4 and IL-13, can induce in these cells the expression of 15-lipoxygenase-1 (15-LOX-1) and in so doing up-regulate the production of 15 hydroxyeicosatetraenoic acid. IL-4 increases 15-LOX-1 protein levels through pretranslational actions. The increased steady-state 15-LOX-1 mRNA is independent of ongoing protein synthesis and involves very modestly increased gene promoter activity. Importantly, IL-4 substantially enhances 15-LOX-1 transcript stability, activity that localizes to a 293-bp sequence of the 3'-untranslated region. IL-4 activates Jak2 in orbital fibroblasts. Interrupting signaling through that pathway, either with the specific chemical inhibitor, AG490, or by transiently transfecting the cells with a Jak2 dominant negative mutant kinase, attenuates the 15-LOX-1 induction. Interferongamma, a Th1 cytokine, could block this induction by attenuating IL-4-dependent mRNA stabilization. 15-LOX-1 protein and its mRNA were undetectable in IL-4-treated dermal fibroblasts, despite comparable levels of cell surface IL-4 receptor and phosphorylated Jak2 and STAT6. Our findings suggest that orbital connective tissues may represent a site of localized 15-hydroxyeicosatetraenoic acid generation resulting from cell type specific 15-LOX-1 mRNA stabilization by IL-4. These results may have relevance to the pathogenesis of orbital Graves disease, an inflammatory autoimmune condition that gives way to extensive fibrosis associated with a Th2 response. PMID- 16675444 TI - A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability. AB - Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-containing RNAs must be exported from the nucleus where they are normally retained, it must be ensured that the unspliced HIV-1 RNA is actively exported from the nucleus and protected from degradation by processes such as nonsense-mediated decay. Here we report the identification of a novel 178-nt-long exon located in the gag-pol gene of HIV-1 and its inclusion in at least two different mRNA species. Although efficiently spliced in vitro, this exon appears to be tightly repressed and infrequently used in vivo. The splicing is activated or repressed in vitro by the splicing factors ASF/SF2 and heterogeneous nuclear ribonucleoprotein A1, respectively, suggesting that splicing is controlled by these factors. Interestingly, mutations in the 5' splice site resulted in a dramatic reduction in the steady-state level of HIV-1 RNA, and this effect was partially reversed by expression of U1 small nuclear RNA harboring the compensatory mutation. This implies that U1 small nuclear RNA binding to optimal but non-functional splice sites might have a role in protecting unspliced HIV-1 mRNA from degradation. PMID- 16675445 TI - Functional analysis of H2B-Lys-123 ubiquitination in regulation of H3-Lys-4 methylation and recruitment of RNA polymerase II at the coding sequences of several active genes in vivo. AB - Previous biochemical studies have demonstrated that Lys-123 ubiquitination of histone H2B is globally required for up-regulation of mono-, di, and trimethylation of Lys-4 of histone H3. However, recent studies have implicated H2B-Lys-123 ubiquitination in the regulation of di- and trimethylation, but not monomethylation, of H3-Lys-4 in vivo. Using a formaldehyde-based cross-linking and chromatin immunoprecipitation assay, we show that H3-Lys-4 trimethylation, but not dimethylation, is up-regulated by H2B-Lys-123 ubiquitination in vivo at the coding sequences of a set of transcriptionally active genes such as ADH1, PHO84, and PYK1. Both the ubiquitination of H2B-Lys-123 and the methylation of H3 Lys-4 are dispensable for recruitment of RNA polymerase II to the coding sequences of these genes, and hence, their transcription is not altered in the absence of these covalent modifications. However, recruitment of RNA polymerase II to the coding sequence of a galactose-inducible gene, GAL1, is significantly reduced in the absence of H2B-Lys-123 ubiquitination but not H3-Lys-4 methylation. Consistently, transcription of GAL1 is altered in the H2B-K123R point mutant strain. Finally, we show that H3-Lys-4 methylation does not regulate H3-Lys-9/14 acetylation. Collectively, our data reveal a "trans-tail" regulation of H3-Lys-4 tri- but not dimethylation by H2B-Lys-123 ubiquitination, and these modifications are dispensable for transcription of a certain set of genes in vivo. PMID- 16675446 TI - Annexin I regulates SKCO-15 cell invasion by signaling through formyl peptide receptors. AB - Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by approximately 2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing. PMID- 16675447 TI - Nup214-Nup88 nucleoporin subcomplex is required for CRM1-mediated 60 S preribosomal nuclear export. AB - The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88. PMID- 16675448 TI - TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis. AB - Transforming growth factor beta-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF kappaB in several cell types. However, the functional role of TAK1 in an in vivo setting has not been determined. Here we have demonstrated that TAK1 is the major regulator of skin inflammation as well as keratinocyte death in vivo. Epidermal specific deletion of TAK1 causes a severe inflammatory skin condition by postnatal day 6-8. The mutant skin also exhibits massive keratinocyte death. Analysis of keratinocytes isolated from the mutant skin revealed that TAK1 deficiency results in a striking increase in apoptosis in response to tumor necrosis factor (TNF). TAK1-deficient keratinocytes cannot activate NF-kappaB or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-kappaB (and possibly JNK)-mediated cell survival signaling. Finally, we have shown that deletion of the TNF receptor can largely rescue keratinocyte death as well as inflammatory skin condition in epidermal-specific TAK1-deficient mice. Our results demonstrate that TAK1 is a master regulator of TNF signaling in skin and regulates skin inflammation and keratinocyte death. PMID- 16675449 TI - Conformational stability and disassembly of Norwalk virus-like particles. Effect of pH and temperature. AB - Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NV-VLPs are highly stable over a pH range of 3-7 and up to 55 degrees C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60 degrees C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner. PMID- 16675450 TI - High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI. AB - Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum. PMID- 16675451 TI - Recoverin binds exclusively to an amphipathic peptide at the N terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase. AB - Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate. PMID- 16675452 TI - Post-transcriptional control of Cited2 by transforming growth factor beta. Regulation via Smads and Cited2 coding region. AB - Cited2 is a transcription factor without typical DNA binding domains. Cited2 interacts with cAMP-responsive element-binding protein-binding protein (CBP)/p300, TFAP2, Lhx2, and nuclear receptors, such as peroxisome proliferator activated receptor and estrogen receptor to function as a transcriptional modulator. Overexpression of Cited2 in Rat1 cells leads to tumor formation in nude mice, suggesting that Cited2 is a transforming gene. Through microarray analysis, Cited2 was found to be down-regulated by transforming growth factor beta1 (TGF-beta) in various cell lines. In this study, we confirmed that both mRNA and protein levels of Cited2 are down-regulated in MDA-MB-231 breast cancer cells. Overexpression of Smad7 or knockdown of Smad4 in MDA-MB-231 cells showed that the Smad pathway is involved in the down-regulation of Cited2. Based on nuclear run-on analysis and Cited2 promoter/reporter assay, Cited2 transcription was not affected by TGF-beta, supporting that down-regulation of Cited2 by TGF beta is most likely through post-transcriptional regulation. By using transcriptional inhibitors, we demonstrated that the turnover of Cited2 transcripts appears to be accelerated during TGF-beta stimulation. Pharmacologic inhibition of translation with cycloheximide attenuated Cited2 down-regulation by TGF-beta. We examined the expression of recombinant Cited2 gene introduced into MDA-MB-231 cells by stable transfection, and we found that mRNA containing the Cited2 protein-coding region controlled by a heterologous promoter indeed responds to TGF-beta-mediated down-regulation. Study from Cited2 deletion mutants showed that the C-terminal conserved region of Cited2 coding sequence is essential for the down-regulation. This is the first demonstration that TGF-beta mediated down-regulation of Cited2 is post-transcriptional, through the Smad pathway, and requires the presence of its coding sequence. PMID- 16675453 TI - MicroRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts. AB - A large number of studies have demonstrated that the expression of the angiotensin II type 1 receptor (AT(1)R) is regulated predominantly by post transcriptional mechanisms. Recently, it has been suggested that 10% of human genes may be regulated, in part, by a novel post-transcriptional mechanism involving microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression primarily through translational repression. The aim of this study was to determine whether miRNAs could regulate human AT(1)R expression. Luciferase reporter assays demonstrated that miR-155 could directly interact with the 3' untranslated region of the hAT(1)R mRNA. Functional studies demonstrated that transfection of miR-155 into human primary lung fibroblasts (hPFBs) reduced the endogenous expression of the hAT(1)R compared with non-transfected cells. Additionally, miR-155 transfected cells showed a significant reduction in angiotensin II-induced extracellular signal-related kinase 1/2 (ERK1/2) activation. Furthermore, when hPFBs were transfected with an antisense miR-155 inhibitor, anti-miR-155, endogenous hAT(1)R expression and angiotensin II-induced ERK1/2 activation were significantly increased. Finally, transforming growth factor-beta(1) treatment of hPFBs resulted in the decreased expression of miR-155 and the increased expression of the hAT(1)R. In summary, our studies suggest that miR-155 can bind to the 3'-untranslated region (UTR) of hAT(1)R mRNAs and translationally repress the expression of this protein in vivo. Importantly, the translational repression mediated by miR-155 can be regulated by physiological stimuli. PMID- 16675454 TI - The human major histocompatibility complex class II HLA-DRB1 and HLA-DQA1 genes are separated by a CTCF-binding enhancer-blocking element. AB - The human major histocompatibility complex class II (MHC-II) region encodes a cluster of polymorphic heterodimeric glycoproteins HLA-DR, -DQ, and -DP that functions in antigen presentation. Separated by approximately 44 kb of DNA, the HLA-DRB1 and HLA-DQA1 encode MHC-II proteins that function in separate MHC-II heterodimers and are diametrically transcribed. A region of high acetylation located in the intergenic sequences between HLA-DRB1 and HLA-DQA1 was discovered and termed XL9. The peak of acetylation coincided with sequences that bound the insulator protein CCCTC-binding factor as determined by chromatin immunoprecipitations and in vitro DNA binding studies. XL9 was also found to be associated with the nuclear matrix. The activity of the XL9 region was examined and found to be a potent enhancer-blocking element. These results suggest that the XL9 region may have evolved to separate the transcriptional units of the HLA DR and HLA-DQ genes. PMID- 16675455 TI - Mg2+ and a key lysine modulate exchange activity of eukaryotic translation elongation factor 1B alpha. AB - To sustain efficient translation, eukaryotic elongation factor B alpha (eEF1B alpha) functions as the guanine nucleotide exchange factor for eEF1A. Stopped flow kinetics using 2'-(or 3')-O-N-methylanthraniloyl (mant)-GDP showed spontaneous release of nucleotide from eEF1A is extremely slow and accelerated 700-fold by eEF1B alpha. The eEF1B alpha-stimulated reaction was inhibited by Mg2+ with a K(1/2) of 3.8 mM. Previous structural studies predicted the Lys-205 residue of eEF1B alpha plays an important role in promoting nucleotide exchange by disrupting the Mg2+ binding site. Co-crystal structures of the lethal K205A mutant in the catalytic C terminus of eEF1B alpha with eEF1A and eEF1A.GDP established that the lethality was not due to a structural defect. Instead, the K205A mutant drastically reduced the nucleotide exchange activity even at very low concentrations of Mg2+. A K205R eEF1B alpha mutant on the other hand was functional in vivo and showed nearly wild-type nucleotide dissociation rates but almost no sensitivity to Mg2+. These results indicate the significant role of Mg2+ in the nucleotide exchange reaction by eEF1B alpha and establish the catalytic function of Lys-205 in displacing Mg2+ from its binding site. PMID- 16675456 TI - Transcriptional profiling of enriched populations of stem cells versus transient amplifying cells. A comparison of limbal and corneal epithelial basal cells. AB - The basal layer of limbal and central corneal epithelium is enriched in stem cells and transient amplifying cells, respectively. This physical separation of stem and transient amplifying cells makes the limbal/corneal epithelium an exceptionally suitable system for isolating basal cells enriched in these two proliferative populations. Prior attempts to isolate epithelial stem cells used methods such as proteolytic tissue dissociation and cell sorting that could potentially alter their gene expression profile. Using laser capture microdissection, we were able to isolate resting limbal and corneal basal cells from frozen sections with minimal tissue processing, thereby improving the yield and quality of RNA. Analyses of RNA isolated from 300 limbal and corneal basal cells from eight mice revealed a set of approximately 100 genes that are differentially expressed in limbal cells versus corneal epithelial basal cells. Semiquantitative reverse transcription-PCR confirmed the up-regulation of three limbal and three corneal genes. LacZ identification of epiregulin from epiregulin null mice and immunohistochemical staining of wild type mice confirmed that epiregulin, one of the limbal epithelium-enriched genes, was associated with the limbal epithelial basal cells. Within the limbal and corneal basal cells, we detected previously unknown genes that were differentially expressed in these two regions that contribute further to our understanding of the unique heterogeneity of these two closely related basal cell populations. Our findings indicate that we can obtain accurate gene expression profiles of the stem cell-enriched limbal basal cell population in their "natural" quiescent state. PMID- 16675457 TI - Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase. AB - The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp 392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu. PMID- 16675458 TI - Up-regulation of the fidelity of human DNA polymerase lambda by its non-enzymatic proline-rich domain. AB - DNA repair pathways are essential for maintaining genome stability. DNA polymerase beta plays a critical role in base-excision repair in vivo. DNA polymerase lambda, a recently identified X-family homolog of DNA polymerase beta, is hypothesized to be a second polymerase involved in base-excision repair. The full-length DNA polymerase lambda is comprised of three domains: a C-terminal DNA polymerase beta-like domain, an N-terminal BRCA1 C-terminal domain, and a previously uncharacterized proline-rich domain. Strikingly, pre-steady-state kinetic analyses reveal that, although human DNA polymerase lambda has almost identical fidelity to human DNA polymerase beta, the C-terminal DNA polymerase beta-like domain alone displays a dramatic, up to 100-fold loss in fidelity. We further demonstrate that the non-enzymatic proline-rich domain confers the increase in fidelity of DNA polymerase lambda by significantly lowering incorporation rate constants of incorrect nucleotides. Our studies illustrate a novel mechanism, in which the DNA polymerase fidelity is controlled not by an accessory protein or a proofreading exonuclease domain but by an internal regulatory domain. PMID- 16675459 TI - Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex. AB - The mouse Hop2 and Mnd1 proteins, which can form a stable heterodimeric complex, ensure the proper synapsis of homologous chromosomes in meiosis by acting in concert with Rad51 and Dmc1 to promote the strand invasion (D-loop formation) step of homologous recombination. Hop2 alone promotes D-loop formation, but Mnd1 and the Hop2-Mnd1 complex do not. Here we show that only the heterodimer complex, but not the individual proteins, can stimulate strand invasion by Dmc1. Furthermore, we demonstrate that the interaction with Mnd1 provokes changes in Hop2 that are responsible not only for abrogating the recombinase activity of Hop2 but also for generating a new molecular interface able to physically interact with and stimulate Dmc1. We also show that coiled-coil motifs in Hop2 and Mnd1 are essential for their interaction with each other and that a clearly delineated region near the COOH terminus of both proteins is necessary for both the DNA binding and single-strand annealing by the Hop-Mnd1 heterodimer. Finally, we describe a point mutation in Hop2 that dissociates its strand invasion activity from its ability to bind and anneal DNA. PMID- 16675460 TI - A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-alpha in budding yeast. AB - Mcm10 is a conserved eukaryotic DNA replication factor that is required for S phase progression. Recently, Mcm10 has been shown to interact physically with the DNA polymerase-alpha (pol-alpha).primase complex. We show now that Mcm10 is in a complex with pol-alpha throughout the cell cycle. In temperature-sensitive mcm10 1 mutants, depletion of Mcm10 results in degradation of the catalytic subunit of pol-alpha, Cdc17/Pol1, regardless of whether cells are in G(1), S, or G(2) phase. Importantly, Cdc17 protein levels can be restored upon overexpression of exogenous Mcm10 in mcm10-1 mutants that are grown at the nonpermissive temperature. Moreover, overexpressed Cdc17 that is normally subject to rapid degradation is stabilized by Mcm10 co-overexpression but not by co-overexpression of the B-subunit of pol-alpha, Pol12. These results are consistent with Mcm10 having a role as a nuclear chaperone for Cdc17. Mutational analysis indicates that a conserved heat-shock protein 10 (Hsp10)-like domain in Mcm10 is required to prevent the degradation of Cdc17. Substitution of a single residue in the Hsp10-like domain of endogenous Mcm10 results in a dramatic reduction of steady state Cdc17 levels. The high degree of evolutionary conservation of this domain implies that stabilizing Cdc17 may be a conserved function of Mcm10. PMID- 16675461 TI - Identification of the RecR Toprim domain as the binding site for both RecF and RecO. A role of RecR in RecFOR assembly at double-stranded DNA-single-stranded DNA junctions. AB - The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR. PMID- 16675462 TI - Alteration of sequence specificity of the type II restriction endonuclease HincII through an indirect readout mechanism. AB - The functional and structural consequences of a mutation of the DNA intercalating residue of HincII, Q138F, are presented. Modeling has suggested that the DNA intercalation by Gln-138 results in DNA distortions potentially used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47). Kinetic data presented here indicate that the mutation of glutamine 138 to phenylalanine (Q138F) results in a change in sequence specificity at the center two base pairs of the cognate recognition site. We show that the preference of HincII for cutting, but not binding, the three cognate sites differing in the center two base pairs has been altered by the mutation Q138F. Five new crystal structures are presented including Q138F HincII bound to GTTAAC and GTCGAC both with and without Ca2+ as well as the structure of wild type HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational changes in the protein, bound DNA, and at the protein-DNA interface, consistent with the formation of adaptive complexes. Analysis of these structures and the effect of Ca2+ binding on the protein-DNA interface illuminates the origin of the altered specificity by the mutation Q138F in the HincII enzyme. PMID- 16675463 TI - Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11). AB - Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group 2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and Fc alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism. PMID- 16675464 TI - The peptidylprolyl cis/trans-isomerase Pin1 modulates stress-induced dephosphorylation of Tau in neurons. Implication in a pathological mechanism related to Alzheimer disease. AB - Deregulation of Tau phosphorylation is a key question in Alzheimer disease pathogenesis. Recently, Pin1, a peptidylprolyl cis/trans-isomerase, was proposed to be a new modulator in Tau phosphorylation in Alzheimer disease. In vitro, Pin1 was reported to present a high affinity for both Thr(P)-231, a crucial site for microtubule binding, and Thr(P)-212. In fact, Pin1 may facilitate Thr(P)-231 dephosphorylation by protein phosphatase 2A through trans isomerization of the Thr(P)-Pro peptide bound. However, whether Pin1 binding to Tau leads to isomerization of a single site or of multiple Ser/Thr(P)-Pro sites in vivo is still unknown. In the present study, Pin1 involvement was investigated in stress induced Tau dephosphorylation with protein phosphatase 2A activation. Both oxidative (H2O2) and heat stresses induced hypophosphorylation of a large set of phospho-Tau epitopes in primary cortical cultures. In both cases, juglone, a Pin1 pharmacological inhibitor, partially prevented dephosphorylation of Tau at Thr 231 among a set of phosphoepitopes tested. Moreover, Pin1 is physiologically found in neurons and partially co-localized with Tau. Furthermore, in Pin1 deficient neuronal primary cultures, H2O2 stress-induced Tau dephosphorylation at Thr(P)-231 was significantly lower than in wild type neurons. Finally, Pin1 transfection in Pin1-deficient neuronal cell cultures allowed for rescuing the effect of H2O2 stress-induced Tau dephosphorylation, whereas a Pin1 catalytic mutant did not. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation on the full-length multiphosphorylated substrate. PMID- 16675465 TI - NIK is involved in nucleosomal regulation by enhancing histone H3 phosphorylation by IKKalpha. AB - The exact physiological role of NF-kappaB-inducing kinase (NIK) in the NF-kappaB activation pathway has not been defined, although it is an upstream kinase of IKKalpha. Recent studies have indicated that IKKalpha is a nucleosomal modifier of NF-kappaB signaling. We hypothesized that NIK generates a proximal signal that contributes to IKKalpha modification of nucleosomal structure through phosphorylation of histone H3 and enhancement of target gene expression. By using a chromatin immunoprecipitation assay, our data show that endogenous IKKalpha is recruited to the promoter site of several NF-kappaB-dependent genes in macrophages. Our data show that immunoreactive NIK is rapidly recruited to nuclear compartment in macrophages in response to treatment with endotoxin where it augments phosphorylation of histone H3 by inducing phosphorylation and kinase activity of IKKalpha. A small interfering RNA knockdown of NIK markedly reduces phosphorylation of histone H3 in endotoxin treated macrophages. These data, together, demonstrate a novel role for NIK as a histone H3 modifier, through an accessory pathway from NIK to IKKalpha, that could play an important role in the endotoxin response through modification of nucleosomal structure. PMID- 16675466 TI - Integrated analysis of transcriptomic and proteomic data of Desulfovibrio vulgaris: zero-inflated Poisson regression models to predict abundance of undetected proteins. AB - MOTIVATION: Integrated analysis of global scale transcriptomic and proteomic data can provide important insights into the metabolic mechanisms underlying complex biological systems. However, because the relationship between protein abundance and mRNA expression level is complicated by many cellular and physical processes, sophisticated statistical models need to be developed to capture their relationship. RESULTS: In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), zero-inflated Poisson (ZIP)-based models were proposed to define the correlation pattern between mRNA and protein abundance. In addition, by assuming that there is a probability mass at zero representing unexpressed genes and expressed proteins that were undetected owing to technical limitations, a Potential ZIP model was established. Two significant improvements introduced by this approach are (1) the predicted protein abundance level values for experimentally detected proteins are corrected by considering their mRNA levels and (2) protein abundance values can be predicted for undetected proteins (in the case of this study, approximately 83% of the proteins in the D.vulgaris genome) for better biological interpretation. We demonstrated the use of these statistical models by comparatively analyzing proteomic and microarray results from D.vulgaris grown on lactate-based versus formate-based media. These models correctly predicted increased expression of Ech hydrogenase and decreased expression of Coo hydrogenase for D.vulgaris grown on formate. PMID- 16675467 TI - A mixture model with random-effects components for clustering correlated gene expression profiles. AB - MOTIVATION: The clustering of gene profiles across some experimental conditions of interest contributes significantly to the elucidation of unknown gene function, the validation of gene discoveries and the interpretation of biological processes. However, this clustering problem is not straightforward as the profiles of the genes are not all independently distributed and the expression levels may have been obtained from an experimental design involving replicated arrays. Ignoring the dependence between the gene profiles and the structure of the replicated data can result in important sources of variability in the experiments being overlooked in the analysis, with the consequent possibility of misleading inferences being made. We propose a random-effects model that provides a unified approach to the clustering of genes with correlated expression levels measured in a wide variety of experimental situations. Our model is an extension of the normal mixture model to account for the correlations between the gene profiles and to enable covariate information to be incorporated into the clustering process. Hence the model is applicable to longitudinal studies with or without replication, for example, time-course experiments by using time as a covariate, and to cross-sectional experiments by using categorical covariates to represent the different experimental classes. RESULTS: We show that our random effects model can be fitted by maximum likelihood via the EM algorithm for which the E(expectation)and M(maximization) steps can be implemented in closed form. Hence our model can be fitted deterministically without the need for time consuming Monte Carlo approximations. The effectiveness of our model-based procedure for the clustering of correlated gene profiles is demonstrated on three real datasets, representing typical microarray experimental designs, covering time-course, repeated-measurement and cross-sectional data. In these examples, relevant clusters of the genes are obtained, which are supported by existing gene function annotation. A synthetic dataset is considered too. AVAILABILITY: A Fortran program blue called EMMIX-WIRE (EM-based MIXture analysis WIth Random Effects) is available on request from the corresponding author. PMID- 16675469 TI - Mechanism of 4-HPR-induced apoptosis in glioma cells: evidences suggesting role of mitochondrial-mediated pathway and endoplasmic reticulum stress. AB - N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid is under clinical evaluation as a therapeutic agent in a variety of cancers. Its mechanism(s) of action involves multiple overlapping pathways that still remain unclear. In glioma cells its mechanism of action is not well elucidated. Here, we show that 4 HPR and not all-trans retinoic acid and 9-cis retinoic acid effectively induce apoptosis in glioma cells. 4-HPR-induced apoptosis is associated with hydroperoxide production and loss of mitochondrial membrane potential (Delta Psi(m)). Ultrastructural changes further indicate 4-HPR-induced mitochondrial swelling, endoplasmic reticulum (ER) dilation as well as close proximity of mitochondria and ER. As suggested by dilated ER, 4-HPR treatment increased the free cytosolic Ca(2+) as well as mitochondrial Ca(2+). Chelation of extracellular Ca(2+) by EGTA did not prevent Ca(2+) elevation, thus suggesting involvement of intracellular calcium stores in the release. Buffering of intracellular calcium by BAPTA-AM did not prevent 4-HPR-induced apoptosis; however, blocking the release of Ca(2+) from ER by heparin inhibited apoptosis, indicating the role of depletion of Ca(2+) from ER stores in apoptosis. 4-HPR treatment also resulted in an increase in Bax levels along with its translocation to mitochondria that promote mitochondrial membrane permeabilization. 4-HPR-induced apoptosis was further associated with the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol and nucleus, respectively, along with caspase 3 and caspase-7 activation. However, AIF nuclear translocation, peripheral chromatin condensation and apoptosis were not completely prevented by general caspase inhibitors, thus suggesting involvement of a caspase-dependent and caspase-independent pathway in 4-HPR-induced apoptosis. Taken together, these results suggest the role of mitochondrial-mediated pathway and ER stress as a key event in 4-HPR-induced apoptosis in glioma cells. PMID- 16675470 TI - MDM2 gene promoter polymorphisms and risk of lung cancer: a case-control analysis. AB - The MDM2 protein negatively regulates p53 expression level in modulating DNA repair, cell-cycle control, cell growth and apoptosis. Polymorphisms in the promoter region of the MDM2 gene have been shown to alter protein expression and may, thus, play a role in carcinogenesis. To test our hypothesis that the MDM2 promoter polymorphisms are associated with risk of lung cancer, we conducted a hospital-based, case-control study of 1026 non-Hispanic white patients newly diagnosed with lung cancer and 1145 cancer-free controls who were frequency matched by age (+/-5 years), sex, ethnicity and smoking status. We genotyped for the MDM2 promoter G2580T (also called SNP309) and G2164A polymorphisms that have a minor allele frequency >0.05. The distributions of the MDM2-2580G variant allele and genotypes were significantly less common among the cases than among the controls (P = 0.038 and 0.045, respectively), but this was not evident for MDM2-2164G (P = 0.865 and 0.614, respectively). Compared with the MDM2-2580TT genotype, the MDM2-2580G variant genotypes were associated with a decreased risk of lung cancer [odds ratio = 0.81 and 95% confidence interval = 0.67-0.98 for GT, 0.83 (0.63-1.08) for GG, and 0.81 (0.68-0.97) for the combined GT/GG genotype]. However, no significant association was observed between the MDM2-2164G variant genotypes and lung cancer risk. Our results suggest that the MDM2-2580G allele may be a marker of reduced genetic susceptibility to lung cancer in the non Hispanic white population, a finding that seems to contradict previous reports. PMID- 16675471 TI - Potent inhibition of Lewis lung cancer growth by heyneanol A from the roots of Vitis amurensis through apoptotic and anti-angiogenic activities. AB - Vitis amurensis Rupr. (Vitaceae) has long been used in Chinese/Oriental herbal medicine for the treatment of cancer, but its active compounds and mechanisms of action have not been well studied. To this end, we isolated from its root heyneanol A (HA), which is a tetramer of resveratrol (RES), and established the in vivo antitumor activity of HA using the mouse Lewis lung carcinoma (LLC) model. We administered HA and RES by daily intraperitonial injection to C57BL/6 mice that were subcutaneously inoculated with LLC cells. HA dose-dependently decreased tumor growth without any adverse effect on body weight and seemed more potent than RES. The tumor inhibitory effects were accompanied by a marked increase in tumor cell apoptosis detected by cleaved caspase-3 and TUNEL assays and decreased tumor cell proliferation index and tumor microvessel density, supporting the involvement of apoptotic and anti-angiogenic activities in the anticancer effects. We next investigated the cellular and molecular processes that mediate the apoptosis and anti-angiogenesis effects using cell culture models. Mechanistically, treatment of LLC cells in vitro with HA or RES significantly increased apoptotic cells. Both HA- and RES-induced cleavage of caspase-9 and caspase-3 and PARP were completely blocked by a pan caspase inhibitor, Z-VAD-FMK. In addition, HA and RES suppressed the basic fibroblast growth factor (bFGF)-induced proliferation and capillary differentiation of human umbilical vein endothelial cells, and inhibited the binding of bFGF to its receptor in a test tube assay and the bFGF-induced vascularization of Matrigel plugs in vivo. Remarkably, HA was fairly stable in cell culture medium and did not undergo intracellular conversion to RES. Therefore, HA is an active anticancer compound that induces caspase-mediated cancer cell apoptosis and inhibits angiogenesis rivaling the potency of RES and merits further evaluation for cancer chemoprevention. PMID- 16675472 TI - A novel anticancer effect of garlic derivatives: inhibition of cancer cell invasion through restoration of E-cadherin expression. AB - Metastatic cancer is one of the main causes of cancer-related death since they rarely respond to available treatments. Recently, certain compounds isolated from the dietary supplement, garlic, have shown anti-proliferation effect on cancer cells. The aim of this study was to investigate whether certain garlic derivatives had any effect on the potentially invasive androgen-independent prostate cancer (PCa) cells. Using colony-forming, wound-closure as well as matrigel-invasion assays, we found that two main water-soluble constituents of the garlic, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), were able to suppress PCa cell proliferation and invasive abilities. This inhibitory effect was associated with induction of mesenchymal to epithelial transition. Most importantly, the SAC and SAMC treatment led to restoration of E-cadherin expression at transcription and protein levels. In contrast, the expression of E cadherin repressor, Snail, was reduced in the SAC- and SAMC-treated cells. Furthermore, examination of cell lines from other types of cancer (ovarian, nasopharyngeal and esophageal carcinomas) also confirmed that the effect of SAC and SAMC on activation of E-cadherin might be a general effect on human cancer cells. Our results demonstrate a novel anticancer effect of garlic and suggest that certain garlic-derived compounds may be potential agents for suppression of invasive growth through restoration of E-cadherin expression in cancer cells. PMID- 16675473 TI - Cancer chemoprevention of intestinal polyposis in ApcMin/+ mice by sulforaphane, a natural product derived from cruciferous vegetable. AB - Sulforaphane (SFN) is an isothiocyanate that is present abundantly in widely consumed cruciferous vegetables and has a particularly high content in broccoli and cauliflower. It has been shown to be an effective inhibitor of some carcinogen-induced cancers in rodents. Here, we investigated the chemopreventive efficacy of SFN in the ApcMin/+ mouse model. ApcMin/+ mice were fed with diet supplemented with two different dose levels of SFN (300 and 600 p.p.m.) for 3 weeks. Our results clearly demonstrated that ApcMin/+ mice fed with SFN supplemented diet developed significantly less and smaller polyps with higher apoptotic and lower proliferative indices in their small intestine, in a SFN dose dependent manner. In addition, immunohistochemical (IHC) staining of the adenomas indicated that SFN significantly suppressed the expression of phosphorylated c Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinases (p-ERK) and phosphorylated-Akt (p-Akt), which were found to be highly expressed in the adenomas of ApcMin/+ mice. In contrast, expression of two important biomarkers of the Wnt signaling pathway, beta-catenin and cyclin-D1 was unaffected by SFN treatment. Measurement of SFN and its metabolite SFN-GSH in the small intestine using LC-MS indicates that the concentrations between 3 and 30 nmol/g are required to prevent, or retard adenoma formation in the gastrointestinal tract of ApcMin/+ mice. PMID- 16675474 TI - Public and private prenatal care providers in urban Mexico: how does their quality compare? AB - OBJECTIVE: To evaluate variations in prenatal care quality by public and private clinical settings and by household wealth. DESIGN: The study uses 2003 data detailing retrospective reports of 12 prenatal care procedures received that correspond to clinical guidelines. The 12 procedures are summed up, and prenatal care quality is described as the average procedures received by clinical setting, provider qualifications, and household wealth. SETTING: Low-income communities in 17 states in urban Mexico. PARTICIPANTS: A total of 1253 women of reproductive age who received prenatal care within 1 year of the survey. MAIN OUTCOME MEASURE: The mean of the 12 prenatal care procedures received, reported as unadjusted and adjusted for individual, household, and community characteristics. RESULTS: Women received significantly more procedures in public clinical settings [80.7, 95% confidence interval (CI) = 79.3-82.1; P < or = 0.05] compared with private (60.2, 95% CI = 57.8-62.7; P < or = 0.05). Within private clinical settings, an increase in household wealth is associated with an increase in procedures received. Care from medical doctors is associated with significantly more procedures (78.8, 95% CI = 77.5-80.1; P < or = 0.05) compared with non-medical doctors (50.3, 95% CI = 46.7-53.9; P < or = 0.05). These differences are independent of individual, household, and community characteristics that affect health-seeking behavior. CONCLUSIONS: Significant differences in prenatal care quality exist across clinical settings, provider qualifications, and household wealth in urban Mexico. Strategies to improve quality include quality reporting, training, accreditation, regulation, and franchising. PMID- 16675475 TI - The influence of observation and setting on community health workers' practices. AB - OBJECTIVE: To determine whether results from an evaluation that involved observation of community health workers while they performed patient consultations in a hospital reflected normal everyday practices. DESIGN: Comparison of two samples of ill-child consultations: (i) consultations performed during an evaluation in which we observed community health workers in a hospital in-patient and outpatient department from February to March 2001 and (ii) consultations performed under no observation in villages and documented in clinical registers within the 90 days before the hospital evaluation. SETTING: Siaya District Hospital and villages in Kenya. STUDY PARTICIPANTS: Community health workers. MAIN OUTCOME MEASURE: Treatment error indicator, defined as the percentage of consultations where at least one recommended treatment (where recommended treatments were those that were indicated based on community health worker assessments of the child's condition) was not prescribed. RESULTS: We analyzed data on 1132 consultations (372 from the hospital evaluation and 760 from the community) performed by 103 community health workers. For all types of consultations combined, the difference between treatment error indicators (hospital minus community) was -16.4 [95% confidence interval (CI): -25.6, -7.1]. CONCLUSIONS: We found that community health workers made treatment errors less frequently when they were observed in a hospital in-patient or outpatient department than when they were not observed in the community. Evaluations that involve the observation of community health workers in a hospital setting might overestimate the quality of care that they normally give in their villages. PMID- 16675476 TI - Male heterosexual crack smokers with multiple sex partners: between- and within person predictors of condom use intention. AB - Little research has examined predictors of condom intention within concurrent partnerships. This study examined predictors of intention among 259 male African American crack smokers with multiple partners. Each participant reported personal condom intention at next sex, condom use self-efficacy, responsibility and outcome expectances for himself and his perceptions for his last two sex partners. Stepwise logistic regressions showed that for both partners one and two, condom use at last sex and personal responsibility for condom use were predictors of intention to use condoms at next sex. Perceived partner responsibility was an additional positive predictor with Partner 2. Hierarchical generalized linear model analyses showed that positive intention was associated negatively with perceived partner responsibility and intimacy, while positively related to situational self-efficacy. Personal responsibility interacted with intimacy such that only men who indicated the highest levels of intimacy were more likely to intend to use condoms. Overall, the findings in this study support the need for examining additional social cognitive constructs that capture the interpersonal aspects of sexual relationships such as personal and perceived responsibility, intimacy and how beliefs may change between multiple partners and across time. Finally, the differences in the valence of perceived partner responsibility across analyses and the interaction of personal responsibility with intimacy suggest the need for studies that include measure of power within the relationship. PMID- 16675477 TI - Compliance with a minimum-age law of 18 for the purchase of tobacco--the case of Sweden. AB - The purpose was 2-fold: first to analyse how compliance with the minimum-age law of 18 for the purchase of tobacco has changed over time, second to determine what factors affect the possibility for adolescents to buy cigarettes. Three series (1996, 1999 and 2002) of test purchases of tobacco were conducted in three regions of Sweden. Altogether, 36 test purchasers carried out 1867 test purchases. In 1996, 93% of all attempted purchases resulted in the purchase of cigarettes. The corresponding figures for 1999 were 80% and for 2002 63%. The results indicate significant regional differences in the test purchasers' opportunities to buy cigarettes. This paper demonstrates that compliance with the minimum-age law has had an effect on the ease with which adolescents purchase cigarettes, and further that such purchase has become more difficult. The study also indicates great potential for local efforts to induce more retailers to comply with the law. PMID- 16675478 TI - Adjuvant chemotherapy with uracil-tegafur for pathological stage III rectal cancer after mesorectal excision with selective lateral pelvic lymphadenectomy: a multicenter randomized controlled trial. AB - BACKGROUND: Although adjuvant radiotherapy was proved to be effective for local control of rectal cancer even after standardized mesorectal excision, the role of adjuvant chemotherapy after such standardized surgery remains to be clarified. We aimed to assess the efficacy of a combination of uracil and tegafur for pathological stage III rectal cancer treated by standardized mesorectal excision with selective lateral pelvic lymphadenectomy. METHODS: We randomly assigned patients with completely resected stage III rectal cancer, who underwent standardized mesorectal excision with selective lateral pelvic lymphadenectomy, to receive either oral uracil-tegafur (400 mg/m2 tegafur per day) for one year or no treatment. Standardization and quality control of the surgery and pathological techniques were ensured by use of the guidelines of the Japanese Society for Cancer of the Colon and Rectum. The primary endpoint was relapse-free survival. The secondary endpoint was overall survival. RESULTS: We enrolled and randomized 276 patients. Excluding two ineligible patients, 274 were included in the analysis. Planned interim analysis 2 years after accrual termination revealed significant prolongation of relapse-free survival (P = 0.001) and overall survival (P = 0.005) in the uracil-tegafur group. The 3-year relapse-free survival and overall survival rates were 78 and 91% in the chemotherapy group and 60 and 81% in the surgery-alone group, respectively. Local recurrence rates were low in both groups. Grade 3 events occurred in 17% of the chemotherapy patients, but no grade 4 or more events occurred. CONCLUSION: Adjuvant chemotherapy with uracil-tegafur improves survival of patients with stage III rectal cancer after standardized mesorectal excision with selective lateral pelvic lymphadenectomy. PMID- 16675479 TI - Biomechanical measurement of post-stroke spasticity. AB - BACKGROUND: spasticity following stroke is common, but clinical measurement is difficult and inaccurate. The most common measure is the modified Ashworth scale (MAS) which grades resistance to passive movement (RPM), but its validity is unclear. AIM: to assess the validity of the MAS. METHODS: spasticity was clinically graded using MAS and RPM measured biomechanically in the impaired arm of 111 patients following stroke. The biomechanical device measured RPM, applied force, angular displacement, mean velocity, passive range of movement (PROM) and time required. RESULTS: the median age was 72 years, and 66 subjects were male. The clinical grading by MAS was '0' in 15, '1' in 15, '1+' in 14, '2' in 13, '3' in 43 and '4' in 11. There was no difference in RPM among '0', '1', '1+' and '2' (P > 0.1). However, grade'4' was higher than '3' and below (P < 0.05). The force required increased with the increasing MAS while velocity and PROM decreased (P < 0.01). We regrouped the data using the algorithm: no stiffness = '0'; mild = '1' and '1+' and '2'; moderate = '3'; severe = '4'. There was no difference between 'no stiffness' and 'mild ' (P > 0.10), but 'mild' and moderate' as well as 'moderate' and 'severe' were different (P < 0.01). CONCLUSION: the MAS is not a valid ordinal level measure of RPM or spasticity. Objective measurement of RPM is possible in the clinical setting. However, additional measurements of muscle activity (electromyography) will be required to quantify spasticity. PMID- 16675480 TI - The synthetic evaluation model for analysis of flooding hazards. AB - BACKGROUND: Although many previous epidemiological studies have reported the incidence of diseases, mortality rate and economic losses after natural disasters, none of these studies has been comprehensive enough. Our aim was to establish a synthetic evaluation model (SEM) that can be used to analyze flood hazards. METHODS: Initial evaluation indicators were selected using systematic and literature data analysis. These indicators were tested with single or multiple variable analyses. Final evaluation indicators and their weights were determined using the Delphi procedure. We established a SEM of flood hazards using the hierarchy method and tested the model using jack-knife analysis. RESULTS: The SEM on flood hazards consists of 6 first-rank indicators and 24 second-rank indicators. First-rank indicators were: direct casualties (w = 0.2123), the increased incidence and prevalence rate of the disease (w = 0.1715), excess mortality rate (w = 0.1745), mental injury (w = 0.1038), epidemic focus expansion (w = 0.1572) and economic loss (w = 0.1807). The agreement of the model reached 98.2% tested with the jack-knife analysis. CONCLUSION: A SEM of flood hazards was established with an agreement of 98.2%, which can be used to evaluate the hazards, and assist public health-care workers provide appropriate flood disaster management. PMID- 16675481 TI - Exposure of pre-school children to passive cigarette and narghile smoke in Beirut. AB - BACKGROUND: Narghile is a resurging smoking device. However, little research has been done to assess passive smoking exposure. The objective of the present study is to evaluate the exposure of pre-school age children in Beirut to parental passive smoking from cigarette and/or narghile. METHODS: Data were collected from 1057 pre-school age children attending 16 day cares and 7 nursery schools in the city of Beirut. RESULTS: The overall prevalence of parental smoking (cigarette and/or narghile) was 53.3%. Ten per cent of respondents reported smoking only narghile. Fathers were significantly more likely than mothers to smoke cigarettes. However, there was no significant difference between fathers and mothers with respect to smoking narghile only. Education was a significant predictor for smoking cigarettes but not for smoking narghile. CONCLUSION: Narghile smoking appears to follow different gender and social patterns than cigarette smoking. Further research is needed to establish the determinants of narghile smoking, in order to develop adequate prevention policies. PMID- 16675482 TI - Gender differences in how men and women who are referred for IVF cope with infertility stress. AB - BACKGROUND: Men and women use a variety of coping strategies to manage stress associated with infertility. Although previous research has helped us understand these coping processes, questions remain about gender differences in coping and the nature of the relationship between coping and specific types of infertility stress. METHODS: This study examined the coping behaviours of 1026 (520 women, 506 men) consecutively referred patients at a University-affiliated teaching hospital. Participants completed the Ways of Coping Questionnaire, Fertility Problem Inventory and the Dyadic Adjustment Scale. RESULTS: Women used proportionately greater amounts of confrontative coping, accepting responsibility, seeking social support and escape/avoidance when compared with men, whereas men used proportionately greater amounts of distancing, self controlling and planful problem-solving. For men and women, infertility stress was positively related to escape/avoidance and accepting responsibility and negatively related to seeking social support, planful problem-solving and distancing. CONCLUSIONS: By analysing relative coping scores, this study identified key gender differences in how men and women cope with infertility. This was particularly true for men's coping processes that had previously remained hidden because of less frequent use of coping strategies when compared with women. PMID- 16675484 TI - Intrauterine insemination catheters for assisted reproduction: a systematic review and meta-analysis. AB - BACKGROUND: Intrauterine insemination (IUI) is the oldest and most practised form of assisted reproduction worldwide. We systematically reviewed the literature so that we could evaluate the use of soft versus firm catheters in subfertile women undergoing IUI. METHODS: Extensive searches were conducted for full-text manuscripts, conference abstracts, ongoing and unpublished trials. Primary outcomes were clinical pregnancy (CPR) and ongoing pregnancy (OPR)/live birth rates (LBRs) per woman. Secondary outcomes were multiple pregnancy rate (MPR) per clinical pregnancy, difficulty cannulating the cervix, bleeding and patient discomfort. Meta-analysis was performed using the Peto-modified Mantel-Haenszel fixed-effect model. RESULTS: Seven randomized trials were identified, and four were excluded. No significant differences were noted for CPR and LBR per woman [OR = 0.96, 95% CI = 0.70-1.32 and OR = 0.82, 95% CI = 0.43-1.58, respectively]. As for the secondary outcomes, MPRs per cycle were also not significantly different. More difficulty was noted with soft catheters and more patient discomfort with firm catheters. Bleeding following the procedure was similar between the two groups. CONCLUSIONS: Catheter choice during IUI does not seem to be a detrimental factor for success, as in other assisted reproduction techniques (ART). More studies are warranted to draw definitive conclusions and support the results of this systematic review. PMID- 16675483 TI - Adiponectin and resistin in PCOS: a clinical, biochemical and molecular genetic study. AB - BACKGROUND: We conducted a cross-sectional case-control study to evaluate the possible involvement of adiponectin and resistin in the pathogenesis of polycystic ovary syndrome (PCOS). METHODS: Seventy-six PCOS patients and 40 non hyperandrogenic women matched for BMI and degree of obesity were included. Serum adiponectin and resistin levels, anthropometrical and hormonal variables, the 45 T-->G and 276 G-->T polymorphisms in the adiponectin gene, and the -420 C-->G variant in the resistin gene, were analysed. RESULTS: Serum adiponectin concentrations were reduced in PCOS patients compared with controls (P = 0.038) irrespective of the degree of obesity, whereas serum resistin levels were increased in overweight and obese women compared with lean subjects (P = 0.016), irrespective of their PCOS or controls status. The adiponectin and resistin polymorphisms were not associated with PCOS and did not influence serum levels of adiponectin, resistin and other clinical and hormonal variables. In a multiple regression model, the waist-to-hip ratio, free testosterone levels and age, but not insulin resistance, were the major determinants of hypoadiponectinaemia. CONCLUSIONS: PCOS patients present with hypoadiponectinaemia, in relation with abdominal adiposity and hyperandrogenism. Our present results suggest that hyperandrogenism and abdominal obesity, by reducing the serum levels of the insulin sensitizer adipokine adiponectin, might contribute to the insulin resistance of PCOS. PMID- 16675485 TI - Differential effects of interleukin-1beta and transforming growth factor-beta1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro. AB - BACKGROUND: The pro-inflammatory cytokine, interleukin-1 beta (IL-1beta) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. METHODS: The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1beta and TGF beta1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. RESULTS: ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1beta increased whereas TGF-beta1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. CONCLUSION: IL-1beta and TGF-beta1 differentially regulate ADAMTS-1 expression in human decidual stromal cells. PMID- 16675486 TI - Fifteen-year results of a randomized phase III trial of fenretinide to prevent second breast cancer. AB - PURPOSE: The synthetic retinoid fenretinide administered for 5 years for prevention of second breast cancer showed no difference after a median of 8 years, but a possible reduction in premenopausal women. We conducted a long-term analysis in a subgroup of women who were regularly followed up in a single center. PATIENTS AND METHODS: We analyzed data after a median follow-up of 14.6 years (IQ range, 12.3-16.3 years) from 1739 women aged 30-70 (872 in the fenretinide arm and 867 in the observation arm), representing 60% of the initial cohort of 2867 women. The main efficacy endpoint was second primary breast cancer (contralateral or ipsilateral). RESULTS: The number of second breast cancers was 168 in the fenretinide arm and 190 in the control arm (hazard ratio = 0.83, 95% CI, 0.67-1.03). There were 83 events in the fenretinide arm and 126 in the observation arm in premenopausal women (HR = 0.62, 95% CI, 0.46-0.83), and 85 and 64 events in postmenopausal women (HR = 1.23, 95% CI, 0.63-2.40). The younger were the women, the greater was the risk reduction associated with fenretinide, which attained 50% in women aged 40 years or younger and disappeared after age 55 (P-age*treatment interaction = 0.023). There was no difference in cancers in other organs, distant metastases or survival. CONCLUSIONS: Fenretinide induces a significant risk reduction of second breast cancer in premenopausal women, which is remarkable at younger ages, and persists several years after treatment cessation. Since adverse events are limited, a trial in young women at high-risk is warranted. PMID- 16675487 TI - A novel mechanism of regulatory T cell-mediated down-regulation of autoimmunity. AB - We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN. PMID- 16675488 TI - Melanoma-derived gangliosides impair migratory and antigen-presenting function of human epidermal Langerhans cells and induce their apoptosis. AB - Gangliosides are ubiquitous, membrane-associated, glycosphingolipids, the composition and production of which is altered in many tumour cells. They have been shown to inhibit the in vitro generation and differentiation of dendritic cells (DCs) from progenitors, but their effect on human tissue-residing DCs is yet to be investigated. In the present study, we analysed the effect of GM3 and GD3 gangliosides purified from human melanoma tumours on the phenotypic and functional maturation of human epidermal Langerhans cells (LCs), the first immune barrier against the tumour cells. We showed that both gangliosides impaired spontaneous LC maturation induced by a short in vitro culture, as assessed by significant down-regulation of co-stimulation (CD40, CD54, CD80, CD86) and maturation markers (CD83, CCR7), which correlated to an impaired ability of the cells to mount allogeneic T cell proliferation. Furthermore, the ganglioside treated cells displayed less ability to migrate towards CCL19/macrophage inflammatory protein 3 beta, the chemokine that specifically binds CCR7 and mediates LC migration to lymph nodes. Lastly, we showed that both GM3 and GD3 gangliosides enhance LC spontaneous apoptosis. Globally, these in vitro results might explain, at least in part, the altered number and distribution of LCs in melanoma-bearing patients. They underscore a new mechanism for gangliosides to impede the host immune response by inducing LC dysfunction in the tumour microenvironment. PMID- 16675489 TI - Requirement of dendritic calcium spikes for induction of spike-timing-dependent synaptic plasticity. AB - Spike-timing-dependent synaptic plasticity (STDP) by definition requires the temporal association of pre- and postsynaptic action potentials (APs). Yet, in cortical pyramidal neurons pairing unitary EPSPs with single APs at low frequencies is ineffective at generating plasticity. Using recordings from synaptically coupled layer 5 pyramidal neurons, we show here that high-frequency (200 Hz) postsynaptic AP bursts, rather than single APs, are required for both long-term potentiation (LTP) induction and NMDA channel activation during EPSP-AP pairing at low frequencies. Furthermore, we find that AP bursts can lead to LTP induction and NMDA channel activation during EPSP-AP pairing at both positive and negative times. High-frequency AP bursts generated supralinear calcium signals in basal dendrites suggesting the generation of dendritic calcium spikes, as has been observed previously in apical dendrites during AP burst firing at frequencies greater than 100 Hz. Consistent with a role of these dendritic calcium spikes in LTP induction, pairing EPSPs with low frequency (50 Hz) AP bursts was ineffective in generating LTP. Furthermore, supralinear calcium signals in basal dendrites during AP bursts were blocked by low concentrations of the T- and R-type calcium channel antagonist nickel, which also blocked LTP and NMDA channel activation. These data suggest an important role of dendritic calcium spikes during AP bursts in determining both the efficacy and time window for STDP induction. PMID- 16675490 TI - Heat shock protein 60 in rostral ventrolateral medulla reduces cardiovascular fatality during endotoxaemia in the rat. AB - The rostral ventrolateral medulla (RVLM) is the origin of a 'life-and-death' signal that reflects central cardiovascular regulatory failure during brain stem death. Using an experimental endotoxaemia model, we evaluated the hypothesis that the 60 kDa heat shock protein 60 (HSP60) reduces cardiovascular fatality during brain stem death via an anti-apoptotic action in the RVLM. In Sprague-Dawley rats maintained under propofol anaesthesia, proteomic or Western blot analysis revealed a progressive augmentation of HSP60 expression in the RVLM after intravenous administration of Escherichia coli lipopolysaccharide (30 mg kg(-1)). Pretreatment with a microinjection of actinomycin D or cycloheximide into bilateral RVLM significantly blunted this HSP60 increase, whereas real-time PCR showed progressive augmentation of hsp60 mRNA. Intriguingly, superimposed on the augmented expression was a progressive decline in mitochondrial, or elevation in cytosolic, HSP60 in ventrolateral medulla. Loss-of-function manipulations in the RVLM using anti-HSP60 antiserum or antisense hsp60 oligonucleotide exacerbated mortality by potentiating the cardiovascular depression during experimental endotoxaemia, alongside intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or augmented cytochromec-caspase-3 cascade of apoptotic signalling in the RVLM. Immunoprecipitation coupled with immunoblot analysis further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during endotoxaemia, alongside a dissociation of the cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 redistributed from mitochondrion to cytosol in the RVLM confers neuroprotection against fatal cardiovascular depression during endotoxaemia via reduced activation of the cytochrome c-caspase-3 cascade of apoptotic signalling through enhanced interactions with mitochondrial or cytosolic Bax or Bcl-2. PMID- 16675491 TI - The carbonic anhydrase inhibitors methazolamide and acetazolamide have different effects on the hypoxic ventilatory response in the anaesthetized cat. AB - We compared the effects of the carbonic anhydrase inhibitors methazolamide and acetazolamide (3 mg kg(-1), i.v.) on the steady-state hypoxic ventilatory response in 10 anaesthetized cats. In five additional animals, we studied the effect of 3 and 33 mg kg(-1) methazolamide. The steady-state hypoxic ventilatory response was described by the exponential function: *Vi= G exp(-D P(O2)) + A where *Vi is the inspired ventilation, G is hypoxic sensitivity, D is the shape factor and A is hyperoxic ventilation. In the first group of 10 animals, methazolamide did not change parameters G and D, while A increased from 0.86 +/- 0.33 to 1.30 +/- 0.40 l min(-1) (mean +/- s.d., P = 0.003). However, the subsequent administration of acetazolamide reduced G by 44% (control, 1.93 +/- 1.32; acetazolamide, 1.09 +/- 0.92 l min(-1), P = 0.003), while A did not show a further change. Acetazolamide tended to reduce D (control, 0.20 +/- 0.07; acetazolamide, 0.14 +/- 0.06 kPa(-1), P = 0.023). In the second group of five animals, neither low- nor high-dose methazolamide changed parameters G, D and A. The observation that even high-dose methazolamide, causing full inhibition of carbonic anhydrase in all body tissues, did not reduce the hypoxic ventilatory response is reminiscent of previous findings by others showing no change in magnitude of the hypoxic response of the in vitro carotid body by this agent. This suggests that normal carbonic anhydrase activity is not necessary for a normal hypoxic ventilatory response to occur. The mechanism by which acetazolamide reduces the hypoxic ventilatory response needs further study. PMID- 16675492 TI - Muscle denervation promotes opening of the permeability transition pore and increases the expression of cyclophilin D. AB - Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria-mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca2+-induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca2+-induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 +/- 12 versus 475 +/- 33 nmol (mg protein)(-1) for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca2+ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham-operated muscles. In contrast, the CypD independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca2+ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles. PMID- 16675493 TI - Post- and prejunctional consequences of ecto-ATPase inhibition: electrical and contractile studies in guinea-pig vas deferens. AB - At sites of purinergic neurotransmission, synaptic ecto-ATPase is believed to limit the actions of ATP following its neural release. However, details of the modulation by this enzyme of the ATP-mediated conductance change and the possible mechanisms mediating this modulation remain unelucidated. We have addressed these issues by studying the effect of ARL 67156, a selective ecto-ATPase inhibitor, on ATP-mediated electrical and contractile activity in the sympathetically innervated guinea-pig vas deferens. ARL 67156 at 100 mum significantly potentiated the amplitude of spontaneous excitatory junction potentials (SEJPs) by 81.1% (P < 0.01) and prolonged their time courses (rise time by 49.7%, decay time constant by 38.2%; P < 0.01). Moreover, the frequency of occurrence of SEJPs was strikingly increased (from 0.28 +/- 0.13 to 0.90 +/- 0.26 Hz; P < 0.01), indicating an additional, primarily presynaptic, effect of ecto-ATPase inhibition. The frequency of occurrence of discrete events (DEs), which represent nerve stimulation-evoked quantal release of neurotransmitter, was also increased ( approximately 6-fold; P < 0.01), along with the appearance of DEs at previously 'silent' latencies. Purinergic contractions of the vas deferens were potentiated significantly (P < 0.01) by ARL 67156; these potentiated contractions were suppressed by the A1 agonist adenosine (P < 0.01) but left unaffected by the A1 antagonist 8-phenyltheophylline (8-PT). Our results indicate (i) that ecto-ATPase activity, in addition to modulating the ATP-mediated postjunctional conductance change, may regulate transmitter release prejunctionally under physiological conditions, and (ii) that the prejunctional regulation may be mediated primarily via presynaptic P2X, rather than A1, receptors. PMID- 16675494 TI - L-Arginine supplementation or arginase inhibition augments reflex cutaneous vasodilatation in aged human skin. AB - Full expression of reflex cutaneous vasodilatation is dependent on nitric oxide (NO) and vasodilatation is attenuated in healthy older humans. NO bioavailability in aged skin may be decreased by an age-related upregulation of arginase, which reciprocally regulates the NO-synthase (NOS) substrate L-arginine (L-Arg). We hypothesized that increased arginase activity contributes to attenuated vasodilatation in aged skin by limiting L-Arg for NOS-mediated NO synthesis. Five microdialysis fibres were placed in forearm skin of 10 young (Y, 23 +/- 1 years) and 9 older (O, 68 +/- 1 years) human subjects, serving as control (C, Ringer solution), NOS-inhibited (10.0 mM NG-nitro-L-arginine), arginase-inhibited (5.0 mM (S)-(2-boronoethyl)-L-cysteine + 5.0 mM Nomega-hydroxy-nor-L-arginine), L-arg supplemented (L-Arg; 10.0 mM L-arginine) and combined arginase-inhibited + L-Arg sites. After 20 min thermoneutral baseline, cutaneous vasodilatation was induced by passive whole-body heating to increase oral temperature (Tor) by 1.0 degrees C. Red blood cell flux was measured by laser-Doppler flowmetry over each microdialysis site. Cutaneous vascular conductance was calculated (CVC = flux/mean arterial pressure) and normalized to maximal CVC (CVCmax, 28.0 mM sodium nitroprusside + local heating to 43 degrees C). Cutaneous vasodilatation during heating was attenuated in O (Y, 42 +/- 1, versus O, 30 +/- 1%CVCmax, P < 0.001) at control sites. NOS inhibition decreased vasodilatation in both age groups compared to C (Y, 22 +/- 2; O, 18 +/- 2%CVCmax; P < 0.001). Arginase inhibition, L-Arg supplementation, and arginase inhibition + L-Arg supplementation augmented vasodilatation in O (arginase-inhibited, 46 +/- 4; L Arg, 44 +/- 4; arginase-inhibited + L-arg, 46 +/- 5%CVCmax; P < 0.001 versus C) but not in Y (arginase-inhibited, 46 +/- 4; L-Arg, 38 +/- 4; arginase-inhibited + L-Arg, 44 +/- 4%CVCmax; P > 0.05 versus C). Increasing L-Arg for NO synthesis by either arginase inhibition or direct L-Arg supplementation restores the age related deficit in reflex cutaneous vasodilatation. PMID- 16675495 TI - Transcapillary fluid balance consequences of missing initial lymphatics studied in a mouse model of primary lymphoedema. AB - To investigate the phenotypic consequences of a deranged lymphangiogenesis in relation to tissue fluid accumulation and the possible role of inflammation in the pathogenesis of lymphoedema, we measured determinants of transcapillary fluid filtration and inflammatory mediators in the interstitial fluid in genetically engineered Chy mice, a model for primary congenital lymphoedema (Milroy's disease). Although initial lymphatics were not present in dermis in any of the areas studied (fore paw, hind paw, thigh and back skin) interstitial fluid pressure (P(if)), measured with micropipettes, and tissue fluid volumes were significantly increased only in the areas with visible swelling - the fore and hind paw, whereas interstitial colloid osmotic pressure (COP(if)) was increased in all the skin areas examined. A volume load of 15% of body weight resulted in a more pronounced increase in P(if) as well as a four-fold increase in interstitial fluid volume in Chy relative to wild-type (wt) mice, showing the quantitative importance of lymphatics for fluid homeostasis during acute perturbations. A similar level of proinflammatory markers in interstitial fluid in early established lymphoedema (3-4 months) in Chy and wt suggests that inflammation does not have a major pathogenetic role for the development of lymphoedema, whereas a reduced level of the immunomodulatory cytokine interleukin (IL)-4 may result in a reduced immunological defence ability and thus lead to the increase in inflammatory cytokines IL-2 and IL-6 observed at a later stage (11-13 months). Our data suggest that primary lymphoedema results in a high interstitial fluid protein concentration that does not induce an interstitial inflammatory reaction per se, and furthermore shows the paramount importance of the initial lymphatics in tissue fluid homeostasis, especially during perturbations of transcapillary fluid balance. PMID- 16675496 TI - Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo. AB - Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS-/-) mice and their wild-type control as experimental animals, platelet activating factor (PAF) at 10(-7) m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 +/- 2.2 to a peak net value of 84.4 +/- 2.7 units, while the corresponding values in cremaster muscle of eNOS-/- mice were 1.0 +/- 0.3 and 15.6 +/- 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS-/- mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10(-7) m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation. PMID- 16675497 TI - Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes. AB - Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia-induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal related kinase (Erk) and c-jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia-induced cardiomyopathy. PMID- 16675498 TI - Upregulation of gene encoding adipogenic transcriptional factors C/EBPalpha and PPARgamma2 in denervated muscle. AB - Muscle denervation induces fatty degeneration in skeletal muscle. However, the possible mechanism(s) remains to be elucidated. To gain insight into the regulation of this process, this study was designed to characterize the expression pattern of genes encoding transcriptional factors that regulate adipogenesis and the terminal differentiation marker of adipocytes in denervated muscle. Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were harvested 5, 10, 20 or 30 days after surgery. The extent of fatty degeneration was assessed as lipid accumulation by Oil Red O staining. The cellular localization of CCCAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma2 (PPARgamma2), which play an important role in the regulation of adipocyte differentiation, was assessed by immunohistochemistry. The mRNA levels were analysed using a real-time polymerase chain reaction. After muscle denervation, most muscle fibres atrophied pathologically, and lipid accumulation was observed in the superficial region of the gastrocnemius muscle, suggesting that fatty degeneration occurs in this model. Both C/EBPalpha and PPARgamma2 proteins were observed in the interstitial space of denervated muscle but detected in small amounts in normal muscle. The expression levels of C/EBPalpha and PPARgamma2 were significantly upregulated 30 days after muscle denervation. The expression levels of fatty acid binding protein 4 (FABP4), which reflects fatty acid metabolism, were decreased slightly at 5 and 10 days and then returned to control levels 30 days after muscle denervation. These findings suggest that muscle denervation-induced fatty degeneration may be mediated through C/EBPalpha and PPARgamma2. PMID- 16675499 TI - The plasma atrial natriuretic peptide response to arm and leg exercise in humans: effect of posture. AB - During arm exercise (A), mean arterial pressure (MAP) is higher than during leg exercise (L). We evaluated the effect of central blood volume on the MAP response to exercise by determining plasma atrial natriuretic peptide (ANP) during moderate upright and supine A, L and combined arm and leg exercise (A + L) in 11 male subjects. In the upright position, MAP was higher during A than at rest (102 +/- 6 versus 89 +/- 6 mmHg; mean +/- s.d.) and during L (95 +/- 7 mmHg; P < 0.05), but similar to that during A + L (100 +/- 6 mmHg). There was no significant change in plasma ANP during A, while plasma ANP was higher during L and A + L (42.7 +/- 12.2 and 43.3 +/- 17.1 pg ml(-1), respectively) than at rest (34.6 +/- 14.3 pg ml(-1), P < 0.001). In the supine position, MAP was also higher during A than at rest (100 +/- 7 versus 86 +/- 5 mmHg) and during L (92 +/- 5 mmHg; P < 0.01) but similar to that during A + L (102 +/- 6 mmHg). During supine A, plasma ANP was higher than at rest and during L but lower than during A + L (73.1 +/- 22.5 versus 47.2 +/- 15.9, 67.4 +/- 18.3 and 78.1 +/- 25.0 pg ml(-1), respectively; P < 0.05). Thus, upright A was the exercise mode that did not enhance plasma ANP, suggesting that central blood volume did not increase. The results suggest that the similar blood pressure response to A and to A + L may relate to the enhanced central blood volume following the addition of leg to arm exercise. PMID- 16675500 TI - Metabolism of perfused pig intercostal muscles evaluated by 31P-magnetic resonance spectroscopy. AB - This study presents a perfused preparation for evaluation of metabolism in pig intercostal muscle in vitro. Preserved vessels and nerves to an intercostal segment including two adjacent ribs allowed for tissue perfusion and electrical stimulation with measurement of contraction force, oxygen consumption and 31P magnetic resonance spectroscopy (31P-MRS). When perfused at rest with Krebs Ringer buffer, the preparation maintained physiological levels of phosphocreatine (PCr), inorganic phosphate (Pi), ATP and pH at a stable oxygen consumption of 0.51 +/- 0.01 micromol min(-1) g(-1) for more than 2 h. Tonic stimulation of the nerve caused anaerobic energy consumption as PCr and pH decreased, and both variables recovered after the contraction with half-time values of approximately 7 min. Force increased to 0.040 N g(-1) (range, 0.031-0.103 N g(-1)) and it gradually decreased by about 70% during the subsequent 5 min of stimulation. The calculated free ADP concentration increased from 7.4 +/- 2.1 nmol g(-1) at rest to 28 +/- 12 nmol g(-1) (mean +/- s.d.) by the end of the stimulation. Thus anaerobic ATP turnover was zero at rest, 6.1 +/- 2 micromol min(-1) g(-1) during the first minute of stimulation and 3.5 +/- 0.5 micromol min(-1) g(-1) during the two last minutes, corresponding to the drop in force. When the preparation was left unperfused, anaerobic ATP turnover averaged 0.40 +/- 0.15 micromol min(-1) g(-1) for the first 10 min. The preparation can also be applied to human intercostal muscles, as demonstrated in one preliminary experiment. The results demonstrate a stable and functional in vitro preparation of intact perfused intercostal muscles in the pig. PMID- 16675501 TI - Raising the antioxidant levels within mouse muscle fibres does not affect contraction-induced injury. AB - A protocol of 75 lengthening contractions (LCP) administered to skeletal muscles of mice causes an initial force deficit owing to the mechanical disruption of sarcomeres and a reduction in calcium release from the sarcoplasmic reticulum. During the 3 days following the LCP, a 'sealing off process' and inflammatory response occurs. The reactive oxygen species (ROS) released by invading inflammatory cells produce a secondary force deficit that is more severe than the initial deficit. The timing of the infiltration of inflammatory cells and increase in force deficit relative to the sealing off process is not well documented. We tested the null hypothesis that following a lifetime of overexpression of the genes for the intracellular antioxidants manganese superoxide dismutase, copper zinc superoxide dismutase or catalase in transgenic mice, the force deficits 3 days following the administration of a 75 LCP to in situ extensor digitorum longus muscles are not different from those of wild-type mice. Following the LCP, the force deficits ranged from 39 to 59% for the muscles of transgenic mice that overexpressed the genes for intracellular antioxidants and were not different from the force deficit of 44% observed for muscles of wild type mice. The results provide evidence that the ROS damage does not occur within the cytosol of the injured fibres. Apparently, the hypercontraction of sarcomeres and accumulation of vesicles seal off and protect the intact portions of damaged fibres, such that the ROS damage and repair occurs in the milieu of the necrotic segments that are continuous with the extracellular matrix. PMID- 16675502 TI - Is frequent attendance in primary care disease-specific? AB - BACKGROUND: Sociodemographic characteristics of frequent attenders in general practice are known. It is not known whether frequent attendance is linked to specific diseases. OBJECTIVE: To investigate whether frequent consultation in primary care is related to specific morbidities and whether this relationship is influenced by the general practice which the patient attends. DESIGN: One-year survey of consultation data. SETTING: Nine general practices in North Staffordshire, UK. PARTICIPANTS: 1000 adults aged 18 years and over who had consulted primary care at least once during the study year were randomly selected from each practice and grouped into frequent (high and very high), medium and low frequency consulters. MAIN OUTCOME MEASURES: Type of morbidity coded at each consultation and number of repeat consultations for each morbidity (based on Read Code Chapters). RESULTS: All morbidity Chapters were associated with frequent consultation. Frequent consultation was also associated with repeated consultation within most morbidities. Stronger associations were seen with mental disorders, blood disorders, circulatory disorders, digestive disorders, endocrine diseases and with causes of injury and poisoning. Some variation between practices in the morbidities associated with frequent consultation were apparent; particularly for skin diseases and unspecified conditions. CONCLUSIONS: Frequent consulters in primary care are not restricted to particular groups of morbidities. There is some aspect of frequent consultation that is a characteristic of individuals regardless of the symptoms with which they consult. Some morbidities are more prominent than others in this group of consulters, and this may help guide practice policies and future research into frequent consulters. PMID- 16675503 TI - Lateral gene transfer of a multigene region from cyanobacteria to dinoflagellates resulting in a novel plastid-targeted fusion protein. AB - The number of cases of lateral or horizontal gene transfer in eukaryotic genomes is growing steadily, but in most cases, neither the donor nor the recipient is known, and the biological implications of the transfer are not clear. We describe a relatively well-defined case of transfer from a cyanobacterial source to an ancestor of dinoflagellates that diverged before Oxyrrhis but after Perkinsus. This case is also exceptional in that 2 adjacent genes, a paralogue of the shikimate biosynthetic enzyme AroB and an O-methyltransferase (OMT) were transferred together and formed a fusion protein that was subsequently targeted to the dinoflagellate plastid. Moreover, this fusion subsequently reverted to 2 individual genes in the genus Karlodinium, but both proteins maintained plastid localization with the OMT moiety acquiring its own plastid-targeting peptide. The presence of shikimate biosynthetic enzymes in the plastid is not unprecedented as this is a plastid-based pathway in many eukaryotes, but this species of OMT has not been associated with the plastid previously. It appears that the OMT activity was drawn into the plastid simply by virtue of its attachment to the AroB paralogue resulting from their cotransfer and once in the plastid performed some essential function so that it remained plastid targeted after it separated from AroB. Gene fusion events are considered rare and likely stable, and such an event has recently been used to argue for a root of the eukaryotic tree. Our data, however, show that exact reversals of fusion events do take place, and hence gene fusion data are difficult to interpret without knowledge of the phylogeny of the organisms--therefore their use as phylogenetic markers must be considered carefully. PMID- 16675504 TI - Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat provides evidence for gene flow from wild tetraploid wheat to hexaploid wheat. AB - All forms of domesticated tetraploid wheat (Triticum turgidum, genomes AABB) are nearly monomorphic for restriction fragment length polymorphism (RFLP) haplotype a at the Xpsr920 locus on chromosome 4A (Xpsr920-A1a), and wild tetraploid wheat is monomorphic for haplotype b. The Xpsr920-A1a/b dimorphism provides a molecular marker for domesticated and wild tetraploid wheat, respectively. Hexaploid wheat (Triticum aestivum, genomes AABBDD) is polymorphic for the 2 haplotypes. Bacterial artificial chromosome (BAC) clones hybridizing with PSR920 were isolated from Triticum urartu (genomes AA), Triticum monococcum (genomes AmAm), and T. turgidum ssp. durum (genomes AABB) and sequenced. PSR920 is a fragment of a putative ATP binding cassette (ABC) transporter gene (designated ABCT-1). The wheat ABCT-1 gene is more similar to the T. urartu gene than to the T. monococcum gene and diverged from the T. urartu gene about 0.7 MYA. The comparison of the sequence of the wheat A genome BAC clone with that of the T. urartu BAC clone provides the first insight into the microsynteny of the wheat A genome with that of T. urartu. Within 103 kb of orthologous intergenic space, 37 kb of new DNA has been inserted and 36 kb deleted leaving 49.7% of the region syntenic between the clones. The nucleotide substitution rate in the syntenic intergenic space has been 1.6 x 10(-8) nt(-1) year(-1), which is, respectively, 4 and 3 times as great as nucleotide substitution rates in the introns and the third codon positions of the juxtaposed gene. The RFLP is caused by a miniature inverted transposable element (MITE) insertion into intron 18 of the ABCT-A1 gene. Polymerase chain reaction primers were developed for the amplification of the MITE insertion site and its sequencing. The T. aestivum ABCT-A1a haplotype is identical to the haplotype of domesticated tetraploid wheat, and the ABCT-A1b haplotype is identical to that of wild tetraploid wheat. This finding shows for the first time that wild tetraploid wheat participated in the evolution of hexaploid wheat. A cline of the 2 haplotype frequencies exists across Euro-Asia in T. aestivum. It is suggested that T. aestivum in eastern Asia conserved the gene pool of the original T. aestivum more than wheat elsewhere. PMID- 16675505 TI - Proteomics: a new diagnostic frontier. AB - BACKGROUND: Analysis of proteins has been an integral part of the field of clinical chemistry for decades. Recent advances in technology and complete identification of the human genome sequence have opened up new opportunities for analysis of proteins for clinical diagnostic purposes. METHODS: Content of a recent conference of proteomics is summarized. RESULTS: New analytical methods allow the simultaneous analysis of a large number of proteins in biological fluids such as serum and plasma, offering partial views of the complete set of proteins or proteome. Plasma presents many analytical challenges, such as the complexity of components, predominance of a few major components, and the large concentration range of components, but the number of proteins that can be detected in plasma has expanded dramatically from hundreds to thousands. At the same time, there is increased capability to detect structural variations of proteins. Recent studies also identified the presence of complex sets of small protein fragments in plasma. This set of protein fragments, the fragmentome or peptidome, is potentially a rich source of information about physiologic and disease processes. CONCLUSIONS: Advances in proteomics offer great promise for the discovery of markers that might serve as the basis for new clinical laboratory tests. There are many challenges, however, in the translation of newly discovered markers into clinical laboratory tests. PMID- 16675506 TI - Inflammation and changes in metabolic syndrome abnormalities in US adolescents: findings from the 1988-1994 and 1999-2000 National Health and Nutrition Examination Surveys. AB - BACKGROUND: Understanding of C-reactive protein (CRP) in adult metabolic syndrome is increasing; however, this relationship in children is less clear. METHODS: We compared the prevalence of metabolic abnormalities and metabolic syndrome in fasting 12- to 19-year-olds from the 1999-2000 and 1988-1994 National Health and Nutrition Examination Survey (NHANES). In the more recent dataset we explored the relationship between metabolic abnormalities and CRP as measured by a high sensitivity assay. RESULTS: The prevalence of central obesity, low HDL cholesterol, and hypertension increased between the 2 surveys. Three or more abnormalities (metabolic syndrome) were found in 12.7% [95% confidence interval (CI), 10.0%-15.4%] of fasting adolescents from the 1999-2000 survey, compared with 9.2% (95% CI, 7.8%-10.6%; P < 0.001) in the 1988-1994 dataset, with increases also seen in sex and ethnic/racial subgroups. Increases in metabolic syndrome were primarily attributable to increasing body mass index (BMI); prevalence of BMI at or above the 85th percentile increased from 25.9% to 30.5%. Metabolic syndrome was much more prevalent in overweight compared with normal weight adolescents (38.6% vs 1.4%; P < 0.001). Median CRP increased with increasing numbers of metabolic abnormalities and was higher in adolescents with metabolic syndrome than in those without. CRP was higher in adolescents with BMI at or above the 85th percentile than those with normal BMI. CONCLUSIONS: Metabolic abnormalities and the metabolic syndrome phenotype are increasingly prevalent in US adolescents, attributable in part to the increasing incidence of overweight. Adolescents with more metabolic abnormalities have higher CRP, which may be an indicator of greater metabolic derangement and future cardiovascular risk. PMID- 16675507 TI - Diagnostic performance of urinary resveratrol metabolites as a biomarker of moderate wine consumption. AB - BACKGROUND: Nutritional biomarkers may be better measures of dietary exposure than self-reported dietary data. We evaluated resveratrol metabolites, potential biomarkers of wine consumption, in humans after moderate consumption of sparkling, white, or red wines. METHODS: We performed 2 randomized, crossover trials and a cohort study. In the first study, 10 healthy men consumed 30 g of ethanol/day as sparkling wine or gin for 28 days. In the second trial, 10 healthy women consumed 20 g of ethanol/day as white or red wine for 28 days. We also evaluated 52 participants in a study on the effects of a Mediterranean diet on primary prevention of cardiovascular disease (the PREDIMED Study). We used liquid chromatography-tandem mass spectrometry to analyze urinary total resveratrol metabolites (TRMs) and predictive values and ROC curve analyses to assess the diagnostic accuracy. RESULTS: We observed significant increases in TRMs [72.4 (95% confidence interval, 48.5-96.2; P = 0.005), 211.5 (166.6-256.3; P = 0.005), and 560.5 nmol/g creatinine (244.9-876.1; P = 0.005)] after consumption of sparkling, white, or red wine, respectively, but no changes after the washout or gin periods. In the cohort study, the reported daily dose of wine consumption correlated directly with TRMs (r = 0.654; P < 0.001). Using a cutoff of 90 nmol/g, we were able to use TRMs to differentiate wine consumers from abstainers with a sensitivity of 72% (60%-84%); and a specificity of 94% (87%-100%). CONCLUSIONS: Resveratrol metabolites in urine may be useful biomarkers of wine intake in epidemiologic and intervention studies. PMID- 16675508 TI - Specificity and clinical utility of methods for the detection of macroprolactin. AB - BACKGROUND: Increased serum concentrations of macroprolactin are a relatively common cause of misdiagnosis and mismanagement of hyperprolactinemic patients. METHODS: We studied sera from a cohort of 42 patients whose biochemical hyperprolactinemia was explained entirely by macroprolactin. Using 5 pretreatments, polyethylene glycol (PEG), protein A (PA), protein G (PG), anti human IgG (anti-hIgG), and ultrafiltration (UF), to deplete macroprolactin from sera before immunoassay, we compared residual prolactin concentrations with monomer concentrations obtained by gel-filtration chromatography (GFC). A monomeric prolactin standard was used to assess recovery and specificity of the pretreatment procedures. RESULTS: Residual prolactin concentrations in all pretreated sera differed significantly (P < 0.001) from monomeric concentrations obtained after GFC. PEG underestimated (mean, 75%), whereas PA, PG, anti-hIgG, and UF overestimated (means, 178%, 151%, 178%, and 112%, respectively) the amount of monomer present. Of the 5 methods examined, PEG correlated best with GFC (r = 0.80) followed by PG (r = 0.78), PA (r = 0.72), anti-hIgG (r = 0.70), and UF (r = 0.61). After UF or pretreatment with anti-hIgG or PEG, recovery of monomeric prolactin standard was low: 60%, 85%, and 77% respectively. In contrast, pretreatment with PA or PG gave almost quantitative recovery. CONCLUSIONS: None of the methods examined yielded results identical to the GFC method. PEG pretreatment yielded results that correlated best and is recommended as the first choice alternative to GFC. PMID- 16675510 TI - Antinociceptive effects of tetrodotoxin (TTX) in rodents. AB - BACKGROUND: Tetrodotoxin (TTX) is a powerful sodium channel blocker extracted from the puffer fish. The analgesic effects of TTX were investigated in different animal pain models. METHODS: Wistar rats were submitted to the formalin test and to partial ligation of the sciatic nerve (Seltzer's model). Swiss Webster mice were used in the writhing test. Rodents were divided into six groups receiving a s.c. injection of either 0.9% NaCl, TTX 0.3, 1, 3, or 6 microg kg(-1), or morphine (5 mg kg(-1)). Substances were injected 30 min before 2.5% formalin injection into the hind paw, acetic acid administration intraperitoneally or neuropathic pain testing consisting of mechanical allodynia (von Frey filament) and thermal hyperalgesia (Plantar test). RESULTS: TTX decreased pain behaviour in the formalin test at the highest dose and in the writhing test at 3 and 6 microg kg(-1). It also diminished mechanical allodynia and thermal hyperalgesia with an ED(50) of 1.08 (0.89) and 0.62 (0.33) microg kg(-1), respectively. Observation of the rats after TTX injection did not show any motor deficit, respiratory distress or sedation. Morphine was also effective in relieving pain in all three tests but with signs of considerable sedation. CONCLUSION: Systemic injections of TTX diminished pain behaviour in a dose-dependent manner in models of inflammatory, visceral and neuropathic pain without causing adverse events, whereas morphine analgesia was associated with heavy sedation. TTX is a very promising substance for the treatment of various types of pain but needs further evaluation. PMID- 16675509 TI - Ondansetron does not reduce the shivering threshold in healthy volunteers. AB - BACKGROUND: Ondansetron, a serotonin-3 receptor antagonist, reduces postoperative shivering. Drugs that reduce shivering usually impair central thermoregulatory control, and may thus be useful for preventing shivering during induction of therapeutic hypothermia. We determined, therefore, whether ondansetron reduces the major autonomic thermoregulatory response thresholds (triggering core temperatures) in humans. METHODS: Control (placebo) and ondansetron infusions at the target plasma concentration of 250 ng ml(-1) were studied in healthy volunteers on two different days. Each day, skin and core temperatures were increased to provoke sweating; then reduced to elicit peripheral vasoconstriction and shivering. We determined the core-temperature sweating, vasoconstriction and shivering thresholds after compensating for changes in mean-skin temperature. Data were analysed using t-tests and presented as means (sds); P<0.05 was taken as significant. RESULTS: Ondensetron plasma concentrations were 278 (57), 234 (55) and 243 (58) ng ml(-1) at the sweating, vasoconstriction and shivering thresholds, respectively; these corresponded to approximately 50 mg of ondansetron which is approximately 10 times the dose used for postoperative nausea and vomiting. Ondansetron did not change the sweating (control 37.4 (0.4) degrees C, ondansetron 37.6 (0.3) degrees C, P=0.16), vasoconstriction (37.0 (0.5) degrees C vs 37.1 (0.3) degrees C; P=0.70), or shivering threshold (36.3 (0.5) degrees C vs 36.3 (0.6) degrees C; P=0.76). No sedation was observed on either study day. CONCLUSIONS: /b>. Ondansetron appears to have little potential for facilitating induction of therapeutic hypothermia. PMID- 16675511 TI - Tissue oxygenation response to mild hypercapnia during cardiopulmonary bypass with constant pump output. AB - BACKGROUND: Tissue oxygenation is the primary determinant of wound infection risk. Mild hypercapnia markedly improves cutaneous, subcutaneous (s.c.), and muscular tissue oxygenation in volunteers and patients. However, relative contributions of increased cardiac output and peripheral vasodilation to this response remains unknown. We thus tested the hypothesis that increased cardiac output is the dominant mechanism. METHODS: We recruited 10 ASA III patients, aged 40-65 yr, undergoing cardiopulmonary bypass for this crossover trial. After induction of anaesthesia, a Silastic tonometer was inserted s.c. in the upper arm. S.C. tissue oxygen tension was measured with both polarographic electrode and fluorescence-based systems. Oximeter probes were placed bilaterally on the forehead to monitor cerebral oxygenation. After initiation of cardiopulmonary bypass, in random order patients were exposed to two arterial CO(2) partial pressures for 30 min each: 35 (normocapnia) or 50 mm Hg (hypercapnia). Bypass pump flow was kept constant throughout the measurement periods. RESULTS: Hypercapnia during bypass had essentially no effect on Pa(CO(2)) , mean arterial pressure, or tissue temperature. Pa(CO(2)) and pH differed significantly. S.C. tissue oxygenation was virtually identical during the two Pa(CO(2)) periods [139 (50-163) vs 145 (38-158), P=0.335] [median (range)]. In contrast, cerebral oxygen saturation (our positive control measurement) was significantly less during normocapnia [57 (28-67)%] than hypercapnia [64 (37-89)%, P=0.025]. CONCLUSIONS: Mild hypercapnia, which normally markedly increases tissue oxygenation, did not do so during cardiopulmonary bypass with fixed pump output. This suggests that hypercapnia normally increases tissue oxygenation by increasing cardiac output rather than direct dilation of peripheral vessels. PMID- 16675512 TI - The comparative toxicogenomics database: a cross-species resource for building chemical-gene interaction networks. AB - Chemicals in the environment play a critical role in the etiology of many human diseases. Despite their prevalence, the molecular mechanisms of action and the effects of chemicals on susceptibility to disease are not well understood. To promote understanding of these mechanisms, the Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org/) presents scientifically reviewed and curated information on chemicals, relevant genes and proteins, and their interactions in vertebrates and invertebrates. CTD integrates sequence, reference, species, microarray, and general toxicology information to provide a unique centralized resource for toxicogenomic research. The database also provides visualization capabilities that enable cross-species comparisons of gene and protein sequences. These comparisons will facilitate understanding of structure-function correlations and the genetic basis of susceptibility. Manual curation and integration of cross-species chemical-gene and chemical-protein interactions from the literature are now underway. These data will provide information for building complex interaction networks. New CTD features include (1) cross-species gene, rather than sequence, query and visualization capabilities; (2) integrated cross-links to microarray data from chemicals, genes, and sequences in CTD; (3) a reference set related to chemical-gene and protein interactions identified by an information retrieval system; and (4) a "Chemicals in the News" initiative that provides links from CTD chemicals to environmental health articles from the popular press. Here we describe these new features and our novel cross-species curation of chemical-gene and chemical protein interactions. PMID- 16675513 TI - Cloning, tissue expression, and regulation of beagle dog CYP4A genes. AB - In addition to its function as a fatty acid hydroxylase, the peroxisome proliferator-activated receptor alpha (PPARalpha) target gene, CYP4A, has been shown to be important in the conversion of arachidonic acid to the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, suggesting a role for this enzyme in mediating vascular tone. In the present study, the cDNA sequence of beagle dog CYP4A37, CYP4A38, and CYP4A39 from the liver was determined. Open reading frame analysis predicted that CYP4A37, CYP4A38, and CYP4A39 each comprised 510 amino acids with approximately 90% sequence identity to one another, and approximately 71 and 78% sequence identity to rat CYP4A1 and human CYP4A11, respectively. PCR analysis revealed that the three dog CYP4A isoforms are expressed in kidney > liver >> lung >> intestine > skeletal muscle > heart. Treatment of primary dog hepatocytes with the PPARalpha agonists GW7647X and clofibric acid resulted in an increase in CYP4A37, CYP4A38, and CYP4A39 mRNA expression (up to fourfold), whereas HMG-CoA synthase mRNA expression was increased to a greater extent (up to 10-fold). These results suggest that dog CYP4A37, CYP4A38, and CYP4A39 are expressed in a tissue-dependent manner and that beagle dog CYP4A is not highly inducible by PPARalpha agonists, similar to the human CYP4A11 gene. PMID- 16675514 TI - Immunomodulatory effects of estradiol and cadmium in adult female rats. AB - A wide range of toxic effects has been associated with cadmium (Cd) exposure in mammals. However, the physiological factors that modulate these effects have received limited attention. We have previously demonstrated that neonatal exposure of rats to Cd during lactation results in sex-specific immunotoxic effects in both juvenile and adult rats. The objectives of this study were to determine the effects of 17beta-estradiol (E(2)) on the immunotoxicity of Cd in female rats. We compared the effects of 28 days of exposure to 0, 5, and 25 ppm cadmium chloride (CdCl(2)) through drinking water on ovariectomized Sprague Dawley rats and on ovariectomized rats with E(2) implant which mimicked the physiological level of E(2) in female rat. Our results clarify the control of important immune functions by E(2) at physiological level and demonstrate significant interactions between Cd and E(2) effects on the cytotoxic activity of natural killer cells and phagocytosis of splenic cells as well as on the total number of thymocytes and of the four subpopulations of the thymocytes as defined by the expression of the cell-surface markers CD4 and CD8. Cd and E(2) share several mechanisms of action that may account for these interactions. The estrogenic potential of Cd could also account for some of the observed effects. These interactions have to be taken into consideration in evaluating the risk of Cd immunotoxicity and the possible interactions with hormonal treatments. PMID- 16675515 TI - Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain. AB - Chlorpyrifos (CPS), a known neurotoxicant, is a widely used agricultural organophosphorus insecticide. The effects of postnatal exposure to CPS on the expression of mRNA for two factors critical to brain development, nerve growth factor (NGF) and reelin, were investigated in the forebrain of rats. In addition, the expression of mRNA for the muscarinic acetylcholine receptor (mAChR) M(1) subtype and cell-specific markers for developing neurons (beta-III tubulin), astrocytes (glial fibrillary acidic protein, GFAP), and oligodendrocytes (myelin associated glycoprotein, MAG) was also investigated. Oral administration of CPS (1.5 or 3.0 mg/kg) or the corn oil vehicle was performed daily from postnatal days (PNDs) 1 through 6. No signs of overt toxicity or of cholinergic hyperstimulation were observed after CPS administration. Body weight was significantly different from controls on PND7 in both males and females exposed to 3.0 mg/kg CPS. Quantitative PCR was performed on the forebrain. The expression of NGF, reelin, and M(1) mAChR mRNA was significantly reduced with both dosages of CPS in both sexes. beta-III Tubulin mRNA expression remained unchanged after exposure, whereas MAG mRNA expression was significantly decreased with both dosages of CPS in both sexes, suggesting effects on the developing oligodendrocytes. In contrast, GFAP mRNA levels were significantly increased with both dosages of CPS in both sexes, suggesting increased astrocyte reactivity. Our findings indicate that dosages of CPS which cause significant cholinesterase inhibition but do not exert overt toxicity can adversely affect the expression levels of critical genes involved in brain development during the early postnatal period in the rat. PMID- 16675516 TI - Mechanisms of 2-butoxyethanol-induced hemangiosarcomas. AB - Chronic exposure to 2-butoxyethanol increased liver hemangiosarcomas in male mice. The mechanism for the selective induction of hemangiosarcomas by 2 butoxyethanol is unknown but has been suggested to occur through non-DNA-reactive mechanisms. The occurrence of liver hemangiosarcomas in male mice has been linked to oxidative damage subsequent to RBC hemolysis and iron deposition and activation of macrophages (Kupffer cells) in the liver, events that exhibit a threshold in both animals and humans. 2-Butoxyethanol is metabolized to 2 butoxyacetaldehyde and 2-butoxyacetic acid, and although the aldehyde metabolite is short lived, the potential exists for this metabolite to cause DNA damage. The present study examined whether 2-butoxyethanol and its metabolites, 2 butoxyacetaldehyde and 2-butoxyacetic acid, damaged mouse endothelial cell DNA using the comet assay. No increase in DNA damage was observed following 2 butoxyethanol (1-10mM), 2-butoxyacetaldehyde (0.1-1.0mM), or 2-butoxyacetic acid (1-10mM) in endothelial cells after 2, 4, or 24 h of exposure. Additional studies examined the involvement of hemolysis and macrophage activation in 2 butoxyethanol carcinogenesis. DNA damage was produced by hemolyzed RBCs (10 x 10(6), 4 h), ferrous sulfate (0.1-1.0 microM; 2-24 h), and hydrogen peroxide (50 100 microM; 1-4 h) in endothelial cells. Hemolyzed RBCs also activated macrophages, as evidenced by increased tumor necrosis factor (TNF) alpha, while neither 2-butoxyethanol nor butoxyacetic acid increased TNF-alpha from macrophages. The effect of activated macrophages on endothelial cell DNA damage and DNA synthesis was also studied. Coculture of endothelial cells with activated macrophages increased endothelial cell DNA damage after 4 or 24 h and increased endothelial cell DNA synthesis after 24 h. These data demonstrate that 2 butoxyethanol and related metabolites do not directly cause DNA damage. Supportive evidence also demonstrated that damaged RBCs, iron, and/or products from macrophage activation (possibly reactive oxygen species) produce DNA damage in endothelial cells and that activated macrophages stimulate endothelial cell proliferation. These events coupled together provide the events necessary for the induction of hemangiosarcomas by 2-butoxyethanol. PMID- 16675517 TI - Diurnal rhythmicity of the clock genes Per1 and Per2 in the rat ovary. AB - Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12 h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0-2 and peaked at Zeitgeber time 12-14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis. PMID- 16675518 TI - Congenic mice provide in vivo evidence for a genetic locus that modulates serum insulin-like growth factor-I and bone acquisition. AB - We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) x C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6.C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i.e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition. PMID- 16675519 TI - Characterization of a novel tonic gamma-aminobutyric acidA receptor-mediated inhibition in magnocellular neurosecretory neurons and its modulation by glia. AB - In addition to mediating conventional quantal synaptic transmission (also known as phasic inhibition), gamma-aminobutyric acidA (GABAA) receptors have been recently shown to underlie a slower, persistent form of inhibition (tonic inhibition). Using patch-clamp electrophysiology and immunohistochemistry, we addressed here whether a GABAA receptor-mediated tonic inhibition is present in supraoptic nucleus (SON) neurosecretory neurons; identified key modulatory mechanisms, including the role of glia; and determined its functional role in controlling SON neuronal excitability. Besides blocking GABAA-mediated inhibitory postsynaptic currents, the GABAA receptor blockers bicuculline and picrotoxin caused an outward shift in the holding current (I(tonic)), both in oxytocin and vasopressin neurons. Conversely, the high-affinity antagonist gabazine selectively blocked inhibitory postsynaptic currents. Under basal conditions, I(tonic) was independent on the degree of synaptic activity but was strongly modulated by the activity GABA transporters (GATs), mostly the GAT3 isoform, found here to be localized in SON glial cells/processes. Extracellular activation of GABAergic afferents evoked a small gabazine-insensitive, bicuculline-sensitive current, which was enhanced by GAT blockade. These results suggest that I(tonic) may be activated by spillover of GABA during conditions of strong and/or synchronous synaptic activity. Blockade of I(tonic) increased input resistance, induced membrane depolarization and firing activity, and enhanced the input output function of SON neurons. In summary, our results indicate that GABAA receptors, possibly of different molecular configuration and subcellular distribution, mediate synaptic and tonic inhibition in SON neurons. The latter inhibitory modality plays a major role in modulating SON neuronal excitability, and its efficacy is modulated by the activity of glial GATs. PMID- 16675520 TI - Evidence of altered brain sexual differentiation in mice exposed perinatally to low, environmentally relevant levels of bisphenol A. AB - Humans are routinely exposed to bisphenol A (BPA), an estrogenic chemical present in food and beverage containers, dental composites, and many products in the home and workplace. BPA binds both classical nuclear estrogen receptors and facilitates membrane-initiated estrogenic effects. Here we explore the ability of environmentally relevant exposure to BPA to affect anatomical and functional measures of brain development and sexual differentiation. Anatomical evidence of alterations in brain sexual differentiation were examined in male and female offspring born to mouse dams exposed to 0, 25, or 250 ng BPA/kg body weight per day from the evening of d 8 of gestation through d 16 of lactation. These studies examined the sexually dimorphic population of tyrosine hydroxylase (TH) neurons in the rostral periventricular preoptic area, an important brain region for estrous cyclicity and estrogen-positive feedback. The significant sex differences in TH neuron number observed in control offspring were diminished or obliterated in offspring exposed to BPA primarily because of a decline in TH neuron number in BPA-exposed females. As a functional endpoint of BPA action on brain sexual differentiation, we examined the effects of perinatal BPA exposure on sexually dimorphic behaviors in the open field. Data from these studies revealed significant sex differences in the vehicle-exposed offspring that were not observed in the BPA-exposed offspring. These data indicate that BPA may be capable of altering important events during critical periods of brain development. PMID- 16675521 TI - Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils. AB - Neutrophils are critical for innate immune defense against microbial invasion but can also cause inflammatory tissue damage if their life span is not tightly regulated. Antiinflammatory glucocorticoids delay spontaneous apoptosis in human, rodent, and bovine neutrophils, but mechanisms involved are unknown. We hypothesized here that glucocorticoids delay neutrophil apoptosis by altering expression of key Bcl-2 apoptosis regulatory proteins, A1 and Bak, via activation of the cell's glucocorticoid receptors. To test this hypothesis, isolated bovine blood neutrophils were exposed to dexamethasone with and without glucocorticoid receptor antagonism (RU486) and aged ex vivo over 0-24 h for assessment of various spontaneous apoptosis pathway indicators and A1 and Bak abundance. Results show that dexamethasone preserved neutrophil mitochondrial membrane integrity, delayed caspase-9 activation, and reduced the rate of spontaneous apoptosis. Also, dexamethasone increased A1 and decreased Bak mRNA abundance. RU486 pretreatment of the cells abrogated each of these dexamethasone effects. Dexamethasone-induced increases in A1 mRNA were reflected in A1 protein increases, which also were observed in circulating neutrophils of dexamethasone treated animals. Bak protein decreases were observed in neutrophils of the dexamethasone-treated animals but not in isolated neutrophils, suggesting that stimuli additional to (and perhaps regulated by) glucocorticoid are required to affect Bak protein expression changes in neutrophils. Collectively, our results are unique in demonstrating a mechanism behind glucocorticoid regulation of spontaneous apoptosis and implicate steroid receptor activation and subsequent regulation of A1 and Bak as contributors to mitochondrial membrane stability, reduced caspase-9 activity, and delayed apoptosis in bovine neutrophils exposed to glucocorticoids. PMID- 16675522 TI - Neuroendocrine stress but not feeding responses to centrally administered neuropeptide Y are suppressed in pregnant rats. AB - Metabolic peptides such as orexin and neuropeptide Y (NPY) exert profound effects on feeding but also act centrally to stimulate the hypothalamo-pituitary-adrenal (HPA) axis. In late pregnancy the HPA axis is hyporesponsive to centrally administered orexin-A, which signals to the HPA axis, in part, via arcuate NPY neurones. We investigated whether reduced HPA axis responses to orexin may be a consequence of down-regulated NPY signaling to the paraventricular nucleus (PVN) in pregnancy. Pregnant (d 21) and virgin rats were blood sampled for ACTH, corticosterone, and oxytocin (also a stress hormone in rats) before and after intracerebroventricular NPY or vehicle. Behavior was monitored. Rats were killed 4 h after NPY and brains removed for in situ hybridization. In another experiment rats were given vehicle or NPY, perfuse fixed 90 min later, and brain sections processed for Fos and oxytocin immunocytochemistry. NPY significantly increased ACTH, corticosterone and oxytocin secretion in the virgins but had no such effect on ACTH or oxytocin in the pregnant rats; the corticosterone response to NPY was markedly attenuated in pregnant rats. NPY increased CRH and vasopressin mRNA expression in the parvocellular PVN and stimulated Fos expression in magnocellular supraoptic and PVN oxytocin neurones of virgin but not pregnant rats. NPY increased food intake and drinking similarly in virgin and pregnant rats. Thus, neuroendocrine stress responses to central NPY are absent in late pregnancy, whereas ingestive behavioral responses are intact. These changes may explain the similarly attenuated HPA response to centrally administered orexin-A and will favor anabolic adaptations in pregnancy. PMID- 16675523 TI - Inhibition of the proteasomal function in chondrocytes down-regulates growth plate chondrogenesis and longitudinal bone growth. AB - The proteasome is a large multiprotein complex that processes intracellular proteins functioning as cell cycle regulators and transcription factors. It has been shown that the chymotryptic component of the proteasome is an important regulator of osteoblast differentiation and bone formation, with inhibitors of the proteasome increasing osteoblast differentiation and bone formation. Yet, little is known about the effects of the proteasomal activity in the growth plate. In the present study, we cultured rat metatarsal bones in the presence of proteasome inhibitor I (PSI), a known inhibitor of the chymotrypsin-like activity of the 20S proteasome. PSI suppressed growth plate chondrocyte proliferation and hypertrophy/differentiation, and induced chondrocyte apoptosis. All these cellular effects led to reduced metatarsal linear growth. In cultured chondrocytes, PSI increased the expression of beta-catenin (a negative regulator of chondrogenesis) and reduced the DNA binding of nuclear factor kappaB, a transcription factor that stimulates growth plate chondrogenesis. In conclusion, our findings suggest that the proteasomal activity facilitates growth plate chondrogenesis and, in turn, longitudinal bone growth. PMID- 16675524 TI - Calcitonin plays a critical role in regulating skeletal mineral metabolism during lactation. AB - The maternal skeleton rapidly demineralizes during lactation to provide calcium to milk, responding to the stimuli of estrogen deficiency and mammary-secreted PTH-related protein. We used calcitonin/calcitonin gene-related peptide-alpha (Ctcgrp) null mice to determine whether calcitonin also modulates lactational mineral metabolism. During 21 d of lactation, spine bone mineral content dropped 53.6% in Ctcgrp nulls vs. 23.6% in wild-type (WT) siblings (P < 0.0002). After weaning, bone mineral content returned fully to baseline in 18.1 d in Ctcgrp null vs. 13.1 d in WT (P < 0.01) mice. Daily treatment with salmon calcitonin from the onset of lactation normalized the losses in Ctcgrp null mice, whereas calcitonin gene-related peptide-alpha or vehicle was without effect. Compared with WT, Ctcgrp null mice had increased circulating levels of PTH and up-regulation of mammary gland PTH-related protein mRNA. In addition, lactation caused the Ctcgrp null skeleton to undergo more trabecular thinning and increased trabecular separation compared with WT. Our studies confirm that an important physiological role of calcitonin is to protect the maternal skeleton against excessive resorption and attendant fragility during lactation and reveal that the postweaning skeleton has the remarkable ability to rapidly recover even from losses of over 50% of skeletal mineral content. PMID- 16675525 TI - Ciliary neurotrophic factor suppresses hypothalamic AMP-kinase signaling in leptin-resistant obese mice. AB - We examined the actions of a second-generation ciliary neurotrophic factor analog (CNTF(Ax15)) on AMP-activated protein kinase (AMPK), a known regulator of food intake. Unlike leptin CNTF(Ax15) has been shown to reduce food intake in obese rodents and humans. Intraperitoneal injection of CNTF(Ax15) acutely (45 min) reduced hypothalamic AMPKalpha2 activity, AMPKalpha2Thr172 phosphorylation, and acetyl-coenzyme A carboxylase phosphorylation, effects not observed 2 or 6 h after injection. Intracerebroventricular CNTF(Ax15) reduced food intake, increased arcuate nucleus (ARC) signal transducer and activator of transcription 3 phosphorylation, and reduced AMPK signaling but not in the paraventricular nucleus (PVN), posterior hypothalamus, or cortex. To compare the effects of leptin and CNTF(Ax15) in a diet-induced model of obesity, mice were fed a control carbohydrate or high-fat diet (HFD) for 12 wk. Leptin treatment ip reduced food intake in control mice but not in mice fed a HFD. In contrast, ip CNTF markedly reduced food intake in both control and HFD animals. Both leptin and CNTF reduced AMPK activity and acetyl-coenzyme A carboxylase phosphorylation in the ARC and PVN of control-fed mice. A HFD blunted leptin but not CNTF effects on AMPK signaling in the ARC and PVN. In summary, these data demonstrate that CNTF(Ax15) bypasses diet-induced leptin resistance to reduce hypothalamic AMPK activity. PMID- 16675526 TI - Regulation of alternative splicing of Slo K+ channels in adrenal and pituitary during the stress-hyporesponsive period of rat development. AB - Stress triggers release of ACTH from the pituitary, glucocorticoids from the adrenal cortex, and epinephrine from the adrenal medulla. Although functions differ, these hormone systems interact in many ways. Previous evidence indicates that pituitary and steroid hormones regulate alternative splicing of the Slo gene at the stress axis-regulated exon (STREX), with functional implications for the calcium-activated K+ channels prominent in adrenal medullary and pituitary cells. Here we examine the role of corticosterone in Slo splicing regulation in pituitary and adrenal tissues during the stress-hyporesponsive period of early rat postnatal life. The sharp drop in plasma corticosterone (CORT) that defines this period offers a unique opportunity to test CORT's role in Slo splicing. We report that in both adrenal and pituitary tissues, the percentage of Slo transcripts having STREX declines and recovers in parallel with CORT. Moreover, addition of 500 nm CORT to cultures of anterior pituitary cells from 13-, 21-, and 30-d postnatal animals increased the percentage of Slo transcripts with STREX, whereas 20 microm CORT reduced STREX representation. Applied to adrenal chromaffin cells, 20 microm CORT decreased STREX inclusion, whereas neither 500 nm nor 2 microm had any effect. The mineralocorticoid receptor antagonist RU28318 abolished the effect of 500 nm CORT on splicing in pituitary cells, whereas the glucocorticoid receptor antagonist RU38486 blocked the effect of 20 microm CORT on adrenal chromaffin cells. These results support the hypothesis that the abrupt, transient drop in CORT during the stress-hyporesponsive period drives the transient decline in STREX splice variant representation in pituitary, but not adrenal. PMID- 16675527 TI - The nuclear hormone receptor farnesoid X receptor (FXR) is activated by androsterone. AB - Farnesoid X receptor (FXR) uses bile acids as endogenous ligands. Here, we demonstrate that androsterone, a metabolic product of testosterone, is also an FXR ligand. Treatment of castrated male mice with androsterone induced expression of the FXR target gene small heterodimer partner (SHP). In mouse AML-12 hepatocytes, chenodeoxycholic acid (CDCA) or androsterone induced SHP expression with a similar kinetic pattern. The FXR antagonist guggulsterone blocked the induction of SHP by androsterone in AML-12 cells. Nuclear magnetic resonance spectroscopy demonstrated the direct binding of androsterone to purified human FXR (hFXR) ligand-binding domain (LBD) protein, resulting in the recruitment of steroid receptor coactivator protein-1 (SRC-1) coactivator peptide. In HEK293 cells, androsterone activated gal4-mouse FXR-LBD and gal4-hFXR-LBD fusion proteins, although in contrast to CDCA, androsterone activation was significantly greater for the mouse FXR-LBD than for the hFXR-LBD. Site-directed mutagenesis of the hFXR-LBD defined amino acids Asn354 and Ser345 as critical for differential species sensitivity to CDCA and androsterone, respectively. Crystal structure studies suggest that the orientation of the steroid nucleus of bile acids within the binding pocket of FXR is reversed from all other nuclear hormone receptors. In support of this model, we show here that mutations M265I or R331H, residues predicted by crystal structure to interact with the carboxylic acid tail of CDCA but not with androsterone, altered CDCA activation but had no effect on androsterone activation. Activation of FXR by androsterone may provide an additional means for physiological or pharmacological modulation of FXR. PMID- 16675528 TI - Molecular characterization and expression of urotensin II and its receptor in the flounder (Platichthys flesus): a hormone system supporting body fluid homeostasis in euryhaline fish. AB - Urotensin II (UII) is a potent vasoconstrictor in mammals, but the source of circulating UII remains unclear. Investigations of the caudal neurosecretory system (CNSS), considered the major source of UII in fish, alongside target tissue expression of UII receptor (UT), can provide valuable insights into this highly conserved regulatory system. We report UII gene characterization, expression of the first fish UT, and responses to salinity challenge in flounder. The 12-aa UII peptide shares 73% sequence identity with pig and human UII. Flounder UT receptor shares 56.7% identity with rat. Although the CNSS is the major site of UII expression, RT-PCR revealed expression of UII and UT in all tissues tested. Around 30-40% of large CNSS Dahlgren cells expressed UII, alone or in combination with urotensin I and/or corticotrophin releasing hormone. Immunolocalization of UT in osmoregulatory tissues (gill, kidney) was associated with vascular elements. There were no consistent differences in CNSS UII expression or plasma UII between seawater (SW)- and freshwater (FW)-adapted fish, although gill and kidney UT expression was lower in FW animals. After acute transfer from SW to FW, plasma UII and kidney and gill UT expression were reduced, whereas UT expression in kidney was increased after reverse transfer. UII appears to be more important to combat dehydration and salt-loading in SW than the hemodilution faced in FW. Potentially, altered target tissue sensitivity through changes in UT expression, is an important physiological controlling mechanism, not only relevant for migratory fish but also likely conserved in mammals. PMID- 16675529 TI - Expression of vesicular glutamate transporter-2 in gonadotrope and thyrotrope cells of the rat pituitary. Regulation by estrogen and thyroid hormone status. AB - Immunocytochemical studies of the rat adenohypophysis identified a cell population that exhibits immunoreactivity for type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype. The in situ hybridization detection of VGLUT2 mRNA expression in adenohypophysial cells verified that VGLUT2 immunoreactivity is due to local synthesis of authentic VGLUT2. Dual-immunofluorescent studies of the hypophyses from male rats showed the presence of VGLUT2 in high percentages of LH (93.3 +/- 1.3%)-, FSH (44.7 +/- 3.9%)-, and TSH (70.0 +/- 5.6%)-immunoreactive cells and its much lower incidence in cells of the prolactin, GH, and ACTH phenotypes. Quantitative in situ hybridization studies have established that the administration of a single dose of 17-beta-estradiol (20 microg/kg; sc) to ovariectomized rats significantly elevated VGLUT2 mRNA in the adenohypophysis 16 h postinjection. Thyroid hormone dependence of VGLUT2 expression was addressed by the comparison of hybridization signals in animal models of hypo- and hyperthyroidism to those in euthyroid controls. Although hyperthyroidism had no effect on VGLUT2 mRNA, hypothyroidism increased adenohypophysial VGLUT2 mRNA levels. This coincided with a decreased ratio of VGLUT2-immunoreactive TSH cells, regarded as a sign of enhanced secretion. The presence of the glutamate marker VGLUT2 in gonadotrope and thyrotrope cells, and its up-regulation by estrogen or hypothyroidism, address the possibility that endocrine cells of the adenohypophysis may cosecrete glutamate with peptide hormones in an estrogen- and thyroid status-regulated manner. The exact roles of endogenous glutamate observed primarily in gonadotropes and thyrotropes, including its putative involvement in autocrine/paracrine regulatory mechanisms, will require clarification. PMID- 16675530 TI - Sprouty2 is involved in male sex organogenesis by controlling fibroblast growth factor 9-induced mesonephric cell migration to the developing testis. AB - Fibroblast growth factor 9 (FGF9) signal has a role in organogenesis of the mammalian testis by controlling migration of mesonephric cells to the XY gonad, but neither it nor the FGF receptors is expressed sex-specifically. Of the Sprouty genes encoding antagonists of receptor tyrosine kinases including FGFr, mSprouty2 expression was confined to the developing testis and mesonephros. Gain of SPROUTY2 function in the male genital ridge and mesonephros malformed the vas deferens and epididymis, and diminished the number of seminiferous tubules and interstitium associating with reduced mesonephric cell migration and Fgf9 expression in embryonic testis, whereas exogenous FGF9 signaling recovered mesonephric cell migration inhibited by SPROUTY2. These phenotypes associated also with the decreased expression of Sox9, Desert hedgehog, Hsd3beta, Platelet/endothelial cell adhesion molecule, and alpha-smooth muscle actin, which are markers of the Sertoli, Leydig, endothelial, and peritubular myoid cells of the developing testis. Based on these data, we propose that the Sprouty proteins are involved normally in mediating the sexually dimorphic signaling of FGF9 and controlling cell migration from the mesonephros during testis development. PMID- 16675531 TI - Exaggerated behavioral phenotypes in Fmr1/Fxr2 double knockout mice reveal a functional genetic interaction between Fragile X-related proteins. AB - Individuals affected by Fragile X syndrome (FXS) experience cognitive impairment, hyperactivity, attention deficits, social anxiety and autistic-like behaviors. FXS results from the loss of expression of the Fragile X mental retardation (FMR1) gene, whose protein product FMRP is thought to play an important role in neuronal function and synaptic plasticity. Two paralogs of FMRP, FXR1P and FXR2P, have been identified, forming the Fragile X-related (FXR) family of proteins. Although the functions of FXR1P and FXR2P are not well understood, there are similarities among all three FXR proteins in gene structure, amino acid sequence, expression pattern and cellular functions. Mouse models have been described for loss of Fmrp, Fxr1p and Fxr2p, the mouse homologs of FMRP, FXR1P and FXR2P. In earlier studies, we found that Fmr1 knockout (KO) mice, which do not express Fmrp, and Fxr2 KO mice, which do not express Fxr2p, show similarities in some behavioral responses such as hyperactivity. To better understand the functional relationship between FMRP and FXR2P, we generated Fmr1 KO, Fxr2 KO, Fmr1/Fxr2 double KO and wild-type control mice as littermates on the same genetic background and examined them in several behavioral assays. Results show that Fmr1/Fxr2 double KO mice have exaggerated behavioral phenotypes in open-field activity, prepulse inhibition of acoustic startle response and contextual fear conditioning when compared with Fmr1 KO mice, Fxr2 KO mice or wild-type littermates. Our findings suggest that Fmr1 and Fxr2 genes contribute in a cooperative manner to pathways controlling locomotor activity, sensorimotor gating and cognitive processes. PMID- 16675532 TI - Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions. AB - Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts. PMID- 16675533 TI - Modeling the relation between socioeconomic status and mortality in a mixture of majority and minority ethnic groups. AB - Ethnic variation in mortality and whether this variation can be explained by socioeconomic status are of substantive interest to social epidemiologists. The authors consider the analysis of mortality data for a mixture of majority and minority ethnic groups. Such data are likely to be coarsely cross-classified by age and socioeconomic status and yet, even then, in some cells of this cross classification the observed mortality rate will be an imprecise estimate of the underlying rate. The authors illustrate conventional and Bayesian approaches to analysis with data from the 1996 census used by the New Zealand Census-Mortality Study. A conventional approach is exploratory data analysis first followed by Poisson regression. The authors use spline smoothing within a generalized additive model framework as an exploratory data analysis, following a strategy of adding just enough model structure to gain a sensible picture. A Bayesian approach is modeling first and then a description of posterior estimates using exploratory data analysis techniques. The authors use hierarchical Poisson regression and then illustrate their posterior estimates of the mortality rate using the same spline smoothing as before. The advantage of the hierarchical Bayesian approach is that it assesses uncertainty about a Poisson regression model proposed a priori; the conventional approach assumes that the fitted Poisson regression model is correct. All analyses use software that is available at no cost. PMID- 16675534 TI - Invited commentary: taking the search for causes of schizophrenia to a different level. AB - In recent years, epidemiologists have established major variations in the incidence of schizophrenia and have begun to investigate the causes of these variations. The report by Pedersen and Mortensen (Am J Epidemiol 2006;163:971-8) in this issue of the Journal examines the contribution of family-level factors to the urban-rural difference in the incidence of schizophrenia. Their results suggest that familial life in urban environments confers some effect that persists after families move to rural settings. Taking these findings together with those of previous studies, it appears that factors operating at the level of the social context, the family, and the individual may all contribute to the urban-rural difference in schizophrenia incidence. This work exemplifies an integrative, multilevel approach to epidemiologic research that employs principles central to eco-epidemiology and other, similar frameworks. PMID- 16675535 TI - Are the cause(s) responsible for urban-rural differences in schizophrenia risk rooted in families or in individuals? AB - Many studies have identified urban-rural differences in schizophrenia risk. Hypothetical underlying cause(s) may include toxic exposures, diet, infections, and selective migration. The authors investigated whether the underlying cause(s) responsible for the urban-rural differences were rooted in families or in individuals. Linking data from the Danish Civil Registration System and the Danish Psychiatric Central Register, a population-based cohort of 711,897 people aged 15 years or more was established. Overall, 2,720 persons developed schizophrenia during the period 1970-2001. The authors evaluated whether the nearest older sibling's place of birth had an independent effect on schizophrenia risk. If the cause(s) responsible for the urban-rural differences are rooted in individuals only, the nearest older sibling's place of birth should have no independent effect. In this analysis, the nearest older sibling's place of birth had an independent effect; among persons who lived in a rural area during their first 15 years of life, the relative risk was 1.59 (95% confidence interval: 1.10, 2.30) if their nearest older sibling had been born in the capital area as compared with a rural area. Some of the cause(s) responsible for the urban-rural differences in schizophrenia risk are rooted in families, but some might also be rooted in individuals. PMID- 16675536 TI - Algorithms for converting random-zero to automated oscillometric blood pressure values, and vice versa. AB - Many surveys and cohort studies have used a random-zero sphygmomanometer blood pressure device (RZS) to measure subjects' blood pressure and to assess the value of blood pressure in predicting cardiovascular events. Recent studies used automated oscillometric blood pressure devices (AODs) that systematically measure higher blood pressure values than RZSs do, hampering comparability of values between these studies. In 2000-2003, the authors randomly used both an RZS and an AOD in an ongoing cohort study in Germany. This analysis aimed to compare blood pressure values by device and to develop an algorithm to convert estimates of blood pressure values from one device to the other. In a randomized subset of 2,365 subjects aged 45-75 years, each subject was measured three times with each device in a randomized order. The mean difference (AOD-RZS) between the devices was 3.9 mmHg for systolic blood pressure and 2.6 mmHg for diastolic blood pressure. The authors found that linear regression models including age, sex, and blood pressure level can be used to convert RZS blood pressure values to AOD blood pressure values, and vice versa. Results may help to better compare blood pressure values in epidemiologic studies that used different blood pressure devices. PMID- 16675537 TI - Environmental risk factors for infection with Mycobacterium avium complex. AB - Infection with Mycobacterium avium complex is acquired from the environment, but risk factors for M. avium complex infection and disease are poorly understood. To identify risk factors for infection, the authors performed a 1998-2000 cross sectional study in western Palm Beach County, Florida, using a population-based random household survey. M. avium complex infection was identified by use of the M. avium sensitin skin test. Of 447 participants, 147 (32.9%) had a positive test reaction, 186 (41.6%) had a negative test reaction, and, for 114 (25.5%), test results were indeterminate. Among the 333 participants with positive or negative M. avium sensitin skin tests, age-adjusted independent predictors of M. avium complex infection in a multivariate model included Black race (odds ratio = 3.8, 95% confidence interval: 2.2, 6.6), birth outside the United States (odds ratio = 2.1, 95% confidence interval: 1.1, 3.9), and more than 6 years' cumulative occupational exposure to soil (odds ratio = 2.7, 95% confidence interval: 1.3, 6.0). Exposure to water, food, or pets was not associated with infection. Results indicate that soil is a reservoir for M. avium complex associated with human infection and that persons whose occupations involve prolonged soil exposure are at increased risk of M. avium complex infection. PMID- 16675538 TI - Risk factors for the incidence of endometrial cancer according to the aggressiveness of disease. AB - There is a wide range of aggressiveness of endometrial tumors, some being indolent and easily treated while others metastasize and prove fatal. The authors used data from three population-based, case-control studies to determine if etiologic factors differ for aggressive disease. Interview data were obtained from 1,304 female residents of western Washington State who were 45-74 years of age and diagnosed with endometrial cancer during 1985-1991, 1994-1995, and 1997 1999 and from 1,779 controls who were of similar ages and selected primarily by random digit dialing. As a means of gauging aggressiveness, tumor characteristics were abstracted from the population-based cancer registry that serves western Washington State. The risk of endometrial cancer among long-term users (> or = 8 years) of unopposed estrogens was particularly high for the least aggressive tumors (odds ratio = 18.6, 95% confidence interval: 12.2, 28.6) but was elevated for moderate and highly aggressive tumors as well (odds ratios = 6.6 and 7.1, respectively). Women who were obese, had a history of diabetes, and had fewer than two children were also at increased risk, regardless of tumor aggressiveness, while oral contraceptive users were at decreased risk of only relatively more aggressive disease. In general, a woman's risk of endometrial cancer appears to be influenced by similar risk factors regardless of disease severity. PMID- 16675539 TI - Sibship characteristics and risk of multiple sclerosis: a nationwide cohort study in Denmark. AB - It has been hypothesized that age at infection with a common microbial agent may be associated with the risk of multiple sclerosis (MS). The authors addressed this hypothesis by using number of older siblings and other sibship characteristics as an approximation of age at exposure to common infections. Data on family characteristics and vital status from the Danish Civil Registration System were used to establish a cohort of all Danes whose mothers had been born in Denmark since 1935. Persons diagnosed with MS during the period 1968-1998 were identified through linkage with the Danish Multiple Sclerosis Register. The cohort of 1.9 million Danes was followed for 28.1 million person-years; during that time, 1,036 persons developed MS. Overall, there was no association between number of older siblings, number of younger siblings, total number of siblings, age distance from the nearest younger sibling, or exposure to younger siblings under 2 years of age and risk of MS later in life. There was no association of MS risk with multiple birth (vs. singleton birth) or with the age of the mother or father at birth. These results do not lend support to the hypothesis that number of older siblings or any of the other sibship characteristics studied is associated with risk of MS. PMID- 16675540 TI - Luteinizing hormone-induced RUNX1 regulates the expression of genes in granulosa cells of rat periovulatory follicles. AB - The LH surge induces specific transcription factors that regulate the expression of a myriad of genes in periovulatory follicles to bring about ovulation and luteinization. The present study determined 1) the localization of RUNX1, a nuclear transcription factor, 2) regulation of Runx1 mRNA expression, and 3) its potential function in rat ovaries. Up-regulation of mRNA and protein for RUNX1 is detected in preovulatory follicles after human chorionic gonadotropin (hCG) injection in gonadotropin-treated immature rats as well as after the LH surge in cycling animals by in situ hybridization and immunohistochemical and Western blot analyses. The regulation of Runx1 mRNA expression was investigated in vitro using granulosa cells from rat preovulatory ovaries. Treatments with hCG, forskolin, or phorbol 12 myristate 13-acetate stimulated Runx1 mRNA expression. The effects of hCG were reduced by inhibitors of protein kinase A, MAPK kinase, or p38 kinase, indicating that Runx1 expression is regulated by the LH-initiated activation of these signaling mediators. In addition, hCG-induced Runx1 mRNA expression was inhibited by a progesterone receptor antagonist and an epidermal growth factor receptor tyrosine kinase inhibitor, whereas amphiregulin stimulated Runx1 mRNA expression, demonstrating that the expression is mediated by the activation of the progesterone receptor and epidermal growth factor receptor. Finally, knockdown of Runx1 mRNA by small interfering RNA decreased progesterone secretion and reduced levels of mRNA for Cyp11a1, Hapln1, Mt1a, and Rgc32. The hormonally regulated expression of Runx1 in periovulatory follicles, its involvement in progesterone production, and regulation of preovulatory gene expression suggest important roles of RUNX1 in the periovulatory process. PMID- 16675541 TI - Diminished growth and enhanced glucose metabolism in triple knockout mice containing mutations of insulin-like growth factor binding protein-3, -4, and -5. AB - IGF-I and IGF-II are essential regulators of mammalian growth, development and metabolism, whose actions are modified by six high-affinity IGF binding proteins (IGFBPs). New lines of knockout (KO) mice lacking either IGFBP-3, -4, or -5 had no apparent deficiencies in growth or metabolism beyond a modest growth impairment (approximately 85-90% of wild type) when IGFBP-4 was eliminated. To continue to address the roles of these proteins in whole animal physiology, we generated combinational IGFBP KO mice. Mice homozygous for targeted defects in IGFBP-3, -4, and -5 remain viable and at birth were the same size as IGFBP-4 KO mice. Unlike IGFBP-4 KO mice, however, the triple KO mice became significantly smaller by adulthood (78% wild type) and had significant reductions in fat pad accumulation (P < 0.05), circulating levels of total IGF-I (45% of wild type; P < 0.05) and IGF-I bioactivity (37% of wild type; P < 0.05). Metabolically, triple KO mice showed normal insulin tolerance, but a 37% expansion (P < 0.05) of beta cell number and significantly increased insulin secretion after glucose challenge, which leads to enhanced glucose disposal. Finally, triple KO mice demonstrated a tissue-specific decline in activation of the Erk signaling pathway as well as weight of the quadriceps muscle. Taken together, these data provide direct evidence for combinatorial effects of IGFBP-3, -4, and -5 in both metabolism and at least some soft tissues and strongly suggest overlapping roles for IGFBP-3 and -5 in maintaining IGF-I-mediated postnatal growth in mice. PMID- 16675542 TI - Molecular mechanism of inhibitory aryl hydrocarbon receptor-estrogen receptor/Sp1 cross talk in breast cancer cells. AB - The trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor alpha (ERalpha)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ERalpha/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ERalpha/Sp1 and the AhR are constitutively bound to the CAD gene promoter and only minor changes are observed after treatment with 17beta-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ERalpha-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ERalpha/Sp1 cross talk is due, in part, to enhanced association of AhR and ERalpha (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ERalpha/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor. PMID- 16675543 TI - Coordinate regulation of neuropeptide Y and agouti-related peptide gene expression by estrogen depends on the ratio of estrogen receptor (ER) alpha to ERbeta in clonal hypothalamic neurons. AB - Neuropeptide Y (NPY) and agouti-related peptide (AgRP) stimulate feeding, whereas NPY also facilitates the estrogen-mediated preovulatory GnRH surge. In addition to regulating reproductive function, estrogen also acts as an anorexigenic hormone, although it is not yet known which hypothalamic neurons are involved in this process. We hypothesize that estrogen may directly control hypothalamic NPY and/or AgRP synthesis to influence energy homeostasis. Using two clonal, murine hypothalamic neuronal cell models, N-38 and N-42, we demonstrate that 17beta estradiol differentially regulates estrogen receptor (ER)alpha and ERbeta levels, as well as NPY and AgRP gene expression in a manner that is temporally coordinated with the changes in ER abundance. The estrogen-mediated repression of NPY and AgRP mRNA levels in N-38 and N-42 neurons require either ERalpha and ERbeta or ERalpha alone, respectively, whereas the induction of NPY and AgRP in N 38 neurons is strictly ERbeta dependent, as assessed by ER-specific agonists and small interfering RNA knockdown of ERalpha or ERbeta. Through transient transfection analysis in N-38 neurons, we have mapped the estrogen-mediated repression of NPY to within -1078 of the 5' regulatory region of the NPY gene. Our results provide the first evidence that NPY and AgRP gene expression is directly regulated by estrogen in specific hypothalamic neurons, and that this regulation is dependent upon the ratio of ERbeta to ERalpha. The biphasic control of neuronal NPY/AgRP transcription may be a mechanism by which estrogen has distinct effects on both energy homeostasis and reproduction. PMID- 16675546 TI - Initial changes in pulpal microvasculature during orthodontic tooth movement: a stereological study. AB - Any alteration in blood flow or vascular pressure caused by a trauma may damage the pulp tissue. The aim of this study was to evaluate the vascular changes during the initial period of tooth movement. These alterations were assessed in coronal molar pulp tissue of 20 male Wistar rats, 90 days of age, submitted to mesial inclination movement by a closed coil spring, placed from the right maxillary first molar to the maxillary incisors. The animals were divided into three experimental groups of 6, 24, and 72 hours of 0.4 N force application, with five animals in each group, and a control group of five animals without tooth movement. The volume density of blood vessels (V(v)) of the coronal pulp tissue in the experimental groups was calculated by stereology and compared with the control group. The results demonstrated a significant increase in V(v) at 6 hours of 10.2 per cent compared with 7.2 per cent for the control group (P 0.05). These results demonstrate the high capacity of adaptation of the pulp tissue to an aggression, provided the biological limits of tolerance of the pulp are respected. PMID- 16675545 TI - Decreased expression of the angiogenic regulators CYR61 (CCN1) and NOV (CCN3) in human placenta is associated with pre-eclampsia. AB - The pregnancy disorder pre-eclampsia (PE) is thought to be caused in part by shallow invasion of the extravillous trophoblast (EVT) leading to uteroplacental insufficiency and hypoxia. Here, we focused on the expressions of cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3), members of the CCN family of angiogenic regulators, in human placenta during normal pregnancy compared with pre-eclamptic and HELLP placentae using quantitative RT-PCR, western blotting and immunocytochemistry. During normal pregnancy, both proteins showed increasing expression levels and were strongly coexpressed in endothelial cells of vessels, stromal cells and interstitial EVT giant cells. However, NOV showed an earlier onset of expression in villous endothelial cells during gestation compared with CYR61, which may signify distinct roles of these proteins in placental angiogenesis. In early-onset pre-eclamptic placentae, both CYR61 and NOV were expressed at a significantly lower level compared with normal matched controls. This decrease of CYR61 and NOV in pre-eclamptic placentae is not associated with a decrease of the endothelial marker CD34 or vimentin. No obvious changes in the localization of CYR61 and NOV in pre-eclamptic placentae were detected but a change in the intracellular distribution in trophoblast giant cells. Our data point to a potential role of both molecules in the pathogenesis of early-onset PE. PMID- 16675544 TI - Androgens, progestins, and glucocorticoids induce follicle-stimulating hormone beta-subunit gene expression at the level of the gonadotrope. AB - FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHbeta subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHbeta has been difficult due to the heterogeneity of the anterior pituitary. Immortalized LbetaT2 cells are a model of a mature gonadotrope cell and express the endogenous steroid receptor for each of the three hormones. Transient transfection of each receptor, along with ligand treatment, stimulates the mouse FSHbeta promoter, but induction is severely diminished using receptors that lack the ability to bind DNA, indicating that induction is likely through direct DNA binding. All three steroid hormones act within the first 500 bp of the FSHbeta promoter where six putative hormone response elements exist. The -381 site is critical for FSHbeta induction by all three steroid hormones, whereas the -197 and -139 sites contribute to maximal induction. Interestingly, the -273 and -230 sites are also necessary for androgen and progestin induction of FSHbeta, but not for glucocorticoid induction. Additionally, we find that all three receptors bind the endogenous FSHbeta promoter, in vivo, and specifically bind the -381 site in vitro, suggesting that the binding of the receptors to this element is critical for the induction of FSHbeta by these 3-keto steroid hormones. Our data indicate that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHbeta gene expression in the pituitary gonadotrope. PMID- 16675547 TI - Glycopeptides as versatile tools for glycobiology. AB - This review describes the recent advances in the field of glycopeptide and small glycoprotein synthesis. The strategies covered include chemical and chemoenzymatic synthesis, native chemical ligation (NCL), and expressed chemical ligation. The importance of glycopeptide synthesis is exemplified by giving the reader an overview of how versatile and important these well-defined glycopeptides are as tools in glycobiology. PMID- 16675548 TI - Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. AB - Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions. PMID- 16675549 TI - Evidence for a clade composed of molluscs with serially repeated structures: monoplacophorans are related to chitons. AB - Monoplacophorans are among the rarest members of the phylum Mollusca. Previously only known from fossils since the Cambrian, the first living monoplacophoran was discovered during the famous second Galathea deep-sea expedition. The anatomy of these molluscs shocked the zoological community for presenting serially repeated gills, nephridia, and eight sets of dorsoventral pedal retractor muscles. Seriality of organs in supposedly independent molluscan lineages, i.e., in chitons and the deep-sea living fossil monoplacophorans, was assumed to be a relic of ancestral molluscan segmentation and was commonly accepted to support a direct relationship with annelids. We were able to obtain one specimen of a monoplacophoran Antarctic deep-sea species for molecular study. The first molecular data on monoplacophorans, analyzed together with the largest data set of molluscs ever assembled, clearly illustrate that monoplacophorans and chitons form a clade. This "Serialia" concept may revolutionize molluscan systematics and may have important implications for metazoan evolution as it allows for new interpretations for primitive segmentation in molluscs. PMID- 16675550 TI - Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation. AB - The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA) differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK 1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP 1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype. PMID- 16675551 TI - An antibody produced in tobacco expressing a hybrid beta-1,4 galactosyltransferase is essentially devoid of plant carbohydrate epitopes. AB - N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human beta-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT. PMID- 16675552 TI - The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation. AB - Filopodia are highly dynamic finger-like cell protrusions filled with parallel bundles of actin filaments. Previously we have shown that Diaphanous-related formin dDia2 is involved in the formation of filopodia. Another key player for the formation of filopodia across many species is vasodilator-stimulated phosphoprotein (VASP). It has been proposed that the essential role of VASP for formation of filopodia is its competition with capping proteins for filament barbed-end interaction. To better understand the function of VASP in filopodium formation, we analyzed the in vitro and in vivo properties of Dictyostelium VASP (DdVASP) and extended our findings to human VASP. Recombinant VASP from both species nucleated and bundled actin filaments, but did not compete with capping proteins or block depolymerization from barbed ends. Together with the finding that DdVASP binds to the FH2 domain of dDia2, these data indicate that the crucial role of VASP in filopodium formation is different from uncapping of actin filaments. To identify the activity of DdVASP required in this process, rescue experiments of DdVASP-null cells with mutant DdVASP constructs were performed. Only WT DdVASP, but not a mutant lacking the F-actin bundling activity, could rescue the ability of these cells to form WT-like filopodia. Our data suggest that DdVASP is complexed with dDia2 in filopodial tips and support formin mediated filament elongation by bundling nascent actin filaments. PMID- 16675553 TI - On the dynamics of molecular conformation. AB - Understanding the mechanism of fast transitions between conformed states of large biomolecules is central to reconciling the dichotomy between the relatively high speed of metabolic processes and slow (random-walk based) estimates on the speed of biomolecular processes. Here we use the dynamical systems approach to suggest that the reduced time of transition between different conformations is due to features of the dynamics of molecules that are a consequence of their structural features. Long-range and local effects both play a role. Long-range molecular forces account for the robustness of final states and nonlinear processes that channel localized, bounded disturbances into collective, modal motions. Local interconnections provide fast transition dynamics. These properties are shared by a class of networked systems with strong local interconnections and long-range nonlinear forces that thus exhibit flexibility and robustness at the same time. PMID- 16675554 TI - Farm-scale evaluation of the impacts of transgenic cotton on biodiversity, pesticide use, and yield. AB - Higher yields and reduced pesticide impacts are needed to mitigate the effects of agricultural intensification. A 2-year farm-scale evaluation of 81 commercial fields in Arizona show that use of transgenic Bacillus thuringiensis (Bt) cotton reduced insecticide use, whereas transgenic cotton with Bt protein and herbicide resistance (BtHr) did not affect herbicide use. Transgenic cotton had higher yield than nontransgenic cotton for any given number of insecticide applications. However, nontransgenic, Bt and BtHr cotton had similar yields overall, largely because higher insecticide use with nontransgenic cotton improved control of key pests. Unlike Bt and BtHr cotton, insecticides reduced the diversity of nontarget insects. Several other agronomic and ecological factors also affected biodiversity. Nevertheless, pairwise comparisons of diversity of nontarget insects in cotton fields with diversity in adjacent noncultivated sites revealed similar effects of cultivation of transgenic and nontransgenic cotton on biodiversity. The results indicate that impacts of agricultural intensification can be reduced when replacement of broad-spectrum insecticides by narrow-spectrum Bt crops does not reduce control of pests not affected by Bt crops. PMID- 16675555 TI - Advanced generation adenoviral virotherapy agents embody enhanced potency based upon CAR-independent tropism. PMID- 16675556 TI - Chk2 molecular interaction map and rationale for Chk2 inhibitors. AB - To organize the rapidly accumulating information on bioregulatory networks related to the histone gamma-H2AX-ATM-Chk2-p53-Mdm2 pathways in concise and unambiguous diagrams, we used the molecular interaction map notation (http://discover.nci.nih.gov/min). Molecular interaction maps are particularly useful for networks that include protein-protein binding and posttranslational modifications (e.g., phosphorylation). Both are important for nearly all of the proteins involved in DNA double-strand break signaling. Visualizing the regulatory circuits underlying cellular signaling may help identify key regulatory reactions and defects that can serve as targets for anticancer drugs. PMID- 16675557 TI - Ectopic expression of the proto-oncogene Mer in pediatric T-cell acute lymphoblastic leukemia. AB - PURPOSE: The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. EXPERIMENTAL DESIGN: Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. RESULTS: Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. CONCLUSIONS: Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy. PMID- 16675558 TI - Presence of circulating CCR10+ T cells and elevated serum CTACK/CCL27 in the early stage of mycosis fungoides. AB - PURPOSE: Mycosis fungoides (MF), a common type of cutaneous T cell lymphoma with an indolent clinical course, has the characteristic that malignant T cell clones are recruited into the skin from the early disease stages. The mechanisms of recruitment have been suggested from our knowledge of various chemokine-chemokine receptor interactions. Recently, CCR10 and CTACK/CCL27 were proposed to play a role in the recruitment of other types of cutaneous T cell lymphoma. We examined the expression of CCR10 in peripheral blood and serum CTACK/CCL27 levels in patients with MF. EXPERIMENTAL DESIGN: Eighteen patients with MF, six patients with atopic dermatitis, and nine healthy volunteers were enrolled in our investigation. We investigated the differences in CCR10+ CD4+ expression in peripheral blood mononuclear cells by flow cytometry. Serum CTACK/CCL27 levels were determined using a CTACK/CCL27 ELISA assay kit. RESULTS: The number of circulating CCR10+ CD4+ cells was significantly higher in MF peripheral blood than in controls, even during the early stages. In lesional MF skin, infiltrating tumor cells also showed extensive expression of CCR10. The serum level of CTACK/CCL27 was higher in patients with MF than normal controls, but no statistical difference was found compared with atopic dermatitis patients. CONCLUSIONS: CCR10-CTACK/CCL27 interactions between circulating T cells and keratinocytes would seem to play an important role in the pathophysiology of MF from the early disease stages. PMID- 16675559 TI - Tumor-driven paracrine platelet-derived growth factor receptor alpha signaling is a key determinant of stromal cell recruitment in a model of human lung carcinoma. AB - Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRalpha ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRalpha was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRalpha mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRalpha in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors. PMID- 16675560 TI - Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma. AB - PURPOSE AND EXPERIMENTAL DESIGN: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2K(d) restricted mouse GPC3(298-306) (EYILSLEEL) peptide prevented the growth of tumor expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2K(d) and HLA-A24 (A*2402), the GPC3(298-306) peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3(298-306) peptide could induce GPC3 reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402)+ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)-restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2+ HCC patients. RESULTS: We found that the GPC3(144-152) (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2+ GPC3+ HCC patients, the GPC3(144-152) peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3(298 306) peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24+ GPC3+ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/severe combined immunodeficiency mice. CONCLUSION: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients. PMID- 16675561 TI - Mechanisms and effects of loss of human leukocyte antigen class II expression in immune-privileged site-associated B-cell lymphoma. AB - PURPOSE AND EXPERIMENTAL DESIGN: Loss of human leukocyte antigen (HLA) expression on tumor cells is frequent in diffuse large B-cell lymphoma (DLBCL) arising in immune-privileged sites, such as the testis and central nervous system, and is associated with small homozygous deletions of HLA-DQ/HLA-DR and larger hemizygous deletions of the MHC region. To better understand the significance of down regulation of HLA class II expression in relation to the homozygous and hemizygous deletions, we analyzed global gene expression patterns in a series of 26 testicular DLBCL after characterization of these deletions. RESULTS: Low levels of HLA-DR mRNA in whole testicular DLBCL samples were associated with a strong down-regulation of numerous immune-related genes specific for T cells, macrophages, antigen presentation and processing, lymphocyte activation, chemokines and chemokine receptors, and the complement system. The number of CD3+ tumor-infiltrating T cells was also significantly lower in low expressors of HLA DR mRNA. Interestingly, hemizygous and homozygous deletions in the MHC region did not have any additional effect on global gene expression. CONCLUSION: In conclusion, we found that loss of HLA class II mRNA expression in testicular DLBCL is associated with a significant change in global gene expression patterns. This effect is independent of the mechanism causing the down-regulation of HLA class II genes in the lymphoma cells. PMID- 16675562 TI - Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells: implications for radiotherapy of hepatocellular carcinoma. AB - PURPOSE: To investigate the clinical benefit of additional radiotherapy to patients with unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization (TACE) and the molecular effects of radiation on gene expression in hepatoma cells. EXPERIMENTAL DESIGN: Between August 1996 and August 2003, 276 and 64 patients with American Joint Committee on Cancer stage T3N0M0 hepatocellular carcinoma receiving TACE alone and TACE followed by three dimensional conformal radiotherapy, respectively, at our institution were studied. Clinical outcome and pattern of failure were analyzed for the association of survival benefit with radiotherapy. The molecular effects of radiotherapy were studied in vitro and in vivo using human hepatoma cells with different p53 mutation and hepatitis B virus infection status. RESULTS: Median follow-up and survival time in the TACE alone and TACE + radiotherapy groups were 39 and 19 months, and 51 and 17 months, respectively. Additional radiotherapy to TACE did not improve overall survival (P = 0.65). However, different failure patterns were noted after TACE and after radiotherapy. Although all irradiated tumors regressed substantially, radiotherapy rapidly enhanced both intrahepatic and extrahepatic tumor progression outside the radiotherapy treatment field in a significant portion of patients, which offset the benefit of radiotherapy on overall survival. In molecular analysis of the radiation effects on human hepatoma cells, radiotherapy rapidly induced p53-independent transcriptional up regulation of vascular endothelial growth factor (VEGF), increased VEGF secretion in a dose-, time-, and cell type-dependent manner, and promoted hepatoma cell growth in vivo with enhanced intratumor angiogenesis, which correlated well with elevated levels of serum VEGF. CONCLUSIONS: Radiotherapy to eradicate a primary hepatocellular carcinoma might result in the outgrowth of previously dormant microtumors not included in the radiotherapy treatment field. Radiotherapy induced VEGF could be a paracrine proliferative stimulus. Therapeutic implications of the study justify the combination of three-dimensional conformal radiotherapy with anti-VEGF angiogenic modalities for the treatment of unresectable hepatocellular carcinoma to reduce relapses. PMID- 16675563 TI - Genomic alterations in human malignant glioma cells associate with the cell resistance to the combination treatment with tumor necrosis factor-related apoptosis-inducing ligand and chemotherapy. AB - PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical development as a cancer therapeutic agent. Many human malignant glioma cells, however, are resistant to TRAIL treatment. We, therefore, investigated the genomic alterations in TRAIL-resistant malignant glioma cells. EXPERIMENTAL DESIGN: Seven glioma cell lines and two primary cultures were first analyzed for their sensitivity to TRAIL and chemotherapy and then examined for the genomic alterations in key TRAIL apoptotic genes by comparative genomic hybridization (CGH), G-banding/spectral karyotyping, and fluorescence in situ hybridization (FISH). RESULTS: CGH detected loss of the chromosomal regions that contain the following genes: 8p12-p23 (DR4 and DR5), 2q33-34 (caspase-8), 11q13.3 (FADD), 22q11.2 (Bid), and 12q24.1-q24.3 (Smac/DIABLO) in TRAIL-resistant cell lines. Spectral karyotyping showed numerical and structural aberrations involving the chromosomal regions harboring these genes. A combination of G banding/spectral karyotyping and FISH further defined the loss or gain of gene copy of these genes and further showed the simultaneous loss of one copy of DR4/DR5, caspase-8, Bid, and Smac in two near-triploid cell lines that were resistant to the combination treatment with TRAIL and chemotherapy. Loss of the caspase-8 locus was also detected in a primary culture in correlation with the culture resistance to the combined TRAIL and chemotherapy treatment. CONCLUSIONS: The study identifies chromosomal alterations in TRAIL apoptotic genes in the glioma cells that are resistant to the treatment with TRAIL and chemotherapy. These genetic alterations could be used to predict the responsiveness of malignant gliomas to TRAIL-based therapies in clinical treatment of the tumors. PMID- 16675564 TI - Expression and nuclear localization of ErbB3 in prostate cancer. AB - PURPOSE: The ErbB1 and ErbB2 receptors have been implicated in prostate cancer progression, but less is known about the role and biology of other ErbB receptor family members in prostate cancer. The aim of this study was to analyze the expression and localization of ErbB3 in prostate tissues and prostate cancer cell lines. EXPERIMENTAL DESIGN: Immunohistochemistry of ErbB3 was done on prostate cancer tissue sections from 143 patients and on a tissue microarray containing 390 cores of radical prostatectomy-derived specimens representing normal, prostatic intraepithelial neoplasia, and malignant tissues from 81 patients. ErbB3 subcellular localization was studied by Western blot analysis in LNCaP, 22Rv1, PC-3, and DU145 prostate cancer cell lines. RESULTS: Immunohistochemistry analysis of prostate cancer tissues revealed that >90% of prostate cancer tissues displayed cytoplasmic ErbB3 staining. Minimal ErbB3 nuclear staining was observed in normal prostate tissues and benign prostatic hyperplasia tissues; in contrast, ErbB3 was frequently localized in the nucleus of cancerous tissues. This nuclear localization was more frequent (P < 0.001) in hormone-refractory tissues (17 of 17, 100%) compared with hormone-sensitive samples (37 of 92, 40.2%). Additionally, in the tissue microarray, increased nuclear ErbB3 was associated with increasing Gleason grade. Interestingly, Western blot analysis of cytoplasmic and nuclear subcellular fractions showed that ErbB3 nuclear localization was more prevalent in hormone-sensitive prostate cancer cell lines (LNCaP and 22Rv1) compared with hormone-insensitive cell lines (PC-3 and DU145). CONCLUSIONS: ErbB3 nuclear localization discriminates normal from malignant prostate tissues and between tumors from hormone-sensitive versus hormone refractory prostate cancer. ErbB3 nuclear staining seems to be associated with risk of disease progression. The high frequency of ErbB3 nuclear localization in hormone-refractory tissues indicates that ErbB3 warrants further study to understand its association with prostate cancer disease progression. PMID- 16675565 TI - Thymidylate synthase expression in colon carcinomas with microsatellite instability. AB - PURPOSE: Colon cancer cells with high-frequency microsatellite instability (MSI H) display resistance to 5-fluorouracil (5-FU) that can be reversed by restoring DNA mismatch repair (MMR) proficiency. Given that thymidylate synthase (TS) is inhibited by 5-FU, we studied the relationship between MSI and TS expression, and the prognostic effect of these and other markers (i.e., p53 and 17p allelic imbalance). EXPERIMENTAL DESIGN: Dukes' stage B2 and C colon carcinomas (n = 320) from participants in 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p allelic imbalance. Expression of MMR (hMLH1, hMSH2), TS, and p53 proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival were determined. RESULTS: Of 320 cancers studied, 60 (19%) were MSI-H. TS expression variables were similar in MSI-H and microsatellite stable/low-frequency MSI (MSS/MSI-L) cancers, and unrelated to MMR proteins. MSI-H tumors had lower stage (P = 0.0007), fewer metastatic lymph nodes (P = 0.004), and improved overall survival (P = 0.01). Loss of MMR proteins was also associated with better overall survival (P = 0.006). None of the TS variables were prognostic. Histologic grade (P = 0.0008) and nodal status (P = 0.0002) were associated with overall survival, in contrast to 17p allelic imbalance or p53. Only MSI status or loss of MMR proteins, histologic grade, and tumor stage were independent markers for overall survival. CONCLUSIONS: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers. PMID- 16675566 TI - Preferential nuclear and cytoplasmic NY-BR-1 protein expression in primary breast cancer and lymph node metastases. AB - PURPOSE: NY-BR-1 is a recently isolated differentiation antigen, which is expressed in normal mammary tissue and in breast cancer. However, current data are based on RT-PCR analysis and nothing is known about the presence of NY-BR-1 on a protein level. We previously generated a monoclonal antibody to NY-BR-1 to study the protein expression of NY-BR-1. METHODS: In our immunohistochemical study, NY-BR-1 was analyzed in normal tissues, various tumor types, 124 primary breast cancers, and 37 paired lymph node metastases. RESULTS: Among normal tissues, NY-BR-1 was present solely in ductal epithelium of the breast. In tumors, carcinoma in situ and invasive carcinoma of the breast were NY-BR-1 positive whereas other tumors and normal tissues were negative. Sixty percent of invasive breast carcinomas were NY-BR-1 positive, displaying cytoplasmic and/or nuclear immunoreactivity. This coexpression was verified by confocal microscopy. Although the monoclonal antibody identified intratumoral heterogeneity, a majority (72%) of NY-BR-1-positive carcinomas revealed immunoreactivity in >50% of the tumor cells. NY-BR-1 expression was more frequent in estrogen receptor positive and lymph node-negative primary carcinomas (P < 0.05 each) and was more common in grade 1 (77%) than in grade 2 (63%) or grade 3 (50%) carcinomas (P < 0.05). This suggests that NY-BR-1 expression is lost with tumor progression. Forty-nine percent of lymph node metastases were NY-BR-1 positive. CONCLUSION: This study supports the notion that NY-BR-1 is a differentiation antigen of the breast, which is present in normal and tumorous mammary epithelium. The organ specific expression of NY-BR-1 and its high prevalence in metastases indicate that it could be a valuable target for cancer immunotherapy. PMID- 16675567 TI - Biological characteristics in bladder cancer depend on the type of genetic instability. AB - PURPOSE: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. EXPERIMENTAL DESIGN: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry. RESULTS: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. CONCLUSIONS: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability. PMID- 16675568 TI - Inflammatory biomarkers for persistent fatigue in breast cancer survivors. AB - PURPOSE: This study seeks to define immunologic and inflammatory variables associated with persistent post-treatment fatigue in breast cancer survivors. EXPERIMENTAL DESIGN: Leukocyte subsets, plasma inflammatory markers, and ex vivo proinflammatory cytokine production were assessed in 50 fatigued and nonfatigued breast cancer survivors recruited > or = 2 years after successful primary therapy. Multivariate statistical analyses were used to define a composite immunologic biomarker of fatigue risk. RESULTS: Fatigued breast cancer survivors were distinguished from nonfatigued survivors by increased ex vivo monocyte production of interleukin (IL)-6 and tumor necrosis factor-alpha following lipopolysaccharide stimulation, elevated plasma IL-1ra and soluble IL-6 receptor (sIL-6R/CD126), decreased monocyte cell-surface IL-6R, and decreased frequencies of activated T lymphocytes and myeloid dendritic cells in peripheral blood (all P < 0.05). An inverse correlation between sIL-6R and cell-surface IL-6R was consistent with inflammation-mediated shedding of IL-6R, and in vitro studies confirmed that proinflammatory cytokines induced such shedding. Multivariate linear discriminant function analysis identified two immunologic markers, the ratio of sIL-6R to monocyte-associated IL-6R and decreased circulating CD69+ T lymphocytes, as highly diagnostic of fatigue (P = 0.0005), with cross-validation estimates indicating 87% classification accuracy (sensitivity = 0.83; specificity = 0.83). CONCLUSION: These results extend links between fatigue and inflammatory markers to show a functional alteration in proinflammatory cytokine response to lipopolysaccharide and define a prognostic biomarker of behavioral fatigue. PMID- 16675570 TI - Class III beta-tubulin overexpression is a marker of poor clinical outcome in advanced ovarian cancer patients. AB - PURPOSE: Overexpression of beta III tubulin has been involved in paclitaxel resistance in several experimental models. We investigated the role of beta III tubulin as predictor of clinical outcome in ovarian cancer patients given platinum/paclitaxel treatment. We also investigated whether beta III tubulin expression could be modified after the selective pressure represented by chemotherapy in vivo. EXPERIMENTAL DESIGN: The study was designed to include a series of consecutive ovarian cancer patients with unresectable disease at time of first surgery, who underwent interval debulking surgery with pathologic assessment of response to treatment with platinum/paclitaxel chemotherapy. Immunostaining was done on formalin-fixed, paraffin-embedded tissue sections from pretreatment and posttreatment tissue biopsies by using the polyclonal rabbit anti-class III beta-tubulin antibody. RESULTS: beta III Tubulin immunoreaction was observed in 51 of 62 (82.2%) cases. beta III Tubulin positivity was neither associated with clinicopathologic variables nor with pathologic response to chemotherapy. Significantly lower percentages of beta III tubulin positivity were observed in posttreatment (range, 5-80%; median, 20%) versus pretreatment (range 10-100%; median, 40%) tissue biopsies (P = 0.0011). Cases with high beta III tubulin expression showed a worse overall survival with respect to cases with low beta III tubulin expression (median overall survival, 25 versus 46 months; P = 0.002). Multivariate analysis showed that high content of beta III tubulin remains independently associated with a worse prognosis. CONCLUSIONS: Assessment of beta III tubulin could be useful to identify poor prognosis ovarian cancer patients candidates to more aggressive and/or targeted therapy. PMID- 16675569 TI - Expression of novel molecules, MICAL2-PV (MICAL2 prostate cancer variants), increases with high Gleason score and prostate cancer progression. AB - PURPOSE: The aim of this study is to identify novel molecular targets for development of novel treatment or diagnostic markers of prostate cancer through genome-wide cDNA microarray analysis of prostate cancer cells purified by laser microdissection. EXPERIMENTAL DESIGN AND RESULTS: Here, we identified molecule interacting with CasL-2 prostate cancer variants (MICAL2-PV), novel splicing variants of MICAL2, showing overexpression in prostate cancer cells. Immunohistochemical analysis using an antibody generated specific to MICAL2-PV revealed that MICAL2-PV was expressed in the cytoplasm of cancer cells with various staining patterns and intensities, whereas it was not or hardly detectable in adjacent normal prostate epithelium or prostatic intraepithelial neoplasia. Interestingly, immunohistochemical analysis of 105 prostate cancer specimens on the tissue microarray indicated that MICAL2-PV expression status was strongly correlated with Gleason scores (P < 0.0001) or tumor classification (P < 0.0001). Furthermore, the expression levels of MICAL2-PVs were also concordant to those of c-Met, a marker of tumor progression, with statistical significance (P = 0.0018). To investigate its potential of molecular therapeutic target for prostate cancers, we knocked down endogenous MICAL2-PVs in prostate cancer cells by small interfering RNA, which resulted in the significant reduction of prostate cancer cell viability. CONCLUSIONS: Our findings suggest that MICAL2-PV is likely to be involved in cancer progression of prostate cancer and could be a candidate as a novel molecular marker and/or target for treatment of prostate cancers with high Gleason score. PMID- 16675571 TI - N-cadherin as a novel prognostic marker of progression in superficial urothelial tumors. AB - PURPOSE: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion and spreading. In bladder cancer, loss or reduced E-cadherin expression has been associated with poor survival, and aberrant expression of N cadherin has been associated with the invasive phenotype of bladder carcinoma cells. The purpose of this study was to investigate whether N-cadherin expression was associated with the bladder tumor progression. EXPERIMENTAL DESIGN: E cadherin and N-cadherin expression was evaluated by immunohistochemistry in 101 tumors (pT1 and pT2-T3) and by reverse transcription-PCR analysis and immunohistochemistry in 28 other fresh frozen tumors (pT(a), pT1, and pT2-T3). RESULTS: N-cadherin expression was absent in normal urothelium, appeared in stage pT1, and increased in pT2-pT3 tumors. In most cases, increased N-cadherin expression in invasive tumors was associated with loss of E-cadherin expression. Progression-free survival and multivariate analyses revealed that N-cadherin expression is an independent prognostic marker for pT1 tumor progression. Analysis of the 28 frozen tumors by immunohistochemistry and reverse transcription-PCR showed a good correlation between protein and gene expression in pT1 and pT2-T3 tumors. Interestingly, in pT(a) tumors, N-cadherin was not immunodetected, whereas mRNA was present in 50% of cases. CONCLUSION: Regulatory defects in the N-cadherin promoter, abnormalities at the translational, or protein processing levels could explain the discrepancies between protein and mRNA expression. Most importantly, this study identified N-cadherin as a novel prognostic marker of progression in superficial urothelial tumors. Clearly, N cadherin acts in an invasive mode in bladder cancer, but whether it has a primary role in urothelial neoplastic progression has yet to be investigated. PMID- 16675572 TI - Prognostic DNA methylation biomarkers in ovarian cancer. AB - PURPOSE: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. EXPERIMENTAL DESIGN: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. CONCLUSION: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease. PMID- 16675573 TI - Overexpression of both CXC chemokine receptor 4 and vascular endothelial growth factor proteins predicts early distant relapse in stage II-III colorectal cancer patients. AB - PURPOSE: CXC chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) are implicated in the metastatic process of malignant tumors. However, no data are currently available on the biological relationship between these molecules in colorectal cancer. We studied whether CXCR4 and VEGF expression could predict relapse and evaluated in vitro the contribution of CXCR4 in promoting clonogenic growth, VEGF secretion, and intercellular adhesion molecule 1 (ICAM-1) expression of colorectal cancer cells. EXPERIMENTAL DESIGN: CXCR4 and VEGF were studied in colorectal cancer tissues and in Lovo, HT29, and SW620 colorectal cancer cell lines by immunohistochemistry. Correlations with baseline characteristics of patients and tumors were analyzed by chi2 test. VEGF secretion induced by CXCL12 was measured by ELISA. The effect of CXCL12 on ICAM-1 expression was evaluated by flow cytometry. Clonogenic growth induced by CXCL12 was determined by clonogenic assays. Functional effects induced by CXCL12 were prevented by the administration in vitro of AMD3100, a bicyclam noncompetitive antagonist of CXCR4. RESULTS: Seventy-two patients, seen between January 2003 and January 2004, were studied. CXCR4 was absent in 16 tumors (22.2%); it was expressed in < or = 50% of cells in 25 (34.7%) tumors and in >50% of cells in 31 (43.0%) tumors. VEGF was absent in 17 (23.6%) tumors; it was expressed in < or = 50% of cells in 16 (22.2%) tumors and in >50% of cells in 39 (54.2%) tumors. There was a significant association between CXCR4 expression and lymph nodal status (P = 0.0393). There were significant associations between VEGF and tumor invasion (P = 0.0386) and lymph nodal involvement (P = 0.0044). American Joint Committee on Cancer stage (P = 0.0016), VEGF expression (P = 0.0450), CXCR4 expression (P = 0.0428), and VEGF/CXCR4 expression (P = 0.0004) had a significant prognostic value for disease-free survival with univariate analysis. The predictive ability of the American Joint Committee on Cancer stage and of the concomitant and high expression of VEGF and CXCR4 was confirmed by multivariate analysis. Prognosis is particularly unfavorable for patients whose primary tumors express CXCR4 and VEGF in >50% of cells (median disease-free survival in relapsed patients, 5.8 months; hazard ratio of relapse, 8.23; 95% confidence interval, 7.24-14.29). In clonogenic assays, CXCL12 (20 ng/mL/d) significantly increased the number of clones in SW620, HT29, and Lovo cells at 7 and 14 days. Again, CXCL12 was able to stimulate VEGF secretion in SW620, HT29, and Lovo cells as well as up-regulated ICAM-1. These effects were prevented by the administration of AMD3100 (1 micromol/L). CONCLUSIONS: We have shown that concomitant and high expression of CXCR4 and VEGF is a strong and independent predictor of early distant relapse in colorectal cancer. CXCR4 triggers a plethora of phenomena, including stimulation of clonogenic growth, induction of VEGF release, and ICAM-1 up-regulation. These data support the inhibition of CXCR4 to prevent the development of colorectal cancer metastasis. PMID- 16675574 TI - Minimal residual disease in hairy cell leukemia patients assessed by clone specific polymerase chain reaction. AB - Cladribine induces long-term complete remission in hairy cell leukemia (HCL) patients but does not clear minimal residual disease (MRD) according to high sensitivity PCR assays. To quantify MRD in patients after anti-CD22 recombinant immunotoxin BL22 and other agents, we used a relative quantitative PCR (RQ-PCR) assay using a primer and probe, both patient specific for the immunoglobulin heavy chain rearrangement. Using this method, we were able to detect one Bonna 12 HCL cell in either 10(6) Jurkat cells or in 10(6) normal mononuclear cells. We studied 84 samples from 10 patients, taken before or after treatment with BL22 and other agents. Patient-specific RQ-PCR was much more sensitive than flow cytometry, which in turn was (as recently reported) more sensitive than PCR using consensus primers. RQ-PCR was positive in 62 of 62 (100%) flow-positive samples in 10 patients and in 20 of 22 (91%) flow-negative samples in six patients. The relative level of MRD as quantified by RQ-PCR correlated with disease status and remission. Thus, patient-specific RQ-PCR is the most sensitive test for MRD in HCL patients and could be used to determine maximal response in patients obtaining multiple cycles of nonmyelotoxic biological treatment for this disease. PMID- 16675575 TI - Effect of calcitriol on prostate-specific antigen in vitro and in humans. AB - BACKGROUND: Calcitriol, the natural ligand for the vitamin D receptor, has significant potential in prostate cancer treatment. Measurement of its antineoplastic activity in prostate cancer clinical trials may be complicated by effects of calcitriol on prostate-specific antigen (PSA) production. We examined the effects of calcitriol at similar concentration on cell proliferation, androgen receptor (AR) expression, and PSA production in vitro and on PSA concentrations in prostate cancer patients. EXPERIMENTAL DESIGN: LNCaP prostate cancer cell proliferation was examined by cell counts 6 days after exposure to a range of concentrations of calcitriol. AR and PSA protein was quantified in LNCaP cells over 96 hours after exposure to 1 nmol/L calcitriol. Serum PSA and free PSA was serially measured by immunoassay over a period of 8 days in patients with hormone-naive prostate cancer after a single dose of 0.5 microg/kg calcitriol. RESULTS: Calcitriol treatment resulted in dose-dependent growth inhibition of LNCaP with approximately 50% growth inhibition at the clinically achievable concentration of 1 nmol/L. Time-dependent up-regulation of AR expression and of PSA production in LNCaP cells was shown at the same concentration. No significant change in serum PSA or free PSA over 8 days was seen in eight subjects treated with a single dose of 0.5 microg/kg calcitriol. The analysis was powered to detect a 1.23-fold change between the baseline and day 8 serum PSA. CONCLUSIONS: At clinically achievable concentrations, calcitriol inhibits growth and induces AR and PSA expression in LNCaP cells. We did not detect similar changes in serum PSA or free PSA in patients exposed to similar concentrations of calcitriol. Thus, a PSA flare, predicted by preclinical systems, is unlikely to occur in patients and therefore unlikely to complicate interpretation of clinical trial outcomes. PMID- 16675576 TI - A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides. AB - Alpha-fetoprotein (AFP) is a self protein expressed by fetal liver at high levels, but is transcriptionally repressed at birth. AFP is up-regulated in hepatocellular carcinomas, and patients with active disease could have plasma levels as high as 1 mg/mL. We previously identified four immunodominant HLA A*0201-restricted peptides [hAFP(137-145) (PLFQVPEPV), hAFP(158-166) (FMNKFIYEI), hAFP(325-334) (GLSPNLNRFL), and hAFP(542-550) (GVALQTMKQ)] derived from human AFP that could stimulate specific T cell responses in healthy donor peripheral blood lymphocytes in vitro. We conducted a phase I/II clinical trial in which HLA A*0201 patients with AFP-positive hepatocellular carcinoma were immunized with three biweekly intradermal vaccinations of the four AFP peptides pulsed onto autologous dendritic cells (DC). DCs were prepared from adherent peripheral blood mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days. Sixteen subjects were enrolled and 10 were treated. Peripheral blood lymphocytes were isolated from these patients before, during, and after AFP peptide/DC immunization and were tested ex vivo with MHC tetramer and IFNgamma ELISPOT analysis. Six of 10 subjects expanded statistically significant levels of AFP-specific T cells postvaccine to at least one peptide by MHC tetramer. Also, 6 of 10 subjects increased IFNgamma producing AFP-specific T cell responses to at least one of the peptides postvaccination, by ELISPOT. We conclude that the human T cell repertoire is capable of responding to the AFP self antigen after the administration of AFP peptide-pulsed DC even in an environment of high circulating levels of this oncofetal antigen. PMID- 16675577 TI - Tumor-specific CD8+ T cell reactivity in the sentinel lymph node of GM-CSF treated stage I melanoma patients is associated with high myeloid dendritic cell content. AB - PURPOSE: Impaired immune functions in the sentinel lymph node (SLN) may facilitate early metastatic events during melanoma development. Local potentiation of tumor-specific T cell reactivity may be a valuable adjuvant treatment option. EXPERIMENTAL DESIGN: We examined the effect of locally administered granulocyte/macrophage-colony stimulating factor (GM-CSF) on the frequency of tumor-specific CD8+ T cells in the SLN and blood of patients with stage I melanoma. Twelve patients were randomly assigned to preoperative local administration of either recombinant human GM-CSF or NaCl 0.9%. CD8+ T cells from SLN and peripheral blood were tested for reactivity in an IFNgamma ELISPOT assay against the full-length MART-1 antigen and a number of HLA-A1, HLA-A2, and HLA-A3 restricted epitopes derived from a range of melanoma-associated antigens. RESULTS: Melanoma-specific CD8+ T cell response rates in the SLN were one of six for the control group and four of six for the GM-CSF-administered group. Only one patient had detectable tumor-specific CD8+ T cells in the blood, but at lower frequencies than in the SLN. All patients with detectable tumor-specific CD8+ T cells had a percentage of CD1a+ SLN-dendritic cells (DC) above the median (i.e., 0.33%). This association between above median CD1a+ SLN-DC frequencies and tumor antigen-specific CD8+ T cell reactivity was significant in a two-sided Fisher's exact test (P = 0.015). CONCLUSIONS: Locally primed antitumor T cell responses in the SLN are detectable as early as stage I of melanoma development and may be enhanced by GM-CSF-induced increases in SLN-DC frequencies. PMID- 16675578 TI - The pharmacokinetics and safety of ABT-751, a novel, orally bioavailable sulfonamide antimitotic agent: results of a phase 1 study. AB - PURPOSE: Microtubules play a critical role in many cellular functions, including cell division and mitosis. ABT-751 is a novel sulfonamide antimitotic that binds to the colchicine site on beta-tubulin that leads to a block in the cell cycle at the G2M phase, resulting in cellular apoptosis. ABT-751 was investigated in this phase 1 trial designed to assess its maximum tolerated dose (MTD), dose-limiting toxicity (DLT), tolerability, and pharmacokinetics. EXPERIMENTAL DESIGN: ABT-751 was administered on a daily (q.d.) or twice daily (b.i.d.) oral schedule for 7 days every 3 weeks to 39 patients with refractory solid tumors. Toxicity was monitored weekly. Plasma and urine ABT-751 and metabolite pharmacokinetics were determined. RESULTS: The MTD for the q.d. schedule was 250 mg/d. DLTs during cycle 1 were abdominal pain, constipation, and fatigue. The MTD on the b.i.d. schedule was 150 mg. Cycle 1 of therapy with the 175 mg b.i.d. schedule was tolerated without DLT. However, six of seven patients reported grade 3 toxicity (ileus, constipation, abdominal pain, or fatigue), which occurred in cycle 2 or 3. ABT-751 was absorbed after oral administration with an overall mean T(max) of about 2 hours. The pharmacokinetics of ABT-751 were dose-proportional and time independent. There was minimal accumulation of ABT-751 after multiple q.d. and b.i.d. doses. Efficacious concentrations, as determined from preclinical models (0.5-1.5 microg/mL), were achieved in all subjects. ABT-751 metabolism occurred primarily by glucuronidation and sulfation. No complete or partial tumor responses were noted, but one patient had a minor response, and four patients had stable disease lasting at least 6 months. CONCLUSIONS: The MTD and recommended phase 2 doses for ABT-751 were 250 mg q.d. and 150 mg b.i.d. on a 7-day schedule given every 3 weeks, due to subsequent cycle toxicities at 175 mg b.i.d. dosing. Toxicities were abdominal pain, constipation, and neuropathy. PMID- 16675579 TI - A phase I study of a new nucleoside analogue, OSI-7836, using two administration schedules in patients with advanced solid malignancies. AB - PURPOSE: To investigate the safety, tolerability, and pharmacokinetic profile of the novel nucleoside analogue OSI-7836 in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: OSI-7836 was initially given as a 60-minute i.v. infusion on day 1 every 21 days. In view of its dose-limiting toxicities, the administration time was amended to a 5-minute bolus, and subsequently, the schedule was amended to weekly for 4 weeks followed by a 2-week rest. Blood and urine samples were collected for pharmacokinetic studies. Analyses of cytokines and lymphocyte subsets were added later in the study to elucidate a mechanism for the severe fatigue and lymphocyte depletion observed in earlier patients. RESULTS: Thirty patients received a total of 61 treatment cycles. Fatigue was the main dose-limiting toxicity. Maximum-tolerated dose was defined as 300 mg/m2 in the 60-minute infusion, (three times per week) schedule; 400 mg/m2 in the 5 minute bolus infusion, (three times per week) schedule; and 100 mg/m2 in the weekly schedule. Other common toxicities were nausea, vomiting, rash, fever, and a flu-like syndrome. There were no clinically significant hematologic toxicities. Following the initial dose, OSI-7836 was eliminated from plasma with a median (range) elimination half-life of 48.3 minutes (22.6-64.8 minutes). Lymphocyte subset analysis showed a significant drop in B cell counts, which persisted to day 14 and beyond. Cytokine analysis showed significant elevations of interleukin 6 and interleukin-10 in all patients who received > or = 200 mg/m2 OSI-7836. Best response was disease stabilization in seven patients. CONCLUSION: OSI-7836 was associated with excessive fatigue, and despite changes in its schedule and duration of administration, we did not observe an improvement in its tolerability. Its potentially selective effect on B lymphocytes could be exploited in further studies in specific hematologic malignancies. PMID- 16675580 TI - Antisense oligonucleotide inhibition of tumor necrosis factor receptor 1 protects the liver from radiation-induced apoptosis. AB - PURPOSE: Liver damage by radiation limits its efficacy in cancer treatment. As radiation can generate apoptotic signals, we wished to examine the potential to protect the liver by inhibiting apoptosis through two key mediators, FAS and tumor necrosis factor receptor 1 (TNFR1). EXPERIMENTAL DESIGN: Radiation-induced liver damage was assessed by serum aspartate aminotransferase and alanine aminotransferase, hepatocyte micronucleus formation, and apoptosis assays (terminal nucleotidyl transferase-mediated nick end labeling and caspase-3 cleavage) in mice. Protection was evaluated by pretreating mice with antisense oligonucleotides (ASO) for FAS or TNFR1 prior to radiation. TNF-alpha production in liver and in Kupffer cells were determined by ELISA. RESULTS: Radiation increased liver FAS and TNFR1 transcription in a dose- and time-dependent manner (maximized at 25 Gy and 8 hours postirradiation). Pretreatment with ASOs for FAS and TNFR1 resulted in the inhibition of liver FAS and TNFR1 by 78% and 59%, respectively. Inductions of serum aspartate aminotransferase and alanine aminotransferase were observed at 2 hours after radiation and could be reduced by pretreating mice with ASO for TNFR1 but not FAS or control oligonucleotide. Radiation-induced liver apoptosis (terminal nucleotidyl transferase-mediated nick end labeling staining and caspase-3 activation on Western blot) and hepatocyte micronucleus formation were reduced by pretreatment with ASO for TNFR1. In addition, radiation stimulated TNF-alpha production both in irradiated liver and in cultured Kupffer cells by >50% and 100%, respectively. CONCLUSION: This study suggests that ionizing radiation activates apoptotic signaling through TNFR1 in the liver, and thus provides a rationale for anti-TNFR1 apoptotic treatment to prevent radiation-induced liver injury. PMID- 16675581 TI - Antitumor efficacy of edotecarin as a single agent and in combination with chemotherapy agents in a xenograft model. AB - The novel indolocarbazole edotecarin (J-107088, formerly ED-749) differs from other topoisomerase I inhibitors both pharmacokinetically and pharmacodynamically. In vitro, it is more potent than camptothecins and has a variable cytotoxic activity in 31 different human cancer cell lines. Edotecarin also possesses greater than additive inhibitory effects on cell proliferation when used in combination with other agents tested in vitro against various cancer cell lines. The present in vivo studies were done to extend the in vitro findings to characterize the antitumor effects of edotecarin when used either alone or in combination with other agents (i.e., 5-fluorouracil, irinotecan, cisplatin, oxaliplatin, and SU11248) in the HCT-116 human colon cancer xenograft model. Treatment effects were based on the delay in onset of an exponential growth of tumors in drug-treated versus vehicle control-treated groups. In all studies, edotecarin was active both as a single agent and in combination with other agents. Combination therapy resulted in greater than additive effects, the extent of which depended on the specific dosage regimen. Toxicity in these experiments was minimal. Of all 359 treated mice, the six that died of toxicity were in the high-dose edotecarin/oxaliplatin group. The results suggest that edotecarin may serve as effective chemotherapy of colon cancer when used as a single agent, in combination with standard regimens and other topoisomerase inhibitors or with novel agents, such as the multitargeted tyrosine kinase inhibitor SU11248. PMID- 16675582 TI - Bisphosphonates inhibit the growth of mesothelioma cells in vitro and in vivo. AB - PURPOSE: Bisphosphonates (such as risedronate and zoledronate) are widely used inhibitors of bone resorption. Despite their in vitro antiproliferative effects in various cancer cells, bisphosphonates have not exhibited significant antitumor efficacy in animal models of visceral cancer, which may be due to their poor bioavailability. The diagnostic use of radioactive bisphosphonates has revealed the accumulation of bisphosphonates in mesothelioma, which prompted us to test the antitumor efficacy of bisphosphonates in this disease. EXPERIMENTAL DESIGN AND RESULTS: Treatment with either risedronate or zoledronate (2 x 10(-4) to 2 x 10(-6) mol/L) inhibited the growth of AB12 and AC29 mouse mesothelioma cells and induced the accumulation of unprenylated Rap1A in these cells. Both these in vitro effects were reversed by geranygeraniol, an end product of the mevalonate pathway that these bisphosphonates inhibit. Both bisphosphonates also induced the phosphorylation of the p38 mitogen-activated protein kinase in AB12 and AC29 cells. The inhibition of p38 augmented bisphosphonate-induced growth inhibition in these cells. Bisphosphonate-induced p38 phosphorylation was not reversible by geranylgeraniol. Risedronate (15 mg/kg) and zoledronate (0.5 mg/kg) inhibited the growth of s.c. tumors and increased the median survival of mice with i.p. mesothelioma tumors in vivo. DISCUSSION: In conclusion, risedronate and zoledronate inhibit the mevalonate pathway and induce p38 activation in mesothelioma cells in vitro. The effects on the mevalonate pathway dominate because the net result is growth inhibition. Both bisphosphonates also inhibit mesothelioma tumor growth in vivo and prolong the survival of mesothelioma bearing mice. These results support further study of bisphosphonates in the management of mesothelioma. PMID- 16675583 TI - Enhanced gene transfer and oncolysis of head and neck cancer and melanoma cells by fiber chimeric oncolytic adenoviruses. AB - PURPOSE: The purpose of this study was to evaluate a fiber knob replacement strategy to improve infectivity and efficacy of Ad5 fiber chimeric oncolytic viruses for treatment of melanoma and head and neck cancers (HNC). EXPERIMENTAL DESIGN: Adenoviral receptors and transduction levels were used to determine the level of infectivity of fiber-modified, green fluorescent protein-expressing, replication-deficient viruses in a panel of melanoma and HNC cell lines in vitro. Virus yield and cytotoxicity assays were used to determine the tumor specificity and virus replication-mediated cytotoxicity of the fiber-modified oncolytic viruses in the same panel of melanoma and HNC in vitro. Xenograft tumor models were used to assess the antitumor activity of those fiber-modified chimeric viruses compared with the parental virus. RESULTS: Marker gene expression following gene transfer of the fiber chimeric vectors in melanoma and HNC cell lines was approximately 10-fold higher than that obtained with parental Ad5 vector. The fiber chimeric oncolytic variants mediated killing of melanoma and HNC cells that was 2- to 576-fold better than with the parental virus. In addition, fiber chimeric variants produced 2- to 7-fold more progeny virus in tumor cells than the parental virus. Moreover, a high multiplicity of infection was needed for the fiber chimeric viruses to produce cytotoxicity in normal cells. A significantly stronger antitumor response and survival advantage were shown in the tested melanoma and HNC xenograft models following i.t. injections. CONCLUSIONS: In vitro and in vivo studies showed the improved transduction, replication, cytotoxicity, antitumor efficacy, and survival advantage in melanoma and HNC tumor models, suggesting a potential use of these oncolytic agents for the treatment of melanoma and HNCs. PMID- 16675584 TI - Nonfucosylated therapeutic IgG1 antibody can evade the inhibitory effect of serum immunoglobulin G on antibody-dependent cellular cytotoxicity through its high binding to FcgammaRIIIa. AB - PURPOSE: Recent studies have revealed that fucosylated therapeutic IgG1s need high concentrations to compensate for FcgammaRIIIa-competitive inhibition of antibody-dependent cellular cytotoxicity (ADCC) by endogenous human plasma IgG. Here, we investigated whether ADCC of nonfucosylated therapeutic IgG1 is also influenced by plasma IgG in the same way as fucosylated IgG1s. EXPERIMENTAL DESIGN: Ex vivo ADCC upon CD20+ human B cells was induced by incubation of human whole blood with nonfucosylated and/or fucosylated anti-CD20 IgG1s rituximab, and quantified by measuring the remaining CD19+ human B cells using flow cytometry. RESULTS: Nonfucosylated anti-CD20 showed markedly higher (over 100-fold based on EC50) ex vivo B-cell depletion activity than its fucosylated counterpart in the presence of plasma IgG. The efficacy of fucosylated anti-CD20 was greatly diminished in plasma, resulting in the need for a high concentration (over 1.0 microg/mL) to achieve saturated efficacy. In contrast, nonfucosylated anti-CD20 reached saturated ADCC at lower concentrations (0.01-0.1 microg/mL) with much higher efficacy than fucosylated anti-CD20 in all nine donors through improved FcgammaRIIIa binding. Noteworthy, the high efficacy of nonfucosylated anti-CD20 was inhibited by addition of fucosylated anti-CD20. Thus, the efficacy of a 1:9 mixture (10 microg/mL) of nonfucosylated and fucosylated anti-CD20s was inferior to that of a 1,000-fold dilution (0.01 microg/mL) of nonfucosylated anti-CD20 alone. CONCLUSIONS: Our data showed that nonfucosylated IgG1, not including fucosylated counterparts, can evade the inhibitory effect of plasma IgG on ADCC through its high FcgammaRIIIa binding. Hence, nonfucosylated IgG1 exhibits strong therapeutic potential through dramatically enhanced ADCC at low doses in humans in vivo. PMID- 16675585 TI - Angiotensin II type 1 receptor antagonist candesartan as an angiogenic inhibitor in a xenograft model of bladder cancer. AB - PURPOSE: There have been several studies on the antitumor activity of angiotensin II type 1 receptor (AT1R) antagonists. In this study, we evaluated the efficacy of the AT1R antagonist candesartan in bladder cancer. EXPERIMENTAL DESIGN: For the study in vitro, human bladder cancer cells (KU-19-19) were cultured with or without angiotensin II and candesartan. Various cytokines and cell viability were analyzed. For the study in vivo, a tumor xenograft model was prepared in nude mice using KU-19-19 cells. Mice were given candesartan daily by oral gavage. Microvessel density, expression of vascular endothelial growth factor (VEGF), and apoptosis were assessed. RESULTS: Candesartan did not induce direct toxicity in KU-19-19 cells, but VEGF and interleukin-8 were significantly lower in candesartan-treated cells (2.55 +/- 0.25 and 6.58 +/- 0.48 pg/10(3) cells) than in the angiotensin II-treated control cells (3.16 +/- 0.42 and 7.91 +/- 0.69 pg/10(3) cells). In mice, candesartan both at doses of 2 and 10 mg/kg/d significantly suppressed tumor growth in mice (35.4% and 33.5% reduction in tumor volume). Microvessel density was significantly decreased by candesartan (9.8 +/- 2.8 per field) compared with the control group (17.6 +/- 6.0 per field), and VEGF expression was significantly suppressed by this AT1R antagonist. However, candesartan did not induce apoptosis of cancer cells in the tumor. CONCLUSIONS: Specific blockade of AT1R prevented bladder tumor growth by inhibiting angiogenesis. However, its antitumor effect was not due to direct toxicity. Because AT1R antagonists are widely used to treat hypertension, and a 2 mg/kg/d dose level of candesartan is clinically achievable, this AT1R antagonist could also be used to treat bladder cancer. PMID- 16675586 TI - A disaccharide-based inhibitor of glycosylation attenuates metastatic tumor cell dissemination. AB - PURPOSE: The binding of hematogenously borne malignant cells that express the carbohydrate sialyl Lewis X (sLe(X)) to selectin adhesion receptors on leukocytes, platelets, and endothelial cells facilitates metastasis. The glycosylation inhibitor, per-O-acetylated GlcNAcbeta1,3Galbeta-O naphthalenemethanol (AcGnG-NM), inhibits the biosynthesis of sLe(X) in tumor cells. To evaluate the efficacy of AcGnG-NM as an antimetastatic agent, we examined its effect on experimental metastasis and on spontaneous hematogenous dissemination of murine Lewis lung carcinoma and B16BL6 melanoma cells. EXPERIMENTAL DESIGN: Tumor cells were treated in vitro with AcGnG-NM, and the degree of selectin ligand inhibition and experimental metastasis was analyzed in wild-type and P-selectin-deficient mice. Conditions were developed for systemic administration of AcGnG-NM, and the presence of tumor cells in the lungs was assessed using bromodeoxyuridine labeling in vivo. The effect of AcGnG-NM on inflammation was examined using an acute peritonitis model. RESULTS: In vitro treatment of Lewis lung carcinoma cells with AcGnG-NM reduced expression of sLe(X)- and P-selectin-dependent cell adhesion to plates coated with P-selectin. Treatment also reduced formation of lung foci when cells were injected into syngeneic mice. Systemic administration of the disaccharide significantly inhibited spontaneous dissemination of the cells to the lungs from a primary s.c. tumor, whereas an acetylated disaccharide not related to sLe(X) in structure had no effect. AcGnG-NM did not alter the level of circulating leukocytes or platelets, the expression of P-selectin ligands on neutrophils, or sLe(X) dependent inflammation. CONCLUSION: Taken together, these data show that AcGnG-NM provides a targeted glycoside-based therapy for the treatment of hematogenous dissemination of tumor cells. PMID- 16675587 TI - The combination of the proteasome inhibitor bortezomib and the bcl-2 antisense molecule oblimersen sensitizes human B-cell lymphomas to cyclophosphamide. AB - PURPOSE: To determine whether the combination of the proteasome inhibitor bortezomib and the bcl-2 antisense molecule oblimersen can sensitize human lymphoma to cyclophosphamide. EXPERIMENTAL DESIGN: Cytotoxicity assays were conducted to determine if there was any additive or synergistic interaction between the combinations of bortezomib, oblimersen, and cyclophosphamide using a standard trypan blue exclusion assay. Based on these experiments, in vivo experiments in severe combined immunodeficiency beige mice were done using human lymphoma xenografts in which different schedules were explored. Bcl-2 and oblimersen levels were determined in treated tumors, some of which were resected at the end of the in vivo experiment and evaluated pathologically. RESULTS: The results suggest that the combination of bortezomib and oblimersen seem to interact in at least an additive fashion, and that the addition of cyclophosphamide to this drug combination can markedly improve tumor cell kill. In addition, it seems that these drug combinations may be schedule-dependent, with a requirement for oblimersen pretreatment. Animals treated with the triplet drug combination in a schedule-dependent manner experienced pathologic complete regression of disease, which was not observed in other treatment cohorts. The addition of bortezomib also seemed to increase the levels of intracellular oblimersen, which resulted in a marked reduction in Bcl-2. Histologic studies confirmed marked necrosis and caspase-3 activation only in the cohort receiving all three drugs. CONCLUSION: The use of Bcl-2-directed therapy and a proteasome inhibitor sensitizes human lymphoma cells to cytotoxic drugs like cyclophosphamide. This combination may offer new opportunities for integrating novel targeted therapies with conventional chemotherapy. PMID- 16675588 TI - In vitro and in vivo radiosensitization induced by the ribonucleotide reductase inhibitor Triapine (3-aminopyridine-2-carboxaldehyde-thiosemicarbazone). AB - PURPOSE: Because ribonucleotide reductase (RR) plays a role in DNA repair, it may serve as a molecular target for radiosensitization. Unlike previously investigated RR inhibitors, Triapine potently inhibits both RR holoenzymes. Therefore, the effects of Triapine on tumor cell radiosensitivity were investigated. EXPERIMENTAL DESIGN: The effects of Triapine on the in vitro radiosensitivity of three human tumor cell lines and one normal cell line were evaluated using a clonogenic assay. Growth delay was used to evaluate the effects of Triapine on in vivo tumor radiosensitivity. The levels of the RR subunits were determined using immunoblot analysis and DNA damage and repair were evaluated using gammaH2AX foci. RESULTS: Exposure of the tumor cell lines to Triapine before or immediately after irradiation resulted in an increase in radiosensitivity. In contrast, Triapine enhanced the radiosensitivity of the normal fibroblast cell line only when the exposure was before irradiation. There were no consistent differences between cell lines with respect to the expression of the RR subunits. Whereas Triapine had no effect on radiation-induced gammaH2AX foci at 1 hour, the number of gammaH2AX foci per cell was significantly greater in the Triapine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Triapine administration to mice bearing tumor xenografts immediately after irradiation resulted in a greater than additive increase in radiation-induced tumor growth delay. CONCLUSIONS: These results indicate that Triapine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair. PMID- 16675589 TI - Systemic oncolytic herpes virus therapy of poorly immunogenic prostate cancer metastatic to lung. AB - PURPOSE: Our goal was to evaluate whether systemic administration of NV1042, an interleukin-12 (IL-12)-expressing oncolytic herpes simplex virus, and its noncytokine parental vector NV1023 are effective against preexisting metastatic prostate cancer in an immunocompetent mice model. EXPERIMENTAL DESIGN: Metastatic TRAMP-C2 lung tumors established in C57Bl/6 or nude mice were treated on day 21 with four i.v. administrations of NV1042 or NV1023 and sacrificed on day 42 to assess virus efficacy and the potential mechanism of efficacy. RESULTS: NV1042 or NV1023 treatment was similarly effective in eliminating extrapleural and hemorrhagic tumors present in mock-treated mice. However, NV1042 was further effective compared with NV1023 in controlling the growth of lung tumors (as determined by mean surface tumor nodule number, lung weights, and surface tumor burden) and in extending survival. NV1042-treated mice exhibited a transient increase of serum IL-12 1 day posttreatment, whereas IL-12 levels in tumor bearing lungs persisted a further 2 days at least. Only splenocytes from NV1042 treated mice secreted IFN-gamma in response to TRAMP-C2 stimulation and displayed natural killer activity. The IL-12-mediated enhancement observed with NV1042 in the syngeneic model was abrogated in athymic mice treated in a similar manner, thus indicating a role for T cells in the augmented efficacy of NV1042 virus. CONCLUSIONS: Systemic administration of the IL-12-expressing NV1042 virus is more effective than its noncytokine parent, NV1023, against preestablished metastatic lung tumors. Given the clinical safety profile of NV1020, the parental vector of NV1023, and NV1042's enhanced efficacy and ability to activate the host immune system, NV1042 merits clinical consideration for treating metastatic prostate cancers. PMID- 16675590 TI - PUMA sensitizes lung cancer cells to chemotherapeutic agents and irradiation. AB - PURPOSE: Lung cancer, the leading cause of cancer mortality worldwide, is often diagnosed at late stages and responds poorly to conventional therapies, including chemotherapy and irradiation. A great majority of lung tumors are defective in the p53 pathway, which plays an important role in regulating apoptotic response to anticancer agents. PUMA was recently identified as an essential mediator of DNA damage-induced and p53-dependent apoptosis. In this study, we investigated whether the regulation of PUMA by anticancer agents is abrogated in lung cancer cells and whether PUMA expression suppresses growth of lung cancer cells and/or sensitizes lung cancer cells to chemotherapeutic agents and irradiation through induction of apoptosis. EXPERIMENTAL DESIGNS: The expression of PUMA was examined in lung cancer cells with different p53 status treated with chemotherapeutic agents. An adenovirus expressing PUMA (Ad-PUMA), alone or in combination with chemotherapeutic agents or gamma-irradiation, was used to treat lung cancer cells. The growth inhibitory and apoptotic effects of PUMA in vitro and in vivo were examined. The mechanisms of PUMA-mediated growth suppression and apoptosis were investigated through analysis of caspase activation and release of mitochondrial apoptogenic proteins. The cytotoxicities of PUMA on cancer and normal/nontransformed cells were compared. The efficacy of PUMA and p53 in suppressing the growth of lung cancer cells was also compared. RESULTS: We showed that the induction of PUMA by chemotherapeutic agents is abolished in p53 deficient lung cancer cells. PUMA expression resulted in potent growth suppression of lung cancer cells and suppressed xenograft tumor growth in vivo through induction of apoptosis. Low dose of Ad-PUMA significantly sensitized lung cancer cells to chemotherapeutic agents and gamma-irradiation through induction of apoptosis. The effects of PUMA are mediated by enhanced caspase activation and release of cytochrome c and apoptosis-inducing factor into the cytosol. Furthermore, PUMA seems to be selectively toxic to cancer cells and more efficient than p53 in suppressing lung cancer cell growth. CONCLUSIONS: Our findings indicate that PUMA is an important modulator of therapeutic responses of lung cancer cells and is potentially useful as a sensitizer in lung cancer therapy. PMID- 16675591 TI - Gefitinib inhibits the growth and invasion of urothelial carcinoma cell lines in which Akt and MAPK activation is dependent on constitutive epidermal growth factor receptor activation. AB - PURPOSE: Abnormally high levels of epidermal growth factor receptor (EGFR) protein are associated with advanced tumor stage/grade. The objective of this study was to evaluate the effects of the specific EGFR tyrosine kinase inhibitor gefitinib on activation of the Akt and mitogen-activated protein kinase (MAPK) pathways in human urothelial cell carcinoma (UCC) cell lines and to identify potential markers of gefitinib responsiveness in biopsy samples of UCC. EXPERIMENTAL DESIGN: Changes in markers of UCC growth and invasion after exposure to gefitinib were studied in six human UCC cell lines expressing various levels of EGFR. The findings were related to activation of Akt and MAPK. We studied the influence of gefitinib on intraepithelial expansion of the responsive 1207 cell line. EGFR, Akt, and MAPK activation was studied by Western blot analysis of a panel of 57 human UCC. RESULTS: Gefitinib had a growth-inhibitory and anti invasive effect in two of six UCC cell lines (i.e., 647V and 1207). Gefitinib was also able to block the expansion of 1207 at the expense of normal urothelial cells. These effects did not depend on the level of expression of EGFR but they were associated with the down-regulation of MAPK and Akt activity; in 1207 cells, gefitinib activity was associated with p27 up-regulation and p21 and matrix metalloproteinase-9 down-regulation. Similarly, the Akt and MAPK pathways were found to be strongly phosphorylated in association with EGFR activation in a subset of human UCC specimens. CONCLUSIONS: Activation of EGFR, Akt, and MAPK defines a subset of UCC which might provide information for the identification of gefitinib responders. PMID- 16675592 TI - Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences. AB - Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 micromol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent. PMID- 16675593 TI - Hepatic stem cells: in search of. AB - The field of stem cell biology has exploded with the study of a wide range of cellular populations involving endodermal, mesenchymal, and ectodermal organs. One area of extensive study has included the identification of hepatic stem and progenitor cell subpopulations. Liver stem cells provide insights into the potential pathways involving liver regeneration that are independent of mature hepatocytes. Hepatic progenitor cells are either bipotent or multipotent and capable of multiple rounds of replication. They have been identified in fetal as well as adult liver. Various injury models have been used to expand this cellular compartment. The nomenclature, origin, and function of the hepatic progenitor cell populations are areas of ongoing debate. In this review, we will discuss the different definitions and functions of hepatic progenitor cells as well as the current research efforts examining their therapeutic potential. PMID- 16675594 TI - Genome-wide transcriptional profiling of human embryonic stem cells differentiating to cardiomyocytes. AB - Mammals are unable to regenerate their heart after major cardiomyocyte loss caused by myocardial infarction. Human embryonic stem cells (hESCs) can give rise to functional cardiomyocytes and therefore have exciting potential as a source of cells for replacement therapy. Understanding the molecular regulation of cardiomyocyte differentiation from stem cells is crucial for the stepwise enhancement and scaling of cardiomyocyte production that will be necessary for transplantation therapy. Our novel hESC differentiation protocol is now efficient enough for meaningful genome-wide transcriptional profiling by microarray technology of hESCs, differentiating toward cardiomyocytes. Here, we have identified and validated time-dependent gene expression patterns and shown a reflection of early embryonic events; induction of genes of the primary mesoderm and endodermal lineages is followed by those of cardiac progenitor cells and fetal cardiomyocytes in consecutive waves of known and novel genes. Collectively, these results permit enhancement of stepwise differentiation and facilitate isolation and expansion of cardiac progenitor cells. Furthermore, these genes may provide new clinically relevant clues for identifying causes of congenital heart defects. PMID- 16675595 TI - Multipotent mesenchymal stromal cell recruitment, migration, and differentiation: what have matrix metalloproteinases got to do with it? PMID- 16675596 TI - Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration. AB - Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5 azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast-conditioned medium resulted in only 1%-2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin-1, 66.1% +/- 5.7% of hfMSCs, but not adult bone marrow-derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin-1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin-1-exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin-1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies. PMID- 16675597 TI - Progenitors systemically transplanted into neonatal mice localize to areas of active bone formation in vivo: implications of cell therapy for skeletal diseases. AB - The potential of cell or gene therapy to treat skeletal diseases was evaluated through analysis of transplanted osteoprogenitors into neonatal homozygous and heterozygous osteogenesis imperfecta mice (oim). The osteoprogenitors used for transplantation were prepared by injection of mesenchymal stem cells (MSCs) marked with the green fluorescent protein (GFP) into normal mice with the subsequent retrieval of the cells at 35 days. The retrieved cells referred to here as osteoprogenitors were expanded in culture and transplanted into the 2-day old oim mice via the superficial temporal vein. The recipient mice were evaluated at 2 and 4 weeks after cell transplantation. Four weeks after transplantation, tissue sections made from femurs and tibias of oim mice showed that the GFP positive (GFP(+)) cells were distributed on the surfaces of the bone spicules in the spongiosa, the area of active bone formation. In the diaphysis, the GFP(+) cells were distributed in the bone marrow, on the endosteal surfaces, and also in the cortical bone. Immunofluorescence localization for GFP confirmed that the fluorescence seen in tissue sections was due to the engrafted donor cells, not bone autofluorescence. Gene expression analysis by polymerase chain reaction of the GFP(+) cells retrieved from the bones and marrow of the recipient mice demonstrated that the cells from bone were osteoblasts, whereas those from bone marrow were progenitors. These data demonstrate that MSCs delivered systemically to developing osteogenesis imperfecta mice engraft in bones, localize to areas of active bone formation, differentiate into osteoblasts in vivo, and may contribute to bone formation in vivo. PMID- 16675598 TI - The effect of overexpression of Pdx1 and Foxa2 on the differentiation of human embryonic stem cells into pancreatic cells. AB - Human embryonic stem cells (HESCs) are pluripotent cells that may serve as a source of cells for transplantation medicine and as a tool to study human embryogenesis. Using genetic manipulation methodologies, we have investigated the potential of HESCs to differentiate into the various pancreatic cell types. We initially created various HESCs carrying the enhanced green fluorescent protein (eGFP) reporter gene under the control of either the insulin promoter or the pancreatic and duodenal homeobox factor-1 (Pdx1) promoter. Our analysis revealed that during the differentiation of HESCs into embryoid bodies (EBs), we could detect green fluorescent cells when eGFP is regulated by Pdx1 promoter but not by insulin promoter. To examine whether we can induce differentiation into pancreatic cells, we have established human embryonic stem cell lines that constitutively express either Pdx1 or the endodermal transcription factor Foxa2. Following differentiation into EBs, the constitutive expression of Pdx1 enhanced the differentiation of HESCs toward pancreatic endocrine and exocrine cell types. Thus, we have demonstrated expression of several transcription factors that are downstream of Pdx1 and various molecular markers for the different pancreatic cell types. However, the expression of the insulin gene could be demonstrated only when the cells differentiated in vivo into teratomas. We conclude that although overexpression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo. PMID- 16675599 TI - Downregulation of connexin 43 expression by high glucose induces senescence in glomerular mesangial cells. AB - Connexin 43 (Cx43) plays an important role in cell differentiation and growth control, but whether it can be regulated by high glucose and whether it can mediate in glomerular mesangial cells (GMC) the phenotype alterations that are induced by high glucose still remain to be explored. In this study, RNA interference and gene transfer techniques were used to knock down and overexpress Cx43 gene in rat GMC to determine the contribution of Cx43 to GMC senescence that was induced by high glucose. The results show that high glucose (30 mM) not only downregulated Cx43 mRNA and protein expression (P<0.05) but also increased the percentage of senescence-associated beta-galactosidase (SA-beta-gal) stained cells and expression of p21cip1 and p27kip1 (P<0.05), indicating that high glucose promoted rat GMC senescence. Knocking down Cx43 gene expression significantly increased the percentage of SA-beta-gal stained cells and p27kip1 and p21cip1 expression in GMC (P<0.05), whereas overexpression of Cx43 significantly decreased the percentage of SA-beta-gal stained cells (P<0.05). These results demonstrate for the first time that downregulation of Cx43 expression by high glucose promotes the senescence of GMC, which may be involved in the pathogenesis of diabetic nephropathy. PMID- 16675600 TI - Angiotensin II upregulates toll-like receptor 4 on mesangial cells. AB - Angiotensin II (AngII) mediates proinflammatory properties by activating NF kappaB transcription factor nuclear translocation and inducing the expression of chemokines. For examination of whether AngII modulates the expression of Toll like receptor 4 (TLR4), a key element of the innate immune system that senses LPS, mouse mesangial cells (MMC) were treated with AngII. AngII upregulated TLR4 mRNA and protein in MMC, and this effect was mediated through AngII type 1 receptors. Reporter gene experiments indicate that an activating protein-1 (AP-1) as well as an E-26 specific sequence (Ets) binding site in the TLR4 promoter are responsible for the AngII-stimulated transcriptional activity of the TLR4 gene. Preincubation of MMC with AngII enhanced LPS-induced NF-kappaB activation and chemokine expression. Immunohistochemical analyses revealed that double transgenic rats that overexpressed human renin and angiotensinogen expressed higher levels of glomerular TLR4 compared with normal Sprague-Dawley rats. In vivo, infusion with AngII but not with norepinephrine into rats for 7 d also enhanced glomerular NF-kappaB activation after systemic application of LPS, suggesting that the effects are independent of concomitantly induced hypertension. Together, these observations suggest that AngII leads to an activation of the innate immune system by a novel mechanism involving the upregulation of TLR4. Our data contribute to a better understanding of how exogenous infections may trigger renal autoimmune processes, particularly in pathophysiologic situations with high renal AngII concentrations. Because TLR4 binds endogenous ligands (e.g., extracellular matrix components) in addition to microbial products, AngII-mediated upregulation of TLR4 also could be relevant for the development of inflammation in many noninfectious renal diseases. PMID- 16675601 TI - Identification of oxalic acid and tartaric acid as major persistent pain-inducing toxins in the stinging hairs of the nettle, Urtica thunbergiana. AB - BACKGROUND AND AIMS: Once human skin contacts stinging hairs of Urtica spp. (stinging nettles), the irritant is released and produces pain, wheals or a stinging sensation which may last for >12 h. However, the existence of pain inducing toxins in the stinging hairs of Urtica thunbergiana has never been systematically demonstrated. Experiments were therefore conducted to identify the persistent pain-inducing agents in the stinging hairs of U. thunbergiana. METHODS: The stinging hairs of U. thunbergiana were removed and immersed in deionized water. After centrifugation, the clear supernatants were then subjected to high-performance liquid chromatography (HPLC), enzymatic analysis and/or behavioural bioassays. KEY RESULTS: The HPLC results showed that the major constituents in the stinging hairs of U. thunbergiana were histamine, oxalic acid and tartaric acid. However, the well-recognized pain-inducing agents, serotonin and formic acid, existed at a low concentration as estimated by HPLC and/or enzymatic analyses. The behavioural tests showed that 2% oxalic acid and 10% tartaric acid dramatically elicited persistent pain sensations in rats. In contrast, 10% formic acid and 2% serotonin only elicited moderate pain sensation in the first 10 min. Moreover, no significant pain-related behavioural response was observed after injecting 10% acetylcholine and histamine in rats. CONCLUSIONS: Oxalic acid and tartaric acid were identified, for the first time, as major long-lasting pain-inducing toxins in the stinging hairs of U. thunbergiana. The general view that formic acid, histamine and serotonin are the pain-inducing agents in the stinging hairs of U. dioica may require updating, since their concentrations in U. thunbergiana were too low to induce significant pain sensation in behavioural bioassays. PMID- 16675602 TI - Distribution of G-actin is related to root hair growth of wheat. AB - BACKGROUND AND AIMS: Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth. METHODS: G-actin was labelled by fluorescein isothiocyanate-DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate-phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy. KEY RESULTS: Thick F-actin bundles did not extend into a region of approx. 5-10 microm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs. CONCLUSIONS: These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips. PMID- 16675603 TI - Fine-scale genetic structure among genetic individuals of the clone-forming monotypic genus Echinosophora koreensis (Fabaceae). AB - BACKGROUND AND AIMS: For rare endemics or endangered plant species that reproduce both sexually and vegetatively it is critical to understand the extent of clonality because assessment of clonal extent and distribution has important ecological and evolutionary consequences with conservation implications. A survey was undertaken to understand clonal effects on fine-scale genetic structure (FSGS) in two populations (one from a disturbed and the other from an undisturbed locality) of Echinosophora koreensis, an endangered small shrub belonging to a monotypic genus in central Korea that reproduces both sexually and vegetatively via rhizomes. METHODS: Using inter-simple sequence repeats (ISSRs) as genetic markers, the spatial distribution of individuals was evaluated using Ripley's L(d)-statistics and quantified the spatial scale of clonal spread and spatial distribution of ISSR genotypes using spatial autocorrelation analysis techniques (join-count statistics and kinship coefficient, F(ij)) for total samples and samples excluding clones. KEY RESULTS: A high degree of differentiation between populations was observed (phi(ST(g)) = 0.184, P < 0.001). Ripley's L(d) statistics revealed a near random distribution of individuals in a disturbed population, whereas significant aggregation of individuals was found in an undisturbed site. The join-count statistics revealed that most clones significantly aggregate at < or = 6-m interplant distance. The Sp statistic reflecting patterns of correlograms revealed a strong pattern of FSGS for all four data sets (Sp = 0.072-0.154), but these patterns were not significantly different from each other. At small interplant distances (< or = 2 m), however, jackknifed 95% CIs revealed that the total samples exhibited significantly higher F(ij) values than the same samples excluding clones. CONCLUSION: The strong FSGS from genets is consistent with two biological and ecological traits of E. koreensis: bee-pollination and limited seed dispersal. Furthermore, potential clone mates over repeated generations would contribute to the observed high F(ij) values among genets at short distance. To ensure long-term ex situ genetic variability of the endangered E. koreensis, individuals located at distances of 10-12 m should be collected across entire populations of E. koreensis. PMID- 16675605 TI - Evolution in caffeoylquinic acid content and histolocalization during Coffea canephora leaf development. AB - BACKGROUND AND AIMS: Caffeoylquinic acids are cinnamate conjugates derived from the phenylpropanoid pathway. They are generally involved in plant responses to biotic and abiotic stress and one of them, chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA), is an intermediate in the lignin biosynthesis pathway. Caffeoylquinic acids, and particularly 5-CQA, are accumulated in coffee beans, where they can form vacuolar complexes with caffeine. Coffea canephora beans are known to have high caffeoylquinic acid content, but little is known about the content and diversity of these compounds in other plant parts. To gain new insights into the caffeoylquinic acid metabolism of C. canephora, caffeoylquinic acid content and in situ localization were assessed in leaves at different growth stages. METHODS: HPLC analyses of caffeoylquinic acid content of leaves was conducted in conjunction with detailed histochemical and microspectrofluorometrical analysis. KEY RESULTS AND CONCLUSIONS: HPLC analyses revealed that caffeoylquinic acid content was 10-fold lower in adult than in juvenile leaves. The most abundant cinnamate conjugate was 5-CQA, but dicaffeoylquinic acids (particularly in juvenile leaves) and feruloylquinic acids were also present. Using specific reagents, histochemical and microspectrofluorometrical analysis showed that caffeoylquinic acids (mono- and di-esters) were closely associated with chloroplasts in very young leaves. During leaf ageing, they were found to first accumulate intensively in specific chlorenchymatous bundle sheath cells and then in phloem sclerenchyma cells. The association with chloroplasts suggests that caffeoylquinic acids have a protective role against light damage. In older tissues, their presence in the leaf vascular system indicates that they are transported via phloem and confirms their involvement in lignification processes. In accordance with the hypothesis of a complex formation with caffeine, similar tissue distribution was observed for alkaloids and this is further discussed. PMID- 16675604 TI - Expressed sequence tags (ESTs) and phylogenetic analysis of floral genes from a paleoherb species, Asarum caudigerum. AB - BACKGROUND AND AIMS: Asarum caudigerum (Aristolochiaceae) is an important species of paleoherb in relation to understanding the origin and evolution of angiosperm flowers, due to its basal position in the angiosperms. The aim of this study was to isolate floral-related genes from A. caudigerum, and to infer evolutionary relationships among florally expression-related genes, to further illustrate the origin and diversification of flowers in angiosperms. METHODS: A subtracted floral cDNA library was constructed from floral buds using suppression subtractive hybridization (SSH). The cDNA of floral buds and leaves at the seedling stage were used as a tester and a driver, respectively. To further identify the function of putative MADS-box transcription factors, phylogenetic trees were reconstructed in order to infer evolutionary relationships within the MADS-box gene family. KEY RESULTS: In the forward-subtracted floral cDNA library, 1920 clones were randomly sequenced, from which 567 unique expressed sequence tags (ESTs) were obtained. Among them, 127 genes failed to show significant similarity to any published sequences in GenBank and thus are putatively novel genes. CONCLUSIONS: Phylogenetic analysis indicated that a total of 29 MADS-box transcription factors were members of the APETALA3(AP3) subfamily, while nine others were putative MADS-box transcription factors that formed a cluster with MADS-box genes isolated from Amborella, the basal-most angiosperm, and those from the gymnosperms. This suggests that the origin of A. caudigerum is intermediate between the angiosperms and gymnosperms. PMID- 16675606 TI - Phylogenetics of Papaver and related genera based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers. AB - BACKGROUND AND AIMS: Representatives from Papaver, Roemeria, Stylomecon and Meconopsis were studied to elucidate phylogenetic relationships between Papaver and these closely allied genera. METHODS: Two molecular data sets were used individually and combined and included sequences from the internally transcribed spacer region (ITS) of 18S-26S nuclear ribosomal DNA and the trnL intron and the trnL-trnF intergenic spacer region of plastid DNA. KEY RESULTS: Parsimony analysis demonstrated that the genus is not monophyletic unless the closely related Roemeria, Stylomecon and Meconopsis cambrica are included in a revised circumscription of Papaver. Three distinct clades are resolved in a combined ITS and trnL-F analysis. Clade 1 consists of Papaver sect. Meconella and Asian Meconopsis. Clade 2 contains a group here identified as Papaver s.s., comprising sections Carinatae, Meconidium, Oxytona, Papaver, Pilosa, Pseudopilosa and Rhoeadium. Clade 3 consists of Papaver sect. Argemonidium and Roemeria refracta. A number of diagnostic indels support these groupings. Within clade 2, sects. Papaver and Rhoeadium are either not monophyletic or lack evidence supporting their monophyly. CONCLUSIONS: The results of this molecular analysis indicate that a number of morphological characters such as valvate capsule dehiscence, dark or light filaments and sessile stigmatic discs have arisen in parallel. The phylogenetic trees are incongruent with the existing taxonomy of Papaver, and a revised classification is suggested. PMID- 16675607 TI - Former diversity of Ephedra (Gnetales): evidence from Early Cretaceous seeds from Portugal and North America. AB - BACKGROUND AND AIMS: The extant species of the seed plant group Gnetales (Ephedra, Gnetum and Welwitschia) have been considered a remnant of a much greater, now extinct, diversity due to the pronounced differences in form and ecology among the genera. Until recently, this hypothesis has not been supported by evidence from the fossil record. This paper adds to the expanding information on Gnetales from the Early Cretaceous and describes coalified seeds from Barremian-Albian localities in Portugal and USA. METHODS: The fossils were extracted from sediment samples by sieving in water. Adhering mineral matrix was removed by chemical treatment. Seeds were investigated using light and scanning electron microscopy. Morphology and anatomy of the seeds were documented and compared with those of extant species. KEY RESULTS: The fossils share characters with extant Ephedra, for example papillae on the inner surface of the seed envelope and in situ polyplicate pollen grains that shed the exine during germination. They differ from extant Ephedra seeds in morphological and anatomical details as well as in their smaller size. Two new species of Ephedra are described together with one species assigned to a new genus of Gnetales. Other Ephedra-like seeds, for which pollen and critical morphological details are currently unknown, are also present in the samples. CONCLUSIONS: These Cretaceous seeds document that key reproductive characters and pollen germination processes have remained unchanged within Ephedra for about 120 million years or more. There is sufficient variety in details of morphology to suggest that a diversity of Ephedra and Ephedra-like species were present in the Early Cretaceous flora. Their presence in Portugal and eastern North America indicates that they were widespread on the Laurasian continent. The fossil seeds are similar to seeds of Erdtmanithecales and this supports the previously suggested relationship between Erdtmanithecales and Gnetales. PMID- 16675608 TI - Achene structure, development and lipid accumulation in sunflower cultivars differing in oil content at maturity. AB - BACKGROUND AND AIMS: Sunflower cultivars exhibit a wide range of oil content in the mature achene, but the relationship between this and the dynamics of oil deposition in the achene during grain filling is not known. Information on the progress, during the whole achene growth period, of the formation of oil bodies in the components of the achene and its relationship with variations in final oil content is also lacking. METHODS: The biomass dynamics of achene components (pericarp, embryo, oil) in three cultivars of very different final oil concentration (30-56 % oil) were studied. In parallel, anatomical sections were used to follow the formation of oil and protein bodies in the embryo, and to observe pericarp anatomy. KEY RESULTS: In all cultivars, oil bodies were first observed in the embryo 6-7 daa after anthesis (daa). The per-cell number of oil bodies increased rapidly from 10-12 daa until 25-30 daa. Oil bodies were absent from the outer cell layers of young fruit and from mature pericarps. In mature embryos, the proportion of cell cross-sectional area occupied by protein bodies increased with decreasing embryo oil concentration. The sclerenchymatic layer of the mature pericarp decreased in thickness and number of cell layers from the low oil cultivar to the high-oil cultivar. Different patterns of oil accumulation in the embryo across cultivars were also found, leading to variations in ripe embryo oil concentration. In the high-oil cultivar, the end of oil deposition coincided with cessation of embryo growth, while in the other two cultivars oil ceased to accumulate before the embryo achieved maximum weight. CONCLUSIONS: Cultivar differences in mature achene oil concentration reflect variations in pericarp proportion and thickness and mature embryo oil concentration. Cultivar differences in protein body proportion and embryo and oil mass dynamics during achene growth underlie variations in embryo oil concentration. PMID- 16675609 TI - Disuse in adult male rats attenuates the bone anabolic response to a therapeutic dose of parathyroid hormone. AB - Intermittent treatment with parathyroid hormone (PTH) increases bone formation and prevents bone loss in hindlimb-unloaded (HLU) rats. However, the mechanisms of action of PTH are incompletely known. To explore possible interactions between weight bearing and PTH, we treated 6-mo-old weight-bearing and HLU rats with a human therapeutic dose (1 microg.kg(-1).day(-1)) of human PTH(1-34) (hPTH). Cortical and cancellous bone formation was measured in tibia at the diaphysis proximal to the tibia-fibula synostosis and at the proximal metaphysis, respectively. Two weeks of hindlimb unloading resulted in a dramatic decrease in the rate of bone formation at both skeletal sites, which was prevented by PTH treatment at the cancellous site only. In contrast, PTH treatment increased cortical as well as cancellous bone formation in weight-bearing rats. Two-way ANOVA revealed that hPTH and HLU had independent and opposite effects on all histomorphometric indexes of bone formation [mineral apposition rate (MAR), double-labeled perimeter (dLPm), and bone formation rate (BFR)] at both skeletal sites. The bone anabolic effects of weight bearing and hPTH on dLPm and BFR at the cortical site were additive, as were the effects on MAR at the cancellous site. In contrast, weight bearing and hPTH resulted in synergistic increases in cortical bone MAR and cancellous bone dLPm and BFR. We conclude that weight bearing and PTH act cooperatively to increase bone formation by resulting in site specific additive and synergistic increases in indexes of osteoblast number and activity, suggesting that weight-bearing exercise targeted to osteopenic skeletal sites may improve the efficacy of PTH therapy for osteoporosis. PMID- 16675610 TI - Sensory-mechanical relationships during high-intensity, constant-work-rate exercise in COPD. AB - During constant-work-rate exercise in chronic obstructive pulmonary disease, dyspnea increases steeply once inspiratory reserve volume (IRV) falls to a critical level that prevents further expansion of tidal volume (Vt). We studied the effects of this mechanical restriction on the quality and intensity of exertional dyspnea and examined the impact of an anticholinergic bronchodilator. In a randomized, double-blind, crossover study, 18 patients with chronic obstructive pulmonary disease (forced expiratory volume in 1 s = 40 +/- 3%predicted; mean +/- SE) inhaled tiotropium 18 mug or placebo once daily for 7 10 days each. Pulmonary function tests and symptom-limited cycle exercise at 75% of each patient's maximal work capacity were performed 2 h after dosing. Dyspnea intensity (Borg scale), operating lung volumes, breathing pattern, and esophageal pressure (n = 11) were measured during exercise. Dynamic hyperinflation reached its maximal value early in exercise and was associated with only mild increases in dyspnea intensity and the effort-displacement ratio, which is defined as the ratio between tidal swings of esophageal pressure (expressed relative to maximum inspiratory pressure) and Vt (expressed relative to predicted vital capacity). After a minimal IRV of 0.5 +/- 0.1 liter was reached, both dyspnea and the effort displacement ratio rose steeply until an intolerable level was reached. Tiotropium did not alter dyspnea-IRV relationships, but the increase in resting and exercise inspiratory capacity was associated with an improved effort displacement ratio throughout exercise. Once a critically low IRV was reached during exercise, dyspnea rose with the disparity between respiratory effort and the Vt response. Changes in dyspnea intensity after tiotropium were positively correlated with changes in this index of neuromechanical coupling. PMID- 16675611 TI - Effect of positional changes of anatomic structures on upper airway dilating muscle shortening during electro- and chemostimulation. AB - Positional changes of anatomic structures surrounding the upper airway are known to affect pharyngeal mechanics and collapsibility. We hypothesized that these alterations also affect the ability of the upper airway dilator muscles to enlarge the pharynx by altering their ability to shorten when activated. Using sonomicrometry, we evaluated in seven anesthetized dogs the effects of changes in tracheal and head position on the length of the genioglossus (GG) and the geniohyoid (GH) and the effects of these positional changes on the magnitude of shortening of the two muscles in response to electro- (ES) and chemostimulation (CS). Caudal traction of the trachea lengthened the GG and GH in all dogs, whereas cranial displacement of the trachea and flexion of the head to a vertical position shortened the muscles. Compared with the magnitude of ES-induced shortening in the neutral position, ES-induced shortening of the GG was 144.7 +/- 14.6, 49.3 +/- 4.3, and 33.5 +/- 11.6% during caudal and cranial displacement of the trachea and during head flexion, respectively. Similar effects of the positional changes were found for the GH, as well as for both muscles during respiratory stimulation with P(CO2) of 90 Torr at the end of CO(2) rebreathing, although inspiratory muscle shortening during CS reached only one-quarter to one third of the magnitude observed during ES. We conclude that positional alterations of anatomic structures in the neck have a dramatic effect on the magnitude of shortening of the activated GG and GH, which may reduce substantially their ability to protect pharyngeal patency. PMID- 16675612 TI - Low-intensity repetitive transcranial magnetic stimulation decreases motor cortical excitability in humans. AB - Repetitive transcranial magnetic stimulation of the motor cortex (rTMS) can be used to modify motor cortical excitability in human subjects. At stimulus intensities near to or above resting motor threshold, low-frequency rTMS (approximately 1 Hz) decreases motor cortical excitability, whereas high frequency rTMS (5-20 Hz) can increase excitability. We investigated the effect of 10 min of intermittent rTMS on motor cortical excitability in normal subjects at two frequencies (2 or 6 Hz). Three low intensities of stimulation (70, 80, and 90% of active motor threshold) and sham stimulation were used. The number of stimuli were matched between conditions. Motor cortical excitability was investigated by measurement of the motor-evoked potential (MEP) evoked by single magnetic stimuli in the relaxed first dorsal interosseus muscle. The intensity of the single stimuli was set to evoke baseline MEPs of approximately 1 mV in amplitude. Both 2- and 6-Hz stimulation, at 80% of active motor threshold, reduced the magnitude of MEPs for approximately 30 min (P < 0.05). MEPs returned to baseline values after a weak voluntary contraction. Stimulation at 70 and 90% of active motor threshold and sham stimulation did not induce a significant group effect on MEP magnitude. However, the intersubject response to rTMS at 90% of active motor threshold was highly variable, with some subjects showing significant MEP facilitation and others inhibition. These results suggest that, at low stimulus intensities, the intensity of stimulation may be as important as frequency in determining the effect of rTMS on motor cortical excitability. PMID- 16675613 TI - Mechanisms for the control of respiratory evaporative heat loss in panting animals. AB - Panting is a controlled increase in respiratory frequency accompanied by a decrease in tidal volume, the purpose of which is to increase ventilation of the upper respiratory tract, preserve alveolar ventilation, and thereby elevate evaporative heat loss. The increased energy cost of panting is offset by reducing the metabolism of nonrespiratory muscles. The panting mechanism tends to be important in smaller mammalian species and in larger species is supplemented by sweating. At elevated respiratory frequencies and body temperatures alveolar hyperventilation begins to develop but is accompanied by a decline in the control of carbon dioxide partial pressure in arterial blood, probably through central chemoreceptors. Most heat exchange takes place at the nasal epithelial lining, and venous drainage can be directed to a special network of arteries at the base of the brain whereby countercurrent heat transfer can occur, which results in selective brain cooling. Such a phenomenon has also been suggested in nonpanting species, including humans, and although originally thought to be a mechanism for protecting the thermally vulnerable brain is now considered to be one of the thermoregulatory reflexes whereby respiratory evaporation can be closely controlled in the interests of thermal homeostasis. PMID- 16675614 TI - Point: Hypoxic pulmonary vasoconstriction is mediated by increased production of reactive oxygen species. PMID- 16675615 TI - Modulation of upper airway muscle activities by bronchopulmonary afferents. AB - Here we review the influence of bronchopulmonary receptors (slowly and rapidly adapting pulmonary stretch receptors, and pulmonary/bronchial C-fiber receptors) on respiratory-related motor output to upper airway muscles acting on the larynx, tongue, and hyoid arch. Review of the literature shows that all muscles in all three regions are profoundly inhibited by lung inflation, which excites slowly adapting pulmonary stretch receptors. This widespread coactivation includes the recruitment of muscles that have opposing mechanical actions, suggesting that the stiffness of upper airway muscles is highly regulated. A profound lack of information on the modulation of upper airway muscles by rapidly adapting receptors and bronchopulmonary C-fiber receptors prohibits formulation of a conclusive opinion as to their actions and underscores an urgent need for new studies in this area. The preponderance of the data support the view that discharge arising in slowly adapting pulmonary stretch receptors plays an important role in the initiation of the widespread and highly coordinated recruitment of laryngeal, tongue, and hyoid muscles during airway obstruction. PMID- 16675616 TI - Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy. AB - Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung. PMID- 16675617 TI - Pulmonary sensory and reflex responses in the mouse. AB - Mouse model research is proliferating because of its readiness for genetic manipulation. Little is known about pulmonary vagal afferents in mice, however. The purpose of this study was to determine whether their pulmonary afferents are similar to those in large animals. Single-unit activity was recorded in the cervical vagus nerve of anesthetized, open-chest, and mechanically ventilated mice. We evaluated airway sensory activity in 153 single units; 141 were mechanosensitive, with 134 inflation receptors and 7 deflation receptors. The remaining 12 receptors were chemosensitive and mechanically insensitive, showing low basal firing frequency and behaving like C-fiber or high-threshold Adelta receptors. In separate studies, phrenic activity was recorded as an index of respiratory drive to assess pulmonary reflexes. Lung inflation produced a typical Hering-Breuer reflex, and intravenous injection of phenylbiguanide produced the typical chemoreflex resulting in apnea, bradycardia, and hypotension. These reflexes were blocked by bilateral vagotomy. We conclude that mice possess a similar set of airway sensors and pulmonary reflexes as typically found in larger animals. PMID- 16675618 TI - Mechanical ventilation promotes redox status alterations in the diaphragm. AB - Oxidative stress is an important mediator of diaphragm muscle atrophy and contractile dysfunction during prolonged periods of controlled mechanical ventilation (MV). To date, specific details related to the impact of MV on diaphragmatic redox status remain unknown. To fill this void, we tested the hypothesis that MV-induced diaphragmatic oxidative stress is the consequence of both an elevation in intracellular oxidant production in conjunction with a decrease in the antioxidant buffering capacity. Adult rats were assigned to one of two experimental groups: 1) control or 2) 12 h of MV. Compared with controls, diaphragms from MV animals demonstrated increased oxidant production, diminished total antioxidant capacity, and decreased glutathione levels. Heme oxygenase-1 (HO-1) mRNA and protein levels increased (23.0- and 5.1-fold, respectively) following MV. Thioredoxin reductase-1 and manganese superoxide dismutase mRNA levels were also increased in the diaphragm following MV (2.4- and 1.6-fold, respectively), although no change was detected in the levels of either protein. Furthermore, copper-zinc superoxide dismutase and glutathione peroxidase mRNA were not altered following MV, although protein content decreased -1.3- and -1.7 fold, respectively. We conclude that MV promotes increased oxidant production and impairment of key antioxidant defenses in the diaphragm; collectively, these changes contribute to the MV-induced oxidative stress in this key inspiratory muscle. PMID- 16675620 TI - Posterior capsular contracture of the shoulder. AB - Posterior capsular contracture is a common cause of shoulder pain in which the patient presents with restricted internal rotation and reproduction of pain. Increased anterosuperior translation of the humeral head occurs with forward flexion and can mimic the pain reported with impingement syndrome; however, the patient with impingement syndrome presents with normal range of motion. Initial management of posterior capsular contracture should be nonsurgical, emphasizing range-of-motion stretching with the goal of restoring normal motion. For patients who fail nonsurgical management, arthroscopic posterior capsule release can result in improved motion and pain relief. In the throwing athlete, repetitive forces on the posteroinferior capsule may cause posteroinferior capsular hypertrophy and limited internal rotation. This may be the initial pathologic event in the so-called dead arm syndrome, leading to a superior labrum anteroposterior lesion and, possibly, rotator cuff tear. Management involves regaining internal rotation such that the loss of internal rotation is not greater than the increase in external rotation. In the athlete who fails nonsurgical management, a selective posteroinferior capsulotomy can improve motion, reduce pain, and prevent further shoulder injury. PMID- 16675621 TI - Complex elbow instability. AB - Complex elbow instability consists of dislocation of the ulnohumeral joint with a concomitant fracture of one or several of the bony stabilizers of the elbow, including the radial head, proximal ulna, coronoid process, or distal humerus. Recurrent instability is not often associated with simple dislocation, but an improperly managed complex dislocation may be a prelude to chronic, recurrent elbow instability. Complex instability is significantly more demanding to manage than simple instability. Radial head, coronoid, and olecranon fracture associated with dislocation each must be assessed and often require surgery. Long-term outcome with surgical management of complex elbow injuries is unknown. A few published series examine combinations of different injury patterns managed with various methods. Recently, however, several well-designed prospective outcome studies have evaluated management of several different individual fracture dislocation patterns with a unified treatment algorithm. Fixation or replacement of injured bony elements, ligamentous repair, and hinged fixation may be used to successfully manage complex elbow instability. PMID- 16675622 TI - Displaced femoral neck fractures in the elderly: hemiarthroplasty versus total hip arthroplasty. AB - The incidence of femoral neck fracture among the elderly in the United States is expected to increase dramatically because of the anticipated explosion in the population aged 65 years and older, increased life expectancy, and the rising incidence of osteoporosis. The resulting public health implications may be significant, with annual hospital admissions resulting from hip fracture projected to increase to 700,000 by 2050, and with annual spending on hip fracture care expected to exceed 15 billion dollars within a few years. The decision to perform internal fixation, unipolar hemiarthroplasty, bipolar hemiarthroplasty, or THA must be based on patient mental status, living arrangement, level of independence and activity, and bone and joint quality. PMID- 16675623 TI - Congenital and developmental deformities of the spine in children with myelomeningocele. AB - The treatment of spinal deformities in children with myelomeningocele poses a formidable task. Multiple medical comorbidities, such as insensate skin and chronic urinary tract infection, make care of the spine difficult. A thorough understanding of the natural history of these deformities is mandatory for appropriate treatment to be rendered. A team approach that includes physicians from multiple specialties provides the best care for these patients. The two most challenging problems are paralytic scoliosis and rigid lumbar kyphosis. The precise indications for surgical intervention are multifactorial, and the proposed benefits must be weighed against the potential risks. Newer spinal constructs now allow for fixation of the spine in areas previously difficult to instrument. Complications appear to be decreasing with improved understanding of the pathophysiology associated with myelomeningocele. PMID- 16675624 TI - Renal osteodystrophy. AB - The incidence of chronic renal disease is increasing, and the pattern of renal osteodystrophy seems to be shifting from the classic hyperparathyroid presentation to one of low bone turnover. Patients with persistent disease also live longer than previously and are more physically active. Thus, patients may experience trauma as a direct result of increased physical activity in a setting of weakened pathologic bone. Patient quality of life is primarily limited by musculoskeletal problems, such as bone pain, muscle weakness, growth retardation, and skeletal deformity. Chronic renal disease also increases the risk of comorbidity, such as infection, bleeding, and anesthesia-related problems. Current treatment strategies include dietary changes, plate-and-screw fixation, and open reduction and internal fixation. PMID- 16675625 TI - Elbow arthroscopy: basic setup and portal placement. PMID- 16675626 TI - Leg-length discrepancy after total hip arthroplasty. PMID- 16675627 TI - Local heat produces a shear-mediated biphasic response in the thermoregulatory microcirculation of the Pallid bat wing. AB - Investigators report that local heat causes an increase in skin blood flow consisting of two phases. The first is solely sensory neural, and the second is nitric oxide mediated. We hypothesize that mechanisms behind these two phases are causally linked by shear stress. Because microvascular blood flow, endothelial shear stress, and vessel diameters cannot be measured in humans, bat wing arterioles (26.6 +/- 0.3, 42.0 +/- 0.4, and 58.7 +/- 2.2 microm) were visualized noninvasively on a transparent heat plate via intravital microscopy. Increasing plate temperature from 25 to 37 degrees C increased flow in all three arterial sizes (137.1 +/- 0.3, 251.9 +/- 0.5, and 184.3 +/- 0.6%) in a biphasic manner. With heat, diameter increased in large arterioles (n = 6) by 8.7 +/- 0.03% within 6 min, medium arterioles (n = 8) by 19.7 +/- 0.5% within 4 min, and small arterioles (n = 8) by 31.6 +/- 2.2% in the first minute. Lidocaine (0.2 ml, 2% wt/vol) and NG-nitro-L-arginine methyl ester (0.2 ml, 1% wt/vol) were applied topically to arterioles (approximately 40 microm) to block sensory nerves, modulate shear stress, and block nitric oxide generation. Local heat caused only a 10.4 +/- 5.5% increase in diameter with neural blockade (n = 8) and only a 7.5 +/- 4.1% increase in diameter when flow was reduced (n = 8), both significantly lower than control (P < 0.001). Diameter and flow increases were significantly reduced with NG-nitro-L-arginine methyl ester application (P < 0.05). Our novel thermoregulatory animal model illustrates 1) regulation of shear stress, 2) a nonneural component of the first phase, and 3) a shear-mediated second phase. The time course of dilation suggests that early dilation of small arterioles increases flow and enhances second-phase dilation of the large arterioles. PMID- 16675628 TI - High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner. AB - Maternal low-protein diet during pregnancy is a risk factor for cardiovascular disease of the offspring in later life. The impact of high-protein diet during pregnancy on the cardiovascular phenotype of the offspring, however, is still unknown. We examined the influence of a high-protein diet during pregnancy and lactation on the renal, hemodynamic, and metabolic phenotype of the F1 generation. Female Wistar rats were either fed a normal protein diet (20% protein: NP) or an isocaloric high-protein diet (40% protein: HP) throughout pregnancy and lactation. At weaning, the offspring were fed with standard diet, and they were allocated according to sex and maternal diet to four groups: normal protein male (NPm, n = 25), normal-protein female (NPf, n = 19), high-protein male (HPm, n = 24), high-protein female (HPf, n = 29). During the experiment (22 wk), the animals were characterized by repeated measurement of body weight, food intake, blood pressure, glucose tolerance, energy expenditure, and kidney function. At the end of the study period histomorphological analyses of the kidneys and weight measurement of reproductive fat pads were conducted. There were no differences in birth weight between the study groups. No influence of maternal diet on energy expenditure, glucose tolerance, and plasma lipid levels was detected. Blood pressure and glomerulosclerosis were elevated in male offspring only, whereas female offspring were characterized by an increased food efficiency, higher body weight, and increased fat pads. Our study demonstrates that a high-protein diet during pregnancy and lactation in rats programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner. PMID- 16675629 TI - The thioredoxin system in aging muscle: key role of mitochondrial thioredoxin reductase in the protective effects of caloric restriction? AB - Cellular redox balance is maintained by various antioxidative systems. Among those is the thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH. In the present study, we examined the effects of caloric restriction (2 mo) on the expression of the cytosolic and mitochondrial thioredoxin system in skeletal muscle and heart of senescent and young rats. Mitochondrial thioredoxin reductase (TrxR2) is significantly reduced in aging skeletal and cardiac muscle and renormalized after caloric restriction, while the cytosolic isoform remains unchanged. Thioredoxins (mitochondrial Trx2, cytosolic Trx1) are not influenced by caloric restriction. In skeletal and cardiac muscle of young rats, caloric restriction has no effect on the expression of thioredoxins or thioredoxin reductases. Enforced reduction of TrxR2 (small interfering RNA) in myoblasts under exposure to ceramide or TNF-alpha causes a dramatic enhancement of nucleosomal DNA cleavage, caspase 9 activation, and mitochondrial reactive oxygen species release, together with reduced cell viability, while this TrxR2 reduction is without effect in unstimulated myoblasts under basal conditions. Oxidative stress in vitro (H2O2 in C2C12 myoblasts and myotubes) results in different changes: TrxR2, Trx2, and Trx1 are induced without alterations in the cytosolic thioredoxin reductase isoforms. Thus aging is associated with a TrxR2 reduction in skeletal muscle and heart, which enhances susceptibility to apoptotic stimuli but is renormalized after short-term caloric restriction. Exogenous oxidative stress does not result in these age-related changes of TrxR2. PMID- 16675631 TI - The link between cardiac autonomic activity and sleep delta power is altered in men with sleep apnea-hypopnea syndrome. AB - We hypothesize that sleep apnea-hypopnea alters interaction between cardiac vagal modulation and sleep delta EEG. Sleep apnea-hypopnea syndrome (SAHS) is related to cardiovascular complications in men. SAHS patients show higher sympathetic activity than normal subjects. In healthy men, non-rapid eye movement (NREM) sleep is associated with cardiac vagal influence, whereas rapid eye movement (REM) sleep is linked to cardiac sympathetic activity. Interaction between cardiac autonomic modulation and delta sleep EEG is not altered across a life span nor is the delay between appearances of modifications in both signals. Healthy controls, moderate SAHS, and severe SAHS patients were compared across the first three NREM-REM cycles. Spectral analysis was applied to ECG and EEG signals. High frequency (HF) and low frequency (LF) of heart rate variability (HRV), ratio of LF/HF, and normalized (nu) delta power were obtained. A coherency analysis between HF(nu) and delta was performed, as well as a correlation analysis between obstructive apnea index (AI) or hypopnea index (HI) and gain, coherence, or phase shift. HRV components were similar between groups. In each group, HF(nu) was larger during NREM, while LF(nu) predominated across REM and wake stages. Coherence and gain between HF(nu) and delta decreased from controls to severe SAHS patients. In SAHS patients, the delay between modifications in HF(nu) and delta did not differ from zero. AI and HI correlated negatively with coherence, while HI correlated negatively with gain only. Apneas-hypopneas affect the link between cardiac sympathetic and vagal modulation and delta EEG demonstrated by the loss of cardiac autonomic activity fluctuations across shifts in sleep stages. Obstructive apneas and hypopneas alter the interaction between both signals differently. PMID- 16675630 TI - Systemic inflammation and remote organ damage following bilateral femur fracture requires Toll-like receptor 4. AB - Extensive soft tissue injury and bone fracture are significant contributors to the initial systemic inflammatory response in multiply injured patients. Systemic inflammation can lead to organ dysfunction remote from the site of traumatic injury. The mechanisms underlying the recognition of peripheral injury and the subsequent activation of the immune response are unknown. Toll-like receptors (TLRs) recognize microbial products but also may recognize danger signals released from damaged tissues. Here we report that peripheral tissue trauma initiates systemic inflammation and remote organ dysfunction. Moreover, this systemic response to a sterile local injury requires toll-like receptor 4 (TLR4). Compared with wild-type (C3H/HeOuJ) mice, TLR4 mutant (C3H/HeJ) mice demonstrated reduced systemic and hepatic inflammatory responses to bilateral femur fracture. Trauma-induced nuclear factor (NF)-kappaB activation in the liver required functional TLR4 signaling. CD14-/- mice failed to demonstrate protection from fracture-induced systemic inflammation and hepatocellular injury. Therefore, our results also argue against a contribution of intestine-derived LPS to this process. These findings identify a critical role for TLR4 in the rapid recognition and response pathway to severe traumatic injury. Application of these findings in an evolutionary context suggests that multicellular organisms have evolved to use the same pattern recognition receptor for surviving traumatic and infectious challenges. PMID- 16675633 TI - Glucose metabolism during lactation in a fasting animal, the northern elephant seal. AB - Fasting is associated with a series of physiological responses that protect body tissues from degradation by efficiently using expendable energy reserves while sparing protein. Lactation requires the mobilization of maternal nutrients for milk synthesis. The rare life history trait of fasting simultaneous with lactation results in the conflicting demands of provisioning offspring while meeting maternal metabolic costs and preserving maternal tissues for her own survival and future reproduction. Certain tissues continue to require glucose for operation during fasting and might constrain tissue mobilization for lactogenesis due to a need for gluconeogenic substrates. This study investigated glucose flux, glucose cycle activity, and the influence of regulatory hormones in fasting lactating northern elephant seals. Measurements were taken early (5 days) and late (21 days) during the lactation period and, as a nonlactating comparison, after the completion of molting. Glucose cycle activity was highly variable in all study groups and did not change over lactation (P > 0.3), whereas endogenous glucose production decreased during lactation (t = -3.41, P = 0.008). Insulin and insulin-to-glucagon molar ratio decreased across lactation (t = 6.48, 4.28; P = 0.0001, 0.002), while plasma cortisol level increased (t = 4.15, P = 0.002). There were no relationships between glucose production and hormone levels. The glucose production values measured exceeded that predicted from available gluconeogenic substrate, indicating substantial glucose recycling in this species. PMID- 16675632 TI - Prenatal and early postnatal dietary sodium restriction sensitizes the adult rat to amphetamines. AB - Acute sodium deficiency sensitizes adult rats to psychomotor effects of amphetamine. This study determined whether prenatal and early life manipulation of dietary sodium sensitized adult offspring to psychomotor effects of amphetamine (1 or 3 mg/kg ip) in two strains of rats. Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) dams were fed chow containing low NaCl (0.12%; LN), normal NaCl (1%; NN), or high NaCl (4%; HN) throughout breeding, gestation, and lactation. Male offspring were maintained on the test diet for an additional 3 wk postweaning and then fed standard chow thereafter until testing began. Overall, blood pressure (BP), total fluid intake, salt preference, and adrenal gland weight were greater in SHR than in WKY. WKY LN offspring had greater water intake and adrenal gland weight than did WKY NN and HN offspring, whereas WKY HN offspring had increased BP, salt intake, and salt preference compared with other WKY offspring. SHR HN offspring also had increased BP compared with other SHR offspring; all other measures were similar for SHR offspring. The low-dose amphetamine increased locomotor and stereotypical behavior compared with baseline and saline injection in both WKY and SHR offspring. Dietary sodium history affected the rats' psychomotor response to the higher dose of amphetamine. Injections of 3 mg/kg amphetamine in both strains produced significantly more behavioral activity in the LN offspring than in NN and HN offspring. These results show that early life experience with low-sodium diets produce long-term changes in adult rats' behavioral responses to amphetamine. PMID- 16675634 TI - Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE. AB - This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE. PMID- 16675635 TI - Water management in nectar-feeding birds. PMID- 16675636 TI - Sex and gender differences in pain and inflammation: a rapidly maturing field. PMID- 16675637 TI - Hypotensive hypovolemia and hypoglycemia activate different hindbrain catecholamine neurons with projections to the hypothalamus. AB - To better understand the involvement of hindbrain catecholamine neurons in hypovolemia-induced secretion of AVP, we injected antidopamine beta-hydroxylase saporin (DSAP) or unconjugated saporin (SAP) control solution into the hypothalamic paraventricular nucleus (PVH) of anesthetized rats to retrogradely lesion catecholamine neurons innervating magnocellular areas of the hypothalamus. Subsequently, hypotensive hypovolemia was induced by remote blood withdrawal (4.5 ml, 1 ml/min) using an intra-atrial catheter. Blood was sampled at 2, 5, 20, and 50 min after onset of blood withdrawal. The AVP response was severely impaired by DSAP. Peak responses at 50 min were 51 pg/ml in SAP control and 17 pg/ml in DSAP lesioned rats, indicating the importance of catecholamine neurons for this response. We also measured AVP responses to osmotic challenge induced by administration of hypertonic saline (1 M, 15 ml/kg, sc) and to insulin-induced hypoglycemia. Osmotic challenge increased AVP levels, but the response was not impaired by DSAP, indicating that AVP neurons were not damaged by the DSAP injection. Insulin-induced hypoglycemia did not increase AVP levels in either DSAP- or SAP-treated rats. However, the same dose of insulin increased food intake and corticosterone secretion in SAP controls, and these responses were profoundly impaired by DSAP. Thus catecholamine neurons are required for both the AVP response to hypotensive hypovolemia and for feeding and corticosterone responses to hypoglycemia. Lack of an AVP response to insulin-induced hypoglycemia in intact rats therefore indicates that responses to hypovolemia and hypoglycemia are mediated by different catecholamine neurons under distinct sensory controls. PMID- 16675638 TI - Pharmacological stimulation of group ii metabotropic glutamate receptors reduces cocaine self-administration and cocaine-induced reinstatement of drug seeking in squirrel monkeys. AB - Group II metabotropic glutamate receptors (mGluRs) have been implicated in regulating the psychopharmacologic effects of cocaine and other drugs of abuse. The present study investigated the interactions between the group II mGluR agonist LY379268 [(-)-2-oxa-4-aminobicyclo [3.1.0] hexane-4,6-dicarboxylate] and cocaine in squirrel monkeys whose operant behavior was maintained under a second order schedule of i.v. cocaine self-administration with or without presentations of a cocaine-paired visual stimulus, extinguished and subsequently reinstated by priming injections of cocaine with or without presentations of a cocaine-paired stimulus, and controlled by cocaine trained as a discriminative stimulus. Antagonism studies with the group II mGluR antagonist LY341495 [2S-2-amino-2 (1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl) propanoic acid] investigated the extent to which the cocaine-modulating effects of LY379268 could be reversed by blocking group II mGluRs. Quantitative observational studies investigated the effects of LY379268 and LY341495 on species-typical behaviors, balance, and muscle resistance. Pretreatment with LY379268 reduced cocaine self-administration and cocaine-induced reinstatement of drug seeking in a dose-dependent, LY341495 reversible manner. Significant effects of LY379268 were observed both in the presence and absence of the cocaine-paired stimulus. LY379268 did not alter the discriminative stimulus effects of cocaine, nor did it markedly affect observed behavior, with the exception of an increase in visual scanning. Emesis frequently was observed after the highest dose of LY379268 (1.0 mg/kg). The results suggest that LY379268, by stimulating group II mGluRs, can attenuate the reinforcing and priming effects of cocaine at doses that do not alter its perceptibility or markedly suppress other behaviors. PMID- 16675639 TI - Desvenlafaxine succinate: A new serotonin and norepinephrine reuptake inhibitor. AB - The purpose of this study was to characterize a new chemical entity, desvenlafaxine succinate (DVS). DVS is a novel salt form of the isolated major active metabolite of venlafaxine. Competitive radioligand binding assays were performed using cells expressing either the human serotonin (5-HT) transporter (hSERT) or norepinephrine (NE) transporter (hNET) with K(i) values for DVS of 40.2 +/- 1.6 and 558.4 +/- 121.6 nM, respectively. DVS showed weak binding affinity (62% inhibition at 100 microM) at the human dopamine (DA) transporter. Inhibition of [3H]5-HT or [3H]NE uptake by DVS for the hSERT or hNET produced IC50 values of 47.3 +/- 19.4 and 531.3 +/- 113.0 nM, respectively. DVS (10 microM), examined at a large number of nontransporter targets, showed no significant activity. DVS (30 mg/kg orally) rapidly penetrated the male rat brain and hypothalamus. DVS (30 mg/kg orally) significantly increased extracellular NE levels compared with baseline in the male rat hypothalamus but had no effect on DA levels using microdialysis. To mimic chronic selective serotonin reuptake inhibitor treatment and to block the inhibitory 5-HT(1A) autoreceptors, a 5 HT(1A) antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2 pyridinylcyclo hexanecarboxamide maleate salt (WAY-100635) (0.3 mg/kg s.c.), was administered with DVS (30 mg/kg orally). 5-HT increased 78% compared with baseline with no additional increase in NE or DA levels. In conclusion, DVS is a new 5-HT and NE reuptake inhibitor in vitro and in vivo that demonstrates good brain-to-plasma ratios, suggesting utility in a variety of central nervous system related disorders. PMID- 16675640 TI - Differential coupling of muscarinic M1, M2, and M3 receptors to phosphoinositide hydrolysis in urinary bladder and longitudinal muscle of the ileum of the mouse. AB - We investigated the coupling of muscarinic receptor (M) subtypes to phosphoinositide hydrolysis in ileum and urinary bladder using muscarinic receptor knockout mice. In urinary bladder from wild-type mice, the muscarinic agonist oxotremorine-M, elicited a robust phosphoinositide response characterized by an EC50 value of 0.22 microM and a maximal response (Emax) of 32.8% conversion of [3H]inositol-labeled phosphoinositides into [3H]inositol phosphates. A similar response was observed in urinary bladder from M2 knockout mice, whereas no measurable response was observed in urinary bladder from M3 and M2/M3 knockout mice. In ilea from wild-type and M2 knockout mice, substantial phosphoinositide responses to oxotremorine-M were measured, characterized by EC50 values of 0.37 and 0.52 microM and Emax values of 35.8 and 34.7%, respectively. Oxotremorine-M also elicited phosphoinositide hydrolysis in ilea from M3 and M2/M3 knockout mice, although these responses were less sensitive (EC50 values of 1.6 and 1.4 microM; Emax values of 31.2 and 20.8%, respectively). The response in ileum from the M2/M3 knockout was significantly smaller than that from the M3 knockout. The muscarinic phosphoinositide response in ilea from M2/M3 knockout mice originated in the smooth muscle and exhibited a profile for competitive antagonism consistent with an M1 mechanism. These data suggest a major role for the M3 receptor in eliciting phosphoinositide hydrolysis in the ileum and urinary bladder and minor roles for the M1 and M2 in ileum. PMID- 16675641 TI - Treatments for metabolic syndrome may expand. PMID- 16675642 TI - Military pharmacy confronts present, future issues. PMID- 16675643 TI - Nigerian pharmacists get involved in HIV treatment. PMID- 16675645 TI - Predicted plasma valproic acid concentrations in patients missing and replacing a full daily dose of extended-release divalproex sodium. PMID- 16675646 TI - Visual compatibility of furosemide with phenylephrine and vasopressin. PMID- 16675647 TI - Educating patients in self-management of epistaxis in an anticoagulation clinic. PMID- 16675648 TI - Cultural competency for new practitioners. PMID- 16675650 TI - Toxicology of oral antidiabetic medications. AB - PURPOSE: The toxicology of oral antidiabetic agents is reviewed. SUMMARY: Type 2 diabetes mellitus is increasing to near epidemic proportions, with a reported 190 million patients worldwide. Use of oral antidiabetic medications is increasing along with a proportional increase in adverse events. Oral antidiabetic medications can be separated by mechanism of action into two groups: hypoglycemics (sulfonylureas and meglitinides) and antihyperglycemics (biguanides and alpha-glucosidase inhibitors). The hypoglycemic agents pose a significant risk of morbidity, mortality, and permanent sequelae secondary to prolonged periods of hypoglycemia. However, outcomes are routinely good if intervention is initiated early, with the primary goal return of euglycemia using supplemental dextrose infusion and octreotide to reduce further insulin secretion. Metformin associated lactic acidosis (MALA) can occur with both acute and chronic metformin exposure. While MALA is not common, its associated rates of morbidity and mortality can be high. Secondary to MALA, the patient may experience changes in the central nervous system, cardiovascular collapse, renal failure, and death. The primary goals of therapy are restoration of acid-base status and removal of metformin, using hemodialysis and bicarbonate therapy. There is no specific antidote for MALA. The alpha-glucosidase inhibitors and thiazolidinediones pose minimal risk of adverse events in acute overdose. However, acarbose and all thiazolidinediones have been reported to produce hepatic injury with chronic therapy. Cessation of therapy with the offending agent and supportive care are the mainstays of overdose management with these drugs. CONCLUSION: The toxicity of oral antidiabetic agents differs widely in clinical manifestations, severity, and treatment. PMID- 16675649 TI - Rasagiline: A second-generation monoamine oxidase type-B inhibitor for the treatment of Parkinson's disease. AB - PURPOSE: The pharmacology, pharmacokinetics, clinical efficacy, and safety of rasagiline are reviewed. SUMMARY: Rasagiline is a novel, investigational propargylamine that irreversibly and selectively inhibits monoamine oxidase type B (MAO-B). Rasagiline demonstrates complete and selective inhibition of MAO-B and is at least five times more potent than selegiline. Unlike selegiline, which is metabolized to amphetamine derivatives, rasagiline is biotransformed to the nonamphetamine compound aminoindan. Clinical studies have revealed that rasagiline is associated with improved outcomes in patients with early Parkinson's disease (PD) and also reduces "off" time in patients with moderate to advanced PD with motor fluctuations. Rasagiline is rapidly absorbed by the gastrointestinal tract and readily crosses the blood-brain barrier. The optimal therapeutic dosage is 0.5-1 mg administered orally once daily. Rasagiline appears to be well tolerated, although elderly patients may be more prone to treatment emergent adverse cardiovascular and psychiatric effects. At the recommended therapeutic dosage of up to 1 mg once daily, tyramine restriction is unnecessary. In addition to MAO-B inhibition, rasagiline has demonstrated neuroprotective properties in experimental laboratory models. The mechanisms whereby rasagiline exerts neuroprotective effects are multifactorial and include upregulation of cellular antioxidant activity and antiapoptotic factors. CONCLUSION: Rasagiline is an investigational selective and irreversible inhibitor of MAO-B that has demonstrated efficacy and safety for the treatment of PD. Whether rasagiline is associated with clinically significant neuroprotection is the subject of ongoing clinical trials. PMID- 16675651 TI - Dosing cards for treatment of children exposed to weapons of mass destruction. PMID- 16675652 TI - Effect of internal reporting criteria on suspected adverse drug reactions submitted to MedWatch. PMID- 16675653 TI - Professional Development Experience: a learning activity for pharmacy residents. PMID- 16675654 TI - Information technology for optimizing the management of infectious diseases. PMID- 16675655 TI - Antipsychotic medication and the death penalty. PMID- 16675658 TI - Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors. AB - Xanomeline is a functionally selective M(1)/M(4) muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M(1) receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M(1) and M(2) receptors expressed in Chinese hamster ovary cells and [(3)H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M(2) receptor with similar affinity as at the M(1) receptor (K(I) = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M(2) receptors was markedly slower than to M(1) receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5'-O-(3-[(35)S]thio)triphosphate binding at M(1) receptors, whereas at M(2) receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M(2) receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M(1) than at M(2) receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M(1) and the M(2) receptor. PMID- 16675657 TI - OSU-03012 in the treatment of glioblastoma. AB - In an article presented in this issue of Molecular Pharmacology, Yacoub et al. (p. 589) examine the actions of 2-amino-N{4-5-(2-phenanthrenyl)-3 (trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-acetamide (OSU-03012) on both primary and glioblastoma cell lines. The authors found that OSU-03012 could induce tumor cell death by itself but also acted as a strong sensitizing agent to radiotherapy induced cell death. Glioblastoma cells were also more sensitive to this compound than nontransformed astrocytes. Radiation-induced cell death was refractory to small interfering RNA-directed inhibition of PDK1 but not OSU-03012. These results indicate that OSU-03012, which has been thought to primarily mediate antitumor effects via the inhibition of PDK1, has actions independent of PDK1. Furthermore, the authors demonstrated that the effects of OSU-03012 were independent of ERB-B1-vIII and PTEN expression. These are important findings because they start to identify a new mechanism to sensitize glioblastoma cells and also suggest that OSU-03012 could be combined with existing inhibitors to further sensitize tumor cells. In glioblastoma cells, OSU-03012 seemed to induce apoptosis via endoplasmic reticulum stress-induced PERK-dependent signaling. OSU 03012-induced death of the glioblastoma was only weakly suppressed by the pan caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp, suggesting that OSU-03012 induced cell death was largely caspase-independent. Overall, these are exciting results and suggest that new more effective treatment options may be obtainable for people suffering from these deadly tumors. PMID- 16675659 TI - Isoliquiritigenin selectively inhibits H(2) histamine receptor signaling. AB - Isoliquiritigenin, one of the major constituents of Glycyrrhiza uralensis (licorice), is a natural pigment with a simple chalcone structure 4,2',4' trihydroxychalcone. In this study, isoliquiritigenin showed selective H(2) histamine receptor (H(2)R) antagonistic effect and remarkably reduced several H(2)R-mediated physiological responses. Preincubation of U937 and HL60 hematopoietic cells with isoliquiritigenin significantly inhibited H(2)R agonist induced cAMP response in a concentration-dependent manner without affecting the viability of cells. Isoliquiritigenin also blocked the binding affinity of [(3)H]tiotidine to membrane receptors in HL-60 cells. Isoliquiritigenin did not affect the elevation of cAMP levels induced by cholera toxin, forskolin, or isoproterenol, indicating that the action site of isoliquiritigenin is not G(s) protein, effector enzyme, adenylyl cyclase, or beta(2)-adrenoceptor. Isoliquiritigenin affected neither H(1)R-nor H(3)R-mediated signaling. In molecular docking studies, isoliquiritigenin exhibited more favorable interactions with H(2)R than histamine. Isoliquiritigenin prominently inhibited H(2)R selective agonist dimaprit-induced cAMP generation in MKN-45 gastric cancer cell. Moreover, isoliquiritigenin reduced gastric acid secretion and protected gastric mucosal lesion formation in pylorus-ligated rat model. Taken together, the results demonstrate that isoliquiritigenin is an effective H(2)R antagonist and provides the basis for designing novel H(2)R antagonist. PMID- 16675660 TI - 5'-O-tritylated nucleoside derivatives: inhibition of thymidine phosphorylase and angiogenesis. AB - Thymidine phosphorylase (TPase) is one of the key enzymes involved in the pyrimidine nucleoside salvage pathway. However, TPase also stimulates angiogenesis, and its expression correlates well with microvessel density and metastasis in a variety of human tumors. We have shown recently that 5'-O-trityl inosine (KIN59) allosterically inhibits TPase enzymatic activity. KIN59 also inhibits TPase-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay. The trityl group was found to be instrumental to preserve both the anti TPase and antiangiogenic effect. We have now synthesized a variety of novel 5'-O trityl nucleoside derivatives. Enzyme activity studies showed that the anti-TPase activity is significantly improved by replacement of the hypoxanthine base by thymine [3.5-fold; i.e., 5'-O-tritylthymidine (KIN6)] and the introduction of chloride on the trityl group [7-fold; i.e., 5'-O-(4-chlorotrityl)-inosine (TP136)], whereas removal of 2'-hydroxyl in the ribose did not significantly alter the anti-TPase activity. Enzyme kinetic studies also demonstrated that 1 (5'-O-trityl-beta-d-ribofuranosyl)-thymine (TP124), like KIN59, inhibits TPase in a noncompetitive fashion both with respect to phosphate and thymidine. Most KIN59 analogs markedly inhibited TPase-induced angiogenesis in the CAM assay. In vitro studies showed that the antiangiogenic effect of these compounds is not attributed to endothelial cell toxicity. For several compounds, there was no stringent correlation between their anti-TPase and antiangiogenic activity, indicating that these compounds may also act on other angiogenesis mediators. The antiangiogenic 5'-O-trityl nucleoside analogs also caused degradation of pre existing, immature vessels at the site of drug exposure. Thus, 5'-O-trityl nucleoside derivatives combine antiangiogenic and vascular-targeting activities, which opens perspectives for their potential use as anticancer agents. PMID- 16675661 TI - Allosteric interactions required for high-affinity binding of dihydropyridine antagonists to Ca(V)1.1 Channels are modulated by calcium in the pore. AB - Dihydropyridines (DHPs) are an important class of drugs, used extensively in the treatment of angina pectoris, hypertension, and arrhythmia. The molecular mechanism by which DHPs modulate Ca(2+) channel function is not known in detail. We have found that DHP binding is allosterically coupled to Ca(2+) binding to the selectivity filter of the skeletal muscle Ca(2+) channel Ca(V)1.1, which initiates excitation-contraction coupling and conducts L-type Ca(2+) currents. Increasing Ca(2+) concentrations from approximately 10 nM to 1 mM causes the DHP receptor site to shift from a low-affinity state to a high-affinity state with an EC(50) for Ca(2+) of 300 nM. Substituting each of the four negatively charged glutamate residues that form the ion selectivity filter with neutral glutamine or positively charged lysine residues results in mutant channels whose DHP binding affinities are decreased up to 10-fold and are up to 150-fold less sensitive to Ca(2+) than wild-type channels. Analysis of mutations of amino acid residues adjacent to the selectivity filter led to identification of Phe-1013 and Tyr 1021, whose mutation causes substantial changes in DHP binding. Thermo-dynamic mutant cycle analysis of these mutants demonstrates that Phe-1013 and Tyr-1021 are energetically coupled when a single Ca(2+) ion is bound to the channel pore. We propose that DHP binding stabilizes a nonconducting state containing a single Ca(2+) ion in the pore through which Phe-1013 and Tyr-1021 are energetically coupled. The selectivity filter in this energetically coupled high-affinity state is blocked by bound Ca(2+), which is responsible for the high-affinity inhibition of Ca(2+) channels by DHP antagonists. PMID- 16675662 TI - Why the cosmological constant is small and positive. AB - Within conventional big bang cosmology, it has proven to be very difficult to understand why today's cosmological constant is so small. In this paper, we show that a cyclic model of the universe can naturally incorporate a dynamical mechanism that automatically relaxes the value of the cosmological constant, including contributions to the vacuum density at all energy scales. Because the relaxation time grows exponentially as the vacuum density decreases, nearly every volume of space spends an overwhelming majority of the time at the stage when the cosmological constant is small and positive, as observed today. PMID- 16675663 TI - CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. AB - Forest trees display a perennial growth behavior characterized by a multiple-year delay in flowering and, in temperate regions, an annual cycling between growth and dormancy. We show here that the CO/FT regulatory module, which controls flowering time in response to variations in daylength in annual plants, controls flowering in aspen trees. Unexpectedly, however, it also controls the short-day induced growth cessation and bud set occurring in the fall. This regulatory mechanism can explain the ecogenetic variation in a highly adaptive trait: the critical daylength for growth cessation displayed by aspen trees sampled across a latitudinal gradient spanning northern Europe. PMID- 16675664 TI - Astronomy. The vacuum energy crisis. PMID- 16675665 TI - Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. AB - Riboswitches are untranslated regions of messenger RNA, which adopt alternate structures depending on the binding of specific metabolites. Such conformational switching regulates the expression of proteins involved in the biosynthesis of riboswitch substrates. Here, we present the 2.9 angstrom-resolution crystal structure of the eukaryotic Arabidopsis thaliana thiamine pyrophosphate (TPP) specific riboswitch in complex with its natural ligand. The riboswitch specifically recognizes the TPP via conserved residues located within two highly distorted parallel "sensor" helices. The structure provides the basis for understanding the reorganization of the riboswitch fold upon TPP binding and explains the mechanism of resistance to the antibiotic pyrithiamine. PMID- 16675666 TI - The high cost of coming to America. PMID- 16675667 TI - Veterans Administration. Texas earmark allots millions to disputed theory of Gulf War illness. PMID- 16675668 TI - Biomedicine. Genes and chronic fatigue: how strong is the evidence? PMID- 16675669 TI - U.S. research funding. Industry shrinks academic support. PMID- 16675670 TI - Quantum optics. A new way to beat the limits on shrinking transistors? PMID- 16675671 TI - Immunology. Differences in immune cell "brakes" may explain chimp-human split on AIDS. PMID- 16675672 TI - Neurobiology. Despite mutated gene, mouse circadian clock keeps on ticking. PMID- 16675673 TI - Energy research. Industry conservation programs face White House cuts. PMID- 16675675 TI - Nuclear physics. Indian angst over atomic pact. PMID- 16675674 TI - Climatology. A tempestuous birth for hurricane climatology. PMID- 16675677 TI - Physics. Tipping the scales--just barely. PMID- 16675676 TI - Neuroscience. The map in the brain: grid cells may help us navigate. PMID- 16675678 TI - Astronomy. Rising from the ashes. PMID- 16675684 TI - Debating sexual selection and mating strategies. PMID- 16675685 TI - Animal research. Harmonization of animal care and use guidance. PMID- 16675686 TI - Planetary science. Linear dunes on Titan. PMID- 16675687 TI - Chemistry. Shuttling polyolefins to a new materials dimension. PMID- 16675688 TI - Social science. The pleasures and pains of information. PMID- 16675689 TI - Planetary science. The primordial porridge. PMID- 16675690 TI - Geochemistry. Follow the nitrogen. PMID- 16675691 TI - Immunology. B cells spread and gather. PMID- 16675692 TI - Retrospective. Nicholas J. Shackleton (1937-2006). PMID- 16675693 TI - Environmental monitoring by wireless communication networks. AB - The global spread of wireless networks brings a great opportunity for their use in environmental studies. Weather, atmospheric conditions, and constituents cause propagation impairments on radio links. As such, while providing communication facilities, existing wireless communication systems can be used as a widely distributed, high-resolution atmospheric observation network, operating in real time with minimum supervision and without additional cost. Here we demonstrate how measurements of the received signal level, which are made in a cellular network, provide reliable measurements for surface rainfall. We compare the estimated rainfall intensity with radar and rain gauge measurements. PMID- 16675694 TI - Catalytic production of olefin block copolymers via chain shuttling polymerization. AB - We report a catalytic system that produces olefin block copolymers with alternating semicrystalline and amorphous segments, achieved by varying the ratio of alpha-olefin to ethylene in the two types of blocks. The system uses a chain shuttling agent to transfer growing chains between two distinct catalysts with different monomer selectivities in a single polymerization reactor. The block copolymers simultaneously have high melting temperatures and low glass transition temperatures, and therefore they maintain excellent elastomeric properties at high temperatures. Furthermore, the materials are effectively produced in economically favorable, continuous polymerization processes. PMID- 16675695 TI - The sand seas of Titan: Cassini RADAR observations of longitudinal dunes. AB - The most recent Cassini RADAR images of Titan show widespread regions (up to 1500 kilometers by 200 kilometers) of near-parallel radar-dark linear features that appear to be seas of longitudinal dunes similar to those seen in the Namib desert on Earth. The Ku-band (2.17-centimeter wavelength) images show approximately 100 meter ridges consistent with duneforms and reveal flow interactions with underlying hills. The distribution and orientation of the dunes support a model of fluctuating surface winds of approximately 0.5 meter per second resulting from the combination of an eastward flow with a variable tidal wind. The existence of dunes also requires geological processes that create sand-sized (100- to 300 micrometer) particulates and a lack of persistent equatorial surface liquids to act as sand traps. PMID- 16675696 TI - Interstellar chemistry recorded in organic matter from primitive meteorites. AB - Organic matter in extraterrestrial materials has isotopic anomalies in hydrogen and nitrogen that suggest an origin in the presolar molecular cloud or perhaps in the protoplanetary disk. Interplanetary dust particles are generally regarded as the most primitive solar system matter available, in part because until recently they exhibited the most extreme isotope anomalies. However, we show that hydrogen and nitrogen isotopic compositions in carbonaceous chondrite organic matter reach and even exceed those found in interplanetary dust particles. Hence, both meteorites (originating from the asteroid belt) and interplanetary dust particles (possibly from comets) preserve primitive organics that were a component of the original building blocks of the solar system. PMID- 16675697 TI - Lower Cambrian vendobionts from China and early diploblast evolution. AB - Ediacaran assemblages immediately predate the Cambrian explosion of metazoans and should have played a crucial role in this radiation. Their wider relationships, however, have remained refractory and difficult to integrate with early metazoan phylogeny. Here, we describe a frondlike fossil, Stromatoveris (S. psygmoglena sp. nov.), from the Lower Cambrian Chengjiang Lagerstatte (Yunnan, China) that is strikingly similar to Ediacaran vendobionts. The exquisite preservation reveals closely spaced branches, probably ciliated, that appear to represent precursors of the diagnostic comb rows of ctenophores. Therefore, this finding has important implications for the early evolution of this phylum and related diploblasts, some of which independently evolved a frondose habit. PMID- 16675698 TI - Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. AB - Fat tissue is the most important energy depot in vertebrates. The release of free fatty acids (FFAs) from stored fat requires the enzymatic activity of lipases. We showed that genetic inactivation of adipose triglyceride lipase (ATGL) in mice increases adipose mass and leads to triacylglycerol deposition in multiple tissues. ATGL-deficient mice accumulated large amounts of lipid in the heart, causing cardiac dysfunction and premature death. Defective cold adaptation indicated that the enzyme provides FFAs to fuel thermogenesis. The reduced availability of ATGL-derived FFAs leads to increased glucose use, increased glucose tolerance, and increased insulin sensitivity. These results indicate that ATGL is rate limiting in the catabolism of cellular fat depots and plays an important role in energy homeostasis. PMID- 16675699 TI - B cell ligand discrimination through a spreading and contraction response. AB - B cells recognize foreign antigens by virtue of cell surface immunoglobulin receptors and are most effectively activated by membrane-bound ligands. Here, we show that in the early stages of this process, B cells exhibit a two-phase response in which they first spread over the antigen-bearing membrane and then contract, thereby collecting bound antigen into a central aggregate. The extent of this response, which is both signaling- and actin-dependent, determines the quantity of antigen accumulated and hence the degree of B cell activation. Brownian dynamic simulations reproduce essential features of the antigen collection process and suggest a possible basis for affinity discrimination. We propose that dynamic spreading is an important step of the immune response. PMID- 16675700 TI - Structure of the multidrug transporter EmrD from Escherichia coli. AB - EmrD is a multidrug transporter from the Major Facilitator Superfamily that expels amphipathic compounds across the inner membrane of Escherichia coli. Here, we report the x-ray structure of EmrD determined to a resolution of 3.5 angstroms. The structure reveals an interior that is composed mostly of hydrophobic residues, which is consistent with its role transporting amphipathic molecules. Two long loops extend into the inner leaflet side of the cell membrane. This region can serve to recognize and bind substrate directly from the lipid bilayer. We propose that multisubstrate specificity, binding, and transport are facilitated by these loop regions and the internal cavity. PMID- 16675701 TI - Signal recognition particle receptor exposes the ribosomal translocon binding site. AB - Signal sequences of secretory and membrane proteins are recognized by the signal recognition particle (SRP) as they emerge from the ribosome. This results in their targeting to the membrane by docking with the SRP receptor, which facilitates transfer of the ribosome to the translocon. Here, we present the 8 angstrom cryo-electron microscopy structure of a "docking complex" consisting of a SRP-bound 80S ribosome and the SRP receptor. Interaction of the SRP receptor with both SRP and the ribosome rearranged the S domain of SRP such that a ribosomal binding site for the translocon, the L23e/L35 site, became exposed, whereas Alu domain-mediated elongation arrest persisted. PMID- 16675702 TI - The embryonic vertebrate heart tube is a dynamic suction pump. AB - The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube. PMID- 16675703 TI - Neurobiological substrates of dread. AB - Given the choice of waiting for an adverse outcome or getting it over with quickly, many people choose the latter. Theoretical models of decision-making have assumed that this occurs because there is a cost to waiting-i.e., dread. Using functional magnetic resonance imaging, we measured the neural responses to waiting for a cutaneous electric shock. Some individuals dreaded the outcome so much that, when given a choice, they preferred to receive more voltage rather than wait. Even when no decision was required, these extreme dreaders were distinguishable from those who dreaded mildly by the rate of increase of neural activity in the posterior elements of the cortical pain matrix. This suggests that dread derives, in part, from the attention devoted to the expected physical response and not simply from fear or anxiety. Although these differences were observed during a passive waiting procedure, they correlated with individual behavior in a subsequent choice paradigm, providing evidence for a neurobiological link between the experienced disutility of dread and subsequent decisions about unpleasant outcomes. PMID- 16675704 TI - Conjunctive representation of position, direction, and velocity in entorhinal cortex. AB - Grid cells in the medial entorhinal cortex (MEC) are part of an environment independent spatial coordinate system. To determine how information about location, direction, and distance is integrated in the grid-cell network, we recorded from each principal cell layer of MEC in rats that explored two dimensional environments. Whereas layer II was predominated by grid cells, grid cells colocalized with head-direction cells and conjunctive grid x head-direction cells in the deeper layers. All cell types were modulated by running speed. The conjunction of positional, directional, and translational information in a single MEC cell type may enable grid coordinates to be updated during self-motion-based navigation. PMID- 16675705 TI - CRM197-conjugated serogroup C meningococcal capsular polysaccharide, but not the native polysaccharide, induces persistent antigen-specific memory B cells. AB - Neisseria meningitidis is one of the leading causes of bacterial meningitis and septicemia in children. Vaccines containing the purified polysaccharide capsule from the organism, a T cell-independent antigen, have been available for decades but do not appear to provide protection in infancy or immunologic memory as measured by antibody responses. By contrast, T cell-dependent serogroup C protein polysaccharide conjugate vaccines protect against serogroup C meningococcal disease from infancy onward and prime for immunologic memory. We compared the magnitude and kinetics of plasma cell and memory B-cell responses to a meningococcal plain polysaccharide vaccine and a serogroup C glycoconjugate vaccine in adolescents previously primed with the conjugate vaccine. Plasma cell kinetics were similar for both vaccines, though the magnitude of the response was greater for the glycoconjugate. In contrast to the glycoconjugate vaccine, the plain polysaccharide vaccine did not induce a persistent immunoglobulin G (IgG) memory B-cell response. This is the first study to directly show that serogroup C meningococcal glycoconjugate vaccines induce persistent production of memory B cells and that plain polysaccharide vaccines do not, supporting the use of the conjugate vaccine for sustained population protection. Detection of peripheral blood memory B-cell responses after vaccination may be a useful signature of successful induction of immunologic memory during novel vaccine evaluation. PMID- 16675706 TI - Oncogenic K-ras cooperates with PML-RAR alpha to induce an acute promyelocytic leukemia-like disease. AB - Most patients with acute promyelocytic leukemia (APL) express PML-RAR alpha, the fusion product of t(15;17)(q22;q11.2). Transgenic mice expressing PML-RAR alpha develop APL with long latency, low penetrance, and acquired cytogenetic abnormalities. Based on observations that 4% to 10% of APL patients harbor oncogenic ras mutations, we coexpressed oncogenic K-ras from its endogenous promoter with PML-RAR alpha to generate a short-latency, highly penetrant mouse model of APL. The APL disease was characterized by splenomegaly, leukocytosis, extramedullary hematopoiesis (EMH) in spleen and liver with an increased proportion of immature myeloperoxidase-expressing myeloid forms; transplantability to secondary recipients; and lack of cytogenetic abnormalities. Bone marrow cells showed enhanced self-renewal in vitro. This model establishes a role for oncogenic ras in leukemia pathogenesis and thus validates the oncogenic RAS signaling pathway as a potential target for therapeutic inhibition in leukemia patients. This mouse model should be useful for investigating signaling pathways that promote self-renewal in APL and for testing the in vivo efficacy of RAS signaling pathway inhibitors in conjunction with other targeted therapies such as ATRA (all trans retinoic acid) and arsenic trioxide. PMID- 16675707 TI - International Working Group (IWG) consensus criteria for treatment response in myelofibrosis with myeloid metaplasia, for the IWG for Myelofibrosis Research and Treatment (IWG-MRT). AB - Myelofibrosis with myeloid metaplasia (MMM) is a clinicopathologic entity characterized by stem cell-derived clonal myeloproliferation, ineffective erythropoiesis, extramedullary hematopoiesis, and bone marrow fibrosis and osteosclerosis. Patients with MMM have shortened survival and their quality of life is compromised by progressive anemia, marked hepatosplenomegaly, and severe constitutional symptoms including cachexia. After decades of frustration with ineffective therapy, patients are now being served by promising treatment approaches that include allogeneic hematopoietic stem cell transplantation and immunomodulatory drugs. Recent information regarding disease pathogenesis, including a contribution to the myeloproliferative disorder phenotype by a gain of-function JAK2 mutation (JAK2(V617F)), has revived the prospect of targeted therapeutics as well as molecular monitoring of treatment response. Such progress calls for standardization of response criteria to accurately assess the value of new treatment modalities, to allow accurate comparison between studies, and to ensure that the definition of response reflects meaningful health outcome. Accordingly, an international panel of experts recently convened and delineated 3 response categories: complete remission (CR), partial remission (PR), and clinical improvement (CI). Bone marrow histologic and hematologic remissions characterize CR and CR/PR, respectively. The panel agreed that the CI response category is applicable only to patients with moderate to severe cytopenia or splenomegaly. PMID- 16675708 TI - Dendritic cells and HIV-specific CD4+ T cells: HIV antigen presentation, T-cell activation, and viral transfer. AB - Human immunodeficiency virus (HIV)-specific CD4+ lymphocytes are preferentially infected in HIV-positive individuals. To study this preferential infection, we have derived several HIV-specific (HS) CD4+ clones. We show that in dendritic cells (DCs), HIV virion capture led to major histocompatibility complex class-II (MHC-II)-restricted viral antigen presentation and to activation of HS cells. In contrast, neither cell-free virions nor infected lymphocytes activated HS cells. In DCs, the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN/CD209), which internalizes virions, promoted MHC-II presentation of HIV antigens. Activation of HS cells by HIV-exposed DCs triggered an efficient viral spread in lymphocytes. CD4+ clones with irrelevant antigenic specificities were not activated by HIV-exposed DCs and poorly supported viral replication under this setting. Our results unravel the mechanisms of MHC-II restricted HIV antigen presentation by DCs and describe how HIV gains access to the very cells designed by the immune system to counteract this pathogen. PMID- 16675709 TI - Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit expressing lineage-negative hematopoietic cells. AB - The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fraction of the bone marrow. In addition, conditional transgene activation also was detected in a very minor population of endothelial cells and in the kidney. However, no activation of the reporter transgene was found in the brain of adult mice. These findings suggested that the expression of tetracycline-responsive reporter genes recapitulated the known endogenous expression pattern of SCL. Our data therefore demonstrate that exogenously inducible and reversible expression of selected transgenes in myeloid, megakaryocytic, erythroid, and c-kit-expressing lineage-negative bone marrow cells can be directed through SCL regulatory elements. The SCL knock-in mouse presented here represents a powerful tool for studying normal and malignant hematopoiesis in vivo. PMID- 16675710 TI - Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders. AB - An acquired gain-of-function mutation in the Janus kinase 2 (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPDs). To test the hypothesis that JAK2-V617F is the disease-initiating mutation, we examined whether all cells of clonal origin carry the JAK2-V617F mutation. Using allele specific polymerase chain reaction (PCR) assays for the JAK2 mutation and for the X-chromosomal clonality markers IDS and MPP1, we found that the percentage of granulocytes and platelets with JAK2-V617F was often markedly lower than the percentage of clonal granulocytes determined by IDS or MPP1 clonality assays in female patients. Using deletions of chromosome 20q (del20q) as an autosomal, X chromosome-independent clonality marker, we found a similar discrepancy between the percentage of cells carrying JAK2-V617F and del20q. Our results suggest that in a proportion of patients with MPDs, JAK2-V617F occurs on the background of clonal hematopoiesis caused by a somatic mutation in an as-yet-unknown gene. PMID- 16675711 TI - Increasing Flt3L availability alters composition of a novel bone marrow lymphoid progenitor compartment. AB - We have recently described a CD19(-) B220(+)CD117(low) bone marrow subpopulation with B, T, and myeloid developmental potential, which we have called "early progenitors with lymphoid and myeloid potential" or EPLM. These cells also expressed Fms-like tyrosine kinase 3, Flt3, or CD135. Treatment of mice with the corresponding ligand, Flt3L, showed a 50-fold increase in EPLM. In addition to the expected increase in dendritic cell numbers, Flt3L treatment had a reversible inhibitory effect on B lymphopoiesis. Limiting dilution analysis of sorted EPLM from Flt3L-treated mice showed that B-lymphocyte progenitor activity was reduced 20-fold, but that myeloid and T-cell progenitor activity was largely preserved. EPLM from treated mice transiently reconstituted the thymus and bone marrow of recipient mice, generating cohorts of functional T and B cells in peripheral lymphoid organs. Thus, Flt3L treatment results in a dramatic increase in a novel bone marrow cell with lymphoid and myeloid progenitor activity. PMID- 16675712 TI - In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes. AB - Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34+ progenitor derived dendritic cells loaded with CMV antigen and its immune complex. CMV pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV+ cultures showed a preferential expansion of CD45RA-/RO+/CD27+ T cells compared to CMV- cultures. CMV-specific IFN-gamma- and TNF-alpha-producing CD4+ (Th1) and CD8+ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later. PMID- 16675714 TI - Family mediators and moderators of treatment outcomes among youths with poorly controlled type 1 diabetes: results from a randomized controlled trial. AB - OBJECTIVE: To determine whether multisystemic therapy (MST) improved family relationships among youths with poorly controlled type 1 diabetes and whether these changes mediated MST effects on health outcomes. The moderating effect of family demographics on study outcomes was also assessed. METHODS: A randomized controlled trial was conducted with 127 youths. Changes in general family relationships and caregiver support for diabetes care from baseline to treatment completion were assessed. Structural equation modeling (SEM) was used to test whether changes in family relations mediated improvements in frequency of blood glucose testing (BGT) and metabolic control. RESULTS: MST increased support for diabetes care from both primary and secondary caregivers in two-parent but not in single-parent families. However, MST had the strongest effects on BGT and metabolic control in single-parent families. SEM did not support family relations as the mediator of improved BGT or metabolic control. Rather, MST had a direct effect on BGT for all participants. BGT mediated improvements in metabolic control among single-parent families. CONCLUSIONS: MST improved family relationships for youths with diabetes in two-parent but not in single-parent families. Objective outcomes related to diabetes were strongest for single-parent families. Other processes such as increased parental monitoring may have been responsible for improved health outcomes among these families. PMID- 16675713 TI - Antileukemia activity of the combination of an anthracycline with a histone deacetylase inhibitor. AB - We studied the cellular and molecular effects of the combination of an anthracycline with 2 different histone deacetylase inhibitors (HDACIs): vorinostat (suberoylanilide hydroxamic acid) and valproic acid (VPA). The 10% inhibitory concentration (IC(10)) of idarubicin was 0.5 nM in MOLT4 and 1.5 nM in HL60 cells. Concentrations above 0.675 microM of vorinostat resulted in at least 80% loss of cell viability in both cell lines. Concentrations of 1.5 to 3 mM of VPA induced 50% to 60% loss in viability in HL60 and 80% in MOLT4 cells. The combination of idarubicin with vorinostat at 0.075 microM or VPA at 0.25 mM resulted in at least an additive loss of cell viability in both lines. Vorinostat (0.35 microM) and VPA (0.25 mM) in combination with idarubicin (0.5 nM) resulted in a significant increase in apoptotic cells in MOLT4 cells. The combination resulted in an increase in histone H3 and H4 acetylation at 24 hours, phosphorylated H2AX, as well as in the induction of p21(CIP1) mRNA. No effect on cell cycle transition was observed. Of importance, the cellular and molecular effects observed were independent of the sequence used. In summary, the combination of an anthracycline with an HDACI should have significant clinical activity in patients with leukemia. PMID- 16675715 TI - Introduction to special issue: Evidence-based assessment in pediatric psychology. PMID- 16675716 TI - Brief report: effect of intravenous methotrexate dose and infusion rate on neuropsychological function one year after diagnosis of acute lymphoblastic leukemia. AB - OBJECTIVE: To compare the effects of two intravenous (IV) methotrexate (MTX) infusion protocols on cognitive function in children newly diagnosed with acute lymphoblastic leukemia (ALL). METHODS: We compared 19 children treated with 1 g/m(2) of IV MTX over 24 hr (Group 1) to 13 children treated with 2 g/m(2) of IV MTX over 4 hr (Group 2) on measures of working memory, nonverbal, and verbal skills shortly after diagnosis (Time 1) and 1 year later (Time 2). RESULTS: A significant GroupxTime interaction was found for a composite measure of working memory with Group 2 declining from Time 1 to Time 2. Group 2 performed significantly worse than Group 1 on a composite measure of nonverbal skills at both time points. CONCLUSIONS: Findings suggest that difficulties in working memory and nonverbal skills may be evident during the first year of treatment for ALL and that severity may be dependent on IV MTX dose and/or infusion rate. PMID- 16675717 TI - A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility. AB - In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2 aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation. PMID- 16675718 TI - Syndecan-4 clustering induces cell migration in a PDZ-dependent manner. AB - Cell migration is a dynamic process involving formation of a leading edge in the direction of migration and adhesion points from which tension is generated to move the cell body forward. At the same time, disassembly of adhesion points occurs at the back of the cell, a region known as the trailing edge. Syndecan-4 (S4) is a transmembrane proteoglycan thought to be involved in the formation of focal adhesions. Recent studies have shown that its cytoplasmic domain can engage in signal transduction, making S4 a bona fide receptor. Here, we show that ligand clustering of cell surface S4 on endothelial cells initiates a signaling cascade that results in activation of Rac1, induction of cell polarization, and stimulation of cell migration that depends on S4 interaction with its PDZ-binding partner. Expression of an S4 mutant lacking its PDZ-binding region (S4-PDZ(-)) leads to decreased cell motility and a failure to form a trailing edge. On clustering S4, but not S4-PDZ(-), targets activated Rac1 to the leading edge of live cells. Cells lacking synectin, a PDZ domain containing protein that interacts with S4, fail to migrate in response to S4 clustering. Both S4-PDZ(-) expressing and synectin(-/-) endothelial cells exhibit elevated basal levels of Rac1. Thus, our data suggest that S4 promotes endothelial cell migration in response to ligand binding by activating Rac1 and localizing it to the leading edge, and that these processes are dependent on its PDZ-binding domain interaction with synectin. PMID- 16675719 TI - Dietary factors related to higher plasma fibrinogen levels of Japanese-americans in hawaii compared with Japanese in Japan. AB - OBJECTIVE: We investigated whether dietary factors explain higher plasma fibrinogen levels in Japanese emigrants living a Western lifestyle in Hawaii compared with Japanese in Japan. METHODS AND RESULTS: Plasma fibrinogen and nutrient intakes were examined by standardized methods in men and women 40 to 59 years of age from a Japanese-American sample in Hawaii (100 men and 106 women) and 4 population samples in Japan (569 men and 567 women). Multiple linear regression models were used to examine the relationship between dietary factors and the plasma fibrinogen difference between Hawaii and Japan. Average plasma fibrinogen was significantly higher in Hawaii compared with Japan (P<0.001 in both genders). In multiple linear regression analyses with each dietary variable considered separately, body mass index reduced the plasma fibrinogen difference between Hawaii and Japan by 20.4%; iron intake (mg/1000 kcal) and estimated total sugar intake (%kcal) reduced this difference by 30.0% and 14.4%, respectively. In a model that included body mass index, iron, estimated total sugars, and caffeine (also age and gender), this difference was reduced by 61.3% (from 42.2 to 16.3 mg/dL). CONCLUSIONS: Higher intake of iron, sugar, and caffeine, in addition to obesity, account largely for higher fibrinogen levels with Westernized lifestyle. PMID- 16675720 TI - Atheroprotective potential of macrophage-derived phospholipid transfer protein in low-density lipoprotein receptor-deficient mice is overcome by apolipoprotein AI overexpression. AB - OBJECTIVE: Using bone marrow transplantation, we assessed the impact of macrophage-derived phospholipid transfer protein (PLTP) on lesion development in hypercholesterolemic mice that expressed either normal levels of mouse apolipoprotein AI (apoAI) or elevated levels of only human apoAI. METHODS AND RESULTS: Bone marrow transplantations were performed in low-density lipoprotein receptor-deficient mice (LDLr-/-) that expressed either normal levels of mouse apoAI (msapoAI) or high levels of only human apoAI (msapoAI-/-, LDLr-/-, huapoAITg). Mice were lethally irradiated, reconstituted with either PLTP expressing or PLTP-deficient bone marrow cells, and fed a high-fat diet over 16 weeks. Macrophage PLTP deficiency increased atherosclerosis in LDLr-/- mice with minimal changes in total plasma cholesterol levels. In contrast, the extent of atherosclerosis in msapoAI-/-, LDLr-/-, huapoAITg mice was not significantly different between groups that had received PLTP-/- or PLTP+/+ bone marrow. In vitro studies indicated that PLTP deficiency led to a significant decrease in alpha-tocopherol content and increased oxidative stress in bone marrow cells. CONCLUSIONS: Our observations suggest an atheroprotective role of macrophage derived PLTP in mice with normal apoAI plasma levels. The atheroprotective properties of macrophage-derived PLTP were not observable in the presence of elevated plasma concentrations of apoAI. PMID- 16675721 TI - Intrinsic fluorescence and diffuse reflectance spectroscopy identify superficial foam cells in coronary plaques prone to erosion. AB - OBJECTIVE: Foam cells perform critical functions in atherosclerosis. We hypothesize that coronary segments with superficial foam cells (SFCs) situated in a region of interest with a depth of 200 mum can be identified using intrinsic fluorescence spectroscopy (IFS) and diffuse reflectance spectroscopy (DRS). This is a key step in our ongoing program to develop a spectroscopic technique for real-time in vivo diagnosis of vulnerable atherosclerotic plaque. METHODS AND RESULTS: We subjected 132 human coronary segments to in vitro IFS and DRS. We detected SFCs in 13 thick fibrous cap atheromas and 8 pathologic intimal thickening (PIT) lesions. SFCs colocalized with accumulations of smooth muscle cells and proteoglycans, including hyaluronan (P<0.001). Two spectroscopic parameters were generated from analysis of IFS at 480 nm excitation and DRS. A discriminatory algorithm using these parameters identified specimens with SFC area >40%, 20%, 10%, 5%, 2.5%, and 0% of the region of interest with 98%, 98%, 93%, 94%, 93%, and 90% accuracy, respectively. CONCLUSIONS: Our combined IFS and DRS technique accurately detects SFCs in thick fibrous cap atheromas and PIT lesions. Because SFCs are associated with histological markers of plaque erosion, our spectroscopic technique could prove useful in identifying vulnerable plaques. PMID- 16675722 TI - Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. AB - OBJECTIVE: Ca2+-influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium dependent vasodilatation. METHODS AND RESULTS: Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca2+ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4alpha-phorbol-12,13 didecanoate (4alphaPDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca2+ measurements, 4alphaPDD increased [Ca2+]i by &140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4alphaPDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by &80% (K(D) 0.3 microL), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3). 4alphaPDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. CONCLUSIONS: Ca2+ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress. PMID- 16675723 TI - Two-phase antithrombotic protection after anti-glycoprotein VI treatment in mice. AB - OBJECTIVE: Collagen and thrombin are the strongest physiological platelet agonists, acting through different receptors, among which glycoprotein VI (GPVI) and protease-activated receptors, respectively, are the essential ones. In mice, targeting of GPVI with the monoclonal antibody JAQ1 induces depletion of the receptor from circulating platelets, resulting in abolished collagen responses and long-lasting antithrombotic protection. METHODS AND RESULTS: Mice were treated with JAQ1, and the early effects of this treatment were analyzed. In addition to the known abolition of the collagen reactivity, this treatment also affected platelet response to thrombin but not other agonists. In platelets from JAQ1-treated mice, thrombin-induced activation of integrin alphaIIbbeta3, the surface expression of P-selectin, and the procoagulant activity were decreased on days 1 and 2, then progressively recovered and returned to normal on day 5. In parallel, the mice were transiently protected from lethal tissue factor-induced pulmonary thromboembolism (100% survivors versus 40% in control group), which appeared to be based on a decreased generation and activity of intravascular thrombin. CONCLUSIONS: Anti-GPVI treatment induces 2-phase antithrombotic protection in mice consisting of a partial and transient inhibition of thrombin responses in platelets and a prolonged and complete loss of the collagen response. PMID- 16675724 TI - Liver-specific inhibition of acyl-coenzyme a:cholesterol acyltransferase 2 with antisense oligonucleotides limits atherosclerosis development in apolipoprotein B100-only low-density lipoprotein receptor-/- mice. AB - OBJECTIVE: The purpose of this study was to determine the effects of liver specific inhibition of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) on the development of hypercholesterolemia and atherosclerosis in mice. METHODS AND RESULTS: Apolipoprotein B100-only low-density lipoprotein (LDL) receptor-/- mice were given saline, a nontargeting control antisense oligonucleotide (ASO), or ASOs targeting ACAT2 biweekly for a period spanning 16 weeks. Mice treated with ACAT2 targeting ASOs had liver-specific reduction in ACAT2 mRNA, yet intestinal ACAT2 and cholesterol absorption was left undisturbed. ASO-mediated knockdown of ACAT2 resulted in reduction of total plasma cholesterol, increased levels of plasma triglyceride, and a shift in LDL cholesteryl ester (CE) fatty acid composition from mainly saturated and monounsaturated to polyunsaturated fatty acid enrichment. Furthermore, the liver-specific depletion of ACAT2 resulted in protection against diet-induced hypercholesterolemia and aortic CE deposition. This is the first demonstration that specific pharmacological inhibition of ACAT2, without affecting ACAT1, is atheroprotective. CONCLUSIONS: Hepatic ACAT2 plays a critical role in driving the production of atherogenic lipoproteins, and therapeutic interventions, such as the ACAT2-specific ASOs used here, which reduce acyltransferase 2 (ACAT2) function in the liver without affecting ACAT1, may provide clinical benefit for cardiovascular disease prevention. PMID- 16675726 TI - Transforming growth factor-betas and vascular disorders. AB - Transforming growth factor-beta (TGF-beta) superfamily members, TGF-beta and bone morphogenetic proteins (BMPs), are potent regulatory cytokines with diverse functions on vascular cells. They signal through heteromeric type I and II receptor complexes activating Smad-dependent and Smad-independent signals, which regulate proliferation, differentiation, and survival. They are potent regulators of vascular development and vessel remodeling and play key roles in atherosclerosis and restenosis, regulating endothelial, smooth muscle cell, macrophage, T cell, and probably vascular calcifying cell responses. In atherosclerosis, TGF-beta regulates lesion phenotype by controlling T-cell responses and stimulating smooth muscle cells to produce collagen. It contributes to restenosis by augmenting neointimal cell proliferation and collagen accumulation. Defective TGF-beta signaling in endothelial cells attributable to mutations in endoglin or the type I receptor ALK-1 leads to hereditary hemorrhagic telangiectasia, whereas defective BMP signaling attributable to mutations in the BMP receptor II has been associated with development of primary pulmonary hypertension. The development of mouse models with either cell type specific or general inactivation of TGF-beta/BMP signaling has started to reveal the importance of the regulatory network of TGF-beta/BMP pathways in vivo and their significance for atherosclerosis, hereditary hemorrhagic telangiectasia, and primary pulmonary hypertension. This review highlights recent findings that have advanced our understanding of the roles of TGF-beta superfamily members in regulating vascular cell responses and provides likely avenues for future research that may lead to novel pharmacological therapies for the treatment or prevention of vascular disorders. PMID- 16675725 TI - Clopidogrel improves systemic endothelial nitric oxide bioavailability in patients with coronary artery disease: evidence for antioxidant and antiinflammatory effects. AB - BACKGROUND: Platelet stimulation and activation are known not only as prerequisite of clot formation but are increasingly recognized as important contributors to inflammation and vascular injury. The present study in patients with symptomatic coronary disease investigated whether platelet adenosine diphosphate receptor blockade by clopidogrel exerts beneficial effects on endothelial nitric oxide bioavailability, oxidative stress, and/or inflammatory status. METHODS AND RESULTS: One hundred three consecutive patients with symptomatic coronary disease and long-term aspirin therapy were studied. Endothelium-dependent and -independent vasodilation was determined measuring forearm blood flow (FBF)-responses to acetylcholine with and without N(G) monomethyl-L-arginin (L-NMMA) and sodium nitroprusside, by using venous occlusion plethysmography. Patients were randomized to receive additional treatment with clopidogrel or placebo. Vascular function tests were repeated after 5 weeks and showed significant improvement of acetylcholine-induced vasodilatation and L-NMMA responses in the clopidogrel-added group (max. FBF from 9.8+/-0.3 to 14.7+/-0.4; L-NMMA-response from 3.7+/-0.1 to 6.8+/-0.3 mL/100 mL/min). In contrast, no significant changes were observed in the placebo group. Sodium nitroprusside induced vasodilation was not changed in either group. Urinary excretion of 8-iso prostaglandin F2alpha and plasma levels of hsCRP, sCD40L, and RANTES were reduced in patients on additional treatment with clopidogrel, but not in patients on placebo. CONCLUSIONS: Clopidogrel improves endothelial nitric oxide bioavailability and diminishes biomarkers of oxidant stress and inflammation in patients with symptomatic coronary artery disease, suggesting that beyond inhibition of platelet aggregation, adenosine phosphate receptor blockade may also have promising vasoprotective effects. PMID- 16675727 TI - Therapeutic potential of a synthetic peptide inhibitor of nuclear factor of activated T cells as antirestenotic agent. AB - OBJECTIVE: The calcineurin/nuclear factor of activated T cells (NFAT) axis plays a pivotal role in the regulation of critical genes in vascular smooth muscle cell (vSMC) proliferation and inflammation, which makes NFAT inhibition an attractive modality in the prevention of restenosis. METHODS AND RESULTS: Synthetic peptide VIVIT potently inhibited NFAT activation in RAW 264.7 macrophages, Ea.Hy.926 endothelial cells and vSMCs, and blocked ionomycin-elicited nuclear import of NFAT. VIVIT, as well as cyclosporine A (CsA) or FK506, completely blunted platelet-derived growth factor-BB (PDGF-BB) and thrombin-induced vSMC proliferation. Moreover, it significantly inhibited PDGF-BB and thrombin-induced interleukin-6, interleukin-8, transforming growth factor-beta1, stromal cell derived factor-1alpha, and monocyte chemotactic protein-1 expression in vSMCs. Unlike FK506 or CsA, VIVIT did not affect nuclear factor kappaB reporter gene activation and did only marginally affect endothelial wound healing in vitro. VIVIT did not intervene in phorbol 12-myristate 13-acetate-stimulated extracellular signal-regulated kinase activation, confirming its specificity for NFAT. Furthermore, our data establish that NFAT is a regulator of PDGF-BB induced vSMC proliferation. CONCLUSIONS: VIVIT appears to be a specific and potent inhibitor of NFAT activation and thus of NFAT-mediated proliferation and inflammation. Unlike FK506 or CsA, synthetic VIVIT therapy will not be accompanied by non-NFAT-mediated side effects on calcineurin signaling and constitutes a promising lead in antirestenotic therapy. PMID- 16675728 TI - Primary prevention of ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council: cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: the American Academy of Neurology affirms the value of this guideline. AB - BACKGROUND AND PURPOSE: This guideline provides an overview of the evidence on various established and potential stroke risk factors and provides recommendations for the reduction of stroke risk. METHODS: Writing group members were nominated by the committee chair on the basis of each writer's previous work in relevant topic areas and were approved by the American Heart Association Stroke Council's Scientific Statement Oversight Committee. The writers used systematic literature reviews (covering the time period since the last review published in 2001 up to January 2005), reference to previously published guidelines, personal files, and expert opinion to summarize existing evidence, indicate gaps in current knowledge, and when appropriate, formulate recommendations based on standard American Heart Association criteria. All members of the writing group had numerous opportunities to comment in writing on the recommendations and approved the final version of this document. The guideline underwent extensive peer review before consideration and approval by the AHA Science Advisory and Coordinating Committee. RESULTS: Schemes for assessing a person's risk of a first stroke were evaluated. Risk factors or risk markers for a first stroke were classified according to their potential for modification (nonmodifiable, modifiable, or potentially modifiable) and strength of evidence (well documented or less well documented). Nonmodifiable risk factors include age, sex, low birth weight, race/ethnicity, and genetic factors. Well documented and modifiable risk factors include hypertension, exposure to cigarette smoke, diabetes, atrial fibrillation and certain other cardiac conditions, dyslipidemia, carotid artery stenosis, sickle cell disease, postmenopausal hormone therapy, poor diet, physical inactivity, and obesity and body fat distribution. Less well-documented or potentially modifiable risk factors include the metabolic syndrome, alcohol abuse, drug abuse, oral contraceptive use, sleep-disordered breathing, migraine headache, hyperhomocysteinemia, elevated lipoprotein(a), elevated lipoprotein-associated phospholipase, hypercoagulability, inflammation, and infection. Data on the use of aspirin for primary stroke prevention are reviewed. CONCLUSIONS: Extensive evidence is available identifying a variety of specific factors that increase the risk of a first stroke and providing strategies for reducing that risk. PMID- 16675729 TI - Thromboembolic complications of endovascular aneurysm occlusion using matrix detachable coils. PMID- 16675731 TI - Lipoprotein(a): involved in events, but not burden of atherosclerotic disease? PMID- 16675732 TI - Classification of cause of death after stroke in clinical research. AB - BACKGROUND AND PURPOSE: Classification of outcome events is essential in clinical research. The Executive Committee of the European/Australasian Stroke Prevention in Reversible Ischaemia Trial (ESPRIT), a secondary prevention trial in patients with cerebral ischemia, repeatedly encountered problems in classifying the cause of death after a stroke if the interval between these events was relatively long. We aimed to develop guidelines for classifying such events. METHODS: Twenty-nine neurologists with a special interest in stroke filled out a questionnaire and audited 5 case vignettes. On the basis of this information, we developed a proposal for classifying causes of death after stroke. This proposal was evaluated in an interobserver analysis in which 10 neurologists or residents in neurology assessed 20 of 100 case vignettes. RESULTS: Initially, there was great variation in classifications of the case vignettes, mainly because the correspondents strongly disagreed about the relative importance of the interval between stroke and death, the degree of disability after stroke, the discharge destination (home or institutional care), and the coexistence of infection. In the new proposal, the main criteria were "interval after stroke" (cutoff point at 1 month) and "best Rankin grade after stroke" (cutoff at 3). In the interobserver analysis, good agreement was obtained among the 5 pairs of neurologists who assessed the 20 case vignettes (kappa 0.80 95% CI, 0.68 to 0.92). CONCLUSIONS: In the absence of guidelines, neurologists show striking variation in the classification of causes of death in patients who die after a stroke. With precise rules, agreement in the classification of death after stroke strongly improved. PMID- 16675733 TI - Nonpulsatile cerebral perfusion in patient with acute neurological deficits. AB - BACKGROUND AND PURPOSE: An extremely low pulsatile cerebral perfusion can result in a massive cerebral infarction and poor outcome. We report a patient who had complete recovery from initial neurological deficits in spite of nonpulsatile perfusion in the middle cerebral artery. METHODS: We used carotid duplex and transcranial Doppler to evaluate cerebral hemodynamics and the National Institutes of Health Stroke Scale (NIHSS) to score the neurological deficits. RESULTS: A 62-year-old man had a sudden chest pain and right hemispheric symptoms with NIHSS score of 18 on arrival. Carotid duplex showed no blood flow in the right common carotid artery. Transcranial Doppler showed a nonpulsatile waveform with slow antegrade flow in right middle cerebral artery. Chest CT angiography revealed type A aortic dissection. After surgical repair for the aortic dissection with brain retroperfusion, the patient had dramatic recovery from the initial neurological deficit, and normal pulsatile cerebral perfusion in the right carotid territory. CONCLUSIONS: Nonpulsatile cerebral perfusion points to a proximal source of arterial flow obstruction that may necessitate interventional treatment or surgery in order to restore brain perfusion and potentially reverse impending stroke. PMID- 16675734 TI - Lipoprotein(a) and incident ischemic stroke: the Atherosclerosis Risk in Communities (ARIC) study. AB - BACKGROUND AND PURPOSE: Numerous case-control and cross-sectional studies have reported higher median lipoprotein(a) [Lp(a)] levels among stroke patients than controls, but existing prospective studies have not consistently shown an association. We sought to examine the relationship between plasma Lp(a) levels and the incidence of ischemic stroke among blacks and whites. METHODS: Between 1987 and 1989, 14,221 men and women (3647 blacks and 10,574 whites) aged 45 to 64 years and free of clinical cardiovascular disease, took part in the first examination of the Atherosclerosis Risk in Communities (ARIC) study cohort. Lp(a) and other risk factors for cardiovascular disease were measured at baseline. RESULTS: During the 13.5-year follow-up, 496 ischemic strokes occurred. Participants with Lp(a) > or =300 microg/mL had a 79% higher age, sex, and race adjusted rate ratio (RR) of ischemic stroke than did those with Lp(a) levels <100 microg/mL. Compared with Lp(a) <100 microg/mL, the multivariate adjusted RRs for Lp(a) > or =300 microg/mL were 1.84 (95% CI, 1.05 to 3.07) in black women, 1.72 (95% CI, 0.86 to 3.48) in black men, 2.42 (95% CI, 1.30 to 4.53) in white women, and 1.18 (95% CI, 0.47 to 2.90) in white men. There was no significant increment in the RRs for 100 to 199 microg/mL and 200 to 299 microg/mL groups. CONCLUSIONS: A high Lp(a) concentration is associated with a higher incidence of ischemic stroke in blacks and white women, but not in white men. PMID- 16675735 TI - Pressor therapy in acute ischemic stroke: systematic review. AB - BACKGROUND AND PURPOSE: Systolic blood pressure (SBP) levels below 140 mm Hg after acute stroke occur in 18% to 25% of patients, and may be associated with adverse outcome, in terms of death and disability. It has thus been proposed that BP elevation in acute ischemic stroke may be beneficial by increasing perfusion to the peri-infarct penumbra, though not only in those with low BP levels. METHODS: All articles studying BP elevation in the context of acute stroke were identified using a structured search strategy. RESULTS: Two reviewers independently searched the databases, and 12 relevant publications were identified. All identified publications related to acute ischemic stroke and no articles on pressor therapy in primary hemorrhagic stroke were found. The review included 319 subjects (age: 42 to 88 years, 46% male), with phenylephrine being the most commonly used pressor agent, though 8 studies incorporated volume expansion. Because of small numbers, and varying entry/outcome criteria, no meta analysis of outcome measures was possible. Overall, in these few studies undertaken, pressor therapy in acute stroke appears feasible and well-tolerated. The benefit and risks in terms of clinical outcomes remains unknown, but intensive monitoring is advised if such therapy is undertaken. CONCLUSIONS: Theoretical arguments exist for inducing BP elevation in acute ischemic stroke to increase blood flow to the ischemic penumbra across patients with a broad BP range. To date, there have only been a few small trials with inconclusive results. Many questions are still unanswered about the safety and potential benefits of pressor therapy in acute stroke. Hopefully, ongoing trials will answer some of these important questions. PMID- 16675736 TI - Stroke information reported on local television news: a national perspective. AB - BACKGROUND AND PURPOSE: Local television news commonly reports on health. This study aimed to characterize local TV news stroke reporting in America. METHODS: Content analysis of stroke stories reported on 122 US local television stations. All stroke stories were coded for main focus and discussion of risk factors, stroke signs and symptoms, recombinant tissue plasminogen activator, treatment within 3 hours, or recommendation to call 911. RESULTS: Of the 1799 health stories, only 13 stroke stories aired, and the median story length was 24 seconds (interquartile range 21 to 48). Stroke was the 22nd most common health topic. Few stroke stories discussed useful information about prevention or treatment of stroke. CONCLUSIONS: Stroke stories were nearly nonexistent in our sample, and those reported failed to discuss important messages needed to improve stroke prevention and treatment. PMID- 16675737 TI - Effects of fractalkine receptor variants on common carotid artery intima-media thickness. AB - BACKGROUND AND PURPOSE: Fractalkine receptor (CX3CR1) plays a key role during atherogenesis. CX3CR1 has 2 common coding polymorphisms, namely V249I and T280M, that have been associated with interindividual differences in susceptibility to atherosclerosis. In the present study, we investigated the possible association between CX3CR1variants and intima-media thickness (IMT). METHODS: We genotyped 1256 samples from the Progression of Lesions in the Intima of the Carotid (PLIC) study (a prospective population-based study) for the presence of the V249 and the M280 variants of CX3CR1. RESULTS: Significantly reduced IMT was observed in subjects with the MM280 genotype (0.57+/-0.12 mm) compared with subjects with the TT (0.65+/-0.14 mm) or the TM (0.65+/-0.13 mm) genotype. No difference in IMT was observed within carrier of the II249, VI249, or VV249 genotype. Subjects with combined genotype VI249/MM280 and II249/MM280 showed a reduced IMT. CONCLUSIONS: The presence of the M280 polymorphism of the fractalkine receptor is associated with a decreased common carotid artery IMT, whereas the presence of the I249 polymorphism does not play a major role on the progression of carotid atherosclerosis. PMID- 16675738 TI - Association of phosphodiesterase 4D polymorphisms with ischemic stroke in a US population stratified by hypertension status. AB - BACKGROUND AND PURPOSE: Phosphodiesterase 4D (PDE4D) underlies the STRK1 linkage peak for stroke on chromosome 5q12 identified in Iceland. We tested association of 13 single-nucleotide polymorphisms (SNPs) and 1 microsatellite in a nested case-control sample of elderly white women (>65 years of age) from the Study of Osteoporotic Fractures (SOF) in the United States. METHODS: The genotypes of 248 women who experienced an incident ischemic stroke during an average of 5.4 years of follow-up were compared with 560 controls. RESULTS: Marginal associations with stroke (P<0.10) were found for 3 polymorphisms. Stratification of the population by hypertension markedly strengthened the association. SNPs 9 (hazard ratio [HR], 0.48; 95% CI, 0.26 to 0.91), 42 (HR, 1.73; 95% CI, 1.10 to 2.70), 219 (HR, 1.73; 95% CI, 1.13 to 2.64), and 220 (HR, 1.56; 95% CI, 1.05 to 2.32) showed significant association with stroke (P<0.05) under a dominant model in subjects without hypertension at baseline, and SNP 175 was significantly associated with stroke under an additive model (HR, 0.76; 95% CI, 0.59 to 0.98) in subjects with hypertension. Furthermore, the microsatellite AC008818-1 showed association with stroke only in the nonhypertensive subjects. Based on results in Iceland, specific haplotypes were tested in SOF, and stratification by hypertension also affected these association results. CONCLUSIONS: These data are consistent with an association of the PDE4D gene with stroke in a non-Icelandic sample and suggest an effect of hypertension status. PMID- 16675739 TI - Hematoma growth and outcome in treated neurocritical care patients with intracerebral hemorrhage related to oral anticoagulant therapy: comparison of acute treatment strategies using vitamin K, fresh frozen plasma, and prothrombin complex concentrates. AB - BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is the most serious and potentially fatal complication of oral anticoagulant therapy (OAT). Still, there are no universally accepted treatment regimens for patients with OAT-ICH, and randomized controlled trials do not exist. The aim of the present study was to compare the acute treatment strategies of OAT-associated ICH using vitamin K (VAK), fresh frozen plasma (FFP), and prothrombin complex concentrates (PCCs) with regard to hematoma growth and outcome. METHODS: In this retrospective study, a total of 55 treated patients were analyzed. Three groups were compared by reviewing the clinical, laboratory, and neuroradiological parameters: (1) patients who received PCCs alone or in combination with FFP or VAK (n=31), (2) patients treated with FFP alone or in combination with VAK (n=18), and (3) patients who received VAK as a monotherapy (n=6). The end points of early hematoma growth and outcome after 12 months were analyzed including multivariate analysis. RESULTS: Hematoma growth within 24 hours occurred in 27% of patients. Incidence and extent of hematoma growth were significantly lower in patients receiving PCCs (19%/44%) compared with FFP (33%/54%) and VAK (50%/59%). However, this effect was no longer seen between PCC- and FFP-treated patients if international normalized ratio (INR) was completely reversed within 2 hours after admission. The overall outcome was poor (modified Rankin scale 4 to 6 in 77%). Predictors for hematoma growth were an increased INR after 2 hours, whereas administration of PCCs was significantly protective in multivariate analyses. Predictors for a poor outcome were age, baseline hematoma volume, and occurrence of hematoma growth. CONCLUSIONS: Overall, PCC was associated with a reduced incidence and extent of hematoma growth compared with FFP and VAK. This effect seems to be related to a more rapid INR reversal. Randomized controlled trials are needed to identify the most effective acute treatment regimen for lasting INR reversal because increased levels of INR were predisposing for hematoma enlargement. PMID- 16675740 TI - Uric acid is a risk factor for myocardial infarction and stroke: the Rotterdam study. AB - BACKGROUND AND PURPOSE: The role of uric acid as a risk factor for myocardial infarction is controversial, and little is known about its role as a risk factor for stroke. Recent evidence suggests that uric acid may be an important causal agent in cardiovascular disease, for example, by inducing renal disease and hence hypertension. We investigated the association between serum uric acid and coronary heart disease and stroke in a large prospective population-based study. METHODS: The study was based on 4385 participants of the Rotterdam Study who, at baseline (1990 to 1993), were > or =55 years of age, free from stroke and coronary heart disease, and had blood taken. Follow-up for incident stroke and myocardial infarction was complete until January 1, 2002. Data were analyzed with Cox proportional hazards models with adjustment for relevant confounders. RESULTS: Average follow-up was 8.4 years. High serum uric acid levels were associated with risk of myocardial infarction and stroke; age- and sex-adjusted hazard ratios (95% CIs) for highest versus lowest quintile of uric acid were 1.68 (1.24 to 2.27) for cardiovascular disease (515 cases), 1.87 (1.12 to 3.13) for myocardial infarction (194 cases), 1.57 (1.11 to 2.22) for stroke (381 cases), 1.77 (1.10 to 2.83) for ischemic stroke (205 cases), and 1.68 (0.68 to 4.15) for hemorrhagic stroke (46 cases). Adjustment for other vascular risk factors only slightly attenuated these associations. Associations were stronger in persons without hypertension than in those with hypertension. CONCLUSIONS: Uric acid is a strong risk factor for myocardial infarction and stroke. PMID- 16675741 TI - Modest MRI signal intensity changes precede delayed cortical necrosis after transient focal ischemia in the rat. AB - BACKGROUND AND PURPOSE: Diffusion-weighted imaging (DWI) hyperintensities and apparent diffusion coefficient (ADC) hypointensities are MRI features of acute stroke. DWI alterations during ischemia recover with early reperfusion, but they can reappear later. Pronounced signal abnormalities early after stroke are associated with infarction, but the significance of subtle changes is unclear. Here we evaluated the degree and time course of regional signal intensity changes during the first 24 hours of reperfusion after transient ischemia, and we related them to the progression of the histopathological damage. METHODS: Rats (n=54) were subjected to 1-hour intraluminal middle cerebral artery occlusion to assess the dynamics of MRI signal intensity changes during the initial 24 hours and their correspondent histopathological features: 2,3,5-triphenyltetrazolium chloride (TTC) staining, and hematoxylin and eosin, and immunoreactivity to 70 kDa heat shock protein and to astroglial and microglial markers. RESULTS: This model of ischemia caused early striatal infarction but delayed necrosis in the cortex. The striatum showed marked MRI changes from 4 hours of reperfusion. By 12 hours, the striatal ADC signal intensity ratio to the homologous contralateral region was 30% reduced, and the TTC staining evidenced infarction. Contrarily, the cortical ADC ratio was only 15% reduced, and TTC staining was normal at 12 hours. After this time, the cortex showed sudden and pronounced (>30%) ADC signal intensity changes coincidentally with the manifestation of infarction, accompanied with severe vacuolation and unambiguous signs of neuronal and astroglial death. CONCLUSIONS: These findings suggest that minor changes in ADC signal intensity early after ischemia should not be underestimated because they may be harbingers of delayed infarction. PMID- 16675742 TI - Embolization of intracranial aneurysms with hydrogel-coated coils versus inert platinum coils: effects on packing density, coil length and quantity, procedure performance, cost, length of hospital stay, and durability of therapy. AB - BACKGROUND AND PURPOSE: The durability of aneurysm coil embolization is thought to depend on packing density. The expansile property of hydrogel coating on coils increases volumetric packing per coil length. We describe our experience using hydrogel-coated coils (HydroCoils) compared with inert platinum coils in intracranial aneurysm embolization. METHODS: Fifty aneurysms embolized primarily using HydroCoils from 2003 to 2004 were compared with 57 volume- and shape matched aneurysms treated with standard platinum coils from 2000 to 2003. Outcome measures included volumetric percentage occlusion (VPO), length and number of coils used, procedure time, fluoroscopy time, contrast volume, coil cost, length of hospital stay, and durability of therapy. RESULTS: Seventeen/26/5 small/medium/large aneurysms treated with HydroCoils were matched with 29/24/4 small/medium/large aneurysms treated with inert platinum. HydroCoil embolization yielded significantly greater VPO (84.8% versus 29.8%; P<0.001), decreased average total coil length used per aneurysm (33.2 versus 44.3 cm), reduced fluoroscopy time (53.2 versus 65.2 minutes; P=0.016), but increased contrast volume used (174.8 versus 112.9 cc; P<0.001). There were no differences in length of hospital stay. Procedure-related morbidity and mortality rates in the HydroCoil cohort were 4% and 0%, respectively. Follow-up angiography at mean 12.3 months revealed lower aneurysm recurrence rates (17% versus 24%; number-needed-to treat [NNT] 14.3). Initial costs associated with HydroCoil embolization were higher ($5835 versus $4017; P=0.004) but countered by lower retreatment rates (10% versus 17%; NNT 14.3). CONCLUSIONS: HydroCoil embolization achieves greater aneurysm packing density with decreased coil length. Initial durability data favor HydroCoils, with lower recurrence and retreatment rates. PMID- 16675743 TI - Repetitive transcranial magnetic stimulation-induced corticomotor excitability and associated motor skill acquisition in chronic stroke. AB - BACKGROUND AND PURPOSE: Although there is some early evidence showing the value of repetitive transcranial magnetic stimulation (rTMS) in stroke rehabilitation, the therapeutic effect of high-frequency rTMS, along with the physiology of rTMS induced corticomotor excitability supporting motor learning in stroke, has not been established. This study investigated high-frequency rTMS-induced cortical excitability and the associated motor skill acquisition in chronic stroke patients. METHODS: Fifteen patients with chronic hemiparetic stroke (13 men; mean age 53.5 years) practiced a complex, sequential finger motor task using their paretic fingers either after 10 Hz or sham rTMS over the contralateral primary motor cortex (M1). Both the changes in the behavior and corticomotor excitability before and after the intervention were examined by measuring the movement accuracy, the movement time, and the motor-evoked potential (MEP) amplitude. A separate repeated-measures ANOVA and correlation statistics were used to determine the main and interaction effects as well as relationship between the changes in the behavioral and corticomotor excitability. RESULTS: High-frequency rTMS resulted in a significantly larger increase in the MEP amplitude than the sham rTMS (P<0.01), and the plastic change was positively associated with an enhanced motor performance accuracy (P<0.05). CONCLUSIONS: High-frequency rTMS of the affected motor cortex can facilitate practice-dependent plasticity and improve the motor learning performance in chronic stroke victims. PMID- 16675744 TI - Carbachol increases Na+-HCO3- cotransport activity in murine colonic crypts in a M3-, Ca2+/calmodulin-, and PKC-dependent manner. AB - The Na(+)-HCO(3)(-) cotransporter (NBC) mediates HCO(3)(-) import into the colonocyte via its pNBC1 isoform. Whereas renal kNBC1 is inhibited by increased cAMP levels, pNBC1 is stimulated. Cholinergic stimulation activates renal NBC, but the effect on intestinal NBC is unknown. Therefore, crypts were isolated from the murine proximal colon by Ca(2+) chelation and loaded with the pH-sensitive dye 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein. Na(+)-HCO(3)(-) cotransport activity was calculated from the dimethylamiloride-insensitive (500 microM) intracellular pH recovery from an acid load in the presence of CO(2)-HCO(3)(-) and the intracellular buffering capacity. Carbachol strongly increased Na(+) HCO(3)(-) cotransport activity compared with control rates. Ca(2+) chelation with BAPTA-AM, blockade of the M(3) subtype of muscarinergic receptors with 4 diphenylacetoxy-N-methylpiperidine methiodide, and inhibition of Ca(2+)/calmodulin kinase II with KN-62 all caused significant inhibition of the carbachol-induced NBC activity increase. Furthermore, PKC inhibition with Go-6976 and Go-6850 significantly reduced the carbachol effect, which may be related to the unique NH(2)-terminal consensus site for PKC-dependent phosphorylation of pNBC1. We conclude that NBC in the murine colon is thus activated by carbachol, consistent with its presumed function as an anion uptake pathway during intestinal anion secretion, but that the signal transductions pathways are distinct from those involved in the cholinergic activation of renal NBC1. PMID- 16675745 TI - Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection. AB - Sphingosine-1-phosphate (S1P) induces an initial Ca(2+)-dependent contraction followed by a sustained Ca(2+)-independent, RhoA-mediated contraction in rabbit gastric smooth muscle cells. The cells coexpress S1P(1) and S1P(2) receptors, but the signaling pathways initiated by each receptor type and the involvement of one or both receptors in contraction are not known. Lentiviral vectors encoding small interfering RNAs were transiently transfected into cultured smooth muscle cells to silence S1P(1) or S1P(2) receptors. Phospholipase C (PLC)-beta activity and Rho kinase activity were used as markers of pathways mediating initial and sustained contraction, respectively. Silencing of S1P(1) receptors abolished S1P stimulated activation of Galpha(i3) and partially inhibited activation of Galpha(i1), whereas silencing of S1P(2) receptors abolished activation of Galpha(q), Galpha(13), and Galpha(i2) and partially inhibited activation of Galpha(i1). Silencing of S1P(2) but not S1P(1) receptors suppressed S1P stimulated PLC-beta and Rho kinase activities, implying that both signaling pathways were mediated by S1P(2) receptors. The results obtained by receptor silencing were corroborated by receptor inactivation. The selective S1P(1) receptor agonist SEW2871 did not stimulate PLC-beta or Rho kinase activity or induce initial and sustained contraction; when this agonist was used to protect S1P(1) receptors so as to enable chemical inactivation of S1P(2) receptors, S1P did not elicit contraction, confirming that initial and sustained contraction was mediated by S1P(2) receptors. Thus S1P(1) and S1P(2) receptors are coupled to distinct complements of G proteins. Only S1P(2) receptors activate PLC-beta and Rho kinase and mediate initial and sustained contraction. PMID- 16675746 TI - Chronic metabolic acidosis stimulated transcellular and solvent drag-induced calcium transport in the duodenum of female rats. AB - Chronic metabolic acidosis results in a negative calcium balance as a result of bone resorption and renal calcium loss. However, reports on the changes in intestinal calcium transport have been controversial. The present investigation therefore aimed to study the effects of chronic metabolic acidosis induced by 1.5% NH(4)Cl administration on the three components of duodenal calcium transport, namely, solvent drag-induced, transcellular active, and passive paracellular components, in rats using an in vitro Ussing chamber technique. The relative mRNA expression of genes related to duodenal calcium transport was also determined. We found that 21-day chronic metabolic acidosis stimulated solvent drag-induced and transcellular active duodenal calcium transport but not passive paracellular calcium transport. Our results further demonstrated that an acute direct exposure to serosal acidic pH, in contrast, decreased solvent drag-induced calcium transport in a pH-dependent fashion but had no effect on transcellular active calcium transport. Neither the transepithelial resistance nor duodenal permeability to Na(+), Cl(-), and Ca(2+) via the passive paracellular pathway were altered by chronic metabolic acidosis, suggesting that widening of the tight junction and changes in the charge-selective property of the tight junction did not occur. Thus the enhanced duodenal calcium transport observed in chronic metabolic acidosis could have resulted from a long-term adaptation, possibly at the molecular level. RT-PCR study revealed that chronic metabolic acidosis significantly increased the relative mRNA expression of duodenal genes associated with solvent drag-induced transport, i.e., the beta(1)-subunit of Na(+)-K(+) ATPase, zonula occludens-1, occludin, and claudin-3, and with transcellular active transport, i.e., transient receptor potential vanilloid family Ca(2+) channels 5 and 6 and plasma membrane Ca(2+)-ATPase isoform 1b. Total plasma calcium and free ionized calcium and magnesium concentrations were also increased, whereas serum parathyroid hormone and 1alpha,25-dihydroxyvitamin D(3) levels were not changed. The results indicated that 21-day chronic metabolic acidosis affected the calcium metabolism in rats partly through enhancing the mRNA expression of crucial duodenal genes involved in calcium absorption, thereby stimulating solvent drag-induced and transcellular active calcium transport in the duodenum. PMID- 16675747 TI - Luminal glucose sensing in the rat intestine has characteristics of a sodium glucose cotransporter. AB - The presence of glucose in the intestinal lumen elicits a number of changes in gastrointestinal function, including inhibition of gastric emptying and food intake and stimulation of pancreatic and intestinal secretion. The present study tested the hypothesis that Na(+)-glucose cotransporter (SGLT)-3, a member of the SGLT family of transport proteins, is involved in detection of luminal glucose in the intestine. Gastric emptying, measured in awake rats, was significantly inhibited by perfusion of the intestine with glucose (60 and 90 mg); this effect was mimicked by alpha-methyl glucose (nonmetabolizable substrate of SGLT-1 and 3) but not 2-deoxy-d-glucose (substrate for GLUT-2) or isoosmotic mannitol. Gastric motility and intestinal fluid secretion, measured in anesthetised rats, were significantly inhibited and stimulated, respectively, by duodenal glucose but not galactose, which has a much lower affinity for SGLT-3 than glucose. Duodenal glucose but not galactose stimulated the release of 5-HT into mesenteric lymph and stimulated the discharge of duodenal vagal afferent fibers. mRNA for SGLT-3 was identified in the duodenal mucosa. Together these data suggest that detection of glucose in the intestine may involve SGLT-3, possibly expressed by enterochromaffin cells in the intestinal mucosa, and release of 5-HT. PMID- 16675748 TI - Contractile activity of lymphatic vessels is altered in the TNBS model of guinea pig ileitis. AB - The ability of the lymphatic system to actively remove fluid from the interstitium is critical to the resolution of edema. The response of the lymphatics to inflammatory situations is poorly studied, so we examined mesenteric lymphatic contractile activity in the 2,4,6-trinitrobenzenesulfonic acid (TNBS) model of guinea pig ileitis, a well-accepted animal model of intestinal inflammation, by videomicroscopy in vivo and in vitro 1, 3, and 6 days after induction of ileitis. Lymphatic function (diameter, constriction frequency, amplitude of constrictions, and calculated stroke volume and lymph flow rate) of isolated vessels from TNBS-treated guinea pigs were impaired compared with sham treated controls. The dysfunction was well correlated with the degree of inflammation, with differences reaching significance (P < 0.05) at the highest inflammation-induced damage observed at day 3. In vivo, significantly fewer lymphatics exhibited spontaneous constrictions in TNBS-treated than sham-treated animals. Cyclooxygenase (COX) metabolites were suggested to be involved in this lymphatic dysfunction, since application of nonselective COX inhibitor (10 microM indomethacin) or a combination of COX-1 and COX-2 inhibitors (1 microM SC-560 and 10 microM celecoxib) markedly increased constriction frequency or induced them in lymphatics from TNBS-treated animals in vivo and in vitro. The present results demonstrate that lymphatic contractile function is altered in TNBS-induced ileitis and suggest a role for prostanoids in the lymphatic dysfunction. PMID- 16675749 TI - First-person accounts: the importance of being honest. PMID- 16675751 TI - Innovations: child & adolescent psychiatry: use of collaborative problem solving to reduce seclusion and restraint in child and adolescent inpatient units. AB - The authors describe "collaborative problem solving," a cognitive-behavioral approach for working with aggressive children and adolescents. The model conceptualizes aggressive behavior as the byproduct of lagging cognitive skills in the domains of flexibility, frustration tolerance, and problem solving. The goal is to train staff to assess specific cognitive skills that may be contributing to challenging behavior and to teach children new skills through collaborative problem solving. The authors present results from an inpatient unit that dramatically reduced rates of seclusion and restraint. PMID- 16675752 TI - Innovations: geriatric psychiatry: diagnosis and treatment of neuropsychiatric symptoms in Alzheimer's disease. AB - The authors review research on the treatment of behavioral disturbances and psychiatric symptoms of patients with dementia, including pharmacological treatment with antipsychotics, antidepressants, cholinesterase inhibitors, and other psychotropic drugs. They conclude that although these medications have some beneficial effects, no intervention is currently able to eradicate behavioral disturbances and psychiatric symptoms of demented patients. Research suggests that multiple interventions for an individual patient are likely to replace the use of a single treatment. Such interventions include caregiver training and support, antipsychotics, antidepressants, and cholinesterase inhibitors, along with other drugs developed for the treatment of Alzheimer's disease. PMID- 16675753 TI - Datapoints: general medical costs of recipients of behavioral health care. PMID- 16675754 TI - A framework to improve the quality of treatment for depression in primary care. AB - New forms of medication and brief psychotherapy have dramatically changed how depressive disorders have been treated over the past two decades. In spite of these changes, the quality of treatment for depression remains poor at the population level. In this article, the authors review current concepts and theory regarding the quality of treatment for depression. They present a conceptual model of four points in the course of a treatment episode when clinicians could deviate from guidelines. Using the model, the authors review research that supports guideline recommendations and that can inform clinicians' decisions. They suggest several areas for future study and action, including extending awareness and recognition outside the medical care setting to schools and workplaces, addressing growing concerns about possible overtreatment, using qualitative research approaches to gain an understanding of patient perspectives on treatment, and improving the measurement for quality of treatment. PMID- 16675755 TI - Treatment seeking for depression in Canada and the United States. AB - OBJECTIVE: Cross-country comparisons of patterns of mental health treatment seeking provide insights into the impact of contextual factors on mental health service use. This study aimed to compare prevalence and predictors of mental health treatment seeking among adults with major depression in Canada and the United States. METHODS: Data for 751 participants with a probable major depressive episode in the past 12 months were drawn from the 2002-2003 Joint Canada/United States Survey of Health: 304 were from Canada and 447 were from the United States. Probable major depressive episodes were ascertained by the Composite International Diagnostic Interview-Short-Form. Patterns of contacts with mental health and general health providers for mental health reasons were compared. RESULTS: Prevalence of contacts with any provider for mental health problems was similar among participants with a probable major depressive episode in Canada and the United States (181 Canadians, or 56 percent, compared with 245 Americans, or 52 percent). Canadian participants were more likely than those in the United States to seek treatment for mental health problems from family doctors and general practitioners, and among participants who sought such treatment, Canadians were more likely to also seek treatment from mental health professionals. In both countries, racial or ethnic minorities were less likely than Caucasians to seek treatment. Depression severity was more closely associated with treatment seeking in Canada than in the United States. CONCLUSIONS: Although studies from the early 1990s showed higher rates of treatment seeking for depression in Canada than in the United States, the more recent data presented here do not show such a gap. However, differences persist in the use of various providers. Compared with the United States, Canada had a closer match between depression severity and treatment, which suggests more efficient allocation of mental health care resources for treatment of depression in Canada. PMID- 16675756 TI - The top ten concerns about recovery encountered in mental health system transformation. AB - The notion of "recovery" has recently taken center stage in guiding mental health policy and practice. However, it is not yet clear what the term means and what is to be entailed in transforming the nation's mental health system to promote it. The authors discuss the various meanings of recovery as applied to mental illness and list the top ten concerns encountered in efforts to articulate and implement recovery-oriented care. These concerns include the following: recovery is old news, recovery-oriented care adds to the burden of already stretched providers, recovery involves cure, recovery happens to very few people, recovery represents an irresponsible fad, recovery happens only after and as a result of active treatment, recovery-oriented care is implemented only through the addition of new resources, recovery-oriented care is neither reimbursable nor evidence based, recovery-oriented care devalues the role of professional intervention, and recovery-oriented care increases providers' exposure to risk and liability. These concerns are addressed through discussion of the two over-arching challenges that they pose, namely the issues of resources and risk. PMID- 16675757 TI - Commentary: all we are saying is give people with mental illnesses a chance. PMID- 16675758 TI - Commentary: disquieting aspects of the recovery paradigm. PMID- 16675760 TI - Pharmacological Treatment Patterns at Study Entry for the First 500 STEP-BD Participants. AB - OBJECTIVE: This study assessed patterns of psychopharmacological treatment for bipolar disorder. METHOD: Intake treatment data were examined for the first 500 patients in the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD) study (1998 to 1999). Diagnoses were assessed by using the Structured Clinical Interview for DSM-IV mood modules. Data on treatments were obtained by interviewing patients during the initial psychiatric examination. RESULTS: Of the 500 participants, 73.6 percent had bipolar I disorder, 23.0 percent had bipolar II disorder, and 3.4 percent had bipolar disorder not otherwise specified. Upon examination, 63.4 percent were euthymic, 24.6 percent were depressed, and 12.0 percent were experiencing manic, hypomanic, or mixed states. Standard mood stabilizers (lithium, valproate, or carbamazepine) were the most commonly prescribed class of drugs that participants were taking at intake (71.9 percent). The next most common class of agents was antidepressants (40.6 percent), followed by novel anticonvulsants (31.8 percent), second-generation neuroleptics (27.2 percent), and benzodiazepines (25.0 percent). Eleven percent of patients were treated with standard mood stabilizer monotherapy. These prescribing patterns were further analyzed by subtype of illness and compared with patterns in other clinical and community settings. CONCLUSION: In a large, well-characterized cross sectional analysis of prescription patterns in the U.S. psychiatric academic setting, patients with bipolar disorder were primarily treated with standard mood stabilizers, followed by moderate use of antidepressants, novel anticonvulsants, and second-generation neuroleptics. Results can be useful in understanding the current clinical standard of care, as well as in guiding research studies toward areas in which there is a relative absence of evidence to inform clinical practice. Studies of longitudinal prescribing patterns in bipolar disorder are also needed. PMID- 16675759 TI - An empirical analysis of cost outcomes of the Texas Medication Algorithm Project. AB - OBJECTIVE: Disease management systems that incorporate medication algorithms have been proposed as cost-effective means to offer optimal treatment for patients with severe and chronic mental illnesses. The Texas Medication Algorithm Project was designed to compare health care costs and clinical outcomes between patients who received algorithm-guided medication management or usual care in 19 public mental health clinics. METHODS: This longitudinal cohort study for patients with major depression (N=350), bipolar disorder (N=267), and schizophrenia (N=309) applied a multi-part declining-effects cost model. Outcomes were assessed by the Inventory of Depressive Symptomatology and the Brief Psychiatric Rating Scale. RESULTS: Compared with patients in usual care, patients in algorithm-based care incurred higher medication costs and had more frequent physician visits, although these differences often became smaller with time. For major depression, algorithm based care achieved better outcomes sustainable with time but at higher agency and non-agency costs (mixed cost-effective). For bipolar disorder, patients in algorithm-based management achieved better outcomes at lower agency costs (cost effective). For schizophrenia, patients in algorithm-based care achieved better outcomes that diminished with time, with no detectable difference in health care costs (cost-effective). CONCLUSIONS: Cost outcomes of algorithm-based care and usual care varied by disorder and over time. For bipolar disorder and schizophrenia, algorithm-based care improved outcomes without higher costs for health care services. For major depression, substantively better and sustained outcomes were obtained but at greater costs. PMID- 16675762 TI - Antidepressant adherence and medical resource use among managed care patients with anxiety disorders. AB - OBJECTIVES: This study evaluated adherence with selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs) among patients who newly initiated therapy for anxiety with or without comorbid depression; the study also quantified the health-related economic consequences of nonadherence. METHODS: A large managed care database was used to gather retrospective data for patients with anxiety disorders who had a prescription for an antidepressant between July 1, 2001, and December 31, 2002. The relationship between antidepressant adherence and medical resource utilization was assessed; the analysis controlled for age, gender, utilization of mental health specialty care, change in medication, whether the dosage was titrated, costs in the six months before the prescription for an antidepressant, and comorbid physical conditions. RESULTS: Of the 13,085 patients with anxiety diagnoses who met the criteria for study inclusion, 57 percent were nonadherent to antidepressant therapy at six months. Patients who received mental health specialty care were more likely than those who did not receive such care to be adherent to therapy (48.5 percent compared with 40.7 percent; p<.001). Those with dual diagnoses of anxiety and depression were more likely than those with anxiety alone to be adherent to therapy (46.8 percent compared with 40.2 percent; p<.001). Those with a coded diagnosis of posttraumatic stress disorder had the highest medical costs. Patients with anxiety and depression had significantly higher total costs than patients with anxiety alone. Adherent patients who did not have a change in medication or a titrated dosage had significantly lower medical costs than nonadherent patients; however, total costs (medical plus pharmacy) were similar. CONCLUSIONS: Nonadherence with antidepressant therapy in anxiety disorders is common, but mental health specialty care may be associated with improved adherence. Lower medical costs for adherent patients who did not have a change in medication or a titrated dosage offset the increase in pharmacy costs, resulting in total costs (medical plus pharmacy) that were similar to those of nonadherent patients. PMID- 16675761 TI - Racial differences in medication switching and concomitant prescriptions in the treatment of bipolar disorder. AB - OBJECTIVE: This study examined whether the practices of switching between classes of medications and prescription of concomitant medications differed between black and non-black patients with bipolar disorders. METHODS: In a retrospective cohort design, data from 1998 to 2004 for patients with diagnoses of bipolar disorders were obtained from a large claims database. Information was obtained on the number of prescriptions for four classes of medications (anticonvulsants, mood stabilizers, and first- and second-generation antipsychotics) as well as on medication switching (between drug classes), concomitant prescriptions, resource use, and outcomes (an emergency department visit or a hospitalization). Logistic models assessed the relationship between outcomes and switching or concomitant prescriptions. RESULTS: The study population consisted of 1,113 adults who received at least one prescription from the four drug classes. Medication switching or concomitant prescriptions were documented for more than one-third of patients (36.6 percent). A significantly greater proportion of black patients received two or more medications from different drug classes (41.1 compared with 34.7 percent). The number of prescriptions was lower for black patients than for non-black patients. Patients for whom switching or concomitant prescriptions were documented were significantly more likely to have an emergency department visit or a hospitalization, and race was a significant predictor of these outcomes. CONCLUSIONS: The prevalence of polypharmacy-medication switching and concomitant prescriptions-was high among patients with bipolar disorder, with a higher prevalence among black patients. Patients who experienced switching or concomitant prescriptions were more likely to visit the emergency department or to be hospitalized. PMID- 16675763 TI - Anticonvulsant treatment for psychiatric and seizure indications among youths. AB - OBJECTIVE: This study compared the prevalence of anticonvulsant treatment for youths with a diagnosis of a psychiatric disorder to youths with a diagnosis of a seizure disorder. METHODS: Administrative claims from outpatient visits and prescriptions were organized for a data set of 258,472 youths who were younger than 18 years of age in a mid-Atlantic state Medicaid program and were continuously enrolled in 2000. Youths dispensed an anticonvulsant were grouped into the following ICD-9 diagnostic categories: a diagnosis of a psychiatric disorder without a seizure disorder, a diagnosis of a seizure disorder without a psychiatric disorder, and a diagnosis of both a psychiatric and a seizure disorder. Anticonvulsant use was analyzed for specific diagnostic classes by age, gender, race or ethnicity, and Medicaid eligibility categories. RESULTS: A total of 4,522 youths in the one-year data set received an anticonvulsant (1.75 percent): 3,061 had a psychiatric disorder only, 251 had a seizure disorder only, and 611 had both psychiatric and seizure disorders. Among anticonvulsant-treated youths with diagnosis information in their records (3,923 of 4,522 youths), 81 percent had a psychiatric diagnosis and 19 percent had a seizure disorder; 71 percent of those with a seizure disorder also had a psychiatric disorder. Anticonvulsant use for seizure control was proportionally greater for those younger than five years. By contrast, a vast majority of anticonvulsant users with a psychiatric diagnosis were between five and 17 years. Among anticonvulsant treated youths with a psychiatric diagnosis, males were approximately twice as common as females. For youths with a seizure disorder, no difference was found for gender. Mood disorders and attention-deficit hyperactivity disorder were the major psychiatric diagnoses associated with anticonvulsant use. Valproic acid products were the most commonly dispensed type of anticonvulsant. CONCLUSIONS: Recent state Medicaid data reveal that youths who use anticonvulsants are far more likely to have a psychiatric diagnosis than a seizure diagnosis. Widespread off-label use of anticonvulsants for psychiatric disorders among youths warrants attention to ensure benefits and minimize risks. PMID- 16675764 TI - Elimination of methadone benefits in the Oregon Health Plan and its effects on patients. AB - OBJECTIVES: This prospective study assessed the impacts of a policy change to Oregon's Medicaid program (Oregon Health Plan; OHP) that eliminated methadone benefits for 60 percent of active methadone patients. Recipients of OHP Standard (expanded Medicaid benefits, which were discontinued after the policy change) self-selected into two groups: those who paid for methadone after the policy change and those who terminated treatment. OHP Plus beneficiaries (traditional Medicaid) did not lose benefits. METHODS: A total of 149 patients participated in the study, and interviews were conducted at baseline (time of policy change) and one, three, and 12 months after the policy change. Patients were assessed with the Addiction Severity Index (ASI), Timeline Follow Back assessment, and chart review. RESULTS: Patients who left treatment because they were unable to pay for methadone services showed significant elevations in ASI composite scores for drug and legal problems at baseline and at two and three months after the policy change. The patients who attempted to self-pay experienced significantly more employment problems than the other two groups. The OHP Standard recipients who paid for their methadone treatment over the year were more likely to have additional resources to pay for methadone, be employed, and have stable housing. CONCLUSIONS: The elimination of methadone treatment benefits in the OHP had substantial negative impacts for patients with the greatest indicators of need. PMID- 16675765 TI - Perceived effectiveness of medications among mental health service users with and without alcohol dependence. AB - OBJECTIVE: No consensus exists on the use of psychiatric medication among patients with co-occurring mental health and alcohol disorders. The authors investigated patterns of use of psychiatric medication and perceived effectiveness of mental health treatment among users of mental health care with and without alcohol dependence. METHODS: Data were obtained from the 2001 to 2003 National Survey on Drug Use and Health. The sample consisted of respondents who reported receiving mental health treatment in the past year (N=11,872). Rates of psychiatric medication use were compared between mental health care patients who were alcohol dependent and those who were not. Patient-reported effectiveness of mental health treatment was examined among alcohol-dependent and non-alcohol dependent patients who did and did not receive psychiatric medication. RESULTS: No statistically significant differences in rates of use of psychiatric medication were found between those with and without alcohol dependence (76.2 percent and 75.9 percent, respectively). Among alcohol-dependent patients, those who received psychiatric medication were significantly more likely than those who did not receive such medication to report that treatment helped a lot or a great deal (OR=2.87, 95 percent CI=1.57 to 2.56, p<.001). Among those who received psychiatric medication, no statistically significant differences were found between alcohol-dependent and nondependent respondents in patients' ratings of treatment effectiveness. CONCLUSIONS: Most alcohol-dependent individuals in mental health treatment received psychiatric medication, despite the lack of guideline support in this area. A large majority of those with alcohol dependence who received psychiatric medication reported that mental health treatment was effective. PMID- 16675766 TI - Psychiatric decision making in the adoption of a new antipsychotic in Germany. AB - OBJECTIVE: Studies in somatic medicine have demonstrated delays in the implementation of new drugs, but data on psychiatric drugs are scarce. This study examined psychiatrists' adoption of a new antipsychotic. METHODS: A prospective survey of 50 hospital psychiatrists in Germany was conducted three months before and three months after the market introduction of aripiprazole. Psychiatrists' awareness and perceptions of the drug and psychiatrist-related data were obtained before the launching of aripiprazole; actual prescription patterns were recorded afterward. RESULTS: Predictors of major concerns about the credibility of statements related to efficacy or tolerability of the drug were older age and longer work experience of the psychiatrists. Three months after market introduction, 79 percent of the psychiatrists had already prescribed the new antipsychotic. Predictors for early adoption were older age, being responsible for patients with mild mental illness, and having a higher tolerance of uncertainty. CONCLUSIONS: Despite concerns as to the credibility of statements about aripiprazole's efficacy and tolerability, the psychiatrists were found to be early adopters of the new compound. The predictors identified for early adoption show the importance of physician-related factors for the quality of everyday care. PMID- 16675767 TI - Psychiatric illness and substance abuse among homeless Asian-American veterans. AB - OBJECTIVES: This study examined the proportion of Asian Americans among homeless veterans and among veterans in the general population to calculate the relative risk of homelessness among Asian-American veterans. It also examined differences in rates of psychiatric and substance use disorders between homeless racial and ethnic subgroups. METHODS: Data were gathered between 1997 and 2001 from the Health Care for Homeless Veterans program and included data from administrative intake, patients' self-reports, and clinicians' diagnostic assessments of substance use disorders and psychiatric illness. RESULTS: Data were examined for 67,441 veterans. Asian-American veterans had a significantly lower risk of homelessness than veterans of other ethnic groups. Alcohol abuse was significantly and consistently less prevalent among Asian Americans compared with blacks and Hispanics. However, drug abuse was less prevalent among Asian Americans than among blacks and Hispanics, but rates were similar to those of whites. CONCLUSIONS: Lower rates of alcohol abuse may protect Asian-American veterans from becoming homeless. PMID- 16675768 TI - Personality disorder and chronicity of addiction as independent outcome predictors in alcoholism treatment. AB - OBJECTIVE: A prospective four-year study examined which components of addiction severity predicted time to relapse among 112 adults with chronic alcoholism who participated in a comprehensive outpatient treatment program. METHODS: Recruited from emergency, inpatient, and outpatient facilities, patients were admitted into the program consecutively between March 1998 and June 2002. Alcohol abstinence was carefully monitored for four years from admission by regular contacts and urine and blood analyses. Alcoholism characteristics and personality disorders were assessed with structured interviews and the International Diagnostic Checklists for Personality Disorders. RESULTS: Among a variety of potential variables, only presence of a personality disorder and chronicity of addiction were independently associated with a decrease of cumulative four-year abstinence probability. CONCLUSIONS: Their high predictive values suggest that chronicity and personality disorder rank among the most important characteristics of addiction severity. PMID- 16675769 TI - Use of video conferencing for psychiatric and forensic evaluations. AB - OBJECTIVE: This study investigated whether the quality of results from video interviews is comparable with that of in-person interviews. METHODS: Interrater reliabilities for two video conference interview conditions were compared with those for in-person interviews with the Brief Psychiatric Rating Scale-Anchored Version and the MacArthur Competence Assessment Tool-Criminal Adjudication, given to 72 forensic inpatients. The video conditions included in-person and remote interviewers. In the first condition, an in-person interviewer administered the instruments, with remote observation and scoring. The second condition entailed remote administration and an in-person observer. The third condition used an in person interviewer and observer. RESULTS: Good to excellent reliabilities resulted from all conditions with intraclass correlations of .69 to .82. CONCLUSIONS: Results suggest that providers can expect remote interviews to provide clinical information similar to that obtained by in-person interviews. PMID- 16675770 TI - Effect of maternal mental health problems on child treatment response in community-based services. AB - OBJECTIVE: Many mothers bringing children for community mental health treatment need mental health services themselves. Moreover, children of mothers with mental health problems enter treatment with more severe symptomatology than do other children. However, little is known about how maternal mental health problems affect children's response to treatment. This study examined the impact of maternal mental health status on the child's treatment response. METHODS: The mental health of 272 mother-child pairs was assessed twice, when children entered treatment at three community mental health clinics and again three months later. Analysis of variance was used to examine the impact of maternal mental health status on children's rate of improvement. RESULTS: Children of mothers with mental health problems had significantly greater behavior problems (F=34.54, p<.001) and demonstrated less improvement over time (F=4.44, p=.04) than children of mentally healthy mothers. CONCLUSION: These findings suggest that treatment for mothers as well as children could be beneficial for this population. PMID- 16675771 TI - Frontline Reports: The Clubhouse Family Legal Support Project for parents with mental illness. PMID- 16675772 TI - Frontline Reports: A peripartum inpatient psychiatric program for mothers and infants. PMID- 16675773 TI - Medicare part D and decompensation. PMID- 16675774 TI - A not-for-profit, managed, single-payer system. PMID- 16675775 TI - Patients' views on suicidality and antidepressants. PMID- 16675776 TI - What do people with schizophrenia think about weight management? PMID- 16675777 TI - Osteoclastogenesis during infective exacerbations in patients with cystic fibrosis. AB - RATIONALE: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations, increased production of proinflammatory cytokines and markers of bone resorption have been reported. OBJECTIVE: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before, during, and after intravenous antibiotic treatment of infective exacerbations in patients with CF. METHODS: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14, CD33, CD34, and CD45 expression. RESULTS: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/10(5) cells plated, p = 0.025), proliferation (28.5%, p < 0.001), and the numbers of circulating potential osteoclast precursors (6.5%, p < 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline. CONCLUSIONS: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF. PMID- 16675779 TI - Macrophage reprogramming by mycolic acid promotes a tolerogenic response in experimental asthma. AB - RATIONALE: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function. OBJECTIVES: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity. METHODS: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function. MEASUREMENTS AND MAIN RESULTS: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC) mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages. CONCLUSIONS: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen presenting cells. PMID- 16675778 TI - Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis. AB - RATIONALE: Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF), especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF. OBJECTIVES: This study investigated ability of NTHi to form biofilms and its impact on airway epithelia using in vivo and in vitro analyses. METHODS: We evaluated bronchoalveolar lavage fluid from young patients with CF for evidence of NTHi biofilms. To further investigate the pathogenesis of NTHi in respiratory infections, we developed a novel in vitro coculture model of NTHi biofilm formation on polarized human airway epithelial cells grown at the air liquid interface. MEASUREMENTS AND MAIN RESULTS: In bronchoalveolar lavage fluid samples from young, asymptomatic patients with CF, we found morphologic evidence suggestive of NTHi biofilm formation. In addition, 10 clinical NTHi isolates from patients with CF formed biofilms on plastic surfaces. NTHi formed biofilms on the apical surface of cultured airway epithelia. These biofilms exhibited decreased susceptibility to antibiotics and were adherent to epithelial surfaces. Airway epithelial cells remained viable throughout 4 d of coculture, and responded to NTHi with nuclear factor-kappaB signaling, and increased chemokine and cytokine secretion. CONCLUSIONS: NTHi formed adherent biofilms on the apical surface airway epithelia with decreased susceptibility to antibiotics, and respiratory cells exhibited inflammatory and host defense responses-evidence of a dynamic host-pathogen interaction. The data presented here have implications both for understanding early CF lung disease pathogenesis and for the treatment of early, asymptomatic colonization of patients with CF with H. influenzae. PMID- 16675780 TI - Atelectasis causes alveolar injury in nonatelectatic lung regions. AB - RATIONALE: Many authors have suggested that the mechanism by which atelectasis contributes to injury is through the repetitive opening and closing of distal airways in lung regions that are atelectatic. However, neither the topographic nor mechanistic relationships between atelectasis and distribution of lung injury are known. OBJECTIVES: To investigate how atelectasis contributes to ventilator induced lung injury. METHODS: Surfactant depletion was performed in anesthetized rats that were then allocated to noninjurious or injurious ventilation for 90 min. MEASUREMENTS: Lung injury was quantified by gas exchange, compliance, histology, wet-to-dry weight, and cytokine expression, and its distribution by histology, stereology, cytokine mRNA expression, in situ hybridization, and immunohistochemistry. Functional residual capacity, percent atelectasis, and injury-induced lung water accumulation were measured using gravimetric and volumetric techniques. MAIN RESULTS: Atelectasis occurred in the dependent lung regions. Injurious ventilation was associated with alveolar and distal airway injury, while noninjurious ventilation was not. With injurious ventilation, alveolar injury (i.e., histology, myeloperoxidase protein expression, quantification, and localization of cytokine mRNA expression) was maximal in nondependent regions, whereas distal airway injury was equivalent in atelectatic and nonatelectatic regions. CONCLUSIONS: These data support the notion that lung injury associated with atelectasis involves trauma to the distal airways. We provide topographic and biochemical evidence that such distal airway injury is not localized solely to atelectatic areas, but is instead generalized in both atelectatic and nonatelectatic lung regions. In contrast, alveolar injury associated with atelectasis does not occur in those areas that are atelectatic but occurs instead in remote nonatelectatic alveoli. PMID- 16675782 TI - Exhaled breath condensate pH and childhood asthma: unselected birth cohort study. AB - RATIONALE: Exhaled breath condensate pH (EBC-pH) may be useful noninvasive marker for evaluation of patients with asthma. OBJECTIVES: To investigate the relationship between EBC-pH and symptoms suggestive of childhood asthma in an epidemiologic setting and examine its relation to lung function, airway hyperresponsiveness (AHR), and airway inflammation. METHODS: Within the context of a prospective population-based birth cohort, EBC was collected from 630 children at age 8 yr using the RTube (pH measured after deaeration with argon). Lung function was measured by spirometry (FEV1; n = 521) and plethysmography (sRaw; n = 567), and AHR by methacholine challenge (n = 498). Airway inflammation was assessed using exhaled nitric oxide (eNO; n = 305). RESULTS: EBC-pH values ranged widely (4.40-8.29), and did not differ between 54 children with parentally reported asthma and 562 nonasthmatic subjects (median [interquartile range]: 7.75 [7.45-7.85] vs. 7.77 [7.59-7.87]; p = 0.35). There was a trend for lower EBC-pH among current wheezers (n = 98; 7.72 [7.50-7.83]) compared with nonwheezers (n = 532; 7.77 [7.60-7.87]; p = 0.07). Wheeze frequency, severity, and use of antiasthma medication were not associated with EBC-pH. There was no consistent association between EBC-pH and lung function, airway reactivity, and airway inflammation (FEV1, sRaw, PD20 methacholine, or eNO). There was no significant difference in EBC-pH between current wheezers receiving asthma medication who had positive methacholine challenge compared with children without any of these features. CONCLUSIONS: In the epidemiologic setting, EBC-pH does not differ between children with and without parentally reported symptoms suggestive of asthma. We found no consistent association between EBC-pH and lung function, AHR, and airway inflammation in this sample from the general population. PMID- 16675781 TI - Moxifloxacin versus ethambutol in the first 2 months of treatment for pulmonary tuberculosis. AB - RATIONALE: Moxifloxacin has promising preclinical activity against Mycobacterium tuberculosis, but has not been evaluated in multidrug treatment of tuberculosis in humans. OBJECTIVE: To compare the impact of moxifloxacin versus ethambutol, both in combination with isoniazid, rifampin, and pyrazinamide, on sputum culture conversion at 2 mo as a measure of the potential sterilizing activity of alternate induction regimens. METHODS: Adults with smear-positive pulmonary tuberculosis were randomized in a factorial design to receive moxifloxacin (400 mg) versus ethambutol given 5 d/wk versus 3 d/wk (after 2 wk of daily therapy). All doses were directly observed. MEASUREMENTS: The primary endpoint was sputum culture status at 2 mo of treatment. RESULTS: Of 336 patients enrolled, 277 (82%) were eligible for the efficacy analysis, 186 (67%) were male, 175 (63%) were enrolled at African sites, 206 (74%) had cavitation on chest radiograph, and 60 (22%) had HIV infection. Two-month cultures were negative in 71% of patients (99 of 139) treated with moxifloxacin versus 71% (98 of 138) treated with ethambutol (p = 0.97). Patients receiving moxifloxacin, however, more often had negative cultures after 4 wk of treatment. Patients treated with moxifloxacin more often reported nausea (22 vs. 9%, p = 0.002), but similar proportions completed study treatment (88 vs. 89%). Dosing frequency had little effect on 2-mo culture status or tolerability of therapy. CONCLUSIONS: The addition of moxifloxacin to isoniazid, rifampin, and pyrazinamide did not affect 2-mo sputum culture status but did show increased activity at earlier time points. PMID- 16675783 TI - Comparison of urokinase and video-assisted thoracoscopic surgery for treatment of childhood empyema. AB - BACKGROUND: Despite increasing incidence and morbidity, little evidence exists to inform the best management approach in childhood empyema. AIM: To compare chest drain with intrapleural urokinase and primary video-assisted thoracoscopic surgery (VATS) for the treatment of childhood empyema. METHODS: Children were prospectively randomized to receive either percutaneous chest drain with intrapleural urokinase or primary VATS. The primary outcome was the number of hospital days after intervention. Secondary end points were number of chest drain days, total hospital stay, failure rate, radiologic outcome at 6 mo, and total treatment costs. RESULTS: Sixty children were recruited. The two groups were well matched for demographics; baseline characteristics; and hematologic, biochemical, and bacteriologic parameters. No significant difference was found in length of hospital stay after intervention between the two groups: VATS (median [range], 6 [3-16] d) versus urokinase (6 [4-25] d) (p = 0.311; 95% confidence interval, -2 to 1). No difference was demonstrated in total hospital stay: VATS versus urokinase (8 [4-17] d and 7 [4-25] d) (p = 0.645); failure rate: 5 (16.6%); and radiologic outcome at 6 mo after intervention in both groups. The mean (median) treatment costs of patients in the urokinase arm US dollars 9,127 (US dollars 6,914) were significantly lower than those for the VATS arm US dollars 11,379 (US dollars 10,146) (p < 0.001). CONCLUSIONS: There is no difference in clinical outcome between intrapleural urokinase and VATS for the treatment of childhood empyema. Urokinase is a more economic treatment option compared with VATS and should be the primary treatment of choice. This study provides an evidence base to guide the management of childhood empyema. PMID- 16675784 TI - The macrophage scavenger receptor SR-AI/II and lung defense against pneumococci and particles. AB - The class A macrophage scavenger receptor SR-AI/II is implicated as a pattern recognition receptor for innate immunity, but its functional role in lung defense has not been studied. We used mice genetically deficient in SR-AI/II and their wild-type C57BL/6 counterparts to investigate the contribution of this receptor to defense against pneumococcal infection and inhaled particles. SR-AI/II deficiency caused impaired phagocytosis of fluorescent bacteria in vivo, diminished clearance of live bacteria from the lungs, and substantially increased pneumonic inflammation. Survival studies also showed increased mortality in SR AI/II-deficient mice with pneumococcal lung infection. Similarly, after challenge of the airways with TiO(2) particles, SR-AI/II-deficient mice showed increased proinflammatory cytokine levels in lung lavage fluid and a more pronounced neutrophilic inflammation. The data indicate that the lung macrophage class A scavenger receptor SR-AI/II contributes to innate defense against bacteria and inhaled particles. PMID- 16675786 TI - Will UK Biobank pay off? PMID- 16675785 TI - Pirfenidone modulates airway responsiveness, inflammation, and remodeling after repeated challenge. AB - We investigated the therapeutic potential of a newly developed antifibrotic agent, pirfenidone, to regulate airway remodeling and the development of allergic airway inflammation and airway hyperresponsiveness after chronic allergen challenge. Administration of pirfenidone after sensitization but during the period of ovalbumin challenge significantly prevented the development of airway hyperresponsiveness and prevented eosinophil and lymphocyte accumulation in the airways. IL-4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid and ovalbumin-specific serum IgE antibody levels were also significantly reduced. Treatment with pirfenidone significantly reduced transforming growth factor-beta1 and platelet-derived growth factor levels in bronchoalveolar lavage fluid. Pirfenidone reduced the expression of transforming growth factor-beta1, the development of goblet cell hyperplasia and subepithelial collagenization, and the increases in contractile elements in the lung. These data indicate that pirfenidone may play an important role in the treatment of asthma and has the potential reduce or prevent airway remodeling. PMID- 16675787 TI - Inhaled insulin. PMID- 16675788 TI - Passive smoking's role in diabetes. PMID- 16675790 TI - Number of uninsured middle class US citizens grows. PMID- 16675789 TI - Community acquired pneumonia in primary care. PMID- 16675794 TI - Dutch want publication of data on heart surgery. PMID- 16675795 TI - UK website publishes cardiac surgery survival rates. PMID- 16675800 TI - Trastuzumab wins backing of European drug regulator. PMID- 16675801 TI - Unicef warns of "epidemic" of childhood malnutrition. PMID- 16675804 TI - Protests and bombing disrupt health care in Nepal. PMID- 16675805 TI - France to pilot testing for prostate cancer. PMID- 16675807 TI - Children worldwide can grow to the same height, says WHO. PMID- 16675811 TI - NICE rules on chemotherapy drugs for colon and breast cancer. PMID- 16675812 TI - Safety and efficacy of rectal compared with intramuscular quinine for the early treatment of moderately severe malaria in children: randomised clinical trial. AB - OBJECTIVE: To compare the safety and efficacy of quinine given by the rectal route with quinine given by the intramuscular route in children with moderately severe Plasmodium falciparum malaria. DESIGN: Randomised, open, clinical trial. SETTING: Health centre in Burkina Faso. PARTICIPANTS: 898 children with moderately severe P falciparum malaria who were unable to take oral treatment. INTERVENTION: Rectal quinine (20 mg/kg diluted to 30 mg/ml in water solution) or intramuscular quinine (12.5 mg/kg) every 12 hours until oral quinine could be taken. MAIN OUTCOME MEASURES: Primary safety outcome was the presence of blood in stools and secondary safety outcome was diarrhoea. Primary efficacy outcome was early treatment failure and secondary efficacy outcomes were late clinical and parasitological failures, fever clearance time, and time to oral intake. RESULTS: Blood in stools and diarrhoea were more common in children given quinine by the rectal route than by the intramuscular route (blood in stools: 5% v 1%, absolute difference 3.9%, 95% confidence interval 1.8% to 6.1%; diarrhoea: 5% v 1%, 3.5%, 1.3% to 5.7%). On anoscopy, inflammatory lesions (9/248, 3%) were associated with bloody striations in stools. Side effects of rectal quinine were rare and transitory. Local pain (90%), inflammation (79%), and transient impairment of mobility (15%) were observed with intramuscular quinine. Early treatment failure was higher in the rectal group (6% v 3%, absolute difference 3.0%, 95% confidence interval 0.2% to 5.9%). All except two children in each group had negative blood slide results at day 5. Fever recurrence at day 7 was higher in the intramuscular group (37/375 v 18/395, absolute difference 5.3%, 1.6% to 8.9%). Other efficacy outcomes (late clinical failure, late parasitological failure, fever clearance time, time to starting oral intake and rate of deterioration to severe malaria) did not differ. CONCLUSION: Quinine given by the rectal route has an acceptable safety profile and could be used in the early management of moderately severe malaria in children in sub-Saharan Africa, halting progression to severe disease. PMID- 16675813 TI - Galactorrhoea may be associated with methadone use. PMID- 16675814 TI - Acute pancreatitis. PMID- 16675815 TI - Pneumonia: update on diagnosis and management. PMID- 16675816 TI - The cost of dichotomising continuous variables. PMID- 16675817 TI - Difficult pain. PMID- 16675819 TI - Research protocols: waiving confidentiality for the greater good. PMID- 16675820 TI - Comparative accuracy: assessing new tests against existing diagnostic pathways. PMID- 16675821 TI - Mortality associated with delay in operation after hip fracture: ... but Italian data seem to contradict study findings. PMID- 16675822 TI - Mortality associated with delay in operation after hip fracture: Scottish data provide additional information... PMID- 16675823 TI - Is any involvement with the UK military unethical? PMID- 16675824 TI - Preterm delivery in primiparous women at low risk: could epidemic chlamydia contribute to rise in preterm births? PMID- 16675825 TI - Preterm delivery in primiparous women at low risk: Preterm birth or delivery? Study authors suggest new terms. PMID- 16675826 TI - Panic disorder: propranolol and behavioural therapy. PMID- 16675827 TI - The cause for quiet celebration. PMID- 16675828 TI - Policing of information from internet breast cancer list: "list mining" raises new issues in research ethics. PMID- 16675829 TI - Policing of information from internet breast cancer list: findings may not be generally applicable. PMID- 16675830 TI - Compensation and complaints in New Zealand. PMID- 16675831 TI - Money for nothing, and your kicks for free. PMID- 16675832 TI - Boom to bust in the NHS: questions on productivity. PMID- 16675833 TI - Boom to bust in the NHS: consultant productivity. PMID- 16675834 TI - Boom to bust in the NHS: income in general practice. PMID- 16675835 TI - Dietary supplements: red wine, ortolans, and chondroitin sulfate. PMID- 16675836 TI - Inner ear troubles: the roar in the forest? PMID- 16675838 TI - Cellular mechanotransduction: putting all the pieces together again. AB - Analysis of cellular mechanotransduction, the mechanism by which cells convert mechanical signals into biochemical responses, has focused on identification of critical mechanosensitive molecules and cellular components. Stretch-activated ion channels, caveolae, integrins, cadherins, growth factor receptors, myosin motors, cytoskeletal filaments, nuclei, extracellular matrix, and numerous other structures and signaling molecules have all been shown to contribute to the mechanotransduction response. However, little is known about how these different molecules function within the structural context of living cells, tissues, and organs to produce the orchestrated cellular behaviors required for mechanosensation, embryogenesis, and physiological control. Recent work from a wide range of fields reveals that organ, tissue, and cell anatomy are as important for mechanotransduction as individual mechanosensitive proteins and that our bodies use structural hierarchies (systems within systems) composed of interconnected networks that span from the macroscale to the nanoscale in order to focus stresses on specific mechanotransducer molecules. The presence of isometric tension (prestress) at all levels of these multiscale networks ensures that various molecular scale mechanochemical transduction mechanisms proceed simultaneously and produce a concerted response. Future research in this area will therefore require analysis, understanding, and modeling of tensionally integrated (tensegrity) systems of mechanochemical control. PMID- 16675839 TI - HIF-1: hypoxia-inducible factor or dysoxia-inducible factor? AB - Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes involved in diverse aspects of cellular and integrative physiology, including energy metabolism, cell growth, survival, invasion, migration or angiogenesis. The activity of this transcription factor is known to be increased by hypoxia, but also by a growing number of apparently unrelated factors that can activate it even in nonhypoxic conditions. Here I propose a model in which an alteration in oxygen metabolism is the key cellular event involved in HIF-1 activation under hypoxic and nonhypoxic conditions. This new perspective unifies previously unrelated observations and predicts cellular processes and therapeutic strategies that may modify HIF-1 activity. This may have relevance, for instance, to cancer, as HIF-1 overexpression is observed in many human cancers and has been associated with increased patient mortality. PMID- 16675840 TI - The fellowship of the rings: distinct pools of proliferating cell nuclear antigen trimer at work. AB - The proliferating cell nuclear antigen (PCNA) is a homotrimeric ring-shaped protein that, by encircling DNA, may function as a sliding platform for proteins participating in various DNA transactions. PCNA plays a fundamental role in DNA replication and repair, but also in postreplicative events, like DNA methylation, chromatin assembly and remodeling, sister chromatid cohesion, and coordinates these activities with cell cycle control. However, relevant aspects of PCNA function are still not well understood, like the role of PCNA in the association with partner proteins, and how multiple protein interactions are orchestrated. Based on emerging evidence, I suggest that 1) PCNA interacting proteins may be reclassified in three major categories, namely, a) cell cycle control; b) DNA replication/repair; c) chromatin regulation/transcription. 2) PCNA is a negative regulator, rather than a processivity/recruitment factor, of chromatin-modifying enzymes. 3) At DNA replication sites, PCNA function may be envisaged with a model of "dynamic hand-off" of interacting partners that rapidly and transiently exchange in a mutually exclusive manner, while cyclin-dependent kinase (Cdk) 2 (CDK2) is stably bound to PCNA. The partner exchange might occur through a conformational change of the PCNA/protein/DNA complex allowing CDK2 to phosphorylate the partner protein, thereby enabling its hand-off from PCNA. PMID- 16675841 TI - The science and ethics of making part-human animals in stem cell biology. AB - The National Academy of Sciences recently issued voluntary guidelines to govern human embryonic stem cell research. Among other restrictions, these guidelines prohibit certain kinds of combinations of human and nonhuman animal cells, and call for ethics review and oversight of any protocol involving the transfer of human embryonic stem cells into nonhuman animals. In this essay, I discuss the history of and scientific rationales for combining human cells with cells of nonhuman animals, and critically assess the most recent attempts to limit such research on moral grounds--and find them lacking. Nonetheless, as I show, this research remains scientifically and morally contested. I then explore whether and how the NAS's recommended Embryonic Stem Cell Research and Oversight committees will allow for scientifically well-informed moral assessment of this controversial, but possibly important, research. PMID- 16675842 TI - Alpha-crystallin expression affects microtubule assembly and prevents their aggregation. AB - The molecular chaperones alphaA- and alphaB-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of alphaA-/- and alphaB-/- lens epithelial cells. However, no report to date has studied the effect of alpha crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of alphaA- and alphaB-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wild-type, alphaA-/-, alphaB-/-, and alpha(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 mum long and approximately 25 nm wide and had a characteristic protofilament structure, but alphaB-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, alpha-enolase, and betaB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, alphaA-/- or alphaB-/- microtubules, extraction of the MAPs gave very long (14-20 microm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous alpha-crystallin (alphaA+ alphaB) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that alpha crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90 degrees light scattering measurements showed that alpha-crystallin suppressed tubulin assembly in vitro. Alpha-crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that alpha-crystallin prevented heat-induced aggregation of tubulin, suggesting that alpha-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin. PMID- 16675843 TI - Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm? AB - Mechanosensing by cells directs changes in bone mass and structure in response to the challenges of mechanical loading. Low-amplitude, high-frequency loading stimulates bone growth by enhancing bone formation and inhibiting disuse osteoporosis. However, how bone cells sense vibration stress is unknown. Hence, we investigated bone cell responses to vibration stress at a wide frequency range (5-100 Hz). We used NO and prostaglandin E2 (PGE2) release, and COX-2 mRNA expression as parameters for bone cell response since these molecules regulate bone adaptation to mechanical loading. NO release positively correlated whereas PGE2 release negatively correlated to the maximum acceleration rate of the vibration stress. COX-2 mRNA expression increased in a frequency-dependent manner, which relates to increased NO release at high frequencies, confirming our previous results. The negatively correlated release of NO and PGE2 suggests that these signaling molecules play different roles in bone adaptation to high frequency loading. The maximum acceleration rate is proportional to omega3 (frequency=omega/2pi), which is commensurate with the Stokes-Einstein relation for modeling cell nucleus motion within the cytoplasm due to vibration stress. Correlations of NO and PGE2 with the maximum acceleration rate then relate to nucleus oscillations, providing a physical basis for cellular mechanosensing of high-frequency loading. PMID- 16675845 TI - Vitreous cryopreservation maintains the viscoelastic property of human vascular grafts. AB - Assess the effects of cryopreservation (cryo) and vitrification (vitro) on the viscoelastic properties of blood vessels. Human external Iliac artery vessels were harvested from liver organ donors (n=8). In each case the vessel was segmented into 3 equal parts, which were randomly placed in one of 3 categories: Fresh (stored in 4 degrees C UW for 6 h), Cryo (Placed in 10% Dulbecco's modified Eagle medium (DMEM) and slowly frozen to -196 degrees C), or Vitro (Placed in 40% DMEM and rapidly cooled to -196 degrees C). A pulsatile flow circuit was used to perfuse arterial segments at physiological pulse pressure and flow. Intraluminal pressure was measured using a Millar Mikro-tip catheter transducer, and vessel wall motion was determined with duplex ultrasonography coupled with a novel echo locked vessel wall tracking system. Diametrical compliance (DC), Petersons elastic modulus (Ep), and stiffness index (beta) were then calculated for each of the three groups over 3 mean pressure ranging from 40 to 80 mmHg. The change in the viscous component of arterial wall (lag phase angle, theta) was calculated from hysteresis plots. No significant changes were observed in the elastic properties of fresh and vitrified vessels (P>0.05 for each of DC, Ep, and beta). Similarly, variation in the wall viscosity between fresh and vitrified vessels appeared to be nonsignificant (theta=12.60+/-4.04 vs. 17.60+/-1.14, respectively). In contrast, statistical analysis of results obtained for cryopreserved vessels to the fresh vessels showed significant reduction in elastic parameter values. There was also a significant increase in the phase angle theta of the cryopreserved vessels (theta=24.30+/-6.32; P<0.001) compared with fresh vessel. Results suggest that vitrification maintains both elastic and viscous components of the mechanical properties of vascular grafts, which is positively correlated with their functional patency. In contrast, damage caused during cryopreservation significantly affects the overall tensile strength and elasticity of the vessel (i.e., Ep and beta), the dynamic properties (DC), and appears to significantly affect the viscous component of the vessel wall (theta), which is likely reduce the patency of the graft for transplantation purposes. PMID- 16675844 TI - Minigenes encoding N-terminal domains of human cardiac myosin light chain-1 improve heart function of transgenic rats. AB - In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (K(D)) for the hVLC-1/1-15-actin complex compared with the K(D) value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 muM) correlated positively with significant (P<0.001) improvements of the intrinsic contractile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart. PMID- 16675847 TI - The neuronal chemokine CX3CL1/fractalkine selectively recruits NK cells that modify experimental autoimmune encephalomyelitis within the central nervous system. AB - Leukocyte trafficking to the central nervous system (CNS), regulated in part by chemokines, determines severity of the demyelinating diseases multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To examine chemokine receptor CX3CR1 in EAE, we studied CX3CR1(GFP/GFP) mice, in which CX3CR1 targeting by insertion of Green Fluorescent Protein (GFP) allowed tracking of CX3CR1+ cells in CX3CR1(+/GFP) animals and cells destined to express CX3CR1 in CX3CR1(GFP/GFP) knockouts. NK cells were markedly reduced in the inflamed CNS of CX3CR1-deficient mice with EAE, whereas recruitment of T cells, NKT cells and monocyte/macrophages to the CNS during EAE did not require CX3CR1. Impaired recruitment of NK cells in CX3CR1(GFP/GFP) mice was associated with increased EAE related mortality, nonremitting spastic paraplegia and hemorrhagic inflammatory lesions. The absence of CD1d did not affect the severity of EAE in CX3CR1(GFP/GFP) mice, arguing against a role for NKT cells. Accumulation of NK cells in livers of wild-type (WT) and CX3CR1(GFP/GFP) mice with cytomegalovirus hepatitis was equivalent, indicating that CX3CL1 mediated chemoattraction of NK cells was relatively specific for the CNS. These results are the first to define a chemokine that governs NK cell migration to the CNS, and the findings suggest novel therapeutic manipulation of CX3CR1+ NK cells. PMID- 16675846 TI - Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides. AB - Human skin is a local source of corticotropin-releasing hormone (CRH) and expresses CRH and CRH receptors (CRH-R) at mRNA and protein levels. Epidermal melanocytes respond to CRH by induction of cAMP with up-regulation of pro opiomelanocortin gene expression and subsequent production of adrenocorticotropin hormone. However, the role of CRH/CRH-R in melanocyte biology is complicated by the significant heterogeneity of cutaneous melanocyte subpopulations, from continuously active and UV-responsive melanocytes in epidermis to UV nonresponsive, hair growth cycle-coupled melanogenesis in hair follicles. In the present study we report that normal human scalp hair follicle melanocytes express CRH at the mRNA level. Furthermore, CRH, urocortin and CRH-R 1 and 2 were differentially expressed in follicular melanocytes, fibroblasts, and keratinocytes depending on anatomic location and differentiation status in situ and in vitro. Stimulation of follicular melanocytes with CRH and CRH peptides, modified for selectivity for CRH-R1 and/or CRH-R2, variably induced cell melanogenesis, dendricity, and proliferation. CRH-peptides also stimulated the expression and activity of Tyrosinase, and expression of Tyrosinase-related protein-1 and-2. However, a modified urocortin peptide highly selective for CRH R2 down-regulated melanocyte differentiation phenotype. This study indicates that CRH peptides can differentially influence hair follicle melanocyte behavior not only via CRH-R1 signaling but also by complex cross-talk between CRH-R1 and CRH R2. PMID- 16675848 TI - Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor. AB - It has been reported that extracellular matrix (ECM) molecules regulate monocyte activation by binding with a 67 kDa nonintegrin laminin receptor (LR). As microgliosis is a pivotal factor in propelling the progress of chronic neurodegeneration in the brain, we hypothesized that LR may regulate the microgliosis and subsequent neurotoxicity. Using 1-methyl-4-phenylpyridinium (MPP+) -treated C57 mice primary mesencephalic neuron-glia cultures as an in vitro Parkinson's disease (PD) model, we observed that MPP+ treatment increased LR expression only in the mixed neuron-glia but not in microglia-enriched or microglia-depleted cultures, indicating that MPP+-induced increase of LR expression is associated with neuron-microglia interaction. Using confocal microscopic examination, we found that LR was localized in the microglia, which were F4/80 positive. Treatment with the antibody (Ab) against LR (LR-Ab) or YIGSR, a synthetic pentapeptide inhibitor for LR, significantly attenuated the MPP+-increased F4/80 immunoreactivity (24 h) and dopaminergic (DA) neurotoxicity. LR-Ab also attenuated MPP+-increased microglial phagocytotic activity (48 h) and the superoxide production (4 days). Further study demonstrated that exogenous laminin (1-10 microg/ml) treatment induced microglial activation and DA neurotoxicity, in a dose-dependent manner, which was partially attenuated by the LR-Ab. We concluded that by regulating cell-ECM interaction, LR plays important roles in mediating microgliosis and subsequent DA neurotoxicity. Laminin is a potential ligand for activating this LR receptor. This study also suggests that laminin/LR is a potential target for developing new therapeutic drugs against neurodegenerative disorders such as PD. PMID- 16675849 TI - Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis. AB - Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors,and not on gene deletion. Here we used ROCK1knockout (ROCK1-/-) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1-/- mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1-/- mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFbeta2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of Gq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli. PMID- 16675850 TI - Estrogen receptor alpha up-regulation and redistribution in human heart failure. AB - Clinical and animal studies suggest that estrogen receptors are involved in the development of myocardial hypertrophy and heart failure. In this study, we investigated whether human myocardial estrogen receptor alpha (ERalpha) expression, localization, and association with structural proteins was altered in end stage-failing hearts. We found a 1.8-fold increase in ERalpha mRNA and protein in end-stage human dilated cardiomyopathy (DCM, n=41), as compared with controls (n=25). ERalpha was visualized by confocal immunofluorescence microscopy and localized to the cytoplasm, sarcolemma, intercalated discs and nuclei of cardiomyocytes. Immunofluorescence studies demonstrated colocalization of ERalpha with beta-catenin at the intercalated disc in control hearts and immunoprecipitation studies confirmed complex formation of both proteins. Interestingly, the ERalpha/beta-catenin colocalization was lost at the intercalated disc in DCM hearts. Thus, the ERalpha/beta-catenin colocalization in the intercalated disc may be of functional relevance and a loss of this association may play a role in the progression of heart failure. The increase of total ERalpha expression may represent a compensatory process to contribute to the stability of cardiac intercalated discs. PMID- 16675851 TI - Enhanced expression of hypersensitive alpha4* nAChR in adult mice increases the loss of midbrain dopaminergic neurons. AB - We describe an inducible genetic model for degeneration of midbrain dopaminergic neurons in adults. In previous studies, knock-in mice expressing hypersensitive M2 domain Leu9'Ser (L9'S) alpha4 nicotinic receptors (nAChR) at near-normal levels displayed dominant neonatal lethality and dopaminergic deficits in embryonic midbrain, because the hypersensitive nAChR is excitotoxic. However, heterozygous L9'S mice that retain the neomycin resistance cassette (neo) in a neighboring intron express low levels of the mutant allele (approximately 25% of normal levels), and these neo-intact mice are therefore viable and fertile. The neo cassette is flanked by loxP sites. In adult animals, we locally injected helper-dependent adenovirus (HDA) expressing cre recombinase. Local excision of the neo cassette, via cre-mediated recombination, was verified by genomic analysis. In L9'S HDA-cre injected animals, locomotion was reduced both under baseline conditions and after amphetamine application. There was no effect in L9'S HDA-control treated animals or in wild-type (WT) littermates injected with either virus. Immunocytochemical analyses revealed marked losses (> 70%) of dopaminergic neurons in L9'S HDA-cre injected mice compared to controls. At 20-33 days postinjection in control animals, the coexpressed marker gene, yellow fluorescent protein (YFP), was expressed in many neurons and few glial cells near the injection, emphasizing the neurotropic utility of the HDA. Thus, HDA-mediated gene transfer into adult midbrain induced sufficient functional expression of cre in dopaminergic neurons to allow for postnatal deletion of neo. This produced increased L9'S mutant nAChR expression, which in turn led to nicotinic cholinergic excitotoxicity in dopaminergic neurons. PMID- 16675852 TI - GFP reporter mouse models of UPS proteolytic function. PMID- 16675853 TI - C. elegans as a model for Friedreich Ataxia. PMID- 16675854 TI - Structural elucidation of phosphoglycolipids from strains of the bacterial thermophiles Thermus and Meiothermus. AB - The structures of two major phosphoglycolipids from the thermophilic bacteria Thermus oshimai NTU-063, Thermus thermophilus NTU-077, Meiothermus ruber NTU-124, and Meiothermus taiwanensis NTU-220 were determined using spectroscopic and chemical analyses to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho) -3'-O-(alpha-N acetyl-glucosaminyl)-N-glyceroyl alkylamine [PGL1 (1)] and the novel structure 2' O-(2-acylalkyldio-1-O-phospho)-3'-O-(alpha-N-acetylglucosaminyl)-N-glyceroyl alkylamine [PGL2 (2)]. PGL2 (2) is the first phosphoglycolipid identified with a 2-acylalkyldio-1-O-phosphate moiety. The fatty acids of the phosphoglycolipids are mainly iso-C(15:0), -C(16:0), and -C(17:0) and anteiso-C(15:0) and -C(17:0). The ratios of PGL2 (2) to PGL1 (1) are significantly altered when grown at different temperatures for three strains, T. thermophilus NTU-077, M. ruber NTU 124, and M. taiwanensis NTU-220, but not for T. oshimai NTU-063. Accordingly, the ratios of iso- to anteiso-branched fatty acids increase when grown at the higher temperature. PMID- 16675855 TI - Optimized conditions for measuring lipolysis in murine primary adipocytes. AB - The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells. PMID- 16675856 TI - Evaluation of a new electromagnetic tracking system using a standardized assessment protocol. AB - This note uses a published protocol to evaluate a newly released 6 degrees of freedom electromagnetic tracking system (Aurora, Northern Digital Inc.). A practice for performance monitoring over time is also proposed. The protocol uses a machined base plate to measure relative error in position and orientation as well as the influence of metallic objects in the operating volume. Positional jitter (E(RMS)) was found to be 0.17 mm +/- 0.19 mm. A relative positional error of 0.25 mm +/- 0.22 mm at 50 mm offsets and 0.97 mm +/- 1.01 mm at 300 mm offsets was found. The mean of the relative rotation error was found to be 0.20 degrees +/- 0.14 degrees with respect to the axial and 0.91 degrees +/- 0.68 degrees for the longitudinal rotation. The most significant distortion caused by metallic objects is caused by 400-series stainless steel. A 9.4 mm maximum error occurred when the rod was closest to the emitter, 10 mm away. The improvement compared to older generations of the Aurora with respect to accuracy is substantial. PMID- 16675857 TI - A unified approach for inversion problems in intensity-modulated radiation therapy. AB - We propose and study a unified model for handling dose constraints (physical dose, equivalent uniform dose (EUD), etc) and radiation source constraints in a single mathematical framework based on the split feasibility problem. The model does not impose on the constraints an exogenous objective (merit) function. The optimization algorithm minimizes a weighted proximity function that measures the sum of the squares of the distances to the constraint sets. This guarantees convergence to a feasible solution point if the split feasibility problem is consistent (i.e., has a solution), or, otherwise, convergence to a solution that minimally violates the physical dose constraints and EUD constraints. We present computational results that demonstrate the validity of the model and the power of the proposed algorithmic scheme. PMID- 16675859 TI - Development of pregnant female, hybrid voxel-mathematical models and their application to the dosimetry of applied magnetic and electric fields at 50 Hz. AB - This paper describes the development of 2 mm resolution hybrid voxel-mathematical models of the pregnant female. Mathematical models of the developing foetus at 8 , 13-, 26- and 38-weeks of gestation were converted into voxels and combined with the adult female model, NAOMI. This set of models was used to calculate induced current densities and electric fields in the foetus from applied 50 Hz magnetic and electric fields. The influence of foetal tissue conductivities was investigated and implications for electromagnetic field guidelines discussed. PMID- 16675858 TI - Volume measurement variability in three-dimensional high-frequency ultrasound images of murine liver metastases. AB - The identification and quantification of tumour volume measurement variability is imperative for proper study design of longitudinal non-invasive imaging of pre clinical mouse models of cancer. Measurement variability will dictate the minimum detectable volume change, which in turn influences the scheduling of imaging sessions and the interpretation of observed changes in tumour volume. In this paper, variability is quantified for tumour volume measurements from 3D high frequency ultrasound images of murine liver metastases. Experimental B16F1 liver metastases were analysed in different size ranges including less than 1 mm3, 1-4 mm3, 4-8 mm3 and 8-70 mm3. The intra- and inter-observer repeatability was high over a large range of tumour volumes, but the coefficients of variation (COV) varied over the volume ranges. The minimum and maximum intra-observer COV were 4% and 14% for the 1-4 mm3 and <1 mm3 tumours, respectively. For tumour volumes measured by segmenting parallel planes, the maximum inter-slice distance that maintained acceptable measurement variability increased from 100 to 600 microm as tumour volume increased. Comparison of free breathing versus ventilated animals demonstrated that respiratory motion did not significantly change the measured volume. These results enable design of more efficient imaging studies by using the measured variability to estimate the time required to observe a significant change in tumour volume. PMID- 16675860 TI - Improving source detection and separation in a spatiotemporal Bayesian inference dipole analysis. AB - Most existing spatiotemporal multi-dipole approaches for MEG/EEG source localization assume that the dipoles are active for the full time range being analysed. If the actual time range of activity of sources is significantly shorter than the time range being analysed, the detectability, localization and time-course determination of such sources may be adversely affected, especially for weak sources. In order to improve detectability and reconstruction of such sources, it is natural to add active time range information (starting time point and ending time point of source activation) for each candidate source as unknown parameters in the analysis. However, this adds additional nonlinear free parameters that could burden the analysis and could be unfeasible for some methods. Recently, we described a spatiotemporal Bayesian inference multi-dipole analysis for the MEG/EEG inverse problem. This approach treated the number of dipoles as a free parameter, produced realistic uncertainty estimates using a Markov chain Monte Carlo numerical sampling of the posterior distribution and included a method to reduce the unwanted effects of local minima. In this paper, our spatiotemporal Bayesian inference multi-dipole analysis is extended to incorporate active time range parameters of starting and stopping time points. The properties of this analysis in comparison to the previous one without active time range parameters are demonstrated through extensive studies using both simulated and empirical MEG data. PMID- 16675861 TI - Resolution enhancement in digital x-ray imaging. AB - We have developed a restoration method for radiographs that enhances image sharpness and reveals bone microstructures that were initially hidden in the soft tissue glare. The method is two fold: the image is first deconvolved using the Richardson-Lucy algorithm and is then divided with a signal modelling the soft tissue distribution to increase the overall contrast. Each step has its own merits but the power of the restoration method lies in their combination. The originality of the method is its reliance on a priori information at each step in the processing. We have measured and modelled analytically the point-spread function of a low-dose gas microstrip x-ray detector at several beam energies. We measured the relationship between the local image intensity and the noise variance for these images. The soft-tissue signal was also modelled using a minimum-curvature filtering technique. These results were then combined into an image deconvolution procedure that uses wavelet filtering to reduce restoration noise while keeping the enhanced small-scale features. The method was applied successfully to images of a human-torso phantom and improved the contrast of small details on the bones and in the soft tissues. We measured a mean 54% increase in signal to noise ratio and a mean 105% increase in contrast to noise ratio in the 70 and 140 kVp images we analysed. The method was designed to facilitate the analysis of radiographs by relying on two levels of visual inspection. The contrast of the full image is first enhanced by division with the signal modelling the soft-tissue distribution. Based on the result, a radiologist might decide to zoom in on a given image section. The full restoration method is then applied to that region of interest. Indeed, full image deconvolution is often unnecessary since enhanced small-scale details are not visible at large scale; only the section of interest is processed which is more efficient. PMID- 16675862 TI - A comparison between objective and subjective image quality measurements for a full field digital mammography system. AB - This paper presents pre-sampling modulation transfer function (MTF), normalized noise power spectrum (NNPS) and detective quantum efficiency (DQE) results for an amorphous selenium (a-Se) full field digital mammography system. MTF was calculated from the image of an angled 0.5 mm thick Cu edge, acquired without additional beam filtration. NNPS data were acquired at detector air-kerma levels ranging from 9.1 microGy to 331 microGy, using a standard mammography x-ray spectrum of 28 kV, Mo/Mo target/filter combination and 4 cm of PMMA additional filtration. Prior to NNPS estimation, the image statistics were assessed using a variance image. This method was able to easily identify a detector artefact and should prove useful in routine quality assurance (QA) measurements. Detector DQE, calculated from the NNPS and MTF data, dropped to 0.3 for low detector air-kerma settings but reached an approximately constant value of 0.6 above 50 microGy at the detector. Subjective image quality data were also obtained at these detector air-kerma settings using the CDMAM contrast-detail (c-d) test object. The c-d data reflected the trend seen in DQE, with threshold contrast increasing at low detector air-kerma values. The c-d data were then compared against predictions made using two established models, the Rose model and a standard signal detection theory model. Using DQE(0), the Rose model gave results within approximately 15% on average for all the detector air-kerma values studied and for detail diameters down to 0.2 mm. Similar agreement was also found between the measured c-d data and the signal detection theory results, which were calculated using an ideal human visual response function and a system magnification of unity. The use of full spatial frequency DQE improved the agreement between the calculated and observer results for detail sizes below 0.13 mm. PMID- 16675863 TI - Wavelet-based feature extraction applied to small-angle x-ray scattering patterns from breast tissue: a tool for differentiating between tissue types. AB - This paper reports on the application of wavelet decomposition to small-angle x ray scattering (SAXS) patterns from human breast tissue produced by a synchrotron source. The pixel intensities of SAXS patterns of normal, benign and malignant tissue types were transformed into wavelet coefficients. Statistical analysis found significant differences between the wavelet coefficients describing the patterns produced by different tissue types. These differences were then correlated with position in the image and have been linked to the supra-molecular structural changes that occur in breast tissue in the presence of disease. Specifically, results indicate that there are significant differences between healthy and diseased tissues in the wavelet coefficients that describe the peaks produced by the axial d-spacing of collagen. These differences suggest that a useful classification tool could be based upon the spectral information within the axial peaks. PMID- 16675864 TI - Simulation of scattering and attenuation of 511 keV photons in a combined PET/field-cycled MRI system. AB - Mixing the imaging modalities of positron emission tomography (PET) and magnetic resonance imaging (MRI) will offer the best soft tissue contrast (MRI) with information about metabolic function (PET). The high magnetic field environment of an MRI system makes the detection of annihilation photons difficult, as the response of standard photo-multiplier tubes is compromised. An approach using field-cycled MRI is discussed here, as field-cycled MRI makes it possible to have long periods of time available for nuclear imaging when there is no magnetic field present. This work focuses upon the effect of the field-cycled MRI upon the nuclear image due to the added material providing additional attenuation of the PET signal, and additional nuclei for scatter. These effects are studied using a Monte Carlo simulation based upon the GEANT libraries. Attenuation effects are shown to be significant, approximately 6% for the RF shield and coil and approximately 24% for the gradients. No significant effect is seen in image quality due to the scattering of the gammas. With these levels of attenuation it is concluded that open gradient coils and shim coils are required around the imaging volume. PMID- 16675865 TI - Automatic online adaptive radiation therapy techniques for targets with significant shape change: a feasibility study. AB - This work looks at the feasibility of an online adaptive radiation therapy concept that would detect the daily position and shape of the patient, and would then correct the daily treatment to account for any changes compared with planning position. In particular, it looks at the possibility of developing algorithms to correct for large complicated shape change. For co-planar beams, the dose in an axial plane is approximately associated with the positions of a single multi-leaf collimator (MLC) pair. We start with a primary plan, and automatically generate several secondary plans with gantry angles offset by regular increments. MLC sequences for each plan are calculated keeping monitor units (MUs) and number of segments constant for a given beam (fluences are different). Bulk registration (3D) of planning and daily CT images gives global shifts. Slice-by-slice (2D) registration gives local shifts and rotations about the longitudinal axis for each axial slice. The daily MLC sequence is then created for each axial slice/MLC leaf pair combination, by taking the MLC positions from the pre-calculated plan with the nearest rotation, and shifting using a beam's-eye-view calculation to account for local linear shifts. A planning study was carried out using two head and neck region MR images of a healthy volunteer which were contoured to simulate a base-of-tongue treatment: one with the head straight (used to simulate the planning image) and the other with the head tilted to the left (the daily image). Head and neck treatment was chosen to evaluate this technique because of its challenging nature, with varying internal and external contours, and multiple degrees of freedom. Shape change was significant: on a slice-by-slice basis, local rotations in the daily image varied from 2 to 31 degrees, and local shifts ranged from -0.2 to 0.5 cm and -0.4 to 0.0 cm in right-left and posterior-anterior directions, respectively. The adapted treatment gave reasonable target coverage (100%, 90% and 80% of the base-of tongue, left nodes and right nodes, respectively, receiving the daily prescription dose), and kept the daily cord dose below the limit used in the original plan (65%, equivalent to 46 Gy over 35 fractions). Most of the loss of coverage was due to one shoulder being raised more superior relative to the other shoulder compared with the plan. This type of skew-like motion is not accounted for by the proposed ART technique. In conclusion, this technique has potential to correct for fairly extreme daily changes in patient setup, but some control of the daily position would still be necessary. Importantly, it was possible to combine treatments from different plans (MLC sequences) to correct for position and shape change. PMID- 16675866 TI - A practical Monte Carlo MU verification tool for IMRT quality assurance. AB - Quality assurance (QA) for intensity-modulated radiation therapy (IMRT) treatment planning and beam delivery, using ionization chamber measurements and film dosimetry in a phantom, is time consuming. The Monte Carlo method is the most accurate method for radiotherapy dose calculation. However, a major drawback of Monte Carlo dose calculation as currently implemented is its slow speed. The goal of this work is to bring the efficiency of Monte Carlo into a practical range by developing a fast Monte Carlo monitor unit (MU) verification tool for IMRT. A special estimator for dose at a point called the point detector has been used in this research. The point detector uses the next event estimation (NEE) method to calculate the photon energy fluence at a point of interest and then converts it to collision kerma by the mass energy absorption coefficient assuming the presence of transient charged particle equilibrium. The MU verification tool has been validated by comparing the calculation results with measurements. It can be used for both patient dose verification and phantom QA calculation. The dynamic leaf-sequence log file is used to rebuild the actual MLC leaf sequence in order to predict the dose actually received by the patient. Dose calculations for 20 patient plans have been performed using the point detector method. Results were compared with direct Monte Carlo simulations using EGS4/MCSIM, which is a well benchmarked Monte Carlo code. The results between the point detector and MCSIM agreed to within 2%. A factor of 20 speedup can be achieved with the point detector method compared with direct Monte Carlo simulations. PMID- 16675867 TI - A difference-matrix metaheuristic for intensity map segmentation in step-and shoot IMRT delivery. AB - At an intermediate stage of radiation treatment planning for IMRT, most commercial treatment planning systems for IMRT generate intensity maps that describe the grid of beamlet intensities for each beam angle. Intensity map segmentation of the matrix of individual beamlet intensities into a set of MLC apertures and corresponding intensities is then required in order to produce an actual radiation delivery plan for clinical use. Mathematically, this is a very difficult combinatorial optimization problem, especially when mechanical limitations of the MLC lead to many constraints on aperture shape, and setup times for apertures make the number of apertures an important factor in overall treatment time. We have developed, implemented and tested on clinical cases a metaheuristic (that is, a method that provides a framework to guide the repeated application of another heuristic) that efficiently generates very high-quality (low aperture number) segmentations. Our computational results demonstrate that the number of beam apertures and monitor units in the treatment plans resulting from our approach is significantly smaller than the corresponding values for treatment plans generated by the heuristics embedded in a widely use commercial system. We also contrast the excellent results of our fast and robust metaheuristic with results from an 'exact' method, branch-and-cut, which attempts to construct optimal solutions, but, within clinically acceptable time limits, generally fails to produce good solutions, especially for intensity maps with more than five intensity levels. Finally, we show that in no instance is there a clinically significant change of quality associated with our more efficient plans. PMID- 16675868 TI - Improvement of radiological penumbra using intermediate energy photons (IEP) for stereotactic radiosurgery. AB - Using efficient immobilization and dedicated beam collimation devices, stereotactic radiosurgery ensures highly conformal treatment of small tumours with limited microscopic extension. One contribution to normal tissue irradiation remains the radiological penumbra. This work aims at demonstrating that intermediate energy photons (IEP), above orthovoltage but below megavoltage, improve dose distribution for stereotactic radiosurgery for small irradiation field sizes due to a dramatic reduction of radiological penumbra. Two different simulation systems were used: (i) Monte Carlo simulation to investigate the dose distribution of monoenergetic IEP between 100 keV and 1 MeV in water phantom; (ii) the Pinnacle3 TPS including a virtual IEP unit to investigate the dosimetry benefit of treating with 11 non-coplanar beams a 2 cm tumour in the middle of a brain adjacent to a 1 mm critical structure. Radiological penumbrae below 300 microm are generated for field size below 2 x 2 cm2 using monoenergetic IEP beams between 200 and 400 keV. An 800 kV beam generated in a 0.5 mm tungsten target maximizes the photon intensity in this range. Pinnacle3 confirms the dramatic reduction in penumbra size. DVHs show for a constant dose distribution conformality, improved dose distribution homogeneity and better sparing of critical structures using a 800 kV beam compared to a 6 MV beam. PMID- 16675869 TI - Commissioning stereotactic radiosurgery beams using both experimental and theoretical methods. AB - The purpose of this investigation is to study the feasibility of using an alternative method to commission stereotactic radiosurgery beams shaped by micro multi-leaf collimators by using Monte Carlo simulations to obtain beam characteristics of small photon beams, such as incident beam particle fluence and energy distributions, scatter ratios, depth-dose curves and dose profiles where measurements are impossible or difficult. Ionization chambers and diode detectors with different sensitive volumes were used in the measurements in a water phantom and the Monte Carlo codes BEAMnrc/DOSXYZnrc were used in the simulation. The Monte Carlo calculated data were benchmarked against measured data for photon beams with energies of 6 MV and 10 MV produced from a Varian Trilogy accelerator. The measured scatter ratios and cross-beam dose profiles for very small fields are shown to be not only dependent on the size of the sensitive volume of the detector used but also on the type of detectors. It is known that the response of some detectors changes at small field sizes. Excellent agreement was seen between scatter ratios measured with a small ion chamber and those calculated from Monte Carlo simulations. The values of scatter ratios, for field sizes from 6 x 6 mm2 to 98 x 98 mm2, range from 0.67 to 1.0 and from 0.59 to 1.0 for 6 and 10 MV, respectively. The Monte Carlo calculations predicted that the incident beam particle fluence is strongly affected by the X-Y-jaw openings, especially for small fields due to the finite size of the radiation source. Our measurement confirmed this prediction. This study demonstrates that Monte Carlo calculations not only provide accurate dose distributions for small fields where measurements are difficult but also provide additional beam characteristics that cannot be obtained from experimental methods. Detailed beam characteristics such as incident photon fluence distribution, energy spectra, including composition of primary and scattered photons, can be independently used in dose calculation models and to improve the accuracy of measurements with detectors with an energy dependent response. Furthermore, when there are discrepancies between results measured with different detectors, the Monte Carlo calculated values can indicate the most correct result. The data set presented in this study can be used as a reference in commissioning stereotactic radiosurgery beams shaped by a BrainLAB m3 on a Varian 2100EX or 600C accelerator. PMID- 16675870 TI - A robust approach to IMRT optimization. AB - Managing uncertainty is a major challenge in radiation therapy treatment planning, including uncertainty induced by intrafraction motion, which is particularly important for tumours in the thorax and abdomen. Common methods to account for motion are to introduce a margin or to convolve the static dose distribution with a motion probability density function. Unlike previous work in this area, our development does not assume that the patient breathes according to a fixed distribution, nor is the patient required to breathe the same way throughout the treatment. Despite this generality, we create a robust optimization framework starting from the convolution method that is robust to fluctuations in breathing motion, yet spares healthy tissue better than a margin solution. We describe how to generate the data for our model using breathing motion data and we test our model on a computer phantom using data from real patients. In our numerical results, the robust solution delivers approximately 38% less dose to the healthy tissue than the margin solution, while providing the same level of protection against breathing uncertainty. PMID- 16675871 TI - A fit method for the determination of inherent filtration with diagnostic x-ray units. AB - A method for the determination of total inherent filtration for clinical x-ray units using attenuation curves was devised. A model for the calculation of x-ray spectra is used to calculate kerma values which are then adjusted to the experimental data in minimizing the sum of the squared relative differences in kerma using a modified simplex fit process. The model considers tube voltage, voltage ripple, anode angle and additional filters. Fit parameters are the thickness of an additional inherent Al filter and a general normalization factor. Nineteen sets of measurements including attenuation data for three tube voltages and five Al-filter settings each were obtained. Relative differences of experimental and calculated kerma using the data for the additional filter thickness are within a range of -7.6% to 6.4%. Quality curves, i.e. the relationship of additional filtration to HVL, are often used to determine filtration but the results show that standard quality curves do not reflect the variety of conditions encountered in practice. To relate the thickness of the additional filter to the condition of the anode surface, the data fits were also made using tungsten as the filter material. These fits gave an identical fit quality compared to aluminium with a tungsten filter thickness of 2.12-8.21 microm which is within the range of the additional absorbing layers determined for rough anodes. PMID- 16675872 TI - Investigation of a 2D two-point maximum entropy regularization method for signal to-noise ratio enhancement: application to CT polymer gel dosimetry. AB - This study presents a new method of image signal-to-noise ratio (SNR) enhancement by utilizing a newly developed 2D two-point maximum entropy regularization method (TPMEM). When utilized as an image filter, it is shown that 2D TPMEM offers unsurpassed flexibility in its ability to balance the complementary requirements of image smoothness and fidelity. The technique is evaluated for use in the enhancement of x-ray computed tomography (CT) images of irradiated polymer gels used in radiation dosimetry. We utilize a range of statistical parameters (e.g. root-mean square error, correlation coefficient, error histograms, Fourier data) to characterize the performance of TPMEM applied to a series of synthetic images of varying initial SNR. These images are designed to mimic a range of dose intensity patterns that would occur in x-ray CT polymer gel radiation dosimetry. Analysis is extended to a CT image of a polymer gel dosimeter irradiated with a stereotactic radiation therapy dose distribution. Results indicate that TPMEM performs strikingly well on radiation dosimetry data, significantly enhancing the SNR of noise-corrupted images (SNR enhancement factors >15 are possible) while minimally distorting the original image detail (as shown by the error histograms and Fourier data). It is also noted that application of this new TPMEM filter is not restricted exclusively to x-ray CT polymer gel dosimetry image data but can in future be extended to a wide range of radiation dosimetry data. PMID- 16675873 TI - Tracking transplanted cells using dual-radionuclide SPECT. AB - The purpose of this study was to characterize the performance of single photon emission computed tomography (SPECT) in tasks associated with tracking transplanted cells. Previous studies identified matters of hardware design, whereas we focus on biological variables impacting system performance, such as cell colony growth and non-specific radiolabelling. Using experimental data, a digital phantom was developed of in vitro 111In-radiolabelled stem cells, transfected with a reporter gene, transplanted into canine infarcted myocardium and interrogated using a peripherally injected 131I-radiolabelled reporter probe. Single- and dual-head SPECT acquisition was simulated. Performance was characterized using an estimation task, where the precision of parameter estimates (111In and 131I radiolabel quantity, cell colony size and location, and background) was tracked as the phantom evolved to simulate 111In-label efflux, cell colony growth and improved reporter probe specificity. In vitro pre labelling of transplanted cells improved precision of parameter estimates via a priori size and location information. Precision of radiolabel quantity estimates improved with cell colony growth, despite 111In radiolabel dilution; size and location parameters were influenced little. Precision of radiolabel quantity estimates improved with reduced reporter probe non-specific uptake. The performance of SPECT in cell tracking is influenced strongly by biological variables. These should be considered when planning experiments or developing SPECT technology for cell tracking. PMID- 16675874 TI - Exact quantification of time signals in Pade-based magnetic resonance spectroscopy. AB - This study reports on the fast Pade transform (FPT) for parametric signal processing of realistically synthesized free induction decay curves whose main spectral features are similar to those encoded clinically from a healthy human brain by means of magnetic resonance spectroscopy (MRS). Here, for the purpose of diagnostics, it is of paramount importance to be able to perform accurate and robust quantification of the investigated time signals. This amounts to solving the challenging harmonic inversion problem as a spectral decomposition of the given time signal by means of reconstruction of the unknown total number of resonances, their complex frequencies and amplitudes yielding the peak positions, widths, heights and phases. On theoretical grounds, the FPT solves exactly this mathematically ill-conditioned inverse problem for any noiseless synthesized time signal comprised of an arbitrarily large (finite or infinite) number of damped complex exponentials with stationary and non-stationary polynomial-type amplitudes leading to Lorentzian (non-degenerate) and non-Lorentzian (degenerate) spectra. Convergent validation for this fact is given via the proof-of-principle which is thoroughly demonstrated by the exact numerical solution of a typical quantification problem from MRS. The presently designed study is a paradigm shift for signal processing in MRS with particular relevance to clinical oncology, due to the unprecedented capability of the fast Pade transform to unequivocally resolve and quantify isolated, tightly overlapped and nearly coincident resonances. PMID- 16675875 TI - A novel photoacoustic tomography based on a time-resolved technique and an acoustic lens imaging system. AB - A novel photoacoustic (PA) tomographic method, which is based on a time-resolved technique and an acoustic lens imaging system, is presented in this paper. A YAG laser operating at 532 nm with a 7 ns pulse width and 10 mJ optical pulse is employed as the excitation source to irradiate the tissue. PA signals generated from the tissue are imaged onto a multi-element linear array transducer with an acoustic lens. A 64 electronic switch is efficiently used for changing the parallel PA signals into a series. The proposed method directly provides PA images without any complex reconstruction algorithms. With the time-resolved technique, tomographic imaging can be achieved successfully. The results show that the images agree well with the original samples. PMID- 16675876 TI - Effects of prolonged surface pressure on the skin blood flowmotions in anaesthetized rats--an assessment by spectral analysis of laser Doppler flowmetry signals. AB - The objective of this study is to assess the effect of prolonged surface compression on the skin blood flowmotion in rats using spectral analysis based on wavelets transform of the periodic oscillations of the cutaneous laser Doppler flowmetry (LDF) signal. An external pressure of 13.3 kPa (100 mmHg) was applied to the trochanter area and the distal lateral tibia of Sprague-Dawley rats via two specifically designed pneumatic indentors. The loading duration was 6 hours/day for 4 consecutive days. Five frequency intervals were identified (0.01 0.04 Hz, 0.04-0.15 Hz, 0.15-0.4 Hz, 0.4-2 Hz and 2-5 Hz) corresponding to endothelial related metabolic, neurogenic, myogenic, respiratory and cardiac origins. The absolute amplitude of oscillations of each particular frequency interval and the normalized amplitude were calculated for quantitative assessments. The results showed that (1) tissue compression following the above schedule induced significant decrease in the normalized amplitude in the frequency interval of 0.01-0.04 Hz both in the trochanter area (p < 0.001) and tibialis area (p = 0.023), (2) prolonged compression induced significant increase in the absolute amplitude (p = 0.004 for the trochanter area and p = 0.017 for the tibialis area) but significant decrease in the normalized amplitude (p = 0.023 for the trochanter area and p = 0.026 for the tibialis area) in the frequency interval of 0.15-0.4 Hz, and (3) at the tibialis area, the flowmotion amplitude (frequency interval 0.15-0.4 Hz) measured prior to the daily tissue compression schedule was found to be significantly higher on day 4 than the measurements obtained on day 1. However, this finding was not observed at the trochanter area. Our results suggested that prolonged compression might induce endothelial damage and affect the endothelial related metabolic activities. PMID- 16675877 TI - Vanishing conflicts on cancer theories. PMID- 16675878 TI - The Fanconi anemia pathway of genomic maintenance. AB - Fanconi anemia (FA), a recessive syndrome with both autosomal and X-linked inheritance, features diverse clinical symptoms, such as progressive bone marrow failure, hypersensitivity to DNA cross-linking agents, chromosomal instability and susceptibility to cancer. At least 12 genetic subtypes have been described (FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA-I have been linked to a distinct gene. Most FA proteins form a complex that activates the FANCD2 protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream of this step. The FA proteins typically lack functional domains, except for FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross-linking agents, the FA proteins are thought to function in the repair of DNA interstrand cross-links, which block the progression of DNA replication forks. Here we present a hypothetical model, which not only describes the assembly of the FA pathway, but also positions this pathway in the broader context of DNA cross-link repair. Finally, the possible role for the FA pathway, in particular FANCF and FANCB, in the origin of sporadic cancer is discussed. PMID- 16675879 TI - The CT-element of the c-myc gene does not predispose to chromosomal breakpoints in Burkitt's lymphoma. AB - BACKGROUND: Chromosomal translocations are causally related to the development of many tumors. In Burkitt's lymphoma, abnormalities involving the c-myc gene are essential. The CT-element of the c-myc promoter adopts non-B-conformation in vivo and in vitro, and therefore provides a potential fragile site. METHODS: We have developed a LM-PCR-based approach to test if chromosomal breakpoints indeed cluster in this region. RESULTS: Amplifying both, wild-type as well as the translocated c-myc gene by LM-PCR, it was shown that chromosomal breakpoints did not cluster within the CT-element. CONCLUSIONS: Therefore, the CT-element is not especially susceptible to the formation of breakpoints leading to chromosomal translocations in Burkitt's lymphoma. PMID- 16675880 TI - Flow cytometry of the Side Population: tips & tricks. AB - BACKGROUND: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. METHODS: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. RESULTS: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red's (because of the weak emission). CONCLUSIONS: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air) and beam shaping optics. PMID- 16675881 TI - The fractal dimension of nuclear chromatin as a prognostic factor in acute precursor B lymphoblastic leukemia. AB - The fractal nature of the DNA arrangement has been postulated to be a common feature of all cell nuclei. We investigated the prognostic importance of the fractal dimension (FD) of chromatin in blasts of patients with acute precursor B lymphoblastic leukemia (B-ALL). In 28 patients, gray scale transformed pseudo-3D images of 100 nuclei (May-Grunwald-Giemsa stained bone marrow smears) were analyzed. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. Whereas FD presented no prognostic relevance, patients with higher R2 values showed a prolonged survival. White blood cell count (WBC), age and mean fluorescence intensity of CD45 (MFICD45) were all unfavorable prognostic factors in univariate analyses. In a multivariate Cox-regression, R2, WBC, and MFICD45, entered the final model, which showed to be stable in a bootstrap resampling study. Blasts with lower R2 values, equivalent to accentuated "coarseness" of the chromatin pattern, which may reflect profound changes of the DNA methylation, indicated a poor prognosis. In conclusion the goodness-of-fit of the Minkowski Bouligand dimension of chromatin can be regarded as a new and biologically relevant prognostic factor for patients with B-ALL. PMID- 16675883 TI - Quantitative histopathological analysis of CIN sections. PMID- 16675885 TI - The search for human osteoporosis genes. AB - Osteoporosis is the most prevalent metabolic bone disease and a major clinical and public health problem. Heredity plays an important and well-established role in determining the lifetime risk of this disease. Major efforts are currently underway to identify the specific genes and their allelic variations that contribute to the heritable component to osteoporosis. A number of laboratories are using quantitative trait locus (QTL) methods of genome scanning in families and animal models to identify candidate genomic regions and, ultimately, the genes and genetic variations that lead to osteoporosis. Several chromosomal regions of the human genome have now been linked to osteoporosis-related phenotypes. Although the specific genes contributing to the majority of these linkage signals have not been identified, two positional candidate genes have now been identified: low density lipoprotein receptor-related protein 5 (LRP5) and bone morphogenetic protein 2 (BMP2). A number of QTL has also been identified by cross-breeding strains of mice with variable bone density and several of these QTL have been fine mapped, providing a rich new base for understanding osteoporosis. Genetic association analyses have also provided evidence for a modest relationship between allelic variants in several biological candidate genes and bone mass and the risk of fracture. These ongoing animal and human studies will provide a continuing source of new insight into the genetic regulation of bone and mineral metabolism and the molecular etiology of osteoporosis. The new insight that will emerge from this ongoing research should lead to new ways of diagnosing, preventing and treating the growing clinical and public health problem of osteoporosis. PMID- 16675886 TI - Identifying genetic risk factors for osteoporosis. AB - Over the past decades epidemiological research of so-called "complex" diseases, i.e., common age-related disorders such as cancer, cardiovascular disease, diabetes, and osteoporosis, has identified anthropometric, behavioural, and serum parameters as risk factors. Recently, genetic polymorphisms have gained considerable interest, propelled by the Human Genome Project and its sequela that have identified most genes and uncovered a plethora of polymorphic variants, some of which embody the genetic risk factors. In all fields of complex disease genetics (including osteoporosis) progress in identifying these genetic factors has been hampered by often controversial results. Because of the small effect size for each individual risk polymorphism, this is mostly due to low statistical power and limitations of analytical methods. Genome-wide scanning approaches can be used to find the responsible genes. It is by now clear that linkage analysis is not suitable for this, but genome-wide association analysis has much better possibilities, as is illustrated by successful identification of risk alleles for several complex diseases. Candidate gene association analysis followed by replication and prospective multi-centred meta-analysis, is currently the best way forward to identify genetic markers for complex traits, such as osteoporosis. To accomplish this, we need large (global) collaborative studies using standardized methodology and definitions, to quantify by meta-analysis the subtle effects of the responsible gene variants. PMID- 16675887 TI - Recent advances in the genetics of osteoporosis. AB - It has been known for over 20 years that osteoporosis is highly influenced by genetic factors. Bone mineral density (BMD) has also been shown to be highly heritable. Other known risk factors for osteoporotic fractures such as reduced bone quality, femoral neck geometry and bone turnover are now also known to be heritable. Susceptibility to osteoporosis is mediated, in all likelihood, by multiple genes each having small effect. Different approaches are being used currently to identify the many genes responsible. These include linkage studies in man and experimental animals as well as candidate gene studies and alterations in gene expression. Linkage studies have identified multiple quantitative trait loci (QTL) for regulation of BMD and, with twin studies, have indicated that the effects of these loci are partly site-dependent and sex-specific. On the whole, the genes responsible for BMD regulation at these QTL have not yet been isolated. Most studies have used the candidate gene approach. The vitamin D receptor gene (VDR), the collagen type I alpha 1 gene (COLIA1) and estrogen receptor gene (ER) alpha have been most widely investigated and found to play a role in regulating BMD, but the effects are modest and together probably account for less than 5% of the heritable contribution to BMD. Genes may vary in their influence of particular intermediate phenotypes, and we now know that not all genes influencing BMD will be important in fracture. In addition, the study of other diseases such as osteoarthritis and metabolic bone syndromes may prove fruitful in highlighting genes which overlap to osteoporosis as well. As large scale genetic testing becomes more cost-effective, recent findings have illustrated the potential of novel approaches. These include combining large multi-national populations for candidate gene analysis, meta-analyses, DNA pooling studies and gene expression studies. PMID- 16675888 TI - Ethnic difference in osteoporosis-related phenotypes and its potential underlying genetic determination. AB - Osteoporosis is a serious health problem in both Caucasians and Asians. Caucasians and Asians are two distinct major ethnic groups, which may have differential genetic determination underlying complex genetic diseases such as osteoporosis. However, to date, there has been no systematic review focusing on the aspect of ethnic difference in risk to osteoporosis and its potential underlying genetic determination between Asians and Caucasians. Here, we firstly review diverse aspects of osteoporosis-related differences, including the differences of epidemiology of osteoporotic fractures, peak bone mass, bone loss, bone area, bone geometry and drug treatment response between Asians and Caucasians. Then, we provide some potential genetic evidence on the different heritability and inheritance mode of bone phenotypes, the different osteoporosis candidate genes and the differential results in related molecular studies between them, to explain the above osteoporosis-related phenotypic differences. The results suggest that the osteoporosis-related phenotypic differences between Asians and Caucasians may be partially the result of the different ethnic genetic background. The present review may increase our understanding of potential different mechanisms related to ethnicity in pathogenesis of osteoporosis for effective and potentially customized treatments in different major ethnic groups. PMID- 16675889 TI - Quantitative genetics of circulating molecules associated with bone metabolism: a review. AB - This paper reviews recent advances in the studies of various biochemical factors (biomarkers) involved in bone metabolism and remodeling. The collected data in this area suggest the existence of complex and multilevel relationships between calciotropic hormones, various cytokines and growth factors. The paper summarizes the data on the magnitude of the familial and genetic effects on the interindividual variation in circulating levels of many of these biomarkers. The majority of the cited heritability estimates are well above 20%, reaching up to 80% for some cytokines (e.g., TNFalpha and VEGF). These estimates point to potential targets for the identification of novel quantitative trait loci involved in the control of the respective molecules variation. This information is of particular importance, because the available data on the association between specific genes/polymorphisms and the respective circulating molecules variation is still very limited. The paper also provides recent findings on the genetics of co-variation between the circulating levels of various biomarkers. It shows that only in a few instances, such as for example, between IGF-I and IGFBP 3, and IGFBP-1 and leptin, significant and substantial genetic (and environmental) correlations were found. It appears that despite the prominent strong genetic effects on variation of each of the numerous biomarkers, the pleiotropic effects are rather limited. We consider briefly some important new data obtained using the gene expression approach and microarray technique. The data, for instance, indicate that the genetic effects on bone metabolism appear to be an open system, which can be activated or modulated by external factors such as drugs, e.g., PTH. Extensive molecular genetic studies in this area are both timely and imperative to detect the specific genes affecting variation (and co-variation) of the circulating factors associated with bone metabolism. PMID- 16675890 TI - Pharmacogenomics of osteoporosis: opportunities and challenges. AB - The genetics of osteoporosis can be considered in two broad areas: disease susceptibility and drug activity. While the former has been studied, the latter is still largely untouched. Pharmacogenomics is the utilization of genetic information to predict outcome of drug treatment, with respect to both beneficial and adverse effects. The pharmacotherapy of osteoporosis is characterized by variability in therapeutic response with limited prediction of response on a patient-by-patient basis. This is particularly problematic in a clinical situation where therapy is typically required for several years before outcomes can be evaluated for an individual. Thus, the emerging field of pharmacogenomics holds great potential for refining and optimising pharmacological treatment of osteoporosis. Key components for future development of the pharmacogenomics of osteoporosis should include improved understanding of mechanisms of drug action, identification of candidate genes and their variants and expansion of clinical trials to include genetic profiling. This approach could provide clinicians and scientists with powerful tools to dissect novel molecular pathways involved in osteoporosis and to identify new drug targets. The iterative combination of innovative genomics with classical endocrinological approaches in osteoporosis research can be examined as a model of biological research and innovate therapeutical approaches in a continuing interaction between clinical science and basic research. PMID- 16675891 TI - Adaptive processes in skeletal muscle: molecular regulators and genetic influences. AB - Skeletal muscle is a highly adaptable tissue. It responds to environmental and physiological challenges by changes in size, fibre type and metabolism. All of these responses are underpinned by our genes and it is therefore generally assumed that genetic variation between individuals may account for the differences in musculature and athletic capabilities between people. Research into the genetic influences of our muscle is at an embryonic stage, but some early insight into potential regulators has recently emerged, which is reflected in this review. Broad heritability, which appears to affect muscle size and strength more than metabolism has been assessed in twin and sibling studies. It appears to account for more inter-individual variation in the young as opposed to older people. However, the studies reported to date do demonstrate a large degree of diversity, which is probably predominantly due to different methodological approaches being adopted as well as distinct populations being studied. At a molecular level, there has been enormous progress in identifying regulators of atrophy and hypertrophy though the study of knock-out and transgenic animals and also through the utilisation of cell culture models. Among others, the insulin like growth factors, calcineurin, desmin, myf5, mrf4, MyoD and myogenin have been identified as positive regulators of muscle size, while TNF-alpha, myostatin and components of the ubiquitin pathway have been recognized as regulators of muscle wasting. However, given the ethical and mechanistic constraints of performing similar studies in humans, difficulties have arisen when attempting to translate the animal and cell culture findings to humans. However, the current search for target "exercise genes" in humans has yielded the first successful results. Variations in the genes encoding for: the angiotensin converting enzyme, alpha actinin 3, bradykinin, ciliary neurotrophic factor, interleukin-15, insulin-like growth factor II, myostatin and the vitamin D-receptor have all been found to account for some of the inter-subject variability in muscle strength or size. However, the influences of these genetic variations are somewhat weak, and not always reproducible and furthermore they are predominantly based in young healthy people. Hence, a key topic, namely the molecular mechanisms of muscle frailty in the elderly still remains to be elucidated. PMID- 16675892 TI - Deletions of genes encoding calcitonin/alpha-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone. AB - It has been suggested that skeletal nerves fibers may play important roles in neuro-osteogenic interactions. This view is partly based upon information obtained from immunohistochemical studies, chemical and surgical denervation experiments and clinical observations in patients with stroke and spinal cord injury, indicating the presence of a network of nerve fibers in the skeleton and that defective signalling in skeletal nerve fibers affects remodelling of bone. This view is also supported by data showing that functional receptors for signalling molecules in skeletal nerve fibers are expressed in bone cells and that activation of these receptors leads to profound effects on bone forming osteoblasts and bone resorbing osteoclasts. Convincing evidence for a role of neuronal signalling in bone metabolism has been provided by gene deletion approaches in which it has been shown that leptin-sensitive and neuropeptide Y sensitive receptors in hypothalamus are important for bone remodelling in mice. Recently, gene deletion experiments have shown that calcitonin gene-related peptide (CGRP), one of the neuropeptides present in skeletal nerve fibers, is an important physiological regulator of bone formation at the level of osteoblast activity. CGRP belongs to the calcitonin (CT) family of peptides also including CT, amylin and adrenomedullin, as well as the recently described intermedin and calcitonin receptor-stimulating peptide. These peptides utilize two seven transmembrane G protein-coupled receptors - the calcitonin receptor (CTR) and the calcitonin receptor- like receptor (CRLR) - which can dimerize with three different single transmembrane proteins, making up the RAMP family. Associations between RAMPs and either CTR or CRLR give rise to seven distinct, molecularly characterized, receptors for CT, CGRP, amylin and adrenomedullin. Deletions of the genes for ligands in the CT family of peptides and for one of the receptors have revealed unexpected findings that have changed our view on the role of these peptides in bone remodelling. It was anticipated that deletions of the CT/alpha CGRP and CTR genes would lead to bone loss, since CT has been shown to inhibit bone resorption in vitro and in vivo and has been used to treat patients with excessive bone resorption. Surprisingly, it was found that CT/alpha-CGRP-/- and CTR+/- mice have increased bone mass due to increased bone formation. Mice with deletion of the amylin gene, however, exhibited bone loss due to enhanced bone resorption. Selective deletion of the alpha-CGRP gene also leads to bone loss, but due to decreased bone formation. Thus, our understanding of the role of the CT family of peptides has been changed dramatically and much more data have to be gained before we fully understand the roles these peptides have in bone biology. PMID- 16675893 TI - Expression of matrix metalloproteinase 2 and heat shock protein-72 in immobilized muscle in rats. AB - Metalloproteinases (MMPs) are proteolytic enzymes that function in the extracellular matrix to degrade connective tissues. While it is clear that certain induced skeletal muscle pathologies promote increased expression of MMP-2 and heat shock protein- 72 (HSP-72), the relationship between muscle disuse and expression of MMP-2 and HSP-72 in muscles is unknown. These experiments tested the hypothesis that knee immobilization induced expression of MMP-2 and HSP-72 is disuse-dependent in a way that short-term joint immobilization increases HSP-72 expression, whereas long-term joint immobilization increases MMP-2 expression in skeletal muscles. Male rats (8 months old) completed 1, 2, 3, and 4 weeks of knee joint immobilization. Muscle mRNA and protein levels of MMP-2 and HSP-72 were assessed in Gastrocnemius (Gast), Superficial and Deep Quadriceps, and Soleus (Sol) muscles by reverse transcriptase-polymerase chain reaction and western blotting, respectively. Results reveal that during the first two weeks of immobilization there is increased protein levels of HSP-72 and expression of mRNA of HSP-72 mainly in slow twitch muscle fibers. However, 3 and 4 weeks of joint immobilization increased both mRNA and protein levels of MMP-2 in skeletal muscles containing a high percentage of fast type II fibers (i.e., Gast and superficial quadriceps). These results support the hypothesis that different periods of muscle disuse induced different proteins expression, and that the influence of joint immobilization on the expression of HSP-72 in the short-term, and MMP-2 in the long ran is associated to fiber types. PMID- 16675894 TI - Yield of sputum microbiological examination in patients hospitalized for exacerbations of chronic obstructive pulmonary disease with purulent sputum. AB - BACKGROUND: Whether sputum microbiological examination should be performed systematically in hospitalized patients with chronic obstructive pulmonary disease (COPD) exacerbations remains unclear. OBJECTIVES: To assess the yield of sputum microbiological examination in COPD patients hospitalized in a medical ward for an acute exacerbation with purulent sputum. METHODS: Two hundred consecutive exacerbations in 118 patients were studied. Patients underwent sputum microbiological examination on admission and baseline lung function tests and CT scans were recorded. Factors associated with positive culture were analyzed. RESULTS: Sputum culture was positive (>or=10(7) CFU/ml) in 59% of samples, Haemophilus influenzae and Streptococcus pneumoniae being the most frequent pathogens. Factors associated with positive culture were bronchiectasis, long term oxygen therapy and low FEV1. Pseudomonas spp. were found in 8.5% of all patients, who all had a FEV1<50% of predicted and were older. Only 25% of sputum samples satisfied all quality criteria. Sputum culture was positive in a high proportion of these samples (80.5%), but also in one half of samples with >25 leukocytes but >10 epithelial cells per field. Microbiological results induced a change in antibiotic therapy in 43.9% of cases with both quality criteria but also in 25.2% of cases with only one quality criterion. Finally, a predominant aspect after Gram stain was found in all positive samples. CONCLUSIONS: These data suggest that sputum microbiological examination with direct examination and leukocyte count should be performed routinely in patients hospitalized for COPD exacerbations with purulent sputum, especially when FEV1 is less than 50% predicted and in patients with bronchiectasis. PMID- 16675895 TI - Usefulness of preoperative endobronchial ultrasound for airway invasion around the trachea: esophageal cancer and thyroid cancer. AB - BACKGROUND: It is important to confirm preoperative tracheobronchial invasion to enable the selection of the most appropriate treatment. OBJECTIVE: This study was performed to compare the usefulness of computed tomography (CT), magnetic resonance image (MRI) and bronchoscopy by endobronchial ultrasonography (EBUS) for the assessment of invasion of thyroid or esophageal cancer in cases with suspected tracheobronchial invasion. METHODS: In cases with suspected contact between the tumor and tracheobronchial wall, CT, MRI and EBUS indicated deformity of the tracheobronchial wall due to the adjacent mass. The final diagnosis was based on surgical and histological results, and/or clinical follow-up. RESULTS: Fifty-four patients were included in this study. Based on the findings of CT, MRI and EBUS, invasion was suspected in 29, 28 and 25 patients, respectively. Seventeen patients did not undergo surgery based on the results of CT, MRI and bronchoscopy with EBUS. Final diagnosis was intact trachea or bronchial adventitia in 26 patients and invasion in 28 patients. The sensitivity and specificity of CT, MRI and EBUS for invasion were 59 and 56, 75 and 73, and 92 and 83%, respectively. The accuracy of EBUS was significantly greater than that of CT in the present study (p = 0.0011). The accuracy of EBUS was significantly different from that of CT and MRI in the surgically treated patients (p = 0.005 and p = 0.032, respectively). CONCLUSION: EBUS is the most useful technique for determining the depth and extent of tumor invasion into the airway wall. The combination of MRI and EBUS will contribute to surgical planning in patients with esophageal and thyroid cancer. PMID- 16675896 TI - Thoracic aortic aneurysm presenting with haemoptysis, weight loss, night sweats, and hilar mass. PMID- 16675897 TI - Association of advanced glycoxidation end products and inflammation markers with thrombosis of arteriovenous grafts in hemodialysis patients. AB - BACKGROUND/AIMS: Intimal hyperplasia at the venous anastomosis of arteriovenous dialysis grafts (AVGs) is a common and costly complication in hemodialysis (HD) patients. Previous studies have shown significant accumulation of advanced glycation end products (AGEs) within the lesions, suggesting a pathogenic role for these compounds in this condition. METHODS: We retrospectively analyzed data from 139 HD patients, including 58 subjects with AVGs, to determine any relationship between serum AGEs and other circulating markers, e.g. C-reactive protein (CRP), tumor necrosis factor (TNF-alpha), vascular cell adhesion molecule 1 (VCAM-1), plasminogen activator inhibitor type 1 and vascular endothelial growth factor, with the presence of graft thrombosis, an index of venous intimal hyperplasia. RESULTS: Patients with previously thrombosed AVGs exhibited significantly higher levels of serum AGEs (42 +/- 18 vs. 28+/- 8 U/ml), CRP (1.4 +/- 1.1 vs. 0.6 +/- 0.4 mg/dl) and VCAM-1 (3,172 +/- 696 vs. 2,447 +/- 1,101 ng/ml) than those with uncomplicated AVGs, variances that were mostly attributed to diabetes difference. There was no difference regarding the levels of the parameters studied between patients with AVGs and other forms of vascular access. CONCLUSIONS: These results support an association between circulating AGEs, markers of inflammation and endothelial function and intimal hyperplasia at the venous anastomosis of AVGs in HD patients. The implication is that there may be a therapeutic role for anti-AGE interventions in the management of this common clinical condition. PMID- 16675898 TI - On the relationship between speech- and nonspeech-evoked auditory brainstem responses. AB - Auditory brainstem response (ABR) reflects activation of the neural generators along the ascending auditory pathway when a sound is heard. In this study, we explored the relationship between brainstem encoding of click and speech signals in normal-learning children and in those with language-based learning problems. To that end, ABR was recorded from both types of stimuli. We found that the normal pattern of correlation between click- and speech-evoked ABRs was disrupted when speech-evoked ABRs were delayed. Thus, delayed responses to speech were not indicative of clinically abnormal responses to clicks. We conclude that these two responses reflect largely separate neural processes and that only processes involved in encoding complex signals such as speech are impaired in children with learning problems. PMID- 16675899 TI - Photoperiod-independent changes in immunoreactive brain gonadotropin-releasing hormone (GnRH) in a free-living, tropical bird. AB - Timing of seasonal reproduction in high latitude vertebrates is generally regulated by photoperiodic cues. Increasing day length in the spring is associated with changes in the brain that are responsible for mediating reproductive activities. A primary example of this is the increased content of gonadotropin-releasing hormone (GnRH) in the preoptic area of the hypothalamus in birds as they enter the spring breeding season. Increased GnRH activity stimulates the release of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary. These gonadotropins induce growth of the gonads and release of sex steroids which act on the brain to mediate reproductive behaviors. By contrast, seasonal breeding in the tropics can occur in the absence of significant changes in photoperiod. To our knowledge, no studies have investigated whether seasonal breeding in free-living tropical vertebrates is associated with seasonal changes in the GnRH system. We studied two populations of rufous-collared sparrows (Zonotrichia capensis) at the equator, separated by only 25 km, but with asynchronous reproductive phenologies associated with local climate and independent of photoperiodic cues. We collected brains and measured GnRH immunoreactivity (GnRH-ir) during each population's breeding and non breeding periods. Breeding males had larger, but not more, GnRH-ir cells than non breeding birds. The plasticity of the GnRH system was associated with local climate, such that the two populations exhibited asynchronous changes in GnRH-ir despite experiencing identical photoperiod conditions. Our results demonstrate that tropical birds can exhibit neural changes similar to those exhibited in higher latitude birds. However, these tropical populations appear to be using supplementary cues (e.g., rainfall, temperature, food availability) in a similar way to higher latitude species using an initial predictive cue (photoperiod). These results raise questions about the evolution of reliance upon photoperiodism and the strength of reproductive responses to other environmental cues in congeners from higher latitudes. The ability to respond to a multitude of environmental cues might be part of the ancestral condition, and the subsequent obligate photoperiodism in high-latitude congeners could reflect a loss of flexibility in response to environmental signals. PMID- 16675900 TI - Highly active antiretroviral therapy-associated metabolic syndrome and cardiovascular risk. AB - The introduction of highly active antiretroviral therapy (HAART) has significantly modified the course of human immunodeficiency virus (HIV) disease, with longer survival and improved quality of life of HIV-infected subjects. However, HAART regimens, especially those including protease inhibitors, have been shown to cause in a high proportion of HIV-infected patients a metabolic syndrome (lipodystrophy/lipoatrophy, dyslipidemia, type 2 diabetes mellitus, insulin resistance) that may be associated with an increased risk of cardiovascular disease (coronary artery disease and stroke). A careful stratification of the cardiovascular risk and cardiovascular monitoring of patients under HAART is needed according to the most recent clinical guidelines. PMID- 16675901 TI - Comparison of the manual Mycobacteria Growth Indicator tube and the Etest with the method of proportion for susceptibility testing of Mycobacterium tuberculosis. AB - BACKGROUND: Clinical microbiology laboratories should provide reliable results on susceptibility testing of Mycobacterium tuberculosis to different agents. METHODS: The manual Mycobacteria Growth Indicator Tube (MGIT) and Etest were compared to the method of proportion (MOP) for susceptibility testing of 88 clinical isolates of M. tuberculosis against isoniazid (INH), rifampin (RIF), streptomycin (STR) and ethambutol (EMB). Isolates were recovered from different patients and were identified at species level by PCR and hybridization. RESULTS: Resistance to INH was detected in 20.5, 29.5 and 12.5% of the isolates, followed by STR resistance (19.3, 26.1 and 1.1%), RIF (9.1, 4.5 and 5.7%) and EMB (2.3, 11.4 and 2.3%) by the MOP, MGIT and Etest, respectively. Sensitivity of the manual MGIT ranged from 37.5% for RIF resistance to 100% for EMB, while Etest sensitivity ranged from 5.9% for STR to 62.5% for RIF. CONCLUSIONS: MOP remains the method of choice, with the manual MGIT showing superior sensitivity at detecting resistance to INH, STR and EMB compared to the Etest. PMID- 16675902 TI - Prospective study of empiric monotherapy with ceftazidime for low-risk grade IV febrile neutropenia after cytotoxic chemotherapy in cancer patients. AB - PURPOSE: It was the aim of this study to evaluate the results of a prospective study in a single medical center using ceftazidime monotherapy in cancer patients with chemotherapy-induced grade IV febrile neutropenia and a low risk for gram negative bacteremia. SUBJECTS AND METHODS: Thirty-eight patients were admitted with low-risk grade IV febrile neutropenia after chemotherapy for solid tumors. The median patient age was 57 years (range 18-74). Sixteen patients (42%) developed febrile neutropenia after the first cycle of current chemotherapy line, 9 patients (24%) received 2-3 cycles and 13 patients (34%) received more than 3 chemotherapy cycles before manifesting febrile neutropenia. Five patients were treated with prophylactic granulocyte colony-stimulating factor commenced 24 h after completion of the chemotherapy cycle. Empiric monotherapy with intravenous ceftazidime was started on admission and administered 2 g every 8 h. RESULTS: The mean polymorphic nuclear cell count on admission was 231 cells/mm(3). Ceftazidime therapy was well tolerated. Twenty-five (66%) patients responded with clinical improvement and complete resolution of fever within 48 h after initiation of ceftazidime therapy. Thirty-two (84%) patients were afebrile after 72 h of therapy. Thirty-three patients (87%) remained on unmodified ceftazidime therapy throughout their hospitalization. Five patients (13%) subsequently required modification of the treatment regimen for various reasons. Mean duration of fever and neutropenia were 2 (1-10) days and 4 (1-11) days, respectively. None of the patients discontinued therapy because of adverse effects. No positive blood cultures were obtained. No events of septic shock were observed. Mean duration of hospitalization was 6 days (range 3-12). CONCLUSION: In our series, monotherapy with intravenous ceftazidime appears safe and effective in cancer patients with low-risk grade IV febrile neutropenia after cytotoxic chemotherapy and may appreciably reduce antibiotics costs. PMID- 16675903 TI - Potential new antimalarial chemotherapeutics based on sphingolipid metabolism. AB - The discovery of new antimalarial drugs is mandatory to improve the effectiveness of antimalarial prophylaxis and treatment. In this review, we focused on sphingolipids as potential new targets for antimalarial drugs. Inhibition of sphingomyelin and/or glucosylceramide synthases leads to increased intracellular concentrations of ceramide and results in growth inhibition of Plasmodium falciparum. In mammalian cells, ceramide mediates death by chemotherapeutic drugs. We demonstrated that ceramide mediates the antimalarial effect of artemisinin and mefloquine by depletion of glutathione levels. Furthermore, ceramide and artemisinin activated p38 mitogen-activated protein kinase in P. falciparum, thus inhibiting its growth, apparently by a non-apoptotic mechanism. In summary, we propose novel options of antimalarials based on ceramide cytotoxic activity. PMID- 16675904 TI - Decreased phosphoactive ERKs and JNKs in Malachite-green-transformed Syrian hamster embryo fibroblasts are associated with increased phosphoactive p38 kinase: possible therapeutic importance. AB - BACKGROUND: Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure of human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. METHODS: Cell transformation assays were carried out as described in the literature. Western blotting and flow cytometry were carried out by standard methods. RESULTS: In this study, we have studied the role of all three isoforms of mitogen-activated protein (MAP) kinases, i.e. extracellular regulated kinases (ERKs), Jun N-terminal kinases (JNKs) and p38 kinase in the MG-transformed SHE fibroblasts compared to controls. Our results showed that transformed cells were associated with decreased expression of ERKs and JNKs as evidenced by Western blotting studies. However, the p38 MAP kinase was found to be upregulated. Flow cytometric DNA histogram analysis indicated an increase in the expression of S phase cells in the transformed cell line as compared to their control counterparts. CONCLUSIONS: The present studies indicate that decreased phosphoactive ERKs and JNKs and increased phosphoactive p38 kinase are associated with increased S phase cells during transformation of SHE cells by MG. PMID- 16675905 TI - Temocapril, an Angiotensin converting enzyme inhibitor, ameliorates age-related increase in carotid arterial stiffness in normotensive subjects. AB - BACKGROUND/AIMS: Oxidative stress plays an important role in vascular ageing. The close relationship of the renin-angiotensin system with mechanisms of oxidative stress suggests its possible involvement in the deleterious effects of ageing. The present study was designed to investigate whether inhibition of the renin angiotensin system improves the age-related increase in arterial stiffness. METHODS: Normotensive elderly subjects in a geriatric nursing home (n = 24; male/female = 7/17; 86 +/- 9 years, mean +/- SD) were randomly assigned to receive either an angiotensin converting enzyme inhibitor, temocapril (n = 12, 1 mg daily) or nothing (n = 12). Carot id arterial stiffness parameter beta and circulating levels of malondialdehyde-modified low-density lipoprotein were measured before and 6 months after randomization. RESULTS: Temocapril decreased beta (from 7.0 +/- 1.0 to 4.9 +/- 0.9, p < 0.05) and malondialdehyde-modified low density lipoprotein levels (from 73.2 +/- 19.9 to 61.3 +/- 18.4 U/l, p < 0.05) without changing the blood pressure. The changes in beta and malondialdehyde modified low-density lipoprotein levels were significantly correlated (rho = 0.600, p < 0.05). CONCLUSION: These results suggest that temocapril ameliorates the age-related increase in carotid arterial stiffness in healthy, normotensive elderly subjects. PMID- 16675906 TI - Mianserin and ventricular tachycardia: case report and review of the literature. AB - Mianserin is a drug frequently used to treat depression and sleep disturbances. Despite documented effects on various cardiac tissues in animal studies, mianserin has a very safe clinical profile. Only one case of ventricular arrhythmias in a patient treated with mianserin has been reported. This patient had a severe cardiac history. Our case is to our knowledge the first report on ventricular arrhythmias in a patient treated with mianserin without previous or present cardiac disease. After discontinuation of mianserin the arrhythmias disappeared within days. The literature on mianserin toxicity is reviewed. PMID- 16675907 TI - Transanal endoscopic microsurgery: where are we now? AB - AIMS: This review of literature aimed to assess the role and establish the current status of transanal endoscopic microsurgery (TEM) in the management of benign and malignant rectal lesions. METHODS: A review of the literature was undertaken through the Medline database and by cross-referencing previous publications, thus identifying 54 relevant publications on TEM in the management of rectal lesions. Aggregated results of various parameters were calculated but statistical comparisons deemed unsuitable due to heterogeneity of data. RESULTS: The TEM procedure is associated with good functional results, morbidity of 4% and zero procedure-related mortality. The local recurrence rates after TEM excision is 4.5% (range 0-14) for benign rectal lesions, 6% (0-13) for T(1) cancers, 14% (range 0-50) for T(2) cancers and 20% (range 14-67%) for T(3) cancers. Local recurrences after TEM can be surgically salvaged with good disease free survival rates. CONCLUSIONS: The TEM procedure clearly offers the benefits of good exposure of the operative field allowing extremely precise dissection and access to high rectal lesions unresectable by other methods. For pTis and low risk pT(1) lesions, the oncological results are comparable to the more traditional formal resection. The routine use of TEM for high-risk pT(1) and higher stage lesions is not an oncologically sound choice at the present moment. PMID- 16675908 TI - The prevalence of endometriosis in women with chronic pelvic pain. AB - BACKGROUND: The 2004 American College of Obstetrics and Gynecology clinical management guideline states that the prevalence of endometriosis is approximately 33% in women with chronic pelvic pain (CPP). This estimate came from a review showing that 28% of adult women with CPP were found to have endometriosis. The prevalence of 28% in adult women was arrived based on a compilation of 11 published studies. Yet even within the 11 studies, the reported prevalence of endometriosis varies wildly, ranging from 2 to 74%. Such an astounding variation or heterogeneity raises the question whether it is appropriate to use a single prevalence of endometriosis for all women with CPP. METHODS: We sought to identify possible sources of heterogeneities in the estimation of prevalence of endometriosis in women with CPP. We included more studies that reported prevalence estimates than the review, and examined the effect of sample size and the year of publication on the heterogeneity. RESULTS: The year of publication is positively associated with the prevalence estimate, which may indicate an increasing awareness of various appearances of endometriosis, or the prevalence of endometriosis may have increased among women with CPP. An alternative analysis with removal of four studies reporting highest prevalence estimates indicated that sample size is negatively associated with the prevalence estimates while the year of publication became only marginally significant. CONCLUSIONS: There are identifiable sources of heterogeneity in prevalence estimates, with the year of publication, sample size, and difference in evaluation of CPP being three apparent sources. Having a single prevalence estimate for all women with CPP may be too simplistic at best. The true prevalence is very likely to be higher than 33%. PMID- 16675909 TI - Obstetric uterine rupture of the unscarred uterus: a twenty-year clinical analysis. AB - BACKGROUND: Rupture of the unscarred uterus is a rare and potentially catastrophic event. We retrospectively reviewed the records of patients with this condition to analyze their obstetric and gynecologic history and evaluate maternal and perinatal morbidity and mortality. METHODS: A total of 11 cases of rupture of the unscarred gravid uterus were managed at Mackay Memorial Hospital from January 1984 to September 2003. Data extracted from the records included the use of uterine stimulants, instrumental delivery, and prior abortion by instrumentation, clinical features, treatment, and maternal and fetal morbidity and mortality. RESULTS: The incidence of unscarred uterine rupture is 0.009% during the 20-year study period. The most common contributing factors were prior abortion by instrumentation and the use of uterotonic agents, in three cases respectively. Fetal distress occurred in six cases and postpartum hemorrhage in two. There was no maternal death, but in two cases, there was intrauterine fetal demise or perinatal death. CONCLUSION: Though unexpected in a woman with an unscarred uterus, rupture should be considered as a possible cause of fetal distress or unusual pain or hypotension in the mother. PMID- 16675910 TI - Validation of a calibrated prediction model for response to growth hormone treatment in an independent cohort. AB - BACKGROUND: Prediction models, e.g. for prediction of response to growth hormone treatment, need validation in appropriate independent cohorts, comparing predicted and observed outcomes. In a previous validation of a model for predicting the first-year response to growth hormone treatment in children with idiopathic growth hormone deficiency, overfitting was observed. We modified the prediction formula and now report validation of this modified model. PATIENTS AND METHODS: The modified and original prediction models were applied to a group of patients selected from Lilly's GeNeSIS database using the same inclusion and exclusion criteria as for the original model. For both prediction methods, observed first-year height velocity was plotted vs. predicted height velocity in a calibration plot. For a valid prediction, the regression line should correspond to the line of identity (observed outcome is equal to predicted outcome); the regression lines for each prediction model were tested for significant differences from this line of identity. RESULTS: The number of patients fulfilling the criteria was 226. The regression line in the calibration plot of the modified model was not significantly different from the line of identity (p = 0.43), in contrast to the original model (p < 0.001). For the modified model the mean (SD) prediction error was -0.11 (2.05) cm/year and for the original model 0.28 (2.11) cm/year. CONCLUSION: The modified prediction method, obtained after calibration of the original model, performs well in an independent patient sample and gives more accurate predictions than the original model. PMID- 16675911 TI - Targeting the epidermal growth factor receptor in colorectal cancer: advances and controversies. AB - Colorectal cancer (CRC) is the second leading cause of cancer death in the western world. Even with the significant improvement in traditional chemotherapy, there remain limitations with this treatment. One of the most promising new targets in the treatment of CRC is the epithelial growth factor receptor (EGFR). Agents that inhibit the EGFR have demonstrated clinical activity as single agents and in combination with chemotherapy and the most promising of these agents is cetuximab, which blocks the binding of EGF and transforming growth factor-alpha (TGF-alpha) to EGFR. Thus, the finding that monoclonal antibodies against EGFR caused a response in patients, and reversed resistance to chemotherapy, was exciting news. However, expression of EGFR did not correlate with clinical benefit. Clearly, the search for markers of response to treatment against EGFR must go on. PMID- 16675912 TI - Germline mutations of the PTCH gene in families with odontogenic keratocysts and nevoid basal cell carcinoma syndrome. AB - BACKGROUND/AIMS: Odontogenic keratocysts (OKC) are aggressive lesions in the jaws, which can occur as isolated cases or in association with nevoid basal cell carcinoma syndrome (NBCCS). Mutations on PTCH gene have been identified in patients with NBCCS. It was hypothesized that PTCH mutations may be causative in isolated OKC. This study aims to investigate germline mutations of PTCH in families with OKC and NBCCS. METHODS: Three Chinese families with OKC and NBCCS were enrolled in the study. The diagnosis was based on examination and medical history. Mutation analysis was performed by amplifying all exons of PTCH and sequencing the products. RESULTS: One family with isolated OKC (family 1) and the other two families with NBCCS were diagnosed. Three novel germline mutations in PTCH were identified, including a missense mutation (p.S1089 > P) in family 1, a nonsense mutation (p.Q160X) in family 2 and a de novo mutation (c.768_777delGACAAACTTC) in family 3. CONCLUSIONS: It is proposed that isolated OKC can be inherited in an autosomal dominant mode. The results suggest that germline mutations on PTCH can cause isolated OKC, and that the PTCH gene responsible for NBCCS plays an important role in the formation of OKCs even when they are not syndrome-related. PMID- 16675913 TI - Expression of the antiapoptotic proteins clusterin and bcl-2 in laryngeal squamous cell carcinomas. AB - Bcl-2 and clusterin genes have been related to the inhibition of apoptosis, an event that plays a key role in malignant transformation and in invasive disease. In this work, we determine the significance of clusterin and bcl-2 expression in a large series of laryngeal carcinomas. We used immunohistochemical methods and in situ hybridization to examine the expression of these proteins. Nontumoral epithelial laryngeal tissues did not express clusterin and bcl-2 proteins. However, 9% (14 out of 154) and 25% of these tumors (39 of 154) had positive clusterin and bcl-2 staining, respectively. Clusterin expression was significantly related to the degree of local invasion and higher bcl-2 expression was found in these clusterin-positive tumors (p < 0.05). Bcl-2 expression was significantly correlated with supraglottic localization, nodal metastases, invasion in depth, and poorly differentiated tumors. However, by multivariate analysis, bcl-2 was shown to be an independent predictor of good prognosis in these tumors (OR = 0.12, 95% CI = 0.02-0.91). These findings indicate that clusterin and bcl-2 are upregulated in laryngeal carcinomas and their expression is related to the invasiveness of these tumors. PMID- 16675914 TI - Molecular markers for discrimination of benign and malignant follicular thyroid tumors. AB - OBJECTIVE: To identify molecular markers useful for the diagnostic discrimination of benign and malignant follicular thyroid tumors. METHODS: A panel of thyroid tumors was characterized with expression profiling using cDNA microarrays. A robust algorithm for gene selection was developed to identify molecular markers useful for the classification of heterogeneous tumor classes. The study included tumor tissue specimens from 10 patients with benign follicular adenomas and from 10 with malignant tumors. The malignant tumors mainly consisted of clinically relevant minimally invasive follicular carcinomas. The mRNA expression level of a candidate gene, FHL1, was evaluated in an independent series of 61 tumors. RESULTS: 22 gene expression markers were identified as differentially expressed. Several of the identified genes, for example DIO1, CITED1, CA12 and FN1, have previously been observed as differentially expressed in various thyroid tumors. FHL1 was significantly underexpressed in carcinomas compared to adenomas in the independent panel of tumors. The results indicate that a small number of genes can be useful to distinguish follicular adenomas from follicular carcinomas. CONCLUSIONS: Our findings clearly corroborate previous studies and identify novel candidate molecular markers. These genes have the potential for molecular classification of follicular thyroid tumors and for providing improved understanding of the molecular mechanisms involved in thyroid malignancies. PMID- 16675915 TI - Functional analysis of bladder cancer-related protein gene: a putative cervical cancer tumor suppressor gene in cervical carcinoma. AB - Our previous study has suggested thatthe bladder cancer-associated protein gene (BLCAP) was among the differentially expressed genes in cervical cancer. We confirm here that BLCAP is expressed in all noncancerous cervical tissues (10/10), but it is greatly lost in primary cervical cancer tissue (31/39). In order to further investigate the functional roles of BLCAP, we stably transfected BLCAP cDNA into HeLa cells. The HeLa cells expressing BLCAP show reduced cell growth and clone genicity compared to the vector-transfected cognate cells. BLCAP expression in HeLa cells leads to growth arrest and significantly enhanced apoptosis in vitro and reduced tumor formation in vivo. Thus, BLCAP might be a potential tumor suppressor gene in cervical carcinoma. PMID- 16675916 TI - Statins. Cholesterol lowering and beyond. PMID- 16675917 TI - Chemotherapy delivered into abdomen may extend ovarian cancer survival. PMID- 16675918 TI - Inherited gene affects tamoxifen's benefit in some breast cancers. PMID- 16675919 TI - Life after treatment. Meeting the emotional challenges of the cancer experience. PMID- 16675921 TI - Shingles. The return of the chickenpox virus. PMID- 16675922 TI - Mayo Clinic office visit. Melanoma. An interview with Mark Pittelkow, M.D. PMID- 16675924 TI - Now that I'm 65, can I stop getting Pap tests? PMID- 16675923 TI - Stretch those muscles. Improve your flexibility. PMID- 16675925 TI - One on one. Are over-the-counter teeth-whitening products safe? PMID- 16675931 TI - Short stature, obesity, and growth hormone deficiency in pseudohypoparathyroidism type 1a. AB - Albright hereditary osteodystrophy (AHO) is a genetic disorder caused by heterozygous inactivating mutations in GNAS, the gene that encodes the alpha chain of Gs (G alpha s). This syndrome is associated with short stature, obesity, brachydactyly, and subcutaneous ossifications. Patients with GNAS mutations on maternally-inherited alleles are resistant to multiple G-protein-coupled hormones, including parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), luteinizing hormone/follicle-stimulating hormone (LH/FSH), and glucagon. This variant of AHO, termed pseudohypoparathyroidism (PHP) type 1a, is due to tissue specific paternal imprinting of G alpha s. We investigated whether patients with PHP type 1a exhibited evidence of resistance to growth hormone releasing hormone (GHRH) (1), another hormone requiring G alpha s function. In addition, G alpha s transcripts are imprinted in the pituitary somatotrophs responsible for growth hormone (GH) secretion which could thereby influence GHRH-dependent stimulation of somatotrophs. We therefore hypothesized that patients with PHP type 1a may be GH deficient which could contribute to the obesity and short stature in this condition. We found that GH deficiency is common in PHP type 1a (69%) with a prevalence that is much greater than in the general population (0.03%). We propose that GH status be evaluated in all patients with this condition. Treatment with recombinant GH could lead to improvements in height in children, as well as other physical (eg, obesity, hyperlipidemia, osteoporosis, reduced renal function) and psychological (fatigue and diminished sense of well-being) parameters in GH-deficient PHP type 1a patients of all ages. PMID- 16675933 TI - Cortisol and growth hormone: clinical implications of a complex, dynamic relationship. AB - Children with Cushing syndrome (CS), whether CS is iatrogenic or caused by endocrine tumors, have short stature and suffer from excess adiposity and lower bone mineral density. These clinical observations are well entrenched in the minds of pediatricians and pediatric endocrinologists. It is then surprising when one discovers that patients with cortisol-secreting adrenal tumors actually do not have completely suppressed growth hormone levels unless they are obese. Adequate adrenocortical glucocorticoid secretion is necessary for normal function of the somatotropic axis, at least in rodents. Furthermore, glucocorticoids appear to play a regulatory role in skeletal bone accumulation that goes far beyond the well known suppressive effects of excess cortisol on osteoblastic function. To add to the complexity, endocrine and paracrine cortisol levels and actions depend much on the activity of metabolizing enzymes, such as 11-beta hydroxysteroid dehydrogenase type-1 and CYP3A. The text that follows reviews the complex and dynamic state of the relationship between two of the most important hormones that regulate growth and development in childhood as revealed from clinical, physiologic, and basic science studies. The message is clear: although excess cortisol inhibits growth and the skeleton, normal cortisol levels are part of the mix that ensures both normal growth hormone secretion and bone accumulation in childhood. PMID- 16675935 TI - Molecular biology on the ICU. From understanding to treating sepsis. AB - Mounting evidence suggests that beside well established factors, such as virulence of pathogens or site of infection, individual differences in disease manifestation are a result of the genetic predisposition of the patient on an Intensive Care Unit (ICU). Specific genetic factors might not only predict the risk to acquire severe infections but also to develop organ dysfunction or ultimately to die. Thus, the advent of molecular techniques allowing screening for a wide variety of genetic factors, such as single nucleotide polymorphisms in genes controlling expression of important mediator systems in patients as well as their purposeful targeting in animal models of sepsis, are revolutionizing understanding of pathophysiology in the critically ill. Molecular tools are about to challenge ''state-of-the-art'' diagnostic tests such as blood culture as they not only increase sensitivity but dramatically reduce time requirements to identify pathogens and their resistance patterns. Similarly, knowledge of genetic factors might in the near future help to identify ''patients at risk'', i.e. those with a high likelihood to develop organ dysfunction or to guide therapeutic interventions in particular regarding resource-consuming and/or expensive therapies (''theragnostics''). While therapeutic options in molecular intensive care medicine, such as stem cells in the treatment of organ failure or therapeutic gene transfer are possible along the road and might become an option in the future, recombinant DNA technology has already a well defined role in the production of recombinant human proteins from insulin to activated protein C. PMID- 16675936 TI - Advances in critical care hepatology. AB - Complications of liver disease are commonly seen in the intensive care unit (ICU). When evaluating patients with liver disease in the ICU, it is important to determine whether it is acute or chronic liver disease. Because the pathophysiological mechanisms differ among acute and chronic liver, they will be consider separately in this review. Significant advances in the management of acute liver failure highlight the importance of intracranial pressure monitoring for Grade III/IV encephalopathy, and suggest that moderate hypothermia may be a promising treatment for these patients with refractory intracranial hypertension. Chronic liver disease is best discussed in terms of the various complications that may ensue such as ascites, hepatorenal syndrome, spontaneous bacterial peritonitis, variceal hemorrhage and hepatic encephalopathy. Each of these conditions will be discussed with specific attention to critical care management. PMID- 16675937 TI - Risks, benefits, alternatives and indications of allogenic blood transfusions. AB - Allogeneic red blood cell (RBC) transfusions are associated with multiple disadvantages, such as limited availability, high costs, multiple risks and side effects. In addition, large outcome studies comparing liberal (hemoglobin transfusion trigger range 9-10 g/dL) and restrictive (hemoglobin transfusion trigger range 7-9 g/dL) transfusion regimens still need to be performed for surgical patients. Different transfusion alternatives are known for the pre-, intra- and postoperative period. Autologous blood donation and erythropoietin are efficacious in the preoperative period. Intraoperatively, acute normovolemic hemodilution (ANH), cell salvage, antifibrinolytics, specific anesthetic and surgical techniques, coagulation monitoring, acceptance of minimal hemoglobin values and hopefully soon artificial oxygen carriers can reduce allogeneic RBC transfusions. In the postoperative period cell salvage, antifibrinolytics, and accepting minimal hemoglobin values represent alternatives to RBC transfusions. When treating a bleeding patient, the initial administration of crystalloids and colloids to restore and maintain normovolemia is important. RBC transfusions are recommended under the following circumstances: for hemoglobin levels <6 g/dL and for physiologic signs of inadequate oxygenation such as hemodynamic instability, oxygen extraction rate >50% and myocardial ischemia, detectable by new ST-segment depressions >0.1 mV, new ST-segment elevations >0.2 mV or new wall motion abnormalities by transesophageal echocardiography. The aim of this article is to review the efficacy, risk and side effects of RBC transfusions, to discuss transfusion alternatives and to summarize current indications for RBC transfusions. This information will help the physician to judiciously use RBC transfusions when they are indeed indicated. PMID- 16675938 TI - Cisatracurium versus vecuronium: a comparative, double blind, randomized, multicenter study in adult patients under propofol/fentanyl/N2O anesthesia. AB - AIM: The aim of this study was to compare the time course characteristics of cisatracurium (C) and vecuronium (V) induced neuromuscular block (NMB) following multiple doses, allowing spontaneous complete recovery (SCRT) and evaluating the influence of age. METHODS: Following institutional approval and signed informed consent, 177 adult ASA 1-2 patients were included in a randomized, double-blind, multicenter study under N20/02/fentanyl/propofol anesthesia. Muscle relaxation was induced with 0.15 mg/kg C or 0.l mg/kg V and was maintained with 0.03 mg/kg of C or 0.02 mg/kg of V injected at T1 25% recovery. Intubating conditions were assessed at 2 min after the initial dose. Time course of NMB was monitored using accelerography (Tofguard) of the adductor pollicis with train-of-four (TOF). Data were analyzed with parametric (Anova) and non parametric statistics (c2, Kruskal Wallis). RESULTS: Both drugs offered good/excellent intubating conditions: duration of action of NMB (mean values +/- SD, minutes) were: dur25 first dose: V 38.20+/-13.2 vs C 51.5+/-11.3 (P<0.02 ); dur25 following repeated boluses (average): V 23.2+/- 8.6 vs C 28.2+/-9.5, ns; dur25 last dose: V 25.1+/-11.5 vs C 31.5+/-11.4, ns: SCRT following last dose: V 50.2+/-23.2 vs C 46.4+/-17.5, ns: t125% to t4/T1 0.80:V 27.1+/-18.7 vs C 18.8+/-10.2, ns. Stratifying for age >or< 65 no differences were noted in the intervals studied following C, while all were longer following V. The duration of block of C was longer than V; the SCRT after the final dose of C was shorter than V albeit not significant. There was a clinically significant increase in duration of block and recovery time in elderly patients for V but not for C. CONCLUSIONS: C and V allow predictable NMB duration and spontaneous recovery even if administered in multiple repeated doses; but in elderly patients duration of block and recovery time is longer following V. PMID- 16675939 TI - Remifentanil vs fentanyl with a target controlled propofol infusion in patients undergoing craniotomy for supratentorial lesions. AB - AIM: Remifentanil hydrochloride is an ultra-short acting m-opioid receptor agonist. This study compared the use of remifentanil with that of fentanyl during elective supratentorial craniotomy in a target controlled infusion (TCI)-propofol anesthesia regimen and evaluated the quality of recovery from anesthesia. METHODS: After written informed consent for this prospective study, 40 adult patients were randomly divided into 2 groups: in group F analgesia was provided with fentanyl 2-3 mg kg(-1) h(-1) and in group R with remifentanil 0.25 mg kg(-1) h(-1). Anesthesia was induced with thiopental and pancuronium bromide, and maintained with propofol-TCI, pancuronium, air and oxygen and fentanyl (group F) or remifentanil (group R), respectively. After tracheal intubation, infusion rate of remifentanil was reduced and then adjusted to maintain stable hemodynamics. Hemodynamics and recovery time were monitored for 60 min after surgery. Analgesic requirements, propofol intraoperative consumption, nausea and vomiting in postoperative period were monitored. Recovery was evaluated according to a modified Aldrete score. RESULTS: Baseline hemodynamics were similar in both groups. Mean arterial pressure differed between the 2 groups (P<0.05) with the greatest decrease in group R during dura opening (P<0.001). Postoperative mean arterial pressure was higher in group R. Patients in group R exhibited a faster recovery. The incidence of nausea and vomiting was similar in the 2 groups. Noteworthy, there was a reduction in the amount of propofol used in group R. CONCLUSIONS: Remifentanil appears to be a reasonable alternative to fentanyl during elective surgery of supratentorial lesions. PMID- 16675940 TI - Are hot-burning sensations produced by the axonal damage of afferent unmyelinated fibres? AB - AIM: Pain resulting from nerve lesions is classically referred to as a ''burning pain''. Both the axonal damage and sensitization of unmyelinated C-fibres have been considered as the possible generators of this sensation. The aim of this study was to verify the hypothesis that hot-burning sensations are produced by the axonal damage of afferent unmyelinated fibres in peripheral nerves. METHODS: A total of 122 patients with pain localised in the distal parts of the upper limbs (hand, forearm) and lower limbs (leg or foot) were enrolled in the study. The intensity of pain and hot-burning sensations was measured using a numerical scale (range 0-10). The relationship between the presence of warm hypoesthesia (related to the loss of afferent unmyelinated fibres) and hot-burning sensations was assessed. Warm hypoesthesia was identified by Quantitative Sensory Testing employing thermal stimulation (QST-t) and the patients were divided into 2 groups: group A, with hypoesthesia and group B with normoesthesia. Patients with a central nervous impairment were excluded. RESULTS: No significant differences in the intensity of pain and hot-burning sensations was observed between the group of patients with warm hypoesthesia and that with warm normoesthesia. CONCLUSIONS: This study does not confirm the hypothesis that hot-burning sensations are produced by the axonal damage of afferent amyelinated fibres in peripheral nerves. It agrees with clinical evidence suggesting that patients with different clinical conditions can complain of hot-burning sensations, independently of the presence of a nerve lesion. PMID- 16675941 TI - Bispectral Index compared to Ramsay score for sedation monitoring in intensive care units. AB - AIM: Continuous monitoring is of paramount importance in order to obtain a correct level of sedation in ICU patients. Clinical scoring systems, although simple and inexpensive, are often inadequate in evaluating the patient level of consciousness. Among instrumental tools for sedation monitoring, Bispectral Index (BIS) is now widely used due to its reliability and applicability. Nevertheless some doubts still exist regarding its usefulness in ICU. METHODS: Sedation, obtained with propofol or midazolam, was monitored with Ramsay scale and BIS in 40 patients admitted in ICU for postoperative monitoring after major abdominal or vascular surgery. A correlation between Ramsay score and BIS values was searched using Pearson test. RESULTS: A good correlation between Ramsay score and BIS values was observed. At the deeper levels of sedation a wide range of BIS values corresponds to Ramsay score 6, indicating various levels of central nervous system depression that are not identified by clinical evaluation. CONCLUSIONS: BIS monitoring is useful in ICU patients and allows a finest differentiation of sedation level in deeply sedated ICU patients. PMID- 16675942 TI - Mitochondrial genome involvement in ischemia/reperfusion-induced adaptive changes in human myocardial cells. AB - AIM: Following previous studies on the ischemia-induced adaptive changes in human cardiac mitochondria, we examined in the present paper the interaction between nitric oxide-induced (NO) partial inhibition of Cyt. c oxidase (Cyt.OX) and mitochondrial encoded subunit 2 expression. Aim of the study was to investigate specific stages of the biochemical and molecular cascade which takes place in cytoprotective mechanisms of ischemic and reperfused cardiac cell. METHODS: We examined human left ventricle samples obtained from 20 patients undergoing elective valve surgery before aortic cross-clamping, 20+/-2 min (prolonged ischemia), 58+/-5 min after cross-clamping (intermittent ischemia) and 21+/-4 min after reconstitution of coronary blood flow (reperfusion). Cyt.OX activity was determined by spectrophotometric method and adenosine triphosphate (ATP) content using bioluminescent assay. Malondialdehyde (MDA) assumed as reactive oxygen species (ROS) generation marker was determined by high-performance liquid chromatography method. On the same cardiac samples mitochondrial encoded Cyt.OX subunit 2 expression was examined by immunoblot analysis and blu native gel electrophoresis method. Statistical study of obtained data was performed using repeated measures analysis of variance (ANOVA). RESULTS: Prolonged as well intermittent ischemia caused reduction of Cyt.OX activity and ATP, a moderate accumulation of ROS and down-regulation of Cyt.OX subunit 2. When reperfused the cardiomyocytes showed a progressive increase of Cyt.OX activity, ATP pools and Cyt.OX subunit 2 expression. ROS generation was significantly increased by the rapid oxygen re-immission in the cardiac cell. CONCLUSIONS: These data confirm the suggestion that prolonged as well as intermittent ischemia induces activation of cytoprotective mechanisms crucial for cardiac cell survival. Indeed, co ordinated down-regulation of Cyt.OX activities, ATP pools and mitochondrial encoded Cyt.OX subunit 2 are in favour of an ischemia-activated adaptive mechanism leading to transient and reversible oxidative injury. This observation is confirmed by reduction of apoptosis molecular markers and by complete recovery of mitochondrial oxidative activities in reperfused cardiac tissue. PMID- 16675943 TI - State prevalence of self-reported doctor-diagnosed arthritis and arthritis attributable activity limitation--United States, 2003. AB - Arthritis is costly (86 billion dollars annually), highly prevalent (affecting 43 million U.S. adults), the leading cause of disability, and associated with substantial disparities in pain, activity limitations, and compromised quality of life. State-based estimates of arthritis prevalence and impact help define the burden of arthritis and provide state arthritis programs with data for program planning. This report summarizes results from the 2003 Behavioral Risk Factor Surveillance System (BRFSS) survey on state-specific prevalence of self-reported doctor-diagnosed arthritis and arthritis-attributable activity limitation in 50 states, the District of Columbia (DC), and three territories. The findings indicated that the prevalence of adults with self-reported doctor-diagnosed arthritis ranged from 17.9% to 37.2% (state median: 27.0%) and with arthritis attributable activity limitation ranged from 6.3% to 16.7% (state median: 9.9%); the proportion of adults with arthritis-attributable activity limitation among those with self-reported doctor-diagnosed arthritis ranged from 30.1% to 49.8% (state median: 37.4%). These high rates of arthritis prevalence and activity limitation are projected to increase with the aging of the population, requiring increased intervention measures to reduce this impact. PMID- 16675944 TI - Mental health in the United States: parental report of diagnosed autism in children aged 4-17 years--United States, 2003-2004. AB - Autism is a lifelong neurodevelopmental disorder characterized by early onset of impairments in social interaction and communication and unusual, stereotyped behaviors. Autism (i.e., autistic disorder) often is classified with two related, although less severe, developmental disorders: Asperger disorder and pervasive developmental disorder--not otherwise specified. These three constitute the autism spectrum disorders (ASDs). Diagnosis of ASDs is based exclusively on developmental pattern and behavioral observation. Two population-based studies conducted by CDC in selected U.S. locations reported ASD prevalence of 3.4 and 6.7 per 1,000 children, respectively. CDC also conducts two nationally representative surveys, the National Health Interview Survey (NHIS) and the National Survey of Children's Health (NSCH), in which parents are asked whether their child ever received a diagnosis of autism. Because of similarities in methodology used by the two surveys, CDC analyzed 2003-2004 data from NHIS and data from the first-ever NSCH (collected during January 2003-July 2004) to 1) estimate the population-based prevalence of parental report of diagnosed autism in the United States and 2) assess parental reporting of child social, emotional, and behavioral strengths and difficulties and special-health care needs among children with and without reported autism. This report describes the results of that analysis, which indicated that the prevalence of parent-reported diagnosis of autism was 5.7 per 1,000 children in NHIS and 5.5 per 1,000 children in NSCH. Prevalence estimates in the two studies were similar across age, sex, and racial/ethnic populations. The consistency in estimates between the two surveys suggests high reliability for parental report of autism. These estimates suggest that, as of 2003-2004, autism had been diagnosed in at least 300,000 U.S. children aged 4-17 years. In addition, parental reports of autism were associated with reported social, emotional, and behavioral symptoms and specialized needs. Thus, these surveys might be useful to assess health, education, and social service needs of children with autism. PMID- 16675945 TI - Worker illness related to ground application of pesticide--Kern County, California, 2005. AB - In California, suspected pesticide and work-related illnesses and injuries are reportable conditions. The Occupational Health Branch (OHB) of the California Department of Health Services (CDHS) conducts surveillance of work-related pesticide illness with support from the National Institute for Occupational Safety and Health (NIOSH) and the U.S. Environmental Protection Agency (EPA). On May 12, 2005, CDHS received a report from the California Department of Pesticide Regulation (CDPR) of a suspected pesticide incident in Kern County involving 27 farmworkers (age range: 21-61 years; median: 32.5 years) and six emergency responders (age range: 28-51 years; median: 33.5 years). CDHS investigated this incident by conducting a site visit; reviewing medical and meteorologic records; and interviewing affected workers, pesticide applicators, and the farmworker employer. Findings indicated that workers became ill from drift of a pyrethroid pesticide (cyfluthrin) that was being applied in a neighboring field. Pyrethroid pesticide applicators should always operate in a manner that ensures workers are not exposed. PMID- 16675946 TI - Brief report: respiratory illness associated with boot sealant products--five states, 2005-2006. AB - During February 2005-February 2006, six regional poison control centers in five states were consulted regarding 172 human and 19 animal (i.e., pet cat or dog) exposures to shoe or boot leather protection or sealant products resulting in respiratory illness. One product was associated with 126 cases of human illness and another product with seven cases. An ongoing investigation, begun in December 2005, is being conducted by the poison centers. The majority of cases occurred in Michigan, where poison control centers are collaborating with the Michigan Department of Community Health to further document exposures and adverse health effects from the products. Results of the investigation of the 150 cases reported during 2005 underscore the continuing need to assess the magnitude of the problem, evaluate the toxic etiology of the products involved, and determine how to prevent further cases of illness resulting from use of the products. PMID- 16675947 TI - Structure of the unusual seryl-tRNA synthetase reveals a distinct zinc-dependent mode of substrate recognition. AB - Methanogenic archaea possess unusual seryl-tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 A resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl-adenylate analogue 5'-O-(N-serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N-terminal tRNA-binding domain and an active site zinc ion. The tetra-coordinated Zn2+ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc-dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial-type SerRSs. Consequently, SerRS represents the only known aminoacyl-tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino-acid substrate. PMID- 16675948 TI - Apoptosis regulation in tetraploid cancer cells. AB - Tetraploidy can result in cancer-associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax-dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl-2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax-proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53-inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and gamma-irradiation. These data indicate the existence of p53-dependent alterations in apoptosis regulation in tetraploid cells. PMID- 16675949 TI - Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide. AB - The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT-rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X-chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA-binding ligands. P9 fed to larvae suppresses the position-effect variegation phenotype of white-mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene. PMID- 16675951 TI - PIASxbeta acts as an activator of Hoxb1 and is antagonized by Krox20 during hindbrain segmentation. AB - The zinc-finger transcription factor Krox20 constitutes a key regulator of hindbrain development, essential for the formation and specification of rhombomeres (r) 3 and 5. It is in particular responsible for the respective activation and repression of odd- and even-numbered rhombomere-specific genes, which include Hox genes. In this study, we have identified PIASxbeta as a novel direct interactor of Krox20. In addition, we found that PIASxbeta is able to activate the r4-specific gene Hoxb1. Binding of Krox20 prevents this activation, providing a molecular basis for the repression of Hoxb1 by Krox20. The same domain in the Krox20 protein, the zinc-fingers, is involved in DNA binding for transcriptional activation and in interaction with PIASxbeta for transcriptional repression, although the actual precise contacts are different. Our findings add an additional level in the complexity of Hox gene regulation and provide an example of how a single regulator can coordinate the activation and repression of a set of genes by very different mechanisms, acting as a molecular switch to specify cell identity and fate. PMID- 16675950 TI - Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase eta on ATR checkpoint signaling. AB - An essential component of the ATR (ataxia telangiectasia-mutated and Rad3 related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation. PMID- 16675952 TI - Distinct functions of the ubiquitin-proteasome pathway influence nucleotide excision repair. AB - The Rad23/Rad4 nucleotide excision repair (NER) protein complex functions at an early stage of the NER reaction, possibly promoting the recognition of damaged DNA. Here we show that Rad4 protein is ubiquitinated and degraded in response to ultraviolet (UV) radiation, and identify a novel cullin-based E3 ubiquitin ligase required for this process. We also show that this novel ubiquitin ligase is required for optimal NER. Our results demonstrate that optimal NER correlates with the ubiquitination of Rad4 following UV radiation, but not its subsequent degradation. Furthermore, we show that the ubiquitin-proteasome pathway (UPP) regulates NER via two distinct mechanisms. The first occurs independently of de novo protein synthesis, and requires Rad23 and a nonproteolytic function of the 19S regulatory complex of the 26S proteasome. The second requires de novo protein synthesis, and relies on the activity of the newly identified E3 ubiquitin ligase. These studies reveal that, following UV radiation, NER is mediated by nonproteolytic activities of the UPP, via the ubiquitin-like domain of Rad23 and UV radiation-induced ubiquitination of Rad4. PMID- 16675953 TI - A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells. AB - In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs. PMID- 16675954 TI - TREK-1, a K+ channel involved in polymodal pain perception. AB - The TREK-1 channel is a temperature-sensitive, osmosensitive and mechano-gated K+ channel with a regulation by Gs and Gq coupled receptors. This paper demonstrates that TREK-1 qualifies as one of the molecular sensors involved in pain perception. TREK-1 is highly expressed in small sensory neurons, is present in both peptidergic and nonpeptidergic neurons and is extensively colocalized with TRPV1, the capsaicin-activated nonselective ion channel. Mice with a disrupted TREK-1 gene are more sensitive to painful heat sensations near the threshold between anoxious warmth and painful heat. This phenotype is associated with the primary sensory neuron, as polymodal C-fibers were found to be more sensitive to heat in single fiber experiments. Knockout animals are more sensitive to low threshold mechanical stimuli and display an increased thermal and mechanical hyperalgesia in conditions of inflammation. They display a largely decreased pain response induced by osmotic changes particularly in prostaglandin E2-sensitized animals. TREK-1 appears as an important ion channel for polymodal pain perception and as an attractive target for the development of new analgesics. PMID- 16675955 TI - ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency. AB - BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full-length Brca1 (Brca1(delta11/delta11)) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1(delta11/delta11)p53+/- mice display cancer susceptibility and premature aging. Here, we show that reduced expression and/or the absence of Chk2 allow Brca1(delta11/delta11) mice to escape from embryonic lethality. Compared to Brca1(delta11/delta11)p53+/- mice, lifespan of Brca1(delta11/delta11)Chk2-/- mice was remarkably extended. Analysis of Brca1(delta11/delta11)Chk2-/- mice revealed that p53-dependent apoptosis and growth defect caused by Brca1 deficiency are significantly attenuated in rapidly proliferating organs. However, in later life, Brca1(delta11/delta11)Chk2-/- female mice developed multiple tumors. Furthermore, haploid loss of ATM also rescued Brca1 deficiency-associated embryonic lethality and premature aging. Thus, in response to Brca1 deficiency, the activation of the ATM-Chk2-p53 signaling pathway contributes to the suppression of neoplastic transformation, while leading to compromised organismal homeostasis. Our data highlight how accurate maintenance of genomic integrity is critical for the suppression of both aging and malignancy, and provide a further link between aging and cancer. PMID- 16675957 TI - Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB. AB - Escherichia coli and other enterobacteria exploit the H+ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5'-phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl- exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values. PMID- 16675959 TI - Molecular basis of Kindler syndrome in Italy: novel and recurrent Alu/Alu recombination, splice site, nonsense, and frameshift mutations in the KIND1 gene. AB - Kindler syndrome (KS) is a rare autosomal recessive disorder characterized by skin blistering in childhood followed by photosensitivity and progressive poikiloderma. Most cases of KS result from mutations in the KIND1 gene encoding kindlin-1, a component of focal adhesions in keratinocytes. Here, we report novel and recurrent KIND1 gene mutations in nine unrelated Italian KS individuals. A novel genomic deletion of approximately 3.9 kb was identified in four patients originating from the same Italian region. This mutation deletes exons 10 and 11 from the KIND1 mRNA leading to a truncated kindlin-1. The deletion breakpoint was embedded in AluSx repeats, specifically in identical 30-bp sequences, suggesting Alu-mediated homologous recombination as the pathogenic mechanism. KIND1 haplotype analysis demonstrated that patients with this large deletion were ancestrally related. Five additional mutations were disclosed, two of which were novel. To date, four recurrent mutations have been identified in Italian patients accounting for approximately approximately 75% of KS alleles in this population. The abundance of repetitive elements in intronic regions of KIND1, together with the identification of a large deletion, suggests that genomic rearrangements could be responsible for a significant proportion of KS cases. This finding has implications for optimal KIND1 mutational screening in KS individuals. PMID- 16675956 TI - Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens specific chromatin. AB - Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA crystallin promoter in lens chromatin and demonstrated that high levels of alphaA crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin remodeling enzymes, and suggest a multistage model for the activation of alphaA crystallin during lens differentiation. PMID- 16675960 TI - Parathyroid hormone hormone-related protein and the PTH receptor regulate angiogenesis of the skin. AB - In developing organs, parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor (PPR) signaling inhibits proliferation and differentiation of mesenchyme-derived cell types resulting in control of morphogenic events. Previous studies using PPR agonists and antagonists as well as transgenic overexpression of the PPR ligand PTHrP have suggested that this ligand receptor combination might regulate the anagen to catagen transition of the hair cycle. To further understand the precise role of PTHrP and the PPR in the hair cycle, we have evaluated hair growth in the traditional K14-PTHrP (KrP) and an inducible bitransgenic PTHrP mice. High levels of PTHrP trangene expression limited to the adult hair cycle resulted in the production of shorter hair shafts. Morphometric analysis indicated that reduced proliferation in the matrix preceded the appearance of thinner hair follicles and shafts during late anagen. CD31 staining revealed that the late anagen hair follicles of the KrP mice were surrounded by reduced numbers of smaller diameter capillaries as compared to controls. Moreover, the fetal skins of the PTHrP and PPR knockouts (KOs) had reciprocal increases in the length, diameter, and density of capillaries. Finally, crossing the KrP transgene onto a thrombospondin-1 KO background reversed the vascular changes as well as the delayed catagen exhibited by these mice. Taken together, these findings suggest that PTHrP's influence on the hair cycle is mediated in part by its effects on angiogenesis. PMID- 16675961 TI - Elastin fragments induce IL-1beta upregulation via NF-kappaB pathway in melanoma cells. AB - In a previous work, we reported the influence of elastin fragments (EFs) on matrix metalloproteinases-2 and -14 expression and activation in melanoma cells in vitro. We hypothesized that EFs might also modulate expression of other mediators involved during melanoma progression. Therefore we investigated the contribution of EFs on IL-1beta expression, a cytokine playing a key role in melanoma cells activation. Our results evidenced that high tumorigenic melanoma cells (M3Da cells) treated with EFs led to IL-1beta mRNA and protein upregulation. The effects of EFs on M3Da cells were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose and reproduced by cell stimulation with the VGVAPG peptide. Binding of EFs to their receptor induced a rapid activation of extracellular signal-regulated kinase 1/2; and p38 mitogen-activated protein kinase pathways. However, these pathways were not associated with IL-1beta mRNA upregulation by EFs. Concomitantly, we demonstrated that EFs stimulation induced NF-kappaB nuclear translocation and DNA binding on IL-1beta promoter region whereas inhibition of NF-kappaB with the specific chemical inhibitor SN-50 or by overexpression of IkappaB, the endogenous inhibitor of NF-kappaB pathway, totally abolished EFs-mediated IL-1beta mRNA overexpression. These results demonstrate that EFs induce NF-kappaB activation, leading to IL-1beta upregulation in invasive melanoma cells. PMID- 16675958 TI - Absence of the steroid receptor coactivator-3 induces B-cell lymphoma. AB - Steroid receptor coactivator 3 (SRC-3/ACTR/AIB-1/pCIP/RAC3/TRAM-1) is a member of the p160 family of nuclear receptor coactivators that plays an important role in mammary gland growth, development, and tumorigenesis. We show that deletion of SRC-3 gene decreases platelet and increases lymphocytes numbers, leading to the development of malignant B-cell lymphomas upon aging. The expansion of the lymphoid lineage in SRC-3(-/-) mice is cell autonomous, correlates with an induction of proliferative and antiapoptotic genes secondary to constitutive NF kappaB activation, and can be reversed by restoration of SRC-3 expression. NF kappaB activation is explained by the degradation of IkappaB, consequent to increases in free IkappaB kinase, which is no longer inhibited by SRC-3. These results demonstrate that SRC-3 regulates lymphopoiesis and in combination with previous studies indicate that SRC-3 has vastly diverging effects on cell proliferation depending on the cellular context, ranging from proliferative and tumorigenic (breast) to antiproliferative (lymphoid cells) effects. PMID- 16675962 TI - Peroxisome proliferator-activated receptors increase human sebum production. AB - Sebum production is key in the pathophysiology of acne, an extremely common condition, which when severe, may require treatment with isotretinoin, a known teratogen. Apart from isotretinoin and hormonal therapy, no agents are available to reduce sebum. Increasing our understanding of the regulation of sebum production is a milestone in identifying alternative therapeutic targets. Studies in sebocytes and human sebaceous glands indicate that agonists of peroxisome proliferator-activated receptors (PPARs) alter sebaceous lipid production. The goal of this study is to verify the expression and activity of PPARs in human skin and SEB-1 sebocytes and to assess the effects of PPAR ligands on sebum production in patients. To investigate the contribution of each receptor subtype to sebum production, lipogenesis assays were performed in SEB-1 sebocytes that were treated with PPAR ligands and isotretinoin. Isotretinoin significantly decreased lipogenesis, while the PPARalpha agonist-GW7647, PPARdelta agonist GW0742, PPARalpha/delta agonist-GW2433, PPARgamma agonist rosiglitazone, and the pan-agonist-GW4148, increased lipogenesis. Patients treated with thiazolidinediones or fibrates had significant increases in sebum production (37 and 77%, respectively) when compared to age-, disease-, and sex-matched controls. These data indicate that PPARs play a role in regulating sebum production and that selective modulation of their activity may represent a novel therapeutic strategy for the treatment of acne. PMID- 16675963 TI - Involvement of alphavbeta5 integrin in the establishment of autocrine TGF-beta signaling in dermal fibroblasts derived from localized scleroderma. AB - Localized scleroderma (LSc) is a connective tissue disorder limited to skin and subcutaneous tissue, which may share pathogenic processes with systemic sclerosis (SSc). We previously demonstrated that upregulated expression of integrin alphavbeta5 might contribute to autocrine TGF-beta signaling in SSc fibroblasts. Based on these data, we presently focused on alphavbeta5 and assessed its involvement in pathogenesis of LSc. We initially demonstrated that LSc fibroblasts might be activated by the stimulation of autocrine TGF-beta. Consistent with SSc fibroblasts, expression levels of alphavbeta5 were elevated in LSc fibroblasts in vitro and in vivo. Anti-alphavbeta5 antibody partially reversed expression levels of type I procollagen and MMP-1 and constitutive DNA Smad3 binding in LSc fibroblasts. In LSc fibroblasts pretreated with antisense TGF-beta1, exogenous latent TGF-beta1 stimulation increased expression of type I procollagen in an alphavbeta5-dependent manner. The luciferase activities of TMLC cells, Mv1Lu cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter, co-cultured with LSc fibroblasts were significantly elevated compared with those co-cultured with normal fibroblasts and were significantly reduced in the presence of anti-alphavbeta5 antibody. Anti alphavbeta5 antibody reversed the myofibroblastic features of LSc fibroblasts. These results indicate that upregulated expression of alphavbeta5 contributes to autocrine TGF-beta signaling in LSc fibroblasts. PMID- 16675964 TI - In vivo monitoring of cutaneous edema using spectral imaging in the visible and near infrared. AB - Tissue inflammation is often accompanied by local interstitial fluid accumulation expressed as edema. Edema can be the manifestation of infection, lymphatic blockage, wound healing, or even cancer, and is typically graded visually. Here we demonstrate that the edema reaction can be objectively quantitated in vivo by the use of spectral imaging. To this end we applied the method on a histamine induced cutaneous edema model. Apparent concentrations of oxy-hemoglobin, deoxy hemoglobin, and water were calculated for each pixel of a spectral image stack. These values were used to construct concentration maps for each of these molecules as well as an intensity map of an optical tissue-scattering parameter. The oxy-hemoglobin and the tissue water maps are two-dimensional quantitative representations of the skin areas involved in erythema and edema, respectively. These maps demonstrated characteristics of the wheal-and-flare reaction and their gray-level intensities were dependent on the applied histamine dose. We conclude that spectral imaging can be a valuable noninvasive tool in the study of edema pathology and can be used to monitor the edema reaction in vivo or follow the efficacy of treatments in a clinical setting. PMID- 16675966 TI - Human mast cells in the neurohormonal network: expression of POMC, detection of precursor proteases, and evidence for IgE-dependent secretion of alpha-MSH. AB - Human mast cells have been shown to release histamine in response to the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH), but it is unknown whether these cells express proopiomelanocortin (POMC) or POMC-derived peptides. We therefore examined highly purified human skin mast cells and a leukemic mast cell line-1 (HMC-1) for their ability to express POMC and members of the prohormone convertase (PC) family known to process POMC. Furthermore, we investigated whether these cells store and secrete alpha-MSH. Reverse transcriptase-PCR (RT-PCR) analysis revealed that both skin mast cells and HMC-1 cells express POMC mRNA and protein. Expression of the POMC gene at the RNA level in HMC-1 cells could be confirmed by Northern blotting. Transcripts for both PC1 and furin convertase were detectable in skin-derived mast cells and HMC-1 cells, as shown by RT-PCR. In contrast, PC2 transcripts were detected only in skin mast cells, whereas transcripts for paired basic amino acid converting enzyme 4 (PACE4) were present only in HMC-1 cells. Radioimmunoassays performed on cell lysates and cell culture supernatants from human skin-derived mast cells disclosed immunoreactive amounts of alpha-MSH in both fractions. Stimulation with an anti-IgE antibody significantly reduced intracellular alpha-MSH and increased extracellular levels, indicating IgE-mediated secretion of this neuropeptide. Our findings show that human mast cells are active players in the cutaneous POMC system. Mast cell-derived alpha-MSH may contribute to cutaneous hyperpigmentation as seen in patients with urticaria pigmentosa. Moreover, IgE-dependent release of alpha-MSH suggests an immunomodulatory role of this neurohormone during inflammatory and allergic reactions of the skin. PMID- 16675965 TI - A chronic contact eczema impedes migration of antigen-presenting cells in alopecia areata. AB - Long-lasting allergen treatment is the most efficient therapy in alopecia areata (AA). The underlying mechanism is unknown. We here asked whether treatment with a contact sensitizer influences leukocyte migration such that dendritic cell (DC) migration or the recruitment of activated T-cells towards the skin become hampered. Allergen treatment of AA mice was not accompanied by a decrease in skin infiltrating leukocytes or draining lymph node cells (LNC). However, the distribution of leukocyte subsets was changed with a dominance of monocytes in the skin and a reduced percentage of DCs in draining nodes. Chemokine and chemokine receptor expression in skin and draining nodes was strikingly increased and LNC from untreated and allergen-treated AA mice showed high migratory activity in vitro and readily homed in draining nodes and skin after intravenous injection. However, FITC labelling of the skin and subcutaneous transfer of dye labelled DC revealed that allergen treatment created a chemokine milieu severely hampering DC migration from the skin towards the draining node. An allergic eczema-induced reduction in DC migration and antigen transfer could well contribute to insufficient T-cell activation and the recovery of hair follicle in AA and possibly be of relevance for other skin-related autoimmune diseases. PMID- 16675967 TI - Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity. AB - Harlequin ichthyosis (HI) is one of the most devastating genodermatoses. Recently, ABCA12 mutations were identified as the cause of HI. A newborn Japanese male demonstrated the typical features of HI. The patient was treated with oral etretinate and his general condition has been good (now aged 1.5 years). This patient with moderate clinical severity was compound heterozygous for a novel de novo missense mutation 1160G > A (S387N) in exon 10 and a maternal deletion mutation 4158_4160delTAC (T1387del) in exon 28 of ABCA12. T1387del was a deletion of a highly conserved threonine residue within the first adenosine 5' triphosphate-binding domain and is thought to seriously affect the function of the ABCA12 protein. Conversely, the residue 387 is located outside the known active sites of ABCA12 and S387N is predicted not to lead to a serious functional deficiency in ABCA12. Electron microscopy revealed abnormal lamellar granules in the granular layer cells and a moderate number of lipid vacuoles in the cornified cells. Disturbed glucosylceramide transport was confirmed in the cultured keratinocytes from the patient. No de novo mutation in ABCA12 has yet been reported either in HI or lamellar ichthyosis. The present case suggested that a de novo ABCA12 mutation might underlie HI. PMID- 16675968 TI - Promotion of human dermal fibroblast migration, matrix remodelling and modification of fibroblast morphology within a novel 3D model by Lucilia sericata larval secretions. AB - Lucilia sericata larvae, or green bottle fly maggots are applied to chronic wounds to aid healing. Previously, our laboratory has characterized the enzymatic activities present within maggot excretions/secretions (ES). Since then, we have related these to the degradation of extracellular matrix components, alteration of human, dermal fibroblast adhesion to surfaces and the stimulation of fibroblast migration within a two-dimensional in vitro assay. In this study, we developed a novel three-dimensional in vitro assay in which to observe fibroblast migration and morphology in response to maggot ES. Here, primary human foreskin fibroblasts were embedded within collagen gels containing fibronectin. Phase contrast and confocal microscopy were used in conjunction with image analysis software to examine and quantify aspects of fibroblast behavior. Our results showed that maggot ES stimulated fibroblast migration through the matrix and induced altered cell morphologies. Remodelling of the extracellular matrix located between individual fibroblasts was also induced, providing a mechanism by which cells may detect each other's presence over considerable distances. Thus, mechanisms by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, acceleration of extracellular matrix remodelling and coordination of cellular responses. PMID- 16675969 TI - A taste of new medicine. PMID- 16675970 TI - Trial set to test how stem cells heal a broken heart. PMID- 16675971 TI - Security fears plague deadly disease lab's move to central London. PMID- 16675973 TI - Despite doubts, containment plans for pandemic take shape. PMID- 16675974 TI - Cell-based tests tackle predicting safety of antibody drugs. PMID- 16675975 TI - AIDS rates in Africa may be overestimated. PMID- 16675976 TI - The body snatchers. PMID- 16675977 TI - Yoshihiro Kawaoka. PMID- 16675979 TI - Your cheatin' heart. PMID- 16675981 TI - Catching the crooks. PMID- 16675982 TI - Focus on fraud. Where are they now? PMID- 16675983 TI - So you suspect someone of fraud. What now? PMID- 16675984 TI - Your own desktop crime lab. PMID- 16675985 TI - Can't hide your lying eyes. PMID- 16675986 TI - Plagiarize or perish? PMID- 16675987 TI - Monkeypox outbreak diagnostics and implications for vaccine protective effect. PMID- 16675989 TI - Will travel restrictions control the international spread of pandemic influenza? PMID- 16675991 TI - Neurons tame T cells. PMID- 16675992 TI - Staying slim on a low-GABA diet. PMID- 16675993 TI - Channeling pain. PMID- 16675994 TI - Virus smuggling, tax evasion and tumor assassination. PMID- 16675996 TI - Personalized medicine progresses. PMID- 16675997 TI - Overcoming insulin resistance with CNTF. PMID- 16676001 TI - Cell death in the absence of Bax and Bak. PMID- 16676002 TI - Emerging role of Mcl-1 in actively counteracting BH3-only proteins in apoptosis. PMID- 16676003 TI - Personal reflections on Stan Korsmeyer and his continuing impact on our work to delineate the role of Bax in female reproduction. PMID- 16676004 TI - Mechanisms of cytochrome c release from mitochondria. AB - In healthy cells, cytochrome c (Cyt c) is located in the mitochondrial intermembrane/intercristae spaces, where it functions as an electron shuttle in the respiratory chain and interacts with cardiolipin (CL). Several proapoptotic stimuli induce the permeabilization of the outer membrane, facilitate the communication between intermembrane and intercristae spaces and promote the mobilization of Cyt c from CL, allowing for Cyt c release. In the cytosol, Cyt c mediates the allosteric activation of apoptosis-protease activating factor 1, which is required for the proteolytic maturation of caspase-9 and caspase-3. Activated caspases ultimately lead to apoptotic cell dismantling. Nevertheless, cytosolic Cyt c has been associated also to vital cell functions (i.e. differentiation), suggesting that its release not always occurs in an all-or nothing fashion and that mitochondrial outer membrane permeabilization may not invariably lead to cell death. This review deals with the events involved in Cyt c release from mitochondria, with special attention to its regulation and final consequences. PMID- 16676005 TI - Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis? AB - Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets. PMID- 16676006 TI - c-Jun promotes cellular survival by suppression of PTEN. AB - Activation of c-Jun, a component of the AP-1 family of transcription factors, leads to either promotion or prevention of apoptosis. However, the molecular determinants of c-Jun-mediated cell survival are still unclear. We show here that inducible expression of c-Jun promotes cellular survival by negatively regulating the expression of the tumor-suppressor PTEN, resulting in the concomitant activation of the Akt survival pathway. Consistently, c-jun-/- fibroblasts, which are sensitive to nutrient deprivation, and human cell lines in which c-Jun expression is silenced, express elevated levels of PTEN. siRNA-mediated silencing of PTEN resulted in the reduction of cell-death owing to c-Jun deficiency. c-Jun was found to suppress PTEN expression by binding to a variant AP-1 site found in the 5' upstream sequences of PTEN promoter. Finally, an inverse correlation between c-Jun and PTEN levels was apparent in a panel of human tumor cell lines, independent of their p53 status. Together, the data demonstrate that c-Jun contributes to the promotion of cellular survival by regulating the expression of PTEN. PMID- 16676007 TI - Mechanisms of apoptosis regulation by viral oncogenes in infection and tumorigenesis. AB - Apoptosis mediated by the proapoptotic BCL-2 family members BCL-2-associated X protein (BAX) and BCL-2 antagonist/killer (BAK) is part of the antiviral response at the cellular level to limit virus replication. Viruses, in turn, have evolved to encode antiapoptotic BCL-2 homologs (v-BCL-2s) to prevent the premature death of the infected host cell to sustain virus replication. These same v-BCL-2 proteins cooperate with loss of retinoblastoma protein and p53 tumor suppressor function, by inactivating the BAX and BAK apoptotic pathway to promote epithelial solid tumor growth and resistance to chemotherapy. Analogously to infected cells, failure of apoptosis in tumors permits the survival of abnormal, damaged cells displaying chromosome instability that may further promote tumor progression. Thus, both infected cells and tumor cells require inhibition of the apoptotic host defense mechanism, the insights from which can be exploited for therapy development. PMID- 16676008 TI - Risk for unplanned hospital readmission of patients with cancer: results of a retrospective medical record review. AB - PURPOSE/OBJECTIVES: To identify potential factors that place patients with cancer at risk for unplanned readmissions after discharge from the hospital. DESIGN: Retrospective, descriptive, medical record review. SETTING: A National Cancer Institute-designated comprehensive cancer center in an urban area of the Northeastern United States. SAMPLE: 78 patients were selected from those readmitted within seven days of discharge. For each readmission case, a nonreadmitted patient was randomly selected and matched on discharge date and reason for prior admission. The age range was 22-87 years, men and women were equally represented, and 88% were Caucasian. METHODS: The Readmission Criteria Record was developed to collect data from medical records about factors associated with readmission, including demographics, severity of illness, support at home, symptoms, and comorbidities. MAIN RESEARCH VARIABLES: Criteria associated with readmission risk. FINDINGS: Patients who had gastrointestinal cancer, nausea within 24 hours of discharge, financial and insurance concerns, or caregiver difficulty or those who lived alone were more likely to be readmitted within seven days of discharge. Patients were more likely to be readmitted on Friday than any other day. Among readmitted patients, 48% were readmitted within one to two days postdischarge. CONCLUSIONS: Knowledge of factors that may place patients with cancer at an increased risk for readmission and subsequent implementation of appropriate interventions during hospitalization may help to decrease risk of readmission. IMPLICATIONS FOR NURSING: The factors identified provide a basis for assessment, planning, interventions, and follow-up of patients to help reduce the risk of readmission and, thus, poor outcomes. PMID- 16676010 TI - Cancer chemotherapy-related symptoms: evidence to suggest a role for proinflammatory cytokines. AB - PURPOSE/OBJECTIVES: To provide an overview of the evidence that supports a role for the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in the etiology of cancer chemotherapy-related symptoms. DATA SOURCES: Electronic nursing, psychology, and medicine databases; online meeting abstracts; and personal experimental observations. DATA SYNTHESIS: Substantial evidence implicates the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 in the etiology of chemotherapy-related anorexia, cachexia, anemia, pain, sleep disturbance, fatigue, and depression. CONCLUSIONS: Further investigation into the role of these cytokines in the genesis of chemotherapy-related symptoms is warranted. The development of appropriate animal models likely will be key to understanding the relationship among cancer chemotherapy, proinflammatory cytokines, and symptoms. IMPLICATIONS FOR NURSING: Nurses traditionally have been leaders in symptom management. The symptoms experienced by patients undergoing chemotherapy have a profound negative impact on quality of life and patients' ability to receive prescribed treatments. An understanding of potential mechanisms underlying the physiologic and behavioral consequences of chemotherapy administration will aid nurses in the development of interventions to effectively manage chemotherapy-related symptoms. PMID- 16676011 TI - Post-traumatic growth and psychosocial adjustment of daughters of breast cancer survivors. AB - PURPOSE/OBJECTIVES: To examine post-traumatic growth, or positive life changes, and its correlates among adult daughters of breast cancer survivors and to compare their psychosocial adjustment to women with healthy parents. DESIGN: Descriptive, cross-sectional survey. SETTING: Outpatient oncology units in two urban hospitals and two breast cancer organizations. SAMPLE: 30 adult daughters of breast cancer survivors (mean age = 38.1 years) and 16 women with healthy parents. METHODS: Participants were recruited by hospital or research staff or responded to an announcement in a newsletter. Respondents completed the Post Traumatic Growth Inventory and standardized assessments of psychosocial adjustment. MAIN RESEARCH VARIABLES: Post-traumatic growth and demographic, stressor, and psychosocial variables. FINDINGS: Women who cared for their mothers following breast cancer diagnosis and perceived their mothers' illness to be stressful reported greater post-traumatic growth. Life satisfaction, social support, emotional processing strategies, and problem-focused coping strategies also were positively associated with growth. Women with maternal histories of breast cancer and those with healthy parents did not differ in psychosocial well being, including affect, life satisfaction, and social support. CONCLUSIONS: Findings suggest that some daughters of breast cancer survivors experience positive life changes following their mothers' illness. IMPLICATIONS FOR NURSING: For nurses seeking to adopt a holistic approach to practice, the personal growth of women following life-threatening familial illness warrants attention. PMID- 16676012 TI - Survivorship issues in ovarian cancer: a review. AB - PURPOSE/OBJECTIVES: To review what is known about survivorship issues for women after ovarian cancer treatment while identifying gaps and controversies. DATA SOURCES: MEDLINE, CINAHL, and CancerLit database searches using the key words ovarian cancer, quality of life (QOL), chronic care, coping, uncertainty, and survivor separately and in combination. DATA SYNTHESIS: Data were categorized into psychosocial, QOL, and physical symptoms and reviewed for design, sample size, method, and outcome. CONCLUSIONS: Ovarian cancer studies focus on women's symptoms and concerns during treatment. Needs and issues of long-term survivors lack exploration. The relationship of ovarian cancer survivorship and physical side effects of treatment or recurrence is insufficient given increasing survival rates. IMPLICATIONS FOR NURSING: The nature and management of physical symptoms, beyond pain, in ovarian cancer survivors need further study. Specifically, QOL and psychosocial issues for long-term survivors require study. Consequences for women who undergo major tissue debulking or multiple and aggressive courses of cytotoxic treatments must be understood to facilitate intervention. PMID- 16676013 TI - A patient orientation program at a comprehensive cancer center. AB - PURPOSE/OBJECTIVES: To determine which method of delivery of a cancer orientation program contributed to higher levels of satisfaction and lower levels of anxiety for newly diagnosed patients with cancer and each patient's support person. DESIGN: A randomized study of patients with cancer and caregivers into one of three delivery methods for an orientation program or a control arm. SETTING: A National Cancer Institute-designated comprehensive cancer center in the midwestern United States. SAMPLE: Newly registered patients with cancer diagnoses and their identified support people. METHODS: The intervention consisted of an orientation video and booklet delivered by three separate methods: class, drop-in sessions, or information mailed to homes. Participants completed questionnaires before the intervention and three weeks after the intervention. MAIN RESEARCH VARIABLES: State and trait anxiety, satisfaction, understanding of the organization, awareness and use of resources, and stress and coping. FINDINGS: The most successful accrual arms were the mailed intervention and control groups. The mailed intervention group compared to the control group reported higher levels of satisfaction with the cancer center, satisfaction with resources, understanding of the cancer center's structure, and satisfaction with healthcare professionals' communication with them. Fewer intervention group participants reported a lack of awareness of specific resources, and a larger percentage of the intervention group used available resources. Fewer benefits were noted with caregivers. CONCLUSIONS: The mailed intervention was successful in improving several patient outcomes. It was shown to be especially helpful to those with high trait anxiety. IMPLICATIONS FOR NURSING: A mailed orientation program can be a useful approach for increasing satisfaction with services. PMID- 16676014 TI - Complementary and alternative medicine: oncology nurses' experiences, educational interests, and resources. AB - PURPOSE/OBJECTIVES: To describe oncology nurses' experiences with patients communicating interest in or use of complementary and alternative medicine (CAM) therapies along with oncology nurses' CAM resources and educational interests. DESIGN: National mailed survey. SETTING: A national medical center and research institute. SAMPLE: A random sample of 850 Oncology Nursing Society (ONS) members who are RNs involved in direct patient care. METHODS: Respondents completed a demographic questionnaire and the Nurse Complementary and Alternative Medicine Knowledge and Attitude Survey. Initial analysis compared the demographics of the sample to the ONS membership. Descriptive analysis was used to further describe nurses' experiences with patients communicating interest in or use of CAM, nurses' interest in CAM education, and nurses' use of CAM resources. MAIN RESEARCH VARIABLES: Experiences, resources, interests, and CAM therapies. FINDINGS: Oncology nurses reported their experiences with patients who communicated interest in or use of CAM therapies. Respondents demonstrated considerable interest in learning more about specific CAM therapies and used a variety of resources to find information on CAM therapies. CONCLUSIONS: Assessing oncology nurses' experiences, resources used, and interest in learning about CAM therapies is the first step in determining the learning needs of oncology nurses in the direct patient care environment. The next step is to obtain baseline information on oncology nurses' CAM knowledge and attitudes for developing and providing appropriate education. CAM education will provide nurses with knowledge to support and advocate for their patients. IMPLICATIONS FOR NURSING: Oncology nurses are the bridge to help patients safely integrate evidence-based CAM therapies into conventional treatment. PMID- 16676015 TI - The relationship of sick leave benefits, employment patterns, and individual characteristics to radiation therapy-related fatigue. AB - PURPOSE/OBJECTIVES: To examine the relationship among sick leave benefits, employment patterns, individual characteristics, and fatigue in patients receiving radiation therapy. DESIGN: Prospective, longitudinal design. SETTING: A community hospital radiation oncology department. SAMPLE: 77 patients receiving radiation therapy to the breast, chest, head and neck, pelvis, or prostate. All were employed at the time of diagnosis. METHODS: The Piper Integrated Fatigue Model guided the study. The Revised Piper Fatigue Scale (PFS), Brief Fatigue Inventory, and a single-item scale were used to measure five dimensions of subjective fatigue. Sick leave, employment, individual characteristics, and fatigue were measured at baseline, weekly during treatment, and at one month post treatment. MAIN RESEARCH VARIABLES: Employment patterns, availability of sick leave benefits, and fatigue. FINDINGS: Mean total fatigue scores on the PFS ranged from 0-4.77 at baseline (mean = 0.46, SD = 0.93), 0-8.77 at the completion of treatment (mean = 2.84, SD = 2.40), and 0-4.82 at one month post-treatment (mean = 0.77, SD = 1.20). Side effects, education, living situation, age, treatment site, and work were associated with fatigue along the trajectory of radiation therapy. Study participants who were working at the end of radiation had lower fatigue scores than those who were not. Availability of sick leave benefits was associated with employment patterns during treatment. CONCLUSIONS: Work may have benefits during radiation therapy but may be affected by radiation therapy-related fatigue. IMPLICATIONS FOR NURSING: Management of treatment side effects, including fatigue, may help patients remain in the workforce during radiation. PMID- 16676016 TI - Prospective assessment of fatigue and health status in Greek patients with breast cancer undergoing adjuvant radiotherapy. AB - PURPOSE/OBJECTIVES: To describe fatigue in women with breast cancer undergoing radiotherapy and to explore the impact of fatigue on their health status. DESIGN: Prospective, descriptive, repeated measures. SETTING: A major oncology center, Saint Savvas Cancer Hospital, in Athens, Greece. SAMPLE: Consecutive sample of 106 women (mean age = 55 + 12), with histologically confirmed diagnosis for stage I or II breast cancer who were receiving adjuvant radiotherapy for approximately six weeks. METHODS: Data were collected with the Revised Piper Fatigue Scale (PFS) and the Short Form-36 (SF-36) Health Survey Scale in the first two days of radiotherapy (T0), during the third week (T1), and during the last week of treatment (T2). MAIN RESEARCH VARIABLES: Fatigue, health status. FINDINGS: Across subjects analysis revealed that fatigue increased during radiotherapy in patients with breast cancer regardless of stage, type of surgery, or whether they received chemotherapy (p 0.05). Between-subject analysis revealed that no differences existed in the PFS between different groups (chemotherapy versus no chemotherapy, breast conservation versus mastectomy, stage I versus stage II) at each measurement point. A negative correlation was found between the subscales of the PFS and all of the subscales of the SF-36. CONCLUSIONS: Fatigue intensity increased significantly during the course of radiotherapy, and patients experienced a significant deterioration in their overall health status. IMPLICATIONS FOR NURSING: Findings contribute to the growing body of evidence regarding fatigue and its impact on health status in Greek patients with breast cancer and provide insights for effective nursing assessment, patient education, and symptom management. PMID- 16676017 TI - Fatigue and physical activity in patients undergoing hematopoietic stem cell transplant. AB - PURPOSE/OBJECTIVES: To examine the patterns of fatigue, physical activity, health status, and quality of life before and after high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) and to examine the feasibility of obtaining real-time fatigue and physical activity data. DESIGN: Prospective, repeated measures. SETTING: Two midwestern academic medical centers. SAMPLE: Convenience sample of autologous or allogeneic patients undergoing HSCT (N = 20 baseline, N = 17 post-transplant). METHODS: Subjects were assessed over a five day period before and after HSCT for a total of 10 days. Subjects rated fatigue intensity three times daily and wore a wrist actigraph to measure physical activity. At the end of both five-day periods, subjects completed measures of perceived health status (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30) and life satisfaction Quality of Life Index). MAIN RESEARCH VARIABLES: Fatigue, physical activity, perceived health status, and quality of life. FINDINGS: Study results indicate that fatigue significantly increased and physical activity decreased following high-dose chemotherapy and HSCT. The decline coincided with diminished physical, emotional, role, and cognitive functioning. The symptoms that patients experienced (i.e., fatigue, pain, nausea and vomiting, sleep disturbances, appetite loss, and diarrhea) increased during the acute post-transplant period. No significant changes in life satisfaction were found. CONCLUSIONS: The study findings suggest that patients receiving high-dose chemotherapy followed by HSCT experience increased fatigue, reduced physical activity, diminished functioning, and poorer quality of life immediately after transplant. Findings demonstrate that real-time fatigue and physical activity data can feasibly be collected in acutely ill patients. IMPLICATIONS FOR NURSING: Patients undergoing HSCT require considerable supportive nursing care immediately following transplant. Clinicians and researchers need to strive for effective symptom management to improve the likelihood of successful outcomes. PMID- 16676018 TI - Quality of life of family caregivers of patients with cancer: a literature review. AB - PURPOSE/OBJECTIVES: To systematically review literature regarding the quality of life (QOL) of family caregivers of patients with cancer and evaluate the instruments measuring family caregivers' QOL. DATA SOURCES: PubMed, CINAHL, PsycINFO, ISI Web of Science, and EBSCO electronic databases; published literature. DATA SYNTHESIS: Overall, the QOL of family caregivers of patients with cancer varied. Influences related to family caregivers' QOL were identified. Global and generic measures have been used because of the lack of specific instruments. Comparisons are complicated because several measures were used at different times along the illness trajectory with caregivers caring for people with various types and stages of cancer. CONCLUSIONS: A more explicit definition of QOL for family caregivers of patients with cancer and specific instruments suitable for different cultures are needed to enhance knowledge. IMPLICATIONS FOR NURSING: Maintaining the QOL of caregivers is important in their ability to provide the care required to keep family members with cancer in the community. Nurses must provide care to maintain caregivers' QOL. PMID- 16676019 TI - Thorough skin self-examination in patients with melanoma. AB - PURPOSE/OBJECTIVES: To examine the feasibility of using Weinstock et al.'s thorough skin self-examination (TSSE) assessment in patients with melanoma, to describe TSSE characteristics of patients with melanoma, and to explore associations of personal and disease variables with TSSE. DESIGN: Cross sectional, descriptive feasibility study; part of a larger study of melanoma in families. SETTING: Outpatient melanoma clinics in a National Cancer Institute designated comprehensive cancer center. SAMPLE: Purposive sample of 70 predominantly white participants (47% women, 53% men), with a mean age of 65 years (SD = 11 years) and pathologically confirmed cutaneous melanoma (any stage). METHODS: Weinstock et al.'s TSSE assessment (self-report of the number of times patients examined the surface of seven specific body areas during the prior two months) and items regarding partnered TSSE and skin examination from healthcare providers. MAIN RESEARCH VARIABLES: Frequency of TSSE and healthcare provider skin examination, partnered TSSE, and reasons for not performing TSSE. FINDINGS: Forty-one (59%) participants reported performing TSSE; by Weinstock et al.'s criteria, only 23 (33%) practiced TSSE. Use of a partner was significantly associated with TSSE (p = 0.001); patients indicated high rates of skin examination by healthcare providers. CONCLUSIONS: Patients with melanoma are at high risk for recurrent disease. TSSE contributes to early detection of melanoma. Although Weinstock et al.'s TSSE assessment is feasible for use among patients with melanoma in a clinical setting, the focus should be on examination of specific body areas, rather than global skin examination. Overall, patients with melanoma had a low frequency of TSSE; however, data regarding previous knowledge or instruction of TSSE were not collected. Involving a partner enhances the frequency of TSSE. IMPLICATIONS FOR NURSING: Patients with melanoma should be informed of the importance of conducting systematic TSSE and using a partner during examination; however, some patients may prefer skin examination by healthcare providers. Measurement of TSSE self-report merits further study. PMID- 16676020 TI - Pain, sleep disturbance, and fatigue in children with leukemia and their parents: a pilot study. AB - PURPOSE/OBJECTIVES: To determine the feasibility of collecting symptom data at home from school-age children with acute lymphoblastic leukemia (ALL) and from their fathers and mothers and to obtain initial descriptions of pain, sleep disturbance, and fatigue experienced by the family members at home. DESIGN: Prospective and descriptive. SETTING: Children's homes in Oregon and southwestern Washington. SAMPLE: 9 children with ALL (aged 8-16 years), 6 fathers, and 7 mothers. The children received vincristine during the maintenance phase of their outpatient chemotherapy treatments. METHODS: With age-appropriate, paper-and pencil diaries and wrist actigraphy, data were collected for three days in the families' homes. Families were reminded by telephone to complete their sleep and activity diaries. MAIN RESEARCH VARIABLES: Pain, sleep disturbance, and fatigue in school-age children and their fathers and mothers. FINDINGS: Most of the families who were approached indicated willingness to participate in the study. After receiving outpatient chemotherapy, the children reported pain, sleep disturbance, and fatigue data over three days. Fathers and mothers also reported symptoms. Actigraphy showed children waking more often during the night than mothers or fathers. CONCLUSIONS: Children's pain, sleep disturbance, and fatigue suggest that the symptoms are influencing families' quality of life. Larger studies are needed to examine the symptom patterns and health outcomes of children, fathers, and mothers over the course of chemotherapy. IMPLICATIONS FOR NURSING: Improving sleep and managing pain and fatigue after chemotherapy treatment for children with ALL may improve health outcomes for children and parents. PMID- 16676021 TI - Measuring subjective side effects and symptoms in palliative photodynamic therapy. AB - PURPOSE/OBJECTIVES: To evaluate the reliability and validity of the Photodynamic Therapy Side Effects and Symptoms Scale (PSES) and to begin to describe patient reported outcomes of photodynamic therapy (PDT). DESIGN: Repeated-measures instrument validation. The PSES uses a 10-point numeric scale to evaluate side effects or symptom trouble or burden on 13 items. SETTING: A Pacific Northwest community hospital. SAMPLE: 14 patients with end-stage lung or esophageal cancer undergoing palliative PDT. METHODS: Participants completed five PSES surveys (i.e., at baseline and once a week for four weeks after PDT). Weekly phone calls were made to assess functional status, operationalized as Karnofsky Performance Status. MAIN RESEARCH VARIABLES: Symptoms and functional status. FINDINGS: The PSES possessed acceptable internal consistency reliability and concurrent validity. Functional status declined in the first week after PDT, concurrent with an increase in side-effect and symptom burden. Photosensitivity became more burdensome over time but was never extremely burdensome. CONCLUSIONS: The feasibility of measuring a diverse set of side effects and symptoms in end-stage cancer with a single-page, large-type instrument essentially was supported. The study provided preliminary information about side effects and symptoms in patients undergoing palliative PDT. IMPLICATIONS FOR NURSING: Nurses often are called on to provide information to patients considering various treatment options. This study offers the first data on patient-reported outcomes of palliative PDT that clinicians can use to help in answering inquiries. The design of the PSES may be replicated by researchers working with other populations with end-stage disease to reduce respondent burden and decrease attrition. PMID- 16676022 TI - Social support and psychological and physical states among Japanese patients with breast cancer and their spouses prior to surgery. AB - PURPOSE/OBJECTIVES: To assess social support and psychological and physical states among Japanese patients with breast cancer and their spouses prior to surgery. DESIGN: Descriptive, comparative, and correlational. SETTING: A general hospital in northern Japan. SAMPLE: 38 Japanese patients with breast cancer and their spouses (N = 76). METHODS: The Japanese versions of three questionnaires were used to collect data before surgery: the Interpersonal Relationship Inventory, the General Health Questionnaire, and the Physical States Interview Form. MAIN RESEARCH VARIABLES: Social support (support, conflict, and reciprocity), social network, and psychological and physical states. FINDINGS: Significant differences were found in support and reciprocity between patients and their spouses. However, no significant differences were found in social network, conflict, or psychological states between patients and their spouses. Moreover, some significant correlations were found in the variables of conflict, social network, and psychological and physical states. CONCLUSIONS: Japanese patients with breast cancer perceived more support and reciprocity than their spouses before their breast surgery. Conflict was significantly correlated with psychological states among Japanese women with breast cancer and their spouses. IMPLICATIONS FOR NURSING: Healthcare professionals need to consider social support as an important factor to help Japanese patients with breast cancer and their spouses cope with the disease. PMID- 16676024 TI - Collimation testing by use of the Lau effect coupled with moire readout. AB - We present an easy, simple, and inexpensive technique for checking the quality of the collimation of optical beams using the Lau effect combined with moire readout. The experimental arrangement consists of a modified Lau-based interferometer in which a white-light incoherent source illuminates a set of two gratings. A collimating lens is placed between the two gratings such that the self-images of the second grating are formed. The third grating is positioned at one of the self-imaging planes forming moire fringes. The type of the moire fringe demonstrates the quality of collimation of the optical beam. The necessary theoretical background is presented and the results of our experimental investigation are reported. The technique can also be used for accurate determination of the focal length of a collimating lens using low-cost components. PMID- 16676023 TI - Profiles of a high-aspect-ratio grating determined by spectroscopic scatterometry and atomic-force microscopy. AB - The new and fast scatterometry method called optical diffraction microscopy is compared with atomic-force microscopy by use of cross-section scanning-electron microscope images as references. The sample is a high-aspect-ratio grating with a period of approximately 1000 nm. To allow the atomic-force microscope to track all parts of the grating profile, the grating is investigated at different tilt angles. The measured quantities of the profile include sidewall angle gamma (approximately 90 degrees), groove height h (approximately 2000 nm), and degree of filling f (approximately 40%). The two methods, which respond to quite different material properties, give consistent results within standard uncertainties of u(gamma) A polymorphism and a variable number of repeats in intron 7 of FVII gene did not significantly influence warfarin dose. The mean warfarin doses increased with the number of (CAA) repeats in the GGCX gene, but the differences were significant only in the CYP2C9*1/*1 subgroup of patients (p = 0.032). Common polymorphism (6484C > T) in intron 1 of the VKORC1 gene led to lower warfarin dose requirement; the means were 5.70 (95% C.I. 4.95-6.45), 3.49 (3.07-3.90) and 2.11 (1.80-2.42) mg/day for 6484 CC, CT and TT genotypes, respectively (p < 0.001). In contrast, 9041G > A polymorphism in 3'UTR of theVKORC1 gene led to higher warfarin dose requirement; the means were 3.09 (2.58- 3.60), 4.26 (3.69-4.82) and 5.86 (4.53-7.19) mg/day for 9041 GG, GA and AA genotypes, respectively (p < 0.001). With a regression model we explained 60.0% of variability in warfarin dose, which was due to gene polymorphisms (CYP2C9, VKORC1), age and body-surface-area. When aiming for individualised warfarin therapy, at least VKORC1 polymorphisms should be included in predictive genotyping besides CYP2C9. PMID- 16676069 TI - Antithrombin reduces reperfusion-induced liver injury in mice by enhancing sensory neuron activation. AB - We recently demonstrated that antithrombin (AT) reduces ischemia/reperfusion (I/R)-induced liver injury in rats by increasing hepatic tissue levels of calcitonin gene-related peptide (CGRP), a neuropeptide released from the sensory nerve endings. In the present study, we examined the effect of AT on I/R-induced liver injury in wild type mice (CGRP+/+) and congenitally alphaCGRP-deficient mice (CGRP-/-). We also investigated any effects of AT on CGRP release from dorsal root ganglion neurons (DRG) isolated from CGRP+/+. Based on results obtained in the present study, we attempted to determine if the anti-inflammatory activity of AT in vivo is dependent mainly on sensory neuron activation. AT enhanced ischemia/reperfusion-induced increases in hepatic tissue levels of CGRP and 6-keto-PGF(1alpha), a stable metabolite of PGI2, in CGRP+/+, but it did not enhance these increases in CGRP-/-. AT inhibited reperfusion-induced increases in serum alanine aminotransferase levels by increasing hepatic tissue blood flow and by attenuating increases in hepatic levels of tumor necrosis factor and myeloperoxidase in CGRP+/+, although it showed neither of these therapeutic effects in CGRP-/-. AT increased CGRP release from cultured DRGs only in the presence of anandamide, and AT-induced increase in CGRP release was not observed in the presence KT5720, an inhibitor of protein kinase A (PKA). AT markedly increased intracellular levels of cAMP in the presence of anandamide. These results strongly suggest that AT might reduce I/R-induced liver injury by enhancing activation of the sensory neurons through activation of PKA in sensory neurons. PMID- 16676070 TI - Anti-beta2-glycoprotein I antibodies are associated with pregnancy loss in women with the lupus anticoagulant. AB - The presence of lupus anticoagulant (LA) predisposes to fetal loss and to venous and arterial thrombosis; however, a subgroup of women is unaffected by pregnancy loss. Currently, no predictive markers are available for the identification of women positive for LA at increased risk for pregnancy loss. It was the aim of our study to investigate whether increased anti-beta2-GPI-antibodies predict pregnancy loss in women positive for LA. We performed a cross-sectional study in a cohort of 39 women with persistent LA, who had in total 111 pregnancies. Fifteen women had exclusively normal pregnancies (30 pregnancies) and 24 women had pregnancy losses (81 pregnancies). Anti-beta2-GPI-antibodies were determined using a semiquantitative enzyme linked immunoassay (QUANTA Lite beta2 GPI IgG and IgM; Inova Diagnostics). Increased levels of anti-beta2-GPI antibodies were significantly associated with pregnancy loss [odds ratio (OR) 9.6, 95% confidence interval (CI) 1.6-56.4]. This risk was even higher in the subgroup of women (n = 16) with more than two miscarriages or fetal loss after the first trimester [OR 13.1, 95% CI 1.4-126.3]. There was no significant association between anticardiolipin antibodies and pregnancy loss [OR 3.5, 95% CI 0.7-17.6]. The co existence of anti-beta2-GPI and anticardiolipin antibodies was also predictive for pregnancy loss [OR 6.1, 95% CI 1.3-29.7]. Interestingly, the prevalence of thrombosis was similar between women with normal pregnancy (87%) and those with pregnancy loss (75%). We conclude that increased levels of anti-beta2-GPI antibodies are predictive for pregnancy loss among women positive for LA, and that prophylactic treatment should be considered in these women even without a history of previous pregnancy loss. PMID- 16676071 TI - Interleukin-6 and interleukin-6 promoter polymorphism (-174) G > C in patients with spontaneous venous thromboembolism. AB - Increased levels of interleukin-6 (IL-6) have been reported in patients with a history of venous thromboembolism (VTE); however, prospective studies did not confirm an association between inflammatory markers that are highly correlated with IL-6 and the risk ofVTE. It was the aim of our study to investigate the association of IL-6 and its promoter polymorphism (-174) G > C with the risk of spontaneousVTE. IL-6 was measured in 128 patients with deep venous thrombosis (DVT,70 w/58 m),105 with pulmonary embolism (PE, 58 w/47 m) and 122 healthy controls (60 w/62 m) with a highly sensitive ELISA (Quantikine HS Human IL-6 Immunoassay, RnDSystems). The promoter polymorphism was determined by genotyping, allele specific PCR was followed by high resolution gel-electrophoresis. Median concentrations [interquartile ranges] were 2.37 [1.51-3.89] (pg/ml) in patients with DVT, 2.83 [1.83-4.87] in those with PE and 2.51 [1.71-4.78] in controls (p = 0.6, p = 0.4). Hetero- or homozygous carriers of the C allele (71% in DVT, 67% in PE and 59% among controls) did not have higher IL-6 levels than homozygous carriers of the G allele (median 2.60 vs. 2.59 pg/ml, p = 0.7). In conclusion, we found no association of IL-6 and its promoter polymorphism (-174) G > C with the risk of spontaneous VTE. PMID- 16676072 TI - Air travel and fatal pulmonary embolism. AB - Although long-distance air travel is commonly regarded as a risk factor for venous thromboembolism, the risk of clinically important events has not been well defined. We estimated the absolute risk of dying from pulmonary embolism following long-distance air travel in a national population-based descriptive study of 121 men and women who were aged 15-59 years (the age range in which the majority of international arrivals are found) and whose underlying cause of death was certified as codes 415.1, 451, or 453 of the International Classification of Diseases (ninth revision). Eleven cases had undertaken long-distance air travel in the four weeks before the onset of the fatal episode. The estimated risks of fatal pulmonary embolism following a flight of at least three hours' duration were 0.5 (95% CI 0.2-1.2) and 0.6 (95% CI 0.2-1.4) per million arrivals for overseas visitors and New Zealand residents, respectively. For air travel of more than eight hours' duration, the risk in New Zealand residents was 1.3 (95% CI 0.4 3.0) per million arrivals. We also conducted a case-control study based on those cases who were normally resident in New Zealand and registered on the electoral roll (n = 99). For each case, four controls matched for sex, age, and electorate, were randomly selected from the electoral roll. In the key analysis (based on 88 cases and 334 controls), the adjusted odds ratio for travellers who had flown for more than eight hours was 7.9 (95% CI 1.1-55.1) compared with those who did not undertake a long-distance flight. Long-distance air travellers have a higher risk of dying from pulmonary embolism than non-travellers, but the absolute risk in people aged 15-59 years appears to be very small. PMID- 16676073 TI - Platelets enhance CD4+ lymphocyte adhesion to extracellular matrix under flow conditions: role of platelet aggregation, integrins, and non-integrin receptors. AB - The purpose of this study was to examine the role of platelets in CD4+ T lymphocyte adhesion to subendothelial extracellular matrix (ECM). Herpesvirus saimiri (HVS)-infected CD4+ T cells were incubated on ECM. An image analysis was used to evaluate T cell adhesion. Under static condition, T cell activation with 4-alpha-Phorbol 12-myristate 13-acetate (PMA) resulted in a 2.6-fold increase in cell adhesion. However, adhesion was not affected by platelets. In contrast, under flow (200s(-1)), platelets markedly enhanced both resting and PMA-activated T cell adhesion (33- and 48-fold), forming lymphocyte-platelet co-aggregates that contain approximately 90% of the adherent T cells. Abrogation of platelet aggregation with tirofiban inhibited formation of platelet-T cell co-aggregates under flow and reduced T cell adhesion by 74%. Separate and combined blockade of CD40L and P-selectin glycoprotein-1 (PSGL-1) on PMA-activated lymphocytes reduced adhesion under flow in the presence of platelets by 28%, 33%, and 55%, respectively. Blockade of beta1-integrins decreased adhesion under both static and flow conditions (by 35% and 44%, respectively), while blockade of beta2 integrin reduced adhesion only under static condition (by 23%). A similar adhesion pattern was observed using CD4+ T cells isolated from normal donor peripheral blood. In conclusion, platelets support CD4+ lymphocyte adhesion to ECM under flow by formation of heterotypic platelet-lymphocyte coaggregates involving alphaIIbbeta3 integrin and beta1-related integrins, as well as CD40L and PSGL-1. PMID- 16676074 TI - The influence of fibrin polymerization and platelet-mediated contractile forces on citrated whole blood thromboelastography profile. AB - Thromboelastography analysis providing a global assessment of coagulation is gaining new interest in clinical practice. MinimalTF triggered whole blood thromboelastography provides a valuable tool for studying the kinetics of clot formation (expressed by the parameters R, K and alpha-angle) and the physical characteristics of the clot, such as its firmness and the elastic modulus shear (expressed by the parameters maximal amplitude MA and G). We studied the influence of fibrin polymerization and platelet functional status on each parameter of thromboelastographic trace obtained by minimalTF activation inWB by employing increasing concentrations of a fibrin polymerization inhibitors (the tetrapeptide Gly-Pro-Arg-Pro-OH.AcOH; Pefabloc-FG) and an inhibitor of actin polymerization (Cytochalasin D). Pefabloc-FG at concentrations higher than 5 mg/ml prolonged the R and K times and decreased the alpha-angle in a concentration-dependent manner but it did not modify MA and G parameters. At the concentration of 5 mg/ml, Pefabloc-FG completely inhibited clot formation. Cytochalasin D had no effect on R time but decreased the alpha-angle, MA and G parameters by reaching a plateau at the concentration of 5 microM. The effect of cytochalasin D was more pronounced on MA and G than on the alpha-angle. A combination of both Pefabloc-FG (0.5 mg/ml) and cytochalasin D (50 microM) significantly decreased alpha-angle compared to control as well as their single effect. However, G value was dramatically reduced in the presence of cytochalasin D exposure, without any additional effect when both inhibitors were combined. This study confirms the importance of fibrin polymerisation on the kinetics of thrombus formation and demonstrates the close association between the quality of the thrombus and the functional status of platelets. Normal platelet contractile forces are of major importance for the maximum amplitude of TEG which is related to the strength and elastic modulus of the thrombus. PMID- 16676075 TI - Plasminogen activator inhibitor-1 potentiates LPS-induced neutrophil activation through a JNK-mediated pathway. AB - Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily, modulates fibrinolysis by interacting with proteolytic mediators, including urokinase plasminogen activator (uPA). Although the roles of uPA and PAI-1 in plasmin generation and the degradation of fibrin are well known, recent evidence also suggests that they can participate in acute inflammatory conditions that involve neutrophil activation. In the present experiments, we found that the addition of PAI-1 to LPS- stimulated neutrophils resulted in enhanced nuclear translocation of NF-kappaB and increased production of the proinflammatory cytokines IL-1beta, Tnf-alpha, and Mip-2. uPA and the kringle domain (KD) of uPA potentiated cytokine expression and NF-kappaB activation by neutrophils cultured with LPS, and had additive effects when combined with PAI-1. The c-Jun N-terminal kinase (JNK) was activated after exposure of resting neutrophils to PAI-1 or the uPA KD. Enhanced JNK activation, but not that of other kinases induced by LPS, was present in neutrophils cocultured with PAI-1 or uPA KD. Inhibition of JNK activation prevented the potentiation of expression of proinflammatory cytokines induced by PAI-1 or uPA KD in LPS stimulated neutrophils. These results demonstrate that PAI-1 and uPA KD enhance LPS-induced neutrophil responses through their effects on JNK mediated pathways. PMID- 16676076 TI - Group B streptococcus isolates from septic patients and healthy carriers differentially activate platelet signaling cascades. AB - Infection with group B streptococcus (GBS) is the most common cause of early onset neonatal sepsis in many countries, leading to neonatal morbidity and mortality. There is much evidence for a direct involvement of platelets in the pathogenesis of inflammation and sepsis. Several bacteria are known to directly interact with platelets leading to activation and aggregation, a phenomenon also observed with GBS. Here, we demonstrate that GBS rapidly bound to platelets; however, only strains isolated from septic patients bound fibrinogen on their surface and induced platelet thromboxane synthesis, platelet aggregation, and P selectin (CD62P) expression. In contrast, GBS strains isolated from healthy newborns or healthy pregnant women induced only shape change, but not platelet thromboxane synthesis, platelet aggregation, or CD62P expression. All GBS strains investigated were able to activate FcgammaRIIA receptor signaling pathways including phospholipase C gamma2 (PLCgamma2), as well as calcium/calmodulin dependent myosin kinase II (CaMKII) and phosphorylation of myosin light chain (MLC). In contrast, protein kinase C (PKC) was exclusively activated by GBS strains isolated from septic patients, and p38 mitogen activated protein kinase (p38 MAP kinase) was preferentially activated by septic GBS strains. Furthermore, stress signaling kinase SEK1/MKK4 and focal adhesion kinase (FAK) were activated by all tested GBS strains in a FcgammaRIIA-independent way. This study demonstrates that septic, but not colonizing, GBS strains bind fibrinogen on their surface, and that septic GBS strains influence platelet function not only via the FcgammaRIIA receptor, but also via pathways distinct from IgG-mediated signalling. These mechanisms lead to platelet aggregation and secretion, thereby possibly modulating the pathophysiologic course of GBS infections. PMID- 16676077 TI - Benefit/risk profile of high-dose antithrombin in patients with severe sepsis treated with and without concomitant heparin. AB - A randomised, prospective, placebo-controlled phase III multicentre clinical trial (KyberSept) has been performed to test the efficacy of high-dose antithrombin therapy in patients with severe sepsis. Concomitant low-dose heparin has been routinely given in two thirds of patients for deep vein thrombosis prophylaxis. This study analyses heparin - antithrombin interactions in terms of long-term mortality, adverse events, and thromboembolic events. From a total of 2,314 patients with severe sepsis (placebo: n = 1,157; antithrombin: n = 1,157) 1,616 patients (placebo: 811, antithrombin: 805) received heparin concomitantly with study drug (antithrombin 30,000 IU) over four days, whereas 698 patients (346 and 352, respectively) did not. In patients with no concomitant heparin, 28 day mortality was lower with antithrombin than with placebo (37.8% vs. 43.6%; absolute reduction: 5.8%; risk ratio: 0.860 [0.725-1.019]), which increased until day-90 (44.9% vs. 52.5%; absolute reduction: 7.6%; risk ratio: 0.851 [0.735 0.987]). In patients with concomitant heparin, no effect of antithrombin on mortality was seen (28-day mortality: 39.4% vs. 36.6%; absolute increase: 2.8%; risk ratio: 1.08 [0.96-1.22]). Frequency of use of concomitant heparin increased during conduct of the study. Increased bleeding incidences were reported with antithrombin plus concomitant heparin as compared to antithrombin alone. Rates of thromboembolic events were similar when antithrombin was given with or without concomitant heparin. In the treatment of severe sepsis, high-dose antithrombin may sufficiently protect against development of venous thromboembolism when no concomitant heparin is given. Combined administration of the two increases bleeding risk and probably abolishes efficacy of antithrombin. PMID- 16676078 TI - Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA. AB - Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI 1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis. PMID- 16676079 TI - In vivo antithrombotic synergy of oral heparin and arginine: endothelial thromboresistance without changes in coagulation parameters. AB - On the basis of suggested clinical efficacy in an uncontrolled study in ninety seven patients with unstable angina, an animal study was conducted to investigate antithrombotic synergy between orally administered heparin and arginine. A rat venous thrombosis model tested the difference in thrombus formation when heparin (7.5 mg/kg) and arginine (113 mg/kg) were administered, alone or in combination, by stomach tube with a minimum of 20 rats/group. Oral heparin, arginine, and heparin plus arginine reduced thrombus formation by 50%, 75%, and 90%, respectively, when compared to saline administration. Heparin was recovered from endothelium, yet there was little or no observable plasma anticoagulant activity. An orally administered low-molecular-weight anticoagulant glycosaminoglycan mixture, sulodexide (7.5 mg/kg), showed an 88% reduction in stable thrombus formation when administered alone but showed no synergy with oral arginine. A 28 day study with oral sulodexide (2.9 mg/kg) and arginine (43.9 mg/kg), 20 rats/group, showed antithrombotic activity with minimal anticoagulant activity indicating suitability for long term treatment. These findings suggest the endothelial localization of heparin and a synergistic antithrombotic effect for orally administered heparin and arginine. PMID- 16676080 TI - Cleavage of human 7-domain VCAM-1 (CD106) by thrombin. AB - Vascular cell adhesion molecule 1 (VCAM-1, CD106) is expressed as a type I transmembrane integrin counter-receptor on activated endothelium and mediates white blood cell attachment. The alternatively spliced 7-domain (7d) form of VCAM 1 contains a potential thrombin cleavage site. Thrombin proteolysis of 7d-VCAM-1 may help regulate adhesive activity ofVCAM-1. We determined whether 7d-VCAM-1 is proteolyzed and rendered inactive by thrombin. Recombinant extracellular domain of 7d-VCAM-1 was cleaved by thrombin to generate 33- and 44-kDa products. Cleavage was in the sequence PGPR/IAAQIG near the N-terminal border of the alternatively spliced fourth immunoglobulin (Ig)-like module. There was no cleavage of 6d-VCAM-1 lacking the fourth module. Expression of full-length 7d VCAM-1 presented on Chinese hamster ovary (CHO) monolayers, as detected by flow cytometry with an antibody directed to Ig-like modules 1-3, was reduced by thrombin treatment whereas there was no reduction in the expression of full length 6d-VCAM-1. Adhesion of blood eosinophils to full-length 7d-VCAM-1 was reduced after treatment of CHO cells with thrombin, whereas adhesion to full length 6d-VCAM-1 was not affected. We conclude that cleavage of 7d-VCAM-1 by thrombin is a potential mechanism for differential regulation of VCAM-1 splice forms in white blood cell adhesion and trafficking. PMID- 16676081 TI - Serum osteoprotegerin in young survivors of myocardial infarction. AB - Osteoprotegerin (OPG) is a member of the tumour necrosis factor superfamily and is involved in the regulation of bone metabolism and vascular calcification. Increased serum OPG levels have been reported in patients with stable angina pectoris and survivors of myocardial infarction with heart failure. The purpose of the present study was to determine serum OPG levels in young survivors of acute myocardial infarction (MI), and the relationship between OPG, homocysteine, sCD40L and coagulation factors in blood. Fifty-eight patients with verified MI, 40-60 years of age, were recruited 1-4 years after the acute event into an age- and sex- matched case control study with controls recruited from the general population. Serum OPG levels were similar in cases (2.41 ng/ml, 2.11-2.77 ng/ml) (mean, 95% CI) and controls (2.43 ng/ml, 2.11-2.79 ng/ml) (p = 0.92). Significant correlation between OPG and homocysteine was found in patients (r = 0.30, p = 0.02) and controls (r = 0.35, p = 0.007). A significant negative correlation was found between OPG and sCD40L in patients (r = -0.51, p < 0.001), but not in controls (r = 0.001, p = 0.96). No associations were found between serum OPG and markers of coagulation activation. The present study shows that serum OPG level was not increased in young survivors of uncomplicated myocardial infarction. Serum OPG levels were not associated with thrombin generation assessed by thrombin-antithrombin complexes (TAT), but a positive association between serum OPG and homocysteine was found. PMID- 16676082 TI - ADAMTS13-binding IgG are present in patients with thrombotic thrombocytopenic purpura. AB - Functional assays are commonly used to measure the antibodies of ADAMTS13 found in patients of thrombotic thrombocytopenic purpura (TTP). In this study we used an enzyme-linked immunoassay to analyze the ADAMTS13-binding IgG levels in six groups of individuals: normal, random hospitalized patients, acute TTP, TTP after receiving plasma therapy, TTP in remission, and other types of thrombotic microangiopathy (TMA). The results showed that ADAMTS13-binding IgG levels were elevated in 100% of the acute TTP group, 75% of the TTP group after receiving plasma therapy, and 40% of the remission group. Overall, the ADAMTS13-binding IgG levels correlated with the inhibitory activity levels againstADAMTS13 (r = -0.69, P < 0.0001). The assay also detected elevated IgG binding levels in 5% - 15% of the normal, random, and other TMA control groups. Addition of purified ADAMTS13 protein to the plasma samples suppressed the IgG binding in each of the acute TTP patients, but in none of the non-TTP groups. Serial measurement in a patient that had two exacerbations of TTP within the first three weeks revealed that the ADAMTS13 activity levels remained <0.1 U/ml during this period, and the ADAMTS13 binding IgG remained elevated, suggesting that ADAMTS13 analysis may provide valuable insight to the disease status during the course of therapy. Analysis of ADAMTS13-binding IgG is helpful for the diagnosis and management of TTP. PMID- 16676083 TI - Combined factor V - factor VIII deficiency (F5F8D): compound heterozygosity for two novel truncating mutations in LMAN1 in a consanguineous patient. PMID- 16676084 TI - Successful pregnancy in a patient with factor V deficiency: case report and review of the literature. PMID- 16676085 TI - Deep venous thrombosis associated with acquired angioedema type II in a patient heterozygous for the mutation of factor V Leiden: effective treatment and follow up for four years. PMID- 16676086 TI - Antiphospholipid syndrome, hyperhomocysteinaemia and normocalcemic hyperparathyroidism. PMID- 16676087 TI - Quantitation of Vervet monkey (Chlorocebus aethiops) plasminogen activator inhibitor-1 in plasma and platelets. PMID- 16676088 TI - Genetic background and inhibitors in previously untreated or minimally treated young patients with severe haemophilia A treated with sucrose-formulated recombinant factor VIII. PMID- 16676089 TI - Increased plasma plasminogen activator inhibitor-1 levels caused by additional aspirin treatment. PMID- 16676090 TI - Marburg I polymorphism of factor VII-activating protease and risk of recurrent venous thromboembolism. PMID- 16676092 TI - Polymorphisms of the protein Z-dependent protease inhibitor (ZPI) gene and the risk of venous thromboembolism. PMID- 16676093 TI - Correlation of a new point-of-care test with conventional optical aggregometry for the assessment of clopidogrel responsiveness. PMID- 16676094 TI - Recurrent venous thromboembolism under anticoagulant therapy: a high risk in adenocarcinoma? PMID- 16676095 TI - High incidence of isolated subsegmental pulmonary emboli on multi-slice spiral CT: a comparative clinical study. PMID- 16676096 TI - Venous thrombosis is not increased in younger women on genuine oestrogen postmenopausal hormonal replacement therapy: results from the Danish Osteoporosis Prevention Study (DOPS). PMID- 16676097 TI - [Pathogenic role of PTEN tumor suppressor gene in ovarian cancer associated to endometriosis]. AB - BACKGROUND: Endometrioid carcinoma and clear cell carcinoma of the ovary are associated to endometriosis. Somatic mutations of PTEN (10q23.3) are present in endometrial endometrioid carcinoma. Therefore, these mutations could be also present in ovarian tumors. Molecular studies show that solitary endometriotic cysts are monoclonal, have aneuploid DNA, have a loss of 9p,11q and 22q heterozygosity (LOH) and a higher cellular proliferation index of the epithelial component. AIM: To determine the cellular proliferation index using Ki 67, the immunohistochemical expression of PTEN and LOH in patients with ovarian endometriosis without atypia (EN), ovarian endometriosis with atypia (EA) and endometriosis with adjacent ovarian carcinoma (ET). MATERIAL AND METHODS: Paraffin embedded samples of 37 endometrioid and clear cell carcinomas of the ovary (CC/CE), 15 solitary ovarian EN and 15 ovarian EA, were studied. Expression of Ki 67 and PTEN was measured by immunohistochemistry. LOH of 10q23.3 locus was measured by polymerase chain reaction. RESULTS: Ki 67 was 5.5 and 2.3% in EA and EN, respectively (p <0.005). There was a histological correlation between EA and a higher cellular proliferation index. PTEN was negative in 5 of 15 EN, 9 of 15 EA and 30 of 37 CE/CC. There was a correlation between LOH and loss of PTEN protein in EN, EA and ET (60%). CONCLUSIONS: Negative expression on PTEN in EN; EA; ET and CE/CC is a manifestation of the inactivation of this gene. The mechanisms that cause this inactivation, must be elucidated. PMID- 16676098 TI - [Effect of oral zinc administration on insulin sensitivity, leptin and androgens in obese males]. AB - BACKGROUND: Zinc is important for insulin synthesis, storage and secretion. When zinc concentration decrease, there is a concomitant reduction in insulin secretion and peripheral insulin sensitivity. AIM: To assess the effects of zinc sulfate on insulin sensitivity, leptin and androgens in obese individuals. MATERIAL AND METHODS: A randomized, double-blind, placebo-controlled clinical trial was performed in 14 obese volunteers between 21 and 30 years old, with body mass index (BMI) (3) 27 kg/m2. During one month, seven subjects received 100 mg/day of zinc sulfate orally (ZnG) and the other seven received placebo, as control group (CG). At baseline and after the intervention, insulin sensitivity was measured using a euglycemic-hyperinsulinemic clamp technique. Blood glucose, serum lipids, zinc, androgens and leptin were also measured in a fasting blood sample. RESULTS: After the intervention, a rise in zinc concentrations from 11.8 to 16.9 umol/L; p=0.001 and in leptin levels from 15.2 to 27.7 ng/mL; p=0.029, was observed in the ZnG. No changes were observed in the CG. There were no significant changes in insulin sensitivity and androgens after the intervention with zinc sulfate. CONCLUSIONS: Zinc increased the leptin concentrations in obese individuals, but did not modify insulin sensitivity and androgens. PMID- 16676100 TI - [Esophageal manometry in patients with non cardiac chest pain]. AB - BACKGROUND: Non cardiac chest pain can be caused by esophageal problems such as gastroesophageal reflux or smooth muscle motor disorders. AIM: To perform esophageal manometry in patients with non cardiac chest pain. MATERIAL AND METHODS: One hundred patients with chest pain in whom coronary problems were discarded, were studied. A computerized esophageal manometry was performed in all and 24 hours esophageal pH measurement was done in 21 patients. RESULTS: Esophageal manometry was normal in eight patients. Nutcracker esophagus was the most common finding, in 36 patients. Twenty eight had a hypotensive sphincter, 16 had unspecific motor disorders, nine had diffuse esophageal spasm, two had a non achalasic esophageal aperistalsis and one had a hypertensive sphincter. CONCLUSIONS: Only eight of 100 patients referred to esophageal manometry for non cardiac chest pain, had a normal study. PMID- 16676099 TI - [Effects of gastric bypass on erosive esophagitis in obese subjects]. AB - BACKGROUND: Obesity is an important risk for pathological gastroesophageal reflux. AIM: To assess the effects of gastric bypass on obese subjects with erosive esophagitis. PATIENTS AND METHODS: Sixty two morbid obese subjects (aged 16 to 70 years, 41 females) with erosive esophagitis at the moment of surgery were studied. These patients were subjected to a gastric bypass with gastric resection. They were followed with upper gastrointestinal endoscopy every one year, looking for the presence of erosions or ulcers in the distal esophagus. RESULTS: The mean follow up period was 21 months. Prior to surgery all patients had heartburn or regurgitation and at two years after surgery, 97% were asymptomatic. Esophagitis was found in 97 and 6.5% before and after surgery, respectively. Two patients had an esophageal ulcer, that healed on follow up endoscopy. There was a 72% reduction of overweight on late follow up. CONCLUSIONS: Gastric bypass is effective to control pathological gastroesophageal reflux in patients with morbid obesity. PMID- 16676101 TI - [Brain natriuretic peptide in primary pulmonary hypertension]. AB - BACKGROUND: Primary pulmonary hypertension (PPH) is a progressive disease leading to right heart failure and death. Right heart catherization and maximal or submaximal tests are employed to assess the course of the disease. A neurohormonal parameter such as pro-brain natriuretic peptide (BNP) would be helpful in the assessment of these patients. AIM: To study the correlation of BNP with functional status and non-invasive hemodynamic determinations in patients with PPH. MATERIAL AND METHODS: Twelve patients (mean age: 48 years; 58% female) were evaluated with 6 minutes walk distance test (6-min WT), plasma BNP, systolic pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR) and cardiac output (CO) determined by echocardiogram. Plasma BNP levels were compared with normal subjects. RESULTS: BNP levels were increased in PPH patients (1270+/-547 vs 48+/-8 pg/ml, p-value <0.01). Mean PAPs was 82+/-27 mmHg and the mean distance walked in 6 minutes was 407+/-113 meters. BNP levels were positively correlated with PVR (r=0.58, p-value=0.006) and negatively correlated with 6-min WT (r= 0.83, p-value <0.001). No correlation was found between BNP levels, PAPs and CO. CONCLUSIONS: In PPH patients, BNP levels are increased and correlate with functional class and PVR. Follow-up studies are needed to evaluate the role of BNP as a marker of progression and therapeutic response in PPH patients. PMID- 16676102 TI - [Renal scars after one year of follow up in children with a first episode of acute pyelonephritis]. AB - BACKGROUND: Acute pyelonephritis in children can leave a kidney scar that eventually can lead to hypertension or renal failure. 99mTc-dimercaptosuccinic acid renal scintigraphy (RC DMSA) is a widely accepted technique to assess children with acute pyelonephritis. AIM: To evaluate the presence of residual kidney scars detected through RC DMSA, in children with a first episode of acute pyelonephritis. PATIENTS AND METHODS: Clinical records of children with a first episode of acute pyelonephritis that were assessed within seven days of the episode with RC DMSA were reviewed. Children were considered eligible if they did not have a new episode of acute pyelonephritis and a second RC DMSA, one year after the first episode, was performed. The presence or absence of a renal scar after one year was associated to demographic, scintigraphy and laboratory variables. RESULTS: Fifty nine children, aged 1 month to 10 years, 35 females, were studied. Thirty nine percent had a renal scar in the scintigraphy perfomed after one year of follow up. The presence of a scar was correlated with a C reactive protein over 130 mg/dl and an altered relative renal function (below 44%), during the acute phase. CONCLUSIONS: A high C reactive protein and alterations of relative renal function during the acute phase of acute pyelonephritis in children, may be risk factors for the development of renal scars in the long term follow up. PMID- 16676103 TI - [Lack of assertiveness in patients with eating disorders]. AB - BACKGROUND: Low self-assertion has been noted as an important feature among patients with eating disorders. AIM: To verify, in a female population, if assertiveness is related or has a predictive capacity for the development of eating disorders. SUBJECTS AND METHODS: An structured clinical interview, the Eating Attitudes Test (EAT-40) and the Rathus Assertiveness Scale (RAS) were administered to 62 patients that fulfilled the DSM-IV diagnostic criteria for eating disorders and to 120 female students without eating problems. RESULTS: Patients with eating disorders ranked significantly higher on the EAT-40 and its factors (p <0.001) and showed a lower level of assertiveness on the RAS (p <0.001). Assertiveness measured by RAS and its factors was inversely related to EAT-40 and its items (r= -0.21). The predictive capability of the lack of self assertion in the development of an eating disorder reached 53%, when patients with eating disorders and subjects at risk were considered together and compared to students without such disorder. CONCLUSIONS: Lack of assertiveness is a significant trait in patients with eating disorders; it may worsen its outcome and even perpetuate symptoms. Low self-assertion may be considered a predictive factor in the development of an eating disorder and must be managed from a preventive or therapeutic point of view. PMID- 16676104 TI - [Infliximab in the treatment of patients with Crohn's disease: preliminary report]. AB - BACKGROUND: Tumor necrosis factor alpha, interleukin 6 and 1 have a pathogenic role in Crohn's disease. Infliximab, a monoclonal antibody against tumor necrosis factor, is useful in the treatment of Crohn's disease, specially when the disease is active and associated to fistula formation. AIM: To assess the effectiveness of Infliximab in patients with Crohn's disease. MATERIAL AND METHODS: Twelve patients with Crohn's disease (aged 16 to 63 years, 10 women) refractory to treatment with steroid, 5-aminosalicylic acid, antimicrobials and immunomodulatory drugs, were studied. A single intravenous 5 mg/kg dose Infliximab was administered. The Crohn's disease activity index (CDAI) was measured before and one week after the administration of the drug. RESULTS: CDAI before and after Infliximab administration was 357+/-62 and 138+/-122 score points, respectively (p <0.005). According to this score, five patients had a severe and seven a moderate disease flare. The disease remitted in 7 (58%) and a clinical response was observed in 75%. CONCLUSIONS: A single dose of Infliximab was effective to induce remission of the disease in 75% of this selected group of patients with Crohn's disease. PMID- 16676105 TI - [Efficacy of infliximab in patients with moderate and severe psoriasis treated with infliximab (Remicade)]. AB - BACKGROUND: Psoriasis has a moderate or severe course in 25% of patients, requiring systemic therapy that is not always successful. Infliximab is a human murine monoclonal anti tumor necrosis alpha (TNF-alpha) antibody. This mediator has a role in the pathogenesis of psoriasis. AIM: To evaluate the use of infliximab in psoriatic patients resistant to conventional therapies. MATERIAL AND METHODS: An open prospective study including eight patients with extensive plaque or erythrodermic psoriasis. They were treated with infliximab 5 mg/kg/dose on weeks 0,2 and 6. Patients were evaluated every 2 weeks for a median lapse of 50.1 weeks. Physical examination, PASI scores (Psoriasis Area Severity Index) and a photographic control, were done in each visit. RESULTS: All the patients responded in the first 10 weeks of follow-up. The mean reduction in PASI score was 86.6%. Six patients received fourth infusion of infliximab at 37.3 weeks, on average. The most common adverse events were pruritus and headache. CONCLUSIONS: This group of patients with extensive plaque or erythrodermic psoriasis resistant to conventional therapies, had a good response to infliximab. PMID- 16676106 TI - [Identification of infectious agents in cases suspicious of Hantavirus infection but with negative serology]. AB - BACKGROUND: The diagnosis of acute respiratory illness caused by Hantavirus is based in the rapid and progressive clinical course, epidemiological background and the serological confirmation of the virus. When the presence of the virus is not confirmed a differential diagnosis must be made with other infections. Between 1999 and 2001, the Chilean Public Health Institute received 1063 blood samples from patients with a suspicious clinical picture, to study the presence of Andes strain of Hantavirus. In 134 of these samples, the presence of the virus was confirmed. AIM: To study the presence of other infections in sera from patients with suspected Hantavirus acute respiratory illness but serologically negative for Hantavirus. MATERIAL AND METHODS: A retrospective study of 98 serum samples, received at the National Public Health Institute, of patients with negative serology for Hantavirus. The presence of antibodies against influenza virus, Mycoplasma pneumoniae, Leptospira and Streptococcus pneumoniae was determined using Latex techniques. RESULTS: Leptospira was detected in 23 patients, influenza virus in 13, Streptococcus pneumoniae in six and Mycoplasma pneumoniae in one case. Leptospira infections were confirmed in four cases by ELISA determination of IgM antibodies. Influenza virus infection was confirmed in three cases by Hemmaglutation Inhibition Assay. CONCLUSIONS: In suspected cases of Hantavirus acute respiratory infection, but with negative serology, other infectious agents such as Leptospira, influenza virus and Streptococcus pneumoniae, must be sought. PMID- 16676107 TI - [Pyoderma gangrenosum: report of three cases in plastic surgery]. AB - Pyoderma Gangrenosum is a rare disease whose etiology is probably autoimmune. We report two males aged 48 and 49 years and one female aged 54 years, with lesions in the right thigh, right leg and in the borders of a surgical incision in the chest. This article gives a brief description of the lesions, tips for diagnosis and help for the initial management and treatment. PMID- 16676108 TI - [Ascites and renal failure caused by a bladder fistula: report of one case]. AB - Bladder fistula to open peritoneum is an uncommon cause of ascites. We report a 50 year-old woman with a history of pain in the lower abdomen and slight weight loss. The patient had a history of a repaired bladder perforation 12 years before, during a labor with forceps. The patient had microscopic hematuria and an abdominal CAT scan showed ascites. Serum creatinine was 2.2 mg/dl. An abdominal Doppler ultrasound showed normal portal and suprahepatic veins. Due to the suspicion that ascites accumulation could be urine, a sample was obtained and urea nitrogen and creatinine were measured. Since both levels were high in the ascitic fluid the patients was subjected to a cystoscopy that disclosed a fistula between the bladder and peritoneum. The patient was operated and the fistula excised. The postoperative period was uneventful, and the serum creatinine normalized. PMID- 16676109 TI - [Thrombotic thrombocytopenic purpura with neurological involvement: report of one case]. AB - Neurological manifestations, secondary to perfusion problems, vasogenic edema or small infarcts, are common in thrombotic purpura. Moreover, they are the first symptoms of the disease in 50% of patients. We report a 50 year-old woman who presented with focal intermittent neurological signs with aphasia and right hemiparesis, who then developed progressive impairment of consciousness with stupor and generalized tonic-clonic seizures. Despite the severe neurological impairment, first neuroimaging studies were normal. A second magnetic resonance imaging showed small cortical infarcts, that were visible only with the technique of diffusion weighted imaging. The standard electroencephalograms showed focal left temporal slowing and low voltage first and then diffuse slowing accordind to the clinical condition of the patients. She was treated with plasmapheresis and had a partial neurological recovery at the fifth day, but died at the twelfth day of therapy. PMID- 16676110 TI - [Application of marketing strategies for the management of public hospitals from the viewpoint of the staff members]. AB - BACKGROUND: The implementation of the marketing strategies in public hospitals provides management advantages and improves the relationship between customers and staff. AIM: To analyze the application of marketing strategies in a public hospital, from the perspective of the staff. MATERIAL AND METHODS: A structured survey that asked about perceptions in 50 items about communication between personnel and customers/users, customer satisfaction, participation in the development of new policies and incentives for efficiency was applied to a stratified sample of the staff. Factorial and regression analyses were performed to define the impact of marketing strategies on the degree of preoccupation and orientation of the organization towards the satisfaction of customer needs. RESULTS: The survey was applied to 74 males and 122 females. The survey showed that the orientation of the hospital towards the satisfaction of its beneficiaries basically depends on the generation of an organizational culture oriented towards them and the implementation of adequate policies in staff management and quality of service. These basic aspects can be accompanied with practices associated to the new marketing approaches such as a market orientation, customer orientation and relational marketing. All these factors presented positive and significant relations. CONCLUSIONS: New marketing strategies should be applied, to achieve an efficient and customer oriented hospital management. PMID- 16676111 TI - [Improving the management of celiac disease: an urgent challenge]. AB - Anti endomysial and anti transglutaminase (EMA/TTG) antibodies measurements in large population groups, show that celiac disease is significantly more common than previously thought and 0.5-1% of the population suffer this condition. The recognition of atypical clinical presentations by active screening of high risk groups is a successful global initiative. In Chile there is little awareness of the enormous changes occurred in this field. Available national information on typical presentation of the disease shows that it is found in 1:1836 live births, figure that is comparable to European societies. Two urgent tasks are required in our country. First, to asses the Chilean population using EMA and/or TTG. Second, to improve the legal aspects that regulate consumer information about gluten content in processed foods. We here review the most relevant aspects to be considered to improve the management of celiac patients and the tasks that require the participation of health authorities for their implementation. PMID- 16676112 TI - [Cystic fibrosis: facing patient transition from the pediatrician to the internist]. AB - The increased survival of patients with cystic fibrosis (CF) resulted in the appearance of new pulmonary and non-pulmonary complications. Even though subjects with CF present with inflammatory pulmonary changes at birth, several pathogens such as Staphyloccocus aureus, Haemophilus influenzae and Pseudomona aeruginosa contribute to the progression of pulmonary injury. Clinical presentations vary according to patient age; even though mild forms exist, patients with severe forms, develop respiratory insufficiency and end-stage disease at an early stage. Today, new diagnostic and therapeutic tools, increase the possibility of an early diagnosis and of greater survival. Successful management will depend on the timely selection of adequate antimicrobials, the use of pancreatic enzyme supplementation and early institution to respiratory physiotherapy. The transition from pediatric care to adult care must occur according to development of each patient. This timing must be flexible and there should be a constant communication and coordination within the different specialists in internal medicine. Thus, it is crucial to recognize the disease progression as a continuous process, giving the appropriate physiologic support and evaluating the needs of the patient and close relatives. PMID- 16676113 TI - [Fatty acid binding protein 2 (FABP-2) polymorphism, obesity and insulin resistance]. AB - The genetic components of insulin-resistance, diabetes and obesity have been largely studied. These conditions are determined by multiple polygenic and environmental factors. Certain candidate genes, that have common functional variants in the general population, may be important determinants of inter individual differences in the response to dietary changes. This review focuses in one of the major candidate genes, the gene encoding for the FABP2, an intracellular protein expressed only in the intestine, involved in the absorption and intracellular transport of dietary long chain fatty acids. Carriers of the Thr54 allele in FABP2 have a 2-fold greater affinity for long chain fatty acids than Ala54 carriers. The increased flux of dietary fatty acids (FA) into the circulation, among carriers of FABP2 Ala54Thr, supports a role of the polymorphism of this allele in the etiology of metabolic disorders. The frequencies of the polymorphism in different populations fluctuate between 18% and 40%. FABP2 Ala54Thr variant has been associated with an increased fasting insulin concentration, fasting fatty acid oxidation and reduced glucose uptake. This evidence, although not conclusive, sustains an association between FABP-2 genotype and metabolic abnormalities. PMID- 16676114 TI - [Medical professionalism: historical and religious aspects]. AB - The essence of the medical profession resides in the medical act, whereupon a sick human being meets another with the power to heal him or her. The source of this power has evolved from the divinity itself through magic to science or acquired knowledge. This power implies acknowledgement of values that are inherent to the profession as well as responsibility toward one's own conscience and toward society, elements considered constitutive of what we now call professionalism. From antiquity these principles have evolved into behavioral codes containing variable components according to the different ages and cultures, but also permenent core values such as respect for life, altruism, and honesty, among others. Scientific and technological advances have magnified medical power but at the same time they have required that the philosophical and ethical principles that ought to inform professional practice be made explicit. This happens at a time when certitudes are questioned or abandoned, relativism and secularism pervade culture, and traditional medical values are challenged. Therefore, consensus attainment appears for some as the only legitimation of the ethics of professional medical acts, while for others the ancestral principles and values of medicine have permanent validity as objective goods based on the dignity of the human person. PMID- 16676117 TI - [Student's scientific journals in Latinamerica]. PMID- 16676115 TI - [Communicating evidence to the patients: physicians as risk communicators]. PMID- 16676119 TI - Taurine improves epidermal barrier properties stressed by surfactants-a role for osmolytes in barrier homeostasis. AB - Epidermal barrier function to water loss is maintained by lipid membrane domains located in the interstices of the stratum corneum. Exposure of the epidermis to a dry environment or UV irradiation stimulates barrier lipid synthesis and accumulation of the organic osmolyte taurine in the outermost granular keratinocyte layer. In this work we studied a possible relationship between these two different epidermal responses to environmental challenges. As a model system we selected anionic surfactant-induced barrier perturbation. Incubation of reconstructed epidermis with taurine inhibited cytotoxic and proinflammatory effects induced by sodium dodecyl sulfate including (i) a decrease in interleukin 1 alpha and prostaglandin E2 release, (ii) stabilization of keratinocyte membrane integrity, and (iii) improvement of keratinocyte viability. Repeated exposure of human skin to sodium dodecyl sulfate induced an increase in transepidermal water loss, inflammation, and hyperplasia. Topical application of taurine significantly decreased transepidermal water loss after repeated exposure to sodium dodecyl sulfate. Moreover, taurine significantly stimulated the synthesis of all three classes of barrier lipids (ceramides, cholesterol, and fatty acids) in reconstructed epidermis. In conclusion, our data suggest a role for taurine in preventing surfactant-induced dry and scaly skin by modulating the proinflammatory response and stimulating epidermal lipid synthesis. PMID- 16676120 TI - New cosmetic agents for skin whitening from Angelica dahurica. AB - To develop a new whitening agent for cosmetics from natural products, Angelica dahurica was selected for its inhibitory effect on melanogenesis in B16 melanoma cells. From the mechanism study, it was clarified that the ethanolic extracts of this plant showed the suppression of tyrosinase synthesis but no inhibition of tyrosinase activity. In order to find the active constituents from this plant, the ethanol extracts were chromatographed repeatedly with silica gel. Two coumarin compounds were isolated from A. dahurica. Their structures were identified by physicochemical and spectral data such as UV, IR, NMR, and MS. It was shown that the active substance was isoimperatorin (10-[(3-methyl-2 butenyl)oxy]-7H-furo[3,2-g][1] benzopyran-7-one) and imperatorin (9-[(3-methyl-2 butenyl)oxy]-7H-furo[3,2-g][1] benzopyran-7-one). They significantly inhibited tyrosinase synthesis in B16 melanoma cells. To elucidate the action mechanism of the active compounds of A. dahurica, we investigated the changes in the mRNA level of tyrosinase using the RT-PCR technique. As a result, the mRNA level of tyrosinase was markedly reduced by active compounds of A. dahurica. From these results, we suggest that these extracts might be useful as a new whitening agent in cosmetics, but the in vitro findings must be verified in in vivo skin lightening studies. PMID- 16676121 TI - Comparative characterization of a wool-wax extract by two complementary chromatographic techniques. AB - A detailed characterization of lipids present in a wool-wax extract, obtained by supercritical fluid extraction, is presented. Thin-layer chromatography coupled to a flame ionization detector (TLC-FID) has been used as a screening technique to identify the different chemical classes of complex mixtures. Furthermore, to confirm peak identification obtained by TLC-FID and to check its suitability as a quality control technique, preparative TLC fractionation has been carried out by re-analyzing the different fractions obtained by TLC-FID and by gas chromatography coupled to mass spectrometry in the electron impact and in the chemical ionization modes. By means of this comparative study, the TLC-FID identification has been confirmed. Also, the complementarity of the different techniques used confirmed the identification of different lipid classes, namely fatty acids, alcohols, sterols, sterane diol, stanone, steryl sulphate, hydroxyacids, alkane diols, aliphatic and steryl esters, hydroxy steryl esters, triacylglycerol, diesters, and ceramides types II and VI. PMID- 16676122 TI - Nanotribological effects of silicone type, silicone deposition level, and surfactant type on human hair using atomic force microscopy. AB - The atomic/friction force microscope (AFM/FFM) has recently become an important tool for studying the micro/nanoscale structure and tribological properties of human hair. Of particular interest to hair and beauty care science is how common hair-care materials, such as conditioner, deposit onto and change hair's tribological properties, since these properties are closely tied to product performance. Since a conditioner is a complex network of many different ingredients (including silicones for lubrication and cationic surfactants for static control and gel network formulation), studying the effects of these individual components can give insight into the significance each has on hair properties. In this study, AFM/FFM is used to conduct nanotribological studies of surface roughness, friction force, and adhesive forces as a function of silicone type, silicone deposition level, and cationic surfactant type. Changes in the coefficient of friction as a result of soaking hair in de-ionized water are also discussed. PMID- 16676123 TI - Partially purified paeoniflorin exerts protective effects on UV-induced DNA damage and reduces facial wrinkles in human skin. AB - Partially purified paeoniflorin (PF), a new cosmetic ingredient from roots of Paeoniae lactiflora, has been developed. Its paeoniflorin content is about 64%, far higher than that of conventional, cosmetic-grade peony root extracts (approximately 10%). In this report, we studied the effects of PF on UV-induced DNA damage in both cultured human keratinocytes and hairless mouse skin. We also investigated the anti-wrinkle effects of PF-containing cosmetic preparations on human skin. From the in vitro and in vivo comet assay, it was revealed that PF protected cells from DNA damage induced by ultraviolet B (UVB) irradiation in both cultured normal human keratinocytes (19.4% decrease at 0.001%) and hairless mouse skin keratinocytes (41% decrease at 0.01%). An eight-week clinical trial using 0.5% PF-containing formulation with 20 volunteers resulted in a statistically significant reduction in facial wrinkles (p < 0.05). These results suggest that the partially purified paeoniflorin has potent anti-aging and anti wrinkle activities and should be a useful ingredient for these purposes. PMID- 16676132 TI - Biomedical engineering and the whitaker foundation: a thirty-year partnership. AB - The Whitaker Foundation, established in 1976, will close in 2006. It will have made awards totaling 805 million US dollars, with over 710 million US dollars in biomedical engineering. Close to 1,500 faculty members received research grants to help them establish academic careers in biomedical engineering, and over 400 graduate students received fellowship support. The Foundation also supported the enhancement or establishment of educational programs in biomedical engineering, especially encouraging the formation of departments. The number of biomedical engineering departments almost tripled during the past 10 years, now numbering close to 75. Leveraging of grants enabled the construction of 13 new buildings. With the field firmly established, the grant program supporting new faculty members will be the one missed the most. New opportunities, however, are emerging as interdisciplinary research is being embraced by both public and private funding sources. The life sciences will be increasingly incorporated into all areas of engineering, and it is expected that such "biofication" will pose both opportunities and challenges to biomedical engineering. PMID- 16676134 TI - Genetic variants in the glucocorticoid receptor gene (NR3C1) and cardiovascular disease risk. The Leiden 85-plus Study. AB - Recently, the ER22/23EK, N363S and BclI polymorphisms in the glucocorticoid receptor (GR) gene have been linked to altered cortisol sensitivity and to cortisol-associated disorders. The aim of this study was to investigate the effect of these genetic variants in the GR gene on cardiovascular disease and mortality in elderly persons aged 85 years and over. In the population-based Leiden 85-plus Study, 552 participants were genotyped for the ER22/23EK, N363S and BclI polymorphisms, and the effects of the polymorphisms on metabolic profile, body composition, and on the prevalence of cardiovascular pathologies at baseline, were assessed. Allcause and cardiovascular disease mortality risks dependent on the SNPs were calculated after a 4.2-year follow-up. The analyses of metabolic profile revealed that carriers of the ER22/23EK polymorphism have higher HbA1C levels (P<0.001) and carriers of the N363S SNP have higher LDL cholesterol (P<0.001) and triglyceride concentrations (P=0.03), compared to the non-carriers. The only significant association between genotype and body composition analyses was for height and the ER22/23EK polymorphism. Men carrying the ER22/23EK polymorphism were taller (P=0.02) compared to non-carriers. No associations with cardiovascular pathologies, allcause and cardiovascular mortality were observed for any of the polymorphisms. We conclude that, in spite the effect of the ER22/23EK and N363S SNPs on metabolism, these polymorphisms together with the BclI SNP, do not affect the risks of cardiovascular disease and survival at old age. PMID- 16676135 TI - Food, fertility and longevity. AB - Some animals live in environments in which the food supply fluctuates. When it is scarce these animals do not breed, but invest resources into survival until food is again available, and they can reproduce. Under these circumstances the lifespan can be increased, just as it is after calorie restriction. Other animals have a fairly constant food supply, and it is predicted that these would not have an extended life span if subjected to calorie restriction. Hibernation is a natural form of calorie restriction, and in some cases may lengthen lifespan. PMID- 16676136 TI - Why calorie restriction would work for human longevity. AB - Experimentally imposed calorie restriction (CR) is shown to result in the most reproducible endpoint of lifespan extension in all animals models tested. In this presentation, the question of CR's effect on human longevity is reviewed by discussing data pertinent to the putative efficacy of CR on humans. Arguments are presented in support of this possibility based on CR's unique abilities to retard biological functional declines and to deter pathological processes, both of which are major targets of deleterious oxidative stress. To delineate the cellular and molecular mechanisms of CR's efficacy on human longevity, this review elaborates on the modulation of CR on the inflammatory process, a common risk factor for many chronic diseases. Discussions also include evidence from human data on the effect of CR in the loss of body weight, known to suppress inflammatory cytokines, subsequently leading to the reduction of chronic diseases known to compromise the functional longevity of humans. PMID- 16676137 TI - Papillary fibroelastoma of mitral papillary chordae in a young patient. AB - Our case is a 38-year-old man, admitted to Cardiology Department with shortness of breath. Echocardiography yields a hyperechogenic mass localized to papillary muscle with severe mitral regurgitation. Coronary angiography demonstrated radiopacity localized to the papillary muscle. The excision of the mass was consistent with elastic tumor, which was reported as papillary fibroelastoma attached to the papillary chordae of the mitral valve. A 29 no St-Jude bileaflet mechanical valve was implanted to mitral position. Papillary fibroelastoma (PF) can be found in young age and originate from the papillary muscle, which the radiopaque angiographic appearance of the mass supports the diagnosis. PMID- 16676138 TI - Optimal imaging protocol for evaluation of aortic coarctation; time for a reappraisal. PMID- 16676141 TI - Correct indication for Ferguson's hemorrhoidectomy. PMID- 16676142 TI - Doppler-guided hemorrhoidal artery ligation: an alternative to hemorrhoidectomy. PMID- 16676146 TI - [Need for care. The role of family care in long-term care]. AB - In Germany, more than 2 million people are currently in need of care, and another 3 million need help below the threshold that entitles them to benefits by social legislation. Thus, the need of care, in contrast to the need of help, is a general risk recognised by legislation. Accordingly, the German long-term care insurance was introduced in 1994, where entitlement to benefits depends on the ability to perform activities of daily living; it neglects the requirements of care arising from gerontopsychiatric impairments. In this article, the concept of "need of care" will be critically discussed and the influence of demographic changes on the state of needing care will be explored. Against this background, future challenges of caregiving will be analysed. This analysis leads to a call for "mixed care arrangements", where family care can only be maintained by relying on professional relief and support of voluntary helpers, and where care institutions would have to define themselves as partners of families. This demand will have to be reflected in changed training concepts. Structurally, it is necessary to develop arrangements, which promote self-determined individual solutions, e.g. by implementing (personal) care budgets. Moreover, target group orientated counselling offers and network structures will have to be established; in this respect, co-operative efforts between professional case management and voluntary work are exemplary approaches. PMID- 16676145 TI - Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. AB - Optimization of reference genes for real-time polymerase chain reaction (PCR) studies in fish is strongly needed. We systematically tested beta-actin (ACTB), 18S rRNA (18S), beta(2)-microglobulin (B2M), elongation factor 1-alpha (EF1A), RNA polymerase I and II (RPL1/2), and glycerol 6-phosphate dehydrogenase (G6PDH) for stability in salmon immune relevant tissues and kidney cells (ASK) infected with infectious salmon anemia virus (ISAV), plus in tissues from fish fed thia fatty acids. Transcription of all genes was unchanged in infected and thia fatty acid-treated tissues versus normal tissues. Between tissues, 18S and EF1A were most stable, RPL1 and RPL2 were intermediate, and G6PDH and ACTB and B2M were the least stable. However, only 18S had constant expression in infected cells; the rest significantly down-regulated. Implications of this finding were demonstrated when normalizing major histocompatibility complex (MHC) class I expression in ASK. Software predictions supported a proper normalization is obtained combining 18S, EF1A, and RPL1 in vivo, but for in vitro viral infection assays we recommend using 18S. PMID- 16676147 TI - [Photoselective vaporization of the prostate: experience with prostate adenomas >80 cm3]. AB - BACKGROUND: We report about our experiences with photoselective vaporization of the prostate (PVP) in patients with symptomatic benign prostatic hyperplasia (BPH) and total prostate volume larger than 80 cm(3). PATIENTS AND METHODS: The study included 201 patients with BPH: 51 (25.4%) patients had a prostate volume larger than 80 cm(3) and 150 (74.6%) patients had a volume smaller than 80 cm(3) in the preoperative transrectal ultrasound. RESULTS: The mean operation time for patients with large prostates was 79 min. Neither TUR syndrome nor severe bleeding was observed. In patients with large adenomas peak urinary flow increased by 135, 136, and 132% after 6, 12, and 24 months, respectively. The overall complication rate was comparable in both groups. CONCLUSION: PVP is characterized by excellent perioperative safety and significant improvement of voiding parameters. PVP is feasible in patients with large prostates. PMID- 16676151 TI - Does caesarean section negatively influence the post-partum prognosis of low back pain and pelvic pain during pregnancy? AB - Low back and pelvic pain (LBPP) is prevalent during pregnancy and also post partum. The aetiology is poorly understood. The aim of this study was to investigate possible associations between epidural or spinal anaesthesia and caesarean section (CS) with persistent LBPP half a year after pregnancy. In a previous questionnaire study (n=891) altogether 639 (72%) women had reported LBPP during pregnancy. We sent these respondents a second questionnaire at approximately 6 months post-delivery. The response rate was 72.6% (n=464). The respondents were divided into three groups reporting 'no pain', 'recurrent pain' and 'continuous pain' in relation to LBPP 6 months after delivery. Pearson's chi square test was used to test the difference between groups and logistic regression analysis was performed. Forty percent of the respondents had received epidural anaesthesia (EDA) or spinal anaesthesia during delivery and 18.5% of women had been delivered by CS. Epidural or spinal anaesthesia was not associated with persistent LBPP. There was no significant difference in CS rates between different sub-groups. The risk of persistent LBPP was increased three- to fourfold in women delivered by elective CS compared with women delivered by emergency CS. Epidural or spinal anaesthesia was not associated with risk of persistent LBPP. Elective CS was associated with an increased risk of persistent LBPP. However, the results must be interpreted with caution because of a relatively small study sample. PMID- 16676152 TI - Comment on: Does caesarean section negatively influence the post-partum prognosis of low back pain and pelvic pain during pregnancy? (Ingrid M. Mogren). PMID- 16676153 TI - Technical note: the swimmer's view for cervical facet joint injections. AB - Image intensifier (C-arm) guided facet infiltration of a mixture of long acting local anaesthetic and corticosteroid is a safe and effective office procedure for cervical spondylosis. Visualisation of lower cervical anatomy using C-arm may not always be possible due to the shadow cast by the shoulders. We describe the successful adaptation of swimmer's view in such cases to carry out the injections. PMID- 16676154 TI - Nerve root decompression without fusion in spondylolytic spondylolisthesis: long term results of Gill's procedure. AB - Nerve root decompression with instrumented fusion is currently most commonly performed in the treatment of patients with spondylolytic spondylolisthesis. The relationship between successful fusion and clinical outcome remains controversial, thereby questioning the necessity of fusion. Nerve root decompression without fusion, i.e. Gill's procedure, might be a less invasive surgical alternative with comparable clinical outcome. The objective of this study is to compare the long-term results of Gills's procedure with data from literature on decompression with fusion, and, moreover, to determine if a future randomised trial is legitimate. We retrospectively reviewed the long-term results of Gill's procedure in patients with grade I or II spondylolytic spondylolisthesis. All patients suffered from leg pain with or without low back pain. No patient had low back pain alone. In 17 patients a bilateral and in 25 patients a unilateral Gill's procedures were performed. The patients were evaluated at three follow-up moments. On moment 1, 38 patients were clinically examined on their last out-patient control (mean follow-up 11 months). On moment 2, 34 patients were interviewed by telephone (mean follow-up 4.4 years). The final long-term follow-up moment 3 (mean follow-up 10.5 years) included a mailed patient-satisfaction questionnaire of 31 patients (response rate 74%). No surgical complication occurred. Ten of the 42 patients were reoperated because of persistent or recurrent radicular pain (mean time interval 2.9 years). Kaplan Meier analysis showed a disease-free survival rate of 79% at 5 years and 72% at 10 years after the index operation. On the three follow-up moments, the improvement of leg pain was 92, 97 and 88%, respectively. The final long-term follow-up showed 71% good result in terms of patient satisfaction. The Gill's procedure is a less invasive surgical technique in the treatment of patients with leg pain due to low-grade spondylolytic spondylolisthesis. This technique can be considered as an alternative to instrumented fusion in selected cases. Preoperative instability, discectomy at the affected level and neuroforaminal nerve root compression seem to be negative influencing factors, increasing the risk for secondary instrumented surgery. The results of this study justify a randomised trial. PMID- 16676156 TI - Anterior versus posterior surgery for osteoporotic vertebral collapse with neurological deficit in the thoracolumbar spine. AB - Despite the increasing number of reports on surgical treatments for thoracolumbar osteoporotic vertebral collapse with neurological deficits, the choice of surgery remains controversial. In this retrospective study, we compared the outcomes of posterior and anterior surgeries for single-level osteoporotic vertebral collapse with neurological deficit in the thoracolumbar spine. Both posterior and anterior surgical approaches were performed with a consistent procedure for a single surgical indication at one institution. Twenty-four patients treated with posterior surgery and 28 patients treated with anterior surgery were followed-up over an average of 5 years after surgery. Radiographic results (kyphotic angle, bony fusion, and instrumentation failure), neurological improvement, and surgical complications were compared between the two groups. The average correction angle after surgery was larger in the posterior group than in the anterior group (P = 0.013), but not at final follow-up (P = 0.755). The average loss of correction was also higher in the posterior group than in the anterior group (P = 0.037). There was no significant difference in neurological outcomes between anterior and posterior approaches (P = 0.080). Two-way analysis of variance (ANOVA) showed that the neurological outcome was better in wedge type than in flat type vertebral collapse, regardless of the type of surgical approach (P = 0.0093). In wedge type vertebral collapse, neurological improvement tended to be greater after anterior than after posterior surgery. In four of six cases with instrumentation failure in the anterior group, a titanium cage subsided more than 5 mm but bony fusion was eventually achieved without causing neurological problems. In the posterior group, six cases experienced instrumentation failure during the postoperative course (two cases with screws loosened from pedicles and bodies, and one case with breakage of a screw neck). None of the patients developed instrumentation-related neurological problems. Two cases in each group developed pseudoarthrosis. In single-level osteoporotic vertebral collapse with neurological deficit, anterior surgery tended to improve neurological deficit in wedge type, but not in flat type collapse, compared with posterior surgery. PMID- 16676157 TI - Is there a role for selective anterior instrumentation in neuromuscular scoliosis? AB - Our objective was to report on the clinical and radiological outcome from a cohort of patients with neuromuscular scoliosis who underwent selective anterior single rod instrumentation for correction of thoraco-lumbar and lumbar scoliosis. Traditionally combined anterior release with long posterior instrumentation has been advocated for the treatment of neuromuscular scoliosis. Neuromuscular curves tend to be long and may have significant pelvic obliquity. However, certain neuromuscular curves with minimal pelvic obliquity may lend themselves to selective anterior correction thereby saving motion segments and allow continued ambulation for those patients. Nine patients with neuromuscular scoliosis underwent selective anterior instrumentation between 1994 and 2000. The mean follow up was 2 years and 9 months (range 24-55 months). The clinical outcome (including parent and caregiver satisfaction), radiological outcome (Cobb angle, apical vertebral translation, pelvic obliquity, truncal shift, thoracic kyphosis, lumbar lordosis, sagittal vertical axis) and complications are reported. Subjective outcome was excellent in six patients and good in three. All nine patients retained their ability to walk. There were no neurological or vascular complications. Supplementary posterior surgery was required in two patients. The mean pre-operative Cobb angle of 52 degrees (range 44-60) improved to 20 degrees (range 10-28) at 3 months, achieving Cobb angle correction of 61% and was 19 degrees (range 7-28) at final follow-up. The mean pre-operative compensatory curve of 31 degrees (range 20-42) spontaneously corrected to 18 degrees (range 14 24) at 3 months and was maintained at 18 degrees (range 10-26) at final follow up. The mean pre-operative pelvic obliquity of 7 degrees (range 0-14) corrected to 4 degrees (range 0-8) at 3 months and was 3 degrees (range 0-8) at final follow up. Selective anterior instrumentation and fusion in carefully selected patients with neuromuscular scoliosis (short flexible curves, minimal pelvic obliquity, pre-operative walkers, slow or non-progressive pathology) appears to have satisfactory clinical and radiological outcome at least in the short-term. PMID- 16676158 TI - Ant patchiness: a spatially quantitative test in coffee agroecosystems. AB - Arboreal ants form patchy spatial patterns in tropical agroforest canopies. Such patchy distributions more likely occur in disturbed habitats associated with lower ant diversity and resource availability than in forests. Yet, few studies have quantitatively examined these patchy patterns to statistically test if ants are non-randomly distributed or at what scale. Coffee agroecosystems form a gradient of management intensification along which vegetative complexity and ant diversity decline. Using field studies and a spatially explicit randomization model, I investigated ant patchiness in coffee agroecosystems in Chiapas, Mexico varying in management intensity to examine if: (1) coffee intensification affects occurrence of numerically dominant ants, (2) numerical dominants form statistically distinguishable single-species patches in coffee plants, (3) shade trees play a role in patch location, and (4) patch formation or size varies with management intensity. Coffee intensification correlated with lower occurrence frequency of numerically dominant species generally and of one of four taxa examined. All dominant ant species formed patches but only Azteca instabilis was patchy around shade trees. Ant patchiness did vary somewhat with spatial scale and with strata (within the coffee layer vs around shade trees). Patchiness, however, did not vary with management intensity. These results provide quantitative evidence that numerically dominant ants are patchy within the coffee layer at different scales and that shade tree location, but not coffee management intensity, may play a role in the formation of patchy distributions. PMID- 16676159 TI - A reassessment of the mating system characteristics of the army ant Eciton burchellii. AB - In a recent study, Denny et al. (2004a) showed that queens of the army ant, Eciton burchellii, mate with multiple males and presented estimates suggesting that they mate with more males than queens of any other ant species so far investigated. They also inferred that data were consistent with queens being inseminated repeatedly throughout their life, which would be exceptional among the social Hymenoptera and contradictory to predictions from kin selection theory. In the present study, we reanalyze these data using new software and supplement them with similar microsatellite data from other colonies of the same species. Mating frequencies in E. burchellii are indeed very high (mean observed and effective queen-mating frequencies of 12.9 each) but considerably lower than the previous estimates. We show that the number of patrilines represented in the first worker offspring of a young queen is lower than in older queens but suggest that this may be due to initial sperm clumping in the queen's sperm storage organ, rather than to repeated inseminations. Moreover, we found no evidence for repeated mating by genotyping sequential worker generations produced by a single old queen, showing that she did not obtain new inseminations despite ample opportunities for mating. PMID- 16676162 TI - Protection effect of piper betel leaf extract against carbon tetrachloride induced liver fibrosis in rats. AB - Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of detoxication, antioxidation, and antimutation. In this study, we evaluated the antihepatotoxic effect of PBL extract on the carbon tetrachloride (CCl(4))-induced liver injury in a rat model. Fibrosis and hepatic damage, as reveled by histology and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were induced in rats by an administration of CCl(4) (8%, 1 ml/kg body weight) thrice a week for 4 weeks. PBL extract significantly inhibited the elevated AST and ALT activities caused by CCl(4) intoxication. It also attenuated total glutathione S-transferase (GST) activity and GST alpha isoform activity, and on the other hand, enhanced superoxide dismutase (SOD) and catalase (CAT) activities. The histological examination showed the PBL extract protected liver from the damage induced by CCl(4) by decreasing alpha-smooth muscle actin (alpha sma) expression, inducing active matrix metalloproteinase-2 (MMP2) expression though Ras/Erk pathway, and inhibiting TIMP2 level that consequently attenuated the fibrosis of liver. The data of this study support a chemopreventive potential of PBL against liver fibrosis. PMID- 16676164 TI - Temporal and region-dependent changes in muscarinic M4 receptors in the hippocampus and entorhinal cortex of adrenalectomized rats. AB - Long-term adrenalectomy induces a dramatic loss of cells in the dentate gyrus and CA1-CA4 fields of the hippocampus resulting in an impairment of cognitive functions such as spatial learning, memory and exploratory behaviour. Muscarinic M1 and M4 receptor levels in the hippocampus and entorhinal cortex of adult male Wistar rats were examined 3, 14, 30, 90, and 150 days after adrenalectomy. Receptor levels in the entorhinal cortex and the hippocampus were determined by quantitative autoradiography using 125I-M1-toxin-1 and 125I-M4-toxin-1, M1 and M4 subtype selective antagonists, respectively. Moreover, the level of hippocampal M1 and M4 muscarinic receptors were evaluated 1 month after adrenalectomy by immunoblot analysis. Adrenalectomy induced apoptotic processes were examined by analysing apoptotic markers using Western blot analysis. No significant changes were observed in the level of muscarinic M1 receptors in the entorhinal cortex, the dentate gyrus and in the different CA fields of the hippocampus of adrenalectomized (ADX) rats. However, M4 receptors showed a significant decrease in the entorhinal cortex (at 3 days), dentate gyrus and CA4 (at 14 days), CA3 (at 30 days), and CA2 and CA1 (at 90 days) after adrenalectomy. Moreover, a decrease in the level of M4 receptors was detected in ADX rats 1 month after adrenalectomy as compared with sham groups using M4 specific antibody. Apoptotic markers such as PARP and p53 were significantly increased whereas Bcl-2 marker was decreased in ADX rat brain homogenates compared to controls. Our results show that M1 and M4 receptors are differentially affected by adrenalectomy and indicate that these subtypes have different functions in the hippocampus. Our data on time and region dependent decreases in hippocampal M4 receptors indicate that the M4 receptor subtype is influenced by adrenal hormones and suggest that the M4 receptor might be linked to memory function in the hippocampus. PMID- 16676163 TI - The drugs don't work-or do they? Pharmacological and transgenic studies of the contribution of NMDA and GluR-A-containing AMPA receptors to hippocampal dependent memory. AB - OBJECTIVE: The aim of this article is to provide a review of studies using N methyl-D-aspartate (NMDA) receptor antagonists to assess the hippocampal long term potentiation (LTP)/learning hypothesis. DISCUSSION: In particular, we will re-examine the validity of both (1) the original hippocampal LTP/spatial learning hypothesis of Morris and (2) the sensorimotor account put forward by Cain, among others, both from the point of view of the pharmacological studies on which they were based and with regard to recent studies with genetically modified mice. More specifically, we will review the pharmacological studies in the light of recent work on the glutamate receptor A (GluR-A or GluR1) L-alpha-amino-3-hydroxy-5 methyl-4-isoxazelopropionate (AMPA) receptor sub-unit knockout mouse. We will argue that neither the original hippocampal LTP/spatial learning hypothesis nor a sensorimotor account can adequately explain all of the available data. We argue instead that hippocampal synaptic plasticity, which requires NMDA receptors for its induction and GluR-A-containing AMPA receptors for its continued expression, contributes to a process whereby appropriate behavioural responses are selected rapidly on the basis of conditional information provided by the context. These contextual cues could include not only the spatial context (i.e. the 'where') and the temporal context (the 'when'), but also other aspects of context, such as internal state cues (hunger and fear state), and can be used to rapidly and flexibly alter valences of specific response options. RECOMMENDATIONS: We also suggest that there is a separate, distinct, NMDA/GluR-A-independent mechanism through which the context can gradually (incrementally or decrementally) alter the valence of a particular response option. PMID- 16676165 TI - The "human visceral homunculus" to pain evoked in the oesophagus, stomach, duodenum and sigmoid colon. AB - The oesophagus, stomach, duodenum and sigmoid colon were electrically stimulated in 12 healthy volunteers with a thin nasal endoscope. The painful cortical evoked potentials (EPs) were recorded from 64 surface electrodes. The early EPs with latencies < 200 ms were studied and the corresponding dipole sources were calculated. The electrical current intensities needed to evoke pain were highest in the stomach and duodenum, compared to the other segments (F = 7.8; P < 0.001; post hoc analysis P < 0.05). The EP latencies after stimulation of the stomach and sigmoid colon were shorter compared with those to stimulation of the oesophagus and duodenum (all P values < 0.001). The EP amplitudes were higher to oesophagus stimulation (all P values < 0.001 except for the early positivity). The potential fields obtained after stimulation of the most distal segments (duodenum and sigmoid colon) were in general distributed more posteriorly compared to those recorded in the more proximal regions. The EP topographies to stimulation of all gut tracts were explained by a bilateral source in the second somatosensory (SII) area, by a dipole in the anterior cingulate cortex (ACC), and by a bilateral generator in the insular cortex. However, the position of the sources significantly changed depending on the stimulated gut tract. Moreover, while the SII and ACC sources were initially activated to oesophagus and stomach stimulation, the ACC and insular activities were the earliest ones after stimulation of the lower gut segments. The findings reflect differences in pathways and brain processing of visceral nociceptive inputs coming from either upper or lower gut and may improve our understanding of the brain-gut axis in health and disease. PMID- 16676166 TI - The effect of task instruction on the excitability of spinal and supraspinal reflex pathways projecting to the biceps muscle. AB - There is controversy within the literature regarding the influence of task instruction on the size of the long-latency stretch reflex (M2) elicited by a joint displacement. The aim of this study was to investigate if the previously reported task-dependent modulation of the M2 is specific to the M2 or can be explained by an early release of the intended voluntary response. We took advantage of the fact that the M2 is absent when the duration of the applied perturbation is less than a critical time period. This allowed us to examine modulation of muscle activity with and without the contribution of the M2. In addition, we applied transcranial magnetic stimulation (TMS) over the primary motor cortex to examine the modulation of corticomotor excitability with task instruction. Elbow joint extension displacements were used to elicit a stretch reflex in the biceps muscle. Subjects were instructed to "do not intervene" (DNI) with the applied perturbation, or to oppose the perturbation by activating the elbow flexors in response to the perturbation (FLEX). Electromyographic (EMG) activity in the time period corresponding to the M2 was significantly facilitated in the FLEX task instruction both with and without the presence of the M2. Motor evoked potentials (MEPs) elicited by TMS were also facilitated during the FLEX condition in the absence of the M2. EMG and MEP responses were not facilitated until immediately prior to the onset of the M2. Paired-pulse TMS revealed a significant reduction in short-interval intracortical inhibition (SICI) during the M2 response, but the level of SICI was not altered by the task instruction. We conclude that the task-dependent modulation of the biceps M2 results, at least in part, from an early release of the prepared movement and is accompanied by an increase in corticospinal excitability that is not specific to the M2 pathway. Task-dependent modulation of the response cannot be explained by an alteration in the excitability of intracortical inhibitory circuits. PMID- 16676167 TI - Thermodynamic properties of hyperpolarization-activated current (Ih) in a subgroup of primary sensory neurons. AB - Ih is a poorly selective cation current that activates upon hyperpolarization, present in various types of neurons. Our aim was to perform a detailed thermodynamic analysis of Ih gating kinetics, in order to assess putative structural changes associated with its activation and deactivation. To select dorsal root ganglia neurons that exhibit large Ih, we applied a current signature method by Petruska et al. (J Neurophysiol 84:2365-2379, 2000) and found appropriate neurons in cluster 4. Currents elicited by 3,000-ms hyperpolarizing pulses at 25 and 33 degrees C were fitted with double exponential functions, yielding time constants similar to those of HCN1. The fast activation and deactivation rates showed temperature coefficients (Q10) of 2.9 and 3.1, respectively, while Q10 of the absolute conductance was 1.3. Using the Arrhenius Eyring formalism we computed heights of voltage-independent Gibbs free energy and entropy barriers for each rate. The free energy barriers of the fast rates were just approximately 2RT units lower than those of the corresponding slow rates (31.3 vs. 33.2RT for activation, and 24.7 vs. 25.8RT for deactivation, at 25 degrees C). Interestingly, the entropy barriers of the slow rates were negative: 15.2R units for activation and -11.9R units for deactivation, compared to 4.6 and 1.3R units, respectively, for the fast component. The equivalent gating charge (zg) (3.75 +/- 0.32, mean +/- SEM, at 25 degrees C) and half-activation potential (V1/2) (-70.0 +/- 1.3 mV at 25 degrees C) did not vary significantly with temperature. PMID- 16676168 TI - The influence of mild traumatic brain injury on the temporal distribution of attention. AB - The purpose of this study was to examine whether the temporal dynamics of attention was deficient in participants who have recently experienced mild traumatic brain injury (mTBI). For this purpose the rapid serial visual presentation (RSVP) task was used and the performance of participants with mTBI was compared to that of controls matched for age, gender, education, and activity type. In the RSVP task a stream of rapidly presented letters is displayed with target and probe letters separated by varying durations. The participant is required to identify the target letter and determine whether the probe letter was present or not. Previous research has shown that healthy participants display an attentional blink: they fail to detect the probe letter when it appears within approximately 500 ms of the target letter. We found that participants with mTBI had a normal attentional blink-it was neither greater in magnitude nor longer in duration than that displayed by the control participants. However, the participants with mTBI did show evidence of attentional competition-making more errors in identifying the target letter when the probe letter was presented-that was not present in the control participants. Taken together, these results suggest that the temporal constraints of attention are subtly but systematically affected by mTBI. PMID- 16676169 TI - Improved differentiation of tactile activations in human secondary somatosensory cortex and thalamus using cardiac-triggered fMRI. AB - Functional magnetic resonance imaging (fMRI) can reveal human brain activations with high precision. The accuracy may, however, be impaired by movement and deformation of brain tissue associated with cardiac pulsations. Here we corrected for such artifacts by time-locking the fMRI data acquisition to the cardiac cycle in ten subjects who received tactile stimuli to their lips, fingers, and toes. The imaged brain areas covered the parietal operculum and the thalamus, including the secondary somatosensory cortex (SII) bilaterally. Variance of the blood oxygen-level-dependent signal decreased on average by 38-40% in the SII cortex and by 26% in the thalamus during cardiac triggering compared with conventional imaging. Consequently, statistically significant responses were seen both in the SII cortex and in the ventroposterior thalamus in a larger number of subjects. At the cortical level, the activation pattern revealed two distinct representations for both fingers and toes in the SII region, and the more medial representations were detected with enhanced clarity during cardiac-triggered imaging. In the group-level analysis, the thalamic response to finger stimulation was seen with cardiac triggering, only. PMID- 16676170 TI - Visuomotor representation decay: influence on motor systems. AB - The contribution of ventral stream information to the variability of movement has been the focus of much attention, and has provided numerous researchers with conflicting results. These results have been obtained through the use of discrete pointing movements, and as such, do not offer any explanation regarding how ventral stream information contributes to movement variability over time. The present study examined the contribution of ventral stream information to movement variability in three tasks: Hand-only movement, eye-only movement, and an eye hand coordinated task. Participants performed a continuous reciprocal tapping task to two point-of-light targets for 10 s. The targets were visible for the first 5 s, at which point vision of the targets was removed. Movement variability was similar in all conditions for the initial 5-s interval. The no-vision condition (final 5 s) can be summarized as follows: ventral stream information contributed to an initial significant increase in variability across motor systems, though the different motor systems were able to preserve ventral information integrity differently. The results of these studies can be attributed to the behavioral and cortical networks that underlie the saccadic and manual motor systems. PMID- 16676171 TI - Threshold control of motor actions prevents destabilizing effects of proprioceptive delays. AB - It is usually assumed that proprioceptive feedback comes to motoneurons too late to contribute to the initial activity of agonist muscles during fast arm movements, leading to the suggestion that this feedback is only efficient in slow movements and postural control. The argument does not take into account that the changes in the motoneuronal membrane potentials and the associated changes in the state of spinal neurons preceding the initial activity of muscles deeply affect, in a forward way, the state of reflex systems by shifting their thresholds, as suggested in the lambda model for motor control. As a result, the initial muscle activity emerges with full contribution of these systems so that the effects of reflex delays become negligible. We tested the hypothesis that threshold control of muscle activation may be instrumental in preventing destabilizing effects of proprioceptive delays in spinal and trans-cortical pathways to motoneurons. The analysis was made by recording fast elbow movements (peak velocity approximately 300-500 degrees/s) and simulating them in a dynamic model that incorporates the notion of threshold control of intrinsic and reflex muscle properties. The model was robust in reproducing experimental movement patterns (R (2)>0.95). It generated stable output despite substantial proprioceptive (up to 100 ms) and electromechanical (40 ms) delays. Stability was thus ensured for delays not only in segmental (about 25-50 ms) but also in trans-cortical loops (50-70 ms). Our study illustrates that a natural physiological process--threshold control--may manifest feed-forward properties hitherto attributed to hypothetical internal neural models. PMID- 16676172 TI - Experimental manipulations of the subthalamic nucleus fail to suppress tonic seizures in the electroshock model of epilepsy. AB - Recently, it has been shown that the subthalamic nucleus (STN) has anticonvulsant effects on epileptic seizures originating from the forebrain. The aim of the present study was to determine whether the anticonvulsant properties of the STN extend to the suppression of tonic seizures originating from the brainstem elicited by electroshock in rats. Three different procedures were used to manipulate activity in the STN and in each case the duration of tonic hindlimb extension elicited by electroshock was used as a measure of seizure-severity. Under general anesthesia, two groups of rats received chronic implants of either bilateral stainless steel guide cannulae or bilateral bipolar stimulating electrodes stereotaxically implanted and aimed at the STN. After 3 days of recovery, each rat in the first group was tested with electroshock on three consecutive days after having received 220 nl bilateral microinjections into the STN of either 200 or 400 pmol of muscimol (a GABA agonist) dissolved in saline or the same volume of normal saline. In the second group the electroshock test was conducted, again on three consecutive days, immediately following high frequency electrical stimulation (HFS) of the STN at 130 or 260 Hz or a no current control condition. In the third group, rats were tested with electroshock before and after bilateral excitotoxic lesions of the STN with either kainic or ibotenic acids. None of these manipulations produced significant suppression of the tonic hind limb extension elicited by electroshock compared with the relevant control conditions. This suggests that, within the limitations of the current procedures, the anticonvulsant properties of the STN appear to be ineffective against tonic seizures originating in the brainstem. PMID- 16676173 TI - Pharmacokinetics and safety of a novel anti-HBs-enriched immunoglobulin in healthy volunteers after subcutaneous and intramuscular administration. PMID- 16676174 TI - Does the composition of voided urine reflect that of the renal pelvis? AB - Studies have shown that the urothelium has a transport function and that urine composition changes on its way through the urinary tract. In this study, we investigated the hypothesis that the composition of voided urine differs from and does not reflect that of the renal pelvis. Urine samples were obtained from the renal pelvis and voided urine of 18 healthy volunteers (mean age 36.2+/-5.1 SD years, 10 men, 8 women). The pH was determined using a pH electrode, osmolality by means of micro-osmometry and Na and K using flame photometry. In comparison to the urine of the renal pelvis, voided urine showed significant increases in pH, osmolality and Na and K concentrations (P<0.05 for each). There were no significant differences in gender and age. This study has demonstrated that the pH, osmolality, Na and K of voided urine differ significantly from the values in the renal pelvis. Urine composition is thus modified as it passes through the urinary tract, which would support the concept of a dynamic urothelium. The composition of voided urine does not seem to compare to renal pelvic urine. This concept needs to be considered in urine analysis evaluation and its relation to renal function. PMID- 16676180 TI - Nitrogen source and mineral optimization enhance D: -xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124. AB - Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61+/-9 g/l) by providing C:N in the vicinity of approximately 57-126:1 and optimizing the combination of urea and amino acids to supply 40-80 % nitrogen from urea and 60-20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others. PMID- 16676175 TI - Simulated data sets for single molecule kinetics: some limitations and complications of data analysis. AB - When the fluorescence intensity of a chromophore attached to or bound in an enzyme relates to a specific reactive step in the enzymatic reaction, a single molecule fluorescence study of the process reveals a time sequence in the fluorescence emission that can be analyzed to derive kinetic and mechanistic information. Reports of various experimental results and corresponding theoretical studies have provided a basis for interpreting these data and understanding the methodology. We have found it useful to parallel experiments with Monte Carlo simulations of potential models hypothesized to describe the reaction kinetics. The simulations can be adapted to include experimental limitations, such as limited data sets, and complexities such as dynamic disorder, where reaction rates appear to change over time. By using models that are known a priori, the simulations reveal some of the challenges of interpreting finite single-molecule data sets by employing various statistical signatures that have been identified. PMID- 16676181 TI - Immunotherapy of renal cell carcinoma. AB - Carcinomas of the kidney generally have a poor prognosis and respond minimally to classical radiotherapy or chemotherapy. Immunotherapy constitutes an interesting alternative to these established forms of treatment, and indeed, cytokine-based therapies have been used for many years, leading to favorable clinical responses in a small subset of patients. During the past few years, immunotherapeutical trials targeting renal cell tumor-associated antigens have also been reported, with diverse passive or active approaches using antibodies or aimed at activating tumor-directed T lymphocytes. The following review presents the results and the progress made in the field, including classical cytokine treatments, non myeloablative stem cell transplantation and antigen specific-based trials, with special focus on T-cell studies. In consideration of the few specific molecular targets described so far for this tumor entity, current strategies which can lead to the identification of new relevant antigens will be discussed. Hopefully these will very soon contribute to an improvement in renal cell carcinoma specific immunotherapy and its evaluation. PMID- 16676182 TI - Immune enhancement and anti-tumour activity of IL-23. AB - Immunotherapy, including the use of cytokines and/or modified tumour cells immune stimulatory cytokines, can enhance the host anti-tumour immune responses. Interleukin-23 (IL-23) is a relative novel cytokine, which consists of a heterodimer of the IL-12p40 subunit and a novel p19 subunit. IL-23 has biological activities similar to but distinct from IL-12. IL-23 can enhance the proliferation of memory T cells and the production of IFN-gamma, IL-12 and TNF alpha from activated T cells. IL-23 activates macrophages to produce TNF-alpha and nitric oxide. IL-23 can also act directly on dendritic cells and possesses potent anti-tumour and anti-metastatic activity in murine models of cancer. IL-23 can also induce a lower level of IFN-gamma production compared with that induced by IL-12. This may make IL-23 an alternative and safer therapeutic agent for cancer, as IL-12 administration can lead to severe toxic side effects because of the extremely high levels of IFN-gamma it induces. PMID- 16676183 TI - DC-based vaccine loaded with acid-eluted peptides in acute myeloid leukemia: the importance of choosing the best elution method. AB - Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4(+) and cytotoxic CD8(+) T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides. PMID- 16676184 TI - Hormone-regulated inflorescence induction and TFL1 expression in Arabidopsis callus in vitro. AB - To study hormone-regulated inflorescence development, we established the in vitro regeneration system of Arabidopsis inflorescences in the presence of cytokinin and auxin. Media containing a combination of thidiazuron (TDZ) and 2,4 dichlorophenoxyacetic acid (2,4-D) were used to induce callus formation. Higher frequencies of calli were obtained by using the inflorescence stems as explants. After transferring the calli to media containing a combination of zeatin and indole-3-acetic acid (IAA), the inflorescences were induced from the calli. The morphology of regenerated inflorescences was similar to that of inflorescences in plants; however, flowers of regenerated inflorescences often lacked a few floral organs. Furthermore, TFL1, a gene involved in floral transition in Arabidopsis, was activated during the inflorescence induction. Our results suggest that the TFL1 gene plays an important role in hormone-regulated inflorescence formation. PMID- 16676186 TI - Continent cutaneous urinary diversion: long-term follow-up of more than 800 patients with ileocecal reservoirs. AB - We report the clinical outcome of more than 800 patients, who underwent continent cutaneous urinary diversion with an ileocecal reservoir (Mainz-pouch I) in two urological tertiary referral centers at a mean follow-up of 7.6 years. Complications related to the continence mechanism (intussuscepted ileal nipple vs. submucosally embedded in situ appendix) and the antirefluxive uretero intestinal anastomosis (submucosal tunnel vs. serosa-lined extramural tunnel) were recorded retrospectively. Stomal stenosis was observed in 23.5% of the patients with appendix stoma and in 15.3% of the patients with intussuscepted ileal nipple. The incidence of calculi was 10.8% in reservoirs with intussuscepted ileal nipple and 5.6% in reservoirs with appendix stoma. Eleven patients (eight with appendix, three with ileal nipple) required reoperation because of ischemic degeneration of the continence mechanism. The overall continence rate (day and night) was 92.8%. Anastomotic strictures of the afferent limb occurred in 6.5% of renal units (RUs) with a submucosal tunnel and in 5.0% of RUs with a serosa-lined extramural tunnel. Continent cutaneous urinary diversion with an ileocecal pouch is a highly satisfactory and safe option for patients, in whom orthotopic urinary diversion is impossible or contraindicated. PMID- 16676187 TI - Results of orchiopexy for the undescended testis. AB - The undescended testis is one of the most common congenital abnormalities of the genitourinary system. Outcomes of orchiopexy include (1) having a viable, palpable testis in the scrotum, (2) fertility, as measured by paternity rates or semen analysis in adulthood and (3) risk of testicular cancer. Multiple operative techniques have been described and are associated with various success rates. In the past decade, success of orchiopexy for inguinal testes has been >95%. For abdominal testes, success for orchiopexy has been >85-90% in most series with single stage orchiopexy or two stage Fowler-Stephens orchiopexy, both with open surgical or laparoscopic technique. However, having a palpable testis in the scrotum does not assure fertility, as there are iatrogenic factors that may adversely affect the outcome. In adult men with a history of unilateral orchiopexy, fertility is nearly normal, but is significantly reduced following bilateral orchiopexy. The risk of testicular carcinoma is increased by a factor of 3.7 to 7.5 times. Tumor type is most commonly seminoma if the testis is undescended, whereas tumors that occur following orchiopexy are much more likely to be nonseminomatous. PMID- 16676188 TI - Temperature-sensitive gating in a descending visual interneuron, DCMD. AB - Activity in neural circuits can be modified through experience-dependent mechanisms. The effects of high temperature on a locust visual interneuron (the descending contralateral movement detector, DCMD) have previously been shown to be mitigated by prior exposure to sub-lethal, elevated temperatures (heat shock, HS). Activity in the DCMD is reduced at high temperature in naive animals (control), whereas HS animals show a maintained spike count at all temperatures. We examined whether this finding was due to direct effects of temperature on visual processing, or whether other indirect feedback mechanisms were responsible for the observed effect in the DCMD. Activity in the DCMD was elicited using a computer-generated looming image, and the response was recorded extracellularly. The temperature of visual processing circuits contributes directly to HS-induced plasticity in the DCMD, as maintaining the brain at 25 degrees C during a thoracic temperature ramp eliminated the high frequency activity associated with HS. Removing ascending input by severing the thoracic nerve cord reduced DCMD thermosensitivity, indicating that indirect feedback mechanisms are also involved in controlling the DCMD response to increased thoracic temperature. Understanding how thermosensitive feedback within the locust affects DCMD function provides insight into critical regulatory mechanisms underlying visually-guided behaviors. PMID- 16676189 TI - Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis). AB - Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a (14)C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates. PMID- 16676190 TI - Identification of the non-specific cytotoxic cell receptor protein 1 (NCCRP1) in regenerating axolotl limbs. AB - The teleost non-specific cytotoxic cells (NCC) are evolutionary precursors of the mammalian natural killer (NK) cells and an important element of innate immunity. The non-specific cytotoxic cell receptor protein (NCCRP1) is a characteristic cell surface protein with main functions in target cell recognition and cytotoxicity with sequence information available for many species of fish. We have isolated a cDNA encoding the Axolotl homologue of fish NCCRP1 out of limb regeneration blastema and analysed its expression by RT-PCR. Sequence analysis revealed a high degree of homology with teleost NCCRP1 on nucleotide and deduced amino acid levels. NCCRP1 contains a conserved C-terminal F-box-associated domain (FBA) and proline-rich motifs (PRM) characteristic for this protein family. NCCRP1 is expressed in multiple tissues with high levels in limb regeneration blastema. The present work describes for the first time the cloning of the NCCRP1 gene in a tetrapod vertebrate providing a valuable link between fish and higher vertebrates. Our findings suggest the existence of NCC in axolotl and a role of the innate immune system in the processes of limb regeneration. PMID- 16676195 TI - The possible preterm birth preventive effect of ampicillin during pregnancy. AB - OBJECTIVE: To study the possible association between oral ampicillin treatment during pregnancy and pregnancy complications, in addition to birth outcomes, particularly preterm birth. METHODS: We evaluated newborn infants without congenital abnormalities born to mothers with or without ampicillin treatment during pregnancy in the population-based large data set of the Hungarian Case Control Surveillance of Congenital Abnormalities. RESULTS: Of 38,151 newborn infants, 2,630 (6.9%) had mothers with ampicillin treatment during pregnancy. Some pregnancy complications, particularly preeclampsia, showed a higher prevalence in pregnant women with ampicillin treatment. Mean gestational age was slightly longer (0.2 week) but it resulted in a significant reduction in the prevalence of preterm birth (7.1 vs 9.3%; adjusted POR with 95% CI: 0.8, 0.7 0.9). The preterm birth preventive effect of ampicillin was most obvious after the use of the drug in the first trimester of pregnancy. Similar difference was not seen in the reduction of low birth weight. The rate of preterm birth was lower in newborn infants born to mothers with acute infectious diseases of both the genitourinary and respiratory systems if they were treated by ampicillin. CONCLUSIONS: Ampicillin treatment, particularly in the first trimester of gestation, can reduce the proportion of preterm birth in pregnant women with acute infectious diseases of the genitourinary and respiratory system. PMID- 16676196 TI - Epithelioid vascular leiomyoma of the uterus mimicking glomangiomyoma. AB - CASE REPORT: An unusual case of epithelioid vascular leiomyoma mimicking glomangiomyoma arising in the uterine corpus of a 55-year-old Japanese woman is presented. The surgically resected uterine mass, measuring 4.0x3.5 cm2, demonstrated a dark red well-circumscribed tumor. Histologically, the rounded epithelioid cells around the dilated vessels showed gradual transition to spindled smooth muscle cells. Immunohistochemistry was positive for smooth muscle actin, but negative for collagen IV. CONCLUSION: The experience in this case emphasizes that glomangiomyoma-like feature is interesting and might indeed be a new histological variant of uterine leiomyomas. PMID- 16676199 TI - Myxoid fibroblastic tumor of the maxillary sinus in a 21-month-old child: An unusual diagnosis. AB - Myxoid tumors of the soft tissue are a heterogeneous group of lesions with significant differences in behavior, from being totally harmless to malignant tumors with metastasizing potential. We describe the unusual case of a 21-month old boy who developed a paranasal low-grade myxoid neoplasm with recurrent potential. The histological findings showed a proliferation of spindle cells within a vascular and myxoid background. The immunohistochemistry had the features of myofibroblasts and fibroblasts with strong Vimentin and light Smooth Muscle Actin reactivity. Close collaboration between clinician and pathologist is necessary to deal with these unusual lesions to predict their local aggressiveness and long-term behavior. Most of these cellular myxomas have been described in adults, involving primarily the limbs, but this case shows that any localization is possible. PMID- 16676201 TI - The dilemma of multiorgan donors with high serum PSA--a pathologist's proposal. PMID- 16676203 TI - Laboratory diagnosis of sulphite oxidase deficiency. PMID- 16676202 TI - Identification of immunoreactive FSH and LH cells in the cichlid fish Cichlasoma dimerus during the ontogeny and sexual differentiation. AB - Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) expressing cells were detected in pituitary, brain and ovary of the Perciform cichlid fish Cichlasoma dimerus. This detection was carried out by immunohistochemistry (IHC) and Western blot techniques using antisera of the Cyprinodontiform Fundulus heteroclitus raised against the conservative region of the teleost betaFSH and the betaLH subunits. The estimated molecular weights were 24 kDa for LH and 19 and 15 kDa for FSH. In the adult pituitary, both cell types were distributed along mid and ventral zones of the proximal pars distalis (PPD, mid immunoreactive cells), and along the ventral and dorsal external border of the pars intermedia (PI, high-immunoreactive cells). Double IHC showed that FSH and LH are mainly expressed in different pituitary cells. FSH cells were detected in the pituitary around day 21 after hatching (ah) (prior to sex differentiation), while LH cells were detected by day 60 ah (during the sexual differentiation period). A correlation between gonadal sex differentiation and FSH was demonstrated in a 15 days organ culture system. FSH and LH neurons were localized in the nucleus lateralis tuberis and their fibers project through the ventral hypothalamus, preoptic area and neurohypophysis. FSH neurons differentiated on day 21 ah, while LH neurons appeared on day 15 ah. In the ovary, the immunoreactivity for both FSH and LH was restricted to the cytoplasm of previtellogenic and early vitellogenic oocytes. PMID- 16676206 TI - Preferential feeding by an aquatic consumer mediates non-additive decomposition of speciose leaf litter. AB - Forest soils and streams receive substantial inputs of detritus from deciduous vegetation. Decay of this material is a critical ecosystem process, recycling nutrients and supporting detrital-based food webs, and has been attributed, in part, to leaf litter species composition. However, research on why speciose leaf litter should degrade differently has relied on a bottom-up approach, embracing interspecific variation in litter chemistry. We hypothesized that preferential feeding by an aquatic detritivore interacts with species-specific leaf palatability and slows decay of speciose leaf litter. We addressed this by offering four single- and mixed-species leaf resources to field densities of a leaf-shredding consumer. Mixing leaf species resulted in slower total leaf decomposition. Decreases in mixed-species decomposition was partly explained by preferential feeding by the consumers in one case, but the lack of preferential feeding in other mixtures suggested an interactive effect of feeding and microbial degradation. Loss of riparian tree biodiversity may have implications for in-stream consumer-resource interactions. PMID- 16676207 TI - A test of three alternative pathways for consumer regulation of primary productivity. AB - The pathways linking consumer effects to primary productivity (PPR) are likely to vary among taxa because of species-specific trophic and functional differences. Thus, it is necessary to understand the dynamics of consumer-PPR interactions so that effects of species loss on ecosystem function can be addressed from a mechanistic approach. In this study, I used three fish taxa (orangethroat darter, Etheostoma spectabile; western mosquitofish, Gambusia affinis; and bullhead minnow, Pimephales vigilax) as model consumers with different trophic and functional characteristics to test alternative mechanisms for consumer regulation of PPR (i.e., trophic cascade, terrestrial nutrient translocation, and sedimentary nutrient translocation). Experiments were conducted in stream mesocosms fitted with a combination of fish and terrestrial insect barriers to address relative importance of consumer-driven top-down and bottom-up control of PPR. A predatory invertivore, orangethroat darter, increased PPR through an apparent trophic cascade by localized reduction of benthic grazing invertebrate densities (i.e., top-down). A surface feeding insectivore, western mosquitofish, consumed terrestrial insects on the stream surface, increasing PPR by enhancing allochthonous nutrients in the mesocosms (i.e., bottom-up). A benthic omnivore, bullhead minnow, consumed benthic food items, resulting in increased PPR by enhancing availability of autochthonous nutrients via translocation of sedimentary nutrients (i.e., bottom-up). However, under specific environmental contexts, this species also consumed terrestrial invertebrates, potentially affecting PPR through terrestrial nutrient translocation as well. In this study, the trophic and functional characteristics of different species resulted in alternative pathways that increased PPR, suggesting that in natural ecosystems multiple consumer-driven pathways may be influencing PPR simultaneously and could potentially be important for temporal persistence of ecosystem function in changing environments. PMID- 16676208 TI - Contrasting mechanisms of secondary metabolite accumulation during leaf development in two tropical tree species with different leaf expansion strategies. AB - Young leaves of most species experience remarkably higher herbivore attack rates than mature leaves. Considerable theoretical effort has focused on predicting optimal defense and tradeoffs in defense allocation during leaf expansion. Among others, allocation to secondary chemistry may be dependent on growth constraints. We studied flavanoid production during leaf development in two species of Inga (Fabaceae: Mimosoideae) with different expansion strategies: Inga goldmanii, a species with slowly expanding young leaves, and Inga umbellifera, a species with fast-expanding young leaves. In these two species, the most abundant and toxic class of defensive compounds is flavanoids (which include tannins). We measured their concentration by leaf dry weight, their total content per leaf, their HPLC chemical profile and their toxicity to a generalist herbivore at different expansion levels. Although in both species the flavanoid concentration decreased with increasing leaf expansion, that decrease was twice as pronounced for I. umbellifera as it was for I. goldmanii. I. umbellifera leaves produced flavanoids only during the first half of their development while I. goldmanii leaves continued production throughout. The changes in flavanoid HPLC profiles and toxicity were also more dramatic for I. umbellifera, which had different flavanoids in young than in mature leaves. Relative to I. umbellifera, I. goldmanii showed smaller changes in both flavanoid composition and toxicity in the transition from young to mature leaves. These results indicate that, even though young leaves suffer higher rates of attack and are predicted to have better chemical defenses than mature leaves, growth constraints may modulate defense allocation and thus, evolution of defense strategies. PMID- 16676210 TI - Structure-olfactive threshold relationships for pyrazine derivatives. AB - Structure-olfactive threshold relationships for 40 pyrazine derivatives have been studied by multivariate statistical analysis. Variable descriptors used to describe the molecules studied were obtained using molecular-mechanics software. A correlation coefficient of 0.82 was obtained when all the molecules were included in the model. When the model was established for closely related subsets of molecules, the correlation coefficients obtained were higher and the established models were bilinear. Such models allow the identification of optimal structures corresponding to low olfactive thresholds for the subsets studied. Surprisingly, we find that the optimum structures are included in the set of 40 molecules. The efficiency of the models was supported by the cross-validation technique, where the correlation coefficients were found to be good with respect to the precision of the values of the olfactive thresholds. PMID- 16676211 TI - Possible dynamic anchor points in a benzoxazinone derivative-human oxytocin receptor system--a molecular docking and dynamics calculation. AB - In this study, we performed a molecular docking and dynamics simulation for a benzoxazinone-human oxytocin receptor system to determine the possible hydrophobic and electrostatic interaction points in the dynamic complex. After the homology modeling, the ligand was docked into the putative active using AutoDock 3.05. After the application of energetic and structural filters, the complexes obtained were further refined with a simulated annealing protocol (AMBER8) to remove steric clashes. Three complexes were selected for subjection to the molecular dynamics simulation (5 ns), and the results on the occurrence of average anchor points showed a stable complex between the benzoxazinone derivative and the receptor. The complex could be used as a good starting point for further analysis with site-directed mutagenesis, or further computational research. PMID- 16676216 TI - Acetylcholinesterase activities in marine snail (Cronia contracta) as a biomarker of neurotoxic contaminants along the Goa coast, West coast of India. AB - The measurement of acetylcholinesterase (AChE) activity is used worldwide as a biomarker of environmental contamination due to neurotoxic substances. In the present study the AChE activities was measured in marine snails (Cronia contracta) collected seasonally from six sampling sites (viz. Arambol, Anjuna, Dona Paula, Vasco, Velsao and Palolem) along the Goa coast during the pre-monsoon (April, 2004), monsoon (September, 2004) and post-monsoon (November, 2004) periods. The AChE activities in C. contracta showed wide variation along the Goa coast. It was found to be quite high at the reference site, Palolem (23.97, 21.72 and 24.85) throughout the sampling period (April-November, 2004). The AChE activities in C. contracta decreased significantly at Vasco (44.6-52.4% reduction) followed by Dona Paula (24.9-36.2% reduction), Velasao (10.8-35.9% reduction), Arambol (12.6-37.3% reduction) and Anjuna (0-12.7% reduction). Such a significant variation of AChE activities in the marine snail along the Goa coast can be attributed to neurotoxic substances prevalent in those regions. The high concentration of different neurotoxic metals (lead, cadmium, copper, manganese and iron) and petroleum hydrocarbons in the tissues of the marine snails at Dona Paula, Vasco and Velsao clearly substantiate reduction of AChE activities in C. contracta. The in vitro studies on the inhibition of AChE by different metals and PHC indicated that lead, cadmium and copper are the most predominant inhibitor. Based on the AChE activities in C. contracta the sampling sites along the Goa coast can be classified into three major clusters such as highly contaminated sites (Dona Paula, Vasco and Velsao), moderately contaminated sites (Arambol, Anjuna) and least contaminated site (Palolem). PMID- 16676217 TI - Biodegradation of dimethyl terephthalate by Pasteurella multocida Sa follows an alternative biochemical pathway. AB - Pasteurella multocida Sa, a bacterial strain isolated from mangrove sediment by enrichment technique, was capable of transforming dimethyl terephthalate (DMT). Biodegradation of DMT was shown to take place as a series of sequential steps involving the hydrolysis of two ester linkages between the carboxyl groups of the terephthalate and the methyl side-chain initially to produce mono-methyl terephthalate (MMT) and then terephthalic acid (TA), respectively. However, with ethanol as the carrying solvent, there was a formation of one metabolite previously not observed. The two metabolites were characterized by high performance-liquid chromatography-electron ionization mass spectrometry as MMT and mono-ethyl terephthalate (MET), suggesting the existence of an alternative biochemical pathway in the degradation of DMT by P. multocida Sa. Since the presence of MMT and ethanol in culture inoculated with P. multocida Sa was prerequisites for the formation of MET, biologically mediated trans esterification was proposed as a mechanism for the novel biochemical process observed. PMID- 16676218 TI - Molecular Biomarkers: their significance and application in marine pollution monitoring. AB - This paper presents an overview of the significance of the use of molecular biomarkers as diagnostic and prognostic tools for marine pollution monitoring. In order to assess the impact of highly persistent pollutants such as polychlorinated biphenyls (PCB), polychlorinated dibenzo-dioxins (PCDD), polychlorinated dibenzo-furans (PCDF), polynuclear aromatic hydrocarbons (PAH), tributyltin (TBT) and other toxic metals on the marine ecosystem a suite of biomarkers are being extensively used worldwide. Among the various types of biomarkers, the following have received special attention: cytochrome P4501A induction, DNA integrity, acetylcholinesterase activity and metallothionein induction. These biomarkers are being used to evaluate exposure of various species of sentinel marine organisms (e.g. mussels, clams, oysters, snails, fishes, etc.) to and the effect of various contaminants (organic xenobiotics and metals) using different molecular approaches [biochemical assays, enzyme linked immuno-sorbent assays (ELISA), spectrophotometric, fluorometric measurement, differential pulsed polarography, liquid chromatography, atomic absorption spectrometry]. The induction of the biotransformation enzyme, cytochrome P4501A in fishes (Callionymus lyra, Limanda limanda, Serranus sp., Mullus barbatus) and mussels (Dreissena polymorpha) by various xenobiotic contaminants such as PCBs, PAHs, PCDs is used as a biomarker of exposure to such organic pollutants. The induction of cytochrome P4501A is involved in chemical carcinogenesis through catalysis of the covalent bonding of organic contaminants to a DNA strand leading to formation of DNA adduct. Measurement of the induction of cytochrome P4501A in terms of EROD (7-ethoxy resorufin O-deethylase) activity is successfully used as a potential biomarker of exposure to xenobiotic contaminants in marine pollution monitoring. In order to assess the impact of neurotoxic compounds on marine environment the evaluation of acetylcholinesterase activity in marine organisms is used as a biomarker of exposure to neurotoxic agents such as organophosphorus, carbamate pesticides etc. Metallothioneins (MTs) are induced by toxic metals such as Cd, Hg, and Cu by chelation through cysteine residues and are used in both vertebrates and invertebrates as a biomarker of metal exposure. The measurement of the levels of DNA integrity in marine organisms such as Sea stars (Asterias rubens) from the North Sea and the marine snails (Planaxis sulcatus) from the Arabian Sea along the Goa coast exposed to environmental xenobiotic contaminants clearly indicated the extent and the nature of pollution at the sampling sites along coastal environment. PMID- 16676223 TI - Gender differences in sexual behaviors, sexual partnerships, and HIV among drug users in New York City. AB - We compared sexual behaviors/partnerships and determined sexual risk correlates associated with HIV by gender among street-recruited drug users using chi-square tests and logistic regression. Men reported higher risk sexual behaviors, yet fewer high-risk sexual partners than women. After adjustment, HIV seropositive men were more likely than seronegatives to be older, MSM, use condoms, and have an HIV-infected partner. HIV seropositive women were more likely to be older, have an HIV-infected partner, and not use non-injected heroin. IDU was not associated with HIV. Prospective studies are needed to determine how gender specific sexual behaviors/partnerships among drug users affect HIV acquisition. PMID- 16676224 TI - Meta-analysis of association between a catechol-O-methyltransferase gene polymorphism and attention deficit hyperactivity disorder. AB - There have been conflicting reports on the association between the Val158/108Met polymorphism of the catechol-O-methyltransferase (COMT) gene and attention deficit hyperactivity disorder (ADHD). Therefore we would like to perform a meta analysis of previous studies to assess the overall magnitude and significance of the association. Family-based and case-control studies of the association between the COMT gene polymorphism and ADHD were searched systematically and comprehensively. Odds ratios (OR) of association were pooled by the fixed effects model if no significant heterogeneity was present among different studies. Subgroup analysis by gender and ADHD subtypes were also performed. Eleven family based and two case-control studies were identified. After pooling the results, no significant association between the COMT Vall58/108Met polymorphism and ADHD was found (OR 0.99 (95% CI: 0.88-1.12), P = 0.87). There was also no significant association when the results were stratified by gender or ADHD subtype. There was no significant statistical heterogeneity (chi2 = 12.27, P = 0.2) although clinical heterogeneity was present in the studies, especially the ethnicity of subjects. Sensitivity analysis demonstrated absence of undue influence of any single study. Standard regression analysis showed no significant publication bias. We concluded that no significant association was present between the most common COMT gene polymorphism and ADHD. Further studies should employ larger sample size in more homogeneous subjects. Further investigations in moderator variables and gene-gene and gene-environment interactions are also warranted. PMID- 16676225 TI - Artificial selection for increased maternal defense behavior in mice. AB - Maternal aggression is directed towards intruders by lactating females and is critical for defense of offspring. Within-family selection for increased maternal defense in outbred house mice (Mus domesticus; Hsd:ICR strain) was applied to one selected (S) line, using total duration of attacks in a 3-min test as the selection criterion. One control (C) line was maintained and both lines were propagated by 13 families in each generation. Prior to selection, heritability of maternal aggression was estimated to be 0.61 based on mother-offspring regression. Duration of attacks responded to selection with a mean realized heritability of 0.40 (corrected for within-family selection) after eight generations. At generation 5, the S and C line also differed significantly for litter size at birth and at mid-lactation (both lower in S), average individual pup mass at midlactation (higher in S), and pup retrieval latency (longer in S), but not for other maternal measures that we studied (e.g., dam mass). Additionally, number of entries to middle and closed plus maze compartments was significantly higher in S mice in Generation 5. This is the first study to select for high maternal defense and these mice will be made available as a tool for understanding the genetic and neural basis of maternal aggression. PMID- 16676226 TI - Four closely related but forgotten species of Rhipidocotyle Diesing, 1858 (Digenea: Bucephalidae) in fishes from European seas. AB - The following species of Rhipidocotyle are described: R. minima (Wagener, 1852) from Chelidonichthys gurnardus, C. lastoviza and Aspitrigla cuculus at various localities off the British Isles; R. nicolli n. sp. from A. cuculus off Plymouth, SW England; R. triglae (van Beneden, 1870) from C. lucernus in the Gulf of Marseilles, western Mediterranean; and R. viperae (van Beneden, 1870) from Echiichthys vipera at various localities off the British Isles. The distinguishing features of the species are discussed in detail. A list of the bucephalid species reported from the Mediterranean Sea is appended. PMID- 16676227 TI - Saturnius minutus n. sp. and S. dimitrovi n. sp. (Digenea: Hemiuridae) from Mugil cephalus L. (Teleostei: Mugilidae), with a multivariate morphological analysis of the Mediterranean species of Saturnius Manter, 1969. AB - Three species of the bunocotyline genus Saturnius Manter, 1969 are described from the stomach lining of mugilid fishes of the Mediterranean and Black Seas. Two of the species are new: S. minutus n. sp. occurs in Mugil cephalus off the Mediterranean coast of Spain; and S. dimitrovi n. sp., a parasite of M. cephalus off the Bulgarian Black Sea coast and the Spanish Mediterranean coast, was originally described as S. papernai by Dimitrov et al. (1998). In addition, S. papernai Overstreet, 1977 is redescribed from M. cephalus off the Spanish Mediterranean coast and from Liza aurata and L. saliens off the Bulgarian Black Sea coast. The three species are distinguished morphometrically using univariate and multivariate analyses. These results were verified using Linear Discriminant Analysis which correctly allocated all specimens to their species designations based on morphology (i.e. 100% successful classification rate) and assigned almost all specimens to the correct population (locality). The following variables were selected for optimal separation between samples: the length of the forebody, ventral sucker and posterior testis, the length and width of the posteriormost pseudosegment, and the width of the muscular flange at ventral sucker level. PMID- 16676228 TI - Morphological and genetic differences among actinosporean stages of fish parasitic myxosporeans (Myxozoa): difficulties of species identification. AB - The occurrence and morphology of actinosporean stages of myxosporeans were studied at a fish farm and in the River Tisza in Hungary. The 43 samples sequenced belonged to 10 'genotypes', from which six were determined as new actinosporean types. Based on DNA sequence analysis, the assumed actinosporean developmental stages of four myxozoan species were identified. The raabeia type collected from the worm Branchiura sowerbyi was identified as Myxobolus cultus Yokoyama, Ogawa & Wakabyashi, 1995. This was the first occurrence of this parasite in Hungary. Aurantiactinomyxon type 'A' was assigned as a developmental stage of Thelohanellus hovorkai Achmerov, 1964, triactinomyxon type 'D' was identified as Myxobolus sp. from the fins of roach Rutilus rutilus, while the DNA sequence of the guyenotia type actinosporean was 99.9-100% identical with Sphaerospora sp. from the kidney tubules of goldfish Carassius auratus auratus. Partial 18S rDNA sequences of the myxosporeans Thelohanellus hovorkai, T. nikolskii Achmerov, 1955 and Myxobolus sp. from Rutilus rutilus were new additions to GenBank. The DNA sequence analysis revealed that, in the case of actinosporeans, different 'morphotypes' can belong to the same 'genotype'. This study confirmed that actinospore classification based solely on traditional morphological features may lead to false conclusions, thus sequence analysis of the 18S rDNA and/or other genes is recommended in species and type descriptions. PMID- 16676229 TI - Synopsis of the species of Ceratomyxa Thelohan, 1892 (Myxozoa: Myxosporea: Ceratomyxidae). AB - A synopsis of the species of Ceratomyxa Thelohan, 1892 (Myxozoa: Myxosporea: Ceratomyxidae), including a total of 147 species, is presented. For each species the relevant morphological and morphometric data are indicated, as well as the site of infection within the host and the original hosts and locality. A diagrammatic illustration of the spores is also provided. PMID- 16676230 TI - Nesippus orientalis Heller, 1868 (Pandaridae: Siphonostomatoida): descriptions of the adult, young and immature females, a first description of the male and aspects of their functional morphology. AB - Nesippus orientalis Heller, 1868, a cosmopolitan species found in the mouth and on the gill-arches of a number of shark hosts, is distinguished from other species by the presence of dorsal plates on the fourth thoracic segment. Specimens were collected from various sharks caught in the nets of the Natal Sharks Board, off the coast of KwaZulu-Natal, South Africa. Collected specimens were preserved in 70% ethanol and studied using the wooden slide technique and scanning electron microscopy. Careful examination of adult females revealed features previously not described in detail. Furthermore, some female specimens were still grasping the placoid scales of their hosts. These specimens showed how the maxillipeds are used to clasp the host. Immature, young females and males, some still attached to the young females, were also collected. The males use their maxillipeds, which have a slightly different structure to those of the female, to hold onto the females. PMID- 16676231 TI - A new canestriniid mite, Haitlingeria longilobata n. g., n. sp. (Acari: Astigmata: Canestriniidae), parasitising Serrognathus platymelus castauicolor Motschulsky (Coleoptera: Lucanidae) from South Korea. AB - Haitlingeria longilobata n. g., n. sp. (Acari: Astigmata: Canestriniidae) is described from Serrognathus platymelus castauicolor Motschulsky (Coleoptera: Lucanidae) from South Korea. Diagnostic features include: the presence of two long lobes on the male opisthosoma; the idiosoma length is similar to the idiosoma width; the hysterosoma is ornamented by distinct reticulate pattern; a suture is present separating the propodosoma from the hysterosoma; the adanal suckers are well developed on the male; there are four pairs of ultralong setae; and solenidion phi on tibia I is very long. PMID- 16676246 TI - [Prehospital life support in trauma patients: basic or advanced trauma life support]. AB - The controversy between Advanced Trauma Life Support (ATLS) and Basic Life Support (BLS) in the prehospital care of trauma patients has not been resolved yet. The purpose of this study was to examine the literature with respect to the type of prehospital care applied to the trauma patients. A total of 76 papers on ATLS and/or BLS for trauma were reviewed regarding the variables such as intravenous catheter application, prehospital fluid resuscitation, transport time, intubation and mortality. As a conclusion, the data in the literature do not support the routine use of on-field ATLS in trauma patients. Prospective randomized trials comparing ATLS and BLS in prehospital management of trauma patients are needed to clarify this issue. PMID- 16676247 TI - [The effects of rapid and slow infusion of fluid on coagulation factors in hemorrhagic shock: an experimental dogs model]. AB - BACKGROUND: The diagnosis of hemorrhagic shock in a patient with tachycardia and hypotension is generally straightforward. The difficulty lies in the selection of optimal approach for the patient. In this study on a canine model of deep hemorrhagic shock induced by bleeding, we used various amounts of fluid infusion to determine the most appropriate amount of fluid resuscitation. METHODS: The study included 30 mongrel dogs. Mongrel dogs were randomized to three equal groups of 10. The control group received no treatment. The rapid infusion group received 60 mL/kg of lactated Ringer's solution in half an hour whereas the third group received 30 mL/kg during the same time period. RESULTS: The lactate decreased in the two treatment groups. The platelet and fibrinogen levels did not vary in the slow infusion group but decreased markedly in the rapid infusion group. CONCLUSION: In conclusion, fluid resuscitation should not be unnecessarily aggressive. The coagulation parameters are diluted in proportion with the amount of fluid given. Blood lactate level can be used as a guiding marker in evaluation of treatment effectiveness of hemorrhagic shock patients. PMID- 16676248 TI - [Intestinal blood flow alterations in postoperative intraabdominal adhesion formation and the role of Endothelin-1 blockade]. AB - BACKGROUND: The current study was planned to investigate intestinal blood flow alterations and the role of ET-1 receptor blockade in the formation of postoperative intraperitoneal adhesion formation. METHODS: Twenty-eight adult Wistar Albino rats weighing between 250 g and 300 g were divided into four groups. Control group (group 1; n=7) did not undergo any operation. Sham group (group 2; n=7) had only laparotomy. In the adhesion group (group 3; n=7), peritoneal patch (1x1 cm) excision from the right abdominal wall and cecal abrasion were done as "adhesion model operation". One week following this, treatment group (group 4; n=7) received a non-selective ET-1 receptor blocking agent bosentan (30 mg/kg, IP) intra-abdominally, once a day for four days. Intestinal blood flow through the superior mesenteric artery was measured, on postoperative seventh day. Adhesion severity and extension as well as myeloperoxidase activity in the adhesion were calculated. RESULTS: Mean intestinal blood flow significantly increased in adhesion group (81.9+/-5.6 ml/100 g) when compared to group 1 (65.5+/-1.2 ml/100 g). Bosentan caused a significant decrease (44.3+/-6.9 ml/100 g) in intestinal blood flow when compared to group 1 and group 2. Sham group (62.2+/-1 ml/100 g) had similar blood flow level with the control group (65.5+/-1.2 ml/100 g). Adhesion scores were similar in adhesion and Bosentan groups. Sham group had almost no adhesions. Myeloperoxidase activity in adhesion tissue was significantly higher in bosentan group. CONCLUSION: Non-selective ET-1 receptor blockade has no effect on prevention of the formation of intra-abdominal adhesion, but causes a decrease in intestinal blood flow. Adhesion formation increases intestinal blood flow. Adhesion formation is accompanied by increased polymorphonuclear infiltration despite bosentan treatment. PMID- 16676249 TI - [Traumatic subarachnoid hemorrhage: a prospective study of 58 cases]. AB - BACKGROUND: We evaluated the prognostic factors in traumatic subarachnoid hemorrhage (tSAH). METHODS: This study was conducted with 58 patients (44 males, 14 females; mean age 39.2; range 17 to 79 years) with tSAH, between 2001 and 2003. The patients who were admitted to the hospital within in the first 6 hours of head injury were included, whereas patients with gun shot wounds, multiple injured patients and postoperative patients were excluded. Fifty-eight patients with tSAH were prospectively followed. The neurological status of the patients and the outcomes were evaluated using Glasgow Coma Scale (GCS) and Glasgow Outcome Scale (GOS), respectively and computed tomography (CT) examinations were done according to the grading criteria by Hijdra and Fisher. RESULTS: The etiology of tSAH included traffic accidents (73%), falls (20%) and others (7%). The GCS scores of patients at admission were mild (9%), moderate (39%) and severe (52%). In the CT scans, the amount and distribution of bleeding was grade 1 (small SAH) in 21 patients, grade 2 (moderate SAH) in 17 patients, and grade 3 (extensive SAH) in 20 patients according to Hijdra grading system and according to Fisher's criteria. The thickness of blood layer was grade 1 (no blood) in 6 patients, grade 2 (bleeding layer less than 1 mm) in 21 patients, grade 3 (bleeding layer more than 1 mm) in 15 patients and grade 4 (ventricular bleeding) in 16 patients. Neurological outcomes of patients were favorable (good recovery or moderate disability) in 59%, and unfavorable (severe disability, persistent vegetative state or death) in 41% according to GOS. CONCLUSION: We have found in our series that the prognosis was poor in patients with poor admission scores of GCS, cysternal or fissural hemorrhage, tSAH with cerebral contusion or acute subdural hematoma, higher than 13 points according to Hidjra's classification and patients of grade 3 or 4 in Fisher's criteria. PMID- 16676250 TI - Procalcitonin is a predictive marker for severe acute pancreatitis. AB - BACKGROUND: Early identification of patients who develop severe acute pancreatitis and those who can benefit from intensive care is important. We studied whether procalcitonin, a marker of systemic inflammation, is important in the differential diagnosis of patients with mild and severe acute pancreatitis. METHODS: Patients were divided into two groups (mild and severe form) prospectively. Procalcitonin levels and the Ranson's and Acute Physiology and Chronic Health Evaluation II scores were determined both at admission and during the follow-up. RESULTS: Of the 65 patients with acute pancreatitis, 46 had mild and 19 had severe pancreatitis. Sensitivity and specificity values for patients calculated using procalcitonin level at 0.5 ng/ml, Ranson's score at 3 and APACHE II score at 8 cut-off levels, were 100%, 84% and 89%; and 84%, 63% and 89% respectively. CONCLUSION: Procalcitonin is a practical, simple parameter that can be used in order to diagnose severe acute pancreatitis earlier and to monitor the clinical prognosis of the disease. PMID- 16676251 TI - Somatotrophic reorganization in the brain after extremity replantation, revascularization and amputations: investigated by SPECT analysis. AB - BACKGROUND: We wanted to investigate the somatotropic reorganization occurring in the motor and somatosensory cortex by using 99mTc-HMPAO SPECT analyses, after the extremity revascularization, replantation or amputation. METHODS: Twelve patients (11 men, 1 female; mean age 38.9+/-14.7 years) and controls (5 men, mean age 32.2+/-7.9 years) were enrolled in this study. After reconstruction, lower, middle and upper orbitomeatal slices with precentral and postcentral slices were obtained. All images were visually and semi-quantitatively evaluated. Mann Whitney U-test was used for statistical analysis. RESULTS: In the revascularization and replantation patients, postcentral and precentral hypoperfusions were seen at dominant hemisphere. In the amputated patients, postcentral (in 3 of 4 cases) and precentral hypoperfusions were seen at non dominant hemisphere and postcentral hypoperfusion (in 1 of 4 cases) was seen at dominant hemisphere. In our patients, most significant difference in regional cerebral blood flow was found in posterior parietal cortex (somatic associated area). CONCLUSION: Changes that take place in precentral and postcentral cortical areas subsequent to the extremity replantation-revascularization of the organ is a good indicator of somatotrophic reorganization. PMID- 16676252 TI - [Pulmonary and systemic fat embolism as a cause of death in forensic autopsy practice]. AB - BACKGROUND: The aim of this study was to evaluate the possible correlations between the grade of fat embolism and age, gender, severity of trauma and post traumatic survival time. METHODS: Thirty-one cases with pulmonary and/or systemic fat embolism, auotopsied at the Morgue Department of Council of Forensic Medicine were evaluated retrospectively. RESULTS: Twenty-eight cases (90%) died due to trauma and its complications. Nineteen cases (61%) were injured in motor vehicle related accidents. Post-traumatic survival time varied between 0-384 hours (61.6+/-86.2 hours). Abbreviated injury scale (AIS) was 8.1+/-3.9 and injury severity score (ISS) was 26.5+/-19.7 SD. Twenty-four (77%) cases were determined as isolated pulmonary fat embolism and 7 (23%) cases as systemic fat embolism on histopathological examination. Possible correlations between the grade of fat embolism and age, gender, severity of trauma and post-traumatic survival time were evaluated statistically with using Chi-square and Spearman's correlation tests. CONCLUSION: There were no correlations between the grade of fat embolism and age, gender, the severity of trauma. Post-traumatic survival time and the severity of trauma had a very weak negative correlation without any statistical significance. PMID- 16676253 TI - [Epidemiological characteristics of electrical injuries of patients applied to the emergency department]. AB - BACKGROUND: In this study, we planned to determine the factors affecting the mortality, and morbidity of electrical injuries. METHODS: Medical records of 165 patients (126 males; 39 females; mean age 21.1 years; range 2.5 to 62 years), who were admitted to the Emergency Department of Dicle University School of Medicine for electrical injuries, between January 2003 and April 2004, were retrospectively reviewed. RESULTS: Among these patients, 60 (36.4%) were children, under 12 years old; 95 (57.6%) were adolescents and adults, and 10 (6%) were aged. Of the victims of electrical accidents, 29 (17.6%) were illiterate and 36 (21.8 %) were educated. Ninety-seven (58.8 %) patients were either graduates or still students of elementary, secondary or high school. The cause of exposure to electricity was accident in 99 (60%), and carelessness and parents' negligence in 66 (40%). Sixty-nine (41.8%) patients were exposed to high voltage, and 96 (58.2 %) to low voltage. Because of electrical injury 16 patients had first degree, 96 patients had second degree, and 86 patients had third degree burns. The most frequent complications were contractures of extremities (10.9%) and compartment syndrome (3.6%). Mortality rate was 9.1% (n=15). Eighty percent of the deaths were due to exposure to high voltage. A positive correlation was demonstrated between mortality and complications (p<0.001). Escarotomy was performed in 10 patients, fasciotomy in 16, and amputation in 9. Two of 5 patients who had intraabdominal hemorrhage underwent surgery. CONCLUSION: A serious education of the society with respect to conscious use of electricity is the most efficient method to decrease electrical accidents. PMID- 16676254 TI - [Surgical coverage technics of pressure sores and their outcomes]. AB - BACKGROUND: We reviewed the outcome of 66 patients with 100 pressure sores between 1984 and 2002. METHODS: In the current study, 100 pressure sores in 66 patients (45 male, 21 female; mean age 39.4; range 13 to 80 years) who underwent surgical repair of pressure sores reconstructed using myocutaneous or fasciocutaneous flaps, skin grafts, excision and closure. RESULTS: The risk factors for pressure sores included acute trauma-induced spinal cord injury in 40 (61%) patients with paraplegia and in 5 (7%) patients with quadriplegia; congenital spina bifida and multiple sclerosis in 7 (11%) patients and prolonged immobilization in 14 (21 patients) patients. We achieved an overall pressure sore recurrence rate of 22% and overall patient recurrence of 24% in a-3-year follow up. The recurrence rates according to anatomic sites; 23% (10 of 43) for the ischial pressure sore, sacral 21% (8 of 37), and trochanteric 20% (4 of 20). Fasciocutaneous and myocutaneous flap reconstructions were the most durable, as they were associated with 17% (6 of 34) and 12% recurrence rates (5 of 39). CONCLUSION: To reduce the recurrence rates the authors advocate the use of myocutaneous and fasciocutaneous flaps instead of skin grafts or direct closure for the coverage of pressure sores. PMID- 16676256 TI - Emergency helical CT scan in acute abdomen: a case of intestinal intussusception. AB - Surgeons are familiar to the nosological entity "partial intestinal obstruction". Intussusception constitutes a rare etiologic factor for this entity but usually remains undiagnosed preoperatively. Several imaging techniques have been proposed as useful in the diagnosis of intussusception but none of these has a remarkable sensitivity and specificity. In the following case of partial intestinal obstruction, we performed an helical CT scan of the abdomen. The method revealed with an excellent accuracy the nature (intussusception) and the location (ascending colon) of the partial intestinal obstruction. Having the extra advantage of the shorter examination time than the classical CT scan, we propose the helical CT as an alternative diagnostic modality for preoperative evaluation of patients with partial intestinal obstruction. PMID- 16676255 TI - Carcinoid tumors of appendix: treatment and outcome. AB - BACKGROUND: The aim of this study is to evaluate the clinical and histopathological features and the treatment of carcinoid tumors of the appendix. METHODS: A retrospective review of medical records and pathology specimens of patients with carcinoid tumor of the appendix has been done. The data derived from this study has been evaluated by descriptive statistical methods (mean, SD, frequency). RESULTS: The histopathological examination of the appendices revealed carcinoid tumor in 11 out of 6777 (0.16%) patients operated for acute appendicitis. Six (54.54%) patients were male, 5 (45.45%) were female and the mean age was 20.2+/-6.7 years (13-35). The tumor was localized in the distal 1/3 region in 10 patients and in the proximal 1/3 region in 1 patient. The mean tumor diameter was 0.73+/-0.36 cm (0.3-1.5). Ten patients had classical type carcinoid tumor whereas goblet cell carcinoid tumor was only seen in one patient. None of the patients underwent an expanded resection. The mean follow up of the patients is 28.5+/-15.2 months (6-48), and no recurrences were seen. CONCLUSION: In tumors with a diameter of 1 to 2 cm, appendectomy is the treatment of choice. No recurrence was detected with tumors smaller than 2 cm with simple appendectomy. PMID- 16676257 TI - [Post-traumatic bronchial stenosis: a case report]. AB - Bronchial rupture due to tracheobronchial trauma is a very rare condition. Early evaluation is necessary in order to avoid irreversible changes such as fibrosis and bronchial stenosis. Cases with post-traumatic hemo-pneumothorax, pneumomediastinum, subcutaneous emphysema and atelectasis require consideration of bronchial laceration with urgent bronchoscopy and early surgery, if needed. A 15 year-old patient with a history of blunt chest trauma two years ago presented with symptoms of pulmonary infection. Radiological and bronchoscopic evaluation revealed right upper lobe atelectasis secondary to fibrosis. Patient underwent right upper lobectomy. PMID- 16676258 TI - Asymptomatic chronic ossified epidural hematoma in a child: a rare entity. AB - A 6-year-old boy presented with an asymptomatic ossified chronic epidural hematoma. He was neurologically intact and had no complaints. This is the first report with a computed tomography image of cerebral compression due to an asymptomatic ossified epidural hematoma. Computed tomography indicated an ossified epidural hematoma in the left frontal region. In children, surgery for asymptomatic ossified chronic epidural hematoma with significant cerebral compression should be considered to relieve cerebral compression and prevent possible future brain damage. PMID- 16676259 TI - [Hepatic portal venous gas: a case report]. AB - Gas in hepatic portal vein is a rare entity. This may be apparent after mesenteric ischemia, blunt abdominal trauma, intestinal obstruction, and intra abdominal infection. Intrahepatic gas was detected by direct abdominal graphy in a 58 year-old man who was admitted to our emergency service with acute abdomen. On computed tomography; portal vein gas, pneumatosis intestinalis, and occlusion of superior mesenteric vein and artery were detected. The patient who had had significant concomittant operative risks, died prior to surgery. Gas in portal vein is a good predictive factor for diagnosis, management, and prognosis. This sign may avoid unnecessary surgery and also it may help to make an early decision for surgery. PMID- 16676260 TI - American Chemical Society--231st National Meeting. PMID- 16676261 TI - American Chemical Society--231st National Meeting. Highlights of 11beta-HSD1, lipid metabolism and immunosuppressant research. PMID- 16676262 TI - American Chemical Society--231st National Meeting. Advances in cancer therapeutics. PMID- 16676263 TI - American Chemical Society--231st National Meeting. Developments in inflammation, siRNA and thrombosis research. PMID- 16676264 TI - American Chemical Society--231st National Meeting. Medicinal Chemistry Awards symposium. PMID- 16676265 TI - American Chemical Society--231st National Meeting. G-protein-coupled receptor structure, modeling and ligand design. PMID- 16676266 TI - American Chemical Society--231st National Meeting. Virtual screening using structure-based and ligand-based methods for lead identification. PMID- 16676267 TI - American Association for Cancer Research--97th Annual Meeting. PMID- 16676268 TI - American Association for Cancer Research--97th Annual Meeting. Drug research developments--part 1. PMID- 16676269 TI - American Association for Cancer Research--97th Annual Meeting. Drug research developments--part 2. PMID- 16676270 TI - Cryptic protein fragments as an emerging source of peptide drugs. AB - This commentary evaluates the newly proposed concept of crypteins - peptides with embedded cryptic activity - as a source of peptide drugs. Although several crypteins are undergoing advanced clinical trials, and one cryptein, adrenocorticotropic hormone (ACTH), is already widely used as a therapeutic, crypteins remain an underappreciated class of biopharmaceuticals. The systematic exploitation of crypteins provides a novel source of peptide drugs, and may also provide templates for developing peptidomimetic compounds. In addition, crypteins appear to offer several advantages over synthetic protein libraries and other protein scaffolds. PMID- 16676271 TI - The impact of parallel chemistry in drug discovery. AB - With the application of parallel synthesis of single compounds to drug-discovery efforts, improvements in the efficiency of synthesis are possible. However, for improvements to occur in effective drug design - a critical requirement to increase productivity in the modern pharmaceutical industry - the implementation of in silico design hypotheses that incorporate comprehensive information on a target, including considerations of absorption, distribution, metabolism and excretion, is also necessary. Concomitantly, the use of automated methods of synthesis and purification is also required to improve drug design. Combining all of these elements allows the possibility to uncover unique insights into a biological target quickly and to therefore accelerate the rate of drug discovery. PMID- 16676272 TI - Drug evaluation: ProCord - a potential cell-based therapy for spinal cord injury. AB - Proneuron Biotechnologies Inc is developing ProCord, an activated macrophage cell therapy, for the potential treatment of neurological conditions. In September 2004, ProCord was granted Orphan Drug status by the US Food and Drug Administration for the treatment of acute spinal cord injury. PMID- 16676273 TI - [Clinical and subclinical neuropathy in patients with human immunodeficiency virus receiving antiretroviral therapy]. AB - INTRODUCTION: There is a clear association between human immunodeficiency virus (HIV) and peripheral neuropathy. Peripheral polyneuropathy (PPN) is the most frequent neurological complication due to both the infection itself and the neurotoxicity deriving from highly active antiretroviral therapies (HAART). AIMS: The aim of this study was to determine the incidence of symptomatic PPN associated to HAART and to find out the true prevalence rate of subclinical neuropathy following over several years' treatment. PATIENTS AND METHODS: In order to evaluate the incidence of symptomatic PPN we conducted a study of patients undergoing treatment with HAART with a combination of didanosine (ddI), lamivudine (3TC) and efavirenz, and its presence was confirmed both clinically and electromyographically. Moreover, to study the prevalence rate of asymptomatic or subclinical PPN we chose patients without PPN who had been receiving this treatment for more than two years, with a viral load that had remained undetectable for over a year and with no further risk factors for PPN, and submitted them to a voluntary electromyographic study for PPN. RESULTS: Of the 108 patients studied, only two cases of symptomatic PPN were found. CONCLUSIONS: The incidence rate of clinical neuropathy following the administration of HAART is low (1.85%); PPN is a rare cause of withdrawal. Nevertheless, the prevalence rate found for subclinical PPN in patients undergoing prolonged therapy is high (66%). We therefore find ourselves with a problem that is little known, rarely suspected and more common than is believed. PMID- 16676274 TI - [Risk factors associated to the presentation of episodes of major depression in a population of outpatients with Parkinson's disease]. AB - INTRODUCTION: Depression is a frequently occurring problem in Parkinson's disease (PD). The purpose of this study is to describe the characteristics of episodes of major depression (EMD) in a population of outpatients with PD and the risk factors linked to their appearance. PATIENTS AND METHODS: We conducted an open study involving 85 patients with PD; their mean age was 65.8 +/- 12.3 years and the mean length of time since disease onset was 7.2 +/- 5 years. Data that was collected included information from the patient record, evaluation with the Motor Examination section of the Unified PD Rating Scale (UPDRS-III), the Parkinson Mini-Mental test, the Neuropsychiatric Inventory (NPI) and the short version of Beck's Depression Inventory (BDI). EMD were diagnosed clinically by means of a semi-structured interview that allowed us to examine the symptoms that are included in the DSM-IV criteria for diagnosis of EMD. RESULTS: In 21 cases (24.7%) DSM-IV criteria for EMD were satisfied, with a BDI score (mean +/- standard deviation) of 17.5 +/- 6.2. In 64 cases (75.3%) who did not fulfil the criteria for EMD, the BDI score was 6.4 +/- 4.7 (p < 0.001). The items on the UPDRS-III related to the dopaminergic response in patients with EMD were 24.9 +/- 11.9 and in patients without EMD the values were 21.2 +/- 10.9 (NS). Those not related to the dopaminergic response in patients with EMD were 9.9 +/- 5.3 and in those without EMD the values were 6.2 +/- 4.6 (p < 0.001). CONCLUSION: In our sample, the presence of motor symptoms that do not respond to levodopa in patients with PD is a risk factor for the appearance of EMD. PMID- 16676275 TI - [Central neuropathic pain and its relation to the quality of life of a person with a traumatic spinal cord injury]. AB - INTRODUCTION AND AIMS: To conduct an exploratory cross-sectional study carried out in the city of Fortaleza (Ceara, Brazil) with the aim of evaluating the relation between central neuropathic pain and the quality of life in individuals with central neuropathic pain due to traumatic injury. PATIENTS AND METHODS: The study examined the cases of 17 adult paraplegic patients with complete traumatic injuries, mainly due to perforations caused by gunshot wounds. The sample was divided into two groups: 1) those with pain, and 2) those with intense pain (more than 20 points according to McGill and eight on the numerical visual scale). The instruments used were the following: the MOS 36-item Short-Form Health Survey (SF 36) and the McGill pain questionnaire, oriented towards the descriptors and the numerical visual scale. RESULTS: The quality of life of patients with central neuropathic pain due to a spinal cord injury is greatly compromised and when the pain is intense the quality of life is even more effected, especially in the following areas: "functional capacity" (p = 0.005), "general state of health" (p = 0.003), "mental health" (p = 0.035), "social aspects" (p = 0.006) and "pain" (p = 0.003). CONCLUSIONS: The quality of life in patients with neuropathic pain due to a traumatic spinal cord injury is severely compromised and the more intense this pain is, the greater its effect on the quality of life will be. It can also be stated that the instruments used were valid for this type of patients (in spite of certain shortcomings) given the complexity and subjectivity of the matter. PMID- 16676276 TI - [Tolosa-Hunt syndrome and orbital pseudotumour. Overlapping conditions in a case with an unusual clinical profile]. AB - INTRODUCTION: The origin of Tolosa-Hunt syndrome (THS) and orbital pseudotumour (OP) is not fully understood. It is acknowledged as having an unspecific granulomatous inflammatory nature in different locations. Although there are differences between the clinical features of the two conditions, they also share a number of physiopathogenetic, therapeutic and, in some cases, iconographic similarities. Possible clinical recurrences are common in the two conditions and a broad differential diagnosis is required in all cases. Yet, the association of both processes in the same patient, with radiological proof of the migration of the inflammatory injury, is not frequent. CASE REPORT: We report the case of a male patient with a long history of recurring unilateral painful ophthalmoplegia that was sensitive to steroids; criteria for THS were fulfilled and there was later development of homolateral OP, six years after the onset of his symptoms. The findings in serial studies conducted with magnetic resonance imaging must be highlighted. The patient was submitted to a surgical intervention to treat the orbital injury and a chronic inflammatory process was observed in the fibrotic phase. CONCLUSIONS: Inflammatory pseudotumour and THS perhaps have more points in common than has traditionally been accepted. To our knowledge few cases of the above-mentioned association have been reported in the same patient. When confronted by cases of painful ophthalmoplegia with excessive recurrences the physician must consider the possibility of other alternative diagnoses. PMID- 16676277 TI - [How does the brain wake up? The nitric oxide blow]. AB - INTRODUCTION: A synthesis of the role of the neuromodulator nitric oxide (NO) on the sleep-wake cycle control is made, emphasizing the function of the activating ascending pathways implicated in arousal. DEVELOPMENT: There are some hypotheses regarding the role of sleep: memory consolidation, ecological factors, cellular repair and nervous system development. The sleep-wake cycle is an active process, modulated by subcortical regions (mesopontine nuclei, diencephalon and basal forebrain) with connections and reciprocal interactions among them. NO is released by neurons and terminals of the sleep-wake cycle modulatory nuclei. The role of NO in this cycle is mainly linked to activation processes: transition to and maintenance of waking and rapid eye movement (REM) sleep. At thalamic level NO is released by cholinergic fibers of the mesopontine nuclei and induces a facilitation of neural responses. In the basal forebrain there exists NO in the cholinergic cells contacting the cortex, suggesting that this ascending pathway can also collaborate in cortical activation through the release of NO. CONCLUSIONS: NO has been identified in neurons of the brain areas controlling the modulation of arousal; hence, this gaseous neuromodulator can have an essential function promoting a quick and global activation of cortical neurons. PMID- 16676278 TI - [Ropinirole in the treatment of Parkinson's disease: an update]. AB - INTRODUCTION: Ropinirole is a non-ergot dopaminergic agonist with a high affinity for D2 dopaminergic receptors which improves the symptoms of Parkinson's disease (PD) and delays the appearance of motor complications. It is different to the first generation of dopaminergic agonists in that, because it lacks an ergolinic structure, it does not have the side effects that usually appear with the use of this pharmacological group. DEVELOPMENT: Recent functional neuroimaging studies suggest a possible neuroprotector effect of the drug, although this aspect is still under discussion. The question as to when and how early treatment of PD must be started has been a controversial issue for many years now. Dopaminergic agonists have been used in monotherapy in patients with de novo disease with the intention of deferring treatment with levodopa and, in consequence, postponing the onset of the complications stemming from its use. Ropinirole has been evaluated in different studies both in monotherapy and as adjunctive therapy with levodopa. CONCLUSIONS: In the numerous clinical trials that were carried out, it would seem clear that ropinirole can be administered for years as sole early treatment for PD and that it offers a notable reduction in the appearance of dyskinesias. Given the linear dose-response relation it presents, the drug has a wide "therapeutic window" that allows the dosage to be increased as the disease progresses. PMID- 16676279 TI - [The neurochemistry and neuropharmacology of diffuse Lewy body disease]. AB - INTRODUCTION AND DEVELOPMENT: The main neurochemical alteration in diffuse Lewy body disease (DLBD) is the cholinergic deficit in the cerebral cortex, which involves mainly cholin-acetyl-transferase. There have been also described dopamine deficiency and alterations affecting other neurotransmitters and neuromodulators, such as serotonin, noradrenaline, neuropeptides, etc. Cerebral perfusion and glucose metabolism studies usually show diffuse hypoperfusion or hypometabolism, with higher alteration of associative cortex, including occipital involvement. Several studies have shown increased markers of oxidative stress in brain and other tissues, suggesting its possible role in the pathogenesis of DLBD. CONCLUSIONS: Acetylcholinesterase inhibitors seem to improve cognitive and conductual symptoms, although their usefulness according evidence-based medicine criteria is weak. Some patients need atypical neuroleptics at low doses to get the symptomatic control of conductual alterations. PMID- 16676280 TI - [The neurochemistry and neuropharmacology of frontotemporal dementia]. AB - AIM: To review the neurochemical features and therapeutic options for frontotemporal dementia (FTD). DEVELOPMENT: The main neurochemical alterations in FTD are the serotoninergic and dopamine depletion. In contrast with Alzheimer's and diffuse Lewy bodies disease, there are not significant alterations of the cholinergic system. Cerebral perfusion and glucose metabolism studies usually show hypoperfusion or hypometabolism, with predominant involvement of temporal and frontal cortices. There have been described some alterations related with oxidative stress and apoptosis, although its pathogenetic role in FTD is not well known. Treatment of FTD is not well established, because there are only a few studies with some drugs. The most studied drugs are serotonin reuptake inhibitors, however, despite the well-known serotoninergic deficiency described in FTD, the results are not conclusive. CONCLUSIONS: The main neurochemical alterations of FTD are serotoninergic and dopaminergic deficiencies. The treatment is not well established, although it should be theoretically ideal to use drugs which modulate these neurotransmitter systems. PMID- 16676281 TI - [Electromyography in distal atrophy of the upper limbs with segmental spinal muscular atrophy: Hirayama's disease]. PMID- 16676282 TI - [Migrainous infarction and clomiphene citrate]. PMID- 16676283 TI - [Isolated vasculitis of the central nervous system resembling Rasmussen's encephalitis]. PMID- 16676284 TI - [Bihemispheric acute stroke associated with large-artery atherosclerosis]. PMID- 16676285 TI - The effects of physical exercise on depressive symptoms among the aged: a systematic review. AB - OBJECTIVE: To determine the effects of physical exercise on depression or depressive symptoms among the aged. METHOD: A literature search covering various medical databases was conducted to identify randomised controlled trials (RCT's) about the effects of exercise treatments on depression or depressive symptoms among the aged. The studies were classified according to the baseline depression status of participants and assessed in relation to allocation concealment, blinding at outcome assessment, follow-up and whether intention to treat analysis was used. Studies meeting the inclusion criteria were accepted. RESULTS: Exercise was effective in treating depression among those suffering from minor or major depression and in reducing depressive symptoms among those with a high amount of depressive symptoms at baseline. However, both the allocation concealment and the blinding method were adequately described in only four studies. Furthermore, intention-to-treat analysis was conducted in half of the studies and some follow up information after the intervention has been published for five studies. CONCLUSIONS: Physical exercise may be efficient in reducing clinical depression and depressive symptoms in the short-term among the aged suffering from depression or a high amount of depressive symptoms. More well controlled studies are needed. PMID- 16676286 TI - Do older adults presenting with memory complaints wish to be told if later diagnosed with Alzheimer's disease? AB - BACKGROUND: Many patients with Alzheimer's disease are not told their diagnosis. Studies have shown that relatives possess mixed views regarding whether or not patients should be told while elderly peers favour disclosure. Recent studies have shown that patients with diagnosed dementia also favour being told. The present study sought the views of patients prior to diagnosis. METHOD: Participants were a consecutive sample of patients aged 65 and over suffering from memory complaints. They were asked what they considered to be causing their memory problems and whether or not they would want to know the cause. They were then specifically asked if they would want to know if diagnosed with Alzheimer's disease and what were their reasons for this. RESULTS: Two-thirds of patients were uncertain regarding the cause of their memory difficulties although the remainder did offer some valid explanations. Eighty-six per cent wanted to know the cause while 69% wanted to know if diagnosed with Alzheimer's disease and a variety of reasons were offered to support their preference. CONCLUSION: The majority of older adults presenting to services with memory complaints had little understanding of the potential cause of their problems. Most were nevertheless keen to know the cause, even if this transpired to be Alzheimer's disease. The clinical implications of the findings are discussed and suggestions made for future research. PMID- 16676287 TI - Do subjective memory complaints predict cognitive dysfunction over time? A six year follow-up of the Maastricht Aging Study. AB - BACKGROUND: Middle-aged and older people often worry that their perceived diminishing memory function may indicate incipient dementia. OBJECTIVES: The present study addresses questions regarding subjective memory complaints as a predictor of lower performance on cognitive tasks. Also, in participants with subjective memory complaints it was investigated, whether trying to keep mentally active improved memory function. Characteristics of the participants who were and were not interested in an intervention to decrease worries and to improve memory in daily life were determined. METHODS: Data were obtained from a large longitudinal study: the Maastricht Aging Study, involving 557 participants aged 55 to 85 years. Follow-up measurement was performed after 6 years. Outcome variables were simple, complex and general information processing speed and immediate and delayed recall. RESULTS: At baseline, forgetfulness was associated with a slower general information processing and delayed recall. At the six-year follow-up, being forgetful was not associated with a significant change in cognitive performance. Taking steps to remain cognitively active was not a predictor of better performance on cognitive tasks at baseline or at the six-year follow-up. CONCLUSION: Being forgetful might be an indicator of slower general information processing speed and delayed recall at baseline but does not predict cognitive change over 6 years in older adults. However, the effects are rather small and cannot directly be generalized to applications in clinical settings. Other factors, such as depression and anxiety might also underlie the cause of the forgetfulness. PMID- 16676288 TI - Determinants of costs of care for patients with Alzheimer's disease. AB - BACKGROUND: Alzheimer's disease (AD), the most common cause of dementia, is a major cause of disability and care burden in the elderly. This study aims to estimate the costs of formal and informal care and identity determinants of care costs. MATERIALS AND METHODS: Two hundred and seventy-two (AD) patients and their caregivers were recruited among patients attending regular visits at six memory clinic in Sweden, Denmark, Norway and Finland. Patients with a diagnosis of AD and with an identifiable primary caregiver were eligible for inclusion. Data was collected by questionnaires at baseline, and at scheduled follow-up visits after 6 months and again after 12 months. Cognitive function was assessed with the Mini Mental State Examination (MMSE) and behavioural disturbances were measured using a brief version of the neuropsychiatric inventory (NPI). RESULTS: Total annual costs were on average 172,000 SEK, ranging from 60,700 SEK in mild dementia to 375,000 SEK in severe dementia. Costs for community care (special accommodation, home help, etc.) constituted about half of total costs of care and increase sharply with increasing cognitive impairment. Informal care costs, valued at the opportunity cost of the caregiver's time, make up about a third of total costs and also increased significantly with disease severity. Medical care costs (inpatient care, outpatient care, pharmaceuticals), on the other hand, were not significantly related to disease severity. Regression analysis confirmed a strong association between costs and cognitive function, between patients as well as within patients over time. There was also a significant influence on costs from behavioural disturbances. Sensitivity analysis showed that the method chosen to value informal care can have considerable impact on results. CONCLUSIONS: Costs of care in patient with AD are high and related to dementia severity as well as presence of behavioural disturbances. The cost estimates presented have implications for future economic evaluation of treatments for Alzheimer's disease. PMID- 16676290 TI - Verbal repetition in patients with Alzheimer's disease who receive donepezil. AB - BACKGROUND: Current outcome measures for Alzheimer's disease (AD) drugs have been criticized as insufficiently patient-centred. One commonly unmeasured goal of patients and caregivers is verbal repetition. OBJECTIVES: We examined how often reducing repetition (of questions, statements or stories) was set as treatment goal, whether and when it responded, and how change in repetition correlated with change in other domains. METHODS: This is a secondary analysis of the open-label Atlantic Canada Alzheimer's Disease Investigation of Expectations study of donepezil for mild-moderate AD in 100 community-dwelling people. Goal Attainment Scaling, an individualized account of the goals of treatment, was the primary outcome measure. RESULTS: Reducing repetition was a treatment goal in 46%, who were not systematically different from others. Of 18 patients in whom repetition improved for 9 months, 83% (15) showed a response at 3 months. Early (3-month) response correlated best with the overall level of goal attainment (r = 0.74) and changes in leisure activities (r = 0.69) and social interactions (r = 0.68) compared with changes in cognition (r = 0.44) or behaviour (r = 0.11). Correlations with the ADAS-Cog and MMSE change scores remained only modest (at 12 months = -0.25 and 0.19, respectively). Correlations with the CIBIC-Plus were higher (-0.47 at 3 months and -0.43 at 12 months). CONCLUSION: Diminution of repetition is common, and appears to mark response to cholinesterase inhibition in some patients. Responders generally also show improved cognition and function, perhaps as an aspect of improved executive function. PMID- 16676291 TI - Standards of care in day hospitals and day centres: a comparison of services for older people with dementia. AB - BACKGROUND: Current policy in England emphasises the importance of caring for highly dependent older people for as long as possible at home. It is therefore crucial that day care services are effective and widely available. AIM: To compare the type and standard of care provided for older people with dementia in day centre and day hospital settings. METHODS: A cross-sectional postal survey design was employed. Representatives from three-quarters of identified specialist day care services for older people with dementia in the North West of England provided information on a range of indicators including: basic structural features; delivery of care; service content; and quality measures. RESULTS: Day hospitals tended to have more day care places and a greater number of attendees, but lower occupancy rates than day centres. Day hospitals reported higher standards of care in relation to systematic assessment and care planning, promotion of rehabilitation, carer involvement and individualised provision of care. They were also more likely to employ building design features to encourage independence and choice for people with dementia. A higher proportion of day centres provided services exclusively to older people with dementia and a greater proportion of staff in day centres had undergone specific training in caring for people with dementia. Day centres were also more likely to have effective transport arrangements in place. CONCLUSION: The standards developed for the study were sufficiently reliable to allow for an acceptable estimate of quality. Day centres and day hospitals appeared to perform two distinct, but complementary functions. These results provide key material for shaping the provision of day care for older people with dementia, especially given the absence of national standards in this area. PMID- 16676292 TI - Screening for dementia in the older Chinese with a single question test on progressive forgetfulness. AB - OBJECTIVES: To assess the utility of asking for presence of progressive forgetfulness (PF) prior to administering the Abbreviated Mental Test (AMT) when screening for dementia in the community-dwelling elderly Chinese. METHODS: This was a two-phased community-based survey of elderly subjects (>or=50 yrs). In phase one, subjects were asked for PF and administered the AMT. Those having PF or an impaired AMT performance were evaluated clinically for dementia in phase two, which also included a randomly selected sample of 35 subjects with no PF and who passed the AMT. RESULTS: 2,566 subjects completed phase one interview, of which 128 subjects completed phase two. Overall prevalence of PF, failed AMT and dementia were 2.4%, 2.2%, 0.9% respectively. The sensitivity of PF for dementia was 95.7% with specificity of 45.1%. PF was significantly associated with depression in the young-old (50-74 yrs) but not in the old-old (>or=75 yrs) age group, after adjusting for dementia. The probability of subjects (%) in the four possible diagnostic combinations of PF and AMT in the young-old and (old-old) age groups were 0 (0.06) in the no PF/ passed AMT, 0 (0.44) in the no PF/failed AMT, 0.23 (9.2) in the PF/passed AMT and 3.6 (43) in the PF/failed AMT groups. CONCLUSION: In screening for the most common dementias, AMT administration is not required if PF is absent. AMT is also of no added utility for diagnosing dementia in older subjects with PF. Younger subjects with PF should be closely evaluated for depression especially if they passed the AMT, and dementia, if they failed the AMT. PMID- 16676293 TI - Screening for depression in older medical inpatients. AB - BACKGROUND: Older people with depression make greater use of healthcare services, but the detection of the disorder is poor. The National Service Framework for Older People recommended screening for depression in acute healthcare settings to improve health outcomes of older people. Previous studies, mainly outside the UK, report widely differing rates for depression that do not usefully inform UK practice. Thus the aim of this study is to estimate, in a large representative sample of older medical inpatients in a UK hospital setting, the prevalence of depressive symptoms and ICD-10 depressive disorder and to examine the sensitivity and specificity of the 15-item Geriatric Depression Scale (GDS-15) as a screening instrument. METHODS: A two-phase prevalence study of depressive disorder was carried out in acute wards of a district general hospital. Six hundred and eighteen (61%) of 1,009 eligible older medical inpatients were screened using the GDS-15. A stratified sample (n = 223) was further assessed using the Geriatric Mental State, from which ICD-10 diagnoses were determined. RESULTS: The weighted prevalence estimate of ICD-10 depressive disorder was 17.7% (95%CI: 12.9-22.5). Forty-four percent of participants scored above the normally recommended cut point of >or=5 on the GDS-15. However, on the basis of ROC, the optimal cut-point of the GDS-15 for screening for depressive disorder in this hospitalised population is two points higher at >or=7 (sensitivity 0.74, specificity 0.81). CONCLUSIONS: This study confirms that depression is common amongst older UK medical inpatients with 1 in 6 suffering from clinical depression. The cut-point for GDS-15 for this population is >or=7. PMID- 16676294 TI - The NEO-FFI is a reliable measure of premorbid personality in patients with probable Alzheimer's disease. AB - AIM: To assess the inter-informant reliability, intra-informant reliability and internal consistency of the NEO-FFI as a measure of premorbid personality in patients with Alzheimer's Disease (AD). SUBJECTS: One hundred and five persons with NINCDS-ADRDA probable AD for the assessment of inter-informant reliability and internal consistency, and 30 for the assessment of intra-informant reliability. METHODS: Premorbid personality was rated retrospectively by close relatives remembering the patient as he/she had been when aged in his/her forties. One hundred and five AD patients were rated by two separate informants. Thirty AD patients were rated by the same informant on separate occasions one year apart. RESULTS: Inter-informant reliability for the five domain scores of the NEO-FFI was shown to range from fair to good when measured using the single measure Intraclass Correlation Co-efficient (ICC) (0.52-0.64), and to range from good to excellent when measured using the average ICC (0.68-0.78). Intra informant reliability for four out of the five domains was shown to be excellent when measured using the single ICC (0.81-0.92), and good for the remaining domain (0.72). Intra-informant reliability was found to be excellent for all five domains when measured using the average ICC (0.84-0.96). Internal consistency of the five domains was good. CONCLUSIONS: The NEO-FFI can be used reliably to measure premorbid personality in patients with probable AD. It may be useful to maximise reliability by using a mean domain score based on questionnaires completed by two or more informants who knew the patient well earlier in life. PMID- 16676295 TI - Family conflict in dementia: prodigal sons and black sheep. AB - OBJECTIVE: To describe family conflict in cases of dementia referred to the Guardianship Tribunal of New South Wales, Australia. METHOD: The file notes of 50 cases of family and systems conflict in cases of dementia presented to the Guardianship Tribunal were examined. Demographics, MMSE score, and type and severity of dementia were recorded. The documents and evidence presented to the Tribunal were coded and subjected to thematic analysis to identify the themes of the conflict, the protagonists and the position of the person with dementia with respect to the conflict. RESULTS: Family conflict was most commonly seen in mild to moderate dementia. Conflict occurred most frequently between siblings (with a group of siblings allied against a 'black sheep' member) and involved other systems such as service providers in 25% of cases. The person with dementia was usually involved in the conflict or in alliance with one or other of the family members in conflict, especially when paranoid ideation was fuelled by family members. Common themes included accusations of neglect, exploitation, lack of communication or sequestration of the person with dementia. No family had received family therapy prior to the application; conciliation during the hearing was successful in 30% of cases. Legal transactions such as Powers of Attorney were frequently made and frequently revoked by persons with dementia involved in family conflict. CONCLUSION: Dementia may be a great family divider, particularly when there are cracks in family solidarity. The understanding of family conflict in dementia has ramifications for both clinical and medico-legal practice. These findings may encourage family-centered interventions which address family dynamics and interpersonal conflict. They may also assist in capacity assessments of persons with dementia who change legal documents because of family conflict. PMID- 16676296 TI - Male gender influences response to an educational package for delirium prevention among older people: a stratified analysis. AB - BACKGROUND: Increasing evidence is pointing towards the efficacy of intervention programmes in decreasing the incidence of delirium among older people admitted to hospital. We have previously shown that an educational package directed at doctors and nurses significantly decreased the point prevalence of delirium among older people on a general medical ward. It is not yet established whether specific and 'fixed' patients' characteristics influence the rate of response to such an intervention. METHODS: A secondary, exploratory stratified analysis was carried out to determine whether age, sex and presence of dementia might influence the effect of the intervention through a delirium educational package. This information is important in order to increase the effectiveness of preventive measures across various patients' subgroups. RESULTS: Male gender (OR 0.17, 0.05-0.65) significantly (p = 0.030) and positively influence the response to the educational package compared to female gender (OR 1.04, 0.38-2.81). Neither age nor the presence of underlying dementia was associated with a significant influence on the rate of response to the delirium prevention package. CONCLUSION: Staff may be more likely to implement an earlier and more effective intervention for males who are perceived as presenting a higher risk to themselves and others. The results reported highlight the need to increase staff's vigilance to female patients whose prodromal symptoms of delirium may be underemphasised. Understanding different sub-group responses to preventive educational packages for delirium is an important consideration if these are to be applied widely. PMID- 16676297 TI - In vitro antiproliferative effect of six Salvia species on human tumor cell lines. AB - This study was designed to examine the in vitro antiproliferative activity of the methanol crude extracts of six Salvia species: Salvia dominica L. leaves, Salvia lanigera Desf. aerial parts, Salvia menthaefolia Ten. roots, Salvia palaestina Benth. aerial parts, Salvia sclarea L. roots and Salvia spinosa L. aerial parts. Extracts were screened for their possible antitumoral activity by MTT test on nine human cancer cell lines: glioblastoma (DBTRG-05MG, T98G, U-87MG), colorectal adenocarcinoma (WiDr and HT-29), prostate adenocarcinoma (MDA Pca2b), choriocarcinoma (JEG-3), endometrium adenocarcinoma (HEC-1A) and B lymphoblast (CIR). IC(50) values were determined for only five extracts and ranged from 90 to 400 microg/mL approximately. Salvia menthaefolia extract exhibited marked antiproliferative activity against all tumor cell lines showing lower IC(50) values, while S. spinosa, S. sclarea and S. dominica extracts showed a degree cytotoxic activity dependent on the cell line type. Finally S. palaestina extract revealed a moderate antiproliferative effect only against three cell lines. Salvia lanigera extract displayed toxic activity at all concentrations tested. The results strengthen the evidence that the genus Salvia could be considered a natural resource of potential antitumor agents. PMID- 16676298 TI - Exploring Allium species as a source of potential medicinal agents. AB - It has been shown that Allium species may help to prevent tumor promotion, cardiovascular diseases and aging; all processes that are associated with free radicals. Therefore the Allium species of both cultivated species (Allium nutans L., Allium fistulosum L., Allium vineale L., Allium psekemense B. Fedtsch, Allium cepa L., Allium sativum L.) and wild species (Allium flavum L., Allium sphaerocephalum L., Allium atroviolaceum Boiss, Allium schenoprasum L., Allium vineale L., Allium ursinum L., Allium scorodoprasum L.) from various locations were investigated for their antioxidative properties. The leaves were examined for activities of antioxidative enzymes (catalase, peroxidase, superoxide dismutase, glutathione-peroxidase), non-enzymic antioxidants (reduced glutathione and total flavonoids), content of soluble proteins, vitamin C, carotenoids, chlorophylls a and b, as well as the quantities of malonyldialdehyde and *OH and O2*- radicals. Using a contemporary spectroscopic fluorescent method, lipofuscin, 'plant age pigments' were determined. ESR spectroscopy was used to follow the decrease of oxygen radicals in the presence of extracts of Allium species in phosphate buffer (pH 7). The results showed that all Allium species had strong antioxidative properties due to their high concentration of total flavonoids, high content of carotenoids and chlorophylls, and very low concentrations of toxic oxygen radicals. ESR signals of DMPO-OH radical adducts, in the presence of Allium extracts in phosphate buffer (pH 7), were reduced by up to 94.3%. PMID- 16676299 TI - Simultaneous quantification of CTN986 and its deglycosylation products in rat serum using liquid chromatography/tandem mass spectrometry. AB - A quantitative method for the simultaneous determination of CTN986, a flavonol triglycoside, and its two deglycosylation products rutin and hirsutin in rat serum was developed and validated for the investigation of the pharmacokinetics of CTN986. Analytes were isolated from the serum samples (200 microL) prior to analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using C(18) solid-phase extraction, and were separated on a Zorbax C(8) reversed-phase column with an isocratic mobile phase consisting of methanol/isopropanol/water/formic acid (20:10:70:0.1, v/v/v/v). The protonated analytes generated in the positive ion mode were monitored through multiple reaction monitoring in an eletrospray ionization source. Calibration was performed by internal standardization with CTN987, a flavonoid structurally similar to CTN986, and regression curves were constructed ranging from 2 to 1000 ng/mL in 200 microL serum samples. The intra- and inter-day precision values were below 11% and accuracy was between -2.37 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of CTN986 in rats following oral and intravenous administration. Rutin and hirsutin were not detected in rat serum. PMID- 16676300 TI - Simultaneous determination of amphetamines and ketamines in urine by gas chromatography/mass spectrometry. AB - A method for the simultaneous determination of amphetamines and ketamines (ketamine, norketamine and dehydronorketamine) in urine samples by gas chromatography/mass spectrometry was developed and validated. Urine samples were extracted with organic solvent and derivatized with trifluoroacetic anhydride (TFAA). The limits of detection and limits of quantification for each analyte were lower than 19 and 30 ng/mL, respectively. Within-day and between-day precisions were within 0.5% and 10.6%, respectively. Biases for three levels of control samples were within -10.6% and +7.8%. The concentration of dehydronorketamine was greater than those of ketamine or norketamine in 19 of 35 ketamine-positive samples. A group of 110 human urine samples previously determined to contain at least one of the target analytes was analyzed using the new method, and excellent agreement was observed with previous results. PMID- 16676301 TI - Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry. AB - Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for the analysis and characterization of protein phosphorylation on the peptide level. In this study, the applicability of ionic liquid matrices (ILM) formed by combination of the crystalline MALDI matrix 2,5-dihydroxybenzoic acid (DHB) with pyridine or n-butylamine was tested for the analysis of phosphopeptides. Low ionization efficiency in both positive and negative ion mode was observed in acid-free sample preparations. Upon addition of 0.1% trifluoroacetic acid (TFA), ion formation was increased, but analogously to the situation described earlier for pure DHB, best results were obtained upon use of 1% phosphoric acid as matrix additive. The samples prepared in this way were significantly more homogeneous than preparations with pure DHB, thus avoiding the need for time-consuming search for hot spots. Other characteristics like metastable fragmentation of phosphopeptides did not differ from that observed in classical preparations. The limits of detection for synthetic phosphopeptides and singly or multiply phosphorylated peptides from tryptic digests of alpha- and beta-casein were comparable with those obtained when using pure DHB; in some cases even higher signal intensities could be observed in the ILM. The use of ILM in combination with 1% phosphoric acid as matrix additive significantly facilitates analysis of phosphopeptides by MALDI-MS. PMID- 16676303 TI - Collision-induced gas-phase smiles rearrangement in phenoxy-N-phenylacetamide derivatives. PMID- 16676305 TI - A study to determine the minimum volume of blood necessary to be discarded from a central venous catheter before a valid sample is obtained in children with cancer. AB - BACKGROUND: The objective of this study was to determine the minimum volume of blood that should be discarded from a range of different types of central venous catheter (CVC), such that the subsequent blood sample was not diluted or contaminated by the residual intra-luminal fluid. PROCEDURE: Seventy children aged 1-19 years with central venous access inserted as part of their standard clinical treatment were recruited to this prospective study. Statistical comparison of the extent of variation in biochemical and haematological parameters observed between two blood samples taken following routine 5 ml discard blood volumes, as compared to the extent of variation between samples drawn following a 5 ml discard volume and <5 ml volumes, was carried out. RESULTS: Data indicate that the measurement error in a clinical sample obtained following a 3 ml discard volume is no different to the measurement error obtained when using a standard 5 ml discard volume. Comparable results were obtained from patients with various different types of CVC or portacath access. CONCLUSIONS: The withdrawal of a 3 ml discard volume is sufficient to ensure that the subsequent blood sample is not diluted or contaminated by residual intra-luminal fluid. This may have a significant clinical impact in paediatric oncology, where patients frequently require blood transfusions due to the haematological toxicities associated with chemotherapy. It is hoped that these results will impact on hospital policies concerning specified discard volumes taken from CVCs prior to the withdrawal of blood samples for research purposes and routine clinical analysis. PMID- 16676307 TI - Disease progression in recently diagnosed patients with inherited marrow failure syndromes: a Canadian Inherited Marrow Failure Registry (CIMFR) report. AB - BACKGROUND: Inherited bone marrow failure syndromes (IMFSs) are genetic disorders characterized by defective single-lineage or multi-lineage hematopoiesis. IMFS patients are at risk for severe cytopenias, development of marrow cytogenetic abnormalities (MCA), myelodysplasia (MDS), and malignancy. The rate of disease progression and proportion of patients at risk for these complications is currently unclear. We examined recently diagnosed IMFS patients to determine distribution of diagnoses, disease progression and development of significant outcomes. METHODS: The CIMFR is a prospective multi-center study established in 2001 to register all IMFS patients in Canada. Analysis was restricted to patients diagnosed after November 30, 1997. Summary statistics were used to depict the study population while survival was described using the Kaplan-Meier method. RESULTS: 74 CIMFR patients were considered recently diagnosed. Median age at diagnosis was 2.7 years (range, birth to 40.6). Annual follow-up data were available for 53 (72%) patients. The five most prevalent diagnoses were Fanconi anemia (FA), Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), dyskeratosis congenita (DKC), and Kostmann's neutropenia (KS). Eighteen (24%) patients were unclassifiable. Twenty-eight (53%) follow-up patients had disease progression as indicated by new or worsening cytopenias, new marrow changes, or initiation of transfusion support and/or medical therapy. Fourteen (19%) fulfilled minimal diagnostic criteria for myelodysplasia. Eleven patients had hematopoietic stem cell transplantation (HSCT) by first follow-up. Five patients have died. Survival at 36 months is 89.8 +/- 5.7%. CONCLUSIONS: IMFS patients are often diagnosed at a young age. The relative distribution of diagnoses is similar to previous reviews of published cases; however, 25% of patients are currently unclassifiable. Disease progression has occurred in approximately 50% of follow up patients. Early mortality is noted. Continued prospective observation of these patients is warranted. PMID- 16676309 TI - Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry. AB - The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two. PMID- 16676310 TI - Investigation of non-covalent complexes of HIV-1 promoter DNA and polyamides containing N-methylpyrrole by electrospray ionization mass spectrometry. AB - Eight novel polyamides containing N-methylpyrrole were designed to target the sequence (5'-CTGCATATAAGCAG-3'/5'-CTGCTTATATGCAG-3') of the TATA box element of the HIV-1 promoter DNA. The non-covalent complexes of the promoter DNA and the polyamides were investigated by electrospray ionization (ESI) mass spectrometry, which provided strong evidence for the binding of the novel polyamides to the sequence of the TATA box element. It also revealed that polyamide 2 (PyPyPyPybetaDp), a potent binder of HIV-1 promoter DNA and a lead molecule for the design of new anti-HIV-1 drugs, had the highest binding affinity with the TATA box element DNA among these polyamides by examining the stoichiometry and the selectivity. PMID- 16676311 TI - Direct determination of acylcarnitines in amniotic fluid by column-switching liquid chromatography with electrospray tandem mass spectrometry. AB - A direct, simple, and simultaneous determination of acylcarnitines in amniotic fluid was developed using column-switching liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analytes can be assayed within 20 min without any sample preparation process, and we monitored separated acylcarnitines with positive electrospray ionization (ESI)-MS/MS. The calibration ranges of acylcarnitines were 1 to 100 nmol/L. The linearity of the method was 0.992 to 0.999, and the limits of detection at a signal-to-noise ratio of 3 were 1 nmol/L. The coefficients of variation were in the range of 5.2 to 13.3% for within-day variation and 6.7 to 11.9% for day-to-day, respectively. We detected acylcarnitines in the amniotic fluid of 22 women in the early stages of their pregnancies in the range of 2.2 to 17.2 nmol/L. The proposed method could be applied to diagnosis, monitoring, and biomedical investigations of inborn errors of the organic acid and fatty acid metabolism of the embryo. PMID- 16676312 TI - A qualitative study of amlodipine and its related compounds by electrospray ionization tandem mass spectrometry. AB - A comprehensive structural analysis of amlodipine and certain related compounds was performed by electrospray ionization tandem mass spectrometry. Triple quadrupole and quadrupole time-of-flight instruments were used to provide collision-induced dissociation and accurate mass measurement for selected product and second-generation product ions. A unique ion rearrangement was observed, which was found to be characteristic of certain dihydropyridines. This study provides a fundamental understanding of the fragmentation of these compounds. The structural elucidation of an unknown impurity is presented as an example. PMID- 16676315 TI - Identification of isocephalomannine in the presence of cephalomannine isomers and alkali metal ion adducts in a paclitaxel active pharmaceutical ingredient using electrospray tandem mass spectrometry. AB - A strategy is developed for the identification of isocephalomannine in the presence of alkali metal ion adducts and other cephalomannine isomers in a paclitaxel active pharmaceutical ingredient. Intact molecular ion analyses and a sub-structural study have been performed for the differentiation of isocephalomannine (2-debenzoylpaclitaxel-2-pentenoate) from cephalomannine and 7 epi-cephalomannine. A comparative study of the cephalomannine isomers was carried out using molecular ions (MS) and fragmentation patterns (MS/MS) for sub structural analysis. An attempt has been made to identify isocephalomannine in Cremophor(R) EL formulations. PMID- 16676316 TI - Study of the phase I and phase II metabolism of nephrotoxin aristolochic acid by liquid chromatography/tandem mass spectrometry. AB - Prolonged exposure to aristolochic acid (AA) was shown to pose rapid progressive renal fibrosis in Belgian women in a slimming regime in the early 1990s. AA was also demonstrated to be strong carcinogen in rats. The carcinogenicity of AA is generally believed to be related to the nitro-reduction of AA, in which the aristolactam-nitriumion ion with a delocalized positive charge is the ultimate carcinogen. In this study, the phase I and phase II metabolism of AA was investigated by using an in vitro system with rat liver S9 and an in vivo animal study with Sprague-Dawley rats. AA was found to have been undergone hydroxylation, lactam formation, and desnitro and desmethyl transformations. Three conjugated metabolites of AA, namely the N- and O-glucuronides of aristolactams, were detected directly in pre-concentrated urine sample, with no acid hydrolysis or enzymatic digestion. Structural elucidation of the metabolites was performed by using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The results indicated that N-glucuronidation was the major phase II metabolic pathway for the aristolactams formed by AA after their nitro-reduction. PMID- 16676317 TI - Mass spectrometry of proton adducts of fucosylated N-glycans: fucose transfer between antennae gives rise to misleading fragments. AB - Fragmentation behavior of fucosylated N-glycans in both protonated and sodiated form was studied by low-energy collision-induced dissociation with an ion trap mass spectrometer as well as by laser-induced dissociation with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI TOF/TOF-MS). Diantennary, core-(alpha1-6)-fucosylated N-glycans with Lewis X (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) and/or fucosylated LacdiNAc antennae (GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) were obtained from the human parasite Schistosoma mansoni and used as model substances, after labeling with 2 aminobenzamide, or as native reducing glycans. While fragment spectra of sodiated as well as protonated species obtained in both mass spectrometers resulted in B- and Y-type ions, fragmentation of proton adducts additionally gave rise to various fragment ions which had acquired fucose residues from other parts of the molecule. In particular, fucose was transferred efficiently to the Lewis X antennae suggesting the occurrence of difucosylated antennae, which could erroneously be interpreted as Lewis Y epitopes. By studying two additional model substances, this fucose gain was shown to occur by transfer of fucose between the antennae, but not by transfer of a core-(alpha1-6)-fucose. Despite the drastically different lifetimes of the ions, protonated species analyzed on the ion trap (millisecond range) and by MALDI-TOF/TOF-MS (microsecond range) showed similar rearrangement patterns, suggesting that the fucose mobility goes hand in hand with decomposition. Notably, permethylation of the model N-glycans seemed to completely preclude fucose migration. This study indicates that caution should be applied with the interpretation of tandem mass spectrometric (MS/MS) data of protonated glycoconjugates, including glycopeptides, because of the potential occurrence of fucose rearrangements. PMID- 16676318 TI - Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids. AB - Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix. PMID- 16676319 TI - Iminodiacetic acid derivatized porous silicon as a matrix support for sample pretreatment and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. AB - Iminodiacetic acid (IDA)-1,2-epoxy-9-decene has been synthesized and covalently linked to the surface of porous silicon wafer through a photochemical reaction. The negatively charged carboxylic acid groups on the porous silicon wafer are capable of binding oppositely charged species from sample solutions through electrostatic interactions. This allows the removal of contaminants prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by simply washing the porous silicon surface. The carboxylic acid end groups on porous silicon can be used to selectively bind and concentrate target species in sample solutions. Furthermore, Fe(3+)-IDA-derivatized porous silicon was prepared to specifically and effectively concentrate phosphopeptides from the tryptic digests of phosphoproteins, followed by MALDI-MS analysis. PMID- 16676320 TI - Chemical study of triterpenoid resinous materials in archaeological findings by means of direct exposure electron ionisation mass spectrometry and gas chromatography/mass spectrometry. AB - A systematic study of standard triterpenes (alpha-amyrine, oleanolic acid, betulin, lupeol, betulinic acid and lupenone) and of raw resinous materials (frankincense resin, mastic resin and birch bark pitch) was performed using direct exposure electron ionisation mass spectrometry (DE-MS) and gas chromatography/mass spectrometry (GC/MS). DE-MS provides a mass spectral fingerprint of organic materials in a few minutes which highlights the compounds that are the main components in the sample. The application of principal component analysis (PCA) on DE-MS data in the mass ranges m/z 181-260 and m/z 331 500, corresponding to the fragmentation of triterpenoid molecules, enabled us to distinguish between different triterpenoid materials such as mastic resin, frankincense resin and birch bark pitch, and to graphically plot the resinous substances in three separate clusters, retaining 89% of the total variance. GC/MS analysis of the same materials has permitted us to elucidate in detail the molecular composition and to identify minor components and species that act as markers of the degradation undergone by the materials. The paper also reports the results for the organic residues contained in an Egyptian censer (5th-7th century AD) which was recovered in the excavation of the Necropolis of Antinoe (Egypt), and for the hafting material found on a Palaeolithic tool recovered at the site of Campitello (Arezzo, Tuscany), dating back to the Mid-Pleistocene period. Although DE-MS was found to be a fast analytical tool, it failed to give any information on the presence of less abundant compounds when applied to mixtures of different materials: only mastic resin was found in the residues from the Roman censer, whereas GC/MS analysis identified the presence of a vegetable oil from Brassicaceae seeds and Pinaceae resin. Birch bark pitch as a pure material was identified in the sample from the Palaeolithic flint flake using both procedures. PMID- 16676321 TI - Reproducible and inducible knockdown of gene expression in mice. AB - RNA interference (RNAi) has emerged as an efficient approach for rapid analysis of gene function. In mammalian cells, vector-based expression of small hairpin RNAs (shRNA) produces potent and stable gene knockdown effects. An inducible RNAi system with reproducible levels of siRNA expression will extend the usefulness of this methodology to the identification of gene functions within the developing or adult mouse. We present evidence that an RNA polymerase III-driven U6 promoter with stuffer sequences flanked by loxP sites inserted at three different sites within the promoter drives shRNA expression in a Cre recombinase-dependent manner. We utilized this approach to develop a generic strategy for the reproducible knockdown of gene expression in mice. By placing the inducible shRNA cassette into the ROSA26 locus of the mouse, we were able to generate reproducible levels of controlled expression of shRNA to produce discernable phenotypes in vitro and in vivo. This approach circumvents the prescreening of random integration in embryonic stem cell clones and further enables conditional gene knockdown with temporal and/or tissue specificity. This methodology should expedite large-scale functional studies. PMID- 16676323 TI - On the N2O correction used for mass spectrometric analysis of atmospheric CO2. AB - To obtain accurate values of delta(13)C(CO(2)) and delta(18)O(CO(2)) on environmental CO(2) by mass spectrometry, the raw isotope data must be corrected for the isobaric N(2)O contribution. This is one of the analytical problems limiting inter-laboratory delta(13)C(CO(2)) data consistency. The key parameter, the N(2)O relative ionisation efficiency (E(N2O)), cannot be determined with sufficient accuracy by direct measurements of pure N(2)O. The determination of (E(N2O)) by analyses on N(2)O--CO(2) mixtures of known isotope composition and mixing proportions has been recently suggested. In this work we propose a new method of N(2)O correction which uses the m/z 30 signal as a measure of the N(2)O/CO(2) ratio, so that determinations of (E(N2O)) and N(2)O content are not required. The method uses the fact that fragment-ion spectra of N(2)O and CO(2) are very specific. The formalism of the correction is considered. Various tests demonstrate that the new method is robust, stable and easy to implement in practice. The effective value (E(N2O)) (the key parameter for the new correction) has to be calibrated on known N(2)O--CO(2) mixtures by measuring (30)R signals only. The method accuracy we presently achieved is around 2.5% and any error which appears to come mostly from our N(2)O--CO(2) mixture preparation. Based on our tests and error considerations, the error of the proposed method that may be achieved is as low as +/-1.5% (relative to the correction magnitude). For tropospheric CO(2) this means +/-0.003 per thousand and +/-0.005 per thousand for delta(13)C(CO(2)) and delta(18)O(CO(2)), respectively. The proposed method may be valuable for small samples where no separate N(2)O determinations are available (e.g. ice core samples and CF-IRMS measurements) as well as for determination of (E(N2O)) and testing the 'traditional' N(2)O correction based on mass balance calculations. PMID- 16676322 TI - Characterization of melanocyte-specific inducible Cre recombinase transgenic mice. AB - Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT) inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma. PMID- 16676324 TI - Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detection. AB - A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50 microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide. PMID- 16676325 TI - Heterozygous P0 deficiency protects mice from vincristine-induced polyneuropathy. AB - Patients with hereditary neuropathies are more susceptible to vincristine (VIN) induced neuropathy than patients without this comorbidity. The heterozygous P0(+/ ) mouse is an animal model of a distinct form of inherited neuropathies. These mice produce only 50% of the major myelin protein protein zero (P0) and display signs of demyelination in motor nerves at 4 months of age. Here we investigated the development of neuropathic signs in P0(+/-) and wild-type (wt) mice after VIN treatment. Neuropathy was induced by daily intraperitoneal injections of VIN (0.5 mg/kg body weight) over 10 days. Behavioral and electrophysiological tests were performed at regular time points. Wt mice developed significant hypersensitivity to heat and mechanical stimuli between days 7 and 38 after the first VIN injection. Surprisingly, P0(+/-) mice did not show sensory or motor signs of neuropathy over the whole testing period. Immunohistochemical analysis showed an increase in macrophage numbers in sciatic nerve sections of wt mice after VIN, whereas P0(+/-) mice had higher baseline levels of macrophages without changes after VIN treatment. Semithin sections revealed a decrease in the number of small diameter myelinated fibers in the sciatic nerves of wt mice after VIN application, whereas P0(+/-) mice had higher baseline values of this fiber subtype that did not change under treatment. Dorsal root ganglion neurons of both genotypes showed an up-regulation of voltage-gated sodium channel immunoreactivity after VIN application without differences between the genotypes. Thus, the P0(+/-) phenotype seems to be protected against VIN-induced neuropathy. The mechanism of this neuroprotection remains elusive. PMID- 16676326 TI - Estrogen-mediated immunomodulation involves reduced activation of effector T cells, potentiation of Treg cells, and enhanced expression of the PD-1 costimulatory pathway. AB - Estrogen (E2)-induced immunomodulation involves dual effects on antigen presenting cells (APC) and CD4(+)CD25(+) regulatory T cells (Treg) but not a direct effect on effector T cells. In this report, we further investigated the effects of E2 on APC and Treg function. We found that E2 treatment in vivo strongly reduced recovery of APC from the peritoneal cavity and inhibited induction of the inflammatory cytokines interleukin (IL)-12 and interferon-gamma but enhanced secretion of IL-10. Moreover, E2-conditioned bone marrow-derived dendritic cells (BM-DC) could both enhance Treg activity and directly inhibit responder T cells in the absence of Treg cells. We examined whether this E2 induced inhibitory activity of BM-DC might involve costimulation through the recently described PD-1 pathway. Both E2 and pregnancy markedly enhanced PD-1 expression in several types of APC, including macrophages, B cells, and especially dendritic cells (DC). Similarly to E2-induced enhancement of FoxP3 expression and experimental autoimmune encephalomyelitis protection, E2-induced enhancement of PD-1(+) cells was also mediated through estrogen receptor alpha (Esr1) in DC and macrophages but not in B cells. Based on antibody inhibition studies, PD-1 interaction with its ligands, PDL-1 and especially PDL-2, could mediate either positive or negative regulatory signaling in both mature and immature E2-conditioned DC, depending, respectively, on a relatively high (10:1) or low (1:1) ratio of T cells:BM-DC. These novel findings indicate that E2 induced immunomodulation is mediated in part through potentiation in BM-DC of the PD-1 costimulatory pathway. PMID- 16676327 TI - Strain difference in susceptibility to experimental autoimmune encephalomyelitis between Albino Oxford and Dark Agouti rats correlates with disparity in production of IL-17, but not nitric oxide. AB - Albino Oxford (AO) rats, unlike Dark Agouti (DA) rats are resistant to the induction of experimental autoimmune encephalomyelitis (EAE). The reason for the resistance could be some restraining mechanism preventing auto-aggressive cell activation at the level of draining lymph nodes (DLN) during the induction phase of the disease. Such a mechanism could be anti-proliferative action of nitric oxide (NO), which has already been shown of importance for the resistance of several rat strains to the induction of the disease. Importantly, number of AO DLN cells (DLNC) is markedly lower and with lower proliferative response to myelin basic protein (MBP) ex vivo in comparison to DA DLNC in the inductive phase of EAE, thus implying that in AO rats DLNC do not proliferate as extensively as in DA rats. We show that AO rats do not produce larger quantities of NO than DA rats after immunization. Further, DLNC of immunized AO rats have significantly lower mRNA expression and synthesis of interferon (IFN)-gamma and interleukin (IL)-17 compared to DLNC of DA rats. Collectively, these results suggest that there is a substantial difference between EAE-resistant AO rats and EAE-prone DA rats in the initiation of autoimmune response. This difference seems to be independent of anti-proliferative actions of NO, but correlates with impaired IL-17 production in AO rats. PMID- 16676328 TI - Repeated administration of the selective kappa-opioid receptor agonist U-69593 increases stimulated dopamine extracellular levels in the rat nucleus accumbens. AB - Reinforcing properties of drugs of abuse are reduced by the coadministration of kappa opioid receptor (KOR) agonists. This effect is related to the inhibition of dopamine (DA) release in the nucleus accumbens (NAc) produced by the acute administration of KOR agonists. The present study was undertaken to investigate the in vivo effect of the repeated administration of KOR agonist on extracellular DA levels in the NAc. Rats were injected once daily with the selective KOR agonist U-69593 (0.16-0.32 mg/kg) or vehicle for 4 days. Microdialysis studies assessing extracellular concentration of DA in the NAc under basal and K(+) stimulatory conditions were conducted 1 day later. The microdialysis studies revealed that preexposure to U-69593 had no effect on basal extracellular DA levels but significantly augmented the amount of extracellular DA induced by high K(+) compared with vehicle pretreated rats. The D2 receptor agonist quinpirole perfused through the dialysis probe in the NAc, although it produced a significant decrease on basal and K(+)-stimulated DA levels in control rats, it did not decrease significantly either basal or K(+)-stimulated DA levels in U 69593 preexposed rats. Preexposure to U-69593 did not alter the expression of tyrosine hydroxylase or dopamine transporter in the ventral tegmental area. These results show that repeated administration of U-696593 increases the amount of extracellular DA induced by high K in the NAc, an effect that may be related to decreased D2 autoreceptor function. It is suggested that repeated activation of KOR changes the response status of dopaminergic neurons in the NAc. PMID- 16676329 TI - Uncaria rhynchophylla, a Chinese medicinal herb, has potent antiaggregation effects on Alzheimer's beta-amyloid proteins. AB - Because the deposition of beta-amyloid protein (Abeta) is a consistent pathological hallmark of Alzheimer's disease (AD) brains, inhibition of Abeta generation, prevention of Abeta fibril formation, or destabilization of preformed Abeta fibrils would be attractive therapeutic strategies for the treatment of AD. We examined the effects of several medicinal herbs used in traditional Chinese medical formulae on the formation and destabilization of Abeta fibrils by using the thioflavin T binding assay, atomic force microscopic imaging, and electrophoresis. Our study demonstrates that several of these herbs have potent inhibitory effects on fibril formation of both Abeta(1-40) and Abeta(1-42) in concentration-dependent manners; in particular, Uncaria rhynchophylla inhibited Abeta aggregation most intensively. Significant destabilization of preformed Abeta(1-40) and Abeta(1-42) fibrils was also induced by Uncaria rhynchophylla as well as some other herb extracts. Three-dimensional HPLC analysis indicated that the water extract of this herb contains several different chemical compounds, including oxindole and indol alkaloids, which have been regarded as neuroprotective. Our results suggest that Uncaria rhynchophylla has remarkably inhibitory effects on the regulation of Abeta fibrils, and we conclude that this medicinal herb could have the potency to be a novel therapeutic agent to prevent and/or cure AD. PMID- 16676331 TI - Discovery of asymmetric autocatalysis with amplification of chirality and its implication in chiral homogeneity of biomolecules. PMID- 16676330 TI - Decreased NR1 phosphorylation and decreased NMDAR function in hibernating Arctic ground squirrels. AB - Heterothermic mammals such as ground squirrels tolerate ischemia and N-methyl-D aspartate (NMDA) better than homeothermic mammals such as rats both in vivo and in vitro, and this tolerance is enhanced in the hibernating state. However, the cellular mechanisms underlying this tolerance remain unclear. NMDA receptors (NMDAR) play a key role in excitotoxicity. The purpose of the current study was therefore to test the hypothesis that NMDAR are down-regulated in hibernating Arctic ground squirrels (hAGS; Spermophilus parryii). To address this hypothesis, we used Western blot analysis to investigate NMDAR phosphorylation, an activator of NMDAR function, and internalization in naive hippocampal tissue from hAGS, interbout euthermic AGS (ibeAGS), and rats. Furthermore, we used fura-2 calcium imaging to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats. We report that phosphorylation of the NMDAR1 (NR1) subunit is decreased in hippocampal tissue from hAGS and that the NMDAR component of Glu induced increase in [Ca(2+)](i) is decreased in hippocampal slices from hAGS. Moreover, the fraction of NR1 in the functional membrane pool in AGS is less than that in rats. PMID- 16676332 TI - The effect of acidic and basic additives on the enantioseparation of basic drugs using polysaccharide-based chiral stationary phases. AB - The enantioseparation of nine commercially available basic drugs was achieved on polysaccharide-based chiral stationary phases with the acidic additive ethanesulfonic acid and the basic additive butylamine. Seven different commercially available CSPs were used for the study (AD, AS, OD, OJ, OG, OB, and OC). Mobile phase additives have been proven to be essential in obtaining satisfactory enantio-resolution in terms of both efficiency and selectivity. Significantly improved selectivities were obtained for the basic probe drugs with the acidic additive, ethanesulfonic acid, rather than the basic additive, butylamine. This is best seen with Chiralpak AS CSP. No enantioseparation for the nine drugs was observed when butylamine was used as an additive; however, satisfactory enantioseparation for the nine drugs was achieved using ethanesulfonic acid. Higher column efficiencies were observed with the acidic additive, especially when isopropanol was used as a modifier. Higher sensitivity was also achieved with ethanesulfonic acid because of the significantly lower background at the UV detection wavelength. The acidic additive was demonstrated to be superior to the basic additive for the enantioseparation of basic drugs using seven different polysaccharide-based CSPs. These results are counterintuitive to the common "rule of thumb" in enantioseparation that states acidic additives work best for acidic analytes and basic additives work best for basic analytes. The beneficial effects of acidic additive in enantioseparations observed in this study could significantly improve the applicability of polysaccharide-based CSPs for the enantioseparation of basic analytes. PMID- 16676333 TI - Lipid signal extraction by SLIM: application to 1H MR spectroscopic imaging of human calf muscles. AB - The measurement of intramyocellular lipid (IMCL) using in vivo (1)H MRS is important for better understanding muscle physiology. However, the accurate measurement of IMCL in muscle adjacent to subcutaneous fat (SF) and bone marrow (BM) is often hampered by contaminations from the fat. In this article a new postacquisition processing method is proposed that selectively removes unwanted lipid signals based on the spectral localization by imaging (SLIM) technique, which can localize spectra from arbitrarily shaped regions. The effectiveness of this lipid extraction method is demonstrated by both computer simulation and in vivo experiments in the human calf. The advantage of this method is that unwanted lipid signal, such as SF signal, can be selectively and completely removed. After the contaminating fat signals are removed, the quality of muscle spectra adjacent to SF improves such that it becomes comparable to that in uncontaminated muscle regions in (1)H MRSI of the calf. PMID- 16676334 TI - High-resolution MAS NMR spectroscopy detection of the spin magnetization exchange by cross-relaxation and chemical exchange in intact cell lines and human tissue specimens. AB - High-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy detects resolved signals from membrane phospholipids and proteins in intact cell and tissue samples. MAS has the additional advantage of quenching spin-diffusion through a mutual "flip-flop" of neighbor spins by time-independent dipolar coupling as long as the dipolar coupling is "inhomogeneous." Under MAS, significant magnetization transfer (MT) was observed between water and each proton site in membrane phospholipid and between water and the NMR-observable protein proton signals. The MT rates between water and membrane phospholipids are lower than those between water and protein proton signals. The interaction of water to other small molecules is selective with the observation of MT from water to creatine, lactate, taurine, and glycine, but not to triglyceride, phosphocholine, choline, or myo-inositol. HR-MAS NMR allows the detection of a complete MT network between water and each proton group of creatine. Two creatine pools (one motion restricted and one motion-free) were identified in skeletal muscle. PMID- 16676335 TI - Propeller EPI in the other direction. AB - A new propeller EPI pulse sequence with reduced sensitivity to field inhomogeneities is proposed. Image artifacts such as blurring due to Nyquist ghosting and susceptibility gradients are investigated and compared with those obtained in previous propeller EPI studies. The proposed propeller EPI sequence uses a readout that is played out along the short axis of the propeller blade, orthogonal to the readout used in previous propeller methods. In contrast to long axis readout propeller EPI, this causes the echo spacing between two consecutive phase-encoding (PE) lines to decrease, which in turn increases the k-space velocity in this direction and hence the pseudo-bandwidth. Long- and short-axis propeller EPI, and standard single-shot EPI sequences were compared on phantoms and a healthy volunteer. Diffusion-weighted imaging (DWI) was also performed on the volunteer. Short-axis propeller EPI produced considerably fewer image artifacts compared to the other two sequences. Further, the oblique blades for the long-axis propeller EPI were also prone to one order of magnitude higher residual ghosting than the proposed short-axis propeller EPI. PMID- 16676336 TI - Isolation and minimization of head motion-induced signal variations in fMRI data using independent component analysis. AB - Task-related head movement during acquisition of fMRI data represents a serious confound for both motion correction and estimates of task-related activation. Cost functions implemented in most conventional motion-correction algorithms compare two volumes for similarity but fail to account for signal variability that is not due to motion (e.g., brain activation). We therefore recently proposed the theoretical basis for a novel method for fMRI motion correction, termed motion-corrected independent component analysis (MCICA), that allows for brain activation present in an fMRI time-series to be implicitly modeled and mitigates motion-induced signal changes without having to directly estimate the motion parameters (Liao et al., IEEE Transactions on Medical Imaging 2005;25:29 44). To explore the effects of non-movement-related signal changes on registration error, we performed several previously proposed test simulations (Freire et al., IEEE Transactions on Medical Imaging 2002;21:470-484) to evaluate the performance of MCICA and compare it with the conventional square-of difference-based measures such as LS-SPM and LS-AIR. We demonstrate that for both simulated data and real fMRI images, the proposed MCICA method performs favorably. Specifically, in simulations MCICA was more robust to the addition of simulated activation, and did not lead to the detection of false activations after correction for simulated task-correlated motion. With actual data from a motor fMRI experiment, the time course of the derived continually task-related ICA component became more correlated with the underlying behavioral task after preprocessing with MCICA compared to other methods, and the associated activation map was more clustered in the primary motor and supplementary motor cortices without spurious activation at the brain edge. We conclude that assessing the statistical properties of a motion-corrupted volume in relation to other volumes in the series, as is done with MCICA, is an accurate means of differentiating between motion-induced signal changes and other sources of variability in fMRI data. PMID- 16676337 TI - Research with families facing cancer: the challenges of accrual and retention. AB - The purposes of this article are: (a) to describe and analyze the accrual and retention patterns in a longitudinal randomized clinical trial with prostate cancer patients and their partners, and (b) to discuss strategies that were used to overcome challenges in conducting this family-based study. Initially, 429 dyads were referred to the study. Of these, 166 were not enrolled due to refusal (n = 120) or ineligibility (n = 46), 21 of whom did not meet one or more of the inclusion criteria, and 25 of whom could not be reached within the 2-month window of eligibility. Of the 383 eligible dyads, 263 dyads were enrolled (enrollment rate of 68.7%). Accrual and retention patterns differed by research site, referral procedures, and phase of prostate cancer. The retention rate was very good with the majority of dyads (n = 218) completing all three follow-up assessments at 4, 8, and 12 months (82.9%). PMID- 16676338 TI - Community-based recruitment and enrollment for a clinical trial on the sensitive issue of fecal incontinence: the Fiber study. AB - The recruitment of community-living participants for clinical trials of sensitive topics, when the population is largely hidden and reluctant to self-identify, and the study protocols and procedures intensive, creates significant challenges to researchers. The Fiber Study is an ongoing randomized controlled clinical trial conducted to compare the effectiveness of three dietary fibers with different levels of fermentability for symptom management in community-living adults with fecal incontinence. The researchers developed a staged approach to recruitment using three primary recruitment methods and a three-phase approach to the enrollment process. We have been successful in reaching, recruiting, and enrolling participants in a clinical trial, as well as in effectively managing study resources and staff time. PMID- 16676339 TI - Recruitment of African American women to a walking program: eligibility, ineligibility, and attrition during screening. AB - The purposes of this study were to identify strategies successful in the recruitment of African American (AA) women to a home-based walking program and to examine factors that contribute to attrition, eligibility, and ineligibility during the recruitment screening protocol. Of the 696 women who contacted the researchers, 281 (40.4%) women enrolled in the study, 227 (32.6%) were lost to attrition, and 188 (27%) were ineligible. Those not enrolled due to attrition during screening or ineligibility reported more family risk for cardiovascular disease (CVD) and lived in neighborhoods with higher poverty. Although our recruitment strategies may have been successful in attracting low-income AA women, we were not as successful in preventing their attrition during the screening protocol, particularly for those living in poorer neighborhoods. PMID- 16676340 TI - Methodological issues in the recruitment of cancer pain patients and their caregivers. AB - Published pain management guidelines recommend that caregivers of cancer pain patients be provided pain management education, but little is known about the utility of providing such information. This study examined recruitment and retention of cancer pain patients and caregivers for a randomized clinical trial that provided psychoeducation and stress management training to caregivers. Of 397 patient/caregiver dyads screened, only 22 (5.5%) were study eligible, consented to participate, and completed the study. A variety of problems hampered successful participation, including a high proportion of non-cancer-related pain syndromes among patients, a high proportion of patients without caregivers, and participants' perception of study burden. Results suggest that researchers should recruit from a broad patient base and limit study burden on participants. PMID- 16676341 TI - Measuring participation in a prevention trial with parents of young children. AB - This paper describes parent participation in a clinical trial of preventive parent training (PT) targeting low-income parents of young children. Participation encompassed enrollment, attendance, and engagement. Average enrollment rate was 34.9%, although enrollment rates were significantly higher in the control (39.1%) than in the intervention (30.6%) centers. Parents attended an average of 39% of the PT sessions. Higher attendance was associated with lower parenting self-efficacy, more parent-reported child behavior problems, and attending the first PT session. Level of engagement in the PT sessions was related to improvements in parent and child outcomes. Findings suggest that attendance is linked to parent perceived need for help and that resources should be focused on ensuring parent attendance at the first intervention session. PMID- 16676342 TI - Family patterns of decision-making in pediatric clinical trials. AB - The decision-making process related to a child's participation in clinical trials often involves multiple family members. The aim of this study was to compare family patterns of decision-making within and across family units in pediatric clinical trials. Participants for this secondary analysis included 14 families from a larger study of informed consent. Four distinct patterns of decision making were identified: Exclusionary, informative, collaborative, and delegated. These patterns varied with regard to three dimensions of parents' decision-making goals, child level of involvement, and the parental role. These patterns of decision-making affect how parents and children communicate with health professionals and influence the effectiveness of health care providers interactions with the family related to the decision-making process. PMID- 16676343 TI - Enacting a theory of caring to recruit and retain vulnerable participants for sensitive research. AB - The recruitment and retention of research participants always presents challenges to researchers. This process is made more complicated when the research being undertaken is socially sensitive and the populations of interest are considered vulnerable. The purpose of this article is to illustrate how Swanson's middle range theory of caring can be used as a framework for recruitment and retention for studies on sensitive topics that involve vulnerable participants. We provide an overview of the theory as well as illustrations from three separate studies that involved in-depth interviews with vulnerable participants. These studies included parents who had either experienced the death of their infant or were involved in life support decisions because of potentially giving birth to an extremely premature infant (22-25 weeks gestation). PMID- 16676344 TI - A research agenda for understanding participation in clinical research. PMID- 16676345 TI - Bursa tract diverticulum in the hermaphroditic land snail Arianta arbustorum (Stylommatophora: Helicidae): morphology, function, and evolutionary implications. AB - A bursa tract diverticulum is widespread in the female part of the hermaphroditic reproductive system of stylommatophoran pulmonates. However, the ultrastructure of the diverticulum is unknown and there is only anecdotal evidence for a spermatophore-dissolving function for this organ. In the present study, we examined the ultrastructure of the diverticulum and investigated histological, histochemical, and morphometric changes at different time intervals after mating in the simultaneously hermaphroditic land snail Arianta arbustorum. The diverticulum in this species of snail is a prominent organ, consisting of a luminal columnar epithelium surrounded by a thick layer of connective tissue. During mating, the diverticulum functions as the site of spermatophore uptake. Within the lumen of the diverticulum the spermatophore wall is dissolved or at least partly broken down. The digested material is taken up by epithelial cells and accumulated in molluscan-specific cells of the connective tissue, the so called rhogocytes. Subsequent to copulation, the total diameter of the diverticulum increases markedly, reaching a maximum size 12 h after mating, while at the same time the thicknesses of the diverticulum wall and diverticulum epithelium decrease. The length of the diverticulum shows a positive allometry and a high phenotypic variation compared to snail size, which suggests that the diverticulum is under directional sexual selection. We propose that the diverticulum in A. arbustorum has evolved in response to selection pressures imposed by divergent evolutionary interests between male and female function. PMID- 16676347 TI - Cancer progression by non-clonal chromosome aberrations. AB - The establishment of the correct conceptual framework is vital to any scientific discipline including cancer research. Influenced by hematologic cancer studies, the current cancer concept focuses on the stepwise patterns of progression as defined by specific recurrent genetic aberrations. This concept has faced a tough challenge as the majority of cancer cases follow non-linear patterns and display stochastic progression. In light of the recent discovery that genomic instability is directly linked to stochastic non-clonal chromosome aberrations (NCCAs), and that cancer progression can be characterized as a dynamic relationship between NCCAs and recurrent clonal chromosome aberrations (CCAs), we propose that the dynamics of NCCAs is a key element for karyotypic evolution in solid tumors. To support this viewpoint, we briefly discuss various basic elements responsible for cancer initiation and progression within an evolutionary context. We argue that even though stochastic changes can be detected at various levels of genetic organization, such as at the gene level and epigenetic level, it is primarily detected at the chromosomal or genome level. Thus, NCCA-mediated genomic variation plays a dominant role in cancer progression. To further illustrate the involvement of NCCA/CCA cycles in the pattern of cancer evolution, four cancer evolutionary models have been proposed based on the comparative analysis of karyotype patterns of various types of cancer. PMID- 16676346 TI - A role of the mitotic spindle checkpoint in the cellular response to DNA replication stress. AB - Replication stress is a frequent and early event during tumorigenesis. Whereas the cellular responses to a persistent block of replication fork progression have been extensively studied, relatively little is known about how cells respond to low-intensity replication stress. However, transient replication fork perturbations are likely to occur even more frequently in tumor cells than a permanent replication arrest. We report here that transient, low intensity replication stress leads to a rapid activation of the DNA replication checkpoint but to a significantly delayed apoptotic response in a small but significant number of cells. This late apoptotic response was independent of p53 and we found evidence for cell death during mitosis in a proportion of cells. To further explore the role of p53 in the response to replication stress, we analyzed mouse embryonic fibroblasts (MEFs) deficient of p53 in comparison to wild-type or p63- or p73-deficient MEFs. We detected a significant increase of apoptosis and morphological signs of failed mitosis such as multinucleation in p53-deficient MEFs following replication stress, but not in wild-type or p63- or p73-deficient cells. Multinucleated p53-deficient MEFs frequently retained cyclin B1 expression indicating a persistently activated mitotic spindle checkpoint. Collectively, our results suggest that the cellular response to replication stress involves the mitotic spindle checkpoint in a proportion of cells. These findings imply that the mitotic spindle checkpoint may act in concert with DNA damage and cell-cycle checkpoints as an early anti-tumor barrier and provide a possible explanation for its frequent relaxation in human cancer. PMID- 16676348 TI - A new hepatocytic isoform of PLZF lacking the BTB domain interacts with ATP7B, the Wilson disease protein, and positively regulates ERK signal transduction. AB - The promyelocytic leukemia zinc finger (PLZF) protein has been described as a transcriptional repressor of the BTB-domain/zinc-finger family, and shown to regulate the expression of Hox genes during embryogenesis and the expression of cyclin A in the cell cycle progression. Here, a 45-kDa isoform of PLZF without a BTB domain was identified via yeast two-hybrid screening using the C-terminal region of ATP7B as bait in our determination of the biological roles of the Wilson disease protein outside of its copper-binding domain. Our immunoprecipitation experiments showed that the hepatocytic isoform of PLZF could specifically interact with the C-terminal region of ATP7B. The immunostaining of HepG2 cells revealed that the ATP7B and PLZF proteins were apparently colocalized into the trans-Golgi complexes. It was also determined that disruption of PLZF expression in the HepG2 cells affected an attenuation of ERK activity in a dose dependent manner. The hepatocytic activities of ERK kinase were found to be enhanced as the result of PLZF or ATP7B expression, but this enhancement was abrogated by the deletion of the C-terminal region of ATP7B. Furthermore, a transgenic Drosophila strain that ectopically expressed the hepatocytic deltaBTB PLZF exhibited phenotypic changes in eye and wing development, and these alterations were fully recovered as the result of ATP7B expression, indicating the obvious in vivo interaction between the two proteins. Those PLZF-induced abnormalities were attributed to the enhancement of ERK signaling, as was shown by phenotypic reversions with loss-of-function mutations in ERK signal transduction in Drosophila. These data suggest the existence of a mechanism that regulates ERK signaling via the C-terminus of ATP7B and the ATP7B-interacting hepatocytic PLZF. PMID- 16676349 TI - Effects of sequential exposure to lipopolysaccharide and heat stress on dental pulp cells. AB - In the present study, we examined the effects of sequential exposure to bacterial lipopolysaccharide (LPS) and heat stress on dental pulp cells. LPS induced the proliferation of pulp cells through the activation of p38 MAPK. HSP27 was expressed in cells with or without LPS during the entire period of heat stress, while transiently phosphorylated by short-term heat stress. In LPS-treated cells, short-term heat stress also induced the phosphorylation of HSF1. The immediate phosphorylation of HSF1 and HSP27 in LPS-treated cells by short-term heat stress occurred dependent on the activation of p38 MAPK. However, with long-term heat stress, the activation of HSF1 and induction of HSP27 occurred independent of p38 MAPK. Further, full activation of Akt in LPS-treated cells was immediately induced by short-term heat stress and lasted during the entire period of heat stress. IkappaB alpha was induced and phosphorylated throughout sequential exposure to LPS and heat stress. These results suggest that LPS has the unique effects on the cytoprotection and the cell death of pulp cells during heat stress through the modification and the activation of heat stress responsive molecules, HSF1 and HSP27, and cell survival molecules, Akt and NF-kappaB/IkappaB alpha. PMID- 16676350 TI - Use of diet pills and other dieting aids in a college population with high weight and shape concerns. AB - OBJECTIVE: The current study examines diet aid use among college women at risk for eating disorders and explores characteristics associated with diet aid use. METHOD: Participants were 484 college women<30 years from 6 universities in the San Francisco Bay Area (SF) and San Diego who were at risk for developing eating disorders. A checklist assessed diet pill, fat blocker, diuretic, laxative, and other diet aid use over the past 12 months. RESULTS: Thirty-two percent of the college women reported using a diet aid. Diet aid use was double the rate in San Diego (44%) compared with SF (22%) (p=.000). Weight and shape concerns were higher among diet aid users than among nonusers across sites. CONCLUSION: A significant number of college women at risk for eating disorders are using diet aids. We recommend that clinicians inquire about diet aid use among college-aged patients. PMID- 16676351 TI - Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis-specific histone H1t gene. AB - Previous work in our laboratory revealed upregulated transcription of the testis specific linker histone H1t gene in pachytene primary spermatocytes during spermatogenesis. Using the H1t X-box as an affinity chromatography probe, we identified Regulatory Factor X2 (RFX2), a member of the RFX family of transcription factors, as a nuclear protein that binds the probe. We also showed that RFX2 activated the H1t promoter in transient expression assays. However, other RFX family members have the same DNA-binding domain and they also may regulate H1t gene expression. Therefore, in this study we examined the distribution of RFX2 and other RFX family members in rat testis germinal cells and in several tissues. Among tissues examined, RFX2 is most abundant in testis. Testis RFX2 is most abundant in spermatocytes where transcription of the H1t gene is upregulated and the steady-state H1t mRNA level is high. RFX2 levels decrease but RFX1 levels increase in early spermatids where H1t gene transcription is downregulated. Antibodies against RFX2 generate a shifted band in electrophoretic mobility shift assays (EMSA) using H1t or testisin X-box DNA probes with nuclear proteins from spermatocytes. These data support the hypothesis that RFX2 expression is upregulated in spermatocytes where it participates in activating transcription of the H1t gene and other testis genes. These data also support the possibility that other RFX family members may bind to the H1t promoter in other testis germinal cell types and in nongerminal cells to downregulate H1t gene transcription. PMID- 16676352 TI - Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family. AB - Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay. PMID- 16676353 TI - Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system. AB - We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non chromosomal context. PMID- 16676354 TI - The impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression. AB - In the normal prostate epithelium, androgen receptor (AR) negative basal epithelial cells adhere to the substratum, while AR expressing secretory cells lose substratum adhesion. In contrast, prostate cancer cells both express AR and adhere to a tumor basement membrane. In this review, we describe the differential expression of integrins, growth factor receptors (GFRs), and AR in normal and cancerous epithelium. In addition, we discuss how signals from integrins, GFRs, and AR are integrated to regulate the proliferation and survival of normal and malignant prostate epithelial cells. While cell adhesion is likely of great importance when considering therapeutic approaches for treatment of metastatic prostate cancer, no data on integrin expression are available from tissues of prostate cancer metastasis. However, several drug targets that are upregulated after androgen ablative therapy regulate cell adhesion and thus novel targeted therapies indirectly interfere with cell adhesion mechanisms in prostate cancer cells. PMID- 16676355 TI - Regulation of insulin response in skeletal muscle cell by caveolin status. AB - Recent studies on the role of caveolin-1 in adipocytes showed that caveolin has emerged as an important regulatory element in insulin signaling but little is known on its role in skeletal muscle cells. In this study, we demonstrate for the first time that caveolin-1 plays a crucial role in insulin dependent glucose uptake in skeletal muscle cells. Differentiation of L6 skeletal muscle cells induce the expression of caveolin-1 and caveolin-3 with partial colocalization. However in contrast to adipocytes, phosphorylation of insulin receptor beta (IRbeta) and Akt/Erk was not affected by the respective downregulation of caveolin-1 or caveolin-3 in the muscle cells. Moreover, the phosphorylation of IRbeta was detected not only in the caveolae but also in the non-caveolae fractions of the muscle cells despite the interaction of IRbeta with caveolin-1 and caveolin-3. These data implicate the lack of relationship between caveolins and IRbeta pathway in the muscle cells, different from the adipocytes. However, glucose uptake was reduced specifically by downregulation of caveolin-1, but not that of caveolin-3. Taken together, these observations suggest that caveolin-1 plays a crucial role in glucose uptake in differentiated muscle cells and that the regulation of caveolin-1 expression may be an important mechanism for insulin sensitivity, implying the role of muscle cells for type 2 diabetes. PMID- 16676356 TI - Overexpression of RGPR-p117 enhances regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells: involvement of TTGGC motif. AB - A novel protein RGPR-p117 was discovered as regucalcin gene promoter region related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct (mutant) with deletion of -523/-435 including TTGGC motif. NRK52E cells (wild type) or stable HA-RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with deletion of -523/-435. Wild type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1-34) (PTH; 10(-7) M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of 523/-435 sequence of regucalcin promoter. This was also seen using the -710/+18 LUC construct with deletion of -523/-503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10(-7) M), Bay K 8644 (10(-6) M), phorbol 12 myristate 13-acetate (PMA; 10(-6) M), or N(6), 2'-dibutyryl cyclic adenosine 3', 5'-monophosphate (DcAMP; 10(-4) M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10(-6) M), staurosporine (10(-9) M), PD 98059 (10(-8) M), wortmannin (10(-8) M), genistein (10(-6) M), vanadate (10(-6) M), or okadaic acid (10(-6) M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. PMID- 16676358 TI - Biological significance and development of practical synthesis of biotin. AB - Biotin (1), a water-soluble B series vitamin, distributes widely in microorganisms, plants, and animals. Biosynthesis of 1 involves five steps sequence starting from pimelic acid. The last step, a transformation from dethiobiotin (DTB) to 1, includes an iron clusters-mediated radical process. The compound 1 is a cofactor of carboxylation enzymes and plays crucial roles in the metabolism of fatty acids, sugars, and alpha-amino acids. In addition to the increasing application to feed additives, recent reports have revealed that 1 enhances insulin secretion in animals, suggesting it for a promising therapeutic candidate for an anti-diabetes drug. The remarkably strong affinity of 1 with avidin and streptavidin has been extensively applied for such technologies as photoaffinity labeling. Among the number of approaches to 1 so far developed in 50 years, a synthesis using L-cysteine and thiolactone as a starting material and a key intermediate, respectively, represents one of the best routes leading to 1, because of short steps, high yield, use of inexpensive reagents, and ease of operation. PMID- 16676357 TI - Hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding. AB - The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2 induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli. PMID- 16676359 TI - Gene-specific mechanisms of p53 transcriptional control and prospects for cancer therapy. AB - The regulation of gene-specific activation is critical to the tumor suppressor function by p53. p53 is a well-characterized transcription factor that responds to DNA damage and other genotoxic stresses by the activation of downstream targets that are involved with repair, differentiation, senescence, growth arrest, and apoptosis. Sequence-specific binding to DNA, conformation, post translational modifications, cofactor binding, stability, and subcellular localization all influence the performance of p53. The purpose of this review is to define features that play a key role in gene-specific activation and to show that these are often incapacitated in cancer cells. Using such knowledge to design selective strategies for the restoration of p53 wild-type function in cancer cells represents a promising cancer therapy. PMID- 16676360 TI - Endothelial cell fatty acid unsaturation mediates cold-induced oxidative stress. AB - Ultraprofound hypothermia (< 5 degrees C) induces changes to cell membranes such as liquid-to-gel lipid transitions and oxidative stress that have a negative effect on membrane function and cell survival. We hypothesized that fatty acid substitution of endothelial cell lipids and alterations in their unsaturation would modify cell survival at 0 degrees C, a temperature commonly used during storage and transportation of isolated cells or tissues and organs used in transplantation. Confluent bovine aortic endothelial cells were treated with 18 carbon fatty acids (C18:0, C18:1n-9, C18:2n-6, or C18:3n-3), C20:5n-3 or C22:6n-3 (DHA), and then stored at 0 degrees C without fatty acid supplements. Storage of control cells caused the release of lactate dehydrogenase (LDH) and a threefold increase in lipid peroxidation (LPO) when compared to control cells not exposed to cold. Pre-treating cells with C18:0 decreased the unsaturation of cell lipids and reduced LDH release at 0 degrees C by 50%, but all mono- or poly-unsaturated fatty acids increased injury in a concentration-dependent manner and as the extent of fatty acid unsaturation increased. DHA-treatment increased cell fatty acid unsaturation and caused maximal injury at 0 degrees C, which was prevented by lipophilic antioxidants BHT or vitamin E, the iron chelator deferoxamine, and to a lesser extent by vitamin C. Furthermore, the cold-induced increase in LPO was reduced by C18:0, vitamin E, or DFO but enhanced by DHA. In conclusion, the findings implicate iron catalyzed free radicals and LPO as a predominant mechanism of endothelial cell injury at 0 degrees C, which may be reduced by increasing lipid saturation or treating cells with antioxidants. PMID- 16676361 TI - Drosophila S2 cells produce multiple forms of carboxypeptidase D with different intracellular distributions. AB - Carboxypeptidase D (CPD) functions in the processing of proteins that transit the secretory pathway, and is present in all vertebrates examined as well as Drosophila. Several forms of CPD mRNA were previously found in Drosophila that resulted from differential splicing of the gene. In the present study, Northern blot, reverse transcriptase PCR, and Western blot analysis showed that each splice variant occurs in a single cell type, the Drosophila-derived Schneider 2 (S2) cell line. The short forms containing a single carboxypeptidase domain were secreted from the S2 cells while the long forms containing three carboxypeptidase domains, a transmembrane domain, and one of two different cytosolic tails were retained in the cell. To investigate the role of the two different C-terminal tail sequences (tail-1 and tail-2) that result from the differential splicing within exon 8, constructs containing a reporter protein (albumin) attached to the transmembrane domain and tail-1 or tail-2 of CPD were expressed in S2 cells and a mouse pituitary cell line (AtT20 cells). Immunofluorescence analysis revealed different intracellular distributions of the two constructs, with the tail-2 construct showing considerable overlap with a Golgi marker. The two C-terminal tail sequences also resulted in different internalization efficiencies from the cell surface in both cell lines. Interestingly, the distribution and routing of the tail-2 form of Drosophila CPD in the AtT20 cells are similar to the previously characterized endogenous mouse CPD protein, indicating that the elements for this trafficking have been conserved between Drosophila and mammals. PMID- 16676362 TI - Proteolytic processing of a sea urchin, ECM-localized protein into lower mol mass species possessing collagen-cleavage activity. AB - The hyaline layer is an apically located extraembryonic matrix, which blankets the sea urchin embryo. Using gelatin substrate gel zymography, we have identified a number of gelatin-cleaving activities within the hyaline layer and defined a precursor-product processing pathway which leads to the appearance of 40- and 38 kDa activities coincident with the loss of a 50-kDa species. Proteolytic processing of the precursor required the presence of both CaCl2 and NaCl at concentrations similar to those found in sea water. The cleavage activities utilized both sea urchin and rat tail tendon gelatins as substrates but demonstrated a species-specific cleavage activity towards sea urchin collagen. The gelatin-cleaving activities were refractory to inhibition by 1,10 phenanthroline but were inhibited by benzamidine. This latter result defines the serine protease nature of the cleavage activities. Both the 40- and 38-kDa activities were found to comigrate with gelatin-cleaving activities present in the sea urchin embryo. PMID- 16676363 TI - 2-(4-Methylphenyl)-1,3-selenazol-4-one induces apoptosis by different mechanisms in SKOV3 and HL 60 cells. AB - We examined the ability of the synthetic selenium compound, 2-(4-methylphenyl) 1,3-selenazol-4-one (hereafter designated 3a), to induce apoptosis in a human ovarian cancer cell line (SKOV3) and a human leukemia cell line (HL-60). Flow cytometry showed that 3a treatment induced apoptosis in both cell lines to degrees comparable to that of the positive control, paclitaxel. Apoptosis was measured by PS externalization, DNA fragmentation and decreased mitochondrial membrane potential (MMP). However, analysis of the mechanism of action revealed differences between the responses of the two cell lines. Treatment with 3a arrested the cell cycle and induced caspase-3 activation in HL-60 cells, but not in SKOV3 cells. In contrast, 3a treatment induced apoptosis through translocation of AIF, a novel pro-apoptotic protein, in SKOV3 cells, but not in HL-60 cells. Collectively, our data demonstrated that 3a induced apoptosis in both cell lines, but via different action mechanisms. PMID- 16676364 TI - The North Carolina-Louisiana Prostate Cancer Project (PCaP): methods and design of a multidisciplinary population-based cohort study of racial differences in prostate cancer outcomes. AB - BACKGROUND: The North Carolina-Louisiana Prostate Cancer Project (PCaP) is a multidisciplinary study of social, individual, and tumor-level causes of racial differences in prostate cancer aggressiveness. METHODS: A population-based sample of incident prostate cancer cases from North Carolina and Louisiana will include 1,000 African Americans and 1,000 Caucasian Americans. Study nurses administer structured questionnaires and collect blood, adipose tissue, urine, and toenail samples during an in-home visit. Clinical data are abstracted from medical records, diagnostic biopsies are reviewed and assayed, and tissue microarrays are constructed from prostatectomy samples. Prostate cancer aggressiveness is classified based on PSA, clinical stage, and Gleason grade. RESULTS: Preliminary data demonstrate between- and within-group differences in patient characteristics, screening, and treatment by race and state. Participation exceeds 70% in all groups. CONCLUSIONS: Preliminary data support the feasibility of this comprehensive study to help determine the focus of public health efforts to reduce racial disparities in prostate cancer mortality. PMID- 16676365 TI - Array-comparative genomic hybridization to detect genomewide changes in microdissected primary and metastatic oral squamous cell carcinomas. AB - Oral squamous cell carcinoma (OSCC) is a common worldwide malignancy. However, it is unclear what, if any, genomic alterations occur as the disease progresses to invasive and metastatic OSCC. This study used genomewide array-CGH in microdissected specimens to map genetic alterations found in primary OSCC and neck lymph node metastases. We used array-based comparative genomic hybridization (array-CGH) to screen genomewide alterations in eight pairs of microdissected tissue samples from primary and metastatic OSCC. In addition, 25 primary and metastatic OSCC tissue pairs were examined with immunohistochemistry for protein expression of the most frequently altered genes. The highest frequencies of gains were detected in LMYC, REL, TERC, PIK3CA, MYB, MDR1, HRAS, GARP, CCND2, FES, HER2, SIS, and SRY. The highest frequencies of losses were detected in p44S10, TIF1, LPL, MTAP, BMI1, EGR2, and MAP2K5. Genomic alterations in TGFbeta2, cellular retinoid-binding protein 1 gene (CRBP1), PIK3CA, HTR1B, HRAS, ERBB3, and STK6 differed significantly between primary OSCC and their metastatic counterparts. Genomic alterations in PRKCZ, ABL1, and FGF4 were significantly different in patients who died compared with those who survived. Immunohistochemistry confirmed high PIK3CA immunoreactivity in primary and metastatic OSCC. Higher FGF4 immunoreactivity in primary OSCC is associated with a worse prognosis. Loss of CRBP1 immunoreactivity is evident in primary and metastatic OSCC. Our study suggests that precise genomic profiling can be useful in determining gene number changes in OSCC. As our understanding of these changes grow, this profiling may become a practical tool for clinical evaluation. PMID- 16676366 TI - Evaluation of volatile low molecular weight insecticides using Drosophila melanogaster as a model. AB - Volatile insecticides, otherwise known as fumigants, have numerous uses in pest control and integrated pest management. Fumigants have been broadly applied in the protection of stored agricultural commodities and also in the elimination of structural pest infestations. However, fumigants have so far been mostly overlooked for use in management of public health pests such as flies and mosquitoes. This paper reports information on the development of a volatility bioassay for use in evaluating volatile insecticides against dipteran insects and on the use of this bioassay to compare the relative efficacy of 30 candidate compounds to Drosophila melanogaster Meig. The findings revealed seven highly effective insecticidal compounds, which include three heterobicyclics (menthofuran, thiophene and coumaran) and four formate esters [butyl, hexyl and heptyl formate and ethylene glycol diformate (EGDF)]. Ethyl formate, a compound previously identified as being a highly effective fumigant against coleopteran stored product pests, caused toxicity in Drosophila but was not as effective as other tested formates. Two volatile fumigant insecticides, dichlorvos (DDVP) and methyl isothiocyanate (MITC), were also tested as positive controls. Both of these volatile standards were significantly more toxic than the seven most effective of the novel compounds. Finally, regression analyses revealed that volatility is not entirely predictive of acute toxicity and that other structural features should be considered when designing and synthesizing volatile insecticidal compounds for future study. PMID- 16676367 TI - Assessment of changes in endometrial and subendometrial volume and vascularity during the normal menstrual cycle using three-dimensional power Doppler ultrasound. AB - OBJECTIVES: To describe changes in endometrial and subendometrial volume and vascularity during the normal menstrual cycle using three-dimensional (3D) power Doppler ultrasonography. METHODS: Fourteen healthy volunteers, 24-44 years old with regular menstrual cycles, underwent serial transvaginal 3D power Doppler ultrasound examinations of the uterus on cycle day 2, 3 or 4, then daily from cycle day 9 until follicular rupture and 1, 2, 5, 7 and 12 days after follicular rupture. Endometrial and subendometrial volume (cm3), vascularization index (VI), flow index (FI) and vascularization flow index (VFI) were calculated using the VOCAL (Virtual Organ Computer-aided AnaLysis) software. RESULTS: Endometrial and subendometrial vascularity indices increased throughout the follicular phase, decreased to a nadir 2 days after follicular rupture and then increased again during the luteal phase. Endometrial and subendometrial volume increased rapidly during the follicular phase and then remained almost unchanged during the luteal phase. CONCLUSIONS: Substantial changes occur in endometrial volume and vascularization during the normal menstrual cycle. There is the potential for 3D power Doppler ultrasonography to become a useful tool for assessing pathological changes associated with female subfertility and abnormal uterine bleeding. PMID- 16676368 TI - Thermal expansion of microstructured DMPC bilayers quantified by temperature controlled atomic force microscopy. PMID- 16676369 TI - Peripheral T-cell lymphoma gene expression profiles. AB - Expression profiling using DNA microarrays has been very helpful to improve our knowledge of the pathobiology of many tumour types, including lymphomas. Peripheral T-cell lymphomas (PTCL) constitute an heterogeneous group of tumours with different morphologic, immunophenotypic, and clinical characteristics. Their complexity and their low frequency in the western countries have made difficult the identification of molecular events responsible of the development of these tumours. The first studies on expression profiling of PTCL have also revealed heterogeneity at this level, mainly regarding the PTCL NOS subgroup. Different molecular subgroups within PTCL unspecified have been identified associated to different expression profiles. However, the clinical significance of this molecular sub-classification remains to be probed in studies involving larger number of samples. In addition, the expression level of NF-kB pathway genes allowed to differentiate two PTCL subgroups, and this difference could have clinical interest. In general, PTCL expression profiles are difficult to interpret due to the significant proportion of other infiltrating cells accompanying the tumour. However, microarrays are being a helpful tool in the initial task of dissecting the PTCL expression profile. PMID- 16676372 TI - Selective oxidation of organic compounds--sustainable catalytic reactions with oxygen and without transition metals? PMID- 16676373 TI - Silica-based mesoporous organic-inorganic hybrid materials. AB - Mesoporous organic-inorganic hybrid materials, a new class of materials characterized by large specific surface areas and pore sizes between 2 and 15 nm, have been obtained through the coupling of inorganic and organic components by template synthesis. The incorporation of functionalities can be achieved in three ways: by subsequent attachment of organic components onto a pure silica matrix (grafting), by simultaneous reaction of condensable inorganic silica species and silylated organic compounds (co-condensation, one-pot synthesis), and by the use of bissilylated organic precursors that lead to periodic mesoporous organosilicas (PMOs). This Review gives an overview of the preparation, properties, and potential applications of these materials in the areas of catalysis, sorption, chromatography, and the construction of systems for controlled release of active compounds, as well as molecular switches, with the main focus being on PMOs. PMID- 16676374 TI - Development of a competitive exclusion product for poultry meeting the regulatory requirements for registration in the European Union. AB - Competitive exclusion treatment is able to increase the pathogen colonization resistance of day-old chicks by applying probiotic bacteria stabilizing the indigenous microflora. In order to develop a safe microbial feed additive, various bacterial strains were isolated out of the gastrointestinal tract of healthy chickens. One hundred twenty-one representatives were selected based on differences in whole-cell protein patterns and screened for antagonistic properties. Five effective strains (Pediococcus acidilactici, Enterococcus faecium, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, and Lactobacillus salivarius ssp. salivarius) exhibited in vitro the ability to inhibit a range of common pathogens and were evaluated with regard to the risks associated with genetic transfer of antibiotic resistances from animals to humans via the food chain. The probiotic strains were sensitive to several clinically effective antibiotics, though some of them showed single resistances. None of the vancomycin-resistant (R) strains carried the enterococcal vanA gene. Two tetracycline R strains were shown to harbor a tet(M)-associated resistance. The strains contained no extrachromosomal DNA and were not able to transfer the resistance by means of conjugation. On basis of the collected data the presence of easy transferable resistances was excluded and the chicken strains were considered to be suitable for the use as feed additive. PMID- 16676375 TI - Monitoring the mycotoxins in food and their biomarkers in the Czech Republic. AB - Testing of the presence of toxigenic microfungi and mycotoxins in foodstuffs in the food chain is an important part of the food safety strategy in The Czech Republic. At the national level, control of their presence in the entire food chain is assured by Public Health Protection Agencies, by the Veterinary Administration and by the Czech Agriculture and Food Inspection Authority. This article summarizes surveillance activities of Public Health Protection Agencies and mycotoxins findings in dietary raw materials and foodstuffs from the 1990s to 2004 in the Czech Republic. At present, the health risk from the mycotoxins exposure from foodstuffs is assessed to be relatively low in the Czech Republic, especially as far as the foodstuffs of the Czech origin are concerned. It may result in late toxic effects (e. g., carcinogenic risk) following a single or repeated ingestion of low mycotoxins doses from foodstuffs. Nevertheless, the overall situation may change due to the globalization of the food market. In order to minimize the risk associated with mycotoxins and eliminate their impact on Czech public health, continuous monitoring of the presence of toxigenic moulds, mycotoxins, and their biomarkers is necessary, in conjunction with strict respect to European Union legislation. PMID- 16676376 TI - The bacterial quality of red meat and offal in Casablanca (Morocco). AB - The present study aimed to evaluate the bacteriological quality of beef (n = 52), lamb (n = 52) and beef offal (n = 52) marketed in Casablanca, Morocco. Meat and offal samples (n = 156), were collected randomly from butcheries, supermarkets, and slaughterhouses. Two sampling periods were considered, one during the hot season and the second one during the cold season. The samples were analyzed for the presence of the following bacteria: Escherichia coli, coagulase-positive Staphylococcus, Clostridium perfringens, Salmonella, and Listeria monocytogenes. Results indicated that counts of the aerobic plate count, and fecal coliforms were particularly high in all the samples analyzed. E. coli, coagulase-positive Staphylococcus and C. perfringens were detected in 37.8, 16, and 4.5% of the meat samples, respectively. Neither Salmonella nor L. monocytogenes were isolated from meat samples. Approximately 26.9% of beef, 34.6% of lamb and 28.8% of beef offal samples contained bacteria above the maximum limits established by the Moroccan regulatory standards for meat and meat products. Seasonality and the distribution location significantly (p < 0.05) affected bacterial populations: the hot season and butcheries appeared to be cases where the highest populations of bacteria in meat were observed. These high levels of microbiological contamination attest the poor hygienic quality of meat and offal, possibly due to uncontrolled processing, storage, and handling of these products. PMID- 16676377 TI - Occurrence of mycotoxins (ochratoxin A, deoxynivalenol) and toxigenic fungi in Moroccan wheat grains: impact of ecological factors on the growth and ochratoxin A production. AB - The aim of the present work was to evaluate the contamination of some samples, taken from Moroccan wheat grains, by ochratoxin A (OTA), deoxynivalenol (DON) and the associated toxigenic fungi. Moreover, we focused on the influence of environmental factors on both the growth and OTA production by three strains of Aspergillus. The results showed that only few samples were contaminated by the two mycotoxins (2 samples for OTA and 7 for DON). The main isolated fungi belong to the Aspergillus, Penicillium and Fusarium genus; 74 Aspergillus and 28 Penicillium isolates were tested for their ability to produce OTA. Only 2 A. alliaceus and 14 A. niger were able to synthesize OTA. However, none of Penicillium isolates can produce this toxin under the conditions mentioned. In respect of the effects of the temperature and water activity (aw), the optimal conditions for the growth and OTA production were different. While the optimal conditions of growth for A. alliaceus and A. terreus are 30 degrees C and 0.98 aw, A. niger preferred 0.93-0.95 aw at 25 degrees C, whereas the optimal production of OTA was observed at 30 degrees C for both A. alliaceus and A. niger at 0.93 and 0.99 aw, respectively. PMID- 16676378 TI - Contamination of free-range chicken eggs with dioxins and dioxin-like polychlorinated biphenyls. AB - Dioxins and dioxin-like (DL) polychlorinated biphenyls (PCB) are persistent organic pollutants that enter the body mainly by food intake. A small margin exists between current exposure levels in the human population and the levels causing biological effects. Therefore, stringent control of concentrations of these contaminants in food and feed is needed. Eggs from free-range chicken are increasingly becoming an important part of the diet. These eggs have a higher risk of being contaminated with increased levels of dioxins and DL-PCB than barn or cage eggs. Ingestion of soil particles from environmentally contaminated areas may contribute to elevated dioxin levels in free-range chicken eggs. Available data show that current soil levels of dioxins and DL-PCB in residential and agricultural areas in Europe often appear to be too high to produce free-range eggs with dioxin levels below the current limit values in the EU. On the other hand, polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans concentrations in eggs from free-range chicken are not necessarily above the limit values. Contamination levels in soil should be kept low and should be controlled in areas with free foraging chicken although all modifying factors that influence uptake of dioxins and PCB from the environment and transfer into eggs are yet not well understood. PMID- 16676379 TI - Biodegradable network elastomeric polyesters from multifunctional aliphatic carboxylic acids and poly(epsilon-caprolactone) diols. AB - Biodegradable elastomeric network polyesters were prepared from multifunctional aliphatic carboxylic acids such as tricarballylic acid (Yt) or meso-1,2,3,4 butanetetracarboxylic acid (Xb) and poly(epsilon-caprolactone) (PCL) diols with molecular weights of 530, 1,250 and 2,000 g.mol-1. Prepolymers prepared by a melt polycondensation were cast from DMF solution and postpolymerized at 280 degrees C for various periods of times to form a network. The resultant films were transparent, flexible and insoluble in organic solvents. The network polyesters obtained were characterized by IR absorption spectra, WAXS, density measurement, DSC, and tensile test. YtPCL1250, and XbPCL1250 network polyester films showed good elastomeric properties with high ultimate elongation (540-590%), and low Young's modulus (2.5-3.3 MPa). The enzymatic degradation was estimated by the weight loss of network films in a buffer solution with Rhizopus delemar lipase at 37 degrees C. The degree and rate of degradation were significantly affected by the molecular weight of PCL diol, chemical structures of multifunctional aliphatic carboxylic acids and the morphology of network films. The changes in the solid states of network films during the degradation were also estimated by the results of DSC and WAXS. [see text] PMID- 16676380 TI - UNICEF statistics about children and AIDS. PMID- 16676381 TI - HIV and gender. National awareness day focuses on HIV and women. PMID- 16676383 TI - Prosecutions of HIV transmission escalate in North America. PMID- 16676382 TI - Woman with HIV, hepatitis C denied SSI benefits. PMID- 16676384 TI - Dismissal affirmed over county HIV-status disclosure. PMID- 16676385 TI - Sentencing. Sentence enhancement upheld for man who bit officers. PMID- 16676387 TI - GAO recommends fund changes for AIDS housing. PMID- 16676386 TI - Testing. Court-mandated HIV test for sex offender upheld on appeal. PMID- 16676388 TI - Prisons. HIV-positive inmate's claim survives dismissal. PMID- 16676390 TI - HIV-positive man's disability claim survives appeal. PMID- 16676389 TI - Inmate's HIV disclosure case against Bureau of Prisons fails. PMID- 16676391 TI - Sentencing. Stiff sentence upheld for HIV-positive sexual offender. PMID- 16676392 TI - Transplants. Woman may be infected with HIV from stolen tissue. PMID- 16676393 TI - Outbreak news. Cholera, southern Sudan--update. PMID- 16676394 TI - Validation of neonatal tetanus elimination in Nepal by lot quality assurance cluster sampling. PMID- 16676395 TI - [Migraine therapy]. PMID- 16676396 TI - Why do we need guidelines for differentiated thyroid cancer? PMID- 16676397 TI - Comparing USA and UK guidelines for the management of differentiated thyroid carcinoma. PMID- 16676398 TI - Double frameshift mutations in APC and MSH2 in the same individual. AB - Heterozygous germline DNA mismatch repair gene mutations are typically associated with HNPCC. Here we report the case of a proband whose father was known for familial adenomatous polyposis. The number of polyps (less than ten) was not typical of polyposis; therefore, the diagnosis of HNPCC was entertained. Microsatellite instability analyses were performed on peripheral blood and biopsy of a right-sided dysplastic adenoma. The tumor tissue showed high-grade instability, and a subsequent, immunohistochemistry showed that neither MSH2 nor MSH6 proteins were expressed in tumor cells. Prophylactic colectomy was performed, and an adenocarcinoma developing within the adenoma was diagnosed (pT1N0). Genomic DNA analysis revealed a novel mutation in MSH2 as a frameshift mutation in exon 7 (c.1191_1192dupG). Both parents of the proband were analysed for MSH2 and APC mutations, and in the father, a truncating mutation in exon 15 of APC was identified as del3471-3473GAGA. This mutation was found to be present in the proband. His mother was found to bear the MSH2 exon 7 mutation. At the follow-up, the proband was diagnosed with fundic, antral and duodenal adenomas (one fundic adenoma showed low-grade dysplasia). Several tubular rectal adenomas with low-grade dysplasia were excised. The patient later developed an intra abdominal desmoid tumor. PMID- 16676399 TI - 2-methoxyestradiol induces apoptosis in cultured human anaplastic thyroid carcinoma cells. AB - Anaplastic thyroid carcinoma (ATC) is one of the most malignant tumors in humans, and currently there is no effective treatment. In the present study we investigated the effect of an endogenous estrogen metabolite, 2-methoxyestradiol (2-ME), on the growth of human ATC cells. 2-ME treatment had a strong growth inhibitory effect on five human ATC cell lines (HTh7, HTh 74, HTh83, C643, and SW1736), but showed no effect on one cell line (KAT-4). Cell cycle analysis of the growth-inhibited cells showed that 2-ME induced a G2/M-arrest, followed by an increased fraction of cells in sub-G1. Analysis of internucleosomal DNA laddering as well as DNA fragmentation in a terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay demonstrated a high number of cells undergoing apoptosis after 2-ME treatment. An increased activation of caspase-3 and caspase-8 by 2-ME was observed, and inhibition of caspase-3 decreased the apoptotic effect. Addition of 2-ME increased activity of p38 mitogen-activated protein kinase (MAPK) in the sensitive HTh7 as well as the refractory KAT-4 cells, however, activation of stress-activated protein kinase/c-jun aminoterminal kinase (SAPK/JNK) was seen only in the HTh7 cells. Inhibitors of p38 MAPK and SAPK/JNK significantly attenuated the 2-ME effect. Taken together, our data demonstrate an antiproliferative and apoptotic effect of 2-ME on ATC cells involving activation of MAPKs. PMID- 16676400 TI - Histone deacetylase inhibitor depsipeptide represses nicotinamide N methyltransferase and hepatocyte nuclear factor-1beta gene expression in human papillary thyroid cancer cells. AB - Nicotinamide N-methyltransferase (NNMT) catalyzes N-methylation of nicotinamide and other structural analogues. NNMT gene expression is enhanced in many papillary thyroid cancer cells and activated by hepatocyte nuclear factor (HNF) 1beta. In this work, we studied the effects of depsipeptide, a histone deacetylase inhibitor, on NNMT gene expression in BHP 18-21 papillary thyroid cancer cells. Depsipeptide reduced NNMT mRNA level in a dose-dependent and time dependent manner as determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). In contrast, expression of the sodium iodide symporter (NIS), a gene with differentiated function, waas enhanced in the treated cells. NNMT protein level determined by Western blot analysis and NNMT catalytic activity was also reduced significantly in the depsipeptide-treated cells. To study the mechanism of NNMT gene repression by depsipeptide, effects of depsipeptide on NNMT promoter activity were determined by luciferase reporter gene assay. NNMT promoter activity was significantly reduced in the HNF-1beta positive BHP 18-21 cells but not in the HNF-1beta-negative BHP 14-9 papillary cancer cells. A mutant reporter construct with mutations in a HNF-1 site in the NNMT basal promoter region did not respond to depsipeptide in both HNF-1beta protein levels, and abolished activity of DNA binding to the HNF-1 site in the NNMT promoter region. Protein synthesis inhibitor cycloheximide and proteasome inhibitor MG-132 enhanced HNF-1beta stability in the depsipeptide-treated cells. In summary, depsipeptide represses NNMT and HNF-1beta gene expression in some papillary thyroid cancer cells. the repression of NNMT by depsipeptide is at the transcription level through downregulation of transcription activator HNF-1beta. PMID- 16676401 TI - Proceedings of the Tenth International Conference on Radiation Shielding and the Thirteenth ANS Topical Meeting on Radiation Protection and Shielding. Part 2. 2004. PMID- 16676402 TI - Gene expression in poorly differentiated papillary thyroid carcinomas. AB - We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PTC) specimens. Seven clinically aggressive carcinomas were included, comprising poorly differentiated PTC and tumors with extensive local invasion or synchronous distant metastases. Ten differentiated (classic) papillary thyroid carcinomas (PTC) and non-neoplastic thyroid tissues were also investigated. TaqMan quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry verified the differential gene expression. The B-Raf gene was mutated with a T-->A transversion at nucleotide 1799 (V600E) in 8 of 10 differentiated PTC, and in 4 of 7 aggressive carcinomas. Among genes markedly and equally over-expressed in carcinomas of both the aggressive and classic PtC groups, compared to normal thyroid tissue, were CBP/p300 transactivator (CItED1), fibronectin, growth/differentiation factor 15, potassium inwardly rectifying channel KCNJ2, glutaminyl peptide cyclotransferase, WNT7A, and dipeptidyl peptidase IV. A marked upregulation in carcinomas of P-cadherin mRNA and protein concomitant with E cadherin downregulation, indicates a possible P-E cadherin "switch" in PTC. The growth factor homologue Nel-like 2, dual specificity phosphatase 5, the serine protease kallikrein 10, and also the tight junction genes claudin 1 and claudin 16, were upregulated in classic PTC but not in aggressive tumors, which may be consistent with altered cell polarity in the dedifferentiated PtC. The aggressive, poorly differentiated PtC group was specifically characterized by marked upregulation of several genes related to cell proliferation such as cell division cycle 2 (CDC2), CDC7, kinesin-like 5, ubiquitin conjugating enzyme E2C, and topoisomerase IIalpha, and by upregulation of genes encoding extracellular matrix proteins such as seprase, extracellular matrix protein 1, and several collagens. These aggressive tumors were also characterized by overexpression of the integrin ligand periostin, and in some biopsies also of osteopontin and of the upstream Rac-regulator dedicator of cytokinesis 10 (DOCK10). These data are interpreted to be consistent with altered cell motility, extracellular matrix remodeling and increased cell proliferation, as important processes in PTC tumor progression. PMID- 16676403 TI - Early sequential changes in serum thyroglobulin after radioiodine ablation for thyroid cancer: possible clinical implications for recombinant human thyrotropin aided therapy. AB - TNM status and posttherapy whole-body scan findings aid in risk stratification of patients with thyroid cancer during initial therapy. Recently, the thyroglobulin (Tg) value measured during hypothyroidism just before 131I therapy (preablation Tg) has proved to be effective for predicting persistent/recurrent disease. In this study, we assessed the changes in serum Tg 48 hours after radioiodine ablative therapy performed in the hypothyroid state in order to evaluate if this parameter could be used in recombinant human thyrotropin (rhTSH)-treated patients. Because rhTSH-stimulated TG is traditionally measured 72 hours after the second injection of rhTSH corresponding to 48 hours post-131I therapy, the time course of serum Tg after radioiodine administration is an important clinical issue. To address this issue, we performed a prospective evaluation of 26 consecutive patients with low-risk differentiated thyroid cancer hypothyroidism for radioiodine ablation (3.7 GBq of 131I). Baseline Tg values were compared to posttherapy Tg values (at 24 and 48 hours). We found that Tg increased after 131I therapy because of the acute radiation effects on residual thyroid cells. Median values at each of the three time points were 1.8 ng/mL (baseline), 3 ng/mL (Tg 24), and 11.3 ng/mL (Tg-48) (Brahms Tg Kryptor assay, Brahms AG, Berlin, Germany). Tg-48 values were not statistically correlated with initial Tg values. Tg-48 remained below 15 ng/mL in 14 of 26 patients. In conclusion, the increase in Tg during the early post-131I therapy period means that first rhTSH-stimulated Tg cannot be used as a corresponding value for preablative hypo-Tg. We discussed whether rhTSH-stimulated Tg value might be useful in a subset of patients. In our opinion, this drawback does not outweight the expected benefits of rhTSH-aided therapy on quality of life of patients and overall cost of the therapy. PMID- 16676404 TI - Integrating multiple views of the cell: DNA repair jumps to higher level at Stowe workshop. PMID- 16676405 TI - Otto Schmitt: an electrocardiographer with multifaceted talents. PMID- 16676406 TI - Old foe learns new tricks. PMID- 16676407 TI - Genetics or environment? PMID- 16676408 TI - Alternative approaches to evaluating cirrhotic patients for laparoscopic cholecystectomy. PMID- 16676409 TI - Detection and surgical treatment of cervical lymph nodes in differentiated thyroid cancer. AB - There is considerable controversy regarding the treatment of patients with cervical metastases from differentiated thyroid cancer. Most have papillary carcinoma and the main areas of contention relate to methods of assessment and staging, surgical management and mode of follow up. there is little evidence to support elective anatomical imaging with CT or MRI in those patients with suspected or proven malignancy at the primary site as indicated by fine needle aspiration cytology (FNAC) but who have no clinical evidence of nodal disease. The role of routine ultrasound (US) in the pre-operative assessment of suspected or known malignancy is developing but is largely unproven. When it is performed, high risk areas for metastatic neck disease (levels II-V) should be assessed. Suspicious nodes on US should be further evaluated by FNAC. Suspected or proven neck disease may be further assessed pre-operatively with CT or MRI and then treated surgically. Disease in the central compartment requires a total thyroidectomy and level VI central compartment neck dissection. Suspected or proven lateral compartment cancer should be treated by selective neck dissection (at least levels III, IV, and V) below the accessory nerve. There is no role for 'Berry picking' and clinically node negative high risk patients should have an elective central compartment level VI neck dissection. Sentinel node biopsy lays no role and neither does elective lateral compartment surgery in patients with no clinical or radiological evidence of disease. For follow up, US represents the most sensitive means of detecting neck recurrences and in the presence of an elevated serum thyroglobulin, imaging may also include whole body iodine-131 scanning and anatomical imaging with CT or MRI. The role of PET remains controversial but is likely to develop further as the technique becomes more widely available. In the future, the concentration of patients with this disease in large center can only improve the way we treat differentiated thyroid cancer. PMID- 16676410 TI - Hurthle cell carcinoma with a giant cystic liver metastasis imaged with 18F labeled fluorodeoxyglucose-positron emission tomography. PMID- 16676411 TI - An interview with Pentti Rautaharju--student, colleague and friend of Otto Schmitt. PMID- 16676412 TI - False-positive 131I whole-body scan and shrinkage of a pulmonary aspergilloma under 131I therapy in a patient with thyroid cancer. PMID- 16676413 TI - Effect of suppressive therapy with levothyroxine on the reduction of serum thyroglobulin after total thyroidectomy. PMID- 16676414 TI - Evaluation of endothelial function in subclinical thyroid dysfunction. PMID- 16676415 TI - Vaccination for vivax malaria: targeting the invaders. AB - Among the surface-exposed antigens of the malaria parasite, those with known essential functions that can be disrupted by antibodies represent the most promising candidates for development as malaria vaccines. Two recombinant protein subunits of the Plasmodium vivax merozoite surface protein 1 have been shown to bind to reticulocytes in enzyme-linked immunosorbent assays. This article discusses the importance of such pre-clinical analyses in the validation of candidate vaccine molecules for P. vivax, given the constraints imposed by the use of primate models and the cost of producing suitable material for human trials. PMID- 16676416 TI - Therapeutic potential of folate uptake inhibition in Plasmodium falciparum. AB - Plasmodium falciparum parasites resistant to the combination sulfadoxine pyrimethamine are spreading in Africa, particularly in East Africa. This is a matter of concern because there are no other affordable drugs available. This article provides the evidence indicating that sulfadoxine-pyrimethamine resistance can be reversed in vitro and discusses how this information might be exploited to extend the therapeutic lifetime of sulfadoxine-pyrimethamine in vivo. PMID- 16676417 TI - Post-survey follow-up for human cystic echinococcosis in northwest China. AB - In Hobukesar, Xinjiang Uygur Autonomous Region, (China), a mass-screening programme for human cystic echinococcosis (CE)was performed during 1995 and 1996. After the survey 51 cases were followed-up using ultrasound scan and serology for 1-8 years(1996-2003). These included 20 CE cases treated by surgery (endocystectomy), four CE cases treated by percutaneous drainage(PAIR), six untreated CE cases, five ultrasound 'query' cases, eight cases of isolated hepatic calcifications, four previous CE surgery cases whose ultrasound was now normal, and four seropositive cases who exhibited a normal hepatic ultrasound. Follow-up results indicated that in the untreated CE patients, one case exhibited a spontaneous cure within 4 years, another case changed from a hepatic hydatid cyst that contained a floating laminated membrane (type CE3) to a cyst that contained daughter cysts (type CE2)within 1 year of follow-up. In the 20 surgically treated cases, two recurrent CE cases occurred within 4 years, and five cases had had residual cavities for more than 4 or 8 years. Of the 4 PAIR treated cases, two recurrent cases of CE occurred within 4 or 8 years of follow up. Serological follow-up data showed that specific IgG antibody levels were persistently positive in most CE cases, but exhibited a decreasing tendency in cases that were effectively treated by surgery; In contrast, serum IgG levels in the PAIR group or in the recurrent CE cases after surgery exhibited an increasing tendency. At least one persistent seropositive case with an isolated hepatic calcification was considered to be abortive CE. PMID- 16676418 TI - [Dispensing advice: glucocorticoids and antibiotics]. PMID- 16676419 TI - Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2. AB - A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text). PMID- 16676421 TI - "Pavlovian dissent" or honesty? PMID- 16676420 TI - [Nobel Prize in Medicine 2005. Helicobacter pylori--the versatile surviving artist ]. PMID- 16676422 TI - FDA approves tipranavir. PMID- 16676423 TI - "End denialism now!" 2nd South African AIDS Conference, June 7-10, 2005, Durban. PMID- 16676424 TI - Traditional healers and HIV care. PMID- 16676425 TI - Exploring antiretroviral interactions: still pioneer days. 6th International Workshop on Clinical Pharmacology of HIV Therapy, April 28-30, 2005, Quebec. PMID- 16676426 TI - Hepatitis C virus and metabolic disturbances. PMID- 16676427 TI - [Science and nation: romanticism and natural history in the works of E. J. da Silva Maia]. AB - The works of physician and naturalist Emilio Joaquim da Silva Maia (1808-59) can be viewed as a scientific project that discovers Brazil and its inhabitants. Maia's nationalism and his romantic view of nature formed the underpinnings of his scientific theories, especially his studies on zoological geography. He subordinated the issue of the biological specificity of different regions of the world to his era's debates on the construction of Brazil as an independent nation. In his interpretations of European natural history, Maia endeavored to understand Brazilian nature as a specific achievement of the Cosmos, in keeping with Alexander von Humboldt's approach. PMID- 16676428 TI - Decreased expression of inhibitory SMAD6 and SMAD7 in keloid scarring. AB - Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterised by an abnormal deposition of extracellular matrix components, in particular collagen. There is evidence that transforming growth factor-beta (TGFbeta) is involved in keloid formation. SMAD proteins play a crucial role in TGFbeta signaling and in terminating the TGFbeta signal by a negative feedback loop through SMAD6 and 7. It is unclear how TGFbeta signaling is connected to the pathogenesis of keloids. Therefore, we investigated the expression of SMAD mRNA and proteins in keloids, in normal skin and in normal scars. Dermal fibroblasts were obtained from punch-biopsies of keloids, normal scars and normal skin. Cells were stimulated with TGFbeta1 and the expression of SMAD2, 3, 4, 6 and 7 mRNA was analysed by real time RT-PCR. Protein expression was determined by Western blot analysis. Our data demonstrate a decreased mRNA expression of the inhibitory SMAD6 and 7 in keloid fibroblasts as compared to normal scar (p<0.01) and normal skin fibroblasts (p<0.05). SMAD3 mRNA was found to be lower in keloids (p<0.01) and in normal scar fibroblasts (p<0.001) compared to normal skin fibroblasts. Our data showed for the first time a decreased expression of the inhibitory SMAD6 and SMAD7 in keloid fibroblasts. This could explain why TGFbeta signaling is not terminated in keloids leading to overexpression of extracellularmatrix in keloids. These data support a possible role of SMAD6 and 7 in the pathogenesis of keloids. PMID- 16676429 TI - Epithelial cells and adipose cells both have their own temporal profile in 72-kd heat-shock protein expression determining their tolerance for ischaemia. AB - Seventy-two-kd heat-shock protein (HSP72) is one of the stress markers induced in cells under stress, such as in the case of ischaemia. Recent studies have suggested that HSP72 is a 'molecular chaperone' to protect cells from various kinds of stress, and that the temporal profile of HSP72 induction is related to ischaemic vulnerability. In this study, we attempted to analyse the temporal profiles of HSP72 induction in epithelial and adipose cells in skin flaps after various periods of transient ischaemia, and we investigated the reason why there were differences in ischaemic tolerance between these cells. We used the abdominal skin flap of Wister rats, which were divided into three groups: the sham control group (n=27), the 2-h ischaemia group (n=25), and the 8-h ischaemia group (n=25). At periods of 8, 24, 48, 96 h, and 7 days after reperfusion, we examined them for any histological changes and performed immunostaining for HSP72 (n=5, each time point). Two animals in the sham control group were sacrificed to harvest the samples immediately after the skin flaps were elevated. As a result, the epithelial cells in all groups revealed positive for HSP72 through the time course, regardless of the ischaemic stresses, and they were alive at 7 days. In the adipose cells, the cells in the sham control group revealed no immunoreactivity after the reperfusion, and they had no change at 7 days. In the 2-h ischaemia group, the adipose cells gradually increased the reactivity for HSP72; consequently they survived beyond 7 days. In the 8-h ischaemia group, the reactivity for HSP72 gradually decreased; consequently they played out a delayed cell death at 7 days. We concluded that these differences of HSP72 expression were related to the cellular vulnerability to ischaemia. PMID- 16676430 TI - Neuromuscular and neuromusculocutaneous flaps in the rat. AB - Since the introduction of flaps based on the vascular structures of the cutaneous nerves, these have gained increasing popularity in reconstructive surgery. The purpose of this study is to describe a new concept in which the flap is supplied solely by the intrinsic vasculature of a motor nerve. A total of 94 Wistar rats weighing 200-250 g were used in this experiment, which was divided into three sections. In section I, the neural anatomy of the posterior thigh region was investigated. In section II, the flap study using experimental and control groups, was performed. In the experimental group neuromuscular and neuromusculocutaneous flaps were created. The biceps femoris muscle was harvested based solely on its motor nerve as a neuromuscular flap, and together with its overlying skin it was similarly raised as a neuromusculocutaneous flap. In the control group, conventional muscle and musculocutaneous flaps were harvested based on the caudal femoral-popliteal artery vascular axis, and a graft subgroup was created ligating both the constant vascular structure and the motor nerve. In section III, with the intention of augmenting the survival areas of neuromuscular and neuroumusculocutaneous flaps, a surgical delay procedure was applied. On postoperative day 7, the viability of all flaps was evaluated using direct observation, microangiography, and tetrazolium blue stain techniques. The results of the anatomic studies demonstrated a consistent motor nerve arising from the sciatic nerve to the biceps femoris muscle with evident perineural vasculature. Average muscle viability levels of neuromuscular, neuromusculocutaneous, delayed neuromuscular, delayed neuromusculocutaneous, conventional muscle and musculocutaneous flaps were 20.6 +/-7.58, 22.4 +/- 4.21, 86.4 +/- 6.14, 85 +/- 4.21, 89.6 +/- 4.48, and 88.0 +/- 5.51%, respectively. Survival levels of the skin paddles of the neuromusculocutaneous, delayed neuromusculocutaneous, and conventional musculocutaneous flaps were calculated as 13 +/- 17.51, 67 +/- 30.29, and 97+/-4.21%, respectively. In the graft subgroup the viability of muscle and skin paddle was almost nil. In conclusion, our new flap model in a favored laboratory animal is of benefit to researchers in providing a means for future various types of investigations into this new concept. The technique might be considered in further experimental research studies and appropriate clinical situations. PMID- 16676431 TI - An ameliorated skin flap model in rats for experimental research. AB - There is a disagreement in the experimental design of random skin flaps owing to their vascular inconsistency. The definition of a reliable axial-pattern skin flap model is needed. The purpose of this study was to describe a new skin flap model to deal with entire drawbacks of existing random and axial pattern skin flap designs. This was accomplished by creating paired skin flaps including both skin and vascular pedicle on the dorsum of the same rat. This design was suitably termed as rando-axial flap. The present study offers a simple and reliable skin flap model with following advantages: (1) it has a predictable necrosis area, (2) it reveals a larger survival area (75 +/- 5%) when compared to other flaps in this study (Mann-Whitney U-test, p<0.001), (3) the vascular pedicle is consistent, (4) control and study flaps are placed on the same animal (5) it can be converted to a random, an axial or a free flap. PMID- 16676432 TI - [Coxitis fugax--the beginning of Perthes' disease?]. PMID- 16676433 TI - Dracunculiasis eradication. PMID- 16676435 TI - [The 76th annual meeting of the Japanese Society for Hygiene. Yamaguchi, Japan. March 25-28, 2006. Abstracts]. PMID- 16676434 TI - Human avian influenza in Azerbaijan, February-March 2006. PMID- 16676436 TI - The 53rd annual meeting of the Japanese Association for Laboratory Animal Science (JALAS). Kobe, Japan. May 11-13, 2006. Abstracts. PMID- 16676437 TI - Ionizing radiation inhibits tumor neovascularization by inducing ineffective angiogenesis. AB - The vascular effects of ionizing radiation were examined in K1735 murine melanoma tumors. Single-fraction and fractionated radiation virtually arrested growth of these tumors for about a week, after which they resumed more rapid growth. Tumor microvessel density (MVD) and blood perfusion was unchanged seven days after radiation but decreased at later time points after irradiation, when they had grown 10-fold or more. Together with the finding of severe tumor hypoxia and VEGF induction in the latter tumors, the evidence pointed to vascular insufficiency and inhibited neovascularization in tumors that had grown substantially after radiation. Endothelial cell (EC) death detected by TUNEL staining only transiently increased the day following radiation, whereas EC proliferation detected by Ki-67 staining was increased in irradiated tumors that had grown substantially. The fact that increased EC proliferative activity produced fewer vessels suggests that angiogenesis is defective or ineffective after radiation. These results complement recent genetic evidence that EC damage from radiation plays a major role in tissue damage and antitumor efficacy to highlight the importance of EC and vasculature in radiation response. Our studies further show that radiation impact on tumor vasculature extends beyond near-term induction of EC death to more prolonged effects on their ability to support angiogenesis. PMID- 16676438 TI - Fabry disease. PMID- 16676439 TI - The black sheep of the Mobitz family. PMID- 16676440 TI - Everyday hazmat risks. The top 10 hazardous chemical every provider needs to understand. PMID- 16676441 TI - A case of fetal anticonvulsant syndrome with severe bilateral upper limb defect. AB - Women with epilepsy have a higher risk of having dysmorphic child. We report on a child exposed prenatally to valproate and carbamazepine presenting with severe bilateral upper limb defect and phenotypic features of fetal valproate syndrome. Anticonvulsant drugs can cause severe birth defects, especially when used in combination. PMID- 16676442 TI - [The ethics of the medical profession and its professionalization (1927-57): a repertory of sources in the constitution of medical associations]. PMID- 16676443 TI - Musculoskeletal injury prevention. Protect you personnel from on-the-job injury. PMID- 16676444 TI - [Haity Moussatche: a homage to the crusader of Brazilian science]. PMID- 16676445 TI - Under pressure. Responding to scuba emergencies. PMID- 16676447 TI - [The frontiers of 'abnormality': psychiatry and social control]. AB - The article examines some of the main aspects governing psychiatry's role in the Brazilian political and social context at the close of the nineteenth century and beginning of the twentieth. It analyzes certain themes - civilization, race, labor, fanaticism, political dissent, sexuality - that were emphasized by specialists in their construction of a very broad notion of 'mental illness'. Through the analysis of texts produced by psychiatrists and legal experts (including dissertations written at the Faculdade de Medicina do Rio de Janeiro, reports from the Servico de Assistencia a Alienados, and works and articles by specialists), the relation between the psychiatric definition of the frontiers of 'abnormality' and efforts to implement new strategies of social control is discussed. PMID- 16676448 TI - [The values and institutional practice of science: Robert Merton's and Thomas Kuhn's conceptions]. AB - While Robert Merton approached the issue form the perspective of "scientific ethos" and Thomas Kuhn, from the perspective of "paradigm/normal science," this comparative analysis of these two author's conceptions of the social nature of science suggests that their views merged particularly when they argued that taking values into account was essential to understanding scientific activities. Placing prime importance on the notion of scientific community, these authors both analyze science as a practice which is defined and developed out of a set of beliefs, principles, and norms shared by a given collectivity. While there were some substantive differences between Merton's and Kuhn's outlooks - each had his own unique way of defining 'social' in the context of science - a comparison of their work underlines the importance of seeing institutionalized beliefs and values as essential in guiding the concrete actions of scientists. PMID- 16676449 TI - House sends deficit-reduction bill to President. PMID- 16676450 TI - Federal government prepares for avian flu epidemic. PMID- 16676451 TI - [Positivism and medical science in Rio Grande do Sul: the Faculdade de Medicina de Porto Alegre]. AB - The article analyzes conflicts and interests at one of Rio Grande de Sul's main centers for medical science, the Faculdade de Medicina de Porto Alegre. It explores the meaning and impact of the emergence of this specific, exclusive field of knowledge in a state where positivist principles of professional freedom were adopted by successive administrations during the early period of the Republic. Physicians there launched an entrenched war to uphold the principles of science over faith and politics, challenging the positivism of the party which held power in Rio Grande do Sul throughout the years. This perspective grew and developed in a climate of conflict and doubts among physicians, within a political context that differed from the rest of Brazil. PMID- 16676453 TI - [The Rockefeller Foundation and the construction of a professional identity in nursing during Brazil's first Republic]. AB - This article deals with the institution of professional nursing in Brazil during the First Republic, based on a study of documents left by the US nursing mission recruited during the 1920s by the Rockefeller Foundation, in association with the National Public Health Department. Certain excerpts from these documents are representative of main events and references within the field and can be considered emblematic of the construction of a professional identity. It is concluded that Brazilian nurses defined their identity around three basic lines: gender, race, and social origin. PMID- 16676452 TI - [Fighting Nazis and mosquitoes: US military men in Northeastern Brazil (1941 1945)]. AB - Malaria-control policies enforced at US military bases set up in Brazil during World War II serve as an example in this analysis of the associations between disease and war. Case studies of the three main US bases (located in Belem, Recife and Natal) show how malaria can be transformed into a matter of political military security. An examination of the Natal case allows the reader to perceive the prejudices, political mistrust, and nationalistic tensions marking relations between the Brazilians and Americans. PMID- 16676454 TI - [Representing and intervening in public health: viruses, mosquitoes and Rockefeller Foundation experts in Brazil]. AB - The attempts by experts from the of the Rockefeller Foundation (RF) to eliminate yellow fever in Brazil were hampered by the pathology's low visibility. Most cases of yellow fever were atypical and easily confused with other fevers. In the 1920s, the RF experts who tried to assess the presence of yellow fever relied mainly on clinical observations. In the 1930s, however, they devised indirect methods of visualizing the presence of the disease agent. Visceroctomy revealed the presence of acute cases of the disease. Mouse protection tests revealed past contacts with the agent. Taken together, these tests enabled the RF specialists to construct maps which indicated zones where the disease was endemic and to target specific anti-yellow fever campaigns based on selective elimination of the yellow fever vector, that is, the mosquito Aedes aegypti. In public health, like in the sciences, representation practices shape intervention. PMID- 16676455 TI - [Images of health, illness, and development: Latin-America's Rockefeller Foundation's photographs]. PMID- 16676456 TI - An American in the tropics. PMID- 16676458 TI - The past and the future of research in the history of science: medicine and technology at the Rockefeller Archive Center. PMID- 16676459 TI - [Yellow fever in Brazil: memories of a medical doctor from the Rockefeller Foundation]. PMID- 16676460 TI - Taking care of your health. PMID- 16676461 TI - [The sciences and education in museums at the close of the nineteenth century]. AB - In the closing decades of the nineteenth century, natural history museums established solid communication networks, and their different audiences formed what became known as the 'museum movement'. It was within this context of exchange that William H. Flower made his 1889 speech on the roles natural history museums should play. The article analyzes his influence on Argentina's Museo da La Plata, a member of this then-expanding circuit of museums. PMID- 16676462 TI - [The personal context of a museum experience: similarities and differences between science and art museums]. AB - The article focuses on the development of research that illuminates not only the socioeconomic profiles of museum visitors and non-visitors but also their cultural habits, general entertainment interests, and their perceptions of art, science, and other topics addressed at these institutes. The more we know about a visitor's personal context, the more we can enhance his or her museum experience, thereby encouraging further museum visits during which his or her expectations, wishes, and needs will be more fully met. The article also focuses on how local culture plays an important part in shaping both personal context as well as each museum experience. Some examples are provided from the literature, above all concerning studies in Brazil and the contributions that research at art museums may have for science museums. PMID- 16676464 TI - [Science museums and psychology: interactivity, experimentation, and context]. AB - The article reflects on the notion of the museum experience from the perspective of a visitor to a science and technology museum. Unlike studies that postulate a generic, abstract 'model visitor', the goal was to discuss the perspectives of the visitor as a psychological being, and to this end the research relied on the notion of interactivity. Using two classic psychology studies analyzing the behavior of children and adolescents, the current study first focused on the notion of experimentation, characterized as an interaction between subject and object. It then explored interactions between subjects and contexts, approaching from the notion of mediated action. My conclusion is that a museum experience should, on the one hand, take into account the visitor's ability to act, ask, and experiment and, on the other, the specific museum contexts that invite and propose but may also limit these very chances to act, question, and experiment. PMID- 16676466 TI - Cross-validated bagged prediction of survival. AB - In this article, we show how to apply our previously proposed Deletion/Substitution/Addition algorithm in the context of right-censoring for the prediction of survival. Furthermore, we introduce how to incorporate bagging into the algorithm to obtain a cross-validated bagged estimator. The method is used for predicting the survival time of patients with diffuse large B-cell lymphoma based on gene expression variables. PMID- 16676465 TI - [The stimulated recall method: a research tool applicable to learning at science museums]. AB - The article reports on preliminary research aimed at developing a methological tool applicable to studies of learning in informal educational settings. The study was conducted at two science museums, using a modified version of the stimulated recall method. Digital photographs replaced the traditional video in the final phase, during which eight interviews were conducted, with satisfactory results. The digital photos efficaciously and efficiently stimulated visitors to narrate their experiences and to convey the meanings they took with them from their interactions with the exhibits. PMID- 16676467 TI - Learning science from museums. AB - This article provides an overview of current understandings of the science learning that occurs as a consequence of visiting a free-choice learning setting like a science museum. The best available evidence indicates that if you want to understand learning at the level of individuals within the real world, learning does functionally differ depending upon the conditions, i.e., the context, under which it occurs. Hence, learning in museums is different than learning in any other setting. The contextual model of learning provides a way to organize the myriad specifics and details that give richness and authenticity to the museum learning process while still allowing a holistic picture of visitor learning. The results of a recent research investigation are used to show how this model elucidates the complex nature of science learning from museums. This study demonstrates that learning form museums can be meaningfully analyzed and described. The article concludes by stating that only by appreciating and accounting for the full complexities of the museum experience will a useful understanding of how and what visitors learn from science museums emerge. PMID- 16676468 TI - Lessons without limit: how free-choice learning is transforming science and technology education. AB - Societies are becoming nations of lifelong learners supported by a vast infrastructure of learning organizations. The centers of this learning revolution are not schools, but a network of organizations and media (museums, libraries, television, books, and increasingly the Internet) supporting the public's ever growing demand for free-choice learning - learning guided by a person's needs and interests. Science learning is an important part of this revolution. Traditional boundaries and roles distinguishing groups of science educators and institutions are disappearing. To not understand and embrace these changes will impede our ability to enhance science learning worldwide. PMID- 16676469 TI - [Educational research and the production of knowledge at science museums]. AB - The article examines the processes by which scientific knowledge is socialized, based on the concepts of museographical/didactical transposition and recontextualization within the realm of science museums. Ways of producing knowledge in museums are analyzed, alongside a discussion of the potential challenges and possibilities involved when applying these concepts to research on dissemination and education in a museum setting. Some considerations are advanced on how these may contribute to the development of pedagogical practice in museums. The processes by which scientific knowledge is transformed for the purpose of educating and disseminating knowledge do not merely simplify ideas and concepts, since new knowledge is constructed inside the realm of museum culture. PMID- 16676470 TI - [Museums, science, and education: new challenges]. AB - The article discusses how the social role of science museums is shaped by scientific and technological endeavor, society's demands, and educational issues, above all in negotiations with a museum's audiences. The text also analyzes the trajectory taken by Brazil's science museums in their process of consolidation and the changes current society has imposed on these institutes. Communication has become the center of the discussion on museum culture, particularly in that it adjusts the educational aspect according to the conception of social practices, which are deemed fundamental resources. Lastly, the article examines the incorporation of the ideas of 'risk' and 'uncertainty', produced by science, into this new way of thinking about museums, which values the public and the communication processes. PMID- 16676471 TI - [Collectors of historical, archeological, and natural science objects at municipal museums in Buenos Aires province, Argentina, during the 1950s]. AB - Through reference to the creation and expansion of municipal museums in the province of Buenos Aires during the 1950s, the article explores some aspects of how archeological collections are compiled. The collections under study came from private hands, having been gathered by collectors who relinquished them so these museums could be formed. At the same time that these collections became public, the collectors themselves became responsible for them in the role of directors of the new institutes. Within this context, the collectors established institutional relations that allowed them to devise common strategies concerning the receipt, selection, and exhibition of archeological collections. The result was the shaping of a network of solidarity in the southern part of Buenos Aires province. PMID- 16676472 TI - [The passing eye: museums, public education, and the visualization of scientific evidence]. AB - In this examination of certain issues related to the history of museums. An emphasis on the functions and missions entrusted to museums does not necessarily reflect the power museums have to create habits or shape meanings. To the contrary, this may reflect the actual fragility of museums and their need to resort to rhetoric to attract governmental favors and funds. Therefore, concentrating on the monumental, representative, or metaphorical aspects of museums obscures the history of these institutions and of their consolidated practices, there by naturalizing the separation between research space and public space and leaving the historian to play the role of an uninitiated observer of the 'cathedrals of science'. PMID- 16676473 TI - Science, society and science centres. AB - The present article analyzes the relation between technological changes and the advance of science, as well as the attitude of individuals in relation to science nowadays. According to researches carried out by the National Science Foundation in the United States, a large number of Americans and Europeans accept the benefits of science and technology, but have complex attitudes in relation to this topic. Contemporary cultures display this contradiction in many different ways. An evidence of this ambiguity is the prevailing image of scientists in popular culture: that of the mad scientist. Interpreting science and technology to a large and diversified audience is the main mission for most science centres. The changes that take place in society demand new approaches in the relationship with visitors. The present article analyzes the economic and intellectual sustainability of science centres and the continuously reinvented ways through which each one of them tires to shape its relationship with the community and with its identity as a science centre in the twenty-first century. PMID- 16676474 TI - The science centre movement in India: a conspectus. AB - The present article is about the development of the science museum net in India started in 1956, when the government of that country created the Industry and Technology Museum in Calcutta. In the 1960's and 1970's, due to the need of simple programs for rural communities and small villages, the idea of Mobile Science Exhibits (MSE) started. In order to take universal scientific concepts to those who could not visit museums, the Museobus was projected. At that time, the educational focus in museums changes from exhibiting artifacts to encouraging learning through "doing". The Exploratorium in San Francisco influenced the approach of museums in India. While the first Science Centre was built in Mumbai, the Planning COmmission of the Indian government created a task force to study the development of Sciences Museums. In 1978, the National Council of Science Museums (NCSM) was created as an independent institution, which later became part of the Ministry of Education and Social Welfare. Twenty-five years after the creation of the Council, the movement for informal science teaching had acquired a solid background. In 1978, their priority was to expand the net of science Centres and Museums. Now, the priority is to achieve better quality and more efficient communication. Nowadays, Indian Science Centres evaluate the impact their activities have on individual, social and economic reality. With new technologies and approaches, they try to emphasize their relevance in a society that is characterized by having a great number of languages and poor education in science. PMID- 16676476 TI - DUR by pharmacists--lessons learned for MTMS. PMID- 16676475 TI - Prospective memory in HIV-1 infection. AB - The cognitive deficits associated with HIV-1 infection are thought to primarily reflect neuropathophysiology within the fronto-striato-thalamo-cortical circuits. Prospective memory (ProM) is a cognitive function that is largely dependent on prefronto-striatal circuits, but has not previously been examined in an HIV-1 sample. A form of episodic memory, ProM involves the complex processes of forming, monitoring, and executing future intentions vis-a-vis ongoing distractions. The current study examined ProM in 42 participants with HIV-1 infection and 29 demographically similar seronegative healthy comparison (HC) subjects. The HIV-1 sample demonstrated deficits in time- and event-based ProM, as well as more frequent 24-hour delay ProM failures and task substitution errors relative to the HC group. In contrast, there were no significant differences in recognition performance, indicating that the HIV-1 group was able to accurately retain and recognize the ProM intention when retrieval demands were minimized. Secondary analyses revealed that ProM performance correlated with validated clinical measures of executive functions, episodic memory (free recall), and verbal working memory, but not with tests of semantic memory, retention, or recognition discrimination. Taken together, these findings indicate that HIV-1 infection is associated with ProM impairment that is primarily driven by a breakdown in the strategic (i.e., executive) aspects of retrieving future intentions, which is consistent with a prefronto-striatal circuit neuropathogenesis. PMID- 16676478 TI - Student researcher on the right track. PMID- 16676477 TI - Sub-par instructors & evasive P waves. PMID- 16676479 TI - The truth surfaces. PMID- 16676480 TI - Bibliography. Current world literature. Neuropsychiatry. PMID- 16676481 TI - Affordable health care: no easy solution. PMID- 16676482 TI - Thoughts on affordable health care. PMID- 16676483 TI - Clinical questions #7. Systemic corticosteroid dosing for acute asthma: is higher better? PMID- 16676484 TI - Improving patient safety through electronic medical records. PMID- 16676485 TI - Medical informatics in population health: building Wisconsin's strategic framework for health information technology. AB - Medicine is increasingly practiced through the application of information sciences. Medical informatics deals with optimal information use within bioinformatics, imaging, clinical, and population health domains. Population health informatics plays an important role in that it critically informs practice in each of the other domains. Proper functioning of health care systems requires an advanced health information network that supports clinical care, personal health management, population health, and research. But this infrastructure does not yet exist in the United States. A number of federal initiatives are underway to address this problem, including the development of a framework for a national health information network and funding for implementation. This network will be facilitated by federal leadership, but public and private partnerships, and state, regional, and local implementation and policy development will play a critical role. In this article, we describe several Wisconsin initiatives that are keys to developing a strategic framework and building the state's electronic health information infrastructure. PMID- 16676486 TI - Pneumonia, mortality, and vaccines: piecing together the puzzle. PMID- 16676487 TI - Community-associated methicillin-resistant Staphylococcus aureus: it's time to pay attention. PMID- 16676488 TI - Hepatitis C virus infection: an overview of Wisconsin's public health resources. AB - Hepatitis C virus (HCV) infection is the most common chronic bloodborne infection in the United States, with an estimated 3.9 million infected persons nationally and 85,000 persons infected in Wisconsin. HCV infection results in significant morbidity and mortality and contributes to high health care costs associated with specialized medical care, medications, and hospitalization. This report is an overview of HCV-related resources for consumers and clinicians caring for persons with HCV infection in Wisconsin. PMID- 16676489 TI - Epidemiologic trends in infection, mortality, and transplants related to hepatitis C in Wisconsin. AB - Hepatitis C virus (HCV) infection is the most common chronic bloodborne infection in the United States. An estimated 3.9 million persons nationally and 85,000 persons in Wisconsin are currently infected. The disease is responsible for approximately 8000 to 10,000 deaths nationally each year. This article summarizes epidemiologic trends in infection, mortality and transplants related to HCV in Wisconsin. It presents surveillance data collected during 1997-2004; HCV-related deaths during 1995-2002, with HCV as an underlying or contributing cause of death; and data for liver transplants related to HCV between 1993 and 2004. During the time periods reviewed, there were 16,668 cases of HCV infection reported, 1186 HCV-related deaths, and 356 HCV-related liver transplants involving HCV in Wisconsin. Infection rates and related adverse health outcomes related to HCV are highest in males, persons ages 35-64, Milwaukee residents, and inmates in the state correctional system. African Americans have high rates of morbidity (24% of cases) and mortality (16% of decedents), but are under represented among recipients of HCV-related transplants (6% of recipients). PMID- 16676490 TI - Wisconsin trends in pneumonia and influenza mortality, 1980-2003. AB - INTRODUCTION: Mortality due to pneumonia and influenza continues to be a serious public health threat, especially among those aged > or = 65. Continued monitoring of these high-risk populations is necessary for evaluating the impact of public health prevention activities, determining vaccine distribution policies, and ensuring that existing guidelines reflect the populations at risk. OBJECTIVES: We characterized pneumonia and influenza mortality in Wisconsin from 1980 to 2003, including trend analysis, identification of high risk populations, and assessment of Wisconsin's progress toward state and national goals for vaccination. METHODS: We examined mortality trends for pneumonia and influenza as underlying causes of death among all Wisconsin residents who died in the state from 1980 to 2003. RESULTS: The pneumonia and influenza (P&I) mortality rate increased from 27/100,000 to 38/100,000 during 1980 through 1988, and then decreased to 26/100,000 through 2003. The decline in the mortality rate after 1988 was temporally associated with improving pneumococcal and influenza vaccination among those > or = 65. By 2003, all age groups except those aged > or = 85 had lower P&I mortality than in 1980. CONCLUSIONS: In Wisconsin, the increase in pneumonia and influenza mortality demonstrated during the 1980s was reversed. However, there was relatively little change in mortality among those aged 65-84 and rates among those > or = 85 years have increased. Novel improvements in public health interventions are needed to improve the focus on the elderly, including efforts to increase vaccination, prevent pneumococcal disease, and explore other evidence based strategies to reduce pneumonia and influenza mortality. PMID- 16676491 TI - How should we approach adolescent and adult pertussis? AB - Previously considered a disease of childhood, pertussis is now also recognized as a significant problem for adolescents and adults; however, diagnosing pertussis remains problematic due to its nonspecific clinical presentation and the time delay, sensitivity/specificity, and expense of testing. To be effective, therapy is best started very early in the illness, when the illness is seldom recognized. Other than chemoprophylaxis in families with a non-immune infant, antibiotic therapy is controversial due to the ubiquitous nature of pertussis, its similarity to other respiratory infections, increased prevalence, prolonged outbreaks, and difficulties in determining true exposures in the general community. If antimicrobial therapy is used extensively for whooping cough prevention, drug reactions and increased bacterial resistance are expected. Likewise, without laboratory confirmation of infection, isolation of individuals is difficult and expensive. Fortunately, 2 new Food and Drug Administration approved vaccines, 1 for adolescents and 1 for adolescents/adults, are now available. Both have been shown to be safe and to produce protective antibody responses. As vaccination of adolescent and adult groups is expanded, the rising incidence of pertussis in all age groups can be curtailed. PMID- 16676492 TI - Community-associated methicillin-resistant Staphylococcus aureus: review of an emerging public health concern. AB - Methicillin-resistant Staphylococcus aureus (MRSA) traditionally has been recognized as a virulent pathogen affiliated with health care institutions. However, community-associated strains of MRSA (CA-MRSA) have emerged over the past several years in young, healthy patients without significant health care contact. These isolates carry a distinct molecular makeup and lack the multidrug resistance pattern harbored by health care strains. CA-MRSA predominantly induces skin and soft tissue infections, though the presence of unique virulence factors may cause potentially lethal necrotizing pneumonia and other invasive infections. In response to this growing public health concern, clinicians must learn to identify risk factors for CA-MRSA, treat infections with judicious use of antimicrobial agents, and facilitate prevention strategies to limit transmission. PMID- 16676493 TI - A young man who could not walk. AB - Infections affecting the central nervous system caused by Blastomyces dermatitidis are rare but curable. We describe a case of a 24-year-old man who presented to the emergency department with progressive bilateral lower extremity weakness over 1 month. On the day of admission, he had minimal muscle strength and was hyperreflexic in the lower extremities. Sensation, however, was intact. Skin examination revealed annular, raised, crusted lesions on his face and legs. A magnetic resonance imaging (MRI) scan showed marrow replacement of the T7 and T8 vertebral bodies and an epidural mass with cord compression. A chest radiograph showed an infiltrate, and a subsequent needle biopsy revealed yeast resembling B. dermatitidis. A skin biopsy was then obtained, and the culture grew out B. dermatitidis. He received 4 weeks of amphotericin B lipid complex (total of 6 grams), followed by oral itraconazole. After 1 week on antifungals, he was able to walk with a walker and the skin lesions virtually resolved. At 5 months he was ambulatory and riding a bicycle daily. Blastomycosis should be included in the differential diagnosis of epidural masses. PMID- 16676494 TI - Pneumocystis carinii pneumonia with pleural effusion in a non-HIV host. AB - Pneumocystis carinii pneumonia (PCP) is a life-threatening opportunistic infection that occurs in immunocompromised hosts, especially patients with the acquired immunodeficiency syndrome (AIDS). However, this infection is increasing in frequency in other immunosuppressed patients, including organ transplant recipients and those with malignancy who are treated with chemotherapeutic regimens. It carries a relatively high mortality in the non-human immunodeficiency virus (HIV) population. Pleural involvement is rare with PCP; all reported cases in the literature are associated with HIV disease and characterized as small effusions. We report a case of a renal transplant recipient with PCP and moderate-sized pleural effusion with pneumocystis cysts. PMID- 16676495 TI - HIV/AIDS interventions and research have significance at home and abroad. PMID- 16676496 TI - Considering the Roth 401(k) plan: what you need to know. PMID- 16676497 TI - The suppression of the bubonic plague. 1906. PMID- 16676498 TI - Chief execs should 'take the rap' if elderly failed on dignity. PMID- 16676499 TI - Reform. 'Not a crisis but an opportunity'. PMID- 16676501 TI - IT programme. Supplier and demands: where NHS IT plans aren't working. PMID- 16676500 TI - Workforce. Trusts fall behind on age discrimination planning. PMID- 16676502 TI - On leading the NHS reforms. PMID- 16676503 TI - Barometer. Acute trusts March 2006. PMID- 16676504 TI - Mental health. 'Death by a thousand pin pricks'. AB - People with mental health problems are subject to a variety of prejudices and inequalities affecting everything from promotion opportunities to social lives. Awareness campaigns in England have been hampered by a lack of funding while better-funded initiatives in Scotland and New Zealand have enjoyed wider success. Tackling stigma will only be effective if backed by legislation. PMID- 16676505 TI - On happiness. PMID- 16676506 TI - Staff survey. The human league. PMID- 16676507 TI - Workforce redesign. Baby boomers. PMID- 16676508 TI - Workforce. Ship shape. PMID- 16676509 TI - [Neurological symptoms as the first sign of prostate carcinoma]. AB - Three male patients aged 82, 56 and 60 years presented with cognitive impairment and hemiparesis, weakness of the tongue and facial muscles, and pain and weakness of the left arm, respectively. They were found to have carcinoma of the prostate with cerebral, skull and cervical spine metastases. They were treated with hormonal therapy and local radiotherapy for bone metastases. The first patient died within 2 weeks, the second after 1.5 year, and the third was still alive after 6 years. The diagnostic work-up in men with unexplained neurological symptoms should probably include a rectal exam and assessment of prostate specific antigen. PMID- 16676510 TI - [The diagnosis of pulmonary embolism based on clinical probability: D-dimer test and spiral CT]]. AB - Recently, an algorithm encompassing a dichotomized clinical score, D-dimer and helical CT has proved to be a valid tool in confirming or rejecting a clinical diagnosis of pulmonary embolism. The diagnosis can be rejected in the context of low clinical suspicion and a negative D-dimer test. In all other situations the diagnostic accuracy of helical CT for the diagnosis of pulmonary embolism compares favourably with pulmonary angiography, with regard to the occurrence of recurrent venous thromboembolism or fatal pulmonary embolism if anticoagulation is withheld in cases of negative findings. For final diagnosis of pulmonary embolism, pulmonary angiography is only necessary if CT-scan results are suboptimal or inconclusive. This new algorithm may increase the compliance in general practice of applying guideline recommendations in patients in whom thromboembolic disease is suspected. However the proposed clinical score must first be validated for use in general practice. PMID- 16676511 TI - [Treatment of patients with diabetes mellitus by means of inhaled insulin]. AB - Good glycaemic control of diabetes mellitus is still hampered by the fear of insulin injections. Particularly in patients with type 2 diabetes, inhaled insulin as a novel therapeutic option for glycaemic control could be an alternative to subcutaneous insulin. Phase III clinical studies have shown glycaemic equivalence between inhaled insulin and conventional subcutaneous insulin. However, no study comparing inhaled insulin with short-acting insulin analogues has yet been published. Thus, methodological problems preclude conclusive remarks concerning quality-of-life issues. Inhaled insulin should be reserved for selected patient groups only. Lengthier studies to evaluate the long term (pulmonary) safety of inhaled insulin and a cost-effectiveness study are needed. PMID- 16676512 TI - [Inhaled insulin: a new route in the optimisation of glucose regulation]. AB - The lungs constitute an attractive route for the administration of insulin in view of their large surface area and the permeability for proteins. Inhaled insulin may greatly reduce the daily nuisance of subcutaneous injections for millions of patients. The acceptance of insulin therapy may therefore be increased, so that better metabolic control can be achieved. For optimal efficacy it is essential that the insulin be precipitated in the alveoli and it is precisely the reproducibility of this process that is variable and dependent upon a variety of factors. Thus, the properties of the particles, technique of respiration, administration system and the presence of pulmonary disease (smoking) all play a role. The pharmacokinetics make inhaled insulin suitable for preprandial administration: the absorption is very rapid, so that it can be taken a very short time before meals. The duration of action, however, is short, so that the use of long-acting insulin usually remains necessary. The biological availability of inhaled insulin is < 20%, so that the required dose is larger. The long-term effects of the intra-alveolar administration of insulin and its immunological consequences are still insufficiently clear, although no permanent negative effects have been demonstrated so far. Partly in view of the favourable pharmacodynamic properties, the use of inhaled insulin is an attractive therapeutic option, not only for the group of patients with a fear of injections but for all diabetics that require insulin therapy. PMID- 16676513 TI - [The practice guideline 'Diagnosis and pharmaceutical treatment of dementia']. AB - The practice guideline 'Diagnosis and pharmaceutical treatment of dementia' emphasizes that a nosological diagnosis should be made and that it is important to assess the extent of need for care. The guideline recommends the use of diagnostic criteria for the various conditions that can cause dementia. With respect to ancillary investigations, the burden to the patient should be weighed against the benefits of increasing diagnostic confidence. Observation of the course of the disease, laboratory and cerebrospinal-fluid investigations, neuropsychological and EEG examinations, and neuroimaging all increase diagnostic confidence. Treatment with a cholinesterase inhibitor or memantine should always be embedded in a comprehensive-treatment protocol that includes explicit discussion of treatment goals and expectations at baseline, in combination with criteria for starting and stopping treatment. Guidelines for evaluating the effects of treatment with cholinesterase inhibitors or memantine are specified. If psychosis, depression or behavioural disturbances occur in patients with dementia, antidepressants, antipsychotics or anticonvulsants may be indicated. PMID- 16676514 TI - [Diagnostic image (270). A confused and restless woman]. AB - A 63-year-old woman suffering from confusion, restlessness, vertigo and nausea had multiple old cortical and subcortical microhaemorrhages on MR gradient echo imaging, consistent with amyloid angiopathy. PMID- 16676515 TI - [Right-ventricular dysfunction and the pulmonary vascular obstruction index: predictable variables of the clinical course over 3 months in patients with acute lung emboli]. AB - OBJECTIVE: To quantify right-ventricular dysfunction and the pulmonary artery obstruction index, in patients with acute pulmonary embolism, using helical CT, and to assess the prognostic value of these parameters. DESIGN: Prospective. METHOD: In 120 consecutive patients with proven acute pulmonary embolism, the extent of right-ventricular dysfunction was assessed by quantifying the ratios of the right to left-ventricular short-axis diameters (RV/LV ratio) and the extent ofobstruction ofthe pulmonary-artery circulation by using helical CT images. Regression analysis was used to correlate these parameters with patient outcome. RESULTS: Right-ventricular dysfunction (RV/LV ratio > 1.0) was seen in 69 patients (57.5%). Seven patients died as a direct result of pulmonary embolism. Both the RV/LV ratio and the obstruction index were significant risk factors for mortality within three months (p = 0.04 and 0.01 respectively). The positive predictive value for pulmonary embolism-related mortality of an RV/LV ratio > 1.0 was 10.1% (95% CI: 2.9-17.4). The negative predictive value for an uneventful outcome of an RV/LV ratio < or = 1.0 was 100% (95% CI: 94.3-100). There was a 11.2-fold risk of dying of pulmonary embolism in patients with an obstruction index > or = 40% (95% CI: 1.3-93.6). CONCLUSION: Markers of right-ventricular dysfunction and pulmonary vascular obstruction, assessed by helical CT examination at baseline, help to predict mortality during follow-up of patients with acute pulmonary embolism. PMID- 16676516 TI - [The first 100 hand-assisted laparoscopic donor nephrectomies at the Academic Medical Center in Amsterdam]. AB - OBJECTIVE: To evaluate the results of the first 100 hand-assisted donor nephrectomies in our hospital. DESIGN: Prospective, descriptive. METHOD: In the period January 2000-July 2004, 100 consecutive donors underwent laparoscopic nephrectomy as part of a living-related kidney transplantation at the Academic Medical Center in Amsterdam, the Netherlands. The operation was performed laparoscopically using a hand port (hand-assisted nephrectomy). The incision made for the hand port was also used for the removal of the kidney. RESULTS: Mean operating time was 168 min (range: 88-285). Median warm ischaemia time was 3 min (1.0-4.5) and median blood loss was 50 ml (20-1500). None of the operations required conversion to an open procedure and there were no deaths among donors or recipients. Postoperatively, 12 complications occurred in 10 donors. Median hospital stay for the donors was 5 days. In 3 recipients, the transplanted kidney had to be removed within 14 days. 1n 3 other recipients, surgical re-intervention was necessary due to urological complications. One-year graft survival was 94%. CONCLUSION: Hand-assisted laparoscopic donor nephrectomy was a safe and minimally invasive procedure for renal transplantation in this patient series. PMID- 16676517 TI - [Neonatal respiratory distress caused by primary ciliary dyskinesia]. AB - Two newborns, both boys, presented with unexplained respiratory distress. One developed recurrent pneumonias in the first neonatal week and was diagnosed with primary ciliary dyskinesia at the age of 2.5 years. The other had respiratory problems besides a situs inversus totalis and was diagnosed with primary ciliary dyskinesia in the neonatal period. Although 65-90% of children with primary ciliary dyskinesia present with neonatal respiratory distress, the disease is often diagnosed after a considerable delay. Primary ciliary dyskinesia should be considered in newborns with unexplained respiratory problems and in children with recurrent respiratory problems. The disease is diagnosed by taking a nasal brush biopsy of the cilia and examining it using electron microscopy or using phase contrast microscopy. Early diagnosis and adequate treatment may prevent further lung damage. PMID- 16676518 TI - [Thrombosis of a coronary stent after discontinuing treatment with clopidogrel]. AB - A drug-eluting stent was implanted in four patients, a man aged 67 and a woman aged 42 with acute myocardial infarction, a woman aged 41 with unstable angina pectoris and a man aged 41 with stable angina pectoris. All suffered stent thrombosis after discontinuation (in three cases prematurely) of clopidogrel therapy. Reasons for discontinuation included allergic reaction, a dental procedure and refusal of reimbursement by the insurer. In order to restore stent patency they were treated by percutaneous coronary intervention and all patients suffered irreversible myocardial damage. Combination therapy using acetylsalicylic acid and clopidogrel during and after angioplasty for the prevention of long- and short-term complications is necessary. Stent thrombosis after drug-eluting stent implantation usually occurs within 1-4 weeks following discontinuation ofantiplatelet medication. These cases stress the importance of antiplatelet therapy after stent implantation. Physicians, dentists and patients must be aware of the risk of the early discontinuation ofantiplatelet therapy. PMID- 16676519 TI - [Psychiatric disorders in pregnant and puerperal women]. PMID- 16676520 TI - [Oesophageal endoscopic ultrasound with fine-needle aspiration biopsy in the staging of non-small-cell lung carcinoma; results from 43 patients]. PMID- 16676521 TI - [Teriparatide: an anabolic drug for the treatment of patients with osteoporosis]. PMID- 16676522 TI - Bromeliad-living spiders improve host plant nutrition and growth. AB - Although bromeliads are believed to obtain nutrients from debris deposited by animals in their rosettes, there is little evidence to support this assumption. Using stable isotope methods, we found that the Neotropical jumping spider Psecas chapoda (Salticidae), which lives strictly associated with the terrestrial bromeliad Bromelia balansae, contributed 18% of the total nitrogen of its host plant in a greenhouse experiment. In a one-year field experiment, plants with spiders produced leaves 15% longer than plants from which the spiders were excluded. This is the first study to show nutrient provisioning in a spider-plant system. Because several animal species live strictly associated with bromeliad rosettes, this type of facultative mutualism involving the Bromeliaceae may be more common than previously thought. PMID- 16676523 TI - Time constraints mediate predator-induced plasticity in immune function, condition, and life history. AB - The simultaneous presence of predators and a limited time for development imposes a conflict: accelerating growth under time constraints comes at the cost of higher predation risk mediated by increased foraging. The few studies that have addressed this tradeoff have dealt only with life history traits such as age and size at maturity. Physiological traits have largely been ignored in studies assessing the impact of environmental stressors, and it is largely unknown whether they respond independently of life history traits. Here, we studied the simultaneous effects of time constraints, i.e., as imposed by seasonality, and predation risk on immune defense, energy storage, and life history in lestid damselflies. As predicted by theory, larvae accelerated growth and development under time constraints while the opposite occurred under predation risk. The activity of phenoloxidase, an important component of insect immunity, and investment in fat storage were reduced both under time constraints and in the presence of predators. These reductions were smaller when time constraints and predation risk were combined. This indicates that predators can induce sublethal costs linked to both life history and physiology in their prey, and that time constraints can independently reduce the impact of predator-induced changes in life history and physiology. PMID- 16676524 TI - 15N in symbiotic fungi and plants estimates nitrogen and carbon flux rates in Arctic tundra. AB - When soil nitrogen is in short supply, most terrestrial plants form symbioses with fungi (mycorrhizae): hyphae take up soil nitrogen, transport it into plant roots, and receive plant sugars in return. In ecosystems, the transfers within the pathway fractionate nitrogen isotopes so that the natural abundance of 15N in fungi differs from that in their host plants by as much as 12% per hundred. Here we present a new method to quantify carbon and nitrogen fluxes in the symbiosis based on the fractionation against 15N during transfer of nitrogen from fungi to plant roots. We tested this method, which is based on the mass balance of 15N, with data from arctic Alaska where the nitrogen cycle is well studied. Mycorrhizal fungi provided 61-86% of the nitrogen in plants; plants provided 8 17% of their photosynthetic carbon to the fungi for growth and respiration. This method of analysis avoids the disturbance of the soil-microbe-root relationship caused by collecting samples, mixing the soil, or changing substrate concentrations. This analytical technique also can be applied to other nitrogen limited ecosystems, such as many temperate and boreal forests, to quantify the importance for terrestrial carbon and nitrogen cycling of nutrient transfers mediated by mycorrhizae at the plant-soil interface. PMID- 16676525 TI - Parasite genotypes identify source populations of migratory fish more accurately than fish genotypes. AB - DNA-based assignment of individuals to their population of origin has many applications such as mixed-stock analysis, identifying individuals from protected populations, and elucidating migration patterns. However, low genetic differentiation among populations will cause misassignments. Thus, an alternative means of determining an individual's population of origin is needed in cases where there is little or no neutral differentiation among source populations. Here, we test the hypothesis that parasite genotypes can be used to identify the origins of hosts more accurately than host genotypes. Using microsatellite markers from steelhead trout and their trematode parasites, we show that the odds of correct assignment are four times greater with the parasite's genotypes than with the host's genotypes. Our analyses show that this result is simply explained by the greater genetic structure among populations of the trematode parasite. Recent studies on the comparative genetic structure of other host and parasite species suggest that our results are not unusual or unique to the host-parasite system we studied. Thus, our work indicates that parasites will be useful for a wide range of applied and basic research that requires the assignment of individuals to source populations. PMID- 16676526 TI - Use of isotopic analysis of vertebrae in reconstructing ontogenetic feeding ecology in white sharks. AB - We conducted stable 13C and 15N analysis on white shark vertebrae and demonstrated that incremental analysis of isotopes along the radius of a vertebral centrum produces a chronological record of dietary information, allowing for reconstruction of an individual's trophic history. Isotopic data showed significant enrichments in 15N with increasing sampling distance from the centrum center, indicating a correlation between body size and trophic level. Additionally, isotopic values verified two distinct ontogenetic trophic shifts in the white shark: one following parturition, marking a dietary switch from yolk to fish; and one at a total length of >341 cm, representing a known diet shift from fish to marine mammals. Retrospective trophic-level reconstruction using vertebral tissue will have broad applications in future studies on the ecology of threatened, endangered, or extinct species to determine life-long feeding patterns, which would be impossible through other methods. PMID- 16676527 TI - Generalized site occupancy models allowing for false positive and false negative errors. AB - Site occupancy models have been developed that allow for imperfect species detection or "false negative" observations. Such models have become widely adopted in surveys of many taxa. The most fundamental assumption underlying these models is that "false positive" errors are not possible. That is, one cannot detect a species where it does not occur. However, such errors are possible in many sampling situations for a number of reasons, and even low false positive error rates can induce extreme bias in estimates of site occupancy when they are not accounted for. In this paper, we develop a model for site occupancy that allows for both false negative and false positive error rates. This model can be represented as a two-component finite mixture model and can be easily fitted using freely available software. We provide an analysis of avian survey data using the proposed model and present results of a brief simulation study evaluating the performance of the maximum-likelihood estimator and the naive estimator in the presence of false positive errors. PMID- 16676528 TI - Estimating species richness and accumulation by modeling species occurrence and detectability. AB - A statistical model is developed for estimating species richness and accumulation by formulating these community-level attributes as functions of model-based estimators of species occurrence while accounting for imperfect detection of individual species. The model requires a sampling protocol wherein repeated observations are made at a collection of sample locations selected to be representative of the community. This temporal replication provides the data needed to resolve the ambiguity between species absence and nondetection when species are unobserved at sample locations. Estimates of species richness and accumulation are computed for two communities, an avian community and a butterfly community. Our model-based estimates suggest that detection failures in many bird species were attributed to low rates of occurrence, as opposed to simply low rates of detection. We estimate that the avian community contains a substantial number of uncommon species and that species richness greatly exceeds the number of species actually observed in the sample. In fact, predictions of species accumulation suggest that even doubling the number of sample locations would not have revealed all of the species in the community. In contrast, our analysis of the butterfly community suggests that many species are relatively common and that the estimated richness of species in the community is nearly equal to the number of species actually detected in the sample. Our predictions of species accumulation suggest that the number of sample locations actually used in the butterfly survey could have been cut in half and the asymptotic richness of species still would have been attained. Our approach of developing occurrence based summaries of communities while allowing for imperfect detection of species is broadly applicable and should prove useful in the design and analysis of surveys of biodiversity. PMID- 16676529 TI - The role of habitat disturbance and recovery in metapopulation persistence. AB - Classical metapopulation theory assumes a static landscape. However, empirical evidence indicates many metapopulations are driven by habitat succession and disturbance. We develop a stochastic metapopulation model, incorporating habitat disturbance and recovery, coupled with patch colonization and extinction, to investigate the effect of habitat dynamics on persistence. We discover that habitat dynamics play a fundamental role in metapopulation dynamics. The mean number of suitable habitat patches is not adequate for characterizing the dynamics of the metapopulation. For a fixed mean number of suitable patches, we discover that the details of how disturbance affects patches and how patches recover influences metapopulation dynamics in a fundamental way. Moreover, metapopulation persistence is dependent not only on the average lifetime of a patch, but also on the variance in patch lifetime and the synchrony in patch dynamics that results from disturbance. Finally, there is an interaction between the habitat and metapopulation dynamics, for instance declining metapopulations react differently to habitat dynamics than expanding metapopulations. We close, emphasizing the importance of using performance measures appropriate to stochastic systems when evaluating their behavior, such as the probability distribution of the state of the metapopulation, conditional on it being extant (i.e., the quasistationary distribution). PMID- 16676530 TI - Quantifying population substructure: extending the graph-theoretic approach. AB - Among the few universal themes in ecology is that resources, energy, and organisms themselves, are patchily distributed. This patchy distribution imposes a need for some level of dispersal or connectivity among spatially separate patches in order to allow organisms to acquire sufficient resources for survival. To date, general patterns of connectivity have not emerged. This is, in part, because different species respond to different scales of patchiness. I propose an extension of the graph-theoretic approach to control for such differences and reveal potential generalities about how natural populations are organized. Using statistical methods and simple applications of graph theory, continuum percolation, and metapopulation models, I demonstrate a pattern of hierarchical clustering among populations in both a plant-pathogen system at an extent of 1000 m and gene flow in a salamander species across a subcontinental range. Results suggest that some patches or populations have a disproportionately high importance to the maintenance of overall connectivity in the system within and across scales. PMID- 16676531 TI - The effect of biodiversity on the hantavirus epizootic. AB - We analyze a mathematical model of the epizootic of Hantavirus in mice populations, including the effect of species that compete with the host. We show that the existence of the second species has an important consequence for the prevalence of the infectious agent in the host. When the two mice species survive in the ecosystem, the competitive pressure of the second species may lead to reduction or complete elimination of the prevalence of infection. The transition between the disappearance of the infection and its presence occurs at a critical value of the competitor's population, resembling a second-order phase transition in a statistical system. The results provide a rigorous framework for the study of the impact of biodiversity in the propagation of infectious diseases, and further lends itself to future experimental verification. PMID- 16676532 TI - Inferring evolutionary signals from ecological data in a plant-pathogen metapopulation. AB - We followed the dynamics of local epidemics in three populations of a natural plant-pathogen system for four sequential years. We characterize the overwintering process with spatial statistics and use a stochastic, spatially explicit, modeling approach with Bayesian parameter estimation to study the spread of the infection during the growing season. Our modeling approach allows us to infer coevolutionary signals from spatiotemporal data on pathogen prevalence. Most importantly, we are able to assess the distribution of resistant hosts within the distribution of all host plants. We show that resistant hosts occur in areas with high pathogen encounter rates, and that the occurrence of resistance correlates with overwintering probability of the pathogen. The estimates for essentially all model parameters are characterized by a large amount of variation over the years and the populations. While the variation in the fraction of resistant hosts and in the force of infection is to a large extent explained by the population, the other model parameters (two parameters describing the shape of the dispersal kernel) vary essentially in an unpredictable manner, suggesting that much of the variation may occur at very fine spatial and temporal scales. PMID- 16676533 TI - The economics of mutualisms: optimal utilization of mycorrhizal mutualistic partners by plants. AB - Can choice of mutualistic partners and the degree of their utilization determine (1) mutualistic partner coexistence, (2) relative abundance of mutualistic partners, and (3) environment-dependent changes in relative abundance? We investigate these questions in the context of the plant-mycorrhizal fungal mutualism by building a biological market model potentially applicable to other mutualisms as well. We examine the situation where a single plant selectively utilizes member(s) of a group of ectomycorrhizal potential trading partners. Under biologically realistic circumstances, the plant may simultaneously utilize multiple partners, its degree of utilization determining the community structure of the fungi. If utilization of multiple partners is optimal, the marginal cost of acquiring additional nitrogen from every trading partner must be equal while the marginal cost of acquiring it from any unutilized partner must be larger. Because the plant's nitrogen demand is light dependent, the composition of the fungal species among its trading partners changes along light-availability gradients. We discuss the design of an experiment to test the key prediction of our model, the equalization of marginal cost. PMID- 16676534 TI - Orchid-fungus fidelity: a marriage meant to last? AB - The characteristics of plant-mycorrhizae associations are known to vary in both time and space, but the ecological consequences of variation in the dynamics of plant-fungus interactions are poorly understood. For example, do plants associate with single fungi or multiple fungi simultaneously, and do the associations persist through a plant's lifetime or do plants support a succession of different fungi? We investigated these and other questions related to plant-fungus interactions in Goodyera pubescens, an evergreen terrestrial orchid of the eastern United States, that interacts with closely related fungi in the genus Tulasnella. Unlike the mycorrhizal associations of other plants, orchid mycorrhizal associations only benefit the orchid, based on current evidence. Many terrestrial orchids have been found to associate with specific groups of fungi. This characteristic could potentially limit orchids to relatively narrow ranges of environmental conditions and may be a contributing factor in the decline of many orchids in the face of changing environmental conditions. We found that G. pubescens protocorms (developing embryos prior to leaf production) and adults associated with only one fungal individual at a time. The orchid-fungus association persists for years, but during a drought period that was associated with the death of many plants, surviving plants were able to switch to new fungal individuals. These results suggest that G. pubescens interacts with the same fungal partner during periods of modest environmental variation but is able to switch to a different fungal partner. We hypothesize that the ability to switch fungi allows G. pubescens to survive more extreme environmental perturbations. However, laboratory experiments suggest that switching fungi has potential costs, as it increases the risk of mortality, especially for smaller individuals. Our findings indicate that it is unlikely that switching fungi is a common way to improve tolerance of less severe environmental fluctuations and disturbances. These findings may have important implications for plant responses to severe climatic events or to more gradual environmental changes such as global warming. PMID- 16676535 TI - Integrating quality and quantity of mutualistic service to contrast ant species protecting Ferocactus wislizeni. AB - Generalized, facultative mutualisms are often characterized by great variation in the benefits provided by different partner species. This variation may be due to differences among species in the quality and quantity of their interactions, as well as their phenology. Many plant species produce extrafloral nectar, a carbohydrate-rich resource, to attract ant species that can act as "bodyguards" against a plant's natural enemies. Here, we explore differences in the quality and quantity of protective service that ants can provide a plant by contrasting the four most common ant visitors to Ferocactus wislizeni, an extrafloral nectary bearing cactus in southern Arizona. The four species differ in abundance when tending plants, and in the frequency at which they visit plants. By adding surrogate herbivores (Manduca sexta caterpillars) to plants, we demonstrate that all four species recruit to and attack potential herbivores. However, their per capita effectiveness in deterring herbivores (measured as the inverse of the number of workers needed to remove half of the experimentally added caterpillars) differs. Using these among-species differences in quality (per capita effectiveness) and quantity (number of workers that visit a plant and frequency of visitation), we accurately predicted the variation in fruit production among plants with different histories of ant tending. We found that plant benefits (herbivore removal and maturation of buds and fruits) typically saturated at high levels of ant protection, although plants could be "well defended" via different combinations of interaction frequency, numbers of ant workers per interaction, and per capita effects. Our study documents variation among prospective mutualists, distinguishes the components of this variation, and integrates these components into a predictive measure of protection benefit to the plant. The method we used to average saturating benefits over time could prove useful for quantifying overall service in other mutualisms. PMID- 16676536 TI - Damage-induced resistance in sagebrush: volatiles are key to intra- and interplant communication. AB - Airborne communication between individuals, called "eavesdropping" in this paper, can cause plants to become more resistant to herbivores when a neighbor has been experimentally clipped. The ecological relevance of this result has been in question, since individuals may be too far apart for this interaction to affect many plants in natural populations. We investigated induced resistance to herbivory in sagebrush, Artemisia tridentata, caused by experimental clipping of the focal plant and its neighbors. We found no evidence for systemic induced resistance when one branch was clipped and another branch on the same plant was assayed for naturally occurring damage. In this experiment, air contact and plant age were not controlled. Previous work indicated that sagebrush received less damage when a neighboring upwind plant within 15 cm had been experimentally clipped. Here we found that pairs of sagebrush plants that were up to 60 cm apart were influenced by experimental clipping of a neighbor. Furthermore, we observed that most individuals had conspecific neighbors that were much closer than 60 cm. Air contact was essential for communication; treatments that reduced airflow between neighboring individuals, either because of wind direction or bagging, prevented induced resistance. Airflow was also necessary for systemic induced resistance among branches within an individual. Reports from the literature indicated that sagebrush is highly sectorial, as are many desert shrubs. Branches within a sagebrush plant do not freely exchange material via vascular connections and apparently cannot rely on an internal signaling pathway for coordinating induction of resistance to herbivores. Instead, they may use external, volatile cues. This hypothesis provides a proximal explanation for why sagebrush does not demonstrate systemic induced resistance without directed airflow, and why airborne communication between branches induces resistance. PMID- 16676537 TI - Scale dependence of reproductive failure in fragmented Echinacea populations. AB - I investigated reproduction in a three-year study of Echinacea angustifolia, purple coneflower, growing in a fragmented prairie landscape. I quantified the local abundance of flowering conspecifics at individual-based spatial scales and at a population-based spatial scale. Regression analyses revealed that pollen limitation increased while seed set and fecundity decreased with isolation of individual plants. Isolation, defined as the distance to the k(th) nearest flowering conspecific, was a good predictor of pollen limitation, for all nearest neighbors considered (k = 1-33), but the strength of the relationship, as quantified by R2, peaked at intermediate scales (k = 2-18). The relationship of isolation to seed set and fecundity was similarly strongest at intermediate scales (k = 3-4). The scale dependence of individual density effects on reproduction (density of flowering plants within x meters) resembled that of isolation. Analyses at a population-based scale showed that pollen limitation declined significantly with population size. Seed set and fecundity also declined with population size, but significantly so only in 1998. Whether quantifying local abundance with population- or individual-based measures, reproductive failure due to pollen limitation is a consistent consequence of Echinacea scarcity. However, individual-based measures of local abundance predicted pollen limitation from a wider sample of plants with a simpler model than did population size. Specifically, the largest site, a nature preserve, is composed of plants with intermediate individual isolation and, as predicted, intermediate pollen limitation, but its large population size poorly predicted population mean pollen limitation. PMID- 16676538 TI - Recruitment limitation constrains local species richness and productivity in dry grassland. AB - Species coexistence and local-scale species richness are limited by the availability of seeds and microsites for germination and establishment. We conducted a seed addition experiment in seminatural grassland at three sites in southern Switzerland and repeated the experiment in two successive years to evaluate various circumstances under which seed limitation and establishment success affect community functioning. A collection of 144,000 seeds of 22 meadow species including grasses and forbs of local provenance was gathered, and seeds were individually sown in a density that resembled natural seed rain. The three communities were seed limited. Three years after sowing, single species varied in emergence (0-50%), survival (0-69%), and establishment rates (0-27%). One annual and 13 perennial species reached reproductive stage. Low establishment at one site and reduced growth at another site indicated stronger microsite limitation compared to the third site. Recruitment was influenced by differences in abiotic environmental conditions between sites (water availability, soil minerals) and by within-site differences in biotic interaction (competition). At the least water limited site, sowing resulted in an increase in phytomass due to establishment of short-lived perennials in the second and third years after sowing. This increase persisted over the following two years due to establishment of longer-lived perennials. After sowing in a wetter year with higher phytomass, however, productivity did not increase, because higher intensity of competition in an early phase of establishment resulted in less vigorous plants later on. Due to the generally favorable weather conditions during this study, sowing year had a small effect on numbers of established individuals over all species. Recruitment limitation can thus constrain local-scale species richness and productivity, either by a lack of seeds or by reduced seedling growth, likely due to competition from the established vegetation. PMID- 16676539 TI - Vegetation structure constrains primary production response to water availability in the Patagonian steppe. AB - Grassland aboveground net primary production (ANPP) increases linearly with precipitation in space and time, but temporal models relating time series of ANPP and annual precipitation for single sites show lower slopes and regression coefficients than are shown by spatial models. The analysis of several ANPP time series showed lags in the ecosystem response to increased water availability, which may explain the difference between spatial and temporal models. The lags may result from constraints that ecosystems experience after drought. Our objective was to explore the structural constraints of the ANPP response to rainfall variability in a semiarid ecosystem, the Patagonian steppe, in southern Argentina. We designed a 3-yr rainfall manipulation experiment where we decreased water input with rainout shelters during two consecutive years, which included three levels of rainfall interception (30%, 55%, and 80%) and a control. In the third year, we irrigated one-half of the plots of each rainfall-interception treatment. We evaluated the immediate effects of drought on current-year ANPP and the effects of previous-year drought on vegetation recovery after water supplementation. ANPP (g x m(-2) x yr(-1)) was linearly related to annual precipitation input (APPT; mm/yr) along the experimental precipitation gradient (ANPP = 0.13 x APPT + 58.3; r2 = 0.34, P < 0.01), and this relationship was mostly accounted for by changes in the ANPP of grasses. Plant density (D; no. individuals/mm2) was related to the precipitation received during the drought period (D = 0.11 x APPT + 18; r2 = 0.39, P < 0.05). The recovery of plants after irrigation was lower for those plots that had experienced experimental drought the previous years relative to controls, and the lags were proportional to the intensity of drought. Therefore, our results suggest that the density of plants may constrain the recovery of vegetation after drought, and these constraints may determine lags that limit the capacity of the ecosystem to take advantage of wet years after dry years. PMID- 16676540 TI - Soil-geomorphic heterogeneity governs patchy vegetation dynamics at an arid ecotone. AB - Soil properties are well known to affect vegetation, but the role of soil heterogeneity in the patterning of vegetation dynamics is poorly documented. We asked whether the location of an ecotone separating grass-dominated and sparsely vegetated areas reflected only historical variation in degradation or was related to variation in inherent soil properties. We then asked whether changes in the cover and spatial organization of vegetated and bare patches assessed using repeat aerial photography reflected self-organizing dynamics unrelated to soil variation or the stable patterning of soil variation. We found that the present day ecotone was related to a shift from more weakly to more strongly developed soils. Parts of the ecotone were stable over a 60-year period, but shifts between bare and vegetated states, as well as persistently vegetated and bare states, occurred largely in small (<40 m2) patches throughout the study area. The probability that patches were presently vegetated or bare, as well as the probability that vegetation persisted and/or established over the 60-year period, was negatively related to surface calcium carbonate and positively related to subsurface clay content. Thus, only a fraction of the landscape was susceptible to vegetation change, and the sparsely vegetated area probably featured a higher frequency of susceptible soil patches. Patch dynamics and self-organizing processes can be constrained by subtle (and often unrecognized) soil heterogeneity. PMID- 16676541 TI - Seasonal variations in plant species effects on soil N and P dynamics. AB - It is well established that plant species influence ecosystem processes, but we have little ability to predict which vegetation changes will alter ecosystems, or how the effects of a given species might vary seasonally. We established monocultures of eight plant species in a California grassland in order to determine the plant traits that account for species impacts on nitrogen and phosphorus cycling. Plant species differed in their effects on net N mineralization and nitrification rates, and the patterns of species differences varied seasonally. Soil PO4- and microbial P were more strongly affected by slope position than by species. Although most studies focus on litter chemistry as the main determinant of plant species effects on nutrient cycling, this study showed that plant species affected biogeochemical cycling through many traits, including direct traits (litter chemistry and biomass, live-tissue chemistry and biomass) and indirect traits (plant modification of soil bioavailable C and soil microclimate). In fact, species significantly altered N and P cycling even without litter inputs. It became particularly critical to consider the effects of these multiple traits in order to account for seasonal changes in plant species effects on ecosystems. For example, species effects on potential rates of net N mineralization were most strongly influenced by soil bioavailable C in the fall and by litter chemistry in the winter and spring. Under field conditions, species effects on soil microclimate influenced rates of mineralization and nitrification, with species effects on soil temperature being critical in the fall and species effects on soil moisture being important in the dry spring. Overall, this study clearly demonstrated that in order to gain a mechanistic, predictive understanding of plant species effects on ecosystems, it is critical to look beyond plant litter chemistry and to incorporate the effects of multiple plant traits on ecosystems. PMID- 16676542 TI - Predator identity and additive effects in a treehole community. AB - Multiple predator species can interact as well as strongly affect lower trophic levels, resulting in complex, nonadditive effects on prey populations and community structure. Studies of aquatic systems have shown that interactive effects of predators on prey are not necessarily predictable from the direct effects of each species alone. To test for complex interactions, the individual and combined effects of a top and intermediate predator on larvae of native and invasive mosquito prey were examined in artificial analogues of water-filled treeholes. The combined effects of the two predators were accurately predicted from single predator treatments by a multiplicative risk model, indicating additivity. Overall survivorship of both prey species decreased greatly in the presence of the top predator Toxorhynchites rutilus. By itself, the intermediate predator Corethrella appendiculata increased survivorship of the native prey species Ochlerotatus triseriatus and decreased survivorship of the invasive prey species Aedes albopictus relative to treatments without predators. Intraguild predation did not occur until alternative prey numbers had been reduced by approximately one-half. Owing to changes in size structure accompanying its growth, T. rutilus consumed more prey as time progressed, whereas C. appendiculata consumed less. The intermediate predator, C. appendiculata, changed species composition by preferentially consuming A. albopictus, while the top predator, T. rutilus, reduced prey density, regardless of species. Although species interactions were in most cases predicted from pairwise interactions, risk reduction from predator interference occurred when C. appendiculata densities were increased and when the predators were similarly sized. PMID- 16676543 TI - Population and community resilience in multitrophic communities. AB - Diversity-stability relationships have long been a topic of controversy in ecology, but one whose importance has been re-highlighted by increasing large scale threats to global biodiversity. The ability of a community to recover from a perturbation (or resilience) is a common measure of stability that has received a large amount of theoretical attention. Yet, general expectations regarding diversity-resilience relations remain elusive. Moreover, the effects of productivity and its interaction with diversity on resilience are equally unclear. We examined the effects of species diversity, species composition, and productivity on population-and community-level resilience in experimental aquatic food webs composed of bacteria, algae, heterotrophic protozoa, and rotifers. Productivity manipulations were crossed with manipulations of the number of species and species compositions within trophic groups. Resilience was measured by perturbing communities with a nonselective, density-independent, mortality event and comparing responses over time between perturbed communities and controls. We found evidence that species diversity can enhance resilience at the community level (i.e., total community biomass), though this effect was more strongly expressed in low-productivity treatments. Diversity effects on resilience were driven by a sampling/selection effect, with resilient communities showing rapid response and dominance by a minority of species (primarily unicellular algae). In contrast, diversity had no effect on mean population-level resilience. Instead, the ability of a community's populations to recover from perturbations was dependent on species composition. We found no evidence of an effect of productivity, either positive or negative, on community- or population level resilience. Our results indicate that the role of diversity as an insurer of stability may depend on the level of biological organization at which stability is measured, with effects emerging only when focusing on aggregate community properties. PMID- 16676544 TI - Metacommunity influences on community richness at multiple spatial scales: a microcosm experiment. AB - Large-scale processes are known to be important for patterns of species richness, yet the ways in which local and larger scale processes interact is not clear. I used metacommunities consisting of five interconnected microbial aquatic communities to examine the manner in which processes at different scales affect local and metacommunity richness. Specifically, I manipulated the potential dispersal rate, whether dispersal was localized or global, and variation in initial community composition. A repeated-measures ANOVA showed that a low dispersal rate and intermediate distance dispersal enhanced local richness. Initial assembly variation had no effect on local richness, while a lack of dispersal or global dispersal reduced local richness. At the metacommunity scale, richness was enhanced throughout the time course of the experiment by initial compositional variation and was reduced by high or global dispersal. The effects of dispersal were contingent on the presence of initial compositional variation. The treatments also affected individual species occupancy patterns, with some benefiting from large-scale processes and others being adversely impacted. These results indicate that the effects of dispersal on species richness have a complex relationship with scale and are not solely divisible into "regional" vs. "local" scales. Finally, predictions of the manner in which dispersal rate structures communities appear dependent upon species compositional variation among communities. PMID- 16676545 TI - Consequences of patch reef spacing for density-dependent mortality of coral-reef fishes. AB - The spatial configuration of habitat patches can profoundly affect a number of ecological interactions, including those between predators and prey. I examined the effects of reef spacing on predator-prey interactions within coral-reef fish assemblages in the Bahamas. Using manipulative field experiments, I determined that reef spacing influences whether and how density-dependent predation occurs. Mortality rates of juveniles of two ecologically dissimilar species (beaugregory damselfish and yellowhead wrasse) were similarly affected by reef spacing; for both species, mortality was density dependent on reef patches that were spatially isolated (separated by 50 m), and density independent on reef patches that were aggregated (separated by 5 m). A subsequent experiment with the damselfish demonstrated that a common resident predator (coney) caused a substantial proportion of the observed mortality, independent of reef spacing. Compared to isolated reefs, aggregated reefs were much more likely to be visited by transient predators (mostly yellowtail snappers), regardless of prey density, and on these reefs, mortality rates approached 100% for both prey species. Transient predators exhibited neither an aggregative response nor a type 3 functional response, and consequently were not the source of density dependence observed on the isolated reefs. These patterns suggest that resident predators caused density-dependent mortality in their prey through type 3 functional responses on all reefs, but on aggregated reefs, this density dependence was overwhelmed by high, density independent mortality caused by transient predators. Thus, the spatial configuration of reef habitat affected both the magnitude of total predation and the existence of density-dependent mortality. The combined effects of the increasing fragmentation of coral reef habitats at numerous scales and global declines in predatory fish may have important consequences for the regulation of resident fish populations. PMID- 16676546 TI - Spatiotemporal variation in survival rates: implications for population dynamics of yellow-bellied marmots. AB - Spatiotemporal variation in age-specific survival rates can profoundly influence population dynamics, but few studies of vertebrates have thoroughly investigated both spatial and temporal variability in age-specific survival rates. We used 28 years (1976-2003) of capture-mark-recapture (CMR) data from 17 locations to parameterize an age-structured Cormack-Jolly-Seber model, and investigated spatial and temporal variation in age-specific annual survival rates of yellow bellied marmots (Marmota flaviventris). Survival rates varied both spatially and temporally, with survival of younger animals exhibiting the highest degree of variation. Juvenile survival rates varied from 0.52 +/- 0.05 to 0.78 +/- 0.10 among sites and from 0.15 +/- 0.14 to 0.89 +/- 0.06 over time. Adult survival rates varied from 0.62 +/- 0.09 to 0.80 +/- 0.03 among sites, but did not vary significantly over time. We used reverse-time CMR models to estimate the realized population growth rate (lamda), and to investigate the influence of the observed variation in age-specific survival rates on lamda. The realized growth rate of the population closely covaried with, and was significantly influenced by, spatiotemporal variation in juvenile survival rate. High variability in juvenile survival rates over space and time clearly influenced the dynamics of our study population and is also likely to be an important determinant of the spatiotemporal variation in the population dynamics of other mammals with similar life history characteristics. PMID- 16676547 TI - Habitat-specific variation and performance trade-offs in shell armature of New Zealand mudsnails. AB - Studies documenting phenotypic variation among populations show that ecological performance in one activity is sometimes traded off against another. Identifying environment-specific costs and benefits associated with performance trade-offs is fundamental to knowing how conflicting selection pressures shape phenotype environment matching in populations. We studied phenotypic variation in shell armature (spininess) of the New Zealand mudsnail, Potamopyrgus antipodarum (Gray), and explored how this variability relates to performance trade-offs between flow resistance and predator deterrence. Smooth- and spiny-shell morphotypes exist in populations in New Zealand streams and lakes, but the patterns and correlates of spatial variation of these phenotypes, and the possible hydrodynamical constraints and antipredatory benefits associated with spiny shell armature, are unknown. Samples from 11 rivers and nine lakes on the South Island showed that, on average, nearly 70% of snails in streams were smooth shelled, whereas >80% of snails in lakes were spiny, suggesting dissimilar selective pressures between habitats. A laboratory flume experiment revealed that spines collected seston (i.e., suspended algae) at current speeds <40 cm/s, making spiny morphs more prone to flow-induced dislodgment than smooth morphs. However, a fish feeding experiment showed that one benefit of spines on shells was a decrease in predation risk from the common bully (Gobiomorphus cotidianus), a widespread predator of mudsnails in both streams and lakes. All snails egested by bullies were dead, further suggesting that these fishes may exert strong lethal effects on mudsnail populations in nature. Spine expression in lakes also appeared to be temperature related. We conclude that functional trade-offs between risk of flow-induced dislodgment and risk of fish predation affect shell armature frequencies of Potamopyrgus in freshwater habitats. PMID- 16676548 TI - Estimation of temporary emigration in male toads. AB - Male boreal toads (Bufo boreas) are thought to return to the breeding site every year but, if absent in a particular year, will be more likely to return the following year. Using Pollock's robust design we estimated temporary emigration (the probability a male toad is absent from a breeding site in a given year) at three locations in Colorado, USA: two in Rocky Mountain National Park and one in Chaffee County. We present data that suggest that not all male toads return to the breeding site every year. Our analyses indicate that temporary emigration varies by site and time (for example, from 1992 to 1998, the probability of temporary emigration ranged from 10% to 29% and from 3% to 95% at Lost Lake and Kettle Tarn, respectively). Although the results provide weak evidence that males are more likely to return after a year's hiatus, a general pattern of state dependent temporary emigration was not supported. We also hypothesized relationships between temporary emigration and a number of weather variables. While some competitive models included weather covariates, imprecise and variable estimates of the effects of these covariates precluded fully defining their impact on temporary emigration. PMID- 16676549 TI - Dispersal depression with habitat fragmentation in the bog fritillary butterfly. AB - Habitat fragmentation is expected to impose strong selective pressures on dispersal rates. However, evolutionary responses of dispersal are not self evident, since various selection pressures act in opposite directions. Here we disentangled the components of dispersal behavior in a metapopulation context using the Virtual Migration model, and we linked their variation to habitat fragmentation in the specialist butterfly Proclossiana eunomia. Our study provided a nearly unique opportunity to study how habitat fragmentation modifies dispersal at the landscape scale, as opposed to microlandscapes or simulation studies. Indeed, we studied the same species in four landscapes with various habitat fragmentation levels, in which large amounts of field data were collected and analyzed using similar methodologies. We showed the existence of quantitative variations in dispersal behavior correlated with increased fragmentation. Dispersal propensity from habitat patches (for a given patch size), and mortality during dispersal (for a given patch connectivity) were lower in more fragmented landscapes. We suggest that these were the consequences of two different evolutionary responses of dispersal behavior at the individual level: (1) when fragmentation increased, the reluctance of individuals to cross habitat patch boundaries also increased; (2) when individuals dispersed, they flew straighter in the matrix, which is the best strategy to improve dispersal success. Such evolutionary responses could generate complex nonlinear patterns of dispersal changes at the metapopulation level according to habitat fragmentation. Due to the small size and increased isolation of habitat patches in fragmented landscapes, overall emigration rate and mortality during dispersal remained high. As a consequence, successful dispersal at the metapopulation scale remained limited. Therefore, to what extent the selection of individuals with a lower dispersal propensity and a higher survival during dispersal is able to limit detrimental effects of habitat fragmentation on dispersal success is unknown, and any conclusion that metapopulations would compensate for them is flawed. PMID- 16676551 TI - [These vocal cords lesions termed sulcus glottidis: clinico-anatomical considerations]. AB - Among the difficult cases which phonosurgeons may encounter, the problem of vergetures is one of the most complex. The primum movens would seem to be a particular vocal fold lesion in the form of more or less pronounced ligament atrophy. About a retrospective study of 52 cases, the authors present their conclusions: This atrophy co-exists anatomically, with (a sometimes considerable amount of atrophied mucosa opposite, "glottal tension" clearly perceptible during surgery, especially along the lower edge); videostroboscopically, with (great vibratory rigidity, more or less severe glottal air-loss). This lesion is completely distinct from a sulcus (the surgical treatment wil be discussed in an other paper). PMID- 16676550 TI - [Vocal fold structure and speech pathologies]. AB - Our knowledge of vocal fold structure has profoundly evolved since the description by M. Hirano in 1974 of the laminar organization of the connective tissue of the human vocal fold, the lamina propria. The molecular composition of the extracellular matrix (ECM) of the lamina propria is essential in determining the viscoelastic properties of the tissue. This is particularly true in the superficial layer of the lamina propria (also called Reinke's space). With the epithelium, Reinke's space constitutes the vocal fold main vibrator. In normal subjects, the jelly like structure of the Reinke's space is due to a very loose fibrous scaffolding and to a balance between the interstitial proteins of the ECM: Proteoglycans like decorin and hyaluronic acid and glycoproteins like fibronectin. Vibratory trauma leads, in disease states, to molecular modification of the extracellular matrix of Reinke's space resulting in benign cordal lesions like nodules, submucosal fibrosis and vocal folds scars. Improving the knowledge of the homeostasis of Reinke's space ECM can lead to improvement of the therapeutic and prevention strategies of vocal fold benign lesions. The main histologic and viscoelastic characteristics, as well as surgical treatment of cordal nodular lesions and scars are described. The recent researches in the field of regeneration of Reinke's space are evoked. PMID- 16676552 TI - [A proposal to adapt the voice handicap index to the singing voice]. AB - OBJECTIVES: The voice handicap index by Jacobson et al doesn't render the difficulties experienced by singers. The aim of this study is to adapt the V.H.I. to the singing voice and evaluate it's reliability and coherence. In order to obtain this first scale, we went through three stages. MATERIAL ET METHOD: The first consisted in the modification of the existing items and the creation of new ones. The second was to measure the stability by a test - retest and the coherence of the adapted and the new items. This was done on groups of dysodic singers and professional voice users. The third stage consisted in evaluating the reliability and the coherence of 31 items on a homogeneous population of 132 classical singers with and without vocal impairment. The subjects were divided in three groups. The first group was composed of 95 normophonic singers, the second included 37 dysphonic singers and the third group was the control group, composed of 20 non singers without vocal complaints. RESULTS: The statistical results show a good stability of the test - retest (ICC=0.949; Rho: 0.912; Bland et Altman [ 010.19, +13.23]). The internal coherence of each sub scale (Cronbach's alpha) is good (>0.70). The correlations between the three sub scales are moderate between the functional and the emotional sub scale: 0.58; between the emotional and the physical: 0.64; and between the functional and the physical: 0.78). Each sub scale is measuring a specific dimension; still they are homogeneous between one another. Four variables have a significative influence on the results: the pathology (p<0.001), being a professional singer (p<0.001), the repetition (p=0.003), being a chorister (p=0.002). CONCLUSION: The VH.I adapted to the singers, appears to be reliable, valid and adapted to the population of dysodic classical singers. PMID- 16676553 TI - [Evaluation of the handicap by means of the EORTC scale in partial glossectomies]. AB - OBJECTIVES: Different treatments for tongue cancer are more and more efficient but there are heavy functional and aesthetic sequels. The aim of this study was to evaluate the quality of life after partial glossectomy and to adapt the rehabilitation program after surgery. METHODS: Our population of 16 patients (5 females and 11 males), who underwent partial glossectomy, had to quote the auto questionnaire on quality of life (EORTC QOL-C30) and the specific head and neck cancer scale (H&N35). RESULTS: In our population, the patient's satisfaction for the global quality of life was 71%; the functioning scale was 83%, and the mean rate of generic symptoms was 13%. On the contrary, the specific Head & Neck symptom rate was high, with 76% of complains. CONCLUSIONS: The QOL scale could be considered as a good tool for patients to express their difficulties in daily life. It is a reliable and reproducible tool for a follow up study of patients. It allows proposing specific rehabilitation procedures in the post operative period. PMID- 16676554 TI - [Perceptive evaluation of substitution voices: the I(I) NFVo rating scale]. AB - OBJECTIVES: Evaluation of a perceptual specific rating scale for specific severe non laryngeral dysphonia. MATERIAL AND METHOD: 113 speech samples from substitution voices were scored perceptually according to the IINFVo scale: overall quality impression (I), impression of Intelligibility (I), additive and unnecessary noise (N), speech fluency (F) and presence of voiced segments (Vo). Each parameter was scored on a visual analogue scale from 0 (minimally deviant) to 10 (maximally deviant substitution voicing). These samples were presented to semi-professional jury-members (second grade speech therapy students) and professional jury-members (phoniatricians and speech therapists, specialised in the oncological field). RESULTS: Interindividual agreement between semi professionals was moderate (0.57-0.68). Interindividual agreement between professionals was higher (0.82-0.86). These figures are similar or even better compared to the classical perceptual evaluation scale for laryngeal speech (GRBAS). CONCLUSION: Our study suggests that the I(I)NFVo rating scale is suitable for perceptual evaluation of substitution voicing and consistency results are comparable with the classical perceptual rating scale (GRBAS). PMID- 16676555 TI - [Determination of oesophageal speech intelligibility using an articulation assessment]. AB - OBJECTIVE: We studied if articulation could determine esophageal speech's intelligibility. MATERIAL AND METHOD: To do so, we proposed 30 subjects distributed in three groups based on their speech proficiency level, two preliminary assessments: an articulation assessment and an intelligibility assessment. RESULTS: The study shows a significantly high correlation (86%) between the results of the articulation assessment and the intelligibility assessment in the whole of group studied. However, we noted differences in correlations according to the level of the subjects: in the novices' group, the percentage reaches 62%, in the medium group, 76%, and in the proficient one: 97%. CONCLUSION: We concluded that if the results of the articulation assessment is very high, intelligibility will be good. On the other hand, if this percentage is moderate, we can't evaluate intelligibility only with the articulation factor. PMID- 16676556 TI - [Contribution of the study of singing in tune in musically non-expert subjects: importance of short term memory of the pitch (19 to 28 year-old subjects)]. AB - INTRODUCTION: In the singing, which requires precise knowledge of the relevant musical code in use, accuracy of intonation plays a central role. Singing in tune requires to perceive pitch precisely and to memorize it before planning and executing the accurate vocal motion, which allows the exact emission of the correct pitch. OBJECTIVES: Our work investigated the role of short term memory of pitch on singing accuracy. MATERIAL AND METHOD: For that purpose, the experimental protocol of Deutsch (1970) was adapted for a perception and a production task. Participants were selected for their singing accuracy and separated into two groups of ten singing in tune and ten out-of-tune. All participants perceived pitch height exactly and were musically non-experts. For the perception and the production tasks, participants had to either compare or reproduce single pitches or two-pitch-sets. For the perception task, participants had to compare either single pitches or two-pitch patterns, all separated by a five seconds delay. For the production task, participants had to reproduce either single pitches or two-pitch patterns after a five seconds delay. The five seconds delay was either filled with intervening numbers, or with intervening tones, or without any disturbing sound. RESULTS: In perception and production task, the presence of intervening tones disturbs deeply the success of the subjects for every trial. Performance of the in-tune singing group is better for all the exercises while the other group had difficulties on single pitches and two-pitch patterns and was more disturbed by the effect of the intervening material. CONCLUSION: The outcome suggests that short term memory of pitch and accuracy of intonation would be closely linked. Further research needs to specify if that would mean that troubles in singing in tune are a consequence of a low-efficient short term memory of pitch, or if that troubles would hold up the right construction of the short term memory of pitch. PMID- 16676557 TI - [Clinical application of synchronised multimedia scanning for the objectification of laryngeal events in stuttering: preliminary study and first results]. AB - OBJECTIVE: We have tried in this preliminary work to observe what kind of mechanical laryngeal events were corresponding to the disfluencies heard while stuttering, especially in the pre-phonatory and phonatory blocks. Basing our observations upon numerised and synchronised multimedia recordings (videonasofibroscopic long duration recordings synchronised to the acoustic recordings of speech corpus) we also tried to figure what happened when an adult speaker used a fluency enhancing method such as the Erasm. Authors advanced the hypothesis of a closed larynx in two or three folds while the stuttering blocks and some even described those folds. MATERIAL AND METHOD: We have recorded the stutterers and non-stutterers (N= 3) as well during speaking tasks as in cough, snuffling (N= 2), swallowing and sustaining a vowel. Secondary, the patients had to use the Erasm method, for the productions they had first stuttered. We wanted to focus rather on the supraglottal components movements. RESULTS: In our study we haven't visualised any laryngeal double or triple folding while the blocks. But we did observe abnormal laryngeal behaviours, which recall spasmodic or myoclonic type of movements with: Tremors of the base of the tongue, a strong lateral pharyngolaryngeal constriction, quick successive up and down involuntary movements of the larynx, anarchic and paradoxal attempts of opening the vocal folds, at the moment of the intention of speaking. We did also objectify a real improvement in those aberrant movements by using the Erasm method. PMID- 16676558 TI - [Increase in the abdominal respiratory movements during the sudden passage from a conversational voice to a loud voice]. AB - The passage of the simple conversational voice to a loud voice is accompanied by an increase in the muscular tensions as well as increase in the mobilized volumes of air. OBJECTIVES: We sought to know if these additional volumes were mobilized rather by a thoracic breathing or an abdominal breathing and if this effort were accompanied by a reduction in the variability of the movements. MATERIAL AND METHOD: The study presented is a prospective experimental study undertaken on healthy subjects (10 subjects) of which we analyzed the spontaneous vocal behavior (conversation with the experimenter) in the medium of vocal spots lures (production of vowels) in a calm environment allowing a conversational voice (condition 1) and in a noisy environment obliging the subjects abruptly to use a strong voice (condition 2). Thoracic and abdominal volumes were recorded using belts provided with pressure pick-ups, while the voice was synchronized with the recordings. RESULTS: The results show a reduction in the number of rheses realized with abdominal volumes and a reduction in the abdominal volumes mobilized in condition 2, testifying to a preferential use of thoracic breathing in loud voice. They also show a reduction in the number of movements of the inspiratory type (increase in the thoracic diameter or reduction in the abdominal diameter) during the phonatory phase in condition 2, which testifies to a reduction in the variability of the strategies used by the subjects. CONCLUSION: The sudden passage to a loud voice because of the appearance of an ambient noise leads to an increase of the use of thoracic breathing, which is in agreement with the concept of voice of distress or of emergency suggested by Le Huche. PMID- 16676559 TI - [Diagnosis of non-organic dysphonia in adult]. AB - Non organic dysphonia or functional voice disorders are the consequence of a vocal misuse or overuse with inefficient oral communication. Any stage of voice production can be altered. A review of physiopathological, aerodynamic and biomechanical mechanisms will help to understand the onset of dysphonia. Organic lesions as a consequence of functional voice disorders are frequent but the link is not easy to establish. It is important to look for various physiologic, anatomic, environmental, behavioural and infectious factors that could induce or aggravate non organic dysphonia, as they can benefit from specific treatment. A thorough functional and organic assessment is the first step of the rehabilitation process, taking into account the patient's expectations about his voice handicap. PMID- 16676560 TI - [Manual care in speech rehabilitation]. AB - The speech therapists work on voice rehabilitation, with different kinds of formation. The dysfunctions of the vocal gesture may have various causes, and manual therapy is some time usefull to correct them. A study shows that using manual therapy is already effective after 10 voice sessions. PMID- 16676561 TI - [Autistic-like behavioural disorders and deafness in children]. AB - There is a co-morbidity between autism spectrum behaviour disorders and deafness in children. Their behavioural difficulties may appear primary and/or secondary to hearing deprivation. In this paper, we present how we manage those patients on the base of our clinical experience. The auditory assessment must be based on subjective tests taking account of their special reactions. It needs objective tests to complete and help the behavioural responses. The hearing aids amplification is increased progressively, to allow the development of a tolerance to the sounds world. The multidisciplinary team tends to open them to the world and to the communication. PMID- 16676562 TI - [Diagnosis and treatment of deafness in autistic children]. AB - The indifference to the sounds world leeds to explore the different levels of what can be a deafness: Including the ability to recognize the sounds, to memorize and the wish to communicate. After that, it is possible to organize an educative, reeducative, psychological program with audiophonology. PMID- 16676563 TI - The use of magnetic resonance in myocardial ischaemia. AB - Despite the efforts that have been made at an international level to identify and control cardiovascular risk factors, cardiopathies and, in particular, coronary artery disease (CAD), remain the principal cause of death in Europe and the United States. These data confirm the importance and necessity of noninvasive, reliable diagnostic imaging of early CAD. Coronary angiography is still the hinge, around which all instrumental and laboratory investigations turn, for cardiac ischaemia today. Indeed, it still holds the role of "gold standard" for the study of the coronary arterial lumina, particularly the smaller vessels due to their complex spatial geometry and because of cardiac motion. At present, with the exception of the study of the coronary arterial lumen, MR is a non-invasive examination, already capable of supplying precise global and regional function, the evaluation of the intra-cardiac flow, myocardial perfusion and the overall viability of the heart. PMID- 16676564 TI - Recovery of physical function and patient's satisfaction after total hip replacement (THR) surgery supported by a tailored guide-book. AB - BACKGROUND AND AIM OF THE WORK: The purpose of this prospective study was to give a customized guide, describing the hospitalization period and the postoperative exercise program, to patients scheduled for total hip arthroplasty (THA) and to show its effectiveness on functional recovery and on patient's satisfaction with the rehabilitation care and with the in-hospital discharge planning after surgery. METHODS: This trial included 365 consecutive subjects with osteoarthritis who underwent THA at the Orthopaedic and Traumatology Clinic of Siena (Italy). The Harris Hip Score (HHS), a disease specific measure, was determined before and after surgery. Postoperative evaluations, associated with a Satisfaction Questionnaire, were carried out at the time of discharge and after 3 months. RESULTS: The overall satisfaction level was very high, both at discharge (81+/-28) and at follow-up (90+/-17). The HHS results showed a significant (p< or =0.05) improvement over time in patients with higher scores 3 months after surgery in comparison with baseline. CONCLUSIONS: Patients reported high levels of satisfaction at the 3 month postoperative follow-up and good levels at discharge. After surgery, the highest improvements were shown in bodly pain and physical function scores. The current study showed that a customized guide was well accepted by patients with THA and satisfie their need of information. It was also effective in improving patient's satisfaction and early recovery of physical function after surgery. PMID- 16676565 TI - Status of immunity against poliomyelitis: a study among European and extra European young immigrants living in Parma. AB - From January 2001 to December 2003 blood specimens obtained from 323 European and extra-European young immigrants were collected for the determination of anti polio antibody levels. They were sent to the Section of Hygiene, Department of Public Health, by the Local Health Authorities. A neutralization assay was performed to detect the anti-polio antibodies against serotypes 1, 2 and 3, using rhesus monkey kidney cells. The results showed 98,1% prevalence of anti-polio 1 antibodies (titre > or =1:2), 99,1% of anti-polio 2, and 98,8% prevalence of anti polio 3 antibodies. The seronegativity against only one or two serotypes (antibody titre <1:2) was found in 9 subjects (2,8%) while no subject was found totally seronegative against all 3 serotypes. According to the total amount of the analyzed samples, the estimated Geometric Mean Titre (GMT) resulted from moderate to low (GMT=29 of serotype 3; GMT=48,4 of serotype 2; GMT=56,5 of serotype 1) and it appeared to be similar to the results obtained in the previous years for all 3 serotypes; the GMT of serotype 3 was still the lowest. No difference was observed in the specimens with respect to the country of origin of the examined subjects and the data collected showed a good level of immunity. PMID- 16676566 TI - Electron microscopy as a reliable tool for rapid and conventional detection of enteric viral agents: a five-year experience report. AB - BACKGROUND AND AIM OF THE WORK: Since the introduction of the electron microscope and its subsequent development, virology has made a great step forward by the improvement of the basic knowledge on viral structure, as well as by broad application of electron microscopy (EM) to viral diagnosis. In this report, we describe a five-year experience in the use of EM for the diagnosis of enteric viral infections. METHODS: Three thousand four hundred and ninety stool specimens were analyzed at the Virology Unit (Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Italy) during a five-year period, from January 1999 to January 2004. The faecal extracts were subjected to EM after negative staining and were simultaneously cultured to evidence the presence of cytopathogenic agents. RESULTS: EM directly applied to the above specimens allowed the detection of several enteric viral agents, particularly evidencing those normally hard to cultivate (thus easily lost with culture methods). It also enabled diagnosis of dual gut infections, such as those from rotavirus and calicivirus. On the other hand, EM-based identification of viral agents after cell culture and ultracentrifugation of cytopathogenic agent containing cellular extracts, allowed the identification of cultivable agents, such as picornaviruses, which can escape the direct EM detection if low concentrated. CONCLUSIONS: A rationalized use of EM on selected samples, such as stool, appears suitable in epidemiological or clinical conditions when a very rapid diagnosis is required to save time, including cases of suspected emerging viral infections. PMID- 16676567 TI - Role of EMG in congenital hypotonia with favorable outcome. AB - BACKGROUND: Since hypotonia is the phenotype of several clinical conditions that do not always lead to a favorable outcome, prompt diagnosis is important. Congenital Hypotonia with Favorable Outcome (CHFO), an underestimated condition, should be rapidly differentiated from other more serious hypotonic states by means of simple, effective and only slightly invasive instrumental diagnostic examinations. AIM: We analyzed the electromyographic data of a group of patients with CHFO and compared them with data taken from their muscle biopsies to evidence the utility of the electromyographic study (EMG) in this condition. METHODS: We performed EMG, nerve conduction study (NCS) and muscle biopsy on 41 subjects with a diagnosis of CHFO (age range 9 months to 12 years, mean age, 6.7 years). RESULTS: No specific EMG findings were observed. Muscle biopsy was normal in all subjects, and we obtained concordance between biopsy and EMG in 85% of the subjects (35/41). CONCLUSION: A normal EMG examination helps investigators to exclude several neurological diseases characterized by hypotonia and it can provide valuable complementary information to confirm the clinical diagnosis of CHFO. PMID- 16676568 TI - Retzius' space haematoma after spontaneous delivery: a case report. AB - We report a case of a haematoma of the Retzius space after spontaneous uncomplicated delivery. In the postpartum period, the patient complained of urinary retention and pain in the hypogastric region radiating to her left hip and leg. The ultrasound examination showed the presence of 160 x 100 x 80 mm confluent solid and liquid areas with peripheral vascularization. At exploratory laparotomy a haemorrhagic infiltration was found in Retzius' space and the anterior wall of the bladder, which appeared thickened and swollen below the peritoneum. We tried to drain the haematoma, however, we failed to drain it completely because of the large blood infiltration in the bladder wall. Clinical and ultrasound follow-up examinations showed a progressive reduction of the haematoma which completely disappeared nine months later. The haemodynamic changes occurring during pregnancy and labour, associated with strong mechanical stress, seem to be among the major causative factors of haematoma formation. Moreover, the venous load in the pelvic vascular system is increased during pregnancy; a stress-induced increase in venous blood pressure might play a prominent role, especially in cases of venous ectasia, where the resistance of blood vessel walls is reduced. Intraoperative evidence seemed to suggest a haemorrhage secondary to the rupture of the venous vessels in the Santorini plexus. The rupture was probably caused by the thrust of the fetal head, associated with abnormality or fragility of the blood vessels, or by some pathologic changes occurring in the anatomical structures during pregnancy, which could not be accurately defined because of the severity and degree of the haematoma infiltration found intraoperatively. PMID- 16676569 TI - Hahn-Steinthal fracture: report of two cases. AB - Isolated capitellar fractures are rare injuries accounting for only 1% of all elbow fractures. Type I or Hahn-Steinthal fractures require internal fixation to restore the anatomic integrity of the elbow joint. Many different fixation methods have been described and, independently of the method chosen, precise reduction is mandatory to maximize articular congruency and to diminish the potential for secondary osteoarthritis. The treatment and the outcome of two type I capitellar fractures are described in this study. One patient (April 1999) underwent open reduction and Kirschner wire fixation while the second patient (May 2004) underwent open reduction and Acutrak standard screw fixation. In both patients no complications occurred and normal elbow function was recovered. No signs of avascular necrosis (AVN), heterotopic ossifications and degenerative osteoarthritis were detected. We conclude that Acutrak screw fixation is a reliable method of treatment because it enables a good interfragmentary compression, earlier mobilization, faster functional elbow recovery, and metal work removal is rarely necessary. PMID- 16676570 TI - [International Institute of molecular and cell biology in Warsaw]. PMID- 16676571 TI - [Education career mobility after the doctorate degree]. PMID- 16676572 TI - [Calreticulin, Ca2+-binding chaperon of the endoplasmic reticulum]. AB - The endoplasmic reticulum (ER) plays a vital role in many cellular processes, including Ca2+ storage and release. Calreticulin is a Ca2+-binding chaperon residing in ER. The protein is a key component of the quality control pathways in ER. In the ER lumen, calreticulin performs two major functions, works as a chaperon and regulates Ca2+ homeostasis. In cardiac muscle, calreticulin plays an important role in cardiac development and pathology. PMID- 16676573 TI - [Complex role of the FA proteins in providing genome stability]. AB - FA is a rare genetic disorder characterized by developmental abnormalities, bone marrow failure and cancer susceptibility. Cells that are derived from patients with FA display spontaneous chromosomal instability and hypersensitivity to DNA crosslinking agents that is used in FA clinical diagnostics. FA is genetically heterogeneous and caused by mutations in at least 11 distinct genes, FANCA, FANCA, B, C, D1, D2, E, F, G, I, J and L. FA proteins interact with various proteins involved in DNA damage response and cell cycle checkpoint regulation, such as: RAD51, BRCA1, BRCA2, ATM or NBS1. Moreover, BRCA2 that plays a crucial role in homologous recombination is one of FA proteins. Collectively, all these data indicate, that the FA pathway is involved in different molecular processes that prevent DNA and control genomic stability, although its precise role still remains undefined. PMID- 16676574 TI - [Molecular mechanisms of biotoxicity of homocysteine--facts and hypotheses]. AB - In the article, the main pathways of homocysteine metabolism are described, i.e. transsulfuration to cysteine and glutathione, as well as remethylation to methionine. Furthermore, formation of homocysteine thiolactone through editing mechanism with methionyl t-RNA syntethase and unusual reactivity of thiolactone against lysine epsilonNH2 groups of proteins as well as calcium dependent enzymatic hydrolysis of thiolactone are discussed. The effects of oxidative stress related to homocysteine are also reviewed. Finally, possible links of homocysteine to NO and arginine metabolism are discussed, including ADMA (N(G),N(G)-dimethylarginine). The links between metabolism of homocysteine, adenosine and other nucleosides are emphasized. In conclusion, the N homocysteilation of proteins with thiolactone changing enormously their properties seems to be the main reason of biotoxicity of homocysteine during atherosclerosis and other diseases. PMID- 16676575 TI - [The influence of nitric oxide on the regulation of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator expression]. AB - Nitric oxide produced in various human tissues by nitric oxide synthase is involved in the regulation of many physiological processes. Mechanism of its action is diverse. The most important physiological activity of nitric oxide is guanylate cyclase activation and an increase of cGMP synthesis. At low concentrations NO plays a pivotal role in vessel relaxation and possesses antithrombotic, antiproliferative and anti-inflammatory features as well. An excessive production of nitric oxide can disturb vascular hemostasis and contribute to development of cardiovascular diseases. Studies provide that NO also participate in fibrynolysis regulation by the influence on the PAI-1 and t PA expression, what may have important clinical implications. The aim of this review is to present current knowledge about the role of nitric oxide in the regulation of these plasminogen activation system factors. PMID- 16676576 TI - [Plasma membrane--order or chaos?]. AB - In the influential "fluid mosaic" model of plasmalemma, transmembrane proteins drift regardless of lipids. Recently researches widen this to a view in which membrane lipids are not randomly distributed but they form liquid-ordered regions with local heterogenity, called lipid rafts. Lipid rafts are subdomains of the plaSma membrane that contain high concentration of cholesterol and glycosphingolipids. They are 50-100 nm distinct liquid-ordered regions of the membrane that are resistant to extraction with nonionic detergents. They are proposed to function as dynamic lipid assemblies which serve as platforms for protein segregation and signaling, protein and lipid sorting during post-Golgi sorting, dynamic of plasmalemma and virial entry budding. Markers for the lipid rafts are flotillin, GPI - linked proteins, Src family kinases, EGF receptors and G proteins. The lifetime, biological relevance and properties of these domains in vivo are still unclear. However the answers will shape our views of signaling and membrane dynamics. PMID- 16676577 TI - [Mechanisms of regulation and function of G-protein coupled receptor kinases]. AB - G protein-coupled receptor kinases (GRKs) are key modulators of G protein-coupled receptor (GPCR) signaling. They constitute a family of seven mammalian serine threonine protein kinases that phosphorylate agonist-bound receptor. GRKs mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization. Activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium sensitive proteins. Moreover, GRK phosphorylation by several other kinases and autophosphorylation have recently been shown to modulate its functionality. This review summarize our current knowledge of GRKs regulatory mechanisms and GRKs physiological function. PMID- 16676578 TI - [Aromatase--key enzyme of estrogen biosynthesis]. AB - Estrogens control a large range pivotal life functions as reproductive development and fertility, bone growth and sexual behavior. Aromatase is a key enzyme of estrogen biosynthesis. The property, structure and reaction mechanism of aromatase as well as detailed structure of human aromatase cytochrome P450 gene (CYP19) was discussed in this article. It was pointed that unique human CYP19 gene expression results from presence of many tissue specific promoters and alternative splicing. The molecular mechanism of control aromatase cytochrome P450 gene expression in various species ovaries, testes and human adipose tissue and placenta was discussed in details. Because of a very important role of estrogen in breast cancer a molecular base of aberrant expression CYP19 gene in breast tumor and adipose tissue proximal to breast tumor and potential possibility of pharmacological silencing of this gene expression was discussed in the article. PMID- 16676579 TI - [Family of pentatricopeptide repeat proteins]. AB - PPR proteins belong to large family of nucleic acid binding proteins, mainly RNA binding proteins. Their name is defined by the presence of so-called pentatricopeptide repeat (PPR), a degenerate 35-aminoacid repeats containing from 2 up to 26 such motifs arrayed in tandem of at least in one pair. PPR motif consists of two a helices A and B forming a superhelix enclosing a groove or tunnel which is likely to be the ligand-binding site. PPR proteins are targeted mainly to mitochondria and chloroplasts where they are mainly involved in posttranscriptional processes and translation. Among PPR proteins they were also found restorer gene products which restorer pollen fertility. Some PPR proteins play roles as adaptors and partner in protein-protein interaction. PPR protein genes were discovered in all analyzed eukariotic genomes. They are especially abundant in plants. PMID- 16676580 TI - [Mitochondrial intermembrane space proteins in apoptosis process]. AB - Mitochondria, despite their function in cellular energy metabolism, play an important role in the apoptotic signaling pathways. These organelles in response to the death signal undergo changes resulting in the release of proteins which are essential to conduct apoptosis via mitochondrial pathway. This article is focused on the properties and functions of apoptogenic proteins released from the mitochondrial intermembrane space, i.e., caspases, cytochrome c, Smac/DIABLO, serine protease Omi/HtrA2, AIF and endonuclease G. PMID- 16676581 TI - [Periplasmatic disulfide oxidoreductases from bacterium Escherichia coli--their structure and function]. AB - The formation of proper structural disulfide bonds is one of the key steps during the folding of many secretory proteins and occurs both in prokaryotes and eukaryotes. In Gram negative bacterium Escherichia coli this process is catalyzed by a set of periplasmic oxidoreductases, termed Dsb. These proteins function in two separate pathways: (1) oxidizing (DsbA/DsbB system), responsible for introducing S-S bonds, and (2) reducing (DsbC/DsbD system, DsbG, CcmG and CcmH) which acts to isomerase wrongly formed disulfide bonds and participates in maturation of cytochrome c. The first system acts in connection with the inner membrane electron transfer system, using quinone molecules as electron acceptors, whereas reducing pathway relies on constant supply of electrons provided by the cytoplasmic thioredoxin system. Majority of Dsb proteins belongs to the thioredoxin superfamily and they contain the conserved Cys-X-X-Cys motif in their active site. The redox properties of Dsb proteins with the particular focus on structure-function dependence are in this review discussed. PMID- 16676582 TI - [Management of human and animal feces is a key element for effective control of epidemic of endemic schistosomiasis in China]. PMID- 16676583 TI - [Call for more extensive research on epidemiology of dementia in China]. PMID- 16676584 TI - [Quantitative analysis of factors affected mortality trend in Chinese, 2002]. AB - OBJECTIVE: To explain trend of death in Chinese by quantitative analysis of demographic and non-demographic factors and estimate the proportion of contribution of non-demographic and demographic factors. METHODS: Using census data and death causes data of National Disease Surveillance Points at 1991 and 2000 to calculate the proportion of contribution of demographic and non demographic factors and to change on various death causes from 1991 to 2000 by methods of decomposing the differences of death rates. RESULTS: The death rate showed a rapid decrease during 1950 - 1975, mainly owing to the contribution of non-demographic factors, including economic development, popularization of education and health service, especially the "patriotic hygiene movement". During 1991 - 2000, the death causes of lung cancer, liver cancer, breast cancer, chronic heart disease, stroke, diabetes and traffic accident had been increasing. The increase of deaths caused by these diseases were contributed to the non demographic factors including 63% of the increase on lung cancer and 88% of increase on death rate of traffic accidents. CONCLUSION: The study showed that the risk factors had contributed to the increase of death rates, including behavioral risk factors described in the preceding 5 papers as smoking and passive smoking, unhealthy diet, sedentary life style, violating traffic regulation etc. In order to reduce the death rates on cancer, heart diseases, diabetes, traffic accidents, emphasis should be also laid on the change of unhealthy behaviors. PMID- 16676585 TI - [Study on the active life expectancy of the elderly and its longitudinal transition in Beijing]. AB - OBJECTIVE: Focus on the Active Life Expectancy (ALE) of elderly in Beijing and the transition in recent years. METHODS: A representative sample of 3257 elderly people who lived in the urban, suburban and rural communities in Beijing that had been followed up for 12 years. Their health and survival status had been surveyed every 2-3 years. Activity Daily Living scale (ADL), recommended by WHO was used to evaluate the physical function capability of the elderly. IMaCH 0.8 was used to estimate life expectancy (LE) and active life expectancy (ALE) for both periods while age, sex and rural/urban residence areas were adjusted. RESULTS: Longitudinally, data showed that the main characters remained unchange throughout the two periods including 1) LE, ALE, ALE/LE of elderly living in urban area were higher than those living in rural area; 2) LE seemed longer in women than men, but ALE/LE was less in women. The transition between two period showed that 1) LE increased modestly in all groups but less prominent in urban residents and in females; 2) ALE was not significantly changed in the rural elderly but declined markedly in women living in the urban area; 3) ALE/LE of the elderly declined in all groups, especially in urban and oldest old groups. CONCLUSION: In Beijing, elderly AL-E did not increase in parallel with the increase of LE while ALE/LE of the elderly declined significantly in recent years. In order to improve quality of life of the elderly and to increase their ALE, emphasis should be given to prevention of cardiovascular, cerebrovascular and other chronic diseases while reducing the occurrence of physical disability and strengthening on rehabilitation would be the basic health care measures. PMID- 16676586 TI - [Analysis on the incidence and relevant risk factors of campus violence among college students]. AB - OBJECTIVE: To study the incidence and risk factors of campus violence and to provide evidence for preventing campus violence among college students. METHODS: 5300 college students in two universities in a province of China were selected to participate in the study and were interviewed with questionnaires. Logistic regression was used for data analysis. RESULTS: (1) In total, 3910 useable questionnaires were gathered to show a response rate of 73.77%. 17.98% of the college students reported they had ever experienced campus violence in the last one year. 29.60% of the male students experienced campus violence so as 7.27% of the female students. The incidence of violence among male students was significantly higher than those of female students (chi2 = 329.89, P = 0.000). (2) Among male students who were victims of campus violence, the incidence rates of threat or blackmail, emotional abuse, physical assault, verbal sexual harassment, sexual assault were 18.03%, 13.97%, 10.77%, 0.85%, 0.48% respectively. Among female students who were victims of campus violence, the incidence rates were 3.64%, 5.84%, 1.38%, 1.33%, 1.13% respectively. The main source of the violence was from their schoolmates. (3) 10.40% of the male students reported they were perpetrators of campus violence in the last year, while 1.47% of the female students reported so. Schoolmates were the main subjects of their aggressive behaviors. (4) Among the college students who were victims of campus violence, logistic regression analysis revealed that smoking, drinking alcohol, frequently getting computer online were important risk factors. The OR values were 1.48, 2.96, 1.66 respectively. Among college students who were perpetrators of campus violence, the OR values were 2.92, 1.88, 2.09 respectively. CONCLUSION: Campus violence among college students was serious, suggesting that intervention measures should be taken. PMID- 16676587 TI - [Study on the risk factors and characteristics on childhood sexual abuses among female students in a college]. AB - OBJECTIVE: To study the risk factors and abusers' characteristics on childhood sexual abuses among female students to provide reference data for preventive measures on child sexual abuse. METHODS: Annonymous questionnaire survey of 701 adult female students in a college about their personal experience on childhood sexual abuses, their home conditions and abusers' characteristics. RESULTS: A total number of 701 female students were surveyed. 115 (22.11%) experienced non physical contact sexual abuses before the age of 18, including 26 (3.71%) who suffered from attempted and 8 (1.14%) were forced with genital or anal sexual intercourse. 117 (16.69%) said they had suffered from sexual abuse before the age of 16. Most abusers were males (99.4%) and only a small percentage of them (3.2%) used violence. Most non-physical contact sexual abuses were from strangers (78.7%), while 71.3% of physical contact abuses were from acquaintances including 12.5% of them were teachers, 17.5% were neighbors and 21.3% were relatives. The risk factors of child sexual abuses were: frequent use of violence, bad hobbies and poor health conditions of parents, tight family relationships, and living in remarried families. Having well-educated fathers was a preventive factor. CONCLUSION: Studies on childhood sexual abuses among females revealed that the incidents were not rare, and family environment played an important role. PMID- 16676588 TI - [Appropriate sample size and molecular marker loci in the study of genetic diversity of Ocomelania hupensis]. AB - OBJECTIVE: To explore the reasonable sample size and the number of molecular marker loci in the study of amplified fragment length polymorphism (AFLP) being used to analyze the genetic diversity of Ocomelania hupensis. METHODS: The ribbed shelled snails coming from Yueyang, Hunan province, were selected to analyze the relationship of the number of AFLP molecular marker loci and sample size with the reliability of information on genetic variation for Ocomelania hupensis by AFLP method. RESULTS: Correlations found among the numbers of AFLP molecular marker loci and the sample size with reliable information on genetic variation for Ocomelania hupensis. When sample size was less than 7 individuals, the total number of AFLP loci, the number of polymorphic loci, Nei's gene diversity and Shannon's information index appeared great changes. However, when sample size was bigger than 30 individuals, the values of these indices tended to be stabilized. When the number of AFLP loci was less than 128, the frequency of polymorphic loci, Nei's gene diversity, Shannon's information index and the standard deviation of these two indices changed greatly. Again, when the number of loci was bigger than 338, the values of these indices tended to be stabilized. CONCLUSION: When the genetic diversity of Ocomelania hupensis were analyzed by AFLP method, the sample size coming from each snail population should not be less than 30 individuals and the number of molecular loci analyzed not less than 338. PMID- 16676589 TI - [Study on time-series analysis and forecast models on lung cancer incidence in Qidong, 1972 - 2001]. AB - OBJECTIVE: To explore the lung cancer incidence rates from 1972 to 2001 and utilize varieties of models in forecasting trend up to 2010 in the city of Qidong, Jiangsu in order to provide baseline data for its control and prevention. METHODS: Using data from the cancer registry office in Qidong, we tried to reveal the trends of lung cancer incidence by analyzing the time-series on trends extrapolation, exponent smoothness, Box-Jenkins model etc. We also compared the prognostication precision, endow differ power, and established assembled forecast model. RESULTS: Data showed that there had been a rising trend of lung cancer from 1972 to 2001 and would still probably be on the increase in the future. The rate of male and female attained to 70 per 100 000 and 20 per 100 000, predicting that there would be a respective 33 percent and 10 percent increase in 2010. CONCLUSIONS: According to analysis of forecast models, it was right to prognosticate lung cancer incidence from time-series and improve forecast precision through developing combination models. The result of combination seemed close to ARIMA models which suggested that it could serve as a chief way to forecast the incidence of lung cancer. PMID- 16676590 TI - [Study on the descriptive epidemiology of pregnancy-induced hypertension from 1995 - 2000 in Jiaxing of Zhejiang province, China]. AB - OBJECTIVE: To describe the epidemiological characters of pregnancy-induced hypertension (PIH) in Jiaxing areas of Zhejiang province of China between 1995 and 2000. METHODS: We analyzed the perinatal health surveillance data that was collected as part of the Sino-American cooperative project on neural tube defects prevention established in 1992. The study population consisted of 136 070 pregnant women with at least 20 weeks of gestational age. National diagnostic criteria were used to identify the cases which were divided into three subgroups: mild, moderate and severe. RESULTS: 15 127 cases were identified and the overall incidence rate of PIH was 11.1% (95% CI : 11.0% - 11.3%). Among all the cases, mild, moderate and severe PIH were accounted for 71.4%, 22.3% and 6.3%, respectively. The proportions of PIH cases that occurred in the second trimester, third trimester and during delivery appeared to be 4.2%, 34.4% and 61.4%, respectively. There was a significant fall in the trend of PIH occurrence every year, which dropped from 10.7% in 1995 to 8.6% in 2000 by 19.6%. More risk of PIH seemed to be related to those mothers living in the urban areas with age under 20 or above 35, being peasants and having little educational, having had multiple gestations, conceiving in spring/summer or delivering in winter or spring etc. Compared with the results of national survey in 1988, the incidence rate of PIH was higher by 18.1%, while the proportion of severe PIH was much lower by 68.8%. Although the incidence rates of PIH in urban and rural areas were somehow similar, the proportion of severe PIH in rural areas was much higher than that in urban areas. CONCLUSION: Overall incidence rate and distribution of PIH were reported. Compared with the results in 1988, incidence rate of PIH was much higher, particularly for mild cases. PMID- 16676591 TI - [Cox regression analysis on influencing factors of coronary heart disease among people over 40 years of age]. AB - OBJECTIVE: To explore the affecting factors on coronary heart disease among people over 40 years of age in Guangxi area, China. METHODS: Baseline data was gathered through the Third National Blood Pressure Survey in 1991 in China. A total number of 11 818 adults over 40 years old had been studied in Guangxi province. Data of morbidity and mortality of coronary heart disease was obtained. RESULTS: Cardiovascular events were related to systolic blood pressure, diastolic blood pressure, pulse pressure, smoking, BMI regardless of their myocardial infarct (MI) history. Cox regression analysis showed that the relative risk for cardiovascular events increased by 21 [95% confidence interval (CI): 9.06-48.44] times for those people having MI history. When pulse pressure, systolic blood pressure, diastolic blood pressure increased by every 10 mm Hg, the relative risk for cardiovascular events increased by 1.29 (95% CI: 1.11-1.49), 1.18 (95% CI: 1.02-1.22), 1.13 (95% CI: 1.05-1.28) respectively. There was 1.23 (95% Cl: 1.05 1.45) times higher in smoker than non-smoker on relative risk for cardiovascular events. When BMI increasing 1, the relative risk for cardiovascular events would increase 1.03 (95% CI: 1.01-1 .05) times. CONCLUSION: Hypertension, smoking, increase of BMI were the risk factors of coronary heart disease among people who were over 40 years of age. PMID- 16676592 TI - [Study on the metabolic health risks of body mass index and waist circumference]. AB - OBJECTIVE: To evaluate the effect of body mass index (BMI) and waist circumference (WC) on metabolic health risks (MHR). METHODS: Based on data from two community populations (Suzhou city and Changshu city) derived from a project on 'multiple metabolic disorders and metabolic syndrome' in Jiangsu province. 1604 subjects were divided into groups as normal weight (BMI: 18-23.9), overweight (BMI: 24-27.9) and obesity (BMI > or = 28) according to BMI measurement. According to the results from WC measurement, they were grouped as abdominal obesity (male: > or = 85 cm; female: > or = 80 cm) and normal. Relative risks (RR) for hypertension, hyperglycemia, dyslipidemia were calculated. RESULTS: In males and females, the mark values of hypertension, hyperglycemia, dyslipidemia as well as the prevalence of MS were related to BMI and WC. The relativity remained true when the confounding factors were under control. Among the overweight group, the aRR of all these metabolic health risks were basically lower than those when the overweight group and the abdominal obesity group were combined. Both in males and females, when groups combining normal weight with abdominal obesity group, the aRR of all these metabolic health risks were higher than overweight group combining with normal WC group in most of the cases. CONCLUSION: As to the effect of BMI and WC on metabolic health risks, WC--the mark of abdominal obesity seemed to be more effective than BMI. PMID- 16676593 TI - [Isolation and sequencing of VP1 region of enterovirus 71 strains in Zhejiang, China]. AB - OBJECTIVE: To study the gene characterization of enterovirus 71 (EV71) virus strains isolated from clinical specimens of children with hand-foot-and-mouth disease (HFMD) in Zhejiang province. METHODS: Virus were isolated from clinical samples including stool, throat swab and vesicle from patients with HFMD. The EV71 isolates were identified by microneutralization assay and reverse transcriptase PCR (RT-PCR) with specific primer pair for VP1 genes of EV71. Complete VP1 gene sequences (891 nucleotides) for recent 6 EV71 isolates were determined and compared with that of A, B, C genotype reference EV71 strains and 11 EV71 China isolates available from GeneBank by homogeneity and phylogenetic tree analyses. RESULTS: 9 strains of EV were isolated from 14 clinical specimens. Data from microneutralization and RT-PCR results indicated that all the strains belong to EV71. The nucleotide and amino acid homogeneity of these 6 Zhejiang strains with the representative isolates of A and B genotypes were 82.9%-85.5% and 94.9%-98.0% respectively; with the representative isolates of C were 89.2% 94.1% and 97.0%-99.0% respectively. There were 91.0%-92.2%, 90.2%-90.3%, 89.2% 89.5%, 96.7%-96.9% nucleotide, homology with representative strains of C1, C2, C3,C4 subgenotypes of EV71. The nucleotide homogeneity of these 6 EV71 isolated strains with 9 previously isolated Chinese strains appeared to be 93.8%-97.1%. These 6 EV71 isolated strains were within genotype C subgenogroup C4 in the phylogenetic tree. CONCLUSION: The recently identified EV71 isolates in Zhejiang province belonged to subgenogroup C4. PMID- 16676594 TI - [Study on the relationship between polymorphisms of Cyp1A1, GSTM1, GSTT1 genes and the susceptibility to acute leukemia in the general population of Hunan province]. AB - OBJECTIVE: Based on the distribution of genetic polymorphisms regarding phase I metabolic enzyme cytochrome P450 1A1 (CYP1A1) and phase II metabolic enzymes glutathione S-transferase GSTM1 and GSTT1 genotypes in acute leukemia patients and health controls among general population of Hunan in China, this study was to explore the relationship between these gene polymorphisms and the susceptibility to acute leukemia. METHODS: Using case-control methodology, we studied 204 healthy controls and 232 patients with acute leukemia, of which 112 patients were suffering acute lymphoblastic leukemia (ALL) and 120 with acute non-lymphoblastic leukemia (ANLL). The frequencies of the genotypes were detected by PCR and PCR RFLP techniques. RESULTS: The variation frequencies of CYP1A1 gene (Msp I polymorphisms, site 3801T-C variation) in ALL and ANLL groups were 74.1% and 70.8% respectively which were higher than 63.3% appeared in the healthy controls. However, the differences between patients (ALL or ANLL) and healthy controls were not statistically significant (P > 0.05 for both). The null genotype of GSTM1 (GSTM1 -/-) in ALL group was 60.7%, which was not significantly different from the controls (55.4%). However, GSTM1 -/- genotype in ANLL group was 68.3%, significantly different from the controls (P < 0.05). The null genotypes among GSTT1 (GSTT1 -/-) in ALL, ANLL and control group were 50.9%, 55.0% and 49.0% but their differences were not statistically significant (P > 0.05). The incidences of GSTM1 -/- and GSTT1 -/- combined genotype in ALL, ANLL and control group were 33.0%, 40.0% and 27.5%, of which the difference between ANLL group and control group was statistically significant (P < 0.05) and CYP1A1 gene heterozygous mutation type or homozygous mutation type combined with GSTM1 -/- and GSTT1 -/- increased the risk of ANLL (OR value 1.890, 95% CI: 1.084-3.295). CONCLUSION: These results indicated that both the variation of CYP1A1 gene or GSTT1 -/- genotype alone might not be associated with the susceptibility of acute leukemia while GSTM1 -/- genotype alone or combined with GSTT1 -/- or the 3801 T-C variation of CYP1A1 gene were correlated with ANLL. These findings suggest that GSTM1 - / - genotype alone or in combination with other defective genotypes might serve as risk factors to the etiology of ANLL. PMID- 16676595 TI - [Analysis on molecular characteristic of VP7 and NSP4]. AB - OBJECTIVE: To explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4. METHODS: Double strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR. RESULTS: Serotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05). CONCLUSION: Obvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7. PMID- 16676596 TI - [Detection and analysis of partial sequences isolated from human and swine in rural area of southern China]. AB - OBJECTIVE: To study the genotype of swines isolated from humans and their relationships with hepatitis E virus (HEV) in the rural areas of southern China. METHODS: Specimens collected from normal people with HEV-IgM positive, acute hepatitis E patients and from swine in the same area were detected for HEV RNA using RT-nPCR with ORF2 primers. The positive PCR products were cloned and sequenced. RESULTS: 13 out of the 132 samples from swine stool, 4 of 26 HEV-IgM positive sera of normal people and 1 of 4 acute hepatitis E patients' stool sample and sera were tested positive for HEV RNA. Data from sequence analysis showed that the identity at nucleotide level was 89.3%-100.0% among the 10 isolates which shared 78.7% - 84.7%, 83.3% - 85.3%, 76.0% - 80.0% and 84.7% - 95.3% nucleotide sequence identity with HEV genotype I, II, III and IV respectively in the region (nt6317- 6466). CONCLUSION: HEV circulating in humans and swine in the area belonged to genotype IV. PMID- 16676597 TI - [Study on the compliance and safety of the oral antifungal agents for the treatment of onychomycosis]. AB - OBJECTIVE: To explore the rate of compliance, influencing factors and the safety of patients with onychomycosis under treatment of oral antifungal agents. METHODS: According to the scoring clinical index of onychomycosis (SCIO), 330 patients with onychomycosis, their target nail's integral of the SCIO were calculated and randomly divided into three groups under the baseline of the SCIO integral range. Patients were treated with intermittent pulse itraconazole (A group), continuous terbinafine (B group) and intermittent terbinafine (C group) respectively. Self-administered questionnaire was applied in the survey on every onychomycosis patient. RESULTS: The average rate of compliance was 55.15%. The cure rate for those compliance with doctors' order was 89.01%, while it was only 30.41% for those noncompliant patients The overall non-compliant rate was 44.85%. Among the noncompliant ones, 29.73% were worried about the side effects of medicine, 22.30% thought that they had already been cured, 15.54% was due to economic reasons and 12.16% could not bear the side effects of medicine. It was found that the compliant rates were significantly correlated to ageing, position of the target nails, the integral of the SCIO and the therapy scheme (P < 0.05), while no significant correlations were seen between male and female, culture degree and course (P > 0.1). The frequency of adverse incident of A, B, C groups were 22.73%, 21.43%, 23.15% respectively, but without statistical significance (P > 0.1). Majority of the adverse incidents happened during the first month of therapy but were mild and reversible. CONCLUSION: Our results showed that the overall compliance was low which exerted a significant influence on the curative effect of onychomycosis patients. Factors as ageing, position of the target nail, integral of the SCIO and the therapy scheme had an influence on the compliant rate. When treating onychomycosis with oral itraconazole, the results seemed to be just as safe as when using terbinafine. PMID- 16676598 TI - [Glutathione S-transferase M1 polymorphism and the risk on colorectal cancer: a multilevel meta regression]. AB - OBJECTIVE: In order to investigate the relationship between Glutathione S transferase M1 (GSTM1) status and the risk on colorectal cancer as well as to detect the related factors to this association. METHODS: A pooled analysis of multilevel Meta-regression was performed to estimate GSTM1 deficiency associated with the risks of colorectal cancer. Then subgroup Meta-regression was undertaken to evaluate the possible relationship between heterogeneity and the related characteristics. RESULTS: The overall pooled odds ratios of colorectal cancer risk associated with GSTM1 deficiency was 1.17 (95% CI: 1.08-1.26). Ethnicity, percent of GSTM1 deficiency in population had significant relationships with heterogeneity across the studies (P < 0.05). Results of subgroup Meta-regression showed that GSTM1 deficiency was significantly associated with colorectal cancer risk in ethnic subgroups of Asians, Caucasians and in low level (lower than 50%) of GSTM1 deficiency population (P < 0.05). The respective pooled ORs were 1.14, 1.25 and 1.29. CONCLUSION: GSTM1 deficiency seemed to be a risk factor for colorectal cancer, while interactions on the characteristics of ethnicity, percentage of GSTM1 deficiency in the studied population were related to this association. PMID- 16676599 TI - [Application of Excel Visual Basic for efficiently complete statistic analysis]. AB - OBJECTIVE: In order to analyze multiple statistic tables more efficiently Excel Visual Basic for Application (VBA) was introduced through the use of an example of calculating standardized mortality rates (SMRs). METHODS: Mortality data of cancer and cardiovascular diseases, by sex and age, have been collected from 1991 to 2003 by the Center for Disease Control and Prevention of Shanghai Huangpu District. Standard population composition was defined as Chinese census statistics in 2000. The male's SMRs were calculated, using Excel VBA for each year and classification of cancers. RESULTS: The male's SMRs were obtained by year and different cancers. At the same time, the results were listed in the cancer's SMRs table for male. CONCLUSIONS: Excel is more flexible than general database on the combination of data and annotation. Excel VBA is better than the basic Excel in operating multiple tables simultaneously and man-machine conversation. Statistic analysis can be efficiently completed by using Excel VBA. PMID- 16676600 TI - [Prevention and control of influenza and human avian influenza in China]. PMID- 16676601 TI - [Studies on the spatial distribution and environmental factors of highly pathogenic avian influenza in Mainland China, using geographic information system technology]. AB - OBJECTIVE: To analyze the spatial distribution of highly pathogenic avian influenza (HPAI) and to explore environmental factors associated with HPAI using geographic information system (GIS) techniques in Mainland China. METHODS: Databases were set up using the information of HPAI during epidemics in 2004, and linked to digital maps at provincial and county administrative layers in the country through the ArcGIS 8.3 software. Spatial cluster analyses, spatial statistics analyses and tracking analyses on epidemic situation of HPAI were implemented. Environmental factors associated with HPAI were also analyzed on data related to weather, vegetation and migratory birds etc. RESULTS: Findings from spatial cluster analyses showed that high incidence area was centralized in 113.261 degrees ordm; east longitude and 23. 119 degrees ordm; north latitude with a radius of 1090.52 kilometers (relative risk= 2.646, P value= 0.001). Spatial statistical analyses showed that HPAI took place mainly in capital cities of provinces and surrounding areas as well as in the circumference areas of arterial rivers, lakes and seacoasts. Results also showed that HPAI occurrences were associated with low air temperature, high relative humidity and high air pressure as well as with east & central migration routes of migratory birds. The average normalized difference vegetation index was 0.36 +/- 0.11 in epidemic areas of HPAI. CONCLUSION: HPAI was unrandomly distributed and geographically clustered in China. PMID- 16676602 TI - [Phylogenetic analysis for H3A1 strain of all human influenza A virus]. AB - OBJECTIVE: Influenza A virus remains an important pathogen which threatens humans. With the help of latest developed bioinformatics tools, all available human Influenza A virus H3A1 strains were explored to deeply understanding its evolution and variation rules. METHODS: All data of H3A1 sequence in NCBI Genbank and Influenza sequence database were downloaded and aligned in ClustalX with two step cluster method used to split the data and Bayesian phylogenetic tree analysis method applied to precisely construct phylogenetic tree for each clusters. RESULTS: Tree topology indicated that H3 strains evolved along a single evolution trunk and tree pattern and model parameter showed obvious variety tendency with time period. However, no geographic distribution features were found for key variation strains and big branch in trees. CONCLUSION: The evolution of human H3 strains were mainly driven by the interaction of human immune barriers and antigenic drift of virus. Since the influenza subtype had already been spread in human population, south China should not be considered as the originated areas of new strains, hence it should be treated as equally as other places in the world. PMID- 16676604 TI - [Analysis for development and impacting factors about HIV/AIDS policy in China]. PMID- 16676603 TI - [A study on the influenza surveillance program in Tianjin, 2004]. AB - OBJECTIVE: To explore the trend of influenza epidemics, predominate strains of the virus in Tianjin city and to analyze the economic impact of vaccine-based interventions for the prevention and control of influenza. METHODS: Data on epidemiological studies and on virus surveillance was gathered. Monte Carlo mathematical simulation modelwas used for data analyses. RESULTS: From 29, December, 2003 to 2, January, 2005, the proportion of influenza-like infection cases was accounted for 8.93% of the total number of patients from the outpatient departments of 4 general hospitals in Tianjin. The proportion reached its peak from November to February and it was accounted for 9.39% in epidemic period, which was significantly different from that in the non-epidemic period (u = 15.53, P< 0.05). There was 56 strains of influenza virus isolated in which 45 were indentified as A(H3N2) and 11 as B with a total positive rate as 13.21%. Predominant strain was found bing type A(H3N2). The positive rate was 15.41% in the epidemic period, which was significantly different from that in the non epidemic period (u = 2.519, P < 0.05). The cost per visit ranged from 475.93 to 581.69 Yuan (RMB) with an average cost of 528.81 Yuan. When the attack rate increased to 30 percent, the positive mean net returns would have been 24 million Yuan among the 0-19 age group. CONCLUSION: Influenza did not seem to be prevalent in Tianjin from 29, December, 2003 to 2, January, 2005. The main type of influenza was type A(H3N2). It is necessary to pay attention to the all-year round surveillance program due to the transformation of influenza type A and the slight increase of incidence in summer. PMID- 16676605 TI - [Analysis on the changing of age patterns among rubella patients after rubella vaccine immunization for children in Shandong Province, China]. AB - OBJECTIVE: To analyze the changing of age patterns among rubella patients after implementing rubella vaccine immunization to children in Shandong province since 1995. METHODS: Epidemiologic data on rubella through surveillance system for suspected measles from 1999 to 2004 and data on rubella vaccination were used and analyzed. RESULTS: The annual average incidence rate of rubella from 1999 to 2004 had been 0.59 per 100 thousands population while 81.17% of cases were concentrated during the outbreaks. 77.77% of the cases were school children between 7-15 years old and 7.93% of the cases were under 7 years old. The age median of cases were 10.37, 11.66, 11.41, 12.81, 14.28 and 13.96 years old from 1999 to 2004, respectively. The estimated coverage of rubella vaccine for pre school children was about 60% but only 20% were for school children. CONCLUSION: The peak age of cases moved from youth towards adolescence which indicated that women with child-bearing age might have been under risk of developing the congenital rubella syndrome (CRS). It is necessary to carry out screening test of rubella antibody and vaccination to women with child-bearing age and the immunization strategy should be established to guide the control of rubella and CRS. PMID- 16676606 TI - [A cross-sectional survey on hepatitis C virus infection among residents aged 18 59 years in a former commercial blood donating community, Shanxi Province]. AB - OBJECTIVE: To determine sero-prevalence of and risk factors for hepatitis C virus infection (HCV) among residents aged 18-59 years in a former commercial blood donating community, Shanxi province. METHODS: A community-based cross-sectional survey was conducted among 660 randomly selected residents aged 18-59 years from 12 villages in a rural county of Shanxi province. Structured questionnaire was administered to collect socio-demographic,medical and risk behavioral information. 7 ml-volume venous blood was collected for HCV antibody testing. RESULTS: The overall HCV sero-prevalence rates were 8.2% and 27.7% among former commercial blood donors in the community. Data from multivariate logistic regression analysis showed that people ever selling blood (adjusted OR = 14.28, 95% CI: 6.83-29.87) and receiving blood transfusion (adjusted OR = 8.66,95% CI: 2.31-32.47) were the primary risk factors for HCV infection. Subgroup analysis in blood donors explored that ever selling plasma (adjusted OR = 8.56,95% CI: 2.87 25.54) was the risk factor for HCV infection. Villagers who stopped selling blood in 1994 and thereafter were less likely to be HCV positive than those stopping selling blood before 1994 (adjusted OR = 0.32,95% CI: 0.11-0.93). Female donors had a lower risk than male ones (adjusted OR = 0.28, 95% CI: 0.10-0.83). CONCLUSION: A HCV epidemic was once existing in rural community residents in the province that former commercial blood/plasma donation was the main reason for HCV epidemic in the community. It is urgent to make efficient measures to prevent HCV secondary transmission and provide patients with care and treatment to this community. PMID- 16676607 TI - [Study on Bartonella species in rodents in western Yunnan, China]. AB - OBJECTIVE: To study the infection status of Bartonella spp. in rodents in western part of Yunnan province. METHODS: Blood samples were collected from four species of rodents captured in four counties in western Yunnan in 2004. Bartomella was isolated through being cultured in brain and heart infusion agar media containing 5% rabbit blood. Suspective Bartomella strains isolates were confirmed by amplification of 379 bp of citrate synthase (gltA) gene with specific primer by polymerase chin reaction (PCR). RESULTS: Fifty-four strains of Bartomella isolates were obtained from 397 samples including four rodent species captured in the fields with an overall isolation-rate of 13.6% (54/397). The rates of isolation among different species were: 22.0% (22/100) in Rattus nitidus, 14.8% (31/210) in Rattus flavipectus and 1.2%(1/87) in Rattus norvegicus while in R. t. yunnanensis it was negative. CONCLUSION: These findings demonstrated that the local rodents in western Yunnan were widely infected by Bartomella spp. It is indispensable to study the vector and the route of transmission to discover the relations between Bartomella and human diseases. PMID- 16676608 TI - [Study on the geographic information system databases regarding the control of schistosomiasis in Zhongxiang, Hubei province, China]. AB - OBJECTIVE: Using geographic information system (GIS) and the remote sensing techniques (RS), we developed a schistosomiasis database and geographic distribution map in Zhongxiang city,Hubei province in order to display and analyze the endemic situation longitudinally after the water conservancy project is completed. METHODS: Epidemiological data of schistosomiasis and the correlated climate and hydrology data for the last 30 years were collected and the relevant GIS databases were established under Artificial Neural Networks(ANN) and network training of Landsat TM images. RESULTS: GIS database of schistosomiasis in Zhongxiang city, Hubei province and its vicinity areas were developed including 1 maps regarding the epidemic situation of schistosomiasis. The areas of snail distributing were 4.4 hm2, 8.2 hm2, 24 hm2, 130.4 hm2, 8.13 hm2 and 7.53 hm2, respectively. CONCLUSION: The maps created by GIS database and RS techniques supported the complicated query on space and property, providing a new way in keeping,updating and analyzing available data. The techniques used should be able to provide evidence for the control of schistosomiasis to this water conservancy project. PMID- 16676609 TI - [Study on the related factors of smoking behavior transition at different stages among undergraduate students]. AB - OBJECTIVE: To explore the influencing factors of smoking behavior transition at different stage of smoking among undergraduate students. METHODS: 8138 undergraduate students from grade 1 to 3 were selected using cluster sampling method from an university in Guangzhou with their smoking behaviors and relevant factors studied through a self-administered questionnaire. Beginners, un-regular smokers and regular smokers were defined as "case group", while non-smokers, beginners and un-regular smokers were defined as "control group" accordingly. Three multivariable logistic regressions were separately conducted to explore the influencing factors of behavioral transition at different stage of smoking. RESULTS: Of the 8138 students investigated, the prevalence rates of beginners, un regular smokers and regular smokers were 19.6%, 2.0% and 1.1%, respectively; while all of the above said three rates increased with grades of the students. Some students might still try to smoke or become regular smoker in the university. In male undergraduate students, determinants for the transition from non-smokers to s mokers were: source of students, father' s educational level, smoking-related knowledge and belief, and smoking habit of the roommates. Risks that influencing the transition from beginners to un-regular smokers were: being non-medical students,smoking-related belief, roommates and classmates who were smokers and transition from un-regular smokers to regular smokers would include father' s educational level and average income of family. In female undergraduate students,influencing factors from non-smokers to beginners were mother's educational level, smoking-related belief, and roommates smokers, while father's educational level, roommates and classmates' smoking habits were significant associated with beginners' change to un-regular smokers. CONCLUSION: The influencing factors related to the transition of different stage on smoking would be different in undergraduate students,and some specific measures should be taken based on the different stage of smoking. PMID- 16676610 TI - [An epidemiological study on mental problems in adolescents in Chengdu, China]. AB - OBJECTIVE: To study the mental health problems in the adolescents aged 11-18 years in Chengdu, China. METHODS: Youth Self-Report (YSR) and the General Information Questionnaire were administered to 1802 students who were randomly selected from schools in Chengdu City. RESULTS: The overall rate of mental problems was 15.1% in the adolescents, 15.2% in boys and 14.9% in girls if 90th percentile of total scores and/or 98th percentile of syndromes scores were assigned as the cutpoint of norms. The overall rate of mental problems was 27.2% in the adolescents, 27.6% in boys and 26.8% in girls if 75th percentile of total scores and/or 98th percentile of syndromes scores were assigned as the cutpoint of norms. The rates of mental problems were not significantly different between boys and girls (chi-squared = 0.042, P = 0.838; chi-squared = 0.126, P = 0.723). Mental problems tended to be increasing with age (P = 0.000). There were 29 items that the percentiles were higher than 50% but 5 items were under 10% in all the 101 items of YSR. 3.1% of the adolescents often had suicidal intention, and 6.1% of the adolescents often thought of becoming opposite sex identity. CONCLUSION: The rates of mental problems in adolescents aged 11-18 years in Chengdu, Chana were 27.2% or 15.1% respectively,if 75th or 90th percentile of the total scores were assigned as the cutpoint of total scores. The mental problems in the adolescents tended to be increasing along with age. PMID- 16676611 TI - [Risk factors for dementia and Alzheimer' s disease-findings from a community based cohort study in Stockholm, Sweden]. AB - OBJECTIVE: It is known that dementia is a multi-factorial disorder, but the etiological factors other than aging remain to be explored, hence we sought to investigate the risk factors of dementia and Alzheimer's disease (AD). METHODS: We followed a community-based dementia-free cohort (n = 1301) aged 75 years and over in Stockholm, Sweden. Baseline data were obtained through a structured interview and extensive clinical examination, or by reviewing the inpatient register database. We used the DSM-III-R criteria to define dementia and AD cases. RESULTS: Over six years of a follow-up program,350 subjects were diagnosed as dementia, including 260 Alzheimer cases. Multiple Cox regression analysis suggested that older age,low education (< 8 years), cognitive impairment, functional disability (ADL > or = 1), low diastolic pressure (< 70 mm Hg), diabetes mellitus, coronary heart disease, and APOEepsilon4 allele were significantly or marginally associated with subsequent development of dementia and AD. Dementia was related also to stroke and atrial fibrillation. Antihypertensive drug use was associated with a lower risk of AD and dementia. CONCLUSIONS: Our study revealed that some sociodemographic features, cognitive and physical dysfunctions, vascular disorders, and genetic susceptibility were major risk factors for dementia and AD. Use of antihypertensive drugs might protect against the dementing disorders in a very old population. PMID- 16676612 TI - [To assess the demands of community-based health care service among 717 elderly]. AB - OBJECTIVE To understand the demands and influencing factors on the community based health care service (CHS) among the elderly and to provide evidence of implementing health service for them METHODS: Cluster sampling method was used on 717 persons aged 60 and above in Yanjiao Developing Area. A survey was carried out, using questionnaire and focus group discussion approaches. RESULTS: Data showed that a two-week prevalence of diseases was 56.3% while the overall prevalence of chronic diseases was 92.1% with 70.0% of them suffering from more than two kinds of chronic diseases. The rates of ADL and IADL loss were 1.8% and 7.0% respectively with 37.0% of them sought medical consultation in the past two weeks. Major reason affecting the use of health service was low income. 57.7% of the elderly expressed their willingness of accepting CHS with the strongest demands as regular physical check-up, health education and consultation, specialized care services, home care service while major reasons hindering the acceptance of CHS could be referred to self-perception on good stamina and lack of awareness to CHS. CONCLUSION: The demands of CHS in the elderly were greater than those in general population that called for CHS programs to be acceptable and relevant to meet the different needs. PMID- 16676613 TI - [Epidemiological study on the occupational noise exposure and hypertension in mechanic factory workers]. AB - OBJECTIVE: To study the relations of noise expose and hypertension in mechanic factory workers. METHODS: A cross-sectional study on 1205 workers (exposed to different noise levels) in Hunan was carried out, using questionnaire, blood pressure of the workers and the exposure level to noise at workplace. RESULTS: The prevalence of hypertension was 12.1% in mechanic factory workers. There was an increasing tendency of hypertension rate along with the increase of accumulative noise doses (tendency chi-squared = 29.932, P < 0.01). Result by logistic regression analysis after adjusting age, history of hypertension in parents and body weight index showed that the risk of hypertension increased about 5% by 1 dB(A) of more noise exposure (OR = 1.047). CONCLUSION: Noise exposure might serve as a risk factor of hypertension. Reducing the sound pressure level in workshops could work as an effective measure to control the incidence rate of hypertension in mechanic factory workers. PMID- 16676614 TI - [Study on hepatitis C virus genotyping in Yixing area, Jiangsu province]. AB - OBJECTIVE: To investigate the distribution of hepatitis C virus (HCV) genotypes in Yixing, Jiangsu province. METHODS: Genotypes identification on sera samples were obtained from 158 donors who had already been anti-HCV positive through PCR method with type specific primer designed according to the sequence of 5'non coding region (5'NCR). 5'NCR was also sequenced and compared with published date. Genotypes distribution was investigated in patients with different sex and clinical types of hepatitis C. RESULTS: Of the total 158 patients, 95 were HCV RNA positive in which 80 patients having genotype 1b (80/95; 84.4%), 5 patients having genotype 2(5/95; 5.3%), 5 patients with 1b/2 mixed genotypes (5/ 95; 5.3%) and another 5 patients whose genotype undetermined. The difference on the distribution of HCV genotypes was significant between female and male patients (P < 0.05) but not in different kinds of hepatitis C patients. CONCLUSION: Type 1b was the predominant HCV genotype in Yixing area. PMID- 16676615 TI - [Study on the mimic epitopes screening program severe acute respiratory syndromes antigen with random phage peptide library]. AB - OBJECTIVE: To screen the severe acute respiratory syndromes (SARS) mimotopes with random phage peptide library and to investigate their immunogenicity. METHODS: Using SARS sera as selective molecule, a 12 mer phage peptide library was biopanned and positive clones containing the mimic epitopes were selected. The immuno-characteriation of the epitopes were then investigated. RESULTS: 2 positive clones that having specific affinity to SARS sera were obtained. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the sequence of SARS. CONCLUSION: SARS mimotopes were obtained by phage peptide library screening. This method might provide a new approach for SARS therapy and vaccine development. PMID- 16676616 TI - [Study on matrix metalloproteinase 1, 9, 12 polymorphisms and susceptibility to chronic obstructive pulmonary disease among Han nationality in northern China]. AB - OBJECTIVE: To study the association between the functional polymorphism of matrix metalloproteinases (MMPs) and the development of chronic obstructive pulmonary disease (COPD). METHODS: 147 COPD patients and 120 healthy smoking controls were selected. Spirometry and chest X-rays had been taken. Questionnaires including sex, age, smoking history, occupational exposure were completed. MMP-9 (-1562 C/T), MMP-1(-1607 1G/2G), MMP-12 (-82 A/G), MMP-12(-357 Asn/ Ser) alleles were determined using PCR-RFLP method. Independent samples T test analysis was carried out to compare patients' age, smoking index, FEV1 /FVC, FEV1 % pred with that of healthy controlled group. The frequencies of genotypes and alleles between groups were analyzed by chi-square tests and multilogistic regression. RESULTS: MMP12 Asn/Asn, CT/AsnAsn were risk factors for smoking-induced COPD. The ORs were 2.361 (95% CI: 1.369-4.017) and 2.433(95% CI: 1.159-5.342) respectively while CC/1G1G/ SerSer seemed to be a protective factor for smoking-induced COPD, with OR as 0.457 and 95% CI as 0.231-0.911. CONCLUSION: Asn/Asn, CT/AsnAsn might be susceptible genotypes while CC/GG/SerSer might serve as protective genotype. PMID- 16676617 TI - [Study on genetic epidemiology on 815 patients with vitiligo in Zhejiang area]. AB - OBJECTIVE: Genetic factors are thought to be involved in the development of vitiligo. The aim of this study is to explore the possible genetic model of vitiligo by analyzing the genetic characteristics of 815 patients from Zhejiang province. METHODS: Data for 815 patients with vitiligo together with their first- and second-degree relatives were obtained using a standardized questionnaire. All these information was requested to confirm the answers about family history in order to reduce the possibility of 'recall' bias. The 815 probands would include 411 (50.43%) males and 404 (49.57%) females with a varied age from 2 months to 71 years old. Since the information on general prevalence of vitiligo in this area was absent, a control group was set up to facilitate the calculations of heritability degree. 468 persons of the control group were from non-vitiligo population with a sex ratio of 241(male): 227(female) with varied age of 4 months to 80 years old. Both gender and age were comparable between the vitiligo and the control population. The inheritance pattern estimation, heritability calculation and complex segregation analysis were performed with Penrose method, Falconer regression method and SAGE-REGTL program. RESULTS: In 815 vitiligo probands, 128 had and 687 had not family histories, with a heritability rate of 15.7%. The vitiligo prevalence in proband's first degree relatives was 2.580%, higher than the prevalence of 0.618% in second degree relatives, and both of them were higher than general prevalence: 0.192%. By Penrose method, the rates on different catagories were as follows: sibling prevalence rates s = 0.080 18; population prevalence rate q = 0.001 92; s/q = 41.76. The ratio of s/q did not approach 1/2q (260.42) or 1/4q (130.21), but approached 1/square root of q(22.82), suggesting vitiligo was consistent with a mode of polygenic inheritance. Using Falconer's method, heritabilities of vitiligo in first-and second degree relatives of probands were 59.61% (95% confidence interval 65.37-53.84) and 55.20% (95% confidence interval 43.88-66.52), respectively. The weighted average of heritability in all relatives was 58.7% (95% confidence interval 53.56-63.83). The results of complex segregation analysis suggested that major gene model including the Mendelian dominant, recessive and additive hypotheses were not rejected (P > 0.05). Purely environmental model and no transmission model were rejected at a 0. 001 significance level. According to AIC, Mendelian dominant inheritance was the best-fitted hypothesis. CONCLUSION: Genetic factors played an important role in the occurrence of vitiligo, and the genetic model of vitiligo could serve as the polygenetic or multifactorial inheritance with major gene trait. PMID- 16676618 TI - [A case-control study on small-for-gestational-age in relation to obstetrical risk factors]. AB - OBJECTIVE: Previous studies suggested that a number of obstetrical factors were associated with small-for-gestational-age (SGA) infant. However, it remained uncertain which obstetrical factors might increase the risk of SGA due to limitations of small sample size and poor study designs in the previous studies in China. We assessed the association of a few maternal factors, such as pregnancy-induced hypertension (PIH), oligohydramnios, infectious diseases of reproductive system, abnormal conditions of placenta or umbilical cord, previous adverse pregnancy outcomes, hysteromyoma and maternal education level, and the risk of SGA. METHODS: We examined the association in a case-control study, in which the SGA group included all of 834 (443 males and 391 females) singleton alive SGA deliveries in our hospital during January 2000 to October 2004. 2502 (1329 males and 1173 females) appropriate-for-gestational-age (AGA) infants (1:3, SGA/AGA) matched by gender- and gestational-age-stratified frequencies of SGA were randomly selected for controls from infants with sex-specific birthweight for gestational age ranged between 10th-90th percentiles. Odds ratios (95% confidence interval, 95% CI) of these factors for SGA were calculated in univariate and multivariate analyses. The maternal risk factors were diagnosed by the relevant criteria used in China. SGA was defined as sex-specific birthweight for gestational age that was less than 10th percentile cut-off of fetal growth reference of singleton alive infants delivered in our hospital during that period. RESULTS: Results from univariate analysis showed that PIH, oligohydramnios, history of adverse pregnancy outcome, poor maternal education and viral hepatitis were significantly associated with SGA. The odds ratios (95% CI) of these five factors for SGA were 3.95(2.79-5.60), 2.94(2.27-3.80), 7.01(3.87-12.70), 3.62(2.92-4.49) and 0.52(0.32-0.87), respectively. In the multivariate logistic analysis, a similar result was shown. The ratios of maternal PIH, oligohydramnios, history of adverse pregnancy outcome and viral hepatitis in the SGA group were 4.00(2.81-5.71), 2.95 (2.27-3.83), 5.95 (3.05 10.64), 0.50(0.30-0.84) folds of those in the AGA group; the ratio of maternal low formal education (< or = 9 years) in the SGA group was 3.46(2.75-4.24) times of the AGA group. CONCLUSION: PIH, oligohydramnios, poor maternal education and a history of adverse pregnancy outcome were significantly associated with an increased risk of SGA while viral hepatitis might be associated with a decreased risk of SGA. PMID- 16676619 TI - [Study on the impact of exposure misclassification on the validity of a study]. AB - OBJECTIVE: To explore the impact of misclassification in binary explanatory variables on the effects associated with 'exposure-disease'. METHODS: Based upon the functions of probabilities on misclassification, effects of association and proportions of exposure, the 'R Project for Statistical Computing' method was used to analyze the impact of misclassification on the validity of a study. RESULTS: To the linear model case, the effect of nondifferential misclassification serves as an attenuating bias. When r = 0.5, the bias is symmetric in both sensitivity and specificity but when r is not equal to 0.5, the bias is not symmetric in sensitivity and specificity. When misclassification is nondifferential, estimated odds ratio tends to be 1 while the exposure prevalence in the control tends to be 0 or 1. Bias seems to be very complex in differential misclassification than in nondifferential misclassification that can make OR tend to or be away from the null value. CONCLUSION: The impact of exposure misclassification on the effect associated with exposure--disease is complicated, hence necessary to understand, to control, and to assess bias of misclassification in order to correctly interpret the results of a study. PMID- 16676620 TI - Belief in the inevitability of suicide: results from a national survey. AB - To examine public opinion regarding the effectiveness of means restriction as an approach to preventing suicide we asked a national sample of 2,770 respondents a hypothetical question about what effect a suicide barrier might have had on the ultimate fate of the more than 1,000 people who have jumped to their death from the Golden Gate Bridge. Thirty-four percent of respondents believed that every single jumper would have found another way to complete suicide and an additional 40% believed that "most" would have completed suicide using other means. The strongest predictors of belief in complete substitution were firearm ownership and cigarette smoking. Belief in the inevitability of suicide may be a political impediment to adopting potentially effective suicide prevention efforts. PMID- 16676621 TI - Suicidal ideation and attitudes toward suicide. AB - Although hopelessness and depression are known risk factors for suicide, most individuals who are hopeless or depressed never make a suicide attempt. In this study, we tested the hypothesis that college students' (n = 230) attitudes toward suicide (the degree to which they see it as an acceptable option under some circumstances) would moderate the link between both hopelessness and depressive symptoms and their levels of suicidal ideation. This moderation hypothesis was supported, but only among men. Specifically, among men, levels of hopelessness and depressive symptoms were significantly related to suicidal ideation among only those with relatively positive attitudes toward suicide. PMID- 16676622 TI - Risky assessments: participant suicidality and distress associated with research assessments in a treatment study of suicidal behavior. AB - The purpose of this study was to examine patterns of self-reported suicidality and distress during research assessments in a sample of 63 women meeting criteria for borderline personality disorder and current and chronic suicidality. The risk management protocol we used during the two-year study period (University of Washington Risk Assessment Protocol; UWRAP) is described. Results indicated that changes in suicidality following assessments were small and relatively infrequent, and were just as likely to reflect decreases in suicidality as increases (17.5% versus 16.4% of sessions, respectively). Further, longitudinal analyses indicated that changes in suicidality became increasingly rare over the course of the 2-year study. Ratings of distress were more changeable than suicidality, underscoring the need for separate measurement of these constructs when assessing risk. With the aid of the UWRAP, our assessors judged 15 participants as high-risk status in 28 assessment sessions (3.7% of all sessions). In comparison to the rest of the sample, these individuals were of significantly greater clinical severity as measured by the HRSD 17-item, GAF scores, number and severity of previous suicide attempts, and number of previous psychiatric hospitalizations. Low-intensity risk intervention strategies (e.g., validating participant's feelings) were typically sufficient to reduce risk in these participants. Overall, our findings indicate that research with highly suicidal individuals can be done safely with the use of well-trained assessors and an appropriate crisis management protocol. PMID- 16676623 TI - The cycle of schizoaffective disorder, cognitive ability, alcoholism, and suicidality. AB - In this study we investigated the putative role of cognitive dysfunction, diagnosis (schizoaffective versus schizophrenia disorder), and alcoholism as risk factors for suicidal behavior among individuals with DSM-TV schizophrenia or schizoaffective disorders. Subjects received cognitive tests and medical records were reviewed for evidence of a history of suicide attempts or suicidal ideation. Discriminant analysis was used to identify cognitive test performance measures that distinguished those with versus those without suicidal behavior. None of the cognitive measures discriminated between the two groups. The rates of suicidal behavior (suicidal ideation and suicide attempts) did not differ between participants with versus those without comorbid alcohol use. An association was found between suicidal behavior and the diagnosis of schizoaffective disorder. It was concluded that the history of prominent mood syndromes characteristic of schizoaffective disorder contributes to increased risk of suicidal behaviors. Cognitive dysfunction and/or alcoholism did not contribute additionally to risk in this study. PMID- 16676624 TI - Violent self-harm in schizophrenia. AB - Patients with schizophrenia have a substantial lifetime suicide risk, especially by violent means. Little published work exists on self-harm (SH) in this population. The goal of this study was to examine whether patients with schizophrenia were also more likely to self-harm in a violent manner. A retrospective analysis performed on method, motive, and suicidal intent in patients with schizophrenia (n = 50) and adjustment reaction (n = 138) who presented with SH over a 3-year period found that there was no significant difference between the two groups in terms of violence of method or suicidal intent. Presence of positive symptoms of schizophrenia was not significantly associated with use of a violent method. This study suggests that the expected pattern of violent SH in schizophrenia is inaccurate and for the majority the acts are of a similar nature to those seen in adjustment reactions. PMID- 16676625 TI - Sex differences in completed suicide by schizophrenic patients: a meta-analysis. AB - A meta-analysis was conducted on all studies of suicide mortality in follow-up studies of schizophrenic patients that presented data for male and female patients separately. The percentage of deaths from suicide was significantly greater for the male schizophrenic patients than for the female schizophrenic patients in studies where both sexes were included. Regression equations devised to predict the percentage of deaths from suicide after all of the sample had died estimated that 0.50% of male schizophrenic patients would die from suicide as compared to 0.20% of female schizophrenic patients. Suggestions are made for future research. PMID- 16676626 TI - Associations between delinquency and suicidal behaviors in a nationally representative sample of adolescents. AB - Suicide was the second leading cause of death for 14-17 years olds in 2002. Prior studies indicate that suicidal behaviors are especially common among juvenile delinquents, yet this association has not been examined in a national sample. The 2003 Youth Risk Behavior Surveillance System was used to examine associations between suicidal behaviors and delinquency in a nationally representative sample of 14-17 year olds. Results indicated that delinquent adolescents were more likely to have seriously considered suicide (adjusted odds ratio [AOR] = 5.44), made a suicide plan (AOR = 4.90), attempted suicide (AOR = 10.08), and required medical treatment after attempting suicide (AOR = 14.61) compared to their nondelinquent counterparts. Delinquency was significantly related to suicidal behaviors for boys and girls, but the magnitudes of effects were larger for girls. This finding suggests that delinquent youth, particularly delinquent girls, should be targeted in interventions to reduce suicidal behaviors. PMID- 16676627 TI - Correlates of suicide among home health care utilizers who died by suicide and community controls. AB - Home health care patients often have several late-life risk factors for suicide and constitute a high risk group for suicidal behaviors. In this study, we examined the characteristics of 14 older adult home health care utilizers who died by suicide and four community controls who used similar services. Both groups of home health care utilizers had multiple health problems and, with one exception, all were functionally impaired. Descriptive comparison suggests that suicide decedents were more likely than their counterparts to suffer from depressed mood, suicidal ideation, prior suicide attempt(s), alcohol dependence, chronic pain, strained family relationships, lack of social support, and financial difficulties. Identifying suicide risk factors that place older adult home health care utilizers at particularly high risk for suicidal behaviors within this population will further help target suicide preventive interventions by home care providers. PMID- 16676628 TI - Suicides in the developing world: case study from Pakistan. AB - There are no official data on suicide from Pakistan, a conservative South Asian Islamic country with traditionally low suicide rates. Both suicide and attempted suicide are illegal acts, as well as socially and religiously condemned, making research in this area difficult. Recent reports suggest an increase in suicide rates. In this study, police data from the Sindh province were examined to provide a unique picture of trends of suicide over 15 years (1985-1999). During this period there were 2,568 reported suicides (71% men, 39% women; ratio 1.8). The lowest number was 90 in 1987 and maximum was 360 in 1999. Poisoning by organophosphates was the most common method followed by hanging. This study, although limited in scope, provides evidence of an increase in suicide rates in Pakistan, from one data source. There is urgent need for further research on suicide in Pakistan; interventions for suicide prevention in the country can then be planned. PMID- 16676629 TI - Predictors of suicide ideation and depression in Hong Kong adolescents: perceptions of academic and family climates. AB - The unique dimensions of perceptions of school and family contributing to depression and suicide ideation in Hong Kong adolescents were examined in two studies. In Study 1, among 327 Hong Kong Chinese female students ages 13-18, 47% reported some suicide ideation. Suicide ideation was significantly associated with depression, test anxiety, academic self-concept, and adolescents' perceived parental dissatisfaction with academic performance. The correlation between test anxiety and depression was especially high (r = .51). Study 2 examined how three different aspects of perceived family relationship were associated with depression and suicide ideation. Among 371 Hong Kong Chinese adolescents ages 14 20, 52.6% reported suicide ideation. Low levels of family cohesion and support and high levels of parent-adolescent conflict were positively related to depression and suicide ideation in both genders. Across both studies, depression mediated associations between academic- and family-related variables and suicide ideation. Findings underscore the importance of both academic and family climate in understanding depression and suicide ideation among Chinese adolescents. PMID- 16676630 TI - Suicide attempts in israel: age by gender analysis of a national emergency departments database. AB - An analysis of all emergency department admissions in Israel classified as an attempted suicide in the years 1996-2002 was done to examine attempted suicide rates by age and gender with particular attention to adolescents and young adults. Gender differences in attempted suicide rates were significant only during adolescence and young adulthood, ages 13 to 26. The highest rate for females was 3 years earlier than the highest rate for males. The results lead to the conclusion that women do not attempt suicide more than men, except for 13-26 year olds. PMID- 16676631 TI - Six year follow-up of health status changes in Danish adults with suicide tendency in 1994. AB - The purpose of this study was to determine whether suicidal ideation and/ or suicide attempts have any long-term health effects. The relationship between suicidal thoughts and/or a previous suicide attempt in 1994 and the presence of suicidal ideation in 2000 was analyzed. We found that health status in 1994 differed significantly between the groups with and without suicidal ideation or a previous suicide attempt and that the differences persisted or even deepened (not significant) after 6 years. Further, people who had suicidal ideation or a suicide attempt in 1994 had a significantly greater risk (OR = 7.4) of having suicidal ideation in 2000 than people who had not. PMID- 16676632 TI - Social inequalities in suicide mortality: Spain and France, 1980-1982 and 1988 1990. AB - In this study we analyzed the socioeconomic differences in mortality from suicide in the economically active male population aged 25-64 years in Spain and France in 1980-1982 and 1988-1990; in the case of Spain the data came from the Eight Provinces Study (Regidor, Gutierrez-Fisac, & Rodriguez, 1995). Individuals were grouped into four categories: professional/managerial, clerical/sales/ service, agricultural, and manual workers. For 1980-1982, among those aged 25-44, professionals and managers had the lowest risk of mortality in Spain, and clerical/sales/service workers in France. These socioeconomic differences in mortality increased in 1988-1990. In 1980-1982, among those aged 45-64, clerical/ sales/service workers had the lowest risk of mortality from suicide relative to the other occupational groups in both countries, but this difference was not maintained in 1988-1990. Thus, differences in suicide mortality for men by occupational status depended in the present study upon both the nation studied and the time period chosen for study. PMID- 16676633 TI - Prevalence of suicidal ideation in patients with atopic dermatitis. AB - The prevalence of suicidal ideation in patients with mild, moderate, and severe atopic dermatitis between the age of 15 to 49 years were 0.21%, 6%, and 19.6%, respectively. In addition, the prevalence of homicide-suicidal ideation in mothers or fathers of patients (aged 0-14 years) with mild, moderate, and severe atopic dermatitis were 0.11%, 0.35%, and 3.28%, respectively. These results indicate that patients with atopic dermatitis or even parents of patients with atopic dermatitis have high prevalence of suicidal ideation. PMID- 16676634 TI - Durkheim's precedence in the use of the terms egoistic and altruistic suicide: an addendum. PMID- 16676635 TI - CMS program P4P precursor. The Physician Voluntary Reporting Program looks to get providers in a pay-for-performance state of mind. PMID- 16676636 TI - A means to an end. The goal is not just to automate, but to make sure that automation improves the quality of healthcare. PMID- 16676637 TI - Nursing and IT: perfect together. Standardized language and nanotechnology line the IT road ahead. PMID- 16676638 TI - Disaster waiting to happen. The national healthcare system has much work to do before the next catastrophe strikes. PMID- 16676639 TI - Give till it hurts. Some industry groups say the proposed relaxation of HHS donation guidelines don't go far enough, others fear they may go too far. PMID- 16676640 TI - Gateway to success. With consumers becoming more responsible for their healthcare choices, portals can help separate tech-savvy providers and payers from the competition. PMID- 16676641 TI - Raising the bar. Barcoding has the potential to dramatically reduce medication errors. PMID- 16676642 TI - Flying high. Kaiser Permanente chairman and CEO George Halvorson pilots the giant payer/provider toward success by embracing it. Interview by Mark Hagland. PMID- 16676643 TI - Nursing takes center stage. Hospitals are realizing that a singular focus on the needs of physicians can leave nurses out in the cold. PMID- 16676644 TI - Spanning the globe. A look at EHR projects in other countries may provide a roadmap for initiatives here at home. PMID- 16676645 TI - Lending rural hospitals a hand. By networking its PACS with eight remote hospitals, Kalispell Regional Medical Center brought radiologists (virtually) to them. PMID- 16676646 TI - Strike up the band. Finding a new way to deal with bandwidth demand helped New York-Presbyterian Hospital put a cap on costs. PMID- 16676647 TI - Better health well in hand. Cell phones have the capacity to more frequently and efficiently connect chronically ill patients with caregivers. PMID- 16676648 TI - Cardiac health for astronauts: coronary calcification scores and CRP as criteria for selection and retention. AB - Due to the limited treatment and return capabilities of most space vehicles, an in-flight cardiac event could result in significant mission impact or even failure. The current literature supports including electron-beam computed tomography (EBCT) and highly selective C-reactive protein (hsCRP) for diagnosis of coronary artery disease (CAD) in asymptomatic, low-pretest probability cohorts. This paper will examine the issues surrounding adding these tests to astronaut retention and selection algorithms. An evidenced-based literature review was performed and consensus obtained from subject-matter experts to create novel cardiac screening algorithms for astronaut applicants and the current astronaut corps. The main focus of this paper is to derive an evidenced-based approach for improving the diagnosis of significant CAD using EBCT and hsCRP testing. The recommended initial astronaut selection and long-duration mission assignment screening algorithms use EBCT-derived calcium scores and serum hsCRP levels to screen for CAD and predict individual cardiac risk. The current medical evidence is compelling for the international space medicine community to consider: (1) Astronaut candidates with a coronary artery calcium score >0 should be disqualified from initial selection; (2) Astronauts with a coronary artery calcium score >100 should be disqualified from selection for long-duration missions; (3) Elevated hsCRP is a reliable risk factor for helping predict future cardiac events that should warrant primary prevention but not necessarily medical disqualification. PMID- 16676649 TI - Head and helmet biodynamics and tracking performance in vibration environments. AB - INTRODUCTION: There are potential effects of vibration on aircrew performance and safety when using helmet-mounted equipment. The objective of this study was to quantify the effects of head orientation and helmet center-of-gravity (CG) on head and helmet biodynamics and tracking performance during exposures to aircraft buffeting and quasi-random vibration. METHODS: Three head orientations, including two off-axis or off-boresight configurations [Side (40 degree elevation, 70 degree azimuth) and Up (40 degree elevation, 0 degree azimuth)], and three helmet CGs were tested. The overall head, helmet, and helmet slippage displacement rotations, and rms tracking error and percent time-on-target were evaluated. RESULTS: For both exposures, the two off-axis orientations produced significantly higher head, helmet, and slippage displacements; a relationship was observed between the orientation and the rotation that was affected (roll, pitch, or yaw). The highest slippage observed was in pitch in the forward (For) and Up orientations. Significantly higher performance degradation occurred with the Side orientation for two of the three CGs during aircraft buffeting, with minimal degradation observed with the quasi-random exposure. Higher head pitch and lower pitch slippage were associated with the CG estimated to produce loading behind the human head CG. CONCLUSIONS: The high off-boresight head movements may influence visual performance in operational vibration environments. Helmet instability appeared to be the greatest in pitch, which could have a significant effect on the design size of the exit pupil. The weight distribution or moments of-inertia of the helmet system may also have a significant influence on both head/helmet biodynamics and tracking performance and should be investigated. PMID- 16676650 TI - Cranial-neck and inhalation rewarming failed to improve recovery from mild hypothermia. AB - INTRODUCTION: Rewarming from hypothermia in a field setting is a challenge due to the typical lack of significant power or heat source, making the targeted application of available heat critical. The highly vascular area of the head and neck may allow heat to be rapidly transferred to the core via blood circulation. At the same time, the warming of only a small skin surface may minimize the rapid rise in skin temperature proposed to attenuate shivering and endogenous heat production. Therefore, we investigated the efficacy of targeting the head and neck for rewarming from mild hypothermia. METHODS: There were 16 participants (9 men, 24.1 +/- 4.5 yr, 15.5 +/- 3.9% body fat; 6 women, 23.0 +/- 5.4 yr, 20.8 +/- 3.2% body fat) who were cooled in 15 degrees C water until rectal or esophageal temperature reached 35.5 degrees C, whereupon they were removed and provided passive (PASS), cranial-neck (CN), or cranial-neck and inhalation (CNIR) rewarming. Heart rate and skin temperature were also measured. RESULTS: The mean cooling times were PASS=83 min (range: 22-295 min), CN=94 min (range: 28-314 min), CNIR=97 min (range: 22-285 min). No significant differences (p > 0.05) were found for magnitude of after-drop (PASS = 0.33 +/- 0.24 degrees C, CN = 0.31 +/- 0.18 degrees C, CNIR = 0.29 +/- 0.28 degrees C esophageal temperature) and duration of afterdrop (PASS = 15.4 +/- 10.2 min, CN = 13.0 +/- 10.1 min, CNIR = 8.8 +/- 6.9 min). No significant differences (p > 0.05) were found for rewarming rate (PASS = 1.85 +/- 1.33 degrees C x h(-1), CN = 1.45 +/- 1.04 degrees C x h( 1), CNIR = 2.24 +/- 1.51degrees C x h(-1) esophageal temperature). DISCUSSION: In summary, neither cranial-neck nor cranial-neck and inhalation rewarming combined have an advantage in reducing the magnitude and duration of after-drop or increasing the rewarming rate over passive rewarming. PMID- 16676651 TI - Three-dimensional spatial skill training in a simulated space station: random vs. blocked designs. AB - BACKGROUND: Astronauts floating inside a spacecraft must be able to recall the direction to surrounding visual landmarks, regardless of their viewing perspective. If 3D orientation skills are taught preflight, should perspective sequences be blocked or randomized? Can standard spatial skill tests predict performance? METHODS: Undergraduates (40 men and 40 women; ages 19-24) learned 3D spatial relationships among landmark pictures in a cubic chamber simulating a space station node. Subjects learned to predict picture directions when told one picture's direction (the one behind them) and the subject's simulated roll orientation, which was changed between trials by rotating pictures. The dependent variable was the proportion of correct predictions. A between group (n=40 per group) independent variable was training type (random vs. blocked sequencing of perspectives). Experiment phase (familiarization, training, transfer, and 2 retention phases) was a within group variable. Subjects also took three standard spatial skill tests: Card Rotation, Cube Comparison, and Group Imbedded Figures. RESULTS: As hypothesized, during training, performance for the random group (0.56) was worse than the blocked group (0.83); during transfer, the random group (0.75) was better than the blocked group (0.56); during retention-i, the random group (0.70) was better than the blocked group (0.55); and during retention-2, the random group (0.76) was better than the blocked group (0.65). Spatial skill tests correlated differently across the two groups, indicating that random sequencing elicits different skills. CONCLUSION: Random presentation enhances 3D spatial skill transfer and retention. Standard spatial tests can predict performance and have the potential to customize training. PMID- 16676652 TI - Hormonal responses of pilots to training flights: the effects of experience on apparent stress. AB - INTRODUCTION: The levels of urinary noradrenaline (NAd), adrenaline (Ad) and salivary cortisol (Cor) were determined in student and instructor pilots during Phase 1 (training with propeller engine; PH1), and Phase 2 (training with jet engine; PH2) flight training. METHODS: The subjects in PH1 were 30 students and 33 instructors, and in PH2 were 17 students and 15 instructors. Urine and saliva were collected approximately 30 min before and 20 min after the flights. The ratio (post/preflight) of the hormonal levels was calculated to compare the students with the instructors and/or PH1 with PH2. RESULTS: In PH1, the levels of all three hormones for postflight were significantly higher than for preflight in students, and the ratios of all three hormones in students were significantly higher than in instructors. In PH2, the ratios of all three hormones for students and instructors did not differ significantly, and the ratios of Ad and Cor levels in students for PH2 were significantly lower than for PH1 (Ad: 1.64 +/- 0.10 vs. 2.23 +/- 0.14; Cor: 0.86 +/- 0.16 vs. 1.68 +/- 0.11, respectively). CONCLUSIONS: The results from PH1 clearly demonstrated that flight stress for students was significantly higher than for instructors. The ratios might be regarded as result of adaptation to flight stress in students. We conclude that the ratios of Ad and Cor levels are a good indicator of stress coping in student pilots. PMID- 16676653 TI - Risk factors for recruit exertional heat illness by gender and training period. AB - INTRODUCTION: Exertional heat illness (EHI) is a recurrent problem for both male and female recruits during basic military training. A matched case control study investigated the effects of fitness and conditioning on EHI risk among Marine Corps recruits during 12 wk of basic training at Marine Corps Recruit Depot, Parris Island, SC. METHODS: Physical fitness and anthropometric measurements at entrance were acquired for 627 EHI cases that occurred during the period 1988 1996 and for 1802 controls drawn from the same training platoons. Conditional logistic regression was used to estimate EHI risk. RESULTS: Slower physical fitness test run times during processing week strongly predicted risk for subsequent EHI in both male and female recruits. A 9% increase in risk for EHI associated with body mass index (BMI = kg x m(-2); weight/height2) was found in male recruits, while BMI was not associated with risk among female recruits. BMI and initial run time were important predictors for EHI in early training, while in late training the initial BMI was no longer as important a risk factor and improvements in fitness reduced risk. CONCLUSION: Tables of estimated absolute risks categorized by BMI and VO2max are provided as a guide for identifying recruits who are at high risk for developing EHI during training. PMID- 16676654 TI - Air traffic controller burnout: survey responses regarding job demands, job resources, and health. AB - INTRODUCTION: There are currently many changes taking place in the aviation system affecting the work of air traffic controllers (ATCOs), and thus it was considered important to assess work-related demands and stress responses among ATCOs. The purpose of this study was to assess the level of burnout among ATCOs compared with other professions; to examine the relationship between job demands, job resources, and burnout; and to examine if burnout could predict both work- and health-related outcomes. METHOD: The participants were 209 Norwegian ATCOs and data were collected using a questionnaire distributed to 500 ATCOs. The study was part of a national survey of both health care and non-health care professions. RESULTS: ATCOs did not score high on burnout compared with the three comparisons groups (police, journalists, and building constructors); in fact, ATCOs scored significantly lower on exhaustion compared with journalists (Hedges g=65) and building constructors (Hedges g=63), and not significantly different from police officers. Both job demands and job resources were related to burnout; work conflict especially was an important predictor for all of the three burnout dimensions. Burnout predicted psychosomatic complaints (r2=0.48), satisfaction with life (r2=0.25), and other work outcomes. DISCUSSION: The overall level of burnout was not high among ATCOs compared with other professions, which may be related to selection procedures for the profession. Burnout was related to important outcome variables such as psychosomatic complaints and attitudes toward work. PMID- 16676656 TI - Decision analysis in aerospace medicine: costs and benefits of a hyperbaric facility in space. AB - INTRODUCTION: Assembly and maintenance of the International Space Station (ISS) requires an unprecedented number of spacewalks, which expose astronauts to the risk of decompression sickness (DCS). We illustrate the use of decision analysis to compare a hyperbaric oxygen (HBO) chamber to currently available therapy for DCS treatment on the ISS. METHODS: A decision-analytic model that simulates events over the lifespan of the ISS was constructed. Inputs to the model for probabilities, costs, and measures of morbidity and mortality were derived from a variety of sources, including a systematic literature review and an iterative consultation process with personnel at the Canadian Space Agency and the National Aeronautics and Space Administration (NASA). The decision model was analyzed using the methods of Monte-Carlo simulation and expected value calculation. Main outcome measures included the present value of costs and quality adjusted life years (QALYs), and the cumulative probability of mission-related events over the life cycle of the ISS. Sensitivity analysis was performed. RESULTS: The HBO chamber strategy is associated with a mean cost of -12.5 million dollarsw (a net cost saving of 12.5 million dollars) with a 95% CI (-112.8 million dollars, 51.3 million dollars). An HBO chamber reduces the likelihood of a premature shuttle return and a premature Soyuz return by 8% and 3%, respectively. The result is sensitive to the lifespan of the ISS. CONCLUSIONS: At a 50 million dollars cost, an HBO chamber is likely, though not certain, to result in cost savings. Decision analysis is a useful tool for use in priority setting in aerospace medicine. PMID- 16676655 TI - Hemodynamic effects of midodrine after spaceflight in astronauts without orthostatic hypotension. AB - INTRODUCTION: Orthostatic hypotension and presyncope are common and potentially serious risks for astronauts returning from space. Susceptible subjects fail to generate an adequate adrenergic response to upright posture. The alpha-1 adrenergic agonist, midodrine, may be an effective countermeasure. We tested the hypothesis that midodrine would have no negative hemodynamic effect on healthy astronauts returning from space. METHODS: Five male astronauts participated in preflight and post-flight tilt testing on a control flight as well as on the test flights, where midodrine (10 mg, orally) was administered after landing approximately 1 h before testing. RESULTS: None of these astronauts exhibited orthostatic hypotension or presyncope before or after either flight. Midodrine did not cause any untoward reactions in these subjects before or after flight; in fact, a modest beneficial effect was seen on postflight tachycardia (p = 0.036). DISCUSSION: These data show that midodrine protected against post-spaceflight increases in heart rate without having any adverse hemodynamic effects on non presyncopal, male astronauts. Among these subjects, midodrine was a safe cardiovascular countermeasure. PMID- 16676657 TI - Recovery from unusual attitudes: HUD vs. back-up display in a static F/A-18 simulator. AB - OBJECTIVE: Spatial disorientation (SD) remains one of the most important causes of fatal fighter aircraft accidents. The aim of this study was to give a recommendation for the use of the head-up display (HUD) or back-up attitude directional indicator (ADI) in a state of spatial disorientation based on the respective performance in an unusual attitude recovery task. METHODS: Seven fighter pilots joining a conversion course to the F/A-18 participated in this study. Flight time will be presented as range (and mean in parentheses). Total military flight experience of the subjects was 835-1759 h (1412 h). Flight time on the F/A-18 was 41-123 h (70 h). The study was performed in a fixed base F/A 18D Weapons Tactics Trainer. We tested the recovery from 11 unusual attitudes and analyzed decision time (DT), total recovery time (TRT), and error rates for the HUD or the back-up ADI. RESULTS: We found no differences regarding either reaction times or error rates. For the HUD we found a DT (mean +/- SD) of 1.3 +/- 0.4 s, a TRT of 9.1 +/- 4.1 s, and an error rate of 29%. For the ADI the respective values were a DT of 1.4 +/- 0.4 s, a TRT of 8.3 +/- 3.8 s, and an error rate of 27%. CONCLUSION: Unusual attitude recoveries are performed equally well using the HUD or the back-up ADI. Switching from one instrument to the other during recovery should be avoided since it would probably result in a loss of time without benefit. PMID- 16676658 TI - Phosphenes in low earth orbit: survey responses from 59 astronauts. AB - INTRODUCTION: It has long been known that many people in space experience sudden phosphenes, or light flashes. Although it is clear that they are related to high energy particles in the space radiation environment, many details about them are still unknown. In an effort to gain more knowledge about the light flashes, a study was initiated to collect information from people who have recently flown in space. METHOD: A survey conducted by anonymous questionnaire was performed among astronauts regarding their experience of sudden light flashes in space. In all, 98 surveys were distributed to current NASA and ESA astronauts. RESULTS: Among the 59 respondents, 47 noticed them sometime during spaceflight. Most often they were noted before sleep, and several people even thought the light flashes disturbed their sleep. The light flashes predominantly appear white, have elongated shapes, and most interestingly, often come with a sense of motion. The motion is described as sideways, diagonal, or in-out, but never in the vertical direction. DISCUSSION: Comparisons with earlier studies of light flashes in space and several ground-based studies during the 1970s are made. One interesting observation from this is that it seems that a small fraction of the light flashes is caused by Cherenkov radiation, while the majority is probably caused by some kind of direct interaction with elements in the retina. PMID- 16676659 TI - A device for sampling arterialized earlobe blood in austere environments. AB - INTRODUCTION: There is currently no effective method of measuring arterial blood gas tensions in austere environments such as in space or at high altitude. An alternative to direct arterial measurement is the sampling of arterialized earlobe blood, an accurate technique that has been in use in clinical medicine and physiology for more than 50 yr. We, therefore, developed an earlobe arterialized blood (EAB) collector for practical use in extreme environments. METHODS: The results from the EAB collector were compared with simultaneous samples of blood drawn from the radial artery. Six healthy subjects breathed a gas mixture of 12.8% O2 in N2 during 15 min of 8 degree head-down tilt. The blood samples were analyzed immediately. RESULTS: The mean differences in Po2 between arterialized earlobe and radial artery samples were 0.25 +/- 1.25 mmHg for Po2 and 1.0 +/- 0.75 mmHg for Pco2; neither difference was significant. There was no difference between the pH values obtained by the two techniques. CONCLUSION: This study suggests that arterialized blood sampled from the earlobe using the EAB collector may provide sufficiently accurate measurements of the Po2, Pco2 and pH of arterial blood for clinical or research use in extreme environments. PMID- 16676660 TI - Aircrew and type 1 diabetes mellitus. AB - The most stringent clinical criteria cannot guarantee that individuals with Type 1 diabetes mellitus (DM) will preserve awareness of an impending hypoglycemic episode or that a hypoglycemic episode can be prevented. Further, cognition may be impaired for a considerable period of time beyond the correction of any episode. These observations and their implications to operational aircrew with Type 1 DM are discussed. PMID- 16676661 TI - Apical hypertrophic cardiomyopathy and arrhythmia in military pilots. AB - Apical hypertrophic cardiomyopathy (ApHCM), a subtype of hypertrophic cardiomyopathy, may be found incidentally in healthy young adults. Arrhythmias are poor prognostic signs, and are the most frequent cause of sudden cardiac death. We present two cases of military aviators with ApHCM. One was a high performance jet weapon system operator, who had asymptomatic non-sustained ventricular tachycardia (NSVT) and subsequently a symptomatic episode of paroxysmal atrial fibrillation. The second was a helicopter pilot, who had asymptomatic NSVT. Both aviators continue their aviation duties without exposure to +Gz under a regime of regular thorough cardiac assessment. PMID- 16676662 TI - You're the flight surgeon. Aviator with perforated tympanic membrane. PMID- 16676663 TI - Rotation direction change hastens motion sickness onset in an optokinetic drum. PMID- 16676664 TI - RFID and aviation medicine. PMID- 16676665 TI - Educational strategies in generational designs. PMID- 16676666 TI - Esophageal Doppler monitoring during organ donor resuscitation: new benefits of existing technology. PMID- 16676667 TI - Requesting the gift of life: courage to ask, courage to give. PMID- 16676668 TI - Preparing a replication study. PMID- 16676669 TI - Living donation decision making: recipients' concerns and educational needs. AB - CONTEXT: Despite the advantages of living donor transplantation, evidence suggests that some potential recipients with living donors have psychological concerns that prevent them from pursuing living donation. Addressing these concerns through education may increase the rates of living donation. OBJECTIVE: To understand the psychological barriers and educational needs of potential kidney recipients regarding living donation. SUBJECTS AND DESIGN: Qualitative focus group study of kidney transplant recipients, donors, and family members to explore their assessment of the advantages of dialysis and deceased donor transplantation over living donation, their concerns about living donation, and what types of living donation education would be most helpful. RESULTS: Kidney recipients reported that they might not pursue living donation because they felt guilty and indebted to the donor, did not want to harm or inconvenience the donor, did not want to accept a kidney that a family member might need later, and did not want to disappoint the donor if the kidney failed. Recipients were generally unaware that donors could personally benefit from donating and would rather wait for donor volunteers than ask anyone directly. Both donors and recipients thought that training on how to make the donation request and education about living donors' motivations for donation and transplant experience could help more renal patients pursue living donation. PMID- 16676670 TI - The dilemma of alcohol use by potential living liver donors. AB - Previous and current alcohol use by potential living liver donors presents ethical challenges for donor selection committees. Discussing these challenges, we offer guidelines for selection and management of these individuals. Donor safety and welfare should be the primary concern, thus relapse potential during the postdonation period for those with a history of alcohol dependence or abuse is of importance, especially because of the potentially severe consequences of mixing pain relievers (eg, acetaminophen) and alcohol during liver regeneration. Psychosocial and chemical dependency evaluations are critical for potential living donors as well as recipients. PMID- 16676671 TI - Trends in adult-to-adult living donor liver transplant organ donation: the Johns Hopkins experience. AB - Adult-to-adult living donor liver transplantation is an increasingly important option for 17000 patients awaiting liver transplantation in the United States. However, adult-to-adult living donor liver transplantation volumes peaked in 2001 (N = 518), and have gradually fallen in 2002 (N = 362), 2003 (N = 321), and 2004 (N = 323). Recent concerns about donor safety and ethical considerations have made careful analysis of donor availability and selection criteria critically important. We conducted a retrospective review of our active liver transplant recipient registry (N = 251) and compared it to our living donor registry (N = 231), which included all potential living donors before the selection process. Fifteen percent of recipients accounted for the majority (53%) of donor evaluations, whereas 42% of recipients did not have even a single donor evaluation. Recipient diagnosis appears to have a significant impact on donor availability, with donors rarely evaluated for patients with alcoholic cirrhosis. Careful and stringent selection criteria rule out 67% of potential donors. PMID- 16676672 TI - Treating tobacco use and dependence in kidney transplant recipients: development and implementation of a program. AB - Tobacco use adversely affects transplant outcomes such as graft survival, patient survival, and other conditions that alter transplant patient longevity. Especially concerning is tobacco's relationship to cardiovascular disease, the number 1 cause of death in kidney transplant recipients. Many authors conclude that tobacco interventions ought to be provided to patients and sometimes lament that there are no tobacco dependence interventions designed for kidney transplant recipients. European Best Practice Guidelines for Renal Transplantation also support tobacco dependence interventions. The purpose of this article is to describe one institution's experience in implementing the clinical practice guideline for treating tobacco use and dependence within a kidney and pancreas transplant program. PMID- 16676673 TI - An exploratory study of the cognitive, academic, and behavioral functioning of pediatric cardiothoracic transplant recipients. AB - CONTEXT: Limited information is available regarding the cognitive, academic, and behavioral outcomes of pediatric cardiothoracic transplant recipients. OBJECTIVE: To examine the cognitive, behavioral, and academic functioning of a group of children who have received heart, heart/lung, or lung transplants. DESIGN, PARTICIPANTS, AND SETTING: An exploratory cross-sectional outcomes study of surviving pediatric cardiothoracic transplant recipients at a Midwestern pediatric hospital. MAIN OUTCOME MEASURES: Transplant recipients completed measures of cognitive and academic functioning. Parents and teachers completed measures of behavioral functioning. Parents and transplant recipients also provided qualitative data regarding their main concerns after transplantation. RESULTS: On measures of cognitive functioning, 46% showed significant delays (> 2 SD below the normative population). Of those eligible for academic testing, 78% fell within the average range (within 1 SD of the normative population) for reading and spelling, and 57% fell within the average range for math. Twenty seven percent of mothers, 11% of fathers, and 17% of teachers rated subjects as having significant behavioral concerns. Results highlight the need for close contact between transplant teams and school personnel to optimize outcomes. In addition, patients and families may benefit from psychological intervention after transplantation as there are ongoing concerns regarding life expectancy and quality of life. PMID- 16676674 TI - Implementation of an intervention plan designed to optimize donor referral in a donor hospital network. AB - CONTEXT: The shortage of donor organs remains the most important factor of waiting list mortality in organ transplantation worldwide. Donor detection is influenced by the legal system, family refusal, and underreporting caused by erroneous knowledge of donation criteria and lack of familiarity with the procedure. OBJECTIVE: To identify possible key factors of donor referral patterns within an existing cooperation with donor hospitals and donor units across the Dutch-speaking part of Belgium, an area of approximately 3 million inhabitants. An intervention plan to optimize the cooperation and procedure quality and efficiency was designed. DESIGN: The intervention plan was based on 3 essential principles in donor referral by donor reporters, information on donor criteria, facilitation of the donor procedure, and communication between donor reporters and the transplant center. The interventions were structured to optimize all 3 of these principles. Two successive periods of 4 years were retrospectively compared. PARTICIPANTS: Data were collected retrospectively on donor referral behavior from a total of 37 donor hospitals and donor units over an 8-year period. MAIN OUTCOME MEASURES: The referrals were reviewed for potential donors, effective donors, percentage of effective donors, refusal rate of relatives, number of tissue donors, impact on local and national transplant programs, and national donor numbers. RESULTS: Data showed a significant positive impact on donor referrals and donor referral behavior (+27% potential donors, +30% effective donors, +172.7% tissue donors, -7% family refusals rates, +9.63% national donors). The results stress the importance of reduced workload and optimization of communication and information availability in an existing donor hospital network. PMID- 16676675 TI - Family experiences of tissue donation in Australia. AB - CONTEXT: Although research exists examining the experiences of organ donor families, little is known about the specific needs of tissue donor families. Understanding family experiences in tissue donation is important to improve the process and potentially increase the number of families who consent to tissue donation. OBJECTIVE: To examine the experiences of recent tissue donor families and to identify potential areas for improvement of the donation process. DESIGN: Cross-sectional survey by mailed questionnaire. SETTING AND PARTICIPANTS: Participants were families who had been approached by DonateWest, the coordinating organization in Western Australia, and had agreed to tissue donation. MAIN OUTCOME MEASURES: Quantitative and qualitative analysis of data with regards to satisfaction with the approach method, reasons for consent, and appropriateness of support services. RESULTS: Seventy-seven of 140 mailed questionnaires were returned. Almost all respondents expressed satisfaction with the approach to donate, and this did not differ with the mode of approach, that is, by phone or in person. The most common reason for agreeing to donation was that it was known to be the donor's wish. The majority of respondents felt they had sufficient time and information to decide to donate, and almost all were happy with their decision and thought they had received adequate support during and after the process. PMID- 16676676 TI - Liver transplantation in Italy: analysis of risk factors associated with graft outcome. AB - OBJECTIVE: To analyze the graft outcome after liver transplantation in Italy in the years 1995 to 2000. METHODS: We performed a longitudinal study with follow-up at 3 months, 1 year, 3 years, and 5 years on 1987 liver grafts. The effect of several variables on graft survival was also analyzed. RESULTS: Several variables affect graft survival: Donor and recipient older age, gender mismatching, prolonged cold ischemia time, acute hepatic necrosis, and retransplantation are reported to significantly affect liver graft survival. Donors older than 60 years show a relative risk of 1.59 (95% CI, 1.23-2.05) compared with donors with an age between 19 and 60 years; recipients older than 50 years show a relative risk of 1.29 (95% CI, 1.04-1.60) compared with recipients aged 19 to 50 years. A cold ischemia time of 12 hours or longer doubled the risk of failure (relative risk = 2.01, 95% CI, 1.36-2.96) compared with a cold ischemia time of less than 6 hours. CONCLUSIONS: The results show that the overall quality of liver transplantation in Italy is satisfying and comparable to the outcome reported by international registries. Follow-up studies on large numbers of liver transplants are useful to define predictors of outcome, and subsequently modify the criteria for organ allocation. PMID- 16676677 TI - Attitudes toward death criteria and organ donation among healthcare personnel and the general public. AB - OBJECTIVE: To examine attitudes toward death criteria and their relation to attitudes and behaviors regarding organ donation. DATA SOURCES: This article reviews empirical studies on the attitudes of healthcare personnel and the general public regarding death criteria and organ donation. STUDY SELECTION AND DATA EXTRACTION: The review was restricted to studies that had as a primary focus attitudes toward 1 or more of the following 3 specific criteria for determining death: (1) brain death, the irreversible loss of all functions of the entire brain; (2) higher brain death, the loss of cerebral cortex function alone; and (3) the circulatory-respiratory criteria commonly used in donation after cardiac death. DATA SYNTHESIS: Studies consistently show that the general public and some medical personnel are inadequately familiar with the legal and medical status of brain death; attitudes toward the dead donor rule are strong predictors of willingness to donate organs using controversial criteria; concerns about donation after cardiac death surround the withdrawal of life support more than the actual death criteria used; and concerns about death criteria correlate with less favorable attitudes toward organ donation. CONCLUSIONS: Both general and ethical education may serve to guide policy and facilitate family member requests and informed consent dialogues. Furthermore, helping families to understand and accept not only medical and legal criteria for determining death, but also ethical criteria for withdrawing life support may help them be more comfortable with their decisions. PMID- 16676678 TI - Cardiac dysrhythmias during donor care. AB - Organ procurement coordinators must treat various cardiac dysrhythmias (arrhythmias), including rhythm disturbances that may cause or follow a cardiac arrest, in about 15% to 50% of donors. Treatment decisions should be based on the particular dysrhythmia and its effect on donor blood pressure. Medications selected should be effective but short acting. In this article, data available in publications located through a PubMed search are reviewed and specific dysrhythmias that are likely to occur during donor care are described. Treatment recommendations are based on guidelines from the American Heart Association. PMID- 16676679 TI - Giving back to donor families: the "thank you letter" package insert project. AB - Family members of organ donors receive anonymous information about the transplant recipients. Also, they often receive written communication from the recipients themselves and or the recipient's family members, expressing gratitude for the generous gifts they donated. The feedback received by donor families serves to affirm that their altruistic decision to donate life-saving and life-enhancing gifts has saved or dramatically improved the lives of the recipients. Feedback allows donor families to assign meaning to the donation itself and make some sense of otherwise tragic circumstances. In contrast, family members of bone and soft tissue donors historically have not received the same level of feedback provided to the family members of organ donors. The tissue banking community, as an extension of the procurement community that provides care to tissue donor families, is in a unique position to facilitate meaningful feedback between bone and soft tissue donor families and their recipients. This article explores the use of tissue packaging as a communication portal to solicit and facilitate the needed feedback loop between bone and soft tissue donor families and their recipients. PMID- 16676680 TI - The decision to donate: a model of decision making among individuals and families. AB - Fifty years ago, the first successful kidney transplantation was performed. Since then significant advances in transplantation have been made. Unfortunately, the number of people needing transplants far surpasses the number willing to donate. The literature addresses a plethora of variables influencing whether individuals will become donors themselves or whether family members will donate a loved one's organs. However, to date, no model has examined similarities and differences in the processes by which individuals and families go about making decisions to donate their own or another's organs. In this article, individual and family donation decisions are discussed using Janis' 5-stage model of decision making. We suggest that the weighty nature of donation decisions levels the playing field for decisions made for self and for others. Although the specifics (eg, micro level) may vary slightly, the processes by which people go about deciding to donate their own organs or those of another individual are similar (eg, macro level). PMID- 16676681 TI - Rule-of-thumb adjustment of sample sizes to accommodate dropouts in a two-stage analysis of repeated measurements. AB - Recent contributions to the statistical literature have provided elegant model based solutions to the problem of estimating sample sizes for testing the significance of differences in mean rates of change across repeated measures in controlled longitudinal studies with differentially correlated error and missing data due to dropouts. However, the mathematical complexity and model specificity of these solutions make them generally inaccessible to most applied researchers who actually design and undertake treatment evaluation research in psychiatry. In contrast, this article relies on a simple two-stage analysis in which dropout weighted slope coefficients fitted to the available repeated measurements for each subject separately serve as the dependent variable for a familiar ANCOVA test of significance for differences in mean rates of change. This article is about how a sample of size that is estimated or calculated to provide desired power for testing that hypothesis without considering dropouts can be adjusted appropriately to take dropouts into account. Empirical results support the conclusion that, whatever reasonable level of power would be provided by a given sample size in the absence of dropouts, essentially the same power can be realized in the presence of dropouts simply by adding to the original dropout free sample size the number of subjects who would be expected to drop from a sample of that original size under conditions of the proposed study. PMID- 16676682 TI - Improving methods of assessing natural killer cell cytotoxicity. AB - Natural killer (NK) cells are a class of lymphocytes important in immune resistance to viral and other serious diseases. The cytotoxic function, or 'killing activity' of NK cells has become important in studies of the effects of stress and other psychosocial factors on physical health. Unfortunately, research on NK cell function has been plagued by discrepancies in the methods of interpreting NK cytotoxicity data. We briefly review some of the variations in measuring NK cell activity and present a new model for interpreting these results, introducing maximal target cell lysis (A) and the slope of the cytolytic curve (k) as parameters that attempt to make full use of the information and the statistical power in NK cell cytotoxicity data. Examples of these interpretation methods are presented using NK cytotoxicity data from a group of metastatic breast cancer patients. This approach will be useful in applications of NK cell measurement in psychoneuroimmunology research. PMID- 16676684 TI - Factorial validation of a French short-form of the Working Alliance Inventory. AB - Evaluation of the therapeutic alliance is crucial for understanding the therapeutic process and its results. However, few instruments are available in French. This article aims to validate a French short form of the Working Alliance Inventory (WAI). Unlike other questionnaires, the WAI is the most widely used in psychotherapy research as well as in social psychiatry. Confirmatory factor analyses were carried out on a sample of 150 client-case manager dyads in order to determine the validity of this short-form instrument. The results of these confirmatory factor analyses allowed us to answer different authors' questions (Horvath and Greenberg, 1989; Tracey and Kokotovic, 1989) regarding the factorial structure of the WAI. The results also indicated a unidimensional solution as being the most valid for the two samples. We suggest that, in future studies, only one score be considered for the evaluation of the WAI. We also suggest modifying two statements in the English and French versions in order to render a faithful comparison between the therapist and client versions. PMID- 16676683 TI - The Italian preadolescent mental health project (PrISMA): rationale and methods. AB - The Italian preadolescent mental health project (PrISMA--Progetto Italiano Salute Mentale Adolescenti) is the first Italian study designed to estimate the prevalence of mental disorders in preadolescents (10-14 years old) living in urban areas, and to analyse the demographic and biological correlates of emotional and behavioural problems. This paper describes the rationale, methods and the analysis plan of the project. The design of the study used a two-stage sampling procedure, one screening stage of emotional and behavioural problems in a large sample of subjects attending public and private schools and a second stage of diagnostic assessment in a sample including all high scorers and a proportion of low scorers. In the screening stage, parents of preadolescents were asked to fill in the Child Behavior Checklist (CBCL), whereas in the second stage preadolescents and their parents were administered the Development and Well Being Assessment for the assessment of mental disorders together with the Strengths and Difficulties Questionnaire and two scales (C-GAS and HoNOSCA) designed to evaluate the functioning of the preadolescent in different areas. Genetic samples were collected during the screening stage, after parents gave their informed written consent. The findings of this study are expected to allow an adequate planning of interventions for the prevention and the treatment of mental disorders in preadolescence as well as efficient health services. PMID- 16676685 TI - A comparison of agency-based and self-report methods of measuring services across an urban environment by a drug-abusing homeless population. AB - The purpose of this paper is to advance the methodology for studying service assessment by comparing self-report and agency-generated methods. This study compares 30-day self-reported service use for homeless individuals (N = 229) randomly recruited from a single urban environment (St Louis, Missouri) with similar data collected from a broad array of service agencies providing homeless, substance abuse and outpatient mental health services across the same environment. Comparisons were made between self-report and agency-based data on shelter use, outpatient mental health service use (case management, psychiatric treatment, group therapy), outpatient substance abuse service use (case management, counselling, group therapy) and drop-in/day treatment use. Consistently low levels of kappa scores (all under 0.4) and correlation coefficients (only shelter use demonstrated significant agreement) were found. Findings demonstrated that the two methods of collecting service data are generally not concordant at the individual level. Certain demographic characteristics (increased age, being male, non-white ethnicity) and diagnoses (cocaine abuse/dependence, mania, schizophrenia) were associated with decreased reliability between the two methods of data collection. The two methods of assessment appeared to capture overlapping but not identical information. Each method of assessment has different utility to researchers and providers wishing to assess service use. PMID- 16676686 TI - An electronmicroscopic study of the interaction of Burkholderia pseudomallei and human macrophages. AB - B. pseudomallei has been shown to persist intracellularly in melioidosis patients until reactivated by decreasing immunocompetence. We have shown by transmission electron microscopy the internalization of B. pseudomallei by human macrophages via conventional phagocytosis enclosed within membrane-bound vacuoles or phagosomes. Ferritin labeled lysosomes provided evidence of phagosome-lysosome fusion. Ingested bacilli were designated as "intact" or "damaged" on the basis of their ultrastructural features. An intact bacterium was seen with low electron opaque central nuclear region surrounded by dense bacterial cytoplasm, bounded externally by bacterial plasma membrane and cell wall. In contrast, B. pseudomallei were considered damaged when seen with cavitation within the central nuclear region, separation of bacterial cytoplasm from the cell wall, herniation of cytoplasmic contents and lamination of bacterial cell wall and its surrounding electron transparent zone. Our observations indicate that the microbicidal mechanism(s) in B. pseudomallei-infected macrophages failed to ensure complete clearance of the organism and this failure probably facilitates intracellular persistence and proliferation, and this may be one of the survival strategies adopted by this organism. PMID- 16676687 TI - A clinical and immunohistochemical study of gastrointestinal stromal tumours. AB - AIM: To study the clinical features, histology and immunohistochemical properties of gastrointestinal stromal tumours (GISTs); and establish any parameters that can help prognosticate the malignant potential. METHODS: Twenty-six patients with GISTs who were seen in Sultanah Aminah Hospital Johor, Malaysia from 1999 to 2003 were selected for study. Patient, clinical characteristics and outcome based on surgical records were analysed. Tumour variables (tumour size, cellularity, mitotic count, necrosis and haemorrhage) were compared between very low to low risk groups and intermediate to high risk groups. The immunohistochemical properties of GISTs were also studied. RESULTS: Patients with GISTs presented mainly with pain, palpable mass or gastrointestinal tract bleeding. The tumours were seen in stomach (50%) followed by small intestine (38.5%) and rectum (11.5%). In the period of study, six patients had metastasis, mainly in the liver or peritoneum. Immunoreactivity for CD117, CD34, vimentin, S100, neuron specific enolase, alpha-smooth-muscle-actin and desmin were observed in 100%, 76.9%, 61.5%, 46.1%, 80.8%, 11.5% and 0% of tumours respectively. The behaviour of GISTs was largely dependent on tumour size and number of mitosis. Necrosis and haemorrhage were seen in tumours with high risk potential. PMID- 16676688 TI - Mantle cell lymphoma--a clinicopathological study of 13 cases. AB - Mantle cell lymphoma is an uncommon non-Hodgkin's lymphoma. In a period of four years, 13 cases of mantle cell lymphoma were diagnosed in our department, comprising 3.1% of all non-Hodgkin's lymphoma diagnosed. The mean age of presentation was 52 years with a slight male preponderance. The disease was nodal in twelve and extra-nodal in tonsil in one. Five patients had bone marrow involvement. Five cases showed a nodular pattern on lymph node biopsy while the remaining eight had a diffuse pattern. Immunophenotyping showed positivity for CD20 and cyclin D1. Despite certain morphological similarity to other low-grade lymphomas, mantle cell lymphoma has a characteristic appearance of its own. It is more aggressive than other low-grade lymphomas and hence needs to be accurately diagnosed. PMID- 16676689 TI - Microwave-stimulated formaldehyde fixation of experimental renal biopsy tissues: computerised morphometric analysis of distortion artefacts. AB - Although microwave irradiation has been used in the histopathology laboratory for several years, there has been minimal published experimental data on its effects on the technical and staining quality of histological sections. Furthermore, it has not been clear whether the advantages gained in reduction of fixation and staining duration has been at the expense of increasing architectural distortion to the tissues. We report here our experience with computerised morphometric analysis to investigate glomerular artifacts caused by microwave-stimulated fixation of renal tissues. 39 rat and 33 human autopsy kidney samples were subjected to (1) fixation in neutral buffered formaldehyde (control), (2) microwave-stimulated fixation followed by neutral buffered formaldehyde, and (3) neutral buffered formaldehyde followed by microwave irradiation. In addition, the effect of post-fixation in 70% ethanol was also investigated. Microwave irradiation was delivered through a dedicated laboratory microwave oven at 80% power and at 55 degrees C for 3 minutes. The different fixation methods were compared with regards to shrinkage (distortion) to glomerular structures (glomeruli and Bowman's spaces) on H and E sections, as determined by morphometric image analysis using a temporary assembled-system consisting of a trinocular microscope, a digital video camera and personal computer. A FlashPoint VGA 3.3 film-grabber card was used to capture images for morphometric analysis by using a Scion Image program. Morphometric analysis of glomerular structures showed that microwaves caused more shrinkage to the area bounded by the Bowman's capsule than the glomerulus proper, but post-fixation with ethanol reduced this shrinkage. These findings have implications on the logistics of tissue preparation of renal biopsies in clinical practice. PMID- 16676691 TI - Haemoglobin Lepore in a Malay family: a case report. AB - A 2-year-old Malay boy was brought to the University Malaya Medical Centre for thalassaemia screening. Physical examination revealed thalassaemia facies, pallor, mild jaundice, hepatomegaly and splenomegaly. Laboratory investigations on the patient including studies on the parents lead to a presumptive diagnosis of homozygous Haemoglobin Lepore (Hb Lepore). The aim of this paper is to increase awareness of this rare disorder, this being the first case documented in Malaysia in a Malay. The case also demonstrates the need for this disorder to be included in the differential diagnosis of patients presenting clinically like thalassemia intermedia or thalassemia major. Accurate diagnosis would provide information necessary for prenatal diagnosis, proper clinical management and genetic counseling. The clinical, haematological and laboratory features of this disorder are discussed in this paper. PMID- 16676690 TI - Thyrotoxic periodic paralysis: a report of 3 Malaysian cases and a review of its pathology. AB - Thyrotoxic periodic paralysis (TPP) is a medical emergency characterised by sudden onset of muscle weakness with hypokalemia that resolves with the treatment of hyperthyroidism. We report three cases of thyrotoxic periodic paralysis seen at the Accident and Emergency Care Department, University of Malaya Medical Centre in a period of four months. We also review the clinical presentation, pathophysiology, biochemical features and management of TPP. All three patients were young Asian males, presenting with muscle weakness of sudden onset. The first patient presented with lower limb weakness and had symptoms of thyrotoxicosis and goitre. He had a previous similar episode which resolved spontaneously. The second patient presented with quadriplegia, respiratory acidosis and had no signs and symptoms of thyrotoxicosis. The electrocardiogram of this patient showed normal sinus rhythm with U wave in V3 and a flat T wave, which are characteristic of hypokalaemia. The third patient, who was a known case of thyrotoxicosis, was admitted thrice for hypokalemic paralysis during the study period. All cases had low serum potassium, suppressed TSH and elevated T4 confirming thyrotoxic periodic paralysis. Potassium therapy was useful during the crisis; however prophylactic potassium has not been shown to prevent attacks as seen in one of our cases. CONCLUSION: Thyrotoxic periodic paralysis should be considered in the differential diagnosis of sudden onset paralysis in young male patients. Determination of the plasma potassium levels and thyroid hormones help in the diagnosis. The definitive treatment for TPP is the achievement of euthyroid state. PMID- 16676692 TI - Congenital dyserythropoietic anaemia type II-like dysplastic anaemia preceding the development of non-Hodgkin lymphoma--a case report. AB - Anaemia is a frequent complication in patients with haematological malignancies and is caused by a variety of mechanisms including neoplastic cell infiltration into the bone marrow, haemolysis, nutritional deficiencies and defect in erythropoiesis or dysplastic anaemia as a result of the disease itself. However, acquired dysplastic anaemia which mimic congenital dyserythropoietic anaemia (CDA) type II morphology in the bone marrow is very rare. A 41-year-old Chinese man presented with refractory symptomatic anaemia in September 2001. He was clinically pale with no other significant physical finding. His initial peripheral blood picture showed normochromic normocytic anaemia with haemoglobin level of 26g/L, with no evidence of haemolysis and a poor reticulocyte response of 0.6%. Bone marrow aspiration was done and showed congenital dyserythropoietic anaemia (CDA) type II-like morphology. He was treated symptomatically with regular blood transfusions approximately every 3 weeks, until August 2002 when he developed multiple cervical lymphadenopathy with loss of appetite, loss of weight and low grade fever. Biopsy of the lymph node confirmed the diagnosis of small lymphocytic lymphoma. Staging with computed tomography and bone marrow aspirate revealed the infiltration of lymphoma cells into the marrow cavity consistent with the staging of IVB. This case report illustrates that CDA type II-like dysplastic anaemia can preceed the development of lymphoma. PMID- 16676693 TI - Complex karyotypic abnormalities in a case of acute myeloid leukaemia--M4Eo. AB - A 25-year-old man was referred to Hospital UKM with a 2-week history of fever, productive cough and loss of appetite. Physical examination revealed an ill looking, tachypnoeic young man. No obvious lymphadenopathy or organomegaly was noted. Examination of the respiratory system revealed right pleural effusion. Full blood picture demonstrated leukocytosis with 90% blasts, and bone marrow examination confirmed the diagnosis of acute myeloid leukemia (AML) French American-British (FAB) classification of M4 with eosinophilia. His chromosome karyotyping showed complex karyotypic abnormalities. Cytological examination of the pleural fluid demonstrated numerous blast cells indicating leukemic infiltration of the lungs, which is a rare presentation in AML. He was then started on induction chemotherapy with intravenous daunorubicin and cytarabine. In the midst of treatment, he developed an episode of seizure and cerebro-spinal fluid cytology confirmed central nervous system (CNS) leukaemic infiltration. Additional intrathecal methotraxate was given. Repeat bone marrow examination done on day 15 of chemotherapy showed persistence of excess blasts indicating refractory AML. He was then reinduced with high dose cytarabine but to no avail. The disease progressed and he succumbed about 8 weeks after the initial diagnosis was made. We highlight here a case of AML-M4Eo with complex karyoyptic abnormalities presenting with leukaemic infiltration of the lungs and CNS which had imparted a bad prognosis for this subtype of AML, AML-M4Eo. PMID- 16676694 TI - Guidelines on retention of pathology records and materials, Part I (version 1/2005). PMID- 16676695 TI - Guidelines on minimum qualification, training and experience of professional personnel working in a pathology laboratory (version 1/2004). PMID- 16676696 TI - Guidelines on laboratory construction and design (version 1/2004). PMID- 16676697 TI - Guidelines on maintenance and operation of equipment in a pathology laboratory (version 1/2004). PMID- 16676698 TI - Guidelines on safe laboratory practice (version 1/2004). PMID- 16676699 TI - Guidelines on sample management (version 1/2004). PMID- 16676700 TI - The prevalence of disordered eating, menstrual dysfunction, and low bone mineral density among US collegiate athletes. AB - The purpose of this study was to examine the prevalence of disordered eating (DE), menstrual dysfunction (MD), and low bone mineral density (BMD) among US collegiate athletes (n = 112) representing 7 different sports (diving, swimming, x-country, track, tennis, field hockey, and softball) and determine differences in prevalence existed between athletes participating in lean-build (LB) and non lean build (NLB) sports. DE and MD were assessed by a health, weight, dieting, and menstrual history questionnaire. Spinal BMD was determined via dual energy x ray absorptiometry. Twenty-eight athletes met the criteria for DE, twenty-nine for MD, and two athletes had low BMDs (using a Z score below -2.0). Ten athletes met the criteria for two disorders (one with disordered eating and low BMD and nine with disordered eating and menstrual dysfunction), while only one athlete met the criteria for all three disorders. Using a Z score below -1.0, two additional athletes met the criteria for all three disorders and three more athletes met the criteria for a combination of two disorders. With the exception of MD, which was significantly more prevalent among LB vs. NLB sports (P = 0.053), there were no differences between the groups in the prevalence of individual disorders or combinations of disorders. These data indicate that the combined prevalence of DE, MD, and low BMD among collegiate athletes is small; however, a significant number suffer from individual disorders of the Triad. PMID- 16676701 TI - The effect of caffeine ingestion on neutrophil oxidative burst responses following prolonged cycling. AB - This study investigated the effect of caffeine ingestion on neutrophil oxidative burst responses to prolonged cycling. In a two part study, 19 endurance trained male cyclists (Part A--11; Part B--8) performed 90 min of exercise at 70% VO2max 1 h after ingesting 6 mg/kg body mass of caffeine (CAF) or placebo (PLA). CAF ingestion had no effect on the PMA-stimulated oxidative burst response (Part A), yet it attenuated the exercise-induced decline in f-MLP stimulated response that occurred with PLA (Part B). CAF ingestion significantly increased serum caffeine concentration and plasma adrenaline concentration following exercise. In addition, circulating lymphocyte count was increased following CAF ingestion whereas there was no effect on neutrophil number. Therefore, although CAF ingestion was associated with an increase in adrenaline, this was not associated with an expected decrease in neutrophil function. This suggests that in the present study, CAF ingestion influenced neutrophil function via alternative mechanisms. PMID- 16676702 TI - Carbohydrate influences plasma interleukin-6 but not C-reactive protein or creatine kinase following a 32-km mountain trail race. AB - Attenuation of exercise-induced interleukin-6 (IL-6) responses by carbohydrate (CHO) has been demonstrated in studies comparing controlled doses (> or = 0.9 g x kg(-1) x h(-1)) to placebo, but not in studies of voluntary intake. This study sought to determine if attenuation of the IL-6 response during a 32.2-km mountain trail race occurs for high compared to low ad libitum CHO intakes. IL-6, C reactive protein (CRP), and creatine kinase activity (CK) were analyzed from blood samples collected 12 h pre-, 0, 4, and 24 h post-race. Subjects were grouped into low (n =14, 0.4 +/- 0.1 g x kg(-1) x h(-1)) and high (n =18, 0.8 +/- 0.2 g x kg(-1) x h(-1)) CHO intake groups. IL-6 0 h post-race (P < 0.05) was higher in the low (40.2 +/- 22.7 pg x mL(-1)) compared to the high CHO group (32.7 +/- 22.1 pg x mL(-1)). CRP and CK both increased post-race, but no differences were observed between groups. Attenuation of exercise-induced IL-6 is apparent across a range of CHO intakes. PMID- 16676703 TI - The effect of bovine colostrum supplementation on salivary IgA in distance runners. AB - Secretory IgA in saliva (s-IgA) is a potential mucosal immune correlate of upper respiratory tract infection (URTI) status. Nutritional supplements may improve mucosal immunity, and could be beneficial to athletes who are at increased risk of URTI. In this study, 35 distance runners (15 female, 20 male, age 35 to 58 y) consumed a supplement of either bovine colostrum or placebo for 12 wk. Saliva samples were taken prior to training at baseline, monthly during supplementation, and 2 wk post supplementation. Median levels of s-IgA increased by 79% in the colostrum group after 12 wk intervention, and the time-dependent change from baseline value was significant (P = 0.0291). This significance was still apparent after adjusting for training volume and self-reporting of upper respiratory symptoms. This study has demonstrated increased s-IgA levels among a cohort of athletes following colostrum supplementation. While this result is statistically significant, its physiological interpretation must be viewed with caution due to the small numbers in this study and the large variability in s-IgA levels. PMID- 16676704 TI - A whey-supplemented, high-protein diet versus a high-carbohydrate diet: effects on endurance cycling performance. AB - This study compared a training diet recommended for endurance athletes (H-CHO) with an isoenergetic high protein (whey supplemented), moderate carbohydrate (H Pro) diet on endurance cycling performance. Over two separate 7-d periods subjects (n = 7) ingested either H-CHO (7.9 +/- 1.9 g x kg(-1) x d(-1) carbohydrate; 1.2 +/- 0.3 g x kg(-1) x d(-1) fat; 1.3 +/- 0.4 g x kg(-1) x d(-1) protein) or H-Pro (4.9 +/- 1.8 g x kg(-1) x d(-1); 1.3 +/- 0.3 g x kg(-1) x d( 1); 3.3 +/- 0.4 g x kg(-1) x d(-1)) diet in a randomized, balanced order. On day 8 subjects cycled (self-paced) for a body weight dependent (60 kJ/bm) amount of work. No differences occurred between energy intake (P = 0.422) or fat intake (P = 0.390) during the two dietary conditions. Performance was significantly (P = 0.010) impaired following H-Pro (153 +/- 36) compared with H-CHO (127 +/- 34 min). No differences between treatments were observed for physiological measures taken during the performance trials. These results indicate an ergolytic effect of a 7-d high protein diet on self-paced endurance cycling performance. PMID- 16676705 TI - Chocolate milk as a post-exercise recovery aid. AB - Nine male, endurance-trained cyclists performed an interval workout followed by 4 h of recovery, and a subsequent endurance trial to exhaustion at 70% VO2max, on three separate days. Immediately following the first exercise bout and 2 h of recovery, subjects drank isovolumic amounts of chocolate milk, fluid replacement drink (FR), or carbohydrate replacement drink (CR), in a single-blind, randomized design. Carbohydrate content was equivalent for chocolate milk and CR. Time to exhaustion (TTE), average heart rate (HR), rating of perceived exertion (RPE), and total work (WT) for the endurance exercise were compared between trials. TTE and WT were significantly greater for chocolate milk and FR trials compared to CR trial. The results of this study suggest that chocolate milk is an effective recovery aid between two exhausting exercise bouts. PMID- 16676706 TI - Active and passive recovery and acid-base kinetics following multiple bouts of intense exercise to exhaustion. AB - The purpose of this study was to profile the effect of active versus passive recovery on acid-base kinetics during multiple bouts of intense exercise. Ten males completed two exercise trials. The trials consisted of three exercise bouts to exhaustion with either a 12 min active (20% workload max) or passive recovery between bouts. Blood pH was lower in the passive (p) recovery compared to active (a) throughout the second and third recovery periods [second recovery: 7.18 +/- 0.08 to 7.24 +/- 0.09 (p), 7.23 +/- 0.07 to 7.32 +/- 0.07 (a), P < 0.05; third recovery: 7.17 +/- 0.08 to 7.22 +/- 0.09 (p), 7.23 +/- 0.08 to 7.32 +/- 0.08 (a), P < 0.05]. Exercise performance times did not differ between recovery conditions (P = 0.28). No difference was found between conditions for recovery kinetics (slope and half-time to recovery). Subsequent performance during multiple bouts of intense exercise to exhaustion may not be influenced by blood acidosis or mode of recovery. PMID- 16676707 TI - Preparation of former heavyweight oarsmen to compete as lightweight rowers over 16 weeks: three case studies. AB - To strengthen the depth of lightweight rowing talent, we sought to identify experienced heavyweight rowers who possessed physique traits that predisposed them to excellence as a lightweight. Identified athletes (n = 3) were monitored over 16 wk. Variables measured included performance, anthropometric indices, and selected biochemical and metabolic parameters. All athletes decreased their body mass (range 2.0 to 8.0 kg), with muscle mass accounting for a large proportion of this (31.7 to 84.6%). Two athletes were able to maintain their performance despite reductions in body mass. However, performance was compromised for the athlete who experienced the greatest weight loss. In summary, smaller heavyweight rowers can successfully make the transition into the lightweight category, being nationally competitive in their first season as a lightweight. PMID- 16676708 TI - Research in sports nutrition: an interview with David Pyne. Interviewed by Louise M. Burke. PMID- 16676709 TI - [Towards a medical consultation for prevention]. PMID- 16676710 TI - [Feasibility of systematic screening for addictive behaviours of hospitalized patients]. AB - BACKGROUND: The national policy against addiction led to the establishment of specialized units with liaison teams trained in addiction specifically to work with the in-patients. The aim of this study has been established within the context of the implementation of one of these addiction liaison units in a Parisian Hospital, the Georges Pompidou European Hospital (HEGP), that is namely to evaluate the feasibility of a systematic screening procedure concerning tobacco and alcohol dependence among inpatients in two of the hospital's specialization departments between December 6, 2001, and March 6, 2002. METHODS: Self-administered questionnaires on addictive behaviours were distributed to each patient admitted to the vascular health department and to the hypertension department They included the Fagerstrom Test for Nicotine Dependence and the AUDIT for evaluating levels of alcohol abuse. RESULTS: The response rate was 30.2% (110/364). Twenty-nine smokers were identified. Among them, 72.5% presented a low level of physical nicotine dependence. Nearly half of them (14/29) were not interested in participating in a smoking cessation intervention with the assistance of relevant hospital staff. Among eleven patients at risk of excessive alcohol abuse, only one accepted to be referred to a specialist. CONCLUSION: This study highlights the difficulties of the systematic screening for addictive behaviours in hospitalized patients. Nevertheless, the obstacles faced in terms of the acceptability of the screening, but the patients as well as by the hospital staff, do not lean against the development of this trend or oppose this type of approach. New methods of individual screening for addictive behaviours should be explored. PMID- 16676711 TI - [Consequences for children involved in a road traffic accident: one year of observation and follow-up in the Rhone county]. AB - Children aged 6 to 11 who have been injured in a traffic accident were observed over a one year period, in parallel with a control group of children. More than one-third of the injured children had been hospitalized (for periods ranging anywhere between 1 and 47 days). One year later, one injured child out of ten was still suffering from pain, and/or still being treated for injuries resulting from the accident. Many other factors were linked to the initial overall level of severity of the injuries, contrary to that of pain, such as the rate and duration of hospitalization, the duration of care provided, the number of medical consultations, and absenteeism from school. Children who had been injured in a road traffic accident were found to be more anxious and nervous, in general, as well as having a high prevalence of sleeping disorders in comparison to the children in the control group. PMID- 16676712 TI - [Health practitioners' evaluation system on risks of contracting cancer due to environmental]. AB - The increase in the number of cancers sprouting from man-made environmental risk factors calls for prevention policies strictly centered on the basis of a fact sharing and transparent, evidence-based knowledge exchange. However, the related areas which are concerned are quite diverse, and given that the risks are of a cumulative nature, they are very difficult to measure. The information system therefore is complex and infinite. A qualitative study was carried out on the representations held by a variety of specialists who have potential links to the public or groups who might be exposed or at risk, including health professionals, engineers, and NGO and association managers and supervisors. The investigation was based upon a questionnaire which was developed with the assistance and participation of those involved in the management of information regarding professional, ecological or other kinds of risks. This method allowed for the study to favour and objectively prioritise the vision of the health professionals who were interviewed. The results show that the depictions vary depending on how and to what extent the different categories of professionals are involved, and that they reflect for the most part society's fears. PMID- 16676713 TI - [An epidemic of unidentified illness experienced by a hospital's staff: a qualitative study]. AB - An outbreak of non-specific complaints of illness and poor health occurred among 40% of the staff at the Archet 2 Hospital in Nice, France, between October 26 and November 26, 2000. This study utilises a psychosocial approach to describe how health-care workers experienced this unusually extensive phenomenon, and aims to contribute useful findings to the management of this type of emergency situation. A qualitative survey was conducted through face to face personal interviews with the first twelve staff members concerned; moreover, articles published in the press during that period were analysed in order to asses the impact of the media on this event. The study demonstrates that, although the source of the problem originated from an issue possibly related to questionable air quality, there were actually an array of factors which contributed to generate a mass psychogenic phenomenon: substantial collective societal complaints and staff members' perceived non-acknowledgement of the reality of their symptoms, lack of an obvious cause to account for those symptoms and collective representation regarding toxic gases, which were all claims promulgated by articles in the press. In addition to the need for technical improvements on the building and premises, the study underlines the relevance of simultaneously addressing related issues with the social structure, represented by the community of concerned hospital personnel, in order to facilitate a solution to the problem and avoid its potential recurrence in the future. PMID- 16676714 TI - [Factors linked to delayed diagnosis of tuberculosis in Conakry (Guinea)]. AB - Untreated smear-positive pulmonary tuberculosis constitutes a reservoir of infection which is highly contagious. The present study was conducted in Conakry, Guinea, to determine the different options which are available when seeking treatment or care, and to ascertain the average delay in diagnosis of pulmonary tuberculosis and the main factors linked to the delay in diagnosis after the initial onset of symptoms. Through a cross-sectional study, 113 consecutive patients with smear-positive pulmonary tuberculosis were interviewed through the use of a questionnaire. The median total delay from the onset of symptoms of pulmonary tuberculosis until the diagnosis was 11 weeks. This delay period exceeded 4 weeks for 90 of the patients (80%). The average delay linked to the conventional health care system was double that of the one at the fault of the patient (6 weeks versus 3 weeks, respectively). 54% of the patients had initially resorted to non-conventional care. To shorten this mean delay period, it is necessary to both strengthen the professional abilities and skills which train for one to better to detect tuberculosis and to sensitize the population to the subject matter and information on the illness and its symptoms. PMID- 16676715 TI - [Estimating mortality attribuable to PM10 particles in 9 French cities participating in the European programme Apheis]. AB - The relationship between air pollution and mortality is now admissible with a sufficiently high level of causality proven. This link allows for health impact assessment to be carried out with a significant degree of accuracy, such as the case for the results which are presented here from the nine French cities involved in the Apheis programme. This health impact assessment is based on the methodology developed by the World Health Organization. The number of avoidable deaths is contained between categories ranging from 2.0 to 4.3, 4.0 to 8.9, and from 15.0 to 31.5 per 100,000 inhabitants according to very short term effects, short term effects and long term effects, respectively. There are two scenarios which can be envisioned for the reduction of fine particles levels which are capable of obtaining similar results for both very short term and short term effects. The first involves diminishing the daily concentrations which are above 20 microg/m3 until they reach this value, and the second entails systematically decreasing the daily levels by 5 microg/m3. The first strategy of reducing values to stabilize at 20 microg/m3 has been shown to be the one most favourable and promising for the long term effects. This strategy therefore confirms the reliability and strength of the recommendation formulated at the national level. PMID- 16676716 TI - [Medical ethics and human rights training in Europe]. AB - In 1999 the World Medical Association (WMA) issued a declaration recommending that there be the teaching of medical ethics and human rights included in the basic medical school curricula across Europe. The study aims to investigate the level of medical ethics and human rights training provided and to take an inventory of the existing programmes in the European Union's medical schools. The study was carried out using a self-administered questionnaire which was disseminated in the year 2002 to 219 schools of medicine in 14 of the 15 European Union member states (Luxembourg did not yet have a school of medicine). One fourth of the medical schools solicited sent back a reply. Medical ethics are taught in 93% of medical schools; its teaching is of a multi-disciplinary and cross-cutting nature in 80% of the schools. Courses on ethics are compulsory in 75% of the cases. Human rights are taught in 63% of the European medical schools, and it is most often the case that the teaching of human rights is incorporated into the ethics courses. Even if the WMA's recommendation is not always fully implemented according to the guidelines of their declaration, this study demonstrates and reveals nonetheless the increasing of awareness and realization by European medical schools of the significance of including such a track in their programme. PMID- 16676717 TI - [Inequalities at the source of infant mortality in Peru]. AB - The principal aim of this paper is to analyze the relationship between social disparities and infant mortality. This research is based on the secondary analysis of the official data concerning socioeconomic and mortality rates for the end of the 1960's and for the mid of 1990's. The infant mortality rate is twice higher amongst the Andean population, particularly true for those living in the South of the Andes, than the average population. In contrast, the babies born in Lima metropolitan area benefit a probability of survival in their first year of life twice higher than the typical Peruvian new born. These inequalities are the result of social exclusion that restrict participation in economic, cultural and political institutions to different social groups in Peruvian society. Illiteracy and distribution of income play a decisive role in the increase of infant mortality rates. PMID- 16676718 TI - [Legal and ethical implications in the protection of data collected from electronic medical records and used for epidemiological statistics and surveillance: a situational analysis]. AB - With the emergence of new information and communication technologies (NICT) in the daily practice of medicine, personal medical data have become exportable. Certainly, they represent an interesting source for epidemiologists who were often lacking complete data sets all the way through to morbidity data; but once these data leave the confines of the structure of a medical office, they must be protected in order to respect the fundamental ethical principles which form the basis of the doctor-patient relationship. Given the fact that medical data are not considered merchandise, there is the need to lead a process of reflection which aims to adapt the existing ethical rules and regulations to norms which conform to this new environment. There is also a need to compile a report on the overall European situation, and more specifically the French case. PMID- 16676719 TI - [Requests for residency permits due to medical reasons from foreigners in the Val d'Oise county between 1999 and 2003]. AB - The requests for residence permits from foreigners who wish to remain in France for health reasons are submitted for review to the county public health medical officer. This article reports on the quantitative and qualitative evolution and progression of these requests in a county located near Paris. The number of requests has drastically increased, from 152 in 1999 to 1,823 in the year 2003. The majority of the applicants were women and individuals from sub-Saharan Africa. HIV infection was the most frequent reason cited for the submission of requests, but its relative proportion has decreased over time from 25% in 1999 to 15% of overall requests made in the year 2003, which is then followed by diabetes (8% of all requests), hypertension (5%), and tuberculosis (4%). The decision handed down from the authorities was favourable for residence based on the need for healthcare in 74% of the cases. The health status of the illegal immigrants remains difficult to determine and systematically track; therefore, the applications for residency granted on medical grounds serve as a practical and efficient means to assess and map the existing situation. PMID- 16676720 TI - [Towards establishing a platform of shared skills sets and competencies for public health doctors]. AB - School physicians, public health medical officers and inspectors, and doctors who work in local public authorities and in the local services of the Ministry of Health all share a common public health skills set even if the activities and functions which they perform are quite different, and in some cases even very specific to their field of practice. After a cross-sectional review of their indispensable roles and tasks, we designed a referential system for shared competencies and have presented it to various active professionals. This tool serves as a starting point which has permitted us to tackle questions relating to the professionalisation, evaluation, and multi-disciplinary nature of these doctors. It also aims to contribute to the current reflection on the level of public health training necessary for all medical practitioners and doctors, but above and beyond that, more specifically for public health professionals. PMID- 16676721 TI - [Planning and assessing the activity of a school health service based on health and social indicators: the experience of Villeurbanne]. AB - In 1998, the municipality of Villeurbanne decided to review its school health service in order to restructure and adjust its activity to better meet the needs of schools which had evolved and developed differently according to their areas of location. In view of the rarity of available information to measure the needs, an information system was built using a participatory and consensual approach. The system has been operational since the year 2000. It was evaluated in 2003 and then adapted according to the results. It has a variety of uses for planning purposes: to classify the schools according to their requirements, to plan the working time and schedules of the nurses, and to programme and evaluate specific actions. It also allows for mapping the health status of the pupils in Villeurbanne and to describe the service's activity. This article presents the design and construction of the information system and serves as a platform to debate the benefits and limitations of this approach in Villeurbanne. PMID- 16676722 TI - [How children with food allergies are accommodated by school cafeterias and dining halls: a survey in Maine-et-Loire]. PMID- 16676723 TI - Conflicting research findings. PMID- 16676724 TI - African American beliefs about diabetes. AB - In this presentation, a diabetes explanatory model of rural, African American adults at high risk for diabetes is described. Kleinman's explanatory model of illness was used as the organizing framework. African American men and women (N=42), between the ages of 18 and 51, participated. Participants described their knowledge and beliefs about diabetes, preventing diabetes, and whether diabetes could be cured or controlled. A common explanatory model of diabetes was not shared among the participants, and gender and age differences were apparent. Common themes included diabetes "running in families", "eating too much sugar", and "not taking care of yourself" as causes of diabetes. Weight and physical activity or exercise were not seen as contributing to the development of diabetes. Participants were not sure if diabetes could be prevented. These results provide information to address primary prevention of diabetes in this at risk group. PMID- 16676725 TI - Self-care and mothering in African American women with HIV/AIDS. AB - African American women are the most rapidly growing group of people in the United States diagnosed with HIV/AIDS. The purpose of this study was to explore experiences of self-care and mothering among African American women with HIV/AIDS. It is important to recognize how culture affects illness management, childrearing, and daily living to design culturally appropriate nursing interventions for African American women. Critical ethnography was used to study 10 African American mothers from the rural Southeast who were HIV positive and mothered children who were HIV positive. Domains derived from the research were disabling relationships, strong mothering, and redefining self-care. The cultural theme was creating a life of meaning. African American mothers with HIV/AIDS in the rural Southeast used culturally specific self-care and mothering strategies reflective of cultural traditions. This study acknowledges strengths of African American women and generates theory that will enhance nursing care to this population. PMID- 16676726 TI - Reactions of hospital staff nurses to recruitment incentives. AB - In response to the nursing shortage, many hospitals offer sign-on bonuses to attract nurses. There are anecdotal reports that the use of such incentives negatively affects staff nurse morale. The present study is the first to empirically examine the reactions of staff nurses who have not received sign-on bonuses. Most of the 800 surveyed nurses were not opposed to the use of incentives, but almost all were concerned about the potential for negative impact on retention. Compared to nurses working in hospitals that did not offer sign-on bonuses, nurses working in hospitals that offer sign-on bonuses reported higher levels of anger and lower levels of optimism. Greater anger and less optimism were associated with a reduced sense of distributive justice. The less fairly treated nurses felt, the greater likelihood of withdrawal from the hospital. The use of recruitment incentives may result in reduced work effort or loss of experienced nurses, exacerbating the nursing shortage. PMID- 16676727 TI - Environmental ambiance as a new window on wandering. AB - This cross-sectional study explored relations between environmental ambiance and locomotion behaviors of elders with dementia (EWD), addressing the larger goal of developing theory explaining how environment affects wandering and other behaviors, thereby illuminating a pathway to intervention. A sample of 47 EWD (older than age 65, Mini-Mental State Examination [MMSE] < or =23) from nursing homes and assisted-living facilities were studied through a random, cluster, sampling strategy. Participants were measured once for stable characteristics and videotaped for 12, randomly chosen, 20-minute periods over 2 separate days at least 48 hours apart. Hierarchical liner modeling was used to analyze data that focused on the observation as the unit of analysis, with repeated observations nested within participants. Results validated an influence of ambiance on locomoting behaviors, highlighting the importance of addressing the emotional valence of social and physical environments in care of EWD. PMID- 16676729 TI - [Public health: from the past toward new frontiers]. AB - The Authors analyse and discuss the contents of some recent international meetings devoted to Public Health, namely the 10th Congress of the WFPHA, the 12th British annual Forum of Public Health and the 12th Conference of the EUPHA. Their efforts to evaluate health services comes off enriched with new scenarios of integration and convergence. Objectives of global interest in the world cannot be faced by individual countries alone: on the contrary, the whole scientific community of public health operators must be involved. The cultural, social and economic development is the unavoidable condition to increase the health level of the populations. No health organization with the characteristics of excellence can exist without a comparable, balanced development regarding the society and its economy. With such considerations in mind, the European Commission in 1997 has began to develop the Health Monitoring Programme, with the aim to build a system for monitoring health in the UE. Within this Programme, the Study ECHI 1 (European Community Health Indicators 1) has been published, which includes 4 large groups of indicators (demographic and socioeconomic indicators, health indicators, determinants of health/disease indicators, health services indicators); such a study was performed with the cooperation of OCSE and WHO/Euro researchers. The following ECHI 2 research did not change the supporting philosophy of ECHI 1. We are trying to evaluate whether the intervention of health operators and health structures could influence - increasing or decreasing - the phenomena described and measured by the indicators, inviting the readers to start a debate with us. PMID- 16676728 TI - Factors related to chronic obstructive pulmonary disease readmission in Taiwan. AB - This study examines the relationships among physiological, psychological, and social factors and hospital readmission to develop a model predicting chronic obstructive pulmonary disease (COPD) readmission for 145 patients with COPD following hospital discharge at 14 days and 90 days in Taiwan. Daily functioning, comorbidity, severity of illness, self-efficacy, depressive symptoms, and perceived informal support were regressed on hospital readmission. Daily functioning was the only significant variable to predict COPD readmission at 90 days in the Taiwan population living in a rural area. Age was significantly correlated with 14 days readmission. Post hoc analyses examined differences in three ethnic groups. Mainlanders perceived less family support, had higher depressive symptoms and lower daily functioning than the majority culture Fukiens and Hakkas, or the Aborigines. The study reinforced the need for identification of cultural differences and low functioning as risk factors for early readmission so they can be addressed in discharge planning. PMID- 16676730 TI - [Antibiotic-resistance in Italy: activity of the first year of the surveillance project AR-ISS]. AB - The antibiotic resistance surveillance project AR-ISS, started in 2001, is based on a network of 62 sentinel microbiological laboratories throughout the country. The laboratories collect and transmit data to the Istituto Superiore di Sanita on the antibiotic susceptibility of bloodstream isolates of 7 species: Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis/faecium, Klebsiella pneumoniae/oxytoca ed Escherichia coli. They also send selected bacterial strains for further characterization. Results of the first year of surveillance are presented and are compared with data from the previous study EARSS-Italia and from other European countries. Oxacillin resistance in S. aureus appears to be stable, however, it remains one of the highest in Europe (41,5%). No strain with intermediate susceptibility or resistance to vancomycin has been isolated. In S. pneumoniae, the level of penicillin resistance is moderate (10,8%), but macrolide resistance has increased greatly (37,6% versus 28,6% of the previous study), following a tendency common to several European countries. Unexpectedly, vancomycin resistance in E. faecium was found to be 18%, the highest in Europe. Presumptive ESBL production in Gram-negative organisms can be estimated at 20% in Klebsiella and 1% in E. coli. Ampicillin and ciprofloxacin resistance in E. coli (respectively 50% and 18%) are among the highest in Europe. In conclusion, the rate of antibiotic resistance in the species studied is worrisome and requires continuing monitoring. Although some activities of AR-ISS need improvements, the surveillance has the potentiality to produce relevant and representative data about antibiotic resistance in Italy that can be used for comparison at the European level. PMID- 16676731 TI - [Reduction of the incidence of nosocomial infections after assessing and revising the quality of assistance in a thoracic surgery unit]. AB - An active surveillance for nosocomial infections has been lead in a Thoracic Surgery with the intention, first to point out their frequency and characteristics, and then to outline all the measures to remove the main risk factors checking the results obtained. A prospective incidence study has been promoted in a Thoracic Surgery in the years 2000, 2001, 2002. The analysis has been lead weekly gathering all necessary data from the health records and making laboratory tests to look for microbes growth in the air of Thoracic Surgery Operating Rooms. A nosocomial infections incidence of 13.3% among surgically treated patients has been registered in 2000. Deep surgical site infections were the most frequent localizations, and microbes isolated were Staphylococcus aureus and coagulase negative Staphylococcus with an high oxacillin resistance (70.6% 76.5%). From the observation of the risk factors the sterilization system has been modified and the assistance and environmental protocols have been improved. In the further evaluation period, a global reduction of nosocomial infections incidence (7.1%), of surgical site infections (from 10.1% to 4.5%) (p = 0.007), of Staphylococcus aureus and coagulase negative Staphylococcus isolations have been obtained even if short results in antibiotic resistances have been registered. Thoracic Surgery has to be considered an area at medium-high risk of nosocomial infections. The quite high incidence of nosocomial infections recorded at the beginning of the study in presence of prevalent deep surgical site infections from staphylococci with an high oxacillin resistance compelled to promote corrections. These lead to a remarkable decrease in incidence of nosocomial infections even if the same results can not be reached in antibiotic resistances. PMID- 16676732 TI - [Accessibility and quality to health social services in Italy for the patients with rare diseases: the opinion of associations]. AB - This paper concerns the first phase of a study about the perception of social and health needs of people with rare diseases. The study was performed by the National Center for Rare Diseases at the Italian National Institute of Health (Istituto Superiore di Sanita - ISS). The project wants to be an example of collaboration between the research and the association worlds. Responsible of Associations of Patients and their relatives were asked their opinion about the accessibility and quality of important features of health and social services (accessibility and quality of diagnostic, pharmacological, psychological and rehabilitative interventions, social support, school and vocational training, information that was given to relatives). An ad hoc questionnaire was developed through focus groups. The questionnaire was completed by 108 associations (26,5% of the associations thar are recorded in the ISS database). Average scores showed satisfaction only for some variables and a negative gradient north-south was observed. The most frequent complaints were about information, quality of school and job training services and availability of psychological support. The study showed an high level of dissatisfaction with availability, quality and integration health and social services. PMID- 16676733 TI - [Opinions of women regarding a planned home birth project in Apulia (Southern Italy)]. AB - A growing desire among women to move away from interventions and hospitals to more "natural" childbirth has emerged over several years. The role and autonomy of midwives, a favourable opinion among women about home childbirth and better cooperation between hospitals and district health facilities are all fundamental in order to implement a home birth project. The opinion of women about "planned" home birth was investigated in the Puglia region through a survey conducted among a sample of women who had just delivered in hospital and a representative sample of the general female population. From 20 to 30% of women in the Puglia region would be basically in favour of home birth project. The main difference between the two groups of women concern the higher proportion of caesarean deliveries among mothers who had just delivered and their demand for more specific training of involved health personnel. Both samples are agreed on the specific training of women during pregnancy and on the presence of a specialist during home delivery. The results of the investigation seem to be in favour of planning a home birth project in the Puglia region. PMID- 16676734 TI - [International review of health educational programs for nutrition in the elderly]. AB - Progressive increase of the elderly may well explain the interest to educational programs at this age. Different chronic conditions such as cardiovascular diseases, diabetes and osteoporosis may be controlled and prevented by proper educational campaigns. In our review, the role of nutritional educational on quality of life has been evaluated, either in healthy subjects or in those with chronic disease. Evaluation tools and outcome index have been described. It is emphasized that applying specific projects may improve nutritional attitudes and lifestyle even in older adults. Proper educational intervention should be implemented, but socio-cultural characteristics of the target population should be taken into account. PMID- 16676735 TI - [Prevention of oral cancer: practice patterns in a dentists' group]. AB - Annually, 3000 people in our country die for oral cancer. Contrarily to other forms of cancer that require the use of instrumental diagnostic tools, the oral cavity is easy to examine and the cancer is usually preceded by precancerous conditions. The problem of the diagnostic delay can be explained through a underestimation of the lesions of the oral cavity by patients and a lack of sensibilization to prevention topics from dentists. Our work has inquired in the activity of a group of dentist with more than ten years than experience on accuracy of the anamnesis, objective exam and appropriate of the resource to specialists, and that with the aim not to measure the clinical abilities but to estimate the attitude towards the Prevention topics, in special way in the field of the malignant tumors of the oral cavity. PMID- 16676736 TI - [Epidemiologic survey of medical and non-medical personnel in a public dental clinic]. AB - Hepatitis B and C and Mycobacterium tuberculosis infection represent an occupational problem for the dentist. Few data are available in literature about the situation in our country. An epidemiological survey was carried out to evaluate the prevalence of Hepatitis B (HbsAb) and C (HCVab) markers and the exposure to Mycobacterium tuberculosis (Tine test) of the whole population of a public dental clinic (247 dental care workers). Furthermore participants were asked to fill a questionnaire to assess their vaccination status. 67,2% of the subjects were found to be positive for HbsAb (77,4% of medical personnel and 28,8% of the auxiliary medical personnel). Only one subject was found positive for anti-HCV (0,4%). 12,5% of the population showed a positive Tine test (15,4% of medical personnel and 1,9% of the auxiliary medical personnel). Our data show a difference between the percentage of subjects who were vaccinated (2,4%) and those who were positive to the Tine test (12,5%). PMID- 16676737 TI - [Health promotion and education in the secondary schools]. AB - To prevent the risks of HIV infection, some meetings have been carried with the students of secondary school; actually the school is the best and the most appropriate place to conduct these health promotion and education meetings. Two questionnaires have been given to the students, a pre-test before the interview, to evaluate their knowledge about drugs, and a re-test after the interview to evaluate whether the knowledge objectives suggested had been reached. After the meeting the students appear to be more informed; differences were statistically significant between the percentages of the correct answers of the pre-test and the re-test. PMID- 16676738 TI - [Decrease of frequency of male lung cancer in Umbria region. 1978-1982 and 1994 1999]. AB - In Umbria, region of Central Italy, lung cancer is the leading cause of death due to cancer in males. The aim of this work is to highlight decrease of mortality from lung cancer in males in Umbria, in the last 20 years and to describe relationship among incidence, mortality and survival of this cancer. The incidence data derived from an ad hoc survey carried out over the period 1978 1982 and for 1994-1999 from the Umbrian Population Cancer Registry (RTUP). The mortality data were derived from official publications of the Italian Central Institute for Statistics (ISTAT) and covered the entire period 1978-1999. Estimated annual percent change (EAPC) was calculated by joinpoint regression analysis. Decrease of mortality from male lung cancer in Umbria began in the 1990 ies due to reduced quantity of tar in cigarettes, decreasing consumption of tobacco and increasing of survival derived from the quality and the effectiveness of the oncological health care system. PMID- 16676740 TI - Holistic nurses group leaves a lasting legacy. PMID- 16676739 TI - Pain can lead to disruptive behaviours. PMID- 16676741 TI - Dialysis cruising. PMID- 16676742 TI - Pre-pouring medications. PMID- 16676743 TI - Advanced nursing practice: enter the nurse practitioner. PMID- 16676744 TI - Practice environments: What's improving? What's not? PMID- 16676745 TI - Putting CRNBC's practice environment guidelines to the test in India. PMID- 16676746 TI - Developing a strategic plan. PMID- 16676747 TI - Boomers: bottlenecked, bored, and burned out. PMID- 16676748 TI - An overview of Medicare reimbursement regulations for advanced practice nurses. AB - The federal government spends nearly 15% of the budget on Medicare services annually, and advanced practice nurses are eligible for reimbursement from that pool. The regulations governing reimbursement are complex because of the social, political, and financial pressures involved in their development. Although economic viability and due diligence considerations make it incumbent on advanced practice nurses to understand the rules, the profession, as a whole, has knowledge deficits in this area. The essentials of regulatory development and structure are reviewed and considerations for optimizing reimbursement are described. PMID- 16676749 TI - Organizational characteristics and their effect on health. AB - There is a complex relationship between work, employee health, and successful business results. At the individual level, multiple factors including physical, psychological, and social aspects of the work environment affect workers' health and well-being. At the organizational level, the consequences of unhealthy work environments may be traced to a decrease in the quality of service and products, and a loss in productivity. A brief overview of the contemporary literature on workplace stressors is presented to help identify future research directions. PMID- 16676750 TI - Minimum nurse staffing ratios for nursing homes. AB - The authors explored minimum nurse staffing ratios for nursing homes using production function simulations. Minimum levels of registered nurse hours per resident day were 0.31, 1.83, and 3.3 at 50%, 75%, and 90% levels of quality respectively. These results suggest that efficiency-oriented minimum nurse staffing points exist, and could be used to inform policymakers and nursing home administrators on better resource allocation and health care delivery. PMID- 16676751 TI - Occupational commitment, education, and experience as a predictor of intent to leave the nursing profession. AB - This national Internet-based study tested occupational commitment in predicting RN intent to leave the nursing profession. Results showed that occupational commitment was increased by educational level and experience. Findings suggest opportunities for policy changes that may increase retention of registered nurses. PMID- 16676752 TI - Promoting nursing as a career choice. AB - Fewer young people are considering nursing as a career. This project was designed to present nursing as a viable career option to middle school students. A questionnaire was administered to six classes of middle school students before and after a presentation on nursing as a career. Results showed the presentation improved student attitudes and perceptions toward nursing as a career. Nurses can use this project as a stepping stone to develop additional programs to interest students in nursing as a career before the students discard the notion altogether. PMID- 16676753 TI - Facility charging and Nursing Intervention Classification (NIC): the new dynamic duo. PMID- 16676754 TI - Paving and resurfacing the road to magnet: the perspective and wisdom of magnet designated coordinators--part I. PMID- 16676756 TI - Staff nurses as masters of their environment. PMID- 16676755 TI - Authentic leadership. PMID- 16676758 TI - "I saw you the other day but I wasn't there". PMID- 16676757 TI - Gyroscope for a new world. PMID- 16676759 TI - Fear, stress, and the well dental office. PMID- 16676760 TI - Small town heart: the 2006 MDA president's interview. PMID- 16676761 TI - When the walls come tumbling down: Part three. AB - As you see, the MDA and HPSP provide a wide variety of services and help for dentists. People needing to contact HPSP can call (651) 643-2120. If you have any questions, please call the MDA office at (651) 646-7454 for help or referral. PMID- 16676762 TI - What's a dentist to do? A call to alms. PMID- 16676763 TI - The professional. 2001. PMID- 16676764 TI - The digital office. Let the transition begin! PMID- 16676765 TI - Oral pathology case: multiple papillomatous lesions. PMID- 16676766 TI - Buccal pit study club #6. PMID- 16676767 TI - Emotional and psychological effects of physician-assisted suicide and euthanasia on participating physicians. AB - This is a review and evaluation of medical and public literature regarding the reported emotional and psychological effects of participation in physician assisted suicide (PAS) and euthanasia on the involved physicians. MATERIALS AND METHODS: Articles in medical journals, legislative investigations and the public press were obtained and reviewed to determine what has been reported regarding the effects on physicians who have been personally involved in PAS and euthanasia. RESULTS AND DISCUSSION: The physician is centrally involved in PAS and euthanasia, and the emotional and psychological effects on the participating physician can be substantial. The shift away from the fundamental values of medicine to heal and promote human wholeness can have significant effects on many participating physicians. Doctors describe being profoundly adversely affected, being shocked by the suddenness of the death, being caught up in the patient's drive for assisted suicide, having a sense of powerlessness, and feeling isolated. There is evidence of pressure on and intimidation of doctors by some patients to assist in suicide. The effect of countertransference in the doctor patient relationship may influence physician involvement in PAS and euthanasia. CONCLUSION: Many doctors who have participated in euthanasia and/or PAS are adversely affected emotionally and psychologically by their experiences. PMID- 16676768 TI - Gonzales v. Oregon. PMID- 16676769 TI - Oral argument in Gonzales v. Oregon. PMID- 16676770 TI - The dissent in Richmond Medical Center v. Hicks in the Fourth Circuit Court of Appeals. PMID- 16676771 TI - Towards an understanding of sleep problems in childhood depression. PMID- 16676772 TI - Optimizing continuous positive airway pressure therapy for obstructive sleep apnea syndrome. PMID- 16676773 TI - Defining insomnia: the role of quantitative criteria. PMID- 16676774 TI - Another chapter in the adenosine story. PMID- 16676775 TI - Dreaming about an open upper airway. PMID- 16676776 TI - Tiagabine is associated with sustained attention during sleep restriction: evidence for the value of slow-wave sleep enhancement? AB - STUDY OBJECTIVES: To evaluate the impact of enhanced slow-wave sleep (SWS) on behavioral, psychological, and physiologic changes resulting from sleep restriction DESIGN: A double-blind, parallel-group, placebo-controlled design was used to compare tiagabine, 8 mg, (a SWS-enhancing drug) to placebo during 4 nights of sleep restriction (time in bed = 5 hours per night). Behavioral, psychological, and physiologic measures of the impact of sleep restriction were compared between groups at baseline, during sleep restriction, and following recovery sleep. SETTING: Two sleep research laboratories. PARTICIPANTS: Thirty eight healthy adults; 9 men and 10 women (mean age: 26.0 +/- 6.1 years) in the placebo group and 8 men and 11 women (mean age: 26.7 +/- 8.1 years) in the tiagabine 8 mg group INTERVENTIONS: Both experimental groups underwent 4 nights of sleep restriction. Each group received either tiagabine 8 mg or placebo on all sleep-restriction nights, and both groups received placebo on baseline and recovery nights. MEASUREMENTS AND RESULTS: Polysomnography documented a SWS enhancing effect of tiagabine. The placebo group displayed the predicted deficits due to sleep restriction on the Psychomotor Vigilance Task and the Multiple Sleep Latency Test. Compared with placebo, the tiagabine group did not demonstrate impairment in sustained attention on the Psychomotor Vigilance Test, performed better on the Wisconsin Card Sorting Task, reported more restorative sleep, and had less of an increase in afternoon-evening salivary free cortisol. Multiple Sleep Latency Test, ratings of sleepiness, recovery sleep, and other measures did not differ between groups. CONCLUSIONS: To our knowledge these findings are the first to be consistent with the hypothesis that pharmacologic SWS enhancement reduces selective aspects of the behavioral, psychological, and physiologic impact of sleep restriction. PMID- 16676777 TI - Identifying critical beliefs about sleep in primary insomnia. AB - SUBJECT OBJECTIVE: Maladaptive beliefs about sleep are associated with insomnia and are assessed with the Dysfunctional Beliefs and Attitudes about Sleep scale (DBAS). Three studies explored which DBAS items (1) maximally differentiated people with insomnia from good sleepers, (2) declined with cognitive behavior therapy (CBT), and (3) were related to other clinical improvement indexes. DESIGN: Data from previous studies were analyzed to evaluate the above 3 hypotheses. PARTICIPANTS: The total sample (N = 332) was comprised of experimental and treatment-seeking people with insomnia and good sleepers ranging from 20 to 79 years of age (mean +/- SD 51.3 +/- 14.7). RESULTS: The analyses of variance of the 30 items of the DBAS in Study 1 suggested that 16 items differentiated insomnia sufferers from good sleepers. In Study 2, 8 items showed significantly greater changes in response to CBT than alternate therapies. However, only 2 of these items were among the 16 items that discriminated insomnia sufferers from good sleepers in Study 1. In Study 3, declining scores on 15 of 30 DBAS items in response to CBT were related to 1 or more indexes of clinical improvement. CONCLUSION: The 16 beliefs of the DBAS-30 that best discriminated insomnia sufferers from good sleepers related to helplessness and hopelessness in the insomnia group. CBT addressed some of these beliefs, although some beliefs relating to helplessness remained relatively elevated. These residual beliefs should be investigated further, as they may confer cognitive risk for future insomnia and imply ways to improve current CBT strategies. PMID- 16676778 TI - Extracellular adenosine in the human brain during sleep and sleep deprivation: an in vivo microdialysis study. AB - STUDY OBJECTIVES: To examine the pattern of extracellular adenosine in the human brain during sleep deprivation, sleep, and normal wake. DESIGN: Following recovery from implantation of clinical depth electrodes, epilepsy patients remained awake for 40 continuous hours, followed by a recovery sleep episode. SETTING: Neurology ward at UCLA Medical Center. PATIENTS OR PARTICIPANTS: Seven male epilepsy patients undergoing depth electrode localization of pharmacologically refractory seizures. INTERVENTIONS: All subjects were implanted with depth electrodes, a subset of which were customized to contain microdialysis probes. Microdialysis samples were collected during normal sleep, sleep deprivation, and recovery sleep from human amygdalae (n = 8), hippocampus (n = 1), and cortex (n = 1). MEASUREMENTS AND RESULTS: In none of the probes did we observe an increase in extracellular adenosine during the sleep deprivation. There was a significant, though very small, diurnal oscillation (2.5%) in 5 of the 8 amygdalae. There was no effect of epileptogenicity on the pattern of extracellular adenosine. CONCLUSIONS: Our observations, along with those in animal studies, indicate that the role of extracellular adenosine in regulating sleep pressure is not a global brain phenomenon but is likely limited to specific basal forebrain areas. Thus, if energy homeostasis is a function of sleep, an increased rate of adenosine release into the extracellular milieu of the amygdala, cortex, or hippocampus is unlikely to be a marker of such a process. PMID- 16676779 TI - Indoor exposure to natural bright light prevents afternoon sleepiness. AB - STUDY OBJECTIVES: The present study examined the effects of indoor exposure to natural bright light on afternoon sleepiness. DESIGN: Participants took part in 3 experimental conditions: (1) a natural bright light condition in which they carried out performance and arousal tests sitting near a window (3260.0 +/- 1812.43 lux) from 12:40 PM to 1:10 PM, (2) a nap condition in which they were provided a nap opportunity for 20 minutes from 12:45 PM, and (3) a control condition in which they performed the tests in less than 100 lux surroundings from 12:40 PM to 1:10 PM. Before and after each treatment, the same series of tests were administered. SETTING: A temperature- and light-controlled sleep laboratory. PARTICIPANTS: Sixteen healthy female paid volunteers aged 33 to 43 (38.1 +/- 2.68) years. INTERVENTIONS: Indoor natural bright light and a short nap. MEASUREMENTS AND RESULTS: Arousal levels were measured by the Psychomotor Vigilance Task, Alpha Attenuation Test, Karolinska Drowsiness Test, and Karolinska Sleepiness Scale. The tests were repeated every 30 minutes from 11:00 AM to 4:10 PM. Ambient light intensity was maintained at less than 100 lux, except during natural bright light exposure. Short-term exposure to natural bright light significantly improved afternoon arousal levels, as measured by the Karolinska Drowsiness Test and Alpha Attenuation Test, the effects of which continued for at least 60 minutes (1:10-2:10 PM). However, no significant differences were observed between conditions for Psychomotor Vigilance Test performance. CONCLUSIONS: Brief indoor exposure to natural bright light may decrease afternoon sleepiness. This technique of light could be used in work settings in which napping is not permitted. PMID- 16676781 TI - Defining insomnia: quantitative criteria for insomnia severity and frequency. AB - STUDY OBJECTIVE: Recent efforts have been made to develop quantitative frequency, duration, and severity criteria for insomnia. The current study was conducted to test a range of frequency and severity criteria sets for discriminating primary insomnia sufferers from normal sleepers. PARTICIPANTS: Seventy-two adults with primary insomnia and 88 age-matched normal sleepers. METHODS: Participants completed 14 consecutive nights of sleep logs to monitor their home sleep patterns. Receiver-operator characteristic curve analyses were used to compare a range of severity and frequency criteria sets for discriminating the insomnia and normal-sleeper groups. In addition, sensitivity and specificity tests were conducted for a range of wake-time severity cutoffs based on 2-week mean sleep log data. RESULTS: Receiver-operator characteristic curve analyses showed that no 1 combination of severity and frequency criteria maximized sensitivity and specificity. Rather, the optimal frequency cutoff decreased as the severity criterion increased. Analyses of mean sleep-log data showed that an average sleep onset latency or middle-of-the-night wake time (ie, time awake between sleep onset and final morning awakening) cutoff of 20 minutes or longer over 2 weeks of sleep-log monitoring appeared to best maximize sensitivity (94.4%) and specificity (79.6%) for insomnia classification. CONCLUSIONS: The optimal quantitative insomnia criteria found herein differ from those previously proposed. Nonetheless, results suggest that quantitative criteria derived from sleep-log data may be useful for classification of primary insomnia. PMID- 16676780 TI - Genioglossal muscle response to CO2 stimulation during NREM sleep. AB - STUDY OBJECTIVES: The objective was to evaluate the responsiveness of upper airway muscles to hypercapnia with and without intrapharyngeal negative pressure during non-rapid eye movement (NREM) sleep and wakefulness. DESIGN: We assessed the genioglossal muscle response to CO2 off and on continuous positive airway pressure (CPAP) (to attenuate negative pressure) during stable NREM sleep and wakefulness in the supine position. SETTING: Laboratory of the Sleep Medicine Division, Brigham and Women's Hospital. PATIENTS OR PARTICIPANTS: Eleven normal healthy subjects. INTERVENTIONS: During wakefulness and NREM sleep, we measured genioglossal electromyography (EMG) on and off CPAP at the normal eupneic level and at levels 5 and 10 mm Hg above the awake eupneic level. MEASUREMENTS AND RESULTS: We observed that CO2 could increase upper-airway muscle activity during NREM sleep and wakefulness in the supine position with and without intrapharyngeal negative pressure. The application of nasal CPAP significantly decreased genioglossal EMG at all 3 levels of PETCO2 during NREM sleep (13.0 +/- 4.9% vs. 4.6 +/- 1.6% of maximal EMG, 14.6 +/- 5.6% vs. 7.1 +/- 2.3% of maximal EMG, and 17.3 +/- 6.3% vs. 10.2 +/- 3.1% of maximal EMG, respectively). However, the absence of negative pressure in the upper airway did not significantly affect the slope of the pharyngeal airway dilator muscle response to hypercapnia during NREM sleep (0.72 +/- 0.30% vs. 0.79 +/- 0.27% of maximal EMG per mm Hg PCO2, respectively, off and on CPAP). CONCLUSIONS: We conclude that both chemoreceptive and negative pressure reflex inputs to this upper airway dilator muscle are still active during stable NREM sleep. PMID- 16676782 TI - Time estimation in chronic insomnia sufferers. AB - STUDY OBJECTIVE: The objective of the present study is 2-fold: (1) compare the time-estimation performance of chronic insomnia sufferers to that of good sleepers and (2) evaluate the severity of the sleep complaint in order to assess its association with the time-estimation performance. DESIGN: Between subjects design. PARTICIPANTS: The sample included 11 individuals suffering from chronic primary insomnia (7 women and 4 men, mean age = 44.64 years, SD = 12.71) and 11 good sleepers (5 women and 6 men, mean age = 48.00 years, SD = 7.86). INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: All participants completed a time estimation task, namely a finger-tapping task. The results indicate no significant between-group differences on time-estimation data, as well as no significant relationship between severity of insomnia complaint and estimation of time. CONCLUSION: These results suggest that the tendency to misestimate sleep difficulties is not linked to impaired time estimation-specific processes in insomnia sufferers, as measured with the present task. PMID- 16676784 TI - Sleep and alertness in children with attention-deficit/hyperactivity disorder: a systematic review of the literature. AB - STUDY OBJECTIVE: To review evidence on sleep and alertness in children with attention-deficit/hyperactivity disorder (ADHD) controlling for potential confounding factors. METHODS: A PubMed search. Studies using ADHD diagnostic criteria other than DSM-III-R or IV and studies not excluding or controlling for psychiatric comorbidity or medication status were not included in the review. Results from objective studies were combined using meta-analysis. RESULTS: From the 46 studies located, 13 were retained. With regard to objective studies, the proportion of subjects who fell asleep during the Multiple Sleep Latency Test, the number of movements in sleep, and the apnea-hypopnea index were significantly higher in children with ADHD than in controls. We found no significant differences in other objective parameters (sleep-onset latency; number of stage changes; percentages of stage 1 sleep, stage 2 sleep, slow-wave sleep, or rapid eye movement sleep; rapid eye movement sleep latency; and sleep efficiency). Limited evidence from subjective studies suggests no significant differences in sleep-onset difficulties and bedtime resistance between children with ADHD and controls, after controlling for comorbidity and medication status. Data on sleep duration, night and morning awakenings, and parasomnias are still very limited. CONCLUSION: Results from our systematic review suggest that children with ADHD have higher daytime sleepiness, more movements in sleep, and higher apnea hypopnea indexes compared with controls. Given the limited number of studies controlling for confounding factors, further subjective and objective studies are needed to better understand alterations in sleep and alertness in children with ADHD. PMID- 16676785 TI - Sleep and aggression in substance-abusing adolescents: results from an integrative behavioral sleep-treatment pilot program. AB - STUDY OBJECTIVE: To examine whether change in total sleep time during an integrative, behavioral sleep intervention is associated with aggression. Specifically, we tested whether adolescents who reported experiencing aggressive thoughts or actions after treatment had worse treatment trajectories (e.g., less total sleep time across treatment) than adolescents with no aggressive thoughts or actions after treatment. DESIGN: Nonpharmacologic open trial with 9 weeks of weekly assessment. SETTING: University of Arizona Sleep Research Laboratory PATIENTS OR PARTICIPANTS: Twenty-three adolescents recently treated for substance abuse in outpatient community centers. INTERVENTIONS: Six-week integrative, behavioral sleep intervention. MEASUREMENTS AND RESULTS: Weekly sleep-summary indexes were calculated from daily sleep diaries and entered as dependent variables in a series of growth-curve analyses. Statistically significant Session x Post-treatment Aggressive Ideation interactions emerged when predicting changes in total sleep time, gamma13 = 9.76 (SE = 4.12), p < .05, and time spent in bed, gamma13 = 10.08, (SE = 4.33), p < .05, even after controlling for aggressive ideation and the frequency of substance use, as assessed at baseline. A similar pattern of results was seen for self-reported aggressive actions occurring during conflicts. CONCLUSIONS: These pilot data suggest that inadequate sleep in substance-abusing adolescents may contribute to the experiencing of aggressive thoughts and actions. Limitations include a small sample size and a restricted assessment of aggression. Nonetheless, these findings lend preliminary support to the breadth of therapeutic effectiveness of an integrative, behavioral sleep therapy program for adolescents with a history of substance abuse and related behaviors. PMID- 16676783 TI - Subjective sleepiness and polysomnographic correlates in children scheduled for adenotonsillectomy vs other surgical care. AB - STUDY OBJECTIVE: To compare a validated subjective measure of childhood sleepiness to an objective determination, assess the frequency of problematic sleepiness among children with suspected sleep-disordered breathing (SDB), and examine what standard or investigational polysomnographic measures of SDB predict subjective sleepiness. DESIGN: Prospective, cross-sectional. SETTING: University based sleep disorders laboratory. PARTICIPANTS: Washtenaw County Adenotonsillectomy Cohort. INTERVENTION: Polysomnography followed by Multiple Sleep Latency Tests (MSLTs) in 103 children aged 5 to 12 years old: 77 were scheduled for clinically indicated adenotonsillectomy, usually for suspected SDB, and 26 for unrelated surgical care. Parents completed the previously validated, 4 item Pediatric Sleep Questionnaire-Sleepiness Subscale (PSQ-SS). RESULTS: Thirty three (43%) of the children scheduled for adenotonsillectomy had high PSQ-SS scores, in comparison with only 3 (12%) of the controls (p = .004). The PSQ-SS scores correlated inversely with mean sleep latencies on the MSLTs (rho = -0.23, p = .006). The obstructive apnea index, apnea-hypopnea index, and respiratory disturbance index (which included respiratory event-related arousals identified by esophageal pressure monitoring) each correlated similarly with PSQ-SS scores, as did investigational quantification of esophageal pressures and respiratory cycle-related electroencephalographic changes (each rho approximately 0.30, p < .02). A stepwise regression identified sigma-frequency respiratory cycle-related electroencephalographic changes as the strongest independent predictor of subjective sleepiness among all subjects and particularly among those without obstructive sleep apnea. CONCLUSIONS: Sleepiness is a frequent problem among children with suspected SDB. Subjective sleepiness (PSQ-SS) reflects MSLT results to a limited extent, as in adults. Standard polysomnographic measures of SDB predict subjective sleepiness, but respiratory cycle-related electroencephalographic changes may offer additional clinical utility. PMID- 16676786 TI - Effects of sleep fragmentation on the arousability to resistive loading in NREM and REM sleep in normal men. AB - STUDY OBJECTIVE: In healthy subjects, arousability to inspiratory resistive loading is greater during rapid eye movement (REM) sleep compared with non-REM (NREM) sleep but is poorest in REM sleep in patients with sleep apnea. We therefore examined the hypothesis that sleep fragmentation impairs arousability, especially from REM sleep. DESIGN: Two blocks of 3 polysomnographies (separated by at least 1 week) were performed randomly. An inspiratory-loaded night followed either 2 undisturbed control nights (LN(C)) or 2 acoustically fragmented nights (LN(F)) SETTING: Sleep laboratory. PARTICIPANTS: Sixteen healthy men aged 20 to 29 years. INTERVENTIONS: In both loaded nights, an inspiratory resistive load was added via a valved facemask every 2 minutes during sleep and turned off either when arousal occurred or after 2 minutes. MEASUREMENTS AND RESULTS: During LN(F), arousability remained significantly greater in REM sleep (71% aroused within 2 minutes) compared with stage 2 (29%) or stage 3/4 (16%) sleep. After sleep fragmentation, arousability was decreased in stage 2 sleep (LN(F): 29%; LN(C): 38%; p < .05) and low in early REM sleep, increasing across the night (p < .01). In stage 3/4 sleep, neither an attenuation nor a change across the night was seen after sleep fragmentation. CONCLUSIONS: Mild sleep fragmentation is already sufficient to attenuate arousability in stage 2 sleep and to decrease arousability in early, compared with late, REM sleep. This means that sleep fragmentation affects the arousal response to increasing resistance and that the effects are different in stage 2 and REM sleep. The biologic reason for this increase in the arousal response in REM sleep across the night is not clear. PMID- 16676787 TI - Which memory processes are affected in patients with obstructive sleep apnea? An evaluation of 3 types of memory. AB - STUDY OBJECTIVE: To investigate which memory processes are affected by obstructive sleep apnea (OSA). DESIGN: Three separate memory systems were investigated in patients with OSA and normal subjects. Verbal episodic memory was tested after forced encoding, in order to control the level of attention during item presentation; procedural memory was tested using a simplified version of a standard test with an interfering task; lastly, working memory was examined with validated paradigms based on a theoretical model. SETTING: Sleep laboratory and outpatient sleep clinic in a French tertiary-care university hospital. PARTICIPANTS: Ninety-five patients with OSA and 95 control subjects matched for age and level of education. Group 1 (54 patients, 54 controls) underwent an extensive battery of tasks evaluating verbal episodic, procedural, and working memory. Group 2 (16 patients, 16 controls) underwent procedural memory tests only, and group 3 (25 patients, 25 controls) working memory tests only. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Compared with matched controls, patients with OSA exhibited a retrieval deficit of episodic memory but intact maintenance, recognition, and forgetfulness; decreased overall performance in procedural memory, although pattern learning did occur; and impairment of specific working memory capabilities despite normal short-term memory. No consistent correlation was found between OSA severity and memory deficit. The long duration of the test session did not negatively impact the patients' performance. CONCLUSIONS: Memory impairment in OSA is mild and does not affect all memory processes but, rather, specific aspects, underscoring the need for extensive and specific memory testing in clinical and research settings. PMID- 16676788 TI - Effects of short-term CPAP withdrawal on neurobehavioral performance in patients with obstructive sleep apnea. AB - STUDY OBJECTIVE: Changes in sleep parameters and neurobehavioral functioning were systematically investigated after an acute (1 night) and short-term (7 nights) period of withdrawal from continuous positive airway pressure (CPAP) treatment and 1 subsequent night of CPAP reintroduction in patients with obstructive sleep apnea. DESIGN: Repeated-measurement within-subject design. SETTING: Sleep laboratory, university teaching hospital. PARTICIPANTS: Twenty participants receiving optimal CPAP therapy for > or = 12 months. INTERVENTIONS: CPAP withdrawal. MEASUREMENTS AND RESULTS: Polysomnograms were performed on Night 0 (with CPAP), Night 1 and Night 7 (without CPAP) and Night 8_R (with CPAP). Acute CPAP withdrawal resulted in the recurrence of sleep-disordered breathing with sleep disruption, hypoxemia, and increased subjective sleepiness. Short-term CPAP withdrawal exacerbated hypoxemia, increased subjective and objective sleepiness and poor mood ratings. Neurobehavioral functioning assessed using the Psychomotor Vigilance Task was impaired following Night 7 and associated with hypoxemia and changes in morning levels of tumor necrosis factor-alpha. However, other neurobehavioral measures were not affected. Autonomic arousals measured via respiratory-related reductions in finger blood volume by peripheral arterial tonometry decreased from Night 1 to Night 7. On Night 8_R, reintroduction of CPAP treatment eliminated most airway obstruction, maintained oxygenation, and reversed daytime sleepiness and some vigilance decrements. CONCLUSION: Despite recurrence of sleep-disordered breathing with increased sleepiness and impaired vigilance, most neurobehavioral variables were unaffected by CPAP withdrawal. The reduction in vigilance appeared to be associated with worsened hypoxemia and changed levels of tumor necrosis factor-alpha. Resumption of CPAP treatment had immediate benefits on sleep consolidation and subjective sleepiness. PMID- 16676789 TI - Heart rate response to respiratory events with or without leg movements. AB - STUDY OBJECTIVE: This study compares the heart rate responses to the termination of respiratory events, both with and without associated leg movements. METHODS: Heart rate was measured for 15 R-R intervals before (T-15 to T-1) and after (T+1 to T+15) the termination of respiratory events as a change from the baseline rate, defined as the average of 10 R-R intervals occurring before the termination of each respiratory event (T-15 to T-6). Individual heart rate changes of the 21 patients were then averaged separately for 10 respiratory events with and 10 without associated leg movements. SETTING: N/A. PARTICIPANTS: Twenty-one patients with obstructive sleep apnea who had respiratory events both with and without associated leg movements. INTERVENTION: N/A. RESULTS: Maximal heart rate rise for respiratory events with leg movements (7.9 beats per minute) was significantly greater than for respiratory events without leg movements (5.1 beats per minute) (p < .0001). The area under the curve for heart rate increase from T-5 to T+9 was 50.1% higher for respiratory events with leg movements than without leg movements. When respiratory events with and without accompanying leg movements were compared, there were no significant differences in mean duration of respiratory events, mean oxygen desaturation after respiratory events, mean duration of electroencephalogram arousal following respiratory events, or mean heart rate during the baseline period. Heart rate rise did correlate with duration of the leg movements (p < .001) in those respiratory events with leg movements. CONCLUSIONS: Cardiac activation is significantly greater when the termination of respiratory events is associated with leg movements compared to those without leg movements. This exaggerated heart rate response may be an independent consequence of the leg movements themselves, as other features of the respiratory events and associated arousal were not different in the two conditions. PMID- 16676790 TI - Prevalence of persistent sleep apnea in patients treated with continuous positive airway pressure. AB - STUDY OBJECTIVE: There are limited data on the prevalence of persistent obstructive sleep apnea (OSA) in patients who are clinically asymptomatic with continuous positive airway pressure (CPAP). Our objectives were to estimate the prevalence of persistent OSA and to explore the parameters that may be capable of discriminating these patients. DESIGN: Prospective survey. SETTING: A tertiary care sleep-disorders clinic. PARTICIPANTS: Consecutive patients treated with single-pressure CPAP for at least 3 months were studied. All had undergone CPAP titrations and were compliant with treatment. They denied snoring or persistent excessive daytime somnolence. Of 114 who qualified, 101 were studied. INTERVENTIONS: Subjects underwent 16-channel polysomnography with electroencephalogram and pneumotachometer while using their CPAP. MEASUREMENTS AND RESULTS: Seventeen of 101 subjects (17%) had an apnea-hypopnea index of over 10. Fifty-one had only split-night protocols for CPAP titration. There was no significant difference between participants with persistent OSA and those with an apnea-hypopnea index < 5 with regard to age, sex, time since diagnosis, reported snoring, change in weight, or quality of life (all p > .10). Mean current CPAP level was higher, with a mean +/- SD 10.6 +/- 2.8 versus 8.6 +/- 2.3 cm H2O (p = .002). Unresolved air leak related to CPAP was more frequent in the patients with persistent OSA. Morning headaches, nonrestorative sleep, and frequent central apneas on the CPAP titration were all associated with persistent OSA. CONCLUSIONS: Persistent OSA is frequent in patients treated with CPAP. This is more frequent in patients with high body mass index, higher prescribed pressures, and unresolved mask leak. PMID- 16676791 TI - Effect of continuous positive airway pressure versus supplemental oxygen on sleep quality in obstructive sleep apnea: a placebo-CPAP-controlled study. AB - STUDY OBJECTIVE: We investigated the short-term effectiveness of continuous positive airway pressure (CPAP) and oxygen in improving sleep quality in patients with obstructive sleep apnea (OSA). DESIGN: Randomized, double-blind, placebo controlled, parallel study. SETTING: General Clinical Research Center at a university hospital. PATIENTS: Seventy-six patients with untreated OSA. INTERVENTIONS: Patients were randomly assigned to 1 of 3 treatments (CPAP, placebo-CPAP, or nocturnal oxygen at 3 L per minute) for 2 weeks. Sleep quality was assessed at baseline and after 1 and 14 days of therapy. Repeated-measures analysis of variance was used to evaluate treatment and time effects, and their interaction. MEASUREMENTS AND RESULTS: Sixty-three patients completed the protocol. When compared with placebo-CPAP and nocturnal oxygen, CPAP increased rapid eye movement (REM) sleep and significantly reduced stage 1 sleep and the number of stage shifts (p < or = .003). CPAP improved, to within normal limits, the apnea-hypopnea index, total arousal index, and mean oxyhemoglobin saturation (p < or = .001). The effects of CPAP were apparent during the first night of therapy. Oxygen improved only mean nocturnal saturation (p = .009). CPAP had no significant effect on stage 2 sleep or slow-wave sleep. CONCLUSIONS: CPAP was associated with an improvement in sleep quality in patients with OSA by consolidating sleep, reducing stage 1 sleep, and improving REM sleep. CPAP was effective in correcting the respiratory and arousal abnormalities of OSA. The effectiveness of supplemental oxygen was limited to oxyhemoglobin desaturation. PMID- 16676792 TI - Sleepiness enhances distraction during a monotonous task. AB - STUDY OBJECTIVES: Although sleepiness appears to heighten distraction from the task at hand, especially if the latter is dull and monotonous, this aspect of sleep loss has not been assessed in any systematic way. Distractions are a potential cause of performance lapses (as are micro-sleeps). Here, we investigate the effects of sleepiness on a monotonous task, with and without distraction. DESIGN: Repeated Measures 2 x 2 counterbalanced design, comprising Sleepiness (night sleep restricted to 5 hours x normal sleep) and Distraction (distraction x no distraction). SETTING: Participants underwent 30-minute sessions on the Psychomotor Vigilance Test (2:00 PM - 3:10 PM), with or without an attractive distraction to be ignored, under normal and sleep-restricted conditions. PARTICIPANTS: Sixteen healthy young adults (mean age 21.10 years; 21-25 years [8 men; 8 women]) without any sleep or medical problems and without any indication of daytime sleepiness. INTERVENTIONS: Normal sleep versus sleep restricted to 5 hours and distraction versus no distraction. Distraction comprised a television in the visual periphery, showing an attractive video that had to be ignored. MEASUREMENTS AND RESULTS: Psychomotor Vigilance Test performance was monitored, as were the participants' head turns toward the television via videocameras. There was a significant increase in both head turns and lapses during sleep restriction plus distraction. Moreover, sleepiness also increased head turns even during no distraction. Distracting effects of sleepiness were clearly evident during the initial 10 minutes of testing. CONCLUSIONS: Distractibility is an important aspect of sleepiness, which has relevance to safety in the real world, eg, sleepy driving. PMID- 16676793 TI - Research on ageing: priorities for the European region. PMID- 16676794 TI - [The realization of the Madrid International Plan of Action on Ageing: role of scientific research]. AB - The article focuses on the role gerontological science is supposed to play in the implementation of the Madrid International Plan of Action on Ageing. Its purpose, structure, the process of development and concordance, as well as problems arising in the course of its realization are discussed by the authors. The contribution of the Russian gerontological science in the realization of the Madrid Plan of Action is debated. PMID- 16676795 TI - [The modified variant of mitochondrial theory of aging]. AB - The central postulate of mitochondrial theory of aging (MTA) says that attenuation of cellular bioenergetics is the leading cause of aging. This attenuation is attributed to the accumulation of injuries into mitochondrial DNA that are exerted by reactive oxygen species (ROS). As the ROS are generated by mitochondrial respiratory chain then vicious cycle arises. As a result the ROS amount and level of injured biopolymers increase progressively and bioenergetics fades. The central postulate is fairly convincing but the mechanism of age dependent bioenergetics attenuation is disproved by many empirical data. The new variant of MTA is suggested in this paper. According to this both the aging and the increase in ROS level are caused by the programmed bioenergetics fade. The mechanism of age-dependent increase in ROS is as follows. Superoxide radical (O2(*-)) generating by respiratory chain is neutralized in two stages: first the enzyme superoxide dismutase transforms O2(*-) into hydrogen peroxide and then H202 is converted into water and oxygen by glutathione peroxidase (GP). The reaction catalyzing by GP is shunted by Fenton reaction that produces extremely aggressive secondary radicals. The programmed bioenergetics decline decreases GP activity, which causes elevation of H202 content and increases hydrogen peroxide flow through Fenton reaction. This leads to the increase in general ROS level and to strengthening their aggressivity. Thus, both the aging and the increase in ROS level are two consequences of programmed bioenergetics decline. PMID- 16676796 TI - [Apoptosis and carcinogenesis in aging: oxygen-peroxide aspect]. AB - New facts are given by the author for the general oxygen-peroxide concept of aging, cancerogenesis and apoptosis. The idea is confirmed that the cell breath dysfunction leads to the oxidative stress firstly in mitochondria, then in cytoplasm and in the cell in general is the starting moment for the induction of the named processes. The superfluous formation of the active oxygen forms, peroxides of lipids and proteins as signal molecules creates disbalance between pro- and antioxidants, which size increases in aging cells and even more in tumor's and apoptosis cells. In the limits of these specialized "disbalanses" the named signal molecules play key role not only on starting "mitochondrial" stages of aging process, carcinogenesis and apoptosis, but also on following "performing" stages. They influence on the work of performing links (telomerase, oncoproteins, transcription factors, proteinkinase, endonuclease, caspase and so on). PMID- 16676797 TI - Muscle wasting in cancer and ageing: cachexia versus sarcopenia. AB - Muscle wasting during cancer and ageing share many common metabolic pathways and mediators. Due to the size of the population involved, both cancer cachexia and ageing sarcopenia may represent targets for future promising clinical investigations. Cancer cachexia is a syndrome characterized by a marked weight loss, anorexia, asthenia and anemia. In fact, many patients who die with advanced cancer suffer from cachexia. The degree of cachexia is inversely correlated with the survival time of the patient and it always implies a poor prognosis. In recent years, age-related diseases and disabilities have become of major health interest and importance. This holds particularly for muscle wasting, also known as sarcopenia that decreases the quality of life of the geriatric population, increasing morbidity and decreasing life expectancy. The cachectic factors (associated with both depletion of fat stores and muscular tissue) can be divided into two categories: of tumour origin and humoural factors. In conclusion, more research should be devoted to the understanding of muscle wasting mediators, both in cancer and ageing, in particular the identification of common mediators may prove as a good therapeutic strategy for both prevention and treatment of wasting both in disease and during healthy ageing. PMID- 16676798 TI - [Genetics aspects of different light regime influence on drosophila life span]. AB - It was analyzed the difference of Drosophila life span in strains with low activity of Sod or excision repair enzyme Mus210 (homolog of yeast RAD4 and mammal XPC) in comparison with wild type strain Canton-S in conditions of 24 h and 0 h light. Mutants having low capacity to detoxify free radicals and repair DNA are characterized by stronger difference between life span at 24 h and 0 h light in comparison with wild type strain. Thus the life span increasing in the dark is due to decline of free radical and DNA lesions production. In wild type strain the effect of 12 h dusky light had not more effect on life span than intensive 24 h light. In most cases the difference between life span at 0 h and 24 h light was too much in males than in females, especially in Sod strain. PMID- 16676799 TI - [Functional characteristics of the cardiovascular system of old people- inhabitants of the north]. AB - The electrocardiographic investigation of healthy old people from three ethnic groups of natives of the Komi Republic at the age of 80+ years revealed increase of QRS-complex, enlargement P-Q and Q-T intervals duration, decrease of amplitude of T-wave. This testifies to age-associated lowering of the depolarization processes in myocardium, increase of atrio-ventricular conductivity and compensative extension of repolarization period. We determined the predominance of the left direction of ORS axis in old people. The increase of amplitude of mode of R-R intervals (AMo 50-90%), low range of variation of cardio intervals characterize heart rate stability in 60% of subject, indicate the limitation of sinus node lability and decrease of cortex component in regulation of cardiac rhythm. These processes in old people are adaptive ones to reduce redundant stress of heart capacity in extreme conditions. Normotensive people amounted more than 50% of investigated Northern Komi (SBP 125 +/- 13, DBP 72 +/- 8 mmHg). In groups of Russian women and Southern Komi the prevalence of hypertension revealed itself and it reached in average 170/97-174/87 mmHg. PMID- 16676800 TI - [Isolated systolic arterial hypertension in elderly and senile patients]. AB - Systolic arterial pressure rises uninterruptedly with age and it appears as a strong independent risk-factor of cardiovascular, cerebral and renal complications. A combined using Norvasc and Egilok gives stable hypotensive effect, provides regression of left ventricular hyper-trophy with improvement parameters of systolic and diastolic function, which is vitally important for researched contingent of patients. A combined using small therapy doses of drugs protects geriatric patients against side effects. PMID- 16676801 TI - [Some aspects of macular degeneration pathogenesis]. AB - Molecular dehydration is a polyetiologic hereditary determinatory disease. Variation in the lipid exchange balance resulting in microcirculation disturbance play an important role in the pathogenesis of molecular dehydration. Free radical damages in retina, synthesis activation of nitrogen oxide and cytokines output cause protein synthesis of apoptosis. Disturbances in apoptosis lead to molecular dehydration. Study of pathogenesis links of molecular dehydration gives the possibility to treat this disease. PMID- 16676803 TI - [Autonomic disbalance in patients of different age with stomach ulcer and ways of its correction]. AB - The study of peculiarity a tone of vegetative nervous system at patients of different age with ulcer defeats of a stomach, types of temperament, a level of neurotics, expressiveness introversion and extraversion. WE USED: Modified Eysenk Personality Questionnaire, EGG, standard vegetative test CITO (SVT CITO). OUR RESULTS: Rise of neurotic level in patients with stomach ulcer, indications of vegetative distones with characteristic apportionment of activity of VNS. Reflexotherapy, carry on counting variation of tone VNS expedite scaring of ulcer defeat. PMID- 16676802 TI - [Study on efficacy and toxicity of new combined regimen "taxoter + cisplatin + 5 fluorouracil" in disseminated and locally-spread stomach cancer. Comparative analysis of tolerance and efficacy in patients younger and older than 65 years]. AB - The aim of our study was to evaluate the efficacy and safety of 3-drugs regimen: T 75 mg/m2 d2 + P 75 mg/m2 d2 + F 500 mg/m2 x 3h d 1-3 every 28 days. 31 patients (pts) with morphologically proven advanced gastric cancer of the age 29-77 years (median 61.0) have been treated with this regimen. They received 138 cycles (1 10, median 4.0 cycles per pt). The response rate was evaluated in pts received > or =2 cycles: CR 1/27 (3.7%), PR 12/27 (44.4%), SD 7/27 (25.9%), PD 7/27 (25.9%). The median duration of CR+PR--4.5 mon (1.1-9.9), of SD--6.8 mon (3.0-10.7). Median TTP--5.5 mon. Overall survival: median--11.5 mon, 1-year--46.6%. PS improvement was observed in 54.8%pts, symptomatic improvement--in 71% pts. Toxicity per pt (per cycle) was moderate. There were 11 elderly among these pts. We didn't receive any significant differences in efficacy and severe toxicity in this group compared to non-elderly pts. We observed 55.6% PR, 33.3% SD, 11.1% PD, TTP--4.6 mon, median OS-7.5 mon. in elderly and 5.6% CR, 38.9% PR, 22.2% SD, 33.3 % PD, TTP--6.1 mon, median OS-12.3 mon for non-elderly pts. But dose reduction was performed more frequently in the elderly then non-elderly: 63.6% vs 30.0% pts (p = 0.07) in 64.8% vs 19.1% cycles (p < 0.0001). We consider this regimen to be effective and well tolerated both for elderly and for non-elderly patients. PMID- 16676804 TI - [Diabetes mellitus in elderly: comorbid characteristics of patients with different ontogenetic forms of the disease]. AB - Diabetes in elderly is the interdisciplinary problem of diabetology and gerontology. Unlike adults the specific feature of these patients is comorbidities. On the other hand well known is the influence both age and aging on clinical sings of diabetes. The aim of the study was to investigate prevalence and structure comorbid chronic diseases in elderly patients with different ontogenetic forms of diabetes mellitus type 2 (DM2). We examined 169 elderly women with clinical diagnosis "DM2" (mean age--69.8 yrs., mean BMI--29.5 kg/m2, mean HbA1c--7.03%). The stratification was made by ontogenetic stage of diabetes onset and there were five ontogenetic forms of DM2: menstrual (Ms), early postmenopausal (EPM), late-postmenopausal (LPM), early-involutional (EI) and late involutional (LI). Anthropometrical, biochemical and immunochemical assays (HbA1c) were made by standard methods. Gognitive index (CGI) and affective index (AFI) were calculated by SCAG scale as mentalmnestic and affective disturbances accordingly. Comorbid index (CI) was calculated as a sum of concomitant diseases. The most comorbid serious was the early-postmenopausal group (CI--6.04 +/- 0.5), mainly by hypertension (92%) coronary heart disease (80%) and osteoarthritis (80%). The lightest comorbid status was in the late-involutional group (CI--4.5 +/- 0.3), with the minimum of gastroenterological diseases (39.5%), kidney diseases (26.3%), thyroid disorders (23.7%) and exclusively the group had valid negative relationship between age and CI (r = -0.550, p = 0.000). As a whole in the elderly diabetic cohort the magnitude of CI correlated positively with BMI (r = +0.344, p = 0.000), frequency of family diabetes (r = +0.204, p = 0.009), AFI (r = +0.161, p = 0.040), menarche (r = +0.175, p = 0.025) and no significantly with CGI (p > 0.05). Thus early ontogenetic forms of DM2 had more comorbidities, especially those with onset DM2 during first 5 years after menopause. And on the contrary, the latest ontogenetic forms with onset DM2 during 20 years after menopause had minimum concomitant diseases. Also early ontogenetic phase (EPM and El) groups had higher magnitudes of CI, frequency of family diabetes and low frequency of familial longevity than late ontogenetic phase (LPM and LI) groups, which allow us to consider early ontogenetic phase DM2 as a phenomena of accelerated aging. PMID- 16676805 TI - [The effect of the bioregulating therapy on the quality of life of elderly patients with retinal pathology]. AB - Involutional eye diseases associated with a decrease of vision functions lead to the deterioration in quality of life of elderly people. Retinalamin application in the course of treatment for these diseases contributes to the increase in vision functions, and thus to the increase in quality of life. PMID- 16676806 TI - [Study on the quality of life of elderly patients with recurrent depressive disorders]. AB - This article presents results of special science research on problem of quality of life of patients suffering from recurrent depressive disorders in advanced age. The authors emphasize the influence of some clinical characteristics of depression and type of personalities upon the rates of quality of life. PMID- 16676807 TI - [Features of varicose veins of the pelvic region of women in premenopause and postmenopause]. AB - The purpose of the research was definition of clinical current and diagnostic criteria of varicose veins of pelvis minor at woman of pre-menopause and post menopause. The varicose veins of pelvis minor at woman of pre-menopause and post menopause is characterized by decrease in a painful syndrome with heavy organic and functional defeat of veins system of a pelvis minor. The diagnostic criteria offered by the author will allow improving diagnostics of disease at women. PMID- 16676809 TI - Case managers can take the lead in pay-for-performance initiatives. PMID- 16676808 TI - [The indices of biological age and early aging in the liquidators of the consequences of radiation accidents]. AB - The researches of biological age and rates of aging of liquidators of the sequels to radiation accidents were carried out. The biological age (BA) index can serve as a characteristic of social-hygienic factors influence on state of health of the officers and veterans of special risk subdivisions, liquidators of the sequels to radiation accidents. Chronic diseases exert influence on the biological age and proper biological age (PBA) index, increase mean value of BA PBA index, bring contribution into acceleration of human organism aging. A direct connection between increase of functional class of the BA and duration of service in the Navy and ships was revealed. It was ascertained that radiation influence was a factor that increased the BA and rates of aging of liquidators of the sequels to radiation accidents. PMID- 16676810 TI - LTACHs offer opportunities for patient contact. PMID- 16676811 TI - Following CF patients throughout continuum. PMID- 16676812 TI - Dashboard helps hospitals focus on core measures. PMID- 16676814 TI - Use technology, creativity to tackle ED overcrowding. PMID- 16676813 TI - Balanced scorecard helps CMs focus on improvement. PMID- 16676815 TI - Assess patient flow; use data to improve. PMID- 16676816 TI - International law, telemedicine & health insurance: China as a case study. PMID- 16676817 TI - Who will protect the "disruptive" dialysis patient? PMID- 16676818 TI - Swing low, sweet chariot: abandoning the disinterested witness requirement for advance directives. PMID- 16676819 TI - The perils of Singleton v. Norris: ethics and beyond. PMID- 16676820 TI - Expandable tracheal stenting for benign disease: worth the complications? AB - OBJECTIVES: To characterize the limitations of self-expandable stents in the management of benign tracheal stenosis, we performed a retrospective review at a tertiary care medical center. METHODS: Patients who underwent tracheal stenting were assessed for the cause and severity of tracheal stenosis, comorbidities, stent-related complications, and follow-up airway procedures. RESULTS: Sixteen adults (12 women, 4 men; mean age, 47 years) had a total of 26 stents placed for benign disease. Intubation-related stenoses were most frequent (81%). The average follow-up time was 20 months (range, 1 to 40 months). Each stent remained functional for an average of 12.4 months. In the study group, 87% had a complication that required surgical intervention to maintain a patent airway. The most common problem was granulation tissue formation at the ends of the stent causing airway restenosis (81%), and 5 patients (31%) required tracheotomy as a result of restenosis around the stent. Fourteen of the stents (56%) were removed or expelled from the patients. CONCLUSIONS: The implantation of self-expandable stents is a minimally invasive method of managing benign tracheal stenosis. Although a small subset of patients may benefit from placement, the majority of patients have complications that require intervention to maintain a patent airway. Thoughtful discretion is critical in selecting patients for this intervention. PMID- 16676821 TI - Photocoagulation of microvascular and hemorrhagic lesions of the vocal fold with the KTP laser. AB - OBJECTIVES: Ectasias and varices of the vocal fold are microvascular lesions that are often due to chronic abuse of the voice, and are occasionally encountered in association with other disorders such as polyps, Reinke's edema, and hematoma. The KTP laser can be used for photocoagulation of small vascular lesions, because the laser beam is well absorbed by hemoglobin, and damage to the epithelium is minimal. The present pilot study examined how the KTP laser could be used for microvascular lesions and their associated lesions. METHODS: Twelve patients who had undergone phonomicrosurgery were enrolled in the present study. The microvascular lesions were treated by photocoagulation with the laser set at a low power of 1.5 W in the continuous mode, while preserving the epithelium, and associated lesions were then treated by microdissection with cold instruments. The postoperative phonatory function was assessed by maximum phonation time, a perceptual test rating (GRBAS scale), and stroboscopy. RESULTS: The procedures were completed successfully in all cases. An exceptional case of a small hemorrhagic polyp allowed treatment with the laser only. The postoperative stroboscopic findings, maximum phonation time, and perceptual test rating all showed significant improvement compared with the preoperative state. No adverse effects, such as scarring or reduction of the mucosal wave, were observed in the current series. CONCLUSIONS: KTP laser photocoagulation is a relatively simple and safe procedure for treating microvascular lesions of the vocal fold. It is not recommended for photocoagulation of hemorrhagic polyps or hematomas, because such lesions have little blood flow inside and thus photocoagulation is usually impossible or requires too much laser energy. However, photocoagulation of perimeter or feeding vessels of such disorders may facilitate the following procedure by avoiding unnecessary bleeding, as well as preventing recurrence of hemorrhagic lesions. PMID- 16676822 TI - Predictors of laryngeal complications in patients implanted with the Cyberonics vagal nerve stimulator. AB - OBJECTIVES: Since its approval by the US Food and Drug Administration in 1997 for management of medically refractory seizures, more than 35,000 patients have been implanted with the Cyberonics vagal nerve stimulator. Preliminary reports described transient vocal changes in the majority of subjects, which were thought to be short-term. However, these reports were for the most part based upon perceptual evaluations by the subjects themselves. Later reports described possibly more permanent recurrent laryngeal nerve injury and recommended measuring the nerve diameter to use the safest spiral cuff electrode. To date, no study has systematically evaluated vocal fold mobility in subjects before and after implantation. The objectives of this study were to determine the true incidence of both short- and long-term recurrent laryngeal nerve injuries and determine whether there are any potential indicators to predict in which patients long-term nerve deficits may develop. METHODS: Thirteen subjects underwent preimplantation laryngeal electromyography, videolaryngoscopy, measurement of the maximum phonation time, Voice Handicap Index determination, and Consensus Auditory-Perceptual Evaluation of Voice. Two weeks after implantation, all subjects underwent videolaryngoscopy. Three months after implantation and activation of the device, all subjects were reevaluated. RESULTS: Six of the 13 subjects had significant vocal fold mobility abnormalities at 2 weeks. Significant electromyographic abnormalities were detected before implantation in 5 subjects. All 5 of these subjects, at 3 months after implantation, had prolonged left vocal fold paresis. CONCLUSIONS: The authors conclude that perioperative vocal fold paresis occurs in approximately 50% of subjects. Further, laryngeal electromyography performed before implantation of the vagal nerve stimulator is a statistically significant predictor (p < .05) of which patients may be at risk for extended vocal fold abnormalities. Possible explanations for this phenomenon are offered. Surgical modifications to limit vagal nerve injury are offered. PMID- 16676823 TI - Multiple auditory steady-state responses in children and adults with normal hearing, sensorineural hearing loss, or auditory neuropathy. AB - OBJECTIVES: We tested the clinical effectiveness of multiple auditory steady state responses (ASSRs) for the objective assessment of hearing thresholds in patients with and without hearing loss, candidates for cochlear implants, and children with auditory neuropathy. METHODS: The study sample included 29 subjects with sensorineural hearing loss (SNHL), 18 candidates for cochlear implants, 11 subjects with auditory neuropathy, and 18 subjects with normal hearing thresholds. Behavioral hearing thresholds and ASSRs to carrier frequencies of 0.5, 1, 2, and 4 kHz were obtained. Special care was taken to minimize possible aliasing and high-intensity multiple stimulation effects. Differences and correlations between the ASSRs and the behavioral thresholds were determined. RESULTS: The ASSR estimation of behavioral thresholds in the normal-hearing group was elevated, whereas very close predictions were found for the SNHL group. The correlations between the two measures ranged from 0.86 at 0.5 kHz carrier frequency to 0.94 at 2 kHz. In the cochlear implant candidates and the auditory neuropathy group, the ASSR thresholds generally overestimated the behavioral audiogram. In these groups the number of detected ASSRs was higher than the number of behavioral responses, especially for the high-frequency carrier stimuli. CONCLUSIONS: Multiple ASSRs may reliably predict the behavioral threshold in subjects with SNHL and may serve as a valuable objective measure for assessing the hearing threshold across different frequencies in candidates for cochlear implants and children with auditory neuropathy. PMID- 16676824 TI - Imaging the human vocal folds in vivo with optical coherence tomography: a preliminary experience. AB - OBJECTIVES: Optical coherence tomography (OCT) and polarization-sensitive OCT (PS OCT) are promising noninvasive methods for in vivo, cross-sectional imaging of the microstructure of the vocal folds. Previous studies in other tissues have shown an axial resolution of less than 10 microm and a maximum imaging depth of about 2 mm. The objectives of this pilot study were to obtain images from the vocal folds of subjects who were being evaluated and/or treated for vocal fold disease and to evaluate how well normal and pathologic microstructure could be seen in these images. METHODS: Twenty-six vocal folds in 13 subjects were imaged with a flexible OCT probe. The images were successfully collected from subjects who were either topically anesthetized or under general anesthesia for microlaryngoscopic procedures. RESULTS: The thickness of the epithelium, the relative collagen content of the subepithelial connective tissue, and certain characteristic features of lesions (including cysts, scarring, and papilloma) were seen in the OCT and PS-OCT images. CONCLUSIONS: "Live microscopy" of the human vocal folds is very promising for improved diagnosis, mapping, and treatment planning. To our knowledge, this study is the first application of PS OCT for in vivo imaging of the human vocal folds. PMID- 16676825 TI - Histological study of acute vocal fold injury in a rat model. AB - OBJECTIVES: We used an acute vocal fold injury in a rat model to characterize vocal fold wound healing by studying the expression pattern of the extracellular matrix components in the vocal fold lamina propria. METHODS: Vocal fold stripping was performed unilaterally in 27 Sprague-Dawley rats. The vocal folds were harvested at 5 time points (1, 3, 5, 7, and 14 days) and histologically analyzed by Alcian blue stain, trichrome stain, and immunofluorescence with antibodies to collagen type I, collagen type III, and fibronectin. RESULTS: Re epithelialization occurred by day 3 and was complete by day 14. Granulation tissue was formed by day 3. Hyaluronic acid and collagen type I appeared in injured vocal folds by day 3, peaked at day 5, and thereafter decreased. Collagen type III and fibronectin appeared by day 1 and continued to be intense at all time points after day 3. CONCLUSIONS: These results suggest that the expression of these extracellular matrix components peaks in the period around days 3 to 5, and that the characteristics of wound healing in the vocal fold are similar to those in the skin in the early phases, but differ during the subsequent remodeling phase. PMID- 16676826 TI - Safety and feasibility of balloon catheter dilation of paranasal sinus ostia: a preliminary investigation. AB - OBJECTIVES: Endoscopic sinus surgery (ESS) is an effective option for managing patients in whom medical therapy for rhinosinusitis fails. However, ESS is not always successful, and serious complications can occur. New techniques and instrumentation that improve outcomes and reduce complications would be seriously welcomed. Innovative catheter-based technology has improved treatment of several conditions such as coronary artery disease, peripheral vascular disease, and stroke. Recently, catheter devices have been developed for the paranasal sinuses. Cadaver studies confirm the potential use of these devices in rhinosinusitis. The objective of this investigation was to ascertain the feasibility and safety of these newly developed devices in performing catheter-based dilation of sinus ostia and recesses in patients with rhinosinusitis. METHODS: A nonrandomized prospective cohort of 10 ESS candidates was offered treatment with a new technique of balloon catheter dilation of targeted sinus ostia. The frontal, maxillary, and sphenoid sinuses were considered appropriate for this innovative catheter-based technology. The primary study end points were intraoperative procedural success and absence of adverse events. RESULTS: A total of 18 sinus ostial regions were successfully catheterized and dilated, including 10 maxillary, 5 sphenoid, and 3 frontal recesses. No adverse events occurred. Mucosal trauma and bleeding appeared to be less with catheter dilation than is typically observed with ESS techniques. CONCLUSIONS: Dilation of sinus ostial regions via balloon catheter-based technology appears to be relatively safe and feasible. Larger multicenter clinical trials are now warranted to further establish safety and to determine the role of this new technique. PMID- 16676827 TI - Allograft tracheoplasty technique for management of refractory tracheal stenosis. AB - OBJECTIVES: Extensive tracheal airway defects represent a clinical dilemma. Although resection and reanastomosis and staged tracheoplasty may prove beneficial in some cases, recurrent or extensive circumferential stenosis remains a reconstructive challenge. We report the use of the allograft tracheoplasty technique for the reconstruction of recurrent, extensive defects of the trachea and cricoid. METHODS: Nine consecutive patients with recurrent tracheal stenosis were treated with the two-stage allograft tracheoplasty technique. A retrospective review was performed to evaluate for prior surgery, length of stenosis, surgical technique, and outcome. All 9 patients underwent multiple surgical procedures for acquired tracheal stenosis (average, 3.4 procedures) before undergoing the allograft tracheoplasty technique. Before surgery, all patients were tracheotomy-dependent. RESULTS: The patients were assessed 8 to 39 months after allograft tracheoplasty. The primary airway disorders included postintubation stenosis (n = 6), surgical resection for malignancy (n = 1), and idiopathic stenosis (n = 2). Three defects involved 30% to 60% of the cricoid cartilage, and 4 defects were complete circumferential tracheal defects. Five patients underwent an island deltopectoral flap for closure of the tracheoplasty site. One patient had a superficial wound infection at the cartilage recipient site, and 1 patient had a hematoma at the deltopectoral flap donor site. All 9 patients were successfully decannulated without shortness of breath, stridor, or recurrent stenosis at the time of follow-up. CONCLUSIONS: Allograft tracheoplasty is a new technique for the reconstruction of recurrent tracheal stenosis. It appears to be reliable for extensive airway defects that are refractory to conventional tracheoplasty techniques. PMID- 16676828 TI - Detection of herpes simplex and varicella-zoster viruses in patients with Bell's palsy by the polymerase chain reaction technique. AB - OBJECTIVES: Infectious causes of peripheral facial paralysis are well known. Bell's palsy, however, is an idiopathic facial paralysis, and the genesis is still unknown. Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) have been suggested as etiologic agents. METHODS: Twenty consecutive adult patients with Bell's palsy were included in the study. Ten adult patients operated on for chronic otitis served as controls. A biopsy specimen from the posterior auricular muscle was resected within 72 hours after the onset of Bell's palsy and was analyzed together with cerebrospinal fluid (CSF) by nested polymerase chain reaction for HSV-1 and VZV DNA. Serum samples were analyzed for antibodies to HSV-1 and VZV. RESULTS: HSV-1 DNA was found in the muscle biopsy specimen from 1 of the 20 patients, but was not found in any of the CSF samples. VZV DNA was detected in the muscle biopsy as well as the CSF from 1 other patient. All controls were negative. Seventeen of 19 patients had stationary serum antibody concentrations to HSV-1, and none displayed an antibody titer rise. A significant antibody titer rise to VZV was found in 1 of 19 patients, whereas 17 of 19 had stationary antibody levels. CONCLUSIONS: HSV-1 or VZV DNA was detected in 10% of patients with Bell's palsy in the present study. Viral replication might already have declined in many cases at the onset of the palsy. Use of an HSV-1/VZV polymerase chain reaction on a muscle biopsy specimen or CSF does not seem to be the method of choice for rapid etiologic diagnosis in the acute phase of Bell's palsy. PMID- 16676829 TI - Electromyography findings of the cricopharyngeus in association with ipsilateral pharyngeal and laryngeal muscles. AB - OBJECTIVES: We reviewed a large series of cricopharyngeal (CP) muscle electromyography (EMG) results and compared them with the EMG results from the inferior constrictor (IC), thyroarytenoid, (TA), cricothyroid (CT), and posterior cricoarytenoid (PCA) muscles. METHODS: We performed a retrospective review of all CP muscle EMG reports from studies performed between January 1996 and June 2003. All of the tested elements from the CP muscle EMG reports were recorded. The EMG results were recorded for the ipsilateral IC, TA, CT, and PCA muscles if they were simultaneously tested. Each muscle result was classified as normal, neurogenic inactive axonal injury (IAI), or neurogenic active axonal injury (AAI), and the muscle findings were compared. A patient chart review was performed to determine a clinical correlation. RESULTS: Fifty-nine patients underwent CP muscle EMG. Eighteen patients had bilateral EMG studies, making a total of 77 CP muscle studies. Nineteen sets of CP muscle results were normal, 43 demonstrated neurogenic IAI, and 15 demonstrated neurogenic AAI. The ipsilateral IC and CP muscles had the same innervation status in 27 of 28 studies (p < .0001). When the ipsilateral TA muscle was studied simultaneously with the CP muscle, 31 of 50 studies had the same innervation status (p = .005). The ipsilateral CT and CP muscles demonstrated the same innervation status in 40 of 50 studies (p < .0001). The correlations between the CP and IC muscle findings and between the CP and CT muscle findings were both stronger than the correlation between the CP and TA muscle findings (p < .0001 and p = .024, respectively). The chart review demonstrated the clinical findings to be consistent with the EMG results. CONCLUSIONS: The EMG studies demonstrated that CP muscle findings have the strongest correlation with IC muscle findings, followed by the CT and TA muscles. This outcome does not support theories indicating that the recurrent laryngeal nerve innervates the CP muscle in all cases. PMID- 16676831 TI - Canada on the Move: a novel effort to increase physical activity among Canadians. PMID- 16676830 TI - Results after revision stapedectomy with malleus grip prosthesis. AB - Revision stapedectomy with a malleus grip prosthesis is a technically challenging otologic procedure. The prosthesis is usually longer and extends deeper into the vestibule than a conventional stapes prosthesis, creating the potential to affect the vestibular sense organs. The prosthesis also bypasses the ossicular joints, which are thought to play a role in protecting the inner ear from large changes in static pressure within the middle ear. The prosthesis is in close proximity to the tympanic membrane, thus increasing the risk for its extrusion. We reviewed our experience with revision stapedectomy with the Schuknecht Teflon-wire malleus grip prosthesis in 36 ears with a mean follow-up of 23 months. The air-bone gap was closed to within 10 dB in 16 ears (44%) and to within 20 dB in 26 ears (72%). The incidence of postoperative sensorineural hearing loss was 8% (3 ears). There were no dead ears. Extrusion of the prosthesis occurred in 1 case (3%). Nearly 50% of patients reported various degrees of vertigo or disequilibrium during the first 3 weeks after surgery. These vestibular symptoms resolved by 6 weeks in all but 1 case. We did not find evidence of damage to the inner ear due to the length of the prosthesis or due to the potential for direct transmission of changes in static pressures within the middle ear to the labyrinth. Our results are similar to those published in the literature for malleus attachment stapedectomy and conventional revision incus stapedectomy. PMID- 16676832 TI - Innovations in population intervention research capacity: the contributions of Canada on the Move. AB - The Canada on the Move project developed within a dynamic context and in response to an expressed need for increased capacity to support research involving population-level interventions. This article describes a) the movement to create an organized approach to chronic disease prevention in Canada, b) the emerging science of population-level intervention, c) the development of Canadian infrastructure to support population intervention science, and d) the contribution of Canada on the Move in developing a health research platform and, opportunistically, instigating a study which included assessment of the population impact of a commercial marketing initiative. PMID- 16676834 TI - Canada on the Move: an intensive media analysis from inception to reception. AB - BACKGROUND: Research evaluating mediated physical activity campaigns uses an unsophisticated conceptualization of the media and would benefit from the application of a media studies approach. The purpose of this article is to report on the application of this type of analysis to the Canada on the Move media campaign. METHODS: Through interviews and document analysis, the press release surrounding Canada on the Move was examined at four levels: inception, production, transmission and reception. Analytic strategies of thematic and textual analysis were conducted. RESULTS AND CONCLUSION: The press release was well received by journalists and editors and was successfully transmitted as inferred from national and local television coverage, although there was no national print pickup. Canada on the Move was perceived by sampled audience members as a useful and interesting strategy to encourage walking. A holistic approach to media analysis reveals the complex and frequently messy process of this mediated communication process. Implications for future media disseminations of Canada on the Move are discussed. PMID- 16676833 TI - Evaluation of quality of commercial pedometers. AB - BACKGROUND: The purpose of this study was to: 1) evaluate the quality of promotional pedometers widely distributed through cereal boxes at the time of the 2004 Canada on the Move campaign; and 2) establish a battery of testing protocols to provide direction for future consensus on industry standards for pedometer quality. METHODS: Fifteen Kellogg's* Special K* Step Counters (K pedometers or K; manufactured for Kellogg Canada by Sasco, Inc.) and 9 Yamax pedometers (Yamax; Yamax Corporation, Tokyo, Japan) were tested with 9 participants accordingly: 1) 20 Step Test; 2) treadmill at 80m x min(-1) (3 miles x hr(-1)) and motor vehicle controlled conditions; and 3) 24-hour free-living conditions against an accelerometer criterion. RESULTS: Fifty-three percent of the K pedometers passed the 20 Step Test compared to 100% of the Yamax. Mean absolute percent error for the K during treadmill walking was 24.2+/-33.9 vs. 3.9+/-6.6% for the Yamax. The K detected 5.7-fold more non-steps compared to the Yamax during the motor vehicle condition. In the free-living condition, mean absolute percent error relative to the ActiGraph was 44.9+/-34.5% for the K vs. 19.5+/-21.2% for the Yamax. CONCLUSIONS: K pedometers are unacceptably inaccurate. We suggest that research grade pedometers: 1) be manufactured to a sensitivity threshold of 0.35 Gs; 2) detect +/-1 step error on the 20 Step Test (i.e., within 5%); 3) detect +/-1% error most of the time during treadmill walking at 80m x min(-1) (3 miles x hr( 1)); as well as, 4) detect steps/day within 10% of the ActiGraph at least 60% of the time, or be within 10% of the Yamax under free-living conditions. PMID- 16676835 TI - Proximal impact of Canada on the Move: the relationship of campaign awareness to pedometer ownership and use. AB - OBJECTIVE: Despite increased participation in leisure-time physical activity, inactivity remains an important public health problem. This study examines the immediate impact of the Canada on the Move initiative to promote walking through pedometer use among adult Canadians. METHODS: Data from a rolling monthly sample were collected via the Canadian Fitness and Lifestyle Research Institute's Physical Activity Monitor between November 2003 and September 2004. Prevalence rates were compared using Bonferroni-adjusted confidence intervals. Correlates of campaign recall and pedometer ownership were estimated using odds ratios adjusted for age, sex, income and education. RESULTS: Message recall and awareness as well as ownership and use of pedometers increased over the campaign and coincided with promotional activity. There was some evidence of a dose response between the number of messages recalled and pedometer use. DISCUSSION: The increased awareness and usage of pedometers among adult Canadians is encouraging; it indicates that these proximal targets are realistic and achievable for health promotion campaigns and contribute to opportunities for increasing physical activity behaviours. PMID- 16676836 TI - Characteristics of participants visiting the Canada on the move website. AB - BACKGROUND: The Internet is a potential medium to attain large groups of Canadians for physical activity monitoring and interventions, however, little is known about the characteristics of participants who could be recruited on a national level. Our objectives were to determine: 1) the extent to which our sample was representative of the Canadian population; 2) the demographic, social cognitive and physical activity behaviour, and environmental characteristics of one-time and multi-time users of the Canada on the Move website; and 3) whether certain recruitment strategies were more likely to reach specific demographic subgroups. METHODS: Web-based self-report measures were collected on demographic, social-cognitive, physical activity behaviour, and environmental factors. Descriptive statistics were employed to address the study's research objectives. RESULTS: Significant differences (p<0.001) were found between our total sample and 2001 Canadian census/Canadian Community Health Survey (CCHS) data on all demographic and physical activity behaviour measures. One-time and multi-time users significantly differed on age (p<0.005) and being responsible for children under age 13 (p=0.01). No differences existed between the groups on any of the social-cognitive, behavioural or environmental variables. Source of knowledge about the Canada on the Move website between one-time and multi-time users was not significantly different. CONCLUSION: The Canada on the Move website presents a viable approach to reaching diverse demographic groups. Further work needs to be undertaken to: 1) develop engaging websites; 2) detail the monitoring of the web-based access information; 3) integrate the website with other organizations promoting physical activity; and 4) expand the number of recruitment sources. PMID- 16676837 TI - Perceived neighbourhood correlates of walking among participants visiting the Canada on the Move website. AB - BACKGROUND: The purposes of this study were to: 1) explore the potential role of sex in the association between the perceived environment and walking; and 2) determine the efficacy of an Internet-based research platform for collecting population-level physical activity and correlates data. METHODS: Visitors to the Canada on the Move website were asked questions about their demographics, physical activity participation and perceptions of their neighbourhood environment. A total of 3,144 Canadians (2,036 women; 609 men) completed the survey. Level of walking was regressed on eight measures of perceived neighbourhood environment in a series of logistic regressions. RESULTS: Individuals who reported interesting scenery (OR=1.41, 95% CI 1.17-1.71) and many places to go that were within easy walking distance (OR=1.39, 95% CI 1.18-1.64) were more likely to report walking at a level sufficient to derive health benefits. For women, interesting scenery in and around their neighbourhood (OR=1.40, 95% CI 1.13-1.74) and the presence of many places to go within easy walking distance (OR=1.42, 95% CI 1.17-1.72) were associated with walking at a sufficient level. Among men, no significant associations were found between the perceived environment measures and walking. CONCLUSIONS: The results from this study provide additional support for the use of models in which sex is treated as a potential moderator of the link between the perceived environment and physical activity. Further, the results support the use of an Internet-based research platform to collect data on the correlates of physical activity. PMID- 16676838 TI - Postscript: Learning from the experience of developing and running Canada on the Move. PMID- 16676839 TI - Toxic solvents in car paints increase the risk of hearing loss associated with occupational exposure to moderate noise intensity. AB - Solvents in car paints are a recognised source of occupational toxicity. In particular, they can cause DNA damage and occupational rhinobronchitis. However, little is known about their toxic effect in noise-induced hearing loss (NIHL) in humans. In this study, a 160 pure tone audiometric test was performed in workers in two independent factories to investigate whether toxic solvents in car paints can result in noise-induced hearing loss in workers exposed to moderate noise levels of less than 85 decibels (dB). It is shown that toxic solvents in car paints increase the risk associated with moderate noise exposure of less than 85 dB, with levels of NIHL being similar to those in workers exposed only to loud noises between 92.5 dB and 107 dB. Tinnitus and spells of dizziness were associated symptoms in all workers with NIHL, and asthma was an associated disease in workers with NIHL exposed to car paints and moderate noises simultaneously. These results may indicate that toxic solvents in car paints act in synergism with moderate noise exposure, damaging the cochlear hair cells. The results also constitute firm grounds for monitoring the hearing of these workers and adherence to strict regulations about wearing special gowns and filtered masks during working hours to protect against this preventable occupational disease. PMID- 16676841 TI - Decannulation in children after long-term tracheostomy. AB - Various approaches and techniques are used in discontinuing tracheostomy in children. The variability in the use of resources is considerable. The objective of the study was to assess decannulation in children attending our ENT department. A retrospective analysis was carried out of the medical records of patients who had had both a tracheostomy (n=7) and a decannulation (n=6) from 1998 to 2003. Tracheostomies placed at a mean age of 4.2 months were discontinued on average 26.4 months later. All the children underwent airway endoscopy in the operating room in preparation for decannulation. They had downsizing, then capping of the tracheostomy as a functional trial. One child failed decannulation and another needed a second trial for successful decannulation. The individualization of tracheostomy decannulation is necessary in children. PMID- 16676840 TI - Wegener's granulomatosis presenting as otomastoiditis. A case report. AB - A 55-year-old male presented with left-sided otorrhoea, hearing loss and tinnitus of 3 months duration. On clinical examination polypoid tissue was seen prolapsing in the external ear canal. A CT scan of the mastoid cells and middle ear showed otomastoiditis with osteolysis. Oral antibiotic therapy and eardrops were started. When a facial nerve paresis appeared one month later, a mastoidectomy was performed. The mastoid cells and middle ear were filled with a connective tissue-like substance. Postoperative corticosteroids were administered. Despite the therapy the facial nerve problem aggravated and the patient developed severe parietotemporal headache, meningeal irritation and somnolence. The diagnosis of neurosarcoidosis was hypothesised. Blood analysis, including c-ANCA's, culture of the otorrhoea and biopsies of the connective tissue were inconclusive. A CT scan of the brain showed thickening of the left tentorium. A biopsy of the dura indicated a diagnosis of Wegener's granulomatosis. The patient was treated with immunosuppressive medication with satisfactory results. PMID- 16676842 TI - Management of an insular thyroid carcinoma invading the larynx and trachea. AB - Management of an insular thyroid carcinoma invading the larynx and trachea. Carcinomas of the thyroid gland are usually classified into four types: papillary, follicular, medullary and anaplastic and into two main groups depending on their clinical behaviour: low and high grade malignancy. Insular carcinomas represent a rare subtype of follicular thyroid carcinoma, which are poorly differentiated. It is an aggressive and often lethal tumour. Thyroid cancers rarely invade the upper airways. We report the case of an insular carcinoma invading the larynx and trachea, which was treated by total laryngectomy, bilateral neck dissection and external beam irradiation. The patient presented no signs of tumour recurrence two years after treatment. Conservative treatment is usually recommended for thyroid carcinomas. Total laryngectomy is reserved for some extensive tumours invading the upper airways. A multidisciplinary approach is necessary to manage these cancers correctly. PMID- 16676843 TI - Chondrosarcoma of the larynx: a report of two cases and a review of the literature. AB - Chondrosarcoma of the larynx: a report of two cases and a review of the literature. This paper describes two cases of low-grade laryngeal chondrosarcoma. In both cases, the tumours were located on the cricoid, and could be visualized with a CT scan and magnetic resonance imaging. The diagnosis was made by a deep wedge biopsy with a CO2 laser, and after subtotal supracricoid laryngectomy. Most of the reported cases have been successfully managed by voice-sparing surgery, but the two cases reported here, needed more radical treatment. PMID- 16676844 TI - Chondrosarcoma of the nasal septum: a report of two cases. AB - Chondrosarcomas of the nasal septum are extremely rare tumours. The clinical presentation, imaging features, histopathological characteristics, and therapeutical options for this uncommon lesion are discussed. In this report, two cases are presented, and the literature regarding this rare pathology is reviewed. PMID- 16676845 TI - Apertura pyriformis stenosis in the newborn. AB - Apertura pyriformis stenosis in the newborn. Respiratory distress in the newborn can have a variety of aetiologies, the best known of which are cardiac and pulmonary diseases. Major nasal airway obstruction is probably often overlooked when acute desaturation of the neonate requires reanimation procedures, although it is well established that the baby is an obligate nose breather at birth. Nasal airway stenosis or atresia could account for a number of unexplained deaths in the delivery room. In the differential diagnosis of major nasal airway obstruction in the newborn, choanal atresia is by far the most common aetiology. However, a few cases of pyriform aperture stenosis have been reported. One child presenting this pathology was recently treated at the Brussels University Children's Hospital. Unexpectedly, the baby survived until 3 months without any medical support but had severe feeding problems. The diagnosis was confirmed by naso-sinusal CT scan. Surgery was performed at 4 months through an unusual endonasal approach which seems to be less traumatic than the classical sublabial approach. The post-operative course was satisfactory. The 9-month-old patient does not now show residual breathing problems. The presentation will focus on this unusual case. PMID- 16676846 TI - Thymic cyst in the differential diagnosis of paediatric cervical masses. AB - Thymic cyst in the differential diagnosis of paediatric cervical masses. Cervical ectopic thymic tissue is rarely reported. However, it should be included in the differential diagnosis of neck masses, especially in children. We present a case of a 7-year-old male with a soft, asymptomatic right-sided cervical mass. The patient underwent complete excision of a 3 x 4 cm cyst, which was derived from a mass of soft tissue and was in close relation with the carotid sheath. Histological examination of the specimen showed a thymic cyst. The child's post operative course was unremarkable and follow-up appointment six months later showed complete recovery. The embryological development, clinical presentation and management of ectopic thymic lesions are discussed together with a brief review of the literature. PMID- 16676847 TI - Autologous costochondral cartilage implant in two cases of velopharyngeal insufficiency. AB - The velopharyngeal sphincter is critical in enabling the functions of speaking and swallowing. Velopharyngeal insufficiency (VPI) results in hypernasal speech and nasal regurgitation. A frequent cause of VPI is congenital cleft palate, but otolaryngologists sometimes encounter iatrogenic VPI after surgery. Treatment of VPI with prostheses is often successful but not always well tolerated. Many surgical procedures have been proposed to correct palatal length or to enlarge the posterior pharyngeal wall. We report two cases in which autologous costochondral cartilage was used as implant augmentation. This approach is indicated and efficient when the velopharyngeal deficit is less than 5 mm. An autologous costochondral cartilage implant procedure is safe and reversible and can be expected to incite minimal host reaction. PMID- 16676848 TI - A historical vignette. "Be proud of yourself: you have a history!". The transethmoidal approach or the way of Anubis. PMID- 16676849 TI - Predictors of mortality and management of patients with traumatic inferior vena cava injuries. AB - The aim of this study was to determine factors that predict mortality in patients with traumatic inferior vena cava (IVC) injuries and to review the current management of this lethal injury. A 7-year retrospective review of all trauma patients with IVC injuries was performed. Factors associated with mortality were assessed by univariate analysis. Significant variables were included in a multivariate regression analysis model to determine independent predictors of mortality. Statistical significance was determined at P < or = 0.05. A literature review of traumatic IVC injuries was performed and compared with our institutional experience. Thirty-six IVC injuries were identified (mortality, 56%; mechanisms of injury, 28% blunt and 72% penetrating). There was no difference in mortality based on mechanism of injury. Injuries with closer proximity to the heart were associated with increased mortality (P < 0.001). Univariate analysis demonstrated that nonsurvivors had a higher injury severity scale, a lower systolic blood pressure in the emergency department, a lower Glasgow coma score (GCS), and were more likely to have thoracotomies performed in the emergency department or operating room. Multivariate analysis revealed that only GCS (P = 0.03) was an independent predictor of mortality. Typical factors predicting mortality were identified in our cohort of patients, including GCS. The mechanism of injury is not associated with survival outcome, although mortality is higher with injuries more proximal to the heart. The form of management by IVC level is reviewed in our patient population and compared with the literature. PMID- 16676850 TI - Total pancreatectomy for the treatment of chronic pancreatitis: indications, outcomes, and recommendations. AB - Total pancreatectomy (TP) for chronic pancreatitis (CP) has not gained widespread acceptance because of concerns regarding technical complexity, diabetic complications, and uncertainty with respect to long-term pain relief. Records of patients having TP from 1997 to 2005 were reviewed. Patient presentation, etiology of disease, and the indication for TP were examined. Operative results were analyzed. Long-term results were critically assessed, including narcotic usage and the need for re-admission. Postoperative quality of life (QOL) was assessed by the SF-36 health survey. During the study period, 7 patients with CP had TP, and 28 had other operations. The etiology of CP was alcohol in four and hereditary pancreatitis in three. The indication for surgery was pain and weight loss. Preoperatively, all patients used narcotics chronically and two had insulin dependent diabetes. Four had TP after failed previous surgical procedures. Endoscopic retrograde cholangiopancreatography and computed tomography demonstrated small ducts and atrophic calcified glands. The mean length of the operation was 468 minutes, and only two patients required transfusion. There were no biliary anastomotic complications. The mean length of stay was 14 days. Major morbidity was limited to a single patient with a leak from the gastrojejunal anastomosis. Thirty-day mortality was zero, with one late death unrelated to the surgical procedure or diabetes. The mean length of follow-up was 46 months. All patients remained alcohol and narcotic free. No patient was readmitted with a diabetic complication. When compared with the general population, QOL scores were diminished but reasonable. We conclude that TP is indicated in hereditary pancreatitis and in those with an atrophic, calcified pancreas with small duct disease; that TP is technically arduous but can be completed with very low morbidity and mortality; and that on long-term follow-up, pain relief and abstinence from alcohol and narcotics was excellent with an acceptable QOL. PMID- 16676851 TI - Gallstone-induced inflammatory fibrosis: a relentless clinical course of retained gallstones after laparoscopic cholecystectomy. AB - Multiple reports in the literature confirm that retained gallstones spilled during laparoscopic cholecystectomy perpetuate chronic inflammation and suppuration long after the initial operation. Two patients who had previously undergone laparoscopic cholecystectomy presented to our institution with complications of retained stones. Patient 1 presented with right upper quadrant pain and a mass involving the right hepatic lobe. Patient 2 presented with a draining right flank abscess. Both underwent exploratory laparotomy at which time multiple abscess cavities were found, many of which contained retained gallstones. Patient 1 required reoperation for recurrent abscesses 7 months after the initial procedure and has been disease free for 6 months. Patient 2 had abscess recurrence that required percutaneous drainage 1 year after the original procedure and has not had recurrence for 4 years. PMID- 16676852 TI - Rib fracture stabilization in patients sustaining blunt chest injury. AB - Conservative management for the majority of patients with severe chest injuries has produced a reduction in mortality, complications, and hospital length of stay. More recently, operative stabilization of rib fractures has been used with the implication of improved outcome. We assessed the impact of operative rib fracture stabilization on outcome among trauma patients. A matched case-control study of patients undergoing operative rib fracture stabilization was performed. Thirty patients undergoing rib stabilization were matched with 30 controls. Length of intensive care unit (controls, 14.1 +/- 2.7 vs cases, 12.1 +/- 1.2, P = 0.51) and total hospital (controls, 21.1 +/- 3.9 vs cases, 18.8 +/- 1.8, P = 0.59) stay were similar for both groups. There was a trend toward fewer total ventilator days for operative patients (6.5 +/- 1.3 days vs 11.2 +/- 2.6 days, P = 0.12). Ventilator days for operative patients from the time of stabilization was 2.9 +/- 0.6 days compared with 9.4 +/- 2.7 days in controls (P = 0.02). Rib fracture fixation may reduce ventilator requirements in trauma patients with severe thoracic injuries. Long-term functional outcomes need to be assessed to ascertain the impact of this procedure. PMID- 16676853 TI - Gender differences in outcomes after off-pump coronary artery bypass graft surgery. AB - Our objective was to assess surgical outcomes between male and female patients undergoing off-pump coronary artery bypass grafting (CABG). The study was conducted from a 10-year hospitalization cohort (n = 11,230) in which the data were collected prospectively. Inclusion criteria included an off-pump CABG-only procedure. There were 526 men and 250 women included in the study. Fourteen potential confounding risk factors and 14 outcome variables were examined. Six potential risk factors were found to be significantly different between men and women. Men were younger (P = 0.014), had a larger body surface area (P < 0.001), a higher creatinine level (P < 0.001), required more grafts (P < 0.001), and were more likely to have a cerebrovascular history (P = 0.020) and a history of tobacco use (P < or = 0.001). Logistic regression analysis showed that even after controlling for age, body surface area, creatinine level, number of grafts, and tobacco history, women had longer length of hospitalization (odds ratio, 1.97; 95% confidence interval, 1.28-3.04, P = 0.002) and more sternal wound complications than men (odds ratio, 1.07; 95% confidence interval, 1.01-2.11, P = 0.028) after off-pump CABG. Although not statistically different, women had lower operative mortality than men after off-pump CABG (0.8% [2 of 10] compared with 1.5% [8 of 10], respectively). Despite women requiring a longer hospitalization and having a greater incidence of sternal wound infections than men, there was no significant difference in mortality. PMID- 16676854 TI - Outcomes of conventional wound treatment in a comprehensive wound center. AB - Conventional wound care is the elementary treatment modality for treating chronic wounds. However, early treatment with topical growth factors may be needed for a subset of chronic wounds that fail to heal with good wound care alone. A prospective nonrandomized case series from a single-community outpatient wound care clinic is presented here in an effort to identify the subset of chronic wounds that may require early adjuvant intervention. There were 378 consecutive patients with 774 chronic wounds of varying etiology. All patients received 4 weeks of conventional wound care, including weekly debridement and twice-daily dressing changes. Wounds not reduced by 50 per cent volume at 4 weeks were nonrandomly treated with human skin equivalent (Apligraf), platelet-derived wound healing factor, or platelet-derived growth factor isoform BB (becaplermin gel, Regranex). A total of 601 of 774 (78%) wounds healed regardless of treatment type. The median time to heal for all wounds was 49 days (interquartile range = 26-93). More women than men healed (85% vs 71%, respectively, P < 0.0001). Diabetic wounds were as likely to heal as nondiabetic wounds (78% vs 80%, P = 0.5675). Wounds that did not heal had larger volumes and higher grade compared with wounds that healed (P < 0.0001 for both variables). The data presented here show that the majority of chronic wounds will heal with conventional wound care, regardless of etiology. Large wounds with higher grades are less responsive to conventional wound care and will benefit from topical growth factor treatment early in the treatment course. PMID- 16676855 TI - Laparoscopic lumbar hernia repair. AB - Lumbar hernias are rare clinical entities that often pose a challenge for repair. Because of the surrounding anatomy, adequate surgical herniorraphy is often difficult. Minimally invasive surgery has become an option for these hernias. Herein, we describe two patients with lumbar hernias (one with a recurrent traumatic hernia and one with an incisional hernia). Both of these hernias were successfully repaired laparoscopically. PMID- 16676856 TI - Splenic abscess: an easily overlooked disease? AB - Splenic abscess is an uncommon but potentially life-threatening disease. Recent advances in radiology have affected the diagnosis and management of this disease entity. The purpose of this study was to review our experience in managing these patients. We retrospectively reviewed the medical records of 51 patients with splenic abscess as seen in a tertiary medical center between 1998 and 2003. We analyzed the demographics, clinical manifestations, etiology, predisposing factors, diagnostic modalities, bacteriologic profile, treatment, and outcome of these patients. The mean age was 59.9 +/- 14.2 years (ranging from 21-89 years). The male:female ratio was 29:22. Common symptoms included fever (82%), abdominal pain (71%), and nausea and vomiting (46%). The majority of these patients (83%) had leukocytosis. Thirty-six patients had associated parenchymal liver diseases and 26 patients had diabetes mellitus. Abdominal sonogram or computed tomography was performed to establish the diagnosis. Most cultures from the abscess cavities grew gram-negative enteric bacilli. Patients were treated with antimicrobial therapy only (n = 33), additional percutaneous drainage with a pigtail catheter (n = 11), or splenectomy (n = 7), and the survival rates were 48 per cent, 45 per cent, and 100 per cent, respectively. Splenic abscess should be considered in a patient with fever, left upper abdominal pain, and leukocytosis. Splenectomy appears to have better treatment outcome than percutaneous drainage or intravenous antibiotics alone. PMID- 16676857 TI - Increased use of computed tomography does not harm patients with acute appendicitis. AB - The increased use of computed tomography (CT) in patients with appendicitis may cause a delay in surgery and, therefore, higher perforation rates. We examined the use of CT, delay in time to surgery, and perforation rates in appendicitis patients operated on in two periods: Phase 1, 1996 through 1998 and Phase 2, 2001 through 2002. CT was performed in 18 per cent of the Phase 1 group compared with 62 per cent in the Phase 2 group. In the Phase 1 group, patients undergoing CT had a delay to surgery compared with those without CT (18.6 hours vs 7 hours; P < 0.0001). In the Phase 2 group, time to surgery was reduced (median time = 12 hours with CT vs 6 hours without CT; P < 0.001). CT was more accurate in the later group; there were less false-negative and equivocal studies. There was no difference in perforation rates between the Phase 1 and 2 groups. Over time, the increased use, efficiency, and accuracy of CT in patients with acute appendicitis were associated with reduced delays to surgery. The use of CT did not harm patients, but did not translate to better overall outcomes in this group of patients. PMID- 16676858 TI - Tension pneumopericardium: a case report and a review of the literature. AB - Pneumopericardium, or air within the pericardial sack, generally occurs after high-speed blunt deceleration injuries. Although it is generally relatively benign, in rare instances, it can become hemodynamically significant. The diagnosis is easily made on plain chest radiography. More recently, chest computed tomography has been helpful in making the diagnosis. Injury to vital structures such as the tracheobronchial tree or esophageal tears require operative fixation. However, in most instances, pneumopericardium is secondary to dissection of air through the adjacent structures to the pericardial space. The air is trapped as a one-way valve. The pneumopericardium is usually self-limited, requiring no specific therapy. In patients where there is a concomitant pneumothorax, chest tube drainage suffices. We present a case of hemodynamically significant tension pneumopericardium that occurred in association with blunt carotid injury and aortic PMID- 16676860 TI - Reversible gastroparesis: functional documentation of celiac axis compression syndrome and postoperative improvement. AB - Celiac axis compression syndrome has generated much controversy since its original description in 1963. The main symptoms are postprandial epigastric abdominal pain, regurgitation of undigested food, and weight loss, all of which are caused by gastric ischemia from impingement of the celiac axis by the median arcuate ligament of the diaphragm. These symptoms are seen in other common disorders such as chronic mesenteric ischemia and gastroparesis. This makes the diagnosis of celiac axis compression syndrome a true challenge for the clinician. We present data on three patients successfully treated. The pre- and postoperative studies clearly demonstrate a resolution of the condition. The duplex ultrasound images clearly show variable compression on the celiac axis. The angiogram presented shows a classic image of the disease. A review of the data has enabled us to develop an algorithm for the diagnosis of this disease. PMID- 16676859 TI - Increased epithelial cadherin expression among Japanese intestinal-type gastric cancers compared with specimens from American patients of European descent. AB - The different patterns of gastric cancer in the Far East and West have evolved to the extent that it has been suggested that the disease in Japan is biologically less aggressive than in the West. We studied paraffin-embedded, formalin-fixed tissue blocks from Japanese patients and American patients of European descent who had undergone gastrectomy for gastric cancer not involving the gastroesophageal junction. Specimens were staged (T stage), graded (Lauren classification), and biomarker expression (epithelial cadherin [E-cadherin], c erbB2, Ki67, and p53) was quantified using immunohistochemistry without knowledge of the country of origin. E-cadherin was expressed in 49 per cent of malignant cells from Japanese specimens compared with 27 per cent of malignant cells from American specimens (P = 0.04). The expression of E-cadherin on diffuse cancers from the two countries was similar (34.4 in Japanese vs 41.5 in American, P = 0.92). E-cadherin expression, however, was significantly higher among intestinal cancers from the two countries: 56.3 per cent of cells from intestinal or mixed cancers from Japan (n = 32) expressed E-cadherin compared with 22.2 per cent of American specimens (n = 12; P = 0.008).-c-erbB2 was expressed on a higher proportion of malignant cells from American specimens (30% vs 22%; P = 0.20). E cadherin expression, a favorable prognostic factor, is more common in Japanese intestinal-type gastric cancer not involving the gastroesophageal junction. If the biology of gastric cancer in the Far East is less aggressive than that in the United States, it is likely that treatments need to be individualized. PMID- 16676861 TI - Spent bullet in the bronchus. AB - Penetrating tracheal trauma, although infrequent, varies from minor to life threatening injuries. Serious injury often results from airway compromise or significant associated esophageal or vascular trauma. Tracheal injuries resulting in a retained ballistic fragment in the airway have been infrequently reported. We report the successful treatment of a patient with a gunshot wound to the anterior cervical trachea resulting in a spent bullet lodged in the left lower lobe bronchus. PMID- 16676862 TI - Biliary-colonic fistula: a case report and literature review. AB - We report the occurrence of common bile duct obstruction and biliary-colonic fistula after open cholecystectomy. Although it is a very unusual complication after cholecystectomy, biliary-colonic fistula should be part of the differential diagnosis for patients presenting with sepsis after open or laparoscopic cholecystectomy. After confirmation and characterization of the injury by endoscopic retrograde cholangiopancreatography and cholangiogram, assessment for undrained collections by computed tomography scan, control of sepsis and coagulopathy, and nutritional support, surgical repair was undertaken. The patient underwent fistula take-down between the common bile duct and the colon at the hepatic flexure, primary closure of the colon enterotomy, and a Roux-en-Y end to-side hepaticojejunostomy at the confluence of the right and left hepatic ducts. Recovery was uneventful and the patient was doing well at the 6-month follow-up. Surgical repair should be undertaken by surgeons with extensive experience in hepatobiliary reconstruction. PMID- 16676863 TI - Carcinosarcoma of the pancreas arising in a mucinous cystic neoplasm. AB - We report a carcinosarcoma of the pancreas in a 67-year-old woman who presented with nausea, vomiting, and painless jaundice. A work-up demonstrated a well circumscribed mass in the head of the pancreas. After pylorus-preserving pancreaticoduodenectomy, the tumor was found to be grossly yellow, and it compressed the common bile duct and pancreatic duct. Histological examination of the neoplasm showed a 4.0 x 4.0 x 3.0-cm mucinous cystadenocarcinoma with invasive poorly differentiated carcinoma, well-differentiated squamous cell carcinoma, and sarcomatous stroma invading into the duodenum. There was no evidence of nodal metastasis (pT3N0M0). Immunohistochemical studies showed that the epithelial cells stained positive for cytokeratin 7, cytokeratin AE1/3, cytokeratin monoclonal antibody 5.2, epithelial membrane antigen, M carcinoembryonic antigen, and low-molecular-weight kininogen, and the sarcomatous component was immunoreactive with vimentin. The patient had an uneventful recovery, but died 4 months later of rapidly progressive metastatic disease to the liver and peritoneum. To the best of our knowledge, this is the second case of carcinosarcoma with invasive epithelial and sarcomatous areas in the background of a mucinous cystic neoplasm of the pancreas. PMID- 16676864 TI - Cecal volvulus in a child with CHARGE syndrome. AB - Cecal volvulus occurring in a child with CHARGE syndrome is presented. This boy was known to have CHARGE syndrome with multiple congenital anomalies, including coloboma, ventricular septal defect, choanal atresia, growth and mental retardation, bilateral cryptorchidism, dysplasia of the right ear, cleft lip, and hydrocephalus. Nissen's fundoplication had been previously performed for severe hiatal hernia and gastroesophageal regurgitation at the age of 1 year. Cecal volvulus occurred with a 540-degree clockwise rotation of terminal ileum to the right transverse colon and a displacement of the rotated loop to the right upper quadrant of the abdomen when he was 10 years old. Right hemicolectomy with divided ileo- and colostomy was performed. A second staged ileocolostomy was performed uneventfully 3 months later. The midline structural defects with nonfixation of the cecum and ascending colon, chronic constipation, and previous abdominal surgery might have been the predisposing factors. PMID- 16676865 TI - A rare case of diffuse neonatal hemangimatosis. AB - Diffuse neonatal hemangiomatosis (DNH) is a rare neonatal condition in which cutaneous and visceral hemangiomas coexist. If left untreated, DNH is usually fatal at an early age. We report a case of a 6-month-old male infant who was brought to our institution with hepatosplenomegaly and a history of anemia and thrombocytopenia since 1 month of age. Cytogenetic analysis and liver biopsy were normal and bone marrow aspirate was nondiagnostic. Congenital red blood cell abnormality was ruled out. Ultrasound confirmed an increase in size of the spleen from 5 to 15 cm, and magnetic resonance imaging demonstrated intense splenic enhancement consistent with a hemangioma or vascular malformation. Despite severe thrombocytopenia, an exploratory laparotomy was done and the patient underwent a splenectomy and omentectomy. The final pathology confirmed hemangiomatosis of the spleen and omentum. In the neonate with unexplained anemia and thrombocytopenia, DNH should be considered as part of the differential diagnosis. In our case, the patient not only exhibited no obvious cutaneous involvement, but also had rare splenic involvement. Although there are risks involved when operating on a thrombocytopenic patient, the benefits of operating on a patient with DNH far outweigh the risks, and operative intervention should proceed without delay. PMID- 16676866 TI - Pancreatic cancer presenting with hematemesis from directly invading the duodenum: report of an unusual manifestation and review. AB - Pancreatic cancer is a rare cause of gastrointestinal bleeding, and it is extremely rare for hematemesis to be the initial manifestation of invasive pancreatic cancer. We report the case of a 67-year-old man with hematemesis who was found to have invasive pancreatic cancer with a bleeding duodenal ulcer. The patient was not icteric, but repeated sudden hematemesis. An urgent pancreatoduodenectomy was performed with a favorable outcome. Microscopic examination revealed that an adenocarcinoma originating from the pancreatic head extended to the muscularis propria of the duodenum. Furthermore, an exposed vessel and narrow fistula were found. The diagnosis, pathological findings, preoperative events, and postoperative outcome in this unusual case are reviewed. PMID- 16676867 TI - Lymphangiomatosis masquerading as metastatic melanoma. AB - A patient undergoing evaluation for malignant melanoma was thought to have a metastatic process involving the anterior mediastinum, axilla, spleen, and possibly liver based on radiologic findings from positron emission tomography and computed tomography scans. The clinical picture did not corroborate this suspicion, and biopsies ultimately confirmed lymphangioma in the accessory spleen and subcutaneous tissues, leading to a diagnosis of lymphangiomatosis. Diagnosis and management of lymphangiomatosis is clinically challenging. This report reviews the literature on the pathology, diagnostic imaging, and management of lymphangiomatosis. PMID- 16676868 TI - There's more than one way to manipulate a spine. PMID- 16676869 TI - Anatomy of the interosseous region of the sacroiliac joint. AB - STUDY DESIGN: Anatomical study of the interosseous region of the sacroiliac joint (SIJ) complex. OBJECTIVES: To document and quantify the surface topography of the interosseous region of the SIJ. BACKGROUND: A review of the literature reveals that little consideration has been given to the interosseous region of the SIJ anatomically, biomechanically, and clinically. METHODS AND MEASURES: The interosseous region of 11 cadaveric specimens (9 formalin embalmed and 2 fresh frozen) were studied. Ten specimens were 55 years of age or older and 1 was 20 years old. To view the interosseous surfaces of the sacrum and ilium the specimens were either axially sectioned (1-cm slices) or disarticulated. One fresh-frozen and 6 embalmed specimens were disarticulated and the remainder axially sectioned. The topography (surface ridging and areas of ossification) of the interosseous region was documented in all specimens and in 2 specimens the surfaces were 3-dimensionally reconstructed using modeling and animation software (MAYA; Autodesk, Inc, San Rafael, CA). RESULTS: Surface characteristics of the SIJ complex observed in specimens 55 years of age or older included moderate to extensive ridging of the interosseous region of the sacrum and ilium in 100% of specimens and ossification of the central interosseous region of the sacroiliac (SI) ligament in 60% of specimens. CONCLUSIONS: Central region ossification of the interosseous SI ligament and the presence of ridges and depressions over the opposing interosseous surfaces of the sacrum and ilium are features common to specimens that are in or beyond their sixth decade. These findings further support the contention that there is little to no movement available at this joint in older individuals. PMID- 16676870 TI - The use of a lumbar spine manipulation technique by physical therapists in patients who satisfy a clinical prediction rule: a case series. AB - STUDY DESIGN: A case series of patients with low back pain (LBP) who satisfy a clinical prediction rule (CPR). BACKGROUND: A CPR that identifies patients with LBP who are likely to respond with rapid and prolonged reductions in pain and disability following spinal manipulation was developed and recently validated. The CPR developed to predict favorable response to manipulation investigated the effects of only 1 manipulation technique. The accuracy of the CPR for predicting outcomes using other manipulation techniques is not known. The purpose of the case series was to describe the outcomes of patients presenting to physical therapy with LBP who met the CPR and were treated with an alternative lumbar manipulation technique. CASE DESCRIPTION: Consecutive patients referred to physical therapy who satisfied the eligibility criteria, including the presence of at least 4 of the 5 criteria on the CPR, were invited to participate in the case series. Patients were treated for 2 visits with a side-lying lumbar manipulation technique, followed by a basic range of motion exercise. Patients who exhibited a 50% reduction or greater in disability, as measured by the Oswestry Disability Index (ODI), were considered to have experienced a successful outcome. OUTCOMES: A total of 12 patients participated in the case series. The mean age of the group was 39 years (SD, 8.9 years) and the median duration of symptoms was 19 days (range, 8-148 days). Of the 12 patients who participated in this case series, the mean reduction in disability as measured with the ODI was 57% (SD, 9%). Only 1 patient did not surpass the 50% reduction in ODI scores. DISCUSSION: Eleven of the 12 patients (92%) in this case series who satisfied the CPR and were treated with an alternative lumbar manipulation technique demonstrated a successful outcome in 2 visits. It is plausible that patients with LBP who satisfy the CPR may obtain a successful outcome with either manipulation technique directed at the lumbopelvic region. PMID- 16676871 TI - Assessment of physical examination and magnetic resonance imaging findings of hamstring injury as predictors for recurrent injury. AB - STUDY DESIGN: Prospective cohort study. OBJECTIVE: To examine clinical and magnetic resonance imaging (MRI) features of hamstring muscle injury to determine if any are predictive for recurrent injury. BACKGROUND: Hamstring muscle strain injury and subsequent recurrent injury are common. Little information exists on factors that may increase the risk for recurrent injury. METHODS AND MEASURES: The subjects were athletes from 3 professional Australian Rules football teams (n = 162). Anthropometric measurements, clinical signs, convalescent interval, and MRI assessment and measurement were undertaken and recorded in athletes with hamstring muscle strain injury. Athletes were followed for the presence, or absence, of recurrent injury to the same-side posterior thigh over the same and subsequent playing seasons. RESULTS: Thirty athletes met criteria for hamstring injury. Twelve (40%) of 30 athletes had recurrent injury within the same season, with an additional 7 athletes having recurrent injury in the subsequent season. None of the features examined were associated with increased recurrent injury risk within the same playing season. Statistical analysis demonstrated that when combining the same with the subsequent playing season a larger size of initial hamstring injury, as measured by MRI, was associated with an increased risk for recurrent injury (P<.01). A measured transverse size of injury greater than 55% of the muscle, or calculated volume of injury greater than 21.8 cm3, resulted in an increased risk for hamstring recurrence of 2.2 (95% CI, 0.88-5.32) and 2.3 (95% CI, 0.94-5.81) times, respectively, when compared to athletes with hamstring injuries below these measurements. CONCLUSIONS: A larger size of hamstring injury was indicative of higher risk for recurrent injury but only after the subsequent playing season was considered along with the same playing season. None of the other parameters tested, including a shorter convalescent interval and clinical features, were associated with an increased risk for recurrent injury. However, due to low sample size the certainty of these conclusions may be limited. PMID- 16676872 TI - The effects of a 3-week use of lumbosacral orthoses on proprioception in the lumbar spine. AB - STUDY DESIGN: Longitudinal, repeated-measures, factorial design. The trunk axial rotation repositioning error was the dependent variable, while the orthoses, test mode (passive versus active), and the testing session were the independent variables. OBJECTIVES: To verify whether lumbosacral orthoses (LSOs) affect proprioception in the lumbar spine and whether these effects change over a 3-week period during which the LSO is consistently worn. BACKGROUND: To date, there is no compelling evidence that lumbar orthoses support the spine. One hypothesis advanced by several authors is that they may enhance position sense (proprioception) in the lumbar spine. METHODS AND MEASURES: Fourteen subjects without low back pain wore lumbosacral orthoses 3 hours a day for 3 weeks. Spine proprioception was tested in a seated posture in 3 sessions (days 0, 7, and 21). RESULTS: A significant 3-way interaction was found between the effects of the orthoses, session, and test mode (P = .03). The ratio of passive to active average error indicated that after 3 weeks of wearing LSO, proprioception in the passive test worsened in relation to the active test with the LSO. In contrast, proprioception in the passive test improved in relation to the active test when performed without the LSO. CONCLUSIONS: The LSO did affect proprioception in the lumbar spine. These effects most likely changed over time due to sensorimotor adaptation. However, no overall proprioceptive benefits could be ascertained from healthy subjects wearing the LSO. PMID- 16676873 TI - The effect of low-Dye taping on kinematic, kinetic, and electromyographic variables: a systematic review. AB - STUDY DESIGN: A systematic review. OBJECTIVE: To determine the strength of evidence of the effect of low-Dye taping on lower limb kinematic, kinetic, and electromyographic variables. BACKGROUND: Low-Dye taping is a foot-taping technique that aims to limit foot pronation and is commonly used to treat a number of foot disorders. METHODS AND MEASURES: Systematic review of randomized or quasi-randomized trials examining the effect of low-Dye taping compared with no taping on kinematic, kinetic, and electromyographic variables. Trials were identified by searching CINAHL, EMBASE, MEDLINE, SPORTDiscus, and CENTRAL, and by recursive checking of bibliographies. Data were extracted from published trials and from mail contact with authors for further information as necessary. Meta analyses were planned for all outcomes using the generic inverse variance method. Sensitivity analyses were planned by pooling data from nonrandomized trials. Statistical heterogeneity was assessed using the quantity I2. RESULTS: Six trials met inclusion criteria and, of these, 5 trials reported sufficient data on kinematic and kinetic variables to be included in the analysis. Results from the 5 randomized trials were considered robust when pooled with data from 7 nonrandomized trials in a sensitivity analysis. When compared to no taping, low Dye taping increased navicular height immediately after application (weighted mean difference [WMD], 5.90 mm; 95% confidence interval [CI], 0.41 to 11.39; P = .04) and had no effect on navicular height post exercise (WMD, 4.70 mm; 95% CI, 0.61 to 10.01; P = .08), maximum rearfoot eversion while walking (WMD, -0.59 degrees; 95% CI, -2.53 to 1.35; P = .55), and total rearfoot range of motion while walking (WMD, 2.3 degrees; 95% CI, -0.64 to 5.24; P = .13). CONCLUSIONS: Low-Dye taping provides a small change in navicular height post application, although it is unclear whether this change is clinically important. There was high heterogeneity between some trials examining other variables, indicating that more research is needed to confirm the results of previous trials. PMID- 16676874 TI - Criterion validity study of the cervical range of motion (CROM) device for rotational range of motion on healthy adults. AB - STUDY DESIGN: This study compared range of motion (ROM) measurements using a cervical range of motion device (CROM) and an optoelectronic system (OPTOTRAK). OBJECTIVES: To examine the criterion validity of the CROM for the measurement of cervical ROM on healthy adults. BACKGROUND: Whereas measurements of cervical ROM are recognized as part of the assessment of patients with neck pain, few devices are available in clinical settings. Two papers published previously showed excellent criterion validity for measurements of cervical flexion/extension and lateral flexion using the CROM. METHODS AND MEASURES: Subjects performed neck rotation, flexion/extension, and lateral flexion while sitting on a wooden chair. The ROM values were measured by the CROM as well as the OPTOTRAK. RESULTS: The cervical rotational ROM values using the CROM demonstrated a good to excellent linear relationship with those using the OPTOTRAK: right rotation, r = 0.89 (95% confidence interval, 0.81-0.94), and left rotation, r = 0.94 (95% confidence interval, 0.90-0.97). Similar results were also obtained for flexion/extension and lateral flexion ROM values. CONCLUSION: The CROM showed excellent criterion validity for measurements of cervical rotation. We propose using ROM values measured by the CROM as outcome measures for patients with neck pain. PMID- 16676875 TI - Performance management and corrective action. PMID- 16676876 TI - And this one is just right... PMID- 16676877 TI - Acceptance of PACS utilizing a PACS QI Program. AB - This article describes the quality improvement program that Mercy Hospital (Alegent Health System) initiated after it implemented a picture archiving and communication system (PACS) in November 2003. The radiology department encountered numerous PACS-related issues that directly affected the quality and workflow of patient care. In order to get a better understanding of the situation, the department developed a quality improvement plan for its PACS program. The first step was to dedicate a resource--in this case, a radiology information technology (RIT) support specialist--who would serve as a PACS subject matter expert while dealing with day-to-day PACS-related issues- specifically, errors. The error data were collected and categorized for consistency using statistical process control (SPC) tools. The information gathered was then traced back to the team members responsible for the errors and used as a training tool to further educate them. As a result of this program, the average error rate was reduced from 12% to 4% because the radiology team developed a better understanding of the errors by identifying the root causes and being accountable for eliminating errors within their control. In addition, the radiology staff learned to accept and trust the PACS, resulting in a positive culture change that benefited teamwork and staff morale as well as improve the workflow and the quality of patient care. PMID- 16676878 TI - High availability web-based image distribution. AB - When a health system implements a picture archiving and communication system (PACS), film is no longer the preferred medium of image distribution from a radiology department's perspective. The goal is for the department to be 100% filmless sometime after the installation. However, implementing change can be difficult, and getting to that goal of 100% is sometimes never achieved. Sutter Health has come close, with 90% of studies being filmless. A primary reason lies with the distribution method of providing access to images. PMID- 16676879 TI - Advances in ultrasound move modality into new fields. AB - For decades, ultrasound has served as an easy-to-use, noninvasive imaging specialty that yielded results both immediately and cost effectively. In the past, medical imaging technology developers focused solely on building ultrasound systems that delivered excellent images. However, over the last decade, as patient and clinical needs evolved, medical imaging manufacturers found themselves adjusting quickly to build systems to meet new market demands. This shift has spurred tremendous technological advancements and enhanced ultrasound's role in the clinical management of a wide range of disease states, including obesity, diabetes, and breast cancer. Ultrasound has become a leader in delivering ergonomically technologies that take into account the needs of clinicians, making this modality one of the most accommodating when it comes to ensuring the long-term safety of today's sonographer. PMID- 16676880 TI - Flying partial panel. PMID- 16676881 TI - National provider identifier update: The impact on imaging centers. PMID- 16676882 TI - Diversity in the imaging workforce. AB - Culture is the coherent, learned, shared view a group of people has about life's concerns that ranks what is important, and instills attitudes about those things which are considered appropriate as well as prescribed behavior. It notes that some things have more significance than others. "Culture shock" is what happens when a person suddenly finds himself or herself in a place where "yes may mean "no," where a fixed price is negotiable, where to be kept in an outer office is not a cause for insult, and where laughter may signify anger Effective leaders should be able to vary their styles of management, such as leading, planning, organizing, motivating, decision making, staffing, and more importantly, communicating with groups of workers from different cultural backgrounds. Leaders need to perceive conflict as both a challenge and an opportunity. Awareness about team members' cultural similarities and differences is key to foster effective conflict resolution. Synergistic organizations transcend the distinct cultures of the team members. They use these differences as a resource in designing and developing organizational systems. Effective leaders must become a champion and capable of acting in many ways, not experts rigidly adhering to a single approach. PMID- 16676883 TI - Culture change. PMID- 16676884 TI - "Managing up". PMID- 16676885 TI - The joy of staffing. PMID- 16676886 TI - Georgia moves forward with Medicaid managed care. PMID- 16676887 TI - Efforts win praise for Katrina volunteers. PMID- 16676888 TI - Don't leave 'em hanging: establish and follow a return call protocol. PMID- 16676889 TI - Pandemic influenza update. PMID- 16676890 TI - Movie doctoring. PMID- 16676891 TI - Steve de Shazer and the future of solution-focused therapy. AB - Steve de Shazer who, along with Insoo Kim Berg, co-founded the Solution-Focused Brief Therapy (SFBT) approach, recently passed away. In this article we will offer a brief biographical sketch and then discuss the current state of the art of SFBT as it applies to practice, training, and research. Future directions for SFBT, such as the emergence of professional associations, the increased research interest in SFBT as evidenced-based practice, the recent focus on process research to determine the mechanisms of change within SFBT, and the application of SFBT to education are discussed. PMID- 16676892 TI - Integrating emotion-focused therapy with the Satir model. AB - The experiential humanistic family systems approach of Virginia Satir lacks a theoretical structure and is thus vulnerable to losing its experiential nature and being reduced to a mere collection of creative techniques. A way to effectively keep Satir's transformative presence alive is to integrate her approach with a model that is solidly grounded in explicit theory, relationship principles, and therapeutic skills and processes. The model proposed as appropriate to this effect is Emotion-Focused Therapy (EFT). Satir's approach, when integrated with EFT, becomes a model for creating change in individual and family systems that is indeed greater than the sum of its parts. PMID- 16676893 TI - Changing emotion: the use of therapeutic storytelling. AB - Even though using metaphors in a therapeutic context is common, there are very few studies that address their effects. This study examines the effects of storytelling in therapy. After discussing a problem in a current relationship, 42 female participants were randomly assigned to receive either a story or psychoeducational information. Results indicated that both treatments were equally successful in reducing amounts of negative affect and negative feelings toward the relationship. In addition, each story was able to facilitate a change in emotional valence. Finally, participants saw no difference between the depth and smoothness of each session. Examples on using storytelling within different models of marriage and family therapy are provided. PMID- 16676894 TI - The chabot emotional differentiation scale: a theoretically and psychometrically sound instrument for measuring Bowen's intrapsychic aspect of differentiation. AB - Bowen's theory (1978) of differentiation is the personality variable most critical to mature development and the attainment of psychological health. The study of psychological differentiation has been confounded by confusion regarding the constructs being studied and the measurement tools used to assess them. The purpose of this review is to introduce and demonstrate the utility and importance of the Chabot Emotional Differentiation Scale (CED; Chabot, 1993), a self-report instrument specifically designed to measure Bowen's intrapsychic aspect of differentiation, or an individual's ability to distinguish between emotional and intellectual functioning and utilize intellect in emotionally charged situations. Psychometric support for the CED, validation and cross-cultural studies to date, and implications for theory, research, and practice are presented. PMID- 16676895 TI - Integrating the older/special needs adoptive child into the family. AB - This qualitative, grounded theory study investigated 11 families who reported having successfully integrated into their family unit at least one older/special needs adoptee. The theory that emerged through the constant comparative methodology consisted of two categories (Decision to Adopt and Adjustment) and a core category (Developing a Sense of Family). The two categories and core category comprised a process that was informed by the Family Narrative Paradigm and culminated in the successful integration of the child or children into the existing family unit. Parental perceptions that appeared to facilitate this process included: (a) finding strengths in the children overlooked by previous caregivers, (b) viewing behavior in context, (c) reframing negative behavior, and (d) attributing improvement in behavior to parenting efforts. PMID- 16676896 TI - Mental health issues and the foster care system: an examination of the impact of the Adoption and Safe Families Act. AB - Although marriage and family therapists are being called on to help at-risk families, some say that clinicians have insufficient knowledge about the impact of policies on families involved in the foster care system. The purpose of this qualitative investigation was to identify how the Adoption and Safe Families Act informs decision making, to recognize trends in decisions regarding termination of parental rights of parents with mental health issues, and to explore treatment issues of families involved in the foster care system. Results indicate that court cases decided after the implementation of the Adoption and Safe Families Act are likely to result in termination of parental rights. Implications for clinicians and researchers are discussed. PMID- 16676897 TI - "Wanting to be a good father": experiences of adolescent fathers of Mexican descent in a teen fathers program. AB - Adolescent fatherhood has received limited attention in research and clinical practice. This article describes the design and implementation of a parenting program for adolescent fathers, largely minority, involved in the juvenile justice system. In the teen fathers groups, adolescent fathers were exposed to therapeutic (e.g., family-of-origin) as well as psychoeducational (e.g., child development and parenting) interventions. Findings from a descriptive phenomenological study with six former group participants indicate that the program is an effective way of assisting teen fathers in increasing their commitment as fathers as well as their involvement with their children. PMID- 16676898 TI - Use of standardized assessment instruments in couple therapy: the role of attitudes and professional factors. AB - The study examined therapeutic approach, professional affiliation, training, seniority, and work setting as predictors of attitudes toward standardized instruments and their use in couple therapy. Data were gathered from 163 certified marriage and family therapists in Israel. Results showed that 27.6% of marital therapists used standardized instruments for assessment. More positive attitudes toward standardized instruments were associated with the extent of training in their use and with a structural-strategic approach. Logistic regression analysis indicated that positive attitudes, training, and work setting best predict the application of standardized assessment instruments. Reasons for the relative lack of use of standardized assessment instruments are discussed. It is recommended that more training in assessment instruments be included in marriage and family therapy training. PMID- 16676899 TI - Postdivorce paternal disengagement: failed mourning and role fusion. AB - In this article, I suggest that postdivorce paternal disengagement may be rooted in the father's tendency to link his children and ex-wife as a single entity in consequence of his failure to adequately mourn the loss of his ex-wife and to redefine his paternal role and identity in distinction from his spousal role and identity. I also suggest that the ex-spousal conflict that disengaged fathers often blame for their disengagement is the product of these failures and shows the progress from conflict through disengagement. These claims are developed on the basis of findings of other authors and illustrated though a case analysis of an absent father. PMID- 16676900 TI - Health risks of enteric viral infections in children. AB - Children are at a greater risk of infections from serious enteric viral illness than adults for a number of reasons. Most important is the immune system, which is needed to control the infection processes. This difference can lead to more serious infections than in adults, who have fully developed immune systems. There are a number of significant physiological and behavioral differences between adults and children that place children at a greater risk of exposure and a greater risk of serious infection from enteric viruses. Although most enteric viruses cause mild or asymptomatic infections, they can cause a wide range of serious and life-threatening illnesses in children. The peak incidence of most enteric viral illnesses is in children <2yr of age, although all age groups of children are affected. Most of these infections are more serious and result in higher mortality in children than adults. The fetus is also affected by enterovirus and infectious hepatitis resulting in significant risk of fetal death or serious illness. In addition to the poliovirus vaccine, the only vaccine available is for hepatitis A virus (HAV). A vaccine for rotavirus has currently been withdrawn, pending review because of potential adverse effects in infants. No specific treatment is available for the other enteric viruses. Enteric viral infections are very common in childhood. Most children are infected with rotavirus during the first 2yr of life. The incidence of enteroviruses and the viral enteric viruses ranges from 10% to 40% in children and is largely dependent on age. On average, half or more of the infections are asymptomatic. The incidence of hepatitis A virus is much lower than the enteric diarrheal viruses. There is no current evidence for hepatitis E virus (HEV) acquisition in children in the U.S. Enteric viral diseases have a major impact on direct and indirect health care costs (i.e., lost wages) and amount to several billion dollars a year in the U.S. Total direct and indirect costs for nonhospitalized cases may run from $88/case for Norwalk virus to $1,193/case for enterovirus aseptic meningitis. Direct costs of hospitalization ran from $887/case for Norwalk virus to $86,899/case for hepatitis A. These costs are based on 1997-1999 data. Generally, attack rates during drinking water outbreaks are greater for children than adults. The exception appears to be hepatitis E virus where young adults are more affected. However, pregnant women suffer a high mortality, resulting in concurrent fetal death. Also, secondary attack rates are much higher among children, probably because of fewer sanitary habits among this age group. Overall, waterborne outbreaks of viral disease have a greater impact among children than adults. To better quantify the impact on children, the literature hould be further reviewed for case studies of waterborne outbreaks where data are available on the resulting illness by age group. The EPA and/or Centers for Disease Control should attempt to collect these data as future outbreaks are documented. PMID- 16676901 TI - Pyrethroid illnesses in California, 1996-2002. AB - This survey summarizes California's recent experience with illnesses related to pyrethroid exposures and augments the data available on pyrethroid inhalation exposure and residue dissipation. We reviewed California Department of Pesticide Regulation (DPR) Pesticide Illness Surveillance Program (PISP) data and DPR Pesticide Use Reporting (PUR) data for 13 pyrethroids used during 1996-2002 and identified 317 illnesses associated with exposure. PUR found a total of 4,629,852 pound (2,100,068 kg) of the 13 active ingredients were applied during the 7 yr. Type II pyrethroids accounted for 1,979,352 (897,820 kg) and 42.7% of the total pounds applied and 220 (69.6%) of the reported illnesses. Cyfluthrin was associated with 122 cases (55% of illnesses related to type II pyrethroids and 38.4% of all pyrethroid illnesses). Agricultural uses accounted for 118 (37.3%) of the reported illness cases, with 116 cases associated with employment. For the 199 cases (62.8%) associated with nonagricultural use, 132 (66.3%) were occupationally related. Overall, approximately equal numbers of illnesses resulted from individual exposures (167 cases) and group exposures (150 cases). The symptom arrays associated with the pyrethroid illnesses included irritant effects or pares- thesias of the eye, skin, or respiratory tract in 269 cases (84.9%). Type II pyrethroids were more frequently associated with isolated irritant symptoms (107 cases) than the type I pyrethroids (26 of 97 cases). Systemic symptoms were reported in 184 illnesses (58% of cases). Isolated systemic effects occurred in 48 cases (15.1%), but systemic effects were also present in 136 (50.6%) of the 269 cases with irritant symptoms. Residue exposures accounted for 158 illnesses (49.8%). Single or multiple violations of pesticide use regulations contributed to exposures in 90 of the 317 illnesses (28.4%); 76 were related to nonagricultural pyrethroid use. We also report results of DPR Worker Health and Safety Branch (WH&S) investigations of three large group illness episodes related to exposure to type II pyrethroids cyfluthrin and lambda cyhalothrin that involved primarily respiratory irritation symptoms. An inhalation monitoring study found cyfluthrin air levels that approached experimentally established irritant thresholds for airborne cyfluthrin, from which a mean estimated absorbed dosage of 1.311 microg/kg/d was calculated. Although additional data are needed to establish threshold levels for both irritant and systemic symptoms for cyfluthrin and other pyrethroids, these observations suggest that field residues can cause irritant respiratory symptoms. DPR conducted a residue dissipation study in seven orange groves and estimated cyfluthrin residue half-lives. The dissipation rates fell into two distinct decay patterns, with more rapid decay in groves 1-4 (overall average half-life = 4.9 d) and a considerably longer decay in groves 5-7. The half-life for groves exhibiting the slower residue dissipation was not constant. The first two half lives for groves 5-7 can be approximated; they are 11 and 32d, respectively. The third investigation involved an illness episode in which 11 raisin harvesters developed acute respiratory irritation symptoms when they were exposed to residues of lambda-cyhalothrin, propargite, and sulfur. Gas chromatography analyses of eight dislodgeable foliar residue (DFR) samples verified mean residues of lambda-cyhalothrin (0.43 +/- 0.10 microg/cm2), propargite (0.35 +/- 0.11 microg/cm2), and sulfur (0.31 +/- 0.28 microg/cm2) on the grape leaves. Subsequent investigation confirmed that the lambda-cyhalothrin product, which was not registered for use on grapes, was mistakenly mixed and applied 45 d earlier at 35 times the highest legal rate for any crop. The effects of exposure to average lambda-cyhalothrin DFR levels of 0.43 microg/cm2 have not been previously documented. PMID- 16676902 TI - Ecological risk assessment of contaminated soil. AB - This review has described three cases of ecological risk assessment. The cases include two heavy metals (Cu and Zn) and an anthropogenic organic chemical (DDT). It concludes that there are at least two major constraints hampering the use of laboratory tests to predict effects under natural field conditions. One key issue is bioavailability, and another is suboptimal conditions or multiple stresses in the field such as climatic stress (drought, frost), predators, competition, or food shortage. On the basis of the presented case studies, it was possible to answer three essential questions often raised in connection to ecological risk assessment of contaminated sites. 1. To what extend does soil screening level (SSL) estimate the risk? The SSL are generally derived at levels corresponding to the lowest observed effect levels in laboratory studies, which often is close to the background levels found in many soils. In the cases of zinc and especially DDT, the SSL seemed quite conservative, whereas for copper they resemble the level at which changes in the community structure of soil microarthropods and the plant community have been observed at contaminated sites. The SSL correspond as a whole relatively well with concentrations where no effects or only minor effects were observed in controlled field studies. However, large variation in field surveys can often make it difficult to conclude to what extent the SSL corresponded to no-effect levels in the field. 2. Do bioassays represent a more realistic risk estimate? Here, there is no firm conclusion. The zinc study in UK showed a better relationship between the outcome of ex situ bioassays and field observations than the SSL. The latter overestimated the risk compared to field observations. However, this would be species dependent, as the sensitivity to metals may vary considerably between recognized test species, even within the same group of organisms, such as Folsomia candida and Folsomia fimetaria or Eisenia fetida and Lumbricus sp. Furthermore, it was demonstrated that bioassays were not useful for predicting general species diversity in the field as they are strongly influenced by natural variation and other factors not related to contamination. In the case of copper, bioassays with springtails and black bindweed seemed to underestimate the risk compared to the Cu concentrations at which significant changes in the community structure of soil microarthropods and plants have been observed at the contaminated site, and this was also the case for the DDT-contaminated soils. Here, bioassays with DDT-contaminated soils showed generally very low toxicity, with EC10 values considerably higher than the levels where clear effects on single species as well as community structure have been detected in the present field study. 3. Is it possible to make sound field surveys or do we lack suitable reference situations? Large natural variation caused by other factors than contaminants were observed in most cases, and this may have particularly hampered the conclusions made in the field surveys. These factors included pH, private and military traffic, age of vegetation, shading effects, and variations in light insensitivity as well as quantity and quality of organic matter. It was therefore concluded that field studies should always be interpreted in concert with similar data from a reference situation. Conclusions should therefore be made with caution in situations where important soil conditions vary between control plots and the contaminated sites. The cases also showed that indices focusing on species richness were unreliable. Estimates of evenness or dominance were recommended instead, and most authors concluded that multivariate analysis of community structure was a sensitive and useful method superior to single-species field data. This review concludes that there is a need for a tiered approach in ecological risk assessment of contaminated soils. Generic soil screening levels are needed as a first tier. Higher tiers of ecological risk assessment should, however, contain some kind of site-specific assessment. It is furthermore important to organize the various studies in a framework or decision support system that is transparent and useful for all stakeholders. A weight of evidence approach may be an obvious choice to deal with these uncertainties. The TRIAD approach, which incorporates and categorizes information in a triangle - chemistry, toxicology, and ecology - is an appropriate tool for handling conceptual uncertainties. PMID- 16676903 TI - Pesticide exposure monitoring databases in applied risk analysis. AB - Faced with the need to evaluate under what conditions chemicals can be used with "reasonable certainty of no harm" to workers and consumers, industry and government agencies have embraced quantitative risk analysis as a science-based approach for product development, regulatory evaluations, and associated risk management decision making. Beginning in the 1990s, a variety of industry sponsored task forces have been formed to develop exposure-related data to support safety evaluations for pesticide chemicals used in agricultural, industrial, institutional, residential, and other settings. Human exposure assessment and the underlying data (e.g., personal exposure and biological monitoring measurements, media-specific residue measurements, product use, and time-activity information) represent a critical component of the risk assessment process and a rapidly advancing science. While task forces have been created to develop databases for supporting the continued safe use of products, the development of these databases has served to advance general understanding of the basic principles underlying exposure assessment methodology and thereby provide the basis for improved science-based risk management by both industry and government. Given that developing chemical-specific data for every product use pattern and associated worker or consumer exposure scenario (e.g., professional mixer, loader and applicator activities associated with the use of a low-pressure sprayer, consumer residential lawn application via a ready-to-use hose-end sprayer product) is prohibitively expensive and time consuming, alternative approaches have been developed based upon meta-analyses and generalizations derived from databases of exposure monitoring studies for multiple chemicals, sorted by significant exposure covariates such as formulation type, method of application, amount of active ingredient applied, site of application, protective equipment and clothing, and task or activity. These generalizations can be used for predictive exposure analyses and have clearly demonstrated the value of "generic databases." Although data in these databases and associated generalizations are subject to interpretation, e.g., during the regulatory decision-making processes, and may be used in conjunction with additional considerations or assessment methods that result in conservative biases, the role of generic databases for risk management decision making, and advancing the science of applied exposure analysis continues to be realized. PMID- 16676905 TI - April fool's commentary: in praise of placebos. PMID- 16676904 TI - Ecotoxicological evaluation of perfluorooctanesulfonate (PFOS). AB - Based on available toxicity data, protective screening-level concentrations of PFOS were calculated for aquatic and terrestrial organisms. Using the Great Lakes Initiative, water concentrations of PFOS were calculated to protect aquatic plants and animals. The screening plant value (SPV) protective of aquatic algae and macrophytes was calculated as 2.3 mg PFOS/L. The secondary chronic value protective of aquatic organisms was 1.2 microg PFOS/L. The screening-value water concentrations less than or equal to 1.2 microg PFOS/L would not pose a potential risk to aquatic organisms. Because the aquatic benchmark is based on the most sensitive species, this benchmark should also be protective of other aquatic organisms, including amphibians. The tissue-based TRV for fish was determined to be 87 mg PFOS/kg ww. For terrestrial plants, a screening benchmark was determined to be 1.3 mg PFOS/kg soil ww or 1.5 mg PFOS/kg soil dw, whereas for soil invertebrates such as earthworms the benchmark value was 39 mg PFOS/kg dw soil or 33 mg PFOS/kgww soil. For avian species, dietary, ADI, and egg yolk-based benchmarks were determined as 0.28mg PFOS/kg diet, 0.021mg PFOS/kg bw/d, and 1.7 microg PFOS/mL yolk, respectively. Benchmarks for serum and liver for the protection of avian species were 1.0 microg PFOS/mL and 0.6 microg PFOS/gww, respectively. However, no-effect levels in laboratory studies suggest actual population-level effects would not be expected to occur until a concentration of 6.0mg PFOS/kg in the diet, 5.0 microg PFOS/gww in the liver, or 9.0 microg PFOS/mL in the serum was exceeded, thus indicating the conservative nature of the benchmarks. PMID- 16676906 TI - The bathhouses of Manhattan. PMID- 16676907 TI - Neurostimulation for epilepsy. PMID- 16676908 TI - Deep brain stimulation surgery for Parkinson's disease: the role of neuropsychological assessment. AB - Neuropsychological assessment has two primary roles in the DBS process. First, assessment of cognitive and emotional functioning ensures that only appropriate candidates undergo this surgical procedure. Patients with dementia, cognitive performance suggestive of an additional neuropathological process, or significant psychiatric impairments should not undergo DBS. Second, neuropsychological assessment is essential to determine the cognitive and emotional outcomes following surgery. At the present time, a disruption in verbal fluency is the only consistent cognitive decline associated with DBS. While worsening of depression and the development of symptoms of mania are potential side effects from DBS, more studies find that DBS is associated with improvements in emotional functioning. Based on the growing understanding of the risk factors and potential side effects to DBS, neuropsychological assessment is necessary to ensure that patients selected to undergo this surgical intervention will likely have positive cognitive and emotional outcomes, in addition to the expected benefits in motor functioning. PMID- 16676909 TI - Surgical aspects of deep brain stimulation. PMID- 16676910 TI - Vagus nerve stimulation and deep brain stimulation for treatment resistant depression. AB - Neurostimulation techniques are potentially useful options for severely depressed patients who have failed trial after trial of medication and psychotherapy. Cervical VNS therapy for chronic or recurrent depression which does not resolve with pharmacotherapy was recently approved by the FDA. DBS for severe intractable depression has been studied in two pilot studies with very few patients to date. Further investigations are currently underway in order to more fully evaluate both of these neurostimulation therapies, with the hope of substantially improving the treatment of refractory depression. PMID- 16676911 TI - Deep brain stimulation for Parkinson's disease: patient selection and motor outcomes. AB - DBS is a safe and effective option for the treatment of patients with advanced PD. To ensure a successful outcome, however, it is important to select the appropriate candidates. The ideal candidate has idiopathic PD, suffers from complications of chronic levodopa therapy despite optimal medical management, and has no cognitive impairment or active psychiatric issues. Although the exact mechanism of how DBS exerts its effects remains under investigation, it is clearly apparent that bilateral stimulation of either the GPi or STN effectively helps the motor symptoms of PD. While many surgical centers favor stimulation of the STN over the GPi, there is accumulating evidence that STN stimulation may result in adverse non-motor outcomes such as depression. Future studies will be needed in order to determine the best site of stimulation, the exact mechanisms of DBS, and the long-term outcomes of both motor and non-motor symptoms. As our understanding of these components becomes clearer, we will be able to optimize the treatment and management for those whose lives are affected by Parkinson's disease. PMID- 16676912 TI - Cerebrovascular disease in Rhode Island: risks and burden. PMID- 16676913 TI - Colorectal algorithm guidelines for screening. PMID- 16676914 TI - Improving nursing home culture pilot. PMID- 16676915 TI - A skeletal etymology, Part II. PMID- 16676916 TI - Chronic pelvic pain in women: Focus on painful bladder syndrome/lnterstitial cystitis. PMID- 16676917 TI - Epidemiology and quality of life. AB - Once thought to be a rare condition, PBS/IC is being increasingly recognized as an important cause of CPP. It can exist either as a solitary disorder or in conjunction with other gynecologic or urologic disorders. The true prevalence of PBS/IC is hard to determine because most patients remain undiagnosed, although it is now thought to occur in up to 7.5% of the general female population and in 38 85% of women who present with CPP. Because the bladder has been insufficiently considered as a component of pelvic pain, many patients with PBS/IC may be misdiagnosed and inappropriately treated for years. It is critical for all clinicians, and especially gynecologists, who are often the first physicians from whom a woman with CPP will seek treatment, to consider PBS/IC in their patients who complain of pelvic pain, particularly when it occurs with urinary symptoms. The hallmark features of PBS/IC are irritative voiding and CPP. A minority of patients have classic ulcers and most have glomerulations, although it is not always necessary to establish these features to make the diagnosis. With careful questioning, most patients, in fact, can be identified by symptomatology, a medical history and a physical examination. Cystoscopy may be useful to rule out other conditions or to evaluate microscopic hematuria, which may be found in these patients. PBS/IC should be correctly diagnosed as early in the disease course as possible in order to avoid debilitating detriments to the patients' QOL in multiple domains. When symptoms of PBS/IC are recognized early on, treatment can be initiated when it is most likely to have a successful outcome. Although evidence-based clinical practice guidelines for IC/PBS are not available, partly because there is a lack of consensus on the definition and etiology of IC, clinicians, both primary care providers and specialists, can be reassured that there are diagnostic and treatment options that are simple to administer and have been demonstrated to be safe and effective. PMID- 16676918 TI - Proposed pathogenesis of painful bladder syndrome/interstitial cystitis. PMID- 16676919 TI - Current issues in the diagnosis of painful bladder syndrome/interstitial cystitis. AB - PBS/IC, which was traditionally thought to be a rare condition, is increasingly thought to be a frequent cause of CPP. Failure to consider the bladder as a component of this pain is common, primarily because of the similarity in symptoms to other urogynecologic conditions. The diagnosis of PBS/IC has been one of exclusion; as a result, PBS/IC is frequently misdiagnosed as urogenital infection, OAB or endometriosis, among other conditions with similar symptomatology. Such misdiagnosis results in unnecessary and ineffective pharmacologic or even surgical interventions. Diagnosis of PBS/IC and appropriate management early in the disease process afford women a better outcome and a better quality of life. Making PBS/IC a diagnosis of inclusion is necessary to attain this goal. Two additions to the diagnostic armamentarium, the PUF Patient Symptom Scale and the PST, can help to identify women whose presenting complaints of CPP might have a bladder component. The high correlation between these 2 tools allows clinicians to administer the noninvasive PUF questionnaire as an initial screening device to identify women suspected of having IC. If PBS/IC is diagnosed early in the disease process, it can be treated successfully in most patients. PMID- 16676920 TI - Multimodal therapy for painful bladder syndrome/interstitial cystitis. AB - Most patients who suffer from PBS/IC can now be simply and effectively treated. The first step to successful management is accurate and timely diagnosis, which has become easier with available and validated screening and diagnostic tools such as PUF and PST. Once PBS/IC is correctly diagnosed, prompt treatment should address the main components of the disease, a dysfunctional urothelium, mast cell activation and neural upregulation. Multimodal treatment that has shown benefit includes oral PPS plus an antihistamine, such as hydroxyzine, and a TCA, such as amitriptyline. Behavioral interventions and intravesical instillation therapy are adjunctive measures that will promote symptom relief. Intravesical "rescue" solutions using lidocaine and heparin or PPS (dissolved in water or in the instillation solution [off-label use of PPS]) can provide immediate relief while patients develop a response to oral PPS. Patient education and support are critical in managing this complex but treatable disorder. PMID- 16676921 TI - Classification of syringomyelia. AB - Syringomyelia poses special challenges for the clinician because of its complex symptomatology, uncertain pathogenesis, and multiple options of treatment. The purpose of this study was to classify intramedullary cavities according to their most salient pathological and clinical features. Pathological findings obtained in 175 individuals with tubular cavitations of the spinal cord were correlated with clinical and magnetic resonance (MR) imaging findings in a database of 927 patients. A classification system was developed in which the morbid anatomy, cause, and pathogenesis of these lesions are emphasized. The use of a disease based classification of syringomyelia facilitates diagnosis and the interpretation of MR imaging findings and provides a guide to treatment. PMID- 16676922 TI - Application of syringosubarachnoid shunt through key-hole laminectomy. Technical note. AB - The authors describe the key-hole laminectomy technique (KHLT) for application of syringosubarachnoid shunts. This technique was used in 29 patients with noncommunicating syringomyelia. In all cases the shunts were inserted successfully without any peri- or late postoperative complications. The late follow-up magnetic resonance examinations revealed the clear collapse of syrinx in all cases. The authors advocate use of the KHLT because it is associated with less surgery-related trauma, epidural fibrosis, infection, and spinal instability. PMID- 16676923 TI - Pain and syringomyelia: a review. AB - The pathophysiological basis of chronic pain syndromes remains poorly defined. Central and dysesthetic pain are probably the most disabling of sensory disturbances associated with syringomyelia, and, unfortunately, effective treatment remains elusive. In this paper, the authors review their institutional experience with both clinical and laboratory studies of patients with syringomyelia, and they review the relevant literature. To date, there is no consensus as to the best treatment for central cord pain syndromes, although there are many promising areas of current research involving the use of neurochemicals in the spinal cord. PMID- 16676924 TI - A genetic hypothesis for Chiari I malformation with or without syringomyelia. AB - In several reports the authors have suggested occasional familial aggregation of syringomyelia and/or Chiari 1 malformation (CM1). Familial aggregation is one characteristic of traits that have an underlying genetic basis. The authors provide evidence for familial aggregation of CM1 and syringomyelia (CM1/S) in a large series of families, establishing that there may be a genetic component to CM1/S in at least a subset of families. The authors observed no cases of isolated familial syringomyelia in their family studies, suggesting that familial syringomyelia is more accurately classified as familial CM1 with associated syringomyelia. These data, together with the cosegregation of the trait with known genetic syndromes, support the authors' hypothesis of a genetic basis for some CM1/S cases. PMID- 16676925 TI - Mechanisms underlying the formation and enlargement of noncommunicating syringomyelia: experimental studies. AB - The pathogenesis of noncommunicating syringomyelia is unknown, and none of the existing theories adequately explains the production of cysts that occur in association with conditions other than Chiari malformation. The authors' hypothesis is that an arterial pulsation-driven perivascular flow of cerebrospinal fluid (CSF) is responsible for syrinx formation and enlargement. They investigated normal CSF flow patterns in 20 rats and five sheep by using the tracer horseradish peroxidase; the effect of reducing arterial pulse pressure was examined in four sheep by partially ligating the brachiocephalic trunk; CSF flow was examined in 78 rats with the intraparenchymal kaolin model of noncommunicating syringomyelia; and extracanalicular cysts were examined using the excitotoxic model in 38 rats. In the normal animals there was a rapid flow of CSF from the spinal subarachnoid space into the spinal cord perivascular spaces and then into the central canal. This flow ceased when arterial pulsations were diminished. In animals with noncommunicating syringomyelia, there was rapid CSF flow into isolated and enlarged segments of central canal, even when these cysts were causing pressure damage to the surrounding spinal cord. Exitotoxic injury of the spinal cord caused the formation of extracanalicular cysts, and larger cysts were produced when this injury was combined with arachnoiditis, which impaired subarachnoid CSF flow. The results of these experiments support the hypothesis that arterial pulsation-driven perivascular fluid flow is responsible for syrinx formation and enlargement. PMID- 16676926 TI - Cerebrospinal fluid flow dynamics study in Chiari I malformation: implications for syrinx formation. AB - Cerebrospinal fluid (CSF) flow abnormalities are known to be present in Chiari I malformation and to underlie the origin and progression of associated syringomyelia. The incidence of syrinx formation, however, is variable for unknown reasons. The aim of this study was to investigate whether differences in CSF flow dynamics in patients with Chiari I malformation may account for the different clinical and radiological presentation. Presurgical and postsurgical phase-contrast magnetic resonance imaging investigations were prospectively conducted in 47 adult patients with symptomatic Chiari I malformation. Patients were divided into two groups according to the presence (32 cases) or absence (15 cases) of syrinx. Cerebrospinal fluid flow patterns were evaluated at four regions of interest: prebulbar cistern, foramen magnum, and the ventral and dorsal spinal subarachnoid spaces at the C-5 level. A temporal analysis of CSF flow waveforms was performed with measurement of cranial- and caudal-directed flow durations. All patients underwent a craniocervical decompressive procedure. Preoperatively, a prolonged caudal- directed (systolic) flow pattern was observed in patients with syringomyelia, as compared with normal control values obtained in 15 healthy volunteers. Conversely, a decreased systolic duration was observed in Chiari I patients who had malformation without syrinx. These trends were not statistically significant because of the considerable degree of overlap with the control values recorded in both groups. Additional comparison of the observed preoperative values obtained in patients with and those without syringomyelia indicated that the difference in systolic flow duration was significant at the ventral spinal subarachnoid space level (p = 0.003) and remarkable at the other levels, although not reaching statistical significance. Cerebrospinal fluid flow was minimal or absent at the foramen magnum (dorsal aspect) due to tonsillar herniation, precluding reliable quantitative measurement at this level. There was no evidence of communication between the fourth ventricle and syrinx in any case. Postoperatively, unobstructed CSF flow was recorded across the enlarged foramen magnum and into the artificial cisterna magna in all patients. A gradual restoration of near-normal flow patterns was observed in both groups. Inside the syrinx, fluid motion gradually tapered, no longer being detectable in 12 patients (37.5%) 1 year postsurgery. In patients with Chiari I malformation and associated syringomyelia different CSF flow patterns were demonstrated as compared with patients in whom syrinx was absent. Analysis of this study's findings supports the hypothesis that in Chiari I malformation an elongated systolic flow may prolong the condition of increased spinal subarachnoid pressure caused by the junctional obstruction, thus favoring CSF penetration into the spinal cord. It may be also proposed that a shortened systolic flow may be insufficient to maintain a hypertensive condition for enough time to induce syrinx formation. PMID- 16676927 TI - The "presyrinx" state: is there a reversible myelopathic condition that may precede syringomyelia? AB - OBJECT: Alteration of cerebrospinal fluid (CSF) flow has been proposed as an important mechanism leading to the development of syringomyelia. We hypothesize that a "presyrinx" condition due to potentially reversible alteration in normal CSF flow exists and that its appearance may be due to variations in the competence of the central canal of the spinal cord. METHODS: Five patients with clinical evidence of myelopathy, no history of spinal cord trauma, enlargement of the cervical spinal cord with T1 and T2 prolongation but no cavitation, evidence for altered or obstructed CSF flow, and no evidence of intramedullary tumor or a spinal vascular event underwent MR imaging before and after intervention that alleviated obstruction to CSF flow. RESULTS: Preoperatively, all patients demonstrated enlarged spinal cords and parenchymal T1 and T2 prolongation without cavitation. Results of magnetic resonance (MR) imaging examinations following intervention in all patients showed resolution of cord enlargement and normalization or improvement of cord signal abnormalities. In one patient with severe arachnoid adhesions who initially improved following decompression, late evolution into syringomyelia occurred in association with continued CSF obstruction. CONCLUSION: Nontraumatic obstruction of the CSF pathways in the spine may result in spinal cord parenchymal T2 prolongation that is reversible following restoration of patency of CSF pathways. We refer to this MR appearance as the "presyrinx" state and stress the importance of timely intervention to limit progression to syringomyelia. PMID- 16676929 TI - Toward a rational treatment of Chiari I malformation and syringomyelia. AB - In patients with Chiari I malformation with and without associated syringomyelia, aberrant cerebrospinal fluid (CSF) dynamics and a spectrum of posterior fossa pathological findings are demonstrated. In this study, the authors test the validity of using prospective cardiac-gated phase-contrast cine-mode magnetic resonance (MR) imaging to define the malformation, delineate its pathophysiology, and assist in implementing a rational treatment plan. Eighty-five cases were prospectively analyzed using cine MR imaging. Sixty-five patients, adults and children, with symptomatic Chiari malformation, with and without syringomyelia, were surgically treated from 1990 to 1999. All patients underwent pre- and postoperative cine MR evaluation. Ten patients were treated after a previous surgical procedure had failed. To establish CSF flow characteristics and normative CSF profiles, 20 healthy volunteers were examined. Compared with normal volunteers, in Chiari I malformation patients with and without syringomyelia, uniformly abnormal craniocervical junction CSF flow profiles were revealed. After intradural exploration, nearly all patients with Chiari I malformation experienced clinical improvement and CSF flow profiles, paralleling those of normal volunteers, were shown. In all patients in whom treatment had failed, abnormal preoperative CSF flow profiles, which correlated with suspected physiological abnormalities and the pathological findings noted at reoperation, were demonstrated. Symptomatic Chiari I malformation is a dynamic process characterized by the impaction of the hindbrain in an abnormal posterior fossa. This compression obstructs the normal venting of CSF in and out of the craniocervical subarachnoid space, throughout the cardiac cycle. Therefore, decompression or enlargement of the posterior fossa to establish normal CSF pathways should be the primary goal of surgical intervention. Aberrant CSF flow appears to be only one aspect of the pathological condition found in patients with Chiari I malformation. Arachnoid scarring in the posterior fossa and selective vulnerability of the spinal cord may also be factors in the pathogenesis and maintenance of associated syringomyelia. Phase-contrast cine MR imaging is a useful tool in defining physiological and anatomical problems in patients with Chiari I and syringomyelia, and it can help guide an appropriate primary or salvage surgical therapy. PMID- 16676930 TI - Primary spinal syringomyelia: a personal perspective. AB - In this paper the author summarizes currently available surgical approaches to spinal syringomyelia that is unrelated to Chiari I malformation or hindbrain descent. Primary spinal syringomyelia is most commonly associated with spinal trauma but is also encountered as a sequela to intradural inflammatory processes (infections or chemical), as a delayed response to surgical procedures, and in association with intra- and extradural neoplasms as well as disc protrusions. The advantages of placing a shunt are its technical simplicity and immediate reduction of syrinx size; its major disadvantages are the high rate of failure observed in long-term follow up and the difficulty in applying this technique in septated cysts. Expansion of the subarachnoid space with resection of scars has better long-term results. Patients in whom a syrinx cavity has caused a kyphotic spinal deformity may need to undergo a procedure in which the kyphotic deformity is corrected to expand the subarachnoid space. Cyst obliteration is an experimental approach that cannot be evaluated at the present time. PMID- 16676932 TI - Treatment of terminal syringomyelia in association with tethered cord syndrome: clinical outcomes with and without syrinx drainage. AB - OBJECT: Current use of magnetic resonance (MR) imaging has led to increased awareness of the frequency of terminal syringomyelia in patients with tethered cord syndrome. However, that the surgical treatment of terminal syringomyelia is necessary remains unclear. In this study the authors attempted to assess the clinical impact, if any, brought after syrinx decompression on the clinical outcome of tethered cord syndrome. METHODS: They randomly assigned 30 cases of pediatric tethered cord into two treatment groups: those in whom an untethering procedure was performed (Group I) and those in whom this procedure was combined with syrinx decompression (Group II). The 1-year follow-up clinical results obtained in the two groups, in correlation with MR imaging findings, were compared to evaluate the benefit of added syrinx drainage. Clinical follow-up evaluation revealed that surgical drainage of the syrinx, when combined with spinal cord untethering, resulted in better outcomes in terms of resolution of sensory deficits (p = 0.036) and bladder dysfunction (p = 0.05). The improvement in clinical outcome correlated with the radiologically documented resolution of the syrinx cavity; however, response rates of symptoms differed for each tethering subgroup. CONCLUSIONS: Preliminary results of this study indicated that terminal syringomyelia should be considered as a comorbidity that contributes to the clinical outcome of patients with tethered cord syndrome. A better clinical outcome is achieved following successful decompression of the syrinx in addition to untethering the spinal cord. These findings emphasize the importance of recognizing, evaluating, and treating this pathological entity. PMID- 16676931 TI - Treatment of posttraumatic syringomyelia with extradural decompressive surgery. AB - The authors review the management of five patients with posttraumatic syringomyelia (PTS) associated with an uncorrected spinal deformity. Patients with evidence of progressive neurological deterioration underwent ventral spinal decompressive surgery. The mean patient age at the time of injury was 39 years, and the time between injury and the diagnosis of PTS ranged from 2 to 22 years. Mechanisms of injury consisted of fracture/subluxations in three patients and burst fractures in two. All patients experienced delayed neurological deterioration consistent with PTS. Magnetic resonance imaging revealed ventral deformities, and the spinal canal stenosis ranged from 20 to 50% (mean 39%). All patients underwent ventral epidural spinal decompressive surgery to correct the bone deformity and restore the spinal canal. The mean follow-up period was 38 months. The decompressive intervention was initially successful in treating the neurological deterioration in all patients. Symptoms resolved completely in four patients, and the other experienced neurological improvement. Postoperative magnetic resonance imaging revealed a reduction in the size of syrinx cavity in the patients whose symptoms resolved and no change in the remaining patient. Two patients required a subsequent second-stage posterior intradural exploration and duraplasty for recurrence of symptoms and/or syrinx. Posttraumatic spinal deformity may cause spinal canal stenosis and alter subarachnoid cerebrospinal fluid (CSF) flow in certain patients. Ventral epidural spinal decompressive surgery may result in neurological improvement and a reduction of the syrinx cavity, avoiding the need for placement of a shunt or other intradural procedures. However, some patients will also require reconstruction of the posterior subarachnoid space with duraplasty if the ventral decompressive procedure achieves only partial restoration of the subarachnoid CSF flow. PMID- 16676933 TI - Crystal structures of extended europium cyanamide-carbodiimide compounds derived from different reaction conditions: temperature-controlled syntheses of In(0.08)Eu4(NCN)3I3, Eu8I9(CN)(NCN)3, and In(0.28)Eu12(NCN)5I(14.91). AB - A different thermal treatment of identical reactants (EuI2, NaCN, NaN3, and InI) leads to the formation of the three title compounds. In(0.08)Eu4(NCN)3I3 is isotypic with the reported LiEu4(NCN)3I3, Eu8I9(CN)(NCN)3 represents the first mixed cyanide-cyanamide rare-earth compound, and In(0.28)Eu12(NCN)5I(14.91) is characterized by a sandwich-like stacking motif involving Eu4-NCN double layers stuffed by a layer of vertex-sharing InI6 octahedra. The redox behavior of In is the main factor that leads to alternative product formation as a function of the temperature. PMID- 16676934 TI - Metal salen derivatives as catalysts for the alternating copolymerization of oxetanes and carbon dioxide to afford polycarbonates. AB - Metal salen derivatives of chromium and aluminum, along with n-Bu4NX (X = Cl or N3) salts, have been shown to be effective catalysts for the selective coupling of CO2 and oxetane (trimethylene oxide) to provide the corresponding polycarbonate with only trace quantities of ether linkages. The formation of copolymer is suggested, based on circumstantial evidence, not to proceed via the intermediacy of trimethylene carbonate, which was observed as a minor product of the coupling reaction. For a reaction catalyzed by (salen)CrCl in the presence of n-Bu4NN3 as the cocatalyst, both matrix-assisted laser desorption ionization time of-flight mass spectrometry and infrared spectroscopy revealed an azide end group in the copolymer. PMID- 16676935 TI - Mechanism of water exchange in aqueous uranyl(VI) ion. A density functional molecular dynamics study. AB - Constrained Car-Parrinello molecular dynamics simulations and thermodynamic integration have been performed for an associative pathway of water exchange between aqueous [UO2(OH2)5]2+ and bulk water. The simulated free energy of activation for this process, 6.7 kcal mol(-1), is significantly lower than that computed for a purely dissociative mechanism, 10.8 kcal mol(-1). Because the transient hexahydrate is indicated to have no chemically significant lifetime, the exchange mechanism can be classified as associative interchange. PMID- 16676936 TI - Polymorphic alternating HNN-cobalt(II) chains both behaving as single-chain magnets (HNN = 4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide). AB - Metal-radical alternating chains [Co(hfac)2HNN]n crystallized in two morphs containing repeating all-cis (alpha) and cis,cis,trans (beta) configurations with respect to the O(HNN)-Co-O(HNN) geometry (HNN = 4,4,5,5-tetramethylimidazolin-1 oxyl 3-oxide). Both phases showed magnetization jumps with hysteresis at 2 K. The alpha phase has a relatively high activation energy of magnetization reorientation (193 K). PMID- 16676937 TI - Tripodal bis(imidazole) thioether copper(I) complexes: mimics of the Cu(B) site of hydroxylase enzymes. AB - Tripodal bis(imidazole) thioether ligands and the corresponding copper(I) complexes [(BIMT-OR)Cu(L)]PF6 [L = CH3CN (2), CO (3); R = H (a), CH3 (b)] have been prepared as models for the Cu(B) site of copper hydroxylase enzymes. The IR (CO) values of 3a and 3b (L = CO) are comparable to those of the carbonylated enzymes. The reaction of 2a with O2 gives dinuclear complex 4 with bridging BIMT O ligands and oxidized -SMe groups, whereas oxygenation of 2b affords [(BIMT OMe)2Cu2O(H)2](CF3SO3)2 (5) and Cu(BIMT-OMe)(DMF)2](PF6)2 (6). PMID- 16676939 TI - Particular topological complexity of lead oxide blocks in Pb31O22X18 (X = Br, Cl). AB - Dark-green platy crystals of the new compound Pb31O22Br10Cl8 (1) have been obtained by rapid quenching of a lead oxide halide melt. The structure of 1 (triclinic, P1, a = 12.1192(7) angstroms, b = 16.2489(10) angstroms, c = 18.3007(11) angstroms, alpha = 93.104(2) degrees, beta = 95.809(2) degrees, gamma = 111.252(1) degrees, V = 3325.4(3) angstroms3, Z = 2) can be viewed as incorporation of [PbX6]4- halide units (X = Br, Cl) into the defect PbO matrix. The latter represents a two-dimensional [O22Pb30]16+ cationic layer of OPb4 tetrahedra that can be derived from the [OPb] tetrahedral layer observed in tetragonal PbO. The layer consists of 22 symmetrically inequivalent OPb4 tetrahedra and represents the topologically most complicated arrangement of tetrahedra known to date. PMID- 16676938 TI - Decarboxylation of 2,2'-bipyridinyl-4,4'-dicarboxylic acid diethyl ester during microwave synthesis of the corresponding trichelated ruthenium complex. AB - Microwave reaction of RuCl3 with 2,2'-bipyridinyl-4,4'-dicarboxylic acid diethyl ester (debpy) in ethylene glycol generated Ru(bpy)3(2+) instead of the expected Ru(debpy)3(2+). Gas chromatography-mass spectrometry analysis of the headspace revealed CO2, and Ru(bpy)3(2+) was recovered from the filtrate. Further experiments suggest that RuCl3 decarboxylates debpy during microwave synthesis. PMID- 16676940 TI - First crystal structure studies of CaAlH5. AB - A new member of the aluminum hydride family, CaAlH5, is formed during the decomposition of Ca(AlH4)2. The crystal structure of this new compound was calculated by density functional theory band-structure calculations and confirmed by X-ray powder diffraction analysis. The structure crystallizes in space group P2(1)/n (No. 14), with a = 8.3797(9) angstroms, b = 6.9293(8) angstroms, c = 9.8138(11) angstroms, beta = 93.78(1) degrees, and Z = 8. PMID- 16676941 TI - Bis[iridium(I)] complex of inverted N-confused porphyrin. AB - The reaction of N-confused tetraphenylporphyrin with IrCl(CO)2(p-toluidine) gave a novel bis[iridium(I)] complex, wherein the confused pyrrole ring took an inverted conformation. PMID- 16676942 TI - Interweaving of triple-helical and extended metal-O-metal single-helical chains with the same helix axis in a 3D metal-organic framework. AB - A novel 3D metal-organic framework [Cd2(m-bptc)(4,4'-bpy)(0.5)(H2O)4].H2O (1; m H4bptc = 1,1'-biphenyl-2,3',3,4'-tetracarboxylic acid, 4,4'-bpy = 4,4' bipyridine) with interweaving of triple- and single-helical chains has been obtained based on hydro(solvo)thermal reactions. PMID- 16676943 TI - Synthesis and structure of dilacunary decatungstogermanate, [gamma-GeW10O36]8-. AB - The dilacunary decatungstogermanate [gamma-GeW10O36]8- (1) has been synthesized and structurally characterized in solution and in the solid state. Reaction of germanium dioxide with sodium tungstate in aqueous acidic medium results in the formation of [beta2-GeW11O39]8- (2), which is then used as a precursor for the synthesis of 1. The (183)W spectrum of 2 shows the expected 11 peaks of equal intensity, whereas that of 1 exhibits the expected three peaks with relative intensities 2:2:1. Polyanion 1 represents a novel lacunary polyoxometalate, giving rise to a multitude of derivatives by reaction with transition metals, lanthanides, and other electrophiles. PMID- 16676944 TI - Synthesis and structure of K3Mg20In14, a stuffed variant of the BaHg11 structure type with a magnesium-indium network. AB - The phase K3Mg20In14 was synthesized via high-temperature reactions of the elements in welded Ta tubes. The cubic crystal structure established by single crystal X-ray diffraction means [space group Pm3m, Z = 1, a = 9.769(1) angstroms] features a 3D Mg-In network formed by K@Mg12In10 units plus cuboctahedral fillers, In@Mg12. This is the first example of a well-ordered stuffed BaHg11 structure (Pearson symbol cP37). On the basis of tight-binding linear muffin-tin orbital, atomic sphere approximation calculations, the electronic structure of the compound shows dominant Mg-In interactions and substantial participation of Mg in the overall network bonding. Both In-In and Mg-In bondings are effectively optimized at the Fermi level. The Fermi energy cuts through substantial densities of states, consistent with the measured metallic property. PMID- 16676945 TI - Thallophilic interactions in aryloxide compounds: the [Tl2(mu2-OAr)4] structural motif in (TlOAr)4 and Tl2Cu(OAr)4 compounds. AB - Two thallium aryloxide compounds TlOC6F5 (TlOAr(F)) and bis-3,5-TlOC6H3(CF3)2 (TlOAr') have been recrystallized from THF and crystallographically characterized in different isomeric forms. The latter compound forms a solvated tetrameric cubane, [TlOAr']4.THF, 1. The TlOAr(F) compound crystallized with a similar stoichiometry, [TlOAr(F)]4.2 THF, 2, but contains a [Tl2(mu2-OAr(F))4] unit that includes a thallophilic interaction at a distance of 3.5943(15) angstroms. Solution 205Tl and 203Tl NMR studies of 1 and 2 support the retention of a cubane structure for 1 in solution and suggest a similar structure for 2 with coupled thallium centers down to -90 degrees C. Fluorescence spectroscopy data for both compounds 1 and 2 in THF are consistent with LMCT. DFT calculations of 1, 2, and three models of the [Tl2(mu2-OAr(F))4] unit show a bonding overlap of the bridged thallium atoms in 2 and are also used to describe the bonding in 1. The structures of two heterobimetallic compounds, Tl2Cu(OAr(F))4, 4, and Tl2Cu(OAr')4, 5, with the [Tl2(mu2-OAr(F))4] structural motif and thallophilic contacts of 3.86(6) and 3.564(1) angstroms, respectively, are described. The crystal structures of the unsolvated of TlOAr(F), 2b, solvated heterobimetallic derivative Tl2Cu(OAr')4.2THF, 5b, and the monomeric (18-crown-6)TlOAr(F), 3, and 205Tl NMR spectra of TlOC6H5, 6, are also reported for comparison purposes. PMID- 16676946 TI - Toward a more general synthetic route to paramagnetic solids containing RCNSSS*+ radical cations. A structure-property correlation for RCNSSS*+ (R = F5C2, Cl3C). AB - Reaction of Cl3CN and F5C2CN with a 1:1 mixture of S4(AsF6)2 and S8(AsF6)2 affords the paramagnetic solids Cl3CNSSSAsF6 (1CCl3AsF6) and F5C2CNSSSAsF6 (1C2F5AsF6). Isotropic electron paramagnetic resonance spectra of 1CCl3AsF6 and 1C2F5AsF6 in SO2 consist of a single line with g = 2.01675 and 2.01580, respectively. The structure of 1CCl3AsF6 contains chains of radical cations with relatively close interchain interactions. In contrast, chains are isolated in 1C2F5AsF6. The magnetic behavior of both compounds was interpreted as that of 1D Heisenberg antiferromagnetic chains (1CCl3AsF6, J = -34 cm(-1), theta = -9 cm( 1), TIP = 0.00082, rho = 0.012; 1C2F5AsF6, J = -21 cm(-1), theta = -4.2 cm(-1), TIP = 0.00092, rho = 0.065). Density functional theory calculated and experimental magnetic coupling constants were in good agreement. The correlation between intermolecular S...S contacts and the strength of magnetic couplings was established. PMID- 16676947 TI - Local structure of actinide dioxide solid solutions Th(1-x)U(x)O2 and Th(1 x)Pu(x)O2. AB - Extended X-ray absorption fine structure (EXAFS) has been utilized to investigate the local atomic structure around Th, U, and Pu atoms in polycrystalline mixed dioxides Th(1-x)M(x)O2 (with M = U, Pu) for x ranging from 0 to 1. The composition dependence of the two first-coordination-shell distances was measured throughout the entire composition range for both solid solutions. The first-shell distances vary slightly across the solid-solution composition with values close to those of the pure dioxide parents, indicating a bimodal cation-oxygen distribution. In contrast, the second-shell distance varies strongly with composition, with values close to the weighted amount average distances. Nevertheless, in both systems, the lattice cell parameters, deduced from the first- and second-shell bond determined by EXAFS, are very close to those measured from X-ray diffraction (XRD). They vary linearly with composition, accurately following Vegard's law. PMID- 16676948 TI - Structural and photophysical properties of coordination networks combining [Ru(Bpym)(CN)4]2- or [[Ru(CN)4]2(mu-bpym)]4- anions (bpym = 2,2'-bipyrimidine) with lanthanide(III) cations: sensitized near-infrared luminescence from Yb(III), Nd(III), and Er(III) following Ru-to-lanthanide energy transfer. AB - Reaction of the cyanoruthenate anions [Ru(bpym)(CN)4]2- and [[Ru(CN)4]2(mu bpym)]4- (bpym = 2,2'-bipyrimidine) with lanthanide(III) salts resulted in the crystallization of coordination networks based on Ru-CN-Ln bridges. Four types of structure were obtained: [Ru(bpym)(CN)4][Ln(NO3)(H2O)5] (Ru-Ln; Ln = Sm, Nd, and Gd) are one-dimensional helical chains; [Ru(bpym)(CN)4]2[Ln(NO3)(H2O)2][Ln(NO3)(0.5)(H2O)(5.5)](NO3)(0.5).5.5H2O (Ru-Ln; Ln = Er and Yb) are two-dimensional sheets containing cross-linked chains based on Ru2Ln2(mu-CN)4 diamond units, which are linked into one-dimensional chains via shared Ru atoms; [[Ru(CN)4]2(mu-bpym)][Ln(NO3)(H2O)5]2.3H2O (Ru2-Ln; Ln = Nd and Sm) are one-dimensional ladders with parallel Ln-NC-Ru-CN-Ln-NC strands connected by the bipyrimidine "cross pieces" acting as rungs on the ladder; and [[Ru(CN)4]2(mu-bpym)][Ln(H2O)6](0.5)[Ln(H2O)4](NO3)(0.5).nH2O (Ru2-Ln; Ln = Eu, Gd, and Yb; n = 8.5, 8.5, and 8, respectively) are three-dimensional networks in which two-dimensional sheets of Ru2Ln2(mu-CN)4 diamonds are connected via cyanide bridges to Ln(III) ions between the layers. Whereas Ru-Gd shows weak triplet metal-to-ligand charge-transfer (3MLCT) luminescence in the solid state from the Ru-bipyrimidine chromophore, in Ru-Nd, Ru-Er, and Ru-Yb, the Ru-based emission is quenched, and all of these show, instead, sensitized lanthanide-based near-IR luminescence following a Ru --> Ln energy transfer. Similarly, Ru2-Nd and Ru2-Yb show lanthanide-based near-IR emission following excitation of the Ru bipyrimidine chromophore. Time-resolved luminescence measurements suggest that the Ru --> Ln energy-transfer rate is faster (when Ln = Yb and Er) than in related complexes based on the [Ru(bipy)(CN)4]2- chromophore, because the lower energy of the Ru-bpym 3MLCT provides better spectroscopic overlap with the low energy f-f states of Yb(III) and Er(III). In every case, the lanthanide-based luminescence is relatively short-lived as a result of the CN oscillations in the lattice. PMID- 16676949 TI - Coordination chemistry of tetradentate N-donor ligands containing two pyrazolyl pyridine units separated by a 1,8-naphthyl spacer: dodecanuclear and tetranuclear coordination cages and cyclic helicates. AB - The tetradentate ligand L(naph) contains two N-donor bidentate pyrazolyl-pyridine units connected to a 1,8-naphthyl core via methylene spacers; L45 and L56 are chiral ligands with a structure similar to that of L(naph) but bearing pinene groups fused to either C4 and C5 or C5 and C6 of the terminal pyridyl rings. The complexes [Cu(L(naph))](OTf) and [Ag(L(naph))](BF4) have unremarkable mononuclear structures, with Cu(I) being four-coordinate and Ag(I) being two-coordinate with two additional weak interactions (i.e., "2 + 2" coordinate). In contrast, [Cu4(L(naph))4][BF4]4 is a cyclic tetranuclear helicate with a tetrafluoroborate anion in the central cavity, formed by an anion-templating effect; electrospray mass spectrometry (ESMS) spectra show the presence of other cyclic oligomers in solution. The chiral ligands show comparable behavior, with [Cu(L45)](BF4) and [Ag(L45)](ClO4) having similar mononuclear crystal structures and with the ligands being tetradentate chelates. In contrast, [Ag4(L56)4](BF4)4 is a cyclic tetranuclear helicate in which both diastereomers of the complex are present in the crystal; the two diastereomers have similar gross geometries but are significantly different in detail. Despite their different crystal structures, [Ag(L45)](ClO4) and [Ag4(L56)4](BF4)4 behave similarly in solution according to ESMS studies, with a range of cyclic oligomers (up to Ag9L9) forming. With transition-metal dications Co(II), Cu(II), and Cd(II), L(naph) generates a series of unusual dodecanuclear coordination cages [M12(L(naph))18]X24 (X- = ClO4- or BF4-) in which the 12 metal ions occupy the vertices of a truncated tetrahedron and a bridging ligand spans each of the 18 edges. The central cavity of each cage can accommodate four counterions, and each cage molecule is chiral, with all 12 metal trischelates being homochiral; the crystals are racemic. Extensive aromatic stacking between ligands around the periphery of the cages appears to be a significant factor in their assembly. The chiral analogue L45 forms the simpler tetranuclear, tetrahedral coordination cage [Zn4(L45)6](ClO4)(8), with one anion in the central cavity; the steric bulk of the pinene chiral auxiliaries prevents the formation of a dodecanuclear cage, although trace amounts of [Zn12(L45)18](ClO4)24 can be detected in solution by ESMS. Formation of [Zn4(L45)6](ClO4)8 is diastereoselective, with the chirality of the pinene groups controlling the chirality of the tetranuclear cage. PMID- 16676950 TI - A new structural motif for biological iron: iron K-edge XAS reveals a [Fe4-mu (OR)5(OR)(9-10)] cluster in the ascidian Perophora annectens. AB - The Phlebobranch ascidian Perophora annectens surprisingly exhibited a biological Fe/V ratio of approximately 15:1 on multichannel X-ray fluorescence analysis of two independent collections of organisms. Iron K-edge X-ray absorption spectroscopy (XAS) indicated a single form of iron. The XAS K-edge of the first collection of blood cells was shifted approximately +1 eV relative to that of the second, indicating redox activity with average iron oxidation states of 2.67+ and 2.60+. The first-derivative iron XAS K-edge features at 7120.5, 7124, and 7128 eV resembled the XAS of magnetite but not of ferritin or of dissolved Fe(II) or Fe(III). Pseudo-Voigt fits to blood-cell iron K-edge XAS spectra yielded 12.4 integrated units of preedge intensity, indicating a noncentrosymmetric environment. The non-phase-corrected extended X-ray absorption fine structure (EXAFS) Fourier transform spectrum showed a first-shell O/N peak at 1.55 angstroms and an intense Fe-Fe feature at 2.65 angstroms. Fits to the EXAFS required a split first shell with two O at 1.93 angstroms and three O at 2.07 angstroms, consistent with terminal and bridging alkoxide ligands, respectively. More distant shells included three C at 2.87 angstroms, two Fe at 3.08 angstroms, three O at 3.29 angstroms, and one Fe at 3.8 angstroms. Structural models consistent with these findings include a [Fe4(OR)13](2-/3-) broken-edged Fe4O5 cuboid or a [Fe4(OR)14](3-/4-) "Jacob's ladder" with three edge-fused Fe2(OR)2 rhombs. Either of these models represents an entirely new structural motif for biological iron. Vanadium domination of blood-cell metals cannot be a defining trait of Phlebobranch tunicates so long as P. annectens is included among them. PMID- 16676951 TI - Structure and magnetism of [M3](6/7+) metal chain complexes from density functional theory: analysis for copper and predictions for silver. AB - The ground-state electronic structure of the trinuclear complex Cu3(dpa)4Cl2 (1), where dpa is the anion of di(2-pyridyl)amine, has been investigated within the framework of density functional theory (DFT) and compared with that obtained for other known M3(dpa)4Cl2 complexes (M = Cr, Co, Ni) and for the still hypothetical Ag3(dpa)4Cl2 compound. Both coinage metal compounds display three singly occupied x2-y2-like (delta) orbitals oriented toward the nitrogen environment of each metal atom, generating antibonding M-(N4) interactions. All other metal orbital combinations are doubly occupied, resulting in no delocalized metal-metal bonding. This is at variance with the other known symmetric M3(dpa)4Cl2 complexes of the first transition series, which all display some delocalized bonding through the metal backbone, with formal bond multiplicity decreasing in the order Cr > Co > Ni. An antiferromagnetic coupling develops between the singly occupied MOs via a superexchange mechanism involving the bridging dpa ligands. This magnetic interaction can be considered as an extension to the three aligned Cu(II) atoms of the well-documented exchange coupling observed in carboxylato bridged dinuclear copper compounds. Broken-symmetry calculations with approximate spin projection adequately reproduce the coupling constant observed for 1. Oxidation of 1 removes an electron from the magnetic orbital located on the central Cu atom and its ligand environment; 1+ displays a much weaker antiferromagnetic interaction coupling the terminal Cu-N4 moieties via four ligand pathways converging through the x2-y2 orbital of the central metal. The silver homologues of 1 and 1+ display similar electronic ground states, but the calculated magnetic couplings are stronger by factors of about 3 and 4, respectively, resulting from a better overlap between the metal centers and their equatorial ligand environment within the magnetic orbitals. PMID- 16676952 TI - Metal-organic architectures of silver(I), cadmium(II), and copper(II) with a flexible tricarboxylate ligand. AB - Three novel metal-organic architectures, [Ag3(bta)].1.5H2O (1), [Cd3(bta)2(H2O)7].5H2O (2), and [Cu11(bta)6(Hbta)2(H2O)10].29H2O (3), were obtained by reactions of the corresponding metal salts with a flexible tripodal ligand, benzene-1,3,5-triacetic acid (H3bta), and their structures were determined by single-crystal X-ray diffraction studies. The results revealed that, in complexes 1 and 2, the carboxylate groups of the bta3- ligand adopted varied coordination modes to link metal atoms and further to form three dimensional structures with open channels occupied by water molecules, while in complex 3, for the first time, the flexible H3bta acted as a secondary building unit to generate a novel nanometer-sized metallocage, which is composed of a Cu(II) paddle wheel (square secondary building units) and bta3-/Hbta2- organic links (triangular secondary building units). The photoluminescence properties of complexes 1 and 2 were investigated, and the results showed that 2 exhibited photoluminescence in the solid state at room temperature. PMID- 16676953 TI - Construction of 3d-4f mixed-metal complexes based on a binuclear oxovanadium unit: synthesis, crystal structure, EPR, and magnetic properties. AB - The oxovanadium(IV)-lanthanide(III) heteronuclear complexes, [[Ce(H2O)7(VO)(TTHA)0.5][(VO)2(TTHA)]].8H2O (2), [Pr(H2O)7(VO)3(TTHA)1.5].10H2O (3), and [Nd(H2O)7(VO)3(TTHA)(1.5)].10H2O (4) (H6TTHA = triethylenetetraaminehexaacetic acid), were prepared based on a binuclear building block of [(VO)2(TTHA)]2- in [VO(H2O)5][(VO)2TTHA].4H2O (1). The X-ray crystallographic studies show that 1 is an ion-pair complex, containing the [(VO)2(TTHA)]2- unit as a useful building block. Adding the light Ln3+ ions to this synthesis system, three new 3d-4f mixed-metal-based complexes were obtained. Although the light lanthanide ions always exhibit similar chemical behavior, the structures of 2-4 are not homologous. 2 is exhibited as a one-dimensional coordination polymer, comprising an unusual Ce2V2 heterometallic lattice in the chain structure, which is the second report of a oxovanadium(IV)-lanthanide(III) coordination polymer. 3 and 4 are isomorphic, every two of the Ln3+ cations linked three [(VO)2(TTHA)]2- anions, forming an interesting linear octanuclear structure. This kind of heteronuclear linear complex is rather rare, which expands the realm of 3d-4f complexes. Further investigations such as IR spectra, UV-vis spectra, magnetic properties, and EPR spectra were studied, and a detailed discussion is given for this system. PMID- 16676954 TI - A computational study of the nondissociative mechanisms that interchange apical and equatorial atoms in square pyramidal molecules. AB - The lowest energy transition state for the nondissociative apical/equatorial atom exchange mechanism for three square pyramidal AEX5 molecular species was calculated (CCSD(T)/pVTZ; B3LYP/pVTZ, aug-cc-pV5Z) to have a hemidirected geometry with C(s) symmetry for BrF5, IF5, and XeF5+. In contrast, holodirected C2v-symmetric transition states for this process were located for the AEX5 square pyramidal molecules ClF5, ICl5, and IBr5. Imaginary frequencies were calculated and examined in a visual/dynamic fashion to gain insight into these fluxional processes. Although both mechanisms exchange one apical for one equatorial atom in each cycle of motion, processes that pass through C2v transition states have characteristic features of the well-known Berry pseudorotation and Lever mechanisms while those which pass through transition states of C(s) symmetry have features that are a mixture of Berry, Lever, and turnstile-like character. Two periodic trends are observed: as the atomic number on the central atom increases (same terminal atoms), the barrier for apical/equatorial exchange and the value of the imaginary frequency both decrease. Similarly, as the atomic number of the terminal atoms increase (same central atom), the barrier for apical/equatorial exchange decreases, as does the computed imaginary frequency. PMID- 16676955 TI - Tetrahedral Co(II) coordination in alpha-type cobalt hydroxide: Rietveld refinement and X-ray absorption spectroscopy. AB - We report a Rietveld refinement analysis and X-ray absorption study on a green color Cl(-)-intercalated alpha-type cobalt hydroxide phase. The refinement clearly demonstrated that one-fifth to one-sixth of the Co(II) at octahedral sites was replaced by pairs of tetrahedrally coordinated Co(II) on each side of the hydroxide plane, represented by a structural formula of [Co(octa)(0.828)Co(tetra)(0.348)(OH)2](0.348+)Cl(0.348).0.456H2O. X-ray absorption spectroscopy also indicated that the divalent cobalt were in local neighboring environments of both octahedral and tetrahedral coordination. Furthermore, UV-vis spectroscopic measurements elucidate the typical green/blue color of an alpha-type cobalt hydroxide. PMID- 16676957 TI - 2D hydrogen-bonded square-grid coordination networks with a substitution-active metal site. AB - Reported here is the preparation and property of 2D coordination networks composed of rodlike ligands with ethylene glycol side chains (1). Two 2D coordination networks, [[Co(1)2(H2O)2](NO3)2.1.5H2O]n and [[Ni(1)2(H(2)O)2](NO3)2.1.5H2O]n, have been synthesized and characterized by single-crystal X-ray diffraction, TG, DSC, UV-vis spectroscopy, and magnetic measurements. The structural analyses clarified that infinite 1D hydrogen-bond arrays composed of ethylene glycol chains contribute to the stabilization of 2D coordination frameworks, keeping the environment of substitution-active metal sites unchanged. They are more stable than a similar square-grid coordination network that does not possess an ethylene glycol chain on the ligand. We also succeeded in the direct observation of a reversible apical-ligand-exchange reaction at the cobalt(II) and nickel(II) ions in a single-crystal-to-single crystal fashion because of the considerable stability as well as moderate flexibility of the framework. The cobalt-containing coordination network crystal showed chromic behavior depending on temperatures. Crystallographic and spectroscopic studies revealed that the color change of the crystal was attributed to the ligand-exchange process between H2O and a NO3 anion on the cobalt metal. Magnetic measurements indicated weak antiferromagnetic nearest neighbor spin coupling between cobalt(II) ions. PMID- 16676956 TI - Aluminum phosphinate and phosphates of salen ligands. AB - A new dealkylation reaction between organophosphate esters and Salen aluminum bromide compounds has been used to prepare three new aluminum salen compounds salen((t)Bu)AlOP(O)Ph2 (1) (salen = N,N'-ethylenebis(3,5-di-tert butylsalicylideneimine)), [(MeOH)Alsalen((t)Bu)[OMePO2(O)]Alsalen((t)Bu)[OMePO2(O)]Alsalen((t)Bu)]Br (2), and [salpen((t)Bu)AlO]2[(BuO)2PO]2 (3) (salpen = N,N'-propylenebis(3,5-di-tert butylsalicylideneimine)). Compounds 1.MeOH, 2, and 3 were characterized by single crystal X-ray diffraction. Compound 1 is the first example of a monomeric aluminum Schiff base phosphinate. Compound 2 is a cationic Salen aluminum phosphate, and compound 3 contains an aluminophosphate ring. This work is the first example of the intentional use of an aluminum-based dealkylation reaction to form new compounds. PMID- 16676958 TI - Porphyrazines peripherally functionalized with hybrid ligands as molecular scaffolds for bimetallic metal-ion coordination. AB - We report the synthesis and physical characterization of a new family of peripherally functionalized porphyrazine (pz) compounds, denoted 1[M1, M2], where metal ion M1 is incorporated into the pz core and metal ion M2 is bound to a salicylidene/picolinamide "hybrid" chelate built onto two nitrogen atoms attached to the pz periphery. The complexes 1[MnCl, Cu], 1[VO, Cu], and 1[Cu, Cu] have been prepared, and crystal structures show 1[MnCl, Cu] and 1[VO, Cu] to be isostructural. These complexes have been subjected to electron paramagnetic resonance and temperature-dependent magnetic susceptibility measurements. The variation of the ligand-mediated exchange splittings (delta) in these complexes is striking: delta/k(B) values for 1[MnCl, Cu] and 1[VO, Cu] are 22 and 40 K, respectively, while delta/k(B) for 1[Cu, Cu] is only 1 K. These coupling results are explained in terms of the relative orientation of the M1 and M2 orbitals and reflect the fact that the ligand set of M2 in the periphery is rotated in-plane by 45 degrees relative to the effectively coplanar pz ligand set of M1. The exchange couplings are essentially the same as those we determined for the Schiff base porphyrazines (pzs). Thus, the hybrid ligand has eliminated the dimerization found to occur when Cu(II) is bound to the periphery of bis(picolinamido) pzs and has created a more robust ligand system than the Schiff base pzs while retaining the ability they show to promote spin coupling between M1 and M2. PMID- 16676959 TI - Structural and spectroscopic characterization of a diruthenium o-dioxolene complex possessing a singly occupied molecular orbital delocalized over the entire molecule, [Ru2(3,6-DTBDiox)4]-. AB - Chemical oxidation of [Ru2(6+)(3,6-DTBCat)4]2- affords [n-Bu4N][Ru2(3,6 DTBDiox)4].acetone (2.acetone). The oxidized species was characterized by X-ray crystallographic analysis and EPR, and the delocalization of the unpaired electron over the entire molecule was indicated. This example showed that the utilization of redox-active ligands into a Ru2 complex expanded the degree of freedom in the electronic structure. DFT calculations support this view, and the spin population was estimated to be approximately 18% and 82% for the Ru2 core and the four dioxolene ligands, respectively. PMID- 16676960 TI - Controllable assembly of metal-directed coordination polymers under diverse conditions: a case study of the M(II)-H3tma/Bpt mixed-ligand system. AB - A series of new metal-organic polymeric complexes, [[Co(bpt)(Htma)(H2O)3].2.25H2O]n (1), [Co(bpt)(Htma)(H2O)]n (2), [Ni(bpt)(Htma)(H2O)]n (3), [Zn(bpt)2(H2tma)2].6H2O (4), [[Cd(bpt)(Htma)(H2O)].(C2H5OH)(H2O)1.5]n (5), and [[Cd(bpt)(Htma)(H2O)2].5.5H2O]n (6), was prepared from solution reactions of 4-amino-3,5-bis(4-pyridyl)-1,2,4 triazole (bpt) and trimesic acid (H3tma) with different metal salts under diverse conditions. All these compounds were structurally determined by X-ray single crystal diffraction, and the bulk new materials were further identified by X-ray powder diffraction. Complexes 1 and 6 show 1-D zigzag or linear Htma-bridged polymeric chains, with the terminal bpt ligands as pendants, which are extended to 2-D hydrogen-bonded arrays with 4.8(2) or (6,3) network topology. Coordination polymers 2 and 3, in which the 2-D corrugated metal-organic frameworks make the interdigitated 3-D packing, are isostructural. Complex 4 has a mononuclear structure, and its subunits are hydrogen-bonded to each other to give a 2-D grid like net. For complex 5, the Cd(II) centers are linked by bpt/Htma ligands to form a 2-D (4,4) coordination layer, and these layers are interdigitated in pairs. Notably, secondary noncovalent forces, such as hydrogen bonds, play an important role in extending and stabilizing these structural topologies. Interestingly, distinct products are obtained for Co(II) (1 and 2) and Cd(II) (5 and 6) under ambient or hydrothermal conditions; however, for Ni(II) and Zn(II), single products, 3 and 4, are generated. The thermal stabilities of 1-6 were studied by thermogravimetric analysis of mass loss. The desorption/adsorption properties of the porous material 5 are also discussed. Solid-state luminescent spectra of the Zn(II) and Cd(II) complexes, 4-6, indicate intense fluorescent emissions at ca. 380 nm. PMID- 16676961 TI - Effects of axial coordination on the Ru-Ru single bond in diruthenium paddlewheel complexes. AB - The 1,8-naphthyridine-based (NP-based) ligands with furyl, thiazolyl, pyridyl, and pyrrolyl attachments at the 2-position have been synthesized. Reactions of 3 MeNP (3-methyl-1,8-naphthyridine), fuNP (2-(2-furyl)-1,8-naphthyridine), tzNP (2 (2-thiazolyl)-1,8-naphthyridine), pyNP (2-(2-pyridyl)-1,8-naphthyridine), and prNP(-1) (2-(2-pyrrolyl)-1,8-naphthyridine) with [Ru2(CO)4(CH3CN)6]2+ lead to [Ru2(3-MeNP)2(CO)4(OTf)2] (1), [Ru2(fuNP)2(CO)4]2[BF4]2 (2), [Ru2(tzNP)2(CO)4][ClO4]2 (3), [Ru2(pyNP)2(CO)4][OTf]2 (4), and [Ru2(prNP)2(CO)4] (5). The molecular structures of complexes 1-5 have been established by X-ray crystallographic studies. The modulation of the Ru-Ru single-bond distances with axial donors triflates, furyls, thiazolyls, pyridyls, and pyrrolyls has been examined. A small and gradual increase in the Ru-Ru distance is measured with various donors of increasing strengths. The shortest Ru-Ru distance of 2.6071(9) angstroms is observed for the axially coordinated triflates in complex 1, and the longest Ru-Ru distance of 2.6969(10) angstroms is measured for axial pyrrolyls in complex 5. The Ru-Ru distances in complexes 3 (2.6734(7) angstroms) and 4 (2.6792(9) angstroms), having thiazolyls and pyridyls at axial sites respectively, are similar. The Ru-Ru distance for axial furyls in complex 2 (2.6261(9) angstroms) is significantly shorter than the corresponding distances in 3, 4, and 5. DFT calculations provide insight into the interaction of the Ru Ru sigma orbital with axial donors. The Ru-Ru sigma orbital is elevated to a higher energy because of the interaction with axial lone pairs. The degree of destabilization depends on the nature of axial ligands: the stronger the ligand, higher the elevation of Ru-Ru orbital. The lengthening of Ru-Ru distances with respect to the axial donors in compounds 1-5 follows along the direction pyrrolyl > pyridyl approximately thiazolyl > furyl > triflate, and the trend correlates well with the computed destabilization of the Ru-Ru sigma orbitals. PMID- 16676962 TI - Influence of steric hindrance of organic ligand on the structure of Keggin-based coordination polymer. AB - Five Keggin-based 3D coordination polymers, namely, [Cu3(pz)3(PW12O40)] (pz = pyrazine) (1), [Cu3(2,3-Me2pz)3(PW12O40)] (2,3-Me2pz = 2,3-dimethylpyrazine) (2), [Cu2(2,5-Me2pz)(1.5)(2,5-HMe2pz)(PW12O40)] (2,5-Me2pz = 2,5-dimethylpyrazine) (3), [Cu3(2,3-Me2pz)3(PMo12O40)] (4), and [Ag3(pz)3(PW12O40)].0.5H2O (5), were synthesized and structurally characterized. Crystal data are as follows: trigonal, space group R3c, a = 18.4070(14) angstroms, c = 22.544(3) angstroms, gamma = 120 degrees, and Z = 6 for 1; orthorhombic, space group Pccn, a = 16.599(2) angstroms, b = 20.470(3) angstroms, c = 14.3757(18) angstroms, and Z = 4 for 2; triclinic, space group P1, a = 10.667(2) angstroms, b = 11.147(2) angstroms, c = 20.207(4) angstroms, alpha = 90.983(4) degrees, beta = 108.128(3) degrees, gamma = 92.150(4) degrees, and Z = 2 for 3; orthorhombic, space group Pccn, a = 16.450(3) angstroms, b = 20.170(4) angstroms, c = 14.244(3) angstroms, and Z = 4 for 4; and rhombohedral, space group R32, a = 18.2047(13) angstroms, c = 23.637(3) angstroms, gamma = 120 degrees, and Z = 6 for 5. Their structural differences were investigated using crystal structure analysis, revealing that the influence of steric hindrance of organic ligand on the structures of Keggin based coordination polymers is realized through changing the number of metal organic units surrounding the POM anion. PMID- 16676963 TI - Synthesis and photochemistry of a two-position Ru(terpy)(phen)(L)2+ scorpionate complex. AB - A dissymmetric 1,10-phenanthroline chelate (N-phen-S) bearing two polyether chains terminated by two monodentate ligands of the benzonitrile (N) and dialkylesulfoxide (S) types was synthesized, characterized, and coordinated to ruthenium. The corresponding Ru(terpy)(N-phen-S)2+ complexes (terpy = 4'-(3,5 ditertiobutylphenyl)-2,2';6',2' '-terpyridine) were fully characterized as being two coordination isomers of the scorpionate type with one of the two tails occupying the sixth position on the coordination sphere. Photoexpulsion of the coordinated tail led to opening of the ruthena-macrocycle and subsequent rearrangement of the bidentate chelate. This rearrangement consisted of a 90 degrees rotation of the phenanthroline around the ruthenium atom. Selective irradiation of one isomer in a mixture of the two was undertaken using band-pass filters; this resulted in an enrichment of the nonirradiated isomer in the mixture. Thermal back-coordination of the tail was investigated in the dark. It took place quantitatively from the corresponding ruthenium chloride complex by trapping of the anion with silver salts. PMID- 16676964 TI - Increasing structure dimensionality of copper(I) complexes by varying the flexible thioether ligand geometry and counteranions. AB - This work focuses on the systematic investigation of the influences of pyrimidine based thioether ligand geometries and counteranions on the overall molecular architectures. A N-containing heterocyclic dithioether ligand 2,6-bis(2 pyrimidinesulfanylmethyl)pyridine (L1) and three structurally related isomeric bis(2-pyrimidinesulfanylmethyl)benzene (L2-L4) ligands have been prepared. On the basis of the self-assembly of CuX (X = I, Br, Cl, SCN, or CN) and the four structurally related flexible dithioether ligands, we have synthesized and characterized 10 new metal-organic entities, Cu4(L1)2I4 1, Cu4(L1)2Br4 2, [Cu2(L2)2I2.CH3CN]n 3, [Cu(L3)I]n 4, [Cu(L3)Br]n 5, [Cu(L3)CN]n 6, [Cu(L4)CN]n 7, [Cu2(L4)I2]n 8, [Cu2(L4)(SCN)2]n 9, and [[Cu6I5(L4)3](BF4).H2O]n 10, by elemental analyses, IR spectroscopy, and X-ray crystallography. Single-crystal X-ray analyses show that the 10 Cu(I) complexes possess an increasing dimensionality from 0D (1 and 2) to 1D (3-5) to 2D (6-9) to 3D (10), which indicates that the ligand geometry takes an essential role in the framework formation of the Cu(I) complexes. The influence of counteranions and pi-pi weak interactions on the formation and dimensionality of these coordination polymers has also been explored. In addition, the photoluminescence properties of Cu(I) coordination polymers 4-10 in the solid state have been studied. PMID- 16676965 TI - Unusual Mn-Mn spin coupling in the polar intermetallic compounds CaMn2Sb2 and SrMn2Sb2. AB - Large single-crystals of two polar intermetallic phases, CaMn2Sb2 and SrMn2Sb2, have been grown using In or Sn as metal fluxes and characterized by single crystal X-ray diffraction. The two compounds are isostructural and crystallize with the CaAl2Si2 structure (space group P3m1, No. 164) with unit cell parameters determined at 120(2) K of a = 4.5204(6) angstroms, c = 7.456(2) angstroms and a = 4.5802(17) angstroms, c = 7.730(5) angstroms for CaMn2Sb2 and SrMn2Sb2, respectively. Temperature- and field-dependent dc- and ac-magnetization measurements suggest complex magnetic ordering of the Mn moments below ca. 250 and 35 K for CaMn2Sb2 and below ca. 265 K for SrMn2Sb2. Resistivity measurements reveal metallic-like temperature dependence with rho(290) = 40 m omega cm for CaMn2Sb2 and rho290 = 100 m omega cm for SrMn2Sb2 with negligible magnetoresistance at 5 K in applied magnetic fields up to 10 kOe. Spin-polarized DFT electronic structure calculations confirm the metallic-like properties and provide further evidence for a magnetic structure where Mn atoms form two magnetic sublattices with ferromagnetic coupling within them and strong antiferromagnetic coupling between them. PMID- 16676966 TI - Construction of [(eta5-C5Me5)WS3Cu3]-based supramolecular compounds from preformed incomplete cubane-like clusters [PPh4][(eta5-C5Me5)WS3(CuX)3] (X = CN, Br). AB - Approaches to the assembly of (eta5-C5Me5)WS3Cu3-based supramolecular compounds from two preformed incomplete cubane-like clusters [PPh4][(eta5-C5Me5)WS3(CuX)3] (X = CN, 1a; X = Br, 1b) have been investigated. Treatment of 1a with LiBr/1,4 pyrazine (1,4-pyz), pyridine (py), LiCl/py, or 4,4'-bipyridine (4,4'-bipy) and treatment of 1b with 4,4'-bipy gave rise to a new set of W/Cu/S cluster-based compounds, [Li[((eta5-C5Me5)WS3Cu3(mu3-Br))2(mu-CN)3].C6H6]infinity (2), [(eta5 C5Me5)WS3Cu3(mu-CN)2(py)]infinity (3), [[PPh4][(eta5-C5Me5)WS3Cu3(mu3-Cl)(mu CN)(CN)].py]infinity (4), [PPh4]2[(eta5-C5Me5)WS3Cu3(CN)2]2(mu-CN)2.(4,4'-bipy) (5), and [[(eta5-C5Me5)WS3Cu3Br(mu-Br)(4,4'-bipy)].Et2O]infinity (6). The structures of 2-6 have been characterized by elemental analysis, IR spectra, and single-crystal X-ray crystallography. Compound 2 displays a 1D ladder-shaped chain structure built of square-like [[(eta5-C5Me5)WS3Cu3(mu3-Br)(mu-CN)]4](mu CN)2(2-) anions via two pairs of Cu-mu-CN-Cu bridges. Compound 3 consists of a single 3D diamond-like network in which each (eta5-C5Me5)WS3Cu3 unit, serving as a tetrahedral node, interconnects with four other nearby units through Cu-mu-CN Cu bridges. Compound 4 contains a 1D zigzag chain array made of cubane-like [(eta5-C5Me5)WS3Cu3(mu3-Cl)(mu-CN)(CN)]- anions linked by a couple of Cu-mu-CN-Cu bridges. Compound 5 contains a dimeric structure in which the two incomplete cubane-like [(eta5-C5Me5)WS3(CuCN)2(mu-CN)]- anions are strongly held together via a pair of Cu-mu-CN-Cu bridges. Compound 6 contains a 2D brick-wall layer structure in which dimers of [(eta5-C5Me5)WS3Cu3Br(4,4'-bipy)]2 are interconnected via four Cu-mu-Br-Cu bridges. The successful construction of (eta5 C5Me5)WS3Cu3-based supramolecular compounds 2-6 from the geometry-fixed clusters 1a and 1b may expand the scope of the rational design and construction of cluster based supramolecular assemblies. PMID- 16676967 TI - Rare Earth coordination polymers with zeolite topology constructed from 4 connected building units. AB - A series of rare earth coordination polymers, M(BTC)(DMF)(DMSO) (M = Tb (1), Ho (2), Er (3), Yb (4), Y (5)), with zeolite ABW topology have been synthesized under mild conditions. They exhibit the same three-dimensional (3D) architecture and crystallize in monoclinic symmetry space group P2(1)/n. Their structures are built up from inorganic and organic 4-connected building units, whose vertex symbols are 4.4.6.6.6.8. The building units link to each other to generate approximate 5 x 8 angstroms2 channels along the [100] direction. The luminescent and magnetic properties of these compounds are investigated, and the results reveal that they could be anticipated to be potential antiferromagnetic and fluorescent materials. PMID- 16676968 TI - A new family of mono- and dicarboxylic ruthenium complexes [Ru(DIP)2(L2)]2+ (DIP = 4,7-diphenyl-1,10-phenanthroline): synthesis, solution behavior, and X-ray molecular structure of trans-[Ru(DIP)2(MeOH)2][OTf]2. AB - A new family of ruthenium complexes of general formula [Ru(DIP)2(L2)]2+, where DIP = 4,7-diphenyl-1,10-phenanthroline, a bidentate ligand with an extended aromatic system, was prepared and fully characterized. When L is a monodentate ligand, the following complexes were obtained: L = CF3SO3(-1) (2), CH3CN (3), and MeOH (4). When L2 is a bidentate ligand, the compounds [Ru(DIP)2(Hcmbpy)][Cl]2 (5) and [Ru(DIP)2(H2dcbpy)][Cl]2 (6) were prepared (Hcmbpy = 4-carboxy-4'-methyl 2,2-bipyridine, H2dcbpy = 4,4'-dicarboxy-2,2'-bipyridine). Complex [Ru(DIP)2(MeOH)2][OTf]2 (4) displayed a trans configuration of the DIP ligands, which is rare for octahedral complexes featuring DIP bidentate ligands. DFT calculations carried out on 4 showed that the cis isomer is more stable by 12.2 kcal/mol relative to the trans species. The solution behaviors of monocarboxylic complex [Ru(DIP)2(Hcmbpy)][Cl]2 (5) and dicarboxylic complex [Ru(DIP)2(H2dcbpy)][Cl]2 (6) were investigated by 1H NMR spectroscopy. VT-NMR, concentration dependence, and reaction with NaOD allowed us to suggest that aggregation of the cationic species in solution, especially for 6, originates mainly from hydrogen bonding interactions. PMID- 16676969 TI - In situ FT-IR and UV-vis spectroscopy of the low-temperature NO disproportionation mediated by solid state manganese(II) porphyrinates. AB - The heterogeneous reaction between NO gas and sublimed layers of manganese(II) porphyrinato complexes Mn(Por) (Por = TPP (tetraphenylporphyrinato dianion), TMP (tetramesitylporphyrinato dianion), or TPP(d20) (perdeuterated tetraphenylporphyrinato dianion)) has been monitored by IR and optical spectroscopy over the temperature range of 77 K to room temperature. These manganese porphyrins promote NO disproportionation to NO2 species and N2O, and the reaction proceeds via several distinct stages. At 90 K, the principal species observed spectrally are the nitric oxide dimer, cis-ONNO, two manganese nitrosyls, the simple NO adduct Mn(Por)(NO), and another intermediate (1) that is apparently critical to the disproportionation mechanism. This key intermediate is formed prior to N2O evolution, and proposals regarding its likely structure are offered. When the system is warmed to 130 K, the disproportionation products, N2O and the O-coordinated nitrito complex Mn(Por)(NO)(ONO) (2), are formed. IR spectral changes show that, upon further warming to 200 K, 2 isomerizes into the N-bonded nitro linkage isomer Mn(Por)(NO)(NO2) (3). After it is warmed to room temperature, the latter species loses NO and converts to the known 5-coordinate nitrito complex Mn(Por)(ONO) (4). PMID- 16676970 TI - New luminescent europium(III) chelates for DNA labeling. AB - The new europium(III) chelate [2,2',2'',2'''-[[4'-(aminobiphenyl-4-yl) 2,2':6',2''-terpyridine- 6,6''-diyl]bis(methylenenitrilo)]tetrakis(acetato)] europium(III) (ATBTA-Eu3+) and its 4,6-dichloro-1,3,5-triazinyl and succinimidyl derivatives (DTBTA and NHS-ATBTA, respectively) were synthesized and characterized. Both labeling complexes DTBTA-Eu3+ and NHS-ATBTA-Eu3+ are luminescent. Especially DTBTA-Eu3+ is strongly luminescent, with a luminescence quantum yield of 9.1%, molar extinction coefficient of 3.1 x 10(4) cm(-1) M(-1) (335 nm), and luminescence lifetime of 1.02 ms. The excitation and emission maximum wavelengths of DTBTA-Eu3+ are 335 and 616 nm, respectively. The complex is very stable in aqueous buffers, with a conditional formation constant log K(DTBTA-Eu) of 25.0 at pH 8, and can be conjugated to DNA and proteins. The chelates are also highly resistant to thermal decomposition, photodegradation, and ozone oxidation. These properties prove that DTBTA-Eu3+ is suitable as a luminescence label in DNA assays. PMID- 16676971 TI - Solvent effects on the structural and formyl substrate reactivity properties of a nitrogen/sulfur-ligated zinc hydroxide complex. AB - The solution structural and formyl substrate reactivity properties of a nitrogen/sulfur-ligated zinc hydroxide complex, [(bmnpaZn)2(mu-OH)2](ClO4)2 (1, bmnpa = N,N-bis-2-(methylthio)ethyl-N-((6-neopentylamino-2-pyridyl)methyl)amine), in acetonitrile and methanol are reported. In CH3CN, 1 has a binuclear cation [(bmnpaZn)2(mu-OH)2]2+ that is stabilized by secondary hydrogen bonding and CH/pi interactions involving the bmnpa chelate ligand. In CH3OH, 1 undergoes reaction with solvent to yield a zinc methoxide species, as determined by 1H NMR and electrospray mass spectral analysis. Treatment of 1 with methyl formate in CH3CN results in stoichiometric hydrolysis of the formyl ester to produce [(bmnpa)Zn(O2CH)]ClO4 (2) and methanol. The formate complex was identified via independent synthesis and characterization (X-ray crystallography, 1H and 13C NMR, FTIR, LRFAB-MS, conductance, and elemental analysis). In the solid state, 2n has a formate-bridged coordination polymer-type structure. However, in CH3CN, 2 behaves as 1:1 electrolyte, indicating cleavage of the polymer structure into mononuclear [(bmnpa)Zn(O2CH)]ClO4 species. Treatment of 1 with a stoichiometric amount of formanilide in CH3CN for 48 h at 45 degrees C results in decomposition of the zinc hydroxide complex to yield the free bmnpa ligand and an inorganic solid, presumably a zinc hydroxide or oxide species. Treatment of 1 with a stoichiometric amount of ethyl formate in CD3OD results in rapid, quantitative transesterification of the formyl carboxylate ester. A control reaction indicates that this transesterification reaction does not occur on the same time scale in the absence of the catalyst. Treatment of 1 with an excess of ethyl formate in CD3OD results in catalytic formyl carboxylate ester transesterification, with approximately 1000 turnovers in 60 min at 22(1) degrees C. Treatment of a CD3OD solution of 1 (0.5 equiv) with formanilide (1 equiv) results in the formation of aniline, d3-methyl formate, and the zinc formate complex 2. While aniline is produced stoichiometrically, the yield of d3-methyl formate varied from 30 to 50%, and the yield of 2 varied from 50 to 70% in repetitive experiments. Formation of both d3-methyl formate and 2 indicates that both methanolysis and hydrolysis reactions take place. PMID- 16676972 TI - Proton reduction and dihydrogen oxidation on models of the [2Fe]H cluster of [Fe] hydrogenases. A density functional theory investigation. AB - Density functional theory was used to compare reaction pathways for H2 formation and H+ reduction catalyzed by models of the binuclear cluster found in the active site of [Fe] hydrogenases. Terminal H+ binding to an Fe(I)-Fe(I) form, followed by monoelectron reduction and protonation of the di(thiomethyl)amine ligand, can conveniently lead to H2 formation and release, suggesting that this mechanism could be operative within the enzyme active site. However, a pathway that implies the initial formation of Fe(II)-Fe(II) mu-H species and release of H2 from an Fe(II)-Fe(I) form is characterized by only slightly less favored energy profiles. In both cases, H2 formation becomes less favored when taking into account the competition between CN and amine groups for H+ binding, an observation that can be relevant for the design of novel synthetic catalysts. H2 cleavage can take place on Fe(II)-Fe(II) redox species, in agreement with previous proposals [Fan, H.-J.; Hall, M. B. J. Am. Chem. Soc. 2001, 123, 3828] and, in complexes characterized by terminal CO groups, does not need the involvement of an external base. The step in H2 oxidation characterized by larger energy barriers corresponds to the second H+ extraction from the cluster, both considering Fe(II) Fe(II) and Fe(II)-Fe(III) species. A comparison of the different reaction pathways reveals that H2 formation could involve only Fe(I)-Fe(I), Fe(II)-Fe(I), and Fe(II)-Fe(II) species, whereas Fe(III)-Fe(II) species might be relevant in H2 cleavage. PMID- 16676973 TI - The reversible and stereoselective N- to C-bonded rearrangement of tris(2 pyridylmethyl)-1,4,7-triazacyclononanecobalt(III). AB - The hexaaminecobalt(III) complex [Co(tmptacn)]3+ (tmptacn = 1,4,7-tris(2' pyridylmethyl)-1,4,7-triazacyclononane) undergoes a novel base-catalyzed N- to C bonded rearrangement in which a tacn nitrogen is displaced by the alpha-carbon which deprotonates and binds to the metal ion as a carbanion. The X-ray structure establishes the configuration for the regio- and stereoselectively (100%) formed product. The reaction involves both ring expansions and ring contraction. The carbanion is part of a strained four-membered ring. The kinetics are reported for the N- to C-rearrangement, shown to be retentive for the optically resolved (+) [Co(tmptacn)]3+ reactant, and also the kinetics for a competitive and somewhat faster base-catalyzed racemization reaction of this complex. The reaction is completely but very slowly reversed in acid, also with retention, and in D2O/D+ there is 1:1 D-incorporation into the two sets of inequivalent tacn carbons. Extensive 1D and 2D NMR studies establish mechanistic details, and alternative mechanisms are proposed for the forward and reverse reactions. In neutral solution, there is a competitive oxidation reaction for the reverse C- to N bonded process, involving the regio- and stereoselective (100%) incorporation of an alpha-OH substituent into the tacn ring. PMID- 16676974 TI - Mixed substituted porphyrins: structural and electrochemical redox properties. AB - To examine the influence of mixed substituents on the structural, electrochemical redox behavior of porphyrins, two new classes of beta-pyrrole mixed substituted free-base tetraphenylporphyrins H2(TPP(Ph)4X4) (X = CH3, H, Br, Cl, CN) and H2(TPP(CH3)4X4) (X = H, Ph, Br, CN) and their metal (M = Ni(II), Cu(II), and Zn(II)) complexes have been synthesized effectively using the modified Suzuki cross-coupling reactions. Optical absorption spectra of these porphyrins showed significant red-shift with the variation of X in H2(TPPR4X4), and they induce a 20-30 nm shift in the B band and a 25-100 nm shift in the longest wavelength band [Q(x)(0,0)] relative to the corresponding H2TPPR4 (R = CH3, Ph) derivatives. Crystal structure of a highly sterically crowded Cu(TPP(Ph)4(CH3)4).2CHCl3 complex shows a combination of ruffling and saddling of the porphyrin core while the Zn(TPP(Ph)4Br4(CH3OH)).CH3OH structure exhibits predominantly saddling of the macrocycle. Further, the six-coordinated Ni(TPP(Ph)4(CN)4(Py)2).2(Py) structure shows nearly planar geometry of the porphyrin ring with the expansion of the core. Electrochemical redox behavior of the MTPPR4X4 compounds exhibit dramatic cathodic shift in first ring oxidation potentials (300-500 mV) while the reduction potentials are marginally cathodic in contrast to their corresponding MTPPX4 (X = Br, CN) derivatives. The redox potentials were analyzed using Hammett plots, and the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) gap decreases with an increase in the Hammett parameter of the substituents. Electronic absorption spectral bands of H2TPPR4X4 are unique that their energy lies intermediate to their corresponding data for the H2(TPPX8) (X = CH3, Ph, Br, Cl) derivatives. The dramatic variation in redox potentials and large red-shift in the absorption bands in mixed substituted porphyrins have been explained on the basis of the nonplanarity of the macrocycle and substituent effects. PMID- 16676975 TI - From one-dimensional chains to three-dimensional networks: solvothermal synthesis of thiogallates in ethylenediamine. AB - Five new thiogallates have been prepared solvothermally in the presence of ethylenediamine and characterized by single-crystal X-ray diffraction, thermogravimetry, and elemental analysis. [enH2][Ga4S7(en)2] (1), which crystallizes in the monoclinic space group P2(1)/c with lattice parameters a = 12.8698(12) angstroms, b = 10.4812(9) angstroms, c = 16.5473(14) angstroms and beta = 102.457(4) degrees (Z = 4), exhibits a layered structure in which both covalently and hydrogen-bonded template molecules coexist. The structures of [M(en)3](0.5)[GaS2] (M = Mn (2) (orthorhombic, Cmcm, a = 9.5555(6) angstroms, b = 15.0696(10) angstroms, c = 12.2893(7) angstroms, Z = 8) M = Co (3) (orthorhombic, Cmcm, a = 9.4660(7) angstroms, b = 15.0990(11) angstroms, c = 12.2540(8) angstroms, Z = 8), M = Ni (4) (orthorhombic, Cmcm, a = 9.4510(10) angstroms, b = 15.1416(15) angstroms, c = 12.2387(11) angstroms, Z = 8)) and Mn(en)2Ga2S4 (5) (monoclinic, C2/c, a = 14.3002(11) angstroms, b = 7.9509(5) angstroms, c = 12.1184(6) angstroms, beta = 100.191(4) degrees , Z = 4) are closely related and contain one-dimensional [GaS2]- chains, which are separated by [M(en)3]2+ counterions in 2, 3, and 4, and linked into a three-dimensional structure by [Mn(en)2]2+ units in 5. PMID- 16676976 TI - Experimental and theoretical investigations of IR spectra and electronic structures of the U(OH)2, UO2(OH), and UO2(OH)2 molecules. AB - Reactions of laser-ablated U atoms and H2O2 molecules produce UO2, H2UO2, and UO2(OH)2 as major products and U(OH)2 and HU(O)OH as minor products. Complementary information is obtained from similar reactions of U atoms with D2O2, with H2 + O2 mixtures, and with H2O in excess Ar. Through extensive relativistic density functional theory calculations, we have determined the geometry structures and ground states of these U species with a variety of oxidation states U(II), U(IV), U(V), and U(VI). The calculated vibrational frequencies, IR intensities, and isotopic frequency ratios are in good agreement with the experimental values, thus supporting assignments of the observed matrix IR spectra. We propose that the reactions proceed by forming an energized [U(OH)4] intermediate from reactions of the excited U atom with two H2O2 molecules. Because of the special stability of the U(VI) oxidation state, this intermediate decomposes to the UO2(OH)2 molecule, which reveals a distinctive difference between the chemistries of U and Th, where the major product in analogous Th reactions is the tetrahedral Th(OH)4 molecule owing to the stable Th(IV) oxidation state. PMID- 16676977 TI - Synthesis and characterization of single-crystal Ce(OH)CO3 and CeO2 triangular microplates. AB - Single-crystal cerium hydroxide carbonate (Ce(OH)CO3) triangular microplates with the hexagonal phase have been successfully synthesized by a hydrothermal method at 150 degrees C using cerium nitrate (Ce(NO3)3.6H2O) as the cerium source, aqueous carbamide as both an alkaline and carbon source, and cetyltrimethylammonium bromide (CTAB) as a surfactant. Single-crystal ceria (CeO2) triangular microplates have been fabricated by a thermal decomposition oxidation process at 650 degrees C for 7 h using single-crystal Ce(OH)CO3 microplates as the precursor. The shape of the Ce(OH)CO3 microplate was sustained after thermal decomposition-oxidation to CeO2. The products were characterized by X-ray powder diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), differential scanning calorimetric analysis (DSC), and thermogravimetric analysis (TG). PMID- 16676978 TI - Synthesis, isolation, and structural characterization of the C4h isomer of metal(1,2-naphthalocyanine) and its one-dimensional conductor of the axially substituted species. AB - The C4h isomer of the 1,2-Nc (1,2-naphthalocyaninato) ligand has been efficiently isolated as a hydrated magnesium complex by the fractional crystallization from the benzene/acetone solution after treating the crude mixture of the four isomers (C4h, C(s), C2v, D2h) with benzene. The C4h symmetry has been confirmed by X-ray structure analysis. The central metal ion has been demetalated and subsequently converted to the Li2 complex followed by conversion to the cobalt(II) complex. Electrochemical oxidation of the Co(III)(1,2-Nc)(CN)2 anion prepared from the cobalt(II) complex with TPP (tetraphenylphosphonium) has yielded a partially oxidized salt, TPP[Co(III)(1,2-Nc-C4h(CN)2]2. The crystal comprises slipped stacked Co(III)(1,2-Nc-C4h)(CN)2 one-dimensional chains and one-dimensional arrays of TPPs. The conductivity at room temperature is 0.1 S cm(-1), and the temperature dependence is semiconducting with a small activation energy of about 0.05 eV. The positive temperature-independent value of about 60 microV deg(-1) observed in the thermoelectric power measurements suggests that the salt is in the correlated hopping regime. PMID- 16676979 TI - Electronic configuration of high-spin imidazole-ligated iron(II) octaethylporphyrinates. AB - The preparation and characterization of two new five-coordinate, imidazole ligated, high-spin iron(II) octaethylporphyrinates is described. [Fe(OEP)(1,2 Me2Im)] and [Fe(OEP)(2-MeHIm)] have been characterized by X-ray structure determinations and temperature-dependent Mossbauer spectroscopy in zero and applied magnetic fields. The distinction between imidazole-ligated and other ligands in high-spin iron(II) porphyrinates, noted for a series of tetraarylporphyrinate derivatives (Hu, C.; Roth, A.; Ellison, M. K.; An, J.; Ellis, C. M.; Schiltz, C. E.; Scheidt, W. R. J. Am. Chem. Soc. 2005, 127, 5675), is seen here as well. The sign of the quadrupole-splitting constant is again negative, which is unique to the imidazole-ligated derivatives and suggests a distinct electronic structure. The derivatives again display a remarkable temperature dependence in the quadrupole splitting, which is also seen for deoxymyoglobin and -hemoglobin. Structural features for the two new derivatives are similar to those seen earlier, although the core conformations show somewhat more doming character. PMID- 16676980 TI - Large-scale synthesis of perpendicular side-faceted one-dimensional ZnO nanocrystals. AB - A novel route to the rational fabrication of narrow one-dimensional ZnO nanocrystals with perpendicular side facets is demonstrated in alcoholic solutions. The synthesis involves no-template or no-substrate solution method, which still allows the oriented growth of ZnO nanostructures at large-scale, low cost, and moderate temperatures. The management consists of monitoring of the nucleation, growth, and aging processes by means of chemical and solvent control of the interfacial free energy. It enables the control of the size of nano-, meso , and microcrystallites with various aspects from cubes to rods, wires, and belts. PMID- 16676983 TI - Template- and pH-directed assembly of diruthenium diphosphonates with different topologies and oxidation states. AB - In the presence of organic templates, six diruthenium diphosphonates, namely, [H3N(CH2)3NH3]2[Ru2(hedp)2] (1), [H3N(CH2)4NH3]2[Ru2(hedp)2].4H2O (2), [H3N(CH2)5NH3]2[Ru2(hedp)2].4H2O (3), [H3N(CH2)3NH3][Ru2(hedp)(hedpH)].H2O (4), [H3N(CH2)4NH3][Ru2(hedpH(0.5))2].2H2O (5), and [H3N(CH2)5NH3]2[Ru2(hedp)2][Ru2(hedpH)2]] (6) [hedp = 1 hydroxyethylidenediphosphonate, CH3C(OH)(PO3)2] have been synthesized under hydrothermal conditions. Compounds 1-3 contain homovalent paddlewheel cores of Ru2(II,II)(hedp)2(4-) that are connected through edge-sharing of the [RuO5Ru] octahedra, resulting in infinite linear chains. Compounds 4-6 contain mixed valent diruthenium(II,III) phosphonate paddlewheel cores of Ru2(II,III)(hedpH(n))2(3-2n)- that are connected by phosphonate oxygen atoms, forming distorted square-grid layers in 4 and 6 or a kagome lattice in 5. Both the templates and the pH values are found to play important roles in directing the final products with particular topologies and oxidation states of the diruthenium unit. The magnetic studies show that weak antiferromagentic interactions are propagated between the homovalent diruthenium units in compounds 1-3. For compounds 4-6, weak ferromagnetic interactions are observed. PMID- 16676982 TI - Water-exchange study revealed unexpected substitution behavior of [(CO)2(NO)Re(H2O)3]2+ in aqueous media. AB - The pH-dependent water-exchange rates of [(CO)2(NO)Re(H2O(cis))2(H2O(trans))]2+ (1) in aqueous media were investigated by means of 17O NMR spectroscopy at 298 K. Because of the low pK(a) value found for 1 (pK(a) = 1.4 +/- 0.3), the water exchange rate constant k(obs)(H2O(trans/cis)) was analyzed with a two-pathway model in which k(Re)(H2O(trans/cis)) and k(ReOH)(H2O)(trans/cis)) denote the water-exchange rate constants in trans or cis position to the nitrosyl ligand on 1 and on the monohydroxo species [(CO)2(NO)Re(H2O)2(OH)]+ (2), respectively. Whereas the rate constants k(ReOH)(H2O)(trans)) and k(ReOH)(H2O)(cis)) were determined as (4.2 +/- 2) x 10(-3) s(-1) and (5.8 +/- 2) x 10(-4) s(-1), respectively, k(Re)(H2O)(trans)) and k(Re)(H2O)(cis)) were too small to be determined in the presence of the much more reactive species 2. Apart from the water exchange, an unexpectedly fast C identical with 16O --> C identical withO exchange was also observed via NMR and IR spectroscopy. It was found to proceed through 1 and 2, with rate constants k(Re)(CO) and k(ReOH)(CO) of (19 +/- 4) x 10(-3) s(-1) and (4 +/- 3) x 10(-3) s(-1), respectively. On the other hand, N identical with 16O --> N identical with *O exchange was not observed. PMID- 16676981 TI - Heterobimetallic activation of dioxygen: characterization and reactivity of novel Cu(I)-Ge(II) complexes. AB - Reaction of the known germylene Ge[N(SiMe3)2]2 and a new heterocyclic variant Ge[(NMes)2(CH)2] with [L(Me2)Cu]2 (L(Me2) = the beta-diketiminate derived from 2 (2,6-dimethylphenyl)amino-4-(2,6-dimethylphenyl)imino-2-pentene) yielded novel Cu(I)-Ge(II) complexes L(Me2)Cu-Ge[(NMes)2(CH)2] (1a) and L(Me2)Cu-Ge[N(SiMe3)2]2 (1b), which were characterized by spectroscopy and X-ray crystallography. The lability of the Cu(I)-Ge(II) bond in 1a and b was probed by studies of their reactivity with benzil, PPh3, and a N-heterocyclic carbene (NHC). Notably, both complexes are cleaved rapidly by PPh3 and the NHC to yield stable Cu(I) adducts (characterized by X-ray diffraction) and the free germylene. In addition, the complexes are highly reactive with O2 and exhibit chemistry which depends on the bound germylene. Thus, oxygenation of 1a results in scission and formation of thermally unstable L(Me2)CuO2, which subsequently decays to [(L(Me2)Cu)2(mu-O)2], while 1b yields L(Me2)Cu(mu-O)2Ge[N(SiMe3)2]2, a novel heterobimetallic intermediate having a [Cu(III)(mu-O)2Ge(IV)]3+ core. The isolation of the latter species by direct oxygenation of a Cu(I)-Ge(II) precursor represents a new route to heterobimetallic oxidants comprising copper. PMID- 16676985 TI - Bimetallic fluorine-substituted anilido-aldimine zinc complexes for CO2/(cyclohexene oxide) copolymerization. AB - Regioselective nucleophilic aromatic substitution of an o-fluorine occurs to afford fluorine-substituted o-phenylene-bridged bis(anilido-aldimine) compounds o C6H4[(C6H2R2)N=CH-C6F4-(H)N(C6H3R'2)]2 when Li(H)N-C6H3R'2 (R' = iPr, Et, Me) is reacted with o-C6H4[(C6H2R2)N=CH-C6F5]2 (R = iPr, Et, Me) in a nonpolar solvent such as diethyl ether or toluene. Successive additions of Me2Zn and SO2 gas to the bis(anilido-aldimine) compounds afford quantitatively dinuclear mu methylsulfinato zinc complexes o-C6H4[[(C6H2R2)N=CH-C6F4-N(C6H3R'2) kappa2N,N]Zn(mu-OS(O)Me)]2 (R = iPr, R' = iPr, 3a; R = iPr, R' = Me, 3c; R = Et, R' = (i)Pr, 3d; R = Et, R' = Et, 3e; R = Et, R' = Me, 3f; R = Me, R' = iPr, 3g; R = Me, R' = Et, 3h; R = Me, R' = Me, 3i). The molecular structure of 3c was confirmed by X-ray crystallography. Fluorine-substituted complexes 3a-i show significantly higher TOF (turnover frequencies) than the unfluorinated analogues for CO2/(cyclohexene oxide) copolymerization. The TOF is highly sensitive to the substituents R and R', and the highest TOF (2480 h(-1)) is obtained with 3g (R = Me, R' = iPr). Complex 3g is less sensitive to the residual protic impurities present in the monomers and shows activity at such a low catalyst concentration as [Zn]:[cyclohexene oxide] = 1:50,000, at which the unfluorinated analogue is completely inactive. By realizing the activity at such an extremely low [Zn]:[cyclohexene oxide] ratio, we achieve a high TON (turnover number) up to 10,100. High-molecular-weight polymers (M(n), 100,000-200,000) are obtained with a rather broad molecular-weight distribution (M(w)/M(n), 1.3-2.5). The obtained polymers are not perfectly alternating, and variable carbonate linkages (65-85%) are observed depending on the N-aryl ortho substituents R and R' and the polymerization conditions. PMID- 16676984 TI - Diastereopure Fe(II) and Zn(II) complexes derived from a tridentate N,N',N bis(methyl-L-prolinate)-substituted pyridine ligand. AB - A series of mononuclear iron(II) and zinc(II) complexes of the new chiral Py(ProMe)2 ligand (Py(ProMe)2 = 2,6-bis[[(S)-2-(methyloxycarbonyl)-1 pyrrolidinyl]methyl]pyridine) have been prepared. The molecular geometry in the solid state (X-ray crystal structures) of the complexes [FeCl2(Py(ProMe)2)] (1), [ZnCl2(Py(ProMe)2)] (2), [Fe(OTf)2(Py(ProMe)2)] (3), [Fe(Py(ProMe)2)(OH2)2](OTf)2 (4), and [Zn(OTf)(Py(ProMe)2)](OTf) (5) are reported. They all show a meridional NN'N coordination of the Py(ProMe)2 ligand. The bis-chloride derivatives 1 and 2 represent neutral isostructural five-coordinated complexes with a distorted geometry around the metal center. Unusual seven-coordinate iron(II) complexes 3 and 4 having a pentagonal bipyramidal geometry were obtained using weakly coordinating triflate anions. The reaction of Zn(OTf)2 with the Py(ProMe)2 ligand afforded complex 5 with a distorted octahedral geometry around the zinc center. All complexes were formed as single diastereoisomers. In the case of complexes 3 5, the oxygen atoms of both carbonyl groups of the ligand are also coordinated to the metal. The stereochemistry of the coordinated tertiary amine donors in complexes 3-5 is of opposite configuration as in complexes 1 and 2 as a result of the planar penta-coordination of the ligand Py(ProMe)2. Complexes 1, 2, and 5 have an overall -configuration at their metal center, while the Fe(II) ion in complexes 3 and 4 has the opposite delta-configuration (crystal structures and CD measurements). The magnetic moments of iron complexes 1, 3, and 4 correspond to that of high-spin d6 Fe(II) complexes. The solution structures of complexes 1-5 were characterized by means of UV-vis, IR, conductivity, and CD measurements and their electrochemical behavior. These studies showed that the coordination environment of 1 and 2 observed in the solid state is maintained in solution. In coordinating solvents, the triflate anion (3, 5) or water (4) co-ligands of complexes 3-5 are replaced by solvent molecules with retention of the original pentagonal bipyramidal and octahedral geometry, respectively. PMID- 16676986 TI - Significance of the molecular shape of iron corrphycene in a protein pocket. AB - The iron complex of a new type of corrphycene bearing two ethoxycarbonyl ( CO2C2H5) groups on the bipyrrole moiety was introduced into apomyoglobin. The reconstituted ferric myoglobin has a coordinating water molecule that deprotonates to hydroxide with a pK(a) value of 7.3 and exhibits 3-10-fold higher affinities for anionic ligands when compared with a counterpart myoglobin with the same substituents on the dipyrroethene moiety. In the ferrous state, the oxygen affinity of the new myoglobin was decreased to 1/410 of the native protein. The anomalies in the ligand binding, notably dependent on the side-chain location, were interpreted in terms of a characteristic core shape of corrphycene that produces the longer and shorter Fe-N(pyrrole) bonds. The spin-state equilibrium analysis of the ferric azide myoglobin containing the new iron corrphycene supported the nonequivalence of the Fe-N(pyrrole) bonds. These results demonstrate that the trapezoidal molecular shape of corrphycene exerts functional significance when the iron complex is placed in a protein pocket. PMID- 16676987 TI - Enhanced solid-state metathesis routes to carbon nanotubes. AB - Ignition of three solids creates multiwalled carbon nanotubes in seconds. A solid state metathesis (exchange) reaction between hexachloroethane (C2Cl6) and lithium acetylide (Li2C2) with 5% cobalt dichloride (CoCl2) added as an initiator produces up to 7% carbon nanotubes, as observed via transmission electron microscopy. Using the concept that sulfur can promote nanotube growth, the reaction yield can be increased to 15% by switching to CoS as the initiator. The more readily available, inexpensive calcium carbide (CaC2) can be substituted for lithium acetylide while maintaining comparable yields. Switching initiators to FeS can be used to further enhance the yield. A systematic study of the C2Cl6/CaC2 reaction system indicates that a yield up to 25% can be realized by using 6% FeS as the initiator. Reaction temperatures for the C(2)Cl6/CaC2 system of up to 3550 degrees C are calculated using thermodynamic data assuming quantitative yield and adiabatic conditions. PMID- 16676988 TI - Molybdocalixarene structure control via rim deprotonation. synthesis, characterization, and crystal structures of calix[4]arene Mo(VI) monooxo complexes and calix[4]arene alkali metal/Mo(VI) dioxo complexes. AB - We report a series of calix[4]arene Mo(VI) dioxo complexes M2RC4MoO2 (M = alkali metal, R = H or Bu(t)) that were fully characterized by NMR, X-ray, IR, UV/vis, and elemental analysis. Molybdocalix[4]arene structures can be controlled via lower rim deprotonation, groups at para positions of calix[4]arene, and alkali metal counterions. Mono deprotonation at the lower rim leads to calix[4]arene Mo(VI) monooxo complexes RC4MoO (R = H, Bu(t), or allyl), and full deprotonation gives rise to calix[4]arene Mo(VI) dioxo complexes. Structural studies indicate that HC4 Mo(VI) dioxo complexes easily form polymeric structures via cation-pi interaction and coordination between different calixarene units. However, Bu(t)C4 Mo(VI) dioxo complexes tend to form dimers or tetramers due to steric hindrance of the tert-butyl groups at para positions in calixarene. The structures of the reduced side products A and C were determined by X-ray diffraction studies. The mechanism of RC4MoO formation from the reaction of calixarene monoanions with MoO2Cl2 appears to include the addition of a calixarene -OH group across a Mo=O bond. PMID- 16676989 TI - Macrocyclic amines as structure-directing agents for the synthesis of three dimensional antimony-sulfide frameworks. AB - A new family of antimony sulfides, incorporating the macrocyclic tetramine 1,4,8,11-tetraazacyclotetradecane (cyclam), has been prepared by a hydrothermal method. [C10N4H26][Sb4S7] (1), [Ni(C10N4H24)][Sb4S7] (2), and [Co(C10N4H24)]x[C10N4H26](1-x)[Sb4S7] (0.08 < or = x < or = 0.74) (3) have been characterized by single-crystal X-ray diffraction, elemental analysis, thermogravimetry, and analytical electron microscopy. All three materials possess the same novel three-dimensional Sb4S7(2-) framework, constructed from layers of parallel arrays of Sb4S8(4-) chains stacked at 90 degrees to one another. In 1, doubly protonated macrocyclic cations reside in the channel structure of the antimony-sulfide framework. In 2 and 3, the cyclam acts as a ligand, chelating the divalent transition-metal cation. Analytical and X-ray diffraction data indicate that the level of metal incorporation in 2 is effectively complete, whereas in 3, both metalated and nonmetalated forms of the macrocycle coexist within the structure. PMID- 16676990 TI - Electronic structure and reactivity of isomeric oxo-Mn(V) porphyrins: effects of spin-state crossing and pKa modulation. AB - The reactivity of the isomeric oxo-Mn(V)-2-tetra-N-methylpyridyl (2-TMPyP) and oxo-Mn(V)-4-tetra-N-methylpyridyl (4-TMPyP) porphyrins has been investigated by a combined experimental and theoretical approach based on density functional theory. The unusual higher reactivity of the more electron-rich 4-TMPyP species appears to be related to both the higher basicity of its oxo ligand, compared to that of the 2-TMPyP isomer, and the smaller low-spin-high-spin promotion energy of 4-TMPyP, compared to that of 2-TMPyP, because of the stabilization of the A2u orbital in the latter isomer. Therefore, in a two-state energy profile involving crossing of the initial singlet and final quintet potential energy surfaces, the 4-TMPyP isomer should be kinetically favored. The calculated differences in the singlet-quintet gaps for the 2-TMPyP and 4-TMPyP systems compare well with the measured differences in the activation energies for two isomeric porphyrins. Both effects, proton affinity and electron-promotion energy, contribute to reduce the reactivity of the more electrophilic oxidant when electron-withdrawing groups are closer to the active site, contrary to the usual expectations based on simple chemical reactivity correlations. These theoretical results are in accord with new experimental data showing O=Mn(V)-O-H pK(a)s of 7.5 and 8.6 for the isomeric 2-TMPyP and 4-TMPyP systems, respectively. PMID- 16676991 TI - EPR and UV-vis studies of the nitric oxide adducts of bacterial phenylalanine hydroxylase: effects of cofactor and substrate on the iron environment. AB - Phenylalanine hydroxylase from Chromobacterium violaceum (cPAH), which catalyzes phenylalanine oxidation to tyrosine, is homologous to the catalytic domain of eukaryotic PAHs. Previous crystallographic and spectroscopic studies on mammalian PAH conflict on whether O2 binds to the open-coordination site or displaces the remaining water ligand to yield either a six- or a five-coordinate iron, respectively. The abilities of nitric oxide to behave as an oxygen mimic and a spectroscopic probe of ferrous iron are used to investigate the geometric and electronic effects of cofactor and substrate binding to cPAH by electron paramagnetic resonance (EPR) and UV-vis spectroscopies. A rhombic distortion observed for the ternary complex is due to two factors: a decrease in the Fe-NO angle and an alteration in the equatorial ligand geometry. Both factors are consistent with NO displacing the sole remaining water ligand to yield a five coordinate iron center. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched water is observed in the absence of substrates or presence of cofactor only (binary complex), demonstrating that water is bound to the Fe(II). However, in the presence of substrate and cofactor (ternary complex), the EPR resonances of the nitrosyl complex are not broadened by 17O enriched water, indicating the displacement of water by NO to afford a five coordinate iron. Furthermore, the increased intensity in the 500-600 nm range of the UV-vis spectrum of the ternary nitrosyl complex indicates an increased overlap between the in-plane NO 2pi and d(x2-y2) and d(xz) orbitals, which corroborates a five-coordinate iron. PMID- 16676992 TI - Assembly of heterometallic clusters and coordination polymers by combining Mo-S based clusters with Mn2+. AB - Addition of [Mo(V)2O2S2(edt)2]2- (edt =1,2-ethanedithiolate) to acetonitrile and/or methanol solutions of MnII containing bipyridines [4,4' trimethylenedipyridine (TDP), 4,4'-bipyridine (4,4'-bpy), 2,2'-bipyridine (2,2' bpy)] or 15-crown-5 produces three new heterometallic cluster coordination polymers, [Mn2[Mo2O2S2(edt)2]2(TDP)3(CH3OH)2(NCMe)2].3CH3OH.0.25MeCN (1), [Mn(TDP)2(H2O)2]2+[Mn[Mo2O2S2(edt)2)2(TDP)2]]2-.6CH3OH (2), [Mn[Mo2O2S2(edt)2](TDP)2(CH3OH)(H2O)].CH3OH (3), and three new multinuclear clusters, [Mn[Mo2O2S2(edt)2](4,4'-bpy)(CH3OH)4].0.5(4,4'-bpy) (4), [Mn[Mo2O2S2(edt)2](2,2'-bpy)2].2CH3OH (5), and (NEt4)2[Mn(15-crown 5)[Mo2O2S2(edt)2]2] (6). All compounds were characterized by X-ray crystallography. The coordination mode of Mn in these compounds depends on the ligands and the crystallization conditions. Compound 2 readily converts to 1 or 3 depending on the reaction and solvent conditions. Compounds 1 and 2 were analyzed using thermogravimetric analysis combined with mass spectroscopy (TG-MS) in the temperature range 25-500 degrees C. The room-temperature magnetic moments for compounds 1-6 were determined. PMID- 16676993 TI - A method for combinatorial fabrication and characterization of organic/inorganic thin film devices in UHV. PMID- 16676994 TI - Quantitative evaluation of HiCore resin for the nonspecific binding of proteins by on-bead colorimetric assay. PMID- 16676995 TI - Three-component, one-pot reaction for the combinatorial synthesis of 1,3,4 substituted pyrazoles. PMID- 16676996 TI - Revisiting benzenesulfonyl linker for the deoxygenation and multifunctionalization of phenols. PMID- 16676997 TI - Divergent structural complexity from a linear reaction sequence: synthesis of fused and spirobicyclic gamma-lactams from common synthetic precursors. PMID- 16676998 TI - Solid-phase synthesis of the 2-aminobenzoxazole library using thioether linkage as the safety-catch linker. AB - An efficient solid-phase methodology has been developed for the synthesis of 2 aminobenzoxazole derivatives. The key step in this procedure involves preparation of polymer-bound 2-mercaptobenzoxazole resins 3 by reaction of the Merrifield resin with 2-aminophenols and CS(2) in the presence of DIC in MeCN. Oxidation of the resulting resins followed by treatment with amines gives the desired 2 aminobenzoxazole products 5. Further diversification can be introduced to the key resin 11, derived from the nitro group containing resin 3c. This process produces the corresponding amine, which upon reaction with acid chlorides and isocyanates can be used to generate various 6-functionalized 2-aminobenzoxazole analogues 13 and 15. PMID- 16676999 TI - MAP: an iterative experimental design methodology for the optimization of catalytic search space structure modeling. AB - One of the main problems in high-throughput research for materials is still the design of experiments. At early stages of discovery programs, purely exploratory methodologies coupled with fast screening tools should be employed. This should lead to opportunities to find unexpected catalytic results and identify the "groups" of catalyst outputs, providing well-defined boundaries for future optimizations. However, very few new papers deal with strategies that guide exploratory studies. Mostly, traditional designs, homogeneous covering, or simple random samplings are exploited. Typical catalytic output distributions exhibit unbalanced datasets for which an efficient learning is hardly carried out, and interesting but rare classes are usually unrecognized. Here is suggested a new iterative algorithm for the characterization of the search space structure, working independently of learning processes. It enhances recognition rates by transferring catalysts to be screened from "performance-stable" space zones to "unsteady" ones which necessitate more experiments to be well-modeled. The evaluation of new algorithm attempts through benchmarks is compulsory due to the lack of past proofs about their efficiency. The method is detailed and thoroughly tested with mathematical functions exhibiting different levels of complexity. The strategy is not only empirically evaluated, the effect or efficiency of sampling on future Machine Learning performances is also quantified. The minimum sample size required by the algorithm for being statistically discriminated from simple random sampling is investigated. PMID- 16677000 TI - Proteomimetic libraries: design, synthesis, and evaluation of p53-MDM2 interaction inhibitors. AB - The p53-MDM2 interaction regulates p53-mediated cellular responses to DNA damage, and MDM2 is overexpressed in 7% of all cancers. Structure-based computational design was applied to this system to design libraries centered on a scaffold that projects side chain functionalities with distance and angular relationships equivalent to those seen in the MDM2 interacting motif of p53. A library of 173 such compounds was synthesized using solution phase parallel chemistry. The in vitro competitive ability of the compounds to block p53 peptide binding to MDM2 was determined using a fluorescence polarization competition assay. The most active compound bound with K(d) = 12 microM, and its binding was characterized by (15)N-(1)H HSQC NMR. PMID- 16677001 TI - Strategy for discovering chemical inhibitors of human cyclophilin a: focused library design, virtual screening, chemical synthesis and bioassay. AB - The discovery of cyclophilin A (CypA) inhibitor is now of special interest in the treatment of immunological disorders. In this work, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, a series of novel small molecular CypA inhibitors have been discovered. First, using the fragments taken from our previously discovered CypA inhibitors (Bioorg. Med. Chem. 2006, 14, 2209-2224) as building blocks, we designed a focused combinatorial library containing 255 molecules employing the LD1.0 program (J. Comb. Chem. 2005, 7, 398-406) developed by us. Sixteen compounds (1a e, 2a-b, 3a-b, and 4a-g) were selected by using virtual screening against the X ray crystal structure of CypA as well as druglike analysis for further synthesis and bioassay. All these sixteen molecules are CypA binders with binding affinities (K(D) values) ranging from 0.076 to 41.0 microM, and five of them (4a, 4c, and 4e-g) are potent CypA inhibitors with PPIase inhibitory activities (IC(50) values) of 0.25-6.43 microM. The hit rates for binders and inhibitors are as high as 100% and 31.25%, respectively. Remarkably, both the binding affinity and inhibitory activity of the most potent compound increase approximately 10 times than that of the most active compound discovered previously. The high hit rate and the high potency of the new CypA inhibitors demonstrated the efficiency of the strategy for focused library design and screening. In addition, the novel chemical entities reported in this study could be leads for discovering new therapies against the CypA pathway. PMID- 16677002 TI - Solution-phase parallel synthesis of a pyridinium pyrazol-3-olate inner salt library using a three-component reaction. AB - We present here the discovery of a novel, versatile, multicomponent reaction leading to various 4-[4-(pyridinium-1-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl] 2H-pyrazol-3-olate inner salts. The structure of the unusual zwitterionic inner salts was elucidated, and the scope of the novel reaction was investigated. After rapid optimization, the reaction was adapted to parallel synthesis, and an 800 membered compound library was produced. PMID- 16677003 TI - Solid-phase synthesis of pyrazolopyridines from polymer-bound alkyne and azomethine imines. AB - Study was made of the 1,3-dipolar cycloaddition of polymer-bound alkynes to azomethine imines generated in situ from N-aminopyridine iodides. Aromatization of the cycloadducts gives polymer-bound pyrazolopyridines that can be released from the resin as carboxylic acids with trifluoroacetic acid or as methyl esters with sodium methoxide. PMID- 16677004 TI - Polystyrene-graft-polyglycerol resins: a new type of high-loading hybrid support for organic synthesis. AB - The preparation of a dendritic graft polymer by a very efficient synthesis of polyglycerol directly on a polystyrene resin is presented. This one-step process can be performed on a multigram scale to provide a chemically stable polymeric support. The resulting hybrid polymers were fully characterized by diverse analytical methods (NMR, IR, ESEM, UV detection of cleaved protecting groups, and mass-spectrometric methods). They combine a high loading capacity (up to 4.3 mmol g(-1)) with good swelling properties in a wide range of solvents (including water), which is the major drawback for many existing solid phase supports. In comparison to the widely employed PEGylated resins, these hybrid materials offer a 10-fold higher loading capacity. Their suitability as supports for organic synthesis and for the immobilization of reagents has been demonstrated. These materials also swell in water, and consequently, it should be possible to use these new hybrid materials for synthesis in protic solvents. PMID- 16677005 TI - Hydrophilic polymer supports for solid-phase synthesis: hydroxyl-functional beads of poly(vinylpyrrolidone). AB - Poly(vinylpyrrolidone) (PVP) has solubility properties that make it an attractive material for polymer-assisted synthesis applications; however, the naked polymer lacks reactive groups upon which to do chemistry. Furthermore, large differences in radical reactivity between 1-vinylpyrrolidin-2-one (NVP) and most other monomers lead to compositional drift during copolymerization, further complicating the introduction of functional groups into the polymer using this method. Monomers that are derivatives of NVP itself are expected to show smaller differences in radical reactivity and therefore provide a way of preparing PVP with adjustable properties. Three monomers introducing hydroxyl-functional groups and a new cross-linker, all derivatives of NVP, were synthesized and used in the preparation of a new type of hydrophilic polymer beads by aqueous suspension polymerization. These lightly cross-linked beads contain hydroxyl groups at a functional loading of 0.21-0.29 mmol/g and swell extensively in a broad range of solvents. PMID- 16677006 TI - Microwave-assisted, boron trichloride mediated acylation of phenols-synthesis of (o-hydroxyaryl)(aryl)methanones and xanthones. AB - A novel and efficient microwave-assisted, BCl(3) mediated coupling reaction to synthesize o-(hydroxyaryl)(aryl)methanone structures from phenols and acyl chlorides is described. This reaction was further incorporated into a two-step synthesis of biologically interesting xanthones. PMID- 16677007 TI - Solution-phase synthesis of a tricyclic pyrrole-2-carboxamide discovery library applying a stetter-Paal-Knorr reaction sequence. AB - The solution-phase synthesis of a discovery library of 178 tricyclic pyrrole-2 carboxamides was accomplished in nine steps and seven purifications starting with three benzoyl-protected amino acid methyl esters. Further diversity was introduced by two glyoxaldehydes and 41 primary amines. The combination of Pauson Khand, Stetter, and microwave-assisted Paal-Knorr reactions was applied as a key sequence. The discovery library was designed with the help of QikProp 2.1, and physicochemical data are presented for all pyrroles. Library members were synthesized and purified in parallel and analyzed by LC/MS. Selected compounds were fully characterized. PMID- 16677008 TI - Design and synthesis of a tetracyclic pyrimidine-fused benzodiazepine library. AB - Method development for a heterocyclic library which entails novel scaffolds of benzodiazepines fused with various heterocycles, such as pyrimidines, indolines, and tetrahydroquinolines, was accomplished. The new synthetic strategy is based on an electrophilic cyclization reaction involving an iminium intermediate formed by the corresponding aminopyrimidine with a carbonyl compound to give the desired heterocycles in high yields. Subsequent replacement of the chloro group in the resulted structures with a nucleophile, such as boronic acids, amines, alcohols and thiols, led to a library of privileged compounds with up to eight accessible diversity points. PMID- 16677009 TI - Straightforward entry to libraries of diversely substituted azinones by a consecutive aza-Michael/palladium-catalyzed functionalization strategy. AB - A highly divergent, flexible, and conceptually simple sequence allowing the parallel solution-phase assembly of functionalized azinone libraries has been developed in a one-pot consecutive fashion. Structural decoration of in situ generated heterocyclic aza-Michael adducts AB was accomplished by exploiting the diversity potential of Heck, Suzuki, Sonogashira, and Stille reactions. PMID- 16677010 TI - Polymer-supported synthesis of pyridone-focused libraries as inhibitors of anaplastic lymphoma kinase. AB - The optimization of screening hits on a promising new target for therapy of certain cancers involving anaplastic lymphoma kinase (ALK) inspired the development of this efficient solid-phase chemistry. A series of novel pyridones have been recently discovered as inhibitors of ALK, which led to the design of focused libraries around the pyridone scaffold. A stepwise process involving iterative template modification based on both medicinal chemistry insights and computational ranking of virtual libraries was employed in the design. The unique solid-phase chemistry has addressed the need for rapid optimization of this "early lead" series. Herein the methodology and scope of the chemistry, as well as its application for library synthesis, are discussed. PMID- 16677011 TI - An efficient and regiospecific strategy to N7-substituted purines and its application to a library of trisubstituted purines. AB - A regiospecific strategy for the preparation of N(7)-substituted purines in an efficient manner was devised. This approach to 6,7,8-trisubstituted purines relies on the cyclization reactions of suitably substituted pyrimidines (1) with either a carboxylic acid or an aldehyde. The method development for the five-step synthetic strategy outlined here was completed using 5-amino-4,6 dichloropyrimidine (4) as the starting material. The utility of this methodology was demonstrated through the preparation of a 40-membered library of 6,7,8 trisubstituted purines (3) in good yields and high purity. PMID- 16677012 TI - Cleavable hydrophilic linker for one-bead-one-compound sequencing of oligomer libraries by tandem mass spectrometry. AB - We have developed a method for the rapid and unambiguous identification of sequences of hit compounds from one-bead-one-compound combinatorial libraries of peptide and peptoid ligands. The approach uses a cleavable linker that is hydrophilic to help reduce nonspecific binding to biological samples and allows for the attachment of a halogen tag, which greatly facilitates post-screening sequencing by tandem mass spectrometry (MS/MS). The linker is based on a tartaric acid unit, which, upon cleavage from resin, generates a C-terminal aldehyde. This aldehyde can then be derivatized with a bromine-containing amino-oxy compound that serves as an isotope tag for subsequent MS/MS analysis of y-ion fragments. We have applied this linker and method to the syntheses of a number of peptoids that vary in sequence and length and have also demonstrated single-bead sequencing of a peptoid pentamer. The linker is also shown to have very low levels of nonspecific binding to proteins. PMID- 16677013 TI - Microwave-assisted three-component synthesis of 7-aryl-2-alkylthio-4,7-dihydro 1,2,4-triazolo[1,5-a]-pyrimidine-6-carboxamides and their selective reduction. AB - Multicomponent reactions (MCRs) and microwave-assisted organic synthesis (MAOS) have been used as key methods for the synthesis of fused dihydropyrimidine derivatives. The three-component condensation of 3-amino-5-alkylthio-1,2,4 triazoles with aromatic aldehydes and acetoacetamides under microwave irradiation was developed as a rapid and efficient solution-phase method for the high yielding preparation of 7-aryl-2-alkylthio-4,7-dihydro-1,2,4-triazolo[1,5 a]pyrimidine-6-carboxamide libraries. In addition, the selective reduction of the formed dihydrotriazolopyrimidines to trans-trans-2-alkylthio-7-aryl-4,5,6,7 tetrahydro-1,2,4-triazolo[1,5-a]pyrimidine-6-carboxamides was established. The described synthetic protocols provide rapid access to novel and diversely substituted dihydroazolopyrimidine libraries. PMID- 16677014 TI - Polymer-supported N-derivatized, o-linked hydroxylamine for concurrent solid phase synthesis of diverse N-alkyl and N-H hydroxamates. AB - We describe parallel/combinatorial, solid-phase, supported synthesis of diverse hydroxamates using a common intermediate, an N-derivatized, O-linked hydroxylamine. The method allows the concurrent synthesis of both N-alkyl and N-H hydroxamates and is compatible with a wide range of chemical transformations. The synthesis of NH hydroxamates includes protection of the nitrogen with a 2,4 dimethoxybenzyl group at the stage of polymer-supported benzyloxyamine. The protecting group eliminates side reactions caused by the presence of a free hydroxamate NH group and is simultaneously removed during cleavage of target compounds from the solid support. The chemical route has been thoroughly tested on model compounds with several linkers, and a high yield and purity synthesis of more than 50 hydroxamates, designed to inhibit cell proliferation of breast cancer cell lines, is described. PMID- 16677016 TI - Poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers that respond sharply to temperature and pH. AB - Temperature- and pH-sensitive random copolymers of N-isopropylacrylamide (NIPAAm) and propylacrylic acid (PAA) were prepared using the reversible addition fragmentation chain transfer (RAFT) polymerization method. The lower critical solution temperatures (LCSTs) (or phase separation temperatures) of the NIPAAm-co PAA copolymer solutions were measured by the cloud-point method. At slightly acidic conditions, the LCST decreased with increase in PAA content, which suggests that the hydrophobic propyl group of PAA has a greater influence on the LCST than the polar carboxylic acid group at those conditions. An increase of pH led to a significant increase in LCST of the copolymers due to the ionization of the -COOH group. The LCSTs were studied as a function of copolymer composition over the pH range from 5.0 to 7.0. Because the pK(a) of the polymers can be tuned to fall close to neutral pH, these polymer compositions can be designed to have phase transitions triggered near physiological pH or at slightly acidic pH values that fall within acidic gradients found in biology. The NIPAAm-co-PAA copolymers thus display tunable properties that could make them useful in a variety of molecular switching and drug delivery applications where responses to small pH changes are relevant. PMID- 16677017 TI - Water soluble nanoparticles from PEG-based cationic hyperbranched polymer and RNA that protect RNA from enzymatic degradation. AB - Recent advances in understanding biological systems have proven that RNA is not merely the carrier of genetic information, but also a key molecule in regulation of gene expression and other crucial metabolic processes. Therefore, it is being considered as an ideal therapeutic candidate both for metabolic and genetic disorders. However, research involving RNA molecules faces a practical limitation since RNA is highly labile. We have developed a novel method to protect RNA from cleavage by complexing it with a hyperbranched cationic polymer. It was found that total cellular RNA isolated from yeast spontaneously interacts with the positively charged polymer to form a spherical nanoparticle morphology. This interaction protects the RNA against enzymatic degradation. This methodology can be easily adapted for long-term storage of RNA, long distance transfer of RNA, and genetic engineering using RNA as a building block. PMID- 16677018 TI - Fluorescent labeling of RAFT-generated poly(N-isopropylacrylamide) via a facile maleimide-thiol coupling reaction. AB - We report a facile labeling technique in which the telechelic thiocarbonylthio functionality of well-defined poly(N-isopropylacrylamide) (PNIPAM) prepared by room temperature RAFT polymerization is first converted to the thiol and subsequently reacted with a maleimido-functional fluorescent dye, N-(1 pyrene)maleimide (PM). Nearly monodisperse PNIPAM (M(n) = 39 500 g/mol, M(w)/M(n) = 1.07) was synthesized using a trithiocarbonate-based CTA, 2 dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP), and a conventional azo-initiator, namely, 2,2'-azobis(4-methoxy-2,4 dimethylvaleronitrile) (V-70), as the primary source of radicals. The key to successful conjugation of PM to PNIPAM is the implementation of a two-step reduction process involving (1) the cleavage of the trithiocarbonate with a strong reducing agent, in this case, NaBH4, to form a mixture of polymeric thiols and disulfides and (2) the conjugation of PM to the pure polymeric thiol in the presence of tris(2-carboxyethyl)phosphine.HCl (TCEP). We show that TCEP efficiently eliminates the formation of polymeric disulfides and thus allows for the desired addition of the free polymeric thiol across the maleimide double bond. This concept is demonstrated using SEC-MALLS and UV-vis spectroscopy measurements. PMID- 16677019 TI - Patterned protein films on poly(lipid) bilayers by microcontact printing. AB - The use of polymerized lipid bilayers as substrates for microcontact printing (muCP) of protein films was investigated. We have previously shown that vesicle fusion of bis-SorbPC, a dienoate lipid, on glass and silica substrates, followed by redox-initiated radical polymerization, produces a planar supported lipid bilayer (PSLB) that is ultrastable(1a) [Ross, E. E.; Rozanski, L. J.; Spratt, T.; Liu, S.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1752] and highly resistant to nonspecific adsorption of dissolved proteins [Ross, E. E.; Spratt, T.; Liu, S.; Rozanski, L. J.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1766].(1b) Here we demonstrate that muCP of bovine serum albumin (BSA) onto a dried poly(bis-SorbPC) PSLB from a poly(dimethylsiloxane) (PDMS) stamp produces a layer of strongly adsorbed protein, comparable in surface coverage to films printed on glass surfaces. Immobilization of proteins on poly(PSLB)s has potential applications in biosensing, and this work shows that direct muCP of proteins is a technically simple approach to create immobilized monolayers, as well as multilayers of different proteins. PMID- 16677020 TI - New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 1. Investigation of the influence of structural parameters on the extent of degradation. AB - Six methyl celluloses (MCs), one with a degree of substitution (DS) of 1.32 and five with DS between 1.83 and 1.88, were thoroughly investigated. Monomer composition and methyl distribution in the polymer chain were analyzed after total or partial random hydrolysis and appropriate derivatization with gas chromatography (GC) and mass spectrometry (MS), respectively, and used as reference data. The same MCs were then hydrolyzed with an enzyme preparation of Trichoderma longibrachiatum and further investigated with size-exclusion chromatography with multiangle light scattering and refractive index detection (SEC-MALS/RI) and MS. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) in combination with various MS analyzers were compared with respect to quantification of the degradation products directly and after perdeuteriomethylation. The methyl group distribution in the oligomeric fractions and the average DS as a function of chain length were calculated from ESI mass spectra. With help of the reference analysis, patterns could be corrected for the unspecific contribution of end groups. By labeling and ESI tandem MS, our knowledge about the tolerance of the enzymes' sub-sites with respect to the number of methyl groups could be improved. PMID- 16677021 TI - New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 2. Comparison of various enzyme preparations. AB - In this part of our studies, dealing with new approaches to the analysis of enzymatically hydrolyzed methyl cellulose, five different enzymes or enzyme preparations containing endoglucanases (from Bacillus agaradhaerens Cel 5A, Trichoderma reesei, Trichoderma viride, and two obtained from Trichoderma longibrachiatum) were used to hydrolyze six different methyl celluloses (MCs). The main goal was to investigate whether enzymes could be used for determination of the heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity, it was necessary to gather information on how the enzymes affect hydrolysis. Size exclusion chromatography with multi-angle light scattering and refractive index detection (SEC-MALS/RI) was used to estimate the molar mass distribution of the MCs before and after hydrolysis. A novel internal standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-ITMS) was used to determine the amount of formed oligomers. Two MCs, one with a degree of substitution (DS) of 1.8 and one with DS 1.3, were hydrolyzed with all of the five enzymes. The yield of summarized di- and trisaccharides was approximately 2% of the hydrolysis products for the MC with DS 1.8, whereas the product mixture, obtained from a MC with a DS of 1.3, contained 7-16% di- and trisaccharides. By a novel sample preparation method in combination with ESI-IT tandem MS, outlined in part 1 of this work, it was shown that the enzymes produced oligomers with the reducing end bearing no or only one substituent. Comparison of the methyl pattern at the nonreducing ends of the dimers and trimers indicated that the -2 subsite of the active complex is less tolerant than subsites -3 and +1. All enzymes had similar general selectivity toward the methyl substituents but also showed some differences. From both SEC-MALS/RI and ESI-ITMS, differences with respect to substituent distribution of MCs could be recognized but not for each enzyme used. Basic considerations for enzymatic hydrolysis and analysis of methyl cellulose were listed as a consequence of the results from the work. PMID- 16677022 TI - Synthesis of a polymer skeleton at the inner leaflet of liposomal membranes: polymerization of membrane-adsorbed pH-sensitive monomers. AB - We describe the synthesis of liposomes with an artificial membrane skeleton as a model of the native cellular cytoskeleton. Similar to natural conditions, a flat polymer network is coupled to the inner membrane leaflet like a suspended ceiling via membrane-inserted anchor monomers with a spacer. The polymer is composed of DMAPMA (N-(3-N,N-dimethylaminopropyl) methacrylamide) and TEGDM (tetraethylene glycol dimethacrylate) as a linker and is coupled to the membrane anchor DOGM (1,2-distearyl-3-octaethylene glycol glycerol ether methacrylate). In the first step of the synthesis, DMAPMA and TEGDM are encapsulated into liposomes composed of egg phosphatidylcholine (EPC), and free monomers are removed by gel chromatography. At pH 10, DMAPMA adsorbs to the inner membrane surface, as demonstrated in parallel studies with lipid monolayers using a Langmuir film balance. The polymerization by UV irradiation was initiated with DEAP (2,2 diethoxyacetophenone) as the initiator and was shown to be complete after 15 min. At pH 6, polymer was desorbed from the inner membrane surface to form a lamellar structure similar to that of the cellular cytoskeleton, as shown by electron microscopy. In comparison to NIPAM (N-isopropylacrylamide), which was used as a monomer in a recent study (Stauch, O.; Uhlmann, T.; Frohlich, M.; Thomann, R.; El Badry, M.; Kim, Y.-K.; Schubert, R. Biomacromolecules 2002, 3, 324-32), DMAPMA shows much slower membrane permeation leading to an essential restriction of the formed polymer to the liposomal interior. The DMAPMA-based composite structure stabilizes the lipid membrane against sodium cholate by a factor of 2.5 as compared to plain EPC liposomes. This is discussed in the context of the situation in the liver, where the cytoskeleton probably plays a crucial role in the stabilization of the membrane against high bile salt concentration. PMID- 16677023 TI - Biocompatible surface preparation using amino-functionalized amylose. AB - Aminopropyl amyloses with various degrees of substitution (DS) were prepared and investigated with respect to their surface modification properties. Poly(acrylic acid) was grafted to plasma-activated PVDF films, and the functional amylose was bound via amide linkage formation. Layer formation was confirmed by X-ray photoelectron spectroscopy. Contact angle measurements and surface MALDI-TOF mass spectrometry indicated a hydrophilic surface and minimization of protein adsorption. PMID- 16677024 TI - Synthesis and characterization of amphiphilic polyphosphates with hydrophilic graft chains and cholesteryl groups as nanocarriers. AB - Amphiphilic polyphosphate graft copolymers with varied densities of cholesteryl esters and hydrophilic graft chains were prepared, and the solution properties of the graft copolymers were evaluated. Polyphosphates were synthesized as backbones by ring-opening polymerization of 2-isopropyl-2-oxo-1,3,2-dioxaphospholane (IPP), 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl-2-bromoisobutyrate) (OPBB), and 2 choresteryl-2-oxo-1,3,2-dioxaphospholane (ChOP) using triisobutylaluminum as an initiator. Three types of polyphosphates (PIBr(x)Ch(y), x = number of OPBB units in a polymer; y = number of ChOP units in a polymer) such as PIBr4, PIBr6Ch1, and PIBr3Ch2 were obtained. The molecular weights of these polymers were 2.4 x 10(4), 2.4 x 10(4), and 2.6 x 10(4) g/mol, respectively. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was grafted from the OPBB sites in PIBr(x)Ch(y) via atom transfer radical polymerization (ATRP) in EtOH. In each polymer system, the molecular weight of the graft polymer was linear with conversion. Furthermore, the polymer radical concentration remained constant during polymerization; that is, the molecular weights of the graft chains were easily controllable with polymerization time. The solution properties of amphiphilic PIBr(x)Ch(y)-g-PMPCs were investigated by the methods of surface tension measurement, light scattering, and fluorescence probe. The transition point (cmc) of the surface tension of the PIBr(x)Ch(y)-g-PMPCs aqueous solution decreased with an increase in the number of ChOP units in a graft polymer. Particularly, PIBr3Ch2-g PMPC14.9K formed nanosized associates (R(h) = 7.5 nm) with 2.2 molecules above 0.1 wt %. v79 cells were used to evaluate the cytotoxicity of the graft polymers, but no cytotoxicity was observed. The graft polymers containing cholesteryl groups effectively enhanced the solubility of paclitaxel in an aqueous solution. PMID- 16677025 TI - Vinyl polymers based on L-histidine residues. Part 2. Swelling and electric behavior of smart poly(ampholyte) hydrogels for biomedical applications. AB - Hydrogels based on the uncharged N-isopropylacrylamide and the ionic ampholyte N acryloyl-L-histidine showed a reversible multiple-responsive volume change and volume phase transition behavior in aqueous solution. The phase transition phenomenon was induced by the temperature, the pH, the salt-type concentration, and the electric potential. The kind of cation (Na+, K+, Cs+, Mg2+, Ca2+, Sr2+) and anion (Cl-, ClO4-, NO3-, SO4(2-)) strongly influenced the critical concentration that improved the phase separation of the gels. The volume of the collapsed gel can be hundred times smaller than that of the swollen one. The oscillatory swelling of the gels in response to temperature and pH (4 and 9) changes was fast and reversible, while the contractile behavior in the electric field showed response only at pH 9, i.e., when the amount of negative charges on the L-histidine residues predominated. The electrically induced anisotropic gel deswelling was attributed to the syneresis of water from the gel. The nontoxicity against the RAW264 cell line and the low osmotic pressure exhibited by the swollen gels make these compounds useful scaffolds for human organs. The ability to load and release an ionizable drug molecular model (ferulic acid) from the hydrogels was shown also at different pH values. PMID- 16677026 TI - Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: attachment strength of the diatom Navicula and the green alga Ulva. AB - To understand the role of surface wettability in adhesion of cells, the attachment of two different marine algae was studied on hydrophobic and hydrophilic polymer surfaces. Adhesion of cells of the diatom Navicula and sporelings (young plants) of the green macroalga Ulva to an underwater surface is mainly by interactions between the surface and the adhesive exopolymers, which the cells secrete upon settlement and during subsequent colonization and growth. Two types of block copolymers, one with poly(ethylene glycol) side-chains and the other with liquid crystalline, fluorinated side-chains, were used to prepare the hydrophilic and hydrophobic surfaces, respectively. The formation of a liquid crystalline smectic phase in the latter inhibited molecular reorganization at the surface, which is generally an issue when a highly hydrophobic surface is in contact with water. The adhesion strength was assessed by the fraction of settled cells (Navicula) or biomass (Ulva) that detached from the surface in a water flow channel with a wall shear stress of 53 Pa. The two species exhibited opposite adhesion behavior on the same sets of surfaces. While Navicula cells released more easily from hydrophilic surfaces, Ulva sporelings showed higher removal from hydrophobic surfaces. This highlights the importance of differences in cell surface interactions in determining the strength of adhesion of cells to substrates. PMID- 16677027 TI - Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune. AB - Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques. This paper reports the first observation of reduction in nanoscale relative surface friction of Sc3 hydrophobin protein modified polymeric surfaces. Relative friction coefficients were dramatically reduced and hydrophilicity increased for polymer surfaces modified with Sc3 hydrophobin thin films. Morphology of the protein films as well as degree of surface modification was observed to be a function of film formation technique and composition of the substrate. PMID- 16677028 TI - Effect of Ca2+, Ba2+, and Sr2+ on alginate microbeads. AB - Microcapsules of alginate cross-linked with divalent ions are the most common system for cell immobilization. In this study, we wanted to characterize the effect of different alginates and cross-linking ions on important microcapsule properties. The dimensional stability and gel strength increased for high-G alginate gels when exchanging the traditional Ca2+ ions with Ba2+. The use of Ba2+ decreased the size of alginate beads and reduced the permeability to immunoglobulin G. Strontium gave gels with characteristics lying between calcium and barium. Interestingly, high-M alginate showed an opposite behavior in combination with barium and strontium as these beads were larger than beads of calcium-alginate and tended to swell more, also resulting in increased permeability. Binding studies revealed that different block structures in the alginate bind the ions to a different extent. More specifically, Ca2+ was found to bind to G- and MG-blocks, Ba2+ to G- and M-blocks, and Sr2+ to G-blocks solely. PMID- 16677029 TI - RGD conjugation to polyethyleneimine does not improve DNA delivery to bone marrow stromal cells. AB - Bone marrow stromal cells (BMSC) modified with therapeutic genes are being actively pursued for gene therapy protocols. To develop safe and effective nonviral methods for BMSC modification, the cationic polymer polyethyleneimine (PEI) has been utilized to condense plasmid DNA for intracellular delivery. This study was conducted to explore the feasibility of increasing the PEI's effectiveness by coupling integrin-binding arginine-glycine-aspartic acid (RGD) peptides to the polymer. BMSC from rats were isolated and expanded in culture for gene transfer studies. In contrast to our expectations, RGD-conjugated PEI did not exhibit an enhanced binding to BMSC. This was the case where the peptides were conjugated to PEI by short, disulfide linkages or long poly(ethylene glycol) linkages. Using a reporter gene for the enhanced green fluorescent protein, the transfection efficiency of RGD-conjugated PEI was also lower than the delivery by the native PEI, which exhibited equivalent transfection efficiency to that of an adenovirus. We conclude that native PEI was sufficient for the transformation of BMSC and that coupling of the integrin-binding RGD-peptides did not improve the effectiveness of this polymer for BMSC transfection. PMID- 16677030 TI - Resilient bioresorbable copolymers based on trimethylene carbonate, L-lactide, and 1,5-dioxepan-2-one. AB - The new combinations of monomers presented in this work were evaluated in order to create an elastic material for potential application in soft tissue engineering. Thermoplastic elastomers (TPE) of trimethylene carbonate (TMC) with L-lactide (LLA) and 1,5-dioxepan-2-one (DXO) have been synthesized using a cyclic five-membered tin alkoxide initiator. The block copolymers were designed in such a way that poly(trimethylene carbonate-co-1,5-dioxepan-2-one) formed an amorphous middle block and the poly(L-lactide) (PLLA) formed semicrystalline terminal blocks. The amorphous middle block consisted of relatively randomly distributed TMC and DXO monomer units, and the defined block structure of the PLLA terminal segments was confirmed by 13C NMR. The properties of the TMC-DXO-LLA copolymers were compared with those of triblock copolymers based either on LLA-TMC or on LLA DXO. Differential scanning calorimetry and dynamic mechanical analysis data confirmed the micro-phase separation in the copolymers. The mechanical properties of the copolymers were evaluated using tensile testing and cycling loading. All of the copolymers synthesized showed a highly elastic behavior. The properties of copolymers could be tailored by altering the proportions of the different monomers. PMID- 16677031 TI - Polystyrene microgel amphiphiles with maltohexaose. Synthesis, characterization, and potential applications. AB - 4-Vinylbenzyl maltohexaoside peracetate (1) was copolymerized with divinylbenzene (DVB) using 1-phenyl-1-(2',2',6',6'-tetramethyl-1'-piperidinyloxy)ethane (2) in m xylene. The copolymerizations were performed at 138 degrees C for 20 h using the mole fraction of 1 in the total feed of 1 and DVB (F1: [1]/[1]+[DVB]) varying from 0.11 to 0.38, affording polymeric products in yields ranging from 32 to 40%. The characterizations by linear PS-calibrated size exclusion chromatography (SEC), dynamic laser light scattering (DLS) measurements, and 1H NMR spectroscopy indicated that the product was assignable to the cross-linked poly(4-vinylbenzyl maltohexaoside peracetate) particle which is able to produce stable solutions, i.e., the PSt microgel with acetyl maltohexaose, 3. The specific rotations ([alpha]D23, c = 1.0 CHCl3) of 3 ranged from +43.3 degrees to +85.6 degrees. The average molar masses determined by the static laser light scattering (SLS) measurement of 3, M(w,SLS)'s, were from 64,700 to 118,000, which were calculated using the respective refractive index increments, dn/dc's, ranging from 0.03387 to 0.08340. The apparent numbers of the 1, 2, and DVB units in 3, N1, N2, and N(DVB), which were estimated from the respective [alpha]D23 values, M(w,SLS)'s, and real yields, ranged from 22 to 35, from 7 to 26, and from 146 to 506, respectively. The deacetylation of 3 was achieved by treatment with sodium methoxide in dry 1,4-dioxane to produce the PSt microgel with maltohexaose as the hydrophilic segment, 4, as a white solid. The solubility of 4 in various solvents was examined, indicating that a hydrophilic property was effectively introduced. Notably, 4 gave clear solutions in the mixed solvent of 1,4-dioxane and H2O. The ability to solubilize fullerite (mixture of fullerenes, C60/C70 = ca. 9/1) in aqueous solutions was examined according to the literature method. Approximately, 100 mg of 4 (1.7 micromol) solubilizes 1.3 mg of fullerite (1.7 micromol). PMID- 16677032 TI - Poly(propylacrylic acid) enhances cationic lipid-mediated delivery of antisense oligonucleotides. AB - The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved 2- to 3-fold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides. PMID- 16677033 TI - Polyelectrolyte nanoparticles with high drug loading enhance the oral uptake of hydrophobic compounds. AB - In the pharmaceutical industry, orally active compounds are required to have sufficient water solubility to enable dissolution within the gastrointestinal tract prior to absorption. Limited dissolution within the gastrointestinal tract often reduces the bioavailability of hydrophobic drugs. To improve gastrointestinal tract dissolution, nonaqueous solvents are often used in the form of emulsions and microemulsions. Here, we show that oil-free polyelectrolyte nanosystems (micellar dispersions and 100-300 nm particles) prepared from poly(ethylenimines) derivatized with cetyl chains and quaternary ammonium groups are able to encapsulate high levels of hydrophobic drug (0.20 g of drug per g of polymer) for over 9 months, as demonstrated using cyclosporine A (log P = 4.3). The polyelectrolytes facilitate the absorption of hydrophobic drugs within the gastrointestinal tract by promoting drug dissolution and by a hypothesized mechanism involving paracellular drug transport. Polyelectrolyte nanoparticle drug blood levels are similar to those obtained with commercial microemulsion formulations. The polyelectrolytes do not promote absorption by inhibition of the P-glycoprotein efflux pump. PMID- 16677034 TI - Negative Poisson ratio of crystalline cellulose in kraft cooked Norway spruce. AB - The tensile properties of kraft cooked Norway spruce were studied by tensile testing with in situ X-ray diffraction (XRD). Samples were of earlywood, cooked for varying times. The total lignin content of the samples was between 21.7% and 9.3%. Tensile tests with XRD were performed on wet samples, without XRD on dry samples. The tensile strength, the modulus of elasticity (MOE), and the elongation at fracture/yield were determined. X-ray diffraction was used to determine the microfibril angle (MFA) and the deformation of crystalline cellulose by monitoring the reflections 200 and 004. The (X-ray) Poisson ratio of crystalline cellulose was calculated, both before and after the yield point. The tensile strength and the MOE of the wet samples were significantly lower than in the dry samples. The tensile properties of dry samples were similar to dry earlywood samples of untreated Norway spruce. The MFA only showed notable changes due to strain when it was initially large, when a diminishing effect was observed. The Poisson ratio of crystalline cellulose was negative. The average values ranged between -0.26 and -1.17 before the yield point and between -0.86 and -1.05 after the yield point. PMID- 16677035 TI - Impact of aldehyde content on amphotericin B-dextran imine conjugate toxicity. AB - The biocompatibility of oxidized dextran (40 kDa) was investigated in vitro. The contribution of aldehyde groups to the toxicity of polymer-drug conjugates, such as dextran-amphotericin B (AmB) was evaluated. Oxidized dextran was proved to be toxic against the RAW 264.7 cell line with an IC50 of 3 micromol/mL aldehydes. Modification of aldehyde groups and their reaction with ethanolamine reduced the toxicity at least 15-fold. Accordingly, the antifungal and antileishmanial dextran-AmB imine conjugate, which contains unreacted aldehyde groups, was modified with ethanolamine and compared to dextran-AmB amine and imine conjugates. Modification of the imine conjugate with ethanolamine reduced its toxicity toward the RAW cell line by 100%. The effect on Leishmania major parasites was 5 times higher than that of the dextran-AmB amine conjugate. The dextran-AmB-ethanolamine conjugate was at least 15 times less hemolytic than free AmB. Stability and drug release profiles in buffer solution were investigated. The imine conjugates released free AmB while the amine conjugate did not. It is concluded that aldehyde groups may contribute to cell toxicity. This toxicity is reduced by converting the aldehyde groups into imine conjugates with ethanolamine. The results have direct implications toward the safety of AmB polysaccharide conjugates used against fungal and leishmanial infections. PMID- 16677036 TI - Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst. AB - The present work attempts to deal with the stability and reusability aspect of nitrilase from Alcaligenes faecalis for the production of (R)-(-)-mandelic acid. Four entrapment matrixes were screened to search for a suitable support, and alginate was found to have significant process advantages over its other counterparts. Thermodynamic analysis allowed us to account for decreased enantioselectivity (E) as a result of immobilization. The system was also characterized based on the Thiele modulus (phi). Efficient reusability of the biocatalyst up to 35 batches was achieved by immobilization as compared to 9 batches for free cells, and cross-linking extended it further to 40 batches. Finally, synthetic utility of the immobilized biocatalyst was demonstrated on a preparative scale to produce 640 g of (R)-(-)-mandelic acid with 97% enantiomeric excess (ee). PMID- 16677037 TI - Molecular blending by polymerization of intercalated solvent. Poly(gamma-benzyl-L glutamate)/benzyl methacrylate as a model system. AB - The aim of the present research is to obtain blending between a polymer and a (polymerized) solvent on the molecular level. Because of its rigid rod structure, poly(gamma-benzyl-L-glutamate) (PBLG) is chosen as the polymer. Benzyl methacrylate (BzMA) has been chosen as the solvent for two reasons. First, the structure of the solvent is very similar to the structure of the side chain of PBLG, favoring interactions between the two materials. Second, the solvent can be polymerized, because of the presence of a C=C bond. In cast films of PBLG and BzMA separate zones of the polymer and solvent are present. Wide-angle X-ray diffraction and Raman results show that upon heating the cast films homogenization occurs and solvent molecules intercalate between the helices of PBLG. At 150 degrees C a hexagonal packing is obtained. The dimensions of the obtained packing depend on the solvent concentration, which confirms that solvent molecules are indeed present within the crystalline lattice. DSC experiments imply that the observed changes upon heating correspond to thermodynamic processes. On cooling the homogeneous samples, disordering of the hexagonal packing occurs. Polymerization of the homogeneous samples results in a disordering of the hexagonal packing and in a contraction of the unit cell. The latter once more confirms that solvent molecules are indeed present within the crystalline lattice. The applied principle of polymerization of a solvent in a molecular homogeneous system can be favorable for many applications, for which morphology control at the molecular level is required. PMID- 16677038 TI - Morphology and properties of soy protein and polylactide blends. AB - Blends of soy protein (SP) and a semicrystalline polylactide (PLA) were prepared using a twin-screw extruder. The melt rheology, phase morphology, mechanical properties, water resistance, and thermal and dynamic mechanical properties were investigated on specimens prepared by injection molding of these blends. The melt flowability of soy-based plastics was improved through blending with PLA. Scanning electron microscopy revealed that a co-continuous phase structure existed in the blends with soy protein concentrate (SPC) to PLA ratios ranging from 30:70 to 70:30. SPC/PLA blends showed fine co-continuous phase structures, while soy protein isolate (SPI)/PLA blends presented severe phase coarsening. At the same SP to PLA ratios, SPC/PLA blends demonstrated a higher tensile strength than SPI/PLA blends. The water absorption of soy plastics was greatly reduced by blending with PLA. The compatibility was improved by adding 1-5 phr poly(2-ethyl 2-oxazoline) (PEOX) in the blends, and the resulting blends showed an obvious increase in tensile strength and a reduction in water absorption for SPI/PLA blends. The compatibility between SP and PLA was evaluated by mechanical testing, dynamic mechanical analysis (DMA), water absorption, and scanning electron microscopy (SEM) experiments. Differential scanning calorimetry (DSC) revealed that PLA in the blends was mostly amorphous in the injection molded articles, and SP accelerated the cold crystallization and could increase the final crystallinity of PLA in the blends. PMID- 16677039 TI - Phase behavior of aqueous solutions of bovine serum albumin in the presence of dextran, at rest, and under shear. AB - The demixing conditions for aqueous solutions of bovine serum albumin (BSA, fraction V) and for joint solutions of BSA plus dextran (DEX, M(w) = 2000 kg/mol) were determined by turbidimetric measurements as a function of composition, temperature, and shear rate. Aqueous solutions of BSA phase separate upon heating. Within the region of BSA concentrations between 0.05 and 32 wt %, the demixing temperature, T1, falls from ca. 65 degrees C to an almost constant value of 45 degrees C. Adding DEX to the BSA solutions reduces the homogeneous region of the mixture drastically where the amount of DEX required to lower T1 to 25 degrees C decreases rapidly as the concentration of BSA is raised. Experiments concerning the influences of shear have been performed for the ternary system up to 500 s(-1). They demonstrate that the content of dextran determines the sign of the effect. At low DEX concentrations, the mechanical field favors the homogeneous state (shear-induced mixing), whereas the opposite effect (shear induced demixing) is observed at high DEX concentrations. Possible reasons for this observation are discussed. PMID- 16677040 TI - Design of biocompatible chitosan microgels for targeted pH-mediated intracellular release of cancer therapeutics. AB - We report the rational design of a chitosan-based drug delivery system. The chitosan derivative N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC) was ionically cross-linked by sodium tripolyphosphate (TPP) to form sub 200-nm microgels that are responsive to pH changes. When these microgels were loaded with methotrexate disodium (MTX), a cytotoxic drug for cancer treatment, and conjugated to the targeting biomolecule apo-transferrin, a protein known to enter cells via receptor-mediated endocytosis, enhanced killing of immortalized HeLa cells was observed. In this intracellular delivery method, the microgel was exposed to low-pH environments that caused the chitosan to swell and release the drug. This rational drug delivery design may be useful in enhancing cancer therapy and reducing side effects. PMID- 16677041 TI - Modeling and experimental investigation of rheological properties of injectable poly(lactide ethylene oxide fumarate)/hydroxyapatite nanocomposites. AB - Injectable multiphasic polymer/ceramic composites are attractive as bioresorbable scaffolds for bone regeneration because they can be cross-linked in situ and are osteoconductive. The injectability of the composite depends on the nanoparticle content and the energetic interactions at the polymer/particle interface. The objective of this research was to determine experimentally the rheological properties of the PLEOF/apatite composite as an injectable biomaterial and to compare the viscoelastic response with the predictions of a linear elastic dumbbell model. A degradable in situ cross-linkable terpolymer based on low molecular weight poly(L-lactide) and poly(ethylene oxide) linked by unsaturated fumarate groups is synthesized. The poly(L-lactide-co-ethylene oxide-co-fumarate) (PLEOF) terpolymer interacts with the surface of the apatite nanoparticles by polar interactions and hydrogen bonding. A kinetic model is developed that takes into account the adsorption/desorption of polymer chains to/from the nanoparticle surface. Rheological properties of the aqueous dispersion of PLEOF terpolymer reinforced with nanosized hydroxyapatite (HA) particles are investigated using mechanical rheometry. To this end, we performed a series of rheological experiments on un-cross-linked PLEOF reinforced with different volume fractions of HA nanoparticles. The results demonstrate that the observed nonlinear viscoelasticity at higher shear rates is controlled by the energetic interactions between the polymer chains and dispersed particle aggregates and by the rate of the adsorption/desorption of the chains to/from the surface of the nanoparticles. PMID- 16677042 TI - Degradation of polyaminophosphazenes: effects of hydrolytic environment and polymer processing. AB - Polyphosphazenes with amino acid ester side groups show potential as hydrolytically degradable materials for biomedical applications. This study focuses on practical aspects of their use as biodegradable materials, such as effects of the hydrolytic environment and sample processing. Poly[di(ethyl glycinato)phosphazene], PEGP, and poly[di(ethyl alaninato)phosphazene], PEAP, were prepared by macromolecular substitution reaction, ensuring the absence of the residual chlorine atoms to avoid their influence on the hydrolysis. The kinetics of polymer degradation was studied by simultaneously measuring polymer mass loss, molecular weight decrease, and the release of phosphates and ammonia. The effect of pH, buffer composition, temperature, casting solvents, and film thickness were investigated. PMID- 16677043 TI - Specific interactions versus counterion condensation. 2. Theoretical treatment within the counterion condensation theory. AB - Polyuronates such as pectate and alginate are very well-known examples of biological polyelectrolytes undergoing, upon addition of divalent cations, an interchain association that acts as the junction of an eventually formed stable hydrogel. In the present paper, a thermodynamic model based on the counterion condensation theory has been developed to account for this cation-induced chain pairing of negatively charged polyelectrolytes. The strong interactions between cross-linking ions and uronate moieties in the specific binding site have been described in terms of chemical bonding, with complete charge annihilation between the two species. The chain-pairing process is depicted as progressively increasing with the concentration of cross-linking counterions and is thermodynamically defined by the fraction of each species. On these bases, the total Gibbs energy of the system has been expressed as the sum of the contributions of the Gibbs energy of the (single) chain stretches and of the (associated) dimers, weighted by their respective fractions 1 - theta and theta. In addition, the model assumes that the condensed divalent counterions exhibit an affinity free-energy for the chain, G(C)(aff,0), and the junction, G(D)(aff,0), respectively. Moreover, a specific Gibbs energy of chemical bonding, G(bond,0), has been introduced as the driving force for the formation of dimers. The model provides the mathematical formalism for calculating the fraction, theta, of chain dimers formed and the amount of ions condensed and bound onto the polyelectrolyte when two different types of counterions (of equal or different valence) are present. The effect of the parameter G(bond,0) has been investigated and, in particular, its difference from G(C,D)(aff,0) was found to be crucial in determining the distribution of the ions into territorial condensation and chemical bonding, respectively. Finally, the effect of the variation of the molar ratio between cross-linking ions and uronic groups in the specific binding sites, sigma0, was evaluated. In particular, a remarkable decrease in the amount of condensed counterions has been pointed out in the case of sigma0 = 1/3, with respect to the value of sigma0 = 1/4, characterizing the traditional "egg-box" structure, as a result of the drop of the charge density of the polyelectrolyte induced by complete charge annihilation. PMID- 16677044 TI - Poly(N-vinylformamide)-A drag-reducing polymer for biomedical applications. AB - Water-soluble drag-reducing polymers (DRPs) were previously demonstrated to significantly increase blood flow, tissue perfusion, and tissue oxygenation when injected intravenously at nanomolar concentrations in various animal models. Turbulent flow drag-reducing ability was proven to be the most important factor defining the potential of polymers to favorably affect blood circulation. Several DRPs were applied in previous in vivo tests, but the search continues for suitable DRPs for biomedical applications. We demonstrated that poly(N vinylformamide) (PNVF) with a molecular weight of 4.5 x 10(6) Da significantly reduced resistance to turbulent flow in a pipe and thus presents a DRP. We also found that the PNVF mechanical degradation is much slower than that of the most commonly used DRP, poly(ethylene oxide). PNVF is known to have low toxicity. Furthermore, our pilot in vivo study showed that PNVF had acceptable biocompatibility and hemodynamic effectiveness and thus could be considered as a DRP candidate for potential clinical use. PMID- 16677045 TI - Laccase-catalyzed polymerization of two phenolic compounds studied by matrix assisted laser desorption/ionization time-of-flight and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with collision induced dissociation experiments. AB - Enzymatic oxidation of two phenolic compounds [syringic acid (3,5-dimethoxy-4 hydroxybenzoic acid) and 2,6-dimethylphenol] was studied. The products of laccase and laccase-mediator-catalyzed oxidation reactions were monitored by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and further analyzed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) MS with collision-induced dissociation (CID) experiments. For the oligomers of syringic acid, some variability was observed in MALDI-TOF analysis. However, the origin of this variability could not be resolved on the basis of MALDI-TOF spectra due to the poor resolution of the instrument in use. The strength of ESI-FTICR MS was the high-resolution data provided from oligomers of syringic acid. The CID experiments were extremely useful for structural studies of oligomers and verified that the variability of the products was due to the end groups; the phenolic hydroxyl group was modified during the oxidation. PMID- 16677046 TI - A blank slate? Layer-by-layer deposition of hyaluronic acid and chitosan onto various surfaces. AB - Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular level interactions defined by the modified surface properties. A promising concept is the so-called "blank slate": essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM. PMID- 16677048 TI - Packing density and structural heterogeneity of insulin amyloid fibrils measured by AFM nanoindentation. AB - A nanoindentation approach based on atomic force microscopy was applied to test the elastic properties of insulin amyloid fibrils. Fibrils exhibited a nearly elastic response to the compressive load. The results, corrected for the finite sample thickness effect, reveal that the fibril Young's modulus is considerably lower than the modulus of protein crystals, suggesting lower packing density in amyloid fibrils. Variation in elasticity among and within fibrils has been studied, showing that the Young's moduli of insulin fibrils have a relatively wide distribution of values, ranging from 5 to 50 MPa. Amyloid fibrils with higher modulus were found to be more wear-resistant during AFM scanning. The measured distribution of elasticity values of different fibrils together with wear-resistance tests indicates structural heterogeneity among fibrils, whereas the structure of individual fibrils appears to be homogeneous. The relative simplicity of the method used in this study can facilitate rapid collection of quantitative information related to the packing density and heterogeneity of fibrils formed by different proteins. PMID- 16677047 TI - Investigation of drug release and matrix degradation of electrospun poly(DL lactide) fibers with paracetanol inoculation. AB - This study was aimed at assessing the potential use of electrospun fibers as drug delivery vehicles with focus on the different diameters and drug contents to control drug release and polymer fiber degradation. A drug-loaded solvent-casting polymer film was made with an average thickness of 100 microm for comparative purposes. DSC analysis indicated that electrospun fibers had a lower T(g) but higher transition enthalpy than solvent-casting polymer film due to the inner stress and high degree of alignment and orientation of polymer chains caused by the electrospinning process. Inoculation of paracetanol led to a further slight decrease in the T(g) and transition enthalpy. An in vitro drug release study showed that a pronounced burst release or steady release phase was initially observed followed by a plateau or gradual release during the rest time. Fibers with a larger diameter exhibited a longer period of nearly zero order release, and higher drug encapsulation led to a more significant burst release after incubation. In vitro degradation showed that the smaller diameter and higher drug entrapment led to more significant changes of morphologies. The electrospun fiber mat showed almost no molecular weight reduction, but mass loss was observed for fibers with small and medium size, which was characterized with surface erosion and inconsistent with the ordinarily polymer degrading form. Further wetting behavior analysis showed that the high water repellent property of electrospun fibers led to much slower water penetration into the fiber mat, which may contribute to the degradation profiles of surface erosion. The specific degradation profile and adjustable drug release behaviors by variation of fiber characteristics made the electrospun nonwoven mat a potential drug delivery system rather than polymer films and particles. PMID- 16677049 TI - Design of mucoadhesive polymeric films based on blends of poly(acrylic acid) and (hydroxypropyl)cellulose. AB - Mixing of aqueous solutions of poly(acrylic acid) and (hydroxypropyl)cellulose results in formation of hydrogen-bonded interpolymer complexes, which precipitate and do not allow preparation of homogeneous polymeric films by casting. In the present work the effect of pH on the complexation between poly(acrylic acid) and (hydroxypropyl)cellulose in solutions and miscibility of these polymers in solid state has been studied. The pH-induced complexation-miscibility-immiscibility transitions in the polymer mixtures have been observed. The optimal conditions for preparation of homogeneous polymeric films based on blends of these polymers have been found, and the possibility of radiation cross-linking of these materials has been demonstrated. Although the gamma-radiation treatment of solid polymeric blends was found to be inefficient, successful cross-linking was achieved by addition of N,N'-methylenebis(acrylamide). The mucoadhesive potential of both soluble and cross-linked films toward porcine buccal mucosa is evaluated. Soluble films adhered to mucosal tissues undergo dissolution within 30-110 min depending on the polymer ratio in the blend. Cross-linked films are retained on the mucosal surface for 10-40 min and then detach. PMID- 16677051 TI - Aging properties of films of plasticized vital wheat gluten cast from acidic and basic solutions. AB - In order to understand the mechanisms behind the undesired aging of films based on vital wheat gluten plasticized with glycerol, films cast from water/ethanol solutions were investigated. The effect of pH was studied by casting from solutions at pH 4 and pH 11. The films were aged for 120 days at 50% relative humidity and 23 degrees C, and the tensile properties and oxygen and water vapor permeabilities were measured as a function of aging time. The changes in the protein structure were determined by infrared spectroscopy and size-exclusion and reverse-phase high-performance liquid chromatography, and the film structure was revealed by optical and scanning electron microscopy. The pH 11 film was mechanically more stable with time than the pH 4 film, the latter being initially very ductile but turning brittle toward the end of the aging period. The protein solubility and infrared spectroscopy measurements indicated that the protein structure of the pH 4 film was initially significantly less polymerized/aggregated than that of the pH 11 film. The polymerization of the pH 4 film increased during storage but it did not reach the degree of aggregation of the pH 11 film. Reverse-phase chromatography indicated that the pH 11 films were to some extent deamidated and that this increased with aging. At the same time a large fraction of the aged pH 11 film was unaffected by reducing agents, suggesting that a time-induced isopeptide cross-linking had occurred. This isopeptide formation did not, however, change the overall degree of aggregation and consequently the mechanical properties of the film. During aging, the pH 4 films lost more mass than the pH 11 films mainly due to migration of glycerol but also due to some loss of volatile mass. Scanning electron and optical microscopy showed that the pH 11 film was more uniform in thickness and that the film structure was more homogeneous than that of the pH 4 film. The oxygen permeability was also lower for the pH 11 film. The fact that the pH 4 film experienced a larger and more rapid change in its mechanical properties with time than the pH 11 film, as a consequence of a greater loss of plasticizer, was presumably due to its initial lower degree of protein aggregation/polymerization. Consequently, the cross-link density achieved at pH 4 was too low to effectively retain volatiles and glycerol within the matrix. PMID- 16677050 TI - Enzymatic polymerization to novel polysaccharides having a glucose-N acetylglucosamine repeating unit, a cellulose-chitin hybrid polysaccharide. AB - A cellulose-chitin hybrid polysaccharide having alternatingly beta(1-->4)-linked D-glucose (Glc) and N-acetyl-d-glucosamine (GlcNAc) was synthesized via two modes of enzymatic polymerization. First, a sugar oxazoline monomer of Glcbeta(1- >4)GlcNAc (1) was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. Monomer 1 was recognized by chitinase from Bacillus sp., giving rise to a cellulose-chitin hybrid polysaccharide (2) via ring-opening polyaddition with perfect regioselectivity and stereochemistry. Molecular weight (M(n)) of 2 reached 4030, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAcbeta(1-->4)Glc (3) was synthesized for the catalysis of cellulase from Trichoderma viride. The enzyme catalyzed polycondensation of 3, providing a cellulose-chitin hybrid polysaccharide (4) in regio- and stereoselective manner. M(n) of 4 reached 2840, which corresponds to 16 saccharide units. X-ray diffraction measurements revealed that these hybrid polysaccharides did not form any characteristic crystalline structures. Furthermore, these unnatural hybrids of 2 and 4 were successfully digested by lysozyme from human neutrophils. PMID- 16677052 TI - Synthesis of copolymers containing an active ester of methacrylic acid by RAFT: controlled molecular weight scaffolds for biofunctionalization. AB - We report the controlled radical copolymerization of N-(2 hydroxypropyl)methacrylamide (HPMA) with a monomer containing an active ester, N methacryloyloxysuccinimide (NMS), by reversible addition fragmentation chain transfer (RAFT). The large difference in the reactivity ratios of HPMA and NMS resulted in significant variations in copolymer composition with increasing conversion during batch copolymerization. The use of a semi-batch copolymerization method, involving the gradual addition of the more reactive NMS, allowed uniformity of copolymer composition to be maintained during the polymerization. We synthesized polymers in a wide range of molecular weights (M(n) = 3000-50,000 Da) with low polydispersities (1.1-1.3). The effect of the ratio of monomer to chain transfer agent (CTA) on the molecular weight of the polymer was investigated. Given the numerous applications of poly(HPMA)-based conjugates in designing polymeric therapeutics, these controlled molecular weight activated polymers represent attractive scaffolds for biofunctionalization. As a demonstration, we attached a peptide to the activated polymer backbone to synthesize a potent controlled molecular weight polyvalent inhibitor of anthrax toxin. PMID- 16677053 TI - Building on the WLF/free volume framework: utilization of the coupling model in the relaxation dynamics of the gelatin/cosolute system. AB - The onset of softening in the glass transition dispersion of the gelatin/cosolute system at 78% solids was examined using the stress relaxation modulus and dynamic oscillatory data on shear. Measurements were made between 5 and -70 degrees C, and isothermal runs were reduced to a master curve covering 21 orders of magnitude in the time domain. The sharpness with which the mechanical properties of our system changed with temperature was reflected in the shift factor a(T) used to pinpoint the glass transition temperature (T(g)). The prevalent analytical framework traditionally employed to follow the transition from the rubbery to glasslike consistency in biomaterials is that of the free volume theory in conjunction with the WLF equation. Increasingly, the combined WLF/free volume approach is challenged by the coupling model, which is able to provide additional insights into the physics of intermolecular interactions in synthetic materials at the vicinity of T(g). The model in the form of the Kohlrausch Williams-Watts function described well the spectral shape of the local segmental motions of gelatin/cosolute at T(g). The analysis provided the intermolecular interaction constant and apparent relaxation time, parameters which depend on chemical structure. Results appear to be encouraging for further explorations of the dynamics of densely packed biomaterials at the glass transition region. PMID- 16677054 TI - Effect of particle size, composition, and thermal treatment on the crystalline structure of polycaprolactone nanoparticles. PMID- 16677055 TI - The case for combination therapy as first-line treatment for the type 2 diabetic patient. AB - The glycosylated hemoglobin (HbA(1c)) goal in patients with type 2 diabetes mellitus should be to achieve as low a value as can be obtained without causing significant or frequent hypoglycemia. This is best achieved by utilizing agents that lower glucose levels without causing hypoglycemia (thiazolidinediones and metformin). To maintain these low HbA(1c) values and avoid the utilization of insulin secretagogues or insulin, which are associated with hypoglycemia and suboptimal dosing leading to higher HbA(1c) values, drugs that maintain or improve pancreatic beta-cell function (thiazolidinediones and possibly incretin based therapies) should be utilized. Restoration of first-phase insulin release, as has been shown with thiazolidinediones, will not only improve postprandial hyperglycemia but will also improve postprandial hyperlipidemia, both of which will decrease cardiac risk. Utilizing small doses of two drugs will also result in a decreased incidence of adverse effects compared with a large dose of a single drug. The use of fixed-dose combination oral antihyperglycemics will not only improve compliance but will often decrease costs compared with individual component dual therapy. PMID- 16677056 TI - Prospects for the development of long-acting formulations of human somatropin. AB - In healthy humans, growth hormone (GH) is secreted in distinct pulses with an underlying nyctohemeral pattern. Current forms of somatropin replacement are unable to closely mimic such a release pattern, but are still able to exert the beneficial action of GH. A limited number of short-term studies in rodents and humans suggest that longitudinal growth may be superior when somatropin is given with a pulsatile mode of administration, whereas hepatic insulin-like growth factor-I generation and beneficial changes in body composition appear to be equal or even enhanced with continuous somatropin administration.Recent developments in drug delivery technology have allowed the use of slow-release preparations of somatropin in humans. The most successful technology so far has been the encapsulation of somatropin molecules in poly(D,L-lactic-co-glycolic acid) biodegradable microspheres. Pharmacokinetic and pharmacodynamic data have been published on two such preparations; Nutropin Depot((R)) and hGH-Biosphere((R)). The latter has a superior release profile, but outcomes data from multicenter trials in both children and adults have been presented for the former: catch-up growth was observed in children, although to a lesser degree than historic comparative data obtained with the use of daily somatropin injections and the effects on metabolic derangements in GH-deficient patients appeared similar to those observed with daily injections. Improved sustained-release somatropin preparations will need further study of their long-term efficacy, but, if successful, will be highly attractive in terms of patient compliance and convenience. PMID- 16677057 TI - Progress in islet transplantation in patients with type 1 diabetes mellitus. AB - More than 500 patients with type 1 diabetes mellitus have now received islet transplants at over 50 institutions worldwide in the past 5 years. Rates of insulin independence at 1 year with current protocols are impressive. However, inexorable decay of islet function over time indicates that there are many opportunities for improvement. Improved control of glycosylated hemoglobin and reduced risk of recurrent hypoglycemia are seen as important benefits of islet transplantation, irrespective of the status regarding insulin independence. For the use of islet transplantation to expand it is essential that the donor-to recipient ratio be reliably reduced to 1 : 1. Enormous opportunities lie ahead for the development of successful living donor islet transplantation, single donor protocols, improved engraftment, islet proliferation in vitro and in the recipient, alternative islet sources, and novel tolerizing drugs. With these emerging opportunities, islet transplantation may expand to include more patients with type 1 diabetes, including children, and will not be restricted to the most unstable forms of the disease, as it is today. PMID- 16677058 TI - Somatropin therapy and cognitive function in adults with growth hormone deficiency : a critical review. AB - Both growth hormone (GH) and insulin-like growth factor (IGF)-I have receptors in the brain, in particular in areas that are involved in cognitive function. Therefore, it has been hypothesized that GH deficiency can lead to cognitive dysfunction, and that somatropin replacement therapy may have beneficial effects on cognitive function in GH-deficient patients. In this review, an overview is given regarding the possible effects of decreased activity of the GH/IGF-I axis and somatropin therapy in GH deficiency in relation to cognitive function. The available data regarding cognitive function in GH-deficient patients are limited, but suggest that this condition can lead to specific cognitive changes, in particular attentional deficits and altered processing speed. The underlying mechanisms and the effects of somatropin treatment on cognitive function in GH deficiency are still unclear. Similar studies to those performed in patients with GH deficiency have been performed regarding the cognitive changes in elderly patients with relatively low GH and/or IGF-I levels. Large controlled studies regarding the effects and safety of somatropin treatment in healthy elderly patients have not been performed. PMID- 16677059 TI - Thiazolidinediones for the therapeutic management of polycystic ovary syndrome : impact on metabolic and reproductive abnormalities. AB - Polycystic ovary syndrome (PCOS) is a diagnosis made between late adolescence and the menopause in 5-10% of women. PCOS is a heterogeneous disorder of unknown etiology characterized by hyperandrogenic chronic anovulation. This syndrome consists of a diverse constellation of signs and symptoms, such as hirsutism, acne, acanthosis nigricans, obesity, menstrual irregularities, anovulation, and/or infertility. Features of the metabolic syndrome, including obesity, insulin resistance, and dyslipidemia, are common in this patient population. Recent insights into the pathophysiology of PCOS have shown insulin resistance and hyperinsulinemia to play a substantial role. Insulin resistance is increasingly recognized as a chronic, low-level, inflammatory state. Recent studies show that serum levels of inflammatory mediators, such as tumor necrosis factor-alpha and interleukin-6, are increased in the insulin-resistant conditions of obesity and PCOS. The optimal modality for long-term treatment should have positive effects on androgen synthesis, sex hormone-binding globulin production, the lipid profile, insulin sensitivity, inflammatory mediators, and clinical symptoms including acne, hirsutism, and irregular menstrual cycles. Treatment with insulin-sensitizing agents is a relatively new therapeutic strategy in women with PCOS. Current research has shown that the use of diabetes mellitus management practices aimed at reducing insulin resistance and hyperinsulinemia (such as weight reduction and the administration of oral antidiabetic drugs) can not only reverse testosterone and luteinizing hormone abnormalities and restore menstrual cycles, but can also improve glucose, insulin, proinflammatory cytokine, and lipid profiles.Clinical treatment with troglitazone, a member of the thiazolidinedione family, for the management of PCOS complications such as insulin resistance, hyperandrogenism, and anovulation was found to have beneficial effects; however, it was taken off the market over concerns of hepatotoxicity. Although troglitazone is no longer available in the US, numerous clinical trials have established the role of thiazolidinediones in the treatment of women with PCOS. Clinical data emerging regarding the utility of two of the newer, safer thiazolidinediones, pioglitazone and rosiglitazone, for this patient population, consistently demonstrate effective improvements of endocrine and ovulatory performance in women with PCOS. The benefit and importance of lifestyle modification and weight reduction, when it can be achieved, is still an important component in the long-term treatment of PCOS. Pharmacologic reduction in insulin levels using thiazolidinediones appears to offer another therapeutic modality for PCOS, which may ameliorate the progress of both hyperinsulinemia and hyperandrogenism. However, additional studies of patients so treated are necessary before these agents can be considered first-line treatment for PCOS. Convincing data from randomized controlled trials with sufficient power to detect both the benefits and risks of long-term treatment with thiazolidinediones in women with PCOS remain to be obtained. PMID- 16677060 TI - Spotlight on pioglitazone in type 2 diabetes mellitus. AB - Pioglitazone (Actos(trade mark)) is an antihyperglycemic agent that, in the presence of insulin resistance, increases hepatic and peripheral insulin sensitivity, thereby inhibiting hepatic gluconeogenesis and increasing peripheral and splanchnic glucose uptake. Pioglitazone is generally well tolerated, weight gain and edema are the most common emergent adverse events, and there are no known drug interactions between pioglitazone and other drugs. In clinical trials in patients with type 2 diabetes mellitus, pioglitazone as monotherapy, or in combination with metformin, repaglinide, insulin, or a sulfonylurea, induced both long- and short-term improvements in glycemic control and serum lipid profiles. Pioglitazone was also effective in reducing some measures of cardiovascular risk and arteriosclerosis. Pioglitazone thus offers an effective treatment option for the management of patients with type 2 diabetes. PMID- 16677061 TI - Opinion and evidence for treatments in endocrine disorders. AB - New treatments and treatment protocols for endocrine disorders are evolving rapidly, and research and development activity in the endocrinology field is high. Optimal therapy remains contentious in some areas. To help you keep up to date with the latest advances worldwide on all aspects of drug therapy and management of endocrine disorders, this section of the journal brings you information selected from the rapid drug news alerting service Inpharma WeeklyInpharma Weekly provides rapid alerts to news on drugs and drug therapy. Summarizing information selected from over 1600 biomedical journals, this newsletter is produced by Adis International Limited and is available in a variety of formats. Please contact your nearest Adis office for subscription details. The use of trade names, identified by ['~'] or the use of a registered (((R))) or trademark (trade mark) symbol, is for product identification purposes only and does not imply endorsement.. Each issue contains easy-to-read summaries of the most important research and development news, clinical studies, treatment guidelines, pharmacoeconomic and adverse drug reaction news, and expert opinion pieces published in the world's top endocrinology journals. PMID- 16677062 TI - Minority status, consumer outcomes, and service inputs in four States. AB - Data from the Center for Outcome Analysis were subjected to independent secondary analysis involving comparisons by ethnic group of eight indicators of service quality for users of adult developmental disability services in four states. Ethnic group membership had very limited or no association with the consumer outcomes and service inputs evaluated. Where significant ethnic group differences were found, they were small, and there was no consistent pattern of a particular ethnic group doing better. These findings may relate to the substantial influence of developmental disability services in the lives of service recipients and to the effect of disability itself, in that adaptive behavior was strongly related to most consumer outcomes. PMID- 16677063 TI - Mandating education of dental graduates to provide care to individuals with intellectual and developmental disabilities. AB - In 2004, The Commission on Dental Accreditation adopted new standards for dental and dental hygiene education programs to ensure the preparation of practitioners to provide oral health services for persons with special health care needs. The course of action leading to the adoption of the new standards, together with the continuing obstacles of limited government support for dental services and the availability of faculty members to provide the needed dental educational experiences is reviewed. Expanding Health Resources and Services Administration definition of medically underserved areas is presented as one approach to improving the delivery of dental services. PMID- 16677064 TI - Supporting aging caregivers and adults with developmental disabilities in future planning. AB - A peer support intervention was developed to support aging caregivers and adults with developmental disabilities in planning for the future. The intervention consisted of a legal/financial training session followed by five additional small group workshops. Pretest and 1-year follow-up surveys were conducted with 29 families participating in the intervention and 19 control families. Outcome measures included future planning activities, caregiving appraisals, discussion of plans with individuals who have developmental disabilities, and choice-making of individuals with developmental disabilities. The intervention significantly contributed to families completing a letter of intent, taking action on residential planning, and developing a special needs trust. Caregiving burden significantly decreased for families in the intervention and daily choice-making of individuals with disabilities increased. PMID- 16677065 TI - Epidemiology of osteoporosis in women with cognitive impairment. AB - Osteoporosis is increasing due to the aging of the population. Women with cognitive impairment from childhood are at disproportionally high risk for osteoporosis and fractures. Suggested explanations for this increased risk include high use of anticonvulsant medications, lower peak bone densities, and higher rates of nonambulation. Down syndrome seems to be an independent risk factor for low bone density and fractures, presumably due to lower muscle tone. Here, the limited information available on the epidemiology of osteoporosis in the general population and in women with cognitive disabilities is briefly reviewed. Health care professionals should consider women with cognitive disabilities at high risk for osteoporosis and fractures. Studies are needed to determine screening regimens and prevention strategies appropriate for women in this population. PMID- 16677066 TI - Injury prevalence among children and adolescents with mental retardation. AB - Childhood injuries lead to increased morbidity and result in significant costs to public insurance programs. People with mental retardation, most of whom are covered by Medicaid, are at high risk for injury, which has implications for community inclusion, a central policy goal. Medicaid data from inpatient, outpatient, and long-term care settings represent an important new resource for injury surveillance in this population. Injury prevalence for 8.4 million Medicaid-eligible children in 26 states was measured using 1999 eligibility and claims data; 36.9% Medicaid beneficiaries ages 1 to 20 with mental retardation had at least one injury claim as compared with 23.5% of those without mental retardation. Prevalence rates are reported by gender and age for a variety of injury types. PMID- 16677068 TI - Reductions continue in average daily populations of large state facilities; nearly 70% decrease between 1980 and 2005. PMID- 16677067 TI - Health outcomes of midlife and older Latina and black American mothers of children with developmental disabilities. AB - The impact of caring for a child with a developmental disability on the physical and mental health of Latina and Black American women was examined. We used the National Health Interview Survey to compare the health of older mothers who were co-residing with a child who had a developmental disability to the health of same age mothers without caregiving responsibilities. Findings show that for both groups, older adult caregivers were more likely to report having limitations from arthritis than their noncaregiving counterparts. Caregiving was associated with more depressive symptoms for Latinas, but this relationship was not found for Black American women. Findings suggest that physical and mental health of caregivers need more attention in research and practice. PMID- 16677070 TI - Working with an Aboriginal community liaison worker. AB - FPA Health (Family Planning NSW) has conducted two integrated clinical and health promotion projects with Aboriginal communities in western NSW, Australia. The first was in Coonamble, a small rural community which had been selected as a pilot site for the Royal Australian College of General Practitioners Women's Aboriginal and Torres Strait Islander Project and was managed by FPA Health with support from the Dubbo/Plains Division of General Practice and Macquarie Area Health Service. The second was in Dubbo, a regional city where FPA Health had an existing centre and which had funding support from the Rio Tinto Aboriginal Foundation. The aim of this article was to share the learning and knowledge gained in managing these projects and to describe the experience of working with Aboriginal Community Liaison Workers who belong to and are supported by, the local Aboriginal community. The article aimed to illustrate the role and value of utilising these workers within a mainstream health service. The beneficial outcomes include improving service provision to Aboriginal women, adding to community knowledge about reproductive and sexual health issues and increasing the cultural knowledge and competency of a mainstream health service organisation. PMID- 16677071 TI - Hydrogen peroxide: a signaling messenger. AB - Hydrogen peroxide (H2O2) is a well-documented component of living cells. It plays important roles in host defense and oxidative biosynthetic reactions. In addition there is growing evidence that at low levels, H2O2 also functions as a signaling agent, particularly in higher organisms. This review evaluates the evidence that H2O2 functions as a signaling agent in higher organisms in light of the known biology and biochemistry of H2O2. All aerobic organisms studied to date from prokaryotes to humans appear to tightly regulate their intracellular H2O2 concentrations at relatively similar levels. Multiple biochemical strategies for rapidly reacting with these low endogenous levels of H2O2 have been elucidated from the study of peroxidases and catalases. Well-defined biochemical pathways involved in the response to exogenous H2O2 have been described in both prokaryotes and yeast. In animals and plants, regulated enzymatic systems for generating H2O2 have been described. In addition oxidation-dependent steps in signal transduction pathways are being uncovered, and evidence is accumulating regarding the nature of the specific reactive oxygen species involved in each of these pathways. Application of physiologic levels of H2O2 to mammalian cells has been shown to stimulate biological responses and to activate specific biochemical pathways in these cells. PMID- 16677072 TI - The diversity of oxidative protein folding. PMID- 16677073 TI - Dynamic retention of Ero1alpha and Ero1beta in the endoplasmic reticulum by interactions with PDI and ERp44. AB - Disulfide bonds are formed in the endoplasmic reticulum (ER) by sequential interchange reactions: Ero1alpha and Ero1beta transfer oxidative equivalents to protein disulfide isomerase (PDI), which in turn oxidizes cargo proteins. Neither Ero1alpha nor Ero1beta contains known ER localization motif(s), raising the question of how they are retained in this organelle. Here the authors show that, unlike endogenous molecules, overexpressed Ero1alpha and Ero1beta are secreted by HeLa transfectants, suggesting saturation of their normal retention mechanism(s). Co-expression of either PDI or ERp44 prevents Ero1 secretion in a KDEL/RDEL dependent way. Covalent interactions between ERp44 and Ero1 are essential for retention. In contrast, a mutant PDI lacking the four cysteines in the two active sites still inhibits secretion, albeit less efficiently. PDI and ERp44 compete for Ero1 binding. PDI also prevents Ero1 aggregation and dimerization, thus chaperoning its own oxidase. This dynamic retention mechanism of Ero1 may be important for fine-tuning the regulation of ER redox homeostasis and quality control. PMID- 16677074 TI - pH dependence of the peptide thiol-disulfide oxidase activity of six members of the human protein disulfide isomerase family. AB - Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds, a process which in vivo is one of the rate limiting steps of protein folding and which is facilitated by the enzyme protein disulfide isomerase (PDI). Higher eukaryotes have multiple members of the PDI family, for example, seventeen human PDIs have been reported to date. With multiple members of the same family being present, even within the same cell, the question arises as to what differential functions are they performing? To date there has been no systematic evaluation of the enzymological properties of the different members of the PDI-family. To address the question of whether different PDI family members have differing thioldisulfide chemistry, we have recombinantly expressed and purified six members of the family, PDI, PDIp, ERp57, ERp72, P5, and PDIr from a single organism, human. An examination of the pH-dependence and nature of the rate limiting step for the peptide thiol-disulfide oxidase activity of these enzymes reveals that, with the exception of PDIr, they are all remarkably similar. In the light of this data potential differential functions for these enzymes are discussed. PMID- 16677075 TI - Differential oxidation of HLA-B2704 and HLA-B2705 in lymphoblastoid and transfected adherent cells. AB - MHC class I molecules are predominantly involved in the presentation of antigens from viral proteins to CD8+ T cells of the immune system. However, MHC proteins can also be linked to autoimmune diseases, and the HLA-B27 allele is expressed by 95% of people with the rheumatic condition ankylosing spondylitis (AS). A precise molecular explanation for the association between HLA-B27 and AS is still lacking, although it is known that inappropriately disulfide bonded HLA-B27 heavy chains can be found at both the cell surface and in the endoplasmic reticulum (ER) of HLA-B27 expressing cells. This papers shows that HLA-B27 heavy chain misfolding does not depend on any unpaired cysteine residue per se when HLA-B27 is highly expressed. Also shown is that major differences exist in the disulfide dependent conformations of two HLA-B27 subtypes, HLA-B2704 and HLA-B2705. The results imply that residues 77, 152, and/or 211 influence the redox potential of the MHC class I heavy chain and suggest that manipulating the redox environment can alter the conformational state of HLA-B27 subtypes. PMID- 16677076 TI - Multidomain flavin-dependent sulfhydryl oxidases. AB - Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs. PMID- 16677077 TI - Extracellular disulfide exchange and the regulation of cellular function. AB - An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin alphaIIbbeta3, from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function. PMID- 16677078 TI - ERp29, an unusual redox-inactive member of the thioredoxin family. AB - Oxidative folding in the endoplasmic reticulum is accomplished by a group of oxidoreductases where the protein disulfide isomerase (PDI) plays a key role. Structurally, redox-active PDI domains, like many other enzymes utilizing cysteine chemistry, adopt characteristic thioredoxin folds. However, this structural unit is not necessarily associated with the redox function and the current review focuses on the interesting example of a loss-of-function PDI-like protein from the endoplasmic reticulum, ERp29. ERp29 shares a common predecessor with PDI; however in the course of divergent evolution it has lost a hallmark active site motif of redox enzymes but retained the characteristic structural fold in one of its domains. Although the functional characterization of ERp29 is far from completion, all available data point to its important role in the early secretory pathway and allow tentative categorization as a secretion factor/escort protein of a broad profile. PMID- 16677079 TI - Fibrillin-1 misfolding and disease. AB - Fibrillin-1 is a 350 kDa calcium-binding protein which assembles to form 10-12 nm microfibrils in the extracellular matrix (ECM). The structure of fibrillin-1 is dominated by two types of disulfide-rich motifs, the calcium- binding epidermal growth factor-like (cbEGF) and transforming growth factor beta binding protein like (TB) domains. Disruption of fibrillin-1 domain structure and function contributes to the pathogenic mechanisms underlying two inherited diseases with very different etiologies: Marfan syndrome (MFS) and homocystinuria (HC). MFS is a connective tissue disease caused by mutations in the fibrillin-1 gene FBN1. Many missense mutations cause variable degrees of fibrillin-1 domain misfolding, which may affect the delivery of fibrillin-1 to the ECM and/or its assembly into microfibrils. HC is a metabolic disorder which affects methionine metabolism and results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Patients with HC often exhibit ocular and skeletal defects resembling the MFS phenotype, suggesting that elevated homocysteine levels may lead to chemical reduction of disulfide bonds within fibrillin-1 domains resulting in the loss of native structure. Protein misfolding therefore is implicated in pathogenic mechanisms underlying MFS and HC. PMID- 16677080 TI - Vitamin K epoxide reductase complex subunit 1 (VKORC1): the key protein of the vitamin K cycle. AB - Vitamin K epoxide, a by-product of the carboxylation of blood coagulation factors, is reduced to vitamin K by an enzymatic system possessing vitamin K epoxide reductase (VKOR) activity. This system is the target of coumarin-derived drugs widely used in thrombosis therapy and prophylaxis. Recently, the key protein of the VKOR system has been identified. The human VKORC1 gene maps to chromosome 16 and consists of 3 exons encoding a 163-amino acid integral ER membrane protein with three or four predicted transmembrane alpha- helices. Expression of human VKORC1 in Spodoptera frugiperda (Sf9) cells and in Pichia pastoris results in enhanced VKOR activity over low endogenous constitutive levels. Sequence based search methods reveal that human VKORC1 belongs to a large family of homologous genes found in vertebrates, insects, plants, protists, archea, and bacteria. All orthologs share five completely conserved amino acids, including two cysteines found in a tetrapeptide motif presumably required for redox function. The recent discovery of the VKORC1 gene has initiated renewed interest in understanding VKOR activity. Analysis of VKORC1 protein structure and function will be crucial in understanding the VKOR catalytic mechanism, how anticoagulant drugs modulate VKOR activity, and the role of VKORC1 in downstream physiological and pathological pathways. PMID- 16677082 TI - Antiaging medicine: antioxidants and aging. PMID- 16677081 TI - Measuring intracellular redox conditions using GFP-based sensors. AB - Recent years have seen the development of methods for analyzing the redox conditions in specific compartments in living cells. These methods are based on genetically encoded sensors comprising variants of Green Fluorescent Protein in which vicinal cysteine residues have been introduced at solvent-exposed positions. Several mutant forms have been identified in which formation of a disulfide bond between these cysteine residues results in changes of their fluorescence properties. The redox sensors have been characterized biochemically and found to behave differently, both spectroscopically and in terms of redox properties. As genetically encoded sensors they can be expressed in living cells and used for analysis of intracellular redox conditions; however, which parameters are measured depends on how the sensors interact with various cellular redox components. Results of both biochemical and cell biological analyses will be discussed. PMID- 16677083 TI - Transcription regulation in NIH3T3 cell clones resistant to diethylmaleate induced oxidative stress and apoptosis. AB - To investigate the molecular mechanisms underlying the induction of cell resistance to oxidative stress, NIH3T3 cell clones (NIH-DEM clones) were isolated and selected for their ability to survive the exposure to diethylmaleate (DEM), a glutathione-depleting agent. The oxidative stress-resistant phenotype of these clones is stable for at least 1 month in the absence of DEM, and includes the resistance also to other apoptosis-inducing stimuli. The expression profile of several antioxidant genes was examined in four of the DEM-resistant clones in the presence and in absence of DEM. The response to the acute exposure to DEM is similar in wild type and DEM-resistant cells, with the exception of the glutathione-S-transferase alpha1 gene, whose expression is highly induced in NIH DEM clones. However, in the absence of an acute stress, the expression of some genes is higher in DEM-resistant clones than in wild-type cells and the gene expression profile significantly varies among the clones. In particular, glutathione-S-transferase alpha1 and cystine/glutamate transporter mRNAs are increased in NIH-DEM-12. In these cells, the promoters of the two genes drive a stronger transcription than in wild-type cells, and this appears to be dependent on the transcription factor Nrf2. PMID- 16677084 TI - Early involvement of ROS overproduction in apoptosis induced by 7 ketocholesterol. AB - Cholesterol oxidation products are increasingly considered as much more bioactive than the parent compound in the multifactor and multistep process that characterizes atherosclerosis. In particular, 7-ketocholesterol has been reported to induce oxidative stress as well as a marked pro-apoptotic effect in vascular cells including macrophages. With the aim to investigate a possible pathogenic correlation between the two events, cultivated murine macrophages were challenged with a concentration of 7-ketocholesterol actually detectable in human vasculature. Conclusive proof was obtained of a primary role of NADPH-oxidase in the overproduction of reactive oxygen species within cells treated with the oxysterol. In addition, such oxidative burst occurred very early after cell intoxication and it was definitely demonstrated as able to lead cells to apoptotic death. In fact, two metabolic inhibitors of NADPH-oxidase and the antioxidant epicatechin very well counteracted 7-ketocholesterol-induced apoptosis by preventing the oxysterol pro-oxidant action. PMID- 16677085 TI - Proteomics analyses of specific protein oxidation and protein expression in aged rat brain and its modulation by L-acetylcarnitine: insights into the mechanisms of action of this proposed therapeutic agent for CNS disorders associated with oxidative stress. AB - Impaired function of the central nervous system (CNS) in aged animals is associated with increased susceptibility to the development of many neurodegenerative diseases. Age-related functional deterioration in brain is consistent with the free radical theory of aging that predicts, among other things, that free radical reactions with and damage to biomolecules, such as proteins and membrane lipid bilayers, leads to loss of neurons and subsequently diminished cognition. These oxidatively modified biomolecules are believed to contribute to the decreased antioxidant content, mitochondrial dysfunction, and impaired plasticity in aged brains. Treatment of rodents with L-acetylcarnitine (LAC; gamma-trimethyl-beta-acetylbutyrobetaine) can improve these functional losses. Although it is well established that administration of LAC can decrease protein oxidation in aged brains, it is not clear which proteins are decreased in their level of oxidation in the brains of aged rats treated with LAC. The current study used a parallel redox proteomics approach to identify the proteins that are oxidized in aged rat cortex and hippocampus of aged rats. Moreover, those proteins that are reduced in oxidation status were identified in aged brains from rats treated in vivo with LAC. The findings are discussed in reference to brain aging and age-related cognitive impairment. PMID- 16677086 TI - Curcumin activates defensive genes and protects neurons against oxidative stress. AB - Spices and herbs often contain active phenolic substances endowed with potent antioxidative properties. We had previously shown that curcumin, the yellow pigment in curry, strongly induced HO-1 expression and activity in rat astrocytes. In the CNS, HO-1 has been reported to operate as a fundamental defensive mechanism for neurons exposed to an oxidant challenge. Treatment of astrocytes with curcumin upregulated expression of HO-1 protein at both cytoplasmic and nuclear levels, as shown by immunofluorescence analysis under laser-scanning confocal microscopy. A significant expression of quinone reductase and glutathione S transferase, two members of phase II detoxification enzymes, was found in astrocytes exposed to 5-15 microM curcumin. Moreover, the effects of curcumin on HO-1 activity were explored in cultured hippocampal neurons. Elevated expression of HO-1 mRNA and protein were detected after 6 h incubation with 5-25 microM curcumin. Higher concentrations of curcumin (50-100 microM) caused a substantial cytotoxic effect with no change in HO-1 protein expression. Interestingly, pre-incubation (18 h) with curcumin resulted in an enhanced cellular resistance to glucose oxidase-mediated oxidative damage; this cytoprotective effect was considerably attenuated by zinc protoporphyrin IX, an inhibitor of heme oxygenase activity. This study gives additional support to the possible use of curcumin as a dietary preventive agent against oxidative stress related diseases. PMID- 16677087 TI - Redox modulation of heat shock protein expression by acetylcarnitine in aging brain: relationship to antioxidant status and mitochondrial function. AB - There is significant evidence to show that aging is characterized by a stochastic accumulation of molecular damage and by a progressive failure of maintenance and repair processes. Protective mechanisms exist in the brain which are controlled by vitagenes and include members of the heat shock system, heme oxygenase-I, and Hsp70 as critical determinants of brain stress tolerance. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders, and the present study reports that treatment for 4 months of senescent rats with acetyl-L-carnitine induces heme oxygenase-1 as well as Hsp70 and SOD-2. This effect was associated with upregulation of GSH levels, prevention of age-related changes in mitochondrial respiratory chain complex expression, and decrease in protein carbonyls and HNE formation. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Particularly, modulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. PMID- 16677088 TI - New insights into structure and function of mitochondria and their role in aging and disease. AB - This review covers some novel findings on mitochondrial biochemistry and discusses diseases due to mitochondrial DNA mutations as a model of the changes occurring during physiological aging. The random collision model of organization of the mitochondrial respiratory chain has been recently challenged on the basis of findings of supramolecular organization of respiratory chain complexes. The source of superoxide in Complex I is discussed on the basis of laboratory experiments using a series of specific inhibitors and is presumably iron sulfur center N2. Maternally inherited diseases due to mutations of structural genes in mitochondrial DNA are surveyed as a model of alterations mimicking those occurring during normal aging. The molecular defects in senescence are surveyed on the basis of the "Mitochondrial Theory of Aging", establishing mitochondrial DNA somatic mutations, caused by accumulation of oxygen radical damage, to be at the basis of cellular senescence. Mitochondrial production of reactive oxygen species increases with aging and mitochondrial DNA mutations and deletions accumulate and may be responsible for oxidative phosphorylation defects. Evidence is presented favoring the mitochondrial theory, with primary mitochondrial alterations, although the problem is made more complex by changes in the cross talk between nuclear and mitochondrial DNA. PMID- 16677089 TI - Friedreich's ataxia: from disease mechanisms to therapeutic interventions. AB - Friedreich's ataxia (FRDA) is the most common inherited ataxia. FRDA is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein that is severely reduced in FRDApatients. The function of frataxin has not been established yet. Studies of the yeast and animal model of the disease as well as of tissues from FRDA patients have demonstrated that deficit of frataxin is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of tissue energy metabolism. Pilot studies have shown the potential effect of antioxidant therapy in this condition and provide a strong rationale for designing larger clinical randomized trials. PMID- 16677090 TI - Redox regulation of heat shock protein expression by signaling involving nitric oxide and carbon monoxide: relevance to brain aging, neurodegenerative disorders, and longevity. AB - Increased free radical generation and decreased efficiency of the reparative/degradative mechanisms both primarily contribute to age-related elevation in the level of oxidative stress and brain damage. Excess formation of reactive oxygen and nitrogen species can cause proteasomal dysfunction and protein overloading. The major neurodegenerative diseases are all associated with the presence of abnormal proteins. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed vitagenes, including the heat shock protein (HSP) system. Of the various HSPs, heme oxygenase-I (HO-1), by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. The HO-1 gene is redox regulated and its expression is modulated by redox active compounds, including nutritional antioxidants. Given the broad cytoprotective properties of the heat shock response, there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. These findings have opened up new neuroprotective strategies, as molecules inducing this defense mechanism can be a therapeutic target to minimize the deleterious consequences associated with accumulation of conformationally aberrant proteins to oxidative stress, such as in neurodegenerative disorders and brain aging, with resulting prolongation of a healthy life span. PMID- 16677091 TI - NF-kappaB: a stress-regulated switch for cell survival. AB - Nuclear factor-kappaB (NF-kappaB), a stress-regulated transcription factor belonging to the Rel family, has a pivotal role in the control of the inflammatory and the innate immune responses. Its activation rapidly induces the transcription of a variety of genes encoding cell adhesion molecules, inflammatory and chemotactic cytokines, cytokine receptors, and enzymes that produce inflammatory mediators. More recently, NF-kappaB activation has been connected with multiple aspects of oncogenesis, including the control of cell proliferation, migration, cell cycle progression, and apoptosis. Interestingly, NF-kappaB is constitutively activated in several types of cancer cells, including hematological and epithelial malignancies. In addition, activation of NF-kappaB in cancer cells by chemotherapy or radiation therapy has been associated with the acquisition of resistance to apoptosis, which has emerged as a significant impediment to effective cancer treatment. Selective cyclopentenone inhibitors of the IkappaB kinase, the key enzyme controlling NF-kappaB activation, were recently shown to be potent inducers of apoptosis in chemoresistant lymphoid malignancies. Increasing evidence, summarized in this review, indicates that the development of selective NF-kappaB inhibitors may represent a promising therapeutic tool to sensitize tumor cells to apoptosis and increase the efficacy of conventional anticancer drugs in a wide spectrum of malignancies. PMID- 16677092 TI - Albumin-bound bilirubin interacts with nitric oxide by a redox mechanism. AB - Bilirubin, the final product of heme catabolism, plays a crucial role in the cellular defense against oxidative and nitrosative stress. This study investigated the interaction of albumin-bound bilirubin, the circulating form of the bile pigment, with nitric oxide (NO), a gaseous modulator involved in many physiological functions but able to induce cytotoxicity and cell death if produced in excess. A short-lived endogenous S-nitrosothiol such as S-nitroso cysteine was used as NO donor. In PBS without chelators, bilirubin was bound to human serum albumin with an apparent affinity of 1.6 +/- 0.2 microM (n = 4). Furthermore, albumin (2-20 microM) dose-dependently increased the half-life of BR (10 microM) exposed to S-nitroso-cysteine (100 microM) of 2.4 +/- 0.4 times (n = 4). Albumin-bound bilirubin was almost completely oxidized by S-nitroso-cysteine derived NO, and biliverdin was the major product formed; this reaction seemed to be rather specific for albumin-bound bilirubin because when free bilirubin was reacted with S-nitroso-cysteine the formation of biliverdin was significantly lower. Uric acid and reduced glutathione, two well-known plasma antioxidants, at physiological concentrations protected albumin-bound bilirubin from NO-mediated oxidation. Taken together, these data suggest that albumin-bound bilirubin maintains its ability to interact with NO also in the bloodstream counteracting extracellular nitrosative reactions. PMID- 16677093 TI - Regulation by the cAMP cascade of oxygen free radical balance in mammalian cells. AB - A study is presented of the effect of the cAMP cascade on oxygen metabolism in mammalian cell cultures. Serum-starvation of the cell cultures resulted in depression of the forward NADH-ubiquinone oxidoreductase activity of complex I, decreased content of glutathione, and enhancement of the cellular level of H2O2. Depressed transcription of cytosolic Cu/Zn-SOD 1, mitochondrial glutathione peroxidase and catalase was also observed. Activation of the cAMP cascade reversed the depression of the activity of complex I and the accumulation of H2O2. The effect of cAMP involved the cAMP-dependent protein kinase. PMID- 16677094 TI - Genetics, redox signaling, oxidative stress, and apoptosis in mammalian aging. PMID- 16677096 TI - Exercise training attenuates age-induced changes in apoptotic signaling in rat skeletal muscle. AB - The aging process in skeletal muscle is characterized by a loss of myocytes and reduction in cross-sectional area of the remaining myocytes, particularly in Type II (fast-twitch) muscle fibers. In multinucleated skeletal muscle, apoptosis may contribute to both fiber atrophy and loss of muscle fibers. Recent evidence suggests that the mitochondrial Bcl-2 family pathway may be a target of aging. Here the authors demonstrated that aging increased DNA fragmentation, cleaved caspase-3, and pro-apoptotic Bax in rat skeletal muscle. Twelve weeks of treadmill exercise training increased anti-apoptotic Bcl-2, while markedly reducing DNA fragmentation, and cleaved caspase-3, Bax, and Bax/Bcl-2 ratio in the white gastrocnemius and soleus muscles of old rats. Upstream anti-apoptotic NF-kappaB activity decreased in aging skeletal muscle, and increased with exercise training. Regulation of NF-kappaB activity with aging and exercise was not related to changes in NF-kappaB subunit protein levels. Instead, changes in post-translational activation of NF-kappaB occurred as a function of altered phosphorylation of IkappaB. These results indicate that treadmill exercise training attenuates fiber atrophy and pro-apoptotic signaling in aging skeletal muscle. PMID- 16677095 TI - Frataxin, iron-sulfur clusters, heme, ROS, and aging. AB - A deficiency in mitochondrial frataxin causes an increased generation of mitochondrial reactive oxygen species (ROS), which may contribute to the cell degenerative features of Friedreich's ataxia. In this work the authors demonstrate mitochondrial iron-sulfur cluster (ISC) defects and mitochondrial heme defects, and suggest how both may contribute to increased mitochondrial ROS in lymphoblasts from human patients. Mutant cells are deficient in the ISC requiring mitochondrial enzymes aconitase and succinate dehydrogenase, but not in the non-ISC mitochondrial enzyme citrate synthase; also, the mitochondrial iron sulfur scaffold protein IscU2 co-immunoprecipitates with frataxin in vivo. Presumably as a consequence of the iron-sulfur cluster defect, cytochrome c heme is deficient in mutants, as well as heme-dependent Complex IV. Mitochondrial superoxide is elevated in mutants, which may be a consequence of cytochrome c deficiency. Hydrogen peroxide, glutathione peroxidase activity, and oxidized glutathione (GSSG) are each elevated in mutants, consistent with activation of the glutathione peroxidase pathway. Mutant status blunted the effects of Complex III and IV inhibitors, but not a Complex I inhibitor, on superoxide production. This suggests that heme defects late in the electron transport chain of mutants are responsible for increased mutant superoxide. The impact of ISC and heme defects on ROS production with age are discussed. PMID- 16677097 TI - Hepatic oxidative stress during aging: effects of 8% long-term calorie restriction and lifelong exercise. AB - Hepatic aging may involve alterations in redox status, resulting in enhanced oxidant production and changes in specific signaling pathways that lead to a pro inflammatory response. The authors investigated whether mild calorie restriction and long-term voluntary exercise could attenuate these changes. Four groups of male Fischer 344 rats were compared: young (6 mo), old (24 mo), old calorie restricted (8% CR, 24 mo) and old CR with daily voluntary wheel running (Exercise; 8% CR, 24 mo). Levels of endogenous reactive oxygen species (ROS), nitric oxide (NO*), and peroxynitrite (ONOO-) were significantly higher in the old ad libitum fed group compared to the young group. Sulfhydryl (-SH) content was significantly reduced and glutathione (GSH) content tended to be lower in the old animals. Old rats had significantly increased nuclear presence of NF-kappaB and in connection, increased levels of regulatory cytosolic phosphorylated I kappaBalpha and decreased dephosphorylated I-kappaBalpha, suggesting an increased inflammatory response. Interestingly, a significant increase in liver RNA oxidation (8-oxo-7,8-dihydroguanosine) in the old ad libitum fed rats was detected and DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine) tended to be increased. The age-associated increase in oxidative stress and upregulation of pro-inflammatory proteins was attenuated in the livers from both the CR and the exercise + CR groups. PMID- 16677098 TI - Moderate caloric restriction increases diaphragmatic antioxidant enzyme mRNA, but not when combined with lifelong exercise. AB - Diaphragmatic antioxidant enzymes are upregulated following acute and long-term treadmill exercise, but the effect of lifelong voluntary exercise (E) on diaphragmatic antioxidants is unknown. Therefore, 10-week old Fisher 344 rats were assigned to either: (a) sedentary ad libitum (AL) fed (24AL; n = 6); (b) E + 8% caloric restriction (24ECR; n = 9); or (c) sedentary + 8% caloric restriction (24CR; n = 9) groups. Diaphragms were harvested from animals at 24 months of age. Heme oxygenase-1 (HO-1) mRNA in addition to catalase (CAT), glutathione peroxidase (GPX), copper-zinc superoxide dismutase (Cu-ZnSOD) and manganese superoxide dismutase (MnSOD) mRNA and protein levels were measured. Reduced glutathione (GSH) and citrate synthase (CS) activity were measured to assess antioxidant status and oxidative capacity, respectively. The 24CR group demonstrated increased GPX, HO-1, MnSOD, and CAT mRNA compared to 24AL and 24ECR. Interestingly, the increased mRNA in 24CR animals did not result in elevated protein levels. No group differences in Cu-ZnSOD mRNA, CS activity, or GSH were observed, although GSH was 30% greater in 24CR animals (p = 0.085). In summary, although CR elevated the mRNA of key antioxidant enzymes in the diaphragm, lifelong CR alone or in combination with voluntary exercise did not alter diaphragm CS activity, antioxidant protein quantity, or GSH levels. PMID- 16677099 TI - Mitochondrial H2O2 production is reduced with acute and chronic eccentric exercise in rat skeletal muscle. AB - Oxidative stress with acute/chronic exercise has been so far examined using exercise involving a combination of concentric and eccentric contractions, but skeletal muscles are likely to be injured to a greater extent by pliometric contractions. In the present study, the effects of acute and chronic bouts of downhill running exercise on mitochondrial hydrogen peroxide (H2O2) generation (fluorimetric detection of a dimer with homovanillic acid in presence of horseradish peroxidase) and oxygen consumption in conjunction with antioxidant enzymes activity were examined. The results show that acute eccentric exercise was accompanied by a significantly reduced mitochondrial H2O2 production that is likely due to a decrease in complex I of the electron transport chain (ETC). On the other hand, eccentric training leads to positive adaptations, reflected by a higher citrate synthase activity and decreased mitochondrial H2O2 production. The decrease in mitochondrial H2O2 cannot be attributed to alterations in antioxidant capacities but rather to changes in mitochondrial membrane composition characterized by an increased polyunsaturated to saturated fatty acids ratio, and decreased contents in arachidonic acid and plasmalogens. These results suggest that changes in mitochondrial membrane properties with eccentric training can affect H2O2 production by muscle mitochondria. It is hypothesized that these changes resulted in a mild uncoupling sufficient to reduce electron back flow through complex I of the ETC, the major generator of reactive oxygen species by skeletal muscle mitochondria. PMID- 16677100 TI - Maintaining genetic integrity in aging: a zero sum game. AB - Aging of somatic cells can be defined as the gradual loss of the information embedded in the global and local properties of complex macromolecular networks. This loss of information may reflect the dynamic interplay between stochastic factors, such as the accumulation of unrepaired somatic damage, and gene-encoded programmatic responses. This would ultimately result in loss of function, impaired response to environmental challenge, and a progressively increased incidence of disease. Here the authors present the case for aging as a continuous battle between maintaining genomic integrity and ensuring sufficient cell functional mass. Focusing on aging of the liver in rodents, evidence is presented that normal aging is associated with a gradual accumulation of random alterations in the DNA of the genome as a consequence of imperfect DNA repair and a decrease in the rate of DNA damage-induced apoptosis. Apoptosis is the cell's genome maintenance mechanism of last resort and an imbalance towards apoptosis can contribute to manifestations of aging-related phenotypes, as exemplified by mouse models of premature aging due to genetic defects in genome maintenance. Prospects to reset the clock in this zero sum game between survival and the maintenance of phenotypic integrity will be discussed. PMID- 16677101 TI - The molecular inflammatory process in aging. AB - Emerging pathological evidence indicates that major chronic aging-related diseases such as atherosclerosis, arthritis, dementia, osteoporosis, and cardiovascular diseases, are inflammation-related. In this review, inflammation is examined as a possible underlying basis for the molecular alterations that link aging and age-related pathological processes. A proposal for the molecular inflammation hypothesis of the aging views the redox derangement that occurs during aging as the major factor for increased risk for age-related inflammation. Accumulated data strongly indicate the activation of redox-sensitive transcription factors and dysregulated gene expression under the age-related oxidative stress seems to be the major culprits. Key players involved in the inflammatory process are the age-related upregulation of NF-kappaB, IL-1beta, IL 6, TNFalpha, cyclooxygenase-2, adhesion molecules, and inducible NO synthase. Furthermore, data are presented on the molecular events involved in age-related NF-kappaB activation and phosphorylation by IkappaB kinase/NIK and MAPKs. Experimental data on anti-aging calorie restriction (CR) for its antiinflammatory efficacy by suppressing the upregulated proinflammatory mediators will be reviewed. Also, the involvement of another super family of transcription factors, PPARs (PPARalpha, gamma) as regulators of proinflammatory responses and NF-kappaB signaling pathway is described as well as a discussion on the physiological significance of a well-maintained balance between NF-kappaB and PPARs. PMID- 16677102 TI - Is the mitochondrial free radical theory of aging intact? AB - The present state of the mitochondrial free radical theory of aging is reviewed. Available studies do not support the hypothesis that antioxidants control the rate of aging because: (a) they correlate inversely with maximum longevity in vertebrates, and (b) increasing their concentration by different methods does not increase maximum lifespan. On the other hand, comparative studies consistently show that long-lived mammals and birds have low rates of mitochondrial reactive oxygen species (ROS) production and low levels of oxidative damage in their mitochondrial DNA. Furthermore, caloric restriction, which extends longevity, also decreases mitochondrial ROS production at complex I and lowers mtDNA oxidative damage. Recent data show that these changes can also be obtained with protein restriction without strong caloric restriction. Another trait of long lived mammals and birds is the possession of low degrees of unsaturation in their cellular membranes. This is mainly due to minimizing the presence of highly unsaturated fatty acids such as 22:6n-3 and emphasizing the presence of less unsaturated fatty acids such as 18:2n-6 in long-lived animals, without changing the total amount of polyunsaturated fatty acids. This leads to lower levels of lipid peroxidation and lipoxidation-derived protein modification in long-lived species. Taken together, available information is consistent with the predictions of the mitochondrial free radical theory of aging, although definitive proof and many mechanistic details are still lacking. PMID- 16677103 TI - Apoptosis and aging: role of p66Shc redox protein. AB - p66Shc was the first mammalian gene whose mutation was demonstrated to increase resistance to oxidative stress and to prolong life span. Many hypotheses have been formulated to explain the biochemical and molecular basis of mammalian aging. Among them the free radical theory of aging, which was first proposed half a century ago by Harman, has received much attention by biomedical scientists. This theory proposed that, because of their high reactivity, reactive oxygen species (ROS) would lead to unavoidable and potentially deleterious by-products, and such an increasingly damaging process could be responsible for degenerative diseases and aging. Recent reports suggest an important role of p66Shc protein in the regulation of cellular responses to oxidative stress, apoptosis, and aging. In this review we discuss what has been discovered about p66Shc in the past 10 years and we focus particularly on its role in ROS regulation, which appears to be extremely promising to define mammalian aging processes. PMID- 16677104 TI - Oxidative damage and platelet activation as new predictors of mobility disability and mortality in elders. AB - Mobility disability is an early phase of the disablement process in older adults, and represents a major risk factor for physical disability and mortality. Pathophysiological mechanisms responsible for the onset of mobility limitation are still largely unknown. Oxidative damage, responsible for the disruption of the equilibrium of biological systems by damaging major constituent molecules, might play an important role in the pathway leading to major health-related events. It has been suggested the existence of a vicious cycle involving oxidative damage, platelet activation, and inflammation as promoter of pathophysiological changes occurring with aging. This hypothesis is based on the following observations: (a) oxidative damage is associated with diseases and clinical conditions potentially leading to disability and mortality; (b) oxidative damage is associated with platelet activation, and a vicious cycle involving oxidative damage, platelet activation, and inflammation has been demonstrated in several metabolic disorders potentially leading to mobility disability; (c) the age-related physical decline may be associated to the oxidative damage due to the excess of free radicals; (d) antioxidant defense and behavioral factors (e.g., physical activity, dietary restriction, smoking cessation) play an important role in the reduction of oxidative damage levels and are associated with improved physical performance and muscle strength. PMID- 16677105 TI - New molecular target for modulation of aging process. AB - Despite many endeavors, no satisfactory strategy has emerged for modulating the aging process, most probably because they were based on faulty rationales. In an extension of the "gate theory of aging" that we proposed recently, we propose here that caveolin, an essential component of caveolae structure, may offer a potential target for modulating the aging process. According to the gate theory, certain biomolecules such as caveolins, amphiphysins, G proteins, and integrins play decisive roles in determining the senescent phenotype and thus provide targets for modulating the aging process. Among these molecules, we chose caveolin, because it can associate with a variety of regulatory and structural molecules via their scaffolding domains and thereby influence a broad spectrum of biological phenomena including both the physiology and morphology of the senescent cells. This is an attempt to review the vast body of evidence available in the literature, both direct and indirect, supporting the accord of this pivotal role to the caveolin in the background of the gate theory for the aging process. PMID- 16677106 TI - Characterization of transgenic mice that overexpress both copper zinc superoxide dismutase and catalase. AB - Transgenic mice overexpressing both Cu/ZnSOD and catalase [Tg(SOD1/CAT) +/o] were used to evaluate the effects of overexpression of both genes against oxidative stress. Characterization of these transgenic mice revealed that catalase or Cu/ZnSOD activities were two- to fourfold higher in the tissues of transgenic mice compared to wild-type mice, and the activities of the other major antioxidant enzymes were not altered in the tissues of the transgenic mice. The murine embryonic fibroblasts (MEFs) from the Tg(SOD1/CAT) +/o and MEFs overexpressing Cu/ZnSOD were more resistant to paraquat cytotoxicity, relative to wild-type MEFs. The MEFs from Tg(SOD1/CAT) +/o tended to be more resistant (up to 2.25-fold) to paraquat cytotoxicity than MEFs overexpressing either Cu/ZnSOD or catalase alone. MEFs from Tg(CAT) +/o and Tg(SOD1/CAT) +/o were equally as resistant to hydrogen peroxide cytotoxicity. However, there were no significant differences in whole animal survival against either paraquat or gamma-radiation. PMID- 16677107 TI - Peroxynitrite induces an alternative NF-kappaB activation pathway in L8 rat myoblasts. AB - The role of peroxynitrite in NF-kappaB activation remains controversial. This study investigated NF-kappaB activation by peroxynitrite in skeletal myocytes. Myocytes were treated with NO and peroxynitrite donors. Both NO and peroxynitrite caused NF-kappaB activation (measured by p65 nuclear translocation and luciferase expression). NO donor-induced NF-kappaB activation was transient, dependent on I kappaB alpha degradation, and was decreased in the presence of I-kappaB alpha super-repressor. Conversely, peroxynitrite donors induced NF-kappaB activation that was dependent on tyrosine nitration of I-kappaB alpha, but independent of its serine phosphorylation and degradation. This activation did not decrease in the presence of I-kappaB alpha super-repressor. Prolonged exposure to peroxynitrite resulted in nontransient NF-kappaB activation and high iNOS expression. Proteasome inhibitor MG-132 did not diminish SIN-1-induced NF-kappaB activation. Tyrosine nitration inhibitor EGCG re-established transient NF-kappaB activation with I-kappaB alpha degradation after SIN-1 treatment. EGCG, but not MG-132 decreased SIN-1- dependent iNOS expression. Peroxynitrite activates NF kappaB in skeletal myocytes through an alternative mechanism, in which I-kappaB alpha is nitrated on tyrosine and dissociated from NF-kappaB, thus enabling its nontransient activation. This resulted in prolonged iNOS expression. Hence, peroxynitrite may exacerbate inflammatory responses mediated by NF-kappaB. PMID- 16677108 TI - Regulation of vascular responses to inflammation: inducible matrix metalloproteinase-3 expression in human microvascular endothelial cells is sensitive to antiinflammatory Boswellia. AB - Endothelial cells are critical elements in the pathophysiology of inflammation. Tumor necrosis factor (TNF) alpha potently induces inflammatory responses in endothelial cells. Recently we have examined the genetic basis of the antiinflammatory effects of Boswellia extract (BE) in a system of TNFalpha induced gene expression in human microvascular endothelial cells (HMECs). Of the 522 genes induced by TNFalpha in HMECs, 113 genes were sensitive to BE. BE prevented the TNFalpha-induced expression of matrix metalloproteinases (MMPs). In the current work, we sought to test the effects of BE on TNFalpha-inducible MMP expression in HMECs. Acetyl-11-ketobeta- boswellic acid (AKBA) is known to be an active principle in BE. To evaluate the significance of AKBA in the antiinflammatory properties of BE, effects of BE containing either 3% (BE3%) or 30% (BE30%, 5- Loxin) were compared. Pretreatment of HMECs for 2 days with BE potently prevented TNFalpha-induced expression and activity of MMP-3, MMP-10, and MMP-12. In vivo, BE protected against experimental arthritis. In all experiments, both in vitro and in vivo, BE30% was more effective than BE3%. In sum, this work lends support to our previous report that BE has potent antiinflammatory properties both in vitro as well as in vivo. PMID- 16677109 TI - Hydrogen sulfide protects HT22 neuronal cells from oxidative stress. AB - Hydrogen sulfide (H2S) is a neuromodulator in the brain and a relaxant for smooth muscle. H2S protects primary cortical neurons from oxidative stress by increasing the intracellular concentrations of glutathione, the major antioxidant in cells. However, changes in glutathione alone are not sufficient to account for full protection in all types of nerve cells. H2S is here shown to protect an immortalized mouse hippocampal cell line from oxidative glutamate toxicity by activating ATP-dependent K+ (KATP) and Cl- channels, in addition to increasing the levels of glutathione. The present study therefore identifies a novel pathway for H2S protection from oxidative stress. PMID- 16677110 TI - The redox-sensitive DNA binding sites responsible for age-related downregulation of SMP30 by ERK pathway and reversal by calorie restriction. AB - It was recently found that age-related changes in SMP30 expression can be modulated by antioxidative action. In the current study, the modulation of SMP30 gene expression was explored by (a) antioxidative calorie restriction (CR), (b) proinflammatory lipopolysaccharide (LPS), in aged rat, (c) oxidative stress promoter, tert-butylhydroperoxide (t-BHP)-injected mouse, and (d) t-BHP-treated Ac2F cells. Utilizing EMSA, particular attention was given to the binding activity of unidentified transcription factor in sites 3 and 5 that are located in -800 bp of the SMP30 promoter. Results showed that CR prevented the age related decrease in SMP30 expression, and also showed that SMP30 gene expression and binding activities of sites 3 and 5 decreased with treatments of t-BHP or LPS. These findings were confirmed by the antioxidant NAC and ERK-specific inhibitor PD098059 that blunted decreased SMP30 gene expression and binding activity of sites 3 and 5 by t-BHP in Ac2F cell system. Our data strongly indicate that the SMP30 transcriptional process is redox-sensitive and its modulation occurs at DNA binding sites 3 and 5 in the promoter region. Perhaps a more significant finding of the present study is that the downregulation of SMP30 is likely involved in ERK signal pathway. PMID- 16677111 TI - Current concepts of redox signaling in the lungs. AB - In the intracellular redox state (GSH/GSSG) the cell plays a key role in the regulation and potentiation of the inflammatory response in lung cells. Glutathione and thioredoxin are the important protective antioxidants in the lungs. Regulation of intracellular redox glutathione and thioredoxin levels in response to reactive oxygen/nitrogen species and in inflammation should have critical effects on different lung cells on the activation of redox sensor/signal transduction pathways and various transcription factors. Possibly via the modification of cysteine residues, oxidative stress activates multiple stress kinase pathways and transcription factors nuclear factor-kappaB and activator protein-1, which differentially regulate the genes for proinflammatory cytokines as well as the protective antioxidant genes. Emerging data suggest that protein-S thiolation, protein-S-nitrosation, and oxidation of protein-SH (formation of sulfenic, sulfinic, and sulfonic acids) are critical in redox signaling during normal physiology and under oxidative stress in controlling the cellular processes. In this review, we discuss the recent findings in the context of redox signaling during inflammation, pathology, and the role of redox modulating agents/dietary interventions either to inhibit abnormal signaling or induce/boost the endogenous antioxidant systems. Furthermore, this also provides information as to how antioxidants are involved in redox signaling to control inflammatory and oxidative stress in the lung. PMID- 16677113 TI - AVMA leaders lobby for more public health veterinarians. PMID- 16677114 TI - HSUS responds to latest letter about feral cats. PMID- 16677115 TI - What is your diagnosis? Pulmonary hypertension. PMID- 16677116 TI - What is your diagnosis? Avulsion of the origin of the gastrocnemius muscle. PMID- 16677117 TI - ECG of the month. Ventricular preexcitation. PMID- 16677118 TI - Theriogenology question of the month. Bleeding varicose veins. PMID- 16677119 TI - Nutrient-gene interactions and their role in complex diseases in dogs. PMID- 16677120 TI - Evaluation of the association between feeding raw meat and Salmonella enterica infections at a Greyhound breeding facility. AB - OBJECTIVE: To investigate Salmonella enterica infections at a Greyhound breeding facility. DESIGN: Cross-sectional study. ANIMAL AND SAMPLE POPULATIONS: 138 adult and juvenile dogs and S. enterica isolates recovered from the dogs and their environment. PROCEDURES: The investigation was conducted at the request of a Greyhound breeder. Observations regarding the environment and population of dogs were recorded. Fecal, food, and environmental specimens were collected and submitted for Salmonella culture. Isolates were serotyped and tested for susceptibility to 16 antimicrobials. Isolates underwent genetic analyses by use of pulsed-field gel electrophoresis and ribotyping. RESULTS: S. enterica was recovered from 88 of 133 (66%) samples of all types and from 57 of 61 (93%) fecal samples. Eighty-three (94.3%) of the isolates were serotype Newport, 77 (87.5%) of which had identical resistance phenotypes. Genetic evaluations suggested that several strains of S. enterica existed at the facility, but there was a high degree of relatedness among many of the Newport isolates. Multiple strains of Salmonella enterica serotype Newport were recovered from raw meat fed on 1 day. CONCLUSIONS AND CLINICAL RELEVANCE: S. enterica infections and environmental contamination were common at this facility. A portion of the Salmonella strains detected on the premises was likely introduced via raw meat that was the primary dietary constituent. Some strains appeared to be widely disseminated in the population. Feeding meat that had not been cooked properly, particularly meat classified as unfit for human consumption, likely contributed to the infections in these dogs. PMID- 16677121 TI - Frequency of and risk factors associated with lingual lesions in dogs: 1,196 cases (1995-2004). AB - OBJECTIVE: To categorize histologic lesions affecting the tongue, determine the frequency with which they develop, and identify risk factors associated with their development in dogs. DESIGN: Retrospective case series. ANIMALS: 1,196 dogs. PROCEDURES: Diagnostic reports of lingual biopsy specimens from dogs evaluated from January 1995 to October 2004 were reviewed. RESULTS: Neoplasia comprised 54% of lingual lesions. Malignant tumors accounted for 64% of lingual neoplasms and included melanoma, squamous cell carcinoma, hemangiosarcoma, and fibrosarcoma. Large-breed dogs, especially Chow Chows and Chinese Shar-Peis, were at increased risk for melanoma. Females of all breeds and Poodles, Labrador Retrievers, and Samoyeds were more likely to have squamous cell carcinomas. Hemangiosarcomas and fibrosarcomas were commonly diagnosed in Border Collies and Golden Retrievers, respectively. Benign neoplasms included squamous papilloma, plasma cell tumor, and granular cell tumor. Small-breed dogs, especially Cocker Spaniels, were at increased risk for plasma cell tumors. Glossitis accounted for 33% of diagnoses; in most cases, the inciting cause was not apparent. Whereas large-breed dogs were more likely to have lingual neoplasia, small-breed dogs were more likely to have glossitis. Calcinosis circumscripta accounted for 4% of lingual lesions and predominately affected young large-breed dogs. The remaining submissions consisted mostly of various degenerative or wound-associated lesions. CONCLUSIONS AND CLINICAL RELEVANCE: The frequency of lingual lesions was not evenly distributed across breeds, sexes, or size classes of dogs. Veterinarians should be aware of the commonly reported lingual lesions in dogs so that prompt diagnosis and appropriate management can be initiated. PMID- 16677122 TI - Evaluation of genetic and metabolic predispositions and nutritional risk factors for pasture-associated laminitis in ponies. AB - OBJECTIVE: To evaluate genetic and metabolic predispositions and nutritional risk factors for development of pasture-associated laminitis in ponies. DESIGN: Observational cohort study. ANIMALS: 160 ponies. PROCEDURES: A previous diagnosis of laminitis was used to differentiate 54 ponies (PL group) from 106 nonlaminitic ponies (NL group). Pedigree analysis was used to determine a mode of inheritance for ponies with a previous diagnosis of laminitis. In early March, ponies were weighed and scored for body condition and basal venous blood samples were obtained. Plasma was analyzed for glucose, insulin, triglycerides, nonesterified fatty acids, and cortisol concentrations. Basal proxies for insulin sensitivity (reciprocal of the square root of insulin [RISQI]) and insulin secretory response (modified insulin-to-glucose ratio [MIRG]) were calculated. Observations were repeated in May, when some ponies had signs of clinical laminitis. RESULTS: A previous diagnosis of laminitis was consistent with the expected inheritance of a dominant major gene or genes with reduced penetrance. A prelaminitic metabolic profile was defined on the basis of body condition, plasma triglyceride concentration, RISQI, and MIRG. Meeting > or = 3 of these criteria differentiated PL- from NL-group ponies with a total predictive power of 78%. Determination of prelaminitic metabolic syndrome in March predicted 11 of 13 cases of clinical laminitis observed in May when pasture starch concentration was high. CONCLUSIONS AND CLINICAL RELEVANCE: Prelaminitic metabolic syndrome in apparently healthy ponies is comparable to metabolic syndromes in humans and is the first such set of risk factors to be supported by data in equids. Prelaminitic metabolic syndrome identifies ponies requiring special management, such as avoiding high starch intake that exacerbates insulin resistance. PMID- 16677124 TI - Evaluation of surgical treatment for signs of acute abdominal pain in draft horses: 72 cases (1983-2002). AB - OBJECTIVE: To determine whether heavy (> or = 680 kg [> or = 1,500 lb]) draft horses undergoing surgical treatment for acute signs of abdominal pain were at a greater risk for anesthetic and postoperative complications and lower postoperative survival rates than light (< 680 kg) draft horses. DESIGN: Retrospective case series. ANIMALS: 72 draft horses. PROCEDURES: Medical records of draft horses that underwent exploratory celiotomy for signs of acute abdominal pain from October 1983 to December 2002 were reviewed. Medical records of draft horses in which a celiotomy was performed for correction of reproductive abnormalities were not included in the study. RESULTS: When compared with light draft horses, heavy draft horses had longer durations of anesthesia, more postoperative complications, and lower survival rates. Seventy-six percent of horses that recovered from anesthesia had postoperative complications. Postoperative complications associated with low survival rates included myopathy and neuropathy, ileus, diarrhea, and endotoxemia. All horses with postoperative myopathy and neuropathy died or were euthanized. The short-term survival rate for horses that recovered from anesthesia was 60%. Horses undergoing small intestinal surgery had a worse prognosis for short-term survival than those undergoing large intestinal surgery. The survival rate for horses for which long-term (> 1 year) follow-up information was available was 50%. CONCLUSIONS AND CLINICAL RELEVANCE: Draft horses weighing > 680 kg that underwent surgery because of acute signs of abdominal pain had longer durations of anesthesia, more postoperative complications, and higher mortality rates than draft horses weighing < 680 kg. PMID- 16677125 TI - Palmar carpal osteochondral fragments in racehorses: 31 cases (1994-2004). AB - OBJECTIVE: To evaluate records of racehorses with palmar carpal osteochondral fragments and determine whether the fragments were indicators of the severity of pathologic joint changes or prognosis. DESIGN: Retrospective case series. ANIMALS: 31 racehorses. PROCEDURES: Medical records, radiographs, and videos of arthroscopic procedures were reviewed. Information gathered included signalment; location, number, and size of the primary lesion; number and size of palmar carpal fragments; and details pertaining to surgical procedures. Outcome variables were obtained from race records. RESULTS: 31 horses met the selection criteria. Multiple palmar fragments were diagnosed in 58% of horses; small fragments (< 3 mm in diameter) were most common (52% of horses). Fifty-two percent of the horses returned to racing, 48% returned to racing and earned money, and 32% had at least 5 more starts. Horses with multiple fragments had significantly less earnings per start and lower performance index values after surgery than those with 1 fragment. Horses with palmar fragments < 3 mm in diameter were significantly less likely to return to racing and have 5 starts or to win money after surgery than horses with larger fragments. CONCLUSIONS AND CLINICAL RELEVANCE: Palmar carpal osteochondral fragments can be used as an indicator of clinically important joint pathology and as a prognostic indicator in racehorses. Horses with multiple small fragments were less likely to successfully return to racing than horses with only dorsally located carpal fragments or horses with 1 or 2 large palmar fragments. When possible, removal of palmar carpal osteochondral fragments should be considered. PMID- 16677126 TI - Effects of modified-live bovine viral diarrhea virus vaccines containing either type 1 or types 1 and 2 BVDV on heifers and their offspring after challenge with noncytopathic type 2 BVDV during gestation. AB - OBJECTIVE: To compare the efficacy of modified-live virus (MLV) vaccines containing either type 1 bovine viral diarrhea virus (BVDV) or types 1 and 2 BVDV in protecting heifers and their offspring against infection associated with heterologous noncytopathic type 2 BVDV challenge during gestation. DESIGN: Randomized controlled study. ANIMALS: 160 heifers and their offspring. PROCEDURES: After inoculation with a placebo vaccine, 1 or 2 doses of an MLV vaccine containing type 1 BVDV, or 1 dose of an MLV vaccine containing both types 1 and 2 BVDV, heifers were bred naturally and challenge exposed with a type 2 BVDV field isolate between 62 and 104 days of gestation. Pregnancies were monitored; after parturition, virus isolation and immunohistochemical analyses of ear-notch specimens were used to determine whether calves were persistently infected. Blood samples were collected at intervals from heifers for serologic evaluation and virus isolation. RESULTS: Persistent infection was detected in 18 of 19 calves from heifers in the control group and in 6 of 18 calves and 7 of 19 calves from heifers that received 1 or 2 doses of the type 1 BVDV vaccine, respectively. None of the 18 calves from heifers that received the type 1-type 2 BVDV vaccine were persistently infected. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the incidence of persistent BVDV infection among offspring from dams inoculated with 1 dose of the MLV vaccine containing types 1 and 2 BVDV was decreased, compared with 1 or 2 doses of the MLV vaccine containing only type 1 BVDV. PMID- 16677128 TI - Efficacy of using an internal teat sealer to prevent new intramammary infections in nonlactating dairy cattle. AB - OBJECTIVE: To determine whether insertion of an internal teat sealer (ITS) at the end of lactation would prevent development of new intramammary infections (IMIs) during the nonlactating period. DESIGN: Controlled clinical trial. ANIMALS: 939 Holstein-Friesian dairy cows from 16 herds. PROCEDURES: Results of bacteriologic culture of milk samples collected 14 days prior to the end of lactation were used to assign cows to groups (group 1 = negative results for all quarters; group 2 = positive results for > or = 1 quarter). Quarters of cows in group 1 were treated with an ITS or a single intramammary dose of cloxacillin; quarters of cows in group 2 were treated with cloxacillin in conjunction with an ITS or with cloxacillin alone. Milk samples were collected at the end of lactation and within 8 days after calving. RESULTS: Regardless of whether the outcome of interest was new IMIs caused by any pathogens, major pathogens, environmental pathogens, or streptococci other than Streptococcus agalactiae, quarters in group 2 treated with both cloxacillin and an ITS were less likely to develop a new IMI than were quarters treated with cloxacillin alone. For cows in group 1, no significant difference in risk of new IMIs was found between treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for dairy cattle with an IMI late in the lactation period, intramammary administration of cloxacillin at the end of lactation followed by insertion of an ITS enhanced protection against development of new IMIs, compared with use of cloxacillin alone. PMID- 16677130 TI - A suicide bomber attack: up close and personal. PMID- 16677129 TI - BCR-bound antigen is targeted to exosomes in human follicular lymphoma B-cells. AB - BACKGROUND INFORMATION: Exosomes are small membrane vesicles secreted by several cell types during exocytic fusion of multivesicular bodies with the plasma membrane. Exosomes from tumour cells can transfer antigens from cell to cell, a property favouring antigen-specific immune responses in vitro and in vivo, and are thus an interesting putative therapeutic tool in human cancers. Exosomes have been well studied in EBV (Epstein-Barr virus)-transformed human B-cell lines; however, biological stimuli regulating exosome secretion quantitatively and/or qualitatively still remain poorly defined. RESULTS: We analysed the effect of the BCR stimulation on exosome release in the human follicular lymphoma B-cell line DOHH2. We found that BCR (B-cell receptor) triggering of DOHH2 cells induced the polarization of CD63(+) MHC class II compartments. Moreover, BCR stimulation increased the release of exosome-associated proteins in the extracellular space. Finally, we found that the BCR was expressed at the surface of exosomes, and could target a bound anti-human IgG to these vesicles. CONCLUSIONS: BCR can modulate the protein content of exosomes upon stimulation, and can target its bound antigen to these vesicles. PMID- 16677131 TI - Posttraumatic stress disorder part II: development of the construct within the North American psychiatric taxonomy. AB - TOPIC: The impairment associated with posttraumatic stress disorder (PTSD) carries with it staggering costs to the individual, to the family, and to society as a whole. Although there is strong evidence that gender plays a significant role in responses to stress and trauma, gender specificity is still not well incorporated into clinical or research work in the area of PTSD. PURPOSE: This is the second of three articles examining the sufficiency of the current PTSD construct to articulate the full spectrum of human responses to trauma. This article chronicles ongoing refinements to the original PTSD criteria and the subsequent controversies. SOURCE OF INFORMATION: Existing bodies of theoretical and research literature related to the effects of trauma. CONCLUSION: In a third article we will review evidence supporting the existence of a more complex posttraumatic stress reaction associated with interpersonal trauma (physical/sexual abuse/assault). PMID- 16677132 TI - Comparing life experiences of college students with differing courses of schizophrenia in Korea: case studies. AB - PURPOSE: The objective of this study was to identify how daily experiences of college students with schizophrenia reflected the course of their disease. METHODS: The sample included 8 college students with schizophrenia. Comparisons of life experiences were made across disease courses using themes established in a prior study. FINDINGS: Subjects had different themes according to the following three disease courses: recovering, deteriorating, and fluctuating. CONCLUSIONS: This finding provides support for developing intervention strategies for college students in each disease course, such as the unique ways that psychiatric nurses can help college students with difficulties due to serious mental illness. PMID- 16677133 TI - An adult with childhood medical trauma treated with psychoanalytic psychotherapy and EMDR: a case study. AB - PROBLEM: Adverse childhood experiences have been found to be a strong predictor of emotional and physical problems in adulthood. However, the long-term sequelae for children who have suffered critical illness and exposure to invasive medical procedures are less well documented. METHODS: This is a case study of an adult client who sought treatment for depression and attention deficit disorder. The psychotherapy treatment is discussed and the use of eye movement desensitization and reprocessing (EMDR) is described targeting a memory of a medical trauma resulting from a tonsillectomy when the client was 8 years old. CONCLUSIONS: Significant healing outcomes were attained as a result of the therapy, i.e., decreased depression, less hypervigilance, and increased ability to concentrate, which resulted in the discontinuation of medication for depression and ADHD as well as significant improvement in overall functioning. PMID- 16677134 TI - Embodiment and embodied engagement: central concerns for the nursing care of contemporary peacekeepers suffering from psychological trauma. AB - TOPIC: Little has been written about the importance of clinicians attending to embodiment and embodied engagement in traumatic clinical situations. By listening and responding to embodied sensations, perceptions, and feelings, embodiment and embodied engagement offer a way to understanding the experience of trauma. PURPOSE: To discuss the concepts of embodiment and embodied engagement as central concerns for the care of contemporary peacekeepers suffering from trauma. SOURCES: Review of the literature on embodiment and embodied engagement, and an exemplar case. CONCLUSIONS: Meanings associated with traumatic events can be understood through embodied approaches, such as interpretive and hermeneutic phenomenology, both for research as well as for reflective practice. The art and science of nursing needs to reinstate the embodied experiences of both the patient and the nurse providing care in order for healing to take place. PMID- 16677135 TI - Partner abuse in physically disabled women: a proposed model for understanding intimate partner violence. AB - PROBLEM: Physically disabled women are a population at significant risk for intimate partner violence. METHOD: This study used a qualitative research design to address the abuse experience of physically disabled women. FINDINGS: The participants identified multiple incidences of abuse, focusing on how the abuse began, proceeded, and terminated. A model was constructed to depict the abuse experience. The progression of abuse triggers, responses of the women, the abuse episode, the after-effects, and the slow return to usual routines were identified. CONCLUSIONS: A model, which is different from Walker's Cycle of Violence (Walker, 1979; 2000), was constructed to demonstrate the journey of women with disabilities through the abuse. PMID- 16677136 TI - We need to talk. Would you please tell me which way to go from here. PMID- 16677137 TI - Perspectives on private practice. Handling the finances: HMO versus private pay. PMID- 16677138 TI - Perspectives on psychiatric consultation liaison nursing. A perspective from the Southern hemisphere. PMID- 16677139 TI - Integrative perspectives. Integrating Bach flower remedies into a therapeutic practice. PMID- 16677140 TI - Biological perspectives. Serotonin and norepinephrine reuptake inhibitors (SNRIs): venlafaxine and duloxetine. PMID- 16677141 TI - The art of prescribing. Antidepressants in late-life depression: prescribing principles. AB - QUESTION: Ms. Antai-Otong, I am a psychiatric nurse practitioner currently employed in a large primary care clinic. My greatest challenge with older adults suspected of being depressed is their hesitancy to admit they are depressed or unwillingness to take antidepressants. I have started some of these patients on antidepressants and had mixed results. Please provide some guidelines for treating depression in older adults with coexisting medical conditions. ANSWER: Depression is a common companion of chronic medical illnesses and frequently goes unrecognized and untreated, resulting in high morbidity and mortality. Depression is unrecognized and underdiagnosed in approximately 16% of older patients seen in primary care settings (Unutzer, 2002). Typically, older adults deny being depressed, minimize symptom severity, fail to recognize common subjective experiences, such as anhedonia, fatigue, and concentration difficulties associated with this disorder, and hesitate to accept their illness due to social stigma and effects of stoicism. Cultural and generational influences also impact how older adults perceive mental health services. Due to the growing number of individuals 65 and older with coexisting medical and psychiatric conditions, particularly depression, seeking health care in vast practice settings, advanced practice psychiatric nurses must collaborate with primary care providers and develop holistic care that addresses coexisting chronic medical and psychiatric conditions. PMID- 16677144 TI - The effectiveness of psychotherapy. PMID- 16677145 TI - Role of probiotics in hepatic ischemia-reperfusion injury. PMID- 16677146 TI - Magnetic resonance imaging of the liver: how I do it. AB - The present paper provides a brief overview of the rationale behind magnetic resonance imaging (MRI) techniques, a description of the most common sequences used, and a general approach to performing liver MRI. PMID- 16677147 TI - Constipation of anorectal outlet obstruction: pathophysiology, evaluation and management. AB - Constipation is a subjective symptom of various pathological conditions. Incidence of constipation fluctuates from 2 to 30% in the general population. Approximately 50% of constipated patients referred to tertiary care centers have obstructed defecation constipation. Constipation of obstructed defecation may be due to mechanical causes or functional disorders of the anorectal region. Mechanical causes are related to morphological abnormalities of the anorectum (megarectum, rectal prolapse, rectocele, enterocele, neoplasms, stenosis). Functional disorders are associated with neurological disorders and dysfunction of the pelvic floor muscles or anorectal muscles (anismus, descending perineum syndrome, Hirschsprung's disease). However, this type of constipation should be differentiated by colonic slow transit constipation which, if coexists, should be managed to a second time. Assessment of patients with severe constipation includes a good history, physical examination and specialized investigations (colonic transit time, anorectal manometry, rectal balloon expulsion test, defecography, electromyography), which contribute to the diagnosis and the differential diagnosis of the cause of the obstructed defecation. Thereby, constipated patients can be given appropriate treatment for their problem, which may be conservative (bulk agents, high-fiber diet or laxatives), biofeedback training or surgery. PMID- 16677148 TI - Protective role of supplement with foreign Bifidobacterium and Lactobacillus in experimental hepatic ischemia-reperfusion injury. AB - BACKGROUND AND AIM: Intestinal microflora play a crucial role in some severe liver diseases. The purpose of this study was to evaluate the effects of a Lactobacillus strain and a Bifidobacterium strain on ischemia-reperfusion (I/R) liver injury. METHODS: Rats were divided into six groups. Each group received either Bifidobacterium Catenulatum ZYB0401; Lactobacillus Fermentum ZYL0401; a mixture of these two bacterial strains; gentamicin; or saline by daily gavage for 7 days. On the sixth day, all rats, except those in the control group, were subjected to 20 min of liver ischemia. After 22 h of hepatic reperfusion, liver enzymes and histology, malondialdehyde (MDA), superoxide dismutase (SOD), endotoxemia, serum tumor necrosis factor-alpha (TNF-alpha), intestinal bacteria, intestinal mucosal ultrastructure, and bacterial translocation were studied. RESULTS: All administered bacteria increased intestinal Bifidobacterium and Lactobacillus, decreased endotoxemia (P < 0.01), alanine aminotransferase (ALT) (P < 0.01), and markedly ameliorated liver histology and intestinal mucosal ultrastructure. Only rats treated with Bifidobacterium Catenulatum ZYB0401 and Lactobacillus Fermentum ZYL0401 showed reduced incidence of bacterial translocation to the kidney (P < 0.05), associated with decreased serum TNF-alpha and liver MDA (P < 0.05) and increased liver SOD (P < 0.05) compared to the I/R group. Gentamicin decreased almost all kinds of intestinal bacteria (P < 0.01) and decreased ALT (P < 0.01) and serum TNF-alpha, but failed to reduce both endotoxemia and the incidence of bacterial translocation and had no effects on liver MDA and SOD. CONCLUSION: Bifidobacterium Catenulatum ZYB0401 in combination with Lactobacillus Fermentum ZYL0401 could be useful in restoring intestinal microflora and in preventing liver injury in hepatic I/R of rats. PMID- 16677149 TI - Efficacy of interferon alpha-2b and lamivudine combination treatment in comparison to interferon alpha-2b alone in chronic delta hepatitis: a randomized trial. AB - BACKGROUND AND AIM: Delta hepatitis is characterized by rapidly progressive liver disease with adverse prognosis in most patients. Patients benefit from high doses and prolonged courses of interferon (IFN) therapy; however, lamivudine as a single agent has been disappointing. Data relating to the efficacy of IFN and lamivudine in combination is limited. The aim of this study was to test the efficacy of IFN-alpha 2b and lamivudine combination treatment in comparison to IFN-alpha 2b alone in patients with chronic delta hepatitis. METHODS: Twenty-six patients with chronic delta hepatitis were randomized into two groups. Twelve patients received IFN-alpha 2b alone (eight men, four women; mean +/- SD age: 43.83 +/- 8.57 years), and 14 patients received IFN-alpha 2b plus lamivudine combination (seven men, seven women; mean +/- SD age: 42.5 +/- 11.02 years). The dose of IFN-alpha 2b was 10 MU t.i.w. and of lamivudine was 100 mg/day. The groups were comparable in reference to serum alanine aminotransferase (ALT), aspartate aminotransferase, bilirubin, albumin levels, histological activity and stage. Four patients (33.3%) in the IFN group and two (14.3%) in the combination group had cirrhosis (P = 0.2). The duration of treatment was 48 weeks with an untreated follow-up period of at least 96 weeks (mean +/- SD, 3.1 +/- 1.9 years). A liver biopsy was performed at the end of treatment. RESULTS: Eight patients from the IFN group and 11 from the combination group completed treatment. Serum ALT values became normal in 8/14 patients (57.1%) treated with IFN plus lamivudine and in 5/12 patients (41.7%) treated with IFN alone (P = 0.43). Serum hepatitis delta virus RNA was no longer detectable in nine of 14 (64.3%) patients treated with IFN plus lamivudine as compared to five of 12 (41.6%) patients treated with IFN alone (P = 0.024). In both groups female patients had significantly better virological response rate (P = 0.007). There was a significant improvement in histological activity in the combination group (mean decrease 5.27 +/- 1.08 score, P = 0.001), but not in the IFN group (mean decrease 1.44 +/- 1.59 score, P = 0.39). No significant improvement was observed in regards to fibrosis. Four of the 14 patients (28.6%) treated with combination therapy as compared to two of 12 patients treated with IFN (16.7%) were sustained virological responders (P = 0.47). The 5-year survival rate was 65% in the IFN group and 85% in the combination group (P > 0.05). CONCLUSION: Interferon and lamivudine in combination is an encouraging treatment method and may be superior to IFN alone in chronic delta hepatitis. PMID- 16677150 TI - Dengue fever related acalculous cholecystitis in a North Indian tertiary care hospital. AB - BACKGROUND AND AIMS: To document the clinical outcome and prognosis of acalculous cholecystitis in dengue fever. METHODS: Prospective analysis of 27 cases of dengue fever presenting to the medical emergency of a tertiary care referral hospital in Chandigarh, India. RESULTS: All patients with dengue fever presenting with abdominal pain in the year 2003 were studied by ultrasound examination for the presence of acalculous cholecystitis. Five cases presented in October and 22 cases in November. Mean age was 29.8 +/- 9.7 years and there were 21 males and six females. Mean duration of fever was 5.07 +/- 1.8 days and mean duration of body ache was 4.06 +/- 2.6 days. The mean platelet counts for all cases at presentation were 29.41 +/- 18.41 x 10(9)/L and at discharge 95.28 +/- 53.01 x 10(9)/L. The mean duration of in-hospital stay was 3.4 +/- 1.7 days. Fourteen patients presented with pain in the abdomen and were confirmed to have acalculous cholecystitis on ultrasound examination of the abdomen (mean gallbladder wall thickness was 5.2 +/- 1.03 mm). Amongst all variables assessed, the only significant difference seen was that the duration of in-hospital stay was 1.68 days more in the group with acalculous cholecystitis. CONCLUSION: Acalculous cholecystitis should be strongly suspected in patients with dengue fever presenting with abdominal pain. Our study has shown that acalculous cholecystitis occurred in 14 out of 27 patients with dengue fever. PMID- 16677151 TI - Rapid induction and activation of Tec tyrosine kinase in liver regeneration. AB - BACKGROUND: Previous studies have indicated that Tec tyrosine kinase is differentially expressed in the regenerating liver. The purpose of the present study was to further investigate the potential involvement of Tec tyrosine kinase in hepatocyte proliferation and liver regeneration. METHODS: Tec kinase gene expression after partial (two-thirds) hepatectomy was examined by representational difference analysis. Tissue distribution and potential involvement of Tec kinase in liver regeneration and hepatocyte proliferation were then determined by northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), and western blotting. Full-length rat Tec cDNA was cloned. RESULTS: Using this cDNA as the probe, northern blotting showed that Tec was specifically expressed in liver and kidney, the highest expression of Tec being detected in embryonic day 15-19 fetal livers. In contrast, the expression level of Tec in adult and neonatal rat livers was significantly decreased. Similar results were obtained from western blotting analyzes. It was thus hypothesized that Tec might be involved in hepatocyte proliferation. To test this hypothesis, Tec expression was examined in regenerating rat livers. An increase in Tec expression and activation of Tec kinase were observed within 1 h after partial hepatectomy. Moreover, it has been shown that hepatocyte growth factor (HGF) dramatically induces Tec expression in primary rat hepatocytes. Additionally, it was observed that Tec gene expression in serum-starved liver tumor cell line HepG2 was substantially decreased. Stimulation with 10% fetal bovine serum and insulin but not epidermal growth factor resulted in dramatic elevation of Tec expression in these cells. CONCLUSION: Tec is an inducible early response gene that might enhance hepatocyte proliferation and liver regeneration. PMID- 16677152 TI - Measurement of parenchymal function and bile duct flow in primary sclerosing cholangitis using dynamic 99mTc-HIDA SPECT. AB - BACKGROUND AND AIM: Primary sclerosing cholangitis is a chronic cholestatic disease characterized by obliterative fibrosis of intrahepatic and/or extrahepatic bile ducts. The unpredictable clinical course of the disease can be relentless, leading to premature death in a large percentage of patients. Prognostic index formulas, the revised Mayo model being the most frequently used, have been developed to predict clinical course and prognosis. The aim of this study was to investigate the potential value of dynamic (99m)Tc-HIDA SPECT in patients with primary sclerosing cholangitis by correlating scintigraphic results with cholangiographic and biochemical findings and prognostic scoring systems used in clinical practice. METHODS: In 18 patients with primary sclerosing cholangitis, segmental parenchymal and bile duct functions were measured using dynamic (99m)Tc-HIDA SPECT. Quantitative scintigraphic results were compared to cholangiographic findings, as graded by a biliary classification system, the Child-Pugh score and revised Mayo prognostic score, as well as the individual biochemical parameters included in the scoring systems. RESULTS: In individual segments, scintigraphic quantitative parameters indicative of bile flow showed a statistically significant correlation with the state of the bile-flow path for the respective segments as assessed by the biliary classification system. The revised Mayo score correlated with the scintigraphic quantitative parameters indicative of parenchymal function and bile flow as calculated for the whole liver. CONCLUSIONS: Dynamic (99m)Tc-HIDA SPECT, capable of assessing different aspects of liver function for the total liver, as well as for individual segments, has potential value in the management of patients with primary sclerosing cholangitis. PMID- 16677153 TI - Blockade of the receptor for advanced glycation end products attenuates acetaminophen-induced hepatotoxicity in mice. AB - BACKGROUND AND AIM: Severe injury to the liver, such as that induced by toxic doses of acetaminophen, triggers a cascade of events leading to hepatocyte death. It is hypothesized that activation of the receptor for advanced glycation end products (RAGE) might contribute to acetaminophen-induced liver toxicity by virtue of its ability to generate reactive oxygen species, at least in part via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and thereby activate downstream signaling pathways leading to cellular injury. METHODS: A model was employed in which toxic doses of acetaminophen (1125 mg/kg) were administered to C57BL/6 mice. To block RAGE, mice received murine soluble (s) RAGE, the extracellular ligand binding domain of the receptor that acts as a decoy to interrupt ligand-RAGE signaling. RESULTS: Animals treated with sRAGE displayed increased survival compared with vehicle treatment, and markedly decreased hepatic necrosis. Consistent with an important role for RAGE-triggered oxidant stress in acetaminophen-induced injury, a significant reduction of nitrotyrosine protein adducts was observed in hepatic tissue in sRAGE-treated versus vehicle treated mice receiving acetaminophen, in parallel with significantly increased levels of glutathione. In addition, pro-regenerative cytokines tumor necrosis factor-alpha and interleukin-6 were increased in sRAGE-treated versus vehicle treated mice. CONCLUSION: These findings implicate RAGE-dependent mechanisms in acetaminophen-induced liver damage and suggest that blockade of this pathway may impart beneficial effects in toxin-induced liver injury. PMID- 16677154 TI - Increasing trend of acute hepatitis A in north India: need for identification of high-risk population for vaccination. AB - BACKGROUND AND AIMS: Hepatitis A (HAV) is endemic in India and most of the population is infected asymptomatically in early childhood with lifelong immunity. Because of altered epidemiology and decreasing endemicity, the pattern of acute HAV infection is changing from asymptomatic childhood infection to an increased incidence of symptomatic disease in the 18-40 age group. The aims of the present study were to assess whether the proportion of adults with acute HAV infection has been increasing over the years and to analyze the seroprevalence of immunoglobulin G (IgG) anti-HAV antibodies in young adults above the age of 15 years as well as in cases of chronic liver disease. METHODS: Sera collected from 3495 patients with acute (1932) and chronic (1563) liver disease attending the Medical Outpatient Department of Lok Nayak Hospital during the previous five years (1999-2003) were tested for various serological markers of acute (HBsAg, HBcIgM, anti-HCV, HEV-IgM, and HAV-IgM) and chronic (HBsAg, HBcIgG, HBeAg, and anti-HCV) hepatitis. In addition, 500 normal healthy attendants of the patients above the age of 15 years were tested for IgG anti-HAV as controls. RESULTS: Of 1932 patients with acute viral hepatitis, 221 (11.4%) were positive for immunoglobulin M (IgM) anti-HAV. The patients who were IgM anti-HAV negative included hepatitis B (321 patients), C (39 patients), E (507 patients) and unclassified (844 patients). Although the frequency of HAV infection among children had increased (10.6% to 22.0%) in the 5-year period, the frequency of HAV infection among adults had also increased (3.4% to 12.3%) during the same period. A total of 300 patients with chronic liver diseases that were etiologically related to hepatitis B (169), C (73) or dual infection (10) and alcoholic liver injury (48) were tested for the presence of IgG anti-HAV antibody; 98% (294/300) were positive for the antibody. CONCLUSIONS: Although universal vaccination against HAV is not currently indicated, selective vaccination of the high-risk population, based on their serological evidence of HAV antibody, would be a rational and cost-effective approach. PMID- 16677155 TI - Educational and health service needs of Australian general practitioners in managing hepatitis C. AB - BACKGROUND: There has been interest in recent years in the role of primary care practitioners in managing hepatitis C, but there has been minimal research to identify educational and health service needs. A national survey of Australian general practitioners (GPs) was therefore conducted to assess their needs and identify areas for service development. METHODS: A self-administered questionnaire was developed that included questions to assess caseload, confidence in patient management, educational needs and approaches to management and prevention. Questionnaires were sent to a random sample of Australian GPs. Returned questionnaires were coded, frequencies tabulated and significant associations identified. RESULTS: A 70% response rate was achieved from 658 eligible GPs. A total of 76% of respondents had managed one patient in the previous year with hepatitis C. While 69% reported feeling more confident about their management of hepatitis C than 5 years previously, 55% identified a high level of need for hospital-based clinics. Financial benefits for case conferences and chronic case management were not considered useful by most GPs. Topics identified for further skills development included therapeutics and diagnostic testing. Only 39% were highly likely to discuss psychosocial issues as part of initial patient management and 37% reported finding it difficult to play a central role in the medical and psychosocial care of patients with hepatitis C. CONCLUSION: These results have significant implications for policy and service development, as well as identifying areas where GPs need support. The findings invite further discussion between health authorities about the source and magnitude of funding for hospital-based services and further consideration of how to provide services to address patients' psychosocial needs. PMID- 16677156 TI - Expression of Ki-67, transforming growth factor beta1, and B-cell lymphoma leukemia-2 in liver tissue of patients with chronic liver diseases. AB - BACKGROUND: The purpose of the present paper was to assess expression of proliferation, fibrosis and apoptosis markers in different phases of chronic liver diseases. METHODS: Sixty-six adults with chronic liver diseases (chronic hepatitis C, n = 48; chronic hepatitis B, n = 10; alcohol chronic liver disease, n = 8) treated at the Department of Infectious Diseases and Hepatology from 1999 to 2001, composed the study group. Liver biopsy specimens were used for immunohistochemical assessment of expression of Ki-67, transforming growth factor beta1 (TGF-beta1) and B-cell lymphoma-leukemia-2 (Bcl-2). Grade of liver inflammation and stage of fibrosis were evaluated according to the Scheuer scale. RESULTS: Expression of Ki-67 in hepatocytes was most intensive in patients with grade 2 and 3 inflammation. The expression in patients with grade 4 inflammation was low. The expression of Ki-67 in lymphocytes was most intensive in patients with grade 2 inflammation. Expression of TGF-beta1 in hepatocytes reached a maximum in patients with grade 2 or 3 inflammation and dropped in patients with grade 4 inflammation. There was a statistically significant correlation between stage of fibrosis and expression of TGF-beta1 in liver stromal cells. A very strong correlation was found between the expression of Bcl-2 in bile ductules epithelium and the grade of inflammation (P = 0.006). The expression of Bcl-2 in hepatocytes was observed only in patients with very intense liver inflammation (grade 3) and in patients with stage 3 or 4 fibrosis. CONCLUSION: Processes of proliferation, fibrosis and apoptosis are not directly correlated to progression of liver disease. Expression of studied markers can be used for analysis of dynamics of these processes. PMID- 16677157 TI - A novel hepatitis B virus X-interactive protein: cytochrome C oxidase III. AB - BACKGROUND: Hepatitis B virus-encoded X protein has been shown to be capable of activating many different viral and cellular promoters through protein-protein interactions and to contribute to the development of hepatocellular carcinoma. As its mechanism has not yet been identified unequivocally, the aim of the present study was to screen the cellular proteins that can interact with X protein. METHODS: The yeast two-hybrid system was used to screen the X-interactive protein. False positive clones were eliminated by segregation analysis, and then putative positive clones were amplified, sequenced and analyzed with bioinformatics. A mating experiment was performed to confirm the binding of putative proteins to X protein in the yeast cells. The specific interaction between X protein and putative proteins in mammalian cells was verified by coimmunoprecipitation. RESULTS: The hepatitis B virus X-interactive protein recovered from a human liver cDNA library was cytochrome C oxidase III. The specific interaction between protein X and cytochrome C oxidase III was verified by mating experiment and coimmunoprecipitation of COS7 cell lysates expressing both proteins. CONCLUSION: These data support the speculation that cytochrome C oxidase III is a novel functional target of hepatitis B virus X protein in cells. PMID- 16677158 TI - Pantoprazole infusion as adjuvant therapy to endoscopic treatment in patients with peptic ulcer bleeding: prospective randomized controlled trial. AB - BACKGROUND AND AIM: Following successful endoscopic therapy in patients with peptic ulcer bleeding, rebleeding occurs in 20% of patients. Rebleeding remains the most important determinant of poor prognosis. We investigated whether or not administration of pantoprazole infusion would improve the outcome in ulcer bleeding following successful endoscopic therapy. METHODS: In this double-blind, placebo-controlled, prospective trial, patients who had gastric or duodenal ulcers with active bleeding or non-bleeding visible vessel received combined endoscopy therapy with injection of epinephrine and heater probe application. Patients who achieved hemostasis were randomly assigned to receive pantoprazole (80 mg intravenous bolus followed by an infusion at a rate of 8 mg per hour) or placebo for 72 h. The primary end-point was the rate of rebleeding. RESULTS: Rebleeding was lower in the pantoprazole group (8 of 102 patients, 7.8%) than in the placebo group (20 of 101 patients, 19.8%; P = 0.01). Patients in the pantoprazole group required significantly fewer transfusions (1 +/- 2.5 vs 2 +/- 3.3; P = 0.003) and days of hospitalization (5.6 +/- 5.3 vs 7.7 +/- 7.3; P = 0.0003). Rescue therapies were needed more frequently in the placebo group (7.8% vs 19.8%; P = 0.01). Three (2.9%) patients in the pantoprazole group and eight (7.9%) in the placebo group required surgery to control their bleeding (P = 0.12). Two patients in the pantoprazole group and four in the placebo group died (P = 0.45). CONCLUSION: In patients with bleeding peptic ulcers, the use of high dose pantoprazole infusion following successful endoscopic therapy is effective in reducing rebleeding, transfusion requirements and hospital stay. PMID- 16677159 TI - Clinical study of 31 patients with primary gastric mucosa-associated lymphoid tissue lymphoma. AB - BACKGROUND: The purpose of the present paper was to investigate the clinical, endoscopic and histological features of 31 patients with gastric mucosa associated lymphoid tissue (MALT) lymphoma to enable correct, early stage diagnosis. METHODS: A retrospective study was undertaken of 31 patients with gastric MALT lymphoma. The cases were examined immunohistologically with anti CD(20CY) and CD(45RO) antibodies for further diagnosis. Helicobacter pylori infection was also detected with modified Giemsa staining. RESULTS: Patients with MALT lymphoma were aged between 22 and 73 years (mean, 45.0 years), and the male:female ratio was 11:20. The patients presented with non-specific symptoms, but chronic epigastric pain was the common symptom in a large proportion of the cases. The gastric smaller curvature was involved in 83.9% of cases (26/31) and in 13/31 cases (41.9%) it was confined the antrum. Under endoscopy, large and deep ulcers were similar to cancers in the majority of patients. Only 29.0% of patients were diagnosed by endoscopy on first examination. CD(20CY) were expressed in all cases and CD(45RO) expressed in only one case among 10 cases of indefinite diagnosis. Helicobacter pylori infection was found in 87.1% of patients. CONCLUSIONS: These findings suggest that primary gastric MALT lymphoma has unique clinical, endoscopic and histological features. The diagnosis for primary gastric MALT lymphoma was delayed not only due to the non-specific symptoms but also due to lack of attention to its features. Endoscopy and submucosal multiple biopsy were the principal diagnostic tools in patients with gastric MALT lymphoma. CD(20CY) and CD(45RO) immunological staining are recommended, especially for patients with indefinite diagnosis of gastric MALT lymphoma. PMID- 16677160 TI - Spectrum of histopathologic findings in patients with achalasia reflects different etiologies. AB - BACKGROUND: The etiology of achalasia is still unknown. The aim of the present study was to elucidate its underlying pathologies and their chronology by investigation of esophageal specimens in patients undergoing surgery (esophageal resection or myotomy) for achalasia. METHODS: In 17 patients with achalasia, histopathologic examinations of the esophageal wall focussing on the myenteric plexus were performed. Preoperative diagnosis was based on clinical evaluation, esophagogastroscopy, barium esophagogram in all, and esophageal manometry in eight patients. The median age at the time of surgery was 54 years (range: 14-78 years). In eight cases, the complete esophageal, body and in nine cases a smooth muscle biopsy including parts of the myenteric plexus from the distal part of the esophagus (high pressure zone) was available. The tissue specimens were fixed in formalin and embedded in paraffin. The staining procedures were hematoxylin and eosin (HE), Elastica van Gieson (EvG), and periodic acid-Schiff (PAS) reaction. Immunohistochemical examinations were performed with antibodies against B and T lymphocytes, neurofilament, protein gene-related product (PGP 9.5), S-100 protein, myosin, desmin, smooth muscle actin and substance P. RESULTS: In 13 of 17 patients, a significant reduction of the number of intramural ganglion cells was present. Common findings were a severe fibrosis of the smooth muscle layer (10/17) and obvious myopathic changes of the smooth muscle cells (5/17). Staining for B and T lymphocytes found signs of inflammation in mucosal and muscular areas. Three patients exhibited a marked invasion of eosinophilic granulocytes of the muscularis propria (eosinophilia). Esophageal carcinoma had developed in three patients (squamous cell carcinoma in two and carcinoma in situ in another patient with Barrett's esophagus and high-grade dysplasia). Severe inflammatory reactions (neural, eosinophilic and mucosal) dominated in patients with a longstanding history of achalasia (>10 years) as well as a marked endomysial fibrosis. CONCLUSIONS: The histopathological investigations of the esophageal wall in 17 patients undergoing esophageal resection or myotomy for achalasia suggest that the reduction of intramural ganglion cells might be a secondary change, probably due to inflammation triggered by autoimmune mechanisms or a chronic degenerative process of the central and/or peripheral part of the vagal nerve. The primary lesion could also be a severe myopathy of the smooth muscle cells. PMID- 16677161 TI - Morbidity and mortality after percutaneous endoscopic gastrostomy in children with neurological disability. AB - BACKGROUND AND AIM: Gastrostomy placement has become an integral mechanism for delivering nutritional support to children with severe neurological disability. Its impact on gastroesophageal reflux and mortality remains contentious. We examined the morbidity and long-term mortality of a group of children with severe neurological disability after percutaneous endoscopic gastrostomy (PEG). METHODS: We retrospectively identified all children with severe neurological disability who had a PEG at the Royal Children's Hospital in Melbourne between 1990 and 1997. Data were obtained from medical records. RESULTS: Ninety-eight children with neurological disability (M:F 1.8:1.0; median age 3.5 years, interquartile range 1.1-8.7 years) had an initial PEG in this period and were able to be followed for 6-14 years. As a group, they were underweight for their age with a Z score at PEG of -3.52 (SD 3.33), but had increased weight-for-age Z scores by 1.05 after a mean period of 6.1 months. Fourteen subsequently required fundoplication for reflux. Mortality rates were 11% after 1 year, 21% after 2 years, 27% after 3 years and 39% after 13 years. Mortality was increased in those children who were older at the time of PEG (P = 0.06). Gastroesophageal reflux, underweight-for-age and gender were not significantly related to mortality. CONCLUSION: Children with severe neurological dysfunction who require gastrostomy feeding have a substantial long-term mortality, but this may be unrelated to PEG placement. PMID- 16677162 TI - Plasma total homocysteine in the active stage of ulcerative colitis. AB - BACKGROUND: Homocysteine, an independent risk factor for thromboembolism, has been recently shown to be elevated in ulcerative colitis (UC). However, its relation to the activity of the disease remain unclear. METHODS: Two groups were studied: group consisted of 1-30 patients with UC (17 men, 13 women, mean age 50.3 +/- 14.7 years), including 15 patients with active disease. Group 2 (controls) consisted of 21 age-, sex-, bodyweight-matched healthy persons (12 men, nine women, mean age 53.1 +/- 12.8 years). Total plasma homocysteine (tHcy) and serum folate and vitamin B12 as well as selected coagulation parameters were assessed. RESULTS: Mean tHcy in UC patients was significantly higher than in healthy controls: 10.8 +/- 3.1 mmol/L versus 6.8 +/- 2.5 mmol/L (P < 0.001). Patients with active disease had higher tHcy than patients in remission: 11.2 +/- 3.5 mmol/L versus 9.0 +/- 2.3 mmol/L (P = 0.048). Patients with > or =4 recurrences of the disease had also higher tHcy than the others: 11.5 +/- 3.6 mmol/L versus 9.0 +/- 2.1 mmol/L (P = 0.035). The tHcy correlated with duration of disease: r = 0.6632 (P < 0.05). Folate and B12 levels were within their reference ranges in all subjects. However, in the patients with active disease the platelet count, fibrinogen and D-dimer were significantly higher than in the patients in remission and the controls. CONCLUSIONS: Ulcerative colitis is associated with elevated tHcy concentration, particularly in the active stage, and in more recurrent types of the disease; this elevation does not seem to be prevented by a normal folate status and might have an enhancing effect on the procoagulation blood profile. PMID- 16677163 TI - Influence of urease activity in the intestinal tract on the results of 13C-urea breath test. AB - BACKGROUND AND AIM: A late rise in (13)CO2 excretion in the (13)C-urea breath test (UBT) should be found when the substrate passes rapidly through the stomach and makes contact with the colonic bacteria. The aim of this study was to evaluate the influence of intestinal urease activity on the results of the UBT. METHOD: A total of 143 subjects who were diagnosed as Helicobacter pylori negative by serology, histology and rapid urease test were recruited. At the end of endoscopy, the tip of the endoscope was placed to the second part of the duodenum and 20 mL of water containing 100 mg of (13)C-urea was sprayed into the duodenum. Breath samples were taken at baseline and at 5, 10, 20, 30 and 60 min after administration. RESULTS: Of 143 subjects, breath Delta(13)CO2 values higher than 2.5 per thousand were detected in six (4.2%), four (2.8%) and five (3.5%) subjects at 20, 30 and 60 min, respectively. There was no subject with high Delta(13)CO2 values at 5 and 10 min. Only one subject had an immediate rise at 60 min. CONCLUSION: Variability derived from urease activity in the intestinal tract appears to be minimal up to 60 min after ingestion of the test urea. PMID- 16677164 TI - Long-term continence in patients with Hirschsprung's disease and Down syndrome. AB - BACKGROUND AND AIM: Hirschsprung's disease is more common in children with Down syndrome, but the outcome for continence in this group is unclear. The aim of the present study was to determine the natural history of bowel function in children with Down syndrome and Hirschsprung's disease. METHODS: We undertook a retrospective study of all patients with both Down syndrome and Hirschsprung's disease diagnosed at the Royal Children's Hospital, Melbourne, between 1974 and 2001 using a structured questionnaire. RESULTS: Ten of the 20 eligible patients were interviewed. Fecal incontinence was common (87%), as were episodes of diarrhea and perianal excoriation (40%). Persistent constipation was relatively unusual (20%). Adverse reactions to food, especially vegetables and fruit, were very common (90%). There was evidence that bowel dysfunction improved with age, particularly sensation of impending stool (P < 0.05), ability to discriminate stool consistency (P = 0.05), constipation (P < 0.05), episodes of diarrhea (P = 0.08) and excoriation (P < 0.05). CONCLUSION: Persistent bowel dysfunction is common in children with Down syndrome and Hirschsprung's disease, but there is evidence of improvement with age. There was an unexpectedly high prevalence of food-related adverse reactions. PMID- 16677166 TI - Hyperosmotic stress enhances interleukin-1beta expression in Helicobacter pylori infected murine gastric epithelial cells in vitro. AB - BACKGROUND AND AIM: Gastric cancer is associated not only with Helicobacter pylori (H. pylori) infection, but also with the intake of a high salt diet. Interleukin-1beta (IL-1beta) is highly expressed in H. pylori-infected gastric mucosa. The aim of the present study was to determine if hyperosmotic stress induces IL-1beta expression in gastric epithelial cells in vitro. METHOD: Murine gastric epithelial cells, GSM06, were cultured with or without H. pylori (Sydney strain-1) at different osmolarities in the range of 300-450 mOsM. Expressions of IL-1beta mRNA and mature IL-1beta protein were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and an IL-1beta enzyme-linked immunosorbent assay (ELISA), respectively. IL-1beta converting enzyme (ICE) activity was measured by an ICE colorimetric assay. Apoptosis was evaluated by a single stranded-DNA assay. RESULTS: Addition of H. pylori at 300 mosM caused significant increases in IL-1beta mRNA, IL-1beta protein, ICE activity and apoptosis. Hyperosmotic stress alone also caused upregulation of IL-1beta mRNA and IL-1beta protein, enhanced ICE activity and accelerated apoptosis. Hyperosmotic stress accentuated the increases in IL-1beta mRNA, IL-1beta protein, ICE activity and apoptosis induced by H. pylori alone. Enhancement of IL-1beta protein release induced by hyperosmotic stress was significantly attenuated by an ICE inhibitor, Z-YVAD-FMK. CONCLUSIONS: Hyperosmotic stress enhances the release of bioactive mature IL-1beta protein in H. pylori-infected gastric epithelial cells, in part by upregulating IL-1beta mRNA expression, and in part by enhancing ICE activity. These results may explain the mechanisms by which chronic intake of a high salt diet increases the risk of gastric cancer among H. pylori-infected human subjects. PMID- 16677165 TI - Influences of Helicobacter pylori on cyclooxygenase-2 expression and prostaglandinE2 synthesis in rat gastric epithelial cells in vitro. AB - BACKGROUND AND AIM: It is known that cyclooxygenase (COX)-2 is over expressed in gastrointestinal neoplasia and Helicobacter pylori (H. pylori) infection is causally linked to gastric cancer. The present study aimed to elucidate the effects of H. pylori on COX-2 expression and prostaglandinE(2) (PGE(2)) production in a gastric epithelial cell line derived from normal rat gastric mucosa (RGM1). METHOD: H. pylori water extracts were prepared from a supernatant of the H. pylori suspension in distilled water. RGM1 cells were cultured with H. pylori water extracts at the final concentration of 2.5, 5, 10 microg/mL for 24 h. For the time sequence study, RGM1 cells were cultured with 10 microg/mL H. pylori water extracts for 0, 6, 12, 24 and 48 h. COX-1 and COX-2 expression in the RGM1 cells was analyzed by western blotting. The levels of PGE(2) in the cultured media were measured by enzyme immunoassay. RESULTS: H. pylori did not affect COX-1 expression; whereas COX-2 expression increased by six-fold at 24 h after incubation of RGM1 cells with 10 microg/mL H. pylori water extracts. The increase in COX-2 expression was evident after 12 h of incubation; reached a peak at 24 h and declined at 48 h. H. pylori dose dependently increased COX-2 expression and PGE(2) synthesis in RGM1 cells. CONCLUSION: H. pylori induces COX 2 expression and increases PGE(2) synthesis in RGM1 cells in vitro. These results indicate that H. pylori-associated gastric carcinogenesis may depend on COX-2 expression. PMID- 16677167 TI - Efficacy of camostat mesilate compared with famotidine for treatment of functional dyspepsia: is camostat mesilate effective? AB - BACKGROUND: Differentiation between functional dyspepsia and early chronic pancreatitis is difficult because these diseases do not produce specific abnormalities in laboratory testing. The aim of this study was to examine the potential efficacy of camostat mesilate, a protease inhibitor, against functional dyspepsia and to characterize patients with favorable responses. METHODS: Dyspeptic patients who exhibited no abnormalities on laboratory blood and urine testing, abdominal ultrasonography and upper gastrointestinal endoscopy were randomized to receive camostat mesilate 200 mg three times daily or famotidine 20 mg twice daily for 4 weeks. Symptoms severity was recorded before and at 2 and 4 weeks after starting treatment using a visual analog scale. RESULTS: Epigastralgia was significantly improved after 2 and 4 weeks of treatment in both groups (P < 0.01); this improvement tended to be more marked in the camostat mesilate group (P < 0.05 at 2 weeks). The beneficial effect of camostat mesilate on epigastralgia was more prominent in chronic alcohol drinkers at 2 weeks (P < 0.05) and 4 weeks (P < 0.01). CONCLUSIONS: Camostat mesilate is superior to famotidine for relieving epigastralgia in patients with functional dyspepsia. Its pain-relieving effect is greater in patients who habitually drink alcohol. PMID- 16677168 TI - Gastrointestinal: pneumatosis coli. PMID- 16677169 TI - Hepatobiliary and pancreatic: agenesis of the dorsal pancreas. PMID- 16677170 TI - Hepatobiliary and pancreatic: anomalous pancreatobiliary ductal junction. PMID- 16677171 TI - Hepatobiliary and pancreatic: anomalous pancreatobiliary junction with choledochal cyst. PMID- 16677172 TI - Corrosive induced carcinoma of esophagus: report of three patients and review of literature. AB - Patients with corrosive induced esophageal strictures have more than a 1000-fold risk of developing carcinoma of the esophagus. We report three cases of corrosion carcinoma seen by us (a team of gastroenterologists, radiologists and a surgeon) in the last 15 years. Two cases were among 156 patients with corrosive induced strictures on our follow-up, and constituted the only corrosion carcinoma out of 650 esophageal carcinomas operated on by us. Nearly all reported patients with corrosion carcinoma in the published literature had consumed an alkali, but two of our three patients had consumed an acid. PMID- 16677173 TI - Primary biliary cirrhosis-autoimmune hepatitis overlap syndrome: complete biochemical and histological response to therapy with ursodesoxycholic acid. PMID- 16677174 TI - Compound heterozygosity for factor V and methylenetetrahydrofolate reductase mutations in a patient with Budd-Chiari syndrome. PMID- 16677175 TI - Migrated double pigtail biliary stent causes small bowel obstruction. PMID- 16677176 TI - Predisposition of antituberculosis drug induced hepatotoxicity by cytochrome P450 2E1 genotype and haplotype in pediatric patients. PMID- 16677177 TI - End of life decisions and care of the elderly. PMID- 16677178 TI - Oral hygiene and the need for treatment of the dependent institutionalised elderly. AB - OBJECTIVE: To assessing the oral hygiene and treatment needs of a geriatric institution in southern France. BACKGROUND: For various reasons, the care demand from elderly people is low and difficult to determine, whereas their oral status would need long and complicated treatments. MATERIALS AND METHODS: From 2003 to 2004, a cross-sectional study of 321 elderly patients was conducted at several geriatric services of Montpellier, France. The clinical evaluation of dental status was recorded together with medical information. Dental and prosthetic hygiene, status of dentures, caries experience, dependence conditions and treatment needs were evaluated. RESULTS: The prevalence of edentulism was 27%, with no gender difference (23% of the men and 29% of the women). Among them, 16.7% (upper jaw) and 18.1% (lower jaw) were totally edentulous with no denture. The mean number of decayed and missing teeth was 3.7 for men and 2.8 for women and 21.5 for men and 21.0 for women, respectively. The mean number of filled teeth was 0.8 for men and 1.3 for women, with no statistical difference according to gender for the three indexes. Most of the subjects needed prostheses (53%), 45.1% extractions and 30.6% conservative treatments. Only 2.4% did not need any treatment. CONCLUSION: The prevalence of edentulism was relatively low, while the need for prosthodontic rehabilitation, especially for men, was still very high. The dental hygiene was globally inadequate. This evaluation emphasises the care demand and the need for help in oral hygiene procedures for the dependent institutionalised elderly. PMID- 16677179 TI - Oral health promotion among older persons and their care providers in a nursing home facility. AB - OBJECTIVES: To assess oral health status and oral health-related quality of life (OHRQoL) of residents in an extended care facility and to assess the care providers' oral health attitudes and knowledge. METHODS: Participants included 137 residents (58.1% female, age range 32-94 years, 91% African-American) and 22 care providers. Residents received an oral examination and completed the Oral Health Impact Profile (OHIP-14), an OHRQoL questionnaire. Care providers completed an oral health knowledge (OHK) questionnaire before and after the on site geriatric oral health education and training programme. RESULTS: Oral examinations showed that 58% of the residents had extensive oral health needs. On the OHIP-14, the mean severity was 9.2 (SD=12.0), extent (number of items rated as 'fairly often' or 'often') was 1.2 (SD=2.6) and prevalence (participants rating at least one item at least 'fairly often') was 37.8%. Most prevalent negative impact items were about 'oral pain', 'appearance' and 'self consciousness'. Regarding OHK, caregivers' knowledge improved following instruction from 65% correct on the pre-test to 90% correct on the post-test (p<0.05). Subsequent to the eight in-service workshops, providers reported that physical limitations, fear of getting bitten and time constraints were barriers to providing oral hygiene to their residents. CONCLUSION: Examination data showed a high level of dental needs among the majority of residents, accompanied by significantly reduced OHRQoL. Although care providers' OHK improved following the geriatric service programme, they reported specific barriers regarding their provision of oral hygiene care to the residents. PMID- 16677180 TI - A study to compare the oral health impact profile and satisfaction before and after having replacement complete dentures constructed by either the copy or the conventional technique. AB - AIMS: To assess the oral health impact profile (OHIP) on edentulous subjects who needed to have their dentures replaced either by a copy technique or a conventional technique, and to determine whether any change in the subjects' assessment of their original and replacement dentures impacted on oral health related quality of life parameters. BACKGROUND: The OHIP is an instrument used to measure subjects' perceptions of the social impact of oral disorders on their well being. Changes in the OHIP may occur when subjects need to have their complete dentures replaced. Furthermore, it is possible that the method by which the dentures are constructed may impact on this. METHODS: A total of 65 edentulous subjects were studied. Thirty-three subjects had their dentures constructed by a copy technique and 32 by a conventional technique. Subjects completed the OHIP-14 questionnaires before and after being provided with a set of replacement complete dentures. They also assessed specific features of the upper and lower dentures. Responses were recorded on a Likert scale. RESULTS: For many subjects, the responses in the before treatment questionnaires were at the lower end of the Likert scale, indicating that there were no major impacts on oral health related quality of life parameters. There were no major differences between the copy denture subjects and the conventional denture subjects in relation to the change in OHIP scores before and after treatment. Generally subjects expressed improved satisfaction with the new lower denture. However, for the copy denture group there were significant improvements for all seven assessments compared with only five out of seven assessments for subjects in the conventional group. CONCLUSION: For these groups of edentulous subjects, although they may need dentures to be replaced after a period of wear, this does not necessarily have significant impacts on oral health related quality of life parameters. It seems likely that this is the main reason why the provision of new dentures by either a copying or conventional technique did not result in major changes to the OHIP. PMID- 16677181 TI - Follow-up visits as a measure of adaptation process to removable prostheses. AB - OBJECTIVES: The paper was aimed to establish the influence of some general and local factors on adaptation process to removable prostheses (RPs). The adaptation process is a complex issue, which is often associated with painful reactions. Those complaints force patients to visit a dentist who makes alterations to reduce the patient's discomfort. MATERIAL AND METHODS: The study involved analysis of 300 dental records of patients who visited our Department for RPs. The authors analysed the influence of gender, age, condition of general health, maintenance of the prosthetic base tissues and the kind of prostheses on the process of adaptation. It was measured by means of a number of follow-up visits of the patients to our polyclinic. The findings were analysed statistically by means of chi-squared test. The level of significance was assumed to be p < 0.05. RESULTS AND CONCLUSIONS: Adaptation to RPs without any correction was revealed by about one-fifth of patients. Men adapted to RPs better than women. The biggest problems with adaptation to RPs were observed in patients using a complete and partial prosthesis simultaneously. The number of follow-up visits by patients who were treated with RPs for the first time or had been treated before was almost the same. Adaptation of RPs on an atrophic muco-osseous ridge was associated with more multiple visits than in the case of a well-preserved ridge. Healthy patients adapt to RPs better than patients with systemic disorders. Taking into account the limitations of the study, the number of follow-up visits may be used as a helpful indicator of the adaptation process. PMID- 16677182 TI - Effects of oral functional training for nutritional improvement in Japanese older people requiring long-term care. AB - OBJECTIVES: The aim of this study was to evaluate the effect of oral functional training and nutrient supplements to improve the nutrition of malnourished elderly people in a nursing home. BACKGROUND: Malnutrition is a frequent problem in the elderly requiring long-tem care; however, it is not clear whether oral functional training can be effective to improve nutrition. SUBJECTS AND METHODS: Fourteen subjects of 82 residents (mean age 85.7+or-6.2 years) in a nursing home, who had a serum albumin level of 21,000) from Belgium, France, Germany, Italy, the Netherlands and Spain were assessed using the Composite International Diagnostic Interview (mental disorders), World Health Organization Disability Assessment Schedule second edition (functional disability) and self-report (physical disorders). Means in different groups were compared using the Mann Whitney U-test and multiple regression analyses. RESULTS: Mental disorders were related to disability in all domains of functioning: anxiety disorders the most, followed by mood disorders, and finally alcohol disorders. The findings suggest that mental disorders are associated with similar or higher levels of disability in all domains, except getting around, than arthritis and heart disease. CONCLUSION: Mental disorders are associated with a similar or higher negative impact on daily functioning than arthritis and heart disease. PMID- 16677227 TI - Outcomes in adulthood for children with foetal growth retardation. A linkage study from the Nord-Trondelag Health Study (HUNT) and the Medical Birth Registry of Norway. AB - OBJECTIVE: The aims were to examine the long-term functional outcome and risk of mood disorders in adulthood in individuals with foetal growth retardation. METHOD: In a prospective cohort study of 7806 individuals aged 20-30 years, using linked data from the Health Survey of Nord-Trondelag (HUNT-2) and the Medical Birth Registry of Norway, we studied the long-term effects of being born with a birth weight below the 10th percentile for gestational age (SGA). RESULTS: SGA individuals had lower educational level (OR: 1.33), lower socioeconomic functioning level (OR: 1.77) and more frequent reported mood disorder in adulthood (OR: 1.26). Analyses of a substratum of infants born at term showed almost identical results. CONCLUSION: Foetal growth retardation measured as SGA shows a moderate risk for lower education and socioeconomic level and for anxiety and/or depression in young adulthood. Issues concerning interventions for children at risk should be considered. PMID- 16677228 TI - Seasonal changes in clinical status in bipolar disorder: a prospective study in 1000 STEP-BD patients. AB - OBJECTIVE: To investigate seasonal and regional effects on bipolar I and II patients. METHOD: The Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD) patients were prospectively examined for monthly change in prevalence rates of depressed and recovered clinical status over the year. General Estimating Equation modeling was used to assess the effect of season on prevalence rates. Additionally, patients were stratified by bipolar subtype and by region. RESULTS: A significantly higher prevalence rate of depression is observed in the northern sites, a significant prevalence by month effect is found only in the bipolar II patients. CONCLUSION: The prevalence of depression is greater in patients from the northern vs. southern STEP-BD sites. Seasonal peak prevalence rates of depression differ by region. Bipolar II patients were more ill year-round and demonstrated greater monthly fluctuation in prevalence rates of being ill than did bipolar I patients. We conclude that seasonal effects upon bipolar patients vary by region and bipolar subtype. PMID- 16677232 TI - Nose and lung, two of a kind? PMID- 16677233 TI - Allergic rhinitis and its impact on otorhinolaryngology. AB - Allergic rhinitis (AR) is a disease with growing impact on everyday medical practice, as its prevalence has steadily increased during the last decades. Immunoglobulin-E (IgE)-mediated airway inflammation may manifest itself as AR, asthma or both. Allergic inflammation in upper and lower airways is now considered as one airway disease, with manifestation of symptoms in upper, lower or global airway. This insight into allergic inflammation of the whole respiratory tract has consequences for the diagnostic and therapeutic approach of affected patients, as highlighted in the ARIA document. In contrast to asthma, the link between AR and associated conditions in the upper airways like rhinosinusitis, nasal polyps, recurrent viral infections, adenoid hypertrophy, tubal dysfunction, otitis media with effusion and laryngitis remains less explored. It is however of utmost importance to consider the aetiological role of IgE-mediated inflammation of the nasal mucosa in several diseases of the upper respiratory tract, as they represent a large body of patient population seen by the general practitioner as well as the paediatrician, allergologist and otorhinolaryngologist. We here aim at reviewing the current literature on the relationship between AR and conditions in upper airways frequently encountered in everyday clinical practice, and highlight the need for further studies exploring the role of allergic inflammation in the development of these diseases. PMID- 16677234 TI - Nasal nitric oxide and nasal allergy. AB - Measurements of nasal nitric oxide (nNO) are attractive because they are completely noninvasive and can easily be performed. The measurements may be useful in the early diagnosis of patients with chronic airway disorders such as Kartager's syndrome and cystic fibrosis. The possible use of nNO measurements in the diagnosis and treatment of allergic rhinitis still needs to be further evaluated because of the variable and also contradicting findings of nNO concentrations in this disease. In this review we will discuss the origin, production and measurement of nNO as well as the effect of allergic rhinitis, nasal allergen challenge and medication on nNO. Subsequently, we examine published data on allergic rhinitis and nNO, and summarize the effect of treatment of rhinitis on nNO. Finally, we discuss the potential future role for nNO in the diagnosis and management of allergic rhinitis. PMID- 16677235 TI - Factors responsible for differences between asymptomatic subjects and patients presenting an IgE sensitization to allergens. A GA2LEN project. AB - The synthesis of allergen-specific IgE is required for the development of allergic diseases including allergic rhinitis and allergic asthma (patients), but many individuals with allergen-specific IgE do not develop symptoms (asymptomatic subjects). Differences may exist between asymptomatic subjects and patients. Whether the presence of allergen-specific IgE translates into clinical allergy most likely depends on a complex interplay of multiple factors. These include a family history of atopy, the levels of total serum IgE and, allergen-specific IgE or IgG, epitope-specificity of IgE and their degree of polyclonality (mono- vs polysensitized), as yet unidentified serum factors, the balance of T regulatory cells (Treg) and Th1/Th2 cells, the polymorphisms of the high affinity receptor for IgE (FcepsilonRI) and other factors regulating the activation of FcepsilonRI bearing cells. Asymptomatic subjects may be more often monosensitized than patients who may be more often polysensitized. There are many unanswered important questions that need to be addressed in order to better understand how IgE sensitization translates into clinical allergy. The assessment of differences between the asymptomatic and symptomatic groups of subjects represent one of the scientific programs of Global Allergy and Asthma European Network funded by the European Union and the hypotheses underlying these differences are presented in this paper. PMID- 16677236 TI - Rhinitis and asthma in athletes: an ARIA document in collaboration with GA2LEN. AB - This consensus document is aimed at reviewing evidence that the rhinitis-asthma links have peculiar features in athletes. Beside a review of epidemological data on the high prevalence of rhinitis and asthma in athletes, the effects on intense physical exercise on the immune system and repiratory functions are discussed, with special reference to the role of allergens and pollutants. In extending the Allergic Rhinitis and its Impact on Asthma (ARIA) recommendations to athletes, the issue is addressed of adapting diagnosis and management to criteria set by the International Olympic Committee (IOC) and regulations adopted by the World Anti-Doping Agency (WADA). PMID- 16677237 TI - Prevalence, classification and perception of allergic and nonallergic rhinitis in Belgium. AB - BACKGROUND: Allergic rhinitis (AR) and noninfectious, nonallergic rhinitis (NINA) are common disorders, which may prompt patients to seek medical help. METHODS: We performed a survey in a representative sample of the Belgian population (n=4959) with an overall prevalence of self-declared recent rhinitis symptoms of 39.3%. Detailed information on patients having experienced nasal symptoms over the past year was then obtained from a random sample of respondents (n=743). RESULTS: The adjusted prevalence was 29.8% for AR and 9.6% for NINA, respectively. According to the ARIA classification, there was significantly more 'persistent' symptomatology in the AR group (40.8%) than in NINA (23.5%) (P<0.001), and more 'moderate/severe' symptom intensity in AR (75.4%) than in NINA (53.1%) (P<0.001). Allergic rhinitis patients suffered from a greater number of symptoms than NINA patients (P<0.001). Asthma, skin and food allergy as co-morbidities were all found to be significantly more prevalent in the AR vs the NINA group (P<0.05 for all). The percentage of consulting patients (total: 66.8%), who subjectively perceived their rhinitis as moderate/severe, was 94.0%, whereas 75.6% of these patients were classified accordingly based on ARIA criteria. CONCLUSIONS: We found a high prevalence of self-declared rhinitis symptoms in the Belgian population, AR being about three times more prevalent than NINA. In addition, AR patients suffered from a greater number of symptoms and displayed a more 'persistent' and 'moderate-severe' ARIA profile than NINA. About 75% of patients seeking medical help suffer from 'moderate to severe' forms of rhinitis. PMID- 16677238 TI - The diagnosis of asthma using a self-questionnaire in those suffering from allergic rhinitis: a pharmaco-epidemiological survey in everyday practice in France. AB - BACKGROUND: All recent guidelines recommend a search for asthma utilizing both specific interrogation and pulmonary function tests in patients suffering from allergic rhinitis. Although the mandatory place of spirometry has not been confirmed, a self-questionnaire containing nine specific questions on asthma symptoms in different daily life situations was found to be capable of discriminating asthmatics from nonasthmatics in a rhinitic population. OBJECTIVE: We addressed the questions of prevalence of asthma using a validated self questionnaire and what might be the risk factors of being asthmatic according to that specific self-questionnaire. METHODS: Between April 2003 and September 2004, nearly 12,000 rhinitis patients were enrolled by more than 2300 physicians (78% general practitioners, 22% ear nose and throat specialists). Patients were consulting for an exacerbation of chronic rhinitis and did not have a previous diagnosis of asthma. Both doctors and patients filled out a specific questionnaire on rhinitis and asthma. RESULTS: Almost 30% of the patients had at least three positive answers to the self-questionnaire and could possibly be considered as asthmatics. We found five independent clinical risk factors for having >or=3 positive answers to the self-questionnaire. Severity of rhinitis (moderate-severe vs mild, OR=1.84; 95% CI=1.68-2.00), diagnosis of allergy (yes vs no) (OR=1.86; 95% CI=1.68-2.00), body mass index (30) (OR=0.51; 95% CI=0.39-0.66), type of rhinitis (persistent vs intermittent) (OR=1.25; 95% CI=1.15-1.37), and patient age (47) (OR=0.73; 95% CI=0.65-0.80). CONCLUSION: Asthma symptoms are frequent in rhinitics without a prior history of asthma. Several variables were shown to be predictive of asthma in these patients. PMID- 16677239 TI - Classification and management of allergic rhinitis patients in general practice during pollen season. AB - BACKGROUND: Allergic rhinitis (AR) represents a major challenge in primary care. The Allergic Rhinitis and its Impact on Asthma (ARIA) group proposed a new classification for AR and developed evidence-based guidelines for the management of this disease. We conducted this study to further characterize the classes of AR described by ARIA, and to evaluate whether the management of AR in general practice is in accordance with the ARIA guidelines. METHODS: During the pollen season of 2003, 95 Belgian general practitioners (GPs) enrolled 804 patients who presented with symptoms of AR. For each patient, a questionnaire comprising the clinical presentation and management was completed. RESULTS: In 64% of the patients, AR was classified as intermittent and in 36% as persistent. Persistent rhinitis caused more discomfort than intermittent rhinitis. Only 50% of the patients had ever undergone allergy testing. Among them, 51% were allergic to both seasonal and perennial allergens. Eighty-two per cent of the persistent rhinitics were allergic to at least one seasonal allergen and 72% of the intermittent rhinitics to at least one perennial allergen. When compared strictly with the ARIA recommendations, 49% of the patients with mild and/or intermittent AR were overtreated, whereas about 30% of those with moderate/severe persistent rhinitis were undertreated. CONCLUSION: This study confirms that the previous classification of AR into 'seasonal' and 'perennial' is not satisfactory and that intermittent and persistent AR are not equivalent to seasonal and perennial AR respectively. Furthermore, persistent rhinitis has been shown to be a distinct disease entity. Further efforts are required to disseminate and implement evidence-based diagnostic and treatment guidelines for AR in primary care practice. PMID- 16677240 TI - Rhinitis symptoms and IgE sensitization as risk factors for development of later allergic rhinitis in adults. AB - BACKGROUND: Rhinitis symptoms and IgE-sensitization often mismatch. Asymptomatic sensitization is an established risk factor for later rhinitis, whereas it is not clear whether rhinitis is a risk factor for later development of IgE sensitization. OBJECTIVE: To investigate whether nonallergic rhinitis is a risk factor for later development of IgE-sensitization in adults during an 8-year follow-up period, and whether asymptomatic sensitization is a risk factor for later development of rhinitis. METHODS: In a population-based study of 15-69 years olds in 1990, 734 subjects were re-examined in 1998. On both occasions questionnaires on rhinitis symptoms were completed and serum IgE (against birch, grass, mugwort, cat, dog, and Dermatophagoides pteronyssinus) were determined (positive if >or=0.35 kUA/l). Asymptomatic sensitization: positive IgE levels without any rhinitis symptoms. Nonallergic rhinitis: rhinitis symptoms and no sensitization. RESULTS: Asymptomatic sensitization to pollens, pets, or house dust mite was significantly associated with onset of rhinitis symptoms, also when changing baseline cut-off for sensitization to >or=0.1 or >or=0.7 kUA/l. The 8 year incidence of pollen-related rhinitis was 15.1% and 2.6% in subjects sensitized and nonsensitized to pollens, respectively (odds ratio 6.1, 95% CI 2.3 16.0). Persistent or intermittent nonallergic rhinitis was not significantly associated with later sensitization, yet a positive trend for development was observed in nonallergic pollen-related rhinitis. CONCLUSION: Asymptomatic sensitization but not nonallergic rhinitis was a significant risk factor for later development of allergic rhinitis. PMID- 16677241 TI - Nasal nitric oxide in objective evaluation of chronic rhinosinusitis therapy. AB - BACKGROUND: The assessment of the response of chronic rhinosinusitis (CRS) to therapy is difficult. Computerized tomographic (CT) scans cannot be repeatedly used so measures such as symptom scores, endoscopic findings, and parallel measures such as saccharin clearance time are employed instead. OBJECTIVE: To study the effect of CRS therapy on nasal nitric oxide and to see whether nasal nitric oxide level changes correlate with other assessments. METHODS: The study was a prospective randomized trial of patients with CRS, with or without polyps, who had failed initial medical therapy with douching and nasal corticosteroids and who then had abnormal CT scans. They were treated either medically or surgically, with follow up at 6 and 12 months whilst still taking nasal corticosteroids. Nasal nitric oxide was measured initially and at 6 and 12 months as well as symptom scores, endoscopy, polyp grading, and saccharin clearance time. RESULTS: Initial absolute nasal nitric oxide levels correlated inversely with CT scan changes, (P<0.001). The percentage rise in nasal nitric oxide seen on both medical and surgical treatment correlated with changes in symptom scores (P<0.001), saccharin clearance time (P<0.001), endoscopic changes (P<0.001), polyp grades (P<0.05 at 6 months, P<0.01 at 12 months) and surgical scores (P<0.01). There was no significant correlation with age, sex, smoking or allergy. CONCLUSION: Nasal nitric oxide, which is easily measured, provides a valuable non invasive objective measure of the response of CRS to therapy. Topical nasal corticosteroids may be needed to reduce the contribution of nasal epithelial nitric oxide and allow that emanating from the sinuses to be measured. PMID- 16677242 TI - Increased expression of lipoxygenase enzymes during pollen season in nasal biopsies of pollen-allergic patients. AB - BACKGROUND: Exposure of patients sensitized to pollen triggers development of seasonal allergic rhinitis symptoms (SAR). Eicosanoids are a group of arachidonic acid metabolites contributing to the symptoms of SAR. The aim of this study was to investigate seasonal changes in the expression of enzymes of the eicosanoid pathway in the nasal mucosa of patients with SAR. METHODS: Twenty SAR patients allergic to birch or grass and eight healthy subjects were included in the study. Patients registered rhinoconjunctivitis symptoms and use of rescue medication before and during the pollen season. Nasal biopsies were obtained before and around the peak of the season, sectioned and stained using markers for eosinophils, mast cells, T cells and neutrophils. Antibodies against the following enzymes were also used: cyclo-oxygenase (COX-1, COX-2), 5-lipoxygenase (5-LO), 5-lipoxygenase-activating factor (FLAP), LTA4 hydrolase (LTA4h) and LTC4 synthase (LTC4s). RESULTS: During the pollen season symptoms of rhinoconjunctivitis and medication score increased significantly (P=0.001; P=0.001 respectively). During the pollen season numbers of eosinophils (P=0.02) and cell positive 5-LO (P=0.02), LTC4s (P=0.04) and LTA4h (P=0.02) increased significantly. During season number of mast cells and cells expressing 5-LO and LTA4h were higher in SAR than in healthy controls group (P=0.02; P=0.01; P=0.03 respectively). CONCLUSION: In sensitized patients exposure to pollen allergen results in increased expression of enzymes of the eicosanoid pathway. PMID- 16677243 TI - Effects of intranasal fluticasone and salmeterol on allergen-induced nasal responses. AB - BACKGROUND: Combination of inhaled steroid and long-acting beta-agonists has synergistic effects in asthma. OBJECTIVE: To investigate whether nasal corticosteroid and long-acting beta-agonists have synergistic effects on allergen induced nasal responses. METHODS: The effects of intranasal treatment with fluticasone p-MDI (50 microg bid), salmeterol p-MDI (25 microg bid), their combination, and placebo, on nasal symptoms, eosinophil differential cell count and albumin in nasal lavage fluid (measures of inflammation and leakage respectively) and nasal electrical potential difference (measure of epithelial integrity) were studied in 11 atopic subjects with rhinitis, in a randomized, partially-blinded, 4-period, cross-over study. The measurements were made at baseline, at the end of 1 week of treatment, and immediately after a nasal allergen provocation. RESULTS: Allergen-induced sneeze, postnasal drip and nasal obstruction were significantly reduced by fluticasone, but not by salmeterol. Eosinophil count in postallergen nasal lavage fluid was significantly less after fluticasone (median 1.9%, IQR 4.6) and salmeterol treatment (median 2.5%, IQR 8.5) compared with placebo (median 12.5%, IQR 27.9). Compared with placebo, both fluticasone and salmeterol attenuated allergen-induced change in nasal potential (mean change from baseline -18.5, +0.4 and -7.2% respectively) and the increase in nasal albumin (median 154, 119 and 130 ng/ml respectively). Combination treatment did not have any additional benefits over the individual therapies. CONCLUSIONS: Although salmeterol has anti-inflammatory properties, intranasal salmeterol or its combination with fluticasone do not offer any added benefit over intranasal fluticasone alone for allergen-induced nasal responses. PMID- 16677244 TI - Effect of montelukast on lung function in asthma patients with allergic rhinitis: analysis from the COMPACT trial. AB - BACKGROUND: The Clinical Outcomes with Montelukast as a Partner Agent to Corticosteroid Therapy (COMPACT) trial demonstrated that montelukast added to budesonide (MNT + BD) was as efficacious as double the dose of budesonide (dBD) in improving morning peak expiratory flow (AM PEF) in adult asthmatics. Recent studies have demonstrated that montelukast is also effective in treating daytime and nighttime allergic rhinitis (AR) symptoms in asthmatic patients. This analysis was designed to examine whether asthmatic patients with comorbid AR respond differently than patients without comorbid AR in terms of asthma control (lung function). METHODS: There were 216 asthmatic patients in the MNT+BD group and 184 patients in the dBD group with AR. Treatment differences in the change from baseline in AM PEF were compared. Least square (LS) mean and 95% confidence interval (CI) were derived from an anova model adjusting for baseline and study site. RESULTS: There was a 9.2% increase in AM PEF from baseline in the MNT+BD group compared with a 6% increase in the dBD group. The LS mean difference [(MNT+BD)-dBD] was 14.2 l/min (P=0.028). Other secondary endpoints were similar between groups. CONCLUSION: In the subgroup of asthmatic patients with AR, a combined treatment approach that included montelukast and budesonide provided significantly greater efficacy in reducing airflow obstruction compared with doubling the dose of budesonide. These results support recommendations by the Allergic Rhinitis and its Impact on Asthma initiative that suggest a unified approach aimed at treating the airway inflammation common to both diseases is beneficial for the large proportion of asthmatics who also suffer from AR. PMID- 16677245 TI - Ocular and airway symptoms related to organic acid anhydride exposure--a prospective study. AB - BACKGROUND: Organic acid anhydrides (OAA) are used as hardeners in epoxy resin systems. They are powerful sensitizers giving frequent rhinitis and asthma in exposed workers. Incidence of symptoms is unknown. Here we present the first prospective study on the associations between OAA exposure, symptoms, and effects of confounding factors. METHODS: All new employees in three plants handling OAA were followed for up to 8.5 years. Before the employment, a questionnaire reporting about symptoms of eyes and airways, smoking habits, and atopy was answered. The subjects were asked at regular medical examinations about work tasks and work-related symptoms. Serum was analysed for specific OAA antibodies. RESULTS: Mean exposures varied between 6 and 39 microg/m3. The incidence for work related symptoms of the eyes, nose, pharynx, and lower airways was 91, 64, 46, and 31 per 1000 years of exposure, respectively. Symptoms were found frequently, even at mean exposure level at <10 microg/m3. Smoking and atopy increased the risk of symptoms. Immunoglobulin (Ig)E sensitized workers had a significant increased risk for symptoms of the eyes and pharynx and for running nose/sneezing. CONCLUSIONS: Organic acid anhydrides exposure is associated with frequent ocular and airway symptoms even at mean exposure levels at <10 microg/m3. There is an important need for establishment of an occupational threshold limit. A limit value of below 5 microg/m3 is proposed. PMID- 16677246 TI - Is outdoor air pollution associated with physician visits for allergic rhinitis among the elderly in Toronto, Canada? AB - BACKGROUND: While a number of studies suggest that air pollution is associated with allergic rhinitis in children, findings among adults have been equivocal. The aim of this study was to examine the relationship between outdoor air pollution and physician visits for allergic rhinitis among individuals>or=65 years of age in Toronto, Canada. METHODS: Physician visits were identified by using data from the Ontario provincial health insurance plan that is made available to all residents. Our analyses are based on 52,691 physician visits for allergic rhinitis among individuals>or=65 years of age in the Toronto metropolitan area between 1995 and 2000. Generalized linear models were used to regress daily counts of physician visits against daily measures of gaseous and particulate components of air pollution after controlling for seasonality, potential confounders (temperature, relative humidity, aeroallergens), overdispersion and serial correlation. RESULTS: A large number of comparisons were undertaken, with most showing no statistically significant associations between daily levels of air pollution and the number of physician visits for rhinitis. In contrast, an interquartile increase in the 10-day average of ragweed particles increased the mean number of daily rhinitis consultations by 6.4% (95% CI=0.7-12.4%). CONCLUSIONS: Our findings suggest that outdoor air pollution is a poor predictor of physician visits for allergic rhinitis among the elderly. PMID- 16677247 TI - Seasonal dynamics of chemokine receptors and CD62L in subjects with asymptomatic skin sensitization to birch and grass pollen. AB - BACKGROUND: Asymptomatic skin sensitization (AS) has been shown to be a risk factor for respiratory allergic disease. CCR4, CXCR1 and CD62L have all been assigned a role in the immunopathogenesis of allergy. Memory T-cell expression of CCR4, CXCR1 and CD62L has not hitherto been investigated in subjects with AS. METHODS: We investigated seasonal CD4 memory T-cell expression of the chemokine receptors CCR4, CXCR1 as well as L-selectin (CD62L) in fresh cultures derived from symptomatic atopics (SAs), subjects with AS and healthy controls (HCs). Peripheral blood mononuclear cells from all three groups were isolated during birch and grass pollination as well as in the following winter. CD4 memory T-cell expression of CCR4, CXCR1 and CD62L was determined by flow-cytometry. RESULTS: During spring and summer, a significantly increased proportion of memory T cells expressed CCR4, CXCR1 and CD62L in SAs when compared with subjects with AS and HCs. Only SAs exhibited seasonal fluctuations in numbers of CCR4, CXCR1 and CD62L positive memory T cells. CONCLUSION: Although clearly IgE sensitized, subjects with AS have significant diminished numbers of CCR4, CXCR1 and CD62L positive memory T cells, during pollination, when compared with SAs. In contrast to SAs, cultures derived from subjects with AS did not display seasonal variation. Our findings explain the lack of clinical symptoms, during pollination, in subjects with AS. PMID- 16677248 TI - Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells. AB - Using human mast cells (MC) derived by culture of CD34+ peripheral blood precursors, a comprehensive study was performed of expression of 11 known Siglecs. Analysis was initially performed at the mRNA level using gene arrays. Positive results were then validated at the protein level using indirect immunofluorescence and flow cytometry, and for some Siglecs, Western blot analysis was also used. Culture-derived MC expressed mRNA for CD22 (Siglec-2), CD33 (Siglec-3), Siglec-5, Siglec-6, Siglec-8 and Siglec-10. Flow cytometry confirmed surface expression of all these molecules except for CD22 and Siglec 10, where levels were low or undetectable. However, Western blotting was able to detect MC expression of CD22 and Siglec-10, suggesting that these proteins were mostly cytoplasmic. CD34+ precursor cells from peripheral blood constitutively expressed surface CD33, Siglec-5 and Siglec-10. As they matured into MC, their constitutive levels of CD33 changed little, Siglec-5 and Siglec-10 declined, and Siglec-6 and Siglec-8 appeared de novo, all in parallel with accumulation of histamine and other MC markers, such as surface expression of FcepsilonRIalpha, and CD51. Phenotypic analysis of LAD-2 MC yielded a similar pattern of Siglec expression except that CD22 expression was particularly prominent. Finally, immunohistochemistry confirmed expression of these same Siglecs by mature tryptase-positive MC in human lung tissues. These data demonstrate an extensive and previously unappreciated pattern of Siglec expression on human MC. Whether engagement and signaling through these inhibitory Siglecs can impact MC biology will require further investigation. PMID- 16677249 TI - A critical role for vesicle-associated membrane protein-7 in exocytosis from human eosinophils and neutrophils. AB - BACKGROUND: Granulocyte exocytosis is proposed to be critically dependent on the interaction of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on granules/vesicles (v-SNAREs) and plasma membrane (t-SNAREs). Previous studies indicated that the v-SNARE, vesicle associated membrane protein (VAMP)-2, as well as t-SNAREs (SNAP-23, syntaxin-4 and -6) are implicated in exocytosis from human granulocytes. Vesicle-associated membrane proteins-7 and -8 have been implicated in endosome/lysosome trafficking, however, their role in granulocyte exocytosis remains obscure. OBJECTIVE: We sought to investigate the expression and functional role of SNARE isoforms in the secretion of different granule-derived mediators in human eosinophils and neutrophils. METHODS: The expression of SNAREs was determined by subcellular fractionation and flow cytometry. SNARE-specific antibodies were examined for their ability to impair mediator release from permeabilized eosinophils and neutrophils. RESULTS: Vesicle-associated membrane proteins-7 and -8 were localized to granule and membrane-enriched fractions in eosinophils and neutrophils, whereas syntaxin-6 was not detectable. In permeabilized cells, anti VAMP-7, but not anti-VAMP-8, antibody impaired the secretion of all mediators examined (in eosinophils, eosinophil peroxidase and eosinophil-derived neurotoxin; in neutrophils, myeloperoxidase, lactoferrin and matrix metalloprotease-9) in a dose-dependent manner. In contrast, anti-VAMP-2 modestly and selectively impaired secretion from small granules and vesicles. Syntaxin-4, but not syntaxin-6, was found to interact with SNAP-23 and was partially involved in mediator secretion from multiple compartments. CONCLUSION: Our observations indicate for the first time a critical role for VAMP-7 in both eosinophil and neutrophil mediator release. PMID- 16677250 TI - Anaphylactic reaction following ingestion of fresh heart of palm (Chamaerops humilis L.). PMID- 16677251 TI - Lichenoid drug eruption due to an antihypertonic drug containing irbesartan and hydrochlorothiazide. PMID- 16677252 TI - Immediate type hypersensitivity to low molecular weight heparins and tolerance of unfractioned heparin and fondaparinux. PMID- 16677258 TI - Vaccination: is it a real problem for anesthesia and surgery? PMID- 16677259 TI - Anesthetic considerations in the management of Wilms' tumor. PMID- 16677260 TI - Immunization and anesthesia - an international survey. AB - BACKGROUND: There is no direct evidence of any major interaction between immunization and commonly used anesthetic agents and techniques in children, but it is possible that immunosuppression caused by anesthesia and surgery may lead to decreased vaccine effectiveness or an increased risk of complications. In addition, diagnostic difficulty may arise if a recently immunized child suffers from postoperative pyrexia or malaise. AIM: The aim of this study was to ascertain anesthetists' attitudes and practices regarding anesthesia and immunization. METHODS: We conducted an international survey of members of the Association of Paediatric Anaesthetists of Great Britain and Ireland (APAGBI) and the Society for Paediatric Anaesthesia of New Zealand and Australia (SPANZA). RESULTS: Two hundred and ninety-six (52.1%) APAGBI and 86 (49.4%) SPANZA responses were analyzed. There was no consensus of approach to this theoretical risk among respondents. In total, 60% of respondents would anesthetize a child for elective surgery within 1 week of receiving a live attenuated vaccine, but 40% would not. Few hospitals have formal policies on this issue and government guidance is based on a lack of evidence for adverse events rather than positive evidence of safety. CONCLUSIONS: There is a theoretical risk associated with anesthesia and surgery in recently immunized children. An international postal survey failed to find a consensus to this risk among pediatric anesthetists. From a risk management perspective, a review of the available evidence suggests that it would be prudent to adopt a cautious approach where the timing of elective surgery is discretionary. We therefore recommend that elective surgery and anesthesia should be postponed for 1 week after inactive vaccination and 3 weeks after live attenuated vaccination in children. PMID- 16677261 TI - Effects of lidocaine and steroids on breast milk-induced lung injury in rabbits. AB - BACKGROUND: Local anesthetics inhibit mediator and free radical release from polymorphonuclear granulocytes and migration to their site of action. In a recent study, lidocaine significantly improved the alveolar-arterial oxygen difference gradients (A-aDO2) after tracheal instillation of acid in rabbits. The purpose of the current study was to evaluate the effects of lidocaine and pulse-dose steroids on human breast milk (HBM)-induced lung injury in rabbits. METHODS: After Animal Care Committee approval, six adult rabbits were assigned to each of three treatments: control, lidocaine, and steroids. After induction of anesthesia and controlled ventilation, acidified HBM at pH 1.8 and volume 1.2 ml.kg(-1) was instilled into the trachea. Rabbits in the lidocaine group received lidocaine 2 mg.kg(-1) i.v. before tracheal instillation and then 2 mg.kg(-1).h(-1) i.v. continuously. Rabbits in the steroid group received 30 mg.kg(-1) methylprednisolone before tracheal instillation. A-aDO2, static compliance and blood for white cell count, and cytokine interleukin-8 (IL-8) concentration were obtained at baseline and at 1 and 4 h postinstillation. After 4 h, the rabbits were killed. The left upper lobe was isolated and excised to determine the wet/dry ratio. The right lung was lavaged with 30 ml normal saline to determine the white cell count and the concentrations of albumin and IL-8. Data were analyzed using one- or two-way anova with repeated measures and an Student-Newman Keuls (SNK) posthoc test (P < 0.05). RESULTS: All rabbits completed the protocol. A-aDO2 and CO2 tensions increased significantly at 1 and 4 h compared with baseline, although there were no differences among the treatments (P < 0.05). Compliance in the control group decreased compared with lidocaine and steroids. CONCLUSION: We conclude that preemptive lidocaine and steroids attenuate in part HBM-induced lung injury in rabbits. PMID- 16677262 TI - Two dimensional ultrasound guidance in central venous catheter placement; a postal survey of the practice and opinions of consultant pediatric anesthetists in the United Kingdom. AB - BACKGROUND: Recent guidelines from the UK National Institute for Clinical Excellence (NICE) recommend the use of ultrasound guidance for central venous catheter (CVC) insertion in children. We conducted a survey of pediatric anesthetists to determine current practice and opinion on the appropriate use of ultrasound guidance. METHOD: A confidential postal questionnaire was sent to all members of the Association of Paediatric Anaesthetists working in the UK. After 4 weeks a follow-up questionnaire was sent to nonrespondents. Members were questioned on availability and use of ultrasound, and its place in clinical practice and training. RESULTS: A total of 250 questionnaires were returned, a response rate of 63%. Of those members who placed CVCs in children (n = 196), 85% had access to ultrasound, and 68% stated that they used ultrasound guidance. Thirty-nine percent of clinicians who used ultrasound did so routinely. The remaining 61% used either a landmark or an ultrasound technique depending on circumstances. Regarding its mandatory use, 76% of responders believed that ultrasound guidance was beneficial in certain circumstances but did not need to be used routinely. Seventy-five percent of responders agreed that all pediatric anesthetists should have training and access to ultrasound for CVC placement. CONCLUSIONS: In the UK most pediatric anesthetists placing CVCs in children currently have access to ultrasound guidance. Despite a lack of widespread support for its routine use, most agree ultrasound is a useful tool, and that all pediatric anesthetists should have access and training in the use of this technology. PMID- 16677263 TI - Relationship between changes in thyroid hormone level and severity of the postoperative course in neonates undergoing open-heart surgery. AB - BACKGROUND: Our aim was to determine whether the changes in thyroid function after open-heart surgery in neonates depend on the postoperative course. METHODS: Twenty neonates undergoing open-heart surgery for congenital heart disease were prospectively studied in the cardiac intensive care unit of a university affiliated children's hospital. The patients were divided into two groups by level of inotropic support (high or mild). RESULTS: The groups were similar in age, bypass time and ultrafiltration volume. In both groups, there was a significant reduction in levels of thyroid-stimulating hormone and FT4 at 24 h postoperatively. However, in the high inotropic support group, FT4 was lower for a longer time. This group also had a significantly higher score on The Pediatric Risk of Mortality (PRISM; P < 0.042) and a longer duration of ventilation (P < 0.014). CONCLUSIONS: Neonates after open-heart surgery undergo changes in thyroid function characteristic of euthyroid sick syndrome. The degree of hypothyroxinemia may be related to the severity of illness and the postoperative course. PMID- 16677264 TI - Pediatric anesthesia practice and training in Japan: a survey. AB - BACKGROUND: Pediatric anesthesia in Japan is in the developing stage. The aim of this study was to review pediatric anesthesia training in Japan and to discuss the future prospects for this field. METHODS: We sent questionnaires to assess current pediatric anesthetic practice and training to all 106 university hospitals [UHs; response rate, 66% (70/106)] and all 17 children's hospitals [CHs; response rate, 87.5% (15/17)] in Japan. We also sent questionnaires to assess attitudes towards pediatric anesthetic training, to all 280 representatives of the Japanese Society of Anesthesiologists [JSA; response rate, 57.9% (162/280)]. RESULTS: The hospital survey revealed the number of pediatric anesthesia cases encountered in 15 CHs (25,009 cases) to be almost equivalent to that in 70 UHs (29,031 cases). In 19 of these UHs, there were no newborn surgical cases. Forty-nine UHs reported that no special training program existed for pediatric anesthesia, and only five UHs mandated training at CHs. Sixty-six percent of the representative JSA members considered it premature for pediatric anesthesia to become a subspecialty, but 87% considered experience in pediatric anesthesia mandatory for anesthesia board qualification. CONCLUSIONS: This survey revealed that although pediatric anesthesia training is considered mandatory, university hospitals lack adequate numbers of pediatric cases and children's hospitals suffer from a shortage of staff positions and anesthesiologists, and hence are unable to satisfy this demand. Most representative members of our society consider it too early to subspecialize pediatric anesthesia in Japan. PMID- 16677265 TI - Ketoprofen and tramadol for analgesia during early recovery after tonsillectomy in children. AB - BACKGROUND: Pain following tonsillectomy is often intense. Nonsteroidal anti inflammatory drugs and opioids are effective, but both can cause adverse effects. Tramadol may be a viable alternative for post-tonsillectomy pain. This study was designed to compare the analgesic effects of ketoprofen and tramadol during the early recovery period after tonsillectomy. METHODS: Forty-five ASA class I children (9-15 years) were randomized to receive either saline, ketoprofen (2 mg.kg(-1)) or tramadol (1 mg.kg(-1)) after induction of anesthesia. Upon completion of surgery, the study treatment was continued as a 6 h intravenous (i.v.) infusion of another dose of saline, ketoprofen (2 mg.kg(-1)) or tramadol (1 mg.kg(-1)). Postoperatively, each patient received rescue analgesia with patient-controlled analgesia (PCA) device programmed to deliver 0.5 microg.kg(-1) bolus doses of fentanyl. Postoperative pain was assessed using Visual Analog Scale (VAS) during swallowing. Intraoperative blood loss was measured. RESULTS: The total number of requests of PCA-fentanyl was significantly less in ketoprofen group compared with tramadol and placebo groups (P = 0.035 and P = 0.049, respectively, in pairwise comparisons) and the VAS scores for pain were significantly lower in ketoprofen group compared with tramadol (P = 0.044) or placebo groups (P = 0.018) during the first six postoperative hours. Measured intraoperative blood loss was greater in ketoprofen-treated patients than in those receiving placebo (P = 0.029). CONCLUSION: A dose of 4 mg.kg(-1) of i.v. ketoprofen provided good pain relief with moderate supplemental PCA-fentanyl requirements during the first six postoperative hours after tonsillectomy in children whereas the effects of 2 mg.kg(-1) of i.v. tramadol did not differ from those of placebo. PMID- 16677266 TI - Clonidine for the prevention of emergence agitation in young children: efficacy and recovery profile. AB - BACKGROUND: Emergence agitation (EA) is a common postoperative problem in young children who have received sevoflurane and isoflurane for general anesthesia. This randomized, double-blinded study evaluated the efficacy of intraoperative clonidine in reducing EA, and describes its recovery profile. METHODS: With Institutional Review Board approval and informed consent, children undergoing brief, minimally painful procedures were studied. All children received preemptive analgesia with acetaminophen and ketorolac, sevoflurane for induction, and isoflurane for maintenance of anesthesia. Children received either 2 microg.kg(-1) clonidine or placebo intravenously (i.v.) following induction of anesthesia. Children were observed postoperatively for behavior and side effects, and their parents were telephoned the next day to determine postdischarge recovery characteristics. RESULTS: One hundred and twenty children were included in this study: 59 of whom received clonidine, and 61 placebo; 41% of those in the placebo group exhibited moderate-severe EA compared with only 22% of those in the clonidine group (P < 0.03). Compared with those who received placebo, children who received clonidine awakened more slowly (22 min vs 14 min), had a longer postanesthesia care unit stay (57 min vs 46 min), and experienced sleepiness more frequently after discharge (75% vs 39%; all comparisons significant at P < 0.03). There were no adverse cardiorespiratory events in either group. CONCLUSIONS: Findings demonstrate that i.v. clonidine administered after induction of anesthesia significantly reduces the incidence of EA in young children, but is associated with sleepiness postoperatively. PMID- 16677267 TI - The effects of exposure to environmental tobacco smoke on pulmonary function in children undergoing anesthesia for minor surgery. AB - BACKGROUND: The objectives of this study were to assess whether children exposed to environmental tobacco smoke (ETS) present for surgery with poorer pulmonary function, and experience a more pronounced deterioration in pulmonary function following anesthesia and surgery, than non-ETS-exposed children. METHODS: Fifty four children aged 5-15 years with a history of ETS exposure from one or both parents and 54 children with no such ETS history were included in the study. All participants were presenting for ambulatory surgery and were judged to conform to American Society of Anesthesiology class I or II. Spirometry was performed preoperatively, postoperatively in the recovery ward when the child met criteria for discharge (Aldrete score 8), and before discharge from the day ward. RESULTS: The ETS-exposed group had a significantly lower mean preoperative peak expiratory flow rate (PEFR) (9.5 points lower percent predicted, 95% confidence interval 18.1 to -1.0, P = 0.03). Although not statistically significant, they also had lower percent predicted baseline mean values of the other spirometric variables that were measured (forced expiratory volume in 1 s -4.5%, P = 0.07; forced vital capacity -4.1%, P = 0.10; forced expiratory flow between 25% and 75%-3.6%, P = 0.44). Pulmonary function tests (PFTs) performed in recovery were between 8% and 14% worse than preoperative values, but the results were similar in the two groups of children. PFTs performed before hospital discharge demonstrated an near complete recovery to baseline values. Again the pattern was similar in exposed and nonexposed children. CONCLUSIONS: Environmental tobacco smoke exposure is associated with lower preoperative PEFR values, but does not impact on recovery from anesthesia for healthy children undergoing ambulatory anesthesia. PMID- 16677268 TI - Transient loss of motor-evoked responses associated with caudal injection of morphine in a patient with spondylolisthesis undergoing spinal fusion. AB - A 7-year-old girl having posterior spinal fusion for Grade 3 anterior spondylolisthesis at the L5/S1 level was administered 2.5 mg of morphine in 10 ml saline via the caudal epidural route before surgery. Motor-evoked responses were markedly diminished in her lower limbs for 1 h following this but returned spontaneously. She suffered no neurological injury. The cause for this is postulated to be transient cauda equina compression from the volume of injectate. This complication of caudal injection has not been reported before. The possible mechanisms for this are discussed. We believe that significant L5/S1 spondylolisthesis should be considered a contraindication to the use of caudal epidural injections. PMID- 16677269 TI - Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections - anesthetic implications and literature review. AB - Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) are rare neurobehavioral disorders in children. Affected children may have a diverse array of perioperative manifestations including compulsive behavior, agitation, and abnormal movements. Perioperative management might be more challenging for anesthesiologists. Series of therapeutic plasmapheresis in these children may complicate their anesthesia management. We describe the first case of a patient with PANDAS and its anesthesia implications. PMID- 16677270 TI - Spinal anesthesia in infants with ventriculoperitoneal shunt: report of five cases and review of literature. AB - We describe five cases of children with ventriculoperitoneal shunt who underwent abdominal and perineal procedures under spinal anesthesia. Four of them had been born prematurely, and all had suffered from severe neonatal complications. All of our patients suffered from severe respiratory impairment, that had required mechanical ventilation, and three of them suffered additionally from apnea of prematurity. Four patients had ventriculoperitoneal shunt inserted because of obstructive hydrocephalus and one because of congenital central nervous system anomalies. Two underwent subsequently shunt revision. The benefits of spinal anesthesia in this high-risk population are described. The risks of spinal anesthesia in the presence of a ventricular shunt device, especially infection and dural leakage, are discussed, and the literature about this topic briefly reviewed. PMID- 16677271 TI - Complete upper airway obstruction after induction of anesthesia in a child with undiagnosed lingual tonsil hypertrophy. AB - We present a case of a 9-year-old patient with previously undiagnosed lingual tonsil hypertrophy (LTH) that caused sudden and complete airway obstruction and inability to ventilate on induction of anesthesia. More frequently described in adults than in children, LTH can complicate mask ventilation, intubation or both, with the potential for catastrophic consequences. PMID- 16677272 TI - Anesthetic management of tracheoesophageal fistula repair in a newborn with hypoplastic left heart syndrome. AB - We present a case of a newborn with hypoplastic left heart syndrome (HLHS) and tracheoesophageal fistula (TEF). The anesthesia management for the repair of the TEF is presented and the management of the unique pathophysiology of the HLHS circulation is discussed. PMID- 16677273 TI - Laparoscopic surgery in the pediatric patient post Fontan procedure. AB - We present two case reports describing laparoscopic surgery in patients who have undergone previous Fontan surgery and discuss the theoretical implications of laparoscopic surgery in these patients. A brief discussion of the late complications of Fontan surgery is included. PMID- 16677274 TI - Choice of optimal anesthesia for transdermal kidney biopsy. PMID- 16677275 TI - Transient effect of epoprostenol and sildenafil combined with iNO for pulmonary hypertension in congenital diaphragmatic hernia. PMID- 16677276 TI - The rotational technique with ProSeal laryngeal mask airway does not improve the ease of insertion in children. PMID- 16677277 TI - Parents should be given better information to manage their child's pain after day surgery. PMID- 16677278 TI - Surgical mask used as a harness. PMID- 16677279 TI - Sevoflurane 12% versus 8% raises concerns. PMID- 16677282 TI - Norepinephrine stimulates calprotectin expression in human monocytic cells. AB - BACKGROUND AND OBJECTIVE: Calprotectin is composed of two proteins, S100A8 and S100A9, which are S100 family members, and is detected in gingival crevicular fluid and gingival tissue with inflammation. The release and production of calprotectin are regulated by lipopolysaccharides of periodontopathic bacteria and cytokines. Emotional or psychological stress, a risk factor of periodontal disease, is transmitted by stress modulators including norepinephrine and cortisol. The aim of the present study was to investigate the effect of stress on calprotectin expression using norepinephrine and cortisol. METHODS: U-937 cells, a human monocytic cell line, were incubated with norepinephrine in the presence or absence of beta- or alpha-adrenergic receptor antagonists, or with cortisol. The expression of S100A8/S100A9 mRNAs was examined by northern blotting and the amount of calprotectin was measured by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha (CCAAT enhancing binding protein), a transcription factor, was examined by electrophoretic mobility shift assay. RESULTS: Norepinephrine stimulated the expression of S100A8/S100A9 mRNAs via beta adrenergic receptors in U-937 cells and significantly increased calprotectin production to about 3.6-fold that of the control. However, cortisol had no effect on calprotectin expression at the mRNA and protein levels. Norepinephrine elevated C/EBPalpha DNA binding activity, but cortisol did not increase the activity. CONCLUSION: Norepinephrine, a stress modulator, stimulated calprotectin expression in human monocytic cells. Calprotectin expression may be regulated by stress in addition to inflammatory factors. PMID- 16677283 TI - The changes in T lymphocyte subsets following periodontal treatment in patients with chronic periodontitis. AB - OBJECTIVE: The aim of this study was to determine whether there was any change in T-lymphocyte subsets in patients with chronic periodontitis after applying different periodontal treatment methods. PATIENTS AND METHODS: Twenty-four patients with chronic periodontitis were included in the study. In every phase of the treatment (pretreatment, initial treatment, curettage and flap operations) the biopsy samples were taken from the gingival tissues at sites of chronic periodontitis. Then CD4(+) and CD8(+) lymphocyte and CD4(+)/CD8(+) ratio values were determined using flow cytometry in the biopsy samples. At the same time, gingival pocket depth, Loe-Silness gingival index, and Silness-Loe plaque index scores were recorded to assess the periodontal status in patients. To determine the correlation between the clinical measurements and the laboratory results obtained before the treatment, after initial treatment, after curettage and after flap operations, we conducted an analysis using a paired t-test. RESULTS: Flow cytometry findings in the patients with chronic periodontitis showed that CD4(+) and CD8(+) lymphocyte values before treatment were under the normal value and the CD4(+)/CD8(+) ratio was within the normal distribution interval. The CD4(+)/CD8(+) ratio decreased postcurettage and postflap operation. This decrease was statistically significant (p < 0.001). The CD4(+) and CD8(+) lymphocyte values were increased postcurettage and postflap operation. This increase was also statistically significant (p < 0.001). CONCLUSIONS: These findings suggest that local immune response was poor in the patients with chronic periodontitis. CD4(+) and CD8(+) T-lymphocytes could play a significant role in chronic periodontitis pathobiology. PMID- 16677284 TI - Selective inhibition of Porphyromonas gingivalis growth by a factor Xa inhibitor, DX-9065a. AB - BACKGROUND: Porphyromonas gingivalis is a causative bacterium of adult periodontitis. However, there is no drug specific for P. gingivalis and for its virulence factor. OBJECTIVES: The objective of this study was to examine the effects of a new selective inhibitor of activated factor X, DX-9065a, on growth of Porphyromonas gingivalis and other periodontopathic bacteria. METHODS: We incubated P. gingivalis and other periodontopathic bacteria in the presence or absence of DX-9065a and examined the effect of DX-9065a on bacterial growth and trypsin-like activity in its cultures. We also examined the effects of DX9065a on amidolytic activity of purified trypsin-like proteinases (gingipains RgpA and RgpB), from P. gingivalis and on trypsin-like activity in gingival crevicular fluids from patients with adult periodontitis. RESULTS: DX-9065a selectively inhibited the growth of P. gingivalis and Prevotella intermedia, and its effect on P. gingivalis was bactericidal. Trypsin-like proteinase activity was detected in P. gingivalis, and the activity was strongly inhibited by DX-9065a. DX-9065a even inhibited amidolytic activity of RgpA and RgpB from P. gingivalis. Furthermore, trypsin-like proteinase activity in gingival crevicular fluids was strongly inhibited by DX-9065a. CONCLUSIONS: DX-9065a inhibits P. gingivalis growth in part through to its ability to inhibit the trypsin-like proteinase activity in P. gingivalis and may be useful for a new drug for treatment of adult periodontitis. PMID- 16677285 TI - Levels of immunoglobulin A1 and messenger RNA for interferon gamma and tumor necrosis factor alpha in total saliva from patients with diabetes mellitus type 2 with chronic periodontal disease. AB - BACKGROUND: Diabetes mellitus and periodontal disease have high incidence in the general population and are associated with various degrees of dysfunction in the immune system. It has been shown that diabetic patients with severe periodontal disease have more complications of diabetes and less effective metabolic control compared with diabetic patients with healthy gingiva. Patients with diabetes and severe periodontal disease present higher levels of serous immunoglobulin A (IgA). Elevation of the IgA1 isotype is thought to contribute to this phenomenon. Another important event in the diabetes-periodontitis association is the disturbance in local and systemic production of inflammatory cytokines. OBJECTIVE: In this study we tested the hypothesis that type 2 diabetic patients with chronic moderate periodontal disease have differences in salivary IgA1 titers and cytokine expression when compared with the chronic severe periodontal disease cases. METHODS: We utilized a jacalin-IgA capture assay to determine the IgA1 titers in total saliva and reverse transcriptase-polymerase chain reaction to detect mRNA for interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in total saliva samples of 13 patients with chronic moderate periodontal disease and 10 with chronic severe periodontal disease. RESULTS AND CONCLUSIONS: We observed a predominance of IgA1 titers of 64 (45.5%) in saliva samples from chronic severe periodontal disease patients and titers averaging 512 (30.8%) in chronic moderate periodontal disease patients. We detected mRNA for IFN-gamma in six out of 10 chronic severe periodontal disease subjects and in two out of 13 chronic moderate periodontal disease patients. TNF-alpha expression was similar in both groups. Our data suggest that higher levels of IgA1 may exert partial protection of the periodontal tissue in chronic moderate periodontal disease diabetic patients when compared to severe periodontal disease. Despite the small number of patients, IFN-gamma expression had a trend association with severity of periodontitis and TNF-alpha gene expression did not correlate with severity of periodontal disease. PMID- 16677286 TI - Effect of smoking on crevicular polymorphonuclear neutrophil function in periodontally healthy subjects. AB - BACKGROUND: Polymorphonuclear neutrophils (PMNs) represent the first line of cellular defences in the gingival crevice. Smoking, as probably the most important environmental risk factor for periodontitis, has been shown to adversely affect many neutrophil functions. OBJECTIVE: The aim of this study was to investigate the influence of smoking on PMN numbers and function in periodontally healthy smokers and non-smokers. METHODS: Sixty subjects were recruited: 15 non-smokers, 15 light smokers (< 5 cigarettes/day), 15 moderate smokers (5-15 cigarettes/day) and 15 heavy smokers (> 15 cigarettes/day). Full mouth plaque index, sulcus bleeding index and probing depths were measured. Crevicular washings were obtained from all subjects to harvest PMNs. Numbers of PMNs, percentage viability, and percentage phagocytosis of opsonized Candida albicans were recorded. RESULTS: Mean plaque scores and probing depths were (non significantly) increased in smokers compared to non-smokers. Mean sulcus bleeding index scores were significantly lower in moderate (0.10 +/- 0.10) and heavy (0.07 +/- 0.11) smokers compared to non-smokers (0.14 +/- 0.13) (p < 0.05). Compared to non-smokers (1.73 +/- 1.08 x 10(6)/ml), the numbers of PMNs were higher in light (1.98 +/- 0.96 x 10(6)/ml) and moderate (2.03 +/- 1.43 x 10(6)/ml) smokers and were lower in heavy smokers (1.68 +/- 1.18 x 10(6)/ml), though there were no significant differences in PMN counts between the groups (p > 0.05). Percentage viability of PMNs was significantly lower in light (77.6 +/- 7.8%), moderate (76.5 +/- 8.2%) and heavy (75.0 +/- 6.5%) smokers compared to non-smokers (85.5 +/- 6.0%) (p < 0.05). Furthermore, the ability of PMNs to phagocytose was significantly impaired in light (58.3 +/- 4.1%), moderate (51.9 +/- 2.33%) and heavy (40.9 +/- 3.5%) smokers compared to non-smokers (74.1 +/- 4.1%) (p < 0.05), with evidence of a dose-response effect. CONCLUSION: Cigarette smoking adversely affected PMN viability and function in this periodontally healthy population. PMID- 16677287 TI - Occlusal interference during mastication can cause pathological tooth mobility. AB - BACKGROUND AND OBJECTIVE: Despite little evidence regarding the relationship between tooth mobility and nonworking contact, the evaluation of occlusion is performed mainly by the detection of premature and/or nonworking contacts during tapping movements and lateral excursion. The hypothesis of this study is that occlusal contact during mastication is potentially traumatic to periodontal tissue. It clarifies the relationship between chewing patterns and the status of periodontal tissue. MATERIAL AND METHODS: Subjects included 73 adults, 20-29 years of age (39 men and 34 women), with complete sets of teeth and no history of orthodontic treatment or periodontal disease. The closing chewing patterns of each subject were classified into three groups by the Masticatory Deviation Index, which depicts the deviation from the normal chewing patterns within 5 mm from the intercuspal position. Periotest was used to diagnose teeth mobility and the values were compared among the three groups. RESULTS: The present study indicates that the chewing movements which deviated from the normal chewing movements increased the mobility of specific types of teeth. CONCLUSION: The results of this study imply a relationship between chewing movements and tooth mobility and indicate that functional evaluation of occlusion is necessary for the examination of periodontal tissue. Occlusal evaluation with border and tapping movements might be insufficient, and functional occlusal evaluation during chewing movements can be clinically useful for using to evaluate periodontal tissue. PMID- 16677288 TI - The 17-kDa sheath protein in enamel proteins induces cementum regeneration in experimental cavities created in a buccal dehiscence model of dogs. AB - BACKGROUND AND OBJECTIVE: Commercially available enamel proteins, such as Emdogain, are clinically used for periodontal regeneration. However, the real mechanisms behind the bioactivities of enamel proteins is still unclear, as enamel proteins have multicomponents. The purpose of this in vivo study was to identify the cementum regeneration-promoting factor in enamel proteins that is clinically used for periodontal regeneration to induce cementum-promotive and osteopromotive activities. MATERIAL AND METHODS: Cementum regeneration, which is an important part of periodontal regeneration, was examined in experimental cavities prepared on a buccal dehiscence model of dogs. The purification of enamel protein with cementum regeneration activity was carried out by gel filtration and ion exchange chromatographies of newly formed secretory enamel. RESULTS: Cementum regeneration activity was found in the aggregate comprising 13 17-kDa sheath proteins along with a small amount of amelogenins, found in the newly formed secretory enamel. In these proteins, cementum regeneration activity was detected upon application of the 17-kDa sheath protein, but not by other lower molecular-weight sheath proteins and amelogenins. However, the purified 17 kDa sheath protein induced cementum regeneration activity only in a small area, although the regenerated cementum was thick. The activity of the 17-kDa sheath protein was believed not to have been a result of contamination by growth factors such as transforming growth factor-beta1 (TGF-beta1) found in the enamel protein, as the application of TGF-beta1 induced weak cementum regeneration activity. CONCLUSION: It is concluded that the 17-kDa sheath protein itself exhibits cementum regeneration activity, although other factors may be needed to demonstrate its full ability. PMID- 16677289 TI - Transvascular dissemination of Porphyromonas gingivalis from a sequestered site is dependent upon activation of the kallikrein/kinin pathway. AB - BACKGROUND AND OBJECTIVE: Epidemiological evidence implicates a connection between human periodontitis and systemic diseases. One possible mechanism involves the direct dissemination of periodontopathogens to the target organs through the circulation. The aim of this work was to define the mechanism used by Porphyromonas gingivalis for dissemination from a sequestered infection site. MATERIAL AND METHODS: BALB/c mice were subcutaneously infected with P. gingivalis via use of a mouse chamber model. Tissue fluids from various sites were collected and cultured to determine the presence of P. gingivalis. Evans Blue dye was used to measure the dissemination ability of P. gingivalis. Kinin-associated molecules were introduced into mice, and their effects on bacterial dissemination and mouse pathology were monitored. RESULTS: P. gingivalis strain A7436 caused remote lesions and septicemia with severe cachexia, resulting in animal death. Intrachamber challenge with A7436 resulted in vascular permeability enhancement (VPE), as measured by the systemic infiltration of Evans Blue dye into chamber fluids. VPE was blocked by kininase and kinin receptor antagonist and enhanced by exogenous bradykinin and kininase inhibitor. Live bacteria were recovered from the subcutaneous perichamber and abdominal spaces (spreading), and from the blood (disseminating) of infected mice. Both kininase and kinin receptor antagonist reduced animal mortality as a result of infection with strain A7436 and decreased the number of bacteria recoverable from the blood, but they were not associated with bacterial spreading. CONCLUSIONS: The results suggest that activation of the kinin system is involved in the breach of the vascular barrier that permits dissemination of P. gingivalis. PMID- 16677290 TI - Anti-inflammatory properties of enamel matrix derivative in human blood. AB - BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro-inflammatory cytokine [tumor necrosis factor (TNF)-alpha], an anti-inflammatory cytokine (interleukin-10) and a chemokine (interleukin- 8) in whole human blood challenged by bacterial cell wall components. MATERIAL AND METHODS: Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8-(4-chlorophenyl)thio-cAMP (8-CPT cAMP). TNF-alpha, interleukin-8 and interleukin-10 were analysed from plasma by enzyme-linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA). RESULTS: We found that EMD attenuated the release of TNF-alpha and interleukin-8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin-10 was unchanged. Enamel matrix derivative also produced a four-fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8-CPT-cAMP attenuated the formation of TNF-alpha, but not of interleukin-10, in blood challenged by lipopolysaccharide. CONCLUSION: Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells. PMID- 16677291 TI - Novel rinse assay for the quantification of oral neutrophils and the monitoring of chronic periodontal disease. AB - BACKGROUND AND OBJECTIVES: The aim of this study was to develop a single, rapid, noninvasive oral rinse assay to enable the accurate quantification of oral neutrophils. Products released by neutrophils are partly responsible for the destruction observed in periodontitis. Quantification of oral neutrophil levels is important for understanding their role in periodontal diseases. Previous studies have relied on time-consuming serial rinses and cumbersome counting techniques for the collection and quantification of oral neutrophils. MATERIAL AND METHODS: Patients with chronic periodontal disease provided rinse samples before and after phase I periodontal treatment. Cells in the rinse samples were stained with acridine orange, and neutrophil counts were carried out using a fluorescence microscope and a hemocytometer. RESULTS: This assay allowed us to detect a significant difference in pretreatment oral neutrophil counts between periodontal disease and healthy control groups (p < 0.001). Patients who responded favorably to phase I therapy demonstrated a 43% reduction in oral neutrophil counts compared with their pretreatment levels (p = 0.019). Patients who did not respond to phase I periodontal treatment showed no significant difference in oral neutrophil levels (p = 0.39). CONCLUSION: Oral neutrophil levels, as determined by a rapid oral rinse, reflect the severity of periodontal disease and treatment response. A single, rapid, oral rinse assay is an effective means of collecting and quantifying oral neutrophil levels and may serve as an excellent research tool for further study of the role of neutrophils in periodontal diseases. PMID- 16677292 TI - Influence of rotating-oscillating, sonic and ultrasonic action of power toothbrushes on abrasion of sound and eroded dentine. AB - OBJECTIVE: This in vitro study aimed to evaluate the susceptibility of sound and eroded dentine to brushing abrasion performed by different rotating-oscillating, sonic and ultrasonic toothbrushes. METHODS: Toothbrushing abrasion (20 cycles, each 30 s) was applied to bovine dentine samples (each subgroup n = 10) exhibiting both a demineralized (each cycle: 1% citric acid, pH: 2.3, 60 s; 30 min remineralization in artificial saliva) and a sound surface area. Toothbrushing was performed in an automatic brushing machine with the rotating oscillating, sonic and ultrasonic toothbrushes either (a) activated, supplemented by 20 strokes/min of the brushing machine, (b) inactivated, supplemented by 20 strokes/min of the brushing machine or (c) inactivated, supplemented by 80 strokes/min of the brushing machine. A manual toothbrush was applied with 20, 80 or 100 linear strokes/min. Specimens of the control group were not brushed after erosion. After each cycle, the samples were stored in artificial saliva for 4 h. After 20 cycles, loss of sound and softened dentine was determined by profilometry. Mann-Whitney-Wilcoxon test and Bonferroni corrections were applied to the data (p < 0.05). RESULTS: For all groups, demineralized dentin areas exhibited significantly higher abrasion values than the respective sound dentine surfaces. However, mean dentine loss of both softened and sound dentine was higher after use of the rotating-oscillating, sonic and ultrasonic brushes with the activated regime [(a) eroded dentine: 9.94-16.45 microm; sound dentine: 3.31 5.47 microm] than after brushing with the inactivated regimes [(b) eroded dentine: 5.10-5.62 microm; sound dentine: 1.16-1.81 microm; (c) eroded dentin: 7.64-8.89 microm; sound dentine: 1.38-1.69 microm]. CONCLUSION: The results indicate that rotating-oscillating, sonic or ultrasonic action of the power toothbrushes leads to an increased loss of demineralized and sound dentine. PMID- 16677293 TI - Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis subgingival presence, species-specific serum immunoglobulin G antibody levels, and periodontitis disease recurrence. AB - BACKGROUND AND OBJECTIVE: The biological and clinical effects of antibody against periodontal pathogenic bacteria are incompletely understood. This study evaluated the inter-relationships among periodontal levels of cultivable Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, species-specific serum immunoglobulin G (IgG) antibody levels, and periodontitis disease activity. MATERIAL AND METHODS: Forty-three adults who had previously been treated for periodontitis and who also harbored cultivable A. actinomycetemcomitans or P. gingivalis were evaluated semiannually for clinical disease recurrence over a 36 month period. Each patient provided subgingival microbial samples, for the recovery of A. actinomycetemcomitans and P. gingivalis, from the two deepest pockets in each dentition sextant. A. actinomycetemcomitans and P. gingivalis serum IgG antibody levels were assessed using enzyme-linked immunosorbent assay (ELISA), together with whole-cell sonicate extracts from A. actinomycetemcomitans serotypes a-c and P. gingivalis ATCC 33277. Data were analyzed using the Mantel Haenszel chi-square and Fisher exact two-tailed tests. RESULTS: Eighteen (60.0%) of 30 A. actinomycetemcomitans-positive subjects, and 10 (76.9%) of 13 P. gingivalis-positive subjects, exhibited recurrent periodontal breakdown within 36 months of periodontal therapy. Nineteen (67.9%) of the 28 patients with active periodontitis had A. actinomycetemcomitans or P. gingivalis serum antibody levels below designated threshold values. In comparison, 10 (66.7%) of 15 culture positive clinically stable subjects showed A. actinomycetemcomitans or P. gingivalis serum antibody levels above threshold values. The difference between specific antibody levels in periodontitis-active and periodontitis-stable patients was statistically significant (p = 0.032). CONCLUSIONS: Serum levels of IgG antibodies against A. actinomycetemcomitans or P. gingivalis in periodontitis stable patients were higher than those in patients with active periodontitis. The results suggest that elevated levels of IgG antibody against A. actinomycetemcomitans and P. gingivalis have a detectable protective effect against periodontal infections with these microorganisms. PMID- 16677294 TI - Orchestrating bacterial cell morphogenesis. AB - MreB proteins are bacterial homologues of actin that directly determine cell shape and are involved in a range of other cellular processes in non-spherical bacteria. Like F-actin in eukaryotes, MreBs self-assemble into dynamic filamentous structures that are essential for cell viability. Recent studies have demonstrated that the MreB cytoskeletal scaffold governs shape determination by controlling functions related to the bacterial cell wall (probably by recruiting and directing peptidoglycan-synthesizing and modifying proteins). Here I consider general implications for bacterial morphogenesis, and the basis for differences in wall expansion and cylindrical cell shape, based on recent studies aimed to determine the role of MreBs in bacteria with different modes of growth. PMID- 16677295 TI - Evolution of mutation rates in bacteria. AB - Evolutionary success of bacteria relies on the constant fine-tuning of their mutation rates, which optimizes their adaptability to constantly changing environmental conditions. When adaptation is limited by the mutation supply rate, under some conditions, natural selection favours increased mutation rates by acting on allelic variation of the genetic systems that control fidelity of DNA replication and repair. Mutator alleles are carried to high frequency through hitchhiking with the adaptive mutations they generate. However, when fitness gain no longer counterbalances the fitness loss due to continuous generation of deleterious mutations, natural selection favours reduction of mutation rates. Selection and counter-selection of high mutation rates depends on many factors: the number of mutations required for adaptation, the strength of mutator alleles, bacterial population size, competition with other strains, migration, and spatial and temporal environmental heterogeneity. Such modulations of mutation rates may also play a role in the evolution of antibiotic resistance. PMID- 16677296 TI - Mobile contingency locus controlling Escherichia coli K1 polysialic acid capsule acetylation. AB - Escherichia coli K1 is part of a reservoir of adherent, invasive facultative pathogens responsible for a wide range of human and animal disease including sepsis, meningitis, urinary tract infection and inflammatory bowel syndrome. A prominent virulence factor in these diseases is the polysialic acid capsular polysaccharide (K1 antigen), which is encoded by the kps/neu accretion domain inserted near pheV at 67 map units. Some E. coli K1 strains undergo form (phase) variation involving loss or gain of O-acetyl esters at carbon positions 7 or 9 of the individual sialic acid residues of the polysialic acid chains. Acetylation is catalysed by the receptor-modifying acetyl coenzyme-A-dependent O acetyltransferase encoded by neuO, a phase variable locus mapping near the integrase gene of the K1-specific prophage, CUS-3, which is inserted in argW at 53.1 map units. As the first E. coli contingency locus shown to operate by a translational switch, further investigation of neuO should provide a better understanding of the invasive K1 pathotype. Minimal estimates of morbidity and economic costs associated with human infections caused by extraintestinal pathogenic E. coli strains such as K1 indicate at least 6.5 million cases with attendant medical costs exceeding 2.5 billion US dollars annually in the United States alone. PMID- 16677297 TI - MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores. AB - MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall. PMID- 16677298 TI - The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis. AB - Soj (ParA) and Spo0J (ParB) of Bacillus subtilis belong to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. Unlike most Par systems, for which intact copies of both parA and parB are required for the Par system to function, inactivating soj does not cause a detectable chromosome partitioning phenotype whereas inactivating spo0J leads to a 100-fold increase in the production of anucleate cells. This suggested either that Soj does not function like other ParA homologues, or that a cellular factor might compensate for the absence of soj. We found that inactivating smc, the gene encoding the structural maintenance of chromosomes (SMC) protein, unmasked a role for Soj in chromosome partitioning. A soj null mutation dramatically enhanced production of anucleate cells in an smc null mutant. To look for effects of a soj null on other phenotypes perturbed in a spo0J null mutant, we analysed replication initiation and origin positioning in (soj spo0J)+, Deltasoj, Deltaspo0J and Delta(soj-spo0J) cells. All of the mutations caused increased initiation of replication and, to varying extents, affected origin positioning. Using a new assay to measure separation of the chromosomal origins, we found that inactivating soj, spo0J or both led to a significant defect in separating replicated sister origins, such that the origins remain too close to be spatially resolved. Separation of a region outside the origin was not affected. These results indicate that there are probably factors helping to pair sister origin regions for part of the replication cycle, and that Soj and Spo0J may antagonize this pairing to contribute to timely separation of replicated origins. The effects of Deltasoj, Deltaspo0J and Delta(soj-spo0J) mutations on origin positioning, chromosome partitioning and replication initiation may be a secondary consequence of a defect in separating replicated origins. PMID- 16677299 TI - Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage-specific expression. AB - Transmission of the malaria parasite depends on specialized gamete precursors (gametocytes) that develop in the bloodstream of a vertebrate host. Gametocyte/gamete differentiation requires controlled patterns of gene expression and regulation not only of stage and gender-specific genes but also of genes associated with DNA replication and mitosis. Once taken up by mosquito, male gametocytes undergo three mitotic cycles within few minutes to produce eight motile gametes. Here we analysed, in two Plasmodium species, the expression of SET, a conserved nuclear protein involved in chromatin dynamics. SET is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. We demonstrated functionally the presence of two distinct promoters upstream of the set open reading frame, the one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter. This transcriptional control, described for the first time in Plasmodium, provides a mechanism by which single-copy genes can be differently modulated during parasite development. In male gametocytes an overexpression of SET might contribute to a prompt entry and execution of S/M phases within mosquito vector. PMID- 16677300 TI - Control of the endonuclease activity of type I restriction-modification systems is required to maintain chromosome integrity following homologous recombination. AB - A type I restriction-modification enzyme will bind to an unmethylated target sequence in DNA and, while still bound to the target, translocate DNA through the protein complex in both directions. DNA breakage occurs when two translocating complexes collide. However, if type I restriction-modification systems bind to unmodified target sequences within the resident bacterial chromosome, as opposed to incoming 'foreign' DNA, their activity is curtailed; a process known as restriction alleviation (RA). We have identified two genes in Escherichia coli, rnhA and recG, mutations in which lead to the alleviation of restriction. Induction of RA in response to these mutations is consistent with the production of unmodified target sequences following DNA synthesis associated with both homologous recombination and R-loop formation. This implies that a normal function of RA is to protect the bacterial chromosome when recombination generates unmodified products. For EcoKI, our experiments demonstrate the contribution of two pathways that serve to protect unmodified DNA in the bacterial chromosome: the primary pathway in which ClpXP degrades the restriction endonuclease and a mechanism dependent on the lar gene within Rac, a resident, defective prophage of E. coli K-12. Previously, the potential of the second pathway has only been demonstrated when expression of lar has been elevated. Our data identify the effect of lar from the repressed prophage. PMID- 16677302 TI - Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C beta-lactamase. AB - The emergence and dissemination of extended-spectrum (ES) beta-lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by third generation beta-lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C beta-lactamase with a wide spectrum of substrates. Unlike the well studied class C ES beta-lactamase from Enterobacter cloacae GC1, the Omega-loop does not affect the active site conformation and the catalytic activity of CMY 10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 A resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of beta-lactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 beta-lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES beta-lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C beta-lactamases to extend their substrate spectrum. PMID- 16677301 TI - Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. AB - In Escherichia coli, chemoreceptor clustering at a cell pole seems critical for signal amplification and adaptation. However, little is known about the mechanism of localization itself. Here we examined whether the aspartate chemoreceptor (Tar) is inserted directly into the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar-GFP, fluorescent spots first appeared in lateral membrane regions, and later cell poles became predominantly fluorescent. Unexpectedly, Tar-GFP showed a helical arrangement in lateral regions, which was more apparent when a Tar-GFP derivative with two cysteine residues in the periplasmic domain was cross-linked to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domain of the double cysteine Tar-GFP mutant was replaced by that of the kinase EnvZ, which does not localize to a pole. Observation of GFP-SecE and a translocation defective MalE-GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested that the general protein translocation machinery (Sec) itself is arranged into a helical array, with which Tar is transiently associated. The Sec coil appeared distinct from the MreB coil, an actin-like cytoskeleton. These findings will shed new light on the mechanisms underlying spatial organization of membrane proteins in E. coli. PMID- 16677303 TI - The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA binding activities on separate domains. AB - The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains. PMID- 16677304 TI - Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation. AB - To investigate Saccharomyces cerevisiae physiology during growth on the conditionally toxic triose dihydroxyacetone (DHA), protein expression was studied in strains overexpressing either of the two dihydroxyacetone kinase isogenes, DAK1 or DAK2, that grow well utilizing DHA as a carbon and energy source. DHA metabolism was found mostly similar to ethanol utilization, involving a strong component of glucose derepression, but also involved DHA-specific regulatory changes. A specific and strong (10- to 30-fold induction of formaldehyde dehydrogenase, Fdhlp, indicated activation of the formaldehyde dissimilation pathway in DHA medium. The importance of this pathway was further supported by impaired adaptation to DHA growth and DHA survival in a glutathione-dependent formaldehyde dehydrogenase (SFA1) deletion mutant. Glutathione synthase (GSH1) deletion led to decreased DHA survival in agreement with the glutathione cofactor requirement for the SFA1-encoded activity. DHA toxicity did, however, not solely appear related to formaldehyde accumulation, because SFA1 overexpression only enhanced formaldehyde but not DHA tolerance. In further agreement with a low DHA to-formaldehyde flux, GSH supplements in the low microM range also fully suppressed the DHA sensitivity of a gsh1Delta strain. Under growth reduction on high (100 mM) DHA medium we report increased levels of advanced glycation end product (AGE) formation on total protein. Under these high-DHA conditions expression of several stress-related proteins, e.g. a heat-shock protein (Hsp104p) and the oxidative stress indicator, alkyl hydroperoxide reductase (Ahp1p) was also found induced. However, hallmark determinants of oxidative stress tolerance (e.g. YAP1, SKN7, HYR1/GPX3 and SOD2) were redundant for DHA tolerance, thus indicating mechanisms of DHA toxicity largely independent of central oxidative stress defence mechanisms. We conclude that mechanisms for DHA growth and detoxification appear complex and that the evolutionary strive to minimize detrimental effects of this intracellular metabolite links to both formaldehyde and glutathione metabolism. PMID- 16677305 TI - A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp. AB - Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e. deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1 and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared with the parent toxins. Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity. PMID- 16677306 TI - AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis. AB - The Pseudomonas syringae pv. glycinea effector protein AvrB induces resistance responses in soybean varieties that contain the resistance gene Rpg1-b and Arabidopsis varieties that carry RPM1. In addition to this avirulence activity, AvrB also enhances bacterial virulence on soybean plants that lack Rpg1-b and induces a chlorotic phenotype on Arabidopsis plants that lack RPM1. We screened a library of avrB mutants for loss of avirulence on soybean and Arabidopsis, and assayed selected avirulence mutants for loss of virulence on both plants. All mutants screened were recognized similarly on both plant species. Nine single site avrB mutations that affected avirulence localized to a solvent-accessible pocket in the protein structure. Seven of these mutated residues are absolutely conserved between AvrB and its nine homologues. Avirulence mutants generally lost virulence enhancement on susceptible soybean varieties and lost the ability to induce a chlorotic response on the rpm1 null Arabidopsis variety Mt-0. Three of four avirulence mutants tested failed to interact with RIN4, an Arabidopsis protein previously shown to be required for RPM1 function. Our results suggest that soybean and Arabidopsis recognize AvrB in the same manner, and that AvrB enzymatic activity is required for its function as an avirulence and virulence effector on two different plant species. PMID- 16677307 TI - Role of FNR and FNR-regulated, sugar fermentation genes in Neisseria meningitidis infection. AB - While it is generally accepted that anaerobic metabolism is required during infection, supporting experimental data have only been described in a limited number of studies. To provide additional evidence on the role of anaerobic metabolism in bacterial pathogens while invading mammalian hosts, we analysed the effect of the inactivation of FNR, the major regulatory protein involved in the adaptation to oxygen restrictive conditions, and of two of the FNR-regulated genes on the survival of Neisseria meningitidis serogroup B (MenB) in vivo. We found that fnr deletion resulted in more than 1 log reduction in the meningococcal capacity to proliferate both in infant rats and in mice. To identify which of the FNR-regulated genes were responsible for this attenuated phenotype, we defined the FNR regulon by combining DNA microarray analysis and FNR-DNA binding studies. Under oxygen-restricted conditions, FNR positively controlled the transcription of nine transcriptional units, the most upregulated of which were the two operons NMB0388-galM and mapA-pgmbeta implicated in sugar metabolism and fermentation. When galM and mapA were knocked out, the mutants were attenuated by 2 and 3 logs respectively. As the operons are controlled by FNR, from these data we conclude that MenB survival in the host anatomical sites where oxygen is limiting is supported by sugar fermentation. PMID- 16677308 TI - A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12. AB - The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart. PMID- 16677309 TI - Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export. AB - The flagellar switch proteins of Salmonella, FliG, FliM and FliN, participate in the switching of motor rotation, torque generation and flagellar assembly/export. FliN has been implicated in the flagellar export process. To address this possibility, we constructed 10-amino-acid scanning deletions and larger truncations over the C-terminal domain of FliN. Except for the last deletion variant, all other variants were unable to complement a fliN null strain or to restore the export of flagellar proteins. Most of the deletions showed strong negative dominance effects on wild-type cells. FliN was found to associate with FliH, a flagellar export component that regulates the ATPase activity of FliI. The binding of FliM to FliN does not interfere with this FliN-FliH interaction. Furthermore, a five-protein complex consisting of FliG, His-tagged FliM, FliN, FliH and FliI was purified by nickel-affinity chromatography. FliJ, a putative general chaperone, is bound to FliM even in the absence of FliH. The importance of the C ring as a possible docking site for export substrates, chaperones and FliI through FliH for their efficient delivery to membrane components of the export apparatus is discussed. PMID- 16677310 TI - Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin. AB - Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors. PMID- 16677311 TI - Proteasome- and SCF-dependent degradation of yeast adenine deaminase upon transition from proliferation to quiescence requires a new F-box protein named Saf1p. AB - In response to nutrient limitation, Saccharomyces cerevisiae cells enter into a non-proliferating state termed quiescence. This transition is associated with profound changes in gene expression patterns. The adenine deaminase encoding gene AAH1 is among the most precociously and tightly downregulated gene upon entry into quiescence. We show that AAH1 downregulation is not specifically due to glucose exhaustion but is a more general response to nutrient limitation. We also found that Aah1p level is tightly correlated to RAS activity indicating thus an important role for the protein kinase A pathway in this regulation process. We have isolated three deletion mutants, srb10, srb11 and saf1 (ybr280c) affecting AAH1 expression during post-diauxic growth and in early stationary phase. We show that the Srb10p cyclin-dependent kinase and its cyclin, Srb11p, regulate AAH1 expression at the transcriptional level. By contrast, Saf1p, a previously uncharacterized F-box protein, acts at a post-transcriptional level by promoting degradation of Aah1p. This post-transcriptional regulation is abolished by mutations affecting the proteasome or constant subunits of the SCF (Skp1-Cullin-F box) complex. We propose that Saf1p targets Aah1p for proteasome-dependent degradation upon entry into quiescence. This work provides the first direct evidence for active degradation of proteins in quiescent yeast cells. PMID- 16677312 TI - Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa. AB - Type IV pili (Tfp) are polar surface structures of Pseudomonas aeruginosa required for twitching motility, biofilm formation and adherence. One protein required for the assembly of tfp is FimX, which possesses both GGDEF and EAL domains characteristic of diguanylate cyclases and phosphodiesterases respectively. In this work we demonstrate that FimX has phosphodiesterase activity towards bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), but does not show diguanylate cyclase activity. Instead, the imperfect GGDEF domain of FimX likely serves to activate phosphodiesterase activity when bound to GTP, as has recently been described for the Caulobacter crescentus composite GGDEF-EAL protein, CC3396. Bacteria expressing FimX in which either the GGDEF or EAL domain is deleted or mutated have phenotypes indistinguishable from a DeltafimX strain, demonstrating the importance of both domains to function. Previous work has shown that FimX localizes to the bacterial pole. In this work we show that restriction of FimX to a single pole requires intact GGDEF and EAL domains. Deletion of the amino-terminal REC domain of FimX, which contains a putative polar localization signal, results in a protein that still supports intermediate levels of pilus assembly and function. RFP-FimXDeltaREC, unlike RFP FimX, is no longer localized to the bacterial pole, while transmission electron microscopy shows that surface pili can originate from non-polar sites in this mutant. Although DeltafimX mutants show limited in vitro cytotoxicity, they are as virulent as the wild-type strain in a murine model of acute pneumonia. PMID- 16677313 TI - Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors. AB - The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family. PMID- 16677314 TI - IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. AB - IscR is an iron-sulphur (Fe-S) cluster-containing transcription factor that represses transcription of the operon containing its own gene and the iscSUA hscBA-fdx genes, whose products are involved in Fe-S cluster biogenesis. In this study, global transcriptional profiling of Escherichia coli IscR(+) and IscR(-) strains grown under aerobic and anaerobic conditions indicated that 40 genes in 20 predicted operons were regulated by IscR. DNase I footprinting and/or in vitro transcription reactions identified seven new promoters under direct IscR control. Among these were genes encoding known or proposed functions in Fe-S cluster biogenesis (sufABCDSE, yadR and yhgI) and Fe-S cluster-containing anaerobic respiratory enzymes (hyaABCDEF, hybOABCDEFG and napFDAGHBC). The finding that IscR repressed expression of the hyaA, hybO and napF promoters specifically under aerobic growth conditions suggests a new mechanism to explain their upregulation under anaerobic growth conditions. Phylogenetic footprinting of the DNase I protected regions of seven promoters implies that there are at least two different classes of IscR binding sites conserved among many bacteria. The findings presented here indicate a more general role of IscR in the regulation of Fe-S cluster biogenesis and that IscR contributes to the O(2) regulation of several promoters controlling the expression of anaerobic Fe-S proteins. PMID- 16677315 TI - Translocation of viable Aeromonas salmonicida across the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum). AB - The pathogenic bacterium Aeromonas salmonicida is the causative agent of the destructive disease furunculosis in salmonids. Horizontal transmission in salmonids has been suggested to occur via the skin, gills and/or intestine. Previous reports are contradictory regarding the role of the intestine as a route of infection. The present study therefore investigates the possibility of bacterial translocation across intestinal epithelia using Ussing chamber technology, in vitro. Intestinal segments were exposed for 90 min to fluorescein isothiocyanate-labelled pathogenic A. salmonicida. Sampling from the serosal side of the Ussing chambers showed that bacteria were able to translocate across the intestinal epithelium in both the proximal and distal regions. Plating and subsequent colony counting showed that the bacteria were viable after translocation. During the 90 min exposure to A. salmonicida, the intestinal segments maintained high viability as measured by electrical parameters. The distal region responded to bacterial exposure by increasing the electrical resistance, indicating an increased mucus secretion. This study thus demonstrates translocation of live A. salmonicida through the intestinal epithelium of rainbow trout, suggesting that the intestine is a possible route of infection in salmonids. PMID- 16677316 TI - Experimental transmission of sleeping disease in one-year-old rainbow trout, Oncorhynchus mykiss (Walbaum), induced by sleeping disease virus. AB - Sleeping disease (SD) is a serious disease of rainbow trout, Oncorhynchus mykiss, reared in fresh water caused by sleeping disease virus (SDV). In this study a detailed clinical, histological, virological and serological description of the experimental reproduction of SD in 1-year-old rainbow trout exposed to SDV was carried out. Two hundred disease-free fish were intraperitoneally inoculated with a SDV isolate and 100 fish were inoculated with an uninfected cell culture lysate as a negative control. Infected and control fish were randomly removed at days 4, 7, 14, 21, 42 and 70 post-infection. Blood and tissues were collected for virus isolation, histopathological examination and serum neutralization. SDV was detected in serum, kidney and brain of infected fish from 4 to 21 days post infection (dpi). Characteristic pathological lesions were observed in infected fish as early as 7 dpi. Lesions were first detected in exocrine pancreas and subsequently observed in heart and skeletal muscle. Neutralizing antibodies to SDV were detected in infected fish from 14 to 70 dpi. Infected fish displayed typical signs of SD 1-month pi and the mortality reached 18.7% within 44 days. This study experimentally reproduced all the pathognomonic features of natural outbreaks of SD in 1-year-old rainbow trout. PMID- 16677317 TI - Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification. AB - A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product. PMID- 16677318 TI - Vibrio vulnificus serovar A: an emerging pathogen in European anguilliculture. AB - The spread of the emerging pathogen Vibrio vulnificus biotype 2 serovar A in Danish anguilliculture is reported. Serovar A was originally isolated in a Spanish eel farm in 2000 and occurred in Denmark in the summer of 2004, affecting eels of 5-10 g body weight cultured in fresh water. The Danish eels showed clinical signs different from those reported for Spanish eels, such as severe haemorrhages in the head and gill region with necrosis of the soft tissues. Danish isolates were biochemically and serologically identical to Spanish serovar A strains and also highly virulent for eels by both intraperitoneal injection and immersion challenges. Vaccination with Vulnivaccine, a vaccine against V. vulnificus serovar E, cross-protected eels against serovar A. The LD(50) for experimentally infected vaccinated animals was significantly higher than for non vaccinated animals. PMID- 16677319 TI - Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool. AB - Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot. PMID- 16677320 TI - A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp. AB - White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years. PMID- 16677322 TI - Pathogenic infections of Gyrodactylus brachymystacis (Monogenea) on Oncorhynchus mykiss (Walbaum) at a fish farm in the Qinling Mountain region of China. PMID- 16677321 TI - A novel systemic granulomatous inflammatory disease in farmed Atlantic cod, Gadus morhua L., associated with a bacterium belonging to the genus Francisella. PMID- 16677324 TI - Periodontology as a recognized dental speciality in Europe. AB - The impetus of the Bologna Process under the auspices of European Union governments has raised enormous expectations. It is the major educational change in Europe within the last 50 years and all the focus from university institutions, learned societies and thematic networks has shifted to this process, with the aim of developing consensus schemes in order to arrive at the expected European Convergence in Higher Education (to be completed by 2010). Dentistry as one of the health professions with clear Educational Standards, as defined by the European Dental Directives, is also reviewing its educational processes within this Bachelor-Master-Doctorate scheme and evaluating how the current and future dental specialities should be accommodated within this framework. Among these specialities, Periodontology is currently considered a formal dental speciality in 11 countries belonging to the EU however it lacks this legal status in the rest of the 14 EU countries. The purpose of this position paper is to provide evidence for the need for a recognized specialty in Periodontology at European level focusing on both the educational and professional perspective, with the hope of providing discussions that may contribute to facilitate its legal establishment as a new dental speciality in Europe. PMID- 16677325 TI - Periodontal disease in the oldest-old living in Kungsholmen, Sweden: findings from the KEOHS project. AB - AIMS: The Kungsholmen Elders Oral Health Study evaluated the oral health status of generally healthy, community-dwelling persons aged 80 years and over living in Stockholm, Sweden. This paper reports periodontal disease findings and evaluates the distribution by sociodemographic factors. METHODS: Eligible persons were identified through the Kungsholmen Project, an ongoing, longitudinal, population based study of older adults. A total of 121 study subjects received a periodontal examination. RESULTS: The mean pocket probing depth was 2.6 mm and the mean clinical attachment loss was 3.7 mm. Gingival bleeding was common. Over half of all study participants met the criteria used for "serious periodontitis" (SP). In the best fit adjusted odds ratio (OR) model, males were 3.1 times more likely than females to have "SP" (OR=3.1, 95% CI 1.2, 8.0), a statistically significant observation. A sub-analysis of the differences in proportion of participants with SP revealed that the difference by sex also increased by age. CONCLUSIONS: These findings document the substantial and ongoing impact of periodontal disease in a sample of generally healthy, community dwelling older adults and underscore the importance of continued periodontal disease prevention and treatment in the oldest-old. PMID- 16677326 TI - Total antioxidant capacity and superoxide dismutase activity levels in serum and gingival crevicular fluid in post-menopausal women with chronic periodontitis. AB - OBJECTIVES: Menopause has been linked with oxidative stress and decreased antioxidant (AO) defence. A connection has been established between menopause and certain periodontal conditions. The objective of this study is to compare serum and gingival crevicular fluid (GCF) total antioxidant capacity (TAOC) and superoxide dismutase (SOD) concentrations in post-menopausal patients with chronic periodontitis (PMCP) with those of pre-menopausal chronic periodontitis patients (CP). MATERIAL AND METHODS: Thirty-two PMCP patients, 31 CP patients, 25 post-menopausal periodontally healthy controls (PMPH) and 26 pre-menopausal controls (PH) were studied. After clinical measurements and samplings, serum and GCF TAOC and SOD concentrations were established in turn using an automated TAOC assay and spectrophotometric end point measurement. The results were analysed statistically. RESULTS: Serum and GCF TAOC and SOD concentrations were significantly lower in menopause and periodontitis (p<0.05). The lowest values were in the PMCP group, whereas the highest values were in the PH group. While the effect of menopause was more evident in serum antioxidant analysis, the effect of periodontitis was observed to be more apparent in GCF. CONCLUSIONS: A decrease in systemic and local AO defence was observed owing to both menopause and periodontitis. The lowest AO values in the PMCP group suggest that menopause may be a risk factor for periodontitis. PMID- 16677327 TI - Heterogeneous presence of myofibroblasts in hereditary gingival fibromatosis. AB - BACKGROUND/AIM: Hereditary gingival fibromatosis (HGF) fibroblasts are characterized by an increased production of collagen and transforming growth factor-beta1 (TGF-beta1), resulting in a fibrotic enlargement of the gingiva of affected patients. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. The goal of this article is to describe the presence of myofibroblasts in HGF in order to elucidate the mechanisms underlying HGF gingival overgrowth. MATERIALS AND METHODS: Fibroblast cell lines and gingival samples from patients of two distinct families affected by HGF and from normal gingiva (NG) were included in this study. To characterize the presence of myofibroblasts, the expression of specific myofibroblast marker smooth muscle isoform of alpha-actin (alpha-SMA) was examined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and flow cytometric analysis. Immunohistochemistry against the alpha-SMA antigen was performed in the gingival tissue samples. RESULTS: Our results demonstrated a significant increase in the expression of the myofibroblast marker alpha-SMA in cells from one HGF family (designed as HGF Family 2), which are also characterized by an elevated expression of type I collagen, TGF-beta1 and connective tissue growth factor (CTGF). Additionally, alpha-SMA-positive cells were broadly detected in the gingival tissue samples from HGF Family 2 patients. In contrast, alpha-SMA expression by HGF Family 1 cells was quite similar to NG cells and no myofibroblasts were detected immunohistochemically, despite the higher levels of TGF-beta1 and type I collagen in HGF Family 1 fibroblasts than in NG cells. The expression of CTGF, which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation, by HGF Family 1 cultures was significantly lower compared with HGF Family 2 and similar to NG control cells. CONCLUSIONS: Our results suggest that the presence of myofibroblasts in HGF could be dependent on CTFG expression levels, and different biological mechanisms may account for the gingival overgrowth observed in HGF patients. This could be an underlying reason for the high variable clinical expressivity of disease. PMID- 16677328 TI - Incidence of bacteremia after chewing, tooth brushing and scaling in individuals with periodontal inflammation. AB - SCIENTIFIC RATIONALE FOR STUDY: Bacteremia occurs with various frequency after oral procedures. Periodontal disease may affect the incidence, magnitude, duration and bacterial spectrum of bacteremia. PRINCIPLE FINDINGS: The incidence and magnitude of bacteremia after scaling was significantly higher in periodontitis than in gingivitis patients and healthy control individuals. In periodontitis patients, the magnitude of bacteremia was associated with gingival index, plaque index and number of sites with bleeding on probing, but not with probing pocket depth measurements. PRACTICAL IMPLICATIONS: The prevention and treatment of periodontal diseases appear to be crucial for the prevention of bacteremia associated with oral procedures. PMID- 16677330 TI - C-reactive protein in patients with coexistent periodontal disease and acute coronary syndromes. AB - AIM: To evaluate periodontal disease (PD) influence on changes in high sensitivity C-reactive protein (hsCRP) concentrations in patients with acute coronary syndromes and coexistent PD. MATERIALS AND METHODS: Dental examinations were carried out in a group of 50 consecutive patients, less than 60 years old, hospitalized as a result of acute coronary syndromes. The patients were divided into two groups on the basis of own-constructed combined PD score (group 2: more advanced; and group 1: less advanced PD) as well as clinical attachment loss (CAL) - group 4: CAL >3 mm; group 3: CAL 18 yr) and from a single-parent household. There was no significant difference in immunosuppressive regimen between non-adherent and adherent patients. Non adherence was significantly (p<0.025) associated with lower socioeconomic status (SES), older age at transplant (p<0.005, 95% CI: -5.5 to -.99, Student's t-test) and episodes of late acute rejection (p<.001). Non-adherence was also significantly associated with re-transplantation and death secondary to chronic rejection by the Fisher exact test (p<0.006 and p<0.05, respectively). Non adherence to immunosuppressive therapy is a prevalent problem that is correlated with certain demographic and medical condition-related risk factors and more frequent adverse consequences in the adolescent liver transplant population. The greater incidence of late acute rejection, death and re-transplantation owing to chronic rejection in non-adherent patients suggests that non-adherence is significantly associated with an increased risk of morbidity and mortality. Further investigation to identify patients at greatest risk for non-adherence is necessary to design the most effective intervention to increase patient survival and well being. PMID- 16677354 TI - Selection of donors for living donor liver transplantation in a single center of a developing country: lessons learned from the first 100 cases. AB - The selection of donors for living donor liver transplantation (LDLT) is one of the most important features in this kind of surgery. The aim of this study is to describe our initial experience in the donor evaluation process. From December 2001 to January 2005, 104 donors were evaluated for 70 recipients (65 potential donors were evaluated for 39 adult recipients, and 39 donors for 31 pediatric recipients). Only 30 donors were able to donate: 13 for the adult group, and 17 for the pediatric one. In general, the utilization rate of potential donors was 28.8% (30/104). For the adult patients, 65 potential donors were seen to perform 13 LDLT, which represents a utilization rate of potential donors of 20%. For the pediatric patients, this rate was 43.6%. The exclusion criteria were clinical in 22 cases (21%), anatomical in 13 cases (13%), psychosocial in nine cases (9%), and others in 12 (12%). Death of recipients led to exclusion 18 of donors (17%). Thirty-three percent of adults and 55% of pediatric recipients who had at least one potential donor to start the evaluation process were able to identify a living donor. In conclusion, the first limit for LDLT is the rigorous donor evaluation. PMID- 16677355 TI - Control of hypertension in children after renal transplantation. AB - The aim of this cross-sectional single-center study was to investigate the efficacy of hypertension control in children who underwent transplantation using ambulatory blood pressure (BP) monitoring, and to determine the risk factors associated with poor control of hypertension. Thirty-six children fulfilled the inclusion criteria. The mean age was 13.9+/-4.4 yr; the mean time after renal transplantation was 2.7+/-2.4 yr (0.5-10.1). Hypertension was defined as a mean ambulatory BP > or =95th centile for healthy children and/or requiring antihypertensive drugs. Hypertension was regarded as controlled if the mean ambulatory BP was <95th centile in children already on antihypertensive drugs, or uncontrolled if the mean ambulatory BP was > or =95th centile in treated children. Hypertension was present in 89% of children. Seventeen children (47%) had controlled hypertension, and 14 (39%) had uncontrolled hypertension. One child (3%) had untreated hypertension, and only four children (11%) showed normal BP without antihypertensive drugs. The efficacy of hypertensive control was 55% (17 of 31 children on antihypertensive drugs had a BP<95th centile), i.e. 45% of treated children still had hypertension. Children with uncontrolled hypertension had significantly higher cyclosporine doses (6.1 vs. 4.3 mg/kg/day, p=0.01) and tacrolimus levels (9.2 vs. 6.1 microg/L, p<0.05), and there was a tendency toward use of lower number of antihypertensive drugs (2.0 vs. 1.5 drugs/patient, p=0.06) and lower use of angiotensin-converting enzyme (ACE) inhibitors (7 vs. 35%, p=0.09) and diuretics (29 vs. 59%, p=0.14) than in children with controlled hypertension. In conclusion, nearly 90% of our children after renal transplantation are hypertensive and the control of hypertension is unsatisfactorily low. The control of hypertension could be improved by increasing the number of prescribed antihypertensive drugs, especially ACE inhibitors, and diuretics, or by using higher doses of currently used antihypertensives. PMID- 16677356 TI - Maturity and adherence in adolescent and young adult heart recipients. AB - BACKGROUND: Pediatric transplant (txp) teams note high rates of non-adherence and risky behaviors linked to morbidity and mortality among adolescent and young adult recipients. Clinicians and parents alike report symptoms of social immaturity and failure to appreciate consequences of risky behavior; relationships between the two have not been studied in this population. METHOD: This two-phase mixed method study examined adherence, high-risk behaviors, and maturity in a sample of 27 heart recipients, aged 15-31, who underwent transplantation in childhood or adolescence at Children's Hospital of Pittsburgh. All subjects completed a projective ego development measure and a questionnaire about adherence to the post-txp regimen. Nine recipients, purposely selected for good or poor adherence according to criteria determined by the transplant team and matched on age, participated in phase 2 qualitative interviews and mood assessments. RESULTS: Sixty-three percent of the phase 1 sample missed medications, 67% missed appointments, 11% smoked, 37% had difficulty with diet, 89% exercised infrequently, 33% had tattoos, 26% had more than two body piercings, and 11% used street drugs. Six themes and a core construct of maturity were identified with qualitative methodology. Poor adherers were less mature on every theme and consistently scored at a less mature level on the projective measure of ego development. Chronological age was not related to the level of maturity in qualitative or projective data. Most interview subjects reported high levels of anxiety, and two reported clinically significant levels of depression and anger; mood was not related to adherence. CONCLUSIONS: Non-adherence and high risk behaviors are prevalent among adolescent and young adult heart recipients. Level of maturity appears to be associated with ability to adhere to the treatment regimen and avoid high-risk behaviors. PMID- 16677357 TI - Mycophenolate mofetil introduction stabilizes and subsequent cyclosporine A reduction slightly improves kidney function in pediatric renal transplant patients: a retrospective analysis. AB - Chronic allograft nephropathy (CAN) is the major cause of late graft loss. Among others, chronic calcineurin inhibitor toxicity (CNI) contributes to the development of CAN. Therefore, reduction in CNI dosage may delay the development of CAN, leading to longer graft survival. It was the aim of the present retrospective analysis to investigate the effect of mycophenolate mofetil (MMF) addition with subsequent cyclosporine A (CSA) reduction on renal function in pediatric kidney allograft recipients. Seventeen patients (aged 8.3-17.6 yr) with monotherapy with CSA and progressive loss of renal function at a median of 3.4 yr after kidney transplantation were enrolled. After at least three months of MMF treatment, CSA dosage was stepwise reduced to trough levels of 100, 80, and 60 ng/mL. In all patients, introduction of MMF prevented a further decrease of glomerular filtration rate (GFR). The mean GFR 12 months before study enrollment was 96.1+/-24.5 and 71.0+/-21.0 mL/min/1.73 m2 at start of MMF. After introduction of MMF and unchanged CSA dosage GFR was stabilized to 71.1+/-23.8 mL/min/1.73 m2. After CSA reduction to trough levels of 60 ng/mL, GFR was slightly ameliorated up to 76.3+/-24.1 mL/min/1.73 m2. Within the follow-up period, one borderline rejection occurred in a patient in whom the CSA trough level was 60 ng/mL since seven months. In pediatric kidney allograft recipients with progressive loss of renal function reduction of CSA after introduction of MMF may stabilize and even slightly ameliorate renal function. PMID- 16677358 TI - Prednisone withdrawal in pediatric kidney transplant recipients on tacrolimus based immunosuppression: four-year data. AB - Corticosteroids have been used in renal transplant immunosuppression for over 40 yr. Despite their adverse effects, steroid therapy continues to be part of early as well as maintenance immunosuppression in most pediatric renal transplant centers. The association of steroids with growth retardation, weight gain, and acne may be particularly distressing during the critical years of adolescence and young adulthood, increasing the risk of medication non-adherence. This study reviews the outcomes of pediatric renal transplant patients treated with low-dose tacrolimus, mycophenolate mofetil, or azathioprine, and planned prednisone withdrawal. Thirty-seven pediatric renal transplant recipients were withdrawn from steroids. The mean follow-up after steroid withdrawal was 42+/-19 months. Graft and patient survival were 100%. The mean serum creatinine levels and calculated creatinine clearances remained stable throughout the period of observation. The mean creatinine clearance was 96+/-24 mL/min/1.73 m2 at steroid withdrawal and 93+/-20 mL/min/1.73 m2 at the latest follow-up. Five patients restarted prednisone; in four (11%) it was for suspected or confirmed acute rejection. Improvements were observed in serum lipid profiles, blood pressure, and body mass index. Most patients experienced catchup or stable growth after prednisone withdrawal. Four patients developed viral infections; all were successfully treated. The potential benefits of steroid withdrawal in pediatric renal transplantation are supported by our results. PMID- 16677359 TI - Pediatric liver transplantation using left hepatic segments from living related donors: surgical experience in 100 recipients at Saint-Luc University Clinics. AB - Living-related liver transplantation was developed in the context of deceased donor organ shortage, which is particularly acute for pediatric recipients. This retrospective study analyzes the surgical technique and complications in the first 100 pediatric liver transplantation using left segmental liver grafts from living donors, performed at Saint-Luc University Clinics between July 1993 and April 2002. Pre-operative evaluation in donors and recipients, analysis of the surgical technique, and postoperative complications were reviewed. After a median follow-up period of 2526 days, no donor mortality was encountered, with a minimal morbidity and no long-term sequelae. At one and five yr post-transplantation, the actuarial patient survival rates were 94% and 92%, the corresponding figures being 92% and 89% for graft survival. The incidences of portal vein and hepatic artery thromboses, and of biliary complications were 14%, 1%, and 27%, respectively. Living-related liver transplantation in children constitutes an efficient therapy for liver failure to face the increased demand for liver grafts. Donor morbidity was kept to acceptable incidence, and surgical technique in the recipient needs to be tailored to minimize postoperative complications. PMID- 16677360 TI - Impact of laparoscopic donor nephrectomy on allograft function in pediatric renal transplant recipients: a single-center report. AB - Laparoscopic donor nephrectomy (LDN) is rapidly becoming the preferred technique for the procurement of living donor kidneys. An association of this technique with delayed graft function and higher risk for rejection has been reported in pediatric recipients. We reviewed our experience of 17 pediatric patients who received a living donor kidney, from 2002 to 2004, procured by LDN, and compared it with a matched group that received living donor kidneys harvested by the open technique. Patient demographics, etiology of renal failure, intra-operative events, length of stay, serum creatinine decline, and graft function were reviewed. Our experience confirmed the findings of earlier reports specifically in small pediatric recipients. The LDN group showed a significantly slower decline in creatinine in the immediate post-operative period and longer intra operative time. However, there was no difference between the two groups in the length of hospital stay, and creatinine clearances at discharge, six, 12 and 24 months post-operatively. The incidence of acute rejection was similar in both groups. LDN is a safe procurement modality for pediatric patients. The risk for prolonged OR time and delay graft function has to be considered during the evaluation process. PMID- 16677361 TI - Sirolimus rescue for tacrolimus-associated post-transplant autoimmune hemolytic anemia. AB - Autoimmune hemolytic anemia (AIHA) has been reported to occur after renal transplantation, and typically does so in the first few weeks post-transplant. We report on a 3-yr-old child who developed cold AIHA nearly 1 yr after an ABO identical, living donor renal transplant from his mother. Numerous transfusions, pulse steroids, repeat plasma exchange treatments, and IVIG were unsuccessful. Nearly 3 wk into his illness, tacrolimus was changed to cyclosporine, and then to sirolimus, and resulted in a prompt response. He currently has a normal renal function and a normal hemoglobin level on sirolimus monotherapy. PMID- 16677362 TI - Successful transplantation in a child with rapid progression of autosomal recessive polycystic kidney disease associated with a novel mutation. AB - Autosomal recessive polycystic kidney disease (ARPKD) is the most common pediatric renal cystic disease with liver involvement. The vast majority of patients with ARPKD carry mutations in the recently characterized PKHD1 gene on chromosome 6p12. A Turkish female demonstrated rapid growth of both kidneys after delivery. Accelerated growth of both kidneys and increasing respiratory distress necessitated right-sided nephrectomy at the age of three months. Because of persistent dyspnea and ongoing growth of the remaining kidney, the second kidney also had to be removed one month later. Biopsies taken from the kidney and the liver confirmed the diagnosis of ARPKD histologically. Renal ultrasound of the patient's consanguineous parents and her older brother showed normal results. PKHD1 mutation analysis yielded a novel homozygous missense mutation (c.1116C >G, F372L) in exon 14, coding for an Ig-like domain (TIG), possibly involved in the increased growth of the kidneys. Peritoneal dialysis was performed for 12 months. The patient had successful transplantation at the age of 15 months and is doing well with actual immunosuppression with cyclosporine, mycophenolate mofetil, and prednisolone. In conclusion, the present case clearly demonstrates the favorable outcome of a child with severe ARPKD after bilateral nephrectomy, pre-emptive dialysis, and successful transplantation. PMID- 16677363 TI - Preventing sensitization with mycophenolate mofetil in a pediatric kidney recipient. AB - Sensitization, as measured by panel reactive antibodies (PRAs), occurs as a result of previous organ transplantation, blood transfusions, or pregnancy. The high-PRA levels increase the risk of complications during transplantation and may result in long time duration for future transplants. Mycophenolate mofetil (MMF) has been shown to decrease lymphocyte proliferation and antibody formation. We report the use of prophylactic MMF in preventing the formation of PRAs in a pediatric renal transplant recipient with multiple donor exposures. A four-yr-old girl received an unsuccessful living-related renal transplant in February 2003 and was subject to multiple blood transfusions in the perioperative period. MMF, 500 mg/m2/day in two divided doses, achieved suppression of PRAs to less than 20% and allowed successful renal transplantation within nine months. No side effects occurred. This approach may reduce the risk of sensitization in future potential organ donor recipients in similar situations. PMID- 16677364 TI - Successful treatment of de novo autoimmune hepatitis and cirrhosis after pediatric liver transplantation. AB - Over a 15-yr period of observation, among the 205 children who underwent liver transplantations, one of them developed a particular type of late graft dysfunction with clinical and histological similarity to autoimmune hepatitis. The patient had alpha1-antitrypsin deficiency and did not previously have autoimmune hepatitis or any other autoimmune disease before transplantation. Infectious and surgical complications were excluded. After repeated episodes of unexplained fluctuations of liver function tests and liver biopsies demonstrating reactive or a biliary pattern, without any corresponding alteration of percutaneous cholangiography, a liver-biopsy sample taken 4 yr after the transplant showed active chronic hepatitis progressing to cirrhosis, portal lymphocyte aggregates, and a large number of plasma cells. At that time, autoantibodies (gastric parietal cell antibody, liver-kidney microsomal antibody, and anti-hepatic cytosol) were positive and serum IgG levels were high. Based on these findings of autoimmune disease, a diagnosis of 'de novo autoimmune hepatitis' was made. The treatment consisted of reducing the dose of cyclosporine, reintroduction of corticosteroids, and addition of mycophenolate mofetil. After 19 months of treatment, a new liver-biopsy sample showed marked reduction of portal and lobular inflammatory infiltrate, with regression of fibrosis and of the architectural disruption. At that time, serum autoantibodies became negative. The last liver-biopsy sample showed inactive cirrhosis and disappearance of interface hepatitis and of plasma cell infiltrate. Presently, 9 yr after the transplantation, the patient is doing well, with normal liver function tests and no evidence of cirrhosis. Her immunosuppressive therapy consists of tacrolimus, mycophenolate mofetil, and prednisolone. In conclusion, the present case demonstrates that de novo autoimmune hepatitis can appear in liver-transplant patients despite appropriate anti-rejection immunosuppression, and triple therapy with tacrolimus, mycophenolate mofetil, and prednisolone could sustain the graft and prevent retransplantation. PMID- 16677365 TI - Rothia dentocariosa sepsis in a pediatric renal transplant recipient having post transplant lymphoproliferative disorders. AB - BACKGROUND: Rothia dentocariosa (RD) is a Gram-positive rod that colonizes the human oral cavity and can cause infective endocarditis. RESULT: We report on a six-yr-old boy who underwent renal transplantation for polycystic kidney disease at the age of eight months. He developed post-transplant lymphoproliferative disorders after four yr and progressive graft failure. Following chemotherapy, the patient presented with neutropenia and sepsis. RD was isolated from blood and treatment with piperacillin/tazobactam was initiated; however, the child died because of multiorgan failure. DISCUSSION: To the best of our knowledge, this is the first case of RD sepsis in a pediatric solid organ transplant recipient. PMID- 16677366 TI - Development of multiple food allergies in children taking tacrolimus after heart and liver transplantation. AB - Angioedema and chronic diarrhea in patients taking immunosuppressants are not always because of side effects and could be a new onset of food allergy. Our aim is to discuss the pathogenesis and treatment of the post-transplant development of food allergies. The first patient was receiving tacrolimus subsequent to heart transplantation and developed angioedema after consumption of dairy products at 12 months after transplantation. He was found to be allergic to multiple foods by both RAST and ImmunoCAP tests. The second patient with argininosuccinic aciduria, post-liver transplant, also received tacrolimus and developed chronic non mucoid/bloody diarrhea at seven months following transplantation. ImmunoCAP test was positive only for egg white and peanuts. Biopsy showed eosinophilic infiltration of the mucosa from the stomach to the rectum. Elimination diets in both patients resolved the symptoms. These cases suggest a direct relationship between tacrolimus and development of food allergy. PMID- 16677367 TI - Antiviral treatment of chronic hepatitis B with lamivudine in pediatric renal transplantation. AB - Renal transplantation in patients with chronic hepatitis B virus (HBV) infection is known to be associated with an increased risk for exacerbation of liver dysfunction. Lamivudine has been proven to be a potent inhibitor of hepatitis B virus replication in adults after kidney transplantation. Little is known about its efficacy and safety in pediatric renal transplant recipients. Three cases serve for the discussion to demonstrate the complexity of the clinical course and effective treatment of chronic hepatitis B in pediatric patients awaiting renal transplantation. Two patients on dialysis with a high HBV replication rate were treated with lamivudine before transplantation. After the viral load had decreased below the detection limit, they underwent transplantation successfully. Despite intensified immunosuppression to treat a rejection episode in one and a relapse of the nephrotic syndrome in the other patient, the viral load remained <2.5 pg/mL. Both patients developed a mutation in the YMDD motif of the HBV genome associated with an increase in the HBV replication rate >10,000 pg/mL without deterioration of the liver function. In a third patient with a chronic HBV infection with a low replication rate, lamivudine was started about nine months after kidney transplantation due to an increasing viral load after treatment of an acute rejection episode. Six months later, the HBV DNA was no longer detectable. The patient had no signs of liver dysfunction. Lamivudine in the treatment of chronic HBV infection in pediatric renal recipients seems to be safe and effective in preventing acute liver deterioration. Three clinical cases are discussed with regard to current options in monitoring and antiviral treatment of chronic HBV in pediatric renal transplant recipients. PMID- 16677368 TI - Post-transplantation lymphoproliferative disorders localizing to the gastrointestinal tract after liver transplantation: report of five pediatric cases. AB - Post-transplantation lymphoproliferative disorder (PTLD) is a life-threatening complication of organ transplantation. PTLD can occur in every kind of organ transplantation. From July 1992 to July 2004, five patients were diagnosed at our transplantation center with PTLD after pediatric liver transplantation. During this period, there were 52 pediatric patients (<18 yr) receiving an orthotopic liver transplantation (OLT) at our center. All five patients had transmural gastrointestinal (GI) PTLD, which occurred mostly in the stomach and duodenum. Epstein-Barr virus (EBV) in situ was demonstrated in each case. EBV viral load was noted to be an important risk factor. Treatment included dose reduction of immunosuppressants and anti-CD20 antibody infusion. Chemotherapy, including cyclophosphamide, doxorubicin, vincristine, and prednisolone, was given to three patients. Four patients have survived more than 10 months until now after treatment. The one who was unresponsive to chemotherapy and anti-CD20 antibody had diffuse metastasis and died of systemic candidiasis. In our series, each PTLD involved the GI tract. The mechanism of this phenomenon is unclear, but these five cases indicate the high incidence of PTLD in pediatric solid organ transplantation. PMID- 16677371 TI - Australian Rheumatology Association and the Rheumatology Health Professional Association 48th Annual Scientific Meeting, Perth, WA, Australia, 20-24 May 2006. Abstracts. PMID- 16677369 TI - Stable mixed chimerism after hematopoietic stem cell transplantation in Wiskott Aldrich syndrome. AB - Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disease characterized by thrombocytopenia, eczema, impaired cellular and humoral immunity, and increased susceptibility to malignancy and autoimmunity. The only curative treatment for WAS is hematopoietic stem cell transplantation, especially in the presence of a matched sibling donor or matched unrelated donor. Here, we report the case of a 2.5-yr-old boy with WAS that resulted in mixed chimerism after having received bone marrow from his phenotypically identical grandfather. Although the patient has persistent thrombocytopenia (platelet counts 50-80 x 10(9)/L), he is currently alive and doing well at 36 months post-transplant and is free of any bleeding episodes. PMID- 16677373 TI - Network motifs: structure does not determine function. AB - BACKGROUND: A number of publications have recently examined the occurrence and properties of the feed-forward motif in a variety of networks, including those that are of interest in genome biology, such as gene networks. The present work looks in some detail at the dynamics of the bi-fan motif, using systems of ordinary differential equations to model the populations of transcription factors, mRNA and protein, with the aim of extending our understanding of what appear to be important building blocks of gene network structure. RESULTS: We develop an ordinary differential equation model of the bi-fan motif and analyse variants of the motif corresponding to its behaviour under various conditions. In particular, we examine the effects of different steady and pulsed inputs to five variants of the bifan motif, based on evidence in the literature of bifan motifs found in Saccharomyces cerevisiae (commonly known as baker's yeast). Using this model, we characterize the dynamical behaviour of the bi-fan motif for a wide range of biologically plausible parameters and configurations. We find that there is no characteristic behaviour for the motif, and with the correct choice of parameters and of internal structure, very different, indeed even opposite behaviours may be obtained. CONCLUSION: Even with this relatively simple model, the bi-fan motif can exhibit a wide range of dynamical responses. This suggests that it is difficult to gain significant insights into biological function simply by considering the connection architecture of a gene network, or its decomposition into simple structural motifs. It is necessary to supplement such structural information by kinetic parameters, or dynamic time series experimental data, both of which are currently difficult to obtain. PMID- 16677372 TI - Analysis of common PTPN1 gene variants in type 2 diabetes, obesity and associated phenotypes in the French population. AB - BACKGROUND: The protein tyrosine phosphatase-1B, a negative regulator for insulin and leptin signalling, potentially modulates glucose and energy homeostasis. PTP1B is encoded by the PTPN1 gene located on chromosome 20q13 showing linkage with type 2 diabetes (T2D) in several populations. PTPN1 gene variants have been inconsistently associated with T2D, and the aim of our study was to investigate the effect of PTPN1 genetic variations on the risk of T2D, obesity and on the variability of metabolic phenotypes in the French population. METHODS: Fourteen single nucleotide polymorphisms (SNPs) spanning the PTPN1 locus were selected from previous association reports and from HapMap linkage disequilibrium data. SNPs were evaluated for association with T2D in two case-control groups with 1227 cases and 1047 controls. Association with moderate and severe obesity was also tested in a case-control study design. Association with metabolic traits was evaluated in 736 normoglycaemic, non-obese subjects from a general population. Five SNPs showing a trend towards association with T2D, obesity or metabolic parameters were investigated for familial association. RESULTS: From 14 SNPs investigated, only SNP rs914458, located 10 kb downstream of the PTPN1 gene significantly associated with T2D (p = 0.02 under a dominant model; OR = 1.43 [1.06-1.94]) in the combined sample set. SNP rs914458 also showed association with moderate obesity (allelic p = 0.04; OR = 1.2 [1.01-1.43]). When testing for association with metabolic traits, two strongly correlated SNPs, rs941798 and rs2426159, present multiple consistent associations. SNP rs2426159 exhibited evidence of association under a dominant model with glucose homeostasis related traits (p = 0.04 for fasting insulin and HOMA-B) and with lipid markers (0.02 = p = 0.04). Moreover, risk allele homozygotes for this SNP had an increased systolic blood pressure (p = 0.03). No preferential transmission of alleles was observed for the SNPs tested in the family sample. CONCLUSION: In our study, PTPN1 variants showed moderate association with T2D and obesity. However, consistent associations with metabolic variables reflecting insulin resistance and dyslipidemia are found for two intronic SNPs as previously reported. Thus, our data indicate that PTPN1 variants may modulate the lipid profile, thereby influencing susceptibility to metabolic disease. PMID- 16677374 TI - Chaos game representation for comparison of whole genomes. AB - BACKGROUND: Chaos game representation of genome sequences has been used for visual representation of genome sequence patterns as well as alignment-free comparisons of sequences based on oligonucleotide frequencies. However the potential of this representation for making alignment-based comparisons of whole genome sequences has not been exploited. RESULTS: We present here a fast algorithm for identifying all local alignments between two long DNA sequences using the sequence information contained in CGR points. The local alignments can be depicted graphically in a dot-matrix plot or in text form, and the significant similarities and differences between the two sequences can be identified. We demonstrate the method through comparison of whole genomes of several microbial species. Given two closely related genomes we generate information on mismatches, insertions, deletions and shuffles that differentiate the two genomes. CONCLUSION: Addition of the possibility of large scale sequence alignment to the repertoire of alignment-free sequence analysis applications of chaos game representation, positions CGR as a powerful sequence analysis tool. PMID- 16677375 TI - Vaccination coverage and reasons for non-vaccination in a district of Istanbul. AB - BACKGROUND: In order to control and eliminate the vaccine preventable diseases it is important to know the vaccination coverage and reasons for non-vaccination. The primary objective of this study was to determine the complete vaccination rate; the reasons for non-vaccination and the predictors that influence vaccination of children. The other objective was to determine coverage of measles vaccination of the Measles Immunization Days (MID) 2005 for children aged 9 month to 6 years in a region of Umraniye, Istanbul, Turkey. METHODS: A '30 x 7' cluster sampling design was used as the sampling method. Thirty streets were selected at random from study area. Survey data were collected by a questionnaire which was applied face to face to parents of 221 children. A Chi-square test and logistic regression was used for the statistical analyses. Content analysis method was used to evaluate the open-ended questions. RESULTS: The complete vaccination rate for study population was 84.5% and 3.2% of all children were totally non vaccinated. The siblings of non-vaccinated children were also non-vaccinated. Reasons for non-vaccination were as follows: being in the village and couldn't reach to health care services; having no knowledge about vaccination; the father of child didn't allow vaccination; intercurrent illness of child during vaccination time; missed opportunities like not to shave off a vial for only one child. In logistic regression analysis, paternal and maternal levels of education and immigration time of both parents to Istanbul were found to influence whether children were completely vaccinated or non-vaccinated. Measles vaccination coverage during MID was 79.3%. CONCLUSION: Efforts to increase vaccination coverage should take reasons for non-vaccination into account. PMID- 16677376 TI - Evaluation of antibodies against human HSP60 in patients with MPO-ANCA associated glomerulonephritis: a cohort study. AB - BACKGROUND: Human Heat Shock Protein 60 (hHSP60) has been implicated in autoimmunity through molecular mimicry, based on the high degree of homology with HSP65 of micro-organisms leading to autoimmune recognition of the human protein. Additionally, sequence homology between hHSP60 and myeloperoxidase (MPO) has been described. MPO is a major autoantigen in vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA). We hypothesized that infections may trigger the ANCA response against MPO through hHSP60. METHODS: In 86 consecutive patients with ANCA-associated vasculitis (AAV), anti-hHSP60 and anti mycobacterial HSP65 were measured by ELISA. Patients were compared with 69 healthy controls (HC). Continuous data between groups were compared using Wilcoxon signed rank test and Kruskal-Wallis test with Dunn's post-test when appropriate. Correlations between data were derived using Spearman correlation. Odds ratios and 95% confidence intervals were obtained using Fisher's exact test. RESULTS: At diagnosis, median anti-mHSP65 level was higher in AAV (median [range]: 42.5 [0-500]), and subsequently in MPO-ANCA (44 [7-500]), compared to HC (22 [0-430]). Anti-hHSP60 levels in AAV were not higher compared to HC (18 [0 319] and 18.5 [0-98], respectively). However, in MPO-ANCA anti-hHSP60 levels were increased (32.5 [0-319]) compared to PR3-ANCA (13 [0-79]) and HC. We could not detect cross-reactivity between hHSP60 and MPO-ANCA. There was a correlation between anti-mHSP65 and anti-hHSP60 levels (r = 0.32, P = 0.003) but not between anti-hHSP60 and MPO-ANCA (r = -0.064, P = 0.69). CONCLUSION: Antibodies against mHSP65 are higher in AAV compared to HC, and anti-hHSP60 antibodies are higher in patients with MPO-ANCA than in patients with PR3-ANCA and HC. Although this finding may be indicative for cross-reactivity between MPO-ANCA and hHSP60, additional assays did not support this hypothesis. PMID- 16677377 TI - Methicillin-resistant Staphylococcus aureus prevalence: current susceptibility patterns in Trinidad. AB - BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most widespread causes of nosocomial infections worldwide. Recently, reports have emerged that S. aureus strains recovered from community-acquired infections are also methicillin-resistant. This study was undertaken to analyze the prevalence of methicillin resistance among isolates at a regional hospital in Trinidad, and document the current resistance profile of MRSA and methicillin sensitive Staphylococcus aureus (MSSA) to the commonly used anti-staphylococcal agents. METHODS: Over a 6-year period we analyzed 2430 isolates of S. aureus strains recovered from various clinical sources, from hospital and community practices. Antimicrobial susceptibility testing was done according to guideline recommendations of the National Committee for Clinical Laboratory Standards. RESULTS: The prevalence of MRSA from surgical/burn wounds, urine and pus/abscess were 60.1%, 15.5% and 6.6%, respectively. The major sources of MSSA were surgical/burn wounds, pus/abscess and upper respiratory tract specimens with rates of 32.9%, 17.1% and 14.3%, respectively. The greatest prevalence of resistance of MRSA was seen for erythromycin (86.7%), and clindamycin (75.3%). Resistance rates among MSSA were highest for ampicillin (70%). Resistance rates for tetracycline were similar among both MRSA (78.7%) and MSSA (73.5%). The MRSA recovery rates from nosocomial sources (20.8%) was significantly higher than that of previous years (12.5%) (p < 0.001), whereas rates among community isolates were relatively similar for the same period (4.1% versus 8.1%). CONCLUSION: The prevalence of MRSA in the hospital increased from 12.5% in 1999 to 20.8% in 2004. Most isolates were associated with infected surgical/burn wounds which may have become infected via the hands of HCPs during dressing exercises. Infection control measures aimed at the proper hand hygiene procedures may interrupt the spread of MRSA. HCPs may also be carriers of MRSA in their anterior nares. Surveillance cultures of both patients and HCPs may help to identify carriers who would be offered antibiotics to eradicate the organisms. Most MRSA are resistant to several non-beta-lactam antibiotics. Frequent monitoring of susceptibility patterns of MRSA and the formulation of a definite antibiotic policy maybe helpful in decreasing the incidence of MRSA infection. PMID- 16677378 TI - Search for QTL affecting the shape of the egg laying curve of the Japanese quail. AB - BACKGROUND: Egg production is of critical importance in birds not only for their reproduction but also for human consumption as the egg is a highly nutritive and balanced food. Consequently, laying in poultry has been improved through selection to increase the total number of eggs laid per hen. This number is the cumulative result of the oviposition, a cyclic and repeated process which leads to a pattern over time (the egg laying curve) which can be modelled and described individually. Unlike the total egg number which compounds all variations, the shape of the curve gives information on the different phases of egg laying, and its genetic analysis using molecular markers might contribute to understand better the underlying mechanisms. The purpose of this study was to perform the first QTL search for traits involved in shaping the egg laying curve, in an F2 experiment with 359 female Japanese quail. RESULTS: Eight QTL were found on five autosomes, and six of them could be directly associated with egg production traits, although none was significant at the genome-wide level. One of them (on CJA13) had an effect on the first part of the laying curve, before the production peak. Another one (on CJA06) was related to the central part of the curve when laying is maintained at a high level, and the four others (on CJA05, CJA10 and CJA14) acted on the last part of the curve where persistency is determinant. The QTL for the central part of the curve was mapped at the same position on CJA06 than a genome-wide significant QTL for total egg number detected previously in the same F2. CONCLUSION: Despite its limited scope (number of microsatellites, size of the phenotypic data set), this work has shown that it was possible to use the individual egg laying data collected daily to find new QTL which affect the shape of the egg laying curve. Beyond the present results, this new approach could also be applied to longitudinal traits in other species, like growth and lactation in ruminants, for which good marker coverage of the genome and theoretical models with a biological significance are available. PMID- 16677379 TI - Interpreting household survey data intended to measure insecticide-treated bednet coverage: results from two surveys in Eritrea. AB - BACKGROUND: As efforts are currently underway to roll-out insecticide-treated bednets (ITNs) to populations within malarious areas in Africa, there is an unprecedented need for data to measure the effectiveness of such programmes in terms of population coverage. This paper examines methodological issues to using household surveys to measure core Roll Back Malaria coverage indicators of ITN possession and use. METHODS: ITN coverage estimates within Anseba and Gash Barka Provinces from the 2002 Eritrean Demographic and Health Survey, implemented just prior to a large-scale ITN distribution programme, are compared to estimates from the same area from a sub-national Bednet Survey implemented 18 months later in 2003 after the roll-out of the ITN programme. RESULTS: Measures of bednet possession were dramatically higher in 2003 compared to 2002. In 2003, 82.2% (95% confidence interval (CI) 77.4-87.0) of households in Anseba and Gash Barka possessed at least one ITN. RBM coverage indicators for ITN use were also dramatically higher in 2003 as compared to 2002, with 76.1% (95% CI 69.9-82.2) of children under five years old and 52.4% (95% CI 38.2-66.6) of pregnant women sleeping under ITNs. The ITN distribution programme resulted in a gross increase in ITN use among children and pregnant women of 68.3% and 48% respectively. CONCLUSION: Eritrea has exceeded the Abuja targets of 60% coverage for ITN household possession and use among children under five years old within two malarious provinces. Results point to several important potential sources of bias that must be considered when interpreting data for ITN coverage over time, including: disparate survey universes and target populations that may include non malarious areas; poor date recall of bednet procurement and treatment; and differences in timing of surveys with respect to malaria season. PMID- 16677380 TI - Identification of consensus RNA secondary structures using suffix arrays. AB - BACKGROUND: The identification of a consensus RNA motif often consists in finding a conserved secondary structure with minimum free energy in an ensemble of aligned sequences. However, an alignment is often difficult to obtain without prior structural information. Thus the need for tools to automate this process. RESULTS: We present an algorithm called Seed to identify all the conserved RNA secondary structure motifs in a set of unaligned sequences. The search space is defined as the set of all the secondary structure motifs inducible from a seed sequence. A general-to-specific search allows finding all the motifs that are conserved. Suffix arrays are used to enumerate efficiently all the biological palindromes as well as for the matching of RNA secondary structure expressions. We assessed the ability of this approach to uncover known structures using four datasets. The enumeration of the motifs relies only on the secondary structure definition and conservation only, therefore allowing for the independent evaluation of scoring schemes. Twelve simple objective functions based on free energy were evaluated for their potential to discriminate native folds from the rest. CONCLUSION: Our evaluation shows that 1) support and exclusion constraints are sufficient to make an exhaustive search of the secondary structure space feasible. 2) The search space induced from a seed sequence contains known motifs. 3) Simple objective functions, consisting of a combination of the free energy of matching sequences, can generally identify motifs with high positive predictive value and sensitivity to known motifs. PMID- 16677381 TI - A universal reference sample derived from clone vector for improved detection of differential gene expression. AB - BACKGROUND: Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs. RESULTS: We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. CONCLUSION: Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes. PMID- 16677382 TI - Identification of gene expression patterns using planned linear contrasts. AB - BACKGROUND: In gene networks, the timing of significant changes in the expression level of each gene may be the most critical information in time course expression profiles. With the same timing of the initial change, genes which share similar patterns of expression for any number of sampling intervals from the beginning should be considered co-expressed at certain level(s) in the gene networks. In addition, multiple testing problems are complicated in experiments with multi level treatments when thousands of genes are involved. RESULTS: To address these issues, we first performed an ANOVA F test to identify significantly regulated genes. The Benjamini and Hochberg (BH) procedure of controlling false discovery rate (FDR) at 5% was applied to the P values of the F test. We then categorized the genes with a significant F test into 4 classes based on the timing of their initial responses by sequentially testing a complete set of orthogonal contrasts, the reverse Helmert series. For genes within each class, specific sequences of contrasts were performed to characterize their general 'fluctuation' shapes of expression along the subsequent sampling time points. To be consistent with the BH procedure, each contrast was examined using a stepwise Studentized Maximum Modulus test to control the gene based maximum family-wise error rate (MFWER) at the level of alphanew determined by the BH procedure. We demonstrated our method on the analysis of microarray data from murine olfactory sensory epithelia at five different time points after target ablation. CONCLUSION: In this manuscript, we used planned linear contrasts to analyze time-course microarray experiments. This analysis allowed us to characterize gene expression patterns based on the temporal order in the data, the timing of a gene's initial response, and the general shapes of gene expression patterns along the subsequent sampling time points. Our method is particularly suitable for analysis of microarray experiments in which it is often difficult to take sufficiently frequent measurements and/or the sampling intervals are non-uniform. PMID- 16677383 TI - Prognostic factors in solitary plasmacytoma of the bone: a multicenter Rare Cancer Network study. AB - BACKGROUND: Solitary plasmacytoma (SP) of the bone is a rare plasma-cell neoplasm. There are no conclusive data in the literature on the optimal radiation therapy (RT) dose in SP. Therefore, in this large retrospective study, we wanted to assess the outcome, prognostic factors, and the optimal RT dose in patients with SP. METHODS: Data from 206 patients with bone SP without evidence of multiple myeloma (MM) were collected. Histopathological diagnosis was obtained for all patients. The majority (n = 169) of the patients received RT alone; 32 chemotherapy and RT, and 5 surgery. Median follow-up was 54 months (7-245). RESULTS: Five-year overall survival, disease-free survival (DFS), and local control was 70%, 46%, and 88%; respectively. Median time to MM development was 21 months (2-135) with a 5-year probability of 51%. In multivariate analyses, favorable factors were younger age and tumor size < 5 cm for survival; younger age for DFS; anatomic localization (vertebra vs. other) for local control. Older age was the only predictor for MM. There was no dose-response relationship for doses 30 Gy or higher, even for larger tumors. CONCLUSION: Younger patients, especially those with vertebral localization have the best outcome when treated with moderate-dose RT. Progression to MM remains the main problem. Further investigation should focus on adjuvant chemotherapy and/or novel therapeutic agents. PMID- 16677384 TI - Maximum static inspiratory and expiratory pressures with different lung volumes. AB - BACKGROUND: Maximum pressures developed by the respiratory muscles can indicate the health of the respiratory system, help to determine maximum respiratory flow rates, and contribute to respiratory power development. Past measurements of maximum pressures have been found to be inadequate for inclusion in some exercise models involving respiration. METHODS: Maximum inspiratory and expiratory airway pressures were measured over a range of lung volumes in 29 female and 19 male adults. A commercial bell spirometry system was programmed to occlude airflow at nine target lung volumes ranging from 10% to 90% of vital capacity. RESULTS: In women, maximum expiratory pressure increased with volume from 39 to 61 cmH2O and maximum inspiratory pressure decreased with volume from 66 to 28 cmH2O. In men, maximum expiratory pressure increased with volume from 63 to 97 cmH2O and maximum inspiratory pressure decreased with volume from 97 to 39 cmH2O. Equations describing pressures for both sexes are: Pe/Pmax = 0.1426 Ln( %VC) + 0.3402 R2 = 0.95 Pi/Pmax = 0.234 Ln(100 - %VC) - 0.0828 R2 = 0.96 CONCLUSION: These results were found to be consistent with values and trends obtained by other authors. Regression equations may be suitable for respiratory mechanics models. PMID- 16677385 TI - Optimizing amino acid substitution matrices with a local alignment kernel. AB - BACKGROUND: Detecting remote homologies by direct comparison of protein sequences remains a challenging task. We had previously developed a similarity score between sequences, called a local alignment kernel, that exhibits good performance for this task in combination with a support vector machine. The local alignment kernel depends on an amino acid substitution matrix. Since commonly used BLOSUM or PAM matrices for scoring amino acid matches have been optimized to be used in combination with the Smith-Waterman algorithm, the matrices optimal for the local alignment kernel can be different. RESULTS: Contrary to the local alignment score computed by the Smith-Waterman algorithm, the local alignment kernel is differentiable with respect to the amino acid substitution and its derivative can be computed efficiently by dynamic programming. We optimized the substitution matrix by classical gradient descent by setting an objective function that measures how well the local alignment kernel discriminates homologs from non-homologs in the COG database. The local alignment kernel exhibits better performance when it uses the matrices and gap parameters optimized by this procedure than when it uses the matrices optimized for the Smith-Waterman algorithm. Furthermore, the matrices and gap parameters optimized for the local alignment kernel can also be used successfully by the Smith-Waterman algorithm. CONCLUSION: This optimization procedure leads to useful substitution matrices, both for the local alignment kernel and the Smith-Waterman algorithm. The best performance for homology detection is obtained by the local alignment kernel. PMID- 16677386 TI - A new extract of the plant Calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation. AB - BACKGROUND: Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS: An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS: The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION: These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells. PMID- 16677387 TI - The generation and gender shifts in medicine: an exploratory survey of internal medicine physicians. AB - BACKGROUND: Two striking demographic shifts evident in today's workforce are also apparent in the medical profession. One is the entry of a new generation of physicians, Gen Xers, and the other is the influx of women. Both shifts are argued to have significant implications for recruitment and retention because of assumptions regarding the younger generation's and women's attitudes towards work and patient care. This paper explores two questions regarding the generations: (1) How do Baby Boomer and Generation X physicians perceive the generation shift in work attitudes and behaviours? and (2) Do Baby Boomer and Generation X physicians differ significantly in their work hours and work attitudes regarding patient care and life balance? Gen Xers include those born between 1965 and 1980; Baby Boomers are those born between 1945 and 1964. We also ask: Do female and male Generation X physicians differ significantly in their work hours and work attitudes regarding patient care and life balance? METHODS: We conducted exploratory interviews with 54 physicians and residents from the Department of Medicine (response rate 91%) and asked about their perceptions regarding the generation and gender shifts in medicine. We limit the analyses to interview responses of 34 Baby Boomers and 18 Generation Xers. We also sent questionnaires to Department members (response rate 66%), and this analysis is limited to 87 Baby Boomers' and 65 Generation Xers' responses. RESULTS: The qualitative interview data suggest significant generation and gender shifts in physicians' attitudes. Baby Boomers generally view Gen Xer physicians as less committed to their medical careers. The quantitative questionnaire data suggest that there are few significant differences in the generations' and genders' reports of work-life balance, work hours and attitudes towards patient care. CONCLUSION: A combined qualitative and quantitative approach to the generation shift and gender shift in medicine is helpful in revealing that the widely held assumptions are not necessarily reflective of any significant differences in actual work attitudes or behaviours of Boomer and Gen X physicians or of the younger generation of women entering medicine. PMID- 16677388 TI - Transmission patterns of smallpox: systematic review of natural outbreaks in Europe and North America since World War II. AB - BACKGROUND: Because smallpox (variola major) may be used as a biological weapon, we reviewed outbreaks in post-World War II Europe and North America in order to understand smallpox transmission patterns. METHODS: A systematic review was used to identify papers from the National Library of Medicine, Embase, Biosis, Cochrane Library, Defense Technical Information Center, WorldCat, and reference lists of included publications. Two authors reviewed selected papers for smallpox outbreaks. RESULTS: 51 relevant outbreaks were identified from 1,389 publications. The median for the effective first generation reproduction rate (initial R) was 2 (range 0-38). The majority outbreaks were small (less than 5 cases) and contained within one generation. Outbreaks with few hospitalized patients had low initial R values (median of 1) and were prolonged if not initially recognized (median of 3 generations); outbreaks with mostly hospitalized patients had higher initial R values (median 12) and were shorter (median of 3 generations). Index cases with an atypical presentation of smallpox were less likely to have been diagnosed with smallpox; outbreaks in which the index case was not correctly diagnosed were larger (median of 27.5 cases) and longer (median of 3 generations) compared to outbreaks in which the index case was correctly diagnosed (median of 3 cases and 1 generation). CONCLUSION: Patterns of spread during Smallpox outbreaks varied with circumstances, but early detection and implementation of control measures is a most important influence on the magnitude of outbreaks. The majority of outbreaks studied in Europe and North America were controlled within a few generations if detected early. PMID- 16677389 TI - An informatics model for tissue banks--lessons learned from the Cooperative Prostate Cancer Tissue Resource. AB - BACKGROUND: Advances in molecular biology and growing requirements from biomarker validation studies have generated a need for tissue banks to provide quality controlled tissue samples with standardized clinical annotation. The NCI Cooperative Prostate Cancer Tissue Resource (CPCTR) is a distributed tissue bank that comprises four academic centers and provides thousands of clinically annotated prostate cancer specimens to researchers. Here we describe the CPCTR information management system architecture, common data element (CDE) development, query interfaces, data curation, and quality control. METHODS: Data managers review the medical records to collect and continuously update information for the 145 clinical, pathological and inventorial CDEs that the Resource maintains for each case. An Access-based data entry tool provides de identification and a standard communication mechanism between each group and a central CPCTR database. Standardized automated quality control audits have been implemented. Centrally, an Oracle database has web interfaces allowing multiple user-types, including the general public, to mine de-identified information from all of the sites with three levels of specificity and granularity as well as to request tissues through a formal letter of intent. RESULTS: Since July 2003, CPCTR has offered over 6,000 cases (38,000 blocks) of highly characterized prostate cancer biospecimens, including several tissue microarrays (TMA). The Resource developed a website with interfaces for the general public as well as researchers and internal members. These user groups have utilized the web-tools for public query of summary data on the cases that were available, to prepare requests, and to receive tissues. As of December 2005, the Resource received over 130 tissue requests, of which 45 have been reviewed, approved and filled. Additionally, the Resource implemented the TMA Data Exchange Specification in its TMA program and created a computer program for calculating PSA recurrence. CONCLUSION: Building a biorepository infrastructure that meets today's research needs involves time and input of many individuals from diverse disciplines. The CPCTR can provide large volumes of carefully annotated prostate tissue for research initiatives such as Specialized Programs of Research Excellence (SPOREs) and for biomarker validation studies and its experience can help development of collaborative, large scale, virtual tissue banks in other organ systems. PMID- 16677390 TI - Bayesian models for pooling microarray studies with multiple sources of replications. AB - BACKGROUND: Biologists often conduct multiple but different cDNA microarray studies that all target the same biological system or pathway. Within each study, replicate slides within repeated identical experiments are often produced. Pooling information across studies can help more accurately identify true target genes. Here, we introduce a method to integrate multiple independent studies efficiently. RESULTS: We introduce a Bayesian hierarchical model to pool cDNA microarray data across multiple independent studies to identify highly expressed genes. Each study has multiple sources of variation, i.e. replicate slides within repeated identical experiments. Our model produces the gene-specific posterior probability of differential expression, which provides a direct method for ranking genes, and provides Bayesian estimates of false discovery rates (FDR). In simulations combining two and five independent studies, with fixed FDR levels, we observed large increases in the number of discovered genes in pooled versus individual analyses. When the number of output genes is fixed (e.g., top 100), the pooled model found appreciably more truly differentially expressed genes than the individual studies. We were also able to identify more differentially expressed genes from pooling two independent studies in Bacillus subtilis than from each individual data set. Finally, we observed that in our simulation studies our Bayesian FDR estimates tracked the true FDRs very well. CONCLUSION: Our method provides a cohesive framework for combining multiple but not identical microarray studies with several sources of replication, with data produced from the same platform. We assume that each study contains only two conditions: an experimental and a control sample. We demonstrated our model's suitability for a small number of studies that have been either pre-scaled or have no outliers. PMID- 16677391 TI - "I really should've gone to the doctor": older adults and family caregivers describe their experiences with community-acquired pneumonia. AB - BACKGROUND: Responding to acute illness symptoms can often be challenging for older adults. The primary objective of this study was to describe how community dwelling older adults and their family members responded to symptoms of community acquired pneumonia (CAP). METHODS: A qualitative study that used face-to-face semi-structured interviews to collect data from a purposeful sample of seniors aged 60+ and their family members living in a mid-sized Canadian city. Data analysis began with descriptive and interpretive coding, then advanced as the research team repeatedly compared emerging thematic categories to the raw data. Searches for disconfirming evidence and member checking through focus groups provided additional data and helped ensure rigour. RESULTS: Community-acquired pneumonia symptoms varied greatly among older adults, making decisions to seek care difficult for them and their family members. Both groups took varying amounts of time as they attempted to sort out what was wrong and then determine how best to respond. Even after they concluded something was wrong, older adults with confirmed pneumonia continued to wait for days, to over a week, before seeking medical care. Participants provided diverse reasons for this delay, including fear, social obligations (work, family, leisure), and accessibility barriers (time, place, systemic). Several older adults and family members regretted their delays in seeking help. CONCLUSION: Treatment-seeking delay is a variable, multi-phased decision-making process that incorporates symptom assessment plus psychosocial and situational factors. Public health and health care professionals need to educate older adults about the potential causes and consequences of unnecessary waits. Such efforts may reduce the severity of community-acquired pneumonia upon presentation at clinics and hospitals, and that, in turn, could potentially improve health outcomes. PMID- 16677392 TI - Reliability, validity and responsiveness of a Norwegian version of the Chronic Sinusitis Survey. AB - BACKGROUND: The Chronic Sinusitis Survey (CSS) is a valid, disease-specific questionnaire for assessing health status and treatment effectiveness in chronic rhinosinusitis. In the present study, we developed a Norwegian version of the CSS and assessed its psychometric properties. METHODS: In the pooled data set of 65 patients from a trial of treatment for chronic sinusitis with long-standing symptoms and signs of sinusitis on computed tomography (CT), we assessed the reliability, validity and responsiveness of the CSS. RESULTS: Test-retest reliability of the two CSS scales and the total scale ranged 0.87-0.92, while internal consistency reliability ranged 0.31-0.55. CSS subscale scores were associated with other items on sinusitis symptoms, and with the Mental health and Bodily pain scale of the SF-36. There was little association of the CSS scale scores with sinus CT findings. The patients with chronic sinusitis had worse scores on all three CSS scales than a healthy reference population (n = 42) (p < 0.001). The CSS sinus symptoms subscale and the total scale were sensitive to improvement in global symptoms during 12 weeks. CONCLUSION: The Norwegian version of the CSS had acceptable test-retest reliability, but lower internal consistency reliability than the accepted standard criteria. The results support the construct validity of the measure and the sinusitis symptoms subscale and the total scales were responsive to change. This supports the use of the questionnaire in interventions for chronic sinusitis, but points at problems with the internal consistency reliability. PMID- 16677393 TI - Promoter prediction and annotation of microbial genomes based on DNA sequence and structural responses to superhelical stress. AB - BACKGROUND: In our previous studies, we found that the sites in prokaryotic genomes which are most susceptible to duplex destabilization under the negative superhelical stresses that occur in vivo are statistically highly significantly associated with intergenic regions that are known or inferred to contain promoters. In this report we investigate how this structural property, either alone or together with other structural and sequence attributes, may be used to search prokaryotic genomes for promoters. RESULTS: We show that the propensity for stress-induced DNA duplex destabilization (SIDD) is closely associated with specific promoter regions. The extent of destabilization in promoter-containing regions is found to be bimodally distributed. When compared with DNA curvature, deformability, thermostability or sequence motif scores within the -10 region, SIDD is found to be the most informative DNA property regarding promoter locations in the E. coli K12 genome. SIDD properties alone perform better at detecting promoter regions than other programs trained on this genome. Because this approach has a very low false positive rate, it can be used to predict with high confidence the subset of promoters that are strongly destabilized. When SIDD properties are combined with -10 motif scores in a linear classification function, they predict promoter regions with better than 80% accuracy. When these methods were tested with promoter and non-promoter sequences from Bacillus subtilis, they achieved similar or higher accuracies. We also present a strictly SIDD-based predictor for annotating promoter sequences in complete microbial genomes. CONCLUSION: In this report we show that the propensity to undergo stress induced duplex destabilization (SIDD) is a distinctive structural attribute of many prokaryotic promoter sequences. We have developed methods to identify promoter sequences in prokaryotic genomes that use SIDD either as a sole predictor or in combination with other DNA structural and sequence properties. Although these methods cannot predict all the promoter-containing regions in a genome, they do find large sets of potential regions that have high probabilities of being true positives. This approach could be especially valuable for annotating those genomes about which there is limited experimental data. PMID- 16677394 TI - Evaluation of pulse-oximetry oxygen saturation taken through skin protective covering. AB - BACKGROUND: The hard edges of adult finger clip probes of the pulse oximetry oxygen saturation (POOS) monitor can cause skin damage if used for prolonged periods in a neonate. Covering the skin under the probe with Micropore surgical tape or a gauze piece might prevent such injury. The study was done to see if the protective covering would affect the accuracy of the readings. METHODS: POOS was studied in 50 full-term neonates in the first week of life. After obtaining consent from their parents the neonates had POOS readings taken directly (standard technique) and through the protective covering. Bland-Altman plots were used to compare the new method with the standard technique. A test of repeatability for each method was also performed. RESULTS: The Bland-Altman plots suggest that there is no significant loss of accuracy when readings are taken through the protective covering. The mean difference was 0.06 (SD of 1.39) and 0.04 (SD 1.3) with Micropore and gauze respectively compared to the standard method. The mean difference was 0.22 (SD 0.23) on testing repeatability with the standard method. CONCLUSION: Interposing Micropore or gauze does not significantly affect the accuracy of the POOS reading. The difference between the standard method and the new method was less than the difference seen on testing repeatability of the standard method. PMID- 16677395 TI - Clinical and immunological effects of Rituximab in patients with lupus nephritis refractory to conventional therapy: a pilot study. AB - We studied the clinical and immunological effects of Rituximab (anti-CD20) therapy in patients with lupus nephritis. In an open clinical trial, 22 patients with active systemic lupus erythematosis and renal involvement (mainly class III and IV according to the WHO classification) that was refractory to conventional therapy were studied. In all these patients, Rituximab (0.5 to 1.0 g at days 1 and 15) was added to the immunosuppressive therapy and its therapeutic effect was evaluated. In addition, the levels and function of regulatory T lymphocytes and the apoptosis of immune cells were assessed. We found a significant reduction in disease activity (p < 0.05, MEX-SLEDAI index), and proteinuria (p < 0.05) at days 60 and 90 of Rituximab therapy. Although most patients showed improvement in creatinine clearance and erythrocyturia, no significant changes in these parameters were detected. In most patients (20/22), B cell depletion was observed, but no clear-cut effect of Rituximab on complement levels or auto antibody titers was detected (p > 0.05 in all cases). One patient died at day 70 with invasive histoplasmosis. No important adverse effects of Rituximab therapy were registered in other patients. A significant enhancement in the levels of different CD4+ regulatory cells (TREG, Th3, Tr1), but not CD8+ Ts lymphocytes, was observed at day 30. This increase was sustained for TREG cells at day 90, and accompanied by an improvement in their regulatory function. In addition, we observed an unexpected increase in the apoptosis of T cells at day 30. Interestingly, the enhancement in the suppressive function of TREG cells was not observed in the two patients that showed the poorest clinical response to Rituximab. We conclude that the data obtained in this open clinical trial suggest that Rituximab is a promising candidate for randomized controlled trials in patients with lupus nephritis refractory to the conventional immunosuppressive therapy. The effects of Rituximab on regulatory cells and apoptosis of T lymphocytes are interesting and its possible role in the putative effect of this biological agent in systemic lupus erythematosis deserves additional studies. PMID- 16677397 TI - A phase II study of LFP therapy (5-FU (5-fluorourasil) continuous infusion (CVI) and Low-dose consecutive (Cisplatin) CDDP) in advanced biliary tract carcinoma. AB - BACKGROUND: Unresectable biliary tract carcinoma is known to demonstrate a poor prognosis. We conducted a single arm phase II study of LFP therapy (5-FU (5 fluorourasil) continuous infusion (CVI) and Low-dose consecutive (Cisplatin) CDDP) for advanced biliary tract malignancies basically on an outpatient basis. METHODS: Between February 1996 and September 2003, 42 patients were enrolled in this trial. LFP THERAPY: By using a total implanted CV-catheter system, 5-FU (160 mg/m2/day) was continuously infused over 24 hours for 7 consecutive days and CDDP (6 mg/m2/day) was infused for 30 minutes twice a week as one cycle. The administration schedule consisted of 4 cycles as one course. RESIST criteria (Response evaluation criteria for solid tumors) and NCI-CTC (National Cancer Institute-Common Toxicity Criteria) (ver.3.0) were used for evaluation of this therapy. The median survival time (MST) and median time to treatment failure (TTF) were calculated by the Kaplan-Meier method. RESULTS: Patients characteristics were: mean age 66.5(47-79): male 24 (54%): BDca (bile duct carcinoma) 27 GBca (Gallbladder carcinoma) 15: locally advanced 26, postoperative recurrence 16. The most common toxicity was anemia (26.2%). Neither any treatment related death nor grade 4 toxicity occurred. The median number of courses of LFP Therapy which patients could receive was two (1-14). All the patients are evaluable for effects with an over all response rates of 42.9% (95% confidence interval C.I.: 27.7-59.0) (0 CR, 18 PR, 13 NC, 11 PD). There was no significant difference regarding the anti tumor effects against both malignant neoplasms. Figure 2 Shows the BDca a longer MST and TTF than did GBca (234 vs 150, 117 vs 85, respectively), but neither difference was statistically significant.The estimated MST and median TTF were 225 and 107 days, respectively. The BDca had a longer MST and TTF than GBca (234 vs 150, 117 vs 85, respectively), but neither difference was statistically significant. CONCLUSION: LFP therapy appears to be useful modality for the clinical management of advanced biliary tract malignancy. PMID- 16677396 TI - Inferring transcriptional modules from ChIP-chip, motif and microarray data. AB - 'ReMoDiscovery' is an intuitive algorithm to correlate regulatory programs with regulators and corresponding motifs to a set of co-expressed genes. It exploits in a concurrent way three independent data sources: ChIP-chip data, motif information and gene expression profiles. When compared to published module discovery algorithms, ReMoDiscovery is fast and easily tunable. We evaluated our method on yeast data, where it was shown to generate biologically meaningful findings and allowed the prediction of potential novel roles of transcriptional regulators. PMID- 16677398 TI - Traditional use of the Andean flicker (Colaptes rupicola) as a galactagogue in the Peruvian Andes. AB - This paper explores the use of the dried meat and feathers of the Andean Flicker (Colaptes rupicola) to increase the milk supply of nursing women and domestic animals in the Andes. The treatment is of preColumbian origin, but continues to be used in some areas, including the village in the southern Peruvian highlands where I do ethnographic research. I explore the factors giving rise to and sustaining the practice, relate it to other galactagogues used in the Andes and to the use of birds in ethnomedical and ethnoveterinary treatments in general, and situate it within the general tendency in the Andes and elsewhere to replicate human relations in the treatment of valuable livestock. The bird's use as a galactagogue appears to be motivated by both metaphorical associations and its perceived efficacy, and conceptually blends human and animal healthcare domains. PMID- 16677399 TI - Antidiabetic and renoprotective effects of the chloroform extract of Terminalia chebula Retz. seeds in streptozotocin-induced diabetic rats. AB - BACKGROUND: Terminalia chebula (Combretaceae) has been widely used in Ayurveda for the treatment of diabetes. In the present investigation, the chloroform extract of T. chebula seed powder was investigated for its antidiabetic activity in streptozotocin-induced diabetic rats using short term and long term study protocols. The efficacy of the extract was also evaluated for protection of renal functions in diabetic rats. METHODS: The blood glucose lowering activity of the chloroform extract was determined in streptozotocin-induced (75 mg/kg, i.p.; dissolved in 0.1 M acetate buffer; pH 4.5) diabetic rats, after oral administration at the doses of 100, 200 and 300 mg/kg in short term study. Blood samples were collected from the eye retro-orbital plexus of rats before and also at 0.5, 1, 2, 4, 6, 8 and 12 h after drug administration and the samples were analyzed for blood glucose by using glucose-oxidase/peroxidase method using a visible spectrophotometer. In long term study, the extract (300 mg/kg) was administered to streptozotocin-induced diabetic rats, daily for 8 weeks. Blood glucose was measured at weekly intervals for 4 weeks. Urine samples were collected before the induction of diabetes and at the end of 8 weeks of treatments and analyzed for urinary protein, albumin and creatinine levels. The data was compared statistically using one-way ANOVA with post-hoc Dunnet's t test. RESULTS: The chloroform extract of T. chebula seeds produced dose-dependent reduction in blood glucose of diabetic rats and comparable with that of standard drug, glibenclamide in short term study. It also produced significant reduction in blood glucose in long term study. Significant renoprotective activity is observed in T. chebula treated rats. The results indicate a prolonged action in reduction of blood glucose by T. chebula and is probably mediated through enhanced secretion of insulin from the beta-cells of Langerhans or through extra pancreatic mechanism. The probable mechanism of potent renoprotective actions of T. chebula has to be evaluated. CONCLUSION: The present studies clearly indicated a significant antidiabetic and renoprotective effects with the chloroform extract of T. chebula and lend support for its traditional usage. Further investigations on identification of the active principles and their mode of action are needed to unravel the molecular mechanisms involved in the observed effects. PMID- 16677400 TI - A possible link between sinusitis and lower airway hypersensitivity: the role of Staphylococcal enterotoxin B. AB - BACKGROUND AND AIMS: The prevalence of asthma has been keeping arising with unknown etiology. The cumulative evidence indicates that chronic rhinosinusitis (CRS) closely relates to asthma, but the detailed mechanisms remain unclear. The present study aimed to take insight into the role of Staphylococcus enterotoxin B (SEB) in a possible association between CRS and asthma. METHODS: 38 patients with both CRS and asthma underwent functional endoscopic sinus surgery. Serum specific IgE and cytokines, clinical symptoms of CRS and asthma were evaluated before and after the surgery. Peripheral blood mononuclear cells (PBMCs) were separated from the patients and cultured. Th2 response of the cultured PBMCs in the presence or absence of specific antigens and SEB was evaluated. RESULTS: Besides the improvement of CRS symptoms, amelioration of asthma was also observed in the patients with both CRS and asthma after the sinus surgery. The preoperatively elevated Th2 cytokines, IL-4 and IL-5, normalized postoperatively. Th2 response was generated with separated PBMCs in the presence of specific antigens. SEB was required for maintaining Th2 response in these separated PBMCs. CONCLUSION: The present results indicate that a possible link exists between CRS and lower airway hypersensitivity. Sinusitis derived SEB may play a role in sustaining Th2 responses in the low airway hypersensitivity related to sinusitis. PMID- 16677401 TI - Derivation of species-specific hybridization-like knowledge out of cross-species hybridization results. AB - BACKGROUND: One of the approaches for conducting genomics research in organisms without extant microarray platforms is to profile their expression patterns by using Cross-Species Hybridization (CSH). Several different studies using spotted microarray and CSH produced contradicting conclusions in the ability of CSH to reflect biological processes described by species-specific hybridization (SSH). RESULTS: We used a tomato-spotted cDNA microarray to examine the ability of CSH to reflect SSH data. Potato RNA was hybridized to spotted cDNA tomato and potato microarrays to generate CSH and SSH data, respectively. Difficulties arose in obtaining transcriptomic data from CSH that reflected those obtained from SSH. Nevertheless, once the data was filtered for those corresponding to matching probe sets, by restricting proper cutoffs of probe homology, the CSH transcriptome data showed improved reflection of those of the SSH. CONCLUSIONS: This study evaluated the relative performance of CSH compared to SSH, and proposes methods to ensure that CSH closely reflects the biological process analyzed by SSH. PMID- 16677402 TI - Duodenum preserving pancreatectomy in chronic pancreatitis: design of a randomized controlled trial comparing two surgical techniques [ISRCTN50638764]. AB - BACKGROUND: Chronic pancreatitis is an inflammatory disease which is characterized by an irreversible conversion of pancreatic parenchyma to fibrous tissue. Beside obstructive jaundice and pseudocyst formation, about half of the patients need surgical intervention due to untreatable chronic pain during the course of the disease. In most of the patients with chronic pancreatitis, the head of the pancreas is the trigger of the chronic inflammatory process. Therefore, resection of pancreatic head tissue must be the central part of any surgical intervention. However, it is unclear to which extent the surgical procedure must be radical in order to obtain a favourable outcome for the patients. DESIGN: A single centre randomized controlled, superiority trial to compare two techniques of duodenum preserving pancreatic head resection. SAMPLE SIZE: 65 patients will be included and randomized intraoperatively. ELIGIBILITY CRITERIA: All patients with chronic pancreatitis and indication for surgical resection and signed informed consent. Cumulative primary endpoint (hierarchical model): duration of surgical procedure, quality of life after one year, duration of intensive care unit stay, duration of hospital stay. Reference treatment: Resection of the pancreatic head with dissection of the pancreas from the portal vein and transsection of the gland (Beger procedure). INTERVENTION: Partial Resection of the pancreatic head without transsection of the organ and visualization of the portal vein (Berne procedure). DURATION: September 2003 October 2007. ORGANISATION/RESPONSIBILITY: The trial is conducted in compliance with the protocol and in accordance with the moral, ethical, regulatory and scientific principles governing clinical research as set out in the Declaration of Helsinki (1989) and the Good Clinical Practice guideline (GCP). The Center for Clinical Studies of the Department of Surgery Heidelberg is responsible for planning, conducting and final analysis of the trial. PMID- 16677404 TI - Sugar, soap and statins--an unlikely recipe for the critically ill. AB - The eagerly awaited SOAP (Sepsis Occurrence in Acutely ill Patients) study is published and its observational data provide much of interest, not least in generating further hypotheses on improving treatment in this challenging group. Glycaemic control in the critically ill is once more the focus of attention, and we discuss three studies in this area. Not least among these reports is that from the van den Bergh group, who provide further data on their intensive insulin protocol in a more heterogeneous group, namely medical intensive care unit patients. Finally, we discuss another good reason to take statins. PMID- 16677403 TI - Effects of overexpression of IL-10, IL-12, TGF-beta and IL-4 on allergen induced change in bronchial responsiveness. AB - BACKGROUND: An increasing prevalence of allergic diseases, such as atopic dermatitis, allergic rhinitis and bronchial asthma, has been noted worldwide. Allergic asthma strongly correlates with airway inflammation caused by the unregulated production of cytokines secreted by allergen-specific type-2 T helper (Th2) cells. This study aims to explore the therapeutic effect of the airway gene transfer of IL-12, IL-10 and TGF-beta on airway inflammation in a mouse model of allergic asthma. METHODS: BALB/c mice were sensitized to ovalbumin (OVA) by intraperitoneal injections with OVA and challenged by nebulized OVA. Different cytokine gene plasmids or non-coding vector plasmids were instilled daily into the trachea up to one day before the inhalatory OVA challenge phase. RESULTS: Intratracheal administration of IL-10, IL-12 or TGF-beta can efficiently inhibit antigen-induced airway hyper-responsiveness and is able to largely significantly lower the number of eosinophils and neutrophils in bronchoalveolar lavage fluid of ovalbumin (OVA) sensitized and challenged mice during the effector phase. Furthermore, the effect of IL-10 plasmids is more remarkable than any other cytokine gene plasmid. On the other hand, local administration of IL-4 gene plasmids before antigen challenge can induce severe airway hyper-responsiveness (AHR) and airway eosinophilia. CONCLUSION: Our data demonstrated that anti inflammatory cytokines, particularly IL-10, have the therapeutic potential for the alleviation of airway inflammation in murine model of asthma. PMID- 16677405 TI - Reducing ventilator-induced lung injury and other organ injury by the prone position. AB - Mechanical ventilation can cause structural and functional disturbances in the lung, as well as other vital organ dysfunctions. Apoptosis is thought to be a histological sign of distant organ damage in ventilator-induced lung injury (VILI). Nakos and colleagues observed a protective effect of prone positioning against VILI in normal sheep. Less alteration in the lung architecture and function and in liver transaminases, and lower indices for apoptosis in the liver, the diaphragm and the lung were noted in the prone position compared with the supine position. If confirmed, these data open a new hypothesis for pathogenesis and prevention of VILI and its extrapulmonary complications. PMID- 16677406 TI - Implementing the International Liaison Committee on Resuscitation guidelines on hypothermia after cardiac arrest. The German experience: still a long way to go? PMID- 16677407 TI - Macrophage migration inhibitory factor: controller of systemic inflammation. AB - Macrophage migration inhibitory factor (MIF) is a cytokine that is secreted by the anterior pituitary and immune cells in response to surgical stress, injury, and sepsis. This cytokine appears to be a critical regulator of the inflammatory pathways, leading to systemic inflammatory response syndrome and subsequent multiple organ dysfunction syndrome. This report provides an integrated scheme describing the manner by which MIF controls the neurohormonal response and the adaptive immune system, namely the T-helper (Th)1 and Th2 lymphocytes, which results in the release of pro-inflammatory cytokines and the anti-inflammatory cytokine interleukin-10. The development of systemic inflammatory response syndrome and subsequent development of multiple organ dysfunction syndrome appear to be related to MIF levels and the balance of Th1 and Th2 function. PMID- 16677408 TI - Strong ion difference in urine: new perspectives in acid-base assessment. AB - The plasmatic strong ion difference (SID) is the difference between positively and negatively charged strong ions. At pH 7.4, temperature 37 degrees C and partial carbon dioxide tension 40 mmHg, the ideal value of SID is 42 mEq/l. The buffer base is the sum of negatively charged weak acids ([HCO3(-)], [A-], [H2PO4( )]) and its normal value is 42 mEq/l. According to the law of electroneutrality, the amount of positive and negative charges must be equal, and therefore the SID value is equal to the buffer base value. The easiest assessment of metabolic acidosis/alkalosis relies on the base excess calculation: buffer base(actual) - buffer base(ideal) = SID(actual) - SID(ideal). The SID approach allows one to appreciate the relationship between acid-base and electrolyte equilibrium from a unique perspective, and here we describe a comprehensive model of this equilibrium. The extracellular volume is characterized by a given SID, which is a function of baseline conditions, endogenous and exogenous input (endogenous production and infusion), and urinary output. Of note, volume modifications vary the concentration of charges in the solution. An expansion of extracellular volume leads to acidosis (SID decreases), whereas a contraction of extracellular volume leads to alkalosis (SID increases). A thorough understanding of acid-base equilibrium mandates recognition of the importance of urinary SID. PMID- 16677409 TI - The validity of a rheumatoid arthritis medical records-based index of severity compared with the DAS28. AB - Outcome measures play an extremely important role in clinical trials and observational research. Outcome measures for rheumatoid arthritis cover a whole array of domains, ranging from measures describing the inflammatory process to measures describing the ultimate consequences of long-term disease, such as joint damage, physical function and quality of life. There is a scientific need to be able to quantify what is called the 'severity of rheumatoid arthritis', so that patients with rheumatoid arthritis can be clustered according to their propensity to develop an unfavourable outcome. It is a challenge to find an appropriate measure for severity. One attempt has been the development of the Rheumatoid Arthritis Medical Record-Based Index of Severity. This commentary elaborates on how such a measure of severity should be validated to determine whether it is appropriate for practical use. PMID- 16677410 TI - Shelling out for genomics. PMID- 16677411 TI - Involution: apoptosis and tissue remodelling that convert the mammary gland from milk factory to a quiescent organ. AB - Involution of the mammary gland is an essential process that removes the milk producing epithelial cells when they become redundant at weaning. It is a two step process that involves the death of the secretory epithelium and its replacement by adipocytes. During the first phase, remodelling is inhibited and apoptotic cells can be seen in the lumena of the alveoli. In the second phase, apoptosis is accompanied by remodelling of the surrounding stroma and re differentiation of the adipocytes. Considerable effort has been directed towards understanding the molecular mechanisms of the involution process and this has resulted in the identification of the principal signalling pathways involved. PMID- 16677412 TI - Is there a positive association between mammographic density and bone mineral density? Authors' response. PMID- 16677413 TI - Clinical review: specific aspects of acute renal failure in cancer patients. AB - Acute renal failure (ARF) in cancer patients is a dreadful complication that causes substantial morbidity and mortality. Moreover, ARF may preclude optimal cancer treatment by requiring a decrease in chemotherapy dosage or by contraindicating potentially curative treatment. The pathways leading to ARF in cancer patients are common to the development of ARF in other conditions. However, ARF may also develop due to etiologies arising from cancer treatment, such as nephrotoxic chemotherapy agents or the disease itself, including post renal obstruction, compression or infiltration, and metabolic or immunological mechanisms. This article reviews specific renal disease in cancer patients, providing a comprehensive overview of the causes of ARF in this setting, such as treatment toxicity, acute renal failure in the setting of myeloma or bone marrow transplantation. PMID- 16677414 TI - Signals and systems. PMID- 16677415 TI - Cancer and kinases: reports from the front line. PMID- 16677417 TI - Reaction to the threat of influenza pandemic: the mass media and the public. PMID- 16677416 TI - The intraductal approach to the breast: raison d'etre. AB - Opportunities for the detection, prediction, and treatment of breast cancer exist at three biological levels: systemically via the blood, at the whole organ level, and within the individual ductal lobular structures of the breast. This review covers the evaluation of approaches targeted to the ductal lobular units, where breast cancer begins. Studies to date suggest the presence of 5 to 12 independent ductal lobular systems per breast, each harboring complex cellular fluids contributed by local and systemic processes. New techniques for accessing and interrogating these systems offer the potential to gauge the microenvironment of the breast and distill biological risk profiles. PMID- 16677418 TI - The alveolar switch: coordinating the proliferative cues and cell fate decisions that drive the formation of lobuloalveoli from ductal epithelium. AB - Massive tissue remodelling occurs within the mammary gland during pregnancy, resulting in the formation of lobuloalveoli that are capable of milk secretion. Endocrine signals generated predominantly by prolactin and progesterone operate the alveolar switch to initiate these developmental events. Here we review the current understanding of the components of the alveolar switch and conclude with an examination of the role of the ets transcription factor Elf5. We propose that Elf5 is a key regulator of the alveolar switch. PMID- 16677419 TI - Ventilator-associated pneumonia: monotherapy is optimal if chosen wisely. AB - Traditionally, ventilator-associated pneumonia (VAP) has been treated either with double drug therapy or with monotherapy. Double drug therapy has been used to increase spectrum, for possible synergy, and to decrease the emergence of resistance. VAP therapy should be directed primarily against Pseudomonas aeruginosa, which also provides aerobic Gram-negative coverage, the usual pathogens in VAP. The potent anti-P. aeruginosa antibiotics available today have sufficient activity that double drug coverage is unnecessary. Double drug therapy does not decrease resistance if a 'high resistance potential' antibiotic is used in the combination. The study by Damas and colleagues in this issue of Critical Care supports monotherapy for VAP. Optimal therapy for VAP involves selecting a potent anti-P. aeruginosa antibiotic with a 'low resistance potential' that minimizes drug-drug interactions, minimizes resistance, and is cost effective. Monotherapy of VAP should be the standard of care. PMID- 16677421 TI - Bone versus breast density. AB - The common link with oestrogen levels suggests that bone mineral density and mammographic density might also be linked. One study found weak support for this, but another study failed to provide confirmation. Overall, the relationship is very weak, if it exists at all. Other factors such as weight-bearing exercise, which have opposing impacts on these variables, may have a more dominant effect. PMID- 16677422 TI - Overdiagnosis and overtreatment of breast cancer: is overdiagnosis an issue for radiologists? AB - Overdiagnosis is diagnosis of cancers that would not present within the life of the patient and is one of the downsides of screening. This applies to low-grade ductal carcinoma in situ and some small grade 1 invasive cancers. Radiologists are responsible for cancer diagnosis, but at the time of diagnosis they cannot determine whether a particular low-grade diagnosis is one to which the definition of overdiagnosis applies. Overdiagnosis is likely to be driven by technological developments, including digital mammography, computer-aided detection and improved biopsy techniques. It is also driven by the patient's fear that cancer will be missed and the doctor's fear of litigation. It is therefore an issue of importance for radiologists, presenting them with difficult fine-tuned decisions in every assessment clinic that are ultimately counted later by those who evaluate their screening. PMID- 16677423 TI - Overdiagnosis and overtreatment of breast cancer: progression of ductal carcinoma in situ: the pathological perspective. AB - Ductal carcinoma in situ (DCIS) is encountered much more frequently in the screening population compared to the symptomatic setting. The behaviour of DCIS is highly variable and this presents difficulties in choosing appropriate treatment strategies for individual cases. This review discusses the current data on the frequency and rate of progression of DCIS, the value and limitations of clinicopathological and biological variables in predicting disease behaviour and suggests strategies to develop more robust means of predicting progression of DCIS. PMID- 16677425 TI - Arginine-vasopressin in catecholamine-refractory septic versus non-septic shock in extremely low birth weight infants with acute renal injury. AB - INTRODUCTION: The aim of this study was to assess the efficacy of arginine vasopressin (AVP) as a rescue therapy in catecholamine-refractory septic and non septic shock in extremely low birth weight (ELBW) infants with acute renal injury. METHODS: Prospective assessment of AVP therapy in three ELBW infants with catecholamine-refractory septic shock and acute renal injury (mean birth weight 600 +/- 30 g) and three ELBW infants with non-septic shock and acute renal injury (mean birth weight 770 +/- 110 g) at a University hospital. The main outcome measures were restoration of blood pressure with adequate organ perfusion and survival at discharge. RESULTS: In all three ELBW infants with catecholamine resistant septic shock, systemic arterial blood pressure increased substantively with restoration of urine output after AVP administration (dosage, 0.035 to 0.36 U/kg/h; length, 70 +/- 21 hours). In the three ELBW infants with non-septic shock, only a transient stabilization in mean arterial pressure with restoration of urine output was observed after AVP therapy (dosage, 0.01 to 0.36 U/kg/h; length, 30 +/- 16 hours). The mortality rate was 1/3 in the sepsis group versus 3/3 in the non-septic group. CONCLUSION: AVP may be a promising rescue therapy in catecholamine-resistant shock in ELBW infants with acute renal injury. Larger prospective clinical trials are warranted to assess the efficacy and safety of AVP as a pressor adjunct in septic versus non-septic shock in ELBW infants. PMID- 16677426 TI - Metabolic cycle, cell cycle, and the finishing kick to Start. AB - Slowly growing budding yeast store carbohydrate, then liquidate it in late G1 phase of the cell cycle, superimposing a metabolic cycle on the cell cycle. This metabolic cycle may separate biochemically incompatible processes. Alternatively it may provide a burst of energy and material for commitment to the cell cycle. Stored carbohydrate could explain the size requirement for cells passing the Start point. PMID- 16677427 TI - Getting to synaptic complexes through systems biology. AB - Large numbers of synaptic components have been identified, but the effect so far on our understanding of synaptic function is limited. Now, network maps and annotated functions of individual components have been used in a systems biology approach to analyzing the function of NMDA receptor complexes at synapses, identifying biologically relevant modular networks within the complex. PMID- 16677428 TI - Circadian rhythms lit up in Chlamydomonas. AB - Recent work on the circadian clock of the unicellular green alga Chlamydomonas reinhardtii strengthens its standing as a convenient model system for circadian study. It was shown to be amenable to molecular engineering using a luciferase based real-time reporter for circadian rhythms. Together with the completed draft genomic sequence, the new system opens the door for genome-scale forward and reverse genetic analysis. PMID- 16677429 TI - TANGOing along the protein secretion pathway. AB - Although the organization and functions of the constitutive secretory pathway have been intensively studied for decades, a recent genome-wide RNAi screen in Drosophila cells has identified about 100 genes encoding novel so-called TANGO proteins (for transport and Golgi organization) that may be direct regulators of various aspects of protein exocytosis or secretion. PMID- 16677430 TI - Plant NBS-LRR proteins: adaptable guards. AB - The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. This large family is encoded by hundreds of diverse genes per genome and can be subdivided into the functionally distinct TIR-domain-containing (TNL) and CC-domain-containing (CNL) subfamilies. Their precise role in recognition is unknown; however, they are thought to monitor the status of plant proteins that are targeted by pathogen effectors. PMID- 16677431 TI - The evolution of human fatness and susceptibility to obesity: an ethological approach. AB - Human susceptibility to obesity is an unusual phenomenon amongst animals. An evolutionary analysis, identifying factors favouring the capacity for fat deposition, may aid in the development of preventive public health strategies. This article considers the proximate causes, ontogeny, fitness value and evolutionary history of human fat deposition. Proximate causes include diet composition, physical activity level, feeding behaviour, endocrine and genetic factors, psychological traits, and exposure to broader environmental factors. Fat deposition peaks during late gestation and early infancy, and again during adolescence in females. As in other species, human fat stores not only buffer malnutrition, but also regulate reproduction and immune function, and are subject to sexual selection. Nevertheless, our characteristic ontogenetic pattern of fat deposition, along with relatively high fatness in adulthood, contrasts with the phenotype of other mammals occupying the tropical savannah environment in which hominids evolved. The increased value of energy stores in our species can be attributed to factors increasing either uncertainty in energy availability, or vulnerability to that uncertainty. Early hominid evolution was characterised by adaptation to a more seasonal environment, when selection would have favoured general thriftiness. The evolution of the large expensive brain in the genus Homo then favoured increased energy stores in the reproducing female, and in the offspring in early life. More recently, the introduction of agriculture has had three significant effects: exposure to regular famine; adaptation to a variety of local niches favouring population-specific adaptations; and the development of social hierarchies which predispose to differential exposure to environmental pressures. Thus, humans have persistently encountered greater energy stress than that experienced by their closest living relatives during recent evolution. The capacity to accumulate fat has therefore been a major adaptive feature of our species, but is now increasingly maladaptive in the modern environment where fluctuations in energy supply have been minimised, and productivity is dependent on mechanisation rather than physical effort. Alterations to the obesogenic environment are predicted to play a key role in reducing the prevalence of obesity. PMID- 16677432 TI - Sex-specific sibling interactions and offspring fitness in vertebrates: patterns and implications for maternal sex ratios. AB - Vertebrate sex ratios are notorious for their lack of fit to theoretical models, both with respect to the direction and the magnitude of the sex ratio adjustment. The reasons for this are likely to be linked to simplifying assumptions regarding vertebrate life histories. More specifically, if the sex ratio adjustment itself influences offspring fitness, due to sex-specific interactions among offspring, this could affect optimal sex ratios. A review of the literature suggests that sex-specific sibling interactions in vertebrates result from three major causes: (i) sex asymmetries in competitive ability, for example due to sexual dimorphism, (ii) sex-specific cooperation or helping, and (iii) sex asymmetries in non competitive interactions, for example steroid leakage between fetuses. Incorporating sex-specific sibling interactions into a sex ratio model shows that they will affect maternal sex ratio strategies and, under some conditions, can repress other selection pressures for sex ratio adjustment. Furthermore, sex specific interactions could also explain patterns of within-brood sex ratio (e.g. in relation to laying order). Failure to take sex-specific sibling interactions into account could partly explain the lack of sex ratio adjustment in accordance with theoretical expectations in vertebrates, and differences among taxa in sex specific sibling interactions generate predictions for comparative and experimental studies. PMID- 16677433 TI - Mechanisms and evolution of deceptive pollination in orchids. AB - The orchid family is renowned for its enormous diversity of pollination mechanisms and unusually high occurrence of non-rewarding flowers compared to other plant families. The mechanisms of deception in orchids include generalized food deception, food-deceptive floral mimicry, brood-site imitation, shelter imitation, pseudoantagonism, rendezvous attraction and sexual deception. Generalized food deception is the most common mechanism (reported in 38 genera) followed by sexual deception (18 genera). Floral deception in orchids has been intensively studied since Darwin, but the evolution of non-rewarding flowers still presents a major puzzle for evolutionary biology. The two principal hypotheses as to how deception could increase fitness in plants are (i) reallocation of resources associated with reward production to flowering and seed production, and (ii) higher levels of cross-pollination due to pollinators visiting fewer flowers on non-rewarding plants, resulting in more outcrossed progeny and more efficient pollen export. Biologists have also tried to explain why deception is overrepresented in the orchid family. These explanations include: (i) efficient removal and deposition of pollinaria from orchid flowers in a single pollinator visit, thus obviating the need for rewards to entice multiple visits from pollinators; (ii) efficient transport of orchid pollen, thus requiring less reward-induced pollinator constancy; (iii) low-density populations in many orchids, thus limiting the learning of associations of floral phenotypes and rewards by pollinators; (iv) packaging of pollen in pollinaria with limited carry-over from flower to flower, thus increasing the risks of geitonogamous self pollination when pollinators visit many flowers on rewarding plants. All of these general and orchid-specific hypotheses are difficult to reconcile with the well established pattern for rewardlessness to result in low pollinator visitation rates and consequently low levels of fruit production. Arguments that deception evolves because rewards are costly are particularly problematic in that small amounts of nectar are unlikely to have a significant effect on the energy budget of orchids, and because reproduction in orchids is often severely pollen-, rather than resource-limited. Several recent experimental studies have shown that deception promotes cross-pollination, but it remains unknown whether actual outcrossing rates are generally higher in deceptive orchids. Our review of the literature shows that there is currently no evidence that deceptive orchids carry higher levels of genetic load (an indirect measure of outcrossing rate) than their rewarding counterparts. Cross-pollination does, however, result in dramatic increases in seed quality in almost all orchids and has the potential to increase pollen export (by reducing pollen discounting). We suggest that floral deception is particularly beneficial, because of its promotion of outcrossing, when pollinators are abundant, but that when pollinators are consistently rare, selection may favour a nectar reward or a shift to autopollination. Given that nectar-rewardlessness is likely to have been the ancestral condition in orchids and yet is evolutionarily labile, more attention will need to be given to explanations as to why deception constitutes an 'evolutionarily stable strategy'. PMID- 16677434 TI - EACTA 2006. Abstracts of the 21st Annual Meeting of the European Association of Cardiothoracic Anaesthesiologists, Venice Mestre, Italy, May 24-27, 2006. PMID- 16677435 TI - New approaches and old controversies to postoperative pain control following cardiac surgery. AB - OBJECTIVE: To evaluate the effect of postoperative pain control in cardiac surgical patients on morbidity, mortality and other outcome measures. BACKGROUND: New approaches in pain control have been introduced over the past decade. The impact of these interventions, either alone or in combination, on perioperative outcome was evaluated in cardiac surgical patients. METHODS: We searched Medline for the period of 1980 to the present using the key terms analgesics, opioid, non steroidal anti-inflammatory drugs, cardiac surgery, regional analgesia, spinal, epidural, fast-track cardiac anaesthesia, fast-track cardiac surgery, myocardial ischaemia, myocardial infarction, postoperative care, accelerated care programmes, postoperative complications, and we examined and discussed the articles that were identified to be included in this review. RESULTS: Pain management in cardiac surgery is becoming more important with the establishment of minimally invasive direct coronary artery bypass surgery and fast-track management of conventional cardiac surgery patients. Advances have been made in this area and encompass specific techniques, such as central neuraxial blockade or selective nerve blocks, and drugs (opioids, sedative-hypnotics and non steroidal anti-inflammatory drugs). Ideally, these therapies provide not only patient comfort but also mitigate untoward cardiovascular responses, pulmonary responses, and other inflammatory and secondary sympathetic responses. The introduction of these newer approaches to perioperative care has reduced morbidity, but not mortality, in cardiac surgical patients. CONCLUSIONS: Understanding perioperative pathophysiology and implementation of care regimes to reduce the stress of cardiac surgery, will continue to accelerate rehabilitation associated with decreased hospitalization and increased satisfaction and safety after discharge. Reorganization of the perioperative team (anaesthesiologists, surgeons, nurses and physical therapists) will be essential to achieve successful fast-track cardiac surgical programmes. Developments and improvements of multimodal interventions within the context of 'fast-track' cardiac surgery programmes represents the major challenge for the medical professionals working to achieve a 'pain and risk free' perioperative course. PMID- 16677436 TI - Falsely increased bispectral index values caused by the use of a forced-air warming device. PMID- 16677437 TI - Atrioventricular sequential pacing causing haemodynamically significant mitral regurgitation immediately following cardiopulmonary bypass. PMID- 16677438 TI - Glutamine and chronic obstructive pulmonary disease. PMID- 16677442 TI - Acute trichinellosis increases susceptibility to Giardia lamblia infection in the mouse model. AB - The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a 'pre existing' nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a 'pre existing' nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease. PMID- 16677444 TI - [Implications of the pathogenic studies in patients with recurrent pulmonary tuberculosis]. PMID- 16677443 TI - Age, intensity of infestation by flea parasites and body mass loss in a rodent host. AB - Parasitism by the flea Synosternus cleopatrae does not affect the body mass of its principal rodent host, Gerbillus andersoni under natural infestation levels. We hypothesized that the lack of negative effects of flea parasitism on rodent body mass could be related either to the low level of natural infestation or to the differential susceptibility of rodent age cohorts to flea parasitism. We tested these hypotheses by measuring body mass change under flea parasitism in (a) adult rodents infested with fleas above the natural infestation level (the first hypothesis) and (b) juvenile rodents infested with fleas at natural infestation levels (the second hypothesis). Adult individuals parasitized by a number of fleas higher than in nature lost body mass at higher rates than non parasitized control individuals. Parasitism significantly affected daily body mass change of juvenile gerbils. Juvenile rodents parasitized by fleas at the natural level of infestation lost body mass faster and gained body mass slower than control animals. We suggest that some regulating mechanisms may limit natural flea densities at a point at which the negative effect on hosts is below the accuracy of our measurements. However, natural flea densities are sufficiently high to harm the more susceptible, juvenile cohort. PMID- 16677445 TI - [Primary drug-resistance is the main cause of drug-resistant tuberculosis]. AB - OBJECTIVE: To investigate the main cause of drug-resistant tuberculosis. METHODS: Clinic isolates of Mycobacterium tuberculosis from 1996 to 2004 in Shanghai were analyzed and the proportion of isolates from new cases (the proxy of primary drug resistance) and retreatment cases (the proxy of acquired drug-resistance) were calculated. Mycobacterial interspersed repetitive units (MIRU) genotyping was performed and analyzed on pairs of isolates of Mycobacterium tuberculosis from 16 recurrent patients with drug-resistant tuberculosis. RESULTS: Of all 8 120 isolates of Mycobacterium tuberculosis from new cases, 707 isolates (8.7%) were drug resistant. Of all 610 isolates from retreatment cases, 150 isolates (24.6%) were drug resistant. Primary drug resistance accounted for 82.5% (707/857) of all drug resistant isolates. Based on the MIRU genotypes, 13 of all 16 recurrent drug resistant patients were reinfected with new drug-resistant strain of Mycobacterium tuberculosis. CONCLUSION: Primary drug-resistance is the main cause of drug-resistant tuberculosis. PMID- 16677446 TI - [Study on the cause of tuberculosis recurrence by Mycobacterium tuberculosis genotyping method]. AB - OBJECTIVE: To study the contribution of exogenous reinfection to tuberculosis recurrence. METHODS: Mycobacterium tuberculosis isolates from patients who had experienced 2 successive tuberculosis episodes during the years from January 1999 to March 2004 were genotyped by mycobacterial interspersed repetitive units (MIRU) method. The cause of tuberculosis recurrence was determined by comparing the MIRU pattern of Mycobacterium tuberculosis isolates responsible for different tuberculosis episodes from patients. RESULTS: Of 37 qualified patients with recurrent tuberculosis, the isolates from 25 patients in their two tuberculosis episodes showed different MIRU patterns, indicating that 68% recurrent patients were due to exogenous reinfection. The exogenous reinfection rate decreased with the age from 3/3 (under 30 years) to 73% (11/15, 30 to 60 years) and 58% (11/19, over 60 years). The frequency of exogenous reinfection increased with the tuberculosis recurrent interval. Within 6 months, the exogenous reinfection accounted for 58% (7/12) of all the recurrence, while for the time more than one year, the percentage increased to 79% (11/14). CONCLUSIONS: Exogenous reinfection was a major cause of tuberculosis recurrence in Shanghai. MIRU genotyping method is useful to study the cause of tuberculosis recurrence. PMID- 16677447 TI - [Study on the sequences of amino-acids and their gene codes of the related proteins of rifampin-dependent Mycobacterium tuberculosis]. AB - OBJECTIVE: To study the sequences of amino-acids and their related genes of rifampin-dependent Mycobacterium tuberculosis. METHODS: The strains of rifampin dependent, rifampin-resistant and rifampin-sensitive Mycobacterium tuberculosis were evaluated by L-J method. The proteins of 134 strains were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of amino-acids of the proteins with differential expressions were determined by Q-TOF2 liquid chromatography-electra mist spray-quadrupole -time of flight cascade mass spectrograph (LC-ESI-MS-MS, Q-TOF2). The corresponding gene codes for the proteins were analyzed. RESULTS: The protein SDS-PAGE profile of 41 of 49 (41/49) rifampin-dependent strains showed a high expression of a protein band (molecular weight 43,000), representative of 30%-50% of the total proteins differed from rifampin resistance and the control strains. The protein profile of H(37)Rv standard strains, 28 (28/30) rifampin-sensitive strains and 44 (44/54) rifampin-resistant strains showed no relevant high expression. The protein of high expression was identified as the hypothetical protein Rv0341 of Mycobacterium tuberculosis H(37)Rv with a molecular weight (M) of 43,894, a iso potential point of 5.25 and a score of 183. No mutation was found in the 1,440 bp encoding sequence in various strains. CONCLUSIONS: The hypothetical protein Rv0341 is related to the rifampin-dependent strains, so it can be regarded as an indicator for rifampin-dependent Mycobacterium tuberculosis. Rifampin upregulated the expression of Rv0341 in rifampin-dependent Mycobacterium tuberculosis. Whether the hypothetical protein Rv0341 is related to virulence and synthesis of the cell wall of Mycobacterium tuberculosis needs further study. PMID- 16677448 TI - [Therapeutic effects and mechanisms of CpG oligodeoxynucleotides in mice infected with Mycobacterium tuberculosis]. AB - OBJECTIVE: To investigate the protective effects and mechanisms of CpG oligodeoxynucleotides (CpG ODN) in mice infected with Mycobacterium tuberculosis. METHODS: Ninety-six female BALB/c mice were randomized into 4 groups, namely CpG ODN treatment (A group), control ODN treatment (B group), infection control (C group) and healthy control (D group) (n = 24 each). A group and B group were treated once with CpG ODN or control ODN (30 microg/mouse) intraperitoneally 2 weeks before infection with Mycobacterium tuberculosis. 1 x 10(6) bacili in a volume of 300 microl saline was injected into the tail vein of mice from A group, B group and C group. Twelve mice from each group were sacrificed at 3 weeks postinfection. Pathologic changes in lung tissues were observed. The expression of Toll-like receptor 9 (TLR9) mRNA and cytokine mRNA was detected by RT-PCR. The numbers of viable bacteria in lung and spleen were counted. The rest 12 mice from each group were monitored for 60 days to observe the mortality. RESULTS: CpG ODN was shown to increase the survival time of mice infected with Mycobacterium tuberculosis. The body weights of mice from A group [(20.37 +/- 1.12) g] were higher than those of B group [(17.50 +/- 0.62) g] and C group [(17.15 +/- 0.97) g, P < 0.01]. The lung weights of mice from A group [(0.25 +/- 0.02) g] were similar to those of B group [(0.27 +/- 0.34) g, P > 0.05], but less than those of C group [(0.28 +/- 0.26) g, P < 0.01]. The spleens of mice from A group [(0.63 +/ 0.37) g] were larger than those of B group [(0.39 +/- 0.05) g] and C group [(0.38 +/- 0.02) g, P < 0.01]. The inflammation in lung tissue of mice from A group was less than that of B group and C group. There was no mycobacterial outgrowth in lungs and spleens of mice from A group. The expression of TLR9 mRNA in lungs and spleens of mice from A group (0.61 +/- 0.29 and 0.72 +/- 0.48) was similar to that in B group (0.58 +/- 0.35 and 0.64 +/- 0.28) and C group (0.60 +/ 0.32 and 0.65 +/- 0.31, P > 0.05), but higher than that in D group (0.11 +/- 0.08 and 0.26 +/- 0.22, P < 0.01). CpG ODN did not affect the expression of TLR9 mRNA. The expression of IFN-gamma mRNA in lungs and spleens of mice from A group (0.44 +/- 0.07 and 0.76 +/- 0.09) was higher than that in B group (0.19 +/- 0.05 and 0.22 +/- 0.05) and C group (0.16 +/- 0.04 and 0.18 +/- 0.08, P < 0.01). The expression of IL-6 mRNA in lungs and spleens of mice from A group (1.56 +/- 0.29 and 8.21 +/- 0.82) was higher than that in B group (0.86 +/- 0.55 and 0.16 +/- 0.09) and C group (0.78 +/- 0.21 and 0.06 +/- 0.04, P < 0.01). The expression of IL-4 mRNA in lungs and spleens of mice from A group (0.18 +/- 0.05 and 0.06 +/- 0.02) was lower than that in B group (0.31 +/- 0.06 and 1.22 +/- 0.01) and C group (0.35 +/- 0.04 and 1.31 +/- 0.31, P < 0.01). The expression of IL-10 mRNA in spleens of mice from A group (0.05 +/- 0.02) was lower than that in B group (0.57 +/- 0.09) and C group (0.65 +/- 0.15, P < 0.01). CONCLUSION: CpG ODN could increase the immunity of mice against tuberculosis through up-regulation of Th1 immunity and down-regulation of Th2 immunity. PMID- 16677449 TI - [Chronic necrotizing pulmonary aspergillosis: diagnosis and treatment]. AB - OBJECTIVE: The purpose of this study was to describe the approaches to the diagnosis and the treatment of chronic necrotizing pulmonary aspergillosis (CNPA). METHODS: The study included 8 patients with CNPA confirmed by histopathological studies. The patients were admitted to the First Affiliated Hospital of College of Medicine of Zhejiang University during 2000-2004. The clinical data were analyzed, and related literature was reviewed. The clinical, radiological and pathological characteristics, and the approaches to the diagnosis and the treatment were described. RESULTS: CNPA was a rare form of pulmonary aspergillosis. Common presentations included productive cough (8/8), haemoptysis (7/8) and constitutional symptoms (5/8). Radiographically, the air crescent sign was suggestive of the diagnosis, which was found in 4 patients. Consolidation was also common in the other 4 cases. The histological feature of CNPA included the presence of tissue invasion by Aspergillus, tissue necrosis and granulomatous inflammation. CONCLUSIONS: The clinical manifestations of CNPA are non-specific. Radiography plays a pivotal role in the diagnosis of CNPA. Treatment with antifungal medications is indicated once the diagnosis is made. Pulmonary resection should be considered when patients have prolonged illness or frequent haemoptysis. PMID- 16677450 TI - [Etiology and a diagnostic protocol for patients with chronic cough]. AB - OBJECTIVE: The spectrum and frequency of causes and the diagnostic protocol for chronic cough were explored. METHODS: A total of 194 patients with at least 3 weeks of chronic cough and normal chest radiographs were recruited from the outpatient clinic of Guangzhou Institute of Respiratory Diseases between July 2003 to June 2004. The causes were investigated using a well-established protocol. The diagnostic protocol included history inquiring and physical examination, pulmonary function tests, induced sputum cell differentials, 24 h esophageal pH monitoring, CT of the paranasal sinuses or chest, fiberoptic rhinoscopy or bronchoscopy. The final diagnosis was made based on clinical manifestation, examination findings and a positive response to therapy. RESULTS: The cause of chronic cough was defined in 95.4% of the patients, with a single cause found in 153 patients (82.7%), and multiple causes in 32 patients (17.3%). The five most important causes of cough were: eosinophilic bronchitis (n = 51, 22.4%), rhinitis and/or paranasal sinusitis (PNDs, n = 39, 17.1%), cough-variant asthma (n = 31, 13.6%), atopic cough (n = 28, 12.3%), and gastroesophageal reflux (n = 27, 11.8%). CONCLUSIONS: The spectrum and frequency of causes of chronic cough in our study is different from the previous reports in western countries. Eosinophilic bronchitis and atopic cough are important causes of chronic cough. A modified diagnostic protocol was established accordingly. PMID- 16677451 TI - [The relationship between the polymorphism of glutamate cysteine ligase modulatory subunit gene and the susceptibility to chronic obstructive pulmonary disease]. AB - OBJECTIVE: To investigate whether glutamate cysteine ligase modulatory (GCLM) subunit gene polymorphism is associated with susceptibility to chronic obstructive pulmonary disease (COPD), and to study the relationship between polymorphism of GCLM gene with plasma gamma-glutamylcysteine synthetase (gamma GCS) activity. METHODS: Blood samples of 104 stable phase COPD smokers (COPD group), 124 healthy smokers (C group) and 132 healthy never-smokers (H group) were collected, and then the genotypes of GCLM -588C/T and GCLM -23C/T polymorphism sites were detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (RFLP). The plasma gamma-GCS activity was measured by coupled enzyme procedure. RESULTS: (1) The distribution of -588CC, -588CT, -588TT genotypes were corresponding to -23GG, -23GT, -23TT respectively in all of the individuals. (2) The frequencies of -588CC genotype and -588 C allele were significantly lower in COPD group (62.3% and 79.2%) than in C group (84.7% and 91.9%) and H group (78.8% and 89.0%, P < 0.01). (3) In smokers, GCLM -588 T allele carried a higher risk to COPD, the odds ratio (OR value) to C allele was 3.0, and with a 95% confidence interval 1.7 - 5.3. (4) The plasma gamma-GCS activity was increased in C group [(282 +/- 58) U/mg.prot] and COPD group [(224 +/- 54) U/mg.prot] as compared with H group [(157 +/- 26) U/mg.prot, P < 0.01], and were higher in healthy smokers than in COPD smokers (P < 0.01). (5) The smokers with -588CC genotype had higher gamma-GCS activity than CT or TT genotype [(292 +/- 54), (225 +/- 45) U/mg.prot, P < 0.01 in C group and (245 +/- 52), (188 +/- 36) U/mg.prot, P < 0.01 in COPD group], but the difference did not exist in H group [(158 +/- 27), (153 +/- 25) U/mg.prot, P > 0.05]. CONCLUSION: The polymorphism of GCLM -588C/T and -23G/T sites were associated with susceptibility to COPD, and were associated with plasma gamma-GCS activity. PMID- 16677452 TI - [Effects of aerosolized perfluorocarbon on gas exchange, respiratory mechanics and hemodynamics in a swine model of acute respiratory distress syndrome]. AB - OBJECTIVE: To investigate the effects of aerosolized perfluorocarbon (PFC) on gas exchanges, respiratory mechanics and hemodynamics in a swine model of acute respiratory distress syndrome (ARDS). METHODS: ARDS was induced by intratracheal instillation of detergent in 16 piglets, and the animals were then randomly assigned to a PFC treated group (n = 8) and a control group (n = 8). Animals of the control group were gas-ventilated with 100% O2 (3 L/min), while those of the PFC treated group received an additional continuous aerosolized PFC at 7 - 8 ml.kg(-1).h(-1). Blood gases, average artery pressure, heart rate, platform pressure, compliance, expiratory tidal volume and intrinsic positive end expiratory pressure (PEEPi) were measured per 15 minutes. RESULTS: Detergent instillation resulted in a marked decrease in arterial oxygen pressure (PaO2) within 60 min, from (383 +/- 53) mm Hg (1 mm Hg = 0.133 kPa) to (49 +/- 12) mm Hg in the control group [fraction of inspired oxygen (FiO2) 100%], and from (377 +/- 55) mm Hg to (56 +/- 13) mm Hg in the PFC group (FiO2 100%). After 2 h treatment, PaO2 was increased from (49 +/- 12) mm Hg to (83 +/- 51) mm Hg in the control group, compliance from (1.4 +/- 0.4) ml/cm H2O to (2.8 +/- 1.8) ml/cm H2O, and expiratory tidal volume from (30.8 +/- 5.5) ml to (50.1 +/- 4.1) ml in the control group; PaO2 from (56 +/- 13) mm Hg to (189 +/- 133) mm Hg, compliance from (1.5 +/- 0.4) ml/cm H2O to (4.1 +/- 1.4) ml/cm H2O, and expiratory tidal volume from (30.8 +/- 3.3) ml to (74.5 +/- 16.9) ml in the PFC group (all P < 0.05). There were no significant differences between groups in arterial carbon dioxide pressure (PaCO2), pH values, blood pressure, heart rates, plat pressure and PEEPi during treatment (all P > 0.05). CONCLUSION: It is suggested that aerosolized PFC increases arterial oxygenation, compliance, and expiratory tidal volume in extended detergent-induced ARDS. PMID- 16677453 TI - [Effects and mechanism of CD4+ CD25+ T cells on the airway inflammation of asthmatic mice]. AB - OBJECTIVE: To investigate the effects and mechanism of CD(4)(+) CD(25)(+) T cells on the airway inflammation of asthmatic mice. METHODS: Sixty mice were divided into 3 groups (20 in each group) including group A [ovalbumin (OVA) sensitized/challenged mice], group B (saline sensitized/challenged mice), group C [CD(25)(+) T cells deleted by anti-CD(25)(+) and OVA sensitized/challenged mice]. Mice of A group were sensitized on days 1 and 13 by OVA and challenged from days 21 to 29 by 2% OVA 10 ml repeatedly to establish a murine model of asthma characterized by airway inflammation. The mice of group B were treated by saline 10 ml. The mice of group C were treated by the same method of group A after deletion of the CD(25)(+) T cells by anti-mouse CD(25)(+) monoclonal antibody (McAb). Lymphocytes were separated from the spleen of the three groups and the number of T cells were calculated by Fluorescence Activated Cell Sorter (FACS). The CD(4)(+) T cells were purified and the expression of interleukin-10 (IL-10), transforming growth factor-beta(1) (TGF-beta(1)) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) mRNA were analyzed by the method of reverse transcriptase-polymerase chain reaction (RT-PCR). The airway inflammatory indices were detected by hematoxylin and eosin (HE) staining. RESULTS: In repeatedly OVA challenged mice (group A), the ratio of CD(4)(+) CD(25)(+) T cells to CD(4)(+) T cells decreased significantly [(3.10 +/- 0.03)%] as compared with group B [(9.60 +/- 0.04)%, P < 0.01]. The expression of IL-10, CTLA-4 mRNA and TGF-beta(1) mRNA of OVA-challenged mice (group A) decreased significantly (0.250 +/- 0.040 vs 0.29 +/- 0.03, 0.28 +/- 0.06 vs 0.480 +/- 0.080, 0.47 +/- 0.05 vs 0.50 +/- 0.03, all P < 0.01). The expression of IL-10, CTLA-4 mRNA and TGF-beta(1) mRNA of the mice treated by anti-CD(25)(+) McAb (group C) decreased as compared with groups A and B (0.080 +/- 0.020, 0.11 +/- 0.04, 0.12 +/- 0.05, P < 0.01). The inflammation in the lungs of mice with depleted CD(4)(+) CD(25)(+) T cells by anti-CD(25)(+) McAb was more marked than that in the OVA-challenged and control group mice. CONCLUSION: The polarization of CD(4)(+) CD(25)(+) T cells in asthmatic mice decreased significantly, which may play an important role in the pathogenesis of asthma. PMID- 16677454 TI - [Differential expression of the three hypoxia-inducible factor alpha subunits in pulmonary artery of rats with hypoxia-induced pulmonary hypertension]. AB - OBJECTIVE: To differentiate the expression patterns of all hypoxia-inducible factor alpha (HIF-alpha) subunits (HIF-1alpha, HIF-2alpha and HIF-3alpha) in pulmonary artery of rats undergoing systemic hypoxia. METHODS: Forty male healthy wistar rats were assigned randomly to 5 groups, 8 rats per group, then exposed to hypoxia [O2, (10.0 +/- 0.5)%] for 0 d (H(0)), 3 d (H(3)), 7 d (H(7)), 14 d (H(14)) and 21 d (H(21)) respectively, 8 h per day intermittently. Mean pulmonary arterial pressure (mPAP), arterial wall area (WA, microm), pulmonary artery medium thickness (PAMT%) and right ventricle hypertrophy index (RVHI) were measured. HIF-1alpha, HIF-2alpha and HIF-3alpha gene expression were determined by immunohistochemistry, in situ hybridization and Western blot. RESULTS: mPAPs in H(7), H(0) and H(14) groups were [(18.40 +/- 0.40) mm Hg, 1 mm Hg = 0.133 kPa], [(14.40 +/- 0.40) mm Hg] and [(21.20 +/- 0.20) mm Hg], respectively, statistically different when H(7) group was compared with H(0) and H(14) groups (all P < 0.05). Arterial morphology showed that WA%, PAMT and RVHI% in H(7) group were (47.8 +/- 0.8)%, (12.3 +/- 0.5) microm, (24.0 +/- 1.0)%, respectively; in H(0) group were (35.5 +/- 1.3)%, (11.9 +/- 0.6)%, (23.6 +/- 0.5) microm, respectively; in H(21) group were (65.0 +/- 0.7)%, (23.0 +/- 0.8) microm, (27.7 +/- 1.0)%, respectively. When H(7) group was compared with H(0) group, only WA% was statistically different; when H(14) group was compared with H(0) group, all the three parameters were statistically different (P < 0.05). In situ hybridization demonstrated that the mRNA levels (absorbance, A) of HIF-1alpha, HIF-2alpha, and HIF-3alpha in H(14) group were 0.200 +/- 0.020, 0.080 +/- 0.010, 0.170 +/- 0.010, respectively; in H(7) group were 0.050 +/- 0.020, 0.160 +/- 0.020, 0.160 +/- 0.020, respectively; in H(0) group were 0.050 +/- 0.010, 0.140 +/- 0.020, 0.060 +/- 0.010, respectively. When H(7) group was compared with H(0) group, only HIF-3alpha was statistically different; when H(14) group was compared with H(0) group, all the three genes were significantly different (P < 0.05). Immunohistochemistry showed that HIF-1alpha, HIF-2alpha and HIF-3alpha protein levels in H(3) group were 0.200 +/- 0.020, 0.020 +/- 0.010, 0.050 +/- 0.010, respectively; in H(14) group were 0.160 +/- 0.010, 0.100 +/- 0.020, 0.160 +/- 0.010, respectively; in H(7) group were 0.220 +/- 0.020, 0.030 +/- 0.010, 0.180 +/- 0.020, respectively; in H(0) group were 0.050 +/- 0.010, 0.020 +/- 0.010, 0.040 +/- 0.010, respectively. HIF-1alpha in H(3) group, HIF-2alpha in H(14) group, HIF-3alpha in H(7) and H(3) group were statistically different with that of H(0) group (P < 0.05). Protein bands of HIF-alpha subunits in lung tissue, measured by Western blot, showed that HIF-3alpha decreased in H(7) group as compared to H(0) group, but the other two proteins showed a marked increase in H(3) group as compared to H(0) group, and increased further corresponding to the duration of hypoxia and peaked in H(14) group as compared to H(7) group. CONCLUSION: The differential expression of the three HIF-alpha subunits may play a role in the development of hypoxia-induced pulmonary hypertension. PMID- 16677455 TI - [Advances in the study of molecular epidemiology of tuberculosis]. PMID- 16677456 TI - [Advances in the study of new methods for the prevention and therapy of Pneumocystis carinii pneumonia]. PMID- 16677470 TI - [Multidisciplinary collaboration and standardization of the management of lung cancer]. PMID- 16677473 TI - [An experimental study on the antitumor effects induced by fusions of dendritic cells with NCI-H460 lung cancer cells]. AB - OBJECTIVE: To study the antitumor effects induced by fusions of dendritic cells (DC) and NCI-H460 cells and compared with antigen pulsed DC. METHODS: (1) DC were induced from human monocytes and were fused with NCI-H460 cells. Three experimental groups were set up, including group fusions cell (FC), group pulsed cell (PC) and group T cell (TC), for which T cells activated by fusions, antigen pulsed DC, and non-activated T cells were used as the effector cells respectively. The killing activity of effector cells to NCI-H460 cells were evaluated by lactate dehydrogenase method. (2) NCI-H460 cells were injected subcutaneously to BALB/c nude mice. Then 18 cancer-bearing mice were divided randomly into 3 groups, group FC, group PC and group TC. Effector cells mentioned above were inoculated subcutaneously. The tumor size and tumor weight were assessed and compared. RESULTS: The killing rates to NCI-H460 cells of group FC, group PC and group TC were 43.54%, 26.57% and 3.25% respectively. Comparison of the killing activity of these 3 groups showed group FC > group PC > group TC (F = 5.47, P < 0.05). The tumor size of group FC was significantly smaller than that of group PC and group TC. The tumor weights of group FC, group PC and group TC were (1,129 +/- 123) mg, (1,709 +/- 160) mg and (3,344 +/- 288) mg respectively (F = 37.05, P < 0.01). CONCLUSION: Fusions of DC and NCI-H460 cells could induce powerful antitumor immunity, which was more effective than antigen pulsed DC. PMID- 16677475 TI - [Involvement of bcl-2 in multidrug resistance in human small cell lung cancer cell subline H446/DDP]. AB - OBJECTIVE: To study the mechanism of bcl-2 involvement in multidrug resistance in human small cell lung cancer (SCLC) cell subline H446/DDP. METHODS: After the construction of pLXSN-bcl-2, in which the full length of bcl-2 cDNA amplified from total RNA of H446/DDP cells was integrated into the mammalian expression vector pLXSN, allowing transcription of a bicistronic mRNA, and the synthesis in vitro of 20-mer ODNs targeting the coding region of bcl-2 mRNA and the scrambled ODNs used as the control, the cationic lipid DOTAP was used to transfect them into H446 and H446/DDP cells, respectively. When H446 cells were transfected with mammalian expression vector pLXSN, the cells were divided into 3 groups, including cells transfected with the recombinant pLXSN-bcl-2, cells transfected with the vector pLXSN and cells untransfected (control); when H446 cells and H446/DDP cells were transfected with PS-ODNs, the cells were divided into 3 groups, including cells transfected with AS-PS-ODNs, cells transfected with NS-PS ODNs and cells untransfected (control), respectively. After transfection, Western blot were carried out to detect the expression level of bcl-2 in the control cells and the transfected cells, respectively. Meanwhile flow cytometry (FCM) was used to detect cell apoptosis in the control cells and the transfected cells. RESULTS: (1) The data from Western blot showed that compared with the control H446 cells (gray-scale value 0.103 +/- 0.005), the expression level of bcl-2 in the pLXSN-bcl-2 transfected H446 cells (gray-scale value 0.854 +/- 0.016) was increased significantly (P < 0.01); and the apoptosis from DNA content analysis decreased significantly in the pLXSN-bcl-2 transfected H446 cells [(20.9 +/- 0.2)%] compared with that of the control H446 cells [(31.1 +/- 0.2)%] by DNA content analysis (P < 0.01). (2) The results from Western blot showed that bcl-2 expression in the AS-PS-ODNs transfected H446/DDP cells (gray-scale value 0.695 +/- 0.014) was significantly reduced compared with that of the control H446/DDP cells (gray-scale value 0.942 +/- 0.018), although not totally reduced to the level of the control H446 cells (P < 0.01); and the data from DNA content analysis indicated that the sensitivity of the AS-PS-ODNs transfected H446/DDP cells to 5 microg/ml DDP-induced apoptosis was strongly increased as compared with that of the control H446/DDP cells (P < 0.01). CONCLUSION: Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression may be one of the important therapeutic approaches to overcoming chemoresistance in human SCLC. PMID- 16677477 TI - [Clinical analysis of 8 cases of paradoxical embolism]. AB - OBJECTIVE: To describe the clinical features of paradoxical embolism and therefore to improve its diagnosis. METHODS: Case analysis and literature review. RESULTS: Eight cases of pulmonary embolism complicated with paradoxical embolism were diagnosed, of whom there were six men and two women (mean age, 47.6 years). Patent foramen ovale with right to left shunt was identified in only 3 cases. Cerebral embolism occurred in three patients, kidney artery embolism in 2 patients, left atrial thrombus in 1 patient, lower limb artery and aortic embolism in 1 patients respectively. The diagnosis of paradoxical embolism can only be confirmed when a venous thrombus was detected lodged at arterial-venous communication; otherwise, paradoxical embolism was considered a clinical diagnosis. Of the 8 cases, 7 were clinically diagnosed, while 1 was confirmed. CONCLUSIONS: Paradoxical embolism is not uncommon. The diagnosis should be considered when venous thromboembolism is complicated with systematic embolism or unknown cause of systematic embolism. PMID- 16677478 TI - [Pulmonary sclerosing hemangioma: report of 15 cases and review of the literature]. AB - OBJECTIVE: To improve the understanding of pulmonary sclerosing hemangioma (PSH). METHODS: The clinical data of 15 cases of PSH were analyzed, and the literature was reviewed. The etiology, clinical manifestations, differential diagnosis, treatment and outcome of PSH were described. RESULTS: The etiology and histological origin of PSH were unclear. Most cases were asymptomatic or only with mild symptoms. The radiology of PSH often showed isolated nodule with distinct margin in the lung field. The characteristics of its pathological manifestation were as follows: (1) background of cell gathering or mucin matrix with scattered white blood cells; (2) proliferation of hemangioma with sclerosis of vessel wall; (3) papillary proliferation of small vessels intruding into the air space; (4) existence of hemorrhage or sclerosis zone. Immunohistological studies had not defined the correct histological origin of PSH. A pre-operation diagnosis of PSH was difficult. Thirteen cases had been misdiagnosed as malignancy. The outcome of the disease was good when early surgical resection was performed. CONCLUSIONS: PSH is an uncommon disease, and can be easily misdiagnosed. More attention should be paid to its clinical features and management. PMID- 16677479 TI - [Clinicopathologic features of 18 cases of cryptogenic organizing pneumonia]. AB - OBJECTIVE: To analyze the characteristics of pathologically proved cases of cryptogenic organizing pneumonia (COP) including manifestation of CT scan, bronchoalveolar lavage (BAL) fluid cytology, pulmonary histopathological appearance. METHODS: Twenty-three consecutive patients from April 1994 to October 2005 in Peking Union Medical College Hospital with pathologic features of organizing pneumonia were enrolled in the study. RESULTS: Eighteen patients were diagnosed as COP. Percutaneous lung biopsy/transbronchial lung biopsy was performed in 10 patients, while open lung biopsy/video-assistant thorascopic biopsy was performed in 8 patients. There were 8 males and 10 females, aged (53.5 +/- 11.0) yr. Dyspnea, cough, inspiratory crackles were the most common symptoms and signs. Lung function tests showed a restrictive ventilatory defect (11/18), and impairment of carbon monoxide transfer factor (T(LCO), 18/18). Chest computed tomography scan in 18 cases mainly showed ground glass opacities (4/18), patchy areas of alveolar consolidation (13/18) with air bronchograms (3/18), and pleural effusion (4/18). BAL fluid cytological analysis showed the percentage of lymphocyte was 0.40 +/- 0.16, neutrophil 0.15 +/- 0.08, eosinophil 0.05 +/- 0.03; the CD(4)/CD(8) ratio was 0.43 +/- 0.21. All patients were treated with corticosteroids. Sixteen were alive and clinically improved or stabilized at the last follow up (8.7 +/- 6.2 months after diagnosis). Two patients died, one of respiratory failure, and another of pulmonary infection. CONCLUSIONS: The diagnosis of COP is usually suggested by clinical-radiological findings, but needs to be confirmed histopathologically. The combination of BAL fluid cytological findings and lung histological appearance obtained during a bronchoscopic procedure appears to be an effective method for diagnosing this disease. PMID- 16677480 TI - [Effects of immunotherapy on the expression of costimulatory molecules on dendritic cells in a mouse model of asthma]. AB - OBJECTIVE: To establish a mouse model of ovalbium in (OVA) specific immunotherapy, and to explore the effects of specific immunotherapy on the expression of CD(80) and CD(86) on spleen-derived dendritic cells (DC) in a mouse asthmatic model. METHODS: A hundred and twenty BALB/c mice were randomly divided into three groups, with 40 mice each. The asthma model (group A) was established by intraperitoneal injection of 10 microg OVA and 1% OVA aerosol challenges. Consecutive subcutaneous injection of 1 mg OVA in the dorsal aspect of the foot was then given to establish the model of OVA specific immunotherapy (group B), while PBS (0.01 mol/L) was given in group C to serve as the control. The pathological changes of lung tissues were studied by HE stain. The serum OVA specific IgE level, and IL-2 and IL-4 production in the splenic T cells were determined by enzyme-linked immunosorbent assay (ELISA). The expression of CD(80) and CD(86) on DC, which were isolated from the spleen, were detected by fluorescein-activated cell sorter. (3)H-thymidine ((3)H-TdR) incorporation assay was used to determine the response of splenic T and the secretion of interleukin 4 (IL-4) and IL-5 was assayed with the use of ELISA after coculture of T cells with DC. RESULTS: (1) The infiltration of eosinophils and lymphocytes within epithelium and lamina propria was present in group B, but was milder than that in group A. The absorbance at 490 nm (A(490)) of serum specific IgE in group A 712 +/- 129, was more than that in group C (20 +/- 13, P < 0.05), but there was no significant difference between group B (124 +/- 59, P > 0.05) and C. The production of IL-2 by splenic T cells in group B (8 +/- 3) pg/ml was less than that in group A [(22 +/- 8) pg/ml, P < 0.05], but there was no significant difference between group B and group C [(6 +/- 4) pg/ml, P > 0.05]. The production of IL-4 by splenic T cells in group B (8.4 +/- 4.3) pg/ml was less than that of group A [(32.4 +/- 12.1) pg/ml, P < 0.05], but there was no significant difference between group B and group C [(5.1 +/- 1.1) pg/ml, P > 0.05]. (2) The expression of CD(86) on splenic DC cells from group B (58.23%) was significantly lower than that from group A (77.59%) and group C (77.84%), while the expressions of CD(80) in group A (96.98%) and group B (95.63%) were higher than that in group C (77.37%). (3) When untreated T cells were cocultured with DC from the individual groups and stimulated by OVA, the secretion of IL-4 from T cells in group B [(10.8 +/- 2.3) pg/ml] was significantly lower than that in group A [(17.3 +/- 4.7) pg/ml, P < 0.05], but there was no significant difference when compared to group C [(5.7 +/- 2.7) pg/ml, P > 0.05], and the secretion of IL 5 from T cells in group B [(18.8 +/- 3.8) pg/ml] was significantly lower than that in group A [(35.7 +/- 7.9) pg/ml, P < 0.05], but there was no significant difference when compared to group C [(11.0 +/- 2.2) pg/ml, P > 0.05]. When untreated T cells were cocultured with DC from the individual groups and stimulated by OVA, the stimulating index of T cells in group B (3.8 +/- 0.7) was significantly lower than that in group A (11.5 +/- 3.2, P < 0.05), but there was no significant difference when compared to group C (5.8 +/- 1.5, P > 0.05). CONCLUSIONS: A mouse model of asthma-specific immunotherapy was successfully established. Downregulation of CD(86) on the surface of DC might be the underlying mechanisms by which specific immunotherapy promotes Th1 to Th2 polarization and induces T cell anergy. PMID- 16677481 TI - [Imbalanced T cell-specific transcription factors T-bet and GATA-3 and relationship to airway inflammation in asthmatic rats]. AB - OBJECTIVE: To identify the change of the mRNA and protein expression of T-bet and GATA-3 in lung tissues, and to investigate the association between the imbalanced T cell-specific transcription factors T-bet/GATA-3 and the airway inflammation in asthmatic rats. METHODS: Twenty-four male SD rats were randomly divided into a control group and an asthmatic group. Airway responsiveness was measured and the change of airway histology was observed. The concentrations of interleukin-4 (IL 4), IL-5, and interferon-gamma (IFN-gamma) in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of IL-4, IL-5, IFN-gamma, T-bet and GATA-3 in the lungs were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. RESULTS: The expiration resistance after injection of acetylcholine chloride (20, 40, 80, 160 microg/ml) in the asthmatic group was (6.26 +/- 0.85), (11.55 +/- 3.09), (28.74 +/- 5.94), (3,710.83 +/- 197.49) cm H2O.ml(-1).s(-1) respectively; and that in the control group was (1.51 +/- 0.18), (2.15 +/- 0.36), (6.08 +/- 1.06), (37.17 +/- 6.12) cm H2O.ml(-1).s(-1) respectively; the difference being significant between the two groups (all P < 0.01). In the asthmatic group, the numbers of eosinophils and lymphocytes, the thicknesses of WA/Pi and ASM/Pi were (26.0 +/- 1.6)/mm(2), (45.2 +/- 3.2)/mm(2), 12.0 +/- 1.4, 6.7 +/- 0.6, respectively; and those of the control group were (2.9 +/- 1.2)/mm(2), (8.8 +/- 1.8)/mm(2), 6.4 +/- 0.8, 2.7 +/- 0.5, respectively; all were significantly different between the two groups (all P < 0.01). In the asthmatic group, the concentrations of IL-4, IL-5, and IFN-gamma in BALF were (23.4 +/- 0.7) pg/ml, (24.8 +/- 0.5) pg/ml, (21.7 +/- 1.1) pg/ml, respectively, and those of the control group were (9.3 +/- 0.3) pg/ml, (12.5 +/- 0.3) pg/ml, (65.8 +/- 2.1) pg/ml, respectively; all were significantly different between the two groups (all P < 0.01). In the control group, the mRNA ratio of T-bet to GATA-3 (0.73 +/- 0.32) was significantly increased compared with the asthmatic group (0.06 +/- 0.09, P < 0.01). There was also a significant difference in the ratio of protein expression of T-bet to GATA-3 between the control group (0.75 +/- 0.25) and the asthmatic group (0.09 +/- 0.04, P < 0.01). The ratio of protein expression of T bet and GATA-3 was correlated negatively with expiration resistance (r = -0.959, 0.919, -0.949, all P < 0.01), the numbers of eosinophils and lymphocytes in lung tissues (r = -0.832, -0.831, all P < 0.01), the thicknesses of WA/Pi and ASM/Pi (r = -0.837, -0.863, all P < 0.01) and the concentrations of IL-4, IL-5 in BALF (r = 0.921, 0.920, all P < 0.01), the mRNA of IL-4, IL-5 in lung tissues (r = 0.964, -0.931, all P < 0.01), but positively with the concentrations of IFN-gamma in BALF and the mRNA of IFN-gamma in lung tissues (r = -0.934, 0.983, all P < 0.01). CONCLUSION: Imbalance of transcription factors T-bet and GATA-3, a reflection of the immune imbalance in asthma, may play a key role in the formation of airway inflammation in the disease. PMID- 16677482 TI - [The relationship between mouth pressure or tracheal pressure and esophagus pressure and diaphragmatic pressure]. AB - OBJECTIVE: To investigate the relationship between mouth pressure (Pmo) or tracheal pressure (Ptr) and esophagus pressure (Pes) or transdiaphragmatic pressure. METHODS: Seventeen patients were involved in the study. Maximal inspiratory pressure (MIP), maximal transdiaphragmatic pressure (Pdi(max)), maximal esophagus pressure (Pes(max)), twitch mouth pressure (TwPmo), twitch transdiaphragmatic pressure (TwPdi) and twitch esophagus pressure (TwPes) were measured before narcotization as a normal procedure for the abdominal operation and twitch tracheal pressure (TwPtr(nar)), twitch esophagus pressure (TwPes(nar)) and twitch transdiaphragmatic pressure (TwPdi(nar)) were dynamically monitored during narcotization. RESULTS: (1) The correlation coefficient (r) values between Pdi(max) and MIP, TwPdi and TwPmo, TwPdi(nar) and TwPtr(nar), Pes(max) and MIP, TwPes and TwPmo, TwPes(nar) and TwPtr(nar) were 0.976 +/- 0.030, 0.816 +/- 0.155, 0.923 +/- 0.446, 0.981 +/- 0.185, 0.829 +/- 0.168 and 0.955 +/- 0.292, respectively. (2) The coefficient variation (CV) of MIP, Pes(max), Pdi(max), TwPmo, TwPes and TwPdi were (14.2 +/- 4.7)%, (15.2 +/- 4.3)%, (15.5 +/- 4.1)%, (30.4 +/- 15.9)%, (10.8 +/- 5.1)% and (9.9 +/- 4.0)%, respectively. The CV of TwPmo was the highest (compare with others, all P < 0.05) and that of TwPes and TwPdi was the lowest (compare with others, all P < 0.05). There was no significant difference among MIP, Pes(max) and Pdi(max) (P > 0.05). (3) The r value between the changing values of TwPtr(nar) and TwPdi(nar) or TwPes(nar) during narcotization were 0.839 or 0.894 (P = 0.000, respectively). CONCLUSION: The measurement of MIP and TwPmo should be repeated and the highest value should be chosen in order to reduce the possibility of underestimating the function of diaphragm, which could be dynamically monitored by TwPtr(nar). PMID- 16677483 TI - [The expression and significance of urotensin II, nitric oxide, and C-type natriuretic peptide in hypoxia-induced pulmonary hypertension in rats]. AB - OBJECTIVE: To investigate the alteration of plasma and lung tissue homogenate urotensin II (U-II), nitric oxide (NO) and C-type natriuretic peptide (CNP) levels in rats with hypoxia-induced pulmonary hypertension (HPH) and to study the role of these factors and oxygen treatment in the development of HPH. METHODS: Thirty male Wistar rats were randomly divided into three groups: a control group, a group with hypoxia for 14 days and a group with oxygen treatment after hypoxia for 14 days, 10 rats per group. Mean pulmonary arterial pressure (mPAP), mean systematic arterial pressure (mSAP) and right ventriculo hypertrophy index (RVHI) were measured. The plasma levels of U-II, NO, CNP, and the lung tissue homogenate levels of U-II and CNP were measured. The structure of the pulmonary arteries was examined using optical microscope. The microstructure and ultrastructure changes in pulmonary small arteries were examined using electron microscope. RESULTS: The mPAP and RVHI in the hypoxia group [(34.1 +/- 2.8) mm Hg, 0.43 +/- 0.11] were higher than those in the control group [(18.9 +/- 2.0) mm Hg, 0.28 +/- 0.04, all P < 0.01]. The plasma levels of U-II, NO, and CNP [(4.4 +/- 0.9) pg/ml, (20.8 +/- 7.0) micromol/L, (6.6 +/- 1.2) pg/ml], and the lung tissue homogenate levels of U II and CNP [(6.3 +/- 0.5), (1.89 +/- 0.43) pg/ml] in the hypoxia group were higher than those in the control group [(0.9 +/- 0.4) pg/ml, (13.2 +/- 2.0) micromol/L, (4.0 +/- 0.6) pg/ml, (2.6 +/- 0.5) pg/ml, (0.69 +/- 0.21) pg/ml, respectively, all P < 0.01]. Compared with the hypoxia group, the mPAP, the plasma levels of U-II, NO, and CNP, and the lung tissue homogenate levels of U-II and CNP in the oxygen treatment group [(31.4 +/- 2.0) mm Hg, (2.1 +/- 0.7) pg/ml, (14.8 +/- 1.7) micromol/L, (4.4 +/- 0.7) pg/ml; (3.5 +/- 0.8) pg/ml, (0.74 +/- 0.17) pg/ml, respectively] were significantly decreased (all P < 0.01). The pulmonary vessel morphology changes of the oxygen treatment group were ameliorated. CONCLUSIONS: U-II, NO and CNP are involved in the pathophysiologic process of HPH. Imbalance of these factors may be partially responsible for the development of the disease. PMID- 16677484 TI - [Investigation of proximal femoral marrow with magnetic resonance imaging in recovered patients with severe acute respiratory syndrome]. AB - OBJECTIVE: To determine the effect of corticosteroid treatment and severe acute respiratory syndrome (SARS) infection itself on the marrow conversion in the proximal femoral marrow in the recovered patients of health care workers with SARS. METHODS: The distribution of proximal femoral marrow on MR imaging of 148 health care workers with recovered SARS including 106 cases treated with varied dosage of steroids and 42 cases without steroid, and 97 age and sex matched health adults as controls were observed. The index of marrow conversion (signal intensity of the femoral neck and intertrochanteric area divided by signal intensity of the greater trochanter multiplied by 100%) was quantitatively measured and compared between SARS patients treated with steroid and without steroid and the normal control group. RESULTS: In 106 cases treated with steroid of the 148 health care workers, femoral head osteonecrosis was found in 4 cases, bilateral femoral marrow edema in 2 cases, femoral marrow infarction in 1 case. The index of marrow conversion in the normal controls, in SARS patients treated without and with steroids was (79.4 +/- 6.8)%, (86.9 +/- 7.4)%, (88.6 +/- 5.9)%, respectively. There was significantly statistical difference between groups (P < 0.05). CONCLUSION: In addition to fat conversion in the proximal femoral marrow due to steroid treatment, the infection of SARS itself may promote an excessively conversion of the red marrow to yellow marrow of the femoral neck in SARS patients. PMID- 16677485 TI - [An epidemic outbreak of respiratory infection caused by Chlamydia pneumoniae in medical workers]. AB - OBJECTIVE: To investigate the clinical manifestations and the chest imaging characteristics of an epidemic outbreak of respiratory infection caused by Chlamydia pneumoniae (CP). METHODS: A prospective study for CP infection in 15 patients from September 2003 was carried out. Sputum and throat swab specimen were obtained and CP DNA was detected by polymerase chain reaction (PCR). Serum samples were obtained and immunoglobulin G and M (IgG and IgM) of antibodies to CP. pneumoniae were studied by microimmunofluorescence test. Chest X-ray and computed tomography were retrospectively analyzed. RESULTS: All patients presented fever, headache, sore throat, hoarseness, muscular ache, and dry cough. Acute cough was often associated with chest pain. The sputum blood was present in 3 patients (20%). Moist rales were heard in 4 patients. Chest imaging abnormalities were present in 67% (10 patients). The organism was demonstrated in 87% (13 patients) by PCR. The most common imaging abnormalities were unilateral and (or) bilateral multi-focal or solitary alveolar nodular opacities (9 patients). The patchy shadows were found in 2 patients, and pulmonary consolidation associated with the local pulmonary edema in 1 patient. Hilar or mediastinal lymphadenopathy and pleural effusion was not found. CONCLUSIONS: The colony occurrences and similar clinical and chest imaging manifestations are characteristics of an outbreak of respiratory infection caused by CP in medical workers. An outbreak of respiratory infection caused by CP should be differentiated from severe acute respiratory syndrome (SARS). PMID- 16677486 TI - [Multidisciplinary approach of minimally invasive treatment of advanced stage of lung cancer]. PMID- 16677487 TI - [Advances in the study of the effects of macrolide antibiotics on Pseudomonas aeruginosa]. PMID- 16677497 TI - [More attention should be paid to the clinical application of computed tomographic (CT) guided technique of interventional radiology]. PMID- 16677498 TI - [Application of computed tomography guided percutaneous fine-needle aspiration biopsy in examination of pancreas and Vater's ampulla]. AB - OBJECTIVE: To evaluate the effects of computed tomography (CT) guided percutaneous fine-needle aspiration biopsy (FANB) in examination of pancreas and Vater's ampulla. METHODS: One hundred and six patients, 71 males and 35 females, aged 21 approximately 74, with lesions located in the pancreas and Vater's ampulla, 1.5 approximately 5 cm in size (< 3 cm in 32 cases and >or= 3 cm in 74 cases), underwent CT guided percutaneous FANB with 22-gauge needle (in 84 cases) or 20-gauge needle (in 22 cases) by anterior approach. The biopsy sites included the head of pancreas and ampulla (n = 70), body of pancreas (n = 24), and tail of pancreas (n = 12). The key points of FANB included selection of appropriate cross section, entry of needle, and multiple insertion in fan-like fashion with fast puncture and aspiration. Pathological examination was done to all biopsy specimens. RESULTS: All lesions were safely accessed with an accuracy rate of 100% and without major complications. Pathological examination showed that 83 lesions were malignant, 5 of which were confirmed as false-positive, and 20 lesions were benign 2 of which were confirmed as false-negative, and 3 lesions were true-negative. The accuracy rates of diagnosis of malignant lesions and of diagnosis of benign lesion were 94% and 90% respectively (P > 0.05). The accuracy rate of diagnosis was 96% for the lesions >or= 3 cm was 96% and 88% for the lesions < 3 cm (P > 0.05). The accuracy rates of diagnosis were 93%, 92%, and 100% for the lesions located in the head of pancreas and ampulla of Vater, body of pancreas, and tail of pancreas (P > 0.05). CONCLUSION: CT guided percutaneous FANB is a safe and effective method for the diagnosis and differential diagnosis of lesions in the pancreas. PMID- 16677499 TI - [CT guided radioactive seed 125I implantation in treatment of pancreatic cancer]. AB - OBJECTIVE: To investigate the clinical value of CT guided radioactive seed (125)I implantation in treatment of pancreatic cancer. METHODS: Twenty-six patients with pancreatic cancer 6.1 cm in size on average, 21 males and 5 females, aged 60 +/- 13 (47 - 73), underwent CT guided radioactive seed (125)I implantation. Of the 26 patients 15 had the symptom of severer abdominal pain and 10 had jaundice. Three of them were in the TNM (pTNM) stage II, 30 in the stage III, and 3 in the stage IV. Treatment plan system was used to design the distribution of the seed (125)I needed. Under CT guidance, the (125)I seeds of (2.2 - 3.3) Mq were implanted into the pancreatic cancer at a distance of 1.0 - 1.5 cm. A follow-up for 3 - 12 months was conducted. RESULTS: Complete relief (CR) of the symptoms was seen in 9 patients, partial relief (PR) in 2 patients, and no change (NC) in 4 cases with an effective rate of 73.4%. The abdominal pain was relieved 3 - 7 days after the implantation. CT performed 2 months after implantation showed CR in 2 cases, PR in 13 cases, NC in 5 cases, and progression in 5 cases with an overall effective rate of 57.7%. Four patients died of local progression and 2 patients died of distant metastasis. Migration of the seeds into the liver was seen in 3 patients and slight decrease of white blood cells was seen in 1 patient during the 2-month follow-up. No severe complications, such as pancreatic leakage, acute pancreatitis, intestinal hemorrhage, etc, were found. CONCLUSION: Safe and with minimal damage and few complications, CT guided radioactive seed (125)I implantation is effective in treatment of pancreatic cancer. PMID- 16677500 TI - [An integral approach for cyanotic congenital heart disease with major aortopulmonary collateral arteries]. AB - OBJECTIVE: To demonstrate the feasibility of combined collateral embolization with surgical repair for tetralogy of Fallot or pulmonary atresia with major aorta pulmonary collateral arteries. METHODS: The clinical, catheterization and surgical data of 15 such patients from November 1992 to September 2003 were analyzed retrospectively. RESULTS: All 15 patients underwent combined therapy of major-aorto pulmonary collateral arteries (MAPCAs) occlusion and complete repair with a mortality of 26.7%. Of the 46 collateral arteries, 35 were occluded with a successful rate of 90.6%. Embolization was performed before corrective surgery in 12 patients. The causes of early death in three patients were low cardiac output syndrome, pulmonary hemorrhage, and pulmonary infarction respectively. Complications included extensive pulmonary infiltration in 1 patient and right ventricular dysfunction in another patient. The other 7 patients recovered smoothly with the arterial oxygen saturation greater than 97% at discharge. Embolization was performed after corrective surgery in 3 patients with postoperative congestive heart failure. One patient died of congestive heart failure. The symptoms and signs of heart failure was significently relieved in the other two cases. CONCLUSIONS: In selective group of patients with dual pulmonary blood supply by both MAPCA and native pulmonary arteries, this combined therapy of collateral embolization and surgical repair simplifies complete surgical operation and enhances surgical results. It is necessary to minimize the error in distinguish sole pulmonary blood supply from dual pulmonary blood supply to avoid postoperative pulmonary infarction. Embolization after surgical correction is an important rescue measurement to congestive heart failure. PMID- 16677501 TI - [Mechanism of reversion of myocardial interstitial fibrosis in diabetic cardiomyopathy by valsartan]. AB - OBJECTIVE: To investigate the mechanism of reversion of myocardial interstitial fibrosis in diabetic cardiomyopathy (DCM) by valsartan. METHODS: Forty male wistar rats were randomly divided into 3 groups: DCM group, n = 16, fed with high fat diet for 4 weeks and injected intraperitoneally with streptozocin (STZ) once to induce hyperglycemia so as to construct a DCM model, and then perfused into the stomach with normal saline; valsartan group, n = 16, to be constructed into DCM model and then perfused into the stomach with valsartan once daily; and control group (n = 8, fed with normal food and perfused into the stomach with normal saline. Four weeks after feeding (i.e., before injection of STZ), 1 week after STZ injection, and by the end of experiment after 12-hour fasting samples of venous blood were collected to detect the contents of triglyceride and fasting blood-glucose and insulin; by the end of experiment miniature cardiac catheter was inserted into the left ventricle to conduct hemodynamic examination. Then myocardium tissues were collected, collagen content was detected by Masson staining, real-time RT-PCR was used to detect the mRNA expression of thrombospondin (TSP)-1 and tumor growth factor (TGF)-beta(1) mRNA, expression, and Western blotting was used to detect the protein expression of TSP-1, active TGF-beta(1) (A-TGF-beta(1)) and latent TGF-beta(1) (L-TGF-beta(1)). RESULTS: By the end of the experiment, the body weights, and insulin sensitivity index were significantly lower and fasting blood-glucose, and serum triglyceride and cholesterol were significantly higher in the DCM group and valsartan group in comparison with those in the control group (all P < 0.01), however, there was no significant differences in fasting insulin among these 3 groups. The values of left ventricular systolic pressure (LVSP) and +/- dp/dt(max) were significantly lower and left ventricular end diastolic pressure were significantly higher in the DCM group in comparison with the control group (all P < 0.01). The LVSP and dp/dt(max) were significantly higher and LVEDP was significantly lower in the valsartan group than in the DCM group (all P < 0.05). The LVEDP was significantly higher and -dp/dt(max) was significantly lower in the valsartan group than in the control group. Electron microscopy showed the distribution of a great amount of collagen in the myocardial interstitial tissue. The collagen content of the DCM group was 17 +/- 3, significantly higher than that of the control group (11 +/- 3, P < 0.05), and the collagen content of the valsartan group was 13 +/- 3, significantly lower than that of the DCM group (P < 0.05). The mRNA expression of TSP-1 and that of TGF-beta(1) were significant higher in the DCM group than in the control group (both P < 0.05), and were significantly lower in the valsartan group than in the DCM group (both P < 0.05); however, the TGF-beta(1) mRNA expression in the valsartan group was significantly higher in the valsartan group than in the control group (P < 0.05). The values of protein expression of TSP-1, A-TGF-beta(1) and L-TGF-beta(1) were all significantly higher in the DCM group than in the control group (all P < 0.05), and the values of protein expression of TSP-1 and A-TGF-beta(1) in the valsartan group were both significantly lower than those in the DCM group (both P < 0.05), however, there was no significant difference in the protein expression of L-TGF-beta(1) between the valsartan group and DCM group. CONCLUSION: Valsartan amelioorates myocardial interstitial fibrosis in DCM via TSP-1/TGF-beta(1) signaling pathway. PMID- 16677502 TI - [Inhibition of nuclear factor kappa B attenuates multiple organ injury following ruptured abdominal aortic aneurysm: an experiment with rats]. AB - OBJECTIVE: To investigate the effect of inhibition of nuclear factor kappa B on multiple organ injury following ruptured abdominal aortic aneurysm. METHODS: Forty-five Wistar rats underwent catheterization to observe the intestinal microcirculation blood flow, and were randomly divided into 3 equal groups. Rats of the ruptured abdominal aortic aneurysm (RAAA) group underwent laparotomy and extraction of blood to cause hemorrhage shock for 1 h (hemorrhagic shock phase), by the end of this phase normal saline at the dose of 50 ml/kg was injected intravenously, after that the abdominal aorta and bilateral common iliac arteries were blocked with artery clamps for 45 min so as to cause lower torso ischemia, and then the extracted blood was reperfused. The lungs, small intestine were taken out to undergo histological examination, and examination of lung polymorphonuclear neutrophilic leukocyte (PMN) sequestration, lung wet tissues/dry (W/D) tissues ratio, and myeloperoxidase (MPO) activity. The rats of pyrrolidine dithiocarbamate (PDTC) group were perfused with PDTC, a specific inhibitor of nuclear factor kappa B (NF-kappaB), by the end of the hemorrhagic shock phase. And the rats of the sham operation group were perfused of normal saline. RT-PCR was used to detect the mRNA expression of NF-kappaB p65 and intercellular adhesion molecule (ICAM). Western blotting was used to detect the protein expression of NF-kappaB p65 and ICAM-1. Immunohistochemistry was used to detect the expression of NF-kappaB p65 and ICAM-1 in the lung and small intestine tissues. RESULTS: Histological examination showed that severe damage could be found in the lung and small intestine of the RAAA group, and damages were significantly mild in the PDTC group. Lung PMN sequestration, W/D ratio, MPO activity were significantly increased in the RAAA group and these changes were relatively mild in the PDTC group (all P < 0.01). The intestinal microcirculation blood flow was 0.25 +/- 0.04 mlxmin(-1)xg(-1) in the RAAA group, significantly less than that of the sham operation group (0.71 +/- 0.11 mlxmin(-1)xg(-1), P < 0.01), and was 0.64 +/- 0.06 mlxmin(-1)xg(-1) in the PDTC group, significantly higher than that of the RAAA group (P < 0.01). The mRNA expression of NF-kappaB p65 in the lung of the RAAA group was 0.68 +/- 0.22, significantly higher than that of the sham operation group (0.11 +/- 0.02, P < 0.01) and that of the PDTC group (0.23 +/- 0.07, P < 0.01). The mRNA expression of NF-kappaB p65 in the intestine of the RAAA group was 0.48 +/- 0.10, significantly higher than that of the sham operation group (< 0.20 +/- 0.05, P < 0.01) and that of the PDTC group (0.27 +/- 0.06, P < 0.01). The mRNA expression of ICAM-1 in the lung of the RAAA group was 0.92 +/- 0.31, significantly higher than that of the sham operation group (0.07 +/- 0.02, P < 0.01) and that of the PDTC group (0.21 +/- 0.04, P < 0.01). The mRNA expression of ICAM-1 in the intestine of the RAAA group was 0.74 +/- 0.15, significantly higher than that of the sham operation group (0.14 +/- 0.05, P < 0.01) and that of the PDTC group (0.25 +/- 0.08, P < 0.01). The protein expression of NF-kappaB p65 in the lung of the RAAA group was 1.04 +/- 0.26, significantly higher than that of the PDTC group (0.52 +/- 0.13, P < 0.01). The protein expression of NF-kappaB p65 in the intestine of the RAAA group was 1.20 +/- 0.30, significantly higher than that of the PDTC group (0.64 +/- 0.21, P < 0.01). The protein expression of ICAM-1 in the lung of the RAAA group was 0.40 +/ 0.12, significantly higher than that of the PDTC group (0.18 +/- 0.06, P < 0.01). The protein expression of ICAM-1 in the intestine of the RAAA group was 0.46 +/- 0.15, significantly higher than that of the PDTC group (0.22 +/- 0.05, P < 0.01). Immunohistochemistry showed that NF-kappaB p65 and ICAM-1 positive cells were widely distributed in the lungs and intestine of the RAAA group and were rarely distributed in the sham operation group. CONCLUSION: PDTC attenuates the multi-organ injury by inhibiting the expression of NF-kappaB p65, thus reducing the mRNA and protein expression of its downstream gene ICAM-1 gene. PMID- 16677503 TI - [Effects of oxidized-low density lipoprotein on expression of urotensin II receptor GPR14 in rat aortic smooth muscle cells]. AB - OBJECTIVE: To investigate the effects of oxidized-low density lipoprotein (ox LDL) and urotensin II (U II) on the proliferation of rat aortic smooth muscle cells (VSMCs) and the effect of ox-LDL on the mRNA expression of U II receptor GPR14 in. METHODS: Rat VSMCs were incubated with UII and ox-LDL at different concentrations. The viability of rat VSMCs was detected by MTT assay. Rat VSMCs were incubated with ox-LDL at the concentrations of 0, 0.1, 1, 10, and 50 microg/ml. Twelve hours later competitive RT-PCR was used to detect the effects of concentration of ox-LDP on the mRNA expression of GPR14 in the VSMCs. Another VSMCs were incubated with ox-LDL at the final concentration of 50 microg/ml, and 0, 2, 6, 12, and 24 hours later competitive RT-PCR was used to detect the mRNA expression of GPR14 in the VSMCs. (125)I-labelled U II was added into the culture fluid of VSMCs, and radioligand binding assay was used to calculate the maximum binding (B(max)). RESULTS: UII of the concentration of 50 nmol/L significantly increased the proliferation of VSMCs. When the concentration of ox-LDL was 10 microg/ml the proliferation of VSMCs (P < 0.01) was 0.678 +/- 0.061, increased by 121% compared with the control group (0.325 +/- 0.052, P < 0.01). 0.01 microg/ml of ox-LDL and 10 nmol/L of UII showed a synergistic effect on the proliferation of VSMCs with the MTT value increased to 162% +/- 29% that of the control group (P < 0.01). When the concentration of ox-LDL increase to 0.1 microg/ml the synergistic effect was attenuated with the MTT value of 153% +/- 22% that of the control group (P < 0.05) and when the concentration of ox-LDL increased to 1 microg/ml the synergistic effect diminished, with a MTT value of 123% +/- 13% that of the control group (P > 0.05). When the concentration of ox-LDL was 50 microg/ml the proliferation of VSMCs the MTT value decreased to 59% that of the of control group, however, ox-LDL of the concentration of 50 microg/ml combined with UII of the concentration of 10 nmol/L the MTT value increased to 68% that of the control group, showing that UII of that concentration significantly inhibited the cytotoxic effect of ox-LDL. Incubation of VSMCs with the ox-LDL at the concentration of 0.1 microg/ml the GPR14 mRNA expression of the VSMCs was 125.1% that of the control group (P < 0.01) as assessed by competitive RT-PCR, however, when the concentration of ox-LDL increased over 1 microg/ml the GPR14 mRNA expression does-dependently decreased as much as 72.6% that of the control group (all P < 0.01), with the maximal effect when the concentration of ox-LDL reached 50 microg/ml. ox-LDL at the concentration of 50 microg/ml time-dependently down regulated the GPR14 mRNA expression with the maximal effect 12 hours after addition of ox-LDL and this effect was sustained for up to 24 hours. After incubation for 12 hours the binding of (125)I-UII to VSMCs was 117.5% that of the control group (P < 0.01) with the ox-LDL at the concentration of 0.1 microg/ml, and was 68.8% that of the control group (P < 0.01) with the ox-LDL at the concentration of 50 microg/ml. CONCLUSION: ox-LDL and with U-II. GPR14 at certain concentrations show synergetic effects on the proliferation of VSMCs. The GPR14 mRNA expression in VSMCs can be upregulated by low concentration of ox-LDL, while downregulated by high concentration of ox-LDL. PMID- 16677504 TI - [Expression and significance of caudal type transcription factor-2 in gastric mucosa at different stages of gastric carcinogenesis]. AB - OBJECTIVE: To investigate the expression and significance of caudal type transcription factor-2 (Cdx2) in gastric mucosa at different stages of carcinogenesis. METHODS: By using ABC immunohistochemistry, the expression of Cdx2 was detected in the biopsy specimens of 104 cases of chronic superficial gastritis, 98 cases of chronic atrophic gastritis, 112 cases of intestinal metaplasia, 106 cases of dysplasia and 97 cases of intestinal-type gastric carcinoma. RESULTS: Cdx2 was expressed in the specimens of intestinal metaplasia with an expression rate of 86.6% (97/112), significantly higher than those in dysplasia (58.5%, 62/106) and intestinal-type gastric carcinoma (56.9%, 53/93) (both P < 0.01), while was not expressed in the specimens of chronic superficial gastritis and chronic atrophic gastritis. No significant difference was found between the expression rate of Cdx2 was between the specimens of dysplasia and those of intestinal-type gastric carcinoma (P > 0.05). CONCLUSION: Cdx2 may be a novel marker of intestinal metaplasia and may play an important role in the carcinogenesis of the intestinal-type gastric carcinoma. PMID- 16677505 TI - [Effects of gastrin receptor antagonist and cyclooxygenase-2 inhibitor on proliferation and apoptosis of gastric cancer cell]. AB - OBJECTIVE: To investigate the regulative roles of the gastrin receptor antagonist proglumide and the specific cyclooxygenase (COX)-2 inhibitor NS-398 on the proliferation and apoptosis of gastric cancer cells. METHODS: Human gastric cancer cells of the line MKN-45 were routinely cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum. Subconfluent cell cultures were treated with proglumide at a final concentration of 5 mmol/L, NS 398 at a final concentration of 10.0 micromol/L, or proglumide in combination with NS-398 for 48 h. The growth and proliferation of MKN-45 cells were analyzed with MTT assay. Flow cytometric analysis was used to detect the apoptosis of the gastric cancer cells. RT-PCR and Western blotting were used to detect the expression of apoptosis-inhibited gene bcl-2 mRNA and protein. RESULTS: The apoptosis rates of the cells treated by proglumide, NS-398, and combination of two agents were 24.7% +/- 3.2%, 26.7% +/- 3.4%, and 36.1% +/- 4.6% respectively, all significantly higher than that in the control group (1.6% +/- 0.6%, all P < 0.01). The apoptosis rates of the MKN-45 cells treated with proglumide combined with NS-398 was significantly greater than those of the cells treated by the two agents alone (both P < 0.05). Treatment with proglumide and NS-398 significantly reduced the bcl-2 mRNA and protein expression in the MKN-45 cells (P < 0.05). CONCLUSION: Both proglumide and NS-398 inhibit the proliferation and induce the apoptosis of human gastric cells. This apoptosis may be mediated by down regulation of the expression of apoptosis-inhibited gene bcl-2. Co-treatment with proglumide and NS-398 have synergistic anticancer role. PMID- 16677506 TI - [Effects of Helicobacter pylori infection on neural expression of stomach and spinal cord: an experiment with mice]. AB - OBJECTIVE: To investigate the effects of Helicobacter pylori (Hp) infection on neural expression in stomach and spinal cord, and to investigate the mechanism of functional dyspepsia after Hp infection. METHODS: Thirty-five female C57BL/6 mice were randomly divided into three groups: Group A (acute infection group, undergoing intragastric gavage of Hp suspension every other day for 3 times and then observed for 2 weeks, 15 mice), Group B (chronic infection group, undergoing intragastric gavage of Hp suspension every other day for 3 times and then observed for 2 weeks, 15 mice) and control group (undergoing intragastric gavage of normal saline every other day for 3 times and then observed for 2 weeks, 5 mice). After the observation the mice were killed and their stomachs were taken out to undergo gastric histology and bacterial colonization by HE staining and Warthin-Starry staining respectively. Their spinal cords of thoracic and lumbar segments were taken out too. Immunohistochemistry was used to detect the expression of Fos, vasoactive intestinal polypeptide (VIP), and calcitonin gene related peptide (CGRP) in the stomach and spinal cord. RESULTS: Three mice died 12 weeks after Hp infection. The rate of Hp colonization, mainly localized in pyloric gland region, was greater in Group B than in Group A, and was 0 in the control group. The severity of inflammation as shown by mononuclear cell infiltration, and activity of inflammation as shown by polymorphonuclear cell infiltration, in the pyloric gland region, proventriculus-glandular stomach region, and corpus gland region were more pronounced in Groups A and B, especially in Group B, than in the control group. The expression values of Fos, VIP, and CGRP in the stomach of Group A were 3.1 +/- 1.4, 4.5 +/- 1.8, and 2.4 +/ 0.8 respectively, all not significantly different from those of Group B (3.1 +/- 1.3, 3.5 +/- 1.6, and 2.2 +/- 0.8, all P > 0.05). The expression values of Fos, VIP, and CGRP in the spinal cord of Group A were 3.8 +/- 1.2, 3.2 +/- 1.5, and 2.2 +/- 0.6, all not significantly different from those of Group B (3.4 +/- 0.7, 2.6 +/- 1.2, and 2.5 +/- 1.1, all P > 0.05 for all). However, the neural expression values in both acute and chronic infection groups were significantly higher than those in the control group (2.4 +/- 0.9, 1.6 +/- 0.9, and 1.2 +/- 0.8 in stomach; and 2.0 +/- 1.6, 1.2 +/- 1.1, and 1.2 +/- 1.1 in spinal cord, P < 0.05 for all). CONCLUSION: Hp infection, both acute and chronic, induces gastric histological changes such as inflammation and activity, and enhances the Fos, VIP, and CGRP expression in stomach and spinal cord, which can be a basis for symptom generation in dyspeptic patients with Hp infection. PMID- 16677507 TI - [pcDPG parathyroid hormone gene therapy of hypoparathyroidism: an experimental study]. AB - OBJECTIVE: To construct recombination eukaryote expression plasmid for human parathyroid hormone (PTH) gene, assay PTH expression and biological activity after transfection in vitro and evaluate gene therapy effect on hypoparathyroidism (HPT). METHODS: (1) PTH gene was amplified from human embryonic parathyroid gland tissue, and plasmid pcDNA3.1-PTH-GFP (pcDPG) was constructed by TOPO recombination technique. Digestion, PCR and sequencing were used to identify the positive vectors. (2) pcDPG was transformed into 293 cells by Lipofectamine 2000(TM), fluorescent inverted microscope was used to observe GFP expression, and PTH gene expression was assayed by RT-PCR technique. (3) PTH protein in supernatant was purified and evaluated biological activity. (4) HPT rabbit models were developed and plasmid pcDPG was injected in skeletal muscles, respectively. Serum calcium, phosphonium and PTH were assayed and pathological changes observed. RESULTS: (1) The findings in digestion and PCR were accorded to anticipation and sequences in report were identified to reference at 99.30%. (2) 24 h after transfection GFP expression could be detected and enhanced with time prolonged arriving to 38.91% and 62.45% at 48 h. PTH gene expression could be detected by RT-PCR. (3) Purified PTH protein made the signs of HPT disappear. (4) Serum calcium and PTH levels were lower than those of pre-operation (P < 0.05) and serum phosphonium enhanced to normal standard 48 h after treatment at the plasmid pcDPG doses of 300 microg/kg and 500 microg/kg. CONCLUSION: Recombination plasmid pcDPG was transformed effectively in vitro and the transfected cells produced PTH protein with biological activity. Besides, the satisfactory therapeutic effect of HPT rabbits was attained by pcDPG plasmid, which provided a foundation for further study of HPT gene therapy. PMID- 16677508 TI - [Effects of inhibition of cyclooxygenase-2 by RNA interference on proliferation and apoptosis of human gastric cancer cells: an experimental study with human gastric cancer cells and mice]. AB - OBJECTIVE: To study whether the expression level of cyclooxygenase-2 (COX-2) is correlated with the proliferation and apoptosis of cancer cells and to study whether the RNA interference technique can be used in anti-cancer gene therapy. METHODS: WBH1, a eukaryotic expression plasmid of shRNA targeting on COX-2, was constructed. Human gastric cancer cells of the line SGC-7901 were cultured and divided into 3 groups: to be transfected with WBH1 or negative control plasmid HK, or used as un-transfected control group. RT-PCR and Western blotting were used to detect the expression of COX-2 mRNA and protein. MTT method was used to detect the proliferation of the cells. The apoptosis of the cells was determined by flow cytometry. Fifteen nude mice were randomly divided into 3 equal groups: 10 to be inoculated subcutaneously with WBH1 plasmid transfected SGC-7901 cells (inhibition group) or negative control plasmid HK transfected SGC-7901 cells, and 5 were used as un-transfected controls. The mice were observed for 4 weeks to observe the survival and the tumorigenesis. Then the mice were killed to take out the tumors. The tumorigenic rate and tumor inhibition rate were evaluated. RESULTS: The proliferation of the SGC-7091 cells transfected with WHB1 plasmid did not changed significantly 24 and 48 hours after the transfection, however, decreased significantly 96 hours and 1 week after (both P < 0.01). The apoptotic rate of the SGC-7091 cells transfected with WHB1 plasmid was 52.28% +/- 17.91%, significantly higher than that of the cells transfected with the control plasmid HK (0.54% +/- 0.16%) and that of the un-transfected cells (0.52% +/- 0.27%, both P = 0.009) without a significant difference between the latter 2 groups (P = 0.998). Four weeks after inoculation the tumorigenic rate was 100% in both the un transfected control mice and the mice inoculated with negative plasmid HK transfected SGC-7901 cells. There was no significant difference in tumor size between these 2 groups (P = 0.965). The tumorigenic rate of the mice in the inhibition group was 0.4 with an inhibition rate of 89.8%. The tumor weight of the inhibition group was 0.050 g +/- 0.003 g, significantly lighter than those of the control group and the group inoculated with negative plasmid transfected SGC cells (0.490 g +/- 0.017 g and 0.490 g +/- 0.013 g respectively, both P < 0.01). CONCLUSION: Construction of a eukaryotic expression vector expressing the specific shRNA targeting on COX-2, closely related to the proliferation and apoptosis of tumor cells, and transfection of it into the tumor cells helps inhibit the expression of COX-1, thus inhibiting the growth and proliferation of the tumor cells. PMID- 16677509 TI - [The trend of clinical characteristics of colorectal cancer during the past 20 years in Guangdong province]. AB - OBJECTIVE: To study the trend of clinical characteristics of colorectal cancer during the past 20 years in Gaungdong province. METHODS: The clinical data of 3870 cases of colorectal cancers confirmed pathologically who were hospitalized to Nanfang Hospital and Huizhou Municipal Hospital, Guangdong province, 1985 - 2004 were divided into 5-year groups and analyzed. RESULTS: The hospitalization number of colorectal cancer in the 2000 - 20004 5-year period was 102%, higher than that of the 1985 - 1989 period with an annual increase of 5.1%. The median age of incidence 2000 - 2004 was 58.6 years, 8.4 years higher than that in 1985 - 1989. The male to female ratio of the 3870 patients was 1.42:1. The male to female ratio 2000 - 2004 was 1.35:1, lower than that 1985 - 1989 (1.50:1). The proportion of rectal cancer 2000 - 2004 was 49.7%, significantly lower than that in 1985 - 1989 (64.8%), while the proportion of right hemi-colon cancer 2000 - 2004 was 28.7%, significantly higher than that in 1985 - 1989 (18.0%). The proportion of moderately and well differentiated cancer 2000 - 2004 was 80.6%, higher than that in 1985 - 1989 (70.1%), and the proportion of poorly differentiated cancer 2000 - 2004 was 19.4%, lower than that in 1985 - 1989 (29.9%). The proportion of colorectal cancer at Dukes A stage 2000 - 2004 was 9.8%, higher than that in 1985 - 1989 (3.2%). CONCLUSION: In the past 20 years, the incidence of colorectal cancer has increased in Guangdong province with a n increase of median age of incidence, The male to female ratio has decreased, and the incidence of right hemi-colon cancer, the rates of higher differentiated cancer and Dukes A stage cancer have increased. PMID- 16677516 TI - [The strategy in management of cervical intraepithelial neoplasia]. PMID- 16677510 TI - [Value of Hounsfield unit on CT in prediction of stone-free rate of upper urinary calculi after extracorporeal shockwave lithotripsy]. AB - OBJECTIVE: To study the value of Hounsfield unit (HU) on CT in prediction of stone-free rate of upper urinary calculi after extracorporeal shockwave lithotripsy (ESWL). METHODS: Fifty-two patients suffering from solitary stone in the upper urinary tract 0.5 - 1.5 cm in size, 34 males and 18 females, aged 15 - 77, underwent non-contrast helical computerized tomography (NCHCT), and then ESWL. One day, 2 weeks, 4 weeks, and 3 months after the ESWL the patients underwent kidney, ureter, and bladder examination and B mode ultrasonography. The patients with the residual fragments less than 3 mm in size were categorized into the stone-free group. The relationship among the HU, number of shock wave, and the characteristics of the patients were analyzed. RESULTS: Thirty-six of the 52 patients were categorized into the stone-free group (69.2%) and 16 of the 52 were categorized into the residual stone group (30.8%). The CT value of the stone-free group was 579.65 +/- 194.65 HU, significantly lower than that of the residual stone group (1032.18 +/- 270.49 HU, t = 6.842, P < 0.01). The number of shock wave of the stone-free group was 1526.67 +/- 723.01, significantly lower than that of the residual stone group (2612.50 +/- 518.81, t = 5.407, P < 0.01). The OR values for the HU and number of shock wave were 0.993 (95% CI: 0.988 - 0.998) and 0.998 (95% CI: 0.997 - 1.000). CONCLUSION: The HU value on pretreatment NCHCT of the upper urinary tract stones can be used to predict the stone-free rate after ESWL. ESWL is not indicated to the patients of upper urinary tract stones with high values of HU. PMID- 16677517 TI - [Choices among the iatrotechnics for cervical intraepithelial neoplasia]. PMID- 16677520 TI - [Cervical carcinoma in situ: a clinical and pathological analysis of 118 cases]. AB - OBJECTIVE: To analyze the clinical characteristics and treatment of cervical carcinoma in situ. METHODS: The clinical data of 118 patients with cervical carcinoma in situ (CIS), aged 38.8 (24-77), were retrospectively analyzed. RESULTS: Cervical cytology showed that most of the lesions were cervical intraepithelial neoplasm (CINIII), high-grade squamous intraepithelial lesion (HSIL), or CIS. Most of the patients underwent colposcopic examination. All the patients underwent conization. 54% of the patients were given cold knife conization, and 46% of the patients received electric knife conization. 71 of the patients undergoing conization (60%) were followed-up. Another 40% received total hysterectomy and/or bilateral salpingo-oophorectomy. 17 cases were found pregnant after the conization. There were no differences in pathologically thorough resection and operational complication between the two surgery methods: cold knife conization and electric knife conization. Follow up study showed that conization had no effect on the subsequent pregnancy. CONCLUSION: Conization is necessary for the final diagnosis of cervical carcinoma in situ. Both cervical cytology and colposcopic examination are very important for screening of CINIII or CIS. For the young patients conization suffices, however, for the postmenopausal women or those unable to receive follow-up examination total hysterectomy and bilateral salpingo-oophorectomy should be considered. PMID- 16677521 TI - [The accuracy of diagnosing cervical intraepithelial neoplasia with colposcopically directed biopsy]. AB - OBJECTIVE: To evaluate the accuracy of colposcopically directed biopsy in the diagnosis of cervical intraepithelial neoplasia (CIN). METHODS: The clinical data of 153 patients with CIN diagnosed by colposcopically directed biopsy and treated with cervical loop electrosurgical procedure (LEEP) shortly after were retrospectively studied. The consistency of pathological examination between colposcopically directed biopsy and LEEP was evaluated. The factors of missed diagnosis of cervical invasive carcinoma were analyzed. RESULTS: The diagnosis of 106 out of the 153 patients by colposcopically directed biopsy was consistent with that by LEEP as confirmed by pathologic diagnosis with a consistence rate of 69.3%, however, the diagnoses of 47 of the 153 patients by colposcopically directed biopsy were not consistent with those by LEEP, with an inconsistent rate of 30.7%, including 22 cases of overdiagnosis (14.4%, 22/153) and 25 cases of underdiagnosis (16.3%, 25/153). Eighteen patients were confirmed as with cervical invasive carcinoma by LEEP/hysterectomy at last with a missed diagnosis rate of 11.8%. The missed diagnosis rate of invasive carcinoma by colposcopically directed biopsy was significantly higher in the patients with cytologic diagnosis of with > or = HSIL than in the patients cytologically diagnosed as with < HSIL (chi(2) = 8.208, P < 0.05). CONCLUSION: The accuracy of diagnosing CIN with colposcopically directed biopsy is still unsatisfying. Attention should be paid on the patients with cytological diagnosis of > or = HSIL so as to avoid missed diagnosis of cervical carcinoma. PMID- 16677522 TI - [Evaluation of gene chip technology for high risk type human papillomavirus in cervical cancer screening]. AB - OBJECTIVE: To investigate the clinical value of gene chips technology for human papillomavirus (HPV) in cervical cancer screening. METHODS: A population-based cross-sectional screening study was conducted among 1137 women aged 15-59 in a community, Shenzhen city. Hybrid capture 2 (hc2) and gene chip technology were performed to examine the high risk type human papillomavirus in the exfoliated cervical cells. Liquid-based cytology test (LCT) was also performed at the same time. The HPV-positive women with LCT > or = atypical squamous cells of undetermined sign (ASCUS) and the HPV-negative women with LCT > or = low grade squamous intraepithelial lesion (LSIL) underwent biopsy under colposcopy. The pathological results were used as the gold standard to evaluate the two HPV test methods. RESULTS: Totally 122 biopsy specimens were obtained. Pathological examination showed no cervical cancer case, 3 cases of grade III cervical intraepithelial neoplasia (CIN), 11 cases of grade II CIN, 36 cases of grade I CIN, 69 cases of chronic cervicitis and metaplasia of squamous epithelium, and 3 cases of normal cervix. The HPV-positive rate was 14.0% by hc2 and 9.8% by gene chips with a HPV-positive rate by hc2 higher than that by gene chips (P < 0.001) and an mediocre accordance rate between these methods (kappa = 0.498). The. HPV positive rate increased along with the increase of the grade of cervical lesions (P < 0.05). The sensitivity, specificity, accuracy, positive prevalue, negative prevalue, positive likelihood ratio and negative likelihood ratio of hc2 for high risk HPV were 100%, 87.1%, 87.3%, 8.8%, 100%, 7.7 and 0.000, respectively; and those of gene chips were 78.6%, 91.1%, 90.9%, 9.9%, 99.7%, 8.8 and 0.235 respectively. CONCLUSION: At present hc2 high risk HPV testing is still the better method for cervical cancer screening. Gene chips technology is able to rival hc2 except that its sensitivity for cervical high grade lesions need be improved. PMID- 16677523 TI - [Expression of key cell cycle markers in squamous cell carcinoma of the cervix positive in human papillomavirus: a comparative study between Chinese and Australian populations]. AB - OBJECTIVE: To investigate the patterns of expression of key cell cycle proteins targeting on the human papillomavirus (HPV) sites E6 and E7 in cervical carcinoma. METHODS: Semiquantitative immunohistochemistry was used to determine the expression of the cell cycle regulatory factor p53, retinoblastoma gene product pRb, cyclin-dependent kinase inhibitors p21(CIP1/WAF1) (p21), p16(INK4A) (p21), and p27(KIP1) (p21), and cyclin D1 targeting on the HPV sites E6 and E7 in 73 HPV 16-positive specimens of cervical squamous cell carcinoma, 35 from Australia patients and 38 age, lymph node status, and grade of tumor-matched Chinese patients. RESULTS: There were no significant differences in age, lymph node status, and grade of tumor between the Chinese and Australian groups, however, the number of the patients in advanced stage (>Stage IIa) was greater in the Chinese group than in the Australian group (19:7). The positive rate of p53 in the Australian group was 3%, significantly lower than that in the Chinese group (23%, P = 0.028). The upregulation rate of pRb, p21, and p27 in the Australian group were 56%, 63% and 34% respectively, all significantly higher than those in the Chinese group (89%, 97%, and 89% respectively, P = 0.01, P < 0.001, and P < 0.001). There were no significant differences in the upregulation rate of p16 and cyclin D1 expression rate between the Australian group and Chinese group (97% versus 100%, and 3% versus 12%, both P > 0.05). In the combined data of both groups, the positive rate of p53 was 13%; and the upregulation rates of pRb, p16, p21, p27, and cyclin D1 were 74%, 99%, 81%, 63% and 7% respectively. CONCLUSION: Cervical carcinoma overexpresses pRb, p16, p21, and p27. Tumors from Chinese patients are significantly more likely to be positive in p53 and to have upregulated pRb, p21, and p27 than their Australian counterparts. The molecular pathways to human papillomavirus-induced cervical cancer may be influenced by ethnicity and further investigation is necessary. PMID- 16677524 TI - [Vagal effects on inducibility of atrial fibrillation at different sites of pulmonary veins after autonomic denervation]. AB - OBJECTIVE: To investigate the vagal effects on the inducibility of atrial fibrillation (AF) at different sites of pulmonary vein after autonomic denervation. METHODS: The bilateral cervical vagal trunks of 10 male mongrel dogs were isolated and decentralized. The ansae subclaviae were exposed, ligated, and cut. Needle electrodes were inserted into the subcutaneous tissue of the 4 extremities to record the myocardiogram. Right ventricle electrode was introduced via femoral vein and an electrode with 4 poles was sutured with the right appendage (RAA), left appendage (LAA), left atrium (LA), left superior pulmonary vein (LSPV), right superior pulmonary vein (RSPV), left inferior pulmonary vein (LIPV), and right inferior pulmonary vein (RIPV) respectively. Local burst stimulation (S1S1 = 80 ms, impulse duration = 0.5 ms) was performed on these sites to record the baseline AF inducibility. When sinus cardiac arrest for 2 s or complete atrio-ventricular block occurred programmed bilateral vagal nerves stimulation (VNS) was performed with the frequency of 12.5 Hz, impulse duration of 0.5 ms, and voltage of 5-8 V. Atropine 0.04 mg/kg was dripped intravenously. The changes of AV inducibility were observed. RESULTS: In the baseline state, S1S1 programmed stimulation on all the sites evoked single or multiple atrial premature beats and short runs of atrial tachycardia, only a few sites induced AF. However, S1S1 programmed stimulation combined with VNS significantly increased the frequencies of induced AV at the sites of 4 PVs (P < 0.05, P < 0.01). When atropine was dripped the AV induction rates at all sites did not changed significantly (all P > 0.05). CONCLUSION: Vagal nerve may play an important role in the initiation of AF originating from pulmonary veins. PMID- 16677525 TI - [Hypoxia upregulates the expression of cytoglobin in lung cancer cells]. AB - OBJECTIVE: To explore the characters of expression of cytoglobin in tumor cells after hypoxia. METHODS: Human pulmonary tumor cells of the line A549 were cultured and divided into 4 groups to be cultured under 5% CO2 and 95% air and exposed to 2% O2, 5% CO2, and 93% N(2) for 4, 12, or 24 hours respectively. The distribution of cytoglobin was examined by immunohistochemistry and the expression of cytoglobin was detected by Western blotting. The mRNA level of cytoglobin in the A549 cells was assayed by reverse-transcription PCR. RESULTS: Immunohistochemistry showed that cytoglobin was located in the plasma of the A549 cells, and the staining strength of cytoglobin was enhanced in the hypoxic groups in comparison with the normoxic group. Western blotting showed significantly stronger expression of protein of cytoglobin in the 3 hypoxic groups than in the normoxic group (all P < 0.05). The expression of cytoglobin was upregulated significantly in the hypoxic 12- and 24-hour groups than in the hypoxic 4-hour group (both P < 0.05). The cytoglobin mRNA levels of the 3 hypoxic groups were all significantly higher than that of the normoxic group (all P < 0.05). The cytoglobin mRNA levels of the hypoxic 12- and 24-hour groups were significantly higher than that of the hypoxic 4-hour group (both P < 0.05), however, without a significant difference between the hypoxic 12- and 24-hour groups (P > 0.05). CONCLUSION: Hypoxia upregulates the expression of cytoglobin in tumor cells. PMID- 16677526 TI - [Reliability of direct probing isthmus of axial vertebrae to defining the screw path in atlas during Magerl technique]. AB - OBJECTIVE: To evaluate the accuracy and reliability of atlantoaxial transarticular screw insertion (Magerl's technique) in atlas under lateral fluoroscopic monitoring without anteroposterior view. METHOD: Seventy-five consecutive patients with atlantoaxial instability, 48 males and 27 females, aged 35.1, were treated by Magerl's technique. The screw path in atlas was achieved by direct probing the isthmus of axial vertebrae then identifying its position through the atlantoaxial joint correlation. Postoperative transoral X ray and CT film were used for analysis of the position of the 150 screws in the mass of atlas. Three areas were delimitated in and around the lateral mass of atlas: area A (inside the joint face), area: B (outside the joint face but still in lateral mass), and area C area (outside the lateral mass) so as to analyze the location of screws. RESULTS: Among the 150 screws 139 (92.7%) were located in area A, 8 in area B(1) and 3 in area B(2) (7.3%), and none in area C. All cases got atlantoaxial union 3 months after operation. CONCLUSION: The method of "direct probing" is reliable for identifying the path of screw in atlas. PMID- 16677527 TI - [Supratentorial cavernous angiomas in children]. AB - OBJECTIVE: To discuss the clinical features, treatment, and prognosis of supratentorial cavernous angiomas in children. METHODS: The clinical data of 25 pediatric patients with supratentorial cavernous angioma, 17 boys and 6 girls, aged 9.6 (1-15), were reviewed retrospectively, and the relationship between the duration of epilepsy before surgery and prognosis was analyzed. RESULTS: The 14 patients with preoperative seizures underwent surgery. All (8/8) of the patients suffering from preoperative seizures with an history of less than one year were seizure free after operation, while only half (3/6) of the patients with an history suffering from preoperative seizures with an history of more than one year were seizure free after operation. Ten of the 11 patients with hemorrhage and hemorrhage-related neurological symptoms as the initial symptoms underwent surgery, and no symptoms of re-bleeding and seizure occurred postoperatively. CONCLUSION: Craniotomy for lesionectomy results in good seizure control and re bleeding avoidance. Better results with regard to seizure control are associated with shorter duration of symptoms before surgery. PMID- 16677528 TI - [A new bioassay model of antibiotic susceptibility by corneofungimetry]. AB - OBJECTIVE: To study the value of a new bioassay model of antibiotic susceptibility by corneofungimetry which is more similar to human internal environment. METHODS: Twenty-one healthy adult volunteers, 12 males and 9 females, aged 32 (22 - 41), were randomly divided into two groups to receive itraconazole 200 mg Bid or 200 mg qd or bid for one week. Stratum corneum strippings were taken from the forearm skin once before administration of itraconazole and 1, 4, 7, 10, 14, 21, 28, and 35 days after administration of itraconazole. Spores of selected fungi (Trichophyton rubrum, T. mentagrophytes, Microsporum canis, Candida albicans, C. glabrata and C. tropicalis) were deposited and cultured on the strippings. PAS stain was used after one-week culture to observe the growth of the fungi by computerized-assisted image analysis to measure the area covered by the yeasts and the dermatophyte. RESULTS: All fungi grew on all the stratum corneum strippings with different numbers of hyphae. The areas covered by hyphae and spores decreased gradually after administration of itraconazole, became the smaller on the seventh day (P = 0.01 or P < 0.01), and began to increased since the 10 th day, and became almost the same sizes as those before the administration of itraconazole on the 35 th day. CONCLUSION: Similar to the human internal environment, corneofungimetry is a useful new model of bioassay for antibiotic susceptibility. PMID- 16677539 TI - [Orientation in research of osteoporosis-related genes]. PMID- 16677540 TI - [Significance of bone remodeling in maintenance of bone strength]. PMID- 16677541 TI - [Do the premenopausal daughters of women with postmenopausal osteoporosis have lower peak bone mass?]. AB - OBJECTIVE: To determine whether premenopausal daughters of women with postmenopausal osteoporosis have lower peak bone mass than the daughters of normal women the same age, and to analyze the related risk factors affecting bone mass variation. METHODS: 126 pairs of mother with postmenopausal osteoporosis and her premenopausal daughter, and 136 pairs of normal postmenopausal mother and her premenopausal daughter selected for 410 core families including one healthy premenopausal daughter aged 20 - 40, all of Han ethnicity living in Shanghai recruited by advertisement and lectures. A questionnaire survey was conducted to investigate their dietary custom, Dual-energy X-ray absorptiometry at lumber spine 1 - 4 (L(1 - 4)) and proximal femur was conducted to measure the values of bone mineral density (BMD). RESULTS: The BMD values in L(1 - 4), femoral neck, and greater trochanter of the daughters of mothers with osteoporosis were 0.68 g/cm(2) +/- 0.07 g/cm(2), 0.59 g/cm(2) +/- 0.08 g/cm(2), and 0.47 g/cm(2) +/- 0.07 g/cm(2) respectively, all significantly lower than those of the daughters of normal mothers (0.86 g/cm(2) +/- 0.14 g/cm(2), 0.70 g/cm(2) +/- 0.11 g/cm(2), and 0.57 g/cm(2) +/- 0.10 g/cm(2) respectively, all P < 0.001). The average body weight of the daughters of mothers with osteoporosis was lighter then that of the daughters of normal mothers by 4.8% (P < 0.05). Multivariate regression analysis showed that age, body height, age of menarche, and milk intake were not influencing factors of BMD value, however, body weight was most significantly associated with BMD of the premenopausal daughters, contributing to the BMD variation at L(1 - 4), femoral neck, and greater trochanter by 9.4%, 16.5%, and 16.6% respectively. When body weight was excluded in the model, lower BMD of mother became the most important factors affecting the BMD variation, contributing to the BMD variation at L(1 - 4), femoral neck, and greater trochanter by 5.1%, 5.3%, and 4.2% respectively. CONCLUSION: The daughters of mothers with osteoporosis have reduced peak bone mass. It is likely due to the lower body weight of the daughter and the lower bone mass of the mother. PMID- 16677543 TI - [Age-related and menopause-related changes of urinary excretion of C- and N terminal cross-linked telopeptides of type I collagen and the relationships thereof with menopause-related bone loss]. AB - OBJECTIVE: To study the age-related and menopause-related changes of urinary excretion of C- and N-terminal cross-linked telopeptides of type I collagen (uCTX/Cr and uNTX/Cr) and the relationships thereof with menopause state, years after menopause, bone mineral density (BMD), and menopause-related bone loss in healthy women. METHODS: ELISA was used to examine the uCTX/Cr and uNTX/Cr of 659 female volunteers aged 20 - 80 in Changsha. Dual energy X-ray absorptiometry (DXA) was used to measure the BMD of various skeletal sites, including the lumbar vertebrae (L1 - L4) at anteroposterior (AP) position, L(2) - L(4) at lateral (LAT) position, hip, and forearm. 339 postmenopausal women among the 659 subjects were divided into 3 groups, osteoporotic, osteopenic, and normal groups according to the WHO criteria of osteoporosis diagnosis. RESULTS: (1) Both the curves of uCTX/Cr and uNTX/Cr with age were fit the best by regression analysis of cubic equation. The coefficients of determination (R(2)) were 0.139 for uCTX/Cr and 0.149 for uNTX/Cr. The levels of uCTX/Cr and uNTX/Cr of the women aged > 35 increased with age. (2) The values of uCTX/Cr and uNTX/Cr were 253 mg/mol +/- 101 mg/mol Cr and 63 nmol +/- 34 nmol BCE/mmol Cr respectively in the postmenopausal women, remarkably higher than those of the premenopausal women (149 mg/mol +/- 80 mg/mol Cr and 33 nmol +/- 17 nmol BCE/mmol Cr respectively), increased by 69.5% and 93.4% respectively. The annual change rates of uCTX/Cr and uNTX/Cr were the highest within the first 5 years after menopause, and these increases were in agreement with the significant decrease of BMD at most skeletal sites by 10.8% approximately 27.6%. (3) After controlled for age and body weight, both uCTX/Cr and uNTX/Cr showed significant negative correlation with BMD (r = -0.078 to 0.283, P < 0.05 or 0.01), and there was a significant positive correlation between uCTX/Cr and uNTX/Cr. (4) The elevation of the levels of uCTX/Cr and uNTX/Cr in the osteoporotic and osteopenic postmenopausal subgroups were significantly higher than those in the postmenopausal women with normal BMD (P < 0.05 or 0.01). For example, the uCTX/Cr levels of the osteoporotic, osteopenic, and normal BMD subgroups according to the DXA results at the anteroposterior lumbar spine were 189 +/- 87, 272 +/- 108, and 366 +/- 135 mg/mol Cr respectively, while the uNTX/Cr levels were 52 +/- 22, 68 +/- 34 and 108 +/- 41 nmol BCE/mmol Cr respectively. CONCLUSION: uCTX/Cr and uNTX/Cr can be used as sensitive markers to determine the bone turnover status, which is changeable with age and menopausal status in women. They present a significantly negative correlation with BMD, and increase significantly in the postmenopausal women with osteopenia or osteoporosis, which indicates that measuring uCTX/Cr and uNTX/Cr can predict age-related and menopause-related bone loss in women. PMID- 16677544 TI - [Relationship between polymorphism of parathyroid hormone and bone mineral density and relevant biochemical indicators in women]. AB - OBJECTIVE: To investigate the distribution of parathyroid hormone (PTH) gene polymorphisms and the relationships of PTH gene polymorphisms with bone mass and serum bone relative biochemical markers. METHODS: Blood samples of 314 normal female volunteers, aged 20 - 80, were collected. Serum PTH, bone alkaline phosphatase (sBAP), cross-linked N-telopeptide of collagen type I (sNTX), cross linked C-telopeptide of collagen type I (sCTX), osteoprotegerin (OPG) and leptin were determined by ELISA. Polymorphisms of PTH gene were detected by polymerase chain reaction fragment length polymorphisms (PCR-RFLP) of restriction enzyme BstBI. BMD (QDR4500A) of the anteroposterior spine (AP), supine lateral spine (Lat), and femoral neck (FN) were measured. RESULTS: (1) The genotype frequency of BB, Bb, and bb were 75.8%, 23.3% and 0.9% respectively in normal females volunteers. The frequencies of RFLP alleles B and b were 87.5% and 12.5% respectively. There was no difference in the polymorphism frequency of PTH gene between the post- and pre menopausal women. (2) There were no significant differences in the BMD of the AP, Lat, and FN and the serum biochemical markers between the BB and Bb genotypes. (3) Multiple stepwise regression analysis showed that PTH did not influence the BMD values. CONCLUSION: PTH gene polymorphism has no relationship with bone mass and serum bone biochemical markers in normal females. PMID- 16677545 TI - [Relationship between smoking and risk of esophageal cancer in 103 areas in China: a large-scale case-control study incorporated into a nationwide survey of mortality]. AB - OBJECTIVE: To examine the relationship between smoking and risk of esophageal cancer (EC), and present a theoretical framework of control selection in population-based case-control study which was incorporated into a nationwide retrospective survey of mortality in China. METHODS: A large-scale population based case-control study was incorporated into the nationwide retrospective survey of mortality conducted 1989 - 1991 in 24 urban cities selected by non random sampling and 79 rural counties selected from 3000 counties included in the 1973 - 1975 cancer distribution survey by random sampling during. A questionnaire survey was conducted by home visit to investigate the death causes and smoking history of 19 734 deceased male adults who died of esophageal cancer during 1986 1988 at the age >or= 35. Two control groups were set up to undergo questionnaire survey by home visit to investigate the smoking history of the deceased persons and the informants. Control group I included the surviving spouses or other informants of 31 989 male adults who died of non-malignant digestive diseases during 1986 - 1988 at the age >or= 35, and control group II included 104 846 male spouses of the deceased female adults who died of different causes during 1986 - 1989 at the age >or= 35. The relative risks and population smoking attributable risks for EC were calculated using non-conditional logistic model, and the results were compared for consistency between the analyses using two different control groups. RESULTS: The EC absolute death rates were higher in the smokers than in the non-smokers in all urban and rural area groups. The total EC absolute death rate per 1000 among the non-smokers vs. smokers was 0.37:0.65 in the urban areas, 0.99:1.29 in the inland rural areas, and 1.09:1.62 in the coastal rural areas in the control group I, and there was a similar trend in the control group II. There was a significant dose-response relation between the period of smoking and the death risk of EC and between the daily cigarette consumption and the death risk of EC. The risk ratios, for example, for cigarette per day < 10, 10-, and 20- in the urban men were 1.42, 1.82, 2.22 in the control group I (trend test P < 0.01), and 1.57, 1.95, and 3.18 in the control group II (trend test P < 0.01). CONCLUSION: Smoking is an important risk factor for mortality from EC in China. Investigating the surviving spouses of the deceased patients is a creative, effective, and feasible trial, with the prerequisite of whole population-based survey, in study of the main types of death and the relevant risk factors. PMID- 16677547 TI - [Comparison of the diagnostic criteria of metabolic syndrome by International Diabetes Federation and that by Chinese Medical Association Diabetes Branch]. AB - OBJECTIVE: To compare the accordance between the diagnostic criteria of metabolic syndrome (MS) by International Diabetes Federation (IDF) and that by Chinese Medical Association (CMA) Diabetes Branch. METHODS: An epidemiological survey on diabetes mellitus (DM) was conducted among 2344 residents in Shijingshan community, Beijing, aged >or= 40. 1870 of these 2344 people, 901 males and 969 females, without DM underwent physical examination, laboratory tests, questionnaire survey and oral glucose tolerance test so as to screen the MS patients by both diagnostic criteria. The accordance of these 2 criteria was analyzed statistically by using the STATA7.0 software. RESULTS: The accordance rate of these 2 MS diagnostic criteria was 79.95%. If the criterion for fasting blood glucose in the China criteria was adjusted to 5.6 mmol/L, the accordance increased to 81.39%. 1091 persons were diagnosed as with central obesity according to the IDF criteria, 626 of which could be diagnosed as central obesity according to the China criteria with an accordance rate of 57.38%. Out of these 1091 persons 578 (92.3%) had the BMI >or= 25 kg/m(2). Out of the 779 persons that failed to be diagnosed as with central obesity 113 persons were diagnosed as with central obesity according to the China criteria. Fifty of these 113 persons had the BMI >or= 25 kg/m(2). 111 persons diagnosed as with MS did not have BMI abnormality. According to the IDF criteria, the morbidity of myocardial infarction (MI) and/or cerebral stroke in those with MS was 19.87%, significantly higher than in those without MS (14.86%, P = 0.004), and according to the China criteria, the morbidity of myocardial infarction (MI) and/or cerebral stroke in those with MS was 19.52%, significantly higher than in those without MS (15.21%, P = 0.013) as well. There was no significant difference in the morbidity of MI and cerebral stroke between the IDF criteria and China criteria (P = 0.092). According to the IDF criteria the morbidity of microalbuminuria in the MS patients was 8.59%, significantly higher than that in the persons without MS (4.28%, P = 0.000). According to the China criteria the morbidity of microalbuminuria in the MS patients was 8.55%, significantly higher than that in the persons without MS (4.49%, P = 0.000). There was no significant difference in the morbidity of microalbuminuria between these 2 criteria (P = 0.976). There were no significant difference in the morbidity of microalbuminuria in the persons with central obesity and those with the BMI >or= 25 kg/m(2) between these 2 criteria (P = 0.77). CONCLUSION: The MS diagnostic criteria of IDF and that of CMA Diabetes Branch are in good accordance. PMID- 16677548 TI - [Serum autoantibodies of patients with chronic hepatitis C and the significance thereof in infection of hepatitis C virus]. AB - OBJECTIVE: To investigate the serum autoantibodies in patients with chronic hepatitis C and to investigate the significance of autoimmune reaction in hepatitis C virus (HCV) infected patients. METHODS: Peripheral blood samples were collected from 69 patients with chronic hepatitis C, 69 patients with chronic hepatitis B (HB), and 69 patients with autoimmune hepatitis (AIH). Indirect immunofluorescence technique was used to detect the serum anti nuclear antibody (ANA), anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (anti-SMA), and anti-liver-kidney antibody (anti-LKM). HCV RNA was detected by PCR. The biochemical indices: alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and gamma-globulin were detected. The relations of autoantibodies to virus load, HCV genotype, cirrhosis, age, sex, and liver function were analyzed. RESULTS: (1) Twenty of the HCV patients were positive in autoantibodies, most being at a low titer, 11 of them being positive in ANA, 7 in anti-SMA, 1 in anti-AMA, and 1 in anti-LKM, with a positive rate of 28.9%, significantly higher than that of the HB patients (4.3%, P < 0.05), and significantly lower than that of the AIH patients (100%, P < 0.05). (2) There were no significant differences between the autoantibody positive group and autoantibody negative group in virus load and HCV genotype. (3) Fourteen of the 18 patients positive in autoantibodies responded to the anti-virus treatment with alpha-interferon with a response rate of 77.8%, significantly higher than that of the autoantibody negative group (53%, P < 0.05). (4) The average age of the autoantibody positive group was 47 +/- 18 years, significantly higher than that of the autoantibody negative group (39 +/- 12 years, P < 0.05). The positive autoantibody rate of the patients aged >or= 40 was 23.1%, significantly higher than that of the patients aged < 40 (5.8%, P < 0.05). There was no significant difference in the autoantibody rate between males and females. (5) The cirrhosis prevalence rate of the autoantibody positive group was 80%, significantly higher than that of the autoantibody negative group (46.9%, P < 0.05). (6) The serum ALT, AST, TBIL and gamma-globulin of the autoantibody positive group were 191 U/L +/- 89 U/L, 169 U/L +/- 80 U/L, 78 micromol/L +/- 50 micromol/L, and 200 g/L +/- 80 g/L respectively, all significantly higher than those of the autoantibody negative group (113 U/L +/- 69 U/L, 98 U/L +/- 62 U/L, 51 micromol/L +/- 30 micromol/L, and 160 g/L +/- 70 g/L respectively, all P < 0.05). (7) There were no significant differences in HCV RNA load and HCV genotype between the autoantibody positive group and the autoantibody negative group (both P > 0.05). CONCLUSION: HCV infection induces the autoimmune reaction resulting in production of autoantibodies. Positive autoantibody rate is related with cirrhosis and age. Titer and type of autoantibody serve as important indices in the diagnosis and treatment of HCV. PMID- 16677549 TI - [Regulation of heat shock protein 27/activating transcription factor-5 complex and its implication in podocyte apoptosis induced by high glucose]. AB - OBJECTIVE: To investigate the regulation of heat shock protein (HSP)27/activating transcription factor (ATF)-5 complex in podocytes induced by high glucose and relevant mechanisms. METHODS: Mice kidney podocytes were cultured in culture fluid with D-glucose at normal concentration (5.5 mol/L) (Group NG) or with D glucose at high concentration (30 mmol/L) (Group HG) cells of these 2 groups were collected at different time points after glucose stimulation to detect the cell apoptosis by Hoechst 33342 staining and fluorescence microscopy and flow cytometry. Western blotting was used to analyze the activation of extracellular signal-regulated kinase (ERK = MAPK) and p38 signaling pathway. The HSP27/ATF5 complex was assessed by co-immunoprecipitation. ERK pathway blocker PD98059 and p38 signal pathway blocker SB203580 were added into the culture fluid of Group HG and Group NG respectively, and then the podocytes were collected at different time points to detect the high glucose-induced HSP27/ATF5 complex and cell apoptosis. RESULTS: The apoptotic rate of the podocytes of Group HG 24 hours after high glucose incubation was 14.3% +/- 6.2%, and that 48 h after was 27.2% +/- 8.9%, significantly higher than that of Group NG (10.6% +/- 2.7%, P < 0.05). HSP27/ATF5 complex could detected in the cells of Group NG too, however, the level of HSP27/ATF5 complex in Group HG 12 hours after incubation was 195% +/- 36% that of Group NG (P < 0.05). Both the ERK signal pathway and p38 signal pathway of Group HG began to be activated 10 min after incubation, peaked 30 min after, remained at the highest level till 1 hour after, and returned almost to the baseline level 2 hours after. No activation of these 2 pathways was observed in Group NG. The HSP27/ATF5 complex level of the PD98059 + high glucose group was 109% +/- 19% that of Group NG, significantly lower than that of Group HG (211% +/ 46% that of Group NG, P < 0.05). The apoptotic rate of the PD98059 + high glucose group was 51% +/- 4%, significantly higher than that of PD98059 + normal glucose group (16% +/- 3%, P < 0.05) and that of Group HG (27% +/- 9%, P < 0.05). The apoptotic rate of the SB203580 + HG group was 16% +/- 6%, significantly lower than that of Group HG (27% +/- 9%, P < 0.05). The HSP27/ATF5 complex level of the SB230580 + HG group was 290% +/- 43% that of Group NG, not significantly different from that of Group HG (231% +/- 20% that of Group NG, P > 0.05). CONCLUSION: High glucose stimulates the formation of HSP27/ATF5 complex in podocytes through ERK signaling pathway but not P38 signaling pathway, and the HSP27/ATF5 complex may have a regulatory effect in podocyte apoptosis induced by high glucose. PMID- 16677550 TI - [Enhancement of invasion of hepatocellular carcinoma cells by lysophosphatidic acid through Rho signal transduction pathway]. AB - OBJECTIVE: To investigate the mechanism of enhancement of invasion of hepatocellular carcinoma (HCC) cells by lysophosphatidic acid (LPA). METHODS: Human HCC cells of the line SMMC7721 were cultured. LPA at different concentrations (2, 5, and 25 micromol/L) was added into the culture fluid. The Rho activity was detected with Rho activity detection kit. Western blotting was used to detect the protein expression of Rho. Adhesion test was conducted to calculate the adhesion percentage of the SMMC7721 cells. The invasion potential of the SMCC7721 cells was observed using transwell cell test. RESULTS: LPA at the concentrations of 5, and 25 micromol/L increases the activity of Rho protein. When the concentration of LPA was 25 micromol/L the activity of Rho protein was 400 times that of the control group (P < 0.01). The Rho protein expression in the SMCC7721 cells increased when stimulated by LPA, peaked 20 approximately 25 hours after stimulation, and then gradually decreased. When the concentration of LPA was 25 micromol/L the Rho protein expression level was 242% higher than that of the control group. LPA at the concentration of 5 micromol/L and over increased the migratory and invading potential of the SMCC7721 cells and increased the adhesiveness of the SMCC7721 cells time-dependently. CONCLUSION: LPA increases the migratory and invading potential of HCC cells through Rho signal transduction pathway. PMID- 16677551 TI - [Repair of skin damage with mesenchymal stem cells-poly (lactic-co-glycolic acid) scaffolds: experimental study with rabbits]. AB - OBJECTIVE: To evaluate the feasibility and effect of mesenchymal stem cells (MSCs)-poly (lactic-co-glycolic acid) (PLGA) scaffold as transplant in repair of skin damage. METHODS: MSCs were isolated from the bone marrow of femur of a one month-old New Zealand rabbit, cultured, and labeled with diamidino-phenyl-indole (DAPI). Porous foam scaffolds were made with PLGA. MSCs of 2 - 3 passages were seeded on the scaffolds. Fluorescence microscopy and scanning electron microscopy were used to observe the growth of the MSCs. Six pieces of skin 2 cm x 2 cm in size were cut from the backs of five 5-month-old new Zealand rabbits and then 4 pieces of MSCs- PLGA scaffolds and 2 pieces of porous foam PLGA scaffolds of the size similar to these of the cut skin were transplanted to the skin wounds. The wound healing was observed. Five days after the operation, samples of newly-grown skin were taken to undergo HE staining, VG staining, and microscopy. Immunofluorescence histochemistry was used to detect the cytokeratin AE1/AE3. RESULTS: Scanning electron microscopy showed that holes were distributed evenly on the surface of and inside the porous foam PLGA scaffolds Fluorescence microscopy and scanning electron microscopy showed that the MSCs grew well on the porous foam PLGA scaffolds and the number of MSCs increased gradually. Animal experiment showed that with the degradation of the polymer scaffolds the wounds were gradually covered by newly grown skin similar to the normal skin. Immunofluorescence histochemistry showed fluorescence positive cells in the stratum corneum and follicles. The wounds transplanted only with porous foam PLGA scaffolds formed new skin too, however, in the dermis of the new skin only thickened fibrous scars and a few follicles were seen. CONCLUSION: The compound of MSCs-PLGA polymer is effective in wound healing. PMID- 16677552 TI - [Clinical evaluation of the heart function early after repair of tetralogy of Fallot: follow-up of 43 patients]. AB - OBJECTIVE: To evaluate the heart function of the patients early after the repair of tetralogy of Fallot (TOF). METHODS: Forty-three patients with TOF, 25 males and 18 females, underwent operation at the age of 2.5 - 52 years (16.7 years on average) and were followed up for 1 - 3.5 years. Twenty-one age-matched healthy persons were used as controls. Tissue Doppler imaging (TDI) was used to measure the values of the peak tricuspid ring velocity during early diastole (Ea), late diastole (Aa), systole, and isovolumic contraction, and isovolumetric contraction acceleration (IVA); and isovolumetric contraction time (ICT), isovolumetric relaxation time (IRT), and isovolumetric contraction velocity (IVV) of the right ventricle. Tei index was calculated using the formula: (ICT + IRT)/ET. Treadmill test was used on the patients aged > 17 to measure the maximal heart rate maximal blood pressure, maximal exercise tolerance (MET), and movement time. RESULTS: The peak tricuspid ring velocity during Ea of the repaired TOF group (rTOF group) was 11.5 +/- 2.6 cm/s, significantly lower than that of the control group (17.1 +/- 2.4 cm/s, P < 0.0001), the peak tricuspid ring velocity during Aa of the rTOF group was 9.6 +/- 1.7 cm/s, significantly lower than that of the control group (12.9 +/- 2.9 cm/s, P < 0.001), the E/A of the rTOF group was 1.16 +/- 0.36, significantly lower than that of the control group (1.36 +/- 0.26, P < 0.05). The IVV of the rTOF group was 7.7 +/- 1.8 cm/s, significantly lower than that of the control group (9.9 +/- 1.4 cm/s, P = 0.0030, and the IVA of the rTOF group was 131.7 +/- 37.6 cm/s(2), significantly lower than that of the control group (222.5 +/- 39.2 cm/s(2), P < 0.001). The Tei index of the rTOF group was 0.58 +/- 0.11, significantly higher than that of the control group (0.52 +/- 0.04, P = 0.029). The maximal heart rate maximal blood pressure, MET, and movement time of the rTOF group were all significantly lower than those of the control group (P < 0.05 or P < 0.01). CONCLUSION: The heart function of the patients undergoing repair of TOF fails to recover to the normal level during a short time after the surgery. PMID- 16677554 TI - [Adult-to-adult living donor liver transplantation using right lobe graft: report of 24 cases]. AB - OBJECTIVE: To investigate the safety and feasibility of adult-to-adult living donor liver transplantation (A-A LDLT) using right lobe graft. METHODS: From January 2005 to December 2005, 24 patients, 22 males and 2 females, aged 20 - 53, with the underlying diseases of liver cirrhosis, hepatocellular carcinoma, and Budd-Chiari syndrome, underwent A-A LDLT using right lobe graft. During the second period, including 22 cases, modified techniques were adopted, e.g. direct anastomosis of the right hepatic vein (RHV) and inferior vena cava was performed in all 22 cases, reconstruction of inferior right hepatic vein was performed on 9 cases, and reconstruction of tributaries of middle hepatic vein by interposing a vein graft on 10 cases so as to provide sufficient venous outflow. RESULTS: No sever complication and death was found in the donors. The graft and recipient weight ratio was 0.72% - 1.17%, being < 1.0% among 19 cases and being < 0.8% among 4 cases. During the first period one of the 2 patients suffered from stenosis at the anastomotic stoma of the right hepatic vein, and the other patient suffered from small-for-size syndrome and died. During the second period modified techniques were adopted and no more small-for-size syndrome occurred. CONCLUSION: With the modified techniques, A-A LDLT has become a relative safe procedure. PMID- 16677563 TI - [Differential diagnosis of hip diseases mimicking osteonecrosis of the femoral head]. PMID- 16677564 TI - [Choice of operational program for ischemic necrosis of femoral head]. PMID- 16677566 TI - [Evaluation of hemodynamic changes by digital subtraction angiography after treatment of avascular osteonecrosis of femoral head with vascularized bone flaps]. AB - OBJECTIVE: To evaluate the effect of digital subtraction angiography (DSA) in evaluation of the hemodynamic changes after treatment of avascular osteonecrosis of femoral head with vascularized bone flaps. METHODS: Fifty-four patients, 33 males and 21 females, 33 hips being at the stage II and 21 hips being at the stage III, were treated with vascularized bone flaps, including iliac bone flaps pedicled with ascending branch of lateral femoral circumflex artery for 23 hips, greater trochanter bone flaps pedicled with gluteal muscle branch of ascending branch of lateral femoral circumflex artery for 13 hips, greater trochanter bone flaps pedicled with transverse branch of ascending branch of lateral femoral circumflex artery for 9 hips, and greater trochanter bone flaps pedicled with gluteal muscle branch and transverse branch of ascending branch of lateral femoral circumflex artery for 9 hips. Selective DSA was performed on all 54 patients pre-operatively and on 19 patients 6-24 months post-operatively. RESULTS: DSA before operation demonstrated abnormal vascularization in all 54 patients. Six months after operation reconstruction of blood supply on the femoral head was seen in 48 cases. Six patients showed poor filling or failure of filling of the vessel pedicles in transplanted bone flaps 6 months post operatively and DSA conducted 8 months postoperatively showed satisfactory filling in one of the 6 cases. The clinical success rate of this group was 91%, and the radiological success rate was 85%. CONCLUSION: Selective DSA is effective in evaluation of the postoperative hemodynamic changes in the necrotic femoral head. Significant blood supply in the bone flap is the key point in the success of treatment of ONFH. PMID- 16677567 TI - [Hematological changes and related gene mutation of post-severe acute respiratory syndrome patients with osteonecrosis]. AB - OBJECTIVE: To investigate the hematological changes and related gene mutation of post-severe acute respiratory syndrome (SARS) patients with osteonecrosis so as to find the sensitive molecular symbols for early screening of the high risk populations. METHODS: Fast peripheral venous blood samples were collected from 61 post-SARS patients with osteonecrosis, 25 males and 36 females, aged 30.4 (20 - 60), and 52 sex and age-matched healthy persons as controls. ELISA was used to detect the coagulation and fibrinolysis indicators: activated partial thromboplastin time (APTT), protein C (PC), antithrombin III (AT-III), plasminogen activator inhibitor (PAI), activated protein C resistance (APC-R), plasminogen (PLG), von Willebrand factor (VWF), D-dimer (D-D), and fibrinogen (Fib). Real-time PCR was used to detect the mutation of factor V G1601A (FV Leiden) and prothrombin G20210A. RESULTS: The levels of PC, AT-III, and PLG of the osteonecrosis group were 85% +/- 34%, 84 +/- 29%, and 69 +/- 23%, significantly lower than that of the control group (109% +/- 20%, 104% +/- 14%, and 94% +/- 15% respectively, all P < 0.01). PAI of the osteonecrosis group was 16 U/ml +/- 14 U/ml, significantly higher than that of the control group (8.0 U/ml +/- 4.3 U/ml, P < 0.01). The percentage of patients with abnormal indicators was 99.5% (54/61) in the osteonecrosis group, significantly higher than that of the control group (36.5%, 19/52, P < 0.01). The percentage of patients with 3 or more abnormal indicators was 72.1% (44/61) in the osteonecrosis group, significantly higher than that of the control group (17.3%, 9/52, P < 0.01). No mutations of F V Leiden and prothrombin G20210A was found in both groups. CONCLUSION: Trends of hypercoagulation and hypofibrinolysis exist in the post SARS patients with osteonecrosis. APTT, PC, AT-III, and PLG can be used as sensitive indicator for screening high risk populations of osteonecrosis. PMID- 16677568 TI - [Perinatal outcomes of intrahepatic cholestasis of pregnancy: analysis of 1210 cases]. AB - OBJECTIVE: To study the perinatal outcomes of intrahepatic cholestasis of pregnancy (ICP). METHODS: The clinical data of 1210 cases of ICP in recent ten years were retrospectively analyzed. RESULTS: The incidence rates of perinatal outcomes of ICP were as follows: 19.0% (230/1210) for threatened premature labor, 24.0% (290/1210) for premature delivery; 23.2% (281/1210) for meconium stained amniotic fluid, 7.1% (86/1210) for neonatal asphyxia, 22.5 per thousand (27/1210) for perinatal mortality, 85.9% (1039/1210) for cesarean section, 0.9% (11/1210) for fetal growth restriction (FGR), 1.4% (17/1210) for postpartum hemorrhage, and 8.1% (101/1210) for preeclampsia. Threatened premature labor occurred beyond the gestation gestation period of 32 weeks in 88.7% (204/230) of the patients, and the fetal death rate in threatened premature labor was 46.7% (7/15). Premature delivery occurred after 34 weeks of gestation in 96.2% of the patients (279/290) 89.7% (260/290) of which were caused by cesarean section because of abnormal fetal monitoring. 41.3% of the cases with meconium stained amniotic fluid (116/281) occurred before the onset of labor. Fetal death accounted for 56% (15/27) of perinatal death, 80% (12/15) of which happened after the gestation week of 35 (36.5 +/- 1.2) with normal fetal heart rate monitoring. 95% (19/20) of the fetal death and stillbirth occurred after threatened premature labor and occasional uterine contractions, or at the early stage of labor. CONCLUSION: The rates of FGR, postpartum hemorrhage, and preeclampsia in ICP are almost the same as those of the normal pregnancy. Routine fetal heart rate monitoring methods cannot predict fetal death. The important measures to decrease the perinatal mortality include paying attention to fetal monitoring when threatened premature labor, occasional uterine contractions and prenatal meconium occur, and at the early stage of labor, and management of threatened premature labor and timely intervention of pregnancy (at the gestation period of 34 - 37 weeks). PMID- 16677569 TI - [The anatomic relationship of the umbilicus to retroperitoneal major vessels]. AB - OBJECTIVE: To study the anatomic relationship of the umbilicus to the retroperitoneal major vessels and the characteristics of such relationships among the Chinese with different body weights so as to provide a clear reference to the operator of laparoscopy. METHODS: Eighty-nine patients without pelvic disease, 57 males and 32 females who accepted digital subtraction angiography (DSA) of aorta were randomly selected and divided into 3 groups: non-obese group, overweight group, and obese group according to body mass index (BMI). All the patients lied supine with a round block of lead 1 cm in diameter located on the umbilicus. Seldinger technique was used to puncture the right femoral artery so as to conduct DSA. Using the bifurcation of the abdominal aorta as reference point the vertical projection relationships of the umbilicus to the retroperitoneal major vessels, abdominal aorta, right common iliac artery, and left common iliac artery were evaluated and the distance from the umbilicus to the aortic bifurcation was measured. The distance was regarded as positive if the umbilicus was cephalic to the aortic bifurcation and as negative if the umbilicus was caudal to the aortic bifurcation RESULTS: There were 32 patients in the normal body weight group, 35 in the overweight group, and 22 in the obese group with the mean distances from the umbilicus to the aortic bifurcation of (14.8 +/- 19.7) mm, (0.04 +/- 2.5) mm, and (-12.6 +/- 15.4) mm respectively. In 50 of the 89 patients (63.9%) the location of umbilicus corresponded to the retroperitoneal major vessels, among which the umbilicus of 47 patients (94%) projected vertically to the abdominal aorta or the right common iliac, and the umbilicus of 3 patients (6%) projected vertically to the left common iliac artery. Compared to the above-mentioned 50 patients, in the other 39 patients (36.1%) the location of umbilicus did not corresponded to the retroperitoneal major vessels (P < 0.05), among which the umbilicus of 32 patients (82.1%) projected vertically to the right side of the aorta or of the right common iliac artery, and the umbilicus of 7 patients (17.9%) projected vertically to the internal side of the right iliac common artery. Along with the increase of body weight the projection of umbilicus gradually moved downward to the inferior side of the bifurcation of the abdominal artery. For example, among the male subjects, the distance were (10.4 +/- 4.0) mm, (-0.51 +/- 5.5) mm, and (-13.1 +/- 2.2) mm respectively in the normal body weight group, overweight group, and obese group (all P < 0.05), and in the females, the distance were (13.7 +/- 2.8) mm, (-0.14 +/- 4.4) mm, and (-11.5 +/- 3.2) mm respectively in the normal body weight group, overweight group, and obese group (all P < 0.05). CONCLUSION: The location of umbilicus was more caudal with the increase of BMI. So once the retroperitoneal major vessels are injured, the incidence of aorta or the right common iliac artery is higher than that of other vessels. PMID- 16677570 TI - [Clinical research of primary intraspinal hemangiopericytoma]. AB - OBJECTIVE: To investigate the effective means of treatment for primary intraspinal hemangiopericytoma. METHODS: The clinical presentation, imaging features, treatment and prognosis of 23 cases of intraspinal hemangiopericytoma, 14 males and 9 females, aged 39.1 (14 - 65), were analyzed retrospectively. RESULTS: Subtotal resection was performed on 11 cases, subtotal resection and radiotherapy on 4 cases, total resection on 5 cases, and total resection and radiotherapy on 3 cases. Spinal stabilization was conducted on 2 cases after total resection with severe damage of the vertebral body or pedicle of vertebral arch. Nineteen cases were followed up for 3.8 years (6 months-10 years). The recurrence rate was 68% and the metastasis rate was 21%. CONCLUSION: Primary intraspinal hemangiopericytoma should be resected en bloc with neighboring dura mater to reduce blood loss and recurrence. Resection of large lesions may be safer after preoperative embolization. The patients undergoing subtotal resection need adjuvant radiotherapy. The patients with evident spinal involvement may benefit from spinal stabilization. Lesions arising from dural mater have better prognosis. Postoperative long-term follow-up is necessary. PMID- 16677571 TI - [The roles of active efflux system overexpression and outer membrane protein OprD deficiency or loss in carbapenem resistance of Pseudomonas aeruginosa]. AB - OBJECTIVE: To investigate the mechanisms of carbapenem resistance in Pseudomonas aeruginosa (PA). METHODS: Forty-nine strains of PA were isolated from surgical intensive care unit during a period of 3 years. The levels of outer membrane protein OprD and OprN were measured by Western blotting. RT-PCR was used to measure the transcription levels of mexA gene. The metallo-beta-lactamase genes IMP and VIM and the negative regulator gene mexR for mexAB-OprM operon were amplified. The DNA fragments were sequenced by automated ABI PRISM 3700 sequencer. RESULTS: 42 of the 49 strains were resistant to carbapenem. 23 of the 42 strains showed loss of OprD and were all resistant to imipenem, but only one strain was resistant to meropenem. 18 of the 42 strains had a decreased OprD expression, 17 of which were resistant to Imipenem, and 3 were resistant to meropenem as well. 7 strains expressed OprD, all of which were sensitive to carbapenem. 27 strains overexpressed the mexAB-OprM. The resistant rate to imipenem of the mexAB-OprM overexpression group was 86.4%, not significantly different from that of the mexAB-OprM low expression group (81.5%, chi(2) = 0.005, P = 0.943). But the resistant rate to meropenem of the mexAB-OprM overexpression group was 44.4%, statistically higher than that of mexAB-OprM low expression group (13.6%, chi(2) = 5.417, P = 0.020). Nucleotide sequences and deduced amino acid sequences analysis revealed that eight strains overexpressed mexAB-OprM carried mutations in mexR gene, 7 of which had amino acid substitutions in MexR protein, and one of which had terminal code at the position of amino acid 32. 14 strains were found expressing OprN. Neither IMP gene nor VIM gene was found in the isolates. CONCLUSION: In the clinical strains from SICU, the imipenem resistance is mainly mediated by OprD deficiency or loss. Overexpression of MexAB-OprM is the primary mechanism of meropenem resistance, which is upregulated by mutations in mexR gene. Metallo-beta-lactamases IMP and VIM are rarely seen. PMID- 16677574 TI - [Correlation of reticular basement membrane thickness and airway wall remolding in asthma patients]. AB - OBJECTIVE: To investigate the correlation between the reticular basement membrane thickness and the airway wall remolding in asthma patients. METHODS: Lung tissues were obtained from 5 patients who died from asthma, 3 males and 2 females, aged 45 +/- 16 (fatal asthma group), 5 asthmatics who died of diseases unrelated to asthma, 3 males and 2 females, aged 47 +/- 13, (non-fatal asthma group), and 5 dead patients without asthma, 3 males and 2 females, aged 24 +/- 14 (control group) to select 41, 38, and 43 transverse sections of tracheae respectively. The samples of tracheae were divided into cartilaginous and membranous airways by light microscopy (x100). The thickness of reticular basement membrane and the dimensions of the airway wall, including the smooth muscle area, submucosal gland area, inner and outer wall areas, and lumen area were measured. The correlations of reticular basement membrane thickness with the airway changes were analyzed. RESULTS: The reticular basement membrane was significantly thicker in both cartilaginous and membranous airways in the fatal and non-fatal asthma groups than in the control group (all P < 0.05). The submucosal gland area of the fatal asthma group was significantly larger than those of the non-fatal asthma group and control group (both P < 0.05). The inner wall area of the cartilaginous airway of the fatal asthma group was significantly larger than that of the non fatal asthma group (P < 0.05); however, the inner wall area of the membranous airway of the fatal asthma group was not significantly different from that of the non-fatal asthma group. The outer wall areas of cartilaginous and membranous airways of the fatal asthma group were both significantly larger than those of the other 2 groups (all P < 0.05). There was no significant difference in the lumen areas of cartilaginous and membranous airways between the fatal and non fatal asthma groups. The reticular basement membrane thickness was correlated with the smooth muscle area (P < 0.05), submucosal gland area (P < 0.05), and inner wall area (P < 0.01) of the corresponding bronchi in regard to the cartilaginous airway; and was correlated with the smooth muscle area (P < 0.05) and inner wall area (P < 0.01) of the corresponding bronchi in regard to the membranous airway, but was not correlated with the airway size, lumen area, and outer wall area in regard to the 2 kinds of airways. CONCLUSION: The reticular basement membrane thickness of central airway reflects the airway remolding of the central airway and the changes of smooth muscle and inner wall of the peripheral airway. It is worthwhile to do end bronchial biopsy to measure the reticular basement membrane thickness so as to assess the pathology of the airway and to conduct long-term follow-up among the asthma patients. PMID- 16677572 TI - [The role of mannose binding lectin in the pathogenesis of systemic lupus erythematosus]. AB - OBJECTIVE: To detect the serum level of mannose binding lectin (MBL) and its genovariation in systemic lupus erythematosus (SLE) patients and to investigate the role of MBL in the pathogenesis of SLE. METHODS: ELISA was used to measure the serum MBL level of 40 SLE patients and 30 healthy blood donors. Tm genotyping method was used for the first timer in China. Primers and specific fluorophore labelled hybridization probes for the exon 1 and promoter regions of MBL gene were designed based on the haplotype MBL2(*) LXPA (GenBank X15422). The genotyping of MBL in these two groups were performed using real-time PCR through Light Cycler Instrument. RESULTS: (1) The serum MBL of the SLE patients was 107.2 microg/L, significantly lower than that of the healthy blood donors (290.2 microg/L, P = 0.0002). (2) MBL mutation in exon 1 region was mainly at codon 54, with a mutation rate of 37.1% in the SLE group, significantly higher than that of the control group (13.3%, p = 0.049). (3) Polymorphisms of H/L in MBL gene were present in both SLE patients and controls, and there was no difference in the L allele frequency between the two groups. (4) The serum MBL level of the SLE patients with MBL mutation in codon 54 was 49.8 microg/L, significantly lower than that of the SLE patients without MBL mutation in codon 54 (141.7 microg/L, P = 0.000 27). The SLE disease activity index (SLEDAI) of the SLE patients with MBL mutation in codon 54 was 7.44, significantly lower than that of the SLE patients without MBL mutation in codon 54 (12.87, P = 0.0029). A negative correlation was observed between SLEDAI score and serum MBL (r = -0.48). CONCLUSION: Mutation occurring in MBL exon 1 region at codon 54 may be a predisposing factor of the pathogenesis of SLE. Serum MBL may be a potential biomarker of disease activity in SLE patients. PMID- 16677575 TI - [Effects of tissue inhibitor of matrix metalloproteinases-4 on the activities of matrix metalloproteinases and collagen of artery: experiment with rats]. AB - OBJECTIVE: To investigate the effects of tissue inhibitor of matrix metalloproteinases-4 (TIMP-4) on the activities of matrix metalloproteinases (MMPs) and the collagen deposition. METHODS: Vascular smooth muscle cells (VSMCs) of rat thoracic aorta were cultured and divided into 3 groups: 2 groups to be transfected with adenovirus vector containing green fluorescence protein (AdGFP) or adenovirus vector containing TIMP-4 (AdTIMP-4), and one group un-transfected. Zymography and reverse zymography were used to detect the activities of MMP-2, MMP-9 and TIMP-4 in the supernatants. Male Wistar rats underwent balloon injury of common carotid artery and then divided into 3 groups: pure injury group, AdGFP transfection group, and AdTIMP-4. transfection group. Four and 28 days later 3 and 6 rats were killed in each group respectively to undergo microscopy and examination of the activities of MMP-2, MMP-9, and TIMP-4, and collagen quantity. RESULTS: The MMP-2 activity in the VSMC culture fluid supernatant of the AdTIMP-4 group was decreased dose-dependently, however, the activity of MMP-9 did not changed significantly among the 3 groups. Bands of activated MMP-2 and MMP-9 could not be examined in the normal vessel tissues. Four hours after the injury, the activity of MMP-2 was significantly increased in the pure injury group and AdGFP group, however, was significantly decreased in the AdTIMP-4 group. Four days later no MMP activity could be detected in either group. The neoformation of tunica intima was inhibited by 66% in the AdTIMP-4 group, The collagen quantity per vessel cell was 12.1 +/- 1.0 in the AdGFP group, not significantly different from that of in the AdTIMP-4 group (11.9 +/- 1, P > 0.05), and the collagen quantity per tunica adventitia cell in the AdTIMP-4 group was 118 +/- 13, not significantly different from that of the pure injury group (135 +/- 11, P > 0.05). CONCLUSION: The regulation of MMP/TIMP balance by TIMP-4 may control the metabolism of collagen and play an important role in the vascular repair process. PMID- 16677576 TI - [Effects of puerarin on proliferation of vascular smooth muscle cells induced by thrombin]. AB - OBJECTIVE: To investigate the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin and the mechanism thereof. METHODS: VSMCs were isolated from the thoracic aorta of a SD rat and cultured, then co-cultured with thrombin of the concentration 0.1, 0.3, 1.0, 3.0, and 10 U/L for 24 h, thrombin of the concentration of 1 U/L for 0, 6, 12, 24, 36, and 48 h respectively, or thrombin of the concentration of 1 U/L combined with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), or 1.5 x 10(-3) mol/L for 24 h. Flow cytometry was used to detect the cell number and cell cycle. Western blotting was used to indicate the protein expression of the oncogenes c fos and bcl-2 RT-PCR was used to evaluate the thrombin receptor (TR) mRNA expression. RESULTS: he numbers of the groups of VSMCs stimulated by 0.1, 0.3, 1.0, 3.0, and 10 U/L thrombin for 24 hours were 4.82 x 10(4)/ml +/- 0.11 x 10(4)/ml, 6.37 x 10(4)/ml +/- 0.09 x 10(4)/ml, 8.78 x 10(4)/ml +/- 0.08 x 10(4)/ml, 7.37 x 10(4)/ml +/- 0.07 x 10(4)/ml, and 5.28 x 10(4)/ml +/- 0.12 x 10(4)/ml respectively, all significantly higher than that of the control group (4.08 +/- 0.054 x 10(4)/ml, all P < 0.05). The effect of thrombin was in a dose dependent manner within a concentration range of 0.1 - 1.0 U/L. The suppression rates of VSMC proliferation in the combination groups with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L were 10.9% +/- 1.6%, 32.1% +/- 3.3%, and 42.6% +/- 5.2% respectively in comparison with the thrombin group (all P < 0.05). The c-fos protein expression of the VSMCs after thrombin stimulation for 24 h increased by 156.0% +/- 11.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 20.7% +/- 2.1%, 31.6% +/- 5.2%, and 44.5% +/- 7.5% respectively (all P < 0.05). The bcl-2 protein expression of the VSMCs after thrombin stimulation for 24 h increased by 96.7% +/ 8.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 7.1% +/- 0.8%, 18.8% +/- 1.2%, and 39.6% +/- 6.4% respectively (all P < 0.05). The stimulation of thrombin increased the TR mRNA expression by 183.9% +/- 9.4%. The puerarin of the concentrations of 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L decreased the increase of TR mRNA expression induced by thrombin, however, without significant differences (both P > 0.05), and puerarin of the concentration of 1.5 x 10(-3) mol/L significantly suppressed the increase of TR mRNA expression induced by thrombin by 17.6% +/- 1.7% (P < 0.05). CONCLUSION: Puerarin suppresses the proliferation and DNA synthesis of VSMC induced by thrombin. The inhibitory effect of puerarin is closely related with the suppression of the protein expression of c-fos and bcl-2n, and partly related with the suppression of the TR mRNA expression. PMID- 16677587 TI - Temporal changes in rates of stillbirth, neonatal and infant mortality among triplet gestations in the United States. AB - OBJECTIVE: The purpose of this study was to examine temporal changes in stillbirth, neonatal and infant mortality rates among triplet births in the US, and to assess the contributions of triplet delivery at < 34 weeks to these changes. STUDY DESIGN: Data on triplet live births, and fetal and infant deaths (1990-2002) delivered at > or = 22 weeks and fetuses weighing > or = 500 g (n = 66,986) were derived from the US linked birth/infant death data files. Relative risk (RR), quantifying changes in triplet stillbirth, neonatal (death within the first 28 days) and infant mortality (death within the first year) rates between 1990 and 1991 and 2001 and 2002, were derived. Temporal changes in triplet births at < 34 weeks, and changes in stillbirth, and neonatal and infant mortality rates were examined through logistic regression models before and after adjusting for confounders. RESULTS: Triplet births at < 34 weeks increased by 25% between 1990 and 1991 (48.7%) and 2001 and 2002 (60.9%). Stillbirth, neonatal and infant mortality rates declined by 52% (RR 0.48, 95% confidence interval [CI] 0.36 0.63), 32% (RR 0.68, 95% CI 0.58-0.80), and 38% (RR 0.62, 95% CI 0.53-0.71), respectively, between 1990 and 1991 and 2001 and 2002. The increase in triplet births at < 34 weeks was not associated with the stillbirth decline, but was associated with an excess 14% and 12% increase in neonatal and infant deaths, respectively. CONCLUSION: Our findings suggest that the increase in triplet births at < 34 weeks' gestation is not associated with changes in triplet stillbirths, but is associated with increases in triplet neonatal and infant mortality. PMID- 16677588 TI - Prior cesarean delivery is not associated with an increased risk of stillbirth in a subsequent pregnancy: analysis of U.S. perinatal mortality data, 1995-1997. AB - OBJECTIVE: An association between cesarean delivery and an increased risk of stillbirth in a subsequent pregnancy has been reported in the United Kingdom. This study investigated the association between prior cesarean delivery and unexplained intrauterine fetal demise at term in the United States. STUDY DESIGN: We conducted a cross-sectional study using the U.S. perinatal mortality data (1995 to 1997). Women aged 15 to 44 years with singleton term (37 weeks or longer) pregnancies were included in the analysis. Study groups were defined as pregnant women with a prior cesarean delivery (prior cesarean delivery) and women with no prior cesarean delivery (no cesarean delivery). Adjustments were made for maternal age, race, underlying medical conditions, and fetal congenital abnormalities. The Cochran-Mantel-Haenszel method was used for relative risk estimation at the 95% confidence interval calculation. RESULTS: A total of 11,061,599 deliveries of singleton pregnancies were recorded in the United States from January 1, 1995, to December 31, 1997. The cesarean delivery rate was 19.6%. The crude term intrauterine fetal demise rate was 1.5 per 1000 births for no cesarean delivery and 1.3 per 1000 births for prior cesarean delivery. After correction for parity greater than 1, congenital anomalies, and underlying maternal medical conditions, term intrauterine fetal demise rates were 0.6 and 0.4 per 1000 births for no cesarean delivery and prior cesarean delivery, respectively. Restriction of the analysis to women with only 1 prior delivery resulted in term intrauterine fetal demise rates of 0.8 and 0.7 per 1000 births for no cesarean delivery and prior cesarean delivery, respectively (relative risk 0.90; 95% confidence interval 0.76-1.06). CONCLUSION: A prior cesarean delivery is not associated with an increased risk of stillbirth in a subsequent pregnancy. PMID- 16677589 TI - Preterm histologic chorioamnionitis: impact on cord gas and pH values and neonatal outcome. AB - OBJECTIVE: The purpose of this study was to further delineate the impact of preterm chorioamnionitis on a spectrum of neonatal outcomes using a large tertiary hospital population. STUDY DESIGN: The perinatal/neonatal and placental pathology databases of St. Joseph's Health Care, London, Ontario, Canada, were used to obtain the umbilical cord gas and pH values, incidence of adverse neonatal outcomes, patient demographics, and placental pathology reports for all preterm (25 to 34 weeks of gestation), singleton, liveborn infants with no major anomalies who were delivered with spontaneous onset of labor or for suspected chorioamnionitis between November 1, 1995, and October 31, 2003. Patient groupings on the basis of placental inflammation and clinical chorioamnionitis were studied by a comparison of mean values and incidences for those neonatal outcomes that were available from the database with the use of linear and logistic regression analysis and controlling for potentially confounding variables. RESULTS: There were 660 infants who met the inclusion criteria and had placental pathology available of whom 368 (56%) had no placental inflammation, 114 (17%) had placental chorioamnionitis, and 178 (27%) had placental funisitis. Umbilical cord partial pressure oxygen and base excess values were generally higher in the placental inflammation/clinical chorioamnionitis groups, in keeping with enhanced oxygen delivery and an overall decrease in the metabolic contribution to acidosis attributed to altered lactate metabolism in these infants. After adjusting for confounders (primarily differences in gestational age), the incidence of respiratory distress syndrome was significantly decreased in the placental inflammation/clinical chorioamnionitis groups, in keeping with cytokine-induced synthesis of surfactant proteins in these infants. Although the incidence of bronchopulmonary dysplasia, intraventricular hemorrhage, and periventricular leukomalacia was generally unchanged among the groups studied, that for intraventricular hemorrhage and periventricular leukomalacia was lowest in the placental inflammation/no clinical chorioamnionitis patients and highest in the placental inflammation/clinical chorioamnionitis patients, suggesting a differential effect of clinical chorioamnionitis for these outcomes. CONCLUSION: Overall, infants born preterm with intrauterine infection were better oxygenated and showed less metabolic acidosis at birth and had incidences of respiratory distress syndrome and intraventricular hemorrhage, which were variably lower. Although there are likely threshold levels of inflammatory cytokines that do give rise to adverse outcome, a minimal level of cytokines may also be beneficial for the transition at birth from intrauterine to extrauterine existence when preterm pending the circumstances (ie, exposure to antenatal steroids) and emphasizing the complex relationship among preterm birth, infection, and adverse neonatal outcome. PMID- 16677590 TI - Dexamethasone, HELLP syndrome, and study design. PMID- 16677591 TI - Mode of delivery and postpartum morbidity in Latin American and Caribbean countries among women who are infected with human immunodeficiency virus-1: the NICHD International Site Development Initiative (NISDI) Perinatal Study. AB - OBJECTIVE: The purpose of this study was to test whether cesarean delivery before labor and before ruptured membranes is associated with a higher risk of postpartum morbidity than vaginal delivery among women who are infected with human immunodeficiency virus-1 in Latin America and the Caribbean. STUDY DESIGN: Data from a prospective cohort study (National Institute of Child Health and Human Development International Site Development Initiative Perinatal Study) were analyzed. The study population consisted of women who were followed for > or = 6 to 12 weeks after delivery, who had singleton infants, and with a known mode of delivery. RESULTS: Of 819 enrollees, 697 women met inclusion criteria (299 vaginal deliveries, 260 cesarean deliveries before labor and before ruptured membranes, 138 cesarean deliveries after labor and/or after ruptured membranes); 36 women (5%) had postpartum morbidity (18 major, 18 minor). Mode of delivery was associated with postpartum morbidity (P = .02). Unadjusted odds ratios (95% CIs) for postpartum morbidity according to mode of delivery were cesarean delivery before labor and before ruptured membranes (odds ratio, 1.16 [95% CI, 0.5, 2.7]), cesarean delivery after labor and/or after ruptured membranes (odds ratio, 2.96 [95% CI, 1.3, 6.7]), and vaginal delivery (reference). These results did not differ appreciably with covariate adjustment. CONCLUSION: The rate of postpartum morbidity was low. Mode of delivery was associated with postpartum morbidity, possibly reflecting the larger proportion of minor postpartum morbidity events among those with cesarean delivery after labor and/or after ruptured membranes. PMID- 16677592 TI - Pathologic expression of p53 or p16 in preoperative curettage specimens identifies high-risk endometrial carcinomas. AB - OBJECTIVE: The purpose of this study was to investigate the prognostic impact of p53 and p16 expression in curettage material from patients with endometrial carcinoma. STUDY DESIGN: Preoperative curettage material from a population-based series of 236 endometrial carcinomas from Norway with long and complete follow-up was studied immunohistochemically for p53 and p16 expression. RESULTS: Pathologic expression of p53 and p16 was seen in 24% and 25%, respectively, and was significantly correlated with high International Federation of Gynecology and Obstetrics (FIGO) stage and serous/clear cell histologic subtypes. Pathologic p53 expression showed significant correlation with postmenopausal status, high grade, high tumor cell proliferation, and aneuploidy. Patients with normal expression had 85% 5-year survival compared with 51% and 50% when pathologic expression of p53 and p16, respectively. Five-year survival for patients with 2 pathologic markers was 13%, compared with 67% and 91% for 1 or no pathologic markers, respectively. CONCLUSION: Pathologic expression of p53 and p16 in curettage material identifies high-risk endometrial carcinoma patients with poor prognosis. PMID- 16677593 TI - Rising cesarean delivery rate in primiparous women in urban China: evidence from three nationwide household health surveys. AB - OBJECTIVE: To examine the trend in the rate of cesarean delivery in primiparous women and to analyse individual socioeconomic factors driving the escalation of cesarean delivery rates in urban China. STUDY DESIGN: Data were drawn from three national household health surveys conducted in 1993, 1998, and 2003. This analysis was based on data from the primiparous women only. RESULTS: The cesarean delivery rate in urban cities of China rose from 18.2% in 1990 to 1992 to 39.5% in 1998 to 2002. The adjusted odds ratio for cesarean delivery associated with living in a large city, having university/colleague education, and having health insurance were respectively 2.39, 4.46, and 1.25 in 1998 to 2002. CONCLUSION: The cesarean delivery rate in urban cities of China has been rising dramatically since 1990. Many nonmedical causes related to individual social and economic factors might have played an important role in such a rapid rise of cesarean delivery rates. PMID- 16677594 TI - Audit of preoperative and early complications of laparoscopic lymph node dissection in 1000 gynecologic cancer patients. AB - OBJECTIVE: Establish the reliability and safety of minimal invasive surgery in gynecologic oncology in a large-scale study. Estimate the complication rate on a large sample size. STUDY DESIGN: From December 1998 to November 2004, 1000 gynecologic cancer patients underwent pelvic and/or aortic lymphadenectomies by laparoscopy. A total of 1192 pelvic and aortic lymphadenectomies have been performed: 777 pelvic (757 transperitoneal, 20 extraperitoneal) and 415 aortic lymphadenectomies (155 transperitoneal, 260 extraperitoneal). Main indications for laparoscopic lymph node dissection were: early cervical carcinoma (n = 456), advanced cervical carcinoma (n = 219), vaginal carcinoma (n = 4), endometrial carcinoma (n = 182), and ovarian carcinoma (n = 139). Surgical laparoscopic management via laparoscopy was achieved during the same operative session in 372 patients. RESULTS: No lethality occurred. Thirteen open surgeries (1.3%) were required as a result of failure to complete a satisfactory laparoscopic procedure. Intraoperative, early postoperative complication rate, and lymphocyst formation rate were 2.0%, 2.9%, and 7.1%, respectively. A laparotomy was required for complication in seven patients (7 per 1000), including five returns to operating room. Eleven significant intraoperative vascular injuries occurred, but none required a laparotomy. The most frequently encountered visceral complications were bowel complications (n = 7), urinary tract complications (n = 5), and nerve injuries (n = 5). CONCLUSION: Evidence is given on a large series that laparoscopic lymph node dissection is safe. Laparoscopic surgery may be considered as the gold standard of assessment of the status of regional lymph nodes in gynecologic malignancies. PMID- 16677595 TI - Detection of peritoneal endometriotic lesions by autofluorescence laparoscopy. AB - OBJECTIVE: This study was undertaken to evaluate the feasibility of autofluorescence laparoscopy in the diagnosis of endometriotic lesions. STUDY DESIGN: Prospective analysis of 83 consecutive patients undergoing laparoscopy for suspected endometriosis under white light illumination and autofluorescence diagnosis. The study measured total number of endometriotic lesions diagnosed under white light illumination and with autofluorescence diagnosis. RESULTS: The biopsy-based sensitivity of white light diagnosis alone and white light illumination and autofluorescence for detecting nonpigmented peritoneal endometriotic lesions was 65% compared with 92% (1.42-fold increase). The corresponding specificity was 68% as opposed to 84%. Occult areas of endometriosis were discovered using autofluorescence diagnosis. Statistical analysis was performed with chi2 test and McNemar test. CONCLUSION: Combination of white light illumination and autofluorescence is significantly superior to white light illumination alone in detecting nonpigmented endometriotic lesions. Autofluorescence diagnosis of nonpigmented endometriotic lesions may become an alternative to fluorescence diagnosis after application of 5-aminolevulinic acid, especially because of no side effects. PMID- 16677596 TI - Can a mast cell subtype (MC(T)) play a role in the progression of endometrial cancer through angiogenesis? PMID- 16677597 TI - Colposcopy at a crossroads. AB - New cervical cancer prevention strategies are arising from rapidly improving insight into human papillomavirus (HPV) natural history and cervical carcinogenesis, challenging the conventional roles of cytology and colposcopically directed biopsy as the reference standards of screening and diagnosis, respectively. HPV testing has high sensitivity but mediocre specificity and positive predictive value, making the role of colposcopy for the accurate identification of patients requiring treatment even more important. We believe that deficiencies of the colposcopically guided biopsy must be addressed, in particular, the inaccuracy of biopsy placement leading to low sensitivity for detection of CIN3. This opinion outlines our concerns and summarizes new data, suggesting possible steps that may lead to improvement in colposcopic accuracy. PMID- 16677598 TI - Enhanced cellular binding of concatemeric oligonucleotide complexes. AB - Interaction of oligonucleotides condensed into long concatemeric complexes with cancer cells was investigated. Pairs of 24- and 25-mer oligodeoxyribonucleotides were designed so that they could hybridize and form concatemeric structures. Pre assembling of the oligonucleotides into concatemers considerably enhanced their ability to bind to human embryo kidney 293 cells and neuroblastoma IMR-32 cells as compared to free oligonucleotides. Efficiency of concatemers binding to the cells is improved with increase of the length and concentration of concatemeric complexes. The obtained results suggest incorporation of pharmacologically active oligonucleotides into concatemeric complexes as an approach to improvement of their cellular interaction. PMID- 16677599 TI - The relation between worrying and concerns: the importance of perceived probability and cost. AB - The relation between worrying and individuals' concerns was examined in a sample of 197 college students. Participants described the five undesirable outcomes that they thought about most often, indicated how likely they thought the outcomes were, and how upset they would be by them. Worry severity was measured using the Penn State Worry Questionnaire. The relation between worry severity and the life domains about which individuals were concerned was quite weak. In contrast, as predicted, greater worry was associated with higher probability and cost estimates. In addition, cost estimates moderated the relation between worry severity and probability estimates. The potential importance of perceived threat for understanding worrying is discussed. PMID- 16677600 TI - Verbal, behavioural and physiological assessment of the generalization of exposure-based fear reduction in a spider-anxious population. AB - Clinical fear of spiders was assessed in 39 patients before and after exposure treatment and at a 3-month follow-up. The assessment included behavioral, physiological and self-report measures. Some patients were tested in the treatment context (group Same), while others were tested in a different context (group Different). Both groups demonstrated a significant fear reduction from pre to post-treatment in all measures, which persisted at the 3-month follow-up. Contrary to our expectations, no clear difference was observed between the groups at any moment. However, a 1-year follow-up test of the self-report measure revealed a return of fear in group Same, but not in group Different. These results show (1) that the effects of exposure treatment persisted over contexts and time in the short term, and (2) that conducting treatment and tests in different contexts enhanced efficiency of exposure treatment in the long term. PMID- 16677601 TI - Transforming growth factor-beta1 induces the non-classical secretion of peroxiredoxin-I in A549 cells. AB - Recent studies found that peroxiredoxin-I (Prx-I) is secreted from A549 cells although it does not contain a signal peptide and is known to be a cytosolic protein. Transforming growth factor-beta1 (TGF-beta1) treatment dramatically enhanced Prx-I secretion from A549 cells, and this effect was not inhibited by brefeldin A. Further investigation revealed that A549 cells constitutively secrete TGF-beta1. Furin, a TGF-beta1-converting enzyme, was also highly activated in A549 cells. Ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-PDX), a potent furin inhibitor, blocked both TGF-beta1 activation and Prx-I secretion. Our findings collectively suggest that non-classical secretion of Prx-I is induced by TGF-beta1, which is constitutively activated by furin in A549 cells. PMID- 16677602 TI - Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration. AB - Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9-12h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis. PMID- 16677603 TI - Generation of variable and fixed length siRNA from a novel siRNA expression vector. AB - Small interfering RNA (siRNA) expression vectors using a Pol III promoter fall into two categories, vectors with a single Pol III promoter that express small hairpin RNA (shRNA) and vectors with two head-to-head (convergent) Pol III promoters that express siRNA. There are technical difficulties in preparing convergent siRNA vectors from cDNA. Here, we report construction of a novel convergent siRNA expression vector, pTHUB. Two XcmI sites were inserted between opposing Pol III promoters. After linearization with XcmI, pTHUB has a single 3' A overhang at each end that allows direct cloning of partially DNase I digested cDNA fragments (20-30 bp) tailed with ddT. A derivative method for generating 19 bp siRNA in pTHUB is also described. The suppression efficiency of the pTHUB vector is comparable to those of conventional shRNA vectors. We have made a siRNA library from a single cDNA. The same approach can be used to construct whole genome siRNA libraries from cellular cDNA. PMID- 16677604 TI - Internalization of CD40 regulates its signal transduction in vascular endothelial cells. AB - The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis. PMID- 16677605 TI - Inhibitory effects of epigallocatechin-3-O-gallate on serum-stimulated rat aortic smooth muscle cells via nuclear factor-kappaB down-modulation. AB - The abnormal growth of vascular smooth muscle cells (VSMCs) plays an important role in vascular diseases, including atherosclerosis and restenosis after angioplasty. Although (-)-epigallocatechin-3-O-gallate (EGCG) has antiproliferative effects on various cells, relatively a little is known about precise mechanisms of the inhibitory effects of EGCG on SMCs. In this study, the inhibitory effects of EGCG on attachment, proliferation, migration, and cell cycle of rat aortic SMCs (RASMCs) with serum stimulation were investigated. Also, the involvement of nuclear factor-kappaB (NF-kappaB) during these inhibitions by EGCG was examined. EGCG treatment resulted in significant (p<0.05) inhibition in attachment and proliferation of RASMCs induced by serum. While non-treated RASMCs migrated into denuded area in response to serum and showed essentially complete closure after 36 h, EGCG-treated cells covered only 31% of the area even after 48 h of incubation. Furthermore, EGCG treatment resulted in an appreciable cell cycle arrest at both G0/G1- and G2/M-phases. The immunoblot analysis revealed that the constitutive expression of NF-kappaB/p65 nuclear protein in RASMCs was lowered by EGCG in both the cytosol and the nucleus in a dose-dependent manner. These results suggest that the EGCG-caused inhibitory effects on RASMCs may be mediated through NF-kappaB down-modulation. PMID- 16677606 TI - The intrinsic mechanisms underlying the maturation of programming sequential spikes at cerebellar Purkinje cells. AB - Cerebellum is involved in the motion coordination and working memory, to which the programming of sequential spikes at Purkinje cells is essential. It is not clear about the intrinsic mechanisms underlying spike capacity and timing precision as well as their postnatal maturation. We investigated the programming and intrinsic property of sequential spikes at Purkinje neurons during postnatal development by whole-cell recording in cerebellar slices. Cerebellar Purkinje neurons demonstrate the increasing of spike capacity and timing precision, as well as the lowering of refractory periods and threshold potentials during the postnatal maturation. In addition, the correlation between spike parameters and intrinsic properties converts to be more linear. This postnatal plasticity of neuronal intrinsic properties improves the timing precision and capacity of spike programming at cerebellar Purkinje neurons. PMID- 16677607 TI - Cloning and characterization of a bacterial iterative type I polyketide synthase gene encoding the 6-methylsalicyclic acid synthase. AB - Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6 methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs. PMID- 16677608 TI - Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene. AB - Neurogranin (Ng), a neuronal protein implicated in learning and memory, contains a TATA-less promoter. Analysis of 5'-deletion mutations and site-directed mutations of the mouse Ng promoter revealed that a 258bp 5'-flanking sequence (+3 to +260) conferred the basal transcription activity, and that the GC-rich sequence (+22 to +33) served as an important determinant of the promoter activity. Transient transfection of the Sp1 expression plasmid transactivated the reporter activity in neuroblastoma N2A cells while knocking down of endogenous Sp1 expression resulted in a 2.5-fold reduction of the reporter activity in HEK 293 cells. Exogenous expression of Sp3 in HEK 293 cells, however, repressed the reporter activity by 50%. Nevertheless, by gel shift assays, Sp1 and Sp3 were not found to be responsible for the protein-DNA complexes formed by the GC-rich sequence. Moreover, a nuclear factor from the mouse brain tissues was discovered to bind to multiple AT-rich regions in Ng promoter. PMID- 16677609 TI - Primary structure-based function characterization of BRCT domain replicates in BRCA1. AB - BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53 binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction. PMID- 16677610 TI - PKC phosphorylates MARCKS Ser159 not only directly but also through RhoA/ROCK. AB - It is well recognized that phorbol 12,13-dibutyrate (PDBu)-activated PKC directly phosphorylates myristoylated alanine-rich C kinase substrate (MARCKS), whose phosphorylation is used as a marker of PKC activation. However, in SH-SY5Y neuroblastoma cells, Western blotting analyses revealed that Rho-associated coiled-coil kinase (ROCK)-specific inhibitor H-1152 inhibited PDBu-induced phosphorylation, and that a small G-protein inhibitor, toxin B, also inhibited MARCKS phosphorylation. Furthermore, in GST pull-down assays, PDBu induced RhoA activation in SH-SY5Y cells, and this activation was inhibited by PKC inhibitor Ro-31-8220. Finally, we showed that the transfection of a dominant negative form of RhoA inhibited PDBu-induced MARCKS phosphorylation in immunocytochemistries. These findings suggest that some PDBu-induced MARCKS phosphorylation includes the RhoA/ROCK pathway in SH-SY5Y cells. PMID- 16677611 TI - Enzymatic properties of human CYP2W1 expressed in Escherichia coli. AB - The human genome project revealed a new member of the P450 family 2, CYP2W1, which has orthologous form in other vertebrate species, suggesting CYP2W1's significant physiological function. Recently, it was reported that CYP2W1 can metabolize arachidonic acid. In this study, we isolated human CYP2W1 cDNA, and successfully expressed truncated CYP2W1 lacking N-terminal 20 amino acids in Escherichia coli cells. In the bicistronic expression system for human CYP2W1 and NADPH-P450 reductase, the formation of blue pigment, indigo, was observed in bacterial cultures. Based on this result, we revealed that CYP2W1 catalyzes the oxidation of indole. In addition, CYP2W1 showed monooxygenase activity towards 3 methylindole and chlorzoxazone. However, no activity was observed towards fatty acids including arachidonic acid. Further analysis using an E. coli expression system will reveal substrate specificity of CYP2W1 and why this P450 isoform is universally conserved in vertebrates. PMID- 16677612 TI - Regulation of the human ADAMTS-4 promoter by transcription factors and cytokines. AB - ADAMTS-4 (aggrecanase-1) is a metalloprotease that plays a role in aggrecan degradation in the cartilage extracellular matrix. In order to understand the regulation of ADAMTS-4 gene expression we have cloned and characterized a functional 4.5kb human ADAMTS-4 promoter. Sequence analysis of the promoter revealed the presence of putative binding sites for nuclear factor of activated T cells (NFAT) and Runx family of transcription factors that are known to regulate chondrocyte maturation and differentiation. Using promoter-reporter assays and mRNA analysis we have analyzed the role of chondrocyte-expressed transcription factors NFATp and Runx2 and have shown that ADAMTS-4 is a potential downstream target of these two factors. Our results suggest that inhibition of the expression/function of NFATp and/or Runx2 may enable us to modulate aggrecan degradation in normal physiology and/or in degenerative joint diseases. The ADAMTS-4 promoter would serve as a valuable mechanistic tool to better understand the regulation of ADAMTS-4 expression by signaling pathways that modulate cartilage matrix breakdown. PMID- 16677613 TI - [(35)S]GTPgammaS binding stimulated by endomorphin-2 and morphiceptin analogs. AB - The ability of several mu-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3 (1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [(35)S]GTPgammaS binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [d-1 Nal(3)]Morphiceptin was more potent in G-protein activation (EC(50) value of 82.5+/-4.5 nM) than DAMGO (EC(50)=105+/-9 nM). [d-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [(35)S]GTPgammaS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists. PMID- 16677614 TI - Gamma-aminobutyric acid (GABA)-C receptor stimulation increases prolactin (PRL) secretion in cultured rat anterior pituitary cells. AB - Gamma-aminobutyric acid (GABA) reportedly inhibits secretion of anterior pituitary hormones by directly acting on GABA-A and GABA-B receptors on anterior pituitary cells, but the roles of GABA-C receptors are little known. In this study, involvement of GABA-C receptors in the secretion of prolactin (PRL) was examined using cultured rat anterior pituitary cells. GABA-C receptor agonist, cis-4-aminocrotonic acid (CACA, 0.1-1 mM) increased PRL secretion dose dependently, while GABA-A receptor agonist, 100 microM muscimol, but not GABA-B receptor agonist, 100 microM baclofen, decreased the secretion. GABA-C receptor antagonist, 15 microM (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA), and GABA-A receptor antagonist, 100 microM bicuculline, not only reversed such an agonist-induced increase or decrease in PRL secretion, but also suppressed or enhanced spontaneous PRL secretion, raising a possibility of GABA-C or GABA-A receptor stimulation by intrinsic pituitary-derived GABA. GABA-C receptor subunits (rho1, rho2, rho3) and GABA synthesizing enzymes (GAD 65 and GAD 67) were shown to be expressed as assayed by RT-PCR, and GABA-C receptor stimulation by CACA obviously increased intracellular Ca2+ concentration in the anterior pituitary cells. Thus, PRL secretion from anterior pituitary cells appears to be enhanced via direct GABA-C receptor stimulation by GABA originating from the anterior pituitary cells besides well-known hypothalamic GABA. PMID- 16677615 TI - The Cdc42 inhibitor secramine B prevents cAMP-induced K+ conductance in intestinal epithelial cells. AB - Cyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl- transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba2+-sensitive K+ channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca2+-mediated chloride secretion pathway, which requires a separate K+ channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K+ conductance in intestinal epithelial cells. PMID- 16677616 TI - Molecular evidence of the neuroprotective effect of Ginkgo biloba (EGb761) using bax/bcl-2 ratio after brain ischemia in senescence-accelerated mice, strain prone 8. AB - In the present studies, we investigated the molecular mechanism of one of the active ingredients of Ginkgo biloba, EGb761, to affect the levels of several apoptotic markers in six brain regions following global ischemia in senescence accelerated mice. A 4-day treatment with EGb761 significantly decreased bax/bcl-2 ratios in all brain regions in both young and aged mice. Our findings indicate that the bax/bcl-2 ratio provides a suitable index of apoptosis and modulation of these markers may explain the neuroprotective action of EGb761. PMID- 16677617 TI - Implicit oculomotor sequence learning in humans: Time course of offline processing. AB - Studies of manual and digital sequence learning indicate that motor memories continue to be processed after training has ended, following a succession of identifiable steps. However, it is not known whether this offline memory processing constitutes a basic feature of motor learning and generalizes to the implicit learning of a sequence of eye movements. To assess this hypothesis, we have created the serial oculomotor reaction time task (SORT). Participants were trained to the SORT then tested after either 30 min, 5 h or 24 h. During training, ocular reaction times decreased monotonically over practice of a repeated sequence, then increased when a different sequence was displayed, demonstrating oculomotor learning of the trained sequence. When tested 30 min after training, a significant gain in oculomotor performance was observed irrespective of the sequence learning. This gain was no longer present after 5 h. Remarkably, a gain in performance specific to the learned sequence emerged only 24 h after training. After testing, a generation task confirmed that most subjects learned implicitly the regularities of the sequence. Our results show that, as for manual or digital sequences, oculomotor sequences can be implicitly learned. The offline processing of oculomotor memories follows distinct stages in a way similar to those observed after manual or digital sequence learning. PMID- 16677618 TI - Prenatal stress in the rat alters 5-HT1A receptor binding in the ventral hippocampus. AB - Exposure of a pregnant woman to physical and/or psychological stress might affect her offspring by promoting the development of various learning, behavioral and/or mood disorders in later life. The 5-HT1A and 5-HT2A receptors are prominently implicated in the modulation of anxiety and mood-related behaviors. Using a semi quantitative radiolabel immunocytochemical analysis (immunobinding), we studied the effect of prenatal stress on binding of these two receptor subtypes in the hippocampus of 4-week-old male and female Fischer 344 rats. Levels of 5-HT1A immunobinding in the ventral hippocampus, which is primarily implicated in emotional processing, were significantly decreased in male offspring after prenatal stress. A trend towards a decrease was observed in the ventral hippocampus of females. In contrast, 5-HT1A immunobinding within the dorsal hippocampus, which is mainly related to learning and memory, was not affected by prenatal stress in offspring of either gender. Likewise, no significant differences between control and prenatally stressed rats were observed for levels of 5-HT2A immunobinding in either part of the hippocampus or gender. The observed reduction in hippocampal 5-HT1A receptor binding in male offspring after prenatal stress may have important consequences for adult anxiety- and depressive-like behavior. PMID- 16677619 TI - Neurone specific regulation of dendritic spines in vivo by post synaptic density 95 protein (PSD-95). AB - Post synaptic density protein 95 (PSD-95) is a postsynaptic adaptor protein coupling the NMDA receptor to downstream signalling pathways underlying plasticity. Mice carrying a targeted gene mutation of PSD-95 show altered behavioural plasticity including spatial learning, neuropathic pain, orientation preference in visual cortical cells, and cocaine sensitisation. These behavioural effects are accompanied by changes in long-term potentiation of synaptic transmission. In vitro studies of PSD-95 signalling indicate that it may play a role in regulating dendritic spine structure. Here, we show that PSD-95 mutant mice have alterations in dendritic spine density in the striatum (a 15% decrease along the dendritic length) and in the hippocampus (a localised 40% increase) without changes in dendritic branch patterns or gross neuronal architecture. These changes in spine density were accompanied by altered expression of proteins known to interact with PSD-95, including NR2B and SAP102, suggesting that PSD-95 plays a role in regulating the expression and activation of proteins found within the NMDA receptor complex. Thus, PSD-95 is an important regulator of neuronal structure as well as plasticity in vivo. PMID- 16677620 TI - Microinjection of muscimol into caudal periaqueductal gray lowers body temperature and attenuates increases in temperature and activity evoked from the dorsomedial hypothalamus. AB - Microinjection of the neuronal inhibitor muscimol into the midbrain lateral/dorsolateral periaqueductal gray (l/dlPAG) suppresses increases in heart rate (HR) and mean arterial pressure (MAP) evoked by microinjection of the GABA(A) receptor antagonist bicuculline methiodide (BMI) into the dorsomedial hypothalamus (DMH) in rats. Injection of BMI into the DMH also increases body temperature (Tco) and motor activity. Here, our goal was to extend previous findings by examining the effect of microinjection of muscimol into the PAG on these thermogenic and behavioral responses in conscious freely moving rats. Microinjection of muscimol (300 pmol and 1 nmol) alone into the l/dlPAG reduced baseline Tco without affecting activity, HR, or MAP. Similar injection of a dose that failed to alter baseline Tco (100 pmol) suppressed the increases in Tco evoked from the DMH and significantly attenuated DMH-induced increases in locomotor activity. Whereas microinjection of 1 nmol muscimol into the ldlPAG abolished the increases in Tco evoked from the DMH and in fact lowered body temperature to a degree similar to that seen after this dose of muscimol alone, 1 nmol muscimol at adjacent sites outside the targeted region of the PAG had no significant effect on DMH-induced increases in Tco or any other parameter. These results indicate a role for neuronal activity in the l/dlPAG in (1) the temperature and behavioral responses to disinhibition of neurons in the DMH, and (2) the maintenance of basal body temperature in conscious freely moving rats. PMID- 16677621 TI - Basic fibroblast growth factor promotes nerve regeneration in a C- -ion-implanted silicon chamber. AB - We reported previously that a silicone tube whose inner surface has been implanted with negatively charged carbon ions (C-) enables a nerve to regenerate across a 15-mm inter-stump gap. In this study, we investigated whether a C- -ion implanted tube pretreated with basic fibroblast growth factor promotes peripheral nerve regeneration. The C- -ion-implanted tube significantly accelerated nerve regeneration, and this effect was enhanced by basic fibroblast growth factor. PMID- 16677622 TI - No effect of prolonged corticosterone over-exposure on NCAM, SGK1, and RGS4 mRNA expression in rat hippocampus. AB - Prolonged over-exposure of rats to corticosterone attenuates 5-HT(1A)-receptor mediated responses in hippocampal CA1 cells through an unknown mechanism, not involving downregulation of 5-HT(1A) receptor expression. We here tested if corticosterone changes 5-HT(1A) receptor function indirectly, by altering hippocampal mRNA expression of NCAM, SGK1, or RGS4, which all modulate 5-HT(1A) receptor function. We found that the expression of none of these candidates was affected by corticosterone treatment. PMID- 16677623 TI - Early syntactic productivity: evidence from dative shift. AB - The abstractness of children's early syntactic representations has been questioned in the recent acquisition literature. While some research has suggested that children's knowledge of basic constructions such as the transitive is robust and abstract at a very young age, other work has proposed that young children only have constructions that are specific to individual lexical items. The present paper seeks to resolve this discrepancy by examining children's abstract knowledge of the English dative alternation via a production study. The studies ask whether young children who hear a sentence like I pilked the cup to Petey know that the same meaning can be expressed with the sentence I pilked Petey the cup. This generalization is well-attested in the language that children hear and represents a strong test-case for determining the nature of children's early syntactic representations. The results indicate that three-year-old children have productive knowledge of the English dative alternation, but that their performance can be influenced by small changes in the nature of the task. A preference for the prepositional dative form is also found and the possible reasons for this preference are discussed. PMID- 16677624 TI - Interlinking physical beliefs: children's bias towards logical congruence. AB - Young children's naive beliefs about physics are commonly studied as isolated pieces of knowledge. The current paper takes a different approach. It asks whether preschoolers interlink individual beliefs into larger configurations or Gestalts. Such Gestalts bring together knowledge such as how an object's mass relates to its sinking speed, how an object's volume relates to its sinking speed, and how mass and volume are correlated. The particular form of organization explored here is referred to as logical congruence, the logical correspondence in directions among three physical relations. Are children's guesses about one physical relation congruent with their beliefs about the other two relations? And can they learn a congruent set of relations more readily than an incongruent set? Two different physical domains were explored, one in which children commonly hold pre-existing beliefs, and one in which they are likely to lack such beliefs. The results in both domains show a strong bias towards congruent knowledge configurations in young children. These findings may explain children's difficulties learning inherently incongruous concepts such as density. PMID- 16677625 TI - Chill sensitivity of honey bee, Apis mellifera, embryos. AB - Improved methods for preservation of honey bee, Apis mellifera L., germplasm would be very welcome to beekeeping industry queen breeders. The introduction of two parasites and the emergence of an antibiotic resistant disease have increased demands for resistant stock. Techniques for artificial insemination of queens are available, and semen has been cryopreserved with limited success. However, cryopreservation of embryos for rearing queens would mesh well with current practices and also provide drones (haploid males). Eggs at five ages between twenty-four hours and sixty-two hours were exposed to 0, -6.6, and/or -15 degrees C for various times, and successful hatch measured. Honey bee embryos show chill sensitivity as do other insect embryos, and the rate of chill injury increases dramatically with decrease in holding temperature. The 48 h embryos in both groups showed the greatest tolerance to chilling, although 44 h embryos were only slightly less so. PMID- 16677627 TI - BMP is an important regulator of proepicardial identity in the chick embryo. AB - The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo. PMID- 16677626 TI - C. elegans Kallmann syndrome protein KAL-1 interacts with syndecan and glypican to regulate neuronal cell migrations. AB - The anosmin-1 protein family regulates cell migration, axon guidance, and branching, by mechanisms that are not well understood. We show that the C. elegans anosmin-1 ortholog KAL-1 promotes migrations of ventral neuroblasts prior to epidermal enclosure. KAL-1 does not modulate FGF signaling in neuroblast migration and acts in parallel to other neuroblast migration pathways. Defects in heparan sulfate (HS) synthesis or in specific HS modifications disrupt neuroblast migrations and affect the KAL-1 pathway. KAL-1 binds the cell surface HS proteoglycans syndecan/SDN-1 and glypican/GPN-1. This interaction is mediated via HS side chains and requires specific HS modifications. SDN-1 and GPN-1 are expressed in ventral neuroblasts and have redundant roles in KAL-1-dependent neuroblast migrations. Our findings suggest that KAL-1 interacts with multiple HSPGs to promote cell migration. PMID- 16677628 TI - Delays in neuronal differentiation in Mash1/Ascl1 mutants. AB - The inactivation of a developmental transcription factor may lead to the complete absence of a specific cell type. More commonly, though, it only partially impairs its generation. The modalities of this partial effect have rarely been documented in any detail. Here, we report a novel function for the bHLH transcription factor Ascl1/Mash1 in the generation of the nucleus of the solitary tract (nTS). In Mash1(-/-) late embryos, the nTS is markedly atrophic. Tracing back the origin of this atrophy, we show that nTS precursors appear in the mutants 1 day later than in the wild type and then accumulate at a slower pace. We also show that the previously reported atrophy of the sympathetic chain in Mash1 mutants is similarly preceded by a delay of 1 to 2 days in the appearance of differentiated ganglionic cells. Finally, we provide evidence that the acceleration imposed by Mash1, regardless of the production of post-mitotic cells, affects differentiation itself, both generic and type-specific. PMID- 16677629 TI - Induction and specification of cranial placodes. AB - Cranial placodes are specialized regions of the ectoderm, which give rise to various sensory ganglia and contribute to the pituitary gland and sensory organs of the vertebrate head. They include the adenohypophyseal, olfactory, lens, trigeminal, and profundal placodes, a series of epibranchial placodes, an otic placode, and a series of lateral line placodes. After a long period of neglect, recent years have seen a resurgence of interest in placode induction and specification. There is increasing evidence that all placodes despite their different developmental fates originate from a common panplacodal primordium around the neural plate. This common primordium is defined by the expression of transcription factors of the Six1/2, Six4/5, and Eya families, which later continue to be expressed in all placodes and appear to promote generic placodal properties such as proliferation, the capacity for morphogenetic movements, and neuronal differentiation. A large number of other transcription factors are expressed in subdomains of the panplacodal primordium and appear to contribute to the specification of particular subsets of placodes. This review first provides a brief overview of different cranial placodes and then synthesizes evidence for the common origin of all placodes from a panplacodal primordium. The role of various transcription factors for the development of the different placodes is addressed next, and it is discussed how individual placodes may be specified and compartmentalized within the panplacodal primordium. Finally, tissues and signals involved in placode induction are summarized with a special focus on induction of the panplacodal primordium itself (generic placode induction) and its relation to neural induction and neural crest induction. Integrating current data, new models of generic placode induction and of combinatorial placode specification are presented. PMID- 16677630 TI - Left and right ventricular contributions to the formation of the interventricular septum in the mouse heart. AB - Mammalian heart development involves complex morphogenetic events which lead to the formation of fully separated left and right atrial and ventricular chambers from a tubular heart. Separation of left and right ventricular chambers is dependent on a single structure, the interventricular septum (IVS), which has both muscular and mesenchymal components. Little is known about the morphogenetic events that lead to the formation of the muscular component of the IVS. We have analyzed two transgenic mouse lines that display complementary nlacZ reporter gene expression patterns in the embryonic ventricles: the Mlc1v-nlacZ-24 transgene is expressed in right ventricular myocardium and the Mlc3f-nlacZ-2 transgene in left ventricular myocardium. Detailed analysis of these transgene expression patterns during IVS formation reveals a symmetric left and right myocardial identity within the developing IVS between embryonic days 9.5 and 11.5. From embryonic day 12.5 onwards, myocytes with a left ventricular identity dominate the IVS, particularly in its dorsal aspect. The T-box transcription factor encoding gene, Tbx18, is expressed in the left ventricle and left side of the developing IVS, providing additional support for the presence of left and right ventricular identities within the IVS. Analysis of clonally related cardiomyocyte clusters confirms that both left and right ventricular myocardial cell populations contribute to the forming IVS, in similar domains to those defined by the Mlc-nlacZ transgenes. Examination of the orientation as well as the distribution of labeled cells in clusters provides new insights into the morphogenesis of the septum. PMID- 16677631 TI - Intravitreal injection of endothelin-1 caused optic nerve damage following to ocular hypoperfusion in rabbits. AB - The purpose of this study was to investigate the time course of the ocular hypoperfusion, retinal damage, and optic nerve damage induced by intravitreal injection of endothelin-1 (ET-1) in rabbits. ET-1, at 5 pmol (20 microL, twice a week for 2 or 4 weeks), was injected from the pars plana into the posterior vitreous of the right eye. Optic nerve head (ONH) blood flow and retinal artery diameter, together with the neurofilament light chain (NF-L) content, retinal morphology, and axon density of the optic nerve, were evaluated at 2, 4, and 8 weeks after the first injection of ET-1 (n=7 or 8). Tissue blood velocity in ONH was measured using a laser speckle method, and the diameter of major retinal arteries on the rim of the ONH was calculated from fundus photographs by a masked observer. Histological analysis and immunoblot evaluation of NF-L in the optic nerve were performed to evaluate optic nerve damage. At 2 weeks after the first ET-1 injection, tissue blood velocity was decreased by approximately 20% (versus the contralateral eye), and the diameter of retinal arteries had decreased by approximately 40%. These changes were sustained at the same level until 8 weeks after the first ET-1 injection. At 4 and 8 weeks after the first ET-1 injection, the amount of NF-L in the optic nerve was significantly less in the ET-1 treated eyes than in the contralateral eyes. At 8 weeks after the first ET-1 injection, a loss of myelinated axons and increases in gliosis and connective tissue were noted in the optic nerve of the treated eye, and the optic nerve-axon number had decreased significantly (each, versus the untreated eye). Retinal ganglion cells in the retina were not observed any damage at 2, 4, and 8 weeks after ET-1 injection. In conclusion, intravitreal injection of ET-1 induced chronic hypoperfusion in the ONH and retina, which presumably caused decreases in NF-L content and axon number in the optic nerve noted in the later part of the observation period. PMID- 16677632 TI - Critical role of tryptophanyl residues in the conformational stability of goose delta-crystallin. AB - Delta-crystallin is the major structural protein in avian and reptilian eye lenses but its sequence is highly homologous with the urea cycle enzyme, argininosuccinate lyase (ASL). In previous studies the multi-step unfolding process of this protein in the presence of GdmCl was sensitively probed using tryptophan fluorescence. In this study the contribution of single tryptophan residues to the stability of the local environment was monitored by mutation of two highly conservative tryptophan residues in goose delta-crystallin, Trp 74 and Trp 169. These residues behaved differently in terms of fluorescence intensity and maxima emission wavelength, consistent with their structural location in buried or solvent accessible regions. No gross changes in the secondary structure after mutation were observed, as judged by far-UV CD. The side chains of tryptophan residues in the structure of wild-type goose delta-crystallin possess both hydrophobic and hydrogen bonding interactions. Replacement of the side chain with phenylalanine or alanine led to expose of a hydrophobic area and a reduction in thermal stability; W169A particularly has a T(m) value that was 10 degrees C lower than the wild type enzyme. In the presence of GdmCl, a sharp red shift in fluorescence wavelength due to subunit dissociation can be sensitively detected using a single tryptophan, with the region surrounding W74 undergoing the first transition with a [GdmCl](1/2) of 0.45 M. Further measurement of unfolding curves by CD revealed that the W169A mutant was most unstable with a [GdmCl](1/2) of 0.22 M. From sedimentation velocity analysis, the unstable conformation of the W169A mutant affected the assembly of the quaternary structure. Our studies demonstrate the critical role for the tryptophan residues in stabilizing protein conformations and subunit assembly in delta-crystallin. PMID- 16677633 TI - Retinal synthesis and deposition of complement components induced by ocular hypertension. AB - Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma. PMID- 16677634 TI - Immunohistochemical study of the serotonergic neuronal system in an animal model of the mood disorder. AB - The monoamine theory is one of the major hypotheses about the biological etiology of major depressive disorders. Recent pharmacological and postmortem investigations suggest that depressed patients have alterations in function of serotonergic neuronal system. However, the exact sites of alterations and the association between these alterations and the etiology of the disorder are still unclear. To elucidate these issues, we immunohistochemically examined vesicle monoamine transporter 2 (VMAT2), serotonin receptor type 1a (5HT1a), and serotonin transporter (5HTT) in the hippocampal region of reserpine-treated rats, an animal model of depression. The results showed more VMAT2-immunoreactive varicose fibers in the pyramidal cell layer of hippocampus and parahippocampal cortexes, and more intense 5HTT-immunoreactivity in the pyramidal cell layer and the area CA4 of hippocampus in the animal models compared to those of the controls. On the other hand, lower density of 5HT1a-immunoreactive deposits in the pyramidal cell layer of hippocampus and the parahippocampal cortex was observed in the animal models compared to those of the controls. These results suggest that a deficit of monoamines induces the alterations in the expression of the storage protein, the receptor and the transporter that are involved in the serotonergic neurotransmission in the hippocampal region. These alterations may underlie the changes of serotonergic system observed in the brains of patients with the depressive disorder. PMID- 16677636 TI - Development and validation of the Infertility Self-Efficacy scale. AB - OBJECTIVE: To develop and validate a scale of perceived self-efficacy for people coping with infertility treatment. DESIGN: Self-efficacy scale development involved: [1] item generation with medical experts in reproductive health; [2] a principal components analysis with varimax rotation to identify underlying item components; [3] test-retest reliability and construct (convergent and discriminant) validity with infertility patients, who were administered the Infertility Self-Efficacy (ISE) scale with other measures; and [4] expert acceptability, as determined by reproductive health professionals. SETTING: Recruitment at a fertility center, a national infertility organization, and medical trials web pages. PATIENT(S): A total of 213 participants (159 women; 54 men) with a diagnosis of infertility in the past 2 years were recruited for the various study phases. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Participants' infertility self-efficacy was assessed with an online assessment battery using the ISE scale; comparison mental health measures assessed current levels of fertility problem distress, perceived stress, and coping style. Reliability, validity, and component structure of the ISE was assessed. RESULT(S): Measurement development yielded a brief 16-item ISE scale that taps an infertility patient's perception about his or her ability to engage in a set of cognitive, emotional, and behavioral skills related to the medical treatment of infertility. The single component of "cognitive/affect regulation" suggests this core set of skills is relevant to successfully managing the experience of infertility. The ISE correlations with comparison mental health measures were as expected, suggesting good convergent and discriminant validity. CONCLUSION(S): The ISE scale appears to be a reliable and valid measure of an individual's self confidence in areas related to health promotion during infertility treatment. Assessing an infertility patients' self-efficacy with the ISE may be useful in clinical research and as a counseling tool to help guide patients in actively managing their fertility treatment. PMID- 16677638 TI - Reanalysis of the data--science at its best and always informative. PMID- 16677639 TI - The future of sperm-derived mRNAs. PMID- 16677640 TI - Use of letrozole versus clomiphene citrate combined with gonadotropins in intrauterine insemination cycles: a pilot study. AB - OBJECTIVE: To compare the clinical outcomes between letrozole and clomiphene citrate (CC) in gonadotropin-combined intrauterine insemination (IUI) cycles. DESIGN: Prospective pilot study. SETTING: One university hospital and two private infertility clinics. PATIENT(S): Ninety-three infertile couples eligible for superovulation and IUI. INTERVENTION(S): A letrozole dose of 2.5 mg/day (n = 66) or a CC dose of 100 mg/day (n = 27) was given on day 3-7 of the menstrual cycle, combined with human menopausal gonadotropin (hMG) at a dose 150 IU every other day starting on day 5. MAIN OUTCOME MEASURE(S): The number of mature follicles, serum estradiol (E2) and progesterone (P) levels, endometrial thicknesses on the day of human chorionic gonadotropin (hCG), and clinical pregnancy rates. RESULT(S): The patients' clinical characteristics were comparable between the two groups. The number of mature follicles (3.2 +/- 1.7 vs. 5.6 +/- 2.4) and serum E2 levels on the day of hCG (231.0 +/- 179.8 vs. 1,371.7 +/- 750.5 pg/mL) were significantly lower in the letrozole group. No significant differences were found in endometrial thickness measured on the day of hCG or clinical pregnancy rates (18.2% vs. 25.9%). The rate of patients with serum P levels > 1.0 ng/mL on the day of hCG was significantly lower in the letrozole group (4.5% vs. 25.9%). CONCLUSION(S): Letrozole produced a comparable pregnancy rate vs. CC in gonadotropin-combined IUI cycles. Our results should be confirmed in larger populations with proper randomization. PMID- 16677643 TI - Preimplantation genetic diagnosis as medical innovation: reflections from The President's Council on Bioethics. AB - In its 2004 report, "Reproduction and Responsibility: The Regulation of New Biotechnologies", The President's Council on Bioethics analyzed ethical issues raised by preimplantation genetic diagnosis (PGD) and made recommendations for improvements. PMID- 16677642 TI - Improving the predictive value of the hypoosmotic swelling test in humans. AB - Using a combined hypoosmotic swelling-eosin (HOS-E) technique in human semen samples, we evaluated the frequency of dead swollen spermatozoa (dHOS) after 10 and 30 minutes of incubation, the correlation between total HOS-reactive (tHOS) and viable HOS-reactive (vHOS) spermatozoa with other seminal parameters, and the possibility that dead spermatozoa react to HOS. We obtained the following results: [1] some dead spermatozoa swell under hypoosmotic conditions and [2] HOS E results correlate strongly with other seminal parameters. We recommend that HOS be performed after 10 minutes of incubation because [1] the increase in the incubation time enhances the percentage of dHOS, [2] there are no differences in vHOS percentages between 10 and 30 minutes, and [3] correlation coefficients between vHOS and tHOS with other parameters are very similar at 10 or 30 minutes of incubation. PMID- 16677645 TI - Spuriously elevated serum estradiol concentrations measured by an automated immunoassay rarely cause unnecessary cancellation of in vitro fertilization cycles. AB - OBJECTIVE: To report a case of a patient in whom serum E2, as measured by an automated E2 assay, remained elevated at levels corresponding to the periovulatory phase (>380 pg/mL), despite 3 weeks of treatment by a GnRH agonist (GnRH-a). DESIGN: Case report. SETTING: Tertiary-care academic center. PATIENT(S): A 39-year-old patient accepted for IVF treatment. In view of controlled ovarian hyperstimulation, down-regulation by a GnRH-a was monitored by serum E2 measurement. INTERVENTION(S): Obvious causes for high serum E2 levels (pregnancy, E2-producing ovarian cysts, and noncompliance of the patient) were excluded. The IVF cycle was canceled. MAIN OUTCOME MEASURE(S): Serum samples were analyzed retrospectively by radioimmunoassay (RIA), and ether extraction of the samples was performed before measurement by the automated E2 assay. RESULT(S): Radioimmunoassay revealed adequately suppressed serum E2 levels under GnRH-a treatment (<15 pg/mL). Ether extraction revealed an interfering component that was soluble in the aqueous phase. CONCLUSION(S): If a patient on a long controlled ovarian hyperstimulation IVF protocol still has high serum E2 levels after 2 to 3 weeks of down-regulation, and obvious reasons for this (pregnancy, E2 producing ovarian cysts, noncompliance of the patient) are excluded, E2 immunoassay interference should be excluded to avoid unnecessary cancellation of the IVF cycle. PMID- 16677646 TI - Filariasis infection is a probable cause of implantation failure in in vitro fertilization cycles. AB - OBJECTIVE: To describe a parasitic infection that probably affected the implantation of good-quality embryos in an in vitro fertilization (IVF) cycle. DESIGN: Case report. SETTING: Tertiary care center in a university hospital. PATIENT(S): A 36-year-old Caucasian female with primary unexplained infertility. The patient underwent two cycles of IVF with good-quality embryos transferred; however, no pregnancy ensued despite adequate luteal support. INTERVENTION(S): In vitro fertilization cycles, CBC, blood smear, evaluation for eosinophilia including serological evaluation for parasitic infections. MAIN OUTCOME MEASURE(S): Pregnancy. RESULT(S): Following treatment for filariasis, a repeat IVF cycle using the same stimulation protocol yielded a full-term pregnancy. CONCLUSION(S): This case is of particular importance because, to our knowledge, it is the first to describe a parasitic infection that probably affected the implantation of good-quality embryos in IVF cycles. PMID- 16677648 TI - Effects of xenobiotics and steroids on renal and hepatic estrogen metabolism in lake trout. AB - Experiments were conducted to (1) elucidate the biochemical pathways of E2 metabolism in the lake trout (Salvelinus namaycush) kidney and liver, and (2) test the hypothesis that specific xenobiotics and endogenous steroids inhibit E2 metabolism by these tissues. Kidney and liver tissue fragments from immature lake trout were incubated in vitro in the presence of radiolabelled E2 plus various xenobiotics or steroids. E2 metabolites were identified by liquid chromatography/mass spectroscopy, and quantified by liquid scintillation spectroscopy. A major metabolite produced by both tissues was an unidentified hydroxylated estrogen metabolite (E2-OH) with a molecular mass of 288 that was not estriol (16-OH-E2), but possibly 7alpha-OH-E2 or 2-OH-E2 (catecholestrogen). Both tissues also produced estradiol-17-glucuronide (E2-17-G), estradiol-17 sulfate (E2-17-S), and estradiol-3-glucuronide (E2-3-G). Compared to the kidney, the liver produced half the amount of conjugated metabolites, but twofold more E2 OH. The following xenobiotics (at a concentration of 100 microM) inhibited the production of water-soluble (i.e., conjugated) E2 metabolites by both the kidney and liver: 4,4'-(OH)2-3,3',5,5'- tetrachlorobiphenyl (4,4'-OH-TCB), bisphenol A (BPA), tetrabromobisphenol A (TB-BPA), tetrachlorobisphenol A (TC-BPA), tribromophenol (TBP), trichlorophenol (TCP), and pentachlorophenol (PCP). The alkylphenols, 4-n-nonylphenol (NP), and 4-octylphenol (OP), and 2,2',4,4' tetrabromodiphenyl ether (TBDE) had no significant effect on E2 metabolism by either tissue. Testosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one inhibited the production of conjugated E2 metabolites by both the kidney and liver. Cortisol and 11-ketotestosterone inhibited E2 metabolism by the liver only. The median inhibitory concentrations (IC50) for 4,4'-OH-TCB ranged from 7 32 microM in the kidney and 0.6-1.6 microM in the liver. For BPA, IC50's ranged from 40-108 microM in the kidney and 11-18 microM in the liver. Low doses (0.1 microM) of 4,4'-OH-TCB and BPA significantly increased estrogen metabolism in the kidney. The results suggest that certain estrogenic xenobiotics and endogenous steroids may inhibit the phase II conjugation of E2 by the kidney and liver of lake trout, and some of the known biological effects of these compounds are likely mediated, at least partially, by this mechanism of action. PMID- 16677647 TI - Effects of acute stress on osmoregulation, feed intake, IGF-1, and cortisol in yearling steelhead trout (Oncorhynchus mykiss) during seawater adaptation. AB - Juvenile steelhead (Oncorhynchus mykiss) acclimated to freshwater (FW) were subjected for 3 h to confinement stress in FW, and subsequently saltwater (SW, 25 ppt) was introduced to all tanks. Fish were sampled immediately after the stress treatment, and 1, 7, and 14 days after introduction of SW. Electrolytes, cortisol, glucose, and lactate showed the typical stress response expected after stress treatment in FW. Fish regained osmotic balance within 24 h. Glucose concentrations were increasing throughout the experiment and lactate levels stayed elevated during the time spent in SW. Insulin-like growth factor-1 (IGF-1) did not show an immediate response to stress but after transfer to SW we detected significantly higher concentrations for control fish at days 1 and 14. The differences in IGF-1 levels between stressed and control fish are not reflected in SW adaptability but positive correlations between IGF-1 and electrolyte levels in control fish may indicate its role for osmoregulation. Confinement stress did not impair feed intake subsequently in SW, but our results suggest that feed intake was suppressed by the change of the media from FW to SW. PMID- 16677649 TI - Progesterone withdrawal effects in the open field test can be predicted by elevated plus maze performance. AB - Allopregnanolone (3alpha-hydroxy-5alpha-pregnane-20-one) is a ring-A-reduced metabolite of progesterone, which is naturally produced during the luteal phase of the menstrual cycle, during pregnancy and by stressful events. The steroid hormone inhibits neural functions through increased chloride ion flux through the GABA(A) receptor. The effects and subsequent withdrawal symptoms are similar to those caused by alcohol, benzodiazepines and barbiturates. This study examined the withdrawal effects of progesterone with regards to the influence of individual baseline exploration and risk taking. Rats were tested on the elevated plus maze (EPM) before hormonal treatment, in order to evaluate differences in risk taking and exploration of open and elevated areas. Treatment consisted of ten consecutive once a day progesterone or vehicle s.c. injections. On the last day of treatment, estradiol was injected in addition to progesterone, followed by a 24-h withdrawal before testing in the open field test (OF). Progesterone treated rats showed a withdrawal effect of open area avoidance in the OF. The vehicle-treated control rats showed strong correlations between the EPM and OF parameters. This relationship was not found for the progesterone group at withdrawal. Rats with greater numbers of open arm entrance in the EPM pretest showed an increased sensitivity to progesterone withdrawal (PWD) compared to rats with low exploration and risk taking. The results indicate that the effects of PWD relate to individual exploration and risk taking. Furthermore, the possible analogy of PWD and PMS/PMDD in relation to individual traits is discussed. PMID- 16677650 TI - Characterization and gene expression of Babesia bovis elongation factor-1alpha. AB - Elongation factor 1 alpha (EF-1alpha) is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1alpha promoter has been utilised to drive exogenous gene expression in transfected cells. In this study, we identified and characterised the ef-1alpha locus of Babesia bovis, a causative agent of bovine babesiosis, and examined the transcriptional activity of the EF-1alpha promoter. The ef-1alpha locus in the T2Bo strain of B. bovis contains two identical ef 1alpha genes ('A' and 'B') arranged in a head to head orientation and separated by a 1.4 kb intergenic (IG) region containing a 260 bp terminal inverted repeat. Both ef-1alpha genes encode identical proteins with 448 amino acids and a calculated molecular mass of 49 kDa. While the B. bovis ef-1alpha-IG sequence is conserved among multiple strains of B. bovis, it is not significantly related to any regulatory sequence in the DNA databases. The IG region promotes expression of both ef-1alpha genes. Both fragment Ig-A containing 730 bp upstream of ef 1alpha open reading frame A and fragment Ig-B containing 720 bp upstream of ef 1alpha open reading frame B were able to promote luciferase in transient transfection. In the 5' to 3' orientation, the Ig-B fragment resulted in the highest level of luciferase activity, 10 times higher than positive control plasmid p40-15-luc containing the rap-1 IG region, suggesting that this fragment contains a very strong promoter. Analysis of ef-1alpha transcripts confirms that both ef-1alpha genes are transcribed in merozoites. Interestingly, in contrast to other related intra-erythrocytic apicomplexans, the ef-1alpha locus of B. bovis contains a 160 bp intron in the 5' untranslated region. PMID- 16677651 TI - Remnant particles are the major determinant of an increased intima media thickness in patients with familial combined hyperlipidemia (FCH). AB - INTRODUCTION: Familial combined hyperlipidemia (FCH) is characterized by several phenotypes, including an atherogenic lipoprotein profile, low grade inflammation, visceral obesity, insulin resistance, and hypertension. In this study, we investigated which of the clinical and biochemical characteristics of FCH patients contributes most to their increased risk of cardiovascular disease, by determining the association of a broad variety cardiovascular risk factors with the intima media thickness (IMT) of the common carotid artery (CCA) in 94 FCH patients and 216 non-affected relatives. METHODS: All subjects filled out a questionnaire about their smoking and drinking habits, medical history and medication use, and venous blood was drawn in the fasting state after discontinuation of lipid lowering medication (if used). IMT of the far wall and near wall of both CCAs was measured by high-resolution B-mode ultrasonography. RESULTS: The mean IMT in FCH patients (adjusted for age and gender) was 33 microm thicker when compared to their non-affected relatives (p=0.006). In multivariate analysis, the waist-to-hip ratio, the apoB concentration and the pulse pressure were significant and independent predictors of IMT in both FCH patients and their normolipidemic relatives. The most important independent predictor of IMT in FCH patients however, was the VLDL-c/TG ratio, explaining 32.8% of the variation in age- and gender-adjusted IMT, whereas this ratio was not an independent predictor of IMT at all in their unaffected relatives. CONCLUSION: Abdominal obesity, a higher blood pressure and dyslipidemia characterised by both an increased number of atherogenic particles and the presence of highly atherogenic remnant lipoproteins play an important role in the development of cardiovascular disease in FCH-patients. PMID- 16677652 TI - Circulating oxidized LDL is associated with parameters of the metabolic syndrome in postmenopausal women. AB - Oxidized low-density lipoproteins (ox-LDL) play a significant role in the development of atherosclerosis. OBJECTIVE: To investigate the relation between circulating ox-LDL and components of the metabolic syndrome (MS) in a sample of 124 postmenopausal women with varying glucose tolerance status. METHODS: This cross-sectional study included postmenopausal women not using hormone therapy. Ox LDL concentrations were measured in plasma by a monoclonal antibody (mAb-4E6) based competition ELISA. LDL peak particle diameter (LDL-PPD) was measured by non denaturating polyacrylamide gradient gel electrophoresis (PAGGE). Presence of the MS was determined according to the definition of the NCEP-ATPIII. RESULTS: Circulating ox-LDL concentrations were significantly associated with some factors of the MS such as triglyceride (r=0.48; p<0.0001), high-density lipoprotein cholesterol (r=-0.34; p=0.0001) and fasting plasma glucose concentrations (r=0.21; p=0.02). Ox-LDL concentrations were also associated with LDL cholesterol (r=0.54; p<0.0001), total cholesterol (r=0.48; p<0.0001), LDL apolipoprotein B (r=0.62; p<0.0001) and LDL-PPD (r=-0.18; p<0.05). Moreover, women with the MS had significantly higher ox-LDL concentrations (79.5+/-28.3 U/l) compared to women without the MS (64.2+/-19.9 U/l) (p<0.05). CONCLUSION: Ox-LDL concentrations are associated with individual components of the MS and are significantly higher in postmenopausal women with MS compared to healthy postmenopausal women. PMID- 16677653 TI - Smoking and atherosclerotic cardiovascular disease in women with lower levels of serum cholesterol. AB - This cohort study of Koreans examines the relationship between smoking on atherosclerotic cardiovascular disease (ASCVD) and whether serum levels of total cholesterol modify the impact of smoking on ASCVD. A 10-year prospective cohort study was carried out on 234,399 Korean women, ranging 40-69 years of age who received health insurance from the National Health Insurance Corporation and had a medical evaluation in 1993. The main outcome measures were hospital admissions and deaths from ischemic heart disease (IHD), cerebrovascular disease (CVD), and total ASCVD. At baseline, 13,696 (5.8%) were current smokers and 105,755 (45.1%) had a total cholesterol <200mg/dl. Between 1994 and 2003, 4534 IHD (176/100,000 person year), 7961 CVD (310/100,000 person year), and 2418 other ASCVD events (94/100,000 person year) occurred. In multivariate Cox proportional hazard models controlling for age, hypertension, hypercholesterolemia, diabetes and alcohol drinking, current smoking increased the risk of IHD [hazard ratio (HR)=1.7 (95% CI: 1.5-1.9)], CVD [HR=1.6 (95% CI: 1.5-1.6)], and total ASCVD events [HR=1.6 (95% CI: 1.5-1.7)]. Throughout the range of serum cholesterol levels, current smoking significantly increased the risk of myocardial infarction and CVD, but not angina pectoris. There was no evidence of an interaction between smoking and serum cholesterol (p for interaction=0.469, 0.612, and 0.905 for IHD, CVD, and total ASCVD, respectively). This study demonstrated that smoking was a major independent risk factor for IHD, CVD and ASCVD in Korean women. A low cholesterol level confers no protective benefit against smoking-related ASCVD. PMID- 16677654 TI - Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. AB - There are increasing needs to develop imaging techniques to study in vivo vascular morphology and function in various mouse models of atherosclerosis. Using ultrasound biomicroscopy (UBM), we developed and validated a new imaging protocol to follow lesion progression in atherosclerotic mice. ApoE and LDL receptor double knockout mice (DKO) with various degree of atherosclerosis and normal control mice were imaged at the level of the ascending aorta using UBM. Average plaque thickness, as well as plaque area were delineated in the short axis images, and were subsequently compared with histological measurements. We showed that plaque area at this vascular site was closely correlated to total plaque burden from en face measurement (p<0.0001). UBM-measured plaque thickness and area correlated with indices for histology measures from the same vascular region (p<0.0001 respective p<0.0001). Furthermore, in 16 DKO mice aged from 32 to 35 weeks, UBM showed significantly weekly increases of IMT in the ascending aorta from 0.106+/-0.108 mm at 32 weeks of age to 0.256+/-0.345 mm at 35 weeks of age (p=0.0002). In conclusion, this novel imaging protocol provides us with a non invasive, accurate and inexpensive way to follow lesion progression in mice in vivo. PMID- 16677655 TI - Inhibition of injury-induced arterial remodelling and carotid atherosclerosis by recombinant human antibodies against aldehyde-modified apoB-100. AB - OBJECTIVE: The immune system plays an important regulatory role in the development of atherosclerotic plaques and neointima formation following various types of angioplasty. In the present study we investigated the effect of antibodies against aldehyde-modified apolipoprotein B-100 (apoB-100), a component of oxidized LDL, on atherosclerosis and response to arterial injury in mice. METHODS: The ability of a high affinity human recombinant antibody (2D03), specific for malondialdehyde-modified apoB-100, to influence formation of atherosclerosis as well as remodelling and neointima formation after a collar induced injury of the carotid artery was studied in LDL receptor(-/-) mice over expressing human apoB-100. RESULTS: The antibody recognized epitopes present in mouse plasma and reduced the plasma level of oxidized LDL by 34%. Antibody treatment inhibited injury-induced restrictive vascular remodelling but did not influence the size of the neointima. Atherosclerosis in the uninjured contra lateral carotid artery was determined by computerized image analysis and the mean plaque area in animals given control IgG1 was 7608+/-10,336 micro m(2). In contrast, essentially no plaques were present in animals treated with the 2D03 antibody (397+/-235 micro m(2), P<0.01 versus control IgG1). CONCLUSIONS: Treatment with antibodies against aldehyde-modified apoB-100 dramatically reduces atherosclerosis and inhibits restrictive vascular remodelling in mice expressing human apoB-100. PMID- 16677656 TI - Do allelic variants of the connexin37 1019 gene polymorphism differentially predict for coronary artery disease and myocardial infarction? AB - A C1019T polymorphism in the human connexin37 (hCx37) gene has been associated with cardiovascular risk, but it remains debatable whether the 1019C or the 1019T allele carries this risk. Here, we investigated whether these allelic variants are differentially predictive of increased risk for coronary artery disease (CAD) and myocardial infarction (MI). A total of 781 Swiss participants, including 597 patients diagnosed with CAD, 50% who reported previous MI, and 184 control subjects were genotyped. Patients in the +CAD group had a higher frequency of the Cx37-1019C allele (70.3% versus 65.0%, p=0.004). Multivariate analysis showed that the hCx37-C1019T polymorphism is an independent predictor of CAD (odds ratio=2.13, confidence interval=1.31-3.46 and p<0.01). Moreover, this polymorphism is not associated with any of the other characteristics examined, including gender, age, body-mass-index, diabetes, total/HDL/LDL-cholesterol, triglycerides, apoA-I, apoB, hypertension and cigarette smoking. In comparison with the -CAD group, we observed an increase of the Cx37-1019C allele in the +MI +CAD subgroup (71.2% versus 65.0%, p=0.002) but not in the -MI +CAD subgroup. Allelic frequency comparisons of these three subgroups predicted that this polymorphism is also an independent risk factor for MI. In conclusion, our results reveal the importance of screening the Cx37-1019C allele for both CAD and MI risk assessments. PMID- 16677657 TI - Effect of peak pressure and pressure gradient on subsurface shear stresses in the neuropathic foot. AB - The pressure distribution on the plantar surface of the foot may provide insights into the stresses within the subsurface tissues of patients with diabetes mellitus and peripheral neuropathy (PN) who are at risk for skin breakdown. The purposes of this study were to (1) estimate the stress distribution in the subsurface soft tissue from a measured surface pressure distribution and determine any differences between values in the forefoot and rearfoot, and (2) determine the relationship between maximum shear stress (MSS) (magnitude and depth) and characteristics of the pressure distribution. The measured in-shoe pressure distributions during walking characterized by the peak plantar pressure and maximum pressure gradient on the plantar surface of the feet for 20 subjects with diabetes, PN and history of a mid foot or forefoot plantar ulcer were analyzed. The effects of peak pressure and maximum pressure gradient at the peak pressure location on the stress components in the subsurface soft tissue were studied using a potential function method to estimate the subsurface tissue stress. The calculated MSSs are larger in magnitude and located closer to the surface in the forefoot, where most skin breakdown occurs, compared to the rearfoot. In addition, the MSS (magnitude and depth) is highly correlated with the pressure gradient (r=-0.77 & 0.61) and the peak pressure (r=-0.61 & 0.91). The peak pressure and the maximum pressure gradient obtained from the surface pressure distribution appear to be important variables to identify where MSSs are located in the subsurface tissues on the plantar foot that may lead to skin break down. PMID- 16677658 TI - Instability of the two-layered thick-walled esophageal model under the external pressure and circular outer boundary condition. AB - The mucosal folding is a phenomenon observed for some biological tissues, including the pulmonary airway and gastrointestinal tract. In order to understand the mechanism of the formation of mucosal folding, a thick-walled two-layered cylindrical mathematical model was developed to investigate the buckling behavior under the external pressure and circular outer boundary condition. With the finite element method, the validity and accuracy of the proposed model was verified. The results showed that the fold number was in the range of 4-6, which was agreed with the experimental observation for the mucosal folding of a porcine esophagus. The fold number was found to decrease with the increase in the ratio of the inner to outer material stiffness. The increase in the thickness of inner layer also caused a slight declination of the fold number. Since the effects of both the material and geometrical nonlinearities have been accounted for, this model is more general to be used for the prediction of the buckling behavior of the layered structure with a wide range of thickness ratios and/or stiffness ratios. PMID- 16677659 TI - Rapid and selective characterization of influenza virus constituents in monovalent and multivalent preparations using non-porous reversed-phase high performance liquid chromatography columns. AB - The characterization of influenza vaccine composition has been approached through a novel methodology suitable for routine analysis. It is based on a two-stage process involving an initial sample processing step followed by analysis by reversed-phase HPLC with UV detection. The sample processing involves an initial concentration step carried out in the presence of a combination of detergents and organic solvents to enhance solubilization and ultimately to provide adequate detection. Conditions that provided fast, reproducible and selective separations of vaccine constituents were investigated by reversed-phase HPLC. The use of non porous silica stationary phases was found to minimize carry-over and non-specific adsorption observed with conventional columns. An evaluation of separation parameters, including mobile phase composition and column temperature, allowed optimization of the selectivity of the method. The optimized method was suitable for the characterization of processed monovalent preparations (containing influenza virus constituents from a single strain). In addition, it allowed the simultaneous detection of the three influenza subtypes in trivalent vaccines in a single analysis. Several influenza constituents were detected including nucleoprotein, the highly hydrophobic matrix protein and the primary surface antigen, haemagglutinin (HA). PMID- 16677660 TI - Formation of hydrotalcite in aqueous solutions and intercalation of ATP by anion exchange. AB - The formation reaction and the intercalation of adenosine triphosphate (ATP) were studied for hydrotalcite (HT), a layered double hydroxide (LDH) of magnesium and aluminum. Hydrotalcite with nitrate ions in the interlayer (HT-NO(3)) was formed (A) by dropwise addition of a solution of magnesium and aluminum nitrates (pH ca. 3) to a sodium hydroxide solution (pH ca. 14) until the pH decreased from 14 to 10 and (B) by dropwise addition of the NaOH solution to the solution of magnesium and aluminum nitrates with pH increasing from 3 to 10. The precipitate obtained with method B was contaminated with aluminum hydroxide and the crystallinity of the product was low, possibly because aluminum hydroxide precipitates at pH 4 or 5 and remains even after HT-NO(3) forms at pH above 8. With method A, however, the precipitate was pure HT-NO(3) with increased crystallinity, since the solubility of aluminum hydroxide at pH above and around 10 is high as dissolved aluminate anions are stable in this high pH region, and there was no aluminum hydroxide contamination. The formed HT-NO(3) had a composition of [Mg(0.71)Al(0.29)(OH)(2)](NO(3))(0.29).0.58H(2)O. To intercalate ATP anions into the HT-NO(3), HT-NO(3) was dispersed in an ATP solution at pH 7. It was found that the interlayer nitrate ions were completely exchanged with ATP anions by ion exchange, and the interlayer distance expanded almost twice with a free space distance of 1.2 nm. The composition of HT-ATP was established as [Mg(0.68)Al(0.32)(OH)(2)](ATP)(0.080)0.88H(2)O. The increased distance could be explained with a calculated molecular configuration of the ATP as follows: An ATP molecule is bound to an interlayer surface with the triphosphate group, the adenosine group bends owing to its bond angles and projects into the interlayer to a height of 1 nm, and the adenosine groups aligned in the interlayer support the interlayer distance. PMID- 16677661 TI - Langmuir-Blodgett monolayers of InP quantum dots with short chain ligands. AB - We demonstrate the organization of nearly monodisperse colloidal InP quantum dots at the air/water interface in Langmuir monolayers. The organization of the particles is monitored in situ by surface pressure-surface area measurements and ex situ by AFM measurements on films transferred to mica by Langmuir-Blodgett deposition. The influence of different ligands on the quality of the monolayer formed has been studied. We show that densely packed monolayers with little holes can be formed using short chain ligands like pyridine and pentamethylene sulfide. The advantage of using short chain ligands for electron tunneling to or from the quantum dots is demonstrated using scanning tunneling spectroscopy. PMID- 16677663 TI - Phase behavior, interfacial composition and thermodynamic properties of mixed surfactant (CTAB and Brij-58) derived w/o microemulsions with 1-butanol and 1 pentanol as cosurfactants and n-heptane and n-decane as oils. AB - Phase diagrams of pseudo-quaternary systems of cetyltrimethylammonium bromide (CTAB)/polyoxyethylene(20)cetyl ether (Brij-58)/water/1-butanol (or 1-pentanol)/n heptane (or n-decane) at fixed omega (=[water]/[surfactant]) of 55.6 were constructed at different temperatures (293, 303, 313, and 323 K) and different mole fraction compositions of Brij-58 (X(Brij-58)=0, 0.5, and 1.0 in CTAB + Brij 58 mixture). Pure CTAB stabilized systems produced larger single-phase domains than pure Brij-58 stabilized systems. Increasing temperature increased the single phase domain in the Brij-58 stabilized systems, whereas the domain decreased in the CTAB stabilized systems. For mixed surfactant systems (with X(Brij)=0.5) negligible influence of temperature in the studied range of 293 to 323 K on the phase behavior was observed. Interfacial compositions of the mixed microemulsion systems at different temperature and different compositions were evaluated by the dilution method. The n(a)(i) (number of moles of alcohol at the interface) and n(a)(o) (number of moles of alcohol in the oil phase) determined from dilution experiments were found to decrease and increase respectively for CTAB stabilized systems, whereas an opposite trend was witnessed for Brij-58 stabilized systems. The energetics of transfer of cosurfactants from oil to the interface were found to be exothermic and endothermic for CTAB and Brij-58 stabilized systems, respectively. At equimolar composition of CTAB and Brij-58, the phase diagrams were temperature insensitive, so that the enthalpy of the aforesaid transfer process was zero. PMID- 16677662 TI - Control on size and adsorptive properties of spherical ferric phosphate particles. AB - Ferric phosphate particles were prepared by aging a solution dissolving Fe(ClO4)(3) and H3PO(4) at 40-80 degrees C for 16 h in a Teflon-lined screw capped Pyrex test tube. The spherical or agglomerated fine particles were only precipitated with an extremely fast rate of reaction. The spherical particles were only produced at a very narrow region in fairly low pH solutions. TEM observation revealed that these particles grew in spherical structure by aggregation of primary small particles. The size of spherical particles was decreased by increase in the solute concentration or raising the aging temperature. Therefore, the formation of spherical particles was explained by a polynuclear layer mechanism proposed by Nielsen. The uniform spherical particles produced are amorphous, but they were crystallized to FePO(4) after calcining above 600 degrees C. It was suggested that the voids between the primary particles within the secondary agglomerated particles constitute mesopores. The Fe/P molar ratio determined and weight loss in TG curves gave the chemical formulas of the particles as Fe(PO4)x(H2PO4)y.nH2O (x: 0.93-1.00, y: 0-0.22, n: 2.4-2.7). The amorphous spherical ferric phosphate particles showed a high selective adsorption of H2O by penetration of H2O molecules into ultramicropores, produced after outgassing pretreatment, of that size is smaller than N2 molecule. The more particles grew, the more adsorption selectivity of H(2)O became remarkable. PMID- 16677664 TI - Adhesion force between calcium oxalate monohydrate crystal and kidney epithelial cells and possible relevance for kidney stone formation. AB - AFM interaction force measurements have been performed between calcium oxalate monohydrate crystal (COM) colloidal probes and monolayers of renal epithelial cells (on a polymer substrate) in artificial urine (AU) solutions. The adhesion force was measured for the COM/MDCK cell interaction, while no adhesion force was found for the COM/LLC-PK(1) cell interaction. Long-range repulsive forces for both lines of cells were measured in the range of 2-3 mum. After removal of the cell from the substrate by the AU flow, the basal membrane (BM), with a thickness of 100-200 nm, remained on the substrate. In this case, the shorter-range repulsive forces were found on the extending (approaching) portion of force/indentation curves. Similar to the COM/MDCK cell interaction, the retracting portions of curves for COM/basal membranes have shown the existence of the attractive force of adhesion for the interaction of COM with a BM of MDCK cells, while no adhesion was found for COM/BM LLC-PK(1) cells interaction. No adhesion force was found for the interaction of a BM (of any cells) with the silicon nitride tip. Besides the hydrodynamic reasons, the adhesion difference between LLC-PK(1) and MDCK cells possibly explains the preferential deposition of crystals only in collecting ducts (lined with MDCK-type cells) and the lack of the crystal deposition in the proximal tubules (lined with LLC-PK(1)-type cells). Previous treatments of cells with oxalate alone increased the adhesion force COM/BM MDCK; however, even after oxalate treatment there was small or no adhesion between COM and BM LLC-PK(1) cells. Note that the adhesion force for COM/BM MDCK is practically independent of the probe velocity, i.e., does not have the viscous origin. Evaluation of the adhesion energy shows that this force should be related to the ionic or hydrogen bonds of samples. PMID- 16677665 TI - Steady electro-optic characteristics of noninteracting colloidal particles. AB - In previous papers on the electro-optic effects of beta-FeOOH particles we proposed a new procedure for analysis of the low frequency behavior of charged particles. The procedure is based on comparison of characteristic field intensity curves on an appropriate scale and helps to test the dependence of the slow effects on particle surface electric polarizability (relaxing in the kilohertz domain). The results stimulated us to test the applicability of the method to other samples and to reconsider the literature data on the electro-optic behavior of charged colloids in the hydrodynamic domain. The aim of the present paper is to demonstrate on a series of samples similar features of the electro-optic responses of charged particles in the relaxation interval of particle rotation. The analysis leads to a new hypothesis for explanation of the complicated low frequency behavior of charged particles. The superposition of two slow effects (linear and quadratic with field intensity), relaxing in the relaxation interval of particle rotation, can explain the complicated frequency curves in this domain. One of the slow effects is observed for all polarizable particles. It is of negative sign and displays the features of an induced dipole effect dependent on the "kilohertz" induced moment. It corresponds to a slow stage of the surface polarization process related to electrokinetic charge. The linear slow effect shows permanent dipole like behavior and appears only at certain ionic content of the medium. It shows no direct dependence on counterion mobility and on the "kilohertz" induced moment and is probably due to surface charge nonuniformity. PMID- 16677666 TI - Bubble motion measurements during foam drainage and coarsening. AB - We have studied bubble motion within a column of foam allowed to undergo free drainage. We have measured bubble motion upward with time and as a function of their initial positions. Depending on the gas used, which sets the coarsening and drainage rates, different bubble upward motion types have been identified (constant speed, acceleration or deceleration) and explained in relation with liquid downward flows. The proofs of the consistency between bubble upward motion and liquid downward flow are obtained both by comparing the bubble motion curves to the liquid drainage ones, and by comparing the time variations of the liquid fraction extracted from bubble motion to direct liquid fraction measurements by electrical conductimetry. The agreement between bubble position tracking and electrical conductivity shows in particular that it is possible to determine the drainage regime from such simple bubble motion measurements. This work also allowed us to demonstrate a special case of foam coarsening and expansion, occurring when the foam gas is less soluble than the outside one, caused by diffusion of this external gas into the foam. All these results allow us to build a picture of drainage and coarsening seen from the bubble point of view. PMID- 16677667 TI - Cloud point curve of nonionic surfactant related to the structures of mesoporous materials. AB - We have investigated the phase behavior of a fluorinated surfactant R(7)(F)(EO)(7) in water. The cloud point is situated at 19 degrees C for 2 wt% of surfactant. Using this surfactant, mesoporous materials have been synthesized with micellar solution prepared either at 10 degrees C (below the cloud point) or at 40 degrees C (above the cloud point). Results show that whatever the syntheses conditions, only wormhole-like structure is recovered. The effect of perfluorodecalin addition on the fluorinated surfactant/water system was also investigated. Swollen micelles, microemulsion, and lamellar (L(alpha)) liquid crystals were identified. When perfluorodecalin is added, the cloud point is shifted toward higher temperature. As regards the mesoporous syntheses, perfluorodecalin plays a dual role. First, incorporation of perfluorodecalin leads to the formation of well ordered materials. Secondly, the pore size enlargement occurs when perfluorodecalin is added. Our results evidence that the ratio between the volume of the hydrophilic headgroup (V(H)) and the hydrophobic part (V(L)) of the surfactant is not an efficiency parameter to explain the ordering improvement of mesoporous materials and that we should rather consider the existence of the cloud point curve, which disturbs the cooperative templating mechanism (CTM). PMID- 16677668 TI - The use of social and salience cues in early word learning. AB - This article explores young infants' ability to learn new words in situations providing tightly controlled social and salience cues to their reference. Four experiments investigated whether, given two potential referents, 15-month-olds would attach novel labels to (a) an image toward which a digital recording of a face turned and gazed, (b) a moving image versus a stationary image, (c) a moving image toward which the face gazed, and (d) a gazed-on image versus a moving image. Infants successfully used the recorded gaze cue to form new word-referent associations and also showed learning in the salience condition. However, their behavior in the salience condition and in the experiments that followed suggests that, rather than basing their judgments of the words' reference on the mere presence or absence of the referent's motion, infants were strongly biased to attend to the consistency with which potential referents moved when a word was heard. PMID- 16677669 TI - The N-terminal 12 residue long peptide of HIV gp41 is the minimal peptide sufficient to induce significant T-cell-like membrane destabilization in vitro. AB - Here, we predicted the minimal N-terminal fragment of gp41 required to induce significant membrane destabilization using IMPALA. This algorithm is dedicated to predict peptide interaction with a membrane. We based our prediction of the minimal fusion peptide on the tilted peptide theory. This theory proposes that some protein fragments having a peculiar distribution of hydrophobicity adopt a tilted orientation at a hydrophobic/hydrophilic interface. As a result of this orientation, tilted peptides should disrupt the interface. We analysed in silico the membrane-interacting properties of gp41 N-terminal peptides of different length derived from the isolate BRU and from an alignment of 710 HIV strains available on the Los Alamos National Laboratory. Molecular modelling results indicated that the 12 residue long peptide should be the minimal fusion peptide. We then assayed lipid-mixing and leakage of T-cell-like liposomes with N-terminal peptides of different length as first challenge of our predictions. Experimental results confirmed that the 12 residue long peptide is necessary and sufficient to induce membrane destabilization to the same extent as the 23 residue long fusion peptide. In silico analysis of some fusion-incompetent mutants presented in the literature further revealed that they cannot insert into a modelled membrane correctly tilted. According to this work, the tilted peptide model appears to explain at least partly the membrane destabilization properties of HIV fusion peptide. PMID- 16677670 TI - Structural and genomic properties of the hyperthermophilic archaeal virus ATV with an extracellular stage of the reproductive cycle. AB - A novel virus, ATV, of the hyperthermophilic archaeal genus Acidianus has the unique property of undergoing a major morphological development outside of, and independently of, the host cell. Virions are extruded from host cells as lemon shaped tail-less particles, after which they develop long tails at each pointed end, at temperatures close to that of the natural habitat, 85 degrees C. The extracellularly developed tails constitute tubes, which terminate in an anchor like structure that is not observed in the tail-less particles. A thin filament is located within the tube, which exhibits a periodic structure. Tail development produces a one half reduction in the volume of the virion, concurrent with a slight expansion of the virion surface. The circular, double-stranded DNA genome contains 62,730 bp and is exceptional for a crenarchaeal virus in that it carries four putative transposable elements as well as genes, which previously have been associated only with archaeal self-transmissable plasmids. In total, it encodes 72 predicted proteins, including 11 structural proteins with molecular masses in the range of 12 to 90 kDa. Several of the larger proteins are rich in coiled coil and/or low complexity sequence domains, which are unusual for archaea. One protein, in particular P800, resembles an intermediate filament protein in its structural properties. It is modified in the two-tailed, but not in the tail less, virion particles and it may contribute to viral tail development. Exceptionally for a crenarchaeal virus, infection with ATV results either in viral replication and subsequent cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, probably facilitated by the virus-encoded integrase and this process can be interrupted by different stress factors. PMID- 16677671 TI - Microsecond dynamics of protein-DNA interactions: direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer. AB - The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)70, were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the approximately 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex. PMID- 16677672 TI - Experimental radiofrequency ablation near the portal and the hepatic veins in pigs: differences in efficacy of a monopolar ablation system. AB - BACKGROUND: We sought to compare the efficacy of a monopolar radiofrequency ablation system in vivo near the portal vein and the hepatic veins in porcine liver. MATERIALS AND METHODS: Radiofrequency ablation of healthy livers near the portal vein and the hepatic veins was performed in 10 pigs with a multitined expandable electrode. Volumes and diameters of zones of ablation were assessed by magnetic resonance imaging. RESULTS: Volumes (16.0 +/- 5.5 mL, P = 0.001) and diameters (4.0 +/- 0.7 cm, 3.3 +/- 0.7 cm, 3.0 +/- 0.6 cm, P or=0.8) were noted for aspects of executive function (category fluency, mental manipulation) and verbal learning. Medium effect sizes (0.5or=100 mg/day DCS acts as a functional NMDAR antagonist and may have antidepressant effects. METHODS: Twenty-two treatment resistant major depression patients participated in a double-blind, placebo-controlled 6 week crossover trial with 250 mg/day DCS added to their ongoing antidepressant medications. RESULTS: DCS treatment was well tolerated and resulted in symptom reductions. However, biweekly-performed clinical assessments, including the Hamilton Depression Rating Scale, Hamilton Rating Scale for Anxiety and Zung Self Rating Depression Scale did not reveal statistically significant therapeutic advantages of DCS vs. placebo adjuvant treatment. LIMITATIONS: Small sample, uneven treatment resistance criteria across subjects. The exposure to DCS (dose/length of treatment) may not have been sufficient. CONCLUSIONS: This exploratory study represents the first attempt to assess the effects of a NMDAR GLY site partial agonist in depression treatment. The findings and limitations of this study should be taken into account in the planning of future clinical trials with NMDAR modulators in depression. PMID- 16677715 TI - Combined dexamethasone/CRF test in remitted outpatients with recurrent major depressive disorder. AB - BACKGROUND: Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is a prominent neurobiological finding during a major depressive episode, reflecting a state dependent factor. An issue under investigation is whether the dysfunction of the HPA axis has also a role to play as a state-independent or trait factor for major depressive disorder (MDD). In relation to this, it is important to examine HPA axis function in patients who are clinically remitted from depression. METHODS: Twenty-three remitted outpatients with recurrent MDD and 23 age- and gender-matched control individuals without a history of MDD participated in the sensitive combined dexamethasone/corticotropin-releasing factor (DEX/CRF) test. RESULTS: Free salivary cortisol responses were not significantly different between the two groups, although three patients (13%) displayed extremely elevated cortisol responses after CRF. LIMITATIONS: Limited sample size. All but one patient were under treatment with an antidepressant. CONCLUSIONS: This study shows no evidence for a disturbed DEX/CRF test as a state-independent factor in recurrent MDD on a group level. However, MDD is a complex and heterogenic disorder. Probably, there is a subgroup of patients who show a disturbed DEX/CRF test due to an inherited and/or acquired predisposition or as a biological scar after previous depressive episodes. PMID- 16677716 TI - Effect of dehydroepiandrosterone on lipopolysaccharide-induced interleukin-6 production in DH82 cultured canine macrophage cells. AB - OBJECTIVE: This study examined the effect of dehydroepiandrosterone (DHEA) on lipopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) production in the DH82 canine macrophage cell line. STUDY DESIGN: Cultured DH82 cells were stimulated with varying concentrations of LPS with or without DHEA for various times. Supernatant IL-6 levels were measured by enzyme-linked immunosorbent assay, and cellular cytoplasmic IkappaBalpha protein expression measured by Western blot analysis. RESULTS: LPS dose-dependently stimulated IL-6 production (p=0.016). Cells stimulated with 20 microg LPS showed a time-dependent increase of IL-6 concentration up to 10 h post-treatment (p=0.007). Co-treatment of DH82 cells with 20 microg LPS and various concentrations of DHEA for 14 h showed that up to 10 microM DHEA dose-dependently decreased the IL-6 concentration (p=0.007). Also, addition of 20 microM DHEA to DH82 cells with 20 microg LPS time-dependently decreased the IL-6 concentration for up to 14 h post-treatment (p=0.018). Stimulation of cultured DH82 cells with 20 microg LPS significantly decreased cellular cytoplasmic IkappaBalpha expression, beginning at 30 min post-treatment and persisting to at least 2 h post-treatment (p=0.012). However, co-treatment of cells with 20 microg LPS and 20 microM DHEA abrogated this effect until 2 h post treatment. CONCLUSIONS: DHEA decreased the IL-6 concentration in the supernatant of LPS-stimulated DH82 cells by inhibiting the sequestration of IkappaBalpha, which is necessary for the activation of nuclear factor-kappa B. These findings provide new insights into the immunomodulatory effects of DHEA. PMID- 16677717 TI - A simple method for preparing synthetic controls for conventional and real-time PCR for the identification of endemic and exotic disease agents. AB - Medical and veterinary diagnostic and public health laboratories world-wide are increasingly being called upon to introduce molecular diagnostic tests for both endemic and exotic diseases. This demand has accelerated following increasing terrorism fears. Ironically these same concerns have lead to tightening of both import and export controls preventing many laboratories, particularly those outside of the United States, from gaining access to positive control material. This in turn has prevented many laboratories from introducing much needed molecular diagnostic tests. We describe here a generic approach for preparing synthetic DNA or RNA control material for use in either TaqMan or conventional PCR assays. The production of synthetic controls using this approach does not require cloning or special equipment or facilities beyond that found in any laboratory performing molecular diagnostics. The approach significantly reduces the possibility of contamination or erroneously reporting false-positive reactions due to contamination from positive control material. Synthetic controls produced using this approach have been employed in all molecular diagnostic tests performed in our laboratory and are used irrespective of whether we possess the organism or not. PMID- 16677718 TI - Quantitative real-time PCR assay for detection of human polyomavirus infection. AB - The human polyomaviruses BK (BKV) and JC (JCV) affect immunosuppressed patients and are associated with urogenital tract (BKV) and CNS disorders (JCV) and in humans, the pathogenic role of the rhesus monkey virus, Simian virus 40 (SV40), is uncertain. These three viruses have somewhat overlapping tissue pathogenicity and detection of all three polyomaviruses is desirable. A broadly targeted, simple, single tube real-time degenerated quantitative PCR (QPCR) technique for detection of JCV, BKV and SV40 DNA was developed. To avoid false positive results, due to contamination with commonly used SV40 T-antigen plasmids, a conserved region of the VP2 gene was targeted. Down to 1-10 copies of target DNA per PCR reaction were detected. The QPCR was compared with a nested PCR on 41 clinical samples (urine, serum and plasma): 24 (58.5%) tested positive by nested PCR, whereas 31 (75.6%) were positive with QPCR. One CSF sample, from a patient with progressive multifocal leukoencephalopathy, was negative with the nested PCR but determined as positive by QPCR. Sera from 24 blood donors were negative with QPCR. The QPCR described had a high sensitivity. Its specificity was confirmed sequencing. The QPCR is simple to perform and is valuable for diagnosis of polyomavirus infection. PMID- 16677719 TI - Compulsive-like effects of repeated administration of quinpirole on drinking behavior in rats. AB - We have previously reported that repeated administrations of quinpirole, a D2/D3 dopamine receptor agonist, facilitate instrumental behavior in rats given the choice between operant and free access to water (contrafreeloading: CFL). The goal of the present study was to investigate the effects of repeated daily administrations of quinpirole (0.5 mg/kg i.p.) on the appetitive versus the consummatory component of water-reinforced behavior, under two experimental conditions. Under one condition, the rats were given access to tap water according to an FR3 schedule of reinforcement. Under the second condition, the rats were given the choice between operant and free access to water. Five major findings were obtained. First, acutely quinpirole suppressed operant behavior and, therefore, water intake for at least 1h. Second, upon repeated administrations tolerance developed to the suppressant effect of quinpirole on instrumental behavior but only to a lesser extent to the antidipsic effect, dissociating the appetitive from the consummatory components of water-reinforced behavior. Third, in CFL conditions quinpirole induced a progressively larger preference for the operant access. Fourth, even when the rats were given the choice between free access to highly palatable saccharine (0.05 or 0.01%) solutions and operant access to tap water, quinpirole shifted the animals towards the operant access. Fifth, repeated quinpirole produced lasting consequences on drinking behavior, since after rehydration and under drug-free conditions quinpirole-pretreated rats ingested larger amounts of water than control rats. In conclusion, the repeated activation of D2/D3 receptors appears to induce the rats to perseverate in performing needless instrumental behavior. PMID- 16677720 TI - Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. AB - Repeated systemic administration of low doses of kainic acid (KA) induces spontaneous convulsive seizures [Hellier JL, Patrylo PR, Buckmaster PS, Dudek FE. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 1998;31:73-84]. In this study, male Sprague-Dawley animals received intranigral transplants of a control cell line M213-2O, or a cell line transfected with human GAD67 cDNA (M213-2O CL4) [Conejero-Goldberg C, Tornatore C, Abi-Saab W, Monaco MC, Dillon-Carter O, Vawter M, et al. Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content. Exp Neurol 2000;161:453-61], or no transplant. Eight weeks after transplantation surgery, KA was administered (5 mg/kg/h) until animals reached stage V seizures as described by Racine [Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281-94]. The group transplanted with CL4 required a larger dose of KA and a longer latency to reach a stage V seizure. In addition, this group exhibited significantly fewer stage III and IV seizures. These results indicate that intranigral transplants of a GABA-producing cell line can decrease the number of kainic acid-induced seizures. PMID- 16677722 TI - Effects of early weaning on anxiety and autonomic responses to stress in rats. AB - Environmental stimuli affect various aspects of the early physical and behavioral development in rats. One of the most important events in the early stage of life is weaning, and we recently reported that precocious weaning augments anxiety and aggressiveness in rats and mice. In the present study, we investigated the autonomic responses to stress in two groups of rats: the early-weaned group (weaned at 16 days of age), and the normally weaned group (weaned at 30 days) as a control. First, the early and normally weaned rats were subjected to an elevated plus-maze test to assess their anxiety levels. It was confirmed that early-weaned male rats, but not the females, showed a lower frequency of entry into and shorter duration of stay in the open arms of the maze compared to the normally weaned rats. Subsequently, the two groups were either placed in a novel clean cage or exposed to an unfamiliar conspecific, and their heart rates and core body temperatures were monitored to evaluate their autonomic stress responses. There was an exacerbation of autonomic responses, such as stress induced hyperthermia and tachycardia, and an alternation of behavioral responses, including increased sniffing, and decreased grooming and resting. These effects of early weaning were significant only in males. In contrast, when rats encountered an unfamiliar individual, no significant differences were observed between the two groups in either sex. This suggests that stimuli emanating from an unfamiliar intruder were too intense to detect the augmentation of stress responses in the early-weaned rat. The results of the present study demonstrate that precocious weaning augments, not only behavioral but also autonomic responses, to stressful conditions with sexually dimorphic patterns, i.e. more profoundly in males than in females. PMID- 16677721 TI - Feeding, exploratory, anxiety- and depression-related behaviors are not altered in interleukin-6-deficient male mice. AB - Interleukin-6 (IL-6) has been implicated in behavioral responses associated with inflammation, sickness behavior and various nervous system disorders. We studied a range of different behaviors in IL-6-knockout (IL-6ko) and wild-type (WT) male mice. No significant differences were observed in ambulatory, exploratory, and stereotypic activities in home or novel cages, in an open field (OF), in the multicompartment chamber (MCC), or in the elevated plus-maze (EPM). IL-6ko mice shed fewer fecal boli than WT mice in the OF, in novel cages and in the MCC although this effect was not statistically significant in the OF. In novel cages, intraperitoneal (i.p.) injection of IL-6 (1 microg) depressed ambulatory activity slightly more in IL-6ko than in WT mice. Restraint and interleukin-1beta (IL 1beta, 100 ng i.p.) decreased exploration of mice in the MCC and EPM, but there was no indication of altered sensitivity in IL-6ko mice. No significant differences were detected in the tail suspension and the Porsolt forced swim tests. IL-1beta and lipopolysaccharide (LPS 1 microg i.p.) injection depressed sweetened milk and solid food intake similarly in IL-6ko and WT mice, but IL-6 had no effect, suggesting that IL-6 is not involved in these effects of IL-1 or LPS. However, IL-1beta and LPS depressed body weight more in WT than in IL-6ko mice. Plasma corticosterone and basal concentrations of catecholamines, indoleamines and their metabolites in several brain regions were similar. The responses in these measures to IL-1beta and LPS were also similar, except that there were no significant changes in tryptophan and serotonin metabolism in IL 6ko mice. This may reflect a role for IL-6 in the tryptophan and serotonin responses to IL-1 and LPS. It is concluded that the lack of IL-6 is not associated with substantial alterations in several different mouse behaviors, and in the responses to restraint, IL-1beta, IL-6 and LPS. PMID- 16677723 TI - Double dissociation between hippocampal and prefrontal lesions on an operant delayed matching task and a water maze reference memory task. AB - The hippocampus and prefrontal cortex have both been implicated in various aspects of the acquisition, retention and performance of delayed matching to position (DMTP) tasks in the rat, although their precise respective contributions remain unclear. In the present study, rats were trained preoperatively on DMTP before receiving excitotoxic bilateral lesions of either the entire hippocampus or the medial prefrontal cortex. Rats with lesions of the prefrontal cortex exhibited a significant delay-dependent impairment on retention of the DMTP task, whereas hippocampal lesions were without effect. Rats were also exposed to a switch in the contingencies to a 'non-matching' rule, as an analogue of switching between decision rules in the human Wisconsin Card Sorting Test, in which human patients with prefrontal damage are impaired. Both lesion groups acquired the new contingency at control levels, providing no evidence towards a role for either of these areas in this type of rule-switching. The same rats were also assessed in a spatial reference memory task in the water maze, which revealed an impairment in escape latencies and path length that was specific to the hippocampal lesions. The results corroborate previous evidence that the hippocampus is not necessary for at least some aspects of working memory performance in the DMTP task, whereas the delay-dependent deficit in the prefrontal lesion group support this task as a potentially powerful tool for assessing the cognitive changes associated with frontal damage and repair. PMID- 16677724 TI - Effects of phencyclidine on spatial learning and memory: nitric oxide-dependent mechanisms. AB - Cognitive deficits of schizophrenia constitute a disabling part of the disease predicting treatment success as well as functional outcome. Phencyclidine (PCP), a non-competitive NMDA receptor antagonist was used to model schizophrenic cognitive dysfunctions of learning and memory using the Morris water maze paradigm for reference memory. In experiment 1 male Sprauge-Dawley rats were acutely administered PCP (0.5, 1.0 and 2.0 mg/kg s.c.) before the first swim session on each of the four acquisition days. Probe test for reference memory was performed 2 days after the last acquisition day; the first probe without drug treatment to assess reference memory and a second probe with prior drug treatment to control for state dependency effects of PCP. In experiment 2 the effects of pre-treatment (10 min before PCP) with the nitric oxide synthase inhibitor, L NAME (10 mg/kg s.c.), on the PCP (2 mg/kg)-induced spatial memory deficit was evaluated in the Morris water maze paradigm for reference memory. The results showed that PCP in a dose of 2 mg/kg disrupts spatial learning as estimated by prolonged search time to find platform during acquisition as well as the reference memory test as measured by less time spent in target quadrant during probe trial. No state dependency effects of PCP were found. Pre-treatment with L NAME completely reversed the PCP-induced disruption of acquisition learning. The reference memory disruption was, however, not completely restored as measured by probe trial. PMID- 16677725 TI - Influence of morphine- or apomorphine-induced sensitization on histamine state dependent learning in the step-down passive avoidance test. AB - Effects of morphine- or apomorphine-induced sensitization on histamine state dependent memory of passive avoidance task were examined in mice. Pre-training intracerebroventricular (i.c.v.) administration of histamine (20 microg/mouse) decreased the learning of a one-trial passive avoidance task. Pre-test administration of histamine (10 and 20 microg/mouse) reversed amnesia induced by pre-training of histamine, with maximum response at 20 microg/mouse. Pre-training histamine-induced amnesia was also reversed in morphine- or apomorphine sensitized mice that had previously received once daily injections of morphine (20 and 30 mg/kg) or apomorphine (0.5 and 1 mg/kg) for 3 days. The reversion of histamine-induced amnesia in morphine-sensitized mice was decreased by once daily administration of naloxone (0.5 and 1 mg/kg), SCH 23390 (0.05 and 0.1 mg/kg) or sulpiride (25, 50 and 100 mg/kg) prior to injection of morphine (30 mg/kg/day, 3 days). Furthermore, once daily administration of sulpiride (50 and 100 mg/kg) but not SCH 23390 (0.01, 0.05 and 0.1 mg/kg) prior to apomorphine (1 mg/kg, for 3 days) decreased the reversion of pre-training histamine-induced amnesia by apomorphine. The results suggest that apomorphine or morphine sensitization affects the impairment of memory induced by histamine and thus it is postulated that opioid and dopamine receptors may play an important role in this effect. PMID- 16677726 TI - Oxytocin null mice ingest enhanced amounts of sweet solutions during light and dark cycles and during repeated shaker stress. AB - Central oxytocin (OT) pathways appear to limit consumption of sweet solutions. Male and female C57BL/6 mice that lack the gene for oxytocin (OT KO mice) displayed an initial and sustained enhanced intake of sucrose solution over water compared to wild type (WT) mice when the solutions were presented as a two-bottle choice [Amico JA, Vollmer RR, Cai HM, Miedlar JA, Rinaman R. Enhanced initial and sustained intake of sucrose solution in mice with an oxytocin gene deletion. Am J Physiol: Regul Integr Comp Physiol 2005;289:R1798-806]. In this study we examined the ingestion of a non-nutritive sweetener, 0.2% saccharin in sucrose-experienced OT KO and WT mice given a two-bottle choice between saccharin solution and water available ad libitum for 4 days. Compared to WT mice, OT KO mice consumed significantly greater volumes of saccharin solution during the dark and light photoperiods on the first day and subsequent days of the study. The results were replicated when the experiment was repeated in the same animals. In another experiment, we determined that daily exposure to platform shaker stress did not alter the marked sucrose consumption in OT KO mice. OT KO mice drank significantly more sucrose than WT mice during periods of stress and non-stress. We conclude that the avid consumption of sweetened solutions by OT KO mice is not restricted to a single photoperiod, occurs independent of caloric content of the sweetened solution, and is not altered by exposure to the daily stress of platform shaker. The cumulative results from our studies of sucrose and saccharin ingestion in OT KO and WT male and female mice suggest a special role for sweet taste in the recruitment of OT neurons. PMID- 16677727 TI - Protein synthesis inhibition in the basolateral amygdala following retrieval does not impair expression of morphine-associated conditioned place preference. AB - Conditioned place preference is an animal model used to evaluate the affective properties of natural rewards and drugs of abuse. This animal model is a kind of classical conditioning that depends on learning and memory. The basolateral amygdala (BLA) plays an important role in the consolidation and extinction of memory for this task. However, there is a lack of evidence demonstrating protein synthesis dependent reconsolidation following retrieval in conditioned animals. In other words, is it possible to observe morphine-associated place preference if recall of this preference is disrupted? Accordingly, we investigated this hypothesis by BLA infusion of protein synthesis inhibitor, anisomycin, immediately after retrieval (test) in conditioned place preference paradigm. In the first experiment, the conditioned animals were exposed to the two sides of the apparatus for 15 min in a drug-free state during retrieval. In the second experiment, the animals received an injection of morphine (7.5 mg/kg, i.p.) and immediately after, they were exposed to the two sides of the apparatus for 15 min. Finally in the third experiment, after habituation and training in the conditioned place preference task, the animals received an injection of the unconditioned stimulus (morphine, i.p.; 7.5 mg/kg) followed by confinement for 10 min in the morphine-paired compartment (conditioned stimulus) during memory retrieval. For the three experiments the animals were subsequently exposed in a free-drug state to the two sides of the apparatus for the retest. Our results show that the protein synthesis inhibition in all of these experimental designs had no effect on conditioned place preference memory under conditions that would initiate reconsolidation, suggesting that if reconsolidation of a conditioned place preference task exists it is not mediated by protein synthesis in basolateral amygdala. The effect of anisomycin on consolidation of contextual fear conditioning was also investigated as a positive control to assure that the negative results were not due to methodological problems. Using the same dose of anisomycin (62.5 microg/1 microl) in morphine-associated place preference procedures, we have found that this anisomycin dose blocks the consolidation of contextual fear memory, ruling out the possibility that these negative results can be attributed to methodological problem of some sort. PMID- 16677728 TI - The influence of fractionated radiation therapy on plasma vascular endothelial growth factor (VEGF) concentration in dogs with spontaneous tumors and its impact on outcome. AB - BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF), a specific pro angiogenic factor is proposed to be involved in cancer progression and resistance to radiation therapy by promoting angiogenesis and by protecting endothelial cells from radiation induced apoptosis. The aim of this study, was first to assess the influence of ionizing radiation on plasma VEGF concentration in spontaneous canine tumors during fractionated radiation therapy with curative or palliative intent and second to analyze plasma VEGF concentration as predictor for treatment outcome. PATIENTS AND METHODS: For plasma VEGF analysis a human VEGF enzyme linked immunosorbent assay was used. Sixty dogs with various tumor types were included in this study. Dogs were irradiated with either low dose per fx (3-3.5 Gy per fraction, total dose: 42-49 Gy, group A: curative intent) or high dose per fx (6-8 Gy per fraction, total dose: 24-30 Gy, group B: palliative intent). Blood samples were taken before and after dose application at certain time points during therapy. Follow-up evaluation was performed for analysis of time to treatment failure and survival. RESULTS: Repeated measures analysis showed no increase of plasma VEGF in dogs treated with fractionated radiation therapy (group A and B). Dichotomizing baseline plasma VEGF into two groups with high and low plasma VEGF, resulted in shorter time to treatment failure in dogs with high plasma VEGF levels (TTF, group A: P=0.038, group B: P=0.041). CONCLUSIONS: This study demonstrated that dogs with a plasma VEGF level higher than 5 pg/ml had a poorer outcome after radiation therapy. It is therefore, suggested, to use plasma VEGF as predictor for treatment outcome in radiation therapy. PMID- 16677729 TI - The impact of dental metal artifacts on head and neck IMRT dose distributions. AB - BACKGROUND AND PURPOSES: To quantify the cold or hot spot induced in IMRT treatment plans due to the presence of metal artifact in CT image data sets stemming from dental work. PATIENTS AND METHODS: Metal artifact corrected image data sets of five patients have been analyzed. IMRT plans were generated using five different planning image data sets: (a) uncorrected (UC) (b) homogeneous uncorrected (HUC), (c) sinogram completion corrected (SCC), (d) minimum value corrected (MVC), and (e) image set (d) subsequently corrected with a streak artifacts reduction algorithm (SAR-MVC). The SAR-MVC data set is assumed to be the closest approximation to the absence of metal artifacts and has therefore been taken as the reference image data set. An IMRT plan was generated for each of the image datasets (a)-(e). The resulting IMRT treatment plans for data sets (a)-(d) were then projected onto the reference data set (e) and recalculated. The reference dose distribution (e) was then subtracted from these recalculated dose distributions. Using dose difference analysis, the cold and hot spots in organs at risk (OARs) and the target volumes (TVs) were quantified. RESULTS: When compared to the reference dose distribution, the UC, HUC, and SCC plans exhibited hot spots showing on average more than 1.0 Gy hot dose in the left and right parotids. For the UC, HUC, and SCC recalculated plans, subvolumes of the clinical target volumes (CTV) were under dosed on average by more than 0.9 Gy. On the other hand, the MVC plan showed less than 0.3 Gy hot dose in both parotids, and the cold dose in the CTVs were reduced by up to 0.8 Gy. CONCLUSIONS: The presence of dental metal artifacts in head and neck planning CT data sets can lead to relative hot spots in OARs and relative cold spots in regions of the TVs when compared to the reference data set that more closely approximates the patient anatomy. This effect can be reduced if a simple minimum value correction (MVC) method for the dental metal artifacts is employed. PMID- 16677730 TI - Prognostic value of response to external radiation in stage IIIB cancer cervix in predicting clinical outcomes: a retrospective analysis of 556 patients from India. AB - BACKGROUND AND PURPOSE: To evaluate the prognostic significance of response to external beam radiation (EBRT) in predicting the clinical outcomes in stage IIIB cancer cervix and to find out factors affecting response to EBRT. PATIENTS AND METHODS: This retrospective study included 556 patients of cancer cervix stage IIIB treated between 1996 and 2001 with EBRT (46Gy/23fx/4.5 weeks) followed by intracavitary radiotherapy (ICRT). At the end of EBRT, response to EBRT was grouped as 'no gross residual tumor'(NRT) or 'gross residual tumor'(GRT). RESULTS: Follow up ranged from 2 to 93 months with a median of 36 months. Median dose to point A was 81Gy. At the end of EBRT, 393 patients (70.7%) attained NRT response. NRT responders had significantly better 5 year pelvic control, disease free survival (DFS) and overall survival (OS) than those who had a GRT response (75.6 vs. 54.6%; 60.6 vs. 31.9% and 62.6 vs. 33.7%, respectively; all P values <0.0001). Apart from response to EBRT, overall treatment time also has emerged as an independent factor to affect all clinical outcomes in multivariate analysis but age had significant impact on pelvic control only. Age was the only factor, which significantly influenced the response to EBRT in univariate as well as multivariate analysis (P=<0.001, OR=1.973, 95% C.I. 1.357-2.868). Patients with age more than 50 years had more NRT response (77%) than patients with age less than 50 years (63.8%). CONCLUSIONS: Patients who attain NRT response to EBRT will have an impressive long term pelvic control, DFS and OS in stage IIIB cancer cervix. Older patients (>or=50 years) attain significantly higher NRT rates than younger patients. PMID- 16677731 TI - Somatosensory evoked magnetic fields to air-puff stimulation on the soft palate. AB - Impairment of sensory input to the soft palate has been reported in patients with obstructive sleep apnea syndrome. To investigate the reaction in the central nervous system related to soft palate perception, we measured the somatosensory evoked magnetic fields following air-puff stimulation in seven healthy volunteers by using a helmet-shaped 122-channel neuromagnetometer. The air-puffs were produced using compressed nitrogen and directed to the middle of the soft palate with an intraoral device. To evaluate the laterality of responses we used another appliance in which the air-puffs were directed to the middle and right side of the soft palate. In all the subjects, responses were identified symmetrically in the bilateral parietotemporal regions with a mean latency of about 130 ms from the soft palate stimulation. Prior to this peak, no distinct early responses were observed. There was no significant difference in the responses between the middle and right side stimulation. Corresponding equivalent current dipoles were estimated around the Sylvian fissures. These results suggested that the responses were derived from the second somatosensory areas. In conclusion, we could record long-latency responses to air-puff stimulation of the soft palate in the bilateral second somatosensory areas. PMID- 16677732 TI - Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167. AB - To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected. PMID- 16677733 TI - Human parainfluenza virus type 1 but not Sendai virus replicates in human respiratory cells despite IFN treatment. AB - Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN-beta into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-beta, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN-beta pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV. PMID- 16677734 TI - Identification of penetration enhancers for testosterone transdermal delivery from spray formulations. AB - The goal of this study was to identify a suitable penetration enhancer-containing formulation for the transdermal delivery of testosterone from a spray. The first step involved in vitro measurement of drug flux from a 1:1 ethanol/water saturated solution across hairless rat skin, which had been pre-treated with a series of penetration enhancers. Isopropyl myristate (IPM) was found to be the most efficient excipient, increasing testosterone transport by more than a factor of 5. The enhancing ability of IPM was also apparent when the drug was formulated in 3:1 ethanol/propylene glycol, a more compatible vehicle for use in a spray. IPM was then incorporated into this formulation directly (as opposed to being used to pre-treat the skin) over a range of concentrations from 10-25% v/v, and testosterone transport was evaluated when delivered from either a solution, or from a mechanical spray, or from an aerosol (which also contained 50% v/v propellant). At the highest level of enhancer, the flux was improved 2.5-fold from both the spray and the aerosol, relative to a control. However, these formulations were far from optimally conceived, in that the amount of drug which eventually contacted the skin represented only approximately 10% of the pulverized quantity from the spray, and approximately 40% of that from the aerosol. Repeated application, especially from the aerosol, improved matters somewhat, but further work is clearly required before the concept can be developed for practical application. PMID- 16677735 TI - Primary surgery revisited in very limited small cell lung cancer: does it have a role? A commentary. PMID- 16677736 TI - Recharge processes drive sulfate reduction in an alluvial aquifer contaminated with landfill leachate. AB - Natural attenuation of contaminants in groundwater depends on an adequate supply of electron acceptors to stimulate biodegradation. In an alluvial aquifer contaminated with leachate from an unlined municipal landfill, the mechanism of recharge infiltration was investigated as a source of electron acceptors. Water samples were collected monthly at closely spaced intervals in the top 2 m of the saturated zone from a leachate-contaminated well and an uncontaminated well, and analyzed for delta(18)O, delta(2)H, non-volatile dissolved organic carbon (NVDOC), SO(4)(2-), NO(3)(-) and Cl(-). Monthly recharge amounts were quantified using the offset of the delta(18)O or delta(2)H from the local meteoric water line as a parameter to distinguish water types, as evaporation and methanogenesis caused isotopic enrichment in waters from different sources. Presence of dissolved SO(4)(2-) in the top 1 to 2 m of the saturated zone was associated with recharge; SO(4)(2-) averaged 2.2 mM, with maximum concentrations of 15 mM. Nitrate was observed near the water table at the contaminated site at concentrations up to 4.6 mM. Temporal monitoring of delta(2)H and SO(4)(2-) showed that vertical transport of recharge carried SO(4)(2-) to depths up to 1.75 m below the water table, supplying an additional electron acceptor to the predominantly methanogenic leachate plume. Measurements of delta(34)S in SO(4)(2 ) indicated both SO(4)(2-) reduction and sulfide oxidation were occurring in the aquifer. Depth-integrated net SO(4)(2-) reduction rates, calculated using the natural Cl(-) gradient as a conservative tracer, ranged from 7.5x10(-3) to 0.61 mM.d(-1) (over various depth intervals from 0.45 to 1.75 m). Sulfate reduction occurred at both the contaminated and uncontaminated sites; however, median SO(4)(2-) reduction rates were higher at the contaminated site. Although estimated SO(4)(2-) reduction rates are relatively high, significant decreases in NVDOC were not observed at the contaminated site. Organic compounds more labile than the leachate NVDOC may be present in the root zone, and SO(4)(2-) reduction may be coupled to methane oxidation. The results show that sulfur (and possibly nitrogen) redox processes within the top 2 m of the aquifer are directly related to recharge timing and seasonal water level changes in the aquifer. The results suggest that SO(4)(2-) reduction associated with the infiltration of recharge may be a significant factor affecting natural attenuation of contaminants in alluvial aquifers. PMID- 16677737 TI - Effects of i.c.v. losartan on the angiotensin II-mediated vasopressin release and hypothalamic fos expression in near-term ovine fetuses. AB - Our previous studies have shown that central administration of angiotensin (ANG II) causes arginine vasopressin (AVP) release in the fetus at 70-90% gestation. This is evidence that the hypothalamic-neurohypophysial system is relatively mature before birth. However, few data exist regarding central ANG receptor mechanisms-mediated AVP response during fetal life. To determine roles of brain ANG receptor subtypes in this response, AT1 and AT2 receptor antagonists, losartan and PD123319, were investigated in the brain in chronically prepared ovine fetuses at the last third of gestation. Application of losartan intracerebroventricularly (i.c.v.) at 0.5 mg/kg suppressed central ANG II stimulated plasma AVP release. Losartan at 5 mg/kg (i.c.v.) demonstrated a significant enhancement of AVP increase to i.c.v. ANG II. Associated with the increase of plasma vasopressin levels, c-fos expression in the hypothalamic neurons was significantly different between the low and high doses of losartan. The low dose losartan markedly reduced the dual immunoreactivity for FOS and AVP in the supraoptic nuclei and paraventricular nuclei after i.c.v. ANG II, whereas the high dose losartan together with ANG II, significantly increased the co localization of positive FOS in the AVP-containing neurons than that induced by i.c.v. ANG II alone. Central ANG II induced fetal plasma vasopressin increase was not altered by PD123319. The data suggest that losartan in the fetal brain has remarkably different effects based on the doses administrated on central ANG II related neuroendocrine effects at the late gestation, and that the AT1 mechanism is critical in the regulation of fetal body fluid homeostasis related to plasma AVP levels. PMID- 16677738 TI - Age-related spatial cognitive impairment is correlated with increase of synaptotagmin 1 in dorsal hippocampus in SAMP8 mice. AB - The age-related decline of learning and memory is a common phenomenon in humans and animals, even though the underlying mechanism is not yet known. In the present study, we propose that synaptotagmin 1 (Syt 1) might be a synaptic protein involved in the loss of learning and memory with aging. To test this hypothesis, the age-related spatial cognitive ability of 36 P8 mice (15 mice aged 4 months, 11 mice aged 8 months and 10 mice aged 13 months) was measured in a Morris water maze. After the behavioral test, both the protein and mRNA levels of Syt 1 were determined in the dorsal hippocampus by means of immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. In the Morris water maze, the latency of the 4-month mice to find the submerged platform was significantly shorter than that of the older mice, while there were no significant differences between the 8- and 13-month-old mice in this respect. Compared to the 4-month-old mice, the Syt 1 protein in the 13-month-old mice was significantly increased in almost all layers of each subfield of the hippocampus. The average level of Syt 1 mRNA in the dorsal hippocampus of the P8 mice had not changed with aging. The latency of the 13-month-old P8 mice tested in the Morris water maze was positively correlated with the Syt 1 immunoreactivity in four circuit-specific regions in the dorsal hippocampus. Interestingly, the latency in the Morris water maze was also positively correlated with the level of Syt 1 mRNA in the dorsal hippocampus in individual aged P8 mouse. These results suggest that increased Syt 1 in the dorsal hippocampus in aged mice might be responsible for the age-related impairment of learning and memory. PMID- 16677739 TI - How different is the dietary pattern in non-alcoholic steatohepatitis patients? AB - BACKGROUND/AIMS: This study aimed at evaluating whether patients with non alcoholic steatohepatitis (NASH) had a specific dietary pattern and how it compared with data representative from the same geographical region individuals. SUBJECTS AND METHODS: Clinical, biochemical and anthropometrics: weight, height, body mass index (BMI) and waist circumference were collected in 45 NASH patients. Diet history was assessed using a validated semi-quantitative food frequency questionnaire, analysed with the Food Processor Plus, and was compared, after adjustment for BMI, with data from a sample of 856 free-living individuals, frequency matched for sex and age. RESULTS: Patients' mean age was 49.6+/-10.6 years, 26 F: 19 M, BMI: 31.2+/-5.0 kg/m2. Comparison of their diet history with control data (C) revealed that carbohydrate consumption was lower in patients (P): P-243.6+/-5.7 g vs. C-261.5+/-1.6 g, P<0.05, and most patients had very low fibre intake. Conversely, total fat consumption was higher in patients: P-79.7+/ 1.7 g vs. 73.0+/-0.4, P<0.01. A significantly higher intake of n-6 fatty acids (P=0.003) and n-6/n-3 ratio was found in patients, P<0.001. CONCLUSIONS: Our results suggest that the quality and combination of carbohydrates and fat intake may be more relevant than their isolated amount; an increased fat intake with an excessive amount of n-6 fatty acids can be implicated in promoting necro inflammation, and provides further grounds for individualized dietary therapy. PMID- 16677740 TI - Effects of soluble fiber on matrix metalloproteinase-2 activity and healing of colon anastomosis in rats given radiotherapy. AB - BACKGROUND AND AIMS: Soluble fiber is fermented by colonic microflora yielding short-chain fatty acids (SCFAs) in the colon. We aimed to investigate the effect of oral administration of soluble fiber on healing of anastomosis and matrix metalloproteinase-2 activity in radiotherapy received colonic anastomosis. METHOD: Eighty-four Wistar rats were divided into six groups. All rats were performed a left colonic resection with end-to-end anastomosis. Group I received rat cow. Group II received soluble fiber orally for five consecutive days preoperatively as well as 3rd and 6th days postoperatively. Group III received SCFAs via rectum for five consecutive days preoperatively. Group IV received irradiation to the pelvis at a total dose of 24 Gy on the 10th and 5th days before the operation. Group V was exposed to irradiation like the rats in Group IV and oral treatment like the rats in Group II. Group VI received irradiation like the rats in Group IV and transrectal treatment like the rats in Group III. On the 3rd and 7th postoperative days, all the rats were anesthetized to evaluate the anastomosis healing clinically, histologically and biochemically. RESULTS: Third and 7th day bursting pressures of the rats that were fed with a normal diet and exposed to radiotherapy were significantly decreased (P<0.001). Bursting pressures of Groups V and VI on the 7th day were significantly higher than the control group's bursting pressures (P<0.05). Hydroxyproline levels of Group IV were significantly decreased (P<0.001). Following oral soluble fiber and transrectal administration of SCFAs, these low levels reached to the levels of control radiotherapy group. Matrix metalloproteinase-2 activity of all the rats that were exposed to radiotherapy was higher than the control group (P<0.001). Matrix metalloproteinase-2 enzyme levels in the Groups V and VI were lower than the ones in the Group IV (P<0.001). The histologic parameters of anastomotic healing such as epithelial regeneration, exudate, necrosis, and fibroblast levels were significantly improved by the use of oral soluble fiber and transrectal SCFAs treatment. CONCLUSION: Undesirable effects of preoperative radiotherapy on mechanical, histological and biochemical parameters can be overcome by oral soluble fiber. Oral soluble fiber administration has similar positive effects like the transrectal administration of the SCFA's. PMID- 16677741 TI - The intestinal bacterial colonisation in preterm infants: a review of the literature. AB - The aim of this study is to review the normal development of the intestinal microflora of preterm infants and the factors influencing its development. Preterm infants have an increased intestinal permeability, which may lead to bacterial translocation to systemic organs and tissues. In combination with immaturity of the immune system the risk to systemic infections might be increased. Especially potential pathogenic bacteria are able to translocate. The intestinal microflora of breast-fed term infants, dominated by bifidobacteria and lactobacilli, is thought to suppress the growth of potentially pathogenic bacteria. Many attemps have been made to stimulate the presence of bifidobacteria and lactobacilli with changes in the diet and ingredients-like prebiotics and probiotics. After selection, six studies were included reviewing the intestinal bacterial colonisation of preterm infants. In general, these studies show that the intestinal bacterial colonisation with beneficial bacteria is delayed in preterm infants. The number of potentially pathogenic bacteria is high. Antibiotics influence the intestinal colonisation. Many preterm infants receive prophylactic antibiotics at birth. As antibiotics delay the normal intestinal colonisation, caution should be given to the treatment with broadspectrum antibiotics in preterm infants at birth and every attempt has to be made to restrict the period of treatment. PMID- 16677742 TI - Camel's milk and gastric emptying. AB - BACKGROUND & AIM: Gastric emptying is determined by food consistency, pH, osmolality, lipid and calorie content as well as the presence of different nutrients in the duodenal lumen. Control of gastric emptying is essential for ensuring optimal digestion. The present study tested the hypothesis that due to its different precipitation properties, gastric emptying of camel's milk may be quicker than that of bovine's milk with the same caloric and fat content. METHODS: Gastric emptying was studied by a scintigraphic technique in a randomized, double blind fashion in 8 volunteers after drinking 450 mL of either cow's milk or camel's milk. RESULTS: No differences in gastric emptying rates were found between the two types of milk. The percentage of retention of the meal after 60 min was 74.6+/-13.2% for cow's milk and 79.8+/-10.8% for camel's milk. The 50% emptying time (T(1/2)) was 131.8+/-37.4 min for cow's milk and 136.8+/ 55.8 min for camel's milk. CONCLUSIONS: Camel's milk is most probably not a useful substitute for other types of milk to shorten gastric emptying. PMID- 16677743 TI - Improved immunogenicity of a H44/76 group B outer membrane vesicle vaccine with over-expressed genome-derived Neisserial antigen 1870. AB - A broadly protective vaccine against meningococcal group B disease is not available. We previously reported that an outer membrane vesicle (OMV) vaccine containing over-expressed genome-derived antigen (GNA) 1870 elicited broader protective antibody responses than recombinant GNA1870 or conventional OMV vaccines prepared from a strain that naturally expresses low amounts of GNA1870. Certain wildtype strains such as H44/76 naturally express larger amounts of GNA1870 and, potentially, could be used to prepare an improved OMV vaccine without genetic over-expression of the antigen. We transformed H44/76 with a shuttle vector to over-express variant 1 (v.1) GNA1870 and compared the immunogenicity in mice of OMV vaccines prepared from wildtype H44/76 (v.1), the mutant, and a recombinant v.1 GNA1870 vaccine. Mice immunized with OMV with over expressed GNA1870 developed broader serum bactericidal and/or greater C3 deposition activity on the surface of encapsulated strains of N. meningitidis than control mice immunized with the OMV vaccine prepared from the wildtype strain, or the rGNA1870 vaccine. When a panel of group B strains from patients in California was tested, sera from mice immunized with the OMV vaccine containing over-expressed GNA1870 were bactericidal against 100% of the v.1 strains. In contrast, only 20% of isolates that expressed subvariants of the v.1 GNA1870 protein were susceptible to bactericidal activity of antibodies elicited by the rGNA1870 or conventional OMV vaccines. Thus, even a modest increase in GNA1870 expression in a strain that naturally is a high producer of GNA1870 results in an OMV vaccine that elicits broader protection against meningococcal disease. PMID- 16677744 TI - Interesting case: Spontaneous exfoliation of an upper central incisor through the nasal floor. PMID- 16677745 TI - Facial nerve morbidity after retrograde nerve dissection in parotid surgery for benign disease: a 10-year prospective observational study of 136 cases. AB - We evaluated the facial nerve function in 136 patients who had had retrograde nerve dissection during parotidectomy for benign disease. One week after the operation 90 patients (66%) had some weakness of the facial nerve. After 1 month 52 (38%) had facial nerve paresis. After 3 months 114 (84%) had recovered fully and 21 (16%) had minor nerve paresis. After 6 months 135 (99%) had normal nerve function. One patient had persistent marginal mandibular nerve paresis. PMID- 16677746 TI - Childbirth policies and practices in Ireland and the journey towards midwifery led care. AB - OBJECTIVE: To describe the dominant policies and practices that have governed childbirth in Ireland, and to outline the progress made towards the introduction of midwifery-led care in one health region. LITERATURE REVIEW: A review of maternity-care policies in Ireland was conducted using government and regional health-authority documents and two historical reviews of government policies. A search was also carried out in PubMed and cinahl databases, using the keywords 'maternity care', 'childbirth', 'policy', 'midwifery-led', 'Ireland/Irish', with relevant Boolean and string operands. Childbirth as a social process is influenced by the model of care, and affects the physical and psychological outcomes for the woman and her family. In Ireland, routine intervention in labour is common, but, since the early 1990s, some changes in the Irish maternity services have taken place. Pilot projects on community midwifery have been introduced in some areas. Challenges to the provision of maternity care in the Health Service Executive, North Eastern area (formerly the North-Eastern Health Board) led to the production of the Kinder report, which included a recommendation to introduce pilot midwifery-led units (MLUs). THE INTRODUCTION OF MIDWIFERY-LED CARE: A Maternity Services Taskforce was established in January 2002 with a wide remit, including facilitation of the establishment of MLUs in Cavan General Hospital, Cavan and Our Lady of Lourdes Hospital, Drogheda, Co. Louth. The MLUs are being evaluated within the context of a randomised trial known as 'the MidU study', which compares midwife-led care with the present system of medical-led care for women who are at low risk of complications during pregnancy and labour. CONCLUSION: The journey to midwifery-led care in Ireland has been a long one. The phased introduction of MLUs, which are subject to rigorous evaluation, will provide quality evidence upon which to base the future development of maternity care across Ireland. PMID- 16677747 TI - Use of the genus Artemia in ecotoxicity testing. AB - Information related to varied uses of several species of the genus Artemia (commonly known as brine shrimp), is dispersed among literature from several scientific areas, such as Ecology, Physiology, Ecotoxicology, Aquaculture and Genetics. The present paper reviews information related to Artemia that may be considered relevant for ecotoxicity testing. Integration of different areas of scientific knowledge concerning biology, life cycle and environmental needs of Artemia is of crucial importance when considering the interpretation of results drawn from tests involving this genus. Furthermore, this paper provides suggestions to overcome problems related to toxicity assessment with the use of Artemia as test organism in bioassays, under the scope of estuarine, marine and hypersaline environments. Aspects related to variability in results, adoptable toxicity end-points, culture conditions, characteristics of species and strains, influence of geographical origins over physiological features and responses to exposure to chemical agents are considered. PMID- 16677748 TI - Corpus callosum size is linked to dichotic deafness and hemisphericity, not sex or handedness. AB - Individuals differ in the number of corpus callosum (CC) nerve fibers interconnecting their cerebral hemispheres by about threefold. Early reports suggested that males had smaller CCs than females. This was often interpreted to support the concept that the male brain is more "lateralized" or "specialized," thus accounting for presumed male predominance in mathematics, as well as for aggressive behavior. Ultimately, meta-analyses of these many reports found no significant overall sex differences in inter-cerebral information carrying capacity. Here, using quantitative MRI, we found the midline CC area of 113 subjects was significantly correlated, not with handedness or sex, but with dichotic deafness, and even more so with redefined hemisphericity, the latter accounting for over 19% of CC variability. That is, both dichotic hearing and right brain-oriented individuals of either sex had significantly larger CCs than dichotically deaf or left brain-oriented persons. Thus, current traditions of brain laterality and gender may benefit from revisions that include redefined hemisphericity. PMID- 16677749 TI - Neurochemical alterations of the brain in bipolar disorder and their implications for pathophysiology: a systematic review of the in vivo proton magnetic resonance spectroscopy findings. AB - OBJECTIVE: To perform systematic analysis of current proton magnetic resonance spectroscopy ((1)H MRS) findings in bipolar disorder (BD). METHOD: We grouped the (1)H MRS studies documenting data on the metabolites of N-acetylaspartate (NAA), Choline (Cho), myo-inositol (mI), Glutamate (Glu)/Glutamine (Gln) and Creatine (Cr) separately, for each of the euthymic, manic, depressed adult and child/adolescent bipolar patients. RESULTS: For NAA resonance, 22 studies involving 328 adult bipolar and 349 control subjects were identified. NAA levels were lower in euthymic bipolar patients in the frontal lobe structures and hippocampus. Lithium seems to have an increasing effect on NAA in those brain regions. Available data in children indicates lower NAA levels in euthymic bipolar patients in dorsolateral prefrontal cortex (DLPFC) and cerebellar vermis. Existing data over 25 studies on 366 adult bipolar and 393 control subjects, although inconsistent, may suggest higher Cho/Cr ratios in the basal ganglia (BG) of euthymic bipolar patients. The metabolite mI seems to be increased both in euthymic and manic bipolar children, while most of the available data does not support such alteration in adults. Glu/Gln levels in adult bipolar patients were higher in all mood states compared to controls. Limited data in children supports such an alteration only in the euthymic state. CONCLUSION: The studies reviewed in this paper suggest regional abnormalities of NAA, Cho and Glu/Gln in BD, with the DLPFC, prefrontal and anterior cingulate cortices, hippocampus, and BG being specifically implicated. Systematic analysis of (1)H MRS findings so far helps to define future strategies in this field for delineation of actual neurochemical framework in BD. PMID- 16677750 TI - [Vertical reduction mammaplasty for gigantomastia with massive fibroadenomatosis: a case report]. AB - Vertical reduction mammaplasty is one of the most debated << short-scar >> breast reduction technique. Advantages and drawbacks of the technique are discussed; most of the authors do not accept it as the technique of choice for high glandular resection weights. In our case report we achieve it for a resection weight up to two kilograms with an areolar transposition distance of more than ten centimetres. We show that it is reasonable to realize it dealing with gigantomastia. The massive fibroadenomatosis is observed following immunosuppressive treatment for kidney transplantation. Cyclosporine intake, even sporadic, is at the origin of the growth of these multiple, bilateral and large fibroadenomas. Drug-induced cytokines stimulate their development. PMID- 16677751 TI - Dimethylarsinic acid: results of chronic toxicity/oncogenicity studies in F344 rats and in B6C3F1 mice. AB - Dimethylarsinic acid (DMA(V), cacodylic acid), a foliar herbicide, was administered in the diet to B6C3F1 mice (at dose levels of 0, 8, 40, 200, and 500 ppm) and to F344 rats (at dose levels of 0, 2, 10, 40, and 100 ppm) for 2 years, according to US EPA guidelines. In mice, there were no treatment-related tumors observed at any site. Treatment-related progressive glomerulonephropathy and nephrocalcinosis were observed in the kidneys in both sexes. The incidence of vacuolation of the epithelium in the urinary bladder was increased in both sexes, but was not associated with cytotoxicity, necrosis or hyperplasia. Based on non neoplastic lesions found in the urinary bladder, the NOEL for mice was assessed to be 40 ppm in males and 8 ppm in females. In rats, treatment-related mortality occurred early in the study in five males in the 100 ppm group and in one male in the 40 ppm group. Papillomas and carcinomas with degeneration of the urothelium, necrosis and urothelial cell hyperplasia, were found in the urinary bladders of both sexes. In male rats, one papilloma was found in each of the 10 and 40 ppm groups; one urothelial cell carcinoma was found in the 2 ppm group and two in the 100 ppm group. Four papillomas and six urothelial cell carcinomas were found in the female 100 ppm group. Non-neoplastic treatment-related kidney lesions were confined to the 40 and 100 ppm levels and included necrosis, pyelonephritis, medullary nephrocalcinosis and tubular cystic dilation, hyperplasia of the epithelial lining of the papilla, and pelvic urothelial cell hyperplasia. All of these kidney changes appear to be related to an increase in the aging nephropathy of the rat. Dose-related increases in the height of the thyroid follicular epithelium were also noted in males and females, however, such changes reflect an adaptive response of the thyroid to decreased levels of circulating thyroid hormone, rather than an adverse effect. Based on the kidney and bladder lesions, the NOEL for non-neoplastic and neoplastic lesions was considered to be 10 ppm in males and females. Based on these studies, DMA(V) is carcinogenic only in rats and only at relatively high doses, with the urinary bladder as the target organ. Female rats appear to be more sensitive to the effects of DMA(V) than male rats. DMA(V) is not carcinogenic in mice. PMID- 16677752 TI - Cardiopulmonary collapse associated with malpositioning of an adjustable flange tracheostomy tube. PMID- 16677753 TI - Anatomical conditions for pelvic floor reconstruction with polypropylene implant and its application for the treatment of vaginal prolapse. AB - OBJECTIVE: The purpose of the surgical treatment of vaginal prolapse is not only the restoration of the anatomy but also of the visceral functioning. To maintain the quality of life for patients with recurrent vaginal prolapse, to reduce the failure rates of operations and to avoid a colpectomy or a colpocleisis at the same time, synthetic materials have been introduced in transvaginal reconstructive surgery of the pelvic floor. The TVM Group from France described the reconstruction of the pelvic floor with polypropylene implants in 2004. The aim of this study is to determine the anatomical position of the polypropylene implants after reconstruction of each compartment of the pelvic floor and to determine the relation of the implants to the major neighbouring neurovascular structures on the basis of corpse dissections. STUDY DESIGN: Following the technique of the TVM Group from France we present the pelvic floor reconstruction using Gynecare Prolift* (Ethicon, Sommerville, NJ, USA). To reach the aims of the study, anatomical dissections of the pelvic floor on three specially preserved anatomical specimens are performed after the placement of the implants. RESULTS: The anatomical dissections show that every defect in all three compartments of the pelvic floor can be repaired by using polypropylene implants. Between the implants and the major neighbouring neurovascular structures a safe distance exists with slight individual differences. CONCLUSION: The pelvic floor reconstruction using polypropylene implants is a treatment option especially for the surgical correction of the recurrent vaginal prolapse. If the surgeon has thorough anatomical knowledge and performs the surgical technique in the recommended manner, injuries of the major neighbouring neurovascular structures will be avoided. Clinical studies will analyze the long-term results after pelvic floor reconstruction using polypropylene implants. PMID- 16677754 TI - A focus group study of factors that promote and constrain the use of satellite derived fire products by resource managers in southern Africa. AB - Semi-structured focus group interviews were employed to examine factors that affect the likelihood that resource managers in southern Africa will use information on vegetation fires provided by two satellite-derived products: an active fire product and a burned area product. The two products are updated regularly and aim to deliver the state-of-the-art in the global monitoring of fires from satellite remote-sensing. Both products are derived from data transmitted by the Moderate Resolution Imaging Spectroradiometer (MODIS) sensors carried onboard NASA's Aqua and Terra satellites. The active fire product can be accessed for free via the internet and on media by users working anywhere in the world; the burned area product will be accessible in a similar manner in 2006. The MODIS fire products provide systematic, near-global coverage and are freely available; as such, they give resource managers new opportunities to obtain or supplement information they need to manage vegetation fires effectively. However, the availability of these products does not mean that resource managers will use them, and many other factors are involved. To understand factors that affect whether southern African resource managers will use the two products, two focus groups were held with members of the Southern African Fire Network (SAFNet) in Malawi, Africa, August 2004. Analysis of the group discussions reveals a number of factors that influence whether they will use the products. The qualitative, in depth nature of the group discussions revealed 12 main factors that influence product use; not least the low international internet bandwidths for African countries outside of South Africa. Analysis of the group discussions also suggests how the uptake of MODIS fire products by resource managers in southern Africa might be enhanced by affecting specific changes to how MODIS products are packaged and delivered. PMID- 16677755 TI - Importance of xeroderma pigmentosum group D polymorphisms in susceptibility to ovarian cancer. AB - The purpose of this study was to evaluate the role of XPD genotypes as genetic indicator of susceptibility to ovarian cancer. We have used a case-control study. We analysed DNA samples from 141 ovarian cancer patients and 202 control subjects, for three XPD polymorphisms using PCR-RFLP. We observed that Asn312Asn XPD genotype carriers have increased susceptibility of ovarian cancer (OR=2.46 95% CI 1.20-5.06; P=0.015). Furthermore, we found that carriers of Gln751Gln XPD genotype have an increased susceptibility of ovarian cancer (OR=3.40 95% CI 1.61 7.15; P=0.001). Asn312Asn and Gln751Gln are particularly associated with an early stage of disease. Our results suggest an important role for Asn312Asn and Gln751Gln XPD polymorphisms in the susceptibility to ovarian cancer. PMID- 16677756 TI - Development of a CA125-mesothelin cell adhesion assay as a screening tool for biologics discovery. AB - Preventing peritoneal implantation of ovarian carcinoma cells could prolong patient remission and survival. CA125 is expressed on most ovarian cancer cells and was reported to be a ligand of mesothelin, a peritoneal protein. We developed a cell adhesion assay with CA125-expresser ovarian cancer cells and human mesothelin-transfected cells and we confirmed that CA125 and mesothelin mediate cell attachment. We also showed that this assay supplies a high-throughput screening system for reagents able to block CA125/mesothelin-dependent cell attachment with a sensitive quantitative readout. We finally demonstrated that a mesothelin chimeric protein and anti-CA125 antibodies block CA125/mesothelin dependent cell attachment. PMID- 16677757 TI - KCNE2, a down-regulated gene identified by in silico analysis, suppressed proliferation of gastric cancer cells. AB - It is important to identify the differentially expressed gene in gastric cancer for elucidating the molecular mechanisms of tumorigenesis of stomach. Here, 38 genes differentially expressed genes between gastric cancer and normal gastric mucosa by in silico approaches. A potassium channel protein KCNE2, identified as a down-regulated gene in gastric cancer, was chosen for further study. We investigated the expression of KCNE2 in gastric cancer tissues and cell lines and examined the effect of KCNE2 on proliferation of gastric cancer. The expression of KCNE2 was markedly down-regulated in gastric cancer tissues and cell lines. Forced overexpression of KCNE2 suppressed the growth of SGC7901 cells and cell cycle progression significantly, which might be related to the down-regulation of Cyclin D1. KCNE2 also inhibited SGC7901 cell growth in soft agar and its tumorigenicity in nude mice. Taken together, our work showed that in silico analysis approaches could be used to identify cancer-related genes effectively. KCNE2, as a novel down-regulated gene in gastric cancer, suppressed cell proliferation and tumorigenesis of stomach. PMID- 16677758 TI - Anomalous transient leaching behavior of metals solidified/stabilized by pozzolanic fly ash. AB - This study presents observations on the transient leaching behavior of chromium, cadmium, and aluminum that were solidified/stabilized by pozzolanic fly ash. These three metals were selected since they were present in a simulated waste stream generated by an evaporator during plutonium purification and also because the minimum solubility of these metals occurs at significantly different pHs. The transient pH behavior of the toxicity characteristic leaching procedure (TCLP) leachate showed a monotonic increase for all cases, but the equilibrium value was affected by process conditions. The transient leachate concentration behavior showed curves with one or two local maxima for some cases and curves with a monotonic increase for other cases. Data from the leaching experiments was compared to the solubility curves for the hydroxides of each metal since it was assumed that the highly alkaline conditions inside the fly ash waste would cause the metals to precipitate as hydroxides after initially dissolving in the acidic leaching solution. It was found that of the three metals, only cadmium followed the solubility curve for pure hydroxide solutions or for fly ash systems currently reported in the literature. PMID- 16677759 TI - Synthesis, characterization and thermolysis studies on triazole and tetrazole based high nitrogen content high energy materials. AB - This paper reports the synthesis, characterisation and thermolysis studies of hydrazinium azotetrazolate (HAZ) and 1,1'-dinitro-3,3'-azo-1,2,4-triazole (N DNAT). TGA and DSC results suggested that HAZ decomposes in the range of 150-180 degrees C and N-DNAT in the range of 160-170 degrees C, respectively. The pattern of decomposition of HAZ dihydrate and N-DNAT has been predicted with the help of pyrolysis GC/MS technique and a probable decomposition mechanism has been proposed. The theoretically predicted performance data suggests the potential nature of HAZ and N-DNAT for their use in propellant/explosive as well as in gas generator formulations. PMID- 16677760 TI - A role for the mTOR pathway in surface expression of AMPA receptors. AB - Delivery of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors (AMPARs) to the synapse is a critical factor controlling synaptic strength. It is now established that blockade of synaptic activity increases the surface expression of AMPARs. Factors modulating the delivery, insertion and expression of AMPARs are not completely known. Using immunohistochemical techniques, we first confirmed rapamycin-mediated inhibition of the mammalian target of rapamycin (mTOR) pathway in cortical neuronal culture. We then demonstrated that acute AMPAR activity blockade increased the synaptic expression of GluR2/3 subunits and rapamycin significantly reduced this expression. Our results suggest a role for the mTOR pathway in surface expression of AMPA receptors on cortical neurons. PMID- 16677761 TI - The effects of Botulinum Toxin type A on capsaicin-evoked pain, flare, and secondary hyperalgesia in an experimental human model of trigeminal sensitization. AB - The trigeminovascular system is involved in migraine. Efficacy of Botulinum Toxin type A (BoNT-A) in migraine has been investigated in clinical studies but the mechanism of action remains unexplored. It is hypothesized that BoNT-A inhibits peripheral sensitization of nociceptive fibers and indirectly reduces central sensitization. We examined the effect of intramuscular injection of BoNT-A on an experimental human model of trigeminal sensitization induced by intradermal capsaicin injection to the forehead. BoNT-A (BOTOX) or saline was injected intramuscularly in precranial, neck and shoulder muscles to 32 healthy male volunteers in a double blind-randomized manner. Intradermally capsaicin-induced pain, flare and secondary hyperalgesia were obtained before and 1, 4 and 8 weeks after the above treatments. A significant suppressive effect of BoNT-A on pain, flare and hyperalgesia area was observed. The pain intensity area was significantly smaller in BoNT-A group (9.16+/-0.83 cm x s) compared to saline group (15.41+/-0.83cm x s) (P=0.011). The flare area was also reduced significantly in BoNT-A group (29.81+/-0.69 cm2) compared to saline group (39.71+/-0.69 cm2) (P<0.001). Similarly, the mean area of secondary hyperalgesia was significantly smaller in BoNT-A group (4.25+/-0.91 cm2) compared to saline group (7.03+/-0.91 cm2) (P=0.040). Post hoc analysis showed significant differences across the trials with a remarkable suppression effect of BoNT-A on capsaicin-induced sensory and vasomotor reactions as early as week1 (P<0.001). BoNT-A presented suppressive effects on the trigeminal/cervical nociceptive system activated by intradermal injection of capsaicin to the forehead. The effects are suggested to be caused by a local peripheral effect of BoNT-A on cutaneous nociceptors. PMID- 16677762 TI - A femtogram resolution mass sensor platform based on SOI electrostatically driven resonant cantilever. Part II: sensor calibration and glycerine evaporation rate measurement. AB - This paper presents mass measurements of glycerine beads performed by means of laterally resonant micro-cantilevers. The transducer architecture is based on a resonant cantilever electrostatically coupled by two parallel placed electrodes. Previous to glycerine measurements, a calibration of the mass sensor has been performed by measuring a standard mass based on latex spheres. From these measurements, a value of the mass responsivity is deduced. In addition, a study of the transducer phase noise has been carried out in order to determine the minimum detectable mass. Mass measurements experiments have been performed by detecting the change on the resonance frequency of the on-plane cantilever resonant mode, produced by locally deposited mass. Additionally, the mass losses detected on the calibrated transducer after glycerine drop deposition allowed determining its evaporation rate. PMID- 16677763 TI - Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM. AB - The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM. PMID- 16677764 TI - A combined optical and atomic force microscope for live cell investigations. AB - We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors. PMID- 16677765 TI - Differential expression of basic fibroblast growth factor-2 in the developing rat brain. AB - Basic fibroblast growth factor-2 is a trophic molecule involved in a number of functions within the CNS, including the regulation of CNS responses to injury. Prior studies suggest that rats recover differently from injury inflicted to different regions and at different ages throughout development, and that basic fibroblast growth factor-2 may, at least in part, underlie this phenomenon. In the present study, we describe the distribution of basic fibroblast growth factor 2 at postnatal days 0, 2, 6, 10, 12, 14, 18, 21 and 30 in the indusium griseum, the external capsule, the hippocampus, the medial prefrontal cortex, the motor cortex, the rostral migratory stream, and the subventricular zone. Our results suggest a differential temporal and spatial expression of basic fibroblast growth factor-2 throughout development, which may explain the differential recovery observed from cortical lesions inflicted at different time points after birth. PMID- 16677766 TI - The Edinger-Westphal nucleus of the juvenile rat contains transient- and repetitive-firing neurons. AB - Classically, the Edinger-Westphal nucleus is described as containing neurons controlling accommodation and pupillary constriction via projections to the ciliary ganglion. However, in several species including rat, some Edinger Westphal neurons have ascending or descending CNS projections suggesting that the Edinger-Westphal nucleus might also have non-ocular functions. To further characterize the function of this nucleus we studied the electrophysiological properties of Edinger-Westphal neurons in a slice preparation from juvenile rats. The position of the Edinger-Westphal nucleus was determined using an immunohistochemical procedure directed at the peptide Urocortin, which is expressed in Edinger-Westphal neurons. Passive and active membrane responses were investigated and two different neuron types were identified. One type had a transient firing response to 400 ms depolarizing current pulses and one type had a repetitive firing response. Transient-firing neurons had an outward rectifying response inhibiting firing, possibly due to slowly inactivating I(D)-like potassium channels since low concentrations (200 microM) of the potassium channel blocker 4-aminopyridine elicited repetitive firing. In all neurons, low threshold Ca(2+) spikes were seen and these were blocked by nickel(II) chloride hexahydrate, suggesting that they are mediated via low voltage-activated Ca(2+) channels. Some biocytin-labeled neurons had axons or axonal collaterals projecting laterally or dorsally, suggesting possible non-ocular targets. In conclusion, the rat Edinger-Westphal nucleus contains two separate types of neurons with distinct electrophysiological properties. PMID- 16677767 TI - Phosphorylation at the hydrophobic site of protein kinase C Apl II is increased during intermediate term facilitation. AB - In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase. PMID- 16677768 TI - Enhanced cyclooxygenase-2 expression in olfactory-limbic forebrain following kainate-induced seizures. AB - Cyclooxygenase-2 is expressed at low levels in a subset of neurons in CNS and is rapidly induced by a multiplicity of factors including seizure activity. A putative relationship exists between cyclooxygenase-2 induction and glutamatergic neurotransmission. Cyclooxygenase-1 is constitutively expressed in glial cells and has been specifically linked to microglia. In this study we evaluated cyclooxygenase-2 protein immunocytochemically and found markedly enhanced immunostaining primarily in olfactory-limbic regions at 2, 6 and 24 h following kainate-induced status epilepticus. Impressive enhanced cyclooxygenase-2 immunoreactivity was localized in anterior olfactory nucleus, tenia tecta, nucleus of the lateral olfactory tract, piriform cortex, lateral and basolateral amygdala, orbital frontal cortex, nucleus accumbens (shell) and associated areas of ventral striatum, entorhinal cortex, dentate gyrus granule cells and hilar neurons, hippocampal CA subfields and subiculum. Alternate sections were processed for dual immunocytochemical analysis utilizing c-Fos and cyclooxygenase 2 antiserum to examine the possibility that the neuronal induction of cyclooxygenase-2 was associated with seizure activity. Neurons that showed a timeline of cyclooxygenase-2 upregulation were found to possess c-Fos immunopositive nuclei. Additional results from all seizure groups showed cyclooxygenase-1 induction in microglia, which was confirmed by Western blot analysis of hippocampus. Western blot and real-time quantitative RT-PCR analysis showed significant upregulation of cyclooxygenase-2 expression, confirming its induction in neurons. These data indicate that cyclooxygenase-2 induction in a neuronal network can be a useful marker for pathways associated with seizure activity. PMID- 16677769 TI - Functional synapse formation between cultured rat accessory olfactory bulb neurons and vomeronasal pockets. AB - To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca(2+) concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7 nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5 phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline 2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture. PMID- 16677770 TI - Decreased susceptibility to oxidative stress underlies the resistance of specific dopaminergic cell populations to paraquat-induced degeneration. AB - The vulnerability of different dopaminergic cell populations to damage caused by the herbicide paraquat was assessed by stereological counts of tyrosine hydroxylase-positive and calbindin-D28k-immunoreactive neurons in A9 (substantia nigra pars compacta) and A10 (ventral tegmental area and other cell groups). In saline-treated control mice, tyrosine hydroxylase-immunoreactive neurons represented 80% and 45% of the total neuronal population in A9 and A10, respectively, and the number of calbindin-D28k-positive neurons was five times greater in A10 than A9. Sequential injections with paraquat resulted in a significant loss of dopaminergic neurons in A9. In contrast, tyrosine hydroxylase positive cells in A10 were spared from paraquat-induced degeneration. Furthermore, expression of calbindin-D28k was consistently associated with neuronal resistance to the herbicide in both A9 and A10. Paraquat exposure also induced oxidative stress as indicated by an increase in the number of midbrain cells positive for 4-hydroxy-2-nonenal, a marker of lipid peroxidation. Co localization studies revealed that calbindin-D28k immunoreactivity overlapped with tyrosine hydroxylase labeling and that, after paraquat administration, (i) the vast majority of midbrain 4-hydroxy-2-nonenal-immunoreactive cells were dopaminergic (tyrosine hydroxylase-immunoreactive), (ii) tyrosine hydroxylase/4 hydroxy-2-nonenal-positive neurons were much more prevalent in A9 than A10, and (iii) all calbindin-D28k-containing neurons were characterized by lack of lipid peroxidation (4-hydroxy-2-nonenal immunoreactivity). Results in this paraquat model emphasize that, despite sharing a similar dopaminergic phenotype, different groups of midbrain neurons vary dramatically in their vulnerability to injury. Data also indicate that these differences are attributable, at least in part, to a varying susceptibility of dopaminergic cell populations to oxidative stress. PMID- 16677771 TI - Behavioral depression in the swim test causes a biphasic, long-lasting change in accumbens acetylcholine release, with partial compensation by acetylcholinesterase and muscarinic-1 receptors. AB - The nucleus accumbens may play a role in acquisition and expression of behavioral depression as measured using the inescapable swim test. Previous work shows that a local injection of a cholinergic muscarinic-1 receptor agonist increases immobility and a specific muscarinic-1 antagonist acts as an antidepressant-like drug by increasing swimming escape efforts. The present study used microdialysis to monitor extracellular acetylcholine levels in the accumbens, fluorescent labeled toxins to monitor changes in acetylcholinesterase and muscarinic-1 receptors, and semiquantitative-polymerase chain reaction to detect changes in gene expression for the muscarinic-1 receptor. Microdialysis showed that acetylcholine levels did not change while an animal was swimming; however, a significant transient decrease occurred when the rat was returned to the dialysis cage, followed by a long-lasting increase that reached a maximum three hours after the test. Acetylcholine levels stayed high even 24 h after the initial test as evidenced by a significant elevation in basal level prior to the second swim. This increase in neurotransmitter may have been partially compensated by a significant increase in the degradative enzyme, acetylcholinesterase, and by a decrease in muscarinic-1 receptors and their gene expression. These results further demonstrate the importance of accumbens cholinergic function in the appearance of a depression-like state. PMID- 16677772 TI - Plasminogen activator inhibitor-1 aids nerve growth factor-induced differentiation and survival of pheochromocytoma cells by activating both the extracellular signal-regulated kinase and c-Jun pathways. AB - Astrocytes are thought to be critical to neurons' surviving damage caused by ischemic stroke or other injury. Plasminogen activator inhibitor-1 is one of the active soluble factors released by astrocytes and regulates plasminogen activator plasmin proteolytic sequence in the CNS as a serpin. In this study, we show that plasminogen activator inhibitor-1 can promote neurite outgrowth and survival of rat pheochromocytoma cells in serum-deprived conditions, and that this neuroprotective activity is correlated with enhanced activation of both extracellular signal-regulated kinases following a direct phosphorylation of nerve growth factor receptor, Trk A, and of c-Jun. Our results suggest that plasminogen activator inhibitor-1 can act as a neurotrophic factor, protecting neurons from serum deprivation-induced neuron death not only by compensating for nerve growth factor functions, but also by activating the c-Jun/activating protein-1 pathway. PMID- 16677773 TI - Phase shift of subthreshold theta oscillation in hippocampal CA1 pyramidal cell membrane by excitatory synaptic inputs. AB - Hippocampal CA1 neurons receive multiple rhythmical inputs with relatively independent phases during theta activity. It, however, remains to be determined how these multiple rhythmical inputs affect oscillation properties in membrane potential of the CA1 pyramidal cell. In order to investigate oscillation properties in the subthreshold membrane potential, we generated oscillations in the membrane potential of the CA1 pyramidal cells in rat hippocampal slices in vitro with a sinusoidal current injection into the pyramidal soma at theta band frequencies (4-7 Hz), and analyzed effect of rhythmically excitatory synaptic inputs. The Schaffer collaterals were stimulated with a cyclic Gaussian stimulation method, whose pulse intervals were distributed at 10 pulses/cycle (5 cycles/s). We found that the cyclic Gaussian stimulations induced membrane potential oscillations and their phase delays from the mean of the pulse distribution were dependent on membrane potential oscillation amplitude. We applied four pairs of cyclic Gaussian stimulations and somatic sinusoidal current stimulations at the same frequency (5 Hz) with varying phase differences (-pi/2, 0, pi/2, pi rad). The paired stimulations induced phase distributions of the oscillation in the membrane potential, which showed a dependency on an increasing membrane potential oscillation amplitude response to cyclic Gaussian stimulation. This membrane potential dynamic was exhibited by the mixture of the membrane potential oscillation-amplitude-dependent phase delay and the linear summation of the two sinusoidal waves. These suggest that phases of the membrane potential oscillation are modulated by excitatory synaptic inputs. This phase-modulation by excitatory synaptic inputs may play a crucial role for memory operation in the hippocampus. PMID- 16677775 TI - Using classification trees to profile adolescent smoking behaviors. AB - The purpose of this study was to explore the interactive nature of various predictor variables in profiling adolescent smoking behaviors characterized by intention to smoke, current, situational, and established smoking using classification trees. The data (n = 3610) were obtained from cross-sectional telephone surveys of the Florida Anti-Tobacco Media Evaluation Program. Three classification trees were constructed, namely, intention versus no intention to smoke among non-smokers, current smokers versus non-smokers, and established versus situational smokers. The tree model for the intention model revealed that social and health risks are important in the context of peer smoking. Certain variables such as peer smoking and alcohol consumption retained their relative importance across the tree classifiers demonstrating that smoking intention may be predictable using some of the same variables as in current or more dependent smoking. PMID- 16677774 TI - Different lengths of times for progressions in adolescent substance involvement. AB - The present study examined Lengths Of Times for important transitions in substance involvement from Initiation to Regular use (LOTIR), first Problem from drug use (LOTIP), and first experience of Dependence (LOTID) for alcohol, tobacco, cannabis, cocaine, and opiates. Data were from a longitudinal study of 590 children (22.2% female) at different levels of risk for substance use disorders based on their fathers' substance use-related diagnoses. Participants' substance involvement was assessed at four ages: 10-12, and follow-ups at two, five, and eight years later. Results suggested that faster transitions were more due to drug-related constructs (including possible social milieus of different drug classes and interactions between drug class and neurophysiology) than intrapersonal constructs. The shortest transition times (and greatest addictive liabilities) were for opiates followed respectively by cocaine, cannabis, tobacco, and alcohol. Females had shorter transition times, though gender differences were small. Some evidence was found for a familial influence on transition times above what was accounted for by differences between substances. PMID- 16677776 TI - Stimulation of proteasome activity as a therapeutic target in schizophrenia: possible usefulness of 17-allylamino-demethoxygeldanamycin. PMID- 16677777 TI - The case against evidence-based principles in psychiatry. AB - There is an organized movement by governmental, academic and commercial interests to make evidence-based practice the standard of care in the United States. There is little proof that this model can be adapted to psychiatry. We examine the diagnostic system, the validity of the data from clinical trials and how these are applied to clinical practice. The discipline of psychiatry relies on imprecise and unstable diagnostic criteria. It divides psychiatric disorders into discrete categories based on discussion and consultations among designated experts in the field. The diagnostic system is based on consensus and not experimental evidence. In fact, psychiatric disorders are not discrete. High co morbidities between disorders and the propensity of one condition to change into another makes the present diagnostic system extremely questionable. Outcomes of clinical trials are defined by fractional reductions in the number and severity of symptoms measured by rating scales and not remission of illness. The data obtained from clinical trials are flawed in design, execution and the selective reporting of outcomes. There is substantial evidence to indicate that both investigators and patients can distinguish between active treatment and placebo in double blind studies. In addition, negative outcomes are frequently not reported. Such evidence impacts not only on the specific study, used as evidence, but invalidates the value of meta analyses. Financial considerations lead to the inclusion of inappropriate subjects into studies and favor newer, patented treatments. When the conclusions derived from evidence-based psychiatry are applied to clinical practice they have little to offer and often produce poor treatment outcomes. In fact, when the data used to support the principles of evidence-based psychiatry are examined, they are unsound. The system itself is best considered an untested hypothesis. The diagnostic system, the manner in which data are gathered, and financial factors combine to produce a system that is misleading and indeed, dangerous. PMID- 16677778 TI - PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation. AB - Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA. PMID- 16677779 TI - Ezrin in osteosarcoma: comparison between conventional high-grade and central low grade osteosarcoma. AB - Ezrin is a cytoskeleton linker protein that is actively involved in the regulation of growth and metastatic capacity of cancer cells. Recently, it has been demonstrated that a significant correlation exists between high ezrin expression levels and the poor outcome of pediatric osteosarcoma patients. The expression of ezrin was compared in conventional high-grade and central low-grade osteosarcoma lesions to investigate the role of ezrin overexpression in the metastasis of osteosarcoma. We compared the expression levels of the ezrin protein in 32 cases of high-grade osteosarcomas and 21 cases of low-grade osteosarcomas using immunohistochemistry. Ezrin protein expression levels were examined in three different human osteosarcoma cell lines by Western blotting. In addition, the mRNA expression levels of ezrin in these osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative PCR. Ezrin immunoreactivity was present in 43.7% of high-grade osteosarcoma specimens. All low-grade osteosarcomas were negative for ezrin. The expression of ezrin was detected by Western blotting in all three osteosarcoma cell lines. The tested osteosarcoma cell lines showed marked amplification of ezrin mRNA compared to control cells. Taken together, ezrin appears to play a role in the progression of tumors, such as the metastasis of osteosarcoma. However, further data are needed before ezrin can be considered in clinical decision-making about osteosarcoma patients. PMID- 16677780 TI - [Identification and management of HNPCC syndrome (hereditary non polyposis colon cancer), hereditary predisposition to colorectal and endometrial adenocarcinomas]. AB - BACKGROUND: The HNPCC syndrome (hereditary nonpolyposis colon cancer) is an inherited condition defined by clinical and genealogical information, known as Amsterdam criteria. In about 70% of cases, HNPCC syndrome is caused by germline mutations in MMR genes, leading to microsatellite instability of tumor DNA (MSI phenotype). Patients affected by the disease are at high risk for colorectal and endometrial carcinomas, but also for small intestine, urothelial, ovary, stomach and biliary tract carcinomas. HNPCC syndrome is responsible for 5% of colorectal cancers. Identification and management of this disease are part of a multidisciplinary procedure. METHODS: Twelve experts have been mandated by the French Health Ministry to analyze and synthesize their consensus position, and the resulting document has been reviewed by an additional group of 4 independent experts. MAIN RECOMMENDATIONS: The lack of sensitivity of Amsterdam criteria in recognizing patients carrying a MMR germline mutation led to an enlargement of these criteria for the recruitment of possible HNPCC patients, and to a 2-steps strategy, asking first for a tumor characterization according to MSI phenotype, especially in case of early-onset sporadic cases. The identification of germline MMR mutations has no major consequence on the cancer treatments, but influences markedly the long-term follow-up and the management of at-risk relatives. Gene carriers will enter a follow-up program regarding their colorectal and endometrial cancer risks, but other organs being at low lifetime risk, no specific surveillance will be proposed. PMID- 16677781 TI - Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 CYP4A15. AB - In the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala (Phascolarctos cinereus), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not ingest eucalyptus leaves as food [Ngo, S., Kong, S., Kirlich, A., Mckinnon, R.A., Stupans, I., 2000. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comp. Biochem. Physiol., C 127, 327-334]. A 1544 bp koala liver CYP4A cDNA, designated CYP4A15, was cloned by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The koala CYP4A15 cDNA encodes a protein of 500 amino acids and shares 69% nucleotide and 65% amino acid sequence identity to human CYP4A11. Transfection of the koala CYP4A15 cDNA into Cos-7 cells resulted in the expression of a protein with lauric acid hydroxylase activity. The koala CYP4A15 cDNA-expressed enzyme catalysed lauric acid hydroxylation at the rates of 0.45+/-0.18 nmol/min/mg protein and 4.79+/ 1.91 nmol/min/nmol CYP (mean+/-SD, n=3), which were comparable to that of rat CYP4A subfamilies. Total CYP content for koala CYP4A15-expressed protein in Cos-7 cells was 0.094+/-0.001 nmol/mg protein (mean+/-SD, n=3) with negligible CYP content in untransfected Cos-7 cells lysate. Immunoblot analysis, using a sheep anti-rat CYP4A polyclonal antibody, detected multiple CYP4A immunoreactive bands in the liver from all species studied. The koala bands were found to be fainter and less confined but appeared much broader as compared to rat, human and wallaby. Northern blot analysis, utilising the koala CYP4A15 cDNA 417 bp probe, detected a mRNA species of approximately 2.6 kb in the koala liver and a mRNA species of approximately 2.4 kb in other species studied. Relative to the intensity of the beta-actin mRNA species, much stronger CYP4A mRNA signal was detected for koala liver relative to rat and human. In Southern blot analysis of EcoR 1-digested genomic DNAs, using the same koala CYP4A15 cDNA probe, the size of CYP4A gene fragments observed for the koala and other species were different, suggested a different CYP4A gene organization across species. Collectively, this study provides primary molecular data regarding koala CYP4A15 gene. The possibility that there may be higher CYP4A15 expression in the koala liver could not be excluded. PMID- 16677782 TI - Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces. AB - Avian intestinal spirochaetosis (AIS) is an infection of the caeca and/or colo rectum of laying and meat breeder hens caused by anaerobic intestinal spirochaetes of the genus Brachyspira. AIS can result in a variety of symptoms, including delayed and/or reduced egg production, and increased faecal water content. The two most commonly reported Brachyspira species involved in AIS are Brachyspira pilosicoli and Brachyspira intermedia, and their detection and identification can be difficult and time consuming. In the current study a two step nested duplex PCR (2S-N-D-PCR) was developed for the detection of these two species, using DNA extracted from washed chicken faeces. In the first step, a duplex PCR (D-PCR) amplifying Brachyspira genus-specific portions of the 16S rRNA and NADH oxidase (nox) genes was undertaken on the washed faeces. In the second step, a nested D-PCR was used that amplified species-specific portions of the 16S rRNA gene of B. pilosicoli and the nox gene of B. intermedia from the amplicons produced in the first step. The 2S-N-D-PCR was rapid and specific, and could be used to detect approximately 10(3) cells of each spirochaete species per gram of washed faeces. When tested on 882 chicken faecal samples from infected flocks, it detected 4-5% more positive faecal samples than did the standard method of selective anaerobic culture followed by individual species-specific PCR assays conducted on the growth on the primary plate. The application of this new technique should improve diagnostic capacity, and facilitate further studies on AIS. PMID- 16677783 TI - The management of multiple pregnancy. AB - The epidemic of multiple pregnancy continues albeit in a different form with twin pregnancies predominating. Determination of chorionicity is the key to management and regular monitoring by ultrasound is a hallmark of quality care. All multiple pregnancies should be offered first trimester screening by nuchal translucency for aneuploidy. MC twins should be scanned at fortnightly intervals to allow complications such as twin-twin transfusion or IUGR to be detected and referral made to a fetal medicine centre. Maternal complications are common and vigilance is required for their detection. Although planned vaginal delivery can often be achieved, mothers must be prepared for the substantial risk of requiring a caesarean section as this currently occurs in the majority. PMID- 16677784 TI - Effects of arsenite on long-term potentiation in hippocampal slices from young and adult rats. AB - The effects of trivalent arsenite were tested at the Schaffer collateral-CA1 synapse of adult (2-4 month) and young (14-21 days) rats. Exposure of 100micromol/l arsenite led to a slight and reversible reduction of the amplitudes of evoked excitatory postsynaptic field potentials in adult and young rats, while exposure of 0.1 and 1micromol/l arsenite had no effects. The long-term potentiation (LTP) was significantly inhibited by arsenite in adult but not in young rats. Exposure of 0.1, 1 and 100micromol/l arsenite to slices of adult rats before and during the LTP stimulus led to a significant reduction in the potentiated amplitudes amounting to a maximum of 50% of the control values. In young animals, however, exposure of 1micromol/l arsenite showed no effect on the LTP potentiated amplitudes, while exposure of 100micromol/l arsenite led initially to a significant reduction in the amplitudes, compared to the control level, which was completely reversible 20min after washout. Exposure of 100micromol/l arsenite did not affect the paired-pulse facilitation, indicating that arsenite does not exert its effects by influencing presynaptic transmitter release mechanisms. PMID- 16677785 TI - Modulation of drug release from glyceryl palmitostearate-alginate beads via heat treatment. AB - Diclofenac calcium alginate (DCA) beads containing glyceryl palmitostearate (GPS) were prepared by ionotropic gelation method. The effect of GPS amount and heat treatment on characteristics of the DCA beads was investigated. Incorporation of GPS into the DCA beads increased particle size and entrapment efficiency of diclofenac sodium (DS), but decreased water uptake in distilled water, and DS release rate. The heat treatment caused the DCA beads to be irregular shape particles and to possess higher water uptake. A slower release rate of DS in distilled water was found because of interaction of DS and alginate polymer matrix, and a restriction of water sorption into the inside region of the beads, which caused by the shrinkage of the beads after heating. However, the heat treatment did not affect particle shape and water uptake in distilled water of the 3%GPS-DCA beads. Differential scanning calorimetric study showed that GPS in the DCA beads was resolidified to different polymorph after cooling. Furthermore, the micro-Raman spectra indicated the existence of DS in the GPS matrix particles in the beads due to the partition of DS into the melted GPS during heat treatment. This led to a decrease in release rate of DS in pH 6.8 phosphate buffer and a change in DS release pattern in distilled water. Thus, not only the calcium alginate matrix, but also the resolidified GPS matrix in the alginate beads controlled the DS release from the 3%GPS-DCA beads with heat treatment. PMID- 16677786 TI - Controlled release of doxorubicin from thermosensitive poly(organophosphazene) hydrogels. AB - Thermosensitive poly(organophosphazenes) bearing hydrophobic isoleucine ethyl esters group and hydrophilic alpha-amino-omega-methoxy-poly(ethylene glycol) of the molecular weight 550 along with hydrolysis-sensitive glycyl lactate ethyl esters have been synthesized for sustained delivery of anticancer drug. The aqueous solution of poly(organophosphazenes) containing doxorubicin, that represents chemotherapeutic agent for cancer treatment, was transformed into hydrogel with good gel strength at body temperature via hydrophobic interactions. Solubility of hydrophobic doxorubicin in the aqueous poly(organophosphazene) solution was dramatically improved as compared with that in PBS (0.01 M, pH 7.4). The hydrogel property of poly(organophosphazenes) was affected on incorporation of doxorubicin, resulting in increase of gelation temperature and decrease of gel strength. The release of loaded doxorubicin from the polymer hydrogel was significantly sustained over 20 days and the effect of gel strength, polymer concentration and drug concentration on the release rate were observed. The release of doxorubicin from the polymer gels was effectively controlled by the gel strength. As a result of investigating anticancer efficacy using cancer cell line of mouse lymphoblast of P388D1, the efficacy of doxorubicin was observed to be constant over a prolonged period of times for more than 30 days, indicating that the release of doxorubicin was sustained for a long time without any initial burst release, and that the delivery system was an excellent candidate for locally injectable gel-depot system. PMID- 16677787 TI - Toxicity studies of Calycopteris floribunda Lam. in calf, rabbit and rat. AB - The toxicological evaluation of the plant Calycopteris floribunda Lam. was done in calf, rabbit and rat. The phytochemical screening revealed the presence of flavonoids, alkaloids, tannins and saponins. Morbidity and mortality was observed in methanol extract-treated rats and rabbits. In rabbits (25 g/kg/day) and calves (35 g/kg/day) fed fresh leaves showed morbidity and mortality with premonitory clinical signs like depression, downer status, polyuria and characteristic forelimb paresis (seen only in rabbits). The serum urea, alanine-amino transferase, glucose and total cholesterol concentrations were significantly (P<0.05) increased compared to control values. Necropsy of calves revealed the congestion of liver, lung and petechiae on epicardium. The present study indicated the toxic nature of the plant in calf, rabbit and rat. PMID- 16677788 TI - Temperature-dependent trade-offs between longevity and fertility in the Drosophila mutant, methuselah. AB - Single gene, hypomorphic mutations which extend the life spans of cold-blooded animals, such as the methuselah (mth) mutation in the fruit fly, Drosophila melanogaster, may have additional, deleterious effects on overall fitness. The hypotheses tested here were: (i) that the extension of life span by mth might be temperature-dependent, and (ii) that it might be associated with depression of reproductive output, physical activity, or the rate of metabolism. The effect of mth on life span was smaller in magnitude than reported previously, and it was both sex-specific and temperature-dependent. Female longevity was increased only at 29 degrees C, whereas for male flies the extension of mean life span diminished progressively from 15-25% 25-29 degrees C to 2% at 18 degrees C, and the survival time at 4 degrees C was decreased by 22-39%. Conversely, the lifetime reproductive output of mth mutants was decreased at 29 degrees C, but increased at 18-22 degrees C. The walking speed of mth flies was significantly elevated, but mth had no effect on the rate of oxygen consumption at 25 degrees C. Collectively, the results demonstrate that where the life span is extended, there is an offsetting effect on reproductive output, suggesting that mth induces trade-off effects and is not a direct, mechanistic regulator of the aging process. PMID- 16677789 TI - Alterations in mitochondrial and cytosolic methionine sulfoxide reductase activity during cardiac ischemia and reperfusion. AB - During cardiac ischemia/reperfusion, proteins are targets of reactive oxygen species produced by the mitochondrial respiratory chain resulting in the accumulation of oxidatively modified protein. Sulfur-containing amino acids are among the most sensitive to oxidation. Certain cysteine and methionine oxidation products can be reversed back to their reduced form within proteins by specific repair enzymes. Oxidation of methionine in protein produces methionine-S sulfoxide and methionine-R-sulfoxide that can be catalytically reduced by two stereospecific enzymes, methionine sulfoxide reductases A and B, respectively. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during conditions of oxidative stress, the fate of this system during ischemia/reperfusion was investigated. Mitochondrial and cytosolic methionine sulfoxide reductase activities are decreased during ischemia and at early times of reperfusion, respectively. Partial recovery of enzyme activity was observed upon extended periods of reperfusion. Evidence indicates that loss in activity is not due to a decrease in the level of MsrA but may involve structural modification of the enzyme. PMID- 16677790 TI - Mitochondrial dysfunction in sporadic and genetic Alzheimer's disease. AB - Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of many common age-related neurodegenerative diseases, including Alzheimer's disease (AD). AD is the most common neurodegenerative disorder characterized by dementia, memory loss, neuronal apoptosis and eventually death of the affected individuals. AD is characterized by two pathologic hallmark lesions that consist of extracellular plaques of amyloid-beta peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated microtubular protein tau. Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein and tau alterations in this type of dementia remains controversial. PMID- 16677791 TI - Techniques in gerontology: cell lines as standards for telomere length and telomerase activity assessment. AB - The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging. PMID- 16677792 TI - Mitochondrial protein oxidation and degradation in response to oxidative stress and aging. AB - Mitochondria are a major source of intracellular reactive oxygen species (ROS), the production of which increases with age. These organelles are also targets of oxidative damage. The deleterious effects of ROS may be responsible for impairment of mitochondrial function observed during various pathophysiological states associated with oxidative stress and aging. An important factor for protein maintenance in the presence of oxidative stress is enzymatic reversal of oxidative modifications and/or protein degradation. Failure of these protein maintenance systems is likely a critical component of the aging process. Mitochondrial matrix proteins are sensitive to oxidative inactivation and oxidized proteins are known to accumulate during aging. The ATP-stimulated mitochondrial Lon protease is a highly conserved protease found in prokaryotes and the mitochondrial compartment of eukaryotes and is believed to play an important role in the degradation of oxidized mitochondrial matrix proteins. Age dependent declines in the activity and regulation of this proteolytic system may underlie accumulation of oxidatively modified and dysfunctional protein and loss in mitochondrial viability. PMID- 16677793 TI - Effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls. AB - The objective of the present study was to characterize the effects of nutrition on circulating concentrations of metabolic hormones, gonadotropins, and testosterone during sexual development in bulls. Nutrition regulated the hypothalamus-pituitary-testes axis through effects on the GnRH pulse generator in the hypothalamus and through direct effects on the testes. Pituitary function (gonadotropin secretion after GnRH challenge) was not affected by nutrition. However, nutrition affected LH pulse frequency and basal LH concentration during the early gonadotropin rise (10-26 weeks of age). There were close temporal associations between changes in insulin-like growth factor-I (IGF-I) concentrations and changes in LH pulse frequency, suggesting a role for IGF-I in regulating the early gonadotropin rise in bulls. The peripubertal increase in testosterone concentration was delayed in bulls with lesser serum IGF-I concentrations (low nutrition), suggesting a role for IGF-I in regulating Leydig cell function. Serum IGF-I concentrations accounted for 72 and 67% of the variation in scrotal circumference and paired-testes volume, respectively (at any given age), indicating that IGF-I may regulate testicular growth. Bulls with a more sustained elevated LH pulse frequency during the early gonadotropin rise (high nutrition) had greater testicular mass at 70 weeks of age relative to the control group (medium nutrition), despite no differences in metabolic hormone concentrations after 26 weeks of age. Therefore, gonadotropin-independent mechanism regulating testicular growth might be dependent on previous gonadotropin milieu. PMID- 16677794 TI - Pituitary progesterone receptor expression and plasma gonadotrophin concentrations in the reproductively dysfunctional mutant restricted ovulator chicken. AB - Female mutant restricted ovulator (RO) chickens of the White Leghorn strain carry a naturally occurring single nucleotide mutation in the very low density lipoprotein receptor (VLDLR) gene. Due to this mutation, RO hens fail to express a functional VLDLR protein on the oocyte membrane, which results in an impaired uptake of circulating yolk precursor macromolecules. Mutant RO hens subsequently develop hyperlipidemia and generally fail to lay eggs due to follicular atresia. Since RO hens also reportedly have three-fold higher basal plasma estrogen concentrations, combined with four-fold lower levels of circulating progesterone as compared to wild-type (WT) hens, we hypothesized that RO hens would have an increased abundance of pituitary progesterone receptor (PR) mRNA and PR isoforms A and B as well as alterations in circulating gonadotrophin levels. Quantitative PCR assays revealed significantly greater (P1.5 or<0.6). Heat shock protein 60 (HSP-60) (ratio>2), Ciclin D1 and topoisomerase I (ratio>1.5) were overexpressed in the ACC cohort, while jun proto-oncogene was down-regulated. cDNA macroarray analysis from paraffin sections of adrenal tumors is feasible, despite with a low success rate. The different expression of HSP-60, Ciclin D1, jun proto-oncogene and topoisomerase I indicates that these genes may play a role in ACC pathogenesis and could represent potential diagnostic/prognostic/therapeutic target markers. Larger series of patients are necessary to confirm the biologic, diagnostic, prognostic and therapeutic implications of these findings. PMID- 16677800 TI - The response to an allegation of scientific misconduct in the Bucci et al. article on DIMP exposure in mink. PMID- 16677801 TI - Biosynthesis and trafficking of seven transmembrane receptor signalling complexes. AB - Recent studies have shown that 7-transmembrane receptors (7TM-Rs), their associated signalling molecules and scaffolding proteins are often constitutively associated under basal conditions. These studies highlight that receptor ontogeny and trafficking are likely to play key roles in the determination of both signalling specificity and efficacy. This review highlights information about how 7TM-Rs and their associated signalling molecules are trafficked to the cell surface as well as other intracellular destinations. PMID- 16677802 TI - Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive growth and overcomes p38 MAPK-mediated apoptosis. AB - Hepatocyte growth factor (HGF) induces invasive growth, a biological program that confers tumor cells the capability to invade and metastasize by integrating cell proliferation, motility, morphogenesis, and survival. We here demonstrate that HGFR activation promotes survival of colorectal carcinoma (CRC) cells exposed to conditions that mimic those met during tumor progression, i.e. nutrient deprivation or substrate detachment, and following chemotherapeutic treatment. In all these conditions, a sustained activation of p38 MAPK delivers a main death signal that is overcome by cell treatment with HGF. HGF-driven survival requires the engagement of the PI3K/Akt/mTOR/p70S6K and ERK MAPK transduction pathways. Abrogation of p38 MAPK activity prevents CRC cell apoptosis also when these transduction pathways are inhibited, and treatment with HGF further increases survival. Engagement of these signaling cascades is also needed for HGF to induce CRC cell scattering, morphogenesis, motility and invasion. Activation of p38 MAPK signaling is therefore a main apoptotic switch for CRC cells in the stressful conditions encountered during tumor progression. Conversely, HGF orchestrates several biochemical pathways, which allow cell survival in these same conditions and promote the biological responses required for tumor invasive growth. Both p38 MAPK and HGF/HGFR signaling constitute potential molecular targets for inhibiting colorectal carcinogenesis. PMID- 16677803 TI - Early-onset of symptoms predicts conversion to non-affective psychosis in ultra high risk individuals. AB - OBJECTIVE: We examined if age of onset of psychiatric symptoms and/or sex predict conversion to non-affective or affective psychosis in individuals considered to be at ultra-high risk for schizophrenia. METHOD: Participants (n=86) were offered treatment and monthly follow-up until transition to psychosis, or for 12 months if they did not meet exit criteria for psychotic disorder. Individuals without transition to psychosis at 12-month were reassessed approximately 3 years after the end of the treatment phase. Ultra-high risk was defined by the presence of subthreshold and/or self-limiting psychotic symptoms and/or having a family history of psychotic disorder combined with functional decline. Cox regressions after adjustment for treatment interventions were applied to investigate associations between age of onset, sex, and other baseline measures with progression to psychotic outcomes. RESULTS: Early age of onset of psychiatric symptoms, in particular onset before age 18 was the only tested variable that significantly predicted non-affective psychosis. Independent significant predictors of affective psychosis were poor functioning, female sex and the presence of a combination of intake criteria (family history of psychosis plus drop in functioning, and attenuated and/or brief limited psychotic symptoms) at baseline. CONCLUSIONS: Age of onset of psychiatric symptoms is the single most important factor associated with conversion to non-affective psychosis in ultra high risk individuals. PMID- 16677804 TI - A novel mutation of the keratin 1 gene in a family with epidermolytic hyperkeratosis. PMID- 16677805 TI - [A nutrition survey among schoolchildren in Brazzaville, Congo]. AB - OBJECTIVE: To identify nutritional characteristics among schoolchildren in Brazzaville, Congo. POPULATION AND METHODS: A 3-day feeding survey was undertaken in 3 primary education schools in the Brazzaville borough. The study was conducted among 1066 schoolchildren (534 boys, 532 girls) ranging from 6 to 14 years. The subjects were divided into 4 age groups: 6-7, 8-9, 10-11 and 12-14 years old. The evolution of the feeding was studied according to age, and the nutritional intakes were compared with recommended dietary allowances. RESULTS AND DISCUSSION: The energy intakes were low, the mean caloric intake was 1984.9+/ 448 Kcal. They increased between 6 and 14 years, especially between 13 and 14 years, where mean increase was twice as important as between 6 and 7 years. Energy provided by the evening meal was equivalent to 67.5% of mean caloric intake per day. The quantities of aliments taken increased with age. In addition, there were excesses and insufficiencies common or specific to the Congolese children of 4 age groups. High natrium intakes were observed at age 12 years, insufficient intakes of phosphore and lipids at 10 years. CONCLUSION: On the basis of the observation of the schoolchildren's behavior at Brazzaville, poverty seems to influence the schoolchildren's nutritional status. But, one could propose dietary advices, in order to improve the problems described here. PMID- 16677806 TI - On the analysis of single versus multiple channels of electromagnetic brain signals. AB - OBJECTIVE: When extracting information from electromagnetic (EM) brain function through recordings such as the electroencephalogram (EEG) it is often assumed that signal processing techniques must be applied to multiple simultaneous recordings in order to obtain useful results. However, sometimes only a single channel of EEG recording is available or desirable. In this paper we objectively assess a novel methodology which exploits only a single measurement channel to extract information of interest relatively independent of channel location (relative to the source of interest). METHODS: The method relies on a combination of a matrix of delay vectors constructed from the single channel measurement, along with constrained independent component analysis, which incorporates prior information into the process. MATERIALS: Here, we use synthetically generated seizure EEG, composed of real, normal multi-channel EEG onto which is superimposed synthetic epileptic "seizure-like" activity, at different signal-to noise (SNR) levels, through an equivalent current dipole model. RESULTS: We show that the method can extract desired information from single channels with a reasonable accuracy even at very small SNR and from channels distant from the focus of the activity. This provides a powerful technique capable of extracting multiple sources underlying single channel recordings and will be useful in situations where only single channel EM recordings of brain function are desirable, such as would be the case in wearable or implantable recording devices. PMID- 16677807 TI - Respiratory muscle energetics during exercise in healthy subjects and patients with COPD. AB - The energy expenditure required by the respiratory muscles during exercise is a function of their work rate, cost of breathing, and efficiency. During exercise, ventilatory requirements increase further exacerbating the potential imbalance between inspiratory muscle load and capacity. High level of exercise intensity in conjunction with contracting respiratory muscles is the reason for respiratory muscle fatigue in healthy subjects. Available evidence would suggest that fatigue of the diaphragm and other respiratory muscles is an important mechanism involved in redistribution of blood flow. Reflex mechanisms of sympathoexcitation are triggered in fatigued diaphragm during heavy exercise when cardiac output is not sufficient to adequately meet the high metabolic requirements of both respiratory and limb musculature. It is very likely that local changes in locomotor muscle blood flow may occur during exhaustive endurance exercise and that changes may have important effect on O2 transport to the working locomotor muscles and, therefore, on their fatigability. In a condition when the respiratory muscles receive their share of blood flow at the expense of limb locomotor muscles, minimizing mechanical work of breathing and therefore its metabolic cost allows a greater amount of cardiac output to be available to be delivered to working limb muscles. Malfunction in any of the multiple components responsible for circulatory flow and O2 delivery will limit the blood supply therefore inhibiting the supply of O2 and the energy substrate to the contracting muscles. Studies are needed to overcome these limitations. PMID- 16677808 TI - The prevalence of osteoporosis in patients with chronic obstructive pulmonary disease: a cross sectional study. AB - Chronic obstructive pulmonary disease (COPD) is a complex disease, where the initial symptoms are often cough as a result of excessive mucus production and dyspnea. With disease progression several other symptoms may develop, and patients with moderate to severe COPD have often multiorganic disease with severely impaired respiratory dysfunction, decreased physical activity, right ventricular failure of the heart, and a decreased quality of life. In addition osteoporosis might develop possibly due to a number of factors related to the disease. We wanted to investigate the prevalence of osteoporosis in a population of patients with severe COPD as well as to correlate the use of glucocorticoid treatment to the occurrence of osteoporosis in this population. Outpatients from the respiratory unit with COPD, a history of forced expiratory volume in 1s (FEV1) less than 1.3 L, with FEV1% pred. ranging from 17.3% to 45.3% (mean 31.4%, standard deviation (sd) 7.3%). Patients between 50 and 70 years were included. Other causes of osteoporosis were excluded before inclusion. At study entry spirometry, X-ray of the spine (to evaluate presence of vertebral fractures), and bone mineral density of lumbar spine and hip were performed. Of 181 patients invited by mail, 62 patients were included (46 females and 16 males). All had symptoms of COPD such as exertional dyspnea, productive cough, limitations in physical activity etc. The mean FEV1 was 0.90 L (sd: 0.43 L) and the mean FEV1% pred. of 32.6% (sd: 14.1%). All had sufficient daily intake of calcium and vitamin D. In 15 patients, X-ray revealed compression fractures previously not diagnosed. Bone density measurements showed osteoporosis in 22 patients and osteopenia in 16. In total, 26 of the COPD patients were osteoporotic as evaluated from both X-ray and bone density determinations. Thus 68% of the participants had osteoporosis or osteopenia, but glucocorticoid use alone could not explain the increased prevalence of osteoporosis. A large fraction of these needed treatment for severe osteoporosis in order to prevent further bone loss and to reduce future risk of osteoporotic fractures. Thus, there is a significant need to screen patients with COPD to select the individuals in risk of fracture and to initiate prophylaxis or treatment for the disease. PMID- 16677810 TI - Neutralization of an extremely acidic sludge and stabilization of heavy metals in flyash aggregates. AB - An extremely acidic, heavy metal-rich sludge (pH=-1.2) was scrubbed with a Class F fly ash in order to simultaneously neutralize the acidity and stabilize the heavy metals contained in both wastes. This paper outlines the leaching behavior of the aggregate material generated by scrubbing. For proper fly ash/sludge ratios, the fly ash acted as an outstanding neutralizer for the acidic waste. Leaching of heavy metals from the aggregate samples was below the environmental limits within a pH range between 3 and 9. Subsequent washing of the leached aggregate with acidic CALWET solutions did not result in an additional release of heavy metals. It is proposed that coordinative bonding of the metal cations onto neutral surface sites and electrostatic adsorption led to stabilization of the heavy metals within the aggregate structure below hydrolysis pHs. PMID- 16677811 TI - Regioselective and stereospecific cleavage of a terminal oxirane system: a novel synthetic approach to lipid mediator congeners--1,2(2,3)-diacyl-3(1)-halo-sn glycerols. AB - Glycidyl esters upon treatment with a mixture of carboxylic acid anhydride (CAA) and trimethylsilyl halide (TMSX) in the presence of tetra-n-butylammonium halide (Bu(4)NX, X=Cl, Br or I) undergo stereospecific and regioselective opening of the oxirane ring to afford mixed-(or mono)-acid 1,2(2,3)-diacyl-3(1)-halo-sn glycerols in high yields. PMID- 16677812 TI - Arylpiperazines with N-acylated amino acids as 5-HT1A receptor ligands. AB - A library consisting of 60 arylpiperazines modified with N-acylated amino acids was prepared on BAL linker SynPhasetrade mark Lanterns and evaluated in vitro for 5-HT(1A) receptor affinity. Biological screening, followed by a simple Fujita-Ban analysis, enabled the description of structure-activity relationships and allowed the selection of some potent, high-affinity ligands for in vivo pharmacological investigations. PMID- 16677813 TI - Design, synthesis and evaluation of novel uracil amino acid conjugates for the inhibition of Trypanosoma cruzi dUTPase. AB - Potential inhibitors of the Trypanosoma cruzi dUTP nucleotidohydrolase were docked into the enzyme using the program FlexX. Compounds that docked selectively were then selected and synthesized using solid phase methodology, giving rise to a novel library of amino acid uracil acetamide compounds which were evaluated for enzyme inhibition and anti-parasitic activity. PMID- 16677814 TI - Synthesis and SAR of novel histamine H3 receptor antagonists. AB - The synthesis and biological evaluation of novel tetrahydroisoquinoline, tetrahydroquinoline, and tetrahydroazepine antagonists of the human and rat H(3) receptors are described. The substitution around these rings as well as the nature of the substituent on nitrogen is explored. Several compounds with high affinity and selectivity for the human and rat H(3) receptors are reported. PMID- 16677815 TI - High-affinity auxin transport by the AUX1 influx carrier protein. AB - In plants, auxin is a key regulator of development and is unique among plant hormones in that its function requires polarized transport between neighboring cells to form concentration gradients across various plant tissues. Although putative auxin-influx and -efflux transporters have been identified by using molecular genetic approaches, a detailed functional understanding for many of these transporters remains undetermined. Here we present the functional characterization of the auxin-influx carrier AUX1. Upon expression of AUX1 in Xenopus oocytes, saturable, pH-dependent uptake of 3H-IAA was measured. Mutations in AUX1 that abrogate physiological responses to IAA in planta resulted in loss or reduction of 3H-IAA uptake in AUX1-expressing oocytes. AUX1-mediated uptake of 3H-IAA was reduced by the IAA analogs 2,4-D and 1-NOA, but not by other auxin analogs. The measured Km for AUX1-mediated uptake of 3H-IAA was at concentrations at which physiological responses are observed for exogenously added IAA and 2,4 D. This is the first report demonstrating detailed functional characteristics of a plant auxin-influx transporter. This biochemical characterization provides new insights and a novel tool for studying auxin entry into cells and its pivotal roles in plant growth and development. PMID- 16677816 TI - A myxobacterial S-motility protein dances with poles. AB - Coordinated movement of packs of S-motile Myxococcus xanthus cells relies on extrusion and retraction of pili that are located at one cell pole. At regular intervals the pili switch their polar location and cells reverse direction. Recently, the FrzS S-motility protein was observed to localize predominantly to the piliated pole. In time, FrzS was redeployed to the opposite pole and its sequestration at the new site coincided with cell reversal. The C-terminal region of FrzS, a response regulator homolog, is rich in coiled-coil motifs and is required for dynamic localization and proper motility. These results raise the possibility that proper spatial control of FrzS has an important role in the regulation of cell reversal and S-motility. PMID- 16677817 TI - Synthesis and biological evaluation of acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl pharmacophore: dual inhibitors of cyclooxygenases and lipoxygenases. AB - A new class of regioisomeric acyclic triaryl (Z)-olefins possessing a 3,5-di-tert butyl-4-hydroxyphenyl (DTBHP) 5-lipoxygenase (5-LOX) pharmacophore that is vicinal to a para-methanesulfonylphenyl cyclooxygenase-2 (COX-2) pharmacophore were designed for evaluation as selective COX-2 and/or 5-LOX inhibitors. The target compounds were synthesized via a highly stereoselective McMurry olefination cross-coupling reaction. This key synthetic step afforded a (Z):(E) olefinic mixture with a predominance for the (Z)-olefin stereoisomer. Structure activity studies for the (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4 methanesulfonylphenyl)-1-phenylalk-1-ene regioisomers showed that COX-1 inhibition decreased, COX-2 inhibition increased, and the COX-2 selectivity index (SI) increased as the chain length of the alkyl substituent attached to the olefinic double bond was increased (Et-->n-butyl-->n-heptyl). In this group of compounds, inhibition of both 5-LOX and 15-LOX was dependent upon the length of the alkyl substituent with the hex-1-ene compound 9c having a n-butyl substituent exhibiting potent inhibition of both 5-LOX (IC50=0.3 microM) and 15-LOX (IC50=0.8 microM) relative to the inactive (IC50>10 microM) Et and n-heptyl analogs. Compound 9c is of particular interest since it also exhibits a dual inhibitory activity against the COX (COX-1 IC50=3.0 microM, and COX-2 IC50=0.36 microM, COX 2 SI=8.3) isozymes. A comparison of the relative inhibitory activities of the two groups of regioisomers investigated shows that the regioisomers in which the alkyl substituent is attached to the same olefinic carbon atom (C-2) as the para methanesulfonylphenyl moiety generally exhibit a greater potency with respect to COX-2 inhibition. The 4-hydroxy substituent in the 3,5-di-tert-butyl-4 hydroxyphenyl moiety is essential for COX and LOX inhibition since 3,5-di-tert butyl-4-acetoxyphenyl derivatives were inactive inhibitors. These structure activity data indicate acyclic triaryl (Z)-olefins constitute a suitable template for the design of dual COX-2/LOX inhibitors. PMID- 16677818 TI - Conditional knockout of Mn superoxide dismutase in postnatal motor neurons reveals resistance to mitochondrial generated superoxide radicals. AB - Mitochondrial dysfunction and oxidative damage are implicated in the pathogenesis of neurodegenerative disease. Mice deficient in the mitochondrial form of superoxide dismutase (SOD2) die during embryonic or early postnatal development, precluding analysis of a pathological role for superoxide in adult tissue. Here, we generated postnatal motor neuron-specific SOD2 knockouts by crossing mice with floxed SOD2 alleles to VAChT-Cre transgenic mice in which Cre expression is restricted to postnatal somatomotor neurons. SOD2 immunoreactivity was specifically lost in a subset of somatomotor neurons resulting in enhanced superoxide production. Yet extensive histological examination revealed no signs of oxidative damage in animals up to 1 year after birth. However, disorganization of distal nerve axons following injury was accelerated in SOD2-deficient motor neurons. These data demonstrate that postnatal motor neurons are surprisingly resistant to oxidative damage from mitochondrial-derived superoxide radicals, but that such damage may sensitize axons to disorganization following nerve injury. PMID- 16677819 TI - Does the Aristotle Score predict outcome in congenital heart surgery? AB - OBJECTIVE: The Aristotle Score has been proposed as a measure of 'complexity' in congenital heart surgery, and a tool for comparing performance amongst different centres. To date, however, it remains unvalidated. We examined whether the Basic Aristotle Score was a useful predictor of mortality following open-heart surgery, and compared it to the Risk Adjustment in Congenital Heart Surgery (RACHS-1) system. We also examined the ability of the Aristotle Score to measure performance. METHODS: The Basic Aristotle Score and RACHS-1 risk categories were assigned retrospectively to 1085 operations involving cardiopulmonary bypass in children less than 18 years of age. Multiple logistic regression analysis was used to determine the significance of the Aristotle Score and RACHS-1 category as independent predictors of in-hospital mortality. Operative performance was calculated using the Aristotle equation: performance = complexity x survival. RESULTS: Multiple logistic regression identified RACHS-1 category to be a powerful predictor of mortality (Wald 17.7, p < 0.0001), whereas Aristotle Score was only weakly associated with mortality (Wald 4.8, p = 0.03). Age at operation and bypass time were also highly significant predictors of postoperative death (Wald 13.7 and 33.8, respectively, p < 0.0001 for both). Operative performance was measured at 7.52 units. CONCLUSIONS: The Basic Aristotle Score was only weakly associated with postoperative mortality in this series. Operative performance appeared to be inflated by the fact that the overall complexity of cases was relatively high in this series. An alternative equation (performance = complexity/mortality) is proposed as a fairer and more logical method of risk adjustment. PMID- 16677820 TI - Malformation of the thoracic aorta associated to aneurysms of the arch: an extraanatomic surgical approach. PMID- 16677821 TI - A polypeptide from Chlamys farreri abolishes UV-induced apoptosis in murine thymocytes in vitro. AB - Previously we reported that a polypeptide from Chlamys farreri (PCF) was a potent photoprotective agent against ultraviolet (UV) irradiation in vitro. To understand the mechanism by which PCF protects cells from irradiation, we studied anti-apoptotic effects of PCF against UV irradiation on the murine thymocytes in vitro. MTT and flow cytometric analysis assays showed that 2h pretreatment with PCF completely abolished UV induced cell death. TEM examination showed that PCF fully protected the ultrastructure of thymocytes exposed to UV irradiation. Lipid peroxidation and intracellular reactive oxygen species assays indicated that PCF efficiently blocked production of reactive oxygen intermediates induced by UV irradiation. Further, PCF protected UV-irradiated thymocytes from losing mitochondrial transmembrane potential and DNA fragmentation. Based on these observations we propose that PCF is a potent anti-apoptotic factor, which protects cells from irradiation at multiple steps. PMID- 16677822 TI - The expression of the chemorepellent Semaphorin 3A is selectively induced in terminal Schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease. AB - Neuromuscular synapses differ markedly in their plasticity. Motor nerve terminals innervating slow muscle fibers sprout vigorously following synaptic blockage, while those innervating fast-fatigable muscle fibers fail to exhibit any sprouting. Here, we show that the axon repellent Semaphorin 3A is differentially expressed in terminal Schwann cells (TSCs) on different populations of muscle fibers: postnatal, regenerative and paralysis induced remodeling of neuromuscular connections is accompanied by increased expression of Sema3A selectively in TSCs on fast-fatigable muscle fibers. To our knowledge, this is the first demonstration of a molecular difference between TSCs on neuromuscular junctions of different subtypes of muscle fibers. Interestingly, also in a mouse model for amyotrophic lateral sclerosis (ALS), Sema3A is expressed at NMJs of fast fatigable muscle fibers. We propose that expression of Sema3A by TSCs not only suppresses nerve terminal plasticity at specific neuromuscular synapses, but may also contribute to their early and selective loss in the motor neuron disease ALS. PMID- 16677823 TI - Head-head coordination is required for the processive motion of cytoplasmic dynein, an AAA+ molecular motor. AB - Cytoplasmic dynein is an AAA(+)-type molecular motor whose major components are two identical heavy chains containing six AAA(+) modules in tandem. It moves along a single microtubule in multiple steps which are accompanied with multiple ATP hydrolysis. This processive sliding is crucial for cargo transports in vivo. To examine how cytoplasmic dynein exhibits this processivity, we performed in vitro motility assays of two-headed full-length or truncated single-headed heavy chains. The results indicated that four to five molecules of the single-headed heavy chain were required for continuous microtubule sliding, while approximately one molecule of the two-headed full-length heavy chain was enough for the continuous sliding. The ratio of the stroking time to the total ATPase cycle time, which is a quantitative indicator of the processivity, was approximately 0.2 for the single-headed heavy chain, while it was approximately 0.6 for the full-length molecule. When two single-headed heavy chains were artificially linked by a coiled-coil of myosin, the processivity was restored. These results suggest that the two heads of a single cytoplasmic dynein communicate with each other to take processive steps along a microtubule. PMID- 16677824 TI - MoxR AAA+ ATPases: a novel family of molecular chaperones? AB - The MoxR AAA+ family is a large, diverse group of ATPases that, so far, has been poorly studied. Members of this family are found throughout the Bacteria and Archaea superkingdoms, but have not yet been detected in Eukaryota. The limited experimental data available to date suggest that members of this family might have chaperone-like activities. Here we present an extensive phylogenetic analysis which builds upon our previously published work, and reveals that the MoxR family can be divided into at least seven subfamilies, including MoxR Proper (MRP), TM0930, RavA, CGN, APE2220, PA2707, and YehL. We also include a comprehensive overview and gene context analysis for each of these subfamilies. Our data reveal distinct conserved associations of certain MoxR family members with specific genes, including further support for our previously reported observation that many members of the MoxR AAA+ family are found near Von Willebrand Factor Type A (VWA) proteins and are likely to function with them. We propose, based on bioinformatic analyses and the available literature, that the MoxR AAA+ proteins function with VWA domain-containing proteins to form a chaperone system that is important for the folding/activation of proteins and protein complexes by primarily mediating the insertion of metal cofactors into the substrate molecules. PMID- 16677825 TI - Crystal structure of a lectin from Canavalia maritima (ConM) in complex with trehalose and maltose reveals relevant mutation in ConA-like lectins. AB - The crystal structure of Canavalia maritima lectin (ConM) complexed with trehalose and maltose revealed relevant point mutations in ConA-like lectins. ConM with the disaccharides and other ConA-like lectins complexed with carbohydrates demonstrated significant differences in the position of H-bonds. The main difference in the ConM structure is the replacement of Pro202 by Ser202, a residue that promotes the approximation of Tyr12 to the carbohydrate-binding site. The O-6' of the second glucose ring in maltose interacts with Tyr12, while in trehalose the interaction is established by the O-2' and Tyr12, explaining the higher affinity of ConM for disaccharides compared to monosaccharides. PMID- 16677826 TI - Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features. AB - The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots. PMID- 16677827 TI - Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity. AB - The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+) chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes. PMID- 16677828 TI - Identification, cloning and characterization of interleukin-17 and its family from zebrafish. AB - Cytokines are one of the major signaling molecules involved in immunity. Many of these cytokines have been isolated in vertebrates and found to play a significant role in host defense mechanism. Interleukin-17 (IL-17) family of genes are known to have pro-inflammatory actions and associated with specific disease conditions, these genes are conserved across vertebrate evolution. In this study, computational screening for the zebrafish (Danio rerio) genome resulted in identification of five contigs harboring IL-17 genes. Zebrafish cDNA encoding five IL-17 genes exhibited percentage identities of 19.3%-61.9% with that of human homologs. The molecules show conservation of cysteines, important for disulphide bonds for IL-17 molecules. The structural composition of these genes shows two introns and three exons except for IL-17D gene that has only one intron and two exons. Phylogenetic analysis using maximum parsimony algorithm showed that zebrafish IL-17 genes clustered well with other IL-17 homologs further proving the structural similarity with IL-17 genes from other organisms. Expression analysis by RT-PCR revealed expression of IL-17 genes in normal and stimulated tissues of kidney, spleen, gills and intestine. The expression of IL 17 in un-stimulated tissues indicates that these genes may play important roles in normal conditions as well. PMID- 16677829 TI - Wrist stabilisation and forearm muscle coactivation during freestyle swimming. AB - The aim of this study was to evaluate the stabilisation of the wrist joint and the ad hoc wrist muscles activations during the two principal phases of the freestyle stroke. Seven male international swimmers performed a maximal semi tethered power test. A swimming ergometer fixed on the start area of the pool was used to collect maximal power. The electromyography signal (EMG) of the right flexor carpi ulnaris (FCU) and extensor carpi ulnaris (ECU) was recorded with surface electrodes and processed using the integrated EMG (IEMG). Frontal and sagittal video views were digitised frame by frame to determine the wrist angle in the sagittal plane and the principal phases of the stroke (insweep, outsweep). Important stabilisation of the wrist and high antagonist muscle activity were observed during the insweep phase due to the great mechanical constraints. In outsweep, less stabilisation and lower antagonist activities were noted. Factors affecting coactivations in elementary movements, e.g. intensity and instability of the load, accuracy and economy of the movement were confirmed in complex aquatic movement. PMID- 16677830 TI - Progressive grey matter atrophy over the first 2-3 years of illness in first episode schizophrenia: a tensor-based morphometry study. AB - Little is known about the structural brain changes that occur over the first few years of schizophrenia, or how these changes differ from those associated with healthy brain development in adolescence and early adulthood. In this study, we aimed to identify regional differences in grey matter (GM) volume between patients with first-episode schizophrenia (FES) and matched healthy controls, both at the time of the patients' first psychotic episode (baseline condition) and 2-3 years subsequently (follow-up condition). Forty-one patients with FES and 47 matched healthy controls underwent a T1-weighted structural MRI scan. Of these participants, 25 FES patients and 26 controls returned 2-3 years later for a follow-up scan. Voxel-based morphometry in SPM2 was used to identify the regions of GM difference between the groups in the baseline condition, while tensor-based morphometry was used to identify the longitudinal change within subject over the follow-up interval. The FES patients exhibited widespread GM reductions in the frontal, parietal, and temporal cortices and cerebellum in the baseline condition, as well as more circumscribed regions of GM increase, particularly in the occipital lobe. Furthermore, the FES subjects were observed to lose considerably more GM over the follow-up interval than the controls, especially in the parietal and temporal cortices. We argue that the progressive GM atrophy we have found to be associated with the onset of schizophrenia arises from a dysfunction in the dramatic period of healthy brain development typically associated with adolescence. PMID- 16677831 TI - Comparison of two Simon tasks: neuronal correlates of conflict resolution based on coherent motion perception. AB - The present study aimed at characterizing the neural correlates of conflict resolution in two variations of the Simon effect. We introduced two different Simon tasks where subjects had to identify shapes on the basis of form-from motion perception (FFMo) within a randomly moving dot field, while (1) motion direction (motion-based Simon task) or (2) stimulus location (location-based Simon task) had to be ignored. Behavioral data revealed that both types of Simon tasks induced highly significant interference effects. Using event-related fMRI, we could demonstrate that both tasks share a common cluster of activated brain regions during conflict resolution (pre-supplementary motor area (pre-SMA), superior parietal lobule (SPL), and cuneus) but also show task-specific activation patterns (left superior temporal cortex in the motion-based, and the left fusiform gyrus in the location-based Simon task). Although motion-based and location-based Simon tasks are conceptually very similar (Type 3 stimulus response ensembles according to the taxonomy of [Kornblum, S., Stevens, G. (2002). Sequential effects of dimensional overlap: findings and issues. In: Prinz, W., Hommel., B. (Eds.), Common mechanism in perception and action. Oxford University Press, Oxford, pp. 9-54]) conflict resolution in both tasks results in the activation of different task-specific regions probably related to the different sources of task-irrelevant information. Furthermore, the present data give evidence those task-specific regions are most likely to detect the relationship between task-relevant and task-irrelevant information. PMID- 16677832 TI - BOLD response during uncoupling of neuronal activity and CBF. AB - The widely used technique of functional magnetic resonance imaging (fMRI) based on the blood oxygenation level-dependent (BOLD) effect is a tool for the investigation of changes in local brain activity upon stimulation. The principle of measurement is based on the assumption that there is a strong coupling between changes in neural activity, metabolism, vascular response and oxygen extraction in the area under investigation. As fMRI is on the way to become a routine tool in clinical examinations, we wanted to investigate whether, generally and under a variety of conditions, there is a strong link between the BOLD signal and neural activity. For clinical and experimental application of the method, it is crucial, whether the absence of changes in BOLD signal intensity upon stimulation can always be interpreted as an absence of changes in brain activity. We approached this question by inhibiting the nitric oxide mediated 'neurovascular coupling' via application of 7 nitroindazole. Before and after inhibition of this neurovascular coupling, we acquired evoked potentials and performed fMRI during somatosensory stimulation in rats. Cerebral blood flow response as well as BOLD signal intensity changes following electrical stimulation were abolished within 10 min after application of 7 nitroindazole, whereas somatosensory-evoked potentials were only slightly affected but still clearly detectable. Even 1 h after injection of 7 nitroindazole, there was still remaining electrical activity. Thus, we observed an uncoupling between electrical, i.e., neural activity and the BOLD signal. According to our results, the absence of BOLD signal changes did not permit the conclusion that there was no neural activity in the area under investigation. Our findings are especially relevant for the clinical application of fMRI in patients suffering from cerebrovascular and other brain diseases. PMID- 16677833 TI - Reproducibility and reliability of MR measurements in white matter: clinical implications. AB - The purpose of this study was to determine the reproducibility and reliability of five MRI-derived measurements, namely, total water content (WC), myelin water content (MWC), mean T2 relaxation time (GMT2), T1 relaxation time (T1) and magnetization transfer ratio (MTR). Five controls were scanned 5 times over 1 year. The five MR measurements were made for 5 white matter regions. All measurements were found to be highly reproducible. MTR had a low reliability coefficient because all individual values were similar. Therefore, MTR would be most sensitive in detecting changes from normal. WC had a high reliability coefficient in all regions. For MWC, GMT2 and T1, the overall reliability coefficients were high but for some individual regions were low. The high coefficients suggest that these measurements, although different between normal subjects, are consistent over time. They could be used to explore natural differences in the normal population, but due to the large spread in normal values, larger sample sizes are needed to detect pathological changes. PMID- 16677834 TI - Expression of the cochaperone HspBP1 is not coordinately regulated with Hsp70 expression. AB - Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70. PMID- 16677835 TI - Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis. AB - Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells. PMID- 16677837 TI - Prognostic factors in first-time care seekers due to acute low back pain. AB - There is limited knowledge on prognostic factors for developing chronic low back pain (LBP) at an early stage of LBP. The objectives of this study were to investigate the clinical course of pain and disability, and prognostic factors for non-recovery after 1-year, in patients seeking help for the first time due to acute LBP. An inception cohort study included 123 patients with acute LBP lasting less than 3 weeks and consulting primary care for the first time. Main outcome measures were pain intensity, Roland-Morris disability questionnaire (RMQ), and sickness absence. Eleven patients (9%) did not return for the 12-month follow-up. There were large and significant reductions in pain intensity (P<0.001) and RMQ scores (P<0.001) during follow-up. Patients with neurological signs showed significantly less improvement in pain (P=0.001) and RMQ (P=0.004) compared with those without neurological signs. The proportions with sickness absence due to LBP at 6, 9, and 12 months were 7%, 8%, and 9%, respectively. At 12 months, 17% of patients had not fully recovered. Multivariate logistic regression analyses showed that high scores on a psychosocial screening (acute low back pain screening questionnaire) and emotional distress (Hopkin's symptom check list) were significantly associated with non-recovery at 12 months, with odds ratios of 4.4 (95% confidence interval 1.1-17.4) and 3.3 (1.1-10.2), respectively. PMID- 16677838 TI - Percutaneous computed-tomography-guided biopsy of the spine: 229 procedures. AB - OBJECTIVES: Percutaneous biopsy of the spine is an effective and well-evaluated procedure. Only very few series containing more than a hundred patients have been reported so far and there is no agreement about the factors affecting the diagnostic rate. We aimed to find out if there is any factor influencing the success rate of the spinal biopsy using our biopsy series. METHODS: Two hundred and twenty-nine procedures were performed in 201 patients between November 2001 and August 2005. All procedures were performed under computed tomography guidance. The side was chosen according to the extension of the lesion. When the lesion was in the midline or extended to both sides, we preferred to obtain biopsy from the right side. The puncture point and the needle trajectory were planned on both lateral computed tomography scout scan and axial scans. RESULTS: We found that the diagnostic rate was not affected by the variables such as age, gender, type and diameter of the biopsy needle, diagnosis as well as lesion localization and level. The success rate of the repeat biopsies was considerably lower than the first procedures. CONCLUSIONS: The diagnostic rate is not affected by any of the variables but the approach, chosen can vary with the level, localization, and lesion characteristics. PMID- 16677839 TI - [Feasibility and limits of laparoscopic treatment of borderline ovarian tumours]. AB - Borderline ovarian tumours (BOT) are mainly diagnosed in young women with early stage disease. Due to the absence of specific pre operative criteria for BOT, a laparoscopy is usually performed. A review of the literature found no pejorative data on laparoscopic approach for BOT. Strict surgical procedures must be performed to avoid incomplete surgical staging, cells dissemination and port-site metastases. The limits of the laparoscopic management are the stage of disease and the tumour size. Laparoscopic treatment of BOT for women with early stage disease is feasible. This treatment should be evaluated in specialized centres for women with advanced stage diseases and/or peritoneal implants. PMID- 16677840 TI - [Chilaiditi's syndrome responsible for an occlusion with necrosis of the small intestine after a caesarean section: first case report]. AB - Chilaiditi's syndrome is the association of a radiological and clinical semiology of the interposition of large colon or small intestine between the lower side of diaphragm and liver. We report the case of a 32-year-old woman, primigravida, who undergone a caesarean section at 39 weeks of amenorrhoea, for a clinical picture of persistent abdominal pain and a beginning hepatic cytolysis. The patient presented one day after the ceasarean section an occlusive syndrome of the small intestine. The etiology of the occlusion of the small intestine in post-partum was a Chilaiditi's syndrome with inter hepatodiaphragmatic incarceration of the small intestine discovered at the computed tomography. An exploratory laparotomy confirmed and permitted to treat Chilaiditi's syndrome complicated by an occlusion. PMID- 16677841 TI - [Update of 'standards, options and recommendations' for FDG-PET in breast and gynaecological cancers]. PMID- 16677842 TI - [Cystocele and stress urinary incontinence: assessment of a total treatment by monoprosthesis]. AB - OBJECTIVES: The aim of this study is to evaluate the effectiveness and the potential complications in the short and medium terms of the transobturator and infracoccygeal hammock, a surgical technique suggested to treat in one sole operating time, by vaginal way, cystocele and stress urinary incontinence. PATIENTS AND METHODS: From February 2002 till August 2004, 45 patients aged 66+/ 11 years on average, presenting a stage 3 or stage 4 cystocele associated with stress urinary incontinence (proven for 40 of them and masked by the prolapse itself for the other five) benefited from this new surgical procedure. RESULTS: With a mean follow-up of 31+/-9 months, the success rate of the technique is estimated at 98% on the anatomical aspect (one single failure) and at 91% on the urinary aspect (73% of the patients were cured, 18% improved and 9% in failure). No patient presented urinary retention except one with an infected pelvic hematoma requiring its evacuation and the partial ablation of the prosthesis. Postvoid residual volume at discharge was 83 ml. Two patients developed de novo bladder overactivity. Unfortunately we had to regret nearly 18% of exposed prosthesis. This result should be improved by the exclusive use of polypropylene monofilament prosthesis with broad meshes, weak weights and by reduced vaginal scars. DISCUSSION AND CONCLUSION: Total treatment by monoprosthesis of cystoceles with associated stress urinary incontinence constitutes an interesting prospect for some high-risk genital urinary prolapses for anatomical repetition or post operative dysvoiding. Patients with recurrent failures of prolapse surgical cure, those with a large cystocele or by extension with a complete prolapse of the three floors, and even those with a post-hysterectomy prolapse have perhaps found a durable solution with their problem. PMID- 16677843 TI - [Endometrioid adenocarcinoma of the uterine cervix associated with mucinous ovarian cystadenocarcinoma]. AB - The authors report the case of a 40-year-old woman, who was operated for an ovarian mucinous cystadenocarcinoma. The pathologic findings of the hysterectomy specimen with bilateral salpingoophorectomy showed an ovarian mucinous cystadenocarcinoma associated with an endometrioid adenocarcinoma of the uterine cervix. The mucinous cystadenocarcinoma represents the third most common type of ovarian carcinoma. In the literature, this tumor had been found in association with endocervical adenocarcinoma or with minimal deviation adenocarcinoma (adenoma malignum) of the uterine cervix. However, its association with an endometrioid adenocarcinoma, to our knowledge, has not been reported. PMID- 16677844 TI - Ultrasound assisted supercritical fluid extraction of oil and coixenolide from adlay seed. AB - Oil and coixenolide are important components of adlay seed (Coix lachrymal-jobi L. var. Adlay) with many beneficial functions to human health. In this work, a novel extraction technique--ultrasound assisted supercritical fluid extraction (USFE)--was studied. Effects of operating conditions on the extraction, including extraction temperature (T), pressure (P), time (t), CO(2) flow rate (F) and ultrasonic power (I) were investigated. There are optimum temperatures which gives the maximum extraction yields (EYs) for the supercritical fluid extractions with and without ultrasound. The effect of pressure on EYs for is similar to that of pressure on CO(2) density. Based on the yield of extraction, the favorable conditions for supercritical fluid extraction (SFE) were: T at 45 degrees C, P at 25 MPa, t at 4.0 h and F at 3.5L/h. While ultrasound was applied as in USFE, the following parameters were preferred: T at 40 degrees C, P at 20 MPa, t at 3.5h and F at 3.0 L/h, respectively. The results show that supercritical fluid extraction with the assistance of ultrasound could reduce the temperature, pressure, CO(2) flow rate, as well as time used in the process. Compared with SFE, USFE could give a 14% increase in the yield for extracting oil and coixenolide from adlay seed with less severe operating conditions. PMID- 16677845 TI - Connexin43: emerging role in erectile function. AB - Connexins, that have their main function as part of gap junction channels, are proteins expressed in a large number of tissues such as endocrine, nervous, vascular, and muscular tissues. Gap junctions are implicated in tissue homeostasis and control of cell proliferation and differentiation. Interestingly, mutations of connexin genes have been reported in several human diseases (peripheral neuropathies, cardiovascular and dermatological diseases, hereditary cataract, and deafness) and altered expression of connexins have been associated with tumoral progression. Today, several lines of study argue for a critical role of gap junctions in corporal smooth muscle relaxation and erectile response. The present review highlights the emerging role of connexin43, one of these membranous proteins, in the physiology and physiopathology of human erectile function and its possible medical application. PMID- 16677846 TI - The variable expression of keratoconus within monozygotic twins: dundee University Scottish Keratoconus Study (DUSKS). AB - INTRODUCTION: As part of a longitudinal observational prospective study (Dundee University Scottish Keratoconus Study, DUSKS) a caucasian female subject was identified with keratoconus affecting both corneas. Of interest was that the subject's mother was a monozygotic twin, however, the mother was reportedly unaffected by keratoconus, although her sister (the subject's aunt) had been diagnosed with keratoconus. METHOD: Computer video-keratoscopy (CVK) was undertaken was on this family to clarify this apparent discordance. RESULTS: CVK demonstrated the variable expression of the keratoconic phenotype, by identifying evidence of subclinical keratoconus in the 'unaffected' twin. CONCLUSION: So far only one set of monozygotic twins with keratoconus have been reported within the UK, this paper reports a second. Since CVK has been available the majority agree on concordance of keratoconus expression but not all, including this study. If monozygotic twin studies demonstrate heterogeneity of keratoconus this may at least explain the contradictory data that exists within the literature and why the inheritance pattern of keratoconus is still unclear in 2005. PMID- 16677847 TI - Obtaining and screening compound collections: a user's guide and a call to chemists. AB - Advances in genetics, proteomics and cell biology over the past 20 years have unearthed a multitude of potential macromolecular targets for the selective treatment of disease. The challenge remains to find appropriate small molecule ligands for these proteins (or nucleic acids), and to use these ligands to validate novel disease targets. The advent of low-cost instrumentation has made industrial-style high-throughput screening possible in academic settings. Unfortunately for many, access to large collections of compounds is still limited and limiting. This article is aimed at the user who has an interest in compound screening but does not have ready access to large collections of small molecules. High-throughput screening need not be the exclusive domain of institutions and centers with vast resources and NIH Roadmap-funded compound repositories. As it turns out, many of the most interesting compounds are probably within arm's reach, in our laboratory freezers and in those of our colleagues. PMID- 16677848 TI - Microfluidic cartridges preloaded with nanoliter plugs of reagents: an alternative to 96-well plates for screening. AB - In traditional screening with 96-well plates, microliters of substrates are consumed for each reaction. Further miniaturization is limited by the special equipment and techniques required to dispense nanoliter volumes of fluid. Plug based microfluidics confines reagents in nanoliter plugs (droplets surrounded by fluorinated carrier fluid), and uses simple pumps to control the flow of plugs. By using cartridges pre-loaded with nanoliter plugs of reagents, only two pumps and a merging junction are needed to set up a screen. Screening with preloaded cartridges uses only nanoliters of substrate per reaction, and requires no microfabrication. The low cost and simplicity of this method has the potential of replacing 96-well and other multi-well plates, and has been applied to enzymatic assays, protein crystallization and optimization of organic reactions. PMID- 16677849 TI - Lip-splitting in transmandibular resections: is it really necessary? AB - Mandibular resection approach (Commando or Composite resection) is one of the fundamental techniques for oral and oropharyngeal large tumour resection. We reviewed the charts of patients who underwent a transmandibular resection for an oral and/or oropharyngeal cancer between 1980 and 2002. Of 700 patients who underwent a mandibular resection for cancer, 332 had been operated without lower lip splitting. A mono or bilateral en-block neck dissection was always performed, except in cases of relapses after a prior surgical treatment with neck dissection. We repaired 307 patients with flaps (pedicled or free flaps, with or without bone). Unsplitting of the lip never complicated resection and reconstruction. Furthermore the procedure was time sparing, as we avoided haemostasis and suture of the lip. The cosmetic results were better than those obtained by traditional technique. We used a non-lip-splitting technique also for pull-through, marginal mandibulectomy and, sometimes, for mandibular-swing approaches. In the latter case, the technique has some advantages and disadvantages and must be applied according to circumstances. We can conclude that lip-splitting in transmandibular resection for oral and oropharyngeal tumours is not necessary. PMID- 16677851 TI - Antioxidant and antimutagenic activity of mannan neoglycoconjugates: mannan-human serum albumin and mannan-penicillin G acylase. AB - The antioxidant and antimutagenic activity of the yeast cell-wall mannan and mannan conjugates--in particular the mannan of Saccharomyces cerevisiae (M-S.c.) and conjugates of mannan S. cerevisiae with human serum albumin (M-HSA1, M-HSA2) and the microbial enzyme penicillin G acylase (M-PGA)--were evaluated in vitro in the unicellular flagellate Euglena gracilis exposed to the genotoxic agents ofloxacin and acridine orange (AO). M-S.c., M-HSA1, M-HSA2 and M-PGA show a statistically significant, concentration-dependent protective antigenotoxic activity against both compounds. M-PGA was the most efficient inhibitor of ofloxacin- and AO-induced chloroplast DNA damage, whereas M-HSA2 and M-HSA1 were less effective and M-S.c. had the lowest antigenotoxic activity. It is suggested that different mechanisms may be involved in their protective effect--antioxidant activity in the case of ofloxacin-induced DNA damage and direct adsorption of AO on mannan conjugates as possible mechanisms of protection, based on spectrophotometric measurements. The important characteristics of yeast cell-wall mannans and mannan conjugates, such as their high water solubility, their broad spectrum of biological activity, low toxicity, stability and their antimutagenic effects via different modes of action, appear to be promising features for their practical application as antioxidants and antimutagenic agents. PMID- 16677850 TI - Assessment of the genotoxic potential of the antipsychotic sigma receptor ligand E-5842. AB - The genotoxic potential of E-5842, a sigma ligand compound being developed as an antipsychotic drug, was evaluated by means of an extensive battery of in vitro and in vivo assays. Negative results were obtained in an Ames test (up to 5000 MUg/plate), a mouse lymphoma assay (up to 535.1 MUg/ml (-S9) and 891.8 MUg/ml (+S9)), an in vivo rat hepatocyte micronucleus assay (up to 100 mg/kg/day on 2 days), and a two-dose mouse micronucleus assay (up to 40 mg/kg/day on 2 days). In a single-dose mouse bone-marrow micronucleus assay (up to 400 mg/kg; 24, 48 and 72 h sampling) a slight and non-statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was observed 48 h after administration of a 200 mg/kg dose, in the absence of bone-marrow toxicity. This minor increase in MNPCE frequency was considered of questionable biological relevance, because it was observed under conditions of marked animal toxicity including mortality. In addition, it occurred in association with a strong hypothermic effect produced by administration of E-5842. A clear increase in the frequency of structural chromosomal aberrations was observed in human lymphocytes at concentrations >=350.6 and 1685.4 MUg/ml in the presence and absence of S9, respectively. Mitotic accumulation was observed at those concentrations at which clastogenic effects were observed, a condition that may have masked toxicity. Concentrations lacking clastogenic effects in this chromosome aberration assay (300.7 and 173.2 MUg/ml in the presence and absence of S9, respectively) were well in excess of maximum human plasma concentrations attained in clinical studies at the maximum tolerated dose (19.1 ng/ml). A weight-of-evidence analysis, taking into consideration the results obtained in the different in vitro and in vivo assays and the conditions of clinical use, suggest that E-5842 would not pose a genotoxic risk under clinical conditions. PMID- 16677852 TI - Spectroscopy, crystal structure, valance molecular orbital energy level diagram and DFT study of cis-[Cr(2,2'-bipy)2Cl2](Cl)0.38(PF6)0.62. AB - A new octahedral chromium(III) complex having 2,2'-bipyridine as ligand system was synthesized in methanol. Single crystal X-ray diffraction analysis shows that it possesses non-stoichiometry in its anionic primary covalency. It has also been studied by elemental analyses, optical spectroscopy (UV-vis, IR) and magnetic susceptibility data. DFT calculations (with B3LYP functional and double-xi quality LANLDZ(D95V) basis set) were carried out to interpret the electronic and infrared spectra of the complex. The DFT optimized geometric structure for the complex is compared with the X-ray crystallographic data; the theory-experiment agreement is satisfactory. PMID- 16677853 TI - ZINDO/S and PM3 calculations for a biscyanide alpha-iminooxime cobalt macrocyclic complex. AB - Molecular modeling and a detailed spectroscopic characterization of the macrocyclic complex [Co(dohpn)(CN)(2)] (dohpn=2,3,9,10-tetramethyl-1,4,8,11 tetraazaundecane-1,3,8,10-tetraen-11-ol-1-olate and py=pyridine) is herein presented. Structural, electronic and vibrational features are discussed and assignments were proposed on the basis of semi-empirical (molecular mechanics, ZINDO/S and PM3) calculations. PMID- 16677854 TI - Vibrational properties of silanol group: from alkylsilanol to small silica cluster. Effects of silicon substituents. AB - Structural and vibrational features of silanol group are investigated in detail by quantum calculations and normal mode analysis. The structural parameters, charge distributions, force fields, vibrational wavenumbers, potential energy distributions of normal modes and derivatives of the electric dipole moment are analyzed in relation to the nature of the substituents adjacent to the silanol group. The calculations results are discussed in light of available experimental data. Although the OH stretching mode has already been well localized in various silanols, both the Si-(OH) stretching and SiOH bending vibrations have not been yet finely analyzed leading to some discrepancies reported in literature. Clarified assignments of these vibrations are proposed on the basis of normal mode analysis and of SiOH-->SiOD isotopic exchange. The following spectral ranges are determined: 790-1030 cm-1 for nuSi-(OH), 790-1010 cm-1 for nuSi-(OD), 790-900 cm-1 for deltaOH and 580-640 cm-1 for deltaOD. The nuSi-(OH)/nuSi-(OD) wavenumbers are highly dependent on silicon substituents: electron-withdrawing groups induce shifts to higher wavenumbers while electron-releasing groups induce shifts to lower wavenumbers. In alkylsilanols, the SiOH bending is observed at higher wavenumber than the stretching vibration. Analysis of infrared intensities and dipole derivatives in internal coordinates gives explanations to spectral "anomalies" observed in experimental measurements such as well defined and intense nuSi-(OD) absorption in contrast with very low intensity for nuSi-(OH). Numerous empirical correlations are established allowing reconstruction of both SiOH force field and SiOH structural parameters with knowledge of few experimental data. PMID- 16677855 TI - Clinical correlates of index values in the focus HerpeSelect ELISA for antibodies to herpes simplex virus type 2 (HSV-2). AB - BACKGROUND: Clinical correlates of HerpeSelect ELISA index values are poorly understood. OBJECTIVES: This study was designed to determine the effects of time of infection, test variability, and antibody avidity on index values. STUDY DESIGN: Sera (N=313) from 81 patients with new HSV-2 infections and 236 sera from 32 patients with long-standing (median 11.3 years) HSV-2 were tested by HerpeSelect HSV-2 ELISA. High positive, low positive and negative controls were run on 42 test plates to establish test variability. RESULTS: Index values tended to rise after infection, peaking a median of 9-10 weeks post-infection (range 8 323 days). Of 32 patients with established HSV-2 infections, 7 (22%) had at least one low index value (>1.1 to < or =3.5), and one had a transient seroreversion event. Test variability of index values was substantially lower than inter- or intra-patient variability. Median antibody avidity was higher in sera with high versus low index values in established infections, but unrelated to index value in patients with early infections. CONCLUSIONS: Index values or index value changes are not absolute indicators of early versus established HSV-2 infection or solely a function of test variability. Low antibody avidity may contribute to low index values once infection is established. PMID- 16677856 TI - Dietary plant sterols accumulate in the brain. AB - Dietary plant sterols and cholesterol have a comparable chemical structure. It is generally assumed that cholesterol and plant sterols do not cross the blood-brain barrier, but quantitative data are lacking. Here, we report that mice deficient for ATP-binding cassette transporter G5 (Abcg5) or Abcg8, with strongly elevated serum plant sterol levels, display dramatically increased (7- to 16-fold) plant sterol levels in the brain. Apolipoprotein E (ApoE)-deficient mice also displayed elevated serum plant sterol levels, which was however not associated with significant changes in brain plant sterol levels. Abcg5- and Abcg8-deficient mice were found to carry circulating plant sterols predominantly in high-density lipoprotein (HDL)-particles, whereas ApoE-deficient mice accommodated most of their serum plant sterols in very low-density lipoprotein (VLDL)-particles. This suggests an important role for HDL and/or ApoE in the transfer of plant sterols into the brain. Moreover, sitosterol upregulated apoE mRNA and protein levels in astrocytoma, but not in neuroblastoma cells, to a higher extend than cholesterol. In conclusion, dietary plant sterols pass the blood-brain barrier and accumulate in the brain, where they may exert brain cell type-specific effects. PMID- 16677857 TI - Tumors localized near the central sulcus may cause increased somatosensory evoked potentials. AB - OBJECTIVE: Median nerve somatosensory evoked potentials (SEPs) may be altered in patients with cerebral tumors. In rare cases cortical responses may be increased, but the significance of this finding remains unclear. METHODS: We investigated 3 patients in whom an epileptic seizure was the only neurological symptom of a cerebral tumor located near the central sulcus. We studied median nerve SEPs, motor evoked potentials in abductor digiti minimi muscle, and long-loop reflexes in abductor pollicis brevis muscle bilaterally. Two patients also underwent intraoperative neurophysiological monitoring. RESULTS: All 3 patients presented with enlarged cortical SEPs on the side of the brain tumor. The responses increased further post-operatively, and the enhancement persisted in follow-up examinations up to 6 months after surgical tumor extirpation. Intraoperative monitoring documented a substantial increase of the enlarged potential N20-P22 during tumor removal in one patient, who also presented with an exaggerated long loop reflex on the tumor side. Transcranial magnetic stimulation revealed unremarkable motor evoked potentials in all 3 patients. CONCLUSIONS: Distinct mechanisms must be considered in order to explain both immediate and long-term changes of neuronal excitability leading to increased cortical SEPs. SIGNIFICANCE: Hyperexcitability of cortical neurons or insufficient cortical inhibitory mechanisms may be responsible for increased SEPs, which may serve as an epileptic marker in patients suffering from a tumor near the central sulcus. PMID- 16677858 TI - What's up? PMID- 16677859 TI - Down syndrome mouse models are looking up. AB - A new mouse model of Down syndrome (DS) carries a copy of human chromosome 21 (Hsa21), in addition to a full complement of mouse chromosomes. In terms of the number of trisomic genes represented, this model, known as 'Tc1', is closer to the genetic background of DS than any previous model. The Tc1 model not only recapitulates several of the DS features present in other mouse models but also exhibits heart defects that are similar to those that make trisomy 21 the leading cause of congenital heart disease in humans. Many cells in adult Tc1 mice show mosaicism - that is, the Hsa21 is lost from some cells during development - increasing the complexity of analyses using this model. Tc1 mice provide a powerful tool for investigation of the pathogenesis of trisomy 21, and a platform for analysis of similarities and differences in the evolution of gene regulation. PMID- 16677860 TI - Is a new tuberculosis vaccine necessary and feasible? A Cuban opinion. AB - Mycobacterium tuberculosis kills more human beings worldwide than any other pathogen. An estimated two billion people are already infected with the bacterium. In 2006, tuberculosis (TB) will kill nearly one million more people than in 1992. In Cuba, TB is not a serious health problem any more and we are striving to eliminate it in the near future. The most widely applied human vaccine in the world is BCG. It is also a safe vaccine except when it is applied to immunocompromised persons. Its protective efficacy is a controversial topic. In spite of this, more than 80 years of experience with this vaccine has demonstrated that BCG is effective, at least in significantly preventing childhood TB, including the meningeal and disseminated forms of the disease, but does not protect against the predominant pulmonary form of the disease in adults, which means that our best TB vaccine now is inadequate; we therefore need a new vaccine. The following facts, apart from the experience with BCG, support discussion about the feasibility of a new and better TB vaccine: Less than 10% of the 2 billion TB infected persons develop active disease. It has been demonstrated that HIV + TB co-infection increases 30 times the risk of contracting active TB and it increases the risk of being killed by the bacterium. Some new vaccine candidates, now under development and evaluation, are showing promising results in preclinical studies, and a few of them have entered clinical trials. There seems to be a consensus that a new TB vaccine will be feasible, but some challenging issues must be positively solved, such as, the lack of universally accepted correlates for protection, improved diagnostics, and final vaccine efficacy evaluation conducted on large phase III clinical trials in underdeveloped countries. The ethical, economical, organizational and scientific questions involved in this global task are enormous, but feasible. PMID- 16677861 TI - Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT 6 protein. AB - We generated several attenuated recombinant influenza A vectors expressing the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein. The ESAT-6 protein was recently identified as one of the most promising protective antigens for cell-mediated immunity. The obtained vectors appeared to be capable of inducing ESAT-6 specific Th1 immune response in mice after intranasal immunization. We found that double immunization with two influenza vectors of different subtypes provided a significant level of protection in mice, when applied as prophylactic vaccine, as well as substantial therapeutic effect in mice with pre-established tuberculosis infection. Moreover, we found a strong synergistic effect when vaccination with Flu/ESAT-6 vectors was combined with isoniazid treatment, resulting in a dramatic reduction of bacterial load in the lungs of infected mice. PMID- 16677862 TI - The effect of the administration of human gamma globulins in a model of BCG infection in mice. AB - The effect of the administration of a commercial preparation of human gamma globulins has been evaluated in a mouse model of intranasal infection with BCG. First, we demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration to mice of human gamma globulins. This treatment of mice inhibited BCG colonization of the lungs (p < 0.01). A similar inhibitory effect was observed after infection of mice with gamma globulin opsonized BCG organisms (p < 0.01). These results are relevant for the development of new strategies for the control and treatment of tuberculosis. PMID- 16677863 TI - Multiplex mapping of CD4 T cell epitopes using class II tetramers. AB - With the advent of class II tetramer technology, a tetramer-guided epitope mapping (TGEM) technique was developed for the identification of CD4+ T cell epitopes. This allowed the direct identification of epitopes recognized by the responding T cells, which were restricted to the single MHC allele of interest. However, as each individual carries multiple class II alleles, it would be advantageous to design an approach to identify CD4+ epitopes presented by different class II alleles at the same time. In the present study, a multiplex TGEM approach was developed to identify antigenic epitopes presented by multiple HLA class II alleles simultaneously. In this new approach, CD4+ T cells were stained with multiple sets of MHC class II tetramers-each labeled with a unique fluorescent label. Using this multiplex approach, novel epitopes from influenza antigens hemagglutinin and matrix protein presented by multiple class II alleles were identified in a single experimental setting. PMID- 16677864 TI - Long-term correction of ammonia metabolism and prolonged survival in ornithine transcarbamylase-deficient mice following liver-directed treatment with adeno associated viral vectors. AB - The purpose of this study was to determine the efficacy of novel recombinant adeno-associated viral (AAV) vector constructs in correcting metabolic defects in the liver in two strains of ornithine transcarbamylase (OTC)-deficient mice (spf and spf-ash). AAV vectors expressing mouse OTC were produced with capsids from AAV2 and the novel serotypes AAV7, 8, and 9. OTC-deficient mice were infused with these vectors as well as a control AAV2/8 vector expressing LacZ. In vivo activity of OTC was assessed by measuring a surrogate marker, urine orotate. The novel vectors restored orotate levels to virtually normal 15 days after infusion, and each persisted to 1 year posttreatment. Liver OTC enzyme activity in spf mice was substantially higher in animals receiving novel vectors compared to those receiving AAV2 vectors. Animals receiving novel OTC-expressing vectors lived longer than those treated with AAV2 OTC or untreated controls, and they were tolerant to a challenge with NH3 at 21 days and beyond, which caused severe morbidity in control OTC-deficient animals. Numerous mice, representative of all treatment groups followed for +250 days, were observed to have either nodules or discrete tumors in the liver, the etiology of which is the subject of a companion paper. PMID- 16677865 TI - A genetic approach for investigating vagal sensory roles in regulation of gastrointestinal function and food intake. AB - Sensory innervation of the gastrointestinal (GI) tract by the vagus nerve plays important roles in regulation of GI function and feeding behavior. This innervation is composed of a large number of sensory pathways, each arising from a different population of sensory receptors. Progress in understanding the functions of these pathways has been impeded by their close association with vagal efferent, sympathetic, and enteric systems, which makes it difficult to selectively label or manipulate them. We suggest that a genetic approach may overcome these barriers. To illustrate the potential value of this strategy, as well as to gain insights into its application, investigations of CNS pathways and peripheral tissues involved in energy balance that benefited from the use of gene manipulations are reviewed. Next, our studies examining the feasibility of using mutations of developmental genes for manipulating individual vagal afferent pathways are reviewed. These experiments characterized mechanoreceptor morphology, density and distribution, and feeding patterns in four viable mutant mouse strains. In each strain a single population of vagal mechanoreceptors innervating the muscle wall of the GI tract was altered, and was associated with selective effects on feeding patterns, thus supporting the feasibility of this strategy. However, two limitations of this approach must be addressed for it to achieve its full potential. First, mutation effects in tissues outside the GI tract can contribute to changes in GI function or feeding. Additionally, knockouts of developmental genes are often lethal, preventing analysis of mature innervation and ingestive behavior. To address these issues, we propose to develop conditional gene knockouts restricted to specific GI tract tissues. Two genes of interest are brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which are essential for vagal afferent development. Creating conditional knockouts of these genes requires knowledge of their GI tract expression during development, which little is known about. Preliminary investigation revealed that during development BDNF and NT-3 are each expressed in several GI tract regions, and that their expression patterns overlap in some tissues, but are distinct in others. Importantly, GI tissues that express BDNF or NT-3 are innervated by vagal afferents, and expression of these neurotrophins occurs during the periods of axon invasion and receptor formation, consistent with roles for BDNF or NT-3 in these processes and in receptor survival. These results provide a basis for targeting BDNF or NT-3 knockouts to specific GI tract tissues, and potentially altering vagal afferent innervation only in that tissue (e.g., smooth muscle vs. mucosa). Conditional BDNF or NT-3 knockouts that are successful in selectively altering a vagal GI afferent pathway will be valuable for developing an understanding of that pathway's roles in GI function and food intake. PMID- 16677866 TI - Pressure gradients and transport in the murine femur upon hindlimb suspension. AB - Interstitial fluid flow (IFF) is important in a number of processes, including stimulation of cells and nutrient and waste transport. In bone, it arises from the vascular pressure gradient between the medullary cavity and the lymphatic drainage at the periosteal surface and is enhanced by mechanical loading events. However, little is known about the pressure gradients experienced by bone cells in vivo and the role of the induced IFF in bone adaptation. This study investigated IFF changes in bone, in a disuse model and in ambulatory mice, from pressure gradients measured by telemetry, and by fluorescent tracers. The role of IFF-mediated transport of oxygen was assessed by the levels of hypoxic osteocytes in mouse femur after disuse by hindlimb suspension and with or without femoral vein ligation. Femoral intramedullary pressures in alert mice decreased to 77% upon hindlimb suspension and increased by 25% upon ligation, relative to baseline. To determine relative perfusion of cortical bone by IFF, the localization of intracardiac-injected fluorescent albumin conjugate with osteocytes was monitored. The number of osteocytic lacunae per bone area positive for Texas Red albumin was increased by 31% within 20-40 s, in the ligated femur compared to the contralateral sham femur. This confirmed that interstitial fluid flow was increased by femoral vein ligation and indicated that the increase was proportional to the pressure increase. Unloaded bone osteocytes were not hypoxic when compared to loaded controls and venous ligation did not alter these levels significantly. These results support the hypothesis that disuse by hindlimb suspension leads to decreased pressure gradients, which indicate lower IFF. Similarly, the increased pressure gradients, seen upon venous ligation, increased IFF from marrow to periosteum. While a decrease in intramedullary pressure in disuse suggests a decrease in IFF, this did not lead to hypoxia in osteocytes. We conclude that decreased oxygen convective transport in the mouse hindlimb disuse model does not account for cortical bone loss. This study is important in increasing our understanding of the mechanotransductory pathways involved in bone loading and unloading. PMID- 16677867 TI - Mitomycin C in the management of pediatric caustic esophageal strictures: a case report. AB - Although the incidence of caustic ingestion is declining, the management of caustic esophageal strictures remains a challenge. Mitomycin C (MMC) inhibits fibroblast proliferation and is effective in reducing scar in animal experiments. We report the case of a child with a distal esophageal stricture from lye ingestion managed with MMC. Despite repeated dilatations, at 1 year post injury, the stricture was 20% of esophageal diameter. Mitomycin C (4 microg/mL) was applied topically and circumferentially by endoscopy and repeated 4 months later. At 20 months follow-up, the child eats normally, and esophagram showed decreased stenosis (stricture was 50% of esophageal diameter). No complications were observed. Although controlled trials are required to confirm its efficacy, MMC should be considered as an adjunct in the management of caustic esophageal strictures in children. PMID- 16677868 TI - Silo pouch stoma: a rescue procedure for intestinal catastrophe in gastroschisis. AB - Silo pouch formation is a standard procedure to prevent compartment syndrome in gastroschisis. Intestinal complications such as perforation and volvulus can occur and their management can be perplexing. We present three such patients in which we formed a stoma through the silo pouch owing to these complications. Creation of stoma through the silo is a novel, safe temporizing technique to decompress the bowel while delayed reduction continues. Subsequently, when the baby and the bowel improve, the stoma can be closed. PMID- 16677869 TI - Unique case of intramural colonic splenosis. AB - Splenosis is usually a sequel of splenic rupture from abdominal trauma but can be associated with elective splenectomy. Recurrence of the hematological disorder for which the patient underwent splenectomy may occur, and splenic nodules can be found anywhere in the thoracic or abdominal cavity, as well as subcutaneously. We are presenting intramural colonic splenosis, a large inoculum of the splenic tissue that has been found to have the capacity to maintain anemia and thrombocytopenia, in a child previously splenectomized because of a hematological problem. PMID- 16677870 TI - Innovative modification of the hepatic portoenterostomy. Our experience of treating biliary atresia. AB - We modified conventional hepatic portoenterostomy by pulling the remnant fibrous mass caudally and placing 5 or 6 numbered and individually clamped absorbable 5-0 double-needle sutures horizontally in the liver surface of the posterior side of the remnant fibrous mass before excision and apposing the posterior and anterior margins of the hepatic portoenterostomy to resemble a flattened isosceles triangle. Our technique was used to treat 14 cases of biliary atresia. Good postoperative biliary excretion (serum total bilirubin, <2 mg/dL) was achieved in 13 (92.9%) of 14 patients. PMID- 16677871 TI - Lymphoplasmacytic sclerosing pancreatitis presenting as a pancreatic head mass in a child: case report and management recommendations. AB - Lymphoplasmacytic sclerosing pancreatitis (LPSP) is an autoimmune form of chronic pancreatitis found most commonly in elderly men and only rarely in children. A 10 year-old boy presented with a 3-week history of obstructive jaundice. Imaging studies showed a pancreatic head mass, hepatic ductal dilatation, and involvement of the portal vein. A preliminary diagnosis of malignancy was based on endoscopic ultrasound characteristics and fine-needle aspiration cytology. The patient underwent a pancreaticoduodenectomy. The patient recovered uneventfully and was discharged home on postoperative day 6. The final pathological diagnosis was LPSP. Lymphoplasmacytic sclerosing pancreatitis is a rare form of chronic pancreatitis in children that is difficult to distinguish from malignancy preoperatively. We discuss the diagnosis and treatment of LPSP. Determination of elevated IgG4 levels in children with pancreatic head masses may allow for the medical treatment of LPSP. PMID- 16677872 TI - Endograft stenting in the adolescent population for traumatic aortic injuries. AB - Traumatic thoracic aortic transections are uncommon in the pediatric population. These injuries are currently treated by open operative repair via thoracotomies. We present 2 adolescent patients with traumatic thoracic aortic transections who were repaired by endovascular techniques. Both adolescents, aged 16 and 17 years, were in high-speed motor vehicle collisions and presented with multisystem trauma. Patient 1 had a transection of the descending aorta and was repaired with two 23-mm (diameter), 3-cm-long endograft cuffs at 48 hours after injury because of her multiple organ injuries. She was hospitalized for 40 days. Patient 2 had a thoracic aortic transection just distal to the aortic arch. He was repaired within 12 hours of injury with a 24-mm and two 22-mm AneuRx endograft cuffs. He was hospitalized for 8 days. Both patients have recovered without complications at 13 and 21 months, respectively. Endovascular stenting, especially in critically ill patients, allows for definitive treatment of the vascular injury without the need for bypass and reduces the recovery time that is associated with thoracotomies. Short-term recovery and follow-up are encouraging for endovascular stenting in the adolescent population; however, further long-term follow-up is required. PMID- 16677873 TI - Bladder prolapse through a patent urachus: fetal and neonatal features. AB - We report a term male neonate who was born with a large, red, tubular, mucosa lined umbilical mass containing a patent lumen. Prenatal ultrasonographic screening at 20 to 28 weeks of gestation revealed a large cyst at the umbilicus, communicating with the urinary bladder. The cyst resolved at 32 weeks, and a small solid mass was newly seen on the fetal abdominal wall, inferior to the umbilical cord insertion. At operation, the mass was discovered to be the prolapsed, open, everted dome of the urinary bladder. The dome was resected, and the bladder was repaired in 2 layers after identification of the ureteral orifices. Bladder prolapse through a patent urachus can be predicted by prenatal ultrasound and has a distinct neonatal appearance. PMID- 16677874 TI - Pulmonary arteriovenous malformation mimicking congenital cystic adenomatoid malformation in a newborn. AB - Congenital pulmonary arteriovenous malformations (AVMs) are rare lesions, usually asymptomatic. We report on the case of a baby who was thought to have a congenital cystic adenomatoid malformation of the left upper lobe based on prenatal and postnatal imaging. Final pathology revealed a congenital pulmonary AVM. Neither the child nor her family have any evidence of hereditary hemorrhagic telangiectasia. To our knowledge, this is the first reported case of a pulmonary AVM mimicking a congenital cystic adenomatoid malformation. PMID- 16677875 TI - A risk-adjusted study of outcome and resource utilization for congenital diaphragmatic hernia. AB - PURPOSE: Perinatal care of infants with congenital diaphragmatic hernia (CDH) is nonstandardized and costly. We examined a risk-adjusted cohort of patients with CDH and hypothesized that (1) among CDH survivors, the cost of the birth admission would be proportional to illness severity, and (2) this cost would be significantly higher compared with a matched non-CDH cohort. METHODS: A retrospective review of costs and outcomes for all patients with CDH admitted to British Columbia Children's Hospital between 1999 and 2003 was performed. Risk grouping of patients with CDH using a validated admission severity score (Score for Neonatal Acute Physiology-version II [SNAP-II]) was conducted, enabling comparison among infants surviving to discharge. Hospital costs were also compared with a contemporaneous, non-CDH cohort matched for birth weight and SNAP II. RESULTS: Thirty-two infants with CDH were included, of who 5 required extracorporeal membrane oxygenation. Twenty-three (72%) infants survived to discharge, with an average length of stay of 46 days. Average cost per survivor to discharge was 54,102 dollars (vs 13,722 dollars for the non-CDH cohort; P < .05). After SNAP-II stratification of survivors into low-, moderate-, and high risk groups, a significant cost difference was noted between the moderate- and low-risk and high- and low-risk groups, respectively. CONCLUSIONS: Infants born with CDH require costly care and can be expected to consume disproportionate resources. Admission SNAP-II score correlates with total cost to discharge. Risk stratification and cost comparison of larger CDH populations may allow identification of cost-efficient treatment strategies. PMID- 16677876 TI - The price of success in the management of congenital diaphragmatic hernia: is improved survival accompanied by an increase in long-term morbidity? AB - BACKGROUND: The impact of "gentle ventilation" (GV) strategies on morbidity and mortality of patients with congenital diaphragmatic hernia (CDH) in our institution has not been determined. This study reviews the primary and secondary outcomes of our patients with CDH treated with the GV approach. METHOD: We performed a retrospective chart review of respiratory, neurologic, nutritional, and musculoskeletal morbidities in patients with CDH treated at a single institution between 1985 and 1989 with conventional ventilation (CV) compared with those treated from 1996 to 2000 with GV. RESULTS: There were 77 CV-treated and 66 GV-treated patients with CDH, with 51% survival in the CV cohort compared with 80% in the GV group (chi(2), P < .05). At 3-year follow-up, we found no statistically significant differences in the frequency of respiratory (38% of CV patients, 50% of GV patients), neurologic (29% of CV patients, 34% of GV patients), or musculoskeletal morbidity (46% of CV vs 29% of GV-treated patients). There was a difference in nutritional morbidity as indicated by the increased frequency of gastrostomy tube use in the GV-treated patients (34%) compared with the CV patients (8%; chi2, P < .05). CONCLUSION: The implementation of GV techniques has significantly decreased mortality in infants with CDH. This has been associated with a documented increase in nutritional morbidity among survivors. PMID- 16677877 TI - Perinatal management of congenital cystic lung lesions in the age of minimally invasive surgery. AB - BACKGROUND: Most congenital cystic lung lesions (CCLLs) do not require in utero or perinatal intervention. The management of asymptomatic lesions is controversial: the theoretical risk of infection and malignancy is offset by whether thoracotomy in asymptomatic children is justified. We examined our recent experience and the role of minimally invasive surgery. METHODS: We analyzed the pre-, peri-, and postnatal findings of all consecutive CCLLs diagnosed between 1997 and 2005. We reviewed records for pre-, and postnatal imaging, management, and outcome. RESULTS: Thirty-five CCLL were diagnosed prenatally. Since 2000, all asymptomatic lesions were removed endoscopically at 6 to 18 months (thoracoscopy for 6 extralobar sequestrations, 3 intralobar sequestrations/congenital cystic adenomatoid malformations, 5 bronchogenic cysts, and retroperitoneal laparoscopy for 2 intraabdominal sequestrations). Congenital cystic adenomatoid malformation elements were present in more than 70%. Two abdominal lesions have regressed, and 2 patients are awaiting intervention. One symptomatic infant underwent thoracotomy for congenital lobar emphysema. CONCLUSIONS: It has been argued that the risks associated with congenital lung lesions (infection and malignancy) justify intervention in the asymptomatic patient. In our experience, all these lesions could be safely removed using endosurgical techniques. Counseling of (future) parents should be updated to include minimally invasive surgery in the management algorithm. PMID- 16677878 TI - The impact of prenatal bowel dilation on clinical outcomes in neonates with gastroschisis. AB - PURPOSE: In fetuses with gastroschisis, the importance of ultrasonographic bowel dilation remains controversial. The outcome of patients with gastroschisis with and without prenatal bowel dilation is reported. METHODS: From 2000 to 2004, 27 neonates with gastroschisis were followed at a single center. Thirteen patients had prenatal ultrasonographic bowel dilation (diameter, > or =6 mm; range, 6-35 mm). Outcomes of those with and without dilation were compared using 2 sample t tests and logistic regression. RESULTS: Time to initiation of enteral nutrition varied significantly between groups (20.4 +/- 11.7 days vs 12.5 +/- 4.3 days, P < .05). A trend toward a reduced rate of primary closure was seen in those with dilation (23% vs 50%, P = .06). No significant difference was found when considering mortality, gestational age, time in the intensive care unit (ICU), time on parenteral nutrition, or length of stay. Prenatal bowel dilation, a longer ICU stay, and later gestational age independently predicted readmission for bowel obstruction (P < .001). CONCLUSION: Infants with gastroschisis and prenatal bowel dilation were significantly slower to initiate enteral feeding and tended to have a reduced incidence of primary closure. This did not translate into increased mortality, time on parenteral nutrition, time in the ICU, or length of stay. However, dilation was associated with readmission for bowel obstruction. PMID- 16677879 TI - Splanchnic perfusion pressure: a better predictor of safe primary closure than intraabdominal pressure in neonatal gastroschisis. AB - BACKGROUND/PURPOSE: Both measured intraabdominal pressure (IAP) and calculated splanchnic perfusion pressure (SPP) have been advocated for use in operative management of gastroschisis. We directly compared these 2 clinical indices. METHODS: Institutional review board-approved multi-institutional retrospective review from 3 centers with 112 subjects. Splanchnic perfusion pressure was recorded as mean arterial pressure-IAP. We compared the clinical utility of IAP and SPP using univariate and multivariate regression analyses. RESULTS: Calculated mean SPP was higher among neonates requiring silo placement compared to those without (39.0 +/- 1.9 vs 33.7 mm Hg, P < .01). Measured IAP levels were similar between groups (11.5 +/- 1.1 vs 10.0 +/- 0.5, mm Hg, P < .4). On a receiver operating characteristic curve, the inflection point for more than 90% specificity for silo placement was at an SPP of 44. In multivariate regression analysis adjusting for all factors below, SPP was independently associated with silo placement (odds ratio 1.2, 95% confidence interval 1.1-1.3, P < .01), and IAP was not (odds ratio 1.2, 95% confidence interval <1.0-1.5, P < .1). CONCLUSIONS: These data suggest that SPP is a stronger predictor than IAP for the ability to achieve primary closure in the management of neonatal gastroschisis. We infer from these data that intraoperative SPP of more than 43 mm Hg may obviate the need for silo placement. PMID- 16677880 TI - Port insertion and removal techniques to minimize premature rupture of the membranes in endoscopic fetal surgery. AB - BACKGROUND: Premature rupture of membranes (PROM) remains a significant complication of fetal surgery. Rates of 40% to 100% have been reported after both open and endoscopic fetal surgery. We describe a technique of endoscopic port insertion and removal that minimizes trauma to the membranes. METHODS: Twenty seven consecutive patients undergoing endoscopic laser ablation for twin-to-twin transfusion syndrome were reviewed. In each case, a minilaparotomy was performed, and the amniotic cavity was entered under direct vision of the uterus using a Seldinger technique. The entry site was carefully dilated to accommodate a 4.0-mm diameter cannula. A gelatin sponge plug was placed at port removal. Postoperative management and outcome were evaluated. RESULTS: Median gestational age at operation was 21.3 weeks. Median operating time was 60 minutes. One patient delivered intraoperatively because of fetal distress. Seventeen (65.4%) patients required postoperative tocolysis (median duration, 12 hours). Median postoperative gestation was 6.5 weeks (range, 1-20 weeks). Only 1 (4.2%) of 24 patients with successful gelatin sponge placement developed PROM. CONCLUSIONS: Meticulous technique and atraumatic insertion and removal of ports help minimize the risk of postoperative amniotic leak after endoscopic fetal surgery. Our PROM rate of 4.2% contrasts sharply with previously reported rates after similar operations. PMID- 16677881 TI - Minimal access surgery in neonates and infants. AB - BACKGROUND: Minimal access surgery (MAS) in small infants carries an important consideration. The tolerance of these small babies and the assumed physiological effect of MAS, in addition to the required anesthetic and surgical skills, have made it difficult to perform these types of procedures in many international centers. The present article reviews our experience with MAS in neonates and infants in the first year of life. METHODS: The medical records of all neonates and infants (<1 year) who underwent MAS over a period of 3 years were retrospectively reviewed for demographic information, procedures, operative time, complications, outcomes, and follow-up. Most of the operations were performed with 3-mm instruments and scopes and mean insufflation pressure of 10 mm Hg (range, 4-15 mm Hg). RESULT: Seventy neonates and infants were included in this study: 19 females and 51 males. The weight ranged from 1.3 to 8.2 kg (mean, 4.3 kg). The mean age was 93 days (range, 1 day to 12 months). Twenty-four (34%) were neonates (first 30 days of life). Procedures performed included repair of tracheoesophageal fistula, lobectomy, repair of diaphragmatic or hiatus hernias, pull-through for imperforated anus and Hirschsprung's disease, plication of the diaphragm, Kasai procedure, excision of choledochal cyst, pyloromyotomy, Ladd's procedure, and reduction of intussusceptions. There were 2 conversions, both in neonates with tracheoesophageal fistula. All patients tolerated the procedure very well, with lesser degrees in neonates undergoing thoracoscopic procedures. Two neonates had postoperative hypothermia (<35 degrees C) and 1 neonate had high PCO2 postoperatively. There was 1 mortality and no morbidities. The follow-up ranged from 1 month to 3 years (mean, 19 months). CONCLUSION: Minimal access surgery in neonates and infants is safe and well tolerated. Intraoperative monitoring of end-tidal CO2 and core temperature is essential in avoiding unwanted effects of performing these procedures, especially in neonates. PMID- 16677882 TI - Esophageal foreign bodies in the pediatric population: our first 500 cases. AB - BACKGROUND: Children with esophageal foreign bodies are frequently seen by pediatric surgeons. Choking and dysphagia are common presentations; however, esophageal perforation has been reported. Historically, rigid esophagoscopy with extraction of the foreign body has been the recommended treatment. Alternatively, Foley balloon extraction is a safe and effective approach. METHODS: Over a 16 year period, 555 children presented with an esophageal foreign body. Retrospective analysis of the medical record was undertaken. Statistics were by univariate analysis. RESULTS: Two hundred ninety-eight boys and 257 girls presented with a mean age of 3.24 years. Dysphagia (37%) and drooling (31%) were the most common symptoms. Foreign bodies were lodged in the superior esophagus in 73%, and 88% of the objects were coins. Balloon extraction with fluoroscopy was performed in 468 children. Eighty percent of the objects were successfully removed with a mean fluoroscopy time of 2.2 min, and 8% were advanced into the stomach. The overall success rate was 88%, with failures necessitating rigid esophagoscopy under general anesthesia. Children younger than 1 year were the most likely to fail (25% failure rate). Airway aspiration never occurred. Significant savings in patient charges were observed with this approach. CONCLUSIONS: Balloon extraction of pediatric esophageal foreign bodies is a safe and cost-effective procedure. This technique is applicable for infants, children, and adolescents. Experienced practitioners should be able to achieve greater than 80% success rate. PMID- 16677883 TI - Total esophagogastric dissociation: 10 years' review. AB - PURPOSE: Neurologically impaired children run a 12% to 45% risk of recurrent gastroesophageal reflux (GER) after fundoplication. Elimination of the reflux by "rescue" total esophagogastric dissociation (TEGD) encouraged us to use it also as a "primary" form of antireflux surgery in this group of patients. METHODS: Twenty-six (14 male, 12 female) patients underwent TEGD between 1994 and 2004, of which 16 were primary and 10 were rescue procedures for failed fundoplication. RESULTS: There was no operative mortality and postoperative complications were limited to one subphrenic collection, one esophagojejunal dehiscence, and one small bowel hernia beneath the jejunal Roux loop. Gastrostomy feeding was usually established by 3 to 5 days and the mean hospital stay was 10.2 days (range, 6-18 days). At follow-up of 7 months to 11 years, there was no recurrence of GER. Four late deaths were unrelated to the surgery. The children's nutritional status improved with the mean weight standard deviation score showing a statistically significant increase from -2.63 preoperatively to -0.96 postoperatively (Wilcoxon's signed rank P value < or =.005). CONCLUSIONS: Total esophagogastric dissociation is a safe definitive solution for GER because it eliminates all risk of recurrent reflux. We therefore feel that TEGD can be used as a primary treatment of choice for severely neurologically impaired patients who are experiencing GER and are completely dependant on tube feeds. PMID- 16677884 TI - The Calgary protocol for bracing of pectus carinatum: a preliminary report. AB - BACKGROUND: The optimal treatment of pectus carinatum (PC) deformities is unclear. We propose a nonoperative approach using a lightweight, patient controlled dynamic chest-bracing device. MATERIAL AND METHODS: With ethical approval, 24 patients with PC were treated at the Alberta Children's Hospital between January 1998 and April 2005. There were 6 (25%) females and 18 (75%) males, with a mean age of 12.9 years at the onset of treatment. Treatment involved fitting of a lightweight, patient-controlled chest brace, worn for 23 hours per day (correction phase [CP]) until the convex deformity was corrected. Following correction of the deformity, bracing was reduced to 8 hours per day (maintenance phase) until axial skeletal maturation ceased. Monitoring was done by measurement of the external pectus carinatum protrusion as well as subjective patient and surgeon appraisal of appearance and exercise tolerance. RESULTS: Nineteen (79.2%) patients have completed initial treatment (mean CP time, 4.3 +/- 2.1 months). There were 3 patients (12.5%) who were noncompliant, and 2 (8.3%) are still in the initial CP phase of therapy. Fourteen (58.3%) patients are presently in maintenance phase, nocturnally braced, and 2 (8.3%) have completed therapy. In patients completing initial treatment, the protrusion pectus carinatum protrusion (pre 22 +/- 6 vs post 6.0 +/- 6.2) and subjective appearance (change + 1.8+/-0.4) showed a significant improvement (P < .001 for both) with no change in exercise tolerance. CONCLUSION: Compressive bracing results in a significant subjective and objective improvement in PC appearance in skeletally immature patients. However, patient compliance and diligent follow up appear to be paramount for the success of this method of treatment. Further studies are required to show the durability of this method of treatment. PMID- 16677885 TI - Characterization of the drug-positive adolescent trauma population: should we, do we, and does it make a difference if we test? AB - PURPOSE: Substance abuse (SA) is a significant cofactor for adolescent injury. Characterization of this population will help define a list of variables that are critical for designing interventions. The purpose of this study is to determine the frequency of SA testing and describe the injury characteristics of the adolescent trauma SA population. METHODS: A urine drug screening (alcohol, cannabis, and opiates) protocol for all injured adolescents (14-17 years old) was used. Four years of data were analyzed. Three groups were defined: not tested (NT), negative (NEG), and positive (POS). Transfers from an outside hospital and those who received opiate pain medication before drug screening were excluded. A patient may have had more than one positive test. Tests were analyzed to compare sex, injury severity score (ISS), survival, mechanism, and length of stay (LOS). RESULTS: Four hundred forty-three patients met the criteria (308 males and 135 females). Mean ISS was 13.5 +/- 0.5 SEM with a 2.9% mortality rate. One hundred ninety-three of 443 (44%) were screened (m = 120, f = 73) with 39% testing positive in each group. Twenty-nine percent of positive tests were opiates, 11.2% alcohol, and 20% cannabis. No sex differences for alcohol, cannabis, or opiate use were noted. There were no differences between the NT and the NEG/POS groups with respect to sex, LOS, or mechanism of injury. Fourteen-year olds were less likely to be drug screened. Mean ISS score was lower in the NT group (11.4 +/- 0.6 vs 15.7 +/- 1.0, P< .005). For the NEG and POS group analysis, a positive drug screen was not predicted by age, sex, ISS score, outcome, or LOS. Bicycle crashes were predictive of a positive drug screen (P < .005). Survival rates were not different between any of the groups. CONCLUSIONS: Forty percent of the patients screened positive, thus, supporting screening in adolescent trauma patients. Selective criteria based on injury characteristics are not justified. PMID- 16677886 TI - All-terrain vehicle injury patterns: are current regulations effective? AB - PURPOSE: All-terrain vehicle (ATV)-related injuries continued to increase in recent years. We aimed to analyze the ATV injury patterns at our institution to help structure public awareness campaign and encourage governmental regulation, with the ultimate goal of injury prevention. METHODS: A retrospective review of all ATV-related admissions at a pediatric trauma center was performed. RESULTS: From 2001 to 2004, 50 ATV-related injuries requiring hospital admission were identified. The annual incidence had increased 2.5-fold from 2001 (8 admissions) to 2004 (20 admissions). The ages ranged from 3 to 17 years (median, 13 years), with equal sex distribution. Fifty-four percent of admissions were traumatic brain injuries, 28% had orthopedic injuries, 14% with facial fractures, and 4% with abdominal injuries. Average length of stay was 6 days (range, 1-47 days); 5 of the 7 intensive care unit admissions occurred in 2004. Eighty-four percent of patients did not wear helmet (97% among those from northern communities). CONCLUSIONS: Both the incidence and severity of ATV-related injuries are increasing in a regional pediatric trauma center. There is a lack of regulation enforcement and public awareness of the danger of ATV use in children. Efforts to ensure helmet use and limit operator age are urgently needed to reduce childhood ATV-related injuries. PMID- 16677887 TI - Necrotizing soft tissue infections--are they different in healthy vs immunocompromised children? AB - PURPOSE: Necrotizing soft tissue infection (NSTI) is rare and often devastating. We sought to define our experience and determine if differences in immune status influenced outcome. METHODS: A retrospective review (1993-2004), with institutional review board approval, was undertaken on children with NSTI. Presentation, laboratory results, and outcome were assessed. RESULTS: There were 19 cases; the median age was 5.9 years (range, 6 days-14 years). Eight were immunocompromised (IC). At presentation, 95% had pain and swelling; fever and tachycardia occurred in 84% and 74%, respectively. Severe tenderness was found in 100% of healthy vs 25% of IC patients. Compared with the healthy, in IC patients, more infections were perineal/buttock (75% vs 32%), polymicrobial (75% vs 58%), and fungal (38% vs 0%). Median intensive care unit stay and length of hospital stay in IC vs healthy were 4 vs 2 and 27 vs 16.5 days, respectively. Mortality rate was 16% (2 healthy and 1 IC). CONCLUSIONS: Most children with NSTI present with fever, tachycardia, pain, and swelling. Compared with healthy children, IC patients are less likely to have severe tenderness and more likely to have polymicrobial perineal/buttock infections. Although IC patients had a longer length of intensive care unit and hospital stay, their mortality (12%) was actually better than that seen in the otherwise healthy children (18%). Coagulopathy developed in 64% of the patients and may be an early marker for the presence of necrotizing soft tissue infections. PMID- 16677888 TI - Vacuum-assisted closure for wound management in the pediatric population. AB - BACKGROUND/PURPOSE: Wound management in children has traditionally consisted of daily dressings. Although vacuum-assisted closure (VAC) is well described in the adult literature, there are few reports about children. We reviewed our experience with VAC. METHODS: A retrospective review from 2003 to 2005 revealed that 16 children underwent VAC. Variables analyzed included demographics, diagnosis, duration and characteristics of VAC, wound closure, recurrence, complications, and cost analysis. RESULTS: Sixteen children received VAC therapy at an average age of 12.1 years (range, 1 month-18 years). Indications included tissue loss after pilonidal sinus excision (n = 8, primary = 5, recurrent = 3) after wound dehiscence of the abdomen (3), the sternum (2), the back (1), the leg (1), and after chronic postoperative perineal fistula. Average length of VAC use was 23 days, with an average pressure of 104 mm Hg. Wound closure occurred in 15 of 16 patients. Patients with primary pilonidal disease obtained wound closure by 45 days, whereas those with recurrent disease required 72 days. Children with wound dehiscence healed by 28 days. Recurrent sinuses developed in all 3 patients with known recurrent pilonidal disease. Pain in 1 patient required cessation of VAC therapy after 7 days. Follow-up after wound closure averaged 8 months. CONCLUSIONS: Vacuum-assisted closure is well tolerated in our pediatric population and offers many advantages including fewer dressing changes and an earlier return to daily activities. PMID- 16677889 TI - Traumatic pediatric bile duct injury: nonoperative intervention as an alternative to surgical intervention. AB - BACKGROUND: Nonoperative management of blunt pediatric liver injuries has become the standard of care in the absence of hemodynamic instability. However, associated bile duct injuries remain as difficult challenges. Few case reports have demonstrated the benefits of conservative approaches, but others have found better outcomes with surgical intervention. In this study, we report on our experience with interventional endoscopic and radiologic management of 5 pediatric patients with bile duct injuries who underwent unsuccessful surgical interventions. METHODS: We conducted a retrospective review of medical records of all pediatric patients who were admitted with major blunt liver trauma and bile duct injuries over a period of 5 years. RESULTS: There were 5 patients (4 boys and 1 girl) whose ages range from 3 to 11 years in this study. All patients had major liver laceration and bilomas. Two had intrahepatic and 3 had extra hepatic bile duct injuries (2 right hepatic ducts and 1 junction of cystic duct with common bile duct). All of them underwent previous laparotomies, once in 2 patients, twice in 2 patients, and thrice in 1 patient. All 5 patients were eventually treated successfully with interventional endoscopic and radiologic techniques. Three underwent endoscopic retrograde cholangiopancreatography stenting with percutaneous drainage. Two patients were managed with percutaneous drainage alone. The follow-up is up to 2.5 years with normal liver function test and bile duct ultrasound. CONCLUSION: With the current advancement in endoscopic retrograde cholangiopancreatography and intervention radiology techniques, we believe that interventional endoscopic and radiologic management of bile duct injuries caused by blunt trauma in children is successful and efficacious even after multiple laparotomies. PMID- 16677890 TI - Longitudinal pancreaticojejunostomy and selective biliary diversion for chronic pancreatitis in children. AB - BACKGROUND: Chronic pancreatitis requiring surgery is rare in children. We review our experience in treating pediatric chronic pancreatitis with longitudinal pancreaticojejunostomy (LPJ). METHODS: Records of children with chronic pancreatitis treated with LPJ between 1997 and 2003 were reviewed. Demographic data, associated conditions, endoscopic interventions, operative procedures, postoperative complications, length and costs of hospitalization, and long-term outcome were recorded. RESULTS: Four patients (one girl), 3 to 16 years old, underwent LPJ. Associated conditions included bile duct obstruction (2), single (1) or multiple (1) pancreatic duct strictures, recurrent familial pancreatitis (1), pseudocyst (1), Down's syndrome (1), and duodenal web (1). Preoperative endoscopic stenting was performed in two patients. All were on restricted diets, one on parenteral nutrition. Pre-LPJ, each child had 3 to 6 admissions for pancreatitis with mean total cost of 39,000 dollars, excluding diet charges. At surgery, two patients required biliary diversion for persistent biliary obstruction in addition to LPJ. Postoperatively, no patient developed fistulas or anastomotic leaks. There were no deaths. The median length of hospitalization post-LPJ was 8 days with mean cost of US37,000 dollars. All patients resumed a normal diet post-LPJ. There were no recurrences of pancreatitis with follow-ups between 2 and 6 years. CONCLUSION: Longitudinal pancreaticojejunostomy is safe and cost-effective for treating pediatric chronic pancreatitis. It has minimal complications and frees patients from pancreatitis-related hospitalizations. PMID- 16677891 TI - Increased CXCR3 expression associated with CD3-positive lymphocytes in the liver and biliary remnant in biliary atresia. AB - BACKGROUND: Lymphocyte-mediated inflammatory damage of the bile ducts has been proposed as a potential mechanism in the pathogenesis of biliary atresia (BA). Chemokines regulate leukocyte migration and act as critical organizers of cell distribution in inflammatory responses. The aim of this study was to analyze the infiltration of T lymphocytes and the expression of a chemokine receptor, CXCR3, predominantly expressed on type 1 polarized T cells (T(H)1, T(C)1) in the liver and excised biliary remnants in infants with BA. METHODS: Immunohistochemistry for CD3, CD8, and CXCR3 was performed using liver biopsy specimens collected from the following 3 age-matched groups of patients: group 1, BA (nonsyndromic) at the time of Kasai portoenterostomy (n = 10); group 2, congenital choledochal dilatation (n = 2); and group 3, other cholestatic diseases including paucity of intrahepatic bile ducts and cholestasis (n = 3) related to total parenteral nutrition. Cellular staining on each section was graded from 0 to 4 and compared using nonparametric statistics. RESULTS: Infiltrating CD3+ and CD8+ lymphocytes in the portal tracts were significantly increased in group 1 (3.1 +/- 0.4, 2.8 +/ 0.4), compared with groups 2 (1.0 +/- 0.0, 1.0 +/- 0.0) and 3 (1.7 +/- 0.3, 1.5 +/- 0.5) (P < .01, P < .05). CXCR3+ mononuclear cells were significantly increased in group 1 (2.6 +/- 0.3) compared with groups 2 (0.5 +/- 0.5) and 3 (0.7 +/- 0.3) (P < .05). They were mainly found in the portal tracts with a similar distribution to CD3+ cells. CXCR3+ cells and CD3+ cells also showed a similar distribution in specimens of biliary remnants from just below the portal plate. CONCLUSIONS: Increased expression of CXCR3 associated with a significantly increased CD3 and CD8 T-cell infiltration suggests that CXCR3+ lymphocytes in a type 1 (T(H)1, T(C)1) cytokine milieu may play a role in the pathogenesis of BA. PMID- 16677892 TI - Isolated liver transplantation in pediatric short bowel syndrome: is there a role? AB - BACKGROUND: The role of isolated liver transplantation in infants with parenteral nutrition-associated cholestasis (PNAC) associated with short bowel syndrome (SBS) is unclear. METHODS: We performed a retrospective descriptive study of infants with PNAC and SBS who received an isolated liver transplant at our institution. Review of the literature was also performed. RESULTS: Three infants (aged 7, 8, and 13 months) with SBS and PNAC received an isolated liver transplant. Etiology of SBS was necrotizing enterocolitis, gastroschisis, and volvulus. Two patients with pretransplant small bowel length of 40 and 80 cm, who were receiving 65% and 79% of intake enterally, demonstrated good graft function (bilirubin, 0 and 7 micromol/L) at 41 and 58 months posttransplant. Despite full tolerance of enteral feeds, both remain on parenteral nutrition (PN) (4 and 7 nights per week) for poor weight gain. One child with 25 cm of small bowel, who received 65% of energy enterally pretransplant, died 7 months posttransplant from PNAC. A literature review revealed 22 cases of isolated liver transplant for PNAC associated with SBS. Overall survival was 77%, with 76% of survivors demonstrating independence from PN. CONCLUSIONS: Isolated liver transplantation is an acceptable option for select infants with PNAC associated with SBS when further intestinal adaptation and freedom from PN are anticipated. PMID- 16677893 TI - Surgical challenges associated with intensive treatment protocols for high-risk neuroblastoma. AB - BACKGROUND: High-risk neuroblastoma (NB; age, >1 year; INSS stage 4) is associated with a poor outcome. At our institution, the current dose-intensive high-risk Children's Oncology Group protocol for advanced NB appears to have a higher surgical complication rate as compared with previous protocols. METHODS: All stage 4 patients (n = 51) entered in high-risk protocols between 1995 and 2005 were analyzed. Patients in the current high-risk protocol, Children's Oncology Group A3973 (n = 22), were compared with those in the 2 previous protocols, CCG 3891 and POG 9341 (n = 29). RESULTS: Patients were comparable in their mean age and tumor markers, including Shimada histology, MYCN amplification, 1p deletion, tumor origin, and extent of metastasis. However, transfusion requirement (86% vs 45%; P = .0019), postoperative infection rate (32% vs 3%; P = .02), and other postoperative issues including nutritional support (45% vs 3%; P = .0001) were significantly higher with the current protocol. No perioperative mortality was noted in either group, and the extent of resectability and margins were similar. Importantly, with the current protocol, the survival rate was higher (P = .0022) and the recurrence rate was significantly lower (P = .0003). CONCLUSIONS: Despite higher surgical morbidity associated with the current high-risk protocol (2.59 vs 0.86 complications/person; P < .01), the recurrence rate is lower and interim survival rate is improved for patients with high-risk NB. Therefore, the higher surgical complication rates associated with the current high-risk protocol are acceptable. PMID- 16677894 TI - Predictors of surgical outcome in Wilms' tumor: a single-institution comparative experience. AB - BACKGROUND: The merits of primary nephrectomy (PN) vs preoperative chemotherapy (PC) for patients with Wilms' tumor (WT) are much debated. Early data from the International Society of Pediatric Oncology suggested decreased intraoperative spillage but increased risk of local recurrence after PC. Patients with WT at our institution were managed with PC until 1996; subsequently, they underwent PN. This study compares these approaches as they affect tumor spillage, local recurrence, and survival. METHODS: Patients with WT diagnosed at the Hospital for Sick Children from 1985 to 2003 were reviewed. RESULTS: One hundred sixty patients were identified (114 PC and 46 PN). Tumor spill occurred in 6 (5.3%) of 114 PC and 2 (4.3%) of 46 PN patients. Tumor inhomogeneity, tumor size, and inferior vena cava compression/clot at diagnosis did not affect incidence of spill. Of 6 PC patients with surgical spill, 1 (17%) had significant tumor shrinkage, compared with 87 (81%) of 108 without spill (P < .001). Preoperative chemotherapy and PN had equal rates of surgical complications. Preoperative chemotherapy resulted in 12 (10.5%) of 114 local recurrences vs 5 (10.8%) of 46 with PN. Event-free survival and overall survival were 80% and 92% for PC at 129 months vs 85% and 96% for PN at 61 months. CONCLUSIONS: Preoperative chemotherapy and PN are equally effective in the treatment of WT with no difference in tumor spillage. Failure of the tumor to shrink in size with PC was significantly associated with an increase in tumor spillage and would suggest that a more cautious surgical approach be undertaken in these cases. PMID- 16677895 TI - Assessment of residual posttreatment masses in Hodgkin's disease and the need for biopsy in children. AB - BACKGROUND: In children with Hodgkin's disease (HD), a significant residual mass may remain after definitive treatment. It may be composed of necrotic/fibrous tissue or resistant HD. Various imaging modalities are available to assess this mass, including chest x-ray (CXR), computed tomographic (CT) scan, and gallium scan. Our aim was to determine the sensitivity and specificity of each modality for residual HD. METHODS: Two hundred fifty-six children with HD during 1985 to 2003 were retrospectively reviewed. RESULTS: Twenty-six patients with HD had residual masses at end of therapy, deemed to be of concern for residual disease. These children had 16 abnormal CXRs, 18 abnormal CT scans, and 9 abnormal gallium scans; all patients underwent biopsy. Ten (38%) showed resistant HD and 16 (62%) had fibrotic and necrotic tissue. The sensitivity and specificity of each imaging modality were 60% and 38% for CXR, 67% and 8% for CT scan, and 71% and 71% for gallium scan, respectively. CONCLUSIONS: These imaging modalities were not sufficiently sensitive or specific to predict which residual masses may be harboring resistant HD. Hence, the need for surgical biopsy will remain important in the assessment of these masses. FDG-PET scan, which was not available to these children, is a modality that may improve this assessment. PMID- 16677896 TI - Alcoholic solution of zein (Ethibloc) sclerotherapy for treatment of lymphangiomas in children. AB - PURPOSE: The aim of this study was to report the experience and efficacy of Ethibloc sclerotherapy as treatment of lymphangiomas. METHODS: Between 1992 and 2004, 63 patients had Ethibloc sclerotherapy for lymphangiomas at our institution. Computed tomographic scan or magnetic resonance imaging and clinical evaluation determined efficacy of the treatment. Results were classified as excellent (> or =95% decrease in lesion volume), satisfactory (> or =50% decrease and asymptomatic), or poor (<50% decrease or symptomatic). RESULTS: Sixty-three patients with 67 lesions underwent sclerotherapy with a median of 2 treatments per patient. Thirty-five involved the neck; 10, the head and face; and 22, the thorax or limb. Thirteen were predominantly microcystic; 28, macrocystic; and 26, mixed. Of the 63 patients, 6 underwent sclerotherapy for postsurgical residual lesions. Results were classified by type: of the 54 macrocystic/mixed cases, 26 (49%) had excellent, 19 (35%) had good, and 9 (16%) had poor results; in the 13 predominantly microcystic lesions, 3 (23%) had excellent, 7 (54%) had good, and 3 (23%) poor results. Five patients (7.7%) required surgery for complications; 2, for scar revision; 2, for persistent drainage; and 1, for a salivary fistula. Infection occurred in 4 patients (6.2%) after sclerotherapy. Follow-up averaged 3.5 years (range, 12 months to 12 years). CONCLUSION: Ethibloc sclerotherapy is a safe and effective alternative to surgical excision of macrocystic lymphangiomas and can be used for postsurgical recurrences. PMID- 16677897 TI - Six thousand three hundred sixty-one pediatric inguinal hernias: a 35-year review. AB - PURPOSE: This study, by its mere size and uniformity (1 pediatric surgeon), aims to corroborate or refute the teachings and myths of the pediatric inguinal hernia. METHODS: From July 1969 to January 2004, 6361 infants and children with inguinal hernias were seen, operated on, and followed by the senior author. A retrospective survey of their charts was carried out to evaluate the demographics and clinical aspects of these patients. The hospital's research ethics board approved of this study. RESULTS: The ages ranged from premature to 18 years (mean age, 3.3 years) with a male-to female ratio of 5:1. There were 59% right, 29% left, and 12% bilateral hernias (almost all indirect). Hydroceles were found in 19%. Incarceration occurred in 12%. A modified Ferguson repair was used. An opposite-side hernia developed in 5%, 95% within the first 5 years, and was not sex or age specific. There were 1.2% recurrences, 96% within 5 years. Thirteen percent had ventriculo-peritoneal shunts, 1.2% wound infections, and 0.3% testicular atrophy. There were no postoperative deaths. One percent had a documented hernia disappearance. CONCLUSIONS: Three of our results have not corresponded with previous teachings and myths: (1) a hernia of a premature baby should be fixed sooner than later; (2) routine contralateral groin exploration is not indicated in any situation; and (3) teenage recurrence rate is 4 times greater than the overall series. PMID- 16677899 TI - Vascular anomalies of the female external genitalia. AB - BACKGROUND/PURPOSE: Vascular tumors and malformations are rare. This large series describes vascular anomalies of the female genitalia and their management. METHODS: An international review board-approved retrospective database and a record review from 1994 through 2004 was conducted. RESULTS: Of the 3186 female patients with a vascular anomaly, 82 (2.6%) had a lesion in the external genitalia. There were 60 malformations and 22 tumors. The most common malformations were combined capillary-lymphaticovenous, venous, and lymphatic. Tumors included 20 infantile hemangiomas, 1 kaposiform hemangioendothelioma, and 1 kaposiform lymphangioendothelioma. The referring diagnosis was incorrect in 56% of patients. Cutaneous stains, swelling, deformity, bleeding, fluid leakage, or infection were the prominent symptoms. Magnetic resonance imaging, ultrasonography, angiography, and computed tomography were used for diagnostic clarification. Malformation treatment consisted of sclerotherapy, embolization, and operative resection. Tumor management included observation, surgical excision, and antiangiogenic pharmacotherapy. CONCLUSIONS: Vascular anomalies of the female external genitalia are uncommon, and the initial diagnosis is often inaccurate. Correct diagnosis using clinical and radiographic data is feasible and leads to meaningful intervention for these frequently devastating lesions. Whereas tumors may respond to excision or antiangiogenic drugs, malformations require ablation or resection. Evaluation and management of these lesions is complex and benefits from interdisciplinary care. PMID- 16677898 TI - Management and outcome of patients with combined vaginal septum, bifid uterus, and ipsilateral renal agenesis (Herlyn-Werner-Wunderlich syndrome). AB - OBJECTIVE: Herlyn-Werner-Wunderlich syndrome (HWWS) is a rare mullerian anomaly consisting of uterine didelphy, hemivaginal septum, and ipsilateral renal agenesis. The purpose of this study was to evaluate the natural history and outcome of patients with HWWS. METHODS: With ethics review board approval, all patients with uterine/vaginal anomalies were reviewed between 1982 and 2004. Patients with cloacal and/or anorectal anomalies were excluded. Presenting symptoms, preoperative investigations, operative management, and long-term follow up were assessed. RESULTS: Of 80 patients identified with uterine/vaginal anomalies, 12 had HWWS. Median age at presentation was 13 years. Most patients (11/12) in this series presented with either abdominal pain and/or pelvic masses. Two patients had intra-abdominal abscesses. Seven patients were menstruating at presentation with 4 of these patients having dysmenorrhea. Symptom duration ranged from 0.5 to 12 months. Diagnosis was confirmed by ultrasound (n = 11), computed tomographic scan (n = 3), and/or magnetic resonance imaging (n = 2). Operative management included vaginal septectomy and drainage of the hematocolpos/hematometrocolpos. One patient required salpingectomy for pyosalpinx. Follow-up ultrasounds revealed no recurrent collections. Median follow-up was 3 years (2 months to 16 years). Eleven patients were asymptomatic after treatment. One patient complained of irregular menses. CONCLUSION: This is one of the largest reviews of HWWS in pediatric patients to date. Good long-term outcome occurs after vaginal septectomy. This diagnosis should be suspected in females with a pelvic mass and ipsilateral renal agenesis. PMID- 16677900 TI - Conjoined twins--past, present, and future. AB - BACKGROUND/PURPOSE: Conjoined twins are rare and complex anomalies of the newborn. They require a highly experienced team and a center equipped to deal with such challenging anatomy. METHOD: A review of 31 sets of conjoined twins was managed by one team over a 15-year period. The spectrum of the anomalies, management strategies, and outcomes will be analyzed with future recommendations. RESULTS: Ten sets of complete conjoined twins and two sets of parasitic conjoined twins were successfully separated. Tissue expanders and prosthetic meshes were not required. A separation of one set of craniopagus parasiticus conjoined twins was attempted, but abandoned owing to major vascular and brain sharing. The remaining 18 sets were not separable owing to major cardiac anomalies and/or sharing, and all died within several weeks of birth. CONCLUSION: Careful planning, a multidisciplinary approach, rehearsal, and experience are important factors in dealing with conjoined twins. Tissue expanders and prosthetic meshes are not required in most cases. Selective abortion or fetal intervention may play a role in the future. PMID- 16677901 TI - Is ultrasonography a good screening test for intestinal malrotation? AB - BACKGROUND: Early diagnosis of malrotation can prevent fatal midgut volvulus. Abnormal orientation of the superior mesenteric artery (SMA) and vein (SMV) on ultrasonography (US) has been described in malrotation. We aimed to determine the accuracy of this technique. METHODS: All children undergoing both upper gastrointestinal series (UGI) and US for possible malrotation over a 3-year period were reviewed. Patients were excluded if US did not include SMV/SMA orientation or if the duodenojejunal flexure was not visualized on UGI. RESULTS: Of 211 eligible patients, UGI and US were both normal in 62% and both abnormal in 15%. Forty-four had abnormal US and normal UGI (false positive, 21%), and 5 patients had normal US and abnormal UGI (false negative, 2%). Of these 5, none were found to have a short mesenteric base, which put them at risk for volvulus. Among abnormal ultrasounds, inversion of SMV/SMA and a "whirlpool" sign were more predictive for malrotation and volvulus than anterior/posterior orientation. CONCLUSIONS: Ultrasonography is a good screening tool that effectively rules out malrotation at risk for volvulus. Children with an abnormal ultrasound should have an UGI or go to the operating room, depending on clinical findings. PMID- 16677902 TI - Utility of hospital admission after successful enema reduction of ileocolic intussusception. AB - BACKGROUND: In-hospital observation of 24 to 48 hours has been the standard practice after successful enema reduction (ER) of ileocolic intussusceptions, but this practice has not been validated. We evaluated retrospectively the safety of short-term emergency department observation. METHODS: Between April 2000 and October 2004, 121 patients presented to the emergency department with ileocolic intussusception, and all had ER attempts. RESULTS: Ninety-six patients had successful reduction, 25 were excluded for failed reduction or unconfirmed diagnosis, and another 16 needed observation anyway for high white blood count or persistent postreduction pain. Of the remaining 80 patients, the mean time from symptoms to reduction was 45.9 hours (4 hours to 10 days). All patients, except one, were admitted for observation for a mean period of 1.6 days (8 hours to 6.5 days). No complications were associated with air ER; however, 6 (7.5%) patients had reintussusception during the observation period and 5 (6.3%) recurred after discharge. The mean intervals for recurrence postreduction were 17.8 hours and 14.5 months with no mortality or morbidity in either. CONCLUSIONS: Short-term emergency department observation could be a safe practice in more than 90% of the selected cases, recurrence of intussusception outside the hospital is not associated with unfavorable outcome, and routine admission is not warranted. PMID- 16677903 TI - Factors determining the need for operative reduction in children with intussusception: a population-based study. AB - AIM: The objective of this study is to determine if children presenting to a nonteaching hospital were at greater risk for operative reduction of intussusception than those presenting to a teaching hospital. METHODS: This population-based 8-year study included all children younger than 6 years with intussusception in the province of Ontario, Canada. Multiple logistic regression was used to model markers for operative reduction including age, sex, coexisting conditions, hospital type, and interhospital transfers. RESULTS: Of the 961 children with intussusception, 25.4% had operative reduction. Risk factors for operative reduction were a diagnosis of Meckel's diverticulum and transfer from one institution to another after the initial diagnosis. One hundred forty-eight (15.4%) were transferred. Of those transferred and initially admitted to a nonteaching hospital, 87.4% (125/143) were transferred to a teaching hospital. Risk of operative reduction was higher in children transferred more than 1 day after admission (52.0% vs 39.0%). Odds ratios after adjusting for age, sex, and hospital type were 1.95 (95% confidence interval, 1.28-2.98; P < . 001) for those transferred on the same day and 3.31 (95% confidence interval, 1.34-7.28; P < .01) for those transferred after 24 hours. CONCLUSION: Children who were presented to a nonteaching hospital and later transferred to a teaching hospital were at greater risk for operative reduction of intussusception, in particular, those who were transferred more than 1 day after admission. These data underline the importance of early diagnosis and timely management of intussusception. PMID- 16677904 TI - A simple and more cost-effective antibiotic regimen for perforated appendicitis. AB - OBJECTIVE: Appendicitis is the most common abdominal emergency in children. When perforation is encountered, postoperative management is grounded upon the use of intravenous antibiotics. The 3-drug regimen of ampicillin, gentamicin, and clindamycin has long been the accepted standard by pediatric surgeons. Although effective and seemingly inexpensive, this regimen produces a cumbersome dosing schedule, which has inspired the search for a simpler regimen that does not compromise efficacy or expense. To this end, we have introduced a 2-drug regimen of ceftriaxone and Flagyl (Pharmacia Corporation, Chicago, Ill) with once-a-day dosing. METHODS: A retrospective review was conducted of the most recent 250 patients treated at our institution with perforated appendicitis. Patients treated since the implementation of this 2-drug regimen were compared with the recent historical cohort treated with triple antibiotic coverage. Parameters analyzed between the 2 groups included temperature curves for the first 5 postoperative days, abscess rate, length of hospitalization, length of intravenous antibiotic treatment, and medication charges. RESULTS: The 2-drug regimen was used in 57 patients (group 1) compared with 193 patients treated with triple antibiotic coverage (group 2). Maximum recorded temperature between the 2 groups was similar upon admission, but the mean maximum temperature in group 1 became significantly lower than group 2 from postoperative day 1 onward (P < .001). Postoperatively, an abscess developed in 8.8% of group 1 compared with 14.2% of group 2, which was not significantly different (P = .37). Mean length of stay was 6.8 days in group 1 and 7.8 days in group 2 (P = .03). Medication charges to the patient were 81.32 dollars per day in group 1 compared with 318.53 dollars per day in group 2, translating to 1186.05 dollars savings for 5 days. CONCLUSIONS: Once-a-day dosing with ceftriaxone and Flagyl provides adequate antibiotic coverage for the postoperative management of perforated appendicitis in children. This regimen allows patients to more rapidly defervesce compared with traditional triple antibiotic coverage; moreover, this simple regimen provides substantial advantages for administration and expense. PMID- 16677905 TI - Carbon dioxide pneumoperitoneum prevents intraperitoneal adhesions after laparotomy in rats. AB - PURPOSE: The aim of this study was to assess whether carbon dioxide insufflation (CDI) pneumoperitoneum prevents intraperitoneal adhesions (IPAs). METHODS: Laparotomy was performed in 40 8-week-old Lewis rats and their bowels delivered through the wound and manipulated. The rats were divided into 4 groups, namely, those that would have laparotomy (Lp group, n = 15), Lp with CDI (Lp-CDI group, n = 15), Lp and bowel anastomosis (LpBA group, n = 5), and LpBA with CDI (LpBA-CDI group, n = 5). LpBA and LpBA-CDI group rats had 1 cm of ileum excised and end-to end anastomosis performed. To accelerate IPA formation, all rats then had their bowels heated to 45 degrees C for 40 seconds and 0.5 mL of Lewis rat blood spilled over them. Rats in the control group (n = 5) had Lp alone without heating or blood spillage. Pneumoperitoneum involved insufflating carbon dioxide into the peritoneal cavity through a cannula at the time of final abdominal closure to create pneumoperitoneum to a pressure of 5 mm Hg. All rats had relaparotomy 10 days after surgery and IPAs were assessed blindly using an IPA severity score (IPASS: 0 = no adhesions, 1 = no serosal tears during adhesiolysis, 2 = serosal tears during adhesiolysis, 3 = bowel perforation during adhesiolysis). Only the worst IPA in each rat was scored. Rats were killed after the peritoneum and bowels were excised for histopathology. RESULTS: Pneumoperitoneum resolved in approximately 5 days. There were no associated side effects. The Lp-CDI group had significantly lower IPASS than the Lp group (0.23 +/- 0.46 vs 1.07 +/- 1.18, P < .05), and the LpBA-CDI group had significantly lower IPASS than the LpBA group (1.50 +/- 0.61 vs 2.40 +/- 0.55, P < .05). Histopathology showed pneumoperitoneum had no effect on the peritoneum or bowels. CONCLUSIONS: Our results suggest that CDI pneumoperitoneum appears to prevent IPA, especially between bowel-bowel IPA. PMID- 16677906 TI - Developmental changes in submucosal nitrergic neurons in the porcine distal colon. AB - BACKGROUND/PURPOSE: As our understanding of the enteric nervous system improves, it becomes clear that it is no longer sufficient to simply determine whether enteric ganglion cells are present but also to determine whether correct number and types of ganglion cells are present. Nitric oxide is recognized as a potent mediator of inhibitory nerves responsible for the relaxation of the smooth muscle of the gastrointestinal tract. The aim of this study was to determine the normal nitrergic neuronal density and morphology in the submucosal plexus of the porcine distal bowel from fetal life to adulthood. METHODS: Distal large bowel specimens were obtained from porcine fetuses of gestational age E60 (n = 5), E90 (n = 5), 1 day-old piglets (n = 5), 4-week-old piglets (n = 5), 12-week-old piglets (n = 5), and adult pigs (n = 5). Whole-mount preparations of the submucosal plexus were made and stained with NADPH diaphorase histochemistry. The ganglia density, the number of ganglion cells per ganglia, and nucleus and cytoplasmic area were measured. RESULTS: Ganglia density decreased progressively and markedly with age until the adulthood (P < .001). On the contrary, ganglion cells increased their size over time predominantly because of increase in cytoplasm (P < .001). The number of ganglion cells per ganglia increased significantly during the fetal life. However, there was a significant reduction in the number of ganglion cells per ganglia during the period from birth to 4 weeks, remaining constant thereafter (P < .001). CONCLUSIONS: The quantitative and qualitative morphometric analysis of the colonic submucous plexus shows that significant developmental changes occur during fetal and postnatal life. These findings indicate that the age of the patient is of utmost importance during histopathologic evaluation of enteric nervous system disorders. PMID- 16677908 TI - Magnets in the stomach. PMID- 16677909 TI - Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. AB - A 4-day embryo-larval zebrafish test, from blastula stage to hatching included, was developed. The observations of embryo developmental were made at different development stages, for which morphological, physiological, and behavioral endpoints were selected and quantified for unexposed and exposed embryos. The sensitivity and the ability of these endpoints to inform about mode of action (MoA) were established in testing three model toxicants with well-known toxic effects (propranolol, malathion, cadmium). Lethal, sublethal (heart rate/edema, spontaneous movements, and hatching rate/time disturbance), and teratogenic effects were detected for all the studied compounds. This bioassay allows characterization of impairments at different biological levels: neuromuscular, physiological, morphological, and behavioral, and brings useful information about the toxic MoA of the chemicals on nontarget organisms. In this sense to answers the chemical industries and international organization (EMEA) requirements for the environmental risk assessment of new chemicals and pharmaceuticals. PMID- 16677910 TI - Differential responses of eight cyanobacterial and green algal species, to carbamate insecticides. AB - In this study, five carbamate insecticides were subjected to 96 h acute toxicity tests to examine their effects on three cyanobacteria, Anabaena flos-aquae, Microcystis flos-aquae, and Mirocystis aeruginosa, and five green algae, Selenastrum capricornutun, Scenedesmus quadricauda, Scenedesmus obliquus, Chlorella vulgaris, and Chlorella pyrenoidosa. The average acute toxicity of the carbamate insecticides to the cyanobacteria and the green algae was in descending order carbaryl>carbofuran, propoxur, metolcarb > carbosulfan. Wide variations in response to the tested carbamate insecticides occurred among the eight individual species of cyanobacteria and green algae. The sensitivity of various species of algae exposed to carbofuran, propoxur, metolcarb, and carbaryl varied over one order of magnitude, and that of algae exposed to carbosulfan varied over two orders of magnitude. With regard to the diffrential sensitivity of cyanobacteria and green algae, the cyanobacteria were less sensitive than green algae to carbosulfan and propoxur. The pollutants may initiate a shift of algal group structure; especially, a shift from dominance by green algae to dominance by cyanobacteria, and may sustain cyanobacterial blooms at particular times. Therefore, the descending order of the ecosystem risk was carbosulfan > propoxur > carbofuran > carbaryl, metolcarb. There was a strong variance between toxicity and ecosystem risk; i.e., "low toxicity" does not imply "low ecosystem risk." PMID- 16677911 TI - Genetic adaptation to metal stress by natural populations of Daphnia longispina. AB - Loss of genetic diversity in natural populations as a result of chemical contamination has been reported in some studies. Here, four field populations of Daphnia longispina, two from sites historically impacted by acid mine drainage (AMD) and two from reference sites, were used to address four objectives: (1) identify differences in sensitivity between the stressed and reference populations; (2) distinguish between the components responsible for those differences (environmental influence vs genetic determination); (3) determine if genetically determined responses of reference and stressed populations converge from lethal to sublethal levels of contamination; and (4) evaluate losses of variability in genetically determined resistance by the stressed populations. Lethal and sublethal assays were carried out by exposing nonacclimated and acclimated neonates to AMD-contaminated waters and to copper dissolved in an artificial medium. Results indicate that both nonacclimated and acclimated individuals from the stressed populations are significantly less sensitive to AMD contaminated waters than those from the reference populations, at both lethal and sublethal levels. The hypothesis of a convergence from lethal to sublethal responses was confirmed. PMID- 16677912 TI - Trace metal concentrations in edible tissue of snapper, flathead, lobster, and abalone from coastal waters of Victoria, Australia. AB - The concentrations of heavy metals in the edible tissue of commonly fished species of the Victorian coast of Australia are reported. The metals studied were As, Cd, Cu, Hg, Pb, Se, and Zn and the fish species examined were snapper (Pagruss auratus), flathead (Platycephalus bassenssis and Neoplatycephalus richardsoni), lobster (Jasus edwardsii), and abalone (Haliotis rubra). None of the fish species studied had average concentrations exceeding the maximum levels specified for As, Cd, Hg, and Pb by the Food Standards Australia and New Zealand Food Standards code. Additionally, the concentrations of Cu, Se, and Zn were close to or below the median values generally expected in these species. Essential trace elements Se and Zn were found to be well regulated by all fish species. Although also essential, Cu was not so well regulated, especially in abalone. Nonessential metals As, Cd, and Hg are not regulated in the studied fish and their concentrations in the fish tissue are dependent on size and fishing zone. Metal concentrations were not largely affected by sex. Surprisingly, the concentrations of metals in fish in Port Phillip Bay, a zone, which includes the major cities of Melbourne and Geelong and is known to have high concentrations of metals in the water and sediment, were not consistently higher than those in other less-populated fishing zones. PMID- 16677913 TI - Salinity effects on activity and expression of glutathione S-transferases in white sturgeon and Chinook salmon. AB - This study evaluated the activity and expression of the glutathione S-transferase (GST) detoxification isoenzymes in juvenile white sturgeon (Acipenser transmontanus) and Chinook salmon (Oncorhynchus tshawytscha) during acclimation from freshwater (2 per thousand) to estuarine (15 per thousand) salinity conditions. In white sturgeon, GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) increased significantly (P = 0.005; n = 5) with elevated salinity, but not for the Chinook salmon (P = 0.174; n = 10). GST activity of both sturgeon and salmon toward ethacrynic acid (ETHA) did not significantly change with elevated salinity (P = 0.516 with n = 3, and P = 0.125 with n = 3, respectively). Expression of the GST classes, and hepatic glutathione (GSH) concentration, as determined by HPLC, also did not significantly change with increased salinity. In conclusion, overall GST activity in white sturgeon, but not Chinook salmon, is stimulated by elevated water salinity, thus electrophilic chemicals such as pesticides may be more effectively detoxified by sturgeon as they undergo seaward migration. PMID- 16677914 TI - Biodegradability of nitrogenous compounds under anaerobic conditions and its estimation. AB - The anaerobic biodegradability of 23 nitrogenous compounds, including nitrogenous heterocyclic compounds and aliphatic amines, was tested and assessed in integration. These nitrogenous compounds were classified into readily, partially, and poorly biodegradable compounds after calculation of their integrated assessment indices (IAIs), Rules for anaerobic biodegradation of these compounds were also drawn. Stepwise regression and backpropagation artificial neural network (BP-ANN) methods were applied to establish quantitative structure biodegradability relationships (QSBRs) based on the assessment results. In QSBR models, three molecular structure descriptors-second-order molecular connectivity index (2chi(V3)chi(v)p), and energy of the highest occupied molecular orbital (EHOMO)--were included. After analysis of the sensitivity of variables in QSBR models, it was found that the key molecular structure descriptor affecting anaerobic biodegradability of nitrogenous compounds is EHOMO, which is directly proportional to the anaerobic biodegradability of nitrogenous compounds. PMID- 16677915 TI - Physiological responses of saplings of Caesalpinia echinata Lam., a Brazilian tree species, under ozone fumigation. AB - The effects induced by long-term (30 day) and short-term (6h) exposures to ozone on the physiological parameters in young plants of Caesalpinia echinata Lam., a Brazilian tree species, were determined. Potted plants were maintained in open top chambers in Valencia, Spain, under charcoal filtered air (mean O3 level: 29 microg m3), nonfiltered air (NF; 43 microg m3), and nonfiltered air plus O3 (NF + O3; 68 microg m3), simulating prevailing concentrations observed in the city of Sao Paulo, Brazil, during spring months (50 microg m3 in 2002). In the plants kept in NF + O3 for 30 days, although no foliar visible injuries were observed, the net carbon assimilation rate was reduced to 50%, stomatal conductance 42%, and transpiration 40%, when compared to the results for the NF plants. No changes in antioxidants, in leaf, stem, and root biomass, and in the root/shoot ratio were observed. Significant reductions were observed in gas exchange and in PSII photochemical efficiency (Fv/Fm) after 6 h of exposure to an O3 peak. The species was shown to be sensitive to ambient O3 concentrations measured in Sao Paulo. PMID- 16677916 TI - Pulmonary effects of deltamethrin inhalation: an experimental study in rats. AB - Deltamethrin is a synthetic pyrethroid widely used as the insecticide of choice especially for local vector mosquitoes in most countries. The application is mostly by cold aerosol spraying using vehicle-mounted equipment with a duration of 3-4 months during the summer. This experimental study aimed to evaluate the morphologic changes in the lungs caused by the inhalation of this insecticide. The study was performed on 30 mature male Wistar rats. While 10 of the rats were used as control group, 20 rats, separated into two groups, were exposed to 1:10 dilution of deltamethrin aerosol spray for 30 min each day for 45 days in doses of 6.0 and 12.0 mg/m3. The animals were sacrificed and tissue samples taken from the lungs were processed for both light microscopy and transmission electron microscopy. Light microscopic examination revealed heavy congestion, marked perivascular edema, and lymphoplasmocytic infiltration with focal nonspecific interstitial pneumonia, foamy macrophage accumulation, emphysema, peribronchial lymphoid tissue hyperplasia, and focal hemorrhage. Ultrastructurally, the ciliated cells of the airways appeared swollen with a few structurally abnormal cilia. Alveolar lining cells revealed mild degeneration and a slight hyperplasia in type II cells. Increases in the number of collagen bundles and edema in the alveolar septa were also noted. PMID- 16677918 TI - Interesting case: Endosteal formation of new bone around a Kirschner wire in long term follow up of a patient with mandibular Ewing sarcoma. PMID- 16677919 TI - Quantitative analyses of sonographic images of the parotid gland in patients with Sjogren's syndrome. AB - The first purpose of this study was to quantify the sonographic images of the salivary gland to differentiate the Sjogren syndrome (SS) group from the non-SS group. We included 132 patients in this study who had been referred to our department because of a suspicion of SS. A total of 91 patients fulfilled the criteria for SS, whereas the remaining 41 patients did not. We placed the regions of-interest within the lesion. The first purpose was to evaluate which indices obtained by the texture analyses were useful for differentiating the SS group from the non-SS group. The second purpose was to evaluate the relationship between the indices and the degree of severity in the SS group. Out of the several indices evaluated, Hurst coefficients, obtained by fractal analysis, of SS group were found to be significantly lower than those of the non-SS group. Moreover, the Hurst coefficient was associated with the degree of destruction of the parotid gland as assessed by sialography. The Hurst coefficient of a globular stage and an advanced stage were both significantly lower than that of a normal pattern, whereas the Hurst coefficient of a punctate stage was almost similar to that of the normal stage. The Hurst coefficient showed a very weak correlation with the results of either the gum test or serologic tests. PMID- 16677920 TI - Three-dimensional power doppler ultrasound is useful to monitor the response to treatment in a patient with primary papillary serous carcinoma of the peritoneum. AB - To date, this is the first report to monitor changes of intratumor vascularization and the response to radiation and Cyberknife therapy in a patient with recurrent primary papillary serous carcinoma of the peritoneum by three dimensional (3D) power Doppler ultrasonography (PDUS). Transvaginal 3D PDUS detected a recurrent presacral tumor with abundant intratumor vascularity. Serial examinations of the tumor volume and serum CA-125 level were studied before, during, and 6 mo after therapy. Meanwhile, the intratumor blood flow was measured and expressed as vascularity indices. All of the tumor volume, intratumor vascularity indices and serum CA-125 level decreased progressively following therapy. A remaining lesion with nearly absent intratumor power Doppler signals suggested a scarring lesion posttreatment. Indeed, CT-guided tissue biopsy confirmed fibrotic change. 3D PDUS is useful to monitor the response to treatments and to differentiate residual tumors from lesions of scarring change posttreatment. It provides more accurate posttreatment information than pelvic computed tomography. PMID- 16677921 TI - 3-D sonography for diagnosis of osteoarthrosis and disk degeneration of the temporomandibular joint, compared with MRI. AB - This study determined the value of three-dimensional (3-D) sonography for the assessment of osteoarthrosis and disk degeneration of the temporomandibular joint (TMJ). Sixty-eight patients (136 TMJ) with clinical dysfunction were examined by 272 sonographic 3-D scans. An 8- to 12.5-MHz motor-angulated transducer positioned inferior-parallel to the zygomatic arch scanned the region-of interest. 3-D condylar morphology was compared with subsequent magnetic resonance imaging (MRI). Fifty-three datasets were complete, i.e., 106 TMJ, 212 examinations. 3-D sonographic examination took 5 min and attained 70% sensitivity/76% specificity/75% accuracy; positive predictive value was 44%%; negative predictive value was 90%. Disk degeneration was diagnosed synonymously with 64%/73%/71%/42%/ 87%. 3-D sonography proved to be reliable for exclusion of osteoarthrosis as disk degeneration compared with MRI, whereas the presence of osteoarthrosis and disk dislocation cannot be reliably diagnosed. Prospective use will include routine screening, using more sophisticated equipment with higher frequency in real-time 3-D viewing. PMID- 16677922 TI - 3-D sonography for diagnosis of disk dislocation of the temporomandibular joint compared with MRI. AB - This study determines the value of three-dimensional (3-D) sonography for the assessment of disk dislocation of the temporomandibular joint (TMJ). Sixty-eight patients (i.e.,136 TMJ) with clinical dysfunction were examined by 272 sonographic 3-D scans. An 8- to 12.5-MHz transducer, angulated by step-motor, was used after picking a volume box on 2-D scan; magnetic resonance imaging followed immediately. Every TMJ was scrutinized in closed- and open-mouth position for normal or dislocated disk position. Fifty-three patients had complete data sets, i.e., 106 TMJ, 212 examinations. Sonographic examination took 5 min, with 74% specificity (62% closed-mouth; 85% open-mouth); sensitivity 53% (62/43%); accuracy 70% (62/77%); positive predictive value 49% (57/41%); and negative predictive value 77% (67/86%). This study encourages more research on the diagnostic capacity of 3-D TMJ sonography, with the advantage of multidimensional joint visualization. Although fair in specificity and negative predictive value, sensitivity and accuracy may ameliorate with future higher-sound frequency, real time 3-D viewing and automated image analysis. PMID- 16677923 TI - Ultrasonographic portography with low mechanical index gray-scale imaging in hepatic VX2 tumor. AB - To evaluate the characteristics of portal blood supply of hepatic tumors by ultrasonographic portography (USP), an in vivo model was studied using SonoVue, a second-generation ultrasound contrast agent (UCA) and low mechanical index (MI), gray-scale harmonic imaging. SonoVue (0.05 mL) was administrated through catheter placed into the main trunk of portal vein at laparotomy, followed by a 0.5 mL saline flush, in 12 rabbits with hepatic VX2 tumor, implanted by VX2 tumor tissue cubes of approximately 1 mm3 from carrier rabbit. Results showed that low MI gray scale imaging delineated clearly the dynamic enhancement of tumors and liver parenchyma. Among 22 tumors, seven tumors were diffusely increased, with the intensity of enhancement weaker than that of the surrounding liver parenchyma. The UCA was washed out earlier from tumors than from surrounding liver parenchyma. Three tumors showed the branches of portal vein. Five tumors showed peripheral contrast enhancement and a central coarse unenhanced hypoechoic area. Seven tumors displayed no actual enhancement. All lesions (100% [22 of 22]) were depicted clearly in the whole duration of enhancement, especially in the early and late phase, regardless of enhancement pattern, and portal blood flow was manifested in 15 of 22 (68%) tumors, by USP. The enhancement pattern of the tumors corresponded to the pathologic findings. The results indicated that ultrasonographic portography, combined with low MI levels and second-generation UCA, is a sensitive and safe method to study portal blood supply for liver cancer. It may contribute to improvement of the detectability and diagnostic ability and assist the choice of a therapeutic strategy for treatment of liver cancer. However, applicability of the method to human may be problematic because of high invasiveness and great difficulty in administering contrast medium. PMID- 16677924 TI - Characterization of in vitro healthy and pathological human liver tissue periodicity using backscattered ultrasound signals. AB - This work studied the periodicity of in vitro healthy and pathologic liver tissue, using backscattered ultrasound (US) signals. It utilized the mean scatterer spacing (MSS) as a parameter of tissue characterization, estimated by three methods: the spectral autocorrelation (SAC), the singular spectrum analysis (SSA) and the quadratic transformation method (SIMON). The liver samples were classified in terms of tissue status using the METAVIR scoring system. Twenty tissue samples were classified in four groups: F0, F1, F3 and F4 (five samples for each). The Kolmogorov-Smirnov test (applied on group pairs) resulted as nonsignificant (p > 0.05) for two pairs only: F1/F3 (for SSA) and F3/F4 (for SAC). A discriminant analysis was applied using as parameters the MSS mean (MSS) and standard deviation (sigmaMSS), the estimates histogram mode (mMSS), and the speed of US (mc(foie)) in the medium, to evaluate the degree of discrimination among healthy and pathologic tissues. The better accuracy (Ac) with SAC (80%) was with parameter group (MSS, sigmaMSS, mc(foie)), achieving a sensitivity (Ss) of 92.3% and a specificity (Sp) of 57.1%. For SSA, the group with all four parameters showed an Ac of 75%, an Ss of 78.6% and an Sp of 66.70%. SIMON obtained the best Ac of all (85%) with group (MSS, mMSS, mc(foie)), an Ss of 100%, but with an Sp of 50%. PMID- 16677925 TI - Serial evaluation of acute cerebral hyperperfusion by transcranial color-coded sonography. AB - Using transcranial color-coded sonography (TCCS), we evaluated the acute changes in the hemodynamics of cerebral hyperperfusion in two cases. The mean flow velocity of the cerebral arteries increased at the onset of clinical symptoms, together with an increase in the regional cerebral blood flow (rCBF). In serial follow-up studies, the flow velocity gradually returned to normal in parallel with the normalization of the rCBF values. TCCS can be useful for evaluation of acute cerebral hyperperfusion. PMID- 16677926 TI - Flow index evaluation of 3-D volume flow images: an in vivo and in vitro study. AB - Three-dimensional (3-D) ultrasound imaging has improved evaluation of organ circulation and might contribute new information on maternal and fetal blood supply. Flow index (FI) of 3-D color images has been proposed as a measure of perfusion. The aim of this study was to evaluate whether the 3-D FI is a parameter of volume flow and flow velocity in a human vessel and in a flow phantom. A 1-cm-long strip of the uterine artery was recorded in 3-D power Doppler (3D-PD) mode in a cross-sectional study of 170 normal singleton pregnancies between 26 and 42 weeks' gestation. A fixed ultrasound system installation was used during the examination. The VOCAL software integrated in the ultrasound unit calculated vessel volume and FI. Reproducibility of the measurements was tested. The method was also tested on a commercially available flow phantom. Reproducibility measurements gave satisfactory results, both in terms of inter- and intraobserver variation. Unexpectedly, in normal pregnancy, the uterine artery FI decreased slightly with gestation. Uterine artery vessel volume increased, however, with gestational age. A poor correlation was found between the FI and both flow velocity and volume flow in the flow phantom. In conclusion, 3D-PD imaging can give impressive anatomical pictures of organ vascular tree. However, the new FI is poorly related to flow velocity or volume of flow. PMID- 16677927 TI - Ultrasound assessment of angiogenesis in a matrigel model in rats. AB - Matrigel, a basement membrane extract, has been extensively used in in vivo angiogenesis. Contrast ultrasound imaging (CUI) of implanted Matrigel plugs with (+bFGF) and without basic fibroblast growth factor (-bFGF) was performed 7 and 14 d after implantation, followed by histologic analysis. Statistically significant differences between +bFGF and -bFGF plugs were apparent at d 7 in both plug size and contrast enhancement (both p < 0.05). Histopathology revealed differences in microvessel density (MVD) between +bFGF and -bFGF at d 7 and d 14. A significant correlation between MVD and both power Doppler contrast-enhanced area (r = 0.65, p < 0.05) and fraction of plug enhanced (r = 0.59, p < 0.05) was present. CUI of Matrigel plugs was shown to be a robust method for distinguishing between two different angiogenic states. Ultrasound measurements of blood flow in the plugs correlated with MVD, a histologic technique used to quantify tumor angiogenesis. PMID- 16677928 TI - Ultrahigh frame rate retrospective ultrasound microimaging and blood flow visualization in mice in vivo. AB - To overcome frame rate limitations in high-frequency ultrasound microimaging, new data acquisition techniques have been implemented for 2-D (B-scan) and color flow visualization. These techniques, referred to as retrospective B-scan imaging (RBI) and retrospective color flow imaging (RCFI) are based on the use of the electrocardiogram (ECG) to trigger signal acquisitions. B-scan and color flow images are reconstructed by retrospectively assembling the processed data on a line-by-line basis. Retrospective techniques are used to produce the first in vivo B-scan and color flow images of mouse carotid arteries at frame rates up to 10,000 fps. Retrospective B-scan images of mouse heart were also produced at frame rates of 1000 fps using a version of RBI implemented on a commercial imaging system (Vevo660, VisualSonics, Toronto, ON, Canada). This technology enables detailed in vivo biomechanical studies of dynamic tissues such as the myocardium of the mouse heart with high temporal resolution. PMID- 16677929 TI - Guided ultrasound wave propagation in intact and healing long bones. AB - Ultrasonic evaluation of bone fracture healing has been traditionally based on the measurement of the propagation velocity of the first arriving signal (FAS). However, the FAS in general corresponds to a lateral wave that propagates along the bone's subsurface. In this work, we study guided ultrasound propagation in intact and healing bones. We developed a 2-D model of a bone-mimicking plate in which the healing process was simulated as a 7-stage process, and we also carried out ex vivo experiments on an intact tibia. Guided waves were represented in the time-frequency (t-f) domain of the signal by incorporating the Lamb wave theory. Three t-f distribution functions were examined, namely the reassigned Spectrogram, the smoothed-pseudo Wigner-Ville, and the reassigned version of it. For the intact plate case, we found that the S2, A3 Lamb modes were the dominant waves for a broadband 1-MHz excitation, and the S2, S0 for a 500-kHz excitation. During the simulated healing process, the mechanical and geometrical callus properties affected the theoretically anticipated Lamb modes. The propagation of guided waves throughout the thickness of the cortical bone and their sensitivity to both the mechanical and structural changes during healing can supplement velocity measurements so as to enhance the monitoring capabilities of ultrasonic evaluation. Nevertheless, the applicability of the Lamb wave theory to real bones has several limitations mostly associated with neglecting the inhomogeneity, anisotropy and irregular geometry of bone. PMID- 16677930 TI - Measuring guided waves in long bones: modeling and experiments in free and immersed plates. AB - Guided waves, consistent with the A0 Lamb mode, have previously been observed in bone phantoms and human long bones. Reported velocity measurements relied on line fitting of the observed wave fronts. Such an approach has limited ability to assess dispersion and is affected by interference by other wave modes. For a more robust identification of modes and determination of phase velocities, signal processing techniques using the fast Fourier transform (FFT) were investigated. The limitations of FFT because of spatial resolution were addressed to improve the precision of the measured modes. An inversion scheme was developed for determining the plate thickness from the measured velocity. Experiments were performed on free and immersed plates, mimicking bone without and with an overlying tissue. With group velocity filtering, modes could be identified reliably with precise phase velocities and thicknesses. These methods were essential for the immersed plates and they should lead to more reliable in vivo measurements. PMID- 16677931 TI - Producing diffuse ultrasound reflections from medical instruments using a quadratic residue diffuser. AB - Simultaneous visualization of tissue and surgical instruments is necessary during ultrasound-guided medical procedures. Standard minimally invasive instruments are typically metallic and act as strong specular scatterers. As a result, such instruments saturate the image or disappear according to the angle of incidence, obscuring nearby tissue and making it difficult to determine the instrument's precise location. The objective of this study was to produce diffusive reflections from the surface of surgical instruments for improved visualization in ultrasound. A surface profile based on a 2D quadratic residue diffuser (QRD) was employed, which has been demonstrated to reduce specular reflection in other acoustic applications. The backscattered echo amplitude from the diffusive surface at various angles of insonation was measured and compared to that from unmodified metal surfaces and heart tissue surfaces. The QRD resulted in an 8 dB reduction of the specular signal. Furthermore, the dynamic range for angles up to 75 degrees was less than 20 dB for the QRD and more than 65 dB for a flat surface. The QRD surface produces two beneficial results for the simultaneous imaging of instruments and tissue. First, the conspicuity of diffusive surfaces in ultrasound images is markedly improved in comparison with unmodified metal surfaces. Secondly, the echo amplitude of diffusive metal surfaces differs in mean and standard deviation from that of tissue facilitating image enhancement and segmentation. PMID- 16677932 TI - Combination of HIFU therapy with contrast-enhanced sonography for quantitative assessment of therapeutic efficiency on tumor grafted mice. AB - The objective was to evaluate treatment efficiency of a new high-intensity focused ultrasound (HIFU) prototype combining a therapeutic transducer with a sonographic probe. The optimal HIFU sequence was defined on ex vivo samples before in vivo evaluation of tumor ablation was performed by perfusion quantification after contrast agent injection. The original feature of this prototype is a 9-MHz sonographic probe in a HIFU device and connected to an Aplio (Toshiba) sonograph. Acoustical power and treatment time were determined on ex vivo livers to generate 1-cm-long lesions. Lesion reproducibility was assessed for the power and treatment time selected. The gap between lesions and HIFU displacement shot procedures were optimized to ablate a 1-cm3 volume. The optimized protocol was applied to five murine tumors in vivo. Tumor ablation was quantified according to (1) contrast uptake (CU) after HIFU using perfusion software (Toshiba) in "vascular recognition imaging" mode and Sonovue (Bracco) contrast agent, and (2) the percentage of necrosis quantified on histologic slides. Ex vivo results: optimized settings, at 442 W/cm2 applied during three cycles (3 s on/5 s off) generated 10 identical elementary lesions measuring 9.78 (+/-0.66) * 2.11 (+/-0.33) mm2. A 4-mm gap between adjacent lesions and a 2-min pause between shot lines were found optimal. In vivo results: 60 % (+/-22) mean reduction in CU after HIFU and tumor necrosis histologically estimated at 58 % (+/-5.7) were quantified for the five animals. The therapeutic potential of this HIFU prototype was demonstrated in vivo through objective quantification of tumor ablation based on CU. PMID- 16677933 TI - Evaluation of nonscanned mode soft-tissue thermal index in the presence of the residual temperature rise. AB - Previously, the temperature rise (deltaT) caused by diagnostic ultrasound and the AUIM/NEMA-defined thermal indices were examined to evaluate whether these indices were reasonable indicators of potential bioeffects due to ultrasound heating in the absence of a residual temperature rise (RTR). In our study, deltaT induced by diagnostic ultrasound exposures was estimated in the presence of an RTR using the Bioheat Transfer Equation. To evaluate deltaT/TIS in the presence of an RTR, 11 frequencies, eight cooling times, eight insonation times for the second ultrasound examination, and three source powers for a circular aperture (A(aprt)< or = 1 cm2) were investigated. In our comparison of the ratios of deltaT/TIS in the absence and presence of an RTR, a higher deltaT/TIS value was obtained in the examination with the RTR. We showed that the deltaT/TIS value is equal to 2.88 in the presence of an RTR, whereas the deltaT/TIS value without the RTR equals 1.90. In the presence of the RTR, although the TIS does not inform the user of higher ultrasound heating due to TIS values that do not exceed 1.00, deltaT reaches 2.62 degrees C, and the deltaT without the RTR reaches 1.68 degrees C in the case of a TIS value that does not exceed 1.00. These results suggest that, for nonscanned mode situations where soft tissue is insonated, the TIS should not be regarded as a reliable indicator of potential bioeffects due to ultrasound heating in the presence of the RTR. Our study also indicates the necessity for a new indicator that provides the clinical user with accurate in vivo temperature rise feedback (possibly even true deltaT), and includes adding an exposure time component to the Bio-Heat Equation model. PMID- 16677934 TI - Effect of 5-fluorouracil, Optison and ultrasound on MCF-7 cell viability. AB - The aim of this study was to analyze cell viability and expression of apoptotic related signaling proteins in MCF-7 breast cancer cells induced by combinations of ultrasound, the anticancer drug 5-fluorouracil (5-FU) and the ultrasound contrast agent Optison. MCF-7 cells were treated with 5-FU and sonicated at the frequency of 3.0 MHz and intensity of 3.0 W/cm2 for 1 min in the presence of Optison. The cells were analyzed for lactate dehydrogenase (LDH) release (a measure of cytotoxicity) and cell proliferation (by MTT assays). The LDH/MTT ratio was used for assessment of cell death. Expression of the apoptotic-related proteins, Bax and p27kip1, as well as phosphorylated forms of ERK and Akt proteins was assessed by Western blot analysis. We demonstrate that, immediately after treatment, cell death was most dependent on Optison; however, 24 h after treatment, cell death was more dependent on 5-FU. Ultrasound duty cycle increased cell death associated with either Optison or 5-FU. Furthermore, we show that treatment with 5-FU and ultrasound increased the levels of the Bax and p27kip1 proteins, but the addition of Optison appears to suppress apoptotic protein expression. PMID- 16677935 TI - Cavitation-enhanced ultrasound thermal therapy by combined low- and high frequency ultrasound exposure. AB - This paper demonstrates a novel approach for enhancing ultrasound-induced heating by the introduction of acoustic cavitation using simultaneous sonication with low and high-frequency ultrasound. A spherical focused transducer (566 or 1155 kHz) was used to generate the thermal lesions, and a low-frequency planar transducer (40 or 28 kHz) was used to enhance the bubble activity. Ex vivo fresh porcine muscles were used as the target of ultrasound ablation. The emitted signals and the signals backscattered from the bubble activity were also recorded during the heating process by a PVDF-type needle hydrophone, and thermocouples were inserted to measure temperatures. Compared with the lesions formed by a single focused transducer, the size of the lesions generated by this approach were as much as 140% larger along the axial direction and 200% larger along the radial direction for combined 566- and 40-kHz sonication. They were 47% and 66% larger along the axial and radial directions, respectively, for combined 1155- and 28-kHz sonication. Cavitation activities enhanced by low-frequency ultrasound were confirmed by the presence of subharmonics in the spectrum and temperature increase as a result of increased tissue absorption. These observations imply that cavitation-enhanced lesions can be generated without reducing the penetration ability; they also show the advantage of producing larger and more uniform thermal lesions by multiple sonications. This technique provides an easy and effective way to achieve cavitation-enhanced heating, and may be useful for generating large and deep-seated thermal lesions. PMID- 16677936 TI - Comparison of ultrasound and electromagnetic field effects on osteoblast growth. AB - This study compares the mechanisms of ultrasound (US) on osteoblast proliferation with those of pulsed electromagnetic field (PEMF), by different signal transduction pathway inhibitors. The cells were stimulated for 15 min under US or for 2 h under PEMF exposure. Twenty-four h after the beginning of stimulation, the cells were harvested and used for mitochondrial activity test (MTT) analysis. The results showed that there are different transduction pathways for US and PEMF stimulation that lead to an upgrade of osteoblast proliferation, although their pathways all lead to an increase in cytocolic Ca2+ and activation of calmodulin. These findings offer a biochemical mechanism to support the process of ultrasound and PEMF-induced enhanced healing of bone fractures. PMID- 16677938 TI - Simultaneous EEG and fMRI in the macaque monkey at 4.7 Tesla. AB - Simultaneous electroencephalography (EEG)/functional magnetic resonance imaging (fMRI) acquisition can identify the brain networks involved in generating specific EEG patterns. Yet, the combination of these methodologies is hampered by strong artifacts that arise due to electromagnetic interference during magnetic resonance (MR) image acquisition. Here, we report corrections of the gradient induced artifact in phantom measurements and in experiments with an awake behaving macaque monkey during fMRI acquisition at a magnetic field strength of 4.7 T. Ninety-one percent of the amplitude of a 10 microV, 10 Hz phantom signal could successfully be recovered without phase distortions. Using this method, we were able to extract the monkey EEG from scalp recordings obtained during MR image acquisition. Visual evoked potentials could also be reliably identified. In conclusion, simultaneous EEG/fMRI acquisition is feasible in the macaque monkey preparation at 4.7 T and holds promise for investigating the neural processes that give rise to particular EEG patterns. PMID- 16677939 TI - Sensitivity of single-voxel 1H-MRS in investigating the metabolism of the activated human visual cortex at 7 T. AB - Proton magnetic resonance spectroscopy (1H-MRS) has been used in a number of studies to noninvasively assess the temporal changes of lactate in the activated human brain. However, the results have not been consistent. The aim of the present study was to test the sensitivity of 1H-MRS during functional experiments at the highest magnetic field currently available for human studies (7 T). Stability and reproducibility of the measurements were evaluated from LCModel analysis of time series of spectra measured during a visual stimulation paradigm and by examination of the difference between spectra obtained at rest and during activation. The sensitivity threshold to detect concentration changes was 0.2 micromol/g for most of the quantified metabolites. The possible variations of metabolite concentrations during visual stimulation were within the same range (+/-0.2 micromol/g). In addition, the influence of a small line-narrowing effect due to the blood oxygenation level-dependent (BOLD) T2* changes on the estimated concentrations was simulated. Quantification of metabolites was, in general, not affected beyond 1% by line-width changes within 0.5 Hz. PMID- 16677940 TI - Tracing neural circuits in vivo with Mn-enhanced MRI. AB - The application of MRI-visible paramagnetic tracers to reveal in vivo connectivity can provide important subject-specific information for multisite, multielectrode intracortical recordings in combined behavioral and physiology experiments. To establish the use of such tracers in the nonhuman primate, we recently compared the specificity of the anterograde tracer Mn2+ with that of wheat-germ-agglutinin conjugated to horseradish peroxidase (WGA-HRP) in experiments tracing the neuronal connections of the basal ganglia of the monkey. It was shown that Mn2+ and WGA-HRP yield the same projection patterns and that the former tracer crosses at least two synapses, for it could be found in thalamus following injections into the striatum. Here we provide evidence that Mn2+ reaches the cortex following striatum injections and, thus, is transferred even further than previously shown. In other words, used as a paramagnetic MRI tracer, Mn2+ can permit the visualization of neural networks covering at least four processing stages. Moreover, unilateral intravitreal injections show that Mn2+ is sufficiently synapse specific to permit visualization of the lamina of the dorsal lateral geniculate nucleus (dLGN). Interestingly, the transfer rate of the substance reflected the well-known axonal size differences between the parvocellular and magnocellular layers of dLGN. After intravitreal injections, we were able to demonstrate transfer of Mn2+ into several subcortical and cortical areas, including the inferotemporal cortex. The specificity of the transsynaptic transfer of manganese that we report here indicates the value of this tracer for chronic studies of development and plasticity, as well as for studies of brain pathology. PMID- 16677941 TI - Development of visually evoked cortical activity in infant macaque monkeys studied longitudinally with fMRI. AB - We studied the development of visual activation longitudinally in two infant monkeys aged 103-561 days using the BOLD fMRI technique under opiate anesthesia and compared the results with those obtained in three adult animals studied under identical conditions. Visual activation in primary visual cortex, V1, was strong and reliable in monkeys of the youngest and oldest ages, showing that functional imaging techniques give qualitatively similar results in infants and adults. Visual activation in extrastriate areas involved in processing motion (MT/V5) and form (V4) was not evident in the younger animals, but became more adult-like in the older animals. This delayed onset of measurable BOLD responses in extrastriate visual cortex may reflect delayed development of visual responses in these areas, although at this stage it is not possible to rule out either effects of anesthesia or of changes in cerebral vascular response mechanisms as the cause. The demonstration of visually evoked BOLD responses in young monkeys shows that the BOLD fMRI technique can usefully be employed to address functional questions of brain development. PMID- 16677942 TI - EEG-fMRI mapping of asymmetrical delta activity in a patient with refractory epilepsy is concordant with the epileptogenic region determined by intracranial EEG. AB - We studied a patient with refractory focal epilepsy using continuous EEG correlated fMRI. Seizures were characterized by head turning to the left and clonic jerking of the left arm, suggesting a right frontal epileptogenic region. Interictal EEG showed occasional runs of independent nonlateralized slow activity in the delta band with right frontocentral dominance and had no lateralizing value. Ictal scalp EEG had no lateralizing value. Ictal scalp EEG suggested right sided central slow activity preceding some seizures. Structural 3-T MRI showed no abnormality. There was no clear epileptiform abnormality during simultaneous EEG fMRI. We therefore modeled asymmetrical EEG delta activity at 1-3 Hz near frontocentral electrode positions. Significant blood oxygen level-dependent (BOLD) signal changes in the right superior frontal gyrus correlated with right frontal oscillations at 1-3 Hz but not at 4-7 Hz and with neither of the two frequency bands when derived from contralateral or posterior electrode positions, which served as controls. Motor fMRI activations with a finger-tapping paradigm were asymmetrical: they were more anterior for the left hand compared with the right and were near the aforementioned EEG-correlated signal changes. A right frontocentral perirolandic seizure onset was identified with a subdural grid recording, and electric stimulation of the adjacent contact produced motor responses in the left arm and after discharges. The fMRI localization of the left hand motor and the detected BOLD activation associated with modeled slow activity suggest a role for localization of the epileptogenic region with EEG-fMRI even in the absence of clear interictal discharges. PMID- 16677943 TI - Metabolic alteration transients during paroxysmal activity in an epileptic patient with fixation-off sensitivity: a case study. AB - The purpose of this study was to investigate short-time metabolic variations related to continuous epileptic activity elicited by fixation-off sensitivity (FOS). Time-resolved magnetic resonance spectroscopy was performed on a patient on whom previous clinical findings clearly indicated presence of FOS. The epileptic focus was localized with a simultaneous electroencephalographic and functional magnetic resonance imaging study. The results showed a linear increase of the sum of glutamate and glutamine with time of paroxysmal activity in epileptic focus and much greater concentration of choline-containing compounds in focus than in the contralateral side. PMID- 16677944 TI - Laminar specificity in monkey V1 using high-resolution SE-fMRI. AB - The lamination of mammalian neocortex is widely used as reference for describing a wide range of anatomical and physiological data. Its value lies in the observation that in all examined species, cortical afferents, intrinsic cells and projection neurons organize themselves with respect to the laminae. The comprehension of the computations, carried out by the neocortical microcircuits, critically relies on the study of the interlaminar connectivity patterns and the intralaminar physiological processes in vivo. High-resolution functional neuroimaging, enabling the visualization of activity in individual cortical laminae or columns, may greatly contribute in such studies. Yet, the BOLD effect, as measured with the commonly used GE-EPI, contains contributions from both macroscopic venous blood vessels and capillaries. The low density of the cortical veins limits the effective spatial specificity of the fMRI signal and yields maps that are weighted toward the macrovasculature, which thus can be significantly different from the actual site of increased neuronal activity. Spin-echo (SE) sequences yielding apparent T2-weighted BOLD images have been shown to improve spatial specificity by increasing the sensitivity of the signal to spins of the parenchyma, particularly at high magnetic fields. Here we used SE-fMRI at 4.7 T to examine the specificity and resolution of functional maps obtained by stimulating the primary visual cortex of monkeys. Cortical layers could be clearly visualized, and functional activity was predominantly localized in cortical layer IV/Duvernoy layer 3. The choice of sequence parameters influences the fMRI signal, as the SE-EPI is by nature sensitive to T2* in addition to its T2 dependency. Using parameters that limit T2* effects yielded higher specificity and better visualization of the cortical laminae. Because the demands of high spatial resolution using SE severely decreases temporal resolution, we used a stimulus protocol that allows sampling at higher effective temporal resolution. This way, it was possible to acquire high-spatial and high-temporal resolution SE fMRI data. PMID- 16677945 TI - An independent component analysis-based approach on ballistocardiogram artifact removing. AB - Interest about simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) data acquisition has rapidly increased during the last years because of the possibility that the combined method offers to join temporal and spatial resolution, providing in this way a powerful tool to investigate spontaneous and evoked brain activities. However, several intrinsic features of MRI scanning become sources of artifacts on EEG data. Noise sources of a highly predictable nature such as those related to the pulse MRI sequence and those determined by magnetic gradient switching during scanning do not represent a major problem and can be easily removed. On the contrary, the ballistocardiogram (BCG) artifact, a large signal visible on all EEG traces and related to cardiac activity inside the magnetic field, is determined by sources that are not fully stereotyped and causing important limitations in the use of artifact-removing strategies. Recently, it has been proposed to use independent component analysis (ICA) to remove BCG artifact from EEG signals. ICA is a statistical algorithm that allows blind separation of statistically independent sources when the only available information is represented by their linear combination. An important drawback with most ICA algorithms is that they exhibit a stochastic behavior: each run yields slightly different results such that the reliability of the estimated sources is difficult to assess. In this preliminary report, we present a method based on running the FastICA algorithm many times with slightly different initial conditions. Clustering structure in the signal space of the obtained components provides us with a new way to assess the reliability of the estimated sources. PMID- 16677946 TI - The effect of artifacts on dependence measurement in fMRI. AB - The study of effective connectivity by means of neuroimaging depends on the measurement of similarity between activity patterns at different locations in the brain, without necessarily presupposing a particular model for this dependence. When these interactions are measured using functional magnetic resonance imaging (fMRI) techniques, however, imaging and physiological artifacts create patterns of dependence that may be unrelated to cortical activity. We demonstrate some of these effects through the measurement of short-range dependencies present in fMRI scans of the primary visual cortex (V1) in the anaesthetized macaque monkey. High field (4.7 T) fMRI scans were conducted to measure responses based on the blood oxygen level-dependent contrast mechanism, during periods of no sensory stimulation and of visual stimulation with rotating polar-transformed checkerboard gratings. Dependence between the haemodynamic activity at different spatial locations (i.e., different voxels) was measured using correlation, mutual information and functional covariance. Particular attention was paid to understanding the sources of spurious dependence that may be observed during such investigations. Two main effects were detected: (a) short-range correlations introduced by the process of image reconstruction and (b) perturbations in the haemodynamic response caused by breathing. The image reconstruction artifacts were shown to create an artificially high short-range dependence in the readout direction of the scan, and the breathing artifacts caused enhanced short-range dependence in both the readout and phase-encode directions. Additional dependence in the phase-encode direction due to image-ghosting is also possible but will not be discussed in this report, as it can be alleviated by fine adjustment of preemphasis (elimination of eddy currents). A technique is described for removing breathing artifacts, and the effect of breathing on the apparent dependence between voxels is illustrated. The correlation of haemodynamic activity with the stimulus was found to be affected by breathing, although this effect can be neutralised by averaging the haemodynamic responses over many repetitions of the stimulus. Nonetheless, patterns of dependent activity between voxels may be lost in this averaging process, which makes the removal of breathing artifacts necessary if statistical dependence and the study of effective connectivity is the primary aim of an investigation. PMID- 16677947 TI - BOLD signal and vessel dynamics: a hierarchical cluster analysis. AB - The aim of the present study was to analyze blood oxygenation level-dependent (BOLD) signal variation during an apnea-based task in order to assess the capability of a functional magnetic resonance imaging (fMRI) procedure to estimate cerebral vascular dynamic effects. We measured BOLD contrast by hierarchical cluster analysis in healthy subjects undergoing an fMRI experiment, in which the task paradigm was one phase of inspirational apnea (IA). By processing the time courses of the fMRI experiment, analysis was performed only on a subclass of all the possible signal patterns; basically, root mean square and absolute variation differences have been calculated. Considering the baseline value obtained by computing the mean value of the initial rest period as reference, particular voxels showed relative important variations during the IA task and during the recovery phase following the IA. We focused our interest on the signal response of voxels that would correspond mainly to white and gray matter regions and that also may be affected by the proximity of large venous vessels. The results are presented as maps of space-temporal distribution of time series variations with two levels of hierarchical clustering among voxels with low to high initial response. Furthermore, we have presented a clustering of the signal response delay, conducting to a partition and identification of specified brain sites. PMID- 16677948 TI - A cluster-based quantitative procedure in an fMRI study of Parkinson's disease. AB - Parkinson's disease is a neurological disorder associated with disfunction of dopaminergic pathways of the basal ganglia. In this study, we report the effects of decreasing plasma concentrations of the dopamine-agonist apomorphine on the size and extents of activity clusters observed with functional magnetic resonance imaging during a simple motor task. Eight patients at advanced disease stage and six healthy volunteers were studied during four consecutive sessions. We observed consistent activations in the primary sensorimotor area of the contralateral side and in the supplementary motor area of both patients and controls during the first session. During subsequent sessions, while the drug concentration gradually decreased in patients, they showed a fragmentation of the activity areas, with an overall decrease of involved volume and a decline of activity in the supplementary motor area. The appearing of activity in the ipsilateral motor area matched a partial recovery of supplementary motor area activation. During the last session, when patients showed severe dyskinesia, a widespread region of positive and negative correlations between signal and task was observed. We conclude that the lack of subcortical circuitry is partially reversible by apomorphine and that when the drug effects are reduced, there is a possible mechanism recruitment of alternate subcortical pathways. PMID- 16677949 TI - Investigation of multicomponent diffusion in cat brain using a combined MTC-DWI approach. AB - In this study, multiple-component water diffusion in the cat brain is investigated using an approach that combines diffusion-weighted images using multiple b values with magnetization transfer contrast (MTC). The MTC allows filter of signal originating from water molecules that rapidly exchange with binding sites on large macromolecular structures, and in brain white matter, it is assumed that a significant portion of the MTC is due to the interaction of water with the extraaxonal myelin sheath. Henceforth, multicomponent analysis of diffusion curves with and without MTC may shed light on the contribution of the extraaxonal water to the diffusion signal and on the relationship between diffusion components and tissue compartments in the brain. When a biexponential model was applied to the data, the volume fractions of the two diffusion components changed significantly in white matter with the application of the MTC. These changes are then discussed in the frame of tissue components and the possible interaction with the myelin sheath. PMID- 16677950 TI - Echo-shifted multislice EPI for high-speed fMRI. AB - The advantages of event-related functional Magnetic Resonance Imaging (fMRI) and the increasing use of fMRI in cognitive experiments are both driving the development of techniques that allow images sensitive to the blood oxygen level dependent effect to be acquired at ever-higher temporal resolution. Here, we present a technique based on the use of echo shifting (ES) in conjunction with a multislice (MS) echo planar imaging (EPI) readout, which allows T2*-weighted images to be generated with a repetition time per slice that is less than the echo time (TE). Using this ES-MS-EPI approach, it is shown that images with a TE of 40 ms can be acquired with an acquisition time per slice of only 27 ms. The utility of the MS-ES-EPI sequence is demonstrated in a visual-motor, event related fMRI study in which nine-slice image volumes are acquired continuously at a rate of 4.1 Hz. The sequence is shown to produce reliable activation associated with both visual stimuli and motor actions. PMID- 16677951 TI - A chemical shift imaging study on regional metabolite distribution in a CADASIL family. AB - A chemical shift imaging (CSI) study was performed to directly assess relative concentrations of N-acetylaspartate (NAA), Cho and Cr metabolites in normal- and abnormal-appearing brain tissue of asymptomatic and symptomatic members of a single family with a neuropathologic, genetic and electrophysiological confirmed diagnosis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. The aim of the investigation was to evaluate clinical findings and metabolite abnormalities as early appearance of axonal injury in this syndrome. The main findings related statistically significant decreases in the mean metabolite ratios for NAA/Cr, NAA/Cho and Cho/Cr in the anterior parts in comparison with the posterior parts of the centrum semiovale in symptomatic and asymptomatic patients. The effect was considerably greater in the symptomatic patients, indicating a strong correlation between CSI and pathology results. No differences were found between the two areas in the control group. Although lactate signals were hardly detectable in individual spectra, there was a trend toward increased Lac/Cr values in the anterior parts with respect to the posterior parts in the patient group, with the effect particularly evident in the asymptomatic subjects with the gene mutation. PMID- 16677953 TI - Complementary aspects of diffusion imaging and fMRI; I: structure and function. AB - Studying the intersection of brain structure and function is an important aspect of modern neuroscience. The development of magnetic resonance imaging (MRI) over the last 25 years has provided new and powerful tools for the study of brain structure and function. Two tools in particular, diffusion imaging and functional MRI (fMRI), are playing increasingly important roles in elucidating the complementary aspects of brain structure and function. In this work, we review basic technical features of diffusion imaging and fMRI for studying the integrity of white matter structural components and for determining the location and extent of cortical activation in gray matter, respectively. We then review a growing body of literature in which the complementary aspects of diffusion imaging and fMRI, applied as separate examinations but analyzed in tandem, have been exploited to enhance our knowledge of brain structure and function. PMID- 16677952 TI - Dynamic imaging with MRI contrast agents: quantitative considerations. AB - Time-resolved MRI has had enormous impact in cognitive science and may become a significant tool in basic biological research with the application of new molecular imaging agents. In this paper, we examine the temporal characteristics of MRI contrast agents that could be used in dynamic studies. We consider "smart" T1 contrast agents, T2 agents based on reversible aggregation of superparamagnetic nanoparticles and sensors that produce changes in saturation transfer effects (chemical exchange saturation transfer, CEST). We discuss response properties of several agents with reference to available experimental data, and we develop a new theoretical model that predicts the response rates and relaxivity changes of aggregation-based sensors. We also perform calculations to define the extent to which constraints on temporal resolution are imposed by the imaging methods themselves. Our analysis confirms that some small T1 agents may be compatible with MRI temporal resolution on the order of 100 ms. Nanoparticle aggregation T2 sensors are applicable at much lower concentrations, but are likely to respond on a single second or slower timescale. CEST agents work at high concentrations and temporal resolutions of 1-10 s, limited by a requirement for long presaturation periods in the MRI pulse sequence. PMID- 16677954 TI - Can MRI reveal phenotypes of multiple sclerosis? AB - The multicontrast capability of magnetic resonance imaging (MRI) is discussed in its role in the search for phenotypes of multiple sclerosis (MS). Aspects of MRI specificity, putative markers for pathogenetic components of disease and issues of spatial and temporal distribution are discussed. While particular reference is made to MS, the concepts apply to common pathological features of many neurologic diseases and to neurodegenerative disease in general. The assessment and dissociation of disease activity and disease severity, as well as the combination of varied metrics for the purposes of inferential and predictive disease modeling, are explored with respect to biomarkers and clinical outcomes. By virtue of its noninvasive nature and multicontrast capabilities depicting multiple facets of MS pathology, MRI lends itself to the systematic search of pathogenetically distinct subtypes of MS in large populations of patients. In conjunction with clinical, immunological, serological and genetic information, clusters of MS patients with distinct clinical prognosis and diverse response profiles to available and future treatments may be identified. PMID- 16677955 TI - Challenges for detection of neuronal currents by MRI. AB - Neuronal current MRI (nc-MRI) is an imaging method that directly maps magnetic field changes caused by neuronal currents with, at the same time, a high spatial and temporal resolution. A viable nc-MRI method would be of great benefit, both for the study of human brain function and for clinical applications in the field of epilepsy, especially for the noninvasive presurgical mapping of epileptogenic foci. A survey of fundamental issues in nc-MRI is reviewed, and challenges for future developments of the method are described within this context. Particularly, an overview of the models for signal generation is given, and the origin and physiology of different sources of neuronal currents are described. Prospects for predicting neuronal currents by electromagnetic field mapping and using this information, both a priori and a posteriori, for nc-MRI are considered. Ways of increasing specificity in nc-MRI by minimizing secondary hemodynamic and metabolic effects are described as well as means of optimizing the nc-MRI method for pushing the detection limit. Previously published works are described within these categories and future directions for nc-MRI are proposed. PMID- 16677956 TI - Illuminating the BOLD signal: combined fMRI-fNIRS studies. AB - Functional magnetic resonance imaging (fMRI) is currently combined with electrophysiological methods to identify the relationship between neuronal activity and the blood oxygenation level-dependent (BOLD) signal. Several processes like neuronal activity, synaptic activity, vascular dilation, blood volume and oxygenation changes underlie both response modalities, that is, the electrophysiological signal and the vascular response. However, accessing single process relationships is absolutely mandatory when aiming at a deeper understanding of neurovascular coupling and necessitates studies on the individual building blocks of the vascular response. Combined fMRI and functional near-infrared spectroscopy studies have been performed to validate the correlation of the BOLD signal to the hemodynamic changes in the brain. Here we review the current status of the integration of both technologies and judge these studies in the light of recent findings on neurovascular coupling. PMID- 16677957 TI - Integration of fMRI, NIROT and ERP for studies of human brain function. AB - Different methods of assessing human brain function possess specific advantages and disadvantages compared to others, but it is believed that combining different approaches will provide greater information than can be obtained from each alone. For example, functional magnetic resonance imaging (fMRI) has good spatial resolution but poor temporal resolution, whereas the converse is true for electrophysiological recordings (event-related potentials or ERPs). In this review of recent work, we highlight a novel approach to combining these modalities in a manner designed to increase information on the origins and locations of the generators of specific ERPs and the relationship between fMRI and ERP signals. Near infrared imaging techniques have also been studied as alternatives to fMRI and can be readily integrated with simultaneous electrophysiological recordings. Each of these modalities may in principle be also used in so-called steady-state acquisitions in which the correlational structure of signals from the brain may be analyzed to provide new insights into brain function. PMID- 16677958 TI - Insights into brain microstructure from the T2 distribution. AB - T2 weighting is particularly sensitive, but notoriously unspecific, to a wide range of brain pathologies. However, careful measurement and analysis of the T2 decay curve from brain tissue promise to provide much improved pathological specificity. In vivo T2 measurement requires accurate 180 pulses and appropriate manipulation of stimulated echoes; the most common approach is to acquire multiple echoes from a single slice. The T2 distribution, a plot of component amplitude as a function of T2, can be estimated using an algorithm capable of fitting a multi-exponential T2 decay with no a priori assumptions about the number of exponential components. T2 distributions from normal brain show peaks from myelin water, intra/extracellular water and cerebral spinal fluid; they can be used to provide estimates of total water content (total area under the T2 distribution) and myelin water fraction (MWF, fractional area under the myelin water peak), a measure believed to be related to myelin content. Experiments on bovine brain suggest that magnetization exchange between water pools plays a minor role in the T2 distribution. Different white matter structures have different MWFs. In normal white matter (NWM), MWF is not correlated with the magnetization transfer ratio (MTR) or the diffusion tensor fractional anisotropy (FA); hence it provides unique information about brain microstructure. Normal appearing white matter (NAWM) in multiple sclerosis (MS) brain possesses a higher water content and lower MWF than controls, consistent with histopathological findings. Multiple sclerosis lesions demonstrate great heterogeneity in MWF, presumably due to varying myelin contents of these focal regions of pathology. Subjects with schizophrenia were found to have significantly reduced MWF in the minor forceps and genu of the corpus callosum when compared to controls, suggesting that reduced frontal lobe myelination plays a role in schizophrenia. In normal controls, frontal lobe myelination was positively correlated with both age and education; this result was not observed in subjects with schizophrenia. A strong correlation between MWF and the optical density from the luxol fast blue histological stain for myelin was observed in formalin-fixed brain, supporting the use of the MWF as an in vivo myelin marker. PMID- 16677959 TI - In vivo 13C NMR spectroscopy and metabolic modeling in the brain: a practical perspective. AB - In vivo 13C NMR spectroscopy has the unique capability to measure metabolic fluxes noninvasively in the brain. Quantitative measurements of metabolic fluxes require analysis of the 13C labeling time courses obtained experimentally with a metabolic model. The present work reviews the ingredients necessary for a dynamic metabolic modeling study, with particular emphasis on practical issues. PMID- 16677961 TI - The challenge of intentional tanning in teens and young adults. AB - Changing UV exposure in adolescents and young adults has the potential to prevent years of excess exposure and subsequent skin cancer. Initial expectations were that an individual or media campaign informational approach would change this behavior. It is being recognized that current efforts have failed. Changing UV behaviors is challenging and has much in common with changing other health risk behaviors. The tanning industry benefits from recruiting new participants in this risky behavior. Research is just beginning to show some promising approaches, and will benefit from our experience attempting to change other risky behaviors. PMID- 16677962 TI - Antiandrogen therapy for skin and hair disease. AB - Androgen hormones play an important role in common skin and hair conditions including acne vulgaris, hirsutism, and androgenetic alopecia. Blocking this androgen effect may lead to significant improvements in these conditions. Several medications that work through a variety of different mechanisms may be prescribed safely and effectively as antiandrogen therapies in the dermatology arena. PMID- 16677963 TI - Vulvar disease pearls. AB - The vulva is a neglected area of the skin, hidden away by ignorance, embarrassment, and taboo with the result that women with vulvar diseases, desperate for help, suffer needlessly and waste millions of dollars on inadequate or inappropriate treatment. As the skin experts, dermatologists are vital for vulvar care. This article covers a range of subjects from normal anatomy to atypical presentations of common infections, such as candidiasis and herpes simplex virus. Tips are presented on the management of common vulvar dermatoses and there is a brief overview of pearls in diagnosis and therapy. Dermatologists are uniquely qualified to care for this area. PMID- 16677964 TI - Human papillomaviruses and genital disease. AB - Human papillomavirus (HPV) infection is causally related to several benign and malignant diseases of the anogenital tract. In this article the authors detail the epidemiology, methods of transmission and risk factors, pathogenesis, and oncogenesis of HPV, and describe clinical manifestations and current treatments. Special attention is given to condyloma acuminatum and non-cervical anogenital intraepithelial neoplasia. The authors conclude with the latest information on prophylactic vaccine development and prospects for future control of HPV-related disease. PMID- 16677965 TI - The use of dermatologic drugs in pregnancy and lactation. AB - Physicians are generally reluctant to prescribe dermatologic drugs to pregnant or nursing women because treatment is often elective and can be harmful to the patient, her fetus, or nursing infant; concerns for potential litigation also give pause. Yet, some effective dermatologic drugs have been determined to be safe during pregnancy and lactation. Of great practicality is an easily accessed reference guide that condenses information on such drugs into a set of tables that list pregnancy and teratogenicity ratings. Indications and contraindications are ordered in relation to the phases and trimesters of pregnancy. A discussion of the necessity of considering stages of childbearing in choosing drug therapies introduces the tables. A summary list of cautions spells out the steps physicians should take in treating women of childbearing age. PMID- 16677966 TI - Nevi and melanoma in pregnancy. AB - The influence of pregnancy on melanocytic nevi and malignant melanoma (MM) continues to be a controversial issue. Over the past 50 years, case reports and uncontrolled studies have suggested that nevi are more likely to become malignant during pregnancy, and MM diagnosed during pregnancy has a poor prognosis. Clinical and laboratory observations suggested that pregnancy-associated hormones may influence nevi and MM. Recent clinical and laboratory evidence suggests, however, that pregnancy does not influence the prognosis of MM, nor does it seem to cause significant changes in nevi. A clear link between hormones and MM has not been established. An analysis of this evidence is presented along with practical recommendations for the patient. PMID- 16677967 TI - Pregnancy and dermatologic surgery. AB - The physiologic changes of pregnancy and risks to the fetus require attention during dermatologic surgery. Elective surgery should be performed in the second trimester or the postpartum period. Cosmetic work should occur after delivery to avoid hypertrophic or hyperpigmented scars. Skin preparatory agents and anesthetics may have fetal implications and should be chosen with care. Antibiotic selection for any infections must take into account possible maternal and fetal risks. Attention to detail and awareness of the changes in pregnancy should lead to safe surgery in the pregnant patient. PMID- 16677968 TI - Allergic contact dermatitis to cosmetics. AB - Cosmetics are used to enhance one's appearance and are used by women worldwide. This article reviews the epidemiology, clinical features, and management of cosmetic allergic contact dermatitis. Additionally, several important cosmetic related allergens are discussed. PMID- 16677969 TI - Nail diseases related to nail cosmetics. AB - The use of nail care products and procedures to beautify and groom the nails is extremely common. Unfortunately, when improperly used, nail cosmetics can lead to nail diseases, such as paronychia, onycholysis, and brittle nails. Understanding the pathophysiology behind these conditions is an essential step toward better treatment and future prevention allowing patients to enjoy nail adornments while maintaining healthy nails. PMID- 16677970 TI - Osteoporosis in health and disease: a dermatologist's perspective. AB - Osteoporosis affects more than 75 million people in the United States, Asia, and Europe. Osteoporosis results from a combination of genetic and environmental factors that contribute to peak bone mass and the rate of bone loss. These factors include medications, diet, race, sex, lifestyle, and physical activity. The management of osteoporosis can be complicated by simultaneous treatment of other diseases. For dermatologists, the use of long-term oral corticosteroids for the management of immunobullous and blistering diseases is of particular importance. Although these treatment modalities are potentially lifesaving for patients, the side effects can be dangerous. This article examines the pathophysiology, clinical course, and treatments for osteoporosis and the special considerations that arise when managing patients using corticosteroids. PMID- 16677971 TI - Body dysmorphic disorder. AB - Body dysmorphic disorder is a relatively common psychiatric disorder among the dermatology patient population. These individuals may present to dermatologists for treatment of normal or minimally abnormal skin findings. Recognizing that these patients have body dysmorphic disorder can avert unnecessary and potentially unsuccessful treatments. Understanding and directing patients toward appropriate treatment options can minimize the distress and morbidities associated with the disorder. PMID- 16677972 TI - Women's occupational dermatologic issues. AB - With almost 65 million women in the workforce, their representation in various occupational sectors is crucial, as is their health and safety. Female-dominated occupations in health care and cosmetology along with their potential cutaneous exposures are reviewed. Proper evaluation and management are necessary to minimize the disability that can result from these illnesses. PMID- 16677973 TI - Dermatologic problems of older women. AB - Women are living longer today, composing the majority of persons aged 65 and over. Their dermatologic needs are unique and cross ethnic and cultural lines. With this increased life expectancy comes an increased occurrence of skin disorders. The identification and treatment of these conditions is important for the practicing clinician. This article reviews some of the more common dermatologic disorders of older women, and discusses the latest treatments and issues facing this geriatric population. PMID- 16677974 TI - Etiologies of acute hypoglycemia in a Taiwanese hospital emergency department. AB - The etiologies of acute hypoglycemia (< 2.8 mmol/L) in adult emergency patients were assessed to provide more proper and prompt management. There were 228 hypoglycemic patients (112 women and 116 men, ranging in age from 22 to 93 years, mean = 69.6 years) identified for the study. These patients had hypoglycemia mainly due to excessive use of sulfonylureas or insulin. There was a diabetic history in 182 patients (79.83%). Other primary etiologies of acute hypoglycemia were sepsis in 13 (5.70%), and extensive liver disease in 13 (5.70%). This study indicates that good diabetic control can dramatically decrease the number of episodes of acute hypoglycemia in Taiwan. For acute hypoglycemic patients without a diabetic history, the possibility of sepsis or extensive liver disease is a problem in Taiwan. PMID- 16677975 TI - Alcohol-related problems: emergency physicians' current practice and attitudes. AB - To determine whether emergency physicians' (EPs) attitudes affect their support and practice of brief intervention in the Emergency Department (ED), EPs completed an anonymous survey. EPs were asked about their attitudes toward patients with alcohol problems, current ED screening, use of brief intervention, and barriers to use of brief intervention. Chi-square analysis was used and a step-wise regression model was constructed. Respondents reported a high prevalence of patients with alcohol-related problems: 18% in a typical shift. Eighty-one percent said it is important to advise patients to change behavior; half said using a brief intervention is important. Attending physicians had significantly less alcohol education than residents, but were significantly more likely to support the use of brief intervention. Support was not associated with gender, race, census, hours of education, or personal experience. EPs who felt that brief intervention was an integral part of their job were more likely to use it in their daily practice. PMID- 16677976 TI - Data-driven quality improvement in the Emergency Department at a level one trauma and tertiary care hospital. AB - To demonstrate how a comprehensive and internally driven Continuous Quality Improvement (CQI) program was designed and implemented in our Emergency Department (ED) in 1999. This program involved monthly data collection and analysis, data-driven process change, staff education in the core concepts of quality, and data reanalysis. Data components collected during the program included census data, physician profiling, and focused clinical audits. CQI measures collected at the beginning of the program and quarterly included: (1) CQI metric data (turnaround times [TAT] and rates of left against medical advice [AMA] or left without being seen [LWOBS]), (2) rates and nature of patient complaints, and (3) results of patient satisfaction surveys performed by an outside consulting firm contracted by hospital administration. During the 4 years since its implementation the program demonstrated improvement in all measured areas. Despite an increase in patient volume of 32% to nearly 37,000 visits/year, and only minimal staffing adjustments, the mean quarterly TAT decreased from 183 min to 165 min (9.8% decrease), the rate of complaints dropped by 56.1% (2.1 per 1000 patients to 0.92), and patients leaving AMA or LWOBS decreased 66.7% from 2.7% to 0.9%. Overall, 44.8% of ED patients rated their care as "excellent." In summary, we demonstrate how a comprehensive quality improvement program was structured and implemented at a tertiary care center and how such a program demonstrated improvement in specific CQI parameters. PMID- 16677978 TI - A case of conversion disorder presenting as a severe acute stroke. AB - Conversion disorders often present with dramatic physical presentations suggestive of severe organic disease. We present the case of a young woman who presented to the Emergency Department with a dense left hemiparesis suggestive of a severe acute stroke. Emergent referral to a regional stroke center facilitated rapid medical evaluation, exclusion of organic disease, and confirmation of conversion disorder as the etiology for the symptoms. This report highlights the dramatic clinical presentations that may result from conversion disorders as well as the benefits of rapid medical evaluation by specialty stroke centers. PMID- 16677977 TI - Diagnostic value of the helical CT scan for traumatic aortic injury: correlation with mortality and early rupture. AB - To evaluate the value of helical computed tomography of the thorax (HCTT) as a definitive tool for diagnosing traumatic aortic injury, this study retrospectively examined 53 patients with blunt thoracic injuries and HCTT during a 5-year period. All CT scans were reviewed for direct signs of aortic injury and correlated with aortography or surgery. Correlations between clinical parameters, as well as combinations of direct signs and individual signs, and mortality were analyzed in all traumatic aortic injury (TAI) cases. Direct signs were seen on the HCTT in 25 cases and 22 had TAI. All false positive results came from the group with only a single direct sign depicted on HCTT. Among individual direct signs examined, intimal flap and luminal thrombus were the most specific (100%), whereas irregular aortic contour was the most sensitive (100%). A combination of > or = 3 direct signs (p = 0.006) and periaortic contrast material extravasation significantly correlated with early rupture and mortality (p = 0.002). In conclusion, intimal flap on HCTT is both the most specific and sensitive sign for TAI. TAI patients with > or = 3 direct signs, including periaortic contrast material extravasation, may not require aortography before immediate surgery. PMID- 16677979 TI - Pelvic tumor presenting as chronic back pain in a young adult. AB - A large proportion of Emergency Department visits are for chronic pain, specifically for chronic back pain. Often this entity requires minimal diagnostics. However, certain populations and presentations require a higher vigilance and a more detailed evaluation. This report describes the clinical presentation of a young adult with chronic back pain who exhibited new symptoms and subtle examination findings that expedited the ultimate diagnosis of pelvic chondrosarcoma. PMID- 16677980 TI - Sudden death in a young woman from medium chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. AB - Medium chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is an inherited disorder of fatty acid metabolism that usually presents in early childhood. This case report describes a 19-year-old woman who presented with lethargy, disorientation, and vomiting. She had a cardiopulmonary arrest from which she could not be resuscitated 24h after the onset of the illness. Pre-mortem blood studies confirmed MCAD deficiency. An MCAD deficiency and other metabolic disorders lie within the differential diagnosis of a patient presenting with acutely altered mental status. The inheritance of MCAD deficiency and its clinical presentation, pathophysiology, treatment, and prevention are discussed. PMID- 16677981 TI - Incidental finding of Chiari I malformation with progression of symptoms after head trauma: case report. AB - Chiari I malformation (CMI) is a neurologic structural anomaly involving cerebellar herniation through the foramen magnum. It is most commonly diagnosed in adult patients who are often asymptomatic. Although there are several case reports of sudden death and neurologic deficits after minor head trauma in patients with CMI, little is known about the effects of trauma on CMI. This case describes a woman with chronic headaches who had an incidental finding of CMI with obstructive hydrocephalus and progression of symptoms after minor head trauma. PMID- 16677982 TI - Antiemetic-related dystonic reaction unmasked by removal of a scopolamine transdermal patch. AB - A case of a dystonic reaction is presented that occurred after the use of prochlorperazine, metoclopramide and ondansetron for the treatment of post operative nausea and vomiting. The onset of dystonia coincided temporally with the removal of a transdermal scopolamine patch used as adjunctive antiemetic therapy. Withdrawal of concurrently administered anticholinergic medication, after recent use of antiemetic medications with dopamine receptor (D2) inhibition, can unmask a dystonic reaction. This case also suggests that transdermal scopolamine may offer an innovative therapy for the treatment of acute dystonic reactions. PMID- 16677983 TI - Mucocele of the appendix: an important clinical rarity. AB - Mucocele is an uncommon pathology of the vermiform appendix that can be confused with acute appendicitis. We present a case of an appendiceal mucocele associated with subacute, intermittent right lower quadrant discomfort. The diagnosis of appendiceal mucocele is an important one in that it can be associated with malignancies and other serious gastrointestinal, ovarian, and urological complications. PMID- 16677984 TI - Pregnancy-associated coronary artery dissection: a case report. AB - We describe the case of a 38-year-old woman who presented with a chief complaint of chest heaviness approximately 2 weeks postpartum. She was otherwise healthy with no cardiac risk factors. Coronary angiography revealed a spiral dissection of the left main coronary artery that extended to the left anterior descending coronary artery. Pregnancy-associated coronary artery dissection is a disease that has in the past carried a high mortality rate. Even today many women die before reaching medical help. Early diagnosis and intervention are crucial. Percutaneous transluminal coronary angioplasty for single vessel disease and coronary artery bypass grafting for multi-vessel disease are becoming the treatments of choice. PMID- 16677985 TI - Acute olanzapine-induced akathisia and dystonia in a patient discontinued from fluoxetine. AB - The patient with acute extrapyramidal signs and symptoms presents a significant clinical challenge. We present the case of a young man who developed an acute akathisia and dystonia after inadvertent overdose of olanzapine (Zyprexa) in the setting of a recent discontinuation of fluoxetine. The receptor chemistry and mechanisms pertinent to his presentation are reviewed. An analysis of the literature indicates that a broad incidence range is cited for the extrapyramidal effects of these medications. We suggest a diagnostic and therapeutic approach to the undifferentiated patient presenting with extrapyramidal signs and symptoms. The possibility of neuroleptic malignant syndrome (NMS), serotonin syndrome (SS), tricyclic overdose, and cocaine abuse should be considered in a patient with extrapyramidal signs and symptoms, given the potential for complications. An emphasis is placed on the need for carefully verbalized discharge instructions to avoid a potential untoward outcome. PMID- 16677986 TI - Elapid snakebite as a cause of severe hypertension. AB - Snakebite is a common medical emergency in tropical countries, however, autonomic dysfunction is an uncommon manifestation of snakebite. The authors present an unusual manifestation of severe neurotoxic snake envenomation: severe hypertension requiring intravenous antihypertensives. A review of the literature on autonomic dysfunction in neurotoxic snake envenomation is also presented. PMID- 16677987 TI - Ischemic colitis in marathon runners: a case-based review. AB - In the United States over 450,000 participants finished a marathon in 2002. Some of them will present to an Emergency Department (ED) with a variety of gastrointestinal complaints. The diagnosis of ischemic colitis should be considered in patients who present with bloody diarrhea. We describe three patients who presented to our ED with abdominal pain and bloody diarrhea after a marathon. Gastrointestinal complaints with emphasis on mesenteric ischemia and ischemic colitis are discussed. PMID- 16677988 TI - Diagnosis of diverticulitis by bedside ultrasound in the Emergency Department. AB - This case report describes a potential novel indication for the use of bedside ultrasound in the Emergency Department. The patient in this case had some of the signs and symptoms of diverticulitis. The "pseudo-kidney" sign, which was thought to represent acute diverticulitis, was appreciated on a rapid, bedside ultrasound and confirmed by computed tomography. Knowledge of the sonographic appearance of diverticulitis may aid emergency physicians in making this diagnosis promptly, and facilitate the appropriate disposition. PMID- 16677989 TI - Synovial fluid analysis. AB - AsA prompt and accurate diagnosis of a painful, swollen joint is imperative, primarily in the case of a septic joint, as delayed therapy may result in progression of disease or permanent loss of function. Procurement and analysis of synovial fluid (SF) are paramount in helping the clinician to determine a patient's clinical condition and further course of treatment. Measurement of white blood cell (WBC) counts, crystal analysis by polarized microscopy, and microbiologic studies including Gram stain and culture are the SF parameters that are collectively most important in the ultimate determination by a clinician of the presence or absence of an infectious or inflammatory joint. It is important for the clinician to understand and recognize the limitations of various SF parameters to minimize under-treating patients with potentially serious joint pathology. PMID- 16677990 TI - Weakness and mental status change. PMID- 16677991 TI - Stingray envenomation. PMID- 16677992 TI - A striking foreign body. PMID- 16677993 TI - Emergency Department overcrowding and ambulance diversion: the impact and potential solutions of extended boarding of admitted patients in the Emergency Department. AB - Emergency Department (ED) crowding and ambulance diversion have been increasingly significant national problems for more than a decade. Surveys of hospital directors have reported overcrowding in almost every state and 91% of hospital ED directors report overcrowding as a problem. The problem has developed because of multiple factors in the past 20 years, including a steady downsizing in hospital capacity, closures of a significant number of EDs, increased ED volume, growing numbers of uninsured, and deceased reimbursement for uncompensated care. Initial position statements from major organizations, including JCAHO and the General Accounting Office, suggested the problem of overcrowding was due to inappropriate use of emergency services by those with no urgent conditions, probably cyclical, and needed no specific policy response. More recently, the same and other organizations have more forcefully highlighted the problem of overcrowding and focused on the inability to transfer emergency patients to inpatient beds as the single most important factor contributing to ED overcrowding. This point has been further solidified by initial overcrowding research. This article will review how overcrowding occurred with a focus on the significance and potential remedies of extended boarding of admitted patients in the Emergency Department. PMID- 16677995 TI - Vasopressin in hypotensive and shock states. AB - Clinical reports and experimental studies support the beneficial effects of low dose vasopressin infusions in vasodilatory shock. Before we can recommend vasopressin for routine clinical use in vasodilatory shock, and particularly septic shock, we must await the results of currently ongoing and recently completed randomized clinical trials to ensure that vasopressin does indeed have beneficial effects on organ function and outcome. PMID- 16677996 TI - Contemporary issues in the pharmacologic management of acute heart failure. AB - Acute heart failure is an evolving syndrome that continues to be defined by ongoing studies and registries. It is associated with significant morbidity and mortality and places a huge economic burden on health care systems. Improved understanding of the underlying pathophysiologic processes has prompted interest into understanding the implications of current and future pharmacologic management strategies beyond hemodynamics. Diuretics, vasodilators, and inotropes remain the mainstays of therapy with several new classes of agents on the horizon. Clinicians should understand the rationale for use and limitations of each therapy to maximize benefit and cost-effectiveness, while minimizing the potential for adverse outcomes. PMID- 16677997 TI - Effect of vasoactive therapy on cerebral circulation. AB - Many questions regarding blood pressure management after acute stroke remain unanswered, resulting in an ongoing debate about whether to treat hypertension acutely and how aggressively blood pressure should be lowered. This review discusses normal and altered cerebrophysiology and provides evidence supporting and opposing the active management of blood pressure within the first 24 hours after stroke. Commonly used intravenous antihypertensive agents and their cerebrovascular effects are reviewed, and therapeutic recommendations are given based on the available evidence. PMID- 16677998 TI - Corticosteroid replacement in critically ill patients. AB - This review addresses the use of corticosteroid replacement in critically ill patients. Low-dose corticosteroid replacement for critically ill patients with septic shock has been shown to reduce the duration of vasopressor-dependent shock, to shorten ICU length of stay, and, in recent trials, to reduce mortality. Numerous questions remain to be fully answered about patient selection, corticotropin-stimulation testing methods, and interpretation of results. PMID- 16677999 TI - Pharmacokinetic changes in critical illness. AB - Physiologic alterations in critically ill patients can significantly affect the pharmacokinetics of drugs used in the critically ill patient population. Understanding these pharmacokinetic changes is essential relative to optimizing drug therapy. This article outlines the major differences seen in the absorption, distribution, metabolism, and excretion of drugs in critically ill patients. Important strategies for drug therapy dosing and monitoring in these patients are also addressed. PMID- 16678000 TI - Principles and practices of medication safety in the ICU. AB - Medication errors are a significant public health problem in United States hospitals. Patients in the ICU are at particular risk for medication errors because of the characteristics of an ICU and the nature of its patients. This article reviews the principles of medication safety and applies these principles to the ICU, and suggests safe practices to improve medication safety in the ICU. PMID- 16678001 TI - Antimicrobial resistance: factors and outcomes. AB - Antimicrobial resistance in the ICU is characterized by increasing overall resistance rates among gram-negative and gram-positive pathogens and increased frequency of multidrug-resistant organisms. In addition to basic principles of appropriate drug selection for empiric and definitive therapy, other specific strategies that may decrease problems of resistance through improved use of antimicrobials include appropriate application of pharmacokinetic and pharmacodynamic principles to antimicrobial use, aggressive dosing of antimicrobials, use of broad-spectrum and combination antimicrobial therapy for initial treatment, decreased duration of antimicrobial therapy, hospital formulary-based antimicrobial restrictions, use of antimicrobial protocols and guidelines, programs for restriction of target antimicrobials, scheduled antimicrobial rotation, and use of antimicrobial management programs. Combinations of various approaches may offer the best potential for effectively intervening in and reducing the spread of resistant pathogens in critically ill patients. PMID- 16678002 TI - Sedative and analgesic medications: risk factors for delirium and sleep disturbances in the critically ill. AB - Sedatives and analgesics are routinely used in critically ill patients, although they have the potential for side effects, such as delirium and sleep architecture disruption. Although it should be emphasized that these medications are extremely important in providing patient comfort, health care professionals must also strive to achieve the right balance of sedative and analgesic administration through greater focus on reducing unnecessary or overzealous use. Ongoing clinical trials should help us to understand whether altering the delivery strategy, via daily sedation interruption, or protocolized target-based sedation or changing sedation paradigms to target different central nervous system receptors can affect cognitive outcomes and sleep preservation in our critically ill patients. PMID- 16678003 TI - Drug-associated disease: cytochrome P450 interactions. AB - Critically ill patients generally are older, frequently have organ failure, and commonly receive multiple medications, all of which make them susceptible to adverse effects of drugs. Drug interactions are a common adverse effect, and many are predictable based on understanding the mechanisms that underlie drug interactions. This article identifies commonly used medications in critically ill patients and the associated drug interactions that may occur with emphasis on the cytochrome P450 enzyme system. PMID- 16678004 TI - Drug-associated disease: hematologic dysfunction. AB - Hematologic dysfunction, including thrombocytopenia, anemia, neutropenia, thromboses, and coagulopathy, occur commonly during critical illnesses. A major challenge is to identify drug-induced causes of hematologic dysfunction. Given the wide variety of drug-induced hematologic effects, clinicians always should consider any concomitant drugs in the differential diagnosis of acquired hematologic dysfunction. The most severe effects include drug-induced aplastic anemia, heparin-induced thrombocytopenia, and drug-induced thrombotic microangiopathy. Certain drugs are associated with multiple hematologic effects. For example, cisplatin can cause hemolytic uremia syndrome and erythropoietin deficiency, and quinine can precipitate immune-mediated thrombocytopenia, immune mediated thrombocytopenia, and thrombotic microangiopathy. PMID- 16678005 TI - Drug-associated renal dysfunction. AB - Acute renal failure (ARF) in patients in the ICU is associated with a high mortality. Drug-induced renal dysfunction is an important, yet often overlooked, cause of ARF in this patient population. A drug use evaluation at the authors' institution, to assess the prescribing patterns of potential nephrotoxins in the adult and pediatric ICUs, found that antibiotics (aminoglycosides, amphotericin B, penicillins, cephalosporins, acyclovir), nonsteroidal anti-inflammatory drugs, contrast dye, and various other nephrotoxic medications are used widely in all of the ICUs. By focusing on several commonly prescribed classes of nephrotoxic medications in the ICU, this article reviews the general mechanisms of drug associated renal dysfunction. PMID- 16678007 TI - Combating oxidative stress at respiratory tract biosurfaces: challenges yet to be resolved, a commentary on "Vitamin supplementation does not protect against symptoms in ozone-responsive subjects". PMID- 16678006 TI - Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases. AB - Obstructive sleep apnea (OSA) has emerged as a major public health problem and increasing evidence indicates that untreated OSA can lead to the development of various cardiovascular disorders. One important mechanism by which OSA may promote cardiovascular diseases is intermittent hypoxia, in which patients are subjected to repeated episodes of brief oxygen desaturation in the blood, followed by reoxygenation. Such cycles of hypoxia/reoxygenation may result in the generation of reactive oxygen species. Some studies have demonstrated the presence of oxidative stress in OSA patients as well as in animals subjected to intermittent hypoxia. Further, modulations of nitric oxide and biothiol status might also play important roles in the pathogenesis of OSA-associated diseases. Reactive oxygen species and redox events are also involved in the regulation of signal transduction for oxygen-sensing mechanisms. This review summarizes currently available information on the evidence for and against the occurrence of oxidative stress in OSA and the role of reactive oxygen species in cardiovascular changes associated with OSA. PMID- 16678008 TI - Oxidative stress, microvascular dysfunction, and scleroderma: an association with potential therapeutic implications, a commentary on "Postocclusive reactive hyperemia inversely correlates with urinary 15-F2t-isoprostane levels in systemic sclerosis". PMID- 16678009 TI - Commentary on "The overlapping of local iron overload and HFE mutation in venous leg ulcer pathogenesis". PMID- 16678010 TI - Apomorphine-induced myocardial protection is due to antioxidant and not adrenergic/dopaminergic effects. AB - Apomorphine (Apo), a dopaminergic agonist used for treatment of Parkinson disease, is a potent antioxidant. In addition to its antioxidative effects, the dopaminergic and adrenergic effects of Apo were studied. Isolated perfused rat hearts were exposed to 25 min of no-flow global ischemia (37 degrees C) and 60 min of reperfusion (I/R, control). Drugs were introduced for the first 20 min of reperfusion. The LVDP of the control group recovered to 54.6 +/- 3.3%. Apo treated hearts had significantly improved recovery (61.6 +/- 5%, p < 0.05). The recovery of the work index LVDP x HR was even bigger: 67.8 +/- 3.7% (Apo treatment) vs 41.7 +/- 4.6% (control, p < 0.001). Haloperidol, a dopaminergic antagonist, did not affect the recovery with Apo. Propranolol, a beta-adrenergic blocker, initially inhibited the effect of Apo. However, the recovery of the combined group (Apo + propranolol) increased and reached significance (LVDP, p < 0.05 vs control group) after cessation of propranolol perfusion. At 60 min of reperfusion this group was superior to Apo-treated hearts (LVDP, p < 0.05). Propranolol (without Apo) did not improve the hemodynamic recovery. The same pattern of recovery applies also to the recovery of the +dP/dt during the reperfusion. L-DOPA was less effective than Apo. I/R caused significant increase in carbonylation of proteins. Apomorphine inhibited the increase in carbonylation. Haloperidol did not affect this beneficial effect of Apo. L-DOPA significantly decreased the carbonylation of proteins. We conclude that the antioxidative effect of Apo is its main mechanism of cardioprotection. PMID- 16678011 TI - Thioredoxin protects against joint destruction in a murine arthritis model. AB - Thioredoxin (TRX) is an oxidative stress-inducible biological antioxidant that is highly expressed in the synoviocytes of rheumatoid arthritis (RA) patients. There is much evidence that oxidative stress plays a key role in the inflammation and destruction of RA joints; the functional relationship between TRX and RA remains unknown, however. We therefore investigated the role played by TRX in the inflammatory and joint-damaging processes of RA using a murine model in which arthritis was induced by administering a mixture of anti-type II collagen monoclonal antibodies (mAb) and lipopolysaccharide (LPS). In Wt mice mAb/LPS injection induced neutrophil infiltration, cartilage destruction, and chondrocyte apoptosis within the joints, all of which were dramatically suppressed in TRX transgenic (TRX-Tg) mice. Moreover, the 8-hydoxy-2'-deoxyguanosine (8-OHdG) expression seen in Wt mice after mAb/LPS injection was almost completely inhibited in TRX-Tg mice. The administration of recombinant TRX also suppressed mAb/LPS-induced joint swelling in Wt mice. Taken together, these results suggest that TRX protects against arthritis and is a plausible candidate with which to develop novel therapies for the treatment of RA. PMID- 16678012 TI - Postocclusive reactive hyperemia inversely correlates with urinary 15-F2t isoprostane levels in systemic sclerosis. AB - Microvascular dysfunction and increased oxidative stress are major hallmarks of the systemic sclerosis disease process. The primary objective of this study was to test whether there is a link between peak postocclusive hyperemia and urinary levels of the F2-isoprostane 15-F2t-IsoP (8-iso-PGF2alpha) in patients suffering from systemic sclerosis. We enrolled 43 patients suffering from systemic sclerosis, 33 patients with primary Raynaud's phenomenon (RP), and 25 healthy volunteers. Microvascular function was assessed using the postocclusive hyperemia monitored by laser Doppler flowmetry. Endothelium-independent response was monitored after 0.4 mg sublingual nitroglycerin. Oxidative stress status was assessed by urinary levels of the F2-isoprostane 15-F2t-IsoP using GC-MS. The peak postocclusive vascular conductance was altered in subjects with systemic sclerosis and primary RP compared to controls (respectively 28 (7-48), 30 (13 48), and 39.9 (13-63) mV/mm Hg, p = 0.01). F2-isoprostanes were increased in the systemic sclerosis group compared to primary Raynaud's phenomenon and healthy controls (respectively 230 (155-387), 182 (101-284), and 207 (109-291) pg/mg, p = 0.006). In patients suffering from systemic sclerosis, there was a significant inverse correlation between F2-isoprostanes and postocclusive hyperemia, expressed as raw data (R = -0.45, p = 0.007) or as an increase over baseline (R = -0.28, p = 0.04). Conversely, no correlation was found with the nitroglycerin response. In conclusion, we provide evidence that there is an inverse correlation between postocclusive hyperemia and urinary F2-isoprostane levels in patients suffering from systemic sclerosis. Whether oxygen free radicals initiate the vascular dysfunction or whether there is an initial trigger that initiates both processes will need to be further clarified in future studies. PMID- 16678013 TI - PI3K-dependent lysosome exocytosis in nitric oxide-preconditioned hepatocytes. AB - We investigated the signal mediators and the cellular events involved in the nitric oxide (NO)-induced hepatocyte resistance to oxygen deprivation in isolated hepatocytes treated with the NO donor (Z)-1-(N-methyl-N-[6-(N methylammoniohexyl)amino])diazen-1-ium-1,2-diolate (NOC-9). NOC-9 greatly induced PI3K activation, as tested by phosphorylation of PKB/Akt. This effect was prevented by either 1H-(1,2,4)-oxadiazolo-(4,3)-quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC), or KT5823, an inhibitor of cGMP-dependent kinase (cGK), as well as by farnesyl protein transferase inhibitor, which blocks the function of Ras GTPase. Bafilomycin A, an inhibitor of the lysosome-type vacuolar H+-ATPase, cytochalasin D, which disrupts the cytoskeleton-dependent organelle traffic, and wortmannin, which inhibits the PI3K-dependent traffic of lysosomes, all abolished the NOC-9-induced hepatocyte protection. The treatment with NOC-9 was associated with the PI3K-dependent peripheral translocation and fusion with the plasma membrane of lysosomes and the appearance at the cell surface of the vacuolar H+-ATPase. Inhibition of sGC, cGK, and Ras, as well as the inhibition of PI3K by wortmannin, prevented the exocytosis of lysosomes and concomitantly abolished the protective effect of NOC-9 on hypoxia-induced pHi and [Na+]i alterations and cell death. These data indicate that NO increases hepatocyte resistance to hypoxic injury by activating a pathway involving Ras, sGC, and cGK that determines PI3K-dependent exocytosis of lysosomes. PMID- 16678014 TI - Iron-binding antioxidant capacity is impaired in diabetes mellitus. AB - Increased lipid peroxidation contributes to diabetic complications and redox active iron is known to play an important role in catalyzing peroxidation reactions. We aimed to investigate if diabetes affects the capacity of plasma to protect against iron-driven lipid peroxidation and to identify underlying factors. Glycemic control, serum iron, proteins involved in iron homeostasis, plasma iron-binding antioxidant capacity in a liposomal model, and non transferrin-bound iron were measured in 40 type 1 and 67 type 2 diabetic patients compared to 100 nondiabetic healthy control subjects. Iron-binding antioxidant capacity was significantly lower in the plasma of diabetic subjects (83 +/- 6 and 84 +/- 5% in type 1 and type 2 diabetes versus 88 +/- 6% in control subjects, p < 0.0005). The contribution of transferrin, ceruloplasmin, and albumin concentrations to the iron-binding antioxidant capacity was lost in diabetes (explaining only 4.2 and 6.3% of the variance in type 1 and type 2 diabetes versus 13.9% in control subjects). This observation could not be explained by differences in Tf glycation, lipid, or inflammatory status and was not associated with higher non-transferrin-bound iron levels. Iron-binding antioxidant capacity is decreased in diabetes mellitus. PMID- 16678015 TI - Antioxidant pyrrolidine dithiocarbamate activates Akt-GSK signaling and is neuroprotective in neonatal hypoxia-ischemia. AB - Pyrrolidine dithiocarbamate (PDTC), an antioxidant and inhibitor of transcription factor nuclear factor kappa-B (NF-kappaB), has been reported to reduce inflammation and apoptosis. Because PDTC was recently found to protect in various models of adult brain ischemia with a wide therapeutic time window, we tested the effect of PDTC in a rodent model of neonatal hypoxia-ischemia (HI) brain injury. T2-weighed magnetic resonance imaging (T2-MRI) 7 days after the insult showed that a single PDTC (50 mg/kg) injection 2.5 h after the HI reduced the mean brain infarct size by 59%. PDTC reduced the HI-induced dephosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta), expression of cleaved caspase-3, and nuclear translocation of NF-kappaB in the neonatal brain. PDTC targeted directly neurons, as PDTC reduced hypoxia-reoxygenation-induced cell death in pure hippocampal neuronal cultures. It is suggested that in addition to the previously indicated NF-kappaB inhibition as a protective mechanism of PDTC treatment, PDTC may reduce HI-induced brain injury at least partially by acting as an antioxidant, which reduces the Akt-GSK-3beta pathway of apoptotic cell death. The clinically approved PDTC and its analogues may be beneficial after HI insults with a reasonable time window. PMID- 16678017 TI - Plasma very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein oxidative modification induces procoagulant profiles in endogenous hypertriglyceridemia. AB - This study was to investigate whether oxidatively modified lipoproteins were associated with changes of pro- and anticoagulant profiles in hypertriglyceridemic subjects. Plasma VLDL, LDL, and HDL were isolated with the one-step density gradient ultracentrifugation method. The oxidation of the lipoproteins was identified. Prothrombin time (PT) and activated partial thrombplastin time (APTT), tissue plasminogen activator and plasminogen activator inhibitor-1, and platelet aggregation rate were determined with a reaction system consisting of mixed fresh normal plasma, in endogenous hypertriglyceridemic (HTG) patients, in in vitro modified lipoproteins from a normolipidemic donor, and in experimental rats. The results indicated that oxVLDL, oxLDL, and oxHDL occurred in the plasma of HTG patients. Compared with the control group, PT and APTT, incubated with plasma VLDL, LDL, or HDL from HTG patients, respectively, were significantly reduced, while platelet maximal aggregation rates were significantly higher (P < 0.05-0.01). Similar procoagulant profiles were observed in in vitro modified lipoprotein components and in rats with intrinsic hypertriglyceridemia as well. These results support our previous finding that LDL, VLDL, and HDL were all oxidatively modified in vivo in the subjects with HTG, and suggest that procoagulation state may result from the abnormal plasma lipoprotein oxidative modification in vivo. PMID- 16678016 TI - NOX1/NADPH oxidase negatively regulates nerve growth factor-induced neurite outgrowth. AB - Reactive oxygen species produced by NADPH oxidase are involved in the neuronal death associated with various neurodegenerative disorders. However, the role of NADPH oxidase in neuronal differentiation has not been well characterized. In nondifferentiated PC12 cells, the mRNA level of NOX1, a catalytic subunit of NADPH oxidase expressed in nonphagocytes, was approximately 10 times higher than that of the phagocyte type subunit, NOX2 (gp91(phox)), while the transcript of another isoform, NOX4, was not detected. Following nerve growth factor (NGF) induced neurite outgrowth, the mRNA level of NOX1 and NOX2 was progressively increased and decreased, respectively. The NGF-induced increase in NOX1 mRNA was mediated by TrkA and accompanied by increased intracellular superoxide, which was suppressed by NADPH oxidase inhibitors. Unexpectedly, these inhibitors and superoxide scavengers significantly enhanced NGF-induced neurite outgrowth. Enhanced neurite outgrowth was similarly demonstrated in cells depleted with the NOX1 transcript by stable expression of ribozymes targeted for the NOX1 mRNA sequence. Furthermore, NGF-induced expression of betaIII-tubulin was significantly augmented in cells treated with NADPH oxidase inhibitors or stably expressing ribozymes. Phosphatidylinositol-3 (PI3) kinase inhibitors, without affecting NGF-induced NOX1 expression, augmented NGF-induced neurite outgrowth but not in clones expressing ribozymes. Taken together, increased superoxide production by up-regulation of NOX1 may negatively regulate neuronal differentiation by suppressing excessive neurite outgrowth. PMID- 16678018 TI - Antioxidant and redox properties of supramolecular complexes of carotenoids with beta-glycyrrhizic acid. AB - Supramolecular complexes between carotenoids and a triterpene glycoside, beta glycyrrhizic acid (GA), were found to exhibit unusual antioxidant activity. Complexation with GA increases a scavenging rate of canthaxanthin and 7',7' dicyano-7'-apo-beta-carotene toward OOH radicals more than 10 times, but has no effect on the scavenging rate of zeaxanthin. Scavenging rate constants were measured in DMSO solution of carotenoids using the EPR spin-trapping technique. EPR parameters of spin adducts were determined as a(H) = 2.3 G, a(N) = 13.9 G for PBN (N-tert-butyl-alpha-phenylnitrone)-OOH, and a(H) = 3.4 G, a(N) = 14.9 G for the PBN-CH3 adduct. Taking into account the previously measured dependence of the scavenging rate constants toward OOH radicals on the oxidation potential of carotenoids, this result can be explained by the hypothesis that the complexation with GA affects the value of oxidation potentials. This hypothesis was confirmed by CV measurements. PMID- 16678019 TI - Heme oxygenase-1 attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation. AB - Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme catabolism, is known to modulate various cellular functions, including cytokine production, cell proliferation, and apoptosis, in stress-related conditions. However, the role of HO-1 in the auditory system remains elusive. Herein, we demonstrate that pharmacologic induction of HO-1 along with catalytic activation significantly suppressed apoptosis of HEI-OC1 cells induced by cisplatin. Studies of ectopic expression of pcDNA3-HO-1 and siRNA of HO-1 further revealed the protective role of HO-1 against cisplatin in HEI-OC1 cells. Among the catabolic metabolites of HO 1, both carbon monoxide (CO) and bilirubin were directly involved in the protective role of HO-1 against cisplatin through inhibition of reactive oxygen species generation. Furthermore, pharmacological induction of HO-1 completely prevented the destruction of outer hair cell arrays by cisplatin through a CO dependent mechanism in organotrophic culture of the rat primary organ of Corti explants. These results suggest that HO-1 may serve as a safeguard of auditory sensory hair cells against a variety of challenges of oxidative stress, including noise trauma, presbycusis, and ototoxic drugs, respectively. PMID- 16678020 TI - Accumulation of macular xanthophylls in unsaturated membrane domains. AB - The distribution of macular xanthophylls, lutein and zeaxanthin, between domains formed in membranes made from an equimolar ternary mixture of dioleoylphosphatidylcholine/sphingomyelin/cholesterol, called a raft-forming mixture, was investigated. In these membranes, two domains are formed: the raft domain enriched in saturated lipids and cholesterol (detergent-resistant membranes, DRM), and the bulk domain enriched in unsaturated lipids (detergent soluble membranes, DSM). These membrane domains have been separated using cold Triton X-100 extraction from membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that xanthophylls are substantially excluded from DRM and remain concentrated in DSM. Concentrations of xanthophylls in DRM and DSM calculated as the mole ratio of either xanthophyll to phospholipid were 0.005 and 0.03, respectively, and calculated as the mole ratio of either xanthophyll to total lipid (phospholipid + cholesterol) were 0.003 and 0.025, respectively. Thus, xanthophylls are over eight times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. It was also demonstrated using saturation-recovery EPR that at 1 mol%, neither lutein nor zeaxanthin affect the formation of membrane domains. The location of xanthophylls in domains formed from unsaturated lipids is ideal if they are to act as a lipid antioxidant, which is the most accepted mechanism through which lutein and zeaxanthin protect the retina from age-related macular diseases. PMID- 16678021 TI - Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction. AB - Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF. PMID- 16678022 TI - BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2. AB - NAD(P)H/NRH:quinone oxidoreductases (NQO1 and NQO2) protect against oxidative stress and neoplasia. Cross-breeding of NQO1-/- with NQO2-/- mice generated double-knockout (DKO) mice. DKO mice were born normal yet showed myelogenous hyperplasia as observed in single-knockout mice. DKO mice also showed bronchial associated lymphoid tissue (BALT) that increased in number and size with age. BALT was absent in wild-type and single-knockout mice. Further analysis demonstrated infiltration of neutrophils and macrophages in BALT and significant increases in the serum cytokines TNFalpha, IL-6, and IL-1beta and increased expression of iNOS and higher nitric oxide in lung macrophages. The development of BALT in DKO mice presumably led to the release of cytokines and higher lung macrophage activation, because histologically spleen, thymus, and blood cultures and urine analysis showed absence of infection. Additionally, the DKO mice upon exposure to hyperoxia demonstrated severe intra-alveolar edema and perivascular inflammation and massive infiltration with neutrophils, compared with wild-type mice. These results suggest that NQO1 and NQO2 combined protect mice against lung inflammation, BALT, and hyperoxic lung injury. PMID- 16678023 TI - Mechanism of metalloid-induced death in Leishmania spp.: role of iron, reactive oxygen species, Ca2+, and glutathione. AB - There is growing evidence that metalloid-induced cell death in protozoan parasites is due to oxidative injury; however, the biochemical changes related to this event are not fully understood. Leishmania spp. demonstrated cross resistance to two related metalloids, arsenic and antimony, and both metalloids induced cell death accompanied by cell shrinkage and DNA fragmentation that was preceded by an increase in reactive oxygen species. Both drugs caused mitochondrial dysfunction in terms of loss of membrane potential and a drop in ATP levels. Arsenic treatment resulted in an elevation of intracellular Ca2+ levels that did not occur with antimony exposure. Cellular glutathione level was reduced after antimony treatment but arsenic did not affect glutathione. Inhibition of Ca2+ influx during arsenic treatment reduced cell death, whereas supplementation of glutathione during antimony treatment rescued cell loss. Under iron-depleted conditions, the cytotoxic effects of arsenic and antimony did not occur and cell survival increased; in contrast, the presence of excess iron resulted in higher cell death. Therefore, this study provides a new possibility that iron can potentiate parasite death induced by metalloids like arsenic and antimony. In addition, an important observation is that the two similar metalloids produce toxicity by very different mechanisms. PMID- 16678024 TI - The overlapping of local iron overload and HFE mutation in venous leg ulcer pathogenesis. AB - Chronic venous stasis determines red blood cell extravasation and either dermal hemosiderin deposits or iron-laden phagocytes. Several authors have suspected that iron could play a role in the pathogenesis of venous leg ulcers. They hypothesized that local iron overload could generate free radicals or activate a proteolytic hyperactivity on the part of metalloproteinases (MMPs) or else down regulate tissue inhibitors of MMPs. However, they were unable to explain why iron deposits, visible in the legs of patients with chronic venous disease (CVD), cause lesions in only some individuals, whereas in others they do not. We hypothesized that such individual differences could be genetically determined and investigated the role of the C282Y and H63D mutations of the HFE gene. C282Y mutation significantly increases the risk of ulcer in primary CVD more than six times (OR = 6.69; 1.45-30.8; p = 0.01). Patients carrying the H63D variant have an earlier age of ulcer onset, by almost 10 years (p > 0.004). The increased risk of skin lesion and the early age of onset of the disease in HFE carriers confirm in a clinical setting that intracellular iron deposits of mutated macrophages have less stability than those of the wild type. We hypothesize that the physiologic iron protective mechanisms are affected by the HFE mutations and should be investigated in all diseases characterized by the combination of iron overload and inflammation. PMID- 16678025 TI - Levitronix as a short-term salvage treatment for primary graft failure after heart transplantation. AB - BACKGROUND: Primary graft failure after heart transplantation is a well recognized catastrophic complication with a high mortality rate. It is becoming more frequent due to the increasing use of marginal donors. In these difficult cases a ventricular assist device (VAD) as a bridge to recovery or as a bridge to re-transplantation can be used. The recently introduced Levitronix Centrimag centrifugal pump might be an ideal device for this purpose. METHODS: In this study we describe 2 patients with primary graft failure who received a Levitronix in the last 2 years, immediately after failure to wean from cardiopulmonary bypass. Biventricular support was necessary in both patients. RESULTS: One patient was successfully re-transplanted after 2 days of support, and subsequently discharged. After 16 months she has good ventricular function with no symptoms of cardiac failure. The second patient showed signs of ventricular recovery after a few days and was weaned from the device after 7 days, with good graft function. No device-related complications were recorded. After 14 days he was discharged from the intensive care unit (ICU), and a post-operative echocardiogram showed normal dimensions, good ejection fraction and no valvular regurgitation. He was discharged home 26 days after the transplant. CONCLUSIONS: In our experience, the Levitronix Centrimag seems to be safe and effective in the treatment of primary graft failure, achieving effective circulatory support and ventricular off-loading. We propose its use in isolated or biventricular graft failure either as bridge to re-transplant or as a bridge to recovery. PMID- 16678026 TI - Initial experience with non-thoracic, extraperitoneal, off-pump insertion of the Jarvik 2000 Heart in patients with previous median sternotomy. AB - BACKGROUND: Successful implantation of left ventricular assist devices (LVADs) in patients with previous median sternotomy remains challenging. METHODS: Seven patients underwent implantation of a Jarvik 2000 Heart by an extrathoracic, extraperitoneal, sub-costal surgical approach, which allows easy exposure of the diaphragmatic surface of the heart and the supraceliac aorta. All patients were at very high risk and were receiving high doses of inotropic agents to maintain their cardiac function. All had undergone prior median sternotomy. We compared data for blood loss and intensive care unit (ICU) stay with those of 15 patients in whom the HeartMate I vented electric LVAD was placed through a re-do sternotomy incision. RESULTS: All 7 patients survived the surgical procedure for implantation of the Jarvik 2000. All 7 patients were rapidly rehabilitated and had a short stay in the ICU (mean 3.3 days, range 1 to 8 days), as compared with the re-do HeartMate patients (mean 10.3 days, range 3 to 33 days) (p = 0.005). The average 12-hour blood loss was 635 ml in the Jarvik patients compared with 2,405 ml in the re-do HeartMate patients (p = 0.028). The cardiac index improved significantly in all Jarvik patients. CONCLUSIONS: The extrathoracic, extraperitoneal, sub-costal surgical approach is less invasive than a median sternotomy and allows the Jarvik 2000 to be implanted quickly and without cardiopulmonary bypass (CPB). By avoiding CPB and an extensive mediastinal dissection, bleeding is decreased in these hypocoagulable patients with compromised end-organ function. The decreased operative morbidity and mortality associated with this technique may allow consideration of the Jarvik 2000 Heart for safe and effective implantation in home-bound New York Heart Association (NYHA) Class III and IV patients. PMID- 16678027 TI - Improved survival after acute myocardial infarction complicated by cardiogenic shock with circulatory support and transplantation: comparing aggressive intervention with conservative treatment. AB - BACKGROUND: The prognosis for patients with myocardial infarction has steadily improved, but remains poor for those developing cardiogenic shock. Utilization of re-vascularization, mechanical circulatory support and transplantation in these patients may improve survival. METHODS: We retrospectively analyzed the clinical outcome of 138 consecutive patients at the Cleveland Clinic from 1992 to 1998 who met the criteria for cardiogenic shock after acute myocardial infarction. All patients received intensive medical therapy and intra-aortic balloon pump support. Forty-three patients received intensive medical therapy (conservative group) and 95 patients were treated aggressively (aggressive group). The aggressive group comprised patients who were treated with percutaneous intervention/coronary artery bypass grafting (n = 77, re-vascularization group), and patients who received circulatory support/cardiac transplantation (n = 18). RESULTS: The baseline demographics and angiographic and hemodynamic features were comparable for the two groups. The in-hospital mortality rate was significantly reduced in the aggressive group compared with the conservative group (54% vs 81%, p = 0.002). The in-hospital mortality rate of the circulatory support/transplant group was markedly reduced compared with the conservative group (33% vs 81%, p < 0.001), and was also significantly lower than that of the re-vascularization group (33% vs 63%, p= 0.03). The aggressive group had a markedly improved 5-year survival compared with the conservative group (30% vs 6.2%, p = 0.003). CONCLUSIONS: These data suggest that an aggressive strategy, particularly left ventricular assist device support as a bridge to heart transplantation, may improve survival in post-myocardial infarction patients with cardiogenic shock. PMID- 16678028 TI - Tissue Doppler imaging detects severely abnormal myocardial velocities that identify children with pre-terminal cardiac graft failure after heart transplantation. AB - BACKGROUND: Children with orthotopic heart transplants (OHT) may die or require retransplantation due to chronic graft failure usually due to severe coronary allograft vasculopathy (CAV). Non-invasive detection of chronic transplant failure has been problematic due to lack of specific echocardiographic findings. Tissue Doppler Imaging (TDI) is a non-invasive ultrasound methodology, which measures myocardial contraction and relaxation velocities. The purposes of this study were to: 1. Determine quantitative changes of longitudinal TDI velocities characteristic to "pre-terminal" patients who subsequently either died or were listed for re-transplantation due to graft failure; 2. to define the time course of these changes, and 3. to show whether RV and LV velocities were equally effected. METHODS: 53 heart transplantation recipients were evaluated. Of these, 45 were "well" patients. They ranged in age at enrollment from 0.5 to 20.1 (mean 10.21) years, age at transplantation from 0.2 to 18 (mean 5.7) years. The time from transplantation to enrollment was 1 day to 14.9 (mean 4.5) years. There were 8 "pre-terminal" (test group) patients who died or were listed for re transplantation within 9 months after TDI echo. These ranged in age from 2.6 to 17 (mean 11.6) years, age at transplant from 1 month to 15.9 (mean 8.1) years, and time from transplant to enrollment was 0.7 to 9.8 (mean 3.6) years. TDI was performed in the apical four-chamber view. Systolic (S, cm/s) and diastolic early (E, cm/s) and late (A, cm/s) velocity. Mitral and tricuspid annular TDI velocities were measured. Tricuspid regurgitation and LV ejection fraction were also compared. RESULTS: Pre-terminal patient's Left Ventricular Ejection Fraction began diverging from controls at 3 to 6 months prior to endpoint (p < 0.001). Tricuspid TDI S velocities of pre-terminal patients diverged by 2.0 cm/sec from controls (p < 0.002) 6 months prior to, and reduced further by 2.9 cm/sec 3 months prior to endpoint (p < 0.001). Tricuspid TDI E velocities diverged 3 to 6 months before endpoint, by 1.9 cm/sec (p < 0.02) and by 3.7 cm/sec 0-3 months prior to endpoint, (p < 0.001). Mitral S velocities diverged from controls by 1.5 cm/sec at 0 to 3 months before terminal endpoints (p = 0.002). Mitral E velocities were statistically similar at all time intervals (p > or = 0.15). Septal S velocities equaled controls 6 months (p = 0.92) and between 3 to 6 months (p = 0.83) but diverged by 1.6 cm/sec 0 to 3 months before terminal endpoints (p = 0.01). Septal E velocities equaled controls. Mortality Prediction: LVEF, tricuspid annulus systolic and diastolic velocities, and tricuspid regurgitation severity were significant in predicting mortality. Coronary angiography was performed in 26 patients, 5 had severe coronary artery disease and all were pre-terminal. DISCUSSION: The TDI data reported here show 3 to 6 months before the terminal graft failure, tricuspid, but not mitral, S and E TDI velocities, deteriorated to uniquely low levels not seen in other clinically well pediatric transplant recipients. Further RV deterioration occurred during the final 3 months before death and severely reduced left ventricular velocities then occurred. Small decreases in LVEF and progressive increases in the severity of tricuspid regurgitation were also detectable and predicted an increased likelihood of mortality. Seven of the 8 preterminal patients had angiograms 5 of which showed severe CAV. These data suggest that there is a critical "pre terminal" window of time in which children demonstrate uniquely reduced right and subsequently left sided myocardial velocities at approximately 6 months prior to graft failure. The practice of annual catheterization and coronary angiography may not allow caregivers an opportunity to intervene early in the process of graft dysfunction. Therefore, a strategy of tissue Doppler echocardiography 2 or 3 times each year might be an appropriate regimen to survey for graft impairment. PMID- 16678029 TI - Early renal benefit of rapamycin combined with reduced calcineurin inhibitor dose in pediatric heart transplantation patients. AB - BACKGROUND: Calcineurin inhibitors such as cyclosporine are effective in preventing rejection in recipients of solid organ transplants. Unfortunately, the prolonged use of calcineurin inhibitors may result in progressive renal injury. METHODS: We studied the renal function of 15 pediatric heart transplant recipients who were taking calcineurin inhibitors. Their renal function was studied before and after rapamycin was introduced to their immunosuppression regimen. With the introduction of rapamycin, the patients were given a lower dose of calcineurin inhibitors, and the calcineurin inhibitor was discontinued in 5 patients. RESULTS: Renal function improved significantly in the patients by 30 days after these changes in the calcineurin inhibitor dose were instituted. Mean levels of blood urea nitrogen and mean serum creatinine decreased, and mean creatinine clearance increased. Pre-rapamycin, the patients' mean level of blood urea nitrogen was 27.1 +/- 12.4 mg/dl and post-rapamycin they decreased to 18.6 +/- 11.1 mg/dl (p = 0.014). Similarly, creatinine decreased from 1.0 +/- 0.5 mg/dl to 0.8 +/- 0.3 mg/dl (p = 0.019). Their creatinine clearance increased from 88 +/- 28 ml/min/1.73 mol/liter2 to 105 +/- 27 ml/min/1.73 mol/liter2 (p = 0.008). The patients' lipid levels did not change after they were prescribed rapamycin. Biopsy-negative rejection developed in 2 patients. CONCLUSIONS: The introduction of rapamycin to the immunosuppressive regimen of patients taking calcineurin inhibitors, with a concomitant reduction of the calcineurin inhibitor dose, may improve renal function within 30 days, without a significant increase in rejection. PMID- 16678030 TI - Comparison of induced sputum and bronchoalveolar lavage in lung transplant recipients. AB - BACKGROUND: Chronic lung rejection is characterized by obliterative bronchiolitis (OB) diagnosed based on spirometric criteria reflecting an already advanced process. Biologic markers such as bronchoalveolar lavage (BAL) neutrophilia or increased levels of chemokines (interleukin-8, RANTES [regulated on activation: normal T cell expressed and secreted]) have been proposed as early diagnosis tools. However, BAL is too invasive to be used as a routine strategy. Induced sputum (IS), however, is a non-invasive method of recovering bronchial cells. METHODS: The aim of this study was to compare BAL and IS differential cellular counts as well as IL-8 and RANTES levels between patients with bronchiolitis obliterans syndrome (BOS), recipients with good outcome and well-preserved lung function (non-BOS) and non-transplanted controls. We compared 34 BAL and IS findings obtained consecutively from 34 lung transplant recipients (LTRs), including 22 non-BOS and 12 BOS patients. RESULTS: IS results were compared with 19 samples from non-transplanted controls. IS was well tolerated. There was no correlation between BAL and sputum cell populations. BAL neutrophils and IL-8 levels were increased in BOS, and these parameters were positively correlated. Moreover, BAL neutrophils and IL-8 levels were both negatively correlated with respiratory function. Sputum evaluation allows discrimination of BOS from non-BOS by the presence of higher neutrophil and eosinophil counts. Moreover, IS neutrophils and eosinophils were both correlated with lung function parameters. In contrast to BAL, IL-8 level in sputum was not a useful predictive marker of BOS development. IS RANTES levels were higher in BOS than in healthy recipients and correlated significantly with IS eosinophils. CONCLUSIONS: IS and BAL provide different but complementary data. In this study, IS appeared to be a useful, non invasive tool for LTR monitoring. Furthermore, IS provides new insights into BOS pathogenesis, especially with regard to implication of eosinophils and its chemokine, RANTES, at the bronchial level. PMID- 16678031 TI - Multiple variables affecting blood usage in lung transplantation. AB - BACKGROUND: A few publications have reported on the role of variables affecting blood component usage during lung transplantation. METHODS: Transfusion records for lung transplantation patients at the Hospital of the University of Pennsylvania (November 1991 to July 2004) were reviewed. Bivariate analyses and regression models were used to correlate usage of packed red blood cells (RBC), fresh-frozen plasma (FFP) and platelets (PLT) with variables such as disease, number of pulmonary lobes (1 or 2), cardiopulmonary bypass (CPB) status and time on bypass. RESULTS: Among 376 patients examined during the study period, blood product usage (in units) was significantly higher in double- than in single-lung recipients (RBC, 5.76 vs 1.21; FFP, 5.55 vs 1.10; PLT, 1.15 vs 0.16; p < 0.001). Patients on CPB also used significantly more units (RBC, 8.28 vs 1.45; FFP, 9.70 vs 0.73; PLT, 1.86 vs 0.14; p < 0.001), correlating with time on bypass. Patients transplanted for Eisenmenger syndrome (ES) and cystic fibrosis (CF) received significantly more blood products than those transplanted for other diseases (RBC, ES = 17.91 vs CF = 7.31 vs all others <2.00; FFP, ES = 19.18 vs CF = 5.72 vs others <2.00; PLT, ES = 4.73 vs CF = 1.22 vs others <0.40; p < 0.001). A regression model identified variables predictive of blood product usage, including the number of lungs transplanted, CPB status, disease and patient age. CONCLUSIONS: Patients receiving double-lung transplantations, on CPB, or transplanted for ES and CF exhibited a very highly statistically significant demand (p < 0.001) for more blood products. Additional selected variables differentially predicted usage. These data will help transplant surgeons and transfusion medicine specialists better anticipate and prepare blood products for use in lung transplantation. PMID- 16678032 TI - Expectant management is safe for cholelithiasis after heart transplant. AB - BACKGROUND: The optimal management of cholelithiasis after heart transplant remains unclear. We use expectant management based on symptoms, without screening studies or prophylactic treatment. We hypothesized that expectant management for cholelithiasis after heart transplant does not result in significant mortality or morbidity from gallstone-associated disease. METHODS: Between November 1985 and August 2004, 409 heart transplants were performed in 402 recipients at the University of Washington. This is a non-concurrent cohort study of these recipients. RESULTS: Among recipients, 24 underwent cholecystectomy before heart transplant. After transplant, in the remaining 378 patients, 34 were found to have gallstones during the observation period. There was no mortality from gallstone-associated disease. Thirty patients developed morbidity from gallstones, including 25 cases of biliary colic, 3 of acute cholecystitis and 2 of pancreatitis, and there was 1 abnormal liver function test. Acute cholecystitis and pancreatitis were treated with conservative management followed by cholecystectomy. Cholecystectomy was performed in 32 patients after transplant. Indications included symptomatic cholelithiasis in 31, and prophylactic cholecystectomy prior to kidney transplant in 1. The laparoscopic approach was performed in 25 of these 32 patients. There was no mortality from cholecystectomy, but there were 4 complications: surgical site infections (n = 2); wound dehiscence (n = 1); and bile duct injury (n = 1). Median hospital stay was 1 day. CONCLUSIONS: Our expectant management for cholelithiasis after heart transplant resulted in no mortality or significant morbidity related to delay in treatment. Symptomatic cholelithiasis was successfully treated with cholecystectomy, mostly with the laparoscopic approach. We believe expectant management is safe for patients after heart transplant. PMID- 16678033 TI - Serum hyaluronan--a potential marker of cardiac allograft rejection? AB - BACKGROUND: The connective tissue component hyaluronan accumulates within the transplanted organ at rejection. Increased tissue content of hyaluronan is seen also in synovial tissue in rheumatoid arthritis and in skin in scleroderma. In these diseases, the elevated hyaluronan levels are reflected by increased concentrations of hyaluronan in serum. The aim of the present study was to study the changes in serum hyaluronan after organ transplantation. METHODS: The experiments were performed in a rat model of heterotopic heart transplantation. Serum hyaluronan was assessed at various times after allogeneic (rejection) and syngeneic (non-rejection) transplantation and correlated with tissue hyaluronan. In addition, serum hyaluronan in animals who had long-term-surviving allogeneic grafts was studied. RESULTS: The hyaluronan concentration in serum was significantly higher in the rejecting than in the non-rejecting group 4 and 6 days after transplantation (p < 0.01). On Day 6, serum hyaluronan had increased by 400% in animals with an allogeneic transplant and by 100% in those with a syngeneic transplant. There was a positive correlation between serum hyaluronan and tissue hyaluronan (p < 0.05). Animals with long-term-surviving grafts displayed normal serum hyaluronan levels. CONCLUSIONS: Rejection of rat heart transplants is associated with strongly increased serum hyaluronan that parallels the hyaluronan accumulation within the transplant. PMID- 16678034 TI - Preventing cardiac allograft vasculopathy: long-term beneficial effects of mycophenolate mofetil. AB - BACKGROUND: The impact of long-term mycophenolate mofetil (MMF) treatment on the development of cardiac allograft vasculopathy (CAV) after heart transplantation is an area of much recent interest. This study analyzed the effects of various immunosuppressive combinations, including cyclosporine (CsA), azathioprine (Aza), tacrolimus (Tac) and MMF, on the time of onset, extent and progression of CAV. METHODS: Two hundred seventy-three consecutive heart transplant recipients (mean age: 51.2 +/- 12.2 years; mean follow-up: 6.8 +/- 1.9 years) were examined by coronary angiography on a yearly basis between 1995 and 2003. The extent of CAV was evaluated using a scoring system based on the severity of vessel stenosis. The onset of CAV was analyzed using Kaplan-Meier estimates and the log rank test for four treatment combinations, CsA/Aza (n = 47, 17.2%), CsA/MMF (n = 26, 9.5%), Tac/Aza (n = 62, 22.7%) and Tac/MMF (n = 138, 50.5%), and for the primary and the secondary immunosuppressants alone. RESULTS: The rate of freedom from CAV at 5 years was 47% with CsA/Aza, 66% with CsA/MMF, 60% with Tac/Aza and 70% with Tac/MMF. After 5 years, the Tac/MMF group showed a significantly lower incidence of CAV than the CsA/Aza group (log rank 7.58, p = 0.0059). CsA (n = 73) was compared with Tac (n = 200) and MMF (n = 164) with Aza (n = 109): the rate of freedom from CAV was 51.2% in CsA patients vs 66.1% in Tac patients (log rank 5.7, p = 0.017), and 54.6% in Aza patients vs 67% in MMF patients (log rank 4.36, p = 0.037). Multivariate Cox regression analysis revealed that MMF decreased the incidence of CAV significantly (p = 0.041). In this patient cohort, Tac or CsA medication was not an independent risk factor for incidence of CAV nor for decreased survival. CONCLUSIONS: The choice of immunosuppression has an impact on the incidence of CAV. In terms of prevention of CAV, MMF is superior to Aza in either combination. A trend toward improved survival in MMF patients was noted. The lower number of rejection episodes in the MMF groups may have contributed to these results. PMID- 16678035 TI - Therapeutic drug monitoring of prednisolone after lung transplantation. AB - BACKGROUND: Despite well-known and serious potential side-effects of corticosteroid therapy, therapeutic drug monitoring (TDM) of prednisolone is rarely performed after lung transplantation (LTx). METHODS: We measured prednisolone exposure using a 6-hour area-under-the-curve (AUC) analysis in 52 LTx recipients (41 bilateral, 9 single and 2 heart-lung), who were 99 +/- 13 (mean +/- SEM) weeks (range 4 to 380) post-LTx. Fourteen of 52 had cystic fibrosis (CF), and 36 of 52 were on cyclosporine and 16 of 52 on tacrolimus. Prednisolone dose was 9.8 +/- 0.7 mg/day (range 1 to 20). RESULTS: Only 9 of 52 LTx patients had a prednisolone AUC within the previously reported reference range for healthy adult control subjects (170 to 260 nmol x hr/liter/milligram prednisolone). Six patients had values below and 37 above this range. Prednisolone AUC was higher in patients with CF compared with non-CF patients (511 +/- 82 vs 349 +/- 27 nmol x hr/liter/milligram, p < 0.02) and 70% of LTx recipients had measurable prednisolone levels at baseline (26.5 +/- 4.5 nmol/liter), unlike normal controls. CONCLUSIONS: LTx recipients show a wide inter-individual variation in prednisolone pharmacokinetics; therefore, many are overdosed on conventional protocols. Side-effects of corticosteroid therapy remain a major clinical problem after transplantation, justifying the use of prednisolone TDM to optimize dosing and minimize morbidity. PMID- 16678036 TI - Cyclosporine profiling with C2 and C0 monitoring improves outcomes after heart transplantation. AB - BACKGROUND: Cyclosporine (CsA) level at 2 hours post-dose (C2) is a more sensitive marker for rejection risk than trough (C0) level. A combination of C2 and C0 monitoring may prove optimal. METHODS: We compared efficacy and safety outcomes among 28 de novo heart transplant patients in whom both C2 and C0 monitoring were undertaken (Group 1), with a single CsA profile at Weeks 2 to 6, versus 28 historic controls monitored by only C0 (Group 2). Patients received anti-thymocyte globulin (ATG) induction with CsA, steroids and azathioprine maintenance therapy. RESULTS: The CsA microemulsion dose was significantly higher in Group 1 than Group 2 up to 3 months post-transplant. Mean C2 values in Group 1 at 3 and 12 months were 1,248 +/- 328 ng/ml and 1,039 +/- 362 ng/ml, respectively. One patient in Group 1 and 7 in Group 2 (25%) discontinued CsA, either due to CsA-related neurotoxicity or two or more episodes of early rejection. At 12 months, graft and patient survival were 100% in both groups. Six patients in Group 1 (21%) and 11 in Group 2 (39%) had at least one episode of biopsy-proven acute rejection (not significant). Over the first 12 months post transplant, the proportion of biopsies showing Grade 3 rejection was 5% in Group 1 and 11% in Group 2 (p < 0.002). Gloerular filtration rate (GFR) was significantly lower in Group 1 than Group 2 at both 3 and 12 months. CONCLUSIONS: Combined use of C2 and C0 monitoring results in improved efficacy versus C0 monitoring alone. Regular measurement of C2 levels should be undertaken in de novo heart transplant recipients. PMID- 16678037 TI - Inhibition of tumor necrosis factor-alpha attenuates myocardial remodeling in rat cardiac allografts. AB - BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) elicits a wide range of pro inflammatory activities on target cells and mediates diverse cardiovascular processes ranging from heart failure to atherosclerosis. Recently, we demonstrated that TNF-alpha regulates the platelelet-derived growth factor (PDGF) A/PDGF-Ralpha activation pathway in rat cardiac allograft arteriosclerosis. The aim of this study was to determine the kinetics and biologic role of TNF-alpha and its receptors, TNF-R1 and TNF-R2, in rat cardiac allografts. METHODS: Heterotopic heart transplantations were performed from Dark Agouti to Wistar Furth rats. In the acute rejection model, recipients were given no immunosuppression and grafts were removed 5 days after transplantation. In the chronic rejection model, cyclosporine (CsA) was administered and grafts were removed at 60 days. To investigate the functional role of TNF-alpha in chronic rejection, recipients received recombinant human soluble TNF receptor p80/IgG1 Fc fusion protein (rhu TNF-R2:Fc). RESULTS: During acute and chronic rejection, an increase in intragraft TNF-alpha and TNF-R2 mRNA expression was recorded, but not TNF-R1 mRNA expression. Prominent induction of TNF-alpha and TNF-R2 immunoreactivity was localized to medial cells of coronary arteries and interstitial inflammatory cells, whereas cardiomyocytes showed moderate immunoreactivity to TNF-alpha and its receptors. Inhibition of the TNF-alpha mediated pathway by TNF-R2:Fc did not affect the incidence or intensity of arteriosclerotic lesions in rat cardiac allografts; however, it significantly inhibited myocardial remodeling with a concomitant decrease in myocardial TNF alpha expression but not intragraft PDGF immunoreactivity. CONCLUSIONS: We conclude that inhibition of TNF-alpha attenuates myocardial remodeling but is not rate-limiting for arteriosclerotic lesion formation. PMID- 16678038 TI - Serial gene expression profiling in the intact human heart. AB - BACKGROUND: In chronic heart failure due to a dilated cardiomyopathy phenotype, the molecular bases for contractile dysfunction and chamber remodeling remain largely unidentified. METHODS: To investigate the feasibility of measuring global gene expression serially in the intact failing human heart, we performed repeated messenger RNA (mRNA) expression profiling using RNA extracted from endomyocardial biopsy specimens and gene chip methodology in 8 subjects with idiopathic dilated cardiomyopathy. In patients treated with beta-blocking agents or placebo, myocardial gene expression was measured in endomyocardial biopsy material and radionuclide ejection fraction was measured at baseline and after 4 to 12 months of treatment. Gene expression was measured for 12,625 gene sequences by using Affymetrix U95 gene chips and commercially available software. For 6 mRNAs, gene chip results were compared with measurements made by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In an unfiltered composite analysis of changes in expression detected in the patients with high signal intensity chips, 241 genes showed an increase and 331 genes a decrease in mRNA abundance. There was good agreement between changes measured by quantitative RT-PCR and those determined by gene chips. There was less variance between differences in phenotype in patients sampled serially as compared between subjects with similar phenotypes sampled at baseline. CONCLUSIONS: Serial gene expression profiling with association to phenotypic change is feasible in the intact human heart and may offer advantages to cross-sectional expression profiling. This study suggests that the intact failing remodeled human heart is in an activated state of gene expression, with a large net reduction in gene expression occurring as phenotypic improvement occurs. PMID- 16678039 TI - Determining the best ventilatory efficiency measure to predict mortality in patients with heart failure. AB - BACKGROUND: Ventilatory efficiency, the relationship between ventilation (VE) and carbon dioxide production (VCO2), predicts mortality in heart failure patients, but its determination has not been standardized. Additionally, it is unclear if the prognostic power of ventilatory efficiency is independent of exercise intensity. Therefore, we investigated the relative prognostic power of different measures of ventilatory efficiency calculated from maximal and sub-maximal exercise in patients with heart failure. METHODS AND RESULTS: Heart failure patients (n = 355, 72% males, age 51 +/- 10 years) had follow-up for at least 5 years from an exercise test. There were 145 events (133 deaths and 12 emergent cardiac transplants). Ventilatory efficiency calculations were not equivalent. Of the different measures of ventilatory efficiency, the VE/VCO2 slope to peak exercise was the most significant predictor of mortality in a multivariable Cox model, including ejection fraction, systolic blood pressure, peak oxygen consumption (VO2), gender, etiology, and heart rate. A 5 U increment of the VE/VCO2 slope to peak exercise corresponded to a 9% increase in mortality risk. When tests were grouped by peak exercise intensity, by quartiles of peak respiratory exchange ratio, the VE/VCO2 slope to peak exercise was always a better predictor than peak VO2. CONCLUSION: Peak and sub-maximal measures of ventilatory efficiency were not equivalent, and the VE/VCO2 slope to peak exercise was the best predictor of mortality in patients with heart failure. Thus, the prognostic power of ventilatory efficiency is enhanced when exercise extends beyond the ventilatory threshold and includes all of the available exercise data. PMID- 16678040 TI - Aminoterminal B-type pro-natriuretic peptide as a marker of recovery after high risk coronary artery bypass grafting in patients with ischemic heart disease and severe impaired left ventricular function. AB - BACKGROUND: Aminoterminal B-type pro-natriuretic peptide (NT-proBNP) is a reliable indicator of heart failure severity. Levels of NT-proBNP are markedly increased in patients with coronary artery disease (CAD) and severely impaired left ventricular (LV) function. The aim of our study was to assess the impact of NT-proBNP levels after high-risk coronary artery bypass grafting (CABG) with regard to recovery potential. METHODS: Between 1998 and 2004, 121 patients with CAD and severely impaired LV function, who were undergoing CABG, were investigated. Their mean age was 64 +/- 11 years. All patients were in New York Heart Association (NYHA) Class III/IV status; LV ejection fraction (EF) was 20 +/ 6%. All survivors underwent follow-up (59 +/- 34 months) spiroergometric, electrocardiographic (ECG) and echocardiographic assessment and were tested for routine blood controls and NT-proBNP levels (Roche, Mannheim, Germany). RESULTS: The survival rate after 8 years was 70%. All survivors received follow-up assessment. Among survivors the median NT-proBNP level at follow-up was 896 (521 to 1,687) pg/ml. The maximum oxygen uptake was 14.6 +/- 4.9 ml/min/kg, and EF increased to 42% at follow-up among all survivors. On dichotomizing survivors into two groups with NT-proBNP levels above and below the median, the post operative body mass index was significantly higher in the high NT-proBNP group (p = 0.036). EF (p = 0.028) and NYHA classification (p < 0.05) improved significantly in both groups, with a tendency toward higher EF in the low NT proBNP group. CONCLUSIONS: Patients undergoing a high-risk CABG procedure have a survival rate comparable to heart transplantation patients and show a potential for clinical and myocardial recovery. NT-proBNP use a useful marker for recovery after a high-risk CABG procedure, with significant correlation with clinical parameters. PMID- 16678041 TI - Disseminated Scedosporium apiospermum infection in a cystic fibrosis patient after double-lung transplantation. AB - Scedosporium apiospermum is a saprophytic ubiquitous filamentous fungus. It can cause a wide spectrum of diseases, from localized to invasive infections. S apiospermum has been described as one of the major fungal agents of chronic colonization of airways in cystic fibrosis (CF) patients. Invasive infections due to S apiospermum are only rarely reported in CF after lung transplantation. A 26 year-old woman with CF and chronic bronchial colonization by S apiospermum developed bilateral chorioretinitis and subcutaneous nodules 4 weeks after double lung transplantation (LTx). Isolates of S apiospermum from sputum samples before and after LTx and from vitreal fluid were typed by random amplification of polymorphic DNA (RAPD). The patient was treated with voriconazole (VRC). The patient improved with VRC given orally for 6 months. Two days after VRC discontinuation, she developed sub-acute meningitis (isolation of S apiospermum from the cerebrospinal fluid). She was again given VRC, but died 23 days later from uncontrolled fungal infection. Molecular typing of clinical isolates of S apiospermum performed by RAPD demonstrated that all isolates belonged to the same genotype. S apiospermum is a frequent, but late colonizing fungal agent in CF patients. In the case of LTx, these patients can develop invasive infection due to the colonizing strain, as confirmed by molecular typing. PMID- 16678042 TI - Administration of rabbit anti-thymocyte globulin during cardiopulmonary bypass: a novel approach to the highly sensitized cardiac transplant patient. PMID- 16678043 TI - Renal toxicity due to inhaled tobramycin in lung transplant recipients. PMID- 16678044 TI - Rupture of chronic thoracic aortic dissection in a Marfan syndrome patient after heart transplantation. PMID- 16678045 TI - Making prostate-specific antigen testing more effective. PMID- 16678046 TI - Treatment of stage I seminoma: a 15-year review. AB - PURPOSE: To review the treatment and outcomes in patients with stage I seminoma after orchidectomy. METHODS: A retrospective chart review of all patients with stage I seminoma referred for initial treatment during the last 15 years was performed. Initial treatment approaches and outcomes were analyzed. Comparisons were made between patients treated with adjuvant radiotherapy and those receiving no adjuvant therapy (surveillance group). RESULTS: A total of 150 patients with stage I seminoma was seen between 1989 and 2003. Median age at diagnosis was 37.5 years (range 19-79), with a median follow-up of 54 months (range 1-162). Of the patients, 71% were treated with adjuvant radiotherapy, and 29% were placed on a surveillance protocol. The 5-year relapse-free survival and overall survival for the entire group were 95% and 100%, respectively. The 5-year relapse-free survival for the adjuvant radiotherapy group was 100% compared with 79% for the surveillance group (P < 0.001). Of the 6 patients who had a relapse, 5 were salvaged with radiation, but 1 required chemotherapy as well. One patient who had a relapse is currently refusing treatment for recurrence. CONCLUSIONS: Our results confirm the excellent prognosis for patients with stage I seminoma and indicate that surveillance does not compromise survival. This result adds to the evidence that surveillance is a good option for many patients and also supports our current approach, which favors surveillance for most patients with stage I seminoma after orchidectomy who are willing to go on our surveillance protocol. PMID- 16678047 TI - International comparisons of prostate cancer mortality rates with dietary practices and sunlight levels. AB - Prostate cancer mortality rates vary widely across the world. The purpose of this study is to identify environmental factors associated with prostate cancer mortality risk. Prostate cancer mortality rates in 71 countries were compared to per capita food intake rates using age-adjusted cancer rates (year 2000) from the International Agency for Research on Cancer, and food consumption data (1990 1992) provided by the Food and Agricultural Organization of the United Nations. Simple regression models were applied to prostate cancer mortality rates and consumption rates for 38 foods (or food categories), and sunlight levels (latitude from the equator and ultraviolet indexes). The analysis found a correlation between increased prostate cancer mortality rates and the consumption of total animal calories, total animal fat calories, meat, animal fat, milk, sugar, alcoholic beverages, and stimulants. The consumption of cereal grains and rice, in particular, correlated strongly with decreasing prostate cancer mortality. The analysis found that increased sunlight levels and consumption of oilseeds, soybeans, and onions also correlate with decreased prostate cancer mortality risk. Stepwise multiple regression analysis was used to build a regression model with minimum colinearity between the variables. Cereals, total animal fat calories, sugar, and onions are the foods that resulted in a model with the best fit. Cereals, ultraviolet index, sugar, and onions were the variables found to provide the best fit in a model when ambient sunlight exposure was included as a factor. PMID- 16678048 TI - Antiprostasome antibodies: possible serum markers for prostate cancer metastasizing liability. AB - Prostasomes are secretory granules synthesized, stored, and secreted by normal and neoplastic human prostate epithelial cells. In prostate cancer, they are anticipated to be released into the blood circulation where they may be immunogenic. The aim of our study was to examine whether prostasome antibody presence in serum bears any prognostic significance for men with prostate cancer. We developed a sensitive and specific immunoassay (enzyme-linked immunosorbent assay) to establish the presence of antiprostasome antibodies in serum. The antiprostasome antibody titer in serum, sampled before any kind of therapy for prostate cancer, was examined together with clinicopathologic variables and outcome over a median follow-up of 350 days in 218 patients with verified prostate cancer. We detected these antibodies in 191 (88%) of these patients. This antibody titer did not correlate to serum values of prostate-specific antigen. Significant, inverse relationships were registered for antiprostasome antibody titer, and metastases to bone and/or lymph nodes (P = 0.035) and pT (P = 0.025). These results indicate that the antiprostasome antibody titer in serum may be a novel marker for prostate cancer liability to metastasize. PMID- 16678049 TI - Clinical symptoms in localized renal cell carcinoma reflect its invasive potential: comparative study between incidentally detected and symptomatic diseases. AB - The objectives of this study were to analyze the association of clinical symptoms with several parameters in patients with organ-confined renal cell carcinoma (RCC), and investigate the difference in biologic characteristics between incidentally detected and symptomatic RCCs. This study included 132 patients who were pathologically diagnosed with organ-confined RCC after radical surgery, and their clinicopathologic features were compared to those in patients with incidentally detected and symptomatic diseases. In this series, 91 patients were incidentally diagnosed with RCC, while the remaining 41 had some clinical symptoms. The cause-specific survival in patients with incidental RCC was significantly higher than that in patients with symptomatic RCC, and the presence of clinical symptoms could be an independent predictor of cause-specific survival in these patients. Immunohistochemical staining of resected specimens from these patients were then performed with Ki-67, Bcl-2, matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor antibodies. Despite the lack of differences in Ki-67, Bcl-2, and vascular endothelial growth factor expression between these 2 groups, significantly increased expressions of MMP-2 and MMP-9 in symptomatic RCCs were observed compared with those in incidental RCCs. Moreover, the presence of clinical symptoms in organ-confined RCC was significantly correlated with immunoreactivities of MMP-2 and MMP-9. These findings suggest that clinical symptoms have a significant impact on the prognosis of patients with organ-confined RCC and that the appearance of clinical symptoms may reflect the increased invasive potentials characterized by overexpression of MMPs. PMID- 16678050 TI - HYTAD1-p20: a new paclitaxel-hyaluronic acid hydrosoluble bioconjugate for treatment of superficial bladder cancer. AB - OBJECTIVE: To report the development of a new water-soluble paclitaxel-hyaluronic acid bioconjugate, HYTAD1-p20, for intravesical treatment of superficial bladder cancer. MATERIALS AND METHODS: HYTAD1-p20 was synthesized by carboxyl esterification of hyaluronic acid with paclitaxel, and its physicochemical and biologic properties were characterized. RESULTS: Paclitaxel loading was optimized at 20% w/w; this procedure increased by 500-fold the paclitaxel concentration in the resulting water-soluble biomaterial. In vitro, HYTAD1-p20 exerted a much higher dose-dependent inhibitory effect against RT-4 and RT-112/84 bladder carcinoma cell growth than that of free drug, and directly interacted with CD44 expressed by bladder tumor cells. In vivo, results of pharmacokinetic studies performed in mice after bladder catheterization and intravesical instillation of HYTAD1-p20 disclosed that drug leakage was negligible during a 2-hour analysis. Histologic examination of drug-instilled bladders revealed that HYTAD1-p20 was extremely well tolerated, while paclitaxel alone produced mucosal disruption and submucosal infiltration of inflammatory cells. Treatment of severe combined immunodeficient mice bearing subcutaneous RT-112/84 tumors with maximum tolerated doses of bioconjugate or paclitaxel showed that HYTAD1-p20 exerted a therapeutic activity comparable to that of free drug. CONCLUSIONS: These data suggest that HYTAD1-p20 significantly improved results obtained with conventional paclitaxel in terms of hydrosolubility, in vitro activity against human bladder cancer cells, and in vivo biocompatibility. This bioconjugate is a potentially useful treatment for superficial urothelial malignancy. PMID- 16678051 TI - Prostate adenocarcinoma manifesting as generalized lymphadenopathy. AB - Generalized lymphadenopathy is a rare manifestation of metastatic prostate cancer. Here, we report the case of a 65-year-old patient with supraclavicular, mediastinal, hilar, and retroperitoneal lymphadenopathy and pulmonary infiltration, which suggested the diagnosis of lymphoma. There were no urinary symptoms, and the serum prostate-specific antigen (PSA) was only mildly increased with a normal free PSA. A biopsy of the supraclavicular lymph node was compatible with adenocarcinoma, whose prostatic origin was shown by immunohistochemical staining with PSA. The origin of the primary tumor was confirmed by directed prostate biopsy. We emphasize that a suspicion of prostate cancer in men with adenocarcinoma of undetermined origin is important for an adequate diagnostic and therapeutic approach. PMID- 16678054 TI - Single nucleotide polymorphisms: early diagnosis and risk assessment in genitourinary malignancy. AB - Genetic screening for malignancy has been limited to high-risk individuals with a strong hereditary predisposition to cancer. With the cloning of the human genome, it has become apparent that genetic anomalies are not limited to high-risk individuals; more than 10 million genetic variants exist. Because the vast majority of these genetic variants have no functional significance, current efforts are focused on identification of which impact cancer development and/or progression. Here, we review the rationale for studying polymorphic variants in urologic malignancies, prior studies in the field, and future avenues of research. PMID- 16678055 TI - Proteomics for the identification of new prostate cancer biomarkers. AB - Molecular profiling studies of human prostate cancer provide great opportunities to identify new prostate cancer biomarkers to improve prostate cancer detection and treatment. Proteomics has distinct advantages over genomic and ribonucleic acid expression studies because it is the proteins that are ultimately responsible for the malignant phenotype. The goal of traditional proteomic studies is to identify disease-specific biomarkers. Two-dimensional (2-D) gel electrophoresis (polyacrylamide gel electrophoresis; PAGE) coupled with mass spectrometry is the most widely used experimental strategy and, to date, has yielded several potentially relevant prostate cancer biomarkers. A promising prostate cancer biomarker identified by 2-D PAGE and mass spectrometry is annexin I. Studies have already confirmed that annexin I is underexpressed in a majority of early stage prostate cancers. Other non-gel-based proteomic technologies that may have improved sensitivity as compared to 2-D PAGE have recently been developed. An example of this is the ProteomeLab PF 2-D (Beckman Coulter, Inc., Fullerton, CA). The goal of most proteomic studies is to identify biomarkers that can be measured by enzyme-linked immunosorbent assay or immunohistochemistry. Improvements in proteomic technology may be changing this paradigm because there are now efforts to develop proteomic technologies directly into clinical diagnostic tests. An example of this technology is surface-enhanced laser desorption ionization time-of-flight mass spectrometry. Using this technology combined with a pattern recognition based bioinformatics tool, discriminatory spectrum proteomic profiles were generated that could help discriminate men with prostate cancer from those with benign prostates. If several technologic hurdles can be overcome, it is possible that methodology will improve the specificity and sensitivity of prostate cancer detection. PMID- 16678056 TI - Cancer immunomics: using autoantibody signatures in the early detection of prostate cancer. AB - Prostate cancer remains the most common malignancy among men and the second leading cause of cancer death of men in the United States. Although measurement of prostate-specific antigen (PSA) has led to earlier detection of many prostate cancers, new serum biomarkers are still needed to improve the accuracy of prostate cancer detection. Considerable evidence has shown that an immune response in the form of autoantibodies to various tumor antigens develops in many patients with cancer. By using phage-epitope microarray analysis, we were able to identify peptides expressed by prostate cancer tissue, which commonly induce formation of autoantibodies in the sera of patients with prostate cancer. Using a panel of 22 peptides, we were able to detect prostate cancer with a specificity of 88.2% and a sensitivity of 81.6%. These results were significantly better than PSA, especially among men with a PSA between 4 and 10 ng/ml. Measurement of the immune response to prostate cancer, as well as other malignancies, has the potential to improve significantly the detection of these cancers and possibly assist in the determination of prognosis. PMID- 16678058 TI - The use of thalidomide in androgen-independent prostate cancer. AB - More than 200,000 men will be diagnosed with prostate cancer during the year 2006. Approximately 20% to 30% of these cases may develop advanced disease, for which there currently is no cure. Although therapy for this disease has improved significantly over the past few years, with docetaxel treatment showing improved survival times in metastatic disease, there remains the need for improved treatment options. Dr. Folkman put forth the idea of angiogenesis in 1971, and, since that time, researchers have been trying to determine the best possible way to inhibit blood vessel formation. This review summarizes the use of thalidomide in androgen-independent prostate cancer and the results of trials conducted at the National Cancer Institute. PMID- 16678059 TI - Angiogenesis inhibition plus chemotherapy for metastatic hormone refractory prostate cancer: history and rationale. AB - Angiogenesis inhibition with bevacizumab and other agents of this class are showing significant activity in a variety of cancers. In prostate cancer, the single agent activity has been low, but the addition of these agents to chemotherapy may be the area in which they provide their greatest clinical benefit. An ongoing study conducted by the Cancer and Leukemia Group B will test this approach in men with metastatic hormone refractory prostate cancer. Future studies may test the efficacy of anti-angiogenic strategies in earlier stage disease as well as in combination with other approaches. This review will highlight the clinical experience to date with angiogenesis inhibitors in prostate cancer and the ongoing studies. PMID- 16678060 TI - Neoadjuvant mitoxantrone and docetaxel for high-risk localized prostate cancer. AB - PURPOSE: Currently available treatment modalities for high-risk clinically localized prostate cancer have limited chances of achieving complete tumor elimination because of either inadequate local or metastatic tumor eradication. The goal of this phase I/II study is to evaluate the safety and efficacy of neoadjuvant docetaxel and mitoxantrone before prostatectomy. MATERIALS AND METHODS: A total of 22 men with high-risk clinically localized prostate cancer underwent weekly treatment with docetaxel (35 mg/m(2)), with increasing doses of mitoxantrone (2-5 mg/m(2)) for a 12 of 16-week treatment cycle before prostatectomy. Testosterone and prostate-specific antigen (PSA) measurements were made before and after chemotherapy. RESULTS: The maximally tolerated dose for mitoxantrone was 4 mg/m(2), and the primary toxicity was neutropenia. Testosterone levels were maintained throughout treatment. PSA reductions were observed in 95% of patients, with a median reduction of 41%. The surgery was well tolerated after chemotherapy, without any major complications. Negative surgical margins were attained in 76% of patients. CONCLUSIONS: Administration of multi agent chemotherapy before prostatectomy was safe in this population. This regimen appeared to have antineoplastic activity as evidenced by PSA reductions in the absence of significant testosterone changes. The benefit of chemotherapy for improving surgical margin rates could not be determined outside of a phase III trial because the effect of patient or surgeon factors could not be dissected from the potential effect of neoadjuvant therapy. Continued study of novel agents in the neoadjuvant setting is warranted because this approach allows for the rapid identification of active agents and for molecular investigation into the mechanism of drug activity. PMID- 16678061 TI - Current status of anti-angiogenesis therapy for prostate cancer. AB - Prostate cancer is the second leading cause of cancer mortality in American men and the single most diagnosed cancer in men. Despite advances in early detection and conventional treatment strategies, prostate cancer progresses and becomes resistant to treatment. Because tumor growth and establishment of metastases are dependent on angiogenesis, interest in the development of anti-angiogenesis therapies has grown. Preclinical studies and early clinical evaluation show promise in the adjunctive use of anti-angiogenesis to overcome the limitations of current therapeutic approaches. In this review, we outline the basic science principles of angiogenesis and their application in the development of anticancer therapies. PMID- 16678073 TI - Genetics of hepatobiliary diseases. AB - With the recent publication of the first human map of genetic variation (ie, Human Haplotype Map), genomic-based discoveries will likely affect not only the research bench but also the bedside. These advances will improve the understanding of the genetics of hepatobiliary diseases, resulting in better prevention measures and diagnosis as well as more effective therapies. Currently, alcoholic liver disease, nonalcoholic fatty liver disease, and symptomatic gallbladder stones affect a sizable portion of the population. On the other hand, chronic cholestatic liver diseases, hepatocellular carcinoma, and polycystic liver disease, although rare, shorten life expectancy and diminish the quality of life of patients. In the genomic era, we have the opportunity to start dissecting the susceptibility genetic variants of liver diseases. We are now in a position to begin elucidating the complex genotype/phenotype relationships of liver diseases with the anticipation to understand disease pathogenesis better. These efforts will require the application of genomic-based approaches in large well organized translational studies in the diseases of interest. PMID- 16678074 TI - Hepatocyte tails. PMID- 16678075 TI - The infant gastroesophageal reflux questionnaire revised: development and validation as an evaluative instrument. AB - BACKGROUND & AIMS: Gastroesophageal reflux disease (GERD) is frequently experienced by infants, and disease-specific measures are needed to evaluate treatment benefits. We revised the Infant Gastroesophageal Reflux Questionnaire (I-GERQ) on the basis of information from parents of infants with GERD and physicians and subjected it to a psychometric evaluation. METHODS: A 3-week, multi-country observational study of 185 caregivers of infants younger than 18 months with GERD and 93 caregivers of control infants was conducted. Caregivers completed the I-GERQ-R weekly and recorded symptoms in a Daily Diary. Caregivers and physicians rated global disease severity and change in overall GERD symptoms. RESULTS: Slightly more than half of infants were male with a mean age of 6.7 months, and most infants had been diagnosed with GERD for a little more than 2 months (mean, 66.7 days). Internal consistency reliability for the I-GERQ-R ranged from 0.86 to 0.87, and test-retest reliability was 0.85. Construct validity was demonstrated by significant differences between cases and controls on all item scores (all P<.01) and the total score (P<.0001), correlations with relevant Daily Diary symptoms, and both physician-rated (P<.05) and caregiver rated disease severity (P<.05). Mean baseline to 3-week I-GERQ-R change scores for those infants whose caregivers reported improvement was -5.7 compared with 0.3 for those whose caregivers reported worse/same (P<.001). Physician ratings of change resulted in similar findings, with mean changes of -5.7 for those rated improved and -0.1 for those rated as worse/same (P<.0001). CONCLUSION: This study demonstrated the I-GERQ-R is a reliable, valid, and clinically responsive measure of infant GERD symptoms. PMID- 16678076 TI - Safety and tolerability of tegaserod in patients with chronic constipation: pooled data from two phase III studies. AB - BACKGROUND & AIMS: Studies show that tegaserod effectively relieves the symptoms of chronic constipation/idiopathic constipation (CC). This pooled analysis assessed the safety and tolerability of tegaserod in a large dataset of CC patients. METHODS: Adverse event (AE) data were pooled from 2 double-blind, placebo-controlled phase III trials of 12 weeks' duration. Post hoc analysis was conducted for the most frequent AEs (incidence, >or=3%). RESULTS: Eight hundred eighty-one, 861, and 861 patients received tegaserod 6 mg twice a day, 2 mg twice a day, or placebo, respectively. Most AEs were mild/moderately severe. AE incidence was similar for the tegaserod 6 mg and 2 mg twice a day (57.1% and 56.3%, respectively) and placebo groups (59.6%) and most frequent in the gastrointestinal system (tegaserod 6 mg twice a day, 25.8%; 2 mg twice a day, 22.5%; placebo, 24.6%). Headache, the most common AE, was slightly more frequent in the placebo group (tegaserod 6 mg twice a day, 11.0%; 2 mg twice a day, 10.1%; placebo, 13.2%). Diarrhea (generally transient and resolved with continued treatment) was the only AE with a statistically significant difference between groups (tegaserod 6 mg twice a day 6.6% vs placebo 3.0%, P=.0005). Serious AE incidence (1.4% overall) was comparable across treatment groups, although abdominal surgery was less common in the combined tegaserod (0.5%) than the placebo group (1.0%). Discontinuation as a result of AEs was slightly higher in tegaserod 6 mg twice a day patients (5.7%; 2 mg twice a day, 3.3%; placebo, 3.7%), mainly because of diarrhea. Laboratory and electrocardiogram parameters were comparable across groups. CONCLUSIONS: Tegaserod is well tolerated by patients with CC during 12 weeks of treatment. PMID- 16678077 TI - Serious infections and mortality in association with therapies for Crohn's disease: TREAT registry. AB - BACKGROUND & AIMS: Long-term safety data for infliximab and other therapies in Crohn's disease (CD) are needed. METHODS: We prospectively evaluated patients for prespecified safety-related outcomes. RESULTS: As of August 2004, 6290 patients were enrolled; 3179 received infliximab (5519 patient-years), 87% of whom received at least 2 infusions, and 3111 received other therapies (6123 patient years). The mean length of follow-up evaluation was 1.9 years. More infliximab treated patients had moderate-to-severe (30.8% vs 10.3%) or severe-fulminant (2.5% vs .6%) CD, and had surgical (17.5% vs 13.8%) or medical (14.4% vs 9.1%) hospitalizations in the previous year. More patients were taking prednisone (27.4% vs 16.1%), immunomodulators (49.4% vs 32.2%), or narcotic analgesics (9.8% vs 5.4%) when compared with those receiving other therapies (P<.001, all comparisons). The mortality rates were similar for infliximab- and non-infliximab treated patients (.53 per 100 patient-years vs .43; relative risk, 1.24; 95% confidence interval [CI], .73-2.10). In multivariate logistic regression analysis, only prednisone was associated with an increased mortality risk (odds ratio [OR], 2.10; 95% CI, 1.15-3.83; P=.016). Although the unadjusted analysis showed an increased risk for infection with infliximab use, multivariate logistic regression analysis suggested that infliximab was not an independent predictor of serious infections (OR, .99; 95% CI, .64-1.54). Factors independently associated with serious infections included prednisone use (OR, 2.21; 95% CI, 1.46-3.34; P<.001), narcotic analgesic use (OR, 2.38; 95% CI, 1.56-3.63; P<.001), and moderate-to-severe disease activity (OR, 2.11; 95% CI, 1.10-4.05; P=.024). CONCLUSIONS: Mortality rates were similar between infliximab- and non-infliximab treated patients. The increased risk for serious infection observed with infliximab likely was owing to disease severity and prednisone use. PMID- 16678078 TI - Capsule enteroscopy and Crohn's disease. PMID- 16678081 TI - Outcome of peptic ulcer bleeding. PMID- 16678082 TI - Learning to lead. AB - A successful research career requires not only an aptitude for science but also the mastering of other skills including communication, management, and grant writing. A growing number of programs at universities and research institutes aim to teach these crucial skills to graduate students, postdoctoral fellows, and junior faculty. PMID- 16678083 TI - The expanding cosmos of nuclear receptor coactivators. AB - About 200 coactivators play a central role in promoting gene expression mediated by nuclear receptors. This diverse group of proteins are key integrators of signals from steroid hormones and have been implicated in cancer and other diseases. PMID- 16678084 TI - Closing the GAP between polarity and vesicle transport. AB - How are tight junctions maintained? In this issue of Cell, Wells et al. (2006) provide intriguing evidence for a new pathway that links polarity proteins and vesicle transport to the maintenance of tight junctions, through the control of Cdc42 by Rich1, a GTPase-activating protein. PMID- 16678085 TI - Descrambling Dscam diversity. AB - Neuronal processes exhibit exquisitely complex branching patterns crucial for the formation of distinct neural circuits. In this issue of Cell, Chen et al. (2006) show that the isoform diversity of the Dscam protein in Drosophila is required to establish stereotypical axonal branching patterns, suggesting that nonrandom expression of Dscam alternative splice variants determines neural connectivity. PMID- 16678086 TI - Circadian transcription: passing the HAT to CLOCK. AB - In animals, the circadian timekeeping mechanism relies on the coordinated activities of activators and repressors to control rhythmic transcription. In this issue of Cell, Doi et al. (2006) reveal that rhythms in histone acetylation are necessary for rhythmic transcription and that the histone acetyl transferase responsible is CLOCK, a key transcription factor that is essential for circadian oscillator function. PMID- 16678087 TI - CAMTA in cardiac hypertrophy. AB - In this issue of Cell, the Calmodulin binding transcription activator 2 (CAMTA2), is shown by Song et al. (2006) to be an indispensable transcription coactivator for cardiac hypertrophy. CAMTA2 is activated by the dissociation of class II histone deacetylase 5 and promotes transcription of genes involved in cardiac hypertrophy through its interaction with Nkx2-5. PMID- 16678088 TI - Forcing the third dimension. AB - One goal of biomedical research is to reliably construct surrogate tissues for replacement therapy and to promote tissue regeneration. In this issue of Cell, Chun et al. (2006) provide insight into the molecular basis of tissue-specific differentiation. The authors show that remodeling of the extracellular matrix by the matrix metalloproteinase MT1-MMP contributes to the three-dimensional development of white adipose tissue in mice. PMID- 16678089 TI - A dedicated Wnt secretion factor. AB - The Wnt family of signaling proteins mediates cell-cell communication during development. In this issue of Cell, Banziger et al. (2006) and Bartscherer et al. (2006) identify Wntless/Evi, a multipass transmembrane protein in the secretory pathway of Wnt-producing cells that promotes Wnt secretion. PMID- 16678090 TI - New "hogs" in Hedgehog transport and signal reception. AB - A recent paper in Cell (Yao et al., 2006) and two papers in Developmental Cell (Tenzen et al., 2006; Zhang et al., 2006) identify a new receptor component for Hedgehog, a key morphogen in embryonic development. Many other proteins that bind to Hedgehog in the extracellular matrix or on the cell surface have been identified. In light of these recent discoveries, we discuss how these factors control the stability, transport, reception, and availability of Hedgehog in modulating Hedgehog-mediated responses. PMID- 16678091 TI - Intraflagellar transport and cilium-based signaling. AB - Cilia are specialized structures that not only play diverse roles in cell motility but also transmit signals to the cytoplasm and nucleus to control gene expression, cell function, animal development, and behavior. Cilia are assembled and maintained by the intraflagellar transport (IFT) machinery, which coordinates rapid, bidirectional transport between the cell body and the distal tip of the cilium. A new study (Wang et al., 2006) illuminates the role of IFT in cilium based signaling during mating in the alga Chlamydomonas. PMID- 16678092 TI - Molecular chaperones and protein quality control. AB - In living cells, both newly made and preexisting polypeptide chains are at constant risk for misfolding and aggregation. In accordance with the wide diversity of misfolded forms, elaborate quality-control strategies have evolved to counter these inevitable mishaps. Recent reports describe the removal of aggregates from the cytosol; reveal mechanisms for protein quality control in the endoplasmic reticulum; and provide new insight into two classes of molecular chaperones, the Hsp70 system and the AAA+ (Hsp100) unfoldases. PMID- 16678093 TI - The transcriptional coactivator CAMTA2 stimulates cardiac growth by opposing class II histone deacetylases. AB - Postnatal cardiac myocytes respond to diverse signals by hypertrophic growth and activation of a fetal gene program. In an effort to discover regulators of cardiac hypertrophy, we performed a eukaryotic expression screen for activators of the atrial natriuretic factor (ANF) gene, a cardiac-specific marker of hypertrophic signaling. We discovered that a family of transcriptional coactivators, called CAMTAs, promotes cardiomyocyte hypertrophy and activates the ANF gene, at least in part, by associating with the cardiac homeodomain protein Nkx2-5. The transcriptional activity of CAMTAs is governed by association with class II histone deacetylases (HDACs), which negatively regulate cardiac growth. Mice homozygous for a mutation in a CAMTA gene are defective in cardiac growth in response to pressure overload and neurohumoral signaling, whereas mice lacking HDAC5, a class II HDAC, are sensitized to the prohypertrophic actions of CAMTA. These findings reveal a transcriptional regulatory mechanism that modulates cardiac growth and gene expression by linking hypertrophic signals to the cardiac genome. PMID- 16678094 TI - Circadian regulator CLOCK is a histone acetyltransferase. AB - The molecular machinery that governs circadian rhythmicity comprises proteins whose interplay generates time-specific transcription of clock genes. The role of chromatin remodeling in a physiological setting such as the circadian clock is yet unclear. We show that the protein CLOCK, a central component of the circadian pacemaker, has histone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme A binding motifs within the MYST family of HATs. CLOCK displays high sequence similarity to ACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity for histones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HAT function. The HAT activity of CLOCK is essential to rescue circadian rhythmicity and activation of clock genes in Clock mutant cells. Identification of CLOCK as a novel type of DNA binding HAT reveals that chromatin remodeling is crucial for the core clock mechanism and identifies unforeseen links between histone acetylation and cellular physiology. PMID- 16678095 TI - Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. AB - Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu. PMID- 16678096 TI - Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. AB - Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion. PMID- 16678097 TI - A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells. AB - Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking. PMID- 16678098 TI - Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas. AB - Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium generated signaling. PMID- 16678099 TI - Specific bacterial suppressors of MAMP signaling upstream of MAPKKK in Arabidopsis innate immunity. AB - Plants and animals possess innate immune systems to prevent infections and are effectively "nonhosts" for most potential pathogens. The molecular mechanisms underlying nonhost immunity in plants remain obscure. In Arabidopsis, nonhost/nonpathogenic Pseudomonas syringae sustains but pathogenic P. syringae suppresses early MAMP (microbe-associated molecular pattern) marker-gene activation. We performed a cell-based genetic screen of virulence factors and identified AvrPto and AvrPtoB as potent and unique suppressors of early-defense gene transcription and MAP kinase (MAPK) signaling. Unlike effectors of mammalian pathogens, AvrPto and AvrPtoB intercept multiple MAMP-mediated signaling upstream of MAPKKK at the plasma membrane linked to the receptor. In transgenic Arabidopsis, AvrPto blocks early MAMP signaling and enables nonhost P. syringae growth. Deletions of avrPto and avrPtoB from pathogenic P. syringae reduce its virulence. The studies reveal a fundamental role of MAMP signaling in nonhost immunity, and a novel action of type III effectors from pathogenic bacteria. PMID- 16678100 TI - A pericellular collagenase directs the 3-dimensional development of white adipose tissue. AB - White adipose tissue (WAT) serves as the primary energy depot in the body by storing fat. During development, fat cell precursors (i.e., preadipocytes) undergo a hypertrophic response as they mature into lipid-laden adipocytes. However, the mechanisms that regulate adipocyte size and mass remain undefined. Herein, we demonstrate that the membrane-anchored metalloproteinase, MT1-MMP, coordinates adipocyte differentiation in vivo. In the absence of the protease, WAT development is aborted, leaving tissues populated by mini-adipocytes which render null mice lipodystrophic. While MT1-MMP preadipocytes display a cell autonomous defect in vivo, null progenitors retain the ability to differentiate into functional adipocytes during 2-dimensional (2-D) culture. By contrast, within the context of the 3-dimensional (3-D) ECM, normal adipocyte maturation requires a burst in MT1-MMP-mediated proteolysis that modulates pericellular collagen rigidity in a fashion that controls adipogenesis. Hence, MT1-MMP acts as a 3-D-specific adipogenic factor that directs the dynamic adipocyte-ECM interactions critical to WAT development. PMID- 16678101 TI - Homeodomain-mediated beta-catenin-dependent switching events dictate cell-lineage determination. AB - While the biological roles of canonical Wnt/beta-catenin signaling in development and disease are well documented, understanding the molecular logic underlying the functionally distinct nuclear transcriptional programs mediating the diverse functions of beta-catenin remains a major challenge. Here, we report an unexpected strategy for beta-catenin-dependent regulation of cell-lineage determination based on interactions between beta-catenin and a specific homeodomain factor, Prop1, rather than Lef/Tcfs. beta-catenin acts as a binary switch to simultaneously activate expression of the critical lineage-determining transcription factor, Pit1, and to repress the gene encoding the lineage inhibiting transcription factor, Hesx1, acting via TLE/Reptin/HDAC1 corepressor complexes. The strategy of functionally distinct actions of a homeodomain factor in response to Wnt signaling is suggested to be prototypic of a widely used mechanism for generating diverse cell types from pluripotent precursor cells in response to common signaling pathways during organogenesis. PMID- 16678102 TI - The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila. AB - Alternative splicing of Dscam generates an enormous molecular diversity with maximally 38,016 different receptors. Whether this large diversity is required in vivo is currently unclear. We examined the role of Dscam in neuron-target recognition of single mechanosensory neurons, which connect with different target cells through multiple axonal branches. Analysis of Dscam null neurons demonstrated an essential role of Dscam for growth and directed extension of axon branches. Expression of randomly chosen single isoforms could not rescue connectivity but did restore basic axonal extension and rudimentary branching. Moreover, two Dscam alleles were generated that each reduced the maximally possible Dscam diversity to 22,176 isoforms. Reduction of Dscam diversity resulted in specific connectivity defects of mechanosensory neurons. Furthermore, the observed allele-specific phenotypes suggest functional differences among isoforms. Our findings provide evidence that a very large number of structurally unique receptor isoforms is required to ensure fidelity and precision of neuronal connectivity. PMID- 16678103 TI - Silencing the transcriptome's dark matter: mechanisms for suppressing translation of intergenic transcripts. AB - Large portions of the genomes of higher eukaryotes are transcribed into RNA molecules that are never destined for translation into proteins. Although some of these transcripts have clearly defined biological roles other than protein coding, most arise from genomic regions devoid of functional genes and many are antisense to regions containing annotated genes. A variety of mechanisms exist to prevent adventitious production of proteins from these transcripts, ranging from degradation within the nucleus to translational silencing in the cytosol. PMID- 16678104 TI - Interaction between c-Abl and Arg tyrosine kinases and proteasome subunit PSMA7 regulates proteasome degradation. AB - Proteasome-mediated proteolysis is a primary protein degradation pathway in cells. The present study demonstrates that c-Abl and Arg (abl-related gene) tyrosine kinases associate with and phosphorylate the proteasome PSMA7 (alpha4) subunit at Tyr-153. Consequently, proteasome-dependent proteolysis is compromised. Notably, cells expressing a phosphorylation mutant of PSMA7(Y153F) display impaired G1/S transition and S/G2 progression, highlighting the biological significance of tyrosine phosphorylation of a proteasome subunit as an important cellular regulatory control. PMID- 16678105 TI - Mitochondrial transcription is regulated via an ATP "sensing" mechanism that couples RNA abundance to respiration. AB - The information encoded in both the nuclear and mitochondrial genomes must be coordinately regulated to respond to changes in cellular growth and energy states. Despite identification of the mitochondrial RNA polymerase (mtRNAP) from several organisms, little is known about mitochondrial transcriptional regulation. Studying the shift from fermentation to respiration in Saccharomyces cerevisiae, we have demonstrated a direct correlation between in vivo changes in mitochondrial transcript abundance and in vitro sensitivity of mitochondrial promoters to ATP concentration (K(m)ATP). Consistent with the idea that the mtRNAP itself senses in vivo ATP levels, we found that transcript abundance correlates with respiration, but only when coupled to mitochondrial ATP synthesis. In addition, we characterized mutations in the mitochondrial promoter and the mtRNAP accessory factor Mtf1 that alter both in vitro K(m)ATP and in vivo transcription in response to respiratory changes. We propose that shifting cellular pools of ATP coordinately control nuclear and mitochondrial transcription. PMID- 16678106 TI - Crystal structures of the vaccinia virus polyadenylate polymerase heterodimer: insights into ATP selectivity and processivity. AB - Polyadenylation of mRNAs in poxviruses, crucial for virion maturation, is carried out by a poly(A) polymerase heterodimer composed of a catalytic component, VP55, and a processivity factor, VP39. The ATP-gamma-S bound and unbound crystal structures of the vaccinia polymerase reveal an unusual architecture for VP55 that comprises of N-terminal, central or catalytic, and C-terminal domains with different topologies and that differs from many polymerases, including the eukaryotic poly(A) polymerases. Residues in the active site of VP55, located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition. VP55's concave surface docks the globular VP39. A model for RNA primer binding that involves all three VP55 domains and VP39 is proposed. The model supports biochemical evidence that VP39 functions as a processivity factor by partially enclosing the RNA primer at the heterodimer interface. PMID- 16678107 TI - Intergenic transcripts regulate the epigenetic state of rRNA genes. AB - Transcripts originating from the intergenic spacer (IGS) that separates rRNA genes (rDNA) have been known for two decades; their biological role, however, is largely unknown. Here we show that IGS transcripts are required for establishing and maintaining a specific heterochromatic configuration at the promoter of a subset of rDNA arrays. The mechanism of action appears to be mediated through the interaction of TIP5, the large subunit of the chromatin remodeling complex NoRC, with 150-300 nucleotide RNAs that are complementary in sequence to the rDNA promoter. Mutations that abrogate RNA binding of TIP5 impair the association of NoRC with rDNA and fail to promote H3K9&H4K20 methylation and HP1 recruitment. Knockdown of IGS transcripts abolishes the nucleolar localization of NoRC, decreases DNA methylation, and enhances rDNA transcription. The results reveal an important contribution of processed IGS transcripts in chromatin structure and epigenetic control of the rDNA locus. PMID- 16678108 TI - The structure of the yFACT Pob3-M domain, its interaction with the DNA replication factor RPA, and a potential role in nucleosome deposition. AB - We report the crystal structure of the middle domain of the Pob3 subunit (Pob3-M) of S. cerevisiae FACT (yFACT, facilitates chromatin transcription), which unexpectedly adopts an unusual double pleckstrin homology (PH) architecture. A mutation within a conserved surface cluster in this domain causes a defect in DNA replication that is suppressed by mutation of replication protein A (RPA). The nucleosome reorganizer yFACT therefore interacts in a physiologically important way with the central single-strand DNA (ssDNA) binding factor RPA to promote a step in DNA replication. Purified yFACT and RPA display a weak direct physical interaction, although the genetic suppression is not explained by simple changes in affinity between the purified proteins. Further genetic analysis suggests that coordinated function by yFACT and RPA is important during nucleosome deposition. These results support the model that the FACT family has an essential role in constructing nucleosomes during DNA replication, and suggest that RPA contributes to this process. PMID- 16678109 TI - The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells. AB - The Per1 gene is a core clock factor that plays an essential role in generating circadian rhythms. Recent data reveal that major biological pathways, including those critical to cell division, are under circadian control. We report here that Per1 provides an important link between the circadian system and the cell cycle system. Overexpression of Per1 sensitized human cancer cells to DNA damage induced apoptosis; in contrast, inhibition of Per1 in similarly treated cells blunted apoptosis. The apoptotic phenotype was associated with altered expression of key cell cycle regulators. In addition, Per1 interacted with the checkpoint proteins ATM and Chk2. Ectopic expression of Per1 in human cancer cell lines led to significant growth reduction. Finally, Per1 levels were reduced in human cancer patient samples. Our results highlight the importance of circadian regulation to fundamental cellular functions and support the hypothesis that disruption of core clock genes may lead to cancer development. PMID- 16678110 TI - Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage. AB - Posttranslational histone modifications play important roles in transcription and other chromatin-based processes. Compared to acetylation, methylation, and phosphorylation, very little is known about the function of histone ubiquitylation. Here, we report the purification and functional characterization of a histone H3 and H4 ubiquitin ligase complex, CUL4-DDB-ROC1. We demonstrate that CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation occurs both in vitro and in vivo. Importantly, CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation is regulated by UV irradiation. Reduction of histone H3 and H4 ubiquitylation by knockdown of CUL4A impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Biochemical studies indicate that CUL4-DDB-ROC1 mediated histone ubiquitylation weakens the interaction between histones and DNA and facilitates the recruitment of repair proteins to damaged DNA. Thus, our studies uncover CUL4-DDB-ROC1 as a histone ubiquitin ligase and demonstrate that histone H3 and H4 ubiquitylation participates in the cellular response to DNA damage. PMID- 16678111 TI - p53 stabilization and transactivation by a von Hippel-Lindau protein. AB - von Hippel-Lindau (VHL) disease is a rare autosomal dominant cancer syndrome. Although hypoxia-inducible factor-alpha (HIFalpha) is a well-documented substrate of von Hippel-Lindau tumor suppressor protein (pVHL), it remains unclear whether the dysregulation of HIF is sufficient to account for de novo tumorigenesis in VHL-deleted cells. Here we found that pVHL directly associates with and stabilizes p53 by suppressing Mdm2-mediated ubiquitination and nuclear export of p53. Moreover, upon genotoxic stress, pVHL invoked an interaction between p53 and p300 and the acetylation of p53, which ultimately led to an increase in p53 transcriptional activity and p53-mediated cell cycle arrest and apoptosis. These results suggest that the tumor suppressor pVHL has an unexpected function to upregulate the tumor suppressor p53. PMID- 16678112 TI - p53 and p21 regulate error-prone DNA repair to yield a lower mutation load. AB - Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability. PMID- 16678113 TI - Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II. AB - Histones are rapidly evicted and deposited during transcription by RNA polymerase (Pol) II, but a factor that mediates histone eviction in vivo has not yet been identified. Here, we show that the histone chaperone Asf1 associates with promoters and coding regions of transcriptionally active genes. Asf1 mediates histone H3, but not H2B, eviction and deposition during Pol II elongation, suggesting that nucleosome assembly and disassembly occur in a stepwise fashion. Lastly, Asf1 inhibits internal initiation from cryptic promoters within coding regions. These results strongly suggest that Asf1 functions as an elongation factor to disassemble and reassemble histones during Pol II elongation. PMID- 16678114 TI - RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi. AB - The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events. PMID- 16678115 TI - Cytotoxic effect of curcumin on Giardia lamblia trophozoites. AB - Giardia lamblia is one of the most important worldwide causes of intestinal infections produced by protozoa. Thus, the search for new alternative therapeutic approaches for this parasitic disease is very important. Common drugs used to control and eradicate this infection, frequently exhibit side effects that force patients to abandon treatment. The present work evaluates the anti-protozoan activity of curcumin, the main constituent of turmeric. Axenic G. lamblia (Portland 1 strain) cultures were exposed to different concentrations of curcumin. Its effects were evaluated on parasite growth, adhesion capacity and parasite morphology. We also evaluated the capacity of curcumin to induce an apoptosis-like effect. All curcumin concentrations inhibited trophozoite growth and adhesion in more than 50% in dose and time dependent manner. Morphological changes were described as protrusions formed under the cytoplasmic membrane, deformation due to swelling and cell agglutination. Curcumin induced apoptosis like nuclear staining in dose and time dependent manner. In conclusion, curcumin exhibited a cytotoxic effect in G. lamblia inhibiting the parasite growth and adherent capacity, induced morphological alterations, provoked apoptosis-like changes. Future in vitro and in vivo experiments are endowed to elucidate the effect of curcumin in an experimental model of G. lamblia infection, analyze the involvement of ion channels in the swelling effect of curcumin during an apparent osmotic deregulation in G. lamblia trophozoites. This will lead to the proposal of the action mechanism of curcumin as well as the description of mechanism involved during the activation process for the apoptotic-like effect. PMID- 16678116 TI - Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress. AB - Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress. PMID- 16678118 TI - Alpha-helical antimicrobial peptides--using a sequence template to guide structure-activity relationship studies. AB - An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, alpha-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the 'sequence template' approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides. PMID- 16678117 TI - Generation of superoxide by the mitochondrial Complex I. AB - Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS controlled processes under physiologically relevant conditions. PMID- 16678119 TI - Peptide-lipid interactions of the beta-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR. AB - The peptide-lipid interaction of a beta-hairpin antimicrobial peptide tachyplesin 1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. (31)P and (2)H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other beta-hairpin antimicrobial peptides are discussed in terms of their molecular structure. PMID- 16678120 TI - Pulling single bacteriorhodopsin out of a membrane: Comparison of simulation and experiment. AB - Mechanical unfolding of single bacteriorhodopsins from a membrane bilayer is studied using molecular dynamics simulations. The initial conformation of the lipid membrane is determined through all-atom simulations and then its coarse grained representation is used in the studies of stretching. A Go-like model with a realistic contact map and with Lennard-Jones contact interactions is applied to model the protein-membrane system. The model qualitatively reproduces the experimentally observed differences between force-extension patterns obtained on bacteriorhodopsin at different temperatures and predicts a lack of symmetry in the choice of the terminus to pull by. It also illustrates the decisive role of the interactions of the protein with the membrane in determining the force pattern and thus the stability of transmembrane proteins. PMID- 16678121 TI - On the measurement of rumination: a psychometric evaluation of the ruminative response scale and the rumination on sadness scale in undergraduates. AB - Rumination is considered a specific cognitive vulnerability factor that is thought to play a prominent role in the maintenance of depressive symptoms. The present study investigated the psychometric properties of two measures of rumination, the ruminative response scale (RRS) and the rumination on sadness scale (RSS) in undergraduates (N=331). A joint factor analysis yielded three factors, 'rumination on causes of sadness', 'symptom-based rumination', and 'rumination on sadness'. The internal consistency of the rumination factors was good and the test-retest stability over a 6-month period of time was moderate. Support was also found for the construct validity of the rumination factors. Finally, the 'rumination on the causes of sadness' factor was found to moderate the relation between depression measured at baseline and at 6-month follow-up. More specifically, baseline depression was a strong predictor of future depression but this was particularly true for high ruminating individuals. Implications of the results and directions for future research are provided. PMID- 16678122 TI - The role of Paraxial Protocadherin in Xenopus otic placode development. AB - Vertebrate inner ear develops from its rudiment, otic placode, which later forms otic vesicle and gives rise to tissues comprising the entire inner ear. Although several signaling molecules have been identified as candidates responsible for inner ear specification and patterning, many details remain elusive. Here, we report that Paraxial Protocadherin (PAPC) is required for otic vesicle formation in Xenopus embryos. PAPC is expressed strictly in presumptive otic placode and later in otic vesicle during inner ear morphogenesis. Knockdown of PAPC by dominant-negative PAPC results in the failure of otic vesicle formation and the loss of early inner ear markers Sox9 and Tbx2, suggesting the requirement of PAPC in the early stage of otic vesicle development. However, PAPC alone is not sufficient to induce otic placode formation. PMID- 16678123 TI - ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion. AB - We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p<0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p<0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p<0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention. PMID- 16678124 TI - Testicular proteins associated with the germ cell-marker, TEX101: involvement of cellubrevin in TEX101-trafficking to the cell surface during spermatogenesis. AB - Recently, we identified a cell-surface marker protein, TEX101, that is unique to male and female germ cells. On/off switching of TEX101 expression in germ cells is closely linked to the kinetics of gametogenesis. In the present study, we isolated testicular proteins by immunoprecipitation with anti-TEX101 antibody and identified the proteins using liquid chromatography/tandem mass spectrometry. Of three proteins identified (annexin 2, ly6k, and cellubrevin), a biochemical association between TEX101 and cellubrevin was confirmed by immunoprecipitation Western blotting experiments. Immunohistochemistry using a cellubrevin-specific antibody indicated that the molecule is abundant on spermatocytes and early-stage spermatids, whereas negligible amounts are found in Sertoli cells, spermatogonia, spermatozoa, and late-stage spermatids. Most of the intracellular cellubrevin appeared to be juxtaposed with intracellular TEX101, and membrane-associated cellubrevin was docked near TEX101-positive plasma membranes on the cytoplasmic side. This close association was never observed on the outer surface of the plasma membrane. From these results we concluded that cellubrevin-dependent membrane trafficking is involved in TEX101-transport to the surface of male germ cells. PMID- 16678125 TI - Adiponectin mediates antiproliferative and apoptotic responses in human MCF7 breast cancer cells. AB - It is well established that obesity is a risk factor for breast cancer and that blood levels of adiponectin, a hormone mainly secreted by white adipocytes, are inversely correlated with the body fat mass. As adiponectin elicits anti proliferative effects in some cell types, we tested the hypothesis that adiponectin could influence human breast cancer MCF-7 cell growth. Here we show that MCF-7 cells express adiponectin receptors and respond to human recombinant adiponectin by reducing their growth, AMPkinase activation, and p42/p44 MAPkinase inactivation. Further, we demonstrate that the anti-proliferative effect of adiponectin involves activation of cell apoptosis and inhibition of cell cycle. These findings suggest that adiponectin could act in vivo as a paracrine/endocrine growth inhibitor towards mammary epithelial cells. Moreover, adipose adiponectin production being strongly reduced in obesity, this study may help to explain why obesity is a risk factor of developing breast cancers. PMID- 16678126 TI - Two distinct ubiquitin-dependent mechanisms are involved in NF-kappaB p105 proteolysis. AB - Generation of the p50 subunit of NF-kappaB is a rare case in which the ubiquitin system processes a longer precursor, p105, into a shorter active subunit: in the vast majority of cases, the target protein is completely degraded. The mechanisms involved in this process have remained elusive. It appears that a Gly rich region (GRR) in the middle of the molecule serves as a "processing stop signal", though under certain conditions, such as after stimulation, p105 can be completely degraded. Since NF-kappaB plays critical roles in a broad array of basic cellular processes, it is important to dissect the mechanisms that regulate its proteolysis-both destruction and processing. We have previously shown that signal induced degradation of p105 requires ubiquitination on multiple lysines. Here we describe a novel region, a Processing Inhibitory Domain-PID, that upon its removal, the molecule is processed in high efficiency, which requires ubiquitination on a single, though non-specific, lysine. PMID- 16678127 TI - Stable knock-down of vomeronasal receptor genes in transgenic Xenopus tadpoles. AB - Xenopus V2R (xV2R), a family of G-protein-coupled receptors with seven transmembrane domains, is expressed in the Xenopus vomeronasal organ (VNO). There are six subgroups of xV2R, one of which, xV2RE, is predominantly expressed in the VNO. To understand the function of xV2R during VNO development, we developed a new method to achieve stable siRNA-suppression of the V2RE genes by introducing siRNA expression transgenes into the genomes of unfertilized eggs. We found that some of the derived transgenic tadpoles lacked VNOs and that their olfactory epithelium was fused. With the exception of one tadpole, expression of xV2RE was not detected in morphologically abnormal mutant tadpoles, although the olfactory marker protein and the olfactory receptors were expressed. These results suggest that we successfully produced transgenic tadpoles in which xV2RE expression was stably suppressed by siRNA, and that xV2RE plays a role in the morphogenesis of olfactory organs. PMID- 16678128 TI - Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist alpha-conotoxin OmIA that discriminates alpha3 vs. alpha6 nAChR subtypes. AB - alpha-Conotoxin OmIA from Conus omaria is the only alpha-conotoxin that shows a approximately 20-fold higher affinity to the alpha3beta2 over the alpha6beta2 subtype of nicotinic acetylcholine receptor. We have determined a three dimensional structure of alpha-conotoxin OmIA by nuclear magnetic resonance spectroscopy. alpha-Conotoxin OmIA has an "omega-shaped" overall topology with His(5)-Asn(12) forming an alpha-helix. Structural features of alpha-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related alpha4/7 subfamily conotoxins. Reduced size of the hydrophilic area in alpha-conotoxin OmIA seems to be associated with the reduced affinity towards the alpha6beta2 nAChR subtype. PMID- 16678129 TI - Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins. AB - Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation. PMID- 16678130 TI - Selective destabilization of soluble amyloid beta oligomers by divalent metal ions. AB - Aggregation of the amyloid beta (Abeta) peptide yields both fibrillar precipitates and soluble oligomers, and is associated with Alzheimer's disease (AD). In vitro, Cu(2+) and Zn(2+) strongly bind Abeta and promote its precipitation. However, less is known about their interactions with the soluble oligomers, which are thought to be the major toxic species responsible for AD. Using fluorescence correlation spectroscopy to resolve the various soluble species of Abeta, we show that low concentrations of Cu(2+) (1 microM) and Zn(2+) (4 microM) selectively eliminate the oligomeric population (within approximately 2h), while Mg(2+) displays a similar effect at a higher concentration (60 microM). This uncovers a new aspect of Abeta-metal ion interactions, as precipitation is not substantially altered at these low metal ion concentrations. Our results suggest that physiological concentrations of Cu(2+) and Zn(2+) can critically alter the stability of the toxic Abeta oligomers and can potentially control the course of neurodegeneration. PMID- 16678132 TI - Effect of photo-immobilization of epidermal growth factor on the cellular behaviors. AB - We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg(2+) although integrin-mediated cell adhesion to natural ECMs is dependent on Mg(2+). Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF. PMID- 16678131 TI - Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F actin in ghost fibers during the actomyosin ATPase cycle. AB - New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin. PMID- 16678133 TI - alpha-Lactalbumin species variation, HAMLET formation, and tumor cell death. AB - HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content. PMID- 16678134 TI - Peroxisomal cholesterol biosynthesis and Smith-Lemli-Opitz syndrome. AB - Smith-Lemli-Opitz syndrome (SLOS), caused by 7-dehydrocholesterol-reductase (DHCR7) deficiency, shows variable severity independent of DHCR7 genotype. To test whether peroxisomes are involved in alternative cholesterol synthesis, we used [1-(14)C]C24:0 for peroxisomal beta-oxidation to generate [1-(14)C]acetyl CoA as cholesterol precursor inside peroxisomes. The HMG-CoA reductase inhibitor lovastatin suppressed cholesterol synthesis from [2-(14)C]acetate and [1 (14)C]C8:0 but not from [1-(14)C]C24:0, implicating a peroxisomal, lovastatin resistant HMG-CoA reductase. In SLOS fibroblasts lacking DHCR7 activity, no cholesterol was formed from [1-(14)C]C24:0-derived [1-(14)C]acetyl-CoA, indicating that the alternative peroxisomal pathway also requires this enzyme. Our results implicate peroxisomes in cholesterol biosynthesis but provide no link to phenotypic variation in SLOS. PMID- 16678135 TI - Biological activity of Tat (47-58) peptide on human pathogenic fungi. AB - Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV 1 regulatory protein Tat, is known for a peptidic delivery factor as a cell penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase. PMID- 16678136 TI - Structure and dynamics of the N-terminal loop of PsbQ from photosystem II of Spinacia oleracea. AB - Infrared and Raman spectroscopy were applied to identify restraints for the structure determination of the 20 amino acid loop between two beta-sheets of the N-terminal region of the PsbQ protein of the oxygen evolving complex of photosystem II from Spinacia oleracea by restraint-based homology modeling. One of the initial models has shown a stable fold of the loop in a 20 ns molecular dynamics simulation that is in accordance with spectroscopic data. Cleavage of the first 12 amino acids leads to a permanent drift in the root means square deviation of the protein backbone and induces major structural changes. PMID- 16678137 TI - Cytosolic heat shock proteins and heme oxygenase-1 are preferentially induced in response to specific and localized intramitochondrial damage by tetrafluoroethylcysteine. AB - Previously, S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC) was shown to mediate cytotoxicity by covalently modifying a well-defined group of intramitochondrial proteins including aconitase, alpha-ketoglutarate dehydrogenase (alphaKGDH) subunits, heat shock protein 60 (HSP60) and mitochondrial HSP70 (mtHSP70). To investigate the cellular responses to this mitochondrial damage, microarray analysis of TFEC treated murine hepatocytes of the TAMH cell line was carried out. Results of these studies revealed a HSP response that was significantly stronger than other well-characterized hepatotoxicants including acetaminophen, diquat and rotenone. Specifically, cytosolic HSP25, HSP40, HSP70, HSP105 and microsomal HSP32 (HO-1) were strongly upregulated within the first few hours of TFEC treatment, while little change was observed among other HSPs that are predominantly localized in the mitochondria and endoplasmic reticulum (ER). Post translational modification of HSP25 was also observed with the appearance of a unique DTT-resistant immunoreactive band at about 50kDa, a putative dimer. The biological significance of HSP responses to TFEC-induced toxicity were subsequently demonstrated using the "gain of function" pretreatment: heat shock. Overall, we report an atypical HSP induction profile that does not conform to changes expected of a classical temperature shock. Furthermore, despite a well defined intramitochondrial origin of toxicity, TFEC rapidly evokes an early and strong upregulation of cytosolic stress proteins. The cytoprotective effects of such HSP responses suggest a plausible role in modulating the progression of TFEC induced cellular injury. PMID- 16678138 TI - Semantics prevalence over syntax during sentence processing: a brain potential study of noun-adjective agreement in Spanish. AB - A review of the literature about the interplay of syntax and semantics, using event-related brain potentials (ERPs), revealed that the results are highly heterogeneous, owing to several possible variables. An experiment was conducted with Spanish sentences that factorially combined syntactic and semantic violations in the same sentence-intermediate adjective and controlled for working memory demands, variables that in previous studies have rarely been taken into consideration. Violations consisted in noun-adjective number or gender disagreements (syntactic violation), noun-adjective semantic incompatibility (semantic violation), or both (combined violation). The N400 to semantic violations was unaffected by additional syntactic violations. The P600/SPS component, considered to reflect syntactic processes, was elicited by both single syntactic and semantic violations but seemed to be diminished in combined violations relative to single syntactic violations. These results suggest that under the conditions of the present experiment semantic information may have a prevailing role over syntactic information. PMID- 16678139 TI - Paeoniflorin attenuates chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rats. AB - Chronic cerebral hypoperfusion, a mild ischemic condition, is associated with the cognitive deficits of AD. Paeoniflorin (PF), a major constituent of peony root, was proved to be neuroprotective in middle cerebral artery occlusion model. In this study, we investigated whether PF could attenuate chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rat. Seven weeks after permanent bilateral occlusion of the common carotid arteries, the rats were tested in the Morris water maze. Subsequently, the animals were sacrificed and neurons, astrocytes and microglias were labeled with immunocytochemistry in hippocampus. PF at the dose of 2.5 mg/kg ameliorated cerebral hypoperfusion related learning dysfunction and prevented CA1 neuron damage. Chronic cerebral hypoperfusion increased the immunoreactivity of astrocytes and microglias in hippocampus. The increase was prevented by PF at the dose of 2.5 mg/kg. Cerebral hypoperfusion also increased expression of nuclear factor-kappaB (NF-kappaB), mostly in astrocytes, but not in neurons. With the treatment of PF (2.5 mg/kg), NF-kappaB immunostaining was diminished in hippocampus. Our results demonstrated that PF could attenuate cognitive deficit and brain damage induced by chronic cerebral hypoperfusion and that suppression of neuroinflammatory reaction in brain might be involved in PF-induced neuroprotection. PMID- 16678140 TI - Low penetrance, subclinical congenital LQTS: concealed LQTS or silent LQTS? PMID- 16678141 TI - Revisiting p53 and its effectors in ischemic heart injury. PMID- 16678142 TI - Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa. AB - Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. Therefore, it is of great interest to get insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this paper we report on the phase behaviour of mixtures of dielaidoylphosphatidylethanolamine (DEPE) with a purified dirhamnolipid (DiRL) fraction from Pseudomonas aeruginosa, as studied by a number of physical techniques such as differential scanning calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering. Our data indicate that the presence of DiRL counteracts the tendency of DEPE to form vesicular aggregates of large size, forming vesicles of smaller diameter which most probably have a lower lamellarity index. The partial phase diagram obtained from calorimetric data shows a complex behaviour with a solid-phase immiscibility. X-ray diffraction shows that DiRL has a bilayer stabilizing effect, impeding formation of the inverted hexagonal-HII phase of DEPE. The presented data are discussed focussing into how DiRL/DEPE interactions could help to explain the membrane perturbing activities of this biosurfactant. PMID- 16678143 TI - Application of Streptomyces and Brevibacterium cholesterol oxidase for total serum cholesterol assay by the enzymatic kinetic method. AB - BACKGROUND: Using non-esterified cholesterol standard, Brevibacterium and Streptomyces are found as suitable sources of cholesterol oxidase for kinetic cholesterol assay. For clinical use, we investigated the suitability of these enzymes for cholesterol determination in human serum. METHODS: We compared the performance of reagents containing 2 enzymes for the kinetic determination of total serum cholesterol with the standardized endpoint method. RESULTS: Reagent containing Streptomyces enzyme was more sensitive than that of Brevibacterium, with linearity up to 20.7 and 2.6 mmol/l, respectively. The analytical reaction for Streptomyces showed a shorter lag phase (148 s) and a steeper slope (absorbance vs. time) than that of Brevibacterium (246 s). The assay using Streptomyces reagent was precise and accurate and compared favorably with the endpoint method (y=1.06x-0.15, r=0.996, bias=0.21 mmol/l). Hemoglobin as high as 7.5 g/l did not interfere while turbidity greater than 2+ (absorbance >0.778 at 670 nm) and bilirubin concentrations >171.0 micromol/l did interfere (in a negative interference). Reagent was stable up to at least 8 weeks. CONCLUSIONS: The Streptomyces cholesterol oxidase, with 3,4-dichlorophenol, proved a suitable source for serum total cholesterol determination by the kinetic method. PMID- 16678144 TI - Assessment of urinary N-telopeptide: point-of-care testing, sample types, and relationship to urinary helical peptide excretion. AB - BACKGROUND: A point-of-care (POC) device would be useful in the space program for measuring N-telopeptide (NTX), a marker of bone resorption. This study was done to establish whether NTX measurements from a POC device compare favorably with standard (ELISA) techniques. We also compared results from fresh and frozen urine samples, samples collected on consecutive days, and second voids (of the day) and 24-h urine pools. Helical peptide (HP), another crosslink, was compared in second voids and 24-h urine pools. METHODS: Ten subjects collected urine for 96 h. NTX was measured with the POC device and by ELISA, and HP measured by ELISA. Seven subjects collected 24-h urine pools, and samples were analyzed fresh and after 27 days of freezing. RESULTS: Excretion of NTX was greater (P<0.05) when measured by the POC device than when measured by ELISA, but was not different between second voids and 24-h urine pools, or among days. HP was similar in second voids and 24 h pools. Less NTX (P<0.05) was found in fresh 24-h pools [mean (S.D.) values, 38.4 (11.6) and 33.6 (9.2) nmol/mmol creatinine for the POC device and ELISA] than in previously frozen 24-h pools [42.7 (17.4) and 41.5 (12.5) nmol/mmol creatinine for the POC device and ELISA]. CONCLUSIONS: Comparisons between NTX measurements from frozen and fresh samples and those analyzed by POC and ELISA techniques should be made with caution. PMID- 16678145 TI - From mere coincidences to meaningful discoveries. AB - People's reactions to coincidences are often cited as an illustration of the irrationality of human reasoning about chance. We argue that coincidences may be better understood in terms of rational statistical inference, based on their functional role in processes of causal discovery and theory revision. We present a formal definition of coincidences in the context of a Bayesian framework for causal induction: a coincidence is an event that provides support for an alternative to a currently favored causal theory, but not necessarily enough support to accept that alternative in light of its low prior probability. We test the qualitative and quantitative predictions of this account through a series of experiments that examine the transition from coincidence to evidence, the correspondence between the strength of coincidences and the statistical support for causal structure, and the relationship between causes and coincidences. Our results indicate that people can accurately assess the strength of coincidences, suggesting that irrational conclusions drawn from coincidences are the consequence of overestimation of the plausibility of novel causal forces. We discuss the implications of our account for understanding the role of coincidences in theory change. PMID- 16678146 TI - Comparison of confirmation measures. AB - Alternative measures of confirmation or evidential support have been proposed to express the impact of ascertaining one event on the credibility of another. We report an experiment that compares the adequacy of several such measures as descriptions of confirmation judgment in a probabilistic context. PMID- 16678147 TI - The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development. AB - Accumulating evidence suggests that in the vertebrate embryo, acquisition of arterial and venous identity is established early by genetic mechanisms, including those regulated by vascular endothelial growth factor (VEGF) and Notch signaling. However, although the COUP-TFII nuclear receptor has recently been shown to regulate vein identity, very little is known about the molecular mechanisms of transcriptional regulation in arterial specification. Here, we show that mouse embryos compound mutant for Foxc1 and Foxc2, two closely related Fox transcription factors, exhibit arteriovenous malformations and lack of induction of arterial markers whereas venous markers such as COUP-TFII are normally expressed, suggesting that mutant endothelial cells fail to acquire an arterial fate. Notably, consistent with this observation, overexpression of Foxc genes in vitro induces expression of arterial markers such as Notch1 and its ligand Delta like 4 (Dll4), and Foxc1 and Foxc2 directly activate the Dll4 promoter via a Foxc binding site. Moreover, compound Foxc mutants show a defect in sprouting of lymphatic endothelial cells from veins in early lymphatic development, due to reduced expression of VEGF-C. Taken together, our results demonstrate that Foxc transcription factors are novel regulators of arterial cell specification upstream of Notch signaling and lymphatic sprouting during embryonic development. PMID- 16678148 TI - Development of the primary mouth in Xenopus laevis. AB - The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure. PMID- 16678149 TI - Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning. AB - Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1 dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains. PMID- 16678150 TI - Proneural gene requirement for hair cell differentiation in the zebrafish lateral line. AB - The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects. PMID- 16678151 TI - C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development. AB - Cytoplasmic polyadenylation element-binding proteins (CPEBs) are well-conserved RNA-binding proteins, which regulate mRNA translation mainly through control of poly(A) elongation. Here, we show that CPB-3, one of the four CPEB homologs in C. elegans, positively regulates multiple aspects of oocyte production. CPB-3 protein was highly expressed in early meiotic regions of the hermaphrodite gonad. Worms deficient in cpb-3 were apparently impaired in germ cell proliferation and differentiation including sperm/oocyte switching and progression of female meiosis. We also show that cpb-3 is likely to promote the meiotic entry in parallel with gld-3, a component of one of the redundant but essential genetic pathways for the entry to and progression through meiosis. Taken together, CPEB appears to have a conserved role in the early phase of meiosis and in the sperm/oocyte specification, in addition to its reported function during meiotic progression. PMID- 16678152 TI - Contrasting effects of age on the plasma/whole blood lead ratio in men and women with a history of lead exposure. AB - We examined the effect of age and sex on the relationship between the concentrations of Pb in blood (Pb-B) and in plasma (Pb-P) in an adult population with a history of lead exposure. Pb-P was determined by inductively coupled plasma mass spectrometry (ICP-MS) and Pb-B by graphite furnace atomic absorption spectrometry (GF AAS). We studied 154 adults (56 men and 98 women) from 18 to 60 year old. Pb-B levels varied from 10.0 to 428.0 microg/L, with a mean of 76 microg/L. Blood lead levels varied from 10.0 to 428.0 microg/L in men (mean, 98.3 microg/L) and from 10.0 to 263.0 microg/L (mean, 62.8 microg/L) in women. Corresponding Pb-Ps were 0.02-2.9 microg/L (mean, 0.66 microg/L) and 0.02-1.5 microg/L (mean, 0.42 microg/L) in men and women, respectively. The relationship between Pb-B and Pb-P was found to be curvilinear (r = 0.757, P < 0.001 Spearman's correlation). The two quantities are related by the line y = 0.0006x(1492) (y = Pb-P, and x = Pb-B). The %Pb-P/Pb-B ratio ranged from 0.03% to 1.85%. A positive association was found between %Pb-P/Pb-B ratio and Pb-B levels. When data were separated by sex, this association was also relevant for men (y = 0.0184x(0.702)) and women (y = 0.0534x(0.5209)) (y = %Pb-P/Pb-B and x = Pb-B). Moreover, we found an interesting positive correlation between Log (Pb-P/Pb-B) and age for women (r = 0.31, P < 0.0001) and a negative correlation for men (r = 0.164, P = 0.07). Taken together, these results suggest contrasting effects of age on the plasma/whole blood lead ratio in men and women with a history of lead exposure. Moreover, sex might play an important role in the metabolism of lead, implying further consideration on the kinetic models constructed of lead toxicity. PMID- 16678153 TI - Detection of triclocarban and two co-contaminating chlorocarbanilides in US aquatic environments using isotope dilution liquid chromatography tandem mass spectrometry. AB - The antimicrobial compound triclocarban (TCC; 3,4,4'-trichlorocarbanilide; CAS# 101-20-2) is a high-production-volume chemical, recently suggested to cause widespread contamination of US water resources. To test this hypothesis, we developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry method for ultratrace analysis of TCC (0.9 ng/L detection limit) and analyzed low-volume water samples (200 mL) along with primary sludge samples from across the United States. All river water samples (100%) collected downstream of wastewater treatment plants had detectable levels of TCC, as compared to 56% of those taken upstream. Concentrations of TCC (mean+/ standard deviation) downstream of sewage treatment plants (84+/-110 ng/L) were significantly higher (P<0.05; Wilcoxon rank sum test) than those of samples taken upstream (12+/-15 ng/L). Compared to surface water, mean TCC concentrations found in dried, primary sludge obtained from municipal sewage treatment plants in five states were six orders of magnitude greater (19,300+/-7100 microg/kg). Several river samples contained a co-contaminant, identified based on its chromatographic retention time, molecular base ion, and MS/MS fragmentation behavior as 4,4' dichlorocarbanilide (DCC; CAS# 1219-99-4). In addition to TCC and DCC, municipal sludge contained a second co-contaminant, 3,3',4,4'-tetrachlorocarbanilide (TetraCC; CAS# 4300-43-0). Both newly detected compounds were present as impurities (0.2%(w/w) each) in technical grade TCC (99%). Application of the new method for chlorocarbanilide analysis yielded TCC occurrence data for 13 US states, confirmed the role of sewage treatment plants as environmental inputs of TCC, and identified DCC and TetraCC as previously unrecognized pollutants released into the environment alongside TCC. PMID- 16678155 TI - Effects of YM218, a nonpeptide vasopressin V(1A) receptor-selective antagonist, on vasopressin-induced growth responses in human mesangial cells. AB - Mesangial cells are centrally-located glomerular pericytes with contractile, endocrine, and immunity-regulating functions. These cells are thought to maintain normal glomerular function, since mesangial cell proliferation and extracellular matrix formation are hallmarks of chronic glomerular disease. Vasopressin causes mesangial cell contraction, proliferation and hypertrophy. Consequently, the effects of YM218, a potent, nonpeptide vasopressin V(1A) receptor-selective antagonist, on the growth responses of human mesangial cells to vasopressin were investigated. YM218 showed high affinity for vasopressin V(1A) receptors, exhibiting a K(i) value of 0.18 nM. Vasopressin concentration-dependently increased intracellular Ca(2+) levels and induced hyperplasia and hypertrophy in cultured mesangial cells, YM218 potently inhibited these vasopressin-induced responses. These results clearly show that YM218 has both strong affinity for human mesangial cell vasopressin V(1A) receptors and great potency in inhibiting the vasopressin-induced growth responses of mesangial cells controlled by the vasopressin V(1A) receptors. The hyperplasia and hypertrophy of mesangial cells in vitro caused by vasopressin indicate its possible in vivo role in glomerular disease pathogenesis. Therefore, YM218 is a potent pharmacologic probe to investigate the physiologic and pathophysiologic roles of vasopressin in the development of renal disease. PMID- 16678154 TI - Mechanisms underlying the impaired EDHF-type relaxation response in mesenteric arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats. AB - We previously reported that in mesenteric arteries from streptozotocin-induced diabetic rats, the endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired, possibly due to a reduced action of cAMP. Here, we observed an impairment of acetylcholine-induced EDHF-type relaxation in mesenteric arteries from a type 2 diabetic model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats [vs. age-matched control Long-Evans Tokushima Otsuka (LETO) rats], and we investigated the mechanism underlying this impairment. In the LETO group, this EDHF-type relaxation was attenuated by 18alpha-glycyrrhetinic acid (a gap-junction inhibitor) and by a protein kinase A (PKA) inhibitor. In both groups (OLETF and LETO), it was enhanced by 3-isobutyl-1-methylxanthine, a cAMP phosphodiesterase (PDE) inhibitor, but following these enhancements it was still weaker in OLETF rats than in LETO rats. The relaxations induced by cilostamide (a selective PDE3 inhibitor) and 8-bromo-cAMP (a cell-permeant cAMP analog) were reduced in OLETF rats, as was PKA activity. The relaxations induced by two activators of Ca(2+)-activated K(+) channels (K(Ca)) [1-ethyl-2-benzimidazolinone (1-EBIO), intermediate-conductance K(Ca) channel (IK(Ca)) activator, and riluzole, small-conductance K(Ca) channel (SK(Ca)) activator] were also impaired in OLETF rats. We conclude that the impairment of EDHF-type relaxation seen in OLETF rats may be attributable not only to a reduction in cAMP/PKA signaling, but also to reduced endothelial K(Ca) channel activities. PMID- 16678156 TI - Stimulation of mitogen-activated protein kinase kinases (MEK1/2) by mu-, delta- and kappa-opioid receptor agonists in the rat brain: regulation by chronic morphine and opioid withdrawal. AB - Opioid addiction modulates the extracellular signal-regulated kinase (ERK) leading to synaptic plasticity in the brain. ERK1/2 are stimulated by mitogen activated protein kinase kinases (MEK1/2), but little is known about the regulation of MEK activity by opioid drugs. This study was designed to assess the acute effects of selective mu-, delta-, and kappa-opioid receptor agonists, as well as those induced by chronic morphine and opioid withdrawal, on the content of phosphorylated MEK1/2 in the rat brain. Sufentanil (1-30 microg/kg, 30-120 min) induced dose- and time-dependent increases in MEK1/2 phosphorylation in the cerebral cortex and corpus striatum (30-177%) through a naloxone-sensitive mechanism. Morphine (100 mg/kg, 2 h) also augmented MEK1/2 phosphorylation in the both brain regions (50-70%). Similarly, the selective delta-opioid receptor agonist SNC-80 (10 mg/kg, 30 min) increased MEK1/2 activity in the cortex (60%) that was antagonized by naltrindole. In contrast, the selective kappa-opioid receptor agonist (-)-U50488H (10 mg/kg, 30-120 min) did not modify significantly MEK1/2 phosphorylation in the cortex. Chronic morphine (10-100 mg/kg, 5 days) was not associated with alterations in the content of phosphorylated MEK1/2 in the brain (induction of tachyphylaxis to the acute effects). In morphine-dependent rats, however, naloxone (2 mg/kg)-precipitated withdrawal (2-6 h) induced robust increases in MEK1/2 phosphorylation in cortex (27-49%) and striatum (83-123%). Spontaneous opioid withdrawal (24 h) in morphine-dependent rats did not alter MEK1/2 activity in the brain. The findings may be relevant in the context of the pivotal role played by the MEK/ERK pathway in various long-lasting forms of synaptic plasticity associated with opioid addiction. PMID- 16678157 TI - Cytoprotection by lithium and valproate varies between cell types and cellular stresses. AB - Despite much evidence that lithium and valproate, two commonly used mood stabilizers, exhibit neuroprotective properties against an array of insults, the pharmacological relevance of such effects is not clear because most of these studies examined the acute effect of these drugs in supratherapeutic doses against insults which were of limited disease relevance to bipolar disorder. In the present study, we investigated whether lithium and valproate, at clinically relevant doses, protects human neuroblastoma (SH-SY5Y) and glioma (SVG and U87) cells against oxidative stress and endoplasmic reticulum stress in a time dependent manner. Pretreatment of SH-SY5Y cells for 7 days, but not 1 day, with 1 mM of lithium or 0.6 mM of valproate significantly reduced rotenone and H2O2 induced cytotoxicity, cytochrome c release and caspase-3 activation, and increased Bcl-2 levels. Conversely, neither acute nor chronic treatment of SH SY5Y cells with lithium or valproate elicited cytoprotective responses against thapsigargin-evoked cell death and caspase-3 activation. Moreover, inhibitors of glycogen synthase kinase-3 (GSK-3), kenpaullone and SB216763, abrogated rotenone induced, but not H2O2-induced, cytotoxicity. Thus the cytoprotective effects of lithium and valproate against H2O2-induced cell death is likely independent of GSK-3 inhibition. On the other hand, chronic lithium or valproate treatment did not ameliorate cytotoxicity induced by rotenone, H2O2, and thapsigargin in SVG astroglial and U87 MG glioma cell lines. Our results suggest that lithium and valproate may decrease vulnerability of human neural, but not glial, cells to cellular injury evoked by oxidative stress possibly arising from putative mitochondrial disturbances implicated in bipolar disorder. PMID- 16678158 TI - Pegaptanib for the treatment of age-related macular degeneration. AB - Although neovascular (wet) age-related macular degeneration (AMD) only accounts for 10-20% of all AMD, the majority (about 90%) of severe vision loss associated with AMD is due to this form. Results from recent studies have implied that vascular endothelial growth factor (VEGF), particularly VEGF(165), plays a predominant role in the development of ocular neovascularization and vascular leakage secondary to AMD. Thus VEGF is an important therapeutic target in neovascular AMD. Pegaptanib, an anti-VEGF aptamer, can selectively bind with VEGF(165) and inhibit both the growth of blood vessels and vascular leakage, and was approved by the Food and Drug Administration in the United States as the therapy for the treatment of all subtypes of neovascular AMD in December 2004. This review summaries the mechanism, preclinical and clinical studies, and adverse events of pegaptanib treatment. PMID- 16678159 TI - Focus on molecules: opticin. PMID- 16678160 TI - The neurotoxicity of amphetamines: bridging drugs of abuse and neurodegenerative disorders. AB - Amphetamine derivatives are the most commonly abused drugs. These compounds have been known for many years to induce neurotoxicity. However, recent findings have highlighted novel alterations produced by amphetamines in the central nervous system consisting of neuronal inclusions and the involvement of proteins belonging to a multi-enzymatic complex known as the ubiquitin-proteasome system. These ultrastructural and molecular changes are similar to those that occur during degenerative processes that affect the basal ganglia, and in particular Parkinson's disease, which is characterized by ubiquitin-containing neuronal inclusions in the subtantia nigra. This is recently confirmed by the occurrence of ubiquitin immunoreactive structures in the substantia nigra of humans abusing methamphetamines. In this article, we propose that the neurotoxicity of amphetamines and degenerative disorders share a number of steps in their mechanism of action involving the ubiquitin-proteasome system. The fine tuning of this ubiquitous proteolytic pathway is now being elucidated because G-protein coupled receptors and signaling proteins such as beta-arrestin regulate access to this catalytic machinery. The identification of the ubiquitin-proteasome pathway and beta-arrestin as molecular targets of neurotoxicity is expected to provide novel therapeutic strategies both for the treatment of drug addiction and the treatment of neurodegenerative disorders. PMID- 16678162 TI - Correlations between neuronal loss, decrease of memory, and decrease expression of brain-derived neurotrophic factor in the gerbil hippocampus during normal aging. AB - It is known that the hippocampus has vital functions in learning and memory, behavioral regulation, and activity-dependent synaptic plasticity, and that the hippocampus contains high levels of brain-derived neurotrophic factor (BDNF). In the present study, we followed age-dependent changes of BDNF immunoreactivity and protein level in the gerbil hippocampus to identify the correlation between BDNF and aging. BDNF immunoreactivity and its protein level significantly increased at postnatal month (PM) 12 in the hippocampus and thereafter reduced. At PM 24, BDNF immunoreactivity in the hippocampal CA1 region and dentate gyrus was similar to that in the PM 1 group, whereas BDNF immunoreactivity in the CA2/3 region at PM 24 was higher than that at PM 1. In the PM 24 group, an age-related neuronal loss and the decrease of reference and working memory were observed. In conclusion, our results suggest that observed reduction in BDNF and reference memory may be associated with age-dependent neuronal loss in the hippocampal CA1 region. PMID- 16678161 TI - Analysis of the neurogenic potential of multipotent skin-derived precursors. AB - Multipotent precursors similar to stem cells of the embryonic neural crest (NC) have been identified in several postnatal tissues, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. We investigated this issue with regard to skin-derived precursors (SKPs), multipotent NC-related precursors isolated from the dermis of skin. SKP cultures follow an appropriate pattern and time-course of neuronal differentiation, with proliferating nestin-expressing SKPs generating post mitotic neuronal cells that co-express pan-neuronal and peripheral autonomic lineage markers. These SKP-derived neuron-like cells survive and maintain their peripheral phenotype for at least 5 weeks when transplanted into the CNS environment of normal or kainate-injured hippocampal slices. Undifferentiated SKPs retain key neural precursor properties after multi-passage expansion, including growth factor dependence, nestin expression, neurogenic potential, and responsiveness to embryonic neural crest fate determinants. Despite undergoing an apparently appropriate neurogenic process, however, SKP-derived neuron-like cells possess an immature electrophysiological profile. These findings indicate that SKPs retain latent neurogenic properties after residing in a non-neural tissue, but that additional measures will be necessary to promote their differentiation into electrophysiologically active neurons. PMID- 16678163 TI - The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB. AB - In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBDeltaN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBDeltaN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment. PMID- 16678164 TI - Heat shock proteins reduce alpha-synuclein aggregation induced by MPP+ in SK-N-SH cells. AB - Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Heat shock proteins (HSPs) can reduce protein misfolding and accelerate the degradation of misfolded proteins. 1-methyl-4-phenylpyridinium ion (MPP+) is the compound responsible for the PD-like neurodegeneration caused by MPTP. In this study, we found that MPP+ could increase the expression of alpha-synuclein mRNA but could not elevate proteasome activity sufficiently, leading to alpha synuclein protein accumulation followed by aggregation. Both HSPs and HDJ-1, a homologue of human Hsp40, can inhibit MPP+-induced alpha-synuclein mRNA expression, promote ubiquitination and elevate proteasome activity. These findings suggest that HSPs may inhibit the MPP+-induced alpha-synuclein expression, accelerate alpha-synuclein degradation, thereby reducing the amount of alpha-synuclein protein and accordingly preventing its aggregation. PMID- 16678165 TI - The logic of TGFbeta signaling. AB - The identification of the TGFbeta cytokine signaling pathway, including membrane receptor serine/threonine kinases and Smad transcription factors as their substrates, has allowed the delineation of a process for conversion of these signals into programs of gene activation and repression that underlie critical cell fate and developmental decisions. The deconstruction of one of these responses - the cell cycle arrest response - into its elemental molecular parts has shed light into the mechanisms used by tumors to evade surveillance and cause metastasis. PMID- 16678166 TI - Macrophage activation increases the invasive properties of hepatoma cells by destabilization of the adherens junction. AB - Tumor-associated macrophages play an important role in tumor progression, but whether they exert a tumor-progressive effect remains controversial. Here, we demonstrated that activated macrophage-conditioned medium (AMCM) obtained from RAW macrophages (RAW/AMCM) induced epithelial-mesenchymal transition (EMT) and stimulated the migratory and invasive activities of HepG2 cells, whereas control conditioned media had no effect. Epithelial-cadherin (E-cadherin) and beta catenin staining patterns were altered at the adherens junctions by RAW/AMCM treatment, with an approximately 50% decrease in E-cadherin and beta-catenin in the cell membrane. Importantly, levels of beta-catenin-associated E-cadherin were also decreased. Following RAW/AMCM treatment, enhanced activation of c-Src was seen prior to increased tyrosine phosphorylation of beta-catenin, and this led to the destabilization of adherens junctions. Pretreatment of HepG2 cells with the Src kinase inhibitor, PP2, completely abolished the effects of RAW/AMCM on the EMT, migration, invasion, and expression and association of E-cadherin and beta catenin. AMCMs obtained from human THP-1 monocytes and mouse peritoneal macrophages also caused disassembly of the adherens junctions and migration of HepG2 cells. Furthermore, inhibition of the epidermal growth factor receptor (EGFR) with gefitinib partially prevented the downregulation of E-cadherin and beta-catenin at the adherens junctions and migration behavior induced by RAW/AMCM. Our results suggest that activated macrophages have a tumor-progressive effect on HepG2 cells which involves the c-Src- and EGFR-dependent signaling cascades. PMID- 16678168 TI - Evidence for p53-like-mediated stress responses in green algae. AB - The tumor suppressor protein, p53, plays a major role in cellular responses to stress and DNA damage in animals; despite its critical function, p53 homologs have not been identified in any algal or plant lineage. This study employs a functional and evolutionary approach to test for a p53 functional equivalent in green algae. Specifically, the study: (i) investigated the effect of two synthetic compounds known to interfere with p53 activity; (ii) searched for sequences with similarity to known p53-induced genes; and (iii) analyzed the expression pattern of one such sequence. The findings reported here suggest that a p53 functional equivalent is present and mediates cellular responses to stress in green algae. PMID- 16678167 TI - Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis. AB - HEN1-dependent methylation of the 3'-terminal nucleotide is a crucial step in plant microRNA (miRNA) biogenesis. Here we report that several viral RNA silencing suppressors (P1/HC-Pro, p21 and p19) inhibit miRNA methylation. These suppressors have distinct effects on different miRNAs. We also show that miRNA* is methylated in vivo in a suppressor-sensitive manner, suggesting that the viral proteins interfere with miRNA/miRNA* duplexes. p19 and p21 bind both methylated and unmethylated miRNA/miRNA* duplexes in vivo. These findings suggest miRNA/miRNA* as the in vivo substrates for the HEN1 miRNA methyltransferase and raise intriguing possibilities regarding the cellular location of miRNA methylation. PMID- 16678169 TI - Identification and characterization of human cardiolipin synthase. AB - The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed. PMID- 16678170 TI - Genetic networks responsive to sodium butyrate in colonic epithelial cells. AB - We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells. PMID- 16678171 TI - Transcription regulation of the alpha-glucanase gene agn1 by cell separation transcription factor Ace2p in fission yeast. AB - During the final stage of the cell division cycle in the fission yeast Schizosaccharomyces pombe, transcription factor Ace2p activates expression of genes involved in the separation of newly formed daughter cells, such as agn1+, which encodes the alpha-glucanase Agn1p. The agn1 promoter contains three copies of the nucleotide sequence motif CCAGCC, whose presence seems to correlate with Ace2p-mediated transcription activation. Here, we describe a simple plate-based assay utilizing as a reporter the secreted glucoamylase of Arxula adeninivorans to investigate the function of this motif. We show that not all three repeats, but only the two most proximal to the transcription start point, act as an upstream activating sequence (UAS). Finally, we demonstrate that this UAS is essential for agn1 promoter activity in vivo. PMID- 16678172 TI - Thermodynamics and stability of the PAAD/DAPIN/PYRIN domain of IFI-16. AB - The PAAD domain is a conserved domain recently identified in more than 35 human proteins that are involved in apoptosis and inflammatory signaling pathways. Structural studies have confirmed that this domain belongs to the death domain superfamily which includes PAAD/CARD/DED/DD families. Recently, the 3D structures determined by NMR of NALP1 and ASC PAAD domain, members of the PAAD family, have shown that it is composed of a 6 helix bundle as with other death domain family members. However, helix-3 in the solved structures is unordered in solution. In this study we compare the thermodynamic, folding and stability properties of different members of the PAAD and CARD families and investigate structural conformational changes induced by the helix inducers trifluoroethanol and SDS on the PAAD domain of IFI16 and on the CARD domain of RAIDD. We show that inside the PAAD and CARD families, members have similar thermodynamic properties, however, the DeltaG of folding for PAAD and CARD members are, respectively, -1.4 and -5.5 kcal mol(-1). This difference is attributed to less alpha helical content for PAAD due to the unfolding of helix-3 that lowers bonded energy and increases disorder when compared to CARD members. Despite identical fold between PAAD and CARD families but limited sequence identity, there are striking differences in the thermodynamics of both families. PMID- 16678173 TI - The alpha-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit. AB - Chloroplasts of higher plants contain a unique signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. CpSRP43 is composed of a four ankyrin repeat domain and three functionally distinct chromodomains (CDs). In this report we confirm previously published data that the second chromodomain (CD2) provides the primary binding site for cpSRP54. However, quantitative binding analysis demonstrates that cpSRP54 binds to CD2 significantly less efficiently than it binds to full-length cpSRP43. Further analysis of the binding interface of cpSRP by mutagenesis studies and a pepscan approach demonstrates that the C-terminal alpha-helix of CD2 facilitates binding to cpSRP54. PMID- 16678174 TI - Interaction of a small heat shock protein with light-harvesting cyanobacterial phycocyanins under stress conditions. AB - Phycobiliproteins such as phycocyanins are the most abundant proteins found in cyanobacteria which are assembled to form the phycobilisome. Here, we showed that a small heat shock protein, HspA, interacts directly with phycocyanins from the cyanobacterium Synechococcus sp. strain PCC 7942 in vitro and suppresses inactivation of their light-harvesting functions due to heat denaturation in the presence of hydrogen peroxide. Under the denaturing conditions, phycobilisomes were de-assembled to lighter complexes and then aggregated. HspA associated with phycocyanins in the dissociated complexes, and suppressed the aggregation. The specific interaction between a small heat shock protein and phycocyanins was further supported by the fact that HspA and alpha-crystallin protected isolated phycocyanins from denaturation, while HtpG and lysozyme did not. The maximum protection was observed at a molar ratio of four HspA monomer per one phycocyanin (alpha beta) monomer. PMID- 16678175 TI - CLIP-region mediated interaction of Invariant chain with MHC class I molecules. AB - An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules. PMID- 16678176 TI - Nischarin as a functional imidazoline (I1) receptor. AB - Gene matching shows that Nischarin is a mouse homologue of human imidazoline receptor antisera-selective (IRAS) protein, a viable candidate of the imidazoline (I1) receptor. Nischarin and IRAS share the functions of enhancing cell survival, growth and migration. Bioinformatics modeling indicates that the IRAS and Nischarin may be transmembrane proteins and the convergence information raises the interesting possibility that Nischarin might serve as the I1-receptor. To test this hypothesis, we developed antibodies against the Nischarin protein, and conducted signal transduction (functional) studies with the I1-receptor agonist rilmenidine in the presence and absence of Nischarin antisense oligodeoxynucleotides (ODNs). NIH3T3 cells transfected with the Nischarin cDNA and incubated with the newly synthesized antibody expressed a 190 kD band. The antibody identified endogenous Nischarin in differentiated PC12 cells around 210 kD, which is consistent with reported findings in other cells of neuronal origin. The immunoflourescence findings showed the targeted protein to be associated with the cell membrane in PC12 cells. Nischarin ODNs abolished the expression of Nischarin in PC12 cells. Equally important, the Nischarin ODNs eliminated the production of MAPK(p42/44), a recognized signal transduction product generated by I1-receptor activation in differentiated PC12 cells. Together, the present findings suggest that Nischarin may serve as the functional I1-receptor or at least share a common signaling pathway in the differentiated PC12 cells. PMID- 16678177 TI - Oocyte degeneration after intracytoplasmic sperm injection: a multivariate analysis to assess its importance as a laboratory or clinical marker. AB - OBJECTIVE: Oocyte degeneration has historically been associated with the intracytoplasmic (ICSI) technique. We sought to determine whether oocyte degeneration rates were associated with the technician performing the procedure, the baseline characteristics of the patient, and/or ovarian stimulation variables. We also evaluated whether the degeneration rate could serve as a surrogate marker for implantation potential. DESIGN: Cohort study. SETTING: Academic medical center. PATIENT(S): Couples undergoing ICSI. INTERVENTION(S): Six thousand six hundred fifty-three injected oocytes were analyzed to determine whether the degeneration rate was technician dependent. Two hundred thirty first entry down-regulated cycles were examined to identify predictors associated with oocyte degeneration. Multivariate analyses were performed using generalized linear model routines. MAIN OUTCOME MEASURE(S): Oocyte degeneration rates and implantation rates. RESULT(S): Neither the ICSI technician nor the stripping technician was associated with the oocyte degeneration rate. However, the day 3 FSH, number of mature oocytes retrieved, and E2 levels on the day of hCG were significant independent predictors of degeneration rate. Physician-adjustable ovarian stimulation variables were not associated with the degeneration rate. The degeneration rate did not appear to be associated with the implantation rate. CONCLUSION(S): These data suggest that oocyte degeneration is not technician or physician dependent. Degeneration is likely a function of the inherent oocyte quality in women who underwent ovarian stimulation. However, the remaining cohort of retrieved oocytes appears to be unaffected by virtue of an uncompromised implantation rate. PMID- 16678178 TI - Factors that affect infertility patients' decisions about disposition of frozen embryos. AB - OBJECTIVE: To describe factors that affect infertility patients' decision making regarding their cryopreserved embryos. DESIGN: Forty-six semistructured in-depth interviews of individuals and couples participating in IVF programs. SETTING: Two major southeastern academic medical centers. PATIENT(S): Fifty-three individuals, including 31 women, 8 men, and 7 couples. MAIN OUTCOME MEASURE(S): Qualitative analysis of interview transcripts. INTERVENTION (S): None. RESULT(S): Seven broad themes informed participants' decisions about embryo disposition: family and personal issues, trust, definition of the embryo, prospective responsibility to the embryo, responsibility to society, adequacy of information, and lack of acceptable disposition options. Many wished for alternative options, such as a ceremony at the time of disposal or placement of embryos in the woman's body when pregnancy was unlikely. CONCLUSION(S): Recent debates regarding embryo disposition do not reflect the range of values that infertility patients consider when deciding about frozen embryos. In addition to questions about the embryo's moral status, decision making about embryos is informed by a range of factors in the lives of individuals who created them. These perspectives may have important implications for the content and timing of informed consent, facilitating embryo disposition, and advancing policy debates about the ethics of frozen embryo use. PMID- 16678179 TI - Behavioral and physiological responses to experimentally elevated testosterone in female dark-eyed juncos (Junco hyemalis carolinensis). AB - Testosterone mediates the expression of many fitness-related traits in male vertebrates and is thought to account for numerous sex differences in trait expression. Testosterone is also secreted by females; however, far less is known regarding its effects on female physiology and behavior. Using a bird species in which the effects of testosterone on males are well characterized, the dark-eyed junco (Junco hyemalis), we tested whether an increase in exogenous testosterone in females would alter the phenotypic expression of a suite of behavioral and physiological traits. We found that increased testosterone levels in female dark eyed juncos led to decreased cell-mediated immune function and increased intrasexual aggression, hypothalamo-pituitary-adrenal (HPA) axis responsiveness, baseline corticosterone and corticosterone-binding globulin (CBG) levels. Furthermore, immunosuppression following testosterone implantation was negatively correlated with total and free testosterone but did not appear to be related to either total or free corticosterone. These results demonstrate that the phenotypic impact of elevated testosterone is not confined to males in dark-eyed juncos, and that the impact in adults can be similar in males and females. We discuss these results in the context of potential endocrine-immune interactions and the evolution of sexual dimorphism. PMID- 16678180 TI - Personal medical documents management--how patients perceive, keep and manage their medical documents: a qualitative study. AB - BACKGROUND: Absence of medical documents damages the quality of treatment and service. Despite the computerized medical folder, its use is still limited, and not all countries have a national health communications systems. That is why the patients are asked to bring with them the documents they hold. However, some patients lose the documents and others are uncooperative due to forgetfulness, and a lack of knowledge. OBJECTIVES: To examine how patients perceive, keep and manage medical documents. DESIGN: The research was conducted in the qualitative method. SETTINGS: A cardiology clinic, in a big medical center in Israel. PARTICIPANTS: A total of 28 patients suffering from ischemic heart disease and/or rhythm disorders. The sample was a convenience one, and were taken from the population visiting the Cardiology Clinic from May to October 2003. METHODS: Semi structured interviews were held with the participants. The data were processed by means of content analysis. RESULTS: The findings indicate that, even though the participants expressed that it is the health system's responsibility to manage medical documents, most of them kept, and some even managed, their medical documents via a personal folder. By keeping documents and deciding to whom and when to show them the patient functions as a self-care manager. This made them more involved in their treatment and, they felt in control and empowered. CONCLUSION: Patient's attention to documents, as keeper and router, results in demonstrations of responsibility and involvement in treatment, consequently empowering the patient. PMID- 16678181 TI - Immunopathogenesis of cerebral malaria. AB - Malaria is one of the most important global health problems, potentially affecting more than one third of the world's population. Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, yet its pathogenesis remains incompletely understood. In this review, we discuss some of the principal pathogenic events that have been described in murine models of the disease and relate them to the human condition. One of the earliest events in CM pathogenesis appears to be a mild increase in the permeability to protein of the blood-brain barrier. Recent studies have shown a role for CD8+T cells in mediating damage to the microvascular endothelium and this damage can result in the leakage of cytokines, malaria antigens and other potentially harmful molecules across the blood-brain barrier into the cerebral parenchyma. We suggest that this, in turn, leads to the activation of microglia and the activation and apoptosis of astrocytes. The role of hypoxia in the pathogenesis of cerebral malaria is also discussed, with particular reference to the local reduction of oxygen consumption in the brain as a consequence of vascular obstruction, to cytokine-driven changes in glucose metabolism, and to cytopathic hypoxia. Interferon-gamma, a cytokine known to be produced in malaria infection, induces increased expression, by microvascular endothelial cells, of the haem enzyme indoleamine 2,3-dioxygenase, the first enzyme in the kynurenine pathway of tryptophan metabolism. Enhanced indoleamine 2,3-dioxygenase expression leads to increased production of a range of biologically active metabolites that may be part of a tissue protective response. Damage to astrocytes may result in reduced production of the neuroprotectant molecule kynurenic acid, leading to a decrease in its ratio relative to the neuroexcitotoxic molecule quinolinic acid, which might contribute to some of the neurological symptoms of cerebral malaria. Lastly, we discuss the role of other haem enzymes, cyclooxygenase-2, inducible nitric oxide synthase and haem oxygenase-1, as potentially being components of mechanisms that protect host tissue against the effects of cytokine- and leukocyte-mediated stress induced by malaria infection. PMID- 16678182 TI - Bovine trypanotolerance: A natural ability to prevent severe anaemia and haemophagocytic syndrome? AB - Trypanotolerance is the capacity of certain West-African, taurine breeds of cattle to remain productive and gain weight after trypanosome infection. Laboratory studies, comparing Trypanosoma congolense infections in trypanotolerant N'Dama cattle (Bos taurus) and in more susceptible Boran cattle (Bos indicus), confirmed the field observations. Experiments using haemopoietic chimeric twins, composed of a tolerant and a susceptible co-twin, and T cell depletion studies suggested that trypanotolerance is composed of two independent traits. The first is a better capacity to control parasitaemia and is not mediated by haemopoietic cells, T lymphocytes or antibodies. The second is a better capacity to limit anaemia development and is mediated by haemopoietic cells, but not by T lymphocytes or antibodies. Weight gain was linked to the latter mechanism, implying that anaemia control is more important for survival and productivity than parasite control. Anemia is a marker for a more complex pathology which resembles human haemophagocytic syndrome: hepatosplenomegaly, pancytopenia and a large number of hyperactivated phagocytosing macrophages in bone marrow, liver and other tissues. Thus, mortality and morbidity in trypanosome-infected cattle are primarily due to self-inflicted damage by disproportionate immune and/or innate responses. These features of bovine trypanotolerance differ greatly from those in murine models. In mice, resistance is a matter of trypanosome control dependent on acquired immunity. However, a model of anaemia development can be established using C57BL/6J mice. As in cattle, the induction of anaemia was independent of T cells but its development differed with different trypanosome strains. Identification of the molecular pathways that lead to anaemia and haemophagocytosis should allow us to design new strategies to control disease. PMID- 16678183 TI - Lipoprotein-associated phospholipase A2 and coronary calcification. The Rotterdam Coronary Calcification Study. AB - OBJECTIVES: Although several studies have recently suggested that lipoprotein associated phospholipase A2 (Lp-PLA2) is an independent predictor of coronary events, only one study has examined the association between Lp-PLA2 and coronary calcification, using young adults. We investigated the association between Lp PLA2 activity and coronary calcification assessed by electron-beam computed tomography (EBT) in a population of older participants. METHODS AND RESULTS: The Rotterdam Coronary Calcification Study is a population-based study in men and women aged >/=55 years. Coronary calcification assessed by EBT was quantified in a calcium score according to Agatston's method. Lp-PLA2 activity measured in samples collected 7 years before scanning (n=520) was associated with coronary calcification in men after adjustment for age. The odds ratio per standard deviation of Lp-PLA2 activity of having a total calcium score >1000 was 1.6 (95% confidence interval: 1.1-2.4), as compared to a total calcium score 95% kinetically complete the assay within a few seconds. In addition, the metallic nanostructures used to facilitate MEF appear to be preferentially heated as compared to the surface assay fluid, advantageously localizing the MEF and heating around the nanostructures. To demonstrate proof of principle, a 96-well plate has been functionalized with silver nanostructures, and a model protein avidin-biotin assay studied. In our findings, a greater than 5-fold fluorescence enhancement coupled with a approximately 90-fold increase in assay kinetics was observed, but with no assay washing steps needed due to the silver-enhanced evanescent field mode of excitation. These findings promise to strongly facilitate high throughput fluorescence-based processes, such as in biology, drug discovery and general compound screening. PMID- 16678197 TI - Development of an immunochromatographic lateral flow test strip for detection of beta-adrenergic agonist Clenbuterol residues. AB - A rapid immunochromatographic lateral flow test strip was developed in a competitive format with the gold-conjugated monoclonal antibody to specifically determine the residues of Clenbuterol (CL), a beta-adrenergic agonist. The test strip is made up of a sample pad, a conjugate reagent pad, a test membrane containing a control line and a test line, and an absorbent pad. CL standard samples of 0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/ml in swine urine were determined by the test strip. It was shown that detection limit of the test strip was as low as 0.1 ng/ml of CL and that the half of maximal inhibition concentration (IC50) in relative optical density was calculated to be 1.78+/-0.17 ng/ml under an optical density scanner. The sensitivity by eye measurement was 1.0 ng/ml. It takes 10 min to accomplish a test. Parallel analysis of urine samples from pigs fed with CL showed comparable results obtained from the test strip and GC-MS. Therefore, the test strip is very useful as a screening method for quantitative, semi quantitative or qualitative detection of CL residues in swine urine. PMID- 16678198 TI - Role of juvenile hormone esterase and epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea. AB - The role of juvenile hormone (JH) esterase (JHE) and epoxide hydrolase (EH) in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were added to males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence (d0). The highest oviposition rates for mated females were noted on d 2-4. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d0 to d8 post-emergence. Maximal JHE activity for virgin females occurred on d2 (1.09+/-0.14(+/-1 SEM) nmol of JH III degraded/min/mg protein), which was approximately twice that of mated females on the same day. The same results were observed for EH where the activity peaked on d2 at 0.053+/-0.003 as compared to 0.033+/-0.003 nmol of JH III degraded/min/mg protein, respectively. By d4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same through d7. The developmental changes and effects of mating on JH degradation were similar when measured per insect. The highest levels of JHE and JH EH activity/min/mg protein in d2 virgin and mated females was found in ovaries followed by the carcass and then haemolymph; no EH activity was found in haemolymph as expected. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed. PMID- 16678199 TI - Enalapril increases ischemia-induced endothelial progenitor cell mobilization through manipulation of the CD26 system. AB - Enalapril, an angiotensin-converting enzyme (ACE) inhibitor, reduces cardiovascular events in patients with acute myocardial infarction. However, whether the beneficial effect of enalapril is mediated in part through endothelial progenitor cells (EPCs) has yet to be elucidated. This study investigated the role of the CD26/dipeptidylpeptidase IV (DPP IV) system in enalapril-modulated EPC mobilization. C57 BL/6 mice were divided into control and enalapril-treated groups. Peripheral EPCs were enumerated before and after ischemic stress. CD26/DPP IV activity and stroma-derived factor-1alpha (SDF 1alpha) levels were measured in the blood and the bone marrow. In response to ischemic stress, the enalapril group displayed a significant increase in circulating EPCs (with a 3.6-fold increase of sca-1+KDR+ cells and a 2.2-fold increase of c-kit+CD31+ cells versus controls at 12 h). Enalapril also caused a sixfold increase in the contribution of bone marrow-derived EPCs to the ischemia induced neovascularization. In the bone marrow, enalapril did not alter CD26+ cell numbers; however, it did amplify DPP IV activity. In the blood, through the anti-inflammatory effect, enalapril significantly decreased CD26+ cell numbers, leading to a decrease in total DPP IV activity. These phenomena were associated with a lower SDF-1alpha concentration in the bone marrow but higher in the blood in the enalapril group, compared to the controls. All these findings were not demonstrated without ischemic stress. The effect of enalapril on EPC mobilization could be substantially blocked by Diprotin-A, a DDP IV antagonist. This study demonstrates that one of the pleiotropic effects of enalapril on the cardiovascular system involves the modulation of circulating EPC numbers via the CD26/DPP IV system, which may serve as a potential target for mobilizing EPCs for therapeutic purposes. PMID- 16678200 TI - Myocardium-targeted delivery of endothelial progenitor cells by ultrasound mediated microbubble destruction improves cardiac function via an angiogenic response. AB - Application of ultrasound-mediated destruction of microbubbles (US + Bubble) to skeletal muscle creates capillary ruptures leading to leakage of the cell components. We studied whether US + Bubble combined with bone-marrow-derived mononuclear cells (BM-MNCs) infusion enables the targeted delivery of endothelial lineage cells into the myocardium and improves cardiac function of the cardiomyopathy model due to the paucity of neocapillary formation. Pulsed US was applied to the anterior chest of BIOTO2 cardiomyopathy hamsters for 90 s after the intravenous injection of microbubble (Optison) followed by infusion of BM MNCs. Cardiac samples from US + microbubble + BM-MNCs (US + Bubble + BM), US + Bubble, US + BM without Bubble, and saline infusion control groups were analyzed 12 weeks after treatment. Labeled BM-MNCs transplanted by US + Bubble were found to be mainly localized in the microvessels, but not by US stimulation without microbubble (121.2 +/- 24.5 vs. 2.80 +/- 1.30 cells/mm2, P < 0.001). Capillary densities in US + Bubble + BM group were increased 1.7-fold (P < 0.05) over the control, and neither US + Bubble nor US + BM enhanced neocapillary formation. 99mTc-Tetrofosmin scintigraphy revealed that blood perfusion area in the US + Bubble + BM group was 48% greater than the control (P < 0.01). US + Bubble stimulation induces the expression of adhesion molecules (VCAM-1 and ICAM-1) in capillaries, and the US + Bubble-mediated supply of BM-MNCs increased the myocardial content of VEGF and bFGF. The left ventricular wt/body wt, area of cardiac fibrosis, and apoptotic cell numbers in the US + Bubble + BM group significantly (P < 0.05) decreased by 82%, 73%, and 64% relative to the control, respectively. The cardiac function in myopathic hamsters (assessed by fractional shortening) was markedly improved 36% (P < 0.05) by US + Bubble + BM treatment. Targeted delivery of BM-MNCs by US + Bubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling, resulting in the prevention of heart failure. This non-invasive cell delivery system may be useful as a novel efficient approach for angiogenic cell therapy to the myocardium. PMID- 16678201 TI - YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407. AB - The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere with methylation at C1962 in 23 S rRNA. Purified recombinant YebU protein retains its specificity for C1407 in vitro, and methylates 30 S subunits (but not naked 16 S rRNA or 70 S ribosomes) isolated from yebU knockout strains. Nucleotide C1407 is located at a functionally active region of the 30 S subunit interface close to the P site, and YebU-directed methylation of this nucleotide seems to be conserved in bacteria. The yebU knockout strains display slower growth and reduced fitness in competition with wild-type cells. We suggest that a more appropriate designation for yebU would be the rRNA small subunit methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification. PMID- 16678202 TI - Protein refolding in silico with atom-based statistical potentials and conformational search using a simple genetic algorithm. AB - A distance-dependent atom-pair potential that treats long range and local interactions separately has been developed and optimized to distinguish native protein structures from sets of incorrect or decoy structures. Atoms are divided into 30 types based on chemical properties and relative position in the amino acid side-chains. Several parameters affecting the calculation and evaluation of this statistical potential, such as the reference state, the bin width, cutoff distances between pairs, and the number of residues separating the atom pairs, are adjusted to achieve the best discrimination. The native structure has the lowest energy for 39 of the 40 sets of original ROSETTA decoys (1000 structures per set) and 23 of the 25 improved decoys (approximately 1900 structures per set). Combined with the orientation-dependent backbone hydrogen bonding potential used by ROSETTA and a statistical solvation potential based on the solvent exclusion model of Lazaridis & Karplus, this potential is used as a scoring function for conformational search based on a genetic algorithm method. After unfolding the native structure by changing every phi and psi angle by either +/ 3, +/-5 or +/-7 degrees, five small proteins can be efficiently refolded, in some cases to within 0.5 A C(alpha) distance matrix error (DME) to the native state. Although no significant correlation is found between the total energy and structural similarity to the native state, a surprisingly strong correlation exists between the radius of gyration and the DME for low energy structures. PMID- 16678203 TI - Common motifs and topological effects in the protein folding transition state. AB - Through extensive experiment, simulation, and analysis of protein S6 (1RIS), we find that variations in nucleation and folding pathway between circular permutations are determined principally by the restraints of topology and specific nucleation, and affected by changes in chain entropy. Simulations also relate topological features to experimentally measured stabilities. Despite many sizable changes in phi values and the structure of the transition state ensemble that result from permutation, we observe a common theme: the critical nucleus in each of the mutants share a subset of residues that can be mapped to the critical nucleus residues of the wild-type. Circular permutations create new N and C termini, which are the location of the largest disruption of the folding nucleus, leading to a decrease in both phi values and the role in nucleation. Mutant nuclei are built around the wild-type nucleus but are biased towards different parts of the S6 structure depending on the topological and entropic changes induced by the location of the new N and C termini. PMID- 16678204 TI - Structural basis for diversity of the EF-hand calcium-binding proteins. AB - The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca2+-binding and target interaction properties. Here, our current understanding of the Ca2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11-14 amino acid residues in the Ca2+-binding loop are analyzed within the framework of the recently proposed two-step Ca2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca2+-binding loops in the two-EF-hand domain. This structure, named EFbeta-scaffold, defines the position of the bound Ca2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca2+-binding loop. It is proposed that the nature of the first ligand of the Ca2+ binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca2+-induced conformational change in the EF-hand proteins. PMID- 16678205 TI - Management of depression relapse: re-initiation of duloxetine treatment or dose increase. AB - Continuation of antidepressant therapy after patients have responded to medication has been shown to greatly reduce but not eliminate relapse. We evaluated response to reinstating or increasing drug doses in patients who relapsed during a long-term, randomized, double-blind, continuation phase study of duloxetine treatment for major depressive disorder (MDD). Patients with HAMD17 scores of >18 and CGI-Severity scores of >4 received duloxetine 60 mg QD for 12 weeks, responders then received 26 weeks of duloxetine 60 mg QD or placebo. The relapsing patients who started 12 weeks of rescue treatment received duloxetine 60 mg QD (if relapsing on placebo) or duloxetine 60 mg BID (if relapsing on duloxetine). In the continuation phase, 87 patients received duloxetine 60 mg QD (n=58) or 60 mg BID (n=29) as treatment for relapse. The percentage of patients who responded to treatment after relapse was 62% of those whose dose was increased from 60 mg QD to 60 mg BID, and 74% for those who switched from placebo to duloxetine 60 mg daily. By the end of the study, 32 patients taking duloxetine 60 mg QD were in remission (57%), compared with 11 patients taking duloxetine 60 mg BID (38%). There was significant improvement in HAMD17 and CGI-Severity scores for both groups at endpoint vs. start of treatment for relapse. Significant improvements were seen for all visual analog scale parameters in the 60 mg QD group, but only Interference with Daily Activities and Back Pain parameters showed significant improvement for patients receiving 60 mg BID. Significant within-group improvement from baseline was seen for both groups in Symptom Questionnaire-Somatic Subscale total and pain subscales. Treatment-emergent adverse events were mild/moderate in severity; no clinically significant changes were noted in laboratory results or vital signs. Reinstatement of duloxetine 60 mg QD was effective for patients who relapsed after discontinuing drug. Patients relapsing on duloxetine 60 mg QD benefited from an increase to 60 mg BID. These duloxetine doses were well tolerated and effective, and appear appropriate for MDD patients requiring treatment of relapse. PMID- 16678206 TI - A behavior based safety approach at a Kuwait research institution. AB - INTRODUCTION: Behavior based safety (BBS) initiatives are the current trend toward improving safety performance. METHOD: This study outlines the framework of the BBS process and summarizes several behavioral safety approaches to accident prevention. RESULTS: Although most of the published research has focused on the improvement of safe behavior in industry, this study is the first to apply the current approach of behavior based safety in a research/educational setting. An experiment conducted at a research institution demonstrated the effectiveness of a well-designed behavior based safety process. A follow-up study indicated that the BBS produced a lasting effect for the experimental group. IMPACT ON INDUSTRY: Results from this study can be the driving force to implement behavior-based safety processes into educational, research, and training organizations. PMID- 16678207 TI - Restoring balance to ataxia with coenzyme Q10 deficiency. PMID- 16678208 TI - Motor assessment of upper extremity function and its relation with fatigue, cognitive function and quality of life in multiple sclerosis patients. AB - OBJECTIVES: The aim of this study was to assess the motor function of upper extremity and its relation with fatigue, cognitive function and quality of life in Multiple Sclerosis (MS) patients. DESIGN: Cross-sectional and controlled study. SETTING: Outpatient clinic in a university hospital. SUBJECTS: Thirty-one patients with MS (25 women, 6 men; mean age 39.74 +/- 10.10 years; mean EDSS, 2.56 +/- 1.91) and 30 healthy subjects (20 women, 10 men; mean age 33.56 +/- 8.85 years) were enrolled into the study. MAIN MEASURES: Nine-hole peg test (9-HPT) and Valpar Component Work Sample Test (VCWS-4), Upper Extremity Index (UEI), Paced Auditory Serial Addition Test (PASAT), Fatigue Severity Scale (FSS), and the Multiple Sclerosis Quality of Life-54 (MSQOL-54). RESULTS: MS patients showed significant impairment in upper extremity motor functions, cognitive function and excessive fatigue compared to controls (p < 0.05). 9-HPT in MS group correlated with EDSS, UEI and MSQOL-54 physical health and cognitive function, whereas VCWS 4 scores (assembly right, assembly left and disassembly) correlated only with EDSS and UEI. No correlation was found between the VCWS-4 and cognitive function and fatigue in both of the groups. Compared to control group, a strong correlation existed between the 9-HPT and VCWS-4 in MS patients (p < 0.05). CONCLUSION: The results indicate that disability level (EDSS), UEI and cognitive function in MS patients are related with impairment in upper extremity motor function. This again contributes to an impairment in physical domain of quality of life. A strong correlation of the 9-HPT with VCWS-4 supports the use of the 9 HPT as a measure of manual dexterity and gross motor functions. PMID- 16678209 TI - Assessment of cognitive dysfunction in multiple sclerosis. AB - Cognitive deficit can be an early feature in the course of multiple sclerosis, may occur clinically isolated, impacts on social activities and could reflect disease progression that is independent of physical disability with potential therapeutical consequences. Neuropsychological tests should be independent of motor coordination or visuo-spatial ability, but sensitive to subtle cognitive changes, exploring attention speed and working memory They could be included in brief batteries with good normative data and alternative forms for future therapeutic trials. We recommend for a brief battery of neuropsychological tests the inclusion of PASAT and two memory tests: the learning stage of CVLT and backward Digit Span with a total time duration of 30 min. A complementary tool could be a cognitive questionnaire for informant. After a relapse, cognitive assessment should be performed at least 8 weeks afterwards. If the patient presents with psychiatric symptoms, a specific assessment should be proposed before starting a complete cognitive assessment. Finally, we propose some red flags warranting neuropsychological screening: patients with a mild physical disability, but unable to maintain their professional activities, pathological laughing-crying, increased age and low educational level. PMID- 16678210 TI - CSF analysis of IgH gene rearrangement in CNS lymphoma: relationship to the disease course. AB - PURPOSE: To assess whether clonal IgH genes in CSF of patients with CNS lymphoma correlates with the disease course. BACKGROUND: It has been shown that the PCR technique, which offers a sensitive test for diagnosis of systemic lymphoproliferative malignancies, can be applied to the CSF. METHODS: Seventy three CSF specimens from 32 patients (27 with primary CNS lymphoma and 5 with an isolated parenchymal CNS relapse of systemic lymphoma) were examined. The results were evaluated retrospectively and compared to conventional cytology, clinical and imaging data, and course of the disease. CNS disease was defined as active when leptomeningeal and/or parenchymal brain involvement was evident on neuroimaging. Patients were considered to have a complete response when imaging confirmed absence of a tumor mass or leptomeningeal seeding. RESULTS: Sixty-three of 73 samples had adequate genetic material for testing. Of the 63, 15 (24%) were positive for clonal IgH rearrangement. In nine (60%) of the 15 patients with active disease, PCR results were positive, while negative results were observed in 19 (95%) of the 20 patients showing clear response to treatment. The sensitivity and specificity of the PCR evaluation were 54% and 97%, respectively. The predictive values of positive and negative tests were 93% and 74%, respectively. CONCLUSIONS: The integrated results of both PCR and cytology evaluations increase the sensitivity of CSF analysis. The PCR study has high specificity and positive results are indicative for the presence of active disease, even when the tumor seems confined to the brain parenchyma. PMID- 16678211 TI - Integrated marine management and administration for an island state-the case for a new Marine Agency for the UK. PMID- 16678212 TI - Imposex in pre-pollution times. Is TBT to blame? PMID- 16678213 TI - Factors affecting outbreaks of Cochlodinium polykrikoides blooms in coastal areas of Korea. AB - We evaluated the causes of the first outbreak of Cochlodinium polykrikoides blooms in Narodo and the Southern coast of Namhaedo on the South Sea, as well as the outbreak of C. polykrikoides blooms in the East Sea and around Wando. From the results of AGP tests using diverse seawater types, we identified seawaters in which C. polykrikoides grow well and those in which they do not, depending on the sampling time and location. The reason for C. polykrikoides blooms initially occurring in Narodo, Namhaedo, and Gujaedo seems to be because the seawater that promotes the growth of C. polykrikoides is transported to the areas of primary generation, such as these three areas, by the influence of the Tsushima Warm Current. The reason that C. polykrikoides blooms occur in the coastal area of Wando and the East Sea is because after the seawater promoting the growth of C. polykrikoides is transported to these areas, the amount of sun radiation increases, and abundant nutrients flow in from heavy rains, resulting in mass propagation of C. polykrikoides. The origin of the seawater that promotes the growth of C. polykrikoides is assumed to be the southern section of the southern coastal area of Narodo, Namhaedo, and Gujaedo, in which C. polykrikoides blooms were initially discovered. The components of the f/2 medium (N, P, Fe, Mn, Co, Cu, Zn, Mo, B12, biotin, thiamine) do not seem to trigger the occurrence of C. polykrikoides blooms. PMID- 16678214 TI - Influence of salinity and fish species on PAH uptake from dispersed crude oil. AB - The use of chemical oil dispersants to minimize spill impacts causes a transient increase in hydrocarbon concentrations in water, which increases the risk to aquatic species if toxic components become more bioavailable. The risk of effects depends on the extent to which dispersants enhance the exposure to toxic components, such as polycyclic aromatic hydrocarbons (PAH). Increased salinities can reduce the solubility of PAH and the efficiency of oil dispersants. This study measured changes in the induction of CYP1A enzymes of fish to demonstrate the effect of salinity on PAH availability. Freshwater rainbow trout and euryhaline mummichog were exposed to water accommodated fractions (WAF), and chemically-enhanced water accommodated fractions (CEWAF) at 0 per thousand, 15 per thousand, and 30 per thousand salinity. For both species, PAH exposure decreased as salinity increased whereas dispersant effectiveness decreased only at the highest salinity. Hence, risks to fish of PAH from dispersed oil will be greatest in coastal waters where salinities are low. PMID- 16678215 TI - Bacteriological water quality along the Tijuana-Ensenada, Baja California, Mexico shoreline. AB - This survey was part of a Binational Program (Mexico-United States) in microbiological water quality, with a goal to assess the shoreline bacteriological water quality from Tijuana to Ensenada, Mexico. Samples were collected at 29 sites (19 beaches and 10 outfalls), from the United States border to Punta Banda, Baja California, during summer (1998) and winter (1999). Total coliforms, fecal coliforms and enterococci were used as bacterial indicators. Standard methods were used for total and fecal coliforms, while the Enterolert quick method (IDEXX) was used for the enterococci. Compared with outfalls, the beaches exceeded water quality standards by a small percent, 25.3% in summer and 17% in winter. For outfalls, the percentage of shoreline that exceeded bacterial indicator thresholds had a minor value in summer (32.7%) than in winter (50%). Sites near wastewater discharges had the lowest quality and did not meet the microbiological water quality criteria for recreational use. PMID- 16678216 TI - Prediction of anti-panic properties of escitalopram in the dorsal periaqueductal grey model of panic anxiety. AB - Escitalopram, the active enantiomer of the selective serotonin reuptake inhibitor (SSRI) citalopram, has effects in animal models for the detection of antidepressant and anxiolytic effect and in patients with major depressive and anxiety disorders. In the present study, the effects of escitalopram in the dorsal periaqueductal grey (dPAG) stimulation model of panic anxiety were investigated and compared to a number of clinically used anti-panic agents. The SSRIs, citalopram (ED(50) 6.6mg/kg), fluoxetine (ED(50) 13.6mg/kg), paroxetine (ED(50) 2.9mg/kg) and the benzodiazepine ligand, alprazolam (ED(50) 0.3mg/kg) all reduced the flight-like escape behaviour produced by electrical stimulation of the dPAG in the rat, this is presumed to indicate a reduction in fear and/or physical sensation and is interpreted as an anti-panic effect of the drugs. Escitalopram (ED(50) 3.1mg/kg) also significantly reduced flight behaviour indicating the anti-panic potential of the compound; it is suggested that escitalopram will be twice as potent as citalopram in this context. PMID- 16678217 TI - 25-Hydroxycholesterol has a dual effect on the proliferation of cultured rat astrocytes. AB - We examined the effects of 25-OH-cholesterol on the growth of cultured rat astrocytes in the presence of lipoprotein-deficient serum (LPDS). 25-OH cholesterol at 0.5-8 microM induced an increase in DNA synthesis as measured by [3H]thymidine incorporation into DNA, staining the cells with crystal violet, or counting the number of cells in different phases of the cell cycle by flow cytometry; however, at higher doses, an inhibition of cell proliferation was produced. Similar dose-dependent effects were found in media containing albumin (alone or with added EGF, PDGF, IGF-I or insulin), fetal bovine serum (FBS), or cholesterol-enriched LPDS. Mevalonate, and partially 25-OH-cholesterol, reversed the decrease in cell viability caused by mevinolin (lovastatin). However, mevalonate did not have any effect on 25-OH-cholesterol-stimulated proliferation. Finally, in media with albumin alone or in the presence of fetal bovine serum, growth factors, insulin or forskolin, 25-OH-cholesterol did not affect the expression of either c-fos mRNA or c-fos protein, as measured by real-time quantitative PCR or by Western blot, respectively. These results suggest that 25 OH-cholesterol has a dual effect on the proliferation of cultured rat astrocytes through an AP-1-independent mechanism. This could be of interest for gaining a better knowledge of the pathophysiological processes occurring in these cells. PMID- 16678218 TI - Reduction of dopaminergic degeneration and oxidative stress by inhibition of angiotensin converting enzyme in a MPTP model of parkinsonism. AB - There is growing evidence indicating that oxidative stress is a key contributor to the pathogenesis and progression of Parkinson's disease. The brain, and particularly the basal ganglia, possesses a local rennin-angiotensin system. Angiotensin activates NAD(P)H-dependent oxidases, which are a major intracellular source of superoxide, and angiotensin converting enzyme inhibitors (ACEIs) have shown antioxidant properties. We treated mice with MPTP and the ACEI captopril to study the possible neuroprotective and antioxidant effects of the latter on the dopaminergic system. Pre-treatment with captopril induced a significant reduction in the MPTP-induced loss of dopaminergic neurons in the substantia nigra and a significant reduction in the loss of dopaminergic terminals in the striatum. Furthermore, captopril reduced the MPTP-induced increase in the levels of major oxidative stress indicators (i.e. lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum. Captopril did not reduce striatal MPP(+) levels, MAO-B activity or dopamine transporter activity, which may reduce MPTP neurotoxicity. Our results suggest that angiotensin-converting enzyme inhibitors may be useful for treatment of Parkinson's disease, and that further investigation should focus on the neuroprotective capacity of these compounds. PMID- 16678219 TI - An AMPA glutamatergic receptor activation-nitric oxide synthesis step signals transsynaptic apoptosis in limbic cortex. AB - We have previously shown that pyramidal neurons engaged in cortico-cortical connectivity in limbic cortex are vulnerable to denervation lesions, i.e. relay pyramidal neurons in layer II of piriform cortex undergo transsynaptic apoptosis after lesions interrupting their inputs from the olfactory bulb (bulbotomies). At least one trigger of this transsynaptic degenerative phenomenon is the activation of inhibitory interneurons in layer I, which are induced to upregulate neuronal nitric oxide synthase (nNOS) and release NO. Thus, we have demonstrated that cortical interneurons play an essential role in transducing injury to apoptotic signaling that selectively targets pyramidal neurons. In the present study, we confirm the role of nNOS with pharmacological inhibition of a significant approximately 30% of transsynaptic apoptosis with the selective nNOS inhibitor BRNI at optimal doses. Outcomes were studied both at the histological and molecular level using DNA blots. We also show that the first-generation competitive non-NMDA (AMPA) antagonist NBQX ameliorates transsynaptic apoptosis by the same margin of difference as BRNI and it also reduces nNOS activation as indicated by a significant decrease in NADPH diaphorase histochemical activity in layer I of piriform cortex. Our findings confirm the role of nNOS activation/NO release in transsynaptic apoptosis and show that glutamatergic agonism at AMPA sites also plays a significant role. In addition, our data suggest that AMPA agonism may occur upstream to nNOS upregulation in inhibitory interneurons of layer I. In concert, our findings indicate that transsynaptic neuronal degeneration in limbic cortex involves complex AMPA-glutamatergic and nitrinergic signaling events. An AMPA-mediated upregulation of nNOS and release of NO by inhibitory interneurons may play a prominent role in this type of injury. PMID- 16678220 TI - Platelet-activating factor antagonists protect amyloid-beta damaged neurons from microglia-mediated death. AB - Neurons treated with sub-lethal concentrations of amyloid-beta1-42 developed phenotypic changes and selectively bound a CD14-IgG chimera; in co-cultures, microglia recognised and killed these amyloid-beta1-42 -damaged neurons. Pre treatment with the platelet-activating factor (PAF) antagonists (Hexa-PAF, CV6209 or ginkgolide B) reduced CD14-IgG binding to amyloid-beta1-42 -damaged neurons, and the presence of PAF antagonists in co-cultures increased neuronal survival in a dose-dependant manner. PAF antagonists also protected neurons treated with HuPrP82-146, a peptide found in prion diseases. Second messenger studies demonstrated that the addition of PAF mimicked some of the effects of amyloid beta1-42 on neurons. PAF-damaged neurons bound CD14-IgG, and PAF-damaged neurons were killed by microglia in a CD14-dependent process. Neuronal death was inversely related to both the concentration of PAF, and the number of microglia added. The effects of PAF were reduced by an antagonist of the prostanoid D receptor (BWA868C) indicating that neuronal damage induced by PAF is partly mediated by prostaglandins. These observations are compatible with the hypothesis that sub-lethal concentrations of amyloid-beta1-42 stimulate a cascade of second messengers including PAF and the prostaglandins. At nanomolar concentrations PAF induces a change in neuronal phenotype that activates microglia via the CD14 molecule, these activated microglia then kill the amyloid-beta1-42 damaged neurons. PMID- 16678221 TI - Antiallodynic effect of etidronate, a bisphosphonate, in rats with adjuvant induced arthritis: involvement of ATP-sensitive K+ channels. AB - Bisphosphonates, pyrophosphate analogues, known as inhibitors of bone resorption, appear to cause analgesia in certain clinical painful situations. To detect clinically relevant analgesic property of etidronate, a non-aminobisphosphonate, we examined and characterized its antiallodynic effect in the rat with adjuvant induced arthritis, in comparison with alendronate, an aminobisphosphonate, as determined by the von Frey test. Repeated systemic administration of etidronate at 10-40 mg/kg/day suppressed the adjuvant-induced mechanical allodynia in rat hindpaw, an effect reaching a plateau in approximately 10 days. Systemic or intraplantar (i.pl.) administration of ATP-sensitive K+ (K+ ATP) channel inhibitors, glibenclamide and/or tolbutamide, completely reversed the antiallodynic effect of etidronate within 1h in the arthritic rats, without affecting the nociceptive scores in naive or arthritic animals that had not received etidronate. Alendronate, administered repeatedly, also revealed similar glibenclamide-reversible antiallodynic effect. In contrast, the antiallodynic effect of repeated systemic indomethacin was resistant to i.pl. glibenclamide in the arthritic rats. Repeated administration of etidronate or alendronate only slightly attenuated the adjuvant-evoked hindpaw edema. Among K+ ATP channel subunits, mRNAs for Kir6.1, SUR1, SUR2A and SUR2B were abundant in rat dorsal root ganglia, while Kir6.2 mRNA was poor. Our data demonstrate that repeated etidronate as well as alendronate exhibits antiallodynic activity in arthritic rats, which might be clinically relevant, and suggest involvement of K+ ATP channels in the underlying mechanisms. PMID- 16678222 TI - Spatial and kinematic features of apraxic movement depend on the mode of execution. AB - Knowledge about the dependency of apraxic movements on the mode of execution may enhance the understanding of apraxia and of the cerebral representation of actions. We examined a common tool-use action in nine patients with left hemisphere damage and apraxia. Arm movements during the use of a handsaw were measured and tested in three different modes of execution: pantomime, pantomime with a bar shaped like the handle of the saw, and actual sawing. Analysis concentrated on spatial and kinematic features of the wrist trajectories during these repetitive movements. In healthy control subjects, both pantomime conditions differed from actual use mainly by larger amplitudes during miming. Apraxic patients executed large proportions of their pantomiming movements in an incorrect direction away from the appropriate anteroposterior direction, while other movement features were normal. The availability of the handle-like bar did not improve performance. During actual use, movement direction was constraint by mechanical demands. In this condition patients moved with moderately decreased velocity. However, this deficit was not related to the errors in movement direction characteristic of pantomiming. These data suggest that pantomiming and actual execution of an action are dictated by different external requirements and constraints, pantomiming serves to convey information, while actual use has to obey the mechanical demands of the task. Due to these differences, spatial and kinematic movement features in healthy subjects vary between the modes of execution, movements are differently vulnerable to apraxia, and deficits in patients may dissociate. PMID- 16678223 TI - Neonatal capsaicin causes compensatory adjustments to energy homeostasis in rats. AB - Several mechanisms involved in ingestive behavior and neuroendocrine activity rely on vagal afferent neuronal signaling. Seemingly contradictory to this idea are observations that vagal afferent neuronal ablation by neonatal capsaicin (CAP) treatment has relatively small effects on glucose homeostasis and long-term regulation of energy balance. It may be proposed that humoral endocrine factors and/or their sensitivities compensate for the loss of vagal afferent information, particularly when subjects face disturbances in ambient fuel levels. Therefore, male adult rats neonatally treated with CAP or with the vehicle (VEH) underwent intravenous glucose tolerance tests (IVGTTs) during which blood fuel levels, and circulating adipose, pancreatic, and adrenal hormones were assessed. CAP rats displayed similar hyperglycemia as VEH rats, but with markedly reduced plasma insulin and corticosterone responses. These results indicate that CAP rats have increased insulin sensitivity during hyperglycemic episodes, and lower plasma levels of corticosterone in CAP rats relative to VEH rats could underlie this effect. After the IVGTT, CAP rats had increased plasma adiponectin and reduced plasma resistin levels, and these alterations in adipose hormones might be relevant for post-ingestive metabolic processes. In a second experiment, anorexigenic efficacies of cholecystokinin and leptin were assessed. While VEH rats, but not CAP rats, responded with reduced food intake to i.p. injected cholecystokinin, only CAP rats responded to i.v. infused leptin with a reduction in food intake. It is concluded that reduced HPA axis activity and/or increased leptin signaling could underlie compensations in fuel handling and energy balance following CAP treatment. PMID- 16678224 TI - Environmental enrichment reduces impulsivity during appetitive conditioning. AB - Although environmental enrichment is presumed to enhance learning, appetitive behavior may also be altered by this experience: anticipatory responding for sucrose is reduced in environmentally enriched (EE) rats [van der Harst, J.E., Baars, A.M. and Spruijt, B.M. Standard housed rats are more sensitive to rewards than enriched housed rats as reflected by their anticipatory behaviour. Behav Brain Res 2003;142:151-156]. To assess the impact of differential environmental experience on learning and appetitive behavior, we trained 17 EE and socially isolated (SI) rats in a three-phase, operant-shaping procedure for sucrose reinforcement. In phase one, a feeder cue was paired with sucrose availability. In phase two, a nose poke to either one of two lit holes on the opposing wall activated the feeder cue. In phase three, the feeder cue was elicited by a poke to a single lit hole. While acquisition rates in phase one and phase two were similar, EE animals reached phase-three criteria [completion of 100 trials in 45 min and 15 or fewer bad pokes] faster than SI animals. These two groups showed similar session completion rates, reinforced and non-reinforced licking responses, and overall behavioral activity during phase three acquisition; however, SI rats performed more bad pokes (responses to the non-lit hole after nose-poke cue onset) and intertrial interval (ITI) pokes during this training period. Because all ITI (and presumably many bad) pokes were initiated before onset of nose-poke cue, this difference indicates greater anticipatory responding in SI animals. This experience-dependent alteration in appetitive behavior may explain, in part, the tendency of SI rats to show attenuated learning rates in appetitive contexts in which complex contingencies exist. PMID- 16678225 TI - Influence of cholinergic system modulators on morphine state-dependent memory of passive avoidance in mice. AB - Memories are shown to be impaired in mice during step-down passive avoidance tasks with substantial residual effects lasting as long as 24 h after the pre training administration of morphine. Administration of the same dose of morphine as a pre-test treatment restored memory. Since the cholinergic system has been reported to be involved in several actions of morphine, e.g.: modulation of memory and analgesia, we have investigated the part played by cholinergic modulator drugs, on the memory recall in mice. The locomotor activity of animals was studied as well. Administration of either atropine, a peripheral-central muscarinic antagonist, or mecamylamine, a peripheral-central nicotinic antagonist, failed to alter memory themselves, but significantly prevented morphine-induced memory recall following co-administration with morphine. Neither hexamethonium, a peripheral nicotinic antagonist, nor neostigmine, a peripheral anticholinesterase, showed intrinsic activity or a significant change in morphine induced memory recall. Finally, physostigmine, a peripheral-central anticholinesterase, not only induced memory recall itself, but also increased morphine-induced retrieval. Memory recall of the step-down passive avoidance task following drug combinations was not related to locomotor activity changes. Thus, morphine-induced memory recall appears to be influenced by central cholinergic activity. PMID- 16678227 TI - The prediction of isotopic patterns in phenylpropanoids from their precursors and the mechanism of the NIH-shift: basis of the isotopic characteristics of natural aromatic compounds. AB - The theoretical 2H-distribution in the aromatic ring of phenylpropanoids can be predicted from that of their precursors--erythrose-4-phosphate, phosphoenolpyruvate and NADPH--and by invoking the mechanism of the NIH-shift and implied deuterium isotope effects. For each position in the non-oxygenated ring, the predicted natural 2H-abundance is in excellent agreement with experimental data obtained from quantitative 2H NMR-measurements on natural compounds, especially concerning the relative 2H-abundances p > o > or = m. For the p hydroxylated derivatives, the experimentally determined 2H-abundance sequence order m > o can also be deduced, assuming an anisotropic migration (intramolecular isotope effect) of the p-hydrogen atom to the two differently 2H substituted m-positions during the NIH-shift (intramolecular hydrogen transfer) and an in vivo deuterium kinetic isotope effect of approximately 1.20 on the final hydrogen elimination from the proposed ketodiene intermediate. The predicted 2H-distribution pattern of methyl salicylate 10, a representative of an o-hydroxylated natural compound, is in excellent agreement with that reported from 2H NMR analyses. However, for salicyl alcohol, minor differences between the theoretical and experimentally determined pattern are found that cannot yet be satisfactorily explained. On the other hand, a very good agreement is found between the theoretical and experimental pattern of coumarin, provided a deuterium kinetic isotope effect of approximately 1.30 is assumed for the elimination of the H-atoms from the ketodiene intermediate. The secondary m hydroxylation of p-coumaric acid in the biosynthesis of vanillin seems to proceed without large isotope effects. Parallel differences are also observed for the 18O kinetic isotope effects on the corresponding monooxygenase-catalysed reactions. The results demonstrate convincingly that the mechanisms of these general reactions of the phenylpropanoid biosynthetic pathway are identical and follow general principles. Small observed differences between the 2H-patterns of individual natural aromatic compounds originating from the same hydroxylation type can therefore be assigned to differences of the patterns of the precursors, the extent and the orientation of the hydrogen migration, and the kinetic isotope effect on the final hydrogen elimination. The evidence for the existence of general systematic rules governing isotopic patterns in the shikimic acid pathway and its subsequent reactions is further supported by the recently reported 13C distribution pattern of vanillin, which is also in agreement with that predicted from the precursors. Hence, it is apparent that the systematics of the isotope patterns of phenylpropanoids are in line with the generally accepted biosynthetic reactions in the shikimic acid pathway and that this knowledge can strengthen their value as an essential support for the distinction of natural and synthetic aromatic compounds. PMID- 16678228 TI - Tetraprenyltoluquinols from the brown alga Cystophora fibrosa. AB - Six cyclised tetraprenyltoluquinols and five stereoisomers with the previously reported amentol skeleton have been isolated from the lipophilic extract of the South African brown alga Cystophora fibrosa. Structures and relative stereochemistry were determined using spectrometric techniques, particularly 1D and 2D NMR, and molecular modelling experiments. The compounds isolated appear to be enantiomeric to compounds with the same skeleton isolated from brown algae of the genus Cystoseira collected in northern Africa and the Mediterranean Sea. The isolation of tetraprenyltoluquinols with the amentol skeleton from this alga suggests that C. fibrosa should be moved from the genus Cystophora into the Cystoseira. PMID- 16678226 TI - Alterations in blood glucose levels under hyperinsulinemia affect accumbens dopamine. AB - Dopaminergic systems have been implicated in diabetes and obesity. Notwithstanding, the most basic relationship between dopamine and plasma insulin as well as glucose levels yet remains unknown. The present experiments were designed to investigate the effects of acute hyperinsulinemia on basal dopamine levels in the nucleus accumbens of the rat under chloral hydrate anesthesia using acute microdialysis in combination with the hyperinsulinemic-glycemic clamping procedure. In Experiment 1, each rat was infused with one of the three concentrations of insulin (2.4, 4.8, or 9.6 mU/kg per min) while plasma glucose levels were maintained at euglycemia (approximately 5.5 mmol/L). Dopamine, dihydroxyphenylacetic acid and homovanillic acid were not significantly different from baseline during either the clamp or post-clamp periods for all insulin concentrations. In Experiment 2, rats were infused with the highest concentration of insulin (9.6 mU/kg per min) and plasma glucose levels were maintained at either hypoglycemia (approximately 3 mmol/L) or hyperglycemia (approximately 14 mmol/L). Dopamine was elevated at 100 min (+113% above basal levels) and 120 min (+117%) in the hypoglycemic condition and at 120 min (+121%) in the hyperglycemic condition. In the hyperglycemic post-clamp period, homovanillic acid was decreased below basal levels (approximately -32%). These results together suggest that short-term blood glucose deviations coupled with acute hyperinsulinemia affect the mesoaccumbens dopamine system. PMID- 16678229 TI - Efficiency of different methods of extraction and purification of cytokinins. AB - The increasing use of advanced methods, such as mass spectrometry, for the determination of cytokinins has raised special requirements for the extraction and purification of this class of plant hormones. Extraction of Arabidopsis thaliana plants with three different solvents, [80% (v/v) MeOH, Bieleski's MCF-7, and modified Bieleski's] provided similar yields of most analyzed cytokinins determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). However, the extraction with a modified Bieleski's solvent (MeOH HCO2H-H2O [15:1:4, v/v/v]) gave the highest responses of deuterated cytokinins (used as test compounds) in plant extracts as compared to the responses of pure deuterated standards (relative internal standard response, RISR). Purification of cytokinins using Oasis MCX sorbent with reversed-phase and cation-exchange characteristics, in comparison to the DEAE Sephadex RP-C18 method, provided higher levels of zeatin riboside monophosphate and similar levels of cytokinin bases, ribosides and glucosides. Using this method the content of UV-absorbing contaminates was decreased by about 90% and the RISR values of all tested cytokinin standards but riboside monophosphates were increased about two-fold. The former method provided preparations more suitable for HPLC/MS/MS analysis with respect to simplicity and sample purity. PMID- 16678230 TI - Relative quantification and detection of different types of infectious bursal disease virus in bursa of Fabricius and cloacal swabs using real time RT-PCR SYBR green technology. AB - In present study, different types of infectious bursal disease virus (IBDV), virulent strain DK01, classic strain F52/70 and vaccine strain D78 were quantified and detected in infected bursa of Fabricius (BF) and cloacal swabs using quantitative real time RT-PCR with SYBR green dye. For selection of a suitable internal control gene, real time PCR parameters were evaluated for three candidate genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA and beta-actin to IBDVs. Based on this beta-actin was selected as an internal control for quantification of IBDVs in BF. All BF samples with D78, DK01 or F52/70 inoculation were detected as virus positive at day 1 post inoculation (p.i.). The D78 viral load peaked at day 4 and day 8 p.i., while the DK01 and F52/70 viral load showed relatively high levels at day 2 p.i. In cloacal swabs, viruses detectable were at day 2 p.i. for DK01 and F52/70, day 8 p.i. for D78. Importantly, the primers set were specific as the D78 primer set gave no amplification of F52/70 and DK01 and the DK01 primer set gave no amplification of D78, thus DK01 and D78 could be quantified simultaneously in dually infected chickens by use of these two set of primers. The method described here is robust and may sever as a useful tool with high capacity for diagnostics as well as in viral pathogenesis studies. PMID- 16678231 TI - Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies. AB - Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection. PMID- 16678232 TI - Fate of coagulant species and conformational effects during the aggregation of a model of a humic substance with Al13 polycations. AB - A model of a humic substance (MHS) obtained from auto-oxidation of catechol and glycine, was aggregated at pH 6 and 8 with Al(13) polycations. The fate of Al(13) coagulant species upon association with MHS functional groups was studied using solid state (27)Al Magic-angle spinning (MAS) NMR and CP-MAS (13)C NMR. Electrophoretic measurements and steady-state fluorescence spectroscopy with pyrene as a fluoroprobe, were combined to investigate structural re-organization of humic material with aluminum concentration. MAS (27)Al NMR revealed that the coagulant species are Al(13) polycations or oligomers of Al(13) units at both pHs. CP MAS (13)C spectra indicated that, at low Al concentration, hydrolyzed aluminum species bind selectively to carboxylic groups at pH 6 and to phenolic moieties at pH 8. At higher coagulant concentrations, the remaining functional groups also interact with hydrolyzed Al to yield similar CP MAS (13)C spectra in the optimum concentration range. Negative values of electrophoretic mobility were obtained at optimum coagulant concentrations even though an overall charge balance was achieved between MHS anionic charge and Al(13) cationic charge at pH 6. The polarity-sensitive fluorescence of pyrene revealed that the interaction of Al(13) coagulant species with MHS functional groups induces the formation of intramolecular hydrophobic microenvironments. Such structural changes were reversed upon further addition of Al(13) polycations. PMID- 16678233 TI - Disinfection of swine wastewater using chlorine, ultraviolet light and ozone. AB - Veterinary antibiotics are widely used at concentrated animal feeding operations (CAFOs) to prevent disease and promote growth of livestock. However, the majority of antibiotics are excreted from animals in urine, feces, and manure. Consequently, the lagoons used to store these wastes can act as reservoirs of antibiotics and antibiotic-resistant bacteria. There is currently no regulation or control of these systems to prevent the spread of these bacteria and their genes for antibiotic resistance into other environments. This study was conducted to determine the disinfection potential of chlorine, ultraviolet light and ozone against swine lagoon bacteria. Results indicate that a chlorine dose of 30 mg/L could achieve a 2.2-3.4 log bacteria reduction in lagoon samples. However, increasing the dose of chlorine did not significantly enhance the disinfection activity due to the presence of chlorine-resistant bacteria. The chlorine resistant bacteria were identified to be closely related to Bacillus subtilis and Bacillus licheniformis. A significant percentage of lagoon bacteria were not susceptible to the four selected antibiotics: chlortetracycline, lincomycin, sulfamethazine and tetracycline (TET). However, the presence of both chlorine and TET could inactivate all bacteria in one lagoon sample. The disinfection potential of UV irradiation and ozone was also examined. Ultraviolet light was an effective bacterial disinfectant, but was unlikely to be economically viable due to its high energy requirements. At an ozone dose of 100 mg/L, the bacteria inactivation efficiency could reach 3.3-3.9 log. PMID- 16678234 TI - Mechanochemical approach to remove phenanthrene from a contaminated soil. AB - Soil samples collected from a cultivated soil of Southern Italy after artificial contamination with phenanthrene (PHE) were ground in a ball mill and compared with spiked (via acetone) sample as control. The mechanochemical treatment was also applied to a simple binary system birnessite (delta-MnO(2))-PHE and to soil added with birnessite to evaluate the oxide role in removing the contaminant. Different extraction methods, such as Soxhlet, sonication, and desorption from resin beads were adopted to estimate the residual PHE analysed with HPLC. X-ray diffraction and TG-DTA analyses were performed to quantify mineralogical phases in soil and their possible modifications after grinding. The results showed that the grinding was more efficient in removal of PHE when the pollutant was in solid phase ( approximately 50% of removal) than when it was spiked via acetone in the same soil ( approximately 20% of removal). Addition of birnessite to soil did not change significantly the removal of PHE through time. Independently of the extraction methods used, the PHE recovered after the mechanochemical treatment in the presence of solid PHE was always lower, suggesting a higher efficiency of such a treatment in degrading PHE or forming bound residues in its original or transformed form. X-ray powder diffraction of milled solid PHE showed that order disorder phase transition occurred in solid phenanthrene as consequence of the mechanochemical treatment. X-ray diffractometry and TG-DTA analyses were crucial in evidencing that interaction between PHE and birnessite occurred. PMID- 16678235 TI - Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea. AB - The Mediterranean mussel, Mytilus galloprovincialis, is a classical sentinel organism for monitoring the biological effects of contaminants through the use of molecular and cellular biomarkers. These biological responses can be modulated also by seasonal changes of both environmental and biological factors, potentially influencing responsiveness and sensitivity to pollutants. The aim of this study was to characterize in a reference mussel population from the Adriatic Sea, the natural fluctuations of several oxidative stress biomarkers widely used in ecotoxicological applications. Analyses of individual antioxidant defenses (catalase, glutathione S-transferases, glutathione reductase, glutathione peroxidases, levels of glutathione) were integrated with those of the total oxyradical scavenging capacity (TOSC-assay), which quantify the overall capability of a tissue to neutralize different forms of oxyradicals. Due to the close relationship between antioxidant efficiency and onset of various cellular alteration, the seasonal characterization was carried out also on lysosomal membrane stability, accumulation of malondialdehyde, neutral lipids and lipofuscin, levels of metallothioneins and activity of peroxisomal enzymes (acyl CoA oxidase and d-aminoacid oxidase). Results indicated a significant seasonality for several biological responses; major variations occurred especially in spring and summer months suggesting the influence of natural factors, such as temperature, reproductive cycle and food availability. The observed seasonal oscillations revealed both similarities and differences with those reported for other Mediterranean mussel populations suggesting that opposite trends can occur when the same environmental prooxidant factors have a different regional influence. PMID- 16678236 TI - Distribution of organochlorine pesticides and heavy metal residues in fish and shellfish from Calicut region, Kerala, India. AB - Fish collected from five different locations from the Calicut region, India were analysed for the levels of organochlorine pesticides (OCPs) and heavy metal (HM) residues in order to elucidate the status of these chemical contaminants in fish and shellfish meant for human consumption. The highest concentrations of OCPs detected in the edible portion of fish were 10.47, 70.57 and 28.35 ng g-1 wet weight, in marine, brackish water and freshwater, respectively. BHC and heptachlor epoxide formed the major share of OCPs in the marine fish while BHCs contributed to the major share in the freshwater and brackish water fish. The DDT (sum of DDT and its metabolites) ranged from 0.05 to 8 ng g-1 in the samples irrespective of the habitat. Among the HMs, Cu, Zn, Mn and Cr were present in the samples irrespective of the place of collection. About 22% of the samples from the freshwater area contained marginally higher lead content above the legal limit of 0.2-0.4 microg g-1. The Cr content ranged from 0.1 to 1.1 microg g-1 in the fish with marginally higher content in the fish from freshwater and marine regions (0.55-1.1 microg g-1). Mercury was detected in the samples (55%) from marine habitat only. The concentrations of OCPs and HMs in the samples, in general, were below the maximum residual level recommended by various organizations. PMID- 16678237 TI - Physiological recovery from episodic acid stress does not mean population recovery of Gammarus fossarum. AB - The physiological responses of the acid-sensitive amphipod Gammarus fossarum exposed in situ to acid stress (pH4.5 and 5.5) and then transferred back to neutral water were investigated. Survival rate and haemolymph [Cl(-)] and [Na(+)] were assessed after 24, 48 and 72h of exposure in acidic streams and after a recovery period of 12, 24, 36, 48 and 60h. After 24h, exposure to slightly acidic (pH5.5) and strongly acidic water (pH4.5) led to a severe and significant depletion in haemolymph [Na(+)] and [Cl(-)] compared to organisms exposed in circumneutral water (pH7.3). However, after only a 12h-period of transfer back in neutral water and whatever the previous exposure time (24, 48 and 72h) in both slightly and strongly acidic water, haemolymph [Na(+)] and [Cl(-)] were equal or superior to the control level without associated mortality. In spite of this fast physiological recovery capacity, populations of G. fossarum living in streams undergoing episodic acid stresses were drastically affected thus, demonstrating the high acid-sensitivity of this species. We discuss the possible reasons of population regression and the absence of population recovery. PMID- 16678238 TI - PCDD/Fs in sprat (Sprattus sprattus balticus) from the Gulf of Finland, the Baltic Sea. AB - The concentrations and congeners pattern of the polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were determined in sprat collected by the commercial catches in the Gulf of Finland, Baltic Sea. Based on the toxic equivalent concentrations 2,3,4,7,8-PeCDF prevailed among the congeners of PCDD/Fs. The significance of age- and season-specific relationship between the concentration of lipids and dioxins was demonstrated. On lipid weight basis the concentration of PCDDs was significantly higher in spring than in autumn. This difference was not statistically significant for PCDFs. On lipid weight basis the concentration of dioxins decreased with rising content of lipids. PCDD/Fs toxic equivalent concentration increased with the age of sprat exceeding the EU maximum limit value at more than 5-year-old fish. PMID- 16678239 TI - Sarcopenia-related apoptosis is regulated differently in fast- and slow-twitch muscles of the aging F344/N x BN rat model. AB - Age-related decreases in muscle mass have been associated with the loss of myonuclei, possibly through a mechanism involving mitochondria. It is unclear if age-related apoptotic mechanisms vary by fiber type. Here we investigate indices of apoptosis along with the regulation of apoptotic mediators in the extensor digitorum longus (EDL) and soleus of adult (6 month), old (30 month), and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia (F344/N x BN) rats. Compared to 6-month muscles, aged muscles exhibited decreases in muscle mass along with increases in the number of nuclei staining positively for DNA fragmentation. The expression of Bax, Bcl-2, caspase-3 and caspase-9 was regulated differently with aging between muscle types and in a manner not consistent with mitochondria-mediated apoptosis. To investigate the potential of calpain involvement in age-related myonuclear loss, the calpain-dependent cleavage of alpha-fodrin was examined. The proteolytic cleavage of alpha-fodrin by calpains was increased in both muscles with only the 36-month soleus exhibiting increased caspase-dependent alpha-fodrin cleavage. Taken together, these data suggest that apoptotic regulatory events differ between fiber types in the aging F344/N x BN and that mitochondrial-dependent apoptosis pathways may not play a primary role in the loss of muscle nuclei with aging. PMID- 16678240 TI - Discovering the importance of lateral CO(2) transport from a temperate spruce forest. AB - Our study investigated the concentration of dissolved carbon at the point when water leaves the pedosphere and whether this amount represents a significant proportion of terrestrial carbon cycling. The investigations were carried out in a temperate forest catchment (Black Forest, Germany) over a period of 1 year. The annual export of dissolved C compounds (14.4 g C m(-2) year(-1)) was dominated by CO(2) (9.7 vs. 4.7 g C m(-2) year(-1) DOC). Even though the direct CO(2) degassing at the spring was inferior (0.4 kg C year(-1)), considerably lower CO(2) concentrations were measured 17 m downstream of the spring. This shows that a large proportion of dissolved CO(2) (93%) originating from the pedosphere is not captured anymore within a short distance from the spring. The measured lateral C-transport was in the same order of magnitude as reported for the net ecosystem exchange (NEE) observed for German spruce forests (-4 to -55 g C m(-2) year(-1)). Therefore, the results clearly demonstrated that the lateral transport of dissolved carbon can be a significant part of terrestrial carbon budgets and for this study site CO(2) was dominating this 'indirect' pathway. However, for generalisation, it is important to extend this investigation to other landscapes and climatic zones. PMID- 16678241 TI - Modelling of the long term fate of pesticide residues in agricultural soils and their surface exchange with the atmosphere: Part I. Model description and evaluation. AB - Sources of pesticides in the atmosphere can be releases of new material through current use, or emission/reemission from soil residues resulting from historical use. It is the latter aspect, soil residues, that is the focus of this study. This paper describes the application of a simple coupled atmosphere-soil pesticide exchange model that can assist in the interpretation of soil residue and air concentration measurements, and in the projection of short period field measurements to larger spatial scales and longer time periods. Only dry gaseous exchange (emission and deposition) between bare agricultural lands and the atmosphere is modelled. Wet deposition and particle associated deposition of pesticide are not included. Model results are compared with published co-located air and soil pesticide concentration measurements made on agricultural lands in the southern U.S. that have soil residues of lindane and the following six highly persistent pesticides: cis-, trans-chlordane, p,p'-DDE, dieldrin, trans-nonachlor and toxaphene. The study results show: (i) that measured air concentrations of toxaphene and p,p'-DDE above agricultural soils in the southern U.S. can be attributed to emissions due to local soil residues of these pesticides rather than to the regional background air concentrations; (ii) that both soil emissions and background air concentrations of dieldrin contribute significantly to the measured air concentrations; (iii) that measured air concentrations of cis- and trans-chlordane as well as trans-nonachlor and lindane are mainly due to the regional background with little contribution from local soil residues. An analysis of modelled summer day and night average soil-air exchange fluxes shows that toxaphene and p,p'-DDE soil residues are strong sources of emission to the atmosphere during both the day and night while the chlordanes, trans-nonachlor, lindane and dieldrin are deposited from the atmosphere to the soil during the night hours and emitted to the atmosphere during the day time. This result illustrates the model's capability to simulate the processes that lead to the 'grasshopper' effect whereby persistent pesticides in soils can be transported in the atmosphere by successive periods of emission and deposition to terrestrial surfaces. In the second part to this paper, the model is used to study the trends of pesticide residues and air concentrations over a twenty year period. PMID- 16678242 TI - Modelling the dispersion of radionuclides following short duration releases to rivers: Part 1. Water and sediment. AB - This paper evaluates and generalises state-of-the-art approaches for modelling short duration liquid discharges of radionuclides ((3)H, (14)C, (60)Co, (134)Cs, (137)Cs, (65)Zn, (89)Sr, (90)Sr, (125)I, (131)I, (241)Am, isotopes of Pu and U) to rivers. An advection-dispersion model was parameterised and used to predict the concentrations of radionuclides in the river environment, i.e. in river water, river bed sediment and fish (Part II of this paper covers uptake to fish). The coupled transport and bio-uptake model was used to predict the concentrations of radionuclides in the River Thames, UK, and one of its tributaries as a result of hypothetical short duration discharges. A simplified version of this model was developed and presented as "look-up" graphs. The influence of various environmental parameters on model output was evaluated by sensitivity analysis. Time-integrated water and sediment concentrations and maximum sediment concentrations may be predicted for all rivers on the basis of the river volumetric flow rate only. Maximum concentration in water is, however, also dependent on other river characteristics. For this latter case, generalised modelling approaches are tested for use in situations where detailed hydrological and dispersion data are not available. PMID- 16678243 TI - Mesenteric fibromatosis masquerading as an ovarian neoplasm twenty years after Chernobyl radiation exposure. AB - BACKGROUND: Since their initial description in 1832, desmoid tumors have been reported to occur in virtually every part of the body. Intra-abdominal desmoid tumors, or mesenteric fibromatosis, are often associated with Familial Adenomatous Polyposis mutation in a syndrome known as Gardner's Syndrome. Although sporadic cases of desmoid tumors do occur, unlike Gardner's Syndrome, they predominantly occur extra-abdominally. CASE: Case report of a 61-year-old female who presented with two months of abdominal pain, progressive lower abdominal distension and a 10-15 pound weight gain accompanied by one week of urinary hesitancy and frequency. Patient underwent a diagnostic workup for an ovarian neoplasm, but was found at surgery to have mesentric fibromatosis. CONCLUSION: Although uncommon, mesentric fibromatosis must be considered in the differential diagnosis when evaluating a patient with an abdominal mass of unknown origin. PMID- 16678244 TI - Is peritoneal cytology a prognostic factor of endometrial cancer confined to the uterus? AB - OBJECTIVE: The goal of this study was to investigate whether intraoperative peritoneal cytology serves as a prognostic factor in patients with the endometrial cancer limited to the disease confined to the uterus. METHODS: From patients with endometrial cancer treated at 2 facilities between 1988 and 2001, 307 patients were selected for retrospective investigation. To be included in this study, patients required (1) full surgical staging including total abdominal hysterectomy/bilateral salpingo-oophorectomy/retroperitoneal lymph node dissection/peritoneal cytology, (2) negative nodes, (3) disease localized to the uterus and (4) endometrioid subtype. RESULTS: The median duration of the follow up period was 61 months (25th to 75th percentiles: 45 to 92 months). Peritoneal cytology was positive in 32 patients (10.4%). The 5-year survival rate of peritoneal-cytology-positive patients was 87%, significantly lower than that (97%) of cytology-negative patients (P = 0.011). The relationship between the clinicopathological factors including peritoneal cytology and the prognosis was investigated by univariate analysis, and peritoneal cytology positivity, age of 60 years or older, histologic grade (Grades 2 and 3), myometrial invasion of 1/2 or more and vascular invasion were significant prognostic factors (P < 0.05 in all). On multivariate analysis of these factors, peritoneal cytology positivity and histologic grade (Grade 2 and 3) were independent prognostic factors (P < 0.05 each). CONCLUSIONS: For the patients with endometrial cancer limited to the disease confined to the uterus in which accurate staging including retroperitoneal lymph node dissection was performed, peritoneal cytology may be an important prognostic factor. PMID- 16678245 TI - Effects of the H3-receptor inverse agonist thioperamide on the psychomotor effects induced by acutely and repeatedly given cocaine in C57BL/6J mice. AB - Previous studies have shown that histamine H(3) blockers potentiate the psychomotor and rewarding effects of cocaine. The present study examined the influence of thioperamide, an inverse H(3) receptor agonist, on the development of psychomotor sensitization and stereotyped activity induced by acute or intermittent cocaine in C57BL/6J mice. In the first experiment, mice were injected i.p. with saline, 10 or 20 mg/kg thioperamide and saline or 8 mg/kg cocaine, 10 min apart, before being tested for their locomotor activity (providing data on the acute effects of thioperamide on cocaine-induced activity). Subsequently, mice were treated in the same manner every other day over six additional sessions. Sensitization was assessed by the responsiveness to a cocaine challenge (8 mg/kg, i.p.) given 2 and 14 days following the intermittent treatment. In experiments 2 and 3, we tested the effects of thioperamide (10 or 20 mg/kg, i.p.) on gnawing and sniffing induced or affected by relatively high doses of cocaine (24 or 32 mg/kg, s.c.), the drugs being given 10 min apart. In the first experiment, both doses of thioperamide amplified cocaine-induced psychomotor hyperactivity almost on all experimental sessions. However, the histamine inverse agonist did not affect the induction of a psychomotor sensitization. All cocaine-treated mice showed similar levels of sensitized activity 2 and 14 days after the intermittent treatments, whether they received thioperamide or not. The second and the third experiments showed that thioperamide did not affect gnawing and sniffing induced by cocaine. Taken together, these results indicate that H(3) receptors clearly contribute to the neurobiological mechanisms of the locomotor component of cocaine-induced psychomotor activation, but less likely to those underlying the development of cocaine behavioral sensitization or the expression of cocaine-induced oro-facial stereotypies. PMID- 16678246 TI - Mercury vapor levels in exhaust air from dental vacuum systems. AB - OBJECTIVE: This study was undertaken to determine mercury (Hg) vapor levels in the air exhausted from dental vacuum systems. METHODOLOGY: Hg vapor concentrations from the dental vacuum system exhaust ports of three dental clinics were measured utilizing the Jerome 431-X mercury vapor analyzer and the United States Occupational Safety and Health Administration's (OSHA) method ID 140 in units of ng Hg/m3. Air velocity measurements and temperatures were determined with a constant temperature thermal anemometer. Hg emissions per unit time were then calculated in ng Hg/min. Ambient Hg concentrations from a location approximately 1000 feet away from the closest clinic sampled in this study were measured with an Ohio Lumex Inc. RA-915+ Hg vapor analyzer. RESULTS: Mean Hg vapor concentrations analyzed with the Jerome 431-X were: 46,526, 72,211, and 36,895 ng/m3 for clinic I (110 chairs), clinic II (30 chairs) and clinic III (2 chairs), respectively. Mean Hg vapor concentrations utilizing OSHA method ID-140 were 45,316, 73,737, and 35,421 ng/m3, respectively. Air flow values were: 11.6, 1.8, and 0.5 standard m3/min, respectively. Hg emission data utilizing air flow measurements were calculated to be 532,684, 131,353, and 18,079 ng/min, respectively, (P<0.001). There was no statistical difference between the two methods used to measure Hg vapor concentrations. The mean Hg concentration in ambient air approximately 1000 feet from the nearest clinic sampled was 13.2 ng/m3. CONCLUSION: The two different methods used to measure Hg vapor concentrations provided similar estimates of Hg concentrations from the exhaust air of three dental vacuum systems. Hg vapor release to the atmosphere from dental vacuums can be substantial and can exceed human exposure limits. PMID- 16678247 TI - Electric device improves bonds of simplified etch-and-rinse adhesives. AB - OBJECTIVES: This study investigated the effects of an electric field produced by a new device for the application of etch-and-rinse adhesives on demineralized dentin surfaces. METHODS: Three simplified etch-and-rinse adhesives (Single Bond, Prime&Bond NT and One-Step) were applied with the electric device and compared with controls prepared with disposable sponges. Specimens were processed for microtensile bond strength test and nanoleakage investigation using high resolution SEM. RESULTS: Microtensile testing revealed higher bond strengths (p<0.05) for all adhesives tested when electricity was used. Adhesive interfaces prepared with electric impulses exhibited very homogenous hybrid layers with minimal nanoleakage compared with the controls. SIGNIFICANCE: The use of electricity produced by a new electronic device during the application of dentin adhesives may increase adhesive adaptation to the dentin substrate and improve dentin hybridization due to the substrate modifications induced by an electric field on the demineralized dentin organic matrix. PMID- 16678248 TI - Profiles and portfolios of adolescent school-based extracurricular activity participation. AB - The current study presented a new description of adolescent school-based activity participation, in the form of mutually exclusive activity portfolios, and described the kinds of youth that participate in each portfolio. These portfolios included (1) Sports Only, (2) Academics Only, (3) School Only, (4) Performance Only, (5) Multiple Activities, and (6) Non-Participation. Findings indicated that youth demographic characteristics and school size differentiated between different kinds of activity participation as well as nonparticipation. More detailed activity portfolios were also identified that were complex and demonstrate the difficulty of examining participation beyond larger, more inclusive groupings. The Multiple Activity portfolio emerged as a unique group worthy of further examination. Characteristics of non-participators included: lower socioeconomic status, lower grades, and attended larger schools. Hispanic adolescents were also less likely to participate in school-based extracurricular activities. Findings from this study inform ecological models of adolescent development as well as school and social policy. PMID- 16678249 TI - Organotin accumulation in an estuarine food chain: comparing field measurements with model estimations. AB - The bioaccumulation model OMEGA (optimal modelling for ecotoxicological applications) is used to explore accumulation of organotins in the Western Scheldt food chain, consisting of herbi-detritivores, primary and secondary carnivorous fish and a piscivorous bird. Organotins studied are tributyltin (TBT) and triphenyltin (TPT) and the respective di- and mono-organotin metabolites. Empirical elimination rate constants are compared to model predictions for organic substances and metals. It is found that field bioaccumulation ratios are higher than predicted based on elimination kinetics relevant for organic compounds. The results indicate that uptake of organotins mainly occurs via hydrophobic mechanisms, whereas elimination may occur via metal-like kinetics. This results in very low elimination rates, which are comparable to model predictions for metals. PMID- 16678250 TI - Molecular aspects on the specific interaction of cytotoxic plant alkaloid palmatine to poly(A). AB - The interaction of the protoberberine alkaloid palmatine with single and double stranded structures of poly(A) was studied by various biophysical techniques. Comparative binding studies were also performed with double stranded DNA, t-RNA, poly(C).poly(G), poly(U) and poly(C). The results of competition dialysis, fluorescence, and absorption spectral studies converge to reveal the molecular aspects of the strong and specific binding of palmatine to single stranded poly(A). The binding affinity of palmatine to natural DNA, t-RNA and double stranded poly(A) was weaker while no binding was apparent with single stranded poly(U), poly(C) and double stranded poly(C).poly(G). The strong affinity of the alkaloid to single stranded poly(A) in comparison to the double stranded structure was also revealed from circular dichroic and viscometric studies. The effect of [Na+] ion concentration on the binding process revealed the significant role of electrostatic forces in the complexation. The presence of bound alkaloid also remarkably affected denaturation-renaturation of stacked helical poly(A). The energetics of the strong binding to poly(A) was studied from thermodynamic estimation from van Hoff' analysis of the temperature dependent binding constants and ultra sensitive isothermal titration calorimertry, both suggesting the binding to be exothermic and enthalpy driven. This study provides detailed insight into the binding specificity of the natural alkaloid to single stranded poly(A) over several other single and double stranded nucleic acid structures suggesting its potential as a lead compound for RNA based drug targeting. PMID- 16678251 TI - Study of the interaction between doxepin hydrochloride and bovine serum albumin by spectroscopic techniques. AB - The binding of doxepin hydrochloride (DH) to bovine serum albumin (BSA) was investigated by spectroscopic (fluorescence, UV-vis absorption and circular dichroism) techniques. The binding parameters have been evaluated by fluorescence quenching method. The thermodynamic parameters, Delta H major, Delta S major, and Delta G major calculated at different temperatures indicated that the hydrogen bond and hydrophobic forces played a major role in the interaction of DH with BSA. Based on the Forster's theory of non-radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (DH) was evaluated and found to be 2.7 nm. Spectral results observed showed that the binding of DH to BSA induced conformational changes in BSA. The effect of common ions on the binding of DH to BSA was also examined. PMID- 16678252 TI - Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen. AB - Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1. PMID- 16678253 TI - Effect of shearing on formation of silk fibers from regenerated Bombyx mori silk fibroin aqueous solution. AB - In this paper, the spinnable regenerated silk fibroin aqueous solution with high concentration was prepared and the regenerated silk fibers were obtained from the aqueous solution by two different spinning processes at ambient temperature. The orientation of these fibers was characterized by polarizing microscope. Their secondary structure was investigated by Raman spectroscopy and related mechanical properties were also measured. These data showed that shearing is an important step for increasing orientation and silk II (beta-sheet) structure, and the mechanical properties of the regenerated silk fibers can also be improved by shearing. PMID- 16678254 TI - The effect of degree of acrylic derivatisation on dextran and concanavalin A glucose-responsive materials for closed-loop insulin delivery. AB - Formulations of dextran methacrylate (dex-MA) and concanavalin A methacrylamide (con A-MA) were photo-polymerized to produce covalently cross-linked glucose responsive materials for the basis of a closed-loop insulin delivery device. The viscoelastic properties of these polymerised materials were tested rheologically in the non-destructive oscillatory mode within the linear viscoelastic range at glucose concentrations between 0% and 5% w/w. The degree of acrylic substitution was varied for the dex-MA and con A-MA, and as the formulation glucose concentration was raised, a graded decrease in storage modulus, loss modulus and complex viscosity when compared at 1 Hz was observed for each cross-linked material. Increasing the degree of substitution (DS) of the derivatised dextran produced viscosity profiles at higher values throughout the glucose concentration range. A comparison with non-polymerised mixtures shows similar rheological properties but at much lower values across the chosen glucose concentration range. High-pressure liquid chromatography analyses and in vitro diffusion experiments showed that there were optimum degrees of derivatisation to minimise dex-MA and con A-MA component leach from the material. The in vitro diffusion experiments also showed that differential delivery of insulin in response to glucose was possible with candidate polymerised glucose-responsive formulations, thus highlighting the potential of such a novel glucose-sensitive material to be used as part of implantable closed-loop insulin delivery device. PMID- 16678255 TI - Porogen-based solid freeform fabrication of polycaprolactone-calcium phosphate scaffolds for tissue engineering. AB - Drop on demand printing (DDP) is a solid freeform fabrication (SFF) technique capable of generating microscale physical features required for tissue engineering scaffolds. Here, we report results toward the development of a reproducible manufacturing process for tissue engineering scaffolds based on injectable porogens fabricated by DDP. Thermoplastic porogens were designed using Pro/Engineer and fabricated with a commercially available DDP machine. Scaffolds composed of either pure polycaprolactone (PCL) or homogeneous composites of PCL and calcium phosphate (CaP, 10% or 20% w/w) were subsequently fabricated by injection molding of molten polymer-ceramic composites, followed by porogen dissolution with ethanol. Scaffold pore sizes, as small as 200 microm, were attainable using the indirect (porogen-based) method. Scaffold structure and porosity were analyzed by scanning electron microscopy (SEM) and microcomputed tomography, respectively. We characterized the compressive strength of 90:10 and 80:20 PCL-CaP composite materials (19.5+/-1.4 and 24.8+/-1.3 Mpa, respectively) according to ASTM standards, as well as pure PCL scaffolds (2.77+/-0.26 MPa) fabricated using our process. Human embryonic palatal mesenchymal (HEPM) cells attached and proliferated on all scaffolds, as evidenced by fluorescent nuclear staining with Hoechst 33258 and the Alamar Blue assay, with increased proliferation observed on 80:20 PCL-CaP scaffolds. SEM revealed multilayer assembly of HEPM cells on 80:20 PCL-CaP composite, but not pure PCL, scaffolds. In summary, we have developed an SFF-based injection molding process for the fabrication of PCL and PCL-CaP scaffolds that display in vitro cytocompatibility and suitable mechanical properties for hard tissue repair. PMID- 16678256 TI - PEGylated albumin-heme as an oxygen-carrying plasma expander: Exchange transfusion into acute anemia rat model. AB - Poly(ethylene glycol) (PEG) conjugated recombinant human serum albumin (HSA) incorporating the synthetic iron-porphyrin (FeP) [PEGylated albumin-heme, PEG(HSA FeP)] is a unique albumin-based oxygen carrier as a red blood cell (RBC) substitute. The physiological responses to an exchange transfusion with PEG(HSA FeP) into an acute anemia rat model were investigated. After a 65% isovolemic hemodilution with HSA, a 30% volume of the circulatory blood was withdrawn, affording a hemorrhaged state. The circulation parameters, blood parameters, renal cortical oxygen partial pressure [PtO(2)(R)], and muscle tissue oxygen partial pressure [PtO(2)(M)] were continuously monitored. The intravenous infusion of PEG(HSA-FeP) restored the reduced levels of the mean arterial pressure, heart rate, respiration rate, mixed venous PO(2), and arterial PCO(2). The increased arterial PO(2) and pH also returned to their basal values. These effects were almost to the same extent as those observed after the administration of the RBC suspension. The relatively low recovery in PtO(2)(R) and PtO(2)(M) might be due to the Langmuir-type oxygen binding profile of PEG(HSA-FeP) (Hill coefficient: 1.0). All the animals survived during the experiments. In contrast, those injected with HSA died within 41 min. The PEG(HSA-FeP) solution is an oxygen-carrying plasma expander which can be used as a resuscitative fluid for hemorrhagic shock. PMID- 16678257 TI - Dense fibrillar collagen matrices: a model to study myofibroblast behaviour during wound healing. AB - Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype. PMID- 16678258 TI - Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells. AB - The ryanodine receptor subtype 3 (RYR3) is expressed ubiquitously but its physiological function varies from cell to cell. Here, we investigated the role of a dominant negative RYR3 isoform in Ca2+ signalling in native smooth muscle cells. We used intranuclear injection of antisense oligonucleotides to specifically inhibit endogenous RYR3 isoform expression. In mouse duodenum myocytes expressing RYR2 subtype and both spliced and non-spliced RYR3 isoforms, RYR2 and non-spliced RYR3 were activated by caffeine whereas the spliced RYR3 was not. Only RYR2 was responsible for the Ca2+-induced Ca2+ release mechanism that amplified Ca2+ influx- or inositol 1,4,5-trisphosphate-induced Ca2+ signals. However, the spliced RYR3 negatively regulated RYR2 leading to the decrease of amplitude and upstroke velocity of Ca2+ signals. Immunostaining in injected cells showed that the spliced RYR3 was principally expressed near the plasma membrane whilst the non-spliced isoform was revealed around the nucleus. This study shows for the first time that the short isoform of RYR3 controls Ca2+ release through RYR2 in native smooth muscle cells. PMID- 16678259 TI - Inhibitory effect of 8-oxo-7,8-dihydro-2'-deoxyguanosine on the growth of KG-1 myelosarcoma in Balb/c nude mice. AB - We previously found that 8-oxo-7,8-dihydro-2'-deoxyguanosine (oh(8)dG) kills KG 1, a human myelocytic leukemic cell line with mutational loss of 8-oxoguanine glycosylase (OGG1) activity in vitro. This observation prompted us to investigate the cytotoxicity of oh(8)dG on KG-1 in vivo. This cytotoxicity was observed by administrating oh(8)dG (3.3-330mg/kgb.w./day) for 14 days into nude mice bearing a KG-1 myelosarcoma. The results were as follows; oh(8)dG inhibited the growth of KG-1 myelosarcoma dose-dependently in terms of tumor size and weight, but had no effect on the growth of myelosarcoma of U937, a human monocytic leukemic cell line possessing wild-type OGG1. 6-Thioguanine (6-TG), an anticancer drug inhibited the growths of KG-1 and U937 tumors. 2'-Deoxyguanosine (dG) had a statistically insignificant anti-growth effect on both tumors. The oh(8)dG treated KG-1 tumor showed the increased expression of apoptosis-processing caspases 8, 9 and 3 together with DNA fragmentation, the increased expression of cell cycle inhibitors, p16 and p27, and the decreased expression of cell cycle accelerator, cyclins and cdks, indicating the nature of cytotoxicity is cell cycle arrest and apoptosis. The genomic DNA of oh(8)dG-treated KG-1 tumors showed an increase in OGG1 sensitive sites, which is consistent with an increase in the 8-oxo-7,8-dihydroguanine (oh(8)Gua) level in the DNA of KG-1 treated with oh(8)dG in vitro. Presumably an increased level of oh(8)Gua in DNA may trigger the cytotoxicity. These findings suggest that oh(8)dG is selectively cytotoxic to KG 1 or tumors that are OGG1-deficient. PMID- 16678261 TI - Water pollution by Cu and Pb can adversely affect mallard embryonic development. AB - The effects of heavy metal pollutants on aquatic birds have been widely studied in ecotoxicological investigations; however, the predominant focus has been on the postnatal period of life. Limited information on the adverse effects of metals to bird eggs is available. The possible toxic effects of lead and copper were studied in mallard eggs. After the accidental severe heavy metal pollution of the Tisa river (Hungary) in March 2000, these metals were detected in the highest concentration in both the water and the sediment, reaching far beyond acceptable concentrations. Pb treatment (2.9 mg/L) significantly increased the rate of mortality after a single immersion of the eggs into polluted water for 30 min. The rate of dead embryos significantly increased after the combined exposure to Cu and Pb (0.86 and 2.9 mg/L, respectively) both in the single- (once for 30 min) and in the multiple- (10s daily during first trimester of incubation) immersion groups. It was concluded that elevated metal concentrations similar to those found in the Tisa river after the tailing dam failure may cause toxic effects (mortality and teratogenicity) upon exposure of mallard eggs. PMID- 16678260 TI - Will MDR-1/P-gp modulators provide clinical benefit in hematologic malignancies? PMID- 16678262 TI - The neural/cephalic phase reflexes in the physiology of nutrition. AB - The cephalic phase of nutrition refers to a set of food intake-associated autonomic and endocrine responses to the stimulation of sensory systems mainly located in the oropharyngeal cavity. These reactions largely occur in the digestive system, but they have also been observed in other structures. Most published data indicate that cephalic responses are mediated by the efferent component of the vagus nerve, although other neurobiological components and brain centers must be involved. The physiological significance of all of these reactions has yet to be fully elucidated, but when the cephalic phase of digestion is obviated major physiological and behavioral dysfunctions can be observed. This has led numerous authors to propose that their function may be essentially adaptive, preparing the digestive system for the reception, digestion, and absorption of the food. Study of the neural/cephalic phase and the consequences of its absence may have clinical relevance in the setting of artificial nutrition, and may explain the difficulties of providing enteral nutrition to many of the patients that require it. PMID- 16678263 TI - Protective effect of 17-beta-estradiol in human neurocellular models of lead exposure. AB - The developing nervous system has long been recognized as a primary target site for lead (Pb)-induced toxicity. Pb-exposure causes cognitive dysfunction, growth retardation, hyperactivity and neurochemical deficits in animals and humans. In the present study the effects of 17-beta-estradiol on human SH-SY5Y neuroblastoma cells in culture exposed to low-levels of Pb were assessed. The cells were exposed to Pb (0.01-10 microM) for 48 h and cell proliferation was determined by the MTT reduction assay. Pb significantly inhibited the proliferation and growth of neuroblastoma cells in a concentration-dependent manner. A 50% inhibition (IC50) in the proliferation of cells was observed with 5 microM Pb. Exposure of cells to Pb (5 microM) for 48 h resulted in a significant increase (+732% of control) in caspase-3 activity, an indicator of apoptosis and total cellular prostaglandin E2 level (+1180% of control), marker of programmed cell death/neuronal cell loss. Pretreatment with 17-beta-estradiol (10 nM) effectively blocked the effects of Pb on caspase-3 activity but not prostaglandin E2 level. Further, Pb but not 17-beta-estradiol in a concentration (0.1-10 microM) dependent manner effectively decreased (38-84%) the cellular concentration of glutathione (GSH), an important intracellular antioxidant. However, the effect of Pb on GSH level was effectively blocked when pretreated with 17-beta-estradiol. The data indicate that even low concentrations of Pb can be detrimental and potentially toxic to the developing brain. In conclusion, these results suggest that at least some of the neurotoxic effects of Pb may be mediated by apoptosis, which by pretreatment with 17-beta-estradiol can be prevented. This study further confirms previous reports of 17-beta-estradiol acting as a neuroprotective and antiapoptotic agent during induced toxic stress conditions. PMID- 16678264 TI - Permethrin, but not deltamethrin, increases spontaneous glutamate release from hippocampal neurons in culture. AB - Pyrethroid insecticide modulation of the voltage-gated sodium channel (VGSC) is proposed to underlie their effects on neuronal excitability. However, some in vitro evidence indicates that target sites other than VGSCs could contribute to pyrethroid disruption of neuronal activity. VGSC-independent, pyrethroid-induced changes in neurotransmitter release were examined to investigate the possibility that target sites other than VGSCs contribute to pyrethroid effects. Using whole cell patch clamp recordings, deltamethrin and permethrin effects on glutamate mediated miniature excitatory postsynaptic currents (mEPSCs) from pyramidal neurons in mixed hippocampal cultures were examined. In the presence of the VGSC antagonist tetrodotoxin, the type I pyrethroid permethrin (10 microM) increased the average frequency of mEPSCs from a basal level of 1.0+/-0.4 to 3.5+/-0.6 Hz, with peak frequency of 9.9+/-1.5 Hz (n=6). Permethrin did not affect the distribution of current amplitudes, indicating that permethrin increased the probability of glutamate release at the presynaptic terminal without effects on postsynaptic responses. Removal of calcium from the extracellular solution following the induction of the permethrin-mediated effect decreased mEPSC frequency (6.8+/-1.8 Hz, n=3) to near control levels (1.9+/-0.8 Hz for control versus 2.5+/-0.6 Hz for permethrin minus Ca(2+), respectively). However, the N- and P/Q-type voltage-gated calcium channel antagonist omega-conotoxin MVIIC had no effect on the permethrin-dependent increase in mEPSC frequency. In contrast to permethrin, the type II pyrethroid deltamethrin (10 microM) failed to affect mEPSC frequency. These results indicate that permethrin causes a calcium dependent increase in glutamate release from hippocampal neurons that is independent of effects on voltage-gated sodium or N- or P/Q-type voltage-gated calcium channels. The data indicate that permethrin increases mEPSC frequency via an alteration in intracellular calcium dynamics at the presynaptic terminal. PMID- 16678265 TI - Developmental lead neurotoxicity: alterations in brain cholinergic system. AB - Developing brain has been shown to be susceptible to the neurotoxic effects of lead (Pb). Our earlier studies (Reddy GR, Riyaz Basha Md, Devi CB, Suresh A, Baker JL, Shafeek A, Heinz J, Chetty CS. Lead induced effects on acetylcholinesterase activity in cerebellum and hippocampus of developing rat. Int J Devl Neurosci 2003;21:347-52) have shown decrease in acetylcholinesterase (AChE) activity in the crude homogenates of cerebellum and hippocampus of rat brain exposed to Pb. In this study, we have further examined in detail, the alterations in AChE activity and acetylcholine (ACh) levels in different brain regions using histochemical and spectrophotometric methods. Rats were lactationally exposed to low level (0.2%) and high level (1%) Pb. The studies were conducted in young (1 month) and adult (3 months) rats. Pb exposure significantly decreased the specific activity of AChE and increased the levels of ACh in the synaptosomal fractions of cerebellum, hippocampus and cerebral cortex in a dose- and age-dependent manner. These alterations in AChE and ACh were more predominant in young rat brain as compared to adult brain. Maximum AChE activity and ACh level as well as maximum alterations following Pb exposure were observed in synaptosomes of hippocampus. Histochemical studies also showed higher AChE activity in the hippocampal region compared to other areas of brain as revealed by the intensity of AChE staining. Though high level Pb exposure remarkably decreased the intensity of AChE staining in the dentate gyrus, CA2 and CA3 areas of hippocampus, and different cell layers of cortex and cerebellum, highly significant loss of AChE activity was observed in the CA3 region of hippocampus, molecular layer of cerebellum and cortical cell layers. These data suggest that Pb exposure may selectively affect cholinergic system in brain areas controlling learning and cognitive behavior. PMID- 16678266 TI - Tributyltin-induced cell death is mediated by calpain in PC12 cells. AB - Tributyltin, an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide and component of antifouling paints. In this study, we investigated whether calpain is involved in tributyltin toxicity in undifferentiated PC12 cells. Tributyltin (2 microM) induced an increase of lactate dehydrogenase release, a marker of cytotoxicity, in PC12 cells in a time dependent manner. It also induced calpain activation in a dose-dependent manner, and a calpain inhibitor, MDL28170 (40 microM), decreased the cellular toxicity, suggesting that calpain is involved in tributyltin toxicity in PC12 cells. Because calpain is a calcium-dependent protease, we examined the effect of EGTA, an extracellular Ca(2+) chelator and BAPTA-AM, an intracellular Ca(2+) chelator. Calpain activation induced by tributyltin was decreased by BAPTA-AM (50 microM), but not by EGTA (1 mM), suggesting that calpain activation is associated with calcium release from intracellular Ca(2+) stores. Further, we investigated the relationship between caspase-3 and calpain. Inhibition of caspase-3 reduced calpain activity induced by tributyltin. In conclusion, we have demonstrated that tributyltin induced cell death through calpain activation, and that intracellular Ca(2+) increase and caspase-3 activation are required for calpain activation by tributyltin. PMID- 16678267 TI - The mechanism of kinetic inhibition of Cu(II)-induced oxidation of low density lipoprotein by lanthanide ions. AB - Oral administration of lanthanum chloride (LaCl(3)) was reported to inhibit atherosclerosis in experimental animals, but the mechanism was not clear. In the present work, the effects of La(III) and other lanthanide ions (Ln(III)) on Cu(II)-induced oxidation of isolated low-density lipoprotein (LDL) and the related mechanism were investigated. By monitoring the formation of conjugated dienes (CD), low concentrations of La(III), Gd(III) and Y(III) were found to inhibit Cu(II)-induced LDL oxidation kinetically, as characterized by the prolongation of the lag time, the decrease of the maximal accumulation of CD, and the maximal rate of CD accumulation. Using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin trapping agents, the electron spin resonance (ESR) results showed that La(III) and Gd(III) at low concentrations significantly decreased the level of free radicals, including alkoxyl radical (LO*), alkyl radical (L*), and a transient radical, alkylperoxyl radical (LOO*), generated during LDL oxidation induced by Cu(II). In addition, Fourier-transform infrared spectroscopy (FT-IR) study revealed that La(III) might cause the conformational change and the less aggregation of apolipoprotein B-100 (apoB) in LDL, as demonstrated by the decreasing contents of alpha-helix, intermolecular beta-sheet, unordered structure and beta-turns, and the increasing contents of intramolecular beta sheet and beta-strands. The inhibitory effect of Ln(III) on Cu(II)-induced LDL oxidation was discussed on the basis of the decreased free radical level and the second structural changes of apoB in LDL. PMID- 16678268 TI - Severe adenovirus pneumonia (AVP) following infliximab infusion for the treatment of Crohn's disease. AB - We report a case of severe AVP three weeks following the administration of infliximab for the treatment of Crohn's disease (CD). Since the introduction of tumor necrosis factor-alpha (TNF-alpha) antagonists, various microbiologic agents have been reported to cause pneumonia following their administration. To our knowledge, AVP following therapy with TNF-alpha antagonists has not been reported in English literature. We present a brief review of the mechanism of action of TNF-alpha antagonists and their ability to predispose to various infections including viral pneumonia by interfering with the immune response. PMID- 16678269 TI - Functional defect of natural immune system in an apparent immunocompetent patient with pulmonary cryptococcosis. AB - We report a case of pulmonary cryptococcosis in a 21-year-old Italian female smoker with no apparent immune disorder. In this study we demonstrated that: (i) patient's neutrophils and monocytes manifested a significant reduction of killing activity against Cryptococcus neoformans as well as Candida albicans; (ii) the suppression was more pronounced in monocytes than in neutrophils; (iii) neutrophils and monocytes showed a significant impairment of TNF-alpha, IL-1beta, and nitric oxide production. These results suggest that the apparent immunocompetent host with pulmonary cryptococcosis could have specific defects in natural immune system mechanisms. PMID- 16678270 TI - n-3 Fatty acids, cardiac arrhythmia and fatal coronary heart disease. AB - n-3 Polyunsaturated fatty acids (n-3 PUFA) are suggested to prevent cardiac death via inhibition of cardiac arrhythmia. In this review we discuss the results of human studies on intake of n-3 PUFAs and heart disease and, more specifically, on cardiac arrhythmia. Observational studies indicate that intake of fish is associated with a lower incidence of fatal coronary heart disease in several populations. These studies are fairly consistent, but people that have a high intake of fatty fish might have a healthier lifestyle in general, and such confounding is difficult to remove completely with statistical adjustments and corrections. Evidence from trials is less clear. In two open label trials in patients with a previous myocardial infarction intake of fish or fish oil prevented fatal coronary heart disease. In contrast, a trial in patients with angina suggested a higher risk of sudden cardiac death in patients taking fish oil. Furthermore, results of trials in patients with an implantable cardioverter defibrillator (ICD) that investigated effects of fish oil on arrhythmia in patients already suffering from ventricular tachycardia are not consistent. Also, studies on relationships between intake of n-3 PUFA from fish and less life threatening forms of arrhythmia, such as atrial fibrillation and premature ventricular complexes (PVCs) are equivocal. Thus, after 35 years of research the question whether fish prevents heart disease remains unanswered, and an anti arrhythmic effect of fish oil remains unproven although the idea is still viable and is being actively tested in further trials. PMID- 16678271 TI - Inflammation and immune regulation by 12/15-lipoxygenases. AB - 12/15-Lipoxygenases (12/15-LOX) are members of the LOX family, which are expressed in mammals by monocytes and macrophages following induction by the T helper type 2 cytokines, interleukins-4 and -13. They oxygenate free polyenoic fatty acids but also ester lipids and even complex lipid-protein assemblies such as biomembranes and lipoproteins. The primary oxidation products are either reduced by glutathione peroxidases to corresponding hydroxy derivatives or metabolized into secondary oxidized lipids including leukotrienes, lipoxins and hepoxilins, which act as lipid mediators. Examination of knockout and transgenic animals revealed important roles for 12/15-LOX in inflammatory diseases, including atherosclerosis, cancer, osteoporosis, angiotension II-dependent hypertension and diabetes. In vitro studies suggested 12/15-LOX products as coactivators of peroxisomal proliferator activating-receptors (PPAR), regulators of cytokine generation, and modulators of gene expression related to inflammation resolution. Despite much work in this area, the biochemical mechanisms by which 12/15-LOX regulates physiological and pathological immune cell function are not fully understood. This review will summarize the biochemistry and tissue expression of 12/15-LOX and will describe the current knowledge regarding its immunobiology and regulation of inflammation. PMID- 16678272 TI - Reactions of 3-isochromanone with aromatic aldehydes--microwave assisted condensations performed on solid basic inorganic supports. AB - An improved Knoevenagel condensation of 3-isochromanone and aromatic aldehydes can be achieved by microwave irradiation on solid supports in the presence of various catalysts. This synthetic method offers some major advantages, especially the possibility to change the ratio of E/Z isomers. PMID- 16678273 TI - Screening and optimization of derivatization in heating block for the determination of aliphatic aldehydes by HPLC. AB - For the study of the derivatization behavior of aliphatic C1-C10 aldehydes with 2,4-dinitrophenylhydrazine (DNPH) in a heating block a gradient elution HPLC method for separation and determination by UV detection at 360 nm was applied. The influence of time, temperature, excess of reagent and stirring onto the reaction yields were examined for conducting the reaction in a thermostated heating block. First, the derivatization procedure had been screened by experiments according to a complete factorial design in order to evaluate the statistically significant variables. Those parameters were used to establish the optimum conditions for the reaction by means of a Box-Behnken design. PMID- 16678274 TI - Demonstration of a common artifact in immunosorbent assays of brain extracts: development of a solid-phase extraction protocol to enable measurement of amyloid beta from wild-type rodent brain. AB - In the process of developing species-specific, immunosorbent assays for brain amyloid-beta (Abeta) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of Abeta in this assay format. Using synthetic peptides, cerebrospinal fluid (CSF), or plasma samples, no nonspecific binding or cross-species immunoreactivity was detected in human or rodent Abeta assays. However, extracts of guinea pig brain (human Abeta sequence) or rat brain (rodent Abeta sequence) demonstrated immunoreactivity regardless of which capture antibody, detection antibody, or reporter method (colorimetric or fluorescent) was used. This immunoreactivity remained even in the absence of a capture antibody. Various blocking conditions failed to resolve the nonspecific binding of detection antibodies in the presence of brain extracts. Fractionation of DEA extracted guinea pig brain over Sephadex G-50 demonstrated the feasibility of separating specific from nonspecific binding components in the brain extracts. Thus, a solid phase extraction method, compatible with multiple extraction buffers, has been developed to isolate and concentrate Abeta from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple Abeta peptides in extracts from wild-type animals. PMID- 16678275 TI - Bipolar or unipolar? - the question for clinicians and researchers. AB - BACKGROUND: Correct diagnosis and criteria of affective disorders is always a subject of interest to researchers and practitioners. METHODS: The study aimed at assessing frequency of various traits and symptoms of bipolar affective disorders (BP-I, BP-II, BP-S spectrum) in patients (n=246) treated for recurrent affective disorders (unipolar-UP). The analysis was based on criteria of affective disorders of Ghaemi et al. and Hirschfeld's Mood Disorders Questionnaire. RESULTS: UP was confirmed in 32.9% of individuals, whereas 19.5% were BP-I, 35% BP-II and 12.6% BP-S. UP patients were significantly more often professionally active than those with BP (37.2% vs. 22.7%). Duration of a disorder was significantly shorter and the number of depressive episodes lower in the UP group. In comparison with UP, BP-I were associated with the previous occurrence of unusual and/or risky behaviour (OR=24.5), excessive, irrational expenditure (OR=21.1), lack of a critical attitude with respect to social behaviour (OR=20.3), increased sex drive (OR=17.7), and excessive self-confidence (OR=12). BP-II were associated with a lack of criticism with regard to social behaviour (OR=12.7) and unusual and/or risky behaviour (OR=10). Spectrum BP were most strongly associated with short term hypomanic episodes, including drug induced episodes (OR=15.8) and lack of criticism (OR=11.8). Early onset of depression (before 25 years of age) increased the risk of all three types of BP (by a factor of 3 to 5). LIMITATIONS: This was a naturalistic study, in which treatment was uncontrolled. CONCLUSIONS: Results of the study are a voice in the discussion on too narrow criteria defining bipolar affective disorders. PMID- 16678276 TI - Lessons from the cat: development of vaccines against lentiviruses. AB - Feline immunodeficiency virus (FIV) is a natural infection of domestic cats, which produces a disease with many similarities to human immunodeficiency virus (HIV) infection in man. The virus is an important cause of morbidity and mortality in pet cats worldwide. As such an effective vaccine is desirable both for its use in veterinary medicine and also as a model for the development of an HIV vaccine. A large number of candidate vaccines have been tested against feline immunodeficiency virus. These include inactivated virus and infected cell vaccines, DNA and viral vectored vaccines, subunit and peptide vaccines and vaccines using bacterial vectors. Ultimately, the development of inactivated virus and infected cell vaccines led to the release of the first licensed vaccine against FIV, in 2002. This review highlights some of the difficulties associated with the development of lentiviral vaccines and some of the lessons that have been learned in the FIV model that are of particular relevance to the development of HIV vaccines. PMID- 16678277 TI - Models of anxiety: responses of rats to novelty in an open space and an enclosed space. AB - Exposure to novelty has been shown to induce anxiety responses in a variety of behavioural paradigms. The purpose of the present study was to investigate whether exposition of naive rats to novelty would result in a comparable or a different pattern of responses in an open space versus enclosed space with or without the presence of an object in the centre of the field. Lewis and Wistar rats of both genders were used to illustrate and discuss the value and validity of these anxiety paradigms. We examined a wide range of measures, which cover several aspects of animals' responses. The results of this study revealed significant differences between the behaviour of animals in an open space and in the enclosed space. It also revealed significant differences in animal's responses to the presence and absence of an object in the open space and in the enclosed space. In the enclosed space, rats spent most of their time in the outer area with lower number of exits and avoided the object area except when there was an object, while in the open space rats displayed frequent short duration re entries in the outer area and spent longer time in the object area in presence of an object. The time spent in the inner area (away from the outer area and the object area) was significantly longer and the number of faecal boli was significantly higher in the open space than in the enclosed space. In the present report, we will discuss the fundamental differences between enclosed space and open space models, and we will examine some methodological issues related to the current animal models of human behaviour in anxiety. In the enclosed space, animals can avoid the potential threat associated with the centre area of a box and chose the safety of walls and corners, whereas, in the open space animals have to avoid every parts of the field from which there was no safe escape. The response of animals to novelty in an open space model appears more relevant to anxiety than in an enclosed space. The present studies revealed no correlations between the measures of behaviour in enclosed space and the measures of behaviour in open space, which suggest that these two models do not involve the same construct. Our results suggest that the enclosed space model involves avoidance responses while the open space model involves anxiety responses. The open space model can be very useful in understanding the underlying neural mechanisms of anxiety responses, and in assessing the effects of potential anxiolytic drugs. PMID- 16678278 TI - Cross-fostering does not alter the differential sensitivity of Fischer and Lewis rats to central neurotensin-induced locomotion and hypothermia. AB - A cross-fostering paradigm was used to determine whether the differential locomotor and hypothermic responses to neurotensin (NT) in Fischer (F344) and Lewis (LEW) rats are mediated by the post-natal environment. From post-natal day (PD) 1 to PD 21, male pups from each strain were assigned to a same-strain dam (in-fostered) or were cross-fostered, and at adulthood were implanted with a guide cannula over the lateral ventricle. They were then tested for locomotion and hypothermia following injection of vehicle, 0.18, 1.8 or 18nmol of NT or D Tyr([11])NT. In-fostered LEW, but not F344, displayed a strong dose-orderly hypothermic response to NT and to D-Tyr([11])NT while in-fostered F344, but not LEW, rats displayed strong locomotor responses to D-Tyr([11])NT. Cross-fostering had no effect on D-Tyr([11])NT-induced locomotor responses in either strain; it had no effect also on NT- and D-Tyr([11])NT-induced hypothermia in F344 rats while it slightly increased the sensitivity to NT in LEW rats. The results show that these NT-mediated actions are not influenced by cross-fostering or the pre weaning environment. PMID- 16678279 TI - The exploratory behavior of rats in an open environment optimizes security. AB - When given a locomotor/exploratory test in the laboratory, rats form one or more home bases, operationally defined as places where they spend a disproportionate period of their time and from which they make excursions. Because exploratory tests in the laboratory necessarily restrict the animals' movements, the cause of exploration (e.g., fear, curiosity, innate disposition) and the extent to which organization is imposed by the restriction of the testing environment has not been fully examined. In the present study, rats received exploratory tests in environments in which restrictions were remote; in a parking lot or on a playing field. Each rat began a test in one of three conditions: in a small refuge, within a transparent open home cage, or beside a landmark. In the parking lot, the rats failed to leave the small refuge, made excursions from the home cage, and left the landmark, usually at a gallop, and made no movements of returning. On the playing field they remained in the small refuge, left and returned to the open home cage, and were more likely to permanently leave the landmark at a gallop. Rats that displayed a strong preference for the landmark over three test sessions in a laboratory, also immediately left the same landmark when tested on the playing field. The pattern of behavior, in which the rats failed to explore from a secure starting position and were increasingly likely to run away as security decreased, suggests that a primary function of locomotor behavior in a novel environment is to optimize security. The results are discussed in relation to the advantages of investigating the influence of neural processes on exploration in terms optimization theory versus motivational theory. PMID- 16678280 TI - ZENK expression in a restricted forebrain area correlates negatively with preference for an imprinted stimulus. AB - Sexual imprinting is an early learning process by which young birds acquire the characteristics of a potential sexual partner. The physiological basis of this learning process is an irreversible reduction of dendritic spines in two forebrain areas, the LNM (lateral nido-mesopallium) and the MNM (medial nido mesopallium). The aim of the present study was to investigate whether these two brain areas are activated if the imprinted stimulus is presented to the adult bird after the end of the sensitive period. One group of zebra finch males was reared by their own parents. These birds, as adults, showed an exclusive preference for their own species in choice tests between a zebra finch and a Bengalese finch female. If exposed as adults to a zebra finch female, LNM and MNM showed lower activation, as measured by ZENK expression, compared to males exposed to a Bengalese finch female. A second group was reared by Bengalese finches and was exposed at day 100 to a zebra finch female for 1 week. As shown earlier, this regime leads to mixed choices, the birds are courting Bengalese and zebra finch females with a fixed ratio (preference score). If these birds were exposed to a zebra finch female as adults, the ZENK expression within LNM was much higher compared to group 1, and it showed a strong tendency to correlate negatively with the preference score: Birds with higher zebra finch preference showed lower activation compared to those with a low zebra finch and a high Bengalese finch preference. We propose that higher ZENK activation in group 2 is due to the rearing by a foster species which may result in a more complex neuronal network. The negative relation between activation and preference score may be explained by special properties of the LNM and MNM networks. PMID- 16678281 TI - Exposure to tetrabromobisphenol-A alters TH-associated gene expression and tadpole metamorphosis in the Pacific tree frog Pseudacris regilla. AB - Presently in the environment, there exist a number of chemical contaminants which share structural similarity with key naturally occurring regulatory hormones. These hormones play pivotal roles in the normal growth and development of wildlife species and humans. In particular, biphenolic chemical compounds may have the potential to act as agonists or antagonists of thyroid hormone (TH) action. We investigated whether there was any biological effect of exposure to low concentrations of the brominated fire retardant, tetrabromobisphenol-A (TBBPA), on the TH-mediated process of metamorphosis of the Pacific tree frog, Pseudacris regilla. Tadpoles exposed to 10nM (5.4microg/L) TBBPA showed an increase in TH-mediated expression of gelatinase B mRNA within 48h in the tadpole tail which was associated with increased tail resorption by 96h. Treatment with 100nM (54.4microg/L) TBBPA resulted in increased TH-mediated thyroid hormone receptor alpha mRNA expression in the tadpole brain and reduced levels of PCNA transcript in the tail. TBBPA alone was also found to alter the mRNA abundance of thyroid hormone receptor alpha in tail, gelatinase B in brain, and PCNA in both tissues of premetamorphic tadpoles. Interestingly, expression of thyroid hormone receptor beta mRNA was not affected by exposure to TBBPA either alone or in the presence of TH. The results suggest that exposure to low levels of TBBPA may act as an agonist of TH action and potentiate TH-mediated gene expression leading to accelerated anuran metamorphosis. PMID- 16678282 TI - Host-generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. PMID- 16678283 TI - Habituation of insulin-induced hypoglycemic transcription activation of lateral hypothalamic orexin-A-containing neurons to recurring exposure. AB - A CNS component of glucose counterregulatory collapse is supported by evidence for nonuniform genomic responsiveness of neurons in characterized central autonomic loci during recurring insulin-induced hypoglycemia (IIH). We have reported that exacerbated hypoglycemia and attenuated patterns of glucagon and epinephrine secretion in rats treated by daily sc injection of the intermediate acting insulin formulation, Humulin NPH (NPH), are correlated with diminished immunodemonstrability of the AP-1 transcription factor, Fos, in several components of the central metabolic regulatory circuitry, including the lateral hypothalamic area (LHA). Neurons that synthesize the potent orexigenic peptide neurotransmitter, orexin-A, are restricted to the LHA and adjacent hypothalamic loci, and project throughout the central neuroaxis to structures that govern autonomic and behavioral motor output. Dual-label immunocytochemical and real time RT-PCR techniques were utilized here to evaluate the functional status of this LHA phenotype during a single versus repetitive exposure to prolonged IIH. Tissue sections were collected at predetermined rostrocaudal levels of the LHA after acute or repeated NPH administration, and processed for nuclear Fos- and cytoplasmic orexin-A-immunoreactivity (-ir). Mean numbers of orexin-A-ir neurons were not different between treatment groups. Colabeling of these cells for Fos was increased relative to controls following a single injection of insulin, but numbers of Fos-ir-positive orexin-A neurons were significantly reduced after treatment with four versus one dose of insulin. Prepro-orexin mRNA levels in microdissected LHA tissue were upregulated during acute hypoglycemia, but were returned to control levels by repeated IIH. These data corroborate previous evidence that IIH is an activational stimulus for orexin-A-synthesizing neurons in the LHA, and further demonstrate that induction of cfos and prepro-orexin gene expression by acute hypoglycemia is attenuated by precedent exposure to hypoglycemia. The current results thus provide unique evidence for neurotransmitter-specific habituation of LHA neuronal sensitivity to IIH. PMID- 16678284 TI - Hypothalamic cardiovascular effects of angiotensin-(1-7) in spontaneously hypertensive rats. AB - The objective of the present work was to study the cardiovascular actions of the intrahypothalamic injection of Ang-(1-7) and its effects on the pressor response to Ang II in spontaneously hypertensive (SH) rats and Wistar Kyoto (WKY) animals. In anaesthetized SH and WKY rats, a carotid artery was cannulated for mean arterial pressure (MAP) measurement and a stainless-steel needle was inserted into the anterior hypothalamus for drug administration. The cardiovascular effects of the intrahypothalamic administration of Ang-(1-7) were determined in SH and WKY rats. In SH rats, the effect of irbesartan and D-Ala-Ang-(1-7) on Ang (1-7) cardiovascular effect was also evaluated. Ang II was administered in the hypothalamus of SH and WKY rats and changes in blood pressure and heart rate were measured followed by the administration of Ang II, Ang II+Ang-(1-7) or Ang II+D Ala-Ang-(1-7). Ang-(1-7) did not the change basal MAP in WKY rats, but induced a pressor response in SH animals. Whilst the co-administration of D-Ala-Ang-(1-7) did not affect the response to Ang-(1-7), the previous administration of irbesartan prevented the effect of the peptide. The intrahypothalamic injection of Ang II induced a significantly greater pressor response in SH animals compared to normotensive rats. The co-administration of Ang-(1-7) with Ang II did not affect the pressor response to Ang II in the WKY group. In SH rats, whilst the co administration of Ang-(1-7) with Ang II reduced the pressor response to Ang II, the concomitant application of D-Ala-Ang-(1-7) with Ang II increased the pressor response to the octapeptide after 5 and 10 min of intrahypothalamic administration. In conclusion, our result demonstrated that the biologically active peptide Ang-(1-7) did not participate in the hypothalamic blood pressure regulation of WKY animals. In SH rats, Ang-(1-7) exerted pleiotropic effects on blood pressure regulation. High dose of the heptapeptide produced a pressor response because of an unspecific action by activation of AT1 receptors. The concomitant administration of lower doses of Ang-(1-7) with Ang II reduced the pressor response to the octapeptide. Finally, the effect of AT(1-7) antagonist on Ang II pressor response suggested that hypothalamic formed Ang-(1-7) are implicated in the regulation of the cardiovascular effects of Ang II. PMID- 16678285 TI - Oxytocin is expressed throughout the human gastrointestinal tract. AB - BACKGROUND/AIM: Several studies have described that oxytocin exerts stimulatory or inhibitory effects on gut functions. Recently, mRNA for oxytocin and its receptor was found throughout the entire human gastrointestinal (GI) tract. The aim of this study was to examine the cellular localization and distribution of the corresponding proteins. MATERIAL AND METHODS: Full-thickness biopsies from 24 patients, covering the entire GI tract, were collected during operations at the Department of Surgery in Malmo and Lund. The biopsies were taken from non_affected margins. The biopsies were fixed by immersion, rinsed in buffered sucrose, and kept frozen at 70 degrees C. Indirect immunofluorescence with primary antibodies to oxytocin and its receptor was used. RESULTS: Oxytocin was expressed in nerve cell bodies and nerve fibres in the myenteric and submucous ganglia all along the GI tract. Immunoreactive nerve cell bodies in myenteric ganglia predominated in the proximal (antrum and duodenum) and distal gut, while those in the submucous ganglia were more numerous in the ileum and colon. The oxytocin receptor was not detectable by two different antibodies in any tissue in the GI tract. CONCLUSION: Oxytocin is expressed in the myenteric and submucous ganglia and nerve fibres along the entire human GI tract. The role for oxytocin in the physiology and pathophysiology of the bowel remains to be settled. PMID- 16678286 TI - Subclinical hypothyroidism increases the risk for depression in the elderly. AB - In order to determine if subclinical hypothyroidism is a risk factor for depression in the elderly, a total of 323 individuals over 60 years old were interviewed using the Structured Clinical Interview for Diagnosis and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) for mood disturbances. Patients were divided into Group I: 252 patients (184 females, 68 males; median age: 67 years, range: 60-89 years) with elevated serum thyrotropin (TSH) levels and Group II: 71 patients (45 females, 26 males; median age: 67 years, range: 60 92 years) with diagnosis of depression. Serum TSH and free thyroxine (fT4) were measured by sensitive assays. Thyroid antibodies were determined by IRMA. Depression was observed in 24 (9.5%) Group I patients and was frequent in subclinical hypothyroidism patients (14/24 = 58.3%). On the other hand, elevated TSH levels were found in 22 (30.9%) Group II patients. Depression was observed more frequently among individuals with subclinical (74/149 = 49.7%) hypothyroidism than among individuals with overt hypothyroidism (21/125 = 16.8%) (p < 0.001). Indeed, subclinical hypothyroidism increased the risk for a patient to present depression more than four times (OR = 4.886; 95% confidence interval = 2.768-8.627). Our results demonstrate that subclinical hypothyroidism increases the risk for depression and emphasize the importance of thyroid screening tests in the elderly. PMID- 16678287 TI - Coronary artery disease in Iranian overweight women. AB - BACKGROUND: Coronary artery disease is the leading cause of mortality in Iran. This study aimed to evaluate coronary risk factors in Iranian overweight and obese women. METHODS AND SUBJECTS: Of all overweight and obese women examined in the main heart clinic in Rasht city, Iran, between 2000 and 2003, those with angiographically approved coronary artery disease (n=180) and overweight normal coronary women (n=224) participated in this study. The subjects in both groups had body mass index above 25 kg/m(2). Data on age, educational level, cigarette smoking, alcohol drinking and current drug therapy were collected using questionnaires. Total serum cholesterol, high density lipoprotein cholesterol, triglyceride, apoprotein B, apoprotein A1, lipoprotein (a), blood glucose, body weight, height, and waist circumference were measured in both groups. RESULTS: The findings indicated that mean age (57.3+/-10.9 vs 40.3+/-10.1 p<0.0001) was significantly higher in coronary artery diseased group than normal overweight and obese subjects. Other measured risk factors including waist circumference, blood lipids and blood pressure were higher in overweight patients with coronary artery disease than overweight normal subjects. Results of logistic regression analysis showed that age (OR=1.16, 95% CI 1.10-1.21) and diabetes (OR=6.31, 95% CI 1.95 20.3) were the only predictors of coronary artery disease in this population of coronary obese patients. The proportion of low educated level was remarkably higher in overweight women with coronary artery disease than normal groups. CONCLUSION: The findings suggested that advancing age and diabetes are independent predictors for development of coronary artery disease in this group of overweight and obese Iranian women. The high proportion of low educated people in these patients with coronary artery disease implicates an important public health message for targeted preventive measures in lower social groups. PMID- 16678289 TI - Rapid and sensitive FAME analysis of bacteria by cold trap injection gas chromatography. AB - Whole cell fatty acid analysis is commonly used to identify bacteria. A cold trap, using a commercially available device that directs a stream of compressed air across a section of the GC column, is used to focus peaks at the head of the column. When combined with a rapid sample processing method that uses smaller volumes of solvents, it becomes possible to correctly identify bacteria from 1 to 2 mg of biomass. PMID- 16678288 TI - The effect of statin on the aortic gene expression profiling. AB - BACKGROUND: Beyond lipid lowering, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) has been found to have anti-inflammatory and anti thrombotic effects. However, the genetic expression pattern changes in atherosclerotic lesions produced by statin are rarely studied. METHODS: Cholesterol-fed apolipoprotein (Apo) E-deficient mice were examined for the treatment effect of statin on aortic gene expression. ApoE-deficient mice were fed with a hypercholesterolemic diet started at 8 weeks of age for a total of 22 weeks. In the statin treatment group (n=25), the ApoE-deficient mice were treated with pravastatin (80 mg/kg/day) dissolved in water by daily oral inoculation from 25 to 30 weeks of age. For the control group (n=25), the ApoE-deficient mice were orally inoculated with water only for the same period of time. The aortic gene expression affected by pravastatin was identified using oligonucleotide microarray technology with Agilent gene chips. RESULTS: The total cholesterol and atherosclerotic lesion/total aortic area were significantly lower in the pravastatin treatment group. Microarray analysis of the expression of 20,281 murine genes in the aortas between the two groups indicated that 94 genes were significantly regulated. Thirty genes were up-regulated and 64 genes were down regulated. The most up-regulated genes were troponin T3, actin alpha1, tubulin alpha1, regulator of G-protein signaling 5 (Rgs5), stathmin-like 2 and myosin light chain kinase. Most of them are related with cytoskeleton organization, while Rgs5 is a G-protein signal transduction molecule. The most down-regulated genes were adenosine deaminase, atrial natriuretic peptide, troponin T2, FXYD domain-containing ion transport regulator 3, and glutathione S-transferase alpha4. CONCLUSIONS: The beneficial effect of the 6-week statin treatment in ApoE deficient mice is largely dependent on its influence on the cytoskeleton organization. Our study results might provide insight into the clinical benefits of chronic statin treatment. PMID- 16678290 TI - Rapid and simple detection of food poisoning bacteria by bead assay with a microfluidic chip-based system. AB - A rapid bead assay for detecting pathogenic bacteria with a simple microfluidic chip-based system was developed. Five oligonucleotide probes corresponding to the 16S rRNA of the targeted bacteria were coupled covalently to fluorescent beads. Four species of bacteria (Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis, Yersinia enterocolitica, and Bacillus cereus) were used as representative food-borne pathogenic bacteria. The RNAs extracted from pure cultures of these microorganisms were fluorescently labeled and hybridized to the oligonucleotide probes-immobilized fluorescent beads (Bead assay). The duplexes of RNAs and the probes-immobilized beads were analyzed with the commercially available microfluidic chip-based system. This bead assay provided results within 3 h following RNA extraction from bacterial cells. PMID- 16678291 TI - Microcarriers in the engineering of cartilage and bone. AB - A major problem in tissue engineering is the availability of a sufficient number of cells with the appropriate phenotype for delivery to damaged or diseased cartilage and bone; the challenge is to amplify cell numbers and maintain the appropriate phenotype for tissue repair and restoration of function. The microcarrier bioreactor culture system offers an attractive method for cell amplification and enhancement of phenotype expression. Besides serving as substrates for the propagation of anchorage-dependent cells, microcarriers can also be used to deliver the expanded undifferentiated or differentiated cells to the site of the defect. The present article provides an overview of the microcarrier culture system, its utility as an in vitro research tool and its potential applications in tissue engineering, particularly in the repair of cartilage and bone. PMID- 16678292 TI - Processing of own versus other names differentially affects startle reflex modification. AB - Previous research suggests that the startle eyeblink response may be differentially modified at very short latencies by words that attract more versus fewer attentional resources. These studies were conducted using threat and non threat words as stimuli. The goal of the present study was to examine whether startle blink modification is similarly affected by differential attention when one's own name versus another name is used as a lead stimulus. Fifty-six undergraduates were presented repeatedly with their own name or another name displayed visually, followed (60, 120, 300, or 2000 ms later) by an acoustic startling noise burst. Startle magnitude was differentially modified by own versus other names, primarily at the 60 and 120 ms lead intervals. Other names demonstrated greater magnitude potentiation at the 60 ms interval and greater magnitude inhibition at the 120 ms lead interval compared with own names, suggesting the startle modification paradigm is sensitive to early attentional processes engaged during the reading of non-threat-related words. PMID- 16678293 TI - Exploring the human resource implications of clinical governance. AB - In 1998, clinical governance was introduced in the National Health Service, UK (NHS) as a major policy initiative to improve the quality of clinical care. The implementation of clinical governance is crucially dependent on the skills, competencies and willingness of the NHS staff. In turn, clinical governance influences the way people work in health care organisations. Therefore, it is no surprise that the introduction of clinical governance has thrown-up new challenges for human resource management. However, what are these human resource management challenges under the clinical governance framework? The current literature on the subject provides no answer. This article attempts to fill this gap in the literature. A qualitative approach influenced by phenomenological case study approach has been adopted. A heterogeneous group of 33 persons identified through a purposive sampling procedure were interviewed using a semi-structured format. The results indicate that the staff members appreciate the crucial role of human resources management in the implementation of clinical governance. However, there is little evidence to suggest that senior management is paying attention to develop the human resources function around the clinical governance agenda. The seven major human resource implications of clinical governance that emerged from the data analysis are discussed. The author argues that a more proactive HR approach is needed to make clinical governance everyone's business in the NHS organisations. PMID- 16678294 TI - Welfare state matters: a typological multilevel analysis of wealthy countries. AB - Building on the social science literature, we hypothesized that population health indicators in wealthy industrialized countries are 'clustered' around welfare state regime types. We tested this hypothesis during a period of welfare state expansion from 1960 to 1994. We categorized data from 19 wealthy countries into 4 different types of welfare state regimes (Social Democratic, Christian Democratic, Liberal and Wage Earner Welfare States). Outcome variables were the infant mortality rate (IMR) and the low birth weight rate (LBW), obtained from the Organization of Economic Co-operation and Development (OECD) Health Data 2000 and from the United Nations Common Statistical Database (UNCSD). A two-level multilevel model was constructed, and fixed effects of welfare state were tested. Through the 39 years analyzed, Social Democratic countries exhibited a significantly better population health status, i.e., lower infant mortality rate and low birth weight rate, compared to other countries. Twenty percent of the difference in infant mortality rate among countries could be explained by the type of welfare state, and about 10% for low birth weight rate. The gap between Social Democracies and other countries widened over the 1990s. Our results confirm that countries exhibit distinctive levels of population health by welfare regime types even when adjusted by the level of economic development (GDP per capita) and intra-country correlations. It implies that countries, as groups, adopt similar policies or through any other ways, achieve similar level of health status. Proposed mechanisms of such process and suggestions for future research directions are presented in the discussion. PMID- 16678295 TI - Panning for gold: an evidence-based tool for assessment of performance indicators in primary health care. AB - It is important that debate occurs between theorists, policy makers, clinicians and service end-users to develop agreement over suitable and appropriate indicators for primary health care. A formal accounting of the relative strengths and weaknesses of any proposed indicator will enable sector commentators from a variety of viewpoints to discuss the relative merits of individual indicators, to understand the political and pragmatic reasons for their inclusion in any set of indicators and to trace the likely organisational impact of any given indicator. This paper details the development of an indicator appraisal tool that combines the assessment of scientific evidence with contextual considerations from the perspective of both the policy environment and the primary health care sector. The use of the tool is discussed in the context of the proposed national implementation of a set of performance indicators in New Zealand. PMID- 16678296 TI - Equity in out-of-pocket payments for hospital care: evidence from India. AB - BACKGROUND: The lack of formal health insurance and inadequate social safety nets cause families in most low-income countries to finance health spending through out-of-pocket (OOP) payments, leaving poor families unable to insure their consumption during periods of major illnesses. OBJECTIVE: To examine how well the Indian healthcare system protects households of differing living standards against the financial consequences of unanticipated health shocks. DATA: The data are drawn from the 52nd round of National Sample Survey, a nationally representative socioeconomic and health survey conducted in 1995-1996. The sample comprises 24,379 (3.84%) households where a member was hospitalized during the 1 year reference period. METHODS: We estimate, using ordinary least squares, the relationship between household consumption (proxy for ability to pay) and OOP payments for hospitalization. We also estimate the relationship between consumption and OOP share in consumption. RESULTS: Our results indicate that both utilization (payments) and the consequent financial burden (payment share) increases with increasing ability to pay (ATP). While this relationship is retained across the different subgroups (e.g., gender, social code, region, etc.), comparisons across groups indicate horizontal inequities including differences in both degrees of progressivity and the redistributive effect. CONCLUSION: The finding that OOP payments do not decline with ATP could be an indication of: (1) the lack of insurance which implies that the better-off must pay from OOP to secure quality health care and (2) the absence of risk-pooling or prepayments mechanisms which poses financial impediments to the consumption of health care by the poor. PMID- 16678297 TI - Resource allocation in public hospitals: is it effective? AB - The allocative inefficiency is a fundamental flaw in the public hospitals of the developing countries. The inefficiencies drain the limited public resources allotted for healthcare. Sri Lanka's public health system faces worsening budget constraints. The resource allocation practices of the Ministry of Health focus on increasing the cadre of hospital staff, consequently crowding out the investments on facility development. The purpose of the study is to assess the impact of resource allocation in the tertiary-care public hospitals that are under the central Ministry of Health. The model is based on the assumption that the hospital managers and other agents of a public hospital pursue the objective of quality maximization (in the absence of a profit motive). The inpatient mortality rate is selected as the indicator of quality. With the use of panel data fixed effects, and first-differencing estimation methods, we study the impact of the resource allocation on the hospital mortality rates. The selected models are statistically significant at 0.1% level. The elasticity effect of the capital is considerably larger than the effects of the human resources, in servicing the patients. The results suggest that the human resource utilization is suboptimal, due to the inadequacy of the capital (i.e. medical equipment, etc.). The reorientation of the resource allocation towards the capital investments may save more lives. PMID- 16678298 TI - Contextual genetics. AB - Complex phenotypes result from multiple inputs from genetic and environmental sources, with intricate subsystems mediating the influence of both sources on the phenotype. Experiments that attempt to describe the influence of a particular gene involve partial isolation of the sub-system in which that gene is an element from other components of the total system influencing the phenotype. Any interactions that exist between the controlled variables and the processes downstream of the gene in the normally operating total system become undetectable; therefore, the results of the experiment can be restricted to the particular configuration of the controlled variables. The inescapable price of the precision of knowledge generated by experiment is a reduction in the generalizability of the results beyond the constrained circumstances of the particular experimental situation. Integrative research, permitting the influence of related subsystems, is required to provide a comprehensive assessment of the influence of a gene. PMID- 16678299 TI - "Ground truth" for selection on CCR5-Delta32. AB - A much-celebrated story of positive selection in the human genome is the 32-bp deletion in the chemokine receptor CCR5, a variant that confers resistance to AIDS. This variant was postulated to be a relatively recent response to plague or smallpox. New research shows that the frequency of CCR5-Delta32 in Bronze Age samples is similar to that seen today, pushing the observed age of the allele back to at least 3000 and possibly 5000 years ago. Interestingly, the extent of heterozygosity, differentiation across populations and linkage disequilibrium in the CCR5 region is not dissimilar to other human genomic regions, challenging claims of recent positive selection. PMID- 16678300 TI - Harvesting the fruit of the human mtDNA tree. AB - Human mitochondrial DNA (mtDNA) studies have entered a new phase since the blossoming of complete genome analyses. Sequencing complete mtDNAs is more expensive and more labour intensive than restriction analysis or simply sequencing the control region of the molecule. But the efforts are paying off, as the phylogenetic resolution of the mtDNA tree has been greatly improved, and, in turn, phylogeographic interpretations can be given correspondingly greater precision in terms of the timing and direction of human dispersals. Therefore, despite mtDNA being only a fraction of our total genome, the deciphering of its evolution is profoundly changing our perception about how modern humans spread across our planet. Here we illustrate the phylogeographic approach with two case studies: the initial dispersal out of Africa, and the colonization of Europe. PMID- 16678301 TI - A new gene for Tourette's syndrome: a window into causal mechanisms? AB - Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder characterized by impairing motor-vocal tics. Locating genetic loci by associating the phenotype with DNA translocations, inversions, gain or losses, State et al. identified SLITRK1 as a candidate gene in an individual with GTS and inv(13) (q31.1; q33.1). This gene was also associated with abnormal axonal-dendritic development in embryonic mouse cells. Although SLITRK1 is not a major causal gene for GTS, it can shed light on our understanding of the gene-based neural correlates of this disease. PMID- 16678302 TI - Dissecting the mammalian genome--new insights into chromosomal evolution. AB - A recent study aligning genomic data from eight mammalian species has provided new and detailed information on the architecture of chromosomes that are thought to comprise the karyotype of the boreoeutherian ancestor. The analyses suggest that evolutionary breakpoints are clustering in "hotspots", that these regions are enriched for centromeres and that the more commonly occurring human cancer associated breakpoints tend to co-localize with evolutionary breakpoints. PMID- 16678303 TI - Where are we going after the first twenty years? PMID- 16678304 TI - Evolved to satisfy our immediate needs: self-control and the rewarding properties of food. AB - Evolutionary explanations of overeating in modern society emphasize that humans have evolved to eat to their physiological limits when food is available. The present paper challenges the idea that eating is driven by the availability of food only and proposes that it is regulated by strategic anticipatory behaviour in service of the most profitable long-term scenario as well. Our alternative explanation emphasizes the interaction between the reward system that regulates the liking and wanting of food and the role of self-control, which is involved in maintaining the best outcome in the long run. PMID- 16678305 TI - Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: toward an understanding of metabolic diversity. AB - Cyanobacteria and particularly Microcystis sp. (Chroococcales) are known to produce a multitude of peptide metabolites. Here we report on the mass spectral analysis of cyanobacterial peptides in individual colonies of Microcystis sp. collected in a drinking water reservoir. A total number of more than 90 cyanopeptides could be detected, 61 of which could be identified either as known peptides or new structural variants of known peptide classes. For 18 new peptides flat structures are proposed. New congeners differed from known ones mainly in chlorination (aeruginosins), methylation (microginins), or amino acid sequences (cyanopeptolins). The high number of peptides and especially the new peptides underline the capability of Microcystis strains as producers of a high diversity of potentially bioactive compounds. PMID- 16678306 TI - Components of the protein quality control system are expressed in a strain dependent manner in the mouse hippocampus. AB - Inbred mouse strains are used in forward-genetic experiments, designed to uncover genes contributing to their highly distinct neurophenotypes and multiple reports of variations in mutant phenotypes due to genetic background differences in reverse-genetic approaches have been published. Information on strain-specific protein expression-phenotypes however, is limited and a comprehensive screen of an effect of strain on brain protein levels has not yet been carried out. Herein a proteomic approach, based upon two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) was used to show significant genetic variation in hippocampal protein levels between five mouse strains. Considering recent evidence for the importance of the intracellular protein quality control system for synaptic plasticity-related mechanism we decided to focus on the analysis of molecular chaperones and components of the ubiquitin-proteasome system. Sixty-six spots, depicting 36 proteins have been unambiguously identified by mass spectrometry. Quantification revealed strain-dependent levels of 18 spots, representing 12 individual gene products. We thus present proteome analysis of hippocampal tissues of several mouse strains as suitable tool to address fundamental questions about genetic control of protein levels and to demonstrate molecular networks of protein metabolism and chaperoning. The findings are useful for designing future studies on these cascades and interpretation of results show that data on brain protein levels cannot be simply extrapolated among different mouse strains. PMID- 16678308 TI - Impairment of lymphocyte function in head-injured rats: effects of standard and immune-enhancing diets for enteral nutrition. AB - BACKGROUND: The metabolic response to head injury (HI) is characterized by a dysimmunity which may be a risk factor of a septic state. The use of immune enhancing diets (IEDs) could be a promising approach to improve immune functions. The aim of the study was to investigate the consequences of HI on lymphocyte function and to determine the effects of an enteral IED comparatively to a standard enteral nutrition. METHOD: A rat model of HI by fluid percussion was used. Twenty-five male Sprague-Dawley rats were randomized into 4 groups: rats receiving standard chow diet ad libitum (AL), rats sustaining HI and receiving standard chow diet and enteral saline (HI), rats receiving the enteral standard diet Sondalis HP (HIS), and rats receiving the IED Crucial (HIC). The two enteral diets were infused continuously during 4 days after the HI and were isocaloric, isonitrogenous and isovolumic. RESULTS: HI induced a thymus atrophy (HI vs. AL, P<0.05), and an impairment in lymphocyte CD25 receptor density responsiveness to stimulation. The IED blunted thymus atrophy and allowed to preserve the stimulation of blood and Peyer patches lymphocytes (HIC: Stimulated vs. Basal, P<0.05). CONCLUSION: IED seems more adapted for preserving lymphocyte function than standard diet in HI patients. PMID- 16678309 TI - Body composition in children with bronchopulmonary dysplasia predicted from bioelectric impedance and anthropometric variables: comparison with a reference dual X-ray absorptiometry. AB - Since children with bronchopulmonary dysplasia often suffer from malnutrition and growth failure, evaluation of body composition is a very important tool to nutritional support. The aim of this study was to compare assessment of fat-mass (FM) and fat-free mass (FFM), evaluated by bio-impedancemetry and anthropometry compared to dual-X-ray-absorptiometry (DXA) in children with bronchopulmonary dysplasia. PATIENTS: Seventy-one children, aged 4-8 years, with bronchopulmonary dysplasia were enrolled. METHODS: FM and FFM measured using anthropometry and bio impedancemetry were compared to FM and FFM obtained by DXA using the Bland-Altman method. RESULTS: Both bio-impedancemetry and anthropometry gave good agreement with DXA to evaluate FM and FFM. Anthropometry method, in general, slightly under estimated FM (mean difference: -0.02 kg, standard deviation: 0.99) and FFM (mean difference: -0.70 kg+/-1.72). Bio-impedancemetry method overestimated FM (mean difference: 0.34 kg+/-2.06) and underestimated FFM (mean difference: -1.24 kg+/ 3.32). CONCLUSION: In children with bronchopulmonary dysplasia aged, 4-8 years, both anthropometry and bio-impedancemetry cannot be used to precisely evaluate body composition. PMID- 16678310 TI - Partially hydrolyzed guar gum increases intestinal absorption of iron in growing rats with iron deficiency anemia. AB - OBJECTIVE: The objective of this study was to evaluate the effect of partially hydrolyzed guar gum (PHGG) dietary fiber towards intestinal iron absorption, for dietary intake and on the growth of rats with iron deficiency anemia in comparison to those fed on a diet with cellulose and without dietary fiber. MATERIALS AND METHODS: Male Wistar rats (n=24) weaned at 21 days were fed with AIN93-G diet without iron for 2 weeks in order to induce iron deficiency anemia. At 36 days old, the anemic rats were divided into three groups: (1) PHGG group 100g of PHGG per kg of diet; (2) Cellulose group-100g of cellulose per kg of diet; (3) Control group-diet without dietary fiber. All the diets had 25mg of elemental iron/kg of diet added to lead to recovery from iron deficiency anemia. RESULTS: The final hemoglobin values in g/dl, for the PHGG group, the cellulose group and the control group were, respectively: 11.3+/-1.2, 8.6+/-0.7 and 8.1+/ 0.9 (P<0.001). The levels of hepatic iron, in mug/g of dry tissue, in the same order, were: 322.2+/-66.6, 217.2+/-59.1 and 203.7+/-42.4 (P<0.001). Apparent iron intestinal absorption was, respectively: 67.5+/-8.9%, 35.4+/-15.3% and 31.3+/ 24.9% (P<0.001). The three groups consumed similar quantities of diet. The changes in weight and in body length were similar in the three groups studied. CONCLUSION: PHGG led to greater intestinal absorption of iron, regeneration of hemoglobin and hepatic levels of iron than diet with cellulose and diet control. PMID- 16678311 TI - Efficacy and functionality of lipoprotein OprI from Pseudomonas aeruginosa as adjuvant for a subunit vaccine against classical swine fever. AB - Bacterial lipoproteins are potent stimulators of innate immune responses and can mediate humoral and cytotoxic T cell responses without additional adjuvants. OprI derived from Pseudomonas aeruginosa was tested in vitro and in vivo for its adjuvant potential in the context of a classical swine fever (CSF) subunit vaccine. OprI activated porcine monocyte-derived dendritic cells (MoDC), upregulating CD80/86 and MHC class II expression, as well as pro-inflammatory cytokines. OprI enhanced CSFV-antigen-specific lymphocyte proliferation and IFN gamma release. An E2/NS3-based subunit vaccine adjuvanted with OprI stimulated specific immune responses and partial protection against CSFV infection. Although, a water-oil-water adjuvanted vaccine was more potent at protecting animals, this study demonstrates that OprI has immunostimulatory properties for porcine DC, and has potential as vaccine immunostimulant. Further studies are necessary to optimize antigen formulation enabling to translate the in vitro efficacy into a potent vaccine in vivo. PMID- 16678312 TI - IC31, a novel adjuvant signaling via TLR9, induces potent cellular and humoral immune responses. AB - IC31, the combination of a novel immunostimulatory oligodeoxynucleotide containing deoxy-Inosine/deoxy-Cytosine (ODN1a) and the antimicrobial peptide KLKL(5)KLK, represents a promising novel adjuvant signaling via the TLR9/MyD88 dependent pathway of the innate immune system. In mice, IC31 induces potent peptide-specific type 1 cellular immune responses, as well as mainly type 1 dominated protein-specific cellular and humoral immune responses. In addition, cytotoxic T lymphocytes were induced, able to kill efficiently target cells in vivo. Activation of murine dendritic cells by IC31 induced efficiently proliferation of naive CD4(+) TCR transgenic T cells (DO.11.10) as well as their differentiation into IFN-gamma- and IL-4-producing T cells in vitro. PMID- 16678313 TI - "One-stop" clinics in the investigation and diagnosis of head and neck lumps. AB - This article considers the issues raised by the NICE guidelines "Improving outcomes in head and neck cancers" (2004) with particular emphasis on the guidelines regarding the creation of "one-stop" neck lump assessment clinics. Tissue sampling is an essential function of the clinics and the relative merits of fine needle aspiration cytology (FNAC) and ultrasound-guided core biopsy (US CB) in the context of a one-stop model are discussed. All cancer service providers in the UK have a duty to implement the NICE guidelines, with the intention of improving cancer services to our patients. The measures against which head and neck cancer teams are to be accredited through the process of peer review will specify such a clinic to be in place. Teams should implement the guidance in such a way that there is a genuine benefit to patients. In many centres this may require clinicians to learn new skills and adopt different ways of working. PMID- 16678314 TI - Gunshot injuries to the parotid gland: Patterns of injury and primary management. AB - We have treated 16 patients with gunshot injuries to the cheek, 10 of whom had damage to the parotid. There were nine men and one woman, mean age 40 (range 15 65). All injuries were high velocity, and eight had other injuries. We followed them up for a month; three patients required further operation, three had facial palsy, and one lost his hearing on that side. PMID- 16678315 TI - Ligamentous reconstruction of scapholunate dislocation through a double dorsal and palmar approach. AB - Six patients were treated for scapholunate dissociation by reconstruction of both the palmar and dorsal parts of the scapholunate interosseous ligament through a combined palmar and dorsal approach. The mean active range of motion of the wrist at final follow-up was 44 degrees of flexion and 58 degrees of extension and the mean hand grip strengths was 88% of that of the contralateral hand. The average time to return to work was 105 days and the mean DASH score at a mean follow-up of 32 months was 18. At follow-up, radiographs showed maintenance of the anatomical reduction of the scapholunate articulation in all cases and the scapholunate angle was normal in five patients. PMID- 16678316 TI - Moment arms of forearm rotators. AB - BACKGROUND: Rotation about a longitudinal axis of the forearm has been a matter of investigation for over 100 years. However, most studies were limited to only a few muscles and to their action in specific set positions of elbow and forearm rotation. This investigation aims at determining the moment arms of muscles that contribute to pronation and supination at three different angles of elbow flexion throughout the entire range of forearm rotation. METHODS: Muscle moment arms were derived from tendon excursions that were recorded on a full-size epoxy model of the radioulnar complex. The results were verified on a fresh cadaver specimen. FINDINGS: Moment arms of all major supinators exhibit peak values in 40-50 degrees of pronation, for all three positions of the elbow. These peak values vary with elbow position, the biceps muscle showing the highest dependency with its greatest moment arm in 90 degrees of elbow flexion. The pronators show a maximum of moment arm about the neutral position, with little dependency on elbow flexion. Brachioradialis brings the pronated, or supinated forearm into the neutral position. The bow of the radius is in function comparable to the 'throw' of a crankshaft, forming a greater lever arm between the point of insertion of the muscles and the axis of rotation of the radius. INTERPRETATION: The observations drawn from this study could be of eminent value in planning rotator muscle transplantation, in understanding functional disorders after injury, and in the physical treatment of forearm rotator muscle deficiency. Reconstruction of the physiological anatomical arrangement in the treatment of injuries is strongly recommended for restoration of function. PMID- 16678317 TI - Predatory insects as bioindicators of heavy metal pollution. AB - Heavy metal concentrations of different predatory insects were studied near by a steel factory and from control sites. Waterstriders (Gerridae), dragon fly larvae (Odonata), antlion larvae (Myrmeleontidae) and ants (Formicidae) were analyzed by AAS. In most cases the metal concentrations were higher near the factory, but e.g. waterstriders had higher cadmium concentrations in control area. Discriminant analysis clearly reveals that all these insect groups can be used as heavy metal indicators. However, the commonly used ants were the least effective in indicating the differences between the factory and control sites. Waterstriders are good in detecting differences in iron and manganese, but seem to be poor in accumulating nickel and lead. Antlions are efficient in detecting differences in iron. Antlions and ants are effective in accumulating manganese; as well antlions are efficient in accumulating cadmium. Waterstriders are poor in accumulating lead, but antlions and ants are effective. PMID- 16678318 TI - Diet-mediated effects of heavy metal pollution on growth and immune response in the geometrid moth Epirrita autumnata. AB - The potential capacity of larval growth and immune response traits of the autumnal moth to adapt to heavy metal polluted environment was tested experimentally. Both the relative growth rate (RGR) and pupal weight were significantly higher in control trees than on polluted trees, indicating that metal pollution prevented the insect from achieving maximal growth on birch leaves. Larval growth rates of different broods differed significantly between metal contaminated and control birches. However, pupal weight of broods, which is considered more important for fitness than growth rate, in response to pollution did not differ. Immune response was significantly higher in moths exposed to pollution than in moths that were exposed to control environment suggesting that pollution enhances the immune defense of defoliators. Encapsulation rate tended to differ between broods indicating that the immune function has potential to respond to selection. PMID- 16678319 TI - Response of Pinus halepensis Mill. seedlings to biosolids enriched with Cu, Ni and Zn in three Mediterranean forest soils. AB - We investigated the response of Pinus halepensis seedlings to the application of biosolids enriched with Cu, Ni and Zn on three Mediterranean forest soils under semiarid conditions. One-year-old seedlings were planted in lysimeters on soils developed from marl, limestone and sandstone which were left unamended, amended with biosolids, or amended with biosolids enriched in Cu, Ni and Zn. Enriched biosolids increased plant heavy metal concentration, but always below phytotoxic levels. Seedlings receiving unenriched biosolids showed a weak reduction in Cu and Zn concentration in needles, negatively affecting physiological status during drought. This effect was alleviated by the application of enriched sludge. Sewage sludge with relatively high levels of Cu, Zn and Ni had minor effects on plant performance on our experimental conditions. Results suggest that micronutrient limitations in these soils may be alleviated by the application of biosolids with a higher Cu, Zn and Ni content than those established by current regulations. PMID- 16678320 TI - Effect of submergence-emergence sequence and organic matter or aluminosilicate amendment on metal uptake by woody wetland plant species from contaminated sediments. AB - Site-specific hydrological conditions affect the availability of trace metals for vegetation. In a greenhouse experiment, the effect of submersion on the metal uptake by the wetland plant species Salix cinerea and Populus nigra grown on a contaminated dredged sediment-derived soil and on an uncontaminated soil was evaluated. An upland hydrological regime for the polluted sediment caused elevated Cd concentrations in leaves and cuttings for both species. Emergence and soil oxidation after initial submersion of a polluted sediment resulted in comparable foliar Cd and Zn concentrations for S. cinerea as for the constant upland treatment. The foliar Cd and Zn concentrations were clearly higher than for submerged soils after initial upland conditions. These results point at the importance of submergence-emergence sequence for plant metal availability. The addition of foliar-based organic matter or aluminosilicates to the polluted sediment-derived soil in upland conditions did not decrease Cd and Zn uptake by S. cinerea. PMID- 16678321 TI - Toxic effects of low concentrations of Cu on nodulation of cowpea (Vigna unguiculata). AB - Although Cu is phytotoxic at Cu(2+) activities as low as 1-2 microM, the effect of Cu(2+) on the nodulation of legumes has received little attention. The effect of Cu(2+) on nodulation of cowpea (Vigna unguiculata (L.) Walp. cv. Caloona) was examined in a dilute solution culture system utilising a cation exchange resin to buffer solution Cu(2+). The nodulation process was more sensitive to increasing Cu(2+) activities than both shoot and root growth; whilst a Cu(2+) activity of 1.0 microM corresponded to a 10% reduction in the relative yield of the shoots and roots, a Cu(2+) activity of 0.2 microM corresponded to a 10% reduction in nodulation. This reduction in nodulation with increasing Cu(2+) activity was associated with an inhibition of root hair formation in treatments containing > or =0.77 microM Cu(2+), rather than to a reduction in the size of the Rhizobium population. PMID- 16678322 TI - Young people of minority ethnic origin in England and early parenthood: views from young parents and service providers. AB - The paper explores the phenomenon of early parenthood in minority ethnic communities in England. The data were collected using focus group interviews, in depth semi-structured interviews and a telephone survey. The sample consisted of 139 participants (41 service providers, 10 grandmothers, 88 young parents). The findings map out the complexity and diversity of experience of early parenthood amongst young people of minority ethnic origin, not least the multiple attachments many experience in relation to their social groups, religious affiliations and the traditional patterns of parenting within their immediate and extended family. Both the young parents and professionals in this study constructed early parenthood in more positive terms than is currently portrayed in the contemporary policy. The findings are analysed and discussed in relation to ethnic identity, social inclusion and exclusion. We explore participants' attempts to counter negative 'deficit' models of early parenthood with reference to perspectives on youth, parenthood and contemporary strategic policy. In conclusion, we suggest an unambiguous focus on the reduction of pregnancy is not a credible message when teenage pregnancy is a social norm for a particular ethnic or cultural group. For young parents of Muslim faith in particular, teenage parenting within marriage is not necessarily considered a 'problem' or seen as a distinctive event. Most participants did not view early parenthood as a barrier to re-establishing career and educational aspirations. A wide diversity of experience amongst young parents is evidenced in the communities studied; this needs to be reflected more comprehensively both in UK policy and in support services. PMID- 16678323 TI - Three studies on configural face processing by chimpanzees. AB - Previous studies have demonstrated the sensitivity of chimpanzees to facial configurations. Three studies further these findings by showing this sensitivity to be specific to second-order relational properties. In humans, this type of configural processing requires prolonged experience and enables subordinate-level discriminations of many individuals. Chimpanzees showed evidence of a composite like effect for conspecific but not human faces despite extensive experience with humans. Chimpanzee face recognition was impaired only when manipulations targeted second-order properties. Finally, face processing was impaired when individual features were blurred through pixelation. Results confirm that chimpanzee face discrimination, like humans, depends on the integrity of second-order relational properties. PMID- 16678325 TI - A reference automated external defibrillator provider course for Europe. AB - BACKGROUND: Scientific evidence is scarce in relation to the effectiveness of different methods of teaching automated external defibrillator (AED) use to laypeople. A reference course is needed in order to test new courses or methods against a comparative standard. OBJECTIVE: To propose a reference AED provider course that can be used as a comparator when testing new courses or teaching methods. METHODS: All national resuscitation councils that are represented in the European Resuscitation Council were sent a questionnaire about the AED provider courses run by them or under their auspices. RESULTS: Sixteen national resuscitation councils responded to the enquiry. Apart from the individual course timetables, there was remarkable consistency amongst the European countries as regards organisation, structure, content and methods. CONCLUSIONS: A reference AED provider course for laypeople, based on a synthesis of existing European courses, is suggested as a tool for research. Prior completion of a basic life support provider course is mandatory. Course duration is 2 h 45 min (excluding breaks), with 1 h 40 min practice time for the participants, 25 min for theory, 20 min for practical demonstrations by the instructor and 20 min for introduction, discussion and closure. A manual is distributed at the start of the course. The ratio of instructors to participants is one to six. Lectures are interactive between the instructor and the class. AED use is practised in groups of six participants. Participants prove their competency by means of a formal test that simulates a cardiac arrest scenario. Using this course as a comparator during research into the methodology of AED teaching would provide a reference against which other courses could be tested. PMID- 16678324 TI - Sevoflurane-induced oxidative stress and cellular injury in human peripheral polymorphonuclear neutrophils. AB - Sevoflurane is an inhalation anesthetic used for general anesthesia. Several studies have demonstrated that reactive oxygen species (ROS) exist in cardioprotection when preconditioned with sevoflurane. Moreover, sevoflurane can also directly trigger the formation of peroxynitrite. Up to now, information pertinent to the effect of sevoflurane on cellular injuries in human polymorphonuclear neutrophils (PMN) is scant. In this study, we demonstrated that sevoflurane significantly increases intracellular H2O2 and/or peroxide, superoxide, and nitric oxide (NO) in PMN within 1h treatment. Intensification of intracellular glutathione (GSH) depletion in PMN has been demonstrated with the presence of sevoflurane. Inhibition of sevoflurane-mediated intracellular H2O2 and/or peroxide in PMN by catalase, mannitol, dexamethasone, N-acetylcysteine (NAC) and trolox, but not superoxide dismutase (SOD) pretreatment, was observed. Among them, catalase has the best effect scavenging intracellular H2O2 and/or peroxide, suggesting that H2O2 is the major ROS during sevoflurane treatment. Two apoptotic critical factors-lowering of the mitochondrial transmembrane potential (DeltaPsim) and activation of caspase 3/7-were significantly increased after 1h of sevoflurane treatment. Apoptosis of PMN were determined by comet assay and flow cytometric analysis of annexin V-FITV protein binding to the cell surface. Exposure of PMN to sevoflurane markedly increased apoptosis in a dose-dependent manner. In summary, these results are important for demonstrating the oxidative stress and cellular injury on sevoflurane-treated human PMN. PMID- 16678326 TI - Laypersons may learn basic life support in 24min using a personal resuscitation manikin. AB - BACKGROUND: Bystander basic life support (BLS) is an important part of cardiopulmonary resuscitation (CPR) and improves outcome after out-of-hospital cardiac arrest. However, the general population has poor BLS skills. Several training initiatives could be used to improve this situation and the challenge is to find the most efficient one. AIMS: To compare the efficiency of a 24 min instruction using a DVD-based self-training BLS course combined with a simple, take-home resuscitation manikin to a conventional 6h course for teaching BLS to laypersons. METHODS: In total, 238 laypersons (age 21-55 years) without previous BLS-training were allocated into two groups: one group received 24 min of instruction using a DVD-based instruction tool on a big screen combined with a BLS self-training device, Laerdal MiniAnne manikin (MAM), before taking home the instruction material for subsequent self-training. The second group attended a conventional 6 h BLS course (6 HR). After 3 months BLS skills were assessed on a Laerdal ResusciAnne manikin using the Laerdal PC Skill reporting System, and a total score was calculated. RESULTS: There was no significant difference between groups in BLS performance using the total score. Assessment of breathing was performed significantly more often in the 6 HR-group (91% versus 72%, P=0.03). In the MAM-group, average inflation volume and chest compression depth were significantly higher (844 mL versus 524 mL, P=0.006, and 45 mm versus 39 mm, P=0.005). CONCLUSIONS: When assessed after 3 months, a 24 min DVD-based instruction plus subsequent self-training in BLS appears equally effective compared to a 6h BLS course and hence is more efficient. PMID- 16678328 TI - Interventions for leg edema and varicosities in pregnancy. What evidence? AB - Leg oedema from venous insufficiency is not dangerous but it can cause women symptoms such as pain, feelings of heaviness, night cramps and paraesthesiae. Leg oedema can be a sign of pre-eclampsia when associated with raised blood pressure or proteinuria. The objective of this review was to assess the effects of treatment to relieve the symptoms associated with varicosity in pregnancy and to reduce leg oedema. We searched the Cochrane Pregnancy and Childbirth Group trials register in October 2004 for randomised trials of any form of treatment for varicosity and or leg oedema in pregnancy. Trial quality was assessed and data were extracted. Four trials of three different treatments were included. In one trial, women given rutoside capsules in the last 3 months of pregnancy noted an improvement in symptoms compared with placebo (relative risk 0.54 95% CI 0.32, 0.89). They had a decrease in ankle circumference at 36 weeks' gestation after 8 weeks of treatment, while women given placebo had a small increase. In one trial, women with ankle oedema had a small non-significant reduction in lower leg volume when treated with external pneumatic intermittent compression for 30 min. In another trial compression stockings prophylactically reduced the emergence of leg symptoms but not venous varicosities (relative risk 0.74 95% CI 0.59, 0.93). Lymphatic reflexology was studied in too few women to draw conclusions. In conclusions, rutosides appear to relieve symptoms of venous insufficiency in late pregnancy. However, it is not known if the drug is safe in pregnancy. External pneumatic compression appears to reduce ankle swelling and compression stockings reduce leg symptoms but not varicose veins. PMID- 16678327 TI - PPAR-gamma2 Pro12Ala polymorphism is associated with weight gain in women with gestational diabetes mellitus. AB - OBJECTIVE: The polymorphisms of peroxisome proliferator-activator receptor-gamma2 (PPAR-gamma2) have been suggested to affect glucose metabolism and weight gain. Both conditions show great variations during pregnancy that makes pregnancy a suitable condition to detect any metabolic abnormalities related to PPAR-gamma2 polymorphisms. The objective of this study is to investigate the prevalence and metabolic impacts of PPAR-gamma2 polymorphism in control pregnant women and in patients with gestational diabetes mellitus (GDM). METHODS: In this case-control study, anthropometric and metabolic variables of 100 non-diabetic pregnant women and of 62 women who were diagnosed as having GDM according to 100 g oral glucose tolerance test (OGTT) were compared on the basis of PPAR-gamma2 polymorphism by univariate analysis of covariance. RESULTS: There were no statistically significant differences in baseline characteristics and the mean 50 g glucose challenge test values of pregnant women in both groups on the basis of PPAR gamma2 genotype, although patients with Pro12Ala polymorphism were significantly taller in GDM group. The Pro12Ala polymorphism had no effect on 100 g OGTT results of patients with GDM. However, patients with GDM who had Pro12Ala polymorphism gained significantly more weight during their pregnancy. CONCLUSION: The PPAR-gamma2 Pro12Ala polymorphism was observed to have no effect on glucose metabolism in normal pregnant women and women with GDM. However, only the patients with GDM who had this polymorphism gained significantly more weight during their pregnancy. It seems that Pro12Ala polymorphism plays a dynamic and interactive role in the regulation of BMI and glucose homeostasis. PMID- 16678329 TI - Superoxide anion production of granulocytes in patients with endometrial cancer at presentation and after treatment. AB - BACKGROUND: In current medical science the reactive oxygen intermediates play an ever more important role. METHODS: The authors analysed superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 30 blood samples from endometrial carcinoma patients. They measured it before the complex treatment and in nine cases at least 1 year after finishing the treatment. The results were compared with healthy controls. Phorbol dibutyrate stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome-c absorbance. RESULTS: The mean superoxide anion production of PMNLs of the 31 healthy controls was 1.541 nM/min/10(5)cells (S.D.=0.201 nM/min/10(5)cells). Superoxide anion production of samples from endometrial cancer patients was much lower. The superoxide anion production of granulocytes in the early stage of endometrial cancer was lower (1.11 nM/min/10(5)cells) as in the controls. There was no essential difference in the superoxide production of patients with different depths of myometrial infiltration. After treatment, the superoxide anion production of the granulocytes of the clinically tumour-free patient had substantially progressed (1.357 nM/min/10(5)cells), but it was henceforth lower than the control. CONCLUSIONS: According to our results, damage to the non-specific immunity is already advanced at the earliest stage of endometrial cancer. Further examinations are needed to decide how to normalise the superoxide production of granulocytes, and whether it has any importance in prevention and therapy. PMID- 16678330 TI - Influence of swim-up time on the ratio of X- and Y-bearing spermatozoa. AB - OBJECTIVE: The objective was to examine the separation of X- and Y-bearing spermatozoa in a modified swim-up procedure using fluorescent in situ hybridization (FISH), and to find out the influence of swim-up time on the ratio of X- and Y-bearing spermatozoa. STUDY DESIGN: Prospective study. SETTING: Reproductive testing laboratory in a university hospital. PATIENTS: Normal spermatozoa samples were obtained from 10 volunteers by masturbation after sexual abstinence for 3-5 days. INTERVENTIONS: Spermatozoa were put into 18 tubes with 0.25 ml in each, then mixed with HTF medium and centrifuged for 5 min (400 x g). The supernatant was removed and discarded and 0.5 ml HTF was added slowly along the tube wall. Motile spermatozoa were collected after swimming up in different times (from 5 up to 150 min, with a total of 17 intervals). The X- and Y-bearing spermatozoa were determined using the FISH technique. The X/Y dual-color CEP probes that were marked by fluorescein isothiocyanate (FITC) and Texas red were applied to analyze the ratio of X- and Y-bearing spermatozoa. The FISH staining slides were analyzed under an immunofluorescence microscope. About 1000-1500 spermatozoa were counted per slide. MAIN OUTCOME MEASURES: The percentages of X- and Y-bearing spermatozoa were calculated. RESULTS: The study results suggested that the total ratio of hybridization was 98.33%. The ratio of X-bearing spermatozoa after swimming up for different amounts of time is 50.03 +/- 0.91% at 0 min, 50.45 +/- 2.06% after 15 min, 50.61 +/- 2.47% after 30 min, 50.16 +/- 2.67% after 60 min, 50.72 +/- 2.64% after 90 min, and 50.56 +/- 2.20% after 150 min. The statistical analysis showed that there were no significant differences among different swim-up times in the ratio of X-bearing spermatozoa. CONCLUSIONS: There was no significant effect of swim-up time on the ratios of X- and Y-bearing spermatozoa using a modified swim-up procedure. No direct evidence was found that the swim-up procedure for separating motile spermatozoa to use for either intrauterine insemination (IUI) or in vitro fertilization (IVF) would lead to an imbalance of boys and girls. PMID- 16678331 TI - Changes in the MRI morphology of the stress continence control system after TVT (tension-free vaginal tape) insertion. AB - OBJECTIVE: Magnetic resonance imaging (MRI) was used to investigate whether tension-free vaginal tape (TVT) insertion (according to Ulmsten) leads to morphologic changes of the stress continence control system. METHODS: Twenty women (mean age 53.4 years) with clinically and urodynamically proven stress urinary incontinence without prolapse were examined by MRI before and 13 months after TVT insertion. RESULTS: Postoperative MRI showed a signal intensity loss of the suburethral portion of the endopelvic fascia in the area of the anterior vaginal wall in 9/20 women with additional signal loss of the paraurethral portion of the fascia in 3/20 women. No morphologic changes of the levator ani muscle and the urethra were seen postoperatively. CONCLUSIONS: TVT insertion does not damage the structures of the stress continence control system or impact on their topographic relationships. MRI identified no excessive scar formation resulting from integration of the TVT. PMID- 16678332 TI - Family history of early-onset cardiovascular disorders is associated with a higher risk of severe preeclampsia. AB - AIM: The aim was to evaluate familial early-onset cardiovascular disorders as potential risk factors for severe preeclampsia. STUDY DESIGN: A case-control study was carried out by interviewing 162 primiparous severely preeclamptic women and 521 primiparous healthy control patients after delivery to determine the frequency of cardiovascular disorders (chronic hypertension, myocardial infarction, stroke) developed before the age of 50 among their parents. The chi2 test was utilized to estimate odds ratios (OR) and 95% confidence intervals (95% CI). The association was adjusted for pre-pregnancy body mass index, maternal age, and smoking habits before pregnancy using logistic regression analysis. RESULTS: Maternal and paternal early-onset chronic hypertension (adjusted OR: 3.84, 95% CI: 2.25-6.54; and adjusted OR: 3.26, 95% CI: 1.76-6.05) as well as paternal early-onset myocardial infarction (adjusted OR: 3.33; 95% CI: 1.51-7.32) were independent risk factors for severe preeclampsia. Early-onset stroke affected only the fathers of severely preeclamptic patients. Among the severely preeclamptic patients a positive family history of cardiovascular disorders developed before the age of 50 increased the risk of early-onset preeclampsia (developing before the 32nd gestational week) by 5.05-fold (95% CI: 3.08-8.31) compared with the control group. CONCLUSION: Our results suggest that the presence of familial early-onset cardiovascular disorders is a predisposing factor for severe preeclampsia. PMID- 16678334 TI - Impact of the FTSE4Good Index on firm price: an event study. AB - This paper examines whether corporate financial performance is affected by public endorsement of environmental and social performance. Event study methodology, which relies on the notion of market efficiency, is used to examine the relationship between positive and negative announcements and changes in share prices or daily returns. Inclusion in and deletion from the FTSE4Good UK Index is used as a proxy measure for good (poor) corporate social responsibility. The abnormal or unexpected daily returns associated with an event are calculated and their significance tested. The results show a trend towards positive and negative announcements having the expected effects on daily returns. But these movements are not significant and the data do not suggest that a firm's presence on the index brings it any significant financial return for signalling its corporate social responsibility. PMID- 16678333 TI - Do transsexuals have micturition disorders? AB - OBJECTIVE: Transsexualism occurs with an estimated prevalence of 2.4:100,000 male to-female (MTF) and 1:100,000 female-to-male (FTM) transsexuals. As sex reassignment surgery involves surgery of the urethra and transsexuals are substituted life-long with the cross gender hormones there could possibly arise micturition disorders. Aim of the study was to determine if transsexuals have an increased risk of micturition disorders and if so which. STUDY DESIGN: Between January and July 2003 we examined 25 transsexuals whereof 18 were MTF and 7 were FTM transsexuals using King's Health Questionnaire, visual analogue scale for patient's well being, perineal and transabdominal ultrasound, urine dipstick and uroflow measurement. RESULTS: 17 out of 25 patients considered themselves very happy. In MTF transsexuals, a diverted stream, overactive bladder and stress urinary incontinence was a common problem. Prostate volume was small with 20 g and palpation did not confirm and solid or suspicious lesions. None of the patients had significant residual urine but MTF transsexuals had a reduced urinary flow. We could not detect a current urinary tract infections in any of the patients. CONCLUSION: Transsexuals have an increased risk for the development of micturition disorders including stress urinary incontinence and overactive bladder compared to age-matched control groups and should be counselled preoperatively. Reasons for the development of incontinence might be surgery including pudendal nerve damage, hormonal reasons and ageing. PMID- 16678335 TI - Understanding self-evaluations of effectiveness by forestry advisory committee members: a case of Ontario's Local Citizens Committee members. AB - Researchers have devised many criteria that could assist with the development of an effective public participation process. Few studies, however, have linked these criteria to the effectiveness of decision-making resulting from these processes. We assess whether several criteria postulated by others for designing an effective decision-making process are associated with evaluations of the effectiveness of decision-making by advisory group members from a forest management planning process in Ontario, Canada. Data were collected by written questionnaires from 197 advisory group members in 2001. Four criteria were linked to the effectiveness evaluations including process control (influence), trust in decision-makers, information credibility, and process efficiency. The criterion of knowledge along with contextual variables related to experience, economic dependence on forestry, and geographical residence were not associated with evaluations of effectiveness. PMID- 16678336 TI - An inexact two-stage mixed integer linear programming method for solid waste management in the City of Regina. AB - In this study, an interval-parameter two-stage mixed integer linear programming (ITMILP) model is developed for supporting long-term planning of waste management activities in the City of Regina. In the ITMILP, both two-stage stochastic programming and interval linear programming are introduced into a general mixed integer linear programming framework. Uncertainties expressed as not only probability density functions but also discrete intervals can be reflected. The model can help tackle the dynamic, interactive and uncertain characteristics of the solid waste management system in the City, and can address issues concerning plans for cost-effective waste diversion and landfill prolongation. Three scenarios are considered based on different waste management policies. The results indicate that reasonable solutions have been generated. They are valuable for supporting the adjustment or justification of the existing waste flow allocation patterns, the long-term capacity planning of the City's waste management system, and the formulation of local policies and regulations regarding waste generation and management. PMID- 16678337 TI - Dynamic modeling of chemical fate and transport in multimedia environments at watershed scale-II: trichloroethylene test case. AB - A multimedia environmental fate model was developed to study the temporal dynamics and spatial distribution of a chemical pollutant at watershed scale. The theoretical considerations and implementation of the model were described in the accompanying paper (Part I). This paper presents the result of a test simulation on the transport of trichloroethylene (TCE) in the Connecticut River Basin. The simulation results were reported as time series of concentrations and inter-media transport fluxes in the compartments of atmosphere, plant, soil, surface water, and sediment. Predicted concentrations from the test simulation were compared with published field data or predictions by validated models. The temporal trends in TCE predictions were evaluated by comparing the simulation results with monthly TCE concentrations in various environmental compartments and monthly fluxes of inter-media transport processes. Results indicated that the simulation results were in reasonable agreement with reported data in the literature. The results also revealed that the mass transport of TCE from the atmosphere compartment to soil and surface water was a major route of TCE dispersion in the environment. PMID- 16678338 TI - Urban Australians using recycled water for domestic non-potable use--an evaluation of the attributes price, saltiness, colour and odour using conjoint analysis. AB - Recycled water use in urban areas is viewed as one part of the solution to Australia's water shortage. The effectiveness of policies designed to promote recycled water systems depends on the acceptance by the community of the price, colour, odour and salt content of the recycled water. In Australia and other countries, limited research has been conducted that investigates community attitudes to and willingness to pay for recycled water, especially in urban settings. Community acceptance of recycled water and the economic feasibility of such projects have not been widely evaluated, even though the long-term feasibility of many projects is dependent on such information. This paper examines the attitudes of an urban Australian community living at Mawson Lakes in South Australia, to using recycled water for non-potable domestic purposes. Conjoint analysis (CA) was used to evaluate participant's (n=136) preferences for various attributes of recycled water (colour, odour, salt content and price) for various uses (garden watering, toilet flushing and clothes washing). The analysis was used to estimate the respondent's willingness to pay (WTP) for quality increases for each of the attributes. Differences in WTP were investigated according to various demographic variables including income and education. Results indicate that for garden watering having 'low salt levels' is the most important attribute of recycled water, for clothes washing 'colourless' is the most important attribute, and for toilet flushing a 'low price' was the most important attribute. Respondents were willing to pay for increases in the quality of recycled water. The amount they were willing to pay varied depending on applied use and the attribute in question. Respondents were most willing to pay for an increase in quality of recycled water when used for clothes washing (willing to pay Australian dollars (A$) 0.07/cubic meter (m(3)) for removal of colour, A$0.065 per cubic meter for an increase in quality of recycled water from 'salty' to 'low salt,' and A$ 0.06/m(3) for removal of odour (1A$=0.75 US $ and 0.62 euro, August 24, 2005)). Segmentation of the price attribute by demographic sectors reveals that those in the higher income bracket, higher education sector, middle age group, and who are male, had a higher marginal valuation on price. The results of this study provide important information for recycled water retailers and water policy developers, helping achieve greater economic feasibility for future recycled water projects, to the satisfaction of the communities involved. PMID- 16678339 TI - Re: Burcin Tuna, Kutsal Yorukoglu, Mehtat Unlu, M. Ugur Mungan and Ziya Kirkali. Association of mast cells with microvessel density in renal cell carcinomas. Eur urol 2006;50:530-34. PMID- 16678340 TI - Influence of the severity of stress urinary incontinence on quality of life, health care seeking, and treatment: A national cross-sectional survey. AB - OBJECTIVES: To determine the prevalence of stress urinary incontinence (SUI) symptoms in women and to evaluate the severity of these symptoms and resulting functional impairment with regard to urinary symptoms, quality of life (QoL) impairment, medical care seeking, and care facilities. METHODS: A national population based, cross-sectional study in France was conducted through an anonymous questionnaire that was completed by telephone. A randomized sample was extracted from the French telephone directory, December 2002 to March 2003. The main outcome measures were symptom severity, functional impairment, non SUI related urinary symptoms, Contilife QoL scale, medical care seeking, and treatment. In total, 6,675 women aged 18-70 were selected and 5,160 questionnaires were completed and analyzed. RESULTS: The point-prevalence of SUI symptoms was 19.5% (1.1% for individuals with severe symptoms; 2.8% for those with severe functional impairment). Symptom severity and functional impairment were associated with older age, longer duration of symptoms, higher frequency of comorbid urinary symptoms, and altered QoL (all p < 0.001). CONCLUSIONS: SUI symptoms are frequent in French women, causing embarrassment and negatively affecting their QoL. Though some could benefit from an effective therapy, only the most severely affected women who report SUI symptoms seek help and receive treatment. PMID- 16678341 TI - Identification of genes responsive to low intensity pulsed ultrasound in a human leukemia cell line Molt-4. AB - We examined the gene expression of human leukemia Molt-4 cells treated with non thermal low intensity pulsed ultrasound. Six hours after 0.3W/cm(2) pulsed ultrasound treatment, apoptosis (24+/-3.3%, mean+/-SD) with minimal cell lysis was observed. Of approximately 16,600 genes analyzed, BCL2-associated athanogene 3 (BAG3), DnaJ (Hsp40) homolog, subfamily B, member 1 (DNAJB1), heat shock 70 kDa protein 1B (HSPA1B), and heat shock 70 kDa protein 6 (HSPA6) showed increased levels of expression while isopentenyl-diphosphate delta isomerase (IDI1) and 3 hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) showed decreased levels in the cells 3h after the ultrasound treatment. The expression levels of these six genes were confirmed by a real-time quantitative polymerase chain reaction. To our knowledge, this is the first report of DNA microarray analysis of genes that are differentially expressed in response to apoptosis induced by non-thermal low intensity pulsed ultrasound in human leukemia cells. The present results will provide a basis for further understanding of the molecular mechanisms of effects of not only low intensity pulsed ultrasound but also that of mechanical shear stress in the cells. PMID- 16678342 TI - Degradation of chemical warfare agent simulants using gas-liquid pulsed streamer discharges. AB - This study determines the effectiveness of pulsed streamer discharges (PSD), a type of advanced oxidation technology (AOT) to clean water contaminated with chemical agents. For the purpose of this study, experiments were conducted with G and H agent simulants to determine the degradation kinetics and to determine the effects of various electrical and chemical parameters in the degradation of these contaminants. The energy efficiency of contaminant degradation shows that pulsed streamer discharges can be an efficient technology in treating water contaminated with chemical agents. The maximum energy yields of degradation of H and G agent simulants by the pulsed corona discharges are 0.029 and 0.008 molecules/100 eV, respectively, in the series configuration with ferrous sulfate salt in solution. PMID- 16678343 TI - Isoflurane disrupts anterio-posterior phase synchronization of flash-induced field potentials in the rat. AB - Consciousness presumes a set of integrated functions such as sensory processing, attention, and interpretation, and may depend upon both local and long-range phase synchronization of neuronal activity in cerebral cortex. Here we investigated whether volatile anesthetic isoflurane at concentrations that produce loss of consciousness (LOC) disrupts long-range anterio-posterior and local anterior synchronization of neuronal activity in the rat. In six rats, deep electrodes were chronically implanted in the primary visual cortex (V1) and in two areas of the motor cortex (M1 and M2) for recording of intracortical event related potentials (ERP). Thirty discrete flashes were presented at random interstimulus intervals of 15-45 s, and ERPs were recorded at stepwise increasing isoflurane concentrations of 0-1.1%. Neuronal synchronization was estimated using wavelet coherence computed from the ERP data band-pass filtered at 5-50 Hz. We found that (1) in the waking state, long-range anterio-posterior coherence in 5 25 Hz and 25-50 Hz frequency bands was significantly higher than local anterior coherence; (2) anterio-posterior coherence in both 5-25 Hz and 26-50 Hz bands was significantly reduced by isoflurane in a concentration-dependent manner; (3) local anterior coherence was not affected by isoflurane at any of the concentrations studied. These findings suggest that a disruption of long-range anterio-posterior rather than local anterior synchronization of neuronal activity precedes the anesthetic-induced loss of consciousness. PMID- 16678344 TI - Using graph theoretical analysis of multi channel EEG to evaluate the neural efficiency hypothesis. AB - Previous studies demonstrated that intelligence is significantly related to an impressive array of psychological, social, biological and genetic factors and that working memory (WM) can be considered as a general cognitive resource strongly related with a wide variety of higher order cognitive competencies and intelligence. Also, evaluating the WM of subjects might allow one to test the neural efficiency hypothesis (NEH). WM typically involves functional interactions between frontal and parietal cortices. We recorded EEG signals to study neuronal interactions during one WM test in individuals who had few years of formal education (LE) as compared to individuals with university degrees (UE). The two groups of individuals differed in the scores they obtained in psychological tests. To quantify the synchronization between EEG channels in several frequency bands, we evaluated the "synchronization likelihood" (SL), which takes into consideration nonlinear processes as well as linear ones. SL was then converted into graphs to estimate the distance from "small-world network" (SWN) organization, i.e., an optimally organized network that would give rise to the data. In comparison to LE subjects, those with university degrees exhibited less prominent SWN properties in most frequency bands during the WM task. This finding supports the NEH and suggests that the connections between brain areas of well educated subjects engaged in WM tasks are not as well-organized in the sense of SWN. PMID- 16678345 TI - Increased phosphorylation of caveolin-1 in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis. AB - The expression of phospho-specific caveolin-1 (p-caveolin-1) was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that p-caveolin-1 was constitutively expressed in normal spinal cords and that it significantly increased in the spinal cord with EAE at both early and peak stages of EAE (P<0.05), and decreased slightly at the recovery stage of EAE. Immunohistochemistry showed that p-caveolin-1 was constitutively expressed in few vascular endothelial cells and glial cells in the spinal cords of normal rats. In EAE lesions, p-caveolin-1 was intensely immunostained in inflammatory T cells and macrophages. Therefore, we postulate that phosphorylation of caveolin-1 occurred in the inflammatory cells of EAE lesions, and that caveolin-associated cell activation is mainly associated with inflammatory cells that appear during early and peak stages of EAE. PMID- 16678346 TI - Enhanced ERK dependent CREB activation reduces apoptosis in staurosporine-treated human neuroblastoma SK-N-BE(2)C cells. AB - Activation of cAMP response element binding protein (CREB) is implicated in neuronal survival. The mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) activates a transcription factor CREB. Previously, we reported that N-acetyl-O-methyldopamine (NAMDA) protects neurons from ischemia via enhancing ERK dependent CREB phosphorylation. To investigate whether NAMDA induces endogenous survival pathways in apoptotic conditions and whether the neuroprotectant enhances a preexisting survival pathway, we determined the degree of ERK-CREB activation and resistance to apoptosis in staurosporine-treated SK-N BE(2)C neurons. Compared to forskolin-treated apoptotic cultures, NAMDA-treated cultures induced a minimum activation on ERK (pERK) or CREB (pCREB). However, NAMDA enhanced the activation of ERK and CREB in the presence of forskolin (1.7 fold increase for pCREB, 2.1-fold increase for pERK2, p<0.05 from forskolin). The effect was completely blocked by a specific MEK inhibitor U0126, suggesting the involvement of ERK dependent CREB signaling. Cleavage of caspase-3 and poly-(ADP ribose)-polymerase was additively reduced in cultures treated with NAMDA and forskolin simultaneously, but not in the presence of U0126. The data showed that NAMDA enhances forskolin-induced ERK-CREB activation and potentiates forskolin induced resistance to apoptosis. The study indicates that enhancing endogenous survival pathways by NAMDA combined with other neuroprotective measure(s) might be a useful strategy to reduce apoptosis. PMID- 16678347 TI - The neuroprotection conferred by activating the mitochondrial ATP-sensitive K+ channel is mediated by inhibiting the mitochondrial permeability transition pore. AB - In order to further explore the mechanisms by which activation of mitochondrial ATP-sensitive potassium channels (mitoKATP) confers neuroprotection, we investigated the role of the mitochondrial permeability transition pore (MPTP) in in vivo and in vitro models. Adult male Sprague-Dawley rats were exposed to 90 min of middle cerebral artery occlusion (MCAO) followed by reperfusion for 22 h, when neurological scores and infarct volumes were evaluated. Activating mitoKATP by infusion of 2 mmol/L diazoxide into the ventricles 20 min before MCAO or inhibiting the MPTP by infusion of 1 micromol/L cyclosporin A 15 min before reperfusion significantly increased functional score and reduced infarction volume. Subsequent intracerebroventricular infusion of 2 mmol/L atractyloside, the MPTP opener, 10 min before reperfusion significantly attenuated the neuroprotective effects of diazoxide and cyclosporin A. The swelling of mitochondria isolated from brain was evaluated by spectrophotometry and served as a measure of MPTP opening. In isolated mitochondria, 100 micromol/L atractyloside attenuated the decrease of mitochondrial swelling induced by 30 micromol/L diazoxide or cyclosporin A (0.5 or 1 micromol/L). Furthermore, 100 micromol/L diazoxide or 1 micromol/L cyclosporin A both attenuated the fluorescence intensity in isolated mitochondria loaded with rhod-2 acetoxymethylester, and 100 micromol/L atractyloside abolished the effects of diazoxide and cyclosporin A. These results suggest that activation of mitoKATP protects the brain against injury, and this is probably mediated by attenuating mitochondrial Ca2+ overload and thus inhibiting MPTP opening during brain ischemia and reperfusion. PMID- 16678348 TI - Characterization of a single molecular QCA cell by Q-control enhanced amplitude modulation atomic force microscopy. AB - Major technical challenges for reduction of device sizes for computation and memory are the interconnection and power dissipation problems. Molecular quantum dot cellular automata (QCA) have been proposed as solutions to this problem. Silicon phthalocyanine (SiPc) is a possible candidate for a molecular QCA element. Therefore, it is important to develop an in situ observation technique to visualize individual SiPc molecules. We report here the first image of a single SiPc dimer in air by using quality factor control (Q-control) enhanced amplitude modulation atomic force microscopy (AFM) and an investigation of the interaction forces between the tip and SiPc dimer. The AFM was operated at 0% relative humidity in an ultrapure nitrogen environment either with or without Q control. Theoretical simulations using the point-mass description of the AFM, demonstrated that Q-enhancement reduced the force exerted from the tip to the sample surface. Our results, consistent with theory, demonstrated that the image force was reduced and a greater height and a larger size were measured. The advantages of this method can be extended to the AFM observation of other "soft" structures, and these results can be useful for a wider community. PMID- 16678349 TI - Estimation of abomasum strongyle nematode infections in sheep at necropsy: tentative proposals for a simplified technique. AB - Several necropsy techniques are available for estimating the abundance of gastro intestinal nematodes in abomasum of ruminants. Standardization of techniques is needed to allow accurate comparisons between laboratories. Here we propose a standardized technique for estimating the abundance of worms. We intend to compare the worms' number estimations in lambs and ewes based on contents and washings, to determine the uniformity of worm counts in aliquots, and to estimate the total worm number from washings. The digesta (or "contents") and the washings of the abomasum are treated separately. The worms of each subsample are diluted with water and the total number of worms is estimated on a small volume (aliquots) of these subsamples. The use of aliquots assumes that the worms are uniformly distributed in the whole volume of each subsample. We first confirmed that the use of aliquots is appropriate in most cases. We then show that the use of the washings alone allows a faster and a suitable estimation of the total worm burden for all strongyle species of the abomasum in both ewes and lambs. The evaluation of our necropsy procedure is a first step to a standardized technique which should be improved by validation in other laboratories. PMID- 16678350 TI - Prophylactic colony-stimulating factors in children receiving myelosuppressive chemotherapy: a meta-analysis of randomized controlled trials. AB - BACKGROUND: The colony-stimulating factors (CSFs) are widely utilized to prevent neutropenic complications in both adults and children, but randomized controlled trials in the pediatric setting have reported varied results. A systematic review of the literature and meta-analysis were conducted to definitively assess the impact of prophylactic CSFs on the risk of febrile neutropenia (FN) in pediatric oncology patients. METHODS: MEDLINE was searched and references hand-searched through July 2004 for randomized controlled trials of prophylactic G-CSF or GM CSF in pediatric oncology patients. Objectives, outcomes, and quality of the 16 included studies were extracted by two reviewers. Weighted summary estimates of relative risks (RR) were calculated for FN and documented infection (DI). Mean differences in hospitalization, antibiotic use, and duration of neutropenia were calculated. RESULTS: FN occurred in 68% of 400 controls and 59% of 404 CSF patients. The estimated RR was 0.88 [0.81-0.97; (P=0.01)] favoring the CSFs for leukemia and high grade lymphoma studies and 0.71 [0.51-0.97; (P=0.03)] for solid tumor studies. DI occurred in 25% of controls and 20% of CSF patients for an estimated RR of 0.80 [0.61-1.06; (P=0.12)]. The mean decrease in duration of neutropenia was 3.5 days [2.2-4.7; (P<0.0001)]. Mean decreases favoring CSF use were also observed for hospital stay of 1.7 days [0.9-2.5 (P<0.01)] and antibiotic use of 2.0 days [0.4-3.6; P=0.02]. CONCLUSIONS: Prophylactic CSFs significantly decrease the incidence of FN and the durations of severe neutropenia, hospitalization, and antibiotic use in pediatric cancer patients, but they do not significantly decrease documented infections. PMID- 16678351 TI - Chronic exposure to ammonia alters basal and NMDA-induced phosphorylation of NMDA receptor-subunit NR1. AB - Hyperammonemia is responsible for many of the neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of brain protein kinase C substrates and impairs N-methyl-d-aspartate (NMDA) receptor-associated signal transduction. The aim of this work was to analyze, in rat cerebellar neurons in culture, the effects of ammonia exposure on NMDA receptor phosphorylation, MK801 binding and surface expression. Ammonia reduces MK801 binding to NMDA receptors and the surface expression of the NR1 and NR2A subunits. As phosphorylation of serines in the NR1 C1 cassette has been implied in receptor trafficking, we assessed whether hyperammonemia alters phosphorylation of these serines. Basal phosphorylation of serines 890, 896 and 897 was increased in neurons exposed to ammonia, while NMDA induced phosphorylation of S896 and S897 was reduced. Exposure to ammonia also increased basal phosphorylation of Akt but reduced NMDA and BDNF stimulation of Akt phosphorylation. These results suggest that alterations in receptor surface expression and possibly the phosphorylation state of the NR1 subunit of NMDA receptors may contribute to the impairment by ammonia of signal transduction pathways modulated by NMDA receptors. PMID- 16678352 TI - Development of a comprehensive measure to assess clinical issues in dual diagnosis patients: The Substance Use Event Survey for Severe Mental Illness. AB - This paper provides a description of The Substance Use Event Survey for Severe Mental Illness (SUESS), a brief (20-30 min) interview that assesses clinical issues and domains that are relevant patients with substance use disorders and severe mental illness. First, we discuss the need for a new clinical measure for dual diagnosis patients, as well as our process of creating domains and items and developing the content of the assessment. Second, we provide a first look at the performance of the SUESS in a large sample of dually diagnosed patients from several large scale studies, including how patients responded to the instrument and their ability to complete the items. Third, we present initial reliability data on the SUESS. Finally, we include some initial validity data, including comparison of the self-report of substance use questions to urinalysis results, and verification of the service use information from medical record review. The SUESS appears to be a useful assessment that is tolerated and understood by dual diagnosis patients, and shows good preliminary reliability and validity. PMID- 16678353 TI - The adaptation of TSH secretion to autonomy in non-toxic goiter may be based-on active regulation of set-point and sensitivity of central TSH-receptors, perhaps by the microRNA (MIR) gene. AB - The hypothalamic-pituitary-thyroid (HPT) feedback system is one of the most complex regulatory systems and it has intrigued researchers for more than 50 years. One of the unsolved problems of this regulation in humans is the hypothalamic and pituitary adaption to autonomous function of the thyroid gland in non-toxic goiter (NTG). A new hypothesis, that TSH secretion is actively regulated in NTG in response to changes in thyroid autonomy in order to keep the patient euthyroid, was discussed in the present study on basis of previous experimental data in NTG patients with different degree of autonomous function. The patients were submaximally suppressed for one month with a fixed daily dose of T3. Group data suggested an inverse correlation between TSH suppression and suppression of thyroid function. Group data also suggested that TSH suppression was significantly correlated to basal TSH. This means that a NTG patient with a low normal TSH in blood before suppression (basal TSH) and a high degree of autonomous function may exhibit a relatively high TSH suppression. This ensures a minimal stimulation of the thyroid gland thereby protecting the gland from developing hyperthyroidism. A patient with a high normal TSH in blood before suppression (basal TSH) and a low degree of autonomous function, may exhibit a relatively low TSH suppression, indicating a high stimulation of the thyroid gland thereby protecting the patient from developing hypothyroidism. It is hypothesized that this adaption of TSH secretion to autonomy in NTG patients may be based on active regulation of both the set- point of the central TSH-receptors and the sensitivity of these receptors as well. Such an active regulation of the HPT axis may be controlled by one of the newly found post-transcriptional genes called MIR (microRNA). PMID- 16678354 TI - Cancer and chronic tissue injury: abnormal repair tissue or functional repair tissue? PMID- 16678355 TI - Where has all the botox gone? (how) will we ever learn... using monoclonal antibodies to track botulinum toxin. AB - Botulinum toxin (BTX) is an important therapeutic tool in the treatment of overactive skeletal and smooth muscles, as well as hypersecretory and painful disorders. Despite advances in our understanding of how BTX works, much remains to be elucidated, such as how BTX ameliorates pain, how it produces weakness remote from the site of injection and the fate of the heavy and light chain components of the BTX molecule following endocytosis into the presynaptic membrane. BTX, conjugated to radionuclides, allows investigators to track the molecule both in vitro and in vivo. However, altering the BTX molecule may cause structural changes or pharmacokinetic and pharmacodynamic alterations, and disrupt its normal action. We propose instead to bind the biomarkers (appropriate dyes, radionuclides or MRI contrast agents) to monoclonal antibodies directed against either heavy or light chain components of BTX, thus allowing administration of native (i.e. unaltered) BTX. PMID- 16678356 TI - Karyomegalic tubulointerstitial nephritis--a case report. AB - Karyomegalic tubulointerstitial nephritis is a rare disease of uncertain etiology, which leads to progressive renal failure. Here, we report on a 39-year old patient who presented with asymptomatic progressive decline of renal function. Two sequential renal biopsies demonstrated chronic tubulointerstitial nephritis with bizarre and dramatic enlargement of proximal tubule epithelial cell nuclei - the hallmark of karyomegalic nephritis. Clinical and pathologic findings of this case are discussed in light of the available literature. The morphologic changes in this disease are highly characteristic if the pathologist is aware of this entity. PMID- 16678357 TI - In patients with Helicobacter pylori gastritis and functional dyspepsia, a biopsy from the incisura angularis provides useful diagnostic information. AB - The aim of this study was to assess whether the taking of an additional biopsy from the incisura angularis increases the chance of detecting maximal degrees of atrophy and intestinal metaplasia (IM) in patients with Helicobacter pylori gastritis and functional dyspepsia. At entry into a randomised trial, biopsies were taken from 328 patients (mean age 48 years), two from both the gastric antrum and corpus, and one from the incisura angularis, and comparative grading of gastritis variables was carried out. Biopsy material from the gastric antrum, corpus, and the incisura angularis revealed no notable differences in atrophy or an incidence of IM and mucosa-associated lymphatic tissue. However, when the incisura biopsies were classified histologically, 58% contained antral mucosa (AM), 18% corpus mucosa (CM), and 24% intermediate zone mucosa. AM at the incisura was associated with considerably more severe gastritis in both the incisura and antrum (14% atrophy, 20% IM) than in CM of incisura (2% atrophy, 6% IM). Corpus atrophy and IM were rare in the AM group and absent from the CM group. Incisura angularis biopsy in patients with H. pylori gastritis and functional dyspepsia does give additional information regarding the severity of gastritis expected in the corpus and antrum. Antral-type mucosa in the incisura angularis region seems to indicate an increased risk for the development of atrophy and/or IM. PMID- 16678359 TI - [Aptamers: selection and scope of applications]. AB - Aptamers are short oligonucleotides selected from large combinatorial pools of sequences for their capacity to bind to many different targets ranging from small molecules (amino acids, antibiotics...) to proteins or nucleic acid structures. Aptamers present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers have been shown in many cases to display an inhibitory activity against their targets. Many aptamers are now being developed against biomedical relevant targets, and one aptamer that inhibits the human VEGF165 already received approval for the treatment of age related macular degeneration. Here we discuss the principles and the practical way of selecting aptamers (SELEX technology) as well as the structural basis for their performance as ligands. A wide scope of applications is described - aptamers have been used as tools for studying nucleic acids/proteins interactions, detecting, purifying or imaging target molecules, regulating gene expression - and includes recent developments of aptamers for therapy and diagnosis. PMID- 16678358 TI - [Detection by flow cytometry of T cell subsets secreting IL-2 and UFNy(Gamma): optimalisation for the technic and the establishment of reference values]. AB - A dysregulation in Th1/Th2 balance has been described for different pathological situations. Knowing the cytokine profile in a given pathology could assist in understanding the disease mechanism and in choosing an immune intervention most effective for the management of this condition. In this work, the production of two Th1 cytokines, IL-2 and IFN- gamma, was analyzed for different T-cell subsets from 20 normal subjects (mean age 33.5 years) and reference values were defined using the flow cytometric analyses. The optimum operating conditions were set as following: mononuclear cells were stimulated with PMA (20 ng/ml) and ionomycin (1 uM) for 6 h in the presence of brefeldin A (10 ug/ml). Cells were fixed with 4% paraformaldehyde and then dually stained, with anti-CD3 or anti-CD4 or anti-CD8 for the membrane and with anticytokine antibody for the intracytoplasma after being permeabilized with 0.5% saponine solution. The frequency determination of cells that produce IL-2 or IFN-gamma revealed large 95% confidence intervals: (CD3-IL-2: 4.60-10.67%, CD8-IL-2: 1.47-23%, CD3-IFN-gamma: 2,97-32,49%, CD4-IFN gamma: 2.83-21%, CD8-IFN-gamma: 4.60-35.28%). CD4+ lymphocytes produce the majority of IL-2 (85 vs 13% for CD8+). For IFN-gamma, the situation is more balanced, but the CD4+ lymphocytes remain the predominant producer cells (63 vs. 41%). PMID- 16678360 TI - Behavioural comparison of human-animal (dog) and human-robot (AIBO) interactions. AB - The behavioural analysis of human-robot interactions can help in developing socially interactive robots. The current study analyzes human-robot interaction with Theme software and the corresponding pattern detection algorithm. The method is based on the analysis of the temporal structure of the interactions by detecting T-patterns in the behaviour. We have compared humans' (children and adults) play behaviour interacting either with an AIBO or a living dog puppy. The analysis based on measuring latencies and frequencies of behavioural units suggested limited differences, e.g. the latency of humans touching the dog/AIBO was similar. In addition other differences could be accounted for by the limited abilities of the robot to interact with objects. Although the number of interactive T-patterns did not significantly differ among the groups but the partner's type (whether humans were playing with dog or AIBO) had a significant effect on the structure of the patterns. Both children and adults terminated T patterns more frequently when playing with AIBO than when playing with the dog puppy, which suggest that the robot has a limited ability to engage in temporally structured behavioural interactions with humans. As other human studies suggest that the temporal complexity of the interaction is good measure of the partner's attitude, we suggest that more attention should be paid in the future to the robots' ability to engage in cooperative interaction with humans. PMID- 16678361 TI - Can humans discriminate between dogs on the base of the acoustic parameters of barks? AB - In this study we tested the often suggested claim that people are able to recognize their dogs by their barks. Earlier studies in other species indicated that reliable discrimination between individuals cannot be made by listening to chaotically noisy vocalizations. As barking is typically such a chaotic noisy vocalization, we have hypothesized that reliable discrimination between individuals is not possible by listening to barks. In this study, playback experiments were conducted to explore (1) how accurately humans discriminate between dogs by hearing only their barks, (2) the impact of the eliciting context of calls on these discrimination performances, and (3) how much such discrimination depends on acoustic parameters (tonality and frequency of barks, and the intervals between the individual barks). Our findings were consistent with the previous studies: human performances did not pass the empirical threshold of reliable discrimination in most cases. But a significant effect of tonality was found: discrimination between individuals was more successful when listeners were listening to low harmonic-to-noise ratio (HNR) barks. The contexts in which barks were recorded affected significantly the listeners' performances: if the dog barked at a stranger, listeners were able to discriminate the vocalizations better than if they were listening to sounds recorded when the dog was separated from its owner. It is rendered probable that the bark might be a more efficient communication system between humans and dogs for communicating the motivational state of an animal than for discrimination among strange individuals. PMID- 16678362 TI - Detection of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen. AB - The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes. PMID- 16678363 TI - Evaluation of Erysipelothrix rhusiopathiae vaccines in pigs by intradermal challenge and immune responses. AB - In a vaccine trial, pigs were challenged intradermally with eight E. rhusiopathiae strains of serovars 1a, 1b or 2 given concurrently. The strains were derived from six herds affected with vaccine breakdowns in 1997-1999, one herd without vaccine breakdown and a serovar 2 reference strain. Responses to two commercial bacterins (one implicated in the vaccine breakdowns), and two experimental bacterins (based on field isolates from affected herds) showed distinct differences in protection, particularly in clinical responses measured at 72 h. Less protection was afforded against serovar 1 challenge by the vaccine implicated in the vaccine breakdowns. Antibody and cell-mediated immune (CMI) responses were significantly different between treatments, and highlighted a similar post-vaccinal antibody response was produced against serovar 2 lysate by all vaccines, but only those providing significant protection against serovar 1 [corrected] produced significantly elevated antiserovar I lysate [corrected] antibodies. Vaccination in general significantly reduced CMI responses to the mitogens concanavalin A and phytohaemagglutinin. This experimental pig challenge system was readily able to confirm suboptimal performance of a commercial bacterin that had passed potency tests in mice but was associated with vaccine failure in commercial herds. This vaccine was also the most immunosuppressive to CMI responses associated with E. rhusiopathiae-specific and non-specific stimulation. The best vaccine response was associated with the highest mean serovar 1 antibody response and the highest CMI response (by lymphoproliferation assay) to serovar 2. PMID- 16678364 TI - Acute centralization of blood flow in compromised human fetuses evoked by uterine contractions. AB - BACKGROUND: During fetal hypoxia blood is redistributed to the brain ('brain sparing'). Sequential changes of the cerebral and placental circulation in parallel in comparisons between basal conditions and acute hypoxic stress have not yet been thoroughly studied in human fetuses. AIM: To explore acute fetal middle cerebral artery (MCA) circulatory changes relative to umbilical artery (UA) blood flow in a clinical experimental model with hypoxic stress provoked by uterine contractions during an oxytocin challenge test (OCT). STUDY DESIGN: Prospective comparative between imminently compromised (OCT positive) and un compromised (OCT negative) fetuses. SUBJECTS AND METHODS: 82 term pregnancies suspected of intrauterine growth restriction were exposed to simultaneous electronic fetal heart rate monitoring and Doppler recordings of pulsatility index (PI) in the UA and MCA during basal conditions and during uterine contractions and relaxations at an OCT. OUTCOME MEASURES: Sequential changes of UA and MCA PI, OCT positive vs. negative cases. Nonparametric statistics with a P < 0.05 considered significant. RESULTS: The UA PI was significantly higher in OCT positive cases (N = 10) compared with OCT negative cases (N = 72) during uterine contractions and relaxations, but not during basal measurements. During contractions and relaxations the MCA PI decreased significantly in both groups (brain-sparing), but significantly more in OCT positive cases. CONCLUSIONS: During acute hypoxic stress, changes towards a centralization of blood flow to the brain develop in imminently compromised (OCT positive) fetuses at an expense of the umbilicoplacental blood flow, and the brain-sparing flow is more pronounced than in un-compromised (OCT negative) fetuses. PMID- 16678365 TI - Updating the EMAS 2004/2005 clinical recommendations on postmenopausal therapy following the recent publications: WHI and Nurses' Health Study. AB - This new statement from EMAS presents the findings reported in recent publications from both WHI and the Nurses' Health Study to consider if these findings require the EMAS position to be modified. In general the reports do not necessitate a revision of the current EMAS advice but do provide further insight into the ongoing controversy around the possibility that estrogen (E) or estrogen progestogen (E-P) use in younger postmenopausal women may supply cardiovascular benefit in the younger group. PMID- 16678366 TI - Nifedipine solid dispersion in microparticles of ammonio methacrylate copolymer and ethylcellulose binary blend for controlled drug delivery. Effect of drug loading on release kinetics. AB - In order to elucidate the controlled-release mechanism of a poorly water-soluble drug from microparticles of ammonio methacrylate copolymer and ethylcellulose binary blend prepared by a phase-separation method, nifedipine-loaded microparticles with different levels of drug loading were evaluated by micromeritic properties, drug physical state, matrix internal structure, drug dissolution, and release modeling. Drug release study indicated that nifedipine release from the microparticles followed the Fickian diffusion mechanism, which supported the study hypothesis that as a result of formation of a nifedipine molecular dispersion, nifedipine dissolution inside the matrix was no longer the rate-limiting step for drug release, and the drug diffusion in matrix became the slowest step instead. Moreover, study results indicated that even though drug loading did not significantly affect the microparticle size distribution and morphology, nifedipine release rate from those microparticles was more or less influenced by the level of drug loading, depending on matrix formulation. At lower levels of drug loading, nifedipine release was well described by the Baker and Lonsdale's matrix diffusion model for microspheres containing dissolved drug and nifedipine had a plasticizing effect on the polymers that caused an increase in drug effective diffusion coefficient with increasing drug loading. However, at higher levels of drug loading, probably due to formation of solid nifedipine domains in microparticles, a change in the release kinetics was observed. PMID- 16678367 TI - alpha-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. AB - Extracts of six selected Malaysian plants with a reputation of usefulness in treating diabetes were examined for alpha-amylase inhibition using an in vitro model. Inhibitory activity studied by two different protocols (with and without pre-incubation) showed that Phyllanthus amarus hexane extract had alpha-amylase inhibitory properties. Hexane and dichloromethane extracts of Anacardium occidentale, Lagerstroemia speciosa, Averrhoa bilimbiPithecellobium jiringa and Parkia speciosa were not active when tested without pre-incubation. Extraction and fractionation of Phyllanthus amarus hexane extract led to the isolation of dotriacontanyl docosanoate, triacontanol and a mixture of oleanolic acid and ursolic acid. Dotriacontanyl docosanoate and the mixture of oleanolic acid and ursolic acid are reported from this plant species for the first time. All compounds were tested in the alpha-amylase inhibition assay and the results revealed that the oleanolic acid and ursolic acid (2:1) mixture was a potent alpha-amylase inhibitor with IC(50)=2.01 microg/ml (4.41 microM) and that it contributes significantly to the alpha-amylase inhibition activity of the extract. Three pure pentacyclic triterpenoids, oleanolic acid, ursolic acid and lupeol were shown to inhibit alpha-amylase. PMID- 16678368 TI - Leads from Indian medicinal plants with hypoglycemic potentials. AB - Diabetes mellitus is caused due to deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. It is a global problem and number of those affected is increasing day by day. The plants provide a potential source of hypoglycemic drugs because many plants and plant derived compounds have been used in the treatment of diabetes. Several medicinal plants have found potential use as hypoglycemic in the Indian system of medicines, including ayurveda. Many Indian plants have been investigated for their beneficial use in different types of diabetes and reports occur in numerous scientific journals. This article aims to provide a comprehensive review on various plant species from Indian biosphere and their constituents, which have been shown to display potent hypoglycemic activity. The use of herbs as hypoglycemic is a major avenue in Indian perspectives particularly for treating diabetes, which require to be explored more effectively as there are so many literatures available on these aspects. This paper describes the chemistry, activity and usage of the constituents isolated from these plants from India for the treatment of diabetes. PMID- 16678369 TI - Search for antibacterial and antifungal agents from selected Indian medicinal plants. AB - A series of 61 Indian medicinal plants belonging to 33 different families used in various infectious disorders, were screened for their antimicrobial properties. Screening was carried out at 1000 and 500 microg/ml concentrations by agar dilution method against Bacillus cereus var mycoides, Bacillus pumilus, Bacillus subtilis, Bordetella bronchiseptica, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus faecalis, Candida albicans, Aspergillus niger and Saccharomyces cerevisiae. Twenty-eight plant extracts showed activity against at least one of the test organisms used in the screening. On the basis of the results obtained, we conclude that the crude extracts of Dorema ammoniacum, Sphaeranthus indicus, Dracaena cinnabari, Mallotus philippinensis, Jatropha gossypifolia, Aristolochia indica, Lantana camara, Nardostachys jatamansi, Randia dumetorum and Cassia fistula exhibited significant antimicrobial activity and properties that support folkloric use in the treatment of some diseases as broad spectrum antimicrobial agents. This probably explains the use of these plants by the indigenous people against a number of infections. PMID- 16678370 TI - Allele frequencies of 15 short tandem repeats (STRs) in three Egyptian populations of different ethnic groups. AB - DNA typing of 15 short tandem repeat (STR) loci included in the AmpFlSTR Identifiler PCR amplification kit (Applied Biosystems), was carried out in three Egyptian populations of different ethnic groups: the Berbers from the Siwa oasis (in the North-Western Egyptian desert), the Muslims and the Copts from Adaima (Upper Egypt). A total of 297 individuals were typed. After Bonferroni's correction, no deviations from the Hardy-Weinberg equilibrium were observed for all samples at the 15 STR loci. All loci are highly polymorphic and population differentiation tests showed that 7, 10 and 8 out of 15 loci have significant differences between the Berbers and the Muslim samples, between the Berbers and the Copts, and between the two samples from Adaima, respectively. Comparative analyses between our population data and other geographically related populations gathered from the literature were performed. PMID- 16678372 TI - Age-related changes in goblet cell density of rat small bowel. Comment on "Age related changes in liver structure and function: Implications for disease?" by DL Schmucker. PMID- 16678373 TI - Detection of hepatic VX2 carcinomas with ferucarbotran-enhanced magnetic resonance imaging in rabbits: Comparison of nine pulse sequences. AB - OBJECTIVE: To compare the diagnostic performance of a variety of magnetic resonance imaging (MRI) sequences, in order to identify the most effective ferucarbotran-enhanced sequence for the detection of multiple small hepatic VX2 carcinomas in rabbits. METHODS: Fifteen rabbits with experimentally induced 135 VX2 carcinomas in the liver underwent ferucarbotran-enhanced MRI using the following nine pulse sequences: a fat-suppressed fast spin-echo (FSE) sequence with two echo times (TE) (proton density- and T2-weighted images), four different T2*-weighted fast multiplanar GRASS (gradient-recalled acquisition in the steady state) (FMPGR) with the combination of three TEs (9, 12, 15 ms, respectively) and two flip angles (20 degrees , 80 degrees, respectively), T2*-weighted fast multiplanar spoiled GRASS (FMPSPGR), T1-weighted FMPSPGR, and dynamic T1-weighted FMPSPGR. All images were reviewed by three radiologists with quantitative and qualitative analysis. RESULTS: Tumor-to-liver contrast-to-noise ratio of the proton density-weighted FSE sequence was significantly higher than those of the others (p<0.05). The lesion conspicuities of proton density- and T2-weighted FSE and T2*-weighted FMPGR (TE/flip angle, 9/20 degrees and 12/20 degrees ) images were better and the image artifacts of T2*-weighted FMPGR (TE/flip angle, 15/20 degrees and 12/80 degrees ) and T2*-weighted FMPSPGR images were more prominent than those of the others (p<0.05). The lesion detection in T2- and proton density weighted FSE and T2*-weighted FMPGR (TE/flip angle, 12/20 degrees ) images were superior to those of the others and for the detection of very small hepatic tumors of less than 5 mm, the sensitivities of these sequences were less than 30%. CONCLUSION: Ferucarbotran-enhanced T2- and proton density-weighted FSE and T2*-weighed FMPGR (TE/flip angle, 12/20 degrees ) images were found to be the most effective pulse sequences for the detection of multiple small hepatic VX2 carcinomas but these sequences were limited in the detection of very small hepatic tumors of less than 5 mm in size. PMID- 16678374 TI - Polymorphism, pseudopolymorphism, and amorphism of peracetylated alpha-, beta-, and gamma-cyclodextrins. AB - Polymorphism, pseudopolymorphism, and amorphism of hexakis(2,3,6-tri-O-acetyl) alpha-cyclodextrin (TAalphaCyD), heptakis(2,3,6-tri-O-acetyl)-beta-cyclodextrin (TAbetaCyD), and octakis(2,3,6-tri-O-acetyl)-gamma-cyclodextrin (TAgammaCyD) were investigated using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), powder X-ray diffractometry (XRPD), Fourier transform infrared spectroscopy (FTIR) and optical microscopy. An anhydrous and a bi-hydrate crystalline forms of TAalphaCyD, two monotropic anhydrous polymorphs and three pseudopolymorphs (i.e. methanolate, hydrate, and isopropanolate-hydrate) of TAbetaCyD, as well as two monotropic anhydrous polymorphs and isostructural pseudopolymorphs (e.g. hydrate and isopropanolate-hydrate) of TAgammaCyD were isolated and characterized. The amorphous forms of each TACyD were also obtained. Thermal data for desolvation of TAalphaCyD.2H2O and TAbetaCyD.CH3OH were reconciled with their crystal packing features. Melting temperatures and enthalpies of the crystalline forms of each TACyD can be referred to for possible solid-state interactions with drugs. PMID- 16678375 TI - Theoretical and experimental study of a praziquantel and beta-cyclodextrin inclusion complex using molecular mechanic calculations and H1-nuclear magnetic resonance. AB - Praziquantel (PZQ) is a broadly effective anthelminthic drug available for human and veterinary use, being the drug of choice for the treatment of all forms of schistosomiasis. Nevertheless, large doses are required in order to achieve adequate concentrations at the target site due to the poor solubility of PZQ and its significant first pass metabolism. To improve it, avoiding efficiency loss, we have designed a controlled-release system, in which PZQ was encapsulated in beta-cyclodextrin (beta-CD). The inclusion complexes between PZQ/beta-CD were studied at two different stoichiometries 1:1 and 1:2, through experimental and theoretical analysis. Molecular modeling calculations were used to foresee the better stoichiometry of the complex formed as well as the possible orientations of PZQ inside the beta-CD cavity. The complexes prepared were analyzed through H1 two-dimensional nuclear magnetic resonance (H1 2D-NMR) experiments, which provide (evidences) for the 1:1 complexation of PZQ/beta-CD. H1 2D-NMR also revealed details of PZQ/beta-CD molecular interaction, in which the isoquinoline ring of praziquantel is located inside the beta-CD cavity. Finally, phase-solubility diagrams revealed a five-fold increase in praziquantel water solubility upon addition of increasing beta-CD concentrations up to 16 mM, corresponding to the solubility of beta-CD itself. The solubilization profile is consistent with 1:1 stoichiometry of the PZQ/beta-CD complex while the solubilization effect will certainly increase the pharmacological activity of praziquantel. PMID- 16678376 TI - Severe osteoarticular infection associated with Panton-Valentine leukocidin producing Staphylococcus aureus. AB - We report here a case of osteoarticular infection associated with Panton Valentine leukocidin-producing Staphylococcus aureus. The 3-year-old child presented osteoarthritis associated with toxic shock syndrome, which was complicated by thrombosis, pneumonia, and diaphysitis, despite appropriate antibiotic treatment. Osteitis associated with toxinogenic S. aureus is rarely described. This case highlights the need of early and aggressive surgical treatment, in addition to appropriate antimicrobial therapy. PMID- 16678377 TI - The rapid evaluation of bacterial growth and antibiotic susceptibility in blood cultures by selected ion flow tube mass spectrometry. AB - We have measured the production of volatile organic compounds (VOCs) by selected ion flow tube mass spectrometry (SIFT-MS) in the headspaces of conventional BacT/ALERT blood culture bottles (Biomerieux, Durham, NC) artificially infected with 5 bacterial strains. Uninfected blood samples were inoculated with Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Neisseria meningitidis. Growth and species identification were determined at 6 h by measuring a panel of 9 VOC products. Two species, E. coli and S. aureus, were also incubated in the presence of gentamicin or flucloxacillin, respectively, above or below their demonstrated MIC. The concentration-dependent antibiotic susceptibility of both strains was demonstrated by the inhibition of VOC production at 22 h (P < .05). These results suggest incorporating SIFT-MS detection of microbial VOCs as a sensitive method for bacterial detection, identification, and determination of antibiotic susceptibility in a conventional blood culture system. PMID- 16678378 TI - Sensitivity and specificity of nested and real-time PCR for the detection of Pneumocystis jiroveci in clinical specimens. AB - A polymerase chain reaction (PCR)-based test for Pneumocystis jiroveci (formerly Pneumocystis carinii f. sp. hominis) might be an alternative to histologic diagnoses of P. jiroveci pneumonia (PCP). However, previously developed nested PCR methods tend to have low specificities (high false-positive rates). In this study, nested and quantitative real-time PCR methods for the amplification of the P. jiroveci DHPS (dihydropteroate synthase) gene were evaluated in a variety of stored clinical samples from Spain, South Africa, and Brazil. The sensitivities of both assays were high, ranging from 62.5% to 100% depending on the type of specimen. In a subset of 71 microscopically confirmed PCP cases and 70 negative cases, the sensitivities and specificities were 94% and 81% for nested PCR and 94% and 96% for real-time PCR, respectively. Real-time PCR has a statistically significantly better specificity than nested PCR (P = .015) and is likely to generate fewer false positives. PMID- 16678379 TI - Epidemiologic surveillance of Clostridium difficile diarrhea in a freestanding pediatric hospital and a pediatric hospital at a university medical center. AB - To describe the epidemiology of Clostridium difficile in children, we cultured stool specimens from patients at the Children's Hospital Central California, Madera, CA (CHCC, n = 676) and at the University of California Davis Medical Center Pediatric Hospital, Sacramento, CA (UCDMC-PH, n = 301) for C. difficile, and toxins A and B genes and strain identity of the isolates were determined by polymerase chain reaction assays. A higher percentage of patients from UCDMC-PH were culture positive (148/301, 49%) and colonized with toxigenic strains (45/301, 15%) compared with CHCC (colonized = 178/676, 26%; toxigenic = 96/676, 14%, P < or = .001). Multiple logistic regression analysis showed decreased colonization with inpatient status (odds ratio [OR] = 0.64; 95% confidence interval [CI] = 0.46, 0.89; P = .007) and use of H-2 antagonists (OR = 0.55; 95% CI = 0.36, 0.84; P = .006), whereas underlying conditions (colonization: OR = 1.42; 95% CI = 1.02, 1.96; P = .04; toxin positive: OR = 1.60; 95% CI = 1.04, 2.44; P = .03) and exposure to > or =2 antiinfectives (colonization: OR = 1.56; 95% CI = 1.10, 2.20; P = .01; toxin positive: OR = 1.71; 95% CI = 1.10, 2.66; P = .02) increased colonization. Most isolates appear to be community acquired, although molecular analysis suggests some nosocomial transmission at UCDMC-PH. These data suggest that the epidemiology of colonization with C. difficile in children is different than previously reported. PMID- 16678380 TI - Phase II studies: International registry of colorectal carcinomatosis. AB - Peritoneal carcinomatosis (PC) is a common manifestation of colorectal cancer and has traditionally been regarded as a terminal disease with a short median survival. Over the last decade, a new local-regional therapeutic approach combining cytoreductive surgery with perioperative intraperitoneal chemotherapy has evolved and promising survival results were reported in large phase II studies. A retrospective multicentric study of 506 patients from 28 institutions was performed to evaluate the international experience with this combined treatment and to identify the principal prognostic indicators. All patients had cytoreductive surgery and perioperative intraperitoneal chemotherapy (intraperitoneal chemohyperthermia and/or immediate postoperative intraperitoneal chemotherapy) within the 7 days following surgery. The morbidity and mortality rates were 22.9% and 4%, respectively. Patients in whom cytoreductive surgery was complete had a median survival of 32.4 months compared to 8.4 months for patients in whom complete cytoreductive surgery was not possible (p < 0.001). Positive independent prognostic indicators by multivariate analysis were complete cytoreduction, treatment by a second procedure, limited extent of PC, age less than 65 years, and use of adjuvant chemotherapy. The use of neoadjuvant chemotherapy, lymph node involvement, presence of liver metastasis and poor histological differentiation were negative independent prognostic indicators. The therapeutic approach combining cytoreductive surgery with perioperative intraperitoneal chemotherapy achieved long-term survival in carefully selected group of patients with PC from colorectal origin and offer a chance for cure or palliation in this condition. Further collaboration between peritoneal surface malignancy treatment centres are needed in order to standardize indications, intraperitoneal chemotherapy and peritonectomy techniques. PMID- 16678381 TI - Streptococcus uberis internalizes and persists in bovine mammary epithelial cells. AB - Streptococcus uberis is one of the most important emerging bovine mastitis pathogens and chronic persistent intramammary infections (IMI) are often described. To define the ability of S. uberis to persist intracellularly, studies on time-dependent internalization and survival of S. uberis strains in bovine mammary epithelial cells were conducted. Two S. uberis strains (UT366 and UT888) and a Staphylococcus aureus strain used as positive control, all isolated from cows with clinical mastitis were cocultured with bovine mammary epithelial cells (MAC-T) and persistent survival in host epithelial cells for extended periods (120 h) studied. Of S. uberis strains tested, UT366 showed highest internalization values at 60 min of incubation whereas at 8 h of incubation the corresponding values for UT888 were the highest. Of both strains of S. uberis tested, UT366 seems to internalize bovine mammary cells more efficiently initially, however, during the first 8 h, UT888 seems to survive intracellularly better than UT366. Results showed that both S. uberis strains could survive intracellularly up to 120 h without apparent loss of host cells viability. S. aureus internalized more efficiently than all strains tested and host cell death was observed after 72 h of incubation. These results indicate that S. uberis can survive within mammary epithelial cells for extended time without apparent loss of host cells viability. Intracellular persistence of S. uberis may be associated with the spread of the infection to deeper tissues and development of persistent IMI. PMID- 16678382 TI - Mycoplasma pneumoniae subtype-independent induction of proinflammatory cytokines in THP-1 cells. AB - Mycoplasma pneumoniae can be divided into two main subtypes depending on the amino acid sequences of the P1 adhesin and the P65 protein, both located in the attachment organelle. Differences between these subtypes in infectivity, virulence and interaction with host cells have not been extensively studied. Using ELISA to measure released protein and real-time PCR to quantify mRNA, we have demonstrated that both M. pneumoniae subtypes significantly increased tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) at comparable levels in THP-1 cells over a 72 h period of time. However, subtype 2 induced a statistically significant increase (P<0.001) in the release of interleukin-1beta at 24 h post-infection compared to subtype 1. These data provide evidence that the induction of proinflammatory cytokine gene and protein expression by M. pneumoniae is not dependent on the infecting subtype. PMID- 16678383 TI - In vitro cytotoxicity assessment of the biocidal agents sodium o-phenylphenol, sodium o-benzyl-p-chlorophenol, and sodium p-tertiary amylphenol using established fish cell lines. AB - The cytotoxicity of three biocidal agents frequently employed as active ingredients in phenolic-based disinfectants, were evaluated in three established fish cell lines (EPC, CHSE and RTG-2). Cell viability was assessed using two fluorescent indicator dyes, Alamar Blue for metabolism and neutral red for lysosomal activity. Total protein content was also quantified as a measure of cell detachment. In order to evaluate the sensitivity of the cell cultures, the results obtained were compared with toxicity data obtained from a previous study with the same three compounds and the in vivo lethality test with rainbow trout. Results from this study established that each of the three cell lines ranked the tested chemicals in the same order of toxicity as the in vivo test; however, the cell cultures were found to be an order of magnitude less sensitive than whole fish studies with the same compounds. The chemical sodium o-benzyl-p-chlorophenol was consistently ranked the most toxic of the tested compounds with each cell line and the endpoints employed. The rank order of toxicity was always sodium o benzyl-p-chlorophenol > sodium p-tertiary amylphenol > sodium o-phenylphenol. The EPC cells were found to be the most sensitive cell line tested based on Alamar Blue IC(50) data, and the Alamar Blue assay was consistently found to be the most sensitive endpoint of the three cytotoxicity assays employed. PMID- 16678384 TI - RETRACTED: Posttranscriptional processing of the ndhCKJ operon of barley chloroplasts: Cotranscription and coprocessing with the trnV gene. AB - This article has been retracted consistent with Elsevier Policy on Article Withdrawal. Please see . The Publisher apologizes for any inconvenience this may cause. PMID- 16678385 TI - Application of transcriptional and biological network analyses in mouse germ-cell transcriptomes. AB - Serial analysis of gene expression (SAGE) provides a global analysis platform for profiling mRNA populations present in cells of interest without the constraint of gene selection and the ambiguous nature of data obtained. However, most of the reports on SAGE and germ cell development are limited to descriptive analyses. Here, we report a series of bioinformatic analyses using recently published SAGE data on the transcriptome of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd). Tags with a total count of > or =20 in three SAGE libraries were examined. Our aim was to identify and discover potential transcriptional regulators and pathways involved at different stages of spermatogenesis. Unsupervised hierarchical clustering based on tag expression and Gene Ontology analysis were applied to identify genes and biological processes overrepresented at a particular stage of development. The 5' cis-regulatory elements were examined for common regulators in different functional clusters. Potential biological networks were also constructed to reveal the link between the gene candidates. Biological pathways related to the three germ cell stages were constructed. A number of known transcription regulators in spermatogenesis, including NF-kappaB, SP1, AP-1, and EGR, were identified. Novel promoter elements such as the E box in Spga-specific genes, GATA in Spcy-specific genes, and GKLF in Sptd-specific genes were also observed. Taken together, our approach is reliable and provides a foundation for the generation of novel biological hypotheses for studying spermatogenesis. PMID- 16678386 TI - Protocol challenges for on-the-job voice dosimetry of teachers in the United States and Finland. AB - The occupational voice use of teachers has been an important research topic over the last 10 years. However, data collection on vocal loading in teachers at and away from the workplace poses significant challenges to the research protocol. The challenges include recruitment of subjects, voice data collection on the job, and subject compliance and retention. The present article offers recommendations for coping with these protocol difficulties. The recommendations are based on the authors' experiences with long-term, on-the-job voice dosimetry of large subject groups of teachers in the United States and Finland. PMID- 16678387 TI - Reliability of speaking and maximum voice range measures in screening for dysphonia. AB - Speech range profile (SRP) is a graphical display of frequency-intensity occurring interactions during functional speech activity. Few studies have suggested the potential clinical applications of SRP. However, these studies are limited to qualitative case comparisons and vocally healthy participants. The present study aimed to examine the effects of voice disorders on speaking and maximum voice ranges in a group of vocally untrained women. It also aimed to examine whether voice limit measures derived from SRP were as sensitive as those derived from voice range profile (VRP) in distinguishing dysphonic from healthy voices. Ninety dysphonic women with laryngeal pathologies and 35 women with normal voices, who served as controls, participated in this study. Each subject recorded a VRP for her physiological vocal limits. In addition, each subject read aloud the "North Wind and the Sun" passage to record SRP. All the recordings were captured and analyzed by Soundswell's computerized real-time phonetogram Phog 1.0 (Hitech Development AB, Taby, Sweden). The SRPs and the VRPs were compared between the two groups of subjects. Univariate analysis results demonstrated that individual SRP measures were less sensitive than the corresponding VRP measures in discriminating dysphonic from normal voices. However, stepwise logistic regression analyses revealed that the combination of only two SRP measures was almost as effective as a combination of three VRP measures in predicting the presence of dysphonia (overall prediction accuracy: 93.6% for SRP vs 96.0% for VRP). These results suggest that in a busy clinic where quick voice screening results are desirable, SRP can be an acceptable alternate procedure to VRP. PMID- 16678388 TI - Facial affect recognition: a mediator between cognitive and social functioning in psychosis? AB - BACKGROUND: Facial affect recognition has been implicated in the relationship between cognition and social functioning. This 1-year longitudinal study tested the hypothesis that facial affect recognition mediates the relationship between cognitive and social functioning. METHOD: Three groups were included: 50 first episode of psychosis (FE) subjects, 53 multi-episode schizophrenia subjects (ME) and 55 non-psychiatric controls (NPC). Subjects were assessed on two facial affect recognition tasks, a comprehensive cognitive battery and a measure of social functioning. FE subjects were assessed on admission to a comprehensive FE program and 1 year later. The ME and NPC groups had two assessments 1 year apart. RESULTS: Both the FE and ME subjects were clearly impaired relative to NPCs in cognition, social functioning and facial affect recognition. There were significant associations among facial affect recognition, cognition and social functioning in all three groups. For ME and FE subjects, but not NPCs, there was evidence that facial affect recognition did partially mediate the relationship between cognitive and social functioning. CONCLUSION: This study provides some first steps in understanding the complex relationship between cognition and outcome and has potential implications for the design of remediation strategies. PMID- 16678389 TI - Aripiprazole and the neuroleptic malignant syndrome. PMID- 16678390 TI - Neuropsychological deficits in nonsmokers with schizophrenia: effects of a nicotinic antagonist. AB - BACKGROUND: Biochemical, physiological and genetic evidence suggests dysregulation of central nicotinic acetylcholine receptor (nAChR) systems in schizophrenia, which may contribute to neuropsychological dysfunction and the high rates of smoking in this disorder. To evaluate the effects of nAChR blockade on neuropsychological performance in schizophrenia without the confounding effects of cigarette smoking, we compared neuropsychological performance in schizophrenia and healthy control nonsmokers after pre-treatment with the centrally-acting nAChR antagonist mecamylamine (MEC). METHODS: Using a within subjects, counterbalanced design, schizophrenia (n = 14) and control (n = 15) nonsmokers were pre-treated for 3 days with MEC (0.0, 5.0, and 10.0 mg/day). Subjects performed repeated neuropsychological assessments including visuospatial working memory (VSWM), Continuous Performance Test (CPT), Wisconsin Card Sorting Test (WCST), Word Serial Position Test (WSPT) and Stroop Color Word Test (SCWT) during three sequential test sessions per week over three test weeks. RESULTS: We found significant main effects of schizophrenia diagnosis on: VSWM 30 and 60 delays (p's < 0.01), CPT (% Hit Rate, Reaction Time, Variability Index; p < 0.01 for all outcomes), WCST (p < 0.01 for all outcomes) and Word Serial Position Test (p < 0.01). However, there were no main effects of repeated test administration (Session) or MEC dose on any of these outcomes, and no significant 3-way (DiagnosisxSessionxMEC dose) interactions. CONCLUSIONS: Our results suggest that there are a broad range of neuropsychological deficits in nonsmokers with schizophrenia. Furthermore, pretreatment with a centrally-acting nAChR antagonist did not alter neuropsychological performance in either nonsmoking patients with schizophrenia or controls. PMID- 16678391 TI - Protein concentration and adsorption time effects on fibrinogen adsorption at heparinized silica interfaces. AB - Heparin was modified with adipic dihydrazide and covalently linked to surface activated silica wafers. X-ray photoelectron spectroscopy was used at each stage of derivatization and showed that successful immobilization had taken place. Surfaces were imaged with atomic force microscopy to determine the uniformity of the heparin layer as well as its thickness. In situ ellipsometry was used to estimate layer thickness as well, and to study protein concentration and adsorption time effects on the adsorption and elution kinetics exhibited by human plasma fibrinogen. The adsorbed amount of fibrinogen increased with time and concentration on each type of surface. Under all experimental conditions, fibrinogen adsorbed at a lower rate and to a lower extent on heparinized as compared to unheparinized silica. In addition, buffer elution experiments showed that fibrinogen was less tightly bound to heparinized silica. In order to examine behavior relative to fibrinogen mobility at these interfaces, the sequential adsorption of fibrinogen was recorded. The difference in adsorption rates between the first and second adsorption cycles, evaluated at identical mass density, indicated that post-adsorptive molecular rearrangements had taken place. In general, higher solution concentration and longer adsorption time in the first adsorption step led to more rearrangement, and these history dependent effects were more pronounced on the heparinized silica. These rearrangements are suggested to involve clustering of adsorbed fibrinogen, in this way increasing the amount of unoccupied area at the interface. These rearrangements were presumably facilitated on the heparinized silica by enhanced lateral mobility of fibrinogen at this negatively charged, highly hydrophilic interface. PMID- 16678392 TI - Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines. AB - Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops (Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6-prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC50 values of 12.3+/-1.1 microM for DU145 and 13.2+/-1.1 microM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC50 of 18.4+/-1.2 microM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC50 of 33.5+/-1.0 and 43.1+/-1.2 microM, respectively), and IX gave comparable activities (IC50 of 45.2+/-1.1 microM for PC-3 and 47.4+/-1.1 microM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro. PMID- 16678394 TI - Is there a role for noninvasive ventilation in acute respiratory distress syndrome? A meta-analysis. AB - The role of noninvasive ventilation (NIV) in the management of acute respiratory distress syndrome (ARDS) is controversial. The aim of this study was to assess the effect of NIV on the rate of endotracheal intubation and intensive care unit (ICU) mortality in patients with ARDS. We searched the MEDLINE database for relevant studies published from 1980 to September 2005, and included studies if (a) the design was a randomized controlled trial; (b) patients had ARDS irrespective of the underlying etiology; (c) the interventions compared NIV and medical therapy with medical therapy alone; and (d) outcomes included need for endotracheal intubation and/or ICU survival. The addition of NIV to standard care in the setting of ARDS did not reduce the rate of endotracheal intubation (absolute risk reduction (RR) 13.5%, 95% confidence interval (CI) -5.2% to 31.3%), and had no effect on ICU survival (absolute RR 4.8%, 95% CI -12.8% to 22.1%). However, the trial results were significantly heterogeneous. Thus, current evidence suggests that patients with ARDS are unlikely to have any significant benefits on outcome when NIV is added to standard therapy. However, this analysis is limited by the presence of significant heterogeneity; hence large randomized controlled trials are required to settle this issue. PMID- 16678395 TI - CFTR gene mutations and asthma in the Norwegian Environment and Childhood Asthma study. AB - BACKGROUND: Several candidate genes have been implicated in the etiology of asthma, including the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR gene result in derangements of mucociliary clearance. Homozygotes for CFTR mutations develop cystic fibrosis (CF), a disorder characterized mainly by lung and pancreas disease. OBJECTIVE: To investigate whether there was an increased frequency of CFTR mutations in asthma patients. METHODS: Seven hundred and three subjects aged 10-11 years from the environment and childhood asthma (ECA) study were included in the present study. Possible associations between asthma, reduced lung function, bronchial hyperresponsiveness (BHR), and increased or decreased nitrogen oxide (NO) levels (based on structural parental interview, spirometry, PD20 methacholine challenge test and exhaled NO measurements), and the five most common CFTR mutations in Norway (DeltaF508, R117H, R117C, 4005+2T-->C, 394delTT), the modulating polymorphisms IVS8(TG)mTn and the IVS8-5T were investigated. RESULTS: No association were found between asthma, reduced lung function, BHR or exhaled NO levels and CF heterozygosity. However, the IVS8(TG)11T7 haplotype was associated with normal lung function. CONCLUSIONS: Our results do not support the hypothesis that CFTR mutations or polymorphisms play a role in the pathogenesis of asthma in children. However, the distribution of Tn(TG)m haplotypes differed between individuals with reduced lung function and individuals with normal lung function. PMID- 16678396 TI - Changes in urinary LTE4 and nasal functions following nasal provocation test with ASA in ASA-tolerant and -intolerant asthmatics. AB - Aspirin-induced asthma (AIA) is a syndrome characterized by intolerance to aspirin (ASA), nasal polyps and bronchial asthma, the metabolic shift of arachidonic acid towards the lipoxygenase pathway and hyper-production of cysteinyl-leukotrienes (cys-LTs) being the current pathogenetic hypothesis. The research for both sensitive indicators and safe diagnostic tests is still attracting. Aim of the study was to measure changes in urinary LTE4 excretion and in nasal function (Resistance-Req, and Volume-Vol, assessed by acoustic rhinomanometry (AR)) following a nasal provocation test (NPT) with ASA:LTE4 measurements have been never previously used to our knowledge for assessing nasal responsiveness to ASA. METHODS: After written consent, 118 mild-to-moderate asthmatics (48 males, mean age 41.8 years+/-11.9SD, range 25-70 years; basal FEV1=80.1% pred.+/-5.8SD) underwent NPT by nasal instillation of ASA (total maximal dose 25 mg). Spirometry, acoustic rhinomanometry (AR; TM Hood Lab., USA) and urinary LTE4 (pg/mg creatinine; Cayman Chemical, MI, USA) were measured in baseline and 2h after the ASA challenge. STATISTICS: t-Test between means+/-sd, assuming P<0.05, and linear regression between all variables considered. RESULTS: In 67 ASA-intolerant asthmatics, FEV1 did not change significantly following NPT (81.7% pred.+/-5.1SD in baseline, 80.5% pred.+/-4.1 after NPT, P=ns) even in the presence of a significant decrease of Vol (11.3 cm3+/-4.1SD in baseline, 5.9 cm3+/-4.2SD after NPT, P=0.003), a substantial increase of Req (0.88 cmH2O/l/min+/-0.11SD in baseline, 2.41 cmH2O/l/min+/-0.77 after NPT, P=0.002), and urinary LTE4 excretion (433.0 pg/mg+/-361.7 in bsln, 858.0 pg/mg+/-471.6 90 min after NPT with L-SA, P=0.04). NPT did not affect FEV1 also in 51 ASA-tolerant asthmatics (89.7% pred.+/-6.9 in bsln, 86.6% pred.+/-4.3 after NPT), but in these subjects also Vol (from 14.9 cm3+/-4.2sd to 14.6 cm3+/-3.8SD), Req (0.38 cmH2O/l/min+/-0.14 in bsln, 0.26 cmH2O/l/min+/-0.2 after NPT, P=ns), and urinary LTE4 (333.1 pg/mg+/-202.8 in bsln, 318.0 pg/mg+/-198.7 after NPT, P=ns) remained unchanged. Only pre-NPT LTE4 values proved related to pre-NPT Req and Vol values (r=0.54 and r=-0.71, respectively), but not to patients' age (R=-0.05), and basal FEV1 (r=0.01). CONCLUSIONS: In ASA-intolerant patients, NPT with lysine-aspirin (L-ASA) only induces a substantial nasal obstruction and enhances urinary LTE4 excretion in the absence of any significant bronchial obstruction. Nasal ASA challenge proves a test absolutely safe for asthma patients suspected of ASA intolerance. Measures of urinary LTE4 excretion contributed significantly to magnify the discriminant and the diagnostic value of NPT. PMID- 16678397 TI - Effects of pre-treatment technologies on quantity and quality of source-sorted municipal organic waste for biogas recovery. AB - Source-sorted municipal organic waste collected from different dwelling types in five Danish cities and pre-treated at three different plants was sampled and characterized several times during one year to investigate the origin of any differences in composition of the pre-treated waste introduced by city, pre treatment technology, dwelling type or annual season. The investigated pre treatment technologies were screw press, disc screen and shredder+magnet. The average quantity of pre-treated organic waste (biomass) produced from the incoming waste varied between the investigated pre-treatment technologies: 59%, 66% and 98% wet weight, respectively (41%, 34% and 2% reject, respectively). The pre-treatment technologies showed differences with respect to distribution of the chemical components in the waste between the biomass and the rejected material (reject), especially for dry matter, ash, collection bag material (plastic or paper) and easily degradable organic matter. Furthermore, the particle size of the biomass was related to the pre-treatment technology. The content of plastic in the biomass depended both on the actual collection bag material used in the system and the pre-treatment technology. The sampled reject consisted mostly of organic matter. For cities using plastic bags for the source-separated organic waste, the expected content of plastic in the reject was up to 10% wet weight (in some cases up to 20%). Batch tests for methane potential of the biomass samples showed only minor variations caused by the factors city, pre-treatment technology, dwelling type and season when based on the VS content of the waste (overall average 459STPm(3)/tVS). The amount of methane generated from 1t of collected waste was therefore mainly determined by the efficiency of the chosen pre-treatment technology described by the mass distribution of the incoming waste between biomass and reject. PMID- 16678398 TI - Survey of solid waste generation and composition in a rapidly growing urban area in Central Nigeria. AB - The objective of this study was to carry out a field survey of the solid waste generation profile in parts of Makurdi, a rapidly growing urban city in north central Nigeria. The areas surveyed covered low, medium and high-density residential quarters, representing high/medium/low income groups in the area. Results of the survey show that the bulk ( approximately 82%) of the solid waste generated in the area originates from households, rather than from commercial, institutional or industrial premises. Of the waste from households, a substantial proportion consists of various putrescible materials (36-57%), with ash, dust and sand (combined) forming another significant proportion (21-41%). From the non household sources, putrescible matter is also significant (23-45%), as is the combined ash/dust/sand fraction (32-36%). The quantity of plastics/cellophane materials from household and non-household sources was, however, comparable (6 10%). There was more paper from commercial and institutional premises (9-12%) than from household or small/medium scale industrial premises (2-4%). Glass (0.1 6.9%), metals (mostly cans and bottle corks, 0.7-3.4%) and textiles (0.3-6%) form only a minor proportion of the waste across generators. Waste generation rates were for households, 0.54kg/cap/day; for commercial, 0.018kg/m(2)/day; institutional, 0.015kg/m(2)/day while for small and medium scale industries, the rate was 0.47kg/m(2)/day. As there is no previous study of this kind in the Makurdi urban area, what is reported here may be taken as baseline for the entire area. The implications of the findings for solid waste management planning are discussed. PMID- 16678399 TI - On-line detection of atmospheric formaldehyde by a conductometric biosensor. AB - Atmospheric formaldehyde (CH(2)O) was detected under continuous flow conditions by an on-line system comprising of a wet scrubber for a continuous transfer of the pollutant to an aqueous solution, a micro-reactor containing immobilized formaldehyde dehydrogenase (FDH) and a conductometric transducer. By this system atmospheric formaldehyde concentrations in the range 0.05-2 ppm were detected with a sensitivity of 20 microS/ppm. In this concentration range the immobilized enzyme oxidized all the sampled formaldehyde molecules to formic acid, avoiding cumbersome calibration procedures. The operational stability of the biosensor was at least 3 months, working continuously 10 h/day at room temperature. PMID- 16678400 TI - Microbial biocatalyst developments to upgrade fossil fuels. AB - Steady increases in the average sulfur content of petroleum and stricter environmental regulations concerning the sulfur content have promoted studies of bioprocessing to upgrade fossil fuels. Bioprocesses can potentially provide a solution to the need for improved and expanded fuel upgrading worldwide, because bioprocesses for fuel upgrading do not require hydrogen and produce far less carbon dioxide than thermochemical processes. Recent advances have demonstrated that biodesulfurization is capable of removing sulfur from hydrotreated diesel to yield a product with an ultra-low sulfur concentration that meets current environmental regulations. However, the technology has not yet progressed beyond laboratory-scale testing, as more efficient biocatalysts are needed. Genetic studies to obtain improved biocatalysts for the selective removal of sulfur and nitrogen from petroleum provide the focus of current research efforts. PMID- 16678401 TI - Learning from stargazin: the mouse, the phenotype and the unexpected. AB - The stargazin gene (also referred to as Cacng2) has been identified by forward genetics in a spontaneous mouse mutant with ataxic gait, upward head-elevating movements (hence the name stargazer for the mouse) and episodes of spike-wave discharges. Stargazin is related to the gamma-1 subunit of skeletal muscle voltage-dependent calcium channel (VDCC), and a deficit in its role as auxiliary VDCC subunit was proposed to underlie the epileptic phenotype of the mouse; yet, a conclusive demonstration of stargazin function in VDCC regulation is still lacking. In contrast, stargazin and its three closely related isoforms gamma-3, gamma-4 and gamma-8 were shown to function as auxiliary subunits for a very different ion channel - the AMPA-type glutamate receptor - prominently regulating early intracellular transport, synaptic targeting and anchoring, and ion channel functions of this major excitatory receptor in the brain. PMID- 16678402 TI - Protein structure comparison: implications for the nature of 'fold space', and structure and function prediction. AB - The identification of geometric relationships between protein structures offers a powerful approach to predicting the structure and function of proteins. Methods to detect such relationships range from human pattern recognition to a variety of mathematical algorithms. A number of schemes for the classification of protein structure have found widespread use and these implicitly assume the organization of protein structure space into discrete categories. Recently, an alternative view has emerged in which protein fold space is seen as continuous and multidimensional. Significant relationships have been observed between proteins that belong to what have been termed different 'folds'. There has been progress in the use of these relationships in the prediction of protein structure and function. PMID- 16678403 TI - Therapeutic potential of adult stem cells. AB - The aim of cell-based therapies is to replace or repair damaged tissues and organs. A diverse number of disorders are amenable to this approach, including haematopoietic, neurological and cardiovascular diseases, as well as bone defects and diabetes. Central to the success of cell therapy is the necessity to be able to identify, select, expand and manipulate cells outside the body. Recent advances in adult stem cell technologies and basic biology have accelerated therapeutic opportunities aimed at eventual clinical applications. Adult stem cells with the ability to differentiate down multiple lineages are an attractive alternative to human embryonic stem cells (hES) in regenerative medicine. In many countries, present legislation surrounding hES cells makes their use problematic, and indeed the origin of hES cells may represent a controversial issue for many communities. However, adult stem cells are not subject to these issues. This review will therefore focus on adult stem cells. Based on their extensive differentiation potential and, in some cases, the relative ease of their isolation, adult stem cells are appropriate for clinical development. Recently, several observations suggest that multipotential adult stem cells are capable of producing a whole spectrum of cell types, regardless of whether or not these tissues are derived from same germ layer; highlighting the opportunity to manipulate stem cells for therapeutic use. PMID- 16678404 TI - Sorption of cadmium and zinc from aqueous solutions by water hyacinth (Eichchornia crassipes). AB - The water hyacinth (Eichchornia crassipes) has been successfully utilized for the removal of Zn(II) and Cd(II) as well as their admixture from samples of aqueous solutions. The growth of the plant after 16 days of exposure to the metal ions showed an increasing trend up to 2.5 ppm of Cd(II) and 6.0 ppm of Zn(II) concentrations, however, the growth became nondetectable or inhibited above these concentrations. The overall metal uptake by the plant was dependent upon the concentration of the metal and the duration of the exposure time. The metal uptake from a mixture of Cd(II) and Zn(II) was reflected by a rate constant quite different from those solutions containing only one metal ion. An analysis of metal in roots and tops of the plants showed that more Zn(II) was accumulated in the root when compared to Cd(II). However, the accumulation factor for the tops and the roots for Cd(II) and Zn(II) was higher than those obtained admixture of Zn(II) and Cd(II). The rate of metal mobility in the root was slower than that in the top of the plant for Zn(II) and Cd(II). A water hyacinth based system can be used to remove Cd(II) and Zn(II) from water/wastewater. PMID- 16678405 TI - Effect of stacking method on Salmonella elimination from recycled poultry bedding. AB - Recycled poultry bedding (RPB) is a protein and mineral supplement for cattle. Concerns regarding this product have arisen because of the perceived risk of transmitting potentially pathogenic organisms to cattle. This study's primary objective was to assess survival of Salmonella in RPB stacked to a recommended height (2.13 m-DS-RPB), or a height of 0.76 m (SS-RPB). Dialysis bags containing RPB and Salmonella typhimurium were placed throughout stacks. Temperature was monitored daily using thermocouples attached to sample bags. After 21 days, sample bags were recovered. Ammonia analysis was performed from multiple sites in the stacks. Bag contents were cultured to determine viability of the salmonella inoculates. This trial demonstrated a wide variation of temperature within the stacks. Temperature near the edge of stacks changed with ambient temperature. Ammonia concentration in the RPB was highest at the top of the DS-RPB. Salmonella was eliminated in 98.7% of sites, with at least a 5-log reduction in the Salmonella organisms in sites where it was still viable. PMID- 16678406 TI - Kinetic and isothermal studies on liquid-phase adsorption of 2,4-dichlorophenol by palm pith carbon. AB - Adsorption studies were conducted to study the removal of 2,4-dichlorophenol (2,4 DCP) from aqueous solution on palm pith carbon under varying experimental conditions such as agitation time, adsorbent dose, pH and temperature. Higher 2,4 DCP was removed with decrease in the initial concentration of 2,4-DCP and increase in amount of adsorbent used. Kinetic study showed that the adsorption of 2,4-DCP on palm pith carbon was a gradual process. Adsorption capacities were 19.16 mg/g for the particle size of 250-500 microm. The equilibrium time was 60 and 80 min for 10 and 20 mg/L and 100 min for both 30 and 40 mg/L phenol concentrations, respectively. Acidic pH was favourable for the adsorption of 2,4 DCP. Studies on pH effect and desorption showed that chemisorption seemed to play a major role in the adsorption process. Thermodynamic study showed that adsorption of 2,4-DCP on palm pith carbon was more favoured. The change in entropy (DeltaS0) and heat of adsorption (DeltaH0) of palm pith carbon was estimated as 30.72 J/mol/k and 7.16 kJ/mol, respectively. The high positive value of change in Gibbs free energy indicated the feasible and spontaneous adsorption of 2,4-DCP on palm pith carbon. The results indicated that palm pith carbon was an attractive candidate for removing phenols from wastewater. PMID- 16678407 TI - Grass mulching effect on infiltration, surface runoff and soil loss of three agricultural soils in Nigeria. AB - Mulching the soil surface with a layer of plant residue is an effective method of conserving water and soil because it reduces surface runoff, increases infiltration of water into the soil and retard soil erosion. The effectiveness of using elephant grass (Pennisetum purpureum) as mulching material was evaluated in the laboratory using a rainfall simulator set at rainfall intensities typical of the tropics. Six soil samples, two from each of the three major soil series representing the main agricultural soils in South Western Nigeria were collected, placed on three different slopes, and mulched with different rates of the grass. The surface runoff, soil loss, and apparent cumulative infiltration were then measured under each condition. The results with elephant grass compared favorably with results from previous experiments using rice straw. Runoff and soil loss decreased with the amount of mulch used and increased with slope. Surface runoff, infiltration and soil loss had high correlations (R = 0.90, 0.89, and 0.86, respectively) with slope and mulch cover using surface response analysis. The mean surface runoff was correlated negatively with sand content, while mean soil loss was correlated positively with colloidal content (clay and organic matter) of the soil. Infiltration was increased and soil loss was reduced greatly with the highest cover. Mulching the soils with elephant grass residue may benefit late cropping (second cropping) by increasing stored soil water for use during dry weather and help to reduce erosion on sloping land. PMID- 16678408 TI - Design and synthesis of noncompetitive metabotropic glutamate receptor subtype 5 antagonists. AB - A series of diaryl amides was designed and synthesized as novel nonethynyl mGluR5 antagonists. The systematic variation of the pharmacophoric groups led to the identification of a lead compound that demonstrated micromolar affinity for the mGluR5. Further optimization resulted in compounds with improved binding affinities and antagonist profiles, in vitro. PMID- 16678409 TI - Array synthesis of progesterone receptor antagonists: 3-aryl-1,2-diazepines. AB - New non-steroidal chemotypes are required for the development of drugs targeting the steroid hormone receptors. The parallel array synthesis of 3-aryl-1,2 diazepines employing solid-supported reagents is described. The resulting compounds demonstrated high affinity binding to the progesterone receptor. PMID- 16678410 TI - Arylamide derivatives as allosteric inhibitors of the integrin alpha2beta1/type I collagen interaction. AB - We herein report a group of allosteric inhibitors of integrin alpha(2)beta(1) based on an arylamide scaffold. Compound 4 showed an IC(50) of 4.80 microM in disrupting integrin I-domain/collagen binding in an ELISA. These arylamide compounds are able to block collagen binding to integrin alpha(2)beta(1) on the platelet surface. Further we find that compound 4 recognizes a hydrophobic cleft on the side of the alpha(2) I-domain, suggesting an alternative targeting site for drug development. PMID- 16678411 TI - A versatile access to new macrocyclic oligoheterocycles (MOH). AB - An efficient and straightforward methodology for the parallel solid-phase synthesis of a variety of new macrocyclic oligoheterocycles is described. Exhaustive reduction of resin-bound cyclic polyamides using borane generates polyamines. Treatment of separated pairs of amines with a variety of bifunctional reagents provides, following cleavage from the solid support, the desired macrocyclic oligoheterocyclic (MOH) compounds in good yields and purities. PMID- 16678412 TI - Nanomolar inhibition of the enterobactin biosynthesis enzyme, EntE: synthesis, substituent effects, and additivity. AB - 2,3-Dihydroxybenzohydroxamoyl adenylate (I) was prepared as a potential product analog inhibitor of EntE (EC# 2.7.7.58), a 2,3-dihydroxybenzoate AMP ligase from Escherichia coli that is required for the biosynthesis of enterobactin. This compound, obtained by the aqueous reaction of imidazole-activated adenosine 5' phosphate and 2,3-dihydroxybenzohydroxamic acid, is a competitive inhibitor with a Ki value of 4.5 x 10(-9)M. Deletion of the catecholic 3-OH group of (I), in compound (II), reduced inhibitory activity by a factor of 3.5, whereas, removal of both the 3-OH and 2-OH groups, in (III), reduced inhibitory activity by a factor of approximately 2000. Acetohydroxamoyl adenylate (IV), in which the entire catechol moiety of (I) is replaced by a hydrogen atom, gave 0 and kurtosis>0). Skewness and kurtosis in MFC data was highly correlated (young: r=0.60, p=0.01; elderly: r=0.95, p<0.01). MFC descriptive statistics provide useful information about basic strategies used by individuals to minimize the likelihood of tripping. Possible strategies to minimize tripping include: (a) increasing MFC height central tendency, (b) reducing MFC variability, and/or (c) increasing right skewness. A low median MFC was often associated with a low IQR or high skewness to compensate. Further research is required to establish how, or if at all, these strategies are modified in populations that are more at risk of falling. PMID- 16678419 TI - Trimeric autotransporter adhesins: variable structure, common function. AB - Trimeric autotransporter adhesins (TAAs) are important virulence factors in gram negative pathogens. Despite the variety of hosts ranging from plants to mammals and the specialized regulation of TAAs, their molecular organization follows surprisingly simple rules: they form trimeric surface structures with a head stalk-anchor architecture. The head and stalk are composed of a small set of domains, building blocks that are frequently arranged repetitively. We propose that this repetitive arrangement facilitates recombination of domains to modulate the specificity of the common function: adhesion to the host. PMID- 16678420 TI - Diversity and functional importance of phytoplasma membrane proteins. PMID- 16678422 TI - Acetate kinase: not just a bacterial enzyme. AB - The bacterial enzymes acetate kinase (AK) and phosphotransacetylase (PTA) form a key pathway for synthesis of the central metabolic intermediate acetyl coenzyme A (acetyl-CoA) from acetate or for generation of ATP from excess acetyl-CoA. Putative AK genes have now been identified in some eukaryotic microbes. In Chlamydomonas reinhardtii and Phytophthora species, AK forms a pathway with PTA. AK has also been identified in non-yeast fungi but these fungi do not have PTA. Instead, AK forms a pathway with D-xylulose 5-phosphate phosphoketolase (XFP), a pathway that was also previously found only in bacteria. In Entamoeba histolytica, neither PTA nor XFP was found as a partner for AK. Thus, eukaryotic microbes seem to have incorporated the 'bacterial' enzyme AK into at least three different metabolic pathways. PMID- 16678421 TI - The structural basis of paramyxovirus invasion. AB - To deliver their genetic material into host cells, enveloped viruses have surface glycoproteins that actively cause the fusion of the viral and cellular membranes. Recently determined X-ray crystal structures of the paramyxovirus fusion (F) protein in its pre-fusion and post-fusion conformations reveal the dramatic structural transformation that this protein undergoes while causing membrane fusion. Conformational changes in key regions of the F protein suggest the mechanism by which the F protein is activated and refolds. PMID- 16678423 TI - Brain donation for research: who donates and why? AB - Understanding what influences people to donate or not donate body organs is critical for the future of transplant surgery and medical research. Are people involved with a brain donor program for research influenced by the same factors, and are they also donors for organ transplantation? Using web-based technology, people involved in an Australian brain donation program (for research) were asked to complete a questionnaire designed to elicit demographic information, motivational factors and information about involvement in organ transplantation programs. The response rate was 82%. The majority of people involved in the program are young, well-educated Australian females. Seventy-eight percent are involved in other organ and tissue donation programs. People involved in the 'Using our Brains' program are the same group as those who are organ and tissue donors. An improvement in the overall donation rate might be possible if the resources of the research and transplant organisations were combined. PMID- 16678424 TI - Circadian blood pressure variation after acute stroke. AB - We aimed to characterise the patterns of circadian blood pressure (BP) variation after acute stroke and determine whether any relationship exists between these patterns and stroke outcome. BP was recorded manually every 4 h for 48 h following acute stroke. Patients were classified according to the percentage fall in mean systolic BP (SBP) at night compared to during the day as: dippers (fall > or = 10-<20%); extreme dippers (> or = 20%); non-dippers (> or = 0-<10%); and reverse dippers (<0%, that is, a rise in mean nocturnal SBP compared to mean daytime SBP). One hundred and seventy-three stroke patients were included in the study (83 men, 90 women; mean age 74.3 years). Four patients (2.3%) were extreme dippers, 25 (14.5%) dippers, 80 (46.2%) non-dippers and 64 (36.9%) reverse dippers. There was a non-significant trend in the proportion of patients who were dead or dependent at 3 months in the extreme dipper (p=0.59) and reverse dipper (p=0.35) groups. Non-dipping and reverse-dipping were relatively common patterns of circadian BP variation seen in acute stroke patients. These patterns were not clearly associated with outcome. PMID- 16678425 TI - Superficial peroneal sensory and sural nerve conduction studies in peripheral neuropathy. AB - The objective of this study was to prospectively evaluate sensory nerve conduction studies (NCS) in the distal lower limbs in the electrodiagnosis of peripheral neuropathy. We prospectively studied 316 consecutive patients with surface stimulation and recording, in comparison with 90 control subjects. A total of 310 patients were found to have lower limb sensory NCS abnormalities. In these patients, the rate of detection of peripheral neuropathy with superficial peroneal NCS (88.5%) was significantly higher (P<0.001) compared with sural NCS (75%). The superficial peroneal NCS appeared to have a higher detection rate for peripheral neuropathy in our study, and its study can be adjunctive to sural NCS. PMID- 16678426 TI - Ethics in medical technologies: the Roman Catholic viewpoint. AB - New medical techniques and novel scientific discoveries bring many basic questions concerning the role of human dignity in medical research as well as in the society of the future. This paper presents the Roman Catholic approach to the use of new technologies, the research of human embryos, the ethical aspects of studies on the human genome. The concept of "human ecology", as proposed by John Paul II, is introduced to reconcile the academic freedom of research with insurmountable ethical barriers which must be recognized to defend human dignity. In critical appraisal of Peter Singer's concept of the quality of life the author points out that it is irrational to try to reduce this quality to the level of biological parameters. Human dignity as well as the sanctity of life express also a quality of life so important for the cultural growth of Homo sapiens. To protect human ecology it is our moral duty to defend human dignity and to recognize the importance of those values that are fundamental in the process of development of the human species. PMID- 16678427 TI - Shunt failure in idiopathic intracranial hypertension presenting with spontaneous cerebrospinal fluid leak. AB - A case of spontaneous cerebrospinal (CSF) fluid leak after ventriculoperitoneal shunt (VPS) failure in a patient with idiopathic intracranial hypertension (IIH) is reported. This is the first report of spontaneous CSF leak in an IIH patient without a history of trauma, sinus surgery, or intracranial surgery. The diagnosis was confirmed using thin-sliced post-contrast computed tomography, which revealed a micro-dehiscence of the cribiform plate at the superior aspect of the ethmoid sinus. The patient underwent VPS revision without complication, resulting in complete amelioration of symptoms and cessation of CSF rhinorrhoea at 1 year follow up. PMID- 16678428 TI - Historical article: DNA polymorphism and the early history of the double helix. AB - Early X-ray diffraction patterns from oriented fibres indicated that DNA must have a simple, repetitious structure and encouraged some researchers, who were already convinced that DNA was the genetic material, to undertake more detailed diffraction analyses and speculative modelling. The pioneering experimental work by Wilkins in the Wheatstone Laboratory at King's College London in the late 1940s first inspired, and then was overtaken by, the conjectural modelling of Watson and Crick in the Cavendish Laboratory at Cambridge. Why this was allowed to happen is still something of a puzzle. Here, I explore the puzzle and expose a peculiar flaw in the details of the original Watson-Crick model that was left for Wilkins to resolve. PMID- 16678429 TI - Synthesis and high-throughput evaluation of triskelion uracil libraries for inhibition of human dUTPase and UNG2. AB - Human nuclear uracil DNA glycosylase (UNG2) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) are the primary enzymes that prevent the incorporation and accumulation of deoxyuridine in genomic DNA. These enzymes are desirable targets for small molecule inhibitors given their roles in a wide range of biological processes ranging from chromosomal rearrangements that lead to cancer, viral DNA replication, and the formation of toxic DNA strand breaks during anticancer drug therapy. To accelerate the discovery of such inhibitors, we have developed a high-throughput approach for directed library synthesis and screening. In this efficient technology, a uracil-aldehyde ligand is covalently tethered to one position of a trivalent alkyloxyamine linker via an oxime linkage, and then the vacant linker positions are derivatized with a library of aldehydes. The resulting triskelion oximes were directly screened for inhibitory activity and the most potent of these showed micromolar binding affinities to UNG2 and dUTPase. PMID- 16678430 TI - 3-Aza-6,8-dioxabicyclo[3.2.1]octanes as new enantiopure heteroatom-rich tropane like ligands of human dopamine transporter. AB - CNS diseases such as Parkinson, schizophrenia, and attention deficit hyperactivity disorder (ADHD) are characterized by a significant alteration of dopamine transporter (DAT) density. Thus, the development of compounds that are able to selectively interact with DAT is of great interest. Herein we describe the design and synthesis of a new set of 3-aza-6,8-dioxabicyclo[3.2.1]octanes having a tropane-like structure with additional heteroatoms at positions 3 and 6. The compounds were evaluated for their in vitro receptor binding properties toward human dopamine (hDAT) and serotonin (hSERT) transporters using [3H]WIN35,428 and [3H]citalopram as specific radioligands, respectively. Biological assays revealed that some compounds having the N-3 atom substituted with aryl groups possess significant affinity and selectivity for monoamine transporters, and in particular, compound 5d displayed an IC50 of 21 nM toward DAT, and a good selectivity toward SERT (IC50=1042 nM). These results suggest that 3-aryl-3-aza-6,8-dioxabicyclo[3.2.1]octanes may represent a new class of DAT ligands. PMID- 16678431 TI - Calculation of coincidence summing corrections for X-ray peaks and for sum peaks with X-ray contributions. AB - In this paper, features of the gamma-ray spectra in the presence of important contributions of X-rays to coincidence summing effects are studied. The experimental results concerning the sum peaks with various X-ray combinations as well as the Ge X-ray escape peaks present in the spectrum of a 133Ba point source measured with an n-type detector are in good agreement with the values computed using the GESPECOR software. PMID- 16678432 TI - Modified Damus-Kaye-Stansel procedure using aortic flap technique for systemic ventricular outflow tract obstruction in functionally univentricular heart. AB - Damus-Kaye-Stansel procedure is a useful method to relieve the systemic ventricular outflow tract obstruction in functionally univentricular heart. Regurgitation of pulmonary valve and recurrence of systemic ventricular outflow obstruction are the major concerns at the late phase of this procedure. Modification of original Damus-Kaye-Stansel procedure that can prevent the use of prosthetic materials is evaluated. The modified Damus-Kaye-Stansel procedure using aortic flap technique was performed in eight patients with functionally univentricular heart. Patients' ages ranged from 3 to 28 months (mean 14 months). Follow-up period was 37 months as a mean (9-71 months), and the follow-up was complete. There was no operative mortality and no late death. In addition, there was no recurrence of systemic ventricular outflow tract obstruction throughout the follow-up period. Regurgitation of the pulmonary valve estimated by echocardiography at the latest follow-up was none to trivial in seven patients and mild in one. The modified Damus-Kaye-Stansel procedure using aortic flap technique is a safe, useful and reproducible technique to solve systemic ventricular outflow tract obstruction in functionally univentricular heart, and it can be an alternative for original technique or the so-called double-barrel modification. PMID- 16678433 TI - Adiponectin and its receptors: partners contributing to the "vicious circle" leading to the metabolic syndrome? AB - Although already described five years ago, it is only from year 2000, following intensive research in the field of genetics that the adiponectin protein was related with insulin sensitivity, type 2 diabetes and the metabolic syndrome. The story began with a paradox as this protein exclusively secreted by fat tissue was dramatically decreased in patients presenting an excess of fat mass. Later this decrease was reported with insulin resistance and metabolic syndrome associated phenotypes. The search for genetic variants in the adiponectin encoding ACDC gene and epidemio genetic investigations allowed to associate genetic variations of the gene and phenotypic traits of the metabolic syndrome. One of the major points was the correlation of the levels of circulating adiponectin with insulin sensitivity, leading to a better knowledge of the role of adiponectin. Indeed it is now clearly admitted that adiponectin is an insulin sensitizing cytokine. Recently two adiponectin receptors were described and genetic variations in their genes were associated with features of the metabolic syndrome. Interactions of adiponectin with various partners are discussed in view of a better understanding of adiponectin resistance and insulin resistance. PMID- 16678434 TI - IL-18 as critical co-stimulatory molecules in modulating the immune response of ITAM bearing lymphocytes. AB - NK cells responses are controlled by inhibitory and activating cell surface receptors. Inhibitory receptors serve to moderate NK activity by dampening cytokine release and cytotoxicity if activating ligands are also triggered by interaction with their receptors on the NK cells. This dampening effect is critical to prevent wide scale self destruction. These studies have focused on cytokine signaling in the context of an environment where ITAM bearing receptors are triggered on NK, NKT and T cells. Our findings indicate that cytokines are an important co-stimulatory signal that is needed to maximize the host innate immune response. PMID- 16678435 TI - Are other protein tyrosine phosphatases than PTPN22 associated with autoimmunity? AB - The discovery that a single amino acid substitution in the PTPN22 protein tyrosine phosphatase can predispose to so many autoimmune diseases (see chapters 2 and 3), even when present in a single copy, raises many questions regarding the broader significance of this observation. Is there something unique about PTPN22 or are genetic variants of other protein tyrosine phosphatases likely also associated with autoimmune disease? If so, will polymorphisms in other phosphatases be found in the same spectrum of diseases? Are protein tyrosine phosphatases like PTPN22 good drug targets for the treatment of human autoimmunity? In this review, I offer some basis for thinking about these questions. PMID- 16678436 TI - Roles of genetic variations in signalling/immunoregulatory molecules in susceptibility to systemic lupus erythematosus. AB - Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a complex genetic basis that includes many susceptibility genes on multiple chromosomes. As complex human diseases like SLE involve multiple, interacting genetic and environmental determinants, identifying genes for complex traits is challenging and has had limited success so far. However, recent advances in genetic resources and technology have been providing new tools to identify the novel pathways or the sequence variants that contribute to autoimmune diseases. During the past several years, several new candidate genes have been implicated in development of SLE though association studies. In this article we describe an overview of the latest findings in the genetics of SLE, especially focusing on the genetic variations in the signalling or immunoregulatory molecules including CD28 and IRF family members. PMID- 16678437 TI - Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms. AB - 14-3-3 proteins are a ubiquitous class of regulatory proteins found in all eukaryotic cells and were the first class of molecules to be recognized as discrete phosphoserine/threonine binding modules. 14-3-3 proteins bind a large number of different substrates to regulate a wide array of cellular signaling events including cell cycle progression and DNA damage responses, programmed cell death, cytoskeletal dynamics, transcriptional control of gene expression, as well as processes directly related to cancer progression. In this review, the structural basis of phosphorylation-dependent binding of 14-3-3 to peptide and protein ligands is discussed along with mechanisms that govern how 14-3-3 regulates the function of its bound ligands. The X-ray crystal structures of all human 14-3-3 proteins bound to peptides have now been solved. Here, we use structural comparisons between isoforms as a framework for discussion of ligand binding by 14-3-3 as well as the mechanisms through which post-translational modification of the different isoforms alters their function. PMID- 16678438 TI - 14-3-3 proteins: a historic overview. AB - This chapter includes a historic overview of 14-3-3 proteins with an emphasis on the differences between potentially cancer-relevant isoforms on the genomic, protein and functional level. The focus will therefore be on mammalian 14-3-3s although many important developments in the field have involved Drosophila 14-3-3 proteins for example and the cross-fertilisation from parallel studies on plant 14-3-3 should not be underestimated. In the major part of this review I will attempt to focus on some novel data and aspects of 14-3-3 structure and function, in particular regulation of 14-3-3 isoforms by oncogene-related protein kinase phosphorylation and aspects of 14-3-3 research with which newcomers to the field may be less familiar. PMID- 16678439 TI - Gaps in scientific knowledge for the post eradication world. AB - There are significant gaps in the understanding of polio and polio vaccines which have programmatic importance even after 50 years of vaccine use. This paper considers four: the effect of strains used in the manufacture of IPV on safety and efficacy, the reasons for the safety of the Sabin strains, the nature and detection of long-term excreters of poliovirus and the basis for the cessation of virus excretion. PMID- 16678440 TI - Polio vaccine: the first 50 years and beyond. Summary of the meeting and next steps. AB - A conference on "Polio vaccine: the first 50 years and beyond" was held in Toronto, Canada, June 2005. The purpose of the conference was to bring together regulators, manufacturers, academics and public health authorities to celebrate the accomplishments of the past 50 years, to consider the challenges of achieving and sustaining polio eradication and to review standardization and regulatory issues around existing and new polio vaccines. In the final session of the conference the following summary of the meeting was presented. PMID- 16678441 TI - Polio vaccine: the first 50 years and beyond. PMID- 16678442 TI - Polio vaccine development in Canada: contributions to global polio eradication. AB - The paper briefly describes Canada's distinctive experience in the control of polio and offers some lessons for governments and health policy leaders in other jurisdictions, particularly as they consider immunization policies for the post polio-eradication era. PMID- 16678443 TI - Surveillance for polioviruses. AB - Clinical case-driven surveillance for poliovirus remains the cornerstone of monitoring the progress of the poliomyelitis eradication initiative (PEI). It includes notification and careful investigation of cases of acute flaccid paralysis (AFP), timely collection of stool specimens from the patient, and virological examination of the specimens in an accredited laboratory. Successful high quality APF surveillance requires painstaking perseverance and smooth interplay of several different groups of health care workers in a country. Environmental surveillance (ES) may in certain situations be more sensitive than AFP surveillance in detecting wild type poliovirus or vaccine derived poliovirus circulation. However, apart from being highly labour intensive, it cannot be optimally applied in large parts of the world because of the lack of converging sewage systems. Under these conditions, the very rationale of ES, examining of individual samples representing large groups (hundreds of thousands) of people cannot be readily exploited. Whatever approach of poliovirus surveillance is used, we will always be monitoring subgroups of potential poliovirus excreting people only. Therefore, several years long poliovirus-free monitoring period is needed to confirm that poliovirus transmission has stopped in a given population. PMID- 16678444 TI - Single-trial discrimination of truthful from deceptive responses during a game of financial risk using alpha-band MEG signals. AB - We studied whether magnetoencephalography (MEG) could detect deceptive responses on a single-subject, trial-by-trial basis. To elicit spontaneous, ecologically valid deception, we developed a paradigm in which subjects in a simulated customs setting were presented with a series of pictures of items which might be in their baggage, and for each item, they decided whether to "declare" (tell the truth) or "smuggle" (lie). Telling the truth involved a small but certain monetary penalty, whereas lying involved both greater monetary risk and greater potential reward. Most subjects showed decreased signal power in the 8-12 Hz (alpha) range during deceptive responses as compared to truthful responses. In a cross-validation analysis, we were able to use alpha power to classify truthful and deceptive responses on a trial-by-trial basis, with significantly greater predictive accuracy than that achieved using simultaneously recorded skin conductance signals. Average predictive accuracy for spontaneous deception was greater than 78%, and for some subjects, predictive accuracy exceeded 90%. Our results raise the possibility that alpha power modulation during deception may reflect risk management and/or cognitive control. PMID- 16678445 TI - A molecular phylogeny of tortoises (Testudines: Testudinidae) based on mitochondrial and nuclear genes. AB - Although tortoises of the family Testudinidae represent a familiar and widely distributed group of turtles, their phylogenetic relationships have remained contentious. In this study, we included 32 testudinid species (all genera and subgenera, and all species of Geochelone, representing 65% of the total familial species diversity), and both mitochondrial (12S rRNA, 16S rRNA, and cytb) and nuclear (Cmos and Rag2) DNA data with a total of 3387 aligned characters. Using diverse phylogenetic methods (Maximum Parsimony, Maximum Likelihood, and Bayesian Analysis) congruent support is found for a well-resolved phylogeny. The most basal testudinid lineage includes a novel sister relationship between Asian Manouria and North American Gopherus. In addition, this phylogeny supports two other major testudinid clades: Indotestudo+Malacochersus+Testudo; and a diverse clade including Pyxis, Aldabrachelys, Homopus, Chersina, Psammobates, Kinixys, and Geochelone. However, we find Geochelone rampantly polyphyletic, with species distributed in at least four independent clades. Biogeographic analysis based on this phylogeny is consistent with an Asian origin for the family (as supported by the fossil record), but rejects the long-standing hypothesis of South American tortoises originating in North America. By contrast, and of special significance, our results support Africa as the ancestral continental area for all testudinids except Manouria and Gopherus. Based on our systematic findings, we also propose modifications concerning Testudinidae taxonomy. PMID- 16678446 TI - Higher-level phylogeny and morphological evolution of tyrant flycatchers, cotingas, manakins, and their allies (Aves: Tyrannida). AB - Despite increased understanding of higher-level relationships in passerine birds in the last 15 years, the taxonomic boundaries and phylogenetic interrelationships of the major groups of the Tyrannida (including the cotingas, manakins, tityrines, and tyrant flycatchers) remain unclear. Here, we present an analysis of DNA sequence data obtained from two nuclear exons, three introns, and one mitochondrial gene for 26 genera of Tyrannida and 6 tracheophone outgroups. The analysis resulted in well-supported hypotheses about the earliest evolution within Tyrannida. The Cotingidae, Pipridae, Tityrinae (sensu) [Prum, R.O., Rice, N.H., Mobley, J.A., Dimmick, W.W., 2000. A preliminary phylogenetic hypothesis for the cotingas (Cotingidae) based on mitochondrial DNA. Auk 117, 236-241], Tyrannidae, and the tyrannid subfamiles Tyranninae and Pipromorphinae (sensu) [Sibley, C.G., Monroe, B. L. Jr., 1990. Distribution and Taxonomy of Birds of the World. Yale University Press, New Haven, CT] were all found to be reciprocally monophyletic (given the present taxon sampling). The Cotingidae and Pipridae form a clade that is the sister group to a well-supported clade including Oxyruncus, the Tityrinae, Piprites, and the Tyrannidae. Oxyruncus is the sister group to the Tityrinae, and Piprites is placed as the sister group to the Tyrannidae. The tyrannid subfamilies Tyranninae and Pipromorphinae are monophyletic sister taxa, but the relationships of Platyrinchus mystaceus to these two clades remains ambiguous. The presence of medial (=internal) cartilages in the syrinx is a synapomorphy for the Oxyruncus-Tityrinae-Piprites-Tyrannidae clade. Although morphological synapomorphies currently support the monophyly of both the Pipridae and the Cotingidae, convergences and/or reversals in morphological character states are common in Tyrannida. The relationship between Oxyruncus and the Tityrinae is congruent with additional syringeal synapomorphies and allozyme distance data. Accordingly, we propose the recognition the family Tityridae within the Tyrannida to include the genera Schiffornis, Laniisoma, Laniocera, Iodopleura, Xenopsaris, Pachyramphus, Tityra, and Oxyruncus. PMID- 16678447 TI - E pluribus unum: A phylogenetic and phylogeographic reassessment of Laevapex (Pulmonata: Ancylidae), a North American genus of freshwater limpets. AB - The North American freshwater limpet genus Laevapex (Walker, 1903) is a ubiquitous inhabitant of lentic and slow-moving lotic habitats east of the Rocky Mountains, but uncertainty clouds its systematic affinities, the phylogenetic validity of its constituent nominal species, and its degree of genetic connectivity among drainages. We addressed these issues by sampling the genus throughout much of its collective range and constructing representative nuclear and mitochondrial (mt) gene trees, in addition to performing morphometric analyses of shell shape variation. Our results identify neotropical Gundlachia and South American Uncancylus as sister lineages for Laevapex and reveal a pronounced sub-familial dichotomy within the Ancylidae, separating these three New World genera from a Holarctic (Ferrissia (Ancylus, Rhodacmea)) sister clade. Five nominal taxa (L. fuscus, L. diaphanus, L. peninsulae, L. sp., and "F."arkansasensis), indistinguishable in our morphometric analyses, were polyphyletic in the mt gene trees, exhibited modest levels (< 3.9%) of genetic divergence in the primary (103 of 109 individuals) mt clade and, with one minor exception, they appeared fixed for a single nuclear ITS-2 genotype. Although complicated by the presence of rare, highly divergent mt lineages (of either introgressive or persistent ancestral polymorphic origin) in some populations, the molecular data were consistent with a taxonomic conclusion that these five nominal taxa represent a single polymorphic lineage of the type species L. fuscus. AMOVA analyses indicated that 56% of the observed mt variation could be attributed to among population differences, only two of 36 haplotypes were detected in more than one sampling location, and estimates of among-population mt gene flow were generally low at both regional and continental scales. Unrooted network analyses revealed a number of mt tip clades, one restricted to the southwestern part of the range, the remainder having overlapping distributions in eastern North America. All of the eastern tip clades occurred in the Mid-Atlantic region, and these samples displayed by far the highest levels of collective mt diversity. However, directional gene flow estimates indicated that this region has been a recipient (especially from Alabama populations), rather than a source of haplotypic diversity, implying that it likely represents a center of overlap, not a primary ice age refugium, for this limpet species. PMID- 16678448 TI - Molecular phylogeny of the Drosophila virilis section (Diptera: Drosophilidae) based on mitochondrial and nuclear sequences. AB - Regardless of the well-documented virilis species group, most groups of the Drosophila virilis section have not been completely studied at molecular level since it was suggested. Therefore, phylogenetic relationships among and within species groups of the virilis section are generally unknown. In present paper, the complete mitochondrial ND2 gene and fragment of COI gene in combination with a nuclear gene, Adh coding region, were used to derive the most extensive molecular phylogeny to date for the Drosophila virilis section. A total of 111 individuals covering 61 species were sampled in this study. Novel phylogenetic findings included (1) support for the paraphyly of the melanica and robusta species group and at least two subgroups of the robusta species group, the lacertosa and okadai subgroups, were distinguished as paraphyletic taxa. In addition, (2) present results revealed the sister relationship between D. moriwakii and the robusta subgroup, conflicting with current taxonomy regarding D. moriwakii, which was shifted from the robusta species group to the melanica group. (3) In contrast to the robusta and melanica species groups, monophyly of the polychaeta species group, the angor group and the virilis group was confirmed, respectively. However, the monophyletic quadrisetata species group was resolved with uncertainty. (4) Our analyses of combined data set suggested close relationship between the quadrisetata species group and the unpublished clefta group, and the okadai subgroup is sister to the clade comprising of the quadrisetata and clefta species groups. Within the virilis section, D. fluvialis and three tropical species groups, the polychaeta group, the angor group and the repleta group, are found to branch off earlier than other ingroup taxa. This suggests that the virilis section might have originated in the Old World tropics. Besides, the derived status of the close affinities of the quadrisetata group, the clefta group, and the melanica and robusta groups is probably the result of their adaptation to forests between subtropical and cool-temperate climate. Based on the consideration of the phylogenetic placement of the species of the virilis section, we suggest that at least five independent migrations occurred from the Old World to the New World. PMID- 16678449 TI - Electrical alternans and hemodynamics in the anesthetized guinea pig can discriminate the cardiac safety of antidepressants. AB - INTRODUCTION: The arrhythmogenic risk of fluoxetine, citalopram, and venlafaxine were evaluated through preclinical assays measuring hERG, blood pressure and electrical alternans over their respective clinical unbound concentration ranges. METHODS: Anesthetized guinea pigs were instrumented with jugular and carotid cannulae for drug infusion and blood pressure monitoring respectively; a thoracotomy was performed for placement of a monophasic action potential probe on the left ventricle and for placement of pacing wires on the left ventricular apex. Drugs were infused as a 5-min loading dose immediately followed by a 10-min maintenance dose to achieve clinically relevant plasma concentrations; blood samples were taken at the end of each maintenance dose. Ventricular pacing was performed twice at baseline and at each dose level as follows: 50 preconditioning beats at S1=220 (or 240) ms immediately followed by 30 test-beats at S2=200 ms. This S1-S2 protocol was repeated for S2=190 to 140 ms. HERG and calcium current measurements were recorded in HEK-293 cells stably expressing hERG potassium currents and freshly isolated guinea pig cardiac myocytes using the whole-cell configuration of the patch clamp technique. RESULTS: Physiologically relevant inhibition (IC(20)) of hERG occurred at concentrations 22-fold (fluoxetine), 9 fold (citalopram), and 11-fold (venlafaxine) beyond their respective clinically effective concentration (C(eff)). At the highest achievable levels, fluoxetine (20-fold C(eff)) and citalopram (28-fold C(eff)) significantly decreased heart rate and/or blood pressure as well as increasing electrical alternans by 5 and 18 ms respectively. Venlafaxine increased blood pressure at only 1.3-fold C(eff), but did not increase electrical alternans at the highest achievable dose (3.1 fold C(eff)). DISCUSSION: These data suggest that evaluating other dose limiting side effects in relation to a drug's therapeutic range may be crucial for accurate assessment of arrhythmia liability. PMID- 16678450 TI - Assessment of joint space narrowing with conventional standing antero-posterior radiographs: relief in mild-to-moderate pain is not a confounder in recent osteoarthritis structure-modifying drug trials. AB - OBJECTIVE: Knee pain relief has been suggested to potentially alter radioanatomic positioning in conventional standing antero-posterior knee radiographs. This study was performed to determine whether this is always the case and in particular if it applied to two recent randomised, placebo-controlled trials showing both symptom- and structure-modification with glucosamine sulfate in knee osteoarthritis. DESIGN: Patients in the two studies were selected if they completed the 3-year evaluations and, irrespectively of treatment, (1) were pain improvers in that they underwent Western Ontario and McMaster Universities (WOMAC) osteoarthritis index (WOMAC) pain decrease at least equal to the mean improvement observed with glucosamine sulfate, or (2) if their baseline standing knee pain (item #5 of the WOMAC pain scale) was "severe" or "extreme" and improved by any degree at the end of the trials. Changes in minimum joint space width were then compared between treatments. RESULTS: Knee pain was of mild-to moderate severity in the two original studies and in all patient subsets identified here. Obviously, there were more pain-improvers in the glucosamine sulfate than in the placebo subsets (N=76 vs 57 in pooling the two studies), but WOMAC pain scores improved to the same extent (over 50% relative to baseline). Notwithstanding such a major pain relief, patients in the placebo subsets of both studies suffered a definite mean (SE) joint space narrowing, that was of -0.22 (0.15)mm in the pooled analysis, and that was not observed with glucosamine sulfate: +0.15 (0.07)mm; P=0.003. Similar evidence was found in the smaller subsets with at least severe baseline standing knee pain improving after 3 years. CONCLUSIONS: Knee pain relief did not bias the report of a structure-modifying effect of glucosamine sulfate in two recent long-term trials, possibly due to the mild-to-moderate patient characteristics. Consensus deliverables should acknowledge that the potential limitations of conventional standing antero posterior radiographs should not be overestimated since they may not apply to all patient populations and to all studies using this gold standard technique. PMID- 16678451 TI - Lead accumulation in tidemark of articular cartilage. AB - OBJECTIVE: Determination of the spatial distribution of the toxic element lead (Pb) and other trace elements in normal articular cartilage and subchondral bone from adult humans with no history of work-related exposure to Pb. METHODS: Four macroscopically normal femoral heads and three patellas were harvested from randomly selected forensic autopsies. All subjects died of acute illnesses, had no history of work-related exposure to Pb and had no metabolic bone disease. The elemental distribution of lead (Pb) together with zinc (Zn), strontium (Sr) and calcium (Ca) in the chondral and subchondral region was detected using high resolution synchrotron radiation induced micro X-ray fluorescence (SR mu-XRF) analysis. SR mu-XRF line scans in conventional and SR mu-XRF area scans in confocal geometry were correlated to backscattered electron (BE) images visualizing the mineralized tissue. RESULTS: In all samples, we found a highly specific accumulation of Pb in the tidemark, the transition zone between calcified and non-calcified articular cartilage. Pb fluorescence intensities in the tidemark, which is thought to be a metabolically active mineralization front, were 13-fold higher when compared to subchondral bone. Pb intensities in the subchondral region were strongly correlated with Zn, but were distinctly different from Ca and Sr. CONCLUSIONS: The finding of the highly specific accumulation of lead in the tidemark of human articular cartilage is novel. However at this point, the exact mechanisms of the local Pb accumulation as well as its clinical implications are unknown. PMID- 16678452 TI - Responsiveness, effect size, and smallest detectable difference of Magnetic Resonance Imaging in knee osteoarthritis. AB - OBJECTIVE: The aim of this study was to determine the responsiveness, effect size (ES) and smallest detectable difference (SDD) of two Magnetic Resonance Imaging (MRI) measures for osteoarthritis (OA) of the knee: a whole-organ semiquantitative evaluation and cartilage volume. DESIGN: This analysis was performed on a dataset from a randomized, double-blind trial (Roche NI-15713) conducted in 1998 of a novel therapy in subjects with mild-moderate knee OA, with MRI at baseline and 6-month follow-up. The trial measurements included (1) cartilage volume measured using a proprietary software method; and (2) semiquantitative scoring of other parameters important for "whole organ" evaluation of OA knee joint pathology, using the Whole-Organ MRI Score (WORMS). The analysis initially examined the distributional characteristics of WORMS items, such as cartilage morphology. Standardized response mean (SRM), ES, and SDD between baseline and 6-month follow-up were then calculated in the whole group and the placebo group alone. RESULTS: In general, the differences were small and this was reflected in the small ESs and SRMs. There was also a suggestion of a treatment effect with reduction in differences between baseline and follow-up in the treatment group. CONCLUSION: Of the MRI semiquantitative measures, cartilage morphology, synovitis and osteophytes appeared to be responsive to change and the focus of repeat measures should highlight these articular features. In general, the ESs and SRMs were small. PMID- 16678453 TI - Examining a whole-organ magnetic resonance imaging scoring system for osteoarthritis of the knee using Rasch analysis. AB - OBJECTIVE: The ability to reliably quantify all the structural abnormalities in osteoarthritis (OA) of the knee is a long-standing goal of OA research. On December 5 and 6, 2002, Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society, International held a Workshop for Consensus on Osteoarthritis Imaging in Bethesda, MD, with the aim of providing a state-of-the art review of imaging outcome measures for OA of the knee. As part of the Workshop, data from previous clinical trials and epidemiological studies of OA were analysed with respect to the metrological properties of the measurement methods used. The following report outlines the results of analyses aimed at evaluating the internal construct validity of a whole-organ, ordinal (semi quantitative) magnetic resonance imaging score (WORMS) using Rasch analysis. The fit of data to the Rasch model offers a measure of the validity of summing different items into a subscale score and the degree to which this score behaves as a unidimensional, interval level measurement tool. METHODS: The Rasch model was applied in two OA studies. The first was a clinical cohort comprising OA knee subjects entering a clinical trial; study entry criteria included patients with at least moderate pain, radiographic osteophytes and a minimum of 1.5mm tibiofemoral joint-space width. The second cohort was from the Boston Osteoarthritis Knee Study, an observational cohort of subjects with symptomatic knee OA with pain on most days and a definite osteophyte in either the tibiofemoral or patellofemoral joints. Baseline WORMS scores from both studies were used for the Rasch analysis, performed with RUMM 2020 software. RESULTS: There was a substantial proportion of subjects in both study populations with zero scores in several of the subscales of WORMS. Few of the subscales met the requirements of the Rasch measurement model when summated across all sites, and summations of some postulated compartmentally based sites also failed to fit the Rasch model. The existing scoring categories also required rescoring at many sites. CONCLUSION: There remain important issues in constructing outcome measurements that summate different features across multiple anatomical sites. The whole-organ scoring system evaluated here is no exception. Resolving these issues will improve the ability of imaging studies to assess complex pathological structural change. PMID- 16678454 TI - Osteoarthritis, imaging and guidance for approval of a structural "indication". PMID- 16678455 TI - Hyaluronan biodegradable scaffold for small-caliber artery grafting: preliminary results in an animal model. AB - OBJECTIVE: To evaluate a new Hyaluronan-based graft. MATERIAL AND METHODS: Hyaluronan-based grafts (HYAFF 11trade mark tube, diameter 2 mm, length 1.5 cm) were implanted in an end-to-end fashion in the abdominal aorta of 15 rats. Histology, immunohistochemistry and electron microscopy were used to evaluate the results at 7, 21, and 90 days. RESULTS: At day 7, new tissue was observed in the graft coming from both the proximal and distal ends of the aorta. The luminal surface of the regenerating tissue was covered by endothelial cells (CD34(+), VEGFR-2(+), vWF(+)). At day 21, regenerating tissue joined at the centre of the tube. The neo-vessel was formed by smooth muscle cells (Myosin Light Chain Kinase) as well as elastic, and collagen fibres. At day 90 a stable artery segment was formed and the biomaterial was almost completely degraded. Infiltration of neutrophils and lymphocytes was not observed. All animals survived the observation period and there were no signs of stenoses or aneurysms. CONCLUSION: The hyaluronan-based graft allowed complete regeneration of a newly formed vascular tube in which all the cellular and extracellular components are present and organized in a well defined architecture similar to native artery. PMID- 16678456 TI - Chronic itch and pain--similarities and differences. AB - Both, pruritus and pain are aversive, but clearly distinct sensations originating in the peripheral and central nervous system. During the last years, many interactions between itch and pain in acute transmission and sensitization processes have been identified. It is common experience that the itch sensation can be reduced by the painful sensations caused by scratching. Vice versa analgesia may reduce this inhibition and thus enhance itch. This phenomenon is particularly relevant to spinally administered mu-opioid receptor agonists, which induce segmental analgesia often combined with segmental pruritus. The peripheral and central sensitization to pain and to itch exhibits striking similarities. Classical inflammatory mediators such as bradykinin have been shown to sensitize nociceptors for both itch and pain. Also regulation of gene expression induced by trophic factors, such as NGF, plays a major role in persistently increased neuronal sensitivity for itch and pain. Finally, itch and pain exhibit corresponding patterns of central sensitization. The knowledge of antagonistic interaction, but also of similar sensitization processes has major implication for antipruritic therapeutic approaches. PMID- 16678457 TI - A temperature-jump design for conventional NMR probes. AB - The design and performance of a simple probe insert for temperature-jump experiments in conventional NMR probes is described. The insert uses the output from conventional NMR amplifiers for heating conductive aqueous samples with a rate of 30-80 K/s for 200 W radiofrequency power. The observed dependence of the heating rate on sample conductivity is explained by the dominance of dielectric heating. Factors governing the temperature gradient within the sample are discussed. PMID- 16678458 TI - Mouth cell collection device for newborn mice. AB - For efficient and accurate genotyping of transgenic and knockout mice, the ability to reduce pain and suffering and to obtain DNA early in life are critical. We have developed a novel method to sample buccal cells from neonatal mice to obtain DNA. Our mouse mouth cell collection process includes an oral speculum and collection device which enables rapid extraction of enough DNA for up to 50 PCRs from each buccal sampling. This cell collection device fills a clear need for buccal sampling from neonatal mice, greatly facilitating research in mouse models of human disease. Eliminating the pain, distress, and death caused by invasive and mutilating procedures lessens the potential for confounding variables between control and experimental animals. In conclusion, our mouse mouth cell collection process can be applied to very small animals for which there exists no current device. PMID- 16678459 TI - Molecular targets and the treatment of myeloid leukemia. AB - Leukemia is a multistep process involving accumulation of genetic alterations over time. These genetic mutations destroy the delicate balance between cell proliferation, differentiation, and apoptosis. Traditional approaches to treatment of leukemia involve chemotherapy, radiation, and bone marrow transplantation. In recent years, specific targeted therapies have been developed for the treatment of leukemia. The success of treatment of acute promyelocytic leukemia with All Trans Retinoic Acid (ATRA) and CML with imatinib have lead to increased efforts to identify targets that can be inhibited by small molecules for treatment of hematological malignancies. In this review, we describe the current advances in the development of targeted therapy in acute myeloid leukemia. PMID- 16678460 TI - Hereditary renal hypouricemia. AB - Hereditary renal hypouricemia (HRH) is an inborn error of renal membrane transport specific for uric acid, resulting in increased renal urate clearance associated with hypouricemia. Apparently in most HRH patients, the disorder is caused by loss of function mutations in the gene SLC22A12 coding for human urate transporter 1 (hURAT1), shown to control urate reabsorption in the proximal tubules. The small group of HRH patients with normal SLC22A12 may be affected with mutations in other not yet identified urate transporters. Patients affected with SLC22A12 mutations exhibit attenuated response of urinary urate excretion to pyrazinamide (PZA) and to probenecid (PBD) loading, attributed previously to reflect defective tubular presecretory urate reabsorption. HRH is inherited in an autosomal recessive mode. Most HRH patients are asymptomatic, but some may form renal tract stones and or be predisposed to exercise-induced acute renal failure. PMID- 16678461 TI - Prevalence and risk factors for Giardia and coccidia species of pet cats in 2003 2004. AB - Prevalence and risk factors for feline coccidia and Giardia species infections were estimated for cats visiting 434 Banfield hospitals in 40 states in 2003 2004. Evaluated were 631,021 cats making 1,456,712 office visits (encounters) and having 211,105 fecal examinations. The overall fecal prevalences of coccidia and Giardia species were 1.4% and 0.58%, respectively. Cats at increased risk of coccidia infection were under 4 years of age, intact, and seen during the summer, fall, and spring months compared to winter. Cats at increased risk of Giardia species infection were under 4 years of age. Those at decreased risk were mixed breed and seen during the summer, fall, and spring. The highest regional risk of coccidia and Giardia species infection was for cats in the East South Central region and Mountain region, respectively, compared to the South Pacific region. PMID- 16678463 TI - Assessing the burden of human rabies in India: results of a national multi-center epidemiological survey. AB - OBJECTIVE: Human rabies has been endemic in India since time immemorial, and the true incidence of the disease and nationwide epidemiological factors have never been studied. The main objectives of the present study were to estimate the annual incidence of human rabies in India based on a community survey and to describe its salient epidemiological features. METHODS: The Association for Prevention and Control of Rabies in India (APCRI) conducted a national multi center survey with the help of 21 medical schools during the period February August 2003. This community-based survey covered a representative population of 10.8 million in mainland India. Hospital-based data were also obtained from the 22 infectious diseases hospitals. A separate survey of the islands of Andaman, Nicobar, and Lakshadweep, reported to be free from rabies, was also undertaken. RESULTS: The annual incidence of human rabies was estimated to be 17,137 (95% CI 14,109-20,165). Based on expert group advice, an additional 20% was added to this to include paralytic/atypical forms of rabies, providing an estimate of 20,565 or about 2 per 100000 population. The majority of the victims were male, adult, from rural areas, and unvaccinated. The main animals responsible for bites were dogs (96.2%), most of which were stray. The most common bite sites were the extremities. The disease incubation period ranged from two weeks to six months. Hydrophobia was the predominant clinical feature. Many of the victims had resorted to indigenous forms of treatment following animal bite, and only about half of them had sought hospital attention. Approximately 10% of these patients had taken a partial course of either Semple or a cell culture vaccine. The islands of Andaman, Nicobar, and Lakshadweep were found to be free of rabies. CONCLUSION: Human rabies continues to be endemic in India except for the islands of Andaman, Nicobar, and Lakshadweep. Dogs continue to be the principal reservoir. The disease is taking its toll on adult men and children, the majority from rural areas, due to lack of awareness about proper post-exposure immunization. The keys to success in the further reduction of rabies in India lies in improved coverage with modern rabies vaccines, canine rabies control, and intensifying public education about the disease. PMID- 16678462 TI - HIV, HBV, HCV, and syphilis co-infections among patients attending the STD clinics of district hospitals in Northern India. AB - OBJECTIVE: The objective of the study was to assess the risk of co-infections with human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis among patients attending sexually transmitted disease (STD) clinics, antenatal clinics (ANC) and Ob-Gyn outpatients department (OPD) clinics which were part of the sentinel surveillance program. METHODS: A serological screening was carried out during the period August-November 2002 to assess the risk of infection with HIV-1/2, and co-infection with HBV, HCV, and syphilis among the outpatients attending STD clinics, Ob-Gyn OPD clinics, and ANC of three district hospitals (Agra, Etawah, and Farrukhabad) of Uttar Pradesh state in Northern India. Unlinked and coded serum samples received from 863 patients (635 females and 228 males) were screened by laboratory tests commonly used for laboratory diagnosis of HIV, HBV, HCV, and syphilis. RESULTS: Among the 863 samples serological reactivity was detected for HIV-1/2 in 21 (2.4%), HBV in 25 (2.9%), HCV in nine (1.0%), and syphilis in 47 (5.4%). The incidence of HBV was higher among males than females, i.e. 10/228 (4.4%) versus 15/635 (2.4%). Co infection was observed for HIV-HBV in two (0.2%), HBV-HCV in one (0.1%), and HIV syphilis in one (0.1%). None were found to have co-infection with HIV-HCV, HBV syphilis, and HCV-syphilis. Age, sex, literacy level, occupation, locality, migration, and presence of different sexually transmitted infections did not significantly influence the rate of HIV positives. CONCLUSION: A substantial percentage of the outpatients seen in the clinics of the district hospital in Uttar Pradesh harbor HIV and viral hepatitis infections, which otherwise would remain undiagnosed without serological screening. PMID- 16678464 TI - Medically attended pediatric influenza during the resurgence of the Victoria lineage of influenza B virus. AB - OBJECTIVES: During the 2002-2003 season, a new variant of influenza B co circulated with influenza A viruses. This study examines the characteristics and outcomes of children with influenza A and B virus infection vs. other acute respiratory illnesses. METHODS: A retrospective chart review was performed on children with laboratory-confirmed influenza infection, and influenza negative acute respiratory illnesses that prompted a hospital visit. RESULTS: Children with influenza were more often previously healthy and presenting with upper respiratory symptoms, while influenza negative patients typically had underlying medical conditions, and lower respiratory tract disease. Children with influenza B were older, were more likely to be in school, and presented with myositis more frequently than those with influenza A. A third of children with influenza A, and 42% with influenza B required hospitalization. The highest hospitalization rates were in infants under one year. No healthy children, and only 15% of those with chronic medical problems, had received influenza vaccine. Vaccine efficacy was estimated to be 82.6%. CONCLUSIONS: Most children with influenza were previously healthy. Overall, a third of children with influenza required hospitalization. Influenza A and B were clinically indistinguishable, except for older age and higher incidence of myositis in patients with influenza B. Influenza vaccine coverage in both healthy and high-risk children was low. PMID- 16678465 TI - Hepatitis delta virus (HDV) genotypes in patients with chronic hepatitis: molecular epidemiology of HDV in Turkey. AB - OBJECTIVE: Analysis of hepatitis delta virus (HDV) isolates from around the world has indicated that there are at least three phylogenetically distinct genotypes with different geographic distributions. The aim of this study was to determine the distribution of HDV genotypes by direct sequencing in patients with chronic delta hepatitis in Izmir, Turkey. DESIGN AND METHODS: Serum samples from 32 chronic hepatitis patients (21 males, 11 females; mean age 44.2 years, range 23 70 years) with anti-delta positivity were analyzed for hepatitis B and C serologies. After reverse transcription, cDNA of partial delta antigen was amplified by in-house nested PCR. The products of the HDV PCR were bidirectionally sequenced with internal primers using Big Dye Terminator DNA Sequencing Kit (Applied Biosystems, CA, USA) and ABI Prism 310 Genetic Analyzer (Perkin Elmer, USA). Nucleotide sequences of HDV were compared with previously reported sequences and aligned by using ClustalW (1.82). RESULTS: HDV-RNA was positive in 26 (81.3%) of 32 anti-delta positive samples. Comparison of the HDV sequences with published sequences of HDV genotypes I, II, and III indicated that all were closely related to HDV genotype I isolates. Similarity among isolated sequences ranged from 84% to 96%. CONCLUSION: HDV genotyping was successfully performed by direct sequencing of the amplicons obtained from routine HDV-RNA screening PCR tests. All of the HDV isolates from the chronic delta hepatitis patients included in this study were found to be genotype I. PMID- 16678466 TI - HIV infection in women utilizing a major hospital in Andhra Pradesh, India, 1993 2004. AB - BACKGROUND: Despite the high prevalence rates of HIV infection in women, epidemiological studies conducted exclusively on HIV-reactive women are very sparse, particularly from the state of Andhra Pradesh, India, where the overall prevalence rate among antenatal women is 2.1%. STUDY POPULATION: Medical records of 2643 HIV-reactive women, identified during a 12-year period, were reviewed for epidemiological and clinical information about HIV in women. RESULTS: The overall prevalence of HIV-reactive women was 1.6% and trends were in parallel with those observed by the HIV sentinel surveillance study from our state. Overall 24.5% of HIV-infected cases were women, ranging from 6.3% in 1993 to 28.9% in 2004. HIV infection was increasingly found in women over 30 years of age. Heterosexual contact (87.3%) was the most common route of HIV transmission. The majority (75.2%) of the women were asymptomatic. Women, compared to men, suffered more from respiratory and bacterial infections, often pneumonia, and from gender specific manifestations like vaginal candidal infections. Following the introduction of antiretroviral (ARV) therapy in our hospital in 1999, only 8.5% of HIV-reactive women have received ARV treatment. CONCLUSION: There is a need for gender- and age-specific HIV surveillance among women, especially from areas of high prevalence. Our study shows that there are good reasons to be concerned about the effects of HIV in women. We stress the importance of increasing the focus of attention on the impact of this pandemic on women as we enter the 25th year since its emergence. PMID- 16678467 TI - Hospital- and community-acquired infections: a point prevalence and risk factors survey in a tertiary care center in Saudi Arabia. AB - BACKGROUND: Point prevalence studies are useful in revealing the prevalence of hospital-acquired infections (HAIs) and community-acquired infections (CAIs). Such information allows prioritization of infection control resources and aids in overall hospital expenditure cut-backs. METHODS: A one-day point prevalence survey was conducted on May 19, 2003 at the King Fahad National Guard Hospital in Riyadh. Since the survey included HAIs and CAIs all patients were included. Data were collected on the underlying diagnosis, infection if present and whether it was hospital-acquired or community-acquired. We identified the presence of a line associated blood stream infection (BSI), ventilator-associated pneumonia (VAP), catheter-associated urinary tract infection (UTI) or a surgical site infection (SSI) based on the United States National Nosocomial Infection Surveillance (NNIS) definitions. RESULTS: Five hundred and sixty-two inpatients were included in the survey. There were 38 patients with 45 (8.0%) HAIs and 76 (13.5%) patients with a CAI. Of the HAIs, 31.1% had a line-related BSI, while 28.9% and 24.4% had a VAP and catheter-related UTI, respectively. Most of the HAIs took place in the intensive care units (ICU) (21 (46.7%)), followed by the medical and surgical wards with six (13.3%) cases in each ward. For all HAIs there was a 12.7-fold increased risk with a hospital stay exceeding eight days (OR: 12.7, CI 3.2-50.6). Most of the 76 CAIs were admitted to the medical ward with community-acquired pneumonia (34.9%) as the most common diagnosis. Among the 89 pathogens isolated, Pseudomonas aeruginosa was the most common (21.3%) followed by Enterococcus spp (16.9%). CONCLUSIONS: The overall rate of HAIs in our hospital was 8%, with significant risk factors including a hospital stay exceeding eight days. A device related infection was more likely in a patient with a venous or bladder catheter in place for more than eight days, or a patient mechanically ventilated for more than eight days. Catheter-related UTIs were identified as an important source of infection, requiring ongoing surveillance. PMID- 16678468 TI - How we interact with objects: learning from brain lesions. AB - Motor deficits are the most common outcome of brain damage. Although a large part of such disturbances arises from loss of elementary sensorimotor functions, several syndromes cannot be explained purely on these bases. In this article, we briefly describe higher-order motor impairments, with specific attention to the characteristic ability of the human hand to interact with objects and tools. Disruption of this motor skill at several independent levels is used to outline a comprehensive model, in which various current proposals for a modular organization of hand-object interactions can be integrated. In this model, cortical mechanisms related to object interaction are independent from representations of the semantic features of objects. PMID- 16678469 TI - Phage as templates for hybrid materials and mediators for nanomaterial synthesis. AB - Genetic engineering of phage provides unprecedented opportunities to build novel nanomaterials by integrating biology, chemistry and materials science principles. By mimicking the evolution process in nature, the phage is used as an information mining tool to identify specific peptide information that can recognize desired materials at the molecular level. The unique recognition peptides of the phage can direct peptide-mediated mineralization processes to grow many useful nanometer-scale electronic and medical materials. The monodispersity and long rod shape of the phage, bearing specific recognition motifs, enable the organization of various nanomaterials into periodically ordered hierarchical structures that could be useful for electronic, optical and biotechnological applications. PMID- 16678470 TI - Nucleic acid aptamers and enzymes as sensors. AB - The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology. PMID- 16678471 TI - Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS. AB - Essential oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba and Cinnamomum camphora are known for their beneficial effects in humans. The present study was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system, the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but caused a significant reduction in revertants and mitotic gene convertants induced among survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same reduced level as the combined treatments with the EOs. This clearly suggests a close relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the EOs appeared to be predominantly linked to the induction of mitochondrial dysfunction. PMID- 16678472 TI - Differential inducibility of rat pulmonary CYP1A1 by cigarette smoke and wood smoke. AB - Mainstream cigarette smoke (CS) and wood smoke (WS) were compared in terms of their pulmonary CYP1A1 inducibility. The inducibility was assessed in pulmonary microsomes from rats exposed to freshly generated CS or WS and in rat lung explants treated with extracts of CS or WS total particulate matter (TPM). Mutagenicity in Salmonella typhimurium TA98 and TA100, an effect established for CS and WS in previous studies, was also examined as a test of the biological activity of the smoke samples in the present study. Pulmonary microsomal CYP1A1 activity (as measured by ethoxyresorufin O-deethylase), was induced 4.4-fold and 8.3-fold following exposure of rats to smoke from a single cigarette and three cigarettes, respectively, relative to the activity in control rats. The induction was paralleled by elevated CYP1A1 mRNA level (by northern blot analysis). WS, in contrast to CS, induced neither pulmonary CYP1A1 activity nor mRNA in exposed rats. CYP1A1 protein (by western blot analysis) was induced in cultured rat lung explants by extracts of CS TPM or by a high concentration (496 nM) of benzo[a]pyrene (B[a]P) but not by extracts of WS TPM or a low concentration (0.110 nM) of B[a]P. The induction by high B[a]P concentration was inhibited by extracts of CS or WS TPM, with the inhibition by extracts of WS TPM (75%) being greater than that by extracts of CS TPM (31%). Extracts of CS TPM were as mutagenic as extracts of WS TPM to Salmonella typhimurium TA98 but were more mutagenic than extracts of WS TPM to Salmonella typhimurium TA100. The results show that CS and WS are mutagenic but that WS differs from CS in its inability to induce pulmonary CYP1A1. PMID- 16678473 TI - Micronucleus test and observation of nuclear alterations in erythrocytes of Nile tilapia exposed to waters affected by refinery effluent. AB - Micronuclei and nuclear alterations tests were performed on erythrocytes of Oreochromis niloticus (Perciformes, Cichlidae) in order to evaluate the water quality from Paraiba do Sul river, in an area affected by effluents from an oil shale processing plant, located in the city of Sao Jose dos Campos, Brazil-SP. Water samples were collected on 2004 May and August (dry season) and on 2004 November and 2005 January (rain season), in three distinct sites, comprising 12 samples. It was possible to detect substances of clastogenic and/or aneugenic potential, as well as cytotoxic substances, chiefly at the point corresponding to the drainage of oil shale plant wastes along the river. The highest incidence of micronuclei and nuclear alterations was detected during May and August, whereas the results obtained in November and January were insignificant. This work shows that the effluent treatment provided by the oil shale plant was not fully efficient to minimize the effect of cytotoxic and mutagenic substances in the test organism surveyed. PMID- 16678474 TI - The mutagenic potential of madder root in dyeing processes in the textile industry. AB - The roots of Rubia tinctorum L. have a long tradition of being used in dyeing processes of textiles from centuries ago until the present time. The colouring principles belong to the class of hydroxyanthraquinones. Concern arose because several of these compounds were recognised as mutagenic in vitro and even carcinogenic in rodents. To assess the possible risk to humans caused by coloured textiles, mutagenicity was investigated with two madder root samples of different origin (Iran and Bhutan) along the entire dyeing process from root extracts to the dyed wool. The Salmonella/microsome test (Ames assay) with the strains TA98, TA100 and TA1537 was used. Significant mutagenic effects could be detected in madder root extracts and also in the final product, the dyed wool. Madder root from Iran showed considerably higher mutagenic responses than samples from Bhutan. Analytical investigations of the extracts by HPLC showed the presence of a spectrum of anthraquinones typical for madder root. Three mutagenic compounds, lucidine, rubiadine and purpuroxanthine, together with the non-mutagenic alizarine could be detected. The mutagenic response of the different samples was positively correlated with the concentration of the mutagenic anthraquinones, and with lucidine in particular. Based on these investigations a risk to dye-house workers and users of textiles dyed with R. tinctorum must be anticipated. PMID- 16678475 TI - Preparation and photoluminescence of a novel beta-diketone ligand containing electro-transporting group and its europium(III) ternary complex. AB - A novel beta-diketone with an electro-transporting oxadiazole group, 1-(4'-(5-(4 methylphenyl)-1,3,4-oxadiazol-2-yl)biphenyl-4-yl)-4,4,4-trifluorobutane-1,3-dione (MPBDTFA), was prepared with high yield. With this synthesized ligand as the first ligand and 1,10-phenanthroline (Phen) as the secondary ligand, a new europium(III) ternary complex, Eu(MPBDTFA)(3)Phen, was synthesized. The new beta diketone and its europium(III) ternary complex were characteristized by elemental analysis, thermo-gravimetric analysis, IR and UV-visible spectroscopies. Photoluminescence measurements indicated that the energy absorbed by the organic ligands was efficiently transfered to the central Eu(3+) ions, and the complex showed intensely and characteristically red emissions due to the (5)D(0)- >(7)F(j) transitions of the central Eu(3+) ions. With an electro-transporting group in molecule and highly thermal stability, the synthesized Eu(III) ternary complex is expected as a red-emitting candidate material for fabrication of organic light-emitting diodes (OLEDs). PMID- 16678476 TI - Absorbance change and static quenching of fluorescence of meso-tetra(4 sulfonatophenyl)porphyrin (TPPS) by trinitrotoluene (TNT). AB - Meso-tetra(4-sulfonatophenyl)porphyrin (TPPS) interacts with trinitrotoluene (TNT) and forms a 1:1 complex with a new absorbance peak at 422 nm. TNT quenches TPPS emission intensity at 645 and 702 nm when excited at 413 nm. The TPPS-TNT complex is formed in the ground state on the basis of a linear Stern-Volmer plot indicative of static quenching. The association constants determined from absorbance and fluorescence studies are in excellent agreement. PMID- 16678477 TI - Signal separation and determination of the enantiomeric purity of primary amines with (-)-myrtenal--a 13C NMR study. AB - The applicability of (-)-myrtenal as a chiral derivatizing agent in combination with (13)C NMR spectroscopy was investigated. (13)C NMR was found to be a valuable tool for the identification and enantiomer differentiation of primary amines including beta-amino alcohols and vicinal diamines. The enantiomeric excess could be determined via automated deconvolution and integration, and was found to be in good accordance with the expected values even in the cases, when enantiomer differentiation was not possible in (1)H NMR spectra. PMID- 16678478 TI - Should aged patients with chronic hepatitis C be treated with interferon and ribavirin combination therapy? AB - The aim of this study was to investigate the efficacy and safety of combination therapy of interferon and ribavirin for aged patients with chronic hepatitis C. METHODS: This study was conducted at Osaka University Hospital and institutions participating in the Osaka Liver Disease Study Group on 329 patients with chronic hepatitis C receiving interferon and ribavirin combination therapy (group A, under 60 year old, n=199; group B, 60-64 year old, n=64; group C, over 65 year old (mean age, 67.8+/-2.2 year old, n=66)). Of the 293 patients who were tested for HCV serotype and HCV viral loads, 215 had HCV-RNA with serotype 1 and high viral loads (1H) and the other 78 had HCV-RNA with serotype 2 or low viral loads (non-1H). RESULTS: In per-protocol analysis, the overall SVR rate of 1H patients was 28% (51/184). Among the 1H patients, the SVR rate was significantly lower in group C (16%) and group B (17%) than in group A (34%) (p<0.05). The overall SVR rate of non-1H patients was 85% (57/67). No significant difference was found in the SVR rate among group C (79%), group B (100%), and group A (84%). On the other hand, the discontinuance of both drugs due to side effects was 29% (19/66) in group C, 20% (13/64) in group B, and 11% (21/199) in group A, with the discontinuance rates being higher in the older group (p=0.002). CONCLUSIONS: In aged chronic hepatitis C patients, interferon and ribavirin combination therapy can be recommended for the non-1H patients who showed a high SVR rate of approximately 65%, but not for the 1H patients. PMID- 16678479 TI - Fucoidan prevents concanavalin A-induced liver injury through induction of endogenous IL-10 in mice. AB - Fucoidan is a complex of sulfated polysaccharides derived from non-mammalian origin such as marine brown algae and induces cytokine expression. We investigated the effect of fucoidan on concanavalin A (Con A)-induced liver injury in mice. Liver injury was induced by an intravenous injection of Con A (18.5mg/kg). Various doses of fucoidan (1-30mg/kg) were intravenously administered 30min before Con A injection. The plasma alanine aminotransferase (ALT) and several cytokines levels were determined, and hepatic histological changes were also assessed. The effect of fucoidan administration by itself on induction of interleukin (IL)-10 in plasma and liver tissue was investigated. Con A administration induced an elevation of plasma ALT level, and fucoidan administration dose-dependently prevented the Con A-induced elevation of plasma ALT. Con A administration increased plasma TNF-alpha and IFN-gamma levels, and fucoidan pretreatment significantly inhibited these alterations and increased plasma IL-10 level. The inhibitory effect of fucoidan on Con A-induced liver injury and production of proinflammatory cytokines were reversed by anti-mouse IL 10 antibody pretreatment. Fucoidan induced the IL-10 production in plasma and liver tissue. These findings suggest that fucoidan prevents Con A-induced liver injury by mediating the endogenous IL-10 production and the inhibition of proinflammatory cytokine in mice. PMID- 16678480 TI - Five-year cohort study of hospitalization for respiratory syncytial virus associated lower respiratory tract infection in African children. AB - OBJECTIVES: To describe the epidemiology of respiratory syncytial virus (RSV) associated lower respiratory tract infection (RSV-LRTI) hospitalizations in South African children over a 5-year period, and determine the impact of gestational age (GA) on the incidence of RSV-LRTI hospitalization. STUDY DESIGN: A cohort of 39,836 children, 6.47% of whom were HIV infected, enrolled into a phase 3 trial were prospectively studied for respiratory viruses when hospitalized for LRTI. RESULTS: The incidence of hospitalization for RSV-LRTI was 19.4 per 1000 in HIV uninfected children and 2.5-fold (95% CI 2.04-3.03) greater in HIV infected children (45.0 per 1000). The incidence of RSV-LRTI was 4.9-fold greater (95% CI 3.9-6.8) in children born at <36 weeks of gestational age (GA) and repeat hospitalizations for RSV-LRTI was 3.7-fold (95% CI 1.4-9.4) more likely in these children (7.3%) than children born at > or =36 weeks of GA (1.9%). The burden of RSV-LRTI was greater in children born at <32 weeks of GA than those born at 32-35 weeks of GA between 6-12 months (P=0.008) and 12-24 months of age (P=0.001). The RSV epidemic occurred at the end of the rainy season and peaked when the monthly temperatures were at its lowest each year. PMID- 16678481 TI - Diversity of cutaneous human papillomavirus types in individuals with and without skin lesion. AB - BACKGROUND: Human papillomavirus (HPV) is ubiquitous on the skin of normal and immunosuppressed hosts. OBJECTIVE: We describe the diversity of HPV types in skin specimens using PCR-sequencing directly and after cloning with FAP59/64 or HVP2/B5 primers. STUDY DESIGN: Cross-sectional analysis of skin swabs. RESULTS: Seventy-five (92.6%) of 81 subjects provided samples that could be analysed with PCR (34 healthy controls <50 years old, 13 healthy controls > or =50 years old, 12 with actinic keratosis (AK), 8 with squamous cell carcinoma (SCC) and 8 renal transplant recipients). HPV DNA was detected more frequently with FAP59/64 (68/75, 91%) than with HVP2/B5 (9/75, 12%) (p<0.001). Agreement of typing results using FAP59/64 primers with both sequencing strategies was fair (mean kappa 0.56+/-0.19, 95% CI: 0.46-0.65). HPV species 1 and 2 of the beta-papillomavirus genus were associated with the presence of AK (OR=24.8, 95% CI: 2.3-262.6). A greater number of HPV types per sample was found in individuals with AK or SCC (p=0.046) or AK alone (p=0.02), than in healthy participants. CONCLUSION: HPV infection on the skin is best evaluated with a combination of primers and sequencing strategies. Novel putative types were frequently detected in SCC. Skin lesions have a greater number of HPV types than normal skin. PMID- 16678482 TI - Bilateral impairment of intracortical inhibition in delayed-onset posthemiplegic dystonia: pathophysiological implications. AB - OBJECTIVE: To study short interval intracortical inhibition (SICI) in a rare patient with segmental dystonia of the left upper limb due to a vascular lesion in the contralateral putamen without corticospinal tract involvement. METHODS: Paired-pulse transcranial magnetic stimulation (TMS) was applied to both hemispheres in a conditioning-test paradigm. Six interstimulus intervals (ISIs) and 4 conditioning stimulation intensities were investigated in two separate sessions. RESULTS: Motor evoked potentials upon single-pulse TMS were within the normal range, whereas paired-pulse TMS revealed major changes in cortical excitability, proving that SICI was bilaterally absent. CONCLUSIONS: The bilateral impairment of SICI cannot be considered the cause of dystonic contractions, but just a predisposing factor. SIGNIFICANCE: The absence of SICI might be regarded as a condition able to promote maladaptive plastic changes triggered by focal lesions in the putamen. PMID- 16678483 TI - F wave study in amyotrophic lateral sclerosis: assessment of balance between upper and lower motor neuron involvement. AB - OBJECTIVE: We sought to record significant F wave variable changes in ALS patients having no advanced disease. Furthermore, an interpretation of these F wave abnormalities in the context of upper (UMN) and lower motor neuron (LMN) dysfunction was attempted. METHODS: Standard motor and sensory conduction study was performed to the ulnar nerves of 23 patients with ALS (13 males and 10 females with mean age 67.2+/-5.3 years), having a clinically predominant LMN syndrome. A series of 40 electrical stimuli were also delivered to both their ulnar nerves in order to obtain F waves. The following F wave variables were estimated: F persistence, F wave latency, amplitude, duration and F chronodispersion. Twenty-three, age-and gender-matched healthy volunteers served as controls. RESULTS: Both the distal and proximal ulnar a-CMAPs (P=0.001) and the MCV (P=0.014) values were significantly decreased in patients, than the controls. The sensory conduction study was normal. The ulnar F wave persistence in the ALS patients was significantly lower than that of the controls (P=0.0007). The mean (P=0.0001), minimal (P=0.0001) and maximal (P=0.0001) F wave latencies were significantly prolonged, the F wave amplitudes (P=0.0001) were significantly higher and the F wave chronodispersion (P=0.014) was significantly increased in the patients than the controls. CONCLUSIONS: Significant F wave abnormalities occur in patients with ALS, even those patients having no advanced disease. Increased F wave amplitudes combined with low persistence is a pattern consistent with ALS. SIGNIFICANCE: Our results show that patients with ALS having predominantly LMN involvement also have electrophysiological UMN dysfunction. PMID- 16678484 TI - A simple model for the generation of the vestibular evoked myogenic potential (VEMP). AB - OBJECTIVE: To describe the mechanism by which the vestibular evoked myogenic potential is generated. METHODS: Vestibular evoked myogenic potential generation is modeled by adding a large number of muscle motor unit action potentials. These action potentials occur randomly in time along a 100 ms long time axis. But because between approximately 15 and 20 ms after a loud short sound stimulus (almost) no action potentials are generated during VEMP measurements in human subjects, no action potentials are present in the model during this time. RESULTS: The evoked potential is the result of the lack of amplitude cancellation in the averaged surface electromyogram at the edges of this 5 ms long time interval. CONCLUSIONS: The relatively simple model describes generation and some properties of the vestibular evoked myogenic potential very well. SIGNIFICANCE: It is shown that, in contrast with other evoked potentials (BAEPs, VERs), the vestibular evoked myogenic potential is the result of an interruption of activity and not that of summed synchronized neural action potentials. PMID- 16678485 TI - Anaemia is an independent predictor of mortality in patients with left ventricular systolic dysfunction following acute myocardial infarction. AB - BACKGROUND: In patients with chronic heart failure (HF), mortality is inversely related to haemoglobin (hgb) concentration. We investigated the prognostic importance of anaemia in patients with acute myocardial infarction (AMI) and left ventricular systolic dysfunction (LVSD) with and without HF. METHODS AND RESULTS: We studied 1731 patients with AMI and left ventricular ejection fraction or =25 years), Australian football players, without a history of hamstring injury in the past 12 months were studied prospectively. Players underwent screening of anthropometric, flexibility and lower extremity range of movement tests during the pre-season period and were followed-up for a full season with respect to injury and match participation. Comparisons of the age groups were performed to identify differences related to age. Logistic regression analysis was undertaken to determine whether the observed differences were predictors of hamstring injury. RESULTS: There were significant differences between the age groups with respect to body weight, body mass index, hip flexor flexibility, hip internal rotation and ankle dorsiflexion range of movement. Body weight and hip flexor flexibility were significant independent predictors of hamstring injury in players aged > or =25 years. None of the observed differences were predictors of injury in the younger age group. CONCLUSIONS: There are age-related changes that are potentially modifiable to reduce injury risk in older athletes and these factors should be considered in the development of hamstring injury prevention programs for this high risk group. PMID- 16678487 TI - Fine-tuning leukocyte responses: towards a chemokine 'interactome'. AB - The extended family of chemokines regulates the activation of leukocytes and coordinates their trafficking to sites of inflammation and during immune surveillance. The heptahelical-receptor-binding and function of chemokines is thought to be governed by their interaction with cell surface proteoglycans, oligomer formation, naturally occurring antagonists and proteolytic processing. Recent studies reveal that heterophilic interactions between chemokines can significantly modify their biological activities and through these we can gain initial insights into the structural basis underlying this novel regulatory mechanism. Here, we propose the concept of a functional 'interactome', constituted by a variety of heterophilic chemokine-chemokine interactions in particular microenvironments. This model could establish how signals conferred by various chemokines are integrated for the combinatorial control of leukocyte responses. PMID- 16678488 TI - Multifaceted antiviral actions of APOBEC3 cytidine deaminases. AB - To defend against external pathogens, metazoan organisms have evolved numerous defenses that generally fall within the innate and adaptive immune responses. Considerable effort continues to focus on developing a vaccine to manipulate the adaptive immune system to protect against or control HIV-1. However, recent advances in our understanding of the innate immune system have revealed that cells have a potent intrinsic antiretroviral defense in the form of APOBEC3G, which is a member of a larger family of cytidine deaminases that are active against HIV-1 and other retroviruses. Insights into how the action of A3G is circumvented by HIV-1 through the action of its Vif protein, and the surprising mechanisms by which A3G is regulated within the cell, offer exciting new opportunities for developing novel anti-HIV-1 therapies that exploit this intrinsic antiretroviral system. PMID- 16678489 TI - TB vaccine strategies--what is needed to solve a complex problem? AB - An estimated 2 billion people are latently infected with Mycobacterium tuberculosis, the majority of which are already BCG vaccinated and repeatedly sensitized to mycobacterial strains from the environment. To be successful in the high endemic regions, any future TB vaccine strategy will have to be tailored in accordance with the resulting complexity of the TB infection and anti mycobacterial immune response. In this review we will discuss some of the most advanced attempts to address this challenge. PMID- 16678490 TI - Spontaneous formation of annular structures observed in molecular dynamics simulations of polyglutamine peptides. AB - Annular structures have been observed experimentally in aggregates of polyglutamine-containing proteins and other proteins associated with diseases of the brain. Here we report the observation of annular structures in molecular level simulations of large systems of model polyglutamine peptides. A system of 24 polyglutamine chains 16 residues long at a concentration of 5 mM spontaneously formed large beta sheets which curved to form tube-like annular structures that resemble beta barrels. This work was accomplished by extending the PRIME model to polyglutamine. PRIME is an off-lattice, unbiased, intermediate-resolution protein model based on an amino acid representation of between three and seven united atoms depending on the residue being modeled. Our results are interesting not only because of the recent discovery of tubular protofibrils in experiments on aggregation of mutant huntingtin fragments containing expanded polyglutamine tracts but also because Perutz predicted that polyglutamine forms water filled nanotubes. PMID- 16678491 TI - Freehand three-dimensional assessment of left ventricular volumes and ejection fraction with ultrasound contrast agent LK565. AB - AIMS: Accurate assessment of left ventricular function by determining left ventricular volumes and ejection fraction is important in evaluating the prognoses of patients with heart failure. Two-dimensional (2D) echocardiography suffers from low correlation with reference methods like ventriculography. Three dimensionally (3D) assessed data have been proved to have better conformity. Endocardial border delineation remains a problem, however, especially in patients with suboptimal recordings. Few data exist on 3D-echocardiographic volumetry with ultrasound contrast agents (UCAs). We evaluated the second-generation UCA LK565 for its boundary-tracing capacities in freehand 3D echocardiography in a phase II clinical trial. Safety and efficacy of the novel contrast agent were also evaluated. METHODS AND RESULTS: Forty patients between the age of 42 and 77 were included in this trial. Left ventricular end-systolic and -diastolic volume (LVESV, LVEDV) and ejection fraction (EF) were determined by either 2D or 3D freehand second harmonic echocardiography with and without use of LK565. Parameters were compared statistically with ventriculography performed in 35 patients. Immune response to LK565 was evaluated by analysing phagocytosis capacity and kinetics of inflammatory cytokines (TNF-alpha, IL-4, IL-10, IFN gamma). Patients were monitored for adverse events up to 72 h after application of the UCA. Calculated values for left ventricular volumes and ejection fraction correlated best for freehand 3D echocardiography in combination with LK565 (r=0.92 for LVEDV; r=0.96 for LVESV; r=0.94 for EF). Excellent left ventricular contrast enhancement was achieved for approximately 8 min. A reversible saturation of phagocytosis capacity for monocytes and neutrophils set in with a maximum peak at 6h. No significant increase in cytokine expression was observed. CONCLUSION: LK565 improves feasibility of endocardial border delineation in 3D echocardiography, leading to better correlation of left ventricular volumetry with reference methods. Efficacy and safety of LK565 are equivalent to those of conventional UCAs. PMID- 16678492 TI - Cryptogenic localization-related epilepsy with childhood onset: The problem of definition and prognosis. AB - BACKGROUND: Up to one-third of children with epilepsy are diagnosed with cryptogenic localization-related epilepsy (CLRE). CLRE is a large nonspecific category within the ILAE classification. For this population no unequivocal prognosis exists. METHODS: Twenty-five articles describing aspects of CLRE were included in this review. RESULTS: As a result of the progress in epilepsy research, as well as more advanced investigation in individual cases, the population with CLRE constantly changes. Also, disagreement on interpretation of the classification has resulted in striking differences between the populations described. High remission rates are reported, but relapse occurs frequently, leaving the long-term prognosis unforeseeable. This is reflected in academic and psychosocial prognosis, which is described to be problematic in CLRE specifically. Possible prognostic factors of CLRE in children have been identified: age at onset, seizure semiology, seizure frequency, intractability, interictal epileptiform activity on EEG, and premorbid IQ. These factors are explored to define subgroups within the CLRE population. DISCUSSION: Prospective studies on well-defined CLRE cohorts are needed to identify factors that distinguish various prognostic subgroups. Specific attention should be focused on course of the epilepsy, scholastic achievement, and psychosocial outcome. PMID- 16678493 TI - Thought disorder: A developmental disability in pediatric epilepsy. AB - This study compared thought disorder (i.e., impaired use of language to formulate and organize thoughts) in 93 children with complex partial seizures (CPSs) and 56 children with primary generalized epilepsy with absence (PGE) and its relationship to age, seizure, cognitive, and linguistic variables. By the use of psychopathology, social competence, academic achievement, and school problem measures, the functional implications of thought disorder in these two groups were compared. When demographic variables were controlled for, there were no significant differences in thought disorder scores between the CPS and PGE groups. However, the profile of age, gender, seizure, and cognitive variables related to thought disorder differed in the CPS and PGE groups. Within each group, different aspects of thought disorder were associated with different seizure variables. Thought disorder was related to psychopathology, school problems, decreased academic achievement, and poor peer interaction in the CPS group, but with school problems in the PGE group. These findings suggest that CPS and PGE affect the normal maturation of children's discourse skills, albeit through different mechanisms. The relationship of thought disorder to behavioral, academic, and social problems implies that these discourse deficits are one component of the developmental disabilities or comorbidities associated with pediatric CPS and PGE. PMID- 16678494 TI - Vasorelaxant effect of euxanthone in the rat thoracic aorta. AB - This study was undertaken to investigate the effect of euxanthone on isolated rat thoracic aorta. Euxanthone concentration-dependently relaxed high K+-induced sustained contractions with IC50 values of 32.28+/-1.73 microM and this inhibition was antagonized by increasing the Ca2+ concentration in the medium. These results indicated that euxanthone may have calcium antagonistic property. Euxanthone also relaxed norepinephrine (NE)-induced sustained contractions with IC50 values of 32.50+/-2.15 microM and this relaxant effect was unaffected by the removal of endothelium or by the presence of propranolol, indomethacin, glibenclamide or N(omega)-nitro-L-arginine. Moreover, euxanthone inhibited both the phasic and tonic contractions induced by NE in a concentration-dependent manner and showed more potent inhibition on phasic contraction (P < 0.01). Pre treatment with euxanthone inhibited vascular contraction induced by phorbol 12, 13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, in either the presence or absence of Ca2+ in the solution with IC50 values of 20.15+/-1.56 and 18.30+/-1.62 microM, respectively. However, when the tissues were treated with euxanthone after the PDBu-induced contraction had reached a steady state, the tension was not affected by euxanthone. This study also showed that the inhibitory effect of pre-treatment of euxanthone was more potent than the post-treatment after the tension had reached a steady state. These results suggested that the vasorelaxation of euxanthone may be through multiple pathways involved PKC mediated signal pathway and calcium-independent pathway besides the direct inhibition of calcium influx and its vasorelaxant effect is more active on calcium-independent pathway and more sensitive to the initial stage of contraction. PMID- 16678496 TI - Familial risk for esophageal cancer: an updated epidemiologic study from Sweden. AB - BACKGROUND AND AIMS: Familial risks for esophageal cancer are not well known, especially for specific histologic types. METHODS: We used the nationwide Swedish Family-Cancer Database to examine familial risks for esophageal cancer in offspring. Standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) were used to calculate the risk. Age standardized incidence rates for specific histology types of esophageal cancer were available from Swedish Cancer Registry. RESULTS: The incidence of male squamous cell carcinoma (SCC) reached its peak rate in 1985 and decreased afterwards. The incidence of adenocarcinoma exceeded that of SCC in 2000 among men. The SIR for offspring esophageal cancer was significantly increased when a parent presented with esophageal cancer (SIR, 2.60). The SIRs for adenocarcinoma were 4.05 and 3.52 when a parent was diagnosed with SCC and any esophageal cancer, respectively; from maternal probands the SIRs were 10.47 and 7.74, respectively. The familial SIR was above unity but not significant for SCC. For associations with other cancer sites, SCC showed a significantly increased risk when a sibling had lung cancer (SIR, 2.52). CONCLUSIONS: The present study showed that adenocarcinoma became the major histologic type of esophageal cancer in Swedish men around 2000. For the first time we could show that familial risks of esophageal adenocarcinoma were increased when parents presented with esophageal cancer (particularly SCC). The high risk for adenocarcinoma in such families might be due to heritable effects. However, because of the limited number of familial cases, the results should be interpreted with caution. PMID- 16678495 TI - Promoter methylation and response to chemotherapy and radiation in esophageal cancer. AB - BACKGROUND & AIMS: Multiple studies have shown that promoter methylation of tumor suppressor genes underlies esophageal carcinogenesis. Hypothetically, methylation resulting in tumor suppressor gene inactivation might result in tumors that are unresponsive to chemotherapy and radiation. Accordingly, our aim was to find methylation markers that could be used to predict response to chemoradiation. METHODS: Tumor specimens were obtained before treatment from 35 patients enrolled in a uniform chemoradiation treatment protocol. Methylation-specific quantitative polymerase chain reaction was performed on all samples. Pathology reports from esophagectomy specimens were used to define response to treatment. RESULTS: Thirteen (37%) of 35 patients were responders, and 22 (63%) of 35 patients were nonresponders. The number of methylated genes per patient was significantly lower in responders than in nonresponders (1.4 vs 2.4 genes per patient; Student t test, P = .026). The combined mean level of promoter methylation of p16, Reprimo, p57, p73, RUNX-3, CHFR, MGMT, TIMP-3, and HPP1 was also lower in responders than in nonresponders (Student t test, P = .003; Mann-Whitney test, P = .001). The frequency (15% of responders vs 64% of nonresponders; Fisher exact test, P = .01) and level (0.078 in responders vs 0.313 in nonresponders; Mann-Whitney test, P = .037) of Reprimo methylation was significantly lower in responders than in nonresponders. CONCLUSIONS: Reprimo methylation occurred at significantly lower levels and less frequently in chemoradioresponsive than in nonresponsive esophageal cancer patients, suggesting potential clinical application of this single-gene biomarker in defining prognosis and management. In addition, increased methylation of a 9-gene panel correlated significantly with poor responsiveness to chemoradiation. PMID- 16678497 TI - Total laparoscopic versus open surgery for stage 1 endometrial cancer: the LACE randomized controlled trial. AB - PURPOSE: Endometrial cancer is the most common gynaecological malignancy in Australia and the US. Current standard treatment involves open surgery to remove the uterus, and both tubes and ovaries (TAH). The Laparoscopic Approach to Cancer of the Endometrium (LACE) trial was designed to assess equivalence of performing this in a total laparoscopic approach (TLH). METHODS: Patient recruitment was designed to proceed along two stages to accommodate for a potential increase in patient requests of laparoscopic surgery. During the first stage, patients are randomised in a 2:1 allocation to receive TLH or TAH, with the primary endpoint quality of life (QoL) at 6 month post-surgery, requiring 180 patients to be enrolled to have 80% power at alpha=0.05 to detect a clinically significant difference of 8 points on the Functional Assessment of Cancer General (FACT-G) QoL instrument. If additional recruitment of patients seems impossible after accrual of 180 patients, this cohort will be followed for 4 years, and disease free survival (DFS) of patients treated by TLH will be compared to DFS within the endometrial cancer population. During the second stage, recruitment will be extended to a total of 590 patients in a 1:1 TLH:TAH allocation, to assess the equivalence with respect to DFS with 80% power and alpha=0.05. Equivalence will be assumed if the difference in DFS does not exceed 7% at 4 years. Secondary outcomes include treatment related morbidity; costs and cost-effectiveness; patterns of recurrence; and overall survival. All data from this multicentre study will be entered using online electronic case report forms (e-CRF), allowing real time assessment of data completeness and patient follow-up. CONCLUSIONS: The LACE trial will establish the equivalence of a TLH approach for patients with stage 1 endometrial cancer following a two stage protocol to accommodate potential threats to patient recruitment through requests for laparoscopic surgery. PMID- 16678498 TI - Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1). AB - We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions. PMID- 16678499 TI - Cloning and developmental expression of the DEC1 ortholog gene in zebrafish. AB - DEC1/STRA13/SHARP2 is a transcription factor of the bHLH family that has been suggested to play key roles in mammalian cell differentiation, the cell cycle and circadian regulation. However, the function of the DEC1 gene during embryogenesis is not well understood. In the present study, we cloned a zebrafish ortholog of the human DEC1 gene and analyzed its expression during development of zebrafish. The predicted protein encoded by zebrafish DEC1 consists of 403 amino acids, and shares 59%, 60% and 59% identity in overall amino acid sequence with human DEC1, mouse STRA13 and rat SHARP2, respectively. Zebrafish DEC1 contains a bHLH domain exhibiting 97% identity with that of the mammalian ortholog. During zebrafish embryogenesis, DEC1 is expressed in a strong ubiquitous manner before early segmentation. At 15-72 hpf, DEC1 shows a specific and dynamic expression in the developing eyes, somites, pineal gland (epiphysis), heart, brain, spinal cord, notochord, pronephric duct, common cardinal vein and blood cells. In older zebrafish, DEC1 also is expressed in multiple tissues including the brain, eye, gut, liver and pancreas. Our data provide evidence that expression of DEC1 is evolutionally conserved in zebrafish. PMID- 16678500 TI - Dipolar ruthenium(II) ammine complexes as electron transfer mediators of amperometric glucose sensors. AB - The mediation of dipolar ruthenium(II) ammine complexes containing pyridinium ions [Ru(NH(3))(5)(L(+))](3+)(L(+): pyridinium ions) in glucose oxidation has been investigated by a voltammetric method. These ruthenium(II) complexes had appropriate redox potentials of 0.10-0.18 V vs. Ag/AgCl and high k(s) values of 5.7-17 x 10(6) M(-1) s(-1) which are the second-order rate constants for electron transfer from glucose oxidase in reduced form to [Ru(NH(3))(5)(L(+))](4+). In particular, the k(s) values for [Ru(NH(3))(5)(L(+))](3+) were greater than those of osmium(II)-polypyridine complexes possessing similar redox potentials which are most commonly used. All the dipolar ruthenium(II) complexes used in this study are therefore concluded to be useful for the electron transfer mediators of amperometric glucose sensors. PMID- 16678501 TI - gamma-H2AX formation in response to interstrand crosslinks requires XPF in human cells. AB - To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (gamma H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, gamma-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced gamma-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO SV and XP3YO) that are also repair-deficient exhibited gamma-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce gamma-H2AX in XP-F cells, although it did induce equivalent levels of gamma-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored gamma-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of gamma-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form gamma-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate. PMID- 16678502 TI - Inhalation solutions: which one are allowed to be mixed? Physico-chemical compatibility of drug solutions in nebulizers. AB - Therapy of chronic respiratory diseases often involves inhalation therapy with nebulizers. Patients often attempt to shorten the time consuming administration procedure by mixing drug solutions/suspensions for simultaneous inhalation. This article considers the issue of physico-chemical compatibility of admixtures of drug solutions/suspensions in nebulizers. A search of databases, prescribing information and primary literature was conducted to locate literature concerning the physico-chemical compatibility of inhalation solutions/suspensions. This was supplemented by telephone interviews. Admixtures of albuterol with ipratropium and/or cromolyn, of albuterol and budesonide, or tobramycin, or colistin are physico-chemically compatible. Physico-chemical compatibility has been demonstrated for admixtures of cromolyn with albuterol and/or ipratropium and for admixtures of cromolyn and budesonide. Admixtures of budesonide with ipratropium and/or fenoterol, and admixtures of budesonide and albuterol, or cromolyn are physico-chemically compatible. Both cromolyn and colistin are incompatible with benzalkonium chloride. Admixtures should be prepared from inhalation solutions/suspensions formulated without preservatives. Besides studies of the physico-chemical compatibility, the aerodynamic behaviour of physico-chemical mixtures needs to be studied before a final recommendation of simultaneous nebulization of compatible admixtures can be made. PMID- 16678503 TI - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Asians with chronic pulmonary disease: a pilot study. AB - BACKGROUND: Little is known about the relationship between cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Asian patients and severe asthma or idiopathic bronchiectasis. We investigated this potential relationship in the Singaporean Chinese. METHODS: Twenty patients with chronic pulmonary disease, 14 with severe asthma and 6 with idiopathic bronchiectasis, were screened for CFTR mutations by direct gene sequencing. The frequencies of identified putative mutations were compared against 40 unaffected controls and 96 unselected population samples. RESULTS: Three missense mutations (I125T, I556V, and Q1352H) and 1 splice site variant (intron 8 12TG5T) were identified in a total of 10 patients, representing a combined mutant/variant allele frequency of 0.25. These alleles were also observed in the controls, but at a significantly lower allele frequency of 0.09 (P<0.01). Furthermore, the I125T mutation was significantly associated with the idiopathic bronchiectasis sub-group (P<0.05). CONCLUSIONS: The significantly higher frequency of CFTR mutations among patients with chronic pulmonary disease compared with unaffected controls suggests that these mutations may increase risk for disease. The association of I125T with idiopathic bronchiectasis alone suggests that different mutations predispose to different disease. PMID- 16678504 TI - Comparison of the IntraLase femtosecond laser and mechanical microkeratome for laser in situ keratomileusis. AB - PURPOSE: To compare clinical outcomes between the IntraLase femtosecond laser and the mechanical microkeratome for creating flaps during laser in situ keratomileusis. DESIGN: Nonrandomized clinical trial. METHODS: The study involved a total of 55 eyes of 30 patients, with 27 eyes of 16 patients comprising the microkeratome group and 28 eyes of 14 patients comprising the IntraLase group. Refractive errors, visual acuity, higher-order aberrations, contrast sensitivity, and corneal sensitivity were compared between the two groups. RESULTS: There were no statistically significant differences between the two groups in terms of refractive errors and postoperative uncorrected visual acuity. There was no loss of best-corrected visual acuity in either group. The higher-order aberrations were similar in both groups (P > .05), except for spherical aberration, which was greater in the microkeratome group (P < .05). In the IntraLase group, the contrast sensitivity value at 12 and 18 cycles per degree under mesopic conditions was significantly improved at three months postoperatively (P < .05). The IntraLase group showed faster corneal sensitivity recovery compared with the microkeratome group, and corneal sensitivity in the peripheral area was nearly normalized at three months postoperatively in the IntraLase group. CONCLUSIONS: The femtosecond laser may have advantages over the microkeratome in the flap making procedure. However, the IntraLase femtosecond laser failed to have significant superiority over the mechanical microkeratome in clinical outcomes, except for faster recovery of corneal sensation, lesser degree of spherical aberration, and some contrast sensitivity value. PMID- 16678506 TI - Bringing the management of accommodative esotropia into sharp focus. PMID- 16678505 TI - Primary transpupillary thermotherapy for 18 small posterior pole uveal melanomas. AB - PURPOSE: To evaluate transpupillary thermotherapy (TTT) for the treatment of small uveal melanomas of the posterior pole. DESIGN: Prospective, nonrandomized interventional case series. METHODS: Eighteen patients underwent TTT for small uveal melanomas located in the posterior pole of the eyes. Tumors were between 2.5 and 4 mm in thickness. TTT was performed with a diode laser at 810 nm. Patients had between one and three TTT sessions, with an intensity adapted to the coloration of the fundus impact. Biomicroscopic examination, ultrasonographic measurements, and angiography were performed before and two months, four months, and six months after treatment, then regularly during follow-up. RESULTS: Eight of the 18 tumors regressed and 10 recurred. The one- and two-year metastasis-free survival rates calculated by the Kaplan-Meier method were, respectively, 61.11% to 44.44% (95% confidence interval). Recurrences were managed with enucleation (three patients), proton beam therapy (six), or additional thermotherapy (one). After treatment, visual acuity was maintained or improved for the eight patients with nonrecurrent tumors. Pathologic analysis of the three enucleated eyes revealed scleral invasion. CONCLUSIONS: Despite encouraging initial short-term results obtained with TTT for the management of small choroidal melanomas, the occurrence of severe complications, especially recurrences and insufficient local tumor control, should raise concern about indications for primary TTT given as isolated treatment for small melanomas of the posterior pole. PMID- 16678507 TI - Medial wall decompression for optic neuropathy but lateral wall decompression with fat removal for non vision-threatening indications. PMID- 16678508 TI - Long-term corneal keratocyte deficits after PRK and LASIK: in vivo evidence of stress-induced premature cellular senescence. PMID- 16678509 TI - Ocular and systemic pseudoexfoliation syndrome. AB - PURPOSE: To provide an update on most recent developments regarding ocular and systemic manifestations and complications, clinical diagnosis and management, and molecular pathophysiology of pseudoexfoliation (PEX) syndrome, and to discuss future tasks and challenges in this field. DESIGN: Perspective. METHODS: Review of recent literature and authors' own clinical and laboratory studies. RESULTS: PEX syndrome is a common age-related generalized fibrotic matrix process of worldwide significance, which may not only cause severe chronic open-angle glaucoma and cataract, but also a spectrum of other serious spontaneous and surgical intraocular complications. Recent progress and advances have led to (1) improvements in clinical management by understanding the effects of the PEX process on ocular tissues, by refining diagnostic criteria, by applying new treatment regimes, and by developing preventive strategies to reduce surgical complications; (2) increasing evidence for systemic associations of PEX with cardiovascular and cerebrovascular morbidity; and (3) new insights into the molecular pathophysiology by analyzing the composition of PEX material, the differential gene expression of affected tissues, and key factors involved in pathogenesis. The current pathogenetic concept describes PEX syndrome as an elastic microfibrillopathy involving transforming growth factor-beta1, oxidative stress, and impaired cellular protection mechanisms as key pathogenetic factors. CONCLUSIONS: Future tasks and challenges comprise epidemiologic prevalence and genetic studies of PEX syndrome, prospective randomized clinical and histopathological screening studies on its systemic manifestations and associations, and intensified basic research on differential protein and gene expression, animal and in vitro models, as well as potential biomarkers for PEX syndrome and its associated glaucoma. PMID- 16678511 TI - Central cone dysfunction in autosomal dominant vitreoretino choroidopathy (ADVIRC). AB - PURPOSE: To describe a patient with clinically documented autosomal dominant vitreoretinochoroidopathy who has had 11 years of progression from initial description and now demonstrates evidence of central cone dysfunction. DESIGN: Case report. METHODS: The patient is a member of a pedigree described in the literature. This is a case report format that follows standard clinical studies. RESULTS: The patient had normal full field electroretinography results but focally reduced multifocal electroretinography results and evidence of macular atrophy on optical coherence tomography. CONCLUSION: Autosomal dominant vitreoretinochoroidopathy may result in central cone dysfunction because of macular atrophy late in the course of the disease, although electroretinography and visual fields remain stable over extended follow-up periods. PMID- 16678510 TI - Intravitreal triamcinolone acetonide in the management of exogenous bacterial endophthalmitis. AB - PURPOSE: To determine the effectiveness of intravitreal triamcinolone acetonide in the management of bacterial endophthalmitis. DESIGN: Interventional case series. METHODS: This pilot case series included five patients (three patients after trauma and two patients after cataract surgery) with culture proven bacterial endophthalmitis. After 48 to 72 hours of the initial surgical procedure, 4 mg/0.1 ml of intravitreal triamcinolone acetonide was injected, after confirmation that the vitreous isolate was sensitive to the initially administered intravitreal antibiotics. All patients received oral ciprofloxacin 750 mg twice each day for seven days. The course of inflammation and the clinical outcome were noted. RESULTS: In all the five cases, there was complete resolution of inflammation with favorable clinical outcome at the end of one month that was maintained at the end of three months. CONCLUSION: Intravitreal triamcinolone acetonide could be a treatment option in the selected cases of bacterial endophthalmitis. PMID- 16678512 TI - Recurrent multiple cavernous hemangiomas of the orbit in association with systemic tumors. AB - PURPOSE: To report the clinical findings and management of multiple, recurrent cavernous hemangiomas of the orbit in a patient with concurrent liver involvement. DESIGN: Interventional case report. METHODS: An analysis of clinical findings, radiology and treatment was conducted with a literature review. RESULTS: A 35-year-old woman with blurry vision and proptosis was found to have multiple cavernous hemangiomas of the orbit. The tumors were excised completely without complication, and the patient subsequently regained her vision. Fifteen years later, the patient had recurrent, multiple cavernous hemangiomas in the same orbit. Further investigations for vague back pain revealed a presumed cavernous hemangioma in the liver and a presumed schwannoma arising from the neural foramen of the spine at the level of T2 through T4. CONCLUSION: Multiple cavernous hemangiomas may recur after complete excision and may exist with concurrent systemic tumors. PMID- 16678513 TI - Resolution of optic nerve edema and improved visual function after optic nerve sheath fenestration in a patient with osteopetrosis. AB - PURPOSE: To describe the outcome of a patient with visual loss and optic nerve edema that resulted from osteopetrosis who underwent an optic nerve sheath fenestration (ONSF). DESIGN: Interventional case report. METHODS: A 33-year-old man with osteopetrosis had bilateral visual field loss and optic nerve edema. Computed tomography and magnetic resonance imaging demonstrated open optic canals. Although a lumbar puncture showed a normal opening pressure, there were other findings that were suggestive of increased intracranial pressure. The patient elected to undergo a unilateral optic nerve sheath fenestration. RESULTS: After ONSF, the patient experienced markedly improved visual acuity, visual field, and optic nerve appearance. CONCLUSION: Individuals with visual loss and optic disk edema that is associated with osteopetrosis may benefit from ONSF if the optic canals appear to be open and the optic nerve edema is thought to be related to increased intracranial pressure. PMID- 16678514 TI - Multiple myeloma manifesting as a salmon patch conjunctival mass. AB - PURPOSE: We report a 70-year-old man with a salmon patch conjunctival mass diagnosed as multiple myeloma. DESIGN: Case report. METHODS: Surgical biopsies of the salmon patch conjunctival mass and bone marrow, as well as hematologic workup for multiple myeloma were performed. RESULTS: Conjunctival biopsy revealed heavy myeloma cells with eccentric nuclei and basophilic cytoplasm infiltrate in the conjunctival stroma. Bone marrow biopsy and aspirate showed interstitial infiltrated by myeloma cells, which stained monoclonally for immunoglobulin G (IgG)-kappa light chains. Hematologic tests confirmed the diagnosis of multiple myeloma. CONCLUSIONS: Multiple myeloma is one of the causes of a salmon patch conjunctival lesion and may be included in the differential diagnosis. PMID- 16678515 TI - Bacterial endophthalmitis after resident-performed cataract surgery. AB - PURPOSE: To determine if there is an increased rate of postoperative bacterial endophthalmitis after resident-performed cataract extraction relative to the reported rates of experienced surgeons. DESIGN: Retrospective, observational case series. METHODS: The operative reports of the resident-performed cataract surgeries at San Francisco General Hospital between 1983 and 2002 were reviewed. Cases of culture-positive bacterial endophthalmitis and vitreous loss were identified. RESULTS: Between 1983 and 2002, three cases (0.11%) of culture positive bacterial endophthalmitis occurred after 2718 resident-performed cataract extractions. The overall vitreous loss rate was 6.7%. Two endophthalmitis cases were acute (Staphylococcus epidermidis, Streptococcus viridans), presenting within five days of surgeries complicated by vitreous loss, and one case was delayed-onset (Corynebacterium species) after Nd:YAG posterior capsulotomy after uncomplicated cataract extraction. CONCLUSIONS: Despite higher rates of vitreous loss, the rate of endophthalmitis following resident-performed cataract surgery remains comparable with the rates of more experienced surgeons. PMID- 16678516 TI - Hemoglobin A1c awareness among patients receiving eye care at a tertiary ophthalmic center. AB - PURPOSE: To describe awareness of hemoglobin A1c (HbA1c) among patients receiving eye care at a tertiary ophthalmic center for diabetic eye disease. DESIGN: Prospective, nonrandomized clinical case series. METHODS: One hundred-fifty adult patients with diabetes mellitus (DM) examined between July and October of 2003 for diabetic retinopathy (DR) were surveyed about HbA1c knowledge. RESULTS: Seventy-six (51%) understood HbA1c, 25 (17%) were not sure if they understood, and 49 (33%) did not understand. Patients who understood HbA1c based on duration of DM were as low as two (29%) of seven with DM < 5 years and as high as 17 (55%) of 31 with DM > 15 years. Seventy-six (51%) had proliferative DR, among whom 33 (43%) understood HbA1c, 69 (46%) had nonproliferative DR, among whom 39 (57%) understood HbA1c, and five (3%) had no DR, among whom four (80%) understood HbA1c. CONCLUSION: Approximately half of diabetic patients surveyed understood HbA1c. PMID- 16678517 TI - Combined 50- and 20-MHz frequency ultrasound imaging in intermediate uveitis. AB - PURPOSE: To evaluate the information provided by multifrequency ultrasound examination in patients with intermediate uveitis. DESIGN: Prospective observational case series. METHOD: High-resolution ultrasonography with 50- and 20-MHz frequency immersion probes was performed in seven eyes of five young patients with clinical diagnosis of intermediate uveitis. RESULTS: Exudative material over the inferior pars plana and peripheral retina (snowbank) was found in all eyes with 50- and 20-MHz probe, although resolution of the latter was poorer. The 50-MHz imaging was superior for visualization of angle structures and details of pars planitis; the anterior vitreous involvement and cyclitic bands were better shown with the 20-MHz probe, which could also evidence cystoid macular edema. CONCLUSIONS: Ultrasound examination with both 50- and 20-MHz frequency probes can detect the typical snowbank in intermediate uveitis and be useful in eyes with small pupil, dense vitreitis, or both, especially before pars plana vitrectomy or cataract surgery. PMID- 16678518 TI - Central corneal thickness in children: stability over time. AB - PURPOSE: To evaluate central corneal thickness (CCT) in children over time. DESIGN: Prospective observational case series. METHODS: CCT was measured by ultrasonic pachymetry in 69 eyes of 38 subjects age 3 to 14 years recruited from the Duke University Eye Center. Subjects included patients with and without glaucoma. RESULTS: For eyes on no glaucoma medication, the mean change in CCT was -1.9 +/- 14 microm (n = 30; mean time between CCT measurements, 567 days). For eyes on stable glaucoma medication, the mean change in CCT was +8.1 +/- 26 microm (n = 27; mean time between CCT measurements, 580 days). For eyes with a change in medical therapy between visits, the mean change in CCT was -3.8 +/- 24 microm (n = 8, mean number of days between visits 723). Four eyes underwent trabeculectomy between measurements and experienced a mean change in CCT of -9.0 +/- 6 microm with a mean of 1257 days between measurements. These CCT changes were not statistically significant. CONCLUSIONS: CCT of children ages 3 to 14 years appears stable over a one- to two-year interval. PMID- 16678519 TI - Implanting a clear intraocular lens in one eye and a yellow lens in the other eye: a case series. AB - PURPOSE: To describe the color vision disturbance reported by patients in whom a clear intraocular lens (IOL) was implanted in one eye and a yellow-tinted (blue light-absorbing) IOL in the other eye. DESIGN: Retrospective interventional case series. METHODS: Data recorded included demographic information, dates of surgery, IOL model and power (manufacturer is the same for all lenses), best corrected visual acuity, and subjective visual complaints. RESULTS: Four of five patients had no spontaneous color vision complaints. When these patients were informed of the unintended mismatch, all remarked that they could perceive a color vision difference, but that it was not bothersome. One of the five patients reported "beige" vision. None of the patients wanted an IOL exchange. CONCLUSION: Many patients can tolerate the color vision imbalance that results when a clear IOL is implanted in one eye and a yellow-tinted IOL is implanted in the other eye. PMID- 16678520 TI - Recurrent bleeding after photodynamic therapy in polypoidal choroidal vasculopathy. AB - PURPOSE: To report a case of recurrent bleeding after photodynamic therapy (PDT) in an eye with polypoidal choroidal vasculopathy (PCV). DESIGN: Interventional case report. METHODS: A 73-year-old man was treated in the left eye for PCV with PDT. RESULTS: Two weeks after PDT, his left eye showed extensive subretinal hemorrhage, with a slight vision loss. Three months after PDT, subretinal hemorrhage was almost absorbed. He received a second session of PDT to the remaining choroidal neovascularization. Two weeks thereafter, his left eye showed massive suprachoroidal hemorrhage with further vision loss. One month after the second PDT, visual acuity was decreased to no light perception as a result of massive vitreous hemorrhage. Although the patient underwent pars plana vitrectomy, visual acuity in the left eye remained hand motion as a result of massive suprachoroidal hemorrhage. CONCLUSIONS: Ophthalmologists and patients should be aware of the risk of massive bleeding after PDT in eyes with PCV. PMID- 16678521 TI - Treatment of traumatic LASIK flap dislocation and epithelial ingrowth with fibrin glue. AB - PURPOSE: To describe a case of a traumatic late dislocation of a laser-assisted in situ keratomileusis (LASIK) flap complicated by epithelial ingrowth. DESIGN: Interventional case report. METHODS: A 50-year-old woman presented 21 months after uncomplicated LASIK with painful vision loss in the right eye after minor trauma. RESULTS: A dislocation of the LASIK flap was noted at examination and was repositioned. One week later, epithelial ingrowth was detected in the flap interface. The ingrowth was treated with flap lifting, debridement, and sealing of the flap with fibrin glue. Visual acuity returned to baseline, and there was no recurrence after 20 months of follow-up. CONCLUSIONS: Traumatic dislocations of LASIK flaps may occur many months after uncomplicated surgery and may be associated with epithelial ingrowth after successful repositioning. The additional use of fibrin glue in conjunction with thorough debridement may be helpful in preventing the recurrence of epithelial ingrowth. PMID- 16678522 TI - Macular hole formation, spontaneous closure, and recurrence in a previously vitrectomized eye. AB - PURPOSE: To report a case of recurrent idiopathic macular hole after spontaneous closure in a previously vitrectomized eye. DESIGN: Case report. METHODS: A 69 year-old woman who had had vitrectomy for vitreous hemorrhage seven years earlier presented with decreased visual acuity and a macular hole. The hole spontaneously resolved within four months. One year later, the macular hole reopened. Internal limiting membrane peeling was performed, resulting in return of visual acuity. Optical coherence tomography (OCT) was obtained during all visits. RESULTS: Initial OCT revealed a macular hole with perifoveal cystoid degeneration and bridging elements. At subsequent visits, OCT showed resolution of the cystoid degeneration. The preoperative OCT demonstrates recurrence of the macular hole with similar perifoveal abnormalities. CONCLUSIONS: After vitrectomy, anteroposterior vitreofoveal traction associated with most macular hole formation does not apply. OCT demonstrates foveal structure and forces coincident with hole formation and resolution. Dynamic interaction between tensile and degenerative forces, and proliferative and reparative glial elements, may lead to hole remodeling. PMID- 16678523 TI - Identification of retained nucleus fragment in the posterior chamber using ultrasound biomicroscopy. AB - PURPOSE: To describe the use of ultrasound biomicroscopy (UBM) to image retained nuclear fragments posterior to the iris plane after uncomplicated phacoemulsification with posterior chamber intraocular lens (PCIOL) implantation in two patients. DESIGN: Interventional case report. METHODS: Two patients presented with an iris elevation after uncomplicated phacoemulsification and lens implantation. RESULTS: Visual acuity was 20/20 in the first patient and 20/30 in the second. There was no anterior chamber inflammation in either eye. UBM revealed a small, retained nuclear fragment between the iris pigment epithelium and the anterior lens capsule, causing localized anterior iris displacement in both patients. The PCIOL was within the capsular bag, and the iris root and ciliary body were normal in both patients. CONCLUSION: UBM can demonstrate retained lens fragments within the posterior chamber. PMID- 16678524 TI - Modification of screening criteria for retinopathy of prematurity in India and other middle-income countries. AB - PURPOSE: To determine the gestational age and birth weight of premature babies who developed vision-threatening severe retinopathy of prematurity. DESIGN: Prospective observational study of babies at risk of blindness due to retinopathy of prematurity. METHODS: Data pertaining to all babies who underwent laser treatment or surgery for acute retinopathy of prematurity and those who had cicatricial retinopathy of prematurity were prospectively collected. The gestational age and birth weight were evaluated to establish screening criteria. RESULTS: In 120 babies, the mean gestational age was 29.6 weeks (range 26 to 36 weeks). The birth weight of 115 babies ranged from 710 to 2000 g (mean 1254.5 g, SD 280.8). Birth weight was not available for five babies. Only one baby had a gestational age of >35 weeks. CONCLUSIONS: Ocular morbidity related to retinopathy of prematurity was seen in bigger and more mature babies. This study provides a scientific basis for establishing screening criteria for retinopathy of prematurity in South India and other middle-income countries. PMID- 16678525 TI - Combining surgical ablation of retinal inflow and outflow vessels with photodynamic therapy for retinal angiomatous proliferation. AB - PURPOSE: To evaluate short-term efficacy of combining surgical ablation of retinal inflow and outflow vessels and photodynamic therapy (PDT) for stage 3 retinal angiomatous proliferation (RAP). DESIGN: Prospective interventional case series. METHODS: Five eyes (five patients) underwent surgical ablation of inflow and outflow vessels and PDT for stage 3 RAP. RESULTS: Inflow and outflow vessels were ablated in four eyes, and only inflow was ablated in one eye. In four eyes starting PDT within six weeks of surgical ablation, choroidal neovascularization (CNV) disappeared or shrank after one PDT session (three months follow-up). However, CNV enlarged again in three of the four (final follow-up) because of reperfusion from newly formed inflow vessels. In one eye starting PDT seven months after ablation, a new retinal inflow vessel feeding the CNV appeared by the time of the first PDT session. The CNV continued to expand, despite two PDT sessions. CONCLUSIONS: Combining surgical ablation and PDT was not useful on account of a high frequency of reperfusion from retinal inflow vessels. PMID- 16678526 TI - Optical coherence tomography measurement of retinal nerve fiber layer after acute primary angle closure with normal visual field. AB - PURPOSE: To detect retinal nerve fiber layer (RNFL) thickness after a single acute primary angle closure (APAC) attack with normal visual field. DESIGN: Prospective, observational case study. METHODS: Twenty-eight patients who had a single unilateral APAC attack associated with normal visual fields at three months after remission and 28 single eyes from normal controls were enrolled. Duration of APAC attack was 5.9 +/- 2.7 hours. Comparisons of average RNFL thickness and RNFL thickness in four quadrants and in 12-o'clock-hour segments of the attacked eyes, unaffected fellow eyes, and normal controls were made using fast RNFL thickness (3.4) of Stratus optical coherence tomography. RESULTS: Significant differences were demonstrated comparing the inferior-quadrant RNFL thicknesses for the attacked eyes with those of the normal controls (P = .02) and fellow eyes (P = .02) using one-way analysis of variance with Tukey's tests. CONCLUSIONS: Inferior region RNFL thickness was significantly decreased in the attacked eyes after an APAC episode associated with normal visual field. PMID- 16678527 TI - Combined en-face optical coherence tomography and confocal ophthalmoscopy findings in active multifocal and serpiginous chorioretinitis. AB - PURPOSE: To describe retinal changes in active multifocal and serpiginous chorioretinitis seen with en-face optical coherence tomography (OCT). DESIGN: Observational case report. METHODS: Two patients, presenting with active multifocal chorioretinitis (MFC) and serpiginous chorioretinitis (SC), respectively, were scanned with a prototype en face OCT ophthalmoscope. RESULTS: The OCT scan in the patient with the active MFC lesion showed a transretinal hyperreflectivity at the site of lesion. The OCT scan in the patient with the active SC lesion showed hyper-reflectivity in the outer retina at the site of the lesion. CONCLUSIONS: OCT allows for better evaluation of intraretinal changes in patients with white dot syndromes. In these two cases, OCT showed different morphologic changes in active lesions. OCT may be helpful in identifying the location of involvement in various white dot syndromes. This may have prognostic and therapeutic significance. PMID- 16678528 TI - Indocyanine green angiography abnormality of the periphery in vitelliform macular dystrophy. AB - PURPOSE: To report the peripheral abnormalities seen only with indocyanine green angiography in patients with vitelliform macular dystrophy (Best disease, caused by a mutation in the bestrophin gene). DESIGN: Observational case report series. METHODS: Eight eyes of four patients, two with only a central macular lesion and two with multifocal lesions, were studied. Results of indocyanine green angiography were compared with findings from ophthalmoscopy and fluorescein angiography. RESULTS: Throughout the fundus periphery, indocyanine green angiography demonstrated a number of hyperfluorescent spots in all eight eyes. The spots were observed in the midperiphery and the periphery in areas with no abnormality visible by ophthalmoscopy or fluorescein angiography. CONCLUSIONS: Although Best disease generally causes lesions visible in the posterior pole, the extensive distribution of the hyperfluorescent spots is consistent with the wide ranging abnormalities of the retinal pigment epithelium, Bruch membrane, and the choroid as seen histopathologically. PMID- 16678530 TI - Risk of retinopathy in children with type 1 diabetes mellitus before two years of age. PMID- 16678531 TI - A prospective randomized clinical trial of laser in situ keratomileusis with two different lasers. PMID- 16678533 TI - Diagnostic testing of vitrectomy specimens. PMID- 16678536 TI - Risk of sudden visual loss after filtration surgery in end-stage glaucoma. PMID- 16678538 TI - How does vitrectomy affect diabetic macular edema? PMID- 16678539 TI - Paracentesis before or after intravitreal injection of triamcinolone acetonide and its necessity? PMID- 16678542 TI - Toward individualized analgesic therapy: functional cyclooxygenase 1 and 2 haplotypes. PMID- 16678543 TI - Genetically mediated interindividual variation in analgesic responses to cyclooxygenase inhibitory drugs. AB - BACKGROUND: Wide interindividual variation in responses to cyclooxygenase (COX) inhibitory drugs limits their clinical utility and safety. METHODS: To better understand the molecular responses to COX inhibition, we analyzed the gene expression level of the genes encoding enzymes related to prostaglandin production including the COX-1 gene (PTGS1) and the COX-2 gene (PTGS2), as well as their genetic polymorphisms, and the analgesic response to COX inhibitory drugs such as ibuprofen or rofecoxib or to placebo after minor surgery. RESULTS: Notable heterogeneity in global gene expression was evident between subjects. At 2 to 4 hours after surgery, PTGS1 expression was slightly decreased (36%, P < .001) and PTGS2 expression was markedly increased (300%, P < .001) with wide interindividual variation; at 48 hours after surgery, little detectable change in PTGS1 and PTGS2 expression was found in the control group. However, ibuprofen and rofecoxib treatment significantly increased PTGS2 expression at 48 hours (P = .001 and P = .049, respectively). At 2 to 4 hours after surgery, patients with the G/G allele at the nucleotide position of -765G>C in PTGS2 showed a significantly higher increase in PTGS2 expression (P = .012) compared with G/C and C/C patients, although all of them showed an increase in PTGS2 expression (P < .001 and P = .043, respectively). Among G/G patients, rofecoxib administration resulted in significantly lower pain intensity on a visual analog scale (7.2 +/- 2.5 mm) (P = .008) at 48 hours after surgery, as compared with ibuprofen administration (31.3 +/- 6.7 mm). The finding regarding pain intensity at 48 hours in G/C and C/C patients was opposite (P = .002), being greater in the rofecoxib group (37 +/- 6.8 mm) compared with the ibuprofen group (7 +/- 1.9 mm). CONCLUSIONS: These results suggest that wide variability in gene expression and functional polymorphisms in PTGS2 may explain part of the interindividual variations in acute pain and the analgesic efficacy of nonsteroidal anti inflammatory drugs and selective COX-2 inhibitors; this may be useful to define individual responders on the basis of genetic variations to predict patient risk and benefit to drugs. PMID- 16678544 TI - Impact of the SLCO1B1 polymorphism on the pharmacokinetics and lipid-lowering efficacy of multiple-dose pravastatin. AB - BACKGROUND AND OBJECTIVE: The polymorphism of SLCO1B1 (solute carrier organic anion transporter family, member 1B1), encoding the hepatic uptake transporter organic anion transporting polypeptide 1B1, has been associated with increased pravastatin concentrations in single-dose studies. We have investigated whether this polymorphism influences the pharmacokinetics and lipid-lowering efficacy of multiple-dose pravastatin. METHODS: A prospective, parallel-group study of 16 healthy volunteers, including 8 carriers of an SLCO1B1 variant haplotype and 8 control subjects, was carried out. Pravastatin pharmacokinetics and reduction in total and low-density lipoprotein (LDL) cholesterol concentrations were analyzed after treatment with 40 mg pravastatin daily for 3 weeks. RESULTS: The mean area under the plasma concentration-time curve of pravastatin was 110% higher (305.0 +/- 149.4 ng . h/mL [mean +/- SD] versus 144.9 +/- 48.2 ng . h/mL, P = .012) and the mean peak concentration in plasma was 231% higher (174.5 +/- 98.6 ng/mL versus 52.7 +/- 22.1 ng/mL, P = .0042) in the SLCO1B1 variant haplotype group than in the control group. Pravastatin significantly reduced the concentrations of total and LDL cholesterol in both groups. The mean percentage reduction in total cholesterol concentration was 13.1% +/- 9.1% and 19.1% +/- 8.3% in the variant and control groups, respectively (P = .19; 95% confidence interval of difference between groups, -15.3% to 3.3%). The mean percentage reduction in LDL cholesterol concentration was 27.7% +/- 8.3% in the variant group and 32.3% +/- 11.2% in the control group (P = .37; 95% confidence interval for difference, 15.1% to 6.0%). CONCLUSIONS: Despite considerably higher plasma pravastatin concentrations in carriers of an SLCO1B1 variant haplotype, there was no significant difference in the lipid-lowering efficacy of pravastatin between the variant haplotype and control groups. However, this pilot study had sufficient statistical power to detect only a large difference in lipid response between the 2 groups. Further clinical studies are warranted to characterize the impact of the SLCO1B1 polymorphism on the lipid response to pravastatin. PMID- 16678545 TI - Effects of organic anion transporting polypeptide 1B1 haplotype on pharmacokinetics of pravastatin, valsartan, and temocapril. AB - OBJECTIVE: Recent reports have shown that genetic polymorphisms in organic anion transporting polypeptide (OATP) 1B1 have an effect on the pharmacokinetics of drugs. However, the impact of OATP1B1*1b alleles, the frequency of which is high in all ethnicities, on the pharmacokinetics of substrate drugs is not known after complete separation of subjects with OATP1B1*1a and *1b. Furthermore, the correlation between the clearances of OATP1B1 substrate drugs in individuals has not been characterized. We investigated the effect of genetic polymorphism of OATP1B1, particularly the *1b allele, on the pharmacokinetics of 3 anionic drugs, pravastatin, valsartan, and temocapril, in Japanese subjects. METHODS: Twenty three healthy Japanese volunteers were enrolled in a 3-period crossover study. In each period, after a single oral administration of pravastatin, valsartan, or temocapril, plasma and urine were collected for up to 24 hours. RESULTS: The area under the plasma concentration-time curve (AUC) of pravastatin in *1b/*1b carriers (47.4 +/- 19.9 ng.h/mL) was 65% of that in *1a/*1a carriers (73.2 +/- 23.5 ng.h/mL) (P = .049). Carriers of *1b/*15 (38.2 +/- 15.9 ng.h/mL) exhibited a 45% lower AUC than *1a/*15 carriers (69.2 +/- 23.4 ng.h/mL) (P = .024). In the case of valsartan we observed a similar trend as with pravastatin, although the difference was not statistically significant (9.01 +/- 3.33 microg.h/mL for *1b/*1b carriers versus 12.3 +/- 4.6 microg.h/mL for *1a/*1a carriers [P = .171] and 6.31 +/- 3.64 microg.h/mL for *1b/*15 carriers versus 9.40 +/- 4.34 microg.h/mL for *1a/*15 carriers [P = .213]). The AUC of temocapril also showed a similar trend (12.4 +/- 4.1 ng.h/mL for *1b/*1b carriers versus 18.5 +/- 7.7 ng.h/mL for *1a/*1a carriers [P = .061] and 16.4 +/- 5.0 ng.h/mL for *1b/*15 carriers versus 19.0 +/- 4.1 ng.h/mL for *1a/*15 carriers [P = .425]), whereas that of temocaprilat (active form of temocapril) was not significantly affected by the haplotype of OATP1B1. Interestingly, the AUC of valsartan and temocapril in each subject was significantly correlated with that of pravastatin (R = 0.630 and 0.602, P < .01). The renal clearance remained unchanged for each haplotype for all drugs. CONCLUSION: The major clearance mechanism of pravastatin, valsartan, and temocapril appears to be similar, and OATP1B1*1b is one of the determinant factors governing the interindividual variability in the pharmacokinetics of pravastatin and, possibly, valsartan and temocapril. PMID- 16678546 TI - Erythromycin breath test results in elderly, very elderly, and frail elderly persons. AB - BACKGROUND AND OBJECTIVE: Although increasing age is often thought to be associated with slower oxidative drug clearance, limited data from old, very old, and frail patients exist. Our objective was to determine the effects of very old age and frailty on erythromycin breath test (ERBT) results in patients and, secondarily, to determine the effects of sex and comedications on ERBT results. METHODS: ERBTs were performed in 60 individuals aged 65 to 101 years, 27 of whom were classified as frail (mean age [+/-SD], 86 +/- 6 years; mean weight, 66 +/- 17.4 kg; 8 men and 19 women; 24 living in a nursing home and 3 living in the community) and 33 of whom were classified as nonfrail (mean age, 79 +/- 8 years; mean weight, 78.5 +/- 14.5 kg; 16 men and 17 women; 17 living in a nursing home and 16 living in the community). RESULTS: ERBT results were faster in women versus men (3.53% +/- 1.15% carbon 14 [14C] excreted/h versus 2.59% +/- 0.99% 14C excreted/h, respectively; ANOVA, P < .002), faster in frail versus nonfrail patients (3.58% +/- 1.2% 14C excreted/h versus 2.81% +/- 1.06% (14)C excreted/h, respectively; ANOVA, P = .01), and faster in those receiving cytochrome P450 (CYP) 3A inducers versus those without inducers (5.02% +/- 0.89% 14C excreted/h versus 3.02% +/- 1.08% 14C excreted/h, respectively; ANOVA, P = .0007). When subjects receiving CYP inducers were excluded, sex affected ERBT results (ANOVA, P < .007) but frailty effects were not detected (P = .09). Age from 65 to 101 years did not affect ERBT results (r2 = 0.001). CONCLUSIONS: Old and very old patients in the clinical setting, especially women and those receiving enzyme inducers, may have preserved medication clearance. Frail elderly persons do not have slower ERBT results compared with nonfrail elderly persons. PMID- 16678547 TI - Effects of ursodeoxycholic acid on P-glycoprotein and cytochrome P450 3A4 dependent pharmacokinetics in humans. AB - BACKGROUND AND OBJECTIVE: On the basis of in vitro studies indicating that ursodeoxycholic acid (UDCA) is a cytochrome P450 (CYP) 3A4 inducer and a pregnane X receptor activator and because the pregnane X receptor is a transcriptional regulator of multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp), we postulated that UDCA might decrease the bioavailability of CYP3A4 and P-gp probe drugs in humans. The main objective of this study was to determine whether UDCA alters the pharmacokinetics of digoxin and midazolam. The secondary objective was to determine whether the intestinal expression of P-gp and CYP3A4 is increased by UDCA. METHODS: The effect of UDCA on MDR1 and CYP3A4 messenger ribonucleic acid (mRNA) expression was investigated in human colon carcinoma cell lines LS174T and Caco-2. Eight healthy volunteers participated in this open, nonrandomized 2 period study, in which the effects of UDCA (13 mg.kg(-1).d(-1) during 2 weeks) versus control on the pharmacokinetics of digoxin (0.5-mg single intravenous infusion), d3-digoxin (3-fold deuterated digoxin, 0.5-mg single oral dose), and midazolam (7.5-mg single oral dose) were compared. Duodenal biopsy specimens were obtained during both periods to quantify MDR1/P-gp and CYP3A4 expression. RESULTS: In vitro UDCA induced MDR1 and CYP3A4 mRNA in Caco-2 cells but not in LS174T cells. In humans UDCA significantly decreased the extent of digoxin absorption from 0.77 to 0.70 and the oral d3-digoxin area under the plasma concentration-time curve from 0 to 4 hours from 6.4 +/- 1.7 ng.h.mL(-1) to 5.3 +/ 1.5 ng.h.mL(-1) (P = .01 and P = .05, respectively). UDCA had no detectable effects on the pharmacokinetics of midazolam or the intestinal mRNA and protein expression levels of MDR1/P-gp and CYP3A4. CONCLUSION: Under the conditions in our study, UDCA only modestly decreased digoxin disposition without detectable changes in midazolam pharmacokinetics. The clinical relevance of these findings remains to be determined. PMID- 16678548 TI - Pharmacokinetics and pharmacodynamics of oral oxycodone in healthy human subjects: role of circulating active metabolites. AB - BACKGROUND: In vitro experiments suggest that circulating metabolites of oxycodone are opioid receptor agonists. Clinical and animal studies to date have failed to demonstrate a significant contribution of the O-demethylated metabolite oxymorphone toward the clinical effects of the parent drug, but the role of other putative circulating active metabolites in oxycodone pharmacodynamics remains to be examined. METHODS: Pharmacokinetics and pharmacodynamics of oxycodone were investigated in healthy human volunteers; measurements included the time course of plasma concentrations and urinary excretion of metabolites derived from N demethylation, O-demethylation, and 6-keto-reduction, along with the time course of miosis and subjective opioid side effects. The contribution of circulating metabolites to oxycodone pharmacodynamics was analyzed by pharmacokinetic pharmacodynamic modeling. The human study was complemented by in vitro measurements of opioid receptor binding and activation studies, as well as in vivo studies of the brain distribution of oxycodone and its metabolites in rats. RESULTS: Urinary metabolites derived from cytochrome P450 (CYP) 3A-mediated N demethylation of oxycodone (noroxycodone, noroxymorphone, and alpha- and beta noroxycodol) accounted for 45% +/- 21% of the dose, whereas CYP2D6-mediated O demethylation (oxymorphone and alpha- and beta-oxymorphol) and 6-keto-reduction (alpha- and beta-oxycodol) accounted for 11% +/- 6% and 8% +/- 6% of the dose, respectively. Noroxycodone and noroxymorphone were the major metabolites in circulation with elimination half-lives longer than that of oxycodone, but their uptake into the rat brain was significantly lower compared with that of the parent drug. Pharmacokinetic-pharmacodynamic modeling indicated that the time course of pupil constriction is fully explained by the plasma concentration of the parent drug, oxycodone, alone. The metabolites do not contribute to the central effects, either because of their low potency or low abundance in circulation or as a result of their poor uptake into the brain. CONCLUSIONS: CYP3A-mediated N-demethylation is the principal metabolic pathway of oxycodone in humans. The central opioid effects of oxycodone are governed by the parent drug, with a negligible contribution from its circulating oxidative and reductive metabolites. PMID- 16678549 TI - Female sex and oral contraceptive use accelerate nicotine metabolism. AB - BACKGROUND: Several studies have reported that female smokers have a higher risk of lung cancer than male smokers. This could be related to sex differences in nicotine metabolism and related smoking behavior. This study tested the hypothesis that women metabolize nicotine more rapidly than men and that, among women, oral contraceptive users metabolize nicotine more rapidly than nonusers of oral contraceptives. METHODS: Two hundred seventy-eight healthy volunteers who were twins and 16 who were siblings of twins, recruited from the Northern California Twin Registry, received an infusion of deuterium-labeled nicotine and cotinine with frequent blood sampling. The plasma clearances of nicotine and cotinine, the clearance of nicotine to cotinine (an index of cytochrome P450 [CYP] 2A6 activity), and the ratio of trans-3'-hydroxycotinine to cotinine (another indicator of CYP2A6 activity) were measured. RESULTS: The clearances of nicotine and cotinine, the clearance of nicotine to cotinine, and the trans-3' hydroxycotinine/cotinine ratio were significantly higher in women than in men (nicotine clearance, 15.6 +/- 4.3 mL.min(-1).kg(-1) in men versus 18.8 +/- 6.6 mL.min(-1).kg(-1) in women; P < .001); they were also higher among women taking oral contraceptives than in those who were not taking oral contraceptives (nicotine clearance, 22.5 +/- 6.6 mL.min(-1).kg(-1) in women taking oral contraceptives versus 17.6 +/- 6.1 mL.min(-1).kg(-1) in those who were not; P < .05). Women who were menopausal or postmenopausal were not different from men. Among oral contraceptive users, nicotine metabolism was accelerated among those taking combined and estrogen-only contraceptives but not progesterone-only contraceptives. CONCLUSIONS: Sex hormones influence nicotine metabolism. Nicotine and cotinine metabolism is faster in women than in men and is faster in women taking oral contraceptives compared with those who are not. Accelerated nicotine metabolism appears to be a result of estrogen. Sex-related differences in nicotine clearance could affect smoking behaviors, as well as response to nicotine medications, and could be a marker for altered metabolism of nicotine derived carcinogens. PMID- 16678550 TI - Liver dysfunction markedly decreases the inhibition of cytochrome P450 1A2 mediated theophylline metabolism by fluvoxamine. AB - BACKGROUND AND OBJECTIVES: In vivo inhibition of cytochrome P450 (CYP) 1A2 by fluvoxamine causes a reduction in the clearance of the high-extraction drug lidocaine, which decreases in proportion to the degree of liver dysfunction. The objectives of this study were (1) to evaluate the effect of liver cirrhosis on the inhibition by fluvoxamine of the metabolic disposition of theophylline, a CYP1A2 substrate with a low-extraction ratio, to assess whether decreased sensitivity to CYP1A2 inhibition in liver disease is a general characteristic of CYP1A2 substrates, regardless of their pharmacokinetic properties, and (2) to investigate the mechanism(s) underlying the effect of liver dysfunction on CYP1A2 inhibition. METHODS: The study was carried out in 10 healthy volunteers and 20 patients with cirrhosis, 10 with mild liver dysfunction (Child class A) and 10 with severe liver dysfunction (Child class C), according to a randomized, double blind, 2-phase, crossover design. In one phase all participants received placebo for 7 days; in the other phase they received one 50-mg fluvoxamine dose for 2 days and two 50-mg fluvoxamine doses, 12 hours apart, in the next 5 days. On day 6, 4 mg/kg of theophylline was administered orally 1 hour after the morning fluvoxamine dose. Concentrations of theophylline and its metabolites, 3 methylxanthine, 1-methyluric acid, and 1,3-dimethyluric acid, were then measured in plasma and urine up to 48 hours. RESULTS: Fluvoxamine-induced inhibition of theophylline clearance decreased from 62% in healthy subjects to 52% and 12% in patients with mild cirrhosis and those with severe cirrhosis, respectively. CYP1A2-mediated formations of 3-methylxanthine and 1-methyluric acid were almost totally inhibited in control subjects, whereas they were only reduced by one third in patients with Child class C cirrhosis. Inhibition of 1,3-dimethyluric acid formation, which is catalyzed by CYP1A2 and CYP2E1, progressively decreased from 58% in healthy subjects to 43% and 7% in patients with mild cirrhosis and those with severe cirrhosis, respectively. CONCLUSIONS: The effect of liver dysfunction on the inhibition of CYP1A2-mediated drug elimination is a general phenomenon, independent of the pharmacokinetic characteristics of the CYP1A2 substrate. Therefore, for any drug metabolized by CYP1A2, the clinical consequences of enzyme inhibition are expected to become less and less important as liver function worsens. Two mechanisms, as follows in order of importance, are responsible for the effect of liver dysfunction: (1) decreased sensitivity to fluvoxamine of CYP1A2-mediated biotransformations in the cirrhotic liver, probably resulting from reduced uptake of the inhibitory drug, and (2) reduced hepatic expression of CYP1A2, which makes its contribution to overall drug elimination less important. PMID- 16678551 TI - Influence of sibutramine treatment on sympathetic vasomotor tone in obese subjects. AB - BACKGROUND: Sibutramine, a serotonin and norepinephrine transporter blocker, is used as adjunctive obesity treatment. Studies in healthy subjects suggested that sibutramine might have opposing effects on peripheral and central sympathetic activity; an increase in blood pressure has been claimed. Direct measurements of muscle sympathetic nerve activity (MSNA) in sibutramine-treated patients have not been conducted. METHODS AND RESULTS: Twenty nondiabetic obese men and women completed the study (mean body mass index, 35 +/- 3 kg/m2; mean age, 42 +/- 8 years). They were treated for 5 days with 15 mg sibutramine per day or matching placebo in a randomized, double-blind, crossover fashion. At the end of each intervention, heart rate, blood pressure, and MSNA were recorded. Patients underwent cold pressor testing and phenylephrine and nitroprusside infusions. RESULTS: The mean blood pressure (systolic/diastolic) was 118 +/- 13 mm Hg/70 +/- 9 mm Hg with placebo and 120 +/- 13 mm Hg/69 +/- 8 mm Hg with sibutramine (P = .29). The mean resting MSNA was 28 +/- 14 bursts/min with placebo and 12 +/- 10 bursts/min with sibutramine (P < .0001). Sibutramine attenuated the rise in blood pressure (25 +/- 9 mm Hg/9 +/- 9 mm Hg versus 31 +/- 12 mm Hg/14 +/- 9 mm Hg, P < .01) and MSNA (0.3 +/- 0.5 arbitrary units/min versus 1.0 +/- 1.1 arbitrary units/min, P = .01) in response to cold pressor testing. Baroreflex heart rate control was similar with sibutramine and with placebo. The sympathetic baroreflex was shifted such that at a given blood pressure, MSNA was substantially decreased (top, 44 +/- 1.23 bursts/min versus 58 +/- 2.99 bursts/min [P < .001]; center point, 65 +/- 0.32 mm Hg versus 67 +/- 0.81 mm Hg [P < .05]). CONCLUSIONS: Sibutramine treatment profoundly and selectively reduces sympathetic nerve traffic at rest and attenuates the responsiveness to sympathetic stimuli. Our data support the idea that sibutramine's peripheral sympathomimetic effect is counteracted by a central sympatholytic mechanism. PMID- 16678552 TI - Effect of CYP3A5*3 on carbamazepine pharmacokinetics in Japanese patients with epilepsy. PMID- 16678553 TI - The functional gastrointestinal disorders and the Rome III process. PMID- 16678554 TI - Fundamentals of neurogastroenterology: basic science. AB - The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs. PMID- 16678555 TI - Applied principles of neurogastroenterology: physiology/motility sensation. AB - Many of the symptoms prominent in the functional gastrointestinal disorders (FGIDs) are consistent with dysfunction of the sensory and/or motor apparatus of the digestive tract. Assessment of these phenomena in man can be undertaken by using a wide variety of invasive and noninvasive techniques, some well established and others requiring further validation. By using such techniques, alterations in both sensory and motor function have been reported in the FGIDs; various combinations of such dysfunction occur in different regions of the digestive tract in the FGIDs. Our understanding of the origins of this gut sensorimotor dysfunction is gradually increasing. Thus, inflammatory, immunologic, and other processes, as well as psychosocial factors such as stress, can alter the normal patterns of sensitivity and motility through alterations in local reflex activity or via altered neural processing along the brain-gut axis. In this context, a potential role of genetic factors, early-life influences, enteric flora, dietary components, and autonomic dysfunction also should be considered in the disease model. A firm relationship between sensorimotor dysfunction and the production of symptoms, however, has been difficult to show, and so the clinical relevance of the former requires continuing exploration. Based on the conceptual framework established to date, a number of recommendations for further progress can be made. PMID- 16678556 TI - Pharmacological and pharmacokinetic aspects of functional gastrointestinal disorders. AB - Medications are commonly used for the treatment of patients with functional gastrointestinal disorders. The general goal of this report is to review the pharmacokinetics and pharmacology of medications used in functional gastrointestinal disorders. Methods included literature review, consensus evaluation of the evidence for each topic assigned originally to 1 or 2 authors, and broader review at a harmonization session as part of the Rome III process. This report reviews the animal models that have been validated for the study of effects of pharmacologic agents on sensation and motility; the preclinical pharmacology, pharmacokinetics, and toxicology usually required for introduction of novel therapeutic agents; the biomarkers validated for studies of sensation and motility end points with experimental medications in humans; the pharmacogenomics applied to these medications and disorders; and the pharmacology of agents that are applied or have potential for treatment of functional gastrointestinal disorders, including psychopharmacologic agents. Clinician and basic investigators involved in the treatment or investigation of functional gastrointestinal disorders or disease models need to have a comprehensive understanding of a vast range of medications. It is anticipated that the interaction between investigators of basic science, basic and applied pharmacology, and clinical trials will lead to better treatment of these disorders. PMID- 16678557 TI - Gender, age, society, culture, and the patient's perspective in the functional gastrointestinal disorders. AB - Patients with functional gastrointestinal disorders (FGID) often experience emotional distress, a perceived lack of validation, and an unsatisfactory experience with health care providers. A health care provider can provide the patient with a framework in which to understand and legitimize their symptoms, remove self-doubt or blame, and identify factors that contribute to symptoms that the patient can influence or control. This framework can be strengthened with the consideration of various important factors that impact FGID but are often overlooked. These include gender, age, society, culture, and the patient's perspective. There is evidence for sex- and gender-related differences in FGID, particularly irritable bowel syndrome (IBS). Whereas the majority of FGID, including IBS, bloating, constipation, chronic functional abdominal pain, and pelvic floor dysfunction, are more prevalent in women than men, functional esophageal and gastroduodenal disorders do not appear to vary by gender. Limited studies suggest that sex differences in visceral perception, cardioautonomic responses, gastrointestinal motility, and brain activation patterns to visceral stimuli exist in IBS. Gender differences in social factors, psychological symptoms, and response to psychological treatments have not been adequately studied. However, there appears to be a greater clinical response to serotonergic agents developed for IBS in women compared to men. The impact of social and cultural factors on the meaning, expression, and course of FGID are important. The prevalence of IBS appears to be lower in non-Western than Western countries. Although further studies are needed, the existing literature suggests that they are important to consider from both research and clinical perspectives. PMID- 16678558 TI - Psychosocial aspects of the functional gastrointestinal disorders. AB - This report reviews recent research on the psychosocial aspects of the functional gastrointestinal disorders (FGIDs). A review and evaluation of existing literature was conducted by a multidisciplinary committee of experts in this field. This report is a synopsis of a chapter published in the Rome III book. The committee reached consensus in finding considerable evidence supporting the association between psychological distress, childhood trauma and recent environmental stress, and several of the FGIDs but noted that this association is not specific to FGIDs. There is also considerable evidence that psychosocial variables are important determinants of the outcomes of global well-being, health related quality of life, and health care seeking. In line with these descriptive findings, there is now increasing evidence that a number of psychological treatments and antidepressants are helpful in reducing symptoms and other consequences of the FGIDs in children and adults. The FGIDs are a result of complex interactions between biological, psychological, and social factors, and they can only be treated satisfactorily when all these factors are considered and addressed. Therefore, knowledge about the psychosocial aspects of FGIDs is fundamental and critical to the understanding, assessment, and treatment of these disorders. More extensive physician training is needed if these aspects of treatment are to be used effectively and widely in clinical practice. PMID- 16678559 TI - Functional esophageal disorders. AB - Functional esophageal disorders represent processes accompanied by typical esophageal symptoms (heartburn, chest pain, dysphagia, globus) that are not explained by structural disorders, histopathology-based motor disturbances, or gastroesophageal reflux disease. Gastroesophageal reflux disease is the preferred diagnosis when reflux esophagitis or excessive esophageal acid exposure is present or when symptoms are closely related to acid reflux events or respond to antireflux therapy. A singular, well-defined pathogenetic mechanism is unavailable for any of these disorders; combinations of sensory and motor abnormalities involving both central and peripheral neural dysfunction have been invoked for some. Treatments remain empirical, although the efficacy of several interventions has been established in the case of functional chest pain. Management approaches that modulate central symptom perception or amplification often are required once local provoking factors (eg, noxious esophageal stimuli) have been eliminated. Future research directions include further determination of fundamental mechanisms responsible for symptoms, development of novel management strategies, and definition of the most cost-effective diagnostic and treatment approaches. PMID- 16678560 TI - Functional gastroduodenal disorders. AB - A numerically important group of patients with functional gastrointestinal disorders have chronic symptoms that can be attributed to the gastroduodenal region. Based on the consensus opinion of an international panel of clinical investigators who reviewed the available evidence, a classification of the functional gastroduodenal disorders is proposed. Four categories of functional gastroduodenal disorders are distinguished. The first category, functional dyspepsia, groups patients with symptoms thought to originate from the gastroduodenal region, specifically epigastric pain or burning, postprandial fullness, or early satiation. Based on recent evidence and clinical experience, a subgroup classification is proposed for postprandial distress syndrome (early satiation or postprandial fullness) and epigastric pain syndrome (pain or burning in the epigastrium). The second category, belching disorders, comprises aerophagia (troublesome repetitive belching with observed excessive air swallowing) and unspecified belching (no evidence of excessive air swallowing). The third category, nausea and vomiting disorders, comprises chronic idiopathic nausea (frequent bothersome nausea without vomiting), functional vomiting (recurrent vomiting in the absence of self-induced vomiting, or underlying eating disorders, metabolic disorders, drug intake, or psychiatric or central nervous system disorders), and cyclic vomiting syndrome (stereotypical episodes of vomiting with vomiting-free intervals). The rumination syndrome is a fourth category of functional gastroduodenal disorder characterized by effortless regurgitation of recently ingested food into the mouth followed by rechewing and reswallowing or expulsion. The proposed classification requires further research and careful validation but the criteria should be of value for clinical practice; for epidemiological, pathophysiological, and clinical management studies; and for drug development. PMID- 16678561 TI - Functional bowel disorders. AB - Employing a consensus approach, our working team critically considered the available evidence and multinational expert criticism, revised the Rome II diagnostic criteria for the functional bowel disorders, and updated diagnosis and treatment recommendations. Diagnosis of a functional bowel disorder (FBD) requires characteristic symptoms during the last 3 months and onset > or =6 months ago. Alarm symptoms suggest the possibility of structural disease, but do not necessarily negate a diagnosis of an FBD. Irritable bowel syndrome (IBS), functional bloating, functional constipation, and functional diarrhea are best identified by symptom-based approaches. Subtyping of IBS is controversial, and we suggest it be based on stool form, which can be aided by use of the Bristol Stool Form Scale. Diagnostic testing should be guided by the patient's age, primary symptom characteristics, and other clinical and laboratory features. Treatment of FBDs is based on an individualized evaluation, explanation, and reassurance. Alterations in diet, drug treatment aimed at predominant symptoms, and psychotherapy may be beneficial. PMID- 16678562 TI - Functional abdominal pain syndrome. AB - Functional abdominal pain syndrome (FAPS) differs from the other functional bowel disorders; it is less common, symptoms largely are unrelated to food intake and defecation, and it has higher comorbidity with psychiatric disorders. The etiology and pathophysiology are incompletely understood. Because FAPS likely represents a heterogeneous group of disorders, peripheral neuropathic pain mechanisms, alterations in endogenous pain modulation systems, or both may be involved in any one patient. The diagnosis of FAPS is made on the basis of positive symptom criteria and a longstanding history of symptoms; in the absence of alarm symptoms, an extensive diagnostic evaluation is not required. Management is based on a therapeutic physician-patient relationship and empirical treatment algorithms using various classes of centrally acting drugs, including antidepressants and anticonvulsants. The choice, dose, and combination of drugs are influenced by psychiatric comorbidities. Psychological treatment options include psychotherapy, relaxation techniques, and hypnosis. Refractory FAPS patients may benefit from a multidisciplinary pain clinic approach. PMID- 16678563 TI - Functional gallbladder and sphincter of oddi disorders. AB - The functional disorder of the gallbladder (GB) is a motility disorder caused initially either by metabolic abnormalities or by a primary motility alteration. The functional disorders of the sphincter of Oddi (SO) encompass motor abnormalities of either the biliary or the pancreatic SO. Dysfunction of the GB and/or biliary SO produce similar patterns of pain. The pain caused by a dysfunction of the pancreatic SO can be similar to that of acute pancreatitis. The symptom-based diagnostic criteria of motility dysfunction of the GB and biliary SO are episodes of moderate to severe steady pain located in the epigastrium and right upper abdominal quadrant that last at least 30 minutes. GB motility disorder is suspected after gallstones and other structural abnormalities have been excluded. This diagnosis should then be confirmed by a decreased GB ejection fraction induced by cholecystokinin at cholescintigraphy and after disappearance of the recurrent biliary pain after cholecystectomy. Symptoms of biliary SO dysfunction may be accompanied by features of transient biliary obstruction, and those of pancreatic SO dysfunction are associated with elevation of pancreatic enzymes and even pancreatitis. Biliary-type SO dysfunction is more frequently recognized in postcholecystectomy patients. SO manometry is valuable to select patients with sphincter dysfunction; however, because of the high incidence of complications, these patients should be referred to an expert unit for such assessment. Thus invasive tests should be performed only in the presence of compelling clinical evidence and after noninvasive testing has yielded negative findings. The committee recommends that division of the biliary or pancreatic sphincters only be considered when the patient has severe symptoms, meets the required criteria, and other diagnoses are excluded. PMID- 16678564 TI - Functional anorectal disorders. AB - This report defines criteria for diagnosing functional anorectal disorders (ie, fecal incontinence, anorectal pain, and disorders of defecation). Functional fecal incontinence is defined as the uncontrolled passage of fecal material recurring for > or =3 months in an individual with a developmental age of > or =4 years that is associated with: (1) abnormal functioning of normally innervated and structurally intact muscles, and/or (2) no or minor abnormalities of sphincter structure and/or innervation insufficient to explain fecal incontinence, and/or (3) normal or disordered bowel habits (ie, fecal retention or diarrhea), and/or (4) psychological causes. However, conditions wherein structural and/or neurogenic abnormalities explain the symptom, or are part of a generalized process (eg, diabetic neuropathy) are not included within functional fecal incontinence. Functional fecal incontinence is a common, but underrecognized symptom, which is equally prevalent in men and women, and can often cause considerable distress. The clinical features are useful for guiding diagnostic testing and therapy. Functional anorectal pain syndromes include proctalgia fugax (fleeting pain) and chronic proctalgia; chronic proctalgia may be subdivided into levator ani syndrome and unspecified anorectal pain, which are defined by arbitrary clinical criteria. Functional defecation disorders are characterized by 2 or more symptoms of constipation, with > or =2 of the following features during defecation: impaired evacuation, inappropriate contraction of the pelvic floor muscles, and inadequate propulsive forces. Functional disorders of defecation may be amenable to pelvic floor retraining by biofeedback therapy (such as dyssynergic defecation). PMID- 16678565 TI - Childhood functional gastrointestinal disorders: neonate/toddler. AB - Recognizing the importance of childhood functional gastrointestinal disorders in understanding adult functional gastrointestinal disorders, and encouraging clinical and research interest, the Rome Coordinating Committee added a pediatric working team to Rome II in 1999. For Rome III, there was an increase from 1 to 2 pediatric working teams. This report summarizes the current consensus concerning functional disorders in infants and toddlers. Another report covers disorders diagnosed more often in school-aged children and adolescents. The symptoms from functional gastrointestinal disorders in children younger than 5 years depend on maturational factors in anatomy, gastrointestinal physiology, and intellectual and affective functioning. There has been little or no change for infant regurgitation, infant rumination syndrome, or infant dyschezia. Cyclic vomiting syndrome may be diagnosed after 2 rather than 3 episodes. The description of infant colic has been expanded, although there was consensus that infant colic does not reflect gastrointestinal malfunction. The greatest change was in functional constipation. Functional constipation and functional fecal retention in the 1999 report were merged into a single entity: functional constipation. Data-driven changes in diagnostic criteria for functional constipation appear to be less rigid and more inclusive than previous criteria. PMID- 16678566 TI - Childhood functional gastrointestinal disorders: child/adolescent. AB - The Rome II pediatric criteria for functional gastrointestinal disorders (FGIDs) were defined in 1999 to be used as diagnostic tools and to advance empirical research. In this document, the Rome III Committee aimed to update and revise the pediatric criteria. The decision-making process to define Rome III criteria for children aged 4-18 years consisted of arriving at a consensus based on clinical experience and review of the literature. Whenever possible, changes in the criteria were evidence based. Otherwise, clinical experience was used when deemed necessary. Few publications addressing Rome II criteria were available to guide the committee. The clinical entities addressed include (1) cyclic vomiting syndrome, rumination, and aerophagia; 2) abdominal pain-related FGIDs including functional dyspepsia, irritable bowel syndrome, abdominal migraine, and functional abdominal pain; and (3) functional constipation and non-retentive fecal incontinence. Adolescent rumination and functional constipation are newly defined for this age group, and the previously designated functional fecal retention is now included in functional constipation. Other notable changes from Rome II to Rome III criteria include the decrease from 3 to 2 months in required symptom duration for noncyclic disorders and the modification of the criteria for functional abdominal pain. The Rome III child and adolescent criteria represent an evolution from Rome II and should prove useful for both clinicians and researchers dealing with childhood FGIDs. The future availability of additional evidence-based data will likely continue to modify pediatric criteria for FGIDs. PMID- 16678567 TI - Design of treatment trials for functional gastrointestinal disorders. AB - This document addresses the design of trials to assess the efficacy of new treatments for functional gastrointestinal disorders (FGID), emphasizing trials in irritable bowel syndrome and dyspepsia, because most research has been undertaken in these conditions. The double-blind, randomized, placebo-controlled, parallel group trial remains the preferred design. Randomized withdrawal designs, although encouraged by the European Agency for the Evaluation of Medicinal Products, have the same potential disadvantages as a crossover design, including carryover effects, unmasking (unblinding), and overestimation of the potential benefit for clinical practice. Innovative trial designs that evaluate intermittent (on demand) treatment are likely to become more common in the future. Investigators should include as broad a spectrum of patients as possible and should report recruitment strategies, inclusion/exclusion criteria, and attrition data. The primary analysis should be based on the proportion of patients in each treatment arm who satisfy an a priori treatment responder definition, or a prespecified clinically meaningful change in a patient-reported symptom improvement measure. Such measures of improvement are psychometrically validated subjective global assessments or a change from baseline in a validated symptom severity questionnaire. It is unethical to change the responder definition after a trial begins. Data analysis should address all patients enrolled, using an intention-to-treat principle. Reporting of results should follow the Consolidated Standards for Reporting Trials guidelines and include an analysis of harms data and secondary outcome measures to support or explain the primary outcome. Trials should be registered in a public location, prior to initiation, and should be published even if the results are negative or inconclusive. PMID- 16678568 TI - The road to rome. PMID- 16678569 TI - Incidence and outcome of cardiopulmonary resuscitation in patients with shunted single ventricle: advantage of right ventricle to pulmonary artery shunt. PMID- 16678570 TI - Controversies in cardiothoracic surgery: should therapeutic cloning be supported to provide stem cells for cardiothoracic surgery research and treatment? PMID- 16678571 TI - Closed heart surgery: back to the future. PMID- 16678572 TI - Measuring the therapeutic efficacy of coronary revascularization: Implications for future management. PMID- 16678573 TI - Surgery for atrial fibrillation: are we heading in the right direction? PMID- 16678574 TI - Prosthesis-patient mismatch and clinical outcomes: the evidence continues to accumulate. PMID- 16678575 TI - Integrated coronary revascularization with drug-eluting stents: immediate and seven-month outcome. AB - OBJECTIVE: We sought to demonstrate the safety and feasibility of an integrated coronary revascularization strategy that combines minimally invasive left internal thoracic artery to left anterior descending coronary artery anastomosis with drug-eluting stent implantation to non-left anterior descending coronary artery lesions. METHODS: Over 18 months, 47 consecutive patients with multivessel coronary artery disease underwent thoracoscopic harvesting of the left internal thoracic artery to graft the left anterior descending coronary artery. Anastomoses were constructed by hand, off-pump, and under direct vision through a 4-cm non-rib-spreading, muscle-sparing chest incision. Non-left anterior descending coronary artery lesions were then treated percutaneously using sirolimus- or paclitaxel-eluting stents. Angiographic follow-up was performed in all patients. RESULTS: Within the first 90 days of hospitalizations, there were no deaths, myocardial infarctions, neurologic events, or wound complications. Forty patients underwent left internal thoracic artery to left anterior descending coronary artery grafting, and 7 patients underwent left internal thoracic artery to left anterior descending coronary artery/diagonal sequential grafting for a total of 54 anastomoses. Angiographic patency scores were FitzGibbon A 96.2% (52/54) and FitzGibbon A + B 100% (54/54). A total of 65 drug eluting stents were implanted in 61 non-left anterior descending coronary artery coronary lesions of which 49.1% (30/61) were type B2 or C lesions, including 5 left main lesions. Diabetes was present in 53.2% of patients (25/47). At a mean follow-up time of 7.0 +/- 4.8 months, the target lesion or vessel repeat revascularization rate was 6.6% (4/61) for drug-eluting stents and 1.9% (1/54) for left internal thoracic artery to left anterior descending coronary artery grafting. One anastomosis required balloon dilation, but no patients have required repeat coronary artery bypass grafting. CONCLUSIONS: Integrated coronary revascularization using drug-eluting stents is feasible and safe. There are sufficient data to justify a randomized comparison of integrated coronary revascularization with standard coronary artery bypass grafting. PMID- 16678576 TI - Fatal pulmonary microthrombi during surgical therapy for end-stage heart failure: possible association with antifibrinolytic therapy. AB - BACKGROUND: Maintaining hemostasis in patients with end-stage heart failure undergoing cardiac surgery is always challenging. These patients have chronic hepatic insufficiency, resulting in derangement of coagulation. In addition, they are commonly receiving both systemic anticoagulation (warfarin or heparin) and antiplatelet therapy. The introduction of antifibrinolytics has had a significant effect on postoperative coagulopathy. We report fatal pulmonary microthrombi in patients receiving antifibrinolytics who developed suprasystemic pulmonary artery pressures and right heart failure that was impossible to overcome despite insertion of a right ventricular assist device. METHODS: We reviewed the surgical procedure and autopsy reports to identify patients with high pulmonary artery pressures caused by pulmonary microthrombi after a cardiac surgical procedure for end-stage heart failure. Patient demographics and preoperative, intraoperative, and postoperative variables were collected from a retrospective review of the patients' medical records. RESULTS: We identified 9 patients (7 men and 2 women; mean age, 45 +/- 16 years) who died of pulmonary microthrombi after cardiac surgery between January 1997 and January 2004. Surgical procedures included 5 left ventricular assist device implantations, 2 heart transplantations, and 2 left ventricular reconstructions with mitral valve repair or replacement. Eight patients received aprotinin, and 1 patient received epsilon-aminocaproic acid immediately before and during cardiopulmonary bypass. All patients had severe suprasystemic pulmonary artery pressures after protamine administration for heparin reversal, a complication that proved fatal in all cases. Intraoperative wedge biopsy of the lungs revealed multiple microthrombi within capillaries and in the small- and medium-sized pulmonary arterioles. CONCLUSION: We report 9 cases for which fatal pulmonary microthrombi might be associated with the use of prophylactic antifibrinolytic therapy. Mortally ill patients with multiorgan failure who are receiving systemic anticoagulation and undergoing surgical procedures require careful perioperative monitoring to identify potential hazards. Anticoagulation and antifibrinolytic therapy protocols may require adjustment in such patients. PMID- 16678577 TI - Importance of endothelial nitric oxide synthase for the hypothermic protection of lungs against ischemia-reperfusion injury. AB - OBJECTIVES: The hypothesis that the protective effects of mild hypothermia against the pulmonary ischemia-reperfusion injury are mediated by endothelial nitric oxide synthase was tested. METHODS: Endothelial nitric oxide synthase knock-out and wild-type mice were sham operated or underwent a 1-hour occlusion of the left pulmonary hilum, followed by 5 hours of reperfusion. Temperature in the left pleural cavity during ischemia was maintained at either 36 degrees C (normothermia) or 32 degrees C (hypothermia). Inflammatory response (myeloperoxidase activity), endothelial barrier function (extravasation of Evans blue-labeled albumin), and endothelial nitric oxide synthase expression and phosphorylation were determined at the end of reperfusion. RESULTS: After normothermic ischemia both strains had a similar mortality (wild-type, 22.9%; knock-out, 15.4%), which was completely abolished by hypothermia. Endothelial barrier function was disturbed after normothermic ischemia in both wild-type and knock-out mice. Mild hypothermia significantly reduced pulmonary Evans blue extravasation in wild-type mice, but not in knock-out mice. Myeloperoxidase activity increased after normothermic ischemia to the same degree in both strains. This response was significantly attenuated by hypothermia in wild-type mice, but not in knock-out mice. In wild-type mice, endothelial nitric oxide synthase expression and phosphorylation were higher after hypothermic ischemia than after normothermic ischemia. No effect of ischemia on expression of inducible nitric oxide synthase was found in wild-type or knock-out mice. CONCLUSION: Hypothermic protection against pulmonary ischemia-reperfusion injury is dependent on endothelial nitric oxide synthase and is associated with increased expression and phosphorylation of endothelial nitric oxide synthase. PMID- 16678578 TI - Restoration of myocardial beta-adrenergic receptor signaling after left ventricular assist device support. AB - OBJECTIVE: Left ventricular assist device support for patients with chronic heart failure can significantly improve beta-adrenergic receptor signaling, which is likely critical to myocardial recovery. The mechanism underlying the restoration of beta-adrenergic receptor signaling is unclear. This study investigates our hypothesis that restoration of cardiac beta-adrenergic receptor signaling by left ventricular assist devices results from inhibition of the G protein-coupled receptor kinase-2, a G protein-coupled receptor kinase that specifically phosphorylates and desensitizes agonist-occupied beta-adrenergic receptors. METHODS: Left ventricular beta-adrenergic receptor signaling was assessed in biopsy specimens taken from patients with chronic heart failure (n = 12) at the time of left ventricular assist device implantation (heart failure group) and again at the time of heart transplantation (left ventricular assist device group). Signaling was also studied in left ventricular biopsy specimens from nonfailing control (n = 8) hearts (nonfailing control group). Signaling was assessed by measuring sarcolemmal membrane beta-adrenergic receptor density, adenylyl cyclase activity, G protein expression, and G protein-coupled receptor kinase-2 expression and activity. RESULTS: Left ventricular beta-adrenergic receptor signaling was severely decreased in the heart failure group versus that seen in the nonfailing control group, as demonstrated by adenylyl cyclase activity. G protein-coupled receptor kinase-2 expression and activity was increased 3-fold in the heart failure group versus that seen in the nonfailing control group. After left ventricular assist device support, beta-adrenergic receptor signaling was restored to levels similar to those seen in the nonfailing control group. G protein-coupled receptor kinase-2 expression and activity were markedly diminished after left ventricular assist device support compared with that seen in the heart failure group and were not different from that seen in the nonfailing control group. CONCLUSION: In chronic heart failure left ventricular assist device support leads to restoration of cardiac beta-adrenergic receptor signaling. The primary mechanism appears to be diminished myocardial G protein coupled receptor kinase-2 activity. This demonstrates the potentially beneficial effects of G protein-coupled receptor kinase-2 inhibition on beta-adrenergic receptor signaling in heart failure and might represent a novel therapeutic strategy for this disease process. PMID- 16678579 TI - The pattern of lymph node involvement influences outcome after extrapleural pneumonectomy for malignant mesothelioma. AB - OBJECTIVE: We sought to examine the distribution and prognostic implications of nodal metastasis in patients undergoing extrapleural pneumonectomy for malignant mesothelioma in a specialist center. METHODS: We have examined the lymphadenectomy specimens from 92 consecutive cases of malignant mesothelioma undergoing extrapleural pneumonectomy from September 1999 through February 2005 inclusive. Nodal stations (Naruke) were assigned to all nodes, and patients were staged according to the current International Union Against Cancer system. The status and number of nodes in each station were recorded, and results were correlated with the results of preoperative mediastinoscopic findings (n = 30) and survival. RESULTS: The nodal distribution was 48 N0, 9 N1, and 35 N2. Single and multistation nodal involvement was present in 20 and 24 cases, respectively. Among the patients undergoing mediastinoscopy, N2 disease after extrapleural pneumonectomy occurred in 10 (33%). Skip N2 metastasis was present in 10 (42%) cases. Positive N2 nodes inaccessible by mediastinoscopy were present in 17 (49%) cases. N2 metastasis was associated with reduced survival (P = .02), but there was no difference between N1 and N2 cases (P = .4). The number of positive nodes correlated with survival (P = .001), although the number of involved stations and their anatomic location did not. There was no difference in survival between skip N2 cases and either other N2 or N1 cases. CONCLUSIONS: The classical anatomic location is not as important as the scatter of nodal involvement. Every effort should be made to obtain biopsy specimens from as many stations as possible before undertaking extrapleural pneumonectomy for malignant mesothelioma. PMID- 16678580 TI - Clinicopathologic study of resected, peripheral, small-sized, non-small cell lung cancer tumors of 2 cm or less in diameter: pleural invasion and increase of serum carcinoembryonic antigen level as predictors of nodal involvement. AB - OBJECTIVE: The number of surgical interventions for small-sized lung cancer has increased with the development of computed tomography. We attempted to identify clinicopathologic characteristics of peripheral, small-sized, non-small cell lung cancer to show the limitation of partial resection or segmentectomy. METHODS: A retrospective analysis of 143 patients who underwent a complete resection for a peripheral non-small cell lung cancer of 2 cm or less in diameter was performed. The relationships between nodal involvement and other clinical factors were also assessed in patients who underwent a lobectomy plus node dissection. RESULTS: The overall 5-year survival rate was 88.1%. The 5-year survival rate was 100% for patients with a tumor of 1.5 cm or less. Survival for patients with adenocarcinoma histology was significantly better than for those with nonadenocarcinoma histology (P = .03). The 5-year survival rate for patients without lymph node metastases was 91.6%, whereas it was 62.5% for those with nodal involvement (P < .01). Increase of prethoracotomy serum carcinoembryonic antigen level was an independent predictor of a poor prognosis. Lymph node metastasis was significantly increased in those with pleural invasion by the primary lesion and increased serum carcinoembryonic antigen level. Fourteen (16.9%) of 83 patients with a tumor diameter of larger than 1.5 cm had nodal metastasis. CONCLUSIONS: Nodal involvement should be considered in patients with non-small cell lung cancer of 2 cm or less in diameter who show pleural invasion or an increased carcinoembryonic antigen level. A lobectomy with node dissection is recommended for patients with a tumor larger than 1.5 cm, suspected pleural invasion, or prethoracotomy carcinoembryonic antigen level increase. PMID- 16678581 TI - Breath-hold single-photon emission tomography and computed tomography for predicting residual pulmonary function in patients with lung cancer. AB - OBJECTIVE: We sought to evaluate the utility of integrated breath-hold single photon emission tomography and computed tomography imaging compared with that of simple calculation with the lung segment-counting technique for predicting residual pulmonary function in patients undergoing surgical intervention for lung cancer. METHODS: A prospective series of 22 patients undergoing anatomic lung resection for cancer were enrolled in this study. Postoperative residual forced expiratory volume in 1 second was predicted by measuring the radioactivity counts of the affected lobes or segments to be resected within the entire lungs by placement of regions of interest on single-photon emission tomography and computed tomography images. Residual forced expiratory volume in 1 second was also estimated by using the segment-counting technique. RESULTS: Both predicted values agreed well with postoperative forced expiratory volume in 1 second. Although the residual forced expiratory volume in 1 second predicted by means of single-photon emission tomography and computed tomography correlated well with that predicted by using segment counting, the values were significantly underestimated by the segment-counting technique in 4 outliers with severe emphysema. There were 2 patients with borderline pulmonary functional reserve whose residual forced expiratory volume in 1 second values were predicted more accurately by means of single-photon emission tomography and computed tomography than by using segment counting. CONCLUSION: Integrated breath-hold single-photon emission tomography and computed tomography images allow the accurate prediction of postoperative pulmonary function but without statistical superiority over the simple segment-counting technique. Further study of the usefulness of single photon emission tomography and computed tomography in patients with severe emphysema and borderline lung function should prove valuable because the segment counting technique underestimates pulmonary functional reserve in these patients. PMID- 16678582 TI - Biologic effects of radiofrequency thermal ablation on non-small cell lung cancer: results of a pilot study. AB - OBJECTIVES: Radiofrequency ablation of lung tumors is an emerging technique with promising results. To achieve more information on its effects on pulmonary parenchyma and primary tumors, and to assess its efficacy in determining complete necrosis of the lesion, we led a pilot study consisting of thermal ablation followed by surgical resection. METHODS: Ten patients with early stage non-small cell lung cancer were enrolled in the study. In 5 patients radiofrequency ablation was achieved through thoracotomy just before the surgical resection. In another 5 patients radiofrequency ablation was performed percutaneously, under computed tomography guidance, and the surgical resection was performed after 15 days. RESULTS: Nine of the 10 patients enrolled in the study were available for analysis. There were 8 men and 1 woman with a mean age of 65.5 years. In all cases histologic diagnosis was available before radiofrequency ablation; adenocarcinoma was identified in 5 patients, and squamous cell carcinoma was identified in 4 patients. Microscopic examination showed complete necrosis of the tumor in 6 of 9 cases. No anatomopathologic alterations were seen in surrounding distant parenchyma. CONCLUSIONS: Our study confirms the safety of radiofrequency ablation, particularly underlining the possibility of performing a thermal lesion in the lung in a controlled manner. It also assesses that radiofrequency ablation may be effective in the local control of primary lung cancer. However, surgery still represents the mainstay of treatment of non-small cell lung cancer, and radiofrequency ablation must be reserved for high-risk patients. PMID- 16678583 TI - Percutaneous cryoablation of small pulmonary malignant tumors under computed tomographic guidance with local anesthesia for nonsurgical candidates. AB - OBJECTIVE: Cryoablation of pulmonary metastases might be a useful therapy for nonsurgical candidates. METHODS: The procedure was performed after achievement of local anesthesia for 35 tumors in 20 patients (12 male and 8 female patients; mean age, 57 years). The primary end point was the safety and feasibility of cryoablation, and the secondary end point was tumor control assessed by follow-up dynamic computed tomographic scanning performed every 3 months. RESULTS: Of the 22 sessions of cryoablation, pneumothorax occurred in 11, hemoptysis occurred in 8, and there was 1 case of phrenic nerve palsy. The mean hospital stay was 2.6 days. There was local recurrence of 7 (20%) tumors in 7 (35%) patients during a 9 to 28-month (median, 21 months) follow-up period. One-year survival according to the Kaplan-Meier method was 89.4%. CONCLUSION: Percutaneous cryoablation therapy for metastatic lung tumors is feasible and minimally invasive, with satisfactory local control. PMID- 16678584 TI - Histologic grade is an independent prognostic factor for survival in non-small cell lung cancer: an analysis of 5018 hospital- and 712 population-based cases. AB - OBJECTIVE: Our objective was to determine whether histologic grade independently contributes to the prognosis of non-small cell lung cancer. METHODS: A total of 5018 hospital-based patients diagnosed from 1997 to 2003 and 712 population-based patients diagnosed from 1984 to 2003 were followed up through the end of 2004. The effect of histologic grade on postdiagnosis survival or postresection recurrence was evaluated by Cox proportional hazards models. Relative risks (RR) were estimated by comparing undifferentiated, poorly differentiated, and moderately differentiated carcinoma with well-differentiated carcinoma. RESULTS: Histologic grade was significantly associated with survival after adjustment for the effects of age, gender, smoking history, tumor stage, histologic cell type, and treatment modality. Patients with undifferentiated carcinoma had an 80% elevated risk of death (RR = 1.83; 95% confidence interval [CI], 1.4-2.4) compared with those with well-differentiated carcinoma; 70% and 40% elevated risks were observed for patients with poorly and moderately differentiated carcinoma, respectively (RR, 1.7 [1.5-2.0] and 1.4 [1.2-1.6]). Similar results were observed for 718 incidence cases in which the relative risks were 1.6 (1.1 2.2) and 1.4 (1.0-1.9) for poorly/undifferentiated carcinoma and moderately differentiated carcinoma, respectively. Patients with less-differentiated carcinoma after tumor resection had a higher risk of recurrence, with adjusted hazard ratios of 2.1 (95% CI: 1.4-2.9) and 1.4 (1.0-1.9) for poorly/undifferentiated and moderately differentiated carcinoma compared with well-differentiated carcinoma. CONCLUSIONS: Histologic grade has significant prognostic value for survival of patients with non-small cell lung cancer. Histologic grade may provide useful information in defining the aggressiveness of tumors and should be considered as an independent factor affecting survival beyond TNM staging. PMID- 16678585 TI - How many arterial grafts are enough? A population-based study of midterm outcomes. AB - OBJECTIVE: Current evidence suggests arterial grafting improves freedom from cardiac events after coronary artery bypass graft surgery. It has been shown that 2 arterial grafts provide improved outcome compared with 1 arterial graft. This population study seeks to understand trends in arterial graft use and midterm outcomes of patients receiving 1, 2, or 3 arterial grafts. METHODS: This study is a retrospective population-based cohort of 53,727 patients (47,214 with 1 arterial graft, 5466 with 2 arterial grafts, and 1047 with 3 arterial grafts) undergoing isolated coronary artery bypass graft surgery in Ontario (1991-2001). The patients were followed by using linked clinical and administrative data, with complete follow-up until December 31, 2003 (average patient years of follow-up: 6 years for those with 1 arterial graft, 5 years for those with 2 arterial grafts, and 4 years for those with 3 arterial grafts). Propensity matching was used to compare outcomes between patients receiving 1 versus 2 arterial grafts, 2 versus 3 arterial grafts, and 1 versus 2 or 3 arterial grafts. The outcomes included death, repeat revascularization (angioplasty or coronary artery bypass grafting), cardiac readmission (readmission for angina, heart failure, and myocardial infarction), and a composite comprising all of these outcomes. Cox proportional hazards models were used to compare outcomes for propensity-matched patients. Subgroup analyses of various patient risk categories defined by the tercile of predicted 30-day mortality risk were conducted between propensity-matched individuals. RESULTS: The use of multiple arterial grafts (defined as >1 arterial graft) increased mainly in the latter part of the study, from 4% in 1991 to 27% in 2001. Four thousand nine hundred sixty-eight patients were propensity matched (91% of patients receiving 2 arterial grafts) to compare outcomes with those of patients receiving 1 arterial graft. One thousand twenty-eight patients were propensity matched (98% of those receiving 3 arterial grafts) to compare outcomes with those of patients receiving 2 arterial grafts. Five thousand four hundred ninety-one patients were propensity matched (84% of those receiving 2 or 3 arterial grafts) to compare outcomes with those of patients receiving 1 arterial graft. Two arterial grafts were shown to be protective for cardiac readmission (0.8; 95% confidence interval, 0.76-0.92) and a composite outcome (0.9; 95% confidence interval, 0.72-0.95) compared with 1 arterial graft. Two or 3 arterial grafts were further found to improve survival (0.8; 95% confidence interval, 0.72 0.99). In all patient operative risk categories, 2 or 3 arterial grafts were protective for cardiac readmission (hazard ratio, 0.7-0.8) and the composite outcome (hazard ratio, 0.8). There was no difference in the Cox hazard ratios of propensity-matched patients in the comparison of the groups receiving 3 versus 2 arterial grafts. CONCLUSIONS: Few patients received more that 1 arterial graft in our region. There was a survival benefit in receiving 2 or 3 arterial grafts. Patients with low, moderate, and high operative risk receiving 2 or 3 arterial grafts had lower rates of cardiac readmission compared with patients receiving only 1 arterial graft. This suggests that the standard of care should include the use of at least 2 arterial bypasses in all categories of operative risk to allow for optimal midterm outcomes. PMID- 16678586 TI - Surgical ablation as treatment for the elimination of atrial fibrillation: a meta analysis. AB - OBJECTIVE: The maze procedure is recognized as the most effective surgical treatment of atrial fibrillation. In the last few years, new surgical ablation techniques were developed involving the left atrium only and modifications of the maze procedure in ablating both atria. For this study, we evaluated the evidence regarding the effectiveness of the surgical ablation procedures (biatrial and left atrial) in reducing postoperative atrial fibrillation and subsequent survival. METHODS: MEDLINE was searched for English-language studies using the terms "maze," "atrial fibrillation," and "surgical treatment" for 1995 through August 2005. Primary outcomes of interest were postoperative survival and postoperative freedom from atrial fibrillation. Survival data were collected at 1 , 2-, and 3-year intervals. Freedom from atrial fibrillation was collected at 3 months and at 1-, 2-, and 3-year intervals. RESULTS: Sixty-nine studies were included in this analysis. Five thousand eight hundred eighty-five total patients were involved. Patients undergoing surgical ablation (range, 90.4-85.4) demonstrated significantly greater rates of freedom from atrial fibrillation compared with those seen in control patients (range, 47.2-60.9). Survival rates among patients with biatrial surgical procedures (range, 94.9-92.8) were similar to those who had left atrial procedures only (range, 93.9-89.4). However, patients undergoing biatrial ablation (range, 92.0-87.1 vs 86.1-73.4) demonstrated superior freedom from atrial fibrillation at all time points. CONCLUSION: Biatrial ablation surgical procedures were more effective in controlling atrial fibrillation than procedures confined to the left atrium. To encourage the use of future meta-analysis within the surgical literature, we suggest the more frequent reporting of either through Kaplan-Meier survival analyses and the reporting of rates for specific time intervals. PMID- 16678587 TI - Prosthesis-patient mismatch after aortic valve replacement predominantly affects patients with preexisting left ventricular dysfunction: effect on survival, freedom from heart failure, and left ventricular mass regression. AB - OBJECTIVE: The effect of prosthesis-patient mismatch on clinical outcome and left ventricular mass regression after aortic valve replacement remains controversial. Data on whether the clinical effect of prosthesis-patient mismatch depends on left ventricular function at the time of aortic valve replacement are lacking. This study examined the long-term clinical and echocardiographic effects of prosthesis-patient mismatch in patients with and without left ventricular systolic dysfunction at the time of aortic valve replacement. METHODS: Preoperative and serial postoperative echocardiograms were performed in 805 adults who underwent aortic valve replacement between 1990 and 2003 and who were subsequently followed up in a dedicated valve clinic (follow-up, mean +/- SD, 5.5 +/- 3.5 years; maximum, 14.2 years). Preoperative left ventricular function was defined as normal (ejection fraction > or =50%) in 548 patients and impaired (ejection fraction <50%) in 257 patients. RESULTS: Patients with impaired preoperative left ventricular function and prosthesis-patient mismatch (indexed effective orifice area < or =0.85 cm2/m2) had a decreased overall late survival (hazard ratio, 2.8; P = .03), decreased freedom from heart failure symptoms or heart failure death (odds ratio of 5.1 at 3 years after aortic valve replacement; P = .009), and diminished left ventricular mass regression compared with patients with impaired preoperative left ventricular function and no prosthesis-patient mismatch. These effects of prosthesis-patient mismatch were not observed in patients with normal preoperative left ventricular function. CONCLUSIONS: Prosthesis-patient mismatch at an indexed effective orifice area of 0.85 cm2/m2 or less after aortic valve replacement primarily affects patients with impaired preoperative left ventricular function and results in decreased survival, lower freedom from heart failure, and incomplete left ventricular mass regression. Patients with impaired left ventricular function represent a critical population in whom prosthesis-patient mismatch should be avoided at the time of aortic valve replacement. PMID- 16678589 TI - Immediate endovascular treatment of blunt aortic injury: our therapeutic strategy. AB - BACKGROUND: Posttraumatic aortic rupture is a potentially lethal injury. Endovascular procedure has recently proved to be a valid option. Timing of the treatment, however, is still a debated issue. We evaluated the feasibility and safety of immediate stent-graft repair of acute posttraumatic aortic injury. METHODS: From 1998 to 2005, 15 patients (11 men and 4 women, mean age 42.3 years) with blunt aortic injury were treated with immediate stent-graft positioning. In patients with clinical and radiologic signs of impending rupture, endovascular treatment was performed in an emergency setting (11 cases). In the 4 remaining patients the aortic lesion was treated after clinical management. When present, immediate life-threatening nonaortic lesions were treated before endovascular stenting (6 cases). In 1 case emergency laparotomy and endovascular procedure were performed simultaneously. Stent positioning was monitored by intraoperative transesophageal echocardiography in all cases. RESULTS: Endovascular procedure was successful in 100% of the patients. Two patients died perioperatively as a consequence of a multiorgan failure. Both patients were in American Society of Anesthetists class V and were in severe intractable hemorrhagic shock before the procedure. Computed tomography scan performed before discharge showed correct positioning of the stent graft and absence of endoleaks in all cases. At a mean follow-up of 29 months (range 1-79) all patients were alive but 1, who died of unrelated cause, and no intervention-related complication had occurred. CONCLUSIONS: Immediate stent-graft repair of posttraumatic aortic injury is a feasible and safe procedure. It allows us to minimize the surgical risks and to treat stable and unstable lesions even when associated lesions would contraindicate traditional surgical intervention. PMID- 16678588 TI - A single nucleotide polymorphism in the matrix metalloproteinase 9 gene ( 8202A/G) is associated with thoracic aortic aneurysms and thoracic aortic dissection. AB - OBJECTIVE: Matrix metalloproteinase 9 plays an important role in the maintenance of the aortic extracellular matrix. Genetic variations that affect protease expression or activity might contribute to thoracic aortic disease. The purpose of this study was to determine whether 3 single nucleotide polymorphisms in the matrix metalloproteinase 9 gene are associated with thoracic aortic aneurysms and dissection. METHODS: Genomic DNA was isolated from blood or aortic tissue from 28 patients with degenerative thoracic aortic aneurysms, 60 patients with thoracic aortic dissection, and 111 control patients. The frequency distributions of 3 matrix metalloproteinase 9 single nucleotide polymorphisms (-8202A/G, IVS4+3G/T, and 2003A/G [Q668R]) were determined by using genotyping accomplished with a real time detection system. Associations between polymorphisms and disease were estimated with odds ratios and their 95% confidence intervals. RESULTS: The frequency of the -8202G allele was significantly higher in patients with thoracic aortic aneurysms and aortic dissection (0.52 and 0.56, respectively) than in control subjects (0.36, P < .001). Patients with thoracic aortic aneurysms or dissection were nearly 5 times more likely than control subjects to have the G allele (adjusted odds ratio, 4.87; 95% confidence interval, 2.04-11.64). There were no significant associations between the IVS4+3G/T or 2003A/G polymorphisms and thoracic aortic disease. CONCLUSIONS: The matrix metalloproteinase 9 -8202A/G polymorphism is associated with thoracic aortic aneurysms and dissection. Further studies are warranted to elucidate the functional role of the -8202A/G variant in matrix metalloproteinase 9 expression. PMID- 16678590 TI - Small aortic annulus: the hydrodynamic performances of 5 commercially available tissue valves. AB - BACKGROUND: In vivo comparison of the performance of heart valve prostheses is confounded by several factors, such as different nominal size, patients' characteristics and hemodynamics, surgical techniques, and study design. The aim of this study was to compare the in vitro hydrodynamic performances of 5 different tissue valves that would fit a 21-mm-diameter valve holder of the Sheffield pulse duplicator. METHODS: Three samples of 5 supra-annular production quality tissue valves, including the sewing ring cuffs, were tested in the aortic chamber of the Sheffield pulse duplicator. The prostheses fitting a 21-mm valve holder, which is comparable with a 21-mm aortic annulus, were as follows: 20-mm Sorin Soprano, 21-mm Carpentier-Edwards Magna, 21-mm SJM-Biocor-Epic-Supra, 21-mm Medtronic Mosaic, and 23-mm Mitroflow. The tests were carried out at a fixed pulse rate (70 beats/min) and at increasing cardiac outputs of 2, 4, 5, and 7 L/min. Each valve was tested 10 times for each different cardiac output. This resulted in a total of 40 tests for each valve and 120 tests for each valve model. Forward flow pressure decrease, effective orifice area, stroke work loss, and total regurgitant, closing, and leakage volumes were recorded while the valve operated under each cardiac output. RESULTS: Pericardial valves showed significantly lower transvalvular gradients than porcine valves, unlike the SJM Biocor-Epic-Supra valve at 2 L/min of cardiac output. Although the Carpentier Edwards Magna valve provided the best performance at 2 and 4 L/min, the Mitroflow valve exhibited the lowest mean and peak gradients at 5 to 7 L/min. Total regurgitant and leakage volumes were higher for the Carpentier-Edwards Magna valve and lower for the SJM-Biocor-Epic-Supra and Mitroflow valves. Between 2 and 4 L/min, the calculated effective orifice area and stroke work loss were better for the Carpentier-Edwards Magna valve, whereas between 5 and 7 L/min, they were significantly superior with the Mitroflow prosthesis. Among the porcine bioprostheses, the SJM-Biocor-Epic-Supra valve showed significantly better results when compared with the Medtronic Mosaic valve at each cardiac output. CONCLUSION: Assuming that the valve holder is comparable with a defined aortic annulus of 21 mm in which a spread of supra-annular tissue valves could be fitted, this hydrodynamic evaluation model allows comparison of the efficiency of currently available bioprostheses with a definite tissue annulus diameter. Pericardial valves exhibited the best performances, and the Mitroflow valve showed the lowest gradients and stroke work loss at increasing cardiac output. PMID- 16678591 TI - Pharmacologic inhibition of vein graft neointimal hyperplasia. AB - Although arterial conduits are widely used and have improved the long-term results of coronary artery bypass grafting, vein grafts remain important additional conduits in coronary surgery. Newer studies show a saphenous vein graft patency of 60% or more at 10 years postoperatively. The pathology of vein graft disease consists of thrombosis, neointimal hyperplasia, and vein graft atherosclerosis, which limit graft longevity. Therapeutic strategies to prevent vein graft disease include external stenting, pharmacotherapy, and gene therapy. The potential benefits of a pharmacologic approach are as follows: (1) Drugs with a broad clinical experience can be used; (2) side effects of systemic application can be minimized by local therapy; and (3) no vascular injury, such as pressurizing the vein for a viral transfection approach, is necessary. The different sites for pharmacotherapy in vein graft disease are reviewed in this article. PMID- 16678592 TI - Impact of follow-up on the success rate of the cryosurgical maze procedure in patients with rheumatic heart disease and enlarged atria. AB - OBJECTIVES: The most important predictors for failure of the maze procedure are long standing atrial fibrillation (AF), rheumatic heart disease (RHD), and enlarged atria. It is well documented, however, that some patients have recurrence of atrial arrhythmia only late in follow-up. The aims of this study are to assess the effectiveness of the maze procedure and late cardioversion (pharmacologic or electrical) in patients wtih an increased risk for procedure failure. PATIENTS AND METHODS: Fifty-five patients with AF, enlarged left atrium (>5 cm), and/or RHD were enrolled in the study. Cryosurgery was performed on all patients and was combined with bipolar radiofrequency in the last four patients. The lesion pattern resembles the maze procedure. A follow-up was completed on all patients (24.5 +/- 9.6 months with a range of 3-39 months). RESULTS: The operative mortality was 3.7% (2 patients), both deaths unrelated to the maze procedure. Ninety-eight percent of patients were free of AF upon discharge, with an 11% incidence of early pacemaker implantation. In the first three months postoperatively, 53% of the patients experienced intermittent atrial arrhythmia. Four patients were recorded with permanent AF in late follow-up. The only predictor for late AF (>3 months of follow-up) was perioperative AF. Atrial size, RHD, and AF duration did not predict late failure. CONCLUSIONS: The maze procedure can be applied with high success rate in patients with RHD, enlarged atria, and long-standing AF. In our experience many of the late recurrences were recorded late in the follow-up and were treated successfully with antiarrhythmic drugs and/or cardioversion. Therefore a close follow-up is required to enhance the success rate of the procedure. PMID- 16678593 TI - Biventricular diastolic filling patterns after coronary artery bypass graft surgery. AB - OBJECTIVE: We sought to study the evolution of biventricular filling properties after coronary artery bypass grafting. BACKGROUND: The evolution of diastolic function as defined with newer echocardiographic modalities after coronary artery bypass grafting surgery is unknown in patients with preoperative left ventricular diastolic dysfunction. METHODS: Transthoracic echocardiography was performed preoperatively and 48 hours and 6 months after coronary artery bypass grafting in 49 patients (randomized to milrinone [n = 25]) or placebo [n = 24]) with preoperative left ventricular diastolic dysfunction classified according to published criteria. Mild right ventricular diastolic dysfunction was defined as the ratio of early to atrial filling velocity of less than 1 in transtricuspid flow or the velocity of reversed atrial flow of greater than 50% of that of systolic flow in hepatic venous flow or the ratio of tricuspid annulus velocity during early and atrial filling of less than 1 if both the ratio of early to atrial filling velocity and the ratio of systolic to diastolic velocity was greater than 1 in hepatic venous flow. Moderate right ventricular diastolic dysfunction was diagnosed when there was a ratio of early to atrial filling velocity of greater than 1 with a ratio of systolic to diastolic velocity of less than 1. Severe right ventricular diastolic dysfunction was defined as a ratio of early to atrial filling velocity of greater than 1 associated with reversed systolic wave in hepatic venous flow. RESULTS: Moderate and severe left ventricular diastolic dysfunction increased from preoperatively to 48 hours after coronary artery bypass grafting from 8.2% to 53.7% and from 2.0% to 9.7%, respectively (P < .0001, 48 hours vs preoperatively for both), and the patterns at 6 months were similar to those observed preoperatively. Similar evolution over time was found for right ventricular diastolic dysfunction. CONCLUSIONS: In patients with preoperative left ventricular diastolic dysfunction, biventricular filling patterns are impaired initially but return to preoperative status 6 months after coronary artery bypass grafting. PMID- 16678594 TI - Thoracic aortic stent grafting: improving results with newer generation investigational devices. AB - OBJECTIVE: Six years ago an endovascular program for repair of descending thoracic aneurysms was established at the University of Pennsylvania. We report on the hypothesis that results are improving with new stent design iterations and describe our experience and lessons learned. METHODS: From April 1999 to March 2005, 99 patients with descending thoracic aneurysms underwent repair with a first or second-generation commercially produced endograft; 24 patients had an early-generation device, and 75 patients had a late-generation device. Each patient was enrolled as part of 3 distinct Phase I or Phase II Food and Drug Administration-approved clinical trials in accordance with strict inclusion and exclusion criteria. RESULTS: Mean age was 73.1 years. Symptomatic aneurysms accounted for 42% of the cohort. Mean aneurysm size was 63.7 mm (range: 30-105 mm). Twenty percent of the patients underwent a subclavian carotid transposition or bypass preoperatively to obtain an adequate proximal landing zone. No procedures had to be aborted. In-hospital or 30-day mortality was 5.0%. The incidence of permanent spinal ischemia was 2%. Perioperative vascular complications requiring interposition graft, stent repair, or patch angioplasty occurred in 27% and seemed to be less frequent in the late-generation cohort than the early-generation cohort (22.7% vs 41.7%, respectively, P = .069). At the 30 day follow-up, 23 endoleaks were detected in 22 patients (14.7% in late generation cohort vs 45.8% in early-generation cohort, P = .001). During the follow-up period, 3 new endoleaks were detected, 3 patients died of aortic rupture, and 10 patients underwent aneurysm-related reintervention. Kaplan-Meier estimated 1, 3, and 5-year survival was 84.5%, 70.5%, and 52.4%, respectively. Freedom from aneurysm-related event, defined as freedom from endoleak, aortic rupture, dissection, or any reintervention on the aorta, was 73%, 69%, and 64% at 1, 3, and 5 years, respectively. CONCLUSION: Thoracic aortic stent grafting is a safe procedure in selected patients with the added benefit of a low incidence of paraplegia. However, there is an incidence of late complications and reinterventions. This risk requires further quantification and must be balanced against the benefits of a minimally invasive approach with low perioperative morbidity and mortality. Results are improving as technology evolves and our level of experience increases. Radiologic follow-up is mandatory. PMID- 16678595 TI - Comparison of Coapsys annuloplasty and internal reduction mitral annuloplasty in the randomized treatment of functional ischemic mitral regurgitation: impact on the left ventricle. AB - BACKGROUND: Functional mitral regurgitation is associated with both annular and ventricular distortion. Aggressive reduction annuloplasty for functional mitral regurgitation acts primarily at the annulus, with variable impact on the left ventricle. The Coapsys device externally reshapes the left ventricle to correct functional mitral regurgitation. Left ventricular reshaping was analyzed in a randomized study. METHODS: The RESTOR-MV study randomizes patients with coronary artery disease and functional mitral regurgitation to either reduction annuloplasty and coronary artery bypass grafting (the RA group) or Coapsys annuloplasty and bypass grafting (the CO group). The Coapsys device consists of epicardial pads connected by a cord. It was placed without cardiopulmonary bypass under echocardiographic guidance and sized to reduce annular dimension and improve leaflet coaptation. Internal reduction annuloplasty was performed by device placement. Intraoperative transesophageal echocardiograms were analyzed in 7 patients having reduction annuloplasty and 7 having Coapsys annuloplasty. RESULTS: Baseline mitral regurgitation (0-4 scale) was similar for the RA (3.0 +/ 0.6) and the CO groups (3.0 +/- 0.6). Intraoperative mitral regurgitation was reduced from 2.86 +/- 0.7 to 0.5 +/- 0.7 (P < .01 pre vs post) for the RA group and from 2.64 +/- 0.9 to 05 +/- 0.7 (P < .01 pre vs post) for the CO group. Annular anteroposterior diameter was reduced with both techniques: RA, 3.45 +/- 0.39 to 2.34 +/- 0.37 cm (P < .01 pre vs post); CO, 3.40 +/- 0.27 to 2.85 +/- 0.34 cm (P < .05 pre vs post). Long-axis dimensions were unchanged with both techniques. Short-axis dimensions measured at three levels were significantly reduced only in the CO patients: basal diameter 4.77 +/- 0.58 to 3.58 +/- 0.38 cm (P < .01 pre vs post); mid diameter 4.88 +/- 0.55 to 3.57 +/- 0.43 cm (P < .01 pre vs post); and apical diameter 4.39 +/- 0.46 to 3.38 +/- 0.34 cm (P < .01 pre vs post). CONCLUSIONS: Coapsys and reduction annuloplasty techniques both acutely reduce functional mitral regurgitation and annular dimension. The Coapsys device provided significantly greater left ventricular reshaping than did reduction annuloplasty. Further evaluation will assess the long-term valvular function and ventricular geometric stability associated with both techniques. PMID- 16678596 TI - Inclusion of oxygen consumption improves the accuracy of arterial and venous oxygen saturation interpretation after the Norwood procedure. AB - OBJECTIVE: Management strategy for the postoperative Norwood neonate has been formulated from models that have estimated oxygen consumption (VO2). Superior vena caval oxygen saturation (SVO2), systemic arterial and superior vena caval oxygen saturation difference (Sa-VO2), and oxygen excess factor (Omega = arterial oxygen saturation/Sa-VO2) have been used as indirect indicators to estimate systemic blood flow (Qs) and oxygen delivery (DO2). We sought to examine the correlation of the indirect indicators to VO2-derived measures of oxygen transport. METHODS: Respiratory mass spectrometry was used to continuously measure VO2 after the Norwood procedure (n = 13). Measured saturations and the direct Fick equation were used to obtain pulmonary blood flow, Qs, DO2, and oxygen extraction ratio (ERO2) values. Correlations to SVO2, Sa-VO2, and Omega were sought. RESULTS: There was a close correlation of SVO2, Sa-VO2, and Omega to ERO2 (r = 0.92, 0.96, and 0.97, respectively; P < .0001). Correlation to Qs and DO2 was variable (r = 0.39 to 0.78, respectively; P < .0001). Correlation to VO2 was poor but significant (r = 0.24 to 0.40, P < .0001). Inclusion of VO2 improved the correlation to Qs and DO2 (r = 0.66 to 0.97, P < .0001). CONCLUSIONS: The close correlation of SVO2, Sa-VO2, and Omega to ERO2 indicates that each is a measure of the balance of DO2 and extraction. The significant but less reliable correlation to DO2 and VO2 indicates the values for SVO2, Sa-VO2, and Omega do not discriminate between the contribution of DO2 and VO2. Measured VO2 and hemodynamics may improve the optimization of postoperative management strategy in the individual neonate. PMID- 16678598 TI - Extracorporeal life support after staged palliation of a functional single ventricle: subsequent morbidity and survival. AB - OBJECTIVE: We sought to review the outcome of infants with a functional single ventricle receiving postoperative extracorporeal life support. METHODS: We reviewed all patients with a functional single ventricle receiving postoperative extracorporeal life support between January 1997 and May 2003. RESULTS: We supported 25 infants (age range, 2-139 days; median age, 15 days; weight range, 1.9-5.9 kg; median weight, 3.4 kg) with extracorporeal life support. Operative procedures were Norwood stage 1 procedure in 18 patients, modified Blalock Taussig shunt in 4 patients, bidirectional superior cavopulmonary shunt in 2 patients, and pulmonary vein repair in 1 patient. Indications for extracorporeal life support included cardiac arrest (14/25) and low cardiac output state (11/25). Extracorporeal membrane oxygenation was initiated in 19 patients, with conversion to a ventricular assist device in 7 patients. Ventricular assist device alone was initiated in 6 patients. Survival to decannulation was 76%, with 5 late deaths from multiorgan failure and 56% intensive care unit survival. Survival to hospital discharge was 44%. On univariate analysis, the presence of arrhythmia before extracorporeal life support (P = .005), renal failure (P = .0007), Candida species-induced sepsis (P = .026), and multiorgan failure (P = .0009) were significant risk factors in the nonsurvivors. Median hospital stay was 43.5 days (range, 6-181 days) for the whole group and 93 days (range, 36-181 days) for survivors. Eight patients completed next stage palliation. CONCLUSIONS: Twenty percent of patients were supported with a ventricular assist device alone, with 50% conversion to a ventricular assist device from extracorporeal membrane oxygenation. Survival to decannulation was encouraging. Multiorgan failure and risk of invasive infection in the post-extracorporeal membrane oxygenation period mitigate against survival to hospital discharge. Use of extracorporeal life support before cardiac arrest might reduce attrition between decannulation and hospital discharge. PMID- 16678597 TI - Contemporary management of right atrial isomerism: effect of evolving therapeutic strategies. AB - OBJECTIVES: Infants with right atrial isomerism have poor outcomes because of a complex combination of cardiac anomalies. Aggressive management of total anomalous pulmonary venous drainage might have a positive effect on the prognosis. METHODS: Outcomes of all children with right atrial isomerism from 1994 to the present were reviewed. Management of total anomalous pulmonary venous drainage evolved from no repair or conventional surgical technique to primary sutureless repair on initial palliation. Cox survival models were used to identify variables associated with reduced survival. RESULTS: There were 55 children enrolled in the study. The median age at the initial visit was 2 days. Fifty-one patients had total anomalous pulmonary venous drainage (obstructive in 22 patients). Withdrawal of treatment occurred in 11 (20%) of 55 patients during an interval of institutional bias toward no treatment. Thirteen (24%) of 55 patients had palliations without total anomalous pulmonary venous drainage repair, and 3 (23%) of 13 survived. Thirty-one (56%) of 55 patients had operations that included total anomalous pulmonary venous drainage repair, of whom 13 (42%) of 31 underwent primary sutureless repair for total anomalous pulmonary venous drainage. Sixteen (52%) of 31 survived, and their current status 1 to 10 years (median, 5.8 years) after repair is post-Fontan (7/16 [44%]), postbidirectional Glenn (6/16 [38%]), and others (3 [20%]). In patients who underwent total anomalous pulmonary venous drainage repair (n = 31), 2 risk factors of decreased survival were identified: drainage site obstruction and infracardiac or mixed-type total anomalous pulmonary venous drainage. After adjustment, sutureless repair appeared to be associated with improved survival (hazard ratio, 0.43), but this beneficial effect did not reach significance (P = .19). CONCLUSIONS: Mortality continues to be high; however, aggressive total anomalous pulmonary venous drainage repair for right atrial isomerism has resulted in improved survival. The role of primary sutureless repair for total anomalous pulmonary venous drainage remains to be defined. PMID- 16678599 TI - Impact of preoperative treatment strategies on the early perioperative outcome in neonates with hypoplastic left heart syndrome. AB - OBJECTIVE: This study was undertaken to determine the impact of specific intensive care procedures on preoperative hemodynamics, incidence of preoperative organ dysfunction, and in-hospital mortality among neonates with hypoplastic left heart syndrome with pulmonary overcirculation and to assess the influence of the change in preoperative management on early postoperative outcome. METHODS: In this retrospective evaluation of 72 neonates with classic hypoplastic left heart syndrome and severe pulmonary overcirculation with different preoperative management strategies from 1992 to 1995 and from 1996 to 2000, univariate and multivariate analyses of risk factors were performed with stepwise logistic regression. RESULTS: Among patients with ventilatory and inotropic support from admission until surgery, degree of metabolic acidosis (lowest recorded and prerepair pH values) was significantly higher than among patients who received systemic vasodilators without ventilation before surgery. Preoperative organ dysfunction occurred in 19 of 72 patients (26%), predominantly before 1996; the most significant was hepatic failure in 13 (68%). Lowest recorded and prerepair pH values did not predict the development of organ dysfunction, whereas inotropic medication, lack of afterload reduction, and especially ventilatory support correlated significantly with organ injury. In-hospital mortality decreased from 65% (13/20) to 13% (6/46) from the first to the second period. According to multivariate analysis, ventilatory support and organ dysfunction were significantly related to in-hospital mortality. CONCLUSION: In neonates with hypoplastic left heart syndrome, systemic afterload reduction can avoid preoperative artificial respiration, identified as a significant risk factor for the development of preoperative dysfunction of end organs and in-hospital mortality. PMID- 16678600 TI - Off-label use of an adjustable gastric banding system for pulmonary artery banding. AB - BACKGROUND: Pulmonary artery banding is proposed as a first palliation in infants with complex congenital heart disease and high pulmonary blood flow. In addition, it may be used to retrain the left ventricle. Optimal tightening may be difficult to obtain, leading to reoperation. An implantable device for pulmonary artery banding with telemetric control was recently developed allowing for repeated adjustments, but it is presently limited to patients weighing less than 20 kg. In large animals, we tested an off-label adjustable gastric banding system for pulmonary artery banding. METHODS AND RESULTS: Fourteen ewes weighing 50 to 75 kg underwent implantation of the Lap-Band device (BioEnterics Corp, Santa Barbara, Calif) around the main pulmonary artery through a left thoracotomy. All had functional evaluation with progressive occlusion and opening of the device at implantation and every 2 weeks until sacrifice immediately after implantation (group 1, n = 8), at 1 month (group 2, n = 3), at 3 months (group 3, n = 3), or death. Invasive pressure measurements in the right ventricle and aorta were carried out each time. Devices were easily implanted in all animals. Progressive occlusion and reopening were possible in all animals during each time point. Two animals died of right heart failure related to excessive tightening of the band. Retrieval of the device without any major damage was possible in 12 of 14 animals. CONCLUSION: With this implantable device, we were able to adjust the pulmonary artery diameter in animals. Patients requiring left ventricle retraining and weighing more than 30 kg would benefit from the device's use in humans. PMID- 16678601 TI - Surgical management of aortopulmonary window associated with interrupted aortic arch: a Congenital Heart Surgeons Society study. AB - OBJECTIVE: The objective was to determine outcomes and risk factors of surgical management of patients with aortopulmonary window associated with interrupted aortic arch. METHODS: From 1987 to 1997, 472 neonates with interrupted aortic arch were enrolled prospectively from 33 institutions. Associated aortopulmonary window was present in 20 patients. Competing risk methodology determined the prevalence of reintervention for postrepair pulmonary artery and aortic arch obstruction. RESULTS: Interrupted aortic arch was type A in 17 patients and type B in 3 patients. Aortopulmonary window morphology was type I (n = 10), type II (n = 5), and type III (n = 5). Associated cardiovascular anomalies were common, including atrial septal defect (n = 13) and systemic venous anomalies (n = 3). Overall survival after initial admission was 91%, 86%, and 84% at 1, 5, and 10 years, respectively. Fifteen patients underwent single-stage repair, and 4 patients underwent staged repair. There was an increased prevalence of patch augmentation of the interrupted aortic arch anastomosis in lower-weight infants (2.3 kg vs 3.1 kg, P = .07). Competing risk analysis estimated that 5 years after repair, 51% had initial arch reintervention, 6% had initial pulmonary artery reintervention, and 43% were alive without reintervention. Reintervention for arch obstruction was more likely for those with interrupted aortic arch type B (P = .08) and for those with higher weight at initial repair (P = .003). CONCLUSIONS: Complete correction of aortopulmonary window in the setting of interrupted aortic arch can be performed with low mortality in the neonatal period. Reinterventions for aortic arch obstruction are the most frequent complication after repair, but pulmonary artery stenosis also occurs. Use of patch augmentation may reduce the need for subsequent arch reintervention. PMID- 16678602 TI - Living donor lobar grafts improve pediatric lung retransplantation survival. AB - OBJECTIVE: Lung retransplantation is a controversial practice due to increased morbidity and mortality and the scarcity of available donor organs. Living donor lobar lung transplantation increases the number of available donor organs and facilitates a more organized procedure than traditional cadaveric donation for this complex reoperation. The purpose of this study was to evaluate our experience with pediatric lung retransplantation and to compare the outcomes of living donor lobar lung transplantation with cadaveric donation. METHODS: Retrospective review of a prospectively collected database identified 39 children who underwent lung retransplantation from 1991 to 2004. Retransplantation was performed with living donor lobar lung transplantation in 13 patients and cadaveric donation in 26 patients. Short- and long-term outcomes were compared between the 2 groups. RESULTS: Perioperative mortality was 1/13 (7.7%) in the patients who had living donor lobar lung transplantation versus 11/26 (42.3%) in the cadaveric donation group (P = .03). Five-year survival for living donor lobar lung transplantation and cadaveric donation was 40.4% and 29.7%, respectively (P = .27). Both groups had a significant improvement in their forced expiratory volume in 1 second 6 months after retransplantation (P < .001). Multivariate analysis identified the use of cadaveric donation (relative risk = 6.16, P = .001) and early graft dysfunction (relative risk = 6.19, P = .001) as the major independent predictors of decreased survival following retransplantation. CONCLUSIONS: Living donor lobar lung transplantation reduces perioperative mortality and is an independent predictor of improved survival following pediatric lung retransplantation. This strategy offers significant benefit for this high-risk group and preserves the limited supply of donor lungs for other children at risk of dying while waiting for lung transplantation. PMID- 16678603 TI - Lung regeneration: implantation of fetal rat lung fragments into adult rat lung parenchyma. AB - OBJECTIVE: The capability of regeneration of lung tissues in adults is limited after chronic destruction. Bone marrow-derived stem cells, retinoic acid, growth factors, and other approaches have been attempted to promote or facilitate this process. We hypothesized that fetal lung tissues, with great potential for growth and differentiation, could be used for lung regeneration. METHODS: Day 17 fetal lung tissue fragments at the pseudoglandular stage of lung development from Lewis rats were implanted into adult Lewis rat lungs. For group 1, fetal lung fragments were injected into the adult left lung parenchyma; for group 2, fetal fragments were injected with the left lung partially resected; for group 3, adult fragments were injected; and for groups 4 and 5, fetal fragments were implanted into the omentum and subcutaneous tissue, respectively. RESULTS: The grafts implanted into pulmonary parenchyma were differentiated with opening of the alveolar space after 4 weeks and were advanced further with morphologic features similar to those of neonatal lungs after 8 and 12 weeks. The implants were connected with pulmonary circulation determined by means of perfusion with India ink. These changes appeared to be further enhanced in animals with partial lung resection that might have facilitated the maturation of implanted fetal lung tissues through mechanical factors, soluble factors, or both. Fetal lung tissues did not mature when implanted into the omentum or subcutaneous tissue. Adult lung fragments did not expand after being reimplanted back into the same animal. CONCLUSIONS: Fetal lung tissue might be an option for further investigation into lung regeneration. PMID- 16678604 TI - Extended donor criteria in lung transplantation: impact on organ allocation. AB - OBJECTIVE: Some reports have documented a higher early mortality with the use of extended criteria donors in lung transplantation. None have evaluated how outcomes compare with the use of these organs for single and bilateral transplantation or whether this practice results in a higher incidence of early bronchiolitis obliterans syndrome. METHODS: We performed a retrospective review of case notes, intensive therapy unit database, and donor details. Between January 1, 2000, and December 31, 2004, 201 patients underwent lung or heart-lung transplantation. RESULTS: Eighty-three (41.3%) patients received organs deemed marginal on the basis of at least one of the following criteria: donor age greater than 55 years, duration of ventilation greater than 5 days, purulent secretions or inflammation at bronchoscopy, smoking of 20 or more cigarettes per day, abnormality on chest roentgenogram, or PO2/fraction of inspired oxygen ratio of less than 300 mm Hg immediately before donor organ procurement. Recipients of marginal lungs had a higher incidence of severe (grade 3) primary graft dysfunction (43.9% vs 27.4%, P = .015) and 90-day organ-specific mortality (15.7% vs 5.1%, P = .012). Bilateral transplantation carried a significantly higher 30 day mortality if performed with marginal organs (17.0% vs 2.7% with standard donor organs, P = .005). Thirty-day mortality was not significantly different for the transplantation of single marginal or standard donor lungs. Cumulative survival and survival free of bronchiolitis obliterans syndrome was not affected by marginal donor status. CONCLUSION: Transplantation of extended criteria donor lungs leads to a higher incidence of primary graft dysfunction. Bilateral transplantation with these organs seems to confer less reserve, resulting in a higher early mortality rate. Medium-term functional outcome is, however, not adversely affected by the relaxation of donor criteria. PMID- 16678605 TI - Clopidogrel reduces the development of transplant arteriosclerosis. AB - BACKGROUND: Transplant arteriosclerosis, the hallmark feature of chronic rejection, is still the major limiting factor for the long-term success of heart transplantation. Platelets have been implicated to play a role in the pathogenesis of this disease. Therefore the aim of this study was to investigate whether platelet inhibition alone has a positive effect on the development of transplant arteriosclerosis. METHODS: Fully major histocompatibility complex mismatched C57BL/6 (H2(b)) donor aortas were transplanted into CBA (H2(k)) recipients, and mice received different doses (1, 10, and 20 mg/kg) of clopidogrel or control saline as a daily intraperitoneal injection for 30 days. Blood was analyzed on days 2, 7, 14, and 30 by using a platelet aggregation test (adenosine diphosphate) for effectiveness of the treatment. Grafts were analyzed by means of histology and morphometry on day 30 after transplantation. RESULTS: When mice were treated daily with 1 mg/kg clopidogrel in the absence of any other immunosuppression, transplant arteriosclerosis was significantly reduced compared with that seen in saline-treated control animals (intimal proliferation of 66% +/ 9% [1 mg/kg clopidogrel] vs 77% +/- 5% [control], n = 7, P < or = .03). Daily application of 10 mg/kg and 20 mg/kg clopidogrel also significantly reduced the development of transplant arteriosclerosis compared with that seen in control animals (intimal proliferation of 61% +/- 11% [10 mg/kg clopidogrel] vs 54% +/- 10% [20 mg/kg clopidogrel] vs 77% +/- 5% [control], n = 8, P < or = .003). There was, however, no additional beneficial effect when compared with mice treated with 1 mg/kg clopidogrel (P = .06). Isografts did not show any signs of vascular lesions on day 30 after transplantation. CONCLUSION: These results demonstrate that monotherapy with clopidogrel can effectively reduce the formation of transplant arteriosclerosis in a murine aortic allograft model. PMID- 16678606 TI - Surgical repair of a large coronary artery aneurysm with arteriovenous fistula. PMID- 16678607 TI - Two distinct clinical presentations in adult unicuspid aortic valve. PMID- 16678608 TI - Thromboembolism from the noncoronary cusp of a patient with a normal aortic valve and protein C deficiency. PMID- 16678609 TI - Lower extremity compartment syndrome after off-pump aortocoronary bypass. PMID- 16678610 TI - Polydioxane (PDS) cord has insufficient reliability to securely close the sternum. PMID- 16678611 TI - Intraoperative finding of structural abnormality of bovine pericardial aortic prosthesis. PMID- 16678612 TI - Valve-sparing excision of aortic valve papillary fibroelastoma. PMID- 16678613 TI - Pulmonary artery myxoma as a rare cause of dyspnea for a young female patient. PMID- 16678614 TI - Fungal prosthetic mitral valve endocarditis caused by Scopulariopsis species: case report and review of the literature. PMID- 16678616 TI - Transaortic correction of tetralogy of Fallot and similar defects. PMID- 16678615 TI - Erosion of a retroesophageal subclavian artery by an esophageal prosthesis. PMID- 16678617 TI - Aneurysmal sac located between right pulmonary artery and left atrium. PMID- 16678618 TI - Use of the endoclamp device in a patient with a native mitral valve endocarditis and a previous arch replacement with a modified Griepp technique. PMID- 16678619 TI - Ventricular assist device use for the treatment of acute viral myocarditis. PMID- 16678620 TI - Off-pump pulmonary valve replacement with the new Shelhigh Injectable Stented Pulmonic Valve. PMID- 16678621 TI - Transapical aortic valve implantation in humans. PMID- 16678622 TI - Nourishing vascularization of a thymoma issued from a left internal thoracic artery graft. PMID- 16678623 TI - Giant gastrointestinal stromal tumor of the esophagus presenting with dyspnea. PMID- 16678624 TI - Stenotic bridging bronchus: a very rare entity. PMID- 16678625 TI - Aseptic mediastinal cyst caused by BioGlue 7 months after cardiac surgery. PMID- 16678626 TI - Hiatal herniation of the pancreas: diagnosis and surgical management. PMID- 16678627 TI - Duropleural fistula as a consequence of Gorham-Stout syndrome: a combination of 2 rare conditions. PMID- 16678628 TI - Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia and a typical carcinoid tumor. PMID- 16678629 TI - Spondyloarthropathies and aortic dissection. PMID- 16678631 TI - Implantable cardioverter-defibrillator after left ventricular reconstruction? PMID- 16678632 TI - Left ventricular assist device in heart failure. PMID- 16678634 TI - Apoptosis in ischemic spinal cord injury. PMID- 16678637 TI - Surgical treatment for congestive heart failure with autologous adult stem cell transplantation. PMID- 16678639 TI - Update of current therapeutic options for the treatment of postmenopausal osteoporosis. AB - BACKGROUND: Osteoporosis is a common chronic condition in elderly women and is associated with decreased bone strength and an increased risk for fractures. As the incidence of osteoporotic fractures continues to rise, it is important to identify the most effective therapies for reducing patients' risk of fracture. OBJECTIVE: This article reviews the medication classes commonly used for treating osteoporosis and the efficacy, tolerability, and drug-interaction potential of specific medications. The evidence for the use of combination therapies is summarized, as are the agents under investigation. METHODS: Relevant articles were identified through a search of MEDLINE (August 1985-August 2005) using the terms osteoporosis, postmenopausal, fracture, and efficacy combined with drug therapy, calcium, vitamin D, estrogen, progesterone, selective estrogen modulators, calcitonin, strontium ranelate, bisphosphonates, alendronate, risedronate, ibandronate, pamidronate, parathyroid hormone, combination therapy, and zoledronic acid. The identified articles were reviewed for suitability, with priority given to meta-analyses. RESULTS: Among the therapeutic options for the treatment of osteoporosis, the bisphosphonates appear to provide the greatest antiresorptive efficacy, with some bisphosphonates providing 7% to 8% increases in bone mineral density and 60% to 70% decreases in markers of bone resorption. Bisphosphonates also may reduce the incidence of new vertebral fractures by 50% to 52%. CONCLUSIONS: Bisphosphonates are currently the first choice for the treatment of osteoporosis. Use of intermittent regimens of the newer bisphosphonates appears to be a promising alternative to administration of daily or weekly treatment. PMID- 16678640 TI - Echinacea in the prevention of induced rhinovirus colds: a meta-analysis. AB - BACKGROUND: The therapeutic effectiveness of Echinacea in the treatment and the prevention of colds has been debated. Studies of naturally occurring colds are hampered by variability in time from onset of symptoms to treatment and by heterogeneity in trial design. Experimental infection studies allow for the standardization of time to initiation of treatment, virus type and dose, and immune competence of volunteers. OBJECTIVE: To determine whether the negative results obtained in previous studies of Echinacea were a consequence of efficacy or of inadequate sample size, we performed a meta-analysis of experimental rhinovirus infection studies on the efficacy of Echinacea extracts to prevent symptomatic development of an experimentally induced cold. METHODS: We carried out a systematic search of English- and German-language literature using the MEDLINE, EMBASE, CAplus, BIOSIS, CABA, AGRICOLA, TOXCENTER, SCISEARCH, NAHL, and NAPRALERT, databases and the search terms Echinacea, black Sampson, coneflower, and Roter Sonnenbut. Matching documents were then searched for > or = 1 of the following terms: rhinovirus, RV, inoculation, Inokulation, induced, induziert, artificial, and artifiziell. Suitable studies were identified and pooled for analysis. The primary end point was the development of symptomatic clinical colds, as defined by the authors of the original studies. Results were reported as differences in the proportion of subjects with symptomatic episodes of a common cold, expressed as odds ratios (ORs) and 95% CIs. The secondary outcome was the difference in total symptom severity scores between treatment groups (assessed daily by integrating the severity scores of 8 individual cold-related symptoms that were rated on a scale from 0 [absent] to 4 [very severe]). RESULTS: A total of 234 articles were identified through the literature search; 231 were excluded from the analysis because they related to studies of spontaneous common colds. Three suitable studies were selected for pooling of data. Based on the analysis, the likelihood of experiencing a clinical cold was 55% higher with placebo than with Echinacea (OR, 1.55 [95% CI, 1.02-2.36]; P<0.043). The absolute difference in total symptom scores between groups was -1.96 (95% CI, -4.83 to 0.90; P=NS). CONCLUSIONS: This meta-analysis suggests that standardized extracts of Echinacea were effective in the prevention of symptoms of the common cold after clinical inoculation, compared with placebo. Further prospective, appropriately powered clinical studies are required to confirm this finding. PMID- 16678641 TI - Entecavir for the treatment of chronic hepatitis B virus infection. AB - OBJECTIVE: This article reviews the pharmacology/pharmacokinetics and therapeutic efficacy of entecavir, which was approved on March 29, 2005, for the management of adult patients with chronic hepatitis B virus (HBV) infection who have active viral replication and/or elevations in liver transaminases or signs of active liver disease on histologic examination. Potential drug interactions and adverse events associated with the use of entecavir are also reviewed. METHODS: Relevant literature was identified through searches of MEDLINE (1996-July 2005) and BIOSIS (1993-July 2005). Search terms included, but were not limited to, entecavir, BMS 200475, hepatitis B, pharmacology, pharmacokinetics, adverse events, and therapeutic use. Further publications were identified from the reference lists of the identified articles and through correspondence with the manufacturer of entecavir. RESULTS: Entecavir is highly selective for the HBV and inhibits all 3 steps of viral replication. Results of early studies indicated a 6% resistance potential after 48 weeks of therapy, although the potential may be higher in patients who harbor lamivudine-resistant mutants. The approved dosage in treatment-naive patients is 0.5 mg/d p.o., administered on an empty stomach; in patients who have failed lamivudine therapy or are known to harbor lamivudine resistant mutants, the approved dosage is 1.0 mg/d p.o.. The oral tablet and solution can be used interchangeably. Entecavir is well absorbed orally, achieving a dose-related Cmax between 0.6 and 1.5 hours after administration. It is metabolized to a small extent and is not a substrate for the cytochrome P450 enzyme system. The mean elimination t(1/2) ranges from 77 to 149 hours in patients with normal kidney function. Entecavir is eliminated primarily in the urine via glomerular filtration and tubular secretion (62%-73%). No dose adjustment appears to be necessary in patients with moderate to severe liver disease alone. The potential for drug interactions with entecavir appears to be minimal, although medications that inhibit tubular secretion of drugs (eg, probenecid) may be expected to prolong serum concentrations of entecavir. One of the Phase III studies of entecavir found statistically significant benefits compared with lamivudine in terms of improvements in liver histology after 48 weeks of therapy (72% vs 62%, respectively; P<0.009), the proportions of patients with undetectable HBV DNA titers on branched DNA signal amplification assay after 48 weeks of therapy (91% vs 65%; P<0.001), and the proportion with undetectable HBV DNA on polymerase chain reaction (PCR) assay after 48 weeks of therapy (69% vs 38%; P<0.001). In another Phase III study, patients who had failed to respond to lamivudine therapy responded to entecavir: after 48 weeks of therapy, significant differences between entecavir and lamivudine were seen in histologic improvement (55% vs 28%; P<0.001) and the proportion of patients with undetectable HBV DNA on PCR assay (21% vs 1%; P<0.001). Adverse events associated with entecavir therapy were similar in character, severity, and incidence to those associated with placebo or lamivudine therapy. The most common adverse events in clinical trials of entecavir were headache (17%-23% of patients), upper respiratory tract infection (18%-20%), cough (12%-15%), nasopharyngitis (9%-14%), fatigue (10%-13%), dizziness (9%), upper abdominal pain (9%-10%), and nausea (6% 8%). CONCLUSIONS: Entecavir is a new antiviral agent for the management of chronic HBV infection. Questions concerning the ideal length of therapy, long term efficacy, and resistance rates over time await the results of ongoing clinical trials. PMID- 16678642 TI - A comparison of valdecoxib and naproxen in the treatment of rheumatoid arthritis symptoms. AB - OBJECTIVES: The primary aim of this work was to compare the efficacy of valdecoxib 10, 20, and 40 mg QD with that of placebo and naproxen 500 mg BID in patients with rheumatoid arthritis (RA). The overall safety and tolerability profiles of valdecoxib and naproxen were also compared. METHODS: A 12-week, multicenter, randomized, double-blind, parallel-group, placebo- and active controlled study was performed in patients with adult-onset RA whose disease was in a flare state after discontinuing NSAIDs or other analgesics. Patients were randomly assigned to valdecoxib 10, 20, or 40 mg QD, naproxen 500 mg BID, or placebo. The primary efficacy measures were the American College of Rheumatology (ACR) 20% responder index (ACR-20), physicians' assessments of tender/painful joint count and swollen joint count, and patients' and physicians' global assessments of disease activity. Adverse events, clinical laboratory data, and vital signs were assessed by the investigator and compared between treatment groups to evaluate overall tolerability and safety. RESULTS: A total of 1093 patients were randomized to receive either valdecoxib 10 mg QD (n=226), valdecoxib 20 mg QD (n=219), valdecoxib 40 mg QD (n=209), naproxen 500 mg BID (n=219), or placebo (n=220). At all time points, the proportion of ACR-20 responders was significantly higher in the valdecoxib groups than the placebo group at weeks 2 (10 mg, P<0.001; 20 mg, P=0.008; 40 mg, P= 0.004), 6 (all, P<0.001), and 12 (10 mg, P=0.006; 20 mg, P=0.004; 40 mg, P<0.001). Similarly, at all time points, the proportion of ACR-20 responders was significantly higher in the naproxen 500-mg group than the placebo group (all time points, P<0.001). In addition, mean changes in the number of tender/painful joint counts were significantly greater in the valdecoxib groups than the placebo group at weeks 2 (all, P<0.001), 6 (10 mg, P=0.002; 20 and 40 mg, P<0.001), and 12 (10 mg, P=0.004; 20 mg, P= 0.012; 40 mg, P<0.001). Naproxen treatment was also associated with greater reductions in tender/painful joint count than placebo (all, P<0.001). Mean changes in swollen joint count decreased at all time points in all groups, with significantly greater changes in the valdecoxib and naproxen treatment groups than the placebo group (valdecoxib 20 and 40 mg: week 6, P= 0.014 and P=0.003, respectively; naproxen: week 2, P=0.014; week 6, P=0.015; week 12, P=0.030). Physicians' global assessments of disease activity scores were significantly lower in the valdecoxib (10 mg: weeks 2 and 6, P<0.001; week 12, P=0.001; 20 and 40 mg: all weeks, P<0.001) and naproxen (all time points, P<0.001) treatment groups than the placebo group. Adverse events were reported by 45.5% patients in the placebo group, 51.8% in the valdecoxib 10 mg QD group, 58.0% in the valdecoxib 20 mg QD group, 56.9% in the valdecoxib 40 mg QD group, and 62.6% in the naproxen 500 mg BID treatment group. CONCLUSIONS: Valdecoxib 10, 20, and 40 mg QD were efficacious for treating the signs and symptoms of RA in these patients. The efficacy of valdecoxib 20 and 40 mg QD was not significantly different from that of naproxen 500 mg BID. Valdecoxib was generally well tolerated in this study. PMID- 16678643 TI - Multicenter, randomized, double-blind, active-controlled, parallel-group trial of the long-term (6-12 months) safety of acetaminophen in adult patients with osteoarthritis. AB - OBJECTIVE: This study evaluated the safety of acetaminophen 4 g/d administered for up to 12 months to adult patients with osteoarthritis pain, using naproxen 750 mg/d as an active comparator. METHODS: This multicenter, multidose, single dummy, randomized, double-blind, active-controlled, parallel-group study enrolled patients with mild to moderate osteoarthritis pain of the hip or knee. Patients received acetaminophen 4 g/d or naproxen 750 mg/d for 12 months (group 1) or 6 months (group 2). Patients in both groups had follow-up visits at months 1, 3, and 6 of treatment (or at the time of study withdrawal). Patients in group 1 also had follow-up visits at months 9 and 12 (or at the time of study withdrawal). Tolerability evaluations consisted of determinations of hepatic (aminotransferase activities) and renal (serum creatinine) function, adverse events, and physical examinations. Adverse events reported by the patient or observed by the investigator during clinical evaluation were recorded. In addition, patients were questioned at each visit regarding the occurrence of adverse events using a nonspecific question. Investigators rated the intensity of adverse events and their subjective assessment of the relationship to study medication while blinded to the treatment group. At all visits, patients completed the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), in visual analog scale format, to assess pain, stiffness, and physical function over the previous 2 weeks. The primary efficacy end point was the mean change from baseline in the WOMAC pain subscale score at 6 months. Data from the 6- and 12-month groups were combined for analysis. RESULTS: Of 581 randomized patients, the safety population included 571 patients who received > or = 1 dose of study medication. The 571 patients had a mean (SD) age of 59.3 (8.6) years, 395 (69.2%) were female, and 480 (84.1%) were white. Of 290 patients randomized to receive acetaminophen, 134 completed 3 months of treatment, 96 completed 6 months, 60 completed 9 months, and 55 completed a full 12 months. The median dose adherence ranged from 95.5% to 98.6% during the trial. The completion and adherence patterns were similar for patients receiving naproxen. Of 291 patients randomized to receive naproxen, 151 completed 3 months, 124 completed 6 months, 85 completed 9 months, and 80 completed 12 months. The median dose adherence ranged from 96.4% to 98.4% during the trial. No patient in either treatment group experienced hepatic failure, hepatic dysfunction, aminotransferase levels > or = 2x the upper limit of the reference range, renal failure, or serum creatinine levels > or = 1.5x the upper limit of the reference range. No statistically significant differences were observed between the 2 treatment groups in the proportion of patients who reported > or = 1 adverse event (206 [71.8%] acetaminophen, 209 [73.6%] naproxen) or in the proportion of patients who discontinued treatment because of adverse events (71 [24.7%] acetaminophen, 63 [22.2%] naproxen). Among adverse events considered to be drug related and reported by > or = 1% of patients, constipation and peripheral edema were reported more frequently in the naproxen group than in the acetaminophen group (9.9% vs 3.1% [P<0.002] and 3.9% vs 1.0% [P<0.033], respectively). No adverse event reported in the acetaminophen group was considered both serious and related to study medication. One subject in the naproxen group had an event that was considered serious and related to study drug: gastrointestinal bleeding. No statistically significant differences were observed between the 2 treatment groups for the primary efficacy end point. CONCLUSION: With physician supervision, acetaminophen was found to be generally well tolerated in these patients for the treatment of osteoarthritis pain of the hip or knee for periods of up to 12 months. PMID- 16678644 TI - Determinants of persistence with bisphosphonates: a study in women with postmenopausal osteoporosis. AB - BACKGROUND: Although bisphosphonates are useful in the management of osteoporosis, patients often discontinue treatment. OBJECTIVES: The aims of this study were to investigate persistence with bisphosphonates, and to assess whether the dose interval influenced persistence, among women with postmenopausal osteoporosis. METHODS: Data were obtained from the PHARMO Record Linkage System, which includes, among other databases, drug-dispensing records from community pharmacies linked to hospital discharge records of > 1 million subjects in defined areas in The Netherlands. Women who were new users of alendronate (daily or weekly), etidronate (daily), or risedronate (daily) during the period from January 2000 through September 2003 were eligible for inclusion in the study if they were aged > or = 55 years or had been hospitalized for a menopausal disorder. One-year rates of persistence with treatment (defined as the percentage of patients who used the drug for > or = 365 days without failure to continue renewals) were determined by using episodes of bisphosphonate treatment. The association between persistence and dose intervals, type of bisphosphonate, and other determinants (including age, occurrence of gastrointestinal adverse events as measured by use of concomitant medications [eg, antacids, proton pump inhibitors, histamine2 (H2)-receptor antagonists, misoprostol, laxatives, antidiarrheals, bowel motility enhancers] and fractures) was assessed. To study whether persistence with bisphosphonates was associated with the former use of other antiosteoporosis medication or the presence of drug-induced osteoporosis, the use of hormone replacement therapy, raloxifene, and systemic corticosteroids in the 6 months before the index date were included as determinants. RESULTS: The study sample included 2124 women who were new users of bisphosphonates. The mean (SD) age of the study population was 71.6 (8.7) years. After 1 year, 51.9% of weekly alendronate users and 30.1% to 42.2% of daily bisphosphonate users were persistent. In the multivariate analysis (which included age, concomitant medication, and fractures), patients using alendronate weekly were significantly more likely to persist than those using alendronate daily (relative risk [RR], 1.56 [95% CI, 1.32-1.85]). The likelihood of persistence was similar among those who used the daily regimens of risedronate, etidronate, and alendronate. The occurrence of gastrointestinal adverse events was associated with decreased persistence with bisphosphonates (H2)-receptor antagonists: RR, 0.71 [95% CI, 0.53-0.94]; bowel motility enhancers: RR, 0.78 [95% CI, 0.65-0.94]). CONCLUSIONS: In this study, dose interval and the occurrence of gastrointestinal adverse events were independent determinants of persistence with bisphosphonate therapy. Although the likelihood of persistence with bisphosphonate use was significantly higher among those who used a less frequently administered regimen, persistence rates were still suboptimal. PMID- 16678645 TI - The association between fibrate use, change in high-density lipoprotein cholesterol, and the risk of cardiovascular disease: a retrospective chart review involving up to 8 years of follow-up. AB - BACKGROUND: Clinical trials have indicated that the use of fibric acid derivatives confers a benefit against cardiovascular disease (CVD) in selected populations. However, whether fibrates provide a CVD risk reduction independent of changes in the traditional lipoprotein fractions and other known CVD risk factors is not clear. OBJECTIVE: This study examined whether the use of fibrate therapy in a general clinical setting provided cardiovascular benefits independent of changes in the traditional lipoprotein fractions. METHODS: This was a matched, retrospective cohort study. From the electronic records of a large health maintenance organization in the northwestern United States, we identified a population that had newly initiated fibrate pharmacotherapy between January 1, 1996, and December 31, 2000. We then identified a comparator group of patients not using fibrates, matching them to fibrate users based on high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels, age, sex, and year of HDL-C and TG measurement. Subjects were followed until a CVD hospitalization, termination from the health plan, or December 31, 2003, whichever came first. We then used multivariate analysis accounting for differences in followup to identify predictors of CVD incidence. RESULTS: The study population included 1722 matched pairs (56.6% male; mean [SD] age, 57.3 [11.1] years). The patients who had newly initiated fibrate pharmacotherapy had low baseline HDL-C levels (mean, 37.4 mg/dL) and very high TG levels (617 mg/dL). The 2 groups were similar overall, with the only significant differences between fibrate users and nonfibrate controls being a greater prevalence of diabetes (37.7% vs 34.3%, respectively; P=0.040) and greater use of angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (56.6% vs 51.6%, respectively; P=0.003), beta-blockers (53.7% vs 49.0%; P=0.006), calcium channel blockers (25.1% vs 20.9%; P=0.004), and niacin (11.7% vs 7.4%; P<0.001). Overall, CVD risk was 26% lower for every 5-mg/dL increment in HDL-C. After adjustment for age, sex, smoking history, diabetes, existing diagnosis of CVD, weight, systolic blood pressure, baseline HDL-C, change in HDL-C, total cholesterol, TG, and use of statins, niacin, and other CVD drugs, fibrate use did not confer an additional CVD risk reduction. CONCLUSIONS: In this cohort with low baseline HDL-C levels and very high TG levels, fibrate use did not confer an independent CVD risk reduction after adjustment for CVD risk factors. Given the current obesity epidemic in the United States and the corresponding rise in the number of patients with the metabolic syndrome, the apparent risk reduction observed in association with higher HDL-C levels should not be ignored. PMID- 16678646 TI - Treatment of psoriatic arthritis with etanercept, methotrexate, and cyclosporin A. AB - BACKGROUND: Psoriatic arthritis (PsA) is seen in approximately 5% to 42% of individuals with psoriasis. CASE SUMMARY: A 37-year-old white male weighing 90 kg presented with erythrodermic psoriasis and PsA. The overall duration of PsA was 3 years. Serum levels of glucose, electrolytes, and tumor markers were normal, as were the results of tests of hepatic and renal function and urinalysis. The findings of posteroanterior radiographic examination of the chest were also normal. However, radiographic examination showed porosis and degeneration in the lumbar vertebrae; narrowing of the L2-L3, L3-L4, and L5-S1 spaces; degenerative changes and narrowing of the proximal interphalangeal and distal interphalangeal (DIP) joints; and osseous ankylosis of the DIP joints of the hands. The cutaneous eruption improved with cyclosporin A (CsA) 3.5 mg/kg p.o., but the severity of PsA did not change. Therefore, parenteral methotrexate (MTX) 15 mg/wk and an indomethacin suppository 100 mg/d were added to the regimen. CsA and MTX were continued for 3 months, during which the patient's PsA symptoms did not abate, based on tender and swollen joint counts, hand-to-floor distance, erythrocyte sedimentation rate, and levels of C-reactive protein (CRP), antistreptolysin O, and rheumatoid factor. Therefore, etanercept 25 mg s.c. twice weekly was added to the regimen. Three weeks after the initiation of this combination, the patient's arthritis had improved. The visual analog scale score decreased from 9 to 4. Tender and swollen joint counts decreased from 28 and 24 to 15 and 10, respectively. The hand-to-floor distance decreased from 20 to 10 cm. The erythrocyte sedimentation rate and levels of CRP, antistreptolysin O, and rheumatoid factor decreased from 72 mm/h, 162 mg/L, 250 IU/mL, and 304 IU/mL at baseline to 23 mm/h, 64 mg/L, 48 IU/mL, and 56.1 IU/mL, respectively. No change was observed in radiographs of the patient's back, hands, and feet. Based on the American College of Rheumatology scoring system, the patient showed 50% improvement in disease severity. Etanercept was discontinued at the end of 4 weeks, and maintenance therapy was continued with MTX alone. No adverse events were reported during or after the completion of etanercept therapy. CONCLUSION: In this patient with PsA that was refractory to CsA and MTX, either alone or in combination, the severity of PsA was reduced after 4 weeks of the combined use of etanercept, CsA, and MTX. PMID- 16678647 TI - Open-label pilot study comparing quantitative ultrasound and dual-energy X-ray absorptiometry to assess corticosteroid-induced osteoporosis in patients with chronic kidney disease. AB - BACKGROUND: Patients with chronic kidney disease (CKD) might be at high risk for bone disease. Decreased functional kidney mass contributes to renal osteodystrophy, which might be exacerbated by certain drug therapies. Long-term (> or = 6 months) corticosteroid treatment is commonly prescribed in patients with glomerular disease, possibly causing bone loss both indirectly and directly, putting the patient at increased risk for fracture. The dual-energy x-ray absorptiometry (DXA) is the current "gold standard" for measuring osteoporosis related fractures and works by passing ultrasound waves through bone to determine the structural anisotropy in the heel. OBJECTIVE: This pilot study was designed to determine whether there is a correlation between DXA and quantitative ultrasound (QUS) in detecting corticosteroid-induced osteoporosis. METHODS: This open-label pilot study was conducted at the Medical Center Nephrology Clinic, Albany Medical College, Albany, New York. Female patients aged > or = 18 years with a diagnosis of CKD and/or a history of kidney transplantation and who were receiving long-term corticosteroid treatment were enrolled. Each patient served as her own control and underwent DXA of the hip and spine (DXA-hip and DXA-spine, respectively) and QUS of the dominant and nondominant heels (QUS-dominant and QUS nondominant, respectively), within 1 week so that conditions were similar in each patient. RESULTS: Eight patients were included in the study (mean [SD] age, 50.2 [11.2] years). A positive correlation was found between DXA-hip and QUS nondominant (r2=0.76; P=0.009); however, no correlation was found with DXA-spine. Similarly, a positive correlation was found between DXA-hip and QUS-dominant (r2=0.75; P=0.009), but no correlation with DXA-spine was found (r2=0.22). CONCLUSION: In this small, selected population, QUS showed a fair correlation with DXA-hip but no correlation with DXA-spine. Further studies are needed to determine the effectiveness in other populations. PMID- 16678648 TI - Efficacy and safety of mixed amphetamine salts extended release (Adderall XR) in the management of attention-deficit/hyperactivity disorder in adolescent patients: a 4-week, randomized, double-blind, placebo-controlled, parallel-group study. AB - BACKGROUND: The ability to recognize and diagnose attention-deficit/hyperactivity disorder (ADHD) has increased in recent years. The persistence of ADHD symptoms puts adolescents with ADHD at risk for long-term adverse psychosocial outcomes. OBJECTIVE: The primary goal of this study was to assess the efficacy and safety of mixed amphetamine salts extended release (MAS XR) in the management of adolescents with ADHD. METHODS: This was a 4-week, randomized, multicenter, double-blind, placebo-controlled, parallel-group, forced-dose-titration study. Adolescents aged 13 to 17 years with ADHD were randomized to 1 of 4 active treatments (MAS XR 10, 20, 30 or 40 mg/d) or to placebo. All doses were given in the morning. This study used a forced-dose-titration design in which patients randomized to the 10-mg/d group received 1 dose of 10 mg/d for 4 weeks. Patients randomized to the 20-mg/d group received 1 dose of 10 mg/d for the first week and 1 dose of 20 mg/d for the remaining weeks; patients randomized to the 30-mg/d group received 1 dose of 10 mg/d for the first week, 1 dose of 20 mg/d for the second week, and 1 dose of 30 mg/d for the remaining 2 weeks; and patients randomized to the 40-mg/d group received 1 dose of 10 mg/d for the first week, 1 dose of 20 mg/d for the second week, 1 dose of 30 mg/d for the third week, and 1 dose of 40 mg/d for the fourth week. The primary efficacy measure was change from baseline to end point in the ADHD Rating Scale-IV (ADHD-RS-IV) score. The secondary efficacy measure was the score on the Clinical Global Impressions Improvement (CGI-I) scale for ADHD. ADHD-RS-IV total scores were analyzed post hoc in patients with low baseline ADHD-RS-IV severity (ie, patients with baseline ADHD-RS-IV total scores less than the median) and high baseline ADHD-RS-IV severity (ie, patients with baseline ADHD-RS-IV total scores greater than the median). Safety was assessed by recording adverse events, vital signs, and body weight at all study visits and 30 days after drug discontinuation. RESULTS: Of the 287 randomized adolescents, 258 completed the study. The intent-to-treat (ITT) population included 278 patients. The majority of patients were male (65.5%) and white (73.7%) The mean weight (57.8 kg [127.1 lb]) at baseline and the mean height (163.8 cm [64.5 in]) at screening were comparable across all MAS XR treatment groups. Patients in the placebo group had a mean weight of 59.8 kg (131.6 lb) and a mean height of 166.1 cm (65.4 in). Most (56.5%) of the patients had ADHD combined inattentive/hyperactive-impulsive subtype. Two hundred nineteen (78.8%) patients were treatment naive, and 59 (21.2%) had received treatment for ADHD within 30 days before screening. ITT analysis of the ADHD-RS-IV revealed statistically significant (P < 0.001) improvement in mean ADHD-RS-IV total scores in all 4 MAS XR treatment groups, compared with placebo, at all weeks throughout the 4-week study; the mean change from baseline to end point was -17.8 in the MAS XR 10- to 40-mg/d groups and -9.4 in the placebo group. Significant treatment effects were observed in both the ADHD-RS-IV inattentive (P < 0.001) and hyperactive-impulsive (P < 0.001) subscales from baseline. In patients with low baseline ADHD-RS-IV severity, statistically significantly (P < or = 0.01) greater improvements were observed in the MAS XR 20-, 30-, and 40-mg/d groups than in the placebo group; in patients with high baseline ADHD-RS-IV severity, statistically significantly (P < or = 0.02) greater improvements were observed in all active treatment groups compared with placebo. On the CGI-I scale at end point, a higher percentage of adolescents in all MAS XR treatment groups were considered improved (MAS XR 10 mg/d, 51.9% [P < 0.01]; 20 mg/d, 66.0% [P < 0.001]; 30 mg/d, 70.7% [P < 0.001]; 40 mg/d, 63.9% [P < 0.001]) compared with adolescents receiving placebo (26.9%). The most common adverse events in patients receiving MAS XR versus placebo were anorexia/decreased appetite (35.6% vs 1.9%), headache (16.3% vs 22.2%), insomnia (12.0% vs 3.7%), abdominal pain (10.7% vs 1.9%), and weight loss (9.4% vs 0%). Most adverse events were mild or moderate in intensity (97.5%); no serious adverse events were reported. CONCLUSIONS: The adolescents with ADHD treated with 10- to 40-mg/d MAS XR up to 4 weeks had significant improvements in ADHD symptoms compared with those who received placebo. Results of this study suggest that once-daily dosing with MAS XR up to 40 mg was effective and well tolerated for the management of ADHD in these adolescents. PMID- 16678649 TI - A post hoc subgroup analysis of an 18-day randomized controlled trial comparing the tolerability and efficacy of mixed amphetamine salts extended release and atomoxetine in school-age girls with attention-deficit/hyperactivity disorder. AB - BACKGROUND: Because the scientific literature on the pharmacotherapy of attention deficit/hyperactivity disorder (ADHD) is almost entirely based on the results of studies in samples consisting primarily of boys, much is unknown about the treatment response in girls. OBJECTIVE: This post hoc analysis compared the efficacy, tolerability, and time course of the effect of mixed amphetamine salts extended release (MAS XR) and atomoxetine in school-age girls with ADHD. METHODS: This was an intent-to-treat subanalysis of the data from girls enrolled in a multicenter, 18-day, randomized, double-blind, parallel-group, forced dose titration, laboratory school study enrolling boys and girls aged 6 to 12 years with ADHD. The study compared the efficacy, tolerability, and time course of the effect of increasing doses of MAS XR (10, 20, and 30 mg/d) and atomoxetine (0.5 and 1.2 mg/kg per day). The laboratory school sessions were organized in cycles to include 12 hours of observation. Efficacy measures included the SKAMP (Swanson, Kotkin, Agler, M-Flynn, and Pelham) deportment rating subscale, the SKAMP attention rating subscale, and academic testing (number of math problems attempted and answered correctly). Adverse events were assessed throughout the study period. Tolerability and efficacy measures were assessed during laboratory school visits on days 7, 14, and 21. RESULTS: This subanalysis included 57 girls (median age, 9 years; 49.1% white, 22.8% black, 17.5% Hispanic) with a diagnosis of ADHD, combined subtype. Twenty-six girls were randomized to receive MAS XR and 31 were randomized to receive atomoxetine. Mean SKAMP deportment and attention subscale scores in the 2 groups were similar at baseline. Mean changes from baseline were significantly greater for MAS XR compared with atomoxetine on the SKAMP deportment score (-0.48 vs -0.04, respectively; P<0.001) and SKAMP attention score (-0.45 vs -0.05; P<0.001). The time course of medication effect, based on change from baseline in SKAMP deportment scores, indicated 12-hour efficacy for MAS XR at hours 2, 4.5, 7, 9.5, and 12 (all time points, P<0.01 vs baseline) but not for atomoxetine. At the end of the study, both treatment groups had a significant increase from baseline in the mean number of math problems attempted and answered correctly (P<0.001). Girls who received MAS XR attempted significantly greater numbers of problems compared with those who received atomoxetine (P=0.04). Both MAS XR and atomoxetine were well tolerated. The most frequently occurring treatment-related adverse events in girls receiving MAS XR were decreased appetite (40.7%), upper abdominal pain (29.6%), insomnia (25.9%), and headache (14.8%). The most frequently occurring treatment-related adverse events in girls receiving atomoxetine were somnolence (28.1%), upper abdominal pain (15.6%), vomiting (15.6%), nausea (12.5%), and decreased appetite (12.5%). CONCLUSION: This post hoc analysis in a subpopulation of girls with ADHD, combined subtype, found that 18-day treatment with MAS XR was significantly more effective than atomoxetine in terms of ratings of classroom behavior, attention, and academic productivity. PMID- 16678651 TI - Comparison of first refill rates among users of sertraline, paroxetine, and citalopram. AB - BACKGROUND: Research suggests high rates of discontinuation of selective serotonin reuptake inhibitors (SSRIs). OBJECTIVE: The aim of this study was to compare the rates of first refill for sertraline, paroxetine, and citalopram, the 3 most commonly used SSRIs that were under patent protection at the time of the study. SSRIs that were available in generic formulations in this period were not included in the analysis because prescribing patterns and compliance rates for generic products may differ from those of branded products. METHODS: This analysis was conducted using claims data from an administrative database. Claims from January 1, 1999, through June 30, 2002, were analyzed. Adults aged 18 to 64 years with a diagnosis of depression, posttraumatic stress disorder, or social anxiety disorder who had initiated one of the SSRIs of interest during this period were included in the analysis. Refill status was determined based on a refill of the SSRI prescription within 1.5 times the days' supply dispensed or 15 days after the dispensing date of the index SSRI. To adjust for the effects of confounding factors, we conducted a multivariate logistic regression on the status of the first refill. The covariates included age (continuous variable), sex, index drugs (with sertraline as the reference group), and a dummy variable for copayment >15 dollars. RESULTS: Recipients of sertraline (n=5590) or citalopram (n=4124) were more likely than recipients of paroxetine (n=5201) to refill their original prescriptions (refill rate, 54.70%, 54.49%, and 50.99%, respectively; both comparisons, P<0.001). In the logistic regression, the effects of age, sex, and copayment were not significant. CONCLUSIONS: This study found that the likelihood of refilling the first prescription varied by SSRI. In the population studied, patients were significantly more likely to refill the first prescription for sertraline or citalopram than the first prescription for paroxetine. PMID- 16678652 TI - Impact of depression on utilization patterns of oral hypoglycemic agents in patients newly diagnosed with type 2 diabetes mellitus: a retrospective cohort analysis. AB - BACKGROUND: Oral hypoglycemic agents (OHAs) are an important component in the management of type 2 diabetes mellitus (DM). Large-scale studies have demonstrated that tight glycemic control with such agents can reduce the frequency and severity of long-term DM-related complications. OBJECTIVES: The main goal of this study was to examine the impact of depression on utilization patterns of OHAs in patients newly diagnosed with type 2 DM. A secondary objective was to estimate the impact of depression on discontinuation and modification of pharmacotherapy for DM in these patients. METHODS: Patients newly diagnosed with type 2 DM during a 3-year period (1998-2000) were identified from a Medicaid claims database. Presence of preexisting depression was determined on the basis of International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes. The patient cohort was followed up until they received their first prescription for an OHA (1998-2001); this date was treated as the index date for the study. Utilization patterns (ie, discontinuation, augmentation, switching, non-modification) for OHAs were computed for a 12-month follow-up period after the index date. A multivariate framework was used to estimate the impact of depression on utilization patterns, controlling for confounders such as demographics, comorbidity, provider interaction, drug regimen complexity, and DM severity. RESULTS: A total of 1237 newly diagnosed type 2 DM patients were identified (depressed, n=446; nondepressed, n=791). A higher number of depressed patients (23.32%) switched or augmented therapy compared with nondepressed patients (16.18%). Also, a higher fraction of depressed patients (39.46%) discontinued OHA therapy compared with nondepressed patients (32.87%). Results of a multinomial logistic regression indicated that, controlling for covariates, patients with depression were 1.72 times more likely to switch (P=0.046) and 1.89 times more likely to augment therapy (P=0.004) compared with nondepressed patients. Logistic regression analysis also indicated that, controlling for confounding covariates, patients with depression were 1.72 times more likely to modify initial OHA therapy compared with patients without depression (P=0.003). CONCLUSION: Depression was significantly associated with utilization patterns of OHAs in these patients newly diagnosed with type 2 DM, thus possibly affecting their disease management. PMID- 16678654 TI - Risk factors for hepatocellular carcinoma in HCV-cirrhosis: what we know and what is missing. PMID- 16678658 TI - IV. Clinical practice recommendations for anemia in chronic kidney disease in transplant recipients. PMID- 16678659 TI - KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for Anemia in Chronic Kidney Disease. PMID- 16678661 TI - II. Clinical practice guidelines and clinical practice recommendations for anemia in chronic kidney disease in adults. PMID- 16678669 TI - III. Clinical practice recommendations for anemia in chronic kidney disease in children. PMID- 16678676 TI - Beyond the aortic bifurcation: branched endovascular grafts for thoracoabdominal and aortoiliac aneurysms. AB - OBJECTIVES: To evaluate the use of novel technology to treat complex aortic aneurysms involving branches that provide critical end-organ blood supply. METHODS: A prospective study was conducted in patients with thoracoabdominal, suprarenal, or common iliac aneurysms (TAA, SRA, or CIA) at high risk for open surgical repair. An endovascular graft using the Zenith platform was customized to fit patient anatomy (TAA or SRA) and combined with Jomed balloon-expandable stent-grafts. Prefabricated hypogastric branches were used with a Zenith abdominal aortic aneurysm (AAA) or Fluency self-expanding fenestrated device in conjunction with a self-expanding stent-graft. Analyses were conducted in accordance with the endovascular aneurysm reporting standards document. Follow-up studies occurred at discharge, 1, 6, and 12 months, and included computed tomography and duplex ultrasound scans, and flat plate radiography. RESULTS: Fifty patients were treated (9 TAA, 20 SRA, 21 CIA). The mean aneurysm size was 7.6 cm (TAA), 7.2 cm (SRA), and 6.1 cm AAA size associated with a mean CIA size of 3.8 cm. Bilateral CIA aneurysms were present in 86% (18/21) of patients with CIA aneurysms. Perioperative mortality was 2% (1/50) and resulted from a myocardial infarction after a planned conduit and iliac endarterectomy required for device access. Five late deaths occurred (2 TAA, 2 SRA, 1 CIA), three of which (2 TAA, 1 SRA) were aneurysm related. Failure to access internal iliac arteries occurred in three cases, and two late hypogastric branch thromboses occurred. No visceral branches were lost acutely or occluded during follow-up. Sac shrinkage (>5 mm) was noted in 65% of patients at 6 months and in all patients (10/10) by 12 months. There were no ruptures or conversions, but nine patients required secondary interventions. CONCLUSIONS: Branch vessel technology has made it technically feasible to preserve critical end-organ perfusion in the setting of CIA, SRA, and TAA aneurysms. The relatively low acute mortality rate and lack of short-term branch vessel loss are encouraging and merit further investigation. These advances have the potential to markedly diminish the complications associated with conventional management of complex aneurysms. PMID- 16678678 TI - Three-dimensional analysis of enlarging aneurysms after endovascular abdominal aortic aneurysm repair in the Gore Excluder Pivotal clinical trial. AB - OBJECTIVE: Recent reports have raised concern about the percentage of enlarging abdominal aortic aneurysms (AAAs) after endovascular repair with the Gore Excluder device. As part of the investigation into this issue, a morphologic analysis was performed on enlarging aneurysms in the Excluder Pivotal clinical trial. METHODS: Computed tomographic scans were evaluated on all patients identified with enlarging aneurysms (5-mm increase by Core laboratory or site) and at least 4 years of follow-up in the Excluder Pivotal clinical trial. Three dimensional reconstruction, a set of 24 standard morphologic measurements, and analysis of potential enlargement mechanisms were performed. RESULTS: Of 112 trial patients with 4 years of follow-up, 38 AAAs (34%) were identified as enlarging. Data were obtained from 196 computed tomographic scans (the mean interval was 47 months from first to last scan). Of the 158 scans with a prior scan for comparison, 41% demonstrated growth relative to the initial scan by diameter criteria, but 79% demonstrated growth relative to the initial scan by 3 dimensional volume criteria (P < .0001 vs diameter; chi2 analysis). This difference was most evident at early time points: at 1 year, diameter criteria indicated that 8% of these AAAs were enlarging, but 56% were already enlarging by volume criteria. On average, enlargement was detected by volume 18 months before it was detected by diameter (P < .0001), and at a smaller diameter (55 +/- 1 mm vs 60 +/- 1 mm; P < .0001). Only 19% of scans (39% of patients) had apparent endoleaks. Scans with apparent endoleaks demonstrated a greater interval rate of growth as compared with those without apparent endoleak (3.6 +/- 0.8 mm vs 1.9 +/ 0.3 mm [P < .02] by diameter; 23 +/- 4 cm3 vs 11 +/- 1 cm3 [P < .001] by volume). Although the etiology of enlargement may be endotension or device permeability in up to 74% of patients, other potential causes of aneurysm enlargement included neck apposition length less than 15 mm (15 patients; 39%), large aortic diameter relative to device (18%), large iliac diameter (5%), and iliac apposition length less than 15 mm (20%). Multiple potential etiologies of enlargement were present in 53% of AAAs. CONCLUSIONS: The etiology of aneurysm enlargement in the Excluder Pivotal trial is likely multifactorial, including endoleak, inadequate attachment site length, and endotension or device permeability. Even by conservative criteria, a substantial percentage of aneurysm growth with the original device is likely due to material permeability. Three dimensional volume criteria detected aneurysm enlargement more frequently, at a smaller diameter, and on average 18 months sooner than standard diameter criteria, thus suggesting a role in further investigation of this issue. PMID- 16678679 TI - Secondary interventions following endovascular abdominal aortic aneurysm repair using current endografts. A EUROSTAR report. AB - OBJECTIVE: The purpose of this study was to evaluate the need for secondary interventions after endovascular abdominal aortic aneurysm repair with current stent-grafts. METHODS: Studied were data from 2846 patients treated from December 1999 until December 2004. The data were recorded from the EUROSTAR registry. The only patients studied were those with a follow-up of at least 12 months or until they had a secondary intervention within the first 12 months. The cumulative incidences of secondary transabdominal, extra-anatomic, and transfemoral interventions during follow-up (after the first postoperative month) were investigated. RESULTS: A secondary intervention was performed in 247 patients (8.7%) at a mean of 12 months after the initial procedure within a follow-up period of a mean of 23 +/- 12 months. Of these, 57 (23%) transabdominal, 43 (16%) involved an extra-anatomic bypass, and 147 (60%) were by transfemoral approach. The cumulative incidence of secondary interventions was 6.0%, 8.7%, 12%, and 14% at 1, 2, 3, and 4 years, respectively. This corresponded with an annual rate of secondary interventions of 4.6%, which was remarkably lower than in a previously published EUROSTAR study of patients treated before 1999. Type I endoleaks (33% of procedures), migration (16%), and rupture (8.8%) were the most frequent reasons for secondary transabdominal interventions. Graft limb thrombosis was the indication for extra-anatomic bypass (60%). Type I endoleak (17%), type II endoleak (23%), device limb stenosis (14%), thrombosis (23%), and device migration (14%) were the most frequent reasons for secondary transfemoral interventions. Operative mortality was higher after secondary transabdominal interventions (12.3%, P = .007) compared with transfemoral interventions (2.7%). Overall survival was lower in patients with secondary transabdominal (P = .016) and extra-anatomic interventions (P < .0001) compared with patients without a secondary intervention. CONCLUSION: Although the incidence of secondary interventions after endovascular aneurysm repair has substantially decreased in recent years, continuing need for surveillance for device-related complications remains necessary. PMID- 16678680 TI - Concomitant unilateral internal iliac artery embolization and endovascular infrarenal aortic aneurysm repair. AB - INTRODUCTION: Endograft limb extension to the external iliac artery with embolization of an internal iliac artery (IIA) may be necessary in patients with abdominal aortic aneurysms (AAAs) extending to the common iliac artery to prevent endoleak during endovascular aortic aneurysm repair (EVAR). Coil embolization of the IIA can be performed at the same operative setting as EVAR or, alternatively, as a staged procedure. Most interventionalists favor the latter approach to avoid excessive contrast material and prolonged operative time. We investigated the clinical outcome of concomitant vs staged unilateral IIA embolization in the setting of EVAR. METHODS: Vascular surgeons at our institution treated 24 patients with infrarenal EVAR and unilateral coil embolization of the IIA from October 1, 2000 to June 30, 2005. All patients had normal renal function. The details of the operative procedure and perioperative complications were compared in patients undergoing concomitant vs staged procedures. Follow up was 1 to 40 months (average, 11 months). RESULTS: Among the 24, 16 underwent concomitant unilateral IIA embolization in the setting of EVAR and eight patients underwent the staged procedure. Average duration of operative time (298 vs 284 minutes), amount of intravenous contrast (215 mL vs 164 mL), and preoperative (1.12 vs 1.26 mg/dL), and postoperative (1.15 v. 1.31 mg/dl) creatinine levels were similar in the concomitant vs staged group, respectively (P > .05 for all factors). More sensitive markers of renal insufficiency such as creatinine clearance were not measured. In the concomitant group, 25% (4/16) of patients reported significant symptoms of buttock claudication ipsilateral to the embolized IIA, which resolved after a mean of 8.8 months (range, 1 to 15 months) vs no cases (0/8) in the staged group (P = .02048). One patient in the staged group developed ischemic colitis, which was treated conservatively. Coil embolizations that were performed as staged procedures were all done on an outpatient basis. All 24 patients were admitted the day of the EVAR and were discharged the next day, except one patient in the concomitant group was discharged the second day after the procedure, and one patient in the staged group was discharged 7 days after the procedure. CONCLUSION: Despite concern of prolonged operative time and the amount of contrast needed to perform concomitant IIA embolization and EVAR, our results showed that in patients with normal renal function, concomitant unilateral IIA embolization in the setting of EVAR was safe and effective and associated with shorter hospitalization compared with staged procedures. The disadvantage of a concomitant procedure is an increased likelihood of transient buttock claudication, but the small number of patients in this series prohibits definite conclusions about this complication. The concomitant procedure may be preferable for infirm patients with normal renal function who would be greatly inconvenienced by two procedures. PMID- 16678681 TI - Cost-effectiveness of screening women for abdominal aortic aneurysm. AB - BACKGROUND: Women are usually not considered for abdominal aortic aneurysm (AAA) screening because of their lower prevalence of disease. This position may, however, be questioned given the higher risk of rupture and the longer life expectancy among women. The purpose of this study was to assess the cost effectiveness of screening 65-year-old women for AAA. METHODS: A systematic review of the literature was conducted to obtain data of importance to evaluate the effectiveness of screening women for AAA. Data were entered into a Markov simulation cohort model. RESULTS: The review suggested some main assumptions for women with AAA. Prevalence is 1.1%. In 6.8%, the AAA is of a size that merits surgery, and the patients are fit for a procedure. For patients with an AAA, the yearly risk for elective surgery and the rupture incidence was 3.1% and 2.4%, respectively, in the invited group and 1.1% and 5.7% in the noninvited group. The operative mortality for elective surgery was 3.5%, and the total mortality for ruptured AAA was 86.3%. The long-term mortality for AAA patients was 3.6 times higher than for an age-matched healthy population. Screening reduced the AAA rupture incidence by 33% and the AAA-related death rate by 35%. The cost per life year gained was estimated at $5911. CONCLUSION: The incremental cost effectiveness ratio was similar to that found for screening men, which reflects the fact that the lower AAA prevalence in women is balanced by a higher rupture rate. Screening women for AAA may be cost-effective, and future evaluations on screening for AAA should include women. PMID- 16678683 TI - Evaluation of the complexity of open abdominal aneurysm repair in the era of endovascular stent grafting. AB - OBJECTIVE: Endovascular repair has proven to be an effective treatment for many abdominal aortic aneurysms (AAA). Aneurysms that require open repair have usually been disqualified from an endovascular approach as a result of a variety of anatomic constraints, which may also make open repair more difficult. Our purpose was to review open AAA repair and assess the complexity of the operative procedure and associated morbidity and mortality data in the era of endovascular stent grafting. METHODS: We retrospectively reviewed the records of 606 patients undergoing elective open AAA repair at a single tertiary care community hospital from January 1, 1996, to December 31, 2004. Patients with ruptured aneurysms and all endovascular repairs were excluded. Patients were grouped into two categories. Group 1 included 301 patients who underwent open repair before the initiation of an endovascular stent grafting program in November 1999. Group 2 included 305 patients who underwent open repair after the initiation of the stent graft program. Operative reports were reviewed to determine the location of the proximal aortic cross clamp, management of the renal vein, associated iliac aneurysmal or occlusive disease, and type of surgical reconstruction. Morbidity, mortality, and disposition data were compared for the two groups and subjected to chi2 analysis. RESULTS: Suprarenal aortic cross-clamp placement was required in 6% of group 1 patients and 20% of group 2 patients (P < .05). Division of the renal vein was necessary in 11% of group 1 patients and 18% of group 2 patients (P < .05). Iliac aneurysms were present in 25% of group 1 patients and 42% of group 2 patients (P < .05). The incidence of associated iliac occlusive disease was 12% in group 1 and 20% in group 2 (P < .05). The type of reconstruction required (aortoaorto, aortoiliac, aortofemoral) was not found to be statistically significant. All major sources of morbidity, including renal insufficiency, myocardial infarction, stroke, and intubation times, were similar between the two groups. The length of stay was 9.2 days in both groups, and 11.3% of group 1 patients and 26% of group 2 patients were discharged to an extended-care facility rather than directly home. The overall mortality rate was 2.0% for patients in group 1 and 3.8% for group 2 patients. This was not a statistically significant difference. CONCLUSIONS: Surgeons performing open repair of AAA in the era of endovascular stent grafting are operating on patients who require more complex repairs, including a greater frequency of suprarenal cross clamping, renal vein division, and management of associated iliac aneurysmal and occlusive disease. Despite this, morbidity and mortality rates are similar to those in patients operated on before the initiation of an endovascular stent grafting program. PMID- 16678684 TI - Factors affecting outcomes of open surgical repair of pararenal aortic aneurysms: a 10-year experience. AB - PURPOSE: Few large series document surgical outcomes for patients with pararenal abdominal aortic aneurysms (PAAAs), defined as aneurysms including the juxtarenal aorta or renal artery origins that require suprarenal aortic clamping. No standard endovascular alternatives presently exist; however, future endovascular branch graft repairs ultimately must be compared with the gold standard of open repair. To this end, we present a 10-year experience. METHODS: Between 1993 and 2003, 3058 AAAs were repaired. Perioperative variables, morbidity, and mortality were retrospectively assessed. Renal insufficiency was defined as a rise in the concentration of serum creatinine by > or = 0.5 mg/dL. Factors predicting complications were identified by multivariate analyses. Morbidity and 30-day mortality were evaluated with multiple logistic regression analysis. RESULTS: Of a total of 3058 AAA repairs performed, 247 were PAAAs (8%). Mean renal ischemia time was 23 minutes (range, 5 to 60 minutes). Cardiac complications occurred in 32 patients (13%), pulmonary complications in 38 (16%), and renal insufficiency in 54 (22%). Multivariate analysis associated myocardial infarction with advanced age (P = .01) and abnormal preoperative serum creatinine (>1.5 mg/dL) (P = .08). Pulmonary complications were associated with advanced age (P = .03), renal artery bypass (P = .02), increased mesenteric ischemic time (P = .01), suprarenal aneurysm repair (P < .0008), and left renal vein division (P = .01). Renal insufficiency was associated with increased mesenteric ischemic time (P = .001), supravisceral clamping (P = .04), left renal vein division (P = .04), and renal artery bypass (P = .0002), but not renal artery reimplantation or endarterectomy. New dialysis was required in 3.7% (9/242). Abnormal preoperative serum creatinine (>1.5 mg/dL) was predictive of the need for postoperative dialysis (10% vs 2%; P = .04). Patients with normal preoperative renal function had improved recovery (93% vs 36%; P = .0002). The 30-day surgical mortality was 2.5% (6/247) but was not predicted by any factors, and in-hospital mortality was 2.8% (7/247). Median intensive care and hospital stays were 3 and 9 days, respectively, and longer stays were associated with age at surgery (P = .007 and P = .0002, respectively) and any postoperative complication. CONCLUSIONS: PAAA repair can be performed with low mortality. Renal insufficiency is the most frequent complication, but avoiding renal artery bypass, prolonged mesenteric ischemia time, or left renal vein transection may improve results. PMID- 16678685 TI - Increased psychiatric morbidity after abdominal aortic surgery: risk factors for stress-related disorders. AB - OBJECTIVE: Research on surgical outcomes has focused on technical results and physical morbidity. However, postoperative psychiatric complications are common and can undermine functional results. High rates of posttraumatic stress disorder and major depressive disorder have been documented after cardiac events or surgery. These complications are also expected after abdominal aortic surgery, but their incidence and relevant risk factors in this population have not been documented. METHODS: We examined the development of posttraumatic stress and depressive symptoms in patients with aortic aneurysms or occlusive disease, comparing surgical with nonsurgical patients and predicting that surgery and a prolonged intensive care stay would contribute to the development of psychiatric morbidity. A consecutive sample of vascular surgery patients (n = 109) was recruited 6 months to 2 years after surgery. Data were analyzed by using group comparisons, regression, and path analyses. RESULTS: Rates of objectively determined postoperative psychiatric morbidity were extremely high (32%). Surgical patients were more than four times more likely to develop psychiatric disorders (odds ratio, 4.8; P = .02). Being younger, having increased preoperative blood pressure, and being intubated at the end of surgery were linked to greater rates of psychiatric morbidity (P < .05), but a longer intensive care stay was not. CONCLUSIONS: New-onset psychiatric symptoms are common after abdominal aortic surgery, and preoperative and surgical factors were more predictive than postoperative complications and stress, as reflected in intensive care unit stays. Prospective examination of vulnerability in this model could identify risk factors for stress-related psychiatric morbidity and help improve surgical outcomes. PMID- 16678686 TI - Activated protein C-protein C inhibitor complex: a new biological marker for aortic aneurysms. AB - OBJECTIVE: The concentration of the complex between activated protein C (APC) and protein C inhibitor (PCI) is a measure of thrombin generation. We studied whether it can provide information useful for the diagnosis and treatment of arterial vascular disease. METHODS: Blood was obtained from 429 vascular patients admitted consecutively during September 2004 to March 2005. The APC-PCI complex was measured by using a sandwich immunofluorometric method. The patients were divided into cohorts according to the planned treatment and compared with a control group of healthy individuals. RESULTS: The APC-PCI complex concentration varied from 0.08 to 2.50 microg/L. In the cohort of patients with aortic aneurysms (n = 78), the median APC-PCI value was 0.45 (10th to 90th percentile, 0.24-1.47), and values were clearly increased compared with all other cohorts (P < .0001). Patients with carotid disease (n = 73) yielded a median of 0.22 (10th to 90th percentile, 0.15-0.48). The median for claudicants (n = 74) was 0.26 microg/L (10th to 90th percentile, 0.15-0.75), which was higher than in those (n = 97) with critical ischemia (0.20; 10th to 90th percentile, 0.13-0.36; P < .0023). The cohort with other forms of atherosclerotic disease (n = 40) had a median of 0.23 (10th to 90th percentile, 0.14-0.42), whereas the value for a cohort of 21 patients with venous disease was 0.19 (10th to 90th percentile, 0.10-0.34). The median was 0.15 (10th to 90th percentile, 0.10-0.23) for the control group (n = 121). CONCLUSIONS: Patients with atherosclerosis had an increased APC-PCI concentration that corresponded to increased generation of thrombin. Patients with aortic aneurysm had a threefold higher median concentration than the control group. We suggest that this remarkable increase is caused by the local activation of coagulation, and we surmise that APC-PCI measurements can be used as a screening tool to identify patients with aortic aneurysms. PMID- 16678687 TI - The posterior approach for repair of popliteal artery aneurysms. AB - OBJECTIVES: Ligation and bypass is the most commonly performed surgical treatment for popliteal artery aneurysm. This approach can be complicated by persistent collateral flow to the excluded aneurysm sac, which may lead to aneurysm growth, the development of compressive symptoms, and in some cases, rupture. Repair of popliteal aneurysms by posterior endoaneurysmorrhaphy and reconstruction with a short prosthetic interposition graft avoids these complications because patent collaterals communicating with the aneurysm sac are oversewn at the time of surgery. We report the early and mid-term outcomes of popliteal artery aneurysm repair using this posterior approach. METHODS: The records of all patients operated on for popliteal artery aneurysm from December 1981 to June 2003 were retrospectively reviewed. Patients who underwent popliteal artery aneurysm repair with a posterior approach were included in the study. RESULTS: From 1981 to 2003, 30 popliteal aneurysms (mean diameter, 3.2 cm; range, 1.9 to 6.2 cm) were repaired in 24 patients using a posterior approach with interposition prosthetic grafting. The median follow up was 21.5 months. Primary patency, primary assisted patency, and secondary patency were 92.2%, 95.8%, and 95.8%, respectively, at 1 and 2 years. The limb salvage rate was 100%. CONCLUSIONS: Popliteal endoaneurysmorrhaphy using a posterior approach with interposition prosthetic grafting is simple, safe, and effective. The patency and limb salvage rates are equivalent to those obtained with ligation and vein bypass. In addition, the posterior approach eliminates the postoperative complications associated with persistent collateral flow into the aneurysm sac. PMID- 16678688 TI - Cerebral ischemia associated with PercuSurge balloon occlusion balloon during carotid stenting: Incidence and possible mechanisms. AB - BACKGROUND: Interruption of antegrade cerebral perfusion results in transient neurologic intolerance in some patients undergoing carotid angioplasty and stenting (CAS). This study sought to evaluate factors that contributed to the development of cerebral ischemia during PercuSurge balloon occlusion and techniques used to allow successful completion of the CAS procedure. METHODS: The PercuSurge occlusion balloon was used in 43 of 165 patients treated with CAS for high-grade stenosis (mean stenosis, 90%). All 43 patients were at increased risk for endarterectomy (7 restenosis, 3 irradiation, 3 contralateral occlusion, and 30 Goldman class II-III); 20% were symptomatic. Symptoms of cerebral hypoperfusion during temporary occlusion of the internal carotid artery occurred in 10 of 43 and included dysarthria (7/10), agitation (6/10), decreased level of consciousness (5/10), and focal hemispheric deficit (3/10). An incomplete circle of Willis or contralateral carotid artery occlusion, or both, was present in 8 of 10 patients. Symptoms resulting from PercuSurge balloon occlusion were managed by balloon deflation with or without evacuation of blood from the internal carotid artery using the Export catheter. All symptoms resolved completely without deficit after deflation of the occlusion balloon. RESULTS: The development of neurologic symptoms after initial PercuSurge balloon inflation and occluded internal carotid artery flow was associated with a decrease in the mean Glasgow Coma Scale (GCS) from 15 to 10 (range, 9 to 14); the GCS returned to normal after occlusion balloon deflation and remained normal during subsequent reinflation. The mean time to spontaneous recovery of full neurologic function was 8 minutes (range, 4 to 15 minutes). No thrombotic or embolic events were present on cerebral angiography or computed tomography scan. Balloon reinflation was performed after a mean reperfusion interval of 10 minutes after full neurologic recovery (range, 4 to 20 minutes). The mean subsequent procedure duration was 11.9 minutes (range, 6 to 21 minutes). No recurrence of neurologic symptoms occurred when the occlusion balloon was reinflated. All 10 patients underwent successful CAS without occlusion, dissection, cerebrovascular accident, or death. CONCLUSION: Several factors may contribute to the development of neurologic intolerance during CAS with balloon occlusion. Elucidation of the protective cellular mechanisms that invoke ischemic tolerance after the initial transient ischemic event may enable CAS with embolic protection in patients who cannot tolerate initial interruption of antegrade cerebral perfusion. PMID- 16678689 TI - Treatment of asymptomatic carotid artery disease: similar early outcomes after carotid stenting for high-risk patients and endarterectomy for standard-risk patients. AB - BACKGROUND: The role of carotid angioplasty and stenting (CAS) in the treatment of asymptomatic patients with carotid disease remains controversial. The purpose of this report is to compare outcomes in asymptomatic patients treated with CAS and carotid endarterectomy (CEA). This was the initial experience performing CAS for most of the surgeons. For comparison, we also report our outcomes in standard risk patients treated concurrently with CEA during the same period of time. METHODS: A retrospective, nonrandomized review of asymptomatic patients undergoing CEA or CAS at Washington University Medical Center in St. Louis was done. Patients with >70% asymptomatic carotid stenosis treated between September 2003 and April 2005 were identified. CEA was the first therapeutic consideration in all patients. CAS was reserved for high-risk patients. Thirty-day outcomes of stroke or death were recorded. During this time interval, 248 patients were treated including with 93 CAS and with 145 CEA. Symptomatic or clinically detected adverse outcomes such as myocardial infarction (MI), arrhythmia, renal failure, or pulmonary complications were noted but were not the primary end points of this review. This study addresses only the periprocedural outcomes of CEA and CAS in asymptomatic patients. No data >30-day follow-up are included. RESULTS: During this period, 93 CAS and 145 CEA procedures were done in asymptomatic patients. Patient characteristics in both groups were similar. Carotid protection devices were used in 91.4% of CAS patients. The results in the CAS group showed one death (1.1%) and one stroke (1.1%). In the CEA group, three strokes occurred (2.1%, P = 0.9999), one associated with death (0.7%, P = 0.9999). The CAS group had 1.34 +/- 0.83 risk factors vs 0.39 +/- 0.58 in the CEA group (P < .0001). Median CAS and CEA length of stay was 1 day. CONCLUSIONS: CAS for asymptomatic carotid stenosis demonstrated equivalent outcomes compared with CEA, despite CAS being reserved for use in a disadvantaged subset of high-risk patients owing to anatomic risk factors or medical comorbidities. These results suggest CAS should be considered a reasonable treatment option in the high-risk but asymptomatic patient. Enthusiasm for CAS should be tempered by the recognition that long-term outcomes in CAS-treated asymptomatic patients remain unknown. PMID- 16678690 TI - A personal experience with coronary artery bypass grafting, carotid patching, and other factors influencing the outcome of carotid endarterectomy. AB - OBJECTIVE: This study was conducted to investigate the influence of coronary artery bypass grafting (CABG), carotid patching, and other factors on the outcome of all carotid endarterectomies (CEAs) performed by a single surgeon at a tertiary referral center. METHODS: The series includes 2262 CEAs (335 bilateral) in 1521 men and 741 women (33%) with median ages of 66 and 68 years, respectively. Surgical indications were asymptomatic stenosis for 1503 procedures (66%), retinal ischemia or cerebral transient ischemic attacks each for 271 (12%), and prior stroke for 217 (9.6%). CEA was done as an isolated operation in 1959 patients and was performed in conjunction with simultaneous CABG in 303 (13%). Primary arteriotomy closure was used for 783 CEAs (35%), vein patching for 1232 (54%), and synthetic patching for 247 (11%). Outcome event rates were assessed by logistic regression analysis, proportional hazards models, and Kaplan Meier estimations. RESULTS: Postoperative mortality (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.7 to 7.5; P = .001), stroke (OR, 3.2; 95% CI, 1.6 to 6.4; P = .001), and combined stroke and mortality rates (OR, 3.4; 95% CI, 2.0 to 5.8; P < .001) were significantly higher for simultaneous CEA/CABG than for isolated CEA. Ipsilateral postoperative stroke rates were similar (2.6% vs 1.7%, P = .41) in both settings. Vein patching had a lower risk for ipsilateral stroke (OR, 0.42; 95% CI, 0.21 to 0.86; P = .015) than primary closure, but was not significantly different from synthetic patching (P = .10). The documented incidence of postoperative carotid thrombosis was 1.5% with primary closure, 0.6% with vein patching, and 2.0% with synthetic patching (P = .088). Overall Kaplan Meier survival was 92% at 1 year, 71% at 5 years, 41% at 10 years, and 20% at 15 years, but long-term mortality rates were higher after simultaneous CEA/CABG (hazard ratio, 1.3; 95% CI, 1.1 to 1.5; P = .002) than after CEA alone. Late strokes or retinal infarctions have been reported after 97 (5.0%) of the 1923 operations for which follow-up was available, 51 (2.3%) of which were ipsilateral to CEA. The incidence of > or = 60% recurrent stenosis was independently influenced by carotid patching (OR, 0.61; 95% CI, 0.40 to 0.92; P = .019) but not by the choice of patch material (P = .11). CONCLUSIONS: These results substantiate the common observation that patients who require simultaneous CEA/CABG have a higher risk for adverse outcomes than patients who undergo isolated CEA. Carotid patching provided significant benefit with respect to the risks for ipsilateral postoperative stroke and > or = 60% recurrent stenosis. PMID- 16678693 TI - High-energy phosphate metabolism during incremental calf exercise in patients with unilaterally symptomatic peripheral arterial disease measured by phosphor 31 magnetic resonance spectroscopy. AB - BACKGROUND: The treadmill exercise test is the most important examination of the functional ability of patients with intermittent claudication or leg pain during exercise, but it does not provide any metabolic information in the calf muscle. The purpose of this study was to investigate the high-energy metabolism in the calf muscle during incremental progressive plantar flexion exercise of a selected peripheral arterial disease (PAD) patient group. METHODS: Using a 1.5-T whole body magnetic resonance scanner, 17 male patients with PAD who had 1 symptomatic and 1 asymptomatic leg and 9 healthy male controls underwent serial phosphor 31 (31P) magnetic resonance spectroscopy during incremental exercise at 2, 3, 4, and 5 W. Furthermore, magnetic resonance angiography was performed, and the ankle brachial pressure index was determined in the patient group. The runoff resistance (ROR) was separately assessed in each patient's leg. RESULTS: The symptomatic legs exhibited significantly increased phosphocreatine (PCr) time constants during the first three workload increments (2-4 W) and the recovery phase compared with the asymptomatic legs and the normal controls. Only two symptomatic legs reached the last increment at 5 W. Compared with the normal controls, the asymptomatic legs showed significantly increased PCr time constants only at 5 W. In the patient group, we detected significant correlations between the PCr time constants and the ROR, as well as the ankle-brachial pressure index. Moreover, the symptomatic legs presented significantly lower PCr levels and pH values at the end of exercise compared with the asymptomatic and control legs. CONCLUSIONS: Our study shows that muscle function in PAD patients can be objectively quantified with the help of 31P magnetic resonance spectroscopy and correlates significantly with hemodynamic parameters such as ROR and ankle brachial pressure index. Consequently, 31P magnetic resonance spectroscopy seems to be a useful method to monitor the muscle function of PAD patients for evaluation of established therapies or new therapeutic strategies during research trials. PMID- 16678691 TI - Role of adhesion molecules in the induction of restenosis after angioplasty in the lower limb. AB - OBJECTIVE: The adhesion molecules P selectin, E selectin, intercellular adhesion molecule, vascular cellular adhesion molecule (VCAM), and monocyte chemoattractant protein 1 play a important role in the development of arteriosclerotic lesions and are considered main contributors to restenosis after angioplasty. We expected that the serum levels of these markers would increase in the early phase of the first few weeks after angioplasty. METHODS: We assessed prospectively the levels of soluble forms of adhesion molecules on the day before and then 24 hours and 2 and 4 weeks after angioplasty in arteries of the lower limb by using enzyme-linked immunosorbent assays. We investigated the distribution pattern of these markers in 44 patients (25 male and 19 female; age, 67.7 +/- 8.5 years [mean +/- SD]) presenting with intermittent claudication (Fontaine stage IIb). Twelve patients (27.3%) underwent diagnostic angiography, 32 (72.2%) received interventional treatment, 22 (68.8%) received balloon angioplasty, and 10 (31.2%) required stent placement. RESULTS: Ten (31.3%) of the treated patients developed restenosis within 6 months. These patients had significantly higher levels of P selectin (P = .034), E selectin (P = .006), and VCAM (P = .050) at all time points. E selectin, VCAM, and monocyte chemoattractant protein 1 levels increased between 24 hours and 4 weeks after angiographic procedures, thus indicating that the angiographic procedure itself leads to activation and inflammation of the endothelium. CONCLUSIONS: This study emphasizes a meaningful role of the adhesion molecules E selectin, P selectin, and VCAM as interesting contributors to restenosis formation after percutaneous transluminal angioplasty. PMID- 16678694 TI - Percutaneous angioplasty of the superior gluteal artery for buttock claudication: a report of seven cases and literature review. AB - BACKGROUND: Buttock claudication due to stenosis or occlusion of the superior gluteal artery is infrequent. The recent development of noninvasive gluteal duplex scanning, combined with aortoiliac angiography using oblique projections and the availability of low-profile devices for percutaneous transluminal angioplasty (PTA), led us to review our recent experience concerning the diagnosis and mid-term results of PTA for superior gluteal artery stenosis or occlusion. METHODS: The files of all patients who had been treated in our department by PTA for superior gluteal artery stenosis or occlusion with buttock claudication were analyzed retrospectively, and any associated arterial lesions, morbidity, restenosis, or recurrent buttock claudication were noted. Outcomes were compared with published reports. RESULTS: Retrospective review identified six patients (5 men, 1 woman; mean age, 64 years) with seven cases of buttock claudication (1 bilateral localization) who had undergone PTA within the past 2 years. There was no case of isolated buttock claudication. Buttock claudication was associated with impotence, thigh claudication, or calf claudication in seven cases. Gluteal duplex scans were performed for three of the patients diagnosed with two stenoses and one occlusion. Aortoiliac angiography revealed five superior gluteal artery stenoses and two occlusions. PTA without stenting was successful in all cases, without morbidity or mortality. During a mean follow-up of 13 months, restenosis occurred in one patient. A repeat PTA without stenting was successful, with resolution of the buttock claudication. CONCLUSIONS: Buttock claudication due to superior gluteal artery stenosis is probably underestimated when gluteal duplex scanning and aortoiliac angiography with oblique projections are not performed. PTA gives good results, and the procedure can be repeated should restenosis occur. PMID- 16678695 TI - Current guidelines produce competent endovascular surgeons. AB - OBJECTIVE: This study was conducted to evaluate the safety of percutaneous endovascular procedures (PEPs) during integration of endovascular skills into an urban academic vascular surgery practice and assess the hypothesis that currently accepted guidelines are a valid benchmark for endovascular competency. METHODS: From 2000 through 2004, an endovascular training paradigm was instituted to integrate endovascular procedures into an academic endovascular practice. The paradigm involved individual mentoring of vascular surgery faculty by a partner with mature endovascular skills. Mentoring continued until each surgeon achieved a procedural experience of 100 diagnostic angiograms and 50 percutaneous endovascular interventions. Once achieved, privileges were granted for independent endovascular practice. To assess the effectiveness of the training process and competency of the newly trained endovascular practitioner, the surgeon-specific 30-day incidence of major complications and deaths for all PEPs performed during and after the mentoring process was determined. Complications and deaths were assigned to the mentor during the training process and to the individual surgeon once endovascular privileges were granted. Complications were classified as local vascular, local nonvascular, or systemic/remote. RESULTS: From 2000 through 2004, 1208 PEPs were performed. During this time, three faculty surgeons achieved sufficient endovascular procedural experience and were granted endovascular privileges. Major complications consisted of 17 local vascular, three local nonvascular, and four systemic/remote. Three deaths occurred. Renal percutaneous transluminal angioplasty/stent procedures had the highest complication and death rate at 9%. The major complication and death rate per year was 1.8% to 4.9% (P = .32) and did not significantly vary. The major complication and death rate for all 1208 PEPs was 2.2%. The surgeon-specific complication and death rate was 1.9% to 3.6% (P = .14) and did not vary between surgeons. CONCLUSION: Endovascular skills can be safely transferred using a vascular surgeon-based training paradigm. When the training paradigm is directed at satisfying currently recommended guidelines for endovascular privileging, competent endovascular surgeons are the result. PMID- 16678696 TI - Continuity of care experience of residents in an academic vascular department: are trainees learning complete surgical care? AB - BACKGROUND: It is widely accepted that exemplary surgical care involves a surgeon's involvement in the preoperative, perioperative, and postoperative periods. In an era of ever-expanding therapeutic modalities available to the vascular surgeon, it is important that trainees gain experience in preoperative decision-making and how this affects a patient's operative and postoperative course. The purpose of this study was to define the current experience of residents on a vascular surgery service regarding the continuity of care they are able to provide for patients and the factors affecting this experience. METHODS: This prospective cohort study was approved by the Institutional Review Board and conducted at the University of British Columbia during January 2005. All patients who underwent a vascular procedure at either of the two teaching hospitals were included. In addition to type of case (emergent, outpatient, inpatient), resident demographic data and involvement in each patient's care (preoperative assessment, postoperative daily assessment, and follow-up clinic assessment) were recorded. Categoric data were analyzed with the chi2 test. RESULTS: The study included 159 cases, of which 65% were elective same-day admission patients, 20% were elective previously admitted patients; and 15% were emergent. The overall rate of preoperative assessment was 67%, involvement in the decision to operate, 17%; postoperative assessment on the ward, 79%; and patient follow-up in clinic, 3%. The rate of complete in-hospital continuity of care (assessing patient pre-op and post-op) was 57%. Emergent cases were associated with a significantly higher rate of preoperative assessment (92% vs 63%, P < .05). For elective cases admitted before the day of surgery compared with same-day admission patients, the rates of preoperative assessment (78% vs 58%, P < .05) and involvement in the decision to operate (16% vs 4%, P < .05) were significantly higher. CONCLUSIONS: The continuity-of-care experiences of vascular trainees are suboptimal. This is especially true for postoperative clinic assessment. Same-day admission surgery accounted for most of the cases and was associated with the poorest continuity of care. To provide complete surgical training in an era of changing therapeutic modalities and same-day admission surgery, vascular programs must be creative in structuring training to include adequate ambulatory experience. PMID- 16678697 TI - Anaphylactoid reactions to Dextran 40 and 70: reports to the United States Food and Drug Administration, 1969 to 2004. AB - BACKGROUND: Clinical dextrans, such as Dextran 40 and Dextran 70, are associated with anaphylactoid reactions caused by dextran-reactive immunoglobulin G antibodies. When infused immediately before clinical dextrans, dextran 1 significantly reduces the incidence of severe anaphylactoid reactions. The objective of the study was to describe the frequency and characteristics of reports submitted to the United States Food and Drug Administration (FDA) for anaphylaxis or anaphylactoid events after clinical dextran administration. METHODS: We searched the FDA's Adverse Event Reporting System for reports associated with a clinical dextran and describing anaphylaxis/anaphylactoid reactions. Our case definition for a probable anaphylaxis/anaphylactoid event required signs or symptoms from at least two body systems, with at least one sign or symptom being hypotension, vasodilation, or respiratory difficulty, and onset within 60 minutes. Other reports were considered possible cases if the reporter specifically described the reaction as anaphylaxis or an anaphylactoid reaction. Premier RxMarket Advisor provided estimates of total US hospitalizations with clinical dextran or dextran 1 administration from 2000 to 2004, based on discharge billing data from a sample of US hospitals. The IMS National Sales Perspective provided estimates of total doses of dextrans sold in the United States from 1999 to 2004, based on volumes of dextrans sold in a sample of retail and nonretail outlets. RESULTS: The FDA received 366 clinical dextran adverse event reports from 1969 to 2004, of which 90 (24.6%) were anaphylaxis/anaphylactoid events. The ratio of hospitalizations where clinical dextran was administered to hospitalizations where dextran 1 was administered was 28.4:1. The expected ratio would be 1:1 if all clinical dextran patients had received dextran 1 pretreatment. The ratio of clinical dextran doses sold to dextran 1 doses sold in the United States was 38.6:1. CONCLUSIONS: A high proportion of adverse event reports for clinical dextrans described anaphylaxis or anaphylactoid reactions. Hospital discharge and product sales data suggest that dextran 1 has not been used consistently before clinical dextran administration in recent years. To reduce the risk of anaphylactoid reactions, physicians should consider routine administration of dextran 1 before the infusion of a clinical dextran. PMID- 16678698 TI - Temporal changes in mouse aortic wall gene expression during the development of elastase-induced abdominal aortic aneurysms. AB - OBJECTIVE: To characterize temporal changes in mouse aortic wall gene expression associated with the development of experimental abdominal aortic aneurysms. METHODS: C57BL/6 mice underwent transient perfusion of the abdominal aorta with either elastase (n = 61) or heat-inactivated elastase as a control (n = 68). Triplicate samples of radiolabeled aortic wall complementary DNA were prepared at intervals of 0, 3, 7, 10, and 14 days, followed by hybridization to nylon microarrays (1181 genes). Autoradiographic intensity data were normalized by conversion to z scores, and differences in gene expression were defined by two tailed z tests at a significance threshold of P < .01. RESULTS: Elastase perfusion caused a progressive increase in aortic diameter up to 14 days accompanied by transmural inflammation and destructive remodeling of the elastic media. No aneurysms occurred in the control group. Compared with healthy aorta, 336 genes exhibited significant alterations during at least 1 interval after elastase perfusion (135 at more than 1 interval and 14 at all intervals), with pronounced increases for interleukin 6, cyclin E2, interleukin 1beta, osteopontin, CD14/lipopolysaccharide receptor, P-selectin glycoprotein ligand 1, and gelatinase B/matrix metalloproteinase 9 (all >20-fold on day 3). Sixty-two genes exhibited synchronous alterations in the elastase and control groups, thus suggesting a nonspecific response. By direct comparisons between the elastase and control groups, there were 384 genes with significant differences in expression for at least 1 interval after aortic perfusion, including 234 with differential upregulation (eg, p44MAPK/ERK1, osteopontin, heat shock protein 84, hypoxia inducible factor 1alpha, apolipoprotein E, monocyte chemotactic protein 3, MIG (monokine induced by gamma interferon), and interleukin 2 receptor gamma) and 163 with differential downregulation (eg, prothrombin, granzyme B, ataxia telangiectasia mutated, and interleukin-converting enzyme). CONCLUSIONS: Development of elastase-induced abdominal aortic aneurysms in mice is accompanied by altered aortic wall expression of genes associated with acute and chronic inflammation, matrix degradation, and vascular tissue remodeling. Knowledge of these alterations will facilitate further studies on the functional molecular mechanisms that underlie aneurysmal degeneration. PMID- 16678699 TI - Lipoplex gene transfer of inducible nitric oxide synthase inhibits the reactive intimal hyperplasia after expanded polytetrafluoroethylene bypass grafting. AB - OBJECTIVE: Intimal hyperplasia (IH) is most commonly the cause of graft occlusion in infrainguinal bypass grafting for arterial occlusive disease. We investigated the influence of nitric oxide on the IH of the arterial vessel wall at the region of prosthetic bypass anastomoses. METHODS: Experiments were performed in 10 Foxhound dogs. We used a technique of inducible nitric oxide synthase (iNOS) overexpression by a non-virus-mediated, liposome-based iNOS gene transfer. The plasmid pSCMV-iNOS, which drives the expression of iNOS under control of the cytomegalovirus promoter, was complexed with cationic liposomes (lipoplexes). Segments of both carotid arteries were pretreated by intramural injection of a lipoplex solution by using an infiltrator balloon catheter (Infiltrator Drug Delivery Balloon System). In each dog, iNOS was administered at one side, and a control vector (pSCMV2) was administered at the contralateral side. Carotid arteries were ligated, and bypass grafts (expanded polytetrafluoroethylene, 6-mm, ring enforced) were implanted on both sides. The proximal and distal anastomoses (end-to-side fashion; running nonabsorbable sutures) were placed in the pretreated regions. After 6 months, the prostheses were excised, and the intimal thicknesses of 50 cross sections (orcein staining) of each anastomosis were measured planimetrically. RESULTS: The average reduction of the neointima thickness of the iNOS side in proximal anastomoses at the prosthetic wall, suture region, and arterial wall was 43%, 52%, and 81%, respectively. In distal anastomoses, the average reduction was 40%, 47%, and 52%, respectively. All differences of neointima thickness between the iNOS and control sides were statistically significant (Wilcoxon test; P < or = .05). CONCLUSIONS: Inducible NOS expression is an efficient approach for inhibition of IH. In contrast to earlier studies, which investigated the efficacy of gene therapeutic NOS expression at 3 to 4 weeks after intervention, the novelty of our findings is that a single local lipoplex-mediated transfection of the vascular wall with iNOS expressing plasmids leads to a reduction of IH in a prosthetic in vivo model even after 6 months. Because all components can be manufactured under Good Manufacturing Practice conditions (the quality-management system of the European pharmaceutical industry based on ISO 9000), this approach is also amenable to human therapy. PMID- 16678701 TI - Use of microcirculatory parameters to evaluate chronic venous insufficiency. AB - BACKGROUND: Microcirculatory impairment caused by chronic venous hypertension is usually not taken into account in chronic venous insufficiency, probably due to lack of practical means to observe it. The objective of this work was to use a new noninvasive technique to access quantitatively the cutaneous microangiopathy in female patients classified according to CEAP from C1 to C5 and matched with healthy controls. METHODS: Forty-four patients and 13 healthy subjects (112 lower limbs), with a mean age +/- SD of 48 +/- 8 years, were evaluated by using orthogonal polarization spectral (OPS) imaging. Films of the internal perimaleolar region were analyzed by the CapImage software. The microcirculatory parameters evaluated were functional capillary density (number of capillaries with flowing red blood cells/mm), capillary morphology (percentage of abnormal capillaries), diameter (microm) of dermal papilla to quantify edema, diameter of capillary bulk (microm) to assess the degree of change, and diameter capillary limb to detect enlargement. A microcirculatory index combining these five parameters was proposed with I, II, and III stages, indicating normal microcirculation, and moderate and severe microangiopathy, respectively. RESULTS: These microcirculatory parameters were significantly different (P < .05) from control values (C): capillary diameter and capillary morphology from C2 to C5, 8.1 +/- 0.8, 3.6 +/- 5.5 (C), and 9.7 +/- 1.3, 27.5 +/- 17.7 (C2); diameter of dermal papilla and diameter of capillary bulk from C3 to C5, 111.4 +/- 13.5, 52.8 +/- 8.8 (C), and 150.5 +/- 31.7, 87.8 +/- 26.9 (C3); and functional capillary density only from C4 to C5, 20.9 +/- 6.1 (C) and 14.5 +/- 4.5 (C4). The microcirculatory index showed good correlation to CEAP classification. CONCLUSION: It was possible to quantify the microangiopathy using OPS imaging and to compare the microcirculatory changes of chronic venous insufficiency patients with healthy controls. Two parameters seemed more important to identify the differences between patients and controls: capillary morphology and capillary diameter. The suggested microcirculatory index can possibly demonstrate, in future studies, a prognostic capability when combined with the CEAP classification. PMID- 16678700 TI - Transforming growth factor-beta1 antisense treatment of rat vein grafts reduces the accumulation of collagen and increases the accumulation of h-caldesmon. AB - BACKGROUND: The main cause of occlusion and vein graft failure after peripheral and coronary arterial reconstruction is intimal hyperplasia. Transforming growth factor beta-1 (TGF-beta1) is a pleiotropic cytokine known to have powerful effects on cell growth, apoptosis, cell differentiation, and extracellular matrix synthesis. METHODS: To investigate the role of TGF-beta1 in intimal hyperplasia, we used adenovirus to deliver to superficial epigastric vein messenger RNA (mRNA) antisense to TGF-beta1 (Ad-AST) or the sequence encoding the bioactive form of TGF-beta1 (Ad-BAT). Infection with "empty" virus was used as a control (Ad CMVpLpA). The treated vein was then used for an interposition graft into rat femoral artery. Grafts were harvested at 1, 2, 4, and 12 weeks and formalin-fixed for histologic studies or placed in liquid nitrogen for mRNA studies. RESULTS: Ad AST treatment resulted in an overall reduction of TGF-beta1 expression (P = .001), and Ad-BAT treatment resulted in an overall increase in TGF-beta1 expression (P = .007). Histologic analysis showed Ad-AST caused reduced collagen build up in the neointima at 12 weeks (P = .0001). Immunohistochemical staining for h-caldesmon at 12 weeks indicated Ad-AST increased smooth muscle cells throughout the vessel wall compared with Ad-CMVpLpA (P = .0024) or Ad-BAT (P = .04). Ad-AST also resulted in reduced CD68-positive cells in the media/adventitia (P = .005 vs Ad-CMVpLpA, P = .01 vs Ad-BAT). To further understand how Ad-AST was influencing the build up of collagen, we performed quantitative polymerase chain reaction on complimentary DNA (cDNA) from homogenates of the vein grafts. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was increased at 1 week by Ad BAT (P = .048 vs Ad-CMVpLpA) and decreased by Ad-AST at all time points (P 90% of bound progesterone) by the end of the hormone-dependent period. The principal steroid in the ovulated egg is 5beta-pregnane 3alpha,17alpha,20beta-triol. There is a rapid efflux of 5beta-pregnane 3alpha,17alpha,20beta-triol into the medium immediately following fertilization and residual steroid levels remain low in the developing blastula. Dissociated blastulae cells prepared from stage 9 1/2 embryos concentrate both pregnenolone and progesterone from the medium with minimal metabolism. The results indicate that the ovarian follicle has the ability to synthesize and metabolize progesterone but that this ability disappears in the ovulated egg. The progesterone metabolites formed during meiosis are largely released at fertilization. PMID- 16678718 TI - Anaesthesia for pituitary surgery. AB - The anaesthetic care of patients undergoing pituitary surgery involves an understanding of the varied presentations of pituitary disease and their implications for the patient's perioperative condition and management. The neuroanaesthetist must also have an appreciation of the issues relevant to the surgical approach (either transsphenoidal or, less commonly, transcranial) and be able to anticipate and manage them accordingly. PMID- 16678719 TI - Venous air embolism and the sitting position: a case series. AB - Venous air embolism is a potentially serious complication of neurosurgery that occurs more commonly when the patient is in the sitting position. In this study, we aimed to quantify the incidence of venous air embolism during neurosurgical procedures performed with the patient in the sitting position in our institution. We performed a prospective audit of venous air embolism in 100 consecutive patients who underwent neurosurgical procedures in the sitting position, as evidenced by a decrease in end-tidal carbon dioxide partial pressure of 5 mmHg or more within 5 min. The incidence of venous air embolism was 9% (95% confidence interval: 3.3-14.7%). These patients did not have a significantly higher rate of hypotension or desaturation. Our results suggest that the sitting position can be safely used in neurosurgery. PMID- 16678720 TI - Need for intrasellar packing in sellar reconstruction of transsphenoidal surgery: less is more? AB - BACKGROUND: This study investigated patients with pituitary macroadenomas who received transsphenoidal surgery using two different sellar reconstruction methods, to determine the need for intrasellar packing. METHODS: The authors reviewed 53 patients with pituitary macroadenomas at a single institution between January 1999 and November 2002. On tumour removal, the group 1 patients underwent traditional sellar reconstruction (intrasellar and sphenoid sinus packing), while the group 2 patients did not undergo intrasellar packing. RESULTS: The two groups did not differ significantly in terms of complications. There was no mortality; notably, no delayed empty sella syndrome occurred in the group 2 patients. CONCLUSIONS: Simplified reconstruction of the sella floor using only sphenoid bone without intrasellar packing is thought to be safe and effective. This technique obviates the need for a second surgical incision, risk of overpacking, complications associated with packing material and interference on postoperative magnetic resonance imaging. PMID- 16678721 TI - Paediatric brainstem gliomas: prognostic factors and management. AB - BACKGROUND: Although most types of tumours involving the brainstem can be surgically removed, patients with diffuse pontine brainstem glioma do not benefit from surgical intervention. The aim of the present study is to establish whether clinical symptoms, their duration prior to diagnosis, the presence of enhancement on MRI scans and the histology of the lesion in paediatric brainstem glioma can guide the surgeon in choosing appropriate management options. METHODS: We retrospectively reviewed the charts and MRI scans of paediatric patients admitted to Turin's Ospedale Regina Margherita with a diagnosis of brainstem glioma. RESULTS: Patients with a diffuse pontine tumour on MRI scan (group 1) had a mean duration of symptoms prior to diagnosis of 2.61 months, 77% had symptoms involving at least one cranial nerve at diagnosis, and no MRI scans showed enhancement with gadolinium. No patients underwent radical surgery and 84.6% died. For all other patients (group 2), the mean duration of symptoms at diagnosis was 10.58 months, cranial nerve involvement was present in only 28.5%, and the MRI scans showed enhancement in 78.6%. Radical surgery was the treatment of choice (100%). In this study, survival of patients with diffuse pontine brainstem glioma was 25% at 2 years and survival of patients with any other brainstem glioma was 90% at 2 years. PMID- 16678722 TI - Recurrence in craniopharyngiomas: analysis of clinical and histological features. AB - The aim of this study was to investigate the recurrence patterns and significance of various clinical and histological features as predictors of recurrence in craniopharyngiomas. A series of 234 craniopharyngiomas (156 males, 78 females; age range 1.6-65 years) was reviewed. The mean follow-up period was 18.53 months (range 1-120 months). Peri-operative mortality was 7.4% and tumor recurrence was observed in 26 patients (20.3%). Of the patients with recurrence, one had total tumor excision (recurrence-free survival (RFS) 14 months), four had near-total excision (mean RFS 18.2 months) and 21 had subtotal tumor excision (RFS for symptomatic recurrence 7.1 months). Histologically, an adamantinous pattern was seen in 81.4% of cases and a papillary pattern was seen in 18.6%. Brain tissue was included in 67 cases and brain invasion was noted in 44 (all were of adamantinous histology). No correlation was noted between histopathological subtyping or brain invasion and recurrence. The significant clinical factors predictive of recurrence included the extent of resection, tumor size greater than 4 cm and cystic tumors. PMID- 16678723 TI - Novel mutations in the arylsulfatase A gene in eight Italian families with metachromatic leukodystrophy. AB - Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. We analysed the ARSA gene in eight unrelated Italian families with different clinical variants of MLD and identified three novel mutations: two Ser406Gly, (Glu329Ter) associated with late infantile MLD and one (Leu52Pro) with juvenile MLD. Only one family carried a pseudodeficiency allele (Asn350Ser). The IVS2+1G>A mutation occurred in four families. We also identified three polymorphisms, all in heterozygosis: Thr391Ser was present in five families, Trp193Cys in four families, and Ala210Ala in one family. We could identify 100% of the alleles causing MLD in the families, involving 12 different mutations, resulting in improved prognosis and genetic counselling. PMID- 16678724 TI - Delayed hypokalemic paralysis following a convulsion due to alcohol abstinence. AB - We encountered three patients with hypokalemic paralysis following a convulsion in the early stages of alcohol abstinence. The transtubular potassium gradient was less than 2.0, suggesting intracellular potassium shift. Hypokalaemic paralysis may result from retention of intracellular cationic potassium bound by anionic phosphorylated compounds, precipitated by an acceleration of the (Na+) (K+) pump in alcohol withdrawal and convulsions. These findings warn of the lethal hypokalemia that may occur after convulsions, particularly soon after alcohol abstinence associated with moderate withdrawal symptoms. PMID- 16678725 TI - Cognitive status of young and older cigarette smokers: data from the international brain database. AB - Previous studies that have examined the impact of cigarette smoking on cognition have revealed mixed results; some studies report no impact and others report detrimental effects, especially in older individuals. Few studies, however, have examined the effects of cigarette smoking on both young and old healthy individuals using highly robust and standardized methods of cognitive assessment. This study draws on an international database to contrast cognitive differences between younger and older individuals who regularly smoke cigarettes and non smokers. Data were sampled from 1000 highly screened healthy individuals free of medical or psychiatric health complications. A cohort of 62 regular smokers (n = 45 < 45 years of age; n = 1745 years) with a Fagerstrom nicotine dependency score of 1 or more were identified and matched to a cohort of 62 healthy nonsmokers (n = 43 < 45 years; n = 1945 years) on demographic variables and estimated intelligence. Performances on cognitive measures of attention, reaction time, cognitive flexibility, psychomotor speed, and memory were considered for analysis. As a group, smokers performed more poorly than nonsmokers on one measure of executive function. A significant age and smoking status interaction was identified with older smokers performing more poorly than older nonsmokers and younger smokers on a measure of long-delayed recall of new information. Cigarette smoking is associated with isolated and subtle cognitive difficulties among very healthy individuals. PMID- 16678726 TI - Neuroprotection with methylaminochroman and lazaroid of embryonic ventral mesencephalic tegmental dopaminergic neurons in cold storage. AB - Embryonic ventral mesencephalic tegmental (EVMT) neurons die off over time in cold storage at 4 degrees C in hibernation buffers (HB). Manipulation of HB conditions may improve the survival of neurons in cold storage. We examined the effect of lipid peroxidation inhibitors, a methylaminochroman (U83836E) and a lazaroid (U74389G) on the viability and survival of embryonic dopaminergic neurons in the co-culture system of embryonic striatal target (EST) cells and EVMT neurons that had been stored for 3 days at 4 degrees C in HB with or without U83836E or U74389G. One-way analysis of variance (ANOVA) was used for analysis of data. The density of tyrosine hydroxylase immunoreactive (THIR)-positive neurons was significantly higher in the groups stored in supplemented HB than in the control (HB alone; P < 0.001). The neuroprotective effect was concentration dependent. We conclude that either U83836E or U74389G-conditioned HB exerted a concentration-dependent neuroprotective effect on embryonic dopaminergic neurons in cold storage for 3 days. Supplementation of U83836E and U74389G or other methylaminochromans and lazaroids in HB may be important for cold storage of donor neuronal cells. PMID- 16678727 TI - Endolymphatic sac carcinoma of the right petrous bone in Von Hippel-Lindau disease. AB - A large endolymphatic sac papillary adenocarcinoma in a patient with Von Hippel Lindau (vHL) disease is reported. A 31-year-old man was treated for a left eye retinal angioma 10 years previously and had been blind in that eye since. He was admitted with progressive tinnitus, lower cranial nerve paresis and ataxia. Investigations revealed a vascular and bone-eroding petrous tumour. The entirely extradural tumour involved a large part of the petrous bone and extended into the cerebellopontine angle. The vascular tumour was radically resected using a presigmoid approach after feeding vessel embolisation. The literature on this rare clinical entity is briefly discussed. PMID- 16678728 TI - Eclamptic subarachnoid haemorrhage without hypertension. AB - Subarachnoid haemorrhage in pregnancy is often the result of aneurysmal rupture or severe hypertension. A young woman with postpartum eclampsia and 'normal' blood pressure developed sudden-onset head pain, and was found to have minor biconvexity subarachnoid hemorrhages. Serial angiograms of the cervicocranial vessels revealed no evidence of aneurysm or arteriovenous malformation. A follow up angiogram revealed diffuse vessel narrowing, consistent with postpartum angiopathy. Treatment consisted only of nimodipine for the prevention of vasospasm. The patient made an excellent recovery, without residual neurological deficits. PMID- 16678729 TI - Carotid cavernous aneurysm presenting as pituitary apoplexy. AB - The authors report an interesting case with a ruptured internal carotid artery aneurysm that presented as a sellar haematoma mimicking radiologically a pituitary adenoma, and clinically a pituitary apoplexy. A 53-year-old woman presented with a 2-week history of episodic severe headache and vomiting associated, 3 days prior to admission, with left ophthalmoparesis and transient right hemiparesis. Brain MRI showed a large intra- and suprasellar mass suggestive of a pituitary macroadenoma. Hormonal profiles showed hyperprolactinaemia and subsequent cerebral angiography demonstrated a carotid cavernous aneurysm. The patient underwent surgery via a subfrontal approach to manage both lesions. At operation, the suspected pituitary adenoma was revealed to be a sellar haematoma; the aneurysm was successfully clipped. Postoperatively, the patient developed hypotension and right hemiparesis which, as well as the third nerve paresis, progressively improved to full recovery. At 12 months follow up the patient is neurologically intact and generally well. The clinical features, the management of such a case and the importance of differential diagnosis in the acute stage are emphasised and discussed along with relevant literature. PMID- 16678730 TI - Ocular flutter induced only by optokinetic stimulation. AB - Ocular flutter (OF) refers to occasional bursts of involuntary horizontal oscillation around the point of fixation, characterised by rapid horizontal repetitive oscillation without intersaccadic interval in electro-ocular recording. In spite of numerous reports of OF, there has been no previous report describing OF developed by simply looking at moving objects. We report a patient with olivopontocerebellar atrophy (OPCA), with OF induced only by optokinetic stimulation (OKS). A 65-year-old man, with OPCA complained of dizziness and oscillopsia occurring only with a specific type of eye movement. The electro oculographic recording showed a decreased gain of pursuit, catch-up saccade, and decreased velocity of saccade, and OF was induced only by OKS of more than 40 degrees /second. In OPCA, partial loss of omnipause neurons and/or abnormal firing of burst neurons, especially those involved in optokinetic movement, could be the cause of OF occurring only with OKS. PMID- 16678731 TI - Nocardial brain abscess: review of clinical management. AB - Nocardiosis has become a significant opportunistic infection over the last two decades as the number of immunocompromised individuals has grown worldwide. We present two patients with nocardial brain abscess. The first patient was a 39 year-old woman with systemic lupus erythematosus. A left temporoparietal abscess was detected and aspirated through a burr-hole. Nocardia farcinica infection was diagnosed. The patient had an accompanying pulmonary infection and was thus treated with imipenem and amikacine for 3 weeks. She received oral minocycline for 1 year. The second patient was a 43-year-old man who was being treated with corticosteroids for glomerulonephritis. He was diagnosed with a ring-enhancing multiloculated abscess in the left cerebellar hemisphere, with an additional two small supratentorial lesions and triventricular hydrocephalus. Gross total excision of the cerebellar abscess was performed via a left suboccipital craniectomy. Culture revealed Nocardia asteroides, and the patient was successfully treated with intravenous ceftriaxone, then oral trimethoprime sulfamethoxazole for 1 year. The clinical course, radiological findings, and management of nocardial brain abscess are discussed in light of the relevant literature, and current clinical management is reviewed through examination of the cases presented here. PMID- 16678732 TI - Brucellar brain abscess and bilateral arachnoid cysts, unilaterally complicated by subdural haematoma. AB - Arachnoid cysts are benign developmental cysts that occur along the cerebrospinal axis. Brucellar abscesses in the brain are relatively uncommon, with only a few cases reported in the literature. We report here a patient with a brucellar brain abscess and bilateral arachnoid cysts (one complicated with subdural haemorrhage), who was successfully managed with craniotomy and antibiotics. PMID- 16678733 TI - Intracranial metastasis of lung adenocarcinoma mimicking colloid cyst of the third ventricle. AB - A patient with intracranial lung adenocarcinoma metastasis mimicking a colloid cyst of the third ventricle is reported. These tumours may be associated with excessive bleeding and may infiltrate into surrounding structures. Open microsurgery rather than endoscopic surgery should be considered for these cases, particularly a transcortical-transventricular or transcallosal approach, in order to avoid serious complications. PMID- 16678734 TI - Heparin-induced transient prolongation of the QT interval during endovascular embolisation of intracranial aneurysm. AB - Prolonged QT interval and increased QT(C) dispersion have been described immediately following rupture of intracranial aneurysm, due to increased sympathetic activity. Here, we report a patient with transient prolongation of the QT interval, probably due to heparin-induced hypocalcaemia, which occurred during coiling of a right internal carotid aneurysm, 54 days after ictus. Correction of the hypocalcaemia led to resolution of the electrocardiogram and blood pressure abnormalities. PMID- 16678735 TI - Paranodal pathology in Tangier disease with remitting-relapsing multifocal neuropathy. AB - Pathological studies of a sural nerve biopsy in a man with Tangier disease presenting as a remitting-relapsing multifocal neuropathy showed abnormalities in the paranodal regions, including lipid deposition (65%) and redundant myelin foldings, with various degrees of myelin splitting and vesiculation (43%) forming small tomacula and abnormal myelin terminal loops (4%). The internodal regions were normal in the majority of myelinated fibres. Abnormal lipid storage was also present in the Schwann cells of the majority of unmyelinated fibres (67%). The evidence suggests that the noncompacted myelin region of the paranode is a preferential site for lipid storage in the myelinated Schwann cell, and that the space-occupying effects of the cholesterol esters leads to paranodal malfunction and tomacula formation as the pathological basis for the multifocal relapsing remitting clinical course. PMID- 16678736 TI - Primary glioblastomas and anaplastic astrocytoma in a glioma family. AB - A 72-year-old man presented with a short duration of symptoms relating to a right fronto-parietal glioblastoma and a family history of children with brain tumours. Analysis of the patient's family tree revealed that out of seven children, he had a living son with anaplastic astrocytoma, a daughter who had died with a glioblastoma, and a son who had died with a histologically undiagnosed intrinsic brain tumour. One niece was also thought to have died from a brain tumour. All of the other affected family members had onset in their third or fourth decades. Tissue was only available from two of the affected individuals, precluding familial genetic analysis at this stage. There is no clinical evidence to support a diagnosis of a multiple cancer or neurocutaneous syndrome in this family. In view of what is known about the genetics of familial glioma, it is interesting to note the clinical evidence of both 'primary' glioblastoma and anaplastic astrocytoma in the same kindred. PMID- 16678737 TI - Recurrent intramedullary sarcoidosis. AB - We present a 47-year-old man with recurrent intramedullary sarcoidosis. This condition, without any other manifestations of the disease, is rare, with less than 20 reports in the literature. Further, to our knowledge our presentation is the only reported case of intramedullary sarcoidosis recurring in a separate, distant region of the cord after a period of treatment with steroids, to which the patient had initially responded. PMID- 16678738 TI - Tongue tremor in brainstem pilocytic astrocytoma. AB - An isolated tremor of the tongue developed in a 22-year-old female patient in a minimally conscious state. The patient was diagnosed with brainstem pilocytic astrocytoma. A widespread lesion of the brainstem and cerebellum was evident on cranial magnetic resonance imaging. The pathophysiology of isolated tongue tremor is discussed with the magnetic resonance findings and the relevant literature. PMID- 16678739 TI - Glucocorticoid-induced osteoporosis: an update. AB - Glucocorticoid-induced osteoporosis occurs in two phases: a rapid, early phase in which bone mineral density is reduced, possibly as a result of excessive bone resorption, and a slower, progressive phase in which bone mineral density declines because of impaired bone formation. Although the indirect effects of glucocorticoids on bone are evident, their direct effects on osteoblasts, osteoclasts and osteocytes are primarily operative in the pathogenesis of glucocorticoid-induced osteoporosis. The management of patients exposed to glucocorticoids includes general health measures, sufficient calcium and vitamin D, and reducing the therapeutic regimen to the minimal effective dose. The gold standard in the pharmacological treatment of glucocorticoid-induced osteoporosis in postmenopausal women involves the use of bisphosphonates, which should be started soon after beginning chronic glucocorticoid therapy. Anabolic and alternative therapeutic strategies are currently under investigation in glucocorticoid-induced osteoporosis. PMID- 16678741 TI - Pediatric ophthalmology and strabismus in Turkey. PMID- 16678742 TI - Incidence, distribution, and duration of birth-related retinal hemorrhages: a prospective study. AB - BACKGROUND: Retinal hemorrhages secondary to birth trauma are part of the differential diagnosis of intraocular hemorrhages seen in the setting of Shaken baby syndrome in very young infants. This prospective study aimed to document the morphology, distribution and, most importantly, the natural history of these hemorrhages using digital imaging. SUBJECTS AND METHODS: Infants were recruited as soon after birth as possible and examined by indirect ophthalmoscopy. Retinal hemorrhages were photographed using the RetCam 120. Birth history was documented from the medical notes. Infants were reexamined and photographed until hemorrhages had resolved. RESULTS: Data were analyzed for a total of 53 neonates. The number of infants with retinal hemorrhage was 18 (34%). The incidence in relation to mode of delivery was as follows: vacuum delivery, 77.8%; normal vaginal delivery, 30.4%; cesarean section, 8.3%; forceps delivery, 30.3%. All hemorrhages were intraretinal and in all but two infants hemorrhages had resolved by 16 days. In two subjects hemorrhages were still present at 31 and 58 days, respectively. Both these infants were delivered by vacuum delivery. CONCLUSIONS: The RetCam 120 provides excellent documentation of retinal hemorrhages and their natural history. We have demonstrated hemorrhages still present at 58 days in a child born by vacuum delivery and this may have important implications for consideration in the differential diagnosis of Shaken baby syndrome. PMID- 16678743 TI - RetCam imaging for retinopathy of prematurity screening. AB - PURPOSE: Indirect ophthalmoscopy is the gold standard for retinopathy of prematurity (ROP) screening. Screening for ROP with digital imaging has been proposed as a possible alternative. Our goal was to evaluate the longitudinal clinical outcomes of employing digital imaging to detect high-risk ROP. METHODS: Serial RetCam imaging and indirect ophthalmoscopy were performed on 43 premature infants. A masked reader evaluated the images and made management recommendations that were compared with indirect ophthalmoscopy results. Successful screening was determined by correctly identifying progression to prethreshold or threshold disease with referral for indirect ophthalmoscopy. Unsuccessful screening was determined by failure to identify prethreshold or threshold disease, inaccurately detecting prethreshold or threshold disease, or inability to evaluate for ROP. RESULTS: No cases of prethreshold or threshold disease were missed by the reader. The reader overestimated prethreshold or threshold disease in 5% of cases. Initial screening in 21% of cases could not be evaluated for ROP secondary to poor image quality. Digital photography had a sensitivity of 100% and specificity of 97.5% in detecting prethreshold and threshold ROP. Positive-predictive value of digital photography was 67% and negative-predictive value was 100%. CONCLUSIONS: Screening and management of ROP using RetCam imaging did not fail to detect prethreshold or threshold disease when images could be obtained. Ophthalmologic examinations were needed in 20% of cases that did not reach threshold or prethreshold disease because of poor image quality or overestimation of ROP. RetCam screening may safely reduce the overall number of indirect ophthalmologic examinations required. PMID- 16678744 TI - Factors affecting sensory functions after successful postoperative ocular alignment of acquired esotropia. AB - PURPOSE: We sought to evaluate the sensory status of patients with acquired esotropia who were able to re-establish stable alignment by optical correction and surgery and to determine the possible predictors of the different sensory outcomes. METHODS: Thirty-four successfully aligned esotropic patients were included in the study. Preoperative evaluation comprised history taking, measurement of visual acuity, evaluation of the sensory status (using the Worth 4 Dot test, and the Titmus Stereo test), measurement of ocular deviation, cycloplegic refraction, and fundus examination. All patients underwent successful surgical alignment to within 10 prism diopters (Delta) of orthotropia. At each postoperative follow-up visit, the sensory functions and ocular alignment were assessed. Statistical analysis of the results was performed. RESULTS: Among the 34 patients included in the study, 62% achieved fusion, 17% had diplopia, 15% had suppression, and 6% had a variable response to the Worth 4-Dot test at 6 months after surgery. Stereopsis was achieved in 32% as determined by the Titmus Stereo test. Statistical analysis revealed a significant relationship between the sensory status and the duration of strabismus (P=.00002), the age at surgery (P=.00289), and postoperative ocular alignment (P=.02211). CONCLUSION: Early surgical and optical ocular alignment of strabismic patients is advisable to achieve fusion and stereopsis. PMID- 16678745 TI - Incidence and risk factors for glaucoma after pediatric cataract surgery with and without intraocular lens implantation. AB - PURPOSE: We sought to report the incidence of glaucoma in the eyes of children who underwent cataract surgery with and without intraocular lens implantation and to report the risk factors for developing glaucoma. METHODS: We undertook a retrospective review of pediatric cataract surgery charts, excluding traumatic cataract, aniridia and Lowe syndrome, steroid-induced cataract, lens subluxation, uveitis, retinoblastoma, radiation-induced cataract, retinopathy of prematurity, secondary IOL implantation, and patients with less than 1 month of postoperative follow-up. RESULTS: After pediatric cataract surgery, 10 (3.8%) of 266 eyes with primary intraocular lens implantation were diagnosed with glaucoma, whereas 8 (17.0%) of 47 aphakic eyes were diagnosed with glaucoma. During the initial analyses, we noted that all of the patients who developed glaucoma underwent cataract surgery when they were 4.5 months or younger. For all patients who underwent surgery during the first 4.5 months of their life, the glaucoma incidence was 24.4% (10/41) in children with pseudophakic eyes and 19.0% (8/42) in age-matched children with aphakic eyes (risk ratio=1.1, CI=0.7-1.9; P=.555). In patients who underwent surgery during the first 4.5 months of their life, the average age of the patients who developed glaucoma was not significantly different than those who did not develop glaucoma in pseudophakic eyes (2.0 months+/-1.4 vs. 1.9 months+/-1.0, P=.700) or aphakic eyes (2.6 months+/-1.5 vs. 1.4 months+/-0.9, P=.070). The corneal diameter of the eyes that developed glaucoma versus eyes that did not was not significantly different in patients with pseudophakic eyes (P=.860) or aphakic eyes (P=.254). Glaucoma was diagnosed in patients at an average of 8.6 months and 117.9 months after cataract surgery in those with pseudophakic eyes and aphakic eyes, respectively. CONCLUSIONS: Patients undergoing cataract surgery at an early age are at high risk for the development of glaucoma with or without an intraocular lens implant. PMID- 16678746 TI - The role of botulinum toxin A in augmentation of the effect of recession and/or resection surgery. AB - PURPOSE: The aim of this study is to assess the effectiveness of intraoperative botulinum toxin A (BTA) injection as an adjunct to the surgical treatment of large-angle esotropia or exotropia. METHODS: Ten patients were included in the study. Mean age of the patients was 27+/-20 years. Of these 10 patients, 7 were esotropic and 3 exotropic. The average preoperative esodeviation was 73.6+/-16.5 prism diopters and exodeviation was 76.7+/-5.8 PD. Five units of BTA were injected intraoperatively into one of the recessed horizontal rectus muscles in all of the patients. RESULTS: The average follow-up was 14+/-10 months (range, 8 to 40 months). The average final deviation in the esotropia group was 13+/-9.6 PD of esotropia. The average final deviation in the exotropia group was 4.7+/-5 PD of exotropia. The final deviation of the 70% patients was within 10 PD of esotropia or exotropia. CONCLUSION: The results of this study suggest that the combination of BTA injection with recession may increase the expected correction of a conventional horizontal rectus muscle surgery. PMID- 16678747 TI - Retinopathy of prematurity in infants with birth weight>or=1250 grams-incidence, severity, and screening guideline cost-analysis. AB - PURPOSE: To determine the incidence and severity of retinopathy of prematurity (ROP) in infants with birth weight (BW) 1250 to 1800 g, to examine the influence of systemic conditions on the development of ROP in this population, and to evaluate the cost-effectiveness of various screening guidelines. METHODS: We reviewed records from 259 consecutive infants with BW 1250 to 1800 g who were screened for ROP over a 3-year period. Extracted data included presence and severity of ROP, and the following potential risk factors (RF) for ROP development: sepsis, meningitis, necrotizing enterocolitis, intraventricular hemorrhage greater than stage I, pneumothorax, direct bilirubin>2 mg/dl, central line placement, antibiotic treatment>14 days, greater than seven red blood cell (RBC) transfusions, and mechanical ventilation>96 hours. RESULTS: The overall incidence of ROP in this population was 4.2%. Two infants had stage 3 ROP, one with plus disease. Infants with stage 3 ROP had significantly lower BW (1299 versus 1484 g, P=0.013) and gestational age (GA) (28 versus 31 weeks, P=0.002) than those with no ROP. No infant with BW>1500 g developed treatable ROP. Conditions that best predicted ROP development in the 1501 to 1800 g BW group were sepsis, ventilation >96 hours, antibiotic use >14 days, RBC transfusions greater than seven units, and central line placement (P=0.001, P=0.001, P=0.012, P=0.014 and P=0.035, respectively). All infants with BW>1500 g who developed ROP had greater than or equal to two of these RF. CONCLUSIONS: All cases of high-risk ROP would have been identified by current screening guidelines. Modified screening criteria of infants with (1) BW2), (1-->3), and (1-->4)-linked C disaccharides in reactions combining them with sugar-derived carbaldehydes. Synthetic methods relying on conjugate nucleophilic additions of these enones, their combination with aluminum reagents and aldehydes (Baylis-Hillman reaction) and modified Takai-Hiyama-Nozaki-Kishi couplings of enol triflates derived from them with sugar-derived aldehydes are reviewed. Highly stereoselective methods have thus been developed. These allow the generation of disaccharide mimetics with a high molecular diversity. PMID- 16678804 TI - Intracarotid injection of granulocyte-macrophage colony-stimulating factor induces neuroprotection in a rat transient middle cerebral artery occlusion model. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to promote collateral flow in patients with coronary artery disease and also to induce arteriogenesis in a rat hypoperfusion brain model. Activated macrophages have been shown to induce vascular proliferation and play an important role in ischemic stroke. In this study, we examined the therapeutic effect of GM-CSF on the ischemic brain by activating microglia/macrophages. Rats were subjected to 1 h intraluminal middle cerebral artery occlusion (MCAO) and received an intracarotid injection of GM-CSF (5 ng) or saline immediately after reperfusion. Infarct volume, neurological function and histological findings were assessed 48 h later. An intracarotid injection of GM-CSF reduced the infarct volume and improved neurological function at 48 h after reperfusion. Histological analysis revealed that the number of activated microglia/macrophages to be increased and the number of apoptotic cells to be decreased in the area of the penumbra. These results suggest that intracarotid injection of GM-CSF may have a therapeutic effect on brain ischemia via activation of microglia/macrophages. PMID- 16678806 TI - Structural elucidation of a neutral fucogalactan from the mycelium of Coprinus comatus. AB - A water-soluble fucogalactan (CMP3), with a molecular mass of 1.03 x 10(4) Da as determined by high-performance size-exclusion chromatography (HPSEC), was obtained from the crude intracellular polysaccharide of Coprinus comatus mycelium. Its chemical structure was characterized by sugar and methylation analysis along with 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments for linkage and sequence analysis. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [structure:see text]. PMID- 16678807 TI - N-glycoside neoglycotrimers from 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl azide. AB - 2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl azide is available on large scale from D-glucose by means of a three-step sequence involving acetylation, activation as the glycosyl bromide, and stereospecific displacement with azide anion. The azide functionality then serves as a convenient anchor upon which to introduce new functionality, usually with retention of the beta-stereochemistry. Here we report the synthesis of an amide-linked N-glycosyl trimer, by employing a Staudinger-aza-Wittig process on the azide, as well as a hybrid N-glycosyl triazole-amide-linked trimer in which the sugars are separated by 1,2,3-triazole heterocycles. Both of these neoglycotrimers are isolated in good yield with high beta-selectivity in each case. PMID- 16678808 TI - Synthesis of thioswainsonine as a potential glycosidase inhibitor. AB - The synthesis of a bicyclic sulfonium-ion analogue of a naturally occurring glycosidase inhibitor, swainsonine, in which the bridgehead nitrogen atom is replaced by a sulfonium ion, has been achieved by a multi-step synthesis starting from 1,4-anhydro-2,3-di-O-benzyl-4-thio-D-lyxitol. The synthetic strategy relies on the intramolecular displacement of a leaving group on a pendant acyclic chain by a cyclic thioether. This bicyclic sulfonium salt will serve as a candidate to test the hypothesis that a sulfonium salt carrying a permanent positive charge would be an effective glycosidase inhibitor. PMID- 16678809 TI - Heparin is an unsuitable anticoagulant for the detection of plasma ammonia. PMID- 16678810 TI - Pancreatic cancer-derived S-100A8 N-terminal peptide: a diabetes cause? AB - BACKGROUND: Our aim was to identify the pancreatic cancer diabetogenic peptide. METHODS: Pancreatic tumor samples from patients with (n=15) or without (n=7) diabetes were compared with 6 non-neoplastic pancreas samples using SDS-PAGE. RESULTS: A band measuring approximately 1500 Da was detected in tumors from diabetics, but not in neoplastic samples from non-diabetics or samples from non neoplastic subjects. Sequence analysis revealed a 14 amino acid peptide (1589.88 Da), corresponding to the N-terminal of the S100A8. At 50 nmol/L and 2 mmol/L, this peptide significantly reduced glucose consumption and lactate production by cultured C(2)C(12) myoblasts. The 14 amino acid peptide caused a lack of myotubular differentiation, the presence of polynucleated cells and caspase-3 activation. CONCLUSIONS: The 14 amino acid peptide from S100A8 impairs the catabolism of glucose by myoblasts in vitro and may cause hyperglycemia in vivo. Its identification in biological fluids might be helpful in diagnosing pancreatic cancer in patients with recent onset diabetes mellitus. PMID- 16678811 TI - Sox9 is required for notochord maintenance in mice. AB - Sox9 encodes a HMG-box transcription factor that has been implicated in numerous developmental processes including chondrogenesis, formation of cardiac valves, and neural crest, testis and spinal cord development. Here we show that Sox9 is expressed in the notochord and the sclerotome during mouse development suggesting that the gene may play additional roles in the development of the axial skeleton. We used ubiquitous mosaic inactivation of a conditional Sox9 allele by Cre/loxP mediated recombination in the mouse to screen for novel functions of Sox9, and revealed that its absence results in severe malformations of the vertebral column. Besides its established role in chondrogenesis, Sox9 is required for maintaining the structural integrity of the notochord. Mutant embryos establish a normal notochord; however, starting from E9.5, the notochord disintegrates in a cranial to caudal manner. The late requirement in notochord development uncovered a function of notochord-derived signals in inducing segmentation of the ventral sclerotome and chondrogenesis. Thus, Sox9 is required for axial skeletogenesis by regulating notochord survival and chondrogenesis. PMID- 16678812 TI - Actin filament organization regulates the induction of lens cell differentiation and survival. AB - The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells. PMID- 16678813 TI - Reduced glutathione levels affect the culmination and cell fate decision in Dictyostelium discoideum. AB - Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate. PMID- 16678814 TI - Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo. AB - After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination. PMID- 16678815 TI - Lef1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland. AB - Inductive reciprocal signaling between mesenchymal and adjacent epithelia gives rise to skin appendages such as hair follicles and mammary glands. Lef1-mediated canonical Wnt signaling is required for morphogenesis of these skin appendages during embryogenesis. In order to define the role of canonical Wnt signaling during early embryonic mammary gland development, we determined the temporal and spatial changes in Wnt signaling during embryogenesis in wild-type and Lef1 deficient embryos harboring a Tcf/Lef1-betagal reporter (TOPGAL) transgene. In contrast to previous studies using TOPGAL mice from a distinct founder, we observe that Wnt signaling acts initially on mesenchymal cells associated with the sequential appearance of mammary placodes. As placode development progresses between 12.5 and 15.5 dpc, Wnt signaling progressively accumulates in the mammary epithelial compartment. By 18.5 dpc, betagal activity is confined to mesenchymal and epithelial cells near the nipple region. In Lef1-deficient embryos, the transition of Wnt signaling from mesenchyme to the mammary epithelia is blocked for placodes #1, 4 and 5 despite the expression of Tcf1 in epithelial cells. These placodes ultimately disappear by 15.5 dpc, while placodes 2 and 3 typically did not form in the absence of Lef1. Progressive loss of placodes 1, 4, and 5 is accompanied by increased apoptosis in mesenchymal cells adjacent to the mammary epithelial placodes. While factors important for embryonic mammary gland development, such as FGF7, are expressed normally in Lef1-deficient animals, one mediator of the Hedgehog (Hh)-signaling pathway is aberrantly expressed. Specifically, Shh, Ihh, and Gli2 are expressed in mammary epithelial cells at levels in Lef1-deficient animals similar to wild-type littermates. However, the signal for Ptc-1 is strongly reduced in mesenchymal cells surrounding the mammary placode in Lef1 mutants relative to wild-type embryos. The loss of Ptc-1, both a receptor for and transcriptional target of Hh signaling, suggests that Hh signaling is blocked in Lef1-deficient embryos. Thus, these data reveal distinct requirements of different mammary placodes for Lef1-dependent Wnt signaling. They further define dynamic changes in which cells integrate Lef1-dependent Wnt signaling during progression of embryonic mammary gland development. PMID- 16678816 TI - The AP-1 transcription factor regulates postnatal mammary gland development. AB - The AP-1 transcription factor is activated by multiple growth factors that are critical regulators of breast cell proliferation. We previously demonstrated that AP-1 blockade inhibits breast cancer cell growth in vitro. Yet a specific role of AP-1 in normal mammary gland development has not been studied. Using a bi transgenic mouse expressing an inducible AP-1 inhibitor (Tam67), we found that the AP-1 factor regulates postnatal proliferation of mammary epithelial cells. Mammary epithelial proliferation was significantly reduced after AP-1 blockade in adult, prepubertal, pubertal, and hormone-stimulated mammary glands. In pubertal mice, mammary cell proliferation was greatly reduced, and the cells that did proliferate failed to express Tam67. We also observed structural changes such as suppressed branching and budding, reduced gland tree size, and less fat pad occupancy in developing mammary glands after AP-1 blockade. We further demonstrated that Tam67 suppressed the expression of AP-1-dependent genes (TIMP 1, vimentin, Fra-1, and fibronectin) and the AP-1-dependent growth regulatory genes (cyclin D1 and c-myc) in AP-1-blocked mammary glands. We therefore conclude that AP-1 factor is a pivotal regulator of postnatal mammary gland growth and development. PMID- 16678817 TI - Protective effect of protocatechuic acid from Alpinia oxyphylla on hydrogen peroxide-induced oxidative PC12 cell death. AB - The neuroprotective effects of protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, on hydrogen peroxide (H(2)O(2)) induced apoptosis and oxidative stress in cultured PC12 cells were investigated. Exposure of PC12 cells to 0.4 mM H(2)O(2) induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCA increased PC12 cellular viability and markedly attenuated H(2)O(2)-induced apoptotic cell death in a dose-dependent manner. By flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against H(2)O(2)-induced apoptosis. In these cells, the levels of glutathione (GSH) and activity of catalase were augmented, while glutathione peroxidase activity remained unchanged. In addition, PCA also protected against cell damage induced by H(2)O(2) and Fe(2+), which generated hydroxyl radicals (OH) by the Fenton reaction. These results suggest that PCA may be a candidate chemical for the treatment of oxidative stress induced neurodegenerative disease. PMID- 16678818 TI - Epidural cortical stimulation enhances motor function after sensorimotor cortical infarcts in rats. AB - This study examined whether epidurally delivered cortical electrical stimulation (CS) improves the efficacy of motor rehabilitative training and alters neuronal density and/or cell proliferation in perilesion cortex following ischemic sensorimotor cortex (SMC) lesions. Adult rats were pre-trained on a skilled reaching task and then received partial unilateral SMC lesions and implantation of electrodes over the remaining SMC. Ten to fourteen days later, rats received daily reach training concurrent with anodal or cathodal 100 Hz CS or no stimulation (NoCS) for 18 days. To label newly generated cells, bromodeoxyuridine (BrdU; 50 mg/kg) was administered every third day of training. Both anodal and cathodal CS robustly enhanced reaching performance compared to NoCS controls. Neuronal density in the perilesion cortex was significantly increased in the cathodal CS group compared to the NoCS group. There were no significant group differences in BrdU-labeled cell density in ipsilesional cortex. Staining with Fluoro-Jade-B indicated that neurons continue to degenerate near the infarct at the time when cortical stimulation and rehabilitation were initiated. These data indicate that epidurally delivered CS greatly improves the efficacy of rehabilitative reach training following SMC damage and raise the possibility that cathodal CS may influence neuronal survival in perilesion cortex. PMID- 16678819 TI - Detecting transient protein-protein interactions by X-ray absorption spectroscopy: The cytochrome c6-photosystem I complex. AB - Reliable analysis of the functionality of metalloproteins demands a highly accurate description of both the redox state and geometry of the metal centre, not only in the isolated metalloprotein but also in the transient complex with its target. Here, we demonstrate that the transient interaction between soluble cytochrome c(6) and membrane-embedded photosystem I involves subtle changes in the heme iron, as inferred by X-ray absorption spectroscopy (XAS). A slight shift to lower energies of the absorption edge of Fe2+ in cytochrome c6 is observed upon interaction with photosystem I. This work constitutes a novel application of XAS to the analysis of weak complexes in solution. PMID- 16678820 TI - Congenital interruption of the ampullary portion of the fallopian tube. AB - We present a rare case of a congenital isolated missing segment of the fallopian tube, including hysterosalpingographic and laparoscopic images. We conclude that when this occurs without concomitant mullerian anomalies, the mechanism of development would not be expected to be associated with an increase in renal abnormalities. PMID- 16678821 TI - Multiple micromanipulations for preimplantation genetic diagnosis do not affect embryo development to the blastocyst stage. AB - To determine the impact of multiple micromanipulation procedures for preimplantation genetic diagnosis (PGD) on embryo development, a retrospective analysis was performed of 9,925 embryos (862 PGD cycles), which were compared with 28,126 nonbiopsied embryos (2,751 consecutive intracytoplasmic sperm injection [ICSI] cycles) from the same time period. Because fertilization rates, the proportion of embryos with > or = 6 cells on day 3, and blastocyst rates were similar in the PGD and control groups, we conclude that multiple micromanipulations on oocytes and embryos can be performed safely for PGD. PMID- 16678822 TI - Detection of an inguinal ovary at controlled ovarian stimulation that was successfully treated by repositioning. AB - OBJECTIVE: To report a rare case of an ectopic ovary placed in the inguinal canal that was detected while performing a controlled ovarian hyperstimulation (COH). DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A couple with primary infertility for 4 years was referred to our infertility clinic. The woman's medical history revealed a left inguinal operation at age 7. On vaginal ultrasound, only the right ovary could be seen. An infertility workup conducted for the man revealed teratospermia. The couple was subsequently admitted to the in vitro fertilization (IVF) program. While having a COH, the woman experienced a painful swelling in the inguinal area, and an ovarian image with follicular growth on the left inguinal region was observed with ultrasound. Afterward, surgery was performed, and the ectopic ovary in the left inguinal region was detected. INTERVENTION(S): Detection of an inguinal ovary with a controlled ovarian hyperstimulation procedure and surgical repositioning of the ectopic ovary. MAIN OUTCOME MEASURE(S): Controlled ovarian hyperstimulation, transabdominal ultrasound, transvaginal ultrasound. RESULT(S): The ectopic ovary was successfully repositioned with surgery. CONCLUSION(S): Patients must be closely monitored while performing COH. In patients who do not have a unilateral ovary, a painful inguinal mass should alert the physician to the possible presence of an ectopic ovary in the inguinal canal. PMID- 16678823 TI - Sex differences in otoacoustic emissions measured in rhesus monkeys (Macaca mulatta). AB - Click-evoked otoacoustic emissions (CEOAEs) and distortion-product OAEs (DPOAEs) were measured in about 60 rhesus monkeys. CEOAE strength was substantially greater in females than in males, just as in humans. DPOAE strength was generally slightly stronger in females, just as in humans. In males, CEOAEs were weaker (more masculine) in the fall breeding season and in winter than in the summer. In females, CEOAEs were slightly stronger (more feminine) in the fall, when sex steroids are elevated in females (and males), than in the summer when rhesus monkeys are reproductively quiescent. Thus, the sex differences in CEOAEs were greater in the fall than in the summer. We presume that the seasonal fluctuations in OAEs reflect activational hormonal effects, while the basic sex differences in OAEs likely reflect organizational effects of prenatal androgen exposure. Some monkeys of both sexes had been treated with additional testosterone or the anti androgen flutamide during prenatal development. In accord with expectations, prenatal androgen treatment weakened CEOAEs in females, and prenatal flutamide treatment strengthened CEOAEs in males. For DPOAEs, the differences between treated and untreated groups were mostly small and often inconsistent. Taken as a whole, the data from both rhesus monkeys and humans suggest that the linear, reflection-based mechanism of OAE production that underlies CEOAEs is more sensitive to prenatal androgenic processes than is the nonlinear distortion mechanism that underlies DPOAEs. PMID- 16678824 TI - Anemia in early pregnancy among Canadian women presenting for abortion. PMID- 16678825 TI - Posterior hemidissection for nonpuerperal uterine inversion. PMID- 16678826 TI - Uterine massage and postpartum blood loss. PMID- 16678827 TI - Postsurgical post-hysterectomy abdominal wound dehiscence treated with Negative Pressure Wound Therapy. PMID- 16678828 TI - The role of parity on neonatal respiratory outcome in patients with preterm premature rupture of membranes. PMID- 16678829 TI - Birth weight at high altitudes in Peru. AB - OBJECTIVE: To determine whether birth weights are lower at high altitudes, and whether gestational age at birth and a population's length of residence mitigate the effect of high altitude. METHODS: The birth weights of 84,173 neonates recorded in the Peruvian Perinatal Information System Database were analyzed between 1995 and 2002 for the cities of Lima (150 m), Huancayo (3280 m), Cuzco (3400 m), and Juliaca (3800 m). RESULTS: Birth weight was lower at high altitude, but there was no linear relation between altitude of residence and birth weight. Mean birth weight was higher in Juliaca than in Huancayo. There were no significant differences between the 4 cities regarding birth weights of infants born between 28 and 35 weeks of gestation. However, for infants born between 36 and 42 weeks, birth weight was lower at higher altitudes. This may be due to inadequate maternal oxygenation later in pregnancy at high altitude. In the multivariate analysis, after controlling for maternal age, marital status, parity, body mass index, pre-eclampsia or hemorrhage during pregnancy, and education, as well as sex of the newborn and gestational age at birth, birth weight was lower in all cities located at a higher altitude than Lima. Yet, longer residence at high altitudes may play a protective role. Juliaca (3800 m), where the population has resided the longest, had the lowest reduction in birth weight compared with Lima (150 m); Cuzco had intermediate values; and Huancayo (3280 m), where the population has resided the shortest, had the highest reduction in birth weight. CONCLUSIONS: Birth weight reduction, which is independent of socioeconomic factors, occurs only in births at term and may be less severe in populations that have resided longer at high altitudes. PMID- 16678830 TI - Inhibin A level in early pregnancy following in-vitro fertilization and embryo transfer. PMID- 16678831 TI - Determinants and definition of abdominal obesity as related to risk of diabetes, metabolic syndrome and coronary disease in Turkish men: a prospective cohort study. AB - We aimed to investigate determinants of abdominal obesity and its clinical impact on metabolic syndrome (MS), diabetes (DM) and coronary heart disease (CHD) in men. METHODS: Prospective evaluation of 1638 male participants (aged 48.5+/ 12.3), representative of Turkey's men who have a high prevalence of MS. For components of MS, criteria of NCEP guidelines were adopted, modified for abdominal obesity. Follow-up constituted 9650 person-years. RESULTS: Insulin level (relative risk [RR] 1.40 for doubling), C-reactive protein (CRP) and heavy smoking (protective) were independent predictors of newly developing abdominal obesity. High triglyceride and low HDL-cholesterol were significantly associated already with waist girth quartile II, apolipoprotein B with quartile III. Waist girth significantly predicted future MS from quartile II on, independent of insulin resistance (IR) by homeostatic model assessment, whereby its hazard ratio (HR, 2.6) exceeded double that of HOMA. CRP independently predicted MS. Age adjusted HR of waist girth (1.59) was significant in predicting DM. Age- and smoking-adjusted top waist quartile conferred significant risk for incident CHD (RR 1.71) but not for overall mortality. As judged by sensitivity and specificity rates for future CHD, DM and MS, abdominal obesity was most appropriately defined with a waist girth of >or=95 cm, and an action level 1 of >or=87 cm was proposed for MS in this population. CONCLUSIONS: Serum insulin, CRP levels and (inversely) heavy smoking are predictors for abdominal obesity in Turkish men. Atherogenic dyslipidemia and elevated blood pressure are associated significantly already with modest rises in waist girth adjusted for age and smoking. Abdominal obesity shows substantial independence of IR in the development of MS. Increasing waist girth was predictive of MS, more strongly than of DM. Risk for CHD imparted by abdominal obesity is essentially mediated by risk factors it induces. PMID- 16678832 TI - A genome scan for loci influencing levels and trends of lipoprotein lipid-related traits since childhood: The Bogalusa Heart Study. AB - Coronary heart disease is the result of life-long processes. Previous genetic linkage analyses of lipid and lipoprotein variables that can be measured throughout life have focused on a single measure at one point in time. Genome wide linkage analyses were performed in the present study to identify loci influencing the long-term levels and trends of high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC) and triglycerides in a longitudinal cohort. Microsatellite markers (n=357) were typed on 779 white and 444 black siblings, ages 14-43 years. Subjects had been examined serially 2-13 times with 6963 serial observations over an average of 22 years from childhood to adulthood. Total and incremental area under the growth curves of lipid traits was calculated and used as measures for long-term levels and trends. After adjusting for age, sex and body mass index, heritability estimates of total area values for all lipid variables were higher than those of a single measurement in either childhood or adulthood. In blacks, significant linkage to LDLC incremental area (peak LOD=3.6 at 50 cM) was observed on chromosome 1; and suggestive linkage for total area of LDLC (LOD=2.9 at 21 cM) on chromosome 19. Only one suggestive linkage (LOD=2.2 at 161 cM) on chromosome 2 was identified in whites for LDLC incremental area. Other suggestive linkage (LOD> or =2.0) was noted for LDLC and HDLC in terms of either total or incremental area on chromosomes 2, 5, 7 and 15 for blacks and whites. Several lipid-related candidate genes such as low-density lipoprotein receptor (LDLR), LDL receptor-related proteins 3 and 8, ApoE, ApoAII and ApoCII are located in these regions. Linkage evidence found in this community-based study indicates that regions on these chromosomes harbor genetic loci that affect the propensity to develop dyslipidemia from childhood. PMID- 16678833 TI - HSF1 and constitutively active HSF1 improve vascular endothelial function (heat shock proteins improve vascular endothelial function). AB - We have been examining the role of heat shock factor 1 (HSF1) in the pleiotropic effects of statins. In parallel studies, we found that statin induces the nuclear translocation of HSF1 and that a decoy oligonucleotide encoding the heat shock element inhibits the statin-induced expression of heat shock protein 70, endothelial nitric oxide synthase (eNOS) and thrombomodulin. Also, in vascular endothelial cells, increases in the expression of human HSF1 corresponded with elevated steady-state levels of eNOS and thrombomodulin and reduced levels of endothelin-1 and plasminogen activator inhibitor-1. We also found that heat shock proteins induced eNOS and thrombomodulin expression and reduced PAI-1 and ET-1 expression. In particular, a combination of HSP70 and HSP90 strongly induced eNOS expression and reduced PAI-1 expression. In the current studies, we generated a constitutively active form of HSF1 and found that it is more effective than the wild-type HSF at inducing thrombomodulin and eNOS expression and decreasing endothelin-1 and plasminogen activator inhibitor-1 expression. These results show that the wild-type and constitutively active forms of HSF1 induce anticoagulation and relaxation factors in vascular endothelial cells and could therefore be used to treat cardiovascular disease. PMID- 16678834 TI - Patient-specific model of brain deformation: application to medical image registration. AB - This contribution presents finite element computation of the deformation field within the brain during craniotomy-induced brain shift. The results were used to illustrate the capabilities of non-linear (i.e. accounting for both geometric and material non-linearities) finite element analysis in non-rigid registration of pre- and intra-operative magnetic resonance images of the brain. We used patient specific hexahedron-dominant finite element mesh, together with realistic material properties for the brain tissue and appropriate contact conditions at boundaries. The model was loaded by the enforced motion of nodes (i.e. through prescribed motion of a boundary) at the brain surface in the craniotomy area. We suggest using explicit time-integration scheme for discretised equations of motion, as the computational times are much shorter and accuracy, for practical purposes, the same as in the case of implicit integration schemes. Application of the computed deformation field to register (i.e. align) the pre-operative images with the intra-operative ones indicated that the model very accurately predicts the displacements of the tumour and the lateral ventricles even for limited information about the brain surface deformation. The prediction accuracy improves when information about deformation of not only exposed (during craniotomy) but also unexposed parts of the brain surface is used when prescribing loading. However, it appears that the accuracy achieved using information only about the deformation of the exposed surface, that can be determined without intra operative imaging, is acceptable. The presented results show that non-linear biomechanical models can complement medical image processing techniques when conducting non-rigid registration. Important advantage of such models over the previously used linear ones is that they do not require unrealistic assumptions that brain deformations are infinitesimally small and brain stress-strain relationship is linear. PMID- 16678835 TI - Multielemental fractionation in pine nuts (Pinus pinea) from different geographic origins by size-exclusion chromatography with UV and inductively coupled plasma mass spectrometry detection. AB - Pine nuts (Pinus pinea) from different geographical origin in Spain and Portugal have been investigated concerning total element content and metal-biomolecules size distribution patterns Mn, Zn, Ni and Cu. All the studied metals were at the highest concentration in pine nuts from Faro and at the lowest from Cataluna. The most abundant element in samples was Mn at concentrations in the range of 26 microg g(-1) (Cataluna) to 559 microg g(-1) (Faro). Zn was also present at high concentration in samples, from 25 microg g(-1) (Cataluna) to 113 microg g(-1) (Faro). To a deeper insight to obtain classification rules for samples, pine nuts were analyzed by size-exclusion chromatography (SEC) with UV detection and inductively coupled plasma mass spectrometry (ICP-MS). Two columns were used covering the molecular weigh range from < 10 to 70 kDa that allowed the discrimination of the studied samples. Data reveal that the most differential UV profile with low molecular weight (LMW) column was obtained with pine nuts from Huelva. This column allows good discrimination in the range of 2126-1352 Da in which a lot of peaks can be used to differentiate samples. The UV profiles obtained with the high molecular weight (HMW) column allows a poorer differentiation of samples, but pine nuts from Huelva, Castilla and Madrid are clearly distinguished to the others. In relation to fractionation patterns of metals, Mn allows a good discrimination between samples (LMW column), Cu was the only one associated to fractions at MW > 70 kDa in sample from Cadiz, and profiles of Ni and Zn are clearly different in terms of abundance of peaks. All these chromatographic profiles for elements give valuable information about the geographical origin of the studied samples and the differences found are discussed in this work. PMID- 16678836 TI - beta-Cyclodextrin as a stationary phase for the group separation of polycyclic aromatic compounds in normal-phase liquid chromatography. AB - The separation of highly alkylated polycyclic aromatic compounds according to the size of their aromatic system is investigated using the polycyclic aromatic sulfur heterocycles in vacuum gas oil. A large number of reference compounds containing several parent ring systems and different alkylation patterns were first investigated to characterize the retention of polycyclic aromatic compounds likely to occur in high-boiling petroleum samples. A beta-cyclodextrin phase, Merck ChiraDex, was found to be more suitable than chemically bonded aminopropanosilane and tetrachlorophthalimide in normal-phase HPLC with respect to a combination of selectivity towards the number of aromatic double bonds and degree of influence of the alkyl groups of the aromatic compounds. Finally the preseparated polycyclic aromatic sulfur heterocycles from a vacuum gas oil were fractionated according to the number of condensed aromatic rings on the ChiraDex phase and were characterized by Fourier transform ion cyclotron resonance mass spectrometry. PMID- 16678837 TI - Automated analysis of 2-methyl-3-furanthiol and 3-mercaptohexyl acetate at ng L( 1) level by headspace solid-phase microextracion with on-fibre derivatisation and gas chromatography-negative chemical ionization mass spectrometric determination. AB - A fast and automated method for the analysis at ng L(-1) level of aroma-powerful polyfunctional thiols has been developed and applied to wine. The sample is just poured in a 20 mL vial and its vapour extracted with a poly(dimethylsiloxane) divinylbenzene (PDMS-DVB) solid-phase microextraction fibre (65 microm thickness) previously exposed to vapours of the reactive (pentafluorobenzyl bromide) and of an alkali (tributylamine). The derivatised compounds are subsequently desorbed in the GC system and determined by negative chemical ionization mass spectrometry. The method is fully automated by using a Combi-Pal autosampler conveniently programmed. The analysis takes 50 min, which contrasts to the long and tedious methods previously proposed. The development of an optimal procedure is constrained by the aggressive character of the reagent (towards the fibre and the chromatographic column), its volatility and the quality of the blanks that can be obtained. Therefore, a critical step was fixing in the fibre a "safe" and repetitive amount of reagent. This was achieved by exposing the fibre (5 min) to the vapours of a water:acetone (9:1) solution containing 200mg L(-1) of reagent. Under these conditions, the extraction-derivatisation of analytes improves with time and temperature, and the best working conditions are dictated by a compromise between sensitivity, speed and chromatographic performance. Although analytes studied were 2-methyl-3-furanthiol, 4-mercapto-4-methyl-2-pentanone, 3 mercaptohexanol, 2-furanmethanethiol and 3-mercaptohexyl acetate, a good analytical performance could be achieved only for these two last compounds. Both of them can be repetitively (10%40,000 CFU/m(3) in both plants. Strains of Escherichia coli, Clostridium perfringens, Staphylococcus aureus and Enterococcus spp. were isolated in some sites of the two plants. Salmonella spp., Yersinia enterocolitica and Legionella spp. were never detected. The activities involving nebulization and mechanical aeration of wastewaters and the sewage inflows have proved to be of greatest potential risk. In both plants, we found a statistically significant dependence of bacterial contamination on the season for many of the analyzed parameters but a clear seasonal trend could not be observed. PMID- 16678883 TI - Treatment of olive oil mill wastewater by combined process electro-Fenton reaction and anaerobic digestion. AB - In this work, we investigated an integrated technology for the treatment of the recalcitrant contaminants of olive mill wastewaters (OMW), allowing water recovery and reuse for agricultural purposes. The method involves an electrochemical pre-treatment step of the wastewater using the electro-Fenton reaction followed by an anaerobic bio-treatment. The electro-Fenton process removed 65.8% of the total polyphenolic compounds and subsequently decreased the OMW toxicity from 100% to 66.9%, which resulted in improving the performance of the anaerobic digestion. A continuous lab-scale methanogenic reactor was operated at a loading rate of 10 g chemical oxygen demand (COD)l(-1) d(-1) without any apparent toxicity. Furthermore, in the combined process, a high overall reduction in COD, suspended solids, polyphenols and lipid content was achieved by the two successive stages. This result opens promising perspectives since its conception as a fast and cheap pre-treatment prior to conventional anaerobic post-treatment. The use of electro-coagulation as post-treatment technology completely detoxified the anaerobic effluent and removed its toxic compounds. PMID- 16678884 TI - Abiotic transformation of DDT in aqueous solutions. AB - Significant concentrations of chlorinated pesticides such as 1,1,1-trichloro-2,2 bis(4-chlorophenyl)ethane (DDT) and its two main transformation products, 1,1 dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) and 1,1-dichloro-2,2-bis(4 chlorophenyl)ethylene (DDE) are still present in soil and sediment systems more than 30 years after DDT use was banned in the United States. DDT enters waterways via the runoff from industrial point sources, agricultural lands and atmospheric deposition. We evaluated zero-valent iron (Fe(0)), ferrous sulfide (FeS), as well as combining them with hydrogen peroxide (H(2)O(2)) as viable treatment technologies for degrading DDT in an aqueous solution. Treatment of DDT with Fe(0) and FeS resulted in approximately 88% and 56% transformation of DDT within 150h, respectively. DDE production was insignificant in all systems. The DDT removal was slower with FeS than with Fe(0), but the amounts of DDD and DDE produced did not exceed baseline. Treatment with a 1:1 mixture of Fe(0)-FeS removed about 95% of the added mass of DDT within 4days and generated significant amounts of DDD and minor amounts of DDMU. When small amounts of H(2)O(2) were introduced halfway through the Fe(0) and FeS treatment times, the mass of DDT decreased by 87% and 96%, respectively, within 2days. Our results demonstrate that mixtures of Fe(0)-FeS in combination with H(2)O(2) can be used for rapid and efficient removal of DDT from aqueous solutions. PMID- 16678885 TI - Patterning of geographic variation in Middle Pleistocene Homo frontal bone morphology. AB - A quantitative assessment of the frontal bone morphology of a sample of Middle Pleistocene hominins was undertaken in order to address questions regarding their population structure and evolutionary history. Outline tracings of the frontal bones of forty-seven fossil crania were obtained, and size-standardized measurements were then computed using an Elliptical Fourier analysis of these tracings. Principal component scores of the Fourier harmonic amplitudes were derived and served as a quantitative representation of the morphology of the frontal bone. Morphological, geographical, and temporal distance matrices were then constructed between each pair of fossils. A partial Mantel matrix correlation test was performed between morphological and geographical distance matrices, controlling for temporal distance, in order to determine if the pattern of geographical differentiation in features of the frontal bone of mid Pleistocene Homo followed that of an isolation-by-distance model of population structure. The results of the partial Mantel tests indicate that the overall patterning of differentiation in the features of the frontal bone cannot best be explained by a population structure shaped by isolation-by-distance. Additionally, various aspects of the frontal bone quantified here follow different patterns of geographical differentiation, suggesting that a mosaic pattern of evolution holds true for characters within one cranial region and not just for those between regions. PMID- 16678886 TI - The impact of sampling techniques on soil pore water carbon measurements of an Icelandic Histic Andosol. AB - The carbon in soil pore water from a Histic Andosol from Western Iceland was studied at three different scales; in the field, in undisturbed outdoor mesocosms and in laboratory repacked microcosms. Pore water was extracted using suction cup lysimeters and hollow-fibre tube sampler devices (Rhizon samplers). There were significant differences in all measured variables, dissolved inorganic carbon (DIC), dissolved organic carbon (DOC) and pH values between the scales of the experiment. Gaseous constituents of soil solution and pH were more susceptible to changes in scale and the type of sampling devices used. Dissolved inorganic carbon concentrations did not differ significantly between field and mesocosm solutions but where up to 14 times lower in microcosms compared to mesocosms solutions. Rhizon samplers yielded solutions with up to 4.7 times higher DIC concentrations than porous cup lysimeters. Mesocosm surface horizon DOC concentrations were 20 and 2 times higher than in field and microcosms respectively. There was difference in DOC concentration between sampling methods (up to 8 times higher in suction cups than rhizon samplers) above 50 cm depth. Soil solution pH values did not differ between field and mesocosms and mesocosms and microcosms respectively down to 80 cm depth. Direct comparison between field and microcosms was not possible due to the nature of sampling devices. Soil solutions sampled with Rhizon samplers yielded lower pH values (up to 1.3 pH units) than those sampled with suction cups. Twenty percent of annually bound organic carbon at the soils surface under field conditions was lost by leaching of DOC and through decomposition to DIC in disturbed non-vegetated microcosms. This percentage increased to 38% in undisturbed vegetated mesocosms highlighting the importance of surface vegetation in importing carbon to soils. Increased influx of nutrients will increase growth and photosynthesis but decrease carbon sequestration in near surface horizons. Although field studies considering long term anthropogenic changes in pedogenesis require considerable experimental duration, more rapid experiments can be conducted with confidence in micro- and mesocosms as in this research. PMID- 16678887 TI - Comparison of operative and functional outcomes of laparoscopic radical prostatectomy and radical retropubic prostatectomy: single surgeon experience. AB - OBJECTIVES: To compare radical retropubic prostatectomy (RRP) and laparoscopic radical prostatectomy (LRP) outcomes in a contemporary series. METHODS: A total of 70 LRP patients operated on between 2001 and 2002 with at least 18 months of follow-up were selected. These patients were compared with a matched cohort of 70 patients who had undergone RRP by the same surgeon from 1999 to 2001. The baseline patient characteristics, perioperative and histologic parameters, recovery time, complications, and 18-month functional data were compared. RESULTS: No significant differences were found in the preoperative characteristics. The mean operative time was 181.8 +/- 18.7 minutes for RRP and 246.4 +/- 46.1 minutes for LRP (P <0.0001). The mean estimated blood loss was 563.2 mL for RRP and 275.8 mL for LRP (P <0.0001). The positive margin rate was 20% and 15.7% for the RRP and LRP groups, respectively (P = NS). The mean pain score on postoperative day 1 was 4.5 in the LRP group and 7.8 in the RRP group on an analog pain score of 0 to 10 (P = 0.02). Full recovery was achieved at 33 +/- 17 days and 45 +/- 20 days for the LRP and RRP groups, respectively (P <0.001). The total perioperative complication rate for LRP and RRP was comparable at 18.5% and 15.7%, respectively. The diurnal continence rate (no pads) for the LRP and RRP groups was 70%, 90%, and 92.8% and 71.4%, 87.6%, and 92% at 6, 12, and 18 months, respectively (P = NS). The potency rate after bilateral neurovascular preservation with or without sildenafil for the LRP and RRP group was 55%, 72.6%, and 79.5% and 43%, 58%, and 72.4% at 6, 12, and 18 months, respectively (P = NS). CONCLUSIONS: LRP is well tolerated and provides short-term oncologic and functional results comparable to those of RRP. PMID- 16678888 TI - Radical prostatectomy as primary treatment modality for locally advanced prostate cancer: a prospective analysis. AB - OBJECTIVES: Locally advanced prostate cancer is frequently treated with radiotherapy and androgen deprivation because of the greater rate of extracapsular disease and the concern that radical prostatectomy (RP) may not be curative in most cases. A case for surgery for locally advanced disease may be made on the basis of a lower rate of local recurrence compared with radiotherapy in our comparative database, data suggesting a survival advantage with pelvic lymph node dissection in those with positive nodes, and the observation of improved survival in those with metastatic disease treated by RP compared with radiotherapy. We report on the feasibility of RP as a primary treatment modality for locally advanced disease. METHODS: A total of 281 consecutive patients treated by RP between January 1998 and June 2004 were reviewed. Locally advanced disease was defined as clinical Stage T2b or worse, prostate-specific antigen level greater than 15 ng/mL, and/or a Gleason score of 8 or greater. Data on the pathologic characteristics, operative complications, and follow-up were obtained from a prospectively maintained institutional review board-approved database. RESULTS: Pathologic examination demonstrated organ-confined disease in 11.7%, extracapsular extension in 56.9%, seminal vesicle involvement in 23.1%, and positive lymph nodes in 8.9%. The overall complication rate was 9.7% compared with 6.9% for all patients undergoing RP. At a mean follow-up of 34 months (range 1 to 78), 198 (70.4%) of 281 patients had an undetectable prostate-specific antigen level at the last follow-up examination. CONCLUSIONS: RP for locally advanced prostate cancer is feasible, with acute morbidity similar to RP for more localized disease. Furthermore, RP results in short-term biochemical recurrence free survival similar to that of combined radiotherapy and androgen ablation. PMID- 16678889 TI - In vitro functional responses of isolated normal human prostatic tissue to compounds interacting with the cyclic guanosine monophosphate pathway. AB - OBJECTIVES: To examine the effects of some nitric oxide-donating agents, as well as the C-type natriuretic peptide (CNP), on isolated human prostatic tissue. To date, guanylyl cyclases and cyclic guanosine monophosphate (cGMP)-degrading phosphodiesterases represent important target proteins for the development of new drugs for the treatment of lower urinary tract symptoms and benign prostatic hyperplasia. METHODS: Using the organ bath technique, the effects of sodium nitroprusside, S-nitrosoglutathione, S-nitrosocysteine, linsidomine, and CNP (1 nM to 1.0/10 microM) on the tension induced by norepinephrine of prostatic tissue strips were investigated. The tissue was also exposed to three different concentrations of the drugs, and the production of cGMP and cyclic adenosine monophosphate (cAMP) was determined. RESULTS: The tension induced by 40 microM norepinephrine of the isolated prostatic tissue was dose dependently reversed by the drugs. The rank order of potency was sodium nitroprusside more than S nitrosoglutathione more than linsidomine more than S-nitrosocysteine, which was equal to CNP (1 microM). The reversal of tension induced by the greatest drug concentrations ranged from 50% relaxation with sodium nitroprusside to 42% relaxation with CNP. The relaxing effects of the drugs were paralleled by a 2 fold to 40-fold and 2-fold to 45-fold increase in tissue levels of cAMP and cGMP, respectively. CONCLUSIONS: Our results provide further evidence that cGMP and cAMP are involved in the control of the normal function of human prostatic smooth muscle. Our findings may provide new strategies for future therapeutics used in the treatment of lower urinary tract symptoms and bladder outlet obstruction secondary to benign prostatic hyperplasia. PMID- 16678890 TI - Transcutaneous high-intensity focused ultrasonography can cure testicular cancer in solitary testis. AB - OBJECTIVES: To report the long-term results in 7 patients (including the 5-year results in 3 patients) after high-intensity focused ultrasonography (HIFU) combined with irradiation to treat testicular tumors in a solitary testis. METHODS: Transcutaneous HIFU ablation of testicular tumors is based on a technique using a piezoceramic transducer operating at 4.0 MHz with a site intensity of 1600 to 2000 W/cm2. In a Phase II trial, 7 patients with the typical sonographic pattern of a tumor in a solitary testis were treated with transcutaneous HIFU, as a minimally invasive organ-preserving approach, followed 6 weeks later by prophylactic testicular irradiation (range 18 to 20 Gy). The aim was to ablate the entire cancer in a single therapeutic HIFU session. In all 7 patients, the contralateral testis had previously been removed because of testicular cancer. RESULTS: One patient received two cycles of chemotherapy for a single suspicious retroperitoneal lymph node diagnosed 6 months after HIFU. The other 6 protocol-treated patients remained tumor free at a mean follow-up of 42 months (range 3 to 93). One patient, who had refused postoperative irradiation, developed a recurrent tumor within 6 months. No patient showed any signs of clinical hypogonadism, and the International Index of Erectile Function score was normal for all patients. No androgen substitution was necessary. The only adverse effect noted was a small thermal lesion of the scrotum in 1 patient. CONCLUSIONS: Despite the lack of tumor histologic examination, transcutaneous HIFU followed by irradiation permits a minimally invasive, organ-preserving, curative treatment for tumors in a solitary testis. PMID- 16678891 TI - Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. AB - OBJECTIVE: Currently available clinical and molecular factors provide still an insufficient prognostic and predictive assessment for patients with epithelial ovarian cancer (EOC). To identify a potential molecular target and prognostic/predictive factor for EOC, we investigated in a retrospective study the prognostic value of Ep-CAM overexpression in EOC. METHODS: We assessed by immunohistochemistry the expression of the Ep-CAM antigen on tissue microarrays containing paraffin-embedded tissue samples of 199 patients with documented EOC. Patients were operated for ovarian cancer in the period between June 1980 and January 2000. RESULTS: We observed a rate of Ep-CAM overexpression of 68.8%. Ep CAM overexpression was significantly related to a decreased overall survival (P = 0.036). The prognostic power of Ep-CAM overexpression was particularly strong in patients with stage III and IV disease. In fact, in this subgroup, median overall survival was twofold higher in patients without as compared to patients with Ep CAM overexpression (46 vs. 23 months, P < 0.01). Univariate analysis revealed a correlation with histologic grade. We observed a significantly higher rate of Ep CAM overexpression (83.5%) in grade 3 tumors. Histologic subtypes associated with a higher rate of Ep-CAM overexpression were serous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, clear cell carcinoma, and endometrioid carcinoma. Cox regression analysis showed Ep-CAM overexpression to be an independent prognostic marker (P = 0.037, RR = 1.64). CONCLUSIONS: This retrospective analysis demonstrates for the first time an independent prognostic value of Ep-CAM overexpression in patients with EOC. Ovarian cancer patients with Ep-CAM overexpressing tumors are frequent and would qualify for treatment with Ep CAM-specific immunotherapeutic approaches. PMID- 16678892 TI - Factor analysis of elevated plus-maze behavior in adolescent and adult rats. AB - The elevated plus-maze (EPM) is among the most popular behavioral models of anxiety. While numerous experiments have validated this test hormonally, pharmacologically, and with factor analysis in adult rodents, few studies have explored use of the EPM to examine ontogenetic differences in anxiety. Given the growing interest in adolescence and the mixed findings to date regarding age related differences in anxiety, validation of the EPM model for use in adolescence is important. Therefore, the present experiment employed factor analysis to examine underlying EPM behavioral components in adolescent and adult male and female Sprague-Dawley rats across three separate data sets. Results of the analyses conducted across both age and sex produced a 3-factor solution, with the primary component of EPM behavior consisting of anxiety-related behaviors in both adolescent and adult males and females. Within the age analyses, the second and third factors were comprised largely of activity- and risk-related behaviors, respectively. Sex analyses revealed a similar pattern in females, with some behaviors comprising the second and third factors reversed in males. Taken together these results confirm use of the EPM in adolescent and adult rats and demonstrate slight differences in the underlying components of EPM behavior in males versus females. PMID- 16678893 TI - Willingness-to-pay for a population program aimed at reducing dietary salt in Denmark. AB - BACKGROUND: High intake of salt increases blood pressure and the risk of cardiovascular disease. Population programs aimed at reducing dietary salt may be cost-effective, but little is known about people's valuation of such programs. METHODS: A random sample (n = 924) of Danish people aged 20-74 was interviewed in person in year 2000. They were asked how much they would be willing to contribute in additional tax for a population program that would half the intake of salt. The respondents were randomized to presentation of effectiveness format (number needed-to-treat (NNT), relative risk reduction (RRR), avoided cases of heart attack or increase in longevity). RESULTS: In total, 57% were unwilling to contribute to the program (63%, 51%, 51% and 63% in the NNT-, RRR-, case- and longevity-groups, respectively). The mean willingness-to-pay among the remaining 43% was $30 per person per month. While in Denmark, the annual net program cost would be $148 millions in the most unfavorable scenario, the aggregate willingness-to-pay was $468 millions. CONCLUSIONS: A public health program may be worthwhile even though only a minority of the population is willing to contribute towards it. People's valuation is moderately sensitive to format of the program information. PMID- 16678894 TI - Relationship between components of leisure physical activity and mortality in Taiwanese older adults. AB - BACKGROUND: Whether the major components of leisure physical activity (number of types, intensity, frequency, and duration) have independent contribution to mortality reduction and there is a minimum amount of activity beneficial for the elderly remain unclear. METHODS: The prospective follow-up study aimed at examining the relationship between exercise components and total mortality in general elderly population. A total of 2113 persons aged 65 and older participating in 2001 Taiwan National Health Interview Survey were studied. Information regarding leisure physical activity, its energy parameters, other factors at baseline, and vital status at the end of 2003 was analyzed with Cox model. RESULTS: A total of 197 deaths occurred during a 2-year follow-up. Regular exercisers reduced 35% risk of death compared with sedentary individuals after adjustment for covariates. Moreover, exercisers with a weekly amount of energy exceeding 1000 kcal had significant benefit of risk reduction when energy expenditure is considered. There was a significant dose-response relationship between number of activity and the reduction in total mortality. The benefit on mortality reduction among the three components of total energy amount was only observed in intensity. CONCLUSIONS: For the amount of energy dedicated to leisure physical activity, older persons are recommended to expend at least 1000 kcal per week through regular exercise for mortality reduction. In addition to energy amount, protection of exercise against death also increases with the number of activities. Among the three components of total amount of energy, only intensity is significantly associated with mortality reduction. PMID- 16678895 TI - Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity. AB - OBJECTIVES: Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. METHODS: PCPCs were formulated from CPC powder+non-aqueous liquid+gelling agent+hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. RESULTS: Setting time (mean+/-S.D.; n=4) for PCPC-Tartaric was 8.2+/-0.8 min, significantly less than the 61.7+/-1.5 min for the Premixed Control developed previously (p<0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5+/ 0.8 MPa, higher than 4.5+/-0.8 MPa of Premixed Control (p=0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p=0.126), indicating that the new PCPCs were as cell compatible as a non premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p>0.1) from that of the non premixed CPC control. SIGNIFICANCE: PCPCs will eliminate the powder-liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs. PMID- 16678896 TI - Assessment of feto-fetal transfusion flow through placental arterio-venous anastomoses in a unique case of twin-to-twin transfusion syndrome. AB - In vivo measurements of blood flow through arterio-venous anastomoses in monochorionic twin placentas have recently been attempted with Doppler ultrasound, but the accuracy is questionable. We present a case of twin-to-twin transfusion syndrome treated with fetoscopic laser surgery. The ex-recipient subsequently became severely anaemic and was treated with an intrauterine blood transfusion at 29 weeks' gestation. After birth, a placental injection study identified residual unidirectional arterio-venous anastomoses from the ex recipient to the ex-donor without arterio-venous anastomoses in the opposite direction. Prospective measurements of decreasing haemoglobin levels between the intrauterine transfusion and birth allowed us to assess the net blood flow through the anastomoses as 27.9mL/24h. This finding may also explain the inaccuracy of Doppler flow measurements, as such low flow velocities cannot possibly be detected with current Doppler techniques. PMID- 16678897 TI - Inhibition of protein tyrosine phosphatase 1B by reactive oxygen species leads to maintenance of Ca2+ influx following store depletion in HEK 293 cells. AB - Depletion of inositol 1,4,5 trisphosphate-sensitive Ca2+ stores generates a yet unknown signal, which leads to increase in Ca2+ influx in different cell types [J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7 (1986) 1-12]. Here, we describe a mechanism that modulates this store-operated Ca2+ entry (SOC). Ca2+ influx leads to inhibition of protein tyrosine phosphatase 1B (PTP1B) activity in HEK 293 cells [L. Sternfeld, et al., Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells, Cell Signal 17 (2005) 951-960]. Since Ca2+ does not directly inhibit PTP1B, we assumed an intermediate signal, which links the rise in cytosolic Ca2+ concentration and PTP1B inhibition. We now show that Ca2+ influx is followed by generation of reactive oxygen species (ROS) and that it is reduced in cells preincubated with catalase. Furthermore, Ca2+-dependent inhibition of PTP1B can be abolished in the presence of catalase. H2O2 (100 microM) directly added to cells inhibits PTP1B and leads to increase in Ca2+ influx after store depletion. PP1, an inhibitor of the Src family tyrosine kinases, prevents H2O2-induced Ca2+ influx. Our results show that ROS act as fine tuning modulators of Ca2+ entry. We assume that the Ca2+ influx channel or a protein involved in its regulation remains tyrosine phosphorylated as a consequence of PTP1B inhibition by ROS. This leads to maintained Ca2+ influx in the manner of a positive feedback loop. PMID- 16678898 TI - NPM1 mutations in myelodysplastic syndromes and acute myeloid leukemia with normal karyotype. AB - Mutations at exon 12 of the nucleophosmin (NPM1) gene are the most frequent acquired molecular abnormalities in adult and pediatric acute myeloid leukaemia (AML) with normal karyotype. We screened 28 patients with new diagnosed primary AML with normal karyotype, 38 patients with myelodysplastic symdromes (MDS) and 19 healthy volunteer for mutations at exon 12 of NPM1 gene. NPM1 mutations were identified in four AML patients and two MDS patients, including one novel sequence variant. As far as we know, this is the first report of NPM1 mutation in patients with MDS in the English literature until now, and our primary data support that NPM1 mutations may be also involved in the pathogenesis of MDS. PMID- 16678899 TI - Intakes of selected food groups and beverages and adult acute myeloid leukemia. AB - Few studies have explored the association between diet and adult acute myeloid leukemia (AML). In a hospital-based case-control study among 111 cases and 439 controls, AML risk was negatively associated with milk intake among women (OR 0.25, 95% CI 0.08-0.73) and tea (OR 0.50, 95% CI 0.23-1.09), and positively associated among women with beer (OR 2.48, 95% CI 1.05-5.85), wine (OR 2.32, 95% CI 1.05-5.09), and beef (OR 4.78, 95% CI 1.35-16.94). Our findings support a role of diet in adult AML; however, further research is needed to explore gender differences in risk. PMID- 16678900 TI - Assessing water quality in rivers with fuzzy inference systems: a case study. AB - In recent years, fuzzy-logic-based methods have demonstrated to be appropriated to address uncertainty and subjectivity in environmental problems. In the present study, a methodology based on fuzzy inference systems (FIS) to assess water quality is proposed. A water quality index calculated with fuzzy reasoning has been developed. The relative importance of water quality indicators involved in the fuzzy inference process has been dealt with a multi-attribute decision-aiding method. The potential application of the fuzzy index has been tested with a case study. A data set collected from the Ebro River (Spain) by two different environmental protection agencies has been used. The current findings, managed within a geographic information system, clearly agree with official reports and expert opinions about the pollution problems in the studied area. Therefore, this methodology emerges as a suitable and alternative tool to be used in developing effective water management plans. PMID- 16678901 TI - Polycyclic aromatic hydrocarbon contamination in coastal sediments of the Izmit Bay (Marmara Sea): case studies before and after the Izmit Earthquake. AB - Izmit Bay and its coastal environment was strongly affected by the August 17th, 1999 Izmit Earthquake. The changes in the Bay ecosystem and its chemical oceanography have been studied in detail previously [Okay, O.S., Tolun, L, Telli Karakoc, F., Tufekci, V., Tufekci, H. And Morkoc, E. 2001. Yzmit Bay ecosystem after Marmara earthquake and subsequent fire: The long-term data. Marine Pollution Bulletin 42, 361-369; Balkys, N. 2003. The effect of Marmara (Izmit ) Earthquake on the chemical oceanography of Izmit Bay, Turkey. Marine Pollution Bulletin 46, 865-878.]. In this study surface sediments collected from the Izmit Bay before and after the earthquake have been analysed for total and individual (14 compounds) polycyclic aromatic hydrocarbons (PAH). Analyses have been performed by high performance liquid chromatography with fluorescence detection (HPLC/FD). Before the earthquake, total PAH concentrations in the Bay sediments ranged from 120 to 8900 ng/g while after the earthquake PAH concentrations varied between 240 and 11,400 ng/g. Molecular indices based on isomeric PAH ratios used to differentiate the pollution sources, clearly indicate the differences in molecular distribution of PAHs before and after the earthquake. Sediment data obtained before the earthquake shows that most of the contamination originated from high temperature pyrolytic inputs while after the earthquake it originated from petrogenic sources. This difference emphasises the environmental impact of uncontrolled discharges from petroleum industries after the earthquake. The LMW/HMW ratio (sum of the low molecular weight PAHs / the sum of higher molecular weight PAHs) predominance also changed after the earthquake as a result of the strong water movements. According to the characteristics of aromatic rings distributed in the bay sediments, the soluble parts of the total PAH were probably transferred to the water column after the earthquake as a result of resuspension process. The TEL/PEL (Threshold Effect Level / Probable Effect Level) analysis suggests that the Izmit Bay sediments were likely contaminated by acutely toxic PAH compounds. PMID- 16678902 TI - Computerized tomography (CT)-guided aspiration of abscesses: outcome of therapy at a tertiary care hospital. AB - OBJECTIVE: To review the experience with percutaneous aspiration of abscesses at a general, tertiary care hospital from 2000 to 2005. METHODS: Computerized medical records of 90 patients who underwent radiologically guided drainage of 92 fluid collections were reviewed for demographic data, co-morbid conditions, location, radiographic imaging characteristics and culture results and to determine the long-term outcome of the procedure. RESULTS: Sixty-seven of 92 (73%) abscesses were cured without the need for open surgical drainage; 44 were cured within 14 days, and 23 had delayed resolution requiring prolonged antibiotics and/or a second drainage. Of the abscesses 85% were drained using a catheter in situ, the remaining aspirated using a needle. Twenty-five (27%) of the 92 abscesses failed percutaneous drainage, requiring open surgical drainage or lead to sepsis and death. Neither co-morbid conditions nor the location of abscesses affected the outcome of drainage. Patients who had sterile cultures of aspirated material after receiving antibiotics prior to the procedure had a significantly higher cure rate than otherwise (P<0.05). Catheter drainage was associated with a significantly higher cure rate than was needle aspiration (P<0.05). CONCLUSION: Percutaneous drainage, along with appropriate antibiotics, is an effective approach to treat deep abscesses. Drainage, itself, is the major determinant of outcome, rather than the location, characteristics of the abscess or condition of the patient prior to the procedure. PMID- 16678903 TI - Micafungin (FK463), alone or in combination with other systemic antifungal agents, for the treatment of acute invasive aspergillosis. AB - BACKGROUND: Micafungin (FK463) is a new lipopeptide compound (echinocandin) with activity against Aspergillus and Candida species. This study evaluated the safety and efficacy of micafungin in patients with proven or probable invasive aspergillosis (IA). METHODS: A multinational, non-comparative study was conducted to examine proven or probable (pulmonary only) Aspergillus species infection in a wide variety of patient populations. The study employed an open-label design utilizing micafungin alone or in combination with another systemic antifungal agent. Criteria for IA and therapeutic responses were judged by an independent panel. RESULTS: Of the 331 patients enrolled, only 225 met diagnostic criteria for IA as determined by the independent panel and received at least one dose of micafungin. Patients included 98/225 who had undergone hematopoietic stem cell transplantation (HSCT) (88/98 allogeneic), 48 with graft versus host disease (GVHD), and 83/225 who had received chemotherapy for hematologic malignancy. A favorable response rate at the end of therapy was seen in 35.6% (80/225) of patients. Of those only treated with micafungin, favorable responses were seen in 6/12 (50%) of the primary and 9/22 (40.9%) of the salvage therapy group, with corresponding numbers in the combination treatment groups of 5/17 (29.4%) and 60/174 (34.5%) of the primary and salvage treatment groups, respectively. Of the 326 micafungin-treated patients, 183 (56.1%) died during therapy or in the 6-week follow-up phase; 107 (58.5%) deaths were attributable to IA. CONCLUSIONS: Micafungin as primary or salvage therapy proved efficacious and safe in high-risk patients with IA, although patient numbers are small in the micafungin-only groups. PMID- 16678904 TI - Reduction of catheter-related colonisation by the use of a silver zeolite impregnated central vascular catheter in adult critical care. AB - Central vascular catheters (CVC) are used extensively in critical care for monitoring and therapy. They can become colonised with viable micro-organisms within 24 h of insertion, which can rapidly form biofilm. This colonisation is a precursor of catheter-related bloodstream infections (CR-BSI), which are associated with substantial morbidity, mortality, prolonged hospital stay and increased cost. Antimicrobials have been incorporated into the bulk material of CVC or applied to their surfaces as a coating in an attempt to reduce the incidence of CVC colonisation and infection. This study examines the effect of a silver zeolite-impregnated catheter on catheter-related colonisation and infection in adult critical care patients. The study was conducted in adult Intensive Care Units (ICU) at three acute hospitals over 14 months and involved 246 CVC insertions (122 silver-impregnated and 124 non-impregnated). CVC tip colonisation was detected by the Maki Roll culture and CR-BSI by differential time-to-positivity of blood cultures. Overall colonisation rate was significantly lower in the silver zeolite-impregnated CVC tips (58%) as compared with the control CVC tips (73%) (p<0.025). In addition, there was a lower rate (34%) of tip colonisation by coagulase negative staphylococci in the silver zeolite impregnated CVC tips as compared with the control CVC tips (47%) (p<0.05). Four episodes of CR-BSI were detected in each arm by differential time-to-positivity in a subset of patients. This study indicates that the silver zeolite-impregnated catheter is superior to non-impregnated catheter in reducing the rate of CVC colonisation but it showed no difference in the rates of CR-BSI in the two arms. Larger prospective randomised control studies are required to evaluate its role in the prevention of CR-BSI. PMID- 16678905 TI - Nosocomial infections in paediatric and neonatal intensive care units. AB - OBJECTIVE: To describe the epidemiological profile of NI in the PICU and NICU, and its related risk factors. DESIGN: A prospective surveillance study from May through October 2000 was performed in the PICU and NICU in a tertiary care university hospital in Barcelona. RESULTS: During the study period, 257 patients were admitted to the PICU and 121 to the NICU. The incidence rate of NI was 26.5 infections per 100 admissions and 1.7 infections per 100 patient-days in the PICU. In the NICU, the incidence rate of NI was 74.3 infections per 100 admissions and 2.7 infections per 100 patient-days. Bacteraemia was the most frequent episode of NI in both units. The most common microorganisms isolated were Gram-positive bacteria (47.2% and 72.7%) in each unit. The factors more frequently associated with NI in the PICU were as follows: patient age under 1 year (RR 5.0; 95% CI 1.6-15.4), class IV of CCS (RR 3.7; 95% CI 1.2-11.1), and mechanical ventilation (RR 2.5; 95% CI 1.0-6.0). In the NICU, the most significant predisposing factors were birth weight less than 1000 g (RR 2.8; 95% CI 1.0-8.0), umbilical arterial catheterization (RR 5.7; 95% CI 1.1-28.5) and parenteral nutrition (RR 2.4; 95% CI 1.2-4.6). The hospital stay was higher in infected patients than in non-infected patients (p<0.001). CONCLUSIONS: This study describes the epidemiological profile of NI in two high-risk paediatric units. These results suggest the need to evaluate the infection control measures with the aim of reducing associated morbidity. PMID- 16678907 TI - Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand. AB - 2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated in 1995 from rat brain and canine gut as an endogenous ligand for the cannabinoid receptors. 2-AG is rapidly formed from arachidonic acid-containing phospholipids through increased phospholipid metabolism, such as enhanced inositol phospholipid turnover, in various tissues and cells upon stimulation. 2 AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, often acts as a partial agonist at these cannabinoid receptors, whereas 2-AG acts as a full agonist in most cases. The results of structure-activity relationship studies suggested that 2-AG rather than anandamide is the true natural ligand for both the CB1 and the CB2 receptors. Evidence is gradually accumulating which shows that 2-AG plays physiologically essential roles in diverse biological systems. For example, several lines of evidence indicate that 2-AG plays an important role as a retrograde messenger molecule in the regulation of synaptic transmission. 2-AG has also been shown to be involved in the regulation of various types of inflammatory reactions and immune responses. In this review, we focused on 2-AG, and summarized information concerning its biosynthesis, metabolism, bioactions and physiological significance, including our latest experimental results. PMID- 16678908 TI - An efficient way of high-contrast, quasi-3D cellular imaging: off-axis illumination. AB - An imaging system enabling a convenient visualisation of cells and other small objects is presented. It represents an adaptation of the optical microscope condenser, accommodating a built-in edge (relief) diaphragm brought close to the condenser iris diaphragm and enabling high-contrast pseudo-relief (quasi-3D) imaging. The device broadens the family of available apparatus based on the off axis (or anaxial, asymmetric, inclined, oblique, schlieren-type, sideband) illumination. The simplicity of the design makes the condenser a user-friendly, dedicated device delivering high-contrast quasi-3D images of phase objects. Those are nearly invisible under the ordinary (axial) illumination. The phase contrast microscopy commonly used in visualisation of phase objects does not deliver the quasi-3D effect and introduces a disturbing 'halo' effect around the edges. The performance of the device presented here is demonstrated on living cells and tissue replicas. High-contrast quasi-3D images of cell-free preparations of biological origin (paper fibres and microcrystals) are shown as well. PMID- 16678909 TI - Comparison of cognitive functions between first- and multi-episode bipolar affective disorders. AB - BACKGROUND: Cognitive impairment has been commonly found in euthymic patients with bipolar affective disorder (BPAD). Information about onset and course of cognitive deficits is, however, scarce. This study examined the cognitive profile of patients following their first episode of BPAD to determine whether cognitive problems are present at such an early stage. METHODS: Executive functions, memory, IQ, attention-concentration and perceptuomotor function were assessed in 16 euthymic patients with BPAD following their first episodes, and compared with a group of 30 euthymic patients with multiple episodes of BPAD and 20 normal controls. Comparisons were controlled for educational status, current IQ and residual symptoms. RESULTS: First-episode patients were significantly impaired, compared to normal controls, on tests of executive function, sustained attention, perceptuomotor function and IQ. Additionally, their performance was significantly worse than patients with multi-episode BPAD on tests of executive functions, sustained attention and perceptuomotor function. Multi-episode patients had impaired memory, compared to normal controls, and performed poorly on a subtest of executive functions compared to first-episode patients. LIMITATIONS: Sample sizes were small, assessments cross-sectional; all confounds could not be controlled for. CONCLUSIONS: Widespread cognitive disturbances following the first episode of BPAD were found in this study. Whether these disturbances progress following repeated episodes was not entirely clear. Since cognitive impairment can have several adverse consequences for patients of BPAD in terms of disability, quality of life, outcome etc., this must remain a priority area for future research. PMID- 16678910 TI - Ocular motor differences between melancholic and non-melancholic depression. AB - BACKGROUND: Major depressive disorder may be a heterogeneous disorder, yet melancholic depression is the most consistently described subtype, regarded as qualitatively different to non-melancholic depression in terms of cognitive and motor impairments. Eye movement studies in depression are infrequent and findings are inconclusive. METHODS: This study employed a battery of saccadic eye movement tasks to explore reflexive saccades, as well as higher order cognitive aspects of saccades including inhibitory control and spatial working memory. Nineteen patients with major depressive disorder (9 melancholic; 10 non-melancholic) and 15 healthy controls participated. RESULTS: Differences were revealed between melancholic and non-melancholic patients. Melancholia was associated with longer latencies, difficulty increasing peak velocities as target amplitudes increased, and hypometric primary saccades during the predictable protocol. In contrast, the non-melancholic depression group performed similarly to controls on most tasks, but saccadic peak velocity was increased for reflexive saccades at larger amplitudes. LIMITATIONS: Most patients were taking antidepressant medication. CONCLUSIONS: The latency increases, reduced peak velocity and primary saccade hypometria with more severe melancholia may be explained by functional changes in the fronto-striatal-collicular networks, related to dopamine dysfunction. In contrast, the serotonergic system plays a greater role in non-melancholic symptoms and this may underpin the observed increases in saccadic peak velocity. These findings provide neurophysiological support for functional differences between depression subgroups that are consistent with previous motor and cognitive findings. PMID- 16678906 TI - Leptin and its role in hippocampal synaptic plasticity. AB - It is well documented that the hormone leptin plays a pivotal role in regulating food intake and body weight via its hypothalamic actions. However, leptin receptors are expressed throughout the brain with high levels found in the hippocampus. Evidence is accumulating that leptin has widespread actions on CNS function and in particular learning and memory. Recent studies have demonstrated that leptin-deficient or-insensitive rodents have impairments in hippocampal synaptic plasticity and in spatial memory tasks performed in the Morris water maze. Moreover, direct administration of leptin into the brain facilitates hippocampal long-term potentiation (LTP), and improves memory performance in mice. There is also evidence that, at the cellular level, leptin has the capacity to convert hippocampal short-term potentiation (STP) into LTP, via enhancing NMDA receptor function. Recent data indicates that leptin can also induce a novel form of NMDA receptor-dependent hippocampal long-term depression. Here, we review the evidence implicating a key role for the hormone leptin in modulating hippocampal synaptic plasticity and discuss the role of lipid signaling cascades in this process. PMID- 16678911 TI - Establishment and characterization of primary canine hepatocellular carcinoma cell lines producing alpha-fetoprotein. AB - A primary cultured cell line named CHKS was established from a hepatocellular carcinoma (HCC) of a dog showing a high level of serum alpha-fetoprotein (AFP). CHKS secreted a 66 KDD AFP into the growth medium regardless of the presence or absence of fetal bovine serum (FBS). Cloning CHKS with limiting dilution produced 4 clones, CHKS-1, -2, -3, and -4, which secreted 826, 471, 70, and less than 10 ng/ml, respectively, of AFP into the culture medium. In culture, these cell lines were similar in morphology and proliferation pattern to epithelial cells and positive to periodic acid-Schiff (PAS) staining. The presence of mRNA for canine albumin was demonstrated by nested PCR. The doubling times of the clone cell lines were 21, 45, 36, and 35 h, saturation densities 34, 18, 22, and 24 x 10(4)/cm(2), and plating efficiencies 18, 45, 46, and 45%, respectively. Chromosome analysis of these cell lines showed near triploidy. These results show that CHKS and its clones have hepatic cell functions and are useful for carcinogenetic and clinical studies of canine HCC. PMID- 16678912 TI - Contribution of respiratory burst activity to innate immune function and the effects of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro. AB - The mechanisms of interaction between phagocytes and different bacteria that help resolve lung infections or contribute to lung pathology are poorly defined. Alveolar phagocytes (resident macrophages and recruited neutrophils) make a major contribution to innate immunity by mounting a respiratory burst that helps kill internalised bacteria. However, this ability may be altered during or after exposure to infection. This review considers the application and limitations of a variety of analytical methods for oxygen-dependent mechanisms of respiratory burst in phagocytes initiated by soluble and particulate activators. Particular reference is given to the study in vitro of phagocytes from healthy and diseased ruminants during either natural infection with Mycobacterium avium paratuberculosis or experimental infection with Pasteurella multocida or Mannheimia haemolytica. PMID- 16678913 TI - PKD at the crossroads of DAG and PKC signaling. AB - Diacylglycerol (DAG) and its primary target protein kinase C (PKC) regulate many important cellular responses, yet the molecular mechanisms that control the specificity of DAG and PKC signaling are not fully understood. As such, targeting the PKC pathway for therapeutic purposes has been challenging. Protein kinase D (PKD), a novel DAG receptor, has been the subject of intense investigation in recent years. DAG regulates the intracellular localization of PKD and also activates PKD through PKC by phosphorylation. The PKC-PKD signaling cascade is crucial to PKD function in cells. Important discoveries have been made regarding the roles of PKD in cell growth, gene expression, survival, motility, protein trafficking and lymphocyte biology. This kinase is implicated in pathological processes such as cardiac hypertrophy, tumor cell proliferation and metastasis. Thus, PKD represents a novel therapeutic target for the DAG-PKC signaling network. PMID- 16678914 TI - Biomechanopharmacology: a new borderline discipline. AB - Flowing blood is more than a drug transporter in pharmacology; its mechanical impact should also be considered. The in vitro pharmacological dose-response pattern of endothelial cellular functions can be significantly modified by in vivo shear stress. A new borderline discipline, biomechanopharmacology, is forming at the boundary between biomechanics and pharmacology. Biomechanopharmacology will probably consist of both the pharmacological intervention of signals induced by biomechanical factors and the biomechanical influence on pharmacokinetics and pharmacodynamics, in addition to the joint effect of biomechanical and pharmacological factors. Recent investigations show that exercise enhances the shear of pulsatile blood flow to stimulate angiogenesis. The benefits of exercise for gaining joint biomechanical and pharmacological effects should be emphasized. PMID- 16678915 TI - IUPHAR: from San Francisco to Beijing. PMID- 16678916 TI - Role of delivery and trafficking of delta-opioid peptide receptors in opioid analgesia and tolerance. AB - Changes in the number of receptors on the cell surface lead to modulations of physiological functions and pharmacological responses of neurons. Recent studies show that delta-opioid peptide (DOP) and mu-opioid peptide (MOP) receptors have distinct subcellular localizations in neurons. In nociceptive small neurons in the dorsal root ganglia, DOP receptors are sorted into neuropeptide-containing secretory vesicles, enabling the stimulus-induced cell surface expression of these receptors. MOP receptors are constitutively expressed on the cell surface. The physical interaction between DOP receptors and MOP receptors seems to be an important mechanism for the modulation of receptor functions. Experiments in animals show that MOP-receptor-mediated spinal analgesia is enhanced and morphine tolerance does not develop when DOP receptor functions are pharmacologically or genetically attenuated. Thus, the delivery and trafficking of DOP receptors are crucial processes that modulate opioid analgesia and tolerance. PMID- 16678917 TI - Using positron emission tomography to facilitate CNS drug development. AB - Positron emission tomography (PET) is a non-invasive technology of nuclear medicine that has sensitivity for tracing low picomolar concentrations of radiolabeled molecules in the human body. Radiolabeling a new drug to high specific radioactivity facilitates a detailed mapping of its distribution to crucial organs in humans after the administration of a "microdose" (< 1 microg), for which limited toxicology documentation is required. For drugs directed at the CNS, this method is particularly useful for confirming exposure to the brain. A different approach is to develop suitable radioligands for quantitative PET studies of drug binding to target proteins and subsequently to correlate receptor occupancy with pharmacodynamic responses. To follow disease progression and to monitor the outcome of new treatments, PEt also facilitates longitudinal studies of biomarkers of pathophysiology such as amyloid plaque load in Alzheimer's disease. Finally, combining genomic knowledge with PET neuroreceptor imaging is expected to facilitate the search for genetic predictors of drug response. PMID- 16678918 TI - Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens. AB - Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample. PMID- 16678919 TI - A new Real-Time-RT-PCR for quantitation of human endogenous retroviruses type K (HERV-K) RNA load in plasma samples: increased HERV-K RNA titers in HIV-1 patients with HAART non-suppressive regimens. AB - Viral components of the human endogenous retroviruses type K (HERV-K) have been largely detected in plasma from HIV-1 infected individuals. A Sybr Green Real Time RT-PCR approach was optimized for detection and quantitation of HERV-K RNA titers in plasma samples using the iCycler technology. The method detected 1000 HERV-K RNA copies/mL of plasma sample. The Intra- and Inter-assay performance revealed a coefficient of variations that ranged from 0.2 to 2.46%, demonstrating accuracy and reproducibility. We quantified the HERV-K RNA load in 20 HIV-1 patients receiving highly active antiretroviral therapy (HAART). We found increased HERV-K RNA titers in patients with non-suppressive HAART (patients who may develop drug-resistance and/or received suboptimal therapeutic doses), compared to suppressive regimens (p < 0.001). HERV-K RNA was not detected in HCV 1 positive or seronegative controls. Sequencing of Real-Time RT-PCR products revealed particular HERV-K subtypes activated in the HIV-1 infection. The application of this assay could expand the understanding of the role of HERV-K in the HIV-1 infection and others pathological conditions. PMID- 16678920 TI - Protection of Balb/c mice against infection with FMDV by immunostimulation with CpG oligonucleotides. AB - Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are potent stimulators of the innate immune system, and are capable of aborting several infections in a non-specific manner. We here report studies of the capacity of such ODN to protect mice against infection with foot and mouth disease virus (FMDV). Susceptibility of Balb/c mice to infection with isolates from the different serotypes of FMDV was investigated, and, at the same time, the capacity of CpG ODN to modulate the infection was evaluated. Treatment with CpG significantly reduced viremia, disease and death in five of six serotypes, when compared to no treatment or treatment with a control ODN. The effect was observed when ODN was administered simultaneously with, or up to 12h after, infection with FMDV, and lasted for 14 days post treatment. The potential application of CpG ODN for control of FMDV during an outbreak is discussed. PMID- 16678921 TI - The metabotropic glutamate 2/3 receptor agonist LY404039 reduces alcohol-seeking but not alcohol self-administration in alcohol-preferring (P) rats. AB - Metabotropic glutamate (mGlu) receptors have been shown to mediate a number of behaviors including emotionality and responsivity to stress as demonstrated by efficacy in preclinical and clinical studies. The objective of this study was to assess the effects of the mGlu2/3 receptor agonist LY404039 (LY) on operant ethanol (EtOH) self-administration during alcohol seeking (pavlovian spontaneous recovery, PSR), alcohol relapse (alcohol deprivation effect, ADE), and maintenance responding for alcohol. Adult alcohol-preferring (P) rats were trained in 2-lever operant chambers to self-administer 15% EtOH (v/v) and water on a concurrent fixed-ratio 5-fixed-ratio 1 (FR5-FR1) schedule of reinforcement in daily 1h sessions. After at least 10 weeks of daily 1 h sessions, rats underwent seven extinction sessions, followed by 2 weeks of no manipulation, and then rats were tested for the expression of an EtOH PSR for four sessions. Rats were then given a week in their home cage before being returned to the operant chambers with access to EtOH and water (alcohol relapse). Finally, the effects of LY upon maintenance EtOH and water responding were assessed once stable responding was reestablished. The mGlu2/3 receptor agonist LY404039 reduced responding on the EtOH in the PSR test. LY also reduced the expression of an alcohol deprivation effect (ADE) during relapse, but did not reduce EtOH responding under maintenance conditions. The results of this study demonstrate that activating mGlu2/3 receptors inhibits the expression of alcohol seeking and relapse behavior without altering alcohol self-administration behavior. PMID- 16678922 TI - Association of cardiorespiratory fitness and levels of C-reactive protein: data from the National Health and Nutrition Examination Survey 1999-2002. AB - BACKGROUND: Elevated C-reactive protein (CRP) is a risk factor for atherosclerosis. Cardiorespiratory fitness, the ability of active skeletal muscle to utilize oxygen during aerobic exercise, can be impaired by atherosclerotic changes peripherally affecting the vasculature or centrally perturbing the heart and coronary arteries. However, the association between cardiorespiratory fitness and CRP has not been adequately examined. METHODS: We examined 1438 adults aged 20-49 years with reliable measures of fitness and non-missing values in CRP from Health and Nutrition Examination Survey 1999-2002. Subjects with major cardiovascular and respiratory conditions were excluded from fitness test which measured the estimated maximal oxygen uptake (VO2max), by a submaximal exercise test. Levels of CRP were quantified by utilizing latex-enhanced nephelometry. RESULTS: In both genders, we observed inverse associations between estimated VO2max and levels of CRP after controlling for age, race, body mass index category, hypertension, diabetes, smoking status, alcohol consumption, and use of medications including aspirin, non-steroidal anti-inflammatory drugs, steroid, lipid-lowering agents, antimicrobials, or estrogen/progesterone (in women). Men in the 2nd, 3rd, and 4th quartiles of CRP concentrations had lower estimated VO2max compared to the first quartile (regression coefficients for quartile 2, 1.54 ml/kg/min, p=0.105; quartile 3, -1.46 ml/kg/min, p=0.130; quartile 4, -2.64 ml/kg/min, p=0.009; p for trend 0.013). Women in the highest quartile of CRP had a significantly lower estimated VO2max (2.40 ml/kg/min, p=0.023) compared to the lowest (p for trend 0.025). CONCLUSIONS: CRP levels, inversely related to cardiorespiratory fitness, are important indicators of exercise tolerance and may be useful in targeting individuals requiring endurance intervention to prevent loss of cardiovascular fitness and function. PMID- 16678923 TI - Extrusion of a regularly stimulating pacemaker. PMID- 16678924 TI - Ill health and retirement in Britain: a panel data-based analysis. AB - We examine the effect of ill health on retirement decisions in Britain, using the British Household Panel Survey (1991-1998). As self-reported health status is likely to be endogenous to the retirement decision, we instrument self-reported health by a constructed 'health stock' measure using a set of health indicator variables and personal characteristics, as suggested by Bound et al. (Bound, J., Schoenbaum, M., Stinebrickner, T.M., Waidmann, M., 1999. The dynamic effects of health on the labor force transitions of older workers. Labour Economics 6, 179 202). Using a range of econometric techniques, we show that adverse shocks to individual health stocks predict individual retirement behaviour among workers aged from 50 until state pension age. We compare responses of economic activity to constructed health measures with that arising using direct indicators of functional limitations and specific health problems. We also examine the dynamics of health shocks and whether adverse and positive health shocks have symmetric effects on transitions in and out of economic activity. PMID- 16678925 TI - Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens. AB - A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections. PMID- 16678926 TI - The effect of transient increase in oxygen level on brain activation and verbal performance. AB - This study aimed to investigate the hypothesis that a transient increase in oxygen level administered to subjects increases the BOLD effect in brain regions associated with verbal cognitive functioning and enhances performance accuracy. A verbal task was presented while brain images were scanned by a 3T fMRI system. The accuracy rate on the verbal task was enhanced during 30% oxygen administration compared to 21% oxygen administration. The neural activations were observed at the occipital, parietal, temporal and frontal lobes, during both 21% and 30% oxygen administration. Increased brain activations were observed in the right middle frontal gyrus, right inferior frontal gyrus, right superior frontal gyrus, cingulate gyrus, left middle temporal gyrus, and left fusiform gyrus with 30% oxygen administration. These results suggest that a higher concentration of breathed oxygen increases saturation of blood oxygen in the brain, and facilitates verbal cognitive performance. PMID- 16678927 TI - The effects of inversion and eye displacements of familiar and unknown faces on early and late-stage ERPs. AB - OBJECTIVES: The objective of this study is to examine whether configural alterations of faces affect early or late processing stages as a function of their familiarity and their level of representation in memory. We then sought to verify whether the structural encoding stage is susceptible to top-down influences. METHODS: Electrophysiologic and behavioral studies were undertaken, during which unknown and familiar faces were presented upright or upside-down with or without feature alterations. The subjects were asked to determine whether the faces were familiar or not. RESULTS: N170 and N360 amplitudes were larger for familiar faces as well as altered ones. A higher degree of familiarity decreased reaction times (RTs) and N360 latencies, but increased N170 latencies, whereas face alterations increased RTs and latencies of both components examined. However, familiarity interacted with altered face configurations only for RTs and the N170. SIGNIFICANCE: In the perceptual stage, familiar faces seem to develop a more elaborate type of processing because of top-down influences linked to the robust nature of their representations in memory. The more elaborate type of processing for familiar faces has advantageous consequences for the following steps of information processing, by facilitating access to structural representations in memory (N360) as well as the final step reflected by RTs. The fact that configural alterations cause different effects for familiar as opposed to unfamiliar faces indicate that these stimuli are processed in a qualitatively different manner and solicit different representations in memory. PMID- 16678928 TI - Contribution of AMPA and NMDA receptors to early and late phases of LTP in hippocampal slices. AB - Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D aspartate (NMDA) receptor mediated responses were investigated in rat hippocampal slices under 4h of long-term potentiation (LTP) expression. A modified medium containing the NMDA receptor antagonist AP5 and low concentration of Mg(2+) was used to monitor isolated AMPA responses. NMDA components were determined from composite excitatory postsynaptic potentials (EPSPs) under brief (15-20 min) wash out of AP5. LTP was induced in a medium with low concentration of AP5, resulting in an about two-fold larger increase of the AMPA component than of the NMDA component at both 1h and 4h after induction. Similar results were obtained if LTP was induced in "normal Mg(2+)" and the NMDA components were assessed at the end of experiment, from either composite or isolated NMDA EPSPs, with or without blockade of GABAergic inhibition. It is generally believed that LTP undergoes biochemical and/or structural conversions during the first few hours. Our study, however, shows constant expression of LTP, at least in terms of AMPA versus NMDA components, during this time. The data support the notion that LTP initiates as a predominant amplification of AMPA receptors and remains so for at least 4h. PMID- 16678929 TI - Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles. AB - A water-insoluble antitumor agent, camptothecin (CPT) was successfully incorporated into polymeric micelles formed from poly(ethylene glycol) poly(benzyl aspartate) block copolymers (CPT-loaded polymeric micelles). Antitumor effects and biodistribution of CPT-loaded micelles were evaluated in mice subcutaneously transplanted by colon 26 tumor cells. Tumor growth was significantly inhibited after a single i.v. injection of CPT-loaded polymeric micelles at doses of either 15 or 30 mg/kg. Efficacy of a single high-dose injection was comparable to low dose multiple injections. CPT loaded in polymeric micelles showed prolonged blood circulation and higher accumulation in tumors compared with CPT in solution. Polymeric micelle systems offer a stable and effective platform for cancer chemotherapy with CPT. PMID- 16678930 TI - Current impact of gene technology on healthcare. A map of economic assessments. AB - OBJECTIVES: It has been claimed that gene technology will induce revolutionary changes in healthcare. This paper investigates how and to what extent these changes have been economically assessed. METHODS: A generic framework was developed to distinguish between methodologically similar evaluations of healthcare technology. Methodological issues and the current state of economic evidence concerning human DNA technology were extracted from publications within these groups of evaluations. RESULTS: Economic evaluations of "healthcare consisting of gene technology" were identified primarily for in vitro diagnostics for hereditary disease and others for pharmacogenetics and molecular pathology. "Healthcare enabled by gene technology" is far more encompassing and includes, e.g., biotechnology drugs for which various health economic evaluations can be found. Yet here, the impact of gene technology intertwines with the impact of other technologies and is therefore hardly susceptible to evaluation. The fields of evaluation may be classified best according to the two dimensions "purpose" and "stage of development". Current evaluations cover screening, diagnostic and treatment technologies in investigational, new and established stages. Apart from prenatal screening, healthcare consisting of gene technology was cost saving only for genotype tests replacing continuous phenotype tests and for one pharmacogenetic test. Conclusive evidence of favourable cost-effectiveness ratios is available only for few conditions. CONCLUSION: Hypotheses about the impact of gene technology on healthcare must be explicit about the definition of "genetic" medicine. A general statement regarding healthcare enabled by gene technology is not possible. Based on current evidence, an era of healthcare consisting of gene technology built on widespread predictive testing is not desirable from a health economic viewpoint. PMID- 16678931 TI - Relationships between community characteristics and municipal smoke-free bylaw status and strength. AB - BACKGROUND: Municipal smoke-free spaces bylaws are a common population-level intervention to address the prominent health risks associated with exposure to second-hand smoke. In Canada, bylaw prevalence is increasing, but inequities in level of protection across communities remain as bylaws vary from place to place. Little is known about the role of place in this policy disparity. To address this gap, this study examined associations between community characteristics and municipal smoke-free spaces policy outcomes to elucidate how ecological conditions are associated with bylaw status and strength. METHOD: Smoke-free public place and workplace bylaws were obtained from all municipalities with a population of >or=5000 in the provinces of Alberta (N=78) and Ontario (N=245), Canada. Bylaws were assigned a strength score (ranging 0-100) rating the degree of bylaw comprehensiveness, restrictiveness and enforcement provisions. These data were then linked to socio-demographic data from the Canadian Census (1996) and the Canadian Community Health Survey Cycle 1.1 (2000/2001). Logistic and multiple linear regression analyses were used to develop models for municipal bylaw status and strength using community factors including: socio-demographics, municipality type, health region smoking rates, and provincial tobacco control environment. CONCLUSIONS: Study findings suggest that community characteristics play a key role in the status and strength of municipal smoke-free bylaws. As smoking bans continue to emerge world-wide under the Framework Convention on Tobacco Control, knowledge about conditions that promote policy success will be critical for those in need of immediate information to maximize policy-making in their own settings. Use of community profiles that consider socio-demographics along with broader contextual factors can aid decision-makers, public health advocates, and researchers in identifying similar jurisdictions with a successful smoke-free spaces policy and communicating with them about their policy-making experiences. Knowledge about the conditions associated with municipal policy development may be translated to other jurisdictions where information is sparse, or evidence is newly emerging. PMID- 16678932 TI - A user-friendly PC-based data acquisition and analysis system for respirometric investigations. AB - We have developed an easy-to-use computer-based system for recording, displaying, storing and analyzing signals generated by Clark-type oxygen electrodes. A user friendly interface of Windows-based program BioMed significantly increases the productivity of investigations. It allows to process, control, present and archive the experimental data in real time. A 12-bit analog-to-digital-converter, analog and digital filters, a possibility to zoom the obtained respiratory curves and calculation of the respiration rates by a linear regression method increase the resolution of the estimated oxygen consumption rates. The new system enables to register even small changes, such as 3-5 ngatoms O/min, in respiration rates of biological objects -- enzymes, mitochondria and permeabilized muscle fibers. The system has been developed and is regularly used for the respirometric investigations at the Laboratory of Biochemistry, Institute for Biomedical Research, Kaunas University of Medicine. PMID- 16678933 TI - Evolutionary perspectives on overeating and overweight. Introduction to the special section of Appetite. PMID- 16678934 TI - CSF neurofilament light chain and tau differentiate multiple system atrophy from Parkinson's disease. AB - BACKGROUND: In early disease stages it can be clinically difficult to differentiate idiopathic Parkinson's disease (IPD) from patients with multiple system atrophy predominated by parkinsonism (MSA-P). METHODS: In CSF of 31 patients with IPD, 19 patients with MSA-P, we analyzed tau, neurofilament light chain (NFL) and heavy chain (NFHp35) and the noradrenergic metabolite 3-methoxy-4 hydroxyphenylethyleneglycol (MHPG). RESULTS: CSF levels of NFL, NFHp35, and tau were significantly increased in MSA-P (all p<0.0001), whereas, MHPG levels were significantly decreased in MSA-P (p<0.0001). Optimal discriminative cut-off values for the differentiation between MSA-P and IPD were calculated resulting in high sensitivity (76-94%) and specificity (83-97%) levels. Multivariate logistic regression resulted in the combination of NFL and tau as independent contributors in differentiating between MSA-P and IPD. DISCUSSION: Higher CSF levels of axonal biomarkers could reflect advanced axonal degeneration in MSA-P. Differentiating MSA-P from IPD could be accurately possible with CSF analysis of a combination of axonal and neurotransmitter biomarkers. PMID- 16678935 TI - [A renal pseudo-tumor]. PMID- 16678936 TI - [Digestive manifestations of systemic vascular diseases]. PMID- 16678937 TI - [Helicobacter pylori gastritis: a connective disease]. PMID- 16678938 TI - [Accelerators of arterial aging]. PMID- 16678940 TI - [Adult-onset Still's disease revealed by a pericardial tamponade: report of two cases]. AB - INTRODUCTION: Adult onset Still's disease is a systemic inflammatory disorder of unknown etiology characterized by the association of a high spiking fever, an evanescent skin rash, arthritis, and hyperleukocytosis. Pericarditis is amongst the most common systemic manifestations of adult onset Still's disease. EXEGESIS: We report on two patients with a pericardial tamponade revealing an adult onset Still's disease in a 52-year-old female and a 31-year-old male. Pericardial fluid was bloody in the two cases, and histopathology only disclosed non specific inflammatory changes. Both patients received corticosteroids and outcome was uneventful with a follow-up of 8 years and 12 months, respectively. CONCLUSION: Pericardial tamponade is an uncommon clinical feature of adult-onset Still's disease and usually occurs at disease onset. It makes the diagnosis of adult onset Still's disease difficult as the other disease manifestations are commonly neglected. Adult onset Still's disease should be added to the differential of acute pericarditis and tamponade. PMID- 16678942 TI - [Celiac disease in adults in 2006]. PMID- 16678943 TI - ESPEN Guidelines on Enteral Nutrition: Pancreas. AB - The two major forms of inflammatory pancreatic diseases, acute and chronic pancreatitis, require different approaches in nutritional management, which are presented in the present guideline. This clinical practice guideline gives evidence-based recommendations for the use of ONS and TF in these patients. It was developed by an interdisciplinary expert group in accordance with officially accepted standards and is based on all relevant publications since 1985. The guideline was discussed and accepted in a consensus conference. In mild acute pancreatitis enteral nutrition (EN) has no positive impact on the course of disease and is only recommended in patients who cannot consume normal food after 5-7 days. In severe necrotising pancreatitis EN is indicated and should be supplemented by parenteral nutrition if needed. In the majority of patients continuous TF with peptide-based formulae is possible. The jejunal route is recommended if gastric feeding is not tolerated. In chronic pancreatitis more than 80% of patients can be treated adequately with normal food supplemented by pancreatic enzymes. 10-15% of all patients require nutritional supplements, and in approximately 5% tube feeding is indicated. PMID- 16678944 TI - Methodology for the development of the ESPEN Guidelines on Enteral Nutrition. AB - Under the auspices of the European Society for Clinical Nutrition and Metabolism (ESPEN) clinical practice guidelines on enteral nutrition were systematically developed between spring 2004 and winter 2005 in a both evidence and consensus based process. A steering committee implemented 13 disease-specific working groups with a total of 88 experts in clinical nutrition from 20 countries. Evidence was gathered by conducting a structured literature search applying a defined search strategy, inclusion criteria and specified keywords. The quality and strength of the supporting evidence was then graded according to published standards. On this basis recommendations were spelled out which were not only based on the evidence levels of the studies but also on the judgement of the working groups concerning the consistency, clinical relevance and validity of the evidence. Preparation of the drafts was accomplished in interactive small group work processes. The voting of statements took place in a consensus conference and final refinements and voting were done using the Delphi technique. The ESPEN guidelines enteral nutrition reflect the current medical knowledge in the field of enteral nutrition therapy and summarize the evidence when enteral nutrition is indicated and which goals can be reached in regard to nutritional state, quality of life and outcome. PMID- 16678945 TI - Cost-effectiveness of influenza vaccination of healthy children. AB - Influenza vaccination of children 6-23 months of age is recommended in the United States and Canada because of high rates of influenza-associated hospitalisations, but few other countries have adopted similar policies. Most children with influenza are treated in the primary care setting, and the cost-effectiveness of influenza vaccination of children has not been fully established. We used a decision analysis model to assess the cost-effectiveness of influenza vaccination of children 6 months to 13 years of age in Finland. The analyses were based on comprehensive clinical data on virologically confirmed influenza infections, hospital medical records, and national registers. We estimated the impact of influenza on outpatient and hospitalised children and their families, and performed the analyses from the health care provider and societal perspective. Influenza vaccination resulted in savings in all programs including children 0.01). Serum lysozyme and myeloperoxidase activities were lower (7.26+/ 0.87mg/ml and, 0.54+/-0.11 OD, respectively) in winter as compared to any other season of the year. The bacterial agglutination titer was higher (p<0.01) in the rainy season (8.70+/-1.70) compared to summer and winter seasons (3.40+/-0.60 and 4.00+/-0.89, respectively). Haemagglutination and haemolytic activities did not vary (p>0.01) throughout the year. In blood smears, lymphocyte percentage was higher (75-80%) as compared to those of neutrophil (10-15%) and monocytes (5-10%) but eosinophilic granulocytes were present only in few cases. The differential leucocyte count did not vary significantly (p>0.05) in any season. This study indicated that certain non-specific immune parameters of this species can be modulated at certain times of the year. PMID- 16679031 TI - An fMRI study of cerebral processing of brush-evoked allodynia in neuropathic pain patients. AB - Previous human imaging studies have revealed a network of brain regions involved in the processing of allodynic pain; this includes prefrontal areas, insula, cingulate cortex, primary and secondary somatosensory cortices and parietal association areas. In this study, the neural correlates of the perceived intensity of allodynic pain in neuropathic pain patients were investigated. In eight patients, dynamic mechanical allodynia was provoked and brain responses recorded using functional magnetic resonance imaging (fMRI). Voxels in which the magnitude of fMRI signal correlated linearly with the ratings of allodynic pain across the group were determined in a whole brain analysis using a general linear model. To ensure that activation reflected only allodynic pain ratings, a nuisance variable containing ratings of ongoing pain was included in the analysis. We found that the magnitude of activation in the caudal anterior insula (cAI) correlates with the perceived intensity of allodynic pain across subjects, independent of the level of ongoing pain. However, the peak of activation in the allodynic condition was located in the rostral portion (rAI). This matches the representation of other clinical pain syndromes, confirmed by a literature review. In contrast, experimental pain in healthy volunteers resides predominantly in the cAI, as shown by the same literature review. Taken together, our data and the literature review suggest a functional segregation of anterior insular cortex. PMID- 16679032 TI - Infant's brain responses to live and televised action. AB - Whether human infants perceive televised stimuli in the same way to live stimuli largely remains unknown. Action observation, which has been extensively confirmed to elicit activation of internal motor representation, provides a promising framework for investigating this issue. This 'mirror-matching' property has been found in the monkey premotor cortex as well as the premotor and primary motor cortices in human adults. Although larger activation in observing a live action compared to a televised action in adult subjects has been reported, it is unknown whether the same neural response is obtained from human infants. To address this issue, we first measured the activity of motor areas in adult subjects while viewing either a live or televised action of other people by using near-infrared spectroscopy. The motor areas that were activated when the subject themselves performed an action were also activated during action observation in the live setting, while this was not evident in the TV setting. We then conducted qualitatively the same experiment with 6- to 7-month-old infants. The infant's motor areas were significantly activated when observing a live person performing an action. Although we also found activation in the same area during action observation in the TV setting, the difference in activity between action observation and object-motion observation was significant only in the live setting. Our results are the first to demonstrate activation in motor areas during action observation in human infants. We suggest that human brain responds differently to the real world and the virtual world. PMID- 16679033 TI - Phylogeny, biogeography, and evolution of two Mediterranean snakes, Malpolon monspessulanus and Hemorrhois hippocrepis (Squamata, Colubridae), using mtDNA sequences. AB - Variation in 815bp of mitochondrial DNA from two gene fragments (300bp of cytochrome b and 395-515bp of 12S rRNA) for 26 Malpolon monspessulanus, and cytochrome b for a further 21 individuals, indicates that this species originated in the Maghreb area of Northwest Africa. Here, an estimated 3.5-6Mya, it divided into the western M. m. monspessulanus, and an eastern clade including M. m. insignitus and M. m. fuscus. The very limited genetic differentiation between Maghreb and Southwest European populations of this form suggests that it arrived in the Iberian Peninsula only recently. Population genetics and demographic tests indicate subsequent expansion in this area around 83,000-168,000 year ago. Because present populations of Malpolon arrived recently, mid-Pliocene and at least some Pleistocene fossils of the genus Malpolon in Southwest Europe are probably derived from an earlier invasion from the Maghreb, possibly as early as the end of the Miocene period, 5.3-5.9Mya, when there was a temporary land bridge across the site of the Strait of Gibraltar and the Mediterranean Sea desiccated. The descendants of this earlier invasion must have eventually become extinct, perhaps during one of the Pleistocene glaciations. In contrast to the western M. m. monspessulanus, the greater genetic divergence found in the eastern clade of M. monspessulanus suggests that it dispersed at an earlier date and probably over a longer period, spreading eastwards through northern Libya and Egypt to Syria, Iraq, and Iran, and around the Mediterranean Sea through Turkey into the Aegean archipelagos and the Balkan peninsula. The western and eastern units of M. monspessulanus have different dorsal color pattern, differences in skull structure and exhibit an 8.4% uncorrected genetic divergence in the combined gene fragments investigated here. It is consequently recommended that they should be treated as separate species: M. monspessulanus (sensu stricto) and Malpolon insignitusstat. nov., the latter including the subspecies Malpolon insignitus fuscuscomb. nov. The same combined mitochondrial gene fragments used in Malpolon were investigated in 20 individuals of Hemorrhois hippocrepis, and of cytochrome b alone in a further 17. They indicate that this species also originated in the Maghreb and again invaded the Iberian Peninsula quite recently. Some of the most recent invasions of the Iberian Peninsula by reptiles and amphibian taxa could probably be anthropogenic in origin. Some other species including M. monspessulanus and H. hippocrepis, may have crossed naturally, by "hopping" across the Strait of Gibraltar via temporary islands on the shallowest parts that were exposed during sea-level fall associated with Pleistocene glaciations. PMID- 16679034 TI - Cardiovascular monkey telemetry: sensitivity to detect QT interval prolongation. AB - INTRODUCTION: Preclinical evaluation of delayed ventricular repolarization manifests electrocardiographically as QT interval prolongation and is routinely used as an indicator of potential risk for pro-arrhythmia (potential to cause Torsades de Pointes) of novel human pharmaceuticals. In accordance with ICH S7A and S7B guidelines we evaluated the sensitivity and validity of the monkey telemetry model as a preclinical predictor of QT interval prolongation in humans. METHODS: Cardiovascular monitoring was conducted for 2 h pre-dose and 24 h post dosing with Moxifloxacin (MOX), with a toxicokinetic (TK) evaluation in a separate group of monkeys. In both studies, MOX was administered orally by gavage in 0.5% methylcellulose at 0, 10, 30, 100, 175 mg/kg. Each monkey received all 5 doses using a dose-escalation paradigm. Inherent variability of the model was assessed with administration of vehicle alone for 4 days in all 4 monkeys (0.5% methylcellulose in deionized water). RESULTS: MOX had no significant effect on mean arterial pressure, heart rate, PR or QRS intervals. MOX produced significant dose-related increases in QTc at doses of 30 (Cmax=5.5+/-0.6 microM), 100 (Cmax=16.5+/-1.6 microM), and 175 (Cmax=17.3+/-0.7 microM) mg/kg with peak increases of 22 (8%), 27 (10%), and 47 (18%) ms, respectively (p7) on the effects of warming up on reducing injury risk in humans. Three of the studies found that performing a warm-up prior to performance significantly reduced the injury risk, and the other two studies found that warming up was not effective in significantly reducing the number of injuries. CONCLUSIONS: There is insufficient evidence to endorse or discontinue routine warm-up prior to physical activity to prevent injury among sports participants. However, the weight of evidence is in favour of a decreased risk of injury. Further well-conducted randomised controlled trials are needed to determine the role of warming up prior to exercise in relation to injury prevention. PMID- 16679063 TI - Sampling and signaling in plasmacytoid dendritic cells: the potential roles of Siglec-H. AB - Plasmacytoid dendritic cells (pDCs) detect viruses through toll-like receptor (TLR)7 and TLR9 and respond by secreting type I interferons (IFNs). Because TLR7 and TLR9 are present in endosomes, a mechanism is required to capture and deliver viruses to TLRs. A member of the sialic acid binding Ig-like lectin (Siglec) family, Siglec-H, has recently been identified as a specific surface marker for pDCs in mice. Siglec-H is endocytosed and can mediate the uptake of antigens for processing and presentation. Thus, Siglec-H might have a role in the capture of viruses or other pathogens for their delivery to intracellular TLRs. Paradoxically, Siglec-H also transmits intracellular signals through the associated adaptor DAP12, which reduces pDC responses to TLR ligands. In this review, we discuss models to explain the potential outcomes of Siglec-H engagement in the pDC secretion of type I IFN. PMID- 16679064 TI - HIV infection: first battle decides the war. AB - The traditional view of HIV-1 infection characterized by the slow decline of CD4+ T cells has radically changed in light of recent observations in rhesus macaques and humans of rapid and extensive infection and removal of memory CD4+ T cells in mucosal tissues within the first three weeks of infection. This initial strike to the immune system seems to be the distinguishing feature of HIV-1 pathogenesis and its extent sets the overall course of the ensuing infection. Qualitatively different mechanisms of CD4+ T-cell depletion prevail during the acute, chronic and advanced phases of infection depending on the availability of the target-cell population and competence of the immune system. The elimination of CD4+ T cells in mucosal lymphoid tissues results in the absence of important regulatory and effector functions that these cells normally perform in controlling immune responses to environmental antigens and pathogens. Ablation of acute HIV-1 viremia limits the initial damage to the CD4+ T-cell compartment and helps to establish a state of equilibrium between the replicating virus, the availability of the target-cell population and the immune control characteristic of long-term non-progression. PMID- 16679065 TI - Disrupted in schizophrenia 1: building brains and memories. AB - Schizophrenia and bipolar affective disorder are common, debilitating, and poorly understood and treated disorders. Both conditions are highly heritable. Recent genetic studies have suggested that the gene disrupted in schizophrenia 1 (DISC1) is an important risk factor. DISC1 seems to have a key role in building the brain and memories by interacting with other proteins, including nuclear distribution E like protein and phosphodiesterase 4B. Here, we review the current knowledge, highlight some key unanswered questions and propose ways forward towards a better understanding of normal and abnormal brain development and function. In the long term, this might lead to the discovery of drugs that are more efficacious and safer than currently available ones. PMID- 16679066 TI - An integrated model of glucose and galactose metabolism regulated by the GAL genetic switch. AB - Glucose and galactose are two alternative carbon sources in yeast for energy production, producing CO2 and alcohol. The yeast needs to switch from glucose to galactose metabolism as required, by transcriptional regulation of the respective metabolic enzymes. This regulation is achieved mainly through the GAL genetic switch, in addition to glucose repression mechanism. This study integrates the two metabolic pathways with the genetic regulatory circuit using the GEPASI 3.30 simulation environment, and investigates the model behavior under various nutrient conditions. Our system is successful in achieving transcriptional upregulation of the galactose metabolizing enzymes as required. Under high glucose and high galactose concentrations, the in silico yeast chooses to metabolize glucose first, after which it resorts to using the galactose available. We also show what the preferred storage macromolecules are in different metabolic pathways. PMID- 16679067 TI - MICB microsatellite polymorphism is associated with ulcerative colitis in Chinese population. AB - The MHC class I-related molecules A and B (MICA and MICB) are stress-inducible cell surface antigens that are recognized by immunocytes bearing the receptor NKG2D, including intestinal epithelial Vdelta1 gammadelta T cells, which may play a role in immunological reaction in intestinal mucosa. The present study was aimed to investigate the association of the microsatellite polymorphisms in the intron 1 of MICB and the MICA-MICB haplotype with the susceptibility to ulcerative colitis (UC) in Chinese population. The microsatellite polymorphisms of MICB were genotyped in unrelated 127 Chinese patients with UC and 193 ethnically matched healthy controls by a semiautomatic fluorescently labeled PCR method. All the subjects were the Chinese with Han nationality. The frequency of MICB-CA18 was significantly higher in UC patients compared with the healthy controls (14.0% vs. 5.8%, P = 0.0016, Pc = 0.024, OR = 2.637, 95%CI: 1.443-4.820) and was increased in the female patients compared with the female healthy controls (18.3% vs. 4.1%, P = 0.0006, Pc = 0.0080, OR = 5.224, 95%CI: 1.940 14.069). Thus, MICB-CA18 is positively associated with UC and female UC patients in Chinese population. PMID- 16679068 TI - Doppler Tissue Imaging positive preejection velocity wave is a sign of non transmural necrosis: comparison with delayed-enhancement cardiac magnetic resonance. AB - AIMS: Our purpose was to test the hypothesis that Tissue Doppler Imaging (TDI) derived positive preejection velocity (PPV) is associated with transmural extent of necrosis in delayed-enhancement cardio-magnetic resonance (DE-CMR) in patients with reperfused myocardial infarction (MI). METHODS AND RESULTS: Longitudinal myocardial velocities were recorded by TDI in 24 patients with MI reperfused with primary angioplasty, using an Acuson-Sequoia equipment. The same day a CMR study was performed, including cine images in short axis and long axis views and DE images in the same views using a 3D-T1-Turbo-field-echo sequence, 15 min after administration of gadodiamide. Transmural extent of hyperenhancement in each segment was compared to presence or absence of PPV wave. A total of 384 segments were evaluated. Normo-hypokinetic segments (100%) showed a PPV wave, whereas it was only present in 53% of akinetic-dyskinetic segments (p=0.0005). One hundred percent of the segments with absent-mild DE showed a PPV wave; this percentage was lower in segments with intermediate and transmural DE (63 and 10%, p=0.001). The presence of PPV wave in an akinetic segment ruled out transmural necrosis with 97% sensitivity and 90% specificity. CONCLUSIONS: The absence of PPV is strongly associated to transmural necrosis in MI and therefore to absence of viability. PMID- 16679069 TI - Responses of dorsal spinal cord blood flow to innocuous cutaneous stimulation in anesthetized rats. AB - In urethane-anesthetized, artificially-ventilated rats, alterations in dorsal spinal cord blood flow (SCBF) were measured with a laser Doppler flowmeter in response to innocuous mechanical cutaneous stimulation. SCBF recorded at the T12 L1 level increased with brushing of the ipsilateral, but not contralateral, upper back, lower back or proximal hindlimb. Brushing of the forepaw, proximal forelimb or hindpaw had no effect on T12-L1 SCBF. SCBF recorded at the L4-6 level increased with brushing of the ipsilateral, but not contralateral, proximal hindlimb and hindpaw. Brushing of the forepaw, proximal forelimb, upper back or lower back had no effect on SCBF at the L4-6 level. None of these brushing stimuli produced significant changes in systemic arterial blood pressure or heart rate. Pretreatment with phenoxybenzamine, an alpha adrenoceptor blocking agent, resulted in a small but statistically significant attenuation in the response of SCBF to brushing. However, pretreatment with propranolol, a beta adrenoceptor blocking agent, or atropine, a muscarinic cholinergic receptor blocking agent, produced no such effect. These results indicate that innocuous mechanical cutaneous input can produce a segmentally-organized increase in regional SCBF, which may be mediated in part, by alpha adrenergic receptors. PMID- 16679070 TI - Modified shuttle test performance in hospitalized children and adolescents with cystic fibrosis. AB - BACKGROUND: The Modified Shuttle Test (MST) is a valid and sensitive measure of exercise capacity in adult CF patients. Recently, its validity in children has been demonstrated. The aim of this study was to demonstrate the utility of the MST as a measure of responsiveness to hospitalisation for i.v. antibiotic and supportive therapy in children and adolescents with CF. METHODS: 28 children and adolescents (40 admissions) performed a MST and lung function within 48 h of admission and discharge to hospital for administration of intravenous antibiotics. Mean age was 12.7 years and antibiotic therapy length was 14.7 days. RESULTS: Upon admission, the mean (S.D.) FEV(1) was 63 (19)% predicted, FVC was 80 (18)% predicted, FEF(25-75) 43 (29)% predicted and MST distance 718 (232) m. FEV(1) increased by 15% (p or = 2 ischemic MR. Only 18% of patients with preserved LV function had a significant ischemic MR, but up to 66% of patients with poor LV function had grade > or = 2 MR. The ratio of early transmitral flow velocity to mitral annulus early diastolic velocity was independently predicted by age, gender, LV ejection fraction, and ischemic MR severity. Even mild ischemic MR was associated with an increase in this noninvasive marker of LV filling pressures. PMID- 16679082 TI - Relation between postexercise abnormal heart rate recovery and myocardial damage evidenced by gated single-photon emission computed tomography. AB - Abnormal heart rate recovery (HRR) after exercise has been associated with increased cardiac mortality. The ability of gated myocardial perfusion single photon emission computed tomography (SPECT) to evaluate myocardial perfusion and function simultaneously might make it helpful in determining possible mechanisms that are involved in this finding. This study investigated the association between abnormal HRR and other indicators of risk for cardiovascular events. Patients (n = 1,296, 784 men; 57 +/- 11 years of age) who underwent exercise/technetium-99m sestamibi gated myocardial perfusion SPECT at rest were prospectively enrolled. Exercise treadmill testing was performed according to a symptom-limited Bruce's protocol. HRR was obtained from the subtraction of heart rate in the first minute of recovery after exercise treadmill testing from maximal heart rate during exercise. Myocardial perfusion SPECT was semi quantitatively analyzed using a 17-segment left ventricular model. Left ventricular ejection fraction was automatically calculated using quantitative gated SPECT software. In our study, patients with abnormal HRR were older, more frequently diabetic, and hypertensive and had previous myocardial infarction and myocardial revascularization, higher heart rate at rest and perfusion defect quantification scores, lower left ventricular ejection fraction, and larger left ventricular volumes than did patients with normal HRR. In multivariable analysis, age (p <0.0001), heart rate at rest (p <0.0001), left ventricular ejection fraction (p <0.0001), and perfusion defect extent and severity at rest (p = 0.038) were independent predictors of abnormal HRR. In conclusion, abnormal HRR was significantly associated with lower left ventricular ejection fraction and with perfusion defect extent and severity at rest, but not with gated SPECT markers of myocardial ischemia. Therefore, abnormal HRR may reflect myocardial damage. PMID- 16679083 TI - Procedural implications of intravascular ultrasound morphologic features of chronic total coronary occlusions. AB - Although the success rates of percutaneous coronary intervention of chronic total occlusions (CTOs) have improved, morphologic features are not well known. We analyzed experience at 4 centers where intravascular ultrasound (IVUS) was performed in 67 native artery CTO lesions (mean CTO duration 6.3 months) just after the lesion was crossed with a guidewire (n = 7) or after dilatation with a 1.5-mm (n = 46) or 2.0-mm (n = 14) balloon. IVUS detected calcium somewhere in the CTO in 96%; however, only 68% had mild calcium. IVUS identified a proximal end of the CTO in all lesions, but a distal end of the CTO in only 50%. An intramural hematoma was observed in 34% of CTOs, suggesting that the guidewire frequently entered the medial space during successful recanalization. CTOs were longer, vessel area was smaller, and total calcium index was greater in lesions with hematomas (p = 0.003, 0.05, and 0.03, respectively). Inadequate reflow after the procedure was observed in 9% and was associated with longer lesions and intralesional calcium. CTO length as measured with angiography was shorter than the length as measured with IVUS (p = 0.02). Calcium was detected on the angiogram in 61% (p = 0.054 vs IVUS). Most typical angiographic findings associated with a low rate of procedural success were not associated with different IVUS morphologies. In conclusion, CTO lesions had multiple small calcium deposits, intramural hematomas were common and were indicative of guidewire penetration into the medial space during the CTO procedure, especially in long calcified lesions in smaller vessels, and inadequate reflow after the procedure was correlated with more complex CTO morphology. PMID- 16679084 TI - Intravascular ultrasonic differences between aorto-ostial and shaft narrowing in saphenous veins used as aortocoronary bypass grafts. AB - Previous studies have reported differences in interventional complication rates that depend on saphenous vein graft (SVG) lesion location. However, little is known about morphologic differences between lesions in different SVG locations. We evaluated preintervention intravascular ultrasound (IVUS) images of 75 de novo SVG lesions (aorto-ostial, n = 15; shaft, n = 60) in 63 patients. IVUS data were measured at the minimal lumen area and at 2 proximal and 2 distal references. Positive remodeling was defined as a lesion site SVG area that was larger than the average of the 2 distal references. Shaft lesions more often contained soft plaque (60.0% vs 26.7%, p = 0.02). Minimal lumen areas were identical (4.5 +/- 2.9 vs 4.3 +/- 1.5 mm2, p = 0.3); however, plaque burden at the minimal lumen area was greater in shaft locations (79.3 +/- 9.4% vs 72.1 +/- 9.2%, p = 0.01). The frequency of positive remodeling in shaft versus aorto-ostial lesions was 70.2% versus 26.7% (p = 0.002). SVG shaft lesions have more soft plaque and larger plaque burdens and undergo positive remodeling more frequently than SVG aorto-ostial lesions. These IVUS differences may account for some of the location specific differences in interventional complications. PMID- 16679085 TI - Comparison of long-term (seven year) outcomes among patients undergoing percutaneous coronary revascularization with versus without stenting. AB - Coronary stents have markedly improved the short- and intermediate-term safety and efficacy of percutaneous coronary intervention by improving acute gains in luminal dimensions, decreasing abrupt vessel occlusion, and decreasing restenosis, yet the long-term benefit of coronary stenting remains uncertain. We examined long-term clinical outcomes of death, myocardial infarction, and repeat target vessel revascularization (TVR) among patients enrolled in the Duke Database for Cardiovascular Disease who underwent revascularization with percutaneous transluminal coronary angioplasty alone or stent placement from 1990 to 2002. Among 6,956 patients who underwent percutaneous revascularization, propensity modeling was applied to identify 1,288 matched patients with a similar likelihood to receive coronary stents according to clinical, angiographic, and demographic characteristics. Significant (p <0.05) predictors of stent placement included multivessel disease, diabetes, hypertension, recent myocardial infarction, decreased ejection fraction, and year of study entry. At a median follow-up of 7 years, although treatment with coronary stenting was associated with a significant and sustained decrease in repeat TVR (18.0% vs 28.1%, p = 0.0002) and the occurrence of death, myocardial infarction or TVR (39.2% vs 45.8%, p = 0.004), long-term survival did not significantly differ between treatment groups (19.9% vs 20.5%, p = 0.72). Outcomes of death and myocardial infarction did not significantly differ between patients who did and did not undergo repeat TVR. In conclusion, compared with angioplasty alone, revascularization with coronary stents provides a significant early and sustained decrease in the need for repeat revascularization, but stents do not influence long-term survival. PMID- 16679086 TI - Comparison of effects of bare metal versus drug-eluting stent implantation on biomarker levels following percutaneous coronary intervention for non-ST elevation acute coronary syndrome. AB - Drug-eluting stents (DESs) deliver biphasic (early and late) elution of anti inflammatory compounds. We therefore hypothesized that DESs would be associated with early reductions in inflammatory biomarker release after percutaneous coronary intervention (PCI). A total of 741 patients with non-ST-elevation acute coronary syndrome underwent PCI in the Randomized Trial to Evaluate the Relative PROTECTion against Post-PCI Microvascular Dysfunction and Post-PCI Ischemia among Anti-Platelet and Anti-Thrombotic Agents (PROTECT) Thrombolysis In Myocardial Infarction 30 study of eptifibatide and reduced-dose antithrombin compared with bivalirudin. Serial biomarkers C-reactive protein, troponin, creatine kinase-MB, soluble CD40 ligand, interleukin-6, prothrombin fragment F1.2, and RANTES (regulated on activation, normal T-cell expressed and secreted) were assessed through 24 hours after PCI. DES use was at the investigator's discretion. Patients treated with DESs (n = 665) versus bare metal stents (n = 139) were more likely to have patent arteries before PCI (92.0% vs 86.6%, p = 0.04), Thrombolysis In Myocardial Infarction myocardial perfusion grade 3 (57.9% vs 47.7%, p = 0.033), and the left anterior descending artery as the culprit artery (38.5% vs 18.3%, p <0.001). The increase in C-reactive protein and troponin was lower among patients undergoing DES implantation (median 2.1 vs 3.5 mg/L for C reactive protein, median 0.11 vs 0.41 ng/ml for troponin), even after adjustment for randomized treatment, clopidogrel before treatment, diabetes mellitus status, epicardial patency, left anterior descending artery location, and myocardial perfusion (p = 0.036 and p = 0.039, respectively). Interleukin-6 was lower with DESs on univariate analysis but not multivariate analysis. Creatine kinase-MB, soluble sCD40 ligand, prothrombin fragment F1.2, and RANTES did not differ by DES use. In conclusion, patients undergoing DES implantation achieved more reductions in periprocedural markers of inflammation and necrosis than patients receiving bare metal stents among those with non-ST-elevation acute coronary syndrome. PMID- 16679087 TI - On- versus off-label use of drug-eluting coronary stents in clinical practice (report from the American College of Cardiology National Cardiovascular Data Registry [NCDR]). AB - Limited data are available on the off-label use of drug-eluting stents (DESs) in clinical practice. We used data from the American College of Cardiology National Cardiovascular Data Registry to describe the rates and outcomes of DES use in 4 common off-label situations: ST-elevation myocardial infarction, in-stent restenosis, coronary artery bypass grafts, and chronic total occlusions. The rates of in-hospital adverse events for each off-label situation were determined and compared with the expected rates calculated from a validated model. From approval of DESs to the end of 2004, a total of 408,033 procedures involved placement of a DES. The use of DESs increased from 19.7% at the start of the study to 78.2% by the end of fourth quarter of 2004. Off-label use of DESs occurred in 24.1% of procedures. The rates of in-hospital adverse events with DES use in ST-elevation myocardial infarction, in-stent restenosis, coronary artery bypass grafting, and chronic total occlusions were lower than expected from the validated model. In conclusion, off-label use of DESs is common and is associated with a low rate of short-term adverse events. Randomized clinical trials are needed to confirm the efficacy of DESs in these clinical and angiographic settings. These data also highlight the importance of multicenter registries in tracking new technologies. PMID- 16679088 TI - Risk and determinants of myocardial injury during off-pump coronary artery bypass grafting. AB - Perioperative myocardial injury (PMI) after coronary revascularization (bypass surgery using cardiopulmonary bypass or percutaneous intervention) is strongly associated with future adverse events, such as death, myocardial infarction, and coronary intervention. The incidence, determinants, and prognostic significance of PMI after bypass surgery without cardiopulmonary bypass (off-pump surgery) are unknown. The study population comprised the patients who were randomized to off pump surgery in the Octopus Study. PMI was defined by a creatine kinase isoenzyme MB/total creatine kinase ratio of >5% during the first 48 hours, postoperatively. PMI occurred in 137 of 260 patients (52%). Using multivariate regression analysis, age, female gender, previous myocardial infarction, preoperative nitrate use, preoperative diuretic use, and number of grafts were independently associated with an increased risk of PMI during off-pump surgery. The presence of preoperative coronary collaterals showed a negative association with PMI. The occurrence of PMI had a crude odds ratio of 7.53 (95% confidence interval 1.59 to 35.63) for an adverse cardiac event at 1 year after off-pump surgery. This odds ratio changed little after adjustment for confounders (odds ratio 6.39, 95% confidence interval 1.41 to 28.93). In conclusion, more severe atherosclerotic disease and female gender were associated with an increased risk of perioperative myocardial injury during off-pump bypass surgery, although the presence of coronary collaterals appeared to be protective. Patients with perioperative myocardial injury during off-pump surgery were at a higher risk of adverse cardiac outcomes at 1 year. PMID- 16679089 TI - Effect of 80 mg/day simvastatin therapy on cardiovascular outcomes in adults with versus without metabolic syndrome. AB - It is not known whether high-dose statins can reduce the excess burden (and consequent adverse cardiovascular outcomes) of coronary artery disease in patients with metabolic syndrome compared with those without metabolic syndrome. The results of our study suggest that high-dose simvastatin may be required to reduce excess events in very high-risk cohorts with metabolic syndrome. PMID- 16679090 TI - Effect of atorvastatin on the recurrence rates of atrial fibrillation after electrical cardioversion. AB - To study the effect of atorvastatin on recurrence of atrial fibrillation (AF) after electrical cardioversion (EC), 48 patients with AF lasting 48 hours who were scheduled for EC were randomized to the atorvastatin (group I) and control (group II) groups. Six patients in group I (25%) and 2 patients in group II (8.3%) had spontaneous conversion before EC (p >0.05). The end point was the recurrence of AF during 3 months of follow-up. Eighteen patients in group I (12.5%) and 11 patients in group II (45.8%) had recurrence (p = 0.01, log-rank test). With the Cox proportional model, the predictors of recurrence included a body mass index of 25 to 30 kg/m2 (relative risk [RR] 0.07, 95% confidence interval [CI] 0.008 to 0.59), body mass index > or = 30 kg/m2 (RR 0.24, 95% CI 0.08 to 0.72), AF duration of > or = 3 months (RR 0.28, 95% CI 0.09 to 0.83), diabetes mellitus (RR 0.34, 95% CI 0.12 to 0.98), and left atrial diameter of > or = 45 mm (RR 0.23, 95% CI 0.07 to 0.74). Atorvastatin was associated with a significantly reduced risk of developing AF (unadjusted RR 0.23, 95% CI 0.064 to 0.82, p = 0.024). This association remained significant after adjustment for these predictors (adjusted RR 0.19, 95% CI 0.052 to 0.72, p = 0.01). High sensitivity C-reactive protein levels at baseline were not different between the 2 groups (p = 0.92). Although the high-sensitivity C-reactive protein levels decreased significantly 48 hours after EC compared with the baseline levels in group I (2.82 +/- 1.46 vs 2.56 +/- 1.3 mg/dl, p = 0.02), no significant change occurred in group II (2.87 +/- 0.8 vs 2.84 +/- 0.8 mg/dl, p = 0.09). In conclusion, atorvastatin decreased the recurrence rate of AF after EC. PMID- 16679091 TI - Usefulness of esophageal leads for determining the strategy of pulmonary vein ablation to avoid complications associated with the esophagus. AB - To avoid fatal complications after extensive pulmonary vein (PV) ablation, it has been proved important to comprehend the anatomic relation between the PVs and the esophagus. In 42 consecutive patients with atrial fibrillation, PV ostial isolation was performed using a basket catheter. The shortest distance and anatomic relation between the esophageal lead and PV ostium, determined by successful PV ostial isolation, was analyzed in biplane fluoroscopic views. In 18 left superior PVs (LSPVs) (43%), 13 left inferior PVs (32%) (LIPVs), and all the right PVs (group A), the shortest distance was > 10 mm in > or = 1 of the biplane fluoroscopic views. In 4 LSPVs (10%) and 2 LIPVs (5%) (group B), the shortest distance was < or = 5 mm in the fluoroscopic views. In the remaining PVs (group C), the esophagus was situated directly behind 10 LSPVs (24%) and 12 LIPVs (29%) (group C1), posteromedial to 1 LSPV (2%) and 9 LIPVs (22%) (group C2), and medial to 9 LSPVs (21%) and 5 LIPVs (12%) (group C3). The risk of esophagus-associated complications with ablation around the left PV ostia was suggested to be high in group B, very low in group A, and relatively low in group C. In group C3, extensive PV ablation might increase the risk of that complication. In conclusion, esophageal leads are useful for determining strategies for PV ablation to avoid esophagus-associated complications, because they enable comprehension of the anatomic relation between the PVs and the esophagus. PMID- 16679092 TI - Utility of plasma N-terminal brain natriuretic peptide as a marker of functional capacity in patients with chronic severe mitral regurgitation. AB - Plasma levels of N-terminal pro-brain natriuretic peptide (NT-pro-BNP) are elevated in severe mitral regurgitation, but their relation to functional capacity and cardiac remodeling is not well defined. We evaluated the role of NT pro-BNP as a marker of functional capacity, symptoms, and cardiac remodeling in 38 patients with severe degenerative mitral regurgitation and preserved left ventricular ejection fraction. The NT-pro-BNP levels increased progressively with New York Heart Association (NYHA) functional class: NYHA class I (geometric mean [GM] 97.1 pg/ml), NYHA class II (GM 169.8 pg/ml), and NYHA III (GM 457.6 pg/ml; p = 0.015). The end-systolic volume index (r = 0.52, p = 0.001), end-diastolic volume index (r = 0.46, p = 0.003), left atrial volume index (r = 0.4, p = 0.01), regurgitant volume index (r = 0.38, p = 0.02), regurgitant fraction (r = 0.46, p = 0.003), and end-diastolic sphericity index (r = 0.56, p <0.001) all correlated significantly with NT-pro-BNP. The NT-pro-BNP levels correlated significantly with the exercise parameters: maximum oxygen uptake (r = -0.6, p <0.001), exercise time (r = -0.52, p <0.001), and oxygen pulse (r = -0.57, p <0.001). In contrast, only weak correlations were obtained between the exercise and echocardiographic variables. NT-pro-BNP was a strong independent predictor of maximum oxygen uptake (p = 0.001). In conclusion, the results of this study have demonstrated that NT-pro-BNP increases progressively with worsening symptoms, is linked to the extent of LV remodeling, and is an independent predictor of functional capacity. NT-pro-BNP may have a role in the optimal treatment of patients with severe mitral regurgitation. PMID- 16679093 TI - Relations of inflammation and novel risk factors to valvular calcification. AB - Investigators have suggested that inflammation may play a role in the pathogenesis of valve calcium. Participants in the Framingham Heart Study's offspring cohort had systemic levels of C-reactive protein, intercellular adhesion molecule-1, interleukin-6, and monocyte chemoattractant protein-1 measured at examination cycle 7. Mitral annular calcium, aortic annular calcium, aortic sclerosis, and aortic stenosis were assessed by echocardiography at examination cycle 6. Logistic regression was used to examine the odds of valvular calcium per 1 unit increase in inflammation (ISUM), a summary statistic of all normalized deviates of the individual markers. Two thousand six hundred eighty three participants (mean age 61 +/- 10 years; 52% women) were analyzed: 8.2% (n = 216) had > or = 1 calcified valve or annulus; 89 had mitral annular calcium, 78 had aortic annular calcium, 135 had aortic sclerosis, and 33 had aortic stenosis. Participants with valvular calcium were older and were more likely to have hypertension and diabetes mellitus. Participants with valve calcium had higher median levels of all markers. For each log unit increase in ISUM, after adjustment for age and gender, there was an associated 1.1-fold increased odds of > or = 1 calcified valve (p = 0.02); the odds ratios were no longer significant after adjustment for cardiovascular disease risk factors (odds ratio 1.0, 95% confidence interval 0.9 to 1.1). Similar results were obtained for the individual markers and the odds of > or = 1 calcified valve. In conclusion, inflammatory markers were elevated in patients with valvular calcium. Our findings suggest that much of the observed association between systemic inflammatory markers and valvular calcium may be due to shared risk factors. PMID- 16679094 TI - Predictive value of noninvasive coronary angiography with multidetector computed tomography to detect significant coronary stenosis before valve surgery. AB - Quantitative coronary angiography (QCA) is routinely performed before valve surgery for severe acquired valvular disease. This technique is relatively invasive, especially in a population with an average risk for significant coronary stenosis. Multidetector computed tomography (MDCT) coronary angiography allows the noninvasive evaluation of the coronary anatomy. The aim of this prospective study was to evaluate the predictive values of 16-slice MDCT in the detection of significant coronary stenosis (> or = 50%) before valve surgery in patients with severe valvular disease without known coronary artery disease and average risk, in comparison with conventional QCA. Forty patients with severe acquired valvular disease (mean age 70 +/- 8.6 years; 20 women; 27 with severe aortic stenosis) underwent coronary MDCT 2 days before cardiac catheterization with QCA. The mean heart rate was 64.7 +/- 8.8 beats/min (range 41 to 78). Four hundred fifty-eight of 600 coronary artery segments (77.3%) were considered assessable by MDCT. In a per-segment analysis, the sensitivity of MDCT for the detection of significant coronary lesions > or = 50% was 77.7%, the specificity was 98%, the positive predictive value was 42.4%, and the negative predictive value was 99%. The main cause of false-positive or false-negative results or nonassessable evaluations was severe coronary calcification. In a per-patient analysis, in comparison with QCA, MDCT correctly classified 33 of 40 patients (82.5%). In conclusion, in patients with an average risk for coronary stenosis before valve surgery, MDCT coronary angiography detected significant obstructive coronary artery disease, with a 99% NPV. PMID- 16679095 TI - Time course of pressure gradient response after first alcohol septal ablation for obstructive hypertrophic cardiomyopathy. AB - Alcohol septal ablation (ASA) causes remodeling of the upper septum and left ventricular outflow tract (LVOT) and reduction in the LVOT gradient. The time course of gradient reduction early after ASA has not been established. This study characterized the time course of gradient response early after ASA. Patients underwent clinical assessment and transthoracic echocardiography at baseline and immediately, 3 days, 3 months, and 1 year after ASA. Forty-seven patients underwent ASA. The baseline LVOT gradient was 98 +/- 48 mm Hg. Three-month echocardiographic success, defined as > or = 50% gradient reduction from baseline, was achieved in 41 procedures (87%); thus, there were 6 failures. On the basis of percentage reduction in LVOT gradient at 3 days, 2 distinct subgroups of the success group were identified. These were monophasic success (> or = 50% gradient reduction at 3 days and 3 months, n = 25) and triphasic success (< 50% gradient reduction at 3 days but > or = 50% gradient reduction at 3 months, n = 16). LVOT gradient in the triphasic success group was similar to that in the failure group at 3 days (81 +/- 28 vs 99 +/- 31 mm Hg, p = NS) but similar to that of the monophasic success group at 3 months (24 +/- 20 vs 12 +/- 16 mm Hg, p = NS) and at 1 year (27 +/- 24 vs 13 +/- 20 mm Hg, p = NS). In conclusion, many patients who undergo ultimately successful ASA demonstrate triphasic LVOT gradient response patterns, with a large gradient 3 days after the procedure. PMID- 16679096 TI - Impact of enzyme replacement therapy on cardiac morphology and function and late enhancement in Fabry's cardiomyopathy. AB - The present study evaluated the evolution of cardiac morphology, function, and late enhancement as a noninvasive marker of myocardial fibrosis, and their inter relation during enzyme replacement therapy in patients with Fabry's disease using magnetic resonance imaging and color Doppler myocardial imaging. Late enhancement, which was present in up to 50% of patients, was associated with increased left ventricular mass, the failure of a significant regression of hypertrophy during enzyme replacement therapy, and worse segmental myocardial function. Late enhancement may predict the effect of enzyme replacement therapy on left ventricular mass and cardiac function. PMID- 16679097 TI - Cardiac involvement in the Churg-Strauss syndrome. AB - Churg-Strauss syndrome (CSS) is a rare systemic disease characterized by necrotizing vasculitis and peripheral eosinophilia. Cardiac involvement is considered common and is given a high rank among the causes of morbidity and mortality. The aim of this study was an update on the cardiac manifestations of this syndrome using a noninvasive approach. Sixteen patients with CSS were compared with a gender- and age-matched group of 20 healthy subjects. All patients but 1 were receiving treatment (steroids and/or immunosuppressive drugs). According to the Birmingham vasculitis activity score, 12 patients were in an active phase, and 4 were in drug-induced remission. All subjects underwent M-B-mode echocardiography and Doppler tissue echocardiography. Heart failure, life-threatening arrhythmias, and other prominent manifestations of heart disease were not observed. No differences were found in left ventricular diameter, volume, mass, or ejection fraction. The 2 groups did not differ in right ventricular diameter and pulmonary pressure. Few and nonspecific changes were detected by 2-dimensional echocardiography, including subclinical pericardial effusion and mitral regurgitation, in fewer than half the subjects. Subjects with CSS showed an impairment of ventricular relaxation. Changes were more prominent in the right ventricle. The peak velocity (PV) of early diastolic tricuspid inflow (E) was about 8% less than in controls, and the velocity of late diastolic inflow (A) was 35% greater. The E/A(PV) ratio was, on average, 33% less. In the left ventricle, E(PV) was 11% less and A(PV) 11% greater. The E/A ratio was decreased by 22%. Doppler analysis of tissue kinetics confirmed these indications. In the right ventricle, E(PV) was decreased by 10% and A(PV) was increased by 20% in the patient group. The E/A(PV) ratio was decreased by 29%. In the left ventricle, in which different sites were sampled, the average changes were -15%, +1%, and -23%, respectively. In the left ventricle, the velocity of systolic contraction was also decreased by 12%. Because of the small group size, only some of these differences were statistically significant. In conclusion, these moderate changes, devoid of clinical correlates, contrast with early reports emphasizing cardiac morbidity and poor prognosis in this syndrome. PMID- 16679098 TI - Relation of angiotensin-converting enzyme inhibitor treatment to insulin-like growth factor-1 serum levels in subjects >65 years of age (the InCHIANTI study). AB - Observational studies have shown that the use of angiotensin-converting enzyme (ACE) inhibitors is associated with the maintenance of greater muscle strength and physical performance in older subjects. However, the mechanism that underlies these beneficial effects remains poorly understood. Because ACE inhibitors block the production of angiotensin II, which is a potent inhibitor of insulin-like growth factor-1 (IGF-1) production, it was hypothesized that treatment with ACE inhibitors is associated with higher levels of IGF-1. This hypothesis was tested in 745 subjects (417 women, 328 men) enrolled in the Invecchiare in Chianti study. Of these, 160 were receiving ACE inhibitors. The association between ACE inhibitor use and serum IGF-1 was tested by linear regression models. After adjusting for multiple potential confounders, serum levels of total IGF-1 were significantly higher in participants receiving ACE inhibitors (mean +/- SD 129.0 +/- 56.1 ng/ml) compared with the rest of the study population (mean +/- SD 116.5 +/- 54.8 ng/ml) (p <0.001). Participants with short (<3 years) and long (3 to 9 years) treatment durations had higher serum IGF-1 levels than participants who were not receiving ACE inhibitor treatment, but the difference was statistically significant only for the short-duration group (p <0.05). In conclusion, in older subjects, treatment with ACE inhibitors for <3 years is associated with significantly higher levels of IGF-1. This may be 1 of the mechanisms by which ACE inhibitors might slow the decreases in muscle strength and physical function that are often observed in older subjects. PMID- 16679099 TI - N-terminal pro-B-type natriuretic peptide and B-type natriuretic peptide for identifying coronary artery disease and left ventricular hypertrophy in ambulatory chronic kidney disease patients. AB - Elevated natriuretic peptide levels are common in patients with chronic kidney disease (CKD), as is the presence of coronary artery disease (CAD) and left ventricular hypertrophy (LVH). It was hypothesized that N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) and B-type natriuretic peptide (BNP) levels could identify CAD and LVH in asymptomatic patients with CKD. Clinical, laboratory, and echocardiographic data were collected prospectively in 54 ambulatory patients with CKD not requiring dialysis. CAD was defined by previous myocardial infarction or coronary revascularization. The median age was 70 years (interquartile range [IQR] 57 to 76). Fourteen patients (26%) had CAD, and 30 (56%) had LVH. Median NT-pro-BNP was 724 pg/ml (IQR 168 to 2,950), median BNP was 137 pg/ml (IQR 31 to 391), and the median glomerular filtration rate (GFR) was 31 ml/min/1.73 m2 (IQR 21 to 42). A strong correlation was found between NT-pro-BNP and BNP levels (R = 0.74, p <0.0001), but only moderate correlations were found between NT-pro-BNP and GFR (R = -0.45, p = 0.0006) and between BNP and GFR (R = 0.38, p = 0.005). There was no trend of an increase in the prevalence of LVH or CAD with decreasing GFR. However, across progressive NT-pro-BNP and BNP quartiles, the prevalences of LVH and CAD increased significantly. Receiver operating characteristic curves showed that these 2 markers are similar and significant predictors for indicating LVH (area under the curve [AUC] 0.72, p = 0.005 for NT-pro-BNP; AUC 0.72, p = 0.007 for BNP) and CAD (AUC 0.80, p = 0.001 for NT-pro-BNP; AUC 0.82, p = 0.0004 for BNP; p = 0.45 for NT-pro-BNP vs BNP). In conclusion, NT-pro-BNP and BNP levels are significant and equivalent indicators of CAD and LVH in asymptomatic patients with CKD. PMID- 16679100 TI - Left ventricular systolic and diastolic function by echocardiogram in pseudoxanthoma elasticum. AB - Pseudoxanthoma elasticum is an inherited systemic disease of connective tissue with possible cardiac involvement. Nineteen patients with pseudoxanthoma elasticum without a history of cardiac disease were investigated by echocardiography using standard measurements and tissue Doppler imaging. Systolic function was normal, but diastolic parameters were abnormal in 7 patients. Explanations for these abnormalities could be silent myocardial ischemia due to early coronary involvement and/or the direct consequences of ultrastructural defects of the elastic tissue of the heart. PMID- 16679101 TI - Cardiovascular disease risk stratification with stress single-photon emission computed tomography technetium-99m tetrofosmin imaging in patients with the metabolic syndrome and diabetes mellitus. AB - The metabolic syndrome represents a constellation of risk factors caused by insulin resistance, dyslipidemia, hypertension, and obesity, resulting in elevated coronary disease risk. From a multicenter prospective registry of 7,849 patients, the relation among the metabolic syndrome, diabetes, and risk stratification with stress technetium-99m tetrofosmin single photon-emission computed tomography (SPECT) was evaluated. The percentage of stress myocardial defects was calculated as < or = 5%, 5.1% to 10%, 10.1% to 15%, and > 15%. A Cox proportional-hazards model was used to estimate cardiovascular death or myocardial infarction (n = 752). Of 7,849 patients, 42% had the metabolic syndrome. Patients with the metabolic syndrome had an 84% 2-year event-free survival rate, lower than patients with normal metabolic status (p <0.0001). In patients with the metabolic syndrome, the percentage of moderate to severely abnormal SPECT findings ranged from 11% to 44% for those with 3 to 5 risk factors for the metabolic syndrome. There was an additive relation between the number of risk factors for the metabolic syndrome and the extent and severity of abnormalities in SPECT findings (p <0.0001). Patients with 5 risk factors for the metabolic syndrome were at the greatest risk, with hazard ratios from 7.8- to 14.1-fold for mild to severely abnormal SPECT findings. For diabetic patients requiring combined oral and insulin therapy, relative risk ratios increased from 15 to 21.4 for patients with > 5% to > 15% stress myocardial perfusion defects. In conclusion, cardiovascular prognosis is affected by the degree of metabolic dysfunction, and stress-induced reductions in myocardial perfusion provide an accurate means for near-term risk stratification. PMID- 16679102 TI - More about yawning. PMID- 16679103 TI - Peripheral arterial disease is a marker of risk for abdominal aortic aneurysms in patients with coronary artery disease. PMID- 16679104 TI - Retinal arterial signs and coronary heart disease. PMID- 16679106 TI - Severe ataxia caused by amiodarone. PMID- 16679105 TI - High iron stores and impaired endothelial function in prediabetic subjects. PMID- 16679107 TI - Does the Veterans Affairs health system have significant racial bearings on the postmyocardial infarction survival outcomes among diabetics? PMID- 16679108 TI - Postoperative atrial fibrillation and obesity. PMID- 16679109 TI - Understanding the internet, website design and intranet development: a primer for radiologists. AB - The internet has become an essential part of daily life for almost all radiologists and yet few fully understand how this works or how best to harness the technology within the workplace. This article will explore the basics of computer networking which has allowed the internet to become a valuable resource. In addition, the process of designing and implementing a website or intranet site for the benefit of radiology departmental administration and education will be discussed. The options of how to develop a website, what to include, and how to achieve this using easy to use, freely available and low-cost software will also be explored. PMID- 16679110 TI - Gastrointestinal and hepatobiliary radiology. AB - This is the fifth in the series of short reviews of internet-based radiological learning resources and will focus on gastrointestinal (GI) and hepatobiliary radiology. Below are details of a few of the higher quality resources currently available. Most of the sites cater for medical students and trainee or non specialist radiologists, but may be also be of interest to specialists, especially for use in teaching. Hyperlinks are available in the electronic version of this article and were all active at the time of going to press (May 2006). PMID- 16679111 TI - The imaging appearances of intracranial CNS infections in adult HIV and AIDS patients. AB - The spectrum of pathology affecting the central nervous system (CNS) in patients suffering from acquired immunodeficiency syndrome (AIDS) is broad and comprises predominantly opportunistic infections and neoplasms. It is estimated that approximately one-third of all patients with AIDS develop neurological complications. The organisms responsible for AIDS are human retroviruses: primarily the human immunodeficiency virus type 1 (HIV). In this review we shall focus on the neurological complications of HIV and AIDS which are applicable to the more frequently occurring intracranial infective organisms. Attention will be paid specifically to those CNS manifestations occurring in the adult HIV and AIDS population as infection in the paediatric HIV and AIDS group, although bearing some similarities, demonstrates some important differences. PMID- 16679112 TI - Salvaging and maintaining non-maturing Brescia-Cimino haemodialysis fistulae by percutaneous intervention. PMID- 16679113 TI - Salvaging and maintaining non-maturing Brescia-Cimino haemodialysis fistulae by percutaneous intervention. AB - AIM: To report our experience of the salvage of non-maturing Brescia-Cimino dialysis fistulae by percutaneous intervention. MATERIALS AND METHODS: Twenty-two patients (15 men, 68%; mean age: 58 years range: 42-79) with non-maturing Brescia Cimino fistulae were treated by percutaneous angioplasty. Fistulae were created a mean of 2.7 months (range, 1-13 months) before intervention. The size of balloons used was 4mm for the arterial and anastomotic stenosis and 5mm or 6mm for the venous stenosis. RESULTS: On initial venography, venous stenosis (17 fistulae) or occlusions (five fistulae) were responsible for non-maturation. Stenoses or occlusions were adjacent to the arterial anastomoses in 18 patients and in the venous outflow (future puncture zone) in four patients. Additionally, a focal arterial stenosis was present in one and occlusion of the innominate vein in one other patient. Clinical success (initiation of dialysis) was achieved in 21/22 patients (95.5%). Twelve patients required 18 repeat angioplasties for recurrent stenosis. Two patients had small extravasation that required no further treatment. Over a follow-up period of 5-40 months (mean 14.6 months) 12 patients required repeat angioplasty. The mean interval between the initial angioplasty and subsequent intervention was 7.5 month (range 3-12 months). Primary patency after intervention at 6 and 12 months was 82 and 28%. Secondary patency at 6 and 12 months was 95 and 85%. CONCLUSION: Percutaneous intervention can effectively salvage non-maturing Brescia-Cimino fistulae. As repeat angioplasty is often necessary to maintain function, careful surveillance is necessary. PMID- 16679114 TI - Diagnosis of angiomyolipoma using computed tomography-region of interest < or = 10 HU or 4 adjacent pixels < or =-10 HU are recommended as the diagnostic thresholds. AB - AIM: To study and compare the diagnostic accuracy of region of interest (ROI) density measurement and pixel mapping [computed tomography (CT) density of individual pixels] for the diagnosis of renal angiomyolipoma (AML) using CT. MATERIALS AND METHODS: A study group of histologically proven AMLs was compared with a control group of histologically proven renal cell cancers, normal renal parenchyma, and simple renal cysts. The mean tissue density (ROI circle) and a pixel density map were recorded. The diagnostic accuracy of various thresholds of ROI and pixel mapping values were compared using receiver operating characteristic curves. RESULTS: Twenty-two AMLs, 16 renal cell carcinomas (RCCs), 30 simple cysts, and 30 sites of renal parenchyma were evaluated. The mean (+/-1 SD) density of the AMLs was significantly lower [-15.2(20.8) units] than the three control groups [+36.0(8.1) units, +5.4(3.4) units and +22.2(46.5) units for RCC, renal cyst and parenchyma respectively; p < 0.001 (analysis of variance)]. The sensitivities and specificities of the ROI diagnostic thresholds of < or =0 units, < or =-10 units and < or =-20 units were 77 and 97%, 73 and 100% and 50 and 100%, respectively. Using pixel mapping [diagnostic thresholds of either a line of 4 pixels < or =-10 units or a square of 4 pixels < or =-10 units] the sensitivity improves to 86% with a specificity of 97%. CONCLUSION: Although a ROI threshold value of < or =-10 units has a very high specificity (100% in the present study) the sensitivity is modest at only 73%. Pixel mapping is more sensitive for recognizing small clusters of fat. In practice, both methods can be recommended for the analysis of suspected AMLs. ROI density measurement is convenient when analysing large areas of suspected fat and < or =-10 units should be used as the diagnostic threshold. When faced with small lucent areas or indeterminate values after ROI analysis, pixel mapping is recommended using a line of 4 pixels < or =-10 units or a square of 4 pixels < or =-10 units as the discriminating thresholds. PMID- 16679115 TI - Myocardial enhancement pattern in patients with acute myocardial infarction on two-phase contrast-enhanced ECG-gated multidetector-row computed tomography. AB - AIM: To evaluate the myocardial enhancement pattern of the left ventricle on two phase contrast-enhanced electrocardiogram (ECG)-gated multidetector computed tomography (MDCT) images in patients with acute myocardial infarction (AMI). METHODS: Two-phase contrast-enhanced ECG-gated MDCT examinations were performed in 16 patients with AMI. The presence, location and pattern of myocardial enhancement were evaluated. MDCT findings were compared with the catheter angiographic results. RESULTS: Subendocardial (n = 9) or transmural (n = 6) area of early perfusion defects of the myocardium was detected in 15 of 16 patients (94%) on early-phase CT images. Variable delayed myocardial enhancement patterns on late-phase CT images were observed in 12 patients (75%): (1) subendocardial residual perfusion defect and subepicardial late enhancement (n = 6); (2) transmural late enhancement (n = 1); (3) isolated subendocardial late enhancement (n=1); and (4) isolated subendocardial residual perfusion defect (n = 2). On catheter angiography, 14 of 15 corresponding coronary arteries showed significant stenosis. CONCLUSION: Variable abnormal myocardial enhancement pattern was seen on two-phase, contrast-enhanced ECG-gated MDCT in patients with AMI. Assessment of myocardial attenuation on CT angiography gives additional information of the location and extent of infarction. PMID- 16679116 TI - Screen-detected mucinous breast carcinoma: potential for delayed diagnosis. AB - AIM: To describe the imaging features of 34 screen-detected mucinous carcinomas lesions. MATERIALS AND METHODS: The BreastScreen Western Australia (WA) database between January 1991 and December 2003 was searched. During this period, 214,507 women were screened and 2745 cases of invasive carcinoma and 45 cases of mucinous carcinoma were recorded. Case notes, radiology films and pathology reports of patients with mucinous carcinoma were reviewed. Thirty-four radiologically detected pure mucinous carcinomas are described. RESULTS: Of the pure mucinous carcinomas, the average age at diagnosis was 65 years (range 48-82 years), which was higher than that of other women with breast cancer (average age 60 years) screened at BreastScreen WA. Characteristic mammographic features of mucinous carcinoma are well-circumscribed masses with lobulated margins (26/34). Only 39% (11/28) of tumours were detected at ultrasound, as the smaller lesions less than 15mm in diameter were often isoechoic with normal fat. Where histological grade was reported at excision, most (25/26) were low to medium-grade tumours (Bloom, Richardson and Elston grade I and II). A significant number of lesions (13/34) were evident on the previous screening examination where they were misinterpreted as benign lesions. However, none of these cases had positive axillary lymph nodes at final diagnosis. CONCLUSION: Although mammographically benign appearances of mucinous carcinoma caused a delay in diagnosis in 38% of the present cases, mucinous breast carcinomas have a favourable prognosis, as they are often low grade tumours and rarely metastasize. Delay in diagnosis for these tumours in a screening programme may not lead to a significant adverse outcome for most women. PMID- 16679117 TI - Is general practitioner access to breast imaging safe? AB - AIM: The aim of this study was to assess the consultant radiologist run open access breast radiology service (OAR) to investigate whether the system was safe or whether cancers were being missed. METHODS: A retrospective review of the national cancer registry database to identify patients presenting with symptomatic breast cancer in the catchment area of the Royal Glamorgan Hospital (RGH) from April 2000 to April 2002 was performed. Pathology, radiology and outpatient records were reviewed to identify patients previously assessed at the RGH. RESULTS: Fifty-four patients with breast cancer were diagnosed via the OAR and 159 by the breast clinic (BC). Twelve patients with breast cancer were diagnosed after their initial presentation. Eight patients had been previously seen for benign breast lesions. Four patients had missed breast cancers (two were initially seen via the BC and two via the OAR). A significant difference in the number of cancers missed by the two referral routes was not observed (p = 0.221). CONCLUSION: OAR is as accurate a means of diagnosing breast cancer as traditional rapid access BCs. Women presenting with discrete lumps with no radiological abnormality should still undergo assessment with clinical fine core-biopsy. PMID- 16679118 TI - A comparison of patient skin doses before and after replacement of a neurointerventional fluoroscopy unit. AB - AIM: After several embolization patients presented with radiation-induced skin injury in our neuroradiology centre, replacement of the centre's interventional fluoroscopy unit was prioritized. The aims of the present study were to compare the maximum skin dose delivered to each patient by the old and new units, to devise a method of estimating skin dose from the displayed dose-area product and to set local reference doses. MATERIALS AND METHODS: On the old unit, skin dose was measured using thermoluminescent dosimeters on 12 patients undergoing Gugliemi detachable coil embolization. Similar skin dose measurements were undertaken and the dose-area product was recorded for a further 12 patients on the new unit. RESULTS: The maximum skin dose measured on each patient on the old and new units had a mean of 2.2 Gy and 0.47 Gy, respectively, and a maximum of 4.1 Gy and 1.0 Gy, respectively. Maximum dose delivered to patients' skin by the new equipment was less than a quarter of the dose from the old equipment (p < 0.0001). CONCLUSION: The large reductions in skin dose reduced the risk of patients suffering radiation injury and confirmed the validity of replacing ageing interventional fluoroscopy equipment with modern equipment that incorporates dose management systems. As patient skin dose was correlated with dose-area product, local reference dose levels were set in terms of dose-area product; this enabled the operator to monitor the likely maximum patient skin dose during embolization procedures. Other centres could use a similar method to set their own reference doses. PMID- 16679119 TI - Accessory extensor carpi radialis brevis muscle, a pseudomass of the distal forearm: ultrasound and MR appearances -case report and literature review. PMID- 16679120 TI - Ultrasound diagnosis of clinically undetected Clostridium difficile toxin colitis. PMID- 16679121 TI - HIV-associated hypervascular primary Burkitt's lymphoma of the liver. PMID- 16679123 TI - Abdominal wall hernia. In brief. PMID- 16679124 TI - Abdominal wall hernia. PMID- 16679125 TI - Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells. AB - Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility. PMID- 16679126 TI - Clinical measurement and evaluation of vertical dimension. 1982. PMID- 16679127 TI - Alveolar distraction osteogenesis and implant placement in a severely resorbed maxilla: a clinical report. AB - Atrophy of maxillary alveolar bone can result in severe defects of the alveolar ridge. Such defects can make it difficult to achieve esthetic and functional restorations. This clinical report describes the use of alveolar distraction osteogenesis to promote the formation of new bone and soft tissue in a large, severely resorbed segment of maxillary alveolus. PMID- 16679128 TI - Provisional anterior tooth replacement using nonimpregnated fiber and fiber reinforced composite resin materials: a clinical report. AB - The loss of anterior teeth is often a serious esthetic concern. While conventional fixed partial dentures and implant-supported restorations may be the treatments of choice, nonimpregnated fibers (NFs) and fiber-reinforced composite (FRC) resins offer a conservative alternative for improving esthetics. This article describes 2 clinical situations in which NF glass ribbon and FRC were successfully used to provisionally restore anterior edentulous areas in an esthetic, functional, and timely manner. PMID- 16679129 TI - Immediate nasal prosthetic rehabilitation following cytomegalovirus erosion: a clinical report. AB - The immediate prosthetic rehabilitation of a patient with erosive cutaneous cytomegalovirus (CMV) is presented. Although CMV is a major cause of morbidity and mortality in immunocompromised patients, skin lesions are rarely discussed in the literature. Recognition of the susceptibility of medically compromised patients to infections that can cause extraoral and intraoral deformity is essential to prevention, early diagnosis, and prompt intervention of a life threatening destructive debilitation. A method for rapidly fabricating a nasal prosthesis is described, and the importance of prompt esthetic and psychological rehabilitation is reviewed. PMID- 16679130 TI - Dynamometric assessment of the mechanical resistance of porcelain veneers related to tooth preparation: a comparison between two techniques. AB - STATEMENT OF PROBLEM: There is little agreement regarding a palatal extension of the preparation for porcelain veneers, as it represents a more invasive technique than a preparation limited to the facial surface of a tooth. PURPOSE: The purpose of this study was to detect the stress in maxillary anterior teeth restored with porcelain veneers and compare the resistance to fracture of porcelain veneers prepared using different preparation designs. MATERIAL AND METHODS: Forty-five maxillary anterior teeth were restored with porcelain veneers and divided into 9 groups as follows: Ca, canines with no preparation; Ca-Ch, canines with palatal chamfer preparation; Ca-W, canines with window preparation; LI, lateral incisors with no preparation; LI-Ch, lateral incisors with palatal chamfer preparation; LI W, lateral incisors with window preparation; CI, central incisors with no preparation; CI-Ch, central incisors with palatal chamfer preparation; CI-W, central incisors with window preparation. Shear-flexural fracture tests were performed. The fractured specimens were subjected to scanning electron microscope (SEM) analysis. Data were statistically analyzed with univariate analysis of variance and the Tukey post hoc test for multiple comparisons (alpha=.05). RESULTS: The following mean fracture load values (N) were recorded: Ca, 395 +/- 6; Ca-Ch, 310 +/- 8; Ca-W, 322 +/- 8; LI, 309 +/- 8; LI-Ch, 242 +/- 6; LI-W, 225 +/- 8; CI, 298 +/- 8; CI-Ch, 255 +/- 8; CI-W, 221 +/- 6. The SEM analysis showed that both adhesive and cohesive fractures were primarily concentrated at the cervical region. Statistical analysis showed that both the type of tooth and the design of the preparation significantly influenced the resistance to fracture of the restored teeth (P<.001). CONCLUSION: The chamfer preparation is recommended for central incisors, whereas the window preparation showed better results for canines. Both preparations can be adopted in the restoration of lateral incisors. PMID- 16679131 TI - Marginal accuracy of interim restorations fabricated from four interim autopolymerizing resins. AB - STATEMENT OF PROBLEM: One of the most important requirements for interim restorations is good marginal adaptation. Polymerization shrinkage of interim restorative materials can jeopardize the marginal integrity of interim restorations. PURPOSE: The purpose of this study was to compare the marginal accuracy of interim restorations made with 4 interim materials. MATERIAL AND METHODS: A direct technique was used to fabricate 44 interim restorations with 4 materials: Protemp 3 Garant, Trim II, Tempron, and Acropars (n=11). The interim restorations were made on a prepared molar-shaped metal die with a vinyl polysiloxane impression as a matrix. Marginal discrepancy of interim restorations was measured at the midpoint of buccal, palatal, mesial, and distal surfaces of metal die finish line with a microscope at x100 magnification. Comparisons were made with 1-way analysis of variance and the Duncan multiple range test (alpha=.05). RESULTS: The mean marginal discrepancies of Protemp 3 Garant, Trim II, Tempron, and Acropars were 0.059, 0.063, 0.068, and 0.102 mm, respectively. Acropars exhibited the most marginal discrepancies and was significantly different from the other materials tested (P<.001). However, there were no statistical differences between the other 3 materials tested. CONCLUSIONS: Interim restorations made from the Bis-GMA and conventional acrylic resins tested produced comparable marginal fit. Acropars demonstrated significant increases in marginal gap size. PMID- 16679132 TI - Influence of dentin bonding agents and polymerization modes on the bond strength between translucent fiber posts and three dentin regions within a post space. AB - STATEMENT OF PROBLEM: Debonding is the most frequent failure encountered with translucent fiber posts and usually occurs along the post space dentin-adhesive interface. PURPOSE: The purpose of this study was to evaluate the effect of different dentin bonding agents and polymerization modes on the bond strength between translucent fiber posts and root dentin in different regions of the post space. MATERIAL AND METHODS: Forty maxillary canines with similar root lengths were selected, sectioned at the cemento-enamel junction, and the roots were endodontically treated. Following post space preparation, the roots were divided into 4 groups of 10 specimens each, and the post spaces were treated with 1 of 4 different dentin bonding agents: light-polymerized, single-bottle bonding agent Excite (Group EX); dual-polymerized, single-bottle bonding agent Excite DSC (Group EX-DSC); self-etching primer Clearfil Liner Bond 2V with a light polymerized bonding agent, Bond A (Group CL-LC); or self-etching primer Clearfil Liner Bond 2V with a dual-polymerized bonding agent, Bond A+B (Group CL-DC). Translucent fiber posts (D.T. Light-Post), 2.2 mm in diameter, were luted (Panavia F) in each specimen after respective dentin bonding procedures. The roots were cut into 3-mm-thick sections, perpendicular to the long-axis in cervical, middle, and apical post space dentin. Push-out tests were performed with a universal testing machine at a crosshead speed of 0.5 mm/min, and bond strength values (MPa) were calculated by dividing the force at which bond failure occurred by the bonded area of the post. The data were analyzed with 1- and 2-way analysis of variance and Tukey multiple comparison tests (alpha=.05). Dentin adhesive bonding mechanisms in different regions of the post spaces were evaluated with a scanning electron microscope. RESULTS: The highest mean bond strength values were obtained for Group CL-LC (18.3 +/- 4.1 MPa). The dual polymerized bonding agent resulted in significantly lower bond strength (P<.001) in combination with self-etching primer (Group CL-DC) (13.2 +/- 2.5 MPa). The light-polymerized and dual-polymerized single-bottle bonding agents provided similar bond strengths (12.7 +/- 5.0 for EX; 13.5 +/- 5.3 for EX-DSC). The regional bond strength values of single-bottle bonding agents were reduced significantly in apical post space dentin (P<.001). Self-etching primers did not demonstrate regional differences in post space dentin bonding and dense resin tags were apparent. CONCLUSION: Data suggests that the self-etching primer system used in this study was unaffected by the morphological variations in the post space dentin compared to the single-bottle bonding agents. Dual polymerization did not improve the bond strength values of the bonding agents tested. PMID- 16679134 TI - Prosthodontic and psychological factors in treating patients with congenital and craniofacial defects. AB - This article addresses treatment of patients with various types of congenital defects, including partial and total anodontia, hypohydrotic ectodermal dysplasia, dentinogenesis imperfecta, and cleft lip and palate. The psychosocial aspects of these patients and rehabilitation with removable, fixed, and implant supported prostheses are discussed. The factors to be considered are altered anatomy, lack of teeth or malformed teeth, teeth in abnormal positions, lack of facial growth, and altered arch development. The short- and long-term treatment of patients in these categories is discussed. PMID- 16679133 TI - A comparison of two solder registration materials: a three-dimensional analysis. AB - STATEMENT OF PROBLEM: Use of a segmental indexing/soldering approach for assembly of complete arch implant frameworks has been advocated by some clinicians to achieve a passively fitting framework. To the authors' knowledge, a mathematical assessment using a 3-dimensional (3-D) distortion analysis has not been performed comparing segmental framework assembly to reassembly of 1-piece complete arch frameworks that required sectioning, indexing, and soldering. PURPOSE: The present study compared the use of 2 indexing materials, autopolymerizing acrylic resin and light-polymerized composite resin, for use in solder registration of complete-arch implant frameworks using 3 framework/indexing material combinations: gold/light-polymerized composite resin, silver palladium/autopolymerizing acrylic resin, and silver palladium/light-polymerized composite resin. MATERIAL AND METHODS: Five frameworks for each alloy/index combination were fabricated, for a total of 15 frameworks. Each framework was fitted to 5 implant abutments. Measurements were made of the initial gold cylinder/abutment positions for each of the 5 sites for the 15 frameworks. The 3 D spatial orientation of each gold cylinder/abutment was calculated prior to the gold cylinders being incorporated into the framework casting and served as the control against which all measurements were compared. After the indexing/soldering procedures, the 3-D spatial orientations of the cylinders were measured, first as 2- and 3-unit solder assembled framework segments and, finally, for complete-arch frameworks. These measurements were recorded in micrometers for linear distortions and degrees for angular distortions using a coordinate measurement machine (CMM). These data were compared and statistically assessed using a 1-way analysis of variance (ANOVA). A Student-Newman-Keuls test was used to determine significance between cylinders within each indexing combination. A repeated-measures ANOVA and a Student-Newman-Keuls test were used to determine significance between alloy/index group and cylinders for each of the measurement variables with statistical significance at alpha=.05 for all statistical analyses. RESULTS: Mean differences and SDs of total linear change for all groups found dR (total linear change) to be 33.56 +/- 27.70 mum for gold/light-polymerized composite resin, 34.87 +/- 27.99 mum for silver palladium/autopolymerizing acrylic resin, and 31.56 +/- 24.37 mum for silver palladium/light-polymerized composite resin. Total angular change, dtheta(R), was calculated to be 0.47 +/- 0.13 degrees for gold/light-polymerized composite resin, 0.46 +/- 0.17 degrees for silver palladium/autopolymerizing acrylic resin, and 0.52 +/- 0.15 degrees for silver palladium/light-polymerizing composite resin. CONCLUSIONS: No significant differences were found between alloy/index combinations, except for the measured variable dtheta(y), angular change about the Y axis. PMID- 16679135 TI - A technique to recement provisional crowns by reactivating residual zinc oxide eugenol cement. PMID- 16679136 TI - Custom-made removable implant analogs for soft-tissue casts. PMID- 16679137 TI - Fabricating a provisional restoration over a healing cap using a 1.25-mm L-shaped Allen key. PMID- 16679138 TI - Global childhood malnutrition. PMID- 16679139 TI - New focus needed for Doha trade talks. PMID- 16679140 TI - Autopsy at the crossroads. PMID- 16679141 TI - Can the chance of having twins be modified by diet? PMID- 16679142 TI - Poliomyelitis eradication and pandemic influenza. PMID- 16679143 TI - A new global immunisation vision and strategy. PMID- 16679144 TI - A secretary by any other name. PMID- 16679145 TI - Prenatal exposure to antiepileptic drugs. PMID- 16679146 TI - Cardiac risk management in severe mental illness. PMID- 16679148 TI - Dora Akunyili: scourge of Nigerian drug counterfeiters. PMID- 16679149 TI - Liquid-based versus conventional cervical cytology. PMID- 16679150 TI - Liquid-based versus conventional cervical cytology. PMID- 16679151 TI - Liquid-based versus conventional cervical cytology. PMID- 16679153 TI - Liquid-based versus conventional cervical cytology. PMID- 16679154 TI - UK medical schools and postgraduate deans. PMID- 16679155 TI - What went wrong in the natalizumab trials Comment? PMID- 16679156 TI - Profile of Lois Jovanovic. PMID- 16679157 TI - Vanished decimal point as cause of weight gain. PMID- 16679158 TI - Book contracts and authors' rights. PMID- 16679159 TI - Greece's tobacco policy: another myth? PMID- 16679160 TI - Sacking of CMAJ editors. PMID- 16679161 TI - Stillbirth rates: delivering estimates in 190 countries. AB - BACKGROUND: While information about 4 million neonatal deaths worldwide is limited, even less information is available for stillbirths (babies born dead in the last 12 weeks of pregnancy) and there are no published, systematic global estimates. We sought to identify available data and use these to estimate the rates and numbers of stillbirths for 190 countries for the year 2000, and provide uncertainty estimates. METHODS: We assessed three sources of stillbirth data according to specified inclusion criteria: vital registration; demographic and health surveys (DHS), based on a new analysis of contraceptive calendar data; and study reports that include published studies identified through systematic literature searches of more than 30,000 abstracts and unpublished studies. A random effects regression model was developed to predict national stillbirth rates and associated uncertainty intervals. FINDINGS: Data from 44 countries with vital registration (71,442 stillbirths), 30 DHS surveys from 16 countries (2989 stillbirths), and 249 study populations from 103 countries (93,023 stillbirths) met the inclusion criteria. Model-based estimates were used for 128 countries. For 62 countries, the observed values were adjusted by a correction factor derived from the model. The resultant stillbirth rates ranged from five per 1000 in rich countries to 32 per 1000 in south Asia and sub-Saharan Africa. The estimated number of global stillbirths is 3.2 million (uncertainty range 2.5-4.1 million). In light of the data limitations and the conservative approach taken, the real number might be higher than this. INTERPRETATION: The numbers of stillbirths are high and there is a dearth of usable data in countries and regions in which most stillbirths occur, with under-reporting being a major challenge. Although our estimates are probably underestimates, they represent a rigorous attempt to measure the numbers of babies dying during the last trimester of pregnancy. Improving stillbirth data is the first step towards making stillbirths count in public-health action. PMID- 16679162 TI - A prognostic index for AIDS-associated Kaposi's sarcoma in the era of highly active antiretroviral therapy. AB - BACKGROUND: AIDS-associated Kaposi's sarcoma remains common in individuals with HIV-1 infection in the era of highly active antiretroviral therapy (HAART). We developed a simple model for predicting mortality on the basis of clinical characteristics present at the time of diagnosis of Kaposi's sarcoma. METHODS: Of 5873 individuals with HIV-1 infection, 326 (6%) developed Kaposi's sarcoma; for 262 (80%) this was their first AIDS-defining illness. We did univariate and multivariate Cox regression analyses to identify covariates predictive of overall survival and validated our model with an independent data set of 446 patients with Kaposi's sarcoma. RESULTS: In the primary model, we developed a prognostic score from 0 to 15 starting at 10. Having Kaposi's sarcoma as the AIDS-defining illness (-3 points) and increasing CD4 count (-1 point for every complete 100 cells per mm3) improved prognosis; age of 50 years or older (2 points) and having another AIDS-associated illness at the same time (3 points) conveyed a poorer prognosis. In individuals with prognostic scores of 0, 5, 10, and 15, probability of survival at 1-year was 0.993, 0.967, 0.834, and 0.378, and at 5 years was 0.984, 0.918, 0.631, and 0.084, respectively. Increasing prognostic score by 1 increased 1-year death hazard ratio by 40% (95% CI 28-53%; bootstrapped hazard ratio 1.39, 1.25-1.51). The index had concordance of 76.8% (71.7-82.3). INTERPRETATION: We identified four prognostic factors that can be used to obtain an accurate prognostic index at diagnosis of AIDS-associated Kaposi's sarcoma. This index is widely applicable and can be used to guide therapeutic options. PMID- 16679163 TI - Non-invasive imaging compared with intra-arterial angiography in the diagnosis of symptomatic carotid stenosis: a meta-analysis. AB - BACKGROUND: Accurate carotid imaging is important for effective secondary stroke prevention. Non-invasive imaging, now widely available, is replacing intra arterial angiography for carotid stenosis, but the accuracy remains uncertain despite an extensive literature. We systematically reviewed the accuracy of non invasive imaging compared with intra-arterial angiography for diagnosing carotid stenosis in patients with carotid territory ischaemic symptoms. METHODS: We searched for articles published between 1980 and April 2004; included studies comparing non-invasive imaging with intra-arterial angiography that met Standards for Reporting of Diagnostic Accuracy (STARD) criteria; extracted data to calculate sensitivity and specificity of non-invasive imaging, to test for heterogeneity and to perform sensitivity analyses; and categorised percent stenosis by the North American Symptomatic Carotid Endarterectomy Trial (NASCET) method. RESULTS: In 41 included studies (2541 patients, 4876 arteries), contrast enhanced MR angiography was more sensitive (0.94, 95% CI 0.88-0.97) and specific (0.93, 95% CI 0.89-0.96) for 70-99% stenosis than Doppler ultrasound, MR angiography, and CT angiography (sensitivities 0.89, 0.88, 0.76; specificities 0.84, 0.84, 0.94, respectively). Data for 50-69% stenoses and combinations of non invasive tests were sparse and unreliable. There was heterogeneity between studies and evidence of publication bias. INTERPRETATION: Non-invasive tests, used cautiously, could replace intra-arterial carotid angiography for 70-99% stenosis. However, more data are required to determine their accuracy, especially at 50-69% stenoses where the balance of risk and benefit for carotid endarterectomy is particularly narrow, and to explore and overcome heterogeneity. Methodology for evaluating imaging tests should be improved; blinded, prospective studies in clinically relevant patients are essential basic characteristics. PMID- 16679164 TI - Effect of B vitamins and genetics on success of in-vitro fertilisation: prospective cohort study. AB - BACKGROUND: There is a need to understand what affects the success of in-vitro fertilisation (IVF) and the rate of resulting twin births so that pregnancy rates can be improved and multiple gestations avoided. Our aim was to assess the role of B vitamins and genetics. METHODS: We did a prospective cohort study of 602 women undergoing fertility treatment. We assessed intake of folate and vitamin B12 with a questionnaire and measured their plasma and red-blood-cell concentrations by radioimmunoassay. We measured five B-vitamin-related gene variants in women who received treatment and in 932 women who conceived naturally. FINDINGS: The likelihood of a twin birth after IVF rose with increased concentrations of plasma folate (1.52, 1.01-2.28; p=0.032) and red-cell folate (1.28, 1.00-1.65; p=0.039). There was no association between folate and vitamin B12 levels and likelihood of a successful pregnancy. Women homozygous for the 1298 CC variant of methylenetetrahydro-folate reductase (MTHFR), rather than the AA variant, were less likely to produce a livebirth after IVF (0.24, 0.08-0.71; p=0.003) or to have had a previous pregnancy (0.42, 0.21-0.81; p=0.008). INTERPRETATION: Our findings suggest that MTHFR genotype is linked to a woman's potential to produce healthy embryos (possibly through interaction with genes related to DNA methylation). In women likely to have a successful IVF pregnancy, high folate status increases the likelihood of twin birth after multiple embryo transfer. Proposals to fortify the UK diet with folic acid could lead to an increase in the number of twins born after IVF. PMID- 16679165 TI - Herodotus, the Scythes and hookworm infection. PMID- 16679166 TI - Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. AB - The three main soil-transmitted helminth infections, ascariasis, trichuriasis, and hookworm, are common clinical disorders in man. The gastrointestinal tract of a child living in poverty in a less developed country is likely to be parasitised with at least one, and in many cases all three soil-transmitted helminths, with resultant impairments in physical, intellectual, and cognitive development. The benzimidazole anthelmintics, mebendazole and albendazole, are commonly used to remove these infections. The use of these drugs is not limited to treatment of symptomatic soil-transmitted helminth infections, but also for large-scale prevention of morbidity in children living in endemic areas. As a result of data showing improvements in child health and education after deworming, and the burden of disease attributed to soil-transmitted helminths, the worldwide community is awakening to the importance of these infections. Concerns about the sustainability of periodic deworming with benzimidazole anthelmintics and the emergence of resistance have prompted efforts to develop and test new control tools. PMID- 16679167 TI - Road-traffic injuries: confronting disparities to address a global-health problem. AB - Evidence suggests that the present and projected global burden of road-traffic injuries is disproportionately borne by countries that can least afford to meet the health service, economic, and societal challenges posed. Although the evidence base on which these estimates are made remains somewhat precarious in view of the limited data systems in most low-income and middle-income countries (as per the classification on the World Bank website), these projections highlight the essential need to address road-traffic injuries as a public-health priority. Most well-evaluated effective interventions do not directly focus on efforts to protect vulnerable road users, such as motorcyclists and pedestrians. Yet, these groups comprise the majority of road-traffic victims in low-income and middle-income countries, and consequently, the majority of the road-traffic victims globally. Appropriately responding to these disparities in available evidence and prevention efforts is necessary if we are to comprehensively address this global-health dilemma. PMID- 16679169 TI - Unusual acute abdomen: to operate or not to operate? PMID- 16679168 TI - 1 year after The Lancet Neonatal Survival Series--was the call for action heard? PMID- 16679171 TI - Influenza vaccination status and influenza-related perspectives and practices among US physicians. AB - BACKGROUND AND OBJECTIVE: The influenza vaccination rate among US healthcare workers (HCWs) remains low. This survey was designed to assess influenza vaccination status and related knowledge, attitudes, and beliefs among a national sample of primary care physicians and subspecialists likely to see patients at high risk for complications from influenza. METHODS: We used a mail survey of a national random sample of 495 family physicians (FPs), 491 internists (IMs), 498 geriatricians (GERs), and 497 pulmonologists (PUDs). RESULTS: The overall response rate was 38%. Almost all respondents (87%) reported receiving an influenza vaccine during the 2003-2004 influenza season, with no significant difference across specialty groups (84% FPs, 87% IMs, 87% GERs, 91% PUDs). In a multivariate model, adjusted for physician specialty and age group, significant predictors of vaccination were: strong agreement that HCWs have professional responsibility to be vaccinated, access to vaccination on site and free of charge, strong worksite recommendation for HCWs to be vaccinated, and strong agreement that benefits of vaccination outweigh risk of side effects. CONCLUSIONS: Physicians reported a high influenza vaccination rate. To improve these rates further, with likely benefits for other HCWs, worksite policies that facilitate access to vaccination and documentation of reductions in nosocomial influenza associated with HCW vaccination should continue to be pursued. PMID- 16679172 TI - A profile of smaller hospitals: planning for a novel, statewide surveillance program, Victoria, Australia. PMID- 16679173 TI - Confidence in controlling a SARS outbreak: experiences of public health nurses in managing home quarantine measures in Taiwan. AB - BACKGROUND: Taiwan experienced one of the most serious outbreaks of severe acute respiratory syndrome (SARS) during the 2003 epidemic. Public health nurses faced unprecedented challenges in implementing an extensive quarantine policy to prevent disease spread. Their professional confidence, however, was shattered during the SARS crisis. This paper assesses factors related to public health nurses' confidence in managing community SARS control programs. METHODS: In May 2003, we sent structured questionnaires to all 361 health centers in Taiwan and asked the public health nurses responsible for epidemic control to complete. A total of 312 completed surveys were returned for a response rate of 86.4%. Descriptive methods and logistic regression were used to analyze the data. RESULTS: Most public health nurses (71.9%) expressed a general lack of confidence in handling the SARS epidemic. Confidence was significantly associated with perceived epidemic severity (OR, 0.58; 95% CI: 0.35-0.99), daily epidemic updates (OR, 2.26; 95% CI: 1.28-3.98), and number of cases in the community (OR, 2.21; 95% CI: 1.13-4.31). CONCLUSION: Nurses' individual risk perception and prompt update of epidemic information significantly affect levels of professional confidence, a key factor influencing quarantine implementation success. Strategies to promote productive interagency collaboration and advocate participatory policy making involving health workers at all levels are needed to control effectively infectious disease outbreaks. PMID- 16679174 TI - The effect of performance feedback on wound infection rate in abdominal hysterectomy. AB - BACKGROUND: In many hospitals, infection of the surgical wound is the most common nosocomial infection. Its presence implies patient morbidity, a mortality risk, and an increase in procedure costs because of prolonged hospitalization. OBJECTIVES: Our objective was to ascertain the effect of an infection control program, using performance feedback, on wound infection (WI) rate in abdominal hysterectomy. METHODS: All patients undergoing abdominal hysterectomy in our center (Hospital A. Marcide, Ferrol, Spain) between 1999 and 2004 were prospectively followed up to determine the WI rate. A complete set of parameters, including age, underlying illnesses, cancer, diabetes mellitus, immunosuppressive therapy, albumin, American Society of Anesthesiologists preoperative assessment score (ASA) risk, days in hospital presurgery, date of surgery, hygiene and perioperative antimicrobial prophylaxis, type of surgical operation, duration of surgery, surgeon, and WI, were collected in each case. After data collection for 1999 concluded, we communicated surgical WI rates to surgeons every year. A logistic regression analysis was performed to compare WI rates with those observed in 1999. RESULTS: A total of 980 females was enrolled in the prospective surveillance: mean age, 50.7 +/- 10.7 years. Cases included 25.8% cancer, 4.9% diabetes, 0.5% immunosuppressive therapy, 26.6% ASA 1, 58.4% ASA 2, 13.9% ASA 3. In 9 patients, emergency surgery was performed and, in 971 patients, surgery was scheduled: Total abdominal hysterectomy, 878; subtotal abdominal hysterectomy, 65; Wertheim-Meigs, 37. The factors associated with WI were albumin (OR, 0.97; 95% CI: 0.94-0.99) and antimicrobial prophylaxis (OR, 0.08; 95% CI: 0.02-0.32). The mean values for albumin and the number of patients with antimicrobial prophylaxis fluctuated from year to year. The WI rate improved from 10.7% (95% CI: 5.8-15.6) in 1999 to 6% (-43.9%) in 2004. CONCLUSION: Performance feedback of surgical wound infection rates to individual surgeons reduces these rates. PMID- 16679175 TI - Clinical, microbiologic, and epidemiologic characteristics of Pseudomonas aeruginosa infections in a University Hospital, Malatya, Turkey. AB - BACKGROUND: Pseudomonas aeruginosa strains are generally resistant to many antibiotics, and nosocomial infections because of this species are one of the major problems in many hospitals. Molecular typing provides very useful information about origin and transmission of the strains. The aims of the present study were to investigate clinical and microbiologic characteristics of the nosocomial infections caused by P aeruginosa strains in a medical center and to bring up the cross-transmission level of this opportunistic pathogen in a university hospital by analyzing the clonal relationship among the isolates. METHODS: A total of 105 P aeruginosa strains had been identified among the 80 inpatients in a 1-year period from August 2003 to August 2004. Demographic, clinical, and epidemiologic data of the patients were prospectively recorded. The standardized disk-diffusion method was used to determine resistance of the strains to imipenem, ceftazidime, aztreonam, amikacin, gentamicin, mezlocillin, cefepime, tobramycin, meropenem, ceftriaxone, and ciprofloxacin. Clonal relatedness of the strains was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Of the 105 P aeruginosa strains identified, 45 (43%) were isolated from the patients hospitalized in intensive care units. Thirteen patients had repeated pseudomonas infection (total 38 infections/13 patients); 26 of these repeated infections in 9 patients showed the same localization. Half of the patients had at least 1 underlying disease such as burn (48%), chronic illness (32%), and malignancy (20%). Fifty-seven patients (71%) had urinary and/or other catheterization. Urinary tract infection (35%) was the most frequent infection encountered, followed by respiratory tract infection (34%) and sepsis (13%). Resistance to the antibiotics tested was in the 12% to 88% range; amikacin was the most effective and ceftriaxone was the least effective antibiotic. The PFGE typing method showed that 28 of the 80 patients' isolates were clonally related, including 23 indistinguishable or closely related strains (29%), and 5 possibly related strains (6%). Epidemiologic data of the 16 patients (20% of the patients) confirmed a clonal relationship among the strains. Of the 26 isolates of the 9 patients having repeated infection in the same location, 18 (69%) were in the clonally related groups, whereas 11 of the 12 strains isolated from repeated infections on different body sites were clonally different. CONCLUSION: Our results indicated that P aeruginosa infections in our hospital mainly affected the patients hospitalized in intensive care units and those having catheterization, burn, and/or chronic illness. Amikacin was the best antibiotic as far as bacterial resistance was considered. Although lack of major PFGE type confirmed no P aeruginosa outbreak, typing results showed that cross transmission and treatment failure are the 2 main problems, which should be consider together to prevent this bacterial infection in medical centers. PMID- 16679176 TI - Evolution of infection control in Egypt: achievements and challenges. AB - BACKGROUND: The high prevalence of hepatitis C virus (HCV) infection in Egypt highlighted the urgent need for implementing infection control (IC) programs in Egypt. OBJECTIVES: The Ministry of Health and Population (MOHP), in collaboration with the US Naval Medical Research Unit No. 3, and the World Health Organization (WHO), developed a national plan to initiate an IC program with the objectives of improving quality of care and reducing transmission of hospital-acquired infections. METHODS: The strategic plan for this program included setting up an organizational structure, developing IC national guidelines, training health care workers, promoting occupational safety, and establishing a system for monitoring and evaluation. Implementation of the program started in late 2001. RESULTS: The achievements to date include developing a national organizational structure, IC guidelines, and a comprehensive IC training program. To date, a total of 72 hospitals in 13 governorates have been enrolled in the program, and 235 IC professionals have been trained. CONCLUSIONS: Many challenges were faced, including administrative, financial, and motivational difficulties. Future plans include expansion of the program to cover all 27 governorates of Egypt and establishment of a surveillance system for hospital-acquired infections. The process of developing the IC program in Egypt may serve as a model for other resource-limited countries that seek to initiate similar programs. PMID- 16679177 TI - Surgical site infection in patients submitted to digestive surgery: risk prediction and the NNIS risk index. AB - BACKGROUND: Some problems have been reported with the power of the National Nosocomial Infection Surveillance (NNIS) risk index to predict the risk of surgical site infections (SSI) for specific procedures. OBJECTIVES: To develop an alternative risk prediction index for SSI and to compare the performance with the NNIS index. METHODS: A prospective cohort study was carried out with all (609) patients submitted to digestive tract surgery in 2 general teaching hospitals in the city of Sao Paulo, Brazil, from August 2001 through March 2002. RESULTS: The final incidence rate of SSI was 24.5%; 149 cases of SSI were identified: 33 (22.1%) during hospitalization and 116 (77.9%) after discharge. Logistic multivariate analysis was used for construction of the model. Obesity, surgery risk, adjusted duration, and video laparoscopic surgery were statistically significant (P < .05) for all previous procedures. The performance of the NNIS model in this study showed a low predictive capacity for the occurrence of SSI as determined by the receiver operating characteristic (ROC) curve (0.627; 95% CI: 0.575-0.678) compared with the alternative model developed with this population (0.732; 95% CI: 0.685-0.779). CONCLUSION: The presence of obesity, adjusted duration, and surgery risk significantly increased the risk for SSI. The NNIS risk index was not significant for SSI in the sample studied, and laparoscopic access was associated with a significant reduction in the risk for SSI. Although the NNIS index is a well-known and simple index, other models depicting variables related to SSI with a better sensitivity and specificity can be developed. Additional studies are required to confirm our results. PMID- 16679178 TI - Blunt and penetrating object injuries in housekeepers working in a Turkish University Hospital. AB - BACKGROUND: Hospitals have been described as hazardous work environments with an increase in job-related injuries. This situation creates great risks and hazards for housekeepers while carrying out their job. METHODS: This descriptive study was performed on 402 housekeepers working in patient-care services in Turkey. The data of this study were collected using a questionnaire form. This form included 26 questions about general features of housekeepers and working units, blunt and penetrating object injuries in the past 3 months and hepatitis B virus immunization. RESULTS: The majority of housekeepers (71.1%) are men, (54%) are graduates of primary school or are illiterate, and (73.6%) are married. Their mean age is 31.5 years; the mean length of employment is 3.2 years. Sixty-two point nine percent of them are working in medical/surgical units, 88.8% of them are working in routine cleaning, and 29.1% of them have been injured with various blunt and penetrating objects while working in hospital in the past 3 months. Only 26.6% of the housekeepers have been administered the hepatitis B vaccination. CONCLUSION: This study showed a high frequency of blunt and penetrating object injuries in housekeepers. Therefore, more efforts are necessary to increase compliance with vaccination in housekeepers. PMID- 16679179 TI - Variation in blood and body fluids exposure when small-gauge needles or peripheral venous catheters were implicated: results of a 4-year surveillance in France. AB - The blood and body fluids exposure (BBFE) risk for health care workers varies according to numerous factors. Based on a needlestick surveillance in 13 French hospitals from 1997 to 2000, we evaluated incidence and temporal trends of BBFE according to medical devices causing needlestick injuries. We observed that the BBFE incidence per 100,000 peripheral venous catheters purchased decreased from 12.9 to 4.9, whereas incidence per 100,000 subcutaneous needles purchased increased from 8.7 to 14.3. PMID- 16679180 TI - Infection control knowledge and practices among dentists and dental nurses at a Jordanian University Teaching Center. AB - BACKGROUND: Information regarding compliance with infection control precautions in the dental settings in the Middle East is scant. OBJECTIVE: To examine the knowledge and practices in infection control among dental staff and dental nurses at a university teaching dental center. METHODS: A self-administered, confidential questionnaire concerning various aspects of infection control knowledge and practices was distributed to 48 dental staff and 28 dental nurses working at the Jordan University of Science and Technology Dental Teaching Center. RESULTS: Responses came from 37 (77%) dental teaching staff members and 23 (82%) dental nurses. Of the dental staff participants, 95% received hepatitis B immunization in comparison with 87% of the dental nurses. Dental nurses were more prone to percutaneous dental injuries (P < .05). In both groups, 100% reported routine wearing of gloves. Although the dental staff reported a statistically higher frequency of washing hands before gloving (46%), dental nurses reported a higher frequency of washing hands after removing the gloves (100%). Dental staff reported statistically higher frequency of routine mask use (43%) than dental nurses (30%). CONCLUSION: Compliance with recommended guidelines for control of cross infection varied among the 2 tested groups. A need exists for proper practice of infection control by both dental staff and dental nurses. PMID- 16679181 TI - National survey of the status of infection surveillance and control programs in acute care hospitals with more than 300 beds in the Republic of Korea. AB - BACKGROUND: This study was conducted to assess the status of infection surveillance and control programs (ISCPs) and to analyze the trends associated with ISCP implementation since the first program was established in Korea in 1991. METHODS: A questionnaire modified from the Study on the Efficacy of Nosocomial Infection Control was mailed 4 times nationwide to acute care hospitals (n = 164) with more than 300 beds between June and October 2003. Eighty five hospitals participated (52%). RESULTS: The mean number of beds (649) in the responding hospitals was significantly greater than in nonresponding hospitals. Of the participating hospitals, 92% had educational functions, 40% to 90% used hand hygiene resources, and 100% had infection control committees; 86% had infection control doctors, 98% had infection control nurses (ICNs), 89% employed only 1 ICN, and 59% employed an ICN only part-time; 68% performed surveillance, undertaking 2.7 epidemic investigations per year and 8.4 teaching programs per year; 88% undertook needlestick prevention programs; 58% performed regular air culture; and 64% discarded ineffective ISCPs. Annual trends analysis of ISCPs indicated that accreditation and legislation impact strongly on Korean ISCPs. CONCLUSION: The figures for ISCPs in this study indicate that improvements have been made since the 1990s. Legislation and accreditation have strongly influenced ISCPs. Much consideration should be given to the weaknesses in Korean ISCPs: surveillance, insufficient hand hygiene resources, and shortage of ICNs. PMID- 16679182 TI - A climatologic investigation of the SARS-CoV outbreak in Beijing, China. AB - The first cases of severe acute respiratory syndrome (SARS) were identified in November 2002, in Guangdong Province, China. The epidemic spread rapidly within China and internationally, with 8454 recorded infections and 792 deaths by June 15, 2003. Temperature, relative humidity, and wind velocity were the three key meteorological determinants affecting the transmission of SARS. The peak spread of SARS occurred at a mean temperature of 16.9 degrees C (95% CI, 10.7 degrees C to 23.1 degrees C), with a mean relative humidity of 52.2% (95% CI, 33.0% to 71.4%) and wind speed of 2.8 ms(-1) (95% CI, 2.0 to 3.6 ms(-1)). In northern China, these conditions are most likely to occur in the spring and suggest that SARS has a seasonal nature akin to viruses such as influenza and the common cold. A regression equation (Y=218.692-0.698X(t)-2.043X(h)+2.282X(w)) was derived to represent the optimal climatic conditions for the 2003 SARS epidemic. Further investigations in other regions are necessary to verify these results. PMID- 16679183 TI - Occupational HIV infection among health care workers exposed to blood and body fluids in Brazil. AB - BACKGROUND: Exposure to bloodborne pathogens poses a serious risk to health care workers (HCWs). Surveillance systems of occupationally acquired human immunodeficiency virus (HIV) infection have been developed in several countries, mainly in the developed world. The purpose of this study was to identify cases of occupationally acquired HIV infection among HCWs in Brazil. METHODS: A systematic literature review was conducted. The databases searched were MEDLINE and LILACS (1981 to 2004), academic dissertations and theses (1987 to 2004), abstracts from national and international meetings during the last 10 years, and local and national bulletins. Reference lists to identify other relevant articles were checked. RESULTS: The database searches generated a total of 60,770 titles. Two hundred and nineteen references were finally analyzed. Four documented cases of occupational HIV infection were identified. All of the cases involved nursing staff and were percutaneous exposures. Seventy-five percent occurred after a procedure involving a needle placed directly into a vein or artery. Most (75%) had source patients with probable high viral load and low CD4 count. Two cases represented HIV seroconversion despite initiation of postexposure prophylaxis. Only one case (1/4; 25%) presented acute retroviral illness. CONCLUSION: After an extensive literature search, 4 documented occupational HIV infection cases were identified, only 1 of which had been published in a scientific journal. Our findings were consistent with the majority of documented infections worldwide. Surveillance systems are indispensable to establish and formulate rational policies for minimizing the risk of occupational infection, not only from HIV but also from hepatitis B and C viruses and other bloodborne pathogens. PMID- 16679184 TI - Prevalence of antibodies against hepatitis B virus and hepatitis C virus among blood donors in Lebanon, 1997-2003. AB - The prevalence of hepatitis B virus (HBV) antigens (HBsAg) and antibody to hepatitis C virus (anti-HCV) was determined among 16,084 blood donors (14,993 males; mean age, 31.7 +/- 8.2 years and 1084 females; mean age, 31.4 +/- 8.2 years) in the period 1997-2003. Of the donors screened, 149 were HBsAg positive (0.926%), and 65 were anti-HCV positive (0.404%). There was a steady decline in HBsAg prevalence from 1.56% (1997) to 0.33% (2003) and in anti-HCV from 1.22% (1997) to 0.16% (2003). Females had a higher prevalence of anti-HCV (P = .031) and HBsAg (P = .047). Results obtained are of value in light of the occurrence of HBV and HCV transmission by nonparenteral routes. PMID- 16679185 TI - Device-associated nosocomial infection rates in intensive care units in four Mexican public hospitals. AB - BACKGROUND: Routine surveillance of nosocomial infections has become an integral part of infection control and quality assurance in US hospitals. METHODS: As part of the International Nosocomial Infection Control Consortium, we performed a prospective nosocomial infection surveillance cohort study in 5 adult intensive care units of 4 Mexican public hospitals using the Centers for Disease Control and Prevention National Nosocomial Infections Surveillance system definitions. Site-specific nosocomial infection rates were calculated. RESULTS: The overall nosocomial infection rate was 24.4% (257/1055) and 39.0 (257/6590) per 1000 patient days. The most common infection was catheter-associated bloodstream infection, 57.98% (149/257), followed by ventilator-associated pneumonia, 20.23% (52/257), and catheter-associated urinary tract infection, 21.79% (56/257). The overall rate of catheter-associated bloodstream infections was 23.1 per 1000 device-days (149/6450); ventilator-associated pneumonia rate was 21.8 per 1000 device-days (52/2390); and catheter-associated urinary tract infection rate was 13.4 per 1000 device-days (56/4184). CONCLUSION: Our rates are similar to other hospitals of Latin America and higher than US hospitals. PMID- 16679186 TI - RE: A brief report on the normal range of forehead temperature as determined by noncontact, handheld, infrared thermometer. PMID- 16679187 TI - Effects of long-term storage on sterility of medical supplies. PMID- 16679191 TI - Educating the orthodontic team. PMID- 16679192 TI - Space for second and third molar eruption. PMID- 16679194 TI - Evidence-based therapy: an orthodontic dilemma. PMID- 16679196 TI - Mandibular changes produced by functional appliances in Class II malocclusion: a systematic review. AB - The aim of this systematic review of the literature was to assess the scientific evidence on the efficiency of functional appliances in enhancing mandibular growth in Class II subjects. A literature survey was performed by applying the Medline database (Entrez PubMed). The survey covered the period from January 1966 to January 2005 and used the medical subject headings (MeSH). The following study types that reported data on treatment effects were included: randomized clinical trials (RCTs), and prospective and retrospective longitudinal controlled clinical trials (CCTs) with untreated Class II controls. The search strategy resulted in 704 articles. After selection according to the inclusion/exclusion criteria, 22 articles qualified for the final analysis. Four RCTs and 18 CCTs were retrieved. The quality standards of these investigations ranged from low (3 studies) to medium/high (6 studies). Two-thirds of the samples in the 22 studies reported a clinically significant supplementary elongation in total mandibular length (a change greater than 2.0 mm in the treated group compared with the untreated group) as a result of overall active treatment with functional appliances. The amount of supplementary mandibular growth appears to be significantly larger if the functional treatment is performed at the pubertal peak in skeletal maturation. None of the 4 RCTs reported a clinically significant change in mandibular length induced by functional appliances; 3 of the 4 RCTs treated subjects at a prepubertal stage of skeletal maturity. The Herbst appliance showed the highest coefficient of efficiency (0.28 mm per month) followed by the Twin block (0.23 mm per month). PMID- 16679197 TI - Biomechanical features of the periodontium: an experimental pilot study in vivo. AB - INTRODUCTION: The orthodontic application of a force or moment leads to a strain distribution in the periodontal ligament. The corresponding local deformation at the interface periodontal ligament-alveolar bone causes a distribution of normal and shearing tension that is thought to be the local stimulus for bone remodeling leading to orthodontic tooth movement. Although researchers have examined initial tooth movement and the biomechanical properties of the periodontal ligament, few have reported human, in-vivo studies. For such examinations, very small tooth displacements must be measured with extremely high resolution and accuracy. METHODS: By a highly sensitive goniometer (resolution <0.001 degrees ), rotations of premolars in 22 subjects with natural spacing and 14 subjects with pronounced anterior crowding were measured in vivo. The rotations followed sets of pure moment-time functions starting at zero and reaching specified final values within +/- 1.43 Ncm. RESULTS: Usually, thresholds were seen that had to be overcome by moment to produce measurable rotations of the tooth in the mesial or distal direction. The subjects with pronounced anterior crowding showed distinct asymmetry: Thresholds for rotations to the distal direction were significantly higher than for those to the mesial direction. CONCLUSIONS: The cases of symmetric thresholds indicated a shear thinning viscosity in the periodontium under increasing moment. In the case of asymmetric thresholds, the findings suggested a mesially directed pretension of the gingival fiber apparatus that might be connected with the mesial drift. PMID- 16679198 TI - A proposal for a new analysis of craniofacial morphology by 3-dimensional computed tomography. AB - INTRODUCTION: Three-dimensional (3D) analysis is essential for making a precise diagnosis of craniofacial morphology. Two-dimensional (2D) x-ray films are used to understand 3D structures. However, 2D images have several limitations. This article proposes a new type of cephalometric analysis by using 3D computed tomography. METHODS: Axial images of 30 subjects (16 men; mean age, 19.2 years; 14 women, mean age, 20.5 years) were reconstructed into 3D models by using Vworks 4.0 (Cybermed, Seoul, Korea). The 3D models were measured with Vsurgery (Cybermed). The zygoma, maxilla, mandible, and facial convexity were analyzed. RESULTS: The measurements were compared with Korean normal averages, and no statistically significant differences were found. Landmark identification was reproducible. CONCLUSIONS: Three-dimensional computed tomography can provide information for use in diagnosis and treatment planning. PMID- 16679199 TI - Plane orientation for standardization in 3-dimensional cephalometric analysis with computerized tomography imaging. AB - INTRODUCTION: The purpose of this study was to propose certain landmarks and planes to standardize 3-dimensional image orientation. METHODS: Cone-beam computerized tomographic images were obtained from 10 adolescent patients and analyzed with AMIRA software (AMIRA, Mercury Computer Systems, Berlin, Germany). RESULTS: Four points (ELSA, right superior external auditory meatus, left superior external auditory meatus, and mid-dorsum foramen magnum) were located on each image. The axial-horizontal plane (x-y plane) was then determined by using both superior external auditory meatus and ELSA; the sagittal-vertical plane (z-y plane) was formed by ELSA and mid-dorsum foramen magnum perpendicular to the x-y plane. CONCLUSIONS: All points had high intrareliability and were adequate for standardizing the orientation of 3-dimensional images. PMID- 16679200 TI - Three-dimensional digital modeling and setup. AB - Obtaining accurate images of the craniofacial region is critical when developing an orthodontic diagnosis and treatment plan. The purpose of this article is to describe a new imaging method that provides complete 3-dimensional views of the maxilla and the mandible, and the model setup with individual anatomic roots. The method uses computed tomography technology and laser scanning; it offers high resolution images with relatively low radiation exposure. Technical aspects of the new procedure and its possible uses for orthodontic diagnosis and treatment planning are discussed. PMID- 16679201 TI - Image analysis and superimposition of 3-dimensional cone-beam computed tomography models. AB - Three-dimensional (3D) imaging techniques can provide valuable information to clinicians and researchers. But as we move from traditional 2-dimensional (2D) cephalometric analysis to new 3D techniques, it is often necessary to compare 2D with 3D data. Cone-beam computed tomography (CBCT) provides simulation tools that can help bridge the gap between image types. CBCT acquisitions can be made to simulate panoramic, lateral, and posteroanterior cephalometric radioagraphs so that they can be compared with preexisting cephalometric databases. Applications of 3D imaging in orthodontics include initial diagnosis and superimpositions for assessing growth, treatment changes, and stability. Three-dimensional CBCT images show dental root inclination and torque, impacted and supernumerary tooth positions, thickness and morphology of bone at sites of mini-implants for anchorage, and osteotomy sites in surgical planning. Findings such as resorption, hyperplasic growth, displacement, shape anomalies of mandibular condyles, and morphological differences between the right and left sides emphasize the diagnostic value of computed tomography acquisitions. Furthermore, relationships of soft tissues and the airway can be assessed in 3 dimensions. PMID- 16679202 TI - Three-dimensional analysis of models articulated in the seated condylar position from a deprogrammed asymptomatic population: a prospective study. Part 1. AB - INTRODUCTION: The seated condylar position (SCP), also known as centric relation (CR), is considered the most reliable and reproducible reference point for accurately recording the relationship of the mandible to the maxilla. Therefore, a determination of the SCP/CR is a prerequisite for the analyses of the dental interarch, condylar position, and skeletal relationships. The purpose of this prospective study was to statistically evaluate the 3-dimensional nature of dental interarch displacement and condylar displacement between the SCP/CR and maximum intercuspation or centric occlusion (MIC/CO). METHODS: The records of 596 consecutive asymptomatic patients having routine orthodontic treatment in a private practice were used. The initial premature occlusal contact and 3 dimensional dental interarch displacement were measured by the analysis of models, mounted on an articulator (Panadent, Grand Terrrace, Calif) in the SCP/CR, by using the modified (nonmanipulated) 2-piece wax SCP/CR recording method with deprogramming. Three-dimensional condylar displacement was measured by analysis of the graphic registrations, produced by condylar position instrumentation, and evaluated for frequency, direction, and magnitude of displacement. RESULTS: The dental interarch discrepancy in the SCP/CR was significantly different from that observed in MIC/CO, with posterior premature contacts (94.0%), increased overjet, decreased overbite, midline differences, and Angle classification changes. A difference in condylar position between the SCP/CR and MIC/CO in at least 1 plane was detected for every asymptomatic patient and every condyle. The most prevalent types of directional change in condylar position were inferior (down) (97.0%) and distal (posterior) (66.7%) when the teeth were brought into MIC/CO. The mean difference in condylar position between the SCP/CR and MIC/CO was .86 mm in the horizontal plane, 1.80 mm in the vertical plane, and .26 mm in the transverse plane. No correlation was found between a patient's age or sex and the magnitude of condylar displacement. CONCLUSIONS: There is a significant difference in the occlusion when it is dictated by the teeth and when it is dictated by the condyles. This difference is quantifiable at both the occlusal and condylar levels. PMID- 16679203 TI - A prospective long-term study on the effects of rapid maxillary expansion in the early mixed dentition. AB - INTRODUCTION: The aim of this prospective longitudinal clinical study was to evaluate the short-term and long-term changes in dental-arch dimensions in patients treated with the acrylic splint rapid maxillary expander in the early mixed dentition followed by fixed appliances in the permanent dentition. METHODS: The dental casts of 51 consecutively treated patients (TG) were compared with those of 26 untreated controls (CG) at 3 different times: pretreatment (T1), after expansion and fixed appliance therapy (T2), and at long-term observation (T3). The mean ages for the TG were 8 years 10 months at T1, 13 years 10 months at T2, and 19 years 9 months at T3. Arch widths, arch depth, arch perimeter, and molar angulation were assessed in all subjects at all observation times. T1-T2, T2-T3, and T1-T3 changes were compared statistically in the TG with respect to the corresponding CG. RESULTS: Treatment with an acrylic splint RME followed by fixed appliances produced significantly favorable short-term and long-term changes in almost all maxillary and mandibular arch measurements. The amount of change in both maxillary and mandibular intermolar and intercanine widths fully corrected the initial discrepancies. Approximately 4 mm of long-term relative increase in maxillary arch perimeter, and 2.5 mm additional maintenance of mandibular arch perimeter were observed in the TG compared with the CG. CONCLUSIONS: These results suggest that this protocol is effective and stable for the treatment of constricted maxillary arches, and can relieve modest deficiencies in arch perimeter. PMID- 16679204 TI - Influence of occlusal plane inclination on ANB and Wits assessments of anteroposterior jaw relationships. AB - INTRODUCTION: Although assessments of anteroposterior relationships are vital for orthodontic treatment planning, they cannot be precisely achieved by current cephalometric and noncephalometric resources. ANB angle and the Wits appraisal are the most popular cephalometric measurements applied in clinical orthodontics, although they have drawbacks. Facial vertical features, especially the occlusal plane angle, play an important role in their assessment, contributing to their unreliability. METHODS: To further understand the influence of occlusal plane inclination, ANB angle and the Wits appraisal were analyzed in cephalo-lateral radiographs of 122 finished orthodontic patients in 2 groups: high occlusal plane angle and low occlusal plane angle. RESULTS: There was a tendency for inconsistency between ANB and Wits assessments in the high occlusal plane angle group and a tendency for consistency in the low occlusal plane angle group. Cranial base influences on ANB and Wits assessments were investigated by subdividing the groups. Any of the considered cranial base factors was decisive for lack or presence of consistency between ANB and Wits assessments. CONCLUSIONS: The clinician should beware of possible misinterpretations when using ANB and Wits to assess the anteroposterior relationship of the jaws, especially in high occlusal plane angle patients. PMID- 16679205 TI - Dentoalveolar compensation in subjects with vertical skeletal dysplasia. AB - INTRODUCTION: The purpose of this cephalometric study was to investigate vertical dentoalveolar compensation in untreated adults with excessive (long-face) and deficient (short-face) lower anterior face heights. METHODS: Vertical and sagittal base relationships, vertical dentoalveolar dimension in the anterior region of the jaws, incisor inclination, overbite, and overjet were assessed in 112 short-face and 95 long-face subjects. The contribution of skeletal and dentoalveolar components to achieve a normal overbite was assessed by means of regression analysis. For the 2 most important independent variables of the regression equation, the values were calculated that would render an overbite of 2 mm. It was subsequently investigated whether the calculated value fell within the range of the sample. RESULTS: In long-face subjects, overbite was mainly related to lower anterior face height; in short-face subjects, it was mainly related to mandibular anterior alveolar and basal heights. Dentoalveolar compensation occurred in both groups mainly by adaptations in mandibular incisor alveolar and basal heights. Molar height was unrelated to overbite. Cutoff values for achieving a positive overbite were calculated for lower face height and mandibular incisor alveolar and basal heights. CONCLUSIONS: The lower face height mainly determines the overbite in long-face subjects, while in short-face subjects, lower dentoalveolar morphology influences overbite. Lower dentoalveolar compensation can maintain a normal overbite in long-face subjects to a limited extent. PMID- 16679206 TI - Influence of buccal segment size on prevention of side effects from incisor intrusion. AB - INTRODUCTION: Deep overbite can be corrected by maxillary incisor intrusion. The purpose of this study was to determine whether the size of the maxillary buccal segment influences the amount of steepening, extrusion, or narrowing of the buccal segments, or the rate of intrusion that occurs with maxillary incisor intrusion. METHODS: Twenty patients, 9 to 14 years of age, seeking treatment at a private practice, were divided into 2 groups. Patients in the long buccal-segment group had maxillary buccal segments that included the canines, both premolars, and the first molars. In the short buccal-segment group, the buccal segments consisted of only the maxillary first molars. Patient records were taken at the beginning and end of maxillary incisor intrusion. RESULTS: Intermolar width increased slightly in the short buccal-segment group and decreased slightly in the long buccal-segment group. More steepening of the buccal segment occurred in the short buccal-segment group, and more proclination of the anterior segment in the long buccal-segment group. The size of the buccal segment had no influence on the rate of incisor intrusion or on the amount of buccal-segment extrusion. In both groups, the mean amount of incisor intrusion exceeded 2 mm. CONCLUSIONS: A buccal segment that extends from canine to first molar will help minimize the side effects of incisor intrusion. PMID- 16679207 TI - Orthodontic treatment time in 2- and 4-premolar-extraction protocols. AB - INTRODUCTION: The purpose of this study was to compare the treatment times of complete Class II malocclusions treated with 2- and 4-premolar-extraction protocols. METHODS: Ninety-seven patients were selected and divided into 2 groups, according to the 2 extraction criteria. Group 1, treated with 2 maxillary premolar extractions, consisted of 49 patients (30 male, 19 female) with a mean age of 14.35 years. Group 2, treated with 4 premolar extractions, consisted of 48 patients (27 male, 21 female) with a mean age of 13.03 years. Treatment times of the groups were compared with the t test. RESULTS: Treatment times were significantly shorter with the 2-premolar-extraction protocol compared with the 4 premolar-extraction protocol. CONCLUSIONS: Treatment time will be shorter and the occlusal results more predictable with a 2-premolar-extraction protocol compared with 4 premolar extractions. PMID- 16679208 TI - Comparison of extraction versus nonextraction orthodontic treatment outcomes for borderline Chinese patients. AB - INTRODUCTION: The purpose of this study was to compare orthodontic treatment outcomes in Chinese patients with borderline problems treated with and without extractions. METHODS: Records of 39 borderline patients treated at the Faculty Clinic of the Peking University Orthodontic Department were evaluated retrospectively by 5 associate professors. Sixteen patients had been treated without extractions, and 23 had 4 first or second premolars extracted. Each judge evaluated the posttreatment records independently for tooth alignment, overbite, overjet, midline symmetry, lateral occlusion, and facial profile, and rated them on a scale from 1 to 5. RESULTS: The only statistically significant difference between the extraction and nonextraction groups was for facial profile, with the judges preferring the extraction profiles. Fifteen soft-tissue cephalometric variables were measured to determine the source of the difference, and 6 showed statistically significant differences. When profile changes from pretreatment to posttreatment were examined, significant differences in treatment-associated changes between extraction and nonextraction groups were all related to the lower lip and chin. CONCLUSIONS: In this sample of Chinese borderline orthodontic patients, Chinese clinicians had a statistically significant preference for the facial profiles of the extraction patients, but no statistically significant preferences for tooth alignment, overbite, overjet, midline symmetry, or posterior occlusion. Extraction treatment increases the inclination of the chin and reduces protrusion of the lower lip compared with nonextraction treatment, and this might explain the difference. PMID- 16679209 TI - Orthodontic treatment provided by general dentists who have achieved master's level in the Academy of General Dentistry. AB - INTRODUCTION: The purposes of this study were to document orthodontic treatment currently provided by general dentists for comparison with past and future studies and to ascertain variables that influence practitioners' orthodontic treatment patterns. METHODS: A 21-item survey was mailed to 750 master's level members of the Academy of General Dentistry. Surveys returned within 8 weeks were included for statistical analysis. RESULTS: The response rate was 62%. Most practitioners spent less than 10% of their practice time providing orthodontic treatment and reported that this would not change in the future. Many provided orthodontic treatment in the permanent dentition, and the most common conditions or malocclusions treated were space maintenance, anterior crossbite, rotation, habits, molar uprighting, and posterior crossbite. The most common orthodontic appliances used were removable Hawley appliances with finger springs, straight wire orthodontic therapy, rapid palatal expanders, and functional appliances. CONCLUSIONS: The number of general dentists providing comprehensive orthodontic treatment has not changed since previous surveys; practitioners also do not expect a change in the next 5 years. Factors that influenced the orthodontic treatment provided included the primary source of orthodontic training, the number of orthodontic continuing education hours earned per year, the practitioner's location, and the proximity to the nearest orthodontist. PMID- 16679210 TI - Forces released during alignment with a preadjusted appliance with different types of elastomeric ligatures. AB - INTRODUCTION: The purpose of this in-vitro study was to compare the forces generated by new nonconventional elastomeric ligatures (NCEL) and conventional elastomeric ligatures (CEL) during leveling and aligning phases. METHODS: The testing model consisted of 5 stainless steel 0.022-in preadjusted brackets for second premolar, first premolar, canine, lateral incisor, and central incisor. The canine bracket was welded to a sliding bar that allowed for different vertical positions. The forces generated by 3 sizes of wires (0.012-, 0.014-, and 0.016-in superelastic nickel-titanium) with the 2 types of elastomeric ligatures at different amounts of upward canine misalignment (1.5, 3, 4.5, and 6 mm) were recorded. RESULTS AND CONCLUSIONS: Significant differences between CEL and NCEL were found for all tested variables (P <.01) with the exception of the 0.014- and 0.016-in wires at canine misalignment of 1.5 mm. A noticeable amount of force was generated with the NCEL at all 4 canine positions with all 3 wire sizes (from about 50 to about 150 g). With 4.5 mm of canine misalignment or more, the average amount of released force with the CEL was approximately zero. PMID- 16679211 TI - Tooth transplantation and cryopreservation: state of the art. AB - Autotransplantation of teeth is useful and has many indications in dentistry. Cryopreservation of teeth creates new possibilities--eg, when autotransplantation is needed, but the recipient site is too small and orthodontic treatment is needed to gain space for the transplant. This review article examines the reactions of various dental tissues after autotransplantation vs after autotransplantation with cryopreservation. Various subjects will be discussed, including periodontal healing, pulp reactions, and root development after autotransplantation with and without cryopreservation. PMID- 16679212 TI - Orthodontic treatment in a patient with Van der Woude's syndrome. AB - Van der Woude's syndrome (VDW; #OMIM 119300) is an autosomal dominant disease characterized by cleft lip and/or palate and lower lip pit (fistula). The precise skeletal characteristics are unclear, and there have been no case reports of orthodontic treatment of patients with VDW. The Japanese girl whose treatment is reported here had VDW, including bilateral cleft lip and palate and bilateral symmetric lower lip pits. Orthodontic treatment started when she was just 3 years old, with a removable maxillary expansion appliance, followed by an edgewise multibracket appliance in both arches. Retention began at 11 years of age, and a secondary bone graft was performed for the alveolar cleft. She received prosthetic treatment and achieved a desirable occlusion at 18 years of age. Early intervention helped achieve a satisfactory treatment result for our patient. In contrast, her mother also had VDW, with a severe Class III skeletal pattern, but she had not been treated orthodontically; she had an anterior and lateral crossbite even after prosthetic treatment. The pretreatment characteristics of 4 other subjects with VDW are discussed; they show wide variations in the sizes of the maxilla and the mandible, suggesting that a common skeletal pattern is not generally seen in VDW. PMID- 16679213 TI - Litigation, legislation, and ethics. A wolf in sheep's clothing. PMID- 16679214 TI - Childhood obesity: is it time for action? PMID- 16679215 TI - Body mass, fat distribution and blood pressure in Southern Italian children: results of the ARCA project. AB - OBJECTIVE: To investigate the association between the degree of adiposity, assessed using the international reference values for body mass index (BMI) of the International Obesity Task Force (IOTF), the fat distribution pattern and the blood pressure (BP) profile in children. METHODS: Anthropometric indices and blood pressure were measured in 3923 children aged 6-11 years in southern Italy. RESULTS: The prevalence of overweight and obesity (by IOTF references) and pediatric hypertension was, respectively: 27, 21 and 10% for boys; 25, 21 and 14% for girls. Body mass index and waist z-scores were the strongest determinants of BP by regression analysis. Overweight and obesity were associated with a greater tendency for central fat deposition and higher BP (waist, cm; boys: 59.2+/-6.0, 69.5+/-7.9, 79.0+/-9.7; girls: 58.8+/-6.5, 68.2+/-7.4, 75.3+/-8.9; SBP/DBP, mmHg; boys: 94/60+/-12/9, 99/62+/-13/8, 103/64+/-15/10; girls: 93/59+/-12/9, 99/62+/ 14/9, 101/63+/-14/9; normal weight, overweight and obese, respectively; P<0.0001; M+/-SD), and a higher risk of hypertension (overweight: RR=2.33; 95% CI 1.76 3.08; obesity: RR=3.69; 95% CI 2.78-4.90), independent of age, physical activity, birth weight, parental adiposity and education. Among normal weight children, 99% had waist <85th percentile and 93% were normotensive. CONCLUSIONS: Overweight and obese children, identified according to the IOTF growth charts, are characterized by a central fat distribution pattern and higher BP. PMID- 16679216 TI - High prevalence of overweight and obesity in 11-15-year-old children from Sicily. AB - BACKGROUND AND AIM: To evaluate the prevalence of overweight and obesity in children and adolescents from Sicily, we carried out a cross-sectional study in a large cohort of 48,897 (24,119 males and 24,778 females) randomly selected 11-15 year-old Sicilian schoolchildren. METHODS AND RESULTS: Anthropometric data (weight and height) were obtained in all children. Urban vs. rural areas were taken into account. Centiles were obtained using the LMS method. Obesity and overweight prevalence were defined using as references both the values of the National Center for Disease Control (CDC 2000) in the United States and those of the International Obesity Task Force (IOTF). Median body mass index (BMI) values in Sicily were comparable to values observed in South and Center-North Italy. BMI cut-off values in Sicilian children were higher than reference values established in the U.S. CDC growth chart 2000. Using both the IOTF or the U.S. CDC 2000 cut off values the prevalence of overweight and obesity in 11-15-year-old Sicilian children was very high: nearly 40% at age 11 and, although progressively decreasing with age increase, still over 25% at age 15. CONCLUSIONS: The prevalence of overweight and obesity in 11-15-year-old Sicilian schoolchildren is one of the highest ever reported. The prevalence is much higher at a younger age; thereafter it progressively decreases and values tend to reconcile with those observed in other geographical areas at age 14-15. PMID- 16679217 TI - Period prevalence of abnormal glucose tolerance and cardiovascular risk factors among obese children attending an obesity centre in Italy. AB - BACKGROUND AND AIM: Several reports have described an increasing prevalence and incidence of type 2 diabetes among children. Limited information is available about the prevalence of impaired glucose tolerance (IGT) and type 2 diabetes in obese children, particularly in Europe. The aim of this study was to examine the prevalence of glucose intolerance and other cardiovascular risk factors in obese children over a 24-year period. METHODS AND RESULTS: The study population consisted of 1376 consecutive subjects who attended a national centre for the study of obesity between 1979 and 2002. Subjects were divided into three successive 8-year cohorts: cohort 1 (period 1979-1986, n=453, male: 39%), cohort 2 (period 1987-1994, n=409, male: 46%), cohort 3 (period 1995-2002, n=514, male: 48%). All subjects underwent an oral glucose tolerance test. Lipids, blood pressure, uric acid, C-reactive protein (CRP), fasting insulin and birth weight were recorded. Insulin resistance was measured by homeostasis model assessment (HOMA-IR). The degree of obesity was higher in the more recent cohorts (standard deviation score of body mass index: 3.3+/-0.04 vs. 3.7+/-0.04 vs. 3.8+/-0.03, P<0.0001). The proportion of subjects with glucose intolerance was lower in the last two cohorts compared with the first one (11.2% vs. 3.9% vs. 6.0%, P<0.0001). This was predominantly due to changes in the frequency of IGT (9.1% vs. 3.2% vs. 5.4%, P<0.001 in cohorts 1, 2 and 3, respectively) while the prevalence of undiagnosed type 2 diabetes and impaired fasting glucose was similar in the three cohorts (0.9% vs. 0% vs. 0.2% and 1.3% vs. 0.7% vs. 0.4%, respectively). After adjustment for differences in age, sex, pubertal status and birth weight the levels of fasting insulin, HOMA-IR, total cholesterol, triglycerides and blood pressure, were significantly lower in cohorts 2 and 3 than in cohort 1 while CRP and uric acid were higher in the last two cohorts. CONCLUSION: Over a recent period spanning 24 years, the degree of obesity has risen but the prevalence of glucose intolerance has fallen in obese children admitted to an obesity centre. This was accompanied by an improvement in traditional but a worsening in non traditional risk factors for cardiovascular disease. PMID- 16679218 TI - Anthropometric predictors of serum fasting insulin in 9- and 15-year-old children and adolescents. AB - BACKGROUND AND AIM: As the prevalence of overweight and obesity increases, the risk of insulin resistance rises. The aim was to study the association between anthropometric measurements and fasting insulin concentration in a population based sample of 9- and 15-year-old children and adolescents. METHODS AND RESULTS: Subjects were randomly selected 9- and 15-year-old pupils (n=262) in a cross sectional, population-based study. Weight and height, waist, hip and mid-arm circumference and subcutaneous skinfolds were measured using standard procedures. Fasting insulin was measured. In general the mean anthropometric measurements increased across insulin quartiles. Higher fasting insulin concentration was seen in overweight children and adolescents than in those of normal weight (8.3+/-4.4 vs. 4.9+/-3.6 mmol/L and 11.0+/-4.4 vs. 9.0+/-4.2 mmol/L in 9- and 15 year-olds, respectively). The odds ratio for having insulin in the highest quartile (age and gender-specific) was, when compared with the lowest quartile, 7.2 (95% CI 3.0 17.2) for body mass index and 6.9 (2.8-16.7) for waist circumference. Other measurements of body fatness were less predictive. About 14-20% of children defined as being of normal weight had high fasting insulin values, i.e., were in the highest quartile of fasting insulin. CONCLUSIONS: Body fatness is positively related to fasting insulin concentration in 9- and 15-year-old children. A large number of normal-weight individuals with high fasting insulin concentration was observed, and these children could be at increased risk of weight gain, compared with normal-weight individuals with normal fasting insulin concentration. PMID- 16679219 TI - Determinants of weight gain in children from 7 to 10 years. AB - BACKGROUND AND AIM: To determine the influence of the family risk factors (parental weight, socioeconomic status and cultural level) on the distribution of overweight or obesity in prepubertal children and the dynamics of their weight gain. METHODS AND RESULTS: Three hundred forty-one children (183 boys) attending the second grade of primary school (age 7.4+/-0.5 years) were enrolled in a longitudinal study in Southern Italy. Overweight and obesity status were defined by a body mass index (BMI) above the percentile having respectively the value of 25 and 30 kg/m(2) at 18 years, according to national reference data; overweight extent was expressed as BMI standard deviation score (BMI-SDS). Information about BMI and socioeconomic conditions (occupation and educational level) of parents were collected. Distribution of overweight and obese schoolchildren and variations of BMI-SDS were evaluated over a 3-year period; relationships with family risk factors were also sought. The distribution of overweight/obese children at baseline was high (40%). Tracking rates of overweight and obesity were 73% and 80%, respectively. The higher the BMI-SDS at 7 years, the higher the BMI-SDS at 10 years (r 0.86, P=0.0001). The highest BMI value and the highest distribution of overweight/obese children were observed in subjects with the highest amount of family risk factors. Children exhibiting accelerated weight gain (delta BM I>1/year) showed higher male/female ratio, higher baseline BMI values, higher maternal BMI values, lower maternal educational level and a dramatic increase in the percentage of overweight/obese children than children with normal weight gain. CONCLUSIONS: Children who are overweight/obese at 7 years tend to maintain this condition during prepubertal age. Paediatricians should be alerted when dealing with a child showing a BMI increase above than 1U/year during primary school. Targeted intervention should be directed at young children with overweight parents and low socio-cultural level. PMID- 16679220 TI - Insulin resistance and impaired glucose tolerance in obese children and adolescents from Southern Italy. AB - BACKGROUND AND AIM: Obesity in children may lead to insulin resistance and impaired glucose regulation over time. The aim of this study was to investigate the insulin resistance status and the frequency of impaired glucose regulation in obese children and adolescents from the Campania region (Southern Italy), where the prevalence of obesity is among the highest in Europe. METHODS AND RESULTS: We studied 100 (62 male) Italian obese children and adolescents (mean age 10.1+/-2.7 years) and 50 (27 male) normal weight healthy subjects (mean age 10.2+/-2.7 years). Anthropometric measures and biochemical tests were performed in all subjects. In obese patients an oral glucose tolerance test was also performed. The estimate of insulin resistance was calculated by a homeostasis model assessment (HOMA) index. A cut-off HOMA level of >2.5 in children and >4.0 in adolescents was used to identify an insulin-resistance status. Insulin resistance was found in 40.8% of obese children and 41.2% of obese adolescents, whereas it was found in 3.0% of normal children and none of the 17 normal adolescents (p<0.0001 and p<0.002, respectively). None of the subjects had impaired fasting glucose or diabetes, while 4 obese patients had impaired glucose tolerance (4%). CONCLUSIONS: Impaired glucose tolerance is still rare whereas insulin-resistance is already detectable in more than 40% of obese children and adolescents in Southern Italy. Our observations confirm that metabolic risk factors can be found at a very early age and strengthen the case for implementing programmes for prevention and treatment of childhood obesity. PMID- 16679221 TI - Aerobic physical fitness in relation to blood lipids and fasting glycaemia in adolescents: influence of weight status. AB - BACKGROUND AND AIMS: We explored the associations between aerobic physical fitness with blood lipids and a composite index of blood lipids and fasting glycaemia in adolescents, analysing possible interactions with weight status. METHODS AND RESULTS: Body mass index and aerobic physical fitness was measured in 2090 adolescents (1034 males and 1056 females) 13-18.5 years by using the 20-m shuttle run test. Plasma glucose, total, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, triglycerides, apolipoprotein (apo) A-I, apo B-100 and lipoprotein(a) [Lp(a)] were measured in 460 of the 2090 subjects. After adjustment for confounding factors, a continuously distributed summary score for blood lipids and fasting glycaemia was significantly related to aerobic fitness in males (P=0.018) and females (P=0.045, from the 2nd to the 4th quartile of aerobic fitness). After adjustment for gender, age, sexual maturation and economic status, aerobic fitness was related to the composite index of blood lipids and glycaemia in both overweight and non-overweight adolescents (P< 0.05). However, for the same level of aerobic fitness, the composite index of blood lipids and glycaemia was significantly higher in overweight adolescents (P=0.001). After setting the minimal aerobic fitness standards to present a healthy lipid profile, about 50% of males did not reach such values. CONCLUSION: Our data suggest that both aerobic fitness and weight management are associated with a composite index of blood lipids and glycaemia in adolescents. Our study also provides the minimal levels of aerobic physical fitness associated with a favourable lipid profile in male adolescents, a new tool which should be adopted by schools as "aerobic fitness standards". PMID- 16679222 TI - Is dietary intake able to explain differences in body fatness in children and adolescents? AB - Obesity is the result of an imbalance between energy intake and energy expenditure. Controversial information exists about what are the strongest energy balance aspects influencing body fatness. This article is focused on food consumption facts that could be related to the risk of being obese in children and adolescents. It reviews whether energy intake, macronutrient composition of diet, eating patterns or other dietary intake factors are able to explain differences in body composition when obesity has been already developed or even in subjects at risk to become obese. There is not enough evidence to clarify the importance of diet on overweight children and adolescents, and conclusions derived are somewhat controversial. Cross-sectional and longitudinal studies do not show clear relationships between energy intake or food composition and body fatness. To find relations between dietary factors and childhood obesity perhaps eating patterns or different types of foods must be considered: meal patterns and meal frequency, snacking and beverage consumption, fast food intake, portion sizes, etc. There is no clear association between different aspects of dietary intake and the development of obesity in children and adolescents. Longitudinal and experimental studies are needed in the future. PMID- 16679223 TI - Understanding and preventing childhood obesity and related disorders--IDEFICS: a European multilevel epidemiological approach. AB - The environment of children has drastically changed in Europe during the last decades as reflected in unhealthy dietary habits and sedentary lifestyle. Nutrition obviously plays a part in the development of overweight in childhood. However, dietary factors and physical activity are also involved in the development of metabolic syndrome, type II diabetes, cardiovascular diseases, osteoporosis, and postural deformities like scoliosis, effects related in part to excessive weight gain. To stop the resulting epidemic of diet- and lifestyle induced morbidity, efficient evidence-based approaches are needed. These issues are the focus of IDEFICS (Identification and prevention of dietary- and lifestyle induced health effects in children and infants), a five-year project proposed under the sixth EU framework. The IDEFICS consortium comprises 25 research centres and SMEs across Europe. The planned prospective study will identify risk profile inventories for children susceptible to any of these disorders with emphasis on obesity and its co-morbid conditions. Genetic and non-genetic factors, psychosocial factors and social settings will be considered. The project will devise tailored prevention strategies that are effective, easy to implement and that account for the needs of different social groups. Population-based studies will investigate the impact of sensory perception and provide results concerning internal and external triggers of food choices and children's consumer behaviour. The ethical implications of a "right not to know" of genetic factors will be addressed. We will propose knowledge-based guidelines on dietary and lifestyle activities for health promotion and disease prevention in children for health professionals, stakeholders and consumers. PMID- 16679224 TI - Surgical treatment of os acromiale with and without associated rotator cuff tears. AB - Nineteen consecutive patients treated surgically for meso-os acromiale and subacromial pathology were reviewed retrospectively, with a mean length of follow up of 40 months (range, 24-94 months). Of the patients, 11 (58%) were treated with acromioplasty in the presence of a stable os acromiale; 8 patients (42%) underwent open reduction-internal fixation for an unstable and painful os fragment. Of the 19 patients, 8 (42%) with an os acromiale had an associated full thickness rotator cuff tear. Overall, only 10 of 19 patients (53%) achieved a satisfactory result. All 8 patients (100%) treated with open reduction-internal fixation achieved union of the os fragment, although only 3 (37.5%) achieved a satisfactory result. Of the 11 patients who underwent acromioplasty, only 7 (64%) achieved a satisfactory result. The outcome of surgical management of symptomatic meso-os acromiale with concomitant rotator cuff pathology was satisfactory in 4 of 8 patients in our study group. The rate of satisfactory results was similar in patients with (50%) and without (55%) associated rotator cuff tears. When we analyzed our results to exclude workers' compensation patients, 80% achieved satisfactory results (compared with only 22% in our workers' compensation group). PMID- 16679225 TI - Pathogenesis of partial tear of the rotator cuff: a clinical and pathologic study. AB - This prospective study investigated the clinical and pathologic results in 66 patients with partial tears of the rotator cuff from January 1996 to December 1998. The pathologic change in the rotator cuff was graded from the magnetic resonance images by using the criteria described by Zlatkin and Iannotti. A modified grading system from Ozaki and Panni was used for pathologic grading of the anterior acromion. The functional score of Constant and Murley was used for clinical assessment. The pathologic change in the rotator cuff revealed by the magnetic resonance imaging study was more severe in patients with articular side tears compared with patients who had bursal side tears. On the contrary, the pathologic changes in the acromion were significantly milder in patients with articular side tears compared with bursal side tears. These observations indicate that articular side tears of the rotator cuff are mainly associated with intrinsic pathologic changes of the rotator cuff, whereas bursal side tears are associated with subacromial impingement on an underlying milder pathologic change of the rotator cuff. PMID- 16679227 TI - Cuff integrity after arthroscopic versus open rotator cuff repair: a prospective study. AB - Arthroscopic rotator cuff repair (RCR) has been reported to have good clinical results but high retear rates by ultrasound. We prospectively assessed postoperative cuff integrity and outcome after arthroscopic RCR (40 patients) and compared these results with open RCR (32 patients). Evaluation preoperatively and at 1 year included a physical examination and magnetic resonance imaging. American Shoulder and Elbow Surgeons and Constant scores improved significantly in both groups (P < .0001). Overall, 69% of repairs in the open group and 53% in the arthroscopic group were intact by magnetic resonance imaging. Of tears less than 3 cm in size, 74% in the open group and 84% in the arthroscopic group were intact. Of tears greater than 3 cm in size, 62% in the open group and 24% in the arthroscopic group were intact (P < .036). In the arthroscopic group, patients with an intact cuff had significantly greater strength of elevation (P = .01) and external rotation (P = .02). We conclude that open and arthroscopic RCRs have similar clinical outcomes. Cuff integrity is comparable for small tears, but large tears have twice the retear rate after arthroscopic repair. PMID- 16679226 TI - One hundred eighteen Bristow-Latarjet repairs for recurrent anterior dislocation of the shoulder prospectively followed for fifteen years: study II-the evolution of dislocation arthropathy. AB - Dislocation arthropathy after surgical treatment of recurrent anterior dislocation of the shoulder has been a subject of discussion over the years. The objective of this prospective study was to evaluate the Bristow-Latarjet repair at 2 and 15 years after surgery with respect to arthropathy and to evaluate factors responsible for this development. At 2 and 15 years after a Bristow Latarjet repair for recurrent anterior dislocation, we prospectively analyzed the radiographs of 115 of 118 shoulders. The majority of the patients also had radiography of the nonindex shoulder (111/118). Dislocation arthropathy was found on ordinary anteroposterior views in 46 of 115 shoulders (mild in 39, moderate in 5, and severe in 2). The anteroposterior view angulated 45 degrees from above gave the best visualization of the humeral osteophyte and, together with the axial view, doubled the incidence of moderate and severe arthropathy at follow-up (mild in 40, moderate in 11, and severe in 5). Of 38 shoulders with a postoperative position of the transferred coracoid process 2 to 4 mm medial to the glenoid rim and the screw and transplant parallel to the glenoid, 3 had moderate arthropathy (8%) compared with 8 shoulders with moderate arthropathy and 5 with severe arthropathy out of 77 shoulders with a nonoptimal position of the coracoid (17%) (P = not significant). The degree of restriction of outward rotation at 2 years did not influence the degree of arthropathy after 15 years. Global assessment of the operative result was not related to arthropathy at follow-up. When the first dislocation occurred before age 23 years, the incidence of arthropathy was significantly lower than when it occurred in age 23 years or older (P = .006). When all views were included, moderate or severe dislocation arthropathy was found in 14% of the shoulders, and a further 35% had mild arthropathy. When follow-up studies of operated series of shoulder dislocations with the Bristow-Latarjet method are done, it is important to have the same radiologic views to compare the incidence of dislocation arthropathy correctly. PMID- 16679228 TI - Clinical and radiologic outcomes of surgical and conservative treatment of type III acromioclavicular joint injury. AB - The management of acute acromioclavicular joint dislocations is controversial. The purpose of this study was to compare the incidence of posttraumatic anatomic alterations after surgical or conservative treatment of type III injuries and to analyze their effect on the outcome. Forty-three patients were evaluated retrospectively, clinically and radiographically, at a 12-month minimum follow up. Thirty-two were treated surgically, using the Phemister technique, and 11 had conservative treatment. A comparison of the overall clinical results in both groups showed no statistically significant differences. The acromioclavicular joint was anatomically reduced in only half of the surgical patients. Those shoulders treated surgically showed a significantly higher incidence of osteoarthritis and coracoclavicular ligament ossification. Differences in clavicular deformity or osteolysis were not significant. None of these abnormalities had any influence on the clinical result. Because operative and conservative treatments achieve equally good clinical results and surgery carries a higher risk of osteoarthritis, we recommend managing this injury conservatively. PMID- 16679229 TI - Does reaching the back reflect the actual internal rotation of the shoulder? AB - To clarify the relationship between the vertebral level reached by the thumb and the internal rotation angle of the humerus, 7 shoulders in healthy volunteers were examined by use of an electromagnetic tracking device. Measurements were repeated in the hanging-arm position with the thumb pointing anteriorly and at the buttock, sacrum, and each vertebral level up to T6. From the hanging-arm position to the buttock, 54.3% of internal rotation occurred (mean, 39.8 degrees ), and from the buttock to the sacrum, 11.7% occurred (mean, 8.6 degrees ). In total, 66% of internal rotation occurred from the hanging-arm position to the sacrum. Above the sacrum, the contribution of elbow flexion to the level of the vertebral spine became much greater, and internal rotation of the shoulder did not change significantly above the T12 level. We recommend that the level of the thumb below the buttock be subdivided for more accurate assessment of internal rotation. PMID- 16679230 TI - Does hand-behind-back range of motion accurately reflect shoulder internal rotation? AB - Shoulder internal rotation (IR) is commonly assessed by an indirect method where the hand is placed behind the back and the distance reached by the tip of the extended thumb is recorded. The aim of this study was to assess the validity of measuring active IR range of motion (ROM) by use of the indirect hand-behind-back (HBB) ROM method in subjects with shoulder pain of mechanical origin. We recruited 137 subjects with unilateral shoulder pain. HBB ROM was determined by measuring the distance between the T1 spinous process and the radial styloid process. Active shoulder IR was measured in the supine position in 45 degrees or 90 degrees abduction. Correlation coefficients adjusted for measurement error were calculated. HBB ROM demonstrated only a low to moderate correlation with active shoulder IR. Active HBB ROM is not an accurate method of measuring active shoulder IR in patients with shoulder pain. PMID- 16679231 TI - Suture anchors for treatment of sternoclavicular joint instability. AB - Instability of the sternoclavicular joint is a difficult problem to treat and can present with gross limitation in activities. Eight sternoclavicular joint stabilization procedures were performed over an 8-year period. The patients' ages ranged from 16 to 48 years (mean, 23.5 years). The indication for stabilization was pain associated with instability of the sternoclavicular joint. The joint was stabilized by use of suture anchors on the manubrium and capsular plication. The functional outcome was evaluated by use of the Constant score and patient-based Oxford Shoulder Questionnaire. At a mean follow-up of 4.5 years (range, 1-7.6 years), none of the patients had instability at the sternoclavicular joint, and all except one had returned to their previous employment. The Oxford score was 16 (range, 12-38). The mean Constant score was 74.88 (range, 33-87). We had only 1 poor result (Constant score of 33). Stabilization of the sternoclavicular joint can safely be performed by use of suture anchors. The technique is recommended for symptomatic sternoclavicular joint instability. PMID- 16679232 TI - Subacromial pressures in vivo and effects of selective experimental suprascapular nerve block. AB - Subacromial impingement has been related to increased subacromial pressures. High subacromial pressures may, therefore, have a negative effect on tendon healing after rotator cuff repair, but avoidance of high pressures during healing is only possible if pressures in different positions of the arm are known. The purpose of this study was to determine the subacromial pressures for different positions of active shoulder movement and to determine the effects of supraspinatus and infraspinatus failure on subacromial pressures, as it is currently held that rotator cuff weakness might increase subacromial pressures. Subacromial pressures were statistically significantly altered by arm position (P = .001). There was an increase in pressure from 17.5 mm Hg at rest up to more than 60 mm Hg during abduction and flexion. Subacromial pressure decreased in external rotation and increased in internal rotation. Generally, patients with strong external rotation (arm at the side) had lower subacromial pressures. Conversely, paralysis of the supraspinatus or infraspinatus muscles had no statistically significant effect on subacromial pressures at rest or during active movements of the shoulder. High subacromial pressures can be prevented by avoiding active abduction, flexion, and internal rotation of the arm. External rotation does not need to be limited to reduce subacromial pressure. PMID- 16679233 TI - Comparison of three stretches for the pectoralis minor muscle. AB - Pectoralis minor adaptive shortening in healthy individuals is associated with altered scapular kinematics similar to the alterations demonstrated in individuals with subacromial impingement. This associative relationship suggests that stretching of the pectoralis minor may improve scapular kinematics and assist in the management of shoulder impingement. Several stretches for the pectoralis minor are used clinically, although it is not known which stretch optimally lengthens the muscle. The purpose of this analysis was to compare the mean length change for 3 pectoralis minor stretches. Fifty subjects without shoulder pathology were examined for the change in length of the pectoralis minor during 3 separate stretches by use of an electromagnetic motion-capture system. The stretches analyzed were a unilateral self-stretch, a supine manual stretch, and a sitting manual stretch. Each stretch was significantly different from the other two (df, 2/98; F ratio, 39.09; P < .00001), with the unilateral self stretch demonstrating the greatest length change (2.24 cm), followed by the supine manual stretch (1.69 cm) and the sitting manual stretch (0.77 cm). Knowledge of the most effective method of elongating the pectoralis minor muscle may improve clinical decision making when targeting this anterior scapulothoracic muscle as part of intervention for or prevention of shoulder impingement. PMID- 16679234 TI - Numerical analysis of cooperative abduction muscle forces in a human shoulder joint. AB - Because some shoulder muscles originate from a wide area, the modeling of such muscles has been a significant problem in a computer simulation. We demonstrated a new method of determining a vector for each of the muscles originating from a wide area. A 3-dimensional musculoskeletal model of a human shoulder was constructed from computed tomography data of a normal volunteer. Numerical analysis of 11 muscle forces and the joint reaction force during shoulder abduction from 10 degrees to 150 degrees was performed from the static equilibrium equations. An optimal origin point for the vector of the muscle with a wide origin area was determined in every analyzed position. Electromyography was carried out to validate the results of the simulation, and a significant correlation with the analyzed force was obtained in each muscle. The anatomic biomechanical model with the new muscle modeling method led to the results reflecting the actual muscle activities in a living body. PMID- 16679235 TI - The effect of scapular protraction on isometric shoulder rotation strength in normal subjects. AB - To measure the effect of scapular protraction on isometric shoulder rotation strength, 20 normal subjects completed 2 maximal isometric internal and external rotation contractions in 2 scapular positions (scapula neutral [SN] and scapula protracted [SP]) from 3 arm positions (90 degrees internal rotation [IR], 45 degrees internal rotation [MR], and 90 degrees external rotation [ER]). Scapular protraction reduced shoulder rotation strength in 5 of 6 test positions (P < .0004), with significant interactions between scapular position and arm position (P < .001) and between scapular position and contraction type (P < .0001). Protraction significantly reduced IR strength by 13% to 24% relative to SN. The effect of SP on ER strength was more position-dependent, increasing strength by 6% in the IR position and decreasing it by 7% in the MR position and 20% in the ER position. In conclusion, acute changes in scapular position affect shoulder isometric IR and ER strength. The potential adverse effects of scapular protraction on shoulder rotation strength should be considered during the evaluation and treatment of shoulder pain. PMID- 16679236 TI - Prospective evaluation of two diagnostic apprehension signs for posterolateral instability of the elbow. AB - Posterolateral rotatory instability (PLRI) of the elbow occurs from attrition of the lateral ulnohumeral collateral ligament of the elbow after elbow dislocation. Diagnosis by physical examination can be difficult in the awake patient. The goals of this study were to define two active apprehension signs for the physical diagnosis of PLRI and to perform a prospective evaluation of the signs in a series of patients with PLRI. Eight patients with PLRI undergoing surgical reconstruction of the lateral ulnocollateral ligament of the elbow were prospectively included in this continuous case series. Preoperative evaluation consisted of physical examination with two active apprehension signs, the chair sign and the pushup sign, as well as the pivot-shift sign. Results were compared with repeat physical examination after reconstruction of the ligaments. Of 8 patients included in the series, 3 demonstrated a positive pivot-shift sign while awake, and all demonstrated a positive pivot-shift sign while under anesthesia. Seven patients demonstrated a positive chair sign, and seven demonstrated a positive pushup sign. At the 2-year follow-up evaluation, 7 patients remained stable and asymptomatic. The pushup sign, chair sign, and pivot-shift sign were negative in all 7 patients. The study demonstrated that both the pushup and chair signs are effective in aiding the diagnosis of PLRI. They are more sensitive than the pivot-shift sign in the awake patient and may be easily performed in the office environment. PMID- 16679237 TI - Debridement of extensors and drilling of the lateral epicondyle for tennis elbow: a retrospective follow-up study. AB - Twenty-one patients treated for tennis elbow with debridement of the extensors, without repair of the affected tendons, and with decortication by drilling of the lateral epicondyle were reviewed retrospectively after a mean follow-up of 15 months (range, 11-33 months). The early postoperative period was characterized by a painful and slow recovery. At latest follow-up, 17 patients (81%) had a satisfactory outcome with no or only mild pain. Of the patients, 20 (95%) felt that they were better or much better compared with preoperatively. There were 2 complications: 1 hematoma that was aspirated and 1 temporary frozen shoulder. Debridement of the extensors and drilling of the lateral epicondyle relieved pain and restored function in the majority of the patients. However, recovery was slow and was never found to be immediate as described in other series of tennis elbow procedures where the extensors were simply released and the lateral epicondyle was not decorticated. PMID- 16679238 TI - Tension-band wiring for olecranon fractures: analysis of risk factors for failure. AB - Thirty-seven consecutive olecranon fractures treated with tension-band wiring were evaluated at a mean follow-up of 4 years. There was no or mild pain in 33 cases, mean elbow extension was 7 degrees , and mean elbow flexion was 131 degrees . According to the Mayo Elbow Performance Score, the results were graded as good or excellent in 32 cases (86%). Most patients had mild residual upper extremity disability (mean Disabilities of the Arm, Shoulder and Hand score, 18 points). In 10 patients, degenerative changes developed. Arthritic changes were significantly associated with elbow instability (P = .014) and length of follow up (P = .031) and were more common in the presence of associated radial head or coronoid fractures (P = .06). Hardware removal was required in 17 cases. Tension band wiring provides satisfactory results in a high percentage of olecranon fractures. However, the outcome may be compromised in the presence of elbow instability and associated fractures of the radial head and coronoid. PMID- 16679239 TI - Functional outcomes and general health status after ulnohumeral arthroplasty for primary degenerative arthritis of the elbow. AB - Ulnohumeral arthroplasty (UHA) is considered a satisfactory surgical treatment option for patients with primary degenerative arthritis of the elbow. Most series have used categoric elbow scoring systems to evaluate the outcome of this procedure. The purpose of our study was to evaluate the outcome of UHA with patient-derived functional and general health status outcome instruments. We evaluated 17 patients (18 elbows) with primary degenerative arthritis of the elbow at a mean of 85 months after UHA. The mean age at the time of surgery was 42 years (range, 26 to 58 years). At the follow-up evaluation, the patients were assessed with a physical examination, outcome assessment tools, and plain radiographs. The mean elbow flexion arc improved by 16 degrees (range, -15 degrees to 60 degrees ; P = .012), and the mean forearm rotation arc (supination/pronation) improved by 35 degrees (range, -20 degrees to 90 degrees ; P < .001). Of the elbows, 11 were painless, 4 were painful with motion, and 3 were painful at rest and with motion. The mean Hospital for Special Surgery elbow score was 70 (range, 40 to 99), and the mean Mayo Elbow Performance Score was 83 (range, 50 to 100). The mean Disabilities of the Arm, Shoulder and Hand score was 9.75 (range, 0 to 43.48). The Short Form-36 scores were better than the mean age- and sex-adjusted normal values. Patient self-assessed outcomes and general health status after UHA for primary degenerative elbow arthritis appear to be better than those determined by some categoric scoring systems. Consequently, the clinical utility of UHA may be underrepresented if physician-derived categoric scoring systems are the only measures of outcome assessment. PMID- 16679240 TI - The posterolateral plica: a cause of refractory lateral elbow pain. AB - Lateral epicondylitis is one of the most common upper extremity pain syndromes. We report the results of patients in whom conservative treatment was unsuccessful and who were finally treated arthroscopically for symptomatic plicae. Ten patients (mean age, 40 years [range, 18-60 years]) who were misdiagnosed as having lateral epicondylitis were included in this study. Examination revealed the site of maximal tenderness to be posterior to the lateral epicondyle and centered at the posterior radiocapitellar joint. Preoperatively, all patients received conservative treatment (physical therapy or corticosteroid injections [or both]). The mean follow-up was 25 months (range, 6-68 months). The mean score on the Disabilities of the Arm, Shoulder and Hand questionnaire was 9 (range, 0 37). Preoperatively, 7 patients had full elbow range of motion; however, in 3 patients, there was a loss of extension at the elbow ranging from 7 degrees to 20 degrees preoperatively. The range of elbow motion was full in all patients postoperatively. No patient demonstrated posterolateral pain after the operation. Synovial plicae of the elbow may be the cause of lateral elbow pain in patients with vague clinical symptoms. Arthroscopic management may provide a successful treatment option for such patients. PMID- 16679241 TI - Expression of growth factors in the early phase of supraspinatus tendon healing in rabbits. AB - Growth factors are known to appear during wound healing. We hypothesized that growth factors would also appear during the healing process of a rotator cuff tear. We determined the expression of various growth factors during healing of acute rotator cuff tears in the rabbit. We made a full-thickness defect in the supraspinatus tendon of 27 Japanese white rabbits. The shoulders were harvested on days 1, 3, 5, 7, 9, 11, 14, 21, and 28 postoperatively (n = 3 at each time point). We assessed the expression of basic fibroblast growth factor, insulin like growth factor 1, platelet-derived growth factor, and transforming growth factor beta. Basic fibroblast growth factor appeared with its peak on days 7 and 9, insulin-like growth factor 1 appeared with its peak on day 5, platelet-derived growth factor appeared with a mild expression between days 7 and 14, and transforming growth factor beta appeared with constant mild expression throughout the observation period. It is likely that each of these growth factors plays a role in the early phase of healing of the supraspinatus tendon in rabbits. PMID- 16679242 TI - Neuropathic elbow arthropathy: a review of six cases. AB - Neuropathic arthropathy, also known as Charcot arthropathy, of the elbow joint is a rare and progressive joint disorder with little available information in the literature. Presentation and treatment data on 6 patients with this pathology are reviewed. In addition, outcome data are presented on all 6 patients at a mean follow-up of 51 months. Three of these patients had undergone surgical procedures for the treatment of their pathology. At the time of their latest follow-up, 2 patients had died but reported good pain relief and functional range of motion before their deaths. At a mean follow-up of 63 months, the remaining 4 patients also had minimal pain. Of these 4 patients, 3 reported moderate to gross instability in their elbows. Nevertheless, all 4 patients had good functional use of their arms with a mean Mayo Elbow Performance Score of 91. Our data suggest that surgical treatment of appropriate patients does not necessarily preclude a successful outcome. However, surgical treatment was also associated with an unpredictable course and a high rate of complications. Therefore, most patients with this pathology should be treated with nonoperative methods, and routine surgical intervention should be avoided. PMID- 16679243 TI - Deltoid compartment syndrome: a result of operative positioning. PMID- 16679244 TI - Progressive massive osteolysis of the humerus. PMID- 16679246 TI - AMTC presentations. PMID- 16679245 TI - Knot-induced glenoid erosion after arthroscopic fixation for unstable superior labrum anterior-posterior lesion: case report. PMID- 16679248 TI - NTSB speaks, but who listens? PMID- 16679249 TI - 3-month-old boy with a liver mass. PMID- 16679250 TI - Clinical research and critical care transport: how to get started. PMID- 16679252 TI - Carilion Life-Guard: a quarter century of air medical service. PMID- 16679253 TI - Small and smaller. PMID- 16679254 TI - Brugada syndrome: silent assassin. PMID- 16679255 TI - Redefining the therapeutic objective in decompensated heart failure: hemoconcentration as a surrogate for plasma refill rate. AB - BACKGROUND: Acute decompensated heart failure is a growing epidemiologic problem about which little consensus exists on guidelines and recommendations for therapy. METHODS AND RESULTS: Available databases suggest that a large percentage of patients are being inadequately decongested while hospitalized, resulting in poor clinical outcomes. This is partly from a lack of an appropriate target to define therapeutic success. The demonstration of a prerenal state by blood work does not indicate adequate decongestion but rather means that the rate of fluid removal has exceeded the plasma refill rate. Hemoconcentration, as evidenced by a rising hematocrit is an appropriate surrogate to indicate that the plasma refill rate has been exceeded by the rate of fluid removal. This surrogate of plasma refill rate can be easily and continuously measured by using an in-line hematocrit sensor during ultrafiltration therapy. CONCLUSION: We propose that the therapeutic objective in acute decompensated heart failure should be redefined and that the rate of volume extraction should be adjusted to approximate the plasma refill rate and that complete decongestion will have occurred only once hemoconcentration is observed at minimal rates of volume extraction. PMID- 16679256 TI - Spironolactone treatment and clinical outcomes in patients with systolic dysfunction and mild heart failure symptoms: a retrospective analysis. AB - BACKGROUND: The effect of spironolactone on clinical outcomes in patients with mild heart failure is unclear. METHODS AND RESULTS: We performed a retrospective analysis of 482 consecutive patients with left ventricular ejection fraction < or =40% and New York Heart Association I-II symptoms. Major cardiac event (MCE) was defined as death, left ventricular assist device implantation, or United Network of Organ Sharing 1 cardiac transplantation. Proportional hazards analysis was used to determine predictors of MCE and to derive an adjusted hazard for spironolactone therapy. Spironolactone was prescribed to 279 (58%) patients and mean follow-up was 1029 days. After controlling for predictors of clinical events, spironolactone treatment was associated with a trend for lower risk of MCE or heart failure rehospitalization (HR, 0.68; 95% CI, 0.43-1.07; P = .095). Exploration of interaction terms between medications revealed that treatment with the combination of spironolactone and thiazide diuretics was associated with lower risk of clinical events (HR, 0.32; 95% CI, 0.12-0.89; P = .029). CONCLUSION: In subjects with mild heart failure treated with a thiazide diuretic, the use of spironolactone is associated with reduced risk of MCE or heart failure rehospitalization. A randomized controlled trial is necessary to accurately define the clinical effects of spironolactone in patients with mild heart failure. PMID- 16679257 TI - Secular trends in renal dysfunction and outcomes in hospitalized heart failure patients. AB - BACKGROUND: Renal dysfunction and worsening renal function (WRF) during heart failure (HF) therapy predict outcomes. We determined whether the severity of renal dysfunction, the incidence of WRF or outcomes have changed over time (secular trends) in patients hospitalized for HF therapy. METHODS AND RESULTS: A total of 6440 consecutive unique patients admitted for HF to Mayo Clinic Hospitals Rochester, MN, January 1, 1987, to December 31, 2002, were identified and data extracted from electronic databases. Over the study period, age and admission creatinine increased, whereas estimated glomerular filtration rate and hemoglobin decreased (P < .0001 for all). The prevalence of hypertension and diabetes among HF patients also increased over time (P < .0001). The incidence of WRF was stable. Renal dysfunction and development of WRF were associated with mortality. When adjusted for the changes in baseline characteristics of HF patients, mortality declined over the study period. CONCLUSION: Hospitalized HF patients are increasingly elderly, have a greater prevalence of diseases that lead to renal dysfunction, and have more severe renal dysfunction and anemia on admission. However, adjusting for these secular trends in patient characteristics, mortality after HF admission is improving. These data enhance our understanding of the changing natural history of HF. PMID- 16679258 TI - Right ventricular dyssynchrony in heart failure: a tissue Doppler imaging study. AB - BACKGROUND: The development of right ventricular dysfunction is a poor prognostic sign in patients with heart failure (HF). Although left ventricular dyssynchrony has been well described, it is not known whether right ventricular dyssynchrony coexists in HF. We used tissue Doppler imaging to determine whether right ventricular dyssynchrony is also present in HF patients. METHODS AND RESULTS: In 34 HF patients (mean age 56 +/- 13 years), we measured longitudinal strain at the right ventricular free wall, interventricular septum, and left ventricular lateral wall. Right ventricular and left ventricular dyssynchrony were defined as the difference in time to peak strain between the right ventricular free wall and the septum and between the left ventricular lateral wall and septum, respectively. Mean right ventricular dyssynchrony was 59 +/- 45 ms and the mean left ventricular dyssynchrony was 80 +/- 62 ms. We found a strong correlation between right ventricular dyssynchrony and pulmonary artery systolic pressure (r = 0.73; P < .001) and a negative correlation between right ventricular dyssynchrony and right ventricular fractional area change (r = -0.43; P < .02). CONCLUSION: HF patients exhibit right ventricular dyssynchrony by strain imaging which correlates with pulmonary hypertension and right ventricular dysfunction. PMID- 16679260 TI - Validation of a risk score for dying within 1 year of an admission for heart failure. AB - BACKGROUND: Development of heart failure greatly reduces life expectancy. Accurate estimates of the risk of dying are needed in clinical practice and for risk adjustment in observational studies. A relatively simple risk score has been developed to determine the risk of dying within 1-year of an admission for heart failure. We wanted to evaluate the risk score's predictive validity. METHODS AND RESULTS: Data were abstracted from the electronic medical records of 769 patients admitted to the Minneapolis Veterans Administration medical center with a primary diagnosis of heart failure. Mortality within 1 year of admission was 25%. The c index for the risk score was 0.71 (95% confidence interval 0.67-0.76). Similar to the original derivation cohort, mortality in risk score groups was 7% for a score lower than 60 (n = 44), 14% for 61 to 90 (n = 246), 26% for 91 to 120 (n = 222), 51% for 121 to 150 (n = 106), and 50% for scores greater than 150 (n = 8). CONCLUSION: A previously developed risk score for 1-year mortality after an admission for heart failure provided a moderate degree of discrimination in a validation cohort from a different setting. Mortality in risk score groups was consistent with the original patient cohort. These results support the validity of the risk score and its application to a different patient population. PMID- 16679259 TI - Strain rate dobutamine echocardiography for prediction of recovery after revascularization in patients with ischemic left ventricular dysfunction. AB - BACKGROUND: The purpose of the present study was to assess the accuracy of quantitative segmental analysis by strain rate imaging (SRI) technique during dobutamine test for detecting myocardial recovery after revascularization in patients with chronic ischemic regional left ventricular (LV) dysfunction and compare results with those of 2-dimensional echocardiography (2D) and tissue Doppler imaging (TDI) as well as rest-4 hours-24 hours redistribution thallium SPECT (Tl SPECT). METHODS AND RESULTS: Forty-one patients with chronic ischemic regional LV dysfunction (EF 29 +/- 8%) underwent dobutamine 2D/TDI/SRI and Tl SPECT before and after myocardial revascularization. The sensitivity, specificity, and accuracy for the recovery of regional LV function were 73%, 81%, and 77% for dobutamine 2D; 77%, 82%, and 80% for dobutamine TDI; 86%, 88%, and 85% for dobutamine SRI; and 94%, 76%, and 84% for Tl tomography. The area under the ROC curve (AUC), which reflects the overall performance for the prediction of recovery, was 0.79 for systolic-SR, 0.81 for Tl SPECT, 0.83 for postsystolic strain, and 0.87 for isovolumic-SR. If both systolic and postsystolic SRI indexes were combined with Tl SPECT, the AUC was improved to 0.94. CONCLUSIONS: Dobutamine SRI is more accurate than TDI in identifying hibernating myocardium. Systo-diastolic values obtained using dobutamine SRI echocardiography and values derived from nuclear perfusion techniques may be complementary in assessing myocardial viability. PMID- 16679261 TI - B-type natriuretic peptide (BNP) levels and ethnic disparities in perceived severity of heart failure: results from the Rapid Emergency Department Heart Failure Outpatient Trial (REDHOT) multicenter study of BNP levels and emergency department decision making in patients presenting with shortness of breath. AB - BACKGROUND: Previous studies have shown that in patients presenting to the emergency department (ED) with heart failure, there is a disconnect between the perceived severity of congestive heart failure (CHF) by physicians and the severity as determined by B-type natriuretic peptide (BNP) levels. Whether ethnicity plays a role in this discrepancy is unknown. METHODS AND RESULTS: The Rapid Emergency Department Heart Failure Outpatient Trial (REDHOT) was a 10 center trial of 464 patients seen in the ED with acute dyspnea and BNP level higher than 100 pg/mL on arrival. Physicians were blinded to BNP levels. Patients were followed for 90 days after discharge. A total of 151 patients identified themselves as white (32.5%) and 294 as black (63.4%). Of these, 90% were hospitalized. African Americans were more likely to be perceived as New York Heart Association class I or II than whites (P = .01). Blacks who were discharged from the ED had higher median BNP levels than whites who were discharged (1293 vs. 533, P = .004). The median BNP of blacks who were discharged was actually higher than the median BNP of blacks who were admitted (1293 vs. 769, P = .04); the same did not hold true for whites. BNP was predictive of 90-day outcome in both blacks and whites; however, perceived severity of CHF, race, and ED disposition did not contribute to the prediction of events. CONCLUSION: In patients presenting to the ED with heart failure, the disconnect between perceived severity of CHF and severity as determined by BNP levels is most pronounced in African Americans. PMID- 16679262 TI - The combined utility of an S3 heart sound and B-type natriuretic peptide levels in emergency department patients with dyspnea. AB - BACKGROUND: Emergency department (ED) patients with undifferentiated dyspnea are a diagnostic dilemma. We hypothesized that electronic detection of an S3 would be more accurate in determining decompensated heart failure than physician auscultation, and that combining electronic heart sounds with B-type natriuretic peptide (BNP) would provide additional decision making information to the emergency physician, especially in the BNP indeterminate range (100-500 pg/mL). METHODS AND RESULTS: We collected demographic, clinical, and laboratory data in a convenience sample of ED patients presenting with signs or symptoms of acute decompensated heart failure between September 2003 and June 2004. The electronic presence of an S3 or S4 was determined using the Audicor system, a validated device that algorithmically detects S3 and S4 heart sounds. Two independent reviewers determined the presence or absence of acute decompensated heart failure (primary HF) based on chart review, while blinded to BNP and Audicor results. Test characteristics were determined with 95% confidence intervals. Of 422 enrolled patients, 343 had complete data and were included in the final analysis. Median age was 61 years, 54% were female, and 48% were white. The sensitivity, specificity, positive and negative predictive value, and diagnostic accuracy of an electronic S3 for primary HF were 34% (26% to 43%), 93% (89% to 96%), 66% (57% to 74%), 7% (4% to 11%), and 70% (65% to 75%) and for physician auscultation were 16% (11% to 24%), 97% (93% to 99%), 84% (76% to 89%), 3% (2% to 7%), and 66% (61% to 71%). The addition of an Audicor S3 to intermediate BNP levels improved the positive LR from 1.3 to 2.9; the positive predictive value from 53% to 80%. CONCLUSION: An S3 is highly specific for primary HF and it is ideally suited for use in combination with BNP to improve diagnostic accuracy in ED patients with dyspnea of unclear etiology. PMID- 16679263 TI - Myocarditis and heart failure associated with hepatitis C virus infection. AB - BACKGROUND: The aim of study is to determine the prevalence of hepatitis C virus (HCV) infection and myocardial injury among patients enrolled in the Myocarditis Treatment Trial. HCV infection has recently been noted in patients with cardiomyopathies and myocarditis. However, prevalence of HCV infection in myocarditis and heart failure remains to be clarified. METHODS AND RESULTS: Patients with heart failure up to 2 years in duration without a distinct cause were enrolled in the trial between 1986 and 1990. Frozen blood samples were available from 1355 among 2233 patients enrolled and examined for presence of anti-HCV antibodies, circulating cardiac troponins I and T, and N-terminal pro brain natriuretic peptide (NT-proBNP). Anti-HCV antibodies were identified in 59 of 1355 patients (4.4%). This higher prevalence of HCV infection than that observed in the general US population (1.8%), varied widely (0-15%) among the different medical centers and regions. The concentrations of circulating cardiac troponin (cTn) I were elevated in 17 of 56 patients (30%), and cTnT was detectable in 28 of 59 patients (48%) with HCV antibodies, suggesting the persistence of ongoing myocardial injury. The concentrations of NT-proBNP were elevated in 42 of 42 patients (100%) with HCV antibodies, (10,000 +/- 5860 pg/mL), a mean value significantly greater than in 1276 patients without HCV antibody (2508 +/- 160 pg/mL, P < .0001). CONCLUSION: Anti-HCV antibodies were identifiable in sera stored for 13 to 17 years and were more prevalent in patients with myocarditis and HF than in the general population. In regions where its prevalence is high, HCV infection may be an important cause of myocarditis and HF. NT-proBNP is a more sensitive marker of myocardial injury than cardiac troponins in patients with heart failure from HCV myocarditis. PMID- 16679265 TI - The symptom of pain with heart failure: a systematic review. AB - BACKGROUND: Pain is one of the most compelling reasons for seeking medical attention. Despite frequent hospitalizations and assessments, the symptom of pain is not often associated with heart failure (HF). The role of pain in exacerbations and hospitalization may be important. A systematic review to synthesize research related to reported pain in patients with HF was undertaken and factors considered to be related to the symptom of pain in this population were identified. METHODS AND RESULTS: Relevant articles were identified using MEDLINE, CINAHL, EMBASE, and the Cochrane Library. Included studies focused on patients with HF and reporting on pain. Nine descriptive studies were identified. Five studies focused specifically on patients with HF. The remaining studies examined a population of seriously ill patients including those with HF as an itemized subset. From 23% to 75% of patients with HF reported pain. Factors identified as related to pain include: anxiety, depression, quality of life rated as poor, dyspnea, and more dependencies in activities of daily living. CONCLUSION: People with HF report having pain but as a complex health group, the symptom of pain is not well understood. Pain could be a contributing factor in the breakdown of self-management and the cycle of exacerbations and hospitalization. PMID- 16679264 TI - Reduced rate of alveolar-capillary recruitment and fall of pulmonary diffusing capacity during exercise in patients with heart failure. AB - BACKGROUND: Patients with chronic heart failure (CHF) have reduced pulmonary diffusing capacity for carbon monoxide (DLCO). Acute pulmonary congestion also causes reduction of DLCO, which is reversible. We hypothesized for patients with CHF that the rate of rise of exercise DLCO is reduced compared to healthy controls and falls near end-exercise consistent with progressive interstitial edema. METHODS AND RESULTS: DLCO and pulmonary blood flow (QC)) were measured by a rebreathe technique in CHF subjects (n = 11) and controls (n = 8) at rest, during constant workload exercise, and after exercise. DLCO of CHF subjects was less than controls at rest (16.5 +/- 1 vs. 21.9 +/- 2 mL/min/mm Hg, P < .01). CHF subjects exercised 11 +/- 2 minutes to 90% peak VO2, whereas controls exercised 17 +/- 2 minutes, reaching 88% peak VO2. In CHF subjects, DLCO increased to 19 +/ 2 mL/min/mm Hg and for controls to 38 +/- 3 mL/min/mm Hg. During the final 3 minutes of exercise, DLCO increased 5% in controls while decreasing 5% in CHF subjects (DLCO/Q(C)) was lower in CHF subjects at rest and progressively lower throughout exercise (P < .01). CONCLUSION: In patients with CHF, DLCO has reduced rate of rise with exercise and falls near end-exercise consistent with limitation of alveolar-capillary recruitment and progressive interstitial edema. PMID- 16679266 TI - Differential expression of MMPs and TIMPs in moderate and severe heart failure in a transgenic model. AB - BACKGROUND: Altered expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) accompanies the development of heart failure (HF). However, changes in MMP and TIMP protein levels or activity during the progression from compensated to decompensated failure remains incompletely examined. METHODS AND RESULTS: Transgenic mice (Tg) with cardiac-specific overexpression of tumor necrosis factor-alpha (TNF1.6) develop a sex-related, progressive cardiac dilation and HF. Echocardiographic measures were used to categorize HF severity in male (M) and female (F) Tg and wild-type (WT) mice between 4 and 50 weeks of age. Cardiac TIMPs-1, TIMPs-2, and MMP-3 (enzyme-linked immunosorbent assay), and potential (APMA-activated) MMP-9 activity were measured at similar ages. In situ zymography assessed tissue gelatinase activity. Systolic function, ventricular dimensions, and presence of pleural effusions identified severe HF in younger M Tg mice (by 18 weeks) and older F Tg (>34 weeks). Regardless of age, sex, or HF severity, Tg mice expressed significantly more TIMP 1 (Tg 119-193 pg/mg vs. WT 13-24 pg/mg, P < .001) and potential MMP-9 activity (Tg 0.41-0.58 ng/mg vs. WT 0.015-0.028 ng/mg, P < .002). M Tg expressed elevated MMP-3 (4 weeks, 0.16 +/- 0.1 ng/mg protein vs. WT 0.04 +/- 0.01 ng/mg, P < .003), which increased with age and HF severity (18 weeks, 0.51 +/- 0.3 ng/mg P < .01). F Tg showed no increase in MMP-3 at 4 weeks but a progressive increase with age and HF severity (18 weeks 0.09 +/- 0.04 ng/mg, P < .02 vs. Tg M or WT; 34 weeks 0.13 +/- 0.02 ng/mg, P < .001 vs. WT). To test the hypothesis that increased MMP 3 may differentially activate MMP-9 in M Tg, in situ zymography was performed and revealed a significant increase in gelatinase activity in M Tg mice relative to both WT and F Tg. CONCLUSION: MMP-3 may regulate activation of MMP-9/gelatinase, the progression of cardiac remodeling, and development of decompensated heart failure. PMID- 16679267 TI - The era of computer-aided viewing of the lung volume. PMID- 16679268 TI - 3D computerized segmentation of lung volume with computed tomography. AB - Three-dimensional (3D)-based detection and diagnosis has an important role for significantly improving the detection and diagnosis of lung cancer upon computed tomography (CT). This report presents a 3D-based method for segmenting and visualizing lung volume by using CT images. An anisotropic filtering method was developed on CT slices to enhance the signal-to-noise ratio, and a wavelet transform-based interpolation method was used combined with volume rendering to construct the 3D volumetric data based on entire CT slices. Then an adaptive 3D region-growing algorithm was designed to segment lung volume, incorporated by automatic seed-locating methods through fuzzy logic algorithms and 3D morphological closing approaches. In addition, a 3D visualization tool was designed to view volumetric data, projections, or intersections of the lung volume at any view angle. This segmentation method was tested on single-detector CT images by percentage of volume overlap and percentage of volume difference. The experiment results show that the developed 3D-based segmentation method is effective and robust. This study lays the groundwork for 3D-based computerized detection and diagnosis of lung cancer with CT imaging. In addition, this approach can be integrated into a picture archiving and communication system serving as a visualization tool for radiologists' reading and interpretation. PMID- 16679269 TI - Scanning systems and protocols used during imaging for acute pulmonary embolism: how much do our clinical colleagues know? AB - RATIONALE AND OBJECTIVES: The imaging systems and protocols used during ventilation-perfusion lung (V-P) scintigraphy and computed tomographic (CT) pulmonary angiography (CTPA) can affect diagnostic performance. We investigated the level of awareness of these factors among US clinicians who refer patients for imaging for suspected acute pulmonary embolism. MATERIALS AND METHODS: Between September 2004 and February 2005, we conducted a mail survey of 855 physicians selected at random from three professional organizations. We asked participants how important the availability of state-of-the-art equipment was in their imaging decisions, whether V-P scintigraphy was performed with planar or single-photon emission CT (SPECT) equipment in their communities, to identify the most advanced type of CT scanner used for CTPA, and whether CT venography (CTV) was performed routinely after CTPA. RESULTS: We received completed surveys from 240 (29.8%) physicians practicing in 44 states. One hundred sixty-six respondents (70.9%) indicated that state-of-the-art equipment was an extremely or very important factor when they made imaging decisions. However, 191 clinicians (80.3%) did not know whether SPECT equipment was used for V-P scintigraphy, and 119 (50.6%) did not know the type of CT scanner used for CTPA in their communities. Of respondents, 39.2% reported access to multidetector row CT technology for CTPA, whereas 10.2% referred patients to facilities using single detector CT. Only 9.3% of respondents indicated that CTV was performed routinely after CTPA. CONCLUSION: Although state-of-the-art equipment is important to them, clinicians practicing in the United States have limited knowledge of the equipment being used during CTPA and V-P scintigraphy scanning in their communities. Radiologists should intensify efforts to familiarize their clinical colleagues with the equipment they use. PMID- 16679270 TI - Theory-based signal calibration with single-point T1 measurements for first-pass quantitative perfusion MRI studies. AB - RATIONALE AND OBJECTIVES: The aim of the study is to develop a theory-based signal calibration approach to be used for the conversion of signal-time curves to absolute contrast concentration-time curves for first-pass contrast-enhanced quantitative myocardial perfusion studies. MATERIALS AND METHODS: A normalization procedure was used to obtain a theoretical relationship between image signal and T1 and perform rapid single-point T1 measurements. T1 measurements were compared with reference T1 measurements. The method also was used in preliminary in vivo contrast-enhanced first-pass perfusion studies, and its applicability for dual delay-time acquisitions was shown. A theory-based error sensitivity analysis was used to characterize the robustness of the method. RESULTS: The normalization procedure was implemented with minimal noise enhancement and insensitivity to small misregistrations through postprocessing techniques. The rapid T1 measurements are in excellent agreement with the reference measurements (R = 0.99, slope = 1.05, bias = -5.96 milliseconds). For in vivo studies, it is possible to simultaneously calibrate the arterial input function and myocardial enhancement curves acquired with different effective trigger delays through appropriate use of the theory-based signal calibration model. With this method, errors of in vivo baseline T1 estimates are large, but the effect of these large errors on the accuracy of contrast agent concentration estimates is limited. CONCLUSION: This theory-based signal calibration approach can be used to perform rapid T1 mapping and provides flexibility for in vivo calibration of signal-time curves resulting from dual-delay-time first-pass contrast-enhanced acquisitions. PMID- 16679271 TI - Multidetector CT angiography using a dual-head power injector and bolus tracking for the diagnosis of aortic aneurysms at reduced contrast material dosage. AB - RATIONALE AND OBJECTIVES: To establish the utility of multidetector computed tomography (CT) angiography using dual-head power injector in the diagnosis of aortic diseases. METHODS: In a prospective study, 151 patients with aortic diseases were examined by four-detector CT. Scanning was performed using bolus tracking technique. In all patients nonionic contrast was injected at the rate of 1.5 mL/sec. One hundred one patients were examined with dual-head power injector using 0.6 mL/kg contrast flushed by 30 mL of saline solution (group D). Fifty patients were examined with single-head power injector using 1.0 mL/kg contrast only (group S). We evaluated CT values at descending aorta, upper abdominal aorta, abdominal aortic bifurcation, and bilateral common femoral arteries. RESULTS: There were no statistically significant difference of CT values at descending aorta, upper abdominal aorta, abdominal aortic bifurcation, and right common femoral artery. At left common femoral artery, CT values in group D were higher than those in group S with statistically difference (P < .05). In group D, about 40% dose reduction was achieved without reducing image qualities. CONCLUSION: Multidetector CT angiography using dual-head power injector was valuable for the contrast dose reduction of aortic diseases. PMID- 16679272 TI - Physical evaluation of the weighted Feldkamp algorithms applied to the 256 detector row CT scanner for volumetric cine imaging. AB - RATIONALE AND OBJECTIVES: To improve effective scan time and image quality in cone-beam computed tomography (CT), Parker's weighting function (half scan [HS] Feldkamp-Davis-Kress [FDK]) extended to a larger range up to 2pi was proposed as new half-scan algorithm (NHS-FDK). We conducted a practical physical evaluation of NHS-FDK and HS-FDK using 256-detector row CT. MATERIALS AND METHODS: Three types of weighting function (full-scan [FS-FDK], HS-FDK, and NHS-FDK) were evaluated by using 256-detector row CT for five variables, ie, point spread function, image noise, CT number uniformity, Feldkamp artifact, temporal resolution, and clinical evaluation. RESULTS: Image noise, Feldkamp artifact, and temporal resolution were dependent on weighting function. Image noise magnitude was independent of projection angle for all regions of interest with FS-FDK, but showed a symmetric pattern with projection angle with HS-FDK and NHS-FDK. With regard to temporal resolution, NHS-FDK did not remove the motion artifact in the heart except in such slower motion organs as the pulmonary vessels, whereas HS FDK reduced the motion artifact in the heart. HS-FDK had an even more incomplete data region in the Radon space than FS-FDK, suggesting that it would provide poor image quality distant to the midplane in the longitudinal direction. In practical testing in human subjects, HS-FDK showed inferior performance in all variables except temporal resolution. CONCLUSION: Despite its inferiority to FS-FDK for static objects, HS-FDK may be useful in chest imaging. Contrary to previous findings using static images, NHS-FDK failed to show advantages over HS-FDK or FS FDK in a moving phantom and human subjects. PMID- 16679274 TI - Inversion recovery MRI in idiopathic Parkinson disease is a very sensitive tool to assess neurodegeneration in the substantia nigra: preliminary investigation. AB - RATIONALE AND OBJECTIVES: Segmented inversion recovery (IR) ratio imaging (SIRRIM) has been established as a sensitive tool to assess neurodegeneration of the substantia nigra pars compacta (SN(C)) in patients with idiopathic Parkinson disease (IPD). The obtained results suggest the possibility of magnetic resonance imaging (MRI) as a biological marker for IPD. The strength and a parsimonious analysis of the technique are discussed to assess the potential of using MRI as a biological marker for IPD and improve the differential diagnosis of sporadic Parkinson disease. Our hypothesis states that the magnetic resonance SIRRIM technique allows direct visualization and quantitation of neural cell loss in the SN(C) and therefore could become a reliable biological marker for Parkinson disease. To achieve this goal, some key aspects of data acquisition and data analysis need to be addressed. The clinical impact of the SIRRIM technique could be considerable, considering that it might become a viable surrogate to other techniques. PATIENTS AND METHODS: Twelve patients with IPD and 12 age-matched control subjects were imaged by using the SIRRIM technique based on two IR imaging sequences that were designed to suppress white and gray matter to assess loss of neural cells in situ by means of a ratio image (white matter suppressed image to gray matter suppressed image). The radiological index was correlated with the Unified Parkinson Disease Rating Scale (UPDRS) for patients with IPD. RESULTS: All patients with IPD were identified correctly, and full dichotomization between healthy volunteers and patients was obtained with our database. Our SIRRIM technique shows that it can be used to rule out Parkinson disease from essential tremor and other forms of Parkinsonism, such as progressive supranuclear palsy and multisystem atrophy. In addition, it is sensitive enough to identify patients with early-stage IPD. CONCLUSION: The hypothesis of using SIRRIM as a biological marker to assess IPD is supported by excellent correlation with clinical UPDRS scoring and has proved useful for the evaluation and quantitation of neurodegeneration with our SIRRIM technique, showing, in addition, that the differential diagnosis of IPD can be improved. Technical aspects of acquisition and data processing that need to be addressed can be overcome. It ultimately confirms that our objectives can be achieved and allows us to expect assessment of the progressive development of neurodegeneration in longitudinal studies and the putative neuroprotective approaches taken during the evolution of the disease. PMID- 16679273 TI - Diagnosis of hepatic tumors with texture analysis in nonenhanced computed tomography images. AB - RATIONALE AND OBJECTIVES: Computed tomography (CT) after iodinated contrast agent injection is highly accurate for diagnosis of hepatic tumors. However, iodinating may have problems of renal toxicity and allergic reaction. We aimed to evaluate the potential role of the computer-aided diagnosis (CAD) with texture analysis in the differential of hepatic tumors on nonenhanced CT. MATERIALS AND METHODS: This study evaluated 164 liver lesions (80 malignant tumors and 84 hemangiomas). The suspicious tumor region in the digitized CT image was manually selected and extracted as a circular subimage. Proposed preprocessing adjustments for subimages were used to equalize the information needed for a differential diagnosis. The autocovariance texture features of subimage were extracted and a support vector machine classifier identified the tumor as benign or malignant. RESULTS: The accuracy of the proposed diagnosis system for classifying malignancies is 81.7%, the sensitivity is 75.0%, the specificity is 88.1%, the positive predictive value is 85.7%, and the negative predictive value is 78.7%. CONCLUSIONS: This system differentiates benign from malignant hepatic tumors with relative high accuracy and is therefore clinically useful to reduce patients needed for iodinated contrast agent injection in CT examination. Because the support vector machine is trainable, it could be further optimized if a larger set of tumor images is to be supplied. PMID- 16679275 TI - Contrast/Noise ratio on conventional MRI and choline/creatine ratio on proton MRI spectroscopy accurately discriminate low-grade from high-grade cerebral gliomas. AB - RATIONALE AND OBJECTIVES: Histopathology is the gold standard to establish the grade of brain tumors but biopsy and/or surgery are not always possible. The aim of this study is to determine whether histological grade of tumors may be predicted by means of conventional gadolinium-enhanced MRI and proton magnetic resonance spectroscopy (MRS). MATERIALS AND METHODS: In this study, we included 35 consecutive patients with single brain tumors and final histopathological verification: 12 had low-grade glioma, 16 had high-grade glioma, and 7 had single metastasis. Initially, we carried out T1 and T2 MRI paying attention to the following features: border definition, mass effect, heterogeneity of signal, perilesional edeme, hemorrhage, necrosis, and corpus callosum invasion. Gadolinium-enhancement was evaluated with the contrast-to-noise ratio (CNR). Next, single-voxel proton MRS was carried out to measure the absolute values of metabolites (N-acetyl-aspartate, creatine, choline, and myo-inositol) and their ratios in the area of maximum contrast enhancement. RESULTS: We found that gadolinium-enhancement measured with the CNR (CNR > 35.86) predicted malignancy at 82.6% sensitivity and 91.7% specificity (area under the curve, 0.88; 95% confidence interval [CI], 0.73-0.97). With regard to MRS a choline/creatine ratio higher than 1.56 predicted malignancy at 88.9% sensitivity and 91.7% specificity (area under the curve, 0.94; 95% CI, 0.78-0.99). When we combined the CNR value, the choline/creatine ratio, and the presence of lactates in a model of discriminant analysis the predictive power improved significantly with an area under the curve of 0.99% (95% CI, 0.87-1). However, the used techniques were unable to distinguish metastases from high-grade gliomas accurately. CONCLUSIONS: The intensity of contrast enhancement measured with the CNR, the choline/creatine ratio, and the presence of lactate were the most powerful variables to predict malignancy in brain tumors. The CNR is a simple, objective, and useful tool in the initial assessment of gliomas and metastases. PMID- 16679276 TI - Temporal lobe perfusion in the deaf: MR measurement with pulsed arterial spin labeling (FAIR). AB - RATIONALE AND OBJECTIVES: Experimental studies in animals have shown that loss of a primary sensory modality early in life may result in substantial alterations in cortical organization. This study was performed to measure cerebral perfusion in auditory cortex in congenitally deaf adults using the FAIR (Flow-sensitive Alternating Inversion Recovery) magnetic resonance imaging technique. Our hypothesis was that there would be relatively intact perfusion in auditory cortex. MATERIALS AND METHODS: Twenty-six profoundly congenitally deaf subjects were compared with 15 control subjects. A FAIR perfusion slice was scanned through the superior temporal gyrus parallel to the Sylvian fissure while subjects were at rest. Perfusion maps were calculated and regions of interest were drawn over the superior temporal gyrus including auditory cortex and the medial occipital lobe. RESULTS: The relative perfusion of the superior temporal gyrus (STG) was slightly less in the deaf (right STG = 0 .79 +/- 0.16, left = 0.93 +/- 0.29) compared with the hearing (right STG = 0.90 +/- 0.14, left = 0.98 +/- 0.31) when normalized to the occipital cortex, but the differences were not statistically significant. Both showed moderate left lateralization; however, only in the deaf did this reach statistical significance (P < .01). CONCLUSIONS: In the resting state, the deaf demonstrate a relatively normal perfusion in the region of cortex usually associated with auditory function. Although the presumed underlying electrical activity may represent some degree of residual auditory function, it is likely that the normal level of perfusion reflects cortical reorganization and the early migration of nonauditory processing into this area. PMID- 16679278 TI - Quantitative analysis of brain asymmetry by using the divergence measure: normal pathological brain discrimination. AB - RATIONALE AND OBJECTIVES: The human brain demonstrates approximate bilateral symmetry of anatomy, function, neurochemical activity, and electrophysiology. This symmetry reflected in radiological images may be affected by pathology. Hence quantitative analysis of brain symmetry may enable the normal and pathological brain discrimination. We propose a method based on the Jeffreys divergence measure (J-divergence), which attempts to quantify "approximate symmetry" and also aids to classify the brain as bilaterally symmetrical/asymmetrical (normal/abnormal). MATERIALS AND METHODS: The dataset included studies of 101 patients (59 without detectable pathologies and 42 with different abnormalities). First, the midsagittal plane is computed for the volume data that divides the head into two hemispheres. The J-divergence is calculated from the density functions of intensities of both the hemispheres. Statistical analysis was conducted to find the best distribution for normal/abnormal datasets. RESULTS: Statistical tests showed that the lognormal distribution best characterizes the values of the J-divergence for both normal and abnormal cases, and the threshold value for the Jeffreys divergence measure to classify the brains with and without detectable pathologies is T = 0.007. The threshold value had a sensitivity of 88.1% and specificity of 90.9%. CONCLUSION: The proposed method is fast and simple to compute. The high sensitivity and specificity indicate the results are encouraging. This method can be used for the initial analysis of data, detection of pathology, classification of dataset as presumably normal/abnormal, and localization of abnormality. PMID- 16679277 TI - Enhancing lesions of the brain: intraindividual crossover comparison of contrast enhancement after gadobenate dimeglumine versus established gadolinium comparators. AB - RATIONALE AND OBJECTIVES: Gadobenate dimeglumine (Gd-BOPTA) possesses a two-fold higher T1 relaxivity compared to other available gadolinium contrast agents. The study was conducted to evaluate the benefits of this increased relaxivity for MR imaging of intracranial enhancing brain lesions. MATERIALS AND METHODS: Forty five patients (31 males, 14 females) with suspected glioma or cerebral metastases were evaluated. Patients received Gd-BOPTA and either Gd-DTPA (n = 23) or Gd-DOTA (n = 22) in fully randomized order at 0.1 mmol/kg body weight and at a flow rate of 2 ml/s. The second agent was administered 1-14 days after the first agent. Images were acquired precontrast (T1wSE, T2wFSE sequences) and at sequential postcontrast time-points (T1wSE sequences at 0, 2, 4, 6, and 8 and 15 min and a T1wSE-MT sequence at 12 min) at 1.0 or 1.5 T using a head coil. Determination of contrast enhancement was performed quantitatively (lesion-to-brain ratio, contrast-to-noise ratio, and percent enhancement) and qualitatively (border delineation, internal morphology, contrast enhancement, and diagnostic preference) by two independent, fully blinded readers. RESULTS: Images from 43/45 patients were available for quantitative assessment. After correction for precontrast values, significantly greater lesion-to-brain ratio (P < .003), contrast-to-noise ratio (P < .03), and percent enhancement (P < .0001) was noted by both readers for Gd-BOPTA-enhanced images at all time-points from 2 min postcontrast. Qualitative assessment of all patients similarly revealed significant preference for Gd-BOPTA for lesion border delineation (P < .004), lesion internal morphology (P < .008), contrast enhancement (P < .0001), and diagnostic preference (P < .0005). CONCLUSIONS: The greater T1 relaxivity of Gd BOPTA permits improved visualization of intracranial enhancing lesions compared to conventional gadolinium agents. PMID- 16679279 TI - Virtual pyeloscopy using volumetric depth peeling. AB - RATIONALE AND OBJECTIVES: The aim of the study is to test a new volume-rendering method, volumetric depth peeling (VDP), for use in virtual pyeloscopy. MATERIALS AND METHODS: VDP was applied to axial contrast-enhanced source computed tomographic (CT) images and coronal reformatted maximum intensity projections of three contrast-filled gloves containing objects of varying density. Similar renderings were performed on CT urograms performed to evaluate hematuria (n = 20). Renderings were assessed for anatomic appearance of ureters and specific calyces in comparison with source images. RESULTS: Objects of soft-tissue and calcific density ranging in size from 4 to 20 mm were identified by using VDP within the glove phantoms. Normal and deformed renal calyces were well visualized by using VDP; however, two stones were not identified. The minimal ureteral width that could be visualized was 3 mm. CONCLUSION: VDP may be a useful technique for virtual pyeloscopy providing that a robust and user-friendly computer interface can be developed. PMID- 16679280 TI - Part I: preparing first-year radiology residents and assessing their readiness for on-call responsibilities. AB - RATIONALE AND OBJECTIVES: The aim of the study is to evaluate the effectiveness of an Emergency Radiology (ER) Core Curriculum training module and a Digital Imaging and Communications in Medicine (DICOM)-based interactive examination system to prepare first-year (postgraduate year 2 [PGY-2]) radiology residents and assess their readiness for taking overnight radiology call. MATERIALS AND METHODS: Institutional review board approval was obtained, and the study was compliant with Health Insurance Portability and Accountability Act (HIPAA) regulations. A dedicated month-long ER curriculum was designed to prepare new radiology residents for overnight radiology call that includes interpretation of off-hour urgent and emergent studies without immediate direct attending supervision. Lectures of the curriculum, provided by department staff, were based on the American Society of Emergency Radiology core curriculum. The lecture series was implemented after PGY-2 residents had completed formal introductory resident rotations during their first 6 months of training. A DICOM-based interactive computer-based testing module was developed and administered at the end of the lecture series. The module consisted of 19 actual emergency department cases with entire series of images, simulating an on-call setting. Tests were scored by two staff members blinded to resident identifying information. Upper level residents also were tested, and comparison was made between first-year and upper-level resident test scores to determine the effectiveness of the test in determining first-year resident preparedness for call. Statistical analysis of results was performed by using t-test (P < .05). RESULTS: All residents in the residency program present during the month (nine PGY-2, six PGY-3, seven PGY-4, seven PGY-5 residents) attended the lecture series and finished the testing module at the end of the lecture series. Of 19 actual emergency cases on the testing module, five cases were neuroradiology, three cases were thoracic imaging, eight cases were body imaging, and three cases were musculoskeletal. PGY 2 residents scored an average of 73.0% (range, 63.2%-81.6%) of total points possible. PGY-3 residents scored an average of 76.8% (range, 68.4%-86.8%); PGY-4 residents scored an average of 77.4% (range, 65.8%-100%), and PGY-5 residents scored an average of 81.2% (range, 68.4%-94.7%). There was no statistically significant difference in scores according to level of training. CONCLUSION: First-year radiology residents who underwent 6 months of formal radiology training followed by an intensive ER lecture series before taking overnight call had scores similar to upper-level colleagues on an interactive computer-based ER simulation module. PMID- 16679281 TI - Part II: preparing and assessing first-year radiology resident on-call readiness technical implementation. AB - RATIONALE AND OBJECTIVES: The effectiveness of using a Digital Imaging and Communications in Medicine (DICOM)-based interactive examination system in evaluating the readiness of first year radiology residents before taking overnight call in the emergency department (ED) was reported in part I of this article. This report describes technical aspects for the design and implementation of this system. MATERIALS AND METHODS: The examination system consists of two modules: Data Collection and Image Viewing. The Data Collection module was a personal computer (PC)-based DICOM storage server based on a free public domain software package, the Mallinckrodt Central Test Node. The Image Viewing module was a Java-based DICOM viewer created using another freeware package: zDicom ActiveX component. RESULTS: The examination takes place once a year at the end of the first 6-month rotation. Cases selected for the examination were actual clinical cases according to the American Society of Emergency Radiology core curriculum. In the 3-hour timed examination, each resident was required to read the cases and provide clinical findings and recommendations. Upper-level residents also participated in the examination to serve as a control. Answers were scored by two staff radiologists. CONCLUSION: We have been using this examination system successfully in our institution since 2003 to evaluate the readiness of the first-year residents before they take overnight call in the ED. This report describes a step-by-step procedure for implementing this system into a PC-based platform. This DICOM viewing software is available as freeware to other academic radiology institutions. The total cost for implementing this system is approximately 2000 US dollars. PMID- 16679282 TI - Digitizing and consolidating mammograms and other images for teaching applications. AB - RATIONALE AND OBJECTIVES: Most mammograms are obtained using screen-film technique and must be digitized for teaching purposes. Digitizing mammograms poses special problems because of high contrast and multiple views. We describe the equipment and process for digitizing and consolidating mammograms for teaching purposes. These techniques can be applied to any type of images where consolidation may be helpful. MATERIALS AND METHODS: Mammograms are digitized using a high optical density scanner. After the window and levels are adjusted, a four-view mammogram may be consolidated into a single image if desired. RESULTS: The high contrast of film screen mammograms is managed by using a high optical density scanner. Consolidation of image sets, such as a four-view mammogram, allows images to be easily inserted into text, slide, or poster documents. CONCLUSIONS: Digitizing mammograms for teaching purposes is facilitated by use of a high optical density scanner and consolidation of image sets into single images. The techniques described are also useful for other areas of radiology in which display of multiple images or modalities is desirable. PMID- 16679284 TI - Deja vu, all over again. PMID- 16679283 TI - Enhanced teaching of screening mammography using an electronic format. AB - RATIONALE AND OBJECTIVES: Most mammography teaching materials provide only diagnostic images, often with obvious findings due to space constraints. In this work, we describe a method of incorporating full resolution digitized screening mammograms in text teaching file documents to enhance teaching of screening mammography. METHODS: Teaching cases are constructed in a word processing document with images inserted. The four view screening mammogram is presented first and may be opened in an image viewing program using an embedded hyperlink to view at high resolution, similar to using a magnifying glass. The screening study is followed by feedback and presentation of diagnostic images. Feedback is again provided, followed by diagnosis, differential diagnosis, discussion, and references. A six-question survey of residents and fellows using 5-point Likert scales was used to assess performance. RESULTS: Teaching cases in this format mimic clinical practice by separately presenting screening and diagnostic views. Mammograms may be viewed at high resolution, allowing incorporation of subtle findings to build detection skills. Inclusion of diagnostic views builds judgment and management skills. Survey results from 15 residents and 2 fellows found a mean overall rating of the teaching file of 1.6 (1 = excellent, 5 = poor). CONCLUSIONS: Screening mammograms with subtle findings can be incorporated into breast teaching cases by embedding hyperlinks to open images at high resolution in an image-processing program. PMID- 16679285 TI - Marvin's boy. PMID- 16679286 TI - Ghrelin--a new player in glucose homeostasis? AB - The hormone ghrelin regulates secretion of growth hormone and energy homeostasis. Sun et al (2006), in this issue of Cell Metabolism, demonstrate that ghrelin inhibits insulin secretion. Deletion of ghrelin increased basal insulin level, enhanced glucose-stimulated insulin secretion, and improved peripheral insulin sensitivity. These effects were not related to changes in food intake or weight, suggesting ghrelin has unique actions on key components of glucose homeostasis. PMID- 16679287 TI - FSH versus estrogen: who's guilty of breaking bones? AB - Bone loss after menopause or gonadectomy has been attributed to the drop in estrogen levels. A recent paper (Sun et al, 2006) challenges this view by showing that the pituitary hormone FSH, previously thought to target only the gonads, also acts on osteoclasts to activate bone resorption. In conjunction with genetic studies, these data raise the possibility that FSH, independent of estrogen, causes hypogonadal bone loss. PMID- 16679288 TI - Fat breakdown: a function for CGI-58 (ABHD5) provides a new piece of the puzzle. AB - The hydrolysis of fat stored in adipose tissues is crucial for providing energy during fasting and exercise, and dysregulation of fat breakdown may contribute to metabolic disease. In this issue of Cell Metabolism, report that CGI-58/ABHD5, a lipid-droplet-associated protein that is mutated in a rare disease characterized by excess lipid storage, activates adipose triglyceride lipase and thus may regulate fat mobilization. PMID- 16679289 TI - Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome. AB - Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS. PMID- 16679290 TI - The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis. AB - Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major phospholipids in mammalian membranes. In liver, PC is synthesized via the choline pathway or by methylation of PE via phosphatidylethanolamine N-methyltransferase (PEMT). Pemt(-/-) mice fed a choline-deficient (CD) diet develop rapid steatohepatitis leading to liver failure. Steatosis is observed in CD mice that lack both PEMT and multiple drug-resistant protein 2 (MDR2), required for PC secretion into bile. We demonstrate that liver failure in CD-Pemt(-/-) mice is due to loss of membrane integrity caused by a decreased PC/PE ratio. The CD-Mdr2( /-)/Pemt(-/-) mice escape liver failure by maintaining a normal PC/PE ratio. Manipulation of PC/PE levels suggests that this ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis. The results have clinical implications as patients with nonalcoholic steatohepatitis have a decreased ratio of PC to PE compared to control livers. PMID- 16679291 TI - Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation. AB - Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1alpha. We could then efficiently knockdown PGC-1beta expression by shRNA expression. Loss of PGC-1alpha did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1alpha or PGC-1beta coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1alpha and PGC-1beta were deficient. These data show that PGC-1alpha is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis. PMID- 16679292 TI - Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKClambda/zeta. AB - Although the class I(A) phosphoinositide 3-kinase (PI3K) pathway is central to the metabolic actions of insulin, its mechanism of action is not well understood. To identify the role of the PI3K pathway in insulin regulation of hepatic function, we ablated the expression of both major regulatory subunits of PI3K by crossing mice lacking Pik3r1 in liver with Pik3r2 null mice, creating liver specific double knockout mice (L-p85DKO). L-p85DKO mice failed to activate PI3K or generate PIP(3) upon insulin stimulation or activate its two major effectors, Akt and PKClambda/xi. Decreased Akt activation resulted in increased gluconeogenic gene expression, impaired glucose tolerance, and hyperinsulinemia, while the defective activation of PKClambda/xi by insulin was associated with hypolipidemia and decreased transcription of SREBP-1c. These data indicate that the PI3K pathway is critical for insulin's actions in the liver in vivo, and that differential regulation by Akt and PKClambda/xi differentially defines specific actions of insulin and PI3K on hepatic glucose and lipid metabolism. PMID- 16679293 TI - Loss of class IA PI3K signaling in muscle leads to impaired muscle growth, insulin response, and hyperlipidemia. AB - The evolutionarily conserved phosphoinositide 3-kinase (PI3K) signaling pathway mediates both the metabolic effects of insulin and the growth-promoting effects of insulin-like growth factor-1 (IGF-1). We have generated mice deficient in both the p85alpha/p55alpha/p50alpha and the p85beta regulatory subunits of class I(A) PI3K in skeletal muscles. PI3K signaling in the muscle of these animals is severely impaired, leading to a significant reduction in muscle weight and fiber size. These mice also exhibit muscle insulin resistance and whole-body glucose intolerance. Despite their ability to maintain normal fasting and fed blood glucose levels, these mice show increased body fat content and elevated serum free fatty acid and triglyceride levels. These results demonstrate that in vivo p85 is a critical mediator of class I(A) PI3K signaling in the regulation of muscle growth and metabolism. Our finding also indicates that compromised muscle PI3K signaling could contribute to symptoms of hyperlipidemia associated with human type 2 diabetes. PMID- 16679294 TI - Identification of the tyrosine phosphatase PTP-MEG2 as an antagonist of hepatic insulin signaling. AB - Insulin resistance is a primary defect in type 2 diabetes characterized by impaired peripheral glucose uptake and insufficient suppression of hepatic glucose output. Insulin signaling inhibits liver glucose production by inducing nuclear exclusion of the gluconeogenic transcription factor FOXO1 in an Akt dependent manner. Through the concomitant application of genome-scale functional screening and quantitative image analysis, we have identified PTP-MEG2 as a modulator of insulin-dependent FOXO1 subcellular localization. Ectopic expression of PTP-MEG2 in cells inhibited insulin-induced phosphorylation of the insulin receptor, while RNAi-mediated reduction of PTP-MEG2 transcript levels enhanced insulin action. Additionally, adenoviral-mediated depletion of PTP-MEG2 in livers of diabetic (db/db) mice resulted in insulin sensitization and normalization of hyperglycemia. These data implicate PTP-MEG2 as a mediator of blood glucose homeostasis through antagonism of insulin signaling, and suggest that modulation of PTP-MEG2 activity may be an effective strategy in the treatment of type 2 diabetes. PMID- 16679295 TI - Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice. AB - Ghrelin and leptin are suggested to regulate energy homeostasis as mutual antagonists on hypothalamic neurons that regulate feeding behavior. We employed reverse genetics to investigate the interplay between ghrelin and leptin. Leptin deficient mice (ob/ob) are hyperphagic, obese, and hyperglycemic. Unexpectedly, ablation of ghrelin in ob/ob mice fails to rescue the obese hyperphagic phenotype, indicating that the ob/ob phenotype is not a consequence of ghrelin unopposed by leptin. Remarkably, deletion of ghrelin augments insulin secretion in response to glucose challenge and increases peripheral insulin sensitivity; indeed, the hyperglycemia exhibited by ob/ob mice is markedly reduced when ob/ob mice are bred onto the ghrelin(-/-) background. We further demonstrate that ablation of ghrelin reduces expression of Ucp2 mRNA in the pancreas, which contributes toward enhanced glucose-induced insulin secretion. Hence, chronically, ghrelin controls glucose homeostasis by regulating pancreatic Ucp2 expression and insulin sensitivity. PMID- 16679296 TI - Combined use of growth hormone and gonadotropin-releasing hormone analogue in short normal adolescent girls: a survey from Iran. AB - Combined therapy with gonadotropin-releasing hormone (GnRH) analogue and growth hormone (GH) has been used to increase the height of adolescents who are not GH deficient and who have normally timed puberty. Its use, however, is still controversial. For 2 years simultaneously, we treated eight healthy girls with very low predicted adult height (PAH < 145 cm) who were entering into normally timed puberty. The GnRH analogue triptorelin pamoate (Decapeptyl, 100 microg/kg intramuscularly every 4 weeks) and GH (0.1 IU/kg/day subcutaneously, 6 days/week) were administered. The mean chronologic age (CA) of our patients was 11.01 +/- 0.95 years, and mean bone age (BA) was 12.25 +/- 1.13 years. With a height of 131.50 +/- 5.83 cm (-2.37 +/- 0.35 SD below the mean) and PAH of 140.87 +/- 3.53 cm, they were all in Tanner stage 2-3 of puberty (except one patient in stage 4). In all cases, GH and thyroid hormone deficiency were ruled out before the study began. Height and BA were measured immediately after discontinuation of therapy. PAH was determined before and at the end of therapy. Combined treatment resulted in a 4.13 +/- 1.19 cm increase in PAH (p < 0.01). Height increased significantly to 142.66 +/- 3.54 cm at the end of treatment (p < 0.01). Height standard deviation score for CA increased from -2.37 +/- 0.35 to -2.32 +/- 0.67, showing no significant improvement (p = 0.5). Height age (HA)/BA ratio and BA/CA ratio both demonstrated significant growth during the treatment (p < 0.03 and p < 0.01, respectively), whereas HA/CA ratio did not improve significantly (p < 0.32). During treatment, puberty was completely suppressed in all cases. Combination therapy with GnRH analogue and GH resulted in significant improvement in height and PAH during therapy. Additional, and perhaps more long-term, studies are required to show whether this kind of treatment is effective in improving final adult height. The cost-benefit of such therapies should also be taken into account. PMID- 16679297 TI - Epidemiology of Blastocystis hominis and other intestinal parasites in a Vietnamese female immigrant population in southern Taiwan. AB - There has been a rapid increase in cross-border female marriage immigrants in Taiwan. In this study, 1,434 Vietnamese female marriage immigrants arriving between July 1998 and June 2001 were examined for intestinal parasites. Most (77.9%) of these women were 20-29 years of age. The prevalence of intestinal parasite infection was 37.7%, and the trend increased from 1999 to 2001 (statistically significant, p < 0.0001), but decreased among age subgroups (p < 0.0001). Among the 20 species of intestinal parasites found in this study, 10 species (27.8%) were transmitted via the fecal-oral route, 5 (14.6%) via the soil mediated route, and 5 (0.7%) by food-borne infection. The prevalence of blastocystosis (20.4%) and hookworm (9.7%) remained high among this population. The results provide unprecedented information on intestinal parasitic infection among these immigrants in southern Taiwan and recommend that appropriate health care be given after parasite infection is confirmed in these migrant communities. PMID- 16679298 TI - Evaluation of ectodermal dysplasia. AB - This case series report outlines possible cranio-maxillofacial deformation consequences associated with ectodermal dysplasia (ED) and embryonic malformations, including dental agenesis. Also described are the oral aspects and rehabilitation. A total of 14 ED patients (7 males and 7 females, aged 5-45 years) underwent clinical examination before assessment and treatment. Lateral cephalometric radiography, Steiner's analysis, and respiratory capacity tests were performed. Most of the patients had sparse or absent hair, a short face with an unusual facial concavity, a maxillary retrusion, and a relative mandible protrusion. Depending on age and orthopedic abnormalities, patients were treated with prosthodontic and orthodontic approaches or implant treatment. Therapists should take a comprehensive and multidisciplinary approach with these patients to improve their dental, masticatory, growth, and orthognathic conditions, as well as esthetic appearance. PMID- 16679299 TI - Lercanidipine and losartan effects on blood pressure and fibrinolytic parameters. AB - Antihypertensive agents may modulate fibrinolysis in addition to reducing blood pressure. We conducted a randomized trial to assess the effects of lercanidipine and losartan on blood pressure (BP) lowering and three fibrinolytic parameters: plasminogen activator inhibitor-1 (PAI-1), D-dimer, and fibrinogen. All patients enrolled had essential hypertension and underwent a placebo run-in period of 2 weeks before randomization to either lercanidipine tablets 10-20 mg once daily or losartan tablets 50-100 mg once daily. Twenty-six patients completed this study. After 8 weeks of treatment, both groups of patients had significantly reduced systolic (SBP) and diastolic BP (DBP) (SBP, p = 0.034 and 0.050, respectively; DBP, p = 0.018 and 0.034 for lercanidipine and losartan, respectively). Both drugs were well tolerated. Only in the group treated with lercanidipine was PAI-1 concentration significantly reduced (57.1 +/- 4.7 to 43.1 +/- 4.8 ng/mL, p = 0.047). No difference was found with D-dimer and fibrinogen in either group. This study shows that both lercanidipine and losartan are effective antihypertensive drugs in patients with essential hypertension. Lercanidipine may provide additional benefit in fibrinolysis. PMID- 16679300 TI - An avoidable complication of percutaneous coronary intervention-entrapment of stent and disconnected balloon catheter. AB - During percutaneous coronary intervention, entrapment of catheter materials is a rare but life-threatening complication that sometimes requires emergency surgical treatment. Coronary artery stents have been developed to prevent acute coronary closure and reduce restenosis after coronary angioplasty. The most frequently reported complications of coronary stents are related to stent thrombosis and anticoagulation problems. This case study describes a 60-year-old female who had stable angina pectoris and underwent stent insertion into the left circumflex artery. Unfortunately, the coronary stent with balloon catheter was entrapped while crossing the angulated segment between the left circumflex and left main coronary artery. The stent catheter was surgically removed, and the patient underwent coronary artery bypass grafting successfully. Physicians should keep in mind that extremely angulated segments may reduce the successful rate of coronary stenting and contribute to the stent entrapment complication. PMID- 16679301 TI - Ischemic stroke following multiple traumas in a child: a case report. AB - Stroke is an uncommon disorder in children but an increasingly recognized cause of disability. Acute stroke may be attributable to trauma, but this topic is seldom discussed. In limited reports, most ischemic strokes following trauma were detected after a considerable delay. Early recognition of stroke following trauma might reduce secondary neurologic complications. We report a case of posterior cerebral artery area infarct following multiple traumas in a child. A comprehensive etiologic survey was undertaken and discussed. Although the definite pathogenesis is still unclear, adequate, individualized, and uncomplicated management can significantly affect the outcome. PMID- 16679302 TI - Ptosis as an initial manifestation of orbital lymphoma: a case report. AB - Ptosis on its own is an infrequent initial manifestation of orbital lymphoma. Orbital lymphoma usually presents as a palpable mass with proptosis, diplopia, and conjunctival ("salmon-pink") swelling. We report here a 62-year-old female patient who presented with right eye ptosis. The initial imaging study showed an indistinct enlargement of the superior rectus-levator muscle complex. After 3-4 months, ptosis and upward gazing movement were further restricted. The imaging study revealed a definite soft-tissue mass in the superior orbit surrounding the superior rectus-levator muscle complex. A tumor biopsy through anterior orbitotomy revealed a large diffuse B-cell lymphoma. With the experience of this case, we suggest that orbital lymphoma should be included in the differential diagnosis of ptosis accompanied by impairment of levator muscle function. PMID- 16679303 TI - Tracheostomy tube ignition during microlaryngeal surgery using diode laser: a case report. AB - Ignition of the tracheal tube during laser microlaryngeal surgery under general anesthesia is an uncommon complication with potentially serious consequences. We present here a case of a patient with glottic stenosis following endotracheal intubation, who experienced this potentially catastrophic combustion during endoscopic arytenoidectomy, using a diode laser under general anesthesia via 60% FiO2, with an airway fire occurring at the tracheostomy tube and causing tubal damage and obstruction. The anesthetic connecting tube was immediately disconnected and the tracheostomy tube replaced. No adverse consequences to this patient's upper airway were noted during follow-up visits. Higher oxygen concentrations, the presence of combustibles, and the narrowness of the surgical field during endolaryngeal diode laser surgery are risk factors for airway fires. PMID- 16679304 TI - Different activation patterns of rat xenobiotic metabolism genes by two constituents of garlic. AB - Diallyl sulfide (DAS) and diallyl disulfide (DADS) are natural components that could account for the anticarcinogenic properties of garlic, at least in part, through the activation of xenobiotic detoxifying metabolism. The aim of this work was to describe the effect of DAS and DADS on xenobiotic-related gene expressions and to study molecular mechanisms relaying DAS effect. We describe the different effects of DAS and DADS on hepatic CYP2B1/2, CYP3A and epoxide hydrolase (EpH) mRNAs in rats, in terms of activation profile, doses and kinetics. The activation profile varied with the mode of chemical administration, i.e. gastric infusion or intraperitoneal (i.p.) injection. Using gastric infusion, DAS and DADS proved different efficiencies at enhancing the mRNA level of the three drug-metabolizing enzymes. After an i.p. administration, we observed a specific activation of CYP2B1/2 gene by DAS. The DAS-mediated CYP2B1/2 activation occurred at transcriptional level and through an okadaic acid-sensitive pathway. In rat livers, a short sequence (NR1) derived from the CYP2B1/2 promoter was stimulated by DAS and we observed a nuclear accumulation of a DNA-protein complex binding NR1. Because constitutively activated receptor (CAR) is a major transcription factor driving the xenobiotic-induced stimulation of CYP2B1/2 through NR1, the role of CAR as a preferential mediator of DAS effect is discussed. PMID- 16679305 TI - K-ras Asp12 mutant neither interacts with Raf, nor signals through Erk and is less tumorigenic than K-ras Val12. AB - Different mutant amino acids in the Ras proteins lead to distinct transforming capacities and different aggressiveness in human tumors. K-Ras Asp12 (K12D) is more prevalent in benign than in malignant human colorectal tumors, whereas K-Ras Val12 (K12V) associates with more advanced and metastatic carcinomas, higher recurrence and decreased survival. Here, we tested, in a nude mouse xenograft model, whether different human K-Ras oncogenes mutated at codon 12 to Val, Asp or Cys would confer NIH3T3 fibroblasts distinct oncogenic phenotypes. We studied tumor histology and growth, apoptotic and mitotic rates, activation of signal transduction pathways downstream of Ras and regulation of the cell cycle and apoptotic proteins in tumors derived from the implanted transformants. We found that the K12V oncogene induces a more aggressive tumorigenic phenotype than the K12D oncogene, whereas K12C does not induce tumors in this model. Thus, K12V mutant tumors proliferate about seven times faster, and have higher cellularity and mitotic rates than the K12D mutant tumors. A molecular analysis of the induced tumors shows that the K12V mutant protein interacts with Raf-1 and transduces signals mainly through the Erk pathway. Unexpectedly, in tumors induced by the K12D oncogene, the K-Ras mutant protein does not interact with Raf 1 nor activates the Erk canonical pathway. Instead, it transduces signals through the PI3K/Akt, JNK, p38 and FAK pathways. Finally, the higher growth rate of the K12V tumors associates with enhanced Rb phosphorylation, and PCNA and cyclin B upregulation, consistent with faster G1/S and G2/M transitions, without alteration of apoptotic regulation. PMID- 16679306 TI - FANCD2 associated with sporadic breast cancer risk. AB - Several components of the Fanconi anaemia (FA) family of proteins allow the formation of the DNA repair complex foci formed by proteins such as BRCA1/2 and RAD51. Because the genes that participate in the DNA repair pathway have been described as low-penetrance breast cancer susceptibility genes, we postulated that variants in FA genes could also be associated with sporadic breast cancer risk. We studied seven SNPs in FANCA, FANCL and FANCD2 in a total of 897 consecutive and non-related sporadic breast cancer cases and 1033 unaffected controls from the Spanish population. We observed a statistically significant association with sporadic breast cancer for the variant rs2272125 (L1366L) located on FANCD2 (OR per allele=1.35; 95% C.I. 1.09-1.67; P=0.005). Both haplotype and diplotype analyses confirmed this association, where one haplotype and pooled diplotypes carrying it were associated with more than 4-fold risk (P=0.007 and P=0.006, respectively). Screening for potential causal variants in FANCD2 was performed, detecting one in the putative promoter region, which is located in a phylogenetically conserved motif with consensus binding sites for some transcriptional factors, suggesting a functional implication. Our data indicate that a relationship between FANCD2 and sporadic breast cancer risk may exist. PMID- 16679307 TI - Molecular epidemiology of human liver cancer: insights into etiology, pathogenesis and prevention from The Gambia, West Africa. AB - Human liver cancer, primarily hepatocellular carcinoma (HCC), is both common and lethal. Notable variation in HCC incidence rates worldwide corresponds to the prevalence and pattern of the primary etiologic factors. In summary of decades of collaborative research centered in The Gambia, West Africa, this review explores the independent and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV) and dietary aflatoxin exposure in the etiology of HCC. Through population surveys, field trials and a series of HCC case-control studies, the patterns and natural history of HBV, HCV and aflatoxin exposures have been defined within this population. These investigations have paralleled and informed the development of molecular biomarkers of these etiologic agents and contributed to understanding the complex mechanisms involved in hepatocarcinogenesis. We discuss preventive approaches to reduce the global burden of HCC, emphasizing The Gambia Hepatitis Intervention Study, a countrywide randomized controlled trial designed to document the efficacy of HB vaccination in preventing HBV infections and HBV-related HCC. By recognizing the synergy of applying molecular techniques to population-based epidemiological studies, the portfolio of Gambian research projects presented provides a model for partnering etiologic and mechanistic investigations with applied research. PMID- 16679308 TI - Activation of the integrin-linked kinase pathway downregulates hepatic connexin32 via nuclear Akt. AB - Gap junctions mediate intercellular communication through channels composed of proteins termed connexins (Cxs). We have shown that Cx32 is downregulated in the liver of female rats exposed to hexachlorobenzene (HCB), an epigenetic environmental carcinogen. This is concomitant with the activation of the integrin linked kinase (ILK) pathway, leading to the activation and nuclear translocation of Akt and the inactivation of glycogen synthase kinase-3beta (GSK3beta). E cadherin, an adhering junction protein, is also downregulated in the liver of these female rats, owing to the inactivation of GSK3beta. Using an in vitro model, the aim of this study was to determine the role of the ILK pathway in the regulation of Cx32. In order to mimic the activation of the ILK pathway, a well differentiated rat hepatoma cell line, MH1C1, was transiently transfected with an expression vector for ILK (ILK+ cells). ILK+ cells displayed significantly lower Cx32 mRNA levels and Akt was also activated and translocated into the nucleus. Using a constitutively active Akt expression vector, we showed that Akt transfected cells had lower Cx32 mRNA levels, indicating a role for Akt in Cx32 regulation. Finally, using an Akt-NES vector, a nuclear-active form of Akt, we showed that Cx32 protein levels were reduced in transfected cells as compared with cell transfected with the wild-type inactive Akt vector, suggesting that the nuclear form of Akt is responsible for the downregulation of Cx32. Overall, these data indicate that Cx32 is downregulated by the ILK pathway activation in rat hepatocytes and that this is mediated via the activation and nuclear translocation of Akt. PMID- 16679309 TI - Chemopreventive effects of alpha-santalol on ultraviolet B radiation-induced skin tumor development in SKH-1 hairless mice. AB - Recent studies from our laboratory have shown the chemopreventive effects of alpha-santalol against 7,12-dimethylbenzanthracene (DMBA) initiated and 12-O tetradecanoylphorbol-13-acetate (TPA) promoted skin tumor development in mice. The objective of the present investigation was to study the effects of alpha santalol on ultraviolet B (UVB) radiation-induced skin tumor development and UVB caused increase in epidermal ornithine decarboxylase (ODC) activity in female hairless SKH-1 mice. For the tumor studies, 180 mice were divided into three groups of 60 mice each, and each group was divided into two subgroups of 30 mice. The first subgroup served as control and was treated topically on the dorsal skin with acetone. The second subgroup served as experimental and was treated topically on the dorsal skin with alpha-santalol (5%, w/v in acetone). The tumorigenesis in the first group was initiated with UVB radiation and promoted with TPA; in the second group it was initiated with DMBA and promoted with UVB radiation; and in the third group it was both initiated and promoted with UVB radiation. In each case, the study was terminated at 30 weeks. Topical application of alpha-santalol significantly (P<0.05) decreased tumor incidence and multiplicity in all the three protocols, suggesting its chemopreventive efficacy against UVB radiation-caused tumor initiation, tumor promotion and complete carcinogenesis. In a short-term biochemical study, topical application of alpha-santalol also significantly (P<0.05) inhibited UVB-induced epidermal ODC activity. Together, for the first time, our findings suggest that alpha-santalol could be a potential chemopreventive agent against UVB-induced skin tumor development and, therefore, warrants further investigations. PMID- 16679311 TI - Pex19p-dependent targeting of Pex17p, a peripheral component of the peroxisomal protein import machinery. AB - Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p. We have identified the corresponding binding sites for Pex14p and Pex19p and demonstrated that a specific loss of the Pex19p interaction resulted in mistargeting of Pex17p. We have shown that a construct consisting only of the Pex19p- and Pex14p-binding sites of Pex17p is sufficient to direct an otherwise cytosolic reporter protein to the peroxisomal membrane in a Pex19p-dependent manner. Our data show that the function of Pex19p as chaperone or import receptor is not restricted to integral membrane proteins but may also include peripheral PMPs. As a consequence of our data, the previous definition of a targeting signal for PMPs (mPTS) as a Pex19p-binding motif in conjunction with a transmembrane segment should be extended to regions comprising a Pex19p-binding motif and a peroxisomal anchor sequence. PMID- 16679310 TI - Structure and function of the voltage sensor of sodium channels probed by a beta scorpion toxin. AB - Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states. PMID- 16679312 TI - Polyamine analogues down-regulate estrogen receptor alpha expression in human breast cancer cells. AB - The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor alpha (ERalpha) and ERalpha target genes in ER-positive human breast cancer cell lines, whereas neither ERbeta nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERalpha in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERalpha transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERalpha minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine inhibited ERalpha expression, suggesting that AP-1 is a positive regulator of ERalpha expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERalpha induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells. PMID- 16679313 TI - Control of chemotactic signal gain via modulation of a pre-formed receptor array. AB - The remarkably wide dynamic range of the chemotactic pathway of Escherichia coli, a model signal transduction system, is achieved by methylation/amidation of the transmembrane chemoreceptors that regulate the histidine kinase CheA in response to extracellular stimuli. The chemoreceptors cluster at a cell pole together with CheA and the adaptor CheW. Several lines of evidence have led to models that assume high cooperativity and sensitivity via collaboration of receptor dimers within a cluster. Here, using in vivo disulfide cross-linking assays, we have demonstrated a well defined arrangement of the aspartate chemoreceptor (Tar). The differential effects of amidation on cross-linking at different positions indicate that amidation alters the relative orientation of Tar dimers to each other (presumably inducing rotational displacements) without much affecting the conformation of the periplasmic domains. Interestingly, the effect of aspartate on cross-linking at any position tested was roughly opposite to that of receptor amidation. Furthermore, amidation attenuated the effects of aspartate by several orders of magnitude. These results suggest that receptor covalent modification controls signal gain by altering the arrangement or packing of receptor dimers in a pre-formed cluster. PMID- 16679314 TI - A novel repressor domain is required for maximal growth inhibition by the IRF-1 tumor suppressor. AB - Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumor suppressor that can regulate gene expression in a manner requiring either its sequence specific DNA binding activity or its ability to bind the p300 coactivator. We show that IRF-1-mediated growth inhibition is dependent on the integrity of a C-terminal transcriptional enhancer domain. An enhancer subdomain (amino acids 301-325) that differentially regulates IRF-1 activity has been identified and this region mediates the repression of Cdk2. The repressor domain encompasses an LXXLL coregulator signature motif and mutations or deletions within this region completely uncouple transcriptional activation from repression. The loss of growth suppressor activity when the Cdk2-repressor domain of IRF-1 is mutated implicates repression as a determinant of its maximal growth inhibitory potential. The data link IRF-1 regulatory domains to its growth inhibitory activity and provide information about how differential gene regulation may contribute to IRF-1 tumor suppressor activity. PMID- 16679315 TI - Of two cytosolic aconitases expressed in Drosophila, only one functions as an iron-regulatory protein. AB - In mammalian cells, iron homeostasis is largely regulated by post-transcriptional control of gene expression through the binding of iron-regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) contained in the untranslated regions of target mRNAs. IRP2 is the dominant iron sensor in mammalian cells under normoxia, but IRP1 is the more ancient protein in evolutionary terms and has an additional function as a cytosolic aconitase. The Caenorhabditis elegans genome does not contain an IRP2 homolog or identifiable IREs; its IRP1 homolog has aconitase activity but does not bind to mammalian IREs. The Drosophila genome offers an evolutionary intermediate containing two IRP1-like proteins (IRP-1A and IRP-1B) and target genes with IREs. Here, we used purified recombinant IRP-1A and IRP-1B from Drosophila melanogaster and showed that only IRP-1A can bind to IREs, although both proteins possess aconitase activity. These results were also corroborated in whole-fly homogenates from transgenic flies that overexpress IRP 1A and IRP-1B in their fat bodies. Ubiquitous and muscle-specific overexpression of IRP-1A, but not of IRP-1B, resulted in pre-adult lethality, underscoring the importance of the biochemical difference between the two proteins. Domain-swap experiments showed that multiple amino acid substitutions scattered throughout the IRP1 domains are synergistically required for conferring IRE binding activity. Our data suggest that as a first step during the evolution of the IRP/IRE system, the ancient cytosolic aconitase was duplicated in insects with one variant acquiring IRE-specific binding. PMID- 16679316 TI - Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis. AB - The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl 3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b(5) (b(5)) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an (18)O-labeling method on the basis that cross linked peptides incorporate twice as many (18)O atoms as non-cross-linked peptides during proteolysis conducted in (18)O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys(428)(CYP2E1) Asp(53)(b(5)) and Lys(434)(CYP2E1)-Glu(56)(b(5)), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1 b(5) interaction, and the stimulatory effect of b(5), was confirmed by site directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b(5) complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations. PMID- 16679317 TI - Aryl hydrocarbon receptor- and calcium-dependent induction of the chemokine CCL1 by the environmental contaminant benzo[a]pyrene. AB - Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8 tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2 aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner. PMID- 16679319 TI - Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway. AB - Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl n-nitrosourea-induced mammary carcinogenesis, to regress 7,12 dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway. PMID- 16679318 TI - A new level of architectural complexity in the human pyruvate dehydrogenase complex. AB - Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is a key metabolic assembly comprising a 60-meric pentagonal dodecahedral E2 (dihydrolipoamide acetyltransferase) core attached to which are 30 pyruvate decarboxylase E1 heterotetramers and 6 dihydrolipoamide dehydrogenase E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organization in which E3 and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 "cross-bridges" linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle x-ray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organization, its catalytic mechanism and regulation by the intrinsic PDC kinase. PMID- 16679320 TI - The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments. AB - The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles. PMID- 16679321 TI - Essential role of the B23/NPM core domain in regulating ARF binding and B23 stability. AB - How cells coordinate inhibition of growth and division during genotoxic events is fundamental to our understanding of the origin of cancer. Despite increasing interest and extensive study, the mechanisms that link regulation of DNA synthesis and ribosomal biogenesis remain elusive. Recently, the tumor suppressor p14(ARF) (ARF) has been shown to interact functionally with the nucleolar protein B23/NPM (B23) and inhibit rRNA biogenesis. However, the molecular basis of the ARF-B23 interaction is hitherto unclear. Here we show that a highly conserved motif in the B23 oligomerization domain is essential for mediating ARF binding in vivo. Mutagenesis of conserved B23 core residues (L102A, G105A, G107A) prevented B23 from interacting with ARF. Modeling of the B23 core indicated that substitutions in the GSGP loop motif could trigger conformational changes in B23 thereby obstructing ARF binding. Interestingly, the GSGP loop mutants were unstable, defective for oligomerization, and delocalized from the nucleolus to the nucleoplasm. B23 core mutants displayed increased ubiquitination and proteasomal degradation. We conclude that the functional integrity of the B23 core motif is required for stability, efficient nucleolar localization as well as ARF binding. PMID- 16679322 TI - Inhibitory interaction of the plasma membrane Na+/Ca2+ exchangers with the 14-3-3 proteins. AB - The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity. PMID- 16679323 TI - Protein phosphatase 2A enhances the proapoptotic function of Bax through dephosphorylation. AB - Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function. PMID- 16679324 TI - Basal level of anti-Mullerian hormone is associated with oocyte quality in stimulated cycles. AB - BACKGROUND: Serum anti-Mullerian hormone (AMH) levels provide a powerful means for predicting ovarian response, which is reflected not only by the size of the primordial follicle pool but also by the quality of the oocytes. Considering a mutual interdependence between AMH-expressing somatic cells and gametes, this prospective morphological study was set up to evaluate whether extreme AMH levels represent diminished oocyte quality and developmental incompetence. METHODS: A total of 141 consecutive ICSI patients were subdivided into three groups using the 25th and 75th percentiles of the serum AMH levels (cycle day 3). In these three groups, morphology of all oocytes and fertilization rate, embryo quality and blastocyst formation were evaluated, and FSH, LH and estradiol (E(2)) levels were also measured. RESULTS: Cycle cancellation rate was correlated with AMH levels (P < 0.05). AMH groups 1 (<1.66 ng/ml) and 3 (>4.52 ng/ml) showed oocytes of lower quality [dark central granulation, aggregation of smooth endoplasmic reticulum (sER)] compared with the median group 2 (1.66-4.52 ng/ml). Basal serum FSH did not allow for adequate prognosis in terms of gamete appearance. Fertilization and further cleavage up to blastocyst stage was not affected by AMH levels. CONCLUSIONS: AMH seems to be superior to FSH in predicting both oocyte number and quality. PMID- 16679325 TI - Auricular electro-acupuncture as an additional perioperative analgesic method during oocyte aspiration in IVF treatment. AB - BACKGROUND: The aim of this study was to compare the pain-relieving effect and the subjective well-being between auricular electro-acupuncture (EA) analgesia, auricular acupuncture (A) and conventional analgesia with remifentanil (CO). METHODS: A total of 94 women undergoing IVF were randomized to auricular acupuncture with (EA, n = 32) or without (A, n = 32) continuous 1 Hz auricular stimulation (using a battery-powered miniaturized stimulator, P-Stim) or with adhesive tapes instead of needles and no electrical stimulation (control group, CO, n = 30) at the auricular acupuncture points 29, 55 and 57. All patients received patient-controlled analgesia (PCA) with remifentanil. Pain intensity and psychological well-being were assessed by means of visual analogue scales (VAS); tiredness, nausea and vomiting and analgesic drug consumption were documented. RESULTS: Pain relief and subjective well-being were significantly greater in group EA during and after the procedure as compared with groups A and CO (P < 0.001). The patients were significantly more tired in group CO than in groups A and EA (P < 0.001). Consumption of the opioid remifentanil was significantly lower in group EA, comparable nausea (P < 0.001). CONCLUSION: Auricular EA significantly reduces pain intensity and analgesic consumption of the opioid remifentanil during oocyte aspiration in IVF treatment. PMID- 16679326 TI - The change of stress distribution on the condyle after mandibular setback surgery. AB - The rigid-body spring model (RBSM) theory was incorporated into a model as a discrete method for analysing problems of limit, such as the stress distribution on the condyle. The purpose of this study was to evaluate the two-dimensional RBSM for determining stress on the temporomandibular joint (TMJ) in patients after orthognathic surgery. Thirty-two patients (five males and 27 females, mean age 21.4 +/- 4.9 years) with mandibular prognathism underwent bilateral sagittal split ramus osteotomy (SSRO) and setback; 48 subjects were recruited as controls. Anatomical landmarks were traced from pre- and post-operative lateral cephalograms and the information was processed using the Fortran analysis program. The force vector on the condyle, its degree, its direction, and the displacement co-ordinates (x, y) and rotation (theta) at the gonial angle were calculated. When muscular power was assumed to be 1, the post-operative degree of the force vector was higher than the pre-operative value (P < 0.05). The X co ordinate, x, and rotation, theta, of the displacement vector in the pre-operative patients with mandibular prognathism were significantly higher than those in the control subjects (P < 0.05). There were still significant differences between the displacement values post-operatively between the patients and controls (P < 0.05). The results suggest that the degree and direction of the force vector and the resulting displacement co-ordinates can be used as parameters in a surgical model. The RBSM may also be useful in evaluating the pre- and post-operative skeletal morphology of jaw deformities. PMID- 16679327 TI - Expression of type X collagen and capillary endothelium in condylar cartilage during osteogenic transition--a comparison between adaptive remodelling and natural growth. AB - Adaptive remodelling of the condylar cartilage in response to mandibular protrusion constitutes the rationale for bite-jumping appliances to solicit growth modification. By investigating the expression of type X collagen and capillary endothelium, this study was designed to evaluate the osteogenic transition of chondrogenesis during adaptive remodelling of condylar cartilage and compare it with that under natural condylar growth. One hundred female Sprague-Dawley rats, 35 days of age, were divided into five experimental groups (n = 15, fitted with bite-jumping appliances) where condylar adaptation was created by forward repositioning of the mandible, and five control groups (n = 5) where the condyles underwent natural growth. The animals were sacrificed at 3, 7, 14, 21 and 30 days and 7 mum serial sections of the condyles were processed for in situ hybridization and immunohistochemical analyses. The expression of type X collagen in the hypertrophic zone and capillary endothelium in the erosive zone of condylar cartilage were examined to evaluate osteogenic transition, a critical programme leading to endochondral ossification. The results showed that (1) The temporal pattern of the expression of type X collagen and capillary endothelium during condylar adaptation coincided with that during natural condylar growth. (2) The amount of the expression of these two factors during condylar adaptation was significantly higher than that during natural growth (P < 0.001). It is suggested that condylar adaptation in growing rats triggered by mandibular forward positioning enhances osteogenic transition which eventually results in increased bone formation. PMID- 16679328 TI - Root growth in the permanent teeth of 45,X/46,XX females. AB - Studies on individuals with sex chromosome anomalies have provided proof of a direct differential promoting effect of the X and Y chromosome genes on tooth crown growth. 45,X/46,XX females are one subgroup of Turner patients who have both normal XX and one X sex chromosome cell lines. Their permanent tooth crown size is reduced, which is mainly due to thin enamel. 45,X females likewise show reduced crown size and enamel thickness, and their root size is reduced. By contrast, the lengths of the roots in 47,XYY males or males with an extra Y chromosome and 46,XY females or females with a male sex chromosome constitution are increased. The aim of the present study was to investigate permanent tooth root lengths in 45,X/46,XX females to obtain additional information on their dental growth. The study group consisted of 15 45,X/46,XX females, mean age 23.4 years; 10 female relatives, mean age 24.5 years; and 47 population control females, mean age 29.8 years, from the Kvantti research project. Root length measurements were made from panoramic radiographs on both sides of the jaw using a sliding digital calliper. The results showed that permanent tooth root lengths in 45,X/46,XX females were, in most cases, significantly shorter than those of the population controls or relatives. It is apparent that a reduced tooth crown size in 45,X/46,XX females is followed by reduced root growth. This begins 3 years after birth and continues to at least 14 years of age. It is suggested that the reduction in crown and root growth in 45,X/4,XX females is due to a deficiency in the amount of dental growth-promoting genes on the sex chromosomes. PMID- 16679329 TI - Development of adiposity in adolescence: five year longitudinal study of an ethnically and socioeconomically diverse sample of young people in Britain. AB - OBJECTIVE: To examine the developmental trajectory of obesity in adolescence in relation to sex, ethnicity, and socioeconomic status. DESIGN: Five year longitudinal cohort study of a socioeconomically and ethnically diverse sample of school students aged 11-12 years at baseline. SETTING: 36 London schools recruited to the study in 1999 by a stratified random sampling procedure. PARTICIPANTS: 5863 students participated in one or more years. MAIN OUTCOME MEASURES: Weight, height, and waist circumference measured annually by trained researchers; overweight and obesity defined according to International Obesity Task Force criteria; adiposity and central adiposity indexed by body mass index (BMI) and waist standard deviation scores relative to 1990 British reference values. RESULTS: In school year 7 (age 11-12), the prevalence of overweight and obesity combined was almost 25%, with higher rates in girls (29%) and students from lower socioeconomic backgrounds (31%) and the highest rates in black girls (38%). Prevalence of obesity increased over the five years of the study at the expense of overweight, but no reduction occurred in the proportion of students with BMIs in the healthy range. Waist circumferences were high compared with 1990 norms at age 11 (by 0.79 SD in boys and by 1.15 SD in girls) and increased further over time. Both BMI and waist circumference tracked strongly over the five years. CONCLUSIONS: Prevalence of overweight and obesity was high in London school students, with significant socioeconomic and ethnic inequalities. Little evidence was found of new cases of overweight or obesity emerging over adolescence, but few obese or overweight adolescents reduced to a healthy weight. The results indicate that persistent obesity is established before age 11 and highlight the need to target efforts to prevent obesity in the early years. PMID- 16679330 TI - Systematic review and meta-analysis of ethnic differences in risks of adverse reactions to drugs used in cardiovascular medicine. AB - OBJECTIVE: To review the evidence for ethnic differences in susceptibility to adverse drug reactions (ADRs) to cardiovascular drugs. DESIGN: Systematic review and meta-analysis. DATA SOURCES: We searched Medline and Embase to March 2005. Reference lists of identified articles were hand searched for further relevant articles. REVIEW METHODS: Studies were eligible for inclusion if they included at least two ethnic groups and one or more ADRs. We excluded case reports and case series. RESULTS: 564 studies contained some description of ethnicity and an ADR, and 132 of them related to cardiovascular therapies. Twenty four studies provided data for ADRs for at least two ethnic groups and were therefore eligible for inclusion. In pooled analyses the relative risk of angio-oedema from angiotensin converting enzyme (ACE) inhibitors in black compared with non-black patients was 3.0 (95% confidence interval 2.5 to 3.7); the relative risk of cough from ACE inhibitors was 2.7 (1.6 to 4.5) in East Asian compared with white patients; and the relative risk of intracranial haemorrhage with thrombolytic therapy was 1.5 (1.2 to 1.9) in black compared with non-black patients. CONCLUSION: Patients from different ethnic groups have different risks for important ADRs to cardiovascular drugs. Ethnic group may therefore be one determinant of harms of a given treatment in the individual patient, either because it acts as a surrogate measure of genetic make up or because cultural factors alter the risk. Data are sparse, and regulators should consider asking for better data before licensing. PMID- 16679331 TI - Effect of family style mealtimes on quality of life, physical performance, and body weight of nursing home residents: cluster randomised controlled trial. AB - OBJECTIVE: To assess the effect of family style mealtimes on quality of life, physical performance, and body weight of nursing home residents without dementia. DESIGN: Cluster randomised trial. SETTING: Five Dutch nursing homes. PARTICIPANTS: 178 residents (mean age 77 years). Two wards in each home were randomised to intervention (95 participants) or control groups (83). INTERVENTION: During six months the intervention group took their meals family style and the control group received the usual individual pre-plated service. MAIN OUTCOME MEASURES: Quality of life (perceived safety; autonomy; and sensory, physical, and psychosocial functioning), gross and fine motor function, and body weight. RESULTS: The difference in change between the groups was significant for overall quality of life (6.1 units, 95% confidence interval 2.1 to 10.3), fine motor function (1.8 units, 0.6 to 3.0), and body weight (1.5 kg, 0.6 to 2.4). CONCLUSION: Family style mealtimes maintain quality of life, physical performance, and body weight of nursing home residents without dementia. TRIAL REGISTRATION: Clinical trials NCT00114582. PMID- 16679333 TI - PROXIMO--a new docking algorithm to model protein complexes using data from radical probe mass spectrometry (RP-MS). AB - The design and implementation of a new algorithm, known as PROXIMO for protein oxidation interface modeller, is described to predict the structure of protein complexes using data generated in radical probe mass spectrometry (RP-MS) experiments. Photochemical radiolysis and discharge sources can be used to effect RP-MS in which hydroxyl radicals are formed directly from the bulk solvent on millisecond timescales and react with surface accessible residues in footprinting like experiments. The algorithm utilizes a geometric surface fitting routine to predict likely structures for protein complexes. These structures are scored based on a correlation between the measured solvent accessibility of oxidizable residue side chains and oxidation shielding data obtained by RP-MS. The algorithm has been implemented to predict structures for the ribonuclease S-protein-peptide and calmodulin-melittin complexes using RP-MS data generated in this laboratory. The former is in close agreement with the high-resolution experimental structure available. PMID- 16679334 TI - BAli-Phy: simultaneous Bayesian inference of alignment and phylogeny. AB - SUMMARY: BAli-Phy is a Bayesian posterior sampler that employs Markov chain Monte Carlo to explore the joint space of alignment and phylogeny given molecular sequence data. Simultaneous estimation eliminates bias toward inaccurate alignment guide-trees, employs more sophisticated substitution models during alignment and automatically utilizes information in shared insertion/deletions to help infer phylogenies. AVAILABILITY: Software is available for download at http://www.biomath.ucla.edu/msuchard/bali-phy. PMID- 16679335 TI - Hip protector use amongst older hospital inpatients: compliance and functional consequences. PMID- 16679336 TI - Mortality in people diagnosed with type 2 diabetes at an older age: a systematic review. AB - OBJECTIVES: To review all published observational studies reporting on all-cause mortality in patients with type 2 diabetes to determine the degree of increased mortality when diagnosed at an older age. DESIGN: Systematic literature search. SETTING: The review included studies carried out in populations from Germany, United Kingdom, United States, Japan, Italy, Western Australia, Netherlands and Sweden. MEASUREMENTS: Medline, CINAHL, EMBASE, National Research Register and Cochrane Reviews were systematically searched from 1975 to 2004. We identified observational studies that reported overall mortality for people diagnosed with type 2 diabetes when they were over the age of 60, compared with a non-diabetic population. Outcome measures were expressed as risk ratios or relative risks. RESULTS: Among 14 eligible studies, one study reported reduced mortality for patients diagnosed with type 2 diabetes over the age of 60, whereas another found virtually no increased risk of mortality. However, 7 of the 14 studies reported increased mortality in all patients diagnosed when older, and 5 studies for certain subgroups only. A meta-analysis showed the combined relative risks (with 95% CI) of increased mortality for men diagnosed between the ages of 60 and 70 to be 1.38 (1.08-1.76) and 1.13 (0.88-1.45) for men diagnosed aged 70 years or older. A similar pattern was found for the same age groups for women, with combined relative risks of 1.40 (1.10-1.79) and 1.19 (0.93-1.52) respectively. CONCLUSION: Increased mortality associated with a diagnosis of type 2 diabetes at an older age is lower than that reported for the general older diabetic population. PMID- 16679337 TI - Exposure to antineoplastic drugs outside the hospital environment. AB - OBJECTIVES: The objectives were (i) to identify occupational populations outside hospitals working with antineoplastic drugs, (ii) to determine the size of the populations 'at risk', (iii) to identify major determinants and routes of exposure outside hospitals and (iv) to estimate exposure levels and frequencies relative to levels found in hospitals. METHODS: The survey consisted of two phases; (i) identification of activities with potential exposure to antineoplastic drugs by literature review, interviews, questionnaires and workplace visits, (ii) exploratory measurements of exposure and surface contamination in selected sectors. RESULTS: Eight sectors were identified with potential exposure to antineoplastic drugs: pharmaceutical industry, pharmacies, universities, veterinary medicine, nursing homes, home care, laundry facilities, and waste treatment. Four sectors were of primary concern: veterinary medicine, home care, nursing homes and industrial laundries. The populations potentially exposed in these sectors vary considerably (from several tens to thousands of workers), as do their levels of exposure. Exposure measurements collected in the veterinary medicine sector showed that workers are indeed exposed to antineoplastic drugs and, in some cases (on gloves after administration), levels were 15 times higher than levels measured during administration in hospitals. Workers sorting contaminated hospital laundry in industrial laundry facilities were exposed to antineoplastic drugs through inhalation. For the home care and nursing homes sectors the highest exposure levels were found when cleaning toilets and washing treated patients. These two sectors are expected to have the largest exposed population (5,000-10,000 individuals). CONCLUSIONS: This study has resulted in a comprehensive overview of populations with potential exposure to antineoplastic drugs. Exposure levels can potentially be high compared with the hospital environment, because exposure routes are complex and awareness of the hazard (and therefore use of protective measures) is low. The number of individuals outside hospitals in The Netherlands exposed to antineoplastic drugs is estimated to be between 5,000 and 15,000. PMID- 16679338 TI - Characterization of endotoxin and mouse allergen exposures in mouse facilities and research laboratories. AB - OBJECTIVES: Researchers and technicians who use mice in research are exposed to complex mixtures containing mouse allergen, endotoxin and particulates from animals, bedding and feed. The particle characteristics of these different exposures, and whether they are encountered together or separately, are important to better understand their adjuvant and allergic effects. Endotoxin and mouse allergen are derived from the same animal source, but have different physicochemical attributes. It is not known if airborne exposures to these agents are correlated in the laboratory animal workplace. METHODS: Side-by-side personal and area samples for airborne endotoxin (52), mouse allergen (46) and total particulates (43) were obtained in the animal facility and laboratories of a medical research institution. Animal handlers and researchers reported time spent on work tasks with mice, symptoms upon exposure to mice and mouse sensitization was determined by skin test or RAST. RESULTS: Mean airborne endotoxin exposure was highest during mouse experiments in the animal facility at 960 pg m(-3), peaked at 3125 pg m(-3), and ranged from 46 to 678 pg m(-3) with work in mouse rooms and research labs. Mouse allergen concentrations were highest during direct mouse work and background in research labs (mean 63-68 ng m(-3), range 41-271 ng m(-3)), but were undetectable during mouse research performed under a hood. Endotoxin and mouse allergen concentrations were correlated during direct research with mice and mouse care activities. Particle counts were low, typically < 1 cm(-3), varied widely, and exhibited peaks and valleys during different work tasks. From 80-90% of particles were < 1 microm in aerodynamic diameter during background measurements. The contribution of respirable particles 1-5 microm in size increased to 25-30% during mouse care and mouse research activities, but we found no association between any particle size and endotoxin or mouse allergen concentrations. Animal handlers and researchers in the mouse facility were exposed to the highest daily endotoxin concentrations, whereas researchers working with mice in the mouse facility and in laboratories were exposed to the highest daily mouse allergen concentrations. CONCLUSIONS: These findings suggest that endotoxin and mouse allergen are co-exposures during mouse handling and research, and that control of exposure peaks may be necessary to limit allergic disease in the laboratory animal workplace. PMID- 16679341 TI - Treatment for glomerulonephritis in HCV-associated mixed cryoglobulinaemia--anti viral therapy vs rituximab. PMID- 16679342 TI - Alcohol consumption does not affect melatonin circadian synchronization in healthy men. AB - Alcohol intake alters melatonin secretion both in healthy volunteers and in alcoholics in a variety of different situations (while drinking, during or after withdrawal, and with neurological complications). This alteration may reduce secretion or affect its circadian rhythm, thus causing daytime secretion in some alcoholics. We sought to determine if daytime melatonin secretion is caused directly by acute alcohol consumption or if it instead indicates a change in circadian synchronization. Because alcohol consumption as it occurs in alcoholics (continuous consumption of large amounts) has never been examined in healthy volunteers, we exposed 11 healthy volunteers to 256 g of alcohol over 24 h to study the circadian profiles of melatonin secretion. Our results demonstrate a lack of daytime secretion in our subjects. This suggests that the disordered circadian melatonin secretion seen in alcoholics indicates a shift in melatonin secretion rather than an acute effect of alcohol on this secretion, or alternatively, that it is a direct effect of chronic rather than acute exposure to high blood alcohol levels. PMID- 16679343 TI - Clinical and genetic characteristics of Korean male alcoholics with and without attention deficit hyperactivity disorder. AB - AIMS: To examine the clinical and genetic characteristics of Korean male alcoholics with and without attention deficit hyperactivity disorder (ADHD). METHODS: The present study included 85 male alcoholics who were diagnosed as having DSM-IV alcohol dependence. A total of 28 (32.9%) alcoholics were diagnosed as having DSM-IV ADHD with ongoing symptoms in adulthood. For the evaluation of their psychiatric conditions, the alcohol dependence scale (ADS), Beck depression inventory (BDI), Beck anxiety inventory (BAI), Barratt impulsiveness scale (BIS), brief anger-aggression questionnaire (BAQ), overt aggression scale (OAS), codependence test, and obsessive compulsive drinking scale (OCDS) were administered. The genotype frequencies of the dopamine type 2 receptor gene (DRD2), aldehyde dehydrogenase type 2 gene (ALDH2), functional polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR), and catechol O-methyltransferase gene (COMT) polymorphisms were examined. RESULTS: Compared with alcoholics without ADHD, the mean ages for the onset of pathological drinking and alcohol withdrawal hallucinations were significantly earlier in alcoholics with ADHD. There was also a significant difference in the history of antisocial behaviour between the two groups. Compared with alcoholics without ADHD, the mean scores of the ADS, BDI, BAI, OAS, and OCDS were significantly higher in alcoholics with ADHD. With regard to the codependence test results, the mean scores of the interpersonal problem, low self-esteem and anxiety/fear subscales, and the mean total score of the codependence test were significantly higher in alcoholics with ADHD when compared with those without ADHD. There were no significant differences in the genotype frequencies of the DRD2, ALDH2, 5 HTTLPR, and COMT polymorphisms between alcoholics with and without ADHD. CONCLUSIONS: The results of this study suggest that the comorbidity of alcohol dependence and ADHD in this Korean sample forms a distinct clinical phenotype that shows an increased severity of alcohol-related symptoms and behavioural/emotional problems and that ADHD is associated with an increased risk for the early onset of alcohol dependence in Korean male alcoholics. PMID- 16679344 TI - Telephone screening for hazardous drinking among injured patients seen in acute care clinics: feasibility study. AB - AIMS: We evaluated the effectiveness of telephoning injured patients after discharge, compared with contacting them in the clinic during the acute care visit, for screening for hazardous drinking and eliciting willingness to participate in a lifestyle intervention trial. METHODS: We conducted a quasi randomized controlled trial among acutely injured adult patients in trauma and acute care clinics, assigning telephone and clinic screening strategies systematically by week. During telephone weeks, we mailed study information to patients identified from computerized records, then telephoned them. During clinic weeks, researchers recruited patients awaiting care. We screened for hazardous drinking using the AUDIT-C (Alcohol Use Disorders Identification Test C). We examined the proportion of all injured adult patients who were screened, the proportion of screened patients with hazardous drinking (AUDIT-C score >or=4), and the proportion willing to participate in a (hypothetical) lifestyle intervention trial. Differences were analysed with non-linear mixed models using generalized estimating equations, controlling for age, sex, and facility. Levers and barriers to screening were explored through structured interviews with research staff. RESULTS: We enrolled 29% (469/1,609) of all injured adult patients and 76% of injured patients contacted and found to be eligible. Of screened patients, 23.1% screened positive for hazardous drinking. Telephone and clinic contact were equally effective for screening patients (OR = 1.05; 95% CI = 0.59-1.87), identifying hazardous drinking (OR=0.97; 95% CI = 0.54-1.74), and eliciting willingness to participate in an intervention trial (OR=1.49; 95% CI = 0.97-2.30). Clinic site modified results: telephone was more effective than clinic contact for screening urban patients (OR=1.99; 95% CI = 1.36-2.93), but less effective for screening suburban patients (OR = 0.70; 95% CI = 0.69-0.71). Barriers to clinic screening included lack of clinic staff support, time constraints, and difficulty recruiting elderly or acutely distressed patients. Barriers to telephone screening included erroneous contact information and failure to answer the telephone. CONCLUSIONS: Telephone screening is a feasible and efficient method for screening moderately injured adult patients for hazardous drinking, but characteristics of the clinical site (including personnel) influence its effectiveness. Trauma and acute care clinics are likely to be fruitful sites for identification of patients with hazardous drinking, whether for enrollment into brief intervention trials or treatment programmes. PMID- 16679346 TI - Accounting for background nucleotide composition when measuring codon usage bias: brilliant idea, difficult in practice. AB - The effective number of codons used in a gene is a commonly used measure of codon usage. It varies between 20 and 61 (standard genetic code) and indicates to which degree the entire genetic code is used. It is a drawback of this method that it does not take background composition into account. This led Novembre to introduce a variant called Nc' (Novembre JA. 2002. Accounting for background nucleotide composition when measuring codon usage bias. Mol Biol Evol 19:1390-4). In this letter, its properties are under the loupe, with special emphasis on phenomena relating to codon homozygosity. A theoretical misunderstanding regarding this estimator is explained in detail, notably Nc varies between 0 and 61 instead of 20 and 61 (with the standard genetic code). Practical examples from the genome of Pseudomonas aeruginosa are given which demonstrate that the problem is not just theoretical. PMID- 16679345 TI - In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations. AB - Viroids are plant subviral pathogens whose genomes are constituted by a single stranded and covalently closed small RNA molecule that does not encode for any protein. Despite this genomic simplicity, they are able of inducing devastating symptoms in susceptible plants. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as branched structures. The shape of these RNA structures is perhaps one of the most characteristic properties of viroids and sometimes is considered their only phenotype. Here we use RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. After characterizing the statistical properties of the distribution of mutational effects on structure stability and the wideness of neutral neighborhood for each viroid species, we show an evolutionary trend toward increased structural robustness during viroid radiation, giving support to the adaptive value of robustness. Differences in robustness among the 2 viroid families can be explained by the larger fragility of branched structures compared with the rodlike ones. We also show that genomic redundancy can contribute to the robustness of these simple RNA genomes. PMID- 16679347 TI - A single oral dose of ethanol can alter transdermal absorption of topically applied chemicals in rats. AB - Topical ethanol is used as a dermal penetration enhancer in some commercial products. Previous studies have demonstrated that chronic ethanol consumption can also disrupt skin barrier function, leading to increased transdermal penetration. This observation becomes much more relevant if a single drinking episode induces similar changes. The purpose of this study was thus to examine the transdermal penetration of three model chemicals after acute ethanol consumption. Wistar rats were gavaged with either 10, 6, 4.3, 3, 1.5 g/kg ethanol or saline and allowed to recover for 2 or 24 h. Blood and skin ethanol levels were determined and in vitro penetration experiments performed. The herbicide paraquat, industrial solvent N,N dimethylformamide (DMF), and insect repellent N,N-diethyl-m-toluamide (DEET) were used as is model chemicals. Absorption was determined and directly compared between ethanol- and saline-treated skin by calculating enhancement ratios. Blood ethanol levels range from 0.25 to 0.015% at 2 h with skin levels at 12-18% of blood values. Ethanol enhances the absorption of paraquat, DMF, and DEET in a dose-dependent fashion. Paraquat and DEET showed no appreciable reduction in enhancement between 2 and 24 h postgavage for the 10-g/kg dose, but DMF did. Enhancement ratios were higher at 24 h for 10 than for 6 g/kg animals, demonstrating a dose-response relationship for recovery time. These studies imply that increased absorption of topical chemical occurs after alcohol ingestion. Both acute and chronic ethanol consumption can compromise the dermal barrier. PMID- 16679348 TI - Cytochrome P450 1A4 and 1A5 in common cormorant (Phalacrocorax carbo): evolutionary relationships and functional implications associated with dioxin and related compounds. AB - The present study characterized cytochrome P4501A (CYP1A) isoforms from common cormorant (Phalacrocorax carbo) with regard to their evolutionary relationships and their roles in disposition of dioxin and related compounds (DRCs). Two clones isolated from a cormorant liver cDNA library were named CYP1A4 and CYP1A5 on the basis of greatest overall amino acid identity shared with chicken (Gallus gallus) CYP1A4 (78%) and CYP1A5 (78%), respectively. Spatial heterogeneity in phylogenetic signal along the sequences strongly indicated that cormorant CYP1A4 and CYP1A5 have undergone partial interparalog gene conversion, similar to chicken and mammalian CYP1As. Phylogenetic analysis of a putatively unconverted region produced a tree topology consistent with the orthology of avian CYP1A5s with mammalian CYP1A2s and avian CYP1A4s with mammalian CYP1A1s. Hepatic CYP1A4 and CYP1A5 mRNA levels in wild cormorants from Lake Biwa, Japan, were quantified to examine the effects of DRCs on isoform-specific expression and to evaluate the toxicokinetics of DRCs in which CYP1A expression is involved. Both CYP1A4 and CYP1A5 mRNA levels were positively correlated with total tetrachlorodibenzo-p dioxin toxic equivalents and concentrations of each congener in most cases in the liver, suggesting the induction of both enzymes through a shared transcriptional mechanism. The lack of correlation of 2,3,7,8-tetrachlorodibenzofuran and 3,3',4,4'-tetrachlorobiphenyl (PCB77) to CYP1A gene expression is likely due to the rapid metabolism of these two congeners. Liver-to-muscle concentration ratios for most DRC congeners except PCB77 and mono-ortho coplanar polychlorinated biphenyls significantly increased with an elevation of CYP1A4 and CYP1A5 mRNA levels. The present data suggest that hepatic sequestration of some DRCs occurs in cormorant via binding to either CYP1A5 or both CYP1A4 and CYP1A5. PMID- 16679349 TI - Effects of Helicobacter pylori on the cadherin-catenin complex. AB - BACKGROUND: The cadherin-catenin complex is the key component of the adherens junction in epithelial cells, and changes in this complex are implicated in gastric adenocarcinoma. Germline mutations in E-cadherin have been described in diffuse-type gastric adenocarcinoma. Helicobacter pylori infection is the first stage in gastric carcinogenesis. AIMS: To determine whether H pylori was associated with changes in the complex, and whether this was affected by virulence of the strain. METHODS: Epithelial cell lines were cultured with H pylori using the wild-type pathogenic and non-pathogenic strains and CagE null and VacA null isogenic mutants. Gastric biopsy specimens at endoscopy were obtained from patients with (n = 17) and without (n = 15) H pylori infection, and E-cadherin and beta-catenin expression was assessed by immunohistochemistry. H pylori was typed by polymerase chain reaction from these patients for CagE and VacA. RESULTS: In vitro studies showed that coculture with a pathogenic strain of H pylori led to disruption of epithelial junctional beta-catenin expression, but without evidence of nuclear translocation or signalling. This effect was independent of a functional Cag pathogenicity island and vacuolating activity, but dependent on live bacteria. No marked differences in beta-catenin or E cadherin expression were seen in gastric biopsy specimens in patients with and without H pylori infection. CONCLUSION: Acute H pylori infection disrupts junctional beta-catenin in vitro, but chronic infection by H pylori has no effect on E-cadherin and beta-catenin expression, as seen in gastric biopsy specimens at the initial gastritis stage of the proposed Correa pathway of gastric carcinogenesis. A later effect at the later stages of atrophy or intestinal metaplasia cannot be ruled out. PMID- 16679350 TI - Expression profiling and prediction of distant metastases in head and neck squamous cell carcinoma. AB - BACKGROUND: For breast and prostate cancer, a gene expression signature of the tumour is associated with the development of distant metastases. Regarding head and neck squamous cell carcinoma (HNSCC), the only known risk factor is the presence of > or =3 tumour-positive lymph nodes. AIM: To evaluate whether a HNSCC gene expression signature can discriminate between the patients with and without distant metastases. METHODS: Patients with HNSCC with and without distant metastases had >3 tumour-positive lymph nodes, and did not differ with respect to other risk factors. Statistical analysis was carried out using Student's t test, as well as statistical analysis of microarrays (SAM), to assess the false discovery rate for each gene. These analyses were supplemented with a newly developed method that computed deviations from gaussian-order statistics (DEGOS). To validate the platform, normal mucosa of the head and neck was included as control. RESULTS: 2963 genes were differently expressed between HNSCC and normal mucosa (t test; p<0.01). More rigorous statistical analysis with SAM confirmed the differential expression of most genes. The comparison of genes in HNSCC with and without metastases showed 150 differently expressed genes (t test; p<0.01), none of which, however, could be confirmed using SAM or DEGOS. CONCLUSIONS: No evidence for a metastasis signature is found, and gene expression profiling of HNSCC has seemingly no value in determining the risk of developing distant metastases. The absence of such a signature can be understood when it is realised that, for HNSCC in contrast with breast cancer, the lymph nodes are a necessary in-between station for haematogenous spread. PMID- 16679351 TI - Liver fluke-associated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes. AB - AIM: To compare cell phenotypes displayed by cholangiocarcinomas and adjacent bile duct lesions in patients from an area endemic in liver-fluke infestation and those with sporadic cholangiocarcinoma. METHODS: 65 fluke-associated and 47 sporadic cholangiocarcinomas and 6 normal livers were studied. Serial paraffin wax sections were stained immunohistochemically with monoclonal antibodies characterising a Brunner or pyloric gland metaplasia cell phenotype (antigens D10 and 1F6), intestinal goblet cells (antigen 17NM), gastric foveolar apomucin (MUC5AC), a gastrointestinal epithelium cytokeratin (CK20) and the p53 protein. RESULTS: 60% of the 112 cholangiocarcinomas expressed antigen D10, 68% MUC5AC, 33% antigen 17NM and 20% CK20; 37% showed overexpression of p53. When present together in a cholangiocarcinoma, cancer cells expressing D10 were distinct from those displaying 17NM or MUC5AC. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinomas displayed 17NM and p53 expression. Most cases of hyperplastic and dysplastic biliary epithelium expressed D10 strongly. Pyloric gland metaplasia and peribiliary glands displayed D10 and 1F6, with peribiliary gland hyperplasia more evident in the livers with fluke-associated cholangiocarcinoma; goblet cells in intestinal metaplasia stained for 17NM. No notable association of expression between any two antigens (including p53) was found in the cancers. CONCLUSIONS: Most cases of dysplastic biliary epithelium and cholangiocarcinoma display a Brunner or pyloric gland cell phenotype and a gastric foveolar cell phenotype. The expression of D10 in hyperplastic and dysplastic epithelium and in cholangiocarcinoma is consistent with a dysplasia carcinoma sequence. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinoma display an intestinal goblet cell phenotype and overexpress p53, indicating differences in the aetiopathology of the cancers in the two groups of patients. PMID- 16679352 TI - A "field change" of inhibited apoptosis occurs in colorectal mucosa adjacent to colorectal adenocarcinoma. AB - BACKGROUND: Colorectal cancer is associated with a "field change" of increased proliferation throughout the colonic and rectal mucosa. Both proliferation and apoptosis are disrupted during carcinogenesis. Whether altered apoptosis contributes to this field change of microscopic abnormality is, however, unclear. Bcl-xL is an anti-apoptotic protein that inhibits apoptosis by preventing release of cytochrome c, a recognised pathway to cell death. AIM: To determine whether Bcl-xL inhibition of apoptosis is increased in colorectal mucosa adjacent to colorectal adenocarcinoma over that in normal non-neoplastic colorectal mucosa. PATIENTS: PATIENTS undergoing surgical resection for neoplastic (adenocarcinoma) or non-neoplastic disease of the colorectum (rectal prolapse, diverticular disease or volvulus). METHODS: Formalin-fixed, paraffin-wax-embedded surgical colorectal resection specimens were immunostained for Bcl-xL protein. Labelling indices were determined by counting the proportion of positively stained cells in mucosal crypts. RESULTS: 85 patients were studied. Bcl-xL immunostaining was most marked in the upper third of mucosal crypts. It occurred in a minority of samples from non-neoplastic colorectal mucosa, but was seen in most mucosal samples adjacent to colorectal adenocarcinoma. Significant increases (p<0.001) were observed in Bcl-xL labelling indices in the mucosa at 1 cm (n = 46, median labelling index 31.8%, interquartile range 8.3-43.9%) and at 10 cm (n = 52, median labelling index 22.0%, interquartile range 0.0-36.3%) from colorectal carcinoma, compared with normal, non-neoplastic colorectal mucosa (n = 22, median labelling index 0.0%, interquartile range 0.0-0.0%). CONCLUSIONS: The findings are consistent with a field change of inhibited apoptosis in mucosa adjacent to colorectal carcinoma. PMID- 16679354 TI - Diagnosis of thymoma. AB - The diagnosis of thymic epithelial neoplasm has been a topic of controversy for many years. Reasons for this include the lack of predictive value associated with the morphology of these tumours and the multiplicity of classification schemes and terminologies proposed over the years. Recently, a new classification schema was introduced by the World Health Organization (WHO) in an attempt to standardise nomenclature and facilitate the diagnosis of primary thymic epithelial neoplasms. This schema, although not originally intended as a new histological classification, but rather as a means for translating equivalent terms from the various existing classifications, has represented a major step forward in this direction. However, problems still exist with the WHO schema, particularly with some of the criteria for the various histological subtypes as well as with issues of interobserver reproducibility. For this reason, we favour using a much more simplified approach to the morphological classification of thymic epithelial neoplasms. A personal approach to the morphological diagnosis of thymoma is described, with a brief explanation for the rationale for simplifying the existing diagnostic categories. PMID- 16679353 TI - An approach to duodenal biopsies. AB - The introduction of endoscopy of the upper digestive tract as a routine diagnostic procedure has increased the number of duodenal biopsy specimens. Consequently, the pathologist is often asked to evaluate them. In this review, a practical approach to the evaluation of a duodenal biopsy specimen is discussed. An overview of the handling of specimens is given and the normal histology and commonly encountered diseases are discussed. Finally, a description of commonly seen infections is provided, together with an algorithmic approach for diagnosis. PMID- 16679356 TI - External quality assurance of molecular analysis of haemochromatosis gene mutations. AB - BACKGROUND: The Royal College of Pathologists of Australasia Quality Assurance Programs has conducted an external quality assurance programme for the testing of the haemochromatosis gene (HFE) mutations C282Y and H63D. METHODS: A total of 10 surveys have been undertaken over a period of 6 years from 2000 to 2005. RESULTS: Of the 3016 responses received, the overall success rate was found to be 99.47% (3000/3016). A total of 16 errors were found, 6 for C282Y and 10 for H63D. Only one sample was associated with more than one error, in which 2 of 23 respondents classified a normal sample as heterozygotic for H63D. Overall performance was observed to vary minimally between surveys, from a low of 91.3% correct (21/23 responses) for a normal sample to 100% correct in most (85/100) samples. Of the 10 complete surveys, four returned a 0% error rate. In one survey in 2004, seven incorrect responses were returned by one laboratory, all of which were secondary to transcriptional errors. Overall success rates per assay were 99.61% (1532/1538) for C282Y and 99.32% (1468/1478) for H63D. Over a period of 6 years from 2000 to 2005, the proportion of respondents using polymerase chain reaction (PCR) and restriction enzyme analysis fell from 85% to around 30%, whereas the proportion of laboratories using real-time PCR rose from 5% to around 55%, as indicated by the questionnaire surveys of methods used by participants. DISCUSSION: Encouraging levels of testing proficiency for two common genetic mutations are indicated by these data, but they also confirm the need for participation of molecular diagnostic laboratories in external quality assurance programmes to ensure the ongoing provision of high-quality genetic testing services. PMID- 16679355 TI - Dynamic behavioural interpretation of cervical intraepithelial neoplasia with molecular biomarkers. AB - The microscopic phenotype of cervical intraepithelial neoplasia (CIN) reflects a fine balance between factors that promote or reduce CIN development. A shortcoming of the current grading system is its reliance on static morphology and microscopic haematoxylin-eosin features of the epithelium alone. In reality, CIN is a dynamic process, and the epithelium may exhibit differing results over time. Functional biomarkers p16, Ki-67, p53, retinoblastoma protein cytokeratin (CK)14 and CK13, help in the assessment of an individual CIN's lesion's potential for progression and regression. The aggregate information provided by these biomarkers exceeds the value of the classic grading system. Consequently, many more CINs that will either regress or progress can be accurately identified. These findings agree with known molecular interactions between HPV and the host. For accurate interpretation of a CIN, it is essential that these biomarkers be determined quantitatively and separately in the superficial, middle and deep layers of the epithelium. Such geography-specific epithelial evaluations of quantitative biomarkers emphasise the dynamic nature of a particular CIN lesion, thereby changing the art of static morphology grading into dynamic interpretation of the diseased tissue, with a strong prognostic effect. PMID- 16679357 TI - Molecular evolution of protein O-fucosyltransferase genes and splice variants. AB - O-Fucose has been described on both epidermal growth factor-like (EGF-like) repeats and Thrombospondin type 1 repeats (TSRs). The enzyme adding fucose to EGF like repeats, protein O-fucosyltransferase 1 (Pofut1), is a soluble protein located in the lumen of endoplasmic reticulum (ER). A second protein O fucosyltransferase, Pofut2, quite divergent from its homolog Pofut1, has recently been shown to O-fucosylate TSRs but not EGF-like repeats. To date, Pofut1 genes have only been characterized in human, mouse, and fly, and Pofut2 in mouse, fly, and partially in the nematode Caenorhabditis elegans. Here, we report cDNA sequences and genomic structures of bovine Pofut1 and Pofut2 genes and describe for the first time five alternative spliced transcripts for each gene. Only one transcript for both Pofut1 and Pofut2 encodes an active bovine O fucosyltransferase. Variant transcript distribution was examined in 13 bovine tissues. Transcripts encoding active forms are ubiquitous, whereas other forms possess a more restricted tissue-expression profile. Sequence comparison and phylogenetic analyses revealed that both Pofut genes are present as a single copy in animal genomes, and their exon-intron organizations are conserved among vertebrates. The last common ancestor of all analyzed bilaterian species would be predicted to possess polyexonic Pofut genes in their genome. PMID- 16679358 TI - Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching. AB - Cells contain many subcellular structures in which specialized proteins locally cluster. Binding interactions within such clusters may be analyzed in live cells using models for fluorescence recovery after photobleaching (FRAP). Here we analyze a three-dimensional FRAP model that accounts for a single spatially localized cluster of binding sites in the presence of both diffusion and impermeable boundaries. We demonstrate that models completely ignoring the spatial localization of binding yield poor estimates for the binding parameters within the binding site cluster. In contrast, we find that ignoring only the restricted axial height of the binding-site cluster is far less detrimental, thereby enabling the use of computationally less expensive models. We also identify simplified solutions to the FRAP model for limiting behaviors where either diffusion or binding dominate. We show how ignoring a role for diffusion can sometimes produce serious errors in binding parameter estimation. We illustrate application of the method by analyzing binding of a transcription factor, the glucocorticoid receptor, to a tandem array of mouse mammary tumor virus promoter sites in live cells, obtaining an estimate for an in vivo binding constant (10(-7) M), and a first approximation of an upper bound on the transcription-factor residence time at the promoter (approximately 170 ms). These FRAP analysis tools will be important for measuring key cellular binding parameters necessary for a complete and accurate description of the networks that regulate cellular behavior. PMID- 16679359 TI - Contribution of the Na+/Ca2+ exchanger to rapid Ca2+ release in cardiomyocytes. AB - Trigger Ca(2+) is considered to be the Ca(2+) current through the L-type Ca(2+) channel (LTCC) that causes release of Ca(2+) from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current (I(Ca)). In this article, we investigate the contribution of the Na(+)/Ca(2+) exchanger (NCX) to the trigger Ca(2+). Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na(+)/Ca(2+) exchange delays the Ca(2+) transient by 3-4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na(+) and Ca(2+) is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na(+) channel is present in the fuzzy space, the NCX is able to bring enough Ca(2+) into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na(+) channel and NCX. PMID- 16679360 TI - Dynamics of DNA ejection from bacteriophage. AB - The ejection of DNA from a bacterial virus (i.e., phage) into its host cell is a biologically important example of the translocation of a macromolecular chain along its length through a membrane. The simplest mechanism for this motion is diffusion, but in the case of phage ejection a significant driving force derives from the high degree of stress to which the DNA is subjected in the viral capsid. The translocation is further sped up by the ratcheting and entropic forces associated with proteins that bind to the viral DNA in the host cell cytoplasm. We formulate a generalized diffusion equation that includes these various pushing and pulling effects and make estimates of the corresponding speedups in the overall translocation process. Stress in the capsid is the dominant factor throughout early ejection, with the pull due to binding particles taking over at later stages. Confinement effects are also investigated, in the case where the phage injects its DNA into a volume comparable to the capsid size. Our results suggest a series of in vitro experiments involving the ejection of DNA into vesicles filled with varying amounts of binding proteins from phage whose state of stress is controlled by ambient salt conditions or by tuning genome length. PMID- 16679361 TI - Mapping complex myoarchitecture in the bovine tongue with diffusion-spectrum magnetic resonance imaging. AB - The ability to resolve complex fiber populations in muscular tissues is important for relating tissue structure with mechanical function. To address this issue in the case of tongue, we employed diffusion spectrum imaging (DSI), an MRI method for determining three-dimensional myoarchitecture where myofiber populations are variably aligned. By specifically varying gradient field strength, molecular displacement in a tissue can be determined by Fourier-transforming the echo intensity against gradient strength at fixed gradient pulse spacing. The displacement profiles are visualized by graphing three-dimensional isocontour icons for each voxel, with the isocontour shape and size representing the magnitude and direction of the constituting fiber populations. To validate this method, we simulated a DSI experiment within the constraints of arbitrary crossing fibers, and determined that DSI accurately depicts the angular relationships between these fibers. Considering the fiber relationships in the whole bovine tongue, we compared the images obtained by DSI with those obtained by diffusion tensor imaging in an anterior slice of the lingual core, a region known to possess extensive fiber crossing. In contrast to diffusion tensor imaging, which depicts the anterior core solely as a region with low anisotropy due to the presence of mixed-orientation fiber populations, DSI shows two distinct fiber populations, with an explicit orthogonal relationship to each other. In imaging the whole lingual tissue, we discerned arrays of crossing and noncrossing fibers involving the intrinsic and extrinsic muscles, which merged at regions of interface. We conclude that DSI has the capacity to determine three dimensional fiber orientation in structurally complex muscular tissues. PMID- 16679362 TI - Maximum likelihood estimation of molecular motor kinetics from staircase dwell time sequences. AB - Molecular motors, such as kinesin, myosin, or dynein, convert chemical energy into mechanical energy by hydrolyzing ATP. The mechanical energy is used for moving in discrete steps along the cytoskeleton and carrying a molecular load. High resolution single molecule recordings of motor steps appear as a stochastic sequence of dwells, resembling a staircase. Staircase data can also be obtained from other molecular machines such as F1 -ATPase, RNA polymerase, or topoisomerase. We developed a maximum likelihood algorithm that estimates the rate constants between different conformational states of the protein, including motor steps. We model the motor with a periodic Markov model that reflects the repetitive chemistry of the motor step. We estimated the kinetics from the idealized dwell-sequence by numerical maximization of the likelihood function for discrete-time Markov models. This approach eliminates the need for missed event correction. The algorithm can fit kinetic models of arbitrary complexity, such as uniform or alternating step chemistry, reversible or irreversible kinetics, ATP concentration and mechanical force-dependent rates, etc. The method allows global fitting across stationary and nonstationary experimental conditions, and user defined a priori constraints on rate constants. The algorithm was tested with simulated data, and implemented in the free QuB software. PMID- 16679363 TI - Chemical potential derivatives and preferential interaction parameters in biological systems from Kirkwood-Buff theory. AB - New expressions for chemical potential derivatives and preferential interaction parameters for ternary mixtures are derived for open, semiopen, and closed ensembles in terms of Kirkwood-Buff integrals, where all three components are present at finite concentrations. This is achieved using a simple approach that avoids the use of the general matrix formulation of Kirkwood-Buff theory. The resulting expressions provide a rigorous foundation for the analysis of experimental and simulation data. Using the results, a simple model is developed and used to investigate the possible effects of finite protein concentrations on the corresponding cosolvent dependent chemical potential and denaturation thermodynamics. PMID- 16679364 TI - Evidence that nystatin channels form at the boundaries, not the interiors of lipid domains. AB - Nystatin (nys) is an antifungal agent that preferentially forms ion channels in membranes containing the sterol, ergosterol (erg). The structure of the nystatin channel is not clear, but it is known that multiple nystatin monomers must aggregate to form channels in a sterol-rich membrane. When nys/erg containing vesicles are fused to a sterol-free bilayer, characteristic spikelike changes in membrane conductance are observed. An abrupt increase in conductance is followed by a decay that is generally stepwise linear and the decay time depends strongly on [erg]. These data are inconsistent with the hypothesis that nys channels form uniformly throughout the membrane and decay independently (which would produce exponential decay). We propose that channels are located at the boundaries of lipid superlattices such that diffusion of erg out of the lattice results in correlated channel decay. This was tested using a statistical mechanical analysis and Monte Carlo simulations, which reveal details of the diffusion process and provide insight into conditions at superlattice boundaries during decay. This analysis predicts the linear decay schemes and the dramatic drop in channel decay time observed at erg mol % = 50. This interpretation also explains puzzling data relating conductance spike height to vesicle diameter. PMID- 16679365 TI - A computational model integrating electrophysiology, contraction, and mitochondrial bioenergetics in the ventricular myocyte. AB - An intricate network of reactions is involved in matching energy supply with demand in the heart. This complexity arises because energy production both modulates and is modulated by the electrophysiological and contractile activity of the cardiac myocyte. Here, we present an integrated mathematical model of the cardiac cell that links excitation-contraction coupling with mitochondrial energy generation. The dynamics of the model are described by a system of 50 ordinary differential equations. The formulation explicitly incorporates cytoplasmic ATP consuming processes associated with force generation and ion transport, as well as the creatine kinase reaction. Changes in the electrical and contractile activity of the myocyte are coupled to mitochondrial energetics through the ATP, Ca2+, and Na+ concentrations in the myoplasmic and mitochondrial matrix compartments. The pseudo steady-state relationship between force and oxygen consumption at various stimulus frequencies and external Ca2+ concentrations is reproduced in both model simulations and direct experiments in cardiac trabeculae under normoxic conditions, recapitulating the linearity between cardiac work and respiration in the heart. Importantly, the model can also reproduce the rapid time-dependent changes in mitochondrial NADH and Ca2+ in response to abrupt changes in workload. The steady-state and dynamic responses of the model were conferred by ADP-dependent stimulation of mitochondrial oxidative phosphorylation and Ca2+ -dependent regulation of Krebs cycle dehydrogenases, illustrating how the model can be used as a tool for investigating mechanisms underlying metabolic control in the heart. PMID- 16679366 TI - Vulnerable window for conduction block in a one-dimensional cable of cardiac cells, 2: multiple extrasystoles. AB - Unidirectional conduction block of premature extrasystoles can lead to initiation of cardiac reentry, causing lethal arrhythmias including ventricular fibrillation. Multiple extrasystoles are often more effective at inducing unidirectional conduction block and reentry than a single extrasystole. Since the substrate for conduction block is spatial dispersion of refractoriness, in this study we investigate how the first extrasystole modulates this dispersion to influence the "vulnerable window" for conduction block by subsequent extrasystoles, particularly in relation to action potential duration restitution and conduction velocity restitution properties. Using a kinematic model to represent wavefront-waveback interactions and simulations with the Luo-Rudy model in a one-dimensional cable of cardiac cells, we show that in homogeneous tissue, a premature extrasystole can create a large dispersion of refractoriness leading to conduction block of a subsequent extrasystole. In heterogeneous tissue, however, a premature extrasystole can either reduce or enhance the dispersion of refractoriness depending on its propagation direction with respect to the previous beat. With multiple extrasystoles at random coupling intervals, vulnerability to conduction block is proportional to their number. In general, steep action potential duration restitution and broad conduction velocity restitution promote dispersion of refractoriness in response to multiple extrasystoles, and thus enhance vulnerability to conduction block. These restitution properties also promote spatially discordant alternans, a setting which is particularly prone to conduction block. The equivalent dispersion of refractoriness created dynamically in homogeneous tissue by spatially discordant alternans is more likely to cause conduction block than a comparable degree of preexisting dispersion in heterogeneous tissue. PMID- 16679368 TI - Application of the thermorheologically complex nonlinear Adam-Gibbs model for the glass transition to molecular motion in hydrated proteins. AB - The nonlinear thermorheologically complex Adam Gibbs (extended "Scherer-Hodge") model for the glass transition is applied to enthalpy relaxation data reported by Sartor, Mayer, and Johari for hydrated methemoglobin. A sensible range in values for the average localized activation energy is obtained (100-200 kJ mol(-1)). The standard deviation in the inferred Gaussian distribution of activation energies, computed from the reported KWW beta-parameter, is approximately 30% of the average, consistent with the suggestion that some relaxation processes in hydrated proteins have exceptionally low activation energies. PMID- 16679367 TI - Vulnerable window for conduction block in a one-dimensional cable of cardiac cells, 1: single extrasystoles. AB - Spatial dispersion of refractoriness, which is amplified by genetic diseases, drugs, and electrical and structural remodeling during heart disease, is recognized as a major factor increasing the risk of lethal arrhythmias and sudden cardiac death. Dispersion forms the substrate for unidirectional conduction block, which is required for the initiation of reentry by extrasystoles or rapid pacing. In this study, we examine theoretically and numerically how preexisting gradients in refractoriness control the vulnerable window for unidirectional conduction block by a single premature extrasystole. Using a kinematic model to represent wavefront-waveback interactions, we first analytically derived the relationship (under simplified conditions) between the vulnerable window and various electrophysiological parameters such as action potential duration gradients, refractoriness barriers, conduction velocity restitution, etc. We then compared these findings to numerical simulations using the kinematic model or the Luo-Rudy action potential model in a one-dimensional cable of cardiac cells. The results from all three methods agreed well. We show that a critical gradient in action potential duration for conduction block can be analytically derived, and once this critical gradient is exceeded, the vulnerable window increases proportionately with the refractory barrier and is modulated by conduction velocity restitution and gap junctional conductance. Moreover, the critical gradient for conduction block is higher for an extrasystole traveling in the opposite direction from the sinus beat than for one traveling in the same direction (e.g., an epicardial extrasystole versus an endocardial extrasystole). PMID- 16679369 TI - A polarized-light spectroscopy study of interactions of a hairpin polyamide with DNA. AB - We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding. PMID- 16679370 TI - Dynamic compression augments interstitial transport of a glucose-like solute in articular cartilage. AB - Solute transport through the extracellular matrix is essential for cellular activities in articular cartilage. Increased solute transport via fluid convection may be a mechanism by which dynamic compression stimulates chondrocyte metabolism. However, loading conditions that optimally augment transport likely vary for different solutes. To investigate effects of dynamic loading on transport of a bioactive solute, triangular mechanical loading waveforms were applied to cartilage explants disks while interstitial transport of a fluorescent glucose analog was monitored. Peak-to-peak compression amplitudes varied from 5 50% and frequencies varied from 0.0006-0.1 Hz to alter the spatial distribution and magnitude of oscillatory fluid flow. Solute transport was quantified by monitoring accumulation of fluorescence in a saline bath circulated around the explant. Individual explants were subjected to a series of compression protocols, so that effects of loading on solute desorption could be observed directly. Maximum increases in solute transport were obtained with 10-20% compression amplitudes at 0.1 Hz; similar loading protocols were previously found to stimulate chondrocyte metabolism in vitro. Results therefore support hypotheses relating to increased solute transport as a mediator of the cartilage biological response to dynamic compression, and may have application in mechanical conditioning of cartilage constructs for tissue engineering. PMID- 16679371 TI - First-principles transversal DNA conductance deconstructed. AB - First-principles calculation of the transverse conductance across DNA fragments placed between gold nanoelectrodes reveals that such conductance describes electron tunneling that depends critically on geometrical rather than electronic structure properties. By factoring the first-principles result into two simple and approximately independent tunneling factors, we show that the conductances of the A, C, G, and T fragments differ only because of their sizes: the larger is the DNA base, the smaller its distance to the electrode, and the larger its conductance. Because the geometrical factors are difficult to control in an experiment, the direct-current measurements across DNA with gold contact electrodes may not be a convenient approach to DNA sequencing. PMID- 16679372 TI - Evidence for lipid/cholesterol ordering in model lipid membranes. AB - It has been postulated that for a binary mixture of phospholipid and cholesterol, phospholipid/cholesterol complexes are formed. Using grazing incidence x-ray diffraction, we have obtained evidence for lipid/cholesterol ordering in model membranes. Scattering features consistent with the existence of lipid/cholesterol complexes persist to high surface pressures even though fluorescence microscopy suggests a homogeneously fluid phase. Contrary to pure phospholipid and cholesterol systems, the resulting lattice spacing, integrated scattering intensity, and coherence lengths of these complexes are almost independent of surface pressure. Furthermore, the single peak observed in these mixed systems is very broad, suggesting that the extent of order for a single scattering structure only persists over a few molecules. This observation is consistent with these complexes being dynamic structures. PMID- 16679373 TI - Kinetic measurement of transient dimerization and dissociation reactions of Arabidopsis phototropin 1 LOV2 domain. AB - Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain 2, was studied with a view to the diffusion coefficients (D) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the D-value decrease from D of the reactant (9.8 +/- 0.4) x 10(-11) m2/s to D of the product (8.0 +/- 0.4) x 10(-11) m2/s. The D-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV2 is the monomeric form in a concentration range of 50-200 microM. According to the Stokes Einstein relationship, the D-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the D-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV2 domain transiently forms a dimer upon photoexcitation. When the sample concentration is increased (>180 microM), a new signal component appears within a few milliseconds. This signal represents a D increase from 8.0 x 10(-11) m2/s to 9.8 x 10(-11) m2/s with a time constant of 300 micros. The completely opposite D change from that observed in a lower concentration, as well as the concentration dependence, implies that a dimer is formed in the ground state in a higher concentration range, even though the fraction of the dimer is still minor in this range. This dimer is photodissociated, with a time constant of 300 micros. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV2 domain is discussed. PMID- 16679374 TI - Structural stability and dynamics of an amyloid-forming peptide GNNQQNY from the yeast prion sup-35. AB - A seven amino acid yeast prion sup-35 fragment (GNNQQNY) forms amyloid fibrils. The availability of its detailed atomic oligomeric structure makes it a good model for studying the early stage of aggregation. Here we perform long all-atom explicit solvent molecular simulations of various sizes and arrangements of oligomer seeds of the wild-type and its mutants to study its stability and dynamics. Previous studies have suggested that the early stage rate-limiting step of oligomer formation occurs in high-order oligomers. Our simulations show that with the increase in the number of strands even from a dimer to a trimer, oligomer stability increases dramatically. This suggests that the minimal nucleus seed for GNNQQNY fibril formation could be small and is likely three or four peptides, in agreement with experiment, and that higher-order oligomers do not dissociate quickly since they have small diffusion coefficients and thus slow kinetics. Further, for the hydrophilic polar GNNQQNY, there are no hydrogen bonds and no hydrophobic interactions between adjacent beta-sheets. Simulations suggest that within the sheet, the driving forces to associate and stabilize are interstrand backbone-backbone and side chain-side chain hydrogen bonds, whereas between the sheets, shape-complementary by the dry polar steric zipper via the side chains of Asn-2, Gln-4, and Asn-6 holds the sheets together, as proposed in an earlier study. Since the polar side chains of Asn-2, Gln-4, and Asn-6 act as a hook to bind two neighboring sheets together, these geometric restraints reduce the conformational search for the correct side chain packing to a two-dimensional problem of intersheet side chain interactions. Mutant simulations show that substitution of Asn-2, Gln-4, or Asn-6 by Ala would disrupt this steric zipper, leading to unstable oligomers. PMID- 16679375 TI - Mechanical deformation of spherical viruses with icosahedral symmetry. AB - Virus capsids and crystalline surfactant vesicles are two examples of self assembled shells in the nano- to micrometer size range. Virus capsids are particularly interesting since they have to sustain large internal pressures while encapsulating and protecting the viral DNA. We therefore study the mechanical properties of crystalline shells of icosahedral symmetry on a substrate under a uniaxial applied force by computer simulations. We predict the elastic response for small deformations, and the buckling transitions at large deformations. Both are found to depend strongly on the number of elementary building blocks N (the capsomers in the case of viral shells), the Foppl-von Karman number gamma (which characterizes the relative importance of shear and bending elasticity), and the confining geometry. In particular, we show that whereas large shells are well described by continuum elasticity-theory, small shells of the size of typical viral capsids behave differently already for small deformations. Our results are essential to extract quantitative information about the elastic properties of viruses and vesicles from deformation experiments. PMID- 16679376 TI - Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations. AB - Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-microm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2+/-2.5 and 22+/-3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 microM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability (NPo)] from 0.31+/-0.19 to 0.60+/-0.21 (P<0.001). The D1 receptor antagonist SCH-23390 (10 microM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not. PMID- 16679377 TI - Mechanisms of activation of eNOS by 20-HETE and VEGF in bovine pulmonary artery endothelial cells. AB - We have demonstrated that VEGF-induced dilation of bovine pulmonary arteries is associated with activation of cytochrome P-450 family 4 (CYP4) enzymes and eNOS. We hypothesized that VEGF and the CYP4 product 20-HETE would trigger common downstream pathways of intracellular signaling to activate eNOS. We treated bovine pulmonary artery endothelial cells (BPAECs) with 20-HETE (1 microM) or VEGF (8.3 nM) and examined three molecular events known to activate eNOS: 1) phosphorylation at serine 1179, 2) phosphorylation of protein kinase B (Akt), which subsequently phosphorylates eNOS, and 3) association of eNOS with 90-kDa heat shock protein (Hsp90). Both 20-HETE and VEGF increase the phosphorylation of eNOS at serine 1179 and Akt at serine 473. The CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) blocks VEGF-induced phosphorylation of eNOS. VEGF had no effect on the binding of Hsp90 with eNOS, whereas 20-HETE decreased the association of the protein partners. Inhibition of Akt-phosphatidylinositol 3 kinase with wortmannin blocks both 20-HETE and VEGF-induced relaxation of pulmonary arteries, supporting the functional contribution of Akt phosphorylation to the vasoactive actions of both agents. Treatment with radicicol had no effect on 20-HETE-induced relaxation of pulmonary arteries, consistent with an absence of effect on association of Hsp90 to eNOS, whereas radicicol partially blocked VEGF-evoked relaxations, possibly secondary to effects on endpoints other than Hsp90 association with eNOS. In conclusion, VEGF and 20-HETE share eNOS activation pathways, including phosphorylation of serine 1179 and phosphorylation of Akt. Unlike aortic endothelial cells, eNOS activation in BPAECs by either VEGF or 20-HETE does not appear to require increased association of Hsp90. PMID- 16679379 TI - VEGF-induced relaxation of pulmonary arteries is mediated by endothelial cytochrome P-450 hydroxylase. AB - The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA rings (1 mm in diameter) relaxed with VEGF (0.1-10 nM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N hydroxy-N'-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6-12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular Ca(2+) in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1-5 microM) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs. PMID- 16679380 TI - Toll-like receptor activation in airway smooth muscle: dual actions via separate MAPK pathways. PMID- 16679378 TI - Extracellular heat shock protein 72 is a marker of the stress protein response in acute lung injury. AB - Previous studies have shown that heat shock protein 72 (Hsp72) is found in the extracellular space (eHsp72) and that eHsp72 has potent immunomodulatory effects. However, whether eHsp72 is present in the distal air spaces and whether eHsp72 could modulate removal of alveolar edema is unknown. The first objective was to determine whether Hsp72 is released within air spaces and whether Hsp72 levels in pulmonary edema fluid would correlate with the capacity of the alveolar epithelium to remove alveolar edema fluid in patients with ALI/ARDS. Patients with hydrostatic edema served as controls. The second objective was to determine whether activation of the stress protein response (SPR) caused the release of Hsp72 into the extracellular space in vivo and in vitro and to determine whether SPR activation and/or eHsp72 itself would prevent the IL-1beta-mediated inhibition of the vectorial fluid transport across alveolar type II cells. We found that eHsp72 was present in plasma and pulmonary edema fluid of ALI patients and that eHsp72 was significantly higher in pulmonary edema fluid from patients with preserved alveolar epithelial fluid clearance. Furthermore, SPR activation in vivo in mice and in vitro in lung endothelial, epithelial, and macrophage cells caused intracellular expression and extracellular release of Hsp72. Finally, SPR activation, but not eHsp72 itself, prevented the decrease in alveolar epithelial ion transport induced by exposure to IL-1beta. Thus SPR may protect the alveolar epithelium against oxidative stress associated with experimental ALI, and eHsp72 may serve as a marker of SPR activation in the distal air spaces of patients with ALI. PMID- 16679381 TI - Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo. AB - Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days+14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1alpha, but not HIF-2alpha, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1 expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD. PMID- 16679382 TI - Three-dimensional imaging and morphometric analysis of alveolar tissue from microfocal X-ray-computed tomography. AB - We evaluated microfocal X-ray-computed tomography (micro-CT) as a method to visualize lung architecture two and three dimensionally and to obtain morphometric data. Inflated porcine lungs were fixed by formaldehyde ventilation. Tissue samples (8-mm diameter, 10-mm height) were stained with osmium tetroxide, and 400 projection images (1,024 x 1,024 pixel) were obtained. Continuous isometric micro-CT scans (voxel size 9 microm) were acquired to reconstruct two- and three-dimensional images. Tissue samples were sectioned (8-microm thickness) for histological analysis. Alveolar surface density and mean linear intercept were assessed by stereology-based morphometry in micro-CT scans and corresponding histological sections. Furthermore, stereology-based morphometry was compared with morphometric semi-automated micro-CT analysis within the same micro-CT scan. Agreement of methods was assessed by regression and Bland-Altman analysis. Comparing histology with micro-CT, alveolar surface densities (35.4 +/- 2.4 vs. 33.4 +/- 1.9/mm, P < 0.05) showed a correlation (r = 0.72; P = 0.018) with an agreement of 2 +/- 1.6/mm; the mean linear intercept (135.7 +/- 14.5 vs. 135.8 +/ 15 microm) correlated well (r = 0.97; P < 0.0001) with an agreement of -0.1 +/- 3.4 microm. Semi-automated micro-CT analysis resulted in smaller alveolar surface densities (33.4 +/- 1.9 vs. 30.5 +/- 1/mm; P < 0.01) with a correlation (r = 0.70; P = 0.023) and agreement of 2.9 +/- 1.4/mm. Non-destructive micro-CT scanning offers the advantage to visualize the spatial tissue architecture of small lung samples two and three dimensionally. PMID- 16679383 TI - Diverse effects of vascular endothelial growth factor on human pulmonary endothelial barrier and migration. AB - Increased endothelial permeability is involved in the pathogenesis of many cardiovascular and pulmonary diseases. Vascular endothelial growth factor (VEGF) is a permeability-increasing cytokine. At the same time, VEGF is known to have a beneficial effect on endothelial cells (EC), increasing their survival. Pulmonary endothelium, particularly, may be exposed to higher VEGF concentrations, since the VEGF level is the higher in the lungs than in any other organ. The purpose of this work was to evaluate the effects of VEGF on barrier function and motility of cultured human pulmonary EC. Using transendothelial resistance measurements as an indicator of permeability, we found that 10 ng/ml VEGF significantly improved barrier properties of cultured human pulmonary artery EC (118.6+/-0.6% compared with 100% control, P<0.001). In contrast, challenge with 100 ng/ml VEGF decreased endothelial barrier (71.6+/-1.0% compared with 100% control, P<0.001) and caused disruption of adherens junctions. VEGF at both concentrations increased cellular migration; however, 10 ng/ml VEGF had a significantly stronger effect. VEGF caused a dose-dependent increase in intracellular Ca2+ concentration; however, phosphorylation of myosin light chain was detectably elevated only after treatment with 100 ng/ml. In contrast, 10 ng/ml but not 100 ng/ml VEGF caused a significant increase in intracellular cAMP (known barrier-protective stimulus) compared with nonstimulated cells (1,096+/-157 and 610+/-86 fmol/mg, respectively; P<0.024). Y576-specific phosphorylation of focal adhesion kinase was also stimulated by 10 ng/ml VEGF. Our data suggest that, depending on its concentration, VEGF may cause diverse effects on pulmonary endothelial permeability via different signaling pathways. PMID- 16679384 TI - Metabolism of nomifensine to a dihydroisoquinolinium ion metabolite by human myeloperoxidase, hemoglobin, monoamine oxidase A, and cytochrome P450 enzymes. AB - Nomifensine is an antidepressant agent that was removed from use because of a high incidence of hemolytic anemia. It contains an N-methyl-8 aminotetrahydroisoquinoline ring which has the potential to be oxidized to quaternary dihydroisoquinolinium and isoquinolinium ions, albeit such a transformation had not been previously observed. In this report, we demonstrate the conversion of nomifensine to a dihydroisoquinolinium ion metabolite by several human enzymes. Human liver microsomes supplemented with NADPH generated the dihydroisoquinolinium ion metabolite along with other hydroxylated metabolites, whereas when supplemented with t-butyl peroxide, only the dihydroisoquinolinium ion metabolite was observed. Monoamine oxidase A, but not monoamine oxidase B, catalyzed this reaction, as well as human hemoglobin supplemented with H2O2. Human myeloperoxidase catalyzed this reaction in the presence of H2O2, and activation of the reaction was observed when incubations were conducted in the presence of acetaminophen at concentrations relevant to those measured in humans. The reaction was also observed in human whole blood. The equilibrium between the dihydroisoquinolinium ion and carbinolamine was shown to have a pK of about 11.7. The dihydroisoquinolinium ion was shown to react with cyanide and borohydride, but not glutathione. These findings suggest that the electrophilic nomifensine dihydroisoquinolinium metabolite, which can be generated by several enzymes, could be behind toxic responses to nomifensine such as hemolytic anemia and hepatotoxicity. PMID- 16679385 TI - Evaluation of time-dependent cytochrome P450 inhibition using cultured human hepatocytes. AB - Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters kinact and KI) which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4'-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1'-hydroxylation in a time- and concentration-dependent manner. Fluoxetine also inhibited CYP2C19-dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLMs, and recombinant CYP2C19 (KI 0.4 microM and kinact 0.5 min(-1)). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, a proprietary AstraZeneca compound; KI 30 microM and kinact 0.02 min(-1)) effectively abolished CYP2C9 activity over 24 h at low (micromolar) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time dependent inhibitors, which may have dramatic inhibitory effects on P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate. PMID- 16679386 TI - Covalent binding of radioactivity from [14C]rofecoxib, but not [14C]celecoxib or [14C]CS-706, to the arterial elastin of rats. AB - Rofecoxib is a cyclooxygenase-2 (COX-2) inhibitor that has been withdrawn from the market because of an increased risk of cardiovascular (CV) events. With a special focus on the arteries, the distribution profiles of radioactivity in rats orally administered [14C]rofecoxib were investigated in comparison with two other COX-2 inhibitors, [14C]celecoxib and [14C]CS-706 (2-(4-ethoxyphenyl)-4-methyl 1 (4-sulfamoylphenyl)-1H-pyrrole), a novel selective COX-2 inhibitor. Whole-body autoradioluminography and quantitative determination of the tissue concentrations showed that considerable radioactivity is retained by and accumulated in the thoracic aorta of rats after oral administration of [14C]rofecoxib, but not [14C]celecoxib or [14C]CS-706. Acid, organic solvent, and proteolytic enzyme treatments of aorta retaining high levels of radioactivity from [14C]rofecoxib demonstrated that most of the radioactivity is covalently bound to elastin. In agreement with this result, the radioactivity was found to be highly localized on the elastic fibers in the aorta by microautoradiography. The retention of radioactivity on the elastic fibers was also observed in the aortic arch and the coronary artery. These findings indicate that [14C]rofecoxib and/or its metabolite(s) are covalently bound to elastin in the arteries. These data are consistent with the suggestion of modified arterial elasticity leading to an increased risk of CV events after long-term treatment with rofecoxib. PMID- 16679387 TI - Inhibition of UDP-glucuronosyltransferase 2b7-catalyzed morphine glucuronidation by ketoconazole: dual mechanisms involving a novel noncompetitive mode. AB - Glucuronidation of morphine in humans is predominantly catalyzed by UDP glucuronosyltransferase 2B7 (UGT2B7). Since our recent research suggested that cytochrome P450s (P450s) interact with UGT2B7 to affect its function [Takeda S et al. (2005) Mol Pharmacol 67:665-672], P450 inhibitors are expected to modulate UGT2B7-catalyzed activity. To address this issue, we investigated the effects of P450 inhibitors (cimetidine, sulfaphenazole, erythromycin, nifedipine, and ketoconazole) on the UGT2B7-catalyzed formation of morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G). Among the inhibitors tested, ketoconazole was the most potent inhibitor of both M-3-G and M-6-G formation by human liver microsomes. The others were less effective except that nifedipine exhibited an inhibitory effect on M-6-G formation comparable to that by ketoconazole. Neither addition of NADPH nor solubilization of liver microsomes affected the ability of ketoconazole to inhibit morphine glucuronidation. In addition, ketoconazole had an ability to inhibit morphine UGT activity of recombinant UGT2B7 freed from P450. Kinetic analysis suggested that the ketoconazole-produced inhibition of morphine glucuronidation involves a mixed-type mechanism. Codeine potentiated inhibition of morphine glucuronidation by ketoconazole. In contrast, addition of another substrate, testosterone, showed no or a minor effect on ketoconazole produced inhibition of morphine UGT. These results suggest that 1) metabolism of ketoconazole by P450 is not required for inhibition of UGT2B7-catalyzed morphine glucuronidation; and 2) this drug exerts its inhibitory effect on morphine UGT by novel mechanisms involving competitive and noncompetitive inhibition. PMID- 16679388 TI - New cytochrome P450 2D6*56 allele identified by genotype/phenotype analysis of cryopreserved human hepatocytes. AB - Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *35, *40, *41, *43, and various multiple copy CYP2D6 alleles (*1xn, *2xn, and *4xn) by the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C 224 T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates that commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested in it, not just those with access to private liver banks. PMID- 16679389 TI - Hepatic and extrahepatic synthesis and disposition of dinitrophenyl-S-glutathione in bile duct-ligated rats. AB - The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 micromol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of gamma-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (-39% and -33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S transferases alpha, mu, and pi, as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis. PMID- 16679390 TI - Selective metabolism of vincristine in vitro by CYP3A5. AB - Clinical outcomes of vincristine therapy, both neurotoxicity and efficacy, are unpredictable, and the reported pharmacokinetics of vincristine have considerable interindividual variability. In vitro and in vivo data support a dominant role for CYP3A enzymes in the elimination of vincristine. Consequently, genetic polymorphisms in cytochrome P450 (P450) expression may contribute to the interindividual variability in clinical response, but the contributions of individual P450s and the primary pathways of vincristine metabolism have not been defined. In the present study, vincristine was incubated with a library of cDNA expressed P450s, and the major oxidative metabolites were identified. CYP3A4 and CYP3A5 were the only P450s to support substantial loss of parent drug and formation of the previously unidentified, major metabolite (M1). The structure of M1, arising as a result of an oxidative cleavage of the piperidine ring of the dihydro-hydroxycatharanthine unit of vincristine, was conclusively established after conversion to suitable derivatives followed by spectroscopic analysis, and a new pathway for vincristine metabolism is proposed. CYP3A5 was more efficient in catalyzing the formation of M1 compared with CYP3A4 (9- to 14-fold higher intrinsic clearance for CYP3A5). The formation of M1 was stimulated (3-fold) by the presence of coexpressed cytochrome b5, but the relative efficiencies of M1 formation by CYP3A4 and CYP3A5 were unaffected. Our findings demonstrate that in contrast to most CYP3A biotransformations, the oxidation of vincristine is considerably more efficient with CYP3A5 than with CYP3A4. We conclude that common genetic polymorphisms in CYP3A5 expression may contribute to the interindividual variability in the systemic elimination of vincristine. PMID- 16679391 TI - Essential role of PKC-zeta in normal and angiotensin II-accelerated neointimal growth after vascular injury. AB - The contribution of atypical protein kinase C (PKC)-zeta to ANG II-accelerated restenosis after endoluminal vascular injury was investigated by using the rat carotid balloon injury model. Exposure of injured arteries to ANG II resulted in an extensive neointimal thickening (1.9 times) compared with vehicle at day 14. Treatment with PKC-zeta antisense, but not scrambled, oligonucleotides reduced neointimal formation observed in the presence or absence of ANG II. Examination of early events (2 days) after injury showed an increase in cellularity in the perivascular area of the artery wall that was transferred to the adventitia and media after exposure to ANG II, events blocked by PKC-zeta antisense, but not scrambled, oligonucleotides. A positive correlation between medial cellularity at day 2 and extent of neointimal growth at day 14 was established. Immunohistochemical analysis showed that upregulation of inflammatory markers after injury, as well as infiltration of ED1(+) monocytes/macrophages from the perivascular area to the adventitia, was accelerated by ANG II. However, ANG II stimulated medial increase in cellularity was proliferation independent, and these cells were monocyte chemoattractant protein-1(+)/vimentin(+) but ED1( )/VCAM(-). PKC-zeta is degraded after injury, and inhibition of its neosynthesis in medial vascular smooth muscle cells or in infiltrating cells with PKC-zeta antisense attenuated medial cellularity and expression of inflammation mediators without reversing smooth muscle cell dedifferentiation. Together, these data indicate that PKC-zeta plays a critical role in normal and ANG II-accelerated neointimal growth through a mechanism involving upregulation of inflammatory mediators, leading to cell infiltration in the media of the vascular wall. PMID- 16679392 TI - Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase. AB - Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis. PMID- 16679393 TI - Changes in activation sequence of embryonic chick atria correlate with developing myocardial architecture. AB - To characterize developmental changes in impulse propagation within atrial musculature, we performed high-speed optical mapping of activation sequence of the developing chick atria using voltage-sensitive dye. The activation maps were correlated with detailed morphological studies using scanning electron microscopy, histology, and whole mount confocal imaging with three-dimensional reconstruction. A preferential pathway appeared during development within the roof of the atria, transmitting the impulse rapidly from the right-sided sinoatrial node to the left atrium. The morphological substrate of this pathway, the bundle of Bachman, apparent from stage 29 onward, was a prominent ridge of pectinate muscles continuous with the terminal crest. Further acceleration of impulse propagation was noted along the ridges formed by the developing pectinate muscles, ramifying from the terminal crest toward the atrioventricular groove. In contrast, when the impulse reached the interatrial septum, slowing was often observed, suggesting that the septum acts as a barrier or sink for electrical current. We conclude that these inhomogeneities in atrial impulse propagation are consistent with existence of a specialized network of fast-conducting tissues. The purpose of these preferential pathways appears to be to assure synchronous atrial activation and contraction rather than rapid impulse conduction between the sinoatrial and atrioventricular nodes. PMID- 16679394 TI - Myocardial dysfunction and neurohumoral activation without remodeling in left ventricle of monocrotaline-induced pulmonary hypertensive rats. AB - In monocrotaline (MCT)-induced pulmonary hypertension (PH), only the right ventricle (RV) endures overload, but both ventricles are exposed to enhanced neuroendocrine stimulation. To assess whether in long-standing PH the left ventricular (LV) myocardium molecular/contractile phenotype can be disturbed, we evaluated myocardial function, histology, and gene expression of autocrine/paracrine systems in rats with severe PH 6 wk after subcutaneous injection of 60 mg/kg MCT. The overloaded RV underwent myocardial hypertrophy (P < 0.001) and fibrosis (P = 0.014) as well as increased expression of angiotensin converting enzyme (ACE) (8-fold; P < 0.001), endothelin-1 (ET-1) (6-fold; P < 0.001), and type B natriuretic peptide (BNP) (15-fold; P < 0.001). Despite the similar upregulation of ET-1 (8-fold; P < 0.001) and overexpression of ACE (4 fold; P < 0.001) without BNP elevation, the nonoverloaded LV myocardium was neither hypertrophic nor fibrotic. LV indexes of contractility (P < 0.001) and relaxation (P = 0.03) were abnormal, however, and LV muscle strips from MCT treated compared with sham rats presented negative (P = 0.003) force-frequency relationships (FFR). Despite higher ET-1 production, BQ-123 (ET(A) antagonist) did not alter LV MCT-treated muscle strip contractility distinctly (P = 0.005) from the negative inotropic effect exerted on shams. Chronic daily therapy with 250 mg/kg bosentan (dual endothelin receptor antagonist) after MCT injection not only attenuated RV hypertrophy and local neuroendocrine activation but also completely reverted FFR of LV muscle strips to positive values. In conclusion, the LV myocardium is altered in advanced MCT-induced PH, undergoing neuroendocrine activation and contractile dysfunction in the absence of hypertrophy or fibrosis. Neuroendocrine mediators, particularly ET-1, may participate in this functional deterioration. PMID- 16679396 TI - Rapid vasoregulatory mechanisms in exercising human skeletal muscle: dynamic response to repeated changes in contraction intensity. AB - We tested the hypothesis that vasoregulatory mechanisms exist in humans that can rapidly adjust muscle blood flow to repeated increases and decreases in exercise intensity. Six men and seven women (age, 24.4+/-1.3 yr) performed continuous dynamic forearm handgrip contractions (1- to 2-s contraction-to-relaxation duty cycle) during repeated step increases and decreases in contraction intensity. Three step change oscillation protocols were examined: Slow (7 contractions per contraction intensityx10 steps); Fast (2 contractions per contraction intensityx15 steps); and Very Fast (1 contraction per contraction intensityx15 steps). Forearm blood flow (FBF; Doppler and echo ultrasonography), heart rate (ECG), and mean arterial pressure (arterial tonometry) were examined for the equivalent of a cardiac cycle during each relaxation phase (FBFrelax). Mean arterial pressure and heart rate did not change during repeated step changes (P=0.352 and P=0.190). For both Slow and Fast conditions, relaxation phase FBFrelax adjusted immediately and repeatedly to both increases and decreases in contraction intensity, and the magnitude and time course of FBFrelax changes were virtually identical. For the Very Fast condition, FBFrelax increased with the first contraction and thereafter slowly increased over the course of repeated contraction intensity oscillations. We conclude that vasoregulatory mechanisms exist in human skeletal muscle that are capable of rapidly and repeatedly adjusting muscle blood flow with ongoing step changes in contraction intensity. Importantly, they demonstrate symmetry in response magnitude and time course with increasing versus decreasing contraction intensity but cannot adjust to very fast exercise intensity oscillations. PMID- 16679397 TI - Right and left ventricular function after chronic pulmonary artery banding in rats assessed with biventricular pressure-volume loops. AB - In many patients with congenital heart disease, the right ventricle (RV) is subjected to abnormal loading conditions. To better understand the state of compensated RV hypertrophy, which could eventually progress to decompensation, we studied the effects of RV pressure overload in rats. In the present study, we report the biventricular adaptation to 6 wk of pulmonary artery banding (PAB). PAB resulted in an RV pressure overload to approximately 60% of systemic level and a twofold increase in RV mass (P < 0.01). Systemic hemodynamic parameters were not altered, and overt signs of heart failure were absent. Load-independent measures of ventricular function (end-systolic pressure-volume relation, preload recruitable stroke work relation, maximum first time derivative of pressure divided by end-diastolic volume), assessed by means of pressure-volume (PV) loops, demonstrated a two- to threefold increase in RV contractility under baseline conditions in PAB rats. RV contractility increased in response to dobutamine stimulation (2.5 microg.kg(-1).min(-1)) both in PAB and sham-operated rats in a similar fashion, indicating preserved RV contractile reserve in PAB rats. Left ventricular (LV) contractility at baseline was unaffected in PAB rats, although LV volume in PAB rats was slightly decreased. LV contractility increased in response to dobutamine (2.5 microg.kg(-1).min(-1)), both in PAB and sham rats, whereas the response to a higher dose of dobutamine (5 microg.kg(-1).min(-1)) was blunted in PAB rats. RV pressure overload (6 wk) in rats resulted in a state of compensated RV hypertrophy with preserved RV contractile reserve, whereas LV contractile state at baseline was not affected. Furthermore, this study demonstrates the feasibility of performing biventricular PV-loop measurements in rats. PMID- 16679398 TI - Growth factor- and heparin-dependent regulation of constitutive and agonist mediated human endothelial barrier function. AB - We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation. PMID- 16679399 TI - NHE-1 inhibition improves cardiac mitochondrial function through regulation of mitochondrial biogenesis during postinfarction remodeling. AB - We have recently demonstrated that mitochondrial respiratory dysfunction and mitochondrial permeability transition pore opening during postinfarction remodeling are prevented by the Na(+)/H(+) exchange-1 (NHE-1)-specific inhibitor EMD-87580 (EMD). One of the mechanisms underlying the beneficial effect of NHE-1 inhibition on mitochondria could result from the drug's ability to regulate transcriptional factors responsible for mitochondrial function. In the present study, the effect of EMD on the expression of nuclear factors involved in mitochondrial biogenesis and expression of nuclear (COXNUCSUB IV) and mitochondrial (COXMITSUB I) encoded cytochrome c oxidase subunits has been studied in rat hearts subjected to either 12 or 18 wk of coronary artery ligation (CAL). Remodeling induced an increase in expression of the hypertrophic marker gene atrial natriuretic peptide, especially 12 wk after CAL. The mRNA level of the peroxisome proliferator-activated receptor-gamma coactivator-1alpha and its downstream factors, including nuclear respiratory factor 1 and 2, mitochondrial transcription factor A, COXNUCSUB IV, and COXMITSUB I, were significantly reduced in hearts both 12 and 18 wk after ligation compared with sham-operated hearts. Dietary EMD provided immediately after ligation attenuated downregulation of mitochondrial transcription factors with a parallel decrease of hypertrophic marker gene expression. Regression analysis demonstrated a strong positive correlation between the transcription factors and mitochondrial respiratory function. Thus our study shows that the downregulation of mitochondrial transcription factors induced by postinfarction remodeling can be significantly attenuated by NHE-1 inhibition with a further improvement of mitochondrial function in these hearts. PMID- 16679400 TI - Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes. AB - To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts. PMID- 16679401 TI - Cytoskeletal networks and the regulation of cardiac contractility: microtubules, hypertrophy, and cardiac dysfunction. AB - The cytoskeleton as classically defined for eukaryotic cells consists of three systems of protein filaments: the microtubules, the intermediate filaments, and the microfilaments. In mature striated muscle such as the heart of the adult mammal, these three types of cytoskeletal filaments are superimposed spatially on the myofilaments, a specialized system of contractile protein filaments. Each of these systems of protein filaments has the potential to respond in an adaptive or maladaptive manner during load-induced hypertrophic cardiac growth. However, the extent to which such hypertrophy is compensatory is also critically dependent on the type of hemodynamic overload that serves as the hypertrophic stimulus. Thus cardiac hypertrophy is not intrinsically maladaptive; rather, it is the nature of the inducing load rather than hypertrophy itself that is responsible, through effects on structural and/or regulatory proteins, for the frequent deterioration of initially compensatory hypertrophy into the congestive heart failure state. As one example reviewed here of this load specificity of maladaptation, increased microtubule network density is a persistent feature of severely pressure overloaded, hypertrophied, and failing myocardium that imposes a primarily viscous load on active myofilaments during contraction. PMID- 16679402 TI - Developmental changes in passive stiffness and myofilament Ca2+ sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth. AB - The giant protein titin, a major contributor to myocardial mechanics, is expressed in two main cardiac isoforms: stiff N2B (3.0 MDa) and more compliant N2BA (>3.2 MDa). Fetal hearts of mice, rats, and pigs express a unique N2BA isoform ( approximately 3.7 MDa) but no N2B. Around birth the fetal N2BA titin is replaced by smaller-size N2BA isoforms and N2B, which predominates in adult hearts, stiffening their sarcomeres. Here we show that perinatal titin-isoform switching and corresponding passive stiffness (STp) changes do not occur in the hearts of guinea pig and sheep. In these species the shift toward "adult" proportions of N2B isoform is almost completed by midgestation. The relative contributions of titin and collagen to STp were estimated in force measurements on skinned cardiac muscle strips by selective titin proteolysis, leaving the collagen matrix unaffected. Titin-based STp contributed between 42% and 58% to total STp in late-fetal and adult sheep/guinea pigs and adult rats. However, only approximately 20% of total STp was titin based in late-fetal rat. Titin-borne passive tension and the proportion of titin-based STp generally scaled with the N2B isoform percentage. The titin isoform transitions were correlated to a switch in troponin-I (TnI) isoform expression. In rats, fetal slow skeletal TnI (ssTnI) was replaced by adult carciac TnI (cTnI) shortly after birth, thereby reducing the Ca2+ sensitivity of force development. In contrast, guinea pig and sheep coexpressed ssTnI and cTnI in fetal hearts, and skinned fibers from guinea pig showed almost no perinatal shift in Ca2+ sensitivity. We conclude that TnI isoform and titin-isoform switching and corresponding functional changes during heart development are not initiated by birth but are genetically programmed, species-specific regulated events. PMID- 16679403 TI - Scaling of diastolic intraventricular pressure gradients is related to filling time duration. AB - In early diastole, pressure is lower in the apex than in the base of the left ventricle (LV). This early intraventricular pressure difference (IVPD) facilitates LV filling. We assessed how LV diastolic IVPD and intraventricular pressure gradient (IVPG), defined as IVPD divided by length, scale to the heart size and other physiological variables. We studied 10 mice, 10 rats, 5 rabbits, 12 dogs, and 21 humans by echocardiography. Color Doppler M-mode data were postprocessed to reconstruct IVPD and IVPG. Normalized LV filling time was calculated by dividing filling time by RR interval. The relationship between IVPD, IVPG, normalized LV filling time, and LV end-diastolic volume (or mass) as fit to the general scaling equation Y = kM beta, where M is LV heart size parameter, Y is a dependent variable, k is a constant, and beta is the power of the scaling exponent. LV mass varied from 0.049 to 194 g, whereas end-diastolic volume varied from 0.011 to 149 ml. The beta values relating normalized LV filling time with LV mass and end-diastolic volume were 0.091 (SD 0.011) and 0.083 (SD 0.009), respectively (P < 0.0001 vs. 0 for both). The beta values relating IVPD with LV mass and end-diastolic volume were similarly significant at 0.271 (SD 0.039) and 0.243 (SD 0.0361), respectively (P < 0.0001 vs. 0 for both). Finally, beta values relating IVPG with LV mass and end-diastolic volume were 0.118 (SD 0.013) and -0.104 (SD 0.011), respectively (P < 0.0001 vs. 0 for both). As a result, there was an inverse relationship between IVPG and normalized LV filling time (r = -0.65, P < 0.001). We conclude that IVPD decrease, while IVPG increase with decreasing animal size. High IVPG in small mammals may be an adaptive mechanism to short filling times. PMID- 16679404 TI - Impaired subendocardial contractile myofiber function in asymptomatic aged humans, as detected using MRI. AB - With aging, structural and functional changes occur in the myocardium without obvious impairment of systolic left ventricular (LV) function. Transmural differences in myocardial vulnerability for these changes may result in increase of transmural inhomogeneity in contractile myofiber function. Subendocardial fibrosis and impairment of subendocardial perfusion due to hypertension might change the transmural distribution of contractile myofiber function. The ratio of LV torsion to endocardial circumferential shortening (torsion-to-shortening ratio; TSR) during systole reflects the transmural distribution of contractile myofiber function. We investigated whether the transmural distribution of systolic contractile myofiber function changes with age. Magnetic resonance tissue tagging was performed to derive LV torsion and endocardial circumferential shortening. TSR was quantified in asymptomatic young [age 23.2 (SD 2.6) yr, n = 15] and aged volunteers [age 68.8 (SD 4.4) yr, n = 16]. TSR and its standard deviation were significantly elevated in the aged group [0.47 (SD 0.12) aged vs. 0.34 (SD 0.05) young; P = 0.0004]. In the aged group, blood pressure and the ratio of LV wall mass to end-diastolic volume were mildly elevated but could not be correlated to the increase in TSR. There were no significant differences in other indexes of systolic LV function such as end-systolic volume and ejection fraction. The elevated systolic TSR in the asymptomatic aged subjects suggests that aging is associated with local loss of contractile myofiber function in the subendocardium relative to the subepicardium potentially caused by subclinical pathological incidents. PMID- 16679405 TI - Oxygen consumption and metabolite concentrations during transitions between different work intensities in heart. AB - Steady-state metabolite (ADP, ATP, P(i), PCr, and NADH) concentrations usually differ little between different workloads with significantly different oxygen consumption rates in the heart. However, during transitions between steady states, metabolite concentrations may in some cases change transiently, exhibiting a significant overshoot or undershoot, whereas in other cases they approach near-exponentially new steady-state values. Oxygen consumption rate usually reaches the new steady-state value very quickly (within a few seconds). The present in silico studies, performed using a previously developed computer model of oxidative phosphorylation in the heart, demonstrate that such a behavior of the oxidative phosphorylation system can be reproduced only under the assumption that ATP usage, substrate dehydrogenation, and (particular steps of) oxidative phosphorylation are directly activated to a similar extend by some cytosolic factor/mechanism during transition from low work to high work (the so called parallel-activation mechanism). Computer simulations show that some differences observed between different experimental systems can be explained by a slightly different balance of the activation of particular components of the system and/or by a delay in time of the activation/inactivation of substrate dehydrogenation and oxidative phosphorylation during low-to-high and high-to-low work transitions. Thus the presented theoretical approach offers a general idea that is able to unify, at least semiquantitatively, different experimental data available in the literature. PMID- 16679407 TI - Functional significance of differential eNOS translocation. AB - Nitric oxide (NO) regulates flow and permeability. ACh and platelet-activating factor (PAF) lead to endothelial NO synthase (eNOS) phosphorylation and NO release. While ACh causes only vasodilation, PAF induces vasoconstriction and hyperpermeability. The key differential signaling mechanisms for discriminating between vasodilation and hyperpermeability are unknown. We tested the hypothesis that differential translocation may serve as a regulatory mechanism of eNOS to determine specific vascular responses. We used ECV-304 cells permanently transfected with eNOS-green fluorescent protein (ECVeNOS-GFP) and demonstrated that the agonists activate eNOS and reproduce their characteristic endothelial permeability effects in these cells. We evaluated eNOS localization by lipid raft analysis and immunofluorescence microscopy. After PAF and ACh, eNOS moves away from caveolae. eNOS distributes both in the plasma membrane and Golgi in control cells. ACh (10(-5) M, 10(-4) M) translocated eNOS preferentially to the trans Golgi network (TGN) and PAF (10(-7) M) preferentially to the cytosol. We suggest that PAF-induced eNOS translocation preferentially to cytosol reflects a differential signaling mechanism related to changes in permeability, whereas ACh induced eNOS translocation to the TGN is related to vasodilation. PMID- 16679408 TI - Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1. AB - Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron (Fe) release. We have shown that NO-mediated Fe efflux from cells required glutathione (GSH), and we have hypothesized that a GS-Fe-NO complex was released. Hence, we studied the role of the GSH-conjugate transporter multidrug resistance-associated protein 1 (MRP1) in NO-mediated Fe efflux. MCF7-VP cells overexpressing MRP1 exhibited a 3- to 4-fold increase in NO-mediated 59Fe and GSH efflux compared with WT cells (MCF7-WT) over 4 h. Similar results were found for other MRP1-overexpressing cell types but not those expressing another drug efflux pump, P-glycoprotein. NO-mediated 59Fe and GSH efflux were temperature- and energy-dependent and were significantly decreased by the GSH-depleting agent and MRP1 transport inhibitor L-buthionine [S,R]-sulfoximine. Other MRP1 inhibitors, MK571, probenecid, and difloxacin, significantly inhibited NO-mediated 59Fe release. EPR spectroscopy demonstrated the dinitrosyl-dithiol-Fe complex (DNIC) peak in NO-treated cells was increased by MRP1 inhibitors, indicating inhibited DNIC transport from cells. The extent of DNIC accumulation correlated with the ability of MRP1 inhibitors to prevent NO mediated 59Fe efflux. MCF7-VP cells were more sensitive than MCF7-WT cells to growth inhibition by effects of NO, which was potentiated by L-buthionine-[S,R] sulfoximine. These data indicate the importance of GSH in NO-mediated inhibition of proliferation. Collectively, NO stimulates Fe and GSH efflux from cells via MRP1. Active transport of NO by MRP1 overcomes diffusion that is inefficient and nontargeted, which has broad ramifications for understanding NO biology. PMID- 16679409 TI - Attenuated spread of X-inactivation in an X;autosome translocation. AB - X inactivation in female mammals involves transcriptional silencing of an entire chromosome in response to a cis-acting noncoding RNA, the X inactive-specific transcript (Xist). Xist can also inactivate autosomal sequences, for example, in X;autosome translocations; but here, silencing appears to be relatively inefficient. This variation has been attributed to either attenuated spreading of Xist RNA at the onset of X inactivation or inefficient maintenance of autosomal silencing. Evidence to date has favored the latter. Here, we demonstrate attenuated spreading of Xist RNA at the onset of X inactivation in the T(X;4)37H X;autosome translocation. Our findings provide direct evidence that underlying chromosome/chromatin features can disrupt spreading of the primary inactivating signal. PMID- 16679410 TI - Hydrodynamic narrowing of tubes extruded from cells. AB - We discuss the pulling force f required to extrude a lipid tube from a living cell as a function of the extrusion velocity L. The main feature is membrane friction on the cytoskeleton. As recently observed for neutrophils, the tether force exhibits a "shear thinning" response over a large range of pulling velocities, which was previously interpreted by assuming viscoelastic flows of the sliding membrane. Here, we propose an alternative explanation based on purely Newtonian flow: The diameter of the tether decreases concomitantly with the increase of the membrane tension in the lipid tube. The pulling force is found to vary as L(1/3), which is consistent with reported experimental data for various types of cells. PMID- 16679411 TI - Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. AB - Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis. PMID- 16679412 TI - Sverdlovsk revisited: modeling human inhalation anthrax. AB - Several models have been proposed for the dose-response function and the incubation period distribution for human inhalation anthrax. These models give very different predictions for the severity of a hypothetical bioterror attack, when an attack might be detected from clinical cases, the efficacy of medical intervention and the requirements for decontamination. Using data from the 1979 accidental atmospheric release of anthrax in Sverdlovsk, Russia, and limited nonhuman primate data, this paper eliminates two of the contending models and derives parameters for the other two, thereby narrowing the range of models that accurately predict the effects of human inhalation anthrax. Dose-response functions that exhibit a threshold for infectivity are contraindicated by the Sverdlovsk data. Dose-dependent incubation period distributions explain the 10 day median incubation period observed at Sverdlovsk and the 1- to 5-day incubation period observed in nonhuman primate experiments. PMID- 16679413 TI - Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert. AB - The minichromosome maintenance (MCM) proteins are essential conserved proteins required for DNA replication in archaea and eukaryotes. MCM proteins are believed to provide the replicative helicase activity that unwinds template DNA ahead of the replication fork. Consistent with this hypothesis, MCM proteins can form hexameric complexes that possess ATP-dependent DNA unwinding activity. The molecular mechanism by which the energy of ATP hydrolysis is harnessed to DNA unwinding is unknown, although the ATPase activity has been attributed to a highly conserved AAA+ family ATPase domain. Here we show that changes to N- and C terminal motifs in the single MCM protein from the archaeon Methanothermobacter thermautotrophicus (MthMCM) can modulate ATP hydrolysis, DNA binding, and duplex unwinding. Furthermore, these motifs appear to influence the movement of the beta alpha-beta insert in helix-2 of the MCM ATPase domain. Removal of this motif from MthMCM increased dsDNA-stimulated ATP hydrolysis and increased the affinity of the mutant complex for ssDNA and dsDNA. Deletion of the helix-2 insert additionally resulted in the abrogation of DNA unwinding. Our results provide significant insight into the molecular mechanisms used by the MCM helicase to both regulate and execute DNA unwinding. PMID- 16679414 TI - Branch development controls leaf area dynamics in grapevine (Vitis vinifera) growing in drying soil. AB - BACKGROUND AND AIMS: Soil water deficit is a major abiotic stress with severe consequences for the development, productivity and quality of crops. However, it is considered a positive factor in grapevine management (Vitis vinifera), as it has been shown to increase grape quality. The effects of soil water deficit on organogenesis, morphogenesis and gas exchange in the shoot were investigated. METHODS: Shoot organogenesis was analysed by distinguishing between the various steps in the development of the main axis and branches. Several experiments were carried out in pots, placed in a greenhouse or outside, in southern France. Soil water deficits of various intensities were imposed during vegetative development of the shoots of two cultivars ('Syrah' and 'Grenache N'). KEY RESULTS: All developmental processes were inhibited by soil water deficit, in an intensity dependent manner, and sensitivity to water stress was process-dependent. Quantitative relationships with soil water were established for all processes. No difference was observed between the two cultivars for any criterion. The number of leaves on branches was particularly sensitive to soil water deficit, which rapidly and strongly reduced the rate of leaf appearance on developing branches. This response was not related to carbon availability, photosynthetic activity or the soluble sugar content of young expanding leaves. The potential number of branches was not a limiting factor for shoot development. CONCLUSIONS: The particularly high sensitivity to soil water deficit of leaf appearance on branches indicates that this process is a major determinant of the adaptation of plant leaf area to soil water deficit. The origin of this particular developmental response to soil water deficit is unclear, but it seems to be related to constitutive characteristics of branches rather than to competition for assimilates between axes differing in sink strength. PMID- 16679415 TI - Differential roles of Arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots. AB - Signaling through heterotrimeric G proteins is conserved in diverse eukaryotes. Compared to vertebrates, the simpler repertoire of G-protein complex and accessory components in Arabidopsis (Arabidopsis thaliana) offers a unique advantage over all other multicellular, genetic-model systems for dissecting the mechanism of G-protein signal transduction. One of several biological processes that the G-protein complex regulates in Arabidopsis is cell division. We determined cell production rate in the primary root and the formation of lateral roots in Arabidopsis to define individually the types of modulatory roles of the respective G-protein alpha- and beta-subunits, as well as the heterotrimer in cell division. The growth rate of the root is in part a consequence of cell cycle maintenance in the root apical meristem (RAM), while lateral root production requires meristem formation by founder pericycle cells. Thus, a comparison of these two parameters in various genetic backgrounds enabled dissection of the role of the G-protein subunits in modulation of cell division, both in maintenance and initiation. Cell production rates were determined for the RAM and lateral root formation in gpa1 (Arabidopsis G-protein alpha-subunit) and agb1 (Arabidopsis G-protein beta-subunit) single and double mutants, and in transgenic lines overexpressing GPA1 or AGB1 in agb1 or gpa1 mutant backgrounds, respectively. We found in the RAM that the heterotrimeric complex acts as an attenuator of cell proliferation, whereas the GTP-bound form of the Galpha subunit's role is a positive modulator. In contrast, for the formation of lateral roots, the Gbetagamma-dimer acts largely independently of the Galpha-subunit to attenuate cell division. These results suggest that Arabidopsis heterotrimeric G protein subunits have differential and opposing roles in the modulation of cell division in roots. PMID- 16679416 TI - The evolutionarily conserved tetratrico peptide repeat protein pale yellow green7 is required for photosystem I accumulation in Arabidopsis and copurifies with the complex. AB - Pale yellow green7-1 (pyg7-1) is a photosystem I (PSI)-deficient Arabidopsis (Arabidopsis thaliana) mutant. PSI subunits are synthesized in the mutant, but do not assemble into a stable complex. In contrast, light-harvesting antenna proteins of both photosystems accumulate in the mutant. Deletion of Pyg7 results in severely reduced growth rates, alterations in leaf coloration, and plastid ultrastructure. Pyg7 was isolated by map-based cloning and encodes a tetratrico peptide repeat protein with homology to Ycf37 from Synechocystis. The protein is localized in the chloroplast associated with thylakoid membranes and copurifies with PSI. An independent pyg7 T-DNA insertion line, pyg7-2, exhibits the same phenotype. pyg7 gene expression is light regulated. Comparison of the roles of Ycf37 in cyanobacteria and Pyg7 in higher plants suggests that the ancient protein has altered its function during evolution. Whereas the cyanobacterial protein mediates more efficient PSI accumulation, the higher plant protein is absolutely required for complex assembly or maintenance. PMID- 16679417 TI - pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene. AB - We recently demonstrated that microRNA399 (miR399) controls inorganic phosphate (Pi) homeostasis by regulating the expression of UBC24 encoding a ubiquitin conjugating E2 enzyme in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing miR399 accumulated excessive Pi in the shoots and displayed Pi toxic symptoms. In this study, we revealed that a previously identified Pi overaccumulator, pho2, is caused by a single nucleotide mutation resulting in early termination within the UBC24 gene. The level of full-length UBC24 mRNA was reduced and no UBC24 protein was detected in the pho2 mutant, whereas up regulation of miR399 by Pi deficiency was not affected. Several characteristics of Pi toxicity in the pho2 mutant were similar to those in the miR399 overexpressing and UBC24 T-DNA knockout plants: both Pi uptake and translocation of Pi from roots to shoots increased and Pi remobilization within leaves was impaired. These phenotypes of the pho2 mutation could be rescued by introduction of a wild-type copy of UBC24. Kinetic analyses revealed that greater Pi uptake in the pho2 and miR399-overexpressing plants is due to increased Vmax. The transcript level of most PHT1 Pi transporter genes was not significantly altered, except PHT1;8 whose expression was enhanced in Pi-sufficient roots of pho2 and miR399-overexpressing compared with wild-type plants. In addition, changes in the expression of several organelle-specific Pi transporters were noticed, which may be associated with the redistribution of intracellular Pi under excess Pi. Furthermore, miR399 and UBC24 were colocalized in the vascular cylinder. This observation not only provides important insight into the interaction between miR399 and UBC24 mRNA, but also supports their systemic function in Pi translocation and remobilization. PMID- 16679418 TI - Molecular cloning and characterization of beta-expansin gene related to root hair formation in barley. AB - Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the beta-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley. PMID- 16679419 TI - DAWDLE, a forkhead-associated domain gene, regulates multiple aspects of plant development. AB - Phosphoprotein-binding domains are found in many different proteins and specify protein-protein interactions critical for signal transduction pathways. Forkhead associated (FHA) domains bind phosphothreonine and control many aspects of cell proliferation in yeast (Saccharomyces cerevisiae) and animal cells. The Arabidopsis (Arabidopsis thaliana) protein kinase-associated protein phosphatase includes a FHA domain that mediates interactions with receptor-like kinases, which in turn regulate a variety of signaling pathways involved in plant growth and pathogen responses. Screens for insertional mutations in other Arabidopsis FHA domain-containing genes identified a mutant with pleiotropic defects. dawdle (ddl) plants are developmentally delayed, produce defective roots, shoots, and flowers, and have reduced seed set. DDL is expressed in the root and shoot meristems and the reduced size of the root apical meristem in ddl plants suggests a role early in organ development. PMID- 16679420 TI - Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. AB - The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor. PMID- 16679421 TI - A novel lipoxygenase in pea roots. Its function in wounding and biotic stress. AB - The genome of pea (Pisum sativum) contains genes encoding a family of distinct lipoxygenases (LOX). Among these, LOXN2 showed eight exons encoding a 93.7-kD enzyme, harboring two C-terminal deletions and an unusual arginine/threonine tyrosine motif in the domain considered to control the substrate specificity. LOXN2, when overexpressed in yeast, exhibited normal enzyme activity with an optimum at pH 4.5, and a dual positional specificity by releasing a 3:1 ratio of C-9 and C-13 oxidized products. The predicted LOXN2 structure lacked a loop present in soybean (Glycine max) LOX1, in a position consistent with control of the degree of substrate access to the catalytic site and for LOXN2's dual positional specificity. The LOXN2 gene was tightly conserved in the Progress 9 and MG103738 genotypes, respectively, susceptible and resistant to the root cyst nematode Heterodera goettingiana. LOXN2 transcription was monitored in roots after mechanical injury and during nematode infection. The message peaked at 3 and 24 h after wounding in both genotypes and was more abundant in the resistant than in the susceptible pea. In nematode-infected roots, transcription of several LOX genes was triggered except LOXN2, which was repressed in both genotypes. In situ hybridization revealed that LOXN2 message was widespread in the cortex and endodermis of healthy roots, but specifically localized at high level in the cells bordering the nematode-induced syncytia of infected roots. However, LOXN2 transcript signal was particularly intense in collapsing syncytia of MG103738 roots, suggesting LOXN2 involvement in late mechanisms of host resistance. PMID- 16679422 TI - Defects in the cytochrome b6/f complex prevent light-induced expression of nuclear genes involved in chlorophyll biosynthesis. AB - Mutants with defects in the cytochrome (cyt) b6/f complex were analyzed for their effect on the expression of a subgroup of nuclear genes encoding plastid localized enzymes participating in chlorophyll biosynthesis. Their defects ranged from complete loss of the cytb6/f complex to point mutations affecting specifically the quinone-binding QO site. In these seven mutants, light induction of the tetrapyrrole biosynthetic genes was either abolished or strongly reduced. In contrast, a normal induction of chlorophyll biosynthesis genes was observed in mutants with defects in photosystem II, photosystem I, or plastocyanin, or in wild-type cells treated with 3-(3'4'-dichlorophenyl)-1,1-dimethylurea or 2,5 dibromo-3-methyl-6-isopropyl benzoquinone. We conclude that the redox state of the plastoquinone pool does not control light induction of these chlorophyll biosynthetic genes. The signal that affects expression of the nuclear genes appears to solely depend on the integrity of the cytb6/f complex QO site. Since light induction of these genes in Chlamydomonas has recently been shown to involve the blue light receptor phototropin, the results suggest that cytb6/f activity regulates a plastid-derived factor required for their expression. This signaling pathway differs from that which regulates state transitions, since mutant stt7, lacking a protein kinase involved in phosphorylation of the light harvesting complex II, was not altered in the expression of the chlorophyll biosynthetic genes. PMID- 16679423 TI - Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C4-specific phosphoenolpyruvate carboxylase promoter in maize. AB - Expression of the C4-specific phosphoenolpyruvate carboxylase (C4-PEPC) gene in maize (Zea mays) is regulated in a tissue-specific manner, but affected by light and nutrient availability. We manipulated these stimuli in a combinatorial manner and analyzed concomitant changes in histone acetylation of the nucleosomes associated with the C4-PEPC gene in relation to transcriptional activity and steady-state mRNA levels. Whereas the transition from the lowest activity to an intermediate activity was observed in the absence of histone acetylation, the light-induced boost to full activity was associated with strong enhancement of the acetylation of both histones H3 and H4 limited to the gene region. Once activated by light, prolonged darkness was necessary to reduce both transcription and, in parallel, histone acetylation. Unexpectedly, histone acetylation was also induced in bundle sheath cells, although the transcriptional activity did not respond to illumination in this tissue. Furthermore, we were able to down regulate the promoter by nitrogen depletion in the light without any decrease in the hyperacetylation of histone H4. When plants kept in prolonged darkness were nitrogen depleted and then exposed to light, transcription was not induced, but the promoter chromatin became hyperacetylated. We suggest a model where inhibition of a histone deacetylase in the light triggers H4 hyperacetylation at the C4-PEPC gene promoter regardless of the transcriptional activity of the gene. Our data indicate that an understanding of the interplay between histone modification and transcription requires analysis of signal integration on promoters in vivo. PMID- 16679425 TI - Considerations in the long-term management of asthma in ambulatory patients. AB - PURPOSE: The goals of treatment and drug therapies used for long-term asthma control, classification of the disease by severity, and treatment based on severity are reviewed, with an emphasis on recent controversies in treatment approach and safety concerns. SUMMARY: Patient education and written asthma self management and action plans are essential components of asthma treatment because of the need for patients to acquire substantial knowledge and skills in self care. Inhaled corticosteroids are the most effective long-term-control therapy and usually suffice as monotherapy for mild persistent asthma. Adding a long acting, inhaled beta2 agonist to the inhaled corticosteroid is preferred for moderate and severe persistent disease despite safety concerns. Omalizumab use is limited to selected patients with moderate-to-severe allergic asthma and an inadequate response to inhaled corticosteroids. CONCLUSION: The long-term control of asthma requires substantial patient knowledge and skill. Persistent disease is best managed by inhaled corticosteroids and if it is moderate or severe, long acting, inhaled beta2 agonists in combination with inhaled corticosteroids. PMID- 16679426 TI - Case studies illustrating the implementation of treatment strategies for acute and chronic asthma. AB - PURPOSE: Considerations that enter into decisions about treatment strategies for acute and chronic asthma are illustrated in two case studies. SUMMARY: A patient's signs, symptoms, vital signs, laboratory test results, recent history of illness or exposure to substances that can provoke asthma symptoms, and response to recently used asthma medications as well as efficacy, safety, and cost are important considerations in choosing drug therapy in the emergency department to treat an acute exacerbation. The need for a rapid onset of action and patient limitations may influence the choice of a dosage form, delivery device, and route of administration. Treatment strategies for patients with chronic asthma may require resolution of problems with adherence to the treatment plan or inhaler technique. Long-term control of asthma symptoms involves the use of preventive measures, including long-term-control medications, efforts to control asthma triggers, and self-monitoring of pulmonary function. Patient education and a written action plan are important elements of the treatment strategy. CONCLUSION: An individualized approach to treating patients with acute or chronic asthma that takes into consideration patient-specific factors as well as efficacy, safety, and cost of drug therapy is needed when devising and implementing a treatment strategy to optimize patient outcomes. PMID- 16679424 TI - PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. AB - Inorganic phosphate (Pi)-signaling pathways in plants are still largely unknown. The Arabidopsis (Arabidopsis thaliana) pho2 mutant overaccumulates Pi in leaves in Pi-replete conditions. Micrografting revealed that a pho2 root genotype is sufficient to yield leaf Pi accumulation. In pho2 mutants, Pi does not repress a set of Pi starvation-induced genes, including AtIPS1, AT4, and Pi transporters Pht1;8 and Pht1;9. Map-based cloning identified PHO2 as At2g33770, an unusual E2 conjugase gene. It was recently shown that Pi deprivation induces mature microRNA (miRNA [miR399]) and that overexpression of miR399 in Pi-replete conditions represses E2 conjugase expression and leads to high leaf Pi concentrations, thus phenocopying pho2. We show here that miR399 primary transcripts are also strongly induced by low Pi and rapidly repressed after addition of Pi. PHO2 transcripts change reciprocally to miR399 transcripts in Pi-deprived plants and in miR399 overexpressers. However, responses after Pi readdition and in beta-glucuronidase reporter lines suggest that PHO2 expression is also regulated by Pi in a manner unrelated to miR399-mediated transcript cleavage. Expression of miR399 was strongly reduced in Pi-deprived Arabidopsis phr1 mutants, and a subset of Pi responsive genes repressed in Pi-deprived phr1 mutants was up-regulated in Pi replete pho2 mutants. This places miR399 and PHO2 in a branch of the Pi-signaling network downstream of PHR1. Finally, putative PHO2 orthologs containing five miR399-binding sites in their 5'-untranslated regions were identified in other higher plants, and Pi-dependent miR399 expression was demonstrated in rice (Oryza sativa), suggesting a conserved regulatory mechanism. PMID- 16679428 TI - Introduction: Asthma epidemiology and economic impact. PMID- 16679429 TI - Asthma pathophysiology and evidence-based treatment of severe exacerbations. AB - PURPOSE: The pathogenesis of asthma and the treatment approach for acute exacerbations are described. The pharmacology, efficacy, safety, and cost of the beta2 agonist, albuterol, a racemic mixture of equal amounts of R- and S enantiomers, and levalbuterol, the R-enantiomer, are compared. SUMMARY: Asthma symptoms are the result of bronchial hyperresponsiveness, bronchospasm, and chronic airway inflammation. Short-acting, inhaled beta2 agonists; oxygen; intravenous fluids; and corticosteroids are the mainstays of treatment for acute exacerbations. The R-enantiomer of albuterol is responsible for bronchodilation. The S-enantiomer exhibits broncho-constricting activity in vitro, which may be mediated by muscarinic receptors and may be opposed by adding the anticholinergic agent ipratropium bromide. Levalbuterol improves pulmonary function to a greater extent than racemic albuterol and reduces the need for costly hospitalizations in patients with acute asthma exacerbations. CONCLUSION: Levalbuterol is an alternative to racemic albuterol with the potential to improve patient outcomes and reduce costs in the treatment of acute asthma exacerbations. PMID- 16679430 TI - Anti-signal recognition particle autoantibodies: marker of a necrotising myopathy. AB - OBJECTIVE: To elucidate the clinical importance of the anti-signal recognition particle (SRP) autoantibody in patients with myositis. METHODS: Retrospective systematic assessment of the clinical, laboratory and histological characteristics of 23 anti-SRP-positive patients from six European centres. Data were compared with a large group of anti-SRP-negative patients with myositis published previously. RESULTS: Clinically, patients with anti-SRP autoantibodies often had a severe symmetric proximal muscle weakness resulting in marked disability, dysphagia and highly elevated levels of serum creatine kinase. Three patients had typical dermatomyositis rashes. The disease was associated with the occurrence of extramuscular signs and symptoms including interstitial lung disease. No association was found with an increased risk of cardiac involvement, and the disease carried a reasonably favourable prognosis with most patients responding to treatment. None of the patients had the typical histological features of myositis. Most muscle biopsy specimens showed the presence of necrotic muscle fibres and no inflammatory infiltrates. CONCLUSIONS: Anti-SRP autoantibodies are associated with a syndrome of a necrotising myopathy in the spectrum of immune-mediated myopathies that differs from typical polymyositis. Further studies are needed to elucidate the pathogenesis and to clarify the role of the anti-SRP autoantibodies in this unique disease. PMID- 16679431 TI - Expression of the peptide C4b-binding protein beta in the arthritic joint. AB - BACKGROUND: C4b-binding protein (C4BP) is a plasma oligomeric glycoprotein that participates in the regulation of complement and haemostasis. Complement regulatory activity depends on the C4BPalpha-polypeptide, whereas the C4BPbeta polypeptide inactivates protein S, interfering with the anti-coagulatory protein C-dependent pathway. OBJECTIVE: To investigate the expression of C4BPbeta in the rheumatoid joint. METHODS: Expression of C4BP was studied in synovial explants from patients with rheumatoid arthritis, osteoarthritis and healthy controls, using immunohistochemistry and in situ hybridisation. C4BP isoforms and free C4BPbeta were studied in synovial effusions from patients with rheumatoid arthritis, osteoarthritis and microcrystalline arthritis (MCA) by immunoblotting; total and free protein S levels were studied by enzyme immunoassay. RESULTS: C4BPbeta was overexpressed in the synovial membranes of patients with rheumatoid arthritis, in close association with the severity of synovitis and the extension of interstitial fibrin deposits. As many as 85% fluids from patients with rheumatoid arthritis contained free C4BPbeta, whereas this unusual polypeptide was present in 50% fluids from patients with MCA and 40% fluids from patients with osteoarthritis. Free protein S at the effusions was pathologically reduced in patients with rheumatoid arthritis and MCA, and remained normal in patients with osteoarthritis. CONCLUSION: C4BPbeta is expressed by the inflamed synovial tissue, where it can participate in processes of tissue remodelling associated with invasive growth. PMID- 16679432 TI - Adalimumab and methotrexate is more effective than adalimumab alone in patients with established rheumatoid arthritis: results from a 6-month longitudinal, observational, multicentre study. AB - OBJECTIVES: To compare the effectiveness of adalimumab monotherapy and adalimumab and methotrexate (MTX) combination therapy in patients with established rheumatoid arthritis. METHODS: Data from an ongoing longitudinal observational study in Norway were used to compare response to treatment with two different adalimumab regimens (monotherapy, n = 84; combination with MTX, n = 99). Patients were assessed with measures of disease activity, health status and utility scores. Within-group changes were analysed from baseline to follow-up at 3 and 6 months and the changes were compared between groups after adjustment for the propensity score. The groups were also compared for the proportions of patients achieving European League Against Rheumatism (EULAR) good response, Disease Activity Score (DAS)28 remission and treatment terminations. RESULTS: The improvement from baseline was significant for all measures in the adalimumab and MTX group, but only for DAS28, joint counts, two Short-form Health Survey with 36 questions (SF-36) dimensions and patient's and investigator's global assessment in the monotherapy group. All between-group differences were numerically in favour of combination therapy and significant for C reactive protein, joint counts, DAS28, Modified Health Assessment Questionnaire, investigator's global assessment, four SF-36 dimensions and Short Form 6D at 6 months. More patients in the combination therapy group reached EULAR good response (p<0.001) and remission (p = 0.07). At 6 months, 80.8% of the patients in the combination therapy group and 59.5% in the monotherapy group remained on treatment (p = 0.002). More withdrawals in the monotherapy group were due to adverse events. CONCLUSIONS: Our results were consistent across several categories of end points and suggest that adalimumab combined with MTX is effective in patients with rheumatoid arthritis treated in daily clinical practice and is superior to adalimumab monotherapy. PMID- 16679435 TI - Potentially inappropriate prescribing for elderly patients in 2 outpatient settings. AB - Research has shown a high prevalence of potentially inappropriate medication prescribing (PIP) for elderly patients in outpatient settings, but little is known about whether a physician's practice setting influences prescribing attitudes. This study examines the prevalence of PIP among elderly patients in 2 out-patient practices, 1 located in a senior citizens center and 1 in a general family medicine clinic. The authors conducted a retrospective chart review of a random sample of 50 individuals aged 65 years or older from each practice. The 2003 version of the Beers criteria was used to identify PIP. Results show that some one fourth of the elderly sampled in both practices had 1 or more incidents of PIP. The most common potentially inappropriate drug classes prescribed were psychotropic agents and anti-inflammatory drugs. Demographic patient variables were not significantly associated with PIP. This study suggests that PIP may be prevalent across physician groups. PMID- 16679433 TI - Joint surgery in the Utrecht Rheumatoid Arthritis Cohort: the effect of treatment strategy. AB - OBJECTIVE: To investigate the prevalence and prognostic factors of joint surgery in a large cohort of patients with rheumatoid arthritis, whose treatment, clinical and radiographic data have been assessed at predefined points in time since disease onset. METHODS: Data on surgical interventions were retrospectively obtained from 482 patients with rheumatoid arthritis whose follow-up data for at least 2 years were available, including treatment and response to treatment during the first 2 years. Survival time until the first surgical intervention and until the first major surgical intervention was determined for the total study population by Kaplan-Meier survival curves. Three separate Cox regression analyses were carried out to determine which variables measured at baseline, during the first year and during the first 2 years were predictors for joint surgery. RESULTS: 27% of the patients underwent surgical interventions. Mean survival time until the first surgical intervention was 10.4 years. The percentage of patients with a surgical intervention was 10% lower in the group with response to treatment when compared with the non-response group. Next to a delayed start with disease-modifying antirheumatic drugs, fast radiographic progression during the first year and first 2 years was a predictor of joint surgery in the multivariate regression analyses. CONCLUSION: Treatment with disease-modifying antirheumatic drugs immediately after diagnosis results in less joint surgery when compared with a delayed start. Furthermore, joint surgery is carried out more often in patients who do not respond to treatment. PMID- 16679436 TI - A systems-based intervention to promote smoking as a vital sign in patients served by community health centers. AB - The frequency of asking and advising adult patients about tobacco use was measured after an intervention to adopt smoking as a vital sign at 7 community health centers. The intervention consisted of training staff, revising forms and vital sign stamps, and disseminating educational materials. Documentation in medical charts was reviewed for 1,571 randomly sampled patients in 2002 and 2003. The point prevalence (last encounter) and period prevalence (any annual encounter) of asking patients about smoking increased significantly from 2002 to 2003 (59% to 85%, and 71% to 97%, respectively) overall and at each health center. On advising smokers to quit, 4 health centers improved, but the overall point prevalence, 26%, and period prevalence, 46%, were unchanged over time. An intervention using multiple strategies may have contributed to improving the rates of asking but did not have as large or consistent an impact on rates of advising smokers to quit. PMID- 16679437 TI - Follow-up of outpatient test results: a survey of house-staff practices and perceptions. AB - Failure to follow up outpatient test results is a potential patient safety concern; however, data about how house-staff physicians follow up on tests are sparse. The authors sought to assess internal medicine house-staff practices and perceptions regarding the follow-up of outpatient tests and identified barriers to timely follow-up. Seventy-five of 111 eligible house staff at a large urban teaching hospital (68%) completed the survey. Seventy-four percent reported they were sometimes unable to follow up on test results, 78% were at least somewhat worried about inadequate follow-up, and 46% stated that they have seen a patient's medical condition worsen due to a delay in test result follow-up at least a few times a year. Barriers to timely follow-up included lack of a reminder system (40%), difficulty accessing results (24%), too many competing demands on time (27%), and uncertainty about who should follow up on results (16%). PMID- 16679438 TI - Quality measurement in medicaid managed care and fee-for-service: the New York State experience. AB - New York State has transitioned 1.7 million Medicaid recipients from a fee-for service delivery system to a managed care model. To evaluate whether managed care has had a positive effect on access and quality, the New York State Department of Health compared rates of performance across standardized measures of quality (ie, childhood immunization, well-child visits, prenatal care in the first trimester, cervical cancer screening, use of appropriate medications for people with asthma, and comprehensive diabetes care) in both systems. For almost all measures, Medicaid managed care rates were statistically higher than Medicaid fee-for service. PMID- 16679439 TI - Physician reactions to quantitative individual performance reports. AB - The purpose of this study was to learn how primary care physicians experienced the introduction and evolution of an individual physician pay-for-performance program. Thirty primary care physicians participated in audiotaped focus groups 13 and 26 months after beginning the program. Transcribed audiotapes were used to group comments into themes. Ten thematic groups were identified. Practitioners reviewed their profiles but found it difficult to use them to change behaviors. They were concerned about the data accuracy, the influence of specialists and patients on their "scores," and, less, the validity of quality measures. They described ways the program changed their practices and consideration of cost, quality, and satisfaction. There were important concerns about the influence of pay-for-performance programs on professionalism. Primary care physicians were skeptical of this pay-for-performance program. On the other hand, physicians described positive influences on making improvements in quality, satisfaction, and practice efficiency. PMID- 16679440 TI - Global satisfaction with perinatal hospital care: stability and relationship to anxiety, depression, and stressful medical events. AB - To evaluate the stability of global maternal satisfaction with perinatal hospital care during the post-partum period and its relationship to anxiety, depression, and stressful medical events, a cohort study of 300 mothers delivering at a university hospital was performed during the first month postpartum. Satisfaction, measured on a 4-point Likert scale (0-3), declined from 2.75 +/- 0.03 (mean +/- standard error) before hospital discharge to 2.48 +/- 0.04 at 1 month postpartum (P = .000), and only 69.5% of mothers very much satisfied predischarge remained so at 1 month (P = .000). Predischarge satisfaction declined with greater medical events (adjusted odds ratio [AOR] = 0.85, 95% confidence interval [CI] = 0.74, 0.97, P < .05) and with concomitant anxiety (AOR= 0.92, CI= 0.89, 0.95, P< .001); reduced satisfaction at 1 month was associated with high anxiety (AOR = 0.97, CI = 0.95, 0.98) and depression (AOR = 0.96, CI = 0.93, 0.99) at that time. Thus, perinatal satisfaction may be time dependent and associated with contemporaneous medical and psychological changes. PMID- 16679441 TI - The Agency for Healthcare Research and Quality's National Healthcare Quality and Disparities Reports: action agendas for the nation. PMID- 16679442 TI - The promising future of proteomics in sarcoidosis. PMID- 16679443 TI - Who are the children with asthma most susceptible to air pollution? PMID- 16679444 TI - Update in chronic obstructive pulmonary disease 2005. PMID- 16679445 TI - Update in diffuse parenchymal lung diseases 2005. PMID- 16679446 TI - Adrenal insufficiency and ventilator weaning: additional study is required. PMID- 16679447 TI - Endotoxin and asthma. PMID- 16679448 TI - Rapid quantification of DNA methylation through dNMP analysis following bisulfite PCR. AB - We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite mediated sequencing) as a validation, deviations were within +/-5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h. PMID- 16679449 TI - Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. AB - Fapy.dG and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5'-TGT and 5'-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy.dG is mutagenic inducing primarily targeted Fapy.G-->T transversions. In the 5'-TGT sequence mutational frequency of Fapy.dG was approximately 30%, whereas in the 5'-TGA sequence it was approximately 8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy.dG, though it also exhibited a similar context effect: 4-fold G-->T transversions (24% versus 6%) occurred in the 5'-TGT sequence relative to 5'-TGA. To investigate a possible structural basis for the higher G-->T mutations induced by both lesions when their 3' neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase beta, which is known to incorporate both dCTP (no mutation) and dATP (G-->T substitution) opposite 8-oxo G. In pol beta, the syn-8-oxo-G:dATP pair showed greater stacking with the 3'-T:A base pair in the 5'-TGT sequence compared with the 3'-A:T in the 5'-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5'-TGT and 5' TGA sequences. Similarly, syn-Fapy.G:dATP pairing showed greater stacking in the 5'-TGT sequence compared with the 5'-TGA sequence, while stacking for anti Fapy.G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3'-A:T base pair in the 5'-TGA sequence might cause lower G-->T mutational frequencies in the 5'-TGA sequence compared to 5'-TGT. The corresponding lesions derived from 2' deoxyadenosine, Fapy.dA and 8-oxo-dA, were not detectably mutagenic in the 5'-TAT sequence, and were only weakly mutagenic (<1%) in the 5'-TAA sequence context, where both lesions induced targeted A-->C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy.dG or Fapy.dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy.G-->T substitutions occur at a higher frequency than 8-oxo-G- >T and that Fapy.dA is very weakly mutagenic, as is 8-oxo-dA. PMID- 16679450 TI - Involvement of DNA curvature in intergenic regions of prokaryotes. AB - It is known that DNA curvature plays a certain role in gene regulation. The distribution of curved DNA in promoter regions is evolutionarily preserved, and it is mainly determined by temperature of habitat. However, very little is known on the distribution of DNA curvature in termination sites. Our main objective was to comprehensively analyze distribution of curved sequences upstream and downstream to the coding genes in prokaryotic genomes. We applied CURVATURE software to 170 complete prokaryotic genomes in a search for possible typical distribution of DNA curvature around starts and ends of genes. Performing cluster analyses and other statistical tests, we obtained novel results regarding various factors influencing curvature distribution in intergenic regions, such as growth temperature, A+T composition and genome size. We also analyzed intergenic regions between converging genes in 15 selected genomes. The results show that six genomes presented peaks of curvature excess larger than 3 SDs. Insufficient statistics did not allow us to draw further conclusion. Our hypothesis is that DNA curvature could affect transcription termination in many prokaryotes either directly, through contacts with RNA polymerase, or indirectly, via contacts with some regulatory proteins. PMID- 16679451 TI - Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell. AB - The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility. PMID- 16679452 TI - Automated extraction and classification of RNA tertiary structure cyclic motifs. AB - A minimum cycle basis of the tertiary structure of a large ribosomal subunit (LSU) X-ray crystal structure was analyzed. Most cycles are small, as they are composed of 3- to 5 nt, and repeated across the LSU tertiary structure. We used hierarchical clustering to quantify and classify the 4 nt cycles. One class is defined by the GNRA tetraloop motif. The inspection of the GNRA class revealed peculiar instances in sequence. First is the presence of UA, CA, UC and CC base pairs that substitute the usual sheared GA base pair. Second is the revelation of GNR(X(n))A tetraloops, where X(n) is bulged out of the classical GNRA structure, and of GN/RA formed by the two strands of interior-loops. We were able to unambiguously characterize the cycle classes using base stacking and base pairing annotations. The cycles identified correspond to small and cyclic motifs that compose most of the LSU RNA tertiary structure and contribute to its thermodynamic stability. Consequently, the RNA minimum cycles could well be used as the basic elements of RNA tertiary structure prediction methods. PMID- 16679453 TI - A novel DNA damage recognition protein in Schizosaccharomyces pombe. AB - Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6 alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N methyl-N-nitrosourea, N-methyl-N'nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6 methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6 methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes. PMID- 16679454 TI - The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD). AB - Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets. PMID- 16679455 TI - Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis. AB - Plants may shed organs when they have been injured or served their purpose. The differential pattern of organ abscission in different species is most likely the result of evolutionary adaptation to a variety of life styles and environments. The final step of abscission-related cell separation in floral organs of wild type Arabidopsis thaliana, which only abscises sepals, petals, and stamens, is controlled by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA). Here, we demonstrate that Arabidopsis 35S:IDA lines constitutively overexpressing IDA exhibit earlier abscission of floral organs, showing that the abscission zones are responsive to IDA soon after the opening of the flowers. In addition, ectopic abscission was observed at the bases of the pedicel, branches of the inflorescence, and cauline leaves. The silique valves also dehisced prematurely. Scanning electron microscopy indicated a spread of middle lamella degradation from preformed abscission zone cells to neighboring cells. A transcript encoding an arabinogalactan protein (AGP) was upregulated in the 35S:IDA lines, and large amounts of AGP were secreted at the sites of abscission. AGP was shown to be a constituent of wild-type floral abscission zones during and soon after cell separation had been completed. We suggest that the restricted expression pattern of IDA precludes abscission of nonfloral organs in Arabidopsis. PMID- 16679456 TI - AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis. AB - Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30 degrees C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS. PMID- 16679457 TI - The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation. AB - Boron (B) is essential in plants but often present at low concentrations in the environment. To investigate how plants survive under conditions of B limitation, we conducted a transcriptome analysis and identified NIP5;1, a member of the major intrinsic protein family, as a gene upregulated in B-deficient roots of Arabidopsis thaliana. Promoter-beta-glucuronidase fusions indicated that NIP5;1 is strongly upregulated in the root elongation zone and the root hair zone under B limitation, and green fluorescent protein-tagged NIP5;1 proteins localized to the plasma membrane. Expression in Xenopus laevis oocytes demonstrated that NIP5;1 facilitated the transport of boric acid in addition to water. Importantly, two T-DNA insertion lines of NIP5;1 displayed lower boric acid uptake into roots, lower biomass production, and increased sensitivity of root and shoot development to B deficiency. These results identify NIP5;1 as a major plasma membrane boric acid channel crucial for the B uptake required for plant growth and development under B limitation. PMID- 16679458 TI - GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis. AB - As a plant shoot matures, it transitions through a series of growth phases in which successive aerial organs undergo distinct developmental changes. This process of phase change is known to be influenced by gibberellins (GAs). We report the identification of a putative transcription factor, GLABROUS INFLORESCENCE STEMS (GIS), which regulates aspects of shoot maturation in Arabidopsis thaliana. GIS loss-of-function mutations affect the epidermal differentiation of inflorescence organs, causing a premature decrease in trichome production on successive leaves, stem internodes, and branches. Overexpression has the opposite effect on trichome initiation and causes other heterochronic phenotypes, affecting flowering and juvenile-adult leaf transition and inducing the formation of rosette leaves on inflorescence stems. Genetic and gene expression analyses suggest that GIS acts in a GA-responsive pathway upstream of the trichome initiation regulator GLABROUS1 (GL1) and downstream of the GA signaling repressor SPINDLY (SPY). GIS mediates the induction of GL1 expression by GA in inflorescence organs and is antagonized in its action by the DELLA repressor GAI. The implication of GIS in the broader regulation of phase change is further suggested by the delay in flowering caused by GIS loss of function in the spy background. The discovery of GIS reveals a novel mechanism in the control of shoot maturation, through which GAs regulate cellular differentiation in plants. PMID- 16679459 TI - The gene controlling the quantitative trait locus EPITHIOSPECIFIER MODIFIER1 alters glucosinolate hydrolysis and insect resistance in Arabidopsis. AB - Glucosinolates are sulfur-rich plant secondary metabolites whose breakdown products have a wide range of biological activities in plant-herbivore and plant pathogen interactions and anticarcinogenic properties. In Arabidopsis thaliana, hydrolysis by the enzyme, myrosinase, produces bioactive nitriles, epithionitriles, or isothiocyanates depending upon the plant's genotype and the glucosinolate's structure. A major determinant of this structural specificity is the epithiospecifier locus (ESP), whose protein causes the formation of epithionitriles and nitriles. A quantitative trait locus (QTL) on chromosome 3 epistatically affects nitrile formation in combination with ESP; this QTL has been termed EPITHIOSPECIFIER MODIFIER1 (ESM1). We identified a myrosinase associated protein as the ESM1 QTL in Arabidopsis using map-based cloning with recombinant inbred lines, natural variation transcriptomic analysis, and metabolic profiling. In planta and in vitro analyses with natural ESM1 alleles, ESM1 knockouts, and overexpression lines show that ESM1 represses nitrile formation and favors isothiocyanate production. The glucosinolate hydrolysis profile change influenced by ESM1 is associated with the ability to deter herbivory by Trichoplusia ni. This gene could provide unique approaches toward improving human nutrition. PMID- 16679460 TI - One of two alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas. AB - Proteins of the YidC/Oxa1p/ALB3 family play an important role in inserting proteins into membranes of mitochondria, bacteria, and chloroplasts. In Chlamydomonas reinhardtii, one member of this family, Albino3.1 (Alb3.1), was previously shown to be mainly involved in the assembly of the light-harvesting complex. Here, we show that a second member, Alb3.2, is located in the thylakoid membrane, where it is associated with large molecular weight complexes. Coimmunoprecipitation experiments indicate that Alb3.2 interacts with Alb3.1 and the reaction center polypeptides of photosystem I and II as well as with VIPP1, which is involved in thylakoid formation. Moreover, depletion of Alb3.2 by RNA interference to 25 to 40% of wild-type levels leads to a reduction in photosystems I and II, indicating that the level of Alb3.2 is limiting for the assembly and/or maintenance of these complexes in the thylakoid membrane. Although the levels of several photosynthetic proteins are reduced under these conditions, other proteins are overproduced, such as VIPP1 and the chloroplast chaperone pair Hsp70/Cdj2. These changes are accompanied by a large increase in vacuolar size and, after a prolonged period, by cell death. We conclude that Alb3.2 is required directly or indirectly, through its impact on thylakoid protein biogenesis, for cell survival. PMID- 16679462 TI - A 2 year old with a rash. PMID- 16679463 TI - Unsteady gait. PMID- 16679461 TI - The Galpha protein controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in Eschscholzia californica. AB - The function of a Galpha protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Galpha content via antisense transformation and the expression of recombinant anti-Galpha single chain antibodies strongly impaired the induction of alkaloid biosynthesis by low elicitor concentrations. All transgenic cell types were deficient in two elicitor triggered early signal events: activation of phospholipase A2 (PLA2) and efflux of vacuolar protons. The lacking H+ efflux could be restored (1) by adding lysophosphatidylcholine (LPC), a product of PLA2 activity, to vacuoles in situ and (2) by exposing intact cells to isotonic, near-neutral HEPES buffers. The latter treatment induced alkaloid biosynthesis in the absence of elicitor and in Galpha-deficient cells. We conclude that Galpha mediates the stimulation of PLA2 by low elicitor concentrations and that the resulting peak of LPC initiates a transient efflux of vacuolar protons. In this way, an acidic peak of the cytoplasmic pH is generated that causes the expression of enzymes of phytoalexin production independent of the hypersensitive response. PMID- 16679464 TI - A wandering fish bone. AB - A unique case is described of an ingested fish bone migrating into the common hepatic duct, without perforation. Most foreign bodies are known to pass through the gastrointestinal tract uneventfully. Sharper objects such as fish bones have been known to cause perforation. This is more common, in countries where fish consumption is considerably higher, such as, south east Asia. However, even in these regions there have been no reports of such a proximal migration. PMID- 16679465 TI - Parkinson's disease and primate research: past, present, and future. AB - Scientific research involving non-human primates has contributed towards many advances in medicine and surgery. This review discusses its role in the progress made towards our understanding of Parkinson's disease and its treatment. Established medical treatments like dopamine agonists continue to need primate models to assess their efficacy, safety, and mechanism of action. The recently developed treatment of deep brain stimulation of the subthalamic nucleus required validation in primates before entering the clinic. Controversies surrounding future treatments such as gene therapy show the need for properly evaluated preclinical research using appropriate animal models before progression to clinical trials. Research on primates has played--and continues to play--a crucial part in deepening our understanding of Parkinson's disease, improving current therapies, and developing new treatments that are both safe and effective. In animal research, the "three Rs" of humane technique--reduction, refinement, and replacement--should be adhered to. PMID- 16679466 TI - Effects of ageing on touch. AB - A decline in the main sensory modalities is well reported to occur with ageing. This article outlines the normal pathways involved in touch sensation and includes a review of available evidence relating to the study of ageing and touch. The authors try to use what is known about the neuroanatomy and neurophysiology of ageing to explain the impact on some broad functional deficits seen in the elderly population. The importance of understanding how the normal ageing process affects touch sensation is emphasised. PMID- 16679467 TI - Acute aortic syndrome: pathology and therapeutic strategies. AB - Acute aortic syndrome (AAS) describes the acute presentation of patients with characteristic "aortic pain" caused by one of several life threatening thoracic aortic pathologies. These include aortic dissection, intramural haematoma, penetrating atherosclerotic ulcer, aneurysmal leak, and traumatic transection. AAS heralds imminent aortic rupture. Highlighting acute aortic pathology as an AAS is therefore important to encourage prompt recognition of this condition and avoid diagnostic delays. The management of AAS remains a therapeutic challenge. The traditional surgical approach to acute "type B" (descending thoracic) aortic pathology is unsatisfactory with high morbidity and mortality. Endovascular aortic stent grafts now represent an alternative minimally invasive approach in these patients who are often poor surgical candidates. Studies show endovascular repair to be technically feasible with fewer complications. This review discusses AAS pathology and in particular assesses the current role for endovascular aortic repair in its treatment. PMID- 16679469 TI - Leflunomide: a possible alternative for gangciclovir sensitive and resistant cytomegalovirus infections. AB - The search for newer more cost effective treatments for infectious diseases remains a challenge. Cytomegalovirus (CMV) infection, which is especially common in the immunosupressed, is an important challenge for treating physicians. Gangciclovir's cost is a major hurdle in developing countries. Leflunomide is cheaper and is easily given orally. It works by a novel mechanism inhibiting virion assembly. It also has immunosuppressive properties. It and has been shown to be effective in both gangciclovir sensitive as well as resistant cases of CMV infection. Given these considerations we believe that leflunomide is an exiting new drug for CMV infection. However, hepatotoxicity and teratogenicity are known side effects. The exact dose and duration of treatment for CMV infection, for secondary prophylaxis, and in situations of gangciclovir resistance need further study. PMID- 16679471 TI - Open and closed chest extrathoracic cannulation for cardiopulmonary bypass and extracorporeal life support: methods, indications, and outcomes. AB - Extrathoracic cannulation to establish cardiopulmonary bypass has been widely applied in recent years and includes: (a) repeat surgery, (b) minimally invasive surgery, and (c) cases with diseased vessels such as porcelain, aneurysmal, and dissecting aorta. In addition, the success and relative ease of peripheral cannulation, among other technological advances, has permitted the development of closed chest extracorporeal life support, in the form of cardiopulmonary support and extracorporeal membrane oxygenation. With this development have come applications for cardiopulmonary bypass based support outside the traditional cardiac theatre setting, including emergency circulatory support for patients in cardiogenic shock and respiratory support for patients with severely impaired gas exchange. This review summarises the approach to extrathoracic cannulation for the generalist. PMID- 16679470 TI - Treatment of non-alcoholic fatty liver disease. AB - Non-alcoholic fatty liver disease (NAFLD) is common and may progress to cirrhosis and its complications. The pathogenesis of steatosis and cellular injury is thought to be related mostly to insulin resistance and oxidative stress. Therefore, management entails identification and treatment of metabolic risk factors, improving insulin sensitivity, and increasing antioxidant defences in the liver. Weight loss and exercise improve insulin sensitivity. Bariatric surgery may improve liver histology in patients with morbid obesity. Insulin sensitising drugs showed promise in pilot trials as have a number of hepatoprotective agents. Further randomised, well controlled trials are required to determine the efficacy of these drugs. PMID- 16679472 TI - Is the incidence and prevalence of inflammatory bowel diseases increasing in Eastern Europe? AB - Limited data are available on the frequency of inflammatory bowel diseases in East European countries. A recent study from Hungary reported an increasing incidence rate for ulcerative colitis (from 1.6 to 11.0) and for Crohn's disease (from 0.4 to 4.7) from 1977 to 2001. A similar trend was seen in Croatia. In contrast, other countries (for example, Czech Republic, Poland, Romania, Slovakia, and Baltic countries) reported low incidence and prevalence rates. This review will discuss the available data on the epidemiology of inflammatory bowel diseases in Eastern Europe, as well as consider the possible factors responsible for the differences seen between countries and epidemiological trends. PMID- 16679473 TI - Clinical outcome of patients with Helicobacter pylori infection: the bug, the host, or the environment? AB - It is well established that only a minority of patients with Helicobacter pylori infection develop severe inflammation leading to peptic ulcer or gastric cancer. Recent evidence suggests that the virulence factors of the organism do not seem crucial in the progression of inflammation towards a more severe disease. It seems probable that other host derived and environmental factors are more significant in determining clinical outcome but additional studies are needed to clarify the underlying mechanisms involved in the pathogenesis of infection. PMID- 16679474 TI - A single centre cohort experience with a new once daily antiretroviral drug. AB - BACKGROUND: Atazanavir, an azadipeptide protease inhibitor (PI) with once daily dosing, a lack of insulin resistance, lipid increase, and gastrointestinal toxicities, is approved in combination with other antiretrovirals for the treatment of patients infected with HIV. Unboosted atazanavir is also used in highly active antiretroviral therapy (HAART) naive patients. METHODS: The study prospectively followed up an established cohort of patients who received atazanavir, and for whom one year of follow up data were available. RESULTS: It was found that use of atazanavir in intent to treat and on treatment analyses, maintained and led to virological suppression and increases in CD4 count in both PI naive and experienced patients. Virological failure occurred in 7% of patients and the main toxicity was hyperbilirubinaemia, which led to treatment withdrawal in 2%. Its efficacy and safety profile was similar to that seen in previous randomised studies investigating its use. CONCLUSIONS: These data should provide reassurance for clinicians wishing to introduce a new antiretroviral into an established cohort. PMID- 16679475 TI - Patients with poorly controlled diabetes in primary care: healthcare clinicians' beliefs and attitudes. AB - OBJECTIVE: To determine doctors' and nurses' attitudes and beliefs about treating patients with type 2 diabetes with less than ideal glycaemic control while receiving maximal oral treatment in primary care. DESIGN: Focus groups. SETTING: Primary care. PARTICIPANTS: Four focus groups of 23 GPs and practice nurses. RESULTS: General practice was thought to be the best setting for managing all patients with type 2 diabetes but there were concerns about a lack of resources and unfamiliarity with starting insulin. Issues around compliance were extensively discussed; the "failing diabetic" had dual meanings of failing glycaemic control and failing compliance and effort by both patient and doctor. Although views about insulin therapy differed, patients were understood to be resistant to starting insulin, representing for them a more serious stage of diabetes, with fears of needles and hypoglycaemia. CONCLUSION: The role of diabetes specialist nurses working in primary care will be crucial in managing such patients to improve knowledge, for extra resources, for their experience of insulin use, and to change attitudes. PMID- 16679477 TI - Use of amino terminal type III procollagen peptide (P3NP) assay in methotrexate therapy for psoriasis. AB - Hepatic fibrosis continues to be a risk in patients receiving methotrexate for psoriasis. Measurement of amino terminal levels of type III procollagen (P3NP) has been advocated as an effective non-invasive test for ongoing hepatic fibrogenesis that could avoid liver biopsies. An audit was conducted to assess the practice of P3NP monitoring using guidelines produced by Manchester and whether the agreed levels correlate with histological severity. Sixty five patients with 174 P3NP assays and 30 liver biopsies were reviewed between the years 1999 and 2003. Total number of patient-methotrexate years was 278.9 and the mean cumulative dose of methotrexate received was 2000 (SD 1838) mg. A higher cumulative dose of methotrexate correlated significantly with high mean and maximum P3NP levels. Of the 30 liver biopsies, 26 (86.6%) showed normal histology or mild to moderate steatosis, three had focal fibrosis, and one had early cirrhosis. A median P3NP value of 5.8 mug/l or higher had a stronger correlation with histological severity. It is concluded that P3NP assay is a valuable adjunct to the clinical management of patients receiving long term methotrexate that can avoid or reduce unnecessary liver biopsies. PMID- 16679476 TI - Chickenpox, chickenpox vaccination, and shingles. AB - Chickenpox in the United Kingdom, where vaccination is not undertaken, has had a stable epidemiology for decades and is a routine childhood illness. Because of vaccination, chickenpox is now a rarity in the USA. In the UK vaccination is not done because introduction of a routine childhood vaccination might drive up the age at which those who are non-immune get the illness (chickenpox tends to be more severe the older you are), and the incidence of shingles may increase. The United Kingdom is waiting to see what happens in countries where vaccination is routine. PMID- 16679479 TI - Prevalence of wet litter and the associated risk factors in broiler flocks in the United Kingdom. AB - A postal questionnaire was sent to the managers of 857 broiler farms in the UK to determine the prevalence and risk factors for wet litter. The response rate was 75 per cent. Wet litter was reported by 75 per cent (95 per cent confidence interval [CI] 71.3 to 78.3) of the respondents in at least one flock during the year 2001 and 56.1 per cent (95 per cent CI 52.0 to 60.0) of them reported that they had an outbreak of wet litter in their most recently reared flock. Wet litter occurred more often during the winter months and farms using side ventilation systems were at an increased risk (odds ratio 1.74; 95 per cent CI 1.09 to 2.76). A multivariable analysis was carried out using two different definitions of wet litter as outcome variables - all cases of wet litter, and cases of wet litter associated with disease. Consistent risk factors for both outcomes were coccidiosis, feed equipment failures and the availability of separate farm clothing for each house. Cases of wet litter associated with disease were reported by 33.7 per cent (95 per cent CI 28.8 to 39.1) of the managers in their last flock and were associated with the use of hand sanitisers and broiler houses with walls made of concrete. PMID- 16679480 TI - Herd-level risk factors for the mortality of cows in Danish dairy herds. AB - The factors affecting the mortality of cows in Danish dairy herds were investigated by analysing data from 6839 herds. The mean risk of mortality during the first 100 days of lactation was 2.5 per cent. The risk of mortality increased with increasing herd size (odds ratio [OR] 1.05 for an increase in herd size of 50 cows), with the proportion of purchased cows (OR 1.05 for an increase in the proportion of purchased cows of 0.1), and with increases in the somatic cell count (OR 1.16 for an increase in average weighted mean somatic cell count of 100,000 cells/ml). The risk decreased with increasing average milk yield per cow (OR 0.93 for an increase in mean yield per cow-year of 1000 kg). The risk was lower in free-stall barns with deep litter (OR 0.79) than in barns with cubicles (OR 1) or tie-stalls (OR 1.04). Herds with Danish Holstein (OR 1) or Danish Jersey (OR 0.93) cows as the predominant breed had a higher risk of mortality than herds of the Danish red dairy breed (OR 0.67). The risk of mortality was lower in organic herds (OR 1) than in conventional herds (OR 1.17), and lower in herds that grazed pasture during the summer (OR 0.78). PMID- 16679481 TI - Detection of lymph node micrometastases in malignant mammary tumours in dogs by cytokeratin immunostaining. AB - A series of 131 local and regional lymph nodes from 40 dogs with malignant mammary tumours were evaluated by staining with haematoxylin and eosin and immunohistochemically for antibodies to pancytokeratin (AE1/AE3) and cytokeratin 14. The immunohistochemical tests detected occult micrometastases in 9.2 per cent of the lymph nodes that were negative by haematoxylin and eosin staining. Under the modified TNM classification of canine mammary tumours, these results raised the clinical stage of 12.5 per cent of the affected dogs. However, if the latest TNM classification of human breast cancer had been applied, none of the animals would have been reclassified. PMID- 16679482 TI - Strawberry disease in rainbow trout in Scotland: pathology and association with Flavobacterium psychrophilum. PMID- 16679483 TI - Circulation of bovine respiratory syncytial virus in Brazil. PMID- 16679484 TI - Gastrointestinal nematode resistance to benzimidazoles on a sheep farm in Algeria. PMID- 16679485 TI - Meningeal cholesterol granulomas in two meerkats (Suricata suricatta). PMID- 16679486 TI - Sheep scab, pyrethroid dips and aquatic life. PMID- 16679487 TI - Eradication of bovine TB: learning from other countries. PMID- 16679488 TI - Tamoxifen and raloxifene differ in their functional interactions with aspartate 351 of estrogen receptor alpha. AB - The bulky side chains of antiestrogens hinder folding of the ligand binding domain (LBD) of estrogen receptors (ERs) into a transcriptionally active conformation. The presence of a tertiary amine in the side chain of raloxifene, which interacts with a negatively charged residue in helix H3 of the ER LBD [Asp351 in human (h)ERalpha], is important for antiestrogenicity in animal and cellular models. To better understand the molecular basis of the differential activity of tamoxifen and raloxifene, we have examined the influence of tertiary amine substituents and of mutations at position 351 in hERalpha on the activity profiles of tamoxifen derivatives. Results obtained in several cellular model systems suggest that the degree of antagonist activity of tamoxifen derivatives does not strictly correlate with the basicity of the side chain but depends on an optimal spatial relationship between the tertiary amine of these antiestrogens and the negative charge at position 351. Although altering the position of the negative charge at residue 351 (mutation D351E) had little effect on transcriptional activity in the presence of tamoxifen, it drastically increased the partial agonist activity of a tamoxifen derivative with improved antagonist activity as well as that of raloxifene. Our results suggest that contrary to raloxifene, tamoxifen and most of its derivatives do not interact with Asp351 in an optimal manner, although this can be improved by modifying tertiary amine substituents. PMID- 16679489 TI - A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro. AB - The enormous capacity of the male silkmoth Bombyx mori in recognizing and discriminating bombykol and bombykal is based on distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed their responses to hydrophobic pheromone compounds dissolved by means of dimethyl sulfoxide. BmOR-1-expressing cells were activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol, indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pheromone receptors and PBPs play an important role in insect pheromone recognition. PMID- 16679490 TI - Survey of the frequency of USH1 gene mutations in a cohort of Usher patients shows the importance of cadherin 23 and protocadherin 15 genes and establishes a detection rate of above 90%. AB - BACKGROUND: Usher syndrome, a devastating recessive disorder which combines hearing loss with retinitis pigmentosa, is clinically and genetically heterogeneous. Usher syndrome type 1 (USH1) is the most severe form, characterised by profound congenital hearing loss and vestibular dysfunction. OBJECTIVE: To describe an efficient protocol which has identified the mutated gene in more than 90% of a cohort of patients currently living in France. RESULTS: The five genes currently known to cause USH1 (MYO7A, USH1C, CDH23, PCDH15, and USH1G) were tested for. Disease causing mutations were identified in 31 of the 34 families referred: 17 in MYO7A, 6 in CDH23, 6 in PCDH15, and 2 in USH1C. As mutations in genes other than myosin VIIA form nearly 50% of the total, this shows that a comprehensive approach to sequencing is required. Twenty nine of the 46 identified mutations were novel. In view of the complexity of the genes involved, and to minimise sequencing, a protocol for efficient testing of samples was developed. This includes a preliminary linkage and haplotype analysis to indicate which genes to target. It proved very useful and demonstrated consanguinity in several unsuspected cases. In contrast to CDH23 and PCDH15, where most of the changes are truncating mutations, myosin VIIA has both nonsense and missense mutations. Methods for deciding whether a missense mutation is pathogenic are discussed. CONCLUSIONS: Diagnostic testing for USH1 is feasible with a high rate of detection and can be made more efficient by selecting a candidate gene by preliminary linkage and haplotype analysis. PMID- 16679491 TI - Fanconi anaemia complementation group B presenting as X linked VACTERL with hydrocephalus syndrome. AB - BACKGROUND: The VACTERL with hydrocephalus (VACTERL-H) phenotype is recognised to be a severe manifestation of autosomal recessive Fanconi anaemia. Several families have been described in which the VACTERL-H phenotype segregates as an X linked syndrome. The mutations which cause X linked VACTERL-H syndrome are not known. OBJECTIVE: To determine if mutations in FANCB, which are known to cause Fanconi anaemia complementation group B, are a cause of X linked VACTERL-H syndrome. METHODS: A three generation pedigree with X linked VACTERL-H syndrome was investigated. X inactivation was tested in carrier females, and fibroblasts from an affected male fetus were analysed for increased sensitivity to diepoxybutane. FANCB coding exons and flanking splice sites were screened for mutations by direct sequencing of polymerase chain reaction (PCR) fragments amplified from genomic DNA. cDNA from affected fetal fibroblasts was analysed by PCR and direct sequencing using specific exonic primers. RESULTS: A FANCB mutation which results in a premature stop codon by causing skipping of exon 7 was identified. Chromosomes from the affected fetus showed increased sensitivity to diepoxybutane, and carrier women were found to have 100% skewed X inactivation in blood. CONCLUSIONS: Mutations in FANCB are a cause of X linked VACTERL-H syndrome. The data presented are of relevance to the genetic counselling of families with isolated male cases of VACTERL-H and Fanconi anaemia. PMID- 16679492 TI - Two cases of severe neonatal hypertrophic cardiomyopathy caused by compound heterozygous mutations in the MYBPC3 gene. AB - BACKGROUND: Idiopathic (primary) hypertrophic cardiomyopathy (HCM) is mainly caused by mutations in genes encoding sarcomeric proteins. One of the most commonly mutated HCM genes is the myosin binding protein C (MYBPC3) gene. Mutations in this gene lead mainly to truncation of the protein which gives rise to a relatively mild phenotype. Pure HCM in neonates is rare and most of the time childhood HCM occurs in association with another underlying condition. OBJECTIVE: To study the presence of mutations in the MYBPC3 gene in idiopathic childhood HCM. METHODS: MYBPC3 coding region and splice junction variation were analysed by denaturing high performance liquid chromatography (DHPLC) and sequencing in DNA isolated from two neonates with severe unexplained HCM, who died within the first weeks of life. RESULTS: Truncating mutations were found in both alleles of the MYBPC3 gene in both patients, suggesting there was no functional copy of the MYBPC3 protein. Patient 1 carried the maternally inherited c.2373_2374insG mutation and the paternally inherited splice-donor site mutation c.1624+1G-->A. Patient 2 carried the maternally inherited frameshift mutation c.3288delA (p.Glu1096fsX92) and the paternally inherited non-sense mutation c.2827C-->T (p.Arg943X). CONCLUSIONS: The findings indicate the need for mutation analysis of genes encoding sarcomeric proteins in childhood HCM and the possibility of compound heterozygosity. PMID- 16679493 TI - Firearm use in intimate partner violence: a brief overview. AB - Readers of this volume are likely to have specific interests in domestic violence or in firearms policy. It is not assumed, however, that the typical reader will know about the interface of the two fields. Thus, the volume begins with a synopsis of the epidemiology of weapon use in intimate partner violence. The purpose of this article is to help readers better understand the nature of the problem, obtain knowledge that will provide a context for the policy, and understand practice implications of the articles that follow. PMID- 16679494 TI - Domestic violence and firearms: reflections of a prosecutor. PMID- 16679495 TI - Disarming batterers through restraining orders: the promise and the reality in California. AB - Laws that prohibit persons under a domestic violence restraining order from purchasing or possessing a firearm are a primary way to keep guns out of the hands of batterers. In July 2005, the California Attorney General's Task Force on the Local Criminal Justice Response to Domestic Violence issued a report called Keeping the Promise: Victim Safety and Batterer Accountability. The report focused, in part, on the extent to which California's domestic violence restraining order system succeeds in disarming batterers. Restraining orders are the principal means by which the criminal justice system can accomplish this objective. The Task Force found that criminal justice agencies and the courts performed poorly in this area. The report strongly recommended that a more strategic, collaborative use of laws already on the books could significantly improve performance, without much additional expense. What follows is a summary of those finding and recommendations. PMID- 16679496 TI - Are temporary restraining orders more likely to be issued when applications mention firearms? AB - Restraining orders, an important legal intervention for victims of domestic violence, have broad potential for injury prevention. Using data from one of the busiest restraining order clinics in the nation, the authors examined 1,354 applicants' descriptions of abuse. Most (89.2%) applicants were issued a restraining order. A total of 16.0% of applicants mentioned a firearm in their descriptions of abuse; doing so was not associated with restraining order issuance. About 1 in 20 applicants, even if issued a restraining order, would not be protected by the federal firearm purchase and possession prohibitions because they had not lived with or had a child with the defendant. However, the proportion of these individuals who report threatened or actual use of firearms against them is similar to that of other applicants. Federal and, when relevant, state law should be modified to include these persons. PMID- 16679497 TI - Gun possession among Massachusetts batterer intervention program enrollees. AB - Batterers with access to firearms present a serious lethal threat to their partners. The purpose of this exploratory study is to estimate the prevalence of and risk markers for gun possession among Massachusetts men enrolled in batterer intervention programs. The authors found that 1.8% of the men reported having a gun in or around their home. Those most likely to report having a gun were White, earned 25,000 US dollars or more per year, had served in the military, engaged in problem gambling, and had attempted homicide or threatened their partner with a firearm. Recommendations for strengthening relevant gun laws both within and outside of Massachusetts are discussed. PMID- 16679498 TI - Separating batterers and guns: a review and analysis of gun removal laws in 50 States. AB - Firearms play an important role in lethal domestic violence incidents. The authors review state laws regarding two policies to separate batterers from firearms: laws authorizing police to remove firearms when responding to a domestic violence complaint ("police gun removal laws") and laws authorizing courts to order guns removed from batterers through a protective order ("court ordered removal laws"). As of April 2004, 18 states had police gun removal laws; 16 states had court-ordered removal laws. The authors examine relevant characteristics of the laws and recommend that these laws be mandatory, apply to all guns and ammunition possessed by an abuser, and include clear procedures to enhance implementation. PMID- 16679499 TI - Do laws restricting access to firearms by domestic violence offenders prevent intimate partner homicide? AB - Domestic violence imposes a large cost on society. The authors exploit state variation in timing to examine the impact of three types of law on intimate partner homicides. These laws restrict access to firearms by individuals who are subject to a restraining order or have been convicted of a domestic violence misdemeanor or allow law enforcement officers to confiscate firearms at a domestic violence scene. The authors find that female intimate partner homicide rates decline 7% after a state passes a restraining order law. They find no effect from the domestic violence misdemeanor or confiscation laws. PMID- 16679500 TI - Understanding and informing policy implementation: a case study of the domestic violence provisions of the Maryland Gun Violence Act. AB - The Maryland Gun Violence Act, enacted into law in 1996, explicitly authorized courts to order batterers to surrender their firearms through civil protective orders. It also vested law enforcement with the explicit authority to remove guns when responding to a domestic violence complaint. In order to assess how these laws were implemented, we designed a case study and collected data from in-depth, key informant interviews, court observations, and relevant documents. We present findings from this study and recommend how to increase the likelihood that policies designed to separate batterers and guns are implemented in a way that will result in greater protections for victims of domestic violence. PMID- 16679501 TI - Taking guns from batterers: public support and policy implications. AB - Federal law prohibits the purchase or possession of a firearm by persons convicted of misdemeanor domestic violence and those under certain domestic violence restraining orders. The purpose of this investigation is to examine public sentiment about the removal of firearms in the absence of a restraining order or misdemeanor conviction following domestic violence. An experimental vignette design was used in a telephone survey of a cross-sectional statewide sample of 522 community-residing adults in California. Study design and population weights were applied; the findings, thus, are a reasonable approximation for the population of California. In more than 3,500 vignettes, the abusive behavior was judged to be wrong, illegal, or should be illegal (98.7%, 73.1%, and 77.7%, respectively). Although only about one half (56.5%) of the scenarios were thought to merit the issuance of a restraining order, three fourths (77.4%) were thought to merit the removal of firearms. Multivariate analyses indicated greater support for firearms removal when the abuse involved sexual or physical abuse (adjusted odds ratio [AOR] ranged from 2.65 to 5.64) or a gun (AOR = 6.54). Men were the sole population group with significantly lower support for firearm removal following domestic violence (AOR = 0.39). The men who wanted firearms to remain did not differ from other men on any of the measured variables. In sum, there is substantial support, especially when a gun is displayed in a domestic violence incident, for policies requiring the removal of firearms from abusers. PMID- 16679502 TI - Pharmacological modulation of plasticity in the human motor cortex. AB - Ischemic cerebral stroke is the leading cause of long-term disability among adults in industrialized countries. One fundamental but still not sufficiently solved question is how to improve disability after stroke. Here, evidence will be reviewed on how pharmacological treatment modulates plasticity and learning in the intact human motor cortex. It will be argued that these data may be useful for advancing the concepts of pharmacotherapy for recovery after stroke. PMID- 16679503 TI - Sensorimotor training in a virtual reality environment: does it improve functional recovery poststroke? AB - OBJECTIVE: To investigate the effectiveness of computerized virtual reality (VR) training of the hemiparetic hand of patients poststroke using a system that provides repetitive motor reeducation and skill reacquisition. METHODS: Eight subjects in the chronic phase poststroke participated in a 3-week program using their hemiparetic hand in a series of interactive computer games for 13 days of training, weekend breaks, and pretests and posttests. Each subject trained for about 2 to 2.5 h per day. Outcome measures consisted of changes in the computerized measures of thumb and finger range of motion, thumb and finger velocity, fractionation (the ability to move fingers independently), thumb and finger strength, the Jebsen Test of Hand Function, and a Kinematic reach to grasp test. RESULTS: Subjects as a group improved in fractionation of the fingers, thumb and finger range of motion, and thumb and finger speed, retaining those gains at the 1-week retention test. Transfer of these improvements was demonstrated through changes in the Jebsen Test of Hand Function and a decrease after the therapy in the overall time from hand peak velocity to the moment when an object was lifted from the table. CONCLUSIONS: It is difficult in current service delivery models to provide the intensity of practice that appears to be needed to effect neural reorganization and functional changes poststroke. Computerized exercise systems may be a way to maximize both the patients' and the clinicians' time. The data in this study add support to the proposal to explore novel technologies for incorporation into current practice. PMID- 16679504 TI - Neural substrates for motor imagery in severe hemiparesis. AB - BACKGROUND: The beneficial effects of imagined movements on motor learning and performance suggest that motor imagery is functionally close to preparatory and executive motor processes. OBJECTIVE: The purpose of this study was to examine the cortical processes associated with imagery of movement of the wrist in subjects with severe hemiparesis. METHODS: During fMRI, subjects with stroke performed alternating blocks of imagining wrist-tracking movements with the hemiparetic hand, active wrist-tracking movements with the unaffected hand, and resting. Control subjects performed the same tasks using an assigned hand. Cortical activation in the primary motor (M1), primary sensory (S1), supplementary motor area (SMA), and pre-SMA regions was determined through a laterality index of active voxels and signal intensity. Ability to imagine was assessed with an Imagery Rating Scale. RESULTS: All subjects displayed primarily contralateral control during the track condition. Healthy subjects demonstrated contralateral control in all areas during the imagine condition, whereas subjects with stroke displayed primarily contralateral activation in S1 but ipsilateral in M1 and SMA. The percentage change in signal intensity was greater in the ipsilateral hemisphere in subjects with stroke than in the ipsilateral hemisphere in healthy subjects during the imagine condition. Additionally, subjects with self-reported low ability to imagine displayed no difference in activation compared to those with high imagery ability. CONCLUSIONS: These findings are consistent with other works demonstrating primarily ipsilateral control of the hemiparetic hand in subjects with functional movement and lay the groundwork for further investigation into the ability of mental imagery to affect functionally relevant cortical control in subjects recovering from stroke. PMID- 16679505 TI - Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord. AB - This study evaluates functional recovery after transplanting human bone marrow derived stromal cells (BMSCs) into contusion models of spinal cord injury (SCI). The authors used a high-throughput process to expand BMSCs and characterized them by flow cytometry, ELISA, and gene expression. They found that BMSCs secrete neurotrophic factors and cytokines with therapeutic potential for cell survival and axon growth. In adult immune-suppressed rats, mild, moderate, or severe contusions were generated using the MASCIS impactor. One week following injury, 0.5 to 1 x 106 BMSCs were injected into the lesioned spinal cord; control animals received vehicle injection. Biweekly behavioral tests included the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB), exploratory rearing, grid walking, and thermal sensitivity. Animals receiving moderate contusions followed by BMSC grafts showed significant behavioral recovery in BBB and rearing tests when compared to controls. Animals receiving BMSC grafts after mild or severe contusion showed trends toward improved recovery. Immunocytochemistry identified numerous axons passing through the injury in animals with BMSC grafts but few in controls. BMSCS were detected at 2 weeks after transplantation; however, at 11 weeks very few grafted cells remained. The authors conclude that BMSCs show potential for repairing SCI. However, the use of carefully characterized BMSCs improved transplantation protocols ensuring BMSC, survival, and systematic motor and sensory behavioral testing to identify robust recovery is imperative for further improvement. PMID- 16679506 TI - Reliability and validity of bilateral thigh and foot accelerometry measures of walking in healthy and hemiparetic subjects. AB - OBJECTIVE: Measures of walking ability in large clinical trials are usually limited to a timed short-distance walk and the distance walked in a fixed time. A new integrated system of 5 accelerometers was tested for reliability and compared to a footswitch system to determine if the accelerometers offered a practical option for the acquisition of spatiotemporal gait parameters. METHODS: Leg accelerations and decelerations were defined in relation to simultaneous kinematic and electromyographic data acquired from a healthy subject. Eight healthy adults and 6 independent ambulators with hemiparetic stroke walked 15 m at 2 different speeds wearing both the accelerometers and footswitches. Twelve healthy subjects walked at 5 different speeds repeated 3 times on each of 2 days wearing the accelerometers. Walking speed, cadence, stride length, and single- and double-limb support, swing, and stance times were calculated. RESULTS: No differences (t test, P > 0.2) were found between footswitch and accelerometer variables when comparing all left or right legs in healthy subjects and all paretic or unaffected legs in stroke subjects. A 2-way nested ANOVA model (speed, left and right legs, trial, and session) with the accelerometers at walking speeds from 0.5 to 1.8 m/s revealed high reproducibility of all measures. CONCLUSIONS: The accelerometry system provided reliable and valid spatiotemporal measures of gait for the upper range of speeds likely to be targeted for rehabilitation interventions in ambulatory subjects. PMID- 16679508 TI - Image in endocrinology: giant mediastinal cystic parathyroid adenoma. PMID- 16679509 TI - Editorial: does lupron dosage make a difference in outcome when treating children with precocious puberty? PMID- 16679510 TI - Editorial: inhibin in bone--new tricks for an old dog. PMID- 16679511 TI - Editorial: MC4R mutations--weight before screening! PMID- 16679512 TI - Editorial: BMP15--the first true ovarian determinant gene on the X-chromosome? PMID- 16679514 TI - Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation. AB - Dentin mineralization requires transcriptional mechanisms to induce a cascade of gene expression for progressive development of the odontoblast phenotype. During cytodifferentiation of odontoblasts there is a constant change of actively transcribed genes. Thus, tissue-specific matrix genes that are silenced in early differentiation are expressed during the terminal differentiation process. Dentin sialophosphoprotein (DSPP) is an extracellular matrix, prototypical dentin, and a bone-specific gene, however, the molecular mechanisms by which it is temporally and spatially regulated are not clear. In this report, we demonstrate that dentin matrix protein 1 (DMP1), which is localized in the nucleus during early differentiation of odontoblasts, is able to bind specifically with the DSPP promoter and activate its transcription. We have identified the specific promoter sequence that binds specifically to the carboxyl end of DMP1. The DNA binding domain in DMP1 resides between amino acids 420 and 489. A chromatin immunoprecipitation assay confirmed the in vivo association of DMP1 with the DSPP promoter. Interactions between DMP1 and DSPP promoter thus provide the foundation to understand how DMP1 regulates the expression of the DSPP gene. PMID- 16679513 TI - Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells. AB - Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3- based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells. PMID- 16679515 TI - Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions. AB - Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs. PMID- 16679516 TI - A single chondroitin 6-sulfate oligosaccharide unit at Ser-2730 of human thyroglobulin enhances hormone formation and limits proteolytic accessibility at the carboxyl terminus. Potential insights into thyroid homeostasis and autoimmunity. AB - We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg CS0) molecules on the basis of their D-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726 2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 ' triiodothyronine (T3) (171%) and 3,5,3',5'-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra thyroidal manifestations of autoimmune thyroid disease are discussed. PMID- 16679517 TI - The glycosaminoglycan attachment regions of human aggrecan. AB - Aggrecan possesses both chondroitin sulfate (CS) and keratan sulfate (KS) chains attached to its core protein, which reside mainly in the central region of the molecule termed the glycosaminoglycan-attachment region. This region is further subdivided into the KS-rich domain and two adjacent CS-rich domains (CS1 and CS2). The CS1 domain of the human is unique in exhibiting length polymorphism due to a variable number of tandem amino acid repeats. The focus of this work was to determine how length polymorphism affects the structure of the CS1 domain and whether CS and KS chains can coexist in the different glycosaminoglycan attachment domains. The CS1 domain possesses several amino acid repeat sequences that divide it into three subdomains. Variation in repeat number may occur in any of these domains, with the consequence that CS1 domains of the same length may possess different amino acid sequences. There was no evidence to support the presence of KS in either the CS1 or the CS2 domains nor the presence of CS in the KS-rich domain. The structure of the CS chains was shown to vary between the CS1 and CS2 domains, particularly in the adult, with variation occurring in chain length and the sulfation of the non-reducing terminal N-acetyl galactosamine residue. CS chains in the adult CS2 domain were shorter than those in the CS1 domain and possessed disulfated terminal residues in addition to monosulfated residues. There was, however, no change in the sulfation pattern of the disaccharide repeats in the CS chains from the two domains. PMID- 16679518 TI - Expression of foreign protein epitopes at the surface of recombinant yellow fever 17D viruses based on three-dimensional modeling of its envelope protein. AB - The yellow fever (YF) 17D vaccine is a live attenuated virus, and its genetic manipulation constitutes a new platform for vaccine development. In this article, we review one of the possible approaches to enable this development, which is the insertion of foreign protein epitopes into different locations of the genome. We describe the three-dimensional (3D) modeling of the YF 17D virus E protein structure based on tick-borne encephalitis (TBE) and the identification of a potential insertion site located at the YF 17D fg loop. Further 3D analysis revealed that it is possible to accommodate inserts of different sizes and amino acid composition in the flavivirus E protein fg loop. We demonstrate that seven YF 17D viruses bearing foreign epitopes that vary in sequence and length show differential growth characteristics in cell culture. The testing of recombinant viruses for mouse neurovirulence suggests that insertions at the 17D E protein fg loop do not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines. PMID- 16679519 TI - The fusogenic state of Mayaro virus induced by low pH and by hydrostatic pressure. AB - Mayaro virus is an enveloped virus that belongs to the Alphavirus genus. To gain insight into the mechanism involved in Mayaro virus membrane fusion, we used hydrostatic pressure and low pH to isolate a fusion-active state of Mayaro glycoproteins. In response to pressure, E1 glycoprotein undergoes structural changes resulting in the formation of a stable conformation. This state was characterized and correlated to that induced by low pH as measured by intrinsic fluorescence, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt fluorescence, fluorescence resonance energy transfer, electron microscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In parallel, we used a neutralization assay to show that Mayaro virus in the fusogenic state retained most of the original immunogenic properties and could elicit high titers of neutralizing antibodies. PMID- 16679520 TI - Expression and purification of rotavirus proteins NSP5 and NSP6 in Escherichia coli. AB - Rotaviruses are one of the worldwide leading causes of gastroenteritis in children under 5 yr old. The rotavirus nonstructural NSP5 is a phosphoprotein implicated in viroplasms formation, whereas NSP6 could have a possible regulatory role of NSP5. It has been reported that N- and C-termini of NSP5 are important for their function. However, no structural information on NSP5 and NSP6 proteins is available. Because a high amount of protein is required for structural analysis, efficient expression systems are required. His-tag fusion at the C terminus and glutathione-S-transferase (GST)-fusion at the N-terminus were used as expression systems, and conditions for recombinant proteins expression were obtained. His-tag fusion was not efficient to produce NSP5 (2% of total protein), but NSP6 was expressed in higher amounts (11% of total protein). In contrast, GSTNSP5 and GST-NSP6 proteins correspond to 34 and 31% of the total proteins, respectively. GST-fusions seem to have a protective effect against nonstructural rotavirus protein toxicity in Escherichia coli; however, in both systems, NSP5 and NSP6 recombinant proteins were expressed as inclusion bodies. Conditions for solubilization and purification of recombinant proteins were achieved. This is the first report of expression and purification of NSP5 and NSP6 recombinant proteins in suitable amounts for further structural analysis. PMID- 16679521 TI - The TACE zymogen: re-examining the role of the cysteine switch. AB - The tumor necrosis factor-alpha-converting enzyme (TACE) is a member of the disintegrin family of metalloproteinases (ADAMs) that plays a central role in the regulated shedding of a host of cell surface proteins. TACE is biosynthesized as a precursor protein with latent proteolytic activity (zymogen). TACE's zymogen inhibition is mediated by its Pro domain, a 197-amino acid region that serves this function as well as aiding in the secretion of this enzyme through the secretory pathway. We have discovered that a conserved "cysteine switch" consensus motif within TACE's Pro domain is, contrary to expectations, not required for maintenance of the inactive precursor state or for the secretion of this metalloproteinase in its functional form. The only role for this motif seems to be in decreasing TACE's susceptibility to proteolytic degradation during its biogenesis and maturation within the secretory pathway. Interestingly, the Pro domain of TACE seems to carry both its inhibitory and secretory functions through the same mechanism: it seems to prevent the Catalytic domain from accessing its native, functional state, resembling the function of true molecular chaperones. Recent evidence suggests that TACE may also be switched out of the active conformation even by small, drug-like molecules such as the synthetic compound SB 3CT. These findings point at the possibility of developing, in the near future, a new generation of antiinflammatory, noncompetitive TACE inhibitors that would exert negative allosteric modulation over the activity of this key enzyme, mediating several inflammatory diseases and certain cancers. PMID- 16679522 TI - Structure-function properties of prolyl oligopeptidase family enzymes. AB - Prolyl oligopeptidase family enzymes regulate the activity of biologically active peptides and peptide hormones, and they are implicated in diseases, including amnesia, depression, diabetes, and trypanosomiasis. Distinctively, these enzymes hydrolyze only relatively short peptide substrates, while large structured peptides and proteins are not usually cleaved. Prolyl oligopeptidase has a C terminal alpha/beta-hydrolase catalytic domain that is similar to lipases and esterases. An N-terminal beta-propeller domain regulates access to the buried active site, explaining the observed oligopeptidase activity. The catalytic and regulatory mechanisms have been investigated using a combination of X-ray crystallography, site-directed mutagenesis, and enzyme kinetic measurements. Crystal structures have now been determined for representative members of three of the four subfamilies and are facilitating a better understanding of the structure-function properties of these physiologically and pharmaceutically important enzymes. PMID- 16679524 TI - Molecular models of tryptophan synthase from mycobacterium tuberculosis complexed with inhibitors. AB - The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the alpha-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases. PMID- 16679523 TI - DBMODELING: a database applied to the study of protein targets from genome projects. AB - Genome sequencing efforts are providing us with complete genetic blueprints for hundreds of organisms. We are now faced with assigning, understanding, and modifying the functions of proteins encoded by these genomes. DBMODELING is a relational database of annotated comparative protein structure models and their metabolic pathway characterization, when identified. This procedure was applied to complete genomes such as Mycobacterium tuberculosis and Xylella fastidiosa. The main interest in the study of metabolic pathways is that some of these pathways are not present in humans, which makes them selective targets for drug design, decreasing the impact of drugs in humans. In the database, there are currently 1,116 proteins from two genomes. It can be accessed by any researcher at http://www.biocristalografia.df.ibilce.unesp.br/tools/. This project confirms that homology modeling is a useful tool in structural bioinformatics and that it can be very valuable in annotating genome sequence information, contributing to structural and functional genomics, and analyzing protein-ligand docking. PMID- 16679525 TI - Molecular modeling of pathogenesis-related proteins of family 5. AB - The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of alpha amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for each protein. PMID- 16679526 TI - Molecular dynamics simulations applied to the study of subtypes of HIV-1 protease common to Brazil, Africa, and Asia. AB - Africa accounts for the majority of HIV-1 infections worldwide caused mainly by the A and C viral subtypes rather than B subtype, which prevails in the United States and Western Europe. In Brazil, B subtype is the major subtype, but F, C, and A also circulate. These non-B subtypes present polymorphisms, and some of them occur at sites that have been associated with drug resistance, including the HIV-1 protease (PR), one important drug target. Here, we report a Molecular Dynamics study of the B and non-B PR complexed with the inhibitor ritonavir to delineate the behavior of each subtype. We compare root mean squared deviation, binding free energy by linear interaction energy approach, hydrogen bonds, and intermolecular contact surface area between inhibitor and PR. From our results, we can provide a basis to understand the molecular mechanism of drug resistance in non-B subtypes. In this sense, we found a decrease of approx 4 kcal/mol in deltaG of binding between B and non-B subtypes. This corresponds to the loss of one hydrogen bond, which is in agreement with our H-bond analysis. Previous experimental affinity studies reported analogous results with inhibition constant values for non-B PR. PMID- 16679527 TI - Determining the structural basis for specificity of ligands using crystallographic screening. AB - Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T cell immune response modulation and has been submitted to extensive structure- based drug design. This methodology may help in the future development of a new generation of PNP inhibitors. PMID- 16679528 TI - Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity. AB - Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX. PMID- 16679529 TI - Effects of bound ts3 on voltage dependence of sodium channel transitions to and from inactivation and energetics of its unbinding. AB - Recently, we proposed a quantitative model to explain the molecular mechanism of action of the Tityus serrulatus Ts3 alpha-toxin on sodium channels. In this model, the toxin acts as a stop that prevents the segment S4 of domain IV from reaching its outermost position, thus impairing the normal fast inactivation without affecting activation. In the present work, we analyze the predictions of the proposed model with regard to the voltage-dependent transitions to and from inactivation. Our results show that the recovery from inactivation was significantly faster in Ts3-bound channels and that there was no significant voltage dependence. The transition to inactivated state from open state in Ts3 modified channels presented a small but significant voltage dependence, which may derive from an intrinsic voltage dependence of inactivation or by a short movement of IVS4 in the presence of bound Ts3. We also studied the thermodynamic parameters of the voltage-dependent displacement of Ts3 from its binding site. We have observed that the activation energy to remove the toxin is 27 kJ/mol, part of which derives from the imposed depolarizing potential and the movement of an equivalent electrical charge of 0.54 e(0). These results support the proposed model. PMID- 16679530 TI - Phospholipid distribution around the plasma membrane calcium pump: a hydrophobic photolabeling study. AB - The functions of membrane proteins are highly dependent on their phospholipid environment. In this article, we have used a hydrophobic photolabeling method to study the noncovalent interactions between plasma membrane calcium pump (PMCA) and surrounding phospholipids. With this approach, we determined (1) the number of lipid molecules in close contact with the transmembrane surface, i.e., the lipid-protein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl 2-[9-[2'-[125I]iodo-4'- (trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl] sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (PC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 +/- 1 molecules of PC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N terminal (1-322), the central (323-660), and the C-terminal (661-1,205) regions was 26, 36, and 38%, respectively. PMID- 16679531 TI - Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes. AB - Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent solubilized enzyme. PMID- 16679532 TI - Biomimetic particles for isolation and reconstitution of receptor function. AB - Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction. PMID- 16679533 TI - Human thyroid receptor forms tetramers in solution, which dissociate into dimers upon ligand binding. AB - Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRbeta1 truncation (hTRbeta1deltaAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRbeta1deltaAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo hTRbeta1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought. PMID- 16679534 TI - CGI-55 interacts with nuclear proteins and co-localizes to p80-coilin positive coiled bodies in the nucleus. AB - The human protein CGI-55 has been described as a chromo-helicase-DNA-binding domain protein (CHD)-3 interacting protein and was also found to interact with the 3'-region of the plasminogen activator inhibitor (PAI)-1 mRNA. Here, we used CGI-55 as a "bait" in a yeast two-hybrid screen and identified eight interacting proteins: Daxx, Topoisomerase I binding RS (Topors), HPC2, UBA2, TDG, and protein inhibitor of activated STAT (signal transducer and activator of transcription) (PIAS)-1, -3, and -y. These proteins are either structurally or functionally associated with promyelocytic leukemia nuclear bodies (PML-NBs), protein sumoylation, or the regulation of transcription. The interactions of CGI-55 with Daxx, Topors, PIASy, and UBA2 were confirmed by in vivo colocalization experiments in HeLa cells, by using green (GFP) and red fluorescence fusion proteins. A mapping study of the CGI-55 binding site for these proteins revealed three distinct patterns of interaction. The fact that CGI-55-GFP has been localized in cytoplasm and nucleus in a dotted manner, and its interaction with proteins associated with PML-NBs, suggested that CGI-55 might be associated with nuclear bodies. Although Daxx and Topors co-localized with promyelocytic leukemia protein (PML), CGI-55 itself as well as PIASy and UBA2 showed only little co localization with PML. However, we observed that CGI-55 localizes to the nucleolus and co-localizes with p80-coilin positive nuclear-coiled bodies. PMID- 16679535 TI - NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome. AB - The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three dimensional solution structure of the p63 sterile alpha-motif (SAM) domain (residues 505-579), a region crucial to explaining the human genetic disease ankyloblepharonectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of freedom of individual residues and discussion of the possible causes for the pathology. PMID- 16679536 TI - Biophysical characterization of interaction between apolipoprotein A-I and bacterial lipopolysaccharide. AB - We studied the effect of bacterial lipopolysaccharide (LPS)-apolipoprotein A-I (apo A-I) interaction on the structure and function of this protein. The micellization process of dimirystoil phosphatidylcholine liposomes (MLV-DMPC) by apo A-I in the presence of LPS was characterized. Apo A-I may interact with MLV DMPC at the lipid transition temperature, forming micellar complexes. The kinetics of MLV-DMPC micellization was studied by turbidimetry. In the absence of LPS, a monoexponential decrease in turbidity is observed. Preincubation of apo A I with LPS impairs the micellization reaction, resulting in biphasic kinetics. The amplitude of the fast phase decreases with increasing concentrations of LPS. In the absence or in the presence of low amounts of LPS (1:0.1 protein:LPS weight ratio), two major micellization products-containing two and three apo A-I molecules per particle-were observed. However, in the presence of higher amounts of LPS (1:1 protein:LPS weight ratio), particles mainly contained two apo A-I molecules. In contrast, a decrease in intrinsic fluorescence intensity of the protein was observed in the presence of an increasing LPS concentration. Finally, we studied the effect of LPS on the transition temperature (Tt) of MLV-DMPC without detecting changes in Tt. In conclusion, the changes found in the micellization process are likely to be mainly caused by changes in the apo A-I conformation by LPS interaction in solution. PMID- 16679537 TI - In-Cell NMR spectroscopy: inhibition of autologous protein expression reduces Escherichia coli lysis. AB - Structural studies by in-cell nuclear magnetic resonance are a developing new field of research, and their objective is to obtain structural information of proteins and other biological macromolecules in the cytoplasm of Escherichia coli cells. The major limitation of in-cell experiments is cell lysis that occurs during the experiments. In this article, we describe how inhibition of autologous expression by rifampicin at a high concentration decreases cell lysis in E. coli. We suggest that rifampicin is acting in the programmed cell death gene system MazEF, which is triggered by stress conditions and ultimately leads to cell lysis. PMID- 16679538 TI - Adsorption of protein GlnB of Herbaspirillum seropedicae on Si(111) investigated by AFM and XPS. AB - The protein GlnB-Hs (GlnB of Herbaspirillum seropedicae) in diazotroph micro organisms signalizes levels of nitrogen, carbon, and energy for a series of proteins involved in the regulation of expression and control of the activity of nitrogenase complex that converts atmospheric nitrogen in ammonia, resulting in biological nitrogen fixation. Its structure has already been determined by X-ray diffraction, revealing a trimer of (36 kDa) with lateral cavities having hydrophilic boundaries. The interactions of GlnB-Hs with the well-known Si(111) surface were investigated for different incubation times, protein concentrations in initial solution, deposition conditions, and substrate initial state. The protein solution was deposited on Si(111) and dried under controlled conditions. An atomic force microscope operating in dynamic mode shows images of circular, linear, and more complex donut-shaped protein arrangement, and also filament types of organization, which vary from a few nanometers to micrometers. Apparently, the filament formation was favored because of protein surface polarity when in contact with the silicon surface, following some specific orientation. The spin-coating technique was successfully used to obtain more uniform surface covering. PMID- 16679539 TI - Cerato-platanin, the first member of a new fungal protein family: cloning, expression, and characterization. AB - The ascomycete Ceratocystis fimbriata, the causal agent of "canker stain disease," secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP similar to proteins of the hydrophobin family. PMID- 16679540 TI - Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor. AB - The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignanttransforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44 degrees C for the full length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58 degrees C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein- protein interaction. PMID- 16679541 TI - Expression and homology modeling of 2-aminobiphenyl-2,3-diol-1,2-dioxygenase from Pseudomonas stutzeri carbazole degradation pathway. AB - The enzyme 2'-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an alpha(2)beta(2) heterotetramer that presents meta cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1,082 base pair polymerase chain reaction product corresponding to carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The alpha(2)beta(2)-heterotetrameric 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5 dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3 dihydroxybiphenyl and not for protocatechuate. PMID- 16679542 TI - Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins. AB - Understanding the energetic and structural basis of protein folding in a physiological context may represent an important step toward the elucidation of protein misfolding and aggregation events that take place in several pathological states. In particular, investigation of the structure and thermodynamic properties of partially folded intermediate states involved in productive folding or in misfolding/aggregation may provide insight into these processes and suggest novel approaches to prevent misfolding in living organisms. This goal, however, has remained elusive, because such intermediates are often transient and correspond to metastable states that are little populated under physiological conditions. Characterization of these states requires their stabilization by means of manipulation of the experimental conditions, involving changes in temperature, pH, or addition of different types of denaturants. In the past few years, hydrostatic pressure has been increasingly used as a thermodynamic variable in the study of both protein folding and misfolding/aggregation transitions. Compared with other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, allowing the stabilization of partially folded states that are usually not significantly populated under more drastic conditions. Much of the recent work in this field has focused on the characterization of folding intermediates, because they seem to be involved in a variety of disease-causing protein misfolding and aggregation reactions. Here, we review recent examples of the use of hydrostatic pressure as a tool to gain insight into the forces and energetics governing the productive folding or the misfolding and amyloid aggregation of proteins. PMID- 16679543 TI - Small molecule inhibitors of lysozyme amyloid aggregation. AB - Protein amyloid aggregation is associated with a number of important human pathologies, but the precise mechanisms underlying the toxicity of amyloid aggregates are still incompletely understood. In this context, drugs capable of blocking or interfering with the aggregation of amyloidogenic proteins should be considered in strategies aimed at the development of novel therapeutic agents. Human lysozyme variants have been shown to form massive amyloid deposits in the livers and kidneys of individuals affected by hereditary systemic amyloidosis. Currently, there are no clinical treatments available to prevent or reverse formation of such amyloid deposits. We have recently described a number of di- and trisubstituted aromatic compounds that block the formation of soluble oligomers and amyloid fibrils of the beta-amyloid peptide (Abeta) and protect hippocampal neurons in culture from Abeta-induced toxicity. Here, we show that some of those compounds inhibit the formation and disrupt preformed amyloid fibrils from both human and hen egg white lysozyme. These results suggest that these small molecule compounds may serve as prototypes for the development of drugs for the prevention or treatment of different types of amyloidoses. PMID- 16679544 TI - Neurotoxicity of depleted uranium: reasons for increased concern. AB - Depleted uranium (DU) is a byproduct of the enrichment process of uranium for its more radioactive isotopes to be used in nuclear energy. Because DU is pyrophoric and a dense metal with unique features when combined in alloys, it is used by the military in armor and ammunitions. There has been significant public concern regarding the use of DU by such armed forces, and it has been hypothesized to play a role in Gulf War syndrome. In light of experimental evidence from cell cultures, rats, and humans, there is justification for such concern. However, there are limited data on the neurotoxicity of DU. This review reports on uranium uses and its published health effects, with a major focus on in vitro and in vivo studies that escalate concerns that exposure to DU might be associated with neurotoxic health sequelae. PMID- 16679545 TI - Effect of high-dose sodium selenite therapy on polymorphonuclear leukocyte apoptosis in non-Hodgkin's lymphoma patients. AB - The present study was undertaken to explore the effect of the administration of high doses of sodium selenite on apoptosis in polymorphonuclear leukocytes in patients with non-Hodgkin's lymphoma. Thirty patients with newly diagnosed non Hodgkin's lymphoma were randomly divided into two groups. Group I was treated with chemotherapy and group II received 0.2 mg/kg/d sodium selenite in addition to chemotherapy. Flow cytometry was used for the monitoring of apoptosis on peripheral blood neutrophils at the time of diagnosis and after treatment in both groups of patients. Sodium selenite administration resulted in a significant reduction in neutrophils apoptosis (82+/-10% vs 32+/-18%, p<0.05) and this was associated with significant reduction in infection rate following chemotherapy (67% vs 20%, p<0.05). Also, significant improvement in cardiac ejection fraction was observed (62+/-4% vs 69+/-5% p<0.05). It is concluded that sodium selenite administration at the dosage chosen acts as a cytoprotective agent, alleviating side effects and immunosuppressive effects of cytotoxic chemotherapeutic agents. PMID- 16679546 TI - Interaction mechanism between Cd2+ ions and DNA from the kidney of the silver crucian carp. AB - Cadmium is one of the most toxic heavy metals and is known to accumulate in freshwater food chains. The underlying mechanism for its genotoxicity has not been investigated for any freshwater fish. It has, however, been suggested that cadmium-induced carcinogenesis might involve either direct or indirect interaction of Cd2+ with DNA. The interaction between Cd2+ and DNA from the kidney of the silver crucian carp (Carassius auratus gibelio) in vitro and in vivo is investigated by spectrophotometric methods and agarose gel electrophoresis methods. Cd2+ could insert into DNA basepairs, bind to nucleic acid, and result in notable hypochromicities. The analysis of agarose gel electrophoresis proves that Cd2+ at different concentrations does not cause DNA cleavage in vitro; however, kidneys display the classical laddering degradation of DNA in vivo, which is the result of the promotion of deoxyribonuclease activity or inhibition of superoxide dismutase and catalyse activity and the accumulation of reactive oxygen species caused by Cd2+ ions in vivo. PMID- 16679547 TI - Combined administration of N-acetylcysteine and monoisoamyl DMSA on tissue oxidative stress during arsenic chelation therapy. AB - The present study deals with the therapeutic potential of combined administration of N-acetylcysteine (NAC) along with monoisoamyl DMSA (MiADMSA) against chronic arsenic poisoning in guinea pigs. Animal were exposed to 50 ppm arsenic in drinking water for 8 mo and subsequently treated for 5 consecutive days with 100 mg/kg NAC (orally) and MiADMSA (intraperitoneally), individually or in combination (50 mg/kg each). Arsenic exposure produced a significant depletion of blood delta- aminolevulinic acid dehydrate (ALAD) activity, increased the blood zinc protoporphyrin (ZPP) level, and reduced blood and liver glutathione (GSH) levels in guinea pigs. Hepatic oxidized glutathione (GSSG) and thiobarbituric acid reactive substance (TBARS) levels showed a marked increase, whereas hepatic alkaline phosphatase (ALP) activity decreased and acid phosphatase (ACP) activity increased on arsenic exposure. Significant depletion of liver transaminase activities on arsenic exposure suggests organ injury. Administration of MiADMSA, alone and in combination with NAC after arsenic exposure, was able to significantly enhance hepatic GSH and to reduce GSSG and TBARS levels compared to the arsenic control. Biochemical variables indicative of liver injury generally remained insensitive to any of these treatments. The recoveries in parameters indicative of oxidative stress were more marked in guinea pigs treated with combined administration of NAC and MiADMSA than monotherapy. Interestingly, there was a more pronounced depletion of arsenic from blood and tissues after combined treatment with NAC plus MiADMSA than MiADMSA. Blood and tissues copper, zinc, iron, and calcium concentrations showed a significant increase after arsenic exposure, which showed improvement, particularly after combined administration of MiADMSA and NAC. Based on these data, a proposal can be made that greater effectiveness in chelation treatment against chronic arsenic poisoning (i.e., turnover in the oxidative stress and removed of arsenic from the system) could be achieved by combined administration of an antioxidant (preferably having a thiol moiety) with MiADMSA. PMID- 16679548 TI - Depleted uranium is not toxic to rat brain endothelial (RBE4) cells. AB - Studies on Gulf War veterans with depleted uranium (DU) fragments embedded in their soft tissues have led to suggestions of possible DUinduced neurotoxicity. We investigated DU uptake into cultured rat brain endothelial cells (RBE4). Following the determination that DU readily enters RBE4 cells, cytotoxic effects were analyzed using assays for cell volume increase, heat shock protein 90 (Hsp90) expression, 3-[4,5-dimethylthiazol- 2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction, and lactate dehydrogenase (LDH) activity. The results of these studies show that uptake of the U3O8 uranyl chloride form of DU into RBE4 cells is efficient, but there are little or no resulting cytotoxic effects on these cells as detected by common biomarkers. Thus, the present experimental paradigm is rather reassuring and provides no indication for overt cytotoxicity in endothelial cells exposed to DU. PMID- 16679549 TI - Dietary zinc effects on zinc, calcium, and magnesium content in bones of growing rats. AB - The aim of the present study was to assess dietary zinc effects on femur weight and mineral content in growing rats. For this purpose, 70 weanling Sprague-Dawley rats were divided into four groups. Each group was subject to a diet containing 2 (BZ), 5 (DZ), 10 (MZ), and 30 (CZ) ppm zinc. The calcium and magnesium content in all diets was 5 g/kg and 507 mg/kg, respectively. The animals were kept on this regime for 28 d and then sacrificed and their femurs were removed for analysis using atomic absorption spectrophotometry. The weights of the BZ and DZ groups were significantly different from the MZ and CZ groups (38.5+/-10.5, 89.9+/-13.7, 118.6+/-13.6, and 134+/-19.9 g, p<0.01) respectively. There were no differences between the MZ and CZ groups. Femur weight also varied with dietary zinc, as it was significantly different among all groups (BZ, 265+/-49 mg; DZ, 380+/-40 mg; MZ, 452+/-54 mg; CZ, 735+/-66 mg; p<0.01). The femur zinc content varied with diets, following a different pattern than the above parameters. Femur zinc from the BZ group (51.5+/-5.4 ppm) was significantly different from the MZ and CZ groups (115.9+/-14.2 and 175.0+/-13.5 ppm, respectively), whereas the DZ group (62.5+/-11.3 ppm) did not differ from the other three groups. The femur content of calcium (BZ, 83.2+/-9.8 mg/g; DZ, 88.0+/-9.2 mg/g; MZ, 90.2+/-13.6 mg/g; CZ, 83.1+/-14.7 mg/g) and magnesium (BZ, 1.82+/-0.13 mg/g; DZ, 1.98+/-0.09 mg/g; MZ, 1.93+/-14 mg/g; CZ, 1.83+/-0.19 mg/g) were not significantly different among the groups, nor was the calcium-magnesium ratio. These results suggest that although dietary zinc deficiency retards growth and causes bone fragility, bone deposition of calcium and magnesium and its ratio are not affected. PMID- 16679550 TI - Contents of metals in some wild mushrooms: its impact in human health. AB - The concentrations of 7 metals (lead, cadmium, manganese, copper, nickel, silver, and chromium) were determined in 32 different species of wild mushrooms. The mushroom samples, which have been using for food and some medical purposes, were collected from Konya, an Inner Anatolian region of Turkey. The highest metal concentrations were determined as 39 mg/kg Pb and 3.72 mg/kg Cd in Trichaptum abietinum, 467 mg/kg Mn in Panaeolus sphinctrinus, 326 mg/kg Cu in Trametes versicolor, 69.4 mg/kg Ni in Helvella spadicea, 6.97 mg/kg Ag in Agaricus campestris, and 84.5 mg/kg Cr in Phellinus igniarius. The maximum contents are 1.52, 2.22, and 60.2 mg/kg in Pleurotus eryngii (for Pb), Amanita vaginata (for Cd), and Helvella leucomelana (for Cu), respectively. These results were compared according to the WHO/FAO standard. PMID- 16679551 TI - Brain-specific small nucleolar RNAs. AB - Small nucleolar RNAs (snoRNAs) are a group of noncoding RNAs that function mainly as guides for modification of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). A subgroup of snoRNAs was found to be predominantly expressed in the brain; and interestingly, these brain-specific snoRNAs (b-snoRNAs) appear not to be involved in modification of rRNAs and snRNAs, raising the question of what their function and targets might be. Expression studies of b-snoRNAs in mice have shown potential involvement of two b-snoRNAs, MBII-48 and MBII-52, in learning and memory. HBII-52, the human homolog of MBII-52, appears to be involved with regulation of 5-HT(2C) receptor subunit mRNA. Furthermore, several reports link the disruption of expression of a specific b-snoRNA, HBII-85, with a neurobehavioral disorder, Prader-Willi syndrome. This paper reviews the current knowledge of the properties, expression, and functions of b-snoRNAs. PMID- 16679552 TI - beta-Amyloid and endoplasmic reticulum stress responses in primary neurons: effects of drugs that interact with the cytoskeleton. AB - In vitro studies designed to probe the cellular mechanisms underlying beta amyloid (Abeta) toxicity in neurons have implicated several processes, including hyperphosphorylation of the microtubule (MT)-associated protein tau, loss of MT stability, and increased cytosolic calcium levels. Given that Alzheimer's disease involves accumulation of aggregates of two different proteins, the potential involvement of the unfolded protein response (UPR) and endoplasmic reticulum (ER) dysfunction has been suggested to lead to cell death. The relationship between these apparently divergent factors and pathways in Abeta toxicity is still unclear. In these studies we investigated the relationship between MT stability and the ER stress response in primary neurons exposed to toxic Abeta peptides in culture. In addition, nocodazole (ND) was used to determine if direct disruption of MT organization activated the UPR. Pretreatment of neurons with MT-stabilizing drugs paclitaxel (Taxol) and epothilone A prevented the induction of three indicators of the UPR induced by Abeta, ND, and thapsigargin, a compound known to inhibit the sarco-ER Ca(2+)-ATPase and deplete ER calcium stores, resulting in initiation of the UPR. In addition, treatment with MT-stabilizing drugs blocked cell death and the cytoskeletal disorganization induced by these insults. The results suggest that loss of cytoskeletal integrity is a very early step in the response to a variety of toxic stimuli and that preservation of MT stability might be important in preventing the induction of ER dysfunction and subsequent cell death by Abeta in neurons. PMID- 16679554 TI - Circadian expression of Bmal1 and serotonin-N-acetyltransferase mRNAs in chicken retina cells and pinealocytes in vivo and in vitro. AB - Unlike mammals, rhythmic changes in serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) transcripts in chicken pineal cells are controlled by an oscillator located in the pinealocytes themselves, which is comprised of clock genes. Asimilar clock-dependent pathway has been postulated to regulate the retinal melatonin rhythm. In chicken retinal photoreceptor cells and pinealocytes, the chicken AANAT gene (cAANAT) is coexpressed with clock genes, including cBmal1 and cClock, which might regulate cAANAT transcription. Here, we have studied the temporal profile of cBmal1, cClock, and cAANAT mRNAexpressions in retinal cells in vivo with chickens housed in a 14/10-h light/dark (LD) cycle for 2 wk and in vitro cultured in a superfusion system for 4 LD cycles. mRNA levels of these genes were analyzed by RT-PCR and compared with their corresponding pineal transcripts. cBmal1 mRNA showed a peak during the light phase between Zeitgeber time (ZT) 8 and 10, preceding the amplitude of the nocturnal increase in cAANAT expression at ZT 16-17. Retinal cBmal1 and cAANAT mRNAs exhibited less robust cycling than their corresponding pineal transcripts in the same animal. cClock mRNAlevels failed to exhibit a well-detectable rhythm. The phase of the rhythms of retinal cBmal1 and cAANAT mRNAs suggests a link between retinal cBmal1 and cAANAT expressions similar to the regulation of pineal cAANAT transcription. Based on the highly conserved nature of the clockwork, it is reasonable to consider that chicken retina and pineal gland might serve as a useful tool for the development of drugs that could influence clock function in man. PMID- 16679553 TI - Coenzyme Q(10) provides neuroprotection in iron-induced apoptosis in dopaminergic neurons. AB - The exact molecular mechanism of progressive loss of neuromelanin containing nigrostriatal dopaminergic neurons in Parkinson's disease (PD) remains unknown, yet evidence suggests that iron might play an important role in PD pathology. In this study we have determined the neuroprotective role of coenzyme Q(10) (CoQ(10)) in ironinduced apoptosis in cultured human dopaminergic (SK-N-SH) neurons, in metallothionein gene- manipulated mice, and in alpha-synuclein knockout (alpha-synko) mice with a primary objective to assess a possible therapeutic and anti-inflammatory potential for CoQ(10) in PD. Iron-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species production, increased metallothionein and glutathione synthesis, caspase- 3 activation, NF-kappaB induction, and decreased Bcl-2 expression, without any significant change in Bax expression. Lower concentrations of FeSO4 (1-10 microM) induced perinuclear aggregation of mitochondria, whereas higher concentrations (100-250 microM) induced CoQ(10) depletion, plasma membrane perforations, mitochondrial damage, and nuclear DNA condensation and fragmentation. FeSO(4) induced deleterious changes were attenuated by pretreatment with CoQ(10) and by deferoxamine, a potent iron chelator, in SK-N-SH cells. 1-Methyl, 4-phenyl, 1,2,3,6- tetrahydropyridine (MPTP)-induced striatal release of free iron, and NF kappaB expression were significantly increased; whereas ferritin and melanin synthesis were significantly reduced in the substantia nigra pars compacta (SNpc) of MT(dko) mice as compared with control(wt) mice, MT(trans) mice, and alpha synko mice. CoQ(10) treatment inhibited MPTP-induced NF-kappaB induction in all of the genotypes. These data suggest that glutathione and metallothionein synthesis might be induced as an attempt to combat iron-induced oxidative stress, whereas exogenous administration of CoQ(10) or of metallothionein induction might provide CoQ(10)-mediated neuroprotection in PD. PMID- 16679555 TI - Seizures induced by in vivo latrunculin a and jasplakinolide microperfusion in the rat hippocampus. AB - The molecular basis for developing epilepsy remains under debate. It is hypothesized that increased excitatory synaptic activity might activate the N methyl-D-aspartate receptor/Ca(2+) transduction pathway, which induces long lasting plasticity changes leading to recurrent epileptiform discharges. To determine if these effects are caused by disruption of F-actin in the dendritic spines, we have perfused the hippocampus of conscious rats with the F-actin depolymerizing agent latrunculin Aand the actin filament stabilizer jasplakinolide. Single perfusions of latrunculin Aand jasplakinolide decrease and increase picrotoxin seizure threshold, respectively. Repeated perfusions of both latrunculin Aand jasplakinolide induce epileptic seizures and a long-term increase in neuronal excitability. These results suggest that actin disruption might not be just a consequence but also a possible cause of epileptic seizures. We propose a new experimental model in rats to study the biochemical changes that might lead to chronic seizures and a method for testing new antiepileptic drugs. PMID- 16679556 TI - alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress. AB - Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity. PMID- 16679557 TI - alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity. AB - Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis. PMID- 16679558 TI - BACE1 gene promoter is differentially regulated: detection of a novel promoter region for its cell type-specific regulation. AB - The amyloid-beta (Abeta) peptide, the proteolytic fragment of Abeta precursor protein (APP), aggregates and forms neuritic plaques, a major hallmark of Alzheimer's disease (AD). The limiting step in generating the Abeta peptide from APP is cleavage by the beta-secretase enzyme, BACE1. Regulation of the BACE1 gene is likely to play an important role in AD etiology and treatment. We therefore studied the activity of a 4.1-kb 5'-flanking region (-3765/+364, +1 being the transcription start site) of the BACE1 gene, both in 5'- and 3'-deletion series and through Northern blotting. We show that the BACE1 promoter has regulatory activity throughout the 4.1-kb length, both positive and negative, and that this activity can be quantitatively modeled according to promoter sequence length, with the specific model depending on the presence of negative regulatory elements as the 5'- most portion of the sequence. We also examined a previously identified 141-bp proximal fragment (+224/+364) of the BACE1 promoter and two constituent (91- and 50-bp) subfragments. We report that the 91-bp fragment (+224/+314) is the most likely seat of neuronal expression of the BACE1 gene and that it is the portion of the 141-bp fragment that accounts for observed DNA-protein interactions in brain extracts. The 50-bp fragment (+315/+364), which showed significant reporter gene activity from the empty vector, binds nuclear proteins in a cell type-specific manner and contains the AP2 site as shown by the electrophoretic mobility shift assay. Overall, the 141-bp fragment had no strong matches within GenBank, and the 91-bp fragment is predicted to have several potential stem-loop sites. Taken together, BACE1 gene promoter activity is differentially regulated, and the 91-bp fragment represents a novel promoter region for cell type-specific regulation. This fragment might be a useful target to regulate BACE1 expression leading to Abeta production and to understand the neuropathogenesis of AD. PMID- 16679560 TI - Regulation of the Pleuronectes americanus Na+/H+ exchanger by osmotic shrinkage, beta-adrenergic stimuli, and inhibition of Ser/Thr protein phosphatases. AB - The ubiquitous Na+/H+ exchanger NHE1 is regulated by protein phosphorylation events, but the mechanisms involved are incompletely understood. We recently cloned NHE1 from the red blood cells of the winter flounder, Pleuronectes americanus (paNHE1), and demonstrated its activation by osmotic cell shrinkage, beta-adrenergic stimuli, and the Ser/Thr protein phosphatase PP1 and PP2A inhibitor calyculin A(CLA) (Pedersen et al. [2003] Am. J. Physiol. 284, C1561 C1576). Here, we investigate the mechanisms involved in paNHE1 activation by these stimuli. Osmotic shrinkage and CLA were only partially additive in their effects on paNHE1 activity, and CLA-mediated paNHE1 activation was inhibited by osmotic cell swelling. Activation by the beta-adrenergic agonist isoproterenol (IP) was fully additive to activation by osmotic shrinkage or CLA. IP-mediated, but neither shrinkage- nor CLA-mediated paNHE1 activation were associated with an increase in cellular cyclic adenosine monophosphate (cAMP) level. IP-mediated activation was partially blocked by the protein kinase A (PKA) inhibitor H89 (10 microM), whereas shrinkage- and CLA-mediated activation were unaffected. All three stimuli activated paNHE1 in a manner unaffected by inhibitors of protein kinase C (calphostin C, 5 microM) and protein kinase G (KT5823, 10 microM) as well as of myosin light chain kinase (ML-7, 10 microM). IP-mediated, but not shrinkage-mediated, paNHE1 activation was associated with an increase in serine phosphorylation of the paNHE1 protein. It is suggested that paNHE1 activation by osmotic shrinkage and by PP1/PP2A inhibition involves partially convergent signaling pathways, whereas activation of paNHE1 by beta-adrenergic stimuli is mediated by a separate pathway. PMID- 16679559 TI - Math1 target genes are enriched with evolutionarily conserved clustered E-box binding sites. AB - The basic helix-loop-helix (bHLH) transcription factor Math1 and its orthologs are fundamental for proper development of various neuronal subpopulations, such as cerebellar granule cells, D1 interneurons in the spinal cord, and inner ear hair cells. Although crucial for neurogenesis, the mechanisms by which Math1 specifically recognizes its direct targets are not fully understood. To search for direct and indirect target genes and signaling pathways controlled by Math1, we analyzed the effect of Math1 knockout on the expression profile of multiple genes in the embryonic cerebellum. Eighteen differentially expressed transcripts were identified and found to belong to a few developmentally-related functional groups, such as transcriptional regulation, proliferation, organogenesis, signal transduction, and apoptosis. Importantly, genomic analysis of E-box motifs has identified a significant enrichment and clustering of MATH1-binding E-boxes only in a subset of differentially expressed genes (Nr2f6, Hras1, and Hes5) in both mouse and man. Moreover, Math1 was shown by chromatin immunoprecipitation (ChIP) to bind, and by a luciferase reporter assay to activate transcription, of an upstream genomic fragment of Nr2f6. Taken together, we propose that when putative direct targets of Math1 are being selected for detailed studies on DNA microarray hybridization, the enrichment and clustering of binding E-boxes in multiple species may be helpful criteria. Our findings may be useful to the study of other bHLH transcription factors, many of which control the development of the nervous system. PMID- 16679561 TI - Biophysical studies on the differentiation of human CD14+ monocytes into dendritic cells. AB - Dendritic cells (DCs), which are the most efficient antigen-presenting cells (APCs) currently known, can be derived from CD14+ monocytes (DC predecessor cells) in vitro. Immature DCs actively take up antigens and pathogens, generate major histocompatability complex-peptide complexes, and migrate from the sites of antigen acquisition to secondary lymphoid organs to become mature dendritic cells that interact with and stimulate T-lymphocytes. During this process, the cells must undergo deformation to translocate through several barriers, including the basement membrane and interstitial connective tissue in the blood vessel wall. To further understand the mechanisms of the activation of immunological responses and the migration from peripheral tissue to secondary lymphoid organs, we have applied biophysical and microrheological methods to study the development processes of DCs in vitro. The results showed that membrane fluidity, osmotic fragility, membrane viscoelastic properties, infrared spectroscopy, and cytoskeleton organization of DCs exhibit significant differences in different developmental stages. PMID- 16679562 TI - New imidazole-coordinated chemotherapeutics with low epithelial toxicity. AB - The new imidazole-coordinated chemotherapeutics with low epithelial toxicity (NICE) presented in this article feature innovative drugs that combine epithelial toxicity comparable with that of carboplatin with novel carrier ligands optimized for DNA interaction. Recent identification of the pivotal role of basolateral organic cation transporters (OCTs) in cisplatin nephrotoxicity by a new model system (electrical resistance breakdown assay) facilitated the search for substances with a favorable organotoxic profile. The assay uses the high transepithelial electrical resistance (TEER) of the C7-clone of Madin-Darby canine kidney (MDCK) cells and the exclusive basolateral expression of OCT2 in these cells. TEER and caspase-3 activity of MDCK-C7-cells grown on microfilter membranes were monitored in response to exposure of either the apical or basolateral plasma membrane to platinum complexes. The impact of complexes on cancer cell lines was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide tests. Effects of substituents on pharmacological properties of NICE were systematically investigated by introducing sterically demanding groups as well as electron-donating and electron-withdrawing groups. Derivatives of NICE showed different renal epithelial toxic profiles and effects on cancer cells. NICE were significantly less toxic than cis- or oxaliplatin. The chlorine substituted NICE had no effect on epithelial integrity but markedly cytotoxic activity against amelanotic melanoma cells. Together, side effect targeted screening for new anticancer drugs with the electrical resistance breakdown assay offers an interesting approach for identifying and investigating new compounds. NICE feature the first group of platinum-based cytostatics discovered by using this system for systematic screening of new chemotherapeutics with low renal epithelial toxicity. PMID- 16679563 TI - Diffusion delays and unstirred layer effects at monolayer cultures of Chinese hamster ovary cells: radioligand binding, confocal microscopy, and mathematical simulations. AB - Cells grown in monolayer culture offer a convenient system for binding and other experiments under conditions that preserve the complexity of the living state. Kinetics experiments, however, may be distorted by the time course of drug penetration into even so simple a "tissue" as the monolayer. The impediments include unstirred layers both above and between the cells, the congregation of receptors within the confined space between cells, and nonspecific binding to membrane components. The contributions of these factors were investigated in cultures of Chinese hamster ovary (CHO) cells either nontransfected or stably transfected with mu opioid receptors. The dissociation of [3H]naloxone was four times faster under displacement than under infinite dilution conditions, clearly demonstrating the "retention effect" of receptors confined in space. Even the penetration of this ligand between nontransfected cells showed salient delays with respect to diffusion into a slab, indicating that nonspecific, low-affinity binding to membrane components was arresting its progress. The optical sectioning capabilities of confocal microscopy demonstrated that the kinetics of two fluorescent antagonists depended on the vertical plane, providing direct evidence for slowed diffusion down a single cell depth. Modeling shows that kinetic errors increase with receptor density, forward rate constant, and the thickness of the unstirred layer. PMID- 16679564 TI - Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates. AB - We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD's broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level. PMID- 16679565 TI - Prolonged high-pressure treatments in mammalian skeletal muscle result in loss of functional sodium channels and altered calcium channel kinetics. AB - Activation and inactivation of ion channels involve volume changes from conformational rearrangements of channel proteins. These volume changes are highly susceptible to changes in ambient pressure. Depending on the pressure level, channel function may be irreversibly altered by pressure. The corresponding structural changes persist through the post-decompression phase. High-pressure applications are a useful tool to evaluate the pressure dependence as well as pressure limits for reversibility of such alterations. Mammalian cells are only able to tolerate much lower pressures than microorganisms. Although some limits for pressure tolerance in mammalian cells have been evaluated, the mechanisms of pressure-induced alteration of membrane physiology, in particular of channel function, are unknown. To address this question, we recorded fast inward sodium (I(Na)) and slowly activating L-type calcium (I(Ca)) currents in single mammalian muscle fibers in the post-decompression phase after a prolonged 3-h, high-pressure treatment of up to 20 MPa. I(Na) and I(Ca) peak amplitudes were markedly reduced after pressure treatment at 20 MPa. This was not from a general breakdown of membrane integrity as judged from in situ high-pressure fluorescence microscopy. Membrane integrity was preserved even for pressures as high as 35 MPa at least for pressure applications of shorter durations. Therefore, the underlying mechanisms for the observed amplitude reductions have to be determined from the activation (time-to-peak [TTP]) and inactivation (tau(dec)) kinetics of I(Na) and I(Ca). No major changes in I(Na) kinetics, but marked increases, both in TTP and tau(dec) for I(Ca), were detected after 20 MPa. The apparent molecular volume changes (activation volumes) deltaV(double dagger) for the pressure-dependent irreversible alteration of channel gating approached zero for Na+ channels. For Ca2+ channels, deltaV(double dagger) was very large, with approx 2.5-fold greater values for channel activation than inactivation (approx 210 A3). We conclude, that in skeletal muscle, high pressure differentially and irreversibly affects the gating properties and the density of functional Na+ and Ca2+ channels. Based on these results, a model of high pressure-induced alterations to the channel conformation is proposed. PMID- 16679566 TI - Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by using fluorescence-based techniques. AB - An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as bimolecular fluorescence complementation (BiFC), can provide information about where and when protein protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein- mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal transduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies. PMID- 16679569 TI - Er5Ni3Al3Ge4: a quaternary variant of the NbCoB type. AB - Single crystals of pentaerbium trinickel trialuminium tetragermanium were synthesized from the elements by arc-melting. The novel compound crystallizes in the space group Pmmn, e6b2a, with all nine crystallographically unique atoms in special positions of site symmetries m.. and mm2. This compound represents a new ordered variant of the NbCoB type. Its two-layer structure is described as an intergrowth of the Er3NiAl3Ge2 (Y3NiAl3Ge2 type) and ErNiGe (TiNiSi type) structures. The coordination polyhedra are distorted pentagonal prisms around Er atoms, tetragonal prisms around Al and Ni atoms, and trigonal prisms around Ge and Ni atoms, capped with eight, four and three extra vertices, respectively. PMID- 16679567 TI - SNARE complex regulation by phosphorylation. AB - SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation. PMID- 16679570 TI - Cu3(CN)4(NH3)2Hg(CN)2: a novel interpenetrating framework formed from Cu(I), Cu(II), Hg(II) and cyanide bridges. AB - The title compound, poly[diamminehexa-mu-cyano-dicopper(I)copper(II)mercury(II)], [Cu3Hg(CN)6(NH3)2]n, has a novel threefold-interpenetrating structure of three dimensional frameworks. This three-dimensional framework consists of two dimensional network Cu3(CN)4(NH3)2 complexes and rod-like Hg(CN)2 complexes. The two-dimensional network complex contains trigonal-planar Cu(I) (site symmetry m) and octahedral Cu(II) (site symmetry 2/m) in a 2:1 ratio. Two types of cyanide group form bridges between three coordination sites of Cu(I) and two equatorial sites of Cu(II) to form a two-dimensional structure with large hexagonal windows. One type of CN- group is disordered across a center of inversion, while the other resides on the mirror plane. Two NH3 molecules (site symmetry 2) are located in the hexagonal windows and coordinate to the remaining equatorial sites of Cu(II). Both N atoms of the rod-like Hg(CN)2 group (Hg site symmetry 2/m and CN- site symmetry m) coordinate to the axial sites of Cu(II). This linkage completes the three-dimensional framework and penetrates two hexagonal windows of two two dimensional network complexes to form the threefold-interpenetrating structure. PMID- 16679571 TI - NaIn(CrO4)2.2H2O, the first indium(III) member of the krohnkite family. AB - Sodium indium(III) chromate(VI) dihydrate, NaIn(CrO4)2.2H2O, synthesized from an aqueous solution at room temperature, is the first indium(III) member of the large family of compounds with krohnkite [Na2Cu(II)(S(VI)O4)2.2H2O]-type chains. The crystal structure is based on infinite octahedral-tetrahedral [In(CrO4)2(H2O)2]- chains along [010], linked via charge-balancing Na+ cations. The slightly distorted InO4(H2O)2 octahedra are characterized by a mean In-O distance of 2.125 A. The CrO4 tetrahedra are strongly distorted (mean Cr-O = 1.641 A). The Na atom shows an octahedral coordination, unprecedented among compounds with krohnkite-type chains. The NaO6 octahedra share opposite edges with the InO4(H2O)2 octahedra to form infinite [001] chains. The hydrogen bonds are of medium strength. NaIn(CrO4)2.2H2O belongs to the structural type F2 in the classification of Fleck, Kolitsch & Hertweck [Z. Kristallogr. (2002), 217, 435 443], and is isotypic with KAl(CrO4)2.2H2O and MFe(CrO4)(2).2H2O (M = K, Tl or NH4). All atoms are in special positions except one O atom. PMID- 16679572 TI - Sr2Cu2TeO6Br2: honeycomb layers of copper(II) ions. AB - Single crystals of distrontium(II) dicopper(II) tellurium(VI) hexaoxide dibromide, Sr2Cu2TeO6Br2, were synthesized via solid state/gas phase reactions in sealed evacuated silica tubes. The building units are irregular SrO4Br3 polyhedra (a new type of coordination polyhedron around Sr(II)), CuO5 square pyramids and TeO6 octahedra. The CuO5 square pyramids and the TeO6 octahedra together form layers in the bc plane bridged by Sr atoms. The Cu atoms are arranged to form puckered honeycomb layers. The Te atom lies on an inversion centre. PMID- 16679573 TI - Dichloro(4,4'-dialkyl-2,2'-bipyridine-kappa2N,N')platinum(II), where alkyl is pentyl and heptyl. AB - Dichloro(4,4'-dipentyl-2,2'-bipyridine-kappa2N,N')platinum(II), [PtCl2(C20H28N2)], adopts a discrete pi-pi stacking structure, where the alkyl chains are located in a random manner. In contrast, dichloro(4,4'-diheptyl-2,2' bipyridine-kappa2N,N')platinum(II), [PtCl2(C24H36N2)], forms a layer structure comprised of alkyl chain layers and paired coordination sites, as observed for analogous complexes with longer alkyl chains. PMID- 16679574 TI - catena-Poly[tetrakis(3-phenylpropylammonium) [iodoplumbate(II)-tri-mu-iodo plumbate(II)-tri-mu-iodo-iodoplumbate(II)-di-mu-iodo]]. AB - The title compound, {(C9H14N)4[Pb3I10]}n, crystallizes as an organic-inorganic hybrid. As such, the structure consists of a two-dimensional inorganic layer of [Pb3I10]n(4n-) ions extending along [100]. The asymmetric unit contains two independent Pb atoms, viz. one in a general position and the other on an inversion centre. Each Pb atom is octahedrally coordinated by six iodide ions and exhibits both face- and corner-sharing with adjacent atoms in the inorganic layer. These anionic layers alternate with 3-phenylpropylammonium cations, which hydrogen bond to the iodides. Simple face-to-edge sigma-pi stacking interactions are observed between the aromatic rings that stabilize the overall three dimensional structure. This net structure has only been observed five times previously. PMID- 16679575 TI - Hydrogen-bonded sheets in racemic cis-(2,2'-bipyridyl-kappa2N,N')oxo(pentane-2,4 dionato-kappa2O,O')(thiocyanato-kappaN)vanadium(IV). AB - The title compound, [V(C5H7O2)(NCS)O(C10H8N2)], crystallizes with Z' = 2 in the space group Pbca. The molecules are linked into sheets by a combination of four C H...O hydrogen bonds and one C-H...N hydrogen bond. The four C-H...O hydrogen bonds generate chains of rings, where each chain contains just a single enantiomer of each of the two independent molecules, while the C-H...N hydrogen bond generates a chain containing both enantiomers of just one of the independent molecules. PMID- 16679576 TI - trans-Dichlorotetrabenzimidazolecadmium(II) tetrabenzimidazole: a three dimensional supramolecular structure built from C-H...pi, N-H...Cl and N-H...N hydrogen bonds. AB - The title compound, [CdCl2(C7H6N2)4].4C7H6N2, consists of a Cd(Bzim)4Cl2 complex (Bzim is benzimidazole) lying on a fourfold rotation axis in the space group P4nc, and four benzimidazole molecules which are linked to the coordinated benzimidazole unit by N-H...N hydrogen bonds. One N-H...Cl and three C-H...pi hydrogen bonds link these units into a three-dimensional supramolecular structure. PMID- 16679578 TI - The twinned crystal structure of mu-2,2'-bipyrimidine-1kappa2N1,N1':2kappa2N3,N3' bis{tris[4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dionato-kappa2O,O']terbium(III)} ethyl acetate solvate. AB - The title compound, [Tb2(C24H12F9O6S3)2(C8H6N4)].C4H8O2, has two terbium(III) centers bridged by the polyazine ligand 2,2'-bipyrimidine (bpm), which is distorted from planarity by 7.0 (2) degrees . The terminal ligand 4,4,4-trifluoro 1-(2-thienyl)butane-1,3-dione (tta) is bidentate, coordinating through the two O atoms, while the bridging ligand is bis-bidentate, coordinating through four equivalent N atoms. Both the complex and the ethyl acetate solvent molecules are disordered. The structure was refined as a non-merohedral twin. PMID- 16679577 TI - cis-Diaquabis[(E)-4-(2-hydroxybenzylideneamino)benzoato-kappa2O,O']cadmium(II): two-dimensional layers built from strong O-H...O hydrogen bonding in the coordination sphere. AB - In the title compound, [Cd(C14H10NO3)2(H2O)2], which crystallizes with Z = 4 in the space group C2c, the Cd atom is located on a twofold rotation axis and coordinated by six O atoms from two water molecules and two carboxylate groups of two planar 4-(2-hydroxybenzylideneamino)benzoate ligands, with a dihedral angle of 85.6 (1) degrees between them. Strong O-H...O hydrogen bonding in the coordination sphere, together with pi-pi stacking interactions, assemble the molecules into two-dimensional layers. PMID- 16679579 TI - Three new enantiomerically pure ferrocenylphosphole compounds. AB - The absolute configurations of three new enantiomerically pure ferrocenylphosphole compounds, namely (2S,4S,S(Fc))-4-methoxymethyl-2-[2-(9 thioxo-9lambda5-phosphafluoren-9-yl)ferrocenyl]-1,3-dioxane, [Fe(C5H5)(C23H22O3PS)], (III), (S(Fc))-[2-(9-thioxo-9lambda5-phosphafluoren-9 yl)ferrocenyl]methanol, [Fe(C5H5)(C18H14OPS)], (V), and (S(Fc))-diphenyl[2-(9 thioxo-9lambda5-phosphafluoren-9-yl]ferrocenylmethyl]phosphine, [Fe(C5H5)(C30H23P2)], (VIII), have been unambiguously established. All three ligands contain a planar chiral ferrocene group, bearing a dibenzophosphole and either a dioxane, a methanol or a diphenylphosphinomethane group on the same cyclopentadienyl. In compound (V), the occurrence of O-H...S and C-H...S hydrogen bonds results in the formation of a two-dimensional network parallel to (001). The geometry of the ferrocene frameworks agrees with related reported structures. PMID- 16679580 TI - Aqua(thiosulfato-kappa2O,S)[2,4,6-tri-2-pyridyl-1,3,5-triazine kappa3N2,N1,N6]zinc(II) hemihydrate. AB - The title compound, [Zn(S2O3)(C18H12N6)(H2O)].0.5H2O, contains two almost identical independent monomeric moieties composed of an octahedral Zn centre coordinated by a tridentate 2,4,6-tri-2-pyridyl-1,3,5-triazine (tpt) ligand, one aqua ligand and an O,S-chelating thiosulfate anion. The structure is stabilized by a solvent water molecule. Multiple strong hydrogen bonds with additional weaker pi-pi interactions between tpt groups define a multiple column spatial organization. PMID- 16679581 TI - Di-mu2-sulfito-kappa8O,O':O',O''-bis[(2,4,6-tri-2-pyridyl-1,3,5-triazine kappa3N2,N1,N6)cadmium(II)] octahydrate. AB - The title compound, [Cd2(SO3)2(C18H12N6)2].8H2O, is a dimer built up around a symmetry center, where the sulfite anion displays a so far unreported coordination mode in metal-organic complexes; the anion binds as a mu2-sulfite kappa4O,O':O',O'' ligand to two symmetry-related seven-coordinate Cd(II) cations, binding through its three O atoms by way of two chelate bites with an O atom in common, which acts as a bridge. The cation coordination is completed by a 2,4,6 tri-2-pyridyl-1,3,5-triazine ligand acting in its usual tridentate mode. PMID- 16679582 TI - Bis(mu6-cis-2,4,6,8,10,12,14,16-octamethylcyclooctasiloxane-2,4,6,8,10,12,14,16 octolato)octakis[(dimethylformamide)copper(II)] dimethylformamide solvate enclosing a pyrazine molecule. AB - The title compound, [Cu8(C8H24O2Si)2(C3H7NO)8].C4H4N2.C3H7NO, features a sandwich like cage enclosing a pyrazine molecule, both situated on a centre of inversion. In addition, the crystal structure contains one dimethylformamide molecule which is disordered over a centre of inversion. The copper layer, containing eight atoms, is located between two siloxanolate fragments. The whole structure of Cu atoms and siloxanolate rings is distorted by the pyrazine molecule, leading to an oval form. As a result, the angles between the Cu atoms differ at the copper layer. The difference in the angles could lead to some deviations in the Cu-Cu exchange interactions within the copper ring, which is of interest for molecular magnetism. PMID- 16679583 TI - Tetrakis(pentafluorobenzenethiolato-kappaS)(triphenylphosphine-kappaP)osmium(IV): a Z' = 3 structure with a supramolecular double-stranded backbone. AB - The title complex, [Os(C6F5S)4(C18H15P)], crystallizes with three independent molecules in the asymmetric unit. Two of these have very similar conformations, while in the third, the axial thiolate ligand has a rotation that differs by ca 21.5 degrees . The coordination around the metal atom is trigonal bipyramidal and the supramolecular structure involves an unusual double-stranded backbone. PMID- 16679584 TI - Poly[di-mu3-chloro-mu2-trans-1,2-di-4-pyridylethylene-dicopper(I)]: a two dimensional organic-inorganic hybrid constructed from linear CuCl clusters and bridging ligands. AB - The structure of the title compound, [Cu2Cl2(C12H10N2)](n), contains infinite CuCl staircase-like chains, which lie about inversion centres. The trans-1,2-di-4 pyridylethylene molecules also lie about inversion centres and connect the CuCl chains through Cu-N coordination bonds into a two-dimensional organic-inorganic hybrid network. The planar sheets are stacked along the c axis and associated through weak C-H...Cl interactions. The results show a reliable structural motif with controllable separation of the CuCl chains by variation of the length of the ligand. PMID- 16679585 TI - catena-Poly[[tetraaquamanganese(II)]-mu-4 (carboxylatomethylsulfanyl)phenoxyacetato]. AB - The title compound, [Mn(C10H8O5S)(H2O)4]n, a one-dimensional manganese(II) complex comprising helical chains bridged by 4 (carboxylatomethylsulfanyl)phenoxyacetate ligands has been characterized by single-crystal X-ray diffraction analysis. Hydrogen-bonding interactions between adjacent chains extend the complex into a three-dimensional supramolecular architecture. PMID- 16679586 TI - Di-mu-acetato-bis[(2-acetylpyridine thiosemicarbazonato)zinc(II)]. AB - The title compound, [Zn2(C2H3O2)2(C8H9N4S)2], is a centrosymmetric dinuclear molecule with two acetate bridging ligands in a syn-syn arrangement. The Zn(II) atom is five-coordinated in a trigonal-bipyramidal configuration by three thiosemicarbazone atoms (two N and one S) and by an O atom from each of the two acetate groups. PMID- 16679587 TI - Gamma-modification of poly[(N,N-diethyldithiocarbamato)silver(I)]. AB - In the title compound, catena-poly[[trisilver(I)-tri-mu3-N,N diethyldithiocarbamato 3'kappaS:1kappaS':2kappaS;1kappaS:2kappaS':3kappaS;2kappaS:3kappa2S,S':1'kappaS'] [Ag3(C5H10NS2)3]n, the trigonally and tetrahedrally coordinated Ag atoms are mu3 bridged by kappa3- and kappa4-S2CNEt2 ligands to form a ribbon structure along the c axis. There is a twofold axis parallel to the b axis and passing through the tetrahedrally coordinated Ag atom. The S2CNEt2 ligands coordinate the Ag atoms in eta1,eta2- and eta2,eta2-fashions, depending on the bridging S atoms. The distances between the trigonal Ag and S atoms are 2.4915 (11)-2.6205 (11) A, while those between the tetrahedral Ag and S atoms are 2.5457 (11) and 2.7145 (10) A. The shortest Ag...Ag distance between trigonal Ag atoms is 2.8336 (7) A, which indicates a weak Ag...Ag interaction, whereas the shortest distance between trigonal and tetrahedral Ag atoms is 3.463 (6) A, which is considered as non bonding. PMID- 16679588 TI - Strong hydrogen bonds in the ionic pair (1,3-diisopropyl-4,5-dimethyl-4 imidazolin-2-ylidene)ammonium trichloro(1,3-diisopropyl-4,5-dimethyl-4-imidazolin 2-ylideneamine)iron(II). AB - The title compound, (C11H22N3)[FeCl3(C11H21N3)], is one of the rare examples where an isolated ionic pair of the type [A]n+ [EMX3]n- (E is any non-metal, M is any transition metal and X is any halogen) could be structurally characterized. Two short N-H...Cl contacts between the two ammonium H atoms and two of the three Cl atoms of the counter-anion generate a six-membered ring. The third Cl atom is involved in a weaker intramolecular hydrogen bond to the neutral 1,3-diisopropyl 4,5-dimethyl-4-imidazolin-2-ylideneamine ligand. PMID- 16679589 TI - cis-Diaqua[(E)-2-(2-oxidobenzylideneamino-kappa2N,O)benzoato-kappaO]copper(II): tubes built from O-H...O hydrogen-bonding, pi-pi and C-H...pi interactions. AB - In the title compound, [Cu(C14H9NO3)(H2O)2], which crystallizes with Z = 18 in the space group R-3, pairs of complexes are linked into dimers by three O-H...O hydrogen bonds. Strong O-H...O hydrogen bonds link the dimers into one dimensional chains that further assemble into tubes through pi-pi and C-H...pi interactions. PMID- 16679590 TI - N-(2-carboxybenzoyl)-L-leucine methyl ester. AB - The title compound (with the systematic name 2-{[(1S)-1-(methoxycarbonyl)-3 methylbutyl]aminocarbonyl}benzoic acid), C15H19NO5, crystallizes in the monoclinic space group P2(1), with two independent molecules per asymmetric unit. The most notable difference between the two molecules is in the dihedral angles between the planes of the carboxyl group and the benzene ring, which are 3.5 (3) and 25.7 (1) degrees . This difference may account for the fact that two competing reactions are observed in aqueous solution, namely cyclization to form the imide N-phthaloylleucine and hydrolysis of N-(2-carboxybenzoyl)-L-leucine methyl ester to phthalic acid and leucine. PMID- 16679591 TI - An orthorhombic polymorph of 10,11-dihydrocarbamazepine. AB - The title compound (systematic name: 10,11-dihydro-5H-dibenz[b,f]azepine-5 carboxamide), C15H14N2O, is shown to crystallize as an orthorhombic polymorph to complement the known monoclinic form. The molecular conformations of both forms are very similar, involving a bent conformation for the seven-membered azepine ring and an overall ;butterfly' shape. The molecules assemble into chains by way of N-H...O bonds and N-H...pi interactions in both crystal modifications. The two polymorphs appear to form due to different van der Waals interactions between the layer-like sheets of molecules. PMID- 16679592 TI - A hydrogen-bonded dimer of 13-hydroxy-13-[(triisopropylsilyl)ethynyl]pentacen 6(13H)-one. AB - The title compound, C33H34O2Si, has been obtained as a product in the synthesis of 6,13-bis[(triisopropylsilyl)ethynyl]-6,13-dihydropentacene-6,13-diol. The solid-state structure reveals a dimer, with strong hydrogen bonds holding the two molecules in a face-to-face arrangement [O...O = 2.746 (2) A and O-H...O = 173 (2) degrees ]. Within each dimer, the pentacene units are pi-stacked (the distance between the mean least-squares planes of 22 C atoms is 3.60 A). PMID- 16679593 TI - 2'-Deoxy-7-propynyl-7-deazaadenosine: a DNA duplex-stabilizing nucleoside. AB - In the title compound, 2'-deoxy-7-propynyl-7-deazaadenosine, C14H16N4O3, the torsion angle of the N-glycosylic bond is anti [chi = -130.7 (2) degrees ]. The sugar pucker of the 2'-deoxyribofuranosyl moiety is C2'-endo-C3'-exo, 2T3 (S type), with P = 185.9 (2) degrees and tau(m) = 39.1 (1) degrees , and the orientation of the exocyclic C4'-C5' bond is -ap (trans). The 7-substituted propynyl group is nearly coplanar with the heterocyclic base moiety. Molecules of the nucleoside form a layered network in which the heterocyclic bases are stacked head-to-tail with a closest distance of 3.197 (1) A. The crystal structure of the nucleoside is stabilized by three intermolecular hydrogen bonds of types N-H... O, O-H... N and O-H... O. PMID- 16679594 TI - Di- and tripeptide segments of zervamicin II-2: Z-Thr(OBn)-Aib-N(Me)Ph and Z-Val Aib-Hyp(OBn)-OMe. AB - The title compounds, O-benzyl-N-(benzyloxycarbonyl)threonyl-2,N dimethylalaninanilide, C30H35N3O5, and methyl (4R)-4-benzyloxy-N (benzyloxycarbonyl)valyl-2-(methylalanyl)prolinate, C30H39N3O7, were obtained from the ;azirine coupling' of the corresponding protected amino acids with 2,2,N trimethyl-2H-azirin-3-amine and methyl (4R)-4-(benzyloxy)-N-(2,2-dimethyl-2H azirin-2-yl)prolinate, respectively. The Aib unit in each molecule has the greatest turn- or helix-inducing effect on the molecular conformation. Intermolecular N-H...O interactions link the molecules of the tripeptide into sheets and those of the dipeptide into extended chains. PMID- 16679595 TI - ent-7alpha,18-Hydroxykaur-16-ene ethanol solvate. AB - The ent-kaurene diterpene in the title compound, 7-epicandicandiol ethanol solvate, C20H32O2.C2H6O, was isolated from the aerial parts of Sideritis ozturkii Aytac & Aksoy. The molecule has the usual conformation and stereochemistry found in related ent-kaurene derivatives. The methyl-substituted ring junction has a trans arrangement and the other junction is cis. The six-membered rings have chair or slightly distorted chair conformations and the five-membered ring has an envelope conformation. Intermolecular hydrogen bonds link the 7-epicandicandiol and ethanol molecules into two-dimensional networks, part of which comprise co operative O-H...O-H...O-H... chains. PMID- 16679596 TI - N-(2-Methoxy-6-oxo-1,6-dihydropyrimidin-4-yl)formamide: hydrogen-bonded sheets of centrosymmetric R(2)2 (8) and R(6)4 (28) rings. AB - Molecules of the title compound, C6H7N3O3, are linked into sheets of centrosymmetric R(2)2 (8) and R(6)4 (28) rings by two nearly linear N-H...O hydrogen bonds [H...O = 1.91 and 1.98 A, N...O = 2.786 (3) and 2.862 (3) A, and N H...O = 175 and 177 degrees ]. PMID- 16679597 TI - Two polymorphic forms of N-(4-chlorophenyl)-5-[(4-chlorophenyl)aminomethyl]-6 methyl-2-phenylpyrimidin-4-amine. AB - Two polymorphic forms of the title compound, C24H20Cl2N4, were obtained and characterized using X-ray crystal structure analysis. Colourless crystals of polymorph (Ia) were obtained from the oily mother residue. Recrystallization of polymorph (Ia) from an acetone-methanol mixture resulted in pale-yellow crystals of polymorph (Ib). The major feature distinguishing the two polymorphic forms is their interaction modes, and hence their packing arrangements. In the crystal structure of polymorph (Ia), there are N-H...N hydrogen bonds and also aromatic pi-pi stacking interactions between molecules. The molecules of polymorph (Ib) are linked by N-H...Cl hydrogen bonds only. PMID- 16679598 TI - Morpholine-2,5-dione. AB - In the title compound, C4H5NO3, the morpholine ring adopts a boat conformation that is distorted towards twist-boat, the boat ends being the two Csp3 atoms of the ring. The molecular packing is stabilized by the establishment of strong intermolecular NH...OC hydrogen bonds, which give rise to centrosymmetric dimers, and a network of weak CH2...OC hydrogen bonds, where each dimer interacts with eight neighbouring morpholinedione rings. PMID- 16679599 TI - Ropinirole hydrochloride, a dopamine agonist. AB - Ropinirole hydrochloride, or diethyl[2-(2-oxo-2,3-dihydro-1H-indol-4 yl)ethyl]ammonium chloride, C16H25N2O+.Cl-, belongs to a class of new non ergoline dopamine agonists which bind specifically to D2-like receptors with a selectivity similar to that of dopamine (D3 > D2 > D4). The N atom in the ethylamine side chain is protonated and there is a hydrogen bond between it and the Cl- ion. In the crystal structure, two cations and two anions form inversion related cyclic dimers via N-H...Cl hydrogen bonds. PMID- 16679600 TI - Symmetric hydrogen bonding in dimethylammonium hydrogen diphenyldiphosphonate. AB - In the title compound, C2H8N+.C12H11O5P2-, pairs of hydrogen diphenyldiphosphonate anions form dimers across a twofold axis, with two symmetric O...H...O hydrogen bonds [O...O = 2.406 (3) and 2.418 (3) A]. The 12 membered ring thus formed has crystallographic 2 and quasi-222 symmetry. Cations on either side of the ring form N-H...O hydrogen bonds to the four extraannular O atoms, with N...O distances of 2.765 (2) and 2.748 (3) A. PMID- 16679601 TI - Hydrogen bonding in isomorphous 2-methyl-4-nitroanilinium bromide and iodide. AB - The isomorphous structures of 2-methyl-4-nitroanilinium bromide, C7H9N2O2+.Br-, and 2-methyl-4-nitroanilinium iodide, C7H9N2O2+.I-, exhibit ionic layers separated by hydrocarbon layers. The hydrocarbon layer stacks head-to-head, while in the ionic layer, the ammonium groups and halide anions interact via hydrogen bonds to form infinite chains. PMID- 16679602 TI - Bis(glycinium) oxalate: evidence of strong hydrogen bonding. AB - In the title 2:1 salt, 2C2H6NO2+.C2O4(2-), the glycine molecule is in the cationic form with a positively charged amino group and an uncharged carboxylic acid group. The doubly charged oxalate anion lies across a crystallographic inversion centre. One of the reasons why the 1:1 glycinium oxalate salt has a higher melting point than the title compound may be the difference in their hydrogen-bonding patterns. A database search for salts formed between amino acids or substituted amino acids and oxalic acid revealed that, in most of the structures, the conformation about the O=C-OH bond is synplanar. D-Tryptophan oxalate is the only example where the OH group of a semi-oxalate adopts an antiplanar conformation. The 2:1 stoichiometry seen in the present salt is observed only in the salts of DL-serine, DL-aspartic acid and betaine with oxalic acid. PMID- 16679603 TI - Conformations of three heterocyclic perhydropyrrolobenzofurans and polymeric assembly via co-operative intermolecular C-H...O hydrogen bonds. AB - In 1-cyclohexyl-6,6,8a-trimethyl-3a,6,7,8a-tetrahydro-1H-1-benzofuro[2,3 b]pyrrole-2,4(3H,5H)-dione, C19H27NO3, (I), and the isomorphous compounds 6,6,8a trimethyl-1-phenyl-3a,6,7,8a-tetrahydro-1H-1-benzofuro[2,3-b]pyrrole-2,4(3H,5H) dione, C19H21NO3, (II), and 6,6,8a-trimethyl-1-(3-pyridyl)-3a,6,7,8a-tetrahydro 1H-1-benzofuro[2,3-b]pyrrole-2,4(3H,5H)-dione, C18H20N2O3, (III), the tetrahydrobenzo-dihydrofuro-pyrrolidine ring systems are folded at the cis junction of the five-membered rings, giving rise to a non-planar shape of the tricyclic cores. The dihydrofuran and pyrrolidine rings in (I) are puckered and adopt an envelope conformation. The cyclohexene rings adopt a half-chair conformation in all the molecules, while the substituent N-cyclohexyl ring in (I) assumes a chair form. Short intramolecular C-H...O contacts form S(5) and S(6) motifs. The isomorphous compounds (II) and (III) are effectively isostructural, and aggregate into chains via intermolecular C-H...O hydrogen bonds. PMID- 16679604 TI - A dimer of alpha- and beta-dihydroartemisinin: bis(3,6,9-trimethyl-3,12-epidioxy 3,4,5,5a,6,7,8,8a,9,10-decahydro-12H-pyrano[4,3-j][1,2]benzodioxepin-10-yl) ether. AB - The title compound, C30H46O9, prepared from a mixture of alpha- and beta dihydroartemisinin, has alpha- and beta-arteether moieties linked via an -O- bridge, so that the molecule is asymmetric about the bridge. The endoperoxide bridges of the parent compounds have been retained in each half of the ether bridged dimer. The rings exhibit chair and twist-boat conformations. PMID- 16679605 TI - 18-Deoxy-13beta,14-dihydrolycoctam: the lycoctamone rearrangement confirmed. AB - The structure of the title compound, C23H35NO4, contains a unique pentacyclic ring system wherein one cyclohexyl ring adopts a chair conformation, two cyclohexyl rings are in boat conformations, and a six-membered heterocyclic ring and a cyclopentyl ring are in envelope conformations. The structures of the lycoctamones, alpha,beta-unsaturated aldehydes produced by acid-catalyzed degradation of lactams of lycoctonine-type alkaloids, previously deduced from the results of extensive chemical investigations have been proven to be correct by the determination of the crystal structure of this compound. PMID- 16679606 TI - N-[1-(Pentafluorophenyl)ethyl]acetamide. AB - The title compound, C10H8F5NO, crystallizes as a racemate with four symmetry independent molecules in the asymmetric unit. The four molecules form two hydrogen-bonded pairs. Each pair is a building unit of an independent C4 chain propagating parallel to the ab plane. PMID- 16679607 TI - Unusual conformations of 1,3-dialkoxythiacalix[4]arenes in the solid state. AB - The structures of three syn-1,3-dialkoxythiacalix[4]arenes with unusual conformations in the solid state are reported. The pinched cone conformation of syn-2(2),4(2)-dihydroxy-1(2),3(2)-bis(prop-2-enyloxy)thiacalix[4]arene, C30H24O4S4, (3a), is stabilized by two intramolecular hydrogen bonds, remarkably formed from both OH groups to the same ether O atom. In syn-2(2),4(2)-dihydroxy 1(5),2(5),3(5),4(5)-tetranitro-1(2),3(2)-bis(prop-2-enyloxy)thiacalix[4]arene acetone disolvate, C30H20N4O12S4.2C3H6O, (3b1), the molecule is found in the 1,3 alternate conformation. The crystallographic C2 symmetry is due to a twofold rotation axis running through the centre of the calixarene ring. The hydroxy groups cannot form intramolecular hydrogen bonds as in (3a) and both are bonded to an acetone solvent molecule. The molecule of the pseudo-polymorph of (3b1) in which the same compound crystallized without any solvent, viz. (3b2), is located on a crystallographic mirror plane. Only one of the two hydroxy groups forms a hydrogen bond, and this is with a nitro group of a neighbouring molecule as acceptor. Molecular mechanics calculations for syn-1,3-diethers suggest a preference of the 1,3-alternate over the usual cone conformation for thiacalix[4]arene versus calix[4]arene and for para-nitro versus para-H derivatives. PMID- 16679608 TI - A three-dimensional framework of pi-stacked hydrogen-bonded chains in benzyl 4 chloro-3-nitrobenzoate, and chains of hydrogen-bonded rings in benzyl 4 nitrobenzoate, redetermined at 120 K. AB - Benzyl 4-chloro-3-nitrobenzoate, C14H10ClNO4, crystallizes with Z' = 2 in the space group P-1. The molecules are linked by three independent C-H...O hydrogen bonds into chains of edge-fused R(4)4 (26) and R(4)4 (34) rings, and these chains are linked into a three-dimensional framework structure by aromatic pi-pi stacking interactions. In benzyl 4-nitrobenzoate, C14H11NO4, the molecules are linked by two independent C-H...O hydrogen bonds into chains containing two types of R(2)2 (10) ring. PMID- 16679609 TI - L-Argininamidium bis(hydrogensquarate). AB - Cations and anions of the title compound {systematic name: 1-[4 (aminocarbonyl)butyl]guanidinium bis(hydrogensquarate)}, C6H17N5O2+.2C4HO4(-), are connected into a three-dimensional network by intermolecular N-H...O hydrogen bonds between the L-argininamidium ammonium, amide and guanidinium functions and the hydrogensquarate carbonyl O atoms. The independent hydrogensquarate monoanions are linked into dimers by pairs of O-H...O' hydrogen bonds. PMID- 16679610 TI - 2-(Methoxycarbonyl)phenylboronic acid. AB - The structure of the title compound, 2(CH3OCO)C6H4B(OH)2 or C8H9BO4, involves two crystallographically independent conformers, A and B, in a 1:2 ratio; molecules of conformer A are located on a crystallographic mirror plane. The most striking difference between the two independent molecules is the opposite orientation of the methoxycarbonyl groups, while the conformations of the boronic acid groups vary more subtly. Molecules of both types are ordered to produce a specific hydrogen-bonding network that can be interpreted in terms of a layer lying parallel to (100). Within the layer, B molecules are linked with each other by two different O-H...O bonds to form an infinite chain where two centrosymmetric dimeric motifs can be distinguished. PMID- 16679611 TI - 2-(4-Chloroanilino)- and 2-(4-methoxyanilino)-1,2-diphenylethanone. AB - The title compounds, C20H16ClNO and C21H19NO2, adopt syn orientations of the C=O and N-H bonds but, like their analogues, form no strong intermolecular hydrogen bonds. PMID- 16679613 TI - [Historical background and recent advance in BMP research]. AB - In 1971, Uurist gave the name bone morphogenetic protein (BMP) to the factor in bone matrix, which retains the activity to induce ectopic bone formation. After that, BMP research has rapidly developed by achieving BMP purification, BMP cloning, and identification of BMP receptors and signal transuding molecules (Smads). In this review, I overview the historical background and recent advance in BMP research. PMID- 16679614 TI - [Regulation of BMP signaling]. AB - Bone morphogenetic proteins (BMPs), members of transforming growth factor-beta (TGF-beta) superfamily, bind to two different serine/threonine kinase receptors, and mediate their signals through Smad-dependent and -independent pathways. Intracellular signaling of BMPs is regulated by various modifiers and post transcriptional modifications including phosphorylation and ubiquitination. PMID- 16679615 TI - [Application of bone morphogenetic protein (BMP) in regenerative medicine of bone]. AB - Main purpose of bone morphogenetic protein (BMP) related research in clinical setting is the development of new efficient technologies to regenerate bone tissue effectively by utilizing BMP's osteoinductivity. Problems to be solved for that purpose are the development of efficient career system for BMP and the exploitation of methods to promote efficacy of BMP's bone forming activity. These new technologies will enable orthopaedic surgeons to replace currently used autografts with BMP combined implants. Additionally, they are also applicable for the acceleration of bone healing, treatment of non-union, spinal fusion, reconstruction of bone defects after the resection of bone tumor or arthroplasty. Thus, these new technologies are expected for the breakthrough in clinical applications of BMP. PMID- 16679616 TI - [Fracture repair and bone morphogenetic protein (BMP)]. AB - The process of fracture healing involves a number of regenerative mechanisms underlying the skeletal systems, and bone morphogenetic protein (BMP) has been believed to be a key molecule during the reaction. Recent investigations showed that several members of BMPs are induced and activated by the impact of fracture, and play important roles via BMP receptors/Smads pathways. BMPs are newly synthesized by callus-forming cells near the fracture site, and form a "BMP network" at the fracture callus. The "BMP-network" contributes to the regulation of callus formation, and the network contains noggin, sonic hedgehog (Shh), hepatocyte growth factor (HGF), and vascular endothelial cell growth factor (VEGF). Potential roles of BMPs during fracture repair open the way towards the formulation of new therapeutic strategies for promising fracture management. Recent molecular technologies with use of recombinant BMP and gene therapy will lead to the development of less-invasive and more successful fracture treatment. PMID- 16679617 TI - [Transcriptional regulation of BMP responsive genes]. AB - Bone morphogenetic proteins (BMPs) bound to specific receptors expressed on cell membrane of target cells, then activate their specific intracellular signaling pathways. These signaling activate transcription of BMP-specific target genes. Recently, various types of genes are identified as the BMP target genes. Some critical signaling molecules are found though studies on early responsive genes of BMPs. PMID- 16679618 TI - [Roles of the BMP family in pattern formation of the vertebrate limb]. AB - In the process of limb development, various signaling molecules are produced in the organizing center of the limb bud. These molecules regulate pattern formation and the morphogenesis of the limb. Members of the bone morphogenetic protein (BMP) family and their receptors are expressed in the limb bud in spatiotemporal specific patterns. BMP signaling regulates chondrogenesis of limb mesenchyme, and promotes apoptosis of the interdigital soft tissue. Recently, this signaling was found to be involved in establishment of dorsoventral polarity of the limb bud and in regulation of limb growth via maintenance of the apical ectodermal ridge. BMPs also regulate digit morphology. In these events, BMPs work together with other signaling molecules, but they sometimes act as negative regulators of these other molecules so that limb morphogenesis can proceed normally. PMID- 16679619 TI - [Roles of BMP in the development of the central nervous system]. AB - Bone morphogenetic protein (BMP) signaling is involved in astrocytic differentiation of neural precursor cells. In the nuclei, BMP-downstream transcription factors, i.e. Smad proteins, induce expression of astrocyte specific genes in cooperation with another cytokine signaling, while inhibiting neuronal and oligodendrocytic differentiation-inducing transcription factors. This cross-inhibitory mechanism contributes to exclusive astrocytic differentiation. Crosstalk among cytokine signaling pathways thus is important for the development of the central nervous system. PMID- 16679620 TI - [Bone morphogenetic proteins and their related molecules in the skeletal tissue]. AB - Biochemical studies revealed bone morphogenetic protein (BMP) receptors and intracelluar Smad proteins that mediate BMP signals. These pathways are regulated extracelluarly by antagonists such as noggin and intracellularly by Smads 6 and 7. Transgenic and conditional knockout mice have been generated and analyzed to clarify function of these molecules in skeletal tissues by using bone- or cartilage-specific promoter sequences. PMID- 16679621 TI - [Experimental spinal fusion in a rabbit model with new drug delivery system for BMP]. AB - Bone morphogenic protein (BMP) is a powerful bone-inducing cytokine and has been used as a new bone graft substitute with bovine-derived collagen sponge as a delivery system. Excellent clinical results have been reported with use of these BMP-retaining implants. However, certain problems remain with use of them, such as the need for high doses of BMP to obtain new bone formation, resulting in high cost, and the potential risks for disease transmission (e.g., variant Creutzfeldt Jacob disease). To avoid use of collagen, we have developed a synthetic polymer/beta-Tricalcium Phosphate composite as a new delivery system for BMP. This new material, when used in combination with low doses of BMP-2, consistently elicited new bone and was successfully used to achieve posterolateral spinal fusion in a rabbit model. PMID- 16679622 TI - [Mechanism of transcriptional regulation by Runx2 in osteoblasts]. AB - Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and the expression is regulated by many factors including bone morphogenetic protein (BMP). Core binding factorbeta (Cbf beta) is required for DNA binding of Runx2 and Runx2-dependent transcription, and Cbf beta regulates Runx2 function isoform-dependently. Further, ability of Runx2 for transcriptional activation is regulated by many molecules. Phosphoinositide 3 kinase (PI3K)-Akt signaling enhances DNA binding of Runx2 and Runx2-dependent transcription, and Runx2 upregulates PI3K subunits (p85 and p110 beta) and Akt. Therefore, Runx2 and PI3K-Akt signaling are mutually dependent each other in the regulation of osteoblast and chondrocyte differentiation and their migration. PMID- 16679623 TI - [BMP and osteoclastogenesis]. AB - Bone morphogenetic protein (BMP)-2 markedly enhanced osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand (RANKL). RANKL expressed by osteoblasts is a requirement for osteoclastogenesis, osteoblasts also play important roles in osteoclastogenesis through offering the critical microenvironment for the action of RANKL. PMID- 16679624 TI - [Role of Wnt in bone formation]. AB - Recently, large body of evidences that Wnt plays an important role in regulation of tissue patterning and skeletogenesis including membranous and enchondral ossification is accumulating. Several genetic studies also indicate that low density lipoprotein receptor related protein (LRP) 5, LRP6, beta-catenin and lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) participate in osteogenic action induced by Wnt family members. In contrast, secreted Frizzled related proteins and dickkopf (Dkk) proteins, natural Wnt inhibitory factors, inhibit Wnt-dependent osteogenesis, presumably functioning as a negative feedback loop. In addition, Wnt signaling interacts with BMP signaling, thereby modulating its osteogenic action. Thus, the network systems of Wnt signaling temporally and spatially controls osteogenic action of Wnt family members. PMID- 16679625 TI - [Effect of recombinant human bone morphogenetic protein on preventing atrophic nonunion]. AB - Severe periosteal and soft tissue disruption at the time of fracture may result in the formation of an atrophic nonunion. The purpose of this study was to evaluate whether the immediate application of recombinant human bone morphogenetic protein (BMP) to the fracture site could rescue the healing process in this nonunion model. In this atrophic nonunion model, we have demonstrated that the immediate application of BMP at the time of injury may rescue the fracture healing process and prevent the development of atrophic nonunion. Early application of BMP may obviate the need for additional surgical treatment, such as autogenous bone grafting, in situations where there is significant periosteal disruption. PMID- 16679626 TI - [Interactions of chondrocytes and osteoblasts during endochondral bone formation]. AB - During endochondral bone formation, mesenchymal condensations, chondrocyte differentiation and proliferation, termination of proliferation, hypertrophic differentiation, and replacement by bone occur sequentially. This sequence is spatially represented by the structure of the growth plate of the embryo, and reflects the evolution of bone. Endochondral bone formation is mainly regulated by the interactions of chondrocytes and osteoblasts; a variety of signals are implicated in this regulation. The roles of factors regulating chondrocyte proliferation and hypertrophy including the Sox trio, PTH related peptide (PTHrP), Indian hedgehog (Ihh), the runt-related transcription factor (Runx) family, fibroblast growth factor receptor 3 (FGFR3) and bone morphogenetic protein (BMP) and other factors including hypoxia-inducible factor 1alpha (HIF 1alpha) and vascular endothelial growth factor (VEGF) will be discussed. PMID- 16679627 TI - [Circadian rhythms in cartilage]. AB - Circadian rhythms in cartilage have been reported repeatedly. However, previous studies used histological analysis or radioisotope-labeled precursors for DNA, collagen and proteoglycan synthesis, and thus it is difficult precisely to evaluate such studies on circadian rhythms in chondrocytes. On the other hand, circadian rhythms in plasma levels of several hormones, which play crucial roles in cartilage metabolism, proved to be significant both in human and animal models. In addition, clock genes--such as Clock, Bmal, Per, Cry and Dec--were identified in suprachiasmatic nucleus (SCN) and some peripheral tissues. These clock genes may be involved in circadian rhythms in cartilage. PMID- 16679628 TI - [Long-term suppressive effect of falecalcitriol on parathyroid hormone secretion in secondary hyperparathyroidism in hemodialysis patients]. AB - Alfacarcidol and calcitriol are widely used to treat secondary hyperparathyroidism associated with chronic renal failure, but it is often not possible to administer doses high enough to sufficiently inhibit parathyroid hormones because of the risk of hypercalcemia and hyperphosphatemia. We administered falecalcitriol (Hornel) Tablets) to patients with poorly controlled secondary hyperparathyroidism. The usefulness of falecalcitriol was demonstrated by the fact that control of intact-PTH was maintained for up to 24 months without a clear increase in serum Ca x serum inorganic phosphorus (iP), iP, and ALP levels. PMID- 16679629 TI - Variations in the cerebral venous anatomy and pitfalls in the diagnosis of cerebral venous sinus thrombosis: low field MR experience. AB - BACKGROUND: Knowledge of variations in the cerebral venous anatomy and apparent signal abnormalities seen on Magnetic resonance (MR) angiography are essential to avoid over-diagnosis of cerebral venous sinus thrombosis (CVST), while interpreting the MR angiograms. AIMS: To evaluate the variations of cerebral venous anatomy and signal abnormalities by using 3D phase contrast MR angiography performed in a 0.5 Tesla MRI scanner. MATERIALS AND METHODS: One hundred patients who underwent MR imaging and MR angiography examinations at our institution from March 2004 to February 2005, with normal MR imaging of brain were studied retrospectively. Patients with clinical suspicion of CVST and patients who underwent color doppler evaluation for suspected deep vein thrombosis were excluded. RESULTS: The superior sagittal, straight sinus and the internal cerebral veins were visualized in all patients. There was hypoplasia of the right transverse sinus in 13 patients, left transverse sinus in 35 patients, right sigmoid sinus in 6 patients and left sigmoid sinus in 19 patients. Absence of transverse sinus on left side was observed in one patient and absence of sigmoid sinus in 2 patients on left side. Flow gaps were observed in non-dominant transverse sinus, sigmoid sinus as well as transverse sigmoid sinus junctions. The occipital sinus was visualized in 17 patients. CONCLUSIONS: MR angiography done at low field strengths is also a reliable method, for assessing cerebral venous sinuses. Awareness of the normal anatomical variations of venous sinuses and apparent MR angiographic flow gaps prevent misdiagnosis of cerebral venous sinus thrombosis. PMID- 16679630 TI - Results of a single blind, randomized, placebo-controlled clinical trial to study the effect of intravenous L-carnitine supplementation on health-related quality of life in Indian patients on maintenance hemodialysis. AB - BACKGROUND: Carnitine insufficiency is responsible for various co-morbid conditions in maintenance hemodialysis (MHD) patients. L-carnitine supplementation is expected to improve the quality of life (QoL) of patients on MHD. AIMS: To study the effect of L-carnitine supplementation on QoL of Indian patients on MHD. SETTING AND DESIGN: This was a single (patient) blind, randomized, placebo-controlled clinical trial conducted on patients on MHD attending hemodialysis unit of the study center. MATERIALS AND METHODS: Twenty patients on MHD suffering from hemodialysis-related symptoms were randomly assigned to receive intravenous L-carnitine 20 mg/kg or placebo after every dialysis session for 8 weeks. SF36 (Short Form with 36 questions) score for QoL, laboratory investigations and dialysis related symptoms were recorded at baseline and after 8 weeks. Improvement in QoL, laboratory parameters and dialysis related symptoms in the two groups after 8 weeks was compared. STATISTICAL ANALYSIS USED: Depending on normality of data, unpaired T test or Mann Whitney U test was used for comparison of change (8 weeks-baseline) in SF36 scores and laboratory parameters observed in the two groups. RESULTS: L-carnitine supplementation increased total SF36 score by 18.29 +/- 12.71 (95% CI: 10.41 to 26) while placebo resulted in reduction in total SF36 score by 6.4 +/- 16.39 (95% CI: -16.59 to 3.73). L-carnitine also resulted in significant increase in hemoglobin and serum albumin and decrease in serum creatinine as compared to placebo. More patients were relieved of dialysis related symptoms in L-carnitine group. CONCLUSION: Intravenous L-carnitine supplementation improves QoL in patients on MHD. PMID- 16679631 TI - A case of herniated gravid uterus through a laparotomy scar. AB - A 36-year-old manual worker presented in her second pregnancy at 34 weeks of gestation with an unusual bulge of her abdomen. The lower abdominal bulge turned out to be her gravid uterus herniated through an anterior abdominal wall incisional hernia which is a rare but serious obstetric situation with complications such as premature labour, intrauterine growth retardation, strangulation, intrauterine death and rupture of the lower uterine segment been reported. We had a successful outcome by conservative treatment till 38 weeks of gestation followed by an elective lower segment Caesarean section with hernia repair. Incisional hernia is a frequent complication of abdominal wall closure and the management of pregnancy with a large incisional hernia with gravid uterus in its sac is challenging. PMID- 16679632 TI - Cefoperazone / sulbactam induced hyponatremia. PMID- 16679633 TI - Extranodal non-Hodgkin's lymphoma of the parapharyngeal space. PMID- 16679634 TI - Stem cells in orthopedics: current concepts and possible future applications. AB - Stem cells are the cells that have the ability to divide for indefinite periods in culture and to give rise to specialized cells. Sources of these cells include embryo, umbilical cord and certain sites in adults such as the central nervous system [CNS] and bone marrow. Its use hold promise of wide spread applications particularly in areas of spinal cord injury, difficult non-unions, critical bone defects, spinal fusions, augmentation of ligament reconstructions, cartilage repair and degenerative disc disorders. This review article contains current information derived from Medline searches on the use in various orthopedic subspecialties. Some issues remain at the forefront of the controversy involving stem cell research - legislation, ethics and public opinion, cost and concentration methods. As is true with any new technology, the enthusiasm for this technology that has potential to influence virtually every orthopedic case management, must be balanced by subjecting it to stringent clinical and basic research investigations. PMID- 16679635 TI - Ethics and neurosurgery. PMID- 16679636 TI - Synergy among the neuro-specialists. PMID- 16679637 TI - Medulloblastomas: new directions in risk stratification. AB - Medulloblastomas (MBs) are the most common malignant brain tumors in children. Current therapeutic approaches combine surgery, radiotherapy, and chemotherapy. Although, there has been significant improvement in long-term survival rates, the tumor remains incurable in about a third of patients while cognitive deficits and other sequelae of therapy are common among long-term survivors. Hence a major challenge remains to differentiate high-from low-risk patients and to tailor therapy based on the degree of biological aggressiveness. A clinical risk stratification system has been widely used in MBs based on age, extent of resection and the Chang staging system. However, recent reports indicate that these clinical variables are inadequate methods of defining disease risk. This has prompted search for new markers for MB stratification. Recent studies indicate that the classification of MBs according to profiles of histopathology and molecular abnormalities possibly help better risk-stratification of patients, thereby rationalizing approaches to therapy, increasing cure rate, reducing long term side effects and developing novel therapeutic strategies. The most accurate outcome prediction till date has been obtained through microarray gene expression profiling. In this article, the current histopathological classification and the recent advances in molecular genetics of MBs are reviewed. Global efforts to translate this knowledge of disease biology into clinical practice especially as outcome predictors are highlighted. PMID- 16679638 TI - Early recombinant factor VIIa therapy in acute intracerebral hemorrhage: promising approach. AB - Intracerebral hemorrhage (ICH) is the most devastating form of stroke with a high morbidity and mortality. ICH constitutes about 20-30% of all strokes, with the prevalence being higher in Asian population. Treatment of ICH is predominantly conservative, which includes control of blood pressure, use of anti-cerebral edema measures such as mannitol and mechanical ventilation. The benefit of early surgery in ICH is debatable. Initial hematoma volume and subsequent growth in its size are important predictors of a poor outcome in ICH. This means that therapies aimed at preventing hematoma enlargement in the earliest possible window period could lead to a better outcome in ICH. Recombinant factor VIIa (rFVIIa) is one such agent, which has been shown to prevent hematoma expansion and improve outcome in acute ICH. The purpose of the current review is to focus on the evidence regarding the usefulness of rFVIIa in acute ICH. PMID- 16679639 TI - A mathematical model for predicting the outcome in moderate head injury. AB - BACKGROUND: Virtually all the literature on head injury has focused on the outcome prediction of severe and mild head injuries and very few studies have been dedicated to patients sustaining moderate head injuries. AIM: To identify the patient following moderate head injury who may die, develop severe disability or significant cognitive and behavioral problems on the first day of injury itself. SETTING: Tertiary teaching hospital. DESIGN: Prospective study divided into two groups. MATERIALS AND METHODS: The study included 85 patients whose Glasgow coma scale score were 9-12 and who had isolated moderate head injury. Among the above patients a preliminary prospective study was conducted in first group of 64 patients using 7 clinical factors, 18 neuro-behavioral sequel and CT brain data in prediction of outcome with moderate head injury. From the results obtained in the above study three statistically significant factors were identified and a mathematical model was developed and used prospectively in the next 21 patients and its accuracy was evaluated. STATISTICAL METHODS USED: Multiple regression analysis and Kendall's tau non- parametric test using statistical package for social sciences (SPSS 11-5-version) were used to find out the predictive factors. RESULTS: Results of these patients showed combination of CT scan brain data, verbal response and neurological signs could provide a reliable prediction in moderate head injury. CONCLUSION: Based upon the above results a mathematical model was developed giving a value for the above-mentioned factors. The mathematical model was "CT brain data x (Verbal response + Neurological Signs)". Its overall accuracy when used on the day of admission was around 80%. PMID- 16679640 TI - Verbal and visual memory in patients with early Parkinson's disease: effect of levodopa. AB - BACKGROUND: The effect of initiation of levodopa therapy on the memory functions in patients with Parkinson's disease remains poorly understood. AIMS: To evaluate the effect of initiation of levodopa therapy on memory, in patients with early Parkinson's disease. SETTINGS AND DESIGN: Prospective case control study. MATERIALS AND METHODS: Seventeen patients with early Parkinson's disease were evaluated for verbal memory using Rey's auditory verbal learning test, and visual memory using the Benton's visual retention test and Form sequence learning test. UPDRS scores, Hoehn and Yahr's Staging and Schwab and England scores of Activities of daily living. Hamilton's depression rating scale and MMSE were also evaluated. Six controls were also evaluated according to similar study protocol. Levodopa was then prescribed to the cases. Same tests were repeated on all the subjects after 12 weeks. RESULTS: The mean age of the patients was 59.8 (+ 12.9 yrs); mean disease duration of 3.26 (+ 2.06 yrs). The mean UPDRS scores of patients were 36.52 (+ 15.84). Controls were of a similar age and sex distribution. A statistically significant improvement in the scores on the UPDRS, Hamilton's depression scale, Schwab and England scale, and a statistically significant deterioration in the scores of visual memory was observed in patients with PD after starting levodopa, as compared to their baseline scores. There was no correlation between degree of deterioration and the dose of levodopa. CONCLUSION: Initiation of levodopa therapy in patients with early and stable Parkinson's disease is associated with deterioration in visual memory functions, with relative preservation of the verbal memory. PMID- 16679641 TI - Intraspinal synovial cysts: a retrospective study. AB - BACKGROUND: We report the clinical presentation, radiographic studies, intraoperative findings, histopathological analysis, and post-treatment outcome in 26 patients diagnosed with spinal synovial cysts (SSCs). AIMS: To describe the clinical presentation, radiographic studies, operative findings, and postoperative follow-up in 26 patients with SSCs. SETTINGS AND DESIGN: The study was retrospective in design, involving chart review. Individual patient data was tabulated and patterns were recognized. MATERIALS AND METHODS: The charts for 26 patients who underwent surgical extirpation of SSC between April 1993 and October 2002 were retrospectively reviewed. Specifically, initial clinical presentation, pertinent radiographs (X-rays, magnetic resonance imaging, computed tomography), intraoperative findings, histopathology, and postoperative follow-up were noted. STATISTICAL ANALYSIS USED: Patient data was tabulated and analyzed for patterns in demographics, symptoms and histopathology. RESULTS: SSCs were more common in females than males (17:9 ratio). Presenting symptoms were back pain with radiculopathy in 13 (50%), radicular pain in the absence of back pain in 10 (38%), and back pain without radicular pain in three (11%). In addition, 17 patients (65%) had sensory deficit, and 9 (35%) had motor deficit. Most SSCs occurred at the lumbar (19/26) or lumbosacral (5/26) regions, with only 2 (2/26) in the thoracic region. One patient had bilateral SSC at the L4-5 level. Intraoperatively, each cyst was located adjacent to a degenerated facet joint. These lesions could grossly be identified intraoperatively and histopathological confirmation was achieved in all the cases. CONCLUSIONS: SSCs are important lesions to consider in the differential diagnosis of lumbar epidural masses and surgical resection leads to significant improvement in the majority of cases. PMID- 16679642 TI - Physical growth and psychomotor development of infants exposed to antiepileptic drugs in utero. AB - OBJECTIVE: To evaluates the physical growth and psychomotor development of infants born to women with epilepsy on regular Anti Epileptic Drugs (AEDs). SETTING: Govt. Stanley Medical College and Hospital, Tertiary care referral centre, Chennai. DESIGN: Open prospective cohort study with a control group. MATERIALS AND METHODS: Consecutive women with epilepsy who were on regular anticonvulsants were followed up from their first trimester. Their babies were examined at birth and anthropometric measurements including anterior fontanelle size were noted. They were followed up till one year and periodically evaluated at 1st, 6th and 12th month of age. Development testing using Griffith scale was done at 2nd, 6th and 12th month. An equal number of control babies were also studied using the same scale for one year at the specified intervals. The results in both the groups were compared. RESULTS: 30 babies were enrolled in the case and control group. The AEDs received by the mothers with epilepsy were Phenytoin, Carbamazepine, and Sodium valproate. At birth and 1st month the weight, head circumference and length of case and control babies were equal. At 6th and 12th month reduction in the above 3 parameters were noted in the case babies ( P < 0.01). Area of anterior fontanelle (AF) was larger in the study group particularly in those exposed to phenytoin in utero (P < 0.001). In the case babies reduction in the sitting, prone and erect progression of the locomotor scores was observed at 2nd month (P < 0.001). Prone progression alone improved by 12th month and other two remained less than the control (P < 0.001). No difference was observed in reaching behaviour and personal/social scores in both groups. Infants exposed to Phenytoin monotherapy had a negative impact on sitting progression. CONCLUSION: Among infants exposed to AEDs in utero physical growth was equal to that of control at birth but reduced at 6th and 12th month probably due to extraneous factors. The Locomotor scores showed reduction in all areas in 2nd, 6th and 12th month except prone progression which alone improved by 12th month. Phenytoin exposure in utero resulted in large AF and it had a negative impact on sitting progression in comparison with Carbamazepine and Sodium valproate. PMID- 16679643 TI - Role of MTHFR C677T polymorphism in ischemic stroke. AB - BACKGROUND: Homozygosity for MTHFR C677T polymorphism can lead to significantly high homocysteine levels and hyperhomocysteinemia is an important risk factor for thrombotic events. AIMS: The aim was to determine role of MTHFR C677T polymorphism in North Indians with ischemic stroke. SETTINGS AND DESIGN: In a prospective study, the subjects of stroke were recruited from the neurology clinic of the hospital. Controls were healthy individuals from the Hematology clinic without any history of stroke. MATERIALS AND METHODS: Plasma homocysteine levels were measured by enzyme immuno assay method after 3 months of acute episode. Serum folate and Vitamin B12 levels were estimated by competitive inhibition radioassay. MTHFR polymorphism was detected by PCR-RFLP using Hinf I enzyme. STATISTICAL ANALYSIS: The analysis of significance of results was done using SPSS software package. A p-value. RESULTS: Thirty-two acute ischemic stroke patients (aged 1-44 years) were studied. Fourteen (43.8%) had recurrent stroke. Nine (28%) had multiple infarcts. Four of 32 patients (12.5%) had high homocysteine levels. Three out of these 4 hyper-homocysteinemia patients were homozygous ( TT ) for MTHFR polymorphism (2 with recurrent stroke). Two of three homozygous cases with TT genotype had low serum folate. Five of 32 stroke cases (18.8%) were heterozygous ( CT ) genotype. CONCLUSIONS: Primary hyper homocysteinemia appears to be an important risk factor for ischemic stroke in North Indians, most due to MTHFR C677T homozygosity. Folate levels may modify the presentation of the MTHFR TT genotype. PMID- 16679644 TI - Treatment of atypical trigeminal neuralgia with microvascular decompression. AB - AIM: To explore the methods for achieving pain relief in patients with atypical trigeminal neuralgia (TN) using microvascular decompression (MVD). STUDY DESIGN AND SETTINGS: Retrospective study of 26 patients treated during the years 2000 to 2004. MATERIALS AND METHODS: Twenty-six patients in whom vascular compression of the trigeminal nerve was identified by high definition magnetic resonance tomographic angiography (MRTA) were treated with MVD for atypical TN in our department. Clinical presentations, surgical findings and clinical outcomes were analyzed retrospectively. RESULTS: In this study, single trigeminal division was involved in only 2 patients (8%) and two or three divisions in the other 24 patients (92%). Of prime importance is the fact that in 46.2% of the patients, several conflicting vessels were found in association. Location of the conflicts around the circumference of the trigeminal root was supero-medial to the root in 53.5%, supero-lateral in 30.8% and inferior in 15.7%. MVD for atypical TN resulted in complete pain relief in 50% of the patients with complete decompression, partial pain relief in 30.8% and poor pain relief or pain recurrence in 19.2% of the patients without complete decompression postoperatively. CONCLUSIONS: Complete decompression of the entire trigeminal root plays an important role in achieving pain relief in patients with atypical TN with MVD. PMID- 16679645 TI - Erythropoietin preconditioning on hippocampus neuronal apoptosis following status epilepticus induced by Li-pilocarpine in rats through anti-caspase-3 expression. AB - Selective neuronal loss following status epilepticus (SE) was first described just under 100 years ago. The acute pathology following SE was shown to be 'ischemic cell change' and was assumed to arise through hypoxia/ischemia. Recently, erythropoietin (Epo) has been shown to have potent anti-apoptosis activity in central nervous system neurons in animal models of ischaemic injury. AIMS: In this report, in order to determine Epo preconditioning on hippocampus neuronal apoptosis, we examined caspase-3 expression following SE caused by Li pilocarpine in rats. SETTINGS AND DESIGN: Animals were classified into three groups: EP group (pilocarpine group), rhEpo-pilocarpine group and control group. Four hours after preconditioning with Epo intraperitoneally, pilocarpine hydrochloride was administered intraperitoneally and observed for behavioral manifestations of SE. The animals were sacrificed at one hour after SE onset. MATERIALS AND METHODS: At the above-mentioned time point, animals were deeply anesthetized and were perfused through the left ventricle. Detection of hippocampus neuronal apoptosis was performed with caspase-3 immunohistochemical technique on three groups. To further confirm which cell population upregulates caspase-3, brain sections were stained for NeuN (green) and caspase-3 (red). STATISTICAL ANALYSIS: ANOVA and Fisher's post hoc test was used. RESULTS: Quatification of hippocampus neurons revealed that the number of caspase-3 positive cells in the CA1/CA3 area and dentate gyrus(DG) of three groups had a significant difference. In comparison with control group, there was an increase by 74% and 534%, 42% and 272% in the CA1/CA3 area and DG of EP group and rhEpo treated group respectively. There was a decrease by 18% and 26% in the CA1/CA3 area and DG of rhEpo-treated group compared with those in EP group. In addition, colocalization of caspase-3 with NeuN was shown. CONCLUSIONS: Systemic rhEpo therapy reduced caspase-3 expression in SE induced by Li-pilocarpine. PMID- 16679646 TI - Microsurgical open mini uniskin incision technique in the surgical treatment of carpal tunnel syndrome. AB - BACKGROUND: Patients who undergo carpal tunnel surgery sometimes complain of the restriction of the grip and pinch function, palmar tenderness, cosmetic problems, and scar formation at the site of the incision. AIMS: We used a modified mini uni skin incision with appropriate hand position for microscopic view in the surgical treatment of carpal tunnel syndrome to prevent cosmetic problems related with scar formation after surgery. SETTINGS AND DESIGN: In this study we used two different skin incision techniques; mini uni-skin incision and standard incision. In mini uni-skin incision technique the hands were positioned in a way that the wrist are hyperextended. A small skin incision one cm long was done from the inferior flexion crease towards the point between the thirth and fourth fingers. MATERIALS AND METHODS: Standard incision and mini uni-skin incision were compared according to their cosmetic result, grip and pinch function, palmar tenderness, and painful scar formation. STATISTICAL ANALYSIS: Student -t test was used for this study. RESULTS: 56 (43%) patients were operated with mini uni-skin incision, and 73 (57%) cases were operated with standard incision. The scores of grip, pinch and cosmetic results were better in the patients who were operated with mini uni-skin incision technique from those of standard incision. CONCLUSION: In this clinical study we used a modified skin incision (mini uni-skin incision) technique in the surgical treatment of carpal tunnel surgery. Our results revealed that mini uni-skin incision is superior from the standard incision. PMID- 16679648 TI - Outcome of "unknown" head injury patients at a tertiary care neurosurgical centre. AB - CONTEXT: A large number of patients are admitted to hospitals in large cities without any identification. These "unknown" patients represent a unique problem in developing countries. There is no systematic study in world literature on this subgroup of patients. AIMS: To elucidate the natural history and outcome of traumatic brain injury patients admitted in the department of neurosurgery as "unknowns". SETTINGS AND DESIGN: Retrospective analysis of all traumatic brain injury patients admitted to the department of neurosurgery as "unknown", between January 2002 and March 2005. RESULTS: Three hundred and twenty five patients were admitted as unknowns over a 3 year and 3 months period. Most of the patients were young males and became known during their hospital stay. Overall, 33 patients stayed for longer than one month, with 4 of them staying for longer than 6 months. Mortality in mild, moderate and severe head injury group was 1%, 6% and 46% respectively. CONCLUSIONS: Unknown patients represent a unique subgroup in metropolitan cities like Delhi. Managing them raises several medico-legal issues. Many of them occupy hospital beds for longer duration than required. There is an acute shortage of rehabilitation homes in Delhi for long term care and rehabilitation of such patients. PMID- 16679647 TI - Carotid artery stenting: results and long-term follow-up. AB - BACKGROUND AND PURPOSE: The role of carotid artery stenting (CAS) as an alternative to carotid endarterectomy in the treatment of for symptomatic carotid artery stenosis is investigated. MATERIALS AND METHODS: Forty-seven patients underwent CAS over 10-year period. Forty-nine vessels were treated. Stenosis quantification was done using North American symptomatic carotid endarterectomy trial method. The mean follow-up period by clinical and Duplex examination ranged is 5.6 years. RESULTS: The technical success rate was 100%. There were four deaths (8.1%) and two (4.1%) minor strokes within thirty days of procedure. There was no major strokes. All patients with minor stroke achieved complete recovery at 1-month follow up. Two deaths occurred probably due to hyperperfusion syndrome (HS) and two due to cardiac arrest. CONCLUSION: CAS is an effective treatment modality of symptomatic carotid artery disease but should be carefully done in high-risk groups having severe medical ailments and those having severe bilateral stenosis of the carotid arteries. PMID- 16679649 TI - Neuropsychiatric manifestations systemic lupus erythematosus: a study from South India. AB - CONTEXT: Neuropsychiatric systemic lupus erythematosus (NPSLE) has varied manifestations. AIMS: To study the pattern of neuropsychiatric (NP) involvement in systemic lupus erythematosus (SLE). SETTINGS AND DESIGN: Hospital based cross sectional and retrospective study. PATIENTS AND METHODS: Patients admitted with a diagnosis of SLE, during a period of 16 months, were evaluated and any NP syndrome present classified as per the American College of Rheumatology (ACR) nomenclature. STATISTICAL ANALYSIS: SPSS software Version 10 was used for descriptive analysis and correlative study. RESULTS: Out of 50 patients with SLE, all the patients with NPSLE [39 (78%)] were females, mean age 25.66 years (range: 11-44). The commonest manifestation was headache [20 (55.6%)]. Seizures were seen in 8 (20.51%) and psychosis in 6 (16.2%). Fine distal tremor was seen in 8 (20.51%) of patients. CONCLUSIONS: Headache is a frequent NP syndrome. Fine distal tremor is a syndrome not included in ACR classification but seen is 20% of our patients with NPSLE. PMID- 16679650 TI - Economics of head injuries. AB - Head injuries account for significant proportion of neurosurgical admissions and bed occupancy. Patients with head injuries also consume significant proportions of neurosurgical resources. A prospective 6-month study has been carried out to evaluate the expenditure incurred on head injury patients in a modern neurosurgical center equipped with state of the art infrastructure. Costing areas included wages / salaries of health care personnel, cost of medicines / surgical items / crystalloids, general store items, stationary, all investigation charges, equipment cost, overhead building cost, maintenance cost, electricity and water charges and cost of medical gases, air conditioning and operation theatre expenses. Expenditure in each area was calculated and apportioned to each bed. The statistical analysis was done using X2 test. The cost of stay in ward was found to be Rs. 1062 / bed / day and in neurosurgical ICU Rs. 3082 / bed / day. The operation theatre cost for each surgery was Rs. 11948. The cost of hospital stay per day for minor, moderate and severe head injury group was found to be Rs. 1921, Rs. 2569 and Rs. 2713 respectively. The patients who developed complications, the cost of stay per day in the hospital were Rs. 2867. In the operative group, the cost of hospital stay per day was Rs. 3804. The total expenditure in minor head injury was Rs. 7800 per patient, in moderate head injury was Rs. 22172 per patient, whereas in severe head injury, it was found to be Rs. 32852 per patient. Patients who underwent surgery, the total cost incurred was Rs. 33100 per operated patient. PMID- 16679651 TI - Post-traumatic dissecting aneurysms of bilateral cervical carotid arteries with delayed complications. AB - Carotid dissection is an uncommon complication of trauma. They can present with immediate or delayed complications. We describe the case of a young patient with bilateral carotid dissections and acute infarcts. Brief review of literature and treatment options are discussed. PMID- 16679653 TI - Hepatic cerebrospinal fluid pseudocyst: a case report and review of the literature. AB - An abdominal pseudocyst is a rare, but important complication in patients with a ventriculo-peritoneal (VP) shunt insertion. Several predisposing factors for this complication have been suggested, including infection, obstruction or dislodgement, but the pathophysiology is still unknown. However, the abdominal inflammatory process is accepted widely as a hypothesis for the formation of an abdominal pseudocyst. In this study, we report the case of a 21-year-old male that presented with a high-grade fever, poor appetite, shortness of breath and unconsciousness 1 week after receiving a VP shunt insertion for obstructive hydrocephalus. Ultrasonography and computed tomographic scans of the abdomen revealed a well-defined large hepatic cyst surrounding the peritoneal tube of the VP shunt. A hepatic cerebrospinal fluid (CSF) cyst was diagnosed and Staphylococcus epidermis was cultured via CSF. After externalization of the VP shunt and adequate antibiotic treatment, the hepatic cyst was resolved. There was no recurrence observed in the regular follow up. PMID- 16679652 TI - Encephalopathy and severe neuropathy due to probable systemic vasculitis as an initial manifestation of mixed connective tissue disease. AB - We described a 69-year-old woman with neurological manifestations due to mixed connective tissue disease (MCTD). The patient demonstrated subacute cognitive decline, seizure and gait disturbance with no connective tissue manifestation. She had been diagnosed with dementia at another hospital, later in our hospital, serological examinations disclosed high titers of anti-RNP antibody. Cognitive dysfunction in this patient was dramatically ameliorated by steroid therapy. Three months later, she developed edema of the hands, synovitis and acrosclerosis. The patient was finally diagnosed as having MCTD. We emphasized MCTD as a rare cause of "treatable dementia". PMID- 16679654 TI - Hemangioblastoma of hippocampus without von Hippel-Lindau disease: case report and review of literature. AB - A rare case of hemangioblastoma located in the region of hippocampus is reported. A 27-year-old female presented with a single episode of generalized convulsion. The vascular and cherry red color hemangioblastoma was resected by a temporo zygomatic approach. There has been no recurrence of tumor at a follow-up of 11 years. PMID- 16679655 TI - Aicardi syndrome: a report of five Indian cases. AB - Five Indian cases of Aicardi syndrome, aged 1-13 years and all with the classic triad of infantile spasm, corpus callosal agenesis and chorioretinal lacunae, were presented. The purpose of this report was to demonstrate the clinical, electroencephalographic (EEG) and radiological spectrum and the treatment outcome of this disorder among the Indians. None of the patient had any family history of similar illness. All had profound psychomotor impairment with no meaningful speech development. There were microphthalmia in 2, optic disc coloboma in 1, interhemispheric cyst in 1, periventricular heterotopia in 2 and thoracolumbar kyphoscoliosis in 2 cases. They all had early onset epilepsy and there were multiple types of seizures. Two cases (40%) had the characteristic dissociated burst-suppression pattern in EEG. Two cases whose antiepileptic medications included vigabatrin had complete control of seizure. PMID- 16679656 TI - Subclavian steal syndrome as the presenting feature of hypervascular thyroid nodule. AB - Subclavian steal syndrome (SSS) is a clinical entity characterized by brachial and basilar insufficiency as a result of critical proximal subclavian artery stenosis or occlusion. We report a patient of giant hypervascular thyroid nodule presenting with features of SSS. The left hand ischemia and symptoms of vertebro basilar artery in our patient were probably related to stealing of blood by the hypervascular thyroid nodule from the subclavian artery. The patient was relieved of the symptoms upon percutaneous subclavian stent placement. PMID- 16679657 TI - Craniofacial surgery for craniometaphyseal dysplasia. AB - Craniometaphyseal dysplasia (CMD) is a rare congenital bone dysplasia with abnormal bony overgrowth leading to characteristic facial features and cranial nerve compression. We present a 10-year-old child with bony swelling at the nasal root since birth along with decreased hearing in both ears. She had normal developmental milestones and intelligence. On examination, she had bossing of forehead with very broad nasal root, short septum, hypertelorism and epicanthic folds. CT scan with 3D reconstruction revealed grossly thickened calvarium and hyperostosis and sclerosis of the cranial base. As the major concern of the parents was cosmetic, craniofacial reconstruction was performed with good cosmetic outcome. PMID- 16679658 TI - 13-year-old boy with cerebellar cystic mass. PMID- 16679659 TI - Cerebral phaeohyphomycosis presenting as an intraventricular mass. PMID- 16679660 TI - Neurofibromatosis type I with occipital encephalocele. PMID- 16679661 TI - Transorbital cerebellar airgun pellet injury. PMID- 16679662 TI - Lumbar spinal dural arteriovenous fistula with a supply from a lumbar multimetameric arterial system. PMID- 16679663 TI - Cervical spinal epidural hematoma with acute Brown-Sequard presentation. PMID- 16679664 TI - Enterococcus avium cerebellar abscess. PMID- 16679665 TI - An unusual cause of hyponatremia: ventricular drainage. PMID- 16679666 TI - CNS demyelination due to hypocupremia in Wilson's disease from overzealous treatment. PMID- 16679667 TI - Cranial nerve lymphomatosis. PMID- 16679668 TI - Disaster and mass casualty management in a hospital: how well are we prepared? PMID- 16679669 TI - Algorithm for recall of HIV reactive blood donors. PMID- 16679670 TI - Hepatitis B or hepatitis C co-infection in individuals infected with human immunodeficiency virus and effect of anti-tuberculosis drugs on liver function. AB - BACKGROUND: Tuberculosis (TB) and hepatitis are the two common co-infections in patients infected with human immunodeficiency virus (HIV). Anti-tuberculosis treatment (ATT) may have an effect on the liver enzymes in these co-infected HIV patients. AIMS: To determine the prevalence of Hepatitis B and C virus coinfection in HIV infected patients in Tamilnadu and assess effects of anti tuberculosis drugs on their liver function. SETTINGS: HIV positive subjects referred to the Tuberculosis Research Centre, Chennai. MATERIALS AND METHODS: All HIV infected patients referred to the Tuberculosis Research centre, from March 2000 to May 2004, were screened for Hepatitis B surface antigen (HBsAg) & Hepatitis C virus (HCV) antibodies by enzyme linked immunoabsorbent assay (ELISA). HIV infection was confirmed using two rapid tests and one ELISA. Patients were given either short-course anti-tuberculosis treatment or preventive therapy for tuberculosis, depending on the presence or absence of active TB, if their baseline liver functions were within normal limits. None of these patients were on antiretroviral therapy during the study period. STATISTICAL ANALYSIS: Paired t-test was used to find the significance between baseline and end of treatment liver enzymes levels, while logistic regression was done for assessing various associations. RESULTS: Of the 951 HIV-infected patients, 61 patients (6.4%) were HBsAg positive, 20 (2.1%) had demonstrable anti HCV antibodies in their blood. Serial estimation of liver enzymes in 140 HIV patients (81 being co infected with either HBV or HCV) showed that 95% did not develop any liver toxicity while they were on anti-tuberculosis treatment or prophylaxis. CONCLUSIONS: The prevalence of hepatitis B and C coinfection was fairly high in this largely heterosexually infected population supporting the use of more careful screening for these viruses in HIV positive persons in this region. Anti tuberculosis therapy as well as TB preventive therapy can be safely employed in HIV and hepatitis coinfected patients, if baseline liver function tests are within normal limits. PMID- 16679671 TI - Prophylactic gabapentin for prevention of postoperative nausea and vomiting in patients undergoing laparoscopic cholecystectomy: a randomized, double-blind, placebo-controlled study. AB - BACKGROUND: Gabapentin is an antiepileptic drug. Its antiemetic effect is demonstrated in chemotherapy-induced acute and delayed onset of nausea and vomiting in breast cancer patients. AIM: To evaluate the antiemetic effect of gabapentin on incidence and severity of postoperative nausea and vomiting in laparoscopic cholecystectomy. SETTINGS AND DESIGN: Double-blind, randomized, placebo-controlled study. MATERIALS AND METHODS: Two hundred and fifty patients of ASA physical status I and II, scheduled for laparoscopic cholecystectomy were randomly assigned into two equal groups to receive 600 mg gabapentin or matching placebo two hours before surgery. Standard anaesthesia technique was used. Fentanyl was used as rescue postoperative analgesic. Ondansetron 4 mg was used intravenously as rescue medication for emesis. The total number of patients who had nausea or vomiting, and its severity and total fentanyl consumption in the first 24 hours were recorded. STATISTICAL ANALYSIS: "Z test" was used to test the significance of severity of post-operative nausea and vomiting between groups. Fentanyl consumed in each group (Mean+/-SD) within 24 hrs was compared using student t test. P value < 0.05 was considered significant. RESULTS: There were no demographic difference between the two groups. Incidence of post-operative nausea and vomiting within 24 hrs after laparoscopic cholecystectomy was significantly lower in gabapentin group (46/125) than in the placebo group (75/125) (37.8% vs 60%; P =0.04). There was a significantly decreased fentanyl consumption in gabapentin group (221.2+/-92.4 microg) as compared to placebo group (505.9+/-82.0 microg; P =0.01). CONCLUSION: Gabapentin effectively suppresses nausea and vomiting in laparoscopic cholecystectomy and post-operative rescue analgesic requirement. PMID- 16679672 TI - Signs of inflammation in children that can kill (SICK score): preliminary prospective validation of a new non-invasive measure of severity-of-illness. AB - BACKGROUND: Signs of Inflammation in Children that can Kill (SICK score) is a new severity-of-illness score. It uses the physical signs of the Systemic Inflammatory Response Syndrome (SIRS) and its continuum--the Multiple Organ Dysfunction Syndrome (MODS). The development of the score used multiple logistic regression model coefficients converted to integer scores that have been published earlier. AIMS: The present study was done to validate the scoring system by predicting outcomes in a fresh data set. SETTING: Intensive care unit in a tertiary referral hospital. DESIGN: Prospective. MATERIALS AND METHODS: 125 admissions to the intensive care unit were evaluated so that the SICK score and the PRISM score could be calculated. In-hospital mortality was noted. STATISTICAL ANALYSIS: Calibration (Hosmer-Lemeshow goodness of fit) and discrimination (area under the ROC curve) were used to measure performance. RESULTS: Of the 125 patients studied 23 died. The area under the ROC curve was 0.76 compared to 0.80 in the development sample. Using PRISM in the validation group, the ROC was 0.78. Calibration was excellent. CONCLUSION: The SICK score can predict severity of illness with nearly the same accuracy as the PRISM score. The SICK score can be calculated immediately on admission and can help to prioritize care for the more sick children who need urgent aggressive management. Larger studies, that includes all admissions to the hospital, will now need to be done. PMID- 16679673 TI - Algorithm for recall of HIV reactive Indian blood donors by sequential immunoassays enables selective donor referral for counseling. AB - BACKGROUND: HIV/AIDS pandemic brought into focus the importance of safe blood donor pool. AIMS: To analyze true seroprevalence of HIV infection in our blood donors and devise an algorithm for donor recall avoiding unnecessary referrals to voluntary counseling and testing centre (VCTC). MATERIALS AND METHODS: 39,784 blood units were screened for anti-HIV 1/2 using ELISA immunoassay (IA-1). Samples which were repeat reactive on IA-1 were further tested using two different immunoassays (IA-2 and IA-3) and Western blot (WB). Based on results of these sequential IAs and WB, an algorithm for recall of true HIV seroreactive blood donors is suggested for countries like India where nucleic acid testing or p24 antigen assays are not mandatory and given the limited resources may not be feasible. RESULTS: The anti-HIV seroreactivity by repeat IA-1, IA-2, IA-3 and WB were 0.16%, 0.11%, 0.098% and 0.07% respectively. Of the 44 IA-1 reactive samples, 95.2% (20/21) of the seroreactive samples by both IA-2 and IA-3 were also WB positive and 100% (6/6) of the non-reactive samples by these IAs were WB negative. IA signal/cutoff ratio was significantly low in biological false reactive donors. WB indeterminate results were largely due to non-specific reactivity to gag protein (p55). CONCLUSIONS: HIV seroreactivity by sequential immunoassays (IA-1, IA-2 and IA-3; comparable to WHO Strategy-III) prior to donor recall results in decreased referral to VCTC as compared to single IA (WHO Strategy-I) being followed currently in India. Moreover, this strategy will repose donor confidence in our blood transfusion services and strengthen voluntary blood donation program. PMID- 16679674 TI - Complex humanitarian emergencies: a review of epidemiological and response models. AB - Complex emergencies (CEs) have been the most common human-generated disaster of the past two decades. These internal conflicts and associated acts of genocide have been poorly understood and poorly managed. This article provides an epidemiological background and understanding of developing and developed countries, and chronic or smoldering countries' CEs, and explains in detail the prevailing models of response seen by the international community. Even though CEs are declining in number, they have become more complex and dangerous. The UN Charter reform is expected to address internal conflicts and genocide but may not provide a more effective and efficient means to respond. PMID- 16679675 TI - Hospital planning for weapons of mass destruction incidents. AB - As terrorists attacks increase in frequency, hospital disaster plans need to be scrutinized to ensure that they take into account issues unique to weapons of mass destruction. This paper reports a review of the literature addressing hospital experiences with such incidents and the planning lessons thus learned. Construction of hospital disaster plans is examined as an ongoing process guided by the disaster planning committee. Hospitals are conceived as one of the components of a larger community disaster planning efforts, with specific attention devoted to defining important linkages among response organizations. This includes the public health authorities, political authorities, prehospital care agencies, and emergency management agencies. A review is completed of six special elements of weapons of mass destruction incidents that should be addressed in hospital disaster plans: incident command, hospital security, patient surge, decontamination, mental health consequences, and communications. The paper closes with a discussion of the importance of training and exercises in maintaining and improving the disaster plan. PMID- 16679676 TI - Understanding community psychosocial needs after disasters: implications for mental health services. AB - The psychosocial impact of disasters has attracted increasing attention. There is little consensus, however, about what priorities should be pursued in relation to mental health interventions, with most controversy surrounding the relevance of traumatic stress to mental health. The present overview suggests that acute traumatic stress may be a normative response to life threat which tends to subside once conditions of safety are established. At the same time, there is a residual minority of survivors who will continue to experience chronic posttraumatic stress disorder (PTSD) and their needs can be easily overlooked. The ADAPT model offers an expanded perspective on the psychosocial systems undermined by disasters, encompassing threats to safety and security; interpersonal bonds; systems of justice; roles and identities; and institutions that promote meaning and coherence. Social reconstruction programs that are effective in repairing these systems maximize the capacity of communities and individuals to recover spontaneously from various forms of stress. Within that broad recovery context, clinical mental health services can focus specifically on those psychologically disturbed persons who are at greatest survival risk. Only a minority of persons with acute traumatic stress fall into that category, the remainder comprising those with severe behavioural disturbances arising from psychosis, organic brain disorders, severe mood disorders and epilepsy. Establishing mental health services that are community-based, family-focused and culturally sensitive in the post-emergency phase can create a model that helps shape future mental health policy for countries recovering from disaster. PMID- 16679677 TI - The challenge of preparation for a chemical, biological, radiological or nuclear terrorist attack. AB - Terrorism is not a new phenomenon, but, in the contemporary scene, it has established itself in a manner which commands the most serious attention of the authorities. Until relatively recently, the major threat has been through the medium of conventional weaponry and explosives. Their obvious convenience of use and accessibility guarantees that such methods will continue to represent a serious threat. However, over the last few years, terrorists have displayed an enthusiasm for higher levels of carnage, destruction and publicity. This trend leads inexorably to the conclusion that chemical, biological, radiological and nuclear (CBRN) methods will be pursued by terrorist organisations, particularly those which are well organised, are based on immutable ideological principles, and have significant financial backing. Whilst it is important that the authorities and the general public do not risk over-reacting to such a threat (otherwise, they will do the work of the terrorists for them), it would be equally ill-advised to seek comfort in denial. The reality of a CBRN event has to be accepted and, as a consequence, the authorities need to consider (and take seriously) how individuals and the community are likely to react thereto and to identify (and rehearse in a realistic climate) what steps would need to be taken to ameliorate the effects of such an event. PMID- 16679678 TI - Systemic lupus erythematosis with antiphospholipid antibody syndrome: a mimic of Buerger's disease. AB - This case report is about a past smoker who presented with history of recurrent ulcers and digital gangrene with claudication pain of the left foot for the past fifteen years. Clinical examination and angiogram showed disease involving the peripheral vessels of lower limb. This patient had been labeled as Buerger's disease 15 years ago based on clinical and demographic profile of the illness. We felt that the progression of the disease despite the patient having stopped smoking 15 years ago along with the presence of elevated inflammatory markers in the blood with proteinuria was not in keeping with the nature of the disease. Further evaluation revealed that the patient had systemic lupus erythematosus with antiphospholipid antibody syndrome. This case highlights the need for a careful search for diseases, which can mimic Buerger's disease in young smokers who present with peripheral vascular disease and who have an atypical clinical presentation or progression. PMID- 16679679 TI - Isolated breast mucormycosis. AB - Mucormycosis is unusual in surgical practice. Awareness of the classical findings leads to early detection. Excisional therapy whenever possible along with systemic antifungal treatment is the key to successful outcome. A 70 year old female, a known case of diabetes mellitus and chronic obstructive pulmonary disease, on inhalational steroids and oral hypoglycemic agents, presented to us with complaints of sudden onset pain, redness and swelling of left breast. A diagnosis of severe mastitis was made and a release incision was taken. The entire breast became gangrenous in next 24 hours and simple mastectomy had to be performed as a life saving measure. Histopathology revealed mucormycosis of breast. To the best of our knowledge this is the first reported case of mucormycosis of breast in the English literature. PMID- 16679680 TI - Skin necrosis in a critically ill patient due to a blood pressure cuff. AB - The non-invasive method of blood pressure measurement is regarded as a safe procedure and the reports of any serious complications are rare. We report a unique case of extensive skin necrosis due to an intermittently inflating blood pressure cuff in a 65-year-old critically ill lady following a third time redo mitral valve surgery. A brief review of the literature on complications associated with noninvasive method of measurement of blood pressure is presented along with possible mechanisms of skin injury and ways to avoid it. PMID- 16679681 TI - Rectal ears. PMID- 16679682 TI - Ultrasound flying pigeon. PMID- 16679683 TI - Solid and cystic papillary epithelial neoplasm of the pancreas. PMID- 16679684 TI - Oguchi disease. PMID- 16679685 TI - A mentally challenged adult with tonic convulsions, dysmorphic face and sebopsoriasis. PMID- 16679686 TI - A shift from significance test to hypothesis test through power analysis in medical research. AB - Medical research literature until recently, exhibited substantial dominance of the Fisher's significance test approach of statistical inference concentrating more on probability of type I error over Neyman-Pearson's hypothesis test considering both probability of type I and II error. Fisher's approach dichotomises results into significant or not significant results with a P value. The Neyman-Pearson's approach talks of acceptance or rejection of null hypothesis. Based on the same theory these two approaches deal with same objective and conclude in their own way. The advancement in computing techniques and availability of statistical software have resulted in increasing application of power calculations in medical research and thereby reporting the result of significance tests in the light of power of the test also. Significance test approach, when it incorporates power analysis contains the essence of hypothesis test approach. It may be safely argued that rising application of power analysis in medical research may have initiated a shift from Fisher's significance test to Neyman-Pearson's hypothesis test procedure. PMID- 16679687 TI - Acute acalculous cholecystitis following the bite of Indian saw-scaled viper. PMID- 16679688 TI - Brainstem and cerebellar hypoplasia associated with osteogenesis imperfecta type 5. PMID- 16679689 TI - Cicatricial ectropion due to herpes zoster ophthalmicus. PMID- 16679690 TI - Spontaneous ovarian hyperstimulation syndrome presenting with acute abdomen. PMID- 16679691 TI - Keratitis due to Cylindrocarpon lichenicola. PMID- 16679692 TI - Relapsing encephalopathy secondary to non-hepatic hyper-ammonemia. PMID- 16679693 TI - Mitochondrial neurogastrointestinal encephalomyopathy and its pathophysiology. PMID- 16679694 TI - Human herpesvirus 6 causes hepatitis in transplant recipients. PMID- 16679695 TI - The JRS guidelines for the management of community-acquired pneumonia in adults: an update and new recommendations. AB - Community-acquired pneumonia (CAP) continues to be a major medical problem. Since CAP is a potentially fatal disease, early appropriate antibiotic treatment is vital. Epidemiologic studies have shown that in the combined cause-of-death category, pneumonia ranks fourth as the leading cause of death in Japan. Therefore, the Japanese Respiratory Society (JRS) provided guidelines for the management of CAP in adults in 2000. Because of evolving resistance to antimicrobials and advances in diagnosis, treatment and prevention of CAP, it is felt that an update should be provided every three years so that important developments can be highlighted and pressing questions can be answered. Thus, the guidelines committee updated its guidelines in 2005. The basic policy and main purposes of the JRS guidelines include; 1) prevention of bacterial resistance and 2) effective and long-term use of medical resources. The JRS guidelines have recommended the exclusion of potential and broad spectrum antibiotics, fluoroquinolones and carbapenems, from the list of first-choice drugs for empirical treatment. In addition, the JRS guidelines have recommended short-term usage of antibiotics of an appropriate dose and pathogen-specific treatment using rapid diagnostic methods if possible. PMID- 16679696 TI - Central neurotranspeptide, alpha-melanocyte-stimulating hormone (alpha-MSH) is upregulated in patients with congestive heart failure. AB - BACKGROUND: Alpha-melanocyte-stimulating hormone (alpha-MSH), a pro opiomelanocortin (POMC) derivative, is a neuropeptide with potent anti inflammatory properties that inhibits tissue injury in a wide array of inflammation models. OBJECTIVE: To determine if alpha-MSH is involved in the development of congestive heart failure (CHF) with the specific aim of examining its peripheral source and one of the mechanisms. METHODS: The circulating levels of alpha-MSH were measured in 115 patients with CHF using a double-antibody radioimmunoassay. To determine one of the sources of circulating alpha-MSH, human peripheral blood mononuclear cells (PBMC) were stimulated with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha. Furthermore, to clarify one of the functions of alpha-MSH, PBMC were cultured in the presence or absence of alpha MSH. RESULTS: Plasma levels of alpha-MSH were significantly higher in NYHA class II patients with CHF than in control subjects (p<0.0001). A significant correlation was found between the levels of alpha-MSH and high-sensitive testing for C-reactive protein in patients with CHF (r=0.41, p<0.0005). PBMC stimulated with LPS or TNF-alpha released alpha-MSH in a concentration-dependent manner. alpha-MSH inhibited LPS-induced TNF-alpha production, and alpha-MSH simultaneously augmented production of interleukin (IL)-10 by PBMC. CONCLUSIONS: Circulating alpha-MSH was increased in patients with CHF. Inflammatory response induced alpha-MSH production in cultured human PBMC. Treatment of alpha-MSH could modify the immunobalance between inflammatory and anti-inflammatory responses in cultured PBMC. These findings suggest that alpha-MSH may play an important role in the pathophysiology of CHF. PMID- 16679697 TI - Relationship between the expression of advanced glycation end-products (AGE) and the receptor for AGE (RAGE) mRNA in diabetic nephropathy. AB - OBJECTIVE: The receptor for advanced glycation end-products (RAGE) is one of several advanced glycation end-product (AGE)-specific cellular receptors. To evaluate the relationship between AGE and RAGE in renal tissues of diabetic nephropathy (DN), we examined the levels of expression of AGE protein and of RAGE mRNA. We also investigated the relationships among the degree of mesangial expansion and the expression of AGE and RAGE mRNA. PATIENTS AND METHODS: Renal biopsy tissues were obtained from 20 patients with DN. We performed immunohistochemical staining using monoclonal anti-AGE antibody and in situ hybridization using non-radioactive oligonucleotide RAGE probe on these tissues. We also examined five control renal samples. We evaluated the intensity of positive anti-AGE antibody staining and the percentage of cells positive for RAGE mRNA. We also measured the total glomerular area and mesangial area in glomeruli using an automatic image analyzer. We then calculated the percentage of mesangial area as a proportion of the total glomerular area (%Mes). RESULTS: Anti-AGE antibody was detected in the expanded mesangial matrix in DN but not in control samples. RAGE mRNA expression was detected mainly in glomerular intrinsic cells, including glomerular mesangial and epithelial cells, in both DN and control. %Mes correlated significantly with both the intensity of anti-AGE antibody positive staining and the percentage of cells positive for RAGE mRNA. CONCLUSIONS: Our findings suggest that both AGE and RAGE are associated with the development and progression of DN. PMID- 16679698 TI - Thymidine phosphorylase gene mutation is not a primary cause of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). AB - OBJECTIVE: The authors identified a patient with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), who completely fulfilled the clinical criteria with low thymidine phosphorylase (TP) activity. However, the same homozygotic S471L TP gene mutation was also found in her unaffected mother, but with normal TP activity. To elucidate the pathogenesis of MNGIE, we performed the analysis below. METHODS: We analyzed the TP gene mutation in the proband and 145 unrelated individuals by direct sequence and restriction fragment length polymorphism (RFLP). TP activity was determined by the spectrophotometric method for each TP S471L genotype. RESULTS: Among 145 normal persons, the S471L homozygote mutants were identified in 2.76% and their enzyme activity was normal. CONCLUSION: TP gene mutation is not a primary cause of MNGIE, but with a mitochondrial deletion mutation, a single nucleotide polymorphism (SNP) of the TP gene may be crucial in the pathogenesis of MNGIE. PMID- 16679699 TI - First impression at stroke onset plays an important role in early hospital arrival. AB - BACKGROUND: Treatment for acute ischemic stroke should be administered as soon as possible after symptom onset. The aim of this study was to investigate whether or not the patient's and bystander's first impression at stroke onset was associated with hospital arrival time. METHODS: To investigate the factors influencing the prehospital delay, we prospectively interviewed consecutive stroke patients and bystanders about their first impression at the stroke onset and assessed the methods of transportation, and clinical characteristics. Early arrival was defined as a hospital arrival of within 2 h from stroke onset. RESULTS: One hundred thirty patients were enrolled: 82% were ischemic stroke and 18% were cerebral hemorrhage. The median interval between symptom onset and the hospital arrival was 7.5 h and 30% of patients presented within 2 h of stroke onset. First impression of stroke (odds ratios [OR] 4.56, 95% confidence interval [CI] 1.54 13.5, p=0.006), presence of consciousness disturbance (OR 4.29, CI 1.39-13.3, p=0.011), arrival through other facilities (OR 0.25, CI 0.08-0.76, p=0.015), a history of diabetes (OR 0.23, CI 0.06-0.80, p=0.028) and nocturnal onset (OR 0.19, CI 0.04-0.88, p=0.042) independently contributed to the early arrival. CONCLUSIONS: The first impression of patients and bystanders at stroke onset is important in order to reach hospital earlier in Japan. Public educational systems such as those, which advertise stroke warning signs, are necessary. PMID- 16679700 TI - Treatment of Japanese restless legs syndrome patients with cabergoline: an open clinical preliminary trial. AB - OBJECTIVE: To determine the effective dose of cabergoline in Japanese patients with restless legs syndrome (RLS). METHODS: Six cases of idiopathic RLS and three of RLS with Parkinson disease (PD) participated in an open clinical preliminary trial. All cases were diagnosed based on the clinical criteria of the International RLS Study Group. Three RLS cases (1.3%) were detected out of 229 consecutive cases with PD. RLS severity was evaluated with International RLS Study Group (IRLSSG) Rating Scale Version 2.2 before and one year after the treatment with cabergoline. RESULTS: For 6 idiopathic RLS patients, the IRLSSG questionnaire scores improved from 25.5+/-3.7 to 10.7+/-8.9 (p=0.028, Wilcoxon test) with 1 mg of daily cabergoline at the endpoint. For 3 RLS cases with PD, the score was 21.7+/-3.7 before the treatment, and RLS symptoms completely disappeared with 1 mg of cabergoline. One of RLS cases with PD required additional cabergoline later because of parkinsonism. No adverse event with cabergoline was reported in this study. CONCLUSION: One mg of daily cabergoline is effective in some Japanese patients of RLS. PMID- 16679701 TI - Right atrial thrombosis as a complication of arrhythmogenic right ventricular cardiomyopathy. AB - A 65-year-old woman was admitted to our hospital due to palpitation. Electrocardiogram (ECG) showed ventricular tachycardia originating from the right ventricle, and transthoracic echocardiography revealed dilatations of the right atrium and ventricle. The diagnosis of arrhythmogenic right ventricular cardiomyopathy was made. Eleven months later, echocardiography revealed a solid thrombus (36x32 mm) attached to the free wall of the right atrium, and it was surgically resected. Four months after the operation, a solid thrombus (48x30 mm) appeared again at the same site despite anticoagulant treatment. The patient died of both left and right heart failure 33 months after the operation. PMID- 16679702 TI - Multifocal fibrosclerosis combined with idiopathic retro-peritoneal and pericardial fibrosis. AB - A 70-year-old man who had been diagnosed with retroperitoneal fibrosis (RPF) was admitted to our hospital complaining of dyspnea. Imaging studies showed massive pericardial effusion. His condition deteriorated and pericardiostomy was performed. A biopsy of the pericardium revealed marked fibrosis with infiltration of lymphocytes, which was identical to RPF findings. A diagnosis of multifocal fibrosclerosis was made. Despite aggressive treatment, he died with clinical signs of cardiovascular failure. The autopsy specimen revealed proliferation of fibrosis with infiltration of lymphocytes in multiple organs. Even after successful decompression of urinary obstruction for RPF, long-term follow-up is necessary in these patients because of the possibility of other fatal complications such as pericardial fibrosis. PMID- 16679703 TI - Scintigraphic evaluation of cardiac metabolism and sympathetic nerve function in alcoholic cardiomyopathy. AB - A 70-year-old man with alcoholic cardiomyopathy underwent 99mtechnetium-sestamibi (MIBI), iodine-123-labeled metaiodobenzylguanidine (MIBG) scintigraphy and Iodine 123-labeled beta-methyl-iodophenyl pentadecanoic acid (BMIPP) scintigraphy. 99mtechnetium-MIBI identified myocardial damage in the inferior wall of left ventricle. (123)I BMIPP showed low uptake in the inferior wall of the myocardium, concordant to perfusion. (123)I BMIPP and (123)I MIBG showed reduced uptake in the inferior segment of the myocardium, indicating impairment of fatty acid metabolism and sympathetic abnormalities. Damaged myocardium was demonstrated in alcoholic cardiomyopathy. Beta blocker (carvedilol) and angiotensin-receptor blocker (valsartan) were started at low doses, then increased gradually, leading to the improvement of cardiac performance. Cardiac sympathetic nerve function, impaired due to alcoholic cardiomyopathy, was improved with beta-blocker therapy. Cardiac scintigraphy may be useful to assess the extent of myocardial improvement and the response to therapy. PMID- 16679704 TI - Rapid onset of glycogen storage hepatomegaly in a type-2 diabetic patient after a massive dose of long-acting insulin and large doses of glucose. AB - We present a case of rapid onset of glycogen storage hepatomegaly, caused by a massive dose of long-acting insulin and large doses of glucose, in a type-2 diabetic patient. A 41-year-old man was admitted to our hospital because of hypoglycemia and unconsciousness following subcutaneous administration of 180 units of insulin glargine in a suicide attempt. Despite continuous hypercaloric infusion with additional intravenous glucose injections, hypoglycemia persisted for 36 hours. Although the hepatic function was normal and no hepatomegaly was detected on admission, the liver function tests became abnormal and hepatomegaly was detected on hospitalization day 3. Plain abdominal computed tomography (CT) scanning confirmed liver enlargement, with hepatic CT attenuation markedly elevated at 83.7 HU. Liver biopsy revealed hepatocytic glycogen deposition with edematous degeneration. Based on these findings, the diagnosis was made as rapid onset glycogen storage hepatomegaly caused by administration of a massive dose of long-acting insulin and supplementation with large doses of glucose. With improved glycemic control, the liver function improved, the CT findings of hepatomegaly improved, and the hepatic CT attenuation decreased. Repeat liver biopsy also confirmed almost complete disappearance of glycogen deposits. When hepatic dysfunction or hepatomegaly is detected during treatment with insulin, the possibility of hepatic glycogen deposition should be considered. CT scanning and liver biopsy were useful in diagnosing this case. PMID- 16679705 TI - Human herpesvirus-6 hepatitis associated with cyclosporine-A encephalitis after bone marrow transplantation for chronic myeloid leukemia. AB - A 31-year-old man referred to our hospital for treatment of his chronic myeloid leukemia (CML) in the first chronic phase by bone marrow transplantation. We pretreated him with cyclophosphamide and total body irradiation and bone marrow transplantation (BMT) was carried out. On day 31, the engraftment was confirmed and on day 52, acute graft versus host disease (GVHD) was observed. On day 189, he lost consciousness due to cyclosporine A-induced leukoencephalopathy and 375 mg cyclosporine A was changed to 100 mg prednisolone. On day 199, liver dysfunction (AST 410 IU/L, ALT 557 IU/L, gammaGTP 385 IU/L, ALP 363 IU/L, D-Bil 0.3 mg/dl) developed and a liver biopsy was performed. PCR analysis of DNA from the liver biopsy specimen was positive for HHV-6 and immunostaining using anti HHV-6 and anti-HHV-6b antibodies showed positive staining in the cytosol of hepatocytes. No other viruses were found to induce hepatitis. From these results, he was diagnosed as having HHV-6 hepatitis and it was successfully treated with gancyclovir (GCV) administration. PMID- 16679706 TI - Autoimmune thrombocytopenic purpura, autoimmune hemolytic anemia and gastric cancer appeared in a patient with myasthenia gravis. AB - We report a case of myasthenia gravis (MG) associated with autoimmune thrombocytopenic purpura (AITP) and autoimmune hemolytic anemia (AIHA), and after that gastric cancer appeared. A 51-year-old man began to suffer from fluctuated muscle weakness in 1985. Muscle weaknesses became exacerbated, and he was admitted to our hospital in 1989. He was diagnosed as MG associated with AITP. After a thymectomy (hyperplasia), prednisolone therapy was started, subsequently his condition was satisfactory. In March 1995, he developed severe anemia and icterus. He was diagnosed as Evans' syndrome (AIHA and AITP) with MG. High-doses of immunoglobulin administration improved the anemia, but thrombocytopenia continued. In November 2002, he suffered marked petechia; the platelet count decreased to 1000/microl. Methylprednisolone pulse therapy and platelet transfusion were started. Gastrofiberscopy was performed and biopsy specimens revealed signet cell-type adenocarcinoma. On December 19, 2002, subtotal gastrectomy and splenectomy were performed. After that, his condition has remained satisfactory, without MG symptoms or thrombocytopenia. This is the first such case report in the literature. PMID- 16679707 TI - Intracerebral hemorrhage in a patient receiving combination therapy of pegylated interferon alpha-2b and ribavirin for chronic hepatitis C. PMID- 16679708 TI - Pure red cell aplasia developing after treatment of pleural recurrence of thymoma, successfully treated with cyclosporin A but not with tacrolimus. PMID- 16679709 TI - Intra-tumor hemorrhage associated with brainstem metastasis from lung cancer. PMID- 16679710 TI - A tethered conus syndrome in an adult woman. PMID- 16679711 TI - A foreign body in the duodenum. PMID- 16679712 TI - Exposure to bright light modifies HRV responses to mental tasks during nocturnal sleep deprivation. AB - This study was intended to determine the effects of continuous bright light exposure on cardiovascular responses, particularly heart rate variability (HRV), at rest and during performance of mental tasks with acute nocturnal sleep deprivation. Eight healthy male subjects stayed awake from 21.00 to 04.30 hours under bright (BL, 2800 lux) or dim (DL, 120 lux) light conditions. During sleep deprivation, mental tasks (Stroop color-word conflict test: CWT) were performed for 15 min each hour. Blood pressure, electrocardiogram, respiratory rate, urinary melatonin concentrations and rectal temperature were measured. During sleep deprivation, BL exposure depressed melatonin secretion in comparison to DL conditions. During sleep deprivation, exposure to BL delayed the decline in heart rate (HR) for 4 h in resting periods. A significant increment of HR induced by each CWT was detected, especially at 03.00 h and later, under DL conditions only. In addition, at 04.00 h, an index of sympathetic activity and sympatho-vagal balance on HRV during CWT increased significantly under DL conditions. In contrast, an index of parasympathetic activity during CWT decreased significantly under DL conditions. However, the indexes of HRV during CWT did not change throughout sleep deprivation under BL conditions. Our results suggest that BL exposure not only delays the nocturnal decrease in HR at rest but also maintains HR and balance of cardiac autonomic modulation to mental tasks during nocturnal sleep deprivation. PMID- 16679713 TI - The influence of size perception and internal modeling on the control process while lifting. AB - The present study sought to verify object size perception through internal modeling while lifting an object. Electromyography (EMG) activity of the upper limb muscles was recorded while 20 healthy females alternately lifted two containers of the same weight, but were unequal in size. When subjects lifted the small container, a significant increase was observed in the EMG activity. Most subjects determined that the small container was heavier than the large container, and predicted that the large container would be heavier than the small container due to size difference. The results may be explained by supporting that subjects predict object weight based on perception of size through internal modeling; however, predictions are cross-checked and modified through sensory feedback based on subjective weight. PMID- 16679714 TI - Differential perceived exertion measured using a new visual analogue scale during pedaling and running. AB - The purpose of this study was to evaluate the differential perceived exertion measured using a new set of Visual Analogue Scales (VAS) during pedaling and running. The subjects were eleven healthy males. They performed an incremental maximal test and then three 4-min stages of exercise, for both pedaling and running. During the tests, VO2, V(CO2), V(E), f, and HR were monitored continuously. Bla and perceptual variables including VAS consisting of four scales (VAS 1-VAS 4) and Borg's RPE were measured at the end of each stage. Although the VO2 (%VO2max)) and HR for both pedaling and running were not significantly different, Bla in pedaling was significantly higher than that in running. A significant interaction (mode, stage) was also obtained. The VAS 1 of pedaling was significantly higher than that of running. A significant interaction in VAS 1 (mode, stage) was obtained. The VAS 2 of pedaling was significantly higher than that of running. The subjects indicated that local pain became stronger than central pain in pedaling, but they were almost equal in running. In both pedaling and running, leg pain became stronger than arm pain (VAS 3). VAS 4 showed that during running, breathing difficulty and heart pain were almost equal in perceived intensity. However, during pedaling, breathing difficulty became greater than heart pain. Thus, a new four-part visual analogue scale was found to be useful for monitoring exercise intensity. In addition, the new VAS gave us more information in relation to the differential perceived exertion reflected in the different physiological responses obtained by different exercise modes. PMID- 16679715 TI - Age-related changes of reaction time and p300 for low-contrast color stimuli: Effects of yellowing of the aging human lens. AB - We measured reaction time (RT), P300, and subjective evaluation for color Landolt Cs with a gray color background presented on a CRT display. Seven young and 7 elderly subjects (mean ages: 21.6 and 68.4 years, respectively) participated, and the young subjects wore glasses with filters simulating spectral transmittance of an aging human lens as a test condition. The results for young subjects not wearing the filters showed that RT and P300 latency are constant among different test colors. In contrast, the results for elderly subjects showed that RT and P300 varied substantially depending upon the test colors and RT and P300 latency became longer than those of young subjects, particularly for gray and blue stimuli. In addition, the results for the young subjects with filters showed tendencies similar to those in elderly subjects. These results indicate that the yellowing of the human lens strongly influences reaction time and cognition time for color targets, suggesting that wearing the filters enables the young to simulate RT qualitatively as well as visibility of the elderly because both the simulated filter and the aging human lens modify the effective luminance, effective luminance contrast and effective color difference between the color target and the background on the retina. We also found that the reciprocal of RT and P300 latency could be expressed in a multiple regression model consisting of effective luminance, effective luminance contrast, effective color difference and age. Absolute values of RT and P300 latency in young subjects with filters, however, did not quantitatively coincide with those of the elderly subjects. There were differences of RT and P300 latency between the young with filters and the elderly, indicating that higher order age-related delay could be involved. PMID- 16679716 TI - Influences of diurnal bright or dim light exposure on urine volume in humans. AB - We investigated with eight healthy females if 8 hr diurnal (0700 to 1500 h) bright rather than dim light (5,000 vs. 80 lx) influenced urine volume. Environmental illuminance was made identical at all other times besides 07:00 to 15:00 h. The participants spent time at strictly regulated schedules in a bioclimatic chamber (26 degrees C, relative humidity 60%) for 57 h. Blood was drawn (2 ml) just before lunch in order to calculate Creatinine clearance (Ccr). Urine volume was significantly higher during wakefulness and the 8-h sleep period with bright rather than dim light. Ccr was significantly higher after bright light. The results were discussed in terms of suppression of the sympathetic nerve system under the influence of diurnal bright light exposure. We also discussed these in terms of physiological polymorphisms. PMID- 16679717 TI - Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa. AB - The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa. PMID- 16679718 TI - Possible roles of intracellular cyclic AMP, protein kinase C and calcium ion in the apoptotic signaling pathway in bovine luteal cells. AB - Structural luteolysis occurs by apoptosis of luteal cells. The present study examined the effects of activators of well-characterized second messengers on Fas and caspase-3 mRNA expression and on P4 production in luteal cells in order to trace the pro- and anti-apoptotic factors in the bovine corpus luteum (CL). Cultured bovine mid luteal cells were treated for 24 h with a cyclic AMP analogue (8-bromo cyclic AMP; 8br-cAMP; 2.5 mM), a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate; PMA; 10 microM), or calcium ionophore (A23187; 10 microM). Fas and caspase-3 mRNA expression was inhibited by 8br-cAMP and PMA but was increased by A23187 (P<0.05). In addition, P4 production by bovine luteal cells was stimulated by 8br-cAMP and PMA, whereas it was inhibited by A23187, compared with untreated controls (P<0.05). The overall results suggest that cAMP and PKC suppress apoptosis in bovine luteal cells through inhibition of Fas and caspase-3 mRNA expression and through stimulation of P4 production. Therefore, substances that activate cAMP or PKC may act as survival factors in the bovine CL. Furthermore, substances that mobilize Ca2+ may act as apoptotic factors by stimulating Fas and caspase-3 expression in the bovine luteal cells. PMID- 16679719 TI - Characterization of Cq3, a quantitative trait locus that controls plasma cholesterol and phospholipid levels in mice. AB - Cq3 was identified in C57BL/6J (B6) x KK-Ay F2 mice as a quantitative trait locus (QTL) that controls plasma cholesterol and phospholipid levels, and normolipidemic B6 allele was associated with increased lipids. Cq3 was statistically significant in F2-a/a, but not in F2-Ay/a; probably because the Cq3 effect was obscured by introduction of the Ay allele, which in itself has a strong hyperlipidemic effect. Because the peak LOD score for Cq3 was identified near D3Mit102 (49.7 cM) on chromosome 3, linkage analyses with microsatellite markers located at 49.7 cM were performed in KK x RR F2, B6 x RR F2, and KK x CF1 F2. However, even a suggestive QTL was not identified in any of the three F2. By testing all pairs of marker loci, I found a significant interaction between Cq3 and the Apoa2 locus, and F2 mice with the Apoa2(KK)/Apoa2(KK); D3Mit102(B6)/D3Mit102(B6) genotype had significantly higher cholesterol levels than did F2 mice with other genotypes. The results showed that the ;round-robin' strategy was not always applicable to the search for QTL genes; probably because specific gene-to-gene interaction limited the validity of the strategy to the utmost extent. PMID- 16679720 TI - Gastrointestinal helminths of feral raccoons (Procyon lotor) in Wakayama Prefecture, Japan. AB - The population and distribution of feral raccoons (Procyon lotor) are expanding in Japan after escape or release from animal-owners. Wakayama Prefecture is one of the most typically devastated areas by this exotic carnivore, particularly in the last five years after a latent distribution for more than ten years. Official control measures of feral raccoons commenced in the summer of 2002 by several municipalities, and 531 animals collected in 12 municipalities between May 2003 and April 2005 were submitted for parasitological examination of gastrointestinal helminths. Detected parasites included six nematodes (Physaloptera sp. [prevalence; 5.1%], Contracaecum spiculigerum [0.9%], Strongyloides procyonis [25.5%], Ancylostoma kusimaense [0.8%], Arthrostoma miyazakiense [0.4%], and Molineus legerae [1.1%]), seven trematodes (Isthmiophora hortensis [4.9%], echinostomatid sp. with 34-39 collar spines [1.7%], Metagonimus takahashii [12.4%], M. yokogawai [0.8%], Plagiorchis muris [0.2%], Macroorchis spinulosus [1.9%], and Consinium ten [0.2%]), one cestode (Mesocestoides sp. [0.2%]), and six acanthocephalan spp. (Centrorhynchus bazalenticus [0.2%], Centrorhynchus teres [5.5%], Sphaerirostris lanceoides [2.4%], Plagiorhynchus ogatai [0.6%], Porrorchis oti [1.5%], and Southwelina hispida [1.9%]). Most of the collected parasites are food-borne, indigenous helminth species. Physaloptera sp. has never been recorded in indigenous wild carnivores in Japan, and resembles closely P. rara, prevalent in raccoons of North America, in morphology. The position of a pair of phasmids in the posteroventral region of the adult male, however, could differentiate it from P. rara. Since Strongyloides procyonis is known to cause creeping eruption as well as intestinal infection in a healthy human volunteer, we should be concerned about the rapid increase in the population and distribution of feral raccoons in Japan from the viewpoint of public health as well. PMID- 16679721 TI - Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos. AB - Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals. PMID- 16679722 TI - Protective effect of botulinum C/D mosaic toxoid against avian botulism. AB - Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross reactivity. Ducks immunized once with the toxoid together with a commercial oil adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism. PMID- 16679723 TI - Life cycle of Eimeria krijgsmanni-like coccidium in the mouse (Mus musculus). AB - Life cycle of Eimeria krijgsmanni-like coccidium isolated from the feces of naturally infected mice purchased from commercial sources was examined. The parasite was purified by single oocyst isolation and maintained by passage in the mice before experiments. The sporulated oocysts were ovoid or ellipsoid, measuring 19.3 x 14.8 microm on average. One or two small polar granules were present. Micropyle and oocyst residuum were absent. Sporocysts were ellipsoid, measuring 11.6 x 7.2 microm on average with a small Stieda body and sporocyst residuum. Six groups of respective 5 mice (4-week-old) were inoculated with doses varying from 2.0 x 10(1) to 10(6) oocysts. All the mice examined began to shed oocysts from 7 day postinoculation (PI) and their maximum number of oocysts per gram of feces were 10(6) on day 8 PI. Patency was 6 or 7 days. This parasite had severe virulence to the mice that is, the mice given 10(6) oocysts showed anorexia, diarrhoea and rough hair from 1 day and all of them died on day 3 PI. The mice given 10(3) or more oocysts showed the clinical signs described above from day 5 and 4 of them received 10(5) died on day 9 or 10 PI. The parasites occurred within the epithelial cells of cecum, colon and rectum of infected mice. Sporozoites, 13.9 x 3.0 microm, with two large refractil bodies on side of the nucleus located subcentrally were observed on day 1 and 2 PI. Merozoites were first observed at 24 hr PI, and sexual stages were found from 4 day PI. No parasites were detected in the small intestine and mecenteric lymph nodes. PMID- 16679724 TI - Development of a neurosurgical operating table for adult cattle and changes in intracranial pressure and blood pressure in adult cattle undergoing long-time isoflurane anesthesia. AB - We developed a neurosurgical operating table for restraining adult cattle in the sternal recumbent position during long-time inhalation anesthesia, and examined intracranial pressure (ICP), blood pressure and blood gases during isoflurane anesthesia. We confirmed that the maintenance of inhalation anesthesia, the restraint of cattle in the sternal recumbent position and bringing the cattle out of anesthesia could all be carried out safely using the operating table we produced. For the purposes of the present experiment, the cattle were divided into 2 groups: the SR group, which underwent sternal recumbency for 8 hr under isoflurane anesthesia using the neurosurgical operating table, and the RR group, which underwent right lateral recumbency for 3 hr under isoflurane anesthesia on a standard operating table. The mean ICP was found to be significantly lower in the SR group than in the RR group during anesthesia, and PaO2 was significantly higher in the SR group. In the SR group, no complications such as regurgitation or ruminal tympany occurred for 8 hr after the induction of anesthesia, and recovery from anesthesia was uneventful. In contrast, all RR cattle showed ruminal tympany and regurgitated ruminal fluid at 3 hr after the induction of anesthesia. Thus, the neurosurgical operating table developed in the present study may be useful for long-time anesthesia and neurosurgery of adult cattle. PMID- 16679726 TI - Changes of blood biochemical values in ponies recovering from hyperlipemia in Japan. AB - Hyperlipemia in horses is a disorder of lipid metabolism peculiar to ponies. This study reports changes of blood biochemical values from the acute to the postconvalescent phases in 3 Shetland ponies with hyperlipemia in Japan. Diseased ponies (all 7 to 9 years old, in late pregnancy, and obese) were fed in the same farm. The periods of their hospitalizations ranged from 30 to 45 days. Twelve well-conditioned ponies (3 to 13 years old) around parturition were used to establish baseline values for blood test results. Main clinical findings in the affected ponies were depression, dysphagia, anorexia, ventral edema and milky appearing plasma. Hypertriglyceridemia (40- to 70-fold rise of controls) was found in the acute phase of the disease in the affected ponies, and was derived from increased very-low density lipoproteins. Aspartate transaminase and gamma glutamyl transpeptidase activities, blood urea nitrogen, and creatinin concentrations were increased in acute ponies compared to controls, suggesting impairment of liver and kidney functions. However, these values gradually recovered until the end of postconvalescent phase. Hyperinsulinemia was observed in the acute phase of the hyperlipemia of all affected ponies. And an exaggerated insulin response to intravenous glucose was observed in the 2 ponies given intravenous glucose tolerance tests. These findings suggest decreased insulin sensitivity in hyperlipemic ponies. PMID- 16679725 TI - PCR-based species-specific amplification of ITS of Mecistocirrus digitatus and its application in identification of GI nematode eggs in bovine faeces. AB - Differential diagnosis of Mecistocirrus digitatus infection relies on morphological examination of either eggs in faecal samples or L3 larvae developed in vitro. Technical limitations hinder the practicability of these approaches. Hence, in order to develop a specific diagnostic measure for M. digitatus infection, we determined the sequence of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA) and designed primers for PCR-based species-specific amplification of the ITS to differentiate between M. digitatus and other common gastrointestinal (GI) nematode species. The newly designed primers amplified a single specific 520 base pair (bp) fragment from the M. digitatus ITS, and its detection limit was as low as 0.001 ng. Further, this sensitivity suggested that the specific fragment could be amplified even from a unicellular egg that collected directly from uteri of an adult M. digitatus female. In fact, we designed a method that employs a small piece of a cover slip and a filter paper by which we could differentially amplify a PCR fragment from a unicellular egg. The reliability of the specific PCR assay was also demonstrated with 10 oval samples that collected from bovine faeces by using sugar flotation method. These data suggested that the specific PCR assay of the ITS region of M. digitatus rDNA could be useful for the identification of GI nematodes. PMID- 16679727 TI - Probenecid: its chromatographic determination, plasma protein binding, and in vivo pharmacokinetics in dogs. AB - Pharmacokinetics (PK) of probenecid including plasma probenecid concentrations, in vitro plasma protein binding properties, and in vivo PK parameters were determined in dogs. Probenecid concentrations were best determined by HPLC, which showed good linearity and good recovery with simple plasma preparation. The quantification limit of probenecid was approximately 50 ng/ml at S/N ratio = 3, by simple procedure with HCl and methanol treatment. Probenecid showed two types of binding characteristics, i.e., high-affinity with low-capacity and low affinity with high-capacity binding. This result indicated 80-88% of probenecid was bound to plasma protein(s) at observed concentrations (< 80 microg/ml) in vivo at an intravenous dose of 20 mg/kg. Plasma probenecid concentration-time profile following i.v. administration in dogs showed biphasic decline and well fitted a two-compartment open model. The total body clearance was 0.34 +/- 0.04 ml/min/kg, volume of distribution at steady-state was 0.46 +/- 0.07 l/kg, elimination half-life was 18 +/- 6 hr, and mean residence time (MRT) was 23 +/- 6 hr. Since probenecid has been known as a potent inhibitor of renal tubular excretion of acidic drugs and highly binds to plasma proteins, our observation in relation to plasma protein binding and PK parameters will serve as the basic information concerning drug-drug interactions in dogs and in other mammalian species. PMID- 16679728 TI - A comparison of biodistribution between 111In-DTPA octreotide and 111In-DOTATOC in rats bearing pancreatic tumors. AB - 111In-DTPA octreotide (DTPAOC) has been used for detecting somatostatin receptor positive tumor for years. In-111 DOTA-Tyr3-octreotide (DOTATOC) is newly developed for diagnostic and therapeutic purposes. In this study, we compared the biodistribution and tumor uptake ratio after injection of In-111 DTPAOC and In 111 DOTATOC in rats. Twelve rats bearing pancreatic tumors were divided into two groups: six rats were sacrificed at 4 hr after injection of 3.7 MBq of In-111 DTPAOC and another 6 rats were sacrificed at the same time after injection of 3.7 MBq of In-111 DOTATOC. Samples of various organs were obtained and counted to calculate the tissue concentration. In addition, 12 rats bearing pancreatic tumors were scanned at 4, 24, and 48 hr after injection of 37 MBq of In-111 DTPAOC or In-111 DOTATOC. The tumor uptake ratios (T/N ratio) were calculated. The biodistribution data showed that the activity in the tumor as well as in the kidney was significantly higher in the In-111 DOTATOC group than in the In-111 DTPAOC group, although both radiopharmaceuticals had the expected high affinity to the tumor. The T/N ratios in the In-111 DOTATOC group were also significantly higher than those in the In-111 DTPAOC group at 24 hr after injection. We conclude that In-111 DOTATOC showed lower clearance than In-111 DTPAOC in the rats bearing pancreatic tumors, although both of these radiopharmaceuticals showed expected high tumor uptake. PMID- 16679729 TI - Therapeutic effects of beta-thujaplicin eardrops on canine Malassezia-related otitis externa. AB - An eardrop solution of beta-thujaplicin was examined for therapeutic effects on canine Malassezia-related otitis externa. Half to one ml of beta-thujaplicin solution of 100 microg/ml including DMSO 2% was injected everyday into both external ear canals of 31 cases for test-of-cure agreement. Fifteen score phases were established from the symptoms and cerumen smear biopsy findings, and score changes were recorded at least once a week. The means of the second and the third inspection day scores decreased significantly more than the previous value of each. In addition, the numbers of yeast-like organisms clearly decreased. These results suggest that beta-thujaplicin eardrops are effective for Malassezia related otitis externa in dogs. PMID- 16679730 TI - Morphometry on lancet flukes found in Japanese sika deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan. AB - Thirty-six flukes were collected from the livers of wild deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan, and were served for morphometry. The length and/or the width of the body, suckers, testes, ovary, vitelline glands, cirrus pouch and eggs in the uterus of the flukes were measured. The distance between anterior end of the body and position of the maximal body-width or upper end of the testes were also determined. A remarked morphological characteristic was that the right and left testes did not lie tandem but lined bilaterally. Also the position of the maximal body-width did not always locate in the posterior part of the body of the fluke. The property was in accordance with those for Dicrocoelium chinensis. PMID- 16679731 TI - Increased expression of osteopontin in the heart tissue of Lewis rats with experimental autoimmune myocarditis. AB - The expression of osteopontin (OPN) in the hearts of rats with experimental autoimmune myocarditis (EAM) was evaluated. Western blot analysis showed that OPN was significantly increased in the hearts with EAM compared with those of complete Freund's adjuvant (CFA) immunized control. Immunohistochemically, OPN was weakly expressed in the cardiomyocytes in the heart with normal and CFA immunized controls. In EAM lesions, OPN was intensely immunostained in some inflammatory cells, mainly ED1 positive macrophages. These findings suggest that OPN is significantly increased in EAM lesions and that OPN mediates the inflammatory process in the course of rat EAM model. PMID- 16679732 TI - Long-term chemotherapy with lomustine of intracranial meningioma occurring in a miniature schnauzer. AB - A 14-year-old male miniature schnauzer was referred to us because it was circling to the right. A mass in the diencephalon was noted on brain magnetic resonance images. The dura was thickened with marked linear enhancement after contrast administration. Based on diagnostic image analysis, this lesion strongly suggested meningioma. The patient's symptoms were well controlled by a combination therapy of prednisolone and lomustine (CCNU), and survived for thirteen months after diagnosis. This case was diagnosed as a meningioma based on histopathological findings. This report describes the clinical findings, imaging characteristics, and pathologic features of a diencephalic and mesencephalic meningioma and long-term survival after lomustine and prednisolone therapy. PMID- 16679733 TI - Occurrence of swine salmonellosis in postweaning multisystemic wasting syndrome (PMWS) affected pigs concurrently infected with porcine reproduction and respiratory syndrome virus (PRRSV). AB - Fourteen diseased pigs from four farms in which there had been an outbreak of salmonellosis were investigated. Granulomatous inflammation with depletion of lymphocytes was observed in the swollen lymph nodes in these pigs. Antigens to porcine circovirus type 2 (PCV2) were immunolabeled in the lesions along with detection of viral DNA as PCV2 by polymerase chain reaction (PCR). In addition, antigens to porcine reproductive respiratory syndrome virus (PRRSV) were immunodetected in the lungs and Salmonella Choleraesuis was isolated from the affected pigs. The nine salmonellosis affected pigs, five (55.6%) with salmonellosis and PMWS concurrently infected with PRRSV were much higher than those infected with salmonellosis and PMWS (22.2%) or with salmonellosis and PPPRV (22.2%). PMID- 16679734 TI - Serum free amino acid concentration in hepatic lipidosis of dairy cows in the periparturient period. AB - Blood samples were taken from eight multiparous cows at a dairy farm on eight occasions between the prepartum period and peak lactation to study the serum concentrations of amino acids and biochemical constituents. The cows were classified as having either severe hepatic lipidosis (HL) or non-hepatic lipidosis (non-HL) according to their clinical condition after calving and changes in serum biochemical parameters. The serum concentrations of beta hydroxybutyric acid were higher in the HL group than in the non-HL group (ANOVA: p<0.01). The serum concentrations of methionine (Met), phenylalanine, and arginine were significantly different between the two groups (ANOVA: p<0.05). In particular, the Met levels were significantly low for 14 days after calving in the HL group (p<0.05), although Met levels in the HL group tended to be lower than the values in the non-HL group until 30 days after calving, starting 14 days before calving. The results suggest that an insufficiency of Met during the periparturient period is related to the development of hepatic lipidosis. PMID- 16679735 TI - A case of protein-losing enteropathy treated with methotrexate in a dog. AB - A 9-year-old female Pug was presented to us with chronic diarrhea. Hematologic findings indicated severe hypoproteinemia and hypoalbuminemia, and endoscopy revealed severe edema of the duodenal mucosa. Based on these results and on additional histopathological findings, we made a diagnosis of protein-losing enteropathy caused by lymphocytic-plasmacytic enteritis with lymphangiectasia. The dog was initially treated with prednisolone and cyclosporine. This treatment regimen was not effective. However, when methotrexate was substituted for cyclosporine, progress was obtained and the diagnosis was confirmed. PMID- 16679736 TI - PCR-based discrimination of Toxoplasma gondii from pigs at an abattoir in Okinawa, Japan. AB - To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in pigs in Okinawa Prefecture, we analyzed lymph node samples that had been collected at an abattoir by PCR analysis using primers specific for the Toxoplasma gondii SAG2 locus. This study revealed the presence of this parasite in 57 out of 101 samples examined. Restriction fragment length polymorphism (RFLP) in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Genotypes I and II were equally predominant, accounting for 22 (44.9%) and 23 (46.9%) of 49 SAG2-positive samples, respectively, while the type III strain was found in only 4 (8.2%) of the 49 samples. The other 8 samples were indistinguishable by PCR-RFLP analysis. Polymorphisms for the 3 genotypes were confirmed at the sequence level for several samples using the sequences from the RH strain, the Beverley strain, and the C56 strain as references. On the other hand, the dihydropteroate synthase gene, which is responsible for sulfonamide resistance, was amplified in 40 of 54 SAG2-positive samples by PCR with the specific primers, and further RFLP and sequence analysis revealed that none of them carried the drug-resistant form of the dhps gene. This is the first report of genotyping of T. gondii distributed in Japan. PMID- 16679737 TI - In vitro efficacies of oseltamivir carboxylate and zanamivir against equine influenza A viruses. AB - To investigate the possibilities of two NA inhibitors [oseltamivir carboxylate (OC) and zanamivir (ZA)] as the clinical agents for equine influenza A virus (EIV) infection, we examined the efficacies of these inhibitors against twelve EIVs in vitro. OC and ZA inhibited NA activities of all EIVs with 50% inhibitory concentrations with ranging from 0.017 to 0.130 and from 0.010 to 0.074 microM, respectively. OC and ZA inhibited plaque-forming of all EIVs in MDCK cells with 50% effective concentrations with ranging from 0.015 to 0.097 and from 0.016 to 0.089 microM, respectively, except for one strain (13.328 microM and 6.729 microM). These results suggest that these inhibitors are effective against most EIVs and might be useful for treatment of EI in horses. PMID- 16679738 TI - Electroencephalography (EEG) spectral edge frequency for assessing the sedative effect of acupuncture in dogs. AB - Electroencephalography (EEG) is an effective method for the evaluation of sedation or anesthesia. The purpose of this study was to examine the sedative effect of acupuncture by electroencephalographic spectral edge frequency (SEF) in Miniature Schnauzer dogs (4.2-6.1 kg, 1-2 years old). The acupuncture points "GV20 and Yintang" were applied for 20 min. Sedation level was assessed before, during, and after acupuncture by spectral edge frequency 95 values and the Ramsay sedation score. The spectral edge frequency 95 values were significantly reduced during acupuncture on GV20 or Yintang point and returned to the baseline values after acupuncture releasing. The Ramsay sedation score (RSS) also showed the acceptable sedation level during acupuncture. It was concluded that an acupuncture application at GV20 or Yintang point used in the present study would be a valuable method to induce the sedation in dogs. PMID- 16679739 TI - [Development of highly sensitive determination of biogenic compounds by high performance liquid chromatography with pre-column fluorescence derivatization]. AB - A sensitive fluorescent labeling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2 methoxyphenylsulfonyl chloride (DMS-Cl), for the determination of amino compounds in HPLC was developed. DMS-Cl reacted with amino compounds in the basic medium to produce the corresponding fluorescent sulfonamides (excition 318 nm, emission 406 nm in aqueous acetonitrile). When amino acids were analyzed using reverse-phase HPLC, the detection limits (signal-to-noise ratio = 3) of almost all amino acids labeled with DMS-Cl were less than 5 fmol/injection. DMS-Cl was utilized for highly sensitive determination of amino compounds in biological samples and HPLC methods for determination of prolyl dipeptides, Pro and Hyp, in serum and urine, pipecolic acid in serum, taurine in plasma, and free and N-acetylated polyamine in urine were established. As these proposed methods are highly sensitive and reproducible and require only a small amount of biological sample, they may be useful for clinical and biochemical research. PMID- 16679740 TI - [Molecular mechanisms underlying renal accumulation of aminoglycoside antibiotics and mechanism-based approach for developing nonnephrotoxic aminoglycoside therapy]. AB - Aminoglycoside antibiotics, such as gentamicin and amikacin, are a class of clinically important antibiotics used worldwide in the treatment of infections caused by Gram-positive and Gram-negative bacteria. However, nephrotoxicity and ototoxicity are serious problems in the use of aminoglycosides and are the major dose-limiting side effects. Most of the intravenously administered dose is excreted into the urine, whereas some of the aminoglycoside injected (about 10% of the dose) is selectively accumulated in the renal cortex, leading to renal injury. Aminoglycosides are taken up into the epithelial cells of the renal proximal tubules by an endocytic pathway. Acidic phospholipids, broadly distributed in the plasma membranes in various tissues, were considered to be the binding site of aminoglycosides. Recently, megalin, a giant endocytic receptor abundantly expressed in renal proximal tubules, has been reported to bind aminoglycosides. Therefore we first examined whether megalin plays an important role in the renal accumulation of aminoglycosides under in vivo and in vitro conditions. We then attempted to develop new strategies for preventing the nephrotoxicity of aminoglycosides based on the molecular mechanisms of aminoglycoside accumulation in the kidney. This review summarizes our recent findings ol the role of megalin in the renal accumulation of aminoglycosides and our approach to develop nonnephrotoxic aminoglycoside therapy. PMID- 16679741 TI - [Study on the regulation of synaptic function by Ca2+/calmodulin-dependent protein kinase II]. AB - Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in the mammalian brain, especially in the hippocampus. Neuronal CaMKII is a multifunctional mediator of activity dependent on an increase in the Ca(2+) level in excitable cells. It plays an important role in synaptic plasticity, including learning and memory, and is recognized as a "memory molecule." The expression of the kinase increases most rapidly during the most active phase in the formation of synapses in the postnatal brain and remains at a high level after synaptic maturation, indicating that the kinase is carefully regulated in the space-temporal gene expression. It is accumulated in the postsynaptic density (PSD), which is central in synaptic transmission. This review presents the gene expression and alternative splicing of CaMKII during neural differentiation, molecular constituents of PSD, and regulation of CaMKII by activity-regulated cytoskeleton-associated protein (Arc) mainly developed in our study. PMID- 16679742 TI - [Roles of nuclear receptors in the gene expression of drug-metabolizing enzymes under various physiological conditions]. AB - The nuclear receptor constitutive androstane receptor (CAR), a key transcription factor for the expression of cytochrome P450 (CYP) 2B genes, resides in the cytoplasm under untreated conditions and translocates into the nucleus upon xenobiotic exposure. CAR forms a multiprotein complex including heat shock protein 90 in the cytoplasm as the glucocorticoid receptor, and it is likely that protein phosphatase 2A plays a critical role in the first step of CAR nuclear translocation. In addition to the xenobiotic induction of CYP2Bs, our recent studies have indicated that CAR is important for sex and strain differences and obesity/diabetes-associated changes in the expression of CYP2B genes. These results have raised the hypothesis that the expression of nuclear receptors varies depending on the physiologic condition, leading to the dysregulation of CYP expression. In obese mice fed a high-fat diet, however, hepatic CYP3A levels are drastically decreased without any significant changes in the expression of nuclear receptors including the pregnane X receptor and hepatocyte nuclear factor 4, which are known to be key transcription factors in the expression of CYP3A genes. These results indicate that it is important to investigate the mechanism of the transcriptional regulation of nuclear receptor genes as well as the activation of nuclear receptors to understand the CYP expression system fully. PMID- 16679743 TI - [Development of the measures to evaluate the similarity of drug brand names]. AB - Brand name confusion is one of the most common causes of drug-related errors. The aim of this study was to develop quantitative measures of similarity among brand names of drugs. We modified the fragmentary pattern-based measure, a measure of similarity for character strings based on the string resemblance system, to develop three novel measures of similarity, i.e., the head and tail-weighted fragmentary pattern-based measure (htfrag), visually weighted htfrag (vwhtfrag), and auditorily weighted htfrag (awhtfrag). The 227 pairs of brand names for which confusion errors have been reported were used as a positive control group. Ten sets of 2270 random pairs of brand names were generated as negative controls. Then we evaluated the measures developed by using the geometric mean of sensitivity and selectivity as an objective function, in comparison with two conventional measures of similarity based on the vector space model (cos1 and htco). The measures developed, htfrag, vwhtfrag, and awhtfrag, provided better discrimination with mean objective function values of 0.953, 0.962, and 0.940, respectively, which were higher than those for the conventional measures cos1 and htco (0.922 and 0.892, respectively). The rates of false-positives and false negatives were 3.3-10.7% and 5.3-11.9% for cos1, respectively, while the rates for vwhtfrag were 4.8-5.9% and 2.2%, respectively. The measures of similarity developed may provide significant information to avoid drug-related errors associated with brand name confusion. PMID- 16679744 TI - [Production of antitumor substances from shochu distillation remnants]. AB - This study aimed to develop a new processing method for the effective use of rice shochu distillation remnants. We examined the inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with various fungi. Interestingly, high inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with Aspergillus oryzae were obtained, although no inhibitory effect was observed in the case of synthetic medium. We therefore fractionated the medium of rice shochu distillation remnants with A. oryzae using anion-exchange and reverse-phase chromatography. Furthermore, we attempted to determine the chemical structure of compounds that showed high inhibitory effects on the growth of tumor cells. The chemical structure of 1-hydroxy-6-(1-methylpropyl)-3-(2-methylproryl)-2(1H) pyrazinone was revealed on the basis of liquid and gas mass spectroscopies. This compound should be completely safe based on toxic test results using model mice. PMID- 16679745 TI - Effect of Lycium barbarum polysaccharide on the improvement of antioxidant ability and DNA damage in NIDDM rats. AB - The effects of polysaccharide extracted from Lycium barbarum (LBP) on blood glucose, oxidative stress and DNA damage in rats with non-insulin dependent diabetes mellitus (NIDDM) were studied. The results show that LBP treatment (10 mg/kg.d) for 4 weeks led to decreased levels of blood glucose, malondialdehyde (MDA) and nitric oxide (NO) in serum of fasting rats; and to increased serum level of superoxidedismutase (SOD). Furthermore, LBP could reduce cellular DNA damage in peripheral lymphocytes of NIDDM rats. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length and tail moment. These results suggest that LBP can control blood glucose and modulate the metabolism of glucose, leading to significant improvement of oxidative stress markers (SOD, MDA) in rats with NIDDM. And that, LBP decreases DNA damage possibly via a decrease in oxidative stress levels. In conclusion, LBP as a dietary supplement may prevent the development of complications or even tendency to carcinogenesis in NIDDM rats. PMID- 16679746 TI - The realities and medical expense of hospitalization that originates in outpatient medicine treatment. AB - Problems associated with outpatient pharmacotherapy may require hospitalization. However, such hospitalization may be prevented if pharmacist's pharmaceutical care (PC) is given. We investigated the reasons for hospitalization in medical institutions and medical expenses were calculated. Inpatient diagnoses, treatment, etc. in the previous year in the past were examined, and cases of hospitalization due to drug therapy were extracted. Next, the possibility of preventing hospitalization with PC practice was examined. Among 1552 cases, outpatient pharmacotherapy was the reason for hospitalization in 27 cases. Noncompliance was the underlying cause in about 40% of hospitalizations. It was thought that in 22 cases hospitalization could have been prevented by pharmacist's PC. The average hospitalization medical expense was 295,805 yen per patient. It is necessary to perform regular consultation recommendations, interventions with the family, home care, etc. for proactive PC. PMID- 16679747 TI - The future in diagnosis and staging of lung cancer: positron emission tomography. AB - Since its introduction in 1974, positron emission tomography (PET) has gained widespread use, especially in diagnosis and staging of lung cancer. In this respect, (18)F-fluorodeoxyglucose (FDG) is by far the most used PET tracer exploiting the increased glucose uptake and metabolism in malignant cells. A large number of studies have suggested that addition of FDG-PET to conventional workup can improve diagnosis and staging in patients with non-small cell lung cancer (NSCLC). In meta-analysis, the sensitivity and specificity of PET in diagnosing single pulmonary nodules and masses is found to be 96 and 78%, respectively. In mediastinal staging, the sensitivity and specificity of PET is estimated to be 83 and 92%. In order to achieve high diagnostic values from PET, it is necessary to pay attention to a number of pitfalls, e.g., the uptake of FDG by inflammatory cells causing false-positive results, as well as size and histology of the tumour in order to avoid false-negative results. In 2001, the first integrated PET/computed tomography (CT) was installed, and since then, the use of this modality has expanded steadily, thereby decreasing examination time and overcoming the lack of anatomical details on PET. Recently, PET and PET/CT have become increasingly integrated in therapy planning and evaluation: response evaluation during and after chemotherapy, restaging after neoadjuvant therapy, planning of radiotherapy and detection of recurrent disease are all examples of emerging indications for PET and PET/CT in managing patients with lung cancer. PMID- 16679748 TI - Chronic obstructive pulmonary disease and asthma in the primary care setting: simple questionnaires can promote patient awareness and provide feedback to physicians. PMID- 16679749 TI - The effects of gender on the development of chronic obstructive pulmonary disease. PMID- 16679750 TI - Mendelian susceptibility to mycobacterial disease. PMID- 16679751 TI - Sirolimus: friend or foe? PMID- 16679752 TI - Prognostic value of pleural fluid pH in malignant epithelial mesothelioma after talc poudrage. AB - BACKGROUND: Current staging schemes for malignant mesothelioma are inadequate. The most accurate staging may require pneumonectomy - a procedure associated with many complications. The pH of pleural fluid (ppH) predicts survival in non mesotheliomatous malignant pleural effusions, suggesting that this noninvasive test might be useful for prognostication in malignant mesothelioma. OBJECTIVE: It was the aim of this study to determine whether baseline ppH correlates with survival in malignant epithelial pleural mesothelioma. METHODS: We reviewed survival data in patients treated with thoracoscopic talc pleurodesis whose final diagnosis was epithelial malignant pleural mesothelioma and whose chart recorded a ppH determination performed just before thoracoscopy. We monitored 26 patients until April 2002 (25 of these patients died), identifying cutoff ppH values that discriminate best for survival; Cox proportional hazards models were recursively run by increasing the ppH cutoff value by 0.02 each time. RESULTS: The mean follow-up time was 19+/-14 months. Mean ppH was 7.30+/-0.09, and median ppH was 7.32. Several cutoff points correlated with a statistically significant difference in survival, but ppH 7.32 was associated with the greatest value for the area under the curve. Patients with ppH>.32 lived a median of 21.2 months (95% confidence interval 16.5-30.0 months) after diagnosis compared with patients who had ppH 55 years at baseline. RESULTS: 8 of 32 patients with amnestic MCI (25%), 22 of 41 patients with multiple-domain MCI (54%), and 3 of 8 patients with single non-memory MCI (37.5%) progressed to dementia. The clinical type of MCI is significantly associated with the likelihood of conversion to dementia. DISCUSSION: When the clinical syndrome of MCI evolves on a neurodegenerative basis, the multiple-domain type of MCI has a less favorable prognosis than the amnestic type and may represent a more advanced prodromal stage of dementia. PMID- 16679763 TI - Impact of behavioural problems on spousal caregivers: a comparison between Alzheimer's disease and frontotemporal dementia. AB - BACKGROUND: Behavioural changes are a key factor in distinguishing frontotemporal dementia (FTD) from Alzheimer's disease (AD), however, little is known about the impact of these changes on caregivers. The aim of this study was to compare caregivers' distress related to behavioural symptoms of AD and FTD. METHODS: 47 spouse caregivers of consecutively referred patients with AD and 27 spouse caregivers of patients with FTD participated in this study. Behavioural disturbances in the patient and caregivers' emotional reactions were measured with the Neuropsychiatric Inventory. RESULTS: Patients with FTD had significantly higher levels of agitation, apathy, disinhibition and aberrant motor behaviour than did patients with AD. High distress scores were found for disinhibition, depression and apathy in caregivers of FTD patients whereas caregivers of AD patients reported patient apathy, depression and anxiety as being severely distressing. Higher mean distress scores were found for disinhibition in the FTD group. Furthermore, caregivers of FTD patients reported higher levels of general burden, and felt less competent than AD caregivers. CONCLUSIONS: Caregivers of FTD patients were overall more distressed by the behaviour of their partners than were the caregivers of AD patients. Findings from this study underscore the importance of differentiating between diagnostic groups and specific behavioural domains when focusing on caregiver reactions to problem behaviour. PMID- 16679764 TI - Of minds and embryos: left-right asymmetry and the serotonergic controls of pre neural morphogenesis. AB - Serotonin is a clinically important neurotransmitter regulating diverse aspects of cognitive function, sleep, mood, and appetite. Increasingly, it is becoming appreciated that serotonin signaling among non-neuronal cells is a novel patterning mechanism existing throughout diverse phyla. Here, we review the evidence implicating serotonergic signaling in embryonic morphogenesis, including gastrulation, craniofacial and bone patterning, and the generation of left-right asymmetry. We propose two models suggesting movement of neurotransmitter molecules as a novel mechanism for how bioelectrical events may couple to downstream signaling cascades and gene activation networks. The discovery of serotonin-dependent patterning events occurring long before the development of the nervous system opens exciting new avenues for future research in evolutionary, developmental, and clinical biology. PMID- 16679765 TI - Effects of neonatal resveratrol exposure on adult male and female reproductive physiology and behavior. AB - Resveratrol (RES) is a phytoestrogen that has the ability to bind to estrogen receptors (ERs) and evoke biological effects that parallel those exerted by endogenous and synthetic estrogens. We have shown in previous studies that adult female rats acutely exposed to RES exhibit estrous cycle irregularity, ovarian hypertrophy, and alterations in sociosexual behavior. The present experiment characterizes the prolonged effects of maternal RES exposure throughout the lactational period on subsequent behavior, reproductive tissues, and brain morphology of the adult offspring. During adulthood, female offspring exposed to RES throughout nursing exhibited reduced body weight and increased ovarian weight, but exhibited normal estrous cyclicity and sociosexual behavior, without changes in the volume of the sexually dimorphic nucleus of the preoptic area or the anteroventral periventricular nucleus of the hypothalamus. During adulthood, males exposed to RES throughout nursing exhibited decreased body weight and plasma testosterone concentration, increased testicular weight, and reduced sociosexual behavior. These males also had significantly smaller sexually dimorphic nucleus of the preoptic area volumes and larger anteroventral periventricular nucleus volumes compared to male controls. These data suggest that postnatal exposure to RES may affect estrogenic activity in specific peripheral tissues (e.g., the gonads), while inducing antiestrogenic effects in the brain. Thus, the present study supports recent in vitro and in vivo findings that RES differs from most other phytoestrogens by acting as a possible mixed ER agonist/antagonist, depending on the tissue-specific availability of ER subtypes that are preferentially localized in specific brain regions and throughout the reproductive tract. More importantly these data indicate that maternal consumption of phytoestrogens during lactation can have lasting effects on the offspring that may not become apparent until they reach adulthood. PMID- 16679766 TI - Polyunsaturated fatty acid supplementation stimulates differentiation of oligodendroglia cells. AB - Dietary polyunsaturated fatty acids (PUFAs) have been postulated as alternative supportive treatment for multiple sclerosis, since they may promote myelin repair. We set out to study the effect of supplementation with n-3 and n-6 PUFAs on OLN-93 oligodendroglia and rat primary oligodendrocyte differentiation in vitro. It appeared that OLN-93 cells actively incorporate and metabolise the supplemented PUFAs in their cell membrane. The effect of PUFAs on OLN-93 differentiation was further assessed by morphological and Western blot evaluation of markers of oligodendroglia differentiation: 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP), zonula occludens-1 (ZO-1) and myelin-associated glycoprotein (MAG). Supplementation of the OLN-93 cells with n-3 and n-6 PUFAs increased the degree of differentiation determined by morphological analysis. Moreover, CNP protein expression was significantly increased by gamma-linolenic acid (GLA, 18:3n-6) supplementation. In accordance with the OLN-93 results, studies with rat primary oligodendrocytes, a more advanced model of cell differentiation, showed GLA supplementation to promote oligodendrocyte differentiation. Following GLA supplementation, increased numbers of proteolipid protein (PLP)-positive oligodendrocytes and increased myelin sheet formation was observed during differentiation of primary oligodendrocytes. Moreover, increased CNP, and enhanced PLP and myelin basic protein expression were found after GLA administration. These studies provide support for the dietary supplementation of specific PUFAs to support oligodendrocyte differentiation and function. PMID- 16679767 TI - Effects of aging and D-glucose on locomotor activity, spontaneous alternation, and plasma glucose levels in preweanling Sprague-Dawley rats. AB - Previous research has shown that developmentally related memory impairments in immature rodents are malleable and may be attenuated using a variety of pharmacological and behavioral treatments. This experiment examined the effects of glucose (10, 100, 250 or 500 mg/kg) or saline on blood glucose level, locomotor activity, and spontaneous alternation (SA) in the T-maze. Studies were conducted with 20-, 22-, and 24-day-old preweanling Sprague-Dawley rats. Results indicated a general decline in blood glucose level with age and size of glucose injection. The largest overall decline in blood glucose was found following the 500 mg/kg dose in 24-day-old animals. An increase in T-maze arm entries with age indicated increased exploratory activity. SA generally improved with age, but glucose had no reliable effect on behavior. The ontogenetic state of the nervous system, including changes in cholinergic system activity, glucoregulation, and proliferation of glucose transporters, is discussed. PMID- 16679768 TI - Cerebellar neurons and glial cells are transducible by lentiviral vectors without decrease of cerebellar functions. AB - Due to the profuse connections of the cerebellum to the rest of the central nervous system, cerebellar dysfunction impacts tremendously on movement coordination, maintenance of equilibrium, muscle tone and motor memory. Efficient gene transfer of therapeutic genes to this central nervous system structure would constitute a relevant step ahead the design of treatments to ameliorate cerebellar dysfunction. Lentiviral vectors (LVs) have been used as efficient vehicles to integrate transgenes into dividing and non-dividing cells, such as postmitotic adult neurons, with minimal toxicity and immune response. This study aimed to use LVs carrying green fluorescent protein (GFP) cDNA for transduction of cerebellar cells in vivo without compromising neurological cerebellar functions. Our results indicate that LVs, injected in the lobulus simplex, transduced different cerebellar neurons including stellate, Purkinje cells, granular neurons and glial cells such as astrocytes, oligodendrocytes, and that this gene transfer approach was not accompanied by cerebellar deficits. PMID- 16679769 TI - Dietary polyunsaturated fatty acids in gestation alter fetal cortical phospholipids, fatty acids and phosphatidylserine synthesis. AB - Docosahexaenoic acid (DHA; 22:6 n-3) is high in brain phosphatidylserine (PS) and phosphatidylethanolamine (PE), but low in phosphatidylcholine (PC). PS is synthesized from PE or PC by exchange of ethanolamine or choline for serine. PS can be decarboxylated to PE, and PC is synthesized from PE by phosphatidylethanolamine N-methyltransferase (PEMT). We characterized the perinatal changes in rat brain cortex phospholipids and metabolism and determined if maternal dietary n-3 fatty acid intake alters newborn brain cortex phospholipids, serine base exchange, PS decarboxylase or PEMT activities. PE became increasingly predominant, with an increase in the cortex PC/PE ratio from 2:1 at gestation day 19 to 1:1 at postnatal day 20. DHA increased and n-6 docosapentaenoic acid (DPA; 22:5 n-6) decreased in all phospholipids during development. [3H]serine incorporation into PS was higher in the fetal than neonatal brain cortex. Newborn rats of dams fed an n-3 fatty acid-deficient diet with 0.02% energy alpha-linolenic acid (ALA; 18:3 n-3) had approximately 50% lower DHA and higher DPA in cortex PE, PS and PC than newborns of rats fed a control diet with 1.5% energy ALA. [3H]serine incorporation into PS was significantly lower in the brain cortex of n-3 fatty acid-deficient than control newborns. n-3 Fatty acid deficiency had no effect on newborn brain PEMT or serine decarboxylase activities. These studies show that maternal dietary n-3 fatty acid deprivation impairs fetal brain DHA accretion and PS metabolism; altered PS metabolism may change release of lipid mediators and neurotransmitter precursors important in brain function. PMID- 16679770 TI - Changes in expression of N-methyl-D-aspartate receptor subunits occur early in the R6/2 mouse model of Huntington's disease. AB - A leading hypothesis of the cause of neuronal death in Huntington's disease (HD) is excitotoxicity, in which subpopulations of striatal neurons are hypersensitive to glutamate release due to changes in postsynaptic N-methyl-D-aspartate receptors (NMDARs). In the present study we used RT-PCR methods on single cells and tissue to compare the expression of NMDAR subunits, NR1, NR2A and NR2B, in the striatum of R6/2 transgenic mice with their wild-type (WT) littermates at three different age groups corresponding to different symptomatic milestones (19 25 days showing no overt evidence of abnormal behavior, 38-45 days at the onset of the overt phenotype and 78-90 days displaying the full behavioral phenotype). Single-cell RT-PCR studies also examined neurons for the expression of substance P and enkephalin to define different subpopulations of medium-sized projection neurons of the striatum. The results showed a significant decrease in the percentage of cells expressing NR2A at all ages examined. The decrease in expression was not associated with any significant change in expression of NR1 or NR2B. Cells that did not express NR2A contained both enkephalin and substance P, but proportionately more cells containing enkephalin displayed decreases in NR2A. Semi-quantitative RT-PCR studies on striatal tissue in the oldest age group confirmed the significant decrease in NR2A and also showed a decrease in NR2B. These results support the hypothesis that changes in the composition of postsynaptic NMDARs occur in the R6/2 model of HD and this effect occurs early in the expression of the phenotype. PMID- 16679771 TI - Why we need a noninvasive diagnostic test for minimal to mild endometriosis with a high sensitivity. PMID- 16679773 TI - Genomic alterations in ectopic and eutopic endometria of women with endometriosis. AB - BACKGROUND/AIMS: Ectopic and eutopic endometria of women with endometriosis have been shown to contain genomic alterations. In this study, we sought to identify genomic alterations in both ectopic and eutopic endometria of 5 women with endometriosis and to examine whether the two tissues share any genomic alterations. We also attempted to classify tissue samples based on the alteration profiles. METHODS: Laser capture microdissection was used to harvest epithelial cells. High-resolution comparative genomic hybridization microarrays were used to identify genomic alterations in eutopic and ectopic endometria from 5 women with endometriosis. The results were validated by real-time RT-PCR and loss of heterozygosity analysis. RESULTS: All 5 patients had genomic alterations in their eutopic and ectopic endometria. The ectopic and eutopic endometria shared a sizable portion of genomic alterations. Cluster analysis of the genomic alteration profile correctly and consistently classified tissue samples from the 5 patients into two groups: peritoneal implants and ovarian cysts. CONCLUSIONS: The correct classification of tissue samples into two groups suggests that these two subtypes of endometriosis may have distinct genomic alteration profiles and are thus distinct entities, as previously proposed. The shared alterations are likely the ones that harbor genes responsible for an increased propensity of endometrial debris to implant to the ectopic sites and for early events that lead to the establishment of lesions. Alternatively, these shared alterations may harbor genes that are dysregulated in both eutopic and ectopic endometria. The identified genomic alterations should help to zero in genes involved in the pathogenesis of endometriosis in future studies. PMID- 16679772 TI - Is endometriosis associated with systemic subclinical inflammation? AB - Endometriosis is a pelvic inflammatory process with altered function of immune related cells and increased number of activated macrophages in the peritoneal environment that secrete various local products, such as growth factors and cytokines. The elevation of cytokines and other factors in the peritoneal fluid is accompanied by the elevation of similar factors, such as CRP, SAA, TNF-alpha, MCP-1, IL-6, IL-8 and CCR1, in the peripheral blood of patients with endometriosis. CD44+ and CD14+ monocytes are significantly increased, while CD3+ T lymphocytes and CD20+ B lymphocytes show modest, but significant decrease in peripheral blood of women with endometriosis. This indicates that endometriosis could be viewed as a local disease with systemic subclinical manifestations. This review provides an overview of data on the changes of various factors in peripheral blood and their potential use as diagnostic tools in patients with endometriosis. PMID- 16679774 TI - Segregation analysis of a complex quantitative trait: approaches for identifying influential data points. AB - BACKGROUND/AIMS: Complex traits pose a particular challenge to standard methods for segregation analysis (SA), and for such traits it is difficult to assess the ability of complex SA (CSA) to approximate the true mode of inheritance. Here we use an oligogenic Bayesian Markov chain Monte Carlo method for SA (OSA) to verify results from a single-locus likelihood-based CSA for data on a quantitative measure of reading ability. METHODS: We compared the profile likelihood from CSA, maximized over the trait allele frequency, to the posterior distribution of genotype effects from OSA to explore differences in the overall parameter estimates from SA on the original phenotype data and the same data Winsorized to reduce the potential influence of three outlying data points. RESULTS: Bayesian OSA revealed two modes of inheritance, one of which coincided with the QTL model from CSA. Winsorizing abolished the model originally estimated by CSA; both CSA and OSA identified only the second OSA model. CONCLUSION: Differences between the results from the two methods alerted us to the presence of influential data points, and identified the QTL model best supported by the data. Thus, the Bayesian OSA proved a valuable tool for assessing and verifying inheritance models from CSA. PMID- 16679775 TI - G-protein-coupled receptor (GPCR)-142 does not contribute to relaxin-3 binding in the mouse brain: further support that relaxin-3 is the physiological ligand for GPCR135. AB - Relaxin-3 is a recently discovered member of the insulin/relaxin superfamily that has been shown to be the endogenous ligand for G-protein-coupled receptor (GPCR)135 (SALPR). In addition, relaxin-3 has demonstrated affinity and functional agonism for GPCR142 (GPR100) and LGR7 receptors in vitro. Recent evidence suggests GPCR142 is the insulin-like peptide 5 (INSL5) receptor and LGR7 is the actual relaxin receptor. We have recently described a chimeric R3/I5 peptide that selectively activates GPCR135 and GPCR142, but lacks affinity for LGR7. GPCR142 is a pseudogene in the rat, which allowed the use of [(125)I]-R3/I5 to show GPCR135-like binding sites in the rat central nervous system by autoradiography. However, mouse GPCR142 is a viable gene. In the present study we explore whether GPCR142 is expressed in the mouse brain and whether it is likely to contribute to or interfere with the pharmacological evaluation of relaxin-3 ligands. Competition binding studies confirmed mINSL5 and [(125)I]-mINSL5 bind to mGPCR142 with high affinity. However, no detectable specific [(125)I]-mINSL5 binding sites were detected throughout the mouse brain and unlabelled INSL5 did not displace [(125)I]-R3/I5 binding sites, indicating an absence of detectable GPCR142 binding sites. Consistent with these findings, neither GPCR142 nor INSL5 mRNA were detectable in mouse brain by in situ hybridization. Overall, the distribution of GPCR135 mRNA overlapped with the distribution of GPCR135 binding sites shown by autoradiography using [(125)I]-R3/I5. GPCR135 mRNA and GPCR135 receptor binding sites are most prominent in the mouse amygdala and hypothalamus. These data suggest that relaxin-3/GPCR135 is the receptor ligand pair with physiological relevance in mouse brain. PMID- 16679776 TI - Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event. AB - Silvering (transition from yellow to silver eel) has been traditionally considered as a metamorphosis in view of the numerous morphological, physiological and behavioral changes preparing the eel for the oceanic migration. However, some changes, such as increases in gonad weight and steroidogenesis, suggest that silvering could also be considered as a pubertal event. In order to assess which endocrine axis may be involved in the induction of silvering, we compared the profiles of pituitary and peripheral hormones during the transition from yellow to silver female eels. A strong activation of the gonadotropic axis was shown during silvering. Follicle-stimulating hormone (FSH) mRNA levels increased during the early stages of silvering, followed by a later increase in luteinizing hormone (protein and mRNA) levels. In addition, plasma levels of sexual steroids (estradiol, E2; testosterone, T, and 11-ketotestosterone) and of vitellogenin significantly increased. In contrast, thyrotropin mRNA levels did not change and no or weak variations in plasma thyroid hormones were observed, indicating no or moderate change of the thyrotropic axis during silvering. Similarly, the somatotropic axis was not activated, as shown by pituitary growth hormone expression (protein and mRNA) and plasma levels. In addition, we studied the effects of chronic treatments of female yellow eels with thyroid hormone (thyroxine, T4) and sex steroids (T and E2) on biometrical parameters characteristics of silvering. T induced an increase in eye size and a reduction of digestive tract, whereas T4 and E2 had no effect. These hormonal profiles and experimental data lead to the conclusion that eel silvering should be considered as an onset of puberty rather than a 'genuine' metamorphosis. PMID- 16679777 TI - Association of mu-opioid receptor gene polymorphism A118G with alcohol dependence in a Japanese population. AB - Ethanol is considered to activate the brain reward system by increasing the release of an endogenous opioid receptor ligand, beta-endorphin. The polymorphism A118G in the mu-opioid receptor gene (OPRM1) causes the amino acid change Asn40Asp and has been reported to affect the affinity of the ligand for the receptor. The association of this polymorphism with the vulnerability to alcohol dependence has been studied in many populations, but not yet in Japanese people. In the present study, we compared the frequencies of the polymorphism OPRM1 A118G between patients with alcohol dependence and healthy control subjects living in a Japanese provincial prefecture. We also genotyped a polymorphism, G1510A, in the acetaldehyde dehydrogenase 2 gene (ALDH2), in which the A allele causes poor metabolism of acetaldehyde, a major metabolite of alcohol. Both OPRM1 118G and ALDH2 1510G were significantly associated with alcohol dependence. These results suggest that OPRM1 118G in addition to ALDH2 1510G might be one of the risk factors for alcohol dependence in Japanese people. PMID- 16679778 TI - Auditory fear conditioning and conditioned stress raise NO(3) level in the Amygdala. AB - BACKGROUND: The conditioned fear response is considered to be acquired by experimental animals when tone information is combined with that of an electrical foot shock (unconditioned stimulus) in the amygdala. Nitric oxide biosynthesized in the brain is reportedly involved in several kinds of learning. METHODS: In this study, we continuously monitored the NO(3)(-) level, as a marker of nitric oxide production, in the amygdala starting before the application of tone and electrical foot shock stimuli together (conditioned group) or the tone stimulus alone (control group) on day 1, until after the tone information was given (both groups) on day 2, using the in vivo microdialysis method. RESULTS: The NO(3)(-) level of the conditioned group was increased on both day 1 and day 2, while that of the control group was not elevated on either day. Freezing behavior was observed in the conditioned but not the control rats. CONCLUSIONS: Although the sources of NO(3)(-) remain uncertain, these results suggest that nitric oxide is associated with auditory fear conditioning and the response to a conditioned stimulus. PMID- 16679779 TI - Should dental amalgam fillings fill us with fear? PMID- 16679780 TI - Tissue microarray analysis of EGFR gene amplification and gain in Bulgarian patients with colorectal cancer. AB - Colorectal cancer is one of the most common neoplastic diseases and a leading cause of cancer-related deaths. Overexpression of epidermal growth factor receptor (EGFR) is commonly associated with this cancer. The aim of our study was to determine the frequency of epidermal growth factor receptor gene amplification and gain in a large number of colorectal carcinomas, arranged in a tissue microarray, in order to assess their role in colorectal cancer development. A tissue microarray of 498 patients with colorectal tumors was constructed, and 239 samples for EGFR copy number changes were successfully analyzed by fluorescence in situ hybridization. No amplification of EGFR was detected in our cohort of patients with colorectal tumors, and the EGFR gene was upregulated in only 2 tumors (0.84%). Therefore, the development of colon cancer in patients cannot be explained by copy number changes of the EGFR gene. PMID- 16679781 TI - Adrenal insufficiency caused by bilateral adrenal metastases -- a rare treatable cause for recurrent nausea and vomiting in metastatic breast cancer. AB - BACKGROUND: Nausea and vomiting are common symptoms in patients with malignant disease. Several, sometimes rare causes have to be considered to decide the right treatment. CASE REPORT: We report of a patient suffering from advanced breast cancer and complaining of severe nausea and vomiting over several weeks without any successful treatment. Later on, she developed marked hyperpigmentation of the skin and hypo-osmolar dehydration. Adrenal enlargement was noted in an abdominal scan. The suspected diagnosis of primary adrenocortical insufficiency due to metastases was confirmed by laboratory tests. After replacement therapy with hydrocortisone and fludrocortisone, the general condition of the patient improved dramatically and the symptoms of nausea and vomiting disappeared completely. CONCLUSION: If a patient with advanced cancer presents with unexplained and protracted nausea, vomiting and weakness, particularly if accompanied by hyponatremia and normal potassium levels, adrenal insufficiency due to adrenal metastases should be considered. PMID- 16679782 TI - Long-term survival after second-line therapy with docetaxel and carboplatin and monthly pamidronic acid in a woman with metastatic non-small cell lung cancer. AB - BACKGROUND: In patients with metastatic non-small cell lung cancer (NSCLC), second-line chemotherapy induces response rates of less than 20% and median survival times between 5 and 8 months. CASE REPORT: In the case described here, a patient with metastatic NSCLC responded with complete remission of the primary tumor and the involved lymph nodes as well as partial remission of bone metastases to a second-line chemotherapy with docetaxel and carboplatin. Since April 2003 (33 months), no tumor progression has been observed. Until present, the patient received monthly infusions of pamidronic acid. CONCLUSION: Our case report indicates that in certain patients with metastatic NSCLC who did not respond to first-line regimens, second-line chemotherapy can induce outstanding tumor response and significantly improve survival. It also indicates that the role of bisphosphonates in the treatment of NSCLC should be further investigated in large clinical trials. PMID- 16679783 TI - Critical appraisal of primary systemic endocrine therapy in receptor-positive postmenopausal breast cancer: an update. AB - Even in elderly patients, greater consideration is now being given to tumor volume reduction in locally advanced breast cancer, with increased subsequent breast-conserving surgery. Neoadjuvant endocrine therapy offers the possibility of testing therapeutic efficacy in vivo, which is of great importance for optimal adjuvant treatment. Resulting therapy modifications can be expected to increase disease-free as well as overall survival. Recent results indicate that remission rates with primary chemotherapy are significantly lower in receptor-positive than in receptor-negative breast cancer and that efficacy parameters in receptor positive tumors tend to favor primary endocrine therapy, highlighting the increased importance of this type of treatment. Aromatase inhibitors are superior to tamoxifen in terms of clinical response as well as breast conservation rate. Results from a small number of studies suggest that prolonged preoperative aromatase inhibitor therapy for up to 12 months can increase the rate of clinical and pathological complete remissions. In conclusion, primary endocrine therapy is a valid therapeutic option for postmenopausal patients with locally advanced hormone receptor-positive breast cancer and significant comorbidity, increased risk of complications with regard to anesthesia and surgery, desire for breast conserving surgery and/or reduced suitability for chemotherapy, as well as in very old patients. PMID- 16679784 TI - Image-guided micro-therapy for tumor ablation: from thermal coagulation to advanced irradiation techniques. AB - Image-guided tumor ablation has been established as a valuable adjunct in oncological treatment concepts. The majority of micro-therapeutic procedures is performed by applying thermal ablation, such as radiofrequency or laser. However, in view of the limitations of thermal ablation regarding tumor size or adjacent risk structures, novel techniques combining brachytherapy with modern interventional techniques have demonstrated favorable outcomes. In addition, advances in non-invasive percutaneous irradiation, such as extracranial stereotaxy, have demonstrated very competitive preliminary results. In this article, state-of-the-art techniques for micro-therapeutic tumor ablation are being described. PMID- 16679785 TI - Dental amalgam fillings and cancer -- consideration of long-term processes. PMID- 16679786 TI - The non-dry 'dry eye' complex. AB - Tactile sensations may derive from non-specific stimuli and lead to erroneous diagnosis and treatment. A perception of dryness may be caused by non-dry conditions of which four are presented here: (1) lid margin touch, (2) conjunctival laxity, (3) cilia touch, and (4) fibrin accumulation in conjunctival inflammation. PMID- 16679787 TI - Long-term outcome after vitrectomy for diabetic macular edema. AB - BACKGROUND: To determine the benefit of vitrectomy on eyes with diabetic macular edema. METHODS: A retrospective institutional case series was used including 66 patients (69 eyes) who had undergone pars plana vitrectomy for diabetic macular edema between 1992 and 2000. Prior to surgery, the patients had been treated with laser coagulation as recommended by the Early Treatment Diabetic Retinopathy Study. In the case of persistent macular edema, vitrectomy with removal of the posterior hyaloid in all cases and the inner limiting mem brane in 51 (74%) of all cases was performed. RESULTS: The mean preoperative best-corrected visual acuity improved from 20/320 to 20/80 at the time of best postoperative best corrected visual acuity (p < 0.0001). The mean increase in Snellen lines was 2.7 +/- 7.9. In 90% of eyes, the macular edema improved. A persistence of the edema was observed in 10%. All eyes had at least 12 months of follow-up with a mean of 55 months and a maximum of 120 months. CONCLUSIONS: Our findings confirm that vitrectomy might represent a therapeutic alternative in the case of persisting diabetic macular edema after laser photocoagulation. PMID- 16679788 TI - Evaluation of indocyanine green toxicity to rat retinas. AB - PURPOSE: To investigate the toxicity of indocyanine green (ICG) on retinal cells using cultured retinal pigment epithelium (RPE) cells and the effects of intravitreous injection of ICG into rat eyes. METHODS: Cultured RPE cells were exposed to various concentrations of ICG for 2 min, a viability assay was performed 1 day after exposure. For an in vivo study, 5 microl of ICG (5 or 25 mg/ml) were injected into the vitreous cavity of rat eyes, which were examined 1, 3 and 7 days after the injection by histological and glutamine synthetase (GS) immunohistological evaluation. RESULTS: Viabilities of RPE cells were decreased dependent on the ICG dose. In the histological evaluation, we observed differences of effects of ICG between the central retinal area and the peripheral area. ICG injection caused degeneration of all retinal layers in the central retinal area. GS immunoreactivities decreased by ICG injection, which corresponded to an area of severe destruction. CONCLUSION: A high concentration of ICG may cause toxic effects on retinal cells. Mueller cell dysfunction may play some role in the retinal toxicity caused by ICG. PMID- 16679789 TI - Scleral resection technique combined with vitrectomy for a macular hole retinal detachment in highly myopic eyes. AB - PURPOSE: To evaluate scleral resection technique combined with vitrectomy for macular hole retinal detachment of highly myopic eyes. MATERIALS AND METHODS: Seventeencases (17 eyes) of macular hole retinal detachment in highly myopic eyes, in which the patient underwent vitrectomy combined with scleral resection technique formacular hole retinal detachment between January 1996 and December 2003 at Fukuoka University Chikushi Hospital, were studied. Following pars plana vitrectomy, as much as possible of the residual vitreous and/or epiretinal membrane was removed. A scleral resection was performed in 2 quadrants of the equatorial region of the temporal sclera. Finally, a fluid-air exchange with SF(6) gas injection was performed to achieve retinal attachment. Pre- and postoperative axial length of the eyeballs were measured by B-scan ultrasonography. RESULTS: All cases had the retina reattached at the initial surgery, and visual acuities were stabilized or improved after the surgery. The posterior staphyloma became obscure in 13 out of 17 eyes (76.8%). The macular hole closed in 14 of 17 eyes (82.4%) ophthalmoscopically. There were no cases in which retinal redetachment occurred during follow-up periods of more than 6 months. CONCLUSION: In cases of macular hole retinal detachment of a highly myopic eye, scleral resection technique combined with vitrectomy changed the shape of the eyeballs and allowed successful retinal reattachment at the initial surgery. PMID- 16679790 TI - Deep lamellar keratoplasty in the treatment of keratoconus. AB - PURPOSE: To present our experience with a series of patients treated with deep lamellar keratoplasty (DLK) for keratoconus (KC). DESIGN: A single surgeon, prospective, consecutive series. METHOD: The study included all patients with KC who underwent DLK between March 1999 and November 2003 at the Royal Adelaide Hospital. The parameters evaluated included patients' demographics, pre- and post operative best corrected visual acuities, post-operative keratometry, and intra- and post-operative complications. RESULTS: There were 22 patients (23 eyes); 10 females and 12 males, with a mean age of 35 +/- 13 years (median, 33.5; range, 17 73). The median follow-up period was 13 months (range 7-38). In 89% (16/18) of eyes which underwent DLK, and in which a visual acuity could be obtained, a best corrected visual acuity of at least 6/12 was recorded. There were two episodes of Descemet's membrane perforation (8.7%), in which the procedure was converted to penetrating keratoplasty without complications. One patient developed a double anterior chamber, which resolved spontaneously without consequences. CONCLUSION: The visual outcomes and complication rates seen in our series are comparable to the recent published literature. Hence DLK can be considered as a suitable alternative to penetrating keratoplasty for the surgical treatment of KC. PMID- 16679791 TI - Instrumentation and technique for delivery of tissue explants to the subretinal space. AB - PURPOSE: The controlled orientation of subretinal or intraretinal delivery of delicate tissue sheets is a critical problem in retinal transplantation research. METHODS: A surgical device (explant injector) was designed and built for the controlled delivery of tissue explants into the subretinal or intraretinal space via a vitrectomy approach. The technical feasibility was demonstrated in enucleated porcine eyes. RESULTS: Using negative pressure, the injector allows for the fixation of an explant, such as a monolayer of cells cultured on a collagen membrane, on the carrier platform. The sheet can then be placed safely without loss of orientation in the subretinal space. CONCLUSIONS: With the aid of the explant injector, it is possible to deliver an explant with the proper orientation to a precise location in the subretinal space. PMID- 16679792 TI - Visual recovery after scleral buckling surgery in macula-off rhegmatogenous retinal detachment. AB - PURPOSE: The primary goal of this study was to investigate the functional results after scleral buckling (SB) surgery in macula-off rhegmatogenous retinal detachment (RRD), with more or less than 7 days' duration of macular detachment (DMD). The secondary outcome measure was to determine the long-term functional results in these two groups 5 years after SB surgery. METHODS: The retrospective studies included 96 eyes of 96 patients with primary, uncomplicated, macula-off RRD. Two studies, one with a short-term follow-up and one with a long-term follow up, were performed, and in both studies the eyes were divided into two groups according to the DMD. In study I, 96 patients were divided into DMD or=4 days and 7 days (n = 11). Additional preoperative predictive factors including DMD, preoperative visual acuity (VA) and patients' age at the time of the RRD were correlated with the postoperative VA. RESULTS: In study I, the mean final VA was significantly better (0.45) after a DMD of less than or equal to 7 days, compared to patients with a DMD of more than 7 days (0.22; Student's t test, p = 0.02). VA >or=0.4 was achieved in 68% with a subacute DMD compared to 52% in patients with prolonged DMD (chi(2) test, p < 0.001). However, the postoperative VA (0.48 +/- 0.09) in eyes with DMD of 1-3 days was similar (0.42 +/- 0.07) to eyes with DMD of 4-7 days (Student's t test, p = 0.455). We divided the 96 patients into 2 groups according to a preoperative VA. Eyes with a preoperative VA 0.1 (n = 34; Student's t test, p < 0.001). Patients 75 years of age or younger were more likely to achieve a VA of 0.4 or better after SB surgery than older patients (more than 75 years; chi(2) test, p = 0.008). In study II, VA improved (by a mean of 1.60 +/- 0.02 lines) during the follow-up period from 0.32 +/- 0.08 at the 3-month follow-up to 0.46 +/- 0.10 at the 5-year follow-up. Eyes with a subacute DMD had a significantly better VA than eyes with a prolonged DMD (Student's t test, p = 0.004). Patients aged 75 years or less were more likely to achieve a VA of 0.4 or better than patients older than 75 (chi(2) test, p = 0.003). CONCLUSION: The time point of SB surgery has no statistical impact on the final visual recovery in patients with an acute primary macular-off RRD of less than or equal to 7 days. A delay of SB surgery within this time frame does not contribute to an impaired final visual outcome. There was no evidence, that primary macula-off RRDs are emergencies, which cannot wait for a systemic evaluation of the RRD and surgical treatment at the next available scheduled day. A preoperative VA of more than 0.1 and patients' age under 60 years at presentation had an additional prognostic value on the final outcome. Surgeons should be aware that visual function after reattachment may continue to improve over a long period. This study provides useful guidelines for the clinical management of macula-off RRD and the assessment of potential visual recovery in patients after successful SB surgery. PMID- 16679793 TI - Ocular findings in Japanese children with nephrotic syndrome receiving prolonged corticosteroid therapy. AB - OBJECTIVE: To examine ocular findings in Japanese children with nephrotic syndrome who receive prolonged corticosteroid treatment. METHODS: Information was retrospectively obtained from each patient's record. RESULTS: A total of 45 children (30 boys and 15 girls) were enrolled. Twenty-one patients (46.7%) had epiblepharon with inverted eyelashes. The incidence of epiblepharon was associated with overweight and obesity. Fifteen patients (33.3%) developed posterior subcapsular cataract in both eyes. Of the 15 patients, 4 had visual acuity less than 0.4 and underwent cataract extraction in both eyes. Nine patients (20%) developed intraocular pressure higher than 22 mm Hg during corticosteroid treatment. The total dose and duration of corticosteroid treatment were associated with cataract formation but were unassociated with elevated intraocular pressure. One boy had elevated intraocular pressure after cessation of corticosteroid therapy and underwent trabeculectomy. One patient had repeated hordeolum in the left eyelid. Three patients had bilateral bacterial conjunctivitis. CONCLUSION: Children with nephrotic syndrome who receive corticosteroid treatment may have epiblepharon, cataract, ocular hypertension, hordeolum and bacterial conjunctivitis. PMID- 16679794 TI - Penetrating keratoplasty combined with vitreoretinal surgery for severe ocular injury with blood-stained cornea and no light perception. AB - OBJECTIVE: To evaluate the effects of treatment of severe ocular injury with blood-stained cornea and no light perception by combined penetrating keratoplasty and vitreoretinal surgery, and to analyze the relevant factors. METHODS: Records of 7 severely injured eyes of 7 patients with blood-stained cornea and no light perception who underwent penetrating keratoplasty combined with vitrectomy using a temporary keratoprosthesis were evaluated retrospectively. The preoperative visual acuity was no light perception in all injured eyes with a mean intraocular pressure of 3 mm Hg and a range from 2 to 5 mm Hg. The average interval from emergency wound closure to vitrectomy was 18 days with a range from 12 to 21 days. The mean follow-up was 28 months with a range from 26 to 30 months. RESULTS: The postoperative visual acuity was better than light perception in 5 eyes with the best corrected visual acuity from light perception to 0.06. The retina was attached in 5 eyes. The postoperative intraocular pressure ranged from 5 to 15 mm Hg with a mean of 12 mm Hg; it was significantly higher than the preoperative one (p < 0.05). The postoperative complications mainly included temporary intraocular elevation (1 eye), corneal neovascularization (4 eyes), corneal rejection (4 eyes), retinal detachment (2 eyes) and ocular atrophy (2 eyes). CONCLUSION: Penetrating keratoplasty combined with vitrectomy using a temporary keratoprosthesis is a safe and effective method in treating severe ocular injury with blood-stained cornea and no light perception. PMID- 16679795 TI - Patent blue: a novel vital dye in vitreoretinal surgery. AB - PURPOSE: To determine a novel vital dye (patent blue; Blueron) for vitreoretinal surgery in a prospective consecutive case series. METHODS: Five patients with either idiopathic epiretinal membrane (ERM; n = 2), proliferative vitreoretinopathy (n = 2), or a macular hole (n = 1) underwent a three-port pars plana vitrectomy. Patent blue assisted staining of the retinal surface followed by a consecutive peeling of the ERM (n = 4) or of the internal limiting membrane (ILM; n = 1) was performed. The main outcome measures were quality of intraoperative visualization of preretinal structures and postoperative visual acuity. RESULTS: The dye induced a moderate staining (++) of the ERM and a mild staining (+) of the ILM. Complete ERM and ILM removal was successfully achieved in all cases. A mean visual improvement of three Snellen lines was observed 6 months postoperatively. No visual field defects or visible retinal pigment epithelial changes were present 6 months postoperatively. CONCLUSION: Patent blue, a novel dye for intraocular applications, may be added as an alternative dye in chromovitrectomy. PMID- 16679797 TI - Eyelid-sparing adjuvant radiation therapy for renal cell carcinoma. AB - PURPOSE: To describe the use of debulking surgery with adjuvant external beam irradiation as an eyelid-sparing treatment for renal cell carcinoma. DESIGN: Interventional case report. METHODS: A 63-year-old male presented with a right upper lid tumor. He had a history of renal cell carcinoma and pulmonary metastasis treated with surgery and systemic chemotherapy. The eyelid tumor was biopsied, followed by debulking surgery and external beam radiation therapy to treat this metastatic tumor. RESULTS: Histopathological evaluation of the excised tumor revealed a metastatic renal cell carcinoma, clear cell type. At 4 months' follow-up, he had no evidence of recurrence or radiation oculopathy. He was pleased with his cosmetic result. CONCLUSIONS: Meta static renal cell carcinoma presenting in the eye and orbit can be the initial manifestation of the primary tumor. It is important to include this tumor in the differential diagnosis of recurrent eyelid lesions. Debulking surgery followed by external beam radiation therapy can be used to control the tumor with an eyelid-sparing cosmetic result. PMID- 16679796 TI - Measurement of choroidal melanoma basal diameter by wide-angle digital fundus camera: a comparison with ultrasound measurement. AB - PURPOSE: To compare the measurement of the basal diameter of choroidal melanoma, an important parameter for planning treatment and as a prognostic factor, by standardized ophthalmic ultrasound versus that obtained using a wide-angle fundus camera. METHODS: The longest and shortest basal diameters of 104 consecutive choroidal melanomas of patients seen at the ocular oncology service of the Hadassah University Hospital were measured by B-scan ultrasound and compared with those measured by a wide-angle fundus camera (Panoret-1000). Each diameter was measured thrice by two ophthalmic photographers, and intra-observer and interobserver reproducibility were calculated as well. RESULTS: The measurements of both the long and short diameters of the choroidal melanomas were significantly larger when measured by Panoret-1000 as compared with the B-scan ultrasound. There was no significant difference among the three measurements of each of the photographers. The interobserver reproducibility between the two photographers was high. CONCLUSIONS: Our findings can be attributed to the larger extent of the pigmentation of the melanoma compared to its recognized elevation. Since pigmentation of choroidal melanoma is easier to recognize than its elevation, we assume that measurement by a wide-angle fundus camera is more accurate in pigmented tumors, and should be adopted for measuring the basal diameter of choroidal melanomas in planning treatment and follow-up. PMID- 16679798 TI - Orbital actinomycosis associated with painful ophthalmoplegia. Actinomycosis of the orbit. AB - PURPOSE: To evaluate a case of orbital actinomycosis presenting as Tolosa-Hunt syndrome in a patient with a history of carcinomas of the kidney and breast. METHODS: A woman with ingravescent painful ophthalmoplegia was brought to our observation. Brain and orbital and total body CT scans showed the presence of two orbital neoformations and a miliary pattern of dissemination in the lung. The initial diagnosis, which pointed to secondary localisations of the previous kidney and breast tumours, was changed to orbital and pulmonary actinomycosis following microbiological analysis of lung biopsy samples. RESULTS: Prolonged antibiotic therapy with synthetic penicillin completely resolved the case. CONCLUSIONS: Actinomycosis is a very rare infection that may also affect the orbit and its association with a pulmonary dissemination is highly unusual. It is important to consider this type of infection among the causes of painful ophthalmoplegia. PMID- 16679799 TI - Vitrectomy for diabetic macular edema with and without internal limiting membrane removal. PMID- 16679801 TI - External dacryocystorhinostomy. PMID- 16679803 TI - Classification of corneal dystrophies. PMID- 16679804 TI - The use of nerve growth factor in surgical wound healing of the cornea. AB - BACKGROUND: Recent studies have shown how the topical application of nerve growth factor (NGF) has led to the repair of neurotrophic corneal ulcers with recovery of corneal surface sensitivity. The biological effect of NGF, at a corneal level, is mediated by the presence of specific receptors localized on the surfaces of the corneal and conjunctival cells. OBJECTIVES: To evaluate the efficacy of NGF to promote corneal wound healing after cataract surgery. METHODS: Thirty patients were divided into two groups (groups A and B) and underwent cataract surgery. After surgery patients in group A received 1 drop of NGF solution (10 microg of NGF dissolved in 50 microl of saline solution, 0.9% of sodium chloride) in the conjunctival fornix every 2 h (from 6 a.m. to 12 p.m.) for 2 weeks and then 4 times a day for another week. The patients in group B received 1 drop of hyaluronic acid 0.2% eye drops in the conjunctival fornix every 2 h for 2 weeks and then 4 times a day for another week. With optical coherence tomography (OCT) we evaluated the corneal thickness at the side of the surgical wound, the endothelial cell count and the incision line in the stroma 1, 7 and 21 days after surgery. RESULTS: Before surgery in group A and in group B, the endothelial cell count was 2,607.4 +/- 261.0 versus 2,602.0 +/- 266.6 (p < 0.991), and the temporal cornea edge thickness was 639.2 +/- 24.7 versus 644.4 +/- 31.9 microm (p < 0.605), respectively. At 24 h after surgery, the results were: 2,523.2 +/- 280.5 versus 2,528.2 +/- 235.7 (p < 0.988) and 804.4 +/- 29.5 versus 802.6 +/- 35.0 microm (p < 0.953). After 7 days the cell count values were: 2,511.4 +/- 229.8 versus 2,490.0 +/- 230.4 (p < 0.361) and corneal thickness 713.6 +/- 16.5 versus 771.4 +/- 36.5 microm (p < 0.047). Finally, 21 days after surgery, the number of endothelial cells was 2,540.2 +/- 237.3 versus 2,503.4 +/- 224.5 (p < 0.382) and corneal thickness 645.2 +/- 22.6 versus 704.2 +/- 11.8 microm (p < 0.002). In the patients treated with NGF, on day 21, we found with OCT a complete wound healing, and the stromal incision was not visible. CONCLUSIONS: This clinical experience shows that the topical administration of NGF is effective in accelerating the healing of surgical corneal wounds. PMID- 16679805 TI - Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases. AB - OBJECTIVE: To determine the levels of interleukin 6 (IL-6) and interleukin 8 (IL 8/CXCL-8) in tears collected from the eyes of normal individuals and of patients with different irritative eye diseases, in order to acquire information on the immunological changes occurring during the early postoperative period following various forms of eye surgery, including penetrating keratoplasty (PKP). METHODS: IL-6 and IL-8 levels were measured with the aid of human ultrasensitive ELISA kits in the non-stimulated tears of patients in the early postoperative period following PKP or cataract operation, and of patients with acute bacterial conjunctivitis or with a corneal foreign body. The IL-6 and IL-8 concentrations, the total amounts released in a given time and the rates of their release were calculated. RESULTS: A significant increase in IL-6 release was observed in all patient groups compared with the normal controls (p < or = 0.003). The IL-8 release levels were significantly higher in the tears of all patient groups (p < or = 0.03), except for the cataract operation group, where the IL-8 release was not significantly higher (p = 0.053) than in the control samples. No significant differences in IL-6 or IL-8 release were observed when the various patient groups were compared with each other. CONCLUSION: The release of IL-6 and IL-8 into the tears is enhanced in various anterior segment eye diseases, and this may be used as an indicator of various inflammatory reactions in the early postoperative period. PMID- 16679806 TI - Detection of PEDF in subretinal fluid of retinal detachment: possible role in the prevention of subretinal neovascularization. preliminary results. AB - PEDF (pigment-epithelium-derived factor) is a member of the serpin family of protease inhibitors. It is considered to be an important regulator of human eye disease and is known to inhibit angiogenesis. We have therefore investigated the presence of PEDF in the subretinal fluid of patients with retinal detachment. METHODS: Eighteen samples from SRF were collected from patients during retinal detachment surgery. Specific ELISA analysis was performed with specific IgG against human PEDF. RESULTS: PEDF was detected in the subretinal fluid of all cases. The mean concentration of PEDF was 33.9 ng/ml (SD 23.7 ng/ml; range 5.3 74.7 ng/ml). The majority of samples had however a concentration of more than 22 ng PEDF/ml fluid. CONCLUSION: PEDF appears to be a constant component of the fluid accumulating in the subretinal space after retinal detachment. The known effects of PEDF, however, suggest that it may be involved in physiological processes of wound healing in the subretinal space. PMID- 16679807 TI - Intraretinal immunohistochemistry findings in proliferative vitreoretinopathy with retinal shortening. AB - To report the major intraretinal pathological changes in retinas with proliferative vitreoretinopathy (PVR) and retinal shortening, 13 human retinal samples from postoperative PVR after primary surgery for retinal detachment were immunostained for vimentin, glial fibrillary acidic protein (GFAP), cytokeratins, and CD68. One more sample was studied with electron microscopy. Retinal disorganization, neuronal loss, and gliosis were observed in 12 out of 13 samples, but all 13 were positive for GFAP. Muller cell processes showed different degrees of intermediate filament hyperplasia. CD68-positive cells were present in 11 of 13 retinal samples. CONCLUSION: A gliotic response plays a major role in retinal shortening in PVR. In addition, the presence of macrophage-like cells in retinal tissues suggests a possible role of these cells in the pathogenesis of this variety of PVR. PMID- 16679808 TI - Effects of chymase on the macular region in monkeys and porcine muller cells: probable involvement of chymase in the onset of idiopathic macular holes. AB - OBJECTIVES: To investigate chymase involvement in idiopathic macular hole onset, the effects of chymase on monkey eyes and cultured Muller cells were investigated. METHODS: Immunohistochemistry using antinestin and antiglial fibrillary acidic protein antibodies was performed in a normal monkey eye. After chymase was injected into the monkey vitreous, histological changes in the retina were evaluated using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Expression of c-kit, a stem cell factor receptor, and nestin was examined in porcine Muller cells cultured with basic fibroblast growth factor. The effects of chymase on proliferation and TUNEL staining in Muller cells were also examined. RESULTS: The number of nestin and glial fibrillary acidic protein-positive cells was higher in the macula than in other regions. Thickening of the posterior hyaloid membrane and some apoptotic cells were found in the macula of chymase treated eyes. The expression of c-kit and nestin in Muller cells was shown and enhanced when cultured with basic fibroblast growth factor. Exposure to chymase inhibited Muller cell proliferation and produced TUNEL-positive cells. CONCLUSIONS: There might be Muller cells possessing atypical properties near the macular region and chymase might cause fibrosis and apoptosis through these cells. These findings suggest that increased chymase activity may result in idiopathic macular hole onset. PMID- 16679809 TI - Photodynamic therapy using verteporfin for choroidal neovascularization associated with angioid streaks--long-term effects. AB - AIM: To investigate the safety and efficacy of photodynamic therapy with verteporfin (PDT) in patients with choroidal neovascularization associated with angioid streaks (CNVAS). METHODS: A nonrandomized, prospective clinical investigation of 12 patients with CNVAS was performed. PDT was based on the criteria concerning the treatment of age-related macular degeneration. RESULTS: The mean follow-up was 41.75 months (range 24-60). The mean number of (re)treatments was 3.3 (range 2-7). Visual acuity improved by at least 1 line in 42%, was stable within +/-2 lines in 33%, decreased by at least 1 line in 58% and by >3 lines in 25% of the patients. The mean visual acuity was 0.30 (range 0.2 0.5) prior to and 0.17 (range 0.03-0.6) after the final PDT. The mean visual acuity of the contralateral eye was 0.1. 75% of contralateral eyes and 25% of the treated eyes had a final visual acuity of < or =0.1 (20/200). At the final follow up, a significant enlargement of the lesion size was noted in 92% of the cases. CONCLUSION: Using the current (re)treatment criteria, PDT does not appear to limit the growth of CNVAS. Compared to the aggressive natural course and to the limited treatment options, PDT may at least in part help to stabilize macular function over a limited period of time. PMID- 16679810 TI - Transtympanic steroids as a salvage therapy in sudden hearing loss: preliminary results. AB - PURPOSE: To understand the real efficacy of transtympanic steroid therapy for sudden sensorineural hearing loss (SSHL) in patients in whom traditional therapies have failed. PROCEDURES: A prospective study was designed in order to evaluate hearing improvement in SSHL patients treated with transtympanic therapy. A solution of methyl-prednisolone (MP) and sodium bicarbonate was administered via a transtympanic injection to 8 patients. Hearing level was evaluated before therapy and at days 1, 7 and 30. RESULTS: Hearing improvement was obtained in 75% of the patients. The patients in this category are usually considered untreatable. CONCLUSION: Transtympanic steroid therapy is an efficacious solution for patients affected by SSHL in whom traditional therapies have failed. Further studies will be required to identify the most favourable dosage, route of administration and type of steroid. Transtympanic steroid application is safe, inexpensive, easy to perform and efficacious in cases of SSHL not responsive to traditional therapy. PMID- 16679811 TI - Quality of life after treatment for laryngeal carcinomas. AB - The objective of this study was to evaluate the impact of different surgical treatments as well as of radiotherapy for laryngeal carcinomas on health-related quality of life (QL). In a prospective, randomized multicenter study (five university hospitals in Germany), a total of 146 patients with laryngeal carcinomas (UICC stages: I-IV) underwent different surgical treatments (32 total laryngectomies, 81 CO(2) laser microsurgical partial laryngectomies, 33 open partial laryngectomies). Postoperative radiotherapy was performed in 44 patients. QL data were obtained by using the EORTC QLQ-C30 questionnaire (developed by the European Organization for Research and Treatment of Cancer). Impaired QL data were seen after total laryngectomy and after radiotherapy. Radiotherapy seemed to have more impact on QL than surgical treatment. Global QL was not affected by any treatment. In general, the QL data were not as discriminating as presumed. To evaluate coping abilities, objective measures (voice, swallowing, breathing) should be obtained for comparison in further investigations. PMID- 16679812 TI - Deep neck infections: a constant challenge. AB - BACKGROUND: Although the advent of antibiotics and improved dental care decreased the incidence and mortality, deep neck infections (DNIs) are not uncommon and present a challenging problem due to the complex anatomy and potentially lethal complications that may arise. OBJECTIVES: This study reviews our experience with DNIs and tries to identify the predisposing factors of life-threatening complications. METHODS: A retrospective review was conducted of patients who were diagnosed as having DNIs in the Department of Otolaryngology and in the Department of Infectious Diseases at Treviso Regional Hospital from 1995 to 2003. Associations between life-threatening complications and other factors were determined by chi(2) test, Fisher's exact test and Student's t test as appropriate. RESULTS: One hundred sixty-seven charts were recorded; 95 (56.9%) were men, and 72 (43.1%) were women, with a mean age of 49.6 +/- 20.4 years (range: 2-96). There were 39 patients (23.4%) who had associated systemic diseases, with 53.8% (21/39) of those having diabetes mellitus. The lateral pharyngeal and submandibular spaces were the most commonly involved spaces. Upper airway infections and odontogenic infections were the two most common causes of DNIs (47.5 and 27.9% of the known causes, respectively). The pathogenesis remained unknown in 45 patients (26.9%). Coagulase-negative staphylococcus (36.9%) and Streptococcusviridans (28.8%) were the most common organisms, identified through cultures. Of the abscess group (77 patients), 42 patients (54.5%) underwent surgical drainage under general anesthesia. Thirty-one patients (18.6%) developed life-threatening complications: airway obstruction (n = 18), descending mediastinitis (n = 6), jugular vein thrombosis (n = 4), and pneumonia (n = 3). Compared with other patients, the unique features of patients with life threatening complications were as follows: older age (p = 0.04), a higher white blood cell count (p = 0.01), abscess formation (p = 0.02), associated systemic disease (p < 0.001), diabetes mellitus (p < 0.001), anterior visceral space involvement (p < 0.001), and multiple-space involvement (p < 0.001). CONCLUSIONS: DNIs continue to occur and these are associated with significant morbidity and mortality even in this era of antibiotics. Furthermore, the widespread and inappropriate use of antibiotics may change the clinical presentation and course of these infections, making them more elusive and less predictable also in complicated cases. The clinical assessment of patients who are older, with abscess formation, underlying systemic diseases, diabetes mellitus, visceral anterior space or multiple-space involvement requires careful consideration of potential complications. PMID- 16679813 TI - Insertion of double bicanalicular silicone tubes after endonasal dacryocystorhinostomy in lacrimal canalicular stenosis: a 10-year experience. AB - To assess the long-term results of double bicanalicular silicone tubes in canalicular (presaccal) stenosis of the nasolacrimal duct system, a retrospective follow-up was performed on patients operated between 1992 and 2002. Forty-five patients were included in the study, 44 of whom had eyes with canalicular stenosis (primary dacryocystorhinostomy), 4 with congenital agenesis of the lacrimal duct system, and 3 cases after primary external dacryocystorhinostomy. Double bicanalicular silicone tubes were inserted, fixed in the vestibulum nasi, and left in place as long-term space holders. Of the 51 endonasal primary dacryocystorhinostomies with canalicular stenosis, 32 (63%) became symptom-free after one procedure. Nineteen (37%) patients had a relapse. Double silicone bicanalicular tube insertion as long-term spacers in canalicular stenoses has a number of advantages, but canalicular stenoses still remain a challenging area. PMID- 16679814 TI - Technical Note: minimal access surgery for cochlear implantation with MED-EL devices. AB - BACKGROUND: Minimally invasive techniques have been described for cochlear implant surgery, but so far this had not been the case for Med-EL devices. OBJECTIVE: To describe a newly developed minimal access approach for the implantation of Med-EL devices and report our results after up to 1 year of follow-up in 52 patients. DISCUSSION: The use of a minimally invasive approach without raising a flap or extensive drilling of a bony well was feasible in all 52 patients of this series. It shortened the surgical time to an average of 45 min and there were no specific postoperative complications. The average follow-up of 8.4 months was uneventful with the implants well covered and fixed in their position. The described approach therefore appears to be a safe, time- and cost effective alternative to the standard procedure in cochlear implant surgery using Med-EL devices. PMID- 16679815 TI - The heart of drug discovery and development: rational target selection. AB - Critical to the discovery and development of drugs and vaccines is the rational selection of biochemical, immunologic or molecular targets. To understand the rationale for target selection, we review strengths and weaknesses of the four main approaches: whole animal disease models; molecular targeting; epidemiology/observation studies, and genomics. After classifying diseases into those with a relatively stable pathophysiology (e.g., hypertension and gout) versus those with an unstable pathophysiology (e.g., AIDS and influenza) to aid in understanding target selection, we provide examples of successful and unsuccessful selection of drug and vaccine targets, focusing on the molecular and epidemiological/observational approaches. We discuss the reasons that molecular targeting has led to successful control of many diseases, whereas the epidemiological/observational approach has had a checkered history. We also assess the potential power of the genomic approach, specifically the curative versus controlling/preventive strategies. With combined genetic and molecular approaches and judicious use of whole animal models and properly performed epidemiology/observation studies to select the appropriate targets, the future for controlling, preventing and even curing many diseases is very bright indeed. PMID- 16679816 TI - The tramadol metabolite, O-desmethyl tramadol, inhibits 5-hydroxytryptamine type 2C receptors expressed in Xenopus Oocytes. AB - PURPOSE: Tramadol is widely used clinically as an analgesic, yet the mechanism by which it produces antinociception remains unclear. O-Desmethyl tramadol, the main metabolite of tramadol, is a more potent analgesic than tramadol. We reported previously that tramadol inhibits the 5-hydroxytryptamine (5-HT) type 2C receptor (5-HT(2C)R), a G-protein-coupled receptor that is expressed widely within brain and that mediates several effects of 5-HT, including nociception, feeding, and locomotion. The effects of O-desmethyl tramadol on 5-HT(2C)R have not been studied. In this study, we investigated the effect of O-desmethyl tramadol on 5 HT(2C)R expressed in Xenopus oocytes. METHODS: We examined the effect of O desmethyl tramadol on 5-HT(2C)R using the Xenopus oocyte expression system. Furthermore, we investigated the effects of O-desmethyl tramadol on the binding of [(3)H]5-HT by 5-HT(2C)R. RESULTS: O-Desmethyl tramadol, at pharmacologically relevant concentrations, inhibited 5-HT-evoked Ca(2+)-activated Cl(-) currents in oocytes that expressed 5-HT(2C)R. The inhibitory effect of O-desmethyl tramadol on 5-HT(2C)R was overcome at higher concentrations of 5-HT. Bisindolylmaleimide I (GF109203X), a protein kinase C inhibitor, increased 5-HT-evoked currents but had little effect on the inhibition of 5-HT-evoked currents by O-desmethyl tramadol. O-Desmethyl tramadol inhibited the specific binding of [(3)H]5-HT by 5-HT(2C)R expressed in oocytes. O-Desmethyl tramadol altered the apparent dissociation constant for binding of [(3)H]5-HT by 5-HT(2C)R without changing maximum binding, which indicated competitive inhibition. CONCLUSION: These results suggest that O desmethyl tramadol inhibits 5-HT(2C)R, which provides further insight into the pharmacological properties of tramadol and O-desmethyl tramadol. PMID- 16679817 TI - Ethnic variation in vellus hair follicle size and distribution. AB - It is a given fact that hair follicles play an important role in the penetration process of topically applied drugs and cosmetics. Since exact knowledge of the potential follicular reservoir is essential for understanding and calculating the penetration process, this knowledge may indeed help to optimize the development of topically applied drugs and cosmetic products. We know that variability in skin properties exists among the human population, but it is the deciphering and quantification of these differences that may help to explain racial disparities in dermatological disorders and provide a basic approach for treatment and prevention. Data pertaining to structural and functional differences within ethnic skin types (reviewed in this paper) support the assumption that hair follicles in the various skin types are not equal. Thus, the need to investigate follicular morphology in non-Caucasian skin types becomes more than apparent. The aim of the present study was to determine the potential reservoir of vellus hair follicles in non-Caucasian skin types in order to estimate their contribution to the penetration of topically applied substances. Cyanoacrylate skin surface biopsies were taken from seven body sites of Asians and African-Americans. To determine the follicular reservoir, the samples were evaluated under the light microscope. Follicular parameters were measured with the help of a digital imaging software program. In the evaluation and comparison of the results with those of Caucasians, significant differences were detected. Compared with Whites, follicular density on the forehead is significantly lower in Asians and African Americans. Smaller values were detected for volume, surface, follicular orifice and hair shaft diameter on the thigh and in calf regions in Asians and African Americans. Furthermore, the follicular reservoir, characterized by follicular volume, is generally higher in Caucasians. In comparison to African-Americans, larger follicular volumes on the forehead, back and on the upper arm were determined in Asians. The fairly similar density in these sites revealed that Asians have a higher follicular reservoir in these sites. Due to significant ethnic differences in the follicular reservoir, particularly on the calf and forehead, prospective skin absorption experiments need to be performed on different skin types in order to be representative. Likewise, the development of topically applied drugs should take place under adequate consideration of ethnic differences in skin physiology. PMID- 16679818 TI - Natural scalp hair regression in preclinical stages of male androgenetic alopecia and its reversal by finasteride. AB - Using contrast-enhanced phototrichogram (CE-PTG) at monthly intervals during 48 months, we measured the duration of the hair cycle, i.e. anagen, catagen and telogen at the exclusion of exogen. Exogen, a recently identified phase of the hair cycle, is characterized by weakening of anchorage of the club hair to the surrounding epithelium. The processing of the club hair terminates at the time of exogen hair release, i.e. hair shedding. We combined a noninvasive exogen sampling before each CE-PTG so that the area contained only anagen, catagen and telogen hair or empty follicular openings. During the first 24 months of this study, natural regression of hair cycling in early i.e. preclinical stages of androgenetic alopecia (AGA) in androgen sensitive areas was documented. Shortening of the hair cycle of thicker hair characterized progression of AGA. During the next 24 months, finasteride (1 mg/day) was introduced into the system. Shortening of the hair cycle was reversed by finasteride in androgen sensitive sites as long as the affected follicle was able to produce a thick hair fiber at the time of treatment initiation. Compared to the baseline period, responding follicles did not produce thicker hair. On average, they initiated active growth more rapidly by reducing the duration of the lag phase by 40%. The duration of the anagen phase of thick hair showed an average 23% increase. In this particular experiment, the already miniaturized follicles producing thinner hair (<40 microm thickness) at the time of finasteride introduction regressed further on treatment. Our results seem to indicate that reversal of 'hair loss' by finasteride probably means that the terminal type follicles that are functionally deficient--a stage of reversible hypotrophy--will be reactivated by two non mutually exclusive mechanisms: faster regrowth followed by extension of the duration of anagen. In our study, there was no clear evidence in favour of reversal of miniaturized hair into terminal hair. This new interpretation indicates that miniaturized hair follicles may be an important diagnostic marker of AGA in males but also that it might be less contributive to the therapeutic response to finasteride. Our results highlight that precise measurement of terminal type hair follicle functionalities opens up avenues for the selection of 'drug-responsive organs' in the human scalp in vivo and these may possibly serve to predict 'quality of response to treatment'. PMID- 16679819 TI - Sodium lauryl sulfate-induced irritation in the human face: regional and age related differences. AB - The particular sensitivity of the human face to care products prompted us to study irritation induced by sodium lauryl sulfate (SLS) in its various regions. We examined regional and age-related differences, correlating basal transepidermal water loss (TEWL) and capacitance to SLS irritation. SLS (2% aq.) was applied under occlusion for 1 h to the forehead, cheek, nose, nasolabial and perioral areas, chin, neck and forearm to two groups of subjects--one with 10 subjects with an average age of 25.2 +/- 4.7 years and another with 10 subjects with an average age of 73.7 +/- 3.9 years. TEWL was measured before and 1 h and 23 h after patch removal. Baseline stratum corneum hydration was also measured. Irritation was assessed by the changes in TEWL (deltaTEWL = TEWL after patch removal - basal TEWL) after corrections to the control. In the younger group, all areas of the face and the neck reacted to SLS, whereas the forearm did not. In the older group, the nose, perioral area and forearm did not react. In both age groups, some significant differences between the regions of the face were detected. The younger group showed higher changes in TEWL than the older group in all the areas studied, but only in the chin and nasolabial area were the differences statistically significant. Significant correlations were found between basal TEWL and deltaTEWL in 5 of the 7 areas which reacted to SLS. Baseline TEWL is one parameter that correlates with the susceptibility of the face to this irritant. PMID- 16679820 TI - Chronobiology: biological clocks and rhythms of the skin. AB - The cyclicity of time affects virtually all aspects of our being and is the basis of the underlying rhythmicity which is typical of our lives. To 'tell time', most living organisms use internal timing mechanisms known as 'biological clocks'. These 'clocks' coordinate our physiological and behavioral functions and interactions with our environment. One of the strongest influences on rhythmicity is the solar day. The study of these temporal rhythms in biological systems has been coined chronobiology. With the present article we aim to give an overview on chronobiology. Examples of chronobiological effects on skin will be described. Particular emphasis will be placed on circadian rhythms (including rhythms that take place within a 24-hour period, including so-called infradian and/or diurnal rhythms) but also on seasonal variations (circaannual rhythms). PMID- 16679821 TI - Influence of nanoencapsulation on human skin transport of flufenamic acid. AB - The effect of the inclusion of flufenamic acid in poly(lactide-co-glycolide) nanoparticles on the transport of flufenamic acid into excised human skin was investigated. Penetration and permeation data were acquired using two different in vitro test systems: the Saarbrucken penetration model, where the skin acts as its own receptor medium, and the Franz diffusion cell, where the receptor medium is a buffer solution. For the stratum corneum, no differences were found between nanoencapsulated and free drug. Drug accumulation in the deeper skin layers and drug transport across human epidermis were slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (<12 h). In contrast, after longer incubation times (>12 h), the nanoencapsulated drug showed a statistically significantly enhanced transport and accumulation (p < 0.05). Additionally, nanoencapsulated flufenamic acid was visualized by multiphoton fluorescence microscopy. Particles were found homogeneously distributed on the skin surface and within the dermatoglyphs, but no nanoparticles were detected within or between the corneocytes. PMID- 16679822 TI - Cluster analysis of the dermal permeability and stratum corneum/solvent partitioning of ten chemicals in twenty-four chemical mixtures in porcine skin. AB - Assumptions based on absorption from single solvent systems may be inappropriate for risk assessment when chemical mixtures are involved. We used K-means and hierarchical cluster analyses to identify clusters in stratum corneum partitioning and porcine skin permeability datasets that are distinct from each other based on mathematical indices of similarity and dissimilarity. Twenty-four solvent systems consisting of combinations of water, ethanol, propylene glycol, methyl nicotinate and sodium lauryl sulfate were used with 10 solutes, including phenol, p-nitrophenol, pentachlorophenol, methyl parathion, ethyl parathion, chlorpyrifos, fenthion, simazine, atrazine and propazine. Identifying the relationships between solvent systems that have similar effects on dermal absorption formed the bases for hypotheses generation. The determining influence of solvent polarity on the partitioning data structure supported the hypothesis that solvent polarity drives the partitioning of non-polar solutes. Solvent polarity could not be used to predict permeability because solvent effects on diffusivity masked the effects of partitioning on permeability. The consistent influence of the inclusion of propylene glycol in the solvent system supports the hypothesis that over-saturation due to solvent evaporation has a marked effect on permeability. These results demonstrated the potential of using cluster analysis of large datasets to identify consistent solvent and chemical mixture effects. PMID- 16679823 TI - An in vivo randomized study of human skin moisturization by a new confocal Raman fiber-optic microprobe: assessment of a glycerol-based hydration cream. AB - BACKGROUND: In a recent study, we demonstrated the ability of the new confocal Raman microprobe to investigate molecular and structural human skin composition under in vivo conditions. Experiments were performed at different anatomical sites, different layers, and with intervolunteer comparison. We also carried out feasibility tests using this probe to determine depth profiles of water content within the skin. OBJECTIVE: In the present investigation we employed this confocal Raman optical microprobe to rigorously objectify the resulting hydration capacities after application of a moisturizing enhancer. METHOD: The in vivo experiments were performed on 26 healthy volunteers and measurements were undertaken on six areas of the volar forearm after a randomized application of hydrating agents. Responses were evaluated by calculating the water/protein band ratio, which determines the water content in the skin. RESULTS: Data collected with the Raman microprobe showed significant changes between baseline values of control and treated skins. Statistical analysis performed on these data revealed an increase in skin moisture after application of a glycerol-based cream, which is the most widely used hydrating agent. CONCLUSION: Our results demonstrate clearly the potentials of this confocal Raman microprobe in the screening of hydrating agents or molecules under in vivo conditions. In the cosmetics field, this promising and suitable technique will undoubtedly offer new opportunities of hydration skin test evaluation. PMID- 16679824 TI - Follicular penetration: development of a method to block the follicles selectively against the penetration of topically applied substances. AB - Investigations into the penetration pathways of topically applied substances through the skin play an important role in dermatological science. Recently, the hair follicles have increasingly been recognized as an important pathway for percutaneous penetration, although the role of the follicles has still not been clarified in detail. Therefore, the aim of the present study was to develop an in vivo method for the analysis of the follicular rate of penetration processes. In order to reach this goal, the follicles were excluded from the penetration process of topically applied substances. Different study designs were evaluated, and we were able to show that the application of nail varnish to each follicular orifice represents an effective method of blocking the follicles selectively against the penetration of topically applied substances. In dermatological science, there is new information on the physiology of skin penetration processes, thus allowing examination of the follicular rate of penetration and improvement of the pharmacokinetics of topically applied substances. PMID- 16679825 TI - Antioxidant supplements improve parameters related to skin structure in humans. AB - In the present study we investigated the influence of two different antioxidant supplements composed of carotenoids, vitamin E and selenium on parameters related to skin health and skin aging. Thirty-nine volunteers with healthy, normal skin of skin type 2 were divided into 3 groups (n = 13) and supplemented for a period of 12 weeks. Group 1 received a mixture of lycopene (3 mg/day), lutein (3 mg/day), beta-carotene (4.8 mg/day), alpha-tocopherol (10 mg/day) and selenium (75 microg/day). Group 2 was supplemented with a mixture of lycopene (6 mg/day), beta-carotene (4.8 mg/day), alpha-tocopherol (10 mg/day) and selenium (75 microg/day). Group 3 was the placebo control. Upon supplementation serum levels of selected carotenoids increased in both verum groups. Skin density and thickness were determined by ultrasound measurements. A significant increase for both parameters was determined in the verum groups. Roughness, scaling, smoothness and wrinkling of the skin were determined by Surface Evaluation of Living Skin (Visioscan). Roughness and scaling were improved by the supplementation with antioxidant micronutrients. In the placebo group no changes were found for any of the parameters. PMID- 16679826 TI - Hair follicles - a long-term reservoir for drug delivery. AB - Nanoparticles represent an important drug carrier system. Recently, we have reported on the penetration and storage behavior of particular and non-particular substances revealing the superiority of particular substances in the range of 300 400 nm. In this regard, it was assumed that the rigid hair shaft acts as a geared pump, moving the particles deeper into the hair follicle. In the present investigation, the storage reservoir capacity of the stratum corneum and the hair follicle infundibulum and canal are compared. Interestingly, we could demonstrate a 10 times longer storage within the hair follicles. These results underscore the importance of the hair follicle for drug delivery purposes, mainly highlighting new possibilities for the future concerning retarded delivery, application frequency, and galenic design. PMID- 16679827 TI - The physiological function of the urothelium--more than a simple barrier. AB - The urothelium, the epithelium lining the surface of the urinary bladder, is a unique cell type with high plasticity and a variety of cellular functions. The urothelium represents the first line of bladder defense and an interface between pathogens and defense mechanisms. Functions of the urothelium include control of permeability, immune responses and cell-cell communication, which seems to play a pivotal role in responding to injuries and infections. The urothelium responds to stretch, during the filling phase of micturition reflex, by increasing the size of apical umbrella cells and by releasing mediators which activate the sensory fibers. For this reason the concept of 'neuron-like properties' was suggested. Finally, despite the fact that the urothelium is a frequent site of cancer formation, few experimental model systems are currently available or well characterized for studying urothelial cancer in the era of genomics and proteomics. The purpose of this review is to give emphasis to urothelial physiology and pathophysiology in different bladder disorders and to offer an up to-date contribution to the field of urothelial research. PMID- 16679828 TI - A new approach for the surgery of large renal masses: abdominal wall flap incision. AB - INTRODUCTION: The type of skin incision affects the course of surgery during radical nephrectomy. We investigated the efficacy of a new type of incision for the surgical approach to large renal masses. PATIENTS AND METHODS: Fourty patients (23 males and 17 females, mean age 63 years) who underwent radical nephrectomy in our department between 2002 and 2004 were evaluated. The outcomes in the groups with chevron incision and abdominal wall flap incision (AFI) were compared in terms of greatest tumor diameter, operation duration, perioperative blood loss, postoperative complications, and duration of hospitalization. Statistical analysis was made by independent-samples t test. RESULTS: Chevron incision was used in the first 15 patients, while subsequent 25 patients were operated via AFI. The operation duration was slightly longer in the AFI group (3.78 vs. 3 h); however, the mean largest tumor diameter was significantly greater in patients operated via AFI (11.3 vs. 7.4 cm, p < 0.05). The mean blood losses were 1,100 and 590 ml in the chevron incision and AFI groups, respectively (p < 0.05). There was no significant difference in terms of duration of hospitalization and postoperative morbidity. CONCLUSION: In our experience, AFI provides the best exposure and improved control of renal vessels and vena cava during radical nephrectomy and enables a safe dissection even in very large tumors with minimal blood loss. PMID- 16679829 TI - When does it hurt? Pain during flexible cystoscopy in men. AB - INTRODUCTION: In this observational study, pain was measured continuously in men undergoing flexible cystoscopy, in order to help to identify which parts of the procedure were the most painful. PATIENTS AND METHODS: Men who were to undergo flexible cystoscopy were recruited. Pain was measured continuously throughout flexible cystoscopy by asking the subject to squeeze a pressure-sensing rubber bulb in proportion to the amount of discomfort experienced giving a score of 0 10. RESULTS: The most painful part of the procedure was as the cystoscope passed through the membranous urethra with a median pain score of 2.82. The initial lidocaine administration gives a median pain score of 0.84. The other parts of the cystoscopy produced median scores of between 0.14 and 0.33. The difference in the pain scores was significant (p = 0.03). There was no statistically significant difference in the pain scores between those who were having their first cystoscopy and those who were having repeat cystoscopy. The subjects' age did not influence the degree of pain experienced. CONCLUSION: This study gives further understanding of how pain is experienced during flexible cystoscopy. It may help explain why previous studies have not reached a clear consensus on the value of lidocaine during flexible cystoscopy. PMID- 16679830 TI - Discontinuation of tamsulosin treatment in men with lower urinary tract symptoms: a pilot study. AB - OBJECTIVES: Since little investigation has been undertaken to determine if alpha1 blockers should be given continuously to sustain their efficacy, we conducted a pilot study to determine symptom change following discontinuation of tamsulosin after an initial improvement in symptoms in men with lower urinary tract symptoms (LUTS). PATIENTS AND METHODS: Thirty-three of 78 patients with mild-to-moderate prostate hyperplasia, who had symptom improvement according to the International Prostate Symptom Score (IPSS) to <10 or the quality of life (QOL) index to < or =3 after initial treatment with tamsulosin, were enrolled in this study. Subjective parameters (IPSS and QOL index) and objective parameters (maximum and mean urinary flow rates) were evaluated at baseline and after initial treatment, and 4, 8, 12 and 24 weeks after discontinuing tamsulosin. RESULTS: The rates of successful discontinuation of tamsulosin were high throughout the follow-up period, i.e., 80.6% at 4 weeks, 80.6% at 8 weeks, 80.0% at 12 weeks, and 68.9% at 24 weeks. Temporary worsening in both subjective and objective parameters was observed only at 4 weeks; however, these parameters recovered to almost post treatment levels at 24 weeks. CONCLUSION: The present study suggests that continuous treatment is not always needed to maintain urinary symptom relief in a specific subset of patients who felt symptom improvement after initial treatment with tamsulosin. PMID- 16679831 TI - Comparative study of international prostate symptom scores and urodynamic parameters in men and women with lower urinary tract symptoms. AB - BACKGROUND: We compared urinary symptoms and urodynamic parameters in men and women with lower urinary tract symptoms. METHODS: A total of 164 individuals (76 men and 88 women) were rated according to the International Prostate Symptom Score and underwent a detailed urodynamic investigation. Patients were stratified by age into 2 groups, namely, those < 50 years (the younger group) and those > 50 years (the elderly group). RESULTS: In the elderly group, scores of voiding symptoms for men were higher than those for women (p = 0.012). Maximum flow rates (Qmax) for women were higher than those for men (p = 0.025 for the younger groups, and p < 0.001 for the elderly groups). Detrusor pressure at Qmax (p < 0.001), opening pressure (p < 0.001) and closing pressure (p = 0.023) were elevated in elderly men vs. in elderly women. A multiple linear regression analysis identified straining (beta = 0.479, p < 0.05) and incomplete emptying (beta = 0.487, p < 0.05) as the only variables influencing quality of life (QOL) in younger men and women, respectively. In elderly men, nocturia (beta = 0.352, p < 0.05) and frequency (beta = 0.287, p < 0.05) were significantly associated with QOL. Frequency (beta = 0.452, p < 0.05) possibly explained a QOL reduction in elderly women. CONCLUSIONS: Our findings suggest that in the elderly patients, storage symptoms are more associated with QOL than voiding symptoms. However, storage symptoms were not associated with QOL in younger men. PMID- 16679832 TI - Questionnaire survey of urologists' initial treatment practices for acute urinary retention secondary to benign prostatic hyperplasia in Korea. AB - BACKGROUND: We undertook a survey of Korean urologists to establish initial treatment practices for acute urinary retention secondary to benign prostatic hyperplasia. METHODS: Probability samples were drawn from the Korean Urological Association Registry of Physicians, and a random sample of 850 Korean urologists were asked to complete a questionnaire that explored practice characteristics and treatment strategies in the management of acute urinary retention. The returned questionnaires were statistically analyzed. RESULTS: Of the 850 questionnaires sent, 302 were returned, and 277 of those were included in the final analysis (response rate 32.6%). Korean urologists preferred urethral catheterization as the initial management. Of respondents, 154 (55.6%) and 109 (39.4%) started their patients on alpha-blockers and alpha-blockers with finasteride, respectively. Nearly half (50.2%) used a trial without catheter 7 days after catheterization. One failed trial without catheter was an indication for surgery for only 69 (24.9%) of the respondents. A second trial without catheter was advocated by 108 (39.0%). Most urologists (82.3%) expected to be successful with the above management in 50% or greater. Physician's demographics or types of initial management did not influence physician's perception of expected success rate when the expected success rate was divided as < or =50% and >50. CONCLUSIONS: These data provide a picture of current practice regarding the management of acute urinary retention secondary to benign prostatic hyperplasia in Korea. There is no uniformity in the management of acute urinary retention in Korea. PMID- 16679833 TI - Larger prostate causes higher frequency of infectious complications in prostate biopsy. AB - INTRODUCTION: Prostate biopsy (PBX) is being performed more often than ever before for the early detection of prostate cancer, but some patients may have febrile infectious complications after biopsy. The risk factors of infectious complications need to be considered. METHODS: We investigated the pre-biopsy risk factors and febrile infectious complications in 120 patients who underwent PBX in our institution. The pre-biopsy risk factors set were prostate volume (> or = 40 ml), transitional zone (TZ) volume (> or = 20 ml), core number of biopsy (> or = 10 cores), and additional performance of TZ biopsy. We analyzed statistically the correlations between these risk factors and C-reactive protein and peripheral white blood cell (WBC) count elevation on day 1 and day 7 after biopsy. RESULTS: Our results showed that a prostate volume > or = 40 ml and a TZ volume > or = 20 ml caused a significantly higher elevation of the C-reactive protein level compared with a prostate volume < 40 ml (p = 0.0055 on day 1 and p = 0.0225 on day 7, respectively) on day 1 and day 7, and a TZ volume < 20 ml (p = 0.0008) on day 1. Regarding WBC count, those patients with a prostate volume > or = 40 ml and a TZ volume > or = 20 ml had a tendency to have an elevated WBC count on day 1 compared with those patients with a prostate volume < 40 ml and a TZ volume < 20 ml, and however these changes were not significant. CONCLUSIONS: This study showed that a larger prostate volume and a larger TZ volume may possibly be risk factors in infectious complications after PBX, although we can perform additional TZ biopsy and increase biopsy core number safely if necessary. PMID- 16679834 TI - Prostatic phyto-oestrogen tissue levels in different Austrian regions. AB - INTRODUCTION: A number of studies suggest that the low incidence of prostate cancer as well as benign prostatic enlargement in Asia depends on the extended consumption of phyto-oestrogens in these parts of the world. In most Asian men, phyto-oestrogen levels are multiple higher compared to Austrian (European) men. The aim of our study was to evaluate, according to the East-West decline, whether there were significant differences within the Austrian population. We compared prostate phyto-oestrogen tissue levels of men living in three different geographical regions of Austria. We further compared men living in rural and urban environments. MATERIAL AND METHODS: Prostatic tissue samples of 103 men undergoing surgery for benign prostatic hyperplasia or prostate cancer were collected and frozen at -40 degrees C. In tissue samples, enterolactone (representative for lignans) and genistein levels (representative for isoflavones) were determined in duplicate by monoclonal antibody-based immunoassays. We subsequently compared tissue levels of men living in rural and urban environments and different geographical regions of Austria. RESULTS: Prostatic enterolactone tissue levels were similar in men living in an urban (median 19.1 ng/g dry weight, range 1.5-76.4) or rural environment (median 15.7 range 0.6-140.6) p = 0.99. The respective values for genistein were 20.5 ng/g dry weight (range 4.6-47.4) and 9.3 (range 0.1-156.7) p = 0.77. Furthermore, enterolactone (p = 0.1) and genistein (p = 0.65) levels were similar in three different geographic regions in Austria. CONCLUSION: No significant differences regarding genistein and enterolactone were found between our study populations. However, we found a wide variation between individual patients. PMID- 16679835 TI - How do patients with familial benign prostatic hyperplasia differ clinically from those with sporadic benign prostatic hyperplasia? AB - INTRODUCTION: The aim of this study was to compare age, prostatic volume, International Prostate Symptom Score (IPSS), maximal flow rate, serum total prostate-specific antigen (PSA), serum free PSA, free/total PSA ratio and PSA density values of familial and sporadic benign prostatic hyperplasia (BPH) patients suffering moderate or severe lower urinary tract symptoms. MATERIALS AND METHODS: Between September 1999 and August 2004, 511 patients with moderate or severe lower urinary tract symptoms (IPSS > or =8) due to BPH were included in the study. Patients with at least 2 first-degree relatives who had undergone surgery or received medication for BPH were classified as having the familial form of the disease, while the remaining patients were taken as sporadic cases. Mean age, prostatic volume, symptom score, maximal flow rate, PSA, free PSA, free/total PSA ratio and PSA density values of the familial and sporadic groups were compared using student's t test. RESULTS: Thirty-eight patients had a positive family history and formed the familial group, while the remaining 473 made up the sporadic group. No significant difference was observed in the parameters studied, except that mean prostate volume of the familial group was found to be greater and the mean age to be lower than those of sporadic patients in accordance with the literature. CONCLUSIONS: Patients with familial BPH need treatment significantly earlier and have larger prostates than those with sporadic BPH. PMID- 16679836 TI - Laparoscopic peritoneal drainage of symptomatic lymphoceles after pelvic lymph node dissection using methylene blue instillation. AB - BACKGROUND: Lymphoceles are frequent complications of pelvic lymph node dissection. While small lymphoceles often remain undetected, larger ones can cause complications and require further treatment, e.g. percutaneous tube drainage alone or in combination with sclerotherapy. However, recurrence rates are considerable, and long-lasting drainage may lead to infection, prolonged hospitalization, and as a consequence, increased overall costs. We report the results of a simplified laparoscopic approach to drain lymphoceles after radical prostatectomy plus pelvic lymphadenectomy using methylene blue instillation. METHODS: 13 patients with large symptomatic pelvic/retroperitoneal lymphoceles refractory to percutaneous tube drainage and doxycycline sclerotherapy received a laparoscopic transperitoneal marsupialization following instillation of a sterile diluted methylene blue solution into the drained cavity to refill and mark the lymphocele. RESULTS: All lymphoceles were sterile and ranged in size from 7 x 6 x 4 to 15 x 12 x 6 cm. Clinical symptoms included lower abdominal swelling, tenderness in the iliac fossa, ipsilateral lymphedema, deep venous thrombosis, wound fistula, and hydronephrosis due to ureteral obstruction. After methylene blue instillation, the lymphoceles were easily identified and opened. Median total operative time was 50 (range 25-70) min; blood loss was negligible. There was one complication in the form of a metachronous infection in the operating field and no relapses. Patients were discharged 1-5 (median 3) days after the surgical procedure. CONCLUSIONS: Laparoscopic peritoneal drainage requires greater operative skill than percutaneous approaches. However, the instillation of a methylene blue solution simplifies this procedure as the extent and location of the lymphoceles can be precisely identified during laparoscopy. We recommend early application of laparoscopic peritoneal drainage following methylene blue instillation for patients with sterile lymphoceles after pelvic lymph node dissection in whom temporary percutaneous drainage and sclerotherapy failed to resolve the lymph fluid collection. PMID- 16679837 TI - Sacral colpopexy with concurrent Burch colposuspension in patients with vaginal vault prolapse. AB - INTRODUCTION: Abdominal sacral colpopexy (SC) is one option in the management of vaginal vault prolapse. In patients who are additionally incontinent an anti incontinence procedure such as a Burch colposuspension or pubovaginal sling is usually performed at the same time. For those patients undergoing SC who are continent there are no clear guidelines for the use of a 'prophylactic' anti incontinence procedure. We describe our experience with SC and concurrent Burch colposuspension. PATIENTS AND METHODS: 47 patients (mean age 65 years) underwent SC and concurrent Burch colposuspension. The preoperative diagnostic check-up included a validated questionnaire, clinical examination, urodynamic tests, ultrasound and colpocystorectography. Patients were also evaluated using Stress, Emptying, Anatomic, Protection and Instability (SEAPI) scores. All patients had a uterine or vaginal vault prolapse in combination with a cystocele, enterocele or rectocele. Thirty-three of 47 (70%) patients were continent and 14 (30%) incontinent. Nineteen (40%) of the 33 'continent' patients were found to have occult incontinence. Clinical examination according to the Halfway system showed 9 of 47 (19%), 21 of 47 (45%) and 17 of 47 (36%) patients with grade 2, 3 and 4 vaginal vault prolapse, respectively. Thirty-five of 47 (74%) patients demonstrated a grade-4 cystocele and 12 of 47 (26%) a grade-3 cystocele. The mean follow-up was 34 months and included a questionnaire (SEAPI), clinical examination and ultrasound. RESULTS: Postoperative SEAPI scores showed a statistically significant improvement in all SEAPI domains (p < 0.001). Ninety four percent of the patients were satisfied, continent and would undergo the surgery again. Three patients were incontinent. No continent patient who underwent concurrent Burch colposuspension had obstructive symptoms or residual urine. Five patients (11%) who had dyspareunia preoperatively were free of this symptom postoperatively. Complications were: dilatation of the upper urinary tract in 2 patients (4%) secondary to distal ureteric deviation by suturing the posterior peritoneum. One patient underwent psoas hitch neoureterocystostomy and 1 patient was successfully treated by insertion of a ureteric stent for 6 weeks. One patient (2%) had a mesh infection necessitating removal of the Gore-Tex mesh. CONCLUSIONS: Sacral colpopexy provides good patient satisfaction, durable pelvic support and restores vaginal function. Due to excellent continence rates concurrent Burch colposuspension should be considered as a joint procedure even in continent patients. PMID- 16679838 TI - Open dismembered tubularized flap pyeloplasty: an effective and simple operation for treatment of ureteropelvic junction obstruction. AB - OBJECTIVE: To evaluate the success rate of dismembered tubularized flap pyeloplasty (DTFP) in the treatment of ureteropelvic junction obstruction (UPJO). PATIENTS AND METHODS: In a prospective study from August 2002 to September 2004, 15 patients with a mean age of 21 years (range 2-47) in whom UPJO had been diagnosed by sonography, excretory urography or diuretic renography and who had a large extrarenal pelvis, underwent operation via flank intercostal incision. The proximal ureter and renal pelvis were dissected and mobilized retroperitoneally, the site of UPJO was excised and the site of insertion of the ureter on the renal pelvis was closed with a stitch. A wide based renal pelvic flap was created and tubularized to bridge the upper ureteral defect. After insertion of a nephrostomy tube, a double-J tube was inserted as an internal ureteral stent and anastomosis of the tubularized flap to the spatulated upper ureter was done and the renal pelvis window was closed. Patients were followed 3, 6 and 12 months postoperatively. RESULTS: Mean operation time was 1 h and mean hospital stay was 3 days. The ureteral stent was removed 4 weeks after operation and at the same time a nephrostogram was done that showed a widely patent ureteropelvic junction with good renal pelvis drainage in 12 ( approximately 80%) of the cases, but in 3 cases (approximately 20%) passage of contrast materials was not seen. In these patients, methylene blue was injected via a nephrostomy tube and in 2 patients (14%) urine color turned blue 20 min later, but in 1 patient (7%) this test was also negative. The latter patient underwent percutaneous endopyelotomy later. Mean patient follow-up was 14 months. Follow-up excretory urography confirmed patent and unobstructed ureteropelvic junction in all patients. The overall success rate of DTFP was 93%. CONCLUSION: DTFP is a simple and effective procedure for patients with UPJO who have long or multiple upper ureteral strictures and a large extrarenal pelvis. PMID- 16679839 TI - Antibiotics-induced acute interstitial nephritis in 6 children. AB - INTRODUCTION: Antibiotics-induced acute interstitial nephritis (AIN) is a rare disorder in children, and the diagnosis is often delayed. However, many commonly prescribed antibiotics seem to be implicated. PATIENTS AND METHODS: We reviewed the medical records of 6 children, age range from 10 months to 14 years, with biopsy-confirmed antibiotics-induced AIN. Clinical presentation, morphological findings, and outcomes are reported. RESULTS: Symptoms of AIN started 2-4 weeks after antimicrobial therapy with beta-lactam antibiotics in 5 children and with gentamicin in 1 child. All patients presented with acute renal failure and fever. The glomerular filtration rate was dramatically reduced in 2 cases and mildly reduced in 4 patients. Two of our patients had supportive treatment, 2 received corticosteroid therapy, and 2 children remained under peritoneal dialysis for 12 and 22 days, respectively. Five patients had a full recovery of their renal function, and 1 child, 2 years later, still presented impairment of the renal function. CONCLUSION: AIN should be considered in case of acute renal failure in children, mostly when other common causes have been excluded, and there is a history of drug exposure. PMID- 16679841 TI - Lack of local anesthetic properties of lidocaine gel in an experimental model. AB - INTRODUCTION: Many reports negate the anesthetic properties of lidocaine gel placed in the urethra during catheterization. The anesthetic action of lidocaine is inseparably associated with the toxic action of this compound on cells. MATERIALS AND METHODS: A primary rabbit urothelial cell culture (PRUCC) was previously established as a monolayer. The effect of 2% lidocaine gel on the viability of the PRUCC was assessed and compared with the cytotoxic effect of decreasing concentrations of lidocaine solution. Cell viability was evaluated after 1-hour exposure using the trypan blue exclusion test. RESULTS: The 2% lidocaine gel did not show cytotoxic properties after 1 h of incubation on a PRUCC. The 2 and 1% lidocaine solutions induced statistically significant decreases in the viability of the PRUCC after 1 h of incubation. CONCLUSIONS: Experimental tests evaluating the cytotoxicity of local anesthetics may prove to be an objective measure of the accessibility of these substances to cells and their anesthetic action. PMID- 16679840 TI - Oxidative and antioxidative status in the testes of rats with acute epididymitis. AB - INTRODUCTION: Epididymitis is an inflammation or infection of the epididymis, a convoluted duct that lies on the posterior surface of the testicle. Oxidative stress due to excessive production of reactive oxygen species in epididymitis, impaired antioxidant defense mechanisms, or both, precipitates a range of pathologies that are currently believed to negatively affect the male reproductive function. How oxidative stress affects the testes is still unknown. We aimed to investigate the oxidative and antioxidative status of testes of rats with unilateral acute Escherichia coli epididymitis. METHODS: The study included 36 male Wistar albino rats which were divided into three groups. In the epididymitis group (n = 12), an E. coli suspension was injected into the right ductus deferens of rats, and the same amount of saline was injected in the saline groups (n = 12). No surgery was performed in the control group (n = 12) for baseline values. Rats were sacrificed after 24 h and the epididymis and testes removed. The infection was confirmed by histopathologic evaluation and microbiological tests. The oxidative status of testes was evaluated by measuring myeloperoxidase (MPO) activity, and antioxidative status was evaluated by measuring total antioxidant response (TAR) and total antioxidant capacity levels (TAC). RESULTS: MPO activity in both the ipsilateral and contralateral testes of the epididymitis group was significantly higher than those of the saline and control groups (p < 0.05). The TAR and TAC levels in both testes were also significantly elevated in the epididymitis group versus the two other groups (p < 0.05). CONCLUSIONS: Acute epididymitis causes an increase of oxidative stress in the ipsilateral and contralateral testes, but this condition is strived for to tolerate the increase of endogenous antioxidants. PMID- 16679842 TI - A case of primary extragonadal seminoma arising in the perineum. AB - We report the first case of primary extragonadal seminoma arising in the perineum. A 34-year-old-man presented with a 2-cm painless mass in the perineum. He underwent surgical resection, and histology showed poorly differentiated adenocarcinoma. Three months later, local recurrence occurred. Whole evaluation including ultrasound and MRI of the testes were normal, so he underwent surgical wide excision, and pathology showed the tumor was pure seminoma. He received two courses of bleomycin, etoposide and cisplatin (BEP) therapy, and there has been no evidence of recurrence for 1 month. PMID- 16679843 TI - Post-operative changes mimicking the radiographic appearance of recurrent renal cell carcinoma. AB - Partial nephrectomy for small renal tumors is associated with excellent long-term outcomes. In the absence of positive surgical margins, local recurrence is uncommon. Although computed tomography is associated with good diagnostic accuracy, the radiographic evaluation of some renal lesions remains difficult. This difficulty can be greater in the period following surgery, when post operative tissue changes can result in abnormal radiographic findings. We report a case of benign post-operative changes mimicking the radiographic appearance of recurrent renal cell carcinoma. PMID- 16679844 TI - Ectopic insertion of a single ureter into the bladder adjacent to the contralateral ureter. AB - We report a 30-year-old female with a single ectopic ureter that opens into the bladder adjacent to the right ureter. PMID- 16679845 TI - Laparoscopic repair of vesicovaginal fistula. AB - Vesicovaginal fistula (VVF) may be a complication of prolonged repair or urogynecologic surgery. Failing conservative management, it may be repaired using an abdominal or vaginal approach. We herein report laparoscopic repair of VVF following vaginal hysterectomy and detail the operative steps. PMID- 16679847 TI - Molecular biology of prostate-cancer pathogenesis. AB - PURPOSE OF REVIEW: The genetic and molecular basis of prostate-cancer pathogenesis is reviewed. RECENT FINDINGS: Several genetic loci have been found that are associated with hereditary predisposition to prostate cancer, but they account for a small fraction of all cases. A number of suppressor genes have been identified that are activated by either complete or partial genetic loss in sporadic prostate cancer. Chromosomal translocation results in transcriptional activation of truncated ETS transcription factors ERG and ETV1, the first candidates for dominant oncogenes for prostate cancer. Lastly, the androgen receptor is active throughout the course of prostate cancer and, in androgen independent prostate cancer, takes on the role of a dominant oncogene as the target of gene amplification, overexpression, and the activation of mutations. SUMMARY: Genetic lesions responsible for familial and sporadic prostate cancer are being revealed and they suggest that prostate cancer often initiates owing to an increased susceptibility to oxidative damage; it then progresses by affecting transcription factors, the PI3 kinase pathway, and other growth stimulatory pathways. The final common pathway after androgen ablation appears to be activation of androgen receptor. PMID- 16679848 TI - An update on chemoprevention strategies in prostate cancer for 2006. AB - PURPOSE OF REVIEW: An increasing volume of research has been directed at the prevention of prostate cancer. This review proposes to summarize the large trials, novel approaches and molecular mechanisms of effect published in 2004 and 2005. RECENT FINDINGS: The impact of the Prostate Cancer Prevention Trial continues and subsequent articles have addressed the increase of high-grade prostate cancers detected in the finasteride arm of the trial, as well as the potential costs and benefits of extrapolating the findings to a public health campaign. Studies of risk have been published warning of excessive vitamin E and cyclooxygenase-2 inhibitor use in chemoprevention. Growing evidence supports the concept of chemopreventative agent combinations and further data on the roles of selenium, lycopene, soy, green tea, anti-inflammatories and statins in prostate cancer prevention are presented. SUMMARY: Level one evidence exists for the preventative effects of finasteride in prostate cancer. The evidence for other agents is less conclusive but a number of large-scale, appropriately designed trials will hopefully address some of the relevant issues in prostate-cancer prevention over the next decade. PMID- 16679849 TI - New drugs in prostate cancer. AB - PURPOSE OF REVIEW: The survival of hormone-refractory metastatic prostate cancer patients has improved with the use of docetaxel-based chemotherapy. The survival benefits, however, are modest suggesting that rationally designed therapeutic approaches are needed. We discuss recent developments in the therapeutic approach to advanced metastatic hormone-refractory prostate cancer, including molecularly targeted therapy, signal transduction inhibitors, stem-cell targeted therapy, anti-angiogenic compounds, vaccines and immunomodulating agents, differentiation agents, cytotoxics, and pro-apoptotic agents. RECENT FINDINGS: Over 200 compounds have entered clinical development for use in advanced prostate cancer, alone or in combination with cytotoxic agents such as docetaxel, or in other combinations. This article will review the results of emerging targets since the approval of docetaxel in 2004, concentrating on some of those compounds that, in our opinion, have the greatest potential and rationale for use. SUMMARY: The growing field of targeted molecular therapy of prostate cancer has opened up numerous opportunities for therapeutic impact. Knowledge of the molecular determinants of progression, relapse after local therapy, chemotherapeutic resistance, and hormone refractoriness remains essential in the rational design of clinical trials of these agents. Given the complexity, heterogeneity, and crosstalk of molecular pathways and the molecular lesions in prostate cancer, combination or sequential therapy may be a necessary step towards significant therapeutic progress. Novel translational clinical trial methodologies may assist in a more rapid identification of active compounds at biologically active doses for phase III testing. PMID- 16679850 TI - What's new in prostate cancer disease assessment in 2006? AB - PURPOSE OF REVIEW: Issues relating to the disease are critical in the diagnosis, management, and prognostication of prostate cancer. RECENT FINDINGS: New data have emerged regarding the disease of prostate cancer and its precursors. The diagnosis of prostate cancer on needle biopsy has been refined because of the recent discovery of alpha-methylacyl-CoA racemase, which preferentially labels adenocarcinoma of the prostate. Modifications and additions to the Gleason grading system were published based on a consensus conference of urological pathologists. Various models have been proposed using Gleason score, clinical findings, as well as measurements of tumor volume on needle biopsy to enhance the prediction in men undergoing radical prostatectomy and to predict "insignificance". Several studies have confirmed that certain findings in radical prostatectomy are adverse, yet conflicting studies were published as to the independent prognosis of tumor volume. The risk of cancer following a diagnosis of high-grade prostatic intraepithelial neoplasia on needle biopsy has decreased to the point at which this author does not recommend a routine repeat needle biopsy within the first year following the diagnosis of high-grade prostatic intraepithelial neoplasia. SUMMARY: This review summarizes developments over the last year in the disease of prostate cancer and its precursors. PMID- 16679851 TI - Update on cryotherapy for prostate cancer in 2006. AB - PURPOSE OF REVIEW: With the recent introduction of novel, minimally invasive procedures for the treatment of prostate cancer, cryotherapy has become a feasible option as a viable alternative to traditional radical surgery and radiation therapy. In this review we update recent data concerning the basic science of cryobiology, technical trends, oncologic results and complications of this procedure. RECENT FINDINGS: As a result of better understanding of tumor cryodestruction at a molecular level, refinements in cryotechniques and improved patient selection, the results of cryotherapy are becoming more promising. Furthermore, the dramatic decrease in the number of complications after modern cryotherapy leads to a better quality of life, which may be a preferable option, especially for elderly patients with comorbidities. Current trends towards nerve sparing and focal cryoablation are also discussed. Recent advances in cryobiology open up new opportunities to apply cryotherapy in combination with chemotherapy or radiotherapy for patients with intermediate or high-risk cancers. SUMMARY: Potential directions for future developments in cryosurgery include concepts to reduce side effects such as minimizing cryodamage of the neurovascular bundles (nerve-sparing procedure), and focal ablation of a specific tumor site in patients in whom saturation biopsy supports unifocal prostate cancer. PMID- 16679852 TI - Update on brachytherapy in localized prostate cancer: the importance of dosimetry. AB - PURPOSE OF REVIEW: Prostate brachytherapy has become a common treatment modality for clinical localized adenocarcinoma of the prostate. This is the result of prostate screening and improved technology that currently allows for an outpatient procedure that generally can be accomplished in 1-2 h. The modern technique of prostate brachytherapy includes three components, (1) treatment planning, (2) placement of the sources and (3) an evaluation of the implant quality. In this review, we will focus on the importance of dosimetric quantifiers in predicting freedom from biochemical recurrence. RECENT FINDINGS: A number of dosimetric quantifiers have been described. These include D90 (dose that covers 90% of the prostate volume) and V100 (fractional volume of the prostate that receives 100% of the prescription dose). The data from four recently reported series are reviewed. Each series looks at various dosimetric cutpoints that predict for freedom from biochemical recurrence making a definitive statement difficult. All series observe a relationship between freedom from biochemical recurrence and one or both of these dosimetric quantifiers. SUMMARY: It is clear that a dose-response relationship exists following treatment with prostate brachytherapy; in other words, dosimetry matters and poor dosimetry will result in fewer men being cured. The lack of consistency in the reports to date precludes any definitive statements about what constitutes a "good" compared with a "bad" implant. Further prospective data from multi-center trials are required. PMID- 16679853 TI - Evidence-based medicine in prostate cancer: where do we stand in 2006? AB - PURPOSE OF REVIEW: A majority of medical decisions relating to the diagnosis, prognosis, and therapy of prostate cancer are based on low levels of evidence. This article reviews the recommendations of currently available evidence-based practice guidelines that relate to the management of patients with newly diagnosed prostate cancer. The article further introduces a selection of recent studies that are relevant to the evidence-based patient care. RECENT FINDINGS: Updated clinical practice guidelines on the management of prostate-cancer patients provide an evidence-based summary of the literature and have recently been made available through the National Cancer Center Network and the European Urological Association. In addition, several recent high-quality studies have made major contributions to our knowledge of the natural history and therapy of prostate cancer. SUMMARY: The efforts of professional associations to generate evidence-based guidelines are reflective of an increased awareness that the care of prostate cancer should--to the greatest possible extent--be evidence-based. Practicing urologists should seek to become involved in the evidence-based medicine process by supporting the accrual to high-quality clinical trials, demanding the highest standards to the timely and unbiased dissemination of research findings and ultimately, strive to apply the best available evidence to the care of individual patients. PMID- 16679854 TI - Update on outcomes research databases in prostate cancer 2006. AB - PURPOSE OF REVIEW: The purpose of this review is to summarize single-institution prostate-cancer-outcomes databases (which are most commonly derived from large academic medical centers, Veterans Affairs medical centers, and military hospitals) to summarize the design and development of three well characterized outcomes databases that combine data from multiple sites (Carcinoma of the Prostate Strategic Urological Research Endeavor, Center for Prostate Disease Research, and the Shared Equal Access Regional Cancer Hospital database) and to use the examples of obesity and prostate-specific antigen changes over time to highlight the importance of these databases in prostate-cancer outcomes. RECENT FINDINGS: Multiple databases have demonstrated that obese men are at greater risk of biochemical progression following radical prostatectomy. In addition, objective data have shown that it is more difficult to operate on obese men leading to greater risk of positive surgical margins, which may contribute to poorer outcomes. Several databases have shown that a rapidly increasing prostate specific antigen, measured either before diagnosis or after failed primary therapy, is associated with increased risk of prostate-cancer-specific mortality. SUMMARY: Outcomes databases are extremely useful tools. They have lead to dramatic improvements of our understanding of prostate cancer. The challenge is to use this information from past patients to help us better manage our current and future patients. PMID- 16679855 TI - Recent progress in hormonal therapy for advanced prostate cancer. AB - PURPOSE OF REVIEW: Primary androgen deprivation therapy and secondary hormonal therapy remain the cornerstones of treatment for advanced prostate cancer. This review outlines the basic evidence for use of hormonal therapy while highlighting major research developments made in the past year. RECENT FINDINGS: Recent research on androgen deprivation therapy has suggested that patients with high risk features may have longer metastasis-free survival with early initiation of androgen deprivation therapy. Fracture risk has been shown to be significantly increased in patients on androgen deprivation therapy and is correlated with duration of treatment. In the treatment of androgen-independent prostate cancer, oral premarin has been shown to induce of prostate specific antigen responses more than 50% in 32% of patients, though thromboembolism remains a risk despite prophylactic low-dose warfarin. Transdermal estradiol has been associated with virtually no cardiovascular toxicity, but induced of prostate specific antigen responses more than 50% in only 12.5% of patients. Clinical studies of nilutamide, flutamide, and ketoconazole have further clarified efficacy of these secondary hormonal treatments. SUMMARY: Optimal timing of androgen deprivation therapy awaits the results of randomized trials, but available evidence indicates that patients with high-risk features may benefit from early androgen deprivation therapy. New estrogen-based therapies have shown promising efficacy in the treatment of androgen-independent prostate cancer, with significantly less cardiovascular toxicity than traditional estrogens. PMID- 16679856 TI - Immunologic targeting: how to channel a minimal response for maximal outcome. AB - PURPOSE OF REVIEW: Drugs that target extracellular molecules and intracellular pathways remain an area of active research in prostate cancer. Although preclinical data suggest that new drugs can modulate or slow prostate tumor proliferation, responses in man, such as disease stabilization or regression, are not as dramatic as those seen in preclinical models. Other approaches, including carbohydrate and cellular product vaccines, cytokines, and monoclonal antibodies either alone or with radiopharmaceuticals, are being used to seek and destroy cancer cells. Although robust in-vitro antibody responses can be generated against a specific immunogen in many vaccines, immunologists would agree that immune responses are suboptimal, as defined by a lack of impact on tumor growth, and insufficient to impact on disease progression. RECENT FINDINGS: Although a preferred result, approaches that maximize the cellular arm of immune responses are limited by technology to detect these responses and by agents that can enhance their activity. DNA vaccines that target prostate-specific antigen and prostate-specific membrane antigen, and drugs that can block inhibitory molecules on T cells, such as cytotoxic T lymphocyte antigen-4, are currently under study. SUMMARY: This article will review state-of-the-art mechanisms by which immunity may be enhanced to elicit antitumor responses against selectively expressed cell surface molecules and to maximize antitumor responses. PMID- 16679857 TI - Update on erectile dysfunction in prostate cancer patients. AB - PURPOSE OF REVIEW: Evolution in the management of prostate cancer includes increased attention being paid to patient quality of life after treatment, specifically with issues related to sexual function. Erectile dysfunction is one of the major concerns of patients undergoing treatment for prostate cancer. There are several recognized factors that determine the postoperative incidence of erectile difficulties, including patient age, degree of cavernosal nerve sparing during surgery, cancer stage, and associated vascular comorbidities. Early initiation of rehabilitation protocols after radical prostatectomy has been advocated to promote the speed and degree of recovery of erectile function. The aim of this communication is to review recent initiatives in erectile dysfunction restoration after prostate cancer therapy. RECENT FINDINGS: In recognition of the neurogenic basis of erectile dysfunction after radical prostatectomy, new strategies have been devised to initiate the rehabilitation process. Type 5 phosphodiesterase inhibitors, vacuum erection devices, and intracavernosal and intraurethral application of vasoactive agents have all been reported in a positive light in recent studies. Developments in cavernous nerve graft interposition procedures, perioperative neuroprotection measures, and postoperative neurotrophic treatments aim to preserve prostate cancer patients' qualities of life. SUMMARY: Data generated from a number of clinical investigations document that pharmacologic rehabilitation programs provide a higher rate of recovery of erectile function following radical prostatectomy. Both intracavernosal and intraurethral applications of vasoactive agents and vacuum devices can speed the recovery period for return of erectile function. Various neuroprotective and neurotrophic approaches are thought to provide integral roles for the maintenance of sexual function in men undergoing prostate cancer therapy. PMID- 16679858 TI - Bibliography. Current world literature. Prostate cancer. PMID- 16679859 TI - Hormone replacement therapy in menopause and lung cancer: an update. PMID- 16679860 TI - Evaluation of health risks caused by radio frequency accelerated carcinogenesis: the importance of processes driven by the calcium ion signal. AB - The acceleration of carcinogenesis, which was induced either by radio frequency radiation from a cellular telephone or by the ferric-ATP complex, was similar in a mouse strain characterized by age-determined carcinogenesis of lymphoid tissues. Organ hypertrophy, the presence of lymphoid blood and ascites, the development of solid tumours, and mortality were very different to those found in control animals. These results emphasize the role of calcium ion signal influx in the activation of oncogenes and the failure of thymus-determined immune defences. PMID- 16679861 TI - Angiotensin I-converting enzyme gene polymorphism modifies the smoking-cancer association: the Hisayama Study. AB - We examined the long-term contribution of smoking and angiotensin I-converting enzyme (ACE) gene I/D polymorphism to total cancer deaths in a prospective study of a general Japanese population. A total of 937 subjects aged 40 years or older were selected from an original cohort of 1621 subjects and were followed up for 32 years. During the follow-up period, 176 subjects died of cancer. Cancer mortality increased significantly with increasing current smoking levels. Although no clear relationship was observed between ACE genotypes and fatal cancer, the interaction term between current smoking and ACE genotype DD was found to be significant. In stratified analysis by ACE genotype after controlling for age, sex, alcohol intake, body mass index, glucose intolerance, serum total cholesterol and systolic blood pressure, the risk of fatal cancer in currently smoking subjects with genotype DD was twofold greater than that in subjects with genotypes II and ID. Among current smokers, subjects with genotype DD also showed a significantly greater risk of death due to cancer compared with those with genotypes II and ID combined (hazard ratio 1.77; 95% confidence interval 1.04 3.00; P=0.03). In conclusion, our findings suggest that ACE genotype DD enhances the association between smoking and cancer death in the general population. PMID- 16679862 TI - Endoscopic follow-up of 383 patients with colorectal adenoma: an observational study in daily practice. AB - Endoscopic removal of colorectal adenomas reduces the incidence and mortality of colorectal cancer (CRC), but follow-up surveillance is recommended. Compliance with the Dutch surveillance guidelines and detection of neoplasia during follow up has been evaluated in daily practice. From 1987 to 1996, 383 consecutive patients with colorectal adenomas (56.4% male, 61.8+/-11.3 years) were included and followed until December 2000. The mean follow-up was 80.5+/-42.5 months with 2.2+/-0.9 follow-up endoscopies. A total of 32.5 and 27.3% of follow-up endoscopies were performed >25% (time between advised and actual endoscopy) too late or too early, respectively. At the end of follow-up, 33.4% of patients had left the follow-up (two-thirds died) and 60.1% were known with co-morbidity. A first, second, third, fourth and fifth follow-up endoscopy had been performed in 327, 238, 132, 64 and 35 patients, respectively. Adenomatous polyps (with high risk polyps) were detected in 100% (42.6%) of the index endoscopies and in 25.1% (17.4%), 23.9% (10.5%), 28.0% (12.1%), 34.4% (25.0%) and 37.1% (17.1%) of the first to fifth follow-up endoscopy, respectively. CRC was diagnosed in seven patients (46.1+/-22.9 months after index endoscopy), resulting in a standardized incidence ratio of 1.4 (confidence interval 0.6-3.0, P=0.4) compared to the general population. In this daily practice, high numbers of total and high-risk adenomatous polyps were found during follow-up surveillance. The incidence of CRC was not significantly different from the general population, which might be due to the intensive follow-up and removal of polyps. These findings support the importance of follow-up surveillance. However, the high overall morbidity and mortality should be taken into account when selecting patients for an intensive follow-up programme. PMID- 16679863 TI - Meat consumption and risk of colorectal cancer in Japan: the Miyagi Cohort Study. AB - The association between meat consumption and risk of colorectal cancer has been controversial. We examined this question in a large prospective cohort study in Japan. From June through August 1990, 47,605 residents, aged 40-64 years, of Miyagi Prefecture in northern Japan completed a self-administered questionnaire, including a food frequency questionnaire. In the study population, we observed 474 incident cases of colorectal cancer during 11 years of follow-up, to March 2001. We used the Cox proportional hazards model to estimate the relative risk of colorectal cancer (colorectum, colon, rectum and proximal colon and distal colon) according to each of the categories of meat intake (total meat, beef, pork, ham or sausage, chicken and liver), with adjustment for sex, age and other potentially confounding variables. The multivariate relative risk of colorectal cancer in the highest category of total meat consumption compared with the lowest was 1.14 [95% confidence interval (CI)=0.85-1.53; P-trend=0.22]. We also found no significant association between total meat consumption and the risk of sub-site of colorectal cancer. In conclusion, our data do not support the hypothesis that meat consumption is a risk factor for colorectal cancer. PMID- 16679864 TI - Do socio-demographic factors influence mammography use of French women? Analysis of a French cross-sectional survey. AB - The aim of this study was to analyse the independent role of socio-demographic factors on the use of mammography according to whether or not an organized breast cancer screening programme exists. The study sample of 2825 women aged 40-74 years was drawn from a cross-sectional population-based survey of French households. Among these women 46% lived in districts that offered a screening programme and 63% reported undergoing mammography in the previous 2 years. Living in a district that offered a screening programme was associated with increased use of mammography. According to both univariate and multivariate analysis, several socio-demographic characteristics, such as high monthly household income or high education level, increased the probability of using mammography. However, three factors had a major positive impact on its use: (1) having had a gynaecological examination in the previous 2 years, (2) living in a district where a screening programme was available, and (3) age. There was a significant interaction between the factors 2 and 3. Between 40 and 60 years, age had the same impact on the use of mammography whether or not women lived in a district with a screening programme. After the age of 60 years, the use of mammography collapsed among women living in a district without a screening programme and remained frequent among women living in the district that offered such a programme. Even if the overall level of mammography screening was high and the existence of a screening programme maintained a high level of mammography use among older women, the programme should target better the women of underprivileged spheres and reinforce the role of the general practitioner; in particular for women not followed by a gynaecologist. PMID- 16679865 TI - Plasma enterolactone and genistein and the risk of premenopausal breast cancer. AB - BACKGROUND: The scientific debate on the role of dietary phytoestrogens for prevention of breast cancer is still ongoing. We previously reported an inverse association between dietary phytoestrogen intake and premenopausal breast cancer risk and now examine the relationship with plasma phytoestrogen concentrations. METHODS: We measured enterolactone (mammalian lignan) and genistein (isoflavone) concentrations in plasma samples of 220 premenopausal cases and 237 age-matched controls from a population-based case-control study in Germany. RESULTS: Median plasma enterolactone concentrations in cases and controls were 6.3 and 9.7 nmol/l, respectively, and median genistein concentrations were 4.5 and 3.7 nmol/l, respectively. Premenopausal breast cancer risk decreased with increasing plasma enterolactone concentrations. Adjusted odds ratios (95% confidence intervals) were 0.42 (0.20-0.90) and 0.38 (0.17-0.85) (P for trend 0.007) for women in the third and fourth quartile of plasma enterolactone compared to those in the lowest quartile. There was no significant association between plasma genistein concentration and premenopausal breast cancer risk. CONCLUSION: Using biomarkers of phytoestrogen intake, we confirmed the strong inverse association between enterolactone and premenopausal breast cancer risk as found with dietary intake estimates. This result gives support to the potential role of mammalian lignans for breast cancer prevention among premenopausal women in Western populations. PMID- 16679866 TI - Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families. AB - Since IGF-1 and IGFBP-3 levels have been implicated in premenopausal breast cancer, we aimed to explore whether there were differences in IGF-1 and IGFBP-3 regulation between women from BRCA1/2 families and from families in which no BRCA1/2 mutations could be detected, that is, from BRCAX families. One hundred and one healthy 18-40-year-old women from hereditary breast cancer families with known BRCA1/2 mutations and 111 women from BRCAX families completed a questionnaire including information on reproductive factors and oral contraceptive use. We measured height, weight, and plasma IGF-1 and IGFBP-3 levels during menstrual cycle days 5-10 and again during cycle days 18-23. IGF1 and IGFBP3 genotypes were determined. Women from BRCAX families had higher IGFBP 3 levels than women from BRCA1/2 families during cycle days 5-10 (P=0.08) and cycle days 18-23 (P=0.0001). The IGFBP3 (A-202C) A allele also segregated at a significantly higher frequency among women from BRCAX families than among women from BRCA1/2 families (40.3 versus 30.3%; P=0.04). The IGFBP3 genotype was highly associated with the IGFBP-3 levels during cycle days 5-10 and 18-23 (Ptrend=0.0004 and Ptrend=0.0003, respectively). After taking the IGFBP3 genotype into account, IGFBP-3 levels were still significantly higher in women from BRCAX families during cycle days 18-23 (P=0.004). No differences were found in IGF1 genotype or IGF-1 levels or the ratio of IGF-1 to IGFBP-3 between women from BRCAX and BRCA1/2 families. In conclusion, we found significant differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families. It is possible that a subgroup of the BRCAX families has an increased risk of breast cancer because of high IGFBP-3 levels. PMID- 16679867 TI - Mammography use and factors associated with its use after the introduction of breast cancer screening programmes in Spain. AB - The aim of this study was to measure the use of mammography and the factors associated with testing among Spanish women, after the introduction of screening programmes in Spain. We conducted a cross-sectional population survey of a representative sample of women aged 40-70 (2409 women). Data collection took place in October 2000, using a questionnaire addressing the dependent variable (mammography use) and the independent variables (socio-demographic and socio health factors, and women's knowledge and attitudes). Mammography use was defined as having received at least one screening test in the previous 2 years. Data analysis consisted of univariate and multivariate analyses. 48.1% (95% confidence interval (CI) 46.0-50.2) of women had received screening mammography. The main factors associated with testing were: mammography invitation from the screening programme (odds ratio (OR) 4.81; 95% CI 3.85-6.01); gynaecologist visit (OR 4.32; 95% CI 3.45-5.41); and intention to have a mammogram (OR 2.94; 95% CI 2.00-4.32). Other test-related factors were: not rejecting test for discomfort or fear of cancer; mammography perceived as necessary; age 56-65; upper/upper-middle socio economic status. In conclusion, after the introduction of screening programmes, almost half of Spanish women aged 40-70 had received mammography. Invitation to screening, gynaecologist visit and women's attitudes are the main reasons for undergoing testing. Women over 65 years of age and/or those in a lower socio economic level warrant special attention. PMID- 16679868 TI - Genetic variation in interleukin 8 and its receptor genes and its influence on the risk and prognosis of prostate cancer among Finnish men in a large cancer prevention trial. AB - The cytokine interleukin 8 (IL-8) may play a role in the pathogenesis of prostate cancer through the modulation of tumour immune response or enhanced angiogenesis. A common polymorphism of the IL-8 (-251) gene, which may affect the production level of the cytokine, has been inversely associated with a number of diseases, including prostate cancer. We examined the most representative single nucleotide polymorphisms (SNPs) for the IL-8 and its receptors (CXCR1 and CXCR2) genes, and conducted a case-control study nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study to examine if these SNPs are associated with susceptibility to and prognosis of prostate cancer. Using incidence density sampling, 584 cases of primary prostate cancer and 584 matched controls were selected. In this population, we observed no strong association between the SNPs for IL-8 -251 (A-->T), CXCR1 +860 (C-->G) and CXCR2 -1010 (A-->G) and either the subsequent risk of prostate cancer or individual prognostic factors among cases. Although none of the SNPs studied are likely to have major effects on prostate cancer susceptibility, a role for other polymorphisms associated within these genes cannot be excluded. PMID- 16679869 TI - Genital and urinary tract diseases and prostate cancer risk. AB - The objectives of this study were to investigate whether venereal diseases, prostatic and urinary infections, and other selected conditions of the genital and urinary tracts may have a role in the development of prostate cancer. We analysed data from a hospital-based case-control study conducted between 1985 and 1992 in Italy, including 280 cases of prostatic cancer and 689 controls, hospitalized for acute, non-neoplastic, non-genital or urinary tract conditions. We calculated odds ratios and 95% confidence intervals using unconditional multiple logistic regression. The odds ratios of prostate cancer were 0.64 for history of selected venereal diseases, 0.53 for prostatitis, 0.67 for benign prostatic hyperplasia, 1.53 for urinary tract stones and 1.76 for cystitis. No relationship, however, was observed for cystitis>or=5 years before prostate cancer. The present study, based on satisfactorily reproducible information on medical history, did not found any association between genital and urinary tract diseases and prostate cancer risk. PMID- 16679871 TI - Increased suicide risk among Danish women with non-melanoma skin cancer, 1971 1999. AB - More suicides than expected (standardized mortality ratio=1.3; 95% confidence interval=1.1-1.6) compared to the population risk were found among Danish women with a diagnosis of non-melanoma skin cancer in the period 1971-1999 but not among men. The increased risk might be associated with a different personality type in this cancer group. PMID- 16679870 TI - Extent, relationship and prognostic significance of apoptosis and cell proliferation in synovial sarcoma. AB - AIMS: To analyse the extent, relationship and clinical significance of apoptosis and cell proliferation in synovial sarcoma. METHODS: Apoptosis was detected by TUNEL, and expression of Ki-67, Bcl-2, Bax and p53 was examined immunohistochemically in 72 synovial sarcomas. Their relation and correlation with clinicopathological parameters and survival rate were analysed. RESULTS: The average values of apoptosis index (AI) and Ki-67 labelling index (LI) were 0.76% and 28.30%, respectively. Both AI and Ki-67 LI in large-volume, high-grade and advanced-stage synovial sarcomas were significantly higher than those in small volume, low-grade and early-stage ones (P<0.05 for all). And there was a linear relationship between AI and Ki-67 LI (r=0.751, P<0.001). All examined synovial sarcomas were positive for Bcl-2 and Bax, and only 20.8% cases showed expression of p53 protein. The expressions of Bcl-2, Bax and p53 were also significantly correlated with AI (P=0.005, P=0.002, P=0.037, respectively). In addition, patients with high AI (>0.76%) had poor prognosis (log-rank test; P=0.007). CONCLUSIONS: Alterations in apoptosis and cell proliferation activity might be responsible for the pathogenesis and behaviour of synovial sarcoma. Increased rate of apoptosis in synovial sarcoma was considered to be an indicator of poor prognosis. In addition, apoptosis in synovial sarcoma may be controlled by multiple apoptosis-regulating mechanisms, including the Bcl-2 family and p53 protein. PMID- 16679872 TI - Effects of ERCC1 expression in peripheral blood on the risk of head and neck cancer. AB - Squamous cell carcinoma of the head and neck (SCCHN) accounted for 3.5% of all cancers registered in Korea during 2001. Although tobacco smoking and alcohol consumption are known risk factors of SCCHN, only a small fraction of the populations exposed to tobacco or alcohol develop SCCHN. Therefore, differences in the susceptibility of SCCHN with respect to DNA repair and xenobiotic metabolisms have focused upon attempts to determine the causes of SCCHN. Here, we investigated factors that affect ERCC1 mRNA expression in peripheral blood. The study subjects were all Koreans (67 patients and 73 control subjects); 72.9% of all subjects were male; 68.4% were current or former smokers; and 62.4% were current or former alcohol drinkers. We also studied the association between ERCC1 mRNA expression and the C8092A polymorphism of ERCC1, and found lower ERCC1 mRNA expression in SCCHN patients than in controls (P<0.01). In the present study, we found that ERCC1 C8092A polymorphism is not related to the risk of SCCHN or expression of ERCC1 mRNA. In addition, we found a positive association between ERCC1 mRNA expression in peripheral blood and tumour tissues and inverse associations between ERCC1 mRNA expression and age or the number of cigarettes smoked. Therefore, our study suggests that ERCC1 mRNA expression is reduced by age and smoking and has a weak effect on SCCHN risk as compared with the effects of age and tobacco-smoking expression. PMID- 16679873 TI - Inter-relation of apoptosis and DNA double-strand breaks in patients with multiple primary cancers. AB - Since the development of multiple primary cancers in an individual is considered an unlikely event, it is suspected that a defect in DNA repair or apoptosis is the underlying cause for some of these patients. Therefore, this study was based on the hypothesis that such patients have increased remaining DNA double-strand breaks (DSBs) and reduced levels of apoptosis after in vitro irradiation. To investigate these mechanisms in cancer patients, 19 with multiple primary cancers were selected out of 25 121 cancer patients. For inclusion in this study, patients had to present with first malignancy at an early age, have a positive family history of cancer and no risk factors. The exclusion criteria were recurrence of cancer or metastasis, haematological tumours and tumours possibly connected to a patient risk factor such as smoking or drinking. Their peripheral blood lymphocytes were tested for proper repair of DNA DSBs and apoptosis after in vitro irradiation. DSBs were measured using constant field gel electrophoresis at 0, 8 and 24 h after irradiation. Apoptotic rates were determined at 24, 48 and 72 h after irradiation using the TUNEL assay. We found that patients' lymphocytes had significantly more initial DNA DSBs compared with controls, but there was no difference in the number of remaining DNA DSBs. Apoptotic rates of lymphocytes were only slightly lower in patients than in controls. These findings show that there are limited differences between patients with multiple cancers and healthy individuals. However, we found a trend towards an inverse correlation between remaining DNA DSBs and apoptotic rates in patients' lymphocytes. This is indicative of DNA DSBs persisting in patients' cells, presumably leading to a higher level of stable chromosomal aberrations that may contribute to tumour formation. PMID- 16679875 TI - 'Fever phobia' in the emergency department: a survey of children's caregivers. AB - OBJECTIVES: To investigate children's caregivers' attitudes towards fever in an emergency department setting. METHODS: A 25-item questionnaire was formulated, on the basis of similar previous published surveys, for administration to a convenience sample of caregivers. It was administered by a medical translator after triage, before assessment by a physician. Most questions were multiple choice, a few open-ended. RESULTS: Three hundred questionnaires were administered to caregivers and 264 were analyzed. A high proportion (82%) of caregivers professed to be 'very worried' about fever. Temperatures that were felt to require treatment were relatively low (one-third treating <37.9 degrees C), but many respondents measured body temperature at the axilla. Similar to previously published studies, the main specific concerns were possible central nervous system damage (24%), seizures (19%) and death (5%), although worries about discomfort and signs of serious illness were also expressed by a significant number of respondents (11%). Similar to older surveys, home treatment of fever was worrisome, with too-frequent dosing (acetaminophen 15. The study was conducted over the period 1998-2000; only patients brought primarily to the trauma centre were included. Statistical tests used include chi, Kruskal-Wallis, Wilcoxon's rank sum and Spearman's rho. RESULTS: Seven hundred and forty-one patients triaged to Aarhus Trauma Centre from which we obtained all information in 596 cases constituted the study group. In 472 cases, an anaesthesiologist was present. On-scene times, median and 95% confidence interval, were as follows: entire study group (n=596) 15.5 min (15 17); ambulance only: 14.0 min (12-15); anaesthesiologist present, no intubation, no entrapment: 15.0 min (14-16); intubation, no entrapment: 21.5 min (16-27); entrapment, no intubation: 21.5 min (17-25); both intubation and entrapment: 22.0 min (16-36). CONCLUSION: The presence of an anaesthesiologist prolonged the median on-scene time by 1 min and in cases of prehospital intubation by 7.5 min. This result was no different from the prolongation caused by entrapment. PMID- 16679881 TI - Relationships among injuries treated in an emergency department that are caused by different kinds of animals: epidemiological features. AB - OBJECTIVE: To analyse animal-related injury features treated in an emergency department. METHODS: Inspection of all reports concerning animal-related injuries treated in the emergency department in 1998-1999, researched through non-codified diagnosis. RESULTS: A total of 988 patients (1.66% of annual emergency department visits) were treated in the emergency department for animal-related injuries. Prevalence of the causing agent was as follows: insects 53.1% (74.9% not identified), mammals 30.9%, marine fauna 7.4%, arachnids 7.1%, reptiles 0.51%, birds 0.51% and intestinal parasites 0.4%. Sex prevalence was also noted: male 59.6%. Age most affected was III-IV decades. Month prevalence: August, except for arachnids, cats and reptiles. Mammals cause a number of lesions higher than any other group from November to May, while insect-related lesions are prevalent from June to October. In dog-related injuries, the upper right extremity prevails over the left (63%, P<0.001). Bites included 28.7% of all cases; cats caused only bite lesions in 50% of cases, and dogs in 95%. Allergic reactions were observed in 18.6% of patients with insect and marine fauna injuries. Vespids show the highest allergic reaction/injury ratio (44%). Hospital admittance was 2.7% of all animal related injury visits (7.9 cases/100 000 residents). CONCLUSIONS: Insects are the prevalent injuring animals but most are not identified; thus, dogs account for most injuries among identified animals (25.2% of total cases). Insects cause most hospitalizations because of allergic and infective complications (erysipelas). Mammalian and bird injuries being excluded, the diagnosis of 'animal lesion' was defined 'probable' in 11.7%; in some cases, a dermatologist's consultation is required. In serious allergic reactions, adrenaline is rarely used. PMID- 16679882 TI - Lipid peroxidation, occupational stress and aging in workers of a prehospital emergency service. AB - BACKGROUND: Stressful conditions lead to formation of excessive free radicals, and lipid peroxidation is one of the major outcomes of free radical-mediated injury that directly damages membranes and generates a number of secondary products. OBJECTIVES: To determine the levels of malondialdehyde, an end product of lipid peroxidation, according to demographic and occupational variables in workers of a prehospital emergency service and to analyse the relationship between malondialdehyde levels and burnout. MATERIAL AND METHODS: One hundred and eleven healthy workers of a prehospital emergency service and eighty aged-matched healthy individuals of both sexes as a control group were surveyed. Malondialdehyde levels were measured by the Bull and Marnett method. To measure burnout, the Maslach Burnout Inventory was used. RESULTS: Professional category is associated with lipid peroxidation and burnout levels (Malondialdehyde levels were: physicians 338.10+/-14.47, nurses 329.17+/-12.62 and technicians 296.74+/ 14.28; burnout levels were: physicians 41.29+/-3.59, nurses 37.38+/-6.05 and technicians 35.33+/-5.87). Working at night and in the evening increased malondialdehyde and burnout levels. Malondialdehyde levels increase with age. No significant variations with respect to sex were detected. Significant variations in malondialdehyde levels were detected between singles (303.13+/-12.74) and married people (344.43+/-13.43) but not with respect to divorcees (326.44+/ 11.74). Significant differences were detected in erythrocyte malondialdehyde levels between smokers (341.37+/-17.09) and nonsmokers (302.21+/-12.38), but not for alcohol consumption. CONCLUSIONS: These findings suggest a positive correlation between malondialdehyde, a biomarker of lipid peroxidation and occupational stress, as estimated by elements of the Maslach Burnout Inventory, and oxidative stress. PMID- 16679883 TI - Life-threatening hemophagocytic syndrome related to mycobacterium tuberculosis. AB - Hemophagocytosis is a classic but uncommon complication of tuberculosis and can lead to a life-threatening condition. We report two cases of severe hemophagocytic syndrome related to mycobacterium tuberculosis. The etiologic diagnosis was made by specific polymerase chain reaction in one patient. These reports illustrate that tuberculosis should be considered for the diagnosis of hemophagocytic syndrome in critically ill patients. PMID- 16679884 TI - Respiratory distress, pneumonic changes on chest X-ray, hypoxaemia, oral candidiasis in a homosexual male: not always Pneumocystis carinii pneumonia. AB - A young homosexual male presented to the emergency department with respiratory distress, oropharyngeal candidiasis, hypoxaemia, and renal failure. Pneumocystis carinii pneumonia was considered to be the likely diagnosis - until 24 h later when the patient divulged the true history of paraquat ingestion some 5 days previously. Ingestion of corrosives should be considered in patients with oropharyngeal candidiasis and pneumonic symptoms, especially in the presence of renal failure as an alternative to HIV infection. PMID- 16679885 TI - Coma due to cannabis toxicity in an infant. AB - In young children, cannabis ingestion resulting in coma is very rare. Only nine cases have been reported in the literature and most have occurred in inquisitive toddlers. We review the cases to date and report the youngest recorded case of coma due to cannabis ingestion in an 11-month-old girl. This case highlights the importance of considering toxicological causes and the parental recreational drug history in young children who present with an altered level of consciousness. Child protection considerations are also discussed. PMID- 16679886 TI - Bicycle handlebar injury in a nine-year old causing a common hepatic duct tear. AB - A case in which a handlebar injury to the abdomen resulted in a common hepatic duct tear is presented. This injury has not been previously documented in the literature. The case emphasizes the diverse and potentially serious consequences of handlebar injuries in children, and reminds us that the presentation may be delayed. PMID- 16679887 TI - Thoracic aneurysm rupture presenting as a rapidly enlarging neck mass. AB - A rapidly enlarging neck mass is an unusual clinical complaint at the emergency department. We report a case of an acute left neck swelling caused by a haematoma from a ruptured thoracic aneurysm. This resulted in dynamic airway distortion and obstruction and posed a great challenge to airway management. PMID- 16679888 TI - Catastrophe for a campanologist. AB - A campanologist was involved in a nonfatal hanging incident. On presentation she had no midline bony tenderness and only subtle abnormalities on plain X-ray films of her cervical spine; however, a CT scan showed a type III odontoid peg fracture. We believe this is the first reported cervical spine fracture sustained while bell-ringing. This case demonstrates the importance of mechanism of injury with regard to decisions about diagnostic imaging and the debate concerning the choice of first-line cervical imaging is highlighted. PMID- 16679889 TI - Traumatic internal carotid artery dissection associated with motor-cross. PMID- 16679890 TI - New perspectives for a new century: implications of pathogen responses for the future of antimicrobial therapy. AB - Although the discovery of new classes of antibiotics has lagged behind in the last three decades, the incidence of life-threatening nosocomial infections that are resistant to multiple antibacterial agents has increased steadily. Recent advances in bacterial pathogenicity through the identification of a number of virulence factors and the bacterial genetics behind it have opened the way to a clearer understanding of the pathogen-host relationship. Bacteria communicate with each other through specific signaling chemicals to act as a community rather than individual cells to achieve a critical density or a "quorum." Establishment of quorum is the initiating signal for turning on a variety of virulence factors essential for the pathogenicity and dissemination of pathogens through the host. Pathogenic bacteria use a variety of biochemical mediators, collectively called "virulence factors," to invade and attack host tissues and to avoid detection and elimination by the host immune system. Delineating the specific responses the host immune system elicits in response to specific virulence factors and quorum sensing molecules is essential to the development of new diagnostic methods for early detection of an infection and the prognosis to a given antibacterial therapy. Identification of inhibitors of virulence factors will represent new antimicrobial therapeutic modalities, and this can be used synergistically with current antibiotic therapy because they act through independent prokaryotic pathways to inhibit bacterial growth and survival. PMID- 16679891 TI - Predictors of survival and length of stay in burn patients older than 80 years of age: does age really matter? AB - Predictors of survival and length of stay (LOS) in the advanced elderly with burn injuries is not well studied. Because of progress in burn wound and critical care, we hypothesized that a contemporary analysis would show improved outcomes. Clinical data were collected on 45 consecutive patients older than 80 years of age that were treated for burn injury at our institution during the past 10 years. Regression analysis was used to identify predictors of LOS and survival. Overall rate of mortality was 29%, and no patient survived a burn more than 60% TBSA. The strongest predictor of survival was percent TBSA burn. LOS of survivors was dependent on presence of inhalation injury and total number of operations. The survival of patients older than 80 years of age with burn injury is better than reported. Modern burn care allows survival in many patients over 80 with less than 60% TBSA burns without significant other co-morbidities. PMID- 16679892 TI - SCORTEN overestimates mortality in the setting of a standardized treatment protocol. AB - Toxic epidermal necrolysis (TEN) is a rare, severe, exfoliative disorder with a high mortality rate. SCORTEN is a recently developed scoring system that estimates severity and predicts mortality in patients with TEN based on seven independent clinical risk factors recorded within the first 24 hours of admission. An increasing SCORTEN level predicts a higher mortality rate. For more than 20 years, the treatment of TEN at our institution has involved the use of a standardized clinical pathway that includes removal of sloughed epidermis, dermal protection with porcine xenograft, early enteral nutrition, and critical care monitoring. We hypothesize that this standardized clinical approach will result in a lower mortality rate than predicted by SCORTEN. A retrospective review was performed on all patients treated for TEN using the standardized pathway from February 1987 to March 2004. SCORTEN was calculated in each patient. One hundred nine patients were treated for TEN during the study period. Overall observed mortality was 20% compared with a SCORTEN predicted mortality of 30%, resulting in a relative reduction in mortality of 33% (P = .011). In addition, observed probability of death was lower than predicted at all levels, except at SCORTEN score of 6 or greater. In conclusion, TEN remains a life-threatening disease with a high mortality rate. Our standardized treatment protocol results in significantly improved outcomes compared to those predicted by SCORTEN. PMID- 16679893 TI - The epidemiology of intentional burns. AB - We sought to present the epidemiology of intentional burns, both deliberate self inflicted burns and assault burns. Patient records from the University of Alabama at Birmingham Burn Center were reviewed retrospectively. Information pertaining to demographic and injury characteristics were obtained. Ninety-six patients with intentional burns were identified. Patients sustaining intentional burn injury had larger mean TBSA burned (26.1% vs 13.8%), longer mean hospital length of stay (19.9 days vs 13.2 days), higher incidence of inhalational injury (20.8% vs 8.7%), higher rate of mortality (20.0% vs 9.8%), and were more likely to have an elevated blood alcohol content (14.6% vs 7%) when compared with all other burn patients. Patients with deliberate self-burns were more likely to be men (85.7% vs 55.7%) and more likely to have a positive drug screen test than assault burn patients (11.4% vs 0%). Patients who suffer intentional burns tend to have more severe burns and experience worse outcomes. PMID- 16679894 TI - Cytomegalovirus and burns: current perceptions, awareness, diagnosis, and management strategies in the United States and Germany. AB - Trauma and immunosuppressed patients are at risk for cytomegalovirus (CMV) disease. Attributing pathogenicity in burn patients remains difficult and controversial because data are sparse. In this work, we surveyed U.S. and German burn centers and evaluated them for awareness, perceptions, diagnosis, and treatment of CMV in patients with burn injury. A total of 58% German (G) and 21% U.S. centers responded. CMV infection incidence was 1:280 G and 1:870 U.S. A total of 41% G and 78% U.S. burn centers considered CMV to be of minor importance and 41% G and 13% U.S. centers considered CMV to be of significant importance. A total of 70% G and 97% U.S. centers performed no admission screening. When testing, 70% G and 19% U.S. centers used serology; 52% G and 25% U.S. centers used body fluid viral isolation; and 43% G and 6% U.S. centers used leukocyte CMV DNA analysis. A total of 72% G and 48% U.S. centers distinguished infection from disease. Human CMV disease was diagnosed by CMV-DNA (82% G, 19% U.S.), direct virus isolation (43% G, 13% U.S.), or RNA detection (26% G, 0% U.S.). A total of 43% G and 19% U.S. would treat the established disease. Establishing consistent guidelines for screening, diagnosis, and treatment seems prudent in caring for the immunocompromised burn patient. PMID- 16679895 TI - Base deficit and alveolar-arterial gradient during resuscitation contribute independently but modestly to the prediction of mortality after burn injury. AB - The main determinants of mortality after burn injury that can be measured on admission include age, total burn size (% burn), and inhalation injury (INHAL). Other variables, measured during resuscitation, may provide additional information about injury severity. We assessed the utility of early arterial blood gas (ABG) data in predicting mortality after burn injury. Data were limited to samples obtained during the first 2 days after burn injury and to those obtained during high-frequency percussive ventilation. Mean values for each patient's ABG data were calculated; subsequent analysis used these derived variables. Logistic regression analysis (LRA) was used to generate a mortality predictor using burn, age (as a cubic age score, AGE), and INHAL. LRA was then repeated with the ABG variables. A total of 162 patients were included. By univariate analysis, death was associated with increased alveolar-arterial gradient (AaDO2), AGE, % burn, full-thickness burn size, INHAL, and with decreased pH and base excess. LRA of % burn, AGE, INHAL, and full-thickness burn size retained the first three variables. The addition of ABG data demonstrated that mean burn excess and mean AaDO2 also contributed independently to mortality. However, there was no difference in accuracy (86%) between the two equations. By Kaplan Meier analysis, AaDO2 but not BE predicted earlier death in those who died. Measured during resuscitation, metabolic acidosis (ie, a base deficit) and oxygenation failure (ie, increased AaDO2) contributed independently, but modestly, to ultimate mortality after burn injury. The inclusion of these variables did not improve predictive accuracy. Whether therapies targeted at these endpoints would improve outcome is unknown. PMID- 16679897 TI - Randomized clinical study of Hydrofiber dressing with silver or silver sulfadiazine in the management of partial-thickness burns. AB - This prospective, randomized study compared protocols of care using either AQUACEL Ag Hydrofiber (ConvaTec, a Bristol-Myers Squibb company, Skillman, NJ) dressing with silver (n = 42) or silver sulfadiazine (n = 42) for up to 21 days in the management of partial-thickness burns covering 5% to 40% body surface area (BSA). AQUACEL Ag dressing was associated with less pain and anxiety during dressing changes, less burning and stinging during wear, fewer dressing changes, less nursing time, and fewer procedural medications. Silver sulfadiazine was associated with greater flexibility and ease of movement. Adverse events, including infection, were comparable between treatment groups. The AQUACEL Ag dressing protocol tended to have lower total treatment costs (Dollars 1040 vs. Dollars 1180) and a greater rate of re-epithelialization (73.8% vs 60.0%), resulting in cost-effectiveness per burn healed of Dollars 1,409.06 for AQUACEL Ag dressing and Dollars 1,967.95 for silver sulfadiazine. A protocol of care with AQUACEL(R) Ag provided clinical and economic benefits compared with silver sulfadiazine in patients with partial-thickness burns. PMID- 16679898 TI - A prospective study of the impact of a critical care pharmacist assigned as a member of the multidisciplinary burn care team. AB - A critical care clinical pharmacy specialist was assigned to the burn center to make scheduled rounds with the physicians and to attend the weekly multidisciplinary burn team rounds. A prospective 6-month study was completed to 1) determine the clinical impact of the pharmacist's interventions and 2) quantify cost savings generated by these interventions. Prospective data concerning clinical interventions by the pharmacist were collected during a 6 month period. Each intervention was independently reviewed by two attending burn surgeons to determine its importance. A total of 165 interventions in 76 patients were documented. Following an independent review of each intervention by burn surgery attendings, 121 of 165 interventions were felt to have improved overall patient care, 42 of 165 prevented possible drug-related toxicity or organ dysfunction, 1 of 165 prevented a possible life-threatening event, and only 1 of 165 was considered insignificant. Drug changes suggested by the pharmacist resulted in a savings of Dollars 11,081.14 for the 6-month period. The integration of the critical care pharmacist into clinical rounds results in significant cost avoidance and improves overall patient care. PMID- 16679899 TI - Adapting adult scald safety standards to children. AB - Scald burns by domestic tap water constitute a painful, potentially debilitating, and sometimes-fatal form of thermal injury. In this setting, the very young and older members of the population are particularly susceptible, owing in part to having thinner skin, which renders them more susceptible to thermal insult. Various codes have set forth a safety standard for maximum delivery temperature of domestic tap water at 120 degrees F (48.9 degrees C), based on adult susceptibility to burns. This work addresses the issue of how the current safety standard for tap water temperature could be adjusted to provide a level of protection to children equivalent to that for an adult at 120 degrees F. A well accepted mathematical model for predicting burn injury as a function of applied surface temperature and time is used to identify these equivalent conditions. Data from the literature of sonographic measurements indicate a representative ratio of child to adult skin thickness of 0.72. The mathematical model shows that the equivalent surface temperature for a threshold scald injury in children is dependent on the depth into the skin at which the injury is identified. For example, the injury produced by a 120 degrees F, 10-second exposure at a depth of 600 microm in an adult is matched in a child at 72% of the depth (432 microm) by an insult of 115.9 degrees F for the same duration. The recommendation is that existing hot water standards be reduced by 3 to 4 degrees F to provide an equivalent level of scald protection to children. PMID- 16679901 TI - Developing a focused scald-prevention program. AB - Scalds account for a high percentage of burn injuries in young children. The purpose of this project was to use a formative evaluation process to design a pilot scald-prevention program for a high-risk population. The burn registry and U.S. Census were used to define a high-risk population. A total of 53 children younger than age 6 were admitted to a local burn center with scalds during a 4 year period. Cooking or food accounted for 84% of these injuries. A total of 21% of the patients resided in one zip code, representing an incidence rate of 23 per 100,000, which was statistically significant. Focus group meetings were conducted with parents in this zip code. They were queried about scald injury knowledge, prevention practices, and attitudes toward interventions. A prevention program was designed based on the findings. Workshops are conducted with high-risk groups in the zip code. Attendees consent to a home visit where prevention practices are assessed and taught. A pre/post test and home risk assessment survey is used to measure change. The Burn Registry, U.S. Census, and focus groups were complimentary formative evaluation measures that assisted in developing a targeted scald prevention project. PMID- 16679902 TI - Surgical reconstruction of attempted suicide charcoal burns. AB - Burning charcoal in an enclosed space is now the second most-common method of committing suicide in Hong Kong. When the suicide is unsuccessful, the patients can still sustain extensive tissue destruction from both direct and indirect thermal burns. We report a series of three patients who sustained deep injuries that, after debridement, left exposed bones and joints needing acute coverage. Free tissue transfer was required in each case to close the wounds. This short series illustrates the extensive destruction that can occur from both contact burns and radiant heat injury and the reconstructive challenges that can result. Of particular note was that serial debridment was necessary in the radiant heat injury because of the evolving and more extensive nature of the wound. PMID- 16679903 TI - The quality of pediatric burn scars is improved by early administration of basic fibroblast growth factor. AB - Pediatric burn wounds can be problematic because an accurate evaluation is difficult as the result of anatomically immature vasculature or immobilization failure, especially in patients with second-degree burns, and because the burn surface areas and the burn depth tend to worsen over the course of time. Delayed wound healing results in unsightly scarring, such as hypertrophic scars, which are problematic both esthetically and functionally. Among cytokines and growth factors, basic fibroblast growth factor (bFGF) is clinically proven, having demonstrated accelerated acute and chronic wound healing. Accelerated wound healing may lead to improved scarring. To elucidate the effects of bFGF on second degree pediatric burn wounds, a comparative study was performed. A total of 20 pediatric patients ranging from 8 month to 3 years (average 1 year, 3 months +/- 6 months) who suffered from the burns by various causes were divided into two groups, conventional (n = 10) and treatment with bFGF (n = 10). A moisture meter, used to objectively measure the stratum corneum and epithelial-mesenchymal functions, was used to assess scars at least 1 year after wound healing. Clinical evaluation of pigmentation, pliability, height, and vascularity demonstrated significant differences between conventional and bFGF-treated scars (1.7 +/- 0.55 vs 0.7 +/- 0.58, 2.4 +/- 0.82 vs 1.1 +/- 0.69, 1.8 +/- 0.66 vs 0.5 +/- 0.57, 1.9 +/- 0.63 vs 0.8 +/- 0.68; conventional vs bFGF-treated, pigmentation, pliability, height, and vascularity, respectively, P < .01). The effective contact coefficient was significantly greater in conventional wounds than bFGF-treated wounds (14.6 +/- 1.68 % vs 8.7 +/- 2.82 %; conventional vs bFGF, P < .01) and bFGF-treated wounds demonstrated significantly less transepidermal water loss values than conventional treatment (8.3 +/- 1.90 g/m/h vs 5.7 +/- 1.85 g/m/hr; conventional vs bFGF, P < .01). Pediatric burn patients treated with bFGF showed less damaging function of the stratum corneum after healing both in clinical assessment and moisture meter analysis. PMID- 16679904 TI - Comparison of positive pressure gloves on hand function in adults with burns. AB - The purpose of this study was to analyze the impact of a standard, custom-made pressure glove vs The NewYork-Presbyterian Dexterity Glove (NYPDG) with silon application on the palmer surface on functional hand use of burn survivors. A standard, custom-made pressure glove and NYPDG were given to 18 participants in a randomized order. Subjects wore each glove for 7 to 10 days during all activities of daily living (ADL). Variables such as hand function, difficulty of fine and gross motor ADL, and participant glove preference were assessed with each glove condition. Data collection of the second glove took place 7 to 10 days later incorporating a quasiexperimental, repeated measure design. A crossover design was used to analyze the data. The NYPDG demonstrated significantly better results in all of the four outcome categories measured: time to complete the Jebsen, the Jebsen Likert scale, fine motor ADL, and gross motor ADL. This study demonstrated that functional tasks took less time to complete and were more easily performed when using the NYPDG. PMID- 16679905 TI - Comparing the Vancouver Scar Scale with the cutometer in the assessment of donor site wounds treated with various dressings in a randomized trial. AB - Cutaneous scarring observed in wounds is, to a significant degree, dependent upon the time it takes for the wounds to heal. Various topical dressings are proposed to influence healing time in donor sites. In this prospective randomized study, we examined the effect of Vaseline gauze (VD; Branolind, Paul Hartmann AG, Germany), Biobrane (BD; Bertek Pharmaceuticals, Inc., Morgantown, WV), an occlusive film dressing Barrier Flex (OD; Moelnlycke Health Care GmbH, Germany), and an equine collagen foil, Tissu Foil E (CD; Baxter, Heidelberg, Germany), on re-epithelialization and scarring in standardized donor site wounds. At 6 months after surgery, donor site scars and normal uninjured mirror sided skin were evaluated in 33 patients using both the Vancouver Scar Scale (VSS) and the cutometer SEM 575 (Courage and Khazaka). The median healing time for OD was 14 days, BD 16 days, CD 19 days, and VD 19 days. The single parameter pliability of the VSS was not significantly different from uninjured skin when all donor site scars were pooled. No difference was found between the four groups. Viscoelastic analysis of all pooled patient data showed a significant difference for Uf (total deformation), Ua (total recovery), Ur (immediate retraction), Ue (immediate distension), Ur/Ue, and Ur/Uf, indicating that donor sites significantly differed from normal uninjured skin. No significant correlation between objective viscoelastic measurements and the subjective pliability assessment of the VSS was found. Viscoelastic differences were greatest in the VD and BD groups. Viscolelastic differences did not significantly correlate with healing time. Various wound dressings had minimal yet significant influence on healing time and scarring. In contrast to the VSS, viscoelastic measurements of skin pliability can objectify scarring when few differences are anticipated. PMID- 16679906 TI - A prototype for an economical vertical microstomia orthosis. AB - One of the complications of thermal injury is the development of scars and contractures during the healing process. This scarring can be devastating, especially when it involves the mouth. The purpose of this report is to describe the construction of an alternative vertical mouth stretching orthosis for vertical microstomia. By using thermoplastic splinting material and a long thread screw, a vertical mouth-stretching orthosis can be custom-fit to any patient's mouth. The device can be fabricated for a pediatric or adult patient for the rehabilitation of a circumferential mouth burn, especially targeting the vertical diameter. When using this device, patients gave positive feedback for comfort and ease of use, with increased mouth mobility and range of motion. This vertical orthotic device provides an economical and comfortable alternative for vertical mouth diameter enlargement to the current five devices available in the literature. PMID- 16679907 TI - Interactions of systemic immune response and local wound healing in different burn depths: an experimental study on rats. AB - This study aimed to clarify the local and systemic immune responses at different burn depths. Thirty female Sprague-Dawley rats were divided into three groups: full-thickness (F), partial-thickness (P), and Sham (S). Burns were induced on three separate areas on the dorsums of rats. Serum levels of interferon (IFN) gamma; tumor necrosis factor-alpha; interleukin (IL)-1, IL-6, and IL-10 were measured once in controls and 1 hour after burn, 48 hours after burn, and 7 days after burn in F and P groups. Neutrophils, CD68-positive macrophages, HLA-DR positive cells, and CD3-positive lymphocytes were graded semiquantitatively, and the wounds were examined once in shams and at 1 hour after burn, 48 hours after burn, and 7 days after burn in F and P groups. IL-6 levels were highest in F group, followed by P group 1 hour after burn. IFN-gamma levels were higher in the F group; IL-1 levels were higher in F and P groups at 1 hour after burn. Local accumulation of macrophages was similar in F and P groups. Lymphocytes were denser in P group at 1 hour after burn, and neutrophils were denser in F group at 7 days after burn. We suggest that early elevations of IL-6 and IFN-gamma prolong inflammation in full-thickness burns. Modulation of proinflammatory cytokines may improve burn wound treatment. PMID- 16679908 TI - Adipocyte apoptosis after burn injury is associated with altered fat metabolism. AB - Burn injury often is associated with the abnormal lipid metabolism, including hyperlipidemia, desensitization to lipolytic responses to catecholamines, and reduction in the size of the white adipose tissue. Understanding the biological mechanisms for the decrease in fat mass despite desensitization to catecholamines is important both for the study of lipid metabolism and for the study of its relationship to concomitant insulin resistance. Using epididymal adipose tissue from adult male Sprague-Dawley rats after burn injury (n = 102) or sham-burn injury (n = 102), we tested the hypothesis that a whole-body burn injury causes apoptosis in that tissue. At 1, 3, and 7 days after 40% to 50% body burn injury to the rat, epidydimal adipose tissue was harvested and studied for apoptotic changes and lipolytic properties. For apoptosis, paraformaldehyde-fixed tissue sections were analyzed by in situ TdT-mediated dUTP-X nick-end labeling (TUNEL) staining, and tissue homogenates were also analyzed for DNA fragmentation by enzyme-linked immunoassay and ligation-mediated polymerase chain reaction ladder assay. Isolated adipocytes were stimulated with isoprotenerol, and glycerol production was measured as a reflector of effectiveness of lipolysis. Epididymal adipose tissue showed increased apoptosis manifested by the positive TUNEL staining and increased DNA fragmentation by enzyme-linked immunoassay at day 3 and 7 after burn injury. The DNA fragmentation was confirmed further by the ligation-mediated polymerase chain reaction ladder assay. This elevated DNA fragmentation persisted in the burned animals from day 3 until day 7 after burn injury, the end of observation period. Increase in apoptosis was correlated with decrease in DNA content and tissue weight in the epidydimis. At the functional level, a significant decrease in isoproterenol-induced lipolytic activity (glycerol production) was observed to almost 50% of control level at day 3 and 7 but was not decreased at day 1. Apoptosis of adipocytes may play a role in the altered lipid metabolism, including hyperlipidemia observed in burned subjects. PMID- 16679909 TI - Closed-loop resuscitation of burn shock. AB - Fluid therapy for burn shock is adjusted to establish a target level of urinary output. However, the means for adjusting infusion rate are not defined. Our objective was to compare the performance of automated computer-controlled resuscitation with manual control for burn resuscitation. Sheep with a 40% TBSA full-thickness burn, administered under halothane anesthesia, were resuscitated to restore and maintain normal sheep urinary outputs in a target range of 1 to 2 ml/kg per hour over the course of 48 hours using closed-loop resuscitation (n = 10) or manual hourly adjustment of infusion rate (n = 11). The automated closed loop resuscitation system is based on a proportional-integral-derivative algorithm, which adjusted infusion rate based on continuous monitoring and changes in urinary output. Mean urinary outputs over the course of 48 hours were in target range and were virtually identical at 1.9 +/- 0.5 ml/kg per hour for the closed-loop group and 2.0 +/- 0.7 ml/kg per hour for the technician group. Mean infusion rates and infused volumes also were similar. The closed-loop group exhibited significantly lower hourly variation for both urinary output and infusion rate compared hourly control. Hourly targets were achieved in 41% of the measurements in technician group compared with 48% for the closed-loop group (P = .23). Hourly urinary output in the technician group was undertarget by 25% as opposed to 16% with the closed-loop group (P = .02). Automated closed-loop control of infusion rates after burn injury produced urinary outputs in target ranges with less variation and less under target values than manual hourly adjustments. Closed-loop resuscitation may provide an improvement over current resuscitation regimens. PMID- 16679910 TI - How relevant and useful is the Cochrane Library as a resource for evidence in burn care, practice, and management? AB - The Cochrane Library, in particular, the Cochrane Database of Systematic Reviews (CDSR), is a compilation of reviews that have collated and assessed all the available clinical research on a particular topic into a summary about its safety and effectiveness. We conducted a study of the CDSR to evaluate the relevance of this database to burns care. Using prespecified criteria, two reviewers independently screened 4041 titles from Issue 3, 2005, of CDSR for possible relevance to burns care. Agreement regarding relevance to was resolved by discussion. Screening of the 4041 titles identified 78 titles as being potentially (unclear) or clearly (clear and directly related) relevant to burns care. Independent scoring of the 78 titles indicated that 9 were directly relevant to burns care and 28 were indirectly relevant to burns care. The remaining 43 reviews were deemed not relevant. The majority of relevant titles in the CDSR were produced by three Cochrane Review Groups. These included five reviews from Injuries, three from Wounds, and one from Pain, Palliative and Supportive Care. In summary, approximately 37, or just less than 1%, of the CDSR, Issue 3, 2005, completed reviews or protocols had some relevance to burns care and management. Increased burn clinician involvement in the Cochrane Library would help to increase the number of reviews conducted on questions relevant to burns care and would enhance the value of this evidence based medicine resource. PMID- 16679911 TI - Attitudes to face transplantation: results of a public engagement exercise at the Royal Society Summer Science Exhibition. AB - Hettiaratchy and Butler (Lancet 2002;360:5-6) framed face transplantation as the next logical step on the reconstructive ladder for severe facial injury, in particular, pan-facial burns. The procedure formed the basis for a Royal College of Surgeons of England working party report. Ethical, surgical, and psychological issues were identified as research priorities before face transplantation is attempted in the United Kingdom. Public engagement is a vital mechanism if people are to be informed about the risks and benefits of the technique and ultimately to be able to consent to either receiving or donating facial tissue. To sample public opinion about the procedure, 304 people attending the Royal Society Summer Science Exhibition were sampled by online questionnaire to determine their attitude toward face transplantation. Although people were more cautious about face transplantation when comparing this with solid-organ transplantation, most of the sample were in favor of the procedure, whether receiving a donor face or donating their own face after their death. Only 10% were against the idea in principle. Concerns about altered identity were not a barrier to face transplantation for 69% of the sample, with most people appropriately identifying the long-term problems of immunosuppression as the issue that most concerned them. There is a substantial population of people who, given the right circumstances, would contemplate face transplantation for themselves and would be willing to donate their own face for transplantation after their death. PMID- 16679912 TI - Pneumatosis intestinalis in a burn patient: case report and literature review. AB - Pneumatosis Intestinalis (PI) is a rare condition characterized by gas in the intestinal wall. PI is not a disease in itself but a sign of an underlying problem. It is associated with a wide variety of underlying disorders, ranging from benign to life-threatening. When found, it should prompt a search for the underlying cause. Diagnostic efforts should be aimed at ruling out catastrophic problems such as intestinal ischemia and necrosis. The presence of peritonitis, rectal bleeding, portomesenteric venous gas, and lactic acidosis are important factors to determine the best course of action. Burn patients have multiple risk factors for developing this condition. Despite this, PI is not well-described in the burn literature. We present here a case report, a brief review of the literature, and clinical considerations. PMID- 16679913 TI - The use of collagen-glycosaminoglycan copolymer (Integra) for the repair of hypertrophic scars and keloids. AB - Integra dermal matrix (Integra Life Sciences Corp., Plainsboro, NJ) was introduced in 1981, and its use in acute surgical burns is well established. However, Integra also has been found to be useful in the surgical treatment of scars. The Integra neodermis is placed at the time of scar excision and then overgrafted several weeks later with a very thin (6/1000-inch) skin graft. The stabilized matrix appears to resist recurrence better than traditional skin grafts, which have a reported recurrence rate of 59%. Many surgeons have had anecdotal success using Integra for both hypertrophic and keloidal scars. This case series presents several patients who underwent reconstructive surgery with the use of Integra to treat their debilitating scar formation. None of the patients developed significant scar morbidity at the donor site when the skin was harvested for grafting during the second stage of the procedure. All patients had documented success with improved appearance, range of motion, and skin quality. PMID- 16679914 TI - Marker turned target? A case to review neprilysin in ALL (or lessons learned at the American Association for Cancer Research Meetings, 2006). PMID- 16679915 TI - Gaucher-like cells in beta-thalassemia patient after bone marrow transplantation. PMID- 16679916 TI - Osteoporosis in children with severe congenital neutropenia: bone mineral density and treatment with bisphosphonates. AB - A high incidence of decreased bone mineral density (BMD) has been described in patients with severe congenital neutropenia (SCN). The objectives of the study are to describe changes in BMD in children with SCN treated with granulocyte colony-stimulating factor and evaluate the response to treatment with bisphosphonates in those who had osteoporosis. A prospective open-label study was performed evaluating BMD and metabolism in 9 Chilean patients with SCN, administrating bisphosphonates in those with osteoporosis. Follow-up ranged between 7 months and 3.5 years. Six out of 9 patients had reduced BMD on initial assessment: 3 had osteoporosis (z score <-2) and 3 had osteopenia (z score <-1). Four children presented vertebral fractures. Two presented osteopenia on follow up without clinical symptoms. Five patients were treated with bisphosphonates, increasing their BMD z score (mean increase 1.2, range 0.27 to 2.62). z Score of hydroxyproline/creatinine ratios, which was elevated in 4 patients with osteoporosis, decreased during treatment (mean decrease 2.18, range 1.56 to 2.53). Four patients remodeled and reexpanded fractured vertebrae during treatment. No side effects of bisphosphonates were seen on follow-up. Osteoporosis is an important comorbidity in SCN patients probably due to increased bone resorption. Bisphosphonates seem to be an effective treatment for osteoporosis in these patients. PMID- 16679917 TI - Hematopoietic stem cell transplantation after first marrow relapse of non-T, non B acute lymphoblastic leukemia: a pediatric oncology group pilot feasibility study. AB - BACKGROUND: Relapsed acute lymphoblastic leukemia (ALL) in children is associated with a poor outcome, especially for those patients whose relapse occurs during the first 36 months after diagnosis. The best therapy for these patients is not known. This study was designed to evaluate the feasibility of enrolling children with recurrent ALL in a standardized treatment protocol that included receipt of a hematopoietic stem cell transplant (HSCT). PROCEDURE: Eligible patients with a bone marrow relapse of non-T, non-B ALL underwent a common induction and consolidation followed by receipt of either an allogeneic HSCT from a human leukocyte antigen (HLA)-identical sibling or an autologous HSCT purged with B-4 blocked ricin. A common conditioning regimen was used for all patients. RESULTS: Twenty-eight patients from eight institutions were enrolled. Fourteen patients did not receive a transplant during the study, because of toxicity (4), relapse (1), inadequate purging (1), and parental or physician preference for an alternative donor transplant (8). Six patients received allogeneic HSCTs. Five of them have remained in remission for a median of 78 months. Eight patients received autologous HSCTs purged with B4-blocked ricin. Four have remained in remission for a median of 94 months. Of the nine patients who received alternative donor transplants, only two remain in remission. CONCLUSION: We conclude that well designed and controlled prospective studies are necessary to define the role of HSCTs in children with recurrent ALL. In order to be successful, such studies must have the full support of participating centers. Autologous HSC transplantation may have a role in the treatment of relapsed ALL, but further studies are needed. PMID- 16679918 TI - Chemokine IL-8 and chemokine receptor CXCR3 and CXCR4 gene expression in childhood acute lymphoblastic leukemia at first relapse. AB - In this study, we examined the gene expression of interleukin (IL)-8, CXCR3, and CXCR4 in leukemic cells from 100 children with relapsed B-cell progenitors (BCP) acute lymphoblastic leukemia (ALL), using quantitative real-time polymerase chain reaction (RT-PCR). IL-8, CXCR3, and CXCR4 were expressed in almost all bone marrow (BM) samples. The CXCR4 expression significantly correlated with known prognostic factors at relapse: time point and site of relapse. Patients who had a combined BM relapse (n=21) had lower IL-8 and CXCR4 expression than those who had an isolated BM relapse (n=79). The CXCR3 expression was higher in female patients (n=39) than in male patients (n=61). However, this did not reach prognostic relevance in relapsed ALL. PMID- 16679919 TI - Developmental therapeutics: review of biologically based CAM therapies for potential application in children with cancer: part I. AB - This article is a review of the status of biologically based complementary and alternative medicine (CAM) therapies that have potential anticancer activity with a focus on those agents that may be active in childhood cancer. The review presented in 2 parts includes the descriptions of the mechanisms that underlie their effects, the manner by which these agents are identified and evaluated and discussion of some of the limitations of current research. The first part introduces the topic of biologically based CAM therapies. The discussion includes a description the discovery biologically based products for cancer, the classification of these compounds and their use from the perspectives of the Ayurvedic and traditional Chinese medicine systems. Issues associated with evidence-based research of compounds used in these practices are addressed. The second part focuses on preclinical and clinical studies of specific herbs or natural products and their bioactive constituents that demonstrate potential in cancer therapeutics. Turmeric, mistletoe, green tea, ginseng, mushrooms, and micronutrients are representative of compounds that exert their effects through mechanisms attributed to specific chemical classes. The section concludes with a discussion of status of clinical trials of biologically based CAM therapeutics for cancer and the future direction of research. PMID- 16679920 TI - Gorham-Stout syndrome in a male adolescent-case report and review of the literature. AB - Gorham-Stout syndrome is a rare disease characterized by vascular proliferation in the bones, effecting osteolysis. When it is complicated by chylothorax, the prognosis is poor. The present case illustrates successful management of chylothorax by a combination of surgery (thoracic duct ligation and excision of lymphangiomatous tissue combined with pleurodesis) and treatment with alpha-2b interferon. Treatment in 38 published cases is reviewed. PMID- 16679921 TI - Hodgkin lymphoma in a child with Diamond Blackfan anemia. AB - Diamond Blackfan anemia (DBA) is a rare disease characterized by aplasia or hypoplasia of erythroid lineage. Normochromic, usually macrocytic, but occasionally normocytic anemia and reticulocytopenia are characteristic findings of DBA. DBA is associated with an increased risk of malignancy. Most of the reported malignancies are acute myeloid leukemia. Solid tumors including hepatocellular carcinoma and osteosarcoma have also been identified. We could find 29 reported cases with DBA and malignancy. Two of them were diagnosed as Hodgkin lymphoma at 15 and 23 years, respectively. Here we report a 7-year-old boy with DBA who developed Hodgkin disease. PMID- 16679922 TI - Successful treatment via chemotherapy and surgical resection of a femoral hemangiopericytoma with pulmonary metastasis. AB - Hemangiopericytoma (HPC) is a soft-tissue neoplasm composed of proliferating capillary pericytes. It has variable and unpredictable malignancy and most commonly occurs in the fifth or sixth decade of life. Diagnosis is based on the histological aspect. HPC is exceedingly rare in childhood. In both adults and children, curative surgery is the most important predictor of survival. The place of chemotherapy in the treatment of HPC is not well established. We describe a case of adult-type metastatic HPC of the thigh in a 13-year-old boy. The response to neoadjuvant chemotherapy was excellent, and local control of this initially unresectable tumor was achieved without radiation therapy or mutilating surgery. The child is alive and well and has had 8 years of follow-up after treatment. PMID- 16679923 TI - Clonality analysis of pediatric multiple tumors: two case reports and laboratory investigation. AB - We examined the possibility of using microsatellite and mitochondrial DNA polymorphisms as markers to detect the clonal origin of tumor cells found in the same patient. We considered two children with complex tumor diseases: one with supratentorial primitive neuroectodermal tumors (PNET) and a hepatic rhabdoid tumor and another with brain and abdominal rhabdoid tumors. In the first patient we found an mtDNA cytosine insertion both in the normal tissue and in the primary tumor, whereas in the hepatic tumor we detected an insertion of 2 cytosine. In the second child, who had a constitutional mutation of hSNF5/INI-1, we identified the same mtDNA pattern both in normal tissue and in the abdominal tumor but not in the brain tumor, which presented three different mtDNA polymorphisms. Thus, we demonstrated the same clonal origin for tumors in the first patient and different clonal origins of the tumors in the second patient. At times it is very difficult to discriminate two neoplastic lesions or metastatic diseases by using only histopathologic techniques. Molecular examination of clonality is a useful tool to obtain information about the origin of synchronous and/or metachronous tumors found in the same patient. PMID- 16679924 TI - Development of severe anemia during fever episodes in patients with hemoglobin E trait and hemoglobin H disease combinations. AB - Globin chain imbalance and tissue hypoxia are important determinants of the clinical severity of thalassemias. Phenotypic expression may be further modified by interactions between alpha- and beta-thalassemia defects. We retrospectively and prospectively studied the clinical and hematologic features in children and adults with hemoglobin (Hb) E trait/Hb H disease (SEA/Pakse) (seven cases) and Hb E trait/Hb H disease (SEA/Constant Spring) (29 cases) and found that they had similar presentations. The severity of these two intermediate thalassemic manifestations ranged from very mild to severe. Severe anemia developed in accordance with very high fever, whereupon the range of Hb and hematocrit (Hct) levels declined to 5.2-5.8 g/dL and 13%-19%, respectively. In one case, during a hemoconcentrated state as occurs in dengue hemorrhagic fever, the Hb and Hct were 10 g/dL and 31%; the latter rose to 35% after fluid therapy. In some patients, the range of Hb and Hct levels was constantly low (4.3-5.8 g/dL and 15%-19%, respectively). (If dengue hemorrhagic fever is misdiagnosed, a fatal outcome may occur for thalassemic patients.) After a hemodiluted condition as in acute post streptococcal glomerulonephritis, the respective Hb and Hct were 5.4 g/dL and 19%. These observations suggest that the instability of Hb E, especially during fever, may play an important role in the clinical manifestations of Hb E trait/Hb H disease with Hb Pakse and with Hb Constant Spring. PMID- 16679925 TI - Successful report of reduced-intensity stem cell transplantation from unrelated umbilical cord blood in a girl with chronic active Epstein-Barr virus infection. AB - We describe an 8-year-old girl with chronic active Epstein-Barr virus (EBV) infection (CAEBV) who was treated successfully by reduced-intensity stem cell transplantation (RIST) from unrelated cord blood (CB). She had been suffering from fever, abdominal pain, and interstitial lymphadenopathy, and CAEBV was diagnosed. After chemotherapy that included etoposide, the amount of EBV decreased transiently below the detection level. However, the disease due to CAEBV worsened despite the chemotherapy, and she finally needed chemotherapy every week. Therefore, instead of conventional myeloablative transplantation, we performed CB transplantation with reduced-intensity conditioning regimens consisting of low-dose total body irradiation, fludarabine, and etoposide. CB, for which human leukocyte antigen (HLA) was 2-loci mismatched on the DR loci from an unrelated donor, was infused after conditioning. Although grade III acute graft-versus-host disease (GVHD) in the gut and chronic GVHD in the lung developed, the symptoms of GVHD disappeared with immunosuppressive therapy. After 15 months, the patient remained a complete chimera, with undetectable levels of EBV in peripheral blood and bone marrow. We conclude that RIST from unrelated CB can be indicated for some cases of CAEBV who are refractory to chemotherapy and have no HLA-matched related and unrelated donors as the source of bone marrow or peripheral blood stem cells. PMID- 16679926 TI - An aggressive childhood tumor mimicking pleural empyema: pleuropulmonary blastoma. AB - We report a case of pleuropulmonary blastoma in a child initially diagnosed with encysted pleural empyema. She was treated with combination chemotherapy including ifosfamide, etoposide, vincristine, adriamycin, actinomycin-D, and cyclophosphamide after surgical excision. PMID- 16679927 TI - Comparative study of chronological, bone, and dental age in Fanconi's anemia. AB - Fanconi's anemia (FA) is a genetic autosomal recessive disorder, characterized by progressive bone marrow failure, congenital abnormalities, growth retardation, and predisposition to leukemia and solid tumors. The purpose of this study was to compare the chronological age (CA), bone age (BA), and dental age (DA) of FA patients and to determine whether the therapy received influenced the BA or DA. Thirty FA patients were evaluated and divided into three groups according to the therapy received. Hand and wrist radiographs for BA estimation and panoramic radiographs for DA estimation were taken. Statistical analysis demonstrated that mean values for CA, BA, and DA were 8.91 years old, 7.90 years old, and 7.75 years old, respectively, indicating low mean values for BA and DA in comparison with CA (P<0.01). The therapy given to the patient did not influence the BA or DA. BA and DA are delayed and are not influenced by the therapy given. PMID- 16679928 TI - N-of-1 trials: innovative methods to evaluate complementary and alternative medicines in pediatric cancer. AB - N-of-1 randomized controlled trials (RCTs) are randomized trials conducted within individuals and may be an attractive methodology for conducting studies of complementary and alternative medicine (CAM) in pediatric oncology. These trials may be used to determine the efficacy of an intervention in an individual, or multiple N-of-1 RCTs may be combined to estimate a population effect. There are many potential advantages to the use of N-of-1 RCTs with CAM in pediatric cancer. These advantages include the ability to determine whether CAM is effective in a specific child. In addition, the N-of-1 RCT allows parents and children to voice preferences about treatment options and allows them to directly participate in balancing adverse events and therapeutic benefits. Also, in estimation of population effects, combining multiple N-of-1 RCTs tends to require smaller sample sizes than do traditional parallel-group designs. However, there also may be several challenges to the conduct of such a trial. The use of N-of-1 RCTs may be very beneficial in evaluating CAM therapies in pediatric cancer. However, careful consideration of the advantages and disadvantages of such a design should be undertaken prior to initiating an N-of-1 RCT. PMID- 16679929 TI - Detection of chemotherapy-induced enterocyte toxicity with circulating intestinal fatty acid binding protein. PMID- 16679930 TI - The kallikrein-kinin system in sickle cell nephropathy: does it play a role? PMID- 16679931 TI - 5q- syndrome in a child with slowly progressive pancytopenia: a case report and review of the literature. AB - 5q- syndrome is a rare myelodysplastic process occurring predominately in middle aged to elderly women. In children, myelodysplasia of all types is rare and 5q- syndrome is exceptionally rare. Only 6 cases of 5q- associated myelodysplasia have been reported in children and all 6 cases had blast counts >5% and/or additional cytogenetic abnormalities. We report a case of 5q- syndrome in a girl who presented with macrocytosis and intermittent pancytopenia at age 5. Cytogenetic studies at age 8 revealed a large interstitial deletion of chromosome 5q without other cytogenetic abnormalities. The patient was clinically stable until age 11, when she became transfusion dependent and severely neutropenic. Subsequently, she underwent a successful unrelated cord blood transplant. To our knowledge, this is the first reported pediatric case meeting the strict criteria for 5q- syndrome. PMID- 16679932 TI - A giant thrombus in the right ventricle of a newborn with Down syndrome: successful treatment with rt-PA. AB - An association between thromboembolic events and Down syndrome (DS) has been increasingly reported in the literature. It is not clear whether patients with DS have increased risk for thrombotic disorders or there is a coincidence. We present a case of giant right ventricular thrombus in a neonate with DS. Risk factors for thrombosis in the present case were neonatal sepsis and dehydration. There was no congenital heart anomaly. The patient was treated successfully using recombinant tissue plasminogen activator. Recombinant tissue plasminogen activator seems to be a safe and effective thrombolytic therapy in neonates. PMID- 16679933 TI - Acute myelomonocytic leukemia in a boy with LEOPARD syndrome (PTPN11 gene mutation positive). AB - The LEOPARD syndrome is a complex of multisystemic congenital abnormalities characterized by lentiginosis, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormalities of genitalia, retardation of growth, and deafness (sensorineural). Mutations in PTPN11, a gene encoding the protein tyrosine phosphatase SHP-2 located on chromosome 12q24.1, have been identified in 88% of patients with LEOPARD syndrome. A missense mutation (836- >G; Tyr279Cys) in exon 7 of PTPN11 gene was identified in this patient and his mother with LEOPARD syndrome. This mutation is one of the two recurrent mutations most often associated with the syndrome. Leukemia has not previously been reported in patients with LEOPARD syndrome. The authors describe a 13-year-old boy diagnosed with both LEOPARD syndrome and acute myelomonocytic leukemia (AML M4). PMID- 16679934 TI - Feasibility and safety of a pilot randomized trial of infection rate: neutropenic diet versus standard food safety guidelines. AB - The neutropenic diet is an intervention that excludes certain foods, especially fresh fruits and vegetables, from the diets of pediatric oncology patients to reduce infection rate. The purpose of this study was to demonstrate a safe and feasible methodology to evaluate the infection rate in pediatric cancer patients randomized to the neutropenic diet or to Food and Drug Administration (FDA) approved food safety guidelines. Pediatric oncology patients receiving myelosuppressive chemotherapy were randomized to the neutropenic diet or to FDA food safety guidelines and followed through one chemotherapy cycle. The primary outcome was febrile neutropenia. Secondary outcomes were adherence and diet tolerability. Nineteen patients were enrolled. Four patients on each diet arm developed febrile neutropenia. The adherence rate was 94% for the neutropenic diet and 100% for the food safety guidelines. Although patients were able to tolerate both diets, there was more reported difficulty adhering to the neutropenic diet. Infection rates for children with cancer on the neutropenic diet were similar to those for patients following food safety guidelines. The results of this study suggest that a larger randomized trial to determine the effectiveness of food safety guidelines in minimizing the risk of food borne infection is safe and feasible in children with cancer. PMID- 16679935 TI - Flow cytometric DNA index, G-band karyotyping, and comparative genomic hybridization in detection of high hyperdiploidy in childhood acute lymphoblastic leukemia. AB - High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic hybridization (HR-CGH) in detecting high hyperdiploid leukemic clones. Twenty-six girls and 34 boys with acute lymphoblastic leukemia diagnosed in 1998 to 1999 were analyzed by FCM, GBK, and HR-CGH. The correlations between DNA indices obtained by FCM, GBK, and HR-CGH were significant (rs=0.61 to 0.77; P<0.001 for all comparisons). However, in 4 of 18 patients, high hyperdiploidy was overlooked by GBK or HR-CGH, and even when FCM was applied, 2 of 18 patients with high hyperdiploidy by GBK and/or HR-CGH were classified as nonhigh hyperdiploid. If high hyperdiploid subclones were included, FCM could detect all high hyperdiploid patients found by either GBK or HR-CGH, but would then in addition classify 15% to 20% of the remaining patients as high hyperdiploid. Thus, both GBK and HR-CGH overlook patients with high hyperdiploidy, and FCM only detects all high hyperdiploid patients if small high hyperdiploid clones are included. In addition, FCM detects patients with high hyperdiploid subclones, not detected by either GBK or HR-CGH, and the challenge remains to determine the prognosis of patients with such high hyperdiploid subclones. PMID- 16679936 TI - Prevalence and characteristics of complementary medicine used by pediatric cancer patients in a mixed western and middle-eastern population. AB - Complementary alternative medicine (CAM) usage and physicians' attitude was evaluated by interviewing all 100 pediatric cancer patients' parents and adolescents seen in the last half of 2003 at Meyer Children's Hospital in Israel. Sixty-one percent of the patients used CAM, an average of 3 different treatments per patient. CAM use was higher: among parents having previous CAM experience (85% vs. 51%, P=0.001) and higher education (79% vs. 50%, P=0.024), and in nonreligious families (71% vs. 50%). Jews and Arabs had similar CAM use rates as with different types of therapies. Socioeconomic status does not affect the rate of CAM use (P=0.9) but does affect the type of treatments used: patients of the higher socioeconomic level mainly used chemi-biological remedies and homeopathy (24% and 28% of interviewees; P=0.002). Middle or lower socioeconomic patients mostly used traditional Arab treatments (32% and 54%, respectively; P=0.012). Only 36% discussed it with their physicians, and most discussions were initiated by parents (79%). Beneficial effect was reported by 69% of CAM users. Most interviewees were interested in getting CAM information and availability in the hospital. Most pediatric cancer patients, regardless of their ethnic origin, use CAM without informing their physicians. Physicians should be aware and know more about it, to be advisors for better integrated care. PMID- 16679939 TI - Treatment strategy in the fracture of an implanted central venous catheter. AB - The breaking of a central venous catheter implanted for chemotherapy is a serious complication. Its removal provides a considerable challenge, especially in immunocompromised and thrombocytopenic patients. We present our experience in the percutaneous transcatheter retrieval of the broken parts of catheters in critically ill onocologic and hematologic patients. Our observation indicates that this transcatheter technique is effective and safe, and may prove to be a good alternative to the surgical retrieval of intravascular foreign bodies. We also cover a short review of the literature in this field. PMID- 16679937 TI - Renal kallikrein: a risk marker for nephropathy in children with sickle cell disease. AB - OBJECTIVE: Although improvements in the management of sickle cell disease (SCD) have increased patient survival into adulthood, morbidity and mortality from end organ damage remain major concerns. One of the most serious complications of SCD is renal failure, affecting about 20% of patients. The clinical manifestations of sickle cell nephropathy (SCN) involve changes in glomerular ultrastructure, albuminuria, and a progressive decline in glomerular hemodynamics. The mechanisms or factors that promote SCN are not fully elucidated. In the present study, the role of renal kallikrein as a risk marker for promoting SCN was explored in a cross-sectional study. METHODS AND RESULTS: We measured the urinary excretion rate of active kallikrein in 73 children with sickle cell anemia (hemoglobin SS, SC, or S thalassemia) and in 30 control healthy African American children. The findings demonstrated that a significant difference in the excretion rate of log kallikrein in male versus female patients with SCD, P<0.0078 was observed. In children with SCD, cross-sectional analysis revealed a positive and significant correlation between the excretion rate of active kallikrein and log albumin excretion rate (AER), P<0.0088. Regression analysis also determined that the excretion rate of active kallikrein negatively correlates with hemoglobin in children with SCD, P<0.0096. In addition, an inverse relationship between log AER and hemoglobin was observed in male patients with SCD, P<0.0143. In children with SCD, cross-sectional analysis revealed a positive and significant correlation between log AER and age, suggesting age as a risk marker for AER in SCD. In multivariate regression analysis, our findings demonstrate a strong association between log AER and age and log kallikrein in children with SCD. About 20% of the variability in log AER in SCD patients is influenced by age and 6% is influenced by log kallikrein, P<0.0001 and P<0.02, respectively. CONCLUSIONS: These findings provide the first evidence that the excretion rate of active kallikrein is positively and independently correlated with log AER in children with SCD, and suggest that kallikrein could be a marker for progressive nephropathy. Longitudinal studies are essential to address this issue. PMID- 16679938 TI - Respiratory virus infections in children with cancer or HIV infection. AB - Most studies focusing on respiratory infections in immunocompromised children have been addressed to bacterial etiology. However, respiratory virus infections in this population can also lead to severe disease. The objective of this study is to evaluate the clinical significance of respiratory virus infections in children with cancer or human immunodeficiency virus (HIV) infection. Retrospective study conducted in a teaching hospital in Madrid. Medical records from children 2,000 U x L(-1) and 51 had values > 10,000 U x L(-1), levels used to diagnose myopathy (e.g., statin myositis) and rhabdomyolysis, respectively. There were no significant increases in any measure of renal function. Despite marked CK and Mb elevations in some subjects, none experienced visible myoglobinuria or required treatment for impaired renal function. CONCLUSIONS: Exertional muscle damage produced by eccentric exercise in healthy individuals can cause profound CK and Mb elevations without renal impairment. PMID- 16679976 TI - How anorexic-like are the symptom and personality profiles of aesthetic athletes? AB - PURPOSE: We tested the hypothesis that aesthetic athletes (AA) have anorexic-like eating attitudes and behaviors, share personality characteristics such as perfectionism and obsessiveness, and are at high risk of eating disorders. METHODS: We compared symptomatology, personality variables typical of anorexia nervosa, and lifetime eating disorder prevalence across four groups of Israeli women: 31 anorexics, 111 AA (mostly dancers), 68 nonaesthetic athletes (NAA), and 248 controls. All participants completed self-report measures of symptomatology, harm avoidance, perfectionism, obsessiveness, self-esteem, and self-rated facial attractiveness and were screened for eating disorders. Those screening positively were interviewed and diagnosed using the structured clinical interview for DSM IV. RESULTS: Scores of the anorexic women differed from those of the three other groups in the expected direction on all variables. NAA scored similarly to controls, but had greater body satisfaction and less drive for thinness. Surprisingly, the AA did not differ from control women on any self-report measure. However, significantly more AA (11.7%) than NAA (5.8%) and controls (4.4%) had a lifetime diagnosis of eating disorder not otherwise specified (EDNOS). The eating attitudes and behavior of the 13 AA with a lifetime history of EDNOS fell intermediately between the anorexic women and the controls, whereas their personality profile resembled that of controls. CONCLUSIONS: Being a nonaesthetic female athlete in Israel appears to promote body esteem and offer some protection from a preoccupation with dieting. AA also appear to enjoy excellent psychological health; however, a subgroup has EDNOS and appears not to receive appropriate treatment for it. These results lend credibility to the existence of the diagnostic entity of anorexia athletica, proposed to be a subclinical, environmentally influenced eating disorder with a favorable prognosis. PMID- 16679977 TI - Strength measurement and clinical outcome after pulley ruptures in climbers. AB - PURPOSE: Ruptures of the finger flexor pulleys are the most frequent injuries in rock climbers. Whereas multiple pulley injuries demand a surgical reconstruction, single ruptures are mainly treated conservatively. Nevertheless, the question of the clinical outcome or a persisting finger strength deficit after conservative therapy arises. METHODS: Twenty-one rock climbers (age 34 +/- 9 yr) with a grade 2-4 pulley injury were reevaluated 3.46 (range: 0.25-18) yr after injury. The clinical evaluation followed a standard questionnaire in combination with an ultrasound examination in extension and forced flexion. In order to determine the finger strengths, the subjects hung with the respective finger in various postures on a ledge attached above a door frame, while standing on a force platform, which measured the relative release. RESULTS: The 21 subjects had old (3.46 yr, range: 0.25-18) pulley injuries in 27 fingers (10 A2, 1 A3, 11 A4, 3 A2/3, 2 A3/4). The clinical outcome was excellent (Buck-Gramcko score of 3) in all cases; the subjects regained their climbing level within a year. There was no difference between the initial ultrasound examination and the follow-up during the study. For 17 finger pairs, data for the relative strength of the injured and the respective healthy finger could be gathered. The finger strength was not significantly different for the injured and the healthy finger in either the hanging or the crimping finger position. CONCLUSIONS: Nonsurgical treatment of single pulley ruptures is recommended. The clinical outcome was good to excellent, and no long-term strength deficit for the injured finger could be observed. PMID- 16679978 TI - Anabolic androgenic steroids: a survey of 500 users. AB - PURPOSE: The use of anabolic androgenic steroids (AAS) to increase muscle size and strength is widespread. Information regarding self-administered AAS used nonmedically to enhance athletic performance or improve physical appearance is sparse and poorly documented. The purpose of this study is to identify current trends in the drug-taking habits of AAS users. METHODS: An anonymous self administered questionnaire was posted on the message boards of Internet Web sites popular among AAS users. RESULTS: Of the 500 AAS users who participated in the survey, 78.4% (392/500) were noncompetitive bodybuilders and nonathletes; 59.6% (298/500) of the respondents reported using at least 1000 mg of testosterone or its equivalent per week. The majority (99.2%) of AAS users (496/500) self administer injectable AAS formulations, and up to 13% (65/500) report unsafe injection practices such as reusing needles, sharing needles, and sharing multidose vials. In addition to using AAS, 25% of users admitted to the adjuvant use of growth hormone and insulin for anabolic effect, and 99.2% (496/500) of users reported subjective side effects from AAS use. CONCLUSIONS: This survey reveals several trends in the nonmedical use of AAS. Nearly four out of five AAS users are nonathletes who take these drugs for cosmetic reasons. AAS users in this sample are taking larger doses than previously recorded, with more than half of the respondents using a weekly AAS dose in excess of 1000 mg. The majority of steroid users self-administer AAS by intramuscular injection, and approximately 1 in 10 users report hazardous injection techniques. Polypharmacy is practiced by more than 95% of AAS users, with one in four users taking growth hormone and insulin. Nearly 100% of AAS users reported subjective side effects. PMID- 16679979 TI - Exercise and vasorelaxing effects of CO-releasing molecules in hypertensive rats. AB - PURPOSE: Because carbon monoxide (CO) has been reported able to induce relaxation, we aimed to investigate the effects of exercise training on the rat thoracic aorta responsiveness to a CO-releasing molecule (CORM), tricarbonyldichlororuthenium ([Ru(CO)3Cl2]2). METHODS: Male Wistar rats (N = 32) were divided in hypertensive and normotensive groups using the two-kidney, one clip model of Goldblatt or SHAM surgery. Hypertensive and normotensive groups were assigned to an exercise training protocol on a level treadmill over a 10-wk period or were assigned to remain sedentary. After the exercise training protocol, blood pressure and cardiac tissue weight were assessed. The responsiveness of endothelium-denuded thoracic aortic rings to [Ru(CO)3Cl2]2 was evaluated by isometric contractions recordings. RESULTS: Systolic, diastolic, and mean blood pressures were significantly increased in hypertensive rats compared with control rats. Exercise training did not significantly alter blood pressure but decreased pulse pressure in hypertensive animals compared with sedentary hypertensive rats. In all groups, application of [Ru(CO)3Cl2]2 induced relaxation in precontracted aortic rings. Compared with normotensive rats, CO-induced relaxation was significantly decreased in hypertensive rats. Nevertheless, training exercise increased relaxation markedly in response to [Ru(CO)3Cl2]2 application in hypertensive rats, whereas it remained without effect in control rings. Pretreatment with TEA, a nonselective K+ channel inhibitor, decreased [Ru(CO)3Cl2]2-induced relaxation in all groups that became similar. In trained hypertensive rats, iberiotoxin had effects similar to those of TEA. CONCLUSIONS: This finding supports the concept that the CORM [Ru(CO)3Cl2]2 can induce relaxation in both normotensive and hypertensive rats with an impairment of the CO-induced relaxation during hypertension. However, exercise training improves the aorta's ability to relax in response to [Ru(CO)3Cl2]2 during hypertension, probably by increasing K+ channel activity. PMID- 16679980 TI - L-citrulline reduces time to exhaustion and insulin response to a graded exercise test. AB - PURPOSE: Oral L-arginine supplementation has been shown to improve treadmill time to exhaustion and resting insulin sensitivity in individuals with peripheral vascular disease and type 2 diabetes, respectively. Furthermore, L-citrulline supplementation increases plasma L-arginine concentration to a level higher than that achieved by oral L-arginine supplementation. The purpose of this investigation was therefore to determine whether time to exhaustion during a graded treadmill test, as well as plasma insulin and glucose profiles, could be improved with oral L-citrulline supplementation in healthy individuals. METHODS: Seventeen young (18-34 yr), healthy male and female volunteers performed incremental treadmill tests to exhaustion following either placebo or citrulline ingestion (3 g 3 h before test, or 9 g over 24 h prior to testing). RESULTS: Steady-state submaximal respiratory exchange ratio and VO2max were not significantly different between placebo and citrulline trials. Treadmill time to exhaustion was lower following citrulline ingestion than during placebo trials (888.2 +/- 17.7 vs 895.4 +/- 17.9 s; P < 0.05; N = 17), which was accompanied by a higher rating of perceived exertion during exercise in the L-citrulline compared with the placebo condition. There was also an increase in plasma insulin in response to this high-intensity exercise in the placebo, but not in the L citrulline, condition (P < 0.05). CONCLUSIONS: It can be concluded that, contrary to the hypothesized improvement in treadmill time following L-citrulline ingestion, there is a reduction in treadmill time following L-citrulline ingestion over the 24 h prior to testing. The normal response of increased plasma insulin following high-intensity exercise is also not present in the L-citrulline condition, indicating that L-citrulline ingestion may reduce nitric oxide mediated pancreatic insulin secretion or increased insulin clearance. PMID- 16679981 TI - Milk ingestion stimulates net muscle protein synthesis following resistance exercise. AB - PURPOSE: Previous studies have examined the response of muscle protein to resistance exercise and nutrient ingestion. Net muscle protein synthesis results from the combination of resistance exercise and amino acid intake. No study has examined the response of muscle protein to ingestion of protein in the context of a food. This study was designed to determine the response of net muscle protein balance following resistance exercise to ingestion of nutrients as components of milk. METHOD: Three groups of volunteers ingested one of three milk drinks each: 237 g of fat-free milk (FM), 237 g of whole milk (WM), and 393 g of fat-free milk isocaloric with the WM (IM). Milk was ingested 1 h following a leg resistance exercise routine. Net muscle protein balance was determined by measuring amino acid balance across the leg. RESULTS: Arterial concentrations of representative amino acids increased in response to milk ingestion. Threonine balance and phenylalanine balance were both > 0 following milk ingestion. Net amino acid uptake for threonine was 2.8-fold greater (P < 0.05) for WM than for FM. Mean uptake of phenylalanine was 80 and 85% greater for WM and IM, respectively, than for FM, but not statistically different. Threonine uptake relative to ingested was significantly (P < 0.05) higher for WM (21 +/- 6%) than FM (11 +/- 5%), but not IM (12 +/- 3%). Mean phenylalanine uptake/ingested also was greatest for WM, but not significantly. CONCLUSIONS: Ingestion of milk following resistance exercise results in phenylalanine and threonine uptake, representative of net muscle protein synthesis. These results suggest that whole milk may have increased utilization of available amino acids for protein synthesis. PMID- 16679982 TI - Bicarbonate reduces serum prolactin increase induced by exercise to exhaustion. AB - PURPOSE: The aim of the study was to examine the effect of acid-base status on serum prolactin (PRL) concentration postexercise. METHODS: Seven male recreational athletes participated in two experimental trials separated by 1 wk. In the respective trial, subjects received either a placebo infusion (normal isotonic saline) or an alkali infusion (isotonic sodium bicarbonate) before and during exercise. Venous and capillary blood samples were drawn at rest, immediately after a 10-min warm-up period, and after a maximal ramp test on a cycle ergometer, as well as at 3, 6, 10, and 15 min postexercise. RESULTS: Power output, HR, capillary blood lactate concentration, carbon dioxide pressure (PCO2), and partial oxygen pressure (PO2) did not differ between trials at any point in time. Capillary PO2 did not change from resting values, but a significant increase (P < 0.05) was found from the end of warm-up to 3 min of the recovery period. Exercise induced a significant (P < 0.01) decrease in capillary blood bicarbonate concentration (HCO3-), pH, base excess (BE), and PCO2 at exhaustion and during the recovery period. Significantly higher HCO3-, pH, and BE were found during bicarbonate infusion and postexercise in comparison with the placebo trial. Serum PRL concentration was significantly increased 3 min postexercise until the end of the placebo trial, whereas after bicarbonate infusion, serum PRL concentration did not change from values at rest. Significant (P < 0.01) differences between trials in serum PRL concentration were found 10 and 15 min postexercise. CONCLUSIONS: The present study suggests that acidosis is a stimulus for exercise-induced PRL secretion. PMID- 16679983 TI - Left ventricular function immediately following prolonged exercise: A meta analysis. AB - PURPOSE: Evidence supporting cardiac fatigue following prolonged endurance exercise remains equivocal. The purpose of this meta-analysis was to quantify all data fulfilling the specified inclusion criteria, examining the short-term effect of prolonged endurance exercise on left ventricular function. METHODS: A random effects meta-analysis of the weighted mean change in ejection fraction (EF), systolic blood pressure/end systolic volume (SBP/ESV) ratio, and early-to-late diastolic filling (E/A) was conducted on 23 studies using the SE of the between subjects SD. HR, SBP, and left ventricular internal diameter during diastole (LVIDd) were also analyzed. Studies were coded according to exercise duration and training status: moderate duration trained (MDt) and untrained (MDu), 60-150 min; long duration (LD), 166-430 min; and ultra duration (UD), 640-1440 min. Relationships were assessed via Pearson's product-moment correlation. RESULTS: A significant (P < 0.05) overall decrease in EF (mean, confidence interval (CI): 1.95%, -1.03 to -2.88%), SBP/ESV (mean, CI: -0.8, -0.63 to -0.97), and E/A (mean, CI: -0.45, -0.39 to -0.51) was observed. Only UD and MDu subgroups demonstrated a reduction in EF. All subgroups demonstrated significant (P < 0.05) decreases in E/A. Alterations in LVIDd and SBP were related to respective decreases in EF and SBP/ESV, but not to E/A. CONCLUSION: The decrease in EF and SBP/ESV observed in UD and MDu indicates a reduction in systolic function, partially explained by altered cardiac loading. A decrease in E/A in all subgroups, unrelated to changes in loading, suggests an intrinsic impairment of left ventricular relaxation. Future investigators should employ load-independent indices of cardiac function and attempt to uncover the mechanisms of this phenomenon. PMID- 16679984 TI - ACE gene polymorphism and erythropoietin in endurance athletes at moderate altitude. AB - PURPOSE: To determine the role of the ACE (I/D) gene polymorphism on erythropoietic response in endurance athletes after natural exposure to moderate altitude. METHODS: Erythropoietic activity was measured in 63 male endurance athletes following natural exposure to moderate altitude (2200 m) during 48 h. Erythropoietin (EPO) levels and hemoglobin (Hb) concentrations were measured at baseline and 12, 24, and 48 h after reaching the set altitude. Reticulocyte counts were determined at baseline and 48 h thereafter. Subjects were grouped into two groups (responders and nonresponders) based on significant increase in EPO levels (median: > 16.5 ng x m(-1)) after 24 h at altitude. ACE gene polymorphism was ascertained by polymerase chain reaction (DD, 31 (49%); ID, 24 (38%); II, 8 (13%)). RESULTS: Overall, EPO levels significantly increased at 12 (70%; P = 0.0001) and 24 h (72%; P = 0.0001) above baseline concentration following exposure to 2200 m. Thereafter, EPO concentration decreased at 48 h, but a significant increase in Hb levels (4.6 +/- 4%; P = 0.0001) and reticulocyte count (50.5 +/- 79%; P = 0.0001) was observed at the end of the experiment, suggesting negative feedback. There were no significant differences in EPO and Hb concentration profiles between subjects with DD genotype and those with other genotypes (ID/II). Moreover, responders (N = 42; DD, 50%; ID/II, 50%) and nonresponders (N = 21; DD, 48%; ID/II, 52%) showed a similar erythropoietic profile during the experiment and the ACE gene polymorphism did not influence the time course of the erythropoietic response. CONCLUSIONS: The ACE gene polymorphism does not influence erythropoietic activity in endurance athletes after short-term exposure to moderate altitude. PMID- 16679985 TI - Bone density in elite judoists and effects of weight cycling on bone metabolic balance. AB - PURPOSE: Weight cycling has been shown to exert negative effects on bone metabolism and bone mass, whereas weight-bearing activity is positively associated with bone mineral density (BMD). Bone health in judoists and effects of weight cycling on bone metabolism have not previously been investigated. To examine potential disrupter and stimulators of bone integrity, this study analyzed bone parameters at baseline and the effects of the first weight cycle of the season on bone metabolic status in 48 male and female elite judoists. METHODS: Body composition and lumbar, femoral, and total body BMD were evaluated by dual-energy x-ray absorptiometry. Cortisol, osteocalcin, C-terminal telopeptide of type I collagen (CTx), and bone uncoupling index (UI) were measured in judoists at normal body weight, after weight reduction, and after regaining weight. As a comparison, a control group of moderately active students was included at baseline. Training, menstrual status, and calcium intake were assessed by questionnaires. RESULTS: EUweighted judoists displayed high BMD and an increased rate of bone formation. Precompetitive weight loss averaged 4 +/- 0.3% of body weight and induced an acute rise in cortisol (81%, P < 0.05) and CTx (33%, P < 0.0001), with a metabolic imbalance in favor of bone resorption. A 4 +/ 0.5% weight regain restored a positive UI in favor of bone formation. Metabolic responses were not dependent on gender. BMD was unaltered by weight cycling. CONCLUSIONS: Increased bone formation rate pertaining to judo athletes lent protection from alterations in bone metabolic balance associated with weight cycling. This observation suggests that powerful osteogenic stimuli provided by judo's unique biomechanical environment may help prevent bone loss associated with weight loss. PMID- 16679986 TI - Population-based reference standards for cardiovascular fitness among U.S. adults: NHANES 1999-2000 and 2001-2002. AB - PURPOSE: To provide reference standards for cardiovascular fitness levels, using population-based estimates of estimated maximal oxygen uptake (VO2max) from a diverse sample of U.S. adults. METHODS: Analysis of data from adults (20-49 yr) who completed a submaximal exercise test in the National Health and Nutrition Examination Survey 1999-2000 and 2001-2002. Estimated VO2max was categorized as low (<20th percentile), moderate (between 20th and 59th percentiles), or high (> or =60th percentile) fitness using age- and sex-specific cut-points. RESULTS: There were 1978 subjects, roughly 29% Mexican-American, 20% non-Hispanic black, and 51% non-Hispanic white. Using population-based sample weights, this was equivalent to 54,078,766 persons with mean (+/- SE) age, 34.4 +/- 0.3 yr; body mass index (BMI), 27.0 +/- 0.2 kg x m(-2); and estimated VO2max, 40.4 +/- 0.3 mL x min(-1) x kg(-1). There was a significant association between race and fitness level and between race and fitness level by sex (P < 0.0001). Low fitness was highest among non-Hispanic blacks (percentage (95% confidence interval): 32%, (26 38%)) compared with Mexican-Americans (22% (18-27%)) and non-Hispanic whites (18% (15-21%)), particularly among females, where 35% (28-42%) non-Hispanic black women had low fitness compared with 22% (16.3-27.4%) Mexican-American and 18% (13.7-22.4%) non-Hispanic white women. CONCLUSIONS: Non-Hispanic black adults have the highest percentage of low cardiovascular fitness among major race groups in the United States. On a population basis, over one third of non-Hispanic black women have low cardiovascular fitness, suggesting this group in particular may be at increased health risk due to poor fitness. PMID- 16679987 TI - Neighborhood environmental factors correlated with walking near home: Using SPACES. AB - PURPOSE: The physical environment plays an important role in influencing participation in physical activity, although the specific factors that are correlated with different patterns of walking remain to be determined. We examined correlations between physical environmental factors and self-reported walking for recreation and transport near home. METHODS: The local neighborhood environments (defined as a 400-m radius from the respondent's home) of 1678 adults were assessed for their suitability for walking. The environmental data were collected during 2000 using the Systematic Pedestrian and Cycling Environmental Scan (SPACES) instrument together with information from other sources. We used logistic regression modeling to examine the relationship between the attributes of the physical environment and the self-reported walking behavior undertaken near home. RESULTS: Functional features were correlated with both walking for recreation (odds ratio (OR) 1.62; 95% confidence interval (CI): 1.20 2.19) and for transport (OR 1.30; 95% CI: 0.97-1.73). A well-maintained walking surface was the main functional factor associated with walking for recreation (OR 2.04; 95% CI: 1.43-2.91) and for transport (OR 2.13; 95% CI: 1.53-2.96). Destination factors, such as shops and public transport, were significantly correlated with walking for transport (OR 1.80; 95% CI: 1.33-2.44), but not recreation. CONCLUSION: The findings suggest that neighborhoods with pedestrian facilities that are attractive and comfortable and where there are local destinations (such as shops and public transport) are associated with walking near home. PMID- 16679988 TI - Reliability of a measure of prediagnosis physical activity for cancer survivors. AB - PURPOSE: This study was conducted to examine the test-retest reliability of a measure of prediagnosis physical activity participation administered to colorectal cancer survivors recruited from a population-based state cancer registry. METHODS: A total of 112 participants completed two telephone interviews, 1 month apart, reporting usual weekly physical activity in the year before their cancer diagnosis. Intraclass correlation coefficients (ICC) and standard error of measurement (SEM) were used to describe the test-retest reliability of the measure across the sample; the Bland-Altman approach was used to describe reliability at the individual level. The test-retest reliability for categorized total physical activity (active, insufficiently active, sedentary) was assessed using the kappa statistic. RESULTS: When the complete sample was considered, the ICC ranged from 0.40 (95% CI: 0.24, 0.55) for vigorous gardening to 0.77 (95% CI: 0.68, 0.84) for moderate physical activity. The SEM, however, were large, indicating high measurement error. The Bland-Altman plots indicated that the reproducibility of data decreases as the amount of physical activity reported each week increases. The kappa coefficient for the categorized data was 0.62 (95% CI: 0.48, 0.76). CONCLUSION: Overall, the results indicated low levels of repeatability for this measure of historical physical activity. Categorizing participants as active, insufficiently active, or sedentary provides a higher level of test-retest reliability. PMID- 16679989 TI - Flexibility enhancement with vibration: Acute and long-term. AB - INTRODUCTION: The most popular method of stretching is static stretching. Vibration may provide a means of enhancing range of motion beyond that of static stretching alone. PURPOSE: This study sought to observe the effects of vibration on static stretching to determine whether vibration-aided static stretching could enhance range of motion acquisition more than static stretching alone in the forward split position. METHODS: Ten highly trained male volunteer gymnasts were randomly assigned to experimental (N = 5) and control (N = 5) groups. The test was a forward split with the rear knee flexed to prevent pelvic misalignment. Height of the anterior iliac spine of the pelvis was measured at the lowest split position. Athletes stretched forward and rearward legs to the point of discomfort for 10 s followed by 5 s of rest, repeated four times on each leg and split position (4 min total). The experimental group stretched with the device turned on; the control group stretched with the device turned off. A pretest was followed by an acute phase posttest, then a second posttest measurement was performed following 4 wk of treatment. Difference scores were analyzed. RESULTS: The acute phase showed dramatic increases in forward split flexibility for both legs (P < 0.05), whereas the long-term test showed a statistically significant increase in range of motion on the right rear leg split only (P < 0.05). Effect sizes indicated large effects in all cases. CONCLUSION: This study showed that vibration can be a promising means of increasing range of motion beyond that obtained with static stretching in highly trained male gymnasts. PMID- 16679990 TI - Dynamic pacing strategies during the cycle phase of an Ironman triathlon. AB - INTRODUCTION: A nonlinear dynamic systems model has previously been proposed to explain pacing strategies employed during exercise. PURPOSE: This study was conducted to examine the pacing strategies used under varying conditions during the cycle phase of an Ironman triathlon. METHODS: The bicycles of six well trained male triathletes were equipped with SRM power meters set to record power output, cadence, speed, and heart rate. The flat, three-lap, out-and-back cycle course, coupled with relatively consistent wind conditions (17-30 km x h(-1)), enabled comparisons to be made between three consecutive 60-km laps and relative wind direction (headwind vs tailwind). RESULTS: Participants finished the cycle phase (180 km) with consistently fast performance times (5 h, 11 +/- 2 min; top 10% of all finishers). Average power output (239 +/- 25 to 203 +/- 20 W), cadence (89 +/- 6 to 82 +/- 8 rpm), and speed (36.5 +/- 0.8 to 33.1 +/- 0.8 km x h(-1)) all significantly decreased with increasing number of laps (P < 0.05). These variables, however, were not significantly different between headwind and tailwind sections. The deviation (SD) in power output and cadence did not change with increasing number of laps; however, the deviations in torque (6.8 +/- 1.6 and 5.8 +/- 1.3 N x m) and speed (2.1 +/- 0.5 and 1.6 +/- 0.3 km x h(-1)) were significantly greater under headwind compared with tailwind conditions, respectively. The median power frequency tended to be lower in headwind (0.0480 +/- 0.0083) compared with tailwind (0.0531 +/- 0.0101) sections. CONCLUSION: These data show evidence that a nonlinear dynamic pacing strategy is used by well trained triathletes throughout various segments and conditions of the Ironman cycle phase. Moreover, an increased variation in torque and speed was found in the headwind versus the tailwind condition. PMID- 16679991 TI - Steadiness training with light loads in the knee extensors of elderly adults. AB - PURPOSE: This study was conducted to determine the effect of steadiness training with light loads in the knee extensors of elderly adults. METHODS: Twenty-one elderly adults (72 +/- 4.6 yr) performed 16 wk of closely supervised knee extensor training that consisted of lifting and lowering 30% of the one repetition maximum (1-RM) load as steadily as possible (10 reps per set, three sets per session, three sessions per week). Nine subjects served in a control group. Unilateral and bilateral maximal voluntary contraction (MVC) force; 1-RM load; force fluctuations during submaximal isometric, concentric, and eccentric contractions; timed functional performance (gait, chair rise, stair ascent and descent); muscle volume via magnetic resonance imaging (MRI); and the electromyogram (EMG) were measured. RESULTS: The training group exhibited modest average gains in MVC force and 1-RM load; muscle volume and EMG were unaltered. Although isometric steadiness was unchanged on average, training elicited the greatest improvements in the least steady subjects. Force fluctuations during concentric and eccentric contractions were significantly reduced. Of 21 subjects, 14 responded to training with gains in 1-RM load greater than the typical change (6%) in the control group. Before training, these responders exhibited greater force during bilateral compared with unilateral contractions. The small changes in physical functional performance were similar for the training and control groups. The training group could lift the pretraining 1-RM load 4.6 times after training (5.6 times for responders). CONCLUSIONS: Steadiness training with the knee extensors thus produced neural adaptations that increased strength in elderly adults who exhibited bilateral facilitation, improved isometric steadiness in unsteady subjects, improved steadiness during concentric and eccentric contractions, and enhanced the ability to lift heavy loads repeatedly. PMID- 16679992 TI - The effect of weighted vest walking on metabolic responses and ground reaction forces. AB - PURPOSE: This study examined how oxygen consumption, relative exercise intensity, vertical ground reaction forces (VGRF), and loading rate (LR) were affected while using a weighted vest (WV) during treadmill walking. METHODS: A sample of 10 (aged 23.4 +/- 1.7 yr) subjects performed a standardized walking test (4-min stages at 0.89, 1.12, 1.34, 1.56, and 1.79 m x s(-1)) on a treadmill under four weighted vest conditions (0, 10, 15, and 20% of body mass (BM)). Dependent variables included oxygen consumption (VO2), relative exercise intensity, first (F1) and second (F2) peaks of the VGRF curve, and LR. RESULTS: Repeated-measures ANOVA revealed significant vest versus speed interaction for V O2 and relative exercise intensity. Follow-up contrasts showed significant vest effect differences between 0% BM and all other WV conditions, and between 10 and 20% BM at all speeds for V O2. Follow-up analysis of relative exercise intensity demonstrated no significant vest effect difference at slower speeds, but significant differences at higher speeds. A significant main effect for WV condition was found for F1, F2, and LR. Follow-up testing revealed F1 and F2 at 0% BM were significantly different than at 10, 15, and 20% BM. F1 and F2 at 10% BM were significantly different than 20% BM, whereas F1 at 15% BM was significantly different than at 20% BM. LR at 0% BM was significantly different than at 15 and 20% BM. CONCLUSION: Using a weighted vest can increase the metabolic costs, relative exercise intensity, and loading of the skeletal system during walking. PMID- 16679993 TI - International Physical Activity Questionnaire: Validity against fitness. AB - PURPOSE: This study was conducted to criterion-validate the short format of International Physical Activity Questionnaire (IPAQ) against health-related fitness. METHODS: Participants included 951 men, aged 21-43 yr. VO2max by ergometer was used to measure cardiorespiratory fitness. Muscular fitness tests included the number of sit-ups, push-ups, and squats performed during 60 s. Physical activity was assessed from IPAQ by categories for health (health enhancing physical activity (HEPA)) and by calculating total and vigorous MET hours per week. The participants were classified in five groups of almost equal size by the IPAQ outcomes. Moreover, the frequency of vigorous activity was assessed by one precoded question (single-item question on leisure-time vigorous physical activity (SIVAQ)). RESULTS: The mean fitness improved from the first through the fourth IPAQ group. VO2max in the most active IPAQ group, however, was lower than in the fourth group (P < 0.05), both with HEPA categories and vigorous METs as the outcome. In contrast, the weekly frequency of vigorous physical activity showed a positive association with fitness through all six categories. The 65 sedentary (according to SIVAQ) and unfit individuals who belonged to the highest 20% (by IPAQ total METs) were older (30.6 +/- 5.1 vs 28.9 +/- 3.8, P = 0.02), more obese (waist 93 +/- 12 vs 89 +/- 10 cm), more often current smokers (59.4 vs 42.0%; P = 0.03), and less educated (<12 yr of education 70.8 vs 52.0%; P = 0.02), compared with the others in the highest IPAQ group. CONCLUSIONS: Almost 10% of young men had poor fitness and apparently low physical activity, but they reported very high physical activity by IPAQ. An evident need therefore exists to develop IPAQ further to solve the apparent overreporting by a considerable proportion of sedentary individuals. PMID- 16679994 TI - Individual calibration for estimating free-living walking speed using the MTI monitor. AB - PURPOSE: This study was conducted to devise a new individual calibration method to enhance MTI accelerometer estimation of free-living level walking speed. METHOD: Five female and five male middle-aged adults walked 400 m at 3.5, 4.5, and 5.5 km x h(-1), and 800 m at 6.5 km x h(-1) on an outdoor track, following a continuous protocol. Lap speed was controlled by a global positioning system (GPS) monitor. MTI counts-to-speed calibration equations were derived for each trial, for each subject for four such trials with each of four MTI, for each subject for the average MTI, and for the pooled data. Standard errors of the estimate (SEE) with and without individual calibration were compared. To assess accuracy of prediction of free-living walking speed, subjects also completed a self-paced, "brisk" 3-km walk wearing one of the four MTI, and differences between actual and predicted walking speed with and without individual calibration were examined. RESULTS: Correlations between MTI counts and walking speed were 0.90 without individual calibration, 0.98 with individual calibration for the average MTI, and 0.99 with individual calibration for a specific MTI. The SEE (mean +/- SD) was 0.58 +/- 0.30 km x h(-1) without individual calibration, 0.19 +/- 0.09 km x h(-1) with individual calibration for the average MTI monitor, and 0.16 +/- 0.08 km x h(-1) with individual calibration for a specific MTI monitor. The difference between actual and predicted walking speed on the "brisk" 3-km walk was 0.06 +/- 0.25 km x h(-1) using individual calibration and 0.28 +/- 0.63 km x h(-1) without individual calibration (for specific accelerometers). CONCLUSION: MTI accuracy in predicting walking speed without individual calibration might be sufficient for population-based studies but not for intervention trials. This individual calibration method will substantially increase precision of walking speed predicted from MTI counts. PMID- 16679995 TI - Pedometer-determined physical activity among multiethnic low-income housing residents. AB - PURPOSE: We sought to characterize pedometer-determined physical activity among a predominantly racial and ethnic minority sample of adults residing in low-income housing. METHODS: Data were collected from 433 participants at baseline in a randomized colon cancer prevention intervention trial conducted within low-income housing communities. Using random effects models to control for clustering within housing sites, we examined variation in daily steps by several sociodemographic characteristics. RESULTS: Participants recorded a mean of 5326 (+/- 3871 SD) daily steps over a 5-d sampling period. Significantly lower levels of pedometer determined physical activity were found among older-aged participants (P < 0.0001), women (P = 0.02), those who were overweight and obese (P = 0.03), those reporting no weekly exercise (P = 0.04), as well as among nonworking individuals (P < 0.0001). No significant differences were found by education or income. In multivariable analyses, age, gender, body mass index, and employment status remained significantly associated with steps. CONCLUSIONS: These findings suggest a high prevalence of physical inactivity among low-income housing residents. These data, derived from a well-characterized sample, provide useful estimates for the investigation of pedometers as measures of total accumulated physical activity among lower-income, racial and ethnic minority populations. PMID- 16679996 TI - Accelerometer-determined physical activity of free-living college students. AB - PURPOSE: This study was conducted to provide descriptive data of accelerometer determined ambulatory physical activity in a sample of free-living college students and to examine college students' ambulatory physical activity patterns. METHODS: All participants (245 females, age = 19.9 +/- 1.6 yr, body mass index (BMI) = 22.9 +/- 3.3 kg x m(-2); 209 males, age = 20.2 +/- 2.0 yr, BMI = 25.2 +/- 4.0 kg x m(-2)) wore an accelerometer for seven consecutive days. Accumulated physical activity (ct.d, ct x min(-1) x d(-1), and min x d(-1) spent at different intensities) and minutes per day spent in moderate or vigorous physical activity in sessions of at least 10 min were analyzed to describe the physical activity of the sample and examine physical activity patterns. RESULTS: The entire sample accumulated 362,750.1 +/- 112,824.1 ct x d(-1) (males = 383,787.2 +/- 112,001.3 vs females = 344,804.1 +/- 110,619.5 ct x d(-1), P < 0.01) and 46.7 +/- 18.9 min x d(-1) in moderate physical activity (males = 51.7 +/- 19.8 vs females = 42.5 +/ 17.0 min x d(-1), P < 0.01). They were more active on weekdays than weekend days (P < 0.05), and they spent 13.6 +/- 12.7 min x d(-1) (males = 13.2 +/- 12.0 vs females = 13.8 +/- 13.3, P > 0.05) in moderate or vigorous physical activity sessions of at least 10 min. CONCLUSION: Participants in this study accumulated a moderate amount of physical activity on most weekdays. Most participants, however, were not meeting the current moderate physical activity recommendation when moderate or vigorous physical activity sessions of at least 10 min were examined. PMID- 16679997 TI - Validation of Omni scale of perceived exertion during prolonged cycling. AB - PURPOSE: The present investigation was conducted to validate the OMNI ratings of perceived exertion (RPE) scale during prolonged cycle exercise when subjects were supplemented with either carbohydrate or placebo. METHODS: Fifteen trained cyclists cycled for 2.5 h at approximately 60% maximal watts on two occasions while receiving 4 mL x kg(-1) x 15 min(-1) carbohydrate (6%) (C) or placebo (P) beverages in a randomized, counterbalanced design. Concurrent and construct validity was established by evaluating perceptual responses with both the OMNI and Borg RPE scales every 20 min throughout the trials. RESULTS: The pattern of change in RPE over time was significantly different between C and P ingestion (P < 0.05), with an attenuated RPE response in C found at the hundredth minute for both the OMNI and Borg scales. The pattern of change in the respiratory exchange ratio and carbohydrate oxidation rates were significantly greater (P < 0.001) in the C than P condition. RPE-OMNI was positively and linearly related to the RPE BORG; r = 0.60-0.98 (P < 0.01) throughout exercise for both the C and P conditions. CONCLUSIONS: Concurrent and construct evidence supports the use of the OMNI-cycle scale by adult men to estimate RPE during prolonged cycle exercise under differing conditions of carbohydrate supplementation. PMID- 16679998 TI - Active commuting to school: Associations with environment and parental concerns. AB - PURPOSE: This study examined the association of objective and perceived neighborhood environmental characteristics and parent concerns with active commuting to school, investigated whether parental concerns varied by environmental characteristics, and compared the association of the perceived environment, parental concerns, and objective environment on the outcome active commuting to school. METHODS: Randomly selected parents of children (aged 5-18 yr), in neighborhoods chosen for their variability in objectively measured walkability and income, completed questionnaires about their neighborhood environment, concerns about children walking to school, and children's behavior (N = 259). Objective measures of the environment were available for each participant and each neighborhood. Logistic regression analyses were used to investigate the relationships among environment, parental concerns, and walking or biking to or from school at least once a week. RESULTS: A parental concerns scale was most strongly associated with child active commuting (odds ratio: 5.2, 95% CI: 2.71-9.96). In high-income neighborhoods, more children actively commuted in high-walkable (34%) than in low-walkable neighborhoods (23%) (odds ratio: 2.1, 95% CI: 1.12-3.97), but no differences were noted in low-income neighborhoods. Parent concerns and neighborhood aesthetics were independently associated with active commuting. Perceived access to local stores and biking or walking facilities accounted for some of the effect of walkability on active commuting. CONCLUSION: Both parent concerns and the built environment were associated with children's active commuting to school. To increase active commuting to school, interventions that include both environmental change and education campaigns may be needed. PMID- 16679999 TI - The female athlete triad myth. PMID- 16680001 TI - Accounting for resting energy expenditure is necessary when bout durations differ. PMID- 16680003 TI - Body checking in youth hockey is dangerous. PMID- 16680006 TI - Modulation of the phosphoinositide 3-kinase signaling pathway alters host response to sepsis, inflammation, and ischemia/reperfusion injury. AB - The phosphoinositide 3-kinases (PI3Ks) are a conserved family of signal transduction enzymes that are involved in regulating cellular activation, inflammatory responses, chemotaxis, and apoptosis. We have discovered that a carbohydrate ligand, glucan, will stimulate the endogenous PI3K/Akt signaling pathway. This article reviews the current data on the role of the PI3K/Akt signaling pathway as a negative feedback mechanism or compensatory regulator of septic and inflammatory responses. Of greater importance, the data reviewed in this article suggest that modulation of the PI3K/Akt signaling pathway can reduce the morbidity and mortality associated with septic and I/R injury. Thus, manipulation of the endogenous PI3K/Akt signaling pathway may represent a new and novel therapeutic approach to management of important diseases. PMID- 16680007 TI - Formation of focal adhesion-like structures in circulating human neutrophils after severe injury: triggering of a tissue-phase response in the vascular space. AB - Neutrophils play a key role in injury to the lung, kidney, liver, and gastrointestinal tract, often seen after major trauma. We evaluated the role of integrin-linked focal adhesions in the primed state, previously identified in peripheral blood neutrophils from severely injured patients. Immunoblot analysis of Triton-insoluble cell fractions revealed that total paxillin content was unchanged in comparison with that found in neutrophils from healthy volunteers, but phosphorylation of paxillin on tyrosine residue 118 was increased by more than 2-fold. Immunoprecipitation with antipaxillin and immunoblotting for proline rich tyrosine kinase 2 (Pyk2) and for fgr showed significantly more colocalization. Densitometric analysis of total phosphotyrosine profiles also demonstrated significantly more in patient cells as compared with healthy cells. When allowed to adhere to fibronectin-coated plates, healthy and patient cells demonstrate a significant increase in tyrosine phosphorylation from that found in suspension-phase cells. Differential interference contrast microscopy of healthy neutrophils adherent to fibronectin matrices demonstrated rounded cells, without evidence of spreading; spreading was induced by addition of TNF-alpha. Patient neutrophils spread spontaneously, a response not further enhanced by TNF-alpha. Confocal imaging using anti-Pyk2 demonstrated aggregation of Pyk2 into punctate structures in patient but not in healthy cells. We conclude that neutrophils from severely injured patients are in a primed state, characterized by formation of focal adhesion-like structures. The identification of such structures in a clinical disease setting where they likely participate in unwanted consequences provides a novel area for study of regulation of neutrophil function. PMID- 16680008 TI - Serum S-100B concentration provides additional information fot the indication of computed tomography in patients after minor head injury: a prospective multicenter study. AB - Ninety percent of patients with minor head injury (MHI) who have cranial computed tomography (CCT) under the present clinical decision rules have normal scans. Serum concentrations of the astroglial protein S-100B were recently found to provide useful information, but these studies were too small to provide a statistically safe basis for changing the present rule. We have investigated whether S-100B concentrations in patients with MHI can provide additional information to improve indication of the need for an initial CCT scan. One thousand three hundred nine patients with MHI were enrolled in this prospective, multicenter study. All had a CCT scan to confirm diagnosis in accordance with the present clinical decision rules. S-100B was measured in serum samples obtained upon admission. Data were analyzed using contingency table and receiver operating characteristic curve and compared with those for healthy donors (n = 540) and with those for patients with moderate to severe head injury (n = 55). Of the 1309 patients studied, 93 exhibited trauma-relevant intracerebral lesions on the CCT scan (CCT+). With a cutoff limit of 0.10-microg/L S-100B (95th percentile of values in healthy volunteers), CCT+ patients were identified with a sensitivity level of 99% (95% confidence interval, 96% - 100%) and a specificity level of 30% (95% confidence interval, 29% - 31%). Adding the measurement of S-100B concentration to the clinical decision rules for a CCT scan in patients with MHI could allow a 30% reduction in scans. A prospective study of the clinical value of S-100B measurement in such patients is now under way. PMID- 16680009 TI - Pretreatment with adult progenitor cells improves recovery and decreases native myocardial proinflammatory signaling after ischemia. AB - Cardiogenic shock from myocardial ischemia is the leading cause of death of both men and women. Although adult progenitor cells have emerged as a potential therapy for heart disease, reports indicate that transplanted adult progenitor cells may not differentiate into heart muscle. We hypothesized that pretreatment with adult progenitor cells may protect myocardium from acute ischemic damage. Treatment immediately before an ischemic event removes the possibility that differentiation to heart muscle may account for the observed effects. In the present study, we determined that adult progenitor cells from three different sources (human bone marrow, rat bone marrow, and human adipose tissue) immediately protect native myocardium against ischemia and decrease myocardial proinflammatory and proapoptotic signaling. Postischemic recovery of adult progenitor cell-pretreated hearts was significantly better than that of control hearts. This was correlated with a 50% decrease in proinflammatory cytokine production. The use of a differentiated cell control had no such effect. Therefore, adult progenitor cell pretreatment improved postischemic myocardial function, decreased myocardial production of inflammatory mediators, and limited proapoptotic signaling. These results represent the first demonstration that pretreatment with progenitor cells is myocardial protective. These findings may not only have mechanistic implications regarding the benefit of progenitor cells but may also have clinical therapeutic implications before planned ischemic events. PMID- 16680010 TI - Lymph from a primate baboon trauma hemorrhagic shock model activates human neutrophils. AB - We have reported that toxic factors in intestinal lymph are responsible for acute lung injury and bone marrow suppression and that they contribute to a systemic inflammatory state based on studies in rodent models of trauma-hemorrhagic shock. Rodent models may not completely reflect the responses of injured patients. Thus, it is important to confirm these findings in primates before applying them to injured human patients with trauma. Thus, we have recently established baboon trauma-hemorrhagic shock (T/HS) and trauma-sham shock (T/SS) models that showed that gut-derived factors carried in the lymph potentiates lung injury and causes human endothelial dysfunction and suppresses human bone marrow progenitor cell growth. Here, we further investigated the effects of these primate lymph samples on human neutrophils. We hypothesized that toxic factors in baboon lymph may prime and/or activate human polymorphonuclear leukocyte (PMN) leading to overproduction of superoxide, thereby contributing to the development of adult respiratory distress syndrome and multiple organ failure. To this effect, we have examined the priming effect of baboon T/HS and T/SS lymph on PMN respiratory burst and expression of adhesion molecule in human neutrophils. The results of these studies indicate that PMN treated with baboon T/HS lymph showed significantly induced respiratory burst responses compared with PMN treated with T/SS lymph or medium when phorbol myristate acetate PMA was applied after lymph pretreatment. Secondly, we found that the expression of CD11b adhesion molecule was increased by incubation with T/HS lymph. These results suggest that baboon lymph from T/HS models can increase respiratory burst and adhesion molecule expression in human PMN, thereby potentially contributing to PMN-mediated organ injury. PMID- 16680011 TI - Tumor necrosis factor receptor regulation of bone marrow cell apoptosis during endotoxin-induced systemic inflammation. AB - Although inflammation-induced release of cells from the bone marrow (BM) is well established, less is known regarding inflammation-induced modulation of bone marrow cell numbers by apoptosis. The purpose of this study is to assess apoptosis of BM immature and mature myeloid cells and peripheral granulocytes, and to elucidate the role(s) of TNFR-p55 and TNFR-p75 as modulators of apoptosis in these cellular compartments in a mouse model of endotoxin-induced systemic inflammation. Gene knockout (p55(-/-), p75(-/-), and p55(-/-)/p75(-/-)), or wild type (WT) mice were injected i.p. with saline (Sal) or LPS (4 microg/g) followed by collection of BM cells and peripheral blood after 24 h. Apoptosis was assessed by propidium iodide staining using two-color flow cytometry with differentiated granulocyte-specific Gr1-fluorescein isothiocyanate. Repeated-measures analysis of variance and Neuman-Keuls post hoc test were used for statistical analyses. After i.p. LPS, apoptosis was induced to the higher level in BM Gr1(-) cells than in BM Gr1(+) cells and was not induced in peripheral Gr1(+) cells. Depletion of cell numbers in both BM Gr1(-) and Gr1(+) subpopulations after LPS treatment was consistent with increase of the apoptotic cell percentages in the groups. LPS induced apoptosis was significantly lower in Gr1(-) cells from the -p55(-/-)/LPS and p55(-/-)/p75(-/-)/LPS mice but not from p75(-/-)/LPS mice as compared with WT/LPS mice, whereas there was no difference in apoptosis of BM Gr1(+) and peripheral Gr1(+) cells among WT groups and knockout groups. Thus, apoptosis of myeloid cells during endotoxemia is minimized because these cells undergo differentiation, which in turn may be because of the attenuation of the proapoptotic effect of TNFR-p55 shown herein to occur with myeloid differentiation. In contrast, TNFR-p75 seems to play a minimal role in apoptosis induction in Gr1(-) myeloid cells during endotoxemia. One explanation for a decrease in BM cell numbers during endotoxemia may be via induction of apoptosis in immature myeloid cells. PMID- 16680012 TI - Key inflammatory signaling pathways are regulated by the proteasome. AB - Lipopolysaccharide (LPS) is a major structural component of all Gram-negative organisms and has been implicated in Gram-negative sepsis and septic shock. In the present study, Affymetrix microarray analysis of RNA derived from murine macrophages treated with LPS in the absence or presence of the proteasome inhibitor lactacystin revealed that the vast majority of genes regulated by LPS is under control of the proteasome. Analysis of the data has revealed that the products of these genes participate in 14 distinct signaling pathways. This represents a novel approach to the identification of signaling pathways that are both toll-like receptor 4- and proteasome-dependent and may lead to the development of new drug targets in Gram-negative sepsis and septic shock. PMID- 16680013 TI - Glycogen synthase kinase-3beta inhibitors protect against the organ injury and dysfunction caused by hemorrhage and resuscitation. AB - Glycogen synthase kinase 3beta (GSK-3beta) is a serine/threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. Dysregulation of GSK-3beta has been implicated in the pathogenesis of several diseases including sepsis. Here we investigate the effects of 2 chemically distinct inhibitors of GSK-3beta, TDZD-8 and SB216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. Male Wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to 35 mmHg for 90 min) and subsequently resuscitated with shed blood for 4 h. Hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. Treatment of rats with either TDZD-8 (1 mg/kg, i.v.) or SB216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. In addition, TDZD-8, but not SB216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin 6 and also of the anti-inflammatory cytokine interleukin 10. Neither of the GSK-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. Thus, we propose that inhibition of GSK-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. PMID- 16680014 TI - Intravenous anesthesia inhibits leukocyte-endothelial interactions and expression of CD11b after hemorrhage. AB - Hemorrhage increases adhesion of leukocytes to the venular endothelium, mediated by increased expression of the Mac-1 integrin complex (CD18/CD11b) present on leukocytes. Anesthetic agents may possess anti-inflammatory properties. Hence, this study determined the effects of i.v. anesthesia on leukocyte adhesion after hemorrhage in relation to expression of CD11b. METHODS: Male Wistar rats were (n = 57) anesthetized i.v. with propofol (Diprivan) and fentanyl, ketamine, or thiopental. During anesthesia, 10% of total blood volume was removed and intravital microscopy used to observe the rat mesentery and measure leukocyte (neutrophils) rolling and adhesion in postcapillary venules (15 - 25 microm). Flow cytometry was also used to determine CD11b expression on neutrophils from blood removed at the end of these experiments (n = 25) or blood incubated with anesthetic agents and activated with platelet activating factor ex vivo (0.1 micromol/L) (n = 24). RESULTS: Hemorrhage increased leukocyte adhesion (stationary count per 150 microm) in rats anesthetized with thiopental (baseline, 3.4 +/- 1.2; hemorrhage, 6.7 +/- 2.0; P < 0.05) but not in those receiving either ketamine (baseline, 3.6 +/- 1.3; hemorrhage, 3.3 +/- 1.3) or propofol/fentanyl (baseline, 6.2 +/- 2.0; hemorrhage, 5.8 +/- 0.8). Neutrophils collected from thiopental-treated rats had elevated CD11b expression with thiopental (mean fluorescence baseline, 67.5 +/- 1.3; hemorrhage, 83.6 +/- 5.3; P < 0.05) but not with propofol/fentanyl (mean fluorescence baseline, 69.1 +/- 1.3; hemorrhage, 65.9 +/- 1.6), and ketamine-treated rats (mean fluorescence baseline, 74.3 +/- 2.1; hemorrhage, 74.8 +/- 1.1). Ketamine also inhibited upregulation of CD11b with platelet activating factor ex vivo. CONCLUSIONS: After hemorrhage, leukocyte adhesion and CD11b expression increased during thiopental anesthesia, but propofol/fentanyl and ketamine protected against hemorrhage-induced leukocyte adhesion. The anti-inflammatory effect of ketamine was mediated by direct inhibition of CD11b expression on leukocytes. PMID- 16680015 TI - Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock. AB - The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited IkappaBalpha liver loss, blunted the augmented nuclear factor-kappaB activity, decreased hepatic tumor necrosis factor (TNF)-alpha mRNA (VGX + SAO + Sham STIM = 1.0 +/- 1.9 TNF-alpha/glyceraldehyde-3 phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 +/- 0.2 TNF alpha/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-alpha (VGX + SAO + Sham STIM = 118 +/- 19 pg/mL; VGX + SAO + STIM = 39 +/- 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 +/- 1 U/g tissue; VGX + SAO + STIM = 3.1 +/- 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 +/- 1.0 U/g tissue; VGX + SAO + STIM = 3.2 +/- 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor kappaB and TNF-alpha in SAO shock. PMID- 16680017 TI - Thalidomide treatment reduces the alteration of paracellular barrier function in mice ileum during experimental colitis. AB - Small intestine permeability is frequently altered in inflammatory bowel diseases and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJs) and adherence. Thus, the aim of the present study was to examine the effects of thalidomide treatment on the permeability and structure of small intestine TJs in an animal model of experimental colitis induced by dinitrobenzene sulfonic acid (DNBS). Four days after colitis induction with DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin and zonula occludens 1. When compared with DNBS-treated mice, thalidomide-treated (200 mg/kg orally starting 30 min after the administration of DNBS) mice subjected to DNBS-induced colitis experienced a significantly reduced rate of the extent and severity of the histological signs of colon injury associated with a significant reduction of plasma and colon tumor necrosis factor alpha levels. After administration of DNBS to the mice induced a significant increase of ileal permeability was observed. Distal colitis in mice induced an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. In particular, we have observed that thalidomide treatment resulted in a significant reduction of the following: (1) the degree of colon injury, (2) the alteration of zonula occludens 1 and occludin localization (immunohistochemistry), and (3) intestinal permeability caused by DNBS in the colon. Taken together, our results clearly show that thalidomide treatment reduced small intestinal permeability in experimental colitis through the regulation of TJ protein. PMID- 16680016 TI - Effect of NOS inhibition on rat gastric matrix metalloproteinase production during endotoxemia. AB - Matrix metalloproteinases (MMPs) degrade the extracellular matrix and contribute to LPS-induced gastric injury. MMPs are closely modulated by their activators, membrane type-MMP (MT-MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). As LPS-induced gastric injury is mediated in part by iNOS, and NO modulates MMP production in vitro, we hypothesized that NOS inhibition would similarly modulate LPS-induced gastric MMP production. Therefore, the purpose of these studies was to compare the effects of selective and nonselective NOS inhibition on LPS-induced gastric MMP production. METHODS: Sprague-Dawley rats were given either the nonselective NOS inhibitor NG nitro-L-arginine methyl ester (L-NAME; 5 mg/kg, s.c.), a selective iNOS inhibitor, aminoguanidine (45 mg/kg, i.p.) or L-N-iminoethyl-lysine (L-NIL; 10 mg/kg, i.p.), or vehicle 15 min before saline or LPS (20 mg/kg, i.p.) and killed 24 h after LPS administration. Stomachs were assessed for macroscopic injury (computed planimetry), and gastric mucosal MMP production was assessed by gelatin zymography, in situ zymography, and Western analysis for MMP-2, MT1-MMP, and TIMP 2. (n > or = 4/group; ANOVA). RESULTS: Aminoguanidine treatment decreased LPS induced macroscopic gastric injury as well as MMP-2 and MT1-MMP protein production while having no effect on TIMP-2 protein levels. L-NIL similarly attenuated the induction of MMP-2 and MT1-MMP by LPS. L-NAME failed to attenuate LPS induced gastric injury or MT1-MMP protein induction and increased MMP-2 levels. L-NAME similarly had no effect on gastric TIMP-2 production. CONCLUSIONS: Selective iNOS inhibition decreases gastric MMP-2 activity after LPS administration, whereas nonselective inhibition increases MMP-2 levels. The ability of selective iNOS inhibition to ameliorate LPS-induced gastric injury may be due in part to its inhibition of active MMP-2 production, whereas nonselective NOS inhibitors increase MMP-2 levels and maintain gastric injury after LPS administration. PMID- 16680018 TI - The inhibition of inducible nitric oxide synthase in ovine sepsis model. AB - Excessive NO has been shown to play a major role in the pathogenesis of multiple organ dysfunctions in septic condition. Burn injury, especially if it is associated with smoke inhalation, is often complicated by subsequent development of pneumonia or sepsis that determine the outcome. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that closely mimics human sepsis and pneumonia. We hypothesized that the inhibition of iNOS-derived excessive NO might be beneficial in treating the cardiopulmonary derangement in this model. Female sheep (n = 18) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (< 40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa (5 x 10(11) colony forming units) into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of BBS-2 (100 microg/kg/h), an iNOS inhibitor, was started 1 h after insult. The administration of BBS-2 improved pulmonary gas exchange (PaO2/FiO2 and pulmonary shunt fraction) and partially reduced airway obstruction and an increase in ventilatory pressures. The lung water content was not affected by iNOS inhibition. The hypotension seen in nontreated animals was not ameliorated either. The increase in plasma concentration of nitrate and nitrite was inhibited by BBS-2. The results of present study show that iNOS may be partially involved in the pathogenesis of acute lung injury induced by smoke inhalation followed by bacterial instillation in the airway. PMID- 16680019 TI - Sesamol delays mortality and attenuates hepatic injury after cecal ligation and puncture in rats: role of oxidative stress. AB - Sesame oil potently protects rats against sepsis, and sesamol appears to be the protective ingredient in sesame oil. The aims of the present study were to examine the effects of sesamol on mortality and reactive oxygen species associated liver injury in Wistar rats with cecal-ligation-and-puncture-induced sepsis (septic rats). After sepsis was induced, sesamol was administered every 6 h. The survival rate was determined during the ensuing 48 h. Hepatic injury was assessed using blood biochemistry and histological examination. Hepatic oxidative stress was assessed by determining the levels of liver lipid peroxidation, hydroxyl radical, and superoxide anion generation, and nitric oxide production 12 h after cecal ligation and puncture. Inducible nitric oxide synthase expression was also determined. Sesamol delayed mortality and attenuated hepatic injury in septic rats. Hepatic lipid peroxidation, hydroxyl radical, and superoxide anion levels were significantly lower in sesamol-treated septic rats. Furthermore, sesamol inhibited the production of nitrite and the expression of inducible nitric oxide synthase in the liver in septic rats. Therefore, sesamol may delay mortality and attenuate oxidative stress-associated liver injury by inhibiting the production of nitric oxide, at least partially, in septic rats. PMID- 16680020 TI - Hydroxyethyl starch 130 kd/0.4 and albumin improve CVVH biocompatibility whereas gelatin and hydroxyethyl starch 200 kd/0.5 lead to adverse side effects of CVVH in anesthetized pigs. AB - Both fluid management and renal replacement therapies play a fundamental role in the treatment of critically ill patients. In a recent in vitro study, we have shown specific interactions of different colloids and the hemocompatibility of hemofilters. The present study was performed to compare the five most common fluids for volume resuscitation, i.e., normal saline (SAL), hydroxyethyl starch 130 kd/0.4 (HES130), hydroxyethyl starch 200 kd/0.5 (HES200), albumin (ALB), and gelatin (GEL) with respect to their interaction with continuous venovenous hemofiltration (CVVH) in anesthetized domestic pigs. METHODS: Animals (n = 63) were allocated randomly to the fluid type and the respective subgroups, which were divided into control and CVVH groups (n = 6 ndash; 7 per group). Bolus infusion of group specific fluid was followed by a bolus of heparin and the initiation of hemofiltration in CVVH groups. Thereafter, fluids were infused at constant rates, and heparin application was adjusted to keep the activated clotting time at 200 to 250 s. Hemodynamics, airway pressures, pulmonary gas exchange, diuresis, creatinine clearance, and blood cell counts were investigated during the entire procedure (10 ndash; 12 hours). RESULTS: Basics of in vivo effects of SAL, HES130, and ALB were not altered during CVVH. HES130 and ALB enabled stable hemocompatibility, diuresis, and hemodynamics in the respective groups. In contrast, organ functions were significantly different between control and CVVH groups when animals were treated with GEL or HES200. In particular, during CVVH, HES200 led to reduced platelet counts, deteriorated hemodynamics, and increasing airway pressures during CVVH. GEL led to increasing airway pressures, a decrease in pulmonary gas exchange, deteriorated hemodynamics, altered renal histomorphology, reduced platelet counts, and reduced hemoglobin. CONCLUSIONS: Direct in vivo effects of colloids in anaesthetized and ventilated pigs are not predictable for their effects during CVVH. Interaction between CVVH and every volume substitute occur in a highly specific manner. This observation could be helpful to explain contradictory study results and should be considered for future study designs. PMID- 16680021 TI - The response of neonatal rat ventricular myocytes to lipopolysaccharide-induced stress. AB - Sepsis induced by exposure to lipopolysaccharide (LPS) can be life-threatening and lead to multiple-organ dysfunction. Sepsis-associated cardiac dysfunction is a primary cause of mortality. The response of isolated cardiac myocytes to LPS exposure is poorly understood. Cultured neonatal rat ventricular cardiomyocytes were used to evaluate the response to LPS exposure. Other authors have reported that LPS exposure at doses sufficient to induce tumor necrosis factor alpha (TNF alpha) production and apoptosis in adult cardiomyocytes do not induce apoptosis in neonatal cardiomyocytes. We therefore hypothesized that neonatal cardiomyocytes have innate protective mechanisms that protect from septic damage. Cultured neonatal rat ventricular cardiomyocytes were stimulated by exposure to LPS for varying lengths of time. NFkappaB signaling pathways, TNF-alpha production, and Akt activation were monitored. We also assessed the induction of apoptosis in these cells by monitoring caspase-3 activity. LPS rapidly stimulates nuclear translocation of NFkappaB and Akt activation. TNF-alpha production is also stimulated. However, high doses of LPS are unable to induce apoptosis in these cells, and protection is not a function of Akt activation. LPS treatment also stimulated the levels of cyclooxygenase-2 and the production of downstream metabolites, specifically PGE2 and 15deoxyDelta12-14PGJ2 (15dPGJ2). Specific inhibition of cyclooxygenase-2 activity induced apoptosis in the presence of LPS, whereas direct exposure to 15dPGJ2 at pharmacological levels induced apoptosis. Neonatal rat ventricular cardiomyocytes have innate protective mechanisms that prevent apoptotic cell death after LPS exposure. Metabolic products of arachidonic acid metabolized by the cyclooxygenase pathway can be potentially apoptotic or antiapoptotic. The balance of these products within these cells may define the cellular response to LPS exposure. PMID- 16680024 TI - The use of chemical cross-linking and mass spectrometry to elucidate the tertiary conformation of lipid-bound apolipoprotein A-I. AB - PURPOSE OF REVIEW: The purpose of this review is to highlight recent advances in mass spectrometry and its use for identifying the lipid-bound conformation of apolipoprotein A-I. Given the current interest in understanding the structure of HDL apolipoprotein A-I, this approach seems ideal in assessing its dual role as mediator of lipid efflux and modulator of cellular inflammation. RECENT FINDINGS: A large number of different technical approaches have been employed over the past 25 years in attempts to solve the lipid-bound conformation of apolipoprotein A-I. Since the X-ray crystal structure of lipid-free Delta43 apolipoprotein A-I was reported in 1997, a 'double belt' model describing lipid-bound apolipoprotein A-I conformation for recombinant HDL has prevailed. Recent studies have focused on determining the exact helix-helix registry and salt-bridging partners found on a two apolipoprotein A-I molecule disc as well as on spherical HDL particles. Investigations are all aimed at defining the conformation of lipid-bound apolipoprotein A-I which may provide an explanation for how specific domains of apolipoprotein A-I interact with important HDL-modifying proteins that ultimately determine the apolipoprotein's fate in circulation. SUMMARY: Recent advances in mass spectrometric sequencing of cross-linked peptides provide an excellent tool to help define protein tertiary structure. This approach has provided refined structural information on apolipoprotein A-I folding which had eluded all previous approaches. PMID- 16680023 TI - The unique role of apolipoprotein A-I in HDL remodeling and metabolism. AB - PURPOSE OF REVIEW: To rationalize the distinctive biological behavior of apolipoprotein (apo)A-I and apoA-II in light of differences in their respective structures, properties, and physico-chemical behavior. RECENT FINDINGS: The distinctive metabolic behavior of apoA-I compared with that of apoA-II, which are revealed as differences in their interactions with the HDL receptor, scavenger receptor class B type I, can be understood in terms of their physico-chemical properties. Detergent and chaotropic perturbation of HDL unmasks properties that distinguish apoA-I from apoA-II and emulate the secondary effects of lecithin: cholesterol acyltransferase, cholesteryl ester transfer protein, and phospholipid transfer protein - the key protein factors in HDL remodeling, that is, formation of lipid-free apoA-I but not apoA-II and particle fusion. Thus, of the two major HDL apolipoproteins, apoA-I is the more plastic and labile and this difference gives apoA-I a unique physiological role that has been verified in mouse models of HDL metabolism. SUMMARY: The compositions, structures, and properties of HDL particles are important determinants of the mechanisms by which these antiatherogenic lipoproteins are metabolized. Although the plasma lipid transfer proteins and lipid-modifying enzymes are important determinants of HDL processing, the distinctive structures and properties of apoA-I and apoA-II, the two major HDL proteins, determine in different ways the thermodynamic stability of HDL - the former through its greater plasticity and the latter by its higher lipophilicity. These distinctions have been revealed by physico-chemical studies of HDL stability in the context of numerous studies of enzyme and lipid transfer activities and of the interaction of HDL with its hepatic scavenger receptor. PMID- 16680025 TI - The role of the Niemann-Pick C1-like 1 protein in the subcellular transport of multiple lipids and their homeostasis. AB - PURPOSE OF REVIEW: In the past 2 years the Niemann-Pick C1-like 1 protein has rapidly emerged as a key regulator of intestinal cholesterol absorption. This review covers the limited number of published reports to develop a hypothesis of the potential function of Niemann-Pick C1-like 1 protein and outlines questions that should be addressed in future studies. RECENT FINDINGS: Considerable disagreement regarding the potential function and subcellular location of Niemann Pick C1-like 1 protein has complicated interpretation of published results and evaluation of their biologic significance. Recent reports suggest, however, that Niemann-Pick C1-like 1 protein plays a key role in modulating the subcellular fate of multiple lipids including cholesterol and sphingolipids. In addition, this function may require Niemann-Pick C1-like 1 protein to move between different cellular locations. SUMMARY: This paper proposes a model of Niemann Pick C1-like 1 protein function based on available data and on knowledge of its close relative and homologue the Niemann-Pick C1 protein, whose precise function, albeit still elusive, is more extensively characterized. It also raises new questions with respect to Niemann-Pick C1-like 1 protein function and discusses potential future directions. PMID- 16680026 TI - ATP-binding cassette transporter G1 and lipid homeostasis. AB - PURPOSE OF REVIEW: This review briefly discusses the ATP-binding cassette transporter G (ABCG) family members and emphasizes recent studies that identify ABCG1 as a key regulator of cellular lipid homeostasis. RECENT FINDINGS: The in vivo importance of ABCG1 has recently been demonstrated with both loss-of function and gain-of-function studies in mice. Administration of a diet high in both fat and cholesterol to ABCG1 mice results in massive cholesterol accumulation in both the liver and lungs. In contrast, lipid accumulation is greatly attenuated in transgenic mice that express both the murine and human ABCG1 genes. Despite the observed tissue lipid accumulation, plasma lipid levels and lipoprotein cholesterol distribution are not significantly different between wild-type, ABCG1, and hABCG1 transgenic mice. Other studies show that ABCG1 expression is induced following activation of the nuclear receptor LXR and that over expression of ABCG1 results in increased efflux of cellular cholesterol to HDL or phospholipid vesicles. SUMMARY: The ABCG1 transporter plays a key role in regulating cellular cholesterol and lipid homeostasis. Elucidation of the molecular mechanism by which ABCG1 controls sterol flux should provide critical information that may link ABCG1 to the reverse cholesterol transport pathway or diseases such as atherosclerosis. PMID- 16680027 TI - Synthetic peptides: managing lipid disorders. AB - PURPOSE OF REVIEW: Recent publications related to the potential use of synthetic peptides for the management of lipid disorders and their vascular complications are reviewed. RECENT FINDINGS: The potential use of synthetic peptides for the management of lipid disorders and their vascular complications has emerged in recent years. These peptides are models of apolipoproteins, but are much smaller in size than the apolipoproteins. Oral peptides that improve the antiinflammatory properties of HDLs have been shown to potently inhibit atherosclerosis in mouse models. Injection of a peptide with a class A amphipathic helix in a rat model of diabetes dramatically reduced endothelial sloughing and improved vasoreactivity. Injected synthetic peptides have also been described that dramatically lower plasma cholesterol and restore endothelial function in a rabbit model of familial hypercholesterolemia. These studies suggest the therapeutic potential for synthetic peptides in the management of lipid disorders and their vascular complications. SUMMARY: Synthetic peptides much smaller than exchangeable human plasma apolipoproteins but with physical and chemical characteristics similar to the plasma apolipoproteins have shown promise in the management of lipid disorders and their vascular complications in animal models. The initial success of these animal studies suggests that synthetic peptides have the potential to emerge as a new therapeutic class of agents in the management of patients with lipid disorders. PMID- 16680028 TI - Diabetic dyslipidaemia. AB - PURPOSE OF REVIEW: Diabetic dyslipidaemia is a cluster of plasma lipid and lipoprotein abnormalities that are metabolically interrelated. The increase of large type 1 very low density lipoprotein particles in type 2 diabetes initiates a sequence of events that generates atherogenic remnants, small dense low-density lipoprotein and small dense high-density lipoprotein particles. Thus, it is of great importance to elucidate the mechanisms behind the overproduction of large very low density lipoprotein particles in diabetic dyslipidaemia. This review discusses the pathophysiology of very low density lipoprotein metabolism in type 2 diabetes and recent concepts of lipid management of diabetic dyslipidaemia. RECENT FINDINGS: Results indicate that triglyceride and apolipoprotein B production in types 1 and 2 very low density lipoprotein are significantly correlated, suggesting a coupling of the two processes governing the metabolism of these lipoprotein subpopulations. Insulin resistance, hyperglycaemia, and liver fat were associated with excess hepatic production of type 1 but not type 2 very low density lipoprotein particles. These data provide support for the independent regulation of types 1 and 2 very low density lipoprotein apolipoprotein B production. SUMMARY: Recent data suggest that the assembly of very low density lipoprotein is fundamentally altered in type 2 diabetes, explaining the overproduction of large type 1 very low density lipoprotein as well as the inability of insulin to suppress production of type 1 very low density lipoprotein in type 2 diabetes. Future discoveries hopefully will delineate the regulatory steps to allow more targeted treatment of diabetic dyslipidaemia. PMID- 16680029 TI - Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages. AB - PURPOSE OF REVIEW: The initial steps of reverse cholesterol transport involve export of cholesterol from peripheral cells to plasma lipoproteins for subsequent delivery to the liver. The review discusses recent developments in our understanding of how these steps occur, with particular emphasis on the macrophage, the major site of cellular cholesterol accumulation in atherosclerosis. RECENT FINDINGS: ATP binding cassette transporter (ABC) A1 exports cholesterol and phospholipid to lipid-free apolipoproteins, while ATP binding cassette transporter G1 and scavenger receptor BI export cholesterol to phospholipid-containing acceptors. ABCA1-dependent cholesterol export involves an initial interaction of apolipoprotein AI with lipid raft membrane domains, although ABCA1 and most exported cholesterol are not raft associated. ABCG1 exports cholesterol to HDL and other phospholipid-containing acceptors. These include particles generated during lipidation of apoAI by ABCA1, suggesting that the two transporters cooperate in cholesterol export. Scavenger receptor BI is atheroprotective, mediating clearance of HDL cholesterol by the liver. The relative contributions of scavenger receptor BI and ABCG to cholesterol export to HDL from macrophages is unclear and may depend on cellular cholesterol status and the cholesterol gradient between cell and acceptor. SUMMARY: The presence of distinct pathways for cholesterol efflux to lipid-free apolipoprotein AI and phospholipid-containing HDL species clarifies our understanding of reverse cholesterol transport, and provides new opportunities for its therapeutic manipulation. PMID- 16680030 TI - New insights into the biogenesis of human high-density lipoproteins. AB - PURPOSE OF REVIEW: The interest for the human HDL system was recently revived by the identification of the ABCA1 as a critical component in the formation and maintenance of plasma HDL levels. The present review focuses on recent progress in our understanding of the basic mechanisms underlying HDL biogenesis pathways. RECENT FINDINGS: Several novel mechanisms governing ABCA1/apoA-I interactions have recently been identified: apolipoprotein A-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway; the majority of ABCA1 exists as a tetramer in human living cell, supporting the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit for the formation of nascent HDL particles; apolipoprotein A-I has been shown to have a recycling retroendocytic pathway with uptake and resecretion of the lipidated nascent HDL particles by the cell, most likely through the ABCA1 transporter pathway; there is evidence that the speciation of nascent HDL into pre-beta and alpha-HDL is linked to specific cell lines, and occurs by both ABCA1-dependent and independent pathways. SUMMARY: The fundamental mechanisms underlying the biogenesis, speciation and maturation of HDL remain complex and not well understood. Understanding the mechanisms governing HDL genesis at the cellular level could provide novel insights into the human atheroprotective system in health and disease. PMID- 16680031 TI - New insights in the pathogenesis of non-alcoholic fatty liver disease. AB - PURPOSE OF REVIEW: The hallmark of non-alcoholic fatty liver disease is hepatic steatosis. This is mostly a benign condition, but for largely unknown reasons it progresses to liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma in about 10% of patients. In this review we discuss recent progress in the understanding of the etiology of non-alcoholic fatty liver disease. RECENT FINDINGS: In the last few years many connections between carbohydrate and triglyceride homeostasis, as well as inflammation, have surfaced. These seemingly unrelated metabolic pathways are linked by the action of diverse nuclear receptors. Many intermediates in lipid metabolism were shown to be activating ligands of these receptors, explaining the dysregulation of intermediary metabolism and induction of insulin resistance by a lipid overload. In addition to invoking a derangement in nuclear receptor regulation, excessive hepatic lipid influx may have direct metabolic consequences, particularly on mitochondrial function. SUMMARY: Non-alcoholic fatty liver disease is a multifactorial disease. Many aspects of the disease and the links to inflammation can be understood when the multiple functions of the regulating nuclear receptors are taken into account. Many of these nuclear receptors seem attractive targets to develop therapy for non-alcoholic fatty liver disease and the closely related metabolic syndrome. PMID- 16680032 TI - Cellular fatty acid uptake: the contribution of metabolism. AB - PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins. PMID- 16680033 TI - Ecto-F1Fo ATP synthase/F1 ATPase: metabolic and immunological functions. AB - PURPOSE OF REVIEW: Until recently, F1Fo ATP synthase expression was believed to be strictly confined to mitochondria where it generates most cellular ATP. This paper reviews the recent evidence for an extra-mitochondrial expression of its components by immunofluorescence, biochemistry and proteomics studies. It discusses its possible implications in an ecto-nucleotide metabolism and its pathophysiological role in normal and tumoral cells. RECENT FINDINGS: F1Fo ATP synthase components have been identified as cell-surface receptors for apparently unrelated ligands in the course of studies carried out on angiogenesis, lipoprotein metabolism, innate immunity, hypertension, or regulation of food intake. SUMMARY: F1Fo ATP synthase is expressed on endothelial cells where it binds angiostatin, regulates surface ATP levels, and modulates endothelial cell proliferation and differentiation. Through binding of apolipoprotein A-I, a similar complex, expressed on hepatocytes, regulates lipoprotein internalization. On tumors, it is recognized in association with apolipoprotein A-I by the antigen receptor of circulating cytotoxic lymphocytes of the gammadelta subtype and thus promotes an innate tumor cell recognition and lysis. It binds enterostatin on brain cells. Biochemistry and proteomics studies indicate an enrichment of F1Fo components in lipid rafts selectively with some other mitochondrial proteins, suggesting intracellular traffic connections between mitochondria and other membrane compartments. Finally, depending on cell type and environment, it can generate ATP or ADP which may transfer a downstream signal to purinergic receptors. PMID- 16680034 TI - Genetics of familial combined hyperlipidemia. AB - PURPOSE OF REVIEW: To provide an overview of recent advances that have defined the first putative genes behind familial combined hyperlipidemia, the most common genetic dyslipidemia and a major risk factor for early coronary heart disease. RECENT FINDINGS: The first locus for familial combined hyperlipidemia on 1q21-23 revealed a gene encoding a transcription factor critical in lipid and glucose metabolism, USF1. All the associated variants represent noncoding single nucleotide polymorphisms, one of which affects the binding site of nuclear proteins with a putative effect on transcript levels of USF1. Transcript analyses of fat biopsies have exposed risk-allele related changes in the downstream genes. Another recent clue to the molecular pathogenesis of familial combined hyperlipidemia is the association of the high triglyceride trait with the APOA5 gene, located on 11q. More familial combined hyperlipidemia genes are expected to be found, since linkage evidence exists for additional loci on 16q24 and 20q12 q13.1. SUMMARY: Genetic research of familial combined hyperlipidemia families has revealed several linked loci guiding to susceptibility genes. The USF1 transcription factor is the major gene underlying the 1q21-23 linkage. Modifying genes, especially influencing the high triglyceride trait, include APOC3 and APOA5, the latter representing a downstream target of USF1 and implying a USF1 dependent pathway in the molecular pathogenesis of dyslipidemias. PMID- 16680036 TI - Membrane and protein interactions of oxysterols. AB - PURPOSE OF REVIEW: Oxysterols, oxidation products of cholesterol, mediate numerous and diverse biological processes. The objective of this review is to explain some of the biochemical and cell biological properties of oxysterols based on their membrane biophysical properties and their interaction with integral and peripheral membrane proteins. RECENT FINDINGS: According to their biophysical properties, which can be distinct from those of cholesterol, oxysterols can promote or inhibit the formation of membrane microdomains or lipid rafts. Oxysterols that inhibit raft formation are cytotoxic. The stereo-specific binding of cholesterol to sterol-sensing domains in cholesterol homeostatic pathways is not duplicated by oxysterols, and some oxysterols are poor substrates for the pathways that detoxify cells of excess cholesterol. The cytotoxic roles of oxysterols are, at least partly, due to a direct physical effect on membranes involved in cholesterol-induced cell apoptosis and raft mediated cell signaling. Oxysterols regulate cellular functions by binding to oxysterol binding protein and oxysterol binding protein-related proteins. Oxysterol binding protein is a sterol-dependent scaffolding protein that regulates the extracellular signal regulated kinase signaling pathway. According to a recently solved structure for a yeast oxysterol binding protein-related protein, Osh4, some members of this large family of proteins are likely sterol transporters. SUMMARY: Given the association of some oxysterols with atherosclerosis, it is important to identify the mechanisms by which their association with cell membranes and intracellular proteins controls membrane structure and properties and intracellular signaling and metabolism. Studies on oxysterol binding protein and oxysterol binding protein-related proteins should lead to new understandings about sterol-regulated signal transduction and membrane trafficking pathways in cells. PMID- 16680035 TI - Apolipoprotein M--a novel player in high-density lipoprotein metabolism and atherosclerosis. AB - PURPOSE OF REVIEW: Apolipoprotein M is a recently described apolipoprotein predominantly associated with high-density lipoprotein, but also found in chylomicrons, very low-density lipoproteins, and low-density lipoprotein. The purpose is to review recent information on the unusual structural properties of apolipoprotein M and its possible role in formation of pre-beta high-density lipoprotein and reverse cholesterol metabolism. RECENT FINDINGS: Apolipoprotein M is a lipocalin having a coffee filter-like structure with a hydrophobic ligand binding pocket. Mature apolipoprotein M retains its signal peptide, which serves as a hydrophobic anchor. In mice, silencing of expression in the liver with siRNA led to disappearance of pre-beta high-density lipoprotein and appearance of unusually large high-density lipoproteins. This suggests that apolipoprotein M is important for the formation of pre-beta high-density lipoprotein and reverse cholesterol transport. In accordance with this idea, hepatic overexpression of apolipoprotein M with an adenovirus in low-density lipoprotein-receptor deficient mice led to an approximately 70% reduction of atherosclerosis. In addition to the liver, apolipoprotein M is also expressed in the kidney. Kidney-derived apolipoprotein M binds to megalin, a member of the low-density lipoprotein receptor family, which interacts with many lipocalins in renal tubuli. Apolipoprotein M is excreted in the urine of mice with a kidney-specific megalin deficiency but not in the urine of normal mice, suggesting megalin-mediated uptake of apolipoprotein M in the tubular epithelium of normal mice. SUMMARY: Apolipoprotein M is a novel apolipoprotein with unusual structural features that appears to play important roles in high-density lipoprotein metabolism and prevention of atherosclerosis. PMID- 16680037 TI - Plasma lipid transfer proteins. AB - PURPOSE OF REVIEW: Plasma cholesteryl ester transfer protein and phospholipid transfer protein are involved in lipoprotein metabolism. Conceivably, manipulation of either transfer protein could impact atherosclerosis and other lipid-driven diseases. RECENT FINDINGS: Cholesteryl ester transfer protein mediates direct HDL cholesteryl ester delivery to the liver cells; adipose tissue specific overexpression of cholesteryl ester transfer protein in mice reduces the plasma HDL cholesterol concentration and adipocyte size; cholesteryl ester transfer protein TaqIB polymorphism is associated with HDL cholesterol plasma levels and the risk of coronary heart disease. In apolipoprotein B transgenic mice, phospholipid transfer protein deficiency enhances reactive oxygen species dependent degradation of newly synthesized apolipoprotein B via a post endoplasmic reticulum process, as well as improving the antiinflammatory properties of HDL in mice. Activity of this transfer protein in cerebrospinal fluid of patients with Alzheimer's disease is profoundly decreased and exogenous phospholipid transfer protein induces apolipoprotein E secretion by primary human astrocytes in vitro. SUMMARY: Understanding the relationship between lipid transfer proteins and lipoprotein metabolism is expected to be an important frontier in the search for a therapy for atherosclerosis. PMID- 16680038 TI - Bibliography. Current world literature. Lipid metabolism. PMID- 16680039 TI - Nutrition and metabolism. PMID- 16680040 TI - Genetics and molecular biology: single nucleotide polymorphism associations and their functional significance. PMID- 16680041 TI - Lipid metabolism: pleiotropic effects of high-density lipoproteins. PMID- 16680042 TI - Hyperlipidaemia and cardiovascular disease. PMID- 16680043 TI - Atherosclerosis: cell biology and lipoproteins. PMID- 16680044 TI - Therapy and clinical trials. PMID- 16680046 TI - Chronic obstructive pulmonary disease resources. PMID- 16680047 TI - Managing breathlessness: providing comfort at the end of life. AB - Dyspnea is a common symptom at the end of life. It occurs as the result of a complex mix of physical, biochemical, and perceptual components. When patients and their healthcare providers focus on the "numbers" related to oxygenation, rather than comfort, the individual's quality of life can suffer. PMID- 16680049 TI - A day in the life of a home care liaison nurse. PMID- 16680050 TI - A new "superbug" leaves the hospital: community-acquired methicillin-resistant Staphylococcus aureus. PMID- 16680051 TI - It takes my breath away end-stage COPD. Part 2: pharmacologic and nonpharmacologic management of dyspnea and other symptoms. PMID- 16680053 TI - Suggestions for preventing musculoskeletal disorders in home healthcare workers. Part 2: lift and transfer assistance for non-weight-bearing home care patients. AB - Home healthcare (HHC) is one of the fastest-growing professions, currently employing more than 1 million workers in the United States. Unfortunately, these workers sustain an exceptionally high rate of musculoskeletal disorders. This is the second article in a two-part series providing information and suggestions for preventing overexertion that can lead to such disorders. PMID- 16680055 TI - Diagnosis: tuberculosis. AB - Home care staff frequently become anxious when a patient with active infectious tuberculosis is referred for home care. Using a home care patient case study, this article reviews tuberculosis epidemiology, pathophysiology, treatment, and infection control. It also discusses home health staff's role in case finding because home care patients with chronic disease and immunosuppression conditions are at risk for active tuberculosis disease. PMID- 16680057 TI - The home care team approach to self-neglecting elders. AB - Elderly patients who neglect their own health present special challenges. This article describes techniques for establishing rapport, understanding the patient's world, using a team approach to care, and achieving closure. At each step the clinician must find a balance between respecting the elder's rights and meeting his or her healthcare needs. PMID- 16680058 TI - Breath as a metaphor for life. PMID- 16680059 TI - VNAA CHAMP: nation's first geriatric practice improvement program enrolls HHAs from coast to coast. PMID- 16680060 TI - A nurse's perspective from the front row of a funeral. PMID- 16680061 TI - Things are not always the way they seem: lesson learned. PMID- 16680062 TI - Role of membrane potential in endothelium-dependent relaxation of isolated mouse main pulmonary artery. AB - The physiology of smooth muscle and endothelial cells of a particular vascular bed and from different species differs from each other. Acetylcholine causes an endothelium-dependent relaxation of preconstricted pulmonary arteries from the rat. This relaxation is mediated by nitric oxide (NO) plus a yet-unidentified endothelium-derived hyperpolarizing factor, which relaxes the smooth muscles by hyperpolarizing them. Our aim is to test whether these observations could be generalized to the smooth muscle cells from the mouse pulmonary artery. Smooth muscle or endothelial cell membrane potential of strips of murine pulmonary artery were measured simultaneously with the force developed by the strip. Acetylcholine hyperpolarized the endothelial cells. However, acetylcholine did not induce an endothelium-dependent hyperpolarization of the smooth muscle, while it relaxed the strip in an endothelium-dependent manner. This relaxation was abolished by an inhibitor of NO synthesis, nitro-L-arginine. Moreover, nitroglycerin relaxed the strips without changing the membrane potential of the smooth muscle cells. Injection of Lucifer yellow into the endothelial cells and the smooth muscle cells did not show heterocellular dye coupling. Furthermore, electron microscopy did not show gap junction plate at the myoendothelial junctions. We conclude that in the mouse main pulmonary artery, NO alone is responsible for the acetylcholine-induced endothelium-dependent vasodilatation, whereas the phenomenon called endothelium-derived hyperpolizing factor is not present. Therefore, caution should be taken when comparing different animal models to study pulmonary circulation and its reactivity. PMID- 16680063 TI - Effect of doxazosin on C-reactive protein plasma levels and on nitric oxide in patients with hypertension. AB - Inflammation has been hypothesized to play a role in the development of hypertension. The high-sensitivity C-reactive protein (hs-CRP) is a well-studied marker of systemic inflammation that has a predictive power with regard to the development of hypertension. This study was designed to test the hypothesis that hs-CRP plasma levels are altered in hypertension. Moreover, the study was to assess whether chronic antihypertensive treatment with doxazosin would normalize hs-CRP and nitrites/nitrates. We measured plasma levels of hs-CRP and nitrites/nitrates in 44 normotensive subjects and in 44 patients with hypertension before and after doxazosin therapy for 4 months. hs-CRP plasma levels were significantly higher (P < 0.007) in untreated hypertensive group compared to controls. Significant decrease was observed for hs-CRP (P < 0.05) in hypertensive patients after antihypertensive treatment. Nitrites/nitrates were significantly lower (P < 0.0001) in the untreated hypertensive group compared to controls. A significant increase was observed for nitrites/nitrates (P < 0.05) in hypertensive patients after antihypertensive treatment. These results suggest that doxazosin treatment exerts anti-inflammatory effects in addition to its antihypertensive properties in hypertensive patients. PMID- 16680064 TI - N-acetylcysteine prevents nitrosative stress-associated depression of blood pressure and heart rate in streptozotocin diabetic rats. AB - Previous studies have indicated that cardiovascular abnormalities such as depressed blood pressure and heart rate occur in streptozotocin (STZ) diabetic rats. Chronic diabetes, which is associated with increased expression of inducible nitric oxide synthase (iNOS) and oxidative stress, may produce peroxynitrite/nitrotyrosine and cause nitrosative stress. We hypothesized that nitrosative stress causes cardiovascular depression in STZ diabetic rats and therefore can be corrected by reducing its formation. Control and STZ diabetic rats were treated orally for 9 weeks with N-acetylcysteine (NAC), an antioxidant and inhibitor of iNOS. At termination, the mean arterial blood pressure (MABP) and heart rate (HR) were measured in conscious rats. Nitrotyrosine and endothelial nitric oxide synthase (eNOS) and iNOS expression were assessed in the heart and mesenteric arteries by immunohistochemistry and Western blot experiments. Untreated diabetic rats showed depressed MABP and HR that was prevented by treatment with NAC. In untreated diabetic rats, levels of 15-F(2t) isoprostane, an indicator of lipid peroxidation increased, whereas plasma nitric oxide and antioxidant concentrations decreased. Furthermore, decreased eNOS and increased iNOS expression were associated with elevated nitrosative stress in blood vessel and heart tissue of untreated diabetic rats. N-acetylcysteine treatment of diabetic rats not only restored the antioxidant capacity but also reduced the expression of iNOS and nitrotyrosine and normalized the expression of eNOS to that of control rats in heart and superior mesenteric arteries. The results suggest that nitrosative stress depress MABP and HR following diabetes. Further studies are required to elucidate the mechanisms involved in nitrosative stress mediated depression of blood pressure and heart rate. PMID- 16680065 TI - 3-HMG-Coenzyme A reductase inhibition and extracellular matrix gene expression in the pressure-overloaded rat heart. AB - The purpose of this study was to determine whether 3-HMG-Coenzyme A (HMG-CoA) reductase inhibition would attenuate the early pressure overload-induced activation of extracellular matrix genes in the left ventricle (LV) of the heart. Sprague-Dawley rats were randomized to 1 of 4 treatment groups: sham operation+vehicle (SH-V), aortic constriction+vehicle (AC-V), AC+rosuvastatin (RSV, 2 mg/kg; AC-LO), and AC+RSV (10 mg/kg; AC-HI). Rats were injected with normal NaCl (V) or RSV once daily, beginning 1 day before surgery, and killed 1 or 3 days after surgery. Hemodynamic measurements were made in the open-chest anesthetized state. LV levels of transforming growth factor beta1 (TGF-beta1), procollagen 1 (C1), and fibronectin (FN) mRNA were measured by Northern blotting. AC induced a approximately 25% increase in LV weight after 3 days that was not altered by RSV treatment. LV expression of TGF-beta1, C1, and FN mRNA was approximately 2-fold, approximately 2.5-fold, and approximately 5-fold greater, respectively, in hearts of AC-V compared to SH-V rats 3 days post-operation, and was not significantly decreased by either dose of RSV. Inhibition of HMG-CoA reductase does not attenuate the pronounced aortic constriction-induced increases in the early expression of TGF-beta1, C1, and FN in this model of acute pressure overload of the rat heart. PMID- 16680067 TI - Differential effect of hormone therapy and tibolone on lipids, lipoproteins, and the atherogenic index of plasma. AB - The aim of our study was to assess the effect of various regimens and doses of hormone therapy and tibolone on the Atherogenic Index of Plasma (AIP). A total of 519 postmenopausal women attending our menopause clinic were studied in a prospective design. Women with climacteric symptoms were randomly assigned to receive 1 of the following regimens: tibolone 2.5 mg, conjugated equine estrogens 0.625 mg plus medroxyprogesterone acetate 5 mg (CEE/MPA), 17beta-estradiol 2 mg plus norethisterone acetate 1 mg (E2/NETA), or 17beta-estradiol 1 mg plus norethisterone acetate 0.5 mg (low E2/NETA). Serum parameters were assessed at baseline and after 6 months and included total cholesterol, LDL-cholesterol, HDL cholesterol, triglycerides, apolipoprotein A1 and apolipoprotein B. The AIP was assessed as the log (triglycerides [mmol/L]/HDL-C [mmol/L]). CEE/MPA treatment associated with lower mean LDL-C but higher mean triglyceride levels (-15.5 mg/dL +/- 3.6, P = 0.0001; 12.6 mg/dL +/- 4.8, P = 0.01). Furthermore, CEE/MPA treatment resulted in higher AIP levels (0.073 +/- 0.021, P = 0.001). On the contrary, both E2/NETA regimens and tibolone associated with lower mean triglyceride and HDL-C levels (E2/NETA, triglycerides: -9.8 mg/dL +/- 5.0, P = 0.049; HDL-C: -4.9 mg/dL +/- 1.8, P = 0.01, low E2/NETA triglycerides: -12.5 mg/dL +/- 4.1, P = 0.003; HDL-C: -4.7 mg/dL +/- 1.3, P = 0.001; tibolone, triglycerides: -21.9 mg/dL +/- 2.7, P = 0.0001; HDL-C: -12.7 mg/dL +/- 1.1, P = 0.0001). None of the 3 regimens had any effect on AIP. The effect of a particular regimen of hormone therapy on the lipid-lipoprotein profile differs depending on the parameter assessed. The use of unified markers such as AIP will be helpful in evaluating the overall effect of lipid-lipoprotein modulation on the cardiovascular system. In fact, the concurrent assessment of the therapy effect on both LDL-C and AIP may be more dependable in evaluating the cardiovascular impact of a given regimen. PMID- 16680066 TI - Quercetin-supplemented diets lower blood pressure and attenuate cardiac hypertrophy in rats with aortic constriction. AB - Quercetin (Q), a flavonoid found in berries and onions, can reduce blood pressure in hypertensive animals and inhibit signal transduction pathways in vitro that regulate cardiac hypertrophy. We hypothesized that quercetin could prevent cardiovascular complications in rats with abdominal aortic constriction (AAC). Rats consumed standard or Q-supplemented chow (1.5 g Q/kg chow) for 7 days before AAC or sham surgery (SHAM, n = 15; AAC, n = 15; SHAMQ, n = 15; AACQ, n = 14). Fourteen days after surgery, plasma and liver Q concentrations were elevated (P < 0.05) and hepatic lipid oxidation was reduced (P < 0.05) in Q-treated versus untreated rats. Carotid arterial blood pressure and cardiac hypertrophy were attenuated (P < 0.05), and cardiac protein kinase C betaII translocation was normalized (P < 0.05) in AACQ versus AAC. Expression of cardiac beta-myosin heavy chain mRNA was also reduced in AACQ versus AAC (P < 0.05). However, extracellular regulated kinase 1/2 phosphorylation was similar in AAC versus AACQ. The level of aortic endothelial dysfunction (wire myography) was also similar between AAC and AACQ, in spite of reduced aortic thickening in AACQ. Importantly, Q-treated rats did not show any deleterious changes in myocardial function (echocardiography). Our data supports an antihypertensive and antihypertrophic effect of Q in vivo in the absence of changes concerning vascular and myocardial function. PMID- 16680068 TI - Effect of homocysteine lowering by 5-methyltetrahydrofolate on redox status in hyperhomocysteinemia. AB - The endothelial dysfunction induced by hyperhomocysteinemia can be reversed by 5 methyltetrahydrofolate (5-MTHF) via homocysteine (Hcy) lowering. An additive antioxidant action of 5-MTHF has been suggested to ameliorate endothelial dysfunction through increased nitric oxide production and superoxide radical scavenging, independent of Hcy lowering. The aim of the study was to assess whether 5-MTHF affects the redox state in hyperhomocysteinemia. We examined the effect of 3 months of oral 5-MTHF treatment (15 mg/day) on the redox pattern in 48 hyperhomocysteinemic subjects compared to 24 untreated hyperhomocysteinemic subjects. By analysis of variance with repeated measures in the 72 subjects, 5 MTHF markedly decreased plasma total Hcy (p-tHcy; P = 0.0001) and blood-total glutathione (GSH; b-tGSH; P = 0.002). By multivariate linear regression in the treated subjects, p-tHcy changes from baseline to 3 months (adjusted by baseline p-tHcy levels) correlated only with changes in reduced cysteinylglycine (P = 0.001). The effects of treatment on Hcy lowering and GSH metabolism were greater in medium than in moderate hyperhomocysteinemia. In conclusion, high-dose 5-MTHF treatment for 3 months ensures marked Hcy lowering to normal values even in subjects with high Hcy levels, and should be the treatment of choice in medium hyperhomocysteinemia. Furthermore, 5-MTHF shows a favorable interaction with GSH metabolism. PMID- 16680069 TI - Bradykinin does not contribute to peripheral vascular tone in patients with cirrhosis and ascites. AB - Bradykinin is an endothelium-dependent vasodilator and inflammatory mediator. The aim of the present study was to examine the effects of bradykinin on peripheral vascular tone in patients with cirrhosis and ascites. Forearm blood flow was measured using venous occlusion plethysmography in 8 patients with biopsy-proven alcohol-induced cirrhosis, ascites, and portal hypertension, and 8 age- and sex matched healthy controls. On 1 occasion, subjects received an intrabrachial infusion of the selective bradykinin antagonist B9340 (1.5-13.5 nmol/min) followed by a control vasoconstrictor norepinephrine (60-540 pmol/min), and on another occasion bradykinin (100-900 pmol/min) followed by the endothelium independent vasodilator, sodium nitroprusside (SNP, 2-8 microg/min). Bradykinin and SNP caused a dose-dependent vasodilatation (P < 0.001 for both) that did not differ between the 2 groups. Although norepinephrine caused a similar dose dependent vasoconstriction in both groups (P < 0.001), B9340 had no effect on forearm blood flow in either group (at 13.5 nmol/min in patients; -5%, 95% CI -13 3). Bradykinin does not provide a major contribution to the maintenance of peripheral vascular tone in patients with cirrhosis and ascites. Our findings also suggest that, in contrast to cardiovascular disease, patients with liver cirrhosis do not have marked endothelial dysfunction. PMID- 16680070 TI - Comparative study of the effects of lacidipine and enalapril on the left ventricular cardiomyocyte remodeling in spontaneously hypertensive rats. AB - Antihypertensive medications are the most efficient drugs in achieving regression of myocardial hypertrophy in both clinical studies and animal models of hypertension. Nevertheless, there is a lack of clear and concise comparative study of their effects on the modulation of cardiomyocyte morphology and function. Here, we assessed the tissue-protective actions of 2 of these drugs, the calcium channel blocker lacidipine (3 mg/kg/day) and the angiotensin converting enzyme-inhibitor enalapril (10 mg/kg/day) in vivo, after 8 weeks of treatment of 12-week-old spontaneously hypertensive rats, as well as in vitro, after short-term (4 min) application to isolated cardiomyocytes. Left ventricular hypertrophy (LVH) was compared at organ, tissue, and single-cell level. Our data showed that both drugs prevented the LVH of 20-week-old spontaneously hypertensive rats, but only lacidipine significantly decreased the cardiomyocyte size. Similarly, the single-cell contractility was significantly lowered in lacidipine-treated rats only. The effect of lacidipine was initiated shortly after exposure to the drug in a dose-dependent manner at 0.5 Hz, as well as at 2 Hz, with EC(50) of 10(-7) mol/L. These results can help in understanding the effects of these drugs on the prevention of LVH. PMID- 16680071 TI - Pentoxifylline improves reoxygenation-induced contractile recovery through a nitric oxide-dependent mechanism in rat papillary muscles. AB - In this study, the protective effect of pentoxifylline against hypoxia reoxygenation injury and the possible involvement of nitric oxide (NO)-mediated pathways in this protection were investigated in isolated rat papillary muscles. Papillary muscles were excised and isolated in Krebs-Henseleit solution aerated with 95% O2 and 5% CO2. Hypoxia was simulated by substituting O2 with argon. Three sets of experiments, testing 30, 60, and 90 min of hypoxia, were performed. The effects of different pentoxifylline concentrations on papillary muscle contractile parameters and responsiveness to isoproterenol were assessed. To investigate the role of NO, N(omega)-nitro-L-arginine methyl ester was added before pentoxifylline treatment. Pentoxifylline did not show any inotropic effect on papillary muscles. Hypoxia caused a profound depression of contractile parameters, which was not affected by pentoxifylline treatment. Reoxygenation resulted in significant partial recovery of contractile parameters after 30 and 60 but not 90 min of hypoxia. In experiments with 30 and 60 min of hypoxia, reoxygenation-induced contractile recovery and responsiveness to isoproterenol were improved by pentoxifylline in a concentration-dependent fashion. These functional improvements were completely blocked by N(omega)-nitro-L-arginine methyl ester pretreatment. No improvement was observed in 90-min hypoxia experiment. In conclusion, pentoxifylline improved contractile recovery during reoxygenation and postreoxygenation responsiveness to beta-adrenergic stimulation through the NO-dependent mechanism. PMID- 16680072 TI - Constitutively active Src tyrosine kinase changes gating of HCN4 channels through direct binding to the channel proteins. AB - Cardiac pacemaker current, if, is generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Our previous studies demonstrated that altered tyrosine phosphorylation can modulate the properties of both if and HCN channels. To assess a hypothesis that the intracellular tyrosine kinase Src may play a role in modulation by tyrosine phosphorylation of if, we cotransfected HEK293 cells with HCN4 and Src proteins. When HCN4 was cotransfected with a constitutively activated Src protein (Src529), the resultant voltage-dependent HCN4 activation was positively shifted (HCN4: V1/2 = -93 mV; Src529: V1/2 = -80 mV). The activation kinetics were accelerated at some potentials but not over the entire voltage range tested (eg, at -95 mV, tau_act(HCN4) = 3,243 ms; tau_act(Src529) = 1,113 ms). When HCN4 was cotransfected with a dominant negative Src protein (Src296), the HCN4 activation was shifted more negative to a smaller degree (HCN4: V1/2 = -93 mV; Src296: V1/2 = -98 mV; statistically insignificant) and the activation kinetics were slowed at most test potentials (eg, at -95 mV, tau_act(Src296) = 7,396 ms). Neither Src529 nor Src296 significantly altered HCN4 current density. Coimmunoprecipitation experiments revealed that Src forms a complex with HCN4 in HEK293 cells and in rat ventricular myocytes. Our data provide a novel mechanism of if regulation by Src tyrosine phosphorylation. PMID- 16680073 TI - Age-dependent acceleration of ischemic injury in endothelial nitric oxide synthase-deficient mice: potential role of impaired VEGF receptor 2 expression. AB - Morbidity and mortality of peripheral arterial occlusive disease significantly increases with age, often exhibiting more severe disease pathology and decreased treatment effectiveness. Therapeutic angiogenesis with angiogenic growth factors may represent a valuable treatment option for the severely ill, older adult patient population. Aging is considered an independent cardiovascular risk factor, but pathomechanistically it is not well understood. Diminished endothelial nitric oxide (EDNO) production has been considered as a major contributor to the aging process. To investigate the effect of age on postischemic revascularization independent of changes in EDNO, we used endothelial nitric oxide synthase-deficient (ecNOS-KO) mice. We found an age dependent acceleration in ischemic injury following unilateral femoral artery ligation in these animals compared to C57BL/J6 mice. Postischemic revascularization, quantified by measuring von Willebrand factor expression, was significantly impaired, suggesting that factors other than progressive EDNO deterioration are also involved in the age-dependent severe disease phenotype. Ischemia led to an increase in the expression of vascular endothelial growth factor receptor-2, KDR, in younger ecNOS-KO; however, this increase in KDR expression was absent in the older animals. Lack of increased KDR expression may provide a mechanistic explanation for the severe ischemic injury and perhaps can be used as a clinical marker to identify severe, vascular endothelial growth factor refractory patient population. PMID- 16680074 TI - U50,488H depresses pulmonary pressure in rats subjected to chronic hypoxia. AB - In this study, we determined the effect of U50,488H (a selective kappa-opioid receptor agonist) on pulmonary artery in rats and investigated its prevention and treatment effects on hypoxic pulmonary hypertension (HPH). Isolated pulmonary arterial rings were superfused and the tension of the vessel was measured. The model of HPH was developed and indexes for hemodynamics and right ventricular hypertrophy were measured. We found that U50,488H relaxed the pulmonary artery rings in a dose-dependent manner and the effect was abolished by nor binaltorphimine, a selective kappa-opioid receptor antagonist. Intravenous administration of U50,488H significantly lowered mean pulmonary artery pressure (mPAP) in normal rats and this effect was also abolished by nor-binaltorphimine. Hypoxia induced severe HPH in rats and intraperitoneal administration of U50,488H (every other day) during chronic hypoxia reduced mPAP and attenuated right ventricular hypertrophy compared with the control group. Moreover, acute intravenous administration of U50,488H after the rats subjected to chronic hypoxia for 4 weeks significantly lowered mPAP. Thus, U50,488H has significant vasorelaxant effect in rat pulmonary artery and has certain preventive and therapeutic application in HPH. PMID- 16680075 TI - Paullinia pinnata extracts rich in polyphenols promote vascular relaxation via endothelium-dependent mechanisms. AB - Paullinia pinnata L. (Sapindaceae) is an African tropical plant whose roots and leaves are used in traditional medicine for many purposes, especially for erectile dysfunction, but its action mechanism is unknown. P. pinnata root and leaf methanolic extracts are rich in phenolic compounds. This study shows that both extracts are highly antioxidative and induce a slight transcriptional activity of peroxisome proliferator activated receptor-alpha. They also increased and decreased endothelial nitric oxide synthase and endothelin-1 mRNA levels in bovine aortic endothelial cells, respectively. In this study P. pinnata methanolic extracts in cumulative doses elicited in a dose-dependent manner the relaxation of phenylephrine precontracted isolated rat aortic rings. N-nitro-L arginine methyl ester significantly attenuated the capacity of both extracts to induce arterial relaxation, indicating that this arterial relaxation was mediated by endothelial nitric oxide release. It could be suggested that the arterial relaxation induced by both extracts could be mainly linked to their capacities to inhibit nitric oxide oxidation through their antioxidant properties. PMID- 16680076 TI - Aldosterone impairs vascular endothelial cell function. AB - Aldosterone is a mineralocorticoid hormone that plays an important role in regulating electrolyte balance and blood pressure and also participates in endothelial dysfunction. We evaluated the direct effect of aldosterone on human umbilical vein cells (HUVEC). Levels of eNOS phosphorylation by vascular endothelial growth factor were diminished, and the amount of NO produced in response to vascular endothelial growth factor measured as NO2+NO3 was significantly decreased in cells previously incubated with aldosterone. Incubation with aldosterone for 24 h dose-dependently increased Nox4 mRNA expression in HUVEC. Although NF-kappaB was not apparently activated by aldosterone, mRNA levels of vascular cell adhesion molecule-1, E-selectin, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in HUVEC were significantly increased after incubation with aldosterone. Thus, aldosterone directly causes the dysregulation of endothelial cell function, which may be partly responsible for high blood pressure and atherosclerosis. PMID- 16680077 TI - Amelioration of ischemia/reperfusion-induced myocardial infarction by the 2 alkynyladenosine derivative 2-octynyladenosine (YT-146). AB - The present study was aimed at determining whether the novel adenosine A2-agonist YT-146 may have cardioprotective effects against ischemia-reperfusion injury. Anesthetized open-chest dogs underwent 90-min occlusion of the left anterior descending artery and subsequent 300-min reperfusion. The animals were randomly assigned to receive vehicle, 3, or 10 microg/kg YT-146 or ischemic preconditioning (4 episodes of 5 min occlusion followed by 5 min of reperfusion). Blood pressure, heart rate, and regional myocardial blood flow throughout the experiment were measured, as was the myocardial infarct size after reperfusion. The infarct size of the vehicle-treated dog was 56.2% +/- 2.7% (n = 5), whereas that of 3 or 10 microg/kg YT-146-treated dog was smaller (ie, 29.5% +/- 8.7% or 20.2% +/- 7.0%, respectively; n = 5). The infarct size of the dog treated with 10 microg/kg YT-146 was reduced to a degree similar to that of the ischemic preconditioning (19.2% +/- 6.3%, n = 5). YT-146 at both doses elicited a dose dependent increase in acute hyperemic coronary flow immediately after reperfusion. The cardioprotective effect may be attributed to the limitation of the infarct size, probably via A2-receptor-mediated coronary artery dilatation during the early period of reperfusion. PMID- 16680079 TI - What do you know about genetics? PMID- 16680080 TI - Negotiation strategies for ACNP practice. PMID- 16680078 TI - Differential effects of chronic treatment with estrogen receptor ligands on regulation of nitric oxide synthase in porcine aortic endothelial cells. AB - In cultured endothelial cells, estrogen increases expression and activity of endothelial nitric oxide synthase (eNOS). This study was designed to determine whether estrogenic treatments increase eNOS similarly in vivo. Aortic endothelial cells were collected from adult ovariectomized pigs which were untreated (8wk OVX) or treated with oral 17beta-estradiol (E2, 2 mg/day), conjugated equine estrogen (CEE, 0.625 mg/day), or raloxifene (60 mg/day) for 4 weeks. Plasma NOx, estrogen receptors (ERalpha and ERbeta), eNOS, eNOS regulatory proteins, and eNOS mRNA in endothelial cells were determined by Griess reaction, Western blot, and real-time polymerase chain reaction, respectively. Ovariectomy decreased, whereas all treatments restored plasma NO(x) to pre-OVX levels. On the contrary, eNOS protein and mRNA increased with ovariectomy; E2 and CEE but not raloxifene reduced mRNA; eNOS protein was reduced by CEE and raloxifene treatments. Tyrosine phosphorylation of eNOS and expression of calmodulin increased, but Hsp90 decreased with all treatments and only raloxifene treatment increased caveolin-1 compared with OVX. Expression of ERalpha/ERbeta increased with ovariectomy and was reversed by treatments such that raloxifene>CEE>E2. Three clinically relevant estrogen treatments restore plasma NO after ovariectomy, but do not affect eNOS mRNA, posttranslational regulation of eNOS or expression of estrogen receptors in the same way. PMID- 16680081 TI - Recognition and management of major depression. PMID- 16680083 TI - Improving outcomes for restless legs syndrome. PMID- 16680085 TI - 2006 Nurse Practitioner salary & practice survey. PMID- 16680086 TI - Anxiety attacks following surgical menopause. PMID- 16680087 TI - Glumetza: once-a-day dosing for management of type 2 diabetes. PMID- 16680092 TI - Two newly identified sites in the ubiquitin-like protein Atg8 are essential for autophagy. AB - Atg8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery in yeast. This protein undergoes reversible conjugation to phosphatidylethanolamine through a multistep process in which cleavage of Atg8 by a specific protease is followed by ubiquitin-like conjugation processes. Here, we identify two essential sites in Atg8, one of them involving residues Phe 77 and Phe 79 and the other, located on the opposite surface of Atg8, residues Tyr 49 and Leu 50. We show that these two sites are associated with different functions of Atg8: Phe 77 and Phe 79 seem to be part of the recognition site for Atg4, a cystein protease that acts also as a deubiquitination enzyme, whereas Tyr 49 and Leu 50 act downstream of the lipidation step. These two newly identified distinct sites that are essential for Atg8 activity provide an explanation for the many protein-protein interactions of this low-molecular-weight protein. PMID- 16680095 TI - On eye analyses. PMID- 16680093 TI - Tumour necrosis factor receptor 1 mediates endoplasmic reticulum stress-induced activation of the MAP kinase JNK. AB - Tumour necrosis factor receptor (TNFR)1 is the main receptor responsible for TNF induced diverse cellular events. In this study, we report that TNFR1 has a crucial role in endoplasmic reticulum (ER) stress-induced Jun amino-terminal kinase (JNK) activation. Although ER stress leads to JNK activation in wild-type mouse embryo fibroblasts, we failed to detect any JNK activation in TNFR1-/- cells. ER stress-induced JNK activation is restored in TNFR1-/- cells when TNFR1 expression is reconstituted. We also found that TNFR1 functions downstream of IRE1 and that IRE1 is present in the same complex with TNFR1 under ER stress condition. Therefore, our study shows a novel role of TNFR1 in mediating ER stress-induced JNK activation. PMID- 16680097 TI - Angle-closure glaucoma in East Asian and European people. Different diseases? PMID- 16680099 TI - Cup-to-disc ratio: agreement between slit-lamp indirect ophthalmoscopic estimation and stratus optical coherence tomography measurement. AB - PURPOSE: To determine agreement between slit-lamp indirect ophthalmoscopy and Stratus optical coherence tomography (OCT) when assessing cup-to-disc ratios (CDRs). METHODS: Twenty-five ocular hypertensive subjects and 56 patients with primary open-angle glaucoma were included. Estimation of vertical (VCDR) and horizontal (HCDR) cup-to-disc ratio with slit-lamp ophthalmoscopy was made by three glaucoma specialists along with OCT scanning of optic nerve head. Agreement between OCT and specialists was measured by intraclass correlation coefficients (ICC), Bland and Altman's scatterplots, and a regression coefficient of the average difference. RESULTS: The mean VCDR and HCDR was significantly higher (P<0.001) with OCT than that estimated by the specialists, with the difference ranging from 0.08 to 0.11, and from 0.13 to 0.18, respectively, depending on the specialist. Difference was higher (P<0.001) for cuppings below 0.3, and looses significance for larger VCDR cuppings (above 0.7). ICC for VCDR was 0.87 among specialists, and ranges from 0.82 to 0.75 when comparing OCT and specialists. ICC for HCDR was 0.83 among specialists and 0.74 between OCT and specialists. When data were plotted according to the Bland-Altman method, as the cupping increased, the agreement also increased. CONCLUSIONS: There is very good agreement among the specialists when estimating CDRs by stereoscopic slit-lamp biomicroscopy. OCT shows higher values than the specialists; the greatest differences occurred when assessing small CDRs and the differences diminished as the cupping increased. These two methods of measurement are not interchangeable, and the difference must be considered, especially in discs with smaller CDRs. PMID- 16680100 TI - Functional loss in early age-related maculopathy: the ischaemia postreceptoral hypothesis. AB - We review proposed models and psychophysical and electrophysiological tests performed in many studies for early age-related maculopathy (ARM). We suggest that ischaemia is the trigger for impaired retinal pigment epithelium function causing imbalance of secretion of vascular growth factors, reduced disc degradation capability and reduced metabolic activity and possible inflammatory response. This results in increased deposition of cell debris, such as drusen and thickens Bruch's membrane causing even more ischaemia of the overlying neurosensory retina. The photoreceptors are more resistant to ischaemia given their proximity to the choroid. Furthermore, being 'upstream' from the inner retinal layers, they act as an oxygen sink depriving retinal layers further from the choroid. Postreceptoral cell layers and especially parts of the inner nuclear layer that are located in the watershed zone between two sources of blood supply are preferentially vulnerable to ischaemia. Based on psychophysical and electrophysiological findings we propose that most of the function impairment in early ARM starts postreceptorally. PMID- 16680101 TI - Iris damage and acute pigment dispersion following photo-epilation. PMID- 16680102 TI - Bilateral ptosis and gaze palsies following radioactive seed treatment of tectal plate tumours. PMID- 16680103 TI - Tacrolimus ointment vs steroid ointment for eyelid dermatitis in patients with atopic keratoconjunctivitis. AB - AIMS: The main objective of this explorative study was to evaluate if tacrolimus ointment could be safer than corticosteroid ointment, with special reference to the intraocular pressure in the treatment of eyelid eczema in patients with atopic keratoconjunctivitis (AKC). Secondary aims were to compare the effects of the treatments on eyelid eczema and their potential impact on ocular surface inflammation. METHODS: Tacrolimus 0.1% ointment and clobetasone butyrate 0.05% ointment were compared in a double-masked explorative crossover study. In total, 25 AKC patients were included. Each ointment was applied twice daily for 3 weeks, with 2 weeks of washout before, between, and after treatments. Efficacy was determined by eye examination and the patients' own symptom scoring. Cytology and cytokine measurements were performed on tear samples. Safety parameters were intraocular pressure, presence of bacteria and fungi, and the patients' reports of adverse events. The validity of the crossover design was explored with analysis of variance, and the effect of each medication was calculated with paired t-test and Wilcoxon paired test. RESULTS: A total of 20 patients completed the study. Both treatments were effective in reducing signs and symptoms of eyelid eczema, with a near superior benefit for tacrolimus in terms of eczema (total skin score) signs (P=0.05). No serious adverse events occurred and interestingly, intraocular pressure was not evidently affected by either treatment. CONCLUSION: Tacrolimus 0.1% ointment is a promising alternative therapy for eyelid eczema in AKC patients. Long-term studies are needed to further determine the value of tacrolimus in this patient group. PMID- 16680104 TI - Epipapillary isolated astrocytoma associated with branch retinal artery occlusion in a single eye. PMID- 16680105 TI - Rapid regression of disc neovascularization in a patient with proliferative diabetic retinopathy following adjunctive intravitreal bevacizumab. PMID- 16680106 TI - Comparison of ultrasound biomicroscopic parameters after laser iridotomy in eyes with primary angle closure and primary angle closure glaucoma. AB - PURPOSE: To study changes in anterior segment morphology after laser peripheral iridotomy (LPI) in primary angle closure (PAC) and primary angle closure glaucoma (PACG) using ultrasound biomicroscopy (UBM). METHODS: Ninety-three eyes of 93 patients underwent anterior segment evaluation including gonioscopy, disc evaluation with + 90D lens, applanation intraocular pressure, and standard achromatic perimetry. UBM was performed before and 2 weeks after Nd:YAG LPI to measure the trabecular-iris angle (TIA), the angle-opening distance (AOD 250/500), and the central anterior chamber depth (ACD). RESULTS: The superior TIA widened from a mean of 7.54+/-3.15 to 15.66+/-6.69 degrees (P=0.0001), the inferior TIA increased from a mean of 9.0+/-4.7 to 15.9+/-6.8 degrees (P=0.0001) after LPI in PAC. In PACG, the mean superior angle changed from 4.55+/-2.5 to 6.12+/-3.8 degrees (P=0.4) and the inferior angle increased from 4.75+/-2.0 to 7.9+/-3.7 degrees (P=0.1). The mean ACD increased from 2.19+/-0.36 to 2.30+/-0.36 mm in PAC group (P=0.0003), with no significant change seen in the PACG group (1.79+/-0.32 vs 1.82+/-0.33 mm, P=0.13). CONCLUSION: LPI leads to a widening of the anterior chamber angle and a deepening of the anterior chamber in eyes with PAC. It does not significantly change any anterior segment parameters in eyes with PACG. PMID- 16680107 TI - Feasibility of bimanual microincision phacoemulsification in hard cataracts. AB - PURPOSE: The purpose of this work was to assess the feasibility of bimanual phacoemulsification in hard cataracts of N3+ using WhiteStar technology of Soveriegn (Advanced Medical Optics, Santa Ana, CA, USA). SETTING: Ain-Shams University Hospitals, Ophthalmology Department, Cairo, Egypt. DESIGN: A randomized prospective noncomparative study. METHODS: A randomized prospective study of 33 consecutive cases (N3+ or more) was conducted, phacoemulsification using a bimanual microincision technique using the Sovereign with WhiteStar technology phacoemulsification machine. One phaco mode was used in all eyes. The ultrasound power was set at 30-25% according to the hardness of the nuclei, duty cycle of 33%, flow rate of 20-28 cm3/min, and vacuum of 240 mmHg. Occlusion mode was on. Nine eyes received rollable intraocular lenses (IOL) of ThinOptx, whereas 24 eyes had been implanted with hydrophobic acrylic foldable IOL (Sensar OptiEdge SA40e of AMO) through a third incision. Study parameters were effective phacotime (EPT), presence of wound burn, degree of immediate postoperative iritis, amount of infusion solution used, and total operating time. RESULTS: The mean EPT was 4.3 s with an average ultrasound used of 5.7%. The mean operating time was 11 min and 20 s. Although the nuclear hardness was of grade 3 or above (in a scale of 5), there were no cases of thermal burn; P=0.005. Only three eyes suffered postoperative iritis 2+, which resolved within 1 week on topical steroids, statistically nonsignificant, P=0.2. The amount of infusion solution was less than that used in conventional coaxial phaco. This technique induced considerably less corneal astigmatism than surgery using conventional corneal incisions. CONCLUSIONS: Hard cataracts of N3 or more could be safely removed through an incision of 1.4 mm incision using bimanual micro-phaco. PMID- 16680108 TI - When the price is right. PMID- 16680109 TI - BIO '06 visible from space? PMID- 16680110 TI - Patently transparent. PMID- 16680111 TI - TeGenero fiasco prompts regulatory rethink. PMID- 16680112 TI - Are small biotechs still underselling themselves? PMID- 16680113 TI - Tuft's report backs FDA fast-track, but post-marketing concerns loom. PMID- 16680114 TI - Stem cell controversy to stall European tissue and cell therapy rules. PMID- 16680115 TI - US-Indian agbiotech deal under scrutiny. PMID- 16680118 TI - Investors bullish in Q1. PMID- 16680117 TI - Alexis Borisy. PMID- 16680119 TI - Chimeras in the crosshairs. PMID- 16680120 TI - TGN1412--a regulator's perspective. PMID- 16680121 TI - Human tissue bank regulations. PMID- 16680123 TI - Transgenic plant science priorities. PMID- 16680124 TI - New pharma-biotech company formation in India. PMID- 16680126 TI - Building biomanufacturing capacity--the chapter and verse. AB - Biopharmaceutical manufacturing capacity has moved through three discrete chapters in its 25-year history. Could the next chapter herald formal manufacturing-capacity sharing among companies? PMID- 16680127 TI - PEGging biotech growth. PMID- 16680129 TI - Gold in the ivory tower: equity rewards of outlicensing. AB - An analysis of life-science initial public offerings from three time periods reveals that the equity share received by universities and their academic researchers has changed over time. PMID- 16680131 TI - Recent patent applications in gene therapy. PMID- 16680130 TI - The human use of humanoid beings: chimeras and patent law. AB - As biotechnology advances, the day may soon come for the creation of a self aware, human-nonhuman chimera. The USPTO has ruled on whether a patent may issue on such an organism, but Congress must still legislate a dividing line between human and non-human patentable subject matter. PMID- 16680132 TI - RNA interference below the immune radar. PMID- 16680133 TI - Replicators lessen transcriptional silencing. PMID- 16680134 TI - A SLAMS dunk for cancer regulators. PMID- 16680135 TI - No longer lost in translation. PMID- 16680137 TI - In silico pharmacogenetics of warfarin metabolism. AB - Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice. PMID- 16680138 TI - Gene prioritization through genomic data fusion. AB - The identification of genes involved in health and disease remains a challenge. We describe a bioinformatics approach, together with a freely accessible, interactive and flexible software termed Endeavour, to prioritize candidate genes underlying biological processes or diseases, based on their similarity to known genes involved in these phenomena. Unlike previous approaches, ours generates distinct prioritizations for multiple heterogeneous data sources, which are then integrated, or fused, into a global ranking using order statistics. In addition, it offers the flexibility of including additional data sources. Validation of our approach revealed it was able to efficiently prioritize 627 genes in disease data sets and 76 genes in biological pathway sets, identify candidates of 16 mono- or polygenic diseases, and discover regulatory genes of myeloid differentiation. Furthermore, the approach identified a novel gene involved in craniofacial development from a 2-Mb chromosomal region, deleted in some patients with DiGeorge-like birth defects. The approach described here offers an alternative integrative method for gene discovery. PMID- 16680139 TI - RNA synthetic biology. AB - RNA molecules play important and diverse regulatory roles in the cell by virtue of their interaction with other nucleic acids, proteins and small molecules. Inspired by this natural versatility, researchers have engineered RNA molecules with new biological functions. In the last two years efforts in synthetic biology have produced novel, synthetic RNA components capable of regulating gene expression in vivo largely in bacteria and yeast, setting the stage for scalable and programmable cellular behavior. Immediate challenges for this emerging field include determining how computational and directed-evolution techniques can be implemented to increase the complexity of engineered RNA systems, as well as determining how such systems can be broadly extended to mammalian systems. Further challenges include designing RNA molecules to be sensors of intracellular and environmental stimuli, probes to explore the behavior of biological networks and components of engineered cellular control systems. PMID- 16680141 TI - Building the Midwest's future. AB - Michigan, Minnesota and Wisconsin have had mixed fortunes in their efforts to build up their research infrastructure. Partnerships may prove to be the strongest construction. PMID- 16680143 TI - Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. AB - Immediate precursors of the many subtypes of dendritic cells (DCs) remain obscure. Here we purified a splenic precursor population that produced all splenic CD8+ and CD8- conventional DCs (cDCs) but not plasmacytoid DCs or other lineages. This 'pre-cDC' population included cells 'precommitted' to form either CD8+ or CD8- cDCs. The pre-cDCs, which comprised 0.05% of splenocytes, expressed a CD11c(int) CD45RA(lo) CD43(int) SIRP-alpha(int) CD4- CD8- major histocompatibility complex class II-negative surface phenotype. The pre-cDCs were not monocytes. Monocytes generated few cDCs in steady-state recipient mice. However, when transferred into mice with an inflammatory milieu dependent on granulocyte-macrophage colony-stimulating factor, monocytes produced a distinct type of splenic DC. Thus, the inflammatory status of the host influences the developmental origin and type of DC present in lymphoid tissues. PMID- 16680144 TI - Transcription factor Pax5 (BSAP) transactivates the RAG-mediated V(H)-to-DJ(H) rearrangement of immunoglobulin genes. AB - Immunoglobulin rearrangement from variable heavy chain (V(H)) to diversity (D) joining heavy chain (J(H)), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage-specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of D(H)-proximal V(H)7183 genes. In exploring the mechanism for Pax5 regulation of V(H)-to-DJ(H) recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse V(H) gene segments. Pax5 bound to those sites in vitro and occupied V(H) genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)-RAG2 complex to enhance RAG-mediated V(H) recombination signal sequence cleavage and recombination of a V(H) gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin V(H)-to-DJ(H) recombination. PMID- 16680146 TI - Multifunctional composites using reinforced laminae with carbon-nanotube forests. AB - Traditional fibre-reinforced composite materials with excellent in-plane properties fare poorly when out-of-plane through-thickness properties are important. Composite architectures with fibres designed orthogonal to the two dimensional (2D) layout in traditional composites could alleviate this weakness in the transverse direction, but all of the efforts so far have only produced limited success. Here, we unveil an approach to the 3D composite challenge, without altering the 2D stack design, on the basis of the concept of interlaminar carbon-nanotube forests that would provide enhanced multifunctional properties along the thickness direction. The carbon-nanotube forests allow the fastening of adjacent plies in the 3D composite. We grow multiwalled carbon nanotubes on the surface of micro-fibre fabric cloth layouts, normal to the fibre lengths, resulting in a 3D effect between plies under loading. These nanotube-coated fabric cloths serve as building blocks for the multilayered 3D composites, with the nanotube forests providing much-needed interlaminar strength and toughness under various loading conditions. For the fabricated 3D composites with nanotube forests, we demonstrate remarkable improvements in the interlaminar fracture toughness, hardness, delamination resistance, in-plane mechanical properties, damping, thermoelastic behaviour, and thermal and electrical conductivities making these structures truly multifunctional. PMID- 16680145 TI - Activation of the integrated stress response during T helper cell differentiation. AB - Adaptive immune responses require clonal expansion and differentiation of naive T cells into cytokine-secreting effector cells. After priming via signals through the T cell receptor, naive T helper cells express cytokine mRNA but do not secrete cytokine protein without additional T cell receptor stimulation. Here we show that primed T cells demonstrated phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha), a 'collapsed' polysome profile, increased expression of stress-response genes and accumulation of cytoplasmic granules associated with RNA-binding proteins, all features of the integrated stress response. Restimulation of the cells resulted in rapid eIF2alpha dephosphorylation, ribosomal mRNA loading and cytokine secretion. Interference with the function of granule-associated proteins or accumulation of phosphorylated eIF2alpha enhanced release of interleukin 4 during T helper type 2 priming. Therefore, T lymphocytes require components of the integrated stress response to uncouple differentiation from the execution of effector functions. PMID- 16680147 TI - Atomic spin structure of antiferromagnetic domain walls. AB - The search for uncompensated magnetic moments on antiferromagnetic surfaces is of great technological importance as they are responsible for the exchange-bias effect that is widely used in state-of-the-art magnetic storage devices. We have studied the atomic spin structure of phase domain walls in the antiferromagnetic Fe monolayer on W(001) by means of spin-polarized scanning tunnelling microscopy and Monte Carlo simulations. The domain wall width only amounts to 6-8 atomic rows. Although walls oriented along <100> directions are found to be fully compensated, detailed analysis of <110>-oriented walls reveals an uncompensated perpendicular magnetic moment. Our result represents a major advance in the field of antiferromagnetism, and may lead to a better understanding of the magnetic interaction between ferromagnetic and antiferromagnetic materials. PMID- 16680148 TI - Carminerin contributes to chondrocyte calcification during endochondral ossification. AB - Endochondral ossification is an essential process not only for physiological skeletal development and growth, but also for pathological disorders. We recently identified a novel cartilage-specific molecule, carminerin (also known as cystatin 10 and encoded by Cst10), which is upregulated in synchrony with cartilage maturation and stimulates the later differentiation of cultured chondrocytes. Although carminerin-deficient (Cst10-/-) mice developed and grew normally, they had a microscopic decrease in the calcification of hypertrophic chondrocytes at the growth plate. When we created experimental models of pathological endochondral ossification, we observed suppression of chondrocyte calcification during formation of osteoarthritic osteophytes, age-related ectopic ossification and healing of bone fractures in Cst10-/- mice. Cultured Cst10-/- chondrocytes showed a reduction in calcification with activation of an SRY site in the promoter of the gene encoding nucleotide pyrophosphatase phosphodiesterase 1 (NPP1, encoded by Enpp1). Functional NPP1 is required for carminerin deficiency to suppress the pathological endochondral ossifications listed above. Carminerin is the first cartilage-specific protein that contributes to chondrocyte calcification during endochondral ossification under physiological and pathological conditions through the transcriptional inhibition of NPP1. PMID- 16680149 TI - Eradication of established tumors in mice by a combination antibody-based therapy. AB - Tumor-cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful antitumor immunotherapies. Here we show that induction of tumor-cell apoptosis by an agonistic monoclonal antibody to DR5, the apoptosis-inducing receptor for TNF-related apoptosis inducing ligand (TRAIL), combined with T-cell activation by agonistic monoclonal antibodies to the costimulatory molecules CD40 and CD137, potently and rapidly stimulated tumor-specific effector CD8+ T cells capable of eradicating preestablished tumors. Primary fibrosarcomas initiated with the carcinogen 3 methylcholanthrene (MCA), multiorgan metastases and a primary tumor containing as many as 90% tumor cells resistant to DR5-specific monoclonal antibody were rejected without apparent toxicity or induction of autoimmunity. This combination therapy of three monoclonal antibodies (trimAb) rapidly induced tumor-specific CD8+ T cells producing interferon (IFN)-gamma in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-gamma in the tumor rejection process. These results in mice indicate that a rational monoclonal antibody-based therapy that both causes tumor-cell apoptosis through DR5 and activates T cells may be an effective strategy for cancer immunotherapy in humans. PMID- 16680150 TI - Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke. AB - Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions. PMID- 16680152 TI - The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity. AB - Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration. PMID- 16680151 TI - Identification of a tumour suppressor network opposing nuclear Akt function. AB - The proto-oncogene AKT (also known as PKB) is activated in many human cancers, mostly owing to loss of the PTEN tumour suppressor. In such tumours, AKT becomes enriched at cell membranes where it is activated by phosphorylation. Yet many targets inhibited by phosphorylated AKT (for example, the FOXO transcription factors) are nuclear; it has remained unclear how relevant nuclear phosphorylated AKT (pAKT) function is for tumorigenesis. Here we show that the PMLtumour suppressor prevents cancer by inactivating pAKT inside the nucleus. We find in a mouse model that Pml loss markedly accelerates tumour onset, incidence and progression in Pten-heterozygous mutants, and leads to female sterility with features that recapitulate the phenotype of Foxo3a knockout mice. We show that Pml deficiency on its own leads to tumorigenesis in the prostate, a tissue that is exquisitely sensitive to pAkt levels, and demonstrate that Pml specifically recruits the Akt phosphatase PP2a as well as pAkt into Pml nuclear bodies. Notably, we find that Pml-null cells are impaired in PP2a phosphatase activity towards Akt, and thus accumulate nuclear pAkt. As a consequence, the progressive reduction in Pml dose leads to inactivation of Foxo3a-mediated transcription of proapoptotic Bim and the cell cycle inhibitor p27(kip1). Our results demonstrate that Pml orchestrates a nuclear tumour suppressor network for inactivation of nuclear pAkt, and thus highlight the importance of AKT compartmentalization in human cancer pathogenesis and treatment. PMID- 16680153 TI - Automated analysis of fluorescence in situ hybridization on fixed, paraffin embedded whole tissue sections in B-cell lymphoma. AB - Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information. PMID- 16680154 TI - TTF-1 and p63 for distinguishing pulmonary small-cell carcinoma from poorly differentiated squamous cell carcinoma in previously pap-stained cytologic material. AB - In histology and cell block sections, antibodies to thyroid transcription factor 1 (TTF-1) and p63 have been demonstrated to be useful markers for distinguishing between small-cell lung carcinoma and poorly differentiated pulmonary squamous cell carcinoma. In this study, we assessed the utility of TTF-1 and p63, as an antibody panel, for differentiating between these two neoplasms in previously Papanicolaou (Pap)-stained cytologic smears and cytospin slides. Twenty-six lung carcinomas (13 small-cell lung carcinomas, 13 poorly differentiated pulmonary squamous cell carcinomas) were evaluated. One or two previously 95% ethanol fixed, Pap-stained smears or cytospin slides were selected from each case. The cytologic material from these slides was transferred to positively charged slides. Unstained recuts were obtained from the corresponding histologic specimens or cell blocks. Immunohistochemical staining for TTF-1 and p63 was performed on the paired samples from each tumor. All (13/13) small-cell lung carcinomas were negative for p63 and 92% (12/13) were positive for TTF-1. Conversely, all (13/13) poorly differentiated pulmonary squamous cell carcinomas expressed p63 and did not express TTF-1. Immunoreactivity for p63 was also noted in bronchial reserve cells and metaplastic squamous cells. The immunostaining results obtained from the cytology slides were concordant with those of the histology or cell block sections in all cases. The results of this study show that TTF-1 and p63 immunostaining can be successfully applied to previously Pap stained cytologic material, as an antibody panel, to facilitate pathologic differentiation between small-cell lung carcinomas and poorly differentiated pulmonary squamous cell carcinomas. p63 immunostaining, however, must be interpreted in conjunction with cytomorphology to distinguish between poorly differentiated pulmonary squamous cell carcinomas and benign cellular constituents of the lung. PMID- 16680155 TI - Novel identification of zyxin upregulations in the motile phenotype of hepatocellular carcinoma. AB - Genome-wide copy number aberrations are common in hepatocellular carcinoma, although the precise genetic events underlying disease progression remain poorly defined. Previous work from our group has indicated several regional chromosomal gains such as chromosome 7q34-q36 that are associated with advanced metastatic tumors. Although the distal chromosome 7q gains have also been implicated in the progression of other malignancies, information on underlying targeted genes is limited. In this study, we have examined the chromosome 7q34-q36 region for involved gene(s) (or genes of interest). An integrated array-based comparative genomic hybridization and transcriptional mapping analyses has enabled us to identify a single candidate, zyxin on chromosome 7q34-q36. This array-derived finding was supported by quantitative reverse transcription-polymerase chain reaction, which also indicated common upregulations of zyxin in hepatocellular carcinoma tumors compared to their corresponding nonmalignant liver tissue (17/52 cases; 33%). Although there was no correlation between zyxin expression and tumor stagings, there was a significant increase in messenger RNA levels in hepatocellular carcinoma cases that presented with multifocal disease (211.5 +/- 936.9-fold) compared to those with solitary lesions (3.5 +/- 6.3-fold). Moreover, recurrence after resection was common in cases that displayed zyxin overexpressions in the initial resected tumor (P = 0.05). Functional examination of zyxin by small interfering RNA-mediated knockdown in Hep3B cell line indicated a significant inhibition on cell migration through porous membrane (P = 0.002) and invasion through matrigel-coated membrane (P = 0.005). In summary, mapping of chromosome 7q34-q36 has led to the identification of frequent zyxin overexpressions in hepatocellular carcinoma, and a potential role for zyxin in conferring a motile phenotype. PMID- 16680156 TI - Expression of CXCL13, a chemokine highly upregulated in germinal center T-helper cells, distinguishes angioimmunoblastic T-cell lymphoma from peripheral T-cell lymphoma, unspecified. AB - The germinal center T-helper cell has been proposed as the cell of origin for angioimmunoblastic T-cell lymphoma. Our recent report of expression of CXCL13, a chemokine critical for germinal center formation and one of the most highly upregulated genes in the germinal center T-helper cell subset, in the majority of angioimmunoblastic T-cell lymphoma cases, provided further support for this theory. To determine the specifity of this marker for angioimmunoblastic T-cell lymphoma, we evaluated CXCL13 expression in 26 nodal-based peripheral T-cell lymphomas and 14 lymph nodes showing paracortical lymphoid hyperplasia. No significant paracortical CXCL13 staining was seen in the reactive lymph nodes. By WHO classification criteria, 20 of the lymphoma cases were considered peripheral T-cell lymphoma, unspecified, and six were reclassified as angioimmunoblastic T cell lymphoma after immunohistochemical detection of disorganized follicular dendritic cell meshworks. Combining the results of our studies, 31 of 35 angioimmunoblastic T-cell lymphoma cases (89%) showed CXCL13 expression, in contrast to two out of 20 peripheral T-cell lymphoma, unspecified cases (10%). The two peripheral T-cell lymphoma, unspecified cases that were positive for CXCL13 showed a Lennert lymphoma-like histology. While these cases did not meet all histologic criteria for angioimmunoblastic T-cell lymphoma, they did show an increase in EBV-positive B cells, suggesting they may be histologic variants of angioimmunoblastic T-cell lymphoma. In conclusion, CXCL13 expression is a distinctive feature of angioimmunoblastic T-cell lymphoma, providing further support for the germinal center T-helper cell as the cell of origin for this neoplasm. Given its specificity when compared to cases of peripheral T-cell lymphoma, unspecified as well as paracortical lymphoid hyperplasia, it may be a useful marker in the diagnosis of angioimmunoblastic T-cell lymphoma. PMID- 16680157 TI - Fibrogenesis in alcoholic chronic pancreatitis: the role of tissue necrosis, macrophages, myofibroblasts and cytokines. AB - Myofibroblasts and cytokines such as transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor (PDGF)-B have been found to play an important role in pancreatitis-associated fibrogenesis. It is still unclear, however, where in the inflamed pancreas and when these fibrogenic cells and cytokines can be detected. In this study we examined pancreatic tissue from patients with alcoholic chronic pancreatitis to determine the localization and distribution of myofibroblasts and the expression of cytokines in relation to the tissue damage and the activity of the inflammatory process. In tissue from pancreatic specimens from 59 patients with alcoholic chronic pancreatitis the inflammatory process was histologically staged. Myofibroblasts and the cytokines latency-associated peptide, a TGF-beta propeptide, TGF-beta receptor II, PDGF-B and the alpha isoform of the PDGF receptor were immunohistochemically identified in 10 selected cases representing the four defined stages of alcoholic chronic pancreatitis. In stage I, the stage with overt tissue injury, myofibroblasts were numerous and especially associated with macrophages around areas of necrosis. In stage II, the stage with cellular fibrosis, myofibroblasts were the main component of the interlobular tissue. In stage III, the stage with dense fibrosis, myofibroblasts were rare, and in stage IV, when calculi were present, myofibroblasts were only detected adjacent to duct ulcerations caused by calculi. Latency-associated peptide and TGF-beta receptor II as well as PDGF-B and PDGF receptor-alpha were mainly expressed by macrophages, myofibroblasts and epithelial cells in stages I and II. The results suggest that the fibrogenic process in alcoholic chronic pancreatitis is initiated by a cytokine-based interplay of macrophages and myofibroblasts that follows tissue injury. PMID- 16680158 TI - Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording. AB - Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays. PMID- 16680159 TI - Identifying off-target effects and hidden phenotypes of drugs in human cells. AB - We present a strategy for identifying off-target effects and hidden phenotypes of drugs by directly probing biochemical pathways that underlie therapeutic or toxic mechanisms in intact, living cells. High-content protein-fragment complementation assays (PCAs) were constructed with synthetic fragments of a mutant fluorescent protein ('Venus', EYFP or both), allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. One hundred and seven different drugs from six therapeutic areas were screened against 49 different PCA reporters for ten cellular processes. This strategy reproduced known structure-function relationships and also predicted 'hidden,' potent antiproliferative activities for four drugs with novel mechanisms of action, including disruption of mitochondrial membrane potential. A simple algorithm identified a 25-assay panel that was highly predictive of antiproliferative activity, and the predictive power of this approach was confirmed with cross-validation tests. This study suggests a strategy for therapeutic discovery that identifies novel, unpredicted mechanisms of drug action and thereby enhances the productivity of drug-discovery research. PMID- 16680160 TI - The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography. AB - Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2 hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase. PMID- 16680161 TI - Exon ligation is proofread by the DExD/H-box ATPase Prp22p. AB - To produce messenger RNA, the spliceosome excises introns from precursor (pre) mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3' splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase. PMID- 16680162 TI - RNA helicase A is necessary for translation of selected messenger RNAs. AB - RNA helicase A (RHA) is a highly conserved DEAD-box protein that activates transcription, modulates RNA splicing and binds the nuclear pore complex. The life cycle of typical mRNA involves RNA processing and translation after ribosome scanning of a relatively unstructured 5' untranslated region (UTR). The precursor RNAs of retroviruses and selected cellular genes harbor a complex 5' UTR and use a yet-to-be-identified host post-transcriptional effector to stimulate efficient translation. Here we show that RHA recognizes a structured 5'-terminal post transcriptional control element (PCE) of a retrovirus and the JUND growth-control gene. RHA interacts with PCE RNA in the nucleus and cytoplasm, facilitates polyribosome association and is necessary for its efficient translation. Our results reveal a previously unidentified role for RHA in translation and implicate RHA as an integrative effector in the continuum of gene expression from transcription to translation. PMID- 16680163 TI - BDNF val66met polymorphism is associated with modified experience-dependent plasticity in human motor cortex. AB - Motor training can induce profound physiological plasticity within primary motor cortex, including changes in corticospinal output and motor map topography. Using transcranial magnetic stimulation, we show that training-dependent increases in the amplitude of motor-evoked potentials and motor map reorganization are reduced in healthy subjects with a val66met polymorphism in the brain-derived neurotrophic factor gene (BDNF), as compared to subjects without the polymorphism. The results suggest that BDNF is involved in mediating experience dependent plasticity of human motor cortex. PMID- 16680164 TI - Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells. AB - Here we demonstrate that cerebellar stellate cells diffusionally isolate synaptically evoked signals in dendrites and are capable of input-specific synaptic plasticity. Sustained activity of parallel fibers induces a form of long term depression that requires opening of calcium (Ca(2+))-permeable AMPA-type glutamate receptors (CP-AMPARs) and signaling through class 1 metabotropic glutamate receptors (mGluR1) and CB1 receptors. This depression is induced by postsynaptic increases in Ca(2+) concentration ([Ca(2+)]) and is limited to activated synapses. To understand how synapse-specific plasticity is induced by diffusible second messengers in aspiny dendrites, we examined diffusion of Ca(2+) and small molecules within stellate cell dendrites. Activation of a single parallel fiber opened CP-AMPARs, generating long-lived Ca(2+) transients that were confined to submicron dendritic stretches. The diffusion of Ca(2+) was severely retarded due to interactions with parvalbumin and a general restriction of small molecule mobility. Thus stellate cell dendrites spatially restrict signaling cascades that lead from CP-AMPAR activation to endocannabinoid production and trigger the selective regulation of active synapses. PMID- 16680165 TI - Eph receptors are negatively controlled by protein tyrosine phosphatase receptor type O. AB - Eph receptors are activated by the autophosphorylation of tyrosine residues upon the binding of their ligands, the ephrins; however, the protein tyrosine phosphatases (PTPs) responsible for the negative regulation of Eph receptors have not been elucidated. Here, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that efficiently dephosphorylates both EphA and EphB receptors as substrates. Biochemical analyses revealed that Ptpro dephosphorylates a phosphotyrosine residue conserved in the juxtamembrane region, which is required for the activation and signal transmission of Eph receptors. Ptpro thus seems to moderate the amount of maximal activation of Eph receptors. Using the chick retinotectal projection system, we show that Ptpro controls the sensitivity of retinal axons to ephrins and thereby has a crucial role in the establishment of topographic projections. Our findings explain the molecular mechanism that determines the threshold of the response of Eph receptors to ephrins in vivo. PMID- 16680166 TI - The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. AB - In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late gestation progenitors, allowing them to remake preplate neurons and then cortical plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed. PMID- 16680167 TI - Medical image. Tussigenic trachea. PMID- 16680168 TI - How many antibiotic prescriptions are unsubsidised in New Zealand? AB - AIMS: To determine the proportion of prescriptions for antibiotics which were unsubsidised, in one town in one year, and to use this to develop a model which could be used to estimate the number of unsubsidised prescriptions. METHODS: Data on all prescriptions for antibiotics during 2002 were extracted from pharmacy computers in one town. Data were obtained from PharmHouse database on all subsidised prescriptions from the town pharmacies during 2002. (The PharmHouse database is a subset of the New Zealand Health Information System database and contains records of all the claims for medicines dispensed within New Zealand.) These were compared and the proportion of unsubsidised prescriptions for each antibiotic calculated. Weighted linear regression was used to develop a model of the relationship between the percentage of each drug subsidised, and patient and prescription characteristics obtainable in PharmHouse. RESULTS: 64.4% of antibiotic dispensings in the study town were subsidised, and therefore captured by the PharmHouse database. The proportion varied substantially between different antibiotics. For particular drugs, the proportion of drugs unsubsidised could be predicted by the price of the drug, the number of days it was prescribed for, and the number of patients aged under six who received subsidised prescriptions. CONCLUSIONS: Previous studies using PharmHouse data are likely to have significantly underestimated the extent of drug use. Further research is needed on whether this model can help to estimate the extent of unsubsidised prescriptions. PMID- 16680169 TI - Inhalant abuse in New Zealand. AB - AIM: To describe patterns of inhalant abuse in New Zealand and discuss management. METHODS: Calls to the National Poisons Centre (NPC) from January 1 2003 to December 31 2004 were analysed. In addition, deaths following inhalational abuse were identified from the Institute of Environmental Science and Research Limited (ESR) database for 2001 and 2002 and available data for 2003. RESULTS: Seventy calls were classified as relating to inhalational abuse incidents. In abusers whose age was known, 83% were between 11 and 20 years, and 61% were male. Over half (44/70) of the calls involved abuse of propane or butane, either alone or in combination with a synthetic pyrethroid. ESR coronial data identified 11 inhalant abuse related deaths, most commonly attributed to cardiac effects. 73% of deaths were in teenagers and all but one fatality involved propane and/or butane. CONCLUSIONS: Inhalant abuse is a persisting problem in New Zealand. NPC and ESR data demonstrate that teenagers are more likely to abuse inhalants than other age groups and butane and propane are the inhalants of choice. Acute management can be difficult, with significant mortality and morbidity. Continued education and other preventive measures are essential to help curb an extremely dangerous practice. PMID- 16680170 TI - New Zealanders' love affair with "alternative" medicine: reason for concern? PMID- 16680171 TI - Complementary and alternative medicines (including traditional Maori treatments) used by presenters to an emergency department in New Zealand: a survey of prevalence and toxicity. AB - AIM: To establish the prevalence of use of complementary and alternative medicines (CAM), including traditional Maori therapies, their perceived benefit, and frequency of adverse effects among presenters to an emergency department (ED) in New Zealand. METHOD: An analytical cross-sectional survey of a convenience sample of patients and relatives presenting to a large tertiary ED (Waikato Hospital, Hamilton) was undertaken. Consenting participants completed a specifically designed questionnaire. RESULTS: 1043 people completed the questionnaire (participation rate 97.2%). 1 in 3 (397 or 38.1%) people had used CAM, including 29 who had used a traditional Maori therapy. CAM use was significantly more likely in females (p<0.0001), those aged 20-60 yrs (p<0.001), and in those of European ethnicity (p=0.01). Only 148 (37.3%) people had told their medical practitioner that they were using CAM, and 103 (25.9%) had used conventional medicines concurrently. Most people (266, 67%) believed that CAM had been beneficial. Adverse effects were reported by 16 people (4% of users). CONCLUSIONS: Many ED presenters in New Zealand use CAM. These may be associated with adverse effects, toxicity, and interactions with conventional medicines, although the incidence of these appears to be low. Doctors in New Zealand should routinely specifically enquire about the use of CAM during patient assessment. PMID- 16680172 TI - Patients consulting outside of funded practices within primary health organisations: implications for utilisation reporting. AB - AIM: To consider two definitions for utilisation reporting in primary care in New Zealand and to assess the affect of two reporting methods on volumes of utilisation in four primary health organisations (PHOs). METHODS: Utilisation data was analysed for a 6-month period from 60 practices across four PHOs. Analysis was based on comparing the expected volumes from two alternative collection and reporting methods, named "matched" and "unmatched" reporting. The "unmatched" method is potentially sensitive to patients consulting outside of the practice in which they are funded. Volumes were grouped into categories based on those used for reporting. RESULTS: There was up to 25% difference in volumes in some reporting groups depending on the matching method used. Several of these were for high deprivation, New Zealand Maori, Pacific Islanders, and Community Service Card (CSC) holders--all potentially target populations within PHOs. Two PHOs were more affected having a total of 7.6% and 6.4% fewer reported encounters using the "unmatched" method. Data implies that some groups of patients may not be receiving continuity of care. CONCLUSIONS: There were differences in reporting volumes between the two methods. The Ministry of Health (MOH), district health boards (DHBs), and PHOs should be aware of how these results may potentially apply to them, especially where they have after-hours services or target groups as minorities. PMID- 16680173 TI - Antibiotic use for upper respiratory tract infections before and after a education campaign as reported by general practitioners in New Zealand. AB - AIM: To assess change in general practitioner (GP) management of upper respiratory tract infections (URTIs) during a nationwide project to reduce antibiotic consumption in a half-decade (1998 to 2002-3). METHOD: Telephone survey of 100 randomly selected Auckland GPs in 1998 and 2002-3. Sixty-five GPs were in both samples. RESULTS: A 69% response rate was recorded for an additional 35 GPs recruited in 2002-3. Of the 65 GPs interviewed at both periods, the number agreeing that most patients who consult for URTIs expect antibiotics decreased from 82% to 57%. Seventy-seven percent of GPs reported they were less likely to prescribe antibiotics, with over a quarter believing this change resulted from both GP and patient education. Common situations where GPs increased their antibiotic prescribing were patient request/expectation; smokers; older; or having sinusitis, purulent sputum, purulent nasal discharge, or imminent overseas travel. Thirty-nine percent of GPs reported an increasing use of delayed prescriptions over the half-decade. Reported use of amoxicillin clavulanate reduced from 21% to 4% (p<0.001). CONCLUSION: The GPs' response that they are less likely to prescribe antibiotics is consistent with the reduction in national antibiotic use. This may be related to the national campaign. The reduction may be a combination of combined GP and patient change. PMID- 16680174 TI - Public views and use of antibiotics for the common cold before and after an education campaign in New Zealand. AB - AIMS: To assess changes in public knowledge, attitudes, and reported behaviour of antibiotic use in the management of the common cold and to compare with results of a 1998 study. The context is a nationwide project to reduce the consumption of antibiotics. METHOD: Cross-section survey: telephone interviews of random sample of consenting Auckland telephone subscribers aged over 15 years comparing 1998 and 2003 responses. RESULTS: A 55% response rate of eligible participants was recorded. No change was noted between 1998 and 2003 in public awareness that antibiotics are not helpful in treating viral infections (38%). However there was a significant reduction in those attending doctor for the common cold (24% to 15%). In 2003, patients were less likely to receive antibiotic prescription and more likely to receive a delayed prescription. CONCLUSIONS: The majority of general public still do not understand that the common cold does not need antibiotic treatment. The advertising campaign may have reduced doctor prescribing hence the reduction in antibiotic use from 1998 to 2003. PMID- 16680175 TI - Ayurvedic medicine: patients in peril from plumbism. AB - Heavy metals are commonly incorporated into Ayurvedic preparations as ashes or 'bhasmas'. A widely disseminated belief within Ayurvedic medicine is that these heavy metals can be valuable therapeutic components. Western toxicology refutes this contention. We report eight cases of lead poisoning occurring in or near the Auckland region of New Zealand. In all cases, poisoning was attributable to consumption of Ayurvedic 'herbal medicines'. Whole blood lead levels ranged from 1.5 to 6.9 micromol/L. Six patients had symptomatic lead poisoning, requiring treatment with chelation therapy. A high index of suspicion is required to detect lead poisoning, which should be suspected in people taking Ayurvedic remedies, especially if they have associated anaemia or abdominal symptoms. PMID- 16680176 TI - Nephrotoxicity of BZP-based herbal party pills: a New Zealand case report. PMID- 16680177 TI - Influenza vaccination among New Zealand healthcare workers: low rates are concerning. PMID- 16680178 TI - Anaphylaxis management: the essential role of adrenaline (epinephrine) auto injectors. Should PHARMAC fund them in New Zealand? AB - Anaphylaxis is an important life-threatening medical emergency. There is extensive evidence supporting the early use of intramuscular adrenaline for first medical responders and for self-initiated treatment, in at-risk individuals. Major patient groups identified as at ongoing risk are children and adults with severe food allergy, patients with venom allergy who have not been desensitised, and those with idiopathic anaphylaxis. Individual anaphylactic events are largely unpredictable. The most effective and safe route of administration for adrenaline is intramuscular, but it is difficult for patients and carers to achieve accurate and timely self-administration using an ampoule, needle, and syringe. The adrenaline auto-injector device which is available in New Zealand (the EpiPen) is not funded by PHARMAC, and thus only available to patients and families who are able to afford the purchase cost. It is difficult to understand the continued unwillingness of PHARMAC to fund an adrenaline auto-injector device to at-risk individuals, given the large body of information supporting its efficacy and use. The Australian model, where authorisation from a relevant specialist is required, could be used. PMID- 16680179 TI - Wait and be seen. PMID- 16680180 TI - Hazardous and beneficial nutritional messages in 858 televised food advertisements during children's viewing hours. PMID- 16680181 TI - The Finsbury dispensary. PMID- 16680182 TI - Immunosuppression for islet transplantation. AB - The development by the Edmonton group of a sirolimus-based, steroid-free, low tacrolimus regimen is a significant breakthrough that allows the rate of insulin independence after islet transplantation to increase from 13% to 80% at 1 year; however, the rate is reduced to 50% at 3 years, attributed to prolonged tacrolimus exposure. Recently, immunosuppression agents such as cyclosporine, mycophenolate mofetil, and the novel agent FTY 720 have been used instead of tacrolimus. Lymphocyte-depleting antibodies such as anti-thymocyte globulin, alemtuzumab, and hOKT3gamma 1 (ala, ala) have been launched, and a costimulatory blockade of anti-CD40 monoclonal antibodies and CTLA4-Ig will be attempted in the near future. Moreover, the potential of a novel immunosuppressing peptide could now be realized using new technology called the protein transduction system. In this review, we show some of the most recent contributions to the advancement of knowledge in this field. PMID- 16680183 TI - Simple surrogate index of the fibrosis stage in chronic hepatitis C patients using platelet count and serum albumin level. AB - This study was conducted to develop a simple surrogate index comprised of routinely available laboratory tests to reflect the histological fibrosis stage. Clinical characteristics and laboratory data from 368 and 249 consecutive patients with chronic hepatitis C, a training cohort and a validation cohort, respectively, were retrospectively evaluated. Platelet (Plt) count and albumin (Alb) level contributed to the discrimination of the respective fibrosis stages. We derived the fibrosis index (FI), FI = 8.0-0.01 x Plt (10(3)/microliter) - Alb (g/dl), from a multiple regression model. FI significantly correlated with the histological fibrosis stage in both the initial and validation cohort at rho=0.691 and rho=0.661, respectively (Spearman's rank correlation coefficient, p<0.0001). The sensitivity and positive predictive value of FI at a cutoff value < 2.10 for predicting fibrosis stage F0-1 were 66.8% and 78.8% in the initial cohort and 68.5% and 63.6% in the validation cohort, respectively. Corresponding values of FI at a cutoff value > or = 3.30 for the prediction of F4 were 67.7% and 75.0% in the initial cohort and 70.8% and 81.0% in the validation cohort. The fibrosis index comprised of platelet count and albumin level reflected the histological fibrosis stage in patients with chronic hepatitis C. PMID- 16680184 TI - Effects of radiofrequency ablation on individual renal function: assessment by technetium-99m mercaptoacetyltriglycine renal scintigraphy. AB - We quantitatively evaluated total and individual renal function by technetium-99m mercaptoacetyltriglycine (Tc-99m MAG3) renal scintigraphy before and after radiofrequency ablation (RFA) of renal tumors. Eleven patients who underwent Tc 99m MAG3 renal scintigraphy 1 week before and after RFA were evaluated (7 men and 4 women; age range: 23-83 years; mean age: 60.6 years). Five patients had solitary kidneys, and five had normally or minimally functioning contralateral kidneys. One patient had a renal cell carcinoma in the contralateral kidney. One patient with a solitary kidney underwent RFA a second time for a residual tumor. In patients with a solitary kidney, MAG3 clearance decreased after 5 of 6 RFAs, and in patients with a normally functioning contralateral kidney, MAG3 clearance decreased after 4 of 5 RFAs, but no significant differences were observed between before and after treatments. In addition to the total MAG3 clearance, the split MAG3 clearance was evaluated in patients with a normally functioning contralateral kidney. MAG3 clearance decreased in 4 of 5 treated kidneys, while it adversely increased in the contralateral kidneys after 4 of 5 RFAs. No significant differences, however, were observed between before and after treatments. The results of our study revealed no significant differences in sCr, BUN, CCr, or MAG3 clearance between pre- and post-RFA values. These results support data regarding the functional impact and safety of renal RFA in published reports. We evaluated total and individual renal function quantitatively using Tc 99m MAG3 renal scintigraphy before and after treatment. This scintigraphy was very useful in assessing the effects of RFA on renal function. PMID- 16680185 TI - Utility of vessel-sealing systems in thyroid surgery. AB - The LigaSure vessel-sealing system (VSS) represents a new approach to intraoperative ligation. This clinical study retrospectively examined the utility of the VSS in thyroid surgery. In this study, we analyzed 56 consecutive patients who underwent thyroid surgery. Characteristics such as operative duration, the volume of intraoperative hemorrhage, and postoperative course were analyzed and compared between thyroid surgery using the VSS or conventional handtie methods. The present results indicate no significant differences in operative duration, volume of intraoperative hemorrhage, postoperative course, or duration of postoperative drainage between surgeries using the VSS or conventional methods. However, the postoperative hospital stay was found to be significantly shorter (p<0.05) with the VSS. No serious postoperative complications were encountered, and no significant differences were observed in the frequency of postoperative complications between methods. The VSS may simplify procedures for thyroid surgery, and hemostasis is effective for both thyroid vessels and thyroid parenchyma. However, further evaluation is warranted to adequately determine the relative merits of the VSS compared to conventional handtie methods. PMID- 16680186 TI - Assessment of total and segmental body composition in spinal cord-injured athletes in Okayama prefecture of Japan. AB - This study assessed total and segmental distribution of fat mass (FM) in athletes with spinal cord injury (SCI) and examined the relationships between segmental distribution of fat mass and age, injury level, athletic history, and training load in order to provide useful information for improvements in their physical strength and training. Twenty-five male athletes with SCI participated in the study. The whole bone composition was measured by a dual-energy X-ray absorptiometry (DXA) method for the calculation of bone minerals, FM, and fat free mass. The percent fat of the trunk, arms, and legs was also calculated. The percent fat in the legs was highest in comparison with that in the trunk and arms (p < 0.001), and the percent fat in the trunk was higher than that in the arms (p < 0.001). The body fat (p < 0.01), waist circumference (p < 0.01), and waist-to hip ratio (p < 0.0001) were higher in the group aged 40 or older in comparison with that aged 39 or younger. Path analysis revealed that training load was a factor decreasing the percent fat on the arms and trunk (p < 0.01), and athletic history was a factor reducing the percent fat on the arms (p < 0.05). Our study suggests that exercise is effective in reducing the waist circumference, waist-to hip ratio, and percent body fat of SCI individuals, and that such effects can help to enhance athletic performance and likely to protect against development of metabolic syndromes resulting from a sedentary lifestyle. PMID- 16680187 TI - Resistance imparted by vitamin C, vitamin E and vitamin B12 to the acute hepatic glycogen change in rats caused by noise. AB - The effects of vitamin C, vitamin E and vitamin B12 on the noise-induced acute change in hepatic glycogen content in rats were investigated. The exposure of rats to 95 dB and 110 dB of noise acutely reduced their hepatic glycogens. Vitamin C (ascorbic acid) and vitamin E (alpha-tocopherol) attenuated the noise induced acute reduction in the hepatic glycogen contents. This result suggests that antioxidants could reduce the change via reactive oxygen species. Vitamin B12 (cobalamin) delayed the noise-induced change, a finding that suggests that vitamin B12 could postpone the acute change via compensating for vitamin B12 deficiency. PMID- 16680188 TI - In vitro evaluation of a newly developed implantable artificial lung. AB - A prototype of an implantable artificial lung without a pump (Prototype II) has been tested. A commercially available membrane oxygenator, MENOX AL6000alpha (Dainippon Ink and Chemicals, Inc., Tokyo, Japan), was used as a basic model. The packing density of the hollow fiber was decreased in order to achieve low resistance through the blood pathway. The configuration of its housing was also re-designed using computational fluid dynamics (CFD). The first prototype, known as Prototype I, was already tested in a 15 kg pig, which showed excellent gas exchange with normal hemodynamics. A second prototype, Prototype II, has a larger membrane surface area than Prototype I. The device was evaluated for resistance through the blood path and gas transfer rate in an in vitro setting by the single pass method using fresh bovine blood. The resistance through the blood path of Prototype II was 2.7+- 0.7 mmHg/(L/min) at Q = 5L/min. The oxygen (O2) transfer rate was 178 +- 5.3 ml/min at Q = 5 L/min, V/Q = 3, and the carbon dioxide (CO2) transfer rate was 149 +- 28 ml/min at Q = 5 L/min, V/Q = 2 (Q: blood flow rate, V: sweep oxygen flow rate through the artificial lung). For the purpose of implantation, this prototype showed sufficiently low resistance in the pulmonary circulation with reasonable gas exchange. PMID- 16680189 TI - The sister-chromatid exchange and acetylcholine esterase enzyme levels among patients with insecticide intoxication in the Cukurova region, Turkey. AB - This study included 45 patients with intentional insecticide intoxication and 21 with accidental intoxication who were treated at the First-Aid and Emergency Department of Balcali Hospital at the Faculty of Medicine in the Cukurova University, Adana, Turkey, while the control group consisted of 25 people selected from university personnel known to be healthy. Patients with a history of X-ray exposure in the last 6 months or of any virus disease as well as continuous drug users and smokers were excluded, leaving a total of 49 patients. Acetylcholine esterase (Pseudocholinesterase) enzyme (AchE), sister-chromatid exchanges (SCE), the mitotic index (MI), and the replication index (RI) were evaluated. Blood samples were cultured for SCE evaluation and sera separated for AchE levels. Insecticide exposure was generally intentional for suicide in adolescents and at older ages, but accidental for children. AchE levels were found to be significantly lower in organophosphorus (OP) and carbamated (CB) insecticide poisoning groups in comparison with the control group (p<0.001), while the pyrethroid (PY) group was not statistically different for the AchE effect (p>0.05). SCE was found to be significantly higher in OP and CB groups (p<0.001), while the PY and control groups were statistically similar for SCE levels (p>0.05). This study showed an increase in SCE in response to orally ingested insecticides. These findings indicate that insecticide exposure results in cell abnormalities, with resulting impediments to the division and replication of cells, as suggested by MI decreases and RI increases, while the speed of the division cycles of stimulated cells increases. PMID- 16680190 TI - Reactivity of blood samples spotted onto filter papers in the WST-8 method for screening of G6PD deficiency. AB - Deficiency of glucose-6-phosphate dehydrogenase (G6PD) causes acute hemolytic anemia triggered by oxidative drugs such as primaquine. It is therefore essential in malaria-endemic areas for malaria patients to be confirmed for their G6PD activity before taking primaquine. The WST-8 method, a newly established screening method for G6PD deficiency, has been demonstrated to be suitable for field conditions, particularly for on-site malaria surveys. Here we report a laboratory evaluation by this method of the reactivity of blood-spotted filters. A time-course experiment was conducted to evaluate the reactivity of blood samples spotted onto 4 types of filter paper, Whatman 31ET Chr (ET), 3MM Chr (3MM), P81, and Advantec No. 2 (AD2). The rank of the relative reaction intensity was ET > 3MM = AD2 > P81. Blood-spotted filters stored at 4 degrees C gradually decreased G6PD reactivity with the passage of storage time, whereas those stored at room temperature rapidly reduced their reactivity. Unexpectedly, saponin supplementation reduced the reactivity of blood-spotted filters. In conclusion, 1) ET is the most suitable filter for the WST-8 method; 2) blood-spotted filters stored in cold condition can be assayed within 14 days, or those stored at room temperature should be tested within 3 days; and 3) reaction mixtures should not contain saponin. PMID- 16680191 TI - Intraatrial extension of thyroid cancer: a case report. AB - A 61-year-old man, who was diagnosed with superior vena cava syndrome by papillary thyroid carcinoma, was referred to our hospital. A bulky thyroid tumor with tracheal invasion extended from the left neck to the right atrium without distant metastases. The risk of sudden death due to airway occlusion, tumor embolism or obstruction of the tricuspid valve led us to elect surgery. Extended resection of thyroid cancer was performed with cardiopulmonary bypass. Peritoneal dissemination was found via laparotomy. A histological diagnosis of anaplastic carcinoma arising from transformation of papillary carcinoma was made. After the operation, bilateral ureteral occlusion by peritoneal dissemination and multiple lung metastases were detected. The patient died with acute renal failure on postoperative day 12. Intraatrial extension of thyroid cancer is rare, and only 12 cases have been reported in the literature. We present a case of thyroid cancer with intraatrial extension. PMID- 16680192 TI - The H1 histamine receptor regulates allergic lung responses. AB - Histamine, signaling via the type 1 receptor (H1R), has been shown to suppress Th2 cytokine production by in vitro cultured T cells. We examined the role of H1R in allergic inflammation in vivo using a murine asthma model. Allergen-stimulated splenic T cells from sensitized H1R-/- mice exhibited enhanced Th2 cytokine production. Despite this Th2 bias, allergen-challenged H1R-/- mice exhibited diminished lung Th2 cytokine mRNA levels, airway inflammation, goblet cell metaplasia, and airway hyperresponsiveness (AHR). Restoration of pulmonary Th2 cytokines in H1R-/- mice by intranasal IL-4 or IL-13 restored inflammatory lung responses and AHR. Further investigation revealed that histamine acts as a T cell chemotactic factor and defective T cell trafficking was responsible for the absence of lung inflammation. Cultured T cells migrated in response to histamine in vitro, but this was ablated by blockade of H1R but not H2R. In vivo, allergen specific WT but not H1R-/- CD4+ T cells were recruited to the lungs of naive recipients following inhaled allergen challenge. H1R-/- T cells failed to confer airway inflammation or AHR observed after transfer of WT T cells. Our data establish a role for histamine and H1R in promoting the migration of Th2 cells into sites of allergen exposure. PMID- 16680193 TI - Carcinoembryonic antigen-related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo. AB - Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease. PMID- 16680195 TI - Genome-wide survey for biologically functional pseudogenes. AB - According to current estimates there exist about 20,000 pseudogenes in a mammalian genome. The vast majority of these are disabled and nonfunctional copies of protein-coding genes which, therefore, evolve neutrally. Recent findings that a Makorin1 pseudogene, residing on mouse Chromosome 5, is, indeed, in vivo vital and also evolutionarily preserved, encouraged us to conduct a genome-wide survey for other functional pseudogenes in human, mouse, and chimpanzee. We identify to our knowledge the first examples of conserved pseudogenes common to human and mouse, originating from one duplication predating the human-mouse species split and having evolved as pseudogenes since the species split. Functionality is one possible way to explain the apparently contradictory properties of such pseudogene pairs, i.e., high conservation and ancient origin. The hypothesis of functionality is tested by comparing expression evidence and synteny of the candidates with proper test sets. The tests suggest potential biological function. Our candidate set includes a small set of long-lived pseudogenes whose unknown potential function is retained since before the human mouse species split, and also a larger group of primate-specific ones found from human-chimpanzee searches. Two processed sequences are notable, their conservation since the human-mouse split being as high as most protein-coding genes; one is derived from the protein Ataxin 7-like 3 (ATX7NL3), and one from the Spinocerebellar ataxia type 1 protein (ATX1). Our approach is comparative and can be applied to any pair of species. It is implemented by a semi-automated pipeline based on cross-species BLAST comparisons and maximum-likelihood phylogeny estimations. To separate pseudogenes from protein-coding genes, we use standard methods, utilizing in-frame disablements, as well as a probabilistic filter based on Ka/Ks ratios. PMID- 16680194 TI - A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss. AB - Activating receptor activator of NF-kappaB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-alpha-induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M CSF-induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-alpha promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF. PMID- 16680196 TI - Fold designability, distribution, and disease. AB - Fold designability has been estimated by the number of families contained in that fold. Here, we show that among orthologous proteins, sequence divergence is higher for folds with greater numbers of families. Folds with greater numbers of families also tend to have families that appear more often in the proteome and greater promiscuity (the number of unique "partner" folds that the fold is found with within the same protein). We also find that many disease-related proteins have folds with relatively few families. In particular, a number of these proteins are associated with diseases occurring at high frequency. These results suggest that family counts reflect how certain structures are distributed in nature and is an important characteristic associated with many human diseases. PMID- 16680197 TI - Coverage and characteristics of the Affymetrix GeneChip Human Mapping 100K SNP set. AB - Improvements in technology have made it possible to conduct genome-wide association mapping at costs within reach of academic investigators, and experiments are currently being conducted with a variety of high-throughput platforms. To provide an appropriate context for interpreting results of such studies, we summarize here results of an investigation of one of the first of these technologies to be publicly available, the Affymetrix GeneChip Human Mapping 100K set of single nucleotide polymorphisms (SNPs). In a systematic analysis of the pattern and distribution of SNPs in the Mapping 100K set, we find that SNPs in this set are undersampled from coding regions (both nonsynonymous and synonymous) and oversampled from regions outside genes, relative to SNPs in the overall HapMap database. In addition, we utilize a novel multilocus linkage disequilibrium (LD) coefficient based on information content (analogous to the information content scores commonly used for linkage mapping) that is equivalent to the familiar measure r2 in the special case of two loci. Using this approach, we are able to summarize for any subset of markers, such as the Affymetrix Mapping 100K set, the information available for association mapping in that subset, relative to the information available in the full set of markers included in the HapMap, and highlight circumstances in which this multilocus measure of LD provides substantial additional insight about the haplotype structure in a region over pairwise measures of LD. PMID- 16680198 TI - High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing. AB - For decades, classical crossover studies and linkage disequilibrium (LD) analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in "hot spots." Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region. PMID- 16680199 TI - The Polycomb group protein EED is dispensable for the initiation of random X chromosome inactivation. AB - The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin. The Polycomb repressive complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing. In the mouse, two different forms of XCI occur. In the preimplantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome (Xp). During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal X-chromosome or the Xp; extraembryonic cells, on the other hand, maintain imprinted XCI of the Xp. PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI. It is therefore thought that PRC2 contributes to the initiation of XCI. Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells. Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally derived proteins in the early embryo. Here we show that Eed-/- embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI. Thus, despite being enriched on the inactive X chromosome, PcGs appear to be dispensable for the initiation and maintenance of random XCI. These results highlight the lineage- and differentiation state specific requirements for PcGs in XCI and argue against PcG function in the formation of the facultative heterochromatin of the inactive X-chromosome. PMID- 16680201 TI - Metabolomics: building on a century of biochemistry to guide human health. AB - Medical diagnosis and treatment efficacy will improve significantly when a more personalized system for health assessment is implemented. This system will require diagnostics that provide sufficiently detailed information about the metabolic status of individuals such that assay results will be able to guide food, drug and lifestyle choices to maintain or improve distinct aspects of health without compromising others. Achieving this goal will use the new science of metabolomics - comprehensive metabolic profiling of individuals linked to the biological understanding of human integrative metabolism. Candidate technologies to accomplish this goal are largely available, yet they have not been brought into practice for this purpose. Metabolomic technologies must be sufficiently rapid, accurate and affordable to be routinely accessible to both healthy and acutely ill individuals. The use of metabolomic data to predict the health trajectories of individuals will require bioinformatic tools and quantitative reference databases. These databases containing metabolite profiles from the population must be built, stored and indexed according to metabolic and health status. Building and annotating these databases with the knowledge to predict how a specific metabolic pattern from an individual can be adjusted with diet, drugs and lifestyle to improve health represents a logical application of the biochemistry knowledge that the life sciences have produced over the past 100 years. PMID- 16680200 TI - Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility. AB - OBJECTIVES: Reactive oxygen species (ROS) induced lipid peroxidation is associated with sperm function. Malondialdehyde (MDA) concentration and glutathione peroxidase (GPx) activity represent the lipid peroxidation and spermicidal antioxidant, respectively. We aimed to evaluate the relationship of MDA and GPx levels with sperm parameters. PATIENTS AND METHODS: Specimens were divided into two groups: group 1. normospermia (n=20); group 2. oligoasthenospermia (n=31). Seminal MDA concentration was measured by thiobarbituric acid reaction method. Seminal GPx activity was measured by oxidation of reduced nicotinamide-adenine dinucleotide. Seminal MDA levels and GPx activities in both groups were compared. RESULTS: MDA concentrations in both groups were significantly different (1.52 +/- 0.75 vs. 2.25 +/- 0.88 nM, p = 0.0021). GPx activities in both groups were non-significantly different (0.48 +/- 0.11 vs. 0.47 +/- 0.12 U/ml). MDA levels were negatively correlated with the sperm motility (MDA = -0.014 x motility + 2.62, p =0.017) and concentration (MDA = -0.0045 x concentration + 2.23, p = 0.0166). GPx activities were positively but non-significantly correlated with the sperm concentration and sperm motility. CONCLUSIONS: Seminal MDA concentrations are negatively correlated with sperm concentration and motility, which might provide a simple and useful tool in predicting sperm parameters. GPx activity is non-significantly correlated with the seminal quality. Roles of seminal MDA upon spermatogenesis merits further surveys. PMID- 16680202 TI - The Timing of Entry Into Fatherhood in Young, At-Risk Men. AB - Timing of first fatherhood was examined in a sample of 206 at-risk, predominantly White men, followed prospectively for 17 years. An event history analysis was used to test a model wherein antisocial behavior, the contextual and familial factors that may contribute to the development of antisocial behavior, and common correlates of such behavior, including academic failure, substance use, and early initiation of sexual behaviors, lead both directly and indirectly to an early transition to fatherhood. Having a mother who was younger at first birth, low family SES, poor academic skills, failure to use condoms, and being in a cohabitating or marital relationship predicted entry into fatherhood. Implications of the findings for prevention of and intervention with early fathering are discussed. PMID- 16680203 TI - Use of diagnostic imaging in Australian general practice. AB - The BEACH program, a continuous national study of general practice activity in Australia, gives us an overview of diagnostic imaging ordered by Australian general practitioners. This provides a backdrop against which the theme articles in this issue of Australian Family Physician can be further considered. PMID- 16680204 TI - Radiological tests in investigations of atypical chest pain. AB - BACKGROUND: Atypical chest pain (ACP) is a diagnostic challenge that often requires multiple laboratory and radiological investigations. While radiological procedures may not be the first line of investigation in most cases, they are invaluable when appropriately applied. Physicians need to have a good understanding of the capabilities and limitations of these constantly evolving imaging modalities. OBJECTIVE: This article provides an overview of radiological modalities as they relate to the investigation of ACP. The focus is on radiological procedures that general practitioners may come across when investigating their patients. A brief outline is provided of specialist radiological investigations. The nature of the procedures, their diagnostic yield and appropriateness are addressed. Nonradiological procedures such as coronary angiography, stress tests, ventilation/perfusion scan, resting myocardial perfusion scan, echocardiography and gastroscopies are not discussed. DISCUSSION: While a thorough history and careful physical examination is paramount, diagnostic imaging is indispensable in patients presenting with ACP. Chest X-ray is almost universally obtained. Other modalities are of value only when appropriately applied. PMID- 16680205 TI - Myocardial perfusion imaging - a validated and mature cardiac imaging modality. AB - BACKGROUND: Nuclear myocardial perfusion imaging (MPI) involves the use of radiotracers to generate scintigraphic images of the myocardium. It is the best validated and most standardised of all cardiac imaging modalities, demonstrating regional perfusion ventricular wall motion and accurately calculating reproducible left ventricular ejection fraction. Physiological or pharmacological stress can be used to uncover myocardial ischaemia. OBJECTIVE: This article provides an update on the use of MPI for the triaging of chest pain, monitoring of known ischaemic heart disease, and cardiac event prediction in the general practice setting. DISCUSSION: A normal stress MPI study is an unambiguous outcome. A perfusion defect on a stress MPI study may be produced by stress ischaemia, stable infarction, hibernating myocardium, or by a variable mixture of all three. Patient access to nuclear MPI is good with most nuclear medicine departments offering nuclear cardiology services. Nuclear cardiology is safe and reliable, and deserves to be a part of the routine diagnostic armamentarium in general practice. PMID- 16680206 TI - What's new in vascular interventional radiology? Aortic stent grafting. AB - BACKGROUND: Medical therapies are moving progressively toward 'minimally invasive' techniques resulting in less physiological disruption, smaller 'entry points', reduced intra- and post-operative complications, and earlier discharge. This is exemplified by the emerging technique of abdominal aortic aneurysm treatment with percutaneous endoluminal stent grafting. OBJECTIVE: This article aims to provide an update on aortic stent grafts. DISCUSSION: Aortic stent grafting carries less risk to the patient, is associated with less morbidity and mortality than traditional surgical procedures, and results in earlier discharge. Care in preoperative assessment to ensure the best match between patient and graft is essential, as is attentive postoperative follow up. PMID- 16680207 TI - Uterine artery embolisation - a treatment alternative for women with fibroids. AB - BACKGROUND: Fibroids are a common benign tumour of the female reproductive tract. Uterine artery embolisation is a minimally invasive catheter based technique that compares favourably with surgical treatments. OBJECTIVE: Uterine artery embolisation is an intervention that is especially suited to women who wish to preserve their uterus. This article briefly outlines the medical and surgical treatment options for fibroids with an in depth focus on fibroid embolisation. DISCUSSION: Australian interventional radiologists have been performing uterine artery embolisation for fibroid disease successfully for a number of years. This procedure - like any intervention - is not without complication, but it does enable women a valid alternative treatment pathway for fibroids. General practitioners can discuss the procedure with their local interventional radiologist and can foster a broader understanding of the process with their female patients when considering treatment options. PMID- 16680208 TI - Vertebroplasty - a new treatment for vertebral compression fractures. AB - BACKGROUND: The lifetime risk of a vertebral body compression fracture is 16% for women and 5% for men. Vertebroplastyn involves the injection of artificial bone cement and an opacifier into the inter-trabecular marrow space of the fractured vertebra. OBJECTIVE: This article describes vertebroplasty as a treatment of vertebral compression fractures. DISCUSSION: Treatment for vertebral compression fractures regardless of aetiology has been largely conservative and directed toward pain control. Vertebroplasty has gained popularity since 1987 for treatment of vertebral compression fractures. Vertebroplasty is a safe, effective, and cost effective procedure when performed in an appropriate technical environment by adequately trained interventional radiologists. PMID- 16680209 TI - Metastatic breast cancer. AB - This fourteenth article in our series on breast disease gives an overview of what to look for in the patient with a past history of early breast cancer and how to assess and support the patient with metastatic breast cancer in the general practice setting. Information about the latest medical oncology treatments, including new chemotherapies and targeted 'biological' therapies is provided. PMID- 16680210 TI - Reforming youth mental health. PMID- 16680211 TI - Emerging psychosis in young people - Part 1 - key issues for detection and assessment. AB - BACKGROUND: A delay in the recognition of first episode psychosis is common and is often associated with psychological distress, social decline, and as recent evidence suggests, lasting brain changes. It is increasingly evident that early intervention potentially alters the course of these serious mental illnesses. General practitioners are ideally placed to identify early changes that may herald an emergent psychotic illness and to bridge the existing gap between patients, their families and specialist mental health services. OBJECTIVE: This article summarises the rationale and importance of early detection of emerging psychotic illness in young people, and provides practical tips regarding its assessment and detection. DISCUSSION: Early warning signs of emerging psychotic illnesses include behavioural, emotional and cognitive disturbances. Risk factors include a combination of a family history of a psychotic illness coupled with a decline in functioning, or a history of a brief self limiting psychotic episode (even if triggered by drugs such as cannabis or amphetamines). It is important that those working in primary care recognise these features in order to facilitate a speedy referral to specialist youth friendly mental health services, or if such services are not available, to initiate treatment and provide appropriate ongoing management. PMID- 16680212 TI - Emerging psychosis in young people - Part 2 - key issues for acute management. AB - BACKGROUND: To optimise the management of emerging psychotic disorders in young people, close collaboration between the general practitioner and youth friendly specialist mental health services is favourable. Ideally, a multidisciplinary team including individual case management with expertise in early intervention should treat these patients for the initial 2-5 years. However, only a few areas across Australia currently provide this type of specialised service. Most GPs will at some point be faced with the reality of personally managing young patients with emerging psychotic disorders on their own, sometimes with very limited support. OBJECTIVE: This article summarises key issues for the optimal management of emerging psychotic disorders, with a particular focus on the role of the GP. DISCUSSION: Once an emerging psychotic disorder is confirmed, the engagement of the young person into therapy is the primary target. Ideally therapy integrates not only the patient, but also their family, carers and friends. General practitioners need to inform patients about the nature of the emerging psychotic illness, manage related comorbidities such as substance abuse, and initiate antipsychotic medication to avoid any unnecessary delay in resolution of symptoms. It is important to monitor the patient on a regular basis even after symptom recovery as up to 80% of cases that cease medication will relapse within 5 years. PMID- 16680213 TI - Emerging psychosis in young people - Part 3 - key issues for prolonged recovery. AB - BACKGROUND: After 18 months of specialist care only about half of all first episode psychosis patients achieve functional recovery, and about 10-20% patients will not respond to initial treatment and have persistent psychotic symptoms. These patients need special attention in order to minimise the burden of disease and prolonged disability. OBJECTIVE: This article reviews the management of young first episode psychosis patients with incomplete recovery, and focuses on the general practitioner's role, how to deal with treatment resistance, vocational rehabilitation, and other aspects of patient care. DISCUSSION: Once the acute episode has been treated, it is important to avoid complacency and address other aspects contributing to a patient's wellbeing including social welfare, physical health and vocational rehabilitation. The prevention of relapse and the psychosocial development of the individual are key in fostering and promoting a healthy lifestyle, leading to improved quality of life. Treatment refractory patients need specialist care. PMID- 16680214 TI - Suicide prevention - targeting the patient at risk. AB - BACKGROUND: Suicide is a major cause of death from adolescence upward. While there has been a concentrated effort to educate families and health workers to be aware of warning signs of pending suicide, there has been limited information or practical strategies available for people at risk of suicide or for those who have suicidal thoughts. Recent acknowledgment of the role of impulsivity in suicide highlights the need for such information. OBJECTIVE: This article identifies those at risk of suicide and outlines strategies for suicide prevention. DISCUSSION: Patients with suicidal thoughts are not an infrequent component of presentations in general practice and present a considerable challenge to the general practitioner because it is very difficult to predict who will make a suicide attempt. There has been much published to assist the GP to identify those at risk, but little to suggest how to assist the patient in such a crisis. Research into the role of impulsivity in suicide would suggest that unless the patient has strategies to confront the suicidal thoughts themselves, they remain at significant risk. PMID- 16680215 TI - Colorectal cancer and CHF - reviewing the evidence for complementary medicine. AB - This is the third of a series of articles looking at the available evidence for complementary medicine. Physical activity is important in attenuating harmful effects of aging and the onset of chronic disease. Benefits of physical activity in preventing or delaying cardiovascular disease and managing obesity and arthritis have mainstream acceptance. However, evidence is emerging of the benefits of physical activity in other conditions such as cancer. PMID- 16680216 TI - Bone mineral density - frequently asked questions. PMID- 16680217 TI - Loss of chance: what loss? AB - A recent New South Wales judgment, Rufo v Hosking, explored the concept of 'loss of a chance' in medical negligence claims. 'Loss of a chance' claims involve an allegation that the patient lost the chance of a better outcome as a result of the negligence of the medical practitioner. This article outlines the case and discusses the implications of the judgment for medical practitioners. PMID- 16680218 TI - Professional autonomy - is it the future of general practice? AB - Internationally, rising financial costs and increasing expectations of health care delivery have increased regulation and decreased the autonomy of general practitioners and other health care professionals. This article explores professional autonomy within Australian general practice, and outlines the importance of autonomy in systems approaches to organisational change in general practice. PMID- 16680219 TI - A teaching ward round in infectious diseases - a pilot module. AB - The ongoing care of patients requires close communication between general practitioners and hospital specialists. In 2005, the General Practice Liaison Office and Department of Infectious Diseases at The Canberra Hospital designed a pilot module to promote interaction between GPs and hospital specialists and to provide an educational opportunity for GPs to be updated on the hospital practice of managing infectious diseases. The simplicity of the module is the key to its generalisability outside Canberra and Australia. PMID- 16680220 TI - Reflecting on research. AB - Reflecting on my first 4 months as a part time academic general practice registrar demonstrates the highs and lows of pursing the challenges of research - particularly after being involved in full time clinical practice. Exploring research in general practice has proved an interesting and rewarding experience. PMID- 16680221 TI - Research papers submitted to Australian Family Physician - types and timelines. AB - BACKGROUND: Articles published in the research section of Australian Family Physician (AFP) are subject to an editorial process comprising several stages. METHOD: Timelines tracking the movement of each research manuscript submitted to AFP from 2002-2004 through all stages of the editorial process were constructed. Of 179 papers, 130 had sufficiently progressed to be included in this study. Manuscripts were grouped by subject matter into eight categories. RESULTS: Waiting for authors' responses to editorial feedback (with reviewers' reports) was the greatest cause of delay to AFP editorial processes. Peer reviewers took 43 (SD: 102) days to return their report. Authors took 67 (SD: 76) days to resubmit their paper following initial feedback, and a further 48 (SD: 79) days after it had been edited. Mean accumulated time between receipt of a manuscript by AFP and sending it to peer review was 15 days. Once the editorial process was completed, articles were usually published within 3 months. Most research (64%) was on the topic of health services research rather than clinical research (36%). The most common research method was observational (78%) rather than experimental (22%). DISCUSSION: There is less clinical research submitted to AFP than expected for a clinical discipline. Authors and reviewers cause the most delay in manuscripts' passage through the editorial process. PMID- 16680222 TI - Do GPs use electronic mental health resources? - a qualitative study. AB - BACKGROUND: The Better Outcomes in Mental Health Care (BOMHC) initiative encourages general practitioners to use electronic mental health resources (EMHRs) during consultation with patients requiring psychological assistance. However, there is little data on GPs' acceptance and use of EMHRs. METHOD: Semistructured interviews were conducted with 27 GPs to determine their attitude toward EMHRs, and their use during consultation with patients. RESULTS: Few GPs reported frequently using EMHRs in consultation. Identified barriers to use included lack of familiarity with information technology, and insufficient knowledge of available resources. Identified advantages of electronic resources included high patient acceptance, time efficiency, and improved quality of information. DISCUSSION: General practitioners recognise several advantages of utilising electronic resources for managing patients with mental illness. However, GPs are not sufficiently familiar with electronic resources to use them effectively. This could be overcome by education. PMID- 16680223 TI - Auditory symptoms in whiplash patients - could earwax occlusion be a benign cause? AB - BACKGROUND: Excess cerumen (earwax) in the external auditory canal is associated with symptoms of earache, fullness in the ears, and diminished hearing. These symptoms, and tinnitus, are commonly associated with whiplash injury. METHODS: Eighty-six whiplash patients were examined to determine if there was a correlation between symptoms of earache, fullness in the ear, diminished hearing, and tinnitus, and the degree of cerumen occlusion. Cerumen occlusion was measured by visualisation of the tympanic membrane and graded according to a 4 point scale. RESULTS: Of 71 subjects reporting no acute onset (within 7 days of the collision that caused their whiplash) earache, fullness in the ears, hearing loss, or tinnitus, 62 had little or no cerenum occlusion. Of seven subjects reporting tinnitus but no other auditory symptoms, none had greater than moderate cerenum occlusion. Of eight subjects reporting one or more of acute onset earache, fullness in the ears, diminished hearing, and tinnitus, seven had complete cerenum occlusion in the affected ear. DISCUSSION: The findings suggest high grade cerumen occlusion frequently occurs in the ear affected by acute auditory symptoms. However, tinnitus alone has no apparent association with cerumen occlusion. It is possible that a significant number of acute onset auditory symptoms reported in whiplash patients have a benign cause. PMID- 16680224 TI - Romanian Journal of Gastroenterology 2006: a new title, look and editorial team. PMID- 16680225 TI - Towards an efficient reprocessing of endoscopes: on the lack of means and machines. PMID- 16680226 TI - The diagnostic value of cytokeratins and carcinoembryonic antigen immunostaining in differentiating hepatocellular carcinomas from intrahepatic cholangiocarcinomas. AB - AIM: To study the differences between the hepatocellular carcinoma (HCC) and peripheral type of cholangiocarcinoma (CHC) using cytokeratin (CK) and carcinoembryonic antigen (CEA) expressions and assessing their accuracy on paraffin sections in the differential diagnosis. MATERIAL AND METHOD: The following antibodies were analyzed: AB1 complex (anti CK9-CK20), AB2 complex (anti CK1-CK8), pCEA, and the monoclonal antibodies against cytokeratins CK7, CK8/18, CK17 and CK19. In the mmunohistochemical studies, 15 selected surgically resected liver tumors, 10 HCCs and 5 CHCs, with well established diagnosis (by morphological criteria) were included. Other markers, such as AFP si CA 19-9, were not available. RESULTS: No CHC, but 50% of HCCs were positive for CEA, presenting a canalicular staining pattern. For CK 7, all but one (which was focally positive), meaning 80% of CHCs were diffusely positive, whereas only two HCCs were positive. For CK 19, 80% of CHCs were diffusely positive, while all but two HCCs (a moderately and a poorly differentiated tumor) were negative. For CK 8/18, 70% of HCCs were diffusely positive, whereas only 20% of CHCs were positive. For CK 17, 60% of CHCs were positive, while all HCCs were negative. 80% of CHCs were positive for AB1 anti-CKs complex, whereas only 50% of HCCs were positive, and relating to AB2 anti-CKs complex, 50% of HCCs were diffusely positive and only 20% of CHCs. CONCLUSION: The immunohistochemical expression of CKs and CEA might be considered helpful in addition to other diagnostic criteria for the differential diagnosis of primary carcinomas of the liver, especially in difficult cases. PMID- 16680227 TI - The epidemiology and clinical findings of colorectal cancer in Iran. AB - BACKGROUND: This study was performed to evaluate the prevalence, clinical features and management of patients with colorectal cancer (CRC) in Iran. METHODS: All cases of CRC presented to a principal referral University hospital (SGH) for cancers in Teheran, Iran between June 20, 2000 and January 3, 2003 were retrospectively reviewed. RESULTS: A total of 200 patients were included. 114 patients (57%) were male and 86 were female (43%). Age ranged from 13-90 years with a mean of 55.15 +/- 14.5. Among these patients, 16.5% were less than 40 years of age. The tumors were categorized according to their distribution as appendix (n=4), cecum ( n=7), right colon (n=1), hepatic flexure (n=2), transverse colon (n=19), splenic flexure (n=3), left colon (n=6), sigmoid ( n=16), rectum (n=117), rectosigmoid and rectal lesions (n=16), and colorectal lesions without known locations (n=9). Non-mucinous adenocarcinoma (AC) was the most common histological type (n=181, 90%), followed by mucinous AC (n=15), squamous cell carcinoma (n=1), carcinoid (n=1), melanoma (n=1) and signet ring carcinoma (n=1). The most common presenting symptom was rectal bleeding (n=68, 34.5%). Younger patients had a greater preponderance of mucinous AC (p=0.008) and generally underwent more extensive chemotherapy as seen with more usage of 5 Fluorouracil (p=0.05). CONCLUSIONS: We have had few reports with reference to the epidemiology of CRC in Iran since 1977. The current data suggest a younger age distribution for CRC. We found no significant difference between age, gender and type of cancer with subsite distribution. Distal CRC was more prevalent. Among patients, 30.5% were diagnosed by screening studies. PMID- 16680228 TI - Mortality risk factors in chronic pancreatitis. AB - BACKGROUND: Mortality in chronic pancreatitis is higher than in the general population, the 10-year survival after diagnosis is estimated between 69-80%. AIM: Evaluation of mortality risk factors in chronic pancreatitis. MATERIAL AND METHOD: Eighty-two patients with chronic pancreatitis were followed-up for an average period of 25 months (median 25 months). None of them had an endoscopic treatment before inclusion in this study. The average age of the patients was 48 years (range 29 to 78, median 49), the ratio men:women being 6.5:1. The etiology was alcoholic in 84.2 % cases, pancreas divisum in 8.5% cases and idiopathic in 7.3% of cases. RESULTS: During the follow-up period the mortality rate was 17%, death occurring at on average 59 months (median 53 months) from the onset of the disease. The most frequent causes of death were: pancreatic cancer (3.6%), complications after surgery (3.6%) and upper digestive hemorrhage (2.4%). The mortality risk factors were presence of diabetes, no alleviation of pain under treatment and unceasing of smoking. The type of treatment applied did not influence survival. The cumulative survival rate estimated at 3 years was 80% and at 5 years 59%. CONCLUSIONS: The mortality rate in chronic pancreatitis was higher than those reported in the literature. Death caused by pancreatic cancer occurred in 3.6 % of the patients. There were no cases of death due to extra pancreatic cancers. The mortality risk factors were unceasing of smoking, no alleviation of pain under treatment and presence of diabetes. PMID- 16680229 TI - A gastroenteritis outbreak due to Norovirus infection in Xanthi, Northern Greece: management and public health consequences. AB - A gastroenteritis outbreak was observed in the town of Xanthi on Jan 28, 2005. A total of 709 patients (350 males and 359 females, mean age 23.8 +/- 19.95 years) visited the local hospital over a period of two weeks with symptoms of fever (22.3%), abdominal pain (78.6%), nausea (85.5%), vomiting (67.1%) and diarrhea (72.5%). PCR for Norovirus in randomly selected stool specimens examined was positive in all cases (8/8). No other pathogen was revealed. As 85.1% of the primary affected individuals had been using water from a single well (supplying approximately 40% of the 34,889 inhabitants), and water specimens from this well were found to carry a high bacterial load, the waterborne transmission of Norovirus was the main suspected cause of the outbreak. People were advised to consume bottled water and to wash their hands carefully especially after toilet use until the remission of the outbreak. Additionally, more chloride was added to the suspected well (10 ppm or 10 mg/l). Fourteen days later, the outbreak waned and all measures were withdrawn. PMID- 16680230 TI - The effect of interferon alpha plus ribavirin on health-related quality of life in chronic C hepatitis. The Romanian experience. AB - BACKGROUND AND AIM: Interferon plus Ribavirin represent the most effective therapy for chronic hepatitis C. Its efficiency is limited by the occurrence of numerous side-effects. A previous study observed in Romanian patients an impairment of quality of life in chronic viral hepatitis C before antiviral therapy. We looked for the effect of this treatment on health-related quality of life in chronic hepatitis C when measured immediately after the antiviral therapy. METHODS: 35 patients with hepatitis C were given Interferon alpha 3MU tiw plus Ribavirin 1000-1200 mg for 48 weeks. Questionnaire-based assessments of health-related quality of life were performed before and immediately after the treatment using the SF-36 questionnaire. Also, in pretest, the health-related quality of life scores were compared in these patients with "normal" subjects (matched on age and sex). In order to compensate for the lack of a specific control group during the longitudinal study, normative comparisons were performed in posttest. RESULTS: Before treatment, patients had significant impairment in all SF-36 concepts compared to "normal" controls (all p < 0.05). The level of transaminases before the treatment was high (out of the normal range) and decreased after the treatment, reaching the normal range (27 of 35). Immediately after the treatment, SF-36 scores did not show any significant differences, as compared to the pretest, no matter what the viral response was (p > 0.10). CONCLUSIONS: Patients with chronic viral hepatitis C have a markedly reduced health-related quality of life both before and after the treatment. The antiviral therapy does not influence the level of the quality of life, when measured immediately after finishing the treatment. PMID- 16680231 TI - Urgent surgery for complicated colonic diverticula. AB - The AIM of this retrospective study was to evaluate the emergency surgical treatment of life-threatening complications of colonic diverticula. MATERIAL AND METHOD: In the last 11 years, 22 of 101 patients with colonic diverticula (22.1%) underwent urgent surgery for acute complications: perforated gangrenous diverticulitis with generalized peritonitis (n=8) or pericolic abscess (n=8), acute bowel obstruction (n=4) and severe diverticular bleeding (n=2). In all patients with diffuse peritonitis or acute obstruction the indication for surgery was decided on clinical basis and the complicated diverticula were recognized only intra-operatively. RESULTS: Emergency surgical strategy differed according to the type of complication and the biologic condition of the patient: segmental colectomy and primary anastomosis for diverticular perforation (n=4), colonic stenosis (n=3) or diverticular bleeding (n=2); Hartmann resection with late reconnecting anastomosis in patients with diverticular perforation (n=5) or colonic obstruction (n=1); diverticulectomy with peritoneal drainage (n=2) and colostomy and drainage followed by secondary colectomy (n=5) for diverticular perforations in patients with poor general condition. Only one patient (4.5%) died post-operatively of multiple organ failure from generalized peritonitis. There was no anastomotic leakage in patients with primary anastomosis. Six patients (27.2%) developed wound infection. Hospital stay ranged between 11 and 60 days, significantly longer in cases with two-stage operations. CONCLUSION: Primary colectomy with immediate or delayed anastomosis is the best surgical procedure for acute divericular complications in patients with good biologic status. Two-stage operations such as colostomy and drainage coupled with late colectomy remain the viable alternative in patients with advanced disease and critical biologic condition. PMID- 16680232 TI - Anorectal incontinence. pathogenesis and choice of treatment. AB - Fecal incontinence represent a complex and multifactorial disorder. Although the condition is widely accepted as belonging to the elderly, it is now becoming apparent that younger adults are also frequently affected. Its incidence is estimated at 2% of the general population, while in the elderly it may increase up to 60%. Despite the considerable advances that have been made in the evaluation of anorectal incontinence during the past decades, the cause of this entity still remains obscure. The patient's history, the physical examination, and specialized investigations are essential for the diagnosis and the selection of the appropriate treatment. In clinical practice the most useful tests are the anorectal manometry, the anal endosonography and the pudendal nerve latency. Complete functional and anatomical assessment of the anorectum, the anal sphincters, and the pelvic floor is mandatory in all patients with fecal incontinence for the appropriate diagnosis and identification of the cause, the type of incontinence and the selection of the appropriate treatment. PMID- 16680233 TI - Spontaneous bacterial peritonitis: pathogenesis, diagnosis, treatment. AB - Due to inadequate defence mechanisms, cirrhotic patients with ascites have an increased susceptibility to infections, the most frequent and the most severe one being spontaneous bacterial peritonitis (SBP). SBP diagnosis is based on testing of the ascitic fluid obtained by paracentesis. A polymorphonuclear cell count of more than 250 cells/mm3 of ascitic fluid is considered diagnostic and from cultures of ascitic fluid only one germ should be isolated. 60% of the SBP episodes are produced by gram negative enteric bacilli - E. coli and Klebsiella spp. being the most frequent isolated microorganisms. The most important pathogenic mechanism for SBP is bacterial translocation. In liver cirrhosis, three mechanisms are proposed for the pathogenesis of SBP: intestinal bacterial overgrowth, the alterations (structural and functional) of the intestinal mucosal barrier and the deficiencies of the local immune response. The most appropriate antibiotic treatment is a third generation cephalosporin (Cefotaxim or Ceftriaxon) which should be administrated for 5 days. With early start of the antibiotic treatment, the short-term prognosis of cirrhotic patients with SBP has improved significantly. Unfortunately, the long term prognosis remains extremely poor due to the severity of subjacent liver disease. PMID- 16680234 TI - Liver involvement in Langerhans' cell histiocytosis. Case report. AB - Langerhans'cell histiocytosis (Histiocytosis X) is a rare disease of unknown cause characterized by oligoclonal proliferation of Langerhans cells. It occurs mostly in children and young adults and involves one or more body systems such as bone, hypothalamus, posterior pituitary gland, lymph nodes, liver or various soft tissues. The diagnosis is always made by a histological approach. We report a case of Langerhans'cell histiocytosis in a young patient with clinical signs of diabetes insipidus and hepatic involvement in whom the immunohistochemical analysis of the liver tissue led to the definitive diagnosis. PMID- 16680235 TI - Diagnostic features of benign pancreatic insulinomas. An analysis of three cases. AB - Insulinoma is a neuroendocrine tumour deriving mainly from the pancreatic islet cells that produces excessive amounts of insulin. Accurate preoperative detection and localization of insulinoma is essential for the selection of appropriate candidates for surgery. We present the case reports of three patients with benign pancreatic insulinoma. Preoperative assessment was performed by using transabdominal ultrasonography, computed tomography and magnetic resonance imaging. Their performances for the effectiveness in detecting and localizing benign insulinoma were compared. We concluded that an appropriate preoperative imaging assessment was in our case impossible, intraoperative inspection and palpation of the pancreas still being superior. PMID- 16680236 TI - Axial torsion and gangrene of a giant Meckel's diverticulum. AB - A variety of complications are related to a Meckel's diverticulum, including hemorrhage, intestinal obstruction and inflammation. Axial torsion and gangrene of Meckel's diverticulum is the rarest of the complications that have been reported, with this being particularly true in case of children. We report a case of axial torsion and gangrene of a giant Meckel's diverticulum in a 6 year old child. PMID- 16680237 TI - Superior mesenteric artery syndrome: an unusual case of intestinal obstruction. AB - Superior mesenteric artery (SMA) syndrome is an uncommon cause of proximal intestinal obstruction, frequently occurring in patients who have had an important weight loss. The diagnosis can be difficult and usually is made by exclusion. The most characteristic symptoms are postprandial epigastric pain, fullness, voluminous vomiting and eructation. These symptoms are due to the compression of the third portion of the duodenum against the posterior structures by a narrow-angled SMA and surgical management is necessary. We report a case of SMA syndrome in a 23 year old patient, with a long history (since childhood) of voluminous vomiting, epigastric pain and an important weight loss. We performed small bowel enteroclysis, upper gastrointestinal series and endoscopy, biopsy of gastric and duodenal mucosa, abdominal computer tomography and ultrasonography to establish the diagnosis. Finally, the patient successfully underwent duodenojejunal anastomosis with a postoperative favourable outcome. PMID- 16680238 TI - Extrahepatic biliary obstruction due to a solitary pancreatic metastasis of squamous cell lung carcinoma. Case report. AB - A 53-year old male, with a history of squamous cell lung carcinoma, was presenting with jaundice. Examinations showed a pancreatic tumor infiltrating the common bile duct and a percutaneous biopsy proved that the lesion was metastatic from the lung carcinoma. The decision was taken to perform a laparotomy. During laparotomy, a palliative operation was performed to relieve the jaundice. According to the literature, symptomatic metastatic lesions of the pancreas from squamous cell carcinoma of the lung are infrequent. PMID- 16680239 TI - Narrow-band imaging endoscopy for diagnosis of malignant and premalignant gastrointestinal lesions. AB - Narrow-band imaging (NBI) is a novel endoscopic technique that may enhance the accuracy of diagnosis by using narrow-bandwidth filters in a red-green-blue (R/G/B) sequential illumination system. Magnifying endoscopy by using NBI has two distinct applications: the analysis of the surface architecture of the epithelium (pit pattern) and the analysis of the vascular network. This new technique allows a better characterization of distinct types of gastrointestinal epithelia (e.g. intestinal metaplasia in Barret's esophagus), as well as the disorganization of the vascular pattern in inflammatory disorders and the irregular pit pattern in early neoplastic lesions of the esophagus, stomach and large bowel. PMID- 16680240 TI - Cleaning and disinfection in gastrointestinal endoscopy: current status in Romania. AB - BACKGROUND: Today, endoscopic procedures have become an important tool in the diagnosis and treatment of gastrointestinal diseases. In order to prevent cross infections, the cleaning and disinfection/sterilisation procedures of the endoscopes and accessories are primordial. The GOAL of this paper was to assess the way and the conditions in which endoscopic disinfection is achieved in our country. METHODS: A study-type questionnaire with eighteen questions, about reprocessing of flexible endoscopes and accessories, was addressed to 37 centres from Romania known to perform digestive endoscopy. RESULTS: Twenty-nine centres (78.37%) answered the questionnaire. In all the cases, the reprocessing is based on pre-established protocols and the endoscopy nurse has the principal responsibility (62.06%). Enzymatic type detergents are available in 75.86% and glutaraldehyde is the most used disinfector (55.17%). The costs (41.38%) and the financial resources (27.6%) are considered when selecting a product. Despite their advantages, automatic-disinfectors are available in only 34.48% of the cases. All the participants in the study (100%) reused endoscopic instruments, but only 31.03% used them after sterilization by steam autoclaving or ethylene oxide. Adequate facilities for manipulating potential toxic solutions are assured in 48.27%, but separate purpose-designed rooms are available in only 27.58%. Most centres (86.20%) regularly control the quality of the cleaning and disinfection by sampling. CONCLUSIONS: In general, the disinfection and sterilization of the endoscopes and accessories are carried out in good conditions. With regard to European Union integration, adequate facilities and funds are required to improve the reprocessing activity. PMID- 16680241 TI - Severe polyneuropathy under interferon therapy. PMID- 16680242 TI - Troponin estimation identifies myocardial infarction patients with different characteristics. AB - OBJECTIVE: To investigate the impact of new myocardial infarction definition based on troponin, the rate of myocardial infarction diagnosis, patients characteristics, and short-term prognosis. METHODS: We enrolled 1,255 consecutive myocardial infarction patients from the Kuwait Registry of Acute Coronary Syndromes from December 2003 to May 2004. Two patient groups were identified, those diagnosed with elevated creatine kinase-MB (CK group) and those diagnosed with elevated troponin with normal CK-MB (Troponin group). RESULTS: The use of troponin have increased the number of myocardial infarctions by 49%. Compared with the CK group, the Troponin group were older [age 60.3 +/- 12.5 years versus 53.7 +/- 12.2 years, p<0.001], more likely to have diabetes (59% versus 41%, p<0.001), hypertension (59% versus 36%, p<0.001), and hypercholesterolemia (37% versus 24%, p<0.001). The Troponin group were more likely to suffer heart failure at presentation than the CK group (32% versus 14%, p<0.001) and, subsequently, increase incidence of heart failure during their hospital stay (17% versus 8%, p<0.001). CONCLUSION: A substantial increase in the rate of myocardial infarction occurred with the adoption of the new diagnostic criteria. The clinical outcome for the additional patients diagnosed was not better than that of patients diagnosed by the old criteria. PMID- 16680243 TI - Avian influenza. AB - A rapidly spreading, highly pathogenic avian influenza virus A H5N1 in the domestic poultry population has crossed the species barrier to humans and other mammalian species, thus, posing an increasing pandemic threat. The World Health Organization, other agencies, and countries worldwide are closely monitoring the prevalent influenza viruses and their related illnesses to detect any increased virulence or transmissibility that might signal the beginnings of any future pandemic. So far, the H5N1 virus has infected birds in more than 30 countries in Asia, Europe and Africa, while further geographical spread remains likely. Human infections are still rare and the virus does not spread easily from birds to humans or readily from person to person. Although antiviral drugs and vaccination are among the most important measures to be used in case of an influenza pandemic, a timely supply of sufficient quantities will not be possible. This review describes various aspects of avian influenza in birds and in humans; epidemiology, transmission, diagnosis and clinical manifestations. Also presented are the global preparedness, the anti-influenza drugs and vaccines. PMID- 16680244 TI - Diabetic foot infection. AB - Diabetic foot infection DFI is simply defined as suspected or documented infection of the tissues that comprise the foot of a diabetic patient. Diabetic foot infection is often caused by introduction of an infection into the otherwise sterile soft tissues of the foot through a minor skin break down. Diabetic foot infection may be mild usually restricted to the uppermost layers of the skin, moderate extending down to the soft tissues of the foot or severe infection associated with systemic toxicity or metabolic instability. The paper reviews the types of DFI, pathophysiology, microbiology of DFI, relevant anatomy of the foot, clinical evaluation, measures of severity of DFI, the role of radiological investigations, and the role of early surgical intervention in the prevention of progressive foot infection and limb salvage. It is concluded that the diagnosis of DFI should be suspected at an early stage based on the presence of local signs of inflammation with or without systemic signs of toxicity or metabolic instability. Optimal treatment of DFI requires a multimodality approach directed at controlling hyperglycemia, administration of systemic antibiotics, and local wound management to prevent the spread and dissemination of infection. PMID- 16680245 TI - The ICAM-1 469 T/C gene polymorphism but not 241 G/A is associated with Behcets disease in the Lebanese population. AB - OBJECTIVE: To investigate the association of the 2 intracellular adhesion molecules-1 (ICAM-1) gene polymorphisms [thymidine/cytidine (T/C) 469 and guanosine/adenosine (G/A) 241] in Behcets disease in Lebanon. METHODS: We initiated the study in July 2003, and carried out the work in the research laboratory of Beirut Arab University, Beirut, Lebanon. We extracted the DNA by glass fiber matrix mini column. We amplified the ICAM gene by polymerase chain reaction (PCR) and tested the PCR products for the presence of the polymorphisms using a restriction enzyme specific for each polymorphism. We analyzed the results by agarose electrophoresis. RESULTS: We demonstrated the association of only one single nucleotide polymorphism (SNP) (K469) with Behcets disease, while we could not detect the other SNP (G241A) in either controls or patients in the Lebanese population. CONCLUSION: The ICAM-1 gene polymorphism 469 T/C, but not 241 G/A, may encode risk for Behcets disease in the Lebanese population. PMID- 16680247 TI - The prevalence of parasites in commonly used leafy vegetables in South Western, Saudi Arabia. AB - OBJECTIVE: To determine the degree of contamination caused by parasites in commonly used leafy vegetables in Abha, Kingdom of Saudi Arabia (KSA). METHODS: We carried out the study in the Department of Clinical Microbiology and Parasitology, King Khalid University, Abha, KSA, during the period September 2004 to May 2005. Five commonly used leafy vegetables, namely, green onion, radish, watercress, lettuce and leek, were washed each in water and Tris-buffer-saline (TBS). The washing solution was then centrifuged and the sediments were examined for parasites. RESULTS: The use of TBS for the extraction, significantly increased the isolation rate (27.2%) of the parasites compared with the use of tap water (7.8%) (z=4.72 p<0.001). The prevalence of the parasites was 28% in green onion, 25% in radish, 17% in watercress, 17% in lettuce, and 13% in leek. The parasites were more common in the months of September to December. Ankylostoma duodenale, Entameba coli, Ascaris lumbricoides and Blastocystis hominis were the most common isolated parasites. We encountered 12 genera of parasites during the study and the least common was iodamoeba butschlii. CONCLUSION: The study shows that parasites are common in leafy vegetables and the use of tap water does little to remove them. The public health implications of our findings will be communicated to the Aseer regional health authorities for appropriate community health education and other necessary actions. PMID- 16680246 TI - The prevalence of extended-spectrum beta lactamase-producing Escherichia coli and Klebsiella pneumoniae in clinical isolates and risk factors. AB - OBJECTIVE: To determine the prevalence of extended-spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), risk factors of ESBL-producing strains and antimicrobial susceptibility pattern of ESBL-producing and non producing strains. METHODS: The study took place at the Faculty of Medicine, Osmangazi University, Eskisehir, Turkey from March to November 2002. We evaluated 100 K. pneumoniae and 100 E. coli strains isolated from various clinical specimens, as well as the patients from whom these strains were isolated. The double-disk synergy test was performed on the isolates for the detection of ESBL. We visited the patients with a growth of E. coli or K. pneumoniae or both from their clinical specimens in their wards if they were hospitalized, while the outpatients with a growth of these microorganisms were evaluated from their hospital records. RESULTS: The prevalence of ESBL-producing K. pneumoniae was 47% and E. coli was found as 12%. The ESBL-producing isolate rates were 50% (14/28) in intensive care units, 36.1% (35/97) in wards and 13.3% (10/75) in outpatients. Foley catheter (p<0.001), intravenous catheter (p<0.001), central venous catheter (p=0.002), intubation (p<0.001), surgery (p<0.001) and mechanical ventilation (p=0.002) were found as the risk factors for the acquisition of E. coli and K. pneumoniae with ESBLs. CONCLUSION: In our study, the prevalence of ESBL-producing isolates was high. The results of the study suggest that an antimicrobial policy and early removal of interventional apparatus be of importance for the control of ESBL-producing K. pneumoniae and E. coli. PMID- 16680248 TI - Anti-hepatitis delta virus seroprevalence and risk factors in patients with hepatitis B in Southeast Turkey. AB - OBJECTIVE: To obtain regional epidemiological data on hepatitis delta virus (HDV, a defective virus) infections, the incidence of anti-HDV positivity and the associated risk factors in asymptomatic hepatitis B virus surface antigen (HBsAg) carriers and in patients with chronic active hepatitis B. METHODS: The study took place at Dicle University Hospital (Diyarbakir, Southeast of Turkey) between January 2002 and July 2004. Anti-HDV screening was performed in asymptomatic hepatitis B carriers (N=889) and in patients with chronic active hepatitis B infection (N=120). We explored the association between anti-HDV positivity and asymptomatic hepatitis B carrier status, presence of active hepatitis B, age, gender, the durations of HBsAg positivity and hepatitis B e antigen (HBeAg) positivity. RESULTS: In 6% of asymptomatic hepatitis B carriers (53/889) and in 27.5% of patients with chronic active hepatitis B (33/120) anti-HDV was positive. The incidence of anti-HDV positivity was significantly higher in patients with chronic active hepatitis B compared with asymptomatic carriers (p<0.001). A significant association between the duration of HBsAg carrier status (3.2 +/- 1.4 years) and anti-HDV positivity was also found (p<0.001). Age, gender, and HBeAg positivity were not significantly associated with anti-HDV positivity (p>0.05). CONCLUSION: Anti-HDV positivity was significantly more common in patients with chronic hepatitis B compared with asymptomatic hepatitis B virus (HBV) carriers in a region with a high prevalence of HBV infection. We found a significant relationship between the duration of HBsAg carrier status and anti-HDV positively, however, age, gender, and presence of HBeAg were not significantly associated with the development of anti-HDV positivity. PMID- 16680249 TI - Prevalence of Helicobacter pylori infection in patients with peptic ulcer diseases. AB - OBJECTIVE: To determine the prevalence of Helicobacter pylori (H. pylori) infection among patients presenting with peptic ulcer disease (PUD) and to establish the relationship between H. pylori infection and its diverse pathology. Secondly, we aimed to examine the effect of age, gender, and geographical distribution on the prevalence of the disease. METHODS: The study included patients with PUD who underwent upper gastrointestinal endoscopy at Hera General Hospital, Makkah, Kingdom of Saudi Arabia between January 2003 and February 2004. We tested the biopsies for the detection of H. pylori by Campylobacter like organism (CLOTM) test, histopathology, immunoglobulin G (IgG) antibodies, culture, and antimicrobial susceptibility testing. RESULTS: The overall prevalence rate of H. pylori infection among 132 patients with PUD was 63%, while it was high among females (70%) as compared with males (58%). The H. pylori were mainly found in chronic active gastritis (89%) and severe active gastritis (96%). Helicobacter pylori were documented by CLOTM test in 73 (55.3%) cases, histopathology in 69 (52.3%) cases, microbiological culture in 59 (44.7%) cases, and IgG antibodies in 61 (46.2%) cases. The highest resistance (31%) was found in metronidazole, while lowest the (3%) in tetracycline and erythromycin. CONCLUSION: The highest prevalence of H. pylori was found in the younger age group with female preponderance. The leading causes of multifocal pathology were chronic and severe active gastritis secondary to H. pylori infection. PMID- 16680250 TI - Intestinal parasitic infections among school children of the Dhahira Region of Oman. AB - OBJECTIVE: To determine the prevalence of intestinal parasitic infections among primary school children in Dhahira region of Oman. METHODS: The study took place in the Dhahira region of Oman from September 2004 to March 2005. We randomly recruited 436 students of 9-10 years of age. Their anthropometric measurements were noted. We collected the stool sample of each student and examined for different intestinal parasites. Stereoscopic microscopy and the World Health Organization's approved KATO-KATZ technique were used. RESULTS: Of the 436 students examined, 65 children (15%) were undernourished (body mass index (BMI) <14 kg/m(2)). The prevalence of intestinal parasitic infections was 38.7% (95% CI 33.2-42.2). The prevalence of protozoan infection was 36% while helminth infection was 9.4%. The prevalence of Entamoeba histolytica/Entamoeba dispar was 24%, Giardia species 10.5% and Escherichia coli 1.4%. The hookworm (Ancylostoma duodenale, Necator americanus), Ascariasis lumbricoides, Trichuris trichiura, Hymenolepiasis nana, Taenia species, Enterobius vermicularis and Strongyloides infections in our study had very low prevalence. Only 29 (6.4%) students had parasitic infection by more than one parasite. The wet zone was significantly associated to the intestinal parasitic infections [Adjusted odds ratio (OR)=3.2 (95% CI 1.9-5.2)]. CONCLUSION: Dhahira region could be classified as low prevalence and low intensity area for soil transmitted helminths infections. However, the magnitude of the protozoan infection was high. The school health program should focus on parasitic infection treatment and addressing the underlying causes of this problem. PMID- 16680251 TI - The pattern of active pulmonary tuberculosis in adults at King Hussein Medical Center, Jordan. AB - OBJECTIVE: To identify the pattern of the clinical, radiological, and diagnostic procedures of the diagnosed cases of active pulmonary tuberculosis (TB) patients presented to the Respiratory Medicine Division at King Hussein Medical Center over the last 10 years. METHODS: This is a retrospective analysis of the medical records and chest radiographs of 137 active pulmonary TB patients who were diagnosed between March 1995 and October 2005. Patient's symptoms were recorded and analyzed. Radiological findings were assessed. Procedures used to identify Mycobacterium TB were identified. RESULTS: One hundred and thirty-seven patients medical records were retrieved and analyzed (84 males and 53 females). The mean age (range) was 48.43 +/- 14.65 (14-83) years. The most common presenting symptoms were cough (79%), weight loss (74%), and fatigue (69%). Other presenting complaints were fever (69%), excessive night sweating (55%), chest pain (41%) and dyspnea (39%). Thirty-one percent of the cohort presented with hemoptysis. Seventy-one patients had different types of opacities and infiltrates in their chest x-ray. Micro- or macro- nodular lung changes were reported in 22 (16%) patients. Lung cavitations and pleural effusions were present in 13% of the studied patients. In 7% of cases, bronchiectasis was noted as a sequelae of long standing lung disease. The right lung was involved in 51% of cases; the left lung in 27% and bilateral lung involvement was noted in 22% of patients. The upper lobes were involved in 63%. Sputum for acid-fast bacilli (AFB) Z-Nielson stain and culture was positive in 51%, bronchial wash was positive in 27% of cases. The diagnosis was made by histopathological examination in 15% of cases. CONCLUSION: This study showed that active pulmonary TB patients vary in clinical presentation. The radiological manifestations of pulmonary TB are heterogeneous. Sputum for AFB remains an important, easy and inexpensive measure for TB diagnosis, but may not be always helpful in early diagnosis. Bronchoscopy with bronchial washings for Mycobacterium stain and culture is an important method, and it helps in confirming the diagnosis in sputum negative patients. In sputum and bronchial wash negative cases histopathological diagnosis can identify an important proportion of active pulmonary TB cases. PMID- 16680252 TI - The role of antifibrinolytic agents in gynecologic cancer surgery. AB - OBJECTIVE: To compare the effects of crystalloid and colloid solutions, tranexamic acid and epsilon-aminocaproic acid on the need for allogenic blood transfusion and on coagulation and fibrinolysis parameters. METHODS: We conducted the study in the Anesthesiology and Reanimation Department of Hacettepe University Medical Faculty, Ankara, Turkey between March 2004 and April 2005. The study included 105 patients, classified by the American Society of Anesthesiology as physical status groups I-II, undergoing gynecologic cancer treatment. We divided them into 5 groups: group I (crystalloid) received crystalloid solutions, group II (colloid) received colloid solutions, group III (tranexamic acid) received 10 mg x kg(-1) tranexamic acid, and group 5 (epsilon-aminocaproic acid) received 100 mg x kg(-1) epsilon-aminocaproic acid. All patients bleeding amount was measured and recorded perioperatively, and at the 12th and 24th hours postoperatively. We then evaluated the patients' hemoglobin, hematocrit, activated thromboplastin time, international normalized ration, fibrinogen, and thrombocyte count and symptoms of pulmonary embolism. RESULTS: In comparing the amount of bleeding, the bleeding in the tranexamic acid group was 30.8% less than the crystalloid group (p<0.05), 33.3% less than the colloid group (p<0.05), and 23.9% less than the epsilon-aminocaproic acid group (p<0.05). CONCLUSION: When the negative effects of blood transfusions were considered, tranexamic acid administration can be recommended for decreasing the need for blood transfusion in gynecologic cancer surgery. PMID- 16680253 TI - The preemptive use of diclofenac sodium in combination with ketamine and remifentanil does not enhance postoperative analgesia after laparoscopic gynecological procedures. AB - OBJECTIVE: To evaluate the preemptive effects of diclofenac sodium, in combination with remifentanil and ketamine. METHODS: A prospective, randomized, double blind, placebo-controlled trial was carried out at the Hacettepe University Hospital, Ankara, Turkey from September to December 2004. Forty-three, American Society of Anesthesiology physical status group I-II women, aged >18 years, who would undergo both diagnostic and operative laparoscopic surgery were randomly assigned into 2 groups. All patients received intraoperative 0.1 microg x kg(-1)min(-1) remifentanil infusion. Diclofenac (1 mg x kg(-1) intramuscular) was administered, 20 minutes before the operation. Ketamine (0.8 mg x kg(-)1 intravenously) was administered 5 minutes before the skin incision and at completion of skin closure. We divided the patients into 2 groups; Group I (diclofenac + remifentanil + ketamine), Group II (remifentanil + ketamine). Pain was evaluated postoperatively using the visual analogue scale (VAS) while global satisfaction by verbal rating scale (VRS). RESULTS: All 43 female patients have a mean +/- SD age of 32.3 +/- 6.5 years, height of 163 +/- 5.3 cm, and weight of 62.9 +/- 9.5 kg. The VAS and VRS scores and also time to first analgesic request were not different between the groups. In all groups, >98% of the patients were satisfied or very satisfied. CONCLUSION: We have not found any preemptive or additive effect of diclofenac sodium with the concomitant use of ketamine. PMID- 16680254 TI - Comparison of the antiemetic effects of ondansetron and dexamethasone on middle ear surgery. AB - OBJECTIVE: To compare the antiemetic efficacy of ondansetron and dexamethasone in adults undergoing middle ear surgery. METHODS: This clinical research took place in the Faculty of Medicine, Gazi University, Turkey between January to December 2004. The study included 60 cases, classified by the American Society of Anesthesiology physical status group I-II, who underwent middle ear surgery. We carried out anesthesia induction with 5 mg x kg(-1) sodium thiopental and performed muscle relaxation with 0.5 mg x kg(-1) atracurium to be followed by orotracheal intubation. Anesthesia was maintained at 5 L x min(-1) gas flows with 2-3% sevoflurane inhalation in 70/30% O(2)/N(2)O. We randomly distributed the cases into 2 groups, and the first group (Group O) was administered with 4 mg ondansetron intravenously (IV) at the stage of surgical skin closure and the second group (Group D) with 5 mg dexamethasone IV immediately after anesthesia induction. In the first 24 hours postoperatively, nausea vomiting score (NVS) and nausea, vomiting frequency, Metamizole-Na and non-steroidal anti-inflammatory drug use, the need for additional antiemetics and cost as well as the number of cases with nausea, vomiting and the need for extra antiemetics during 0-4, 4-12 and 12-24 hours were recorded, and their distribution to groups was evaluated. RESULTS: The NVS was 0 (0-0) in group O compared with 1 (0-3) in group D (p=0.003). The use of additional antiemetics was found to be significantly lower in group O (1 +/- 0.6) compared with group D (3.70 +/- 1.02) (p=0.028). In comparing the cost, group O (9.8 dollars) was found to have a significantly higher cost compared with group D (1.1 dollars) (p<0.0001). CONCLUSION: Ondansetron had a more significant effect on nausea and vomiting in the early period, however, no difference was found after 4 hours of administration. Furthermore, dexamethasone was found to cost less compared with ondansetron. PMID- 16680255 TI - Suture versus mesh repair for incisional hernia. AB - OBJECTIVE: To compare suture with mesh repair, for incisional hernia in terms of early and late outcomes. METHODS: We reviewed the records of all the patients who presented with primary or recurrent incisional hernia in the Department of General Surgery, Riyadh Medical Complex, Riyadh, Kingdom of Saudi Arabia, from January 2000 to December 2004. We divided patients, who underwent repair, in 2 groups: Group A (suture repair) and Group B (mesh repair). The information recorded for both groups included gender, age, associated systemic illness, site of hernia, initial surgery, number and type of previous hernia repairs, size of hernial defect, techniques of repair, and hospital stay. The principal early and late outcome measures studied were septic complications and recurrence. RESULTS: A total of 123 patients qualified for the study, 72 in group A and 51 in group B. Wound infection was 5.5% in group A versus 3.9% in group B (p=0.51). Follow up ranged between 6-58 months (mean 37.5 months) for both groups. Fifteen patients (20.8%) developed recurrence in group A, while the recurrence rate in group B was only 5.8% (p=0.04). CONCLUSION: Mesh repair resulted in a lower recurrence rate, and is not associated with increased incidence of wound complications compared with suture repair. PMID- 16680256 TI - Outcome of laparoscopic cholecystectomy in acute and chronic cholecystitis. AB - OBJECTIVE: To evaluate and compare the outcome of laparoscopic cholecystectomy (LC) in acute and chronic cholecystitis in terms of complications, conversion rates, reason of conversion and hospital stay. METHODS: Between April 1999 and March 2004, we retrospectively evaluated all patients admitted for symptomatic gall bladder disease in the Department of General Surgery, Lahore General Hospital, Lahore, Pakistan. All patients who underwent LC for symptomatic cholelithiasis were included. They were classified as group A [having acute cholecystitis (AC)] and group B [with chronic cholecystitis (CC)] based on the operative findings and histological diagnosis. RESULTS: Out of 725 patients who underwent LC, 173 patients were included in group A and 552 patients in group B. Conversion rate was 6% for group A and O.7% for group B (p<0.0001). Two patients (0.3%) in group B, while one patient (0.5%) in group A sustained bile duct injury (p>0.05). Two patients (1.1%) in group A and 4 patients (0.7%) in group B developed sub-hepatic bile collections (p>0.05). The mean hospital stay was 1.2 +/- 1.1 days for group A and 1.7 +/- 2.1 days for group B (p<0.001). CONCLUSION: Laparoscopic cholecystectomy is a safe and effective procedure in almost all patients presenting with symptomatic cholelithiasis. PMID- 16680257 TI - Results of total knee replacement using a cemented stemmed prosthesis. AB - OBJECTIVE: To present the results of total knee replacement at the King Abdul Aziz University Hospital in Jeddah, Kingdom of Saudi Arabia (KSA). METHODS: The medical records of 205 patients who received 309 prostheses was reviewed. The study took place at King Abdul-Aziz University Hospital, Jeddah, KSA between May 1989 and August 2005, where patients were either examined in the outpatient clinic or interviewed on the phone. Seventeen patients (22 prostheses) were lost for follow up. The scores were registered according to the Knee Society Clinical Rating System. RESULTS: There was a significant improvement in all-functional scores postoperatively in the first year, which continues without significant change throughout the follow up period. Symptomatic deep vein thrombosis was observed in 5 patients, 3 of them had pulmonary embolism, which was fatal in 2 cases. Early deep infection occurred in 3 cases. Superficial wound infection was seen in one patient. One patient suffered peroneal nerve palsy, while 4 patients had aseptic loosening of the femoral implant. One patient had rupture of the patellar tendon. Metal breakage was observed once and one patient is diagnosed to have polyethylene wear. In the early phase, when a prosthesis type without dislocation protection was used, 7 dislocations occurred. This type was not used anymore and no such complication was observed. CONCLUSION: Total knee replacement relieves pain and improves quality of life significantly. The observed complications compare well with the results reported in the literature. PMID- 16680258 TI - The spectrum of bone disease in Jordanian hemodialysis patients. AB - OBJECTIVE: To evaluate the spectrum of mineral abnormalities and bone disease (BD) in hemodialysis patients at Jordan University Hospital (JUH), Amman, Jordan. METHODS: A cross-sectional study was conducted among 63 patients (38 males and 25 females), mean age 44.19 years (range 17-76 years), with chronic kidney disease (CKD) on regular hemodialysis at JUH between November 2004 and April 2005. All patients have undergone complete blood count, chemistry profile, alkaline phosphatase, serum albumin, intact parathyroid hormone (iPTH) and plain x-rays. RESULTS: Bone disorders were identified in 45 patients on x-rays (70%). Osteopenia was found in 43 patients (68.3%), subperiosteal resorption in 24 patients (38.3%) and metastatic calcification in 22 patients (35%). Hypocalcemia was found in 28.6% and hypercalcemia in 7.9%. All patients were taking calcium carbonate, and 55.5% of patients were on vitamin D supplements. The calcium levels in 63.5% and the phosphorus levels in 50.8% of patients were within the recommended guidelines of the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (K/DOQI). Serum i-PTH level was above 300 pg/ml high turnover bone disease in 24.6% of patients, 21.3% had iPTH of 150-300 pg/ml target, and 44.3% had i-PTH levels below 100 pg/mL suggesting a dynamic bone disease. Patients with severe bone disease had a statistically significant higher iPTH levels (p<0.005). CONCLUSION: Bone disease and mineral abnormalities are common in hemodialysis patients at JUH. Earlier detection of bone disease and better overall management strategy may reduce the frequency and severity of bone disease in CKD patients in Jordan. PMID- 16680259 TI - Diagnostic accuracy of clinical diagnosis versus echocardiography in evaluating heart murmurs in Iraqi children. AB - OBJECTIVE: To assess the diagnostic accuracy of clinical diagnosis versus echocardiography in evaluating childhood heart murmurs. METHODS: We investigated 107 children under the age of 10 years with heart murmurs admitted to 3 hospitals in Baghdad (Al-Khadimyiah Pediatric Hospital, Al-Karama Teaching Hospital/Pediatric Department and Al-Noor Teaching Hospital/Pediatric Department) during the period of January 1998 to January 2002. Data on age, gender, provisional clinical diagnosis and final echocardiographic diagnosis were taken. Statistical analysis through Chi-square was carried out. RESULTS: There were more males than females (58.9% versus 41.1%, p<0.05). Provisionally, diagnoses of pathological murmurs were made in 88.8% of patients compared with 11.2% for innocent murmurs. The most common pathological murmur was that of ventricular septal defect (58.9%) and the least was aortic stenosis (1.9%). Echocardiographically, pathological findings were seen in 62.6% of patients compared with 37.4% for normal results. Provisional and echocardiographic diagnoses were concordant in 52.3% of patients and discordant in 47.7%, a difference of highly statistical significance (p<0.001). The most common concordant and discordant diagnoses were ventricular septal defect and innocent murmurs. CONCLUSION: Though echocardiography can be used to precisely evaluate heart murmurs, the role of comprehensive history taking and meticulous physical examination must not be neglected. Defect in specific accuracy of clinical acumen noted by the pediatricians who cared for the studied patients probably reflects their different standards in medical education and clinical training. Developing and implementing advanced physical diagnosis curriculum, establishing training programs in cardiology for pediatricians and provision of well-trained pediatric echocardiographers seem crucial. These options will eventually contain medical expenses. PMID- 16680260 TI - Estimated risk of coronary heart disease in obese adult males in Northern Jordan. AB - OBJECTIVE: To examine the relationship between obesity, lipid profile and blood pressure, and to quantify the risk of coronary heart disease (CHD) for the next 10 years, using the Framingham risk scoring scheme among Jordanian adult males. METHODS: We conducted this study in Al-Sarieh, Jordan during the period March to May 2001. A total of 306 apparently healthy adult males, aged 30-50 years completed all the study procedures. We selected the participants using a multi stage cluster sampling design. Dietary history and smoking habits were obtained using a pre-tested questionnaire and interview. Blood samples were obtained and examined for lipid profiles. We measured the blood pressures, as well as the weight and height to calculate the body mass index (BMI). The sample was categorized into 3 groups using the World Health Organization classifications for BMI. The risk of CHD was calculated using a scoring scale according to Framingham scheme. Analyses of data were carried out using the Chi-square test, and the Analysis of Variance. RESULTS: The mean age of the subjects was 39 years with a mean BMI of 28.2 kg/m2. The percentage of current smokers was 44.1%. The mean of serum total cholesterol, triglycerides, low density lipoprotein cholesterol and systolic blood pressure, increased significantly with increasing BMI categories, whereas the mean of high density lipoprotein cholesterol decreased with increasing BMI categories. Prevalence of medium and high risk of CHD significantly increased as BMI categories increases. CONCLUSION: The prevalence of estimated CHD risk for the next 10 years in moderate and high CHD categories increases as the BMI categories increases among Jordanian adult men in Al-Sarieh area. PMID- 16680261 TI - The effect of loss of body weight on lipid profile in overweight individuals. AB - OBJECTIVE: To determine the effect of reducing body weight and body mass index on serum lipid. METHODS: This is a prospective longitudinal study, which was implemented in Naim Primary Health Care Center in the Kingdom of Bahrain during May and June of 2004. The study included 55 individuals who were willing to participate. The criteria for the sample selection included being overweight, having high serum lipid and patients' willingness to reduce their weight. The patients were followed up for a 26 weeks period consisting of continuous health education program, maintenance on low caloric diet and exercise to help them reduce their body weight. This period included a lifestyle enhancement awareness program that included 4 health education sessions on weight loss, 4 sessions on weight loss maintenance and frequent and regular visits to the clinic to make sure that those patients were compliant with the given instructions. RESULTS: The study showed that patients who completed the designed program had a reduction in body weight, body mass index, and lipid level and an increase in physical activity and dietary readiness to control over-eating. Although in both genders there was a significant drop in all the parameters, only body mass index (p<0.002) and cholesterol (p=0.007) showed a statistically significant difference between men and women. In men, cholesterol level at the initial stage of the study was strongly related to the body weight (r=0.65, p<0.002) and body mass index (r=0.83, p<0.0005). Despite that the cholesterol level reduced more in women than men; women decreased less in weight (p<0.0005) and body mass index (p<0.0005) than men. CONCLUSION: Overweight individuals would benefit from a life enhancement program that increases their awareness on the danger of obesity and helps them in reducing body weight and serum lipid level. PMID- 16680262 TI - Physicians perception of health insurance in Saudi Arabia. AB - OBJECTIVE: To assess physicians views on health insurance and its implication on the health care system. METHODS: We carried out a cross-sectional study in 2 major hospitals in Riyadh, Kingdom of Saudi Arabia. Data were collected from January to December 2002 through self-administered questionnaires that were distributed to a total sample of 400 physicians. The instrument consisted of 28 items that focused on assessing physicians' perception towards health insurance and its effect on health services. We performed a descriptive statistics and analysis of variance using the Statistical Package for Social Sciences. RESULTS: Overall, 151 physicians (38%) completed the survey. This study clearly shows that access to health care services is a major concern; more than 94% of the respondents agree that everyone in the Kingdom should have access to healthcare services. Respondents also agree that health insurance will improve access to healthcare services for all citizens. Physicians also believed that health insurance would lead to more regulations and utilization review of services, create more competition between healthcare providers, and create new jobs in the healthcare sector. Saudi physicians reported a higher mean score for 11 items with significant p-values as compared with non-Saudi physicians. CONCLUSION: Physicians in this survey believed that accessibility is a major policy concern, and that health insurance will have a positive effect on access to the health care system. Yet, accessibility is an illusive term with many aspects that go beyond the identification of need for health care to the actual delivery of health care services and the organizational structures to match the needs of society. Cooperation as a national health system should be built on collaborative efforts rather than market competition in itself. It has been suggested that markets are stronger in the role of delivery than in the financing of health care, that markets tend to promote more expenditure on technological innovation rather than producing the most desired set of social outcomes. Cooperative health insurance can be an answer to the current problems facing the health care system in the Kingdom of Saudi Arabia as long as it remains cooperative rather than competitive. PMID- 16680263 TI - Smoking habits among medical students in Central Saudi Arabia. AB - OBJECTIVE: To estimate the prevalence of smoking habits among male medical students at the College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia (KSA). METHODS: We carried out this cross-sectional study using Arabic questionnaires distributed to the medical students in the College of Medicine, King Saud University, Riyadh, KSA at different educational levels from September 13 to September 25, 2005. A total of 322 medical students completed the questionnaires (response rate 80.5%). RESULTS: The study shows that 13% of male medical students were currently active smokers, 5.3% were ex-smokers, and 38.2% were passive smokers. The types of smoking included sheesha 44.1%, cigarette 32.2%, and both 23.7%. The common reason given for the smoking behavior was the influence of friends (35.6%). The study shows that 57.1% of current smokers were motivated to stop smoking. CONCLUSION: There is an urgent need to promote multi disciplinary health education activities at different age groups in order to prevent young age students from smoking, and to help smokers to quit. PMID- 16680264 TI - A rare foreign body into the male penile urethra. AB - A wide variety of urethral foreign bodies have been described in the literature. In adults, this is commonly caused by the insertion of objects used for masturbation, and is often associated with a mental disorder as in our case described herein. We report a 28-year-old man, hospitalized for self-insertion of a pen into the urethra. The pen was removed successfully by surgery. PMID- 16680266 TI - Airway management of a rare huge-size supraglottic mass. AB - Laser excision of a huge-sized supraglottic mass nearly obstructing the airway passage is a real challenge to anesthesiologists. Upper airway obstruction due to neoplasm in supraglottic region, is traditionally managed by preoperative tracheostomy, however, such a common procedure can potentially have an impact on long-term outcome. A 26-year-old patient presented with dysphagia caused by left cystic vallecular synovial sarcoma. The airway was successfully secured via fiberoptic bronchoscopy, followed by excision of the supraglottic tumor with CO(2) laser surgery. Tracheostomy was not required. The patient was discharged from the hospital on the 4th day of surgery. This case, highlights the possibility to secure the airway passage without performing preoperative tracheostomy resulting in good outcome and short hospital stay. PMID- 16680265 TI - Staphylococcus aureus endocarditis complicated by bilateral pneumothorax. AB - Staphylococcus aureus is the most common cause of endocarditis in intravenous drug users. The organism gains access by intravenous injection or from the direct invasion of skin at injection sites. Known for its aggressiveness, the right sided endocarditis that ensues can lead to complications such as pulmonary abscesses and even death. We report the unusual case of an intravenous drug abuser, who following the occurrence of extensive pulmonary abscesses, developed bilateral pneumothoraces within a few days. PMID- 16680267 TI - Perioperative management of a patient with Henoch-Schonlein purpura for appendectomy. AB - Henoch-Schonlein purpura HSP is a multisystem disease and immunoglobulin A mediated vasculitis with a self-limited course affecting the skin, joints, gastrointestinal tract, and kidneys. It is the most common form of acute small vessel vasculitis primarily affecting children. Severe renal and central nervous system disease may lead to life-threatening conditions, and immunosuppressive agents and plasmapheresis may be needed. The cause of HSP is unknown; immunizations, certain food allergies, insect bites, infection, and some medications may play a role in the development of the disease. Perioperative management for liver and kidney functions is very important for anesthetized patients with HSP. We report the perioperative management of a patient with HSP for appendectomy. PMID- 16680268 TI - Persistent sciatic artery. Radiologic features and patient management. AB - Persistent sciatic artery PSA represents the persistence of the sciatic vessel in adult life that is responsible for the major blood supply to the lower limb in early embryologic development. The incidence of PSA has been estimated as low as 0.025-0.04%. We present 2 cases of PSA, one of which was complicated by an aneurysm that led to a life-threatening hemorrhage. PMID- 16680269 TI - Oral mega pulse methylprednisolone in alopecia universalis. AB - Intravenous pulse corticosteroids is an alternative method of corticosteroids delivery, which proved to be safe with rapid and potent efficacy. It is an effective treatment of alopecia areata, but not for the totalis, universalis or ophiasis types, for which no effective therapy is available yet. Recently, it has been confirmed that oral and intravenous pulse methylprednisolone MP have comparable efficacy. Here, we report a 9-year-old Saudi boy with alopecia universalis who was treated with MP sodium succinate 15 mg/kg ideal body weight orally for 3 consecutive days bimonthly for 12 sessions. Complete hair regrowth was obtained without toxic effects. When the interval was increased to 4 weeks, he showed partial relapse. So, 2 more pulses were given with an interval of 3 weeks in between. This maintained his regrown hair for a year without treatment. PMID- 16680270 TI - Benign lateral pharyngeal band pathologies as a cause of sore throat. PMID- 16680271 TI - Adipsic hypernatremia with a reset osmostat. PMID- 16680272 TI - A 29-year-old man with bouts of tachycardia. A case of WPW syndrome. PMID- 16680274 TI - Profile of baseline CD4 T-lymphocyte and viral load levels in HIV infected treatment naive patients in Lagos, Nigeria. PMID- 16680273 TI - 3-Methylcrotonyl-CoA carboxylase deficiency. A long-term outcome. PMID- 16680275 TI - Cytotoxic potential of cervical mucus on Trichomonas vaginalis to prevent upper genital tract infections. PMID- 16680276 TI - Cutaneous anthrax as an occupational disease in Central Anatolia, Turkey. PMID- 16680277 TI - Congenital renal anomalies. PMID- 16680278 TI - Acute surgical abdomen in pregnancy. PMID- 16680279 TI - Comparison of early results of limited thoracotomy versus complete sternotomy in atrial septal defect closure. PMID- 16680280 TI - Open tibial fractures. PMID- 16680281 TI - Atheromatous disease and latent prostate cancer. A correlation or a coincidence. PMID- 16680282 TI - Effect of Urtica Dioica on bacterial translocation in mechanic icter model. PMID- 16680283 TI - Ethical guidelines in genetics and genomics. An Islamic perspective. PMID- 16680286 TI - [Sirolimus-eluting stents for the treatment of stenoses in small coronary arteries: what have we learned?]. PMID- 16680285 TI - The therapeutic effects of music in children following cardiac surgery. AB - OBJECTIVE: To investigate, both objectively and subjectively, the effect of music on children in a pediatric cardiac intensive care unit following heart surgery, in conjunction with standard care. METHODS: Randomized clinical trial with placebo, assessing 84 children, aged 1 day to 16 years, during the first 24 hours of the postoperative period, given a 30 minute music therapy session with classical music and observed at the start and end of the session, recording heart rate, blood pressure, mean blood pressure, respiratory rate, temperature and oxygen saturation, plus a facial pain score. Statistical significance was set at 5%. RESULTS: Five of the initial 84 patients (5.9%) refused to participate. The most common type of heart disease was acyanotic congenital with left-right shunt (41% of cases: 44.4% of controls). Statistically significant differences were observed between the two groups after the intervention in the subjective facial pain scale and the objective parameters heart rate and respiratory rate (p < 0.001, p = 0.04 and p = 0.02, respectively). CONCLUSIONS: A beneficial effect from music was observed with children during the postoperative period of heart surgery, by means of certain vital signs (heart rate and respiratory rate) and in reduced pain (facial pain scale). Nevertheless, there are gaps to be filled in this area, and studies in greater depth are needed. PMID- 16680287 TI - [Predicting therapy duration and recurrence in patients with vasovagal syncope: is there light at the end of the tunnel?]. PMID- 16680288 TI - [Clinical applications of B-type natriuretic peptide assays]. PMID- 16680289 TI - [Clinical outcome of patients with neurocardiogenic syncope (NCS) after therapy interruption]. AB - OBJECTIVE: To evaluate the outcome of patients with NCS after interruption of pharmacological therapy and to investigate the possible clinical variables predicting recurrence. METHODS: Thirty-seven patients (age 31+/-16 years) with refractory recurrent NCS being 19 females where prospectively studied. All patients became asymptomatic and had a negative tilt table test (TT) after pharmacological therapy. The treatment was interrupted and one month later, a new TT with no medication was carried out. The probability free of symptoms recurrence was analyzed according to sex, age, number of syncope episodes previously to the treatment, clinical history time, treatment time, drug free from treatment time and TT result. RESULTS: Twenty-two patients (59%) presented recurrence during a mean follow-up of 21+/-19.7 months. The variables related to greater recurrence were number of previous syncope (p=0.0248), positive TT after interruption of the therapy (p=0.0002) and female gender (p=0.0131). CONCLUSIONS: Most of the very symptomatic patients with NCS present recurrence after the suppression of a specific therapy. A TT carried out after treatment discontinuation can identify patients with higher risk of recurrence, specially in the first year of follow-up. PMID- 16680290 TI - [Technical aspects of coronary sinus catheterization based on the atrial component of the intracavitary electrogram and radiological anatomy during the implantation procedure of a biventricular pacemaker]. AB - OBJECTIVE: To present a technical proposal based on the experience of 130 implantations using a simplified technique for coronary sinus catheterization, based on the atrial component of the intracavitary electrogram and radiological anatomy. METHODS: From October, 2001 to October, 2004, 130 biventricular pacemaker implantations were performed, using radiological anatomy and observation of the intracavitary electrogram, focusing on the atrial component. RESULTS: The implantation of the system using left ventricular pacing via coronary sinus was not possible in 8 patients. Difficulties on the cannulation of the coronary ostium were felt in 12 patients and difficulties of lead advancement through the coronary sinus were felt in 15 patients. The mean time of radioscopy utilization was 18.69 min. CONCLUSION: The implantation technique, using the atrial component morphology of the intracavitary electrogram and radiological anatomy showed to be workless, safe and effective for the cannulation of the coronary sinus ostium requesting reduced time of radioscopy. PMID- 16680291 TI - [Comparative analysis of intimal hyperplasia after sirolimus-eluting stent and thin-strut bare-metal stent implantation in small coronary arteries]. AB - OBJECTIVE: This study aimed at evaluating reduction in intimal hyperplasia volume following angioplasty using sirolimus-eluting stents (Cypher) compared with thin strut bare-metal stents (Pixel) in patients with small vessels. METHODS: Eighty patients with coronary artery disease were prospectively included in two consecutive series, the first using sirolimus-eluting stents (50) and the second using bare-metal stents (30). RESULTS: The use of sirolimus-eluting stents reduced: in-stent net volume obstruction [5.0% (SE = 0.77) x 39.0% (SE = 4.72), p < 0.001], in-stent late loss [0.25 mm (SE = 0.03) x 1,11 mm (SE = 0.13), p < 0.001], in-segment late loss [0.30 mm (SE = 0.04) x 0.83 mm (SE = 0.11), p < 0.001], in-stent restenosis (0% x 33.3%, p < 0.001) and in-segment restenosis (4% x 36.7%, p < 0.001). The event-free survival rate was 96% in the sirolimus eluting stent group versus 86.7% in the bare-metal stent group (BMS) (p = 0.190). CONCLUSION: Sirolimus-eluting stents are superior to thin-strut bare-metal stents in reducing intimal hyperplasia (less in-stent obstruction and less late lumen loss) in patients with small vessels. The use of these stents significantly reduced angiographic restenosis at eight months. PMID- 16680292 TI - [Cigarette smoke exposure intensifies ventricular remodeling process following myocardial infarction]. AB - OBJECTIVE: To evaluate the role of cigarette smoke exposure (CSE) on ventricular remodeling following acute myocardial infarction (AMI). METHODS: Rats were submitted to myocardial infarction and divided into two groups: C (control, n = 31) and F (CSE: 40 cigarettes/day, n = 22). After 6 months, the survivors were submitted to echocardiogram, functional study with isolated heart, and morphometric analysis. For comparison purposes, we used the t test (mean +/- standard deviation) or the Mann-Whitney test (with median and 25th and 75th percentiles). RESULTS: The CSE animals tended to have larger diastolic (C = 1.5 +/- 0.4 mm2, F = 1.9 +/- 0.4 mm2; p = 0.08) and systolic (C = 1.05 +/- 0.3 mm2, F = 1.32 +/- 0.4 mm2; p = 0.08) left ventricular(LV) areas. The systolic function of the LV, assessed according to the fractional area change, tended to be impaired in CSE animals (C = 31.9 +/- 9.3%, F = 25.5 +/- 7.6%; p = 0.08). The dp/dt values for CSE animals were statistically lower (C = 1474 +/- 397 mmHg, F = 916 +/- 261 mmHg; p = 0.02) than for control animals. The CSE animals presented higher right ventricle (RV) weight adjusted for body weight (C = 0.8 +/- 0.3 mg/g, F = 1.3 +/- 0.4 mg/g; p = 0.01), higher content of water in lungs (C = 4.8 (4.3-4.8)%, F = 5.4 (5.1-5.5); p = 0.03), and larger LV myocyte cross-sectional areas (C = 239.8 +/- 5.8 microm2, F = 253.9 +/- 7.9 microm2; p = 0.01). CONCLUSION: Cigarette smoke exposure intensifies ventricular remodeling following acute myocardial infarction. PMID- 16680293 TI - [Correlation and concordance between echocardiographic measurements obtained during echocardiography and digitized image measurements--transversal study]. AB - OBJECTIVE: To evaluate the correlation and concordance between the measurements of echocardiographic analysis of cardiac dimensions obtained through the Echo off line applicative (software for obtaining digitized image measurements in a dedicated workstation) available to public domain, and those obtained through the conventional method. METHOD: Transversal contemporary study, of 56 randomized patients whose images were obtained during examinations. The measurements of the M mode and 2D, were done in the ventricles, left atrium, and aorta by the Echo off-line program. These measurements were compared to those obtained by another professional, through Pearson's correlation test (r), alpha = 0.05 and by concordance analysis (Bland and Altman). RESULT: The measurements carried out by the Echo off-line system showed r that varied from 0.85 to 0.98. The analysis of concordance showed that for most measurements, the mean difference between the methods was approximately zero. The variation of absolute values did not show, in average, a clinical significance. The Echo off-line applicative allows a reduction of approximately 30% in the time spent to obtain the measurements. CONCLUSION: This study demonstrated the accuracy of the Echo off-line program to measure cardiac dimensions in a dedicated workstation, showing that it can be routinely used in echocardiography labs. PMID- 16680294 TI - [The effects of nonsupervised exercise program, via internet, on blood pressure and body composition in normotensive and prehypertensive individuals]. AB - OBJECTIVE: To verify the effects of a six-month non-supervised physical training program followed via the Internet on blood pressure and body composition in normotensive and borderline hypertensive individuals. METHODS: One hundred and thirty five individuals were divided into two groups: 1) normotensive individual (n = 57), 43 +/- 1 years of age, systolic blood pressure (SBP) < 120 and diastolic blood pressure (DBP) < 80 mmHg (GI); and 2) borderline hypertensive individual (n = 78), 46 +/- 1 years of age, SBP 120 to 139 and DBP 80 to 89 mmHg (GII). RESULTS: After a three and six-month physical training, GII individuals showed a significant reduction in SBP (-3.6 +/- 0.94 and -10 +/- 0.94 mmHg, p < 0.05, respectively) and PAD (-6.5 +/- 1 and -7.1 +/- 0.9 mmHg, p < 0.05, respectively), body weight (-1.12 +/- 0.26 and -1.25 +/- 0.31 kg, p < 0.05, respectively), BMI (-0.79 +/- 0.4 and -0.84 +/- 0.41 kg/m2, p < 0.05, respectively) and waist circumference (-1.12 +/- 0.53 and -1.84 +/- 0.56 cm, p < 0.05, respectively). In the GI group, the physical training led to a decrease in waist circumference at the sixth month (-1.6 +/- 0.63 cm, p < 0.05). CONCLUSION: This program decreases blood pressure, body weight, BMI, and waist circumference in borderline hypertensive individuals, and is therefore a safe and low-cost strategy in the prevention of cardiovascular diseases and improvement of health status of the population. PMID- 16680295 TI - [Clinical and therapeutics aspects of heart failure due to Chagas disease]. AB - OBJECTIVE: Describe the clinical and therapeutic characteristics of patients with heart failure (HF) secondary to chronic chagasic cardiomyopathy and evaluate if these characteristics are different from those found in other etiologies. METHODS: A prospective analysis of the patients treated between August 2003 and June 2004 at a HF referral outpatient clinic was conducted. RESULTS: Three hundred and fifty six patients diagnosed with HF were included in the study. Chagasic cardiomyopathy was the most common etiology (48% of the cases). Other etiologies included hypertensive cardiomyopathy in 19% of the patients, idiopathic dilated in 11% and ischemic in 9%. Patients with HF secondary to chagasic cardiomyopathy were more frequently from non-white ethnic groups (88 vs. 75%; p = 0.002), had a family history of Chagas disease (57 vs. 21%; p = 0.001), had the disease for a longer length of time (71 vs. 56 months; p = 0.034), had lower levels of education (4.4 +/- 4.1 vs. 5.7 +/- 4.2 years of study; p = 0.004), had a lower heart rate (69 +/- 12 vs. 73 +/- 13; p = 0.03) and a lower systolic blood pressure (121 +/- 25 vs. 129 +/- 28 mmHg; p = 0.006). There was also a higher incidence of the use of amiodarone (22 vs. 13%; p = 0.036) and artificial pacemakers (15 vs. 1%; p = 0.001). There was a lower usage of beta blockers (39 vs. 59%; p = 0.001). CONCLUSION: In this sample of HF outpatients, in a state with a high prevalence of Chagas disease, chagasic cardiomyopathy was the most common etiology and they presented some unique clinical and therapeutic characteristics in comparison to other heart failure patients. PMID- 16680297 TI - [Real time tridimensional echocardiography in a patient with hypertrophic cardiomyopathy]. PMID- 16680296 TI - [Atrogenic pseudoaneurysm of axillary artery]. PMID- 16680298 TI - [Spontaneous coronary artery dissection causing acute coronary syndrome]. PMID- 16680299 TI - [Heart failure in a 33 year-old woman in the late follow-up after myocardial infarction]. PMID- 16680300 TI - Potential bird dispersers of Psychotria in a area of Atlantic forest on Ilha Grande, RJ, Southeastern Brazil: a biochemical analysis of the fruits. AB - The present study assessed the fruiting pattern, bird foraging behavior, and sugar content of ripe fruits of two sympatric species of Rubiaceae (Psychotria brasiliensis and P. nuda). This study was carried out in an Atlantic forest area on Ilha Grande, RJ, between August 1998 and July 1999. Fruit production occurred year round, with a peak of mature P. brasiliensis fruits in December 1998 and another of P. nuda in February of 1999. Lipaugus lanioides (Cotingidae), Baryphtengus ruficapillus (Momotidae) and Saltator similis (Emberizidae) made the most frequent foraging visits to fruiting P. brasiliensis, so that L. lanioides and B. ruficapillus removed the fruits with sallying maneuvers while S. similis gleaned the fruits. Lipaugus lanioides was by far the most important consumer, and potentially the main disperser of P. brasiliensis. Birds of this genus are heavy frugivores in the tropical forests and are widely assumed to be important seed dispersers. The fruits were analyzed quantitatively and qualitatively in relation to the amounts of sucrose and starch. Psychotria brasiliensis (the visited species) showed the smallest quantity of sucrose and the highest amount of starch. These findings suggest that what may influence the birds' choice of fruit is the proportion of starch in the Psychotria species studied here rather than the carbohydrate composition. PMID- 16680301 TI - Dispersal of the egg parasitoid Gryon gallardoi (Brethes) (Hymenoptera: Scelionidae) in tobacco crops. AB - A mass marking-recapture experiment was carried out to study the dispersal of Gryon gallardoi (Brethes) (Hymenoptera: Scelionidae) in tobacco crops. Parasitoids emerged in the laboratory were marked with dye powder. Six separate field trials were conducted in Jan/Feb 2002 involving the simultaneous release of marked parasitoids. Dispersal was determined by the recapture of individuals in sets of sticky and Moericke traps arranged in three concentric circles arranged at 1.4 m intervals from the central point of release. Traps were checked 4, 8, 24, 28, 32 and 48 h after parasitoid release and weather data were recorded at 2 h intervals from 9 to 17 h at the site. Of the 699 marked parasitoids released, 91 were recaptured (13.02%). Fewer females were recaptured in the late afternoon, suggesting they are less active than males after dusk. G. gallardoi recaptures were not associated with average wind direction in any trials. After eight hours of release, recaptures occurred mostly in the traps farthest from the release point, suggesting that the experimental area was small in relation to the dispersal capacity of the parasitoid. Fitting a 4-h-after-release density distance curve to a geometric model and considering a daily activity of 12 h, the female dispersal capacity was estimated to be at least 7.6 m/day. The values reported here must be considered as indicative of the potential for active dispersal under the particular experimental conditions of these trials. It is possible that G. gallardoi may disperse downwind much farther than the distance recorded in this study. PMID- 16680302 TI - Reproductive parameters and longevity of Gryon gallardoi (Brethes) (Hymenoptera: Scelionidae) parasitizing Spartocera dentiventris (Berg) (Hemiptera: Coreidae) eggs. AB - The fecundity, daily progeny and longevity of Gryon gallardoi (Brethes) (Hym.; Scelionidae) were determined under laboratory conditions, using Spartocera dentiventris (Berg) (Hem.; Coreidae) eggs as host. Nineteen G. gallardoi females and 34 males were reared at 25 +/- 1 degrees C, with a 12 h photophase, fed on a 10% aqueous honey solution and provided with 25-30 S. dentiventris eggs daily. The average pre-oviposition period was 1.3 +/- 0.35 days, although some females began laying from the day of emergence. On average, oviposition lasted for 10.1 +/- 1.74 days, reaching a peak on the second day, with 67.5 +/- 11.29 eggs laid. The post-oviposition period was short (2.4 +/- 0.48 days). G. gallardoi females lived significantly longer than males: 13.7 +/- 1.94 and 10.6 +/- 1.78 days, respectively. The overall sex ratio was 0.79. The results reported here on the reproductive capability of the species suggest it may have a good potential as an agent for the control of S. dentiventris. PMID- 16680303 TI - Diet and capture of Hypostomus strigaticeps (Siluriformes, Loricariidae) in a small Brazilian stream: relationship with limnological aspects. AB - The purpose of this study is to ascertain whether variations in the limnological parameters of the Corumbatai river resulting from the discharge of a variety of wastes into its waters may be responsible for spatial shifts in the diet and capture of the armored catfish Hypostomus strigaticeps (Regan, 1907). Individuals were collected over a period of two years from two sites with similar physical, albeit distinct limnological characteristics. As a whole, the environmental variables (temperature, pH, dissolved oxygen, electrical conductivity, and total coliforms and fecal coliforms) of the two sites were found to vary significantly. The food items found in the guts of these armored catfish (sediments, diatoms, fungi hyphae, chlorophytes, cyanophytes and non-identified material) ranked differently in samples from the two sites. In the more polluted (site B), diatoms and chlorophytes ranked higher in the diet than in that of individuals caught in the more preserved location (site A). This fact may be related to the greater amount of organic material found at site B, which provides favorable environmental conditions for such algae and, consequently, for algivorous fishes. Even so, fewer fish were captured at site B than at site A, suggesting that although food is more abundant in the more polluted site, its limnological conditions appear, on the whole, to be less beneficial than the conditions at site A. PMID- 16680305 TI - Expression pattern of glycoconjugates in the Bidderian and ovarian follicles of the Brazilian toad Bufo ictericus analyzed by lectin histochemistry. AB - The Bidder's organ and ovary of the Brazilian toad Bufo ictericus were studied by light microscopy, using hematoxylin-eosin (HE) and periodic acid Schiff (PAS) staining. The expression and distribution of carbohydrate moieties was analyzed by lectin histochemistry, using 8 lectins with different carbohydrate specificities: Ulex europaeus (UEA I), Lens culinaris (LCA), Erythrina cristagalli (ECA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Aleuria aurantia (AAA), Triticum vulgaris (WGA), and Glycine maximum (SBA). The results showed that the Bidderian zona pellucida presented alpha-mannose, alpha-L-fucose, beta-D-galactose, N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. The Bidderian follicular cells showed the presence of beta-D-galactose and N-acetyl-D-glucosamine. In the extracellular matrix, alpha-mannose and alpha/beta-N-acetyl-galactosamine residues were detected. The ovarian zona pellucida showed alpha-L-fucose, N-acetyl-D-glucosamine, alpha/beta-N-acetyl galactosamine residues, and alpha-mannose and N-acetyl-D-glucosamine residues were detected in the follicular cells. Thus, the zona pellucida in both organs contains N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. alpha-L-fucose residues were detected in the zona pellucida of both organs, using different lectins. Considering that beta-D-galactose residue was absent from ovary but present in the Bidder's organ, this sugar residue may play an important role in follicle development, blocking the Bidderian follicles and preventing further development of the Bidder's organ into a functional ovary. PMID- 16680304 TI - Effects of an oil spill and discharge of domestic sewage on the insect fauna of Cururu stream, Manaus, AM, Brazil. AB - Assemblages of aquatic and edaphic insects in three streams in Central Amazonia were used to evaluate the impact on one of the streams (Cururu) caused by an oil spill that occurred in August 1999. The Cururu stream had already been impacted by domestic sewage. These three streams were evaluated during four different periods of the regional hydrological cycle (falling-, low-, rising- and high water), from September 2000 to May 2001. An Eckman dredge, an aquatic entomological net and a manual auger were used to collect insects, which were used to estimate the taxonomic richness (at the family level) and insect density in each stream. Anoxic and eutrophic conditions were determined in each stream based on the mean concentration of dissolved oxygen, total nitrogen and phosphorus. Richness and abundance of insect fauna were higher in the Cristalino stream (not impacted) than in the Bom Jardim stream (impacted by domestic sewage) and in the Cururu stream, with the exception of samples collected along the stream banks (littoral fauna). The mean dissolved oxygen was lower in the Cururu stream than in the other two, while the mean concentrations of total nitrogen and phosphorus were higher. These elements showed intermediate values in the Bom Jardim stream, while the opposite pattern was observed in the Cristalino stream, with higher concentrations of dissolved oxygen and lower concentrations of total nitrogen and phosphorus. The concentrations of these variables reflect the modifications resulting from anthropogenic eutrophication caused by the discharge of domestic sewage into Bom Jardim and Cururu and by the oil spill in Cururu, which negatively affected the richness and abundance of insects in these streams. PMID- 16680306 TI - Dental development of Didelphis albiventris (Marsupialia): I--incisors and canines. AB - The formation of incisors and canines in marsupials of D. albiventris was studied at various stages of development. Seventy-six specimens, with ages varying from 0 to 100 days, were used in this investigation. Serial sections of the maxilla were obtained in the transverse plane and stained with hematoxylin and eosin. Histological analyses were made to verify the pattern of teeth development, as well as their chronology of eruption. The period of time from birth to 100 days comprised the entire process of teeth development, from epithelial bud formation to early eruption of the teeth. Oral epithelium thickening gave rise to the functional incisors and canines. In addition, a secondary dental lamina emerged in different phases of development in the outer epithelium of incisors and canines, which degenerated when it reached the bud stage. No evidence of deciduous dentition was observed. The results of this investigation suggest that secondary dental lamina represents remnants of a primitive condition in which secondary dentition used to be present. PMID- 16680307 TI - Distribution patterns of Neotropical primates (Platyrrhini) based on Parsimony Analysis of Endemicity. AB - The Parsimony Analysis of Endemicity (PAE) is a method of historical biogeography that is used for detecting and connecting areas of endemism. Based on data on the distribution of Neotropical primates, we constructed matrices using quadrats, interfluvial regions and pre-determinated areas of endemism described for avians as Operative Geographic Units (OGUs). We codified the absence of a species from an OGU as 0 (zero) and its presence as 1 (one). A hypothetical area with a complete absence of primate species was used as outgroup to root the trees. All three analyses resulted in similar groupings of areas of endemism, which match the distribution of biomes in the Neotropical region. One area includes Central America and the extreme Northwest of South America, other the Amazon basin, and another the Atlantic Forest, Caatinga, Cerrado and Chaco. PMID- 16680308 TI - Ichthyofauna diversity in a protected area in the State of Sao Paulo, southeastern Brazil. AB - The study site is located in an environmentally protected area known as an "APA" in Sao Pedro and Analandia, State of Sao Paulo, southeastern Brazil, whose watercourses are under strong anthropogenic pressure. Two basins were studied (sub-basin of the Corumbatai River and basin of the Jacare-pepira River) with the purpose of characterizing the ichthyofauna of various streams, comparing fish diversity among assemblages. The Passa-cinco River showed the highest diversity (H'), and the Jaccard and Morisita-Horn indices showed low similarity among sites and between the basins. Diversity was correlated with the number of available habitats and with the environmental conditions. PMID- 16680309 TI - Seasonal maturation of Glypthelmins vitellinophilum (Trematoda: Digenea) in Lysapsus limellus (Anura: Pseudidae) from an Argentinian subtropical permanent pond. AB - From December 1995 to November 2000, the seasonal maturation of Glypthelmins vitellinophilum Dobbin, 1958, in its definitive host, the frog Lysapsus limellus Cope, 1862, was studied in a subtropical permanent pond in northeastern Argentina. The objectives of this study were: 1) to determine the infrapopulation dynamics of the parasite, analyzing the seasonal maturation cycle throughout the years; and 2) to examine the relationship between the intensity of trematode infection in different developmental stages (recruitment, growth and maturation) and the host's body length. Of a total of 1,400 frogs examined over 60 months (5 years), 38% were found to be infected with G. vitellinophilum, and the intensity of infection was 1-15 trematodes per frog. Specimens of G. vitellinophilum were present in L. limellus throughout the years, but did not show a pronounced seasonal maturation cycle. Possible reasons for these findings are discussed with reference to climatic fluctuations and biotic factors. The infective period of the parasite (stage I) occurred in summer, autumn and spring, coinciding with the time each frog cohort appeared. These infections were found principally in small body sizes (classes 1 and 2) of L. limellus. Juvenile and nongravid specimens of worms (stage II and III) were found in frogs of different body sizes throughout the period of investigation. Gravid specimens of the parasite (stage IV) were generally recorded in autumn, winter and spring, mainly in the bodies of larger frogs. The body length of Trematodes in stages I and IV was significantly and positively correlated with that of the frogs. PMID- 16680310 TI - Diversity of Calliphoridae (Diptera) in Brazil's Tingua Biological Reserve. AB - The Calliphoridae are flies of great ecological, medical and sanitary importance because they are decomposers of organic matter, mechanical vectors of pathogenic agents, and causers of myiasis. This paper attempts to ascertain the diversity of Calliphoridae in the Tingua Biological Reserve and correlate meteorological data (temperature, relative humidity of the air, and precipitation) with the occurrence of these flies. The study was conducted at a site in the Tingua Biological Reserve, located in the municipality of Nova Iguacu, state of Rio de Janeiro, Brazil. Four traps were set up using chicken viscera as bait. The experiment was conducted over the period of May 2001 to April 2002, with two monthly collections. The captured flies were killed with ether and conserved in 70% alcohol and identified in the Laboratory of Diptera Studies (UNIRIO) and the Laboratory of Vector Insect Biology and Control (FIOCRUZ), both headquartered in the city of Rio de Janeiro, RJ, Brazil. A total of 1,987 insects were captured, 37.5% belonging to the family Calliphoridae, 29.4% to Muscidae, 16.0% to Sarcophagidae, and 17.1% to other families. The most representative species found was Phaenicia eximia (47.0%), followed by Hemilucilia semidiaphana (23.6%), Mesembrinella bellardiana (13.7%), Hemilucilia segmentaria (7.5%), Chloroprocta idioidea (4.9%), Chrysomya albiceps (1.2%), Chrysomya megacephala (0.9%), Phaenicia sericata (0.6%), Eumesembrinella sp. (0.5%), and Chrysomya putoria (0.1%). Large numbers of Calliphoridae were noted in May, June, September and January, coinciding with low rainfall and an average temperature of 21.8 to 27.0 degrees C. A negative correlation was found between the number of Calliphoridae captured and meteorological factors. PMID- 16680311 TI - Mercury contents in aquatic macrophytes from two reservoirs in the Paraiba do Sul: Guandu river system, SE Brazil. AB - This paper reports on a study to determine the Hg content in the five most abundant aquatic macrophyte species (Elodea densa, Sagittaria montevidensis, Salvinia auriculata, Pistia stratiotes and Eichhornia crassipes) in two artificial reservoirs flooded by water diverted from the Paraiba do Sul river, SE Brazil. The potential of these species for Hg accumulation and their role in Hg transport along the river system due to macrophyte management were evaluated. Mercury concentrations were higher in free-floating than in rooted species. Roots were also richer in Hg than were leaves. Dry weight Hg concentrations in leaves and roots from all species varied from 46-246 ng.g(-1) to 37-314 ng.g(-1), respectively. These values are higher than those reported for uncontaminated lakes in Brazil and in other tropical areas and similar to those reported for moderately contaminated sites. Mercury concentrations can be attributed to fluvial transport from the heavily industrialized Paraiba do Sul river basin. Intensive sampling of Pistia stratiotes from two sites in the Vigario reservoir was performed to evaluate the capacity of Hg incorporation in short periods of time. The results showed a significant negative correlation between Hg content and size class of individual plants, demonstrating the importance of juveniles, fast growing plants in absorbing Hg. The foremost impact related to Hg contents in the studied area concerns the periodic removal of macrophytes for reservoir management, followed by disposal in nearby areas. This results in the mobilization of 0.52 to 1.3 Kg of Hg per year, a significant fraction of the Hg burden present in reservoir waters. Disposal of such material may result in Hg leaching to river systems, affecting the Hg transfer throughout the basin. PMID- 16680312 TI - Body size, diet and endoparasites of the microhylid frog Chiasmocleis capixaba in an Atlantic Forest area of southern Bahia state, Brazil. AB - We analyzed the diet composition, endoparasites and sexual size dimorphism of the microhylid frog Chiasmocleis capixaba (Microhylidae) from a "mussununga" habitat in the municipality of Nova Vicosa, southern Bahia state, Brazil. All the 119 specimens analyzed were collected in a single night of heavy rainfall. Females (mean snout-vent length = 15.7 + 3.0 mm) were significantly larger than males (mean snout-vent length = 13.2 + 2.1 mm), and specimens of both sexes were smaller than those of a conspecific population previously reported in Aracruz, state of Espirito Santo state. The diet of C. capixaba was dominated by mites, ants and collembolans. Seventy-nine frogs (66.4% of the total) were infected by helminths, all belonging to a single species, Cosmocerca ornata, an intestinal nematode parasite. PMID- 16680313 TI - Larval development of Brachidontes solisianus (Bivalvia, Mytilidae), with notes on differences between its hinge system and that of the mollusk Perna perna. AB - This work, which is part of a study program on meroplankton larvae, aims to gain more in-depth knowledge about planktonic larvae. This study began with the mollusk Brachidontes solisianus (Bivalvia-Mytilidae), which is abundant on the rocky shores of the Cabo Frio region (state of Rio de Janeiro, Brazil). Brachidontes solisianus larvae were grown under controlled conditions for a period of 26 days and were fed with Isochrysis galbana and Tetraselmis chui. The temperature was kept at 26 degrees C and the saltiness at 28. Images of the larvae were taken daily with a light camera and measured with a micrometric lens until settlement occurred. The average size of the first D-shaped veliger stage was 90 microm in length and 70 microm in height, while the size in the last stage before settlement (pediveliger) was 273 microm in length and 257 microm in height. The comparative study of the hinge system involved the most abundant intertidal species of the study area: Brachidontes solisianus and Perna perna. The B. solisianus species were found to have more visible denticles at the extremities of the provinculum, whereas the denticles of the P. perna species occur along the entire provinculum. PMID- 16680314 TI - The immature stages of Paramallocera hirta Kirby, 1818 (Coleoptera: Cerambycidae: Elaphidionini). AB - Last instar larva and pupa of Paramallocera hirta Kirby, 1818 are described and illustrated based on specimens reared in the laboratory from neonate larvae on Eucalyptus globulus ssp. globulus logs and on an artificial diet. Characteristics of possible diagnostic value are also presented in this paper. PMID- 16680315 TI - Feeding dynamics and ecomorphology of Oligosarcus jenynsii (Gunther, 1864) and Oligosarcus robustus (Menezes, 1969) in the Lagoa Fortaleza, southern Brazil. AB - Oligosarcus jenynsii and Oligosarcus robustus are fishes of Characidae family that occur in Rio Grande do Sul, Uruguay and northern Argentina. This work purported to study the feeding dynamics (repletion and hepatosomatic indexes and condition factor) over time, and to investigate the coexistence of these two species by evaluating the partition of resources using qualitative and quantitative analyses of diet, temporal and spatial segregation throughout the water column and some ecomorphological aspects of the species in the Lagoa Fortaleza. Specimens were sampled monthly, from May 2000 to April 2001 during 24 h/month, using stationary gill nets of different mesh sizes. The records of each individual included total and standard length; total, stomach and liver weight; sex and stomach repletion. The variation of the mean values of repletion index and relative frequencies of stomach repletion stages indicate that O. jenynsii and O. robustus do not present seasonal differences in feeding intensity. The hepatosomatic index shows an allocation of energy to the liver during every period except reproduction, when part of the energy is used for gonad maturation. The estimated condition factor for both species reveals an increase in the reproductive period, evidencing the influence of gonads upon the condition of the fish. The diet analysis revealed that O. robustus is piscivorous, whereas O. jenynsii is a generalist carnivore, tending to piscivory as well. The active period of O. robustus is more concentrated at sunrise and sunset, whereas O. jenynsii is continually active, a characteristic related to hunting for prey. The ecomorphological analysis revealed differences between the two species in the dimensions of the mouth. Evidence suggests that the species coexist, sharing food sources, differing in oral morphology but ingesting similar prey, possibly because food is not a limiting factor in the environment. PMID- 16680316 TI - Abundance and frugivory of the Toco toucan (Ramphastos toco) in a gallery forest in Brazil's southern Pantanal. AB - Unlike other toucan species, the Toco toucan (Ramphastos toco)--the largest Ramphastidae--usually inhabits dry semi-open areas. This conspicuous canopy frugivore uses a large home range that includes a variety of vegetation types, among which gallery forests are widely cited as important to this species. However, the factors relating to the occurrence of Toco toucans in such habitats are unclear. I studied the abundance of Toco toucans as well as the availability of fleshy fruit in a gallery forest in the southern Pantanal (sub-region of Miranda, Brazil), in order to assess the relationship between these parameters. Also, I examined toucan foraging activity to analyze its relationship with both toucan abundance and fruit availability. The presence of the Toco toucan was more common in the gallery forest from the middle to the end of the dry season and during the middle of the wet season. Toucans foraged for fleshy fruits, mainly Genipa americana, Ficus luschnatiana, and Cecropia pachystachya fruits, feeding mostly on G. americana (by far the favorite food resource) and F. luschnatiana fruits during the dry season, while C. pachystachya fruits were important in the wet season. Toco toucans foraged particularly heavily (> 80% of foraging activity) on G. americana fruits during the latter part of the dry season, when fleshy fruit availability declined sharply. Toco toucan abundance in the gallery forest was associated with the availability of the most commonly consumed fleshy fruits, and also with its foraging activity. This finding suggests that the Toco toucan moved to the gallery forest periodically in response to the availability of abundant food resources, especially the G. americana fruits widely available and exploited during the severely dry season. Therefore, these fruits potentially contribute to Toco toucan persistence in the South Pantanal during the harshest period of the year. PMID- 16680317 TI - Testicular maturation of Oligosarcus hepsetus (Cuvier) (Actinopterygii, Characidae) in a Brazilian tropical reservoir. AB - Six reproductive classes of male Oligosarcus hepsetus (Cuvier, 1829), a medium sized carnivorous Characiform species, are described based on macroscopic and histological techniques. A total of 175 individuals were caught monthly between April 2001 and June 2002 in the Lajes Reservoir, Brazil, one of the largest impoundment areas in the state of Rio de Janeiro. The reproductive classes were based upon changes in the testicular morphology and stages of germinative cells, i.e., resting, early maturing, late maturing, mature, partially spent and totally spent. Fish in the resting class showed testes with spermatogonia and spermatocytes along the wall of seminal lobules, while spermatids were present in the lumina of the lobules. During early maturing, active spermatogenesis occurs throughout the testis; in the late maturing and mature classes, the lobules are swollen with sperm that are typical of fish in breeding condition. Spent testes presented seminal lobules with residual spermatozoa, coinciding with decreasing GSI and greatly reduced sperm production. Overall, the testicular morphology and class of maturity development of O. hepsetus in the Lajes reservoir did not differ significantly from those of closely related species in other lentic environments. Lower GSI values in the oligotrophic Lajes reservoir than in other eutrophic natural lakes suggest that this species may be modifying this aspect of its reproductive strategy in response to the artificial environment. PMID- 16680318 TI - Reproductive studies on ipecac (Cephaelis ipecacuanha (Brot.) A. Rich; Rubiaceae): meiotic behavior and pollen viability. AB - Reproductive studies were carried out on Brazilian accessions of ipecac, Cephaelis ipecacuanha. Meiotic behavior was studied using the squashing technique. Irregular chromosome segregation in meiosis I and II, many sets of chromosomes in telophase II, micronuclei, incorrect cytoplasm division, incomplete cytokinesis and anomalous post-meiotic products, mainly polyads, were observed. The mean meiotic index was lower than 72%. Pollen viability was analyzed using Alexander solution, and the percentages ranged between brevistylous and longistylous floral morphs (85.3 to 93.1%), and among different localities (82.5 to 92.6%) analyzed. The size of pollen ranged between viable and sterile, and empty and shrunken sterile. In its natural habitat, this species is known to propagate by vegetative multiplication, but sexual reproduction seems to be as important as the vegetative propagation to this species. PMID- 16680319 TI - Chromosomal analysis of Astyanax fasciatus (Pisces, Characidae) from the Araguari river, Uberlandia, MG, Brazil. AB - The genus Astyanax is one of the most numerous of the family Characidae, comprising a large number of similar-shaped species, but displaying innumerable karyotypic variations in its chromosome number and/or structure. The literature describes A. fasciatus populations with diploid chromosome numbers varying from 2n = 45 to 2n = 48. In this study, A. fasciatus specimens captured in the Araguari River (Alto Parana basin) were cytogenetically characterized, revealing a diploid chromosome number of 2n = 46. The nucleolar organizing regions (NORs), detected with silver nitrate staining, showed a multiple system with two pairs of marked chromosomes. These findings are congruent with those of other studies involving populations of the same species. PMID- 16680320 TI - Considerations on the effect of anti-sandfly antibodies on biological parameters of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae). AB - The immunization of vertebrate hosts with vector components may be an alternative for the control of diseases transmitted by insects. In the present study we evaluated the effects of anti-sandfly antibodies on some of the biological parameters of female Lutzomyia longipalpis, a vector of visceral leishmaniasis. Rabbits were immunized with extracts of gut from blood-fed (GB) or sugar-fed (GS) females, carcass of sugar-fed (CS) or blood-fed (CB) females, and with repeated sandfly bites (BITE). Immune sera showed increased antibody titers compared to pre-immunized animals, and specific bands were detected by Western Blot. An analysis of biological parameters revealed a decline in fecundity in the group of females fed on rabbits immunized with GB and BITE. Longevity and mortality were studied in females with oviposition (parous) and without oviposition (nulliparous). Nulliparous females that fed on rabbits immunized with bites died in the highest percentage. A mortality analysis after egg laying revealed a peak on the fifth day in all the groups, but females fed on rabbit subjected to repeated bites showed a shift towards the third day. PMID- 16680321 TI - Second record of Lasiurus ega (Gervais) (Mammalia, Chiroptera, Vespertilionidae) over the south Atlantic. PMID- 16680322 TI - Onychophora in humid forests of northeastern Brazil. PMID- 16680324 TI - Dietary intake of female bariatric patients after anti-obesity gastroplasty. AB - PURPOSE: Roux-en-Y gastric bypass is a popular and successful operation for the treatment of morbid obesity. However, it greatly restricts ingestion and moderately interferes with absorption of food, thus potentially paving the way for undernutrition, especially during the first year before patients adapt to the new condition. Aiming to document actual dietary intake during this period, a prospective observational study was performed. METHODS: Forty consecutive patients were investigated using a 24-hour dietary recall technique every 3 months after surgery for 1 year. Females only were accepted for greater homogeneity of the sample. All received a vitamin and mineral supplement on a daily basis as a postoperative routine. A questionnaire was employed regarding general, nutritional, and gastrointestinal changes as well as consumption of medications. Dietary intake was analyzed after data processing using the Virtual Nutri software package (Sao Paulo, SP, Brazil). RESULTS: The surgical response was within the expected range, with about 67% excess weight loss at the end of the 1st year, and the same occurred with gastrointestinal symptoms and drug requirements. Daily energy intake on the 4 analyzed occasions was 529.4 +/- 47.4, 710.9 +/- 47.6, 833.2 +/- 72.0, and 866.2 +/- 95,1 kcal/day (mean +/- SEM); protein intake was increased in the same proportion at 6 and 9 months, but reduced at 12 months. Thus, patients did not meet standard recommendations regarding calories and proteins, even at the end of the 1st year; iron and zinc intake were also inadequate, although deficiencies were probably staved off by the prescribed supplement preparation. CONCLUSIONS: 1) The risk for postoperative undernutrition was evidenced up to 1 year, while spontaneous improvement in food intake was slow and inefficient; 2) Specific protocols should be devised to improve nutrition and health during the postoperative phase until successful dietary adaptation is achieved. PMID- 16680325 TI - Comparative analysis between osteosarcoma and Ewing's sarcoma: evaluation of the time from onset of signs and symptoms until diagnosis. AB - OBJECTIVE: The purposes of this study were to describe the early signs and symptoms of osteosarcoma and Ewing's sarcoma, identify symptoms that could be used to help differentiate the two types of tumors, and determine the time elapsed between the onset of signs and symptoms and the definitive diagnosis in our service, providing information and imputus for earlier diagnosis of these tumors. METHODS: A retrospective analysis of the medical dossiers of 365 patients under 30 years of age diagnosed with osteosarcoma or Ewing's sarcoma was performed, and the aspects of the clinical diagnosis were statistically analyzed and compared. RESULTS: The time between the onset of signs and the symptoms was 5.25 months for osteosarcoma and 8.1 months for Ewing's sarcoma, and the most frequent (89.5%) early symptom of osteosarcoma and Ewing's sarcoma was local pain. Symptoms that might aid diagnosis included early local volume increase and the presence of fever. CONCLUSION: The time until diagnosis of both neoplasias was higher than that reported for North America and Europe. Education of the lay public and medical professionals regarding suspicious early signs and symptoms might shorten the delay of diagnosis. PMID- 16680326 TI - Follow-up of the aneurysmal sac after exclusion and bypass of popliteal artery aneurysms. AB - Popliteal artery aneurysms are frequent and may lead to thromboembolic events and limb loss. PURPOSE: To evaluate clinical and ultrasonographic follow-up of patients who underwent exclusion of a popliteal artery aneurysm using the technique proposed by Edwards. METHODS: Data of all patients who underwent surgery to repair a popliteal artery aneurysm at Hospital das Clinicas, the Sao Paulo University Medical School between 1996 and 2004 were reviewed. Inclusion criteria were repair with aneurysm exclusion and bypass using the technique proposed by Edwards, as well as the existence of preoperative and postoperative measurements of the aneurysmal sac. RESULTS: Data of 16 patients who underwent 20 procedures for popliteal artery aneurysm exclusion and bypass were available to analysis. The preoperative diameter of the popliteal artery aneurysms ranged from 1.3 cm to 6.1 cm (mean = 3.1 cm). Patients underwent duplex ultrasound scanning 1 month to 7 years after surgical repair. Follow-up of the 20 cases revealed that 10 aneurysms exhibited decreased mean transverse diameters, ranging from 0.2 to 2.3 cm, while 7 had increased in diameter, ranging 0.3 to 3.3 cm, and 3 remained unchanged. Flow was observed only in 5 out of the 20 procedures, 3 of which (60%) had increased diameters. CONCLUSION: Although exclusion is a widely accepted procedure for the repair of popliteal artery aneurysms, data in the literature and the results of this study, which did not include cases of rupture or compression, suggest that strict follow-up of patients who undergo aneurysm exclusion is necessary. PMID- 16680327 TI - Cerebral and cardiac amyloidosis in autopsied elderly individuals. AB - INTRODUCTION: Amyloidosis in elderly individuals can be an independent alteration and a characteristic of aging. However, the clinical, pathophysiologic, and biochemical characteristics of amyloidosis related to age remain uncertain. OBJECTIVE: The purpose of this study was to determine the extent to which the heart and/or the brain of individuals aged 60 years or over exhibits amyloid deposits. MATERIALS AND METHODS: The autopsy findings of individuals who were at least 60 years old were studied. The autopsies took place between the years of 1976 and 2000. A total of 10 cases were selected that had hearts without cardiopathies, had negative serology for Chagas' disease, and had brains without morphological changes related to encephalopathies. Slides with fragments of heart and brain were processed and analyzed using polarized and common light microscopy. RESULTS: Of the 10 cases, 4 were positive for amyloidosis. All had positive findings in the brain, and 1 case also had positive findings in the heart. Among the positive cases, 50% were of people aged 60 to 69 years. There appeared to be a relationship between the presence of amyloid deposits and the ratio of brain and body weight, with the ratio in the positive cases being smaller than in the negative cases. CONCLUSIONS: The analysis of amyloid deposits in the brains and hearts of elderly individuals shows that such deposits may lead to a systemic attack of senility, common to natural aging. It is not certain that beta-amyloid deposits would alone bring such drastic repercussions to the individual. Some additional disorders of the organism could cause the breakdown of the natural balance related to the accumulation of these proteins, leading the way to the pathological contexts of amyloidosis. PMID- 16680328 TI - Multilocus genotyping of Cryptosporidium hominis associated with diarrhea outbreak in a day care unit in Sao Paulo. AB - A number of species of Cryptosporidium are associated with diarrhea worldwide. Little data exists regarding the genotypes and species of Cryptosporidium associated with cases of infections in Brazil. PURPOSE: In the present study, we ascertained by molecular methods the species and the genotype of Cryptosporidium sp from a diarrhea outbreak diagnosed in a day care at the Hospital Clinicas, Sao Paulo University Medical School. MATERIALS AND METHODS: Specific identification and typing of the isolates associated with the outbreak was done by DNA sequencing analysis of fragments amplified by polymerase chain reaction (PCR) from 3 different Cryptosporidium loci: the SSUrRNA coding region, the Cryptosporidium oocyst wall protein (COWP) gene, and the microsatellite locus 1 (ML1), a tandem GAG-trinucleotide repeat containing substitutions that differentiate the genotypes of Cryptosporidium parvum and Cryptosporidium hominis. RESULTS: A total of 29 positive samples from the outbreak were studied by the molecular methods described. Our study revealed the presence of a single genotype of Cryptosporidium hominis in all samples. CONCLUSION: The molecular analysis reinforced the hypothesis that the transmission of Cryptosporidium hominis during the period the samples were collected occurred in an outbreak pattern, possibly by person-to-person contact through the fecal-oral route. As far as we know, this is the first time that molecular tools have been used to identify the species and the genotype of isolates showing the presence of the ML1 genotype in samples from Brazilian patients. PMID- 16680329 TI - Association between maternal-fetal genetic histocompatibility and maternal undernutrition in mice: influence on intrauterine growth. AB - OBJECTIVE: The purpose of this study was to evaluate the effects of maternal fetal genetic histocompatibility and the association of that condition with maternal undernutrition regarding fetal growth and litter size. STUDY DESIGN: Fetuses that were either syngeneic or allogeneic with the mothers were bred, using mice of well-defined syngeneic strains (A/J and Balb/c). Pregnant mice were fed using either unrestricted normal diet with 22% protein, consumed ad libitum, or a diet containing 14% protein, with intake restricted to 70% of that consumed by the unrestricted group. At the end of gestation, the number of fetoplacental units and fetal losses, the fetal and placental weight, and the weights of fetal brain and liver were recorded. RESULTS: Fetuses from undernourished mothers showed a reduction in body, placental, and brain weight (P < 0.01); the association of undernourishment with maternal-fetal genetic compatibility resulted in a greater impairment of fetal growth (P < 0.01). A reduction in the number of viable fetuses per female in the dietary-restricted groups was observed. (P < 0.01). Although the concurrence of maternal-fetal genetic compatibility resulted in a trend towards a greater reduction in the number of viable fetuses as well as in a higher rate of fetal loss, these differences did not reach statistical significance. CONCLUSIONS: In mice, the occurrence of maternal-fetal genetic histocompatibility alone did not change fetal growth; maternal undernutrition during pregnancy resulted in growth stunting of progenies with reduction of litter size; and the association of these 2 factors produced greater reduction of fetal weight. PMID- 16680330 TI - Results of meniscectomy for treatment of isolated meniscal injuries: correlation between results and etiology of injury. AB - PURPOSE: To evaluate the results of the treatment of patients with isolated meniscal injuries of different etiologies. MATERIALS AND METHODS: 435 patients of both sexes and different age groups underwent meniscectomy after their isolated meniscal injuries were clinically diagnosed and confirmed by nuclear magnetic resonance imaging. RESULTS: Most patients achieved good results and were able to return to the activities they had practiced prior to surgery without major limitations. CONCLUSIONS: Meniscectomy for the treatment of traumatic meniscal injury provides better results than meniscectomy for the treatment of degenerative meniscal injury. The results of meniscectomy for the treatment of meniscal injury due to fatigue are similar to those of meniscectomy for the treatment of traumatic meniscal injury, although the risk of osteonecrosis development is higher. PMID- 16680331 TI - Oncologic progression of bone plasmacytomas to multiple myeloma. AB - PURPOSE: To evaluate the clinical aspects, diagnoses, prognostic factors, and percent progression of plasmacytoma to multiple myeloma. MATERIALS AND METHODS: 103 medical records of patients suspected of plasmacytoma were surveyed covering the period between 1950 and 1998, and 30 were selected for analysis. Patients were classified into 2 groups: patients who did (n = 17) and did not (n = 13) progress to multiple myeloma. Comparative statistics regarding a variety of clinical aspects were developed. RESULTS: Patients who progressed to multiple myeloma were younger than those who did not (52.3 +/- 2.6 vs 62.6 +/- 3.4 years; mean +/- SEM; P = 0.02). There were no significant differences in gender between groups. A higher incidence of multiple recurrence was observed in patients who progressed to multiple myeloma (75%, P = 0.049). Both groups showed a prevalence of vertebral column injuries. No significant differences were found between groups regarding the disease period (from the onset of symptoms until diagnosis) (P = 0.20) and survival (P = 0.34). The average time to progression from plasmacytoma to myeloma was 41 +/- 39 months (mean +/- SD), and the progression rate was 57%. CONCLUSION: Patients who progressed to multiple myeloma were younger than those who did not. No significant differences were found between groups regarding sex, time from symptom onset to diagnosis, and survival time. In both groups, the most affected anatomic location was the vertebral column, and most affected sex was male. The average time to progression to multiple myeloma was 41 months. It was not possible to determine the factors that influenced the survival of patients with plasmacytoma or for those who progressed to multiple myeloma. PMID- 16680332 TI - Varicella zoster in children attending day care centers. AB - OBJECTIVE: To describe morbidity associated to varicella in children attending day care centers. METHODS: Descriptive study carried out through inquiries with parents of 664 children who acquired varicella after admission to day care centers in Taubate (population: 244,165, census of 2004), a prosperous city in the State of Sao Paulo. RESULTS: The median age was 36 months (range 6 to 80 months); 8.4% of the children had varicella before 1 year of age. The main symptoms were: exanthema (100.0%), fever (85.4%) anorexia (39.6%), and headache (15.3%). 517 children (77.9%) had at least 1 medical visit, and 80.6% received at least 1 medication; 73 (11.0%) received nonsteroidal antiinflammatory drugs, and 52 (7.8%) received antibiotics. Complications occurred in 38 children (5.7%; 95% confidence interval: 3% - 8%); 8 (1.2%) were hospitalized, and 5 (0.7%) had sequelae. Complications and hospitalizations rates were 3 times more frequent in children with less than 1 year of age than in older children. More than half of the children and of the working parents were absent from their regular activities for more than a week. CONCLUSIONS: Varicella was associated with significant morbidity, affected younger children, was complicated in more than 5%, and left sequelae in 0.7% of children. More than 10% of the children received nonsteroidal antiinflammatory drugs, highlighting the need to warn the population about the risks of these drugs. Although varicella vaccination is not recommended for children younger than 12 months, vaccination of the children older than a year could avoid by herd immunity the transmission to babies. Brazilian public health authorities should be alerted to this issue and offer varicella vaccine to children attending day care centers. PMID- 16680333 TI - Analysis of the immunogenicity and stability of a porcine pulmonary surfactant preparation administered in rabbits. AB - PURPOSE: To study the immunogenicity and the stability of the porcine pulmonary surfactant preparation produced by the Instituto Butantan. METHOD: Immunogenicity assay: Sixteen New-Zealand-White rabbits (1000 g body weight) were divided into 4 study groups. Each group was assigned to receive either a) Butantan surfactant, b) Survanta (Abbott Laboratories), c) Curosurf (Farmalab Chiesi), or d) no surfactant. The surfactants were administered intratracheally, and the animals were collected immediately before and 60 and 180 days after surfactant administration. Sera were assayed for the presence of antisurfactant antibodies by enzyme-linked immunosorbent assay (ELISA). Stability assay: The Butantan surfactant used in this assay had been stored for one year in the refrigerator (4 to 8 degrees C) and its stability was evaluated in distinct assay conditions using a premature rabbit model. RESULTS: Immunogenicity assay: None of the surfactants analyzed triggered antibody immune responses against their components in any of the animals. Stability assay: The results of this study demonstrate that Butantan surfactant was as effective as Curosurf when both were submitted to the adverse circumstance of short- and long-term storage at room temperature. A similar level of efficacy for the Butantan surfactant, as compared to Curosurf was demonstrated by the pulmonary dynamic compliance, ventilatory pressure, and pressure-volume curve results. CONCLUSION: The results of our study demonstrate that Butantan surfactant may be a suitable alternative for surfactant replacement therapy. PMID- 16680335 TI - Importance of angiographic study in preoperative planning of conjoined twins: case report. PMID- 16680334 TI - Oral manifestations of syphilis. AB - The past decade has shown a significant rise in the prevalence of infective syphilis in the developed world, and striking increases in its frequency have occurred in Eastern Europe, particularly the UK, and in the US. Although oral manifestations of syphilis are most likely to be observed during secondary disease, all stages of the disease can give rise to oral lesions. Significant oral lesions such as gumma-associated bony destruction and a possible predisposition to oral squamous cell carcinoma are associated with tertiary disease. Since the prevalence of infective syphilis in heterosexuals has been increasing, there has now been a gradual rise in the number of children born with congenital syphilis. Consequently, the congenital disease gives rise to dental anomalies as well as bone, skin, and neurological anomalies of the face. The aim of this report is to review syphilis-related oral lesions, as well as to summarize the relations between human immunodeficiency virus (HIV) and syphilis. PMID- 16680336 TI - Surgical treatment for eyelid deformity in Crouzon syndrome associated with acanthosis nigricans: case report. PMID- 16680337 TI - Pancreatic carcinoid: a rare cause of diarrheogenic syndrome. PMID- 16680338 TI - Meta-analysis of aspirin for the prevention of preeclampsia: do the main randomized controlled trials support an association between low-dose aspirin and a reduced risk of developing preeclampsia? PMID- 16680339 TI - Is there any scientific evidence for the prevention of preeclampsia with low-dose aspirin?: Meta-analysis versus randomized controlled trial to answer this question. PMID- 16680340 TI - Technical modification of the Nuss operation for the correction of pectus excavatum. PMID- 16680341 TI - Growth in Brazilian scientific output in public health dentistry. PMID- 16680342 TI - Coffee and gastric cancer: systematic review and meta-analysis. AB - We systematically reviewed the literature on the association between coffee consumption and gastric cancer and performed a meta-analysis of the results. Published cohort and case-control studies were identified in PubMed and reference lists. Random effects meta-analysis was used to pool effects from 23 studies, and heterogeneity was explored by stratification and meta-regression. The odds ratio (OR) for the overall association between coffee and gastric cancer (highest vs. lowest category of exposure) was 0.97 (95%CI: 0.86-1.09), similar for cohort (OR = 1.02; 95%CI: 0.76-1.37) and case-control studies (population-based: OR = 0.90; 95%CI: 0.70-1.15; hospital-based: OR = 0.97; 95%CI: 0.83-1.13). The OR was 1.26 (95%CI: 1.02-1.57) when considering five studies conducted in the USA, 0.97 (95%CI: 0.82-1.14) for the five Japanese studies, 0.98 (95%CI: 0.81-1.17) for the six studies from Europe, and 0.64 (95%CI: 0.47-0.86) for the two studies from South America. In this meta-analysis we found no adverse effect of coffee associated with gastric cancer. Knowledge on the level of exposure to different coffee constituents may provide a deeper understanding of this reassuring result and the real role of coffee on cancer risk. PMID- 16680343 TI - [Public health research output related to males and health: a bibliographical review]. AB - This study focuses on the research output in the relationship between males and health in the public health field. The objective is to critically analyze the most recurrent ideas on the specificities of maleness in the health-disease process. The method consisted of a literature review in public health, based on a qualitative research approach. Male sexuality, masculinity, and reproduction and masculinity and power were the themes found in the analysis. The study concludes that further research on masculinity is needed with a focus on gender, nationality, class, age, ethnicity, and sexual orientation. PMID- 16680344 TI - [Craniofacial anomalies: description and evaluation of treatment under the Brazilian Unified Health System]. AB - The first initiative for treating craniofacial anomalies under the Brazilian Unified Health System was in 1993. An important step was the creation of the Reference Network for Craniofacial Treatment. There are now 29 services listed in this Network. The current study aimed to describe and assess the general characteristics of healthcare in this Network. Data were collected by a questionnaire, sent to the centers. Response rate was 86.2%. The results showed an increase in services in Southeast Brazil, in universities, and in relation to cleft lip and palate; public financing was prevalent; team composition was largely in accordance with North American standards; routine care occurred in 90%; and 70% used clinical protocols. The Network's name does not appear to entirely reflect its scope. The results show the need to review the Network's definition, aims, and achievements and the standards for inclusion of craniofacial centers. PMID- 16680345 TI - [Problem-posing as a nutritional education strategy with obese teenagers]. AB - Obesity is a public health issue with relevant social determinants in its etiology and where interventions with teenagers encounter complex biopsychological conditions. This study evaluated intervention in nutritional education through a problem-posing approach with 22 obese teenagers, treated collectively and individually for eight months. Speech acts were collected through the use of word cards, observer recording, and tape-recording. The study adopted a qualitative methodology, and the approach involved content analysis. Problem-posing facilitated changes in eating behavior, triggering reflections on nutritional practices, family circumstances, social stigma, interaction with health professionals, and religion. Teenagers under individual care posed problems more effectively in relation to eating, while those under collective care posed problems in relation to family and psychological issues, with effective qualitative eating changes in both groups. The intervention helped teenagers understand their life history and determinants of eating behaviors, spontaneously implementing eating changes and making them aware of possibilities for maintaining the new practices and autonomously exercising their role as protagonists in their own health care. PMID- 16680346 TI - [Reliability of the final dengue diagnosis in the epidemic occurring in Rio de Janeiro, Brazil, 2001-2002]. AB - This study analyzed the reliability of the final diagnosis in the 155,242 dengue reports during the 2001-2002 epidemic in the city of Rio de Janeiro, Brazil, using the official information system on communicable diseases (SINAN). The system allows the following options for the final diagnosis: classic dengue, dengue hemorrhagic fever, discarded, inconclusive, and unknown. We built a classification routine in Epi Info to compare the final diagnosis from SINAN with Ministry of Health criteria. According to the final diagnosis, the case breakdown was: 52.4% classic dengue; 0.6% dengue hemorrhagic fever; 0.9% discarded; 46% inconclusive and unknown. The revised diagnosis showed that 78% of classic dengue, 69% of dengue hemorrhagic fever, and 21.1% of discarded cases met the classification criteria. Although the reliability of the SINAN final diagnosis was generally satisfactory (kappa = 0.681; 95%CI: 0.685-0.677), it was worse for fatal cases (kappa = 0.152; 95%CI: 0.046-0.258). Considering the epidemic's magnitude, the final diagnosis of classic dengue and dengue hemorrhagic fever was satisfactory, but the high proportion of inconclusive or unknown cases and the poor quality of information for fatal cases limit the usefulness of SINAN in this context. PMID- 16680347 TI - [Women, Medicine, and technology in the discourse of medical residents in Obstetrics/Gynecology]. AB - This study focused on revealing the cultural meanings assigned to womanhood and the health-disease process in women according to the discourse of medical residents in Obstetrics/Gynecology at the Fernandes Figueira Institute, a public reference hospital specializing in maternal-child care in Rio de Janeiro. The research had two components: participatory observation in Ob-Gyn meetings and recording of oral sources. The sign-based method was used to analyze the data. The methodology included qualitative analytical coding of interviews and subsequent semiotic analysis. According to the results: (a) women are seen essentially as mothers, and their illness focuses primarily on their childbearing function; (b) technological evolution, mainly with the increased use of imaging, has reduced the importance of semiology; and (c) within the biotechnological context medicalization is part of the material and semiotic practices. PMID- 16680348 TI - [Primary care in European Union countries: configurations and organizational reforms in the 1990s]. AB - During the 1990s, proposals to contain health expenditures and demands raised by changes in the epidemiological profile led to organizational reforms in primary healthcare services in European Union countries, aimed at promoting the coordination of services provided by the various levels of care. Based on a literature review, document analysis, and interviews with key informants, the current study analyzes the institutional configurations for first-level healthcare and discusses the pre-coordination organizational reforms in the European Union countries. First-level outpatient care is provided in these countries through a wide variety of institutional configurations, observing the different financing mechanisms, range of healthcare professionals, range of clinical services provided, and the position occupied by physicians in primary care within the healthcare system. Reforms in the last decade were accompanied by a diversification in the outpatient care organizational models, with an expansion in the clinical, healthcare coordination management, and financing functions of primary care professionals, indicating a process in transition, with a redefinition of the roles played by general practitioners as the leaders of patient care. PMID- 16680349 TI - [Inequalities in the conditions of infection control in dentistry offices in a southeastern Brazilian city]. AB - The study analyzed infection control conditions in dentistry offices in Cariacica, Espirito Santo, Brazil. Data pertaining to interviewees, location and type of office, setting, equipment, and materials were obtained by direct observation in all the registered services and in twenty unlicensed establishments operated by "non-graduate dentists". For grouped analysis of infection control measurements in each establishment, a composite indicator based on 19 variables was developed, such that the lower its value, the better the infection control. The results referred to 113 offices. Infection control in offices of "non-graduate dentists" (xICI = 23.05, dp = 11.86) was four times worse than for private offices. Public dental care services (xICI = 16.27; dp = 6.96) occupied an intermediate position. Conditions were also four times worse in dentistry offices located in peripheral urban areas. There was a clear-cut division in sanitary conditions within dental care as a whole. A large proportion of the population depending on dental care from unlicensed dentists is exposed to increased risk of cross-infection. PMID- 16680350 TI - [Construction workers' reactions to educational materials: a debate on relations between health and work]. AB - This study focuses on the reactions of construction workers to educational materials (posters, folders, and leaflets) produced by the Brazilian Ministries of Health and Labor. The authors conducted two focal groups at construction sites in the city of Rio de Janeiro to analyze four educational materials, employing thematic analysis. The article presents the workers' perspective on the themes discussed in the leaflets, classified in two groups of meaning: health and social control. They go on to discuss how workers' display an expanded vision of health to the extent that it includes social determinants of the health-disease process. The study identified the workers' limited participation in work-process surveillance activities and their lack of knowledge concerning workers' health measures adopted by the Ministry of Health. The authors conclude that the analysis of educational materials by their target public represents a privileged place for negotiating meanings and grasping ways of life in specific groups. They suggest educational activities using leaflets as a means to motivate critical analysis of the health-disease process. PMID- 16680351 TI - [Dengue control in an urban area of Brazil: impact of the Family Health Program on traditional control]. AB - A study was performed in different areas of Sao Jose do Rio Preto which include the Family Health Program (FHP) and the Dengue Control Program, and the results of integration between the two programs were evaluated. In addition to other responsibilities, community health agents instructed residents on dengue control measures, encouraging the population to adopt the practices in areas with the FHP. Vector control agents were responsible for breeding site control and instructed local residents on the Dengue Control Program. From 2001 to 2003, surveys were conducted to measure residents' dengue control knowledge and practices. The proportions of residents in the FHP area that reported health services as a source of information increased significantly as compared to the other area. There were significant changes concerning the increase in information about the disease and reduction in vector breeding sites. The results show that integration between the programs is possible and could help optimize resources, avoiding duplicity of procedures and fostering greater community involvement in dengue control. PMID- 16680352 TI - [Factors associated with minor psychiatric disorders among women selected from a healthcare center in Rio de Janeiro, Brazil]. AB - The aim was to investigate factors potentially associated with minor psychiatric disorders, including maternal nutritional status variables. A cohort was studied with 479 women 15-45 years of age. The reduced General Health Questionnaire (GHQ 12 items) was applied at nine months post-partum with the 312 women who had completed follow-up. Minor psychiatric disorder was defined as a GHQ score of > or = 4 points and was treated as the response variable. Statistical analysis used hierarchical multivariate logistic regression models. The prevalence of minor psychiatric morbidity was 54.2% (95%CI: 48.6-59.7). According to the final model, the following variables remained statistically associated with minor psychiatric morbidity: level 1: total family income (1st quartile: OR = 2.71, 95%CI: 1.42 5.19; 2nd quartile: OR = 2.13; 95%CI: 1.13-4.04); level 3: body fat > or = 30% (OR = 1.66; 95%CI: 1.03-2.65). In conclusion, low income and obesity were the only factors potentially associated with minor psychiatric disorders, even after adjusting for confounding variables, while there are few studies relating maternal nutritional status and minor psychiatric morbidity. PMID- 16680353 TI - [Frequency of multiple neonatal malformations in Pelotas, Rio Grande do Sul, Brazil, and associated socio-demographic factors]. AB - This study aimed to estimate the frequency and to correlate the possible causal agents and monitor the occurrence of multiple neonatal malformations in Pelotas, Rio Grande do Sul, Brazil. The study included all births from 1990 to 2002 in the local maternity hospitals with birth weight > 500 g. Each newborn presenting a malformation (case) was assigned a live matched neonate (control) without any malformation and of the same sex. A database was established by filling out the ECLAMC-MONITOR forms, 1982 edition, tabulated with SPSS. Statistical analysis used Student t and chi2. During the study period, 71,500 children were born, of whom 0.11% presented multiple malformations. Significant results were found for birth weight, twin births, parents' ethnic background, paternal age, and number of previous abortions and stillbirths. In Pelotas, the proportion of newborns with malformations during the study period was 1.37%. Of these, 8.1% presented multiple malformations, predominantly in females and in births occurring during the winter. PMID- 16680354 TI - [Factors associated with burnout's syndrome: an epidemiological study of teachers]. AB - This article presents the results of an epidemiological study on burnout syndrome among private school teachers in Greater Metropolitan Porto Alegre, Rio Grande do Sul, Brazil, testing possible associations with demographic, work-related, and stress-related variables. The target population consisted of 217 teachers, of whom 190 participated (87.5%). The Maslach Burnout Inventory (MBI) was used to measure burnout, together with a questionnaire to record the other variables. The results showed that teachers have a low score in the three dimensions comprising burnout: emotional exhaustion, depersonalization, and decreased personal fulfillment at work. Demographic variables did not show any association with burnout, while occupational variables, workload, and number of students were associated with emotional exhaustion. Bad conduct by students, family expectations, and limited participation in institutional decisions were the stress factors associated with burnout. PMID- 16680355 TI - [Clinical and epidemiological profile of patients with Chagas disease at the University Hospital in Maringa, Parana, Brazil]. AB - This paper describes the clinical and epidemiological profile of patients with Chagas disease treated in outpatient and inpatient departments at the Maringa University Hospital (UHM) in Parana State, Brazil, from May 1998 to May 2003. Mean age was higher among hospitalized patients (p < 0.000). Hospitalized patients were predominantly males, as opposed to inpatients who were mostly females (p = 0.0033). Of the 95 patients, 60% were born in the States of Minas Gerais and Sao Paulo and 25.3% in Parana. Family history for Chagas was positive in 68.9%, while 53.3% reported the presence of triatomine vectors in the household. In decreasing order, the most common clinical forms were cardiac, digestive, indeterminate, and cardiodigestive. The indeterminate form predominated among outpatients, as opposed to the cardiac and digestive forms among inpatients. Chronic cardiac and digestive complications were the principal complaints at hospital admission. This study showed particularly high percentages of cardiac (38.9%) and digestive disease (26.3%) as compared to other geographic areas. The Maringa University Hospital provides symptomatic care for these complications and does not prioritize etiological treatment, even for patients in the indeterminate phase. PMID- 16680356 TI - [Declining caries rate in a municipality in northwestern Sao Paulo State, Brazil, 1998-2004]. AB - This study analyzes the DMF and DEF indices, SiC index, and percentage of caries free public schoolchildren in the municipality of Bilac, Sao Paulo State, Brazil, in 1998, 2000, 2002, and 2004. The same methodology was employed (WHO-1997) in all the surveys. Kappa test was carried out at each stage, and the minimal and maximum inter-examiner agreement values obtained were 0.86 and 0.89, respectively, while minimal intra-examiner agreement values were 0.91. DEF indices decreased slightly in the years 1998 and 2004. There was a continuous reduction in the DMF index, as well as the phenomenon of polarization at 12 years of age. The rate was 5.28 in 1998, decreasing to 4.11 in 2000, 3.47 in 2002, and 2.62 in 2004. Inversely, the proportion of caries-free 5-year-olds increased from 37.9% in 1998 to 40% in 2000 and 2002 and 45.3% in 2004. In conclusion, there has been a decrease in dental caries in 12 year-old public schoolchildren in the municipality. PMID- 16680357 TI - [Workers' contribution to the consolidation of municipal health surveillance services]. AB - This study aimed to expand knowledge on the role and possible contribution of workers in the consolidation of health surveillance services in three municipalities in Greater Metropolitan Porto Alegre, Rio Grande do Sul, Brazil. The research used a qualitative methodology based on focus groups and participatory observation. The resulting data were categorized by content analysis. The results pointed to precarious management of the health surveillance services. The main problems were deficiencies in staff education and training, precarious work conditions, lack of resource allocation autonomy and self management, in addition to prioritization of actions with a limited scope, focused on the control and oversight of products and services, to the detriment of broader activity with other determinants of the health-disease process. Despite this situation, most staff members seek further training on their own and oscillate between the pursuit of alternatives to overcome the difficulties and moments of frustration and discouragement. PMID- 16680358 TI - [Public health and mental health: methodological tools to evaluate the Brazilian Network of Referral Centers for Psycho-Social Care (CAPS) in the Brazilian Unified Health System]. AB - This article presents a preliminary discussion of potential methodological tools for qualitative research on the Network of Referral Centers for Psycho-Social Care (CAPS) in the Brazilian Unified Health System (SUS). The relevance of mental health within the field of public health is examined. The study focuses on the high prevalence of mental disorders and the disproportionate lack of studies on the interface between mental health and public health. The establishment of an interdisciplinary field between public health and mental health is proposed to meet common needs by achieving similar perspectives in knowledge and practice. A particular group of tools is proposed, emphasizing the importance of reclaiming and guaranteeing the roles of various social actors to shape the assessment process, the need for collecting and standardizing academic studies on the topic, and the importance of promoting a new research field focusing on public health policies to support policymakers, managers, and health teams in reshaping their practices. PMID- 16680359 TI - [Neonatal mortality in Londrina, Parana State, Brazil, in 1994, 1999, and 2002]. AB - Despite technological progress in recent decades, neonatal mortality accounts for some two-thirds of infant deaths where the infant mortality rates are low. This study analyzes neonatal deaths in Londrina, Parana, Brazil, during three periods, beginning with 1994, the year when pediatric and neonatal intensive care beds were created in the city. The data were collected from live birth certificates in the National Information System on Live Births (SINASC) and individual analysis of neonatal death certificates. Births declined in the city, but the low birthweight rate increased from 7.7 to 8.8% and the preterm birth rate from 6.3 to 8.4%. Multiple births also increased. Caesarian sections varied from 48 to 52%. The percentage of deaths from congenital malformations increased. The vast majority of neonatal deaths are preventable, mainly by providing specialized care during pregnancy. The neonatal mortality rate has declined recently, from 10.1 to 6.4 per 1,000 live births. The authors conclude that neonatal care is improving in Londrina. PMID- 16680360 TI - [Prevalence and factors associated with dental pain that prevents the performance of routine tasks by civil servants in Rio de Janeiro, Brazil]. AB - The aims of this study were to estimate the prevalence of dental pain preventing the performance of routine tasks and to assess its association with socioeconomic factors, minor psychiatric disorders, number of missing teeth, and dental consultation patterns. A cross-sectional study was conducted using a self completed questionnaire answered by 4,030 administrative employees at a university in Rio de Janeiro, Brazil (the Pro-Saude Study). Data were analyzed using multiple logistic regression. Prevalence of toothache preventing the performance of routine tasks in the two weeks prior to the interview was 2.9% (95%CI: 2.5-3.6). Men (OR = 1.6; 95%CI: 1.1-2.4), individuals with minor psychiatric disorders (OR = 1.7; 95%CI: 1.2-2.6), individuals with extensive tooth loss (OR = 3.4; 95%CI: 1.5-7.8), and those failing to appear for regular dental checkups (OR = 2.5; 95%CI: 1.8-17.3) showed increased odds of experiencing dental pain. Dental pain was an important problem in this population. Unfavorable living conditions and lack of regular dental checkups increased the odds of dental pain. PMID- 16680361 TI - [The impact of changes in age-related and cause-related mortality on life expectancy at birth in the State of Santa Catarina, Brazil, in the 1990s]. AB - This study aimed to analyze the impact of changes in age-related and cause related mortality on life expectancy at birth in the State of Santa Catarina, Brazil, in the 1990s. Data were obtained from the Mortality Information System (SIM) for the three-year periods 1989/1990/1991 and 1998/1999/2000. Infant mortality rates and specific mortality rates for individuals > or = 1 year of age were calculated by gender and cause of death. The Pollard method was used to disaggregate the contributions by mortality from different causes and in different ages to life expectancy at birth. The age groups that contributed the most to the increase in life expectancy at birth were 0-1 year and > or = 70 years. The causes of death that most contributed to this increase were chronic non-communicable diseases, such as cardiovascular diseases, in the age brackets above 35 years, and neonatal diseases in the < 1-year bracket. Finally, there were a high proportion of deaths from ill-defined causes, indicating that the true cause-related mortality structure in the State is still unknown. PMID- 16680362 TI - [Intestinal parasitosis in Mbya-Guarani populations from Misiones Province, Argentina: epidemiological and nutritional aspects]. AB - Intestinal parasite infestation in indigenous Mbya-Guarani communities in Misiones, Argentina, was described and associated with nutritional status and environmental and cultural factors. The results were compared with those from Takuapi, a neighboring indigenous population, and the nearest urban population, Aristobulo del Valle. The Ritchie, Willis, and Kato Katz techniques were used to analyze the stool samples. Anthropometric parameters were analyzed and earth samples processed. From a total sample of 296 individuals analyzed in the four populations, 100 (87.7%), 63 (88.7%), 49 (96.1%), and 50 (82%) were infested in Kaaguy Poty, Yvy Pyta, Takuapi, and Aristobulo del Valle, respectively. 84% of infested individuals had multiple parasites. The 43% of the individuals presented malnutrition, and 87% of these were infested. There was an association between use of latrines and Giardia lamblia (p < 0.01); open-air defecation, lack of footwear, and hookworms (p < 0.01); and housing type and total helminthes (p < 0.01). Earth samples were contaminated with parasites. The results suggest the relationship between environmental contamination and high prevalence of intestinal parasites in these human populations. PMID- 16680363 TI - [Use of contraceptive methods by sexually active women in Sao Leopoldo, Rio Grande do Sul, Brazil]. AB - In 2003, a population-based cross-sectional study was conducted in the urban area of Sao Leopoldo, Rio Grande do Sul, Brazil. The sample included 867 sexually active women from 20 to 60 years of age. The objective was to describe the use of contraceptive methods. Data were collected using a standardized questionnaire. The study analyzed the prevalence of contraceptive use and socioeconomic variables in women reporting an active sex life (84.5%), stratified by age groups. Some 627 (61.1%) women reported use of contraceptive methods. In the 20 49-year old group, 48.8% reported using oral contraceptives, 18.7% tubal ligation, 17.3% condoms, and 7.3% IUDs. In the 50-60-year old group, the most widely used method was tubal ligation (79.6%). Regarding tubal ligation, the schooling variable showed a linear trend, that is, women with less schooling showed a higher prevalence. Prevalence of oral contraception was higher in low income women. PMID- 16680364 TI - Microbiological evaluation of sugarcane juice sold at street stands and juice handling conditions in Sao Carlos, Sao Paulo, Brazil. AB - Fresh sugarcane juice is sold by street vendors without any heat treatment in Sao Carlos, Sao Paulo, Brazil. Twenty-four samples of point-of-sale juice were tested by standard methods to determine heterotrophic bacteria, total and thermo tolerant coliform counts, Salmonella, and parasites in the juice. 25% of samples showed poor sanitary conditions, with thermo-tolerant coliform levels higher than allowed by Brazilian standards. Salmonella spp. and parasites were absent in all samples. Thermo-tolerant coliforms were detected on the hands of 37% of juice handlers, and heterotrophic bacterial counts reached 2.0 x 10(3) cfu/per hand. Escherichia coli was detected in one hand sample, and no Salmonella spp. was detected. Screening questionnaires were used to interview the vendors, and 62% of interviewees were either unfamiliar with or failed to adopt adequate hygiene for food handling. PMID- 16680365 TI - Students of physical therapy. A comparative study of student profiles at a college and university in Israel. AB - This study focuses on the attributes of students of physical therapy in order to compare the profiles of students of physical therapy (PT) in two institutions of higher learning in Israel: Ben Gurion University (BGU) and the Academic College of Judea and Samaria (ACJS). This study focuses on a department where studies have an occupational/ applicative/practical orientation and high status in the higher education system. Findings of this study indicate broad similarities in the profiles of students at both institutions in relation to their age, family status, country of origin, number of siblings, parental education, and financial status of student family of origin. On the other hand, students at both institutions differed in terms of gender composition, students' employment status, the source of payment for tuition, and in their academic attainments prior to admission. Specifically, students of physical therapy at ACJS had lower academic achievements prior to their admission and reported having been rejected by other physical therapy programs. Students at ACJS placed higher importance on factors relating to the quality of instruction including teacher involvement, competitiveness, organization, control, and orientation to the study material. Students at BGU attributed greater importance to teachers' support. Findings support a thesis of a converging system of higher education in Israel, traditionally dominated by national universities and regional colleges, a relatively recent phenomenon. PMID- 16680366 TI - Incidence of galactorrhea in young women using Depot-Medroxyprogesterone Acetate. AB - Galactorrhea is rarely mentioned as a possible side effect of the use of Depot Medroxyprogesterone Acetate (DMPA). Over the last few years, we have noticed an increased number of patients complaining of galactorrhea. A review of clinical data showed that between 1999 and 2005, 360 adolescents in our clinic used DMPA for at least 6 months. After medical follow-up, 13 (3.6%) of these patients were found to have developed galactorrhea. The mean age of the patients was 19.4 years with a range from 13-24. Prolactin levels in these patients were normal, and in all subjects, the galactorrhea resolved spontaneously within the next year in both patients who continued use and those who discontinued use of DMPA. It appears that galactorrhea is a benign side effect and as previous reports have suggested, it did not seem to be related to changes in Prolactin levels in our patients. It is thought that this is a progesterone-mediated effect. We believe that reassurance and education of patients is sufficient and there is no evidence of need for further intervention. Since the sample size is small in this study, additional research is recommended as to validate the presence of progesterone mediated effects secondary to the use of DMPA. PMID- 16680367 TI - The expression of MMP-2 following immobilization and high-intensity running in plantaris muscle fiber in rats. AB - The effect of 2-week, high-intensity running and a 2-week immobilization on muscle fiber type composition of the plantaris muscle from 18 female, 6-month-old Wistar rats (running, n = 6; immobilization, n = 6; sedentary control, n = 6) was bio- and histochemically investigated. The high-intensity treadmill running began with 20 min (32 m/min, 0% gradient, 75% VO2 max), up to 50 min/day. Right hind limbs were immobilized by an external fixation procedure for 13 days. Muscle mass of the plantaris muscle in the immobilized groups was reduced by 16% in comparison with the sedentary control group. High-intensity running and immobilization increased both mRNA and protein levels of matrix metalloproteinase type 2 (MMP-2) in plantaris. Running and immobilization decreased the percentages of transverse sectional area of fast-twitch glycolytic (FG) type IIb fibers, running increased relative cross-sectional area of fast-twitch oxidative glycolytic (FOG) type IIa muscle fibers, whereas immobilization increased relative cross-sectional area of slow-twitch oxidative (SO) muscle fibers (type I). Our results suggest that both high-intensity running and immobilization are enough to induce overwhelming changes in plantaris. PMID- 16680368 TI - Partial rescue of pos5 mutants by YEF1 and UTR1 genes in Saccharomyces cerevisiae. AB - Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs. PMID- 16680369 TI - Dynamics of CD4+CD25+ T cells in spleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum. AB - CD4+CD25+ T cells play a major role in modulating immune response, but few reports have been published about schistosomiasis. Here, we investigated the changes in CD4+CD25+ T cell populations in spleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum. The proportions of CD4+CD25+ T cells in total CD4+ T cells were analyzed by flow cytometry. CD25 and Foxp3 expression was measured by real-time quantitative polymerase chain reaction. The suppressive activities of CD4+CD25+ T cells were detected by in vitro proliferation of splenocytes. Evidence showed that the percentage of CD4+CD25+ T cells was the same as controls 3 weeks post-infection. At the acute stage of infection, the percentage decreased significantly. However, at the chronic stage of infection, it rebounded to normal levels or even higher. The expression of the CD25 and Foxp3 showed gradual increase along with the infection progress. In vitro experiment also showed the strong suppressive effect of CD4+CD25+ T cells, isolated during the chronic stage, on proliferation of the CD25- splenocytes. This is the first time that the dynamics of CD4+CD25+ T cell populations was demonstrated in mice infected with schistosomiasis. In conclusion, our data indicated that CD4+CD25+ cells might be involved in the immune modulation during S. japonicum infection, which enhances current knowledge of the mechanisms of the immuno-downregulation and re-infection in schistosomiasis. PMID- 16680370 TI - Production of the polyclonal anti-human metallothionein 2A antibody with recombinant protein technology. AB - Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the cDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli. Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays. PMID- 16680371 TI - Energy transfer among chlorophylls in trimeric light-harvesting complex II of Bryopsis corticulans. AB - A study on energy transfer among chlorophylls (Chls) in the trimeric unit of the major light-harvesting complex II (LHC II) from Bryopsis corriculan, was carried out using time-correlated single photon counting. In the chlorophyll Q region of LHC II, six molecules characterized as Chlb628, Chlb646, Chlb652(654,657), Chla664(666), Chla674(677,680) and Chla682(683) were discriminated according to their absorption spectrum and fluorescence emission spectrum. Then, excited by pulsed light of 628 nm, fluorescence kinetics spectra in the chlorophyll Q region were measured. In accordance with the principles of fluorescence kinetics, these kinetics data were analyzed with a multi-exponential model. Time constants on energy transfer were obtained. An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97 picoseconds (ps), which shows that, before transferring energy to another Chl, the excited Chl might convert energy to vibrations of a lower state with different multiplicity (intersystem crossing). Energy transfer at the level of approximately 10 ps was also obtained, which was interpreted as the excited Chls may go through internal conversion before transferring energy to another Chl. Although with a higher standard deviation, time constants at the femtosecond level can not be entirely excluded, which can be attributed to the ultrafast process of direct energy transfer. Owing to the arrangement and direction of the dipole moment of Chls in LHC II, the probability of these processes is different. The fluorescence lifetimes of Chlb652(654,657), Chla664(666), Chla674(677,680) and Chla682(683) were determined to be 1.44 ns, 1.43 ns, 636 ps and 713 ps, respectively. The percentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in the trimeric unit of LHC II. These results are important for a better understanding of the relationship between the structure and function of LHC II. PMID- 16680372 TI - Nuclear factor-kB signaling pathway constitutively activated in esophageal squamous cell carcinoma cell lines and inhibition of growth of cells by small interfering RNA. AB - Although constitutive nuclear factor (NF)-kappaB activation has been reported in many human tumors, the role of the NF-kappaB pathway in esophageal squamous cell carcinoma (ESCC) has not been known. In this study, NF-kappaB pathway in two ESCC cell lines was investigated using immunocytochemistry, Western blot and reverse transcription-polymerase chain reaction. The activation of NF-kappaB DNA binding was determined by electrophoretic mobility-shift assay. RNA interference was used to specifically inhibit the expression of p65. Growth of cells was evaluated by 3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that p50, p65, IkappaBalpha p-IkappaBalpha and IkappaB kinase beta were expressed and mainly localized in the cytoplasm. Reverse transcription-polymerase chain reaction results showed the constitutive expressions of p50, p65 and IkappaBalpha mRNA in the two ESCC cell lines. Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the nucleus had DNA-binding activity. Finally, small interfering RNA of p65 decreased the expression of p65, and the viability of cells transfected with p65 small interfering RNA was significantly suppressed at the same concentration of 5-fluorouracil (P < 0.05) compared to untransfected cells. The results of this study showed that there was the constitutively activated NF-kB signaling pathway in the ESCC cell lines. RNA interference targeting at p65 increased the sensitivity of the ESCC cell lines to 5-fluorouracil, suggesting that NF-kappaB might be a good target for cancer treatment. PMID- 16680373 TI - Characterization of CD4+ T cell responses in mice infected with Schistosoma japonicum. AB - To better understand the interaction between Schistosoma japonicum and its murine host, we characterized the immune response of CD4+ T cells generated during an experimental S. japonicum infection based on different key aspects, from gene expression to cell behavior. Mouse oligonucleotide microarrays were used to compare gene expression profiles of CD4+ T cells from spleens of mice at 0, 3, 6 and 13 weeks post-infection. Flow cytometry analysis was used to determine type 1 and type 2 cytokine-secreting CD4+ T cells, to test apoptosis of CD4+ T cells and to count CD4+CD25+ T cells, a kind of regulatory subpopulation of CD4+ T cells. The percentage of interleukin-4-producing CD4+ T cells was found to be much higher than that of gamma-interferon-producing cells, especially after stimulation with S. japonicum egg antigen, which was consistent with type 1 and type 2 cytokine gene expression in the genechip. Microarray data also showed that S. japonicum induced the increased expression of Th2 response-related genes, whereas some transcripts related to the Th1 responsive pathway were depressed. Flow cytometry analysis showed a marked increase in the apoptotic CD4+ T cells from 6 weeks post-infection and in the ratio of CD4+CD25+ to CD4+ T cells in infected mice after 13 weeks. We therefore concluded that experimental infection of mice with S. japonicum resulted in a Th2-skewed immune response, which was to a great extent monitored by the immune regulatory network, including cytokine cross-modulation, cell apoptosis and the subpopulation of regulatory cells. PMID- 16680374 TI - Characterization of ATPase activity of recombinant human Pif1. AB - Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione S-transferase (GST)-fused N terminal truncated human Pif1 protein (hPif1deltaN) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 microM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1. PMID- 16680375 TI - Analysis of the resveratrol-binding protein using phage-displayed random peptide library. AB - Resveratrol, a plant polyphenol, is found in significant amounts in the skin of grapes and in some traditional herbs. It is reported to exert different biological activities, such as inhibiting lipid peroxidation, scavenging free radicals, inhibiting platelet aggregation, and anticancer activity. In order to screen the resveratrol-binding proteins, we synthesized biotinylated resveratrol, purified by liquid chromatography and immobilized it into streptavidin-coated microplate wells. 3-(4,5-Demethylthiazol-)-2,5-diphenyl tetrazolium bromide assay showed little change in the anticancer activity of biotinylated resveratrol in vitro. A random library of phage-displayed peptides was screened for binding to immobilized resveratrol to isolate resveratrol-binding proteins. Several peptides were found to bind to resveratrol specifically, which was proven by enzyme-linked immunosorbent assay. Through amino acid sequence analysis of the selected peptides and human proteins using the BLAST program, the results showed that resveratrol has an affinity for various proteins such as breast cancer-associated antigen, breast cancer resistance protein, death-associated transcription factor, and human cyclin-dependent kinase. These results demonstrate that our study provides a feasible method for the study of binding proteins of natural compounds using a phage-displayed random peptide library. PMID- 16680376 TI - Genetic and epigenetic alterations of DLC-1, a candidate tumor suppressor gene, in nasopharyngeal carcinoma. AB - The DLC-1 gene, located at the human chromosome region 8p22, behaves like a tumor suppressor gene and is frequently deleted in diverse tumors. The deletion of 8p22 is not an uncommon event in nasopharyngeal carcinoma (NPC), therefore we explored the expression levels of the DLC-1 gene in NPCs and NPC cell lines by reverse transcription-polymerase chain reaction. The results showed the mRNA level of DLC 1 was downregulated. To identify the mechanism of DLC-1 downregulation in NPC, we investigated the methylation status of the DLC-1 gene using methylation-specific PCR, and found that 79% (31 of 39) of the NPC tissues and two DLC-1 nonexpressing NPC cell lines, 6-10B and 5-8F, were methylated in the DLC-1 CpG island. Microsatellite PCR was also carried out, and loss of heterozygosity was found at four microsatellite sites (D8S552, D8S1754, D8S1790 and D8S549) covering the whole DLC-1 gene with ratios of 33% (4 of 12 informative cases), 18% (2 of 11), 5% (1 of 18), and 25% (3 of 12), respectively. Taken together, our results suggest that DLC-1 might be an NPC-related tumor suppressor gene affected by aberrant promoter methylation and gene deletion. PMID- 16680377 TI - Effects of heat stress on yeast heat shock factor-promoter binding in vivo. AB - Heat shock factor-DNA interaction is critical for understanding the regulatory mechanisms of stress-induced gene expression in eukaryotes. In this study, we analyzed the in vivo binding of yeast heat shock factor (HSF) to the promoters of target genes ScSSA1, ScSSA4, HSP30 and HSP104, using chromatin immunoprecipitation. Previous work suggested that yeast HSF is constitutively bound to DNA at all temperatures. Expression of HSF target genes is regulated at the post-transcriptional level. However, our results indicated that HSF does not bind to the promoters of ScSSA4 and HSP30 at normal temperature (23 degrees C). Binding to these promoters is rapidly induced by heat stress at 39 degrees C. HSF binds to ScSSA1 and HSP104 promoters under non-stress conditions, but at a low level. Heat stress rapidly leads to a notable increase in the binding of HSF to these two genes. The kinetics of the level of HSF-promoter binding correlate well with the expression of target genes, suggesting that the expression of HSF target genes is at least partially the result of HSF-promoter binding stability and subsequent transcription stimulation. PMID- 16680378 TI - Prevention of disability at work. PMID- 16680379 TI - Life-expectancy estimations and the determinants of survival after 15 years of follow-up for 81 249 workers with permanent occupational disabilities. AB - OBJECTIVES: This study attempts to estimate life expectancy and explore the determinants of survival for workers with permanent occupational disabilities. METHODS: A database on permanent occupational disabilities occurring between 1986 and 2000 was linked with the national death registry database to construct the survival function. A method with Monte Carlo simulation was used to extrapolate survival for up to 600 months to derive the life expectancy for different disability grades (N=81249). A Cox (proportional hazard) regression was carried out to explore the determinants and to estimate the hazard ratios. Demographic variables, including age, gender, insured wage, severity of disability, injury causes, and organ-system disability, were included in the model as covariates. RESULTS: The results indicate that the survival period for workers suffering permanent occupational disabilities is shorter than that of the general population, amounting to an estimated loss of life expectancy ranging from 5 to 19 years. After adjustment for age and gender, a higher severity of disability, impairment of vital organs or lower extremities, and a lower insured wage had a significant association with shorter survival. Injury types, including transportation incidents, being struck by sliding objects, or a trip, slip or stumble, and collapse injury, indicated hazard ratios of between 1.24 and 1.34, as compared with injuries such as being trapped or caught in machinery. CONCLUSIONS: The findings identify major determinants for predicting survival for workers with permanent occupational disabilities; these determinants may be of use in improving the equity of the compensation system for workers. PMID- 16680380 TI - Natural course of nontraumatic rotator cuff tendinitis and shoulder symptoms in a working population. AB - OBJECTIVES: This study assessed the prevalence, incidence, and persistence of nontraumatic rotator cuff tendinitis and shoulder symptoms over a 1-year period in a working population and the predictive value of symptoms and physical findings. METHODS: A 1-year prospective study of 436 active workers was conducted at 12 different worksites. Detailed health interviews, psychosocial questionnaires, and physical examinations were conducted at baseline and again after 1 year, with shorter evaluations at 4 and 8 months. Individual observed exposure assessment of shoulder posture, arm-hand activity, and hand forces was conducted. RESULTS: The prevalence of rotator cuff tendinitis at baseline was 7.6% [95% confidence interval (95% CI 5.1-10.1%)] for the right and 4.8% (95% CI 3.0-7.0%) for the left, compared with shoulder symptoms of 18.6% (95% CI 14.9 22.3%) (right) and 11.2% (95% CI 8.2-14.2%) (left). The incidence of rotator cuff tendinitis was 5.5% (95% CI 2.8-6.8%) and 2.9% (95% CI 1.0-3.8%), respectively. Higher proportions of participants with current symptoms or physical findings at baseline became clinical cases after 1 year than those without symptoms or findings. The 1-year persistence of clinical case status was 31.3% (95% CI 26.9 35.7%) (right) and 31.6% (95% CI 27.2-36.0%) (left). There were significant differences at baseline between the asymptomatic participants and the clinical cases with respect to physical health on the 12-item Short-form Health Survey (P=0.0002), the perception of general health (P=0.0027), and the frequency of high hand force exposure (P=0.0177). CONCLUSIONS: Considerable movement occurs between different stages of shoulder problems. Symptoms and physical findings alone appear to predict clinical case status within 1 year. Frequent follow-up is necessary to capture changes in health and exposure status in prospective studies. PMID- 16680381 TI - Salivary cortisol and self-reported stress among persons with environmental annoyance. AB - OBJECTIVES: Increased vulnerability to stress has been suggested as a possible mechanism behind medically unexplained conditions such as sensitivity to electricity and common smells. This study examined whether subjective environmental annoyance among the general population is associated with increased physiological reactivity or subjective stress scores. METHODS: Four groups were studied (N=141): an electrically annoyed (N=17), a smell-annoyed (N=29), and a generally annoyed group (N=39) and a reference group matched for age, gender, and socioeconomic status (N=56). Over 5 days, the participants collected saliva for cortisol determination at awakening, 30 minutes after awakening, 8 hours after awakening, and at 9 o'clock in the evening. On the evening preceding the fifth day, the participants ingested a 0.5-mg dexamethasone tablet so that possible differential suppression of the hypothalamic-pituitary-adrenal (HPA) axis could be assessed. Each day, the participants also rated their subjective stress and health complaints. RESULTS: No significant differences were found between the groups regarding cortisol secretion over 5 days. The dexamethasone suppression test showed inhibited cortisol secretion in all four groups. No associations were found between the cortisol concentrations and the self-reported stress scores or subjective health complaints. CONCLUSIONS: Although the environmentally annoyed groups showed no signs of increased HPA-axis activation, being annoyed by both electrical devices and smells seems to be related to increased psychological activation in terms of self-reported stress. Because the participants were otherwise healthy and recruited from the general population, the results imply that subtle psychological stress processes may be important in the early development of environmental annoyance. PMID- 16680382 TI - Disturbed sleep and fatigue in occupational burnout. AB - OBJECTIVES: The purpose of this study was to investigate sleep with polysomnography and self-ratings and the diurnal pattern of sleepiness and fatigue in a group suffering from severe occupational burnout. METHOD: Twelve white-collar workers on long-term sick leave (>3 months) and 12 healthy controls with high and low scores on the Shirom Melamed Burnout Questionnaire (SMBQ) were included. A 1-night polysomnographic recording (after habituation) was carried out at home, and sleepiness and mental fatigue were rated at different times of the day for weekdays and the weekend. Precipitating factors at the time of the illness at work and real life were considered, and different dimensions of occupational fatigue were described. A repeated-measures analysis of variance using two or three within group factors was used to analyze the data. RESULTS: The main polysomnographic findings were more arousals and sleep fragmentation, more wake time and stage-1 sleep, lower sleep efficiency, less slow wave sleep and rapid eye movement sleep, and a lower delta power density in non-rapid eye movement sleep in the burnout group. The burnout patients showed pronounced sleepiness and mental fatigue at most times of the day for weekdays without reduction during weekends. The precipitating factor was occupational stress (psychiatric interview), and work stress indicators were increased. CONCLUSIONS: Occupational burnout is characterized by impaired sleep. It is suggested that impaired sleep may play a role in the development of fatigue or exhaustion in burnout. PMID- 16680383 TI - Adolescents' jobs and the course of dermatitis symptoms throughout puberty. AB - OBJECTIVES: This study evaluated the course of dermatitis symptoms throughout puberty taking into account occupational exposures in a population-based study. METHODS: Participants enrolled in the ISAAC-II (International Study of Asthma and Allergies in Childhood) study in Munich and Dresden in 1995 and 1996 were sent a postal questionnaire in 2002 (age at follow-up 16 to 18 years). The questionnaire included items on atopic diseases, jobs, including holiday jobs and vocational training, and potential confounders. The most recent of the adolescents' jobs held for at least 8 hours a week, and for at least 1 month, were coded according to the ISCO-88 system. RESULTS: Overall, data of 3785 adolescents were included in the analyses. The incidence of dermatitis symptoms during puberty among those without such symptoms at baseline was 7%. Altogether 31% of the participants reported an employment history. Those already employed were more likely to report a new onset of dermatitis symptoms. Jobs associated with a new onset of symptoms were work in the health care sector, vocational training in bakeries, and cleaning. The first 9 months of exposure were particularly relevant for new cases of dermatitis symptoms (odds ratio 3.7, 95% confidence interval 1.5-9.6). CONCLUSIONS: Early occupational exposure is associated with the development of symptoms of dermatitis. The types of skin alterations need to be assessed in the next stage of the study. PMID- 16680384 TI - Changes in ocular and nasal signs and symptoms among air crew in relation to air humidification on intercontinental flights. AB - OBJECTIVE: This study evaluates the influence of air humidification in aircraft on symptoms, tear-film stability, nasal patency, and peak expiratory flow. METHODS: Commercial air crew (N=71) were given a medical examination during eight flights from Stockholm to Chicago and eight flights in the opposite direction. Examinations were done onboard one Boeing 767 aircraft equipped with an evaporation humidifier in the forward part of the cabin. The investigators followed the air crew, staying one night in Chicago and returning with the same crew. Four of the flights had the air humidification device active in-flight to Chicago and deactivated when returning to Stockholm. The other four flights had the inverse humidification sequence. The humidification sequence was randomized and double blind. Hygienic measurements were performed. RESULTS: The humidification increased the relative air humidity by 10% in the 1st row in business class, by 3% in the last row (39th row) in tourist class, and by 3% in the cockpit. Air humidification increased tear-film stability and nasal patency and decreased ocular, nasal, and dermal symptoms and headache. The mean concentration of viable bacteria [77-108 colony-forming units (cfu)/m(3)], viable molds (74-84 cfu/m(3)), and particulate matter (1-8 microg/m(3)) was low, both during the humidified and non-humidified flights. CONCLUSIONS: Relative air humidity is low (10-12%) during intercontinental flights and can be increased by the use of a ceramic evaporation humidifier, without any measurable increase of microorganisms in cabin air. Air humidification could increase passenger and crew comfort by increasing tear-film stability and nasal patency and reducing various symptoms. PMID- 16680385 TI - Effect of respirators equipped with particle or particle-and-gas filters during exposure in a pig confinement building. AB - OBJECTIVES: This study compared the protective effect of two respiratory protection devices during exposure in a pig confinement building. METHODS: Thirty six healthy persons were exposed for 3 hours in the building, 12 without any protection, 12 with a particle-filter mask, and 12 with a mask filtering both particles and gases. Symptoms, body temperature, nasal lavage fluid, exhaled nitric oxide, and bronchial responsiveness to methacholine were assessed before and after the exposure. Pre- and postexposure urine and blood samples were collected. RESULTS: After the exposure, the participants with respirators reported fewer symptoms than those without. Wearing a mask also reduced the inflammatory response assessed with nasal lavage (cell concentration, interleukins 6 and 8) and peripheral blood (cell number). Lung function was significantly impaired only in the unprotected group; postexposure vital capacity and forced expiratory volume in 1 second showed a decrease of 3-4% from the preexposure levels (P=0.006 and P=0.002, respectively). Bronchial responsiveness (P<0.01) and body temperature (P<0.001) increased similarly in the three groups. Bronchial responsiveness to methacholine increased 2.7, 2.4, and 2.1 doubling concentration steps for those unprotected, those using a particle-filter mask, and those using a mask with particle and gas filters, respectively. The prostaglandin D2 metabolite, 9a, 11b-PGF2 increased significantly (P=0.003) only in those unprotected. CONCLUSIONS: Wearing a respirator in a pig confinement building reduces the inflammatory reaction but does not influence the increase in bronchial responsiveness, with no difference between the use of a particle-filter mask or a mask with a particle-gas filter combination. PMID- 16680386 TI - Hemoglobin adducts in the assessment of potential occupational exposure to acrylamides -- three case studies. AB - OBJECTIVES: Workers in three types of occupations with potential exposure to acrylamide were examined for the purpose of excluding or confirming exposure, evaluating actions for reducing exposure, or investigating the possible cause of ill health. METHODS: Workers were examined through the measurement of adducts from acrylamide (and N-methylolacrylamide) to the N-terminal valines in hemoglobin. RESULTS: The first case concerned workers transporting acrylamide contaminated waste soil. The measured acrylamide-adduct levels were in the range of the normal background levels (ie, any potential occupational exposure was too low to be detected). The second case included workers handling a sealing product containing acrylamide and N-methylolacrylamide. One worker had an acrylamide adduct level of 0.3 nmol/g globin, close to the level at which acrylamide-exposed persons have shown mild reversible symptoms of the peripheral nervous system. After actions to reduce exposure, the adduct levels were still elevated, and, as a precautionary measure, the sealing product was replaced. The third case concerned a man with observed neurotoxic symptoms, working with a sealing product containing acrylamide. This worker had an extremely high acrylamide-adduct level (23 nmol/g globin) (ie, the acrylamide exposure was probably the cause of his ill health). The acrylamide product was replaced, and after 5 months the adduct level had decreased to 2.4 nmol/g globin, and after another 6 months it was about 0.4 nmol/g globin. CONCLUSIONS: These studies illustrate the usefulness of in vivo dose measurement through the use of hemoglobin adducts for occupational surveillance and as a basis for the health risk assessment of electrophilically reactive compounds. PMID- 16680387 TI - Preventing injury, illness and disability at work. AB - This discussion paper is intended to start a dialogue about prevention among those who are interested in making workplaces safer and healthier. The following four key themes are presented to build a framework for further discussion and activity: theme 1 -- with multiple causes for workplace injury, illness, and disability, preventing these problems requires multiple solutions, operating in synergy; theme 2 -- efforts to reduce workplace injury, illness, and disability must build on both primary and secondary prevention approaches, merging these to create a more effective strategy; theme 3 -- before which strategies work and which do not can be agreed upon, a shared understanding is needed of how the effectiveness of occupational health interventions should be evaluated; and theme 4 -- building relationships between those who do research and those who use research knowledge is important if relevant research is to be produced that is readily taken up and applied to improve occupational health and safety. PMID- 16680388 TI - Treatment with anti-TNF-alpha antibody infliximab reduces serum IL-15 levels in patients with rheumatoid arthritis. AB - The aim of this study was to analyze the change of serum cytokines and pentosidine levels in patients with rheumatoid arthritis (RA) by infliximab treatment. Twenty-three patients with RA were studied for 30 weeks on the effects of infliximab treatment. Serum levels of IL-15, IL-16, IL-17, and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured with ELISA methods and pentosidine levels were determined using high-performance liquid chromatography, both at baseline and at 14 and 30 weeks after the initial treatment with infliximab. In addition, the patients also underwent physical and routine blood examinations. The higher levels of serum IL-15 in RA patients before treatment with infliximab significantly decreased at 14 and 30 weeks after the initial treatment with infliximab, but serum IL-16, IL-17, GM-CSF, and pentosidine levels did not decrease. The serum IL-17 and GM-CSF levels remained to be a limited detectable level at the pre- and posttreatment with infliximab. Infliximab treatment significantly lowered the serum levels of IL-15 in patients with RA. IL-15 is one of the crucial cytokines affected by infliximab. PMID- 16680389 TI - Ethnicity may be a reason for lipid changes and high Lp(a) levels in rheumatoid arthritis. AB - There are so many studies that suggest the changes in lipid profiles and lipoprotein (a) [Lp(a)] are associated with early atherosclerosis in rheumatoid arthritis (RA). But there are some opposite studies also. Because of marked ethnicity differences in the distribution of Lp(a), we aimed to investigate the associations of Lp(a) levels and lipid changes in Turkish RA patients. There were 30 women and 20 men, a total of 50 patients with RA (mean age 47.6 +/- 13.2 years), included and 21 healthy women and 14 healthy men (mean age 45.7 +/- 14.5 years) were recruited as a control (C) group. Serum Lp(a), total cholesterol (TC), triglyceride (TG), HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C) levels were analysed for each group. Analysis of six different studies was performed. In the RA and C groups, mean serum Lp(a) levels were 39.7 +/- 64.4 and 10.5 +/- 13.4 mg/dl, respectively (P=0.001). Mean TC levels were 189.2 +/- 142.5 and 174.0 +/- 29.3 mg/dl (P=0.294), mean TG levels were 121.4 +/- 65.4 and 106.5 +/- 80.0 mg/dl (P=0.030), mean HDL-C levels were 44.5 +/- 10.0 and 47.7 +/- 4.8 mg/dl (P=0.014) and mean LDL-C levels were 94.3 +/- 35.3 and 102.0 +/- 24.6 mg/dl (P=0.98), respectively. Analysis of the six studies showed Lp(a) level was higher and HDL level was lower in RA patients than in healthy controls. Patients with RA may have altered lipid profiles from one country to another one. Especially in Turkey, higher serum Lp(a), lower HDL-C and higher TG levels may be found in RA patients instead of some findings of other countries showing different results. Ethnicity may be a reason for these findings. PMID- 16680390 TI - Rheumatologic aspects of lysosomal storage diseases. AB - Lysosomal storage diseases are rare metabolic disorders, some of which can now be treated using enzyme replacement therapies. Because the time point of treatment initiation significantly influences the outcome in Gaucher disease, Fabry disease, and mucopolysaccharidosis type I, early diagnosis is of utmost importance. All three disorders can present with musculoskeletal symptoms in early stages, therefore, the rheumatologist may be the first to be contacted by these patients. Here, we present three characteristic lysosomal storage disease cases to increase awareness in the rheumatological community of the typical symptom constellations associated with these rare but treatable disorders. PMID- 16680391 TI - Scalp necrosis: a rare complication of temporal arteritis. PMID- 16680392 TI - A hidradenitis suppurativa related SAPHO case associated with features resembling spondylarthropathy and proteinuria. AB - We present a 53-year-old man with synovitis-acne-palmoplantar pustulosis hyperosteosis-osteitis (SAPHO) syndrome who is HLA-B27 positive with a history of uveitis and complicated by proteinuria and osteoporosis. Interesting, yet unreported features of SAPHO syndrome and the etiology of proteinuria are further discussed. PMID- 16680393 TI - A supraomohyoidal plexus block designed to avoid complications. AB - Interscalene blocks of the brachial plexus are used for surgery of the shoulder and are frequently associated with complications such as temporary phrenic block, Horner syndrome or hematoma. To minimize the risk of these complications, we developed an approach that avoids medially directed needle advancement and favors spread to lateral regions only: the supraomohyoidal block. We tested this procedure in 11 cadavers fixed by Thiel's method. The insertion site is at the lateral margin of the sternocleidomastoid muscle at the level of the cricoid cartilage. The needle is inserted in the axis of the plexus with an angle of approximately 35 degrees to the skin, and advanced in lateral and caudal direction. Distribution of solution was determined in ten cadavers after bilateral injection of colored solution (20 and 30 ml) and followed by dissection. In an eleventh cadaver, computerized tomography and 3D reconstruction after radio contrast injection was performed. In additional five cadavers we performed Winnie's technique with bilateral injection (20 and 30 ml). Concerning the supraomohyoidal block the injection mass reached the infraclavicular region surrounded all trunks of the brachial plexus in the supraclavicular region and the suprascapular nerve in all cases. The solution did not spread medially beyond the lateral margin of the anterior scalene muscle into the scalenovertebral triangle. Therefore, phrenic nerve, stellate ganglion, laryngeal nerve nor the vertebral artery were exposed to the injected solution. Distribution was comparable with the use of 20 and 30 ml of solution. Injections on five cadavers performing the interscalene block of Winnie resulted in an extended spread medially to the anterior scalene muscle. We conclude that our method may be a preferred approach due to its safety, because no structures out of interest were reached. Solution of 20 ml is suggested to be enough for a successful block. PMID- 16680399 TI - Characteristics of taurine release in slices from adult and developing mouse brain stem. AB - Taurine has been thought to function as a regulator of neuronal activity, neuromodulator and osmoregulator. Moreover, it is essential for the development and survival of neural cells and protects them under cell-damaging conditions. Taurine is also involved in many vital functions regulated by the brain stem, including cardiovascular control and arterial blood pressure. The release of taurine has been studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release have not been systematically characterized in the brain stem. The properties of release of preloaded [(3)H]taurine were now characterized in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice, using a superfusion system. In general, taurine release was found to be similar to that in other brain areas, consisting of both Ca(2+)-dependent and Ca(2+)-independent components. Moreover, the release was mediated by Na(+)-, Cl(-)-dependent transporters operating outwards, as both Na(+)-free and Cl(-) -free conditions greatly enhanced it. Cl(-) channel antagonists and a Cl(-) transport inhibitor reduced the release at both ages, indicating that a part of the release occurs through ion channels. Protein kinases appeared not to be involved in taurine release in the brain stem, since substances affecting the activity of protein kinase C or tyrosine kinase had no significant effects. The release was modulated by cAMP second messenger systems and phospholipases at both ages. Furthermore, the metabotropic glutamate receptor agonists likewise suppressed the K(+)-stimulated release at both ages. In the immature brain stem, the ionotropic glutamate receptor agonists N-methyl-D aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) potentiated taurine release in a receptor-mediated manner. This could constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions. PMID- 16680400 TI - Production of hypotaurine, taurine and sulfate in rats and mice injected with L cysteinesulfinate. AB - We studied in vivo production of taurine, hypotaurine and sulfate following subcutaneous administration of L-cysteinesulfinate (CSA) to rats and mice. When 5.0 mmol/kg of body weight of CSA was injected to rats, increased urinary excretions of taurine, hypotaurine and sulfate in 24 h urine were 617, 52 and 1,767 micromol/kg, respectively. From these results together with our previous data, sulfate production was calculated to be 1.6 times greater than taurine production. Increased contents (micromol/g of wet tissue) over the control of taurine and hypotaurine in mouse tissues at 60 min after the injection of 5.0 mmol/kg body weight of CSA were: liver, 3.5 and 9.9; kidney, 0.3 and 5.2; heart, 3.7 and 0.2; blood plasma, 0.4 and 0.2, respectively. Upon loading of hypotaurine or taurine, tissue contents of these amino acids in liver and kidney increased greatly. Our results indicate that liver is the most active tissue for taurine production, followed by kidney, and that external CSA, hypotaurine and taurine are easily taken up by these tissues. PMID- 16680401 TI - Non-irradiation-derived reactive oxygen species (ROS) and cancer: therapeutic implications. AB - Owing to their chemical reactivity, radicals have cytocidal properties. Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalysed reactions. Although these developments are currently still in their infancy, they nevertheless deserve consideration. There are now numerous examples known of conventional anti-cancer drugs that may at least in part exert cytotoxicity by induction of radical formation. Some drugs, such as arsenic trioxide and 2-methoxy-estradiol, were shown to induce programmed cell death due to radical formation. Enzyme-catalysed radical formation has the advantage that cytotoxic products are produced continuously over an extended period of time in the vicinity of tumour cells. Up to now the enzymatic formation of toxic metabolites has nearly exclusively been investigated using bovine serum amine oxidase (BSAO), and spermine as substrate. The metabolites of this reaction, hydrogen peroxide and aldehydes are cytotoxic. The combination of BSAO and spermine is not only able to prevent tumour cell growth, but prevents also tumour growth, particularly well if the enzyme has been conjugated with a biocompatible gel. Since the tumour cells release substrates of BSAO, the administration of spermine is not required. Combination with cytotoxic drugs, and elevation of temperature improves the cytocidal effect of spermine metabolites. The fact that multidrug resistant cells are more sensitive to spermine metabolites than their wild type counterparts makes this new approach especially attractive, since the development of multidrug resistance is one of the major problems of conventional cancer therapy. PMID- 16680406 TI - 1H, 15N and 13C assignments of the cysteine protease inhibitor chagasin from Trypanosoma cruzi. PMID- 16680408 TI - Depression amongst Nigerian university students. Prevalence and sociodemographic correlates. AB - BACKGROUND: This study aimed to estimate the prevalence and examine the socio demographic correlates of depressive disorder among university students in Western Nigeria. METHODS: A representative sample of students living in the halls of residence of a federal university (n = 1,206) completed sets of questionnaires on socio-demographic details, problems encountered in the university, alcohol use and smoking. Depressive disorder was assessed using the Mini International Neuropsychiatric Interview (MINI). RESULTS: A total of 101 (8.3%) students met the criteria for depressive disorder with 68 (5.6%) having minor depressive disorder and 33 (2.7%) having major depressive disorder. The factors that were significantly associated with depressive disorders in the students include problems with accommodation (OR 2.72, 95% CI 1.79-4.16), very large family size (OR 2.8, 95% CI 1.42-5.73), female gender (OR 2.21, 95% CI 1.46-3.35), heavy cigarette smoking (OR 3.67, 95% CI 2.23-6.05) and high level of alcohol consumption (OR 9.44, 95% CI 3.32-26.89). CONCLUSIONS: Depression is common among Nigerian university students and significantly associated with sociodemographic factors. An effective model for the prediction of the development of depression in university students need to be developed and evaluated and interventions aimed at reducing the incidence of depression among this population need further research. PMID- 16680409 TI - Long-acting medications for the hyperkinetic disorders. A systematic review and European treatment guideline. AB - A systematic review of published and unpublished data on the use of long-acting medications in ADHD and hyperkinetic disorder is reported, giving effect sizes and numbers-to-treat for extended-release stimulant preparations and atomoxetine (ATX). A panel of experts from several European countries used the review to make recommendations about the use of these drugs in practice, and conclusions are reported: (1) Long-acting preparations should be available and used; (2) They should not replace short-acting drugs (which will be the initial treatment for many children for reasons of cost and flexibility of dosing). Individual clinical choice is needed. (3) Both ATX and extended-release preparations of stimulants should be available. The choice will depend upon the circumstances, and detailed recommendations are made. PMID- 16680410 TI - Violent deliberate self-harm amongst adolescent refugees. AB - Investigation of frequency and characteristics of violent deliberate self-harm (DSH) amongst adolescents. Study was retrospective, over 3-year period, based on records of attendees at child and adolescent mental health clinics in North West London. Nine adolescents showed violent self-harm out of 982 DSH cases (frequency 0.92%). The nine cases included five female asylum seekers, who experienced war related events, were significantly more likely to have affective disorders and post-traumatic stress disorder (PTSD), compared with the four males (none asylum seekers), who were significantly more likely to have conduct and substance misuse disorders. PMID- 16680412 TI - Ab initio and DFT study on the electrophilic addition reaction of bromine to tetracyclo[5.3.0.0(2,6).0 (3,10)]deca-4,8-diene. AB - The electronic and geometric structures of tetracyclo[5.3.0.0(2,6).0(3,10)]deca 4,8-diene (hypostrophene) have been investigated by ab initio and DFT/B3LYP methods using the 6-31G* and 6-311G* basis sets. The double bonds of hypostrophene are endo-pyramidalized. The cationic intermediates and products formed in the addition reaction have been investigated using the HF/6-311G*, HF/6 311G**, and B3LYP/6-311G* methods. The bridged bromonium cation was more stable than the U-type cation. Considering that the bridged cation does not isomerize to the less stable U-type cation, it is not possible for the U-type product to be obtained in the reaction. The bridged bromonium cation transformed into the more stable N-type cation and the N-type product was obtained via this cation. The thermodynamic stability of the exo, exo and exo, endo isomers of the N-type dibromide molecule were almost identical. The N-type product was 16.6 kcal mol( 1) more stable than the U-type product. PMID- 16680414 TI - [Ultrasonography of the thyroid and parathyroid gland]. AB - Ultrasonography is the most important imaging tool in the diagnosis of thyroid disease. The results of real-time B-imaging of the thyroid gland along with physical signs and basal TSH can aid in the diagnosis of thyroid dysfunction, of for instance, a small, hypoechogenic gland in Hashimoto's and radiation thyroiditis, or an enlarged, hypoechogenic and pulsating gland in Graves' disease. Although recent improvements in technology have increased sensitivity of colored duplexsonography, certain sonographic differentiation of benign and malignant lesions as well as of active (hormone secreting) and inactive nodules is not yet possible. Diagnostic interpretation of ultrasonographic findings is feasible only when the history of the patient, physical examination and the laboratory evaluation are taken into account. Ultrasound detects thyroid nodules, is useful for following nodule size, in guiding fine needle biopsies and in the aspiration of cysts. Highly experienced investigators in ultrasound can assist preoperatively in the localization of parathyroid adenomas in primary and tertiary hyperthyroidism (when followed by (99m)Tc szintigraphy plus SPECT). PMID- 16680416 TI - CT endoscopy for the follow-up of Cronkhite-Canada syndrome. PMID- 16680422 TI - Problems in the metabolic evaluation of renal stone disease: audit of intra individual variation in urine metabolites. AB - Preliminary metabolic assessment of patients with renal stones includes measurement of urine metabolites. This paper reports on the degree of intra individual variation in some key urine metabolites. Over 80 medically untreated patients under initial metabolic investigation were audited from whom 24-h urine results were available as three separate urine pairs collected at intervals not less than 1 month apart. Ranking patients by intra-individual variation, above the 75th centile, the highest calcium was at least 216% of the lowest calcium, the respective figures for phosphate, urate, oxalate, citrate, creatinine and sodium were 207, 190, 271, 412, 175 and 233%. In order to estimate pre-treatment excretion within 30% of a true mean at the 95% confidence limit, for calcium and oxalate, the number of 24-h samples required were 3 and 4 respectively with 6 and 9 required to be within 20%. These observations illustrate significant practical clinical problems in assessing patients with renal stones when assessing these basic parameters. Regimens based on small numbers of urine collections are flawed, hence evidence based protocols should be devised. A minimum of three pairs of 24-h urine samples based upon predicting metabolite output within 20-30% or less of the true mean is recommended. PMID- 16680423 TI - Time to task failure varies with the gain of the feedback signal for women, but not for men. AB - Varying the gain of the feedback signal during a target-matching task alters the synaptic input onto the motor neuron pool. The purpose was to determine the influence of the gain of the feedback signal on the time to failure for men and women when maintaining arm position while supporting a submaximal inertial load with the elbow flexor muscles. While seated with the upper arm vertical, 15 women and 14 men maintained a constant elbow angle (1.57 rad) and supported a load equal to 15% of maximal voluntary contraction (MVC) force until failure. The task was performed on separate days with either a low gain or a high gain for the joint-angle signal. The percent decline in MVC force after the fatiguing contraction was similar for the low- and high-gain conditions (P = 0.24), and did not differ for men and women (P = 0.11). The discharge of motor units in biceps brachii declined at a greater rate during the high-gain condition for men and women, but only the women experienced a briefer time to failure for the high-gain session (8.7 +/- 2.3 min) compared with the low-gain session (11.9 +/- 4.8; P = 0.003). The men had similar times to failure for the low- (6.0 +/- 2.2 min) and high-gain conditions (5.9 +/- 2.1 min; P = 0.35). Linear and stepwise, multiple regression analyses revealed that the time to failure for the men was associated with the absolute target force, the standard deviation (SD) for the resultant wrist acceleration, and the brachialis aEMG (P T, 190C>T, 559C>T/560G>A, 640T>G and 752A>T). In addition, a "composite" NAT1 genotype was defined as a function of the genotyped SNPs. Descriptive analyses of the SNPs and of the composite genotype indicated that heterozygous parents were more likely to transmit the common allele than the rare allele to their affected offspring. Furthermore, matings of mothers homozygous for the common allele and heterozygous fathers were more common than the reciprocal matings. Log-linear analyses confirmed that both the maternal (P = 0.008) and offspring (P = 0.003) composite NAT1 genotypes were significantly related to the risk of spina bifida. NAT1 variants that reduce or abolish enzyme activity appear to protect against spina bifida, and to exert their influence via both the maternal and the offspring genotypes. These associations may be attributable to a decrease in either folate catabolism or the conversion of exogenous agents to teratogenic derivatives in women and/or developing embryos with a NAT1 genotype that includes a loss of function allele relative to those who do not. PMID- 16680437 TI - Comparison of accuracy of lag screw placement in cephalocondylic nails and sliding hip screw plate fixation for extracapsular fractures of the neck of femur. AB - This study compared the accuracy of lag screw placement between extracapsular femoral fractures fixed with sliding hip screw plate systems and those fixed with cephalocondylic nails. It involved 75 retrospective radiographs of fractures fixed with either a cephalocondylic nail (32) or a sliding hip screw plate system (43). Postoperative anteroposterior and lateral radiographs of the hip were scanned using a digital X-ray scanner and measured using computer software. Measurements were conducted by two independent observers, and the radiographs were calibrated to correct for magnification. Accuracy of lag screw placement was determined by "tip apex distance," described by Baumgaertner et al., and by the ratio method described by Parker. The mean tip apex distance was 24.0 mm in sliding hip screw plate systems and 21.1 mm in cephalocondylic nails. This was found to be statistically significant. Lag screw placement through cephalocondylic nails is more accurate and therefore has less chance of cut-out compared with sliding hip screw plate systems. There was no statistically significant difference using Parker's ratio method because this method quantifies the direction of the screw rather than the depth of penetration. PMID- 16680434 TI - The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light signaling, but can also modulate phytochrome A responses. AB - Phytochrome photoreceptors enable plants to perceive divergent light signals leading to adaptive changes in response to differing environmental conditions. However, the mechanism of light signal transduction is not fully understood. Here we report the identification of a new signaling intermediate from Arabidopsis thaliana, Scarecrow-like (SCL)13, which serves as a positive regulator of continuous red light signals downstream of phytochrome B (phyB). SCL13 antisense lines exhibit reduced sensitivity towards red light, but only a distinct subset of phyB-mediated responses is affected, indicating that SCL13 executes its major role in hypocotyl elongation during de-etiolation. Genetic evidence suggests that SCL13 is also needed to modulate phytochrome A (phyA) signal transduction in a phyB-independent way. The SCL13 protein is localized in the cytoplasm, but can also be detected in the nucleus. Overexpression of both a nuclear and cytoplasmic localized SCL13 protein leads to a hypersensitive phenotype under red light indicating that SCL13 is biologically active in both compartments. SCL13 is a member of the plant-specific GRAS protein family, which is involved in various different developmental and signaling pathways. A previously identified phytochrome A signaling intermediate, PAT1, belongs to the same subbranch of GRAS proteins as SCL13. Although both proteins are involved in phytochrome signaling, each is specific for a different light condition and regulates a different subset of responses. PMID- 16680438 TI - Total knee arthroplasty without patellar resurfacing. AB - One hundred and eleven total knee replacements without patellar resurfacing were followed-up for a minimum of 48.8 months (range 48.8-108.2 months; average 78.2 months). Evaluation was performed using the Knee Society Clinical Rating System (KS-CRS). Preoperatively, the mean knee score was 34.3 points and the mean function score was 42.2 points. Postoperatively, this knee score improved to a mean of 91.1 points and the function score improved to mean of 89.6 points at the most recent follow-up. There were no significant differences among the knees with mild, moderate, or severe degenerative change to the patella with regard to the preoperative (p=0.83) and postoperative (p=0.39) knee pain score. It seems likely that the postoperative knee pain is not related to the severity of degenerative change to the patella in total knee arthroplasties performed without patellar resurfacing, and none of those patients required patellar resurfacing to achieve knee pain relief. PMID- 16680441 TI - [Infection-inflammation-sepsis: state of the art and future perspectives. Tubingen Intensive Symposium, 08.10.2005]. PMID- 16680442 TI - [PROWESS, ENHANCE and ADDRESS: clinical implications for the treatment with drotrecogin alfa (activated)]. AB - Drotrecogin alfa (activated) (DrotAA) represents a therapeutic advance in the treatment of severe sepsis. In the pivotal PROWESS trial DrotAA had demonstrated a significant decrease in 28-day mortality, most evident in the subgroup of patients at higher risk of death. Thus, DrotAA was licensed throughout Europe for treatment of adult patients with severe sepsis with multiple organ failure when added to best standard care. The ADDRESS trial was mandated by the FDA to investigate prospectively the treatment effect of DrotAA in patients at low risk of death, e.g. single organ failure. The trial was prematurely stopped due to futility, because no reduction in mortality was observed in this non-indicated patient population. The ENHANCE open-label trial enrolled similar patients to the PROWESS trial and the observed 28-day mortality was consistent with the results seen in the PROWESS trial. Survival rates for patients receiving DrotAA early within 24 h from the first sepsis-induced organ dysfunction were significantly higher than in patients treated later. In this overview we will discuss the results of the ENHANCE and ADDRESS trials in the context of the PROWESS study and clinical implications for the treatment with DrotAA. PMID- 16680443 TI - Intra-observer and inter-observer agreement of the manual examination of the lumbar spine in chronic low-back pain. AB - Examination is a cornerstone in the manual procedures leading to mobilisation/manipulation of the low back. The observer variation of the more specific segmental tests remains to be investigated. Two skilled specialists in manual medicine examined the segmental changes in the lumbar spine. The patients were unknown to the examiners and no information of the case history was given. All test results were recorded by an observer present in the room who ensured that no conversation was allowed during the examination. The primary outcome measures were the kappa values for each test. The matching was defined as acceptable (acc) within two neighbouring levels and perfect (per) on the same level. Intra-observer variation (tested in 33 patients and 10 subjects without low-back pain): The agreement between first and second segmental diagnosis examination was 70% (per) and 82% (per + acc). Kappa values were: segmental diagnosis 0.60 (per) and 0.70 (per + acc), multifidus test 0.51 (per) and 0.60 (per + acc), sideflexion 0.57 (per) and 0.69 (per + acc), and ventral flexion 0.31 (per) and 0.45 (per + acc). Inter-observer variation (tested in 60 patients): The agreement for segmental diagnosis between the examiner A and B was 42% (per) and 75% (per + acc). Kappa values were: segmental diagnosis 0.21 (per) and 0.57 (acc), multifidus test 0.12 (per) and 0.48 (acc), sideflexion 0.22 (per) and 0.45 (acc), and ventralflexion 0.22 (per) and 0.44 (acc). By manual tests, skilled examiners seem to be able to diagnose segmental dysfunctions in the low back. The clinical implication of these dysfunctions remains to be clarified. PMID- 16680445 TI - Letter to the Editor regarding "Scheuermann's kyphosis: surgical management" (V. Arlet, D. Schlenzka). PMID- 16680446 TI - Comment on: efficacy and safety of balloon kyphoplasty in the treatment of vertebral compression fractures: a systematic review (C. Bouza et al.). PMID- 16680447 TI - Validity of pressure pain thresholds in female workers with and without recurrent low back pain. AB - Recurrent low back pain (LBP) is a common pain condition in elderly workers in a variety of occupations, but little is known about its origin and the mechanisms leading to an often disabling sensation of pain that may be persistent or intermittent. In the present study we evaluated the pressure pain thresholds (PPTs) in subjects suffering from recurrent LBP, as well as in healthy controls, to investigate if recurrent LBP is associated with an increased sensitivity of the muscular and ligamentous structures located on the lower back. One hundred and six female workers, aged between 45 and 62 years and working either in administrative or nursing professions were examined. The subjects were classified into LBP cases and controls based on the Nordic questionnaire. Subjects indicating 8-30 or more days with LBP during the past 12 months were graded as cases. PPTs were measured on 12 points (six on each side of the body) expected to be relevant for LBP (paravertebral muscles, musculus quadratus lumborum, os ilium, iliolumbar ligament, musculus piriformis and greater trochanter), as well as on a reference point (middle of the forehead) using a digital dolorimeter. The PPTs on all points on the lower back highly correlated with each other and a high internal consistency was found with a Cronbach alpha coefficient > 0.95. There was a moderate and significant correlation of the PPT on the forehead with the PPT on the lower back with correlation coefficients ranging from 0.36 to 0.49. In LBP cases from administrative professions, the PPT on the forehead was significantly decreased (P < 0.05). The PPT on the lower back did not significantly differ between the four groups studied, namely nurses and administrative workers with and without recurrent LBP. These results give evidence that recurrent LBP is not associated with an altered sensitivity of the muscular and myofascial tissues in the lumbar region. Furthermore, they raise questions about the value of reference point measurements in recurrent LBP. PMID- 16680448 TI - Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure. AB - Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and are at their minimum during lying or relaxed sitting and at maximum during lifting weights with a round back. We hypothesize that these different loading magnitudes influence the intervertebral disc (IVD) by alteration of disc matrix turnover depending on their magnitudes. Therefore the aim of this study was to assess changes in gene expression of human nucleus cells after the application of low hydrostatic pressure (0.25 MPa) and high hydrostatic pressure (2.5 MPa). IVD cells isolated from the nucleus of human (n = 18) and bovine (n = 24 from four animals) disc biopsies were seeded into three-dimensional collagen type-I matrices and exposed to the different loading magnitudes by specially developed pressure chambers. The lower pressure range (0.25 MPa, 30 min, 0.1 Hz) was applied with a recently published device by using an external compression cylinder. For the application of higher loads (2.5 MPa, 30 min, 0.1 Hz) the cell-loaded collagen gels were sealed into sterile bags with culture medium and stimulated in a newly developed water-filled compression cylinder by using a loading frame. These methods allowed the comparison of loading regimes in a wide physiological range under an equal three-dimensional culture conditions. Cells were harvested 24 h after the end of stimulation and changes in the expression of genes known to influence IVD matrix turnover (collagen-I, collagen-II, aggrecan, MMP1, MMP2, MMP3, MMP13) were analyzed by real-time RT-PCR. A Wilcoxon signed-rank test(1) and a Wilcoxon 2 sample test(2) were performed to detect differences between the stimulated and control samples(1) and differences between low and high hydrostatic pressure(2). Multiple testing was considered by adjusting the p value appropriately. Both regimes of hydrostatic pressure influenced gene expression in nucleus cells with opposite tendencies for the matrix forming proteins aggrecan and collagen type-I in response to the two different pressure magnitudes: Low hydrostatic-pressure (0.25 MPa) tended to increase collagen-I and aggrecan expression of human nucleus cells (P < 0.05) but only to a small degree. High hydrostatic pressure (2.5 MPa) tended to decrease gene expression of all anabolic proteins with significant effects on aggrecan expression of nucleus cells (P = 0.004). Low hydrostatic pressure had no influence on the expression of matrix metalloproteinases (MMP1, MMP2, MMP3 and MMP13). In contrast, high hydrostatic pressure tended to increase the expression of MMP1, MMP3 and MMP13 of human nucleus cells with high individual-individual variations. The decreased expression of aggrecan (P = 0.008) and collagen type II (P = 0.023) and the increased MMP3 expression (P = 0.008) in response to high hydrostatic pressure could be confirmed in additional experiments with bovine nucleus cells. These results suggest that hydrostatic pressure as one of the physiological stimuli of the IVD may influence matrix turnover in a magnitude dependent way. Low hydrostatic pressure (0.25 MPa) has quite small influences with a tendency to anabolic effects, whereas high hydrostatic pressure (2.5 MPa) tends to decrease the matrix protein expression with a tendency to increase some matrix-turnover enzymes. Therefore, hydrostatic pressure may regulate disc matrix turnover in a dose-dependent way. PMID- 16680452 TI - Decomposition of reactive oxygen species by copper(II) bis(1-pyrazolyl)methane complexes. AB - Two bis(1-pyrazolyl)alkane ligands, bis(3,5-dimethyl-1-pyrazolyl)methane and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methane, and their copper(II) complexes, bis(3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL1(NO3)2] and bis(4 iodo-3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL2(NO3)2] x 2H2O, were prepared. Physiochemical properties of the copper(II) complexes were studied by spectroscopic (UV-vis, IR, EPR) techniques and cyclic voltammetry. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:copper(II) ion and a bidentate coordination mode for the nitrate ions in both of the complexes. According to experimental and theoretical ab initio data, the copper(II) ion is located in an octahedral hexacoordinated environment. Both complexes were able to catalyze the dismutation of superoxide anion (O2*-) (pH 7.5) and decomposition of H2O2 (pH 7.5) and peroxynitrite (pH 10.9). In addition, both complexes exhibited superoxide dismutase (SOD) like activity toward extracellular and intracellular reactive oxygen species produced by activated human neutrophils in whole blood. Thus, these complexes represent useful SOD mimetics with a broad range of antioxidant activity toward a variety of reactive oxidants. PMID- 16680451 TI - Superoxide dismutase 1 modulates expression of transferrin receptor. AB - Copper-zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system. PMID- 16680453 TI - Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies. AB - Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E0 =455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the kcat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15-0.05% for the non phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity. PMID- 16680454 TI - Binding of tetrakis(pyrazoliumyl)porphyrin and its copper(II) and zinc(II) complexes to poly(dG-dC)2 and poly(dA-dT)2. AB - Interactions of cationic porphyrins bearing five-membered rings at the meso position, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP; M is H2, Cu(II) or Zn(II)), with synthetic polynucleotides poly(dG-dC)2 and poly(dA-dT)2 have been characterized by viscometric, visible absorption, circular dichroisim and magnetic circular dichroism spectroscopic and melting temperature measurements. Both H2PzP and CuPzP are intercalated into poly(dG-dC)2 and are outside-bound to the major groove of poly(dA-dT)2, while ZnPzP is outside-bound to the minor groove of poly(dA-dT)2 and surprisingly is intercalated into poly(dG dC)2. The binding constants of the porphyrin and poly(dG-dC)2 and poly(dA-dT)2 are on the order of 10(6) M(-1) and are comparable to those of other cationic porphyrins so far reported. The process of the binding of the porphyrin to poly(dG-dC)2 and poly(dA-dT)2 is exothermic and enthalpically driven for H2PzP, whereas it is endothermic and entropically driven for CuPzP and ZnPzP. These results have revealed that the kind of the central metal ion of metalloporphyrins influences the characteristics of the binding of the porphyrins to DNA. PMID- 16680456 TI - Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger. AB - Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(-)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane. PMID- 16680457 TI - Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. AB - Escherichia coli FBR5 containing recombinant genes for ethanol production on plasmids that are also required for anaerobic growth was cultivated continuously on 50 g/l xylose or glucose in the absence of antibiotics and without the use of special measures to limit the entry of oxygen into the fermenter. Under chemostat conditions, stable ethanol yields of ca. 80-85% of the theoretical were obtained on both sugars over 26 days at dilution rates of 0.045/h (xylose) and 0.075/h (glucose), with average plasmid retention rates of 96% (xylose) and 97% (glucose). In a continuous fluidized bed fermenter, with the cells immobilized on porous glass beads, the extent of plasmid retention by the free cells fell rapidly, while that of the immobilized cells remained constant. This was shown to be due to diffusion of oxygen through the tubing used to recirculate the medium and free cells. A change to oxygen-impermeable tubing led to a stable high rate of plasmid retention (more than 96% of both the free and immobilized cells) with ethanol yields of ca. 80% on a 50 g/l xylose feed. The maximum permissible level of oxygen availability consistent with high plasmid retention by the strain appears to be of the order of 0.1 mmol per hour per gram dry biomass, based on measurements of the rate of oxygen penetration into the fermenters. Revertant colonies lacking the ethanologenic plasmid were easily detectable by their morphology which correlated well with their lack of ampicillin resistance upon transfer plating. PMID- 16680458 TI - Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation. AB - The use of inert absorbent polymeric supports for cellular attachment in solid state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL Cyan416 produced acremonidins A-E when grown on agar bearing moist polyester cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A-E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester-cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative metabolite yields, as determined by HPLC analysis and measurement of antimicrobial activity. The application of such immobilized-cell fermentation methods under solid and liquid conditions facilitated the discovery of new antibiotic compounds, and offers new approaches to fungal fermentation for natural product discovery. PMID- 16680460 TI - Epigenetic regulation of immune escape genes in cancer. AB - According to the concept of immune surveillance, the appearance of a tumor indicates that it has earlier evaded host defenses and subsequently must have escaped immunity to evolve into a full-blown cancer. Tumor escape mechanisms have focused mainly on mutations of immune and apoptotic pathway genes. However, data obtained over the past few years suggest that epigenetic silencing in cancer may be as frequent a cause of gene inactivation as are mutations. Here, we discuss the evidence that tumor immune evasion is mediated by non-mutational epigenetic events involving chromatin and that epigenetics collaborates with mutations in determining tumor progression. Since epigenetic changes are potentially reversible, the relative contribution of mutations and epigenetics, to the gene defects in any given tumor, may be a factor in determining the efficacy of treatments. We review new developments in basic chromatin mechanisms and in this context describe the rationale for the current use of epigenetic agents in cancer therapy and for a novel epigenetically generated tumor vaccine model. We emphasize that epigenetic cancer treatments are currently a 'blunt-sword' and suggest future directions for designing chromatin-based programs of potential value in the diagnosis and treatment of cancer. PMID- 16680459 TI - Global transcriptome response of recombinant Escherichia coli to heat-shock and dual heat-shock recombinant protein induction. AB - Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid tRNA genes were elevated for the dual cultures compared to the induced cultures. Since, tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. PMID- 16680461 TI - Monocyte fructose 1,6-bisphosphatase and cytidine deaminase enzyme activities: potential pharmacodynamic measures of calcitriol effects in cancer patients. AB - PURPOSE: To determine, in peripheral blood monocytes (PBM), whether the enzymatic activities of fructose 1,6-bisphosphatase (FBPase), cytidine deaminase (CDDase) and 24-hydroxylase (CYP24), enzymes regulated by calcitriol are useful pharmacodynamic (PD) measures of calcitriol effects in cancer patients. METHODS: Cancer patients enrolled in a phase I clinical trial of calcitriol and carboplatin were studied. Baseline and calcitriol-induced changes in FBPase, CDDase and CYP24 activities were measured in PBM collected before, 6, 24, and 48 h after administration of calcitriol, prior to carboplatin, in doses ranging from 4 to 11 mug daily for 3 consecutive days (QDx3). Normal FBPase, CYP24 and CDDase activities were measured in PBM from untreated healthy volunteers. RESULTS: Baseline activities in PBM from cancer patients and healthy volunteers were (median and range): 1.0 (0.0-43.5) and 4.4 (3.1- 8.2) nmol/min/mg protein for FBPase (P = 0.002); 2.5 (0.9-9.3) and 0.8 (0.4-2.0) fmol/h/10(6) cells for CYP24 (P = 0.016), and 5.6 (2.5-22.3) and 6.6 (1.1-47.4) nmol/min/mg protein for CDDase (P > 0.05), respectively. All calcitriol doses achieved peak serum calcitriol levels > x3 the physiological levels, increased cancer patient PBM FBPase activity to normal levels and decreased CDDase activity to undetectable levels within 48 h, with no significant change in CYP24 activity. These enzyme activity changes were not associated with hypercalcemia. CONCLUSIONS: Calcitriol treatment induced increase in FBPase and decrease in CDDase activities in cancer patient PBM are potential early and sensitive non-hypercalcemia PD measures of calcitriol effects. PMID- 16680462 TI - A pharmacokinetic and safety study of a novel polymeric paclitaxel formulation for oral application. AB - PURPOSE: To investigate the pharmacokinetics, safety, and tolerability of a new oral formulation of paclitaxel containing the polymer polyvinyl acetate phthalate in patients with advanced solid tumors. PATIENTS AND METHODS: A total of six patients received oral paclitaxel as single agent given as a single dose of 100 mg on day 1, oral paclitaxel 100 mg in combination with cyclosporin A (CsA) 10 mg/kg both given as a single dose on day 8, and i.v. paclitaxel (Taxol) 100 mg as a 3-h infusion on day 15. RESULTS: The AUC (mean +/- standard deviation) values of paclitaxel after oral administration without CsA and with CsA were 476 +/- 254 and 967 +/- 779 ng/ml h, respectively. T (max) was 4.0 +/- 0.9 h after oral paclitaxel without CsA, and 6.0 +/- 3.1 h after oral paclitaxel with CsA. The mean AUC after oral administration as single agent was 13% of the AUC after i.v. administration of paclitaxel, and increased to 26% after co-administration with CsA. No haematological toxicities were observed, and only mild (CTC-grade 1 and 2) non-hematological toxicities occurred after oral intake of paclitaxel with or without CsA. CONCLUSION: The AUC of the new polymeric paclitaxel formulation increased a factor 2 in combination with CsA, which confirms that CsA co administration can also improve exposure to paclitaxel after oral administration of a polymeric formulation. Because of the delayed release of paclitaxel from this formulation, we hypothesize that a split-dose regimen of CsA where it is administered before and after paclitaxel administration will further increase the systemic exposure to paclitaxel up to therapeutic levels. The formulation was well tolerated at the dose of 100 mg without induction of severe toxicities. PMID- 16680464 TI - Prediction of HELLP syndrome with assessment of maternal dual hepatic blood supply by using Doppler ultrasound. AB - OBJECTIVE: Early structural and functional changes in the systemic vasculature have been proposed to play a major pathogenetic role in HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome. Our objective was to assess whether the evaluation of maternal hepatic blood supply is instructive to the prediction of onset of HELLP syndrome. DESIGN: Prospective observation study. POPULATION: Fifty-eight women with severe preeclampsia and 60 healthy pregnant controls at 25-36 weeks gestation. METHODS: Angle-corrected time-averaged flow velocity and the cross-sectional area of common hepatic artery and portal vein were measured by using Doppler ultrasonography in 58 women with severe preeclampsia and in 60 healthy pregnant controls at 25-36 weeks gestation. Intravascular flow volumes were calculated from the product of the time-averaged velocity and the cross-sectional area. The total liver blood flow was taken as the sum of flow volumes in the hepatic artery and portal vein. RESULTS: The total liver blood flow decreased significantly to about 40% of control in 9 women with severe preeclampsia who developed HELLP syndrome within 4 days after the examination, but not in 49 women with severe preeclampsia without HELLP syndrome. CONCLUSION: The results indicated that the decrease in dual hepatic blood supply preceded the onset of HELLP syndrome. PMID- 16680463 TI - Irinotecan in combination with thalidomide in patients with advanced solid tumors: a clinical study with pharmacodynamic and pharmacokinetic evaluation. AB - PURPOSE: Recent clinical studies have demonstrated a reduction of irinotecan (CPT 11) gastrointestinal toxicities when the CPT-11 is administered in combination with thalidomide in patients with diagnosis of colorectal cancer. The main purpose of this study was to investigate possible interactions between CPT-11 pharmacokinetics and thalidomide to explain the previously described gastrointestinal toxicity reduction. METHODS: In our clinical trial, advanced cancer patients were treated with CPT-11 on a dose of 350 mg/m2 at day 1 every 3 weeks. Only at the first cycle, CPT-11 was administered in association with thalidomide on a dose of 400 mg/day given from day 1 to day 14. From the second cycle, the treatment was continued with irinotecan alone at the same dose. Pharmacokinetics analysis of irinotecan and its metabolites, SN-38 and SN-38 glucuronide, were performed at the first and second cycle. RESULTS: A total of 19 patients entered the study. The pharmacokinetic analysis were performed on 16 patients. Pharmacokinetic data suggested a decreased metabolism of irinotecan into SN-38 and SN-38-glucuronide when it was administered with thalidomide. Indeed, area under the time-concentration curve (AUC) of SN-38 was significantly lower at the first cycle than the second cycle (0.99+/-0.45 hxmicrog/ml vs 1.34+/ 0.65, respectively, P=0.027) whereas AUC of irinotecan and SN-38-glucuronide were higher at first cycle than second cycle (34.53+/-11.38 hxmicrog/ml vs. 28.42+/ 12.23 hxmicrog/ml, P=0.064 and 2.39+/-1.21 h(microg/ml vs. 1.86+/-1.11 hxmicrog/ml, P=0.018, respectively). CONCLUSIONS: Our study demonstrates a significant decreased metabolism of CPT-11 into the active metabolite SN-38 when CPT-11 is administered in association with thalidomide. These observations strongly suggest an interaction of thalidomide with CPT-11 metabolism and, at least in part, it might explain the previously described improvement in tolerability. PMID- 16680465 TI - Correlation between radiographic findings of osteoarthritis and arthroscopic findings of articular cartilage degeneration within the patellofemoral joint. AB - OBJECTIVES: To correlate radiographic findings of osteoarthritis on axial knee radiographs with arthroscopic findings of articular cartilage degeneration within the patellofemoral joint in patients with chronic knee pain. SUBJECTS AND METHODS: The study group consisted of 104 patients with osteoarthritis of the patellofemoral joint and 30 patients of similar age with no osteoarthritis of the patellofemoral joint. All patients in the study group had an axial radiograph of the knee performed prior to arthroscopic knee surgery. At the time of arthroscopy, each articular surface of the patellofemoral joint was graded using the Noyes classification system. Two radiologists retrospectively reviewed the knee radiographs to determine the presence of marginal osteophytes, joint-space narrowing, subchondral sclerosis, and subchondral cysts. The sensitivity and specificity of the various radiographic features of osteoarthritis for the detection of articular cartilage degeneration within the patellofemoral joint were determined. RESULTS: The sensitivity of marginal osteophytes, joint-space narrowing, subchondral sclerosis, and subchondral cysts for the detection of articular cartilage degeneration within the patellofemoral joint was 73%, 37%, 4%, and 0% respectively. The specificity of marginal osteophytes, joint-space narrowing, subchondral sclerosis, and subchondral cysts for the detection of articular cartilage degeneration within the patellofemoral joint was 67%, 90%, 100%, and 100% respectively. CONCLUSION: Marginal osteophytes were the most sensitive radiographic feature for the detection of articular cartilage degeneration within the patellofemoral joint. Joint-space narrowing, subchondral sclerosis, and subchondral cysts were insensitive radiographic features of osteoarthritis, and rarely occurred in the absence of associated osteophyte formation. PMID- 16680466 TI - Evaluation of hearing loss after spinal anesthesia with otoacoustic emissions. AB - The aim of this study was to determine the degree and reversibility of hearing loss (HL) following spinal anesthesia with an objective audiometric test: otoacoustic emissions (OAE). Eleven patients (22 ears) who were undergoing surgery under spinal anesthesia were included in this study. Transient-evoked otoacoustic emissions (TEOAE) and distortion product otoacoustic emissions (DPOAE) were evaluated 1 day before the operation and postoperative day 1, 2, and 15. DPOAE were recorded as DPgram and input/output functions (I/O). The emission amplitudes of the TEOAE and DPOAE of right and left ears were found to be affected immediately after the surgery and progressive improvement detected with full recovery within postoperative 15 days. These changes were mainly at around 1,500-3,000 Hz. None of the patients had permanent OAE amplitude deterioration. Transient HL may occur more often than it is generally assumed, and the symptoms might not be recognized. OAE is an effective and objective way of evaluating the HL in this particular group of patients. We suggest informing patients about this transient HL for medicolegal issues. PMID- 16680467 TI - Occupational noise exposure and sensorineural hearing loss among workers of a steel rolling mill. AB - Hearing loss is one of the most common chronic health conditions and has important implications for the patient's quality of life. However, hearing loss is substantially underestimated and under treated. The purpose of this study was to determine the prevalence of sensorineural hearing loss among the workers in a steel rolling mill in Nigeria. Each of the 150 randomly selected subjects had a structured questionnaire administered to them, followed by a full otological examination. Of these, 116 had tympanometry and pure-tone audiometry. Also a noise mapping of their respective work units was done. The workers were exposed to noise levels varying from 49 to 93 dBA. About 28.2% of the 103 who had their audiogram analysed had mild to moderate sensorineural hearing loss in their better ear and 56.8% of them had mild to moderate sensorineural hearing loss in their worse ear. The pure-tone average and the average hearing thresholds at 4 kHz for the groups significantly increased with an increasing noise exposure level. The prevalence of sensorineural hearing loss among the study population is high; and noise exposure is at least contributory. Pre-employment and regular audiometry while on the job is highly recommended. PMID- 16680468 TI - Increased protein carbonyl groups in the serum of patients affected by thalassemia major. AB - High oxidative stress status is known to be one of the most important factors determining cell injury in thalassemic patients and causing other serious medical complications, including a continuous proinflammatory status. The quantification of protein carbonyl groups in peripheral blood is widely used to measure the extent of oxidative modification. Thus, we measured serum concentrations of protein carbonyl groups in 30 patients affected by thalassemia major and in 15 healthy subjects. Strongly higher levels of protein carbonyl groups were measured in the blood from thalassemic patients than in that from healthy controls. Our findings evidence that thalassemic patients suffer from protein oxidative stress; the possibility of a role for carbonyl stress in the progression and severity of the disease needs further investigation. PMID- 16680470 TI - A resource-based model of microbial quiescence. AB - To analyze the ecological features of microbial quiescence, a model is proposed that involves "wake-up" rate and "sleep" rate at which the population transitions from a quiescent to an active state and back, respectively. These rates depend continuously on the resources and turn on and off at resource thresholds which may not coincide. The usual dichotomy is observed: the population is washed out under environmental stress and a single "survival" steady state exists otherwise. Proportional nutrient enrichment is used to explore analytically as well as numerically the nature of the steady state which bifurcates from the washout state. PMID- 16680471 TI - A diffusion approach to approximating preservation probabilities for gene duplicates. AB - Consider a haploid population and, within its genome, a gene whose presence is vital for the survival of any individual. Each copy of this gene is subject to mutations which destroy its function. Suppose one member of the population somehow acquires a duplicate copy of the gene, where the duplicate is fully linked to the original gene's locus. Preservation is said to occur if eventually the entire population consists of individuals descended from this one which initially carried the duplicate. The system is modelled by a finite state-space Markov process which in turn is approximated by a diffusion process, whence an explicit expression for the probability of preservation is derived. The event of preservation can be compared to the fixation of a selectively neutral gene variant initially present in a single individual, the probability of which is the reciprocal of the population size. For very weak mutation, this and the probability of preservation are equal, while as mutation becomes stronger, the preservation probability tends to double this reciprocal. This is in excellent agreement with simulation studies. PMID- 16680472 TI - A comparative study of five fatal cases of Taxus poisoning. AB - The study presents five fatal cases of poisoning with Taxus spp., all of which were suicides of young people aged between 16 and 26 years. Yew leaves were consumed in four fatalities, whereas a mash from Taxus was ingested in one case. No relevant concentrations of alcohol, narcotic drugs, and pharmaceuticals were determined in postmortem toxicological screening. At forensic autopsy, a widely dilated pupil was found in two decedents. Furthermore, autopsy showed unspecific findings of intoxication in all cases: acute blood congestion of lungs, liver, kidney, and brain as well as dilated cardiac ventricles. No signs of violence could be found in any of the fatalities. Yew leaves were identified in four cases in the stomach and duodenum. 3,5-Dimethoxyphenol, the aglycon of the Taxus ingredient taxicatine, was determined as toxicological evidence for the absorption of yew ingredients. Taxus intoxication could be confirmed by 3,5 dimethoxyphenol concentrations in cardiac blood between 31 and 528 ng/ml for all cases. 3,5-Dimethoxyphenol was also detected in stomach contents as well as in urine, liver, kidneys, and brain samples. Based on the different concentrations of 3,5-dimethoxyphenol determined in the cardiac blood samples, it was concluded that the form of ingestion plays a decisive role in the process of poisoning. Finally, a toxic range for Taxus poisoning based on 3,5-dimethoxyphenol as marker substance is proposed as orientation. PMID- 16680473 TI - The iliopsoas abscess: aetiology, therapy, and outcome. AB - BACKGROUND AND AIMS: The treatment strategy for patients with a retroperitonally localised abscess is controversial as it remains open which fluid collections should be drained by open access or by percutaneously inserted drainage. PATIENTS: Therefore, the data of 40 consecutively treated patients with an iliopsoas abscess were analysed retrospectively. RESULTS: Ten patients suffered from a primary abscess and ten from a post-operative abscess; further, in 20 patients, the aetiology of the abscesses were due to Crohn's disease, neoplasia, spondylitis or other relevant concomitant diseases. Eight of 40 patients were initially treated by image-guided percutaneous drainage (PD), the other by open access drainage. Six patients died (15%), all of them had been operated; 15 (37.5%) patients had a recurrence of their abscess and needed re-operation. Factors predicting a poor outcome were age, APACHE II score, bi-lateral abscesses and a post-operative or bony cause, but the bacteriological findings did not influence the outcome. CONCLUSIONS: We suggest an algorithm for treatment of iliopsoas abscesses depending on number and volume of the abscesses. PMID- 16680474 TI - Prospective randomised comparison of organ-preserving pancreatic head resection with pylorus-preserving pancreaticoduodenectomy. AB - BACKGROUND AND AIMS: In a prospective, randomised, control trial organ-preserving pancreatic head resection (OPPHR) was compared with pylorus-preserving pancreaticoduodenectomy (PPPD) to assess the advantages and disadvantages of each type of operation. PATIENTS AND METHODS: Forty patients were allocated randomly to either the OPPHR (n=20) or the PPPD (n=20) group. The surgical data, postoperative complications, induction of diabetes mellitus, postoperative pain and quality of life 1 year, postoperatively, were considered. RESULTS: The two study groups of 20 patients were well balanced with regard to sex, age, chronic pancreatitis history and indication for surgery. The duration of the operation for OPPHR and PPPD was 142.5+/-4.9 and 278+/-6.9 min, respectively (P<0.05). The postoperative mortality in each group was zero. After OPPHR and PPPD, the morbidity was 0 and 40%, respectively (P<0.05). The duration of hospital stay was also significantly different: 8.05+/-0.9 vs 13.8+/-3.9 days (P<0.05). After 1 year the pain relief was effective in both groups, but three patients acquired diabetes mellitus after PPPD; the body weight had increased by 7.8+/-0.9 and 3.2+/-0.3 kg after OPPHR and PPPD, respectively (P<0.05). CONCLUSION: The two procedures are equally safe and effective with regard to pain relief, but OPPHR is superior to PPPD not only in the operation data and morbidity, but also in the quality of life 1 year postoperatively. OPPHR should be regarded as a recommended procedure in the treatment of chronic pancreatitis. PMID- 16680475 TI - Hydrodissection with adrenaline-lidocaine-saline solution in laparoscopic cholecystectomy. AB - AIM: This investigation examined the effects of a solution injected to the gallbladder bed on operative time, bleeding, incidence of gallbladder perforation, and postoperative pain. METHODS: One hundred sixty-four consecutive patients with cholelithiasis were randomized into two clinically comparable groups. In group 1 (84 patients), 40 ml of saline-adrenaline-lidocaine solution was injected between the gallbladder and liver. In group 2 (80 patients), laparoscopic cholecystectomy was performed without hydrodissection. The time taken to dissect the gallbladder from the liver, bleeding from the liver bed, incidence of gallbladder perforation and spillage of bile and stones, duration of operation, amount of gas used for the laparoscopic cholecystectomy, conversion to open cholecystectomy, postoperative pain and pain localization were recorded. RESULTS: The mean dissection time, amount of gas used, incidence of gallbladder perforation, spillage of stones, and liver bed bleeding were not significantly different between the groups. There also was no significant difference between the groups regarding postoperative pain and pain localization. CONCLUSION: Hydrodissection did not reduce time to dissect the gallbladder from the liver or risk of gallbladder perforation. Similarly, adrenaline and lidocaine injection between the gallbladder and the liver did not effect bleeding from the dissection area and did not alter postoperative pain or pain localization. PMID- 16680477 TI - Management of cervical paragangliomas: review of a 15-year experience. AB - BACKGROUND AND AIMS: Cervical paragangliomas are highly vascular neoplasms and should be considered in the evaluation of all lateral neck masses. The aim of this study is to review an institutional experience in the management of these tumors. MATERIALS AND METHODS: Thirteen patients with 14 paragangliomas were treated in our institution during a period of 15 years. There were eight women (61.5%) and five men (38.5%) with a mean age of 41.3+/-15 years. A painless lateral neck mass was the main finding in 69.2% of patients. There was no evidence of a functional tumor. Carotid angiography was performed in all patients to define the vascular anatomy of the lesion. The 78.6% of paragangliomas underwent selective embolization of the major feeding arteries. Surgical resection followed within the next 48 h. RESULTS: The majority of the lesions were paragangliomas of the carotid bifurcation (85.7%), while one patient was diagnosed with a jugular and one with a vagal paraganglioma. In one patient, bilateral paragangliomas in the carotid bifurcation were detected. There was no evidence of malignancy in any case. Preoperative embolization has proven successful in reducing tumor vascularity. Vascular reconstruction was necessary in one patient. The main postoperative complication was transient cranial nerve deficit in seven (53.8%) patients, and a permanent Horner's syndrome was documented in one patient. No stroke occurred. The jugular paraganglioma was treated with irradiation due to skull base extension with significant symptomatic relief. CONCLUSION: Combined therapeutic approach with preoperative selective embolization followed by surgical resection by an experienced team offers a safe and effective method for complete excision of the tumors with a reduced morbidity rate. PMID- 16680479 TI - Assessment of risk-independent follow-up to detect asymptomatic recurrence after curative resection of colorectal cancer. AB - BACKGROUND AND AIMS: Colorectal cancer is one of the leading causes of cancer death. We analyzed the value of standardized, risk-independent postoperative surveillance. MATERIALS AND METHODS: Between 1995 and 2001, 564 patients with colorectal cancer underwent standardized oncologic resection. One hundred thirty four were unable to take part in the surveillance program, while 430 patients were grouped as follows: group I (n=272, risk-independent follow-up), group II (n=113, follow-up at other departments), and group III (n=45, no follow-up). RESULTS: The 5-year cancer-specific survival rate for UICC III and IV was significantly higher in group I (87%) as compared to group II (35%). In group I, the 5-year disease-free survival rate was 70%. Cancer recurrence occurred at mean 17 (+/-12) months after colorectal resection and yielded a 5-year survival rate of 63%. Reresection was performed in 17 (35%) patients, of whom ten remained disease-free (5-year survival rate, 91%). The money spent for one patient's 5 year follow-up was 1665. CONCLUSIONS: A standardized, risk-independent follow-up program allows early diagnosis of asymptomatic recurrence of colorectal cancer. Reresection improves the 5-year survival rate in this setting. PMID- 16680481 TI - The endoproteolytic maturation of progastrin and procholecystokinin. AB - The homologous brain-gut propeptides, procholecystokinin (proCCK) and progastrin, both undergo extensive posttranslational maturation in specific neuroendocrine cells. The process comprises multiple endoproteolytic cleavages at mono- and dibasic sites, in addition to exoproteolytic trimmings and amino acid derivatizations. Knockout of prohormone convertases (PCs) in mice and studies in cell lines indicate that PC1, PC2 and, to a minor extent, PC5, are responsible for most of the endoproteolytic cleavages of both prohormones. Progastrin in antral G-cells is cleaved by PC1 at two di-Arg sites, R36R37 and R73R74, whereas, PC2 only cleaves at the single di-Lys site, K53K54. Pituitary corticotrophs and intestinal TG-cells, both of which express gastrin, do not cleave K53K54 due to lack of PC2. In proCCK five monobasic (R25, R44, R50, K61 and R75) as well as a single dibasic site (R85R86) can all be cleaved by both PC1 and PC2. But the cleavage differs in a cell-specific manner in that PC1 is responsible for the entire endoproteolytic cleavage in intestinal endocrine I-cells, except for perhaps the K61 site. In contrast PC2 is responsible for most endoproteolysis of proCCK in the cerebral CCK-neurons, which do not express PC1 in significant amounts. Moreover, PC5 appears to contribute to a minor extent to the neuronal proCCK and to the antral progastrin processing. This review emphasizes that prohormone convertases play a decisive but substrate and cell-specific role in the biosynthetic maturation of gastrin and CCK. PMID- 16680483 TI - From cardiac cation channels to the molecular dissection of the transient receptor potential channel TRPM4. AB - In 2006, we celebrate not only the milestone paper on the patch-clamp technique but also the publication of the first single-channel measurements in cardiac cells revealing a Ca(2+)-activated, nonselective cation channel. Considerable effort has been undertaken since this time to identify molecular candidates for this class of cation channels that can be found in a variety of tissues. Recent work has shown that this channel is very likely TRPM4, a member of the TRPM ion channel family. The current review links the epochal Colquhoun et al. paper to the detailed molecular knowledge and structure function aspects of this TRP channel. It will be shown that TRPM4 is a Ca(2+)- and voltage-activated channel, which is dramatically modulated by the phospholipid phosphatidyl inositol bisphosphate (PIP(2)) and belongs to the heat-activated thermoTRPs. A functional hallmark of TRPM4, as for several TRP channels, is a dramatic shift of its voltage dependence towards negative, physiologically meaningful potentials. PMID- 16680484 TI - Signaling pathways involved with the stimulatory effect of angiotensin II on vacuolar H+-ATPase in proximal tubule cells. AB - It has been documented that angiotensin II (ANG II) (10(-9) M) stimulates proton extrusion via H(+)-adenosine triphosphatase (ATPase) in proximal tubule cells. In the present study, we investigated the signaling pathways involved in the effects of ANG II on H(+)-ATPase activity and on the cytosolic free calcium concentration in immortalized rat proximal tubule cells, a permanent cell line derived from rat proximal tubules. The effects of ANG on pH(i) and [Ca(+2)](i) were assessed by the fluorescent probes, 2',7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy methyl ester and fluo-4-acetoxy-methyl ester, in the absence of Na(+) to block the Na(+)/H(+) exchanger. In the control situation, the pH recovery rate following intracellular acidification with NH(4)Cl was 0.073+/-0.011 pH units/min (n=12). This recovery was significantly increased with ANG II (10(-9 )M), to 0.12+/-0.015 pH units/min, n=10. This last effect was also followed by a significant increase of Ca(+2) (i), from 99.72+/-1.704 nM (n=21) to 401.23+/ 33.91 nM (n=39). The stimulatory effect of ANG II was blocked in the presence of losartan, an angiotensin II subtype 1 (AT(1)) receptor antagonist. H89 [protein kinase A (PKA) inhibitor] plus ANG II had no effect on the pH recovery. Staurosporine [protein kinase C (PKC) inhibitor] impaired the effect of ANG II. Phorbol myristate acetate (PKC activator) mimicked in part the stimulatory effect of ANG II, but reduced Ca(+2) (i). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N' tetraacetic acid (intracellular calcium chelator) alone reduced the pH(i) recovery rate below control levels and impaired the effect of ANG II, in a way similar to that of trimethoxy benzoate (a blocker of Ca(+2) (i) mobilization). We conclude that ANG II regulates rat proximal tubule vacuolar H(+)-ATPase by a PKA independent mechanism and that PKC and intracellular calcium play a critical role in this regulation. PMID- 16680485 TI - Expression and regulation of alphaB-crystallin in the kidney in vivo and in vitro. AB - alphaB-crystallin, a major component of the mammalian eye lens, is a small heat shock protein and molecular chaperone that is also abundant in the mammalian kidney. The present study aimed to characterize more closely the intrarenal expression and regulation of alphaB-crystallin in vivo and in vitro. In normal rat kidney, the expression of alphaB-crystallin mRNA and protein were both close to the detection limit in cortex, but increased steeply from the outer to the inner medulla where alphaB-crystallin constitutes approximately 2% of total tissue protein. Immunohistochemistry disclosed papillary collecting duct cells and thin limbs as the major sites for intrapapillary alphaB-crystallin immunoreactivity. In rats subjected to sucrose diuresis for 3 days, alphaB crystallin mRNA expression was reduced by 27 and 46% in outer and inner medulla, respectively. In agreement with the results obtained in vivo, in Madine-Darby canine kidney cells, alphaB-crystallin mRNA and protein were induced significantly by elevating the medium osmolality to 500 mosm/kg H(2)O by the addition of NaCl and raffinose, and also by urea. The NaCl-induced increase in alphaB-crystallin expression was concentration-dependently blunted by SP600125, a specific JNK inhibitor. Overexpression of alphaB-crystallin in 293 cells resulted in increased tolerance to acute osmotic stress. These results indicate that alphaB-crystallin may be regulated by papillary interstitial tonicity in a JNK dependent process. Moreover, the high abundance of alphaB-crystallin in the renal medulla may be important for cell survival in an environment characterized by extreme interstitial solute concentrations as present during antidiuresis. PMID- 16680486 TI - Exposure to traffic pollutants and effects on 17-beta-estradiol (E2) in female workers. AB - OBJECTIVE: The aim of the study is to evaluate whether the occupational exposure to urban pollutants including endocrine disruptors (EDs) could cause alterations in plasma 17-beta-estradiol (E2) levels and related diseases (adverse pregnancy outcome and mental health disorders) in female traffic police compared to a control group. METHODS: After excluding the subjects with the principal confounding factors, traffic police and controls were matched by age, years of police work, age of menarche, menstrual cycle day, body mass index (BMI), drinking habit, cigarette smoking habit, habitual intake of soy or liquorice in diet and habitual consumption of Italian coffee. Thirty-seven traffic police and 31 controls (seventh day; follicular phase of the ovarian cycle); 38 traffic police and 42 controls (14th day; ovulatory phase of the ovarian cycle); and 25 traffic police and 28 controls (21st day; lutheal phase of the ovarian cycle) were included in the study and then matched for the above-mentioned variables. RESULTS: In follicular and in lutheal phases, mean E2 levels were significantly lower in traffic police compared to controls. The distribution of E2 values in traffic police and controls was significant in follicular, ovulatory and lutheal phases. In ovulatory phase, mean E2 levels were lower but not significant in traffic police compared to controls. An increase was found concerning mental health disorders referred to in the questionnaire items in traffic police compared to controls, although the difference was not significant. CONCLUSIONS: Considering that the potential confounding effect of extraneous factors was controlled for by restricting the study population and by matching traffic police and controls on the above-mentioned variables, our results suggest that occupational exposure to urban pollutants, EDs included, might alter E2 plasma concentrations. E2 could be used in occupational set as an early biomarker of exposure to urban pollutants, valuable for the group, even before the onset of the related pathologies (adverse pregnancy outcome and mental health disorders). PMID- 16680487 TI - Stress monitoring of ambulance personnel during work and leisure time. AB - OBJECTIVES: The aim of the present study was to assess physiological and subjective stress markers during a 24-h ambulance work shift and during the next two work-free days, and relate these parameters to self-reported health complaints. METHODS: Twenty-six ambulance personnel were followed during a 24-h work shift and during the next two work-free days with electrocardiogram, cortisol assessments and diary notes. The ambulance personnel also performed tests of autonomic reactivity before and at the end of the work shift. The subjects were categorized into two groups according to their number of health complaints. RESULTS: In general, stress markers did not show differences between the work shift and leisure time. However, a modest deviation in heart rate variability pattern and higher morning cortisol values during work in comparison with work-free days were observed in personnel with many health complaints. CONCLUSIONS: Subjective and physiological characteristics of ambulance personnel did not indicate distinctive stress during the 24-h work shift. Relationships between frequent health complaints and specific work-related factors require further prospective studies. PMID- 16680490 TI - Early foetal programming of hepatic gluconeogenesis: Glucocorticoids strike back. PMID- 16680491 TI - Peptide YY ablation in mice leads to the development of hyperinsulinaemia and obesity. AB - AIMS/HYPOTHESIS: Obese people exhibit reduced circulating peptide YY (PYY) levels, but it is unclear whether this is a consequence or cause of obesity. We therefore investigated the effect of Pyy ablation on energy homeostasis. METHODS: Body composition, i.p. glucose tolerance, food intake and hypothalamic neuropeptide expression were determined in Pyy knock-out and wild-type mice on a normal or high-fat diet. RESULTS: Pyy knock-out significantly increased bodyweight and increased fat mass by 50% in aged females on a normal diet. Male chow-fed Pyy (-/-) mice were resistant to obesity but became significantly fatter and glucose-intolerant compared with wild-types when fed a high-fat diet. Pyy knock-out animals exhibited significantly elevated fasting or glucose-stimulated serum insulin concentrations vs wild-types, with no increase in basal or fasting induced food intake. Pyy knock-out decreased or had no effect on neuropeptide Y expression in the arcuate nucleus of the hypothalamus, and significantly increased proopiomelanocortin expression in this region. Male but not female knock-outs exhibited significantly increased growth hormone-releasing hormone expression in the ventromedial hypothalamus and significantly elevated serum IGF I and testosterone levels. This sex difference in activation of the hypothalamo pituitary somatotrophic axis by Pyy ablation may contribute to the resistance of chow-fed male knock-outs to late-onset obesity. CONCLUSIONS/INTERPRETATION: PYY signalling is important in the regulation of energy balance and glucose homeostasis, possibly via regulation of insulin release. Therefore reduced PYY levels may predispose to the development of obesity, particularly with ageing or under conditions of high-fat feeding. PMID- 16680498 TI - Lifetime sport and leisure activity participation is associated with greater bone size, quality and strength in older men. AB - INTRODUCTION: It remains uncertain whether long-term participation in regular weight-bearing exercise confers an advantage to bone structure and strength in old age. The aim of this study was to investigate the relationship between lifetime sport and leisure activity participation on bone material and structural properties at the axial and appendicular skeleton in older men (>50 years). METHODS: We used dual-energy X-ray absorptiometry (DXA) to assess hip, spine and ultradistal (UD) radius areal bone mineral density (aBMD) (n=161), quantitative ultrasound (QUS) to measure heel bone quality (n=161), and quantitative computed tomography (QCT) to assess volumetric BMD, bone geometry and strength at the spine (L(1)-L(3)) and mid-femur (n=111). Current (>50+ years) and past hours of sport and leisure activity participation during adolescence (13-18 years) and adulthood (19-50 years) were assessed by questionnaire. This information was used to calculate the total time (min) spent participating in sport and leisure activities and an osteogenic index (OI) score for each participant, which provides a measure of participation in weight-bearing activities. RESULTS: Regression analysis revealed that a greater lifetime (13-50+ years) and mid adulthood (19-50 years) OI, but not total time (min), was associated with a greater mid-femur total and cortical area, cortical bone mineral content (BMC), and the polar moment of inertia (I (p)) and heel VOS (p ranging from <0.05 to <0.01). These results were independent of age, height (or femoral length) and weight (or muscle cross-sectional area). Adolescent OI scores were not found to be significant predictors of bone structure or strength. Furthermore, no significant relationships were detected with areal or volumetric BMD at any site. Subjects were then categorized into either a high (H) or low/non-impact (L) group during adolescence (13-18 years) and adulthood (19-50+ years) according to their OI scores during each of these periods. Three groups were subsequently formed to reflect weight-bearing impact categories during adolescence and then adulthood: LL, HL and HH. Compared to the LL group, mid-femur total and cortical area, cortical BMC and I (p) were 6.5-14.2% higher in the HH group. No differences were detected between the LL and HL groups. CONCLUSIONS: In conclusion, these findings indicate that long-term regular participation in sport and leisure activities categorized according to an osteogenic index [but not the total time (min) spent participating in all sport and leisure activities] was an important determinant of bone size, quality and strength, but not BMD, at loaded sites in older men. Furthermore, continued participation in weight-bearing exercise in early to mid adulthood appears to be important for reducing the risk of low bone strength in old age. PMID- 16680502 TI - Clinical and physiologic outcomes after transvaginal rectocele repair. AB - PURPOSE: This study was designed to evaluate the clinical and physiologic outcomes after transvaginal rectocele repair. METHODS: Between June 2000 and January 2003, 30 females (mean age, 62 (range, 45-78) years) with a symptomatic large rectocele (>3 cm) underwent transvaginal rectocele repair (anterior levatorplasty). Six months after surgery, a physiologic evaluation was performed by using defecography (depth of rectocele) and anorectal manometry (maximum resting pressure, maximum squeeze pressure, rectal threshold, and maximum tolerable volume). Using a questionnaire, a clinical evaluation was performed one year after surgery to analyze symptoms, including difficult evacuation, digital support, sexual discomfort, as well as patient satisfaction. Follow-up of all patients was conducted during a median duration of 38 (range, 23-54) months. RESULTS: There were no operative complications, such as hematoma, wound infection, or rectovaginal fistula. Difficult evacuation improved in 27 of 30 patients (90 percent) and completely disappeared in 9 patients. Postoperatively, digital support was no longer necessary during evacuation in 15 of 21 patients (71 percent). Overall patient satisfaction reached 25 of 30 (83 percent). Although mild sexual discomfort was observed in nine patients, it disappeared gradually and only one patient complained of persistent symptoms. No patient reported symptomatic recurrences at the end of the follow-up. The radiologic mean depth of the rectocele was significantly reduced: preoperative, 3.9 cm; postoperative, 0.5 cm. None of the physiologic parameters significantly changed after surgery. CONCLUSIONS: Transvaginal rectocele repair can provide excellent long-term symptomatic relief and a high rate of patient satisfaction without any alteration in anorectal physiologic function. PMID- 16680499 TI - Epidemiology of hip fracture in Iran: results from the Iranian Multicenter Study on Accidental Injuries. AB - INTRODUCTION: The incidence of hip fracture varies substantially between countries. As a result of improving life expectancy, the number of elderly people susceptible to hip fractures is increasing rapidly in the developing world. Little is known, however, about the epidemiology of hip fractures in the Middle Eastern countries. In this study, our objective was to estimate the incidence of hip fracture in Iran and compare it with other populations. METHODS: The data used were obtained from the Iranian Multicenter Study on Accidental Injuries, a large-scale population-based study conducted in nine provinces across the country. All of the hospitals in these provinces, which provide services to about 9.5 million people, were prospectively surveyed for any incident injury resulting from accidental events occurring in the study period of 135 days (4.5 months). All patients aged >or=50 with radiographically confirmed proximal femur fractures were included in this study. A total of 555 new cases of hip fracture (284 male, 271 female) were recorded during the study period. The annual incidence of hip fracture per 100,000 person-years was 115.2 (95% CI: 107.2-123.7) in men and 115.6 (95% CI: 107.4-124.3) in women; of these,73.2 and 89.2%, respectively, were fall-related fractures. The female-to-male ratios for fall-induced and total hip fracture rates were 1.2 and 1.0, respectively. RESULTS: The incidence rates increased exponentially after the age of 60 years in both genders and nearly tripled with each successive decade. When these results are compared to those of other studies, the Iranian age-standardized incidence rates of 127.3 (men) and 164.6 (women) per 100,000 person-years are considerably lower than those of all Western countries when standardized to data on the U.S. population in 2000. When compared with incidence rates reported for other Asian countries, those of Iranian females are the lowest next to China. CONCLUSION: The low incidence rate of hip fracture for older Iranian women may be the result of several potential factors related to genetic or lifestyle differences between Iranians and people of other countries. Further studies are required to investigate contributing factors in more detail. PMID- 16680506 TI - Lymphotrophic nanoparticle enhanced MR imaging (LNMRI) for lymph node imaging. AB - Nodal staging is an integral part of the pretreatment staging of any patient with malignancy and has therapeutic and prognostic implications. Currently used imaging techniques used for nodal evaluation are limited in accuracy because they rely on size criteria for the detection of metastases. This has led to the emergence of lymphotropic nanoparticle enhanced magnetic resonance imaging as a promising tool for nodal characterization. This article reviews the properties of lymphotropic iron oxide nanoparticles and the technique, image interpretation, and initial clinical experience with lymphotropic nanoparticle enhanced magnetic resonance imaging. PMID- 16680507 TI - MRCP of congenital pancreaticobiliary malformation. AB - BACKGROUND: Congenital pancreaticobiliary malformations are sometimes associated with acute or chronic pancreatitis and biliary carcinoma. Currently, magnetic resonance cholangiopancreatography (MRCP) is one of the first choices for investigating and diagnosing pancreaticobiliary diseases noninvasively. We compared the accuracy of conventional MRCP and endoscopic retrograde cholangiopancreatography (ERCP) in making the diagnosis of congenital pancreaticobiliary malformations. METHODS: In patients with pancreas divisum (n = 17), pancreaticobiliary maljunction (n = 12), choledochocele (n = 2), and annular pancreas (n = 1) who underwent ERCP and MRCP, the diagnostic accuracy and findings on MRCP were compared with those on ERCP. RESULTS: Of the 32 patients with congenital pancreaticobiliary malformations diagnosed on ERCP, 23 (72%) presented the same diagnosis on MRCP. Complete pancreas divisum was diagnosed in 73% on MRCP based on the finding of a dominant dorsal pancreatic duct crossing the lower bile duct and emptying into the duodenum without communicating with the ventral pancreatic duct. Pancreaticobiliary maljunction was diagnosed in 75% on MRCP based on the finding of an anomalous union between the common bile duct and the pancreatic duct and the existence of a long common channel. CONCLUSIONS: Conventional MRCP is a useful, noninvasive tool for diagnosing congenital pancreaticobiliary malformations; and the diagnostic accuracy can be increased with three-dimensional MRCP or dynamic MRCP with secretin stimulation. PMID- 16680508 TI - Cholecysto-urachal fistula. AB - The urachus is an embryonic remnant that connects the primitive bladder to the allantois. Multiple urachal anomalies have been described, including the urachal sinus, urachal cyst, patent urachus, and urachal diverticulum. We describe an unusual case in which a patient with urinary tract infection and dysuria was ultimately found to have urinary gallstones from a urachal connection with the gallbladder. PMID- 16680509 TI - Unusual presentation of acute pancreatitis: an irreducible inguinoscrotal swelling mimicking a strangulated hernia. AB - We report an unusual presentation of acute pancreatitis as a tender, irreducible, inguinoscrotal swelling mimicking a strangulated hernia. Lack of abdominal symptoms or signs can lead to misdiagnosis and unnecessary surgery. PMID- 16680511 TI - Three-dimensional microorganization of the soil-root-microbe system. AB - Soils contain the greatest reservoir of biodiversity on Earth, and the functionality of the soil ecosystem sustains the rest of the terrestrial biosphere. This functionality results from complex interactions between biological and physical processes that are strongly modulated by the soil physical structure. Using a novel combination of biochemical and biophysical indicators and synchrotron microtomography, we have discovered that soil microbes and plant roots microengineer their habitats by changing the porosity and clustering properties (i.e., spatial correlation) of the soil pores. Our results indicate that biota act to significantly alter their habitat toward a more porous, ordered, and aggregated structure that has important consequences for functional properties, including transport processes. These observations support the hypothesis that the soil-plant-microbe complex is self-organized. PMID- 16680510 TI - Isolation and genetic analysis of haloalkaliphilic bacteriophages in a North American Soda Lake. AB - Mono Lake is a meromictic, hypersaline, soda lake that harbors a diverse and abundant microbial community. A previous report documented the high viral abundance in Mono Lake, and pulsed-field gel electrophoresis analysis of viral DNA from lake water samples showed a diverse population based on a broad range of viral genome sizes. To better understand the ecology of bacteriophages and their hosts in this unique environment, water samples were collected between February 2001 and July 2004 for isolation of bacteriophages by using four indigenous bacterial hosts. Plaque assay results showed a differential seasonal expression of cultured bacteriophages. To reveal the diversity of uncultured bacteriophages, viral DNA from lake water samples was used to construct clone libraries. Sequence analysis of viral clones revealed homology to viral as well as bacterial proteins. Furthermore, dot blot DNA hybridization analyses showed that the uncultured viruses are more prevalent during most seasons, whereas the viral isolates (Aphi and phi2) were less prevalent, confirming the belief that uncultured viruses represent the dominant members of the community, whereas cultured isolates represent the minority species. PMID- 16680512 TI - Changes in organic matter biodegradability influencing sulfate reduction in an aquifer contaminated by landfill leachate. AB - In situ experiments were conducted to measure sulfate reduction rates and identify rate-limiting factors in a shallow, alluvial aquifer contaminated with municipal landfill leachate. Single-well, push-pull tests conducted in a well adjacent to the landfill with > 8 mM dissolved organic carbon (DOC) exhibited a sulfate reduction rate of 3.2 mumol SO4(-2) (L sediment)(-1) day(-1), a value in close agreement with laboratory-derived estimates. Identical tests conducted in wells located 90 m downgradient where DOC levels remained high (> 3 mM) showed no detectable sulfate consumption, and laboratory assays confirmed this observation. However, the rates of sulfate reduction in sediment samples obtained from this site were three times larger when they were amended with filter-sterilized groundwater from the upgradient location. The effect of various amendments on sulfate reduction rates was further examined in laboratory incubations using sediment collected from the downgradient site amended with 35S sulfate. Unamended sediments showed only weak conversion of the tracer to 35S sulfide (5 to 7 cpm/cm2), whereas the addition of Desulfovibrio cells increased 35S sulfide production to 44 cpm/cm2. However, the application of heat-killed Desulfovibrio had a similar stimulatory effect, as did a lactate amendment. Collectively, these findings indicate that the lack of measurable sulfate reduction at the downgradient site was not due to the absence of the necessary metabolic potential, the presence of lower sulfate concentration, or the quantity of electron donor, but by its biodegradability. The findings also indicate that field bioaugmentation attempts should be interpreted with caution. PMID- 16680513 TI - Radiologists' preferences for just-in-time learning. AB - BACKGROUND: Effective learning can occur at the point of care, when opportunities arise to acquire information and apply it to a clinical problem. To assess interest in point-of-care learning, we conducted a survey to explore radiologists' attitudes and preferences regarding the use of just-in-time learning (JITL) in radiology. MATERIALS AND METHODS: Following Institutional Review Board approval, we invited 104 current radiology residents and 86 radiologists in practice to participate in a 12-item Internet-based survey to assess their attitudes toward just-in-time learning. Voluntary participation in the survey was solicited by e-mail; respondents completed the survey on a web based form. RESULTS: Seventy-nine physicians completed the questionnaire, including 47 radiology residents and 32 radiologists in practice; the overall response rate was 42%. Respondents generally expressed a strong interest for JITL: 96% indicated a willingness to try such a system, and 38% indicated that they definitely would use a JITL system. They expressed a preference for learning interventions of 5-10 min in length. CONCLUSIONS: Current and recent radiology trainees have expressed a strong interest in just-in-time learning. The information from this survey should be useful in pursuing the design of learning interventions and systems for delivering just-in-time learning to radiologists. PMID- 16680519 TI - Retraction. Purification and characterization of three novel plasmid-born bacteriocins (curvalicins 28) produced by Lactobacillus curvatus CWBI-B28. PMID- 16680515 TI - NOTES: Gathering momentum. PMID- 16680520 TI - Oxidative stress and heat-shock responses in Desulfovibrio vulgaris by genome wide transcriptomic analysis. AB - Sulfate-reducing bacteria such as Desulfovibrio vulgaris have developed a set of responses that allow them to survive in hostile environments. To obtain further knowledge of the protective mechanisms employed by D. vulgaris in response to oxidative stress and heat shock, we performed a genome-wide transcriptomic analysis to determine the cellular responses to both stimuli. The results showed that 130 genes were responsive to oxidative stress, while 427 genes were responsive to heat-shock. Functional analyses suggested that the genes regulated were involved in a variety of cellular functions. Amino acid biosynthetic pathways were induced by both oxidative stress and heat shock treatments, while fatty acid metabolism, purine and cofactor biosynthesis were induced by heat shock only. The rubrerythrin gene (rbr) was up-regulated in response to oxidative stress, suggesting an important role for this protein in the oxidative damage resistance response in D. vulgaris. In addition, thioredoxin reductase (trxB) was also responsive to oxidative stress, suggesting that the thiol-specific redox system might also be involved in oxidative protection in this organism. In contrast, the expression of rubredoxin oxidoreductase (rbo), superoxide dismutase (sodB) and catalase (katA) genes were not regulated in response to oxidative stress. Comparison of cellular responses to oxidative stress and heat-shock allowed the identification of 66 genes that showed a similar drastic response to both environmental perturbations, implying that these genes might be part of the general stress response (GSR) network in D. vulgaris. This hypothesis was further supported by the identification of a conserved motif upstream of these stress responsive genes. PMID- 16680521 TI - Identification of genes necessary for jinggangmycin biosynthesis from Streptomyces hygroscopicus 10-22. AB - A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I (jinggangmycin). Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. An ordered cosmid library of the 300 kb AseI-F fragment was made and one of the cosmids conferred jinggangmycin productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids (18G7, 5H3 and 9A2) also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. This gene resembles acbC from Actinoplanes sp. 50/110, which encodes a C7-cyclitol synthase that catalyses the transformation of sedoheptulose 7-phosphate into 2-5-epi valiolone for acarbose biosynthesis. The valA/acbC-homolog (orf1) of S. hygroscopicus 10-22 was shown to be essential for jinggangmycin biosynthesis as an engineered mutant with a specific in-frame deletion removing a 609 bp sequence internal to orf1 completely abolished jinggangmycin production and the corresponding knock-out mutant (JXH4) could be complemented for jinggangmycin production by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of jinggangmycin and validamycin, which led to a clear demonstration that they are identical. PMID- 16680522 TI - Will dietary restriction work in primates? AB - This issue of Biogerontology addresses whether dietary restriction (DR) "...can increase longevity in all species, particularly in human beings". The possibility that DR can increase longevity in all species seems a trivial issue compared to that of DR's potential efficacy in people. The striking phylogenetic breadth of DR's longevity increasing effect supports the notion of human translatability. The available evidence in primates (human and nonhuman) suggests that it is highly probable that DR will increase the span of good health (and the average lifespan) but the magnitude of this predicted increase is unknown. Robust survival data for monkeys subjected to highly controlled DR will be available in approximately 25 years; similar data for large numbers of human practitioners of DR appear to be many decades away. PMID- 16680524 TI - Antibiotic resistance and plasmid profile of environmental isolates of Vibrio species from Mai Po Nature Reserve, Hong Kong. AB - In this study, three environmental isolates of Vibrio species were isolated from water and sediment samples of Mai Po Nature Reserve, Hong Kong SAR. They were identified to be Vibrio cholerae MP-1, Vibrio aestuarianus MP-2 and Vibrio vulnificus MP-3 by both biochemical test and subsequent full-length 16S rDNA sequencing. Both disc diffusion susceptibility test and microplate dilution technique were used to investigate the antibiotic resistance profile of these three bacteria. Results from both assays were in generally good agreement. All three isolates were strongly resistant to beta-lactam family of antibiotics while susceptible to tetracycline, chloramphenicol, nalidixic acid and streptomycin. In addition, V. cholerae MP-1 was very tolerant to high concentration of vibriostatic agent O/129 to 40 microg/ml, at which the growth of V. aestuarianus MP-2 and V. vulnificus MP-2 was completely inhibited. A small naturally occurring plasmid was found in V. cholerae MP-1 while no plasmid was detected in the other two bacteria. This study suggests that Mai Po Nature Reserve harbors bacteria of unique characteristics that warrant further investigation. PMID- 16680530 TI - Uncertainty of illness relationships with mental health and coping processes in fibromyalgia patients. AB - Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain condition poorly understood in terms of etiology and treatment by both physicians and patients. This condition of "uncertainty of illness" was examined as a variable involved in the adjustment of FMS patients, relating it to their depression, anxiety, affect, and coping styles. Fifty-one community-residing FMS patients provided self-report information on subsets of adjustment variables. Both cross-sectional and more dynamic longitudinal analyses showed that illness uncertainty was significantly associated with anxiety, negative affect, and avoidant and passive coping. Its positive relationship with depression was eliminated when a control variable, pain helplessness, was included as a covariate. Longitudinally, illness uncertainty interacted with interpersonally stressful daily events in predicting reports of reduced positive affect, suggesting that illness uncertainty acts as a risk factor for affective disturbances during stressful times. Implications of these results for therapeutic interventions are discussed. PMID- 16680531 TI - Midwives' approach to genetic diseases and genetic counseling in Denizli, Turkey. AB - The purpose of this study was to evaluate Denizli midwives' self-reported knowledge of genetic diseases and genetic counseling. Data was collected on forms that obtained information about midwives approaches to basic genetics, genetic disorders, and genetic counseling (response rate = 70.1%). The highest response rate of midwives describing themselves as "knowledgeable" about basic genetic information was for mitosis and meiosis with 4.8%, about genetic disorders was for phenylketonuria with 61.1%, and about genetic counseling was for recommending ultrasonography during pregnancy with 98.1%. The source for basic genetics information for 56.4% of participants was in their school level classes. None of the midwives felt that they had sufficient knowledge about genetic counseling or screening and 76.4% would like to attend an educational course. Even though the midwives have recognized their knowledge deficit they occasionally give genetic counseling. As a result of this study a genetics course is planned for midwives so they can actively participate in the prevention and early diagnosis of genetic diseases. PMID- 16680532 TI - Beyond diversity: toward a cultural community psychology. AB - We are increasingly considering culture as the context of diversity and, in the process, becoming a cultural community psychology. Our recent progress is discussed and a suggestion to accelerate our progress through greater collaboration with cultural psychology(4) is offered. The development of a cultural community psychology has implications for the traditional distinction between basic and applied psychology, our understanding of context, partnerships between academicians and practitioners, and the place of community psychology within the discipline of psychology. These implications are presented and discussed. PMID- 16680533 TI - Reaching higher in community psychology: social problems, social settings, and social change. AB - Forty years after the founding of community psychology, we have yet to deliver on the full promissory note of our birth, where we were poised to address social problems, social settings, and social change. Despite some success, we are at risk for selling ourselves short, for dying out in the discipline of psychology, and for failing to improve the common good. Given changes in demographics and in the safety net, the problem of entrenched disparities is even more urgent-in perception of the other, in the provision of opportunities for development, and in outcomes. Envisioning and enabling will be critical as we work with unified purpose toward a cumulative science where failure will not be predictable. PMID- 16680534 TI - A community practice model for community psychologists and some examples of the application of community practice skills from the Partnerships for Success initiative in Ohio. AB - This paper provides an opportunity to consider the concept of community practice from the vantage point of community psychology. The author argues that community psychology has significant potential to change organizations, communities, and other settings to benefit setting occupants. However, it is the author's contention that the full realization of this potential is contingent upon an organized effort to engage in formal community practice. The author defines community practice in terms of four skill sets related to mobilization, planning, implementation, and evaluation. The author also describes settings that might support community practice and discusses implications for training and the field of community psychology in general. Finally, the author illustrates several community practice skills and roles in the context of a local community-based initiative in Ohio called Partnerships for Success. PMID- 16680535 TI - Revealing the cues within community places: stories of identity, history, and possibility. AB - Place-based approaches to community change have become increasingly popular strategies for addressing significant social problems. With their intentional focus on 'place,' most efforts have sought to gain greater understanding into how neighborhood contexts affect people. However, while both aggregate characteristics and social dynamics of neighborhoods have been subject to scrutiny in the literature, less attention has been paid to understanding how the environmental characteristics of neighborhoods and communities as places have meaning for residents. The present study used an innovative methodology called Photovoice to obtain a greater understanding of the meanings residents ascribe to the salient characteristics of their neighborhoods and communities. As part of a place-based initiative, 29 adult and youth residents in seven distressed urban neighborhoods photographed and dialogued about the meaningful physical attributes of their community. According to participants, place characteristics provided cues about their personal histories as members of the community; communicated messages about the value and character of the community and its residents; defined social norms and behavior within the community; and provided markers that could remind residents of who they are and inspire a sense of possibility for who they could become. Implications for practice are discussed. PMID- 16680536 TI - Homelessness in the United States: assessing changes in prevalence and public opinion, 1993-2001. AB - A national survey was administered in 1993-1994 (N = 360) and repeated in 2001 (N = 435) to assess the prevalence of homelessness as well as attitudes, opinions and knowledge regarding homelessness. No significant changes in prevalence were found, despite a strong US economy during most of the 7-8 year period. Respondents in 2001 had less stereotyped views of homeless people and were more supportive of services, but came to see homelessness as a less serious problem that was less often due to economic factors. This "mixed" set of findings may reflect both beliefs on the benefits of a good economy and an increased awareness of the complexity of homelessness. Across the surveys, younger, female, liberal, and less wealthy respondents demonstrated more sympathetic attitudes towards homeless people. PMID- 16680537 TI - Cross-national variations in behavioral profiles among homeless youth. AB - Cross-national comparisons of homeless youth in Melbourne, Australia, and Los Angeles, CA, United States were conducted. Newly (n = 427) and experienced (n = 864) homeless youth were recruited from each site. Compared to Australia, homeless youth in the United States were younger, more likely to be in school or jail, demonstrated fewer sexual and substance use risk acts, fewer suicidal acts, and reported less need for social services. Across sites, experienced homeless youth were more likely to be older, male, engage in sexual and substance use, report greater need for social services, and make greater use of work, substance use, and health-related services. Homeless youth have different behavioral profiles in Australia and the United States, reflecting differences in the effectiveness of service systems in the two countries in keeping youth with fewer problems out of homelessness. PMID- 16680538 TI - Promoting Hmong refugees' well-being through mutual learning: valuing knowledge, culture, and experience. AB - Refugees who resettle in a new country face numerous struggles, including overcoming past traumas and coping with post-migration stressors, such as lack of meaningful social roles, poverty, discrimination, lack of environmental mastery, and social isolation. Thus, in addition to needing to learn concrete language skills and gain access to resources and employment, it is important for refugees to become a part of settings where their experiences, knowledge, and identity are valued and validated. The Refugee Well-Being Project (RWBP) was developed to promote the well-being of Hmong refugees by creating settings for mutual learning to occur between Hmong adults and undergraduate students. The RWBP had two major components: (1) Learning Circles, which involved cultural exchange and one-on-one learning opportunities, and (2) an advocacy component, which involved undergraduates advocating for and transferring advocacy skills to Hmong families to increase their access to resources in their communities. The project was evaluated using a mixed quantitative and qualitative approach. This article discusses data from qualitative interviews with participants, during which the importance of reciprocal helping relationships and mutual learning emerged as significant themes. PMID- 16680539 TI - Intimate partner violence, welfare receipt, and health status of low-income african american women: a lifecourse analysis. AB - Although the association among intimate partner violence (IPV), welfare receipt, and health status has been well-established, little is known about the temporal sequencing of these events. In a random sample of low-income African American women in an urban Midwestern county, lifecourse data about IPV and welfare receipt were obtained using the Life History Calendar method (D. Freedman, A. Thornton, D. Camburn, D. Alwin, & L. Young-DeMarco, 1988), along with data about mental and physical health status. Controlling for relevant factors, longitudinal analyses found that previous experience of IPV increased women's odds of receiving welfare benefits in a given year, but previous welfare receipt did not. Cumulative IPV, but not cumulative welfare receipt, was associated with current and past-year health problems. These findings are consistent with the hypothesis that IPV leads women to turn to welfare assistance, and that compromised physical and mental health due to past cumulative IPV interferes with women's gainful employment. PMID- 16680540 TI - Community social organization, parents, and peers as mediators of perceived neighborhood block characteristics on delinquent and prosocial activities. AB - Research on contextual and neighborhood effects has grown exponentially over the past decade as researchers have reacknowledged that community matters. Although empirical findings have consistently documented the significant influence of neighborhood context, the field is just beginning to investigate the varied and multiple pathways through which this influence is transmitted. The current study found support for both a direct and indirect influence of perceived neighborhood structural characteristics (i.e., neighborhood stability, income), measured at the block level, on neighborhood and youth outcomes. Directly, perceived neighborhood advantage led to significantly lower neighborhood rates of official delinquency and higher rates of prosocial activity. Indirectly, perceived neighborhood advantage significantly impacted outcomes by influencing more proximal constructs such as community social organization, informal social control, parenting practices, and affiliation with delinquent peers. Different pathways were significant across neighborhood and youth outcomes, yet perceived neighborhood advantage, in particular neighborhood stability, consistently exerted substantial effects, highlighting the need for more comprehensive and multilevel prevention efforts. PMID- 16680541 TI - The social ecological model and physical activity in African American women. AB - Little is known about the social and contextual correlates (e.g., social norms, environment, social networks, and organizational support) influencing the adoption and maintenance of regular physical activity among minority and underserved populations. The purpose of this review was to apply the social ecological model to better understand physical activity among African American women. A review of the literature pertaining to correlates of physical activity among African American women was conducted and applied to a social ecological perspective. Understanding and addressing social and contextual correlates of physical activity behavior among African American women are necessary to establish comprehensive programs, particularly within community settings. The social ecological model provides a strong theoretical basis to guide physical activity intervention in this population. PMID- 16680542 TI - Neighborhood poverty, social capital, and the cognitive development of African American preschoolers. AB - In this investigation, we examine the impact of the ecological context of the residential neighborhood on the cognitive development of children by considering social processes not only at the family-level but also at the neighborhood-level. In a socioeconomically diverse sample of 200 African American children living in 39 neighborhoods in Baltimore, we found that neighborhood poverty was associated with poorer problem-solving skills over and above the influence of family economic resources and level of positive parent involvement. Sampson has theorized that neighborhood poverty affects child well-being by altering levels of neighborhood social capital as well as family social capital. Although we found that indicators of neighborhood and family social capital were associated with cognitive skills, these factors did not explain the association between neighborhood poverty and problem-solving ability. Implications for future research in the area of neighborhoods and child development are discussed. PMID- 16680543 TI - Real time online imaging for interventional electrophysiologists: many directions and extraordinary opportunities. PMID- 16680544 TI - Rationale and design of a prospective study of the clinical significance of atrial arrhythmias detected by implanted device diagnostics: the TRENDS study. AB - BACKGROUND: Sustained atrial fibrillation (AF) is a common risk factor for stroke. While intermittent AF also appears to pose a substantial stroke risk, the quantitative relationship between the percentage of time spent in AF and stroke risk is poorly specified and "intermittent" AF is not the same as paroxysmal AF. Improved assessment of the impact of AF burden on stroke risk will allow more targeted and safer use of antithrombotic therapy. METHODS AND RESULTS: The primary objective of this study is to determine if AT/AF (all device detected atrial tachyarrhythmias, including atrial flutter, atrial fibrillation, and atrial tachycardia) burden over a 30 day period is an independent predictor of the occurrence of ischemic stroke, transient ischemic attack (TIA) and/or systemic embolism in subjects not receiving anticoagulation therapy. TRENDS is a prospective, post-market, non-randomized, multicenter study designed to enroll 3100 subjects who have an independent Class I/II indication for cardiac rhythm device implantation and who have demographic features suggestive of an increased risk for thromboembolic complications related to AT/AF. All implanted devices will have the ability to collect long-term AT/AF burden trending data and will be equivalently programmed to ensure consistent data collection. All subjects will be followed with device interrogations every 3 months and clinic visits every 6 months for 1 year. Subjects with a documented history of AT/AF prior to enrollment and those who develop AT/AF during the 12-month follow-up will be followed until the last subject enrolled in the study has completed their 24 month follow-up. CONCLUSIONS: The results of the TRENDS study should help clarify the implications of data retrieved from an implantable device with regard to the risk for thromboembolic complications from atrial arrhythmias, even in the absence of symptoms. PMID- 16680545 TI - Pulmonary vein isolation under direct visual identification of the left atrium pulmonary vein junction using intra-cardiac echography. AB - INTRODUCTION: Intra-cardiac echocardiography (ICE) which has some benefits, can be used to obtain detailed anatomy of the heart chambers or large vessels, and the catheter positions, and it has been considered useful for improving the outcome of the ablation. In the present study, we performed pulmonary vein isolation (PVI) under real time monitoring of ICE imaging utilizing an ICE catheter placed at the junction of the left atrium (LA) and PVs (LA-PV junction). METHODS: PVI for atrial fibrillation (AF) was performed in 30 cases with drug resistant AF (mean age: 66-years-old; including 22 males). An ICE catheter utilizing a 9 MHz frequency was inserted into the LA via the atrial septum, and placed at the LA-PV junction. Circumferential ablation was performed in the LA outside of the PV ostium, encircling both the superior and inferior ostia together under ICE imaging. RESULTS: The anatomy of the LA to the PVs and catheter sites were clearly identified by the ICE during the procedure, which enabled a precise and safe catheter manipulation with minimal fluoroscopy. Further, the wall thickness of the PV and LA, and position of the esophagus could be obtained by ICE, facilitating care in adjusting the power and/or duration of the current delivery. CONCLUSION: ICE imaging of the LA-PV junction permitted real time monitoring of the target sites for PVI during the ablation procedure, and was considered a useful technique for performing PVI. PMID- 16680546 TI - Coronary sinus morphology in different types of supraventricular tachycardias. AB - BACKGROUND: Atrioventricular nodal reentry tachycardia (AVNRT) is based on the concept of dual AV node pathways that are functionally and anatomically distinct. The bigger coronary sinus ostium (CSO) in patients with AVNRT compared to other supraventricular tachycardias (SVTs) may produce separation of atrial inputs into the AV node or create anisotropic conduction, thus giving rise to a different AV nodal physiology. Previous studies measuring the size of the CSO using CS angiography between patients with AVNRT and other SVTs showed conflicting results. Besides, no previous studies have compared the CS morphology of the different forms of AVNRT. OBJECTIVES: This study compares the size and morphology of the CS among patients with typical AVNRT, atypical AVNRT and accessory pathways mediated reentrant tachycardia (AVRT). METHODS: Ninety-six patients with clinically documented SVTs were divided into three groups. The diameter of the CS was measured in LAO projection during end ventricular systole (by choosing the last ventricular inward motion). The CSO as well as 5, 10 and 15 mm inside the CS were measured. CS morphology is defined as either wind-sock shape or tubular shape. RESULTS: The size of the CS ostium was 13.58 +/- 3.98, 15.93 +/- 4.86 and 12.50 +/- 2.83 mm for the atypical AVNRT, typical AVNRT and AVRT, respectively (p = 0.03). There was significant difference in the size of the CS from the ostium until 15 mm into the CS between 1) typical AVNRT and AVRT, 2) typical AVNRT and atypical AVNRT. Typical and atypical AVNRT patients had more windsock morphology CS (13/32, 40.6% and 10/32, 31.2%) compared to AVRT which had only one (1/32, 3.1%) windsock morphology (p = 0.002). CONCLUSION: The easier CS cannulation in patients with typical AVNRT could be due to a bigger CS size and to a more windsock morphology. The CS size and morphology may be a very important substrate of tachycardia in patients with AVNRT. PMID- 16680548 TI - Radiofrequency catheter ablation within the coronary sinus eliminates a macro reentrant atrial tachycardia: importance of mapping in the coronary sinus. AB - We describe a patient who underwent radiofrequency (RF) catheter ablation of symptomatic atrial fibrillation. After left atrial (LA) catheter ablation and pulmonary vein isolation, a macro-reentrant atrial tachycardia (AT) with a critical isthmus at the mitral isthmus was induced by incremental atrial pacing from the coronary sinus. Extensive RF energy applications from endocardial sites using ablation catheters with 4 mm- and 8 mm- tips resulted in no discrete potentials being recorded from the endocardial sites of the isthmus, but the tachycardia could not be terminated. However, discrete potentials were recorded within the CS, and epicardial RF energy applications from the CS eliminated the tachycardia. Thus, mapping in the CS is useful for detecting residual conduction at epicardial sites along the mitral isthmus. RF catheter ablation within the CS should be considered when no distinct electrograms are recorded after extensive ablation from the endocardial sites and when distinct electrograms are recorded within the CS. PMID- 16680547 TI - Targets and endpoints in ablation therapy for atrial fibrillation in the light of pathophysiological mechanisms. AB - Atrial fibrillation (AF), an important public health problem is a complex and multifactorial arrhythmia. Non-pharmacological treatment for symptomatic patients is of increasing importance. The different catheter ablation techniques in AF treatment developed during recent years, all based on different pathophysiological insights, are discussed. The non-standardized use of different follow-up methods after ablation make interpretation and comparison of results difficult. PMID- 16680549 TI - Two atrial reentrant tachycardias originating from the superior vena cava: electrophysiological characteristics and radiofrequency ablation. AB - A case with two different types of atrial reentrant tachycardia of superior vena cava (SVC) origin is presented. Recent clinical studies have shown that the origin of focal atrial tachycardia typically lies in the venous structures connecting to both atria--the coronary sinus, the superior and inferior vena cava, and the pulmonary vein. These foci have atrial muscle fiber extensions which have electrophysiological characteristics essential to generation of focal ectopic firing. However, little is known about reentrant mechanism of these venous structures. In this report, we present a case of two atrial tachycardias (SVT1 and SVT2) independently originating from the SVC. SVT1 had 430 ms of tachycardia cycle length, and SVT2 had 390 ms of tachycardia cycle length. Both of them showed the character of reentry, and their earliest activations were recorded in the SVC. They were successfully eliminated by focal radiofrequency ablation in the SVC. PMID- 16680550 TI - Effects of cardioactive medications on retrograde conduction: continuing relevance for current devices. AB - INTRODUCTION: Retroconduction (ventriculo-atrial conduction) remains a problem for patients with implanted cardiac rhythm devices. Pacemaker algorithms can detect and terminate endless loop tachycardia (ELT), but actual prevention of ELT may require anti-arrhythmic drugs (AADs). Similarly, AADs can affect ICD rhythm discrimination algorithms that depend on atrio-ventricular ratios. There is concern whether these drugs remain effective during stress situations. METHODS: Electrophysiologic studies that included retroconduction testing using slow ramp pacing were done in 1332 patients. The presence or absence of retroconduction at baseline and with drug was recorded, as was the rate at block. As a stress surrogate, isoproterenol was used to test retroconduction and reversal of drug induced block. RESULTS: Procainamide, mexiletine, phenytoin, disopyramide, quinidine, beta-blockers, encainide, and amiodarone caused complete retrograde block or decreased the rate at which block occurred (mean 76% of patients, p < 0.008), whereas digoxin, lidocaine, diltiazem, and verapamil did not. Isoproterenol (in the absence of AADs) increased the rate at block in 82% of 404 patients with retroconduction at baseline (p < 0.005). Of 319 patients without retroconduction at baseline, 134 (42%) developed retroconduction after isoproterenol. Isoproterenol reversed retrograde block in 39% of patients with block on an AAD. Amiodarone, digoxin, and the combination of digoxin plus a beta blocker were most effective at resisting this reversal of ventriculo-atrial block (80%, 68%, and 75% respectively). CONCLUSION: Most of the AADs reviewed increase the cycle length at block or abolish retroconduction, while isoproterenol has the opposite effect. Anti-arrhythmic medications, particularly amiodarone, digoxin, and the combination of digoxin plus a beta-blocker may be considered for a patient with multiple ELT episodes or certain ICD detection problems. PMID- 16680551 TI - Epicardial radiofrequency ablation of ventricular myocardium: factors affecting lesion formation and damage to adjacent structures. AB - We evaluated the factors affecting epicardial radiofrequency (RF) lesion formation in normal ventricular myocardium. In 16 dogs, a minithoracotomy was made and a sheath was placed in the pericardial space. Standard ablation lesions (4-mm tip catheter; 70 ( composite function) C/60 seconds) were created in each ventricle under fluoroscopy guidance (n = 7) or hand-held with direct visualization of the catheter to assure optimal electrode-tissue contact (n = 6). In the latter, thermally-shielded (TS) electrodes (50% tip surface along its 4 mm length) were used in 3/6 dogs. Catheter tip (4 mm) irrigation (13 mL/minutes; 40 ( composite function) C/60 seconds) was employed with conventional techniques in 3 additional dogs. RESULTS: With optimal electrode-tissue contact (11 lesions), power (3.4 +/- 2.3 W vs. 16 +/- 13 W; p < 0.001) and pacing thresholds (0.2 +/- 0.0 mA vs. 3.6 +/- 5.7 mA; p = 0.004) were lower than standard RF (25 lesions). However, lesion dimensions were similar and transmural lesions did not occur (depth 2.8 +/- 1.1 mm vs. 3.0 +/- 1.5 mm). Catheter irrigation allowed high power outputs (43 +/- 6.1 W; p < 0.001) generating transmural lesions, 5/9 (55%), depth 6.4 +/- 2.1 mm. At constant power (2 W), catheter-tip temperature (52 +/- 5.2( composite function) C vs. 57 +/- 6.6( composite function) C; p = NS) and lesion (10 in each group) dimensions were similar for conventional and TS electrodes, but damage to parietal pericardium and lungs occurred with conventional electrodes only (70% vs. 0% p = 0.02). CONCLUSION: Standard epicardial RF ablation does not produce deep lesions and exhibits a significant energy loss probably due to poor electrode-tissue contact. Catheter irrigation allows delivery of high power outputs to the epicardium consistently creating deeper lesions than standard ablation. TS electrodes may reduce damage to neighboring structures during epicardial RF ablation. PMID- 16680552 TI - Inappropriate ICD discharge due to T-wave oversensing in a patient with the Brugada syndrome. AB - Accurate sensing is an essential requirement for appropriate functioning of implantable cardioverter-defibrillator (ICD). T-wave oversensing remains as an annoying problem in currently available ICD. The Brugada syndrome with its inherent dynamic variations in electrophysiologic phenomena may complicate ICD therapy. We report a patient with diagnosis of Brugada syndrome who presented with frequent inappropriate therapy due to intermittent T-wave oversensing. This problem could not be eliminated by device reprogramming and necessitated implantation of a new sense/pace lead. PMID- 16680553 TI - Koch and the "ultimum moriens" theory--the last part to die of the heart. PMID- 16680555 TI - Asymmetrical (ADMA) and symmetrical dimethylarginine (SDMA) as potential risk factors for cardiovascular and renal outcome in chronic kidney disease - possible candidates for paradoxical epidemiology? AB - BACKGROUND: Asymmetrical dimethylarginine (ADMA) is an inhibitor of nitric-oxide synthase. It has been linked to atherosclerotic risk in the general population as well as in end-stage renal disease patients (ESRD), whereas symmetrical dimethylarginine (SDMA) is thought to be biological inactive. Prospective data concerning the role of both dimethylarginines are rare in patients with chronic kidney disease. METHODS: 200 Patients with chronic kidney disease (mean age 57.6 +/- 13.0 years, 69 female, 131 male); 82 with chronic renal failure (CRF), 81 on maintenance haemodialysis (HD) and 37 renal transplant recipients (RTR) were prospectively followed for 24 months. ADMA and SDMA were measured by HPLC. The relation of plasma levels of ADMA and SDMA together with conventional risk factors for the cardiovascular and renal outcome was investigated with Cox proportional hazards model. RESULTS: Mean serum levels of SDMA were significantly increased in all groups compared to the control group (P 1.65-fold increase) and 139 were downregulated (> 1.58-fold decrease) with a B value > 4.68 (99.08% probability of real differential expression). When SPH was compared to normal parathyroid tissue, 50 genes were differentially expressed: 42 were upregulated (> 1.89) and 8 were downregulated (> 1.7) with a B > 4.26 (98.6% probability of real differential expression). At least 17 genes were differentially expressed and able to discriminate SPH from adenoma or normal tissue. Upregulated genes were related to apoptosis inhibition, cell proliferation, transcriptional activity and cell adhesion, among other activities. Downregulated genes were mainly related to ion channel activity, lipopolysaccharides, prostaglandin-d synthase, and integral membrane proteins. CONCLUSIONS: Our data suggest that SPH and adenoma have a singular molecular signature that, theoretically, could be used for the differential diagnosis of these entities and normal parathyroid tissue. PMID- 16680587 TI - The effects of retinoic acid on the insulin-like growth factor axis in primary tissue culture from hyperparathyroidism. AB - BACKGROUND: The importance of the IGF system in HPT has been previously demonstrated. Additionally, the role of vitamin A in HPT has been reported. Retinoic acid (RA), a derivative of vitamin A, is a ligand for the IGF II receptor (IGF2R). We have evaluated the interactions of RA with the IGF system in a primary parathyroid cell culture model. MATERIALS AND METHODS: Primary cell cultures were prepared from nine patients. Following adhesion, the cells were transferred to serum-free medium and dosed once with growth factors +/- RA for 96 hours. Proliferation was assessed by measuring tritiated thymidine incorporation. RESULTS: Compared with the control group (100%), both IGF I and II increased DNA synthesis significantly. Retinoic acid significantly reduced the basal DNA synthesis to 82.2% +/- 4.2% compared with control (P < 0.05). Retinoic acid x10( 5) M completely abrogated the proliferative actions of IGF II (70.2% +/- 9.7%, P < 0.05) but had no significant effect on the IGF I response (P > 0.05). To evaluate the role of IGF2R or IGFBPs in mediating the actions of RA, the IGF II analogs [Leu27]IGF II (10-20-fold reduced IGF I receptor affinity) and des(1-6) IGF II (lower IGFBP binding affinity) were used. The IGF II inhibitory effect of RA was enhanced in the presence of analogs [Leu27]IGF II (P = 0.052) but not with des(1-6)IGF II (P > 0.05), compared with wild-type IGF II. CONCLUSIONS: These data implicate a novel antiproliferative role for RA in enhancing the pericellular clearance of IGF II via the IGF2R preventing ligand activation of the IGF I receptor. This may have broader implications for RA effects in other tumors. PMID- 16680588 TI - Indian primary hyperparathyroidism patients with parathyroid carcinoma do not differ in clinicoinvestigative characteristics from those with benign parathyroid pathology. AB - INTRODUCTION: No foolproof preoperative diagnostic indicators of parathyroid carcinoma (PC) exist in absence of nonskeletal metastases. Palpable parathyroid tumor, advanced skeletal and renal manifestations, and very high serum calcium and parathyroid hormone levels are considered strong predictors. Most of these features are common in Indian primary hyperparathyroidism (PHPT) patients although only few have PC. The aim of this study was to identify dependable clinicoinvestigative predictors of PC in Indian PHPT patients. MATERIALS AND METHODS: Clinical, biochemical, radiological, and densitometric attributes of 100 PHPT patients who underwent successful parathyroidectomy (1990-2004) were studied. Various parameters of patient groups with parathyroid adenoma (n = 84), primary hyperplasia (n = 12), and carcinoma (n = 4) were compared using ANOVA, with P value < 0.05 considered significant. RESULTS: Mean age of patients was 37.4 years, with no difference in the 3 groups (P = 0.92). Patients in 3 groups had comparably severe bone disease; 36 had coexistent renal disease. Two patients with PC and 27 (32%) with adenoma had palpable parathyroid tumor. None of the biochemical parameters predicted malignant pathology. Mean tumor weight (milligram) in carcinoma patients (15,080 +/- 5,638.02) was significantly higher than those with adenoma (5,724 +/- 1,257.9) (P = 0.002). Postoperative course and recovery in carcinoma patients were similar to those with adenoma. In follow-up (mean: 33 months), none of the adenoma patients were found to have persistent/recurrent PHPT attributable to missed PC. CONCLUSION: Indian patients with parathyroid adenoma, hyperplasia, and carcinoma were not found to differ in their clinical, biochemical, and pathological characteristics except for significantly higher tumor weight in the carcinoma group. PMID- 16680589 TI - Total parathyroidectomy without autotransplantation for renal hyperparathyroidism: experience with a qPTH-controlled protocol. AB - BACKGROUND: Controversy regarding the optimal surgical treatment for secondary hyperparathyroidism (sHPT) continues. Subtotal parathyroidectomy (PTX) with a small remnant and total parathyroidectomy with autotransplantation prevail, although impaired by considerable recurrence rates. Concerns about postoperative management and long-term supplementation prevent broader acceptance of total parathyroidectomy without autotransplantation. MATERIALS AND METHODS: The standardized surgical procedure with intraoperative PTH assessment (qPTH) included cervical thymectomy, histological proof of four parathyroid specimens and obligatory cryopreservation of parathyroid tissue in all 23 patients undergoing total PTX without autotransplantation. Whenever qPTH did not normalize, complete cervical exploration of ectopic sites was performed. Another 64 patients with subtotal PTX for sHPT served as comparison for the postoperative course. RESULTS: There were 13 primary and 10 completion (5 persistent, 5 recurrent sHPT) total PTX with 14 concurrent thyroid resections performed. Mean preoperative PTH was 1.351 pg/ml (12-72 pg/ml) and serum calcium was 2.5 mmol/l (2.25-2.5 mmol/l). PTH showed intraoperative normalization in 15 patients and a 50% PTH reduction from preoperative values in all. Postoperative course was not significantly different from the subtotal PTX group and showed PTH within the normal range for 5 patients (4 < 35 pg/ml), 7 with PTH < 12 pg/ml, and 4 without measurable PTH. In 4 patients PTH did not normalize postoperatively. Serum calcium levels were below normal in all patients: < 2.25 mmol/l in 9, < 2.00 mmol/l in 7, and <1.8 mmol/l in 6 patients. Only 1 patient required intermittent early postoperative i.v. calcium supplementation, 6 patients received oral calcium and vitamin D supplement for low calcium levels, but no severe hypocalcemic symptoms were encountered. Mean postoperative hospital stay was 5 days. No recurrent laryngeal nerve palsies were encountered. Complications were two cervical bleedings following postoperative hemodialysis requiring evacuation. CONCLUSIONS: Total PTX without autotransplantation proves to be an equally safe and successful procedure for sHPT as subtotal PTX or total PTX with autotransplantation. Measurable PTH after total PTX as demonstrated in this study, supports the idea of uncontrollable isolated cell nests that are inevitably prone to stimulated growth with time. Therefore, total PTX is superior with regard to prevention of recurrence. Adequate supplementation with calcium and vitamin D, often necessary after subtotal PTX to suppress inadequate PTH and protect from recurrence, will prevent severe hypocalcemia and with the modern aluminium-diminishing dialysis regimen, development of adynamic bone disease appears less likely than feared. If necessary, cryopreserved parathyroid tissue can be autotransplanted on demand. PMID- 16680590 TI - Classical and follicular variant of papillary thyroid carcinoma: a comparative study on clinicopathologic features and long-term outcome. AB - INTRODUCTION: The follicular variant of papillary thyroid carcinoma (FVPTC) is the most common histologic subtype of papillary thyroid carcinoma (PTC). However, it is still controversial whether FVPTC should behave differently from classical PTC (CPTC). The present study aimed at evaluating any potential difference in clinicopathologic features and long-term outcome of FVPTC as compared with CPTC. PATIENTS AND METHODS: Of 568 patients with PTC managed from 1973 to 2004, 308 were shown to have CPTC (54.2%) and 67 (11.8%) FVPTC after histologic review. The mean (+/- SD) follow-up period was 11.3 (+/- 8.9) years. The two groups were compared in terms of clinicopathological features, treatment received, and outcome regarding recurrence and disease-specific survival. RESULTS: There was no difference in age and gender ratio between the CPTC and FVPTC patients. Both groups had similar tumor characteristics in terms of tumor size, presence of multifocality, capsular invasion, lymphovascular permeation, and perineural infiltration. However, FVPTC patients had significantly fewer histologically confirmed cervical lymph node metastases (P = 0.027) and extrathyroidal involvement (P = 0.005). The proportion of bilateral resection, adjuvant radioactive iodine, and lymph node dissection did not differ significantly between the two groups. The FVPTC patients had a more favorable tumor risk by DeGroot classification (P = 0.003) and MACIS (Metastasis, Age, Completeness of excision, Invasiveness, and Size) score (P = 0.026). The 10- and 15-year actuarial disease-specific survivals did not differ significantly between FVPTC and CPTC patients (96.2% versus 90.7% and 96.2% versus 89.1%, respectively). CONCLUSIONS: Although patients with FVPTC had more favorable clinicopathologic features and a better tumor risk group profile, their long-term outcome was similar to that of CPTC patients. PMID- 16680591 TI - Papillary microcarcinoma: is there any difference between clinically overt and occult tumors? AB - INTRODUCTION: Papillary microcarcinoma (PMC) is a subtype of papillary thyroid carcinoma (PTC) associated with excellent prognosis. However, clinical and biologic behaviors of PMC may vary considerably between tumors that are clinically overt and those that are occult. MATERIALS AND METHODS: From 1964 to 2003, 185 of 628 patients with PTC were identified as having PMC, based on tumor size < or = 1 cm. There were 110 overt and 75 occult PMCs detected based on clinical presentation. The clinicopathologic features, treatment, and long-term outcome of PMCs were evaluated and compared between the two groups. RESULTS: There were 37 men and 148 women with a median age of 45 years (range: 11-84 years). The median tumor size was 6.2 mm. Thirty-eight (21%) patients presented with cervical nodal metastases. Three (1.6%) had distant metastases and 5 (2.7%) underwent incomplete resection. Bilateral procedures were performed for 129 patients (70%) and 53 (29%) received postoperative I131 treatment. During a mean follow-up of 8.2 years, 4 patients died of the disease and 13 developed recurrence. Clinically overt PMCs were significantly larger, were more likely to be multifocal, and more likely to lead to bilateral thyroidectomy. Extrathyroidal or lymphovascular invasion, nodal metastases, I131 ablation, high-risk tumors, and postoperative recurrence occurred in overt PMC only. Patients with nodal metastases had a decreased survival and an increase in locoregional recurrence. CONCLUSIONS: Despite a relatively good prognosis in PMC, a distinction should be made between clinically overt and occult PMCs in which clinically overt PMC should be managed according to tumor risk profile and clinical presentation. PMID- 16680592 TI - Mutational analysis of the APC gene in cribriform-morula variant of papillary thyroid carcinoma. AB - INTRODUCTION: Familial adenomatous polyposis (FAP) is an inherited autosomal dominant syndrome caused by germline mutations in the adenomatous polyposis coli (APC) gene. Papillary thyroid cancer is one of the extracolonic manifestations of FAP. A characteristic histologic feature of this type of thyroid tumor is the cribriform-morula variant of papillary thyroid carcinoma (CMVPTC). METHODS: To investigate roles of the APC and beta-catenin genes in the development of CMVPTC, we examined germline and somatic mutations of these genes in a female patient with CMVPTC and FAP. The patient had undergone total colectomy at the age of 19 years and total thyroidectomy at age 25 years. RESULTS: Numerous tumors were disseminated in both lobes of the thyroid gland, and histopathologic examination revealed typical CMVPTC. DNA was extracted from peripheral blood leukocytes and 12 CMVPTC tumors, and exons 1-15 of the APC gene and exon 3 of the beta-catenin gene were examined. A germline mutation was detected in exon 13 of the APC gene, and this mutation generated a premature stop codon. Six somatic mutations (922delC, 1602delA, 1821delT, 1920delG, 2706del20, 2804insA) were found in the CMVPTC specimens. All mutations were truncating mutations in the N-terminus of the APC protein. Loss of heterozygosity was not observed in the remaining tumor tissues without somatic APC mutations. There were no mutations of the beta catenin gene in peripheral blood leukocytes or 12 CMVPTC specimens. CONCLUSIONS: These results suggest that APC mutations play an important role in the development of CMVPTC and occur predominantly in the 5' side of the APC gene between codons 308 and 935. PMID- 16680593 TI - Video-assisted thyroidectomy: report on the experience of a single center in more than four hundred cases. AB - BACKGROUND: We report on our series of patients selected for video-assisted thyroidectomy (VAT) over a 7-year period and discuss the results obtained. METHODS: Video-assisted thyroidectomy is a gasless procedure performed under endoscopic vision through a single 1.5-2.0-cm skin incision, using a technique very similar to conventional surgery. Eligibility criteria were these: thyroid nodules < 35 mm; thyroid volume < 30 ml; no previous conventional neck surgery. Small, low-risk, papillary thyroid carcinomas (PTC) were considered eligible. RESULTS: A total of 473 VATs were attempted on 459 patients. Locoregional anesthesia was used in 15 patients. Conversion was necessary in 6 (difficult dissection in 1 case, large nodule size in 3, gross lymph node metastases in 2). Thyroid lobectomy was successfully performed in 110 cases, total thyroidectomy in 343, and completion thyroidectomy in 14. In 66 patients with carcinoma, central neck nodes were removed through the same access. Concomitant parathyroidectomy was performed in 14 patients. Pathology showed benign disease in 277 cases, PTC in 175, and medullary microcarcinoma in 1. Postoperative complications included 8 transient recurrent nerve palsies, 64 transient hypocalcemias, 3 definitive hypocalcemias, 1 postoperative hematoma, and 2 wound infections. Postoperative pain was minimal and the cosmetic result excellent. In patients with PTC no evidence of recurrent or residual disease was shown. CONCLUSIONS: Indications for VAT are still limited (20% of patients who require thyroidectomy). Nonetheless, in selected patients, it seems a valid option for thyroidectomy and it could be considered even preferable to conventional surgery because of its significant advantages, especially in terms of cosmetic result. PMID- 16680595 TI - Endoscopic lateral approach thyroid lobectomy: safe evolution from endoscopic parathyroidectomy. AB - INTRODUCTION: Endoscopic thyroid surgery has been shown to be feasible. Most minimal access procedures have been performed via a midline approach. Based on our experience of more than 500 endoscopic parathyroidectomies via a lateral approach we have used the same method for thyroid lobectomy. METHODS: We present our experience of endoscopic thyroid lobectomy via a lateral approach (ETLA) and review of the results over a 1-year period (2004). Inclusion criteria for ETLA were (1) solitary nodule with atypical/suspicious fine-needle biopsy (FNB) or solitary toxic nodule; (2) lesions with a diameter of < 3 cm. Patients with a history of previous neck surgery or radiation exposure were excluded. All patients underwent postoperative vocal cord checks and plasma calcium evaluation. RESULTS: A total of 742 thyroid procedures were performed during 2004. Among them, 38 patients (5.1%) underwent ETLA. Indications for surgery were suspicious FNB results (36 patients) and a toxic nodule (2 patients). Mean nodule size was 19.2 mm. Mean +/- SD operating time was 102 +/- 27 minutes. All recurrent laryngeal nerves were identified (including one that was nonrecurrent). Of the 38 patients, the superior parathyroid gland was identified in 36 and the inferior parathyroid gland in 33. There were two conversions due to difficulty with the dissection. Two operations were converted because malignancy was diagnosed on frozen section examination. Two patients underwent a delayed completion thyroidectomy when definitive histology necessitated it. There were no permanent operative complications, and all patients were discharged on the first postoperative day. CONCLUSIONS: ETLA offers excellent intraoperative visualization of the vital structures and is a safe alternative to conventional thyroid lobectomy in selected cases. PMID- 16680596 TI - Pitfalls of intraoperative neuromonitoring for predicting postoperative recurrent laryngeal nerve function during thyroidectomy. AB - INTRODUCTION: Intraoperative neuromonitoring has been widely adopted to facilitate the identification and preservation of recurrent laryngeal nerve (RLN) function during thyroid surgery. The present prospective study validated the ability of this technique to predict postoperative RLN outcomes in a single endocrine surgical unit. METHODS: Neuromonitoring was performed using Neurosign 100 with laryngeal surface electrodes in 171 patients with 271 nerves at risk during thyroidectomy. Vocal cord function was routinely documented perioperatively. Patients were also stratified to low risk (primary surgery for benign disease) and high risk (malignancy and recurrent disease) for subgroup analysis. RESULTS: Unilateral vocal cord palsy occurred in 15 patients (5.5%) postoperatively. The incidence of postoperative nerve palsy in the low risk and high risk groups was 4.4% and 7.8%, respectively. All but two patients had recovery of function within a median period of 4 months after the operation. The rates of transient and permanent RLN palsy based on nerves at risk were 4.8% (n = 13) and 0.7% (n = 2), respectively. There were 241 true-negative (positive signal and no cord palsy), 15 false-positive (negative signal but no cord palsy), 8 true positive (negative signal and cord palsy), and 7 false-negative (positive signal but cord palsy) results, as correlated with the postoperative assessment. The sensitivity, specificity, and positive and negative predictive values were 53%, 94%, 35%, and 97%, respectively. For the high risk group, the sensitivity and positive predictive value increased to 86% and 60%, respectively. CONCLUSIONS: There are pitfalls associated with the use of intraoperative neuromonitoring during thyroid surgery. Routine application is not recommended except for selected high risk patients. PMID- 16680597 TI - Hypocalcaemia and parathyroid hormone assay following total thyroidectomy: predicting the future. AB - BACKGROUND: Ambulatory surgery (23:59-hour hospital stay) is gaining popularity in endocrine surgery. Hypocalcaemia is common following total thyroidectomy. Identifying patients with low risk of hypocalcaemia may facilitate early discharge (24-hour stay). METHODS: We conducted a prospective study including all patients undergoing total thyroidectomy. Blood samples were taken immediately following skin closure and the following morning for parathyroid hormone (PTH) and calcium measurement. Calcium supplements were routinely given when serum calcium was below 2.0 mmol/l. RESULTS: Thirty patients (27 females, 3 males) underwent total thyroidectomy (including 4 nodal dissection) for multinodular goitre (14), Graves' disease (11), papillary (4) and follicular (1) thyroid carcinoma. Twelve patients developed symptomatic transient hypocalcaemia. Based on morning calcium of < 2.0 mmol/l as trigger for calcium supplementation, 8 patients received calcium supplement with 4 false negatives, resulting in a specificity of 94.4%, sensitivity of 66.7%, positive predictive value (PPV) of 88.9% and negative predictive value (NPV) of 81%. Based on PTH levels (< 1.5 pmol/l) immediately following skin closure, 11 patients would receive calcium supplement, with 1 false negative resulting in a specificity of 83.3%, sensitivity of 91.7%, PPV of 78.6% and NPV of 93.8%. If supplementation is based on PTH levels (< 1.5 pmol/l) immediately following skin closure and morning calcium level (< 2.0 mmol/l), all 12 symptomatic patients will be correctly treated, with 4 false positives resulting in a combined specificity of 77.8%, sensitivity of 100%, PPV of 75% and NPV of 100%. CONCLUSIONS: Combining the immediate postoperation PTH levels (< 1.5 pmol/l) and morning serum calcium (< 2.0 mmol/l) can accurately identify patients at risk of hypocalcaemia following total thyroidectomy, allowing safe, early discharge. PMID- 16680598 TI - Sentinel lymph node biopsy may be used to support the decision to perform modified radical neck dissection in differentiated thyroid carcinoma. AB - BACKGROUND: The prognostic significance of lymph node metastases (LNM) in follicle cell-derived differentiated thyroid carcinoma (DTC) is still controversial. The management of cervical lymph nodes varies from "berry picking" to modified radical neck dissection (MRND). The incidence of LNM in papillary thyroid carcinoma varies from 27% to 80%. The importance of sentinel lymph node (SLN) biopsy for decisions about the surgical management of lymph nodes in DTC has been the subject of several previous studies. PATIENTS AND METHODS: In 40 patients with DTC methylene blue dye was applied peritumorally. Both SLN and non SLN in the lower third of the jugulo-carotid chain were dissected prior to total thyroidectomy and routine dissection of the central neck compartment and examined by frozen-section and standard histology. MRND was performed in 9 cases of LNM in the lateral neck compartment. RESULTS: The SLN identification rate was 92.5%. Metastases in SLNs were revealed by frozen-section histology in 7 cases, leading to immediate MRND. The findings were confirmed on standard HE examination. In 2 false-negative cases SLN metastases were revealed on standard histology and MRND was performed 1 week later. The specificity of the method was 100%, sensitivity 77.7%, negative predictive value 94%, positive predictive value 100%, with overall accuracy of 95%. CONCLUSION: Our results seem to imply that SLN biopsy in the jugulo-carotid chain using methylene blue dye mapping may be a feasible and valuable method for estimating lymph node status in the lateral neck compartment. It may be helpful in the detection of true-positive but nonpalpable lymph nodes, and in such cases may support the decision to perform MRND in patients with DTC. PMID- 16680599 TI - Prognostic significance of disseminated tumor cells in the connective tissue of patients with medullary thyroid carcinoma. AB - INTRODUCTION: Disseminated tumor cells in the connective tissue (CT-DTCs) do not have any connection to a primary tumor or the lymph nodes. They are identified quite often in patients with medullary thyroid carcinoma (MTC), but nothing is known regarding their prognostic significance. METHODS: Among 450 patients with MTC, 69 (15%) were identified as having CT-DTCs. A case-control group of patients without CT-DTCs was selected. The two groups were matched concerning TNM classification, age, heredity, and sex. Because many patients with CT-DTCs had extrathyroidal tumor extension (pT4 category), distant metastases (M1 category), or both, only 35 matched pairs could be identified. The TNM classification in both groups was as follows: pT1, n = 8; pT2, n = 15; pT3, n = 4; pT4, n = 8; pN0, n = 4; pN1, n = 31; M0, n = 30; M1, n = 5. The mean age was 46.8 +/- 17.0 years in the CT-DTC group and 44.4 +/- 15.0 years in the case-control group (NS). RESULTS: In both groups, 23 patients had sporadic MTC, and 12 patients had hereditary MTC. Neither mean basal preoperative nor postoperative calcitonin levels differed significantly between the two groups. In contrast, none of the patients with CT-DTCs was biochemically cured (normal calcitonin level after pentagastrin stimulation) compared to eight patients without CT-DTCs (P < 0.005). The two groups did not differ concerning other parameters (basal calcitonin level > 3000 pg/ml, more than 10 lymph node metastases, more than two involved locoregional lymph node compartments, mediastinal lymph node metastases) that have been reported to correlate with the lack of or almost (< 10%) lack of biochemical cure. CONCLUSIONS: In patients with MTC, disseminated tumor cells in the connective tissue correlate with advanced tumor stages and appear to be of prognostic significance. PMID- 16680600 TI - Desmoplastic stromal reaction in medullary thyroid cancer-an intraoperative "marker" for lymph node metastases. AB - BACKGROUND: Medullary thyroid cancer (MTC) disseminates early to lymph nodes (LN). There is no pre- or intraoperative marker to exclude LN involvement and thereby avoid lateral neck dissection in LN-negative patients. MATERIALS AND METHODS: This study was intended to verify the observation that patients with MTC lacking desmoplastic stromal reaction (DSR) never have LN metastases. In 120 patients undergoing primary operation for sporadic MTC the prognostic value of DSR with respect to LN involvement was evaluated. RESULTS: Thirty-two (27%) of 120 tumors were DSR negative, and 88 (73%) were DSR positive. All 32 (100%) DSR negative tumors were LN negative (N0), and all patients were biochemically cured. Of 88 DSR-positive tumors, 57 (65%) were staged N0, and 31 (36%) were staged N1(LN positive; Fisher's exact test: P = 0.0001). In a comparison of the LN involvement to the DSR characteristics, 32 (36%) of the 89 N0 patients and none of the 31 N1 patients were DSR negative. The sensitivity of DSR in predicting N0 is 38% (95% confidence interval: CI 95% = 27 %-55%), the specificity is 100% (CI 95% = 88%-100%). The sensitivity and specificity for the parameter DSR to differentiate between N0 or N1 were 100% and 36%, respectively. CONCLUSIONS: The desmoplastic stromal reaction appears to be an excellent intraoperative marker to predict LN involvement with a high specificity but low sensitivity. We therefore propose to avoid initial lateral neck dissection in MTC patients without DSR. PMID- 16680601 TI - Isthmus-preserving total bilobectomy: an adequate operation for C-cell hyperplasia. AB - BACKGROUND: Autopsy studies show that C cells deriving from the ultimobranchial body and migrating into the thyroid do not reach the isthmus region and are distributed along the vertical axes of thyroid lobes. This was confirmed in a surgical series of 58 patients (34 with preoperatively normal and 24 with elevated serum calcitonin) where no calcitonin-positive cells were demonstrable immunohistochemically within separately investigated isthmi. Consequently, isthmus-preserving total bilateral lobectomy (IPTB) may be regarded as an adequate surgical procedure for C-cell hyperplasia (CCH). PATIENTS AND METHODS: IPTB was performed from October 2001 to December 2004 in 64 patients, 59 patients with nodular goiter and slightly to moderately elevated serum calcitonin (stimulated under 500 pg/ml) (group A, apparently sporadic cases) and in 5 patients undergoing prophylactic surgery for hereditary medullary thyroid carcinoma (MTC) with intermediate- or low-risk RET mutations (non-634) (group B). The surgical procedure focused on meticulous total extracapsular resection of both thyroid lobes, preservation of an isthmus remnant of about 3 ml (smaller in children), and histologic workup of the border zones of resection in addition to that of the completely removed lobes. When malignancy could be proven intraoperatively (7 patients) or when the isthmus turned out to contain nodular lesions (4 patients), completion total thyroidectomy (plus lymphadenectomy) was performed as a one-stage procedure. Second-stage total thyroidectomy was performed in 3 cases. Thus, IPTB was the definitive surgical procedure in 50 patients (45 of group A and all 5 of group B). RESULTS: In all of the 50 definite IPTB cases, postoperative serum calcitonin was below the measurable limit (2 pg/ml); stimulated calcitonin was below the measurable limit in 47 (including all of group B) and was measurable in 3 sporadic cases in a lower-normal range between 2.4 and 3.5 pg/ml. Genetic screening of the apparently sporadic cases with CCH was positive in one (codon 791). The risk of recurrent laryngeal nerve paralysis seems not to be elevated (0% permanent); permanent hypocalcemia occurred in 1 patient (2%). Follow-up data of 37 patients, median 18 (6-36) months, showed continuously nonmeasurable serum calcitonin with one exception, where it was in the normal range after 18 months. All IPTB patients are still under substitution therapy with L-thyroxine (median 125 mug/day) with decreasing tendency in all 3 children after prophylactic operation, the latter also showing an increasing volume of well-vascularized isthmi (from 1.5 to 2.5 ml). CONCLUSION: IPTB reliably removes all C cells. There may not be need for total thyroidectomy (TTx) in cases with CCH. When necessary, completion TTx can be performed easily without additional risk. IPTB leaves a functionally relevant remnant, corresponding to that of a subtotal resection. This might be of importance especially for prophylactic surgery in children where the isthmus can compensate for the loss of thyroid function with time. PMID- 16680602 TI - Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress? AB - BACKGROUND: Adrenocortical carcinoma (ACC), a rare and aggressive malignancy, accounts for up to 14% of adrenal incidentalomas. The only chance of cure for ACC is diagnosis at an early stage; therefore, a main indication for adrenalectomy in patients with adrenal incidentaloma has been the potential risk of ACC. Recent studies suggest that this has led to earlier stage of ACC at diagnosis, more curative operations, and better survival. METHODS: We analyzed data on ACC from The National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database. Four equal time quartiles (1973-1979, 1980-1986, 1987-1993, and 1994-2000) were compared for changes in demographics, pathology, treatment, and cause-specific mortality. RESULTS: The average age was 51.2 years (range: 1-97), and 45.9% of patients were men. The average tumor size was 12 cm (range: 2-36 cm), and only 4.2% were < or = 6 cm. Most (88%) patients had surgical resection of their tumor, and external beam radiotherapy was used in only 12% of patients. Between the time quartiles compared (as well as annually), there was no significant difference at presentation in age at diagnosis, sex, race/ethnicity, tumor size, tumor grade, the frequency of distant metastasis, and overall TNM stage. Low tumor grade, lower stage of ACC, later time quartile, and surgical resection were associated with a lower cause-specific mortality by univariate analysis (P < or = 0.002) and by multivariate analysis (P < or = 0.031). CONCLUSIONS: Although adrenal incidentalomas have become a common indication for adrenalectomy, this has not resulted in patients with ACC being diagnosed earlier or treated at a lower stage of disease at the national level. The most important predictors of survival in these patients are tumor grade, tumor stage, and surgical resection. PMID- 16680603 TI - Selective use of adrenal venous sampling in the lateralization of aldosterone producing adenomas. AB - INTRODUCTION: It has been suggested that routine adrenal venous sampling (AVS) is necessary to lateralize an aldosterone-producing adenoma in patients with primary hyperaldosteronism. However, the success rate of AVS is variable, with potential risks. We review our experience at University of California San Francisco (UCSF), where AVS is used only selectively, to determine outcomes with this approach. METHODS: All patients undergoing adrenalectomy for aldosteronoma at UCSF from January 1995 to October 2004 were included. Outcome after adrenalectomy was determined based on plasma levels of aldosterone and potassium, rates of persistent hypertension, and reduced use of antihypertensive medications. RESULTS: Altogether, 65 patients were included in the study, 52 (80%) of whom had their adrenal tumors lateralized based on computed tomography scans, magnetic resonance imaging, or both. The remaining 13 (20%) patients had doubtful localization of their lesions on imaging. We did not routinely perform AVS in patients with definitive imaging findings. Thus, only 4 (8%) patients with definitive imaging findings underwent AVS, and one was unsuccessful. Of the 13 patients with doubtful lateralization on imaging, 8 underwent AVS. With this practice, biochemical cure rates after adrenalectomy were up to 100%, and hypertension resolved or was improved in 85% of patients. CONCLUSIONS: AVS may be performed selectively only when preoperative imaging cannot definitively lateralize the aldosteronoma. This practice in our center has resulted in high cure rates. During the era of improved imaging resolution and experience, mandatory routine AVS is not necessary to achieve high cure rates for aldosteronomas. PMID- 16680605 TI - Long-term outcome following laparoscopic adrenalectomy for large solid adrenal cortex tumors. AB - INTRODUCTION: Laparoscopic adrenalectomy (LA) is the procedure of choice for small benign adrenal tumors. In the absence of local invasion or metastases, the preoperative diagnosis of an adrenocortical carcinoma (ACC) is difficult, often leaving size as the principal predictor of malignancy. Large tumors are resectable laparoscopically, but the long-term outcome and therefore appropriateness of LA for cortical tumors > 6 cm is not known. METHODS: We reviewed the LA experience in our institution since its introduction in June 1994. Patients who underwent LA for solid cortical tumors > or = 60 mm in diameter without preoperative or intraoperative evidence of malignancy were reviewed. Follow-up data, including clinical examination, biochemical analysis, and repeat scans, were reviewed for evidence of local or systemic recurrent disease. RESULTS: Between 1994 and 2004 a total of 462 adrenalectomies were performed, 391 of which were done laparoscopically. Among them, 19 were solid cortical tumors > or = 60 mm in diameter with no overt malignant preoperative or intraoperative characteristics: 9 nonsecreting tumors, 8 Cushing's syndrome tumors (including 2 virilizing variants), 1 virilizing tumor, and 1 aldosteronoma. The mean age of the patients was 49.9 years (range 22-77 years), and the mean tumor size was 69.0 mm (range 60-80 mm). Histology confirmed a cortical adenoma in eight patients, malignant tumors in three, and indeterminate tumors in eight. The mean follow-up was 34 months (range 4-108 months). Two patients died of systemic recurrent disease (liver metastases) at 10 and 19 months, respectively, following surgery; two other patients died 12 and 21 months, respectively following surgery owing to unrelated cardiovascular and cerebrovascular pathology. One patient underwent surgery for local recurrence 54 months after primary surgery; the remaining 14 patients are well with no clinical or radiologic evidence of recurrent disease. CONCLUSIONS: Laparoscopic adrenalectomy for large solid cortical tumors without pre- or intraoperative evidence of malignancy is not contraindicated, and it is unlikely to have a deleterious effect on long-term outcome. Each case should be considered individually. We provide an algorithm for the approach to adrenocortical tumors > or = 6 cm. PMID- 16680606 TI - In reappraisal of the bidet, nearly half a century later. PMID- 16680607 TI - Comparison of POSSUM scoring systems and the surgical risk scale in patients undergoing surgery for complicated diverticular disease. AB - PURPOSE: This study was designed to evaluate the accuracy of the Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity, Portsmouth-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity, colorectal-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity, and the Surgical Risk Scale for the treatment of patients with complicated diverticular disease. METHODS: Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity variables were prospectively recorded for 324 patients undergoing colorectal resections in 42 hospitals in the United Kingdom from January to December 2003. The accuracy of each model was evaluated by measures of discrimination, calibration, and subgroup analysis. RESULTS: The overall operative mortality was 10.8 percent (Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity-estimated mortality rate, 21.9 percent; Portsmouth Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity-estimated mortality rate, 10.5 percent; colorectal-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity-estimated mortality rate, 10 percent; Surgical Risk Scale-estimated mortality rate, 38.2 percent). Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity and the Surgical Risk Scale over-predicted mortality in young patients (P < 0.001) and Portsmouth-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity underpredicted mortality in elderly patients (P < 0.001). Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity and the Surgical Risk Scale overpredicted mortality in patients with generalized peritonitis (Hinchey III and IV). There was no significant difference between the observed and colorectal-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity predicted mortality across patient subgroups and when the overall sample was considered. CONCLUSIONS: The study suggested a lack of calibration of Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity, Portsmouth-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity, and the Surgical Risk Scale at the extreme of age and for patients with severe peritoneal contamination. Colorectal-Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity was found to accurately evaluate mortality arising from complicated diverticular disease. PMID- 16680608 TI - Rehabilitation Medicine Summit: Building Research Capacity-Executive Summary. AB - The general objective of the "Rehabilitation Medicine Summit: Building Research Capacity" was to advance and promote research in medical rehabilitation by making recommendations to expand research capacity. The five elements of research capacity that guided the discussions were (1) researchers; (2) research culture, environment, and infrastructure; (3) funding; (4) partnerships; and (5) metrics. The 100 participants included representatives of professional organizations, consumer groups, academic departments, researchers, governmental funding agencies, and the private sector. The small group discussions and plenary sessions generated an array of problems, possible solutions, and recommended actions. A post-Summit, multiorganizational initiative is called to pursue the agendas outlined in this report. PMID- 16680609 TI - Robotic upper-limb neurorehabilitation in chronic stroke patients. AB - This pilot study tested the effectiveness of an intense, short-term upper-limb robotic therapy for improvement in motor outcomes among chronic stroke patients. We enrolled 30 subjects with upper-limb deficits due to stroke of at least 6 mo duration and with a Motor Power Assessment grade of 3 or less. Over 3 wk, 18 sessions of robot-assisted task-specific therapy were delivered with the use of a robotic exercise device that simulates a conventional therapy known as skateboard therapy. Primary outcome measures included reliable, validated impairment and disability measures of upper-limb motor function. Statistically significant improvements were observed for severely impaired participants when we compared baseline and posttreatment outcomes (p < 0.05). These results are important because they indicate that improvement is not limited to those with moderate impairments but is possible among severely impaired chronic stroke patients as well. Moderately and severely impaired patients in our study were able to tolerate a massed-practice therapy paradigm with intensive, frequent, and repetitive treatment. This information is useful in determining the optimal target population, intensity, and duration of robotic therapy and sample size for a planned larger trial. PMID- 16680610 TI - Response to upper-limb robotics and functional neuromuscular stimulation following stroke. AB - Twelve moderately to severely involved chronic stroke survivors (>12 mo) were randomized to one of two treatments: robotics and motor learning (ROB-ML) or functional neuromuscular stimulation and motor learning (FNS-ML). Treatment was 5 h/d, 5 d/wk for 12 wk. ROB-ML group had 1.5 h per session devoted to robotics shoulder and elbow (S/E) training. FNS-ML had 1.5 h per session devoted to functional neuromuscular stimulation (surface electrodes) for wrist and hand (W/H) flexors/extensors. The primary outcome measure was the functional measure Arm Motor Ability Test (AMAT). Secondary measures were AMAT-S/E and AMAT-W/H, Fugl-Meyer (FM) upper-limb coordination, and the motor control measures of target accuracy (TA) and smoothness of movement (SM). ROB-ML produced significant gains in AMAT, AMAT-S/E, FM upper-limb coordination, TA, and SM. FNS-ML produced significant gains in AMAT-W/H and FM upper-limb coordination. PMID- 16680611 TI - Recovery of cohesion in narrative discourse after left-hemisphere stroke. AB - Little is known about the recovery of narrative discourse after stroke. While several studies have analyzed the recovery of language in individuals with aphasia, few known studies exist on the recovery of narrative discourse in stroke survivors, particularly those who have not been diagnosed with aphasia. In this study, we evaluated the cohesiveness of narrative discourse in a cohort of individuals who had suffered a left-hemisphere stroke and had not been diagnosed with expressive language impairment. We analyzed their narrative discourse at 1, 6, and 12 months poststroke. Our findings indicate that, while the mean number of cohesive ties in narrative discourse remained generally constant during the first year poststroke, the percentage correct use of cohesive ties increased significantly during the same time period. These findings suggest that subtle disruptions in expressive language can be present initially in narrative discourse, and recovery from these disruptions can occur naturally over time. PMID- 16680612 TI - Quality-of-life predictors for caregivers at 1 and 6 months poststroke: Results of path analyses. AB - In this study, weused path analyses to test a theoretical model of influences on quality of life (QOL) for stroke caregivers at 1 and 6 months poststroke. We examined data from two points in time to determine the influence of stroke survivor and caregiver characteristics on QOL for stroke caregivers. Stroke survivor and caregiver characteristics had some direct influence on QOL outcomes for stroke caregivers at both 1 and 6 months poststroke. However, the most influential factor at both time points on each QOL component was sense of coherence (SOC). SOC is a relatively new factor in QOL and caregiver literature and is essentially the ability of caregivers to mobilize their coping resources during periods of stress. Caregivers who were able to mobilize these resources effectively experienced less burden in four QOL components and fewer symptoms of depression. PMID- 16680613 TI - Key characteristics of walking correlate with bone density in individuals with chronic stroke. AB - Several recent studies of ambulatory stroke survivors have shown decreased bone mineral density (BMD) in the lower limbs and an elevated risk of hip fracture. Because bone mass is linked to skeletal loading, weight-bearing activities of daily living such as walking are considered critically important for maintenance of femoral BMD in ambulatory individuals. Little is known about the relationships between walking characteristics, skeletal loading, and bone maintenance in individuals who have experienced a stroke. This study determined whether certain gait-related parameters correlate with proximal femoral BMD in ambulatory individuals with poststroke walking deficits. We analyzed data from 33 individuals with chronic stroke and found that a recently introduced metric, the Bone Density Index, which incorporates body weight, number of steps per day, and ground reaction force magnitude, predicted proximal femoral BMD better than other commonly measured demographic and gait-related parameters that we examined. PMID- 16680614 TI - Constraint-induced movement therapy for recovery of upper-limb function following traumatic brain injury. AB - A volunteer sample of 22 participants with chronic traumatic brain injury (TBI) (onset >1 year) and relative hemiplegia that revealed moderate disability in the more-affected upper limb (UL) participated. Constraint-induced (CI) movement therapy (CI therapy) was employed for a 2-week period; treatments included massed practice, shaping of the more-affected UL, behavioral contracts, and other behavioral techniques for affecting transfer to a real-world setting. We used the Wolf Motor Function Test, the Fugl-Meyer Motor Performance Assessment, and the Motor Activity Log to measure outcomes. All outcome measures improved significantly as a result of the intervention. More-adherent participants had more improvement compared with less-adherent participants. These preliminary results suggest that CI therapy may be effective for improving UL motor function following chronic TBI. PMID- 16680615 TI - Efficacy of multidisciplinary treatment program on long-term outcomes of individuals with Parkinson's disease. AB - We examined the impact of multidisciplinary clinical management of the Parkinson's Disease Research, Education, and Clinical Center program on Parkinson's disease progression. Initial and follow-up scores on the Part III Motor Examination subscale of the Unified Parkinson's Disease Rating Scale (UPDRS) were examined. Overall, 37 (75.5%) of the 49 patients demonstrated stable or improved UPDRS motor scores at 1- to 3-year follow-up; in the 1-year group (n = 28), 22 patients (78.6%) improved, while 6 (21.4%) worsened. In the 2-year group (n = 15), 10 (66.7%) improved, while 5 (33.3%) worsened. In the 3-year group (n = 6), 5 (83.3%) improved, while 1 (16.7%) worsened. Multidisciplinary interventions included neurology (95.9% of patients), physiatry (93.9%), nursing (87.8%), psychology (42.9%), medication changes (59.2% increases, 18.4% decreases), rehabilitation therapies (physical, occupational, speech-language, 67.3%), functional diagnostic testing (18.4%), support group (16.3%), home exercise instruction (85.7%), and disease and wellness education (81.6%). Improved and worsened patients did not significantly differ on the individual program components. Clinical implications and study limitations are discussed. PMID- 16680616 TI - Effectiveness of muscle vibration in modulating corticospinal excitability. AB - This study explored the effect of vibration of the forearm extensors on motor cortical excitability and the influence of stimulus duration. Sixteen healthy volunteers between 23 and 42 years old participated in one or two studies. We applied 15 or 30 min of 100 Hz, 0.5 mm-amplitude vibration to the extensor carpi radialis longus (ECRL) muscle. Cortical excitability was measured as the magnitude of the motor-evoked potentials (MEPs), and the size of the representation area associated with ECRL and flexor carpi ulnaris muscles was determined with the use of transcranial magnetic stimulation. A 33% increase in MEP size and enlarged area of cortical excitability was detected 5 min after 15 min of vibration in the ECRL only. No changes were associated with 30 min of vibration in either muscle. These findings indicate that the facilitatory effects of vibration in healthy subjects depend on stimulus duration and provide impetus for testing the extent to which short-duration vibration augments corticospinal excitability to improve muscle function in people with central motor disorders. PMID- 16680617 TI - Efficacy of shock-absorbing versus rigid pylons for impact reduction in transtibial amputees based on laboratory, field, and outcome metrics. AB - Prosthetic manufacturers have marketed shock-absorbing pylons (SAPs) for attenuation of injurious loads from foot-ground contact. In this study, we compared a commonly prescribed SAP with a conventional rigid pylon, using a within-subject design (n = 15 unilateral transtibial amputees), to assess effect on gait mechanics, measure transmitted accelerations in situ, and determine functional outcomes using step counts and questionnaires. No differences were found across pylons for self-selected walking speed, prosthetic-side step length, prosthetic-side loading rate and decelerative peak of the vertical ground reaction force, peak pylon acceleration, step count per week, or questionnaire results that examined pylon performance and subjects' pain and fatigue levels. The only statistically significant finding was for the prosthetic-side knee angle at initial contact, where subjects displayed an average of 2.6 degrees more flexion with the rigid pylon than the SAP while walking at a controlled speed (p = 0.004); this result indicates that transtibial amputees are able to modulate the effective stiffness of their residual limb in response to changes in prosthetic component stiffness. The results from the laboratory, field, and subjective outcome measurements suggest that the SAP in this study is as effective as a rigid pylon for unilateral transtibial amputees. PMID- 16680618 TI - Foot and ankle ligament morphometry. AB - This study primarily measured and established a morphometric spectrum of cross sectional areas for foot and ankle ligaments with the use of a new freeze fracture technique that was independent of ligament cross-sectional shape. Ligament morphometry measurements were made on a total of 121 bone-ligament-bone preparations that were harvested from 26 fresh (unembalmed) male cadaver feet. We used the traditional digital caliper method to measure length and the freeze fracture technique to measure cross-sectional area. Cross-sectional area values ranged from 21.36 to 170.48 mm(2) and length values from 5.01 to 37.45 mm. In addition, area-to-length ratios ranged from 0.60 to 27.02 mm. Compared with the freeze-fracture technique, the digital caliper method, which assumed that ligament cross-sectional shape was rectangular, resulted in an approximately 35% difference in cross-sectional area. PMID- 16680619 TI - Nerve conduction topography in geriatric hand assessment. AB - Motor nerve conduction is a noninvasive clinical test used to diagnose nerve problems such as carpal tunnel syndrome or peripheral neuropathy. Current techniques use a single-site recording over a superficial muscle. This traditional approach does not account for the electrical contributions from the other muscles innervated by the nerve being stimulated, which need to be considered with thumb carpometacarpal (CMC) degenerative joint disease (DJD) because these electrical contributions may change the anatomic relationship of the thenar muscles. This study recorded from 15 sites over the thenar eminence during motor nerve conduction studies of the median nerve of 12 young subjects with normal thenar anatomy and 25 elderly subjects with thumb CMC DJD. The maximum compound muscle action potential (CMAP) values did not occur in the same electrode position for the two groups, and traditional single-site recording would have resulted in smaller amplitudes and longer latencies for the elderly than the values noted with the multiple-site recordings. This pilot study of nerve conduction topography mapping with multiple-site recording illustrates that single-site studies may be misleading and supports further exploration of multichannel grid electrodes for topographic display and analysis of the CMAP. PMID- 16680620 TI - Neurocognitive enhancement therapy with work therapy: Productivity outcomes at 6- and 12-month follow-ups. AB - Neurocognitive enhancement therapy (NET), which involves computerized cognitive training and other methods, has been shown to improve working memory and executive function in schizophrenia. In the present study, 145 outpatients with diagnoses of schizophrenia or schizoaffective disorder recruited from a Department of Veterans Affairs mental hygiene clinic and from a community mental health center were ran domized to 6 months of paid work therapy (WT) or to NET+WT. Mixed random effects analyses revealed significant increase in hours worked and money earned over time for both conditions (p < 0.0001). NET+WT worked more hours than WT (p < 0.03), with differences emerging after rehabilitation. Responders to NET+WT worked the most during follow-up and tended to have more competitive-wage employment. Results indicate that work outcomes were enhanced by NET training. Effects were greatest for NET responders. Findings support the efficacy of cognitive training when it is integrated into broader rehabilitation programs. PMID- 16680621 TI - Use of 35 words for evaluation of hearing loss in signal-to-babble ratio: A clinic protocol. AB - Data from earlier studies that presented 70 words at 24 to 0 dB signal-to-babble (S/B) ratios indicated that most young listeners with normal hearing required 0 to 6 dB S/B ratios to attain 50% correct word recognition. Older listeners with hearing loss often required a >12 dB S/B ratio to attain 50% correct word recognition. In our study, we converted the Words in Noise test from one 70-word list into two 35-word lists for quicker administration by clinicians. Using baseline data from previous studies, we used two strategies to randomize the 35 word lists: based on recognition performance at each S/B ratio and based on recognition performance only. With the first randomization strategy, the 50% correct word-recognition points on the two lists differed by 0.5 dB for 72 listeners with hearing loss. With the second randomization strategy, 48 listeners with hearing loss performed identically on the two lists. PMID- 16680622 TI - Issues in maintenance and repairs of wheelchairs: A pilot study. AB - In this pilot study, we assessed wheelchair durability and its effect on user satisfaction. Specifically, we examined the characteristics of the participants' wheelchairs, the types of maintenance and repairs completed, and whether the participants' satisfaction was affected by problems with their wheelchairs. A convenience sample of 130 participants who used wheelchairs as their primary means of mobility was recruited. Participants completed a questionnaire about their wheelchairs, the maintenance and repair history, and their satisfaction levels. Results showed that 26% of the participants had completed a wheelchair repair in the past 6 months, 16% had completed general maintenance, and 27% had completed tire repairs. Neither hours of wheelchair use nor wheelchair age affected repair or maintenance frequency. Participants were generally satisfied with their wheelchairs. Better understanding of wheelchair maintenance and repair issues will guide improvements in wheelchair design and enhance the community participation of individuals who use wheelchairs. PMID- 16680624 TI - [The link between forensic and general psychiatry]. PMID- 16680625 TI - [For and against: increase of compulsory admissions of psychiatric patients]. PMID- 16680623 TI - [Involuntary medication in psychiatry]. AB - OBJECTIVE: A systematic review should take into account epidemiological, clinical, ethical, and legal aspects. METHOD: Search for literature was performed by Medline and Medpilot, guidelines were obtained via the internet or from own ongoing research projects. RESULTS: Epidemiological data on the frequency of involuntary medication is only scarcely available. The incidence on psychiatric wards seems to be between 2 % and 8 % of patients, predominantly with schizophrenic or bipolar disorder. In general, the efficacy of involuntary medication has been poorly evaluated in inpatients, two randomised controlled trials from the U. S. are available regarding involuntary outpatient commitment including involuntary medication. Therefore, the empirical database on aspects of clinical safety of involuntary medication is quite small. However, a considerable body of literature exists with respect to patients' opinions on involuntary medication, mostly demonstrating a highly critical attitude. Some European countries already have guidelines, but the overall situation suggests the need for further improvement. In 2004, the EU Council ratified a guideline for future European legislation on involuntary treatment of people with serious mental illness (white paper). DISCUSSION: A very controversial discussion of former years has become more reasonable, ideological anti-psychiatric criticism being increasingly replaced by search for evidence, development of guidelines, clearer legislation, and a tendency of European harmonization. PMID- 16680626 TI - [Burden of siblings of inpatients with schizophrenia]. AB - OBJECTIVE: Previous studies dealing with effects of psychiatric illness on patients' families mostly focussed on concerns of the parents. The siblings' role as a caregiver, however, has been mostly disregarded or forgotten about. METHOD: 37 narrative interviews with siblings of schizophrenia patients were analysed using a summarizing content analysis. The global statements and categories were quantitatively analysed to assess their relative importance. RESULTS: Analysis of the 492 individual statements of the siblings revealed 26 global types of statements which were assigned to five categories: 1. "Burden arising in the daily contact with the sibling" (36.2 %), 2. "Burden with respect to the healthy sibling's privacy" (26.8 %), 3. "Burden with respect to the contact with the family" (15.7 %), 4. "Burden with respect to the contact with institutions and professionals" (14.2 %), 5. "Burden with respect to the siblings' own social contacts (friends/public)" (7.1 %). The three types of burden most reported by the healthy siblings are: "Handling the symptoms of illness" (100 %), "Emotional burden due to the illness of the sibling" (100 %) and "Uncertainty in judging what amount of stress the schizophrenia patient can cope with" (81.1 %). CONCLUSIONS: Siblings of schizophrenia patients are burdened in many aspects. More attention should be given to their outpatient and inpatient medical treatment. PMID- 16680627 TI - [Patient knowledge regarding Clozapine-induced agranulocytosis]. PMID- 16680628 TI - [Compulsory treatment under guardianship legislation]. PMID- 16680629 TI - [100 years Albert Hofmann: LSD--miracle drug and problem child, Basel, January 13 15, 2006]. PMID- 16680631 TI - Three-dimensional endorectal ultrasound using a new freehand software program: results in 35 patients with rectal cancer. AB - BACKGROUND AND STUDY AIMS: This paper describes experience in the staging of rectal cancer using a new software program for three-dimensional endoscopic ultrasonography (EUS) that works without electromagnetic sensors and can be used even with electronic radial or linear rectal probes. MATERIALS AND METHODS: From May 2003 to March 2004, 35 three-dimensional endorectal ultrasound (ERUS) examinations were carried out using this program. The indication for ERUS was local staging of rectal cancer in all cases. The three-dimensional software imaging program forms part of a new ultrasound scanning system (Hitachi 6500 or 8000) and allows reconstruction of the two-dimensional EUS images in six different scans. RESULTS: Thirty-five rectal cancers were assessed using two dimensional and three-dimensional EUS. Using two-dimensional imaging, it was not possible to assess precisely the degree of involvement of the mesorectum (more or less than 50%). No differences were evident with three-dimensional EUS for superficial tumors (T1 and T2N0), but in six of 15 patients classified as having T3N0 lesions, three-dimensional EUS revealed malignant lymph nodes, a finding that was confirmed surgically in five of the six cases. Three-dimensional EUS also made it possible to assess the degree of infiltration of the mesorectum precisely in all cases, demonstrating complete invasion of the mesorectum in eight cases. These findings were confirmed in all cases by the surgical data. Two dimensional EUS correctly assessed 25 of the 35 rectal tumors (71.4%) in relation to the T and N classifications, and three-dimensional EUS increased this figure to 31 correct evaluations out of 35 (88.6%). CONCLUSION: Three-dimensional ERUS is easy to carry out using this new software program. There is no need for an external sensor mounted at the tip of the probe, and manipulation of the rectal probe is facilitated. Three-dimensional ERUS can be carried out using linear and radial electronic probes with the same ultrasound equipment. Three-dimensional ERUS allows more precise staging of lesions and better definition of the mesorectal margins, and this has a direct impact on therapeutic decision-making in patients with rectal cancer. PMID- 16680632 TI - Endoscopic ultrasound elastography: the first step towards virtual biopsy? Preliminary results in 49 patients. AB - BACKGROUND AND STUDY AIMS: It is well known that some diseases, such as cancer, lead to changes in the hardness of tissue. Sonoelastography, a technique that allows the elasticity of tissue to be assessed during ultrasound examination, provides the ultrasonographer with important additional information that can be used for diagnosis. The aim of this study was to evaluate the ability of endoscopic ultrasound elastography to differentiate between benign and malignant pancreatic masses and lymph nodes. PATIENTS AND METHODS: During a 12-month period, 49 patients underwent endoscopic ultrasound (EUS) examinations with elastography, conducted by a single endoscopist. Twenty-four patients underwent evaluation of a pancreatic mass (mean diameter 24.7 +/- 11.1 mm) and 25 underwent evaluation of 31 lymph nodes. The mean diameter of the lymph nodes was 19.7 +/- 8.6 mm, and they were found in the cervical area (n = 3), mediastinum (n = 17), celiac arterial trunk region (n = 5), and aortocaval region (n = 6). RESULTS: The sonoelastography images of pancreatic masses were interpreted as benign in four cases and malignant in 20. The sensitivity and specificity of sonoelastography in the diagnosis of malignant lesions were 100% and 67%, respectively. The sonoelastography images of the lymph nodes were interpreted as showing malignancy in 22 cases, benign conditions in seven, and indeterminate status in two. The sensitivity and specificity of sonoelastography for evaluating malignant lymph node invasion were 100% and 50%, respectively. CONCLUSIONS: EUS elastography is potentially capable of further defining the tissue characteristics of benign and malignant lesions but specifity has to be improved. It can be used to guide biopsy sampling for diagnosis. PMID- 16680633 TI - Early morbidity of endoscopic ultrasound: 13 years' experience at a referral center. AB - BACKGROUND AND STUDY AIM: Endoscopic ultrasonography (EUS) now has an important place in the diagnosis of gastroenteropancreatic diseases. However, prospective data on the morbidity and mortality related to its use are sparse and often retrospective. We attempted to assess the acute and immediate complications of both diagnostic and interventional EUS. PATIENTS AND METHODS: At our university affiliated tertiary care referral center, immediate (occurring during the procedure) and acute (occurring within 24 hours) complications of EUS were prospectively investigated. Over a first period, spanning 10 years, complications of diagnostic EUS involving 3207 consecutive patients were assessed. During the second period of 3 years, complications observed after EUS-guided fine-needle aspiration (FNA) biopsy were evaluated from 224 procedures. EUS was mostly done with the patient under sedation with intravenous propofol and spontaneous ventilation, and complications were evaluated by both the operator and the anesthesiologist. RESULTS: There were no deaths, and no surgery was required over the two periods of assessment. Three mild complications occurred among patients who underwent standard diagnostic EUS: two immediate complications were related to anesthesia and one to the procedure. There were five complications associated with interventional EUS; all were related to the procedure (acute pancreatitis, duodenal perforation, upper digestive bleeding, cyst, and mediastinal infection), with a mean delay of occurrence of 30 hours, and mean duration of hospitalization of 7 days. CONCLUSION: In our experience, which is the longest reported in Europe, the morbidity rates of diagnostic EUS and EUS-guided FNA biopsy were 0.093% and 2.2%, respectively, with no mortality. PMID- 16680634 TI - Endoscopic ultrasound drainage of pancreatic pseudocyst: a prospective comparison with conventional endoscopic drainage. AB - BACKGROUND AND STUDY AIMS: Pancreatic pseudocysts are a complication in up to 20% of patients with pancreatitis. Endoscopic management of pseudocysts by a conventional transenteric technique, i. e. conventional transmural drainage (CTD), or by endoscopic ultrasound-guided drainage (EUD), is well described. Our aim was to prospectively compare the short-term and long-term results of CTD and EUD in the management of pseudocysts. PATIENTS AND METHODS: A total of 99 consecutive patients underwent endoscopic management of pancreatic pseudocysts according to this predetermined treatment algorithm: patients with bulging lesions without obvious portal hypertension underwent CTD; all remaining patients underwent EUD. Patients were followed prospectively, with cross-sectional imaging during clinic visits. We compared short-term and long-term results (effectiveness and complications) at 1 and 6 months post procedure. RESULTS: 46 patients (37 men) underwent EUD and 53 patients (39 men) had CTD. The mean age of the entire group was 50 +/- 13 years. There were no significant differences between the two groups regarding short-term success (93% vs. 94%) or long-term success (84% vs. 91%); 68 of the 99 patients completed 6 months of follow-up. Complications occurred in 19% of EUD vs. 18% of CTD patients, and consisted of bleeding in three, infection of the collection in eight, stent migration into the pseudocyst in three, and pneumoperitoneum in five. All complications but one could be managed conservatively. CONCLUSIONS: No clear differences in efficacy or safety were observed between conventional and EUS-guided cystenterostomy. The choice of technique is likely best predicated by individual patient presentation and local expertise. PMID- 16680635 TI - Propofol sedation during endoscopic procedures: safe and effective administration by registered nurses supervised by endoscopists. AB - BACKGROUND AND STUDY AIMS: Propofol has several attractive properties, including a rapid onset of action and rapid recovery. However, the administration of propofol sedation in the absence of anesthesiologists remains controversial. This report describes the safety profile of propofol sedation for endoscopy when administered by registered nurses under the supervision of endoscopists. PATIENTS AND METHODS: The study was conducted in the endoscopic center of a Japanese private hospital. With assistance from an anesthesiologist, a protocol for administration of propofol by registered nurses was developed. Over the past 6 years, 27,500 patients received nurse-administered propofol sedation. The safety and patient satisfaction with this sedation procedure were evaluated. RESULTS: Among the participating patients, 6.7% developed hypoxemia (Sp(O2) < 90%); 6.2% required oxygen administration via a nasal cannula. Severe hypoxemia (Sp(O2) < 85%) occurred in 121 patients (0.62%) during upper gastrointestinal endoscopy and 20 patients (0.25%) during colonoscopy, but neither mask ventilation nor endotracheal intubation was necessary. A decline in blood pressure (systolic blood pressure < 90 mm Hg) was seen in 3.5% of the colonoscopy patients and 1.2% of the upper endoscopy patients. However, hypotension was corrected immediately using an intravenous saline solution. Patients who received propofol sedation expressed overall satisfaction on a 10-point visual analogue scale (with an average of 9.4 points). Among patients who had previously received a combination of midazolam and pethidine for colonoscopy, 85% preferred propofol sedation. The mean time from the end of the procedure to full recovery was 14.6 min. CONCLUSIONS: Administration of propofol by registered nurses under the supervision of endoscopists was safe, and resulted in high rates of patient satisfaction. PMID- 16680636 TI - Deep sedation with propofol for upper gastrointestinal endoscopy in children, administered by specially trained pediatricians: a prospective case series with emphasis on side effects. AB - BACKGROUND AND STUDY AIMS: The need to administer procedural sedation to children has increased in recent years, as has experience in this field among nonanesthesiologists. Using propofol makes it easier to achieve sufficiently deep sedation. There is a considerable literature on the administration of propofol by nonanesthesiologists for gastroscopy in adults, but very few data are available on this issue in children. The aim of the present study was to assess the safety and efficacy of procedural sedation with propofol for gastroscopy in a pediatric ward with trained personnel and monitoring facilities. PATIENTS AND METHODS: A training protocol was developed to educate nurses and residents. Children requiring gastroscopy were included in the study prospectively and underwent procedural sedation with propofol administered by nonanesthesiologists. RESULTS: A total of 811 upper gastrointestinal endoscopies were carried out with procedural sedation. Sedation was achieved in all procedures, and all but three (0.4%) were conducted successfully. None of the patients required intubation. Stridor with signs of upper airway obstruction occurred in 14 of the 811 procedures (1.7%). Laryngoscopy was required to manage difficulties in introducing the gastroscope in 16 of the 811 procedures (2.0%). Major desaturation requiring a short course of ventilation occurred in six procedures (0.7%), and transient desaturation that resolved spontaneously occurred in 97 of the procedures (12%). CONCLUSIONS: Administration of propofol by nonanesthesiologists for gastroscopy examinations in children was successful in this study, but was associated with a small risk of potentially severe complications. Although the residents were generally able to administer procedural sedation alone, constant and immediate availability of anesthesiological support continues to be mandatory. PMID- 16680637 TI - Accuracy of laryngeal examination during upper gastrointestinal endoscopy for premalignancy screening: prospective study in patients with and without reflux symptoms. AB - BACKGROUND AND STUDY AIMS: Gastroesophageal reflux disease may be associated with laryngeal damage caused by reflux material. The aim of this study was to investigate the accuracy of laryngeal examinations during routine upper gastrointestinal endoscopy as a method of screening for major laryngeal injury in a series of patients with reflux symptoms. PATIENTS AND METHODS: A total of 100 consecutive patients with reflux symptoms and 100 control individuals underwent upper gastrointestinal endoscopy with standard or high-resolution magnifying video endoscopes. Any laryngeal abnormalities were initially identified by the gastroenterologist before the scope was inserted into the esophagus. All of the examinations were recorded on video and subsequently reevaluated by an otorhinolaryngologist. All of the patients underwent standard laryngoscopy as a reference procedure. RESULTS: All of the patients completed the study. The sensitivity, specificity, negative predictive value, and positive predictive value of the preliminary laryngeal exploration for detecting laryngeal abnormalities (such as laryngitis and vocal leukoplakia) were 90%, 90%, 92%, and 89%, respectively. High-resolution magnifying endoscopy had a higher sensitivity and specificity than standard endoscopy for detecting laryngeal pathology. Two patients were found to have laryngeal leukoplakia. CONCLUSIONS: This study shows that preliminary diagnosis of laryngeal disorders can be accurately carried out by the gastroenterologist when patients are undergoing upper endoscopy for reflux symptoms. This approach could also be helpful for the timely diagnosis of major reflux-related laryngeal disease. PMID- 16680638 TI - Direct-vision stenting: the way forward for malignant oesophageal obstruction. AB - BACKGROUND AND STUDY AIMS: Self-expanding metallic stents now form the mainstay of treatment for palliation of dysphagia in oesophageal cancer. These stents are generally inserted under fluoroscopic guidance. However, both the internal and external marking of the tumour can be inaccurate and time-consuming, and access to fluoroscopic facilities is sometimes limited. We prospectively assessed the use of a method of stent insertion under direct vision without the aid of fluoroscopy. PATIENTS AND METHODS: A total of 50 consecutive patients presenting with obstructive symptoms secondary to inoperable oesophageal cancers were included in the study. We used either the 7-cm or the 11-cm covered Choo stent (MI-Tech Ltd., Seoul, South Korea). RESULTS: A total of 52 stents were inserted under direct vision. The procedure generally took less than 15 minutes and good palliation was achieved without complications. Fluoroscopic assistance was required in only one patient. CONCLUSIONS: Direct-vision stent insertion is simple, safe, effective, and only rarely requires fluoroscopic assistance. The technique may be of particular use in centres with limited access to fluoroscopy. PMID- 16680639 TI - Video capsule endoscopy in peutz-jeghers syndrome: a blinded comparison with barium follow-through for detection of small-bowel polyps. AB - BACKGROUND AND STUDY AIMS: Video capsule endoscopy has shown promise for the surveillance of significant small-bowel polyps in patients with Peutz-Jeghers syndrome. In this study the yield of significant small-bowel polyps by capsule endoscopy was compared to the yield of these polyps by barium follow-through in adults with Peutz-Jeghers syndrome. PATIENTS AND METHODS: A total of 19 adults with Peutz-Jeghers syndrome who were undergoing surveillance underwent both capsule endoscopy and barium follow-through examinations, and the number of small bowel polyps of at least 1 cm in diameter that were detected was recorded for each method. The two methods were assessed in terms of comfort and convenience for the patients and their preferred future surveillance method, using a questionnaire. RESULTS: Capsule endoscopy detected a median of four significant small-bowel polyps while barium follow-through detected a median of one (P = 0.008). There was a trend towards more participants having at least one significant small-bowel polyp detected by capsule endoscopy than was the case with barium follow-through, though capsule endoscopy seemed to be less reliable for accurate sizing of polyps that were at the limit of clinical significance. Most participants expressed a preference for capsule endoscopy for future surveillance. CONCLUSIONS: Capsule endoscopy should replace barium follow-through for the surveillance of small-bowel polyps in adults with Peutz-Jeghers syndrome, but the findings of this study should be interpreted with caution until a more reliable means of sizing polyps by capsule endoscopy is available. PMID- 16680640 TI - Clinical and endoscopic characterization of depressed gastric adenoma. AB - BACKGROUND AND STUDY AIM: Depressed gastric adenoma remains poorly characterized because it is rare, and is infrequently detected by endoscopy. The aim of this study was to elucidate clinical and endoscopic characteristics of depressed adenoma of the stomach. PATIENTS AND METHODS: 95 consecutive patients who underwent endoscopic resection of gastric adenomas were studied. Gastric adenomas, diagnosed according to the Vienna classification, were endoscopically classified into two types: depressed and protruding adenomas. In order to clarify endoscopic features of gastric adenomas, we performed indigo carmine chromoendoscopy as well as magnifying endoscopy with narrow band imaging, which yields clear images of mucosal microvasculature. RESULTS: 12% of 100 gastric adenomas resected from 95 patients were depressed adenomas. Age and gender were comparable between patients with each type. Depressed adenomas (15.9 +/- 6.2 mm) were significantly larger in diameter than protruding adenomas (10.6 +/- 8.0 mm) (P = 0.01). Half of depressed adenomas were reddish in color, whereas only 18% of protruding adenomas were reddish. Magnifying endoscopy with narrow band imaging showed that 71% of depressed adenomas had a regular ultrafine network pattern of mucosal microvasculature, which was not seen in protruding adenomas. Intramucosal carcinomas were more frequently found in depressed adenomas (25%) than in protruding adenomas (4.5%). CONCLUSIONS: In comparison with protruding adenomas, depressed adenomas were rare and appeared endoscopically as large and reddish with a specific regular ultrafine network pattern of mucosal microvasculature. Depressed adenomas should be endoscopically resected because intramucosal carcinomas were found in a quarter of them. PMID- 16680641 TI - Push-and-pull enteroscopy using a single-balloon technique for difficult colonoscopy. AB - BACKGROUND AND STUDY AIMS: Several factors predict difficult or incomplete colonoscopy with a standard colonoscope, including female gender, low body mass index, extensive diverticulosis, and prior pelvic surgery. PATIENTS AND METHODS: A push-and-pull enteroscopy device and a single-balloon technique was used in 14 patients (six men, eight women; mean age 62 +/- 15 years) after failure of ileocolonoscopy or endoscopic therapy using either a standard or a pediatric colonoscope. The original ileocolonoscopy had been performed to investigate colon polyps (n = 6), an obscure inflammatory process in the ileocecal region (n = 6), or iron deficiency anemia with a positive fecal occult stool test (n = 2). RESULTS: Ileocolonoscopy was successfully performed using this push-and-pull technique in all 14 patients without technical problems or complications. All the endoscopic therapeutic interventions that were required were also performed without complications, with the exception of one instance of bleeding. Multiple polyps were found in six patients; a colon cancer was found in one patient; appendicitis combined with a carcinoid tumour of the appendix was diagnosed in one patient; ileocolitis was observed in two patients; one patient with Crohn's disease had stenoses in the region of an anastomosis; and in three patients no relevant pathological finding was seen. CONCLUSION: It was possible to perform ileocolonoscopy with therapeutic interventions using this new thin push-and-pull enteroscopy device with a single-balloon technique in patients who had previously undergone incomplete colonoscopy using a standard colonoscope. PMID- 16680643 TI - Video capsule endoscopy in small-bowel malignancy: a multicenter Belgian study. AB - BACKGROUND AND STUDY AIMS: Early diagnosis of small-bowel tumors is crucial for therapy. Video capsule endoscopy has improved the diagnosis of small-bowel diseases, but data concerning the role of this technique in detecting small-bowel malignancy are scarce. The aim of this paper was to review all capsule endoscopy findings at Belgian hospitals, in order to evaluate the diagnostic yield of capsule endoscopy in the field of small-bowel malignancy. PATIENTS AND METHODS: For this retrospective study, the seven Belgian academic hospitals where the device was being used were asked to review the findings obtained by means of video capsule endoscopy, and to collect information about the cases of small bowel malignancy. RESULTS: In total, 443 capsule endoscopies were performed up to November 2004, and 11 malignant small-bowel processes were detected (2.5%). The most frequent indications for performing capsule endoscopy in those 11 cases were intestinal bleeding of undefined origin or iron-deficiency anemia. The mean number of diagnostic procedures performed before capsule endoscopy was 3.6. The capsule endoscopy results had a diagnostic yield of 1.6% after classical work-up. In 55% of these cases, capsule endoscopy findings had an influence on therapy. CONCLUSIONS: Tumors of the small bowel remain a rare condition. Video capsule endoscopy is able to detect tumors undiagnosed by classical procedures in about 1.6% of cases and has an impact on the therapy in 55% of the tumor cases. PMID- 16680642 TI - Endoscopic ultrasound-guided interstitial brachytherapy of unresectable pancreatic cancer: results of a pilot trial. AB - BACKGROUND AND STUDY AIMS: Intraoperative interstitial brachytherapy has been found to be effective when used during laparotomy to improve local control in patients with locally advanced pancreatic cancer. In this study, we report the results of using endoscopic ultrasound- (EUS-)guided interstitial brachytherapy in patients with advanced pancreatic cancer, with respect to tumor response, clinical response, safety, and complications. PATIENTS AND METHODS: Fifteen patients (eight men, seven women; median age 61 years) with unresectable pancreatic adenocarcinoma were enrolled into the study, eight patients with stage III disease and seven patients with stage IV disease. A mean number of 22 radioactive seeds per patient were implanted into the tumors by EUS-guided needle puncture. The mean total implanted activity was 20 mCi, the minimum peripheral dose was 14,000 cGy, and the mean volume of implants was 52 cm3. Patients were followed-up by examination and by imaging tests every 2-3 months: clinical end points included the Karnofsky performance status and pain responses, tumor response (assessed by computed tomography and/or EUS), and survival. RESULTS: During a median follow-up period of 10.6 months, the objective tumor response was classified as "partial" in 27% of patients (with a median duration of partial response of 4.5 months), "minimal" in 20% patients, and indicative of "stable disease" in 33% of patients. Clinical benefit was shown in 30% of patients, mostly due to reduction in pain, but this lasted for a limited time. Local complications (pancreatitis and pseudocyst formation) occurred in three patients; grade III hematologic toxicity occurred in three patients without serious clinical sequelae. CONCLUSIONS: EUS-guided intraoperative interstitial brachytherapy had a moderate local tumor effect and showed some clinical benefit in 30% of the patients in this study. Combination of this form of treatment with external radiation and/or chemotherapy should be tested in future trials. PMID- 16680645 TI - Blue clouds and green clouds: virtual biopsy via EUS elastography? PMID- 16680644 TI - Histopathologic characteristics of gastric ulcers created by endoscopic submucosal dissection. AB - BACKGROUND AND STUDY AIM: We have previously reported that gastric ulcers artificially created by endoscopic submucosal dissection (ESD) would heal within 8 weeks regardless of size and location. The details of the healing process remain unclear, and we aimed to clarify the mechanism by histopathological investigation. PATIENTS AND METHODS: 21 post-ESD ulcers were examined histopathologically, using surgically resected specimens from patients who had subsequently undergone gastrectomy due to submucosal invasion and/or lymphovascular infiltration by the tumor. The grade of ulcer, appearance of regenerative mucosa, scar formation, and extent of fibrosis were evaluated. RESULTS: Fibrosis and wall thickening were observed from 2 weeks after ESD, but regenerative mucosa was not observed until 5 weeks. Among 12 patients who underwent gastrectomy later than 8 weeks after ESD, a mucosal defect was still observed in two patients. In these two patients the lesion was associated with severe fibrosis due to previous peptic ulcer or submucosal invasion by the lesion. CONCLUSION: Size reduction in these ulcers occurs by contraction in the early phase, then regenerative mucosa covers the remaining mucosal defect within 8 weeks. If there is fibrosis under the lesion before ESD, there is a possibility that the artificially created ulcer will not heal within 8 weeks. PMID- 16680646 TI - Transanal endoscopic microsurgery: a review. PMID- 16680647 TI - Endoscopic clip application for postoperative residual esophageal duplication cyst. PMID- 16680648 TI - Live video demonstrations. PMID- 16680649 TI - Causes of non-attendance at an open-access endoscopy outpatient clinic. PMID- 16680650 TI - Iatrogenic perforation at therapeutic colonoscopy: should the endoscopist attempt closure using endoclips or transfer immediately to surgery? PMID- 16680651 TI - Controversy concerning the cutoff value for depth of submucosal invasion after endoscopic mucosal resection of early gastric cancer. PMID- 16680653 TI - Fatal pancreatitis after endoscopic balloon dilation for extraction of common bile duct stones in an 80-year-old woman. PMID- 16680654 TI - Splenic infarction: an unusual complication of ethanolamine oleate injection. PMID- 16680655 TI - Newly developed endotriptor for patients with basket impaction in the distal common bile duct. PMID- 16680656 TI - A novel maneuver for colonic polypectomy at the 6-o'clock position. PMID- 16680657 TI - Fatal superior vena cava syndrome after endoscopic variceal sclerotherapy. PMID- 16680658 TI - Combined endoscopic transgastric and transpapillary drainage of an infected biloma. PMID- 16680659 TI - A new technique for releasing a stuck loop. PMID- 16680660 TI - Endoscopic band ligation of active cardiac variceal bleeding. PMID- 16680661 TI - [Jorg Bohler, December 15, 1917 to December 11, 2005]. PMID- 16680662 TI - [Experiences with the distally based dorsal metacarpal artery (DMCA) flap and its variants in 41 cases]. AB - Distally based DMCA flaps are an established procedure for soft tissue reconstruction in the hand. Since the introduction of the DMCA flaps, several variants have been developed to match specific defect requirements. The aim of this retrospective study is to give an overview of the experience with the DMCA flaps based on a single center's experience. 41 distally based DMCA flaps have been performed from 2000 to 2004. Twenty-nine times a distally based DMCA flap has been performed, nine times the extended distally based DMCA flap and three times a distally based DMCA fascial flap. In summary, 34 flaps showed no complications, five had a partial necrosis, but were successfully treated with split-thickness skin graft, and two flaps were lost due to infections. The distally based DMCA flaps II to IV have proven to be reliable flaps, but nevertheless require sufficient surgical experience. They are suitable for any kind of defects, including burn reconstruction. Defects of the entire finger can be covered by the various variants of the DMCA flaps. PMID- 16680663 TI - [Open reduction and internal fixation of distal radius extension fractures in women over 60 years of age with the dorsal radius plate (pi-plate)]. AB - PURPOSE: The goal of this study was to review the clinical and radiological results and to correlate them with the functional outcome score DASH in women over 60 years who underwent open reduction and internal fixation with a pi-plate for displaced distal radius fractures. METHOD AND CLINICAL MATERIAL: In this retrospective study, forty-nine women over 60 years old with mainly intra articular distal radius fractures underwent open reduction and internal fixation with a pi-plate. The dorsal approach was used permitting an anatomical reduction of the articular surface under direct vision of the radio-carpal joint. Patient follow-up was for a minimum of twelve months with a mean of 32 months. RESULTS: For 71 % of patients, there were no limitations in their daily activities and only one patient complained of a major disability. Overall, the flexion of the wrist was reduced by 15 degrees compared to the opposite side (mean 50 degrees). The DASH score (14.4) was not higher than that of a comparable age controlled group without radius fracture. The pi-plate was demonstrated to be a stable implant in osteoporotic bone and there was only one secondary dislocation. Most patients were painfree with two patients describing tolerable pain, one because of a progressing radio-carpal osteoarthritis and the second due to a neuroma of the superficial branch of the radial nerve. The other major complication was a late rupture of the extensor tendon of the index finger in one of the twelve patients where the pi-plate had not been removed. CONCLUSION: We recommend anatomical reduction and internal fixation for displaced intra-articular distal radius fractures using a dorsal approach to achieve a good wrist function including painless load bearing with the implant removed four months after surgery. PMID- 16680664 TI - [Interposition arthroplasty for the treatment of primary osteoarthritis: comparison of results using the technique of Epping and Weilby]. AB - We compare clinically and radiologically two groups of total trapezectomy performed as treatment for the primary osteoarthritis of trapeziometacarpal joint combined in 83 cases (2000 to 2003) with tendon interposition arthroplasty type Weilby, in 51 cases (1987 to 1991) with Epping tendon interposition and ligament reconstruction arthroplasty in a retrospective study. In both groups we used a strip of the flexor carpi radialis tendon. The mean follow-up time in both groups is 33 months. Overall, both methods achieved similar results. The relief of pain and the patient satisfaction are better, stability and grip force are worse in the Weilby group. At least one year (12 to 57 months) after operation with the technique of Weilby, 88 % of the patients would prefer this method for the other side. It appears that there is no clinical and radiological benefit of ligament reconstruction. However, the two procedures studied yield equally good results in most cases, but we prefer the simple technique of Weilby and guess that the (missing) long-term results would be superior to the results after trapezectomy alone. Long-term results need to be evaluated. PMID- 16680665 TI - [Long-term results after resection arthroplasty in patients with arthrosis of the thumb carpometacarpal joint: comparison of abductor pollicis longus and flexor carpi radialis tendon suspension]. AB - PURPOSE/BACKGROUND: This retrospective analysis focused on a comparison of long term results in patients who underwent resection of the trapezium with subsequent arthroplasty and tendon suspension using either the abductor pollicis longus (APL) or the flexor carpi radialis (FCR) tendon. METHOD AND MATERIAL: Based upon a positive history along with a clinical and radiological examination, 20 patients underwent suspension arthroplasty using the APL tendon (APL group) and 21 patients suspension arthroplasty using the FCR tendon (FCR group) after trapeziectomy. In both groups mean age (APL group: 60.4 +/- 5.3; FCR group: 61.7 +/- 6.8 years), pain severity according to the Visual Analogue Scale (VAS; APL group: 6.7 +/- 1.9; FCR group: 6.9 +/- 1.7), severity of arthrosis in the thumb carpometacarpal joint according to the Eaton-Littler classification (APL group: 3 +/- 0.7; FCR group: 3.2 +/- 0.6) and time interval from onset of symptoms to surgery (APL group: 27 +/- 8.1; FCR group: 41.5 +/- 14.1 months) did not significantly differ. Each patient of both groups was treated surgically and reviewed by one experienced hand surgeon. Both groups received the same standardized postoperative treatment. RESULTS: In the APL group the mean operative time was significantly shorter (31.7 +/- 9.5 min) than in the FCR group (48.7 +/- 7.9 min). The follow-up period from surgery to the final examination was similar in both groups (APL group: 23.1 +/- 12.2; FCR group: 31 +/- 17.6 months). At the time of the final examinations, no statistically significant differences were found when analyzing the results of the DASH score (APL group: 20.1 +/- 15.1; FCR group: 29.3 +/- 15.7), the self-administered hand ability score (APL group: 1.7 +/- 0.6; FCR group: 2.1 +/- 0.6) and the VAS (APL group: 1.1 +/- 1.6; FCR group: 0.8 +/- 1.5). The time period from surgery to the offset of postoperative pain was also comparable in both groups (APL group: 5 +/- 1.8; FCR group: 5.3 +/- 2.5 months). The range of abduction in the first carpometacarpal joint after arthroplasty, parallel and perpendicular to the dorsum of the hand, was also similar in both groups (APL group: 63.4 +/- 14.3 degrees /62.1 +/- 11 degrees ; FCR group: 67.8 +/- 12.7 degrees /66 +/- 12.1 degrees ). However, patients enrolled in the APL group revealed significantly better results compared to patients in the FCR group regarding grip-strength, key and pinch grip (APL group: 23.9 +/- 9.7/6.6 +/- 2.4/6.2 +/- 2.8 kg; FCR group: 17 +/- 7.2/4.5 +/- 1.5/3.6 +/- 1.5 kg). CONCLUSION: Both techniques led to highly satisfactory results as seen in DASH and VAS data together with a near normal range of abduction in the first carpometacarpal joint in all enrolled patients. However, in direct comparison the APL procedure is technically easier to perform with significantly shorter surgery time recorded and significantly higher values in all force parameters compared to the FCR procedure. PMID- 16680666 TI - [Arthroscopy of the wrist: compared results of MRT and arthrography and outcome in the arthroscopy -- an examination 1998 to 2003]. AB - Between 1998 and 2004, 62 arthroscopies of the wrist were performed in the Vienna Danube Hospital. We examined if preoperative arthrography or magnet resonance tomography showed the same results concerning lesions of the discus ulnocarpalis or the scapholunate ligaments as did the arthroscopy and whether these two examinations are a good preoperative diagnostic instrument. PMID- 16680667 TI - [The potential of high-resolution sonography in the follow-up of surgically treated flexor tendons]. AB - Sonography of the hand is frequently used for diagnostics of hand diseases. Advantages are the lack of side effects (e. g., X-ray contamination) and easy accessibility. Soft tissues of the hand are easily examined using a linear-array transducer with a high frequency (7.5 to 15 MHz) and, in some cases, a pre positioned device (water or gel). Also there is the possibility to record functional motility of ligaments or joints non-invasively. The beneficial use of sonography is demonstrated on patients with injured flexor tendons of the hand or fingers. Ultrasound was used to examine ligaments after suture, for the diagnosis of suspected re-ruptures and in cases of fibrotic adhesions. In this study, the sonomorphology of previously injured flexor tendons with consecutive surgical reconstruction was examined in 47 patients. The average observation period was eight months post-operatively. 15 patients were submitted to a second operation. In 86.5 % of the cases, the sonographic results acquired prior to surgery were in agreement with macroscopic morphology seen during the operation. PMID- 16680668 TI - [Xanthoma disseminatum: a rare case involving the upper extremity]. AB - We describe a dramatic case of class II non-Langerhans cell histiocytosis, xanthoma disseminatum, in a 30-year-old male patient with progressive involvement of the skin, vocal cords, eyes, bones and nerves in spite of chemotherapy with Vespesid and immunotherapy with interferon-gamma. At the age of 43 years, the patient required surgical clearance of airways, eyelids and peripheral nerves, but at present exhibits stable disease on a combination of lipid-lowering drugs including thiazolidinedione. PMID- 16680669 TI - [Tourniquet syndrome -- case report]. AB - Tourniquet syndrome involves fibres of hair or thread wrapped around an appendage producing tissue necrosis. The result in this case and a review of the literature show that prompt recognition and aggressive treatment are necessary. PMID- 16680670 TI - [Chronic compartment syndrome of the flexor muscles in the forearm due to motocross]. AB - A case of a mechanic and motorcyclist is reported who developed unilateral chronic exertional compartment syndrome of the flexor muscles in the forearm. After years of discomfort and medical check-ups, a subcutaneous fasciotomy of the superficial compartments of the flexor muscles in the forearm led to a complete relief of symptoms, which allowed the patient unrestricted activity. PMID- 16680671 TI - [Ulnar sesamoid bone of the small finger causing painful trigger finger]. AB - We report on a 38-year-old woman suffering from painful trigger finger. Contrary to the expected intraoperative finding of a simple stenosing pulley and ganglion cyst on a thickened flexor tendon sheath, we found fibrotic cords between an abnormal ulnar sesamoid bone at the fifth finger and the A1-pulley to be responsible for distortion of the tendon sheath and a consecutive "klicking" phenomenon. A coherence between sesamoid bones and trigger finger has repeatedly been found on the thumb but there is no such description regarding the long fingers. PMID- 16680672 TI - [Painful tumour at the knee joint: case report and review of the literature]. AB - PURPOSE/BACKGROUND: Glomangiomas are perivascular tumours which, on the whole, are rare. They are most frequently located subungually on the fingers and toes. Diagnosis is difficult when they are atypically located at the knee joint. This may frequently result in a course of disease extending over many years. MATERIAL AND METHODS: The case of a 75-year-old male patient with a tumour which had existed for two years, located at the knee joint, is reported. RESULTS: Symptomatology, diagnostics, histology and therapy are presented and discussed in the context of the currently available literature relevant to this disease. In the case presented here, following removal of the tumour, the patient remained free of complaints and did not suffer a relapse. CONCLUSION: Glomangiomas exhibit local, invasive growth, metastases have not been reported. The clinical picture is characterised by the classical trilogy of sensitivity to pain, pressure and temperature. In cases associated with the knee joint, misdiagnoses such as meniscus lesions or degenerative changes, are frequently made. Diagnostic procedures include sonography and magnetic resonance investigations. A resection of the tumour should be carried out well into the healthy tissue, as local relapse is a frequent occurrence in this disease. PMID- 16680673 TI - [Limits of insight]. PMID- 16680674 TI - [Evidence-based medicine and psychotherapy: what are adequate research methods?]. AB - Evidence-based Medicine (EbM) was accepted during the last decade both as a method for systematically evaluating risks and benefits and a decision aid for therapists in practice. Randomized controlled trials (RCT) have become the gold standard for medical research. The medical community is sceptical about RCTs because many therapists are not convinced that care can be mirrored adequately enough, though. The doubts are especially strong among psychiatrists and psychotherapists. The paper argues in favour of EbM in two ways. Firstly, from a scientific point of view there are no reasons why RCTs could not contribute to improve the standards of care. Secondly, health services research doesn't amount to much more than RCTs. The importance of qualitative methods is a black box as large within health services research as the meaningful use of RCTs. PMID- 16680675 TI - [Methodology, method, and quality of qualitative research]. AB - Over the last ten years qualitative research approaches have been increasingly recognized and applied to the fields of psychotherapy and psychosomatic medicine in German-speaking countries, although this development has lagged behind what has occurred at the same time in English-speaking countries. In the former, especially, a misunderstanding prevails that qualitative research methods are at best preliminary to real science. This misunderstanding has roots in the philosophy of science and the sociology of knowledge. It comes from the lack of recognition that the human sciences, unlike the natural sciences, entail a double rather than single hermeneutic and that qualitative research methods are logically coherent ways of working systematically and rigorously with this double hermeneutic. Indeed, this nature of qualitative research is generally not well understood by many qualitative researchers themselves. In this article, the need for qualitative research is shown and its methodology is seen as an integration of method and hermeneutics, or methodical hermeneutics, which in turn is distinguished from alternative qualitative research methodologies. The article closes with considerations of quality criteria and publication standards. PMID- 16680676 TI - Crystal structure of the single-domain rhodanese homologue TTHA0613 from Thermus thermophilus HB8. PMID- 16680677 TI - Microspectroscopic fluorescence analysis with prism-based imaging spectrometers: review and current studies. AB - BACKGROUND: Fluorescence imaging spectroscopy is a powerful but under-utilized tool. This article gives perspective on the use of imaging spectroscopy, and provides two examples of imaging spectroscopy done with a prism-based system. The intent is to give insight into the power of imaging spectroscopy when used in combination with other imaging techniques. In particular, studies of intact coral photobleaching and beads designed to show energy transfer are reported. In the bead study, spectroscopic lifetime imaging was performed at each photobleaching step. RESULTS: Spectroscopic photobleaching of the hard coral, Montastrea annularis, revealed two spectral regions. A region in the red portion of the spectrum bleached rapidly while progressively increasing fluorescence was observed over a wide portion of the spectrum. This behavior is consistent with current theories for the role of fluorescent proteins in corals. Following a photobleaching study of beads designed to exhibit energy transfer with imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM) allowed unambiguous assignment of fluorescence resonance energy transfer (FRET). The data in this experiment indicated that most of the commonly used markers of FRET would have been inconclusive. The ability of the ISFLIM to look at all regions of the spectrum, particularly the acceptor region, allowed FRET to be assigned. CONCLUSIONS: Fluorescence imaging spectroscopy is a rapidly advancing technology, uniquely suited to the flexible detection of dyes over a wide range of wavelengths. PMID- 16680678 TI - Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane. AB - BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose. PMID- 16680679 TI - Use of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl as an EPR oximetry probe: potential for in vivo measurement of tissue oxygenation in mouse brain. AB - Measurement of oxygen concentration and distribution in the brain is essential for understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that potentially can be used to map O(2) concentration in the brain. We examined two nitroxides, 3-methoxycarbonyl-2,2,5,5 tetramethyl-1-pyrrolidinyloxyl [2] and 3-acetoxymethoxycarbonyl-2,2,5,5 tetramethyl-1-pyrrolidinyloxyl [3], as pro-imaging agents to deliver 3-carboxy 2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] across the blood-brain barrier (BBB). In primary cultured neurons, nitroxide [3] but not [2] was hydrolyzed by intracellular esterases to [1], which, being anionic at physiologic pH, was well retained intracellularly. In contrast, [2] was not well retained by neurons. In vivo pharmacokinetic and pharmacodynamic studies in mice suggested that esterase labile nitroxide [3] crossed the BBB, and was converted to [1] and retained. Retention occurred in brain tissue and not in the extensive vasculature, as evidenced by the fact that removal of blood by whole-body saline perfusion did not eliminate the nitroxide EPR signal from the brain. The EPR linewidths of [1] and [3] were more O(2)-sensitive than that of the commonly-used oximetry probe 4 oxo-2,2,6,6-tetramethylpiperidine-d(16)-1-(15)N-oxyl [4]. Moreover, we used [3] in vivo to estimate O(2) concentration in mouse brains. These results indicate that nitroxide [3] could be useful for mapping O(2) distribution in the brain following stroke. PMID- 16680680 TI - Biodegradation process of alpha-TCP particles and new bone formation in a rabbit cranial defect model. AB - The purpose of the present study was to observe the biodegradation process of pure alpha-tricalcium phosphate (alpha-TCP) particles and to determine the efficacy of alpha-TCP as a space maintainer in a bone defect. We used 14 rabbits and prepared two cranial bone defects in each rabbit. One defect was left empty as a control, whereas the other was filled with alpha-TCP particles about 300 mum in diameter. Animals were sacrificed at 1 week, 4 weeks, and 8 weeks. The cranial bone was then embedded either in paraffin wax for the preparation of decalcified specimens, or in polyester resin for the preparation of nondecalcified specimens. All specimens were evaluated histologically and histomorphometrically. As a consequence of the degradation of alpha-TCP, a "reticulate structure" appeared in the particles at 1 week and new bone was observed in this structure at 8 weeks. The amount of new bone between the control and experimental groups was not significantly different at any of the time points. However, in the experimental group, new bone at the surface of alpha-TCP was evident even in the center of the defect whereas fibrous connective tissue was dominant in the control group. These results indicate that alpha-TCP is a degradable osteoconductive material that is able to act as a space maintainer for bone regeneration when applied to a bone defect. While there was no significant difference in total bone formation between the experimental and negative control groups, the space-maintaining and osteoconductive properties of the particles may result in more complete bone formation in longer-term studies. PMID- 16680681 TI - Quantification of regional pulmonary blood flow using ASL-FAIRER. AB - Pulsed arterial spin labeling (ASL) techniques have been theoretically and experimentally validated for cerebral blood flow (CBF) quantification. In this study ASL-FAIRER was used to measure regional pulmonary blood flow (rPBF) in seven healthy subjects. Two general ASL strategies were investigated: 1) a single subtraction approach using one tag-control pair acquisition at an inversion time (TI) matched to the RR-interval, and 2) a multiple-subtraction approach using tag control pairs acquired at various TIs. The mean rPBF averaged 1.70 +/- 0.38 ml/min/ml when measured with the multiple-subtraction approach, and was approximately 2% less when measured with the single-subtraction method (1.66 +/- 0.24 ml/min/ml). Assuming an average lung density of 0.33 g/ml, this translates into a regional perfusion of approximately 5.5 ml/g/min, which is comparable to other measures of pulmonary perfusion. As with other ASL applications, a key problem with quantitative interpretation of the results is the physical gap between the tagging region and imaged slice. Because of the high pulsatility of PBF, ASL acquisition and data analysis differ significantly between the lung and the brain. The advantages and drawbacks of the single- vs. multiple-subtraction approaches are considered within a theoretical framework tailored to PBF. PMID- 16680682 TI - Anisotropic polyvinyl alcohol hydrogel for cardiovascular applications. AB - Polyvinyl alcohol (PVA) is a hydrophilic polymer with various characteristics desired for biomedical applications and can be transformed into a solid hydrogel by physical crosslinking, using a low-temperature thermal cycling process. As with most polymeric materials, the mechanical properties of the resultant PVA are isotropic, as oppose to most soft tissues, which are anisotropic. The objective of this research is to develop a PVA-based hydrogel that not only mimics the nonlinear mechanical properties displayed by cardiovascular tissues, but also their anisotropic behavior. By applying a controlled strain to the PVA samples, while undergoing low-temperature thermal cycling, we were able to create oriented mechanical properties in PVA hydrogels. The oriented stress-strain properties of porcine aorta were matched simultaneously by a PVA hydrogel prepared (10% PVA, cycle 3, 75% initial strain). This novel technique allows the controlled introduction of anisotropy to PVA hydrogel, and gives a broad range of control of its mechanical properties, for specific medical device applications. PMID- 16680683 TI - Real-time monitoring of the setting reaction of brushite-forming cement using isothermal differential scanning calorimetry. AB - The setting behavior of a brushite-forming cement (beta-tricalcium phosphate/mono calcium monophosphate) was investigated using an indentation technique (the Gillmore needles method) and isothermal differential scanning calorimetry (DSC). The two objectives of the study were to investigate whether DSC could be used to real-time monitor a fast-setting calcium phosphate cement (CPC) and to determine if it is possible to correlate DSC results directly with conventional setting time measurements. Best-fit linear correlation analysis revealed that both the initial and final setting time (T(i) and T(f)) measured by indentation were strongly correlated to the maximum heat flow measured with DSC. It seems therefore possible to predict the setting times, usually achieved with user dependent indentation methods, of this specific fast setting CPC on the basis of objective DSC measurements. The drawbacks of DSC, however, are its overall complexity and expense and the fact that only exothermal reactions can be investigated in comparison to the Gillmore needles method, furthermore, it is not possible to monitor the complete reaction as the first 2 or 3 min are lost due to sample preparation and apparatus set up. PMID- 16680684 TI - Surface integrity and removal mechanism in simulated dental finishing of a feldspathic porcelain. AB - Dental abrasive finishing of a fine-grained feldspathic porcelain was performed on a computer-assisted apparatus for simulation of a 2-degrees-of-freedom restorative operation with a dental handpiece and a coarse diamond bur of grit size of 106-125 mum. Finishing forces, surface roughness, and morphology were investigated as functions of finishing conditions. The tangential and normal forces were measured using a piezoelectric dynamometer and a data processing system. The results indicated that these forces increased with either the depth of cut or with the feed rate, in the ranges of 0.12-0.31 N and 0.45-1.09 N, respectively. However, an increase in either depth of cut or feed rate affected neither the surface roughness measured using a stylus profilometer nor the morphology observed under a scanning electron microscope. The finished porcelain surfaces were found to consist of the microfracture and chipping areas, ductile removal areas, smeared areas, and debris. Irregular fracture and chipping resulted from the extension of lateral/median cracks; ductile micromachining was attributed to the plastic deformation accompanied by distributed microcracks. It was determined that a combination of the microfracture and ductile micromachining was the primary mechanism for material removal. PMID- 16680685 TI - Contact stress assessment of conventional and highly crosslinked ultra high molecular weight polyethylene acetabular liners with finite element analysis and pressure sensitive film. AB - Stress magnitude and distribution of both conventional polyethylene versus a crosslinked polyethylene in the liner of a total hip replacement (THR) were examined using finite element analysis and pressure sensitive film. Both types of polyethylene were assessed against head sizes of 22 and 28 mm with 5-mm thick polyethylene liners and head sizes of 28, 38, and 46 mm with 3-mm thick polyethylene liners. Liners with 5-mm conventional polyethylene represented successful combinations with long track records. Our hypothesis was that although the combination of the large head and the lower modulus of the highly crosslinked polyethylene would lead to lower stresses, the stresses would be excessive if the liner was extremely thin at 3 mm. Von Mises stresses at the articulating surface of the highly crosslinked liners were lower, when compared to conventional polyethylene, in every THR size examined. Specifically, however, the 38- and 46 mm inner diameter (ID) highly crosslinked polyethylene even at the extreme of only 3-mm thick had lower stresses than the 22-mm ID conventional liner of 5-mm thickness. These data indicate that the use of a large head against highly crosslinked material even at 3-mm thickness results in lower stresses than in an existing conventional 22-mm head and 5-mm thick combination. Obviously, other considerations will influence the minimum thickness to be recommended. PMID- 16680686 TI - Calcium-deficient apatite synthesized by ammonia hydrolysis of dicalcium phosphate dihydrate: influence of temperature, time, and pressure. AB - In this work, calcium-deficient apatites (CDA) were synthesized by ammonia hydrolysis reaction of dicalcium phosphate dihydrate (DCPD; CaHPO4 x 2 H2O) to obtain biphasic calcium phosphates (BCP) without any extraionic substitution. The influence of three parameters was studied: temperature of the reaction (70 and 100 degrees C), time of the reaction (4 and 18 h), and the pressure (open and closed system). Experiments were made according to a factorial design method (FDM) allowing optimization of the number of samples as well as statistical analysis of results. Moreover, the influence of temperature (until 200 degrees C) was investigated. The crystal size of CDA was determined according to the Scherrer's formula and from Rietveld refinements taking the CDA anisotropy into account. The last method seems to be a reliable method to determine crystallite sizes of CDA, since crystallite sizes of CDA along <00l> and directions were accessible. The results describe the hydroxyapatite % (HA%) in BCP by a first-order polynomial equation in the experimental area studied and the HA content was found to increase by raising time and temperature of the reaction. Moreover, the type of reaction system (open/closed vessel) appeared to have little influence on HA%. PMID- 16680687 TI - System-wide analysis of hepatotoxicological responses: tissomics is key. AB - BACKGROUND: Combining diverse data streams across different levels of biological observation, such as molecular, cellular, and clinical chemistry responses, support a system-wide diagnostic approach. Recent progress in slide-based cytometry contributes to the development of tissomics, a high-throughput and high content phenotyping methodology that provides data-rich profiles of cellular heterogeneity in tissues enabling correlative statistical treatments over multiple scales of biological hierarchies. METHODS: Phenotypical data are covariants that can be used as biomarkers to identify relevant candidate genes by associating initiating molecular events with phenotypical changes and adverse outcomes. We introduce a procedure of combined statistical and analytical tools to identify and visualize such associations for nonpooled entities. The new utility is applied to a time-controlled, low-dose toxicological study including a control and two xenobiotic compounds. RESULTS: An integrated analysis identified specific molecular and phenotypical biomarkers, which support the classification of animals in the absence of any visual indicators from pathology readings. DISCUSSION: The introduction of controlled perturbations to tissues provides a prototypical setting to develop a sensitive, systems-based analysis methodology suitable for a broader range of biomedical applications. PMID- 16680688 TI - BOLD contrast sensitivity enhancement and artifact reduction with multiecho EPI: parallel-acquired inhomogeneity-desensitized fMRI. AB - Functional MRI (fMRI) generally employs gradient-echo echo-planar imaging (GE EPI) to measure blood oxygen level-dependent (BOLD) signal changes that result from changes in tissue relaxation time T(*) (2) between activation and rest. Since T(*) (2) strongly varies across the brain and BOLD contrast is maximal only where the echo time (TE) equals the local T(*) (2), imaging at a single TE is a compromise in terms of overall sensitivity. Furthermore, the long echo train makes EPI very sensitive to main field inhomogeneities, causing strong image distortion. A method is presented that uses accelerated parallel imaging to reduce image artifacts and acquire images at multiple TEs following a single excitation, with no need to increase TR. Sensitivity gains from the broadened T(*) (2) coverage are optimized by pixelwise weighted echo summation based on local T(*) (2) or contrast-to-noise ratio (CNR) measurements. The method was evaluated using an approach that allows differential BOLD CNR to be calculated without stimulation, as well as with a Stroop experiment. Results obtained at 3 T showed that BOLD sensitivity improved by 11% or more in all brain regions, with larger gains in areas typically affected by strong susceptibility artifacts. The use of parallel imaging markedly reduces image distortion, and hence the method should find widespread application in functional brain imaging. PMID- 16680689 TI - Removal of noncollagenous components affects dentin bonding. AB - The structural integrity of fibrillar type I collagen is critical for effective dentin bonding. Since most noncollagenous matrix components in dentin are closely associated with collagen, we hypothesized that they may also contribute to dentin bonding. To test this hypothesis, bovine dentin was acid-etched, treated with chondroitinase ABC (C-ABC), endo-beta-galactosidase (Endo-beta), or trypsin. Controls were prepared in the same manner but without the enzymes. All control and experimental specimens were then bonded with One-Step. Bond strength data were analyzed by one-way ANOVA and Fisher's PLSD test (p < 0.05). When dentin was treated with C-ABC or trypsin, bond strengths significantly decreased for the rewetted groups (p < 0.05). The treatment with Endo-beta showed no effects on bond strengths (p > 0.05). When the treated dentin surfaces were observed under SEM, the C-ABC and trypsin treated groups revealed significant loss of collagen fibril architecture. The results indicate that chondroitin sulfate glycosaminoglycans and trypsin-digestible noncollagenous proteins play roles in maintaining the open dimensions of the collagen fibril scaffold, which is essential for optimal dentin bonding. PMID- 16680690 TI - Hydrothermal crystallization of carbonate-containing hydroxyapatite coatings prepared by radiofrequency-magnetron sputtering method. AB - Carbonate-containing hydroxyapatite (HA) films were prepared by low-temperature hydrothermal annealing from carbonate-containing calcium phosphate amorphous coatings on titanium substrates. The biocompatibility of the carbonate-containing HA layers was estimated by in vitro tests using simulated body fluid (SBF). Precursory amorphous coatings were deposited with rf-magnetron sputtering apparatus, using calcium phosphate glass target in Ar/CO2 atmosphere. The carbonate-containing HA coatings were successfully formed by the annealing at above 130 degrees C for 20 h. On the basis of SEM observation, about 2-microm thickness films coated rigidly were durable enough for the hydrothermal treatment. The coating layer was revealed to consist of single phase of PO4(-) and OH- partially carbonated HA by XRD and IR analyses. Overgrowing of bone-like apatite layers on the carbonate-containing HA surfaces in the SBF implied that the obtained films acquired a sufficient osteoconductivity, while it was still unclear that activity was enhanced, compared to pure HA coatings. The low temperature hydrothermal annealing method was effective for preparation of rigid HA coatings on titanium as well as modification of their chemical compositions. PMID- 16680691 TI - Surface analysis and biocorrosion properties of nanostructured surface sol-gel coatings on Ti6Al4V titanium alloy implants. AB - Surfaces of biocompatible alloys used as implants play a significant role in their osseointegration. Surface sol-gel processing (SSP), a variant of the bulk sol-gel technique, is a relatively new process to prepare bioreactive nanostructured titanium oxide for thin film coatings. The surface topography, roughness, and composition of sol-gel processed Ti6Al4V titanium alloy coatings was investigated by atomic force microscopy (AFM) and X-ray electron spectroscopy (XPS). This was correlated with corrosion properties, adhesive strength, and bioreactivity in simulated body fluids (SBF). Electroimpedance spectroscopy (EIS) and polarization studies indicated similar advantageous corrosion properties between sol-gel coated and uncoated Ti6Al4V, which was attributed to the stable TiO2 composition, topography, and adhesive strength of the sol-gel coating. In addition, inductive coupled plasma (ICP) and scanning electron microscopy with energy dispersive spectrometry (SEM-EDS) analysis of substrates immersed in SBF revealed higher deposition of calcium and phosphate and low release rates of alloying elements from the sol-gel modified alloys. The equivalent corrosion behavior and the definite increase in nucleation of calcium apatite indicate the potential of the sol-gel coating for enhanced bioimplant applications. PMID- 16680692 TI - Protein adsorption onto polyester surfaces: is there a need for surface activation? AB - Surface hydrolysis of polyester scaffolds is a convenient technique suggested to promote protein adsorption for improving cell attachment. We have, therefore, investigated the effect of hydrolysis of polyester surfaces for protein adsorption to clarify the conditions needed. Three polyesters, poly(ethylene terephthalate) (PET), poly(lactic acid) (PLA), and poly(glycolic acid) (PGA), were selected. Adsorption was investigated by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and quartz crystal microbalance (QCM). Hydrolyzed PET adsorbed significantly more proteins than nonhydrolyzed. Degradable polymers adsorbed at higher rates when the polymers were hydrolyzed prior to adsorption, but the same amount as nonhydrolyzed, suggesting spontaneous hydrolysis during the adsorption. XPS shows that hydrolysis prior to absorption for PET results in a surface nitrogen composition of approximately 14%, similar to pure protein (16%). Nonhydrolyzed PET surfaces showed only approximately 7% nitrogen, indicating protein layers thinner than approximately 10 nm. Adsorption to PLA and PGA shows nitrogen contents of 14-15% in both cases. SEM revealed striking differences in morphology of the protein coating. Hydrolyzed or spontaneously hydrolyzable surfaces display a pronounced fibrous structure while nonhydrolyzed surfaces give smooth structures. In combination, the results show that surface hydrolysis increase adsorption rate, but not the amount of proteins on polyesters that degrades in vivo. Surface treatment of nondegradable polyester increases the total amount of proteins and induces the formation of fibrous protein structures. Post hydrolysis treatment by acetic acid, replacing the counter-ion to a proton, further enhances protein attachment. Finally, cell attachment experiments verifies that protein adsorption increase the cell attachment to polyester surfaces. PMID- 16680693 TI - Changes in scattering and absorption properties of esthetic filling materials after aging. AB - OBJECTIVES: Assuming that color changes after aging are related to changes in translucency of materials, the purpose of the present study was to determine the correlation between the changes in color and the changes in scattering and absorption properties after accelerated aging with representative dental esthetic restorative materials: glass ionomer, resin-modified glass ionomer, compomer, and resin composite. METHODS: Color was measured according to the CIELAB color scale in the transmittance and reflectance modes and used to calculate changes in color (deltaE*(ab)), color coordinates (deltaL*, delta a*, and delta b*), translucency parameter (deltaTP), scattering coefficient (deltaS), absorption coefficient (deltaK), and light reflectivity (deltaRI) after accelerated aging. Simple correlations between each pair of the changes in optical values were calculated, and multiple regression analysis was used to determine the parameters influencing the changes in color and color coordinates (p = 0.05). RESULTS: In the resin composite and compomer, deltaS, deltaK, and deltaRI values were approximately zero, whereas deltaS was as high as 8.9 in the glass ionomer. For most comparisons, correlation coefficient (r) was between 0.700 and 0.997. DeltaL* was found to have a major influence on color changes, and deltaS, deltaTP, and deltaRI influenced deltaL*. Therefore, changes in scattering and absorption properties, after aging, were closely correlated with changes in color and color coordinates, especially in glass ionomer-based filling materials. PMID- 16680694 TI - Hybrid layer seals the cementum/4-META/MMA-TBB resin interface. AB - Although 4-META/MMA-TBB resin has adhesive properties to dentin, and has been clinically used for the bonding treatment of vertically fractured roots and apicoectomy, there has not been any investigation on the adhesion of 4-META/MMA TBB resin to cementum. The purpose of this in vitro study was to evaluate the bonding and the sealing ability of 4-META/MMA-TBB resin to cementum. Bovine root cementum and dentin surfaces were treated with a citric acid and ferric chloride solution, and the 4-META/MMA-TBB resin was applied on the treated surfaces before testing. The microtensile bond strength and the leakage levels obtained for the cementum were almost equal to those for the dentin. In SEM and TEM observations, a hybrid layer approximately 2-3 microm in thickness was observed at the interface between the resin and the cementum. It is concluded that 4-META/MMA-TBB resin adhered to cementum via a hybrid layer on cementum, as previously reported for dentin. PMID- 16680695 TI - Surface modification of titanium-based alloys with bioactive molecules using electrochemically fixed nucleic acids. AB - A new method of surface modification for titanium (alloys) with bioactive molecules was developed with the intention of providing a new basis of implant adaptation for particular requirements of certain medical indications. Nucleic acid single strands are fixed electrochemically via their termini (regiospecifically) by growing an oxide layer on Ti6Al7Nb anodically. It could be shown that they are accessible to subsequent hybridization with complementary strands at physiological pH. Amount of nucleic acids immobilized and hybridized were determined radioanalytically using 32P-labelled nucleic acids. Stable fixation was attained at and above potentials of 4 V(SCE). Up to 4 pmol/cm2 of nucleic acid single strands could be immobilized and hybridization efficiencies up to 1.0 were reached. Hybridization efficiency was found to depend on surface density of immobilized oligonucleotides, while hybridization rates increased when MgCl2 was added. A conjugate consisting of an oligonucleotide complementary to the immobilized strand and the hexapeptide GRGDSP with RGD as an integrin recognition site was synthesized. This conjugate was able to bind to integrins on osteoblasts. It was shown that this conjugate binds to the anchor strand fixed on Ti6Al7Nb to an extent comparable with the unconjugated complementary strand. PMID- 16680696 TI - Infiltration/evaporation-induced shrinkage of demineralized dentin by solvated model adhesives. AB - During dentin bonding, solvated adhesive comonomers are applied to water saturated decalcified dentin matrices. When alcohol-solvated hydrophilic or hydrophobic methacrylate monomers are applied, they chemically remove water and cause matrix shrinkage during comonomer infiltration. Evaporation of solvent induces further shrinkage. The purpose of this work was to compare the shrinkage of water-saturated dentin matrices infiltrated with ethanol- or methanol-solvated 2-hydroxyethyl methacrylate (HEMA), 2,2-bis[4(2-hydroxy-3-methacryloyloxy propyloxy)-phenyl] propane (BisGMA), or triethyleneglycol dimethacrylate (TEGDMA) at 90/10, 70/30, 50/50, and 30/70 mass fraction % alcohol/monomer before and after evaporation of alcohol. Thin (ca 0.2 mm) disks of human mid-coronal dentin were demineralized and placed in a well beneath the contact probe of a linear variable differential transformer (LVDT). The height of the matrix was measured before and after random application of one of the twelve alcohol/monomer mixtures. Matrix height was measured during infiltration and during solvent evaporation. Between trials, residual monomer was extracted using ethanol. These studies were repeated on specimens in which 100% alcohol was used to substitute for water in the matrix. Both studies revealed that matrices shrink 30-50% but that pretreatment of matrices with alcohol prevents BisGMA phase separations from occurring. Wet bonding with ethanol instead of water permits infiltration of relatively hydrophobic alcohol/monomers. PMID- 16680697 TI - Detecting microcirculatory changes in blood oxygen state with steady-state free precession imaging. AB - Recently, it has been demonstrated that oxygen-weighted images of whole blood can be obtained with steady-state methods. In this article, based on computational and experimental models, we investigate the potential for employing this technique to monitor oxygen changes in microcirculation. Results show that oxygen sensitive images of rabbit kidney and muscle may be obtained at high signal-to noise ratio within a few seconds. The results also show that in steady-state free precession imaging, in addition to the exchange mechanism that generates oxygen contrast in blood, there are additional mechanisms that provide oxygen-sensitive contrast in microcirculation. PMID- 16680698 TI - Equivalences between refractive index and equilibrium water content of conventional and silicone hydrogel soft contact lenses from automated and manual refractometry. AB - PURPOSE: The purpose of the present study was to develop mathematical relationships that allow obtaining equilibrium water content and refractive index of conventional and silicone hydrogel soft contact lenses from refractive index measures obtained with automated refractometry or equilibrium water content measures derived from manual refractometry, respectively. METHODS: Twelve HEMA based hydrogels of different hydration and four siloxane-based polymers were assayed. A manual refractometer and a digital refractometer were used. Polynomial models obtained from the sucrose curves of equilibrium water content against refractive index and vice-versa were used either considering the whole range of sucrose concentrations (16-100% equilibrium water content) or a range confined to the equilibrium water content of current soft contact lenses (approximately 20 80% equilibrium water content). RESULTS: Values of equilibrium water content measured with the Atago N-2E and those derived from the refractive index measurement with CLR 12-70 by the applications of sucrose-based models displayed a strong linear correlation (r2 = 0.978). The same correlations were obtained when the models are applied to obtain refractive index values from the Atago N-2E and compared with those (values) given by the CLR 12-70 (r2 = 0.978). No significantly different results are obtained between models derived from the whole range of the sucrose solution or the model limited to the normal range of soft contact lens hydration. CONCLUSIONS: Present results will have implications for future experimental and clinical research regarding normal hydration and dehydration experiments with hydrogel polymers, and particularly in the field of contact lenses. PMID- 16680699 TI - Radial artery as an autologous cell source for valvular tissue engineering efforts. AB - To create a viable tissue-engineered aortic valve, it is important to identify suitable autologous cell sources that may be seeded onto a biocompatible scaffold. This study focused on the radial artery (RA) as one possible source, investigated optimal culture conditions, and determined the usefulness of small intestinal submucosa (SIS) as a scaffold for tissue-engineering. Porcine RA cells were cultured on either two-dimensional (2D) 100-mm dishes or three-dimensional (3D) 1-cm(2) SIS sheets, producing cell-scaffold composites (CSCs). Both 2D and 3D cultures were maintained in either Medium 199 (M199) or endothelial growth media (EGM) to determine optimal growth conditions. Cellular phenotype and matrix metalloproteinase (MMP) profiles were determined by immunoblotting of cell lysates and zymography of conditioned media, respectively. Cellular invasion was analyzed immunohistochemically on CSC tissue sections. We show that the RA contains phenotypes consistent with those found in the normal aortic valve. EGM, compared with M199, promotes the invasion and remodeling of SIS by RA cells, which is crucial in the process of replacing the foreign tissue scaffold prior to implantation. To our knowledge, this is the first study to show that the RA is a suitable source for the generation of a tissue-engineered valve. PMID- 16680700 TI - Biocompatibility of subsieve-size capsules versus conventional-size microcapsules. AB - Biocompatibility of cell-enclosing capsules, defined as suppression of pericapsular cellular reactions, is one of the factors governing the success of enclosed cell transplantation in in vivo cell therapy. Agarose capsules of subsieve size, less than 100 microm in diameter, and conventional size, approximately 300-1,000 microm in diameter, were implanted into the peritoneal cavity and epididymal fat pads of mice and rats, respectively, to determine the effect of a reduction in diameter to subsieve size. The degree of cellular reaction to the subsieve-size capsules was much lower than that of the conventional-size microcapsules, independent of implantation site. The frequency of overgrown subsieve-size capsules retrieved from the peritoneal cavities was less than 5% in contrast to approximately 20% for capsules 387 microm in diameter. In addition, no increase in floating cells, which are generated through capsule stimulation, was observed in the peritoneal cavity only with subsieve size capsules. From these results, we concluded that subsieve-size capsules are more biocompatible than microcapsules of conventional size. PMID- 16680701 TI - Personal cytometers: slow flow or no flow? AB - BACKGROUND: Although some manufacturers have optimistically described instruments with prices in the 40,000 US dollars range as "personal cytometers", analogy with the personal computer suggests that the target price for a true "personal" cytometer should be under 5,000 US dollars. Since such an apparatus could find a wide range of applications in cytomics in both developing and developed countries, it seemed desirable to consider its technical and economic feasibility. METHODS: Using resolution targets and a variety of fluorescent bead standards immobilized on filters and/or slides, we evaluated high-intensity LEDs as fluorescence excitation sources, relatively inexpensive CCD cameras as detectors, and 35 mm camera lenses and plastic low-power microscope optics for light collection in a simple, inexpensive low-resolution imaging cytometer. RESULTS: The components tested could be combined toproduce an instrument capable of detecting fewer than 10,000 molecules of cell-associated fluorescent label, and thus applicable to a broad range of cytometric tasks. CONCLUSIONS: Given the requirements for light sources, detectors, optics, mechanics, electronics and data analysis hardware and software, and the components presently available, it should be easier to reach the desired 5,000 US dollars price point with an image cytometer than with a flow cytometer. PMID- 16680702 TI - Extracting quantitative information from tissue--an industrial perspective. AB - The focus of this article is to provide an overview of the current technologies for the pharmaceutical and biotech industry. Disease processes express themselves in the functional and structural disturbance of cellular systems. Cells and their metabolites constitute the building blocks of tissues and entire organisms. Studying the spatial and temporal phenotype of disease processes in tissues at the cellular level reveals a multitude of information about the progress and status of a disease. Detailed exploration of tissues by slide-based cytometry is an important source of information about disease processes. Technological and analytical advances allow us to shed a new light on tissues and to come to a better understanding of the complexity of disease processes. Dealing with complex multidimensional datasets from tissue samples requires an advanced approach to image processing and data management. The increase in computing power and the continuing research into imaging algorithms allow us to improve the exploration of the data content of tissues. PMID- 16680703 TI - Spectral imaging perspective on cytomics. AB - BACKGROUND: Cytomics involves the analysis of cellular morphology and molecular phenotypes, with reference to tissue architecture and to additional metadata. To this end, a variety of imaging and nonimaging technologies need to be integrated. Spectral imaging is proposed as a tool that can simplify and enrich the extraction of morphological and molecular information. Simple-to-use instrumentation is available that mounts on standard microscopes and can generate spectral image datasets with excellent spatial and spectral resolution; these can be exploited by sophisticated analysis tools. METHODS: This report focuses on brightfield microscopy-based approaches. Cytological and histological samples were stained using nonspecific standard stains (Giemsa; hematoxylin and eosin (H&E)) or immunohistochemical (IHC) techniques employing three chromogens plus a hematoxylin counterstain. The samples were imaged using the Nuance system, a commercially available, liquid-crystal tunable-filter-based multispectral imaging platform. The resulting data sets were analyzed using spectral unmixing algorithms and/or learn-by-example classification tools. RESULTS: Spectral unmixing of Giemsa-stained guinea-pig blood films readily classified the major blood elements. Machine-learning classifiers were also successful at the same task, as well in distinguishing normal from malignant regions in a colon-cancer example, and in delineating regions of inflammation in an H&E-stained kidney sample. In an example of a multiplexed ICH sample, brown, red, and blue chromogens were isolated into separate images without crosstalk or interference from the (also blue) hematoxylin counterstain. CONCLUSION: Cytomics requires both accurate architectural segmentation as well as multiplexed molecular imaging to associate molecular phenotypes with relevant cellular and tissue compartments. Multispectral imaging can assist in both these tasks, and conveys new utility to brightfield-based microscopy approaches. PMID- 16680704 TI - Digital image analysis of cytoskeletal F-actin disintegration in renal microvascular endothelium following ischemia/reperfusion. AB - BACKGROUND: Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function. METHODS: Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phalloidin. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line scans. RESULTS: Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phalloidin fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine. CONCLUSIONS: The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF). PMID- 16680705 TI - Chemical images: technical approaches and issues. AB - Chemical mapping techniques using Raman microscopy are introduced, and using an example of a pharmaceutical tablet, the practical aspects of data collection and processing to produce a chemical image of the sample are detailed. Issues related to data processing, instrument standards, chemical image reportable errors, and the interpretation of chemical images are presented to encourage debate, develop solutions, and promote use in other challenging scientific applications. applications. PMID- 16680706 TI - Object-oriented image analysis for high content screening: detailed quantification of cells and sub cellular structures with the Cellenger software. AB - BACKGROUND: Detailed image analysis still is a considerable bottleneck for many cellular assays, and automated solutions to the problem are desirable. However, dealing with the complexity and variability of structures in cellular images makes detailed and reliable analysis a nontrivial task. METHODS: Therefore, based on the object-oriented image analysis approach, a novel image analysis technology, a flexible and reliable system for image analysis in cellular assays was developed. It contains a library of predefined, adaptable modules, each of them developed for a specific analysis task. The system can be configured easily by combining appropriate modules and adapting them interactively to the specific image data, if necessary. By representing cells and sub cellular structures within a network of interlinked image objects, a large number of parameters can be derived that describe shape, intensity, and relevant structural and relational aspects of any chosen class of structures. RESULTS: Thus, multi-parameter analysis and multiplexing are supported. A sample application based on this approach demonstrates that GFP signals can be distinguished based on their properties and the relative location within the cell. PMID- 16680707 TI - Multispectral/hyperspectral image enhancement for biological cell analysis. AB - BACKGROUND: Microscopes form projected images from illuminated objects, such as cellular tissue, which are recorded at a distance through the optical system's field of view. A telescope on a satellite or airplane also forms images with a similar optical projection of objects on the ground. Typical visible illuminations form a displayed set of three-color channels (Red Green Blue [RGB]) that are combined from three image sensor arrays (e.g., focal plane arrays) into a single pixel coding for each color present in the image. Analysis of these RGB color images develops a qualitative image representation of the objects. METHODS: Independent component analysis (ICA) is used for analysis and enhancement of multispectral images, and compared with the similar and widely used principal component analysis. RESULTS: The data examples indicate that the ICA enhancement, and the resulting RGB image combination display, can be useful in processing datacubes of cellular data where isolation of unknown subtle image elements representing objects is desired. CONCLUSIONS: ICA image enhancement can aid processing of datacubes of cellular data by clarifying subtle image elements. These parallelizable algorithms can be implemented for real-time, online analysis. PMID- 16680708 TI - Automated neurite labeling and analysis in fluorescence microscopy images. AB - BACKGROUND: To investigate the intricate nervous processes involved in many biological activities by computerized image analysis, accurate and reproducible labeling and measurement of neurites are prerequisite. We have developed an automated neurite analysis method to assist this task. METHODS: Our approach can be considered as automated with certain user interaction in setting initial parameters. Single and connected centerlines along neurites are extracted. The computerized method can also generate branching and end points. Owing to its multi-scale flexibility, both thick and thin neurites are simultaneously detected. RESULTS: We employ the relative neurite length difference (defined as the difference between the lengths obtained by automated and manual analysis divided by the total length of the latter) and neurite centerline deviation (defined as the area of the regions enclosed by different paths between automated and manual analysis divided by the total length of the former) to evaluate the performance of our algorithm, which is of great interest in neurite analysis. The average of the relative length difference is about 0.02, while the average of the centerline deviation is about 2.8 pixels. The probabilities of the distributions being the same from the Kolmogorov-Smirnov (KS) test of the automatic and manual results are 99.79%. The KS test also shows no significant bias between different observers based on the proposed new validation scheme. CONCLUSIONS: With the accurate and automated extraction of neurite centerlines and measurement of neurite lengths, the proposed method, which greatly reduces human labor and improves efficiency, can serve as a candidate tool for large-scale neurite analysis beyond the capability of manual tracing methods. PMID- 16680709 TI - Hyperchromatic cytometry principles for cytomics using slide based cytometry. AB - BACKGROUND: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. METHODS AND RESULTS: We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), (3) differential photobleaching (differentiating fluorochromes by their different photostability), (4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and (5) photodestruction (destruction of FRET dyes). Based on the ability to relocate cells that are immobilized on a microscope slide with a precision of approximately 1 microm, identical cells can be reanalyzed on the single cell level after manipulation steps. CONCLUSION: With the intelligent combination of several different techniques, the hyperchromatic cytometry approach allows to quantify and analyze all components of relevance on the single cell level. The information gained per specimen is only limited by the number of available antibodies and sterical hindrance. PMID- 16680710 TI - 3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry. AB - BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging. PMID- 16680711 TI - Distal symmetric polyneuropathy: limitations of the proposed case definition. PMID- 16680712 TI - Interactions of the C2 domain of human factor V with a model membrane. AB - Activated coagulation Factor V is an important cofactor of the coagulation cascade that catalyzes the formation of the prothrombinase complex on the surface of membranes rich in phosphatidyl-L-serine (PS). Here we report molecular dynamics simulations of the two crystallographic structures (the open and closed conformations) of domain C2 of coagulation Factor V (FaVC2). The calculations were performed in water (1.5 ns for each conformation) and in the presence of a neutral phospholipid bilayer model (POPE; 10 ns for each conformation) in order to describe the dynamics of the free (plasma circulating) and membrane bound forms of FaVC2. Water simulations confirmed the hypothesis that the plasma circulating form is in the closed conformation. In contrast, the membrane simulations showed that both conformations are energetically compatible with membrane binding. We have investigated the mechanism, the dynamics, and the energetics of the binding process. Our data are consistent with published estimates of the immersion depth of the aromatic residues (W26 and W27), and with mutagenesis studies involving specific residues located on the spikes at the bottom of the FaVC2 structure. Electrostatic interactions between the phospholipid head groups and hydrophilic residues at the bottom of the structure play a key role in the binding process by creating a large number of hydrogen bonds that anchor the protein to the membrane. The simulations identified a stable phospholipid binding pocket reminiscent of a previously suggested PS interaction site. Our structural data could contribute to the design of potential inhibitors able to disrupt membrane association. PMID- 16680713 TI - Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution. AB - Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general. PMID- 16680714 TI - Effect of nanostructure on biodegradation behaviors of self-setting apatite/collagen composite cements containing vitamin K2 in rats. AB - Apatite cement and collagen were combined by a mechanochemical method to create a new self-setting apatite/collagen composite cement, and menatetrenone (VK2) was loaded into a drug-delivery system to test biocompatibility in rats. Powder X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), and electron probe microanalyzer (EPMA) were performed to characterize the physicochemical properties of apatite/collagen composite cements. The XRD results suggested that ground apatite/collagen cement was completely transformed into bone-like hydroxyapatite, but that without grinding was incomplete. The SEM and EPMA results suggested that ground apatite/collagen cement was homogeneously dispersed of nanoapatite crystals in collagen matrices, similar to that in natural bone. In contrast, the cement without grinding was heterogeneously distributed. To evaluate in-vivo cement density (CMM), microradiograms were measured for 72 days after implanting apatite/collagen composite cements in intramuscular tissue on the backs of rats, and cross sections of the cements and surrounding soft tissues were observed by microscope. The CMM results of the apatite/collagen composite cements suggested that the biodegradation rate was dependent on the cement quality and nanogeometrical structure. The CMM result of VK2-loaded apatite/collagen cements suggested that the biodegradation rates of the cements were significantly dependent on their formulation. The CMM of ground apatite/collagen cement increased until 7 days and then decreased, and bone-like cells penetrated deeply in the center. The microphotograph and CMM results of apatite/collagen without grinding indicated that a lot of bone-like cells penetrated into the cement and the cement shape was totally deformed. PMID- 16680715 TI - Biomechanical and surface physico-chemical analyses of used osteosynthesis plates and screws--potential for reuse in developing countries? AB - Reprocessing of single-use devices is an upcoming issue in the Western world, but has been for many years in developing countries. In developing countries, the number of bone fractures due to traffic or industrial accidents is high. Patients often need an osteosynthesis with plates and screws, but most patients, however, cannot afford this because of the costs involved and have to rely on the application of used plates and screws. This study aims to determine whether used plates can be safely reused in another patient from a biomechanical, surface physico-chemical, and biological point-of-view. Osteosynthesis plates weakened in a predictable way during use, regardless of the history, presumably because tests were conducted under extreme conditions in the absence of clinically applied load sharing between bone and plate. Surface physico-chemical analyses indicated that used plates and screws were more hydrophilic than new ones; had increased amounts of calcium-phosphates at their surfaces; and possessed higher number of scratches. Pitting corrosion could be seen on SEM micrographs. Simple cleaning methods, as available in developing countries, including toothbrush, water, detergent and bleach yielded elemental surface compositions, and hydrophobicities similar to those of new ones, while biologically thus cleaned screws were not cytotoxic according to ISO-10993-5 and endotoxin release according to USP-27-NF 22 was within the requirements of the FDA. It is concluded that the reuse of osteosynthesis plates and screws is not necessarily unsafe, although preferably a register should be kept of all previous users to limit the number of reuses as mechanical weakening does occur. PMID- 16680716 TI - Noninvasive diagnostic assessment of brain tumors using combined in vivo MR imaging and spectroscopy. AB - To determine the potential value of multimodal MRI for the presurgical management of patients with brain tumors, we performed combined magnetic resonance imaging (MRI) and proton MR spectroscopy (MRS) in 164 patients who presented with tumors of various histological subtypes confirmed by surgical biopsy. Univariate statistical analysis of metabolic ratios carried out on the first 121 patients demonstrated significant differences in between-group comparisons, but failed to provide sufficiently robust classification of individual cases. However, a multivariate statistical approach correctly classified the tumors using linear discriminant analysis (LDA) of combined MRI and MRS data. After initial separation of contrast-enhancing and non-contrast-enhancing lesions, 91% of the former and 87% of the latter were correctly classified. The results were stable when this diagnostic strategy was tested on the additional 43 patients included for validation after the initial statistical analysis, with over 90% of correct classification. Combined MRI and MRS had superior diagnostic value compared to MRS alone, especially in the contrast-enhancing group. This study shows the clinical value of a multivariate statistical analysis based on multimodal MRI and MRS for the noninvasive evaluation of intracranial tumors. PMID- 16680717 TI - The polyvinyl alcohol-bacterial cellulose system as a new nanocomposite for biomedical applications. AB - Finding materials suitable for soft tissue replacement is an important aspect for medical devices design and fabrication. There is a need to develop a material that will not only display similar mechanical properties as the tissue it is replacing, but also shows improved life span, biocompatibility, nonthrombogenic, and low degree of calcification. Polyvinyl alcohol (PVA) is a hydrophilic biocompatible polymer with various characteristics desired for biomedical applications. PVA can be transformed into a solid hydrogel with good mechanical properties by physical crosslinking, using freeze-thaw cycles. Hydrophilic bacterial cellulose (BC) fibers of an average diameter of 50 nm are produced by the bacterium Acetobacter xylinum, using a fermentation process. They are used in combination with PVA to form biocompatible nanocomposites. The resulting nanocomposites possess a broad range of mechanical properties and can be made with mechanical properties similar to that of cardiovascular tissues, such as aorta and heart valve leaflets. The stress-strain properties for porcine aorta are matched by at least one type of PVA-BC nanocomposite in both the circumferential and the axial tissue directions. A PVA-BC nanocomposite with similar properties as heart valve tissue is also developed. Relaxation properties of all samples, which are important for cardiovascular applications, were also studied and found to relax at a faster rate and to a lower residual stress than the tissues they might replace. The new PVA-BC composite is a promising material for cardiovascular soft tissue replacement applications. PMID- 16680718 TI - Handedness control of peptide helices by amino acid side-chain chirality: Ile/aIle peptides. AB - A set of four hexapeptide sequences, each characterized by four strongly helicogenic Aib residues and all combinations of two isomeric Ile/aIle residues at positions 2 and 5, was synthesized by solution methods and fully characterized. A detailed solution (by FT-IR absorption, NMR, and CD techniques) and solid/crystalline state (by X-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well developed 3(10)-helical structures. However, the most relevant conformational conclusion extracted from the present 3D-analysis is that the handedness of the 3(10)-helical structures formed does not seem to be sensitive to the configurational change at the beta-carbon atom of the constituent Ile versus the diastereomeric aIle residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid alpha-carbon atom). These results complement published findings on the diverging relative stabilities of the intermolecularly H-bonded beta-sheet structures generated by Ile versus aIle homo-oligopeptides. PMID- 16680719 TI - A robust hybrid peptide crystal formed with weak hydrogen bonds. AB - In the course of our work relating to the design of a bihelical structure (I) from diphenic anhydride by tethering with cystine di-OMe, stable, hard, and rigid crystals, mp 215-218 degrees C were isolated in low yields ( approximately 2%). The crystal structure established that it was a bis amide (II) arising from diphenic acid and cystine di-OMe [(II), C(22)H(22)N(2)O(6)S(2) (a = 9.897 (1) A, b = 12.210 (1) A, c = 18.192 (1) A, sp. gr. P2(1)2(1)2(1))]. An authentic sample of (II) was subsequently prepared in 47% yields by condensation of diphenic acid dichloride with cystine di-OMe. A most surprising feature of II was, despite its high density, rigidity, and hardness, it did not exhibit any normal hydrogen bonds. The nearest approximation to a "usual" hydrogen bond was the single NH...OC linkage that occurred between molecules along a twofold screw axis. In this linkage, N...O = 3.265 A and H...O = 2.43 A, values that are at least 10% longer than those usually observed in peptides. The rigidity of the crystals appears to depend upon many weak hydrogen bonds of the type CH...O, CH...pi, CH...S, and NH...S working in concert. Even these attractions have separations that are at the high end of the range of previously observed values, although some of the weak hydrogen bonds have been rarely reported and have poorly defined ranges. The attractive effect of each of these weak bonds may be enhanced by the occurrence of a number of them in a parallel fashion like rungs in a ladder. PMID- 16680720 TI - Immunoinformatics may lead to a reappraisal of the nature of B cell epitopes and of the feasibility of synthetic peptide vaccines. PMID- 16680721 TI - Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled coil template. AB - Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS 1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels. PMID- 16680722 TI - Recent advances in craniofacial morphogenesis. AB - Craniofacial malformations are involved in three fourths of all congenital birth defects in humans, affecting the development of head, face, or neck. Tremendous progress in the study of craniofacial development has been made that places this field at the forefront of biomedical research. A concerted effort among evolutionary and developmental biologists, human geneticists, and tissue engineers has revealed important information on the molecular mechanisms that are crucial for the patterning and formation of craniofacial structures. Here, we highlight recent advances in our understanding of evo-devo as it relates to craniofacial morphogenesis, fate determination of cranial neural crest cells, and specific signaling pathways in regulating tissue-tissue interactions during patterning of craniofacial apparatus and the morphogenesis of tooth, mandible, and palate. Together, these findings will be beneficial for the understanding, treatment, and prevention of human congenital malformations and establish the foundation for craniofacial tissue regeneration. PMID- 16680723 TI - Novel gene ashwin functions in Xenopus cell survival and anteroposterior patterning. AB - The novel gene ashwin was isolated in a differential display screen for genes activated or up-regulated early in neural specification. ashwin is expressed maternally and zygotically, and it is up-regulated in the neural ectoderm after the midgastrula stage. It is expressed in the neural plate and later in the embryonic brain, eyes, and spinal cord. Overexpression of ashwin in whole embryos leads to anterior truncations and other defects. However, a second Organizer does not form, and the secondary axial structures may result from splitting of the Organizer, rather than axis duplication. Morpholino oligonucleotide-mediated reduction in ashwin expression leads to lethality or abnormalities in gastrulation, as well as significant apoptosis in midgastrula embryos. Apoptosis is also observed in midgastrula embryos overexpressing ashwin. Coexpression of ashwin with the bone morphogenetic protein-4 antagonist noggin has a synergistic effect on neural-specific gene expression in isolated animal cap ectoderm. Ashwin has no previously characterized domains, although two nuclear localization signals can be identified. Orthologues have been identified in the human, mouse, chicken, and pufferfish genomes. Our results suggest that ashwin regulates cell survival and anteroposterior patterning. PMID- 16680724 TI - MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field. AB - Targeted deletion of the mef2c gene results in a small left ventricle and complete loss of the right ventricle (Lin et al. [1997] Science 276:1404-1407). Absence of the right ventricle is from defective differentiation of cells from the secondary heart field. Our studies of the dysmorphogenesis of the left ventricle uncovered morphological and transcriptional abnormalities at the transition from the cardiac crescent to the linear-tube stage heart. Use of the cgata6LacZ transgene demonstrated that lacZ-positive cells, which normally mark the precursors to the atrioventricular canal and adjacent regions of the left ventricle and atria, remain in the sinoatrial region of the mutant. This, along with the absence of a morphologically distinct atrioventricular canal, indicates a misapportioning of cells between the inflow and outflow segments. The underlying genetic program was also affected with altered expression of mlc2a, mlc2v, and irx4 in outflow segment precursors of the primary heart field. In addition, the sinoatrial-enriched transcription factor, tbx5, was ectopically expressed in the primitive ventricle and ventricle-specific splicing of mef2b was lost, suggesting that the mutant ventricle had acquired atrial-specific characteristics. Collectively, these results suggest a fundamental role of MEF2C in ventricular cardiomyocyte differentiation and apportioning of cells between inflow and outflow precursors in the primary heart field. PMID- 16680725 TI - Identification, characterization, and expression pattern of the chicken EKLF gene. AB - EKLF/KLF1 was the first of the Kruppel-like factors (KLFs) to be identified in mammals and plays an important role in primitive and definitive erythropoiesis. Here, we identify and characterize EKLF in the chicken (cEKLF). The predicted amino acid sequence of the zinc finger region of cEKLF is at least 87.7% similar to mammalian EKLF proteins and is 98.8% and 95% similar to the EKLF orthologues in Xenopus and zebrafish, respectively. During early embryonic development, cEKLF expression is seen in the posterior primitive streak, which gives rise to hematopoietic cells, and then in the blood islands and in circulating blood cells. cEKLF mRNA is expressed in blood cells but not in brain later in chicken embryonic development. cEKLF mRNA is increased in definitive compared with primitive erythropoiesis. The conserved sequence and expression pattern of cEKLF suggests that its function is similar to its orthologues in mammals, Xenopus, and zebrafish. PMID- 16680726 TI - Analysis of origin and growth of the thyroid gland in zebrafish. AB - The zebrafish thyroid gland shows a unique pattern of growth as a differentiated endocrine gland. Here, we analyze the onset of differentiation, the contribution of lineages, and the mode of growth of this gland. The expression of genes involved in hormone production and the establishment of epithelial polarity show that differentiation into a first thyroid follicle takes place early during embryonic development. Thyroid follicular tissue then grows along the pharyngeal midline, initially independently of thyroid stimulating hormone. Lineage analysis reveals that thyroid follicle cells are exclusively recruited from the pharyngeal endoderm. The ultimobranchial bodies that merge with the thyroid in mammals form separate glands in zebrafish as visualized by calcitonin precursor gene expression. Mosaic analysis suggests that the first thyroid follicle differentiating at 55 hours postfertilization corresponds later to the most anterior follicle and that new follicles are added caudally. PMID- 16680727 TI - Establishment of the epaxial-hypaxial boundary in the avian myotome. AB - Trunk skeletal muscles are segregated into dorsomedial epaxial and ventrolateral hypaxial muscles, separated by a myoseptum. In amniotes, they are generated from a transient structure, the dermomyotome, which lays down muscle, namely the myotome underneath. However, the dermomyotome and myotome are dorsoventrally continuous, with no morphologically defined epaxial-hypaxial boundary. The transcription factors En1 and Sim1 have been shown to molecularly subdivide the amniote dermomyotome, with En1 labeling the epaxial dermomyotome and Sim1 the hypaxial counterpart. Here, we demonstrate that En1 and Sim1 expression persists in cells leaving the dermomyotome, superimposing the expression boundary onto muscle and skin. En1-expressing cells colonize the myotome initially from the rostral and caudal lips, and slightly later, directly from the de-epithelializing dermomyotomal center. En1 expression in the myotome is concomitant with the appearance of Fgfr4/Pax7-expressing mitotically active myoblasts. This finding suggests that Fgfr4+/Pax7+/En1+ cells carry their expression with them when entering the myotome. Furthermore, it suggests that the epaxial-hypaxial boundary of the myotome is established through the late arising, mitotically active myoblasts. PMID- 16680728 TI - Consideration of magnetically-induced and conservative electric fields within a loaded gradient coil. AB - We present a method to calculate the electric (E)-fields within and surrounding a human body in a gradient coil, including E-fields induced by the changing magnetic fields and "conservative" E-fields originating with the scalar electrical potential in the coil windings. In agreement with previous numerical calculations, it is shown that magnetically-induced E-fields within the human body show no real concentration near the surface of the body, where nerve stimulation most often occurs. Both the magnetically-induced and conservative E fields are shown to be considerably stronger just outside the human body than inside it, and under some circumstances the conservative E-fields just outside the body can be much larger than the magnetically-induced E-fields there. The order of gradient winding and the presence of conductive RF shield can greatly affect the conservative E-field distribution in these cases. Though the E-fields against the outer surface of the body are not commonly considered, understanding gradient E-fields may be important for reasons other than peripheral nerve stimulation (PNS), such as potential interaction with electrical equipment. PMID- 16680729 TI - Microindentation test for assessing the mechanical properties of cartilaginous tissues. AB - Mechanical properties of the fresh control, frozen, and vitrified cartilaginous (cartilage and meniscus) samples were measured by microindentation. Indentation depth, elastic modulus, and indentation yield strength were obtained from the microindentation loading curves. Indentation deformation behavior was studied using Hertz contact model. The stress distribution of cartilaginous tissues under indentation loading was analyzed by finite element technique. It was found that fresh tissue shows the lowest indentation depth and the highest elastic modulus and indentation yield strength, followed by vitrified and frozen tissues. The vitrified tissue shows slightly lower but comparable mechanical properties with control tissue. The vitrification technique used in this study can preserve live cells with superior mechanical properties that make it an ideal technique for use in orthopedic and other biomedical applications. The microindentation tests and corresponding analysis methods used in this study offer a simple way to evaluate the mechanical properties of cartilaginous tissues. It suits small sample sizes and it may be used for other biological tissues. PMID- 16680730 TI - Entrapment of retroviral vector producer cells in three-dimensional alginate scaffolds for potential use in cancer gene therapy. AB - We explored the possibility of entrapping retroviral vector producing cells (VPC) within porous 3D matrix to induce a local and sustained release of viral particles to the malignant milieu. PA317/STK, which constantly shed retroviral vectors, was used to transduce cancer cells with the herpes simplex virus thymidine kinase (HSV-tk) gene. Once HSV-tk is expressed, it preferentially phosphorylates nucleoside analog prodrugs, such as ganciclovir (GCV) and N methanocarbathymidine (N-MCT), to their active triphosphate metabolites, which when incorporated into cellular DNA cause cell death. PA317/STK cells were seeded within 3D alginate scaffold at two different cell densities via static seeding procedure. In vitro assays determined that PA317/STK seeded at high-cell density in scaffolds maintained constant cell number, low cell leakage, and spheroid morphology with viral vector transfection activity. Postcell-seeding viral vector activity was confirmed by transfection of murine colon cancer cells (MC38) with conditioned media originated from VPC-containing scaffolds and the subsequent ability to generate N-MCT triphosphate. Preliminary in vivo transplantation of VPC-containing scaffolds into the peritoneal cavity of mice bearing intraperitoneal MC38 tumors with 2 weeks subsequent GCV administration resulted in a significantly higher survival rate relative to control groups. Our results demonstrate the feasibility of employing alginate scaffolds to efficiently entrap and support PA317/STK cells for cancer gene therapy. PMID- 16680731 TI - Fast spectroscopic imaging using online optimal sparse k-space acquisition and projections onto convex sets reconstruction. AB - Long acquisition times, low resolution, and voxel contamination are major difficulties in the application of magnetic resonance spectroscopic imaging (MRSI). To overcome these difficulties, an online-optimized acquisition of k space, termed sequential forward array selection (SFAS), was developed to reduce acquisition time without sacrificing spatial resolution. A 2D proton MRSI region of interest (ROI) was defined from a scout image and used to create a region of support (ROS) image. The ROS was then used to optimize and obtain a subset of k space (i.e., a subset of nonuniform phase encodings) and hence reduce the acquisition time for MRSI. Reconstruction and processing software was developed in-house to process and reconstruct MRSI using the projections onto convex sets method. Phantom and in vivo studies showed that good-quality MRS images are obtainable with an approximately 80% reduction of data acquisition time. The reduction of the acquisition time depends on the area ratio of ROS to FOV (i.e., the smaller the ratio, the greater the time reduction). It is also possible to obtain higher-resolution MRS images within a reasonable time using this approach. MRSI with a resolution of 64 x 64 is possible with the acquisition time of the same as 24 x 24 using the traditional full k-space method. PMID- 16680733 TI - Description of patients with squamous cell carcinoma in the atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion triage study. AB - BACKGROUND: The Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS) accumulated information regarding conventional and liquid-based Papanicolaou (Pap) cytology, 2 kinds of human papillomavirus (HPV) DNA testing, cervicography, and colposcopically directed biopsy. The prevalence of squamous cell carcinoma in these women, the efficacy of tests, and the time to detection were reviewed. METHODS: The ALTS data base was reviewed for all women with invasive carcinoma. All results of colposcopy, HPV testing, cytology, biopsies, and cervigrams were reviewed for all women in the ALTS trial who were diagnosed with squamous cell carcinoma. RESULTS: There were 7 diagnoses of invasive cancer (all squamous cell) during the 2 years of the ALTS trial. Although the enrollment studies isolated many high-grade lesions, none of those results were diagnostic of the underlying carcinoma. CONCLUSIONS: The prevalence of squamous cell carcinoma in the setting of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion cytology interpretation appears to be low (approximately 1 per 1000 women in the ALTS trial). Many of the carcinomas were not visible on the ectocervix by cervicography or colposcopy, which may explain in part the paucity of atypical cells detected on the Pap tests and the finding that the presenting cytology, although abnormal, was never diagnostic of cancer. HPV DNA tests were positive in all 7 cancers. Type-specific testing identified HPV type 16 in 6 of 7 cancers and HPV type 18 in 1 of 7 cancers. PMID- 16680734 TI - Adrenal enlargement and insufficiency: a common presentation of intravascular large B-cell lymphoma. AB - Intravascular large B-cell lymphoma (IVLL) is a rare subtype of extranodal diffuse large B-cell lymphoma and is very difficult to diagnose in patients antemortem due to nonspecific presentation of the disease. We report a case of IVLL in a 68-year-old white female who presented with 3-month history of fever of unknown origin (FUO), night sweat, and weight loss. Laboratory tests revealed bilateral adrenal enlargement and adrenal insufficiency. IVLL was diagnosed only at autopsy. A review of the English language medical literature indicates that it is not uncommon for IVLL presented initially with FUO, bilateral adrenal enlargement, and adrenal insufficiency. We present this case to call for heightened awareness of the possibility of IVLL, particularly in an elderly woman who presents with FUO, bilateral adrenal enlargement, and adrenal insufficiency. The pathophysiologic mechanisms of adrenal insufficiency in IVLL are also discussed. PMID- 16680735 TI - Prolonged overall survival with second on-demand autologous transplant in multiple myeloma. AB - Between August 1993 and March 2003, 130 consecutive multiple myeloma (MM) patients eligible for high-dose treatment were offered a program including up front autologous stem cell transplantation (ASCT) after conditioning with 200 mg/m(2) melphalan followed by a second ASCT in case of relapse or progression. A total of 107 (82%) patients completed the first ASCT. The best response obtained after ASCT was complete response (CR) 23%, very good partial response (VGPR) 28%, partial response (PR) 42%, and minimal response (MR) 7%. Median overall survival (OS) and event-free survival (EFS) were 65.4 and 27.7 months, respectively. Relapse or progression occurred in 70 patients; 26 received a second ASCT (with a median time of 20.4 months from first ASCT). A major response (> or =PR) was obtained in 69% of these patients. Median OS and EFS after the second ASCT were 38.1 and 14.8 months. Treatment-related mortality was 1.9% after the first ASCT but no deaths occurred after the second. Our experience suggests that elective up front single ASCT followed by second ASCT after relapse or progression is a safe and effective global strategy to treat MM patients. PMID- 16680737 TI - Chronic eosinophilic leukemia presenting with autoimmune hemolytic anemia and erythrophagocytosis by eosinophils. AB - Eosinophils function primarily as secretory cells and phagocytosis by eosinophils is rarely seen. We describe a case of chronic eosinophilic leukemia (CEL) in a 72 year-old male with a history of previously treated non-Hodgkin's lymphoma (NHL) presenting with erythrophagocytosis by eosinophils and an associated autoimmune hemolytic anemia (AIHA). This patient did not show evidence of relapsed NHL. The patient's blood showed a markedly elevated eosinophil count of 16 x 10(9)/L [normal 0-0.45 x 10(9)/L] on a background of myelodysplasia and features of AIHA. Prominent erythrophagocytosis by eosinophils was visualized in the blood and in the bone marrow. Numerous Charcot-Leyden crystals were also seen in the bone marrow amid increased numbers of eosinophils and the presence of dysplastic granulopoiesis. AIHA is rarely described in the setting of CEL. More significantly, this represents the first case report to describe erythrophagocytosis by eosinophils. PMID- 16680736 TI - Non-Hodgkin's lymphoma presenting as multiple bone lesions and hypercalcemia. AB - We report a rare case of a patient with non-Hodgkin's lymphoma who developed multiple bone lesions and hypercalcemia. A 50-year-old woman complained of drowsiness and multiple bone pain on admission. Radiographic examination revealed multiple bone fractures and osteolytic lesions. She was diagnosed with diffuse large B cell lymphoma by biopsy of an inguinal lymph node. Elevation of parathyroid hormone-related protein (PTHrP) and hypercalcemia were confirmed pretreatment, and those serum levels decreased during chemotherapy for lymphoma. However, the disease was resistant to chemotherapy combined with rituximab. These findings suggest that hypercalcemia is associated with PTHrP and the prognosis of patients with bone lymphoma in advanced stage is poor, although it is thought to be a relatively favorable prognosis in localized primary lymphoma of bone. PMID- 16680738 TI - Structured telephone-based outreach using nonmedical personnel can improve adherence to comprehensive care in families of children with sickle cell disease. AB - Comprehensive medical care of patients with sickle cell disease decreases morbidity, mortality, and health care resource utilization. Nonadherence to comprehensive care is a barrier to further improvement in the outcomes of these patients. We investigated the feasibility and acceptability of telephone delivered structured follow-up, support, and education provided by nonmedical personnel and its impact on adherence to comprehensive measures. A semistructured script was designed that included a series of questions relating to the patient's well-being and health-related behaviors and was administered by a graduate student researcher in genetics. Families of all 202 children followed at Children's Hospital of Pittsburgh were attempted to be contacted at 3 monthly intervals from their last contact. A total of 76% [147] of those called were available and willing to talk. Eighteen months after initiation of the study, the number of patients who had not attended comprehensive care clinic for 2 years or more decreased from 46 (19.7%) to 23 patients (9.9%) (P = 0.0019). Similarly, the proportion of eligible patients who had undergone transcranial Doppler screening within the past 12 months increased from 34 to 49% (P = 0.0501). Structured telephone-based follow-up is feasible and well accepted and can lead to improved adherence with comprehensive care measures. PMID- 16680739 TI - High prevalence of inherited prothrombotic risk factors in 134 consecutive patients with von Willebrand disease. AB - We screened 134 consecutive patients with von Willebrand disease (VWD) (106 type 1, 26 type 2, and 2 type 3 VWD) for the most important inherited prothrombotic risk factors. One hundred eight patients (80.6%) were positive for at least one of the prothrombotic risk factors screened for. A high prevalence of prothrombin G20210A (10.5%) and factor V Leiden (11.9%) mutations was found with allele frequencies of 5.2 and 6%, respectively. Three carriers of multiple prothrombotic gene mutations experienced a thrombotic event. Our study suggests that the recent evidence of an association between inherited thrombotic and hemorrhagic disorders also holds true in VWD patients. PMID- 16680740 TI - Myelodysplastic syndrome and acute myeloid leukemia after treatment with fludarabine, mitoxantrone, and dexamethasone. PMID- 16680741 TI - Morphologic heterogeneity of acute promyelocytic leukemia: therapy-related acute promyelocytic leukemia presenting with FAB-M2 morphology. PMID- 16680742 TI - Diagnosis and prognosis of overt disseminated intravascular coagulation in a general hospital -- meaning of the ISTH score system, fibrin monomers, and lipoprotein-C-reactive protein complex formation. AB - The meaning, the utility, and the prognostic significance of the International Society of Thrombosis and Hemostasis overt disseminated intravascular coagulation (DIC) score and other parameters of coagulation activation including soluble fibrin monomer complexes (SFMC), antithrombin and protein C consumption, and formation of lipoprotein-C-reactive protein (LP-CRP) complexes (MDA slope 1 and flag A2) were evaluated in 165 inpatients from a general hospital for whom DIC testing was required by the attending physicians. Of these 165 patients, 148 had an underlying disease that clearly justified the laboratory request from our systematic post hoc review of the clinical charts. Of these 148 patients, 28 had a positive overt DIC score, 19 had an A2 flag, and 4 had both. The DIC score was strongly related to several major markers of coagulation activation such as D dimers, thrombin-antithrombin complexes, and soluble fibrin and was inversely related to antithrombin and protein C levels, which began to fall from DIC score 4 or higher. The formation of LP-CRP complexes was only related to Gram-negative sepsis and these patients had a strong inflammatory reaction. Independent risk factors for death were high creatininemia, positive overt DIC score, and/or presence of SFMC. In patients with positive DIC score, SFMC positivity and low levels of antithrombin and/or protein C were additional risk factors. The ISTH overt DIC score proves useful and adequate as a marker for clinically significant DIC. Illness severity is further defined by SFMC, antithrombin, and protein C levels. LP-CRP complexes are related to sepsis but not to actual overt DIC and lethal prognosis. PMID- 16680743 TI - Efficacy of prophylactic warfarin for prevention of thalidomide-related deep venous thrombosis. AB - BACKGROUND: Deep venous thrombosis (DVT) is a common complication of thalidomide treatment. There is little information to guide clinicians in selecting effective preventive treatments and physician practice varies. We sought to determine whether prophylactic anticoagulation with warfarin prevents DVT related to thalidomide treatment. METHODS: We reviewed the records of 131 patients receiving thalidomide for a variety of indications. Fifty-five patients were prescribed warfarin with the intent of preventing DVT. Thirty-seven patients received warfarin at a dose of 1-2 mg per day (low dose) and 18 received a dose intended to raise the INR to 2-3 (high dose). RESULTS: Twenty-one of the 131 patients developed venous thrombosis during thalidomide treatment. Eighteen of the 76 patients (23.7%) who were not prescribed prophylactic anticoagulation developed DVT compared to 3 of the 55 patients (5.5%) who were prescribed any dose of prophylactic warfarin (P = 0.010). Only 1 of the 37 patients who received low dose warfarin developed DVT (P = 0.011). Bleeding complications occurred in 4 patients, all of whom were receiving high-dose warfarin. CONCLUSION: Prophylactic anticoagulation with warfarin reduces the risk of thrombosis during thalidomide treatment. Low-dose warfarin may be as effective as higher dose treatment and may result in fewer bleeding complications. PMID- 16680744 TI - Vincristine-loaded platelet infusion for treatment of refractory autoimmune hemolytic anemia and chronic immune thrombocytopenia: rethinking old cures. AB - We report our experience with vincristine-loaded platelet infusion in patients with refractory immune thrombocytopenia (ITP), autoimmune hemolytic anemia (AIHA), and Evans syndrome. Ten patients with symptomatic thrombocytopenia and/ or hemolytic anemia who failed to respond to two to six different treatment modalities, including corticosteroids and splenectomy, were treated with infusion of vincristine-loaded platelets. Platelets were harvested by plateletpheresis from a healthy ABO compatible blood donor and incubated with 5 mg vincristine. Excess of vincristine was removed, and platelets were resuspended in 50 ml plasma and infused over 30 min. All 10 patients responded, and 6 of them achieved complete remission. The response was prompt, occurring 3-8 days after vincristine loaded platelet infusion. Two patients with AIHA are still in remission 9 and 8 years posttreatment with no maintenance treatment. Three ITP patients achieved persisted partial response for 6 years, 5 years, and 11 months; in the remaining 5 patients the response lasted for 2-5 months. No side effects were seen. Our results suggest that this inexpensive and well-tolerated treatment modality may be a useful approach in patients with ITP and AIHA refractory to primary therapy. PMID- 16680746 TI - Could the C677T mutation in the MTHFR gene be another genetic cause of arrhythmogenic right ventricular dysplasia? PMID- 16680745 TI - Pulmonary hypertension and beta-thalassemia major: report of a case, its treatment, and a review of the literature. AB - Pulmonary hypertension is a common complication of beta-thalassemia major. We report a case of successful treatment of pulmonary hypertension in a patient with beta-thalassemia major and review the literature on pulmonary hypertension and beta-thalassemia major. A 28-year-old man with beta-thalassemia major, splenectomy, hepatitis C, and hemosiderosis who presented with increasing dyspnea on exertion was diagnosed with pulmonary hypertension. After receiving continuous epoprostenol infusion and desferoxamine, his functional capacity and hemodynamic status improved. To our knowledge, this is the first case of pulmonary hypertension associated with beta-thalassemia treated with continuous epoprostenol infusion and desferoxamine. Epoprostenol, beneficial in the treatment of other types of pulmonary hypertension, may ameliorate the morbidity and mortality of pulmonary hypertension associated with thalassemia. PMID- 16680747 TI - Acute promyelocytic leukemia with secondary myelofibrosis -- case report and review of the literature. PMID- 16680748 TI - Flow cytometric assessment of hematopoietic cell subsets in cryopreserved preterm and term cord blood, influence of obstetrical parameters, and availability for transplantation. AB - OBJECTIVE: The aim of this study was to characterize the lymphocyte and the hematopoietic stem and progenitor cell (HPC) subsets of cryopreserved premature cord blood (PCB) compared to term cord blood (TCB) by flow cytometry, to study the influence of birth conditions, and to assess its availability for transplantation. MATERIALS AND METHODS: Four-color flow cytometric analysis was performed on 43 PCB and 40 TCB cryopreserved samples using a panel of 24 different mAbs, directed against lymphoid and HPC surface markers. The CB volume was estimated by the weight of the newborn to determine the absolute MNC and CD34(+) cell content/CB sample. Clinical and obstetrical data were recovered. Statistical comparisons and a multiple regression analysis were performed. RESULTS: No consistent differences were found in the mononuclear cell (MNC) or CD34(+) cell concentration (x10(6)/L) between PCB and TCB. The percentage of primitive HPC (CD34(+)CD38(-), CD34(+)CD38(-)CD90(-)HLA-DR(-), CD34(+)CD38( )CD90(-)HLA-DR(+)) and primitive lymphoid progenitors (CD34(+)CD7(+), CD34(+)CD7(+)CD19(-)CD117(-)) were higher in PCB than in TCB. Correspondingly, TCB had an increased percentage of committed HPC. No sample of PCB contained >2 x 10(7) MNC/kg (and only 48% had >1 x 10(5) CD34(+) cells) for a recipient of 20 kg body wt, as the minimum threshold recommended for CB transplantation. Obstetrical factors modulated mainly lymphocyte subsets and fewer HPC subpopulations. Acute fetal distress increased CD34(+) cells, especially the immature subsets. Maternal treatment with dexamethasone and intrauterine growth retardation decreased CD3(+) cells. No other obstetrical factors played a detrimental effect on CB cells if used for transplantation. CONCLUSION: PCB is richer in immature cells both in lymphocyte and HPC populations, and its use for transplantation, at least in special cases, should be reconsidered. PMID- 16680749 TI - Primary cutaneous follicle center lymphoma of the arm with a novel chromosomal translocation t(12;21)(q13;q22): a case report. AB - We report a reciprocal translocation between the long arms of chromosomes 12 and 21, t(12;21)(q13;q22), in a patient with primary cutaneous follicle center lymphoma. Follicle center lymphoma of the skin and follicle center cell lymphoma of the lymph node are morphologically and immunophenotypically very similar. However, the clinical behavior and prognosis of these tumors are different due to the molecular basis of these malignancies. Follicle center cell lymphoma of the lymph node is determined by the presence of a unique translocation between chromosomes 14 and 18, t(14;18)(q32;q21), BCL-2-JH gene rearrangement, that is not present in primary cutaneous follicle center lymphomas. Chromosomal translocations in the primary skin lymphomas have not been previously reported. We hope that our discovery of a new translocation t(12:21)(q13q22) will encourage further investigation into the molecular basis of this translocation and other cytogenetic abnormalities in primary cutaneous B-cell lymphomas. PMID- 16680750 TI - Disseminated intravascular coagulation due to cytomegalovirus infection in an immunocompetent adult treated with plasma exchange. AB - We report the case of an immunocompetent woman who developed disseminated intravascular coagulation (DIC) due to acute cytomegalovirus (CMV) infection and was successfully treated with plasma exchange. Extensive investigations revealed no other cause for her DIC, and she did not have any recurrence after more than 1 year of follow up. Clinically evident DIC due to CMV infection is most common in immunosuppressed patients. The one previously reported immunocompetent patient with DIC due to acute CMV infection died from the illness despite traditional supportive measures. Plasma exchange is a novel therapy that should be considered in this situation. PMID- 16680751 TI - Pseudo-Gaucher cells in light chain plasma cell myeloma. PMID- 16680752 TI - Induction chemotherapy and post-remission imatinib therapy for de Novo BCR-ABL positive AML. PMID- 16680753 TI - Antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) and antiphospholipid syndrome (APS). AB - Antiphospholipid antibodies (APLA) are associated with anti-phospholipid syndrome (APS), a thrombotic disorder, but they are also frequently detected in immune thrombocytopenic purpura (ITP), a bleeding disorder. To investigate possible differences of APLA between these two disorders, we assayed IgG and IgM APLA by ELISA in 21 patients with ITP and 33 with APS. The APLA reacting against two protein target antigens, beta(2)-glycoprotein 1 (beta2GP1) and FVII/VIIa, and four phospholipids [cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] as well as lupus anticoagulant (LA) were analyzed. We made the following observations: (i) IgG and IgM antibodies to beta2GP1 and IgM antibodies to FVII/VIIa were more common in APS than ITP, P < 0.05, while IgG antibodies against the phospholipids (aCL, aPC, aPS, aPE) were more common in ITP than APS, P < 0.05; (ii) multiple APLA > or =3 antigens) were more frequent in APS than ITP, P < 0.05; (iii) LA was frequently associated with APS but was absent in ITP; (iv) APLA is quite common in ITP: two thirds were positive for at least one APLA. In summary, APLA are prevalent in ITP but their profile differs from APS. In APS, antibodies were predominantly against beta2GP1 and 80% had positive LA, while in ITP the APLA reacted most often with the phospholipids without LA. The difference in APLA may result in opposite clinical manifestations in two disorders. PMID- 16680754 TI - Another case of myelodysplasia with monosomy 7 following fludarabine-based chemotherapy. PMID- 16680755 TI - Folic acid deficiency: main etiological factor of megaloblastic anemia in Kazakhstan? PMID- 16680756 TI - Successful treatment of chronic myeloid leukemia with imatinib mesylate in a patient with chronic renal failure on hemodialysis. PMID- 16680757 TI - Use of breath carbon monoxide measurements to assess erythrocyte survival in subjects with chronic diseases. AB - Anemia is very common in patients with chronic diseases. To determine the role of increased red blood cell (RBC) turnover in such subjects, we estimated RBC survival in three groups of chronically ill patients using a simple technique in which RBC life span is estimated via measurements of breath carbon monoxide concentration. The study groups consisted of subjects with: (1) osteoarthritis, (2) rheumatoid arthritis, and (3) anemia who were hospitalized for treatment of a variety of chronic illnesses. None of the anemic subjects had evidence of hemorrhage, a deficiency state, or a marrow abnormality to account for their reduced hemoglobin concentration. Subjects with osteoarthritis had a mean RBC life span (127 +/- 25 days) that did not differ significantly from normal (122 +/ 23 days). In contrast, RBC life span was significantly reduced (P < 0.001) in both the rheumatoid arthritis subjects (90 +/- 15 days) and the anemic, hospitalized patients (87 +/- 33 days). The hemoglobin concentration of the rheumatoid patients was near normal (13.5 +/- 1.5 g/dl), indicating that the marrow was compensating for the reduced RBC life span, whereas no such compensation was apparent in the anemic, chronically ill subjects. We conclude that a modest (approximately 25%) reduction in RBC life span commonly occurs in patients with chronic disease, and this reduction becomes clinically relevant in subjects whose marrow cannot respond with increased RBC output. PMID- 16680758 TI - Bovine prion (PrP) and Doppel (Dpl) proteins expression after in vitro leukocyte activation or Dpl/PrP blocking. AB - It has been postulated that Doppel (Dpl) and Prion (PrP) proteins have yet undetermined interactions, since Dpl is overexpressed in transgenic PrP-deficient mice. In this study we investigated the expression levels of Dpl and PrP on lymphocytes, monocytes and neutrophils (PMNs) isolated from bovine blood and incubated (2 and 18 h) with TNFalpha, IL-1, IL-2, IL-8, C5a, IFNgamma, anti-PrP, and anti-Dpl antibodies by flow cytometry. The isolation procedures yielded cell populations with high purity, viability and recovery rates. After 2 h incubation, expression of PrP or Dpl was altered only in PMNs. These cells overexpressed PrP when incubated with TNFalpha and IFNgamma, and both PrP and Dpl when incubated with C5a; incubation with TNFalpha, IL-8 and IFNgamma led to down-regulation of Dpl. Lymphocytes incubated for 18 h with IL-2 and with IFNgamma overexpressed Dpl. Incubation with the anti-PrP antibody induced down-regulation of Dpl in PMNs after 2 h and overexpression of Dpl in lymphocytes after 18 h. The differences recorded after 2 h were likely due to redistribution of pre-existing PrP or Dpl molecules, while those seen at 18 h were most probably due to increased protein synthesis. The variations seen using the different activators depend on different receptors and/or signaling pathways. These results demonstrate that is possible to alter the expression of Dpl and PrP in blood cells in vitro by incubation with either cytokines or anti-PrP antibodies. This opens an interesting opportunity to study the biology of these proteins using in vitro systems. PMID- 16680759 TI - Dynamic expression of neurotrophic factor receptors in postnatal spinal motoneurons and in mouse model of ALS. AB - Neurotrophic factors support the survival of spinal motoneurons (MNs) and have been considered as strong candidates for treating motoneuron diseases. However, it is unclear if the right combination of neurotrophic factor receptors is present in postnatal spinal MNs. In this study, we show that the level of c-ret expression remains relatively stable in embryonic and postnatal spinal MNs. In contrast, the mRNA and protein of GFRalpha1 and -2 are progressively down regulated in postnatal life. By 3 and 6 months of age, both receptors are barely detectable in spinal MNs. The down-regulation of GFRalpha1 appears accelerated in transgenic mice expressing mutant SOD1(G93A). Despite the progressive loss of GFRalpha1 and -2, phosphorylation of c-ret shows no detectable reduction on tyrosine residues or on serine 696. In addition to the GFRalpha subunits, expression of TrkB also shows a dynamic change. During embryogenesis, there is twice as much full-length TrkB as the truncated TrkB isoform. However, this ratio is reversed in postnatal spinal cord. Expression of the mutant SOD1(G93A) appears to have no effect on the TrkB receptor ratio. Taken together, our data indicate that the expression of neurotrophic factor receptors, GFRalpha1, -2, and TrkB, is not static, but undergoes dynamic changes in postnatal spinal MNs. These results provide insights into the use of neurotrophic factors as therapeutic agents for ALS. PMID- 16680760 TI - Adverse neurologic effect of methylene blue used during parathyroidectomy. PMID- 16680761 TI - Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. II. Human cerebral white matter and cortex. AB - This report is the second of a two-part evaluation of developmental differences in alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunit expression in cell populations within white matter and cortex. In part I, we reported that, in rat, developmental expression of Ca2+-permeable (GluR2 lacking) AMPARs correlated at the regional and cellular level with increased susceptibility to hypoxia/ischemia (H/I), suggesting an age-specific role of these receptors in the pathogenesis of brain injury. Part II examines the regional and cellular progression of AMPAR subunits in human white matter and cortex from midgestation through early childhood. Similarly to the case in the rodent, there is a direct correlation between selective vulnerability to H/I and expression of GluR2-lacking AMPARs in human brain. For midgestational cases aged 20-24 postconceptional weeks (PCW) and for premature infants (25-37 PCW), we found that radial glia, premyelinating oligodendrocytes, and subplate neurons transiently expressed GluR2-lacking AMPARs. Notably, prematurity represents a developmental window of selective vulnerability for white matter injury, such as periventricular leukomalacia (PVL). During term (38-42 PCW) and postterm neonatal (43-46 PCW) periods, age windows characterized by increased susceptibility to cortical injury and seizures, GluR2 expression was low in the neocortex, specifically on cortical pyramidal and nonpyramidal neurons. This study indicates that Ca2+-permeable AMPAR blockade may represent an age-specific therapeutic strategy for potential use in humans. Furthermore, these data help to validate specific rodent maturational stages as appropriate models for evaluation of H/I pathophysiology. PMID- 16680762 TI - Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection. AB - Recent evidence suggests that substance P (SP) is up-regulated in primary sensory neurons following axotomy and that this change occurs in larger neurons that do not usually produce SP. If this is so, then the up-regulation may allow normally neighboring, uninjured, and nonnociceptive dorsal root ganglion (DRG) neurons to become effective in activating pain pathways. By using immunohistochemistry, we performed a unilateral L5 spinal nerve transection on male Wistar rats and measured SP expression in ipsilateral L4 and L5 DRGs and contralateral L5 DRGs at 1-14 days postoperatively (dpo) and in control and sham-operated rats. In normal and sham-operated DRGs, SP was detectable almost exclusively in small neurons (< or =800 microm2). After surgery, the mean size of SP-positive neurons from the axotomized L5 ganglia was greater at 2, 4, 7, and 14 dpo. Among large neurons (>800 microm2) from the axotomized L5, the percentage of SP-positive neurons increased at 2, 4, 7, and 14 dpo. Among small neurons from the axotomized L5, the percentage of SP-positive neurons was increased at 1 and 3 dpo but was decreased at 7 and 14 dpo. Thus, SP expression is affected by axonal damage, and the time course of the expression is different between large and small DRG neurons. These data support a role for SP-producing, large DRG neurons in persistent sensory changes resulting from nerve injury. PMID- 16680763 TI - Analysis of direct hippocampal cortical field CA1 axonal projections to diencephalon in the rat. AB - The hippocampal formation is generally considered essential for processing episodic memory. However, the structural organization of hippocampal afferent and efferent axonal connections is still not completely understood, although such information is critical to support functional hypotheses. The full extent of axonal projections from field CA1 to the interbrain (diencephalon) is analyzed here with the Phaseolus vulgaris-leucoagglutinin (PHAL) method. The ventral pole of field CA1 establishes direct pathways to, and terminal fields within, the anterior hypothalamic nucleus, ventromedial hypothalamic nucleus, lateral hypothalamic and lateral preoptic areas, medial preoptic area, and certain other hypothalamic regions, as well as particular midline thalamic nuclei. These results suggest that hippocampal field CA1 modulates motivated or goal-directed behaviors, and physiological responses, associated with the targeted hypothalamic neuron populations. PMID- 16680764 TI - Semaphorins and their receptors in lamprey CNS: Cloning, phylogenetic analysis, and developmental changes during metamorphosis. AB - The large, conserved semaphorin gene family encodes axon guidance molecules in both invertebrates and vertebrates. The primitive vertebrate lamprey diverged near the time of vertebrate origins and is useful for understanding the gene duplication events that led to the increased complexity of the vertebrate genome. We characterized the sequence and expression pattern of semaphorins and their receptors genes in the sea lamprey, Petromyzon marinus. We uncovered two members of the semaphorin family in sea lamprey. The first encodes a diffusible class 3 type semaphorin protein that is most similar to the human and mouse Sema3F (71% amino acid identity). The second encodes a transmembrane class 4 type semaphorin that is most similar to mouse Sema4D and human Sema4G, with 38% amino acid identity within the Sema domain. We also identified in lamprey two members of the semaphorin receptor family, lamprey Plexin A1 and Plexin A2. Phylogenetic analysis indicates that lamprey Sema3 and Sema4 represent precursor genes existing prior to the origin of the vertebrate Sema3A-G and Sema4A-G subfamilies. Therefore, the gene duplication event that gave rise to those subfamilies must have occurred after the divergence of jawed vertebrates from jawless fish. These semaphorins and plexins are expressed in unique and dynamic patterns in lamprey spinal cord and brain during development. PMID- 16680766 TI - Sox3 expression identifies neural progenitors in persistent neonatal and adult mouse forebrain germinative zones. AB - Neural precursors persist throughout life in the rodent forebrain subventricular zone (SVZ) and hippocampal dentate gyrus. The regulation of persistent neural stem cells is poorly understood, in part because of the lack of neural progenitor markers. The Sox B1 subfamily of HMG-box transcription factors (Sox1-3) is expressed by precursors in the embryonic nervous system, where these factors maintain neural progenitors in an undifferentiated state while suppressing neuronal differentiation. Sox2 expression persists in germinative zones of the adult rodent brain, but Sox3 expression in the postnatal brain remains largely unexplored. Here we examine Sox3 expression in the neonatal and adult mouse brain to gain insight into its potential involvement in regulating persistent neural stem cells and neurogenesis. We also investigate Sox3 expression during expansion and neural differentiation of postnatal mouse SVZ neural stem cell and human embryonic stem cell (hESC) cultures. We find that Sox3 is expressed transiently by proliferating and differentiating neural progenitors in the SVZ-olfactory bulb pathway and dentate gyrus. Sox3 immunoreactivity also persists in specific postmitotic neuronal populations. In vitro, high Sox3 protein expression levels in undifferentiated, SVZ-derived neurospheres decline markedly with differentiation. Sox3 immunoreactivity in hESCs appears upon differentiation to neural progenitors and then decreases as cells differentiate further into neurons. These findings suggest that Sox3 labels specific stages of hESC-derived and murine neonatal and adult neural progenitors and are consistent with a role for Sox3 in neural stem cell maintenance. Persistent Sox3 expression in some mature neuronal populations suggests additional undefined roles for Sox3 in neuronal function. PMID- 16680765 TI - Neonatal deafness results in degraded topographic specificity of auditory nerve projections to the cochlear nucleus in cats. AB - We previously examined the early postnatal maturation of the primary afferent auditory nerve projections from the cat cochlear spiral ganglion (SG) to the cochlear nucleus (CN). In normal kittens these projections exhibit clear cochleotopic organization before birth, but quantitative data showed that their topographic specificity is less precise in perinatal kittens than in adults. Normalized for CN size, projections to the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) subdivisions are all significantly broader in neonates than in adults. By 6-7 postnatal days, projections are proportionate to those of adults, suggesting that significant refinement occurs during the early postnatal period. The present study examined SG projections to the CN in adult cats deafened as neonates by ototoxic drug administration. The fundamental organization of the SG-to-CN projections into frequency band laminae is clearly evident despite severe auditory deprivation from birth. However, when normalized for the smaller CN size in deafened animals, projections are disproportionately broader than in controls; AVCN, PVCN, and DCN projections are 39, 26, and 48% broader, respectively, than predicted if they were precisely proportionate to projections in normal hearing animals. These findings suggest that normal auditory experience and neural activity are essential for the early postnatal development (or subsequent maintenance) of the topographic precision of SG-to-CN projections. After early deafness, the basic cochleotopic organization of the CN is established and maintained into adulthood, but the CN is severely reduced in size and the topographic specificity of primary afferent projections that underlies frequency resolution in the normal central auditory system is significantly degraded. PMID- 16680767 TI - Organization of frontoparietal cortex in the tree shrew (Tupaia belangeri). I. Architecture, microelectrode maps, and corticospinal connections. AB - Despite extensive investigation of the motor cortex of primates, little is known about the organization of motor cortex in tree shrews, one of their closest living relatives. We investigated the organization of frontoparietal cortex in Belanger's tree shrews (Tupaia belangeri) by using intracortical microstimulation (ICMS), corticospinal tracing, and detailed histological analysis. The results provide evidence for the subdivision of tree shrew frontoparietal cortex into seven distinct areas (five are newly identified), including two motor fields (M1 and M2) and five somatosensory fields (3a, 3b, S2, PV, and SC). The types of movements evoked in M1 and M2 were similar, but M2 required higher currents to elicit movements and had few connections to the cervical spinal cord and distinctive cyto- and immunoarchitecture. The borders between M1 and the anterior somatosensory regions (3a and 3b) were identified primarily from histological analysis, because thresholds were similar between these regions, and differences in corticospinal neuron distribution were subtle. The caudal (SC) and lateral (S2 and PV) somatosensory fields were identified based on differences in architecture and distribution of corticospinal neurons. Myelin-dense modules were identified in lateral cortex, in the expected location of the oral, forelimb, and hindlimb representations of S2, and possibly PV. Evidence for a complex primate-like array of motor fields is lacking in tree shrews, but their motor cortex shares a number of basic features with that of primates, which are not found in more distantly related species, such as rats. PMID- 16680768 TI - Fine-needle aspiration of chondrosarcoma. AB - Fine-needle aspiration (FNA) is a reliable, safe and cost-effective procedure with an established role in the diagnosis of various solid tissue neoplasms. However, the role of FNA in the diagnosis of primary bone tumors, including chondrosarcoma (CS) is controversial. To determine the accuracy of FNA as a diagnostic procedure, the author reviewed the institutional experience of a series of patients with CS who underwent FNA for diagnosis. The author's objectives were to determine the accuracy of the technique as well as possible limitations to sensitivity and specificity, and perhaps to suggest the most appropriate use for this procedure. Computer records and then subsequently archives of the department were searched for patients diagnosed and treated for CS between 1993 and 2003. Patients without adequate clinical follow-up, missing materials or records otherwise unavailable for review were eliminated from study. All patients who underwent FNA for a diagnosis had to have a subsequent histological confirmation to be included in the study. FNAs were largely performed with image-guided assistance. In those that were palpable, the aspiration was performed by the aspiration cytologist using standard methods. Histologic materials were processed according to standard methods. All cytological and histologic materials were reviewed for accuracy and appropriateness of diagnosis by the author. There were 34 aspirates from 32 patients with CS (2 patients with 2 aspirates each). Attempts at diagnoses were made from 27 primary lesions, 6 recurrent lesions, and one metastatic lesion. There were an additional two patients who were assigned a diagnosis of CS on FNA who ultimately were proven to have chondroblastic osteosarcoma. Of the primary CS, 18 were definitively diagnosed as CS or "malignant chondroid neoplasm," 8 of the aspirates were considered equivocal in that an additional diagnostic procedure was required to clarify or confirm the diagnosis. Two aspirates were diagnosed as negative. Both of the false negatives were due to inadequate sampling of the lesion on FNA. Diagnostic accuracy of FNA for primary CS in this series was 67% (18/27). Accuracy for recurrent or metastatic lesions was higher at 86% (6/7). FNA appears to be a reliable means of diagnosis of recurrent and/or metastatic CS in patients with a documented history. In primary lesions, however, the accuracy of the technique is lower. In addition, there are problems of sampling chondroid components of non-CS lesions such as this study's experience with chondroblastic osteosarcoma. PMID- 16680769 TI - Tyrosine crystals in pleomorphic adenoma. PMID- 16680770 TI - Cytomorphology of yolk sac tumor of the liver in fine-needle aspiration: a pediatric case. AB - Yolk sac tumors (YST) in extragonadal locations are rare. Cytologic diagnosis of YST on fine-needle aspiration (FNA) smears may be a challenge to the cytopathologist. Further neo-adjuvant therapy may be based on cytologic diagnosis making accuracy important. We studied FNA material from a hepatic mass in a pediatric patient to further define the cytomorphologic features of hepatic YST. Features include large pleomorphic balls of tumor cells with high nuclear to cytoplasmic ratios, papillary or microglandular pattern of growth, cytoplasmic and intranuclear vacuoles, and prominent nucleoli. Positive immunohistochemical studies included alpha-fetoprotein, cytokeratin AE1/AE3, and CAM 5.2, which are useful in supporting the diagnosis. We report a pediatric patient in whom the diagnosis of hepatic YST was made by cytologic, histologic, and immunohistochemical studies. The subsequent liver biopsy was consistent with the FNA diagnosis. Our findings may further help to characterize the cytomorphologic features of this rare lesion. PMID- 16680771 TI - Cytological evaluation of head and neck tumors in children--a pattern analysis. AB - A total of 135 pediatric head and neck tumors diagnosed in our institute were reviewed with a view to elucidate the overall cytological patterns and analyze the important cytological features. Ninety-four tumors (69.6%) were aspirated for a primary diagnosis, and in 41 (30.4%) cases, fine-needle aspiration cytology was performed to document relapse, recurrence or a metastasis. Among the 94 tumors aspirated for a primary diagnosis, 66 cases (70.2%) were accurately diagnosed, in 22 cases (23.4%) a broad working diagnosis was offered, and 6 cases (6.4%) were misdiagnosed. The accuracy rate was higher (79.3%) when relapse-recurrent and metastatic tumors were included. The smears were broadly divided into six patterns, viz. round cell, epithelial, anaplastic, giant cell, mixed inflammatory, and spindle cell patterns. The round cell pattern was the most frequent one encountered in this group. The cytological features that stood the test of variability were lymphoglandular bodies and a noncohesive cell population in hematolymphoid malignancies, pale chromatin and cytoplasmic vacuoles in primitive neuroectodermal tumor/Ewing's sarcoma (PNET/ES), neuropil and rosettes in neuroblastoma, and plasmacytoid rhabdomyoblasts in rhabdomyosarcoma. A fairly good accuracy was seen in the diagnosis of metastatic undifferentiated carcinoma and anaplastic lymphoma, but the giant-cell and spindle-cell tumors continued to pose a problem in diagnosis. Ancillary techniques such as immunocytochemistry and electron microscopy applied in limited cases helped evaluate Langerhans cell histiocytosis, alveolar rhabdomyosarcoma, and the PNET/ES family of tumors. PMID- 16680772 TI - Critical values in cytology. AB - The concept of critical values (CVs) is well established in clinical pathology, and has only recently been suggested in surgical pathology. To evaluate CVs in cytology, we reviewed 2,000 cytology reports at two large academic medical centers. Cases considered CV included unexpected malignancy, disagreement between immediate interpretation and final diagnosis in fine-needle aspirations (FNAs), and evidence of microorganisms in non-gynecology (non-GYN) and FNA specimens. We identified 52 CV cases (2.6%), including 0.25% (1/400) GYN, 1.88% (15/800) non GYN, and 4.5% (36/800) FNA. Most of these (42 cases) were unexpected malignancies. Documentation of physician notification was present in 30 out of 52 cases. We also did a survey with 22 cytopathologists and 13 clinicians at large academic medical centers. The participants were asked to rate 18 different possible CVs from 1 to 3 as follows: (1) no phone call necessary, (2) phone call within 24 hr, (3) phone call as soon as possible (ASAP). Participants could also list additional diagnoses they believed constituted a CV. Most respondents agreed on the need for a phone call ASAP in many situations, and important additional CV cases were suggested. We suggest that a consensus conference of leaders in anatomic pathology and clinicians might prove useful to propose guidelines for CVs in cytology. PMID- 16680774 TI - Fine-needle aspiration diagnosis of Hodgkin lymphoma using current WHO classification--re-evaluation of cases from 1999-2004 with new proposals. AB - With the advent of modern therapy, the differences in prognoses and treatment regimens among different subtypes of Hodgkin lymphoma (HL) have largely vanished. Stage and the presence of systemic symptoms are much more important than histologic subtypes as predictive factors. The current (2001) WHO classification markedly de-emphasizes spatial relationships as critical to the diagnosis of lymphoma and emphasizes cell morphology, immunophenotype, genetic features, and clinical information to define the disease states. This classification, thus, greatly enhances the capability of fine-needle aspiration (FNA) to accurately diagnose HL. We searched all the FNA cases in our institute in years 1999 through 2004 and found 42 cases, for which 13 were primarily diagnosed (31.0%), 2 were recurrent (4.8%), 5 were highly suspicious (11.9%), and 22 were suspicious (52.3%) for HL. On follow-up tissue biopsy, all the primarily diagnosed, recurrent, and highly suspicious cases were confirmed to be HL (100% agreement). For the 22 suspicious cases, 13 were HL (59.1%), 5 were other lymphomas (22.8%), 1 was lymphoma unclassifiable (4.5%), and 3 were reactive processes (13.6%). The effect of immunostains on the diagnosis of HL was examined, and its importance was emphasized. Analysis of demographic data and the distribution of HL subtypes demonstrate that the study sample is representative of the general HL patient population. On the basis of these results, we propose: (1) If the FNA diagnosis of HL is confirmed both by morphology and immunostains, no further tissue confirmation, subclassification and grading is necessary, and appropriate treatment regimens should follow. (2) The nodular lymphocyte predominant HL and classical HL can be differentiated by adequate immunostaining. (3) If a definitive diagnosis cannot be achieved by FNA, a second FNA or a tissue biopsy should be recommended. PMID- 16680773 TI - Testing automated liquid-based cytology samples with a manual liquid-based cytology method using residual cell suspensions from 500 ThinPrep cases. AB - We report a technical improvement upon a previously disclosed manual liquid-based cytology (MLBC) method; and, we use the improved method to prepare slides from residual ThinPrep specimens in order to see how often ThinPrep diagnoses correspond to diagnoses derived from exhaustive examination of their parent sample suspensions. Residual cell suspensions from 500 ThinPrep cases comprising (1) 20 low-grade squamous intraepithelial lesions (LSILs); (2) 200 high risk (HR) negatives and 20 ASC-US; and (3) 260 screening cytology specimens were studied. Institutional review committee guidelines allowed us to know diagnoses by groups of specimens, but did not allow us to know individual patient diagnoses, so we could not perform case-by-case matched outcome-comparisons. Cells were concentrated by conventional centrifugation and sedimented into a polymer gel that was then vortex-mixed and converted into a viscous cell-rich suspension. The cell suspension was smeared between two clean glass slides, which were air-dried and stained with the Papanicolaou stain. Two study-sets were created, comprising one slide from each case. Each of the two study sets was examined by two cytopathologists, and discordant diagnoses were adjudicated. Because of the ambiguity involved in the "atypical" (ASC-US, ASC-H, AGC) diagnosis categories, only outcomes at the level of LSIL or greater were recorded. All MLBC SILs were digitally imaged and abnormal slides plus digital images were sent to the laboratory that provided the residual automated liquid-based cytology (ALBC) suspensions. The final diagnoses were confirmed by the laboratory that provided the residual ALBC specimens. MLBC slides of the 20 LSIL cases afforded 2 high grade squamous intraepithelial lesions (HSILs) and 18 LSILs. Those of the 200 HR Negatives showed 3 HSILs and 30 LSILs; and those of the 20 HR-ASC-US showed 3 HSILs and 9 LSILs. MLBC slides of the 260 screening cytology specimens showed 1 Carcinoma, 3 HSILs and 20 LSILs; affording 3 HSILs and 14 LSILs more than originally diagnosed. The MLBC method of this report is useful for preparing cell suspensions for cytological examination. Our analytical method was exhaustive and used nearly all of the cell material that was provided to us for analysis; therefore, we conclude that this approach is useful for determining how well ALBC instruments represent their parent sample suspensions. It appears that "rare events" may be overlooked when limited sample aliquots are analyzed by ALBC instruments, and this probably accounts for our increased discovery of SILs by the MLBC method. Also, SILs often present as aggregates of cohesive cells which, if overlooked or ineffectively transferred to ALBC slides, would not be diagnosed. PMID- 16680775 TI - Occult hepatocellular carcinoma metastasising to the left temple diagnosed by fine-needle aspiration biopsy. AB - Although hepatocellular carcinoma (HCC) is relatively rare in Western countries, it is one of the most prevalent cancers worldwide. Metastatic spread outside the liver is common and typically occurs in the setting of advanced disease. Metastatic disease is the initial presentation of HCC in less than 5% of cases and even fewer are diagnosed by cytology. We present the cytological findings in an 85-yr-old man of a soft tissue mass in the left temple, which suggested the diagnosis of an occult HCC. Fine-needle aspiration biopsy showed moderately cellular smears with large malignant epithelial cells present in fragments, with apposition of endothelial cells to the tumour cells. Deposits of non-refractile green pigment consistent with bile were present. Investigations revealed abnormal liver function tests, and a large mass replacing much of the liver. In view of the cytology and clinico-radiological correlation, a diagnosis of metastatic HCC was made. PMID- 16680776 TI - Metastatic granulosa cell tumor: diagnosed by fine-needle aspiration cytology. PMID- 16680777 TI - Diagnostic pitfalls in fine-needle aspiration cytology of temporomandibular chondroblastoma: report of two cases. AB - Chondroblastoma is a benign bone neoplasm, which usually presents in the epiphysis of long bones, but can occur in unusual locations. This report describes the clinical, radiologic, and cytologic features on FNA of two chondroblastomas of the temporomandibular region that were only recognized by histopathologic study. This emphasizes the diagnostic pitfalls of this entity and expands the cytologic differential diagnosis of tumors of the parotid region. PMID- 16680778 TI - Synchronous ordinary lipoma and spindle-cell lipoma diagnosed by fine needle aspiration. PMID- 16680779 TI - Fine-needle aspiration of neurilemoma (schwannoma). A clinicocytopathologic study of 116 patients. AB - The preoperative fine-needle aspiration cytology (FNAC) diagnoses in 116 surgically excised neurilemomas were reviewed and compared with the corresponding histopathologic diagnoses made on surgical specimens and with clinical data. In addition, the utility of adjunctive techniques was analyzed and other spindle cell lesions in the differential diagnoses were discussed. An unequivocal, benign diagnosis was rendered by FNAC in 80 cases, 67 of which were correctly labelled as neurilemoma in a review of the original cytology reports. There were 6 false positive malignant diagnoses while 23 smears were considered insufficient and 7 inconclusive as to whether benign or malignant. On reevaluation, the diagnostic smears in most cases contained spindle cells with wavy nuclei embedded in a fibrillar, occasionally collagenous, and/or myxoid matrix and Antoni A/Antoni B tissue fragments. A moderate to abundant admixture of round to oval cells was also frequent. Nuclear palisading was seen in 41 smears with distinctive Verocay bodies in 10. Markedly pleomorphic nuclei were seen in smears from 8 ancient and 6 conventional neurilemomas, and slight to moderate nuclear pleomorphism was observed in 38 additional cases. Thus most neurilemomas have distinct cytomorphologic features that allow correct diagnosis. The major problem in FNAC of neurilemoma is to obtain sufficient material. Furthermore aspirates showing predominantly Antoni A features, nuclear pleomorphism, and/or myxoid changes can easily be confused with other types of benign or malignant soft-tissue tumors. PMID- 16680780 TI - Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds. AB - The presence of one or more calcium-dependent ecto-ATPases (enzymes that hydrolyze extracellular 5'-triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto-ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double-label assays were performed with antisera directed against the glial glutamate/aspartate transporter (GLAST), the transduction pathway proteins phospholipase Cbeta2 (PLCbeta2) or the G-protein subunit alpha-gustducin, and serotonin (5HT) as markers of type I, II, and III taste cells, respectively. Analysis of the double labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto-ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system. PMID- 16680781 TI - Subbarrel patterns of thalamocortical innervation in rat somatosensory cortical barrels: Organization and postnatal development. AB - Barrel hollows in the posteromedial barrel subfield of adult rat somatosensory cortex typically encompass two or three metabolically and structurally distinct regions, termed subbarrels. We used immunohistochemical staining for vesicular glutamate transporter 2 and the neuronal serotonin transporter, in conjunction with cytochrome oxidase (CO) histochemistry, to investigate the distribution of thalamocortical (TC) axon terminals in relation to subbarrel domains. We found, first, that CO-dark subbarrels are more intensely immunoreactive for thalamocortical terminals than the CO-light clefts that separate them. Second, during the first postnatal week, immunoreactivity for markers of TC terminals is relatively homogeneous throughout the barrel hollow; subbarrel patterns of distribution only become recognizable between P-8 and P-10. These observations extend previous findings that subbarrels denote barrel regions enriched in synaptic contacts. The data also indicate that allocation of TC terminals into subbarrel domains does not occur immediately upon thalamic axon ingrowth. Instead, refinement of TC arbors into subbarrels is a gradual process, the outcome of which is not manifest until the second week of postnatal life. PMID- 16680783 TI - The phosphine-stabilized gold-arsenic clusters [Au19(AsnPr)8(dppe)6]Cl3, [Au10(AsnPr)4(dppe)4]Cl2, [Au17(AsnPr)6(As2nPr2)(dppm)6]Cl3, and [Au10(AsPh)4(dppe)4]Cl2: synthesis, characterization, and DFT calculations. PMID- 16680785 TI - A small-molecule-protein interaction system with split-ubiquitin as sensor. AB - The identification of receptors for small molecules is of great pharmaceutical importance for drug-discovery research. Several systems for the identification of protein-small-molecule interactions have been developed in the past. These were modifications of the classical yeast two-hybrid system, relying on a transcriptional read-out following nuclear translocation of the complex. Here we present a novel three-hybrid technology based on the split-ubiquitin system for the analysis of protein-small-molecule interactions independently of a nuclear translocation of the complex. The performance of the system is compared to a method based on the classical yeast two-hybrid system by using a chemical inducer of dimerization (CID) comprised of methotrexate linked to dexamethasone. Steric issues are addressed by varying the linker length of the compounds, as well as by comparing the orientation of fusion proteins. The system is further extended to the analysis of a small-molecule inhibitor of human PCTAIRE protein kinase 3, which is related to cyclin-dependent kinases (CDKs), an important class of pharmaceutical targets. PMID- 16680784 TI - How to control NO production in cells: N(omega),N(omega)-dimethyl-L-arginine dimethylaminohydrolase as a novel drug target. PMID- 16680786 TI - Experimental and theoretical studies of magnetic exchange in silole-bridged diradicals. AB - Five bis(tert-butylnitroxide) diradicals connected by a silole (7 a-d) or a thiophene (12) ring as a coupler were studied. Compound 12 crystallizes in the orthorhombic space group Pna2(1) with a = 20.752(5), b = 5.826(5), and c = 34.309(5) A. X-ray crystal structure determination, electronic spectroscopy, variable-temperature EPR spectroscopy, SQUID measurements and DFT computations (UB3LYP/6-31+G*) were used to study the molecular conformations and electronic spin coupling in this series of molecules. Whereas compounds 7 b, 7 c, and 7 d are quite stable both in solution and in the solid state, 7 a and 12 undergo a partial electronic rearrangement to both a diamagnetic quinonoidal form and a monoradical species owing to the fact that they correspond to the open form of a pi-conjugated Kekule structure. In the solid state, magnetic measurements indicate that the diradicals are all antiferromagnetically coupled, as expected from their topology. These interactions are best reproduced by means of a "Bleaney-Bowers" model that gives values of J = -142.0 cm(-1) for 7 a, -1.8 cm( 1) for 7 b, -1.3 cm(-1) for 7 c, -4.2 cm(-1) for 7 d, and -248.0 cm(-1) for 12. The temperature dependence of the EPR half-field transition in frozen dichloromethane solutions is consistent with singlet ground states and thermally accessible triplet states for diradicals 7 b, 7 c, and 7 d with DeltaE(T-S) values of 3.48, 2.09, and 8 cm(-1), respectively. No evidence of a populated triplet state was found for diradicals 7 a and 12. Similarities between the DeltaE(T-S) and J values (DeltaE(T-S) = -2 J) clearly show the intramolecular origin of the observed antiferromagnetic interaction. Analyses of the data with a "Karplus-Conroy"-type equation enabled us to establish that the silole ring, as a whole, allows a more efficient magnetic coupling of the two nitroxide radicals attached to its 2,5-positions than the thiophene ring. This superiority probably originates from the nonaromaticity of the silole which thus permits a better magnetic interaction through it. DFT calculations also support the experimental results, indicating that the magnetic exchange pathway preferentially involves the carbon pi system of the silole. PMID- 16680787 TI - Mild copper-catalyzed vinylation reactions of azoles and phenols with vinyl bromides. AB - An efficient and straightforward copper-catalyzed method allowing vinylation of N or O-nucleophiles with di- or trisubstituted vinyl bromides is reported. The procedure is applicable to a broad range of substrates since N-vinylation of mono , di-, and triazoles as well as O-vinylation of phenol derivatives can be performed with catalytic amounts of copper iodide and inexpensive nitrogen ligands 3 or 8. In the case of more hindered vinyl bromides, the use of the original bidentate chelator 8 was shown to be more efficient to promote the coupling reactions than our key tetradentate ligand 3. The corresponding N-(1 alkenyl)azoles and alkenyl aryl ethers are obtained in high yields and selectivities under very mild temperature conditions (35-110 degrees C for N vinylation reactions and 50-80 degrees C for O-vinylation reactions). Moreover, to our knowledge, this method is the first example of a copper-catalyzed vinylation of various azoles. Finally, this protocol, practical on a laboratory scale and easily adaptable to an industrial scale, is very competitive compared to the existing methods that allow the synthesis of such compounds. PMID- 16680782 TI - Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex. AB - This is the first part of a two-part study to investigate the cellular distribution and temporal regulation of alpha-amino-3-hydroxy-5-methyl-4 isoxazole-propionic acid receptor (AMPAR) subunits in the developing white matter and cortex in rat (part I) and human (part II). Western blot and immunocytochemistry were used to evaluate the differential expression of AMPAR subunits on glial and neuronal subtypes during the first 3 postnatal weeks in the Long Evans and Sprague Dawley rat strains. In Long Evans rats during the first postnatal week, GluR2-lacking AMPARs were expressed predominantly on white matter cells, including radial glia, premyelinating oligodendrocytes, and subplate neurons, whereas, during the second postnatal week, these AMPARs were highly expressed on cortical neurons, coincident with decreased expression on white matter cells. Immunocytochemical analysis revealed that cell-specific developmental changes in AMPAR expression occurred 2-3 days earlier by chronological age in Sprague Dawley rats compared with Long Evans rats, despite overall similar temporal sequencing. In both white and gray matter, the periods of high GluR2 deficiency correspond to those of regional susceptibility to hypoxic/ischemic injury in each of the two rat strains, supporting prior studies suggesting a critical role for Ca2+-permeable AMPARs in excitotoxic cellular injury and epileptogenesis. The developmental regulation of these receptor subunits strongly suggests that Ca2+ influx through GluR2-lacking AMPARs may play an important role in neuronal and glial development and injury in the immature brain. Moreover, as demonstrated in part II, there are striking similarities between rat and human in the regional and temporal maturational regulation of neuronal and glial AMPAR expression. PMID- 16680788 TI - Toward the complete prediction of the 1H and 13C NMR spectra of complex organic molecules by DFT methods: application to natural substances. AB - The NMR parameters (1H and 13C chemical shifts and coupling constants) for a series of naturally occurring molecules have been calculated mostly with DFT methods, and their spectra compared with available experimental ones. The comparison includes strychnine as a test case, as well as some examples of recently isolated natural products (corianlactone, daphnipaxinin, boletunone B) featuring unusual and/or crowded structures and, in the case of boletunone B, being the subject of a recent revision. Whenever experimental spectra were obtained in polar solvents, the calculation of NMR parameters was also carried out with the Integral Equation-Formalism Polarizable Continuum Model (IEF-PCM) continuum method. The computed results generally show a good agreement with experiment, as judged not only by statistical parameters but also by visual comparison of line spectra. The origin of the remaining discrepancies is attributed to the incomplete modeling of conformational and specific solvent effects. PMID- 16680789 TI - Postmetallocene lanthanide-hydrido chemistry: A new family of complexes [{Ln{(Me3Si)2NC(NiPr)2}2(mu-H)}2] (Ln = Y, Nd, Sm, Gd, Yb) supported by guanidinate ligands-synthesis, structure, and catalytic activity in olefin polymerization. AB - The new family of Lewis base free hydrido complexes of rare-earth metals supported by guanidinate ligands [{Ln{(Me3Si)2NC(NiPr)2}2(mu-H)}2] (Ln = Y, Nd, Sm, Gd, Yb) was synthesized and structurally characterized. Single-crystal X-ray and solution NMR studies revealed that these complexes are dimeric in both solid state and in [D6]benzene. The dimeric hydrido complexes can adopt eclipsed (Nd, Sm, Gd) or staggered (Y, Yb, Lu) conformations depending on the metal-atom size. Catalytic activity of these [{Ln{(Me3Si)2NC(NiPr)2}2(mu-H)}2] complexes in the polymerization of ethylene, propylene, and styrene has been investigated. Complexes of Sm and Y have high catalytic activity in ethylene polymerization (1268 and 442 g mmol(-1) atm(-1) h(-1), respectively). PMID- 16680790 TI - Messages in molecules: ligand/cation coding and self-recognition in a constitutionally dynamic system of heterometallic double helicates. AB - Double helicates are known to exhibit self-recognition characteristics determined by the coordination geometry of the metal involved as well as by the topicity of the ligands. Combining tridentate (terpyridine, T) or bidentate (bipyridine, B) subunits in a tritopic strand affords a set of ligands able to assemble by pairs to form double helicates, homo- or heterostranded, homo- or heterotopic, depending on the coordination properties of the metals involved. The four ligand strands, BBB, TTT, BBT, and TBT form constitutionally dynamic sets of double helicates with the metal ions Cu(I), Cu(II), and Zn(II); these helicates correspond to the correct coding of the BB, BT, and TT pairs for tetra-, penta-, and hexacoordinate Cu(I), Cu(II), and Zn(II) cations, respectively. PMID- 16680791 TI - The trinuclear gallium-bridged Ferrocenophane [{Fe(eta5-C5H4)2}3Ga2]: synthesis, bonding, structure, and coordination chemistry. AB - The trinuclear ferrocenophane [{Fe(eta(5)-C(5)H(4))(3)}(2)Ga(2)] (3) featuring two sp(2)-hybridized gallium atoms in bridging positions between three ferrocene 1,1'-diyl units represents a novel type of ferrocene derivative. Compound 3 is obtained by thermal treatment of 1,1'-bis(dimethylgallyl)ferrocene (1) in nondonor solvents or in diethyl ether as solvent and subsequent thermal decomplexation. The [1.1]ferrocenophane [{Fe(eta(5)-C(5)H(4))(2)}(2){GaMe}(2)] (2) is an intermediate in the formation of 3. The reaction of 3 with an excess of trimethylgallium leads back to 1 and proves the reversibility of the multistep reaction sequence. Theoretical calculations reveal a carousel-type D(3h) structure for 3. The compound can best be described as being composed of three only weakly interacting ferrocenediyl units covalently connected by gallium atoms without any pi-bond contribution in the Ga--C bonds. Owing to steric constraints 3 cannot be reduced to the dianion 3(2-), which would feature a Ga--Ga bond. Compound 3 represents a stereochemically rigid difunctional Lewis acid allowing the formation of the adducts 3 a-3 d possessing linear donor-aceptor-aceptor donor arrangements. Crystal structure data for 3 a-3 d show a symmetry-reduced chiral ferrocenophane core (D(3h)-->D(3)). A polymeric rodlike structure is observed for 3 b and 3 d caused by pi-stacking effects (3 b) or by a difunctional donor-acceptor interaction (3 d). In solution, the chirality of the adducts is lost by rapid interconversion of the enantiomers. A cyclic voltammogram of 3 b in pyridine reveals three quasi-reversible oxidation steps at -356, -154, and 8 mV, indicating only weak electron delocalization in the cationic species. The redox potentials of the pyridine adduct 3 b are compared with those of other pyridine stabilized gallyl-sustituted ferrocene derivatives and with ferrocene itself. PMID- 16680792 TI - Determining the intermolecular structure in the S0 and S1 states of the phenol dimer by rotationally resolved electronic spectroscopy. AB - The rotationally resolved UV spectra of the electronic origins of five isotopomers of the phenol dimer have been measured. The complex spectra are analyzed using a fitting strategy based on a genetic algorithm. The intermolecular geometry parameters have been determined from the inertial parameters for both electronic states and compared to the results of ab initio calculations. In the electronic ground state, a larger hydrogen-bond length than in the ab initio calculations is found together with a smaller tilt angle of the aromatic rings, which shows a more pronounced dispersion interaction. In the electronically excited state, the hydrogen-bond length decreases, as has been found for other hydrogen-bonded clusters of phenol, and the two aromatic rings are tilted less toward each other. PMID- 16680793 TI - Electronic conduction in the chiral nematic phase of an oligothiophene derivative. PMID- 16680794 TI - Membrane binding and structure of de novo designed alpha-helical cationic coiled coil-forming peptides. AB - We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level. PMID- 16680795 TI - Experimental and theoretical study of 2,6-difluorophenylnitrene, its radical cation, and their rearrangement products in argon matrices. AB - 2,6-Difluorophenylnitrene was reinvestigated both experimentally, in Ar matrices at 10 K, and computationally, by DFT and CASSCF/CASPT2 calculations. Almost-pure samples of both neutral rearrangement products (the bicyclic azirine and the cyclic ketenimine) of a phenylnitrene were prepared and characterized for the first time. These samples were then subjected to X-irradiation in the presence of CH2Cl2 as an electron scavenger, which led to ionization of the neutral intermediates. Thereby, it was shown that only the phenylnitrene and the cyclic ketenimine yield stable radical cations, whereas the bicyclic azirine decays to both of these compounds on ionization. The cyclic ketenimine yields a novel aromatic azatropylium-type radical cation. The electronic structure of the title compound is discussed in detail, and its relation to those of the iso-pi electronic benzyl radical and phenylcarbene is traced. PMID- 16680796 TI - A parameter-free quantum-mechanical approach for calculating electron-transfer rates for large systems in solution. PMID- 16680797 TI - Gas-phase spectroscopy of the unstable sulfur diisocyanate molecule S(NCO)2. AB - Sulfur diisocyanate is generated from a heterogeneous reaction of gaseous sulfur dichloride with silver cyanate and studied for the first time in the gas phase. Combined with quantum chemical calculations, the electronic structure is characterized by photoelectron spectroscopy (PES). Simultaneously, an investigation of the possible ionization and dissociation processes for the molecular cation is presented based on experimental soft ionization mass spectrometry. From the calculated bond-dissociation energies, the dissociation pathway is determined. S(NCO)2+ undergoes 1,3-sigmatropic rearrangement with a smaller barrier height (9.9 kcal mol(-1)) than the neutral counterpart. Thus, the 1,3-sigmatropic rearrangement is preferred for the molecular cation, and OCNCO+ and NS+ is produced by subsequent dissociation of the rearrangement product. The analysis agrees very well with the experimental mass spectrum. PMID- 16680798 TI - Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein. AB - Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively. PMID- 16680799 TI - Novel monocyclic and bicyclic loop mimetics of brain-derived neurotrophic factor. AB - Brain-derived neurotrophic factor (BDNF) is a protein that promotes the survival of neurons. It is widely thought to possess clinical potential for the treatment of neurodegenerative diseases, and in recent years, has been found to play a role in the pathogenesis of some tumours. BDNF is thought to bind to its cellular receptors trkB and p75(NTR) primarily by way of solvent-exposed loops on the BDNF dimer. In this paper, we describe our recent progress towards the development of small peptides as mimetics and inhibitors of BDNF. Two classes of peptides were prepared: disulphide-constrained monomeric monocyclic peptides designed to mimic a single solvent-exposed loop; and homo- and heterodimeric bicyclic peptides designed to mimic pairs of loops. Each peptide was examined in cultures of embryonic chick dorsal root ganglion sensory neurons, both alone, and in competition with BDNF. All peptides were found to inhibit BDNF-mediated neuronal survival, while one--a dimeric peptide based on the two loop 4 regions of BDNF- behaved as a partial BDNF-like agonist. The work described in this paper supports the proposed receptor-binding role of loops 1, 2, and 4 of BDNF, and provides valuable steps towards our long-term goal of developing BDNF mimetics and inhibitors for clinical use. PMID- 16680802 TI - The effect of provider-level ascertainment bias on profiling nursing homes by J. Roy and V. Mor, Statistics in Medicine 2005; 24(23):3609-3629. PMID- 16680804 TI - Generating survival times to simulate Cox proportional hazards models by Ralf Bender, Thomas Augustin and Maria Blettner, Statistics in Medicine 2005; 24:1713 1723. PMID- 16680807 TI - Fibrous membranes electrospinning from acrylonitrile-based polymers: specific absorption behaviors and states of water. AB - Fibrous membranes with a fiber diameter ranging from 80 to 800 nm are prepared from polyacrylonitrile and poly[acrylonitrile-co-(N-vinyl-2-pyrrolidone)] by the electrospinning process. The parameters can be controlled to fabricate fibrous membranes with similar fiber diameters (between 600 and 800 nm) for further studies on the swelling behaviors and water states. Water swelling experiments indicate that the fibrous membrane has a great capacity for water sorption, which reaches a maximum in a few minutes because of its extremely high porosity. Furthermore, a remarkable overshoot occurs as a result of polymer chain relaxation and the non-compact structure of the fibrous membranes. Contrary to the dense membrane, the equilibrium water content in the fibrous membrane decreases with the content of hydrophilic NVP though the maximum is almost the same. Results from DSC experiments demonstrate that only non-freezable bound water and free water can be distinguished in the fibrous membrane. On the basis of the results of water swelling and DSC experiments, it is concluded that the specific behaviors of the fibrous membranes are induced by the non-compact and pore-fiber discontinuous structure, which is different from either dense membranes or hydrogels. [GRAPHS: SEE TEXT] DSC curves of fully swollen electrospun fibrous membranes and of fully swollen dense membranes with different NVP contents. PMID- 16680808 TI - Cytocompatibility and antibacterial activity of a PHBV membrane with surface immobilized water-soluble chitosan and chondroitin-6-sulfate. AB - A water-soluble chitosan (WSC)/chondroitin-6-sulfate (ChS) polyelectrolyte complex (PEC) is covalently immobilized onto the surface of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) membranes via ozone-induced oxidation and poly(acrylic acid) (PAA) graft polymerization. To characterize the modified membranes, X-ray photoelectron spectroscopy (XPS) and water contact angle measurements are performed. It is shown that by coupling WSC as a spacer, the amount of ChS immobilized can be significantly increased. The water contact angle decreases with the amount of PAA, WSC, and ChS immobilized, which indicates the improving hydrophilicity. After WSC- and PEC-immobilization modification, the PHBV membranes possess antibacterial activity against S. aureus, E. coli, P. aeruginosa, and Methicilin resistant Staphylococus aureus (MRSA). According to the L929 fibroblast cell growth inhibition index, the as-prepared PHBV membranes are non-cytotoxic. In addition, the in-vitro evaluation of L929 fibroblast attachment, proliferation, and viability of PEC-immobilized PHBV membranes are ascertained to be superior to those of immobilized WSC or ChS alone. The overall results demonstrate that WSC/ChS PEC immobilization can not only improve the hydrophilicity and cytocompatibility of the PHBV membrane, but also endows antibacterial activity. [GRAPH: SEE TEXT] The bacterial survival ratio of as prepared PHBV membranes (n=3). PMID- 16680809 TI - Missing: entrepreneurial women in biotech. PMID- 16680810 TI - The hermit crab solution. PMID- 16680811 TI - [Inflammatory diseases of the vulva and vagina]. PMID- 16680812 TI - Intentional attunement: a neurophysiological perspective on social cognition and its disruption in autism. AB - A direct form of experiential understanding of others, "intentional attunement", is achieved by modeling their behavior as intentional experiences on the basis of the activation of shared neural systems underpinning what the others do an feel and what we do and feel. This modeling mechanism is embodied simulation. In parallel with the detached sensory description of the observed social stimuli, internal representations of the body states associated with actions, emotions, and sensations are evoked in the observer, as if he/she would be doing a similar action or experiencing a similar emotion or sensation. Mirror neuron systems are likely the neural correlate of this mechanism. By means of a shared neural state realized in two different bodies, the "objectual other" becomes "another self". A defective intentional attunement caused by a lack of embodies simulation might cause some of the social impairments of autistic individuals. PMID- 16680813 TI - [Bilateral insertion of cochlear implants]. PMID- 16680814 TI - Metallothionein and anti-metallothionein, complementary elements of cadmium induced renal disease. PMID- 16680815 TI - Debating sexual selection and mating strategies. PMID- 16680816 TI - Trophoblast cells exhibit differential responses to laminin isoforms. AB - Extracellular matrix (ECM) has specific effects on cell behavior that influence many aspects of early development. In the early postimplantation mouse embryo the ECM component laminin promotes polarization and survival of the embryonic ectoderm and formation of Reichert's membrane. In addition, dynamic patterns of laminins 1 and 10/11 expression in the embryo and the uterus correlate with the progression of implantation. In the implanting blastocyst, laminin 1 is strongly expressed in the trophectoderm basement membrane, whereas laminin 10/11 is expressed only in the inner cell mass and polar trophectoderm. In the uterus, laminin 10/11 is strongly expressed in the decidualizing matrix of the stroma. We show here that laminins 1 and 10/11 have distinct effects on trophoblast cell behavior that influence the process of implantation. Laminin 1 promotes random migration and decreases spreading, whereas laminin 10/11 promotes both spreading and persistent migration. When presented as adjacent substrates, cells stop at the boundary and do not enter the region containing laminin 1. Laminin 1 also affects cell-cell adhesion through changes in the localization of vascular endothelial (VE) cadherin. Cultured cells and primary trophoblast explants become single cells or very small groups on laminin 1 and VE-cadherin localization at regions of cell-cell contact decreases dramatically. In contrast, trophoblast cells maintain strong cell-cell contacts on substrates of laminins 10/11, and exhibit strong staining of VE-cadherin in all regions of cell-cell contact. These effects, and the localization of laminin 1 in Reichert's membrane and laminin 10/11 in the surrounding decidual matrix, suggest that these laminin isoforms influence the direction and quality of invasion of trophoblast cells during implantation, and provide epigenetic cues that drive the morphogenesis of the yolk sac placenta. PMID- 16680817 TI - Debating sexual selection and mating strategies. PMID- 16680818 TI - Debating sexual selection and mating strategies. PMID- 16680819 TI - Debating sexual selection and mating strategies. PMID- 16680820 TI - Debating sexual selection and mating strategies. PMID- 16680821 TI - Debating sexual selection and mating strategies. PMID- 16680822 TI - Debating sexual selection and mating strategies. PMID- 16680823 TI - Debating sexual selection and mating strategies. PMID- 16680824 TI - Debating sexual selection and mating strategies. PMID- 16680825 TI - Genome restructuring in mouse embryos during reprogramming and early development. AB - Although a growing number of studies investigates functional genome organization in somatic cell nuclei, it is largely unknown how mammalian genome organization is established during embryogenesis. To address this question, we investigated chromo center formation and the peculiar arrangements of chromosome domains in early mouse embryos. At the one-cell stage, we observed characteristic arrangements of chromosomes and chromo center components. Subsequently, starting with the burst of zygotic genome transcription major rearrangements led to the establishment of somatic type chromo centers with a defined spatio-temporal organization. These processes appeared to be completed at the blastocyst stage with the onset of cell differentiation. During the same developmental period, a fraction of pericentric heterochromatin that was late replicating in the first cycle underwent switches in replication timing, spatial organization and epigenetic marks. Cloning experiments revealed that the genome organization typical for more advanced stages was quickly reverted into the one-cell stage specific form after nuclear transfer, supporting the idea that reprogramming associated genome remodeling in normal and cloned embryos is determined by cytoplasmic factors. Together, the results suggest that distinct but characteristic forms of nuclear genome organization are required for genome reprogramming in early embryos and for proper regulation of differential gene expression patterns at later stages. PMID- 16680826 TI - NFATc1 expression in the developing heart valves is responsive to the RANKL pathway and is required for endocardial expression of cathepsin K. AB - NFATc1 is necessary for remodeling endocardial cushions into mature heart valve leaflets and is also an essential effector of receptor activator of NFkappaB ligand (RANKL) signaling required for transcriptional activation of bone matrix remodeling enzymes during osteoclast differentiation. Therefore, developing heart valves were examined to determine if NFATc1 functions in the RANKL pathway during leaflet remodeling. Key components of RANKL signal transduction including RANKL, its receptor RANK, and the downstream remodeling enzyme cathepsin K (Ctsk) are expressed in the heart during valve remodeling and colocalize with NFATc1 in developing valve endocardium. However, the absence of tartrate-resistant acid phosphatase (TRAP) activity and the lack of F4/80-positive macrophage lineage contribution to the remodeling valves demonstrate that certain aspects of osteoclast RANKL function are not shared during valve formation. Analysis of NFATc1-/- mouse embryos shows that NFATc1 is specifically required for endocardial expression of RANKL and Ctsk during valve formation. In addition, RANKL treatment augments expression of NFATc1 and Ctsk in embryonic heart cultures, and the RANKL-mediated increase in Ctsk expression is dependent on NFATc1. Together, these results support a role for RANKL signaling during heart valve development and suggest that valve leaflet morphogenesis involves NFATc1 dependent expression of remodeling enzymes including Ctsk. PMID- 16680827 TI - The RNA-binding protein, Vg1RBP, is required for pancreatic fate specification. AB - Signaling mechanisms underlying the induction of the pre-pancreatic tissue within the endoderm remain poorly understood. Through an expression cloning strategy, we have identified a previously uncharacterized pancreatic factor that we named Shirin. Interestingly, the non-coding RNA regulatory sequence (3 UTR) of Shirin is sufficient to induce insulin expression in Xenopus embryos. Biochemical studies demonstrate that this RNA sequence is able to bind directly to a trans acting factor, Vg1RBP, which was previously shown to be involved in the localization of endodermal determinant factors. Loss-of-function analysis indicates that Vg1RBP is required for establishment of pancreatic fate within the endoderm, suggesting a synergism between Vg1RBP and Shirin in the embryo. This study argues for a central role of post-transcriptional mechanisms in establishing pancreatic fate, where a 3 UTR may recruit factors necessary for pancreatic development, and highlights an unknown embryological activity of Vg1RBP. PMID- 16680828 TI - Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra. AB - The regulation of the glutamatergic projection from the subthalamic nucleus (STN) to the substantia nigra (SN) was investigated using dual-probe microdialysis in the awake behaving rat. Reverse dialysis of the cholinergic receptor agonist carbachol (1 mM) into the STN caused an increase in the extracellular concentrations of glutamate and dopamine in the SN. The increase in glutamate was transient and returned toward basal values despite the continued perfusions of the STN with carbachol. Carbachol-stimulated glutamate release was prolonged by perfusion of the selective D2 dopamine receptor antagonist raclopride (100 microM) into the SN and was attenuated by the perfusion of the selective D2-like receptor agonist quinpirole (10 microM). In contrast, perfusion of the D1 dopamine receptor antagonist SCH-23390 (100 microM) did not alter the carbachol stimulated glutamate release even though it increased basal glutamate concentrations. Perfusion of the GABAA receptor antagonist bicuculline (10 microM) into the SN prolonged the carbachol-stimulated glutamate release in similar fashion as raclopride. The present findings suggest that somatodendritically released dopamine in the SN regulates glutamate release from subthalamic axon terminals by differentially activating dopamine D2 and D1 receptors. Activation of D2 heteroreceptors, located on STN axon terminals, provides a negative feedback control on stimulated subthalamic glutamate release, while D1 receptor activation preferentially regulates basal glutamate concentrations. The findings of the present study also indicate that GABA exerts an inhibitory control on glutamate release in the SN through GABAA receptors. PMID- 16680829 TI - BMP and FGF regulatory pathways control cell lineage diversification of heart valve precursor cells. AB - The atrioventricular heart valve leaflets and chordae tendineae are composed of diverse cell lineages and highly organized extracellular matrices that share characteristics with cartilage and tendon cell types in the limb buds and somites. During embryonic chicken valvulogenesis, aggrecan and sox9, characteristic of cartilage cells, are observed in the AV valve leaflets, in contrast to tendon-associated genes scleraxis and tenascin, present in the chordae tendineae. In the limb buds and somites, cartilage cell lineage differentiation is regulated by BMP2, while FGF4 controls tendon cell fate. The ability of BMP2 and FGF4 to induce similar patterns of gene expression in heart valve precursor cells was examined. In multiple assays of cells from prefused endocardial cushions, BMP2 is sufficient to activate Smad1/5/8 phosphorylation and induce sox9 and aggrecan expression, while FGF4 treatment increases phosphorylated MAPK (dpERK) signaling and promotes expression of scleraxis and tenascin. However, these treatments do not alter differentiated lineage gene expression in valve progenitors from fused cushions of older embryos. Together, these studies define regulatory pathways of AV valve progenitor cell diversification into leaflets and chordae tendineae that share inductive interactions and differentiation phenotypes with cartilage and tendon cell lineages. PMID- 16680830 TI - The role of bone markers in metastatic bone disease. AB - Patients with advanced cancer often develop bone metastases that lead to significant skeletal morbidity and substantially reduced functionality and autonomy [Coleman RE, Rubens RD. Bone metastases. In: Clinical Oncology, 2nd edn. New York: Churchill Livingstone. 2004, p. 1091-128]. As current methods of diagnosing bone metastases are cumbersome and not cost-effective, there is great interest in exploring the potential of biochemical assays that measure bone turnover activity. These markers may have value in measuring metastatic bone disease (MBD) progression and identifying patients at higher risk for metastases. Bone markers are also being evaluated as a possible guide to optimize treatment of bone complications. In the reports included in this supplement, experts at the frontiers of bone marker research offer insights on their progress. PMID- 16680831 TI - Optimizing patient therapy: the role of bone markers? AB - A reduction in bone turnover activity, assayed by bone markers, appears to correlate with improved outcomes. Thus, use of bone markers to measure abnormal bone turnover may help identify patients at increased risk for bone complications or skeletal-related events (SREs). In addition, bone markers may prove useful in monitoring response to treatment and customising future treatment. Although many questions remain concerning use of bone markers in the treatment of metastatic cancer, these markers are expected to have a valuable role in managing metastatic bone disease (MBD). This review examines SREs and the burden they impose on patients, the relationship between SREs and bone turnover markers, and the potential role of bone markers in guiding treatment and improving patients' quality of life (QOL). PMID- 16680832 TI - Bone markers and current laboratory assays. AB - Metastasis of cancer to bone leads to significant alterations in normal bone remodelling that are reflected in changes in bone turnover markers. These markers are classically defined as markers of bone resorption or formation; markers of bone resorption are measures of osteoclastic activity, whereas markers of bone formation are measures of osteoblastic activity. Recently, there has been growing interest in the use of these markers in metastatic bone disease (MBD), and an increasing number of studies have investigated the potential use of these markers in diagnosis, monitoring of disease progression and treatment, and prediction of outcome. In this review, we briefly discuss the biology of bone metastases as well as describe the bone turnover markers and their possible role in aiding clinicians in the treatment of patients with MBD. PMID- 16680833 TI - Biochemical markers of bone metabolism in multiple myeloma. AB - Osteolytic bone disease is a major clinical feature of multiple myeloma (MM). Mechanisms of bone destruction are related to increased osteoclastic activity, which is not accompanied by a comparable increase in bone formation, as osteoblasts are functionally exhausted. Thus the lesions rarely heal and bone scans are often negative in myeloma patients with extensive lytic lesions, offering very little in the follow-up of bone disease. Biochemical markers of bone resorption, such as N- and C-terminal cross-linking telopeptide of type I collagen (NTX, CTX/ICTP, respectively), tartrate resistant acid phosphatase isoform-5b, bone formation (bone-specific alkaline phosphatase [BAP]), and osteocalcin provide useful information on bone dynamics. Several studies have shown that NTX, CTX, and ICTP are elevated in myeloma patients, reflect the extent of bone disease, and correlate with survival. Furthermore, they are useful in monitoring bone destruction during antimyeloma or bisphosphonate treatment. Markers of bone formation have produced conflicting results in trials. However, BAP correlates with bone pain, lytic lesions, and fractures in quite a few studies of MM. Novel markers, such as bone sialoprotein, receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin, osteopontin, dickkopf-1, and soluble Frizzle-related protein-2 have been found of value in assessing bone lytic disease in MM, but their promising results must be confirmed in large trials. In conclusion, although no marker provides optimal analysis of MM or of MM treatments, combinations of markers have at times helped in assessing MM stages and lytic bone disease and in monitoring specific treatment modalities. The need for further research in this field is clear. PMID- 16680834 TI - Biochemical bone markers in breast cancer. AB - Bone metastases, the most common metastatic manifestation of many cancers, including breast cancer and prostate cancer, contribute significantly to the pain and disability often associated with later stages of malignant disease. Although bisphosphonates have demonstrated efficacy in the treatment of metastatic bone disease (MBD), identifying patients most likely to develop bone metastases (and thus to benefit from treatment) is a challenge. In recent years, advances in understanding the pathophysiologic underpinnings of bone metastases have led to the discovery of several potential markers for dysregulation of bone coupling. Trials have been conducted to validate these biochemical markers and to explore their possible role in measuring efficacy of bisphosphonate treatment and of other metastatic bone therapies. In no area is this research more important than in the management of breast cancer. Although the value of these bone turnover markers is still unclear, and further research is necessary, results from these recent trials indicate some correlation. PMID- 16680835 TI - Markers of bone metabolism in prostate cancer. AB - Although bone metastases from prostate cancer are described as osteoblastic, markers of both osteoblastic and osteoclastic activity are strikingly elevated in men with metastatic prostate cancer. Elevated markers of osteoblastic and osteoclastic activity are associated with adverse clinical outcomes in men with prostate cancer--outcomes including shorter time to skeletal complications, disease progression, and death. Bone marker measurement appears to be a promising method for monitoring the efficacy of bone-targeted therapy. Additional studies are needed to assess the potential role of bone markers in identifying men at highest risk for development of bone metastases. PMID- 16680836 TI - Conclusion: Bone markers in metastatic bone disease. AB - Bone metastases occur frequently in patients with multiple myeloma and breast, prostate, and lung cancer. Presence of metastatic bone disease (MBD) is correlated with abnormal bone remodelling, as high levels of biochemical markers of bone turnover can be detected in serum or urine from patients with these malignancies. The idea of using bone markers in the diagnosis and follow-up of patients with MBD is undergoing validation and gaining acceptance. In this conclusion, we summarise data from leading studies relating to the search for useful markers of bone resorption and bone formation in MBD. PMID- 16680837 TI - Keeping weight-loss drugs in perspective. If you're dangerously overweight, diet pills may help--but not without major lifestyle changes. PMID- 16680838 TI - Hormone therapy heart benefit may depend on timing. PMID- 16680839 TI - Insulin by inhaler: new option for diabetes treatment. PMID- 16680840 TI - "Narrow angles" a tip-off to eyesight risk. Proper eye care can limit vision loss from most glaucoma--and prevent one of its most serious forms. PMID- 16680841 TI - Soy: not so miraculous? PMID- 16680842 TI - By the way, doctor. Recently, I read about a new vaccine against cancer of the cervix. Is this something I should consider? PMID- 16680843 TI - Minding your mind: 12 ways to keep your brain young with proper care and feeding. PMID- 16680844 TI - Facing the fat--and the facts. PMID- 16680845 TI - Prostate cancer in younger and older men. PMID- 16680846 TI - On call. I am 47 years old and I've always been healthy. My main problem is insomnia. Sleeping pills don't work well, and they make me feel groggy. Finally, my doctor gave me a prescription for Desyrel, and he seems to have hit the bull's eye. I've been sleeping much better and haven't noticed any side effects. But my pharmacist warned me about abnormal erections. Is it a serious concern? PMID- 16680847 TI - Atrial fibrillation: beyond drug therapies. Medications are the first treatment for atrial fibrillation, but if drugs don't do the job, there are other options. PMID- 16680848 TI - Inflammation: a unifying theory of disease? Research is showing that chronic inflammation may be the common factor in many diseases. PMID- 16680849 TI - Body heat: older is colder. The normal temperature of 98.6 degrees F is anything but, especially as we get older. PMID- 16680850 TI - Get a good grip while toiling in the soil. Ergonomic tools for gardeners are not what they're cracked up to be. PMID- 16680851 TI - By the way, doctor. I've read recently that organic coconut oil has health benefits. Could coconut oil improve lipid profiles if consumed every day? PMID- 16680852 TI - [The relation between blood pressure level in prehypertensives and multiple metabolic disorders]. AB - OBJECTIVE: To study the relation between blood pressure level in prehypertensives and multiple metabolic disorders. METHODS: Based on the populations in Jiangsu province from the project of Multiple Metabolic Disorders and Metabolic Syndrome, a case-control study was conducted including 585 cases of isolated systolic prehypertensive,746 cases of diastolic-systolic prehypertensive, 340 cases of isolated diastolic prehypertensive and 1755 individuals with normal blood pressure. RESULTS: The occurrence of triglyceride(TG), body mass index(BMI) and waist circumference(WC) disorders were significantly higher in the three case groups than in the normal blood pressare group. Among case groups, 50% of them had at least one more metabolic abnormalities as compared to 35% in the controls. CONCLUSION: Among patients in prehypertensive stage, TG disorder was most popular in multiple metabolic disorders. Prehypertensive was associated with WC, BMI and age, indicating that there had already been collections of cardiovascular risk factors in the prehypertensive stage. PMID- 16680853 TI - [Optimisation of the myocardial infarction pharmacotherapy--important insights from the COMMIT Trial]. PMID- 16680854 TI - [Dangers and hopes connected with the evidence based catalog in 2006. Part I]. PMID- 16680855 TI - [Against: "Every person with schizophrenia should be treated as early as possible"]. PMID- 16680856 TI - [Serratia marcescens bacteremia in a patient with choledocholithiasis]. PMID- 16680857 TI - [Kaposi sarcoma in a patient with AIDS in the syndrome of immune reconstitution]. PMID- 16680858 TI - [Pleural effusion in systemic primary amyloidosis]. PMID- 16680859 TI - [Antiphospholipid syndrome in a patient with Salmonella enteritidis bacteremia]. PMID- 16680861 TI - [Focal nodular hyperplasia in a young woman]. PMID- 16680860 TI - [Acute viral myositis in the adult]. PMID- 16680862 TI - [Lymphocytic infundibuloneurohypophysitis as a cause of diabetes insipidus]. PMID- 16680863 TI - [Internet application in oncology patients]. PMID- 16680864 TI - [Levofloxacin-induced tendon rupture with fatal outcome]. PMID- 16680865 TI - [Children of mentally ill]. PMID- 16680866 TI - [Supporting mental health and prevention mental illness--luxury or necessity?]. PMID- 16680867 TI - [Decompressive craniectomy in acute encephalitis]. AB - We report a patient with acute encephalitis of unknown causation who underwent hemicraniectomy because of threatening herniation. The patient recovered uneventfully and three weeks later, findings on neurological examination were normal. Hemicraniectomy should be considered in all patients with encephalitis and threatening herniation. PMID- 16680868 TI - [Patients with life-threatening brain diseases]. PMID- 16680869 TI - The "total" museum, a tool for social change. AB - Science museums encourage not only scientific knowledge and methodology, but also people's opinion about scientific issues. This has been the main concern of Barcelona's Museo de Ciencia de la Fundacion "la Caixa" throughout its twenty years of existence. According to the author of the present article, the goals of "total museology" comply with the new trend some museums have been following. So that this new trend becomes more sound and widespread, it is necessary to create new concepts for museology. The first science museums were natural history and tools and machinery museums, which displayed artifacts in glass cases to visitors. Their mission was also that of preserving collections for the use of scientists. Science museums of today display real phenomena and provide visitors' interaction with them. Whatever the topic it focus, a science museums is "concentrated reality" either of objects or phenomena. This is probably the main distinctive feature of museology and of other forms of scientific communication. For teachers and lecturers, words are the basic element of communication, for books and magazines, the written language. There are no films without images, as there is no radio with sounds. In a museum, there are no restrictions as to the use of stimulation, models, graphic images or new technology, but just as accessories to reality, never as reality itself. PMID- 16680870 TI - The effect of cimetidine on dextromethorphan O-demethylase activity of human liver microsomes and recombinant CYP2D6. AB - Clinically, cimetidine therapy impairs the clearance of various drugs metabolized by CYP2D6, such as desipramine and sparteine. Cimetidine is known to reversibly inhibit CYP2D6 in vitro; however, Ki values are greater than plasma concentrations observed in vivo. There is evidence suggesting that this drug may act as an inactivator of cytochrome P450 (P450) enzymes after metabolic activation. Therefore, the purpose of this study was to determine whether cimetidine acts as a mechanism-based inactivator of CYP2D6. Dextromethorphan O demethylation was used as a probe of CYP2D6 activity. The Vmax and Km of this reaction were 0.82 +/- 0.06 nmol/min/nmol of P450 and 4.1 +/- 0.1 microM, respectively, in pooled human liver microsomes; and 15.9 +/- 0.8 nmol/min/nmol P450 and 1.4 +/- 0.6 microM, respectively, with recombinant CYP2D6. With human liver microsomes, cimetidine competitively inhibited CYP2D6 (Ki = 38 +/- 5 microM) and was a mixed inhibitor of recombinant CYP2D6 (Ki = 103 +/- 17 microM). Preincubation of human liver microsomes with cimetidine and NADPH did not increase the inhibitory potency of cimetidine; however, preincubation with recombinant CYP2D6 resulted in enzyme inactivation that could be attenuated by the CYP2D6 inhibitor quinidine. The KI and kinact were estimated to be 77 microM and 0.03 min-1, respectively, and the half-life of inactivation was 25 min. Therefore, cimetidine may represent a class of compounds capable of inactivating specific cytochromes P450 in vivo, but for which conditions may not be achievable in vitro using human liver microsomes. PMID- 16680872 TI - The need to understand IRB deliberations. PMID- 16680873 TI - Part II: HIPAA and disclosure risk issues. PMID- 16680874 TI - "You may have already won..": an examination of the use of lottery payments in research. PMID- 16680875 TI - [The Museums of Morphological Sciences: a different place at the Universidade Federal de Minas Gerais]. AB - This article introduces a new science museum, one that works actively to engage its audience in learning about the structure and functioning of the human organism, so that it can be understand life and thus protect its quality. PMID- 16680876 TI - Addiction and autonomy: can addicted people consent to the prescription of their drug of addiction? AB - It is often claimed that the autonomy of heroin addicts is compromised when they are choosing between taking their drug of addiction and abstaining. This is the basis of claims that they are incompetent to give consent to be prescribed heroin. We reject these claims on a number of empirical and theoretical grounds. First we argue that addicts are likely to be sober, and thus capable of rational thought, when approaching researchers to participate in research. We reject behavioural evidence purported to establish that addicts lack autonomy. We present an argument that extrinsic forces must be irresistible in order to make a choice non-autonomous. We argue that heroin does not present such an irresistible force. We make a case that drug-oriented desires are strong regular appetitive desires, which do not compromise consent. Finally we argue that an addict's apparent desire to engage in a harmful act cannot be construed as evidence of irrational or compulsive thought. On these arguments, a sober heroin addict must be considered competent, autonomous and capable of giving consent. More generally, any argument against legalisation of drugs or supporting infringement of the liberty of those desiring to take drugs of addiction must be based on considerations of harm and paternalism, and not on false claims that addicts lack freedom of the will. PMID- 16680877 TI - [Three views from overseas: the museum as a setting for educational outreach in the sciences]. AB - During their first visit to Brazil, in 2001, Michel Van Praet, Jean Davallon, and Daniel Jacobi - French researchers in the field of museology - discussed the complex nature of the museum experience. Professor Van Praet underscores the unique character of natural history museums and offers an evaluation of his work as head of the restoration project at Paris's Grande Gallery of Evolution, inaugurated in 1994. Professor Davallon discusses the contribution of semiotics and reception theory in analyzing how an exhibit communicates and how meaning is constructed within it. Dr. Jacobi defines some characteristics of the dissemination of science at museums and points to problems encountered in achieving this end. PMID- 16680878 TI - Addiction, autonomy and ego-depletion: a response to Bennett Foddy and Julian Savulescu. PMID- 16680879 TI - Autonomy, addiction and the drive to pleasure: designing drugs and our biology: a reply to Neil Levy. PMID- 16680882 TI - [Lights, art, science - action!]. AB - The article offers some reflections on the main interactions between theater, science, and technology down through the history of theater. Based on our experience at "Science in the Spotlight", part of the Casa de Oswaldo Cruz's Museum of Life, we discuss how these interactions can be part of a science museum's daily activities. We use the word 'science' in its broad sense, encompassing not only the natural but human sciences as well; likewise, we use the word 'technology' as it relates to applied science. Art and science are understood here as creative processes, as ways of representing the world and expressing human knowledge. PMID- 16680883 TI - It's time to go public with neuroethics. PMID- 16680884 TI - [Latin American science museums and equity]. AB - Latin America and the Caribbean form a region of great variations yet marked by cultural resemblances. The origins of the region's countries, of their wealth, and also of their problems are quite similar. Indeed, there are clear points of contact in the most basic aspects, such as each society's worldview. Throughout the region there is a very strong democratizing trend that seeks more just and more educated societies. Science plays a key role in the development as a powerful weapon for tolerance and equity and therefore should be disseminated among the greatest possible number of Latin Americans. Red POP the network for the Popularization of Science and Technology in Latin America and the Caribbean - was created to help reach this goal. Under the auspices of ORCYT-UNESCO, Red POP is an interactive network of centers and programs that work to bring science and technology to the public at large. It fosters exchange, skill-acquisition, and resource use among its members. Based on the network's experience, we explore to what extent science museums favor equity in their home societies. PMID- 16680885 TI - [Poison and the mosquito: epistemological aspects of the etiology and prophylactics of yellow fever]. AB - The strategies against yellow fever developed by Argentina and Brazil were discussed at the Second Medical Congress of Latin America which was held in Buenos Aires in 1904. The study of the controversy between physicians from Argentina and Brazil around the existing explanatory models of this illness and the international prophylactic strategies in use at the time enables an epistemological understanding of the breakthrough brought about by the emergence of medicine of vectors. This chapter of Latin American medicine history constitutes a unique opportunity to analyze that reorganization of knowledge, which permitted the inclusion of intermediary living beings into the medical and epidemiological discourse. PMID- 16680887 TI - Meeting the challenges of teaching and research: AVTRW's 60th annual conference. PMID- 16680888 TI - [The construction of the "sick-subject" in RSI]. AB - There is a running debate over the relationship between work and disease, namely the disease known as repetitive strain injury (RSI), or else work-related osteomuscular disturbances (in Portuguese DORT), as it was renamed in 1997 by the Instituto Nacional de Seguridade Social (INSS) in accordance with the Disability Assessment Norms. Standing out among the signs brought about by this new infirmity process is its restricted focus on the subjective aspects in the analysis of the disease. Underlying the INSS stance is the view of a sickness prone subject and the notion of health as capacity for work. This paper analyzes the historical context which characterizes these health/work and body/disease notions. It also discusses the construction of the 'sick-subject' and the way in which such a subjectivizing process entraps the subject itself. Finally, we show the way in which the INSS in Rio de Janeiro has managed to stop this disease of its work-related characteristics, fighting and reducing the occurrences of RSI as well as the costs with work compensation claims. PMID- 16680889 TI - [Water pipelines conduits and urban sanitation in Cartagena in the beginning of the twentieth century]. AB - Throughout its history, Cartagena de Indias, a seaport in the Colombian Caribbean, has been handicapped for not offering salubrious conditions to its people and visitors. The lack of an aqueduct and a sewerage system was an impairment to progress. For nearly forty years (1890-1930) these problems have caused a myriad of medical discourses formulated by scientists, technicians and politicians. Cartagena's contribution to solve the sanitation problem in cities has consisted in making use of engineers' knowledge. The construction of urban facilities in the beginning of the twentieth century required a more technical knowledge, one which would advance a comprehensive solution to the water problem, ensure sufficient supply and efficient drainage. Thus, in the last turn of the century, the medical doctor is no longer the only authoritative voice when it comes to the management of urban life. The construction works which require an engineer, involving him in public health, have drawn a distinction between "hygiene" and "sanitary science". PMID- 16680890 TI - [Richard Spruce, botanist-South America's explorer]. AB - Between 1849 and 1864, the English botanist and explorer Richard Spruce carried out a detailed study of the Amazon flora and the costumes of the peoples who inhabited the region. To date a large part of the existing knowledge about several botanical families in the region stems from this scientist's efforts. His comprehensive interests, his detailed and precise descriptions were outstanding: nothing seems to have been left out of his scrutiny and recording aptitude. Not only was Spruce a remarkable botanist but he was also a distinctive anthropologist, linguist (he knew French, Spanish and Portuguese), geologist and geographer, as well as an acute sociological observer of the political systems and habits of the Amazonian and Andean trips in which has has been. He could thus make a considerable contribution to the understanding of indigenous beliefs and practices, as well as to the knowledge and uses of plants within the Amazonian context. Also important was his participation in the economic exploration of local species, particularly as regards the Hevea and the Cinchona genera. PMID- 16680893 TI - [Videographies of the heart: an ethnographic study of the cardiac catheterization]. PMID- 16680894 TI - [GMOs on the menu: crack them before eating!]. PMID- 16680895 TI - [Much ado about nothing]. PMID- 16680896 TI - [The scientific community is in doubt]. PMID- 16680897 TI - [Transgenics and controlled evolution]. AB - Mutation events are responsible for the generation of genetic variability in the populations enabling the occurrence of natural selection which favors the better adapted types. The exploitation of this variability, though carried out empirically, dates from ten thousand years ago with the domestication of the first cultivated crops. With the advent of genetics, rational selection procedures were adopted with a view of the genetic breeding of plants, animals and microorganisms which might be of interest to men. Recently, new DNA manipulation techniques came up enabling the transference of genes between organisms, cutting across barriers which hindered crossing between the vegetable, animal, protist and fungus kingdoms. The generation of genetically modified organisms, or transgenics, has aroused a heated and controversial debate in various sectors of our society. Yet we must be cautious before generalizing the use of transgenics since each one should be analyzed at a time for its particular advantages and drawbacks, and for its contribution to the improvement of life quality. This paper also considers recent methods of mutation and in vitro genic recombination. PMID- 16680898 TI - [A technology with multiple applications]. AB - Plant breeding has been a human practice for some thousands of years. However, this process of domestication has made plants more vulnerable to pests and diseases. Classic plant breeding has allowed the genetic manipulation of plants through crossings with a resulting increase in crop productivity. Recently, the recombinant DNA technology has increased the possibilities of integration of exogenous genes to the plant genome, resulting in the production of transgenic plants. Despite the great debate on this issue, such plants represent to date a promising avenue for plant breeding. There are many examples of gene transference strategies which have been successful in promoting resistance to herbicides, viruses, fungi, bacteria and insects, or in producing an increase in food quality. In addition to biotechnological applications, transgenic plants have made a significant contribution to the study of gene functioning, such as the analysis of genic expression regulation and the study of protein functions codified by distinct plant genes. PMID- 16680899 TI - [Implications of transgenics for environmental and agricultural sustainability]. AB - The potential risks of GMOs, their impact on human and animal health, and on the environment, as well as their socioeconomic effects, have generated a worldwide discussion which is far from drawing to a close for lack of sufficient information. Part of this information supports risk-hypotheses previously put forward. Thus the presence of transgenic plant genes in other plants and in other organisms has been confirmed in several occasions. Therefore, gene dissemination to plants of the same species as well as to widely different species is already regarded as an actual risk. The principle of substantial equivalence has opened the way for the liberation of transgenic plants for commercial crops, despite short-term tests, which are quantitatively and qualitatively insufficient to certify that the foods deriving from those plants are healthy and safe. Thus, the adoption of the so-called precautionary principle (PP) has turned out to be the most adequate safety measure to date, or else until scientific data should be able to demonstrate the actual impact of transgenic plants on human and animal health, and on the environment. PMID- 16680900 TI - [Transgenics without Manichaeism]. AB - We live in an era characterized by the hegemony of science and technology, an era fraught with questions awaiting answers which would enable a safe and sustainable future for humankind. The development of agro-industrial processes - food products in particular - through recombinant DNA technology has enhanced the profit prospects of the few big biotechnology companies and of large-scale farmers who have access to the latest technological developments. We thus oppose a moratorium on recombinant DNA technology. Moreover, hasty statements about risk free transgenics may be misleading in the absence of extensive safety tests. There is a pressing need for the establishment of biosafety policy in this country involving the organized civil society and every government agency responsible for monitoring such matters. There is also the need to put in place a bio-surveillance and a code of ethics regarding genetic manipulation. PMID- 16680901 TI - Chromosome 2q24.2 is lost in sporadic but not in BRCA1-associated ovarian carcinomas. AB - AIMS: Comparison between BRCA1-associated and sporadic ovarian carcinomas is a potential method to identify candidate modifier gene/s involved in the carcinogenic pathway of either or both groups. A previous study identified a significant difference in the frequency of copy number gain at 2q24-q32 by comparing BRCA1-associated and sporadic ovarian tumour specimens using comparative genomic hybridisation (CGH). The present study aimed to investigate the reported allelic imbalance at 2q24-32 by amplification of several microsatellite markers at the region by quantitative microsatellite analysis (QuMA) using Taqman at the same region identified as a site of allelic imbalance. METHODS: The copy number of the genomic region in 2q24-32 was established in 21 BRCA1-associated ovarian carcinomas and 14 sporadic cases using quantitative microsatellite polymerase chain reaction (PCR). Statistical analysis was performed using permutation test analysis. RESULTS: A significant loss at D2S156 marker (2q24.2) (p = 0.026) compared with the other three markers at 2q24-32 was found in the sporadic cohort but not in the BRCA1-associated group (p = 0.385). CONCLUSIONS: Our data do not support the association between copy number gain at 2q24-32 and BRCA1 mutation status in ovarian cancers reported previously. The novel finding of the present study was significant loss at 2q24.2 in sporadic ovarian cancers. PMID- 16680902 TI - Primary immunodeficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005. AB - Although relatively rare, primary immunodeficiency diseases (PIDs) provide an excellent window into the functioning of the immune system. In the late 1960s, observations on these diseases, with their associated infections and genetics, bisected the immune system into humoral immunity and cell-mediated immunity. These diseases also represent a challenge in their diagnosis and treatment. Beginning in 1970, a unified nomenclature for the then-known PIDs was created by a committee convoked by the World Health Organization. Since then, and later under the aegis of the International Union of Immunological Societies, an international committee of experts has met every 2 to 3 years to update the classification of PIDs. During the past 15 years, the molecular basis of more than 120 PIDs has been elucidated. This update results from the latest meeting of this committee in Budapest, Hungary, in June 2005, which followed 2 1/2 days of scientific discussions. As a result of this work, new entities have been included, and the nomenclature of some PIDs (specifically of the various forms of class-switch recombination defects, previously known as hyper-IgM syndromes) has been changed. PMID- 16680903 TI - Identification of cellular cooperation in antibody production. PMID- 16680904 TI - Thymocytes and bone marrow cells in 1966: where did we go from there? PMID- 16680905 TI - Returning genetic research results to individuals: points-to-consider. AB - This paper is intended to stimulate debate amongst stakeholders in the international research community on the topic of returning individual genetic research results to study participants. Pharmacogenetics and disease genetics studies are becoming increasingly prevalent, leading to a growing body of information on genetic associations for drug responsiveness and disease susceptibility with the potential to improve health care. Much of these data are presently characterized as exploratory (non-validated or hypothesis-generating). There is, however, a trend for research participants to be permitted access to their personal data if they so choose. Researchers, sponsors, patient advocacy groups, ethics committees and regulatory authorities are consequently confronting the issue of whether, and how, study participants might receive their individual results. Noted international ethico-legal guidelines and public policy positions in Europe and the United States are reviewed for background. The authors offer 'Points-to-Consider' regarding returning results in the context of drug development trials based on their knowledge and experience. Theses considerations include: the clinical relevance of data, laboratory qualifications, informed consent procedures, confidentiality of medical information and the competency of persons providing results to participants. The discussion is framed as a benefit to-risk assessment to balance the potential positive versus negative consequences to participants, while maintaining the integrity and feasibility of conducting genetic research studies. PMID- 16680906 TI - Neuroethics. AB - Neuroimaging, psychosurgery, deep-brain stimulation, and psychopharmacology hold considerable promise for more accurate prediction and diagnosis and more effective treatment of neurological and psychiatric disorders. Some forms of psychopharmacology may even be able to enhance normal cognitive and affective capacities. But the brain remains the most complex and least understood of all the organs in the human body. Mapping the neural correlates of the mind through brain scans, and altering these correlates through surgery, stimulation, or pharmacological interventions can affect us in both positive and negative ways. We need to carefully weigh the potential benefit against the potential harm of such techniques. This paper examines some of these techniques and explores the emerging ethical issues in clinical neuroscience. PMID- 16680907 TI - Syndromic Surveillance: is it a useful tool for local outbreak detection? PMID- 16680908 TI - Users fees and worst off: it's time to find a solution. PMID- 16680909 TI - Young adults' reluctance to seek help and use medications for mental distress. PMID- 16680910 TI - Reliability and validity of the Safety Assessment Scale for people with dementia living at home. AB - BACKGROUND: With the recent funding changes in health care services, more seniors with dementia are cared for in their homes and the nature of their condition puts them at higher risk for accidents and injuries. PURPOSE: The Safety Assessment Scale (SAS) was developed for use by community healthcare providers to evaluate and lower the risk of accidents, provide recommendations to family caregivers and enhance case management. Method. The scale was assessed in terms of the reliability as well as content, criterion and construct validity with 176 community-residing people with dementia in Quebec, Alberta and British Columbia. The Safety Assessment Scale was validated in French and English. RESULTS: The SAS demonstrated an excellent test-retest (ICC=.91) and inter-rater (ICC=.88) reliability. Results for validity were also extremely good. PRACTICE IMPLICATIONS: The scale is available in French and English. The short version of the SAS is a screening tool and the longer version provides an in-depth evaluation of safety and intervention planning. The SAS can be used by a variety of health care professionals who work with individuals who have dementia. PMID- 16680911 TI - Developing empathy as a foundation of client-centred practice: evaluation of a university curriculum initiative. AB - BACKGROUND: The foundation of client-centred practice is the therapist's capacity to view the world through the client's eyes and to develop an understanding of the lived experience of disability. PURPOSE: This paper describes the evaluation of an educational initiative promoting student empathy to the lived experience of disability. METHODS: Pairs of first-year occupational therapy students visited adults with disabilities who shared their knowledge and experience of living with a disability. Students reflected on their visits in journals, which were later analyzed using pattern matching. FINDINGS: Students appeared to appreciate the co existence of health and disorder and demonstrated a holistic understanding of living with a disability. Little attention was focused on cultural and institutional environments. Students struggled to define the nature of their relationship with their tutors. Practice Implications. The evaluation confirmed our belief that this educational initiative could facilitate student empathy, consistent with critical features of client-centred practice. PMID- 16680912 TI - An argument for pragmatism as a foundational philosophy of occupational therapy. AB - BACKGROUND: In 1986, Estelle Breines argued that occupational therapy identity problems could be resolved by acquiring tools that enable therapists to analyze their profession in a sophisticated manner. Breines identified philosophical inquiry as one of these tools. PURPOSE: This study examined how pragmatism can provide a philosophical framework for occupational therapy practice and knowledge. METHOD: An analysis of a sample of occupational therapy literature, philosophy of pragmatism literature, and Slagle lectures delivered between 1955 and 1995 was completed. Themes underlying the assumptions, principles, and values articulated in the literature and the Slagle lectures were identified and compared. RESULTS: Findings of the analysis indicate that some of the core assumptions, principles, and values of occupational therapy as identified in occupational therapy literature and the Slagle lectures are conceptually consistent with those of the philosophy of pragmatism. PRACTICE IMPLICATIONS: Pragmatism may provide a philosophical framework to organize the knowledge and practice of occupational therapy and assist with clarifying the profession's identity. PMID- 16680913 TI - Effects of workplace policy on continuing professional development: the case of occupational therapy in Nova Scotia, Canada. AB - BACKGROUND: Continuing professional development is essential for professionals to remain competent, and for effective recruitment and retention. PURPOSE: This paper reports a qualitative study of the effects of workplace policy on continuing professional development on a small, dispersed profession in a resource-challenged province, using the case example of occupational therapy in Nova Scotia. METHODS: The study used a multi-methods design, theoretically based on institutional ethnography. Methods were critical appraisal of the literature, interview and focus group data collection with 28 occupational therapists and 4 health services administrators, and a review of workplace policy. RESULTS: The study identified a policy wall. Notable policies were those which defined who is responsible for continuing professional development, and which limited employee benefits and work flexibility options for those with family duties. It appears that a female-dominated profession, such as occupational therapy, may also face gender-based challenges. PRACTICE IMPLICATIONS: Suggestions are offered for workplace policy makers, unions, provincial regulatory organizations, and health professionals. The findings are generally applicable to any small, dispersed health profession operating in resource-challenged conditions. PMID- 16680914 TI - Occupational therapy research on assistive technology and physical environmental issues: a literature review. AB - BACKGROUND: To determine future directions for research in the area of assistive technology and physical environmental issues, it is important to have an understanding of prior research. PURPOSE: This literature review examined how assistive technology and physical environmental issues have been studied in the research published in international peer-reviewed occupational therapy journals. METHOD: Five recent volumes of nine journals were manually searched utilizing specific criteria. The publications were classified according to their perspective, application of the Person-Environment-Occupation (PEO) model, and the research design. RESULTS: Both research fields demonstrated use of different research methods and they displayed equal needs with regard to improved research methodologies. PRACTICE IMPLICATIONS: There are a lack of studies involving all three PEO components indicating a lack of research in occupational performance issues. Further research on occupational performance is important for developing occupational therapy practice in the area of assistive technology and physical environmental issues. Furthermore, study designs reflecting the societal level in all three PEO components are required. Finally, there is a strong need for conceptual and theoretical development in both fields. PMID- 16680915 TI - Canadian Association of Occupational Therapists position statement. Occupational therapy and home and continuing care. PMID- 16680916 TI - Canadian Association of Occupational Therapists position statement. Occupational therapy and primary health care. PMID- 16680917 TI - Cedar elm. PMID- 16680918 TI - Over-the-counter intranasal corticosteroids: empowering the patient or putting them in harm's way? PMID- 16680919 TI - Allergy, nonallergy, and the role of the allergist. PMID- 16680920 TI - The link between the Hippocratic Oath and evidence-based medicine. PMID- 16680921 TI - Concerns about intranasal corticosteroids for over-the-counter use: position statement of the Joint Task Force for the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology. AB - The Joint Task Force for the American Academy of Allergy, Asthma and Immunology and the American College of Allergy, Asthma and Immunology was charged with formulating a position paper regarding the potential release of intranasal corticosteroids for over-the-counter use. We took the position that safety issues regarding this proposal would be our sole concern. We reviewed the literature to evaluate the frequency and severity of potential adverse events related to the administration of intranasal corticosteroids. We limited this review to 5 areas: (1) effects on growth, (2) ocular effects, (3) effects on bone, (4) effects on the hypothalamic-pituitary-adrenal axis, and (5) local adverse effects. After review of the available data, we concluded that intranasal corticosteroids should remain prescription-only drugs. Patients receiving an intranasal corticosteroid should be instructed in its use and that use should be monitored by a physician or an appropriately trained medical provider (eg, nurse practitioner or physician assistant) under the direct supervision of a physician. This conclusion was reached based on the evidence that corticosteroids administered by any route, including the intranasal route, have the potential to cause adverse effects in all the areas noted herein. Our conclusion was strengthened by the fact that these adverse effects can be insidious and therefore not evident for many years; there is the potential for overuse; patients could also have access to other forms of topically administered corticosteroids, thus increasing their total dose; and individuals vary in their susceptibility to corticosteroid-induced adverse effects. We were also influenced to take this position knowing that generally reassuring data regarding the use of respiratory tract-administered corticosteroids are based on mean data and that all such studies have shown outliers in whom adverse effects were evident. Thus, as stated, we recommend that intranasal corticosteroids remain prescription-only drugs. PMID- 16680922 TI - Questionnaire evaluation and risk factor identification for nonallergic vasomotor rhinitis. AB - BACKGROUND: Chronic rhinitis, a prevalent primary care disorder associated with significant comorbidities, is often incorrectly diagnosed. As a result, inappropriate treatment leads to higher health care costs and poor clinical outcomes. OBJECTIVE: To verify whether responses to specific questions may help in the diagnosis of nonallergic vasomotor rhinitis (NAVMR). METHODS: A questionnaire listing allergic and nonallergic triggers was blindly distributed to 100 random new patients with chronic rhinitis in an allergist's office. Questionnaire and physician diagnoses were compared. Allergic triggers included cat, dog, feathers, other furry animals, and symptoms during the spring, summer, and fall seasons. Nonallergic triggers included temperature changes, diesel and car exhaust, tobacco smoke, perfumes and fragrances, incense, cleaning products, newsprint, hairspray, and alcoholic beverages, spicy foods, or eating. RESULTS: The construct validity and internal consistency indicated that the questionnaire was a good tool for diagnosing NAVMR. Multivariate analysis revealed that the absence of outdoor symptoms in the spring (odds ratio [OR], 7.76; 95% confidence interval [CI], 1.18-51.22; P = .03), no parental history of allergy (OR, 5.16; 95% CI, 1.05-25.20; P = .04), no symptoms around cats (OR, 3.82; 95% CI, 0.59 24.83; P = .16), the presence of symptoms around perfumes and fragrances (OR, 4.875; 95% CI, 1.05-22.60; P = .04), and age at symptom onset (OR, 1.08; 95% CI, 1.02-1.14; P = .008) were predictive of NAVMR. CONCLUSIONS: Specific questionnaire responses may help primary care physicians accurately diagnose NAVMR. Questions asked of patients by physicians to diagnose allergic rhinitis do not correlate well with physician diagnosis. PMID- 16680923 TI - Once-daily evening administration of mometasone furoate in asthma treatment initiation. AB - BACKGROUND: In a previous study, a 200-microg once-daily evening dose of mometasone furoate dry powder inhaler (DPI) was effective in patients with asthma previously taking inhaled corticosteroids. No studies have been conducted to test the effect of a once-daily evening dose in patients previously using only short acting beta2-adrenergic agonists (SABAs) for symptom relief. OBJECTIVE: To evaluate the effectiveness of mometasone furoate DPI administered once daily in the evening as initial controller therapy in patients previously using SABAs alone for asthma. METHODS: Patients with mild-to-moderate persistent asthma from 18 US centers participated in a 12-week, randomized, double-blind, placebo controlled study. Patients received either mometasone furoate DPI, 200 microg, or placebo once daily in the evening. The primary efficacy variable was the change in forced expiratory volume in 1 second from baseline to the end point (last evaluable visit). Other measurements included forced vital capacity, forced expiratory flow between 25% and 75%, morning and evening peak expiratory flow, asthma symptoms, use of albuterol, nocturnal awakenings, physicians' evaluation of response to therapy, and time to asthma worsening. RESULTS: At the end point, the mean increase in forced expiratory volume in 1 second relative to baseline for the mometasone furoate DPI group of 0.43 L (16.8%) was significantly greater than that for the placebo group of 0.16 L (6.0%) (P < .01). Morning peak expiratory flow, forced vital capacity, and forced expiratory flow between 25% and 75% also significantly improved with mometasone furoate DPI treatment relative to placebo (P < .01). Once-daily dosing with mometasone furoate DPI was well tolerated. CONCLUSION: Mometasone furoate DPI (200 microg) administered once daily in the evening significantly improves pulmonary function in patients previously using SABAs alone for asthma control. PMID- 16680924 TI - Effect of montelukast on peripheral airflow obstruction in children with asthma. AB - BACKGROUND: Montelukast is a widely used controller agent in childhood asthma. It is modestly effective in reducing symptoms, decreasing the need for rescue albuterol, and improving forced expiratory volume in 1 second (FEV1). OBJECTIVE: To determine whether montelukast therapy improves peripheral airway obstruction as measured by lung volumes, air trapping, airway resistance (Raw), and specific conductance (Sgaw). METHODS: Twenty-one children aged 9 to 18 years with mild-to moderate asthma were randomized into a double-blind, placebo-controlled study to receive montelukast (5 or 10 mg) or matching placebo daily for 8 weeks. Symptoms and albuterol use were recorded twice daily, and exhaled nitric oxide measurement, forced oscillometry, spirometry, and body box plethysmography (before and after beta-agonist use) were performed at randomization and at 2, 4, 6, and 8 weeks. Circulating eosinophil counts and serum eosinophil cationic protein (ECP) levels were obtained at randomization and at 8 weeks. RESULTS: Montelukast-treated patients had lower residual volume (P = .05), residual volume total lung capacity ratio (P = .04), Raw (P = .02), Sgaw (P = .03), and serum ECP levels (P = .02) at 8 weeks compared with those treated with placebo. There was a trend toward reduced daytime and nighttime albuterol use, although the difference did not reach statistical significance. There were no significant differences in FEV1, FEV1-forced vital capacity ratio, exhaled nitric oxide levels, or daytime and nighttime symptom scores between the 2 groups. CONCLUSIONS: Montelukast therapy was associated with less air trapping, hyperinflation, and Raw and better Sgaw compared with placebo. Lower serum ECP levels, a surrogate measure of airway inflammation, were associated with improvements in lung function. PMID- 16680926 TI - Isolation of the main allergen Fra e 1 from ash (Fraxinus excelsior) pollen: comparison of the natural and recombinant forms. AB - BACKGROUND: Fra e 1 is a major allergen for ash pollen-sensitized individuals in northern and central Europe. It belongs to the Ole e 1-like family and displays high cross-reactivity with taxonomically related members. OBJECTIVES: To isolate and characterize natural Fra e 1 (nFra e 1) from ash pollen and to compare its structural, antigenic, and allergenic properties with those of its recombinant form (rFra e 1). METHODS: The allergen was isolated by means of gel permeation chromatography and reverse-phase high-performance liquid chromatography columns. Molecular characterization was performed by means of Edman degradation, mass spectrometry, circular dichroism, concanavalin A lectin reaction, and anti horseradish peroxidase polyclonal antibody. Immunologic characterization was performed using immunoblotting and enzyme-linked immunosorbent assay, inhibition experiments, and histamine release assays with serum samples from allergic patients with well-known reactivity to Fra e 1 or Ole e 1 and with polyclonal antiserum and monoclonal antibodies against Ole e 1. The protein used as a reference was rFra e 1, which was produced in the yeast Pichia pastoris. RESULTS: Purified nFra e 1 appeared as 5 variants with different glycosylation degrees. Both nFra e 1 and rFra e 1 were equivalently folded as deduced from the spectroscopic analysis using circular dichroism. Both molecules share the antigenic and allergenic epitopes after the purification process, and the glycan group of nFra e 1 is a potential epitope. Natural Fra e 1 displayed strong cross reactivity with Ole e 1. CONCLUSIONS: Natural Fra e 1 is a heterogeneously glycosylated protein with high allergenic relevance. It displays structural, antigenic, and allergenic similarity with rFra e 1. Both proteins could be used for clinical purposes. PMID- 16680925 TI - Effect of sodium sulfite on mast cell degranulation and oxidant stress. AB - BACKGROUND: Sulfur dioxide is 1 of 6 environmental pollutants monitored by the Environmental Protection Agency. Its ability to induce bronchoconstriction is well documented. It is highly soluble, initially forming sulfite ions in solution. Sulfur oxides are important constituents of other pollutants, such as diesel exhaust and fine particulates. OBJECTIVE: To investigate the cellular responses of sulfite on cultured mast cells (rat basophilic leukemia [RBL-2H3] cells) and human peripheral blood basophils. METHODS: Sulfite-induced mast cell degranulation and intracellular production of reactive oxygen species were evaluated in the presence and absence of antioxidants and inhibitors of redox metabolism. Degranulation was determined using beta-hexosaminidase, serotonin, and histamine release assays. Induction of intracellular reactive oxygen species generation was determined using the redox-sensitive dye 2',7'-dichlorofluorescein diacetate. RESULTS: Sodium sulfite induced degranulation and the generation of intracellular reactive oxygen species in RBL-2H3 cells. These responses were inhibited by the free radical scavenger tetramethylthiourea and the flavoenzyme inhibitor diphenyliodinium but not by depletion of extracellular calcium. Peripheral blood basophils also showed histamine release after exposure to sodium sulfite CONCLUSIONS: Sulfite, the aqueous ion of sulfur dioxide, induces cellular activation, leading to degranulation in mast cells through a non-IgE-dependent pathway. The response also differs from IgE-mediated degranulation in that it is insensitive to the influx of extracellular calcium. The putative pathway seems to rely on activation of the reduced form of nicotinamide adenine dinucleotide phosphate oxidase complex, leading to intracellular oxidative stress. PMID- 16680927 TI - Allergenicity of 10 different apple varieties. AB - BACKGROUND: More than 100 apple varieties are consumed worldwide. However, the allergenic composition of most apple varieties has not been fully characterized. OBJECTIVE: To analyze the antigenic and allergenic profiles of 10 different commercially available apple varieties. METHODS: Golden, Golden Perlim, Reineta, Reineta Parda, Fuji, Verdedoncella, Granny-Smith, Pink Lady, Royal Gala, and Starking apple varieties were carefully peeled. Peels were individually extracted and centrifuged, and the supernatants were collected, dialyzed, filtered, frozen, and freeze-dried. The extracts were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblots. Protein content was measured using the Lowry-Biuret method. Twenty-two Spanish patients with oral allergy syndrome after apple ingestion were included in the study. Specific IgE to all apple varieties and birch pollen was evaluated. Eleven patients underwent skin prick testing using extracts of Golden, Starking, and Reineta Parda varieties to determine differences in their in vivo allergenicity. RESULTS: The antigenic profile of the 10 varieties showed differences using sodium dodecyl sulfate polyacrylamide gel electrophoresis. A 9-kDa band, which could correspond to Mal d 3, was visualized in all the extracts. The Starking variety had the highest protein content. Higher specific IgE values were also obtained for the Starking variety, and the lowest values were for Reineta Parda and Royal Gala. The largest wheals were obtained in all patients with the Starking variety, and the smallest with Reineta Parda. Fourteen patients (64%) recognized Mal d 3 in the Golden variety using immunoblots; 6 patients (27%), who also had specific IgE to Betula, recognized bands with higher molecular weights. CONCLUSIONS: We demonstrated differences in the antigenic and allergenic profiles of the analyzed varieties and a significant variation in Mal d 3 content. PMID- 16680928 TI - Effect of animal contact and microbial exposures on the prevalence of atopy and asthma in urban vs rural children in India. AB - BACKGROUND: Environmental factors, including microbial exposures and close animal contact, are implicated in the lower prevalence of asthma and allergy in rural vs urban children. OBJECTIVES: To determine (1) the prevalence of asthma, rhinitis, eczema, and atopic sensitization in rural and urban children in India; (2) differences in microbial and animal exposures in these locales; and (3) whether differences in environmental exposures account for the different rates of asthma and atopy in these locales. METHODS: One child from each of 50 urban (Mysore) and 50 rural (Vinobha) households in southern India was randomly selected for data analysis. Allergy, asthma, health, environment, and lifestyle information was obtained using a questionnaire and household inspections. Atopy was determined via skin prick testing for common allergens. Endotoxin content was measured in house dust samples. RESULTS: Children from rural vs urban areas had lower prevalences of self-reported asthma (8% vs 30%; P = .005), rhinitis (22% vs 42%; P = .03), and atopic sensitization (36% vs 58%; P = .03). Higher median dust endotoxin loads were found in rural vs urban households (6.50 x 10(4) EU/m2 vs 1.27 x 10(4) EU/m2; P < .001). In multivariate analysis, close indoor animal contact (adjusted odds ratio [OR] 0.2; 90% confidence interval [CI], 0.05-0.9), outdoor animal contact (OR, 0.3; 90% CI, 0.1-0.8), and exclusive breastfeeding for at least 6 months (OR, 0.2; 90% CI, 0.1-0.5) were associated with lower atopic sensitization; mud flooring was associated with lower self-reported wheezing (OR, 0.1; 90% CI, 0.02-1.0). CONCLUSION: Children in India who live with close animal contact and mud flooring and who were exclusively breastfed in infancy are less likely to develop asthma, rhinitis, and atopic sensitization. PMID- 16680929 TI - Occupational allergic rhinoconjunctivitis and asthma to goat and cross-reactivity with cow epithelium. AB - BACKGROUND: The development of sensitization and symptoms after the inhalation of epithelial allergens is common. OBJECTIVES: To investigate the allergic response (in vivo and in vitro) of 3 individuals clinically sensitive and occupationally exposed to cow and goat and to evaluate the allergenic cross-reactivity between cow and goat epithelium extracts. METHODS: Three patients--a butcher and 2 doctors in veterinary medicine--were evaluated. These patients reported allergic respiratory symptoms after occupational exposure to goats and cows. Extracts were prepared from epithelia of both animals. All the patients underwent organ specific allergen challenges with cow and goat extracts. Four nasal and 2 bronchial challenges were conducted following standardized procedures. RESULTS: All 3 patients had positive challenge results with the offending allergen extract. Specific IgE to goat allergens was detected in all patients. Several IgE binding bands were detected. Inhibition assays (enzyme allergosorbent test and immunoblots) confirmed moderate-to-high cross-reactivity between goat and cow extracts. CONCLUSIONS: We confirm clinical sensitivity and specific IgE binding to goat and cow allergens in occupationally exposed individuals. There was good correlation among the clinical history, exposure, and the laboratory findings. PMID- 16680930 TI - Hypersensitivity to black locust (Robinia pseudoacacia) pollen: "allergy mirages". AB - BACKGROUND: The allergenicity of the ornamental tree Robinia pseudoacacia, or black locust, is unknown. OBJECTIVE: To evaluate the prevalence of sensitization to R. pseudoacacia pollen, its possible allergenic cross-reactivity with other common pollens, and the potential implication of pollen panallergens (profilin, polcalcin, and 1,3-beta-glucanase) as a cause of sensitization to R. pseudoacacia pollen. METHODS: Skin prick testing with R. pseudoacacia pollen was performed in 149 patients with pollinosis. Nasal challenge with R. pseudoacacia pollen was performed in 10 patients. The prevalence of sensitization to the recombinant forms of profilin (rChe a 2), polcalcin (rChe a 3), and the N-terminal of the 1,3 beta-glucanase (rNtD of Ole e 9) was investigated. Immunoblotting, enzyme-linked immunosorbent assay, and competitive inhibition assays were performed with R. pseudoacacia pollen and recombinant pollen allergens. RESULTS: Sixty-four patients (43%) had positive skin prick test reactions to R. pseudoacacia pollen. Nasal challenge results were positive in 5 sensitized patients and negative in 4 controls and 1 sensitized patient. The allergenic profile of R. pseudoacacia pollen comprises at least the panallergen profilin, a calcium-binding protein, and a 1,3-beta-glucanase. The prevalence of sensitization to rChe a 2, rChe a 3, and rNtD of Ole e 9 was 60%, 33%, and 87%, respectively, among patients sensitized to R. pseudoacacia pollen. Binding of IgE to R. pseudoacacia extract was completely inhibited by Robinia, Chenopodium, Olea, Cupressus, and Lolium extracts. CONCLUSIONS: The high prevalence of R. pseudoacacia pollen sensitization in patients with pollinosis is likely to be due to cross sensitization to panallergens (profilin, polcalcin, and 1,3-beta-glucanase) from other common pollens. This phenomenon may lead to a diagnosis of "allergy mirages." PMID- 16680932 TI - Effect of pretreatment with fexofenadine on the safety of immunotherapy in patients with allergic rhinitis. AB - BACKGROUND: Allergen-specific immunotherapy, although not a cure, remains the only treatment available that can alter the natural course of an allergic disease. However, the risk of allergen specific immunotherapy-related systemic reactions (SRs), reported to occur in approximately 1% to 14% of patients and which can range from mild to fatal in seriousness, represents a barrier to implementing this unique and effective treatment option. OBJECTIVE: To explore the possibility that pretreatment with the H1-antihistamine fexofenadine could prevent the occurrence of severe SRs induced by immunotherapy in Japanese patients with allergic rhinitis. METHODS: In this open-label, multicenter study, 134 patients receiving immunotherapy for allergic rhinitis were randomized 1:1 to a group receiving pretreatment with fexofenadine hydrochloride (60 mg) 2 hours before immunization injection (n = 67) or to a control group receiving no pretreatment (n = 67). Patients were further grouped into those who received cedar pollen immunotherapy and those who received dust mite immunotherapy. RESULTS: Pretreatment with fexofenadine 2 hours before immunotherapy significantly reduced the occurrence of severe SRs (P = .03), significantly increased the proportion of patients receiving cedar pollen immunotherapy who achieved the target maintenance dose (TMD) (P = .03), and significantly reduced the length of time to attain the TMD (P = .047 and P = .003 for patients receiving cedar pollen and dust mite immunotherapy, respectively). CONCLUSIONS: This study suggests a novel role for fexofenadine in enhancing the safety of immunotherapy and increasing the proportion of patients achieving the TMD. PMID- 16680931 TI - Survey of asthma care in Taiwan: a comparison of asthma specialists and general practitioners. AB - BACKGROUND: Asthma is a common disease in Taiwan. The promotion of quality care for asthmatic patients should focus not only on new treatment remedies but also on patient adherence to treatment and a continuous education program integrated into treatment plans. One reason for patients' poor response to treatment is the lack of asthma knowledge on the part of physicians in terms of attitudes toward treatment. OBJECTIVE: To investigate the current status of asthma treatment among asthma specialists and general practitioners and their relative acceptance of and adherence to treatment guidelines. METHODS: One thousand questionnaires were distributed to physicians throughout Taiwan using a randomized sampling procedure. The questionnaire asked about the use of different kinds of medications, including inhaled corticosteroids, to treat asthma; adherence to asthma treatment guidelines; the use of a peak flow meter for monitoring asthma status; and self-efficacy in the treatment of asthma. RESULTS: A total of 526 questionnaires were returned. Of these, 90.4% of specialists and 63.2% of general practitioners would follow the guidelines for patient care (P = .002). It was significant that 79.8% of specialists but only 41.9% of general practitioners would instruct patients to use a peak flow meter to monitor symptoms (P < .001). Asthma specialists also seemed to be significantly more competent than general practitioners regarding asthma knowledge, instruction of inhalation techniques, use of peak flow meters to monitor symptoms, and making an action plan. CONCLUSIONS: To minimize the knowledge gap between specialists and general practitioners regarding asthma treatment, recognized treatment guidelines need to be popularized or simplified. Furthermore, the continuing education of general practitioners in asthma knowledge and management skills is important. PMID- 16680934 TI - Milk allergy and vitamin D deficiency rickets: a common disorder associated with an uncommon disease. AB - BACKGROUND: Cow's milk allergy is one of the most common allergies in infancy. It has an excellent prognosis since most cases resolve by 4 years of age. The complications associated with milk allergy include delayed growth and atopic conditions, such as asthma, allergic rhinitis, atopic dermatitis, and other food allergies. OBJECTIVE: To report a case of vitamin D deficiency rickets in a 2 year-old boy with cow's milk allergy. METHODS: We describe a patient with clinical and biochemical evidence of rickets, including decreased serum calcium, phosphate, and 25-hydroxy vitamin D levels and an elevated alkaline phosphatase level. A dietary history revealed the prolonged absence of dietary vitamin D because the child did not tolerate cow's milk. Skin prick testing and measurement of specific IgE to cow's milk were performed to determine whether there was an allergy to cow's milk. RESULTS: Results of skin prick testing and measurement of specific IgE to cow's milk confirmed an IgE-mediated sensitivity to cow's milk. Introduction of appropriate supplementation into the child's diet resulted in complete resolution of his symptoms. CONCLUSIONS: This case emphasizes that the management of cow's milk allergy involves strict avoidance of the allergenic food while also ensuring that essential dietary requirements are met. A dietary history is crucial at all pediatric visits, and inquiry about supplementation of vitamins and minerals is important, especially in children with food allergies. PMID- 16680933 TI - Efficacy of diphenhydramine vs desloratadine and placebo in patients with moderate-to-severe seasonal allergic rhinitis. AB - BACKGROUND: Previous studies have shown that diphenhydramine and desloratadine effectively relieve symptoms of seasonal allergic rhinitis (SAR). OBJECTIVE: To compare the relative efficacy of 50 mg of diphenhydramine hydrochloride, 5 mg of desloratadine, and placebo in relieving symptoms in patients with moderate-to severe SAR. METHODS: In this 1-week, multicenter, parallel-group, randomized, double-blind, double-dummy, placebo-controlled study, 610 patients with moderate to-severe SAR received 50 mg of diphenhydramine hydrochloride 3 times daily, 5 mg of desloratadine once daily, or placebo. Daily 24-hour reflective total nasal symptom scores (TNSSs) (primary end point), total symptom scores, and individual symptom scores were evaluated. A global evaluation of response to treatment was conducted at 2 posttreatment visits. RESULTS: The mean reduction from baseline in 24-hour reflective TNSSs relative to the placebo response was 77.6% for the diphenhydramine group (P < .001) and 21.0% for the desloratadine group (P = .12). A TNSS between-treatment difference of -1.81 (46.7%; P < .001) was observed when comparing diphenhydramine with desloratadine. A similar between-treatment difference was observed for the 24-hour reflective total symptom score comparing diphenhydramine to desloratadine (-3.35; 45.5%; P < .001). Diphenhydramine provided clinically and statistically significant reductions vs placebo and desloratadine in all individual symptoms, including nasal congestion. Desloratadine had a tendency toward improvement compared with placebo for most individual symptom scores. However, a statistically significant result was reached only for sneezing (-0.27; 33.9%; P = .04). CONCLUSIONS: Diphenhydramine, 50 mg, given for 1 week provided statistically significant and clinically superior improvements in symptoms compared with 5 mg of desloratadine in patients with moderate-to-severe SAR. Somnolence occurred more frequently with diphenhydramine (22.1%) compared with desloratadine (4.5%) and placebo (3.4%). PMID- 16680935 TI - Amprenavir-induced maculopapular exanthem followed by desensitization in a patient with late-stage human immunodeficiency virus. AB - BACKGROUND: Amprenavir, a human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is approved for the treatment of HIV infection in combination with other antiretroviral agents in treatment-naive and experienced patients. Amprenavir is generally well tolerated. However, cutaneous hypersensitivity reactions to amprenavir occur in up to 28% of patients, with treatment discontinuation required in 3% of cases. OBJECTIVE: To report successful desensitization to amprenavir after the occurrence of a maculopapular exanthem in an HIV-infected patient with late-stage disease and limited antiretroviral treatment options. METHODS: Incremental doses of 0.025, 0.1, 0.25, 1, 2.5, 7.5, 25, 50, 100, 300, 600, and 1,200 mg of amprenavir oral solution were administered via percutaneous endoscopic gastrostomy tube at 20- to 30-minute intervals. RESULTS: The patient successfully tolerated amprenavir desensitization and has continued therapy without recurrence of rash at 19 months of follow-up. CONCLUSION: Desensitization may permit the continued use of amprenavir in HIV positive patients with a history of amprenavir-induced maculopapular eruptions who have limited alternate treatment options. PMID- 16680936 TI - Characterization of an anaphylactoid reaction to omalizumab. AB - BACKGROUND: The novel humanized murine monoclonal antibody omalizumab prevents binding of human IgE to its high-affinity receptor. A contraindication to therapy with omalizumab is allergy to the medication or previous immediate-type hypersensitivity or anaphylaxis to omalizumab or similar medications. OBJECTIVE: To determine whether a 32-year-old woman with asthma, allergic rhinitis, and idiopathic chronic urticaria and angioedema with anaphylactoid reactions to omalizumab could tolerate the medication in a desensitization protocol. METHODS: Omalizumab was administered after pretreatment with nonsteroidal anti inflammatory drugs (ibuprofen, 600 mg) while the patient was closely monitored in an intensive care unit. RESULTS: Omalizumab was well tolerated using this protocol, but a serum sickness-like reaction developed that required discontinuation of the medication after the seventh dose. CONCLUSIONS: Our experience suggests that some patients with anaphylactoid reactions to omalizumab can tolerate the medication when pretreated with nonsteroidal anti-inflammatory drugs but that a serum sickness-like illness may develop, requiring discontinued use of the medication. PMID- 16680937 TI - An allergist's role in a natural disaster. PMID- 16680938 TI - Allergy to horse. PMID- 16680939 TI - Detection of a novel nonsense mutation in the interleukin 2 receptor gamma gene causing X-linked severe combined immunodeficiency. PMID- 16680940 TI - Gutta-percha does not include main latex allergens. PMID- 16680941 TI - The chalice of silence and the case that refuses to go away. PMID- 16680942 TI - Ethics, culture, and taboos-organized psychoanalysis and the Cabernite affair. PMID- 16680943 TI - The Lobo-Cabernite affair revisited: and commentary on articles by Villela and Rubin. PMID- 16680944 TI - The history of membership and certification in the APsaA: old demons, new debates. PMID- 16680945 TI - Determinants of licensing in psychoanalysis: exclusivity and pluralism. PMID- 16680946 TI - Politics masquerading as science: Ralph Greenson, Anna Freud, and the Klein wars. PMID- 16680947 TI - Transforming trauma into tragedy: Oedipus/Israel and the psychoanalyst as messenger. PMID- 16680948 TI - Hitler's aesthetics: a psychoanalytic perspective on art and fascism. PMID- 16680949 TI - [To be a qualified burn surgeon--a senior old doctor's words]. PMID- 16680950 TI - [Grow with care and efforts]. PMID- 16680952 TI - [Burn clinical research--a field deserves emphasizing]. PMID- 16680951 TI - [Perfection and regret, dream and reality--the current situation and future of tissue engineering in the field of wound repair]. PMID- 16680953 TI - [Study on the optimal ratio of autoskin and alloskin during mixed microskin grafting]. AB - OBJECTIVE: To investigate the influence of different amount of allogeneic microskin in mixed grafting with certain quantity of autologous microskin on wound healing in rats. Methods Male Wistar rats served as alloskin donor rats. Forty female SD rats with full thickness skin defect were enrolled in the study, and they were randomly divided into four groups, i.e. group I (n=10, with allogeneic microskin graft at area expansion rate of 10:3); group II (n=10, with autologous microskin graft at area expansion rate of 10:1); group III (n=10, with mixed grafting of autologous and allogeneic microskin at area expansion rate of 10:1, respectively); group IV (n=10, with mixed grafting of autologous and allogeneic microskin at area expansion rate of 10:1 and 10:3, respectively). The wound healing rate, wound contraction rate and histological changes were observed at the 2, 3 and 4 post graft weeks (PGW). RESULTS: (1) In group I, there was mainly granulation tissue with some de novo epithelial cells appearing at the wound edge along with the rejection of grafted allogenous skin in the rat wound. In group II, there was still some granulation tissue remaining at 2 PGW due to insufficient amount of microskin. However, the wounds in the mixed grafting group appeared almost totally epithelialized. (2) Various amounts of mononuclear inflammatory cell infiltration and different degrees of angiectasis were observed in the dermal layer after the skin grafting in all groups, especially in group II and IV. There was thickening of the epithelial layer in all groups except group I. (3) The wound healing rate decreased obviously along with the development of rejection in group I at 2 to 4 PGWs. The wound healing rate was (55 +/- 26)% in group II, which was obviously lower than that in group III (88 +/- 6)% and in group IV (76 +/- 10)% at 3 PGWs (P < 0.01). (4) The contraction rate of the wound in group IV (69 +/- 7)% was much higher than that in group I (58 +/- 11)% at 3 PGWs (P < 0.05), and there was no difference among all the other groups. CONCLUSION: Wound healing can be obviously accelerated by mixing some autologous microskin with appropriate amount of alloskin. Moreover, certain amount of autologous microskin (expansion rate 10:1) mixed with the same proportion of allogeneic microskin seems to be more beneficial in promoting wound healing. PMID- 16680954 TI - [Multi-center clinical study of acticoat (nanocrystalline silver dressing) for the management of residual burn wounds]. AB - OBJECTIVE: To investigate the clinical efficacy and safety of Acticoat (nanocrystalline silver dressing) for the treatment of residual burn wounds. METHODS: Ninety-eight patients with 166 residual burn wounds were enrolled in the multi-center randomized clinical trials. In addition to the routine treatment, Acticoat was applied onto the wounds of the trial group once a day if there was much exudation from the wound, or the dressing change was made every other two days when the wounds were clean. Silver sulfadiazine (SD-Ag) was used in the control group of patients. The healing time was observed up to 20 days. The healing rate on the 15th day after treatment was taken as the percentage of healing. RESULTS: The average healing time was (12 +/- 5) days after the application of Acticoat, which was significantly shorter than that in control wounds with SD-Ag (16 +/- 6) days, (P = 0.005 < 0.01). The total effective rate of the wounds for trial was 97.05%, which was higher than that in control (94.17%) group, but there was no statistically significant difference. The bacterial clearing rate of the Acticoat group on the 6th and 12th post treatment day was 21.7% and 43.5% respectively, which was significantly higher than that in control group. No side-effect was observed in the two groups during the study. CONCLUSION: Acticoat with nanocrystalline silver can promote the healing of residual burn wounds effectively. PMID- 16680955 TI - [Clinical management of deep facial burn]. AB - OBJECTIVE: To explore the better clinical methods for the management of deep facial burn with optimal quality. Methods Fifty-four patients with deep facial burns were enrolled in the study and were divided into delayed skin grafting group (n=48) and early escharectomy group (n=6). In delayed grafting group, after the erosion of new born granulation tissue to the basal layer with blade holder or with peel or eschar shaving method at 3 postburn weeks (PBW) according to the eschar separation and granulation growth status, the whole face of the patients were divided into 10 regions and were then covered by split thickness auto skin. The same treatment was performed on the patients in early escharectomy group at 1 PBW. Physical therapy and plastic surgery were applied after skin grafting, and the patients were followed up from 3 month to 11 years. The first operation time, postburn facial operation time, operation times to repair the whole face, blood content of Hb, the amount of blood transfusion and hemorrhage and the prognosis were compared between the two groups. RESULTS: There was no difference between the two groups in regards to the first operation time, the total operation times,blood concentration of Hb before and after operation,and the amount of blood transfusion during the operation (P > 0.05). The operation time in delayed grafting group (21.9 +/- 3.2) d was obviously later than that in early escharectomy group (12.6 +/- 1.3) d, (P < 0.05). And there was evidently less amount of hemorrhage during operation(98 +/- 52) ml/100 cm2 than that in early escharectomy group (331 +/- 121) ml/100 cm2 (P < 0.01). The facial appearance of the patients in delayed grafting group was plump with more elasticity and richer expression compared with those in early grafting group. There exhibited different degrees of microstomia and both eyebrow defect in both groups during and after 1 postoperative year. In addition, mild to moderate ectropion and hypertrophic scar on the conjunction of grafted skin could appear in 80% of these patients. These deformities might be corrected by several times of plastic surgery. CONCLUSION: Based on the principle of arranging skin grafts according to the cosmetic and functional area units, split thickness skin grafting can provide satisfactory results for the repair of deep burn injury involving whole face when the wounds were treated with eschar peeling, tangential excision, escharectomy, granulation tissue scaling, or early escharectomy. In comparison with early escharectomy, eschar peeling, tangential excision, escharectomy, or granulation tissue scaling can get better results with less bleeding, full and round facial appearance, more elasticity of grafted skin and richer facial expression appearance after the operation. Meanwhile, effective physical therapy and scheduled plastic surgery after skin grafting can also be very important in achieving cosmetic results in the repair and reconstruction of whole facial deep burn. PMID- 16680956 TI - [Early transplantation of double rectus abdominis musculo-cutaneous flaps from upper abdomen for the repair of patients with electrical injury in the wrist]. AB - OBJECTIVE: To observe the effect of the early transplantation of double rectus abdominis musculo-cutaneous flaps on the repair of electrical injury of the wrist. METHODS: The study involved six patients suffering from circumferential deep electrical burn with only small amount of normal skin left on the dorsal side. The wounds were covered with double rectus abdominis musculo-cutaneous flaps raised from the upper abdomen with pedicles in both proximal and distal ends at an early postburn stage. The postoperative recovery of wrist function and wound repair were evaluated. RESULTS: The wrist wounds in all the 6 patients were primarily healed, with perfect function and appearance. CONCLUSION: Early application of double rectus abdominis musculocutaneous flaps on the electrically injured wrists can promote the wound healing processes and plays important roles in the preservation of wrist function. PMID- 16680957 TI - [Clinical observation of the effects of FE combined enzymes on the infection of the granulation burn wound during late post burns stage]. AB - OBJECTIVE: To observe the effects of combined FE enzymes on the infection of the granulation burn wound during late postburn stage in controlling burn wound infection caused by common antibiotic resistant bacteria. METHODS: Thirty patients in our burn ward were enrolled and were randomly divided into A [treated with combined FE enzymes (50 ml dissolved in 0-150 ml normal saline to reach the final concentration of 1-3 U/ml)] and B (treated with gentamicin) groups, with 15 patients in each group. Several layers of gauze, either soaked with combined FE enzyme in A or gentamicin in B group, were used to cover the burn wounds once to twice a day. Bacterial culture from the burn wound exudation before and after drug administration was done before the application of the agents. The bacteria in the burn wounds and their susceptibility to antibiotics were identified. The healing time of the burn wounds was recorded. Furthermore, the healing rate of the burn wound was recorded on the 3rd, 5th, 8th, 10th and 12th post skin grafting days (PSGD). RESULTS: The dominating bacteria in the burn wounds in both groups were Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae and MRSA. The susceptibility rate of bacteria ( MRSA, Staphylococcus epidermidis, Staphylococcus saprophyte, Pseudomonas aeruginosa, Escherichia coli and Enterobacter cloacae) to combined FE enzyme was 93.8%, 100.0%, 100.0%, 100.0%, 100.0% and 95.0% respectively, which were much higher than those in B group (17.6%, 31.3%, 28.6%, 44.0%, 33.3%, 28.0% respectively, P < 0.1. The wound healing time after skin grafting in A group (10.6 +/- 1.5 days) was significantly shorter than that in B group (15.3 +/- 1.7 days, P < 0.01). The wound healing rate on 10 PSGD in A group was (85.4 +/- 2.4)%, and which was only (51.3 +/- 1.5% in B group (P < 0.01) CONCLUSION: Combined FE enzyme can effectively control burn wound infection, so that the interval between skin grafting and wound healing can be shortened and success rate of skin grafting be improved. PMID- 16680958 TI - [Pathomorphological observation of the hypertrophic scar induced by injury to conical structure in female red Duroc pig]. AB - To further explore the relationship between hypertrophic scar and injury to conical structure of skin and the pathogenesis of hypertrophic scar, and to reproduce an optimal animal model of hypertrophic scar. METHODS: The back of two FRDP pigs were shaved, and a piece of normal skin was harvested for the observation of conical structure of skin. Skin wounds with depth of 0.38 mm, 0.76 mm, 1.14 mm and 1.52 mm, respectively, were created by gas-driven dermatome. Eight wounds measuring 7.0 cm x 7.0 cm were created on each pig. The wounds were divided into 4 groups according to the wound depth with 4 wounds in each group, i.e. 0.38 mm group, 0.76 mm group, 1.14 mm group and 1.52 mm group. The 0.38 mm and 0.76 mm groups were designated as superficial wound groups and 1.14 mm and 1.52 mm groups as deep wound groups. The wounds were allowed to heal without treatment. Tissue samples from the wound were harvested on 0, 10, 30, 60, 90 and 150 post injury day (PID) , and they were sectioned for HE staining and staining for elastic fibers (VVG). The wound healing and the scar formation were observed with naked eye. The skin conical structures in normal and injured skin were also observed. The morphology of hypertrophic scar was observed, and the thickness of the scar tissue was determined and scored. RESULTS: The wounds in superficial wound groups healed within 3 weeks with flat surface without scar formation. The wounds in deep wound groups healed later than 4 weeks with thick, hairless, hard in texture, with depigmentation or pigmentation, finally forming contracture. The skin conical structure could be found on the back of FRDP with HE and VVG staining, and it was similar to that of human in terms of the structure. In superficial wound groups, the upper part of the skin conical structure was injured, but fat fornix and glands were intact. In deep wound groups, the lower part of the skin cone, together with the fat fornix and gland were all injured. On the 150th post injury day, the histological picture of the tissue in superficial wound groups was similar to that of normal skin. But the skin conical structure could not be found in deep wound groups, and the wounds were filled by a large accumulation of disarrayed and irregularly arranged collagen fibers. With passage of time, the scar became thicker and thicker, and the scar hypertrophy reached the zenith in 150th PID. CONCLUSION: The injury of skin conical structure can lead to the formation of hypertrophic scar. FRDP can be used to reproduce and ideal model of hypertrophic scar. PMID- 16680959 TI - [Influence of some topical antibiotics and FGF2, EGF and rhGH on the biological characteristics of fibroblasts in vitro]. AB - OBJECTIVE: To investigate the influence of some topically used antibiotics (amikacin, gentamicin, chloromycetin and sulfamylon), basic fibroblast growth factor (FGF2) , epithelial growth factor (EGF) and recombinant human growth hormone (rhGH) on the growth of fibroblasts in vitro. METHODS: Fibroblasts were cultured and passaged. The cultured cells were then divided into control (routine culture of fibroblasts), amikacin (amikacin in respective dose of 0.021, 0.210, 2.100 mg/L), gentamicin (in respective dose of 5, 50, 500 mg/L) , chloromycetin (in respective dose of 0.01, 0.10, 1.00 mg/L), sulfamylon (in respective dose of 5, 10 g/L), FGF2 (2400 U/ml), EGF (2000 U/ml) and rhGH (0.016, 0.160, 1.600 g/L) groups. After the above agents were added to the culture medium respectively, the proliferation of the cultured fibroblasts was determined with MTT method, and the result was expressed as A (absorption) value. The cell cycle was determined with flow cytometry and the morphology of the cells was observed with inverted microscope. RESULTS: (1) MTT method: The A value of fibroblasts cultured with amikacin, gentamicin, chloromycetin and sulfamylon in various doses was obviously lower than that in control group (0.4553 +/- 0.0217, P < 0.05 or 0.01) , and the A value of sulfamylon group was the lowest in two doses (0.1013 +/- 0.0011 for 5 g/L and 0.0950 +/- 0.0041 for 10 g/L, P < 0.01). On the other hand,the A value in FGF2 and rhGH group(0.016 g/L) was much higher than that in the control (P < 0.05). However,theA value in EGF (both doses) and rhGH groups (0.160, 1.600 g/L) was close to that in control (P > 0.05). (2) Cell cycle determination: The proliferation index (PI) of fibroblasts cultured with amikacin in dose of 0.210 mg/L showed no difference compared to that in control (9.63 +/- 0.45)%, (P > 0.05). But the PI of fibroblasts cultured with FGF2, EGF and rhGF in dose of 0.016 g/L was increased significantly (46.76 +/- 2.33)%, (42.30 +/- 1.41)%, and (13.29 +/- 0.47)%, respectively, (P < 0.05 or 0.01). (3) Histological examination of the cells: The number of fibroblasts in elongated or spindle shape was larger, showing a blur contour but high transparency in control as well as in EGF and rhGH groups (both 0.160 and 1.600 g/L doses groups). The number of cells was lower in amikacin, gentamicin, chloromycetin and sulfamylon groups with sharp but irregular contour and lower transparency, and more granule-like materials and vacuoles in the cytoplasm. The cells in the FGF2 and rhGH (in dose of 0.016 g/L) groups exhibited dense with even distribution and slender or spindle shape and with more mitotic figures but blur contour and high transparency. CONCLUSION: Different kinds of the topically used therapeutic agents for burn wounds exert different influence on the biological characteristics of fibroblasts in vitro. The topically used agents for burn wounds should be carefully selected so that wound healing will be promoted and scar formation inhibited. PMID- 16680960 TI - [Analysis of the quality of papers dealing with clinical trails in "Chinese Journal of Burns" during 2000-2004 by the standard of evidence-based medicine]. AB - OBJECTIVE: To investigate the quality of reports of clinical results concerning burn injury, in order to raise the standard of clinical study of burn care in accordance to the standard of evidence-based medicine (EBM), with the aim of improving clinical research in burn care of this country. METHODS: All the papers of clinical study published in Chinese Journal of Burns (CJB) from 2000 to 2004 were evaluated according to EBM standard. RESULTS: There were 89 papers about clinical trials published in the past 5 years, in which 43 (48.3%) of the studies were carried out with random control trials (RCT), and 46 (51.7%) were clinical control trials (CCT). RCT papers increased year by year, while the number of CCT papers fluctuated greatly. The disparity in the quality of research was found as follows. In all the RCT and CCT papers, blinded research was adopted only in 5 papers (5.6%). Strict diagnostic standard including inclusion and exclusion standards were reported in 53 articles (59.6%). The comparison with baselines was not provided in 64 articles (71.9%). P value was given in 10 papers but statistical method was not mentioned (11.2%). Follow-up visits and lost information were only recorded in 2 articles, but no detailed follow-up visiting data were provided. Side effects were reported in 10 articles (11.2%). There were analysis and explanation of mixed interfering factors only in 5 papers (5.6%). There was no explanation of the evaluation of sample size in any one paper. CONCLUSION: In summary, the literature concerning clinical studies published in CJB in the past five years has become more extensive. However, the present study indicates that many clinical trials are not designed and ethical consideration is often missing. Therefore, it is deemed imperative to improve the quality of the clinical studies by improving the planning of the protocols of the study and statistical analysis of the research results in future. PMID- 16680961 TI - [The biological characteristics of dermal fibroblasts of the diabetic rats with deep-partial thickness scald]. AB - OBJECTIVE: To investigate the biological characteristics of dermal fibroblasts of the diabetic rats with deep partial thickness scald, and to explore its relationship with delayed wound healing due to diabetes. METHODS: Sprague-Dawley rats weighing 250 g were randomly divided into control (NM, n=40) and STZ-induced diabetic (DM, n=50) groups, and then deep partial thickness scald involving 10% TBSA were reproduced in the two groups. Skin samples were harvested from the wounds on 0, 3, 7, 14 and 21 post scald day (PSD) for the determination of certain histological characteristics. RESULTS: The thickness of dermis layer in DM group before injury was obviously thinner than that in NM group (P < 0.01). There was an infiltration of a large amount of chronic inflammatory cells and increased content of cutaneous glucose in the dermal tissue in DM group (2.77 mg/g) compared with 0.85 mg/g in NM group, (P < 0.01). An accumulation of advanced glycation end products (AGEs) was found in the dermal tissue in DM group. After the scalding, the percentage of fibroblasts in S phase and hydroxyproline synthesis in DM group was evidently lower than those in NM group. But the apoptosis rate of fibroblasts was much higher in DM group than that in NM group (P < 0.05 or 0.01). CONCLUSION: It is found that the high contents of glucose and AGEs in diabetic skin exert untoward effects on biological characteristics of dermal fibroblast, probably constituting one of the underlying mechanisms of delay wound healing of scald in diabetic rats. PMID- 16680962 TI - [Prolongation of survival time of allogenous skin graft after transfection of Molluscum contagiosum virus 148 gene in rats]. AB - OBJECTIVE: To explore the immunosuppressive effect of local transfection of Molluscum contagiosum virus 148 (MC148) gene to allogenous skin graft against rejection. METHODS: MC148 gene was cloned from molluscum contagiosum virus (MCV), and was employed to construct recombinant adenovirus vector (Ad-MC148). The recombinant Ad-MC148 was then locally transfected into a part of the tail skin of eight Lewis rats, which served as skin donors for grafting. The wounds (1 cm x 1 cm) were produced on the tails of 16 Wistar rats, and they were then randomly divided into control (C, n=8, with grafting of skin from donor rats without transfection), and transfection (T, n=8, with grafting of skin from donor rats with transfection of the recombinant Ad-MC148) groups. The expression of MC148 mRNA gene in T group was detected on 6 post operation hour( POH) and 2, 3, 7 and 10 post operation day (POD), and the results were expressed by the ratio of absorption value (A) between MC148 gene and beta-actin. The survival time of skin grafts in both groups was compared. Gross examination of grafted skin was carried out from 7 POD on in both groups, and the pathomorphological changes were examined in both groups on 7 POD. RESULTS: The MC148 gene expression in rat skin of T group could be identified in 6 POH, and it reached the peak on 3 POD (A(MC148 mRNA) / A(beta_actin) = 0.86), and then subsided thereafter, but it maintained for 10 days. The survival time of the grafts in T group was (15.0 +/- 2.0) days, and it was significantly longer than that in C group (8.5 +/- 3.4) days, (P < 0.01). Gross and microscopic examination showed that the tail skin of T group appeared ruddy on 7 POD, with little leukocytic infiltration in subcutaneous tissue; it began to turn black after 12 to 20 PODs. On the other hand, the tail skin of C group began to turn black and to shed off on 7 POD, with evident leukocytic infiltration in subcutaneous tissue and dermis. CONCLUSION: Local transfection of MC148 gene may promote immunosuppression by inhibiting leukocytic infiltration after allogenous skin transplantation. PMID- 16680963 TI - [Clinical analysis of 135 patients with severe eye burn]. AB - To investigate the clinical characteristics, prognosis, and complications of acute severe burn injury of the eyes. METHODS: One hundred and thirty-five patients (155 eyes) with acute severe burn injury of the eyes admitted to our hospital from 1977 to 2002 were analyzed retrospectively. The recovery rate and time, causes and incidence of blindness, and complications of burn injury of different depth and causes of burns were analyzed statistically. RESULTS: (1) The recovery time of patients with full-thickness burn of the eye ball was obviously shorter than that with IV degree eye burn, and the recovery rate of those with III degree burn was also evidently higher than that with IV degree burns (P < 0.01). Among the patients with non-repaired IV degree eye burn, the incidence of corneal perforation (6 eyes) in patients injured by acid was markedly higher than that by alkali (1 eye, P < 0.05). There were 38 III degree injured eyes with the visual acuity more than 0.05 and 51 with that less than 0.05. While there was no eye with the visual acuity more than 0.05 but 66 with that less than 0.05. Fifty two out of 78 eyes injured by acid and alkali went blind with occoecatio ratio of 66.7%. But 65 out of 77 eyes injured by heat went blind with occoecatio ratio of 84.4%. The complication in patients with III degree eye burn was lower than that with IV degree burns. The incidence of corneal perforation and symblepharon in patients with III degree eye burn was 1.1% and 36.0%, which was obviously lower than that with IV degree eye burn (27.3% and 59.1%, P < 0.01). The highest incidence of corneal perforation and symblepharon was in those with eye burn injured by acid (29.2%, 37.5%). While the highest incidence of the symblepharon in those with thermal eye burn was 53.2%. CONCLUSION: Among the patients with IV degree burns, those with acid, alkali and heat burns have bad prognosis, with high blindness rate. Current therapies cant improve eye function and prognosis of the patients thoroughly, which need further study in the future. PMID- 16680964 TI - [Study on the regular pattern of the distribution of skin epidermal stem cells in the different parts of a healthy human body]. AB - OBJECTIVE: To investigate the regular pattern of the distribution of skin epidermal stem cells (ESCs) in the different parts of a healthy human body, and to evaluate the feasibility of the identification of ESCs by P63 and CD29 with single and double labeling. METHODS: Full-thickness skin samples from 21 parts (including scalp, dorsum of foot, sole of foot, pubic region, and scrotum) of 5 healthy persons were harvested for the study. Immunohistochemistry method with biotin-streptavidin-horseradish peroxidase (SP) was employed with P63 and CD29 as the first antibody to carry out single and double labeling. The staining results were subjected to image analysis. The distribution of the ESCs in the skin from the above parts was observed and expressed as positive unit (PU) value. RESULTS: It was found by P63 single labeling and P63 and CD29 double labeling that the PU value in the dorsum of foot was the lowest while that in the scalp was the highest among all the parts of a healthy body. It was also found by CD29 single labeling that the PU value in the dorsum of foot was the lowest [(11.9 +/- 1.5)%] while highest in the scalp [(29.1 +/- 5.0)%]. The PU value in the hairy region of a human body was evidently higher than that in the non-hairy region (P < 0.01), when examined by P63 and CD29 single and double labeling. But there was no difference in the PU values between the trunk and limbs by means of P63 and CD29 single and double labeling (P > 0.05). CONCLUSION: There are more ESCs in the skin from the scalp, mons pubis and scrotum than other parts of the body. Single P63 or CD29 labeling exhibits higher sensitivity but lower specificity in the identification of ESCs. While the double labeling method exhibits higher specificity but lower sensitivity. Above all, it seems that the double labeling may be a simple and effective method for the identification of ESCs. PMID- 16680965 TI - [Clinical observation of the efficacy of MEBO in the treatment of burn patients with burn area over 50% TBSA]. AB - OBJECTIVE: To observe the effectiveness of MEBO in the treatment of burn patients with burn area over 50% TBSA. METHODS: Two hundred and ninety-eight patients hospitalized in our hospital from May of 1991 to December of 2003 with burn area over 50% TBSA, who had MEBO treatment before hospitalization, were enrolled in the study as the experiment (E) group. Another group of 300 burn patients with burn area over 50% TBSA that treated with SD-Ag cream were enrolled in the study as the control (C) group. Bacterial culture results, major changes in injury and mortality were compared between the two groups. RESULTS: There were 1 506 bacteria strains isolated from wounds in E group, and 9 main changes in injury (1679 cases) occurred with 20.8% mortality in this group. There were 353 bacteria strains isolated, with occurrence of 9 changes in injury (518 cases) and 4.7% mortality in the SD-Ag group. CONCLUSION: MEBO is much less effective for the treatment of the burn patients with large burn area compared with SD-Ag cream treatment. PMID- 16680966 TI - [Regional characteristics of population health in Russia: an analytical review]. AB - Analysis of state and trends of processes defining population health in various regions of Russia based both on official administrative statistic releases from various regions of Russian Federation and proper data is presented. PMID- 16680967 TI - [Dehumanization of medical education]. PMID- 16680968 TI - [Murders and suicides as problem of public health]. AB - Role of violence in public health deflection on international and regional levels is discussed. Data of public and sectoral statistics and sociological surveys is used. Social economic factors contributing to increase of victimization of Russian citizen is analyzed. PMID- 16680969 TI - [Attitude of students to health and healthy life-style]. AB - Results of sociological survey of attitude of academic first-year students to health and healthy life-style are presented. Concurrence of respondents' opinions with used in scientific literature notions "health and healthy life-style" is established. Respondents emphasized significance of dependence of health from such most vital medical social factors as bad habits, nutrition characteristics and passing leisure. Respondents expressed their opinions about means of health promotion, need of preventive check-ups, importance of being informed on issues of health maintenance. Need to include courses on healthy life-style into academic curriculum is emphasized. PMID- 16680970 TI - [Invalidity of adolescents of rural areas]. AB - Analyzed data demonstrates that both in rural areas and all over the Russian Federation in spite of that certain decreasing of particular invalidity indicators begins to show, still it is not possible to consider these indicators as stabilized. The reason being in impact on invalidity formation of overall complex of climatic geographic, demographic, social, medical and economic factors. Lower effectiveness of rehabilitation activities, including reluctance of invalids themselves, contributes to retention of higher incidence of general invalidity. Lacking of adequate and statistically reliable registration of invalidity of adolescent population breaks system of medical demographic indicators. In view of the aforesaid it is needed to carry out systematic analysis of cause-and-effect relations to ensure differentiated approach in managing activities and administrative decision-making to reduce invalidity rate. PMID- 16680971 TI - [Factors enhancing spread of sexually transmitted diseases among adolescents]. PMID- 16680972 TI - [Comparative characteristics of physical development of first year infants]. AB - Physical development data of 3740 first year infants born in 2002-2004 as compared with their 2782 coevals born in 1992-1994 is presented. Study results allowed to elaborate local standards of physical development of first year infants. It is established that in infants born in 2002-2004 increase of body mass and body height values occurs more steady than in their coevals born 10 years before. The reason is in altered approaches to infant feeding mode. Today, asthenization of first year infants relates to increase of chest circumference predetermining disharmonic development. PMID- 16680973 TI - [Study of quality of life in diabetes mellitus patients]. PMID- 16680974 TI - [Drug addiction morbidity in Republic of Dagestan]. AB - In Republic of Dagestan, analysis of drug addiction morbidity rate for last ten years refers to that its level in urban population is four times higher than in rural areas. Portion of opiate addicts detected for the first time consists 46.8% in urban areas and 22.1% in rural areas. In the morbidity structure of age specific drug addiction highest level is in the age group of 20-39 years and 40 59 years. In rural areas, as compared with urban areas, drug addiction cases among women were detected for the first time only in 2004. In rural areas and towns of Republic, there are territories with higher rates of drug addiction morbidity. PMID- 16680975 TI - [Analysis of dynamics of medical social situation in Novgorodskaya oblast based onsociological monitoring]. PMID- 16680976 TI - [Prevalence of chronic bronchitis among adult population]. PMID- 16680977 TI - [On effectivenes of mass prevention of iodine defficiency]. PMID- 16680978 TI - [Process approach in management of medical organization]. PMID- 16680979 TI - [Significance of marketing in public health]. AB - Issues of new marketing relations being formed in national public health system are discussed. Presence and essence of such relationships is defined. Their possible formalized notions, logic of interplay of elements of public health marketing systems and elements of general marketing and medical services marketing is offered for discussion. PMID- 16680980 TI - [Innovative forms and methods in management of curative preventive establishments in departamental dental service]. PMID- 16680981 TI - [Monitoring of health status of rural population in Lipetskaya oblast]. PMID- 16680982 TI - [Age-specific and gender characteristics of patterns of development of malignant malformations in Tomskaya oblast]. AB - Malignant malformations morbidity in population of Tomskaya oblast in 1980-2003 is examined. The structure of various localizations of oncological pathology depending on age-specific and gender aspects is presented. Nature of cancer diseases in compliance with genetic, ontogenetic, accumulative and ecologic patterns is discussed. Mean and peak life span, rate of dying out of oncological patients with various localizations of cancer process is established. It is demonstrated that in Tomskaya oblast over studied period on average mean life span came to 5.42 years and peak life span came to 10.8 +/- 0.2 years with annual rate of dying out of patients 9.249%. Study results consider as foundation for in depth investigation of the role of risk factors in development of oncological pathology, life quality and life span of cancer patients, enhancement of management of medical preventive and curative diagnostic care to patients of this particular profile. PMID- 16680983 TI - [Role of state in contemporary public health of France]. AB - By the example of France, issue of increasing role of state in public health is examined. It is shown that in France gradual broadening of system of medical insurance and enhancement of state influence in it resulted in institution of mandatory medical insurance in 2000. This act provided accessibility of medical care in conditions of coexistence in public health both of public and private sectors. Role of state in determination of French health policy and expenses extent in both sectors is analyzed. Forms and certain results of state impact on material technological and personnel support of public health of France and particular legislative measures approved recently as well are discussed. Comparison of public health data from France and U.S.A. permitted to conclude that reinforcement of role of state in national public health permits to achieve better results with lesser expenses. PMID- 16680984 TI - [Opthalmologic disease in Education Program of Medical Faculty of Moscow University in XVIII century. Report II: teaching of opthalmologic diseases in Europe in XVIII century]. PMID- 16680985 TI - [Formation and development of medical education in Bashkiria]. PMID- 16680986 TI - [Emmigration of Russian physicians in Kingdom of Serbs, Croats and Slovenes (Yugoslavia)]. PMID- 16680987 TI - [Public health in Yakutya (1920-1930): the financing]. PMID- 16680988 TI - [Conference "medicine and public health during Great Patriotic War"]. PMID- 16680989 TI - The legal framework for meeting surge capacity through the use of volunteer health professionals during public health emergencies and other disasters. PMID- 16680990 TI - Prescribing a legislative response: educators, physicians, and psychotropic medication for children. PMID- 16680991 TI - Optional or optimal?: The Medicaid Hospice Benefit at twenty. PMID- 16680992 TI - Restaurants, bars and workplaces, lend me your air: smokefree laws as private property exactions--the undiscovered country for Nollan and Dolan? PMID- 16680994 TI - Urban legend: dispelling the myth that rural hospitals require increased federal funding at the expense of urban hospitals. PMID- 16680993 TI - Stopping the practice of authorized generics: Mylan's effort to close the gaping black hole in the Hatch-Waxman act. PMID- 16680995 TI - [Image of the month. Bronchogenic cyst]. PMID- 16680996 TI - [How I treat...topical treatment of plaque type psoriasis by the combination of calcipotriol and betamethasone dipropionate]. AB - The combination of calcipotriol and betamethasone dipropionate in a single topical formulation is a recent advance in treatment of plaque type psoriasis. Studies rooted on evidence-based medicine have been recently published. We report a synthesis and a critical analysis, critical analysis of these investigations, and translate their data with a view to apply them in daily practice. PMID- 16680997 TI - [Clinical case of the month. Pierre Marie Bamberger syndrome]. AB - We report the case of a lung adenocarcinoma revealed by clubbing and secondary hypertrophic osteoarthropathy or Pierre-Marie Bamberger syndrome. PMID- 16680998 TI - [Clinical case of the month. Fatal pertussis infection in a 2 month old infant]. AB - The incidence of B pertussis has increased by 50% from the 1980s to the 1990s, primarily among those aged 4 months and younger. Worldwide, pertussis is a significant cause of infectious mortality with 40 million cases and 400.000 deaths. Most of these cases and deaths occur in infancy. Symptoms vary from common cold in adults to respiratory distress in infants. Non immune babies with respiratory disease and significant lymphocytosis should be considered to have pertussis until proven otherwise. The onset of severe pulmonary hypertension during B pertussis pneumonia is frequenly rapid and relentless. Exchange transfusion can be life-saving by reducing the leucocyte mass. Classic vaccination or boosters given to adults and adolescents would reduce the spread from parents tho infants, but a new vaccination schedule is under investigation at Vanderbilt Children's Hospital to give baby's first pertussis vaccination at birth? PMID- 16680999 TI - [The 2005 AHA selection of the "top ten advances in heart disease and stroke research"]. AB - The American Heart Association has once again released its "top ten" research advances in heart disease and stroke for the past year. As always, the choice is rather heterogeneous. Besides the quality of the selected contributions, it also reflects some areas of concern which primarily interest the Unites States, or the support that the AHA wishes to bring to some particular research programmes; Reading this list is however always rich of enlightment for anyone working in the field. PMID- 16681000 TI - [Anemia and heart failure]. AB - The prevalence of heart failure is increasing. Anemia is often present, particularly in severe cases. The Fick equation illustrates the relationship between anemia and heart failure. The concepts of cardiorenal syndrome and erythropoietin resistance are parts of the physiopathology. Anemia is associated with a bad prognosis in heart failure. Nevertheless, it is too early to know precisely whether anemia represents a simple marker of severity or if specific treatment, especially with erythropoietin, is useful in terms of morbidity and mortality. Current guidelines recommend the correction of secondary causes when anemia is present in a heart failure patient. PMID- 16681001 TI - [The skin and menopause]. AB - The estrogen deficit occurring at menopause manifests itself under various modalities in the diverse organs and their functions. Skin does not escape this global involutive process. In addition to the effects of chronological ageing, sunlight exposure and other environmental and endogenous stimuli, the climacteric appears to exert some dramatic consequences on skin biology and aspect. The epidermis, its adnexae and the dermis are altered by this process. The epidermis may become xerotic and exhibits altered functions. The dermis thins out and its elasticity decreases in concert with the decline in bone mass. The skin microcirculation is impaired. These aspects are some of the better-worked out skin climacteric changes of which in turn seem to be stabilized or in part reversible with hormone replacement therapy (HRT). PMID- 16681002 TI - [Acute colitis in Wegener's disease: a case report]. AB - We report the case of a 52 year old man who was hospitalized within a context of a persistent deterioration of his general condition. He was suspected of having a chronic inflammatory colitis. A pulmonary radiography revealed the presence of voluminous bilateral excavated masses with hydro-aerical levels. After having refuted among others a suspicion of tuberculosis, the results of a thoracic percutaneous transpleural lung aspiration by needle under tomodensitometric control steered our diagnosis towards a vascularitis of the Wegener disease type. A treatment with corticotherapy in large doses completed with cyclophosphamid allowed for clinical, biological and radiological improvement. Wegener's granulomatosis usually starts in an insidious manner with febrile episodes and an impairment of the general condition associated with inflammatory biological signs, as observed in our patient. After these warning symptoms, come ORL and/or pulmonary and/or renal impairment, which represent the classical triad of diffused GW. However a certain number of particularities unusual for that diagnosis characterized our patient and prompted the discussion of this case. PMID- 16681003 TI - [Glucosamine as a pain-modifying drug in osteorthritis. What's new in 2006]. AB - Glucosamine is widely used as a symptom-modifying drug in osteoarthritis. New clinical trials, from Europe and the United-States bring some clarification regarding the optimal formulation and doses to be used in knee osteoarthritis. Their results are supported by new pharmacokinetic and preclinical studies, explaining the mode of action of glucosamine in osteoarthritis. PMID- 16681004 TI - [Post-radiotherapy and atherosclerotic stenosis of the internal carotid artery treated by angioplasty and stenting]. AB - We report the case of a 70-year old man treated by surgery and radiotherapy for a neoplasic lesion of the tongue in whom severe symptomatic stenosis of the left internal carotid artery occurred a few years later. This lesion was successfully treated by percutaneous carotid angioplasty and stenting with distal embolic protection (PCAS). After a period of experimenting with angioplasty and stenting, PCAS is becoming a serious alternative for carotid thrombo-endarterectomy (TEA). Indications for PCAS are currently patients who, due to several reasons, are no good candidates for surgery. Several of large PCAS versus TEA randomized trials are underway and final results will help us determine the best indication for TEA or PCAS for both low and high risk patients with symptomatic or asymptomatic carotid stenosis. PMID- 16681005 TI - [A Tunisian case of pachydermoperiostosis]. AB - Clubbed fingers are classically associated with chronic cardiac and pulmonary affection. Pachydermoperiostosis, a rarer aetiology, is a hereditary affection which is difficult to diagnose in its incomplete form. We relate a Tunisian case report. Our patient consulted for joint manifestations. The diagnosis of Pachydermoperiostosis was set up on 3 out of the 4 Borochowitz criteria. The osteoarticular manifestations mainly led to differential diagnosis with secondary hypertrophic osteoarthropathy. The underlying pathogenic mechanism of this disease still remains unclear. PMID- 16681006 TI - [Epidemiological analysis of drug self-poisonings treated by a mobile intensive care unit]. AB - PURPOSE: Study the epidemiology, prehospital emergency care and short-term evolution of the drug self poisonings. METHODS: Retrospective analysis from January 1999 to December 2000 of drug self poisonings requiring Grenoble (France) intensive care unit action and patient follow up. RESULTS: 325 patients, 39.3 years old +/- 14.5 have been treated. The drug self poisonings are representing 4.6% of Grenoble mobile intensive care unit activity. The sex-ratio was, for the 15 to 24 years old population, 2.3 females for 1 male. Mobile intensive care unit has been decided in first intention on 56.3%. 556 toxic substances have been reported, psychotropics are prevailing (79.4% of all drug self poisonings). Intubation has been necessary in 24%. 64.4% of the treated population has been hospitalized into intensive care units. The main exit step of the first admission unit was returning home. Death rate was 0.9%. CONCLUSION: Drug self poisoning is an important part of mobile intensive care unit activity. The very low death rate could be related to several criteria: a high efficient rescue unit setting up a symptomatic based medical treatment and the current decreased toxicity of the ingested drugs. PMID- 16681007 TI - [Basic initiatory glossary to clinical research]. AB - We present a glossary of terms frequently used in medico-scientific studies. PMID- 16681008 TI - [How do you investigate an elevated calcitonin level?]. AB - This article describes a case of lung cancer with associated hypercalcitoninemia in a man with a goitre. The case raises the problem of the differential diagnosis between medullary thyroid carcinoma and a neuroendocrine neoplasm with ectopic calcitonin secretion. The article first reviews the physiology of calcitonin, then outlines the diagnostic tests that are required to investigate hypercalcitoninemia and finally discusses the interpretation of test results. PMID- 16681010 TI - [Nursing, a matter of management?]. PMID- 16681009 TI - [Etanercept (Enbrel) for the treatment of moderate to severe plaque type psoriasis]. AB - Etanercept (Enbrel) is a soluble tumour necrosis factor (TNF) receptor drug. By this mechanism, it inhibits the effects of TNF-alpha involved in the pathogenesis of psoriasis. Enbrel is approved for the treatment of moderate to severe chronic plaque psoriasis, and also for psoriatic arthritis. PMID- 16681011 TI - [Harry Potter and obesity]. PMID- 16681012 TI - [Body weight and illness]. PMID- 16681013 TI - [Obesity and the search for thinness: between the two sides of as similar problem]. PMID- 16681014 TI - [Complications after the surgery. Not as planned]. PMID- 16681015 TI - [Chronically ill children. Complex work for the family]. PMID- 16681016 TI - [Benin. When poverty involves vision problems]. PMID- 16681018 TI - [Avoiding stress]. PMID- 16681017 TI - [Recognizing aggression and relieving stress]. PMID- 16681019 TI - [Only a clean wound is capable of healing]. PMID- 16681020 TI - [Weight, a real problem]. PMID- 16681021 TI - [The kilos of illness]. PMID- 16681022 TI - [The moments of appeasement and comfort]. PMID- 16681024 TI - [Male or female: each has its problems]. PMID- 16681023 TI - [Too many women currently live in danger]. PMID- 16681025 TI - [A special private school opens its doors]. PMID- 16681026 TI - [Mechanic and humanist]. PMID- 16681027 TI - [Obesity: a health problem]. PMID- 16681028 TI - [Problem of obesity]. PMID- 16681029 TI - [Physicians establish their own schedules]. PMID- 16681030 TI - Blood-brain barrier interfaces and brain tumors. AB - In the developing brain, capillaries are differentiated and matured into the blood-brain barrier (BBB), which is composed of cerebral endothelial cells, astrocyte end-feet, and pericytes. Since the BBB regulates the homeostasis of central nervous system (CNS), the maintenance of the BBB is important for CNS function. The disruption of the BBB may result in many brain disorders including brain tumors. However, the molecular mechanism of BBB formation and maintenance is poorly understood. Here, we summarize recent advances in the role of oxygen tension and growth factors on BBB development and maintenance, and in BBB dysfunction related with brain tumors. PMID- 16681031 TI - Synthesis and cytotoxic activities of 8-alkyl or 8-aryl-8,9-dihydro-7H isoindolo[5,6-g]quinoxaline-7,9-diones. AB - A series of 8-alkyl- and 8-aryl-8,9-dihydro-7H-isoindolo[5,6- g]quinoxaline-7,9 diones were synthesized using sultine chemistry as a key step in good yield. These compounds were evaluated for their in vitro cytotoxicity against six human cancer cell lines (HCT15, SK-OV-3, A549, SNB19, MCF7 and MCF7/ADR). PMID- 16681032 TI - Naturally occurring biflavonoid, ochnaflavone, inhibits cyclooxygenases-2 and 5 lipoxygenase in mouse bone marrow-derived mast cells. AB - Ochnaflavone is a medicinal herbal product isolated from Lonicera japonica that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC50 values of 0.6 microM. Western blotting probed with specific anti COX-2 antibodies showed that the decrease in quantity of the PGD2 product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 6.56 microM. These results demonstrate that ochnaflavone has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound strongly inhibited degranulation reaction in a dose dependent manner, with an IC50 value of 3.01 microM. Therefore, this compound might provide a basis for novel anti-inflammatory drugs. PMID- 16681033 TI - Isorhamnetin glycosides with free radical and ONOO-scavenging activities from the stamens of Nelumbo nucifera. AB - In this study, we isolated two new isorhamnetin glycosides, designated as nelumboroside A (3) and nelumboroside B (4), as well as the previously characterized isorhamnetin glucoside (1) and isorhamnetin rutinoside (2), from the n-BuOH fraction of Nelumbo nucifera stamens. The structures of the two new compounds were then determined, using chemical and spectroscopic techniques. All isolated isorhamnetin glycosides 1-4 showed marked antioxidant activities in the DPPH, and ONOO- assays. PMID- 16681034 TI - Anti-inflammatory principles from the fruits of Evodia rutaecarpa and their cellular action mechanisms. AB - The fruits of Evodia rutaecarpa Benth (Rutaceae) has long been used for inflammatory disorders and some anti-inflammatory actions of its constituents such as dehydroevodiamine, evodiamine and rutaecarpine were previously reported. Since the pharmacological data is not sufficient to clearly establish the scientific rationale of anti-inflammatory medicinal use of this plant material and the search for its active principles is limited so far, three major constituents (evodiamine, rutaecarpine, goshuyuamide II) were evaluated for their anti-inflammatory cellular action mechanisms in the present study. From the results, evodiamine and rutaecarpine were found to strongly inhibit prostaglandin E2 synthesis from lipopolysaccharide-treated RAW 264.7 cells at 1-10 microM. Evodiamine inhibited cyclooxygenase-2 induction and NF-kappaB activation, while rutaecarpine did not. On the other hand, goshuyuamide II inhibited 5-lipoxygenase from RBL-1 cells (IC50 = 6.6 microM), resulting in the reduced synthesis of leukotrienes. However, these three compounds were not inhibitory against inducible nitric oxide synthase-mediated nitric oxide production from RAW cells up to 50 micorM. These pharmacological properties may provide the additional scientific rationale for anti-inflammatory use of the fruits of E. rutaecarpa. PMID- 16681035 TI - Bactericidal effects of CaO (scallop-shell powder) on foodborne pathogenic bacteria. AB - This study was investigated the bactericidal effects of calcium oxide (CaO) on three common foodborne pathogenic bacteria: Escherichia coli, Listeria monocytogenes, and Salmonella typhimurium. Each bacteria level was determined in a CaO solution (0.01, 0.03, 0.05, 0.10, 0.15, and 0.20% [w/v]) exposed for either 15 sec, 30 sec, 1 min, 2 min, 3 min, 5 min, 10 min, or 30 min. All three bacteria were not greatly affected by CaO solutions at concentrations of 0.01 and 0.03%, however, the decline of E. coli (99%; 2.78 log10 CFU/mL), L. monocytogens (45%; 1.44 log10 CFU/mL), and S. typhimurium (70%; 2.08 log10 CFU/mL) was greatest when they were exposed to 0.05% CaO solution for 10 min. Moreover, the bactericidal action of CaO was maintained for at least 24 h of storage. The results of this study provide evidence that CaO, as a substitute for synthetic chemical substances has potential for use in the disinfection and sanitization of foods and food processing equipment. PMID- 16681036 TI - Endotoxin induces late increase in the production of pulmonary proinflammatory cytokines in murine lupus-like pristane-primed model [corrected]. AB - Lupus-like syndrome is characterized by multiple organ injuries including lungs and kidneys. Endotoxin induces a transiently intent systemic inflammatory response and indirectly transient acute lung injury in normal condition. However, whether endotoxin may trigger the persistent development of lung injury in chronic, inflammatory lupus-like syndrome compared with normal condition remains unclear. We examined the pulmonary vascular permeability and production of proinflammatory cytokines, such as TNF-alpha, IL-6, IL-10 and IFN-gamma, which play prominent roles in the pathogenesis of lupus-like tissue injury, 6 h and 72 h after i.p. lipopolysaccharide (LPS; endotoxin) injection in pristane-primed chronic inflammation ICR mice characterized by a lupus-like syndrome. These results demonstrated that levels of serum IL-6, IL-10 and IFN-gamma and bronchoalveolar lavage (BAL) IL-6 and IFN-gamma were remarkably increased 6 h in LPS-exposed pristane-primed mice compared with pristane-primed controls, while pulmonary vascular permeability and levels of serum and BAL TNF-alpha were not. And levels of BAL TNF-alpha, IL-6 and IL-10 were significantly enhanced 72 h in LPS-exposed pristane-primed mice compared with pristane-primed controls. Also, LPS significantly induced the increased in vitro production of TNF-alpha, IL-6 and IL-10 by lung cells obtained from LPS-exposed pristane-primed mice compared with LPS-exposed normal mice. Our findings indicate that LPS may trigger persistent progression of lung injury through late overproduction of BAL TNF alpha, IL-6, and IL-10 in lupus-like chronic inflammation syndrome compared with normal condition. PMID- 16681037 TI - Chemical modification of the human ether-a-go-go-related gene (HERG) K+ current by the amino-group reagent trinitrobenzene sulfonic acid. AB - We investigated the effects of trinitrobenzene sulfonic acid (TNBS), an amino group reagent, on the human ether-a-go-go-related gene (HERG) K+ channels expressed in Xenopus oocytes. TNBS neutralizes the positively charged amino groups of peptide N-terminal and lysine residues. External application of TNBS at 10 mM for 5 min irreversibly shifted the curves for currents at the end of the pulse and tail currents of HERG to a more negative potential and decreased the maximal amplitude of the I(tail) curve (I(tail,max)). TNBS had little effect on either the activated current-voltage relationship or the reversal potential of HERG current, indicating that TNBS did not change ion selectivity properties. TNBS shifted the time constant curves of both activation and deactivation of the HERG current to a more hyperpolarized potential; TNBS's effect was greater on channel opening than channel closing. External H+ is known to inhibit HERG current by shifting V(1/2) to the right and decreasing I(tail,max). TNBS enhanced the blockade of external H+ by exaggerating the effect of H+ on I(tail,max), not on V(1/2). Our data provide evidence for the presence of essential amino-groups that are associated with the normal functioning of the HERG channel and evidence that these groups modify the blocking effect of external H+ on the current. PMID- 16681038 TI - Mechanism of intestinal transport of an organic cation, tributylmethylammonium in Caco-2 cell monolayers. AB - Many quaternary ammonium salts are incompletely absorbed after their oral administration and may also be actively secreted into the intestine. However, the underlying mechanism(s) that control the transport of these cations across the intestinal epithelium is not well understood. In this study, the mechanism of absorption of quaternary ammonium salts was investigated using Caco-2 cell monolayers, a human colon carcinoma cell line. Tributylmethyl-ammonium (TBuMA) was used as a model quaternary ammonium salts. When TBuMA was administrated at a dose of 13.3 imole/kg via iv and oral routes, the AUC values were 783.7 +/- 43.6 and 249.1 +/- 28.0 micormole x min/L for iv and oral administration, indicating a lower oral bioavailability of TBuMA (35.6%). The apparent permeability across Caco-2 monolayers from the basal to the apical side was 1.3 times (p < 0.05) greater than that from the apical to the basal side, indicating a net secretion of TBuMA in the intestine. This secretion appeared to be responsible for the low oral bioavailability of the compound, probably mediated by p-gp (p-glycoprotein) located in the apical membrane. In addition, the uptake of TBuMA by the apical membrane showed a Na+ dependency. Thus, TBuMA appears to absorbed via a Na+ dependent carrier and is then secreted via p-gp related carriers. PMID- 16681039 TI - Altered pharmacokinetics and hepatic uptake of TBuMA in ethynylestradiol-induced cholestasis. AB - The objective of this study was to examine the pharmacokinetics of organic cations in intrahepatic cholestatic rats. A pretreatment with 17alpha ethynylestradiol was used to induce intrahepatic cholestasis, and tributylmethylammonium (TBuMA) was used as a representative model organic cation. When [3H]TBuMA was intravenously administered1 the AUC value for TBuMA was significantly increased by 79% in cholestasis, and its total systemic clearance was consequently decreased by 46%. In addition, the in vivo hepatic uptake clearance of TBuMA from the plasma to the liver was decreased by 50% in cholestasis. The concentration of bile salts in plasma was increased by 2.1 fold in cholestatic rats. Since TBuMA forms ion-pair complexes with anionic components such as bile salts, the decreased hepatic uptake of TBuMA in cholestasis may be due to a change in endogenous components, e.g., bile salts in the plasma. In isolated normal hepatocytes, the uptake clearance for TBuMA in the presence of cholestatic plasma was decreased by 20% compared with normal plasma. Therefore, we conclude that the inhibition of the hepatic uptake process by the cholestasis may be in part due to the increased formation of ion-pair complexes of TBuMA with bile salts in the plasma. PMID- 16681040 TI - Bioequivalence and pharmacokinetics of 70 mg alendronate sodium tablets by measuring alendronate in plasma. AB - The bioequivalence and pharmacokinetics of alendronate sodium tablets were examined by determining the plasma concentration of alendronate. Two groups, consisting of 24 healthy volunteers, each received a 70 mg reference alendronate sodium tablet and a test tablet in a 2x2 crossover study. There was a 6-day washout period between doses. The plasma alendronate concentration was monitored for 7 h after the dose, using HPLC-Fluorescence Detector (FD). The area under the plasma concentration-time curve from time 0 to the last sampling time at 7 h (AUC(0-7h) was calculated using the linear-log trapezoidal rule. The maximum plasma drug concentration (Cmax) and the time to reach Cmax (Tmax) were derived from the plasma concentration-time data. Analysis of variance was performed using logarithmically transformed AUC(0-7h) and Cmax, and untransformed Tmax. For the test medication versus the reference medication, the AUC(0-7h) were 87.63 +/- 29.27 vs. 102.44 +/- 69.96 ng x h x mL(-1) and the Cmax values were 34.29 +/- 13.77 vs. 38.47 +/- 24.39 ng x mL(-1), respectively. The 90% confidence intervals of the mean differences of the logarithmic transformed AUC(0-7h) and Cmax values were log 0.8234-log 1.1597 and log 0.8222-log 1.1409, respectively, satisfying the bioequivalence criteria guidelines of both the U.S. Food and Drug Administration and the Korea Food and Drug Administration. The other pharmacokinetic parameters for the test drug versus reference drug, respectively, were: t(1/2), 1.87 +/- 0.62 vs. 1.77 +/- 0.54 h; V/F, 2061.30 +/- 986.49 vs. 2576.45 +/- 1826.05 L; CL/F, 835.32 +/- 357.35 vs. 889.48 +/- 485.87 L x h(-1); K(el), 0.42 +/- 0.14 vs. 0.40 +/- 0.18 h(-1); Ka, 4.46 +/- 3.63 vs. 3.80 +/- 3.64 h(-1); and Tlag, 0.19 +/- 0.09 vs. 0.18 +/- 0.06 h. These results indicated that two alendronate formulations (70-mg alendronate sodium) were biologically equivalent and can be prescribed interchangeably. PMID- 16681041 TI - Enhanced nimodipine bioavailability after oral administration of nimodipine with morin, a flavonoid, in rabbits. AB - The aim of this study was to investigate the effect of morin on the bioavailability of nimodipine after administering nimodipine (15 mg/kg) orally to rabbits either co-administered or pretreated with morin (2, 10 and 20 mg/kg). The plasma concentrations of nimodipine in the rabbits pretreated with morin were increased significantly (p < 0.05 at 10 mg/kg, p < 0.01 at 20 mg/kg) compared with the control, but the plasma concentrations of nimodipine co-administered with morin were not significant. The areas under the plasma concentration-time curve (AUC) and the peak concentrations (Cmax) of the nimodipine in the rabbits pretreated with morin were significantly higher (p < 0.05 at 10 mg/kg, p < 0.01 at 20 mg/kg), but only the Cmax of nimodipine coadministered with morin 10 mg/kg was increased significantly (p < 0.05). The absolute bioavailability (A.B%) of nimodipine in the rabbits pretreated with morin was significantly (p < 0.05 at 10 mg/kg, p < 0.01 at 20 mg/kg) higher (54.1-65.0%) than the control (36.7%). The increased bioavailability of nimodipine in the rabbits pretreated with morin might have been resulted from the morin, which inhibits the effiux pump P glycoprotein and the first-pass metabolizing enzyme by cytochrome P-450 3A4 (CYP 3A4). PMID- 16681042 TI - HPLC determination of tolperisone in human plasma. AB - A simple high performance liquid chromatographic (HPLC) method was developed for the determination of tolperisone in human plasma. Tolperisone and internal standard (chlorphenesin) were isolated from 1 mL of plasma using 8 mL of dichlormethane. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with 300 mL aliquot of mobile phase and a 100 mL aliquot was injected onto the C18 reverse-phased column. The mobile phase, 45% methanol containing 1% glacial acetic acid and 0.05% 1-hexanesulfonic acid was run at a flow rate of 1 mL/min. The column effluent was monitored using UV detector at 260 nm. The retention times for tolperisone and the internal standard were approximately 7.1 and 8.4 min, respectively. The standard curve was linear with minimal intra-day and inter-day variability. The quantification limit of tolperisone in human plasma was 10 ng/ mL. The proposed method has been applied to the determination of pharmacokinetic profile of tolperisone in Koreans. The Tmax of tolperisone in Koreans (0.94 +/- 0.42 h) was not significantly differ from that reported in Europeans (0.5-1 h), but the mean half-life in Koreans (1.14 +/- 0.27 h) was shorter than that in Europeans (2.56 +/- 0.2 h). The proposed HPLC method is simple, accurate, reproducible and suitable for pharmacokinetic study of tolperisone. PMID- 16681043 TI - Surgical techniques of cataract surgery and subsequent postoperative endophthalmitis. AB - OBJECTIVES: To compare the incidence and characteristics of patients with endophthalmitis after extracapsular cataract extraction (ECCE) to those after phacoemulsification MATERIAL AND METHOD: Records of patients receiving intravitreal antibiotic injection to treat endophthalmitis after cataract surgery between Jan 2001 and Dec 2004 were reviewed. Demographic data and other characteristics including associated diseases, details of cataract surgical procedure and intraoperative complication, onset of endophthalmitis after cataract surgery, presenting symptoms and signs of endophthalmitis, how endophthalmitis was managed, causative organisms, duration of hospitalization and results of treatment were collected. This information was compared between those of endophthalmitis patients after ECCE and those after phacoemulsification. RESULTS: There were 5 cases who developed endophthalmitis after ECCE and 31 cases after phacoemulsification. The incidence was 0.365% after ECCE and 0.279% after phacoemulsification (p = 0.589). Visual acuity (VA) before cataract surgery in ECCE group was worse than the phacoemulsification group (median VA: counting fingers vs 6/36, p = 0.001). Median onset of endophthalmitis was 8 days after ECCE and 6 days after phacoemulsification. Presenting symptoms and signs were similar. Causative agents were identified in 4 (80%) and 14 (45%) cases in the ECCE and phacoemulsification groups respectively. Gram-positive bacteria were the major cause of infection in both groups. Endophthalmitis caused by citrobacter sp. in ECCE group and enterococcus or streptococcus sps. the phacoemulsification in the group ended up with enucleation or no light perception. CONCLUSION: The present study has not demonstrated an apparent difference between endophthalmitis after ECCE and those after phacoemulsification. Endophthalmitis after either procedure can be managed as the same condition. PMID- 16681044 TI - Sterility of non-preservative eye drops. AB - OBJECTIVES: To assess microbial contamination of common non-preservative eye drops stored at 4 degrees C and non-preservative fortified antibiotic eye drops used in a hospital inpatient setting. MATERIAL AND METHOD: A prospective study of the sterility of non-preservative eye drops was examined by dividing the patients into 2 groups. Group 1 composed of 5 samples of each ophthalmic preparation, which consisted of fortified antibiotics (vancomycin, cefazolin, gentamicin, and amikacin), non-preservative steroid eye drops (1% methylprednisolone and 0.01% dexamethasone), and 20% autologous serum stored at 4 degrees C. Group 2 was composed of 28 samples of fortified vancomycin and 23 samples of fortified amikacin used in an inpatient setting for 1 week and kept in 4 degrees C for 28 days. The contamination was assessed at the day of preparation and then at day 4, 7, 14, 21, 28 of all medications except autologous serum was done up to day 84. The sterility was examined by the culturing on tryptic soy broth and thioglycolate broth. RESULTS: In group 1 and 2, no contamination was detected in any samples within 4 weeks and up to 12 weeks for autologous serum. CONCLUSION: Non- preservative topical eye drops can be safely kept at 4 degrees C without contamination for 4 weeks and 12 weeks for 20% autologous serum drop. For topical antibiotics, under a strict protocol for preparation and usage might be used without significant risk of bacterial contamination for 4 weeks. PMID- 16681045 TI - Eyelid tumors in Siriraj Hospital from 2000-2004. AB - OBJECTIVES: To determine the relative incidence of eyelid lesions seen in Siriraj Hospital from January 2000-April 2004. STUDY DESIGN: Retrospective charts review MATERIAL AND METHOD: Two hundred and ninety-seven cases of eyelid lesions seen in Siriraj Hospital from 2000 to 2004 were analyzed. RESULTS: There were 53 (17.8%) inflammatory conditions, 212 (71.4%) benign eyelid tumors and 32 (10.8%) malignant eyelid tumors. These 32 malignant eyelid tumors included 13 sebaceous gland carcinomas, 12 basal cell carcinomas, 3 malignant melanomas, 2 squamous cell carcinomas, 1 apocrine adenocarcinoma and 1 metastatic carcinoma. Various flaps techniques or primary closures were used for reconstruction in 20 cases. Six cases needed exenteration. CONCLUSION: The majority of eyelid lesions were benign eyelid tumors while malignant eyelid tumors contributed 10.8% of the total eyelid lesions. Sebaceous gland carcinoma was the most common eyelid tumor found in their present study that was consistent with other studies from Asian countries. PMID- 16681046 TI - Treatment of cytomegalovirus retinitis in AIDS patients with intravitreal ganciclovir. AB - BACKGROUND: Cytomegalovirus (CMV) retinitis is the most common opportunistic ocular infection in AIDS patients, and frequently leads to blindness if untreated. Intravitreal ganciclovir proved to be effective in stopping the progression of the disease. OBJECTIVES: To determine the efficacy and complications of intravitreal ganciclovir (2 mg in 0.1 ml per injection) to control CMV retinitis. STUDY DESIGN: A retrospective non-randomized interventional case series. MATERIAL AND METHOD: The participants were 363 consecutive patients with CMV retinitis treated at the CMV Retinitis Clinic, Maharaj Nakorn Chiang Mai Hospital over the period from June 2001 to December 2003. The affected eyes received weekly intravitreal injections of 2 mg of ganciclovir until the lesions were inactive, then 2-4 weeks each time continuously or until relapse. If the lesions relapsed, then the weekly schedule was re-started. RESULTS: In 568 treated eyes at the time of last follow up, visual acuity remained stable in 343 (60%), improved in 76 (13%), and decreased in 149 (26%). Of these, 33 retinal detachments, 6 intravitreal hemorrhages, 6 endophthalmitis, and 2 cataract occurred. Bilateral disease occurred in 22% of patients who first came with unilateral involvement. CONCLUSION: Intravitreal ganciclovir appeared to be a worthwhile therapeutic alternative for CMV retinitis patients with unaffordable or intolerant to systemic anti-CMV therapy, but the complications of intravitreal injections should also be recognized. PMID- 16681047 TI - Long-term intraocular pressure change after clear corneal phacoemulsification in Thai glaucoma patients. AB - OBJECTIVES: To evaluate long-term intraocular pressure (IOP) changes after sutureless clear corneal phacoemulsification in eyes with preoperatively controlled glaucoma. DESIGN: Retrospective study. SETTING: Eye Clinic, Ramathibodi Hospital, Faculty of Medicine, Mahidol University. MATERIAL AND METHOD: The medical records of 218 patients who had uneventful sutureless clear corneal phacoemulsification with acrylic foldable lens (IOL) implantation were retrospectively reviewed. Included were 58 patients with medically controlled open-angle glaucoma and 160 normal controls. Follow-up was 12 to 18 months. Outcome measures were postoperative IOP and number of glaucoma medications. RESULTS: Postoperatively, there was an insignificant decrease in IOP in the glaucoma group; the mean decrease was 1.4 mm Hg +/- 3.8 (SD) at 12 months and 1.6 +/- 4.2 mm Hg at 18 months. The mean number of medications decreased significantly at 12 months (0.51 +/- 0.75) and at 18 months (0.41 +/- 0.83) (P = .04). The control group also had a significant decrease in IOP, with a mean decrease of 0.83 +/- 2.8 mm Hg at 12 months (P = .01) and 1.26 +/- 2.9 mm Hg at 18 months (P < .0001). The decrease in IOP was more pronounced in eyes with a higher preoperative IOP in both the glaucoma and control groups. CONCLUSION: These findings suggest that sutureless clear corneal phacoemulsification with foldable acrylic IOL implantation is a relatively safe and simple surgical option in patients with cataract and well-controlled glaucoma. The approach provided favorable long-term IOP change and led to rapid visual rehabilitation in both the glaucoma and control group. PMID- 16681048 TI - Vogt-Koyanagi-Harada disease in Thai patients. AB - OBJECTIVES: To study the demographic, clinical manifestations, treatment and visual outcome of Vogt-Koyanagi-Harada disease (VKH) in a group of Thai patients. DESIGN: Retrospective case series. MATERIAL AND METHOD: Patients with VKH disease in Pramongkutklao Hospital from January 1998 to December 2002 were identified. Demographic data, clinical manifestations, treatment, and outcome were analyzed. RESULTS: Among the 33 patients, there were 12 males and 21 females with a mean age of onset of 35 +/- 13.4 year-old (range 17-67). Serous retinal detachment was detected in 48%. Transient hearing loss, vitiligo, poliosis and alopecia occurred in 18%, 24%, 15% and 33% respectively. Forty two percent of the patients were treated with systemic corticosteroids alone and 58% with a combination of steroids and immunosuppressive drugs. At the end of the follow-up, visual acuity (VA) improved in 55%, remained unchanged in 36% and worsened in 9% of the eyes. Final VA of equal or better than 20/40 was obtained in 76%. The mean follow-up time was 21.6 +/- 14.9 months (range 2-64). CONCLUSION: Demographic data, clinical manifestations, and treatment outcome of this group of Thai patients are comparable with studies from other countries. PMID- 16681049 TI - Unfocused effect on the measurement of ocular aberrations. AB - OBJECTIVES: To evaluate the effect of unfocused measurement in the value of ocular aberrations. MATERIAL AND METHOD: The present study was conducted at Siriraj Hospital, Mahidol University. Ocular aberrations of 20 eyes in 10 myopic patients (myopia less than -6.00 diopters (D) and astigmatism less than -2.00 diopters (D)) were analyzed by using Zywave aberrometer (Bausch and Lomb, USA). The measurements were done at the pupillary plane (focus) and 6 other different planes (unfocus); 5, 10 and 15 mm inside and outside pupillary plane respectively. The value of each measurement was analyzed to find the effect of unfocused measurement on ocular aberrations. RESULTS: The magnitude of error (root mean square, RMS) of the spherical component of refraction at 5, 10, 15 mm inside and outside the pupillary plane was 0.16, 0.44, 0.57 D and 0.21, 0.38, 1.35D respectively. The RMS of astigmatic component of refraction inside and outside the pupillary plane at the same distance was 0.19, 0.50, 0.80 and 0.18, 0.52, 1.55 D respectively. The RMS of higher order aberrations inside and outside the pupillary plane at the same distance was 0.05, 0.13, 0.15 and 0.06, 0.1, 0.1 microns respectively. CONCLUSION: There was more effect of outward unfocused measurement in the value of refraction (spherical and astigmatism) than inward unfocused measurement. Higher order aberration showed a similar error in both inward and outward defocus. The magnitude of error was 10 - 12% within 5 mm unfocused planes. PMID- 16681050 TI - Pseudophakic retinal detachment with ruptured posterior lens capsule. AB - The relationship between retinal detachment and posterior lens capsule rupture in pseudophakic patients was studied. Records of patients with pseudophakic retinal detachment who were admitted to Siriraj Hospital from 1992 to 2004 were reviewed. The patients were divided into 2 main groups: 1) The study group (pseudophakic retinal detachment) 79 eyes (78 cases) which was subdivided into group 1a) pseudophakic retinal detachment with intact posterior lens capsule (50 eyes), and group 1b) pseudophakic retinal detachment with ruptured posterior lens capsule (29 eyes); 2) The control group 99 eyes (90 cases) which was subdivided into group 2a) pseudophakic patients with intact posterior lens capsule (83 eyes), and group 2b) pseudophakic patients with ruptured posterior lens capsule (16 eyes). The relationship between pseudophakic retinal detachment and posterior lens capsule rupture was analyzed by Chi-square test, and risk factors were analyzed by multiple logistic regression. Retinal detachment in pseudophakic patients was found more often in males more than in females, and the average age was 57.9 +/- 11.8 years, the retinal detachment was related to posterior lens capsule rupture (p = 0.003). Patients with pseudophakic retinal detachment with a ruptured posterior lens capsule had a risk 5.7 times greater than pseudophakic patients with an intact posterior lens capsule (adjusted Odds ratio = 5.716, 95% CI = 2.118-15.427). The present study showed that posterior lens capsule rupture increased the risk of retinal detachment. Ophthalmologists should be aware of retinal detachment development after YAG capsulotomy, especially in patients who have other risk factors such as myopia, lattice degeneration, retinal break, or previous retinal detachment surgery. PMID- 16681051 TI - Current trends in cataract surgery in Thailand--2004 survey. AB - OBJECTIVES: To survey and investigate the current trend of cataract surgery in Thai ophthalmologists in 2004. MATERIAL AND METHOD: Questionnaires were sent to 600 ophthalmologists who were the members of the Ophthalmological Society and Royal Colleges of Ophthalmologists of Thailand. Data received from 248 (41.3%) of the recipients were analyzed and compared with those from the previous survey. RESULTS: The majority of respondents were male (63.3%) while 36.7% were female. In cataract surgery, 99.2% were still doing cataract surgery, the average number of cataract surgery procedures per surgeon per month was 25.6, 89.8% preferred phacoemulsification, and 42.5% preferred acrylic lens. The posterior capsular tear was the most common complication. CONCLUSION: There are trends toward more cataract surgical procedures performed by a surgeon, increasing preference of topical anesthesia, small incision wound and foldable acrylic lenses which reflected the popularity of phacoemulsification. PMID- 16681052 TI - The fundoscopic features for differentiation between polypoidal choroidal vasculopathy and choroidal neovascularization from age-related macular degeneration. AB - OBJECTIVES: To evaluate the fundus features that can differentiate polypoidal choroidal vasculopathy (PCV) from choroidal neovascularization (CNV) due to age related macular degeneration (AMD). DESIGN: Retrospective, case-control study. MATERIAL AND METHOD: The fundoscopic features of AMD-related CNV and PCV were compared for evaluation of relative risks for PCV. The odds ratio, sensitivity and specificity of each feature were also calculated. RESULTS: A total of 30 patients, 15 PCV and 15 age-matched non-PCV cases, approved by indocyanine green angiography (ICG), were enrolled. The odds ratio is highest for subretinal polyp like structure and lowest in scar formation (28 and 0, respectively) and these ratios are statistically significant differences (p = 0.001 and 0.032 respectively). The ratio for either circinate exudates, or larger than 4-disc area of subretinal fluid, or retinal pigment epithelial detachment is 1.75. The ratio for peripapillary location is 1.63. Subretinal membrane has a ratio of 1 while subretinal hemorrhage and subretinal fibrin have less than 1; they are 0.31 and 0.23 respectively. All these ratios are not statistically significant differences (p > 0.05). CONCLUSION: The orange polyp-like structures beneath retina are hallmarks for diagnosis of PCV. The "serosanguineous retinopathy", described as serous retinal deatchment or retinal pigment epithelial detachment with circinate exudates with or without subretinal hemorrhage at the posterior pole or peripapillary region, may also suggest the condition. PMID- 16681053 TI - The results of amniotic membrane transplantation for symptomatic bullous keratopathy. AB - OBJECTIVES: To evaluate the outcomes of amniotic membrane transplantation for symptomatic relief in patients with bullous keratopathy. MATERIAL AND METHOD: This retrospective study included 17 eyes (17 patients) with bullous keratopathy presenting with intractable pain or discomfort. The patients were enrolled from January 2000 to December 2004. Amniotic membrane transplantation was performed. Symptomatic relief epithelial healing, and visual changes were analyzed. RESULTS: During the follow up period of 14.1 +/- 11.9 months (range 1-36 months) after amniotic membrane transplantation, 14 eyes of 17 eyes (82.4%) with intolerable pain preoperatively had pain relief postoperatively. Corneal epithelial healing was complete in all except 2 eyes one of which had evisceration because of severe corneal ulcer, and the other underwent penetrating keratoplasty soon after amniotic membrane transplantation. CONCLUSION: Amniotic membrane transplantation is a safe and effective treatment modality for pain relief associated with chronic bullous keratopathy. It can be an alternative to conjunctival flap, with better cosmetic appearance for the management of patients with bullous keratopathy. PMID- 16681054 TI - The high-dose, alternate-week intravitreal ganciclovir injections for cytomegalovirus retinitis in acquired immune deficiency syndrome patients on highly active antiretroviral therapy. AB - OBJECTIVES: To evaluate the efficacy, and complications of the high-dose, alternate-week, intravitreal ganciclovir injection for cytomegaloviral retinitis (CMVR) in acquired immune deficiency syndrome (AIDS) patients on highly active antiretroviral therapy (HAART). DESIGN: Retrospective case series. PARTICIPANTS: AIDS patients with CMVR and on HAART MATERIAL AND METHOD: The high-dose, 4 mg/0.1 ml, ganciclovir was injected intravitreally to the enrolled patients on an alternate-week basis. The patients were monitored clinically until the retinitis was inactive, then the injections were withdrawn. The injections were re initiated if relapse occurred. MAIN OUTCOME MEASURES: The number of eyes achieved inactive retinitis and corresponded to the number of injections, number of relapses and corresponded duration, visual acuity during the injection, and complications of the injection. RESULTS: Inactive lesions were found in 42/51 eyes (82.40%), the corresponding mean number of injections was 5.4 (1-18) per eye. There was no relapse and the corresponded duration of follow-up was 5.1 months (1-16). The final visual outcomes were improved or stable in 26 eyes (50.9%). These visual outcomes were statistically related to initial visual acuity (p = 0.022) but not statistically related to the number of injections (p = 0.929). Complications were found in 7/51 eyes (13.7%). They were vitreous haze, immune recovery uveitis, rhegmatogenous retinal detachment, and infectious endophthalmitis. CONCLUSION: The high-dose, alternate-week, intravitreal injection of ganciclovir may be an alternative for the treatment of CMVR in AIDS patients who are on HAART However, the induction course is longer than the weekly regimen and close monitoring of patients is essential. PMID- 16681055 TI - Surgical outcomes in rhegmatogenous retinal detachment at Srinagarind Hospital. AB - OBJECTIVES: To evaluate the surgical outcome for patients with rhegmatogenous retinal detachment and to find out the factors influencing the anatomic reattachment and visual results. MATERIAL AND METHOD: Retrospective review of the medical records of patients for the following details: age, sex, time to diagnosis, time to surgery, phakic status, types, numbers and locations of the retinal breaks, macular status, presence of proliferative vitreoretinopathy (PVR), types of surgical procedures, anatomic reattachment and visual improvement. RESULTS: Two hundred and twelve patients were surgically treated by pneumatic retinopexy, scleral buckling, pars plana vitrectomy or combined procedures. Initial anatomic reattachment was found in 157 cases (74.06%) and final reattachment in 193 cases (91.04%). Several factors including age, phakic status, number of the retinal breaks, macular detachment, PVR and types of surgical procedures have been identified to have a correlation with the anatomic success rate. Types of surgical procedures and time to surgery within 8 weeks of detachment have also been demonstrated to have a correlation with final visual improvement. However, only types of surgical procedures had statistically significant difference. Pneumatic retinopexy had a lower anatomic reattachment rate, but obtained a higher visual improvement than did other surgical procedures. Moreover, after repeated pneumatic retinopexy or further surgical procedures, the final anatomic success rate improved from 65.52% to 92.24%. CONCLUSION: Surgical outcomes for rhegmatogenous retinal detachment are comparable to other studies. Types of surgical procedures had a significant correlation with anatomic reattachment and final visual improvement. PMID- 16681056 TI - Translimbal fixation of posterior chamber lenses. AB - OBJECTIVES: To evaluate the result of a surgical technique to insert and secure a standard posterior chamber lens through a simple limbal incision in eyes that had no capsular support or whose capsule was inadvertently ruptured during extracapsular cataract extraction. MATERIAL AND METHOD: Retrospective review of the medical records of patients who underwent translimbal fixation of posterior chamber lenses according to age, gender, preoperative visual acuity, postoperative visual acuity, follow-up period and any complications, especially those that may be related to the surgical technique. RESULTS: Twelve patients were recruited in the present study. The patents' age ranged from 31 to 77 years, with a mean of 62.58 years. There were 9 male and 3 female patients. The preoperative visual acuity ranged from hand motion to counting fingers, and the postoperative visual acuity ranged from 6/6 to 6/24 except one patient who had postoperative visual acuity of counting fingers due to a pre-existing macular hole. The length of follow-up period ranged from 3 to 12 months, with a mean of 6.17 months. There was no clinically significant lens-related complication. CONCLUSION: This simple surgical technique controls the location of the needle entry and exit to decrease the risk of bleeding and increase the chance of ciliary sulcus placement. An inferior groove was made to bury the suture knot and was sutured closed to prevent the knot erosion through the conjunctiva. PMID- 16681057 TI - Outcomes of cataract surgery in senile cataract patients at Siriraj Hospital: a prospective observational study. AB - The present study was done to assess the results of cataract surgery performed in Siriraj Hospital by comparing phacoemulsification and extracapsular cataract extraction techniques in senile cataract patients within a 3-month period. The prospective observational study of a representative sample of 379 patients (409 eyes) who underwent cataract surgery in Siriraj Hospital from January 1, 2004 to March 31, 2004. Identical clinical assessment protocol was used to compare the improvement of postoperative visual acuity and surgical complications between two surgical techniques and different surgeons. Of the 409 eyes, phacoemulsification was performed on 373 eyes (91.2%), and extracapsular cataract extraction was performed on 36 eyes (8.8%). Departmental staff operated on 326 eyes (79.7%) and ophthalmology residents operated on 83 eyes (20.3%). The primary outcome was postoperative visual acuity. Visual acuity was improved > or = 2 Snellen chart lines in 278 of 373 eyes (74.5%) in the phacoemulsification group, and > or = 2 lines in 31 of 36 eyes (86.1%) in the extracapsular cataract extraction group. A small risk of surgical complications was found in the present study. Posterior lens capsule rupture was the most frequent complication found (10/ 409 eyes), followed by corneal injury (9 of 409 eyes). Endophthalmitis was the most severe complication in the present study, occurring in 1 of 409 eyes. There was a significant difference in postoperative visual improvement between phacoemulsification and extracapsular cataract extraction techniques. Final visual acuity in the phacoemulsification group was better than in the extracapsular cataract extraction group. PMID- 16681058 TI - Vitreous loss during phacoemulsification learning curve performed by third-year residents. AB - OBJECTIVES: To assess the resident's phacoemulsification learning curve as a risk factor for vitreous loss and to determine the incidence of vitreous loss among the residents performing phacoemulsification. DESIGN: Retrospective matched case control study MATERIAL AND METHOD: A case-control study comparing all consecutive cases of attempted phacoemulsification with intraocular lens (IOL) implantation from January 1st, 1998 to December 31st, 1999. The surgeon variable will be categorized into two groups, the third year ophthalmology residents, representing resident's phacoemulsification learning curve, and faculty staffs. The study group consisted of eyes that had had intraoperative complication of vitreous loss. The control group consisted of eyes that had not had vitreous loss. Continuous variables were compared with the 2-sided unpaired t-test. Categorical variables were compared between groups using analytical matched case-control study with relative risk or odd ratio, Mc Nemar's (Marginal) o 2 test and 95% confident interval of relative risk. RESULTS: The odds that the eyes in the resident group would have an intraoperative complication of vitreous loss were 4 times the odds that the eyes in the faculty staff group would have such complication (P = 0.0052, 95% confidential interval (CI) of relative risk (RR) = 1.516-10.556). The incidence of vitreous loss among residents was 6.93% (28/404) and 2.06% (28/1358) among the faculty staffs. The overall incidence of vitreous loss was 3.18% (56/1762). CONCLUSION: The incidence of intraoperative complication of vitreous loss, the relative risk of such complication performed by the learning curve surgeon in the present study serve as benchmarks for residents-in-training, beginning and surgeon-in-practice converting to phacoemulsification. PMID- 16681059 TI - The outcomes of horizontal strabismus surgery and influencing factors of the surgical success. AB - OBJECTIVES: To evaluate the surgical outcomes of horizontal strabismus and identify the influencing factors of the surgical success. MATERIAL AND METHOD: The medical records of 304 patients who underwent their first strabismus surgery for treatment of horizontal deviation at Siriraj Hospital were reviewed retrospectively from January 1998 to December 2003. The main outcome measure for successful surgery was defined as eye deviation 10 prism diopters or less at 6 weeks post-surgery. The outcomes of postoperative binocular function and visual acuity improvement were also evaluated. The pre operative deviation, diagnosis, binocular function, visual acuity and age were analyzed as influencing factors of surgical success. The patients were divided into two groups, the first group was 6 years or less (161 cases) and the second group was older than 6 years (143 cases). The statistical methods for analysis the data were Chi-square test, Mann Whitney U test and Logistic regression. RESULTS: The follow- up period was from 3 to 12 months. 187 patients (61.5%) for esotropia and 117 patients (38.5%) for exotropia were identified. Mean patient's age was 10.5 +/- 10.2 years (median 6, min 0.5, max 53), mean preoperative deviation was 44.9 +/- 16.6 prism diopters and mean VA was 20/30 (no PL, 20/20). The success rate was 62.0% for esotropia and 57.0% for exotropia. There was no statistical difference between the two groups of diagnosis. The average final deviation postoperative in the success group was 6 +/- 4.9 prism diopters. The comparison of difference visual acuity between pre and post-operation showed no statistical significance but the presence of binocular vision was increased in number of patient by two times. The complication rate was 0.7% (2/304), one case showed conjunctival wound dehiscence and the other had globe perforation. Re-operative rate was 12.9% (39/304) after one year. CONCLUSION: The surgical success rate of horizontal strabismus surgery was 60.2% and revealed no statistically significant improvement of visual acuity after surgery. The successful surgery was related to the age younger than 6 years and preoperative deviation less than 30 degrees. PMID- 16681060 TI - The efficacy of unoprostone isopropyl as an adjunct to topical beta-blocker in patients with open angle glaucoma: a-6-month study. AB - OBJECTIVES: To assess the efficacy and safety of unoprostone isopropyl as an adjunctive treatment to topical beta-blocker in patients with primary open angle glaucoma (POAG). STUDY DESIGN: This was a prospective, open-label clinical study. MATERIAL AND METHOD: A total of 44 eyes of 22 eligible patients whose intraocular pressure (IOP) was inadequately controlled by topical beta-blocker were enrolled. Inclusion criteria consisted of patients with primary open angle glaucoma who either had IOP measurements > or = 22 mmHg while on topical beta-blocker monotherapy or had IOP measurements > or = 18 mmHg while on dual therapy (topical beta-blocker and a second drug of a different class which was to be discontinued prior to the study to allow washing out of its effects). INTERVENTION: Baseline IOP, pupil size, blood pressure and pulse rate were initially measured; the patients were then examined at 2nd, 4th, 8th, 12th, 18th and 24th weeks of following commencement of topical unoprostone isopropyl therapy (given twice daily). MAIN OUTCOME MEASURES: IOP pupil size, blood pressure and pulse rate were measured and were compared to baseline values. RESULTS: In 44 eyes of 22 eligible patients, unoprostone isopropyl resulted in a statistically significant IOP reduction of 24.6% (p < 0.02). The mean systolic blood pressure decreased from 132.79 +/- 22.11 mmHg (range 100-180 mmHg) at baseline to 125.77 +/- 18.40 mmHg (range 80-160 mmHg) at 24th week after unoprostone isopropyl administration. This reduction was statistically significant (p = 0.002) but was unlikely to have clinical importance. Both mean diastolic blood pressure (p = 0.344), pulse rate (p = 0.306), and pupil diameter (p = 0.107) were not significantly affected. CONCLUSION: Topical unoprostone isopropyl beneficially provides additive IOP lowering effect to topical beta-blocker in patients with primary open angle glaucoma. No serious systemic side effects were found in the present study. PMID- 16681061 TI - Assessment of the anterior structures of eyes in a normal Northern Thai group using the Orbscan II. AB - OBJECTIVES: To evaluate the anterior structures of the eyes in normal Northern Thais include the corneal topography and thickness, as well as the anterior chamber depth (ACD) and the white-to-white (WTW) using the Orbscan II system. MATERIAL AND METHOD: One hundred and six eyes of 56 normal subjects were investigated using the Orbscan II. The simulated keratometry (SimK), astigmatism, corneal thickness, ACD and WTW were collected. The axial power maps as well as anterior and posterior elevation maps were read and categorized. Corneal thickness was measured in different regions and the pachymetry patterns were classified. RESULTS: The mean SimK was 44.18 (1.41)/43.30 (1.46) diopters (D) and the mean astigmatism was 0.93 (0.58) D. Symmetric bow tie was the most common axial power pattern in the anterior cornea (57.6%), followed by asymmetric bow tie (19.8%), irregular patterns (12.3%), round (9.4%), and oval (0.9%). Incomplete ridge (40.6%) and island (34%) were common elevation patterns observed in the anterior corneal surface, and island (92.5%) was the most common topographic pattern in the posterior. The thinnest point on the cornea had an average thickness of 512.49 (35) micron and was located at an average of 0.43 (0.24) mm from visual axis. In the pachymetry maps, round (47.2%) and oval (45.3%) were common patterns. The mean ACD and WTW was 2.79 (0.35) and 11.61 (0.36) mm, respectively. CONCLUSION: The present study demonstrated the normal variations of anterior segment structures of the eyes in normal Northern Thais using the Orbscan II topography system. This information may be useful for comparison with further quantitative studies of various abnormal states. PMID- 16681062 TI - Prevalence of abnormal vision in one-year-old Thai children, based on a prospective cohort study of Thai children (PCTC). AB - OBJECTIVES: To collect preliminary data on the prevalence of abnormal vision in one-year-old Thai children. MATERIAL AND METHOD: A retrospective study was conducted using data collected from a prospective cohort study of Thai children (PCTC) carried out during 2000-2002, to examine the prevalence of abnormal vision in one-year-olds. Data from five districts in five provinces were examined. One year-old children in the present study underwent vision screening and eye examination performed by non-medical research assistants. RESULTS: There were records from 3,898 children in five districts, 49.7% females and 50.2% males. Their eye examinations showed very good vision in 77.8% (9.8 cy/cm at 38 cm), good vision in 21.5% (6.5 cy/cm at 38 cm), and fair vision in 0.7% (< or = 1.6 cy/cm at 38 cm). Normal ocular motility was found in 99.7% and 99.9% had normal anterior segment and lens. No strabismus was found in 99.4%, 99.9% had normal pupil light reflex, 99.8% had normal red reflex, 93.6% could fix and follow at 3 months of age, 85.7% could detect a falling object at 6 months, and 78.5% of mothers and 46.5% of fathers regularly played with the children. Logistic regression analysis indicated that some factors had statistical significance, such as "fix and follow" by 1 month of age, and lack of father-child interaction, but these were not clinically significant. Using Kappa analysis, the authors combined groups 1 (very good vision) and 2 (good vision) to create a "normal vision" category. If children had 2 abnormal eye examinations (eye exam for strabismus, cornea, anterior chamber, lens, pupil and red reflex), the authors recommended sending them to a specialist. The sensitivity and specificity of the visual screening and eye examination instruments were 19.23% and 99.38%, respectively. The Kappa statistic was 0.17. These instruments are not appropriate for eye screening in one-year-old children. CONCLUSION: More than 99% of the children examined had normal results. The majority of children could fix and follow from 2-3 months of age. The early fix and follow development may be related to better visual acuity. The interaction between father and child may be associated with eye development. However, eye screening of one-year-old children by assistant researchers may be inappropriate due to lack of expertise and experience. PMID- 16681063 TI - Intraocular pressure control following phacoemulsification in patients with chronic angle closure glaucoma. AB - OBJECTIVES: To evaluate intraocular pressure (IOP) and glaucoma control following phacoemulsification with posterior chamber intraocular lens implantation in patients who had chronic angle closure glaucoma (CACG) whose peripheral anterior synechiae (PAS) were less than 270 degrees. DESIGN: Non-randomized consecutive cases series. PARTICIPANTS: CACG cases at the glaucoma service, Ramathibodi Hospital who underwent Neodymium YAG laser peripheral iridotomy (PI) and subsequently received or did not receive anti-glaucoma medication. MATERIAL AND METHOD: Retrospective analysis of CACG patients who had PAS 270 degrees or less and underwent YAG-PI with or without anti-glaucoma medication to control IOP at 21 mmHg or less. The IOP and number of anti-glaucoma medication used at 1, 3 and 6 months were measured. Patients were classified into 2 Groups according to degree of PAS: Group 1 were patients who had PAS 180 degrees or less and Group 2 were those whose PAS was between 181 degrees and 270 degrees. MAIN OUTCOME MEASURES: The IOP and number of anti-glaucoma medication at baseline and postoperatively at 1, 3 and 6 months were compared by nonparametric statistics. RESULTS: There were 28 patients (48 eyes) in the present study. Twenty two were females and 6 were males. Patients' age ranged from 45 to 76 years old with a mean of 55 +/- 6.5 years. Of the 48 eyes, 34 were in Group 1 and 14 were in Group 2. In Group 1, the mean baseline IOP was 20 +/- 2.5 mmHg. and the average number of preoperative anti-glaucoma medication used was 0.08. At 1, 3 and 6 months postoperatively, the IOPs were 16.2 +/- 2.2, 17.1 +/- 2.0, 18.1 +/- 1.4 mmHg. respectively with an average number of anti-glaucoma medications of 0, 0.3 and 0.3, respectively, whereas in Group 2, the mean baseline IOP was 22 +/- 3.8 mmHg and mean preoperative number of anti-glaucoma was 1.8, whereas postoperatively, the IOPs at 1, 3 and 6 months were 17.1 +/- 2.2, 17.3 +/- 1.8, and 17.1 +/- 1.7 mmHg, with an average number of 1.1, 1.4 and 1.4 anti-glaucoma medications used, respectively. When compared between the 2 Groups, Group 1 had a significant difference in IOP control at 1 and 3 months and less use of antiglaucoma medications than Group 2 up to at least 6 months. CONCLUSION: Phacoemulsification in CACG helped control of glaucoma. There was a statistically significant difference in IOP reduction and number of anti-glaucoma medication used before and after phacoemulsification in the CACG patients whose PAS did not exceed 270 degrees at least up to 6 months. PMID- 16681064 TI - A traumatic bitemporal hemianopia with macular sparing. AB - Traumatic chiasmal syndrome is a rare complication of closed head trauma. It often presents as bitemporal hemianopia and may be associated with other neurological signs. The authors report a case of a 47-year-old man who had sustained severe frontal head trauma from a motor vehicle accident that caused multiple cranial fractures and prolonged loss of consciousness. He was subsequently diagnosed with traumatic chiasmal syndrome. Tangent field testing revealed bitemporal hemianopias with some macular sparing. Macular sparing was not found on the central 30-2 pattern of Humphrey visual field test. PMID- 16681065 TI - Ocular myasthenia gravis and auto-immune thyroiditis in children. AB - A 7-year-old girl presented at a university hospital with ptosis of the left eye. This resolved spontaneously within 4 weeks but then the right eye became similarly affected but responded to prostigmine. Left hypertropia with restriction of the right inferior rectus, mild exophthalmos, non-tender diffuse enlargement of the thyroid, normal thyroid function tests, anti-thyroglobulin, and anti-microsomal antibodies indicated an association of autoimmune thyroiditis and ocular myasthenia. The ptosis was remedied with pyridostigmine and short course oral prednisolone, but the hypertropia persisted. PMID- 16681066 TI - Isolated corneal intraepithelial neoplasia. AB - OBJECTIVES: To report two cases of isolated corneal intraepithelial neoplasia. MATERIAL AND METHOD: The present study included 2 cases presenting with isolated corneal intraepithelial neoplasia. Both patients were treated by corneal epithelial scraping. RESULTS: The authors present the clinical picture and histopathological findings of 2 patients suffering from corneal intraepithelial neoplasia that did not arise from the corneoscleral limbus. Both patients showed no recurrence after treatment for a period of time. CONCLUSION: Isolated corneal intraepithelial neoplasia is rare. Corneal scraping may be an effective treatment. PMID- 16681067 TI - Transitional cell carcinoma of the lacrimal sac. AB - Lacrimal sac tumor is a rare tumor, for which we do not know the exact incidence. Malignant tumors account for 70% of all cases. Epithelial tumors are the most common pathological type and squamous cell carcinoma is the most common of these, a transitional cell carcinoma is rare and has a poor prognosis. The authors report a rare case of transitional cell carcinoma of the lacrimal sac. A 57-year old woman had tearing on her right eye. The pre-operative diagnosis was possible nasolacrimal duct obstruction with signs of dacryocele. Intraoperatively the authors found a lacrimal mass and a frozen section showed squamous papilloma with dysplasia. The tumor was completely removed and DCR was done. The subsequent pathological report of the right lacrimal sac was papillary transitional cell carcinoma, so medial maxillectomy and resection of the medial inferior orbit with ethmoidectomy were performed. She received radiation and has been tumor-free for 2 years. PMID- 16681068 TI - Sheathotomy without separation of venule overlying arteriole at occlusion site in uncommon branch retinal vein occlusion. AB - Two cases of uncommon branch retinal vein occlusion (BRVO) with vein overlying artery at occlusion site that can be found in less than 1% who underwent retinal venule sheathotomy without separation of retinal vessel for decompression of BRVO were reported. Both patients had macular hemorrhage, edema, and area of macular capillary nonperfusion. Visual acuity (VA) worsened to 6/60 in both cases. The retinal venules were dissected around the crossing site without separation of retinal vessels. Intraoperative dilation, pulsation and restoration of downstream blood flow of the involved venules were observed. In the first patient, at 1 day, 2 weeks, and 6 weeks postoperatively, VA improved to 6/36, 6/9, and 6/6, respectively, and remained unchanged at 12 months postoperatively. In the second patient, VA improved to 6/24 on the first day postoperatively and improved to 6/18 at 1 week follow up and continuously improved to nearly normal at 2 months postoperatively then patient lost contact. Postoperative fundus fluorescein angiogram showed dilated and improved perfusion with decreased macular edema in both cases. Optical coherent tomography confirmed remarkable reduction of retinal thickness (from 874 microns preoperatively to 420 microns at 1 week postoperatively) in the second patient. Retinal venule sheathotomy without separation of retinal vessel for decompression of BRVO with venule overlying arteriole at occlusion site could be effective for improving VA and decreased macular edema. PMID- 16681069 TI - Late postoperative capsular block syndrome. AB - The authors report the case of a 61-year-old man with late postoperative capsular block syndrome who had undergone phacoemulsification and posterior chamber intraocular lens (IOL) implantation 3 years earlier. He presented with a decreased vision in the right eye caused by the accumulation of liquefied milky fluid in the space between the posterior surface of IOL optic and the posterior capsule. Neodymium: YAG laser posterior capsulotomy allowed a resolution of the milky fluid and the improvement of the visual acuity to 20/20. PMID- 16681070 TI - Health related quality of life instruments for glaucoma: a comprehensive review. AB - Health related quality of life (HRQOL) outcome is becoming important and of interest for clinicians and patients alike. HRQOL can be affected immediately after the initial diagnosis of the disease through anxiety of blindness. Further impairment in various aspects of HRQOL is expected over time as the disease progresses, reducing daily activities. Without a gold standard for HRQOL construct in this population, a number of instruments have become available with different characteristics and foci. This article reviews published HRQOL instruments and their psychometric properties in glaucoma patients. Of the 10 instruments reviewed, 2 were generic, 4 were vision-specific and 4 were glaucoma specific instruments. Overall, vision- and glaucoma-specific instruments appear to be more sensitive than generic instruments in detecting potential changes of HRQOL in the patients. The shortcoming of existing instruments, however, arises from being predominantly focused on physical functions while omitting other aspects relevant to patients HRQOL such as psychological and social well-being. In addition, many vision-specific instruments have inadequate coverage of important issues, such as peripheral and color vision, which are affected by glaucoma disease. Validation of the instruments using various magnitudes of visual field is warranted and further investigation of their responsiveness is required for them to be more useful for outcome evaluation in the clinical setting. Refinement of an instrument to enhance the incorporation of HRQOL in routine management of patients with glaucoma is briefly described. PMID- 16681071 TI - Guidelines for prescribing optical correction in children. AB - As the eye grows, the axial length increases while the cornea and lens flatten. High refractive errors which are common in the neonatal period, reduce rapidly during the first year of life through the process called emmetropization. The possibility that long-term full- time glasses wear may impede emmetropization must be considered. Hyperopia greater than 5.00 diopters (D) in young children is associated with an increased risk of amblyopia and strabismus, therefore optical correction should be prescribed. When hyperopia is associated with esotropia, full correction of the cycloplegic refractive error should be prescribed. Myopia greater than 8.00 D and astigmatism greater than 2.50 D are common causes of isometropic amblyopia. Patients with hyperopic anisometropia with as little as l D difference between the eyes may develop amblyopia while the difference should reach 3-4 D for myopic anisometropia to develop amblyopia. Full cycloplegic refractive difference between two eyes should be given to the anisometropic child in spite of age, strabismus and degree of anisometropia. Myopia control is the attempt to slow the rate of progression of myopia such as cycloplegic agents, plus lenses at near, and rigid contact lenses. PMID- 16681072 TI - The history of the Royal College of Ophthalmologists of Thailand. PMID- 16681073 TI - Clinical and financial opportunities abound in maternity DM. PMID- 16681074 TI - Generous provider incentives deliver dramatic returns. AB - A stunning success in the use of provider incentives to boost care. A physician incentive program established a decade ago by CA-based Inland Empire Health Plan has turned into a powerhouse for quality improvement. In fact, the approach has worked so well that the plan has now turned its attention to chronic care management, where early evidence suggests that generous bonus payments will produce improvements in clinical outcomes as well as process measures. PMID- 16681075 TI - Impedence cardiography: a new tool in the treatment of hypertension. AB - Help for physicians in zeroing in on the cause of high BP. Impedance cardiography (ICG) has been used to guide treatment decisions for heart failure and shortness of breath for many years. However, now there is new evidence that the approach can help physicians target the underlying cause of high BP, and technology is available to facilitate non-invasive ICG testing in primary care settings. It's a new tool that could potentially make a big difference in tackling one of the major risk factors for heart disease and strokes. PMID- 16681076 TI - Using performance feedback to change provider behavior. AB - Using performance feedback to overcome clinical inertia. For a variety of reasons, physicians have a strong tendency to under-treat, particularly with respect to diseases like diabetes, which are often asymptomatic. However, researchers at Emory University have a developed an intervention that can successfully overcome this problem, and ultimately improve clinical outcomes as well. PMID- 16681077 TI - Tear osmolarity. PMID- 16681078 TI - Accessory cell populations in the cornea. AB - According to the "classical" doctrine, resident cells of the cornea include the stratified epithelial cells, quiescent keratocytes, and a single layer of nondividing endothelial cells. However, it has become increasingly evident that other cell types are involved in the homeostasis of the cornea. The presence of various cell types from different lineages has raised concern among researchers as to what we are actually "seeing" in the cornea. Although definitive conclusions cannot yet be drawn, this review attempts to clarify the various accessory cell types reported in the human and murine cornea. The epithelial layer of the limbal area includes melanocytes, as well as antigen-presenting cells that are also present in the peripheral clear cornea. The most debated tissue currently is perhaps the corneal stroma, where resident keratocytes are not as large a population as was previously believed. Bone marrow-derived cells are found in the cornea, and these may not express the typical HLA molecules usually found on the surface of antigen-presenting cells. Nerve fibers extend though the superficial stroma to form a plexus beneath the epithelium. Although the cell body of the neuron itself is not in the cornea, neural glial cells, such as Schwann cells, are present in the stroma. The use of specific molecular markers and high-quality imaging techniques will be required to fully elucidate the various accessory cells of the cornea and their function. PMID- 16681079 TI - In vivo confocal microscopy of the ocular surface. AB - Over the past two decades, the applications of in vivo confocal microscopy to the investigation of ocular surface diseases in the living eye have been greatly extended. Confocal microscopy enables detailed investigation of tarsal and palpebral conjunctiva, central and peripheral cornea, tear film, and lids, and it allows evaluation of the ocular surface at the cellular level. High-quality imaging in both contact and noncontact modes has allowed new understanding of the functions of the ocular surface system, and in the coming years, such knowledge will become increasingly comprehensive and specific. Confocal microscopy may provide a link between well-established ex vivo histology and in vivo study of ocular pathology, not only in clinical science but also in clinical practice. The purpose of this review is to summarize the current knowledge about in vivo confocal microscopy of the ocular surface. PMID- 16681080 TI - Ocular effects of cosmetic products and procedures. AB - Cosmetic products and procedures can produce adverse effects on the ocular surface, ranging from mild discomfort to vision-threatening conditions. Complications of skin and eye products can be related to allergy or toxicity, often attributable to perfumes. Complications of blepharoplasty may be associated with overcorrection, scarring, or uneven contour of the lid margins, conditions that can cause significant ocular surface disease until they are corrected. Ocular surface effects of botulinum toxin injection include dry eye syndromes and also epiphora. More serious complications occur (rarely) and are dose- and location-related. Adverse effects of micropigmentation procedures involve pathological processes or, simply, poor cosmetic results. In certain youth cultures, special-effect contact lenses and even decorative conjunctival implants have gained popularity, presenting a wide variety of dangers, often attributable to poor education about care and hygiene. PMID- 16681081 TI - Research ethics. PMID- 16681082 TI - Juan Murube, MD, PhD. PMID- 16681083 TI - Argon versus selective laser trabeculoplasty. AB - PURPOSE: To compare conventional argon laser trabeculoplasty (ALT) with selective laser trabeculoplasty (SLT) in terms of their efficiency in lowering the intra ocular pressure. METHODS: In this retrospective study, 56 eyes from 44 patients with primary open angle glaucoma, ocular hypertension, pseudo-exfoliative (PXF) or pigment dispersion glaucoma (PDG) were included. Patients underwent either ALT (n=18) or SLT (n=38). The intraocular pressure (IOP) was measured immediately prior to and 3 to 5 weeks after the theraPY. RESULTS: At 3 to 5 weeks the lOP reduction was 22.4% after ALT and 15.5% after SLT (p = 0.141). Of note, of the four patients with PDG 2 underwent ALT and 2 SLT. Remarkably, both patients who had had SLT showed a paradoxical rise in lOP after the procedure (+15.5%). When these patients were excluded from the analysis, a similar hypotensive efficacy was found between ALT (-19%) and SLT (-17.9%) (p = 0.836). A small additional study with lower energy levels (< 0.9 mJ) confirmed the paradoxical IOP rise in 6 patients with heavily pigmented angles (2 with PDG and 2 with PXF) (+19.2%). It occurred in the absence of steroid treatment and persisted until 12 weeks after treatment. CONCLUSIONS: The short term efficacy of ALT and SLT was similar. In this study, the patients with PDG who underwent SLT showed a paradoxical rise in IOR This finding may indicate that even lower energies (0.4 to 0.6 mJ) are required when performing SLT in patients with heavily pigmented trabeculae. PMID- 16681084 TI - Late opacification of hydroview intraocular lenses: report of 11 cases. AB - PURPOSE: To report on the clinical and light microscopic analyses of Hydroview intraocular lenses (lOLs) explanted for late postoperative IOL opacification. MATERIAL AND METHODS: Retrospective study of all cases with secondary lensimplantation performed at the University Hospital Leuven during a period of one year (2003-2004) for postoperative opacification of Hydroview IOLs. Further analyses on some of the explanted IOLs included gross and light microscopic evaluation. RESULTS: Lens exchange for late-onset IOL opacification was performed in eleven eyes of eight patients. IOL opacification became apparent between 12 and 24 months after uneventful phacoemulsification and lens implantation. Visual acuity varied from < 0.05 to 0.6 before explantation. In 9 out of the 11 eyes postoperative visual acuity improved to the pre-opacification level. Light microscopic analyses showed a granular anterior lenssurface morphology composed of calcium. CONCLUSION: Late postoperative opacification of Hydroview IOLs may cause severe visual impairment, requiring explantation. Calcium is involved in the formation of these opacifications. Reporting on this type of complications is mandatory according to the Belgian and European directives for medical devices. PMID- 16681085 TI - [Primary palpebral tuberculosis (case report)]. AB - Cutaneous tuberculosis is an uncommon disease and tuberculosis affecting the eyelid alone is even more rare. The clinical presentation is polymorphous which explains a diagnosis delay. Its treatment is based on antitubercular drugs chemotherapy. Authors report through an observation the case of a child with eyelid tuberculosis and discuss a difficult diagnosis, a physiopathogeny of eyelid tuberculosis, its diagnosis and treatment. PMID- 16681086 TI - Side effects of glaucoma medications. AB - The safety profile of the different glaucoma medications is an important issue when initiating therapy in glaucomatous patients. The decision on which medication to prescribe depends not only on the type of glaucoma, but also on the patient's medical history and needs a detailed knowledge of the potential side effects of each medication. Medications side effects may be an important cause of non adherence for the individual patient The properties of the drugs, the composition of the glaucoma eyedrops and the dynamics of ocular drug absorption must be considered. The ocular surface changes induced by long-term antiglaucomatous treatment especially by their preservatives are a major cause of intolerance or poor tolerance to glaucoma eyedrops. Moreover topically applied ophthalmic medications can attain sufficient serum levels through absorption into conjunctival and nasal mucosas to have systemic effects and to potentially interact with other drugs. Then this presentation will deal with the ocular and systemic side-effects which can be encountered with the different classes of the currently available glaucoma topical medications. Recommendations than can be applied to reduce both frequency and severity of side-effects of glaucoma medications will be stressed on. Concurrently patients should be fully informed not only about their disease but also the medications they used and what side effects they have to expect. PMID- 16681087 TI - Direct carotid-cavernous fistula: a case report and review of the literature. AB - A 21-year-old man presented with severe proptosis, chemosis, diplopia and an orbital bruit three weeks after a motor vehicle accident. The intraocular pressure was increased. The suspected diagnosis of a direct carotid-cavernous fistula (CCF) was confirmed by digital substraction arteriography. Placement of a covered stent in the internal carotid artery was performed with rapid resolution of the symptoms and normalization of the intraocular pressure. The epidemiology, pathogenesis, symptomatology, differential diagnosis, treatment and outcome of carotid-cavernous fistulas are discussed. PMID- 16681088 TI - Photodynamic therapy with verteporfin in Belgian patients with subfoveal choroidal neovascularization secondary to age-related macular degeneration. AB - This is a retrospective, uncontrolled study of 55 patients (61 eyes) treated between January 2002 and January 2004 with Photodynamic Therapy with Verteporfin for subfoveal choroidal neovascularization (CNV) secondary to age-related macular degeneration. The eyes were divided into three groups: predominantly classic CNV (21 eyes), minimally classic CNV (7) and occult with no classic CNV (4 eyes). For 31 patients (32 eyes) the follow-up period was at least one year. The following parameters were studied: visual acuity, progression of the CNV, and activity of the CNV expressed as leakage on fluorescein angiography and edema on OCT. In the group of predominantly classic CNV at 12 month the treatment resulted in an inactive lesion in 43% of the eyes, however in 48% the visual acuity had decreased. For the group of minimally classic CNV, regression of activity was noted in 71% but vision further decreased in 57%. In the group of occult with no classic CNV one eye developed classic CNV, whereas the lesion regressed on 3 eyes. Two of the 4 eyes lost vision. These results suggest that although PDT is capable of stabilizing the lesion, further visual loss is to be expected in a large number of patients. PMID- 16681089 TI - Refractive outcome after bilateral implantation of an apodized diffractive intraocular lens. AB - PURPOSE: Evaluation of the results of the first 30 patients, bilaterally implanted with the bifocal Acrysof Restor Apodized Diffractive intraocular lens (IOL) (SA60D3). The optics of this lens are designed to restore near and distance vision. SETTING: Department of Ophthalmology, Aalsters Stedelijk Ziekenhuis, Aalst, Belgium. METHODS: Sixty eyes of 30 patients had implantation of the Alcon Acrysof Restor SA60D3 IOL in this prospective nonrandomized clinical study. Fourty-eight eyes were evaluated at 6 months and 60 eyes at 3 months. Patient selection included no other eye disease besides cataract and corneal astigmatism of one diopter or less. Uncorrected distance visual acuity (UCVA-D), uncorrected near visual acuity (UCVAN), best corrected distance visual acuity (BCVA-D) and distance corrected near visual acuity (DCVA-N) were recorded. Glare phenomena, halos and night vision difficulties were evaluated as well as patient satisfaction. RESULTS: Postoperative UCVA-D was better than 20/ 40 at 3 months and better than 20/30 at 6 months in all eyes. UCVA-N was equal or better than Jaeger (J) 3 at 3 months in 91% and all eyes achieved J3 or better at 6 months. BCVA-D was 20/30 or better in all eyes at 3 months and 20/25 or better at 6 months. DCVA-N was J2 or better in all eyes at 3 and 6 months. Patient satisfaction was excellent (76%), good (10%) or acceptable (14%). No patient was dissatisfied with the result. Twenty percent of patients mentioned glare or halos when specifically asked. CONCLUSION: 3- and 6-month data indicate that the Acrysof Restor Apodized diffractive IOL (SA60D3) provides excellent near visual acuity without compromising distance visual acuity. The incidence of visual disturbances is low and patient satisfaction high. PMID- 16681090 TI - [25G transconjunctival vitrectomy for epiretinal membranes and vitreo-macular traction syndromes: results after 6 months]. AB - PURPOSE: to evaluate the safety and efficacy of the 25G transconjunctival vitrectomy in the treatment of epimacular membranes and vitreo-macular traction syndromes. METHODS: in this prospective study dealing with a consecutive series of 38 eyes with an epimacular membrane or a vitreo-macular traction syndrome, we recorded mainly the visual acuity the day before surgery, at day 1, 8 and 6 months after surgery, duration of surgery, and complications peroperatively, at day 1, 8, and at 6 months. RESULTS: 23 eyes had a 6-month follow-up (60%). Mean preoperative, day 1, day 8 and 6-month distance visual acuities were 0.3, 0.2, 0.4 and 0.5, respectively. Mean preoperative and 6-month near visual acuities were P4 and P3, respectively. At day 1 and day 8, 14/38 eyes (31%) and 33/37 eyes (89%), respectively, had a distance visual acuity equal or superior to the preoperative visual acuity. At 6 months, 16/23 eyes (70%) had a distance visual acuity improvement of at least 2 lines. 13/23 eyes (57%) had a near visual acuity improvement of at least 2 levels. Mean duration of surgery was 19 minutes (7 - 40'). Short-term complications were very few, and none were encountered afterwards. CONCLUSION: the 25G transconjunctival vitrectomy is a safe, effective, and convenient technique in the treatment of epimacular membranes and vitreomacular traction syndromes. PMID- 16681091 TI - Five-year experience with non penetrating deep sclerectomy. AB - PURPOSE: To assess the long-term safety and effectiveness of non penetrating deep sclerectomy (DS) and to compare the incidence and the severity of postoperative complications and the IOP results according to surgical adjuvants (implant device, antimetabolite or both) were used or not. MATERIAL-METHODS: Retrospective non randomised study including 171 eyes (136 patients), mean age: 63.9 years) with medically uncontrolled open-angle glaucoma and without previous filtering surgery. 81 eyes (48.2%) had severe glaucomatous damage. All procedures were performed according to the Kozlov's and Memoud's technique. Except for 8 eyes, they were associated with the placement of an implant device (SKGEL or T-FUX) and/or intra-operative application of low dose antimetabolite (5-FU in 58 eyes and mitomycine C in 53 eyes). RESULTS: Mean follow-up was 39.6 +/- 18.3 months. According to surgery, DS were categorized in 4 groups: Group 1: DS with Healon GV (n=8)(4.7%); Group 2: DS with antimetabolite application (n=26) (15.2%); Group 3: DS with placement of an implant (n=53) (31%). Group 4: intraoperative antimetabolite +implant device (n=84 eyes) (49.1%). Peroperative microperforations without iris hernia occurred in 35 eyes (21%). 1st month postoperative complications were observed in 90 eyes (52.6%) with mild to moderate hyperfiltration in 27 eyes, excessive scarring of filtration bleb in 38 eyes, and iris incarceration in 10 eyes. 5-FU injections were given in 58 eyes (34%). YAG gonioperforation was needed in 107/171 eyes (63%) and was complicated by iris incarceration in 9 eyes. Early and late spontaneous iris incarceration was observed in 10 eyes. A second filtering procedure was needed in 10 eyes. PMID- 16681092 TI - [Drug-induced arrhythmia a difficult to catch adverse effect. New guidelines for increased cardiovascular safety]. PMID- 16681093 TI - [The time of the uncritical oxygen treatment is over]. PMID- 16681094 TI - [Treatment of erectile dysfunction with PDE-5 inhibitors. Difficult for the physician to choose between the preparations--the patient should be given the opportunity to try out all three]. PMID- 16681095 TI - [Citalopram is not safe from the cardiac point of view--SSRI preparations can cause prolonged QTc time]. PMID- 16681096 TI - [Hypertension trials--for whom?]. PMID- 16681097 TI - [Quinolones should not be administered in monotherapy for Pseudomonas aeruginosa infection]. PMID- 16681098 TI - [Confidentiality threatened if physicians are to report drivers who pose a danger to traffic]. PMID- 16681099 TI - [Doctor Glass--still an unsettling physician's novel]. PMID- 16681100 TI - Thromboembolic disease in hip replacement surgery. AB - Total hip replacement surgery carries the risk of thromboembolic complications, which could be fatal. Over the last three decades however, the risk has decreased considerably thanks to progress made in the understanding of the physiopathogenetic mechanism of thromboembolic disease and perioperative prophylaxis. It is the purpose of this study to discuss the main medical and surgical preventive measures that must be carried out before, during and immediately after surgery. The old concept of deciding thromboembolic prophylaxis after surgery is now obsolete. PMID- 16681101 TI - Complications following anterior cervical spine surgery for disc diseases: an analysis of ten years experience. AB - The purpose of this study is to define, analyse and discuss the incidence and severity of the complications associated with anterior cervical spine surgery for degenerative disc diseases. The results and the management of complications of anterior spine surgery are discussed in relation to numerous previous published reports: precise knowledge of all potential accidents and pitfalls related to the surgical approaches and of their aetiology may contribute to preventing failures. The most common complication was a recurrent laryngeal nerve injury that developed in 7.9% of the cases. Dysphagia occurred in 5.6%, hoarseness in 5.2%, transient sore throat in 4.8%, worsening of pre-existing myelopathy in 3%, graft extrusion in 1.7%; root injury, haematoma, and wound infection developed in 0.87%. There was one case of oesophageal injury (0.43%) and there were no deaths related to the surgical approach. The rate of complications in our series has been reduced in the past years by 1) better patients selection: all of the patients in fact had previously received conservative treatment for at least four weeks; 2) more care in correct positioning of the patient during the operation; 3) meticolous removal of all harmful structures. PMID- 16681102 TI - The modified Burton-Pellegrini method for the treatment of trapeziometacarpal arthritis. Medium-term results. AB - An original modification in the Burton-Pellegrini technique was used to treat 20 cases of trapeziometacarpal arthritis between 1992 and 2001. A retrospective evaluation concerned 13 cases and was based on functional and radiographic criteria measuring the height of the column of the thumb at rest and under stress. Mean follow-up was 54.2 months (range 24-144). The height of the column of the thumb was decreased in relation to that of healthy hands and shortening under stress was significantly correlated with an increase in pain under stress (R2 = 44.7; p = 0.012). The observation of hyperextension of the MP (> 20 degrees) at follow-up was correlated with worse functional results. The method proposed has proven to be reliable, safe, capable of guaranteeing greater force as compared to other surgical methods described in the literature. The study emphasizes the need for a careful preoperative evaluation and possible treatment of MP joint hyperextension. PMID- 16681103 TI - The metal prosthesis in radial head fracture: indications and preliminary results. AB - Radial head displaced irreparable fracture is an indication for prosthesis; it becomes elective also when there are associated injuries of the skeletal and ligamentous stabilization systems. A retrospective study was conducted to evaluate the functional and radiographic results in 10 patients (mean age 39.6 yrs; minimum 20, maximum 80) submitted to radial head replacement. There were 4 Mason-Johnston type IV Rochwerger subtype "b" fractures, 7 type III fractures (1 associated with an Essex-Lopresti injury and 1 with fracture of the ulnar proximal metaepiphysis). Mean follow-up was 24.6 months (minimum 18, maximum 32). Postoperative functional evaluation of the elbow and ipsilateral wrist was carried out using the ESSSE/SECEC form (mean score 80.7/100; minimum 63, maximum 96) and the PRWE (mean score 11.1; minimum 0, maximum 36) respectively. Radiographically there were postoperative calcifications in 30% of cases and periprosthetic lucency in 40%. The results of this study encourage the use of a metal prosthesis, but a longer follow-up is needed for a better evaluation. PMID- 16681104 TI - Two-stage revision surgery to treat an infected hip implant. A comparison between a custom-made spacer and a pre-formed one. AB - A retrospective study was conducted to evaluate the results of two-stage surgical treatment for infected hip implant using two different spacers in antibiotic impregnated cement, one custom-made and one pre-formed. Out of a total of 20 patients treated between 1995 and 2003, the temporary implant of one custom-made spacer was carried out in 8 cases, while in the remaining 12 cases we resorted to using a pre-formed spacer (Spacer G). In both groups we observed one recurrence of infection, for an overall 90% success rate in terms of eradication of the infection. Custom-made spacers had a greater incidence of local complications (1 breakage, 1 dislocation, 1 sinking of the revision stem) as compared to pre formed ones (1 dislocation). The clinical results evaluated using the Harris Hip Score showed a statistically significant difference (p < 0.05) in both groups as compared to the preoperative score. The use of a pre-formed spacer, although more costly as compared to that custom-made one, was advantageous in some ways, for example in terms of standardization of the implant method, the lower incidence of mechanical complications and the better functional results. PMID- 16681106 TI - Prevalence and treatment of carpal tunnel syndrome in renal haemodialysis. AB - The purpose of this study was to investigate the prevalence and aetiology of carpal tunnel syndrome (CTS) and the effect of open carpal tunnel decompression (CTD) in a group of patients on renal haemodialysis. In 91 patients attending a Renal Unit who were assessed only by clinical means, the prevalence of CTS was 7.1%. CTS development was not significantly correlated with known risk factors, in particular duration of dialysis and presence of an artero-venous fistula on the symptomatic limb. CTD led to a mean symptom-free period of 12 years. In summary, CTS is a frequent and serious disease affecting renal dialysis patients, but its aetiology remains uncertain. Nevertheless, CTD is an effective and lasting treatment for this condition. PMID- 16681105 TI - Influence of the diameters of tendon graft and bone tunnel in hamstring ACL reconstruction. A bovine model. AB - In an animal model of Anterior Cruciate Ligament reconstruction by hamstring tendons and 8mm diameter biodegradable interference screws, we aimed to investigate whether pull out forces and failure modes were influenced by changing tendon and bone tunnel diameters. Three groups of 10 calf tibiae each were prepared with 8, 9 and 10mm diameter proximal tunnels fitted respectively with 8, 9 and 10mm diameter tendon grafts. Mean +/- SD pull out force was 607.9 +/- 191.5N in the 8mm group, 494.2 +/- 206.2N in the 9mm group and 530.4 +/- 212.5N in the 10mm group. These differences were not significant at t-test. 8mm specimens failed by tendon rupture, 10mm specimens failed by screw pull out and 9mm specimens failed in both ways. In this ACL reconstruction model, safe pull out forces were achieved by fixing tendon grafts to bone by 8 mm absorbable interference screws regardless of tendon and bone tunnel diameter. PMID- 16681107 TI - Bone metastasis in hepatocellular carcinoma. A report of five cases and a review of the literature. AB - Hepatocarcinoma occurs frequently throughout the world. Bone metastases are rare although incidence has increased because of progress in diagnosis and treatment. The authors report 5 cases of bone metastases and review the literature. The spine is the most frequent localization of bone metastases. Radiotherapy is the treatment of choice for this lesion. Surgery should be used to prevent and treat complications such as nerve compression and pathologic fracture, only if the coagulative pattern and the conditions of the patient allow it. The authors recommend the use of long intramedullary nailing when localization of the disease is in the femur, with prophylactic stabilization of the neck in diaphyseal metastasis. PMID- 16681108 TI - Pyle's disease. A description of two clinical cases and a review of the literature. AB - The authors present the case of a sister and a brother who came to our observation at different times and both affected with Pyle's disease with marked valgus of the knee. The clinical cases reported demonstrate how in Pyle's disease with significant valgus of the knee temporary asymmetrical tibial epiphysiodesis was able to correct the deformity, not only by provoking the momentary arrest of growth in the growth cartilage on the internal tibial aspect, but also by allowing for bone remodeling in the tibial metaphysis. PMID- 16681109 TI - Spondyloepiphyseal dysplasia tarda: description of one case. AB - The authors describe the case of an Italian male aged 19 years who came to their observation for severe limping with reduction in hip movement and spondyloepiphyseal radiographic modifications of an osteochondrodysplastic origin. The studies carried out led to a diagnosis of spondyloepiphyseal dysplasia tarda (SEDT). PMID- 16681110 TI - Competence by consequence: ambiguity and incoherence in the law. AB - There is an ambiguity of principle delivered by those legal judgements which have considered the issue of whether the level of patients' decision-making capacity should vary in accordance with the seriousness of risks associated with the procedure subject to decision, particularly in decisions to refuse life-saving treatment. There appears to be support for (1) a risk-related standard of capacity, although some judgements could also be interpreted as (2) a procedural standard, subject to a rigorous standard of evidence of capacity. I argue that a risk-related standard of capacity itself is incoherent. This would lend weight to the second interpretation. However, I argue that the wording of the judgements belies an allegiance to the first interpretation, which is also consistent with how medical practitioners generally view capacity in these situations. I consider the implications of the dissonance between the two ways of interpreting the judgments, and make suggestions aimed at clarifying the issue of risk-related competence for clinicians. PMID- 16681111 TI - World without borders: integrating clinical perspectives into the coronial jurisdiction in Victoria, Australia. AB - A professional world without borders would allow for more appropriate collaboration between the Coroner's office and the healthcare sector in their endeavours to improve patient safety. In Victoria (Australia), the Clinical Liaison Service draws on the distinct experiences and expertise of medical, nursing and research personnel to evaluate clinical evidence for the investigation of healthcare deaths reported to the State Coroner's Office. This approach allows for greater intersectoral collaboration between the Coroner's office and healthcare sector than a traditional English-style coronial system that relies on the expertise of coroners, forensic pathologists and police officers to investigate unexpected deaths. Encouraging collaboration between these two sectors may have averted or at least mitigated the atrocities caused by Harold Shipman or the events at Bristol's Royal Infirmary. This paper describes the work processes employed by the Clinical Liaison Service (CLS) to investigate healthcare deaths in a coronial setting. To describe how this work has fostered the advancement of patient care. PMID- 16681112 TI - Post-Perruche: what responsibilities for professionals? AB - Damage compensation relating to handicapped new-borns, the wrongful life law, was put in place after a great controversy in France by the Perruche ruling of November 13th 2000. The French parliaments have voted a special law against wrongful life action. But questions about medical liability are not answered by the new law. For wrongful life, what are now the medical errors likely to directly provoke a handicap or aggravate it or obstruct measures to alleviate it? For a wrongful birth claim it limits parental damages to psychological damages only, due to a lack of mental preparation, whilst denying economic losses, thus preventing complete compensation arising from medical malpractice for all the other types of difficulty suffered by the parents. Why does it give a new definition of medical malpractice as a "characterized fault" and insist upon the qualification 'blatant" in the field of medical liability? For health professionals, it is a question of knowing and being acquainted with the errors that fall under their liability with regard to handicapped persons and a "characterized fault" with regard to parents. It is through a review of past and present case law that we will try to answer these medical questions. All court rulings that awarded monetary damages to the child were reviewed. We include only three decisions after the new law, which do not allow damage compensation in respect of a handicapped new-born. The rulings incorporate decisions taken by the French Supreme Court, of the council of state as well as by courts of appeal. Failure of voluntary termination of pregnancy, missing rubella immune antibody, omission of mandatory tests during pregnancy, typical medical fetal malpractice, were and are professional faults. PMID- 16681113 TI - Legal rights, constitutional controversies, and access to health care: lessons from Canada. AB - This paper provides a critical analysis of the use of legal claims to assert rights to access health care. Using Canada's system of public health insurance as an example, the paper discusses two significant Supreme Court of Canada cases in which claimants use legal mechanisms to influence health care reform. While one case seeks to expand the range of services covered by public health insurance, the other challenges the government "monopoly" over health care and advocates an expanded role for private health care. These legal claims play out in an adversarial setting where the focus is on the rights claims advanced by individual litigants. Yet, the outcomes of these cases involve broad implications regarding allocation of scarce health care resources and the very structure of the health care system. This paper discusses the benefits and limits of using legal claims in this context and also considers the role of courts in making decisions that may have the effect of constraining policy options available to government decision-makers. PMID- 16681114 TI - Can you ask that over the telephone? Conducting sensitive or controversial research using random-digit dialing. AB - Social science, medical, and legal researchers often study sensitive or controversial topics and behaviors. This research raises methodological and ethical issues. Using examples from the literature and a recent statewide telephone prevalence survey on sexual assault, we focus on the relative merits of various survey methods, especially those employing new technologies; developing instrumentation that includes explicit behavioral questions; obtaining an appropriate sample in a cost efficient way; gaining informed consent and inquiring about sensitive topics while protecting participants from harm or retraumatization; presenting findings in a way that does not further stigmatize participants; and responding to the media. PMID- 16681115 TI - Monitoring long-term court order psychiatric hospitalization: a pilot project in Israel. AB - BACKGROUND: In Israel, the rules of compulsory psychiatric hospitalization, including hospitalization under a court order, are set out in the Israel Mental Health Act, 1991 (MHA). The MHA does not specifically define the time limits of hospitalization by Court Order, though every patient, by law has to be brought before the Regional Psychiatric Board (RPB) once every six months for reevaluation. The Supreme Court recently addressed this issue and suggested that by having no specific time span, the way lies open for infringement of individual rights (Criminal Appeals 3854?02). Consequently the Supreme Court suggested that some court committed patients should be moved from the criminal to the civil track which inflicts less severe infringement of the mentally ill patient's rights. This ruling generated rethinking at the ministerial level aimed at improving the monitoring of the care of long-term psychiatric hospitalization in criminal cases. The Ministry of Health initiated a project designed to study this issue. OBJECTIVES: The main objective of this project, which is described below, is to monitor the type and incidence of forensic mental patients hospitalized in Israel for more than 10 years, and to propose alternatives to replace this untenable situation. METHODS: All the 12 psychiatric hospitals in Israel which hospitalize forensic patients were sent written requests for data on criminal patients hospitalized under court orders, including demographic data, diagnosis and type of offense. We identified in all 65 such patients. The data received were compared with the National Psychiatric Register databank of the Ministry of Health and divided in subgroups according to diagnosis, type of offence, demographic variables and length of hospitalization. RESULTS: Most of the subjects of the sample (89%) suffered from psychotic disorder mainly schizophrenia of the paranoid type. 95.5% were male. The most prominent type of offense was assault against family members (37%), which is in keeping with statistics reported in the relevant literature. The profile of the typical patient of this sample is: male, aged 45-65, unmarried, with 8 years of education and suffering from paranoid schizophrenia. DISCUSSION: No correlation between type and severity of offense and length of involuntary forensic hospitalization was found. We suggest some possible alternatives to improve the current handling of the group of long term hospitalized forensic patients. We also feel that a further study should be carried out on forensic patients hospitalized for a period of five to ten years. PMID- 16681116 TI - Law and practice in relation to coronial post mortems--a social perspective. AB - The coronial post mortem, might be described as a coercive process because it takes place by force of law and, subject to the safeguards which the law now recognizes, irrespective of the wishes of the family of the deceased or, indeed, any wishes expressed by the deceased in his or her lifetime. That process should therefore be considered in the context of the competing social and consequent legal imperatives that impact upon the decision to undertake an autopsy, the means by which it is undertaken and the extent of the process. The purpose of this paper is to examine those social imperatives, the legislative framework and case law including recent legislative reforms designed to meet these social imperatives, some of which have been recognised only in recent times. Recent developments in law and practice have had a significant impact on the social issues involved. Important scientific research has now been undertaken that demonstrates the value of radiological techniques particularly CAT scans and MRIs as a means of avoiding some of the social difficulties involved in the process and, at the same time, proving valuable forensic tools in many cases. The role of such processes will be discussed. References to legislation are references to the statutes of New South Wales unless otherwise stated. PMID- 16681117 TI - Naming, blaming and shaming? AB - Few doctors at the centre of complaints or disciplinary proceedings wish to be publicly named. Publication of a doctor's name can adversely affect his or her reputation, patients, and family members, even if the allegation is ultimately not upheld. Yet, there is a strong public interest in freedom of speech and transparency of complaints and disciplinary processes. In determining whether to grant name suppression, complaints agencies and disciplinary tribunals are required to balance competing public and private interests. In New Zealand, the Health and Disability Commissioner has responsibility for investigating complaints about the quality of medical care. The Commissioner's current practice is not to publicly name doctors under investigation, or even those who are found to have breached a patient's rights. This approach fits well the non-punitive, rehabilitative focus of New Zealand's medical regulatory system. In the rare cases where a matter reaches the threshold for disciplinary action, the balance tips in favour of disclosure. PMID- 16681118 TI - The recent Italian Consolidation Act on privacy: new measures for data protection. AB - In the light of the new Consolidation Act on privacy, in effect since January 2004, the author has made some observations in relation to the protection of data concerning health. Special focus has been given to an overall view of the new regulations. "Sensitive data" are seen as differing from health data, while always respecting the rights, the fundamental freedom and the dignity of the person involved. A central role continues to be played by the Controller, who has regulating and control powers. PMID- 16681119 TI - Judicial sentencing in Canadian intimate partner sexual assault cases. AB - This article examines judicial descriptions of and responses to intimate partner sexual assaults in the Canadian context. Drawing on data from sexual assault sentencing judgments, we investigated 186 cases across four categories of perpetrator-victim relationship. Offence and case-related characteristics, as well as sentencing outcomes were analyzed. The data indicated notable similarities between intimate partner and stranger sexual assaults in terms of the occurrence of penetration, force, and injury. At the same time, our analyses revealed that strangers received longer sentences than intimate perpetrators. Some significant differences were also found between intimate partner and authority figure relationship groups across the variables examined. PMID- 16681120 TI - Submissions from non-existent claimants: the non-identity problem and the law. AB - Some medical interventions (or indeed omissions) determine the identity of a person. Those interventions or omissions may themselves cause harm, for which a child subsequently sues. Even if they do not themselves cause harm they may result in the birth of a child who subsequently brings a claim for being allowed to exist in the state caused by the intervention. The obvious objection to a claim of either class is that in the absence of the medical intervention or omission of which the claimant complains, the claimant would never have existed at all. This objection arises from what philosophers have called the "non identity problem". This article examines the attitude that the English courts would be likely to have to such a claim. It concludes that the English law has so far failed to appreciate the significance of the non-identity problem, but will have to grapple with it soon. PMID- 16681121 TI - The British Abortion Act (1967) and the interests of the foetus. AB - This article examines ethical issues on the rights and interests of the unborn foetus, an issue that remains highly contentious. Furthermore, it attempts to investigate how well the British legislation fits with the foetus and pregnant woman's rights and interests. "Pro-life" and "pro-choice" groups have provided extensive arguments for and against. One important theoretical issue rests on whether foetuses are human beings in the moral sense, in which all human beings have full and equal moral rights. What constitutes personhood is a matter of moral decision and is not one of scientific fact and thus it consists of all persons, rather than all genetically human entities. It is persons who invent moral rights and who are capable of respecting them. Legislators in Britain have sidestepped the ethical debate on abortion by opting for the pragmatic course of permitting abortion in a limited range of circumstances and thus the Abortion Act 1967 has failed to address the status of the foetus, or indeed the rights of any of the parties concerned. Thus, although the Act supports the interests of the foetus capable of free existence by lowering the foetal age to 24 weeks after which termination is not permissible, the legislators have accepted that the rights of the woman outweigh those of the foetus and if a woman's own life or health is in danger then even a late abortion is the best choice. PMID- 16681122 TI - Euthanasia--he illusion of autonomy. AB - The paper deals with some of the more common arguments used for the legalisation of voluntary euthanasia. It looks at these arguments from an ethical and philosophical point of view. First, the argument that to offer a person the possibility of euthanasia is to respect that person's autonomy is questionable. Can a person's decision on euthanasia be really autonomous? If euthanasia were legal everybody would be conscious of this option: the patient, the doctor, the family and the nursing staff. Thus, there could be indirect pressure on the patient to make a decision. The choice is meant to be free but the patient is not free not to make the choice. Secondly, a choice that seeks to alleviate suffering and thus improve life by annihilating it is irrational. Thirdly, autonomy as to one's own death is hardly exercised freely. Even an otherwise competent person may not be competent in deciding on his own death on account of despair, hopelessness, fear or maybe a feeling of being weak, superfluous and unwanted. This is a very uncertain base for decision-making, especially in the irrevocable decision of euthanasia. Finally, a competent person usually makes any choice in a responsible way and after due consideration; a 'good' decision should consider and respect the wishes and feelings of others. This will be no less the case in making a decision on the so-called free choice of euthanasia. Thus 'normal' behaviour in decision making will only add to the tendency of the already depressed person to feel a burden on his family, the staff and even on society. PMID- 16681123 TI - A model for decision making at the end-of-life: Queensland and beyond. AB - This paper addresses when it is legal to withdraw or withhold medical treatment that is needed to keep a patient alive. It draws on cases and legislation from the common law world (including Australia, England and New Zealand) and considers the various legal tests applied in the different jurisdictions. Two of the most common tests employed in this situation are the "best interests of the patient" test and the "substituted judgment" test. Some jurisdictions also include other criteria as well, such as a requirement that withdrawing or withholding of medical treatment is "not inconsistent with good medical practice". This paper analyses these different legal tests, and after identifying the factors that are judged to be legally relevant to consider when deciding to withdraw or withhold treatment, outlines a preferred model. This model addresses who the relevant decision maker should be, and the criteria that should govern their decision. It suggests that family members are better equipped and more appropriate to act as decision makers than health professionals, and also questions the appropriateness of responsible medical opinion as the decisive factor in such cases, preferring instead an approach more consistent with the principles of self determination. The model also proposes a method for resolving any disputes that arise. PMID- 16681124 TI - Attitudes of medical practitioners towards "Do Not Resuscitate" orders. AB - When the desires of a patient are unknown or cannot be ascertained, cardio pulmonary resuscitation (CPR) is the default procedure. Explicit, Do Not Resuscitate (DNR), orders are required to prevent implementation of CPR. We studied the response of general medical internists in specific clinical situations demanding consideration of DNR orders and respect for patient preferences; their current practice regarding slow codes and participation in CPR attempts considered futile provide information as to how often they discuss DNR issues with patients or families. Eighty-five internists attending the monthly meeting of the Internal Medicine Forum participated in the study. The physicians demonstrated their consent to participate by accepting a remote transmitter that elicited a response 2-3 minutes following the presentation of case vignettes or practice-related questions. The survey showed that 73% of the physicians agreed to assign a DNR order for a terminally ill patient unable to express her preferences. Only 55% agreed to do the same for a competent patient who specifically requested that CPR be withheld in the event of a cardiopulmonary arrest (p<0.05). 77% reported to have performed CPR, at least three times, in situations where they expected no benefit. 59% affirmed that their team had performed a partial CPR (slow code) at least once. Only 28% discussed the subject of DNR with patients or family more than 5 times a year. Paternalism, disregard for patients' preferences and poor communication skills influence normative behaviour in end-of-life decision-making. PMID- 16681125 TI - Building an academic-community partnered network for clinical services research: the Community Health Improvement Collaborative (CHIC). AB - OBJECTIVE: Community-based participatory research is recommended for research on health disparities and to improve uptake of clinical research findings. We describe the development of a multicenter consortium designed to support a community agency-academic partner infrastructure to support community-based, health-services research on multiple sources of health and healthcare disparities in local communities. DESIGN: We describe the development of the Los Angeles Community Health Improvement Collaborative (CHIC). RESULTS: The CHIC partners examined the research capacity and health priorities of its partners and developed a research agenda focused on four tracer conditions (depression, violence, diabetes, and obesity) and four areas for development of research capacity: public participation in all phases of research; understanding community and organizational context for clinical services interventions; practical clinical services trial methods; and advancing health information technology for clinical services research. The partners pooled resources to develop these areas for the tracer conditions. CONCLUSIONS: The challenges of a participatory approach to community-based clinical services research go beyond the significant methodologic and operational issues for specific projects and include building a sustainable capacity for research, community programs, and partnership across diverse communities and stakeholder organizations even when funding sources are not fully aligned with these goals. PMID- 16681126 TI - Witness for Wellness: preliminary findings from a community-academic participatory research mental health initiative. AB - Quality improvement programs promoting depression screening and appropriate treatment can significantly reduce racial and ethnic disparities in mental-health care and outcomes. However, promoting the adoption of quality-improvement strategies requires more than the simple knowledge of their potential benefits. To better understand depression issues in racial and ethnic minority communities and to discover, refine, and promote the adoption of evidence-based interventions in these communities, a collaborative academic-community participatory partnership was developed and introduced through a community-based depression conference. This partnership was based on the community-influenced model used by Healthy African-American Families, a community-based agency in south Los Angeles, and the Partners in Care model developed at the UCLA/RAND NIMH Health Services Research Center. The integrated model is described in this paper as well as the activities and preliminary results based on multimethod program evaluation techniques. We found that combining the two models was feasible. Significant improvements in depression identification, knowledge about treatment options, and availability of treatment providers were observed among conference participants. In addition, the conference reinforced in the participants the importance of community mobilization for addressing depression and mental health issues in the community. Although the project is relatively new and ongoing, already substantial gains in community activities in the area of depression have been observed. In addition, new applications of this integrated model are underway in the areas of diabetes and substance abuse. Continued monitoring of this project should help refine the model as well as assist in the identification of process and outcome measures for such efforts. PMID- 16681127 TI - Innovative approaches to obtaining community feedback in the Witness for Wellness experience. AB - BACKGROUND: Awareness of the need for innovative approaches to obtaining feedback in community-based participatory research (CBPR) is increasing. These innovative approaches should incorporate the core principles of CBPR, including equity and co-learning. Additionally, the methods should be culturally appropriate and inclusive of the community and academic partners. OBJECTIVE: To develop and implement two separate methods of obtaining community feedback for two activities in a CBPR initiative: 1) discussion of three work-group plans during a leadership council meeting; and 2) feedback from the work groups to the target community in a public setting. METHODS: In order to facilitate a feedback process for the discussion of 3 separate group action plans, an adapted version of the modified Delphi technique was used during which 42 community and academic partners voted and evaluated each plan both before and after group discussion. Results were immediately posted on a projection screen for the group to process. The second community feedback method incorporated the use of an audience response system (ARS) in order to obtain responses from 187 community participants after hearing summaries of the Witness for Wellness work-group action plans. More than 60% of the respondents added that the use of the handheld device made research seem more relevant and less intimidating. CONCLUSIONS: Both the use of the adapted modified Delphi process and ARS were effective in capturing community feedback related to two group activities in the Witness for Wellness initiative. Both methods also allowed participants to understand the role of research in a community setting. PMID- 16681128 TI - Supporting wellness through policy and advocacy: a case history of a working group in a community partnership initiative to address depression. AB - This paper describes the implementation of a collaborative project with community and academic partners in Los Angeles, focusing on the experiences of the Supporting Wellness (SW) working group in a depression outreach/ education initiative (the Witness for Wellness project). The first-year activities of the SW group involved infrastructure development to prepare for policy and advocacy work. Scribe notes from monthly meetings along with the authors' observations are the primary source of data for this article. One of the major accomplishments of the group was to obtain a position on a countywide stakeholder committee that makes recommendations to policymakers on mental health spending for the county. One significant challenge for the academic group members has been providing guidance and support for potential research projects. Although community partnerships with academia are a potentially useful approach for developing health interventions to address community needs and priorities, the experiences of this group illustrate that a substantial amount of time is required to develop the infrastructure for efforts to be successful. In addition, we learned the "community perspective" is essential to developing interventions that can address barriers to depression care that are unique to communities with high numbers of under-served, unserved, and inappropriately served residents. PMID- 16681129 TI - The Building Wellness project: a case history of partnership, power sharing, and compromise. AB - INTRODUCTION: The Institute of Medicine has recommended development of community focused strategies to alleviate the disproportionate burden of illness on minorities, including depression. So far, limited data exist on the process of developing such partnerships within diverse racial/ethnic environments as they strive to develop community-driven, evidence-based action plans to improve the quality of outreach services. We describe such an effort around depression in south Los Angeles and explore the issues of the process in the hopes of informing future partnership development. METHODS: Community meetings, presentations, feedback, discussion groups, and consensus-based action items were implemented over an 18-month period. A writing subcommittee was designated to develop a description of the group's work and process, as well as the diverse perspectives in the partnership. Data sources included meeting minutes, materials for members and community feedback presentations, scribe notes, and the reflections of the authors. RESULTS: Development was seen on the formal group level, in the process, and on the realization of three categories of action plans. Designed to assist social service caseworkers in the recognition of and referral for depression, the action plans included developing a website, a tool kit (modified Delphi process), and a one-page depression "fact sheet" with region-specific referrals. CONCLUSION: Through the process of developing a means to combat depression in a racially/ ethnically diverse population, the community is not only better informed about depression but has become a true partner with the academic element in adapting these programs for local service providers, resulting in improved understanding of the partnership process. PMID- 16681130 TI - Talking Wellness: a description of a community-academic partnered project to engage an African-American community around depression through the use of poetry, film, and photography. AB - The design, implementation, and preliminary evaluation of an enhanced community engagement program that uses poetry, film, and photography at a film festival in south Los Angeles is described. This project is one of several Talking Wellness projects designed to develop social capital and enhance community engagement in projects designed to improve the community's capacity to communicate effectively about depression, to decrease the associated stigma, and to participate in the design and evaluation of research interventions. The high degree of collaboration in the development and evaluation of this community participatory research model is illustrated by describing the selection and design of the intervention and the development of the survey questionnaires used for data collection. The project is described from the perspective of community members involved in the process. PMID- 16681131 TI - Seeking community input to improve implementation of a lifestyle modification program. AB - OBJECTIVES: Obesity increases a person's risk for diabetes, which is becoming the most common chronic disease in the United States. Latina and African-American women in disadvantaged communities are at higher risk for becoming overweight and subsequently developing diabetes. The purpose of this focus-group study was to guide our adaptation of an evidence-based lifestyle intervention and implementation of the Community-Based Lifestyle Balance program (CLSB). DESIGN, SETTING, AND PARTICIPANTS: We conducted 11 focus-group discussions with 87 African-American and Latina women in disadvantaged communities, including schools, senior centers, subsidized housing communities, and churches. We also conducted informal key informant interviews with community service providers and leaders. RESULTS: Discussions revealed high knowledge of healthy behavior and strong interest in making lifestyle changes. However, barriers such as competing demands on these women prevented long-term practice of healthy behaviors. Women frequently expressed feelings of guilt and self-blame in their attempts and failures to make healthy changes in their daily routine. Some patterns were identified that varied by age and race/ethnicity. These findings suggest guidelines for implementing this lifestyle intervention in a variety of community settings. CONCLUSIONS: Community-level changes such as safer streets and better access to quality grocery stores or markets, with affordable, healthy, fresh food can take years to accomplish. In the interim, CLSB can provide women with skills and strategies that can help improve their health and the health of their families. PMID- 16681132 TI - A community participatory research partnership: the development of a faith-based intervention for children exposed to violence. AB - When an inner city Latino immigrant faith community in Los Angeles identified mental health care as an area of need, a community-research partnership was formed that resulted in the adaptation of an intervention for children who have trauma-related symptoms from violence exposure. This participatory research partnership includes St. Thomas the Apostle School and Church community; QueensCare Health and Faith Partnership, an organization that provides health services and outreach to faith communities; and mental health researchers from UCLA. During the planning phase of this project, parent focus groups were conducted, and an evidence-based intervention for traumatized students was adapted for this community. Focus group participants described significant concerns about community violence and multiple ways in which this ongoing violence has affected their children's functioning and child-parent relationships. The partnership has collaborated on each aspect of the research study, from design and adaptation, implementation, data analyses, and identification of areas for future research. This paper, a participatory process written in the words of the community and research partners, describes the experience of and challenges met by this partnership in adapting the Cognitive Behavioral Intervention for Trauma in Schools program for use in this Catholic school. PMID- 16681133 TI - Community-research collaboration between researchers and acupuncturists: integrating a participatory research approach in a statewide survey of licensed acupuncturists in California. AB - The Licensed Acupuncture Collaborative Study, a job analysis of licensed acupuncturists in California, provides a model for building community-research partnerships between university researchers and communities of non-physician clinicians. The study design used a project-management approach based on the core principles of community-based participatory research: 1) mobilizing shared expertise and resources to address issues of concern; 2) sharing power in the decision-making process; and 3) promoting mutual ownerships of resources and products derived from the collaboration. A project infrastructure involving the sharing of study responsibilities across university researchers, individual acupuncturists, and state community organizations was developed and cultivated over a three-year project period. Essential factors in the success of this project included shared objectives, addressing the concerns about collaboration among academic and community partners, inclusion of nontraditional viewpoints about healthcare policy, and participation by the acupuncturist community in performing the research. These activities helped to overcome mistrust and perceived power differences between researchers and the acupuncturist community. PMID- 16681134 TI - Developing systems interventions in a school setting: an application of community based participatory research for mental health. AB - OBJECTIVES: The goal of this study was to develop systems interventions in a public school district using community-based participatory research (CBPR) methods to improve the social and academic functioning of children from racial and ethnic minority populations. DESIGN: The study used qualitative methods in the process of problem definition and intervention planning, including in-depth qualitative interviews and stakeholder dialogue groups. The study was conducted at three levels--the school system as a whole, two individual schools, and a multiple-stakeholder participatory group. SETTING: The study took place in a public school system in an urban city with a population of 101,355 and in two public schools located in this city. PARTICIPANTS: The CBPR team included two researchers, a researcher/consulting psychiatrist in the schools, the director of the special education office, her management team, four teachers, and two school based administrators. INTERVENTIONS: The CBPR group engaged in a process of problem definition and intervention planning at all three levels of the system. In addition, both schools initiated systems interventions to target the needs of their school environments. RESULTS: The project led to system interventions at both schools, clarity about the policy constraints to effective collaboration, and increased awareness regarding the behavioral and academic needs of minority children in the schools. The process produced a series of questions to use as a framework in CBPR partnership development. CONCLUSIONS: The CBPR approach can expand the scope of mental-health services research, particularly related to services for racial and ethnic minorities. PMID- 16681135 TI - Ethics of clinical research within a community-academic partnered participatory framework. AB - Recommendations for reducing racial and ethnic disparities in health and health care suggest that clinical researchers try community-based participatory research (CBPR). While the body of literature discussing the ethics of CBPR continues to grow, we are not aware of a specific attempt to provide a structure for analyzing the ethics of clinical research using a CBPR approach. We adapt a framework developed by Emanuel, Wendler, and Grady articulating seven requirements for ethical clinical research to clinical research using a CBPR approach. We incorporate findings from the literature on CBPR and identify some of the ethical and practical challenges from our experiences working in CBPR as academics and community members. We find Emanuel et al's framework easily adaptable for CBPR. Six of the requirements are flexible enough to accommodate the needs of CBPR; they are: social or scientific value, scientific validity, fair subject selection, favorable risk-benefit ratio, independent review, and informed consent. We suggest that the seventh requirement, respect for potential and enrolled participants, be amended to respect for potential and enrolled participants, community, and research partners to acknowledge that separate attention should be paid to relationships on these three levels. This adapted framework can guide community-academic partnerships as they evaluate whether to proceed with potential clinical research studies and as they work to enhance the ethics of clinical research studies using a CBPR approach. PMID- 16681136 TI - Assessing organizational readiness and change in community intervention research: framework for participatory evaluation. AB - This paper describes a study currently underway that uses a collaborative approach to assess organizational capacity to form partnerships around mental health and substance abuse care. Employing many of the principles of community based participatory research, the study's primary objective is to collaboratively develop a conceptual understanding and generalizable, practical measures of organizational capacity. The intent of this collaborative approach is to increase the rigor and relevance of the assessment framework while strengthening the ability of health partnerships and stakeholders to understand and track community organizational capacity. The study investigators developed an initial model of community dissemination based on the research literatures on organizations and the diffusion of innovations. Through the collaborative process, the specific goals of the project shifted substantially to match the partnership interests and concerns of community agencies. One of the benefits of a collaborative approach has been to use researchers' academic knowledge to catalogue potential factors and the wealth of community coinvestigators' experiential knowledge of interagency dynamics to identify specific relevant dimensions of capacity. This initial exploratory study represents a first step toward developing a general approach to conceptualizing and tracking the organizational capacity of communities. The model and measurement framework may have wider applicability to capacities to partner around and implement a variety of health-related interventions within communities. PMID- 16681137 TI - Commentary: challenges for the field in overcoming disparities through a CBPR approach. PMID- 16681138 TI - Commentary: building partnerships between schools and academic partners to achieve a health-related research agenda. PMID- 16681139 TI - Commentary: community-academic partnership for research to improve health in communities: a foundation officer's perspective. PMID- 16681140 TI - Commentary: community partnered research: driving sensemaking, managing knowledge, and moving mental health care to new heights. PMID- 16681141 TI - Antimicrobial activity of 1,2-bis-[2-(5-R)-1H-benzimidazolyl]- 1,2-ethanediols, 1,4-bis-[2-(5-R)-1H-benzimidazolyl]- 1,2,3,4-butanetetraols and their FeIII, CuII, and AgI complexes. AB - 1,2-Bis-[2-(5-H/Me/Cl/NO2)-1H-benzimidazolyl]-1,2-ethanediols (L1-L4), 1,4-Bis-[2 (5-H/Me/Cl)-1H-benzimidazolyl]-1,2,3,4-butanetetraols (L5-L7) and their complexes with FeCl3, CuCl2, and AgNO3 were synthesized; antibacterial activity of the compounds was determined toward Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Proteus mirabilis, and antifungal activity against Candida albicans. The AgI complexes have considerable activity toward the microorganisms. Some AgI complexes show higher activity toward S. epidermidis than AgNO3 and cefuroxime. Cu(L3)Cl2 and Fe(L3)Cl3 show an antifungal effect on C. albicans but L3 itself has no activity. PMID- 16681142 TI - Antimicrobial activity of ZnII, CdII, and HgII complexes of 1,2-bis-[2-(5-R)-1H benzimidazolyl]-1,2-ethanediols and 1,4-bis-[2-(5-R)-1H-benzimidazolyl]-1,2,3,4 butanetetraols. AB - 1,2-Bis-[2-(5-H/Me/Cl/NO2)-1H-benzimidazolyl]-1,2-ethanediols (L1-L4), 1,4-bis-[2 (5-H/Me/Cl)-1H-benzimidazolyl]-1,2,3,4-butanetetraols (L5-L7) and their complexes with ZnCl2, CdCl2 and HgCl2 were synthesized and antibacterial activity of the compounds was tested toward Staphylococcus aureus, S. epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Proteus mirabilis and antifungal activity against Candida albicans. HgII complexes have a considerably higher antimicrobial activity against all microorganisms. Some HgII complexes show higher antifungal activity than clotrimazole toward C. albicans. Zn2(L3)Cl4, Zn2(L4)Cl4, and Cd(L3)Cl2 were moderately effective against S. aureus and S. epidermidis; Cd(L4)Cl2 exhibited a weak activity only against S. epidermidis. PMID- 16681143 TI - New antimycobacterial S-alkylisothiosemicarbazones. AB - In connection with a systematic study of antimycobacterial agents against potentially pathogenic strains the series of 12 S-alkylisothiosemicarbazones was evaluated for in vitro antimycobacterial activity against Mycobacterium tuberculosis, M. kansasii, M. fortuitum, two strains of M. intracellulare and three strains of M. avium. Quinoline-4-carbaldehyde-S-hexyl-isothiohydrazone was found to be more active against potentially pathogenic strains than isoniazide. PMID- 16681144 TI - Evidence for apparent gene instability in the rifamycin-producing oligoketide synthase. Implications for combinatorial biosynthesis and heterologous gene expression. AB - The oligoketide ('polyketide') synthase leading to the formation of proansamycin X in Amycolatopsis mediterranei also prematurely releases a range of acyclic intermediates from the enzyme complex. We intended to study the chemical biology of this ectopic chain release using RifA as a model protein system; however, we were unable to clone the rifA gene in its entirety. Restriction analysis of cosmid clones revealed that rifA is subject to random deletions at high frequency, especially in central regions of the locus. Examination of the gene sequence in this region reveals a high concentration of inverted repeats; we suggest that these sequences are subject to alteration in secondary structure when cloned outside the environment of the A. mediterranei genome, leading to recombination and deletion. PMID- 16681145 TI - A rapid screening method for detecting active compounds against erythromycin resistant bacterial strains of Finnish origin. AB - A rapid and simple microdilution technique on 96-well microplate based on turbidimetry was optimized and validated for screening of antimicrobial activity against erythromycin-resistant bacterial strains of Streptococcus pyogenes and Staphylococcus simulans isolated from Finnish patients. Using S. pyogenes ATCC 12351 as reference strain the developed method was evaluated by reproducibility measurements and using parameters typically employed for screening methods, i.e. signal-to-background, signal-to-noise and a screening-window coefficient, the Z' factor. The method was further used for screening a group of natural compounds and their synthetic derivatives against resistant bacterial strains. Of these, octyl and dodecyl gallates, and usnic and ursolic acids were the most active. The described method is a rapid, homogeneous, cost-effective and easy-to-perform system for screening of new potential antimicrobial agents in drug discovery. PMID- 16681146 TI - Taxonomic reclassification of Candida stellata DBVPG 3827. AB - We present data demonstrating that the strain DBVPG 3827 does not belong to C. stellata. From the results of physiological analysis, electrophoretic karyotyping, the PCR-RFLP of the ITS1-5.8S-ITS2 region and the nucleotide sequence of the D1/D2 domain of the 26S rDNA it can be concluded that DBVPG 3827 is a strain of Starmerella bombicola. This finding, and the recent observation that C. stellata can easily be confused with C. zemplinina in tests of conventional taxonomy, urges a critical revision of the enological role(s) attributed by researchers to C. stellata. PMID- 16681147 TI - Multiplex PCR for detection of three exfoliative toxin serotype genes in Staphylococcus aureus. AB - Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control. PMID- 16681148 TI - Effect of phosphate on the expression of protein-Ser/Thr kinase pkg2 in Streptomyces granaticolor. AB - A time-correlated expression of eukaryotic-like protein Ser/Thr kinase Pkg2 of Streptomyces granaticolor was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by transcriptional fusion experiments. In a complex medium the activity of pkg2 promoter was constant during the life cycle. Direct RNA analysis proved the presence of corresponding pkg2 transcript. S1 nuclease protection analysis of the transcription initiation site showed that pkg2 gene is expressed as a leaderless mRNA. Under phosphate starvation the promoter activity was detectable merely in the early exponential phase. Under these conditions turning off of pkg2 promoter and cessation of pkg2 transcript level coincided with the start of granaticin production. PMID- 16681149 TI - Evaluation of abundance of aerobic bacteria in the rhizosphere of transgenic and non-transgenic alfalfa lines. AB - Fourteen genetically modified lines of alfalfa (Medicago sativa) containing the gene Ov from Japanese quail, coding for a methionine-rich protein ovalbumin, were evaluated for nodulation ability and concentration of aerobic bacteria in the rhizosphere. The transgenic lines were derived from a highly regenerable genotype Rg9/I-14-22, selected from cv. Lucia. On selective media, a higher concentration of ammonifying bacteria, bacterial spores, denitrifying and nitrifying bacteria were observed in the rhizosphere of transgenic clonesand, on the other hand, lower concentration of cellulolytic bacteria and Azotobacter spp. compared with the rhizosphere of non-transgenic clone SE/22-GT2. A statistically significant difference in the concentration of all the bacterial types was found between samples taken from two types of substrates (i.e. sterile vs. nonsterile). Higher bacterial concentration (measured as colony forming units per g soil dry mass) were observed for all tested groups of culturable bacteria in the non-sterile substrate. The presence of Azotobacter spp. was found only in the rhizosphere of plants grown in non-sterile soil in which the highest number of fertile soil particles (97 %) was observed in transgenic clones SE/22-9-1-12 and SE/22-11-1 1S.1. Concentration of bacteria involved in the N cycle in the soil was increased in the rhizosphere of transgenic clones and decreased in the rhizosphere of non transgenic plants compared with the average value. In spite of some differences in colony numbers in samples isolated from the root rhizosphere of transgenic and nontransgenic alfalfa plants, we could not detect any statistically significant difference between individual lines. PMID- 16681150 TI - Listeria monocytogenes protein p60 affects hemolytic activity and uptake of bacteria by macrophages. AB - Bacillus subtilis strains expressing listeriolysin O (LLO) and simultaneously LLO and p60 protein were constructed. The effect of p60 protein on hemolytic activity and on the invasion of professional phagocytes was demonstrated in the absence of other virulence factors of L. monocytogenes. The hemolytic activity of LLO in the presence of p60 protein decreased which indicates that p60 promoted adhesion and subsequent invasion of professional phagocytes. PMID- 16681151 TI - The virulence markers of Salmonella enterica serovar Typhimurium. Different phage type strains isolated in Slovakia. AB - Phage-typing determination of cell-surface hydrophobicity, motility, and serovar specific virulence plasmid was performed in a collection of 154 clinical isolates of S. enterica serovar Typhimurium (SeT) isolated in Slovakia. All isolates were also examined in PCR for the presence of both stn (enterotoxin) and iroB (siderophore) genes. The DT104 was the definitive phage type most frequently identified (37.7 %), the second most frequently isolated phage type was DT41 (5.8 %); the occurrence of other phage types was not epidemiologically significant. On the basis of virulence-marker investigation, 46.1 % of isolates were hydrophobic in the assay of bacterial adherence to xylene, and 97.4 % were hydrophobic in salt-aggregation test. Motility of more than 50 mm was expressed by 20.8 % isolates. The serovar-specific 90-kb virulence plasmid was contained in 138 (89.6 %) of isolates. All SeT isolates were found (according to PCR) to carry the Salmonella-enterotoxin (stn) gene and the siderophore (iroB) gene. The increasing incidence of SeT DT104 human strains in Slovakia requires continuous attention; this can be markedly improved by surveillance efficiency and made possible by determining relationships between sporadic isolates. PMID- 16681152 TI - Identification of Staphylococcus piscifermentans from dog feces. AB - The AD 2 strain isolated from feces of a healthy dog in Slovakia was characterized phenotypically by the conventional tests and commercial identification kits API Staph and ID32 Staph. Results of biochemical tests identified the strain as S. piscifermentans, fully corresponding with the species description. Further characterization by whole-cell protein profile analysis (SDS PAGE) confirmed the identification based on biochemical tests and showed that the AD 2 strain is S. piscifermentans; lactic acid production, urease activity, bacteriocin production and the antibiotic susceptibility of it were also determined. S. piscifermentans AD 2 isolated first from an animal source was deposited in the Czech Collection of Microorganisms as Staphylococcus piscifermentans CCM 7165. PMID- 16681153 TI - [Parvovirus B19]. PMID- 16681154 TI - [Sonographic diagnosis of post-interventional pneumothorax and hydropneumothorax- prospective study of 100 patients]. AB - In order to answer the question, if transthoracic sonography may replace chest radiographs in diagnosing post-interventional pneumothorax/hydropneumothorax, this prospective study was conducted. A total of 100 patients (38 females, 62 males; median age 63 years), biopsy and 37 had an ultrasound-guided tube thoracostomy, were enrolled in the study. Transthoracic sonography was performed three hours after the intervention, followed by postero-anterior chest radiograph. One (1%) of the 100 patients developed a pneumothorax after transbronchial biopsy. Eight of 37 patients suffered from hydropneumothorax due to tube thoracostomy detected by sonography. In one patient, hydropneumothorax was missed by posteroanterior chest radiography. Sensitivity, specificity and accuracy of transthoracic sonography were 100%. Transthoracic sonography is a safe bed-side-method, allowing an immediate exclusion/diagnosis of postinterventional pneumothorax/hydropneumothorax. The results suggest that chest radiography may only be required in patients with pneumothorax diagnosed by transthoracic sonography so as to assess its extension, if full sonographic assessment is not possible or if any discrepancy exists between TS-results and clinical presentation. PMID- 16681155 TI - [Sonography in acute diverticulitis of the sigmoid colon]. AB - Sonography is the primary diagnostic method in the painful left lower quadrant. Experienced investigators can diagnose an acute diverticulitis in more than 90%. In unclear cases or when complications are suspected, CT should be performed. Sonography can diagnose most differential diagnosis of the painful left lower quadrant as colitis, appenagitis or haematomas. PMID- 16681156 TI - [Sonography of acute appencitis and the main differential diagnoses]. AB - Today sonography is the first line imaging method for diagnosing acute appendicitis. Experienced investigators will have an accuracy of more than 90%. Sonography can diagnose many conservatively managed diseases. The most important differential diagnoses are infectious ileocoecitis, right sided diverticulitis, appendagitis, adnexitis, ruptured or torque ovarian cysts, ectopic pregnancies. Ureterolithiasis, cholecystitis, haematomas in the psoas muscle or in the rectus muscle are rarer causes of right lower quadrant pain. Sonography can reduce the high rate of false positive clinical examinations concerning acute appendicitis. It has to be stated that an exclusion of appendicitis can only be made sonographically if the normal appendix can be seen in its full length and/or an other differential diagnosis can be depicted that explains the clinical symptoms. Mucoceles are rare cystoid lesions of the appendix. They exhibit a typical onion skin sign structure caused by different mucus viscosities. In large mucoceles a tumor causes this lesion. PMID- 16681157 TI - [Emergency ultrasound on the chest]. PMID- 16681158 TI - [Medical literature puzzle. About unbelievable trips. Ilse Aichinger: Unbelievable trips]. PMID- 16681159 TI - ["A risk for life and health?" Alternative medicine practitioners, pharmacologists and drug regulation in the 1970's]. PMID- 16681161 TI - Public health. PMID- 16681160 TI - It's time for physicians to "weigh in" on this issue. PMID- 16681162 TI - Professionalism: a challenge to medical education in a changing society. PMID- 16681163 TI - Professionalism, medical education and our social contract. PMID- 16681164 TI - A review of current management of endometriosis in 2006: an evidence-based approach. AB - Endometriosis, a common cause of morbidity in reproductive age females, results in pelvic pain and infertility. Effective, evidence-based treatments of endometriosis-associated infertility include conservative surgical therapy and assisted reproductive technologies. In early stage endometriosis ovulation induction, with or without intrauterine insemination, improves pregnancy rates. In early stage disease in vitro fertilization reduces time to pregnancy as compared to controls, but does not increase the chance of pregnancy after three years. Endometriosis-associated pain can be approached with surgical or medical therapies. Conservative surgery maintains the reproductive organs and is an effective mode of treatment for endometriosis-associated pain. A more radical surgical approach of hysterectomy with bilateral salpingo-oophorectomy, not investigated in randomized controlled trials, remains a mainstay of therapy for endometriosis-associated pain in patients who have completed child-bearing. Current medical therapies rely upon interruption of normal cyclic, ovarian hormone production resulting in an environment not conducive to the growth of endometriosis. The current accepted therapies for endometriosis include danazol, progestational agents, oral contraceptive agents, and gonadotropin-releasing hormone analogues which all function similarly in relieving pain. The new era of genomics promises to help characterize endometriosis and allow one to tailor therapies based on a woman's symptoms and reproductive goals. PMID- 16681165 TI - Generic drugs: an overview. PMID- 16681166 TI - Take advantage of iHealth services. PMID- 16681167 TI - Physicians Voluntary Reporting Program (PVRP). PMID- 16681168 TI - A doctor's story. PMID- 16681169 TI - Hands: reflections of a retired surgeon. PMID- 16681171 TI - A profile of Ecstasy (MDMA) use in Sao Paulo, Brazil: an ethnographic study. AB - The use of Ecstasy in Brazil has gained visibility in recent years. Nevertheless, scientific data on this issue remain scarce. The present study focused on clarifying this phenomenon by means of qualitative and ethnographic research. Observational studies (N = 8), information obtained from key informants (N = 14) and interviews with Ecstasy users (N = 32) from the city of Sao Paulo were part of this study. The results pointed to night clubs and rave parties where electronic music is played as the main context of use. Interviews with users revealed a sample of young adult members of the highest layers of society, with a good educational background and access to the labor market. Furthermore, polydrug use was manifested. Users' typology was established regarding the pattern of drug use, showing two profiles of users: the "Rave Generation," consisting of more recent users who initiated this use with the arrival of the large-sized rave parties at the end of the 1990s. The second profile, the "Sons of the Hell's Club," consisted of more experienced users (beginning in the early 1990s) and showed a richer history of use. Neither profile group had sought help regarding their Ecstasy use, and both showed positive beliefs related to the safety of use. PMID- 16681170 TI - Ecstasy (MDMA), methamphetamine, and date rape (drug-facilitated sexual assault): a consideration of the issues. AB - The term "date rape drug" has traditionally been applied by the media to powerful sedatives, such as gamma hydroxybutyrate (GHB) and flunitrazepam (Rohypnol), which can render a person unconscious and hence unable to resist and/or recall an assault. However, some law enforcement agents and others have recently obtained convictions by arguing that the empathy-generating and sensual effects of MDMA, and an occasional increase in disinhibition and sexual desire linked with methamphetamine use, remove a person's ability to give a reasoned consent, turning the person into "a helpless slave" to their own sexual desires and those of the alleged perpetrator. The argument holds that the victim becomes part of the assault because they may appear to be cooperating and colluding with activity which they would not have consented to without taking these drugs. This interpretation of the term "date rape" has been fed by data that sometimes finds MDMA and amphetamines in samples taken from sexual assault victims, and hence these prosecutions sometimes rely on expert testimony from toxicologists, pathologists and police officers rather than psychologists and psychiatrists who are expert in the human effects of these drugs. Some of those in the latter group have dismissed claims that MDMA is an aphrodisiac or a date rape drug as myths propagated by the media. In this article, these arguments and their respective strengths and weaknesses will be examined to assist professionals and others who may become involved in these cases. PMID- 16681172 TI - Psychiatric disorders and their correlates among young adult MDMA users in Ohio. AB - This study describes the lifetime prevalence, correlates, and age of onset of selected psychiatric disorders among a community sample of MDMA users (n = 402), aged 18 to 30, in Ohio. Participants responded to interviewer-administered questionnaires, including sections of the computerized Diagnostic Interview Schedule for DSM-IV. Fifty-five percent of the sample had at least one lifetime disorder, with major depression (35.3%) and antisocial personality disorder (ASPD) (25.4%) the most common. Proportionately more women were diagnosed with depression, generalized anxiety disorder, and posttraumatic stress disorder (PTSD), while proportionately more men were diagnosed with ASPD. Proportionately more non-White participants had attention deficit/hyperactivity disorder (AD/HD). Higher levels of education were associated with proportionately less PTSD, ASPD, and AD/HD. Higher frequencies of MDMA use were associated with proportionately more ASPD and AD/HD. Comparing the age of first MDMA use with the age of onset for selected psychiatric disorders revealed that for most participants disorders preceded use. Multivariate analysis revealed participants with more than a high school education were less likely to have experienced a lifetime disorder, while those who had used MDMA more than 50 times were more likely. Variations in the prevalence of psychiatric disorders have practical implications for drug abuse prevention and treatment programs. PMID- 16681173 TI - Exploring the validity of self-reported Ecstasy use among club rave attendees. AB - While several empirical studies have focused on Ecstasy use among rave attendees, only one study has explored the validity of self-reported Ecstasy use within this population. To address this limitation, the authors collected self-report drug use information and oral fluid (OF) specimens from 96 club rave attendees within the Baltimore-Washington corridor between August and October 2000. The Kappa statistic is 0.59, suggesting a moderate relationship between the self-report and OF measures. Such accurate reporting bodes well for researchers interested in eliciting sensitive information from this population. PMID- 16681174 TI - Alcohol, drugs, and substance use among Asian-American college students. AB - Two hundred and forty eight self-identified Asian-American college students participated in this study that examined the prevalence rates and sociodemographic factors of substance use among Asian Americans in college. Using a Basic Demographic Questionnaire, Family of Origin Measure, Acculturation Lifestyle Survey, and Substance Use Checklist (all instruments were in English), prevalence rates were found to be comparable to or higher than a national sample: 94.5% lifetime prevalence and 78.6% current prevalence (past 30 days) of alcohol use; and higher current prevalence (past 30 days) of illicit drug use (9.5%) and of cigarette use (22.8%) than other Asians aged 12 and older (3.5% for illicit drugs and 17.7% for cigarettes) in a national survey. Male students and those who were employed were more likely to be current users (past 30 days) of drugs in general (15%) and marijuana (13.2%), and users of wine coolers (76.0%) and cigarettes (61.1%) in their lifetime. Being born overseas, years in the U.S., and preference for American TV/movies are associated with substance use. Asian Americans are not immune from substance use (or abuse) while in college. Culture specific prevention is necessary. PMID- 16681176 TI - Axis I psychiatric comorbidity among adult inhalant dependents seeking treatment. AB - The present study aimed to investigate the prevalence of Axis I disorders in adult inhalant-dependent patients in comparison to other substance-dependent patients and subjects without substance use disorders. The inhalant-dependent group consisted of 83 male inpatient and outpatient adults diagnosed according to DSM-IV criteria. This group was compared with 74 other substance-dependent patients and with 70 subjects without alcohol and substance use disorder diagnoses. Ninety-three percent of the inhalant dependents had a lifetime history of at least one type of comorbid Axis I disorder, while 77% of the same subjects had at least one type of any affective disorder and 75.9% of them had at least one type of anxiety disorder. Prevalence of Axis I disorders among inhalant dependents was 72.3% for lifetime major depression, 41% for major depression during the past month, 24% for dysthymic disorder, 20.5% for inhalant-induced depressive disorder, 27.7% for panic disorder, 30% for PTSD, 36.1% for social phobia and 20.5% for generalized anxiety disorder. The rate of lifetime axis I disorders was higher in patients with inhalant dependency in comparison to the other two groups. This finding suggests that inhalant-dependent adults have high rates of comorbid psychiatric problems, and that it is important to determine Axis I disorder comorbidity in this population before making an inpatient or outpatient treatment plan. PMID- 16681175 TI - Medical use, illicit use and diversion of prescription stimulant medication. AB - The objective of this study was to examine the prevalence and factors associated with the illicit use of prescription stimulants and to assess the relationship between the medical and illicit use of prescription stimulants among undergraduate college students. A Web survey was self-administered by a random sample of 9,161 undergraduate students attending a large public midwestern university in the spring of 2003. A total of 8.1% reported lifetime illicit use of prescription stimulants and 5.4% reported past year illicit use. The number of undergraduate students who reported illicit use of prescription stimulants exceeded the number of students who reported medical use of prescription stimulants for ADHD. The leading sources of prescription stimulants for illicit use were friends and peers. Multivariate logistic regression analyses revealed several risk factors for illicit use of prescription stimulants such as being male, White, member of a social fraternity or sorority, Jewish religious affiliation, and lower grade point average. All of these characteristics were also related to medically prescribed use of prescription stimulants. Those who initiated medically prescribed use of prescription stimulants for ADHD in elementary school were generally not at increased risk for illicit use of prescription stimulants or other drugs during college as compared to those who were never prescribed stimulant medication. The present study provides evidence that the illicit use of prescription stimulants is a problem among undergraduate college students, and certain subgroups appear to be at heightened risk. PMID- 16681177 TI - An exploratory investigation of marijuana and other drug careers. AB - In-depth interviews and a qualitative methodology are used to examine the drug careers of a nonrandom sample of 31 current or former marijuana smokers. Three user typologies were developed to describe the complex development of marijuana careers over time: Druggies, Weekend Warriors, and Grown-ups. Druggies and Weekend Warriors tend to use marijuana (and alcohol) fairly heavily during the early phases of their careers; Druggies pass this stage quickly and eventually move on to harder drugs and periods of chronic substance abuse. Gradual assumption of adult identities and roles allow Weekend Warriors to moderate their marijuana (and other drug use), which steadily declines throughout the remainder of their careers. Grown-ups tend to use marijuana and all drugs moderately during the beginning phases of their careers, eschew experimentation with harder drugs, and generally progress to very controlled use of marijuana and other intoxicants (or abstinence) in the later stages of their careers. The social process of using marijuana teaches the majority of users to quit or how to integrate its use into a lifestyle that can reasonably be described as normal or conventional. Lastly, two future directions for drug career research are suggested. PMID- 16681178 TI - The rise of the mobile phone in the hard drug scene of Rotterdam. AB - The rise of mobile phone dealing in the retail market of heroin and cocaine in the city of Rotterdam is described. Multiple methods were used for the study, including analysis of street survey data (1998, 2000, 2003), qualitative and quantitative analysis of fieldwork data, and semi-open interviews with drug users and key informants. In 2000, 70% of the respondents to a street survey bought drugs from a mobile dealer. Qualitative data showed that the majority of mobile dealers have an ethnic Moroccan background; the reasons for this may include the ambiguous attitude of the Moroccan community towards drug crime, and repressive legislation causing the market to find alternatives for basic street dealing. The rise of mobile dealing is discussed as a form of reshaping of the drug market under prohibition. PMID- 16681179 TI - Dissemination and feasibility of a cognitive-behavioral treatment for substance use disorders and posttraumatic stress disorder in the Veterans Administration. AB - This article describes a small dissemination effort and provides initial efficacy data for use of Seeking Safety, a cognitive-behavioral treatment for comorbid substance use disorders (SUD) and posttraumatic stress disorder (PTSD), in a VA setting. After providing a daylong interactive training in Seeking Safety to front-line clinicians, a cotherapist group practice model was implemented. Following 14 months of clinician training and an uncontrolled pilot study of four groups with 18 veterans, initial efficacy data indicate significant symptom reduction for patients and acceptability to clinicians. Findings are encouraging in that Seeking Safety treatment appears to have the potential to be beneficial for veterans with SUD-PTSD and also appeal to clinicians. Dissemination of Seeking Safety is feasible in the VA, yet there are likely barriers to sustaining it as a routine treatment. Recommendations for future dissemination are proposed, including ways VA administration can facilitate this process. PMID- 16681180 TI - Predicting treatment retention with a brief "Opinions About Methadone" scale. AB - It is important to identify social conditions, program factors, and client characteristics that predict retention because time in substance abuse treatment is associated with improved health, mood, and social functioning. Treatment dropouts also are at high risk for serious harms, including relapse. Most opioid dependent persons require long-term stabilization in methadone maintenance treatment (MMT) to normalize brain function and control withdrawal symptoms. The purpose of this study was to determine whether a client characteristic, namely, attitude toward methadone, was related to retention. Analysis of 14 opinion statements about methadone identified a reliable five-item scale with factorial validity. This scale (Opinions About Methadone; OAM-5) also predicted retention in MMT. Of 338 clients followed for one year after MMT enrollment, 48% dropped out of treatment; those who were more likely to remain had more favorable opinions. A supplementary qualitative study with MMT counselors demonstrated face validity for the OAM-5; counselors found the items easy to interpret and relevant to client attitudes towards MMT. The findings suggest that it would be worthwhile for MMT staff to assess client attitudes at intake, using the OAM-5. This would help them to identify and intervene promptly with those in greatest need of support for remaining in treatment. PMID- 16681181 TI - Obviation of opioid withdrawal syndrome by concomitant administration of naltrexone in microgram doses: two psychonautic bioassays. AB - Two psychonautic bioassays (self-experiments) in stepwise and abrupt cessation of long-term daily oral ingestion habits of 800 mg of codeine phosphate are presented. Concomitant administration of minute doses (about 0.5 mcg) of the opioid antagonist naltrexone with each dose of codeine was found in both cases to obviate the expected opioid withdrawal syndrome, resulting in asymptomatic and uneventful transitions from physical opioid dependency states to exogenous opioid free metabolism. These experiments are analyzed in the context of a conjectured, rapid, iterative reduction and complete elimination of opioid tolerance, once acquired. It was found that coadministration of naltrexone with codeine phosphate obviated the development of both tolerance and physical dependency over several months of four daily oral doses of 200 mg, allowing abrupt ("cold turkey"), asymptomatic and uneventful withdrawal. This points the way to the biochemical substrate of opioid tolerance itself, and shows that this can easily and inexpensively be blocked, even over months of iterative oral administration of substantial doses of opioid analgesics. Finally, it suggests the opioid withdrawal syndrome is directly related to the physiology of opioid tolerance, and can be prevented by blocking tolerance itself. Even when tolerance has been acquired, this can be reduced stepwise over a matter of days, with no symptoms of opioid withdrawal syndrome. PMID- 16681182 TI - Heroin addicts show impairment of information processing at the iconic level. AB - Heroin addicts (n = 100) and healthy controls (n = 100) were examined for their information processing ability at iconic (100 ms, 125 ms) as well as relatively deeper (250 ms, 500 ms) levels. Subjects were asked to identify briefly exposed photographs of familiar objects through an electronic tachistoscope. Addicts showed significant impairment of visual information processing at iconic but not at deeper levels. PMID- 16681183 TI - United States life tables, 2003. AB - This report presents period life tables for the United States based on age specific death rates in 2003. Data used to prepare these life tables are 2003 final mortality statistics; July 1, 2003, population estimates based on the 2000 decennial census; and data from the Medicare program. Presented are complete life tables by age, race, and sex. In 2003, the overall expectation of life at birth was 77.5 years, representing an increase of 0.2 years from life expectancy in 2002. Between 2002 and 2003, life expectancy increased for males and females and for both the white and black populations. Life expectancy increased by 0.3 years (from 77.7 to 78.0) for the white population and by 0.4 years (from 72.3 to 72.7) for the black population. Both males and females in each race group experienced increases in life expectancy between 2002 and 2003. The greatest increase was experienced by black females with an increase of 0.5 years (from 75.6 to 76.1). Life expectancy increased by 0.2 years for black males (from 68.8 to 69.0), white males (from 75.1 to 75.3), and for white females (from 80.3 to 80.5). PMID- 16681184 TI - Mystery of seasonality: getting the rhythm of nature. AB - Seasonality, a systematic periodic occurrence of events over the course of a year, is a well-known phenomenon in life and health sciences. Understanding seasonal fluctuations in diseases patterns presents us with a major challenge. To develop efficient strategies for disease prevention and control, we need to grasp the main determinants of temporal variations and their interactions. This paper will introduce the notion of seasonality by outlining several of its factors, using as illustrations respiratory and enteric water- or food-borne infections. PMID- 16681185 TI - Preparation and response in case of natural disasters: Cuban programs and experience. AB - Inadequate preparation for national disasters is frequently particularly devastating in lower income countries. The Cuba's location has a diversity of potential natural disasters, including hurricanes, non-tropical depressions, tropical storms, tropical cyclones, and severe local storms, all with intense rains and winds, earthquakes and droughts. Cuban preparation, at all levels, is geared to these predominant threats. Planning for natural disasters is integral to the political and economic life of Cuba, nationally and locally. On several occasions, United Nations (UN) officials have pointed to Cuba as a model for developing countries preparing for hurricanes and other natural disasters. A global policy for managing the risks of natural disasters could improve continuity of assistance for development and reduce the necessity of humanitarian aid. Planning in advance of disasters is a feasible way of helping people, by reducing expenses of emergencies, recuperation, and reconstruction. As climate changes accelerate, many researchers fear a period of irreversible and uncontrollable change. While the atmosphere continues to warm, it generates more intense rains, more frequent heat waves, and more ferocious storms. Thus, achieving better protection of developing countries from an increasing onslaught of natural disasters will only grow in importance. Even though Cuba's contribution to know-how has been recognized by United Nations' officials, progress toward more adequate preparation worldwide has been slow. To support other countries beyond conveying the lessons, Cuba now offers specially trained personnel to cooperate immediately with any country suffering a natural disaster. PMID- 16681187 TI - Vaccine innovation: lessons from World War II. AB - World War II marked a watershed in the history of vaccine development as the military, in collaboration with academia and industry, achieved unprecedented levels of innovation in response to war-enhanced disease threats such as influenza and pneumococcal pneumonia. In the 1940s alone, wartime programs contributed to the development of new or significantly improved vaccines for 10 of the 28 vaccine-preventable diseases identified in the 20th century. This article examines the historical significance of military organizations and national security concerns for vaccine development in the United States. PMID- 16681186 TI - Quebec's approach to population health: an overview of policy content and organization. AB - While Canada's international leadership in the area of health promotion has been widely acknowledged in the past, Quebec's approach could be better known. Canada's second largest province has indeed developed a comprehensive public health infrastructure and adopted a population health approach which features an integrated set of legislative, organizational and programmatic policy instruments. These instruments not only ensure the core functions of public health, but also foster public intervention on the social determinants of health. In addition, Quebec's policy is supported by a solid research infrastructure, networked expertise and a mobilized workforce among health professionals. In spite of the interest it represents for the larger public health community in Canada and elsewhere, this largely French-speaking province's approach remains little known because of language and cultural barriers between Quebec and Anglo Saxon countries, and it has yet to be systematically discussed in the English language literature. This article provides an overview of policies and administrative structures in Quebec to support public health and address socially determined inequalities in health. It analyzes the development of these policies over the past decade and offers insight to their core content. PMID- 16681188 TI - Environmental and occupational causes of cancer re-visited. AB - We recently completed a review of scientific evidence, particularly epidemiologic evidence, regarding the contribution of environmental and occupational exposures to the overall cancer burden in the US. We evaluated the efforts to estimate the proportion of cancer due to these involuntary exposures, including the ambitious effort by Doll and Peto and an update by a group of authors at the Harvard Center for Cancer Prevention. In this paper, we critique these efforts, and their resulting estimates of the proportion of cancer due to various factors. We also provide an alternative interpretation of the evidence and a caution against the very idea of attributing specific fractions or proportions of cancer to particular factors. We conclude by recommending that environmental and occupational links to cancer be given serious consideration by individuals and institutions concerned with cancer prevention, particularly those involved in research and public education. We support the new initiative in the European Union to evaluate chemicals more fully before they reach the market. PMID- 16681189 TI - Fighting a tobacco-tax rollback: a political analysis of the 1994 cigarette contraband crisis in Canada. AB - We identify factors that led a regional government (Quebec, Canada) to opt for a reduction of its tobacco tax to combat tobacco smuggling. Then we explore the fallout of Quebec's tobacco-tax rollback on its tobacco control policy. We conducted qualitative research using a case-study design and multiple sources of data. We applied the Advocacy Coalition Framework in respect of data collection and analysis. Advocates of the tobacco-tax rollback framed the contraband problem in a way that won the support of an array of actors. However, anti-tobacco activists succeeded in convincing the government to invest more in tobacco control. The new resources were instrumental in enhancing the activists' ability to promote legislative measures. Our approach sheds light on the tobacco industry's strategy to have governments reducing their tobacco tax. Quebec offers an example of how tobacco control activists can transform defeat into the cornerstone of a comprehensive tobacco control policy. PMID- 16681190 TI - Letter to the editors responding to Gould article in Journal issue no. 26. PMID- 16681191 TI - Supporting real advances in public health through FDA regulation. PMID- 16681192 TI - Access all areas. PMID- 16681193 TI - Who decides? AB - Changes to the law on mental capacity will be implemented next year, giving people more rights in decisions about their treatment and care. The new law will assist people in disagreements about their care, welfare or finances. Independent mental capacity advocates will support and represent people when important decisions have to be made. PMID- 16681194 TI - Accidental hero. Interview by Carol Davis. AB - Sarifa Kabir took on an accident prevention role and now provides health education to black and minority ethnic groups in Preston. She has covered burns prevention and basic first aid, road safety, misuse of paracetamol products and alcohol, diet and exercise, and falls prevention. PMID- 16681195 TI - Best banking bets. AB - Nursing students' unique circumstances and particular problems mean they are often under great financial pressure. We surveyed student bank packages. PMID- 16681197 TI - Development of a practice learning team in the clinical setting. AB - This article demonstrates how an action learning approach was used to implement and develop a practice learning team in a clinical setting. The team linked a higher education institution and a practice setting to improve the placement experience for nursing students and practice mentors. The practice learning team has developed initiatives to improve mentoring for nursing students. PMID- 16681198 TI - A pelvic floor muscle exercise programme for urinary incontinence following childbirth. PMID- 16681199 TI - Benchmarking the nurse consultant role in rheumatology. AB - This article demonstrates how the four core standards for the nurse consultant role have been developed and applied in the field of rheumatology. The authors suggest that the standards: expert practice, leadership and service redesign, research and education, are relevant for consultant nurses in all specialties. The standards can be used by other nurse consultants to benchmark their practice. PMID- 16681200 TI - Assessment and management of obsessive compulsive disorder. AB - This article outlines the nature of obsessive compulsive disorder and sets out an evidence-based approach to assessment and treatment. The article also examines the key elements of the National Institute for Health and Clinical Excellence (2005) guideline on this condition. PMID- 16681201 TI - Mechanical ventilation. PMID- 16681202 TI - Registration blues. PMID- 16681203 TI - New York State urban and rural measurements of continuous PM2.5 mass by FDMS, TEOM, and BAM. AB - Field evaluations and comparisons of continuous fine particulate matter (PM2,5) mass measurement technologies at an urban and a rural site in New York state are performed. The continuous measurement technologies include the filter dynamics measurement system (FDMS) tapered element oscillating microbalance (TEOM) monitor, the stand-alone TEOM monitor (without the FDMS), and the beta attenuation monitor (BAM). These continuous measurement methods are also compared with 24-hr integrated filters collected and analyzed under the Federal Reference Method (FRM) protocol. The measurement sites are New York City (the borough of Queens) and Addison, a rural area of southwestern New York state. New York City data comparisons between the FDMS TEOM, BAM, and FRM are examined for bias and seasonality during a 2-yr period. Data comparisons for the FDMS TEOM and FRM from the Addison location are examined for the same 2-yr period. The BAM and FDMS measurements at Queens are highly correlated with each other and the FRM. The BAM and FDMS are very similar to each other in magnitude, and both are approximately 25% higher than the FRM filter measurements at this site. The FDMS at Addison measures approximately 9% more mass than the FRM. Mass reconstructions using the speciation trends network filter data are examined to provide insight as to the contribution of volatile species of PM2.5 in the FDMS mass measurement and the fraction that is likely lost in the FRM mass measurement. The reconstructed mass at Queens is systematically lower than the FDMS by approximately 10%. PMID- 16681204 TI - The measurement of fine particulate semivolatile material in urban aerosols. AB - Ammonium nitrate and semivolatile organic material (SVOM) are significant components of fine particles in urban atmospheres. These components, however, are not properly determined with methods such as the fine particulate matter (PM2.5) Federal Reference Method (FRM) or other single filter samplers because of significant losses of semivolatile material (SVM) from particles collected on the filter during sampling. The R&P tapered element oscillating microbalance (TEOM) monitor also does not measure SVM, because this method heats the sample to remove particle bound water, which also results in evaporation of SVM. Recent advances in monitoring techniques have resulted in samplers for both integrated and continuous measurement of total PM2.5, including the particle concentrator Brigham Young University organic sampling system (PC-BOSS), the real-time total ambient mass sampler (RAMS), and the R&P filter dynamics measurement system (FDMS) TEOM monitor. Results obtained using these samplers have been compared with those obtained with either a PM2.5 FRM sampler or a TEOM monitor in studies conducted during the past five years. These studies have shown the following: (1) the PC-BOSS, RAMS, and FDMS TEOM are all comparable. Each instrument measures both the nonvolatile material and the SVM. (2) The SVM is not retained on the heated filter of a regular TEOM monitor and is not measured by this sampling technique. (3) Much of the SVM is also lost during sampling from single filter samplers such as the PM2.5 FRM sampler. (4) The amount of SVM lost from single filter samplers can vary from less than one-third of that lost from heated TEOM filters during cold winter conditions to essentially all during warm summer conditions. (5) SVOM can only be reliably collected using an appropriate denuder sampler. (6) A PM2.5 speciation sampler can be easily modified to a denuder sampler with filters that can be analyzed for semivolatile organic carbon (OC), nonvolatile OC, and elemental carbon using existing OC/elemental carbon analytical techniques. The research upon which these statements are based for various urban studies are summarized in this paper. PMID- 16681205 TI - Comparison of particle light scattering and fine particulate matter mass in central California. AB - Particle light scattering (Bsp) from nephelometers and fine particulate matter (PM2.5) mass determined by filter samplers are compared for summer and winter at 35 locations in and around California's San Joaquin Valley from December 2, 1999 to February 3, 2001. The relationship is described using particle mass scattering efficiency (sigmasp) derived from linear regression of Bsp on PM2.5 that can be applied to estimated PM2.5 from nephelometer data within the 24-hr filter sampling periods and between the every-6th-day sampling frequency. An average of sigmaSp = 4.9 m2/g was found for all of the sites and seasons; however, sigmasp averaged by site type and season provided better PM2.5 estimates. On average, the sigmasp was lower in summer than winter, consistent with lower relative humidities, lower fractions of hygroscopic ammonium nitrate, and higher contributions from fugitive dust. Winter average sigmasp were similar at non source-dominated sites, ranging from 4.8 m2/g to 5.9 m2/g. The sigmasp was 2.3 m2/g at the roadside, 3.7 m2/g at a dairy farm, and 4.1 m2/g in the Kern County oilfields. Comparison of Bsp from nephelometers with and without a PM2.5 inlet at the Fresno Supersite showed that coarse particles contributed minor amounts to light scattering. This was confirmed by poorer correlations between Bsp and coarse particulate matter measured during a fall sampling period. PMID- 16681206 TI - Use of the aerodynamic particle sizer to measure ambient PM10-2.5: the coarse fraction of PM10. AB - The Aerodynamic Particle Sizer (APS 3321, TSI, Inc., Shoreview, MN) rapidly measures particle number concentration by size from 0.5 to 20 microm. This work used simple assumptions for particle shape factor and density to estimate ambient coarse mode particulate matter, PM10-2.5, from APS number concentration data. This estimate was compared with that measured with time-integrated, filter-based federal reference method (FRM) samplers in four U.S. field studies: two in Phoenix, AZ; one in Gary, IN; and one in Riverside, CA. Near one-to-one agreement and a strong linear relationship were observed between APS-estimated and FRM measured PM10-2.5 in the first Phoenix, AZ study (slope = 0.90, R2 = 1.00); the second Phoenix, AZ study (slope = 0.99, R2 = 0.99); and the Riverside, CA study (slope = 1.00, R2 = 0.84). In the Gary, IN study, PM10-2.5 estimates made with data from the APS tended to be less than that measured with the FRM samplers (slope = 0.57), but the linear relationship between the two methods was still strong (R2 = 0.90). Particle-bound water associated with wet atmospheric conditions may account for these differences. Additional testing is required to resolve this issue. PMID- 16681207 TI - Nanoparticle and ultrafine particle events at the Fresno supersite. AB - Continuous measurements of particle size distributions of 3-407 nm were collected from August 2002 to July 2004 at the Fresno Supersite to understand their number concentrations, size distributions, and formation processes. Measurements for fine particulate matter (PM2.5) mass, sulfate (SO4(2-)), nitrate (NO3-), black carbon (BC), particle-bound polycyclic aromatic hydrocarbons (PAHs), nitrogen oxides (NOx), carbon monoxide (CO), ozone (O3), and meteorological data (wind speed, wind direction, temperature [T], relative humidity [RH], and solar radiation) were used to determine the causes of nanoparticle (3-10 nm) and ultrafine (10-100 nm) particle events. These events were found to be divided into four types: (1) 3- to 10-nm morning nucleation; (2) 10- to 30-nm morning traffic; (3) 10- to 30-nm afternoon photochemical; and (4) 50- to 84-nm evening home heating, including residential wood combustion. Intense examples of the first type (>10(4) number [#]/cm3) were observed on 29 days, nearly always during the summer. The second type of event was observed on more than 73 days and occurred throughout the year. The third type was observed on 36 days, from spring through summer. The fourth type was found on 109 days, all of them during the winter. Although sulfur dioxide (SO2) emissions in Central California are low, the small residual amounts in gasoline and diesel fuel are apparently sufficient to initiate nucleation events. These were measured in the morning, soon after the shallow surface inversion coupled with layers aloft where nucleation probably was initiated. PM2.5 concentrations were poorly correlated with nanoparticle number. PMID- 16681208 TI - Influence of atmospheric dispersion and new particle formation events on ambient particle number concentration in Rochester, United States, and Toronto, Canada. AB - Continuous measurements of particle number concentrations were performed in Rochester, NY, and Toronto, Ontario, Canada during the 2003 calendar year. Strong seasonal dependency in particle number concentration was observed at two sites. The average number concentration of ambient particles was 9670 +/- 6960 cm(-3) in Rochester, whereas in Toronto the average number of particles was 28,010 +/- 13,350 cm(-3). The particle number concentrations were higher in winter months than in summer months by a factor of 1.5 in Rochester and 1.6 in Toronto. In general, there were also distinct diurnal variations of aerosol number concentration. The highest weekdays/weekends ratio of number concentration was typically observed during the rush-hour period in winter months with a ratio of 2.1 in Rochester and 2.0 in Toronto. The correlation in the total particle number concentrations between the two urban sites was stronger in winter because of the common urban traffic patterns, but weaker in summer because of local sulfur dioxide (SO2)-related particle formation events in Rochester in the summer. Strong morning particle formation events were frequently observed during colder winter months. Good correlations between particle number and carbon monoxide (CO) as well as temperature suggested that motorvehicle emissions lead to the formation of new particles as the exhaust mixes with the cold air. Regional nucleation and growth events frequently occurred in April. Local SO2-related particle formation events most frequently occurred in August. SO2 and UV-B were highly correlated with particle concentration, suggesting a high association of photochemical processes with these local events. A high directionality in a northerly direction was observed for particle number and SO2, indicating the influence of point sources located north of Rochester. PMID- 16681209 TI - Water-based condensation particle counters for environmental monitoring of ultrafine particles. AB - TSI Inc. (Shoreview, MN) has introduced three new water-based condensation particle counters (WCPCS) that were designed to detect airborne particles larger than 2.5 nm (model 3786), 5 nm (model 3785), and either 10 or 20 nm (model 3782). These WCPCs are well suited for real-time, environmental monitoring of number concentration of airborne ultrafine particles. Their unique design incorporates the use of water as the working fluid instead of alcohol. Water is odor free, readily available, and eliminates the problem of water condensation and absorption into alcohol working fluids during operation in humid environments. In this study, the performance of three TSI WCPCs was characterized for several aerosol compositions, including sucrose, salt (NaCl), dioctyl sebacate (DOS), dioctyl phthalate (DOP), emery oil (poly-alpha-olefin), silver, impurity residue particles, and ambient aerosol particles. All particles were size selected using a nano differential mobility analyzer (nano-DMA; model 3085, TSI Inc.) to create monodisperse challenge aerosols. The challenge aerosol was mixed uniformly with clean makeup flow and split into a WCPC and a reference instrument to determine the counting efficiency of the WCPC. For the model 3785 WCPC, the D50 (i.e., the particle diameter with 50% counting efficiency) was determined to be 3.1 nm for salt particles, 4.7 nm for sucrose and ambient particles, 5.6 nm for silver particles, and >50 nm for ultrapure oil particles. The sensitivity to oil droplets increased dramatically (D50 < 10 nm) when the oil was slightly contaminated. The D50 of model 3786 ultrafine water-based CPC (UWCPC) was 2.4 nm for impurity residue particles. The D50 of the model 3782 WCPC was 10.8 (with a nominal setting of 10 nm) or 19.8 nm (with a nominal setting of 20 nm) for sucrose particles. All three WCPCs have response times of less than 2 or 3 sec and are therefore able to detect fast-changing events. PMID- 16681210 TI - An overview of the gulf coast aerosol research and characterization study: the Houston fine particulate matter supersite. AB - The Gulf Coast Aerosol Research and Characterization Study ([GC-ARCH], also known as the Houston Fine Particulate Matter [PM] Supersite) examined the spatial and temporal variability in fine PM source contributions and composition and the physical and chemical processes that govern PM formation and transformation in southeastern Texas. This was accomplished through the analysis of data collected in a 16-month field sampling program (August 2000 through November 2001). Three core sites and approximately 15 peripheral sites, jointly operated by the study team and the Texas Commission on Environmental Quality (TCEQ), were used. Key scientific findings related to spatial and temporal variability in fine PM concentrations, sizes and composition of the fine PM, the strength of primary emission sources and causes of secondary fine PM formation are reported. PMID- 16681211 TI - Comparison of two winter air quality episodes during the California Regional Particulate Air Quality Study. AB - The duration, strength, spatial extent, and chemical makeup of particulate matter (PM) are compared for two winter air quality episodes captured during the California Regional Particulate Air Quality Study (CRPAQS). Each episode, from the beginning of the buildup through dissolution, lasted about 3 weeks. The first episode occurred from December 14, 1999, through January 1, 2000, with peak 24-hr average fine particulate matter (PM2.5) concentrations reaching 129 microg/m3. The second episode occurred a year later, from December 18, 2000, through January 8, 2001, with peak 24-hr average PM2.5 concentrations reaching 179 microg/m3. Although similar in duration, each episode exhibited unique characteristics. One significant difference was the episode buildup rate; rapid in 1999, but slow and steady in 2000. The rapid buildup of the first episode resulted in more days with PM2.5 concentrations above the 24-hr federal standard, whereas the slow and steady increase of the second episode produced higher peaks. Spatial extent and progress also differed between the two episodes. The Northern Valley was impacted more during the December 1999 episode, and the Southern Valley during the December 2000 episode. The differences carried over into chemical composition. Ammonium nitrate dominated the PM2.5 mass during the December 1999 episode. The second episode reflected a dichotomy typical to the San Joaquin Valley, with Fresno concentrations dominated by organic and elemental carbon and the rest of the Valley concentrations dominated by ammonium nitrate. Each episode showed a regional as well as a local component. Ammonium nitrate concentrations, which result from more regional-scale secondary formation and mixing of emissions, were fairly uniform among the urban and rural sites. Carbon concentrations were always higher at urban sites than at rural sites, corresponding to the higher emissions density of primary carbon sources in urban areas. PMID- 16681212 TI - Comparison of continuous and filter-based carbon measurements at the Fresno supersite. AB - Results from six continuous and semicontinuous black carbon (BC) and elemental carbon (EC) measurement methods are compared for ambient samples collected from December 2003 through November 2004 at the Fresno Supersite in California. Instruments included a multi-angle absorption photometer (MAAP; lambda = 670 nm); a dual-wavelength (lambda = 370 and 880 nm) aethalometer; seven-color (lambda = 370, 470, 520, 590, 660, 880, and 950 nm) aethalometers; the Sunset Laboratory carbon aerosol analysis field instrument; a photoacoustic light absorption analyzer (lambda = 1047 nm); and the R&P 5400 ambient carbon particulate monitor. All of these acquired BC or EC measurements over periods of 1 min to 1 hr. Twenty four-hour integrated filter samples were also acquired and analyzed by the Interagency Monitoring of Protected Visual Environments (IMPROVE) thermal/optical reflectance carbon analysis protocol. Site-specific mass absorption efficiencies estimated by comparing light absorption with IMPROVE EC concentrations were 5.5 m2/g for the MAAP, 10 m2/g for the aethalometer at a wavelength of 880 nm, and 2.3 m2/g for the photoacoustic analyzer; these differed from the default efficiencies of 6.5, 16.6, and 5 m2/g, respectively. Scaling absorption by inverse wavelength did not provide equivalent light absorption coefficients among the instruments for the Fresno aerosol measurements. Ratios of light absorption at 370 nm to those at 880 nm from the aethalometer were nearly twice as high in winter as in summer. This is consistent with wintertime contributions from vehicle exhaust and from residential wood combustion, which is believed to absorb more shorter-wavelength light. To reconcile BC and EC measurements obtained by different methods, a better understanding is needed of the wavelength dependence of light-absorption and mass-absorption efficiencies and how they vary with different aerosol composition. PMID- 16681213 TI - PM2.5 speciation trends network: evaluation of whole-system uncertainties using data from sites with collocated samplers. AB - The objectives of this paper are to contrast the relative variability of replicate laboratory measurements of selected chemical components of fine particulate matter (PM) with total variability from collocated measurements and to compare the magnitudes of the uncertainties determined from collocated sampler data with those currently being provided to U.S. Environmental Protection Agency (EPA)'s Air Quality System (AQS) database by RTI International (RTI). Pointwise uncertainty values are needed for modeling and data analysis and should include all the random errors affecting each data point. Total uncertainty can be decomposed into two primary components: analytical measurement uncertainty and sampling uncertainty. Analytical measurement uncertainties are relatively easy to calculate from routine quality control (QC) data. Sampling uncertainties, on the other hand, are comparatively difficult to measure. In this paper, the authors describe data from collocated samplers to provide a snapshot of whole-system uncertainty for several important chemical species. The components of uncertainty were evaluated for key species from each of the analytical methods employed by the PM2.5 Speciation Trends Network (STN) program: gravimetry, ion chromatography (IC), X-ray fluorescence (XRF), and thermal-optical analysis for organic carbon and elemental carbon. The results show that the laboratory measurement uncertainties are typically very small compared with uncertainties calculated from the differences between samples collected from collocated samplers. These differences are attributable to the "field" components uncertainty, which may include contamination and/or losses during shipping, handling, and sampling, as well as other distortions of the concentration level due to flow and sample volume variations. Uncertainties calculated from the collocation results were found to be generally similar to the uncertainties currently being loaded into EPA's AQS system, with some exceptions described below. PMID- 16681214 TI - Retained nitrate, hydrated sulfates, and carbonaceous mass in federal reference method fine particulate matter for six eastern U.S. cities. AB - Material balance of fine particulate matter (PM2.5) measured with the Federal Reference Method (FRM) is developed for one rural and five urban locations in the eastern half of the United States using routine Speciation Trends Network (STN) and FRM chemical measurements and thermodynamic models. The Aerosol Inorganics Model is used to estimate retained particle bound water, and an ammonium nitrate evaporation model is used to estimate nitrate concentrations retained on the Teflon-membrane filter of the FRM. To address large uncertainties in carbonaceous mass calculated from STN carbon measurements, retained carbonaceous mass is derived by material balance between PM2.5 FRM mass and estimates of its non carbon constituents. The resulting sulfate, adjusted nitrate, derived water, inferred carbonaceous material balance approach (SANDWICH) is compared with reconstructed fine mass (RCFM) using the Interagency Monitoring of Protected Visual Environments monitoring program equation. For this study, the SANDWICH method resulted in approximately 21-27% higher sulfate mass and approximately 24 85% lower nitrate mass. The combined mass associated with sulfates and nitrates, however, are well within +/- 10% of the proportion derived using the more traditional RCFM method. The discrepancies between SANDWICH and measurement derived carbonaceous mass vary from -21% to +56% on an annual basis and are attributed in part to urban-rural source influences and uncertainties in estimating FRM-retained carbonaceous mass. PMID- 16681215 TI - Stationary sources of airborne lead: a comparison of emissions data for southern California. AB - Estimates for the air releases of lead from stationary point sources are considered for the South Coast Air Basin of California. We have examined four databases published by U.S. Environmental Protection Agency, the California Air Resources Board, and the South Coast Air Quality Management District. Our analysis indicates that none of the databases includes every emitting facility in the South Coast Air Basin of California and that other discrepancies among the databases exist. Additionally, the data have been analyzed for temporal variation, and some of the California Air Resources Board data are not current. The South Coast Air Quality Management District inventory covers 12 times more facilities in 2001 than in 1996. From this analysis, we conclude that all four of the databases would benefit by sharing data, increasing transparency, analyzing uncertainty, and standardizing emission estimation methods. PMID- 16681217 TI - Cost-benefit analysis in the selection of efficient multipollutant strategies. AB - Pollution control efforts are motivated by the desire to protect human health and the environment. Often, those efforts involve selecting among multiple options for attaining air quality objectives. For example, state and local decision makers must choose the mix of control strategies for meeting the requirements of the National Ambient Air Quality Standards (NAAQS) and the Regional Haze Rule. We demonstrate that including assessments of the human health and environmental benefits when evaluating alternative strategies may help decision-makers to identify multipollutant attainment strategies that achieve greater net benefits than would accrue under strategies optimized for cost alone. This paper presents a conceptual framework that decision-makers could use to choose among alternative multipollutant control strategies, accounting for the benefits and the costs of different types and locations of emissions reductions. PMID- 16681216 TI - A comprehensive particulate matter monitoring system and dosimetry-based ambient particulate matter standards. AB - A numerical particulate matter (PM) measurement model is developed to characterize and evaluate PM sampling methods. Simulations are conducted using the model to evaluate currently widely used PM samplers, including Federal Reference Method (FRM) samplers. The simulations show that current PM samplers are very vulnerable to both changes in measurement target (i.e., natural variability of particle size distribution) and the sampler's design, manufacturing, and operating conditions, potentially resulting in significant errors in the monitoring data. The numerical model is used in conjunction with two types of commercially available PM monitoring devices to form a Comprehensive Particulate Matter Monitoring System (CPMMS). The first type of device can be any mass-based PM monitor with a well-defined sampling efficiency curve. The second type of device is one capable of measuring particle size distribution with a reasonably good relative accuracy between size categories but not necessarily accurate in measuring absolute mass concentrations. This study shows that CPMMS can produce much higher quality PM monitoring data than the current PM samplers under the same conditions. In addition, unlike past and current PM monitoring data such as total suspended particulates, coarse PM (PM10), fine PM (PM2.5), etc., the CPMMS monitoring data will survive changes in PM regulatory definition. A new concept, dosimetry-based PM metrics and standards, is proposed to define ambient PM level based on the deposition fraction of particles in the human respiratory tract. The dosimetry-based PM metrics is more meaningful because it correlates the ambient PM level with the portion that can be deposited in the respiratory tract without an arbitrary cutoff particle diameter. CPMMS makes dosimetry-based PM metrics and standards feasible. PMID- 16681218 TI - [More responsibility and higher qualification for nurses of people with chronic diseases]]. PMID- 16681219 TI - [Ongoing breastfeeding with breast abscess]. AB - Mastitis puerperalis can frequently be complicated by a breast abscess. Immediate diagnosis and treatment are crucial if breastfeeding is to be continued and for the prevention of further complications. Somatic and psychological interventions are of importance for recovery. Specialised nurses and midwives have an important impact on the promotion of well-being of affected mothers. PMID- 16681220 TI - [Quality criteria that are important to residents of elders' homes and their relatives--a literature analysis]. AB - This literature analysis attempts to identify quality criteria which are important for residents of nursing homes and their relatives. To this end, 13 research articles published in international scientific journals were analysed. For the residents themselves, the ability to manage their daily lives autonomously as well as their ability to structure social contacts and relationships appears to be of special importance. In contrast, relatives emphasise criteria that refer to the quality of the care provided and the structure quality of the institutional setting. For them it is of specific importance to know that residents feel at home in the facility. PMID- 16681221 TI - [The satisfaction of relatives with service delivery of nursing homes for the elderly. Development of the screening-questionnaire ZUF-A-7]. AB - PURPOSE: To enable the measurement of satisfaction with the service delivery of residential homes, a practicable and statistical proven screening-test was supposed to educe. In order to identify critical quality-related situations a specific cut-off should be allocated. METHODS: Based on literature research a questionnaire was developed and responded by 3581 relatives of residents of 45 nursing homes for the elderly. The questionnaire was analysed and compressed based on scale, item and factor analysis. For validation, demographic variables and separate quality-ratings were available. Finally a ROC analysis was performed to recognize specificity and sensitivity of possible cut-offs. RESULTS: The Angehorigen-Screening-Fragebogen (relatives-screening-questionnaire) ZUF-A-7 was generated consisting of seven items. The scale is reliable (Cronbach's Alpha). Like other satisfaction measurements it displays a skew distribution. Middle size up to large correlations with the quality-ratings exists but there were no relevant coherences to the demographic variables. The ROC analysis suggested a critical cut-off of 21 (range = 7-28). PMID- 16681222 TI - [Survey of work schedules of Swiss intensive care units and assessment of their possible impact on nurses]. AB - Work schedules impact significantly on the health and well-being of health care professionals. The prevailing schedules can place the care providers 'at risk both psychologically and physically as well as in their social and private sphere. The added burden of daily activities in intensive care units (ICUs) intensifies these effects. The present quantitative study was designed to determine the range of work schedules currently in practice in intensive care units in Switzerland and to assess their possible health impact on nurses. Furthermore, it assessed how the organizational context differs between intensive care units with better and worse work schedules. A questionnaire was developed, pilot tested, and distributed to 84 ICUs in Switzerland. All 84 professionals responsible for designing the work schedules of nurses on ICUs were requested to fill out the questionnaire. The collected data were analyzed using descriptive statistical methods. To assess the health impact of the work schedules, a method was developed which considers the most important health relevant dimensions of the work schedules for a global estimate. Finally, the most extreme groups regarding the health impact of the working schedules were compared regarding their organizational context using non-parametric and parametric tests. The findings show that the working schedules in ICUs could be considerably improved, particularly regarding duration and density of working hours as well as regarding supportive structures of the hospitals. PMID- 16681223 TI - [Nurse turnover: A differential analysis of the predictors for intent to change the job and intent to leave the profession]. AB - In a cross-sectional questionnaire study predictors for the intent to leave the nursing profession on the one hand and predictors for the intent to change the job within the nursing profession on the other hand are examined. Data were collected by a national survey of nursing personnel in Germany. The sample of the study consists of 454 nurses working in acute care hospitals. As possible predictors work-related psychological stress, the strain due to illness and behaviour of the patients, psychosomatic pains, organisational, structural as well as sociodemographic variables are taken into account. The relevant predictors are identified by two multiple, linear regression analyses. Psychosomatic pains, low decision latitude, high psychological job demands, short job tenure, the strain due to aggressive and nagging patients, bad cooperation between doctors and nurses, low support by the supervisor and professional status (supervising position) turn out to be predictive for the intent to leave the nursing profession (R2 = 30%). For the intent to change the job within the profession significant predictors are low social support by colleagues, low support by the supervisor, psychosomatic pains, age, short job tenure, and the number of beds in the unit (R2 = 44%). The results indicate, that for the prevention of professional exit early individual measures should be taken where as the improvement of communication and cooperation among colleagues could keep nurses from job rotation within the profession. PMID- 16681224 TI - [The conduct of deaconesses during the NS-dictatorship and the efforts to overcome these experiences. A case study on historical research]. AB - The article is an approach to the conduct of nurses organisations and their members during the time of National Socialism as well as the following attempts to come to terms with these events. Both themes are dealt with rarely. The present state of research is outlined. In a case-study, which relies on source material from an archive of a deaconesses mother house and covers the period from 1943 to 1990, central problems are discussed exemplarily. Questions for further research are formulated and methodological problems are discussed. In this connection the special relevance of the analysis of personal files of the nurses is emphasized. PMID- 16681225 TI - [Comments on "Urinary incontinence as a risk factor for pressure sores does not withstand a critical examination"]. PMID- 16681227 TI - A pandemic flu: not if, but when. SARS was the wake-up call we slept through. AB - If an influenza pandemic struck today, borders might close, the global economy would be severely impacted, international vaccine supplies and health are systems would be overwhelmed, and some people might panic. To limit the fallout, the industrialized world must create a detailed response strategy involving the public and private sectors. Some experts feel we are overdue for a flu pandemic and the SARS pandemic of 2003 could have been the wake up call to begin preparations. Fortunately there is some assistance coming from the federal government. On January 12, U.S. Department of Health & Human Services Secretary Mike Leavitt announced funding to assist in the preparation for a pandemic flu response. $100 million is being provided initially with another $250 million due later this year to assist states in pandemic flu preparedness. Texas' initial allocation is $5,875,044. While some believe that the AI (H5N1) causing illness and deaths in Asia and Turkey will be the pandemic flu strain, there is no guarantee that will occur. Thus, without knowing which strain may lead to a pandemic, development and manufacturing of a vaccine is delayed. PMID- 16681228 TI - Trust. PMID- 16681229 TI - When you cannot say no! "A wounded snake": why COBRA does not work. PMID- 16681230 TI - It's go time! Get in the game with TMA's 2006 legislative agenda. PMID- 16681231 TI - TMA president's project: heart health for women. PMID- 16681232 TI - Failure to diagnose, treat or refer. PMID- 16681233 TI - Volunteering: gratitude in action. PMID- 16681234 TI - Coding changes for 2006. PMID- 16681235 TI - Family security with a comprehensive financial plan. PMID- 16681236 TI - Streptococcus intermedius: an unusual cause of a primary empyema. AB - Despite the evolution of modern antibiotics, pleural space infection remains a common illness with significant morbidity and mortality. Every year up to 60 percent of the 1.2 million patients in the United States who present with community-acquired pneumonia develop parapneumonic pleural effusions. Few of these parapneumonic effusions progress to an empyema. Interestingly, in about 10 percent of the cases of empyema, no underlying pulmonary infection can be identified. These pleural space infections are called primary empyemas. We present a patient with a primary empyema caused by Streptococcus intermedius. Relatively few studies have investigated primary empyema caused Streptococcus intermedius. This case presentation serves as a reminder to the clinician about primary empyema caused by this micro-organism. PMID- 16681237 TI - Posttraumatic stress disorder following military deployment in Iraq and Afghanistan. PMID- 16681238 TI - The dance of 1805--A belated review of Felix Robertson: an essay on Chorea Sanctis Viti, Philadelphia, 1805. PMID- 16681239 TI - Twenty-four years later and things are changing. . . some good, some not. PMID- 16681240 TI - Use of oral health care services and oral findings in children with special needs in Lagos, Nigeria. AB - This study was carried out to determine the use of oral healthcare services by children with special needs in Lagos, Nigeria. In this study, 125 structured questionnaires were sent to parents of children in two public day schools for CSN in Lagos. Once the questionnaires were returned, oral examinations were scheduled. The findings were compared to a control group of healthy children from two public schools in the same environment. One hundred and nine CSN and 134 controls, aged 5 to 19 years, participated in the study. The majority of the children had parents who had a low educational level. Fewer than 5% of the children had ever been to the dentist, and had only attended because of some symptoms. Non-attendance in 59.6% and 58% of CSN and controls respectively was reported to be due to absence of dental problem, 28% and 33.5% expressed ignorance about dentists and 8% of the parents of CSN identified finance and transportation as a problem. There were no significant differences between the two groups (p>0.05). It was found that 54% of CSN and 9% of controls had poor oral hygiene (p<0.05); dmft/DMFT was 0.25 in the controls and 0.12 in CSN (p>0.05). Untreated fractured teeth and missing teeth were more prevalent in CSN (p<0.05), while enamel hypoplasia was more prevalent in the controls (p<0.05). This study concluded that CSN and children from lower socioeconomic levels did not adequately utilize dental facilities in Lagos. Preventive programs in schools and the development of dental clinics in or near their schools could help. PMID- 16681241 TI - Successful treatment of mucous membrane pemphigoid with tacrolimus. AB - Mucous membrane pemphigoid (MMP) is a heterogeneous group of autoimmune, chronic inflammatory ubepithelial vesiculobullous disorders, predominantly affects the mucous membranes of the oral cavity and eyes of the elderly population. Oral manifestations of pemphigoid include desquamative gingivitis, ulcers, erythematous patches, erosions, vesicles and bullae located on the attached gingiva, palate, buccal mucosa, labial mucosa, and tongue. Diagnosis is based on history, clinical features and a biopsy stained with hematoxylin and eosin (H&E), and also direct and indirect immunofluorescence. Topical and systemic corticosteroids are the most commonly used medications for managing pemphigoid. Recently, topical tacrolimus has been successfully used in the treatment of ocular and skin pemphigoid. In this report we present a patient with longterm recalcitrant MMP that did not respond to conventional treatment but as treated successfully with tacrolimus ointment. PMID- 16681242 TI - Oral health status of children with treacher Collins syndrome. AB - There is a lack of data on the oral health status of individuals with craniofacial syndromes. A group of 15 children with Treacher Collins syndrome, aged 5 to 15 years old, was examined and evaluated for plaque, caries and gingival problems. The ability of the patients to clean their teeth was also investigated. A high plaque index and poor efficacy of tooth-brushing was recorded. The caries and gingival indexes were not proportionally as high as the plaque accumulation. There was no association between the gingival index and presence of mouth breathing. There was predominance of the D component in both the dmft and DMFT indexes; this was associated with a need for restorative dental treatment in 60% of the patients, which indicated the need for dental care for these patients. Caretakers should be informed of the importance of oral health and oral hygiene and encouraged to take responsibility for the oral care of the children living at home. PMID- 16681243 TI - Metastatic lesion of the anterior mandible with an occult primary: a case report. AB - Metastatic tumors to the oral cavity are relatively uncommon and account for about 1% of all oral cancers. Distant metastases to the jawbone are associated with a poor prognosis and a survival rate that is usually less than one year. The leading common primary sites for these lesions are the breast in females and the lung in males followed by the adrenals, kidneys, prostate, thyroid and colon. In 30% of all cancers, a metastatic lesion could be the first sign of a primary tumor elsewhere in the body. Metastatic lesions to the jaws are known to simulate periodontal and pulpal disease and other radiolucent lesions that can occur in the jaws. Microscopic evaluation with concurrent radiographic skeletal survey is warranted in patients where a metastatic lesion is suspected. PMID- 16681244 TI - Interdisciplinary approach to treat dyskeratosis congenita associated with severe aplastic anemia: a case report. AB - This paper reports on a 4-year-old male who had dyskeratosis congenita and who acquired severe aplastic anemia. The patient developed hyperpigmentation of the face, neck and chest region, arms, shoulders and legs. In addition, he had dry skin, deformed fingernails and toenails, sparse hair and eyebrows and hyperkeratosis of the dorsum of the hands and feet. Laboratory and histological analysis revealed severe pancytopenia and dyserythropoiesis of red blood cells, hypocellularity of white blood cells and decreased megakaryocytes with dysplasia. The intraoral examination identified bleeding gums; petechiae of the palate, tongue and cheek mucosa; and an atrophic, smooth and shining dorsal surface of the tongue. There were deep carious lesions in the deciduous mandibular molars and maxillary anterior teeth; as well as mobility of mandibular left canine, which had bone loss. The treatment for oral lesions included diet changes, improved oral hygiene, and extraction of the deciduous teeth destroyed by caries. PMID- 16681245 TI - [Longitudinal decline of forced expiratory volume in one second in non-smoking Japanese women]. AB - We prospectively studied 1,696 clinically healthy non-smoking women to assess the influence of aging on longitudinal decline in forced expiratory volume in one second (FEV10). All the subjects took an annually medical examination involving a pulmonary function test from 1995 to 2001. The mean value of annual decrease in FEV10 (slope) was 19.6 mL/year. Multiple regression analysis revealed that initial age, height, and FEV10 were significant parameters determining the slope. This study clearly showed the aging-related longitudinal decline in pulmonary function in non-smoking Japanese women. PMID- 16681246 TI - [Effectiveness of pneumococcal polysaccharide vaccine in older patients with chronic respiratory diseases]. AB - To evaluate effectiveness of pneumococcal polysaccharide vaccine, we recommended 1378 outpatients aged over 60 with chronic respiratory diseases to be vaccinated from August to October 2002, and 647 patients were vaccinated from August to November 2002. In the 1229 patients without respiratory failure, the incidence of antimicrobial treatment for bacterial respiratory infections in 547 vaccinated patients significantly decreased from 7.9% in the 2001/02 winter season to 5.7% in the 2002/03 winter season, although that in the 682 unvaccinated patients increased from 3.8% to 5.7%. The incidence of antimicrobial treatment for bacterial respiratory infections in 229 vaccinated patients with pneumococcal and influenza vaccines together significantly decreased from 10.5% in the 2001/02 winter season to 5.2% in 2002/03 winter season although that in 110 subjects vaccinated with influenza vaccine only increased from 2.7% to 7.2%. These findings suggest the effectiveness of the pneumococcal polysaccharide vaccine for the prevention of bacterial respiratory infections and the additive effectiveness of pneumococcal and influenza vaccines together. PMID- 16681247 TI - [A questionnaire report concerning the actual situation of respiratory physicians in Japan]. AB - A nation-wide questionnaire was carried out at the end of 2004 to elucidate the actual situation of respiratory physicians in Japan. The questionnaires were characterized by simultaneously looking into the actual situation in the departments of respiratory medicine, cardiology and gastroenterology. The number of the surveyed institution was 168, from which questionnaire replies were obtained from 79 general hospitals, 39 university hospitals and 10 hospitals mainly for tuberculosis. In general hospitals, compared to the departments of cardiology and gastroenterology, the full-time physician staff in the department of respiratory medicine per 10 inpatient beds was only 60.9% and 79.4%, while specialist staff were 54.9% and 68.4%, respectively. The numbers of deaths in the department of respiratory medicine per 10 inpatient beds were 186.9% and 132.6%, compared with in the departments of cardiology and gastroenterology, while those per full-time physicians were 271.9% and 148.5%, respectively. Thus, it was concluded that there was an obvious shortage in number of not only in respiratory specialists but also of all respiratory physicians. Furthermore, respiratory physicians were taking care of more severe patients than other colleagues. PMID- 16681248 TI - [A case of primary fibrosarcoma of the pulmonary artery]. AB - A 38-year-old-man was admitted to our hospital complaining of exertional dyspnea. Chest radiography and CT showed a huge mass that completely occluded the left main pulmonary artery and spread into the right main pulmonary artery. The tumor was diagnosed as sarcoma by transbronchial biopsy. Radiation therapy obtained temporary partial response. He died of respiratory insufficiency after 14 months. The autopsy revealed primary pulmonary artery sarcoma: fibrosarcoma arising from the left pulmonary artery. To our knowledge, this is the first case report of fibrosarcoma of the pulmonary artery in Japan. PMID- 16681249 TI - [A case of large cell carcinoma of the lung with rhabdoid phenotype]. AB - A 37-year-old man was admitted to our hospital because of suspicion of the lung cancer in November 2003. Transbronchial tumor biopsy revealed a small number of tumor cells with rhabdoid features, which had eosinophilic cytoplasmic globules. However, a definitive histological diagnosis was not obtained. We considered that a diagnosis of lung malignant tumor was likely according to the findings of chest CT scan and pathology. Although radiotherapy reduced the tumor size, he started to have abdominal pain and tarry stool one month after radio therapy. Multiple small intestine metastases were detected by gastroenterological endoscopy. The patient died due to bleeding from these metastatic lesions in May 2004. Immunohistologic staining of the cervical lymph node showed that rhabdoid cells were positive for epithelial membrane antigen (EMA), vimentin, and anticytokeratin antibody (CAM5.2), but not for thyroid transcription factor-1 (TTF-1). From the autopsy findings and clinical course, he was finally diagnosed with large cell carcinoma of the lung with rhabdoid phenotype. Because of its aggressive clinical course, early diagnosis and decision on therapy would be very important for this disease. PMID- 16681250 TI - [Pleurodesis using autologous blood plus OK-432 for intractable spontaneous pneumothorax with high surgical risk]. AB - Air leakage persisted from the lung in three cases of spontaneous pneumothorax. Pleurodesis with autologous blood ended in failure. Autologous blood plus OK-432 was instilled into the thoracic cavity from the chest drainage tube. Air leakage was stopped without serious side effects and the chest tube was removed uneventfully. Although the long-term outcome of this treatment is not known, pleurodesis using autologous blood plus OK-432 may be an effective way of treatment for spontaneous pneumothorax in cases with high surgical risk. PMID- 16681251 TI - [A case of hepatitis and pneumonitis caused by Bofutsusyo-san herbal medicine]. AB - We report a case of drug-induced pneumonitis and hepatitis associated with the herbal medication Bofutsusyo-san. A 41-year-old woman was admitted to our hospital with pneumonitis and hepatitis. She complained of fever and dyspnea. Fine crackles were heard on chest auscultation and a chest X-ray film revealed diffuse reticulo-nodular shadow in both lung fields. Cessation of the medication improved the patient' s clinical and X-ray findings. Bronchoalveolar lavage showed an increase of the lymphocytes and a diminished CD4/CD8 ratio. A drug induced lymphocyte stimulation test was positive. Challenge tests for Bofutsusyo san were positive. A diagnosis of drug-induced pneumonitis and hepatitis was made. Our findings suggested an association with Ou-gon. PMID- 16681252 TI - [A case of multiple pulmonary arteriovenous fistulae associated with hereditary hemorrhagic telangiectasia that deteriorated during pregnancy]. AB - A 29-year-old woman was admitted to our hospital because of persistent breathlessness on exertion after the delivery of her second child. Although at the age of 26 she had been given a diagnosis of multiple pulmonary arteriovenous fistula (PAVF), treatment was not done because of the absence of symptoms. An intrauterine growth retardation (IUGR) due to hypoxemia occurred during the second pregnancy, and then she delivered a 1,283g baby by cesarean section in the 36th gestation week. Her symptoms and hypoxemia improved after transcatheter embolization. Hereditary hemorrhagic telangiectasia was diagnosed on the basis of recurrent epistaxis since her childhood, pulmonary and hepatic vascular abnormality (PAVF and hepatic arterioportal shunt) and telangiectasis of the buccal mucosa and tongue. We should consider an early treatment of PAVF for young female because pregnancy may induce increase of arteriovenous shunt, fatal hemoptysis, infertility, miscarriage and IUGR. PMID- 16681253 TI - [A case of sarcoidosis with diabetes insipidus]. AB - A 23-year-old man was admitted because of polydipsia and polyuria. As chest radiographs and computed tomography showed mediastinal and bilateral hilary lymph node swelling, and diffuse small nodules in bilateral lung fields, we suspected sarcoidosis. Sarcoidosis was diagnosed by biopsy of a cervical lymph node, and central diabetes insipidus was diagnosed by fluid restriction test and vasopressin load test. A pituitary mass was also revealed by magnetic resonance imaging. Prednisolone therapy improved all of his clinical findings except diabetes insipidus. He had to continue intranasal 1-desamino-8-D-arginine vasopressin (DDAVP) therapy. PMID- 16681254 TI - [Three cases of spontaneous pneumomediastinum]. AB - We encountered 3 male patients with spontaneous pneumomediastinum. The patients were a 16-year old and a 17-year old and a 24-year old. Predisposing episodes for the development of spontaneous pneumomediastinum could be identified in all 3 patients: throwing a ball during a baseball game in 1, lifting a heavy load during work in 2. However, they were healthy and suddenly developed symptoms in the absence of any underlying disease. The presenting complaint was chest pain in all 3 patients. Chest X-ray films and chest CT images revealed pneumomediastinum. A diagnosis of spontaneous pneumomediastinum was made based on chest X-ray films and chest CT images. After conservative treatment, all 3 patients recovered. PMID- 16681255 TI - [A case of Swyer-James syndrome with interesting chest radiographs]. AB - A 28-year-old man was admitted for further examination after overinflation of the right lung was observed on a screening chest radiograph. The chest radiograph on admission showed increased radiolucency of the left lung and overinflation of the right lung, associated with a shift of the mediastinum toward the midline on expiration. High-resolution CT on expiration showed air trapping in the left lung and in part of the right lower lobe. Pulmonary perfusion scintigraphy showed a markedly decreased perfusion in these areas. Pulmonary angiography revealed small left pulmonary arteries with diminished peripheral vascular branches. Bronchoscopy showed no abnormality of either bronchus. Swyer-James syndrome was diagnosed on the basis of these findings. Although bronchial damage due to the lower respiratory tract infection during childhood is considered a very important factor in the pathogenesis of this syndrome, many patients have no history of airway infection, as in this case. This is a rare case of Swyer-James syndrome presenting with marked overinflation of the unaffected lung in an asymptomatic adult. Progression of this compensative overinflation probably involved asymptomatic damage caused by the weakness of the affected lung. PMID- 16681256 TI - [CyberKnife robotic radiosurgery]. PMID- 16681257 TI - [Pathophysiology and therapeutic approaches on Binswanger's disease]. PMID- 16681258 TI - [New evidence in the field of white matter changes]. PMID- 16681259 TI - [Decompressive craniectomy for ischemic stroke]. AB - We analysed retrospectively 15 consecutive patients with cerebral infarction undergoing decompressive craniectomy. Ninety-three percent of patients survived, and 53% of them were partially dependent (Barthel Index > 0). We defined the partially dependent patients as good outcome group, and totally dependent as poor outcome group. In good outcome group, compared with poor outcome group, there are more frequent left hemispheric lesion (50%: 0%, p = 0.029), and pre-operative JCS < or = II-30 (62.5%: 14.3%, p = 0.057). Although many patients were severely disabled, 79% of the patients and their family answered that having operation was correct choice. All the patients in good outcome group acquired the ability of oral feeding and communication skill. 87.5% of the patient in good outcome group was satisfied with the outcome. Based on these results, we emphasized that comprehensive evaluations, including satisfaction scale and QOL assessment necessary to decide the indication of decompressive craniectomy for ischemic stroke. PMID- 16681260 TI - [The research of the pathogen responsible for Pleurocybella porrigens related encephalopathy: investigation of 3-nitropropionic acid hypothesis]. AB - Pleurocybella porrigens related encephalopathy exhibits consciousness disturbance and convulsion in the patients after taking and patients show bilateral basal ganglia lesion resulted in high mortality rate. This encephalopathy is a very similar to the moldy sugarcane encephalopathy epidemic in China in the past. We investigated the relationship between Pleurocybella porrigens related encephalopathy and 3-nitropropionic acid which had caused the moldy sugarcane encephalopathy. We have tried to detect 3-NPA in the various specimens from patients and Pleurocybella porrigens, but failed. Further examinations for elucidating the causation of Pleurocybella porrigens related encephalopathy are needed. PMID- 16681261 TI - [A case of recurrence of cerebral hemorrhage in a patient with adult moyamoya disease in the recovery period rehabilitation ward]. AB - A 61-year-old woman with a history of repeated cerebral hemorrhage due to moyamoya disease was admitted to our recovery period rehabilitation ward for walking training. She suddenly noticed paresthesia in her left forearm and the palm seven days later. Brain computed tomography on that day and magnetic resonance imaging on the next day after the onset of paresthesia revealed right thalamic hemorrhage. It was suggested that the hematoma localized to a small part of the central portion of the ventroposterior lateral nucleus caused paresthesia limited to the forearm and the palm. In a recovery period rehabilitation ward, we should carefully listen to the complaints of patients who are in advanced age and have a risk like our patient. Then adequate neurological examination should be performed for the pertinent inspection diagnosis, if needed. PMID- 16681262 TI - [A case of acute bromvalerylurea intoxication: clinical course and alteration in serum bromvalerylurea concentration]. AB - Bromvalerylurea is one of the non-barbiturates products and has been used as analgesics and hypnotics in Japan. A 20-year-old woman was admitted to our hospital for loss of consciousness. She had a 6-month history of transient delirium and drunken gait. Physical examination revealed erythema less than thumb's head size at her face, shoulder and thigh. Neurologically, she had a state of coma and low muscle tonus. EEG showed the pattern of burst-suppression. The level of her serum chloride was not elevated. The erythema made us check up her state of acute bromvalerylurea intoxication. High blood concentration of bromvalerylurea led to diagnosis of the bromvalerylurea intoxication. The maximum value of her serum bromvalerylurea concentration was 107 microg/ml on the second hospital day, while the concentration in cerebrospinal fluid were also increased and remained for several days. She was treated with respiration control and drip infusions. She gradually improved and recovered to be alert. She was complicated severe liver dysfunction and disseminated intravascular coagulation resulting from bromvalerylurea intoxication, also treated with intensive care and gradually recovered. We should take notice to bromvalerylurea, easily available over the counter, as one of the drugs which may cause severe loss of consciousness or coma, and general complications. And if the bromvalerylurea intoxication is prospective, we should consider whether the option of gastric irrigation is available regardless of the elapsed time. PMID- 16681263 TI - [Adult bacterial meningitis complicated by cerebral infarction: report of three cases]. AB - Fatality rates and the number of patients suffering from the after sequelae of bacterial meningitis have still remained high despite the development of new antibiotics. Cerebrovascular complications have been reported less frequently in adult cases than in child cases. We experienced 3 consecutive cases of bacterial meningitis in adults complicated by brain infarction. Primary causative organism were the Group B Streptococci in one case and the Streptococcus pneumoniae in other two cases. MRI, especially with FLAIR method and of coronal view, disclosed abnormal intensity areas near the brain surface. Conventional angiography and MR angiography in the two cases showed tapering and stenosis of arteries. These cases suggest that routine use of MRI/ MRA in bacterial meningitis reveal neuro vascular complications more frequently and easily. The benefits of adjunctive steroid therapy in the treatment of bacterial meningitis might be beneficial to suppression of these neurovascular complication cased by inflammatory vasculitis. PMID- 16681265 TI - [A case of meningeal carcinomatosis due to gastric signet-ring cell carcinomatosis which occurred after delivery]. PMID- 16681264 TI - [Kanji-predominant alexia with agraphia in opticospinal multiple sclerosis]. AB - Alexia with agraphia is very rare symptom in multiple sclerosis. We present a patient of opticospinal multiple sclerosis with kanji-predominant alexia with agraphia. A 55-year-old, right-handed man was admitted to our hospital because of difficulty in reading and writing in August 2001. The patient had been diagnosed as having relapsing-remitting opticospinal multiple sclerosis eight years prior to admission. Language examination showed alexia with agraphia predominantly affecting kanji and also mild naming difficulties, but a good comprehension and a normal repetition. T2-weighted MRI demonstrated hyperintensity area in the left temporo-parietal lobe, involving the white matter beneath the postero-inferior temporal lobe and inferior parietal lobule. On brain SPECT, low blood perfusion was observed in the left temporo-parietal regions. Although agraphia for kana and alexia for both kana and kanji improved after steroid therapy, agraphia for kanji did not improve. After the treatment, high intensity area of inferior parietal lobule was disappeared on MRI, and the hypoperfusion of inferior parietal lobule on brain SPECT was also improved, but the lesion of left postero-inferior temporal lobe did not show any remarkable changes. We considered that the kanji predominant alexia with agraphia was due to the lesions of left inferior parietal lobule and postero-inferior temporal lobe, and agraphia for kanji was due to the lesion of left postero-inferior temporal lobe. PMID- 16681266 TI - [Intracerebral intravascular papillary endothelial hyperplasia mimicking a metastatic brain tumor: a case report]. PMID- 16681267 TI - [An autopsied case of a 39-year-old man with an onset of left hemiplegia, followed by various changes of head imaging]. PMID- 16681268 TI - Paying the plumber. PMID- 16681270 TI - Story short on science. PMID- 16681271 TI - Cash is king. PMID- 16681272 TI - Above and beyond. PMID- 16681273 TI - Driven to doubt. PMID- 16681274 TI - Lean machine. PMID- 16681275 TI - Quick-change artists. PMID- 16681276 TI - Harsh words on high costs. PMID- 16681277 TI - Toward a better, simpler system. PMID- 16681278 TI - Physicians spend a day at the Capitol. PMID- 16681279 TI - Recasting the lowly formulary. PMID- 16681280 TI - Million dollar miracles. PMID- 16681281 TI - When ends don't meet. PMID- 16681282 TI - A different path. PMID- 16681283 TI - Paying physicians and protecting the poor. PMID- 16681284 TI - Access to health care: differences between insured and uninsured patients in south Minneapolis. AB - This article reports the results of a survey of 575 patients who were seen at 2 clinics in south Minneapolis. About half had health insurance. We found a strong connection between not having insurance and going without medical care. We also found that when the uninsured do go for care, they often fail to take prescribed medications because of the cost. Our findings also contradicted some prevalent thinking about the uninsured: that they are unemployed, that they refuse employer sponsored insurance, and that they can easily get care at free clinics. PMID- 16681285 TI - Not paid to talk. PMID- 16681286 TI - Getting older, getting better. PMID- 16681287 TI - Intrathecal baclofen therapy: ten steps toward best practice. AB - Practitioners from around the country who have extensive experience in intrathecal baclofen (ITB) therapy gathered in early 2004 to develop best practice guidelines for ITB therapy. Discussion focused on the idea that ITB therapy is a program rather than a procedure. Key recommendations were made in areas including team coordination, patient selection and goals, patient education, patient screening, implant technique, long-term management, individualized dosing options, ongoing evaluation of patient response, appraisal of the integrity of the catheter and infusion system, and appropriate practice resources. PMID- 16681288 TI - A critical pathway for meeting the needs of families of patients with severe traumatic brain injury. AB - Family members of patients recovering from severe traumatic brain injuries have needs that are best met through early assessment and proactive intervention. A critical pathway addressing the needs of families during the acute and postacute phases of hospitalization was developed and implemented in the neuroscience intensive care and step-down units of a university hospital. A team of 10 neurosurgical nurses used and evaluated the program. PMID- 16681289 TI - Influence of prior cognitive impairment on the severity of delirium symptoms among older patients. AB - Delirium is common among hospitalized elderly patients with prior cognitive impairment. Detecting delirium superimposed on dementia is a challenge for nurses and doctors. As a result, delirium among demented elderly patients is of increasing interest to healthcare professionals. So far, studies have failed to describe how symptoms of delirium are altered by severity of dementia. This would be valuable information to improve the rate of detection by nurses of delirium among demented patients. However, until now no research has examined the effect of severity of prior cognitive impairment on the severity of delirium symptoms among institutionalized older patients. This study describes the effect of severity of prior cognitive impairment on the severity of delirium symptoms among institutionalized older patients with delirium at the time of their admission to an acute care hospital. One hundred four institutionalized elderly people were included in this study and screened for delirium using the confusion assessment method. Patients with delirium (n = 71) were evaluated with the delirium index to determine the severity of the symptoms of delirium. The results showed that the severity of prior cognitive impairment influences the severity of most of the symptoms of delirium, particularly disordered attention, orientation, thought organization, and memory. Thus, taking into account the severity of prior cognitive impairment could help nurses to detect delirium among older patients. PMID- 16681290 TI - Serotonin syndrome. AB - Serotonin syndrome is a preventable, drug-related complication that results from increased brainstem serotonin activity, usually precipitated by the use of one or more serotonergic drugs. Its clinical presentation consists of autonomic dysfunction, alteration in mental status, and neuromuscular disorder. Early recognition and treatment is important, because this condition is potentially fatal. Management includes withdrawal of causative agents and supportive measures such as hemodynamic stabilization, sedation, temperature control, hydration, and monitoring for complications. Serotonin antagonists, specifically cyproheptadine, have been used, but the documented benefits are purely anecdotal. PMID- 16681291 TI - Palliative care in Parkinson's disease: implications for neuroscience nursing. AB - Parkinson's disease (PD) is a chronic, progressive neurological disease affecting 1.5 million Americans. The modern success of pharmacology and deep-brain stimulation surgery to treat the motor symptoms of tremor, rigidity, and bradykinesia provide PD patients with longer lives and increased motor functioning. However, in the moderate and advanced stages of disease, the therapeutic benefits of pharmacology diminish and motor symptoms are more complicated to treat. The nonmotor symptoms of PD receive little attention in clinical settings, although they can lead to disability and caregiver burden. The Center to Advance Palliative Care advocates applying the principles of palliative care to chronic disease. Likewise, the World Health Organization has redefined palliative care to include life-threatening illness. The Parkinson's Disease Model of Care (PDMC) takes the precepts of palliative care and presents a model for the neuroscience nurse to use in individual care planning across the trajectory of disease. The PDMC guides the nurse in providing relief from suffering for PD patients and their families, from diagnosis through bereavement, with an emphasis on advance care planning. PMID- 16681292 TI - Conversion disorder in a pediatric transgender patient. AB - Somatoform disorders are difficult to diagnosis and often present as a neurological illness in pediatric populations. Conversion disorder is the somatoform disorder most commonly seen in children, particularly adolescents, who have anxiety related to sexual behaviors and orientation. In a transgender patient, the risk of conversion disorder is even higher. The patient described in this article presented with multiple neurological symptoms that disappeared after she began presenting herself as a male. There is a significant need for research into somatoform disorders as well as research into the transgender population. PMID- 16681293 TI - Practical considerations in administering intravenous medications. PMID- 16681294 TI - Understanding mentoring relationships. AB - Nursing embodies a universal mission to care--to comfort, heal, and be hopeful toward those entrusted to our charge through acts of compassion. Responding to this call includes the necessity to hold our nurse colleagues in high esteem. Paradoxically, the latter remains a matter of debate. Vance (1982) believes that nurses, as members of a helping profession, trained in nurturing and being aware of the benefits of working together can be able mentors. Vance (1982, 2000) further reflects that a willingness to honor and support colleagues through strong mentor connections can forge both individual and collective power in our professional endeavors. Implicit in the essence of mentoring is the resonating phenomenon, also known as the ripple effect (Stewart and Krueger, 1996; Yoder, 1990). Experiencing the process firsthand, proteges are likely to pave the way, and, in turn, their proteges will follow and leave their own imprints or legacies. Mentors set a precedent for passing along all good works. They want to share that which is heartfelt, enriching career life by embracing optimism and unity through lifelong loyalty to organized nursing and, ultimately, the profession at large. PMID- 16681295 TI - Organizational climate: implications for the home healthcare workforce. AB - Organizational climate generally refers to staff members' perceptions of organizational features like decision making, leadership, and norms. It is widely acknowledged that these perceptions influence patient, employee, and system outcomes. However, there has been little consensus on how best to measure these important relationships. This article examines how organizational climate has been defined and measured in health services research and identifies factors most important in home healthcare delivery. Standardization of climate measures will facilitate the ultimate goal of translating results into evidence-based management practices to improve the quality of care delivery. PMID- 16681296 TI - Knowledge transfer and utilization: implications for home healthcare pain management. AB - Implementation of research evidence into clinical practice is a complex and dynamic process that has become the subject of investigation in the field of "translation science" or "knowledge utilization." Research shows how individuals, units, and organizations all influence the rate and extent of adoption of research evidence. Environmental factors also play an important role in this process. This article summarizes key lessons from translation science and examines the implications for the organization and delivery of home healthcare. The implementation of pain management guidelines is used as an example. PMID- 16681297 TI - The science of improving function: implications for home healthcare. AB - Since implementation of the Outcome and Assessment Information Set and publicly reported outcome indicators, a focus of home healthcare necessarily includes efforts to improve older patients' functional abilities. This article presents results from selected home-based research studies providing strategies for evidence-based practice to efficiently and effectively improve functional outcomes. Assessments and interventions are suggested, as well as administrative and clinical processes of care that have been found to successfully translate research into practice. Results suggest that if systems of care are redesigned, the payoff in patient and staff satisfaction and in improved functional and economic outcomes can be substantial. PMID- 16681298 TI - Exploring the utility of automated drug alerts in home healthcare. AB - Computerized drug utilization review (DUR) can potentially reduce adverse drug events. We examined automated DUR for home healthcare patients with diabetes or hypertension. Sixty-eight percent of diabetes patients and 50.7% of hypertension patients triggered severe, moderate, or duplicative alerts. Among diabetes patients, 74.3% of duplicative alerts were trivial or inappropriate, compared with 3.9% among hypertension patients. Experts judged that 40.5% of high-risk diabetes patients and 53.6% of hypertension patients had alerts requiring nurse follow-up. Adequate follow-up was significantly lower for the former. The relationship between inappropriate alerts and poorer follow-up reinforces the need for more specific alert systems to focus clinicians' attention on clinically important alerts. PMID- 16681299 TI - Effective pain management: lessons from a nursing home research study. AB - Persistent pain affects at least half of all older adults. The negative consequences of persistent pain are numerous and include depression, anxiety, and lower overall quality of life: However, pain is often underrecognized, underreported, underassessed, and undertreated, and multiple barriers to optimal pain practices exist. Although a solid evidence base exists for optimal pain practices, little progress has been made. This article reviews a study that developed and implemented a multifaceted intervention to improve pain practices in nursing homes and then examines the implications of those study findings for the home healthcare setting. PMID- 16681301 TI - Unwarranted caution from the EU will affect MRI use and patient care. PMID- 16681300 TI - Transitional care: a critical dimension of the home healthcare quality agenda. AB - Focusing on the critical transitions of patients and their caregivers across healthcare settings and among providers is a promising approach to enhancing care coordination and improving quality. This article describes the research base for the transitional care of older adults and offers recommendations to advance the science, translate best practices into home healthcare settings, and improve the transitions of high-risk older adults to and from home healthcare. Home healthcare is a component of the healthcare industry uniquely positioned to improve transitional care and outcomes for the growing population of older adults with continuous complex needs. PMID- 16681302 TI - Failing older hearts? Of myths and left-overs. PMID- 16681303 TI - Cutaneous T-cell lymphoma. PMID- 16681304 TI - Biological therapy for psoriasis. PMID- 16681305 TI - Epidermolysis bullosa. PMID- 16681306 TI - Chronic pelvic pain: the enigma of gynaecological practice. PMID- 16681307 TI - ST elevation and atypical chest pain. PMID- 16681308 TI - Foundation programme assessment: the trainee's guide. PMID- 16681309 TI - Blood transfusion: a practical guide. PMID- 16681310 TI - Interpretation of cervical spine radiographs. PMID- 16681311 TI - Femoral shaft fracture. PMID- 16681312 TI - Recognizing and managing severe community-acquired pneumonia. PMID- 16681313 TI - Pathogen recognition by human lung epithelial cells. PMID- 16681314 TI - So you want to be ... an ophthalmologist. PMID- 16681315 TI - Preimplantation genetic diagnosis. PMID- 16681316 TI - Recent advances in adult cardiac surgery. PMID- 16681317 TI - Consultation skills training for hospital doctors: an evaluation of a pilot workshop for ophthalmology. PMID- 16681318 TI - Chromobacterium violaceum infection. PMID- 16681319 TI - Barium appendicitis. PMID- 16681320 TI - Unilateral ocular myasthenia gravis in an acute medical take: is a Tensilon test useful? PMID- 16681321 TI - Tuberculosis of the prostate. PMID- 16681322 TI - Laryngeal tuberculosis: not the usual suspect. PMID- 16681324 TI - Assessment of perioperative fluid balance. PMID- 16681325 TI - Reduce morbidity by balancing your fluids. PMID- 16681326 TI - Estate planning. PMID- 16681327 TI - Should I wheel out the heliox with non-invasive ventilation? PMID- 16681328 TI - Tracing 8,600 participants 36 years after recruitment at age seven for the Tasmanian Asthma Study. AB - OBJECTIVE: To trace all participants 36 years after the original Tasmanian Asthma Study (TAS). METHODS: In 1968, the TAS investigated asthma in 8,583 children who were born in 1961. We attempted to trace these participants in 2002-04 using names, dates of birth and gender. Current addresses were sought by computer linkage to the Commonwealth Electoral Roll, the Medicare database and the Tasmanian marriage records. Computer linkage was conducted with the National Death Index (NDI). Siblings of participants were also linked to the Commonwealth Electoral Roll and those identified were sent a letter requesting the participant's address. The Australian Twin Registry (ATR) and the 1991-93 TAS substudy were used to locate participant addresses. RESULTS: After three rounds of electoral roll linkage, 56% of all cohort members were traced. Name changes were identified for 49% of the 3,477 females not initially matched to the electoral roll using linkage to marriage records. NDI linkage yielded a 0.7% match. Medicare linkage identified addresses for 27% of the 1,982 remaining participants. Writing to siblings located 60% of 1,661 participants. One hundred and eighty-three participants were matched to the 1991-93 TAS and 23 twins matched to the ATR. Overall, 81.5% of the cohort members were identified. CONCLUSIONS: With these methods, we have been able to trace a possible address for a large portion of the original participants, with the electoral roll linkage being the most useful. IMPLICATIONS: It is possible to trace Australians for follow-up studies using electronic linkage, although without unique identifiers it is labour and resource intensive and requires matching to several databases. PMID- 16681329 TI - Recruitment to mammography screening: a randomised trial and meta-analysis of invitation letters and telephone calls. AB - OBJECTIVES: To determine the effectiveness of three recruitment strategies to encourage women to attend for an initial mammography screen, and to compare results with similar service studies. Interventions were: (1) an invitation letter; (2) two invitation letters; and (3) an invitation letter plus a follow-up telephone call. METHODS: All women aged 50-54 years in two BreastScreen New South Wales (BSNSW) Screening and Assessment Service catchment areas (n=3,144) were recruited from the Australian Electoral Roll and randomised to the four groups. Response rates for each intervention were compared relative to standard practice (one invitation letter) at 12-weeks follow-up. Marginal cost-effectiveness for each condition was calculated. Other similar randomised trials were also meta analysed. RESULTS: The screening rate for two letters was 8.5% (OR=1.61, 95% CI 1.08-2.40) and 7.8% (OR=1.46, 95% CI 0.97-2.18) for one letter plus a telephone call, compared with 5.5% for standard practice (one letter) (OR=1.00). The response rate in the one letter plus a phone call group was 13.3% (OR=2.65, 95% CI 1.76-4.00) for women where a phone number was located. CONCLUSION: Initial screening rates after a 12-week follow-up were significantly higher in the women receiving a second invitation letter, compared with standard practice (one letter). Marginal cost-effectiveness favoured the two-letter approach. IMPLICATIONS: A follow-up invitation letter is more cost-effective than one invitation letter plus a follow-up telephone call in the BSNSW program. However, an invitation letter plus follow-up phone call is more cost-effective in recruiting women to BSNSW only if a phone number is located. PMID- 16681330 TI - Mental health expenditure in Australia: time for affirmative action. AB - In Australia, there is a clear relationship (r=-0.56) between the ratio of health expenditure to burden and the ratio of mortality to disability across illness groups. Those illnesses with high mortality relative to disability (e.g. cancer and cardiovascular) are associated with lower health expenditure, relative to the burden they impose. Conversely, illnesses with low mortality but high disability (e.g. musculoskeletal disorders) demonstrate higher expenditure. The notable exception is mental health, which has low mortality, high disability and very low health expenditure. Indeed, the relationship between the mortality/disability and health expenditure/burden ratios is stronger (r=-0.72) when mental health is excluded. As this disparity cannot be explained by a lack of cost-effective treatments for mental disorders, we propose a process of affirmative action for mental health. Urgent investments in cost-effective modes of care are required and these should prioritise increased access among those subpopulations disadvantaged by other social, economic or geographic factors. PMID- 16681331 TI - ECHO: the Western Australian Emergency Care Hospitalisation and Outcome linked data project. AB - OBJECTIVE: To describe and assess the quality of the data resources linked for the Western Australian Emergency Care Hospitalisation and Outcome (ECHO) project. METHODS: The ECHO project links electronic records from the WA Emergency Department Information System to the St John Ambulance Service Pre-Hospital Care Database, the WA Hospital Morbidity Data System and the WA Mortality Database. Linkages are created using standard probabilistic matching techniques with extensive clerical review. Commencing with all metropolitan Perth public emergency departments from July 2000, these linkages will be updated annually for at least five successive years. The proportion of actual linkages between emergency department records and ambulance, admission and death records was assessed in comparison to expected linkage rates. RESULTS: Of 578,200 total emergency department records, there were 144,897 emergency presentations recorded as arriving by ambulance, of which 135,332 (93.4%) were linked to an ambulance record pertaining to the same episode. Of the 165,650 presentations recorded as admitted, 162,216 (97.9%) were linked to a hospital morbidity record relating to the same episode. Furthermore, 96.2% of the 2,084 cases recorded as 'dead on arrival' and 98.9% of the 624 cases recorded as 'died in emergency' were linked to a corresponding death record. CONCLUSIONS: Linkage quality consistent with international standards has been achieved, resulting in an information infrastructure capable of supporting an extensive research agenda focusing on the interaction and outcomes of both pre-hospital and within-hospital emergency medical care services. PMID- 16681332 TI - Test agreement for classifying diabetes in indigenous Australians. AB - OBJECTIVE: To evaluate for Indigenous Australians the agreement between a fasting plasma glucose (FPG) criterion of 7.0 mmol/L and diabetes test results using a two-hour oral glucose tolerance test (OGTT), comparing relationships between test agreement and prevalence to similar studies. METHODS: Screening was undertaken in 25 remote settlements. Agreement between FPG and OGTT results was evaluated using the kappa coefficient (chance-corrected agreement). RESULTS: Participants (n=3,249) ranged from 15-94 years. Kappa ranged from 0.70-0.77 for diabetes prevalence of 5-30%, with overall agreement of 0.76. In comparison studies, the relationship between kappa and diabetes prevalence was similar, but with kappa 0.21-0.48. CONCLUSIONS AND IMPLICATIONS: A FPG test with a 7.0 mmol/L cut-off is a reliable screening method, relative to the OGTT, for remote Indigenous settlements. The level of agreement between the FPG test and the OGTT for the Indigenous population surveyed is superior by far to agreement reported for non Indigenous samples. PMID- 16681333 TI - Evaluation of a multi-component community tobacco intervention in three remote Australian Aboriginal communities. AB - OBJECTIVES: To assess the effect of community tobacco interventions in Aboriginal communities. METHODS: The study consisted of a pre- and post-study of the effect of a multi-component tobacco intervention conducted in six Aboriginal communities in the Northern Territory (NT). The intervention included sports sponsorship, health promotion campaigns, training health professionals in the delivery of smoking cessation advice, school education about tobacco, and policy on smoke free public places. The study was conducted in three intervention communities and three matched control communities. Surveys were used to measure changes in prevalence of tobacco use, changes in knowledge, and attitudes to cessation in intervention communities. RESULTS: Tobacco consumption decreased in one intervention community compared with the matched control community; the trends of consumption (as measured by tobacco ordered through points of sale) in these communities were significantly different (t = -4.5, 95% CI -33.6 - (-12.5), p < or = 0.01). Community samples in intervention communities included 920 participants. There was no significant change in the prevalence of tobacco use, although knowledge of the health effects of tobacco and readiness to quit increased. CONCLUSIONS: Although it is difficult to demonstrate a reduction in tobacco consumption or in the prevalence of tobacco use as a result of multi component community tobacco interventions delivered in Aboriginal communities, such interventions can increase awareness of the health effects of tobacco and increase reported readiness to cease tobacco use. PMID- 16681334 TI - Screenieboppers and extreme screenies: the place of screen time in the time budgets of 10-13 year-old Australian children. AB - OBJECTIVES: Excessive 'screen time' has been associated with a range of psychosocial disturbances and increasing pediatric obesity. This study describes the magnitude, distribution, composition and time-distribution of children's screen use; examines correlates of screen use; and characterises 'extreme' screen users (top quartile). METHODS: 1,039 South Australian children aged 10-13 years old completed a multimedia 24-hour activity recall diary on 2-4 occasions in 2002, including at least one school day and one non-school day. RESULTS: The median screen time was 229 minutes.d(-1). This was higher in boys (264 vs. 196 minutes; p<0.001) and on non-school days (260 vs. 190 minutes; p<0.001), increased with age (p=0.003), and decreased with socio-economic status (SES; p=0.003). Television consumed 73% of all screen time, video games 19%, non-game computer use 6%, and cinema 2%. The top quartile of screen users were more likely to be boys (OR=3.8), have low physical activity (OR=4.3), spend >25% of screen time playing video games (OR=1.8), sleep less, and be of lower SES. CONCLUSIONS AND IMPLICATIONS: Interventions to reduce screen time should target inactive, low SES boys, encourage earlier bedtimes, and limit video game use. PMID- 16681335 TI - In-patient hospital use in the last years of life: a Western Australian population-based study. AB - OBJECTIVES: To estimate the likelihood and costs of in-patient care in the last three years of life. METHODS: A population-based retrospective cohort study using linked hospital and death records to evaluate in-patient use by Western Australians who died in 2002. RESULTS: Age was unrelated to the likelihood of in patient admission and inversely related to in-patient costs, after adjustment for sex, cause of death and proximity to death. In-patient costs increased in the final three quarters before death. In the last quarter before death, the predicted average quarterly in-patient cost increased 2.8 fold from quarter two and 3.8 fold from quarter three. CONCLUSIONS: Older decedents were not more likely to be hospitalised than younger decedents in the final three years of life. Moreover, once hospitalised, their in-patient costs were lower. In-patient costs were heavily concentrated in the three last quarters of life. IMPLICATIONS: Remaining lifetime is a significant predictor of in-patient costs. Failure to account for proximity to death will overemphasise the impact of population ageing on health care expenditure, because older people have a higher probability of dying. PMID- 16681336 TI - Community senior first aid training in Western Australia: its extent and effect on knowledge and skills. AB - OBJECTIVE: To define the extent of Senior First Aid training in a sample of the Western Australian community, and to evaluate the effect of previous training on first aid knowledge and skills. METHODS: A telephone survey of a random sample from suburban Perth and rural Western Australia; and practical assessment of first aid skills in a subsample of those surveyed. RESULTS: 30.4% of respondents had completed a Senior First Aid certificate. Trained individuals performed consistently better in theoretical tests (p=0.0001) and practical management of snakebite (p=0.021) than untrained. However, many volunteers, both trained and untrained, demonstrated poor skills in applying pressure immobilisation bandaging and splinting the limb adequately despite electing to do so in theory. CONCLUSIONS AND IMPLICATIONS: Overall knowledge and performance of first aid skills by the community are poor, but are improved by first aid training courses. PMID- 16681337 TI - Trends in postpartum haemorrhage. AB - OBJECTIVE: To assess trends and outcomes of postpartum haemorrhage (PPH) in New South Wales (NSW). METHODS: A population-based descriptive study of all 52,151 women who had a PPH either during the hospital stay for the birth of their baby or requiring a re-admission to hospital between 1994 and 2002. Data were obtained from the de-identified computerised census of NSW hospital in-patients and analysed to examine trends over time. The outcome measures included maternal death, hysterectomy, admission to intensive care unit (ICU), transfusion and major maternal morbidity, including procedures to reduce blood supply to the uterus, acute renal failure and postpartum coagulation defects. RESULTS: From 1994 to 2002 both the number and adjusted (for under-reporting) rate of PPH during the birth admission increased from 8.3% of deliveries to 10.7%. The rate of PPH adjusted for maternal age and mode of delivery was similar to the unadjusted rate. There was a sixfold increase in the rate of transfusions from 1.9% of women who haemorrhaged to 11.7%. Hospital readmissions for PPH declined from 1.2% of deliveries to 0.9%. These were statistically significant changes. There were no significant changes in the rate of hysterectomies, procedures to reduce blood supply to the uterus, admissions to ICU, acute renal failure or coagulation defects. CONCLUSION: The increased rate of PPH during the birth admission is concerning. The increase in PPH could not be explained by increasing maternal age or caesarean sections. Linked birth and hospital discharge data could determine whether the increase in PPH is caused by other changes in obstetric practices or PMID- 16681338 TI - Demographic, migration status, and work-related changes in Asian female sex workers surveyed in Sydney, 1993 and 2003. AB - OBJECTIVE: To compare demography, sexual health awareness, migration and workplace conditions of Asian female sex workers in Sydney in 1993 and 2003. METHOD: A Chinese interpreter and a Thai-speaking health education officer (HEO) were used to administer a questionnaire survey to Thai- and Chinese-speaking sex workers attending sexual health clinics in 1993. A follow-up survey, which included some women contacted at work as well as clinic attenders, was administered by Thai- and Chinese-speaking HEOs in 2003. RESULTS: Ninety-one female sex workers were surveyed in 1993 and 165 in 2003. Median age increased (26 years vs. 33 years, p=0.000), as did numbers of Chinese- versus Thai-speakers (1993, 25.3% Chinese vs. 2003, 58.2% Chinese, p=0.000). In 2003, the women reported more years of schooling and better English skills. Fewer reported previous sex work (48.4% vs. 17.6%, p=0.000). Numbers currently or ever on a contract decreased sharply (27.5% vs. 9.1%, p=0.000) and the majority were apparently working legally. Condom use at work for vaginal (51.6% vs. 84.8%) and oral sex (39.6% vs. 66.1%) increased significantly (p=0.001). Chinese-speaking sex workers were less informed about HIV transmission and safer sex practices than were Thai sex workers. Drug and alcohol use was low. CONCLUSIONS AND IMPLICATIONS: Positive changes have occurred in the conditions of Asian female sex workers surveyed over 10 years in Sydney. Maintaining current levels of health service delivery will ensure continued improvements in health and workplace conditions and address inequalities between language groups. PMID- 16681339 TI - Risk factors for preterm, low birth weight and small for gestational age birth in urban Aboriginal and Torres Strait Islander women in Townsville. AB - OBJECTIVES: To assess the characteristics of Indigenous births and to examine the risk factors for preterm (<37 weeks), low birth weight (<2,500 g) and small for gestational age (SGA) births in a remote urban setting. DESIGN: Prospective cohort of singleton births to women attending Townsville Aboriginal and Islander Health Services (TAIHS) for shared antenatal care between 1 January 2000 and 31 December 2003. MAIN OUTCOME MEASURES: Demographic, obstetric, and antenatal care characteristics are described. Risk factors for preterm birth, low birth weight and SGA births are assessed. RESULTS: The mean age of the mothers was 25.0 years (95% CI 24.5-25.5), 15.8% reported hazardous or harmful alcohol use, 15.1% domestic violence, 30% had an inter-pregnancy interval of less than 12 months and 9.2% an unwanted pregnancy. The prevalence of infection was 50.2%. Predictors of preterm birth were a previous preterm birth, low body mass index (BMI) and inadequate antenatal care, with the subgroup at greatest risk of preterm birth being women with a previous preterm birth and infection in the current pregnancy. Predictors of a low birth weight birth were a previous stillbirth, low BMI and an interaction of urine infection and non-Townsville residence; predictors of an SGA birth were tobacco use, pregnancy-induced hypertension and interaction of urine infection and harmful alcohol use. CONCLUSION: The prevalence of demographic and clinical risk factors is high in this group of urban Indigenous women. Strategies addressing potentially modifiable risk factors should be an important focus of antenatal care delivery to Indigenous women and may represent an opportunity to improve perinatal outcome in Indigenous communities in Australia. PMID- 16681340 TI - Cervical smear participation and prevalence of sexually transmitted infections in women attending a community-controlled Indigenous health service in north Queensland. AB - OBJECTIVE: To increase Pap smear participation and to assess the prevalence of sexually transmitted infection (STI) in urban Indigenous women in Townsville. DESIGN: Convenience sample of women attending Townsville Aboriginal and Islanders Health Services (TAIHS) for health care participating in the Trichomonas vaginalis (TV) project, coordinated by an Aboriginal Health Worker (AHW) trained in women's health and cervical screening. PARTICIPANTS: 198 women, aged 20-69 years of age, attending TAIHS between March 2002 and 31 January 2004. MAIN OUTCOME MEASURES: Pap smear participation and prevalence of STIs. RESULTS: Pap smear participation among eligible women at TAIHS increased from 20.9% pre TV project to 28.6% during the project and 35.6% in the 12 months post completion of the TV project (p<0.0001), an increase of 70.3%. Of 196 smears performed during the study, 20 (10.6%, 95% CI 6.2-15) were abnormal; abnormality was more common in non-Indigenous 8/50 (16.0%) than in Indigenous women 12/139 (8.6%), but did not reach significance (p=0.180). The prevalence of Chlamydia trachomatis was low, with no differences between the Indigenous and non-Indigenous groups, and highest in younger women: 6/44 (13.6%) in women less than 25 years; Trichomonas vaginalis was more common in the Indigenous group. CONCLUSION: An AHW trained to perform and advocating for Pap smear participation has significantly increased the smear participation at TAIHS. The overall prevalence of STI was low. PMID- 16681341 TI - Improving access to medicines among clients of remote area Aboriginal and Torres Strait Islander Health Services. AB - Despite unequivocally worse health, expenditure on Indigenous people through the Pharmaceutical Benefits Scheme (PBS) is considerably less than for other Australians. We report on the effectiveness of a program to supply PBS medicines to remote Aboriginal and Torres Strait Islander Health Services (ATSIHSs) under section 100 (s. 100) of the National Health Act 1953. THE PROGRAM: Under the special PBS arrangements (SPBSAs), approved ATSIHSs are able to order PBS medicine in bulk through local pharmacies and supply them as needed to patients on-site. The usual co-payment associated with PBS medicine is not charged and the pharmacist remuneration structure is different. METHODS: The project involved consultation with the evaluation reference group and other stakeholders at all stages. There were six main data collection components: public submissions; interviews with government and other key stakeholders; pharmacist survey; medicine utilisation and expenditure data; national ATSIHS minimum dataset; and case studies of ATSIHSs. RESULTS: These SPBSA potentially benefit 36% of the Aboriginal and Torres Strait Islander population. They have resulted in improved access to much-needed medicines, representing an increase of dollar 36.5 million in expenditure on Aboriginal and Torres Strait Islander people through the PBS between 2000/01 and 2002/03. They have further ensured that dollar 8.3 million of State and Territory expenditure formerly directed at medicine can be spent on prevention and primary care. CONCLUSION: Overall, the SPBSAs have been very successful and demonstrates an effective model for the development of Indigenous health policy. PMID- 16681342 TI - A survey of CVD risk factors in the general, Chinese and low socio-economic populations of a small LGA in Victoria. PMID- 16681343 TI - Indigenous identification in hospital dental separation data. PMID- 16681344 TI - Little evidence of effectiveness for screening in new Aboriginal and Torres Strait Islanders health check. PMID- 16681345 TI - Mortality rates among nuclear industry workers at Lucas Heights Science and Technology Centre. PMID- 16681346 TI - Simple, cost-effective process IDs ideal candidates for DM. PMID- 16681347 TI - Is capitated nurse case management more cost-effective for delivering Medicare services? PMID- 16681348 TI - HMOs show greater pricing efficiency, but consumers opting for more flexibility. PMID- 16681349 TI - Intracranial endoscopy. AB - The authors' intention is to reduce the invasiveness of intracranial procedures while avoiding traumatization of brain tissue, to decrease the risk of neurological and mental deficits. Intracranial endoscopy is a minimally invasive technique that provides rapid access to the target via small burr holes without the need for brain retraction. Craniotomy as well as microsurgical brain splitting and dissection can often be avoided. Furthermore, because obstructed cerebrospinal fluid pathways can be physiologically restored, the need for shunt placement is eliminated. The ventricular system and subarachnoid spaces provide ideal conditions for the use of an endoscope. Therefore, a variety of disorders, such as hydrocephalus, small intraventricular lesions, and arachnoid and parenchymal cysts can be effectively treated using endoscopic techniques. With the aid of special instruments, laser fibers, and bipolar diathermy, even highly vascularized lesions such as cavernomas may be treated. Moreover, during standard microsurgical procedures, the endoscopic view may provide valuable additional information ("looking around a corner") about the individual anatomy that is not visible with the microscope. In transsphenoidal pituitary surgery, transseptal dissection can be avoided if an endonasal approach is taken. In the depth of the intrasellar space, the extent of tumor removal can be more accurately controlled, especially in larger tumors with para- and suprasellar growth. The combined use of endoscopes and computerized neuronavigation systems increases the accuracy of the approach and provides real-time control of the endoscope tip position and approach trajectory. In the future, the indications for neuroendoscopy will certainly expand with improved technical equipment. PMID- 16681350 TI - "Picture-in-picture" endoscopic images in the microscope. AB - The authors describe a new piece of equipment that enables simultaneous monitoring of the endoscopic image and unilateral microscopic image; that is, "picture-in-picture" images for endoscope-assisted microsurgery. The system is simple and purely optical, consisting of light-weight semi-rigid fibers and a special adapter incorporated in the microscope. Throughout the initial neurosurgical procedures in which it was tested, the system proved particularly useful for inspecting the facial nerve behind the acoustic neurinoma and for observing the contact between arteries and the facial nerve during decompressive procedures for hemifacial spasm. PMID- 16681351 TI - Virtual endoscopic environments in modern neurosurgical practice. AB - Modern radiographic techniques have allowed the creation of high-definition planar images that can provide important anatomical as well as physiological data. Planar imaging sets can be reformatted into three-dimensional (3-D) data sets that can then be manipulated to demonstrate important anatomical or gross pathological features. Three-dimensional data sets have been used with success in modern image-guided or frameless stereotactic surgery. Another potential application is so-called "virtual endoscopy" or "scopeless endoscopy," in which a 3-D anatomical data set is reformatted into a volume-rendered image that can then be viewed. By reformatting images in this way, a "surgeon's-eye" view can be obtained, which can aid in presurgical planning and diagnosis. The use of virtual endoscopy has the potential to increase our understanding of the appropriate anatomy and the anatomical relationships most apparent during neurosurgical approaches. In so doing, virtual endoscopy may serve as an important means of planning for therapeutic interventions. On the other hand, one must always be cognizant of the technical limitations of these studies regardless of the quality of the reconstructed images. Prospective, correlative, clinical studies in which the anatomical advantages of virtual-based endoscopy are evaluated in large cadaver or patient series must be performed. Until then, the only potential ways to compensate for errors that exist in the algorithms and reconstructions of 3-D endoscopic images are based on the surgeon's understanding of the clinical state of the patient and prior experience with the anatomy in the region of question. PMID- 16681352 TI - Concurrent three dimensional neuroendoscopy: initial descriptions of application to clinical practice. AB - Applications of endoscopic technique neurosurgery are becoming increasingly popular as greater evidence of the safety and efficacy of these techniques is reported. Nevertheless, significant technical limitations need to be solved before neuroendoscopy can achieve widespread popularity. One limitation is the surgeon's difficulties in becoming anatomically oriented in a two-dimensional (2 D) environment. The lack of appropriate visual cues to orient oneself in three dimensional (3-D) space makes relatively simple anatomical regions difficult to navigate. The authors describe an endoscopy system that allows for stereoscopic visualization during minimally invasive procedures and that acts as an adjunct to conventional open craniotomies. Four cases are described in which stereoendoscopy was used as either a primary means of visualization or as an adjunct to the operating microscope in conventional open neurosurgical procedures. The authors believe that stereoendoscopic vision is a significant advance in endoscope technology and will play a large role in the popularization of minimally invasive techniques in neurosurgery. PMID- 16681353 TI - Improved safety of neuroendoscopic third ventriculostomy by using an operative Doppler ultrasound probe. Technical note. AB - In this paper the authors describe the use of a Doppler device, originally built for intravascular use, that is passed through a flexible neuroendoscope during a neuroendoscopic third ventriculostomy (NTV) procedure to treat for hydrocephalus. There was no morbidity associated with the use of this Doppler probe, and the procedure was not significantly lengthened. The Doppler probe was very accurate in locating the position of the basilar artery tip and assured the safety of NTV. PMID- 16681354 TI - Neuroendoscopic approach to tectal tumors: a consecutive series. AB - The authors report a consecutive series of 10 patients who presented with signs and symptoms caused by tectal tumors. Clinical findings, radiographic features, neuroendoscopic management strategies, and histological findings are reported and discussed. Since January 1990, 11 neuroendoscopic procedures were performed in 10 patients who harbored tectal tumors. The patients were followed for an average of 5 years (range 2 months to 12 years), and a retrospective study was conducted in which case notes, radiological findings, operative notes, and histopathological findings were assessed. Magnetic resonance (MR) imaging was performed, and the images were used to classify patients into three groups: those with hypertrophy of the tectum in whom isointensity appeared on T1-weighted images (Group 1); those with a tectal tumor occupying the cerebral aqueduct in whom decreased signal intensity appeared on T1-weighted images, as well as no enhancement after gadolinium administration (Group 2); and those with a tectal tumor in whom mixed signal intensity appeared on T1-weighted images and conspicuous evidence of contrast enhancement (Group 3). The results of histological examination were consistent with MR imaging features: in Group 1, glial tissue or gliosis; in Group 2, benign astrocytoma; and in Group 3, malignant astrocytoma. Cerebrospinal fluid diversion was the only surgical treatment that provided relief from obstructive hydrocephalus. One patient in Group 3 underwent radiotherapy and subsequent partial tumor removal under neuroendoscopic guidance. Thereafter, the tumor remained in decline. All patients had normal intellectual status after undergoing surgery in which a neuroendoscope was used. Neuroendoscopic procedures can provide histological diagnosis, define the tumor-midbrain interrelationship, and be highly effective in treating obstructive hydrocephalus and in removing tectal tumors. This procedure may receive clinical application as a new management strategy for tectal glioma. PMID- 16681355 TI - Endoscopy of the posterior fossa and endoscopic dissection of acoustic neuroma. AB - The objective of this study was to evaluate the importance of endoscopes in eliminating the disadvantage of posterior fossa approach-that is, the lack of adequate visualization obtained of the lateral aspect of the internal acoustic canal (IAC). Over a 10-year period 32 patients underwent removal of acoustic neuroma (AN) via a combined retrosigmoid retrolabyrinthine approach (CRSRL). Endoscopes were used at different stages of the operation, and their use was evaluated with regard to elimination of the disadvantages of the posterior fossa approach. All patients in whom AN had been diagnosed underwent surgery. A standard combined retrosigmoid retrolabyrinthine approach was performed. Standard sinus endoscopes of 0, 30, and 70 degrees were introduced into the cerebellopontine angle before debulking the tumor, and the IAC was inspected at the end of the operation. Neurovascular integrity as well as the relationship between AN and surrounding structures were evaluated. The IAC was inspected for residual tumor, and if any was found, endoscopic tumor dissection was performed. Endoscopes have facilitated the understanding of the relationship between AN and neighboring neurovascular structures. The disadvantage of posterior fossa approach can be eliminated by using endoscopes. In surgery in which the posterior fossa approach is performed, endoscopes can be used to make operations safer. In addition to allowing inspection of the fundus, it is possible to perform tumor dissection within the IAC by using the endoscopes. PMID- 16681356 TI - Neuroendoscopic third ventriculostomy. AB - Neuroendoscopic third ventriculostomy is becoming increasingly popular as the primary mode of therapy for patients with noncommunicating hydrocephalus. In this article the author reviews the procedure and its indications, and highlights its complications. It can, without doubt, be recommended as the first line treatment for hydrocephalus and also in cases in which shunt malfunction or infection occurs. PMID- 16681357 TI - Failure of third ventriculostomy in the treatment of aqueductal stenosis in children. AB - Object. The goal of this study was to analyze the types of failure and long-term efficacy of third ventriculostomy in children. Methods. The authors retrospectively analyzed clinical data obtained in 213 children affected by obstructive triventricular hydrocephalus who were treated by third ventriculostomy between 1973 and 1997. There were 120 boys and 93 girls. The causes of the hydrocephalus included: aqueductal stenosis in 126 cases; toxoplasmosis in 23 cases, pineal, mesencephalic, or tectal tumor in 42 cases; and other causes in 22 cases. In 94 cases, the procedure was performed using ventriculographic guidance (Group I) and in 119 cases by using endoscopic guidance (Group II). In 19 cases (12 in Group I and seven in Group II) failure was related to the surgical technique. Three deaths related to the technique were observed in Group I. For the remaining patients, Kaplan-Meier survival analysis showed a functioning third ventriculostomy rate of 72% at 6 years with a mean follow-up period of 45.5 months (range 4 days-17 years). No significant differences were found during long-term follow up between the two groups. In Group I, a significantly higher failure rate was seen in children younger than 6 months of age, but this difference was not observed in Group II. Thirty-eight patients required reoperation (21 in Group I and 17 in Group II) because of persistent or recurrent intracranial hypertension. In 29 patients shunt placement was necessary. In nine patients in whom there was radiologically confirmed obstruction of the stoma, the third ventriculostomy was repeated; this was successful in seven cases. Cine phase-contrast (PC) magnetic resonance (MR) imaging studies were performed in 15 patients in Group I at least 10 years after they had undergone third ventriculostomy (range 10-17 years, median 14.3 years); this confirmed long-term patency of the stoma in all cases. Conclusions. Third ventriculostomy effectively controls obstructive triventricular hydrocephalus in more than 70% of children and should be preferred to placement of extracranial cerebrospinal shunts in this group of patients. When performed using ventriculographic guidance, the technique has a higher mortality rate and a higher failure rate in children younger than 6 months of age and is, therefore, no longer preferred. When third ventriculostomy is performed using endoscopic guidance, the same long-term results are achieved in children younger than 6 months of age as in older children and, thus, patient age should no longer be considered as a contraindication to using the technique. Delayed failures are usually secondary to obstruction of the stoma and often can be managed by repeating the procedure. Midline sagittal T2-weighted MR imaging sequences combined with cine PC MR imaging flow measurements provide a reliable tool for diagnosis of aqueductal stenosis and for ascertaining the patency of the stoma during follow-up evaluation. PMID- 16681358 TI - Fatal subarachnoid hemorrhage after endoscopic third ventriculostomy. Case report. AB - In recent years, endoscopic third ventriculostomy has become a well-established procedure for the treatment of various forms of noncommunicating hydrocephalus. Endoscopic third ventriculostomy is considered to be an easy and safe procedure. Complications have rarely been reported in the literature. The authors present a case in which the patient suffered a fatal subarachnoid hemorrhage (SAH) after endoscopic third ventriculostomy. This 63-year-old man presented with confusion and drowsiness and was admitted in to the hospital in poor general condition. Computerized tomography scanning revealed an obstructive hydrocephalus caused by a tumor located in the cerebellopontine angle. An endoscopic third ventriculostomy was performed with the aid of a Fogarty balloon catheter. Some hours postoperatively, the patient became comatose. Computerized tomography scanning revealed a severe perimesencephalic-peripontine SAH and progressive hydrocephalus. Despite emergency external ventricular drainage, the patient died a few hours later. Although endoscopic third ventriculostomy is considered to be a simple and safe procedure, one should be aware that severe and sometimes fatal complications may occur. To avoid vascular injury, perforation of the floor of the third ventricle should be performed in the midline, halfway between the infundibular recess and the mamillary bodies, just behind the dorsum sellae. PMID- 16681359 TI - Neuroendoscopic approach to intraventricular lesions. AB - Object. The purpose of this study was to determine the efficacy of endoscopic treatment in patients with intraventricular tumors. Methods. A series of 30 patients with endoscopically treated intraventricular lesions is reported. The lesions included seven colloid cysts, six astrocytomas, three subependymomas, two ependymomas, and one each of the following: pineoblastoma, pineocytoma/pineoblastoma (intermediate type), epidermoid cyst, pineal cyst, medulloblastoma, arteriovenous hemangioma, cavernoma, choroid plexus papilloma, pituitary adenoma, craniopharyngioma, melanoma, and germinoma. Total tumor resections, partial resections, biopsies, stent implantations, septostomies, and third ventriculostomies were performed. In two cases (two subependymomas > 2 cm in diameter), piecemeal endoscopic resection was ineffective because of the very firm consistency of the tumors. Therefore the endoscopic procedure was discontinued and the tumors were removed microsurgically. In the remaining cases the procedures were completed as planned. Even in the presence of difficulties such as poor orientation or significant bleeding, there was no need to abandon the endoscopic procedure. A total of 28 strictly endoscopic interventions were performed, in which the average duration was 85 minutes (range 35-170 minutes). All colloid cysts and the epidermoid lesion were completely evacuated and the capsules were widely resected. Total extirpation of solid tumors was achieved in five cases, whereas most astrocytomas were partially resected. The hydrocephalus related symptoms resolved in all of the 22 patients with cerebrospinal fluid pathway obstruction. There were no endoscopy-related deaths. In two cases, major bleeding occurred and was controlled endoscopically. The authors observed one case of meningitis, one of mutism, two of memory loss attributed to forniceal injury, one of transient trochlear palsy after a biopsy specimen of an aqueductal tumor was obtained, and one of transient confusion after a biopsy specimen of a germinoma was obtained. Conclusions. In the authors' preliminary experience, the endoscopic approach was found to be safe and effective. In this series, it was possible to achieve relief of noncommunicating hydrocephalus, tumor resections, and even complete tumor removals by using endoscopic techniques. Based on the results, the authors believe that endoscopic techniques should be considered in the treatment of selected intraventricular lesions. PMID- 16681360 TI - Endoscopic surgery for intracranial cerebrospinal fluid cyst malformations. AB - Endoscopic surgery represents a new and very useful modality of treatment for intracranial cysts. The authors review the cases of 19 patients with intracranial malformative CSF cysts (seven intraventricular, six paraventricular, and six arachnoid) who underwent endoscopic fenestration by using a burr-hole approach. The various endoscopic approaches and techniques of fenestration, according to the type and location of the cyst, and the causes of unsuccessful outcome are critically discussed. The authors recommend endoscopic fenestration as the treatment of choice for patients with para- and intraventricular cysts, in whom the procedure may help to avoid the microsurgical approach and shunt placement in nearly all patients. In patients with arachnoid cysts, the endoscopic procedure, although associated with a lower rate of successful outcome, may be performed as the primary procedure in most cases because it is a minimally invasive procedure; the traditional surgical treatment may be performed without additional risk in which endoscopic surgery has failed. PMID- 16681361 TI - Endoscopic management of cysticercal cysts within the lateral and third ventricles. AB - In this report the authors review their 3-year experience with the endoscopic management of patients with hydrocephalus who harbored cysticercal cysts within the third and lateral ventricles. The management plan was to utilize an endoscopic approach to remove the cysts and to incorporate techniques useful in treating obstructive hydrocephalus. The ultimate goals were to avoid having to place a complication-prone cerebrospinal fluid shunt and to eliminate the risk of complications related to cyst degeneration. A retrospective analysis of 10 endoscopically managed patients with hydrocephalus and cysticercal cysts within the third or lateral ventricles was performed. A general description of the instrumentation and the technique used for removal of the intraventricular cysts is given. At presention, neuroimaging revealed findings suggestive of obstructive hydrocephalus in eight patients. Seven of the 10 patients treated endoscopically were spared the necessity of shunt placement. Three successful third ventriculostomies and one therapeutic septum pellucidotomy were performed. Despite frequent rupture of the cyst walls during removal of the cyst, there were no cases of ventriculitis. The endoscopic approach successfully allowed removal of a cyst situated in the roof of the anterior third ventricle. One patient suffered from recurrent shunt obstructions secondary to a shunt-induced migration of cysts from the posterior fossa to the lateral ventricles. The endoscopic removal of third and lateral ventricle cysticercal cysts, combined with a third ventriculostomy or septum pellucidotomy in selected cases, is an effective treatment in patients with hydrocephalus and should be considered the primary treatment for this condition. PMID- 16681362 TI - Endoscopic removal of cysticercal cysts within the fourth ventricle: technique and results. AB - There is no consensus as to the optimum management of patients who harbor cysticercal cysts within the fourth ventricle. Compared with the alternative treatment options of anthelmintic medication and/or cerebrospinal fluid shunting, the surgical removal of the cyst has the advantage of eliminating the inflammatory nidus and potentially obviating the need for a complication-prone shunt. Here, an endoscopic surgical approach is described and proposed as a superior alternative to the suboccipital craniotomy for removal of a fourth ventricle cyst. A retrospective analysis was conducted to compare five consecutive endoscopic cases with eight previous consecutive cases in which a suboccipital craniotomy was performed to remove fourth ventricle cysticercal cysts. Comparisons are made with regards to success in removing the cyst, length of operation, surgical blood loss, postoperative discomfort, and complications. A detailed description of the endoscopic technique is given. Endoscopic removal of all cysts within the fourth ventricle was successful in each case. The mean length of operative times was shorter and the blood loss was reduced with the endoscopic procedure compared with the suboccipital craniotomy (2.5 +/- 0.8 hours and 5.3 +/- 2.2 hours [p = 0.02], 19 +/- 4ml and 269 +/- 203 ml [p = 0.02], respectively). The endoscopic procedure was safe and associated with less postoperative discomfort in most patients. In properly selected patients, the endoscopic removal of cysticercal cysts located within the fourth ventricle is superior to the suboccipital craniotomy and should be considered as the primary treatment for this condition. PMID- 16681363 TI - Complete endoscopic removal of colloid cysts: issues of safety and efficacy. AB - The ideal management of colloid cysts is controversial. Treatment options include shunting procedures, stereotactic cyst aspiration, open craniotomy for microsurgical removal, and endoscopic removal. Although recent literature would suggest endoscopic removal is a reasonable approach, issues of safety and efficacy have dampened the universal acceptance of this surgical modality. The author performed a retrospective anaylsis to address these controversial issues. The charts of all patients in whom endoscopic removal of colloid cysts was performed by the primary author at the University of Arkansas for Medical Sciences were reviewed. Eighteen patients underwent this procedure over the last 5 years. The mean patient age was 32 years, and the mean follow-up period was 32 months. In all patients complete tumor removal was macroscopically and radiologically confirmed, and there were no permanent deficits. Two patients suffered aseptic meningitis without long-term sequelae. There were no deaths and no incidence of tumor recurrence. The results of this series support those previously published that underscore the advantages of endoscopic removal of colloid cysts. The procedure is safe and effective. Longer follow-up review is required to address the issue of duration of tumor-free survival. PMID- 16681364 TI - The nucleotide switch of tubulin and microtubule assembly: a polymerization driven structural change. AB - GTP-binding proteins from the tubulin family, including alphabeta-tubulin, gamma tubulin, bacterial tubulin, and FtsZ, are key components of the cytoskeleton and play central roles in chromosome segregation and cell division. The nucleotide switch of alphabeta-tubulin is triggered by GTP hydrolysis and regulates microtubule assembly dynamics. The structural mechanism of the switch and how it modulates assembly are beginning to be understood. A conserved structural change between the active and inactive states, different from other GTPases, may be extracted from recent tubulin and FtsZ structures. From these and the biochemical properties of tubulin, the new concept emerges that, contrary to what was thought, unassembled tubulin-GTP is in the inactive, curved conformation as in tubulin-GDP rings, and it is driven into the straight microtubule conformation by the assembly contacts; binding of the GTP gamma-phosphate only lowers the free energy difference between the curved and straight forms. PMID- 16681366 TI - Calcium is a cofactor of polymerization but inhibits pyrophosphorolysis by the Sulfolobus solfataricus DNA polymerase Dpo4. AB - Y-Family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus serves as a model system for eukaryotic translesion polymerases, and three-dimensional structures of its complexes with native and adducted DNA have been analyzed in considerable detail. Dpo4 lacks a proofreading exonuclease activity common in replicative polymerases but uses pyrophosphorolysis to reduce the likelihood of incorporation of an incorrect base. Mg(2+) is a cofactor for both the polymerase and pyrophosphorolysis activities. Despite the fact that all crystal structures of Dpo4 have been obtained in the presence of Ca(2+), the consequences of replacing Mg(2+) with Ca(2+) for Dpo4 activity have not been investigated to date. We show here that Ca(2+) (but not Ba(2+), Co(2+), Cu(2+), Ni(2+), or Zn(2+)) is a cofactor for Dpo4-catalyzed polymerization with both native and 8-oxoG-containing DNA templates. Both dNTP and ddNTP are substrates of the polymerase in the presence of either Mg(2+) or Ca(2+). Conversely, no pyrophosphorolysis occurs in the presence of Ca(2+), although the positions of the two catalytic metal ions at the active site appear to be very similar in mixed Mg(2+)/Ca(2+)- and Ca(2+)-form Dpo4 crystals. PMID- 16681365 TI - Participation of the tRNA A76 hydroxyl groups throughout translation. AB - The free 2'-3' cis-diol at the 3'-terminus of tRNA provides a unique juxtaposition of functional groups that play critical roles during protein synthesis. The translation process involves universally conserved chemistry at almost every stage of this multistep procedure, and the 2'- and 3'-OHs are in the immediate vicinity of chemistry at each step. The cis-diol contribution affects steps ranging from tRNA aminoacylation to peptide bond formation. The contributions have been studied in assays related to translation over a period that spans at least three decades. In this review, we follow the 2'- and 3'-OHs through the steps of translation and examine the involvement of these critical functional groups. PMID- 16681367 TI - Iodination of proteins by IPy2BF4, a new tool in protein chemistry. AB - Iodination is a very useful method for protein characterization and labeling. However, derivatization chemistries used in most conventional iodination procedures may cause substantial alterations in protein structure and function. The IPy(2)BF(4) reagent [bis(pyridine)iodonium (I) tetrafluoroborate] has been shown to be an effective iodinating reagent for peptides. Herein we report the first application of IPy(2)BF(4) in protein iodination in an aqueous medium using three representative substrates: insulin, lysozyme, and the enzyme 1,3-1,4-beta-d 4-glucanohydrolase. Our results show that IPy(2)BF(4) has clear advantages over existing methods in that the reaction is quantitative, fast, and selective for the most accessible Tyr residues of a protein, and it preserves the functional integrity of the protein when moderate Tyr labeling levels are pursued. PMID- 16681368 TI - Structure-guided design of peptide-based tryptase inhibitors. AB - Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity. PMID- 16681369 TI - Catalytically active monomer of class mu glutathione transferase from rat. AB - Although rat glutathione transferase M1-1 is crystallized as a homodimer (GST M1 1), we have generated monomers (GST M1) of the enzyme by adding potassium bromide to buffer solutions containing the wild-type enzyme and by introducing point mutations in the electrostatic region of the subunit interface. The wild-type enzyme was evaluated in 0.05 M MES (pH 6.5) containing up to 3 M KBr. We report that the addition of KBr greatly influences the monomer-dimer equilibrium of the wild-type enzyme and that at 3 M KBr GST M1 has a specific activity close to that of GST M1-1. Since the effect of KBr is likely due to charge screening at the subunit interface, the influence on the monomer-dimer equilibrium exerted by the amino acid residues in the electrostatic region of the interface (Arg77, Asp97, Glu100, Asn101) was investigated. Mutations introduced at positions 97, 100, and 101 promote monomerization, resulting in enzymes that exhibit a decreased weight average molecular weight in comparison to that of the wild-type enzyme. However, only mutations at position 97 result in enzymes that have catalytic activity in the monomeric form. The mutations introduced at positions 100 or 101 result in enzymes whose activity can be accounted for by the amount of dimeric enzyme present. Our results indicate that the electrostatic region of the interface is important in the monomer-dimer equilibrium of glutathione transferase and that, although GST M1-1 may be more active than GST M1, the dimer is not required for catalytic function. PMID- 16681370 TI - Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase. AB - Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M( )(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors. PMID- 16681371 TI - Characterization of urotensin-II receptor structural domains involved in the recognition of U-II, URP, and urantide. AB - Urotensin-II (U-II) and urotensin-II-related peptide (URP) are potent vasoconstrictors, and this action is mediated through a G protein-coupled receptor identified as UT. This receptor is expressed abundantly in the mammalian cardiovasculature, and the effects of U-II and URP can be blocked with urantide, a selective antagonist. Thus, we carried out a study with the aim to characterize the conformational arrangement of the three extracellular loops of UT as well as the transmembrane domains III and IV. Secondary structures of the synthetic receptor fragments were determined using circular dichroism (CD) spectroscopy in a variety of solvent and micelle conditions. Spectra showed that all receptor segments but not the extracellular loop I exhibited a propensity for adopting the alpha-helix folding. Furthermore, using surface plasmon resonance (SPR) technology, we measured the binding affinities of the ligands, U-II, URP, and urantide toward the UT extracellular segments. SPR data showed that both U-II and URP bind extracellular loops II and III with similar affinities, whereas none of these two ligands were able to interact with the extracellular loop I. Moreover, the binding of urantide was observed only with the second extracellular loop. These results imply that U-II and URP but not urantide would bind to UT according to a common pattern. Also, the correlation of the CD spectral information with the affinity data suggested that the adoption of a helical geometry in UT, by extracellular loops II and III, might be essential for favoring the binding of ligands. PMID- 16681372 TI - Purification and spectroscopic characterization of Ctb, a group III truncated hemoglobin implicated in oxygen metabolism in the food-borne pathogen Campylobacter jejuni. AB - Campylobacter jejuni is a food-borne bacterial pathogen that possesses two distinct hemoglobins, encoded by the ctb and cgb genes. The former codes for a truncated hemoglobin (Ctb) in group III, an assemblage of uncharacterized globins in diverse clinically and technologically significant bacteria. Here, we show that Ctb purifies as a monomeric, predominantly oxygenated species. Optical spectra of ferric, ferrous, O(2)- and CO-bound forms resemble those of other hemoglobins. However, resonance Raman analysis shows Ctb to have an atypical nu(Fe)(-)(CO) stretching mode at 514 cm(-)(1), compared to those of the other truncated hemoglobins that have been characterized so far. This implies unique roles in ligand stabilization for TyrB10, HisE7, and TrpG8, residues highly conserved within group III truncated hemoglobins. Because C. jejuni is a microaerophile, and a ctb mutant exhibits O(2)-dependent growth defects, one of the hypothesized roles of Ctb is in the detoxification, sequestration, or transfer of O(2). The midpoint potential (E(h)) of Ctb was found to be -33 mV, but no evidence was obtained in vitro to support the hypothesis that Ctb is reducible by NADH or NADPH. This truncated hemoglobin may function in the facilitation of O(2) transfer to one of the terminal oxidases of C. jejuni or, instead, facilitate O(2) transfer to Cgb for NO detoxification. PMID- 16681373 TI - Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects. AB - dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R) (+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants. PMID- 16681374 TI - Role of distal arginine in early sensing intermediates in the heme domain of the oxygen sensor FixL. AB - FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O(2), NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O(2), a 5-8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase ( approximately 10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O(2) and arginine 220 away from the heme pocket as a second step in the signal pathway on the 50 ps time scale. PMID- 16681375 TI - Phosphate analogues as probes of the catalytic mechanisms of MurA and AroA, two carboxyvinyl transferases. AB - The role in catalysis of phosphate with AroA (enolpyruvyl shikimate 3-phosphate synthase) and MurA (enolpyruvyl UDP-GlcNAc synthase) was probed using phosphate analogues and an AroA mutant. Phosphate, the second reaction product, increases the reactivity of the enolpyruvyl products (EP-OR's) approximately 10(5)-fold in the reverse reaction, forming phosphoenolpyruvate and R-OH (shikimate 3-phosphate or UDP-GlcNAc). Phosphate is intrinsically unreactive with EP-OR, raising the question of how AroA and MurA promote EP-OR reactivity. Eleven phosphate analogues were examined. All those with tetrahedral geometries bound with AroA, except sulfate, while no nontetrahedral analogues did. Arsenate, vanadate, and fluorophosphate caused reactions of AroA and MurA with EP-OR's, yielding pyruvate and R-OH. Their k(cat)/K(M) values relative to phosphate were similar for both enzymes, ca. 100-fold worse for arsensate, 200-fold worse for vanadate, and 5000 fold worse for fluorophosphate, implying similar interactions with both enzymes. Examination of the arsenate-promoted reactions using [3'-(3)H]EP-OR's, (2)H(2)O, and H(2)(18)O provided evidence of an arseno-tetrahedral intermediate, analogous to the natural tetrahedral intermediate, proceeding to arsenoenolpyruvate, which spontaneously broke down to pyruvate and arsenate. The only physicochemical property that appeared to be essential for reactivity of the analogues was the presence of a proton. Titration of the intrinsic tryptophan fluorescence of the weakly active AroA mutant, Asp313Ala (D313A), demonstrated a fluorescence decrease upon enolpyruvyl shikimate 3-phosphate (EPSP) binding, and a further decrease upon binding of phosphate or arsenate to AroA_D313A.EPSP, suggesting a further conformational change. We are hopeful that understanding enzyme-phosphate interactions will make it possible to design inhibitors that can use the high endogenous phosphate concentration in bacteria to enhance inhibitor binding. PMID- 16681376 TI - Catalysis of electron transfer by selenocysteine. AB - Selenium is an essential element that is involved in biological redox processes. The electrode potentials of the selenocysteine half-reactions RSe(*) + e(-) --> RSe-, (RSeSeR)(*)(-) + e(-) --> 2 RSe(-), and RSeSeR + 2 e(-) --> 2 RSe(-) [E degrees' (pH 7)] are +0.43, +0.18, and -0.38 V, respectively, at pH 7. The spectra of RSe(*) and (RSeSeR)(*)(-) are characterized by absorption maxima at 460 nm (epsilon = 560 M(-)(1) cm(-)(1)) and 455 nm (epsilon = 7100 M(-)(1) cm( )(1)), respectively. The bond dissociation energy of RSe-H has been calculated, and the value of 310 kJ/mol is in agreement with literature values. In comparison with the sulfur analogue cysteine, the more facile accessibility of the radical oxidation state is striking and may have biological implications, such as in mediation of one-electron- and two-electron-transfer processes, as illustrated by catalysis by selenocysteine of the electron transfer between dithiothreitol and benzyl viologen. PMID- 16681377 TI - Catalytic, noncatalytic, and inhibitory phenomena: kinetic analysis of (4 hydroxyphenyl)pyruvate dioxygenase from Arabidopsis thaliana. AB - (4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) incorporates both atoms of molecular oxygen into 4-hydroxyphenylpyruvate (HPP) to form homogentisate (HG). This reaction has direct relevance in both medicine and agriculture. In humans, the specific inhibition of HPPD alleviates the symptoms of diseases that arise from tyrosine catabolism defects. However, in plants, the inhibition of HPPD bleaches, stunts, and ultimately kills the organism. The reason for this is that in mammalian metabolism the product HG does not feed into other pathways, whereas in plants it is the precursor for the redox active portion of tocopherols and plastoquinones. There are a number of commercially available herbicides that directly target the inhibition of the HPPD reaction. Plant HPPD however is largely uncharacterized in terms of its catalysis and inhibition reactions. In this study, we examine the catalysis and inhibition of HPPD from Arabidopsis thaliana (AtHPPD). We have expressed AtHPPD and purified the enzyme to high specific activity. This form of HPPD accumulates two transient species in single turnover reactions with the native substrate HPP. These transients appear to be equivalent to intermediates I and III observed in the enzyme from Streptomyces (Johnson-Winters et al. (2005), Biochemistry, 44, 7189-7199). The first intermediate is a relatively strongly absorbing species with maxima at 380 and 490 nm. This species decays to a second intermediate that is fluorescent and has been assigned as the complex of the enzyme with the product, HG. The decay of this intermediate is rate-determining in multiple turnover reactions. The reaction of the enzyme with the analogue of the substrate, phenylpyruvate (PPA), is noncatalytic. A single turnover reaction is observed with this ligand that renders the enzyme oxidized to the ferric form, consumes a stoichiometric amount of dioxygen, and yields 66% phenylacetate as a product. Additional absorbance features at 365 and 670 nm accumulate during inactivation and give the inactivated enzyme a green color but has the same molecular mass as the active enzyme as determined by mass spectrometry. PMID- 16681379 TI - Fluorescent PNA probes as hybridization labels for biological RNA. AB - Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts. Noncovalent strategies for labeling RNA rely on attaching fluorescent dyes to hybridization probes which deliver the dye to a specific region of the RNA through Watson-Crick base pairing. This report demonstrates the use of high-affinity peptide nucleic acid (PNA) probes in labeling mRNA transcripts with thiazole orange donor and Alexa-594 acceptor fluorophores. The PNA probes were targeted to sequences flanking splice sites in a pre-mRNA such that before splicing the PNAs were separated by >300 nucleotides (nts) whereas after splicing the separation decreased to Fe(II) > Zn(II) > Ni(II). The identity of the catalytic metal ion influences both the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the Michaelis constant, with Fe(II)- and Co(II)-HDAC8 having K(M) values that are over 5-fold lower than that of Zn(II)-HDAC8. These data suggest that Fe(II), rather than Zn(II), may be the in vivo catalytic metal. In further support of this hypothesis, recombinant HDAC8 purified from E. coli contains 8 fold more iron than zinc before dialysis, and the HDAC8 activity in cell lysates is oxygen-sensitive. Identification of the in vivo metal ion of HDAC8 is essential for understanding the biological function and regulation of HDAC8 and for the development of improved inhibitors of this class of enzymes. PMID- 16681390 TI - A gene cluster for biosynthesis of the sesquiterpenoid antibiotic pentalenolactone in Streptomyces avermitilis. AB - Streptomyces avermitilis, an industrial organism responsible for the production of the anthelminthic avermectins, harbors a 13.4 kb gene cluster containing 13 unidirectionally transcribed open reading frames corresponding to the apparent biosynthetic operon for the sesquiterpene antibiotic pentalenolactone. The advanced intermediate pentalenolactone F, along with the shunt metabolite pentalenic acid, could be isolated from cultures of S. avermitilis, thereby establishing that the pentalenolactone biosynthetic pathway is functional in S. avermitilis. Deletion of the entire 13.4 kb cluster from S. avermitilis abolished formation of pentalenolactone metabolites, while transfer of the intact cluster to the pentalenolactone nonproducer Streptomyces lividans 1326 resulted in production of pentalenic acid. Direct evidence for the biochemical function of the individual biosynthetic genes came from expression of the ptlA gene (SAV2998) in Escherichia coli. Assay of the resultant protein established that PtlA is a pentalenene synthase, catalyzing the cyclization of farnesyl diphosphate to pentalenene, the parent hydrocarbon of the pentalenolactone family of metabolites. The most upstream gene in the cluster, gap1 (SAV2990), was shown to correspond to the pentalenolactone resistance gene, based on expression in E. coli and demonstration that the resulting glyceraldehyde-3-phosphate dehydrogenase, the normal target of pentalenolactone, was insensitive to the antibiotic. Furthermore, a second GAPDH isozyme (gap2, SAV6296) has been expressed in E. coli and shown to be inactivated by pentalenolactone. PMID- 16681392 TI - Size and orientation of the lipid II headgroup as revealed by AFM imaging. AB - In this study, we investigated the size and orientation of the bacterial Lipid II (L II) headgroup when the L II molecule is present in liquid-crystalline domains of DOPC in a supported DPPC bilayer. Using atomic force microscopy, we detected that L II causes the appearance of a 1.9 nm thick layer, situated over the DOPC headgroup region. With an increased scanning force, this layer can be penetrated by the AFM tip down to the level of the DOPC bilayer. Using different L II precursor molecules, we demonstrated that the detected layer consists of the headgroups of L II and that the MurNAc-pentapeptide unit of the headgroup is responsible for the measured 1.9 nm height of that layer. Monolayer experiments provided information about the in-plane dimensions of the L II headgroup. On the basis of these results and considerations of the molecular dimensions of L II headgroup constituents, we propose a model for the orientation of the L II headgroup in the membrane. In this model, the pentapeptide of the L II headgroup is rather extended and points away from the bilayer surface, which could be important for biological processes, in which L II is involved. PMID- 16681391 TI - Translesion synthesis past equine estrogen-derived 2'-deoxyadenosine DNA adducts by human DNA polymerases eta and kappa. AB - Hormone replacement therapy (HRT) increases the risk of developing breast, ovarian, and endometrial cancers. Equilin and equilenin are the major components of the widely prescribed drug used for HRT. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equilin and equilenin, promotes 4-OHEN-modified dC, dA, and dG DNA adducts. These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT. We have recently found that the 4-OHEN dC DNA adduct is a highly miscoding lesion generating C --> T transitions and C - > G transversions. To explore the mutagenic potential of another major 4-OHEN-dA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) eta and kappa that are highly expressed in the reproductive organs. Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products. With the major 4-OHEN-dA-modified templates (Pk-2 and Pk-3), primer extension was retarded prior to the lesion and opposite the lesion; a fraction of the primers was extended past the lesion. Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa. The bypass frequency past Pk-2 was more efficient than that past Pk-3. Thus, 4-OHEN-dA is a miscoding lesion generating A --> T transversions and A --> G transitions. The miscoding frequency and specificity of 4-OHEN-dA varied depending on the stereoisomer of the 4-OHEN-dA adduct and DNA polymerase used. PMID- 16681393 TI - Localization of multidrug transporter substrates within model membranes. AB - Active extrusion of drugs from the cell interior by primary and secondary efflux pumps is an essential mechanism underlying the phenomenon of multidrug resistance. The first discovered and best characterized primary efflux pump found in humans is the ABC transporter P-glycoprotein (PGP), which shows very broad substrate specificity. Many of these molecules are lipophilic, and binding most likely takes place within the membrane. PGP could either translocate them from the inner to the outer leaflet (flippase) or extrude them from the membrane into the extracellular environment (hydrophobic vacuum cleaner). Recognition and binding of such a diverse set of substrates must be associated with a preferred membrane location, determined by molecular properties and lipid interactions. Therefore, a systematic study of the interaction among seven PGP substrates (phenazine, doxorubicin, cephalexin, ampicillin, chloramphenicol, penicillin G, and quercetin) and two modulators (quinidine and nicardipine) and 1,2-dimyristoyl sn-glycero-3-phosphocholine (DMPC) model membranes is reported here. The location profile of these molecules across the membrane was determined by (1)H NOESY MAS NMR based on (1)H-(1)H cross-peaks between their aromatic fingerprint region and lipid resonances. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains. Our findings are consistent with PGP substrate and modulator binding from the membrane interface region. PMID- 16681395 TI - The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity. AB - The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1) ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP. PMID- 16681394 TI - Regulatory and catalytic domain dynamics of smooth muscle myosin filaments. AB - Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 +/- 79 micros for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 +/- 4 degrees and 24 +/- 9 degrees as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40, 8283-8291), the latter displayed higher regulatory domain mobility, tau(r)= 40 +/- 3 micros, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (approximately 4 micros) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 +/- 4 degrees to 36 +/- 7 degrees ) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 micros) or the catalytic domain (24 to 17 micros). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides. PMID- 16681396 TI - Pathway of oxidative folding of secretory leucocyte protease inhibitor: an 8 disulfide protein exhibits a unique mechanism of folding. AB - Secretory leucocyte protease inhibitor (SLPI) is a 107-amino acid protein with a high density of disulfide pairing (eight). The mechanism of oxidative folding of reduced and denatured SLPI has been investigated here. Despite an exceedingly large number of possible folding intermediates ( approximately 46 million disulfide isomers) and their potential to complicate the refolding process, oxidative folding of SLPI turns out to be surprisingly simple and efficient. Complete oxidative folding and a near-quantitative recovery of the native SLPI can be achieved in a simple buffer solution using air oxidation without any supplementing thiol catalyst or redox agent, a phenomenon that has not yet been observed with other disulfide proteins. Because of the heterogeneity and extensive overlapping of folding intermediates, identification of the predominant intermediate was unfeasible. Nonetheless, studies of reductive unfolding of native SLPI and oxidative folding of a six-disulfide variant of SLPI enable us to propose an underlying mechanism accounting for the unique folding efficiency of SLPI in the absence of a redox agent. Our studies indicate that oxidative folding of SLPI undergoes heterogeneous populations of one-, two-, three-, four-, five-, six-, and seven-disulfide isomers, including two nativelike isomers, SLPI-6A and SLPI-7A, as transient intermediates. Formation of the last two native disulfide bonds leading to the conversion of SLPI-6A --> SLPI-7A --> N-SLPI is relatively slow and represents the final stage of oxidative folding. Most importantly, free cysteines of SLPI-6A and SLPI-7A also act as a thiol catalyst in promoting the disulfide shuffling of diverse non-native intermediates accumulated along the folding pathway. This explains why a near-quantitative recovery of N-SLPI can be achieved in the absence of any thiol catalyst and redox agent. Properties of SLPI 6A and SLPI-7A were investigated and compared to those of other documented kinetic intermediates of oxidative folding. The correlation between the mechanism of SLPI folding and the three-dimensional structure of SLPI is also elaborated. PMID- 16681397 TI - A multicenter outcomes assessment of five-year-old patients with unilateral cleft lip and palate. AB - OBJECTIVE: Compare 5-year-old dental arch relationships of patients from three centers with differing primary protocols. DESIGN: Retrospective study of treatment outcomes using blinded evaluation of dental study casts. SETTING: Three major cleft-craniofacial centers; one (center A) is a free-standing institution, and two (centers B and C) are university hospitals. PATIENTS: 118 (A = 41; B = 33; C = 44) consecutively treated 5-year-old patients with complete, nonsyndromic unilateral cleft lip and palate. INTERVENTIONS: Centers A and C completed primary repair without presurgical orthopedics by 18 months (center A in three surgeries and center C in two surgeries). Center B used passive presurgical orthopedics with lip/soft palate repair at 6 months and gingivo-alveoloplasty/hard palate repair at 18 to 36 months. MAIN OUTCOME MEASURE: Averaged ratings of dental casts using the 5-year yardstick were computed for each patient. The Wilcoxon two sample test was used to compare means; a chi-square test was used to compare distributions. RESULTS: Intra- and interexaminer reliability tests showed excellent reliability (>.90). Mean scores were not significantly different. Distribution of scores differed significantly. Center A had the highest percentage of good scores and the lowest percentage of poor scores (72% versus 6.5%), followed by center B (63% versus 6.6%) and center C (59% versus 16.3%). CONCLUSIONS: Centers A and B had comparable scores and completely different protocols in surgical technique, timing, sequencing, and nonuse/use of appliances. Center C's results were slightly lower than those of 1 and 3, but the center had the protocol with the least burden of treatment (only two surgeries, without use of appliances). PMID- 16681398 TI - Hard palate repair timing and facial morphology in unilateral cleft lip and palate: Before versus after pubertal peak velocity age. AB - OBJECTIVE: To investigate whether timing of hard palate repair, before versus after pubertal peak velocity age, had a significant effect on facial growth in patients with unilateral cleft lip and palate. DESIGN: Retrospective cross sectional study. SETTING: Sri Lankan Cleft Lip and Palate Project. PATIENTS: A total of 125 adult patients with nonsyndromic unilateral cleft lip and palate were recruited and their last cephalometric radiographs were used. MAIN OUTCOME MEASURES: Clinical notes were used to record surgical treatment histories. Cephalometry was used to determine facial morphology. RESULTS: The patients who had hard palate repair after pubertal peak velocity age had a deeper bony pharynx (Ba-PMP), a longer alveolar maxilla (PMP-A), a longer effective length of the maxilla (Ar-ANS, Ar-A), and as a result had a more favorable anteroposterior jaw relation (ANS-N-Pog, ANB, NAPog) and larger overjet, compared with those who had hard palate repair before pubertal peak velocity age. CONCLUSION: Timing of hard palate repair significantly affects the growth of the maxilla in patients with unilateral cleft lip and palate. Hard palate repair after (versus before) pubertal peak velocity age has a smaller adverse effect on the forward growth of the maxilla. This timing affects the forward displacement of the basal maxilla and the anteroposterior development of the maxillary dentoalveolus. PMID- 16681399 TI - Three-dimensional computer graphics for surgical procedure learning: Web three dimensional application for cleft lip repair. AB - OBJECTIVE: In surgical procedures for cleft lip, surgeons attempt to use various skin incisions and small flaps to achieve a better and more natural shape postoperatively. They must understand the three-dimensional (3D) structure of the lips. However, they may have difficulty learning the surgical procedures precisely from normal textbooks with two-dimensional illustrations. Recent developments in 3D computed tomography (3D-CT) and laser stereolithography have enabled surgeons to visualize the structures of cleft lips from desired viewpoints. However, this method cannot reflect the advantages offered by specific surgical procedures. To solve this problem, we used the benefits offered by 3D computer graphics (3D-CG) and 3D animation. DESIGN AND RESULTS: By using scanning 3D-CT image data of patients with cleft lips, 3D-CG models of the cleft lips were created. Several animations for surgical procedures such as incision designs, rotation of small skin flaps, and sutures were made. This system can recognize the details of an operation procedure clearly from any viewpoint, which cannot be acquired from the usual textbook illustrations. This animation system can be used for developing new skin-flap design, understanding the operational procedure, and using tools in case presentations. The 3D animations can also be uploaded to the World Wide Web for use in teleconferencing. PMID- 16681400 TI - The cleft audit protocol for speech-augmented: A validated and reliable measure for auditing cleft speech. AB - OBJECTIVES: To develop an assessment tool for use in intercenter audit studies of cleft speech and to test its acceptability, validity, and reliability. The tool is to be used systematically to record and report speech outcomes, providing an indication of treatment needs and continuing burden of care. SETTING: Regional Cleft Center, U.K. METHODS: The Cleft Audit Protocol for Speech-Augmented (CAPS A) was developed by three cleft speech experts who identified the key features required from existing assessment measures. Criterion validity was assessed by comparing the Cleft Audit Protocol for Speech-Augmented outcomes reported for 20 cases with clinical assessment results and other investigations. Intra- and interrater reliability were tested following the training of specialist speech and language therapists who used the Cleft Audit Protocol for Speech-Augmented on two occasions to assess 10 cases. The raters evaluated acceptability and ease of using a questionnaire. RESULTS: The mean percentage agreement for criterion validity in each section was 87% (range 70% to 100%). Both intra- and interexaminer reliability were rated as good/very good (Kappa 0.61 to 1.00) for seven sections and moderate (Kappa 0.41 to 0.60) for three sections. Raters reported that the Cleft Audit Protocol for Speech-Augmented was acceptable and easy to use with appropriate training. CONCLUSION: An acceptable, valid, and reliable cleft speech audit tool has been developed based on a small sample. The Cleft Audit Protocol for Speech-Augmented is recommended for use in intercenter audit studies in the U.K. and Ireland and could be used in other English-speaking countries. In addition, it has wider applicability for use in reporting speech outcomes of surgical procedures. PMID- 16681401 TI - Effect of nasal decongestion on nasalance measures. AB - OBJECTIVE: The purpose of this study was to evaluate the effect of nasal decongestant on nasalance scores for a group of 100 individuals. PARTICIPANTS: Forty-one subjects with hypernasality and 59 subjects without hypernasality underwent nasometric assessment at the Hospital for Rehabilitation of Craniofacial Anomalies, University of Sao Paulo, Bauru, Brazil. DESIGN: Nasalance scores were obtained for each subject before the application of a nasal decongestant and again 10 minutes after subjects received a topical nasal decongestant applied into both nostrils. RESULTS: The nasalance scores obtained after the application of the nasal decongestant were significantly higher than those obtained before the decongestant. CONCLUSIONS: Nasal decongestion had a small but statistically significant effect on nasalance scores, suggesting that in some individuals, nasal congestion should be a variable of concern when using the Nasometer. Both nasal congestion (i.e., due to nasal rhinitis) and the effects of nasal decongestant sprays may influence Nasometer test results. Interpretation of nasalance scores, therefore, should be done carefully. Furthermore, nasometry, with and without nasal decongestant, can be a valuable clinical tool for screening anterior nasal obstruction, helping to isolate obstruction due to nasal congestion from structural obstruction in the nasal cavities. PMID- 16681402 TI - Speech in children with an isolated cleft palate: A longitudinal perspective. AB - OBJECTIVE: To describe articulation and speech symptoms related to velopharyngeal impairment in children born with an isolated cleft palate. DESIGN: Blind assessment of speech at 3, 5, 7, and 10 years of age was performed. Two subgroups were formed based on the results at age 5 years, the no-VPI group and the VPI group, and they were compared with controls. SETTING: A university hospital. PATIENTS: Twenty-six children born with isolated cleft palate. Seventeen children served as controls. INTERVENTIONS: Soft palate closure at 7 months and hard palate closure at a mean age of 3 years and 11 months if the cleft extended into the hard palate. MAIN OUTCOME MEASURES: Perceptual assessments of four variables related to velopharyngeal function and of articulation errors were performed at all ages. Phonetic transcriptions of target speech sounds were obtained at 5, 7, and 10 years and nasalance scores were obtained at age 10 years. RESULTS: The no VPI group continued to have no or minor difficulties. The VPI group improved but continued to have moderate velopharyngeal impairment. Both groups differed significantly from the controls at age 10 years. Persistent velopharyngeal impairment, as well as glottal misarticulation, were mostly found in children with the cleft as a part of a syndrome or together with multiple malformations. CONCLUSION: Small changes in velopharyngeal impairment were found across ages. Improvement seemed to be related to surgical intervention, and persistent problems seemed to be related to the presence of additional multiple malformations or syndromes. PMID- 16681403 TI - A mutation in RYK is a genetic factor for nonsyndromic cleft lip and palate. AB - OBJECTIVE: The RYK, EPHB2, and EPHB3 genes are attractive candidates for cleft lip and/or palate and cleft palate only pathogenesis. Both the Ryk-deficient mouse and Ephb2/Ephb3 (genes for interaction molecules with RYK) double-mutant mouse show cleft palate. SETTING: Mutation searches for RYK, EPHB2, and EPHB3 were carried out in a large number of Japanese and Vietnamese patients with cleft lip and/or palate and cleft palate only. Case-control study and transmission disequilibrium tests were performed also, using three single nucleotide polymorphisms within a linkage disequilibrium block in RYK. Seven haplotypes were constructed from the single nucleotide polymorphisms. RESULTS: A missense mutation, 1355G>A (Y452C), in RYK was identified in one Vietnamese patient with cleft lip and/or palate. This mutation was not found among 1646 Vietnamese, Japanese, and Caucasians, including 354 cleft lip and/ or palate and cleft palate only patients. Colony formation assay using NIH3T3 cells transfected with mutant cDNA revealed that mutant RYK had significantly reduced protein activity, compared with those with wild-type RYK, implying that the transformation ability of RYK is depleted by this mutation. Although a case-control study and transmission disequilibrium tests on three individual single nucleotide polymorphisms provided no evidence for association with oral clefts, a case control study on one rare haplotype suggested a positive association in Japanese patients with cleft lip and/or palate and cleft palate only. No mutations in EPHB2 and EPHB3 were found in any patients examined. CONCLUSION: The findings suggested that a missense mutation, 1355G>A, and one rare single nucleotide polymorphisms haplotype may play a role in the development of cleft lip and/or palate in the Vietnamese, and cleft lip and/ or palate and cleft palate only in the Japanese. PMID- 16681404 TI - Pierre Robin sequence with esophageal atresia and congenital radioulnar synostosis. AB - A wide spectrum of anomalies can be associated with Pierre Robin sequence. This report presents a 3-day-old infant with micrognathia, U-shaped cleft palate, low set right ear with microtia, glossoptosis, esophageal atresia, and right congenital radioulnar synostosis. The association of congenital radioulnar synostosis and esophageal atresia with Pierre Robin sequence has not been previously described. PMID- 16681405 TI - Presurgical nasoalveolar molding therapy for the treatment of bilateral cleft lip and palate: A preliminary study. AB - OBJECTIVE: To evaluate the outcome of presurgical nasoalveolar molding therapy in the treatment of patients with bilateral cleft lip and palate. DESIGN: A prospective study with blinded measurements. SETTING: The Cleft and Craniofacial Clinic at the University of Texas at Houston Medical School, Houston, Texas. PATIENTS: Eight patients with bilateral cleft lip and palate, treated between 2002 and 2004. INTERVENTIONS: The starting age for presurgical nasoalveolar molding therapy was 34.9 days and the average length of the therapy was 212.5 days. MAIN OUTCOME MEASURES: Measurements of intraoral and extraoral casts were performed, and statistical analyses were used to compare the differences between measurements before and after therapy. RESULTS: Intraoral measurements demonstrated that there was a statistically significant reduction of the premaxillary protrusion and deviation. There was also a significant reduction in the width of the larger cleft. Extraoral measurements revealed that there was a significant increase in the bi-alar width and in the columellar length and width. Moreover, there was a significant improvement in columellar deviation. Finally, the nostril heights of both sides were increased. CONCLUSION: The authors have quantitatively shown that presurgical nasoalveolar molding therapy has significant advantages in the treatment of bilateral cleft lip and palate patients. It improves the nasal asymmetry and deficient nasal tip projection associated with bilateral cleft lip and palate. It also forces the protruded premaxillary segment into alignment with the dental alveolar segments, improving the shape of the maxillary arch. As a result, the changes associated with presurgical nasoalveolar molding therapy help decrease the complexity of subsequent surgeries. PMID- 16681406 TI - Results of randomized controlled trial of soft palate first versus hard palate first repair in unilateral complete cleft lip and palate. AB - OBJECTIVE: To compare the outcomes for primary repair of unilateral cleft lip and palate, operating on the soft palate first versus the hard palate first. DESIGN: Randomized controlled trial. SETTING: The Regional Cleft Service of West Nepal. PATIENTS: Forty-seven consecutive patients with nonsyndromic unilateral cleft lip and palate, of whom 37 were assessed 4 to 6 years after completing primary surgical repair. INTERVENTIONS: Primary repair of unilateral cleft lip and palate by two differing sequences: (1) soft palate repair, with hard palate and lip repair 3 months later; and (2) lip and hard palate repair, followed by the soft palate repair 3 months later. MAIN OUTCOME MEASURES: Analysis of dental study models, weight gain, and speech recordings. RESULTS: Four to 7 years after completing the cleft closure, there was no significant difference in facial growth between the two types of repair sequencing. Completing posterior repair first had no effect on anterior alveolar gap width. It narrowed the hard palate gap by reducing the intercanine distance. Anterior repair dramatically closed the anterior alveolar gap, and narrowed the intercanine distance. Comparing anterior alveolar gap width with age at first presentation demonstrated that there was no spontaneous narrowing of the cleft in older children. Completing posterior closure first had a weight gain advantage over anterior closure first. Improved oropharyngeal closure, and thus swallowing, is the likely explanation. CONCLUSION: Changing the sequencing of cleft closure has no demonstrable difference in facial growth at 4 to 7 years after completion of the primary surgery. PMID- 16681407 TI - Soft tissue facial angles in individuals with ectodermal dysplasia: A three dimensional noninvasive study. AB - OBJECTIVE: To supply quantitative information about the facial soft tissues of patients with hypohidrotic ectodermal dysplasia. DESIGN: Prospective assessment. SETTING: National meetings of hypohidrotic ectodermal dysplasia patients and families. PATIENTS AND MAIN OUTCOME MEASURES: Facial and mandibular corpus convexities in the horizontal plane; facial convexity in the sagittal plane; interlabial, naso-labial, nasal convexity, and left and right soft tissue gonial angles were calculated from the three-dimensional coordinates of 11 soft tissue facial landmarks obtained in 18 male and 17 female hypohidrotic ectodermal dysplasia patients aged 3 to 41 years and in 504 reference healthy individuals. In addition, z-scores were computed and the patients were grouped by cluster analysis. RESULTS: Male and female z-scores did not differ. In the pooled group, facial convexities in the horizontal and sagittal planes were significantly (Student's t, p < .01) increased (flatter) in hypohidrotic ectodermal dysplasia patients, compared with normal controls. The naso-labial angle was significantly reduced (more acute). Upper and lower facial convexity and mandibular corpus convexity in the horizontal plane deviated less from the norm with increasing age. Facial convexity in the horizontal and sagittal planes, soft tissue gonial angles, and naso-labial and interlabial angles deviated less from the norm with increasing number of teeth present in the mouth. Cluster analysis identified three homogeneous groups, all characterized by a peculiar facial phenotype. Modifications in facial convexity and gonial and interlabial angles differentiated each cluster. CONCLUSIONS: Patients with hypohidrotic ectodermal dysplasia had flatter faces in the horizontal and sagittal planes than normal controls had. Cluster analysis revealed patterned differences in facial phenotype. PMID- 16681408 TI - Digital surface photogrammetry for anthropometric analysis of the cleft infant face. AB - OBJECTIVE: To analyze the three-dimensional morphology of the cleft infant face with digital surface photogrammetry. DESIGN: Fifty plaster casts of unoperated infants with cleft lip and palate were imaged three-dimensionally with digital surface photogrammetry. Twenty-one standard craniofacial measurements were taken. The plaster casts were divided into 4 groups with unilateral, bilateral, complete, and incomplete clefts of the lip and palate. The measurements were compared with standard values for healthy infants. RESULTS: Significant differences (p < .0025) were found for the alar base width (33% to 55%), the alar base root width (59% to 103%), the width of the nose (7% to 25%), the length of the alar wing (18% to 25%), and the intercanthal (6% to 17%) and biocular (4% to 12%) width, depending on the cleft type. The vertical dimensions of the nose and the upper lip did not differ significantly from the controls. CONCLUSION: This study describes preliminary data on the cleft infant facial deformity. The obtained results were mainly in agreement with data in the limited literature. Three-dimensional photogrammetry has proven to be reliable and can be applied more readily to potentially uncooperative patients. PMID- 16681409 TI - Structural birth defects associated with oral clefts in Hawaii, 1986 to 2001. AB - OBJECTIVE: To identify structural birth defects that occur in association with oral clefts. METHODS: Data were obtained from a birth defects registry and included all infants and fetuses with cleft palate without cleft lip or cleft lip with or without cleft palate delivered from 1986 to 2001. For 47 specific structural birth defects, rates among oral cleft cases were compared with the rates among all infants and fetuses with major birth defects, excluding those with oral clefts. RESULTS: Among cleft palate only cases, the rates were significantly higher than expected for encephalocele, microcephaly, and syndactyly. Among cases of cleft lip with or without cleft palate, the rates were significantly higher than expected for anophthalmia/microphthalmia, single ventricle, reduction deformity of upper limbs, and reduction deformity of lower limbs. When cases of cleft palate only and cleft lip with or without cleft palate were compared as to the rates for particular birth defects, the rates of the defects were either higher or lower than expected in both oral cleft categories for 38 (81%) of the defects. CONCLUSIONS: Certain birth defects were more frequently associated with oral clefts than might be expected. For the majority of defects, their patterns of association were similar between cleft palate without cleft lip and cleft lip with or without cleft palate. PMID- 16681410 TI - Congenital fenestration of the palate: A case of embryologic syzygy. AB - Congenital fenestration of the secondary palate is the rarest type of facial cleft. Of the 26 putative cases in the literature, only 5 had confirmation of the cleft during the neonatal period. This report documents such a cleft in an infant and presents the likely pathogenesis. PMID- 16681411 TI - Surgical repair of unidirectional palatopharyngeal epignathus: Case report and review of literature. AB - Epignathus is an extremely rare, benign, congenital teratoma that arises from within the oral cavity and may be attached to the mandible, palate, or base of the skull. Because of its location, it can cause airway and feeding problems, as well as secondary defects due to the presence of an intraoral tumor. This tumor usually causes death in neonates, due to airway obstruction. Surgical removal is usually impossible, especially in large tumors (Stone, 1951; Bennett, 1970). A patient with a neonatally diagnosed nonobstructive palatopharyngeal epignathus is described. It was associated with a shortened palate, cleft uvula, and velopharyngeal insufficiency, and was successfully excised surgically. PMID- 16681412 TI - Using a modified nutrition plate for early intervention in a newborn infant with Pierre Robin sequence: A case report. AB - Pierre Robin sequence is a triad of micrognathia, glossoptosis, and cleft palate that results in upper airway obstruction and feeding problems. It is a serious condition with potentially severe, life-threatening airway obstruction. Approaches for treatment of airway obstruction among neonates with Pierre Robin sequence include prone positioning of the infant, tongue-lip adhesion, mandibular distraction, and tracheostomy. This case report describes a modified nutrition plate and a baby treated using this appliance. PMID- 16681413 TI - Treatment of nasopharyngeal stenosis by prosthetic hollow stents: Clinical experience in eight patients. AB - OBJECTIVE: A series of nasopharyngeal appliance designs is presented that represents our evolving experience over a 20-year period in the adjunctive use of prosthetic stents in the surgical correction of nasopharyngeal stenosis. DESIGN: Retrospective assessment of effectiveness of two nasopharyngeal stenosis hollow stent designs in a consecutive series of patients for relief of nasal obstructive symptoms. SETTING: Tertiary academic medical center, Craniofacial Program at Children's Hospital. PATIENTS: Four patients with nasopharyngeal stenosis were treated with a preoperatively fabricated stent made from a clasped palatal appliance onto which hollow acrylic conduits were extended through surgically re created pharyngeal ports. A subsequent set of four patients with nasopharyngeal stenosis were treated with intraoperatively-fashioned silastic grommets, as opposed to palatal appliances. INTERVENTIONS: Postoperative intraoral stenting of nasopharyngeal ports. MAIN OUTCOME MEASURES: Maintenance of pharyngeal port opening after 1 year, improvement in nasal airway obstructive symptoms. RESULTS: The palatal appliance stents were less well tolerated and had a lower maintenance of port patency after device removal (4 of 8, 50%). The silastic grommets provided better retention into the ports and increased patient tolerance, as well as better 1-year port maintenance (6 of 8, 75%). CONCLUSIONS: The grommet stent appliance offers numerous advantages over a conventional dental-clasped appliance for prosthetic nasopharyngeal stenting, including obviation of extensive preoperative preparation, ease of insertion and removal, and exchange of air during the stenting period. Improved nasopharyngeal patency with this device may be due to greater patient tolerance and subsequent longer use. PMID- 16681414 TI - The commission on social determinants of health: tackling the social roots of health inequities. PMID- 16681415 TI - The limits of reductionism in medicine: could systems biology offer an alternative? PMID- 16681418 TI - Factor V Leiden, prothrombin G20210A substitution and hormone therapy: indications for molecular screening. AB - Venous thromboembolism is a well-known complication of oral contraception and hormonal replacement therapy. Inherited thrombophilia is viewed as an important determinant in modulating the effects of estrogens on thrombotic risk. An increasing number of kits for thrombophilic mutations [factor V Leiden, G20210A prothrombin and methylenetetrahydrofolate reductase (MTHFR) C677T genes] are becoming commercially available, and screening for inherited thrombotic risk is among the most requested genetic tests in molecular diagnostic laboratories. However, the question of routine genetic screening for thrombophilia before prescribing hormones is still a matter of debate. The purpose of this article is to discuss the usefulness and practical applications of thrombotic genetic testing to identify which women should be tested to improve both the safety and efficacy of individualized estrogen therapy. PMID- 16681419 TI - Immunochemical quantification of free immunoglobulin light chains from an analytical perspective. AB - Immunoglobulin light chains are components of antibodies, but some exist in a free form in serum and urine as a result of their excess production over heavy chains. Free light chain (FLC) levels are of the order of milligram per liter in normal serum and urine, but marked increases have been observed in various disease conditions. It has now been established that the measurement of FLC levels contributes to diagnosis and clinical management in monoclonal gammopathies. Recent developments in FLC assays have been adapted to several automated platforms and they have now become available in laboratories. There have, however, been some concerns regarding the analytical aspects. The current assay specificity appears to be insufficient to prevent the influence of intact light chains of several orders of magnitude greater than FLCs in serum. Moreover, the heterogeneous nature of light chains makes accurate quantification unreliable. FLC assays have never been standardized because of the lack of an international reference calibrator. In this review, we summarize the reports on FLC measurements and examine the specificity of anti-FLC antibodies and the reliability of FLC assays. We also discuss difficulties in the standardization and setting of normal reference intervals for FLC assays. PMID- 16681420 TI - De novo deletion removes a conserved motif in the C-terminus of ABCA4 and results in cone-rod dystrophy. AB - BACKGROUND: Mutations in the retina-specific ABC transporter (ABCA4) gene are associated with different types of macular degeneration, including Stargardt disease, cone-rod dystrophy, Fundus flavimaculatus, Retinitis pigmentosa and probably age-related macular degeneration. METHODS: Screening for mutations in the ABCA4 gene was performed using denaturing high-performance liquid chromatography and direct sequencing. RESULTS: We describe the identification of a new de novo 44-bp deletion in an Italian patient affected by cone-rod dystrophy. The mutation, located in intron 48 of the ABCA4 gene, is predicted to cause exon 49 skipping, resulting in loss of the C-terminus of the ABCA4 protein. Interestingly, exon 49 also codes for a highly conserved VFVNFA motif, which has been demonstrated to be essential for the activity of ABCA1, another gene of the ABC transporter family. The presence of CT repeats at the breakpoints might have facilitated the generation of the deletion through a slippage mispairing mechanism. CONCLUSIONS: The new 6730-16del44 deletion is the first de novo mutation associated with cone-rod dystrophy and may contribute to a better understanding of the role of ABCA4 mutations in macular dystrophies. PMID- 16681421 TI - Molecular detection of squamous cell carcinoma antigen transcripts in peripheral blood of cancer patients. AB - The squamous cell carcinoma antigen (SCC-Ag) has been widely applied as a serum marker in different kinds of cancer and was reported as a target gene for the detection of tumor cells in peripheral blood in cervical cancer. Nucleic acids released into the circulation are non-invasive diagnostic tools for cancer detection. The objective of this study was to determine the utility of SCC-Ag mRNA as a cancer detection marker in blood of cancer patients. For this purpose, 77 blood samples from five gastric cancer, 23 laryngeal cancer, 31 lung cancer, nine esophageal, and nine cervical cancer patients were analyzed. The SCC gene was amplified by reverse transcription-polymerase chain reaction. SCC-Ag mRNA was detected in two patients with gastric cancer, six patients with laryngeal cancer, 17 patients with lung cancer, three patients with esophageal cancer, and two patients with cervix cancer. The detection rate was highest (54.83%) in patients with lung cancer. SCC-Ag transcripts were not detectable in the control group, indicating that molecular analysis showed no false positives. Our results indicate that this approach could be useful in a considerable number of patients and could improve the lower diagnostic yield of conventional tests. PMID- 16681422 TI - Influence of human haptoglobin polymorphism on oxidative stress induced by free hemoglobin on red blood cells. AB - BACKGROUND: An in vitro study was conducted to determine whether haptoglobin phenotypes differed in their protective effect against oxidative stress induced by extracellular hemoglobin on red blood cells. METHODS: Conjugated dienes and thiobarbituric acid-reactive substances (TBARS) were determined in human red blood cell membranes in the presence of hemoglobin and various concentrations of each type of purified haptoglobin. In addition, the release of K+ and lactate dehydrogenase from red blood cells was measured. RESULTS: A protective effect of haptoglobin was observed in terms of results obtained for the four parameters examined, with significant differences (p<0.001) between the three haptoglobin types; type 1-1 was the most active and type 2-2 the least active. A proportion of oxidative damage was not sensitive to haptoglobin, but to desferrioxamine (an iron chelator), indicating the participation of two actors, hemoglobin and free iron, in the oxidative stress of membrane lipids. CONCLUSIONS: The antioxidant role of haptoglobin and the phenotype dependence were confirmed for preventing possible oxidative damage induced by free hemoglobin and iron release during its catabolism. PMID- 16681423 TI - Real-time RT-PCR quantification of PRAME gene expression for monitoring minimal residual disease in acute myeloblastic leukaemia. AB - BACKGROUND: Specific gene rearrangements are used for minimal residual disease (MRD) assessment, but are frequently lacking in leukaemias. In these cases, the quantification of PRAME (preferentially expressed antigen of melanoma) transcripts could be useful. METHODS: PRAME transcripts were quantified by real time RT-PCR in normal and leukaemic samples, and the results were compared with those of conventional RT-PCR. Basal expression of PRAME was determined in 25 blood samples and 25 bone marrow samples from healthy donors, as well as in 12 haematological cell lines (Jurkat, K562, HL60, DOHH2, IM9, Daudi, CEM, KG1, DG75, 8226, U937, Raji). RESULTS: In paediatric acute myeloid leukaemia (AML) (n=22) and acute lymphoblastic leukaemia (ALL) (n=17), and in adult AML (n=20), abnormal PRAME expression was found in 41%, 35% and 40% of cases, respectively. To assess the sensitivity of PRAME for monitoring MRD, PRAME-positive t(8;21) AML samples with detectable AML1/ETO expression by conventional RT-PCR (n=17) were assessed for quantitative expression of AML1/ETO and PRAME. The expression of these genes was closely correlated. To confirm that PRAME expression was correlated with clinical data, the expression of PRAME was also sequentially followed in patients (n=13) from onset to cytological remission or relapse. The cytological and molecular data were highly correlated in all patients. CONCLUSIONS: Our data confirm that PRAME quantification by real-time RT-PCR appears suitable for monitoring MRD in PRAME-positive leukaemia. PMID- 16681424 TI - Association of high-sensitive C-reactive protein with advanced stage beta-cell dysfunction and insulin resistance in patients with type 2 diabetes mellitus. AB - BACKGROUND: Type 2 diabetes mellitus is associated with increased cardiovascular risk. One laboratory marker for cardiovascular risk assessment is high sensitivity C-reactive protein (hsCRP). METHODS: This cross-sectional study attempted to analyze the association of hsCRP levels with insulin resistance, beta-cell dysfunction and macrovascular disease in 4270 non-insulin-treated patients with type 2 diabetes [2146 male, 2124 female; mean age +/-SD, 63.9+/ 11.1 years; body mass index (BMI) 30.1+/-5.5 kg/m(2); disease duration 5.4+/-5.6 years; hemoglobin A(1c) (HbA(1c)) 6.8+/-1.3%]. It consisted of a single morning visit with collection of a fasting blood sample. Observational parameters included several clinical scores and laboratory biomarkers. RESULTS: Stratification into cardiovascular risk groups according to hsCRP levels revealed that 934 patients had low risk (hsCRP <1 mg/L), 1369 patients had intermediate risk (hsCRP 1-3 mg/L), 1352 patients had high risk (hsCRP >3-10 mg/L), and 610 patients had unspecific hsCRP elevation (>10 mg/L). Increased hsCRP levels were associated with other indicators of diabetes-related cardiovascular risk (homeostatic model assessment, intact proinsulin, insulin, BMI, beta-cell dysfunction, all p<0.001), but showed no correlation with disease duration or glucose control. The majority of the patients were treated with diet (34.1%; hsCRP levels 2.85+/-2.39 mg/L) or metformin monotherapy (21.1%; 2.95+/-2.50 mg/L hsCRP). The highest hsCRP levels were observed in patients treated with sulfonylurea (17.0%; 3.00+/-2.43 mg/L). CONCLUSIONS: Our results indicate that hsCRP may be used as a cardiovascular risk marker in patients with type 2 diabetes mellitus and should be evaluated in further prospective studies. PMID- 16681425 TI - A longitudinal evaluation of urinary glycosaminoglycan excretion in normoalbuminuric type 1 diabetic patients. AB - BACKGROUND: Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A(1c) (HbA(1c)) > or =8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA(1c) < 8.0% at baseline who maintained better metabolic control over time. METHODS: We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA(1c) levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (+/-SD) follow-up of 6.8+/-1.1 years. RESULTS: All patients remained normoalbuminuric at follow-up, and had normal urinary alpha(1)-microglobulin levels. In patients with HbA(1c) <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA(1c) > or =8.0% and were significantly related to both HbA(1c) levels and the duration of poor glycemic control. CONCLUSIONS: Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes. PMID- 16681426 TI - National survey on the execution of the oral glucose tolerance test (OGTT) in a representative cohort of Italian laboratories. AB - BACKGROUND: Recently revised diagnostic criteria for diabetes mellitus and the lack of universal agreement on the methodology for the screening and diagnosis of gestational diabetes mellitus (GDM) still generate inconsistency in execution of the oral glucose tolerance test (OGTT). The aim of the present survey was to evaluate the adherence of Italian laboratories to the internationally accepted guidelines in carrying out the OGTT for the diagnosis of diabetes in the general population and for the screening of GDM. METHODS: A questionnaire was designed to investigate the following issues related to the OGTT: 1) the relationship between laboratories and diabetes centres for the definition of standard protocols; 2) the amount of glucose administered; 3) the number and timing of blood samples; 4) the procedures used for the screening and diagnosis of GDM; and 5) reference to WHO guidelines for the interpretation of the results. The questionnaire was administered to 400 specialists in laboratory medicine working in public or private laboratories nationwide participating in the "Italian External Evaluation of Quality in Laboratory Medicine" Study Group. RESULTS: The survey was completed in the period from June to September 2003. In the observation period, 241 questionnaires were returned by specialists working in laboratories scattered throughout 15 out of the 20 Italian regions. Only 50% of the laboratories performed the OGTT according to protocols defined in agreement with local reference diabetes centres. OGTT using 75 g of glucose in adults and 1.75 g/kg for children as recommended by WHO was performed by 87.1% of the laboratories. WHO indications to collect samples at baseline and at 120 min were followed by 33.2% of the centres. Higher variability was highlighted with respect to the methodology for GDM screening: 49.8% of the laboratories always adopted the two step procedure consisting of a glucose challenge test (GCT) and subsequent OGTT in positive cases; 4.9% performed the 100-g OGTT with four blood samples; 1.6% the 75-g OGTT with two blood samples; and 2.7% the 75-g OGTT with four blood samples. More than 30% of the centres referred to different diagnostic schemes, 62% of which used individually chosen procedures amongst those reported above, 19% used only the GCT and no subsequent OGTT in positive cases, and 18.4% used a variety of completely different, arbitrarily chosen methods. Finally, only 25.6% of the laboratories referred to the WHO limits for interpretation of the results. CONCLUSIONS: For the Italian laboratories investigated, relevant variability was highlighted for performance of the OGTT in general and GDM screening in particular. A variable relationship between laboratories and diabetes centres was also detected, which might represent a relevant indicator for the need for rationalisation or standardisation of the method for performing an OGTT. These data highlight the need for greater collaboration between these different bodies. We suggest that other similar investigations should be carried out in other countries within the framework of the IFCC Global Campaign on Diabetes Mellitus. PMID- 16681427 TI - The reduction of cholesteryl linoleate in lipoproteins: an index of clinical severity in beta-thalassemia/Hb E. AB - BACKGROUND: Oxidative modification of lipoproteins has been reported in beta thalassemia and has been suggested to relate to atherogenesis-risk. This study focused on the change in cholesteryl esters in plasma lipoproteins under oxidative stress resulting from iron overload in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients. METHODS: Markers of oxidative damage and cholesteryl esters (CEs) were measured in plasma and lipo-proteins from 30 beta-thal/Hb E patients and compared to those from 10 healthy volunteers. CEs in plasma, low density lipoprotein (LDL) and high-density lipoprotein (HDL) were separated and identified using HPLC. RESULTS: beta-Thal/Hb E patients presented iron overload, a precipitous decrease in alpha-tocopherol and increased lipid peroxidation (thiobarbituric acid-reactive substances; TBARs) in both plasma and lipoproteins. Cholesteryl linoleate, the most abundant CE in lipoproteins, showed a reduction of 70% in LDL, while other CEs showed a lower reduction (50%). An inverse relationship between the cholesteryl linoleate/cholesteryl oleate ratio (CL/CO) and the degree of clinical severity suggested that the CL/CO ratio is an index of damaged lipoproteins and could be used as a pathologic marker of underlying iron overload. Good correlation of non-transferrin-bound iron (NTBI) and TBARs (r=0.8, p<0.01) in LDL strongly supported the contention that iron overload is responsible for initiating the lipid peroxidation in beta-thal/Hb E. CONCLUSIONS: This study suggests that cholesteryl linoleate is the primary target of oxidative modification induced by NTBI in beta-thal/Hb E patients and that reduction in cholesteryl linoleate in lipoproteins could be used as a severity index for beta thal/Hb E. PMID- 16681428 TI - Alterations in serum glycosaminoglycan profiles in Graves' patients. AB - BACKGROUND: Qualitative and quantitative analyses of glycosaminoglycans (GAGs) in serum obtained from Graves' disease (GD) patients without extrathyroidal complications were carried out to provide a clearer understanding of the role of these macromolecules in the disease pathogenesis. METHODS: GAGs were isolated from-SIMEL (Italian Society of Laboratory Medicine) Inter-associative Study Group on Diabetes Mellitus serum of 17 GD patients before treatment and after attainment of the euthyroid state, as well as from 20 healthy individuals. GAGs were quantified using the hexuronic acid assay and subjected to electrophoretic fractionation. RESULTS: Increased amounts of total GAGs were found in GD patients. Attainment of euthyroidism led to a decrease in, but not normalization of, total serum GAGs level. Electrophoretic analyses of GAGs before and after treatment identified the presence of chondroitin sulfate (CS), heparan sulfate/heparin (HS/H) and dermatan sulfate (DS) in serum from healthy subjects and GD patients. CS was the predominant serum GAG constituent in all subjects investigated. Enhanced CS levels in both GD patient groups were accompanied by increased structural heterogeneity of these compounds. Normalization of thyroid function did not change CS levels. DS levels in serum of untreated GD patients were elevated in comparison to healthy subjects. Anti-thyroid treatment led to a significant decrease in DS to levels below those in controls. DS in all serum samples investigated displayed a similar structure. HS/H levels in serum of untreated GD patients was seven-fold higher than in healthy subjects. In addition, HS/H in untreated GD patients were characterized by higher structural heterogeneity than those isolated from control subjects and euthyroid GD patients. Anti-thyroid therapy led to a decrease in HS/H concentrations towards normal values. CONCLUSIONS: Our results indicate that in the course of GD, the metabolism of particular types of GAGs is regulated by different mechanisms, including a hyperthyroid state and immunological abnormalities. Furthermore, qualitative and quantitative changes in serum GAGs seem to reflect GD-associated systemic changes in extracellular matrix properties. PMID- 16681430 TI - Sandwich ELISAs for soluble immunoglobulin superfamily receptor translocation associated 2 (IRTA2)/FcRH5 (CD307) proteins in human sera. AB - BACKGROUND: The immunoglobulin superfamily receptor translocation-associated 2 (IRTA2) gene encodes both membrane and secreted forms of the B-cell differentiation antigen (also identified as FcRH5). The membrane form is highly expressed on the surface of hairy cell leukemia (HCL) cells from patients. This study aimed to develop immunoassays for soluble IRTA2/FcRH5 proteins in human serum. METHODS: Two sandwich ELISAs for soluble IRTA2/FcRH5 were designed using two pairs of monoclonal antibodies specific to four different epitopes on IRTA2/FcRH5. RESULTS: In both ELISAs, the lower limit of quantitation for soluble IRTA2/FcRH5 in human serum was 30 ng/mL. The analytical recovery of 0.3-2.1 ng/mL of IRTA2/FcRH5-Fc used as the standard, from a 100-fold dilution of IRTA2/FcRH5 free sera, was 94-106% for ELISA #1 and 89-97% for ELISA #2. Between-assay coefficients of variance were 7.7-17.6% for ELISA #1 and 7.7-32.7% for ELISA #2. Both ELISAs detected soluble IRTA2/FcRH5 protein in sera from normal donors (median 169 ng/mL in ELISA #1 and 146 ng/mL in ELISA #2, n=123) without correlations to gender or age. A marked increase in soluble IRTA2/FcRH5 levels was observed in samples from patients with HCL (medians 719 ng/mL in ELISA #1 and 754 ng/mL in ELISA #2, n=44). CONCLUSIONS: These ELISAs may be useful for monitoring soluble IRTA2/FcRH5 in HCL and other B-cell malignancies. PMID- 16681429 TI - Alterations in anti-oxidative defence enzymes in erythrocytes from sporadic amyotrophic lateral sclerosis (SALS) and familial ALS patients. AB - BACKGROUND: Overproduction of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) may be an important factor in the pathogenesis of amyotrophic lateral sclerosis (ALS). Owing to their ability to permeate through biological membranes, excess NO and H(2)O(2) may be present in the media surrounding motor neurones. Anti oxidative defence enzymes (ADEs) in erythrocytes are capable of detoxifying reactive oxygen species (produced endogenously or exogenously), but may also be structurally modified and inactivated by reactive oxygen and nitrogen species. Both balanced and coordinated ADE activities are of utmost importance for their correct physiological function. METHODS: We determined activity of the following ADEs: copper-zinc superoxide dismutase (CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in erythrocytes from sporadic ALS patients [SALS (-/+)], familial ALS patients with the Leu144Phe mutation in the SOD1 gene [FALS (+/+)], asymptomatic carriers with the Leu144Phe mutation in the SOD1 gene (+/-), and control subjects (-/-). We also examined the in vitro effect of diethyldithiocarbamate (DDC) on CuZn SOD activity in erythrocytes from FALS patients, SALS patients and control subjects. RESULTS: The influence of the Leu144Phe mutation and/or disease was apparent for ADE activities measured in all three patient groups. The SOD1 gene mutation decreased CuZn SOD and GSH-Px activity (two-way ANOVA, significant mutation effect). We noted that the disease also contributed to decreased CuZn SOD activity in SALS patients in comparison with the control group (two-way ANOVA, mutation and disease effect). The disease also influenced CAT and GR activity. CAT activity was decreased in both SALS and FALS patients. In all three patient groups, GR activity was higher than in the control group. Finally, DDC inhibited CuZn SOD activity in erythrocytes from control subjects, FALS (Leu144Phe) patients and SALS patients; however, its effect was more pronounced and significant in FALS patients. CONCLUSIONS: Changes in erythrocyte ADE activities suggest that oxidative stress, involved in the motor neurone pathogenesis of SALS and FALS, also has systemic effects. Differences in ADE systems between the study groups revealed the presence of different types of oxidative pressure, indicating the potential additional benefit of individually designed anti-oxidant cocktail therapies. PMID- 16681431 TI - Utilizing ultrafiltration to remove alkaline phosphatase from clinical analyzer water. AB - Alkaline phosphatase (ALP) conjugated to antibodies is often used in enzyme immunoassays (EIAs). These assays are notably sensitive to experimental conditions. A possible source of interference is bacterial ALP, which is released when bacterial contamination occurs in clinical analyzers. Preliminary experiments led to the selection of a detection kit, ALP source, and specific types of tubes for collecting water samples and performing assays. The release of ALP from various strains of bacteria identified in pure water was demonstrated (10-30 x 10(6) cfu/mL released 6-10 microU/microL). It was shown that ultrafiltration is totally efficient in removing ALP from water, while residual ALP activity (2.21 microU/microL after filtration of an ALP solution of 6.22 microU/microL) was observed after filtration using a 0.22-mum filter. PMID- 16681432 TI - Measurement of serum monoclonal components: comparison between densitometry and capillary zone electrophoresis. AB - Quantitative measurement of serum monoclonal protein (M-protein) is one of the most important tools for monitoring disease activity in monoclonal gammopathies. The aims of this study were to evaluate serum M-protein quantification by capillary zone electrophoresis (CZE) and to compare results with those obtained by densitometric scanning of high-resolution agarose gel electrophoresis (HRE AGE). The evaluation was carried out on 82 samples from patients with various monoclonal gammopathies. All the suspected M-proteins were confirmed and characterised by immunofixation on agarose gel (IFE). CZE was performed on a Paragon CZE 2000 system (Beckman Coulter). Passing-Bablok regression was: y (CZE)=1.27 x(HRE-AGE)-5.21 g/L. The correlation coefficient was 0.92. Bland Altman analysis demonstrated a mean difference of -1.83 g/L (95% CI -0.76 to 2.90) with clear evidence of a concentration-related bias. Densitometry gave higher values at low M-spikes (<20 g/L), whereas CZE gave higher values at large M-spikes (>20 g/L). The concentration-related bias was found to be independent of the immunoglobulin isotype. In conclusion, to compare previous results obtained by M-protein densitometric scanning with those obtained by direct measurement of CZE peaks, the calculation of a univocal transforming factor appears to be unreliable. PMID- 16681433 TI - Salivary aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase: possible markers in periodontal diseases? AB - BACKGROUND: Saliva can be used as a diagnostic fluid in medicine. Components of saliva proposed as disease markers include enzymes (alkaline phosphatase, esterase, glucuronidase, aminopeptidase), immunoglobulins (IgA, IgG), and hormones (steroid hormones). Many of these salivary components appeared to be useful biochemical markers of the evolution of periodontal disease, for which salivary analysis can offer a cost-effective approach for monitoring the disease. The salivary components proposed as markers for periodontal disease activity are aspartate aminotransferase (AST), alkaline phosphatase (ALP), aminopeptidases, and glucuronidases. The purpose of our study was to illustrate the influence of periodontal disease on the level of salivary AST, alanine aminotransferase (ALT) and ALP. METHODS: All clinical periodontal examinations were performed by the same periodontist. All patients included in the study presented a probing depth >5 mm, bleeding on probing and alveolar bone loss >40%. Salivary AST, ALT and ALP activities were measured using DiaSys analysis kits from Diagnostic Systems. The methods were adapted for saliva. RESULTS: Salivary AST activity in patients with periodontal disease was significantly increased (p<0.01) (median 81.75+/-23 U/L) compared with controls (15.25+/-10.5 U/L). Salivary ALT activity was not significantly modified in saliva from patients with periodontal disease compared with the control group. Our results showed a significant (p<0.01) increase in salivary ALP activity (34.38+/-1.5 U/L) in patients with periodontal disease compared with controls (6.6+/-4.2 U/L). CONCLUSIONS: Our results revealed that periodontal destruction such as periodontal pockets, gingival bleeding and suppuration are related to higher ALP and AST levels in saliva. Salivary AST could be used as a useful marker for monitoring periodontal disease. The increase in salivary ALP activity in periodontitis demonstrated could be associated with alveolar bone loss, a key feature of periodontal disease. More studies are necessary to evaluate which specific clinical, microbiological and histological characteristics of periodontal disease are associated with elevated levels of AST and ALP in saliva. PMID- 16681434 TI - Reticulocyte count, mean reticulocyte volume, immature reticulocyte fraction, and mean sphered cell volume in elite athletes: reference values and comparison with the general population. AB - BACKGROUND: The role of measurement of reticulocytes and their parameters is growing in sports medicine. The use of reticulocyte counts in protocols for evaluating and screening for the suspected abuse of hormones that stimulate the bone marrow is an example. Reticulocytes are also important for evaluation of the performance and general health status of athletes, especially for monitoring therapies and diets. The current availability of fully automated haematological systems that can measure reticulocyte numbers and characteristics (volume, density) increases the potential use of these parameters in laboratory and sports medicine. Few studies have considered the application of these parameters in athletes and a lack of specific reference ranges means that their valid clinical use is difficult. METHODS: Using a Coulter LH700 instrument, we measured reticulocyte count (Retics), mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF), and mean sphered cell volume (MSCV) in 106 male professional elite athletes (football and rugby players and skiers). Reference intervals for the athletes were compared with the intervals found for a control group of 73 age-matched males. RESULTS: We calculated the following reference intervals: 0.30-1.54% for Retics, 93.1-114.8 fL for MRV, 0.18-0.39% for IRF, and 76.8-94.5 fL for MSCV. CONCLUSIONS: No statistically significant differences were observed for Retics, MRV, IRF, and MSCV between elite athletes and controls. Significant differences were observed for haemoglobin (Hb), erythrocytes, haematocrit (Ht), and mean corpuscular volume. Moreover, no statistical differences were observed among different sports, whereas differences were remarked in football and rugby players between the samples drawn before the start of competitive season and the samples drawn during the season, demonstrating that reticulocyte counts and parameters are useful for monitoring sportsmen. PMID- 16681436 TI - The effects of adrenocorticotrophic hormone and cortisol on homocysteine and vitamin B concentrations. AB - BACKGROUND: Homocysteine metabolism is mainly governed by serum concentrations of folate and vitamin B(12), renal function and genetic factors. It is also well documented that endocrinological disturbances influence homocysteine metabolism. However, studies on the hormones of the hypothalamic-pituitary-adrenal axis have given conflicting results. METHODS: A total of 30 healthy young males were randomised into three groups of equal size; one group received adrenocorticotrophic hormone (ACTH)(1-24) 1 mg i.m. daily for 4 days, another group was treated with cortisol 50 mg i.m. t.i.d. for 4 days, while a control group was observed for 4 days. Fasting blood samples were collected before and after treatment or observation. RESULTS: The pattern of changes was the same for the ACTH and cortisol groups; there were significant decreases in serum concentrations of folate (23% and 24%) and cobalamines (13% and 19%) and decreases in plasma total homocysteine concentrations that did not reach significance. There were no changes in the control group. CONCLUSIONS: The virtually identical pattern of changes in both treatment groups suggests that the effects were mediated by cortisol. The reductions in serum concentrations of folate and cobalamines call for further research. PMID- 16681435 TI - Serum homocysteine levels and paraoxonase 1 activity in preschool aged children in Greece. AB - BACKGROUND: Over the past decade, it has been well established that elevated total serum homocysteine (tHcy) in adults is associated with increased risk of cardiovascular and thromboembolic diseases. Since risk factors for such diseases are established at a young age, the aim of the present study was to measure serum tHcy levels in 134 (71 boys, 63 girls) randomly selected healthy preschool children aged 4-6 years (mean 5.1), and to investigate possible correlation with paraoxonase 1 (PON1) activity, an antioxidant enzyme that contributes to the antiatherogenic properties of high-density lipoprotein (HDL). METHODS: tHcy was determined using an IMX tHcy assay (FPIA). PON1 was measured by a spectrophotometric method at 412 nm. RESULTS: Mean serum tHcy was 7.71+/-2.35 mumol/L. A relatively significant percentage (15.6%) of boys and girls had elevated serum tHcy levels (>10 mumol/L). tHcy levels were slightly higher in girls compared to boys (8.20+/-2.80 vs. 7.29+/-1.79 mumol/L, respectively; p<0.11). There was no significant interaction between age and tHcy levels. Mean PON1 activity was 124.86+/-66.62 U/L. No statistical difference in enzyme activity was observed between boys and girls (126.81+/-69.99 vs. 121.74+/-64.78 U/L) was observed. On the contrary, a weak negative relationship between tHcy concentration and PON1 activity was detected, with Pearson's correlation coefficient of r=-0.27. CONCLUSIONS: The significant percentage of elevated tHcy levels observed in healthy preschool cases and the negative tHcy correlation with PON1 activity are reported for the first time. Since children with a family history of cardiovascular disease have higher levels of serum Hcy, tHcy screening in children, even of this age, in relation to other parameters, such the protective PON1, might prove a useful prevention procedure for the genetic risk of premature atherosclerosis. PMID- 16681437 TI - Plasma, salivary and urinary cotinine in non-smoker Italian women exposed and unexposed to environmental tobacco smoking (SEASD study). AB - BACKGROUND: The aim of this study was to compare cotinine determinations in three biological fluids for assessing environmental tobacco smoke (ETS) exposure in female non-smokers (n=1605) in Italy. METHODS: Information about ETS exposure at home, in the workplace, and in other places within the previous week was collected via questionnaire. Plasma, salivary and urinary cotinine levels were measured. Cotinine levels of > or =0.1 ng/mL for plasma, > or =0.2 ng/mL for saliva, and > or =0.5 ng/mL for urine were used to determine biochemical exposure. RESULTS: Median cotinine levels were significantly higher in exposed than in unexposed women (0.21 vs. 0.05 ng/mL in plasma, 0.80 vs. 0.41 ng/mL in saliva, and 9.74 vs. 5.30 ng/mL in urine). Self-reported ETS exposure was significantly related to biochemical exposure [odds ratio 2.99, (95% CI 2.40 3.72) for plasma; 1.90 (1.51-2.39) for saliva; and 2.67 (2.14-3.34) for urine]. Cotinine significantly increased with increasing exposure level, regardless of the exposure source. Among self-reported exposed subjects, higher percentages of cotinine level above the cut-off, i.e., indicating exposure, were found in saliva (76%) and urine (75%) than in plasma (52%). CONCLUSIONS: In general, women correctly reported their ETS exposure status. Both non-invasive salivary and urinary cotinine determinations seem preferable in epidemiological studies, in view of their higher sensitivity, when compared to plasma cotinine. PMID- 16681438 TI - Cut-off values for total serum immunoglobulin E between non-atopic and atopic children in north-west Croatia. AB - BACKGROUND: The aim of this study was to determine cut-off values for total serum immunoglobulin E (IgE) between non-atopic and atopic children with respiratory symptoms. Children of 0-16 years of age were evaluated for respiratory symptoms of >4-week duration. METHODS: Children were divided into two groups: non-atopic children (n=3355) who were non-IgE-sensitized with undetectable allergen-specific IgE (<0.35 kIU(A)/L), and atopic children (n=4620) who were sensitized to > or =1 allergens (specific IgE > or =0.35 kIU(A)/L). Upper and lower centiles were determined and cut-off values calculated using receiver operating characteristic (ROC) analysis. RESULTS: Serum total IgE increased with age in both groups, although at a variable level and rate, and reached a plateau at 9 and 10 years in non-atopic and atopic children, respectively. Atopic children had on average 14 fold higher serum total IgE compared to non-atopic children. In both groups, the median was lower than the corresponding mean and the distribution skewness was always positive (group I, 0.87; group II, 0.91). In almost all age groups, the 95th percentile for non-atopic children corresponded to the calculated cut-off values, whereas the 10th percentile for atopic children corresponded to the respective cut-off values only until the age of 8 years, after which greater differences between the cut-off values and the 10th percentile were recorded. Cut off values for total serum IgE in children up to 16 years were determined with diagnostic sensitivity, specificity and area under the ROC curve of 96%, 91% and 0.950, respectively. CONCLUSIONS: The 95th percentile for total IgE in non-atopic children and the 10th percentile in atopic children could be taken as cut-off values in children up to 8 years of age, after which significant percentile discrepancies between non-atopic and atopic children were recorded. Since atopic subjects show a more irregular centile distribution, cut-off values are best determined by ROC analysis. PMID- 16681439 TI - Thyroglobulin assay during thyroxine treatment in low-risk differentiated thyroid cancer management: comparison with recombinant human thyrotropin-stimulated assay and imaging procedures. AB - BACKGROUND: Circulating human thyroglobulin (TG) measurement has a pivotal role in the management of patients affected by differentiated thyroid cancer (DTC). Undetectable thyrotropin (TSH)-stimulated serum TG after thyroid ablation (i.e., thyroidectomy and radioiodine) implies the absence of residual or relapsing DTC. Recently, high-cost recombinant human TSH (rhTSH) was proposed for TG stimulation to avoid uncomfortable thyroxine (T(4)) withdrawal. However, only a small fraction of relapsing DTC patients showed undetectable TG under T(4) treatment (onT(4)-TG) by high-sensitivity assays. The present study was undertaken to compare onT(4)-TG with the rhTSH-stimulated TG assay (rhTSH-TG), (131)I scanning and neck ultrasound (US) with fine-needle aspiration biopsy. METHODS: We enrolled 117 patients with histologically proven DTC treated by total thyroidectomy and radioiodine. Inclusion criteria were: complete tumour excision, no radioiodine uptake outside of the thyroid bed at post-treatment scan and undetectable onT(4) TG 3 months after primary treatment. At 1 year after radioiodine treatment, all patients underwent onT(4)-TG assay, rhTSH-stimulated TG assay, (131)I scanning and neck US. Based on histology, clinical data and long-term follow-up, persistent/relapsing disease was confirmed in 14 patients. RESULTS: onT(4)- and rhTSH-TG were positive in 10 and 12 patients, respectively and two patients converted from undetectable to detectable TG after rhTSH administration. Neck US was positive in 10 patients and a combination of US with onT(4)- and rhTSH-TG assays showed positivity in 13 and 14 out 14 patients, respectively. A radioiodine scan was positive in six patients, all with positive onT(4)- and rhTSH-TG levels. Globally, the negative predictive value of the onT(4)- and rhTSH TG assays was 99% and 100%, respectively, and 104 rhTSH stimulations had to be performed to detect one local recurrence with negative onT(4)-TG. CONCLUSIONS: Our preliminary data need further confirmation on larger groups of patients, but seem to indicate that onT(4)-TG assay by a high-sensitivity method combined with neck US may avoid rhTSH stimulation in low-risk DTC patients after surgery and radioiodine thyroid ablation. PMID- 16681440 TI - Evaluation of serum levels of p53 in hepatocellular carcinoma in Egypt. AB - Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-induced death. Somatic mutation of the p53 gene is the most common genetic abnormality so far described in human cancer and there is evidence that supports a high level of p53 alterations in HCC. The aim of this study was to investigate serum levels of p53 in Egyptian patients with HCC, and its relation to other prognostic factors such as tumor grade, alpha-fetoprotein (AFP), and liver function tests in an attempt to clarify their significance in the pathogenesis of the disease. Liver function tests were carried out and AFP and p53 levels were measured for all individuals studied. Our results show that detection of p53 increased the frequency of HCC prediction from 79.5% to 86.3%. Moreover, significant positive correlation between p53 and tumor size (cm) for tumor grade II and III was identified. In conclusion, serum concentration of p53 protein may be a convenient and useful non-invasive screening test for prediction of HCC. PMID- 16681441 TI - Insufficient filling of vacuum tubes as a cause of microhemolysis and elevated serum lactate dehydrogenase levels. Use of a data-mining technique in evaluation of questionable laboratory test results. AB - Experienced physicians noted unexpectedly elevated concentrations of lactate dehydrogenase in some patient samples, but quality control specimens showed no bias. To evaluate this problem, we used a "latent reference individual extraction method", designed to obtain reference intervals from a laboratory database by excluding individuals who have abnormal results for basic analytes other than the analyte in question, in this case lactate dehydrogenase. The reference interval derived for the suspected year was 264-530 U/L, while that of the previous year was 248-495 U/L. The only change we found was the introduction of an order entry system, which requests precise sampling volumes rather than complete filling of vacuum tubes. The effect of vacuum persistence was tested using ten freshly drawn blood samples. Compared with complete filling, 1/5 filling resulted in average elevations of lactate dehydrogenase, aspartic aminotransferase, and potassium levels of 8.0%, 3.8%, and 3.4%, respectively (all p<0.01). Microhemolysis was confirmed using a urine stick method. The length of time before centrifugation determined the degree of hemolysis, while vacuum during centrifugation did not affect it. Microhemolysis is the probable cause of the suspected pseudo-elevation noted by the physicians. Data-mining methodology represents a valuable tool for monitoring long-term bias in laboratory results. PMID- 16681442 TI - Evaluation of three different specimen types (serum, plasma lithium heparin and serum gel separator) for analysis of certain analytes: clinical significance of differences in results and efficiency in use. AB - BACKGROUND: There is a lack of consensus regarding the most appropriate specimen type for analysis of many biochemistry analytes. The aim of this study was to compare renal and lipid analyte profiles and phenytoin values in plain serum (S), serum gel (G) and plasma (lithium heparin, P) tubes and to investigate the stability of these analytes after prolonged contact with cells or gel at room temperature (RT, 20 degrees C) and as aliquoted and stored at 4 degrees C. METHODS: Primary specimens were centrifuged once, maintained at RT and analysed within 2 h (T(0)) and after 24 h (T(24)) and 48 h (T(48)). For assessment of stability at 4 degrees C, two cell-free aliquots were separated from each of the primary tubes and stored at 4 degrees C and then analysed at T(24) and T(48). Differences in analyte concentrations between tubes at T(0) and following storage (at T(24) and T(48)) were evaluated for both statistical and clinical significance. RESULTS: At T(0) all analytes, except potassium, demonstrated equivalence between serum, gel and plasma tubes. Potassium and creatinine were more stable in gel tubes than in serum/plasma tubes. In contrast, phentytoin was stable in plain serum and plasma up to T(48) at RT, but showed a progressive and clinically significant decrease in concentration in gel tubes at T(24) and T(48) at RT. All analytes except CO(2) were stable up to T(48) when aliquoted and stored at 4 degrees C. CONCLUSIONS: We concluded that the serum gel tube has advantages over plain serum and plasma tubes for measurement of the analytes investigated in this study, with the exception of phenytoin. In practice, the gel tubes demonstrate enhanced analyte stability and reduce the need to aliquot specimens, with greater protection against possible contamination. Further investigation would be required to evaluate a broader range of analytes. PMID- 16681443 TI - Comparative evaluation of a new immunoradiometric assay for corticotropin. AB - We have characterized the performance of a commercial two-site immunoradiometric assay for manual in vitro diagnostic measurement of plasma corticotropin from Scantibodies Laboratory. We compared the results with those of a similar commonly used assay from Nichols Institute Diagnostics that has recently been withdrawn from production. The lower detection limit, range of the standard curve, cross reactivity, and intra-assay and inter-assay imprecision of the two assays are very similar. Measurement of clinical samples and a series of samples from an experimental subject demonstrate high correlations between the two assays. These factors, together with recent clearance by the United States Food and Drug Administration for manual in vitro diagnostic measurement, make the Scantibodies corticotropin immunoradiometric assay an appropriate replacement for the Nichols assay. PMID- 16681444 TI - Mast cells in atherosclerosis as a source of the cytokine RANKL. PMID- 16681445 TI - Falsely increased total serum protein due to dextran interference. PMID- 16681446 TI - Towards optimization of the thrombogenic potential of blood recirculating cardiovascular devices using modeling approaches. PMID- 16681447 TI - New device to detect breath metabolites indicative of breast cancer. PMID- 16681448 TI - Merci Retriever in acute stroke treatment. AB - The Merci Retriever is a device used for mechanical clot extraction in cerebral arteries. It obtained US FDA clearance in August 2004 for recanalization of cerebral arteries in acute stroke. Previously, intravenous recombinant tissue plasminogen activator administered within 3 h from symptom onset was the only other FDA-approved treatment in acute stroke. Stroke from large brain artery occlusion, which has the highest morbidity and mortality rate, is inefficiently treated with intravenous recombinant tissue plasminogen activator and has a high likelihood of hemorrhagic complication. In the multicenter prospective Mechanical Embolus Removal in Cerebral Ischemia trial that led to FDA clearance, the Merci Retriever achieved 48% vessel recanalization when used within 8 h of stroke onset, and resulted in lower morbidity and mortality in revascularized patients. Clinical efficacy trials are needed to determine the place of this device in the treatment of stroke patients. PMID- 16681449 TI - ArteFill: a permanent skin filler. AB - The ideal soft-tissue filler for wrinkles and skin defects should be safe, biocompatible, resistant to phagocytosis, persist and maintain its volume without being resorbed or degraded. ArteFill, an improved, next-generation derivative of Artecoll, is expected to become the first and only FDA-approved permanent filler for use in the USA in 2006 and will be available worldwide. ArteFill consists of polymethylmethacrylate microspheres suspended in a 3.5% solution of bovine collagen containing 0.3% lidocaine. In this article, the pathophysiology, efficacy and safety of ArteFill are discussed and details of its injection technique are provided. Insight into the pharmacoeconomic value of ArteFill over nonpermanent fillers and ArteFill's unique role in the growing world market of dermal fillers is provided. PMID- 16681450 TI - Amplatzer Duct Occluder device: a new technology for the closure of the moderate to-large-sized patent ductus arteriosus. AB - The Amplatzer Duct Occluder is a novel device designed to allow for the transcatheter closure of virtually all sized patent ductus arteriosuses. Prior technologies have been cumbersome and challenging to deploy with somewhat less than ideal closure rates associated with their use. This article summarizes the present knowledge about the Amplatzer Duct Occluder device and its various indications. With the introduction of this device as well as the use of embolization coils, all but the neonatal patent ductus arteriosuses can now be safely, consistently and successfully closed in the cardiac catheterization laboratory. PMID- 16681451 TI - Regulatory issues facing the development of drug-eluting stents: a US FDA perspective. AB - Coronary drug-eluting stents (DES) are a breakthrough technology that has changed the standard of care for many patients undergoing percutaneous intervention for coronary artery disease. Initial trials of two DES demonstrated significant clinical benefit with respect to the need for reintervention when compared with bare metal stents. However, more recent studies of DES involve in-patients with more complex disease, such as bifurcation lesions, chronic total occlusions and multiple-vessel disease. Additionally, DES are now being evaluated in patients previously only considered for surgical intervention. Assessment of DES in these complicated patient populations can lead to challenges in trial design, but the US FDA is willing to consider alternative clinical trial designs and statistical analysis plans. Other complex issues associated with DES include duration of clinical trials to determine safety, and the appropriate dose and duration of concomitant antiplatelet therapy. Finally, the FDA acknowledges that DES are complex products to produce and we believe that through interaction with the FDA during development, difficulties with test methodologies, animal studies and clinical trial designs can be addressed. The future of DES likely involves new stent and carrier materials, including biodegradable materials and new drugs and biologicals. The FDA anticipates continued collaboration with physicians, manufacturers, academic institutions and professional societies. PMID- 16681452 TI - Diabetic retinopathy: the unmet needs for screening and a review of potential solutions. AB - Diabetic retinopathy remains the leading cause of severe vision loss and blindness in the developed world, in spite of recognized ocular treatments that are successful at reducing the rate of vision impairment. Retinal photography appears a promising method to perform screening in such a setting utilizing new 45 degrees + retinal cameras that do not require pupil dilation and can be operated by a trained, nonophthalmic technician. Certain developments may make the photography more successful including the conversion to electronic chip camera sensors that allow each picture as it is taken to be immediately projected onto a monitor for evaluation and assessment. Utilizing a nonmydriatic camera, studies of single-field photography through a dilated pupil have demonstrated superior or equal sensitivity to fundus examination by an ophthalmologist in a number of studies. However, photography without pupil dilation, especially in the older age group may result in poor-quality photographs owing to intense bilateral pupil constriction after the first images and also due to the presence of cataracts. Computer analysis of the retinal images allows extraction of quantitative data, not only of the diabetic lesions but also of vascular changes that, up until now, have been impossible by human grading and potentially allows a much more detailed and quantitative evaluation of the progression of retinopathy over time. When success of image processing algorithms is demonstrated for a large number of images taken under screening conditions, the benefits of retinal photography and image processing to provide timely, reliable, quantitative and cost-effective results, will make this the preferred method over physician examination or human grader evaluation of the images. PMID- 16681453 TI - Visual prosthetics 2006: assessment and expectations. AB - This report provides a brief overview of blinding eye diseases for which prosthetic vision may hold promise as a treatment modality, and of current and near-term technological approaches towards the creation of prosthetic interfaces with the remaining visual system. Principal anatomical, physiological, technological and functional obstacles and possible solutions are outlined, and references are provided to pioneering work by over a dozen groups on four continents. PMID- 16681454 TI - Novel lumbosacral axial fixation techniques. AB - For patients with low back pain secondary to pathological motion of an unstable lumbar motion segment, interbody fusion may be indicated. Numerous open and minimally invasive techniques have been traditionally used, but all suffer from shortcomings related to biomechanics or inherent iatrogenic destabilization. A novel transaxial approach to the lumbosacral junction has recently been described which appears to obviate many of the limitations of previous techniques. Preliminary results of the transaxial approach to lumbosacral fixation appear promising. PMID- 16681455 TI - Robotic cardiovascular surgery. AB - Cardiovascular surgery has traditionally been performed through a median sternotomy, allowing the surgeon generous access to the heart and surrounding great vessels. Recently, less invasive methods have been developed to allow the surgeon the same amount of dexterity and accessibility to the heart, thus resulting in a paradigm shift in cardiac surgery. Originally, long instruments without pivot points were used, however; with the application of robotic telemanipulation systems that allow for improved dexterity, the surgeon is able to perform cardiac surgery from a distance not previously possible. In this rapidly evolving field, this article reviews the recent history and clinical results of robotics in cardiovascular surgery. PMID- 16681456 TI - Currently available biomaterials for use in cardiopulmonary bypass. AB - Cardiopulmonary bypass (CPB) represents one of the most important technical innovations in healthcare history, yet the systemic responses to CPB remain a fundamentally unresolved problem. Study of the blood-biomaterial interaction and development of biocompatible materials is intimately related to efforts to optimize patient outcome following CPB. This article reviews the design innovations in biomaterial surfaces that have been introduced into clinical practice in an attempt to ameliorate the detrimental consequences of CPB, contrasting the actual clinical improvements and patient benefits achieved against those predicted on the basis of theory and in vitro testing. Some discussion of the underlying mechanisms of action as presently understood is provided and the current limitations of biomaterial-dependent strategies to improve outcome following CPB are addressed. PMID- 16681457 TI - Aminolevulinic acid photodynamic therapy: medical evidence for its expanded use. AB - Photodynamic therapy (PDT) has gained much popularity over the past several years as clinicians and researchers have expanded upon its indications and how one should utilize PDT in everyday practice. PDT is not a new field within dermatology or medicine in general, but for all practical reasons, PDT is considered to still be in its infancy. This article will review the history of PDT, explore PDT in both the US and European market place, and show how PDT is best utilized today in the USA. Finally, future ideas/thoughts on where PDT is heading will be surmised. PMID- 16681458 TI - Biological skin substitutes for wound cover and closure. AB - In the past 30 years, the development and use of artificial skin in the treatment of acute and chronic wounds has advanced from an experimental concept to a working reality. However, while there have been an increasing number of artificial skin substitutes licensed for clinical use, they have yet to supplant the current gold standard of an autologous tissue graft for most applications. This article reviews the advantages and disadvantages of the currently available, biologically based substitutes, with special emphasis on their relative efficacy and suitability for treatment of particular wound types. Economic considerations, desirable improvements of currently available materials and the potential impact of future advances in the field will also be discussed. PMID- 16681459 TI - Design and development of mechanical embolic protection devices. AB - Carotid angioplasty and stenting (CAS) is rapidly becoming an acceptable alternative to carotid endarterectomy in many patients. Much of the success of CAS is credited to advances in equipment dedicated to this minimally invasive approach. Lower profile delivery systems, tapered stent designs and most importantly, development of mechanical cerebral embolic protection devices (EPDs) have contributed to a reduction in periprocedural neurological complications that now rival results of carotid endarterectomy. Despite a lack of level one evidence in support of EPDs, a meta-analysis as well as two recent institutional reports of CAS with and without EPDs suggest a reduced stroke risk when a mechanical protection device is incorporated as part of the procedure. Since the original description by Theron and colleagues, embolic protection systems are markedly improved in ease of use and effectiveness. The three primary groups of EPDs are distal balloon occlusion, distal filtration and proximal occlusion. Although the ideal EPD has yet to materialize, this review provides insight into current design systems and the accompanying strengths and weaknesses of each. PMID- 16681460 TI - New developments in ureteral stent design, materials and coatings. AB - Ureteral stents are used in a variety of urological diseases and procedures. The majority of patients with indwelling ureteral stents experience bothersome symptoms and are at increased risk for urinary tract infection. Stent encrustation and the associated complications can also result in significant patient morbidity. The development of new stent designs using novel biomaterials, stent coatings and drug-eluting technologies are being applied to reduce the disadvantageous features of ureteral stents. PMID- 16681461 TI - Testosterone and vascular reactivity. AB - The mechanisms by which male sex hormones modulate cardiovascular function are a subject of contemporary interest. Several lines of evidence indicate that androgens can exert acute vasorelaxing effects. On the other hand, chronic exposure to androgens has been shown to promote increases in blood pressure and compromise renal function. In the present issue of Clinical Science, Malkin and co-workers show that testosterone replacement impairs vascular reactivity in men with androgen deficiency. These studies may shed light on the functional and therapeutic significance of the diverging acute and chronic cardiovascular effects of androgens. PMID- 16681463 TI - Fabrication and detection of tissue-engineered bones with bio-derived scaffolds in a rotating bioreactor. AB - In order to explore the methods for commercialized bone tissue engineering, engineered bones should be cultivated in bioreactors to realize three-dimensional culture under well-defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1-month-old Zelanian rabbits were inoculated on to the BDBS (bio-derived bone scaffolds) to investigate the three-dimensional fabrication of engineered bone in an RWVB (rotating-wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent protein, were respectively seeded at 2 x 10(6) and 1 x 10(6) cells x ml(-1) on to the BDBS and then cultured in a T-flask and an RWVB for 1 week. The morphologies and structure of the fabricated bone were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von-Kossa staining on mineralized nodules, type I collagen and bone morphogenetic protein-2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell-seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T-flask. Bone tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three-dimensional fabrication of engineered bone could be realized. PMID- 16681462 TI - Evolution of two alanine glyoxylate aminotransferases in mosquito. AB - In the mosquito, transamination of 3-HK (3-hydroxykynurenine) to XA (xanthurenic acid) is catalysed by an AGT (alanine glyoxylate aminotransferase) and is the major branch pathway of tryptophan metabolism. Interestingly, malaria parasites hijack this pathway to use XA as a chemical signal for development in the mosquito. Here, we report that the mosquito has two AGT isoenzymes. One is the previously cloned AeHKT [Aedes aegypti HKT (3-HK transaminase)] [Han, Fang and Li (2002) J. Biol. Chem. 277, 15781-15787], similar to hAGT (human AGT), which primarily catalyses 3-HK to XA in mosquitoes, and the other is a typical dipteran insect AGT. We cloned the second AGT from Ae. aegypti mosquitoes [AeAGT (Ae. aegypti AGT)], overexpressed the enzyme in baculovirus/insect cells and determined its biochemical characteristics. We also expressed hAGT for a comparative study. The new cloned AeAGT is highly substrate-specific when compared with hAGT and the previously reported AeHKT and Drosophila AGT, and is translated mainly in pupae and adults, which contrasts with AeHKT that is expressed primarily in larvae. Our results suggest that the physiological requirements of mosquitoes and the interaction between the mosquito and its host appear to be the driving force in mosquito AGT evolution. PMID- 16681464 TI - The power of the written word. PMID- 16681465 TI - Are androgens critical for penile erections in humans? Examining the clinical and preclinical evidence. AB - Androgens are deemed critical for penile-tissue development, growth, and maintenance of erectile function, however, their role in erection, especially in humans, remains controversial. In this review, we summarize information from clinical and animal model studies to provide a comprehensive and rational argument for the role of androgens, or lack thereof, on penile erection ability in humans. The goal of this review is to present the clinical and preclinical evidence available in the literature with regard to testosterone and erectile physiology and engage the reader in this discussion. Ultimately, each reader will have to form his or her own conclusions based on the existing evidence. In humans, androgen-deficiency manifestations are noted in clinical situations such as: (i) inadequate development of the penis; and (ii) loss of erectile function in prostate cancer and benign prostatic hyperplasia patients managed with medical or surgical castration or antiandrogen therapy. Androgen treatment causes: (i) improvement in sexual function in hypogonadal patients treated with androgen supplementation; (ii) improvement in nocturnal penile tumescence in hypogonadal patients treated with androgens; (iii) improvement in erectile function with androgen supplementation in patients who did not respond to phosphodiesterase type 5 inhibitor therapy initially; and (iv) improvement in the well-being, mood, energy, and sexual function in aging men who have testosterone deficiency treated with androgen therapy. In contrast to animals, especially rodents in which the adrenal cortex does not synthesize androgens, the human adrenal is a source of peripherally circulating androgen precursors, thus, complete androgen insufficiency may not be observed in men at a younger age. Furthermore, in light of the concept that a threshold of androgen levels exists in animals and humans below which sexual function is diminished, further contributes to the complexity of understanding androgens role in erections, especially in humans. Nevertheless, based on the preclinical and clinical data available in the literature, to date, we infer that androgens play a critical role in maintaining erectile physiology in humans. PMID- 16681466 TI - Female hypoactive sexual desire disorder: History and current status. AB - INTRODUCTION: Hypoactive sexual desire disorder (HSDD) is a common problem that is often treatment refractory. This refractoriness to treatment is reflective of our lack of knowledge concerning the determinants of sexual libido in women. AIM: To review the development and current status of information concerning the diagnosis and treatment of HSDD. METHODOLOGY: Literature on HSDD published between 1950 and 2005 was reviewed. RESULTS: Historically, HSDD was considered to be a psychiatric disorder. Thus, the diagnostic criteria utilized in studies of interventions for this disorder are based on the Diagnostic and Statistical Manual of the American Psychiatric Association. This system was never designed to encompass organic causes of sexual dysfunction and has evolved by expert opinion. If the syndrome is poorly defined by these diagnostic criteria, this could limit progress in this field. Epidemiological studies have found that approximately 24 43% of women complain of low sexual desire in the preceding year. The percentage of the population meeting diagnostic criteria for HSDD is probably much lower. There has been considerable progress in the development of psychometrically sound instruments for the assessment of libido. The development of approaches to treatment was reviewed. Approaches to treatment have evolved in three major areas: psychological, hormonal, and use of psychopharmacological agents. There is some evidence of efficacy utilizing all three approaches. The major evidence of efficacy concerns the use of testosterone therapy. Long-term safety data concerning this treatment are absent. CONCLUSION: There is a rapidly expanding knowledge base concerning the diagnosis and treatment of HSDD. However, the contemporary clinician is faced with the absence of an approved treatment for this disorder and the lack of clear guidelines concerning the indications and safety of the use of non-approved agents. PMID- 16681467 TI - Effect of chronic tadalafil administration on penile hypoxia induced by cavernous neurotomy in the rat. AB - BACKGROUND: Numerous men develop postprostatectomy erectile dysfunction (PPED), due to surgery-related nervous damage. PPED is often refractory to phosphodiesterase type 5 (PDE5) inhibitors therapy. AIM: To verify whether chronic tadalafil (CT) preserves bilateral cavernous neurotomy (BCN)-induced penile damage and hypo-oxygenation. METHODS: In a rat model of BCN we evaluated in vitro and ex vivo effect of CT treatment (2 mg/kg, daily for 3 months). RESULTS: Bilateral cavernous neurotomy induced massive hypoxia and decreased muscle/fiber ratio, completely restored by CT. Hypersensitivity of hypoxic tissues to the relaxant effect of the endothelin type B receptor (ETB) agonist IRL-1620 was observed, along with ETB mRNA and protein overexpression. CT restored sensitivity to IRL-1620, and normalized ETB expression. Hypoxic penis showed increased sensitivity to the relaxant effect of the nitric oxide donor sodium nitroprusside (SNP), while acute tadalafil (100 nM) did not amplify the SNP effect. Accordingly, PDE5 mRNA and protein were reduced in BCN penile tissues. By restoring PDE5, CT decreased SNP-induced relaxation and rescued sensitivity to acute tadalafil. However, in hypoxic penis, CT normalizes neither acetylcholine hyporesponsiveness nor neuronal nitric oxide synthase-endothelial nitric oxide synthase expression. CONCLUSION: Chronic tadalafil restores some of the investigated BCN-induced alterations, including PDE5 and tadalafil efficacy. PMID- 16681468 TI - Effect of the 5-HT receptor agonist DOI on female rat sexual behavior. AB - INTRODUCTION: Female rats display a repertoire of behaviors during a sexual encounter with a male, including sexually receptive (the lordosis response) and proceptive (darts and hops, and ear wigglings) behaviors. AIM: We investigated the effects of subcutaneous injection of the 5-HT(2A/2C) receptor agonist (2,5 dimethoxy-4-idophenyl)-2-aminopropane hydrochloride (DOI) on sexual behaviors of ovariectomized female rat hormonally supplemented with estradiol benzoate (10 microg) and progesterone (250 microg). METHODS: Both female and male sexual behaviors were observed for 10 minutes (pretest). Then females were injected with the treatment and after a 10-minute delay replaced with the same male for a 30 minute mating test (posttreatment period). RESULTS: DOI (0.5 and 1 mg/kg) significantly increased the number of darts and hops/mounts. In contrast, no significant differences in ear wigglings/mounts were observed. In addition, DOI failed to modify sexual receptivity. CONCLUSION: These data suggest that 5 HT(2A/2C) receptors are important in the regulation of female proceptivity. PMID- 16681469 TI - Moderate and severe erectile dysfunction equally affects life satisfaction. AB - PURPOSE: To explore the life satisfaction of patients with erectile dysfunction (ED) and to examine the relation between severity of ED and life satisfaction. MATERIALS AND METHODS: The study sample was recruited from patients who presented in an andrologic outpatient clinic complaining of ED. All patients underwent the basic clinical evaluation and were assessed by the International Index of Erectile Dysfunction (IIEF) and the Life Satisfaction Inventory (LSI). RESULTS: The study included 69 patients with ED (age range 22-71 years, mean 49.5, SD 13.7). The LSI appeared to have satisfactory internal consistency (Cronbach's alpha = 0.82). Men with ED had significantly lower satisfaction with their sexual life (t = -13.756, d.f. = 68, P = 0.000), but also significantly lower total score of satisfaction with their life (t = -2.793, d.f. = 68, P = 0.007) compared with available normative data from healthy population. However, when compared with controls, men with ED showed significantly higher satisfaction scores on their financial status (t = 5.075, d.f. = 68, P = 0.000) and on their leisure time activities (t = 4.029, d.f. = 68, P = 0.000). Regarding ED severity, mild ED affects patients' satisfaction with sexual life less than do moderate and severe ED. Interestingly, no difference was identified between moderate and severe ED groups. CONCLUSIONS: Men who seek help for ED have lower satisfaction with sexual life and lower satisfaction with their overall life compared with healthy people. Severity of ED seems to be an important factor, as men with moderate ED perceive the impact of ED on their life satisfaction equally with those with severe ED. ED patients gain more satisfaction from other domains of their life, provided they adopt coping behaviors that help compensate for their low overall life satisfaction. Furthermore, as ED patients do not differ from healthy people in their expressed subjective rating of quality of life (QoL), life satisfaction may be more sensitive than QoL assessment in the evaluation of the impact of ED on our patients' life. PMID- 16681470 TI - Nipple/Breast stimulation and sexual arousal in young men and women. AB - INTRODUCTION: The role of nipple/breast stimulation in influencing sexual arousal in men and women during lovemaking has only been the subject of opinion-based comment rather than evidence-based study. No attempt to question people about such sexual behavior has ever been undertaken. AIM: The study was designed to ascertain the effects of nipple/breast manipulation in young men and women on their sexual arousal. METHODS: A short questionnaire about nipple/breast stimulation during sexual activity was administered to 301 (148 men; 153 women) sexually experienced undergraduates (age range 17-29 years, 95% between 18 and 22). MAIN OUTCOME MEASURES: Replies to questions in questionnaire. RESULTS: The major findings in regard to the women were that 81.5% reported that stimulation of their nipples/breasts caused or enhanced their sexual arousal, 78.2% agreed that when sexually aroused such manipulation increased their arousal, 59.1% had asked to have their nipples stimulated during lovemaking, and only 7.2% found that the manipulation decreased their arousal. In regard to the men, 51.7% reported that nipple stimulation caused or enhanced their sexual arousal, 39% agreed that when sexually aroused such manipulation increased their arousal, only 17.1% had asked to have their nipples stimulated, and only 7.5% found that such stimulation decreased their arousal. CONCLUSION: Manipulation of the nipples/breasts causes or enhances sexual arousal in approximately 82% of young women and 52% of young men with only 7-8% reporting that it decreased their arousal. PMID- 16681471 TI - A methodology study to develop and validate a screener for hypoactive sexual desire disorder in postmenopausal women. AB - INTRODUCTION: Current methods for diagnosing hypoactive sexual desire disorder (HSDD) can be complicated and time-consuming. A previous study reported validity and reliability of a structured diagnostic method created for clinical trials that can be performed in approximately 1 hour. METHODS: A more succinct tool is needed for incorporation into busy physician practices. Therefore, a brief HSDD screening tool was developed consisting of four self-report questions with an interpretable cut-score and concise confirmatory physician interview. MAIN OUTCOME MEASURES: Accuracy of the HSDD screener cut-score alone, and in combination with physician interview, was then separately evaluated when compared with in-depth interview diagnosis. RESULTS. The results showed good agreement between the two diagnoses (kappa of 0.669 and 0.562 for cut-score alone and cut score in combination with physician interview, respectively). CONCLUSIONS: The HSDD screener can reliably detect the likely presence of HSDD in postmenopausal women. PMID- 16681472 TI - Development and validation of a new questionnaire to assess sexual satisfaction, control, and distress associated with premature ejaculation. AB - INTRODUCTION: No validated questionnaires to assess the subjective aspects of premature ejaculation (PE) are currently in use. Clinical trials have generally only considered time, measured by ejaculatory latency, as an indicator of efficacy, but ejaculatory control, sexual satisfaction, and distress are important aspects of PE, which impact both the patient and his partner. The objective of this study was to develop and validate a new questionnaire to measure the overall experience of PE. METHODS: The questionnaire was developed using four stages: item pool development, initial psychometric analyses, patient interviews, and final psychometric analyses. RESULTS: An item pool of 17 was generated and reduced to 10 items through the initial psychometric analyses. Patient interviews did not require addition of further items and resulted in only minor modifications to item wording for clarity. Final psychometric analyses of the 10-item measure confirmed a three-factor solution: sexual satisfaction, control, and distress. Reliability was good, both internal consistency and test retest reliability. Convergent validity using intravaginal ejaculatory latency time was excellent: control domain (0.75), sexual satisfaction domain (0.60), and distress domain (0.68). Known-groups validity was very good, all domain mean scores being statistically significantly worse in men with PE compared with the men reporting no PE problems. CONCLUSION: The Index of Premature Ejaculation is a reliable and valid questionnaire for the assessment of control over ejaculation, satisfaction with sex life, and distress in men with PE. This tool has the potential to add value to interpretations of improvements in ejaculation latency resulting from new treatments of PE. PMID- 16681473 TI - Chocolate and women's sexual health: An intriguing correlation. AB - INTRODUCTION: Historically chocolate has been reported to exert several effects on human sexuality, mainly acting as an effective aphrodisiac, increasing sexual desire, and improving sexual pleasure. AIM: The aim of our study was to assess whether there is an association between daily chocolate intake and sexual function in a convenience sample of Northern Italian women. METHODS: A convenience sample of 163 women (mean +/- SD age: 35.3 +/- 9.2 years; body mass index [BMI]: 22.5 +/- 3.5 kg/m2), recruited through advertising, completed an anonymous semistructured interview on recreational habits and questionnaires to assess sexual function (Female Sexual Function Index [FSFI]), sexual distress (Female Sexual Distress Scale), and depression (Beck Depression Inventory and Center for Epidemiological Survey Depression Scale). RESULTS: Complete data were available for 153/163 (93.8%) women. Participants who reported daily chocolate intake (Group 1: 120 women) were significantly younger than those (Group 2: 33 women) who did not report to eat chocolate (33.9 +/- 0.8 years vs. 40.4 +/- 1.6 years, respectively) (P = 0.0003), despite a similar BMI. Participants in Group 1 had significantly higher total (P = 0.002) and desire domain (P = 0.01) FSFI scores than participants in Group 2. No differences between the two groups were observed concerning sexual arousal and satisfaction, sexual distress and depression. Our data also confirm that aging has a high statistically significant impact on women's sexual function. CONCLUSIONS: It is alluring to hypothesize that chocolate can have either a psychological or a biological positive impact on women's sexuality. In our sample women reporting chocolate consumption have higher FSFI scores than women who do not eat chocolate. However, when data are adjusted for age FSFI scores are similar, regardless of chocolate consumption. PMID- 16681474 TI - The psychosocial outcomes of total and subtotal hysterectomy: A randomized controlled trial. AB - INTRODUCTION: Current controversies involve the adverse effects of hysterectomy on women's psychosocial functioning and whether subtotal as opposed to total hysterectomy mitigates these effects. AIM: To investigate the psychosocial effects of hysterectomy by examining sexual, pain, and psychological outcomes of total vs. subtotal hysterectomy in a randomized controlled trial. METHODS: Patients suffering from benign gynecological conditions were randomly assigned to one of two groups: (i) total hysterectomy, that is, laparoscopic assisted vaginal hysterectomy (TOT, N = 32); or (ii) subtotal hysterectomy, that is, supracervical laparoscopic hysterectomy (SUB, N = 31). Both groups were premenopausal and underwent hysterectomy without concurrent oophorectomy. Two premenopausal control groups: (i) minor gynecological surgery (SURG-CON, N = 30); and (ii) healthy nonsurgical controls (NORM-CON, N = 40), were also tested. All surgical groups were assessed 2-3 weeks before surgery and then 6-7 months afterward; the nonsurgical control group was assessed at the time of recruitment and 6-7 months later. OUTCOME MEASURES: Assessments included semistructured interviews, standardized questionnaires, and standardized gynecological examinations. RESULTS: For the TOT group, sexual drive, arousal, and sexual behavior significantly improved postoperatively. For the SUB group, sexual behavior and overall sexual functioning significantly improved. For both TOT and SUB groups, unprovoked pain in the abdomen and pain in the abdomen during gynecological examinations was significantly reduced. For both TOT and SUB groups, overall psychological functioning did not significantly change postoperatively. Although between 3% and 16% of women undergoing hysterectomy reported adverse changes in psychosocial well-being after surgery, similar percentages of women in the control groups reported such effects. CONCLUSIONS: Hysterectomy resulted in a consistent reduction in abdominal pain, some improvement in sexual functioning, but no change in overall psychological functioning. There was no evidence supporting the idea that subtotal hysterectomy produced more favorable psychosocial outcomes than total hysterectomy nor was there any evidence that either type of hysterectomy resulted in adverse effects. PMID- 16681475 TI - Efficacy and safety of tadalafil across ethnic groups and various risk factors in men with erectile dysfunction: Use of a novel noninferiority study design. AB - AIM: This U.S. multicenter open-label study used a noninferiority trial design to assess the efficacy of tadalafil 20 mg to treat erectile dysfunction (ED) in the black American and Hispanic groups compared with a reference group of Caucasians. A secondary objective was to demonstrate the efficacy and safety of tadalafil 20 mg in various populations of men with ED. METHODS: A total of 1,911 patients with ED were enrolled into eight predefined groups: (1-3) Caucasian (Reference group), Black American, or Hispanic patients, < or =65 years of age with no diabetes mellitus or depression; (4) patients with depression, < or =65 years of age, no diabetes; (5) patients with diabetes, < or =65 years of age, no depression; (6) patients >65 years of age, no diabetes or depression; (7) patients who met enrollment criteria but were not included in any other group; and (8) patients with ED due to traumatic spinal cord injury. The study had a 4-week run-in period, followed by a 12-week treatment period with tadalafil taken as needed, up to one dose/day, prior to sexual activity. Change from baseline to endpoint in the Erectile Function (EF) domain of the International Index of Erectile Function (IIEF) was used to determine noninferiority of groups 2 and 3 from group 1. Secondary efficacy measures included other IIEF domains and Sexual Encounter Profile (SEP). RESULTS: Noninferiority analyses based on the EF domain score showed that tadalafil was as efficacious in the Hispanic and Black American groups as in the Reference group. Patients in each of the eight groups had a significant change from baseline (P < 0.001) in the IIEF EF domain score and positive responses to SEP Questions 1-5. The most common treatment-emergent adverse events reported by patients in all eight groups were headache, nasal congestion, dyspepsia, flushing, and back pain. CONCLUSION: Tadalafil 20 mg was as efficacious in the Hispanic and black American groups as in the Caucasian/Reference group. Tadalafil was efficacious and well tolerated in each of the groups studied in this trial. PMID- 16681476 TI - Tolerance to the therapeutic effect of tadalafil does not occur during 6 months of treatment: A randomized, double-blind, placebo-controlled study in men with erectile dysfunction. AB - INTRODUCTION: Tolerance can cause a decrease in drug efficacy during chronic therapy, possibly leading to treatment failures. AIM: The aim of this article is to determine whether tolerance developed to the effects of tadalafil on erectile function (EF) over a 6-month treatment period. METHODS AND MAIN OUTCOME MEASURES: Post hoc analysis of data from a multicenter, double-blind, randomized, placebo controlled, parallel group study was performed. Men (> or =18 years of age) with erectile dysfunction (ED) were randomized to treatment with placebo (N = 47) or 20-mg tadalafil (N = 93) taken as needed for 6 months. This report focuses on efficacy assessed with the Sexual Encounter Profile (SEP) diary (diaries were collected after a 4-week treatment-free run-in period [baseline], and monthly for 6 months), and with the International Index of Erectile Function (IIEF) (administered at baseline, and at 3 and 6 months). RESULTS. The mean per-patient percentage "yes" response on SEP question 3 (SEP3, successful intercourse) was 33 +/- 4% at baseline, 74 +/- 4% after 1 month, and 78 +/- 4% after 6 months of tadalafil treatment. The IIEF EF domain score was 16.2 +/- 0.7 at baseline, 24.3 +/- 0.8 after 3 months, and 24.3 +/- 0.9 after 6 months of tadalafil treatment. In a subgroup of patients who took tadalafil > or =3 times per week (N = 24), the SEP3 score was 87 +/- 4% after 1 month and 93 +/- 3% after 6 months of treatment, and the IIEF EF domain score was 27.3 +/- 0.9 after 3 months and 28.5 +/- 0.4 after 6 months. Of 16 tadalafil-treated patients who discontinued, three cited a lack of efficacy. CONCLUSIONS: Tadalafil treatment significantly improved SEP3 and IIEF EF domain scores. The efficacy of tadalafil, taken as needed, was maintained over a 6-month treatment period in men with ED. PMID- 16681477 TI - Efficacy and safety of two dosing regimens of tadalafil and patterns of sexual activity in men with diabetes mellitus and erectile dysfunction: Scheduled use vs. on-demand regimen evaluation (SURE) study in 14 European countries. AB - AIM: The aim of this article is to evaluate the efficacy and safety of 20-mg tadalafil taken on demand or three times per week and its effect on the sexual activity of patients with diabetes mellitus and erectile dysfunction (ED). METHODS: The scheduled use vs. on-demand regimen evaluation (SURE) was a randomized, crossover, open-label study with 4,262 patients in 14 European countries. The efficacy measures for the 762 patients with diabetes and ED included changes from baseline in the erectile function (EF) domain of the International Index of Erectile Function (IIEF), and the proportion of "yes" responses to patient Sexual Encounter Profile (SEP) questions 2 (SEP2) and 3 (SEP3). The treatment satisfaction was measured with responses to SEP question 4 (SEP4) and SEP question 5 (SEP5), and sexual attempts data were collected. Patient preference for either regimen was determined by the treatment preference question (TPQ). RESULTS: At end point on both regimens, the mean IIEF EF domain score was 22, and >40% of the patients had a normal EF domain score (> or =26). The proportion of "yes" responses was > or =73% for SEP2 (penetration), > or =58% for SEP3 (successful intercourse), >46% for SEP4 (hardness of erection), and > or =45% for SEP5 (overall satisfaction). Efficacy was maintained up to 36 hours post dosing. More than 70% of sexual attempts while on the three-times-per-week regimen and approximately 50% of the attempts on the on-demand treatment occurred >4 hours post-dosing. Tadalafil was well tolerated, with dyspepsia and headache as the most frequent adverse events reported. Treatment preference was 57.2% for on demand and 42.8% for three times per week. CONCLUSIONS: Tadalafil, when taken on demand or three times per week, is efficacious and safe in men with diabetes and ED. PMID- 16681478 TI - Sildenafil citrate improves self-esteem, confidence, and relationships in men with erectile dysfunction: Results from an international, multi-center, double blind, placebo-controlled trial. AB - INTRODUCTION: Erectile dysfunction (ED) can significantly impact a man's relationships and well-being. AIM: We assessed changes in self-esteem, confidence, sexual relationship satisfaction, and overall relationship satisfaction in men with ED using the validated Self-Esteem And Relationship questionnaire (SEAR). METHODS: This was a 12-week, double-blind, placebo controlled, flexible-dose (25, 50, 100 mg, as needed) international study of sildenafil in men > or =18 years of age in Mexico, Brazil, Australia, and Japan. MAIN OUTCOME MEASURES: The primary study outcome was change in self-esteem from baseline to the end of treatment. Secondary study measures were changes in other SEAR components, International Index of Erectile Function (IIEF) domains, percentage of intercourse attempts that were successful, and the response to a global efficacy question at the end of treatment. RESULTS: Patients were well balanced for age and duration of ED (placebo = 149 and sildenafil = 151). Compared with placebo, sildenafil significantly improved self-esteem, confidence, sexual relationship satisfaction, and overall relationship satisfaction (P < 0.0001). The psychosocial measures of well-being assessed with the SEAR were positively correlated (range 0.60-0.86, P < 0.0001) with erectile function, the frequency of achieving erections that allowed satisfactory sexual intercourse, the percentage of successful sexual intercourse attempts, and global treatment efficacy. CONCLUSIONS: Significant improvements in self-esteem, confidence, sexual relationship satisfaction, and overall relationship satisfaction after treatment of ED with sildenafil were consistent among countries. These data suggest a substantial cross-cultural improvement in well-being after successful treatment of ED with sildenafil. PMID- 16681479 TI - Treatment-seeking behavior of erectile dysfunction patients in Europe: Results of the Erectile Dysfunction Observational Study. AB - INTRODUCTION: The Erectile Dysfunction Observational Study (EDOS) is a 6-month, pan-European prospective, observational study of health outcomes designed to assess patients' profiles and characteristics and the effectiveness of erectile dysfunction (ED) treatment in routine clinical practice. AIM: To present baseline characteristics and treatment-seeking behavior of a large sample of ED patients recruited in real-life clinical settings. METHODS: Men aged 18 years and older who visited a physician to initiate or change any ED treatment were enrolled in EDOS. They were assessed at baseline, 3 months, and 6 months as part of their normal course of care in nine European countries. MAIN OUTCOME MEASURES: Sexual health outcomes using the short form of the Psychological and Interpersonal Relationship Scales. Treatment effectiveness and satisfaction were assessed using the International Index of Erectile Function questionnaire, Global Assessment Questions, and further single-item questions. RESULTS: Of the 8,186 patients enrolled by 904 investigators (69% general practitioners [GPs]) across nine European countries, 8,055 patients were eligible for analysis at baseline; 63.9% were ED treatment-naive. Of the total patient population, mean age was 56.5 years, mean body mass index (BMI) was 27.2 kg/m2, 18.3% were obese (BMI > 30 kg/m2), 42.5% had severe ED, and there was a high frequency of comorbidities and concomitant medication use. A similar proportion of the treatment-naive patients were seen by GPs (62.9%) and specialists (65.8%). In the treatment-naive group, there was a higher frequency of severe ED among ex-smokers, obese patients, and in those who drank no alcohol or excessive amounts of alcohol. CONCLUSIONS: Unmet need of treatment in ED is high; 66% of patients had experienced ED symptoms for 1 year or longer when they were looking for treatment. Severity seems to be related to treatment seeking. PMID- 16681481 TI - Surgical technique to maintain penile length after insertion of an inflatable penile prosthesis via infrapubic approach. AB - INTRODUCTION: The aim of this study was to provide an overview of the principal author's experience in maintaining penile length after implantation of a three piece inflatable penile prosthesis (IPP). For many patients with erectile dysfunction, who choose surgical treatment, loss of penile length after implantation of an IPP is a common concern. In the principal author's experience, release of the suspensory ligament during IPP implant surgery can maintain penile length, alleviating this concern. METHODS: After implantation with an IPP, the principal author released the patient's suspensory ligament. Main Outcome Measures. The main outcome measure was patient satisfaction with penile length after IPP surgery. A second outcome measure was a substudy measuring the patient's penile length after IPP implantation both before and after suspensory ligament release. RESULTS: From August 1997 through September 2002, the principal author implanted a total of 303 Alpha 1 IPPs into men who suffered from erectile dysfunction (ED). All had their suspensory ligament released. Postoperative complications were minimal and for the most part transitory. Ninety-three percent reported satisfaction with IPP performance, penile length, and willingness to undergo the IPP surgery again. None of the patients reported penile shortening, with some of them reporting an increase in penile length, as compared with preoperative measurements. CONCLUSION: Release of the suspensory ligament appears to maintain or even increase penile length, with a minimum of complications, and a high degree of patient satisfaction. PMID- 16681480 TI - Vaginal application of testosterone: A study on pharmacokinetics and the sexual response in healthy volunteers. AB - INTRODUCTION: Androgen substitution is advocated to improve sexual functioning in women with androgen insufficiency. Nevertheless, the role of androgens in female sexual functioning is not yet unraveled. Even less is known about changes in androgens and the female sexual response. AIM: The aim of the study is to describe the pharmacokinetics of a single dose of vaginally applied testosterone. In addition, the study aims to gain more insight into the relation between acute changes in testosterone levels and the sexual response in women. METHODS: A randomized, double-blind, crossover study design was used to compare a single vaginal dose of testosterone propionate (2 mg) with placebo. Ten healthy premenopausal women participated. Serum levels of testosterone, free testosterone, and estradiol were measured. The sexual response was measured before application of medication and 4 and 8 hours after application. Erotic video fragments and erotic fantasies were used as stimuli. The genital sexual response was measured using vaginal plethysmography. The subjective sexual response was measured using a visual analog scale. RESULTS: Vaginal administration of testosterone propionate induced a significant rise in serum testosterone levels and free testosterone levels, but not in serum estradiol levels. Peak levels were reached after 5.5 hours (range 2-12 hours). Mean peak levels of testosterone were 7.71 nmol/L after testosterone propionate and 2.99 nmol/L after placebo (P < 0.005). Mean peak levels of free testosterone were 0.12 nmol/L after testosterone propionate and 0.04 nmol/L after placebo (P < 0.005). Despite marked elevated levels of androgens this study was unable to detect a direct effect on the genital or subjective sexual response. CONCLUSIONS: A single dose of vaginally applied testosterone propionate elevates serum levels of testosterone and free testosterone within 6 hours. Nevertheless, this acute rise in androgens has no effects on the female sexual response. PMID- 16681482 TI - The penile vasculature in systemic sclerosis: A duplex ultrasound study. AB - INTRODUCTION: Systemic sclerosis is a connective tissue disease characterized by Raynaud's phenomenon, degenerative changes and vascular lesions in the presence of thickened, sclerotic skin lesions determined by cellular proliferation, and excess of extracellular matrix production. The role of ultrasound in the investigation of penile pathology is well established as vasculogenic impotence accounts for more than 30% out of overall causes. AIM: In this article, we report for the first time the extent of penile vascular damage in a series of 15 sclerodermic patients (mean age 47 +/- 12.5 SD) under current treatment for their disease irrespective of their sexual dysfunction complaints. METHODS: After disease classification (mean duration of disease 7.2 +/- 5.1 years), all patients were interviewed about the presence or absence of erectile dysfunction (ED) by using the Sexual Health Inventory for Men (SHIM) questionnaire, and then blood flow velocity in the cavernous artery following standardized pharmacostimulation was determined with Duplex ultrasonography along with the intima media thickness (IMT) of the common carotid artery, a valid index for atherosclerosis. RESULTS: Mean SHIM scores revealed the presence of moderate-to-severe ED (mean 13.3 +/- 6.3). Interestingly, in all patients diffuse hyperechoic "spots" inside the corpora cavernosa along with thickening of the tunica albuginea were found. Severely impaired mean peak systolic velocities (20.2 +/- 5.5 cm/second) in the presence of mild venous leakage as expressed by mean end diastolic velocities (4.6 +/- 2.9 cm/second) were found along with normal IMT (0.065 +/- 0.010 cm) and acceleration time (92.3 +/- 32.7 cm/second). CONCLUSION: Penile fibrosis almost invariably occurs in sclerodermic patients and this determines incomplete penile arterial and smooth muscle cell relaxation and ED despite the absence of indirect signs of early atherosclerosis, that is, abnormal IMT and acceleration time. PMID- 16681483 TI - Surgical techniques: Surgery for Vulvar Vestibulitis syndrome. PMID- 16681485 TI - Do oral contraceptives produce irreversible effects on women's sexuality? PMID- 16681488 TI - Scalloped dental implants: a retrospective analysis of radiographic and clinical outcomes of 17 NobelPerfect implants in 6 patients. AB - BACKGROUND: The scalloped dental implant (NobelPerfect, Nobel Biocare, Yorba Linda, CA, USA) is designed to biologically guide and facilitate interproximal bone remodeling during healing and to maintain bone height and papillae during functional loading. The design features of the scalloped implant include hard and soft tissue apposition areas, which are parallel to each other and mirror the cementoenamel junction. The hard tissue surface area is intended for osseointegration. The soft tissue surface area is meant to support the connective tissue zone and to provide a space for the subgingival margin of the restoration. Current literature on the clinical performance of the scalloped dental implant is limited. PURPOSE: The aim of this study was to evaluate whether the scalloped dental implant maintains interproximal bone and the overlying soft tissue. MATERIALS AND METHODS: Radiographs for six patients (mean age 40.5 years) treated with 17 implants (NobelPerfect) were reviewed for an 18-month follow-up evaluation of bone response. Orthodontic movement and/or autogenous bone augmentation had been provided to obtain the best possible soft and hard tissue dimensions prior to implant placement. A surgical guide was used for an optimal implant placement. No surgical flap was reflected, and implants were placed a minimum of 2 mm and a maximum of 3 mm apical (midbuccally) to the most apical portion of the surgical guide. Final optimal rotational alignment was achieved in all cases by not exceeding 45 Ncm. Implants were immediately restored with provisional crowns. Photographic documentation provided the basis for analysis of papillary response. Radiographic change in the interproximal bone level was obtained by computer analysis (ImageJ for Windows, National Institutes of Health, Bethesda, MD) by measuring the distance from the interproximal shoulder of the scalloped implant to the crest of the bone. RESULTS: When the scalloped implants were placed adjacent to existing natural dentition, the average bone level at placement and at 6, 12, and 18 months was -1.7, -3.5, -3.8, and -3.9 mm, respectively, compared with -1.0, -3.6, -4.3, and -4.4 mm respectively, when placed adjacent to other scalloped implants. Papillae formation exhibited no differences from the configuration that typically results after placement of conventional dental implants. Moreover, bone loss around the scalloped implants was notably greater than that expected after traditional implant placement. CONCLUSION: This chart review of 17 scalloped implants, followed for 18 months, determined that the scalloped implant design resulted in bone loss that was more severe than that associated with properly placed conventional dental implants. Further, the design showed no evidence of exceptional capacity to increase or maintain soft tissue height. PMID- 16681489 TI - Bone reformation and implant integration following maxillary sinus membrane elevation: an experimental study in primates. AB - BACKGROUND: Recent clinical studies have described maxillary sinus floor augmentation by simply elevating the maxillary sinus membrane without the use of adjunctive grafting materials. PURPOSE: This experimental study aimed at comparing the histologic outcomes of sinus membrane elevation and simultaneous placement of implants with and without adjunctive autogenous bone grafts. The purpose was also to investigate the role played by the implant surface in osseointegration under such circumstances. MATERIALS AND METHODS: Four tufted capuchin primates had all upper premolars and the first molar extracted bilaterally. Four months later, the animals underwent maxillary sinus membrane elevation surgery using a replaceable bone window technique. The schneiderian membrane was kept elevated by insertion of two implants (turned and oxidized, Branemark System, Nobel Biocare AB, Goteborg, Sweden) in both sinuses. The right sinus was left with no additional treatment, whereas the left sinus was filled with autogenous bone graft. Implant stability was assessed through resonance frequency analysis (Osstell, Integration Diagnostics AB, Goteborg, Sweden) at installation and at sacrifice. The pattern of bone formation in the experimental sites and related to the different implant surfaces was investigated using fluorochromes. The animals were sacrificed 6 months after the maxillary sinus floor augmentation procedure for histology and histomorphometry (bone-implant contact, bone area in threads, and bone area in rectangle). RESULTS: The results showed no differences between membrane-elevated and grafted sites regarding implant stability, bone-implant contacts, and bone area within and outside implant threads. The oxidized implants exhibited improved integration compared with turned ones as higher values of bone-implant contact and bone area within threads were observed. CONCLUSIONS: The amount of augmented bone tissue in the maxillary sinus after sinus membrane elevation with or without adjunctive autogenous bone grafts does not differ after 6 months of healing. New bone is frequently deposited in contact with the schneiderian membrane in coagulum-alone sites, indicating the osteoinductive potential of the membrane. Oxidized implants show a stronger bone tissue response than turned implants in sinus floor augmentation procedures. PMID- 16681490 TI - Report of a case receiving full-arch rehabilitation in both jaws using immediate implant loading protocols: a 1-year resonance frequency analysis follow-up. AB - BACKGROUND: Immediate occlusal implant loading has been documented as a viable treatment option for various indications. However, documentations related to full arch rehabilitation are usually limited to treatment of one jaw at a time, thereby leaving the opposing dentition unchanged. Furthermore, clinical documentation using traditional, well-accepted measuring techniques may not be adequate when it comes to short-term evaluation of the success or failure of implants subjected to immediate occlusal loading. PURPOSE: The purpose of this case report is to (1) present an implant stability follow-up of a patient receiving an immediate, implant-supported full-arch rehabilitation in both jaws and (2) evaluate the patient's acceptance of this rehabilitation. MATERIALS AND METHODS: A 68-year-old patient scheduled for implant treatment was selected for an immediate implant loading protocol in both jaws. During two surgical events 3 weeks apart, eight maxillary and four mandibular Branemark System Mk IV TiUnite fixtures (Nobel Biocare AB, Goteborg, Sweden) were inserted and subsequently used to immediately support a cross-arch fixed prosthesis in the maxilla and a bar retained overdenture in the mandible. Implant stability was recorded from the day of surgery periodically during a 1-year follow-up using resonance frequency analysis (RFA). RESULTS: At the 1-year follow-up, based on clinical, RFA, and radiographic evaluations, all implants and the reconstructions were classified as successful. All maxillary implants showed a decrease in the implant stability quotient (ISQ) value from the measurement at the time of surgery to the first follow-up, whereas two of four mandibular implants revealed an initial drop in stability. Irrespective of a specific ISQ level measured at implant surgery (ISQ range 53-74) and despite an initial decrease in stability, measurements recorded at the 12-month follow-up indicated similar stability levels for all maxillary implants (ISQ range 64-68) or the group of mandibular implants (ISQ range 72-75) but with a higher ISQ level for mandibular implants. Furthermore, the patient's acceptance of the immediate full-arch rehabilitation in both jaws was high. CONCLUSIONS: The present case report demonstrates that a slightly staged approach for full-arch rehabilitation in both jaws using immediate implant loading protocols is a realistic treatment option. Furthermore, RFA follow-up indicates that immediately occlusally loaded implants placed in reduced bone quality and quantity are more prone to loose stability in the early healing period compared with implants placed in dense bone quality. PMID- 16681491 TI - Influence of titanium surface charge on fibroblast adhesion. AB - BACKGROUND: Although dental implants have a high success rate, failure owing to the absence of adhesion between the gingival connective tissue and the implant surface is still being reported. PURPOSE: This study was designed to evaluate the effect of a titanium surface charge on fibroblast adhesion. MATERIAL AND METHODS: An electrical chamber was custom-made to generate negative and positive surface charges on commercially pure titanium cylinders with a potential difference of 4.5 V. Twenty-seven titanium cylinders were divided into three experimental groups. In each group, cell attachment to a positively charged titanium cylinder, a negatively charged titanium cylinder, and a titanium cylinder (control) was studied at three time intervals of 15, 30, and 60 minutes. NCTC clone 929 fibroblasts were used in these experiments. The effect of the potential difference in the pH of Dulbecco's Modified Eagle Medium (DMEM) was also evaluated using two new specimens at time intervals of 15, 30, 60, and 80 minutes. RESULTS: The fibroblast cell attachment was more statistically significant to the positively charged titanium cylinder than the negatively charged titanium cylinder (p =.002) and the control (p=.000), whereas the cell adhesion difference between the control and the negatively charged titanium cylinder was not statistically significant (p=.808). The range of pH difference of the DMEM in the negative and positive parts of the electrical chamber was 0.46 and 0.30, respectively. CONCLUSION: Within the limitations of this in vitro study, the positive surface charge of the titanium cylinder results in significantly favorable cell adhesion. PMID- 16681492 TI - Patient evaluation after treatment with maxillary implant-supported overdentures. AB - PURPOSE: To evaluate and compare outcome among patients after implant overdenture treatment in the maxilla. MATERIALS AND METHODS: The study sample comprised two groups of patients: group 1, in which the patients were planned for overdenture treatment, and group 2, in which the patients originally were planned for a fixed prosthesis in the maxilla but had overdenture treatment owing to implant failures, resulting in an insufficient number of implants to support a fixed prosthesis. All patients treated with maxillary implant-supported overdentures in the Department of Prosthetic Dentistry, Central Hospital, Skovde, Sweden, between 1993 and 2002 received a questionnaire at their yearly follow-up visit with nine questions related to their treatment. All questions had visual analogue scale response alternatives ranging from a negative to a positive opinion. RESULTS: Nineteen patients, 10 in group 1 and 9 in group 2, completed the questionnaire, yielding a response rate of 86%. Both groups expressed a high satisfaction rate, and few regretted their choice of treatment. Patients planned for overdenture treatment (group 1) reported significantly fewer speech problems after treatment compared with those originally planned for a fixed prosthesis (group 2, p < .05). No other significant differences between the two groups were seen. CONCLUSION: Within the limitations of the present study, it can be concluded that maxillary implant overdenture treatment may be considered a viable option among patients with an insufficient number of implants for a fixed prosthesis. PMID- 16681493 TI - Histologic evaluation of Branemark clinic oral implants retrieved from grafted sites. AB - PURPOSE: The aim of this report is to quantitatively and qualitatively describe the bone tissue response to Branemark implants retrieved from grafted sites in patients. MATERIALS AND METHOD: The material consists of consecutively received Branemark implants retrieved from grafted sites. Thirty-five of these implants, retrieved from 16 patients, were suitable for the histologic evaluation of undecalcified sections in the light microscope. RESULTS: The unloaded implants were mainly lined with soft tissue, and sparse bone-implant contact was observed only in some sections. The loaded implants, with the exception of one implant removed due to mobility, had mature and new bone-implant contact. Resorption of graft through cutting cone structures was detected. Cement lines were found separating bone-like tissue albeit no cellular content and bone tissue with detectable osteocytes. CONCLUSION: In this heterogeneous group of implants from grafted sites, the unloaded implants showed limited bone-implant contact. The autografts showed seemingly mixed viability as judged by the cell content in the osteocyte lacunae and cement lines separating areas with filled and empty lacunae. PMID- 16681494 TI - Commentary on "Scalloped dental implants: a retrospective analysis of radiographic and clinical outcomes of 17 NobelPerfect implants in 6 patients". PMID- 16681496 TI - Kudos. PMID- 16681497 TI - Marginal adaptation of Cerec 3 CAD/CAM composite crowns using two different finish line preparation designs. AB - PURPOSE: The purpose of this study was to compare marginal discrepancies of Cerec 3 CAD/CAM composite crowns, fabricated on human prepared teeth with two different finish line designs, chamfer and shoulder. MATERIALS AND METHODS: Sixteen human molar teeth were used to prepare full crowns. Eight teeth were prepared with a 1 mm-wide chamfer finish line and the other eight with a 1.2- to 1.5-mm circumferential shoulder. Cerec 3 crowns were fabricated from optical impressions using Paradigm MZ100 composite polymer. Marginal adaptation was evaluated in two ways: (1) using modified United States Public Health Service (USPHS) criteria to evaluate eight preselected sites on each crown margin, and (2) using scanning electron microscopy (SEM) to measure marginal gaps on all four axial walls with 15 measurements on each wall (60 measurements per crown). An evaluation of the number of acceptable crowns, determined by having all measured sites per tooth with margin gap size less than 100 microm, as a function of finish line design was also conducted. RESULTS: In both chamfer and shoulder groups, there were only two crowns (out of eight) with clinically acceptable ratings for all eight measurement sites according to USPHS criteria. Fisher's chi-square analysis showed that there was no statistically significant difference in marginal adaptability as a function of finish line design ( p>0.05). With SEM imaging, overall mean marginal gaps for the chamfer group were 65.9+/-38.7 microm (range 35.0 to 130.0 microm), and for the shoulder group were 46.0+/-9.2 microm (range 26.3 to 55.6 microm); this difference was not found to be statistically significant ( p>0.05). While crown assessment based on mean marginal discrepancy measurements indicated that both the chamfer and shoulder groups were considered clinically acceptable (<100 microm); crown acceptability based on all measurement sites being less than 100 mum indicated that in the chamfer and shoulder groups there were four and three acceptable crowns out of eight, respectively. The Fisher's chi-square test indicated no statistically significant difference between the groups ( p>0.05). An agreement rate of 81.2% was calculated between the two evaluation methods, modified USPHS criteria and SEM measurements. CONCLUSIONS: Based on mean marginal discrepancy measurements, the typical marginal assessment technique, Cerec 3 Paradigm MZ100 crown restorations appear to have acceptable marginal adaptability (mean discrepancies <100 microm). Thus, the evidence from this investigation would suggest that the finish line preparation design had no effect on marginal adaptation for Cerec 3 composite crowns. PMID- 16681498 TI - Preloads generated with repeated tightening in three types of screws used in dental implant assemblies. AB - PURPOSE: Abutment screw loosening, especially in the case of cemented single tooth restorations, is a cause of implant restoration failure. This study compared three screws (titanium alloy, gold alloy, and gold-coated) with similar geometry by recording the preload induced when torques of 10, 20, and 35 Ncm were used for fixation. MATERIALS AND METHODS: Two abutment types were used prefabricated preparable abutments and cast-on abutments. A custom-designed rig was used to measure preload in the abutment-screw-implant assembly with a strain gauge. Ten screws of each type were sequentially tightened to 10, 20, and 35 Ncm on ten of the two abutment types. The same screws were then loosened and re tightened. This procedure was repeated. Thus, each screw was tightened on three occasions to the three insertion torques. A linear regression model was used to analyze the effects on preload values of screw type and abutment type for each of the three insertion torques. RESULTS: The results indicated that the gold-coated screw generated the highest preloads for all insertion torques and for each tightening episode. Further analysis focused on the effects of screw type and abutment type for each episode of tightening and for each fixation torque. The gold-coated screw, fixed to the prefabricated abutment, displayed higher preloads for the first tightening at 10, 20, and 35 Ncm. Conversely, the same screw fixed to the cast-on abutment showed higher values for the second and third tightening for all fixation torques. All screws showed decay in preload with the number of times tightened. Given the higher preloads generated using the gold-coated screw with both abutment types, it is more likely that this type of screw will maintain a secure joint when tightened for the second and third time. CONCLUSION: All screw types displayed some decay in preload with repeated tightening, irrespective of abutment type and insertion torque. The gold-coated screw showed markedly higher preloads for all insertion torques and for all instances of tightening when compared with the uncoated screws. PMID- 16681499 TI - Effect of core diameter and surface treatment on the retention of resin composite cores to prefabricated endodontic posts. AB - PURPOSE: With advances in adhesive dentistry and current emphasis on esthetic restorations, dowel systems have been developed to take advantage of these new techniques. Of interest when using these systems is the interaction between core materials and post materials. This investigation compared the tensile retentive force of two resin composite core materials to two metallic and one non-metallic prefabricated endodontic posts. Two dimensions of core build-up and two post surface treatments were tested. MATERIALS AND METHODS: One hundred twenty posts (stainless steel, titanium alloy, and glass fiber-impregnated resin) were secured in a jig with 4 mm of the post extending into a cylindrical matrix. The matrix formed cylinders with diameters of 3 and 5 mm into which resin composite was inserted. The posts were treated or not treated with a bonding agent. After storage for 24 hours at 100% humidity, five specimens per condition were tested in an Instron testing machine at a crosshead speed of 0.5 mm/min. Failure loads were recorded in kilograms and failure modes were observed under light microscopy (40 x). Four-way analysis of variance and multiple comparison testing were used to compare means at the 0.05 level of significance. RESULTS: The means and standard deviations of tensile loads were calculated. All variables were significant in either main effects or interactions ( p<0.05). Fisher's PLSD intervals for post, core, treatment, and diameter were 2.0, 1.6, 1.6, and 1.6 kg, respectively. In most cases, the retentive force recorded for metallic posts was higher than that of glass fiber posts. Titanium posts had higher retentive forces than did the stainless steel posts. For metallic posts, 5-mm cores provided higher forces than 3-mm cores. In the glass fiber group, core diameter was not significant. For core materials, Build-It gave higher results with stainless steel posts, and FluoroCore gave higher results with the titanium ones. The surface treatment results were mixed. In the metallic post groups the adhesive failure data ranged between 80% and 100%, whereas in the glass fiber post groups, adhesive failures ranged between 60% and 70%. CONCLUSIONS: Within the limitations of this study, the metallic post groups always provided higher tensile retentive forces, with the titanium post groups showing higher retentive forces than the stainless steel ones. In the glass fiber post groups, different core diameters did not affect retention values. PMID- 16681500 TI - Surface roughness of denture base acrylic resins after processing and after polishing. AB - PURPOSE: Circumstances exist in which the need to adjust denture base acrylic resins is necessary. This process obviously alters the surface of the polished denture base. The purpose of this study was to compare the effects of three chairside polishing kits and conventional polishing on four denture acrylic resins. MATERIALS AND METHODS: Twenty-four 30 x 30 x 2 mm acrylic resin specimens were fabricated with each of four acrylic resins: autopolymerizing, heat processed, injection molded, and microwaveable. One side was polished conventionally with pumice and polishing compound. The other side was polished with one of three chairside polishing kits: Axis, Brasseler, and Shofu. Each side was evaluated by a Dektak 8 Programmable Stylus Profiler to determine the surface roughness (Ra). RESULTS: One-way analysis of variance (ANOVA) revealed that: (1) There was no significant difference in the time it took to polish the specimens with the chairside polishing kits (F=2.118, p=0.14). (2) There was a significant difference in surface roughness between the acrylic resins before any polishing, with the injection-molded and heat-processed being less rough than the autopolymerizing (F=4.588, p=0.005). (3) There was a significant difference in surface roughness between the acrylic resins when conventionally polished, with the injection-molded and microwavable being less rough than the autopolymerizing (F=4.503, p=0.005). Factorial ANOVA revealed that: (1) There was no significant difference in the surface roughness among the chairside polishing kits (F=1.209, p=0.30). (2) There was a significant difference between the acrylic resins, with the heat-processed, injection-molded, and microwaveable being significantly less rough than the autopolymerizing (F=6.610, p=0.0001). (3) There was no significant interaction between the acrylic resins and the chairside polishing kit in the amount of surface roughness (F=1.728, p=0.12). An independent t-test revealed that conventional polishing was significantly smoother than polishing with the chairside polishing kits (t=3.847, p=0.0001). CONCLUSIONS: It was concluded that time was not a factor in using any of the chairside polishing kits. It is recommended that conventional polishing be used after adjustments to the cameo surface of denture acrylic resin. PMID- 16681501 TI - Immediate occlusal loading of Osseotite implants in mandibular edentulous patients: a prospective observational report with 18-month data. AB - PURPOSE: To evaluate the efficacy of treatment consisting of placement and immediate occlusal loading of implants in 27 patients with edentulous mandibles. MATERIALS AND METHODS: Twenty-seven patients were treated in two private practice settings. One hundred fifty-one implants were placed and immediately occlusally loaded with fixed implant prostheses (15 cement-retained, 12 screw-retained) on the day of implant placement. The implant-retained prostheses were inserted within 5 hours of implant placement. Patients were followed for at least 18 months. The required criteria for immediate occlusal loading was primary implant stability of at least 30 Ncm of insertion torque. The implant prostheses were removed at least 12 months post-placement and the implants were evaluated for primary clinical stability and radiographic bone apposition to implants. RESULTS: At the 12-month follow-up appointments, cumulative survival rates of 98.0% and 100% were recorded for implants and prostheses, respectively. Three implants failed within 3 months. All other implants were clinically successful. CONCLUSIONS: Immediate occlusal loading of multiple, splinted mandibular implants is an effective treatment when implants are stable at insertion and are rigidly splinted with implant-retained prostheses. PMID- 16681502 TI - Facial moulage fabrication using a two-stage poly (vinyl siloxane) impression. AB - Traditional techniques for the fabrication of a facial moulage have relied on materials with various shortcomings. This article describes a two-stage Poly(vinyl siloxane) impression technique that avoids some of these shortcomings and provides a predictable and accurate facial moulage. PMID- 16681503 TI - Screw-retained prosthesis for Straumann implant sites with limited interocclusal clearance. AB - This article reports a technique that addresses the problem of the restricted interocclusal distance when screw-retained prostheses are selected. PMID- 16681504 TI - Dentist communication with the dental laboratory for prosthodontic treatment using implants. AB - PURPOSE: A questionnaire was sent to U.S. dental laboratories to evaluate the level of communication between dentists and laboratory technicians and to determine trends in procedures and materials used in fixed and removable implant restorations. METHODS AND MATERIALS: Dental laboratories were randomly chosen from the National Association of Dental Laboratories for each of the 50 states. The questionnaire was mailed to the laboratory directors for 199 dental laboratories. One hundred fourteen dental laboratories returned the survey, yielding a response rate of 57%. Of those laboratories, 37 indicated that they did not participate in the fabrication of fixed implant restorations, yielding a response rate of 39%. Forty-two dental laboratories indicated that they did not participate in the fabrication of implant-retained overdenture prostheses, yielding a response rate of 36%. RESULTS: Results from this survey show inadequate communication by dentists in completing work authorization forms. Custom trays are used more frequently for implant-retained overdenture impressions and stock trays for impressions of fixed implant prostheses. Poly(vinyl siloxane) is the material most commonly used for both fixed and removable implant-supported prostheses. Two implants with stud attachments are used more widely than those with bar attachments for implant-retained overdentures. CONCLUSIONS: Most laboratories working on implant prosthodontic cases report inadequate communication between the laboratory and dentists related to materials and techniques used in fabrication of implant restorations. PMID- 16681505 TI - Marketing for prosthodontists: become a master of digital photography. PMID- 16681510 TI - Ground water is not an educational priority. PMID- 16681514 TI - The response of playa and sabkha hydraulics and mineralogy to climate forcing. AB - Dry playa lakes and sabkhat often represent the terminus of large ground water flow systems and act as integrators of both upgradient (recharge) and downgradient discharge (evaporation). Ground water levels beneath playa/sabkha systems show a variety of surprising responses driven by large evaporation demands and chemical processes not typically encountered in more humid regions. When the water table is very close to the land surface, almost instantaneous rises can be observed with little observed change in either upgradient ground water recharge or potential evaporation. Conversely, when water tables are several meters below the playa surface, water table responses to interannual variability of recharge can be damped and lag significantly behind such changes. This review of the dynamics of shallow water tables in playa lakes and sabkhat discusses the pertinent hydraulic and solute processes and extracts a simple but comprehensive model based on soil physics for predicting the water table response to either upstream recharge changes or changes in potential evaporation at the playa/sabkha. Solutes and associated authigenic minerals are also shown to be important in discriminating both the causes and effects of water level fluctuations. PMID- 16681515 TI - MODFLOW/MT3DMS-based simulation of variable-density ground water flow and transport. AB - This paper presents an approach for coupling MODFLOW and MT3DMS for the simulation of variable-density ground water flow. MODFLOW routines were modified to solve a variable-density form of the ground water flow equation in which the density terms are calculated using an equation of state and the simulated MT3DMS solute concentrations. Changes to the MODFLOW and MT3DMS input files were kept to a minimum, and thus existing data files and data files created with most pre- and postprocessors can be used directly with the SEAWAT code. The approach was tested by simulating the Henry problem and two of the saltpool laboratory experiments (low- and high-density cases). For the Henry problem, the simulated results compared well with the steady-state semianalytic solution and also the transient isochlor movement as simulated by a finite-element model. For the saltpool problem, the simulated breakthrough curves compared better with the laboratory measurements for the low-density case than for the high-density case but showed good agreement with the measured salinity isosurfaces for both cases. Results from the test cases presented here indicate that the MODFLOW/MT3DMS approach provides accurate solutions for problems involving variable-density ground water flow and solute transport. PMID- 16681516 TI - Springflow hydrographs: eogenetic vs. telogenetic karst. AB - Matrix permeability in the range of 10(-11) to 10(-14) m(2) characterizes eogenetic karst, where limestones have not been deeply buried. In contrast, limestones of postburial, telogenetic karst have matrix permeabilities on the order of 10(-15) to 10(-20) m(2). Is this difference in matrix permeability paralleled by a difference in the behavior of springs draining eogenetic and telogenetic karst? Log Q/Q(min) flow duration curves from 11 eogenetic-karst springs in Florida and 12 telogenetic-karst springs in Missouri, Kentucky, and Switzerland, plot in different fields because of the disparate slopes of the curves. The substantially lower flow variability in eogenetic-karst springs, which results in the steeper slopes of their flow duration curves, also makes for a strong contrast in patterns (e.g., "flashiness") between the eogenetic-karst and telogenetic-karst spring hydrographs. With respect to both spring hydrographs and the flow duration curves derived from them, the eogenetic-karst springs of Florida are more like basalt springs of Idaho than the telogenetic-karst springs of the study. From time-series analyses on discharge records for 31 springs and published time-series results for 28 additional sites spanning 11 countries, we conclude that (1) the ratio of maximum to mean (Q(max)/Q(mean)) discharge is less in springs of eogenetic karst than springs of telogenetic karst; (2) aquifer inertia (system memory) is larger in eogenetic karst; (3) eogenetic-karst aquifers take longer to respond to input signals; and (4) high-frequency events affect discharge less in eogenetic karst. All four of these results are consistent with the hypothesis that accessible storage is larger in eogenetic karst aquifers than in telogenetic-karst aquifers. PMID- 16681517 TI - Scale effects of hydrostratigraphy and recharge zonation on base flow. AB - Uncertainty regarding spatial variations of model parameters often results in the simplifying assumption that parameters are spatially uniform. However, spatial variability may be important in resource assessment and model calibration. In this paper, a methodology is presented for estimating a critical basin size, above which base flows appear to be relatively less sensitive to the spatial distribution of recharge and hydraulic conductivity, and below which base flows are relatively more sensitive to this spatial variability. Application of the method is illustrated for a watershed that exhibits distinct infiltration patterns and hydrostratigraphic layering. A ground water flow model (MODFLOW) and a parameter estimation code (UCODE) were used to evaluate the influence of recharge zonation and hydrostratigraphic layering on base flow distribution. Optimization after removing spatial recharge variability from the calibrated model altered base flow simulations up to 53% in watersheds smaller than 40 km(2). Merging six hydrostratigraphic units into one unit with average properties increased base flow residuals up to 83% in basins smaller than 50 km(2). Base flow residuals changed <5% in watersheds larger than 40 and 50 km(2) when recharge and hydrostratigraphy were simplified, respectively; thus, the critical basin size for the example area is approximately 40 to 50 km(2). Once identified for an area, a critical basin size could be used to guide the scale of future investigations. By ensuring that parameter discretization needed to capture base flow distribution is commensurate with the scope of the investigation, uncertainty caused by overextending uniform parameterization or by estimating extra parameter values is reduced. PMID- 16681518 TI - Stream-stage response tests and their joint interpretation with pumping tests. AB - Hydraulic head response to stream-stage variations can be used to explore the hydraulic properties of stream-aquifer systems at a relatively large scale. These stream-stage response tests, also called flooding tests, are typically interpreted using one- or two-dimensional models that assume flow perpendicular to the river. Therefore, they cannot be applied to systems that are both horizontally and vertically heterogeneous. In this work, we use the geostatistical inverse problem to jointly interpret data from stream-stage response and pumping tests. The latter tests provide flow data (which are needed to resolve aquifer diffusivity into transmissivity and storage coefficient) and may supply supplementary small-scale information. Here, we summarize the methodology for the design, execution, and joint numerical interpretation of these tests. Application to the Aznalcollar case study allows us to demonstrate that the proposed methodology may help in responding to questions such as the continuity of aquitards, the role and continuity of highly permeable paleochannels, or the time evolution of stream-aquifer interaction. These results expand the applicability and scope of stream-stage response tests. PMID- 16681519 TI - Interpreting velocities from heat-based flow sensors by numerical simulation. AB - We have carried out numerical simulations of three-dimensional nonisothermal flow around an in situ heat-based flow sensor to investigate how formation heterogeneities can affect the interpretation of ground water flow velocities from this instrument. The flow sensor operates by constant heating of a 0.75-m long, 5-cm-diameter cylindrical probe, which contains 30 thermistors in contact with the formation. The temperature evolution at each thermistor can be inverted to obtain an estimate of the ground water flow velocity vector using the standard interpretive method, which assumes that the formation is homogeneous. Analysis of data from heat-based flow sensors installed in a sand aquifer at the Former Fort Ord Army Base near Monterey, California, suggested an unexpected component of downward flow. The magnitudes of the vertical velocities were expected to be much less than those of the horizontal velocities at this site because the sensors were installed just above a clay aquitard. Numerical simulations were conducted to examine how differences in thermal conductivities may lead to spurious indications of vertical flow velocities. We found that a decrease in the thermal conductivity near the bottom of the sensor can perturb the temperature profiles along the instrument in such a manner that analyses assuming homogeneous thermal conductivity could indicate a vertical flow component even though flow is actually horizontal. This work demonstrates how modeling can be used to simulate instrument response to formation heterogeneity and shows that caution must be used in interpreting data from such devices. PMID- 16681520 TI - Simulations to verify horizontal flow measurements from a borehole flowmeter. AB - This paper reports on experiments and simulations of subsurface flow from a slotted acrylic tube deployed in a sand-tank flow chamber for two different purposes. In the first instance, the slotted tube is used to represent a single fracture intersected by an uncased well. In the second instance, the slotted tube is used to represent a multislot well screen within a porous medium. In both cases, the scanning colloidal borescope flowmeter (SCBFM) measures ground water velocity within the well by imaging colloids traveling through a well to measure their speed and direction. Measurements are compared against model simulations. For the case of a slotted tube representing a single fracture, SCBFM and model results agree with respect to the flow direction and to within a factor of 1.5 for the speed near the well's center. Model and experimental agreement lend confidence that for an uncased well drilled in a fractured-rock medium, a calibrated SCBFM could be used to identify and quantify flowing features. Next, the SCBFM was deployed in a four-column multislotted casing with slots aligned with the flow direction. Another numerical model was developed to estimate the flow field within this well screen to evaluate the potential usefulness of employing the SCBFM in a screened well to estimate flow speed and direction in the surrounding porous medium. Results indicate that if the slots are not aligned with the flow, the SCBFM may only provide order-of-magnitude speed measurements and direction measurements with an uncertainty of approximately +/-25 degrees . PMID- 16681521 TI - Pure-phase transport and dissolution of TCE in sedimentary rock saprolite. AB - The objective of this study was to experimentally determine the influence of pore structure on the transport and dissolution of trichloroethylene (TCE) in clay rich saprolite. In order to simulate a "spill," pure-phase TCE containing a water insoluble fluorescent dye was injected into two heterogeneous 24-cm-diameter by 37-cm-long undisturbed columns of water-saturated saprolite. TCE entry occurred at capillary pressures of 2.7 and 4.0 kPa. Ten or 28 d after injection, the column was sliced horizontally into three sections and visually examined. The distribution of fluorescent dye indicated that pure-phase TCE migrated mainly through fractures in the shale saprolite and through fine root holes or other macropores in the limestone saprolite residuum. Analysis of saprolite subsamples indicated that TCE was present throughout much of the saprolite column but usually at concentrations less than the solubility of TCE. This spreading was caused by diffusion, which also contributed to the rapid dissolution of TCE in the fractures and macropores. Modeling was carried out using previously published dissolution and diffusion equations. The calculations confirm that rapid disappearance of immiscible TCE can occur in this type of material because of the small size of fracture or macropore openings and the high porosity of the fine grained material. This study indicates that industrial solvents can readily enter fractures and macropores in otherwise very fine-grained subsoils and then rapidly dissolve and diffuse into the fine-pore structure, from which they may be very difficult to remove. PMID- 16681522 TI - Change of collision efficiency with distance in bacterial transport experiments. AB - Understanding the mechanisms of bacterial transport in aquifers is important in developing bioremediation strategies. Collision efficiency (alpha) is one important parameter used in modeling bacterial transport. This study was undertaken to measure change in alpha with distance by performing a bacterial transport experiment in Oyster, Virginia. Following injection of a bacterium, Comamonas sp., into a well, water samples were collected at various distances along the flowpath and injected into columns packed with homogenized South Oyster focus area sediment. Zeta potentials of the bacteria in the samples were measured. Values of alpha were determined at various locations in the field in two ways: based on field breakthrough concentrations at the sampling points and based on column breakthrough concentrations. The alpha values estimated from field breakthrough decreased with distance, whereas those estimated from column breakthrough increased with distance. Bacterial cell surface charge became progressively more negative with distance in the field. We hypothesize that the apparent contradiction between field and column alpha values was caused by differences in the flow of the two systems. Flow in the columns was forced to occur through fine-grained zones of iron and aluminum hydroxide coatings that selectively removed the most negatively charged bacteria. In contrast, in the field, the injected cells did not come into contact with the positively charged coatings because the bulk solution bypassed them due to heterogeneous hydraulic properties. These results suggest that laboratory-based models may underestimate bacterial transport distance in the field. A more realistic approach may be necessary to capture the degree of heterogeneity. PMID- 16681523 TI - Geophysical monitoring of a field-scale biostimulation pilot project. AB - The USGS conducted a geophysical investigation in support of a U.S. Naval Facilities Engineering Command, Southern Division field-scale biostimulation pilot project at Anoka County Riverfront Park (ACP), down-gradient of the Naval Industrial Reserve Ordnance Plant, Fridley, Minnesota. The goal of the pilot project is to evaluate subsurface injection of vegetable oil emulsion (VOE) to stimulate microbial degradation of chlorinated hydrocarbons. To monitor the emplacement and movement of the VOE and changes in water chemistry resulting from VOE dissolution and/or enhanced biological activity, the USGS acquired cross-hole radar zero-offset profiles, travel-time tomograms, and borehole geophysical logs during five site visits over 1.5 years. Analysis of pre- and postinjection data sets using petrophysical models developed to estimate VOE saturation and changes in total dissolved solids provides insights into the spatial and temporal distribution of VOE and ground water with altered chemistry. Radar slowness difference tomograms and zero-offset slowness profiles indicate that the VOE remained close to the injection wells, whereas radar attenuation profiles and electromagnetic induction logs indicate that bulk electrical conductivity increased down-gradient of the injection zone, diagnostic of changing water chemistry. Geophysical logs indicate that some screened intervals were located above or below zones of elevated dissolved solids; hence, the geophysical data provide a broader context for interpretation of water samples and evaluation of the biostimulation effort. Our results include (1) demonstration of field and data analysis methods for geophysical monitoring of VOE biostimulation and (2) site-specific insights into the spatial and temporal distributions of VOE at the ACP. PMID- 16681524 TI - Cross-borehole flowmeter tests for transient heads in heterogeneous aquifers. AB - Cross-borehole flowmeter tests have been proposed as an efficient method to investigate preferential flowpaths in heterogeneous aquifers, which is a major task in the characterization of fractured aquifers. Cross-borehole flowmeter tests are based on the idea that changing the pumping conditions in a given aquifer will modify the hydraulic head distribution in large-scale flowpaths, producing measurable changes in the vertical flow profiles in observation boreholes. However, inversion of flow measurements to derive flowpath geometry and connectivity and to characterize their hydraulic properties is still a subject of research. In this study, we propose a framework for cross-borehole flowmeter test interpretation that is based on a two-scale conceptual model: discrete fractures at the borehole scale and zones of interconnected fractures at the aquifer scale. We propose that the two problems may be solved independently. The first inverse problem consists of estimating the hydraulic head variations that drive the transient borehole flow observed in the cross-borehole flowmeter experiments. The second inverse problem is related to estimating the geometry and hydraulic properties of large-scale flowpaths in the region between pumping and observation wells that are compatible with the head variations deduced from the first problem. To solve the borehole-scale problem, we treat the transient flow data as a series of quasi-steady flow conditions and solve for the hydraulic head changes in individual fractures required to produce these data. The consistency of the method is verified using field experiments performed in a fractured-rock aquifer. PMID- 16681525 TI - Boron isotopes as an artificial tracer. AB - A field study was conducted using a combination of intrinsic and artificial tracers to estimate travel times and dilution during transport of infiltrate from a reclaimed water infiltration basin to nearby monitoring wells. A major study objective was to validate boric acid enriched in (10)B as an artificial tracer. Basin 10E at the Rio Hondo Spreading Grounds in Whittier, California, was the site of the test. The basin normally receives a mixture of treated municipal waste water, purchased State Project water, and local runoff from the San Gabriel River. Approximately 3.5 kg of (10)B-enriched boric acid was dispersed among 2.05 x 10(5) m(3) of basin water to initiate the experiment. The resultant median delta(11)B in the infiltration basin was -71 per thousand. Prior to tracer addition, the basin water had an intrinsic delta(11)B of +2 per thousand. Local monitoring wells that were used to assess travel times had delta(11)B values of +5 per thousand and +8 per thousand at the time of tracer addition. Analytic results supported an assumption that boron is conserved during ground water transport and that boron enriched in (10)B is a useful artificial tracer. Several intrinsic tracers were used to reinforce the boric acid tracer findings. These included stable isotopes of oxygen (delta(18)O) and hydrogen (deltaD), sulfate concentration, and the boron to chloride ratio. Xenon isotopes, (136)Xe and (124)Xe, also supported boron isotope results. Xenon isotopes were added to the recharge basin as dissolved gases by investigators from the Lawrence Livermore National Laboratory. PMID- 16681526 TI - Interpretation of transmissivity estimates from single-well pumping aquifer tests. AB - Interpretation of single-well tests with the Cooper-Jacob method remains more reasonable than most alternatives. Drawdowns from 628 simulated single-well tests where transmissivity was specified were interpreted with the Cooper-Jacob straight-line method to estimate transmissivity. Error and bias as a function of vertical anisotropy, partial penetration, specific yield, and interpretive technique were investigated for transmissivities that ranged from 10 to 10,000 m(2)/d. Cooper-Jacob transmissivity estimates in confined aquifers were affected minimally by partial penetration, vertical anisotropy, or analyst. Cooper-Jacob transmissivity estimates of simulated unconfined aquifers averaged twice the known values. Transmissivity estimates of unconfined aquifers were not improved by interpreting results with an unconfined aquifer solution. Judicious interpretation of late-time data consistently improved estimates where transmissivity exceeded 250 m(2)/d in unconfined aquifers. PMID- 16681527 TI - Analysis of depth-dependent pressure head of slug tests in highly permeable aquifers. AB - A curve-matching method is developed for the analysis of depth-dependent pressure head responding to a slug test in a highly permeable aquifer. The depth dependency is due to the fact that the pressure transducer is placed at depth relatively far from the initial water level. The Springer and Gelhar solution and a depth correction relation are used to generate theoretical curves of pressure head vs. time. A trial-and-error procedure is established to find the theoretical curve best fitting the field data by adjusting the two unknown parameters, the horizontal hydraulic conductivity, and the effective length of the water column. Analytic relations for some oscillation characteristics of the converted pressure head are derived. A field example indicates that the theoretical relations and the curve-matching method developed herein are suitable to deal with the oscillatory head data dependent on depth of measurement. Nevertheless, it is recognized that placing the pressure transducer close to the initial water level and using a small initial water displacement can effectively avoid complicating the data analysis, such that previous, simpler data analysis methods can be used. PMID- 16681528 TI - Where do periodic variations in the discharge of a well become negligible? AB - A rule of thumb is presented to determine where variations in the discharge of a pumping well have a significant influence on the flow in an aquifer. The rule of thumb relates the period of the variation of the discharge to the distance from the well beyond which the transient effect on the flow in the aquifer is insignificant. For example, when an irrigation well pumps intermittently during the growing season, the rule may be applied to determine the distance from the well beyond which flow in the aquifer can be simulated with an average discharge during the growing season; the distance from the well beyond which flow can be simulated with a steady, yearly averaged discharge can also be computed. PMID- 16681529 TI - Basic concepts for the linear model of ground water level recession. AB - Basic concepts are illustrated for the display of ground water level recession as a linear plot on a semilog graph, as first described by Rorabaugh. This exponential decay function can be achieved if there is a definable outflow boundary such as a lake or river and if water levels are expressed relative to the altitude of the boundary. The model can be used to estimate aquifer hydraulic diffusivity. Concepts are illustrated using three finite-difference simulations. One represents the ideal case as described by Rorabaugh, in which the altitude of the outflow boundary is uniform along its length. Another simulation includes a sloping boundary with simple geometry and demonstrates that the model can be used accurately. Based on this simulation, it appears that the ground water level must be expressed relative to the closest point on the outflow boundary. The third simulation includes a sloping boundary and complex boundary shape, and demonstrates departures from the linear model of recession and errors in the estimate of hydraulic diffusivity. Another cause of nonlinearity is the instability of the ground water head profile soon after a recharge event. The nature of these early-time departures will vary depending on the location of the water level observation site relative to the outflow boundary and the hydrologic divide of the ground water flow system. PMID- 16681530 TI - On storage coefficient and vertical strain. PMID- 16681531 TI - Antireflux surgery: raising the bar? PMID- 16681532 TI - Resuscitating the autopsy in Australian hospitals. AB - Previous studies have consistently shown that autopsy examination after surgery can identify a significant rate of discrepant and/or erroneous diagnoses. Findings gleaned from autopsies can be invaluable for the medical community and science as a whole, ultimately leading to improvements in quality surgical care. Despite the proven benefits of this underused tool, the number of autopsies carried out in Australian hospitals is declining dramatically, with autopsy rates plummeting from 21% in 1992-1993 to 12% in 2002-2003. This notable decline ought to be a genuine source of concern and deserves further examination. This review seeks to address the reasons for decline, while reasserting the value and relevance of autopsies within modern surgical practice Down Under. PMID- 16681533 TI - Australian Vascular Quality of Life Index (AUSVIQUOL): a pilot study of a disease specific quality of life measure. AB - BACKGROUND: To develop and test a quality of life (QOL) index specific for patients with vascular disease and appropriate for patients with abdominal aortic aneurysm (AAA) in the clinical setting. METHODS: The questions and domains of the Australian Vascular Quality of Life Index (AUSVIQUOL) were determined by examination of a prospective database for frequency of symptoms and an in-depth interview of a sample population. The validity of the AUSVIQUOL was tested by comparing it with the Medical Outcomes Short Form Health Survey (SF-36) in a study involving 60 patients who underwent endovascular AAA repair and 48 open AAA repair. A subpopulation of 22 patients representative of the two groups was then reassessed using the SF-36 and the AUSVIQUOL, to compare the reliability of the two indices. RESULTS: Similar domains of the SF-36 and the AUSVIQUOL measured common QOL elements. The correlation between the two indices was moderate; the AUSVIQUOL measured additional disease-specific QOL factors. The AUSVIQUOL showed better reliability than the SF-36 in all domains and statistically better in the physical function domain (P < 0.05). CONCLUSION: The AUSVIQUOL is an appropriate tool for the QOL assessment of patients with AAA in the clinical setting. PMID- 16681534 TI - Surgical management of carotid body tumours: a 24-year surgical experience. AB - BACKGROUND: Carotid body tumours (CBT) are rare tumours, best treated by complete surgical resection. However, there is no uniform agreement on the method of resection. The aim of this study was to review our 24 years' experience of meticulous subadventitial excision of CBT. METHODS: A retrospective study, from March 1980 to September 2004 of patients with CBT was undertaken, detailing presentation, diagnosis and treatment and postoperative complications. RESULTS: Twenty-five patients (six men and 19 women) with an age range of 23-72 years had been operated on, and all were treated by surgical excision. All patients had neck mass. No patient had a positive family history. Angiography was the main method of diagnosis. All of the patients had unilateral tumours. There was no postoperative permanent neurological deficit. Temporary neurological problems developed in only four patients. External carotid artery was ligated in three patients to facilitate excision of the tumour. Surgical care limited blood loss to an average of 480 mL. CONCLUSIONS: Subadventitial excision, carried out meticulously, allowed complete resection to be achieved in all of the patients with minimal morbidity and no surgical mortalities. This method is therefore recommended. Facilities for shunting and arterial repair should always be available. PMID- 16681535 TI - Implications of low great toe pressures in clinical practice. AB - INTRODUCTION: The aim of this study was to observe the outcome of patients with a great toe pressure (GTP) reading of less than 40 mmHg. METHODS: Between 2002 and 2004, of 4,714 patients assessed in the Westmead Vascular Laboratory, 365 (7.7%) had GTP measurements for assessment of possible critical limb ischaemia. There were 56 limbs in 40 patients with a GTP measurement of less than 40 mmHg, and this group was studied to assess outcome factors of death, requirement for major or minor amputation, arterial reconstructive surgery, clinical stability, or documented improvement from the initial assessment. RESULTS: Patients requiring a major amputation had an average GTP of 13 mmHg and a toe brachial index (TBI) of 0.08, whereas those not undergoing a major amputation had an average GTP of 23.6 mmHg and TBI of 0.15. This association was weaker when an initial single evaluation was used as opposed to two or more serial assessments. Patients with and without a major amputation had an average ankle brachial index of 0.16 and 0.53, respectively. Diabetes mellitus requiring insulin and cerebrovascular disease were risk factors for major amputations. CONCLUSIONS: Low GTP was associated with a greater risk of major amputations. Two or more serial assessments were found to be of greater value than an initial single assessment. PMID- 16681536 TI - Weight change 1 year following total knee or hip arthroplasty. AB - BACKGROUND: Pain secondary to osteoarthritis (OA) of the hip or knee is often used as a reason to not lose weight prior to total knee or hip arthroplasty (TKA, THA). This study followed the weight change of patients who subjectively increased their activity levels 1 year following TKA or THA. METHODS: We reviewed the records and prospectively followed the weight of 84 patients 1 year following surgery. The pre and postoperative weight were compared, including separate 10 kg categories (e.g. 60.1-70.0 kg). RESULTS: All patients had improved mobility as evaluated by either the clinical notes or a patient-based questionnaire. At 1 year, there was no significant change in weight; only an insignificant small increase in weight was seen. CONCLUSIONS: This study suggests that lower preoperative activity levels are not the cause for the inability to lose weight or that the gain in mobility achieved by joint replacement, of its own, does not result in weight loss. PMID- 16681537 TI - Postoperative pain following coblation tonsillectomy: randomized clinical trial. AB - BACKGROUND: Tonsillectomy is one of the commonest surgical procedures, with postoperative pain being an important source of morbidity. Coblation (cold ablation) is a new technique for tonsillectomy, promoted by claims of reduced postoperative pain levels. This study was designed to compare postoperative pain after tonsillectomy using coblation and tonsillectomy using the standard dissection techniques. METHODS: Twenty adult patients underwent tonsillectomy, each having one randomly selected tonsil removed by dissection and the other removed by coblation. For each side, subjective pain levels were recorded on a daily basis for 10 postoperative days, using a visual analogue scale. RESULTS: Coblation tonsillectomy was significantly less painful than dissection tonsillectomy on day 1 (P < 0.001), day 2 (P = 0.003) and day 3 (P = 0.018). For all subsequent postoperative days, there was no significant difference in pain levels between the techniques. CONCLUSION: Coblation tonsillectomy causes significantly less pain during the first three postoperative days, when compared with dissection tonsillectomy. No demonstrable benefit was shown on days 4-10. The beneficial effects of coblation on early postoperative pain make it a potentially attractive technique for day-case tonsillectomy in adults with recurrent or chronic tonsillitis. PMID- 16681539 TI - Diagnostic and treatment challenges of inframammary crease breast carcinomas. AB - BACKGROUND: To review one surgeon's experience with tumours of the inframammary crease. METHODS: Individually reviewed cases were identified from a prospective database. RESULTS: Of the 550 patients treated between January 2000 and January 2005, six patients were found to have carcinomas of the inframammary crease (IMC). The features identified from these cases included diagnostic confusion with sebaceous cysts delaying diagnosis and early involvement of the dermis and chest wall resulting in management issues related to local T staging. Lymphoscintigraphy showed drainage to bilateral, contralateral or ipsilateral axillae as well as the internal mammary chain. CONCLUSION: The anatomy and histology of the IMC are reviewed, as is the available published work on IMC breast tumours. By virtue of their location, IMC tumours invade the dermis and chest wall early. Despite this, they do not seem to behave as T4 cancers. Their lymphatic drainage is a cross between patterns observed for breast parenchymal and skin primaries, making sentinel node lymphoscintigraphy essential. The clinical cases presented highlight these peculiarities of IMC breast cancer. PMID- 16681540 TI - Mammary tuberculosis: analysis of thirty-eight patients. AB - Thirty-eight patients with mammary gland tuberculosis were evaluated over a 5 year period presenting to the surgical unit of our institution. Unilateral involvement of the breast in a woman presenting at an average age of 29 years was the commonest observation. A lump in the breast with or without discharging sinuses was the most common clinical presentation. Ten (26%) of these patients had breast pain with or without increased breast nodularity. Axillary lymph nodal involvement was evident in 14 (36%) of our patients. Only five patients had associated pulmonary tuberculosis, the rest having an isolated involvement of the breast. Fine-needle aspiration cytology was the most reliable diagnostic modality. Medical therapy with antitubercular drugs ranging from 6 to 9 months was the mainstay of treatment. Surgical intervention was reserved for selected refractory cases. PMID- 16681541 TI - Thyroidectomy is safe and effective for retrosternal goitre. AB - BACKGROUND: Retrosternal goitre was defined as any thyroid enlargement identified below the thoracic inlet at operation, with the patient's neck held in extension. The aim of this study was to determine the characteristics of the patients, the goitres, the surgery and its morbidity (including tracheomalacia, recurrent laryngeal nerve palsy and hypocalcaemia) and the incidence of malignancy in order to establish guidelines for managing patients with a retrosternal goitre. METHODS: Data were collected prospectively on all thyroidectomies carried out by a single surgeon over 14 years. Patients underwent appropriate preoperative assessment and thyroidectomy was carried out using a standardized capsular dissection technique. There were 199 cases of retrosternal extension. RESULTS: Retrosternal extension was significantly more common on the left side than on the right side (ratio 3:2, P < 0.05). Most patients (83.4%) had significant symptoms that were relieved by surgery. Of the 199 thyroidectomies, none required a sternal split. The rate of malignancy was low (2.5%). Postoperative morbidity was 30%, the majority being asymptomatic temporary hypocalcaemia. There were no patients with permanent recurrent laryngeal nerve palsies or permanent hypoparathyroidism. There was one case of tracheomalacia. There was no death. CONCLUSION: Retrosternal goitre is a frequently symptomatic condition, with a low but definite rate of malignancy. Surgery is usually possible through a cervical incision and with an acceptable risk of significant morbidity. Thyroidectomy should be recommended as the treatment of choice. PMID- 16681542 TI - Post caesarean vesicouterine fistulae-- Youssef syndrome: our experience and review of published work. AB - OBJECTIVE: To analyse the incidence, symptomatology, diagnosis and therapeutic aspects of Youssef syndrome (post caesarean vesicouterine fistula), and to review relevant published work. METHODS: A retrospective study from the urosurgical unit of a tertiary care referral hospital was carried out. In a retrospective analysis of urogenital fistulae over 10 years, we identified 14 patients with uterovesical fistulae, resulting from caesarean section. All the patients were evaluated by history, physical examination, radiological tests and cystoscopy. All patients underwent transperitoneal repair of these fistulae with omental interposition. Results of surgery were evaluated by absence of cyclic haematuria, stoppage of urinary incontinence, and achievement of fertility. RESULTS: A total of 12 patients who had minimum follow up was included in the present study. The results showed that 50% of the fistulae resulted from emergency caesarean operation with 58% of patients presenting after their second caesarean section. The mean age of the patients was 19 years (range 15-29) and mean duration of symptoms was 7 months (range 3-16). Menouria and amenorrhoea were predominant presenting symptoms. The results of surgical treatment were excellent with good continence and resolution of the cyclic haematuria. Three pregnancies (37.5%) which resulted in elective caesarean section were recorded. CONCLUSION: Vesicouterine fistulae, despite being infrequent, are no longer a rare diagnosis and are most commonly secondary to lower segment caesarean section. With patient history and selected investigations diagnosis is relatively easy. The surgical repair of these fistulae is standard treatment, especially with delayed fistulae with achievement of total continence, and complete resolution of cyclic haematuria. Meticulous practice of obstetric and surgical principles during caesarean section can prevent the formation of these fistulae. PMID- 16681543 TI - Hepatectomy for metastatic noncolorectal gastrointestinal, breast and testicular tumours. AB - BACKGROUND: The indications for hepatectomy for colorectal or neuroendocrine metastases are becoming clear with increasing experience reported. For other primary diseases, however, the overall number of cases is relatively small, and it is more difficult to derive clear guidelines. This paper reviews the reported experience of hepatectomy for metastases from non-colorectal gastrointestinal primary cancers, breast cancer and testicular teratoma. The aim is to determine for each whether and under what circumstances hepatectomy is indicated. METHODS: A Medline search was used to identify papers reporting hepatectomy for metastases from non-colorectal gastrointestinal carcinomas, breast carcinomas and testicular teratomas. The data collected included the primary disease, the number of cases reported, the survival post-hepatectomy and any prognostic factors associated with outcome. RESULTS: Of the gastrointestinal malignancies, hepatectomy for gastric metastases yields a 5-year survival, roughly half that reported for colorectal disease, and further elucidation of prognostic factors would be desirable. Results were poor for other gastrointestinal malignancies. Good results were reported for breast and testicular teratoma. CONCLUSION: Of the non colorectal gastrointestinal primaries, at present only hepatectomy for gastric metastases can be cautiously recommended. For nongastrointestinal primaries, hepatic metastases probably represent widespread dissemination even if occult, and therefore, hepatectomy may only be of use when effective adjuvant treatments are available. PMID- 16681544 TI - Mirizzi syndrome: history, present and future development. AB - BACKGROUND: Mirizzi syndrome was reported in 0.3-3% of patients undergoing cholecystectomy. The distortion of anatomy and the presence of cholecystocholedochal fistula increase the risk of bile duct injury during cholecystectomy. METHODS: A Medline search was undertaken to identify articles that were published from 1974 to 2004. Additional papers were identified by a manual search of the references from the key articles. RESULTS: A preoperative diagnosis was made in 8-62.5% of cases. Open surgical treatment gave good short term and long-term results. There was a lack of good data in laparoscopic treatment. Conversion to open surgery rates was high, and bile duct injury rate varied from 0 to 22.2%. CONCLUSION: A high index of clinical suspicion is required to make a preoperative or intraoperative diagnosis, which leads to good surgical planning to treat the condition. Open surgery is the gold standard. Mirizzi syndrome should still be considered as a contraindication for laparoscopic surgery. PMID- 16681545 TI - Optimization of outcome after laparoscopic antireflux surgery. AB - Laparoscopic fundoplication has emerged as an effective treatment for gastro oesophageal reflux disease. The majority of patients who have undergone antireflux surgery report an improvement in reflux symptoms and in quality of life. However, some patients are dissatisfied with the outcome of antireflux surgery, and attempts have been made by surgeons to improve the results of this surgery. Careful case selection based on objective evidence of acid reflux, refinement of the surgical technique and 'tailoring' the wrap to suit the patient by selective use of a partial fundoplication may help to optimize the outcome from laparoscopic antireflux surgery. PMID- 16681546 TI - Pulsating varicose veins!! The diagnosis lies in the heart. AB - Right heart failure is associated with increased systemic venous pressure, which can be diagnosed clinically with the findings of elevated jugular venous pressure, pulsatile liver and distinctive cardiac murmurs (precordial systolic). Severe tricuspid regurgitation (TR) has occasionally been known to lead to marked pulsation of varicose veins. We report three cases that were referred to the vascular clinic of Royal Perth Hospital in which the patients involved had unilateral (right leg) varicose veins and chronic venous ulcers. On clinical examination all three patients had pulsations along the course of the varicose long saphenous vein up to the mid calf. The main differential diagnosis was arterio-venous malformation, which was excluded by compression of the sapheno femoral junction and demonstrating absence of pulsation in the long saphenous vein. A venous duplex scan showed a grossly incompetent sapheno-femoral junction with abnormal wave forms. Two of the cases were managed conservatively with compression dressing. The option of sapheno-femoral junction ligation was reserved in one patient who had unsettling cellulitis and oedema of the lower limb in spite of compression dressing and optimal conservative management. All three patients had improvement in ulcer size at 3-month follow up with compression therapy. This article highlights that in cases of right heart failure the venous pressures can be felt as low as the mid calf level and that can be a cause of the venous ulcers. There should be a high suspicion of right heart failure in patients with late onset venous insufficiency. PMID- 16681547 TI - Maxillary tumours and bilateral reconstruction of the maxilla. AB - Reconstruction of maxillectomy defects following intraoral tumour excision presents a challenge to the plastic surgeon. The defect left after maxillectomy requires a formal reconstruction in order to minimize problems associated with speech and swallowing. A multidimensional approach is required to achieve a structural and functional restoration. The maxillectomy defects are complex, which often requires composite tissue replacement. A variety of reconstructive techniques have been used for the repair of the maxillary and palatal defects, including prosthetics, skin grafts, local and free flaps using the iliac crest, scapula, fibula, latissimus dorsi, radial forearm and abdominis rectus free flaps. We have used a lateral-arm osteocutaneous free flap for reconstruction of a bilateral maxillary defect, with excellent aesthetic, functional and structural support results. This type of reconstruction for this defect has never been reported previously. PMID- 16681548 TI - Management of colorectal anastomotic membrane by an endoscopic double illumination technique. AB - Two cases of postoperative colorectal anastomotic mucosal membrane completely occluding the lumen of the bowel are described. Although an unusual event, patency may be readily re-established by an endoscopic double-illumination technique. PMID- 16681549 TI - Coeliac artery trunk thrombosis in acute pancreatitis causing total gastric necrosis. PMID- 16681550 TI - Bilateral congenital radial head dislocation presenting as Monteggia's fracture. PMID- 16681551 TI - Keratoacanthoma centrifugum marginatum. PMID- 16681552 TI - Surgical masks may reduce serious postoperative cataract complications. PMID- 16681553 TI - Diabetes mellitus as a contributory factor in oral candidosis. AB - It has been reported that poor glycaemic control predisposes to oral candidal infection in diabetic patients. For instance, the carriage of Candida species and the density of candidal growth in the oral cavity is frequently claimed to be increased in patients with diabetes mellitus. However, the validity of these observations remains controversial. Hence, we review and discuss here the clinical data in the literature on the relationship between diabetes and oral candidal carriage and infection, and possible mechanisms associated with its pathogenicity. PMID- 16681554 TI - Comparison of international and New Zealand guidelines for the care of pregnant women with diabetes. AB - OBJECTIVE: To compare international guidelines for the care of women with diabetes and pregnancy with reported current practice among New Zealand tertiary centres. RESEARCH DESIGN AND METHODS: A literature review of national and international guidelines for the care of women with diabetes in pregnancy was undertaken. Guideline activities were placed within nine facets of care, from preconception advice, through pregnancy from screening to follow-up. New Zealand tertiary centres guidelines were obtained and placed in the same framework. RESULTS: International guideline consensus was inconsistent across most facets of care. Those for the detection and diagnosis of gestational diabetes mellitus (GDM) were particularly discordant internationally, although intranational agreement has occurred. CONCLUSIONS: International guidelines for the care of women with diabetes in pregnancy remain fragmented. The development of one set of guidelines based on the consensus of international best practice could overcome many of the misconceptions associated with diabetes in pregnancy. PMID- 16681555 TI - Metabolic syndrome--a new world-wide definition. A Consensus Statement from the International Diabetes Federation. AB - AIMS: To establish a unified working diagnostic tool for the metabolic syndrome (MetS) that is convenient to use in clinical practice and that can be used world wide so that data from different countries can be compared. An additional aim was to highlight areas where more research into the MetS is needed. PARTICIPANTS: The International Diabetes Federation (IDF) convened a workshop held 12-14 May 2004 in London, UK. The 21 participants included experts in the fields of diabetes, public health, epidemiology, lipidology, genetics, metabolism, nutrition and cardiology. There were participants from each of the five continents as well as from the World Health Organization (WHO) and the National Cholesterol Education Program-Third Adult Treatment Panel (ATP III). The workshop was sponsored by an educational grant from AstraZeneca Pharmaceuticals. CONSENSUS PROCESS: The consensus statement emerged following detailed discussions at the IDF workshop. After the workshop, a writing group produced a consensus statement which was reviewed and approved by all participants. CONCLUSIONS: The IDF has produced a new set of criteria for use both epidemiologically and in clinical practice world wide with the aim of identifying people with the MetS to clarify the nature of the syndrome and to focus therapeutic strategies to reduce the long-term risk of cardiovascular disease. Guidance is included on how to compensate for differences in waist circumference and in regional adipose tissue distribution between different populations. The IDF has also produced recommendations for additional criteria that should be included when studying the MetS for research purposes. Finally, the IDF has identified areas where more studies are currently needed; these include research into the aetiology of the syndrome. PMID- 16681556 TI - Pregnancy in women with diabetes--after the CEMACH report, what now? AB - The second of three studies being undertaken by the Confidential Enquiry into Maternal and Child Health (CEMACH) has recently reported its findings and recommendations. The standards of diabetes and maternal care and the outcomes of 3808 pregnancies in England, Wales and Northern Ireland are described. Pre pregnancy planning and care before conception is poor. Stillbirth rate (26.8 per thousand) and perinatal mortality (41.8 per thousand) were 4-5 times higher than the background population, and congenital anomaly (41.8 per thousand) double the background rate. Type 2 diabetes now represents 27.3% of pre-gestational diabetes and more often of ethnic minority and deprived background than Type 1 diabetes. The ideals of the Diabetes National Service Framework and the target of the Saint Vincent declaration are far from being achieved. Much can be done to improve the outcomes of pregnancy within existing resources with better systems and organization of care. However, if significant progress is to be made, it is incumbent upon health-care professionals and health-care commissioners to direct resources specifically at improving pre-pregnancy and maternity services. Research is needed firstly to analyse in greater detail the wealth of data collected in the CEMACH survey and consider the implications of health-care costs. Further research to discover ways of reducing the adverse outcomes is urgently required. The need to educate, motivate and bring about improved pre pregnancy care in women with diabetes is a priority. PMID- 16681557 TI - The diabetes educator: trying hard, but must concentrate more on behaviour. AB - Current guidelines state that education is fundamental to help people with diabetes modify their lifestyle and prevent ill health and early death. However, many people with diabetes are not receiving adequate education. There is a widespread assumption that transferring knowledge will improve health outcomes but there is little empirical support for this assertion. Indeed, knowledge and behaviour are poorly correlated. Knowledge may be a necessary condition but is rarely a sufficient condition for behaviour change. Single interventions, cognitive or behavioural, have had disappointing results, unsurprisingly given the complexity of human behaviour. The most effective interventions are multifaceted and include education, behavioural and psychosocial elements, and target lifestyle change and factors such as self-efficacy and empowerment. We advocate that educational interventions should have multiple components. They should aim to improve patients' sense of self-efficacy and empowerment, and build attitudes towards diabetes that will support the lifestyle changes needed for successful self-management. These conclusions have implications for future research and clinical practice. PMID- 16681558 TI - Functional erythropoietin deficiency in patients with Type 2 diabetes and anaemia. AB - AIMS: Anaemia is a common finding in patients with diabetic nephropathy. Impaired production of erythropoietin is thought to be the predominant cause, as a result of renal microvascular disease. This study aims to determine the prevalence of functional erythropoietin deficiency in a cross-sectional survey of patients with Type 2 diabetes. METHODS: Clinical data on 604 patients with Type 2 diabetes were obtained, including a full blood count, iron indices and detailed history of diabetic complications. Erythropoietin levels were correlated with the presence of anaemia, iron deficiency and renal dysfunction. Functional erythropoietin deficiency was defined by erythropoietin levels in the normal range despite the presence of anaemia. RESULTS: Nineteen per cent of patients (n = 112) were anaemic, among whom erythropoietin deficiency (76%) and reduced iron availability (58%) were common findings. Over 90% of patients had erythropoietin deficiency, once those with reduced iron stores or availability were excluded. Most of these patients had moderate renal impairment (60%, n = 67). However, even in the absence of renal impairment, 71% of anaemic patients (n = 32/45) had functional erythropoietin deficiency, although most had other evidence of nephropathy. In addition, two-thirds of patients with reduced iron availability were unable to increase erythropoietin above the normal range. CONCLUSIONS: These findings confirm the failure of the kidney to produce erythropoietin in response to a falling haemoglobin is a key component to anaemia in diabetes. The likelihood of functional erythropoietin deficiency as a cause of anaemia is not dependent on the severity of renal impairment or excluded in diabetic patients with reduced iron stores or availability. PMID- 16681559 TI - Smoking enhances absorption of insulin but reduces glucodynamic effects in individuals using the Lilly-Dura inhaled insulin system. AB - AIM: To quantify the pharmacokinetic (PK) and glucodynamic (GD) impact of smoking on inhaled and subcutaneous (SC) insulin administration in healthy subjects. METHODS: This study employed the euglycemic clamp procedure in a four-period, four-way randomized crossover design. Eight smoking and eight non-smoking healthy males were given SC insulin on two occasions and human insulin inhalation powder (HIIP) on two other occasions. RESULTS: Smokers exhibited greater insulin exposure (AUC(0-t')) than non-smokers, following both routes of insulin administration (HIIP, P = 0.003, 58% increase; SC, P = 0.006, 24% increase). The maximum insulin concentration (C(max)) following HIIP was greater in smokers by 172% (P = 0.001) compared with non-smokers. The glucodynamic effects were greater in smokers following HIIP, consistent with the insulin concentration difference observed. However, maximum glucose response (R(max)) following SC was decreased by 36% (P = 0.001) and obtained later [time of maximum glucose response (TR(max)); P < 0.001] in smokers than in non-smokers. Smokers appeared less sensitive to insulin [total glucose infused during the clamp procedure normalised by total insulin exposure (G(tot))/AUC(0-t')] than non-smokers following both SC (P = 0.001) and inhaled (P = 0.011) routes of administration. CONCLUSION: Smokers had substantially increased peak HIIP insulin concentration, but the glucodynamic effect was partially offset, most likely because of increased insulin resistance. PMID- 16681560 TI - Mortality in people with type 2 diabetes in the UK. AB - AIMS: Under-reporting of diabetes on death certificates contributes to the unreliable estimates of mortality as a result of diabetes. The influence of obesity on mortality in Type 2 diabetes is not well documented. We aimed to study mortality from diabetes and the influence of obesity on mortality in Type 2 diabetes in a large cohort selected from the General Practice Research Database (GPRD). Methods A cohort of 44 230 patients aged 35-89 years in 1992 with Type 2 diabetes was identified. A comparison group matched by year of birth and sex with no record of diabetes at any time was identified (219 797). Hazards ratios (HRs) for all-cause mortality during the period January 1992 to October 1999 were calculated using the Cox Proportional Hazards Model. The effects of body mass index (BMI), smoking and duration of diabetes on all-cause mortality amongst people with diabetes was assessed (n = 28 725). Results The HR for all-cause mortality in Type 2 diabetes compared with no diabetes was 1.93 (95% CI 1.89 1.97), in men 1.77 (1.72-1.83) and in women 2.13 (2.06-2.20). The HR decreased with increasing age. In the multivariate analysis in diabetes only, the HR for all-cause mortality amongst smokers was 1.50 (1.41-1.61). Using BMI 20-24 kg/m(2) as the reference range, for those with a BMI 35-54 kg/m(2) the HR was 1.43 (1.28 1.59) and for those with a BMI 15-19 kg/m(2) the HR was 1.38 (1.18-1.61). CONCLUSIONS: Patients with Type 2 diabetes have almost double the mortality rate compared with those without. The relative risk decreases with age. In people with Type 2 diabetes, obesity and smoking both contribute to the risk of all-cause mortality, supporting doctrines to stop smoking and lose weight. PMID- 16681561 TI - Insulin detemir under steady-state conditions: no accumulation and constant metabolic effect over time with twice daily administration in subjects with Type 1 diabetes. AB - AIMS: This study investigated the pharmacodynamic and pharmacokinetic characteristics of the novel long-acting insulin analogue insulin detemir (IDet) under single-dose and steady-state conditions in comparison with those of NPH insulin at steady state. METHODS: Twenty-five subjects with Type 1 diabetes [seven females, 18 males, mean age (+/- sd) 39 +/- 12 years, body mass index 24 +/- 3 kg/m(2)] participated in three 24-h glucose clamps. IDet or NPH were given at 12-h intervals in fixed, individualized doses. The first clamp assessed the metabolic effect of NPH at steady state, the second investigated the effect of two single injections of IDet. Subjects continued IDet treatment for 7-14 days, after which the third clamp was performed to investigate IDet at steady state. RESULTS: At steady state, the metabolic effect of IDet was constant over 24 h while a clear peak in the metabolic effect [expressed as glucose infusion rates (GIR)] was observed with NPH after each injection. The fluctuation in the metabolic effect (maximum GIR divided by the average of the GIR values at the interval ends) was significantly lower in the second 12 h of the experiments with IDet under steady-state conditions compared with NPH (fluctuation(12-24 h) 1.27 +/- 0.17 vs. 1.56 +/- 0.72, P < 0.05). The overall metabolic effect of IDet at steady state was comparable with that of NPH [GIR-area under curve (AUC)(0-24 h): 5697 +/- 1861 vs. 5929 +/- 1965 mg/kg] whereas a significantly lower effect (5187 +/- 1784 mg/kg, P = 0.01 vs. steady state) was observed following the first two IDet injections. GIR values at the end of clamp day 2 (first doses) and clamp day 3 (steady state) were comparable [GIR(trough 24 h) 3.7 +/- 1.7 vs. 3.8 +/- 1.6 mg/(kg x min) NS], indicating that IDet had reached steady state after the first two injections. CONCLUSIONS: IDet administered twice daily reached steady state after the second injection and showed a constant metabolic effect over time under steady-state conditions. This should facilitate basal insulin substitution and decrease the risk of hypoglycaemia in insulin-treated subjects. PMID- 16681562 TI - The MTHFR CT polymorphism confers a high risk for stroke in both homozygous and heterozygous T allele carriers with Type 2 diabetes. AB - OBJECTIVE: Individuals with Type 2 diabetes are at increased risk of stroke. Plasma homocysteine (tHcy) is an independent risk factor for cardiovascular (CV) disease. The methylene-tetrahydrofolate reductase (MTHFR) gene polymorphism (thermolabile variant C(677)T) is associated with CV risk, partly as a result of increased Hcy, especially in homozygous subjects. AIM: To relate the occurrence of the MTHFR polymorphism with stroke prevalence by examining allelic frequency and genotype distribution in 165 subjects with Type 2 diabetes studied for the presence of thermolabile C(677)T MTHFR mutation. RESULTS: Mean age was 67.7 years, and tHcy 18.2 micromol/l. T allele frequency was 38.5%. MTHFR genotypes were: normal (CC) 40%; heterozygous (CT) 43%; homozygous (TT) 17%. Serum levels of folic acid and B12 vitamin were within normal limits. Stroke prevalence was 14%. Sixty-four per cent of stroke-free subjects had the normal C allele vs. 46% in stroke subjects. The frequencies of genotypes (CC-CT-TT) were (%): 44-41-15 in stroke-free vs. 17-57-26 in stroke patients. Coronary (CAD) and peripheral artery disease (PAD) were common in all groups, with no differences according to genotypes. Stroke prevalence was markedly higher in genotypes CT and TT (18 and 21%) compared with CC (6%). Mean tHcy levels were higher in TT subjects. CONCLUSION: The allelic frequency of C(677)T MTHFR mutation in Type 2 diabetes subjects with stroke is markedly different from that of subjects without stroke. Genotypic characteristics suggest that C(677)T MTHFR mutation confers a higher risk for stroke to both homozygous and heterozygous T allele carriers that cannot be ascribed solely to raised tHcy and/or lower folate status in CT subjects, nor to phenotypic expression of conventional risk factors for stroke. The impact of the MTHFR polymorphism on stroke may result from T allele-linked deleterious effects, or C allele-linked protection. Confirmatory studies are warranted, as this cohort was not randomly selected, and a type 1 error cannot be ruled out. PMID- 16681563 TI - Effect of pioglitazone on insulin sensitivity, vascular function and cardiovascular inflammatory markers in insulin-resistant non-diabetic Asian Indians. AB - AIMS: To determine the effects of pioglitazone (30 mg once daily for 16 weeks) on insulin sensitivity, insulin-mediated vasodilation, vascular inflammatory markers, fat distribution and lipids in Asian Indians and Caucasians of European ancestry. METHODS: Cross-sectional study. Eighteen non-diabetic Asian Indians and 17 Caucasians of comparable age (34 +/- 3 vs. 36 +/- 3 years) and body mass index (26.0 +/- 1.2 vs. 24.7 +/- 1.0 kg/m(2)) had measurements of insulin sensitivity (M, insulin clamp at 6 pmol/kg per min), abdominal fat (computed tomographic scan at L4-L5), endothelial-dependent (reactive hyperaemia, RH) and -independent (0.4 mg sublingual nitroglycerin, TNG) vasodilation using brachial artery ultrasound before and after the 2-h clamp at baseline and after pioglitazone therapy. RESULTS: Asian Indians were insulin resistant compared with Causasians during the baseline clamp (M = 25.6 +/- 1.7 vs. 41.1 +/- 2.2 micromol/kg per min, P < 0.0001) and improved significantly after pioglitazone (to 33.9 +/- 1.7 micromol/kg per min, P < 0.001). Vasodilatory responses to RH and TNG were similar in Asian Indians and Caucasians at baseline and did not change. Insulin mediated vasodilation improved after pioglitazone in Asian Indians, but not in Caucasians, and correlated with the change in insulin sensitivity (r = 0.52, P = 0.03). C-reactive protein (CRP) was higher in Asian Indians vs. Caucasians (1.6 +/- 0.4 vs. 0.9 +/- 0.2 mg/l) and was negatively correlated with insulin sensitivity (r = -0.53, P = 0.02). In the Asian Indian group, CRP and plasminogen activator inhibitor-1 decreased and adiponectin increased after pioglitazone, but there were no significant changes in total or visceral fat. CONCLUSIONS: These results demonstrate that insulin-resistant Asian Indians respond favourably to an insulin sensitizer with improvements in insulin sensitivity, cardiovascular and inflammatory risk markers, and vascular responses to insulin. These agents may have a role in decreasing the risk of diabetes and cardiovascular disease in this high-risk population. PMID- 16681564 TI - Effectiveness of community health workers in the care of persons with diabetes. AB - AIMS: The purpose of this systematic review was to examine the effectiveness of community health workers in supporting the care of persons with diabetes. METHODS: Computerized searches were conducted of multiple electronic bibliographic dababases until March 2004. We identified studies in any language and of any design that examined the effectiveness of diabetes-related interventions involving community health workers and reported outcomes in persons with diabetes. Results were synthesized narratively. RESULTS: Eighteen studies were identified, including eight randomized controlled trials. Most studies focused on minority populations in the USA. The roles and duties of community health workers in diabetes care were varied, ranging from substantial involvement in patient care to providing instrumental assistance in education sessions taught by other health professionals. Participants were generally satisfied with their contacts with community health workers and participant knowledge increased. Improvements in physiological measures were noted for some interventions and positive changes in lifestyle and self-care were noted in a number of studies. There were few data on economic outcomes, but several studies demonstrated a decrease in inappropriate health care utilization. CONCLUSIONS: Diabetes programmes include community health workers as team members in a variety of roles. There are some preliminary data demonstrating improvements in participant knowledge and behaviour. Much additional research, however, is needed to understand the incremental benefit of community health workers in multicomponent interventions and to identify appropriate settings and optimal roles for community health workers in the care of persons with diabetes. PMID- 16681565 TI - Weight gain is associated with improved glycaemic control but with adverse changes in plasma lipids and blood pressure isn Type 1 diabetes. AB - AIMS: To assess the effects of weight gain on metabolic control, plasma lipids and blood pressure in patients with Type 1 diabetes. METHODS: Patients in the EURODIAB Prospective Complications Study (n = 3250) were examined at baseline and 1800 (55%) were re-examined a mean of 7.3 years later. Patients had Type 1 diabetes, defined as a diagnosis made before age 36 years and with a need for continuous insulin therapy within a year of diagnosis. Patients were aged 15-60 years at baseline and were stratified for age, sex and duration of diabetes. RESULTS: The change in HbA(1c) from baseline to follow-up examination was significantly more favourable in those who gained 5 kg or more during follow-up ('marked weight gain') than in patients who gained less or no weight or lost weight ('less or no weight gain'). In those with marked weight gain, there was a significantly greater rise in plasma triglycerides and total cholesterol and significantly less favourable changes in low-density lipoprotein and high-density lipoprotein cholesterol compared with those with less or no weight gain, with or without adjustment for HbA(1c). Systolic and diastolic blood pressure also rose significantly more in the group with marked weight gain. CONCLUSION: Weight gain in patients with Type 1 diabetes has adverse effects on plasma lipids and blood pressure, despite a small improvement in glycaemic control. PMID- 16681566 TI - Glucose tolerance of offspring of mother with gestational diabetes mellitus in a low-risk population. AB - AIMS: To describe the prevalence of impaired glucose tolerance and obesity in offspring of mothers whose pregnancies were complicated by gestational diabetes mellitus (GDM) in a low-risk population and to investigate the effect on these outcomes of minimal intervention compared with tight control for management of GDM. METHODS: Eighty-nine children (mean age 9.1 years, 93% Caucasian) were recruited through a follow-up study of women previously involved in a randomized controlled trial of minimal intervention (control group) vs. tight glycaemic control (treatment group) for GDM. Fasting blood glucose (FBG) and 2-h glucose tolerance tests (2hGTT) were performed on offspring and body mass index (BMI) calculated. Glucose tolerance and BMI of treatment groups were compared using non inferiority tests (non-inferiority margin -15%). RESULTS: Of those offspring, 6.9% (5/72) had abnormal glucose metabolism [four children had impaired glucose tolerance (IGT) and one had Type 2 diabetes mellitus (DM) (all Caucasian)]. Of the four children with IGT, three were male, three had normal BMI, and three had a family history of Type 2 diabetes. Of the 71 offspring who underwent 2hGTT, 25/25 (100%) of the control offspring and 46/46 (100%) of the treatment offspring had normal FBG (FBG < 5.7 mmol/l). Twenty-five of 25 (100%) of control and 42/46 (91.3%) of the treatment offspring had normal glucose tolerance (2hGTT < 7.8 mmol/l) (% difference 8.7, 95% CI -5.6, 20.3). BMI < 85th percentile was found in 25/33 (75.8%) of the treatment group and 44/52 (84.6%) of the control group (difference in percentage -8.9, 95% CI -27.2, 7.8). CONCLUSIONS: School-age children of mothers with GDM are at risk of IGT and overweight, even if from a low-risk ethnic population. FBG was not adequate for screening this population. Minimal intervention for glycaemic control in GDM pregnancies appears to be as effective as tight control for preventing IGT in childhood but not for preventing obesity. PMID- 16681567 TI - Simultaneous pulmonary and cerebral oedema, and multiple CNS infarctions as complications of diabetic ketoacidosis: a case report. AB - We present a case of a 29-year-old woman with known Type 1 diabetes who presented with diabetic ketoacidosis (DKA). Despite appropriate treatment and initial improvement, 12 h after initiation of treatment she deteriorated rapidly and developed pulmonary oedema, cerebral oedema and multiple infarctions of the brain and cervical spinal cord. This resulted in spastic quadraparesis and she has remained wheelchair-bound. These complications of DKA are rare and unpredictable. In this case report we discuss the proposed aetiologies of these complications with reference to our case report and highlight the importance of vigilance for early signs of these complications during the treatment of all patients with DKA. PMID- 16681568 TI - Ergotamine use in severe diabetic autonomic neuropathy. AB - BACKGROUND: Symptomatic postural hypotension in diabetes is uncommon. When it does occur, it can prove debilitating and difficult to treat. We report here the therapeutic challenges encountered in managing a patient with severe postural hypotension secondary to diabetes-related autonomic neuropathy. CASE REPORT: A 69 year-old gentleman with a 23-year history of Type 1 diabetes mellitus and multiple microvascular complications was admitted with symptoms of severe postural hypotension. Cardiovascular autonomic testing confirmed the presence of severe autonomic neuropathy. He failed to respond to non-pharmacological measures, fludrocortisone, midodrine, octreotide, erythropoietin and increased caffeine intake. Eventually he was commenced on half a Cafergot suppository (giving him a dose of ergotamine 1 mg and caffeine 50 mg) which resulted in dramatic clinical improvement. CONCLUSION: Ergotamine may be considered in refractory cases of postural hypotension. PMID- 16681569 TI - Is there a role for antileukotrienes in urticaria? AB - In vitro and in vivo clinical and experimental data have suggested that leukotrienes play a key role in inflammatory reactions of the skin. Antileukotriene drugs, i.e. leukotriene receptor antagonists and synthesis inhibitors, are a new class of anti-inflammatory drugs that have shown clinical efficacy in the management of asthma. We searched the MedLine database and carried out a manual search on journals specializing in allergy and dermatology for the use of antileukotriene drugs in urticaria. Montelukast might be effective in chronic urticaria associated with aspirin or food additive hypersensitivity or with autoreactivity to intradermal serum injection when taken with an antihistamine but not in moderate chronic idiopathic urticaria. Evidence for the effectiveness of zafirlukast and the 5-lipoxygenase inhibitor, zileuton, in chronic urticaria is mainly anecdotal. In addition, there is anecdotal evidence of effectiveness of antileukotrienes in primary cold urticaria, delayed pressure urticaria and dermographism. No evidence exists for other physical urticarias, including cholinergic, solar and aquagenic urticarias, vibratory angio-oedema, and exercise-induced anaphylaxis. PMID- 16681570 TI - Malignant melanoma in Chile: an unusual distribution of primary sites in men from low socioeconomic strata. AB - BACKGROUND: Mortality from malignant melanoma (MM) has increased in Chile in the past decade. The location of MM lesions on the body has been correlated with prognosis and survival. AIM: To review body site and gender relationships with histopathologically confirmed primary MM in Chile. METHODS: Records of 575 cases presenting to 5 state hospitals from 1992 to 2001 were analysed. RESULTS: There were 360 women and 215 men. Women showed a significantly higher number of MM on the legs, cheeks and arms, and in the genital area, whereas men showed a significantly higher number on the ears, backs of the hands, soles and feet. Men had a predilection for MM with a poor prognosis. CONCLUSION: The different body site distribution of primary MM in men and women may be explained by a different pattern of sun exposure. Ethnic and genetic factors may also be involved. The predominant location of MM in women in Chile is similar to white populations, whereas the location in men is similar to that observed in black and Asian populations. These observations may be relevant to the high mortality of MM in Chilean men. PMID- 16681571 TI - Clinicoepidemiological features and course of 43 cases of bullous pemphigoid in Kuwait. AB - BACKGROUND: The clinicoepidemiological characteristics and course of bullous pemphigoid (BP) have not been described in populations from the Arabian Gulf. Hypothesis. Ethnic and regional variations can influence the clinical behaviour and course of autoimmune diseases. METHODS: In this study, 43 patients with BP, registered in our autoimmune bullous diseases (ABD) clinic over a span of 14 years, were studied to determine the clinicoepidemiological features and course of the disease in our region. RESULTS: BP was observed to be the second commonest ABD in our clinic (27%), with a minimum estimated incidence of 2.6 cases/million/year among the referral population. The largest proportion (93%) of the patients was of Arab ethnicity and the female to male ratio was 5.1 :1. Mean +/- SD age at diagnosis was 65.20 +/- 18.80 years. Most of the patients (96%) had moderate to severe disease, and mucosal involvement was seen in 37% of the patients. Systemic steroids (prednisolone 20-60 mg daily) alone or in combination with azathioprine, intravenous immunoglobulins, tetracyclines, mycophenolate mofetil or dapsone were used to treat theses cases. At the last follow-up, 32% of patients were in complete remission and off treatment. The first-year mortality was 30%. Old age and poor general medical condition were the significant risk factors (P < 0.05) contributing to the mortality. CONCLUSION: The study highlights the regional variations of BP and thus a need to uncover the ethnic, genetic and geographical influences, if any, responsible for these variations. PMID- 16681572 TI - UVA1 phototherapy for genital lichen sclerosus. AB - BACKGROUND: Lichen sclerosus (LS) is characterized histologically by an inflammatory T-cell infiltrate, sclerosis and thickening of the dermis, and epidermal atrophy. Ultraviolet (UV) A1 therapy has been shown to be effective in the management of morphea and scleroderma, diseases that have some histological and clinical similarities with LS, and more recently in extragenital LS. AIM: To determine the effectiveness of UVA1 therapy for genital LS. METHODS: Seven women with severe genital LS uncontrolled by ultrapotent topical corticosteroids, with a median age of 62 years (range 48-78) and disease duration of 6-47 years, were treated with UVA1 therapy from a high output source. After completion of UVA1 therapy, a clinician and the patient graded the overall response of symptoms and physical signs. RESULTS: Five patients improved with therapy. Three obtained moderate improvement in overall disease severity and two had minimal improvement. Of these five, one relapsed within 3 months and another after a year. Both had a further course of UVA1 therapy, resulting in minimal improvement in one and moderate improvement in the other. In the remaining three, disease severity had improved to a point where intermittent use of topical corticosteroids resulted in acceptable control. DISCUSSION: UVA1 therapy may be of benefit in the management of vulval LS, a disease that is often poorly responsive to standard therapies. The therapy is well tolerated and could provide an acceptable therapeutic option for patients with severe disease. PMID- 16681573 TI - A randomised controlled trial on photo(chemo)therapy of subacute prurigo. AB - BACKGROUND: Psoralen ultraviolet A (PUVA) is the standard photo(chemo)therapeutic regimen for patients suffering from subacute prurigo (SP). HYPOTHESIS: Regarding efficacy, bath PUVA is not superior to medium-dose ultraviolet-A1 (MD-UVA1) and narrowband ultraviolet-B (NB-UVB), which may be considered the new photo(chemo)therapeutic options for SP. METHODS: We performed a prospective randomised, controlled, three-arm photo(chemo)therapeutic study. Patients suffering from histopathologically proven SP with a clinical score (PIP score; papules, infiltration and pruritus) of at least 5 points were enrolled into the study. Treatment with bath PUVA was performed 4 times weekly and MD-UVA1 and NB UVB 5 times weekly. Photo(chemo)therapy was administered over a 4-week period. Outcome measure was the severity of SP investigated by means of the PIP score after 4 weeks of therapy. RESULTS: In total, 33 patients with SP were randomly allocated to photo(chemo)therapy. Bath PUVA (n = 9), MD-UVA1 (n = 11) and NB-UVB (n = 13) resulted in a significant reduction of the baseline PIP score as assessed on the basis of intention-to-treat (ITT) analysis (P = 0.003). However, ITT analysis revealed significantly higher PIP score reduction in patients who were treated with bath PUVA and MD-UVA1 compared with NB-UVB (P < 0.01, 95% CI 1.1-3.63 and P < 0.05, 95% CI 0.42-2.70, respectively). CONCLUSIONS: Photo(chemo)therapy, including bath PUVA, MD-UVA1 and NB-UVB, appears to be an effective and safe treatment option for patients suffering from SP. UVA1 and particularly PUVA seem superior to NB-UVB in the management of SP. PMID- 16681574 TI - Skin-cancer awareness in Scottish cardiac transplant recipients. AB - BACKGROUND: Cardiac transplant recipients have a greatly increased risk of nonmelanoma skin cancer, with a relative risk of up to 108. Skin cancer is more aggressive in transplant patients and results in substantial morbidity and mortality. It is therefore important that these patients understand this risk and take adequate sun-protection measures. AIM: To assess awareness of skin cancer risk and sun protection measures used by cardiac transplant recipients and determine the impact of patient education. METHODS: Using a detailed questionnaire, we surveyed 118 patients attending the cardiac transplant clinic at our centre to quantify knowledge of skin cancer risk (maximum total score 10) and behaviour in the sun (maximum total score 15). Of these patients, 50 were then seen by a dermatologist for education about skin cancer risk, sun protection measures and skin cancer screening. Six months later, we asked them to complete the same questionnaire again. RESULTS: The mean knowledge score was 7.3/10 and the mean behaviour score was 11.2/15. In the group that received education, the mean knowledge score improved from 7.2/10 before the dermatology consultation to 7.8/10 after the consultation (P < 0.03). The mean score for the behaviour questions improved even more, from 11.2/15 before to 13.5/15 after the consultation (P < 0.0001). CONCLUSIONS. This study demonstrates that specialist advice can improve self-reported knowledge of skin cancer risk and sun protective behaviour in cardiac transplant recipients. It is hoped that this may reduce the risk of nonmelanoma skin cancer in these patients. PMID- 16681575 TI - Lupus band test and disease activity in systemic lupus erythematosus: does it still matter? AB - BACKGROUND: Lupus band test still has no clearly defined position within either diagnostic or disease activity measuring tools for systemic lupus erythematosus (SLE). OBJECTIVES: We tested the hypothesis that positive LBT correlates with global activity of SLE measured by the SLE Disease Activity Index (SLEDAI) score. METHODS: In total, 90 SLE patients who underwent biopsy of sunprotected non lesional (SPNL) skin were studied prospectively. The skin specimen was processed for standard direct immunofluorescence. The patients were classified into groups as negative and positive LBT, and the latter were further subdivided on the basis of the type and morphology of the deposits. Every patient was thoroughly examined and assigned a SLEDAI score. The relationship between LBT findings and SLEDAI score was analysed. RESULTS: The disease was significantly more active in patients with positive LBT and in those with a higher number of deposited immunoreactants. Almost all patients with renal involvement had a positive LBT. CONCLUSIONS: LBT on SPNL skin may be a good marker of severe disease at presentation, particularly when three immunoglobulins are found at the dermoepidermal junction. PMID- 16681576 TI - Drug eruptions to contrast media in Japan. AB - BACKGROUND: In Japan, drug eruptions to nonionic iodinated contrast media have been reported since the products appeared on the market in 1986. OBJECTIVES: To evaluate this clinical finding, we analysed the number of patients with drug eruption to contrast media in our hospital from 1989 to 2003. METHODS: In total, 117 patients suspected of such drug eruptions were patch and intradermal tested with contrast media (as commercially sold). Those who tested positive were evaluated. RESULTS: Of the 117 patients, 69 patients (19 men; 50 women, mean +/- SD age 51.4 +/- 16.5 years, range 17-86) showed positive reactions to contrast media. The number was 6-13 annually from 1989 to 1995, and 1-4 annually from 1996 to 2003. CONCLUSION: Although our data suggest (but do not prove) that patients with drug eruption to contrast media decreased in number, this condition is still not rare in Japan. Higher annual exposure to contrast media, including pretesting, could play an important role in this observation. PMID- 16681577 TI - A case of Rothmann-Makai panniculitis successfully treated with tetracycline. AB - Rothmann-Makai syndrome (lipogranulomatosis subcutanea) is a rare variant of Weber-Christian disease, but lacks visceral involvements and systemic manifestations. We herein report the case of a 56-year-old Chinese woman with this disorder who complained of subcutaneous tender nodules over her extremities, trunk, and face of 2 years' duration. She was firstly treated with 300 mg of oral roxythromycin for 8 weeks and subsequently treated with 400 mg of oral clarithromycin for the next 8 weeks. However, no significant effect was observed. She was then administered 200 mg of oral minocycline hydrochloride. After 1 week, her skin symptoms rapidly improved and no relapse has been observed in a follow up period of 3 months. Considering that tetracycline antibiotics, but not macrolide antibiotics, inhibit the activity of pancreatic lipase in vitro, the efficiency of tetracycline antibiotics may be a clue to clarifying the pathogenesis of this disorder. PMID- 16681578 TI - Multiple Spitz naevi: a report of both variants with clinical and histopathological correlation. AB - Spitz naevi are usually solitary lesions. Multiple Spitz naevi are extremely rare and reported as widespread (disseminated) or grouped (agminated). We report two cases of multiple Spitz naevi and review their aetiology and treatment. PMID- 16681579 TI - Necrotizing stomatitis in the developed world. AB - We present a case of a malnourished 68-year old man with occult hypothyroidism who presented with malaise, pyrexia, tongue swelling, oral ulceration and dysphagia after a 6-month period of increasing lethargy and failing self-care. Severe necrotic oral ulcerative lesions were accompanied by cutaneous purpura, blood-filled blisters and bedsores. It was concluded that the patient's clinical condition reflected necrotizing stomatitis on a background of malnutrition with scorbutic skin lesions and hypothyroidism. The patient made a good recovery with scrupulous oral hygiene, debridement, intravenous metronidazole and nutritional support. Healing occurred with marked fibrosis and trismus, which has slowly improved with mouth-opening exercises. Necrotizing stomatitis is more commonly encountered in malnourished children in developing countries, and may subsequently result in devastating facial defects and death. Patients in the developed world with poor oral hygiene, malnourishment and immunosuppression are also at risk, but early diagnosis and treatment is life-saving and reduces subsequent disability. PMID- 16681580 TI - Development of lichen planus and psoriasis on lesions of vitiligo vulgaris. AB - This paper reports a unique case of coexistence of vitiligo vulgaris, lichen planus and psoriasis vulgaris in a 53-year-old man. Five years after the onset of vitiligo, lichen planus developed on his lower lip. Another 4 years after the onset of lichen planus, he also exhibited psoriasis on his upper arms and trunk. Both the lichen planus and psoriasis occurred on lesions of the preceding vitiligo vulgaris. We discuss potential mechanisms for association of these three dermatoses, including Koebner phenomenon and photodamage. PMID- 16681581 TI - Treatment of vulval Crohn's disease with infliximab. AB - Anti-tumour necrosis factor-alpha therapy of Crohn's disease (CD) with infliximab has proved a major advance in the treatment of patients with difficult disease. We report our experience of the use of infliximab in a patient with recalcitrant vulval CD. The introduction of infliximab has improved disease control, although a search for an optimum remittive dosing schedule is ongoing. PMID- 16681583 TI - Behcet's disease-like presentation of bullous pyoderma gangrenosum associated with Crohn's disease. AB - A 47-year-old woman presented with a 2-month history of generalized arthralgia and a 10-day history of oral aphthous ulcers. After hospitalization, papulopustular lesions and perianal ulcerations developed. Pathergy test was positive and ophthalmological examination was normal. The presence of oral aphthous ulcers, genital ulcerations, papulopustular lesions and arthralgia, and the positive pathergy test suggested the diagnosis of Behcet's disease (BD). In a few days, positive pathergy reactions and papulopustular lesions evolved into bullous lesions, which were diagnosed dermatopathologically as pyoderma gangrenosum. Two days after the presentation of papulopustular lesions, the patient experienced diarrhoea accompanied by bloody stools and mucus. Histopathological examination of biopsy specimens showed no vasculitis but revealed findings suggestive of Crohn's disease. The patient responded well to treatment with systemic steroids and 5-aminosalicylic acid. Our case demonstrates that the differential diagnosis of BD and inflammatory bowel disease may be perplexing and that these two diseases may be closely related. PMID- 16681582 TI - Pseudoscleroderma associated with cancer. AB - In the period 1994-2002, we saw 71 patients with sclerotic skin changes; 66 were diagnosed with systemic sclerosis (SSc) while five (7%) were diagnosed with pseudoscleroderma associated with various malignancies. The mean duration of disease in these five patients was significantly shorter than that of patients with SSc. The incidence of positive antinuclear antibodies, Raynaud's phenomenon or oesophageal involvement in patients with pseudoscleroderma was significantly lower than that in patients with SSc. The distributions of skin sclerosis varied in each case. Serum basic fibroblast growth factor (bFGF) levels in the five patients with pseudoscleroderma were very elevated compared with levels in controls. Elevated expression of bFGF was detected on fibroblasts of affected skin and in one lung-cancer tissue sample obtained by excision. PMID- 16681584 TI - Systemic sarcoidosis presenting in a tattooed man undergoing treatment for hepatitis C. AB - A heavily tattooed man presented with nodular, ulcerated sarcoidal lesions distributed throughout all the colours of his tattoos. The lesions developed within the confines of his tattoos 4 months into therapy with ribavirin and interferon-alpha for chronic hepatitis C. This appears to be the first case of sarcoidosis in association with numerous pigment colours as well as dual therapy for hepatitis C. PMID- 16681585 TI - Epidermodysplasia verruciformis and generalized verrucosis: the same disease? AB - We report a patient with epidermodysplasia verruciformis (EV) who had severe generalized verrucous skin lesions for 50 years without any immunological abnormality. Microscopic examination showed two histopathological features, including seborrhoeic keratosis and common warts. The detected human papilloma virus (HPV) types were found to be HPV 3, 50, 5, and 76, using a degenerate PCR method. EV and generalized verrucosis are distinguished by slight differences in clinical symptoms or HPV types, so there should be no apparent differential points common to both diseases. Therefore, we propose that an abnormal susceptibility specific to HPV, which is the most characteristic feature in EV, should be regarded as a differential point in these two diseases. PMID- 16681586 TI - Topical 5-fluorouracil has no additional benefit in treating common warts with cryotherapy: a single-centre, double-blind, randomized, placebo-controlled trial. AB - The role of topical 5-fluorouracil (5-FU) in treating common warts is not well defined. We tried to evaluate the efficacy and adverse effects of combination cryotherapy and topical 5% 5-FU ointment in the treatment of common warts. The study was a single-centre, double-blind randomized placebo-controlled trial. In the study, 80 patients with common warts were randomized into two groups and underwent two 10-second freeze/thaw cycles of cryotherapy with liquid nitrogen once every three weeks for a maximum of five treatments. Between treatments, patients applied either topical 5% 5-FU ointment (group A) or placebo aqueous cream (group B) twice daily. The mean +/- SD reduction in wart area was 58.57 +/- 0.06% in group A and 65.29 +/- 0.06% in group B. In total, 19 patients in group A and 24 patients in group B had wart size reduced by 75% or more (P = 0.50), while 12 patients in group A and 17 patients in group B had clearance of their warts (P = 0.245). Logistic regression with age, sex, smoking status, immune status, site, duration and number of warty lesions, history of previous treatment, and treatment group found that only a history of previous treatment and acral lesions were significant adverse predictors of improvement. There was no significant difference in the number of adverse events between the two groups, although there was a trend towards more pain and blistering associated with topical 5-FU. We concluded that topical 5-FU has no added benefit in treating common warts with cryotherapy. PMID- 16681587 TI - A case of melanonychia due to Candida albicans. AB - Candida species rarely cause black pigmentation of infected nails and only a few cases have been reported in the literature. We describe a 53-year-old white man who had diffuse melanonychia of the fourth right fingernail due to C. albicans. A progressive dark pigmentation of his nail appeared over 6 months, following paronychial inflammation. The melanonychia was associated with brittleness. There was no onycholysis or hyperkeratosis. Direct examination with potassium hydroxide demonstrated round yeast cells in the specimen. The samples were cultured on Sabouraud glucose agar containing chloramphenicol at 27 degrees C and showed white growth after a few days. The patient was successfully treated with systemic itraconazole. PMID- 16681588 TI - An unusual patient with Rothmund-Thomson syndrome, porokeratosis and bilateral iris dysgenesis. AB - Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive genodermatosis characterized by poikiloderma and the variable presence of other features including skeletal and ocular abnormalities, ectodermal defects, and susceptibility to certain malignancies. We report a 40-year-old woman with known RTS who developed porokeratoses on her limbs in adulthood, an association that has not previously been reported. In addition, she had bilateral iris dysgenesis, which has only been described once before in RTS. PMID- 16681589 TI - Multiple myxoid cysts secondary to occupation. AB - We report the case of a 50-year-old woman who presented with eight digital myxoid cysts (DMCs) involving the fingers of both hands. They developed within 12 months of the patient starting a job that involved pushing a garment into an embroidery mould, thus exerting a downward force on the fingertips. The pressure exerted from this force could have potentially damaged the joint synovial capsule, leading to rupture and loss of synovial gel, thus inducing myxoid cysts. This case suggests that DMCs may be related to occupation, and to our knowledge, this is only the second reported case of occupationally induced DMCs. PMID- 16681590 TI - Proopiomelanocortin (POMC): the cutaneous roles of its melanocortin products and receptors. AB - The precursor protein proopiomelanocortin (POMC) produces many biologically active peptides via a series of enzymatic steps in a tissue-specific manner, yielding the melanocyte-stimulating hormones (MSHs), corticotrophin (ACTH) and beta-endorphin. The gene for alpha-MSH is encoded for by the POMC gene, but alpha MSH cannot be produced from POMC gene transcription and translation without these specific post-translational proteolytic steps taking place. The MSHs and ACTH bind to the extracellular G-protein-coupled melanocortin receptors (MCR), of which there are five subtypes. Two (MC1R and MC5R) show widespread cutaneous expression. ACTH and alpha-MSH bind to MC1R to influence both pigmentation and the immune system. MC5R regulates the sebaceous glands. Mutations in the MC1R gene lead to fair skin and red hair in humans, which is also seen with inactivating human POMC gene mutations. MC1R mutant receptor expression can also correlate with an increased incidence of the three commonest forms of skin cancer. Other mutations can occur in the POMC system or parallel interacting pathways, such as in prohormone convertase 1 and agouti signalling protein, a human homologue of murine agouti protein. However, they do not necessarily affect skin colour or function in humans, and further studies are needed to clarify these observations. PMID- 16681591 TI - Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in human malignant melanoma and their relation to angiogenesis. AB - BACKGROUND: Angiogenesis is the major and key factor for growth and invasion of tumours, including malignant melanoma (MM), but the factors that contribute to tumour angiogenesis are still unclear. OBJECTIVE: To study expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in human MM and their relation to angiogenesis. To investigate the correlation between eNOS and VEGF and the role of nitric oxide (NO) generated by eNOS in the process of mediating angiogenesis by VEGF. METHODS: Tissue sections from 31 patients with MM were examined using immunohistochemistry and morphological quantitative analysis for protein expression of eNOS and VEGF. Microvessel density (MVD) was counted in endothelial cells in immunostained by anti-FVIII:RAg antibody. RESULTS: Positive eNOS and VEGF immunostaining were observed in 77.4% and 83.9% of MM lesions, respectively, whereas pigmented naevi never expressed eNOS and VEGF. A positive correlation between eNOS and VEGF in MM was observed. Expression of eNOS and VEGF was positively correlated with MVD expression in MM, and MVD expression in MM was stronger than in pigmented naevi. Expression of eNOS and VEGF was not correlated with lymph node metastasis. CONCLUSIONS. On the basis of the current data showing that malignant melanocytic tumours displayed strong VEGF and eNOS expression, whereas benign melanocytic proliferations showed no immunoreactivity for VEGF and eNOS, such expression may be used as a discriminating factor to distinguish malignant melanoma from pigmented naevi. Expression of eNOS and VEGF may contribute to angiogenesis of MM, eNOS probably plays an important role in mediating VEGF-induced angiogenesis. PMID- 16681592 TI - No association of cytokine gene polymorphisms in Chinese patients with atopic dermatitis. AB - BACKGROUND: Atopic dermatitis (AD) is a common chronically relapsing skin disease associated with the activation of T-helper 2 cells. Recent studies have shown that polymorphisms in the genes for interleukin (IL)-4, the IL-4 receptor, IL-13, and signal transducer and activator 6 (STAT6) may contribute to susceptibility of AD. To date, no cytokine gene polymorphism study has been conducted on Chinese patients with AD. AIMS: To determine whether genetic polymorphisms of the cytokine genes might influence the development of AD. METHODS: DNA samples were obtained from 94 patients and 186 control subjects. Using direct sequencing and microsatellite genotyping, we examined 22 polymorphisms in eight cytokine genes including the genes for IL-4, -10, -12B and -13, the IL-4 receptor, tumour necrosis factor (TNF)-alpha, STAT6, and interferon (IFN)-gamma. RESULTS: No significantly different allelic and genotypic distributions of the cytokine gene polymorphisms could be found between patients and controls. Moreover, no association was observed with disease onset, gender, the presence of elevated serum total IgE level or blood eosinophilia. CONCLUSION: Our study suggests that the analysed genetic polymorphisms of cytokine genes do not appear to be associated with AD susceptibility in our Chinese population. PMID- 16681593 TI - HLA class I and class II antigens in Turkish patients with chronic ordinary urticaria. AB - BACKGROUND: Chronic urticaria is a common disease with an unclear pathogenesis, which may be resistant to therapy. Recent studies have focused primarily on a possible autoimmune basis. AIM. To investigate HLA class I and II antigens in a Turkish population with chronic ordinary urticaria (COU; not physical, vasculitic or contact), and identify susceptible HLA antigens. METHODS: HLA antigens were investigated in 55 patients diagnosed with COU, using a two-stage microdroplet lymphocytotoxicity test, with 104 healthy and genetically unrelated individuals evaluated as the control group. RESULTS: HLA Bw4 and HLA DQ1 antigens were significantly higher in the study group (odds ratio (OR) = 2.93, 95% CI 1.47 5.85, P = 0.003 and OR = 7.81, 95% CI 1.96-28.50, P = 0.001, respectively) whereas HLA-A24 antigen was higher in controls (OR = 0.36, 95% CI 0.15-0.86, P = 0.03). CONCLUSION: We propose that HLA-Bw4 and DQ1 antigens may be responsible for susceptibility to COU while HLA-A24 may have a protective role in the Turkish population. PMID- 16681594 TI - Does the plasma level of vitamins A and E affect acne condition? AB - BACKGROUND: Vitamin A and E are lipid soluble antioxidants that are necessary for our health. Deficiency in these vitamins can cause serious diseases. Administration of vitamin A and E to patients with acne was shown to improve their acne condition. AIMS: To test the relationship between plasma vitamin A and E levels and acne. METHODS: Plasma vitamin A and E concentrations were determined by high performance liquid chromatography in 100 newly diagnosed untreated patients with acne and were compared with those of 100 age-matched healthy controls. Patients were carefully graded using the Global Acne Grading System. RESULTS: We found that plasma vitamin A concentrations in patients with acne were significantly lower than those of the control group (336.5 vs. 418.1 mug/L, respectively) P = 0.007. We also found that plasma vitamin E concentrations in patients with acne were significantly lower than those of controls (5.4 vs. 5.9 mg/L) P = 0.05. In addition, we found that there is a strong relationship between decrease in plasma vitamin A levels and increase in the severity of acne condition. Patients with severe acne had significantly lower plasma concentrations of vitamins A and E than did those with lower acne grade and the age-matched healthy controls. DISCUSSION: Based on our results, we conclude that low vitamin A and E plasma levels have an important role in the pathogenesis of acne and in the aggravation of this condition. PMID- 16681595 TI - A homozygous missense mutation in PEPD encoding peptidase D causes prolidase deficiency associated with hyper-IgE syndrome. AB - BACKGROUND: Prolidase deficiency is a complex disease characterized by various skin manifestations accompanied by mental retardation, facial dysmorphism and susceptibility to pyogenic infections. METHODS: We assessed a patient presenting a peculiar phenotype combining manifestations of prolidase deficiency with features typical of hyper-IgE syndrome. Mutation analysis was performed using direct PCR amplification and PCR restriction fragment length polymorphism analysis. RESULTS: We identified a novel homozygous recessive mutation in the PEPD gene, which was found to segregate in the family of the patient with the disease and was not found in a panel of DNA samples representative of all major Druze families living in northern Israel. DISCUSSION: Our results suggest that prolidase deficiency associated with hyper-IgE syndrome, a rare disorder, can be caused by mutations in PEPD. PMID- 16681596 TI - Analysis of gene mutations in four cases of dermatofibrosarcoma protuberans. AB - Fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor B-chain (PDGFB) gene has been described in dermatofibrosarcoma protuberans (DFSP). Various exons of the COL1A1 gene have been shown to be involved in the fusion with exon 2 of the PDGFB gene. We examined the breakpoints of the COL1A1 gene using the tumour specimens from four patients with DFSP. The COL1A1-PDGFB fusion transcripts were detected from the cultured tumour cells by reverse transcriptase polymerase chain reaction. Sequence analysis revealed that the ends of exons 23, 25, 26 and 36 in the COL1A1 gene were fused with the start of exon 2 in the PDGFB. This study identified three novel COL1A1 breakpoints: exons 23, 26 and 36 of the COL1A1 gene. In one case, the tumour was composed of two areas that differed in cytological atypia, cellularity and mitotic activity, indicating the dedifferentiation of the tumour. In tumour cells from two different areas the same aberrant fusion transcripts were identified. These results suggest that the dedifferentiation of tumour cells has nothing to do with the specific breakpoints of the COL1A1 gene, but depends on other unknown factors. PMID- 16681597 TI - Proteomic analysis of suction blister fluid isolated from human skin. AB - The regulation of cutaneous immune responses in health and disease is mediated locally by proteins such as cytokines and chemokines. We used a novel approach involving proteomic profiling of fluid drawn from suction blisters to compare and contrast protein expression in normal skin with that in nonlesional skin from a patient with plaque psoriasis. We also examined the impact of exogenous interleukin-1beta, a proinflammatory cytokine, on protein expression in these tissues. Described here are the results of proteomic profiling of 670 proteins from blister fluid, and the identification by differential expression of nine proteins between one volunteer with psoriasis and one normal volunteer. Although the apparent disease association of these nine proteins will require validation using additional volunteers, the identification of candidate protein biomarkers through proteomic analyses of blister fluid represents a promising approach for monitoring the disease activity and efficacy of therapeutic intervention in human skin diseases. PMID- 16681598 TI - Fibrillar pattern of a plantar acquired melanocytic naevus: correspondence between epiluminescence microscopy and transverse section histology. AB - The main epiluminescence microscopy (ELM) patterns observed on volar acquired melanocytic naevi are the parallel-furrow, the lattice-like and the fibrillar patterns. Because of the peculiar arrangement and configuration of epidermal rete ridges in the glabrous skin, the traditional histological picture does not provide an exact correlation with ELM features. In particular, the fibrillar pattern lacks a histopathological correlate. To clarify the microscopic features of the fibrillar pattern, we used transverse histological sectioning to study an acquired compound melanocytic naevus of the sole, characterized by a mixed parallel-furrow/fibrillar pattern on ELM. Low-magnification images of transverse sections allowed us to highlight the typical anatomical arrangement of the volar skin clearly, while high-magnification images demonstrated that the fibrillar pattern corresponded to parallel striae of intensely pigmented corneocytes arranged obliquely to the cutaneous surface. PMID- 16681599 TI - Treatment of multiple giant molluscum contagiosum in a renal transplant patient with imiquimod 5% cream. PMID- 16681600 TI - Juvenile plantar dermatosis responding to topical tacrolimus ointment. PMID- 16681601 TI - Dowling-Degos disease, hidradenitis suppurativa and arthritis in mother and daughter. PMID- 16681602 TI - A case of 'giant' aneurysmal benign fibrous histiocytoma. PMID- 16681604 TI - Laser hair removal for peristomal skin. PMID- 16681605 TI - Micronucleus frequency in the oral mucosa and lymphocytes of patients with Behcet's disease: comment. PMID- 16681606 TI - Hepatitis: a rare, but important, complication of infliximab therapy for psoriasis. PMID- 16681607 TI - Elastotic striae associated with striae distensae after application of very potent topical corticosteroids. PMID- 16681608 TI - Progressive idiopathic atrophoderma of Pasini and Pierini: the evaluation of cutaneous atrophy by 13-MHz B-mode ultrasound scanning method. PMID- 16681610 TI - Nailfold fluconazole fluid injection for fingernail onychomycosis. PMID- 16681609 TI - Olanzapine therapy for subacute prurigo. PMID- 16681611 TI - An unusual association of pigmentary mosaicism (hypomelanosis of Ito) with generalized hypertrichosis. PMID- 16681612 TI - Mycobacterium leprae in untreated lepromatous leprosy: more than skin deep. PMID- 16681613 TI - Stasis ulcer and dermatitis caused by artificial arteriovenous fistula created 33 years previously for the treatment of poliomyelitis. PMID- 16681614 TI - Chronic cutaneous graft-versus-host disease associated with multiple cutaneous squamous cell carcinomas. PMID- 16681615 TI - Acute urticaria and dry cough with interstitial pneumonia: a clue for the diagnosis of primary parvovirus B19 infection. PMID- 16681616 TI - Successful treatment of calcific uraemic arteriolopathy with wide local excision. PMID- 16681617 TI - Symmetrical scrofuloderma with tuberculosis verrucosa cutis. PMID- 16681618 TI - Pustular exacerbation of psoriasis due to fexofenadine. PMID- 16681619 TI - Interferon alpha-2B in juvenile hyaline fibromatosis. PMID- 16681620 TI - Fixed cutaneous sporotrichosis: an unusual diagnosis in West Europe. PMID- 16681621 TI - Drug-induced delayed multiorgan hypersensitivity (DIDMOHS). PMID- 16681622 TI - The confusing nosology of incontinentia pigmenti. PMID- 16681623 TI - An asymptomatic pink plaque on the cheek. PMID- 16681624 TI - Blistering disease in a child. PMID- 16681625 TI - Axillary sinuses and cervical swellings. PMID- 16681626 TI - Multiple pustulogranulomatous plaques in the elderly. PMID- 16681627 TI - A patient presenting with breast nodules. PMID- 16681628 TI - Verrucous and crusty lesions since childhood. PMID- 16681629 TI - An orange-tinted baby! PMID- 16681630 TI - Arsenicosis from homeopathic medicines: a growing concern. PMID- 16681632 TI - Leukaemic infiltration of the retina at onset of Philadelphia-positive acute lymphoblastic leukaemia revealed by stratus optical coherence tomography. PMID- 16681633 TI - Blindness due to angioid streaks in congenital dyserythropoietic anaemia type I. PMID- 16681634 TI - Insights into von Willebrand factor proteolysis: clinical implications. AB - The proteolysis of von Willebrand factor (VWF) by the recently discovered metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin repeats), is a normal processing step in VWF biochemistry. Emerging data indicate that this step may be influenced by a variety of factors, some of which favour increased proteolysis and some of which compromise proteolysis. The former may predispose to bleeding, whilst the latter appears to be the underlying mechanism for thrombotic thrombocytopenic purpura (TTP). The new insights support the concept of "risk" in bleeding, particularly in the case of type 1 von Willebrand disease (VWD), in much the same way that risk is considered in venous thrombosis. This review presents relevant current knowledge of VWF proteolysis by ADAMTS13, and a novel model of how this may be implicated in type 1 VWD is proposed, based on events at the vessel wall at a time of haemostatic challenge. PMID- 16681635 TI - Eosinophilia: secondary, clonal and idiopathic. AB - Blood eosinophilia signifies either a cytokine-mediated reactive phenomenon (secondary) or an integral phenotype of an underlying haematological neoplasm (primary). Secondary eosinophilia is usually associated with parasitosis in Third World countries and allergic conditions in the West. Primary eosinophilia is operationally classified as being clonal or idiopathic, depending on the respective presence or absence of a molecular, cytogenetic or histological evidence for a myeloid malignancy. The current communication features a comprehensive clinical summary of both secondary and primary eosinophilic disorders with emphasis on recent developments in molecular pathogenesis and treatment. PMID- 16681636 TI - Joe Burchenal and the birth of combination chemotherapy. AB - When Joe Burchenal started studying medicine at the University of Pennsylvania in 1934, antibiotics had not been discovered and the survival of patients diagnosed with acute leukaemia was < 4 months. By the time he retired in 1983, 58% of children with acute lymphoblastic leukaemia survived 5 years with the majority being cured of their disease. His early work in infectious diseases and antimicrobials equipped him well, both clinically and scientifically. The approach to developing antibiotics to conquer previously incurable infection was an inspiration and model for his pioneering work when searching for drugs with activity against cancer. Trials of sequential and then combination chemotherapy followed. Success in treating lymphoid malignancies in children led him to develop treatment regimens for other more resistant cancers, and as an advocate of collaborative working he introduced multimodal therapy to tackle bulky or metastatic cancers, replacing inevitable relapse with a chance of true cure. PMID- 16681637 TI - Somatic hypermutation and VH gene usage in hairy cell leukaemia. AB - To examine the usage and mutational status of VH genes in hairy cell leukaemia (HCL), we analysed 24 immunoglobulin heavy chain (IgH) sequences expressed in 23 patients. None had premature stop codons. VH3-23 was the most common gene and a VH6 gene was observed for the first time in HCL. Although the mean mutation frequency was 6.1%, slightly higher than in previous HCL series, four patients had 99.6-100% homology to germline sequences, three of whom had high tumour burdens and poor outcomes. Despite the high mutation frequency, only two of 24 rearrangements had clear statistical evidence of antigen-dependent somatic mutation. Our results increase the database of reported functional HCL rearrangements to 94 in 92 patients. Overall, both gene usage and mutation frequency are very similar to mucosa-associated lymphoid tissue-type marginal zone lymphoma. The data are consistent with HCL originating from post-germinal centre marginal zone B cells, although the heterogeneity observed suggests that HCL may originate differently in some patients, and this could have implications for prognosis and treatment. PMID- 16681638 TI - High-dose darbepoetin alpha in the treatment of anaemia of lower risk myelodysplastic syndrome results of a phase II study. AB - An open-label, phase II non-randomised trial was conducted with darbepoetin (DAR), an erythropoiesis-stimulating factor with prolonged half-life, at a weekly dose of 300 mug subcutaneously in 62 anaemic patients with myelodysplastic syndrome (MDS) with an endogenous erythropoietin (EPO) level <500 mU/ml. Most of the patients were classified as low or intermediate 1 according to the International Prognostic Scoring System. After 12 weeks, 44 (71%) patients had an erythroid response (34 major and 10 minor), including eight of 13 patients who were previous non-responders to conventional EPO. Two additional responses (one minor and one major) occurred, in 10 non-responders, after the addition of granulocyte colony-stimulating factor (G-CSF). Thirty-six of the 46 total responders (31/35 major and 5/11 minor) continued to respond on maintenance DAR after a median of 40 weeks (range 4-84). Median dose of DAR required to maintain response was 300 microg every 14 d. The only prognostic factors of favourable response were low endogenous EPO level and low or absent red blood cell transfusion requirement. Those results suggest that high-dose DAR alone yields high erythroid response rates in anaemia of lower risk MDS, possibly equivalent to those obtained with conventional EPO + G-CSF, although this will need to be confirmed in larger and randomised trials. PMID- 16681639 TI - Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cells. AB - Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients' resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome. PMID- 16681640 TI - Serum pleiotrophin levels are elevated in multiple myeloma patients and correlate with disease status. AB - Pleiotrophin (PTN), a tightly regulated angiogenic and mitogenic heparin-binding protein, is markedly elevated in a variety of aggressive solid tumours. The role of PTN in haematological malignancies, however, has not been previously evaluated. This study demonstrated that PTN serum levels were elevated in multiple myeloma (MM) patients when compared with healthy subjects (P < 0.0001). Serum levels of this protein significantly increased during progression of disease, and decreased during response to anti-MM therapy (P < 0.001). These results suggest that serum PTN may be a new biomarker for monitoring the disease status and therapeutic response of MM patients. PMID- 16681641 TI - Diverse niches within multiple myeloma bone marrow aspirates affect plasma cell enumeration. AB - A basic criterion for the diagnosis of multiple myeloma is plasma cell enumeration within the bone marrow (BM). This report showed that flow cytometry under-estimated the number of plasma cells in BM aspirates by an average of 60%, compared with morphological evaluation. The discrepancy was partially because BM smears contain cells associated with the lipid-enriched spicules. In contrast, flow cytometry is performed on the BM fluid, which is depleted of the lipid adhesive plasma cells. This discrepancy may point to different plasma cell subpopulations associated with diverse niches within the BM. PMID- 16681642 TI - Fixed-dose single agent pegfilgrastim for peripheral blood progenitor cell mobilisation in patients with multiple myeloma. AB - High-dose chemotherapy and autologous peripheral blood progenitor cell transplantation is an effective treatment for multiple myeloma. Progenitor cells are generally mobilised into the peripheral blood by administration of filgrastim. Pegfilgrastim is a covalent conjugate of filgrastim with a longer half-life. The results of a phase II study of pegfilgrastim, administered as a single injection to mobilise autologous peripheral blood progenitor cells in patients with multiple myeloma, is reported. All patients (n = 19) received 12 mg of pegfilgrastim. Leukaphaeresis was started when the peripheral blood CD34(+) count was >0.015 x 10(9)/l. Daily, leukaphaeresis was performed until the target progenitor cell dose was obtained. The median number of leukaphaeresis procedures required to collect the target CD34(+) cell dose was 2 (range 1-5). A median of 8.4 x 10(6) CD34(+) cells/kg (range 4.1-15.8) was collected. The most common toxicity was bone pain/myalgia. Sustained haematological recovery occurred in all the patients who underwent high-dose chemotherapy followed by autologous peripheral blood progenitor cell transplantation with pegfilgrastim-mobilised cells. A single fixed dose of pegfilgrastim was effective in mobilising adequate peripheral blood progenitor cells in patients with multiple myeloma. The efficacy and toxicity profile was similar to that described with filgrastim treatment. PMID- 16681643 TI - A possible role for CCL27/CTACK-CCR10 interaction in recruiting CD4 T cells to skin in human graft-versus-host disease. AB - Graft-versus-host disease (GvHD) is a serious complication of allogeneic stem cell transplantation (SCT) affecting the skin, gut and liver. The involvement of distinct organs suggests a role for tissue-specific chemokines and their receptors in directing activated donor T cells to these sites. In this study the potential involvement of the skin-specific CCL27/CTACK-CCR10 interaction was investigated in 15 paediatric SCT patients with skin GvHD. During the course of skin GvHD, peripheral blood T cells from these patients contained a high proportion of CD4+ CCR10+ T cells that disappeared after the GvHD was resolved. These cells were CD45RO+, expressed additional skin homing markers (cutaneous lymphocyte-associated antigen and CCR4), and produced the T-cell helper type 1 cytokines tumour necrosis factor-alpha and interleukin-2. The increase in CD4+ CCR10+ T cells was absent in SCT patients without GvHD. Immunohistochemical investigations showed CD4+ CCR10+ T cells in the GvHD skin biopsies of the same patients, but not in the gut biopsies of patients also suffering from gut GvHD. The infiltration of CD4+ CCR10+ T cells in the GvHD-affected skin correlated with an enhanced epidermal expression of CCL27/CTACK, the ligand for CCR10. These findings support the involvement of CCL27/CTACK-CCR10 interaction in recruiting CD4+ T cells to the skin, thus contributing to the pathogenesis of acute GvHD. PMID- 16681644 TI - Recombinant human activated protein C upregulates the release of soluble fractalkine from human endothelial cells. AB - Fractalkine is a unique endothelial cell-derived chemokine that functions both as a chemoattractant and as an adhesion molecule. Recent findings suggest that fractalkine plays an important role in inflammatory diseases by modulating leucocyte endothelial cell interactions. A modulating effect on the immune system in severe sepsis has been suggested for recombinant human activated protein C (rhAPC). However, a little is known about the effect of rhAPC on the endothelial release of soluble fractalkine. The effect of rhAPC (50 ng/ml to 10 microg/ml) and protein C (in equimolar concentrations) on the synthesis of fraktalkine-mRNA and release of soluble protein in human umbilical vein endothelial cells (HUVEC) was determined by reverse transcription-polymerase chain reaction and by an enzyme-linked immunosorbent assay. rhAPC at supra-pharmacological concentrations (1-10 microg/ml) stimulated fractalkine-messenger RNA-gene transcription and release of soluble fractalkine in a time- and dose-dependent manner, whereas the zymogen protein C was ineffective. As shown by experiments using monoclonal antibodies against the thrombin receptor, protease-activated receptor-1 (PAR-1), PAR-2 and against the endothelial protein C receptor (EPCR), the effect of rhAPC on fractalkine upregulation was mediated by binding to the EPCR-receptor and signalling via PAR-1. These in vitro data demonstrate that induction of fractalkine release is an important response of HUVEC to stimulation with rhAPC and may lead to a better understanding of the molecular pathways involved in the mode of action of rhAPC. Further clinical trials are needed to confirm the in vivo relevance of these data. PMID- 16681645 TI - Shwachman-Diamond syndrome: an inherited model of aplastic anaemia with accelerated angiogenesis. AB - Bone marrow angiogenesis is increased in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but has not been studied in inherited or acquired marrow failure syndromes. Shwachman-Diamond syndrome (SDS) carries a high risk of MDS/AML and is characterised by marrow stromal dysfunction. Compared with controls, SDS patients without MDS/AML had higher marrow microvessel density. Stromal VEGF gene expression, stromal vascular endothelial growth factor (VEGF) secretion and VEGF levels in serum and marrow mononuclear cells were normal. Future studies should investigate the mechanism for increased angiogenesis in SDS, and whether SDS marrow, with its increased angiogenesis, promotes progression of malignant clones. PMID- 16681646 TI - von Willebrand factor propeptide in malaria: evidence of acute endothelial cell activation. AB - The pathogenicity of Plasmodium falciparum is thought to relate to the unique ability of infected erythrocytes to adhere to and subsequently activate the vascular endothelium. To study the state of endothelial activation during falciparum malaria, we measured plasma levels of both von Willebrand factor (VWF) and its propeptide, indices of chronic and acute endothelial cell perturbation, respectively. Results were correlated with clinical and biochemical markers of disease severity, including plasma lactate. Our data show that acute endothelial cell activation is a hallmark of malaria in children, indicated by a significant rise in VWF and VWF propeptide. The highest VWF and propeptide levels were seen in cerebral and non-cerebral severe malaria, and associations found between VWF propeptide level and lactate (P < 0.001). Mean VWF propeptide levels (nmol/l) were in cerebral malaria 33.4, non-cerebral severe malaria 26.3, mild malaria 22.1, non-malaria febrile illness 10.2, and controls 10.1. Differences between patient and control groups were highly significant (P < 0.005). Follow-up of 26 cerebral malaria cases showed that levels of VWF propeptide, but not VWF fell by 24 h, following the clinical course of disease and recovery. These novel findings potentially implicate acute, regulated exocytosis of endothelial cell Weibel Palade bodies in the pathogenesis of Plasmodium falciparum malaria. PMID- 16681648 TI - One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis. AB - The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP-9TH, similar to the previously reported Xenopus laevis MMP-9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP-9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone-treated tail-derived myoblastic cultured cells, while MMP-9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP-9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP-9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis. PMID- 16681647 TI - Sickle cell leg ulcers: associations with haemolysis and SNPs in Klotho, TEK and genes of the TGF-beta/BMP pathway. AB - Cutaneous leg ulcers are common in sickle cell anaemia and their risk might be genetically determined. Sickle cell anaemia patients were studied to examine the relationship of leg ulcers with haemolysis and with single nucleotide polymorphisms (SNPs) in candidate genes that could affect sickle vasoocclusion. Leg ulcer patients had lower haemoglobin levels and higher levels of lactate dehydrogenase, bilirubin, aspartate transaminase and reticulocytes than did control patients with sickle cell anaemia but without leg ulcers. Age-adjusted comparisons showed that sickle cell anaemia-alpha thalassaemia was more frequent among controls than cases. These results strongly suggested that the likelihood of having leg ulcers was related to the intensity of haemolysis. 215 SNPs in more than 100 candidate genes were studied. Associations were found with SNPs in Klotho, TEK and several genes in the TGF-beta/BMP signalling pathway by genotypic association analyses. KL directly or indirectly promotes endothelial nitric oxide (NO) production and the TEK receptor tyrosine kinase is involved in angiogenesis. The TGF-beta/BMP signalling pathway modulates wound healing and angiogenesis, among its other functions. Haemolysis-driven phenotypes, such as leg ulcers, could be improved by agents that reduce sickle erythrocyte density or increase NO bioavailability. PMID- 16681649 TI - Expression and function of Wnt5a in the development of the glandular stomach in the chicken embryo. AB - The epithelium of the chicken embryonic glandular stomach (proventriculus) differentiates into both a glandular and a luminal epithelium, the cells of which express specific marker genes. The subsequent formation and differentiation of the glands then proceed under the influence of the mesenchyme. To search for possible candidates for the mesenchymal factors involved, we have now investigated the expression and function of Wnt5a in this process. Our current results show that Wnt5a is expressed in the mesenchyme during active gland formation and that overexpression of this gene in ovo results in the increased and ectopic expression of some of the marker genes of the luminal and glandular epithelia. In particular, the overexpression of Wnt5a markedly enhances the expression of the embryonic chicken pepsinogen gene, a marker of the glandular epithelium, indicating its role as a mesenchymal factor that regulates the differentiation of the proventricular epithelium. PMID- 16681650 TI - Sperm mitochondrial DNA transmission to both male and female offspring in the blue mussel Mytilus galloprovincialis. AB - In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type-specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance. PMID- 16681651 TI - In vitro osteogenesis from human skin-derived precursor cells. AB - Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity. PMID- 16681652 TI - Effects of glypican-1 on turkey skeletal muscle cell proliferation, differentiation and fibroblast growth factor 2 responsiveness. AB - The heparan sulfate proteoglycan, glypican-1, is a low affinity receptor for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and an inhibitor of differentiation. Heparan sulfate proteoglycans like glypican-1 are required for FGF2 to transduce an intracellular signal. Understanding the role of glypican-1 in the regulation of FGF2-mediated signaling is important in furthering the understanding of the biological processes involved in muscle development and growth. In the current study, a turkey glypican-1 expression vector construct was transfected into turkey myogenic satellite cells resulting in the overexpression of glypican-1. The proliferation, differentiation, and responsiveness to FGF2 were measured in control and transfected cell cultures. The overexpression of glypican-1 in turkey myogenic satellite cells increased both satellite cell proliferation and FGF2 responsiveness, but decreased the rate of differentiation. The current data support glypican-1 modulation of both proliferation and differentiation through an FGF2-mediated pathway. PMID- 16681653 TI - Abortive second meiosis detected in cytochalasin-treated eggs in androgenetic diploid Corbicula fluminea. AB - The hermaphroditic diploid clam Corbicula fluminea reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as two first polar bodies and subsequently second meiosis did not occur. In eggs treated with cytochalasin D (CD) to inhibit the polar body extrusion, the second meiosis was abortive. After the first meiosis, two centrosomes at the spindle poles remained in the cytoplasm because of the effect of CD. The chromosomes divided into two groups at anaphase I as observed in the control eggs. Two centrosomes divided into four just after the first meiosis but did not separate completely. The microtubules from the centrosomes were rather short. So at the second meiosis, two monoasters or tetrapolar spindles were formed. The fluorescence signal from microtubules of the monoaster or tetrapolar spindle was weak compared with the spindle at the first meiosis. The maternal chromosomes on the monoaster or tetrapolar spindle did not move, and became large female pronuclei. The pronuclei became the metaphase chromosomes on the spindle for the first cleavage. The present study suggests that second meiosis regulating factors may be abortive in androgenetic diploid C. fluminea. PMID- 16681654 TI - Mesotherapy and phosphatidylcholine injections: historical clarification and review. AB - BACKGROUND: Mesotherapy was originally conceived in Europe as a method of utilizing cutaneous injections containing a mixture of compounds for the treatment of local medical and cosmetic conditions. Although mesotherapy was traditionally employed for pain relief, its cosmetic applications, particularly fat and cellulite removal, have recently received attention in the United States. Another treatment for localized fat reduction, which was popularized in Brazil and uses injections of phosphatidylcholine, has been erroneously considered synonymous with mesotherapy. Despite their attraction as purported "fat dissolving" injections, the safety and efficacy of these novel cosmetic treatments remain ambiguous to most patients and physicians. OBJECTIVE: To distinguish mesotherapy from phosphatidylcholine injections by reviewing their history and the relevant experimental or clinical findings. METHODS: A comprehensive search of Medline indexed literature and conference proceedings. RESULTS: All the published studies evaluating the clinical efficacy of traditional mesotherapy currently originate from Europe. These reports focus primarily on musculoskeletal pain and vascular disease, rather than cosmetic applications. Although experimental data suggest that a number of traditional mesotherapy ingredients may theoretically reduce fat, these effects have not been supported in peer-reviewed studies. An increasing number of reports demonstrate that subcutaneous injections of a formula containing phosphatidylcholine combined with its emulsifier, deoxycholate, are effective in removing small collections of adipose tissue. Cell lysis, resulting from the detergent action of deoxycholate, may account for this clinical effect. CONCLUSIONS: Mesotherapy is distinct from a method of treating adipose tissue with subcutaneous injections of deoxycholate alone or in combination with phosphatidylcholine. Additional clinical and experimental studies are necessary to more definitively establish the safety and efficacy of these treatments. PMID- 16681655 TI - Role of sun exposure in melanoma. AB - BACKGROUND: Malignant melanoma is the third most common skin cancer in the United States. It is commonly thought that sun exposure is causative in these tumors. Recently, however, the significance of the role of sun exposure in melanoma has come into question. Some have suggested that other factors, such as genetics, play a larger role, and that sun protection may even be harmful. OBJECTIVE AND METHODS: To investigate the role of sun exposure in melanoma etiology. An extensive review of basic science and clinical literature on this subject was conducted. RESULTS: Although exceptions exist, sun exposure likely plays a large role in most melanomas. The pattern of this exposure, however, is not fully known, and controversy exists, especially in the use of sunscreens. Sun exposure may interact with genetic factors to cause melanomas, and sun protective measures appear to be prudent. CONCLUSIONS: The cause of melanoma is probably variable and multifactorial. Sun exposure may play a primary or supporting role in most melanoma tumors. PMID- 16681656 TI - Lentigo maligna/lentigo maligna melanoma: current state of diagnosis and treatment. AB - BACKGROUND: Lentigo maligna (LM) is a subtype of melanoma in situ that typically develops on sun-damaged skin. Presentation may be quite subtle and delayed diagnosis is common. Clinical margins are often ill defined. Histologic evaluation can be difficult due to the widespread atypical melanocytes that are present in the background of long-standing sun damage. Recurrence following standard therapies is common. OBJECTIVE: To review the clinical features, histopathology, and treatment options for LM. Emphasis is placed on recent advances in the treatment of LM. METHODS AND MATERIALS: Literature review. RESULTS: The estimated lifetime risk of LM progressing to LM melanoma is 5%. Standard excision of LM with 5 mm margins is insufficient in 50% of cases. The recurrence rate with standard excision ranges from 8 to 20%. Mohs surgery and staged excision may offer better margin control and lower recurrence rates (4 5%). Estimates of recurrence rates following nonsurgical therapies such as cryosurgery, radiotherapy, electrodessication and curettage, laser surgery, and topical medications range from 20 to 100% at 5 years. CONCLUSIONS: Adequate treatment of LM requires a comprehensive knowledge of the diagnostic features, histopathology, and treatment options. Surgical modalities with meticulous evaluation of tissue margins appears to offer the lowest rates of disease recurrence. PMID- 16681657 TI - Tumescent liposuction with dermal curettage for treatment of axillary osmidrosis and hyperhidrosis. AB - BACKGROUND: Axillary osmidrosis and hyperhidrosis are distressing social problems, particularly in Asian societies. Various treatment methods have been developed for removal of the apocrine and eccrine glands. But conventional surgical methods often lead to significant scarring and frequent recurrence. OBJECTIVE: To evaluate the effect of liposuction with curettage using a new device, the Fatemi cannula, in the treatment of axillary osmidrosis and hyperhidrosis. METHODS: From August 2003 through December 2004, 25 patients (15 women and 10 men) with axillary osmidrosis or hyperhidrosis were treated by tumescent liposuction and curettage. The results of malodor elimination were graded by the patients as excellent, good, fair, and poor. Changes in axillary sweating and hair growth, postoperative scarring, patient satisfaction degree, and complications were also evaluated. Biopsies of the axillary skin were performed in 14 patients. RESULTS: Of the 50 axillae, 38 (76.0%) were graded as excellent results, 11 (22.0%) were good, and one (2.0%) was fair. No serious complications were noted except temporary ecchymosis and local infection in minor cases. The preoperative histologic examinations showed large and numerous apocrine glands and postoperative significant decrease and degeneration of them. CONCLUSION: Tumescent liposuction with dermal curettage using the Fatemi cannula is an effective and safe method in the treatment of axillary osmidrosis and hyperhidrosis. PMID- 16681658 TI - Refractory venous leg ulcers: a study of risk factors. AB - BACKGROUND: Although certain risk factors for poor healing of leg ulcers have been identified, data concerning the characteristics of refractory ulcers have not been specifically studied in the literature. OBJECTIVE: To study the characteristics of refractory venous leg ulcers. METHODS: We retrospectively studied prognostic factors for healing in patients with refractory venous leg ulcers followed and treated in our dermatology department between January 1993 and January 2000. Each patient included in this study was compared with two patients matched for age and gender and presenting leg ulcers with normal healing, followed during the same period. RESULTS: Thirty-two of 571 patients with leg ulcers were included. The study population consisted of 20 females and 12 males with a mean age 73.5 years. The control population comprised 64 patients, 40 females and 24 males, with a mean age of 73 years. Univariate analysis demonstrated the negative prognostic impact of several previously identified factors (including surface area and history of the ulcer). In particular, multivariate analysis identified four main risk factors for refractory ulcer that are often associated in these patients: associated arterial disease, presence of post-thrombotic popliteal sequelae, recurrence of the ulcer, and disability. CONCLUSION: Four main risk factors that are often associated were identified, indicating the multifactorial nature of these refractory ulcers. PMID- 16681660 TI - Clinical and instrumental evaluation of skin improvement after treatment with a new 50% pyruvic acid peel. AB - BACKGROUND: Pyruvic acid is an alpha-keto acid that presents keratolytic, antimicrobial, and sebostatic properties as well as the ability to stimulate new collagen production and elastic fibers formation. Because of its low pKa and its small dimension, it penetrates rapidly and deeply through the skin, so far as to be considered a potent chemical peel agent. It has proven its efficacy for the treatment of many dermatological conditions such as acne, superficial scarring, photodamage, and pigmentary disorders. Pyruvic acid application usually induces intense burning, and the postpeeling period is characterized by erythema, desquamation, and, sometimes, crusting. OBJECTIVE: The aim of the study is to assess the efficacy and tolerability of 50% pyruvic acid in a new non erythematogenic formulation (pyruvic acid 50%, dimethyl isosorbide, propylene glycol, ethyl alcohol, dimethyl sulfone, ethyl lactate, water) for the treatment of photodamage, superficial scarring, and melasma. MATERIALS AND METHODS: Twenty subjects affected by photodamage, superficial scarring, and melasma, but otherwise healthy, entered the study. Four peeling sessions were performed once every 2 weeks. The patients were evaluated clinically and by means of several noninvasive methods in order to monitor the following parameters: hydration, color (erythema and pigmentation), elasticity, skin smoothness, skin roughness, scaliness, and wrinkles. RESULTS: The patients did not report any discomfort either during the peeling session or during the postpeeling period, without any impact on their social life. We did not observe any case of persistent erythema as well as any case of postinflammatory hyperpigmentation. Instrumental evaluations showed a significant reduction in the degree of pigmentation in patients with melasma, a significant increase in skin elasticity, and an improvement of the degree of wrinkling in all the patients. CONCLUSION: This innovative formulation of 50% pyruvic acid peel has been shown to be safe and effective to treat photodamage, melasma, and superficial scarring, allowing the patients to carry out regularly their working life as well as their social life. Furthermore, the results have been evaluated by means of noninvasive devices, which have permitted one to quantify the improvements. PMID- 16681659 TI - Comparison of the surgical outcomes of punch incision and elliptical excision in treating epidermal inclusion cysts: a prospective, randomized study. AB - BACKGROUND: Punch incision has been introduced as an alternative method to surgical excision for troublesome epidermal inclusion cysts. To date, there is no randomized study directly comparing the long-term results of these two methods. OBJECTIVE: To compare the surgical outcomes of these two procedures and to identify the characteristics of a lesion most suitable for the punch incision technique. METHODS: In a 16-month period, 60 patients with noninfected epidermal inclusion cysts were randomly treated with either punch incision or elliptical excision. Demographic data, size, and location of lesion, length of wound, operative time, complications, recurrence, and patient satisfaction were compared statistically. RESULTS: The mean lengths of the wounds in the punch incision and elliptical excision groups were 0.73 and 2.34 cm, respectively (p<.001). Mean operative time was significantly shorter in the punch group (12.7 minutes) as compared with the surgical group (21.6 minutes) (p<.001). No complication occurred in the punch incision group. There was no significant difference in the recurrence rate. CONCLUSION: Punch incision produces a superior cosmetic result while keeping a low recurrence rate of cysts. Epidermal inclusion cysts measuring 1 to 2 cm that are located on the face or in an area of cosmetic concern are best treated with punch incision. PMID- 16681661 TI - Treatment of dermatofibroma with a 600 nm pulsed dye laser. AB - BACKGROUND: Dermatofibroma (DF) is one of the most basic and common dermatologic diseases treated by practicing dermatologists on a daily basis. Although benign, it can be pruritic or tender. Furthermore, it is difficult to treat effectively with optimal cosmetic outcomes. OBJECTIVE: We report a safe, effective, and cosmetically superior method of treating DF with the 600 nm pulsed dye laser (PDL). METHODS: We used a 600 nm PDL to treat 20 lesions from 18 Caucasian patients. The laser parameter was set at a fluence of 7 J/cm2, a spot size of 7 mm and a pulse duration of 1.5 ms. Each lesion was treated three times at a 6- to 8-week interval. For each treatment, the lesion was double pulsed with a 20 to 30% overlap. Clinical improvement was graded by a single examiner in evaluating three clinical parameters: color, size/volume, and symptoms. For each parameter, improvement was ranked as no improvement, partial improvement, and complete response. RESULTS: All 18 patients (17 women) completed the study. For the volume/size parameter, 15 of 20 lesions (75%) showed complete response. For improvement in color, 12 of 20 patients (60%) showed complete response. Only six lesions were symptomatic (i.e., tender and irritating), and all six lesions showed complete resolution of symptoms after the PDL treatments. After each treatment, all patients experienced blistering, crusting, and purpura that usually resolved after 10 days. CONCLUSIONS: We have demonstrated for the first time that PDL (600 nm and 1.5 ms pulse duration) is an effective and safe treatment of DF. It may provide superior cosmetic outcomes compared with other modalities such as surgical excision. PMID- 16681662 TI - Stable vitiligo treated by a combination of low-dose oral pulse betamethasone and autologous, noncultured melanocyte-keratinocyte cell transplantation. AB - BACKGROUND: Various surgical procedures are in use to treat stable vitiligo. The possibility of Koebner phenomenon always exists with surgical treatment. Partial or complete failure to repigment is observed in spite of clinical stability. AIM: To evaluate the usefulness of a combination treatment of low-dose oral betamethasone and melanocyte-keratinocyte transplantation. METHODS: Oral betamethasone was given to patients who failed to respond either completely or partially to melanocyte-keratinocyte cell transplantation, and the procedure was repeated for previously treated and nontreated area. A simpler and modified method described by Mulekar was performed. RESULTS: Seventeen patients with vitiligo vulgaris and eight patients with segmental vitiligo were retransplanted after giving oral betamethasone for 2 to 10 months after the initial procedure. Two patients of vitiligo vulgaris and one of segmental vitiligo failed to respond completely even after repeat transplantation. Fifteen vulgaris and seven segmental patients showed excellent to good repigmentation after repeat transplantation. CONCLUSION: Combined treatment of oral betamethasone and melanocyte cell transplantation has a potential to produce complete repigmentation in patients with large vitiliginous areas. PMID- 16681663 TI - Basal cell carcinoma with mixed histology: a possible pathogenesis for recurrent skin cancer. AB - BACKGROUND: Basal cell carcinoma with mixed histology (BCC-MH) demonstrates more than one pathologic pattern of tumor. Appropriate therapy for the nonaggressive tumor subtype diagnosed from a superficial biopsy may not be adequate to treat the unsuspected aggressive tumor subtype, resulting in recurrent skin cancer. OBJECTIVE: We prospectively evaluated the incidence and characteristics of BCC MH. METHODS: Mohs surgical sections were evaluated for BCC-MH. RESULTS: BCC-MH was observed in 43% (49/144) of cancers. Only 10% (5/49) of the biopsy reports from these cancers diagnosed BCC-MH. Most BCC-MH were previously untreated (90% [44/49] of tumors) and were located on the nose. CONCLUSION: More than 40% of basal cell carcinomas referred for removal of the residual tumor were BCC-MH. An unsuspected aggressive pathologic pattern of growth may not be detected after a superficial biopsy. Subsequently, the cancer may recur if the initial treatment for the diagnosed nonaggressive tumor subtype is inadequate for the undiscovered aggressive carcinoma. Mohs micrographic surgery is recommended for BCC-MH treatment. PMID- 16681664 TI - Prevention of nosocomial infection during dermoscopy? AB - BACKGROUND: Dermatoscopes are applied directly to cutaneous or mucocutaneous surfaces with immersion fluid (IF) such as oil or alcohol to reduce light reflection. Recently, Staphylococcus aureus has been isolated from dermatosopes that used mineral oil as the IF. Thus, dermatoscopes might be a potential source of nosocomial infection. OBJECTIVE: In this study we propose the use of an alcohol-based antibacterial gel to reduce nosocomial infection transmission while optimizing optical resolution during dermatoscopic examination. MATERIALS AND METHODS: Aerobic bacterial cultures were performed on three dermatoscopes used in an outpatient setting after routine examination of 31 patients with an alcohol based antibacterial gel as IF. RESULTS: There was no bacterial growth after using the antibacterial gel with the dermatoscopes. The optical resolution for the antibacterial gel appeared equal to the 'dermatoscopy oil' and superior to alcohol wipes. CONCLUSIONS: Alcohol-based antibacterial gel appears to inhibit bacterial colonization while offering excellent optical resolution during dermoscopic examination. The use of alcohol-based IF appears to obviate the risk of nosocomial infections. PMID- 16681665 TI - Tophaceous lesion of the middle third of the nose. PMID- 16681666 TI - Lexicon of areas amenable to liposuction. AB - BACKGROUND: Areas amenable to liposuction are identified by a variety of terms. Both common and professional language is used. OBJECTIVE: This manuscript identifies colloquial terms for these body areas and suggests a standardized nomenclature. RESULTS: Physicians need to be aware of the lay terms so they can communicate with their patients. The careful use of a standardized vocabulary provides precision and accuracy. PMID- 16681667 TI - Chemoprevention of nonmelanoma skin cancer with systemic retinoids: practical dosing and management of adverse effects. AB - BACKGROUND: In patients with nonmelanoma skin cancer that is high risk or characterized by numerous tumors, chemoprevention with systemic retinoids may effectively decrease the number of new tumors whereas the chemosuppressive effects may reduce the risk of recurrence or disease progression. A patient's intolerance of the mucocutaneous effects of retinoid therapy or abnormal laboratory findings may hamper continuous therapy. OBJECTIVE: To present a method for optimizing tolerance of systemic retinoids for chemoprevention and for monitoring and managing adverse events. METHODS: After reviewing the data on the use of systemic retinoids for chemoprevention, we developed a simplified approach for administering oral retinoids for chemoprevention of nonmelanoma skin cancer as well as basic guidelines for the prevention and management of adverse effects and appropriate laboratory monitoring. RESULTS: Chemoprevention with systemic retinoids in patients with a history of numerous tumors or high-risk skin cancer can be optimized with graduated dose escalation and preventive strategies for the most common adverse effects. Routine laboratory monitoring may assist in detecting adverse effects, which can be managed in most cases. CONCLUSION: In our experience, the effective use of systemic retinoids for chemoprevention of nonmelanoma skin cancer in high-risk patients can be optimized through a standardized, proactive approach. PMID- 16681668 TI - Dermoscopic pattern of pigmented basal cell carcinoma, blue-white variant. PMID- 16681669 TI - Nasal cavity squamous cell carcinoma presenting as a nasal sidewall nodule: treatment with Mohs surgery, partial rhinectomy, and adjuvant radiotherapy. PMID- 16681670 TI - Regressed subungual melanoma simulating cellular blue nevus: managed with sentinel lymph node biopsy. AB - BACKGROUND: Subungual melanoma, a not uncommon presentation of cutaneous melanoma in Asian populations, is easily overlooked as benign and thus is improperly treated. OBJECTIVE: To present two cases with clinical suspicion of subungual melanoma. Skin biopsies failed to demonstrate the diagnostic features of malignancy. METHODS: Lymphoscintigraphy and sentinel lymph node (SLN) biopsies were performed to determine regional lymph node status. RESULTS: Both hematoxylin eosin and HMB45 staining revealed melanoma cells in the SLN of the patient. The second patient's SLN was negative for malignant cells, but her excised primary lesion showed extensive regressed melanoma. CONCLUSION: Regression phenomena are not uncommon for subungual melanoma. An extention biopsy techniques are useful for determining nodal basin status in regressed subungual melanoma. PMID- 16681671 TI - Atypical cellular neurothekeoma treated with Mohs micrographic surgery. AB - BACKGROUND: Atypical cellular neurothekeoma is a rare neoplasm generally regarded as a benign tumor with locally aggressive behavior. Recurrence is common with inadequate excision, but metastatic disease has yet to be reported. OBJECTIVE: The objective was to report a case of atypical cellular neurothekeoma of the nasal ala in a young woman treated by Mohs micrographic surgery. The biologic behavior and histopathology will be reviewed. METHODS: Case report and review of the literature. RESULTS: The neoplasm was extirpated in a three-stage, five section Mohs surgery procedure. The resulting defect was repaired by a two-stage, pedicled nasolabial transposition flap with a cartilage graft taken from the ipsilateral ear. No clinical recurrence was noted after 6-months of follow-up. CONCLUSION: Mohs micrographic surgery is unsurpassed in its efficacy in treating a wide variety of nonmelanoma skin cancers. Although most commonly used to address basal and squamous cell carcinoma, it has also been reported as a successful treatment for melanoma and a wide variety of cutaneous malignancies. We discuss a case of atypical cellular neurothekeoma removed with the Mohs technique. Debate in the literature is ongoing regarding the true histogenesis of this rare tumor. Because of this tumor's local destructive behavior and propensity to recur with inadequate resection, we recommend Moths micrographic surgery for the treatment of cellular neurothekeomas. PMID- 16681672 TI - Multiple recurrent atypical fibroxanthomas/superficial malignant fibrous histiocytomas of the forehead excised with Mohs micrographic surgery. PMID- 16681673 TI - Subungual metastasis from a rectal primary: case report and review of the literature. AB - BACKGROUND: Subungual metastases from colorectal cancer are unusual and have mainly been reported in patients with lung, genitourinary, and breast cancer. OBJECTIVE: We present the case of a 72-year-old man with rectal adenocarcinoma and a subungual metastasis to the left thumb 5 years later. METHODS: A case report and a brief review of the literature of subungual metastases are given. RESULTS: The thumb was amputated and the patient died 6 months later with extensive metastatic disease. CONCLUSION: Metastatic carcinoma should be considered in the differential diagnosis of persistent subungual masses, particularly in patients with a history of cancer. The prognosis with such lesions is generally poor. PMID- 16681674 TI - Satellite repigmentation (remote reverse Koebner phenomenon). PMID- 16681675 TI - Medical versus surgical dermatology: how much training do residents receive? PMID- 16681676 TI - Cardiac devices and electromagnetic interference revisited: new radiofrequency technologies and implications for dermatologic surgery. PMID- 16681677 TI - In response to commentary on new methodology and instrumentation for follicular unit extraction: lower follicle transection rates and expanded. PMID- 16681678 TI - Prognostic and predictive factors in endocrine tumours. AB - This review encompasses the diagnostic features of malignancy, the routinely observable prognostic features and the prognostic and predictive features emerging from research techniques in the principal endocrine neoplasms: pancreatic and extrapancreatic endocrine cell tumours, thyroid and parathyroid neoplasia, adrenal cortical neoplasms and adrenal and extra-adrenal paragangliomas. While each endocrine tissue has its own set of diagnostic features for malignancy, and prognostic features once a diagnosis of malignancy has been established, there are a few common themes. For several endocrine neoplasms, definite recognition of malignancy can be difficult and may depend upon frank invasive or metastatic growth at presentation. Endocrine tissues are dynamic, with hyperplastic and regressive phenomena, some of which may mimic malignancy. Even when unequivocal features of malignancy are available for observation, their distribution in tissue may be very focal, necessitating thorough sampling. The accurate documentation of routinely observable histological features interpreted in the light of current literature has not been superseded by special techniques in the statement of diagnosis or prognosis in the vast majority of endocrine neoplasms. PMID- 16681679 TI - A UK-based investigation of inter- and intra-observer reproducibility of Gleason grading of prostatic biopsies. AB - AIMS: The frequency of prostatic core biopsies to detect cancer has been increasing with more widespread prostate specific antigen (PSA) testing. Gleason score has important implications for patient management but morphological reproducibility data for British practice are limited. Using literature-based criteria nine uropathologists took part in a reproducibility study. METHODS: Each of the nine participants submitted slides from consecutive cases of biopsy diagnosed cancer assigned to the Gleason score groups 2-4, 5-6, 7 and 8-10 in the original report. A random selection of slides was taken within each group and examined by all pathologists, who were blind to the original score. Over six circulations, new slides were mixed with previously read slides, resulting in a total of 47 of 81 slides being read more than once. RESULTS: For the first readings of the 81 slides, the agreement with the consensus score was 78% and overall interobserver agreement was kappa 0.54 for Gleason score groups 2-4, 5-6, 7, 8-10. Kappa values for each category were 0.33, 0.56, 0.44 and 0.68, respectively. For the 47 slides read more than once, intra-observer agreement was 77%, kappa 0.66. The study identified problems in core biopsy interpretation of Gleason score at levels 2-4 and 7. Patterns illustrated by Gleason as 2 tended to be categorized as 3 because of the variable acinar size and unassessable lesional margin. In slides with consensus Gleason score 7, 13% of readings were scored 6 and in slides with consensus 6, 18% of readings were scored 7. CONCLUSIONS: Recommendations include the need to increase objectivity of the Gleason criteria but limits of descriptive morphology may have to be accepted. PMID- 16681680 TI - A study of Gleason score interpretation in different groups of UK pathologists; techniques for improving reproducibility. AB - AIMS: To test the effectiveness of a teaching resource (a decision tree with diagnostic criteria based on published literature) in improving the proficiency of Gleason grading of prostatic cancer by general pathologists. METHODS: A decision tree with diagnostic criteria was developed by a panel of urological pathologists during a reproducibility study. Twenty-four general histopathologists tested this teaching resource. Twenty slides were selected to include a range of Gleason score groups 2-4, 5-6, 7 and 8-10. Interobserver agreement was studied before and after a presentation of the decision tree and criteria. The results were compared with those of the panel of urological pathologists. RESULTS: Before the teaching session, 83% of readings agreed within +/- 1 of the panel's consensus scores. Interobserver agreement was low (kappa = 0.33) compared with that for the panel (kappa = 0.62). After the presentation, 90% of readings agreed within +/- 1 of the panel's consensus scores and interobserver agreement amongst the pathologists increased to kappa = 0.41. Most improvement in agreement was seen for the Gleason score group 5-6. CONCLUSIONS: The lower level of agreement among general pathologists highlights the need to improve observer reproducibility. Improvement associated with a single training session is likely to be limited. Additional strategies include external quality assurance and second opinion within cancer networks. PMID- 16681681 TI - The combination of millimetres of cancer and Gleason index in core biopsy is a predictor of extraprostatic disease. AB - AIMS: The Gleason index (GI) is related to several pathological endpoints in radical prostatectomy (RP) specimens, including the risk of extraprostatic disease (ED). The amount of tumour (TM) in core biopsy (CB) specimens also correlates with staging. The aim was to determine whether the sum of the relative statistical weights of GI and TM in CBs strengthens the prediction of ED in RPs. METHODS AND RESULTS: A series of 290 RPs and their previous respective CBs were reviewed. TM and the GI were blindly evaluated in CBs and statistically correlated with ED in RPs. A total of 60 (20.6%) RPs showed ED. The logistic regression model indicated that all cases with > 22 mm of cancer in CB showed ED in RP. All cases with a GI > 7 and > 12 mm of cancer in CB displayed ED. Finally, Pearson's chi2 revealed that 80% of cases with a GI > 7 and > or = 5 mm of cancer in CB showed ED in RP. CONCLUSIONS: The combined evaluation of GI and TM in CB is a useful method to strengthen the prediction of ED, is based on two simple morphological criteria and may be used as an additional tool to choose the best treatment modality. PMID- 16681682 TI - Detection of prostate cancer by alpha-methylacyl CoA racemase (P504S) in needle biopsy specimens previously reported as negative for malignancy. AB - AIMS: To investigate the possibility of detecting small focal prostatic cancer by alpha-methylacyl CoA racemase (AMACR)/P504S immunohistochemistry on needle biopsy specimens that were previously interpreted as negative for carcinoma on routine haematoxylin and eosin (H&E)-stained sections. METHODS: Prostate needle biopsy specimens (n = 793) previously interpreted as benign prostatic tissue by conventional morphology from 239 patients with prostatic cancer diagnosed in other biopsy cores taken at the same biopsy session were stained with the P504S monoclonal antibody. If a biopsy specimen stained positively, two pathologists independently reviewed the original corresponding H&E-stained sections to establish the malignant diagnosis. RESULTS: Eighty-four of the 793 biopsy specimens showed AMACR immunoreactivity; nine of these (9/793, 1.1%) contained previously unrecognized small focal prostatic carcinoma (Gleason 6, N = 8; Gleason 8, N = 1). Six of nine (67%) carcinomas showed foamy/pseudohyperplastic (N = 3) or atrophic (N = 3) features. Additionally, five biopsy specimens (5/793, 0.6%) with positive AMACR staining that did not meet the criteria for prostatic cancer on the original H&E slides were considered to be atypia. CONCLUSIONS: In this study, we found a 1.1% false-negative rate for carcinoma on routine H&E stained sections. AMACR immunohistochemical staining has shown the ability to improve detection of small focal prostatic carcinoma that could be missed by conventional histological examination. PMID- 16681683 TI - Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassification: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index. AB - AIMS: Counting mitotic figures is considered to be a reliable prognosticator, but evaluation of Ki67 immunohistochemistry has become more popular in evaluating proliferation. Our previous studies suggested an occasional discrepancy between mitotic figures and Ki67 fraction. The aim of this study was to investigate this more closely and also to study the associations between bcl-2 and p53 expression and proliferation. METHODS AND RESULTS: Two hundred and sixty-five infiltrating breast carcinomas were immunostained for Ki67, p53 and bcl-2. The standardized mitotic index (SMI) was determined. Four proliferation groups were based on Ki67 positivity fraction and SMI at optimal cut-off points. Cox's multivariate model was used to test the power of the prognosticators. SMI and nodal status were the most powerful individual prognosticators. Ki67 was an independent prognosticator if nodal status, tumour size, age and histological grade were included in the analysis but not if analysed with SMI. The group with low SMI and low Ki67 fraction had the best prognosis. Groups with high SMI had the poorest prognosis. The group with low SMI and high Ki67 fraction had a favourable prognosis. Bcl-2 negativity and p53 positivity correlated with proliferation. CONCLUSIONS: We have found a 'wrong positive' Ki67 group with favourable prognosis. SMI cannot be replaced by Ki67 because of the danger of misclassification of some patients. PMID- 16681684 TI - Immunohistochemistry increases the accuracy of diagnosis of benign papillary lesions in breast core needle biopsy specimens. AB - AIMS: Recent studies have suggested that benign papillary lesions without atypia [benign papilloma (BP)] diagnosed on breast core needle biopsy (CNB) may not require excision. However, most have studied only small numbers of cases and scant data are available on the utility of immunohistochemistry in the categorization of papillary lesions on CNB. In the largest published series of BP identified on CNB, we studied the impact of immunohistochemistry on the accuracy of a CNB diagnosis of BP. METHODS AND RESULTS: Breast CNBs (n = 129) with a diagnosis of papillary lesion were immunostained for calponin, p63 and cytokeratin 5/6. Haematoxylin and eosin and immunostained slides were independently reviewed by four breast pathologists. BP was the final excision diagnosis in 35 cases. With the use of immunohistochemistry, the positive predictive value (PPV) of BP diagnosis by the four individual pathologists increased from 72.7-83.3% (mean 79.2%) to 77.8-87.5% (82.1%), the negative predictive value (NPV) increased from 77.8-98.5% (88.6%) to 100% for all four participants and overall accuracy increased from 78.7-92.6% (84.7%) to 90.7-95.4% (92.8%). No case of invasive carcinoma was diagnosed as BP on CNB by any participant. The frequency of ductal carcinoma in situ following a BP diagnosis on CNB ranged from 2.5% to 4.8% (4%) but was only 0-3% (2.3%) after excluding cases that were radiologically suspicious for malignancy. CONCLUSIONS: Immunohistochemistry increases accuracy of BP diagnosis in CNB specimens. Benign papillary lesions diagnosed on CNB do not require excision in the absence of suspicious clinical/radiological findings. PMID- 16681685 TI - Different patterns of inflammation and prognosis in invasive carcinoma of the breast. AB - AIM: Inflammation in carcinoma of the breast may represent an immune response to the tumour, but there is evidence that this response is impaired. Inflammation may also stimulate tumour growth by releasing proteolytic enzymes and angiogenic factors. Prognostic studies have produced conflicting results, but most investigators have not evaluated the different patterns of inflammation. The aim of this study was to test the hypothesis that moderate or marked diffuse inflammation is associated with a better prognosis. We also tested the 'danger model', which suggests that necrosis is necessary for an effective immune response. METHODS AND RESULTS: On multivariate analysis of women with stage 1 and 2 tumours (n = 679, median follow-up of 9.8 years), survival was independently associated with diffuse inflammation (relative risk 0.43, 95% confidence interval 0.24, 0.77, P =0.005) in addition to histological grade, axillary lymph node status, tumour size and oestrogen receptor status. The presence or absence of tumour necrosis did not have a clear effect on the relationship between survival and diffuse inflammation. CONCLUSIONS: Moderate or marked diffuse inflammation in breast cancer is associated with a better prognosis, suggesting that the immune effects of the inflammation predominate over the protumour effects. PMID- 16681686 TI - HER2 status in pure ductal carcinoma in situ and in the intraductal and invasive components of invasive ductal carcinoma determined by fluorescence in situ hybridization and immunohistochemistry. AB - AIM: To determine the HER2 status of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. The increased prevalence of HER2 amplification and overexpression in DCIS is considered to be maintained in the intraductal component of IDC; however, HER2 amplification and overexpression are detected much less in IDC. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed to detect HER2 in 270 IDCs with an intraductal component and in 50 pure DCIS samples; IHC was also performed in 116 metastatic nodes. HER2 was found to be amplified in 77 cases (28.5%) and overexpressed in 79 (29.3%) of the 270 IDCs. HER2 amplification was similar between intraductal and invasive components of the same tumour. The concordance for HER2 status between invasive and intraductal components of individual tumours was 98.5% and 99.3% by FISH and IHC, respectively. HER2 was amplified in 25 (50%) of the 50 pure DCIS samples. HER2 overexpression in metastatic nodes resembled the HER2 status in the primary tumour for 108 (93.1%) of 116 cases (kappa =0.831). CONCLUSION: Our study indicates that the intraductal component of IDC may differ biologically when compared with pure DCIS. HER2 appears to lack a critical role in the progression from DCIS to IDC and HER2 status is maintained in metastatic lesions. PMID- 16681688 TI - Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinization. AB - Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinizationAims : To examine invasive head and neck squamous carcinomas for expression of GLUT1, a glucose transporter and marker of increased glucose uptake, glycolytic metabolism and response to tissue hypoxia; p63, a p53 homologue that is a marker of the undifferentiated proliferative basaloid phenotype; and phospho-histone H1, a marker of activation of the cell cycle promoting cyclin-dependent kinases 1 and 2. Methods : Routinely processed slides from 34 invasive squamous carcinomas, including 25 with intraepithelial components, were immunostained with anti-GLUT1 (Chemicon), anti-p63 (4A4, Santa Cruz), and antiphospho-histone H1 (monoclonal 12D11). Results : In keratinizing carcinomas, all three markers were most commonly immunodetected peripherally, with loss of expression in central keratinized zones. In contrast, in non keratinizing carcinomas, p63 and phospho-histone H1 expression was most commonly observed throughout tumour nests and anti-GLUT1 stained in a pattern suggestive of hypoxia-induced expression ('antistromal' staining), in which cells at the tumour-stromal interface were GLUT1- and cells in central, perinecrotic zones showed progressive induction of GLUT1. Intraepithelial components also displayed basal and 'antibasal' GLUT1 staining patterns, homologous to the pro- and antistromal patterns in invasive carcinoma; basal patterns in intraepithelial lesions appeared to be more predictive of keratinizing invasive carcinoma and antibasal intraepithelial staining more predictive of non-keratinizing poorly differentiated carcinomas. Conclusions : Keratinizing and non-keratinizing squamous carcinomas differ in expression patterns of GLUT1, p63 and phospho histone H1. In the former, all three markers were typically suppressed in conjunction with keratinization; in the latter, GLUT1 expression was more likely to occur in a hypoxia-inducible pattern and expression of p63 and phospho-histone H1 was unsuppressed. GLUT1 expression patterns in intraepithelial lesions may be predictive of the differentiation status of the associated invasive carcinoma. PMID- 16681687 TI - Immunohistochemical detection of GLUT1, p63 and phosphorylated histone H1 in head and neck squamous intraepithelial neoplasia: evidence for aberrations in hypoxia related, cell cycle- and stem-cell-regulatory pathways. AB - AIM: Most epithelial malignancies are characterized by multistep progression from preinvasive/intraepithelial neoplasia to invasive malignancy. Detection and grading of early squamous intraepithelial neoplasia may at times be problematic. The aim of this study was to examine the ability of immunomarkers GLUT1, phospho histone H1 and p63 to detect such early lesions. METHODS: Sections of formalin fixed paraffin-embedded tissue from 27 cases of squamous intraepithelial neoplasia, 26 associated with invasive carcinoma, were immunostained with anti p63 (4A4; Santa Cruz), anti-GLUT1 (Chemicon) and anti-phospho-histone H1 (monoclonal 12D11) and compared with normal, hyperplastic and immature squamous metaplastic epithelium. RESULTS: Normal epithelium displayed phospho-histone H1 in scattered parabasal cells; p63 in the basal one-quarter to one-half of epithelium; and GLUT1 negativity or weak/equivocal mid-epithelial GLUT1+ foci. In hyperplasia phospho-histone H1+ cells were also limited to the parabasal layer; p63 positivity was essentially identical to that in normal epithelium; GLUT1 characteristically stained basal cells in rete-like areas. p63 staining in squamous intraepithelial neoplasia (grade 1) was indistinguishable from normal epithelial staining; in contrast, squamous intraepithelial neoplasia (grade 3) was readily apparent, with up to full-thickness p63 positivity. Squamous intraepithelial neoplasia (grade 1) was readily distinguishable from normal epithelium with both phospho-histone H1 and GLUT1 immunostaining; both markers were detected in cell layers above the parabasal layer. With both, progressively higher cell layers stained in proportion to the severity of the intraepithelial neoplasia, up to full thickness positivity in grade 3 squamous intraepithelial neoplasia. Squamous metaplasia and grade 3 squamous intraepithelial neoplasia were not distinguishable with p63 (both showed full-thickness staining) but were readily distinguishable with GLUT1 and phospho-histone H1 stains. GLUT1 appeared to be the most sensitive marker for all grades of intraepithelial neoplasia. CONCLUSION: Altered expression of all three markers was a common finding in squamous intraepithelial neoplasia, hence, dysregulation of cell cycle-promoting cyclin-dependent kinases (phospho-histone H1), altered stem cell regulatory pathways (p63) and enhancement of hypoxia-sensing pathways (GLUT1) are all early alterations in the progression of squamous malignancy of head and neck origin. A panel of all three may be a useful means of detecting squamous intraepithelial neoplasia. PMID- 16681689 TI - Extramammary Paget's disease--a proliferation of adnexal origin? AB - AIM: To investigate a possible follicular origin of extramammary Paget's disease (EPD). EPD is a predominantly intraepidermal tumour with extensive involvement of adnexal structures and high recurrence rates suggesting a follicular stem cell origin. Cytokeratin (CK) 15 and CK19 are considered markers for follicular stem cells located in the hair follicle bulge region. METHODS AND RESULTS: Formalin fixed paraffin-embedded tissues of 12 cases of primary EPD (three anal, nine vulvar) were studied immunohistochemically with antibodies to CK15 and CK19. All cases of EPD showed polygonal Paget cells in the interfollicular epidermis, hair follicles, sebaceous and apocrine glands distributed individually, in nests and in gland-like areas. The polygonal Paget cells were intimately associated with small, flat, mitotically active, 'compressed' keratinocytes. The large Paget cells uniformly expressed CK19 in 12/12 EPD. The small 'compressed' keratinocytes showed strong cytoplasmic CK15 staining in 9/12 EPD with focal accentuation, while the polygonal Paget cells were negative. CONCLUSIONS: These histological and immunohistochemical observations allow the following conclusions: (i) the small, flat, 'compressed' keratinocytes are an integral part of EPD; (ii) the dual cell population is reminiscent of sebaceous glands with mature sebocytes and germinative keratinocytes; (iii) since both cell types express cytokeratins typical for follicular differentiation, EPD may be a proliferation of adnexal stem cells residing in the infundibulo-sebaceous unit of hair follicles and adnexal structures. PMID- 16681690 TI - Detailed analysis of the T-cell lymphocytic infiltrate in penile lichen sclerosus: an immunohistochemical and molecular investigation. AB - AIM: To determine the extent of clonal outgrowth in the lymphocytic tissue infiltrate of lichen sclerosus (LS). The presence of T cells with a monoclonally rearranged T-cell receptor gamma-gene (TCRgamma) has been described in up to 50% of biopsies of vulvar and penile LS. MATERIAL AND RESULTS: We analysed 33 foreskin specimens with LS for the presence of clonal T cells by conventional polymerase chain reaction (PCR) analysis and with TCRgamma-PCR-based fluorescent fragment analysis. Eighteen of 33 patients revealed a band indicating a monoclonally rearranged TCRgamma on conventional PCR analysis. Subsequent TCRgamma-PCR-based fluorescent fragment analysis identified 8/18 patients with monoclonal T-cell DNA ranging from 1.4% to 23.1% of total T-cell DNA analysed and a size range from 56 to 72 base pairs. Four of 18 patients had an oligoclonal and 6/18 patients revealed a polyclonal banding pattern. The lymphocytic infiltrate contained low numbers of gammadelta T cells and cytotoxic T cells in comparable numbers to the low percentage of clonal TCRgamma DNA. CONCLUSIONS: The low percentage of clonal TCRgamma DNA argues against a systemic neoplastic disease, but rather for a local immune disorder. The target antigen of the clonal outgrowth is unknown, but an exaggerated antigen-dependent proliferation of T cells due to chronic local antigen exposure, probably an infectious antigen, is the most likely explanation. PMID- 16681691 TI - Interstitial fibrosis in the heart: differences in extracellular matrix proteins and matrix metalloproteinases in end-stage dilated, ischaemic and valvular cardiomyopathy. AB - AIMS: To investigate whether or not there are differences in the distribution of extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs) in end stage heart failure underlying different cardiomyopathies. METHODS AND RESULTS: Thirty-nine explanted human hearts were investigated: 15 with dilated cardiomyopathy (DCM), 17 with ischaemic cardiomyopathy (ICM) and seven with valvular cardiomyopathy (VCM). Transmural samples from four different sites were investigated. Frozen sections were processed for immunohistochemistry for collagens type I, III, IV, laminin and fibronectin, as well as MMP-1, -2 and -9. Volume densities were determined. All ECM components were expressed more frequently in DCM than in ICM. Comparing ICM with VCM, all proteins were found more frequently in VCM than in ICM except for type III collagen, which was significantly more frequent in ICM. Comparing DCM and VCM, VCM showed significantly higher volume densities for type III collagen and laminin. MMPs showed only slight variations between the cardiomyopathies. CONCLUSION: The distribution of ECM proteins differs between DCM, ICM and VCM, which suggests that they can be morphologically discriminated by interstitial fibrosis, especially by their expression of matrix proteins. PMID- 16681693 TI - Pigment synthesizing melanoma (so-called animal type melanoma): a clinicopathological study of 14 cases of a poorly known distinctive variant of melanoma. AB - AIMS: Pigment synthesizing melanoma (so-called animal type melanoma) (PSM) is a rare histopathological variant of melanoma so termed because of prominent melanin production and its similarity to a variant of melanoma seen in grey horses. The aim of this study was to report the clinicopathological characteristics of 14 cases of animal type melanoma. METHODS AND RESULTS: Six patients were female and eight were male with ages ranging from 5 to 52 years (mean 31 years, median 39 years). The head and neck represented the most common site. The clinical diagnosis was of melanoma in seven cases, blue naevus in three cases, benign naevus in three cases and a pigmented basal cell carcinoma in one case. The histological diagnosis of PSM was predicated on the basis of an asymmetrical, predominantly intradermal tumour formed of deeply pigmented, round or short, spindle-shaped dendritic melanocytes with some degree of hyperchromatism and a single nucleolus. Cytological atypia was always present but was not pronounced. A prominent population of macrophages was invariably present. Four tumours were compound and 10 tumours were predominantly intradermal. The mitotic count was usually low, ranging from 1 to 5 per 10 high-power fields (mean 2). Perineural and lymphovascular invasion was not seen. The Breslow thickness ranged from 1.1 to 7.5 mm (mean 3.3 mm). Follow-up was available in 13 patients. The median follow-up period was 5 years. Six patients had no recurrence, three had local recurrence in the form of satellite nodules adjacent to the scar, four had spread to the regional lymph nodes and one patient had distant metastases to the liver. There were no deaths. CONCLUSIONS: This study demonstrates that PSM is a distinctive, possible low-grade variant of melanoma usually lacking the histological features predictive of aggressive behaviour seen in ordinary melanoma. It should be managed in the same way as other melanomas with wide local excision. PMID- 16681692 TI - Expression of ETV6-NTRK in classical, cellular and mixed subtypes of congenital mesoblastic nephroma. AB - AIM: Congenital mesoblastic nephroma (CMN) is the commonest renal tumour of infancy, with classical, cellular and mixed histological subtypes described. A specific ETV6-NTRK3 fusion-gene product is reported in association with the cellular variant. The aim was to investigate the relationship between the presence of this product and morphological phenotype using paraffin-embedded archival material. METHODS AND RESULTS: Cases of CMN from a single centre during a 15-year period (1989-1994) were identified, anonymized and blindly classified using morphological criteria. RNA was extracted from frozen and paraffin sections for both conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. Fifteen samples were analysed; two were non informative and three expressed ETV6-NTRK3 using both techniques, two showing similar expression, whilst one showed expression two orders of magnitude lower, from a cellular tumour. All fusion positive cases were previously classified as cellular subtype. Six patients had mixed-subtype tumours in which the cellular components, morphologically indistinguishable from cellular tumours, were fusion negative, as were all classical cases. CONCLUSIONS: Real-time PCR Taqman assays, using both fixed and frozen tissue, provide highly reproducible detection and quantification of fusion transcript expression. Differences in expression levels may explain previous conflicting data on fusion gene detection in these tumours. PMID- 16681695 TI - TTF-1 expression in primary ovarian epithelial neoplasia. PMID- 16681696 TI - A graphic approach to normal placental development. PMID- 16681697 TI - The expression of p16 is not correlated with HPV status in CINI. PMID- 16681698 TI - The use of the monoclonal antibody mesothelin in the diagnosis of malignant mesothelioma in pleural biopsies. PMID- 16681700 TI - A TFE3+ gastrointestinal tumour: report of a case. PMID- 16681699 TI - Tuberous sclerosis complex presenting as a pulmonary solitary nodule. PMID- 16681701 TI - Up-regulation of macrophage inhibitory factor in infants with acute neonatal necrotizing enterocolitis. PMID- 16681702 TI - Cytokeratins 1, 7 and 14 immunoexpression are helpful in the diagnosis of basaloid squamous carcinoma. PMID- 16681703 TI - Thrombotic microangiopathy in an allograft kidney: a diagnostic challenge. PMID- 16681704 TI - Expanding acute care nurse practitioner practice: focus on emergency department practice. PMID- 16681705 TI - Common complaints, difficult diagnosis: multiple myeloma. AB - PURPOSE: To review the presenting signs and symptoms of multiple myeloma, its pathophysiology, diagnostic evaluation, and treatment options. DATA SOURCES: A literature review of research articles and publications by oncology experts who specialize in multiple myeloma, supplemented by a case study. CONCLUSIONS: Multiple myeloma is a cancer of the geriatric population, with the average age at diagnosis between 65 and 68 years. As the population of those over age 65 is predicted to double by the year 2050, the incidence of myeloma is expected to increase. Nurse practitioners (NPs) must become familiar with the signs, symptoms, and complications of myeloma for patients to be diagnosed and referred to specialists in a timely manner. IMPLICATIONS FOR PRACTICE: Patients with multiple myeloma often present with vague, common symptoms such as back pain, bony pain, fatigue, and anemia. These symptoms may be treated as separate medical conditions if NPs fail to include multiple myeloma in their differential diagnosis. If NPs are educated on this malignancy, they will have the expertise to look for other signs of the disease such as hypercalcemia, pathological fractures, osteopenia, or renal failure. Without early recognition of multiple myeloma and referrals to oncology specialists, patients are left with a delayed diagnosis and poor symptom control. PMID- 16681706 TI - Uremic syndrome and end-stage renal disease: physical manifestations and beyond. AB - PURPOSE: This review summarizes data concerning the incidence, definition, pathophysiology, and physical manifestations of patients with uremic syndrome. DATA SOURCES: Data sources utilized in writing this article included the National Kidney Foundation Guidelines, the United States Renal Data System, textbooks of medicine and pathophysiology, and medical care and nursing journals. CONCLUSIONS: Early identification of kidney disease in the early stages is essential to preserving kidney function for as long as possible. The progression of chronic kidney disease (CKD) and the manifestations of uremic syndrome leading to end stage renal failure (ESRF) are often not addressed in the literature for nurse practitioners. IMPLICATIONS FOR PRACTICE: Patients with progressing CKD and ESRF often present in the primary care setting for treatment of acute and chronic conditions not pertaining to their renal status (e.g., viral upper respiratory infections, diabetes, hypertension). Nurse practitioners need to be knowledgeable about the subtle early presentation of uremic syndrome and ESRF, risk factors for kidney disease, assessment tools to make the diagnosis and stage the disease, treatment of this disease, as well as psychological, economic, and the social impact that ESRF imposes on individuals, families, communities, and the healthcare system as a whole when the chronic disease has progressed to end stage. PMID- 16681707 TI - Management of chronic hepatitis B virus infection: current perspectives for the nurse practitioner. AB - PURPOSE: To address the clinical management of chronic hepatitis B virus (HBV) infection. DATA SOURCES: Studies from the National Library of Medicine that examine the natural history, prevention, and antiviral therapy of chronic HBV infection, with emphasis on recent studies. CONCLUSIONS: Chronic infection with HBV is a frequent cause of cirrhosis, liver cancer, and liver-related mortality worldwide. Strategies to prevent infection, screen for liver cancer in HBV carriers, and treat chronic hepatitis B are all important in managing this disorder. IMPLICATIONS FOR PRACTICE: Much can be done to prevent and treat infection. Both classes of drugs to treat hepatitis B, nucleoside analogues and interferons, have advantages and disadvantages. Selection of therapy should be based on biochemical, histological, and virological parameters as well as consideration of several practical issues. PMID- 16681708 TI - Effective case presentations--an important clinical skill for nurse practitioners. AB - Effective case presentations are an important component of the nurse practitioner's skills, yet very little literature exists to guide the development of this skill, and frequently little priority is given to teaching this skill during the education of the nurse practitioner. This report discusses the importance of effective case presentations, describes the organization of the presentation, and outlines the appropriate information to be included. The main components of a case presentation-introduction, history of the present illness, physical examination, diagnostic studies, differential diagnosis, management, and summary of the case-are discussed in detail. Examples of a formal and an informal case presentation are presented and used to illustrate key points in the text. PMID- 16681709 TI - Association of parental smoking behaviors and absences in school-age children: implications for the nurse practitioner. AB - PURPOSE: To examine the association between reported absences and parental smoking in school-age children, aged 6-17, and to specifically explore the impact of maternal smoking on the health and attendance of school-age children. DATA SOURCES: The sample of 7488 parent-child dyads was randomly selected from the 2002 National Health Interview Survey, a multipurpose cross-sectional household interview survey conducted by the Centers for Disease Control and Prevention. The parent sample included 2673 men and 4375 women. Children's ages ranged from 6 to 17 with a mean of 11.7 years. CONCLUSIONS: Maternal, but not paternal, present and past smoking behavior significantly impacts the child's wellness and school attendance. The reasons for this disparity are unclear but may relate to synergistic effects of pre- and postnatal nicotine exposure, the traditional role of mother as caregiver, or specific smoking habits that increase environmental tobacco exposure. IMPLICATIONS FOR PRACTICE: Assessment and educational strategies for families regarding the hazards of childhood exposure to environmental tobacco smoke are indicated. Specific implications for the role of the nurse practitioner across diverse specialties are addressed with emphasis on the need for development of gender, age, and culturally sensitive smoking cessation programs and support networks. PMID- 16681710 TI - Prescriptive patterns of nurse practitioners and physicians. AB - PURPOSE: To compare the prescriptive patterns of nurse practitioners (NPs) and physicians in a primary care setting for the treatment of sinusitis, bronchitis, musculoskeletal injury, and back pain. DATA SOURCES: One hundred charts were reviewed for each condition for a total of 400 charts (200 of the charts were for NP providers, and 200 were for physician providers). CONCLUSIONS: While the mean scores of the differences in prescriptive patterns were found to be statistically significant in only one instance, the overall findings indicate slight differences in the administration of medications for patients. Additionally, NPs prescribed more over-the-counter medications and provided more nonpharmacotherapeutic interventions for their patients than the physicians. IMPLICATIONS FOR PRACTICE: The results, although statistically significant in only one category, indicate that NPs may be more cautious in their prescriptive interventions and provide more teaching for patients. PMID- 16681711 TI - Comparison of the traits of physically active and inactive women. AB - PURPOSE: To explore the determinants of physical activity for working women and to compare traits of active with inactive women. DATA SOURCES: Original research articles, books, government reports, and self-report survey data from 373 university employees who attended an employer-sponsored health screening. CONCLUSIONS: Active women had lower body mass indexes and more beneficial high density lipoprotein cholesterol when compared to inactive women. They were more likely to have a past history of sustained physical activity. Significant psychosocial determinants to physical activity included time constraints, confidence in finding an activity that can be enjoyed in bad weather, and not being too tired at the end of the day to engage in physical activity. IMPLICATIONS FOR PRACTICE: Nurse practitioners are in key positions to help women increase their levels of physical activity. Knowing the traits of active women can help busy clinicians focus interventions that address those traits, leading to greater efficiency and effectiveness. PMID- 16681712 TI - Progress in ovarian cancer research: proceedings of the 5th Biennial Ovarian Cancer Research Symposium. AB - Ovarian cancer remains the most lethal gynecological malignancy. The 5th Biennial Symposium overviewed the progress of ovarian cancer research over the last few years. Molecularly based technologies have allowed the identification of multiple biomarkers to aid in ovarian cancer diagnosis and treatment. Furthermore, data analysis systems evaluating the behavior of these markers have been designed. Therapeutic use of ovarian cancer protein markers has been fueled by the development of animal models that more closely simulate the pathogenesis of ovarian cancer, and multiple new therapies are being developed that may have impact against the disease. Finally, the design of clinical trials both for ovarian cancer treatment and prevention are key in advancing the science of ovarian cancer into the clinic. The need for strategies that would optimize patient participation in clinical trials is paramount. PMID- 16681713 TI - Guidelines for gynecological cancer-an audit of current network documents in England and Wales. AB - The objective of this study was to assess the adequacy of network cancer guidelines paying particular attention to referral criteria, referral routes, tumor diagnosis, staging, and suggested management and care pathways for ovarian and endometrial cancer. Guidelines from 15 regions in England and Wales were analyzed quantitatively and qualitatively as a prospective audit of predefined data items and subsequently agreed management recommendations. Details of unit and center clinicians were included in a minority of documents (2 to 5/15). Multidisciplinary team membership was not usually offered (6/15). Among the least reported data items were histopathology minimum dataset for endometrial cancer and an algorithm for management or summary and clinical symptoms and signs for both cancers. Among the most reported data items were hysteroscopy and ultrasound scanning for endometrial cancer and CA125 and chemotherapy for ovarian cancer. Qualitative analysis revealed differing criteria for the use of endometrial biopsy and radiotherapy in endometrial cancer, for lymphadenectomy and management of recurrent disease in ovarian cancer, and for referral pathways and the use of computed tomography/magnetic resonance (MR) scanning in the assessment of either disease. This study concludes that consideration should be given to the development of national guidelines or templates to ensure consistency of management for gynecological malignancy in England and Wales. PMID- 16681714 TI - Symptomatic osteoradionecrosis of pelvic bones in patients with gynecological malignancies-result of a long-term follow-up. AB - The aim of this study was to assess the incidence and risk factors of pelvic fractures as a result of radiation therapy in women with gynecological cancer. We retrospectively reviewed 3530 female patients treated at our institute between 1980 and 1998 with megavoltage radiation with or without brachytherapy for cancer in the pelvic area. Eligible were patients with vulvar, vaginal, cervical, endometrial, and fallopian tube cancer. Median follow-up was 88 months (range 0 240). Emphasis was put on treatment-related and patient-related risk factors. Of the eligible 3155 patients, 15 developed symptomatic bone fracture caused by osteoradionecrosis, which makes an overall incidence of 0.44% The diagnosis was based on anamnesis, clinical course, and X-ray or computed tomography images. Median time of onset was 44 months (range 6-197). All patients had pain as the first symptom. The only independent predictive factor for developing osteoradionecrosis seemed to be preexistent osteoporosis. Other risk factors that are related to osteoporosis include higher age, postmenopausal status, or steroid treatment. We did not find any significant treatment-related predictive factor for pelvic osteoradionecrosis. Patients with osteoporosis are probably at the highest risk for developing osteoradionecrotic fractures after pelvic radiotherapy. More studies are needed to find out other endogenous predictive factors. PMID- 16681715 TI - Long-term survival after paclitaxel plus platinum-based combination chemotherapy for extraovarian peritoneal serous papillary carcinoma: is it different from that for ovarian serous papillary cancer? AB - The purpose of this study was to compare the effect of paclitaxel plus platinum based chemotherapy in the treatment of extraovarian peritoneal serous papillary carcinoma (EPSPC) and ovarian serous papillary cancer (OSPC). Only the patients treated with initial surgery plus postoperative adjuvant chemotherapy and having FIGO stage IIIC disease with omental and/or peritoneal involvement were analyzed. Thirty-two patients with EPSPC and 43 with OSPC were included in this study. The median age, mean CA-125, and volume of ascitis were higher in patients with EPSPC. There was no significant difference between the two groups with respect to other prognosticators. The median overall survival (OS) durations were 30 months (95% CI 24.8-35.3) in patients with EPSPC and 28 months (95% CI 21.1-34.9) in those with OSPC (P= 0.35). The 3-year OS rates in the patients and controls were 28% and 31%, respectively (P= 0.84). In patients with EPSPC, only optimal cytoreduction was significantly related to progression-free survival and OS durations as a prognostic factor. In the EPSPC group, 65.5% of the patients (19/29) had lymphatic involvement, compared to 88.4% (38/43) in the OSPC group (P= 0.02). As an adjuvant therapy, the paclitaxel plus platinum-based combination regimen had similar effects on survival in the EPSPC and OSPC groups. PMID- 16681716 TI - Long-term survival in advanced ovarian carcinoma following cytoreductive surgery with standard peritonectomy procedures. AB - The impact of cytoreductive surgery with standard peritonectomy procedures has not been extensively assessed in the treatment of advanced ovarian cancer. The aims of the study are to report the long-term results of patients with advanced ovarian cancer undergoing cytoreductive surgery with standard peritonectomy procedures and to identify the prognostic indicators that may affect outcome. The records of 74 women with advanced ovarian cancer were retrospectively reviewed. Clinical indicators were correlated to survival. The hospital mortality and morbidity rates were 13.5% and 28.4%, respectively. Complete or near-complete cytoreduction was possible in 78.4% of the patients. Overall 10-year survival rate was 52.5%. Complete cytoreductive surgery, small-volume tumor, low-grade tumor, the absence of distant metastases, the use of systemic adjuvant chemotherapy, performance status >70%, and limited extent of peritoneal carcinomatosis were favorable indicators of survival. Complete cytoreduction (P= 0.000) and treatment with systemic chemotherapy (P= 0.001) independently influenced survival. Recurrence was recorded in 37.8% of the patients and was independently influenced by the tumor grade (P= 0.037). Cytoreductive surgery with standard peritonectomy procedures followed by adjuvant chemotherapy offers long-term survival in women with advanced ovarian cancer who have limited peritoneal carcinomatosis and no distant and irresectable metastases. PMID- 16681717 TI - The utility of presurgical CA125 to predict optimal tumor cytoreduction of epithelial ovarian cancer. AB - The objective of this study was to evaluate the ability of a preoperative serum CA125 to predict whether optimal debulking (OD) could be achieved for patients with stage III and IV epithelial ovarian cancer (EOC). The records of consecutive patients who underwent primary surgery for EOC at Indiana University Hospital between January 1997 and January 2003 were reviewed. Eligibility criteria included FIGO stage III/IV disease, surgery by gynecologic oncology faculty, preoperative CA125, and an operative note clearly defining volume of residual disease. The Medcalc software statistical package was used to generate a receiver operating characteristic (ROC) curve. Two hundred and eighty-nine cases of stage III/IV EOC were identified, of which 164 met the eligibility criteria. Serum CA125 /=75% of the time. Conversely, OD was achieved in /=4500. The area under the ROC curve for CA125 was .670. The OD rate for those with and without ascites was 49% and 79%, respectively (P < 0.001). In a multivariate analysis using CA125, age, and ascites, the area under the curve was 0.686. We conclude that preoperative serum CA125 did not reliably predict OD in patients with stage III-IV EOC. PMID- 16681718 TI - Clinicopathologic study of the putative precursor lesions of epithelial ovarian cancer in low-risk women. AB - Possible precursor lesions for epithelial ovarian cancer (EOC) have been defined in the ovaries of women with contralateral EOC, with breast cancer susceptibility gene (BRCA)-1 mutations, or with positive family history. We aimed to investigate the prevalence of these lesions in women without any recognizable risk and to correlate these lesions with clinical ovulatory markers. The study group consisted of 184 women who were operated for benign gynecological conditions. Patients were requested to fill a questionnaire about anthropometric characteristics and medical and reproductive history. Oophorectomy specimens were examined for presence of epithelial inclusion cysts (EIC), cortical invaginations (CI), stromal hyperplasia (SHPP), epithelial pseudostratification (EPS), and surface papillomatosis (SP). Women with EIC were older, had lower age at menarche, and had higher menarche-to-pregnancy and menarche-to-operation time. SHPP was found to be related with age, menarche-to-operation time, history, and the duration of oral contraceptive use. Women with SP had lower age at menarche, lower menopausal age, and longer duration of hormone replacement therapy. No significant correlations were established between CI and any clinical parameters. Only one patient had EPS. Our findings suggest that these lesions correlate closely with reproductive features. Exact mechanisms that lead to development of these lesions should be clarified before implying them as precursor lesions of EOC. PMID- 16681719 TI - Optimized sequence of drug administration and schedule leads to improved dose delivery for gemcitabine and paclitaxel in combination: a phase I trial in patients with recurrent ovarian cancer. AB - We examined appropriate sequence, schedule, and doses of gemcitabine (G) and paclitaxel (T) in patients with persistent or recurrent epithelial ovarian cancer. Patients received a maximum of six cycles of gemcitabine on days 1 and 8 (starting 1000 mg/m(2)), and paclitaxel (starting 135 mg/m(2)) on day 8 (groups A and B) or day 1 (group C). Drug sequences (G-->T and T-->G) were tested in group A. In group A, changing sequences of gemcitabine and paclitaxel infusion were evaluated. Sequence G-->T raised grade 3 alanine transaminase in two of three patients leading to use of T-->G sequence for remainder of study. In group B, maximum tolerable dose was reached at gemcitabine 1000 mg/m(2) and paclitaxel 175 mg/m(2). Reducing paclitaxel to 150 mg/m(2) allowed escalation of gemcitabine to 1250 mg/m(2), but neutropenia-related treatment delays occurred. Giving paclitaxel on day 1 (group C) enabled administration of paclitaxel 175 mg/m(2) and gemcitabine 1250 mg/m(2) with minimal dose adjustments. The overall response rate was 41.0%, with 2 complete responses and 14 partial responses in 39 eligible patients. The schedule of paclitaxel 175 mg/m(2) (day 1) and gemcitabine 1250 mg/m(2) (days 1 and 8), with sequence of T-->G, appears most suitable with tolerable toxicity and promising activity. PMID- 16681720 TI - Unfavorable prognostic value of CD24 expression in sections from primary and relapsed ovarian cancer tissue. AB - Expression of CD24 represents a poorly recognized, unfavorable prognostic factor. Expression of the protein is supposed to facilitate extravasation of tumor cells. Our study aimed at examination of prognostic significance of CD24 estimation in samples obtained from primary surgeries (PS) and secondary cytoreductions (SCR) (after chemotherapy) in ovarian cancer patients. The analyses were performed on sections originating from 73 tumor samples. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD24. Kaplan-Meier's analysis showed that a significantly shorter overall survival time and progression-free time was demonstrated to characterize cases with cytoplasmic membranous expression of CD24 (CD24c-m) (P < 0.001). The calculations performed demonstrated also a significantly higher proportion of CD24c-m positive cases in sections from SCR as compared to that from PS (P= 0.04) and in cases of progressive disease as compared to complete response at PS and SCR (P= 0.002 and P= 0.05, respectively). Summing up, in this study, we have demonstrated a negative prognostic significance of a cytoplasmic membranous expression of CD24 in cases of ovarian cancer. PMID- 16681721 TI - Suppression of human ovarian carcinoma metastasis by the metastasis-suppressor gene, BRMS1. AB - Metastasis-suppressor genes, by definition, suppress metastasis without affecting tumorigenicity and, hence, present attractive targets as prognostic or therapeutic markers. BRMS1 (breast cancer metastasis suppressor) has recently been identified as a metastasis-suppressor gene for human breast cancer and melanoma. Expression of BRMS1 messenger RNA (mRNA) in multitissue including normal prostate, ovarian, testis, and colon has been detected by northern blot analysis. We hypothesize that the role of BRMS1 in tumor progression may not be limited to breast cancer and melanoma. We previously found that BRMS1 mRNA levels in primary ovarian epithelial carcinomas were significantly lower than that in normal ovarian and benign tumors (P < 0.05), and statistical analysis of BRMS1 mRNA levels revealed that BRMS1 mRNA levels were significantly higher in early tumor stages (I, II) compared with advanced tumor stages (III, IV) in which lymph node or distant metastases were present (P < 0.01). Our data showed that reduced BRMS1 mRNA seems to influence ovarian carcinoma metastatic ability. Therefore, we transfected BRMS1 plasmid into highly malignant ovarian carcinoma cell line, HO 8910PM, and examined cell biologic behaviors including proliferation, adhesion, invasion, and metastasis in vitro and in vivo. BRMS1 expression did not alter the proliferation of HO-8910PM cells in vitro and primary tumor formation in vivo. But, BRMS1 expression significantly suppressed the cell adhesion to extracellular matrix components and in vitro cell invasion in BRMS1-transfected HO-8910PM cells compared to parental HO-8910PM and vector-only transfectants (HO-8910PM-vect). Furthermore, motility of BRMS1 transfectants was inhibited. lung colony formation of intravenously injected BRMS1 transfectants in nude mice was significantly reduced. Also, BRMS1 transfectants form significantly less metastatic to organs of peritoneal cavity in orthotopically implanted ovarian tumor nude models. We further discovered that BRMS1 expression did downregulate expression of an actin bundling protein associated with cell motility -fascin, which perhaps is the mechanism underlying BRMS1 suppression of metastasis. These data suggested that in addition to its already described role in breast cancer and melanoma, BRMS1 functions as a metastasis-suppressor gene in ovarian carcinoma by modifying several metastatic-associated phenotypes, offering a new target for therapeutic intervention. PMID- 16681722 TI - Arsenic trioxide-loaded, microemulsion-enhanced cytotoxicity on MDAH 2774 ovarian carcinoma cell line. AB - The antiproliferative effect of As(2)O(3)-loaded microemulsion (As(2)O(3)-M) on human MDAH 2774 ovarian cancer cells was compared with a regular solution of the As(2)O(3). We used MDAH 2774 as model cell lines for ovarian cancer. The (2,3 bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) (XTT) and trypane blue dye exclusion tests were used to evaluate cytotoxicity. Apoptotic effect of solutions was evaluated using cell death detection kit. Standard microemulsion formulation used in this experiment contains 5 x 10(-6) M As(2)O(3). It was clearly demonstrated that As(2)O(3)-M had a significant cytotoxic effect on MDAH 2774 cell line, and the cytotoxic effect of As(2)O(3)-M was significantly higher than that of regular As(2)O(3) solutions. Even approximately 6000 times diluted microemulsion formulation loaded with 5 x 10(-6) M As(2)O(3) showed a cytotoxic effect. As a result, this diluted concentration (approximately 8 x 10(-10) M) was found to be approximately 6000 times more effective than regular As(2)O(3) solutions (5 x 10(-6) M). Moreover, this diluted concentration resulted in 1.5-fold enhancement of apoptosis. According to the in vitro cytotoxicity studies, we concluded that by incorporating As(2)O(3) into the microemulsion (As(2)O(3)-M), which is a new drug carrier system, it is possible to increase antiproliferative effect of regular As(2)O(3) on MDAH 2774 cells. Translating these results to in vivo conditions would open new windows in the treatment of ovarian cancer. PMID- 16681723 TI - Synergistic antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand combined with cisplatin in ovarian carcinoma cell lines in vitro and in vivo. AB - Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert selectively cytotoxic activity against many tumor cells but not normal cells. In this study, we evaluated the antitumor activity of TRAIL and cisplatin (CDDP) both separately and combined in the human ovarian cancer cell lines. In vitro study showed that TRAIL elicited significant cell apoptosis of cell lines 3AO, SKOV3, and OVCAR3 in a dose- and time-dependent manner (P < 0.05), while normal ovarian epithelial cells were resistant; this toxicity-free effect may be the result of upregulation of TRAIL receptors DcR1 and DcR2. Combined TRAIL and CDDP therapy produced more profound cell killing in 3AO cells than each alone (P < 0.05), and CDDP could upregulate the expression of both death and decoy TRAIL receptors. To further evaluate the apoptosis-inducing effects of TRAIL and the combination therapy, the abdominally and subcutaneously spread tumors in nude mice via inoculation of 3AO cells were established, and treatment of TRAIL resulted in a dose- and time-dependent inhibition of tumor growth while slight damage was observed in normal tissues. Furthermore, combined TRAIL and CDDP therapy had a synergistic effect in the regression of established ovarian cancer xenografts than TRAIL treatment alone (P < 0.05). We also examined the apoptosis related gene expression in the transplantation tumors after TRAIL treatment, and the data suggested that the intracellular mechanism of TRAIL may be associated with downregulation of Bcl-2 and upregulation of CD95 and Apo2.7. PMID- 16681724 TI - Expression of cyclooxygenase-2 and inducible nitric oxide synthase in ovarian surface epithelial carcinomas: is there any correlation with angiogenesis or clinicopathologic parameters? AB - Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) products have been implicated in the regulation of immune system, tumor cell apoptosis, and angiogenesis in many human tumors. In this study, we investigated the expression of COX-2 and iNOS in ovarian carcinomas by immunohistochemistry and correlated the results with other prognostic parameters. Specimens from 100 ovarian carcinomas were studied by immunohistochemistry for COX-2 and iNOS expression, and angiogenesis microvessel density (MVD) was evaluated by CD34-stained microvessels. High COX-2 expression was observed in 85% of carcinomas. No correlation was found between COX-2 expression and clinicopathologic variables. Patients with high COX-2-expressed tumors had shorter overall survival, but it is not statistically significant. Expression of iNOS in serous and low-grade carcinomas was significantly higher than that in nonserous and high-grade carcinomas (P < 0.05). There was a positive correlation between COX-2 and iNOS expression (P= 0.009). No correlation of COX-2 and iNOS expression with MVD was found. Expression of iNOS showed no effect on survival of the patients. We found that iNOS expression might act in the first steps of carcinogenesis, whereas COX 2 expression was seen in more advanced tumors. Shorter overall survival of patients with high COX-2 expression might indicate new targets for therapy. PMID- 16681725 TI - Computerized nuclear morphometry for the prediction of inguinal lymph nodes metastases in squamous cell carcinoma of the vulva. AB - This study evaluated the sensitivity and specificity of computerized morphometry in predicting lymph nodes metastases (LNM) in patients with squamous cell carcinoma (SCC) of the vulva. Histologic samples obtained from 20 consecutive cases of SCC of the vulva with positive inguinal LNM were morphometrically assessed and compared with samples from 20 consecutive cases of vulvar SCC negative for LNM. Computerized morphometry was performed on tumor cells and on adjacent nonneoplastic epithelial cells located 2-4 mm from the tumor margins. Computerized morphometric variables of tumor cell nuclei in patients with negative LNM significantly differed from those in patients with positive LNM. Morphometric differences in nuclear size and contour regularity were detected when comparing the nonneoplastic nuclei adjacent to the tumor of both groups. Multivariate analysis showed that the only independent predictors of LNM were the depth of the invasion (P= 0.005) and the mean nuclear roundness of the nonneoplastic nuclei adjacent to the tumors (P= 0.008). Using these variables, a discriminant score revealed a sensitivity of 90% and a specificity of 86.4% for predicting LNM in SCC of the vulva. Our data suggest that cells from the primary tumors with LNM differ morphometrically from primary tumors with no LNM. In addition, normal epithelial cells adjacent to the tumor express morphometric changes between the two groups. The results of our study justify the need for a prospective study of a larger number of patients to evaluate the reproducibility and the clinical use of the data. PMID- 16681726 TI - Gene expression patterns in advanced human cervical cancer. AB - The purpose of this study was to evaluate gene expression patterns in human cervical tumors by extent of lymph node metastases at diagnosis. Pretreatment whole-body fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging was performed in eight patients with invasive squamous cell carcinoma of the cervix to evaluate the extent of lymph nodes metastases. Pretreatment tumor tissue samples were subjected to laser-capture microdissection, and isolated RNA was linearly amplified and hybridized to Affymetrix Human U95A GeneChip microarrays. Molecular FDG-PET imaging revealed that three patients had lymph node involvement in the supraclavicular region and five patients did not. Microarray data were segregated into two groups based on the extent of regional lymph node involvement. Supervised clustering analysis identified 75 of about 12,000 gene transcripts represented on the array whose average expression was at least threefold different. We identified 12 of the 75 transcripts that demonstrated a statistically significant difference in expression between the two patient groups (P < 0.05). Five transcripts were upregulated and seven downregulated. Both overall and cause-specific survivals were different between these two patient groups (P= 0.006). This limited data set identified candidate biomarkers of extent of lymph node metastases that correlated with poor survival outcome. PMID- 16681727 TI - Risk factors of having high-grade cervical intraepithelial neoplasia/invasive carcinoma in women with atypical glandular cells of undetermined significance smears. AB - The objective of this study was to find the risk factors of having high-grade cervical intraepithelial neoplasia/invasive carcinoma in women with atypical glandular cells of undetermined significance (AGUS) smears. A retrospective study of the women with AGUS smears during the 3-year period was performed to determine the correlation between the suspected variables and the histopathologic diagnoses. Among 44,071 smears performed, 119 (0.27%) smears were reported as AGUS. Colposcopies were performed in 102 (88.7%) cases, and high-grade cervical intraepithelial neoplasia/invasive carcinoma was found in 18 (17.6%) cases. Among the following variables, which included age, menopausal status, hormonal contraception, history of previous Pap smears, medical diseases, clinical symptoms, and subclassification of AGUS, both hormonal contraception and AGUS favor neoplasia were risk factors with an odds ratio of 5.4 and 5.0, respectively. Although clinical symptoms seemed to be a significant variable in univariate analysis, it appeared as a confounding factor in multivariate analysis. PMID- 16681728 TI - Angiogenesis in cervical intraepithelial neoplasia and early-staged uterine cervical squamous cell carcinoma: clinical significance. AB - The objective of this study is to evaluate angiogenesis in cervical intraepithelial neoplasia (CIN), microinvasive squamous cell carcinoma (MIC), and early-staged squamous cell carcinoma (SCC), stage IB-IIA of the cervix. Microvessel density (MVD) was evaluated and correlated with other pathologic prognostic factors and disease outcomes. Four hundred seventy-four cervical specimens were studied. Among these, 100 were designated normal cervix, 30 CIN1, 32 CIN2, 178 CIN3, 74 MIC, and 60 early-staged SCC. MVD per high-power field (x400) of early-staged SCC, MIC, and CIN3 were significantly higher in comparison to CIN2, CIN1, and control subjects (P<0.05). There was no statistically significant difference in MVD between control group, CIN1, and CIN2. In early staged SCC, no correlation between MVD and pelvic lymph node status, parametrial involvement, depth of stromal invasion, and lymphovascular space invasion was found. Patients with bad outcomes (recurrence or death) showed no statistically different MVD from the ones who had unremarkable clinical courses. PMID- 16681729 TI - Angiomyofibroblastoma of the vagina in a postmenopausal breast cancer patient treated with tamoxifen: clinicopathologic analysis of a case and review of the literature. AB - Angiomyofibroblastoma is a rare mesenchymal tumor. This study presents the clinical, histologic, and immunohistochemical features of an angiomyofibroblastoma of the vagina occurring in an 80-year-old breast cancer patient under prolonged treatment with tamoxifen. Histologically, the tumor was characterized by alternating hypercellular and hypocellular edematous zones and small- to medium-sized blood vessels, which were characteristically thin walled. The tumor cells were spindle shaped (mainly) or round shaped (occasionally) arranged in cords and nests. The stroma was edematous and contained inflammatory cells, especially lymphocytes and mast cells. Immunohistochemistry of the tumor cells revealed diffuse and intense immunoreactivity for vimentin and desmin. The staining for estrogen receptors and progesterone receptors was positive, with a percentage of 70% and 40%, respectively. In conclusion, the tumor was diagnosed as an angiomyofibroblastoma based on its typical histologic and immunohistochemical features. The expression of estrogen and progesterone receptors suggests that it might arise as a neoplastic proliferation of hormonally responsible mesenchymal cells. Tamoxifen may exert stimuli effects upon mesenchymal cells. PMID- 16681730 TI - Investigation of human papillomavirus by hybrid capture II in cervical carcinomas including 113 adenocarcinomas and related lesions. AB - Hybrid capture is an easy and highly sensitive technique for screening population due to its capacity to detect malignant and premalignant lesions of the cervix. To evaluate its sensitivity, we investigated the frequency of high-risk human papillomavirus (HPV) infection and its correlation with glandular malignant lesions, analyzing a total of 113 cases of adenocarcinomas and related lesions. High-risk HPV was investigated using a hybrid capture II (HC2) assay. Samples were collected in two different ways: either brushed directly from surgical specimens before fixation or collected from the patients. We also investigated the frequency of HPV in squamous malignant lesions, 65 squamous cell carcinomas (SCC) and 66 in situ squamous cell carcinomas (ISSCC), to compare the occurrence of HPV for these lesions. The 113 glandular lesions comprised 62 invasive adenocarcinomas (IAC), 8 in situ adenocarcinomas (ISAC), 26 IAC plus SCC, and 17 adenosquamous cells carcinomas (ASCC). The HPV-positive reactions were as follows: 51 (82.2%) in IAC, 8 (100%) in ISAC, 25 (96.1%) in IAC plus SCC, and 14 (82.3%) in ASCC. HC2-positive results in the squamous malignant lesions were as follows: 58 of 63 (89.0%) for SCC and 94 of 103 (91.2%) for ISSCC. High-risk HPV infection was quite similar for glandular and pure squamous invasive malignant lesions, 82.2% and 89.0%, respectively, indicating that high-risk HPV is also highly prevalent in glandular lesions. Although hybrid capture proved to be an excellent adjunctive technique, we do not believe its results merit replacing the Pap smear as a screening tool. PMID- 16681731 TI - Human papillomavirus testing improves the accuracy of colposcopy in detection of cervical intraepithelial neoplasia. AB - To assess the performance of human papillomavirus (HPV) testing and colposcopy in detection of cervical pathology. A series of 389 women referred for colposcopy due to an abnormal Pap smear had cervical swabs analyzed for oncogenic (high-risk [HR]) HPV types using Hybrid Capture II (HC2) assay. Loop electrical excision procedure cone biopsy (88%) or colposcopic biopsy (11%) was used as the gold standard. Of the atypical squamous cells of undetermined significance (ASCUS) smears, 48% were positive for HR HPV, as compared to 76.3% of low-grade squamous intraepithelial lesions (LSIL) smears. HR HPV was detected in 66.7% and 90% of patients with cervical intraepithelial neoplasia (CIN) 1 and CIN2 (or higher), respectively. The sensitivity of the Pap smear using an ASCUS threshold in detecting high-grade CIN was 94.5% (95% confidence intervals (CI): 91-97%) and that of colposcopy 98.5% (95% CI: 95-99%). The respective specificities were 30% (95% CI: 17-28%) and 35.6% (CI: 29-42%). HC2 test had comparable sensitivity, 90% (95% CI: 85-93%), but higher specificity, 54.3% (95% CI: 47-61%). Combining HC2 test with Pap increased specificity, 66.7% and 41.3% for ASCUS and LSIL cutoff, respectively. The minor-abnormality threshold together with HC2 increased specificity of colposcopy with no changes in sensitivity. High viral load (>100 relative light unit/positive control) was associated with significant disease. HPV DNA testing improves the accuracy of colposcopy in the detection of high grade CIN in women with ASCUS or LSIL smears. PMID- 16681732 TI - Knowledge and attitudes about human papillomavirus, Pap smears, and cervical cancer among young women in Brazil: implications for health education and prevention. AB - The objective of the study was to assess knowledge and attitudes about human papillomavirus (HPV), cervical cancer, and Papanicolaou (Pap) smears among young women. A questionnaire was administered to 204 women aged 16-23 years, attending a public clinic. Data were gathered on sociodemographic characteristics, knowledge, and attitudes related to HPV. Overall, 92% of women reported current/previous sexual activity, 42% perceived themselves at high risk of acquiring a sexually transmitted disease, 67% did not know that HPV can cause cervical cancer/warts, and only 10% acknowledged that HPV might lead to cervical cancer. In general, women had a poor knowledge on HPV diagnosis/treatment, condyloma signs, and Pap smear test. The main reasons for not having a Pap smear test done before were embarrassment (63%) and fear of pain (61%). Knowledge of HPV infection and cervical cancer was low in this urban young population. Our findings recommend for greater HPV education of the public and health care practitioners. PMID- 16681734 TI - Correlation between MRI and histopathologic findings in stage I cervical carcinomas: influence of stromal desmoplastic reaction. AB - Although the effectiveness of magnetic resonance imaging (MRI) in depicting cervical carcinoma has been reported, whether MRI can detect early-stage or stage IB "occult"-type cervical carcinoma remained undetermined. We examined the correlation between MRI and pathologic findings in 38 stage I (IB 28 cases, IA 10 cases) cervical carcinoma patients, with special reference to the influence of desmoplastic stromal reaction around the tumor. The results demonstrated that the tumor was detected by MRI in none of stage IA patients but in 21 (75%) stage IB patients. The image was clearly demonstrated in 15 of 18 (83%) tumors of more than 2 cm in diameter and in 6 of 10 (60%) tumors of 2 cm or less. The tumor image was evident in 21 of 22 (95%) tumors with prominent (>200 micron) stromal reaction but in none of 6 tumors with minimal (50%, and lymph node involvement were important prognostic factor responsible for survival. Neoadjuvant chemotherapy followed by surgery seems to be tolerated and active in the treatment of locally advanced cervical cancer and might be an alternative choice of therapy to chemoradiation. A prospective randomized trial with a larger number of cases is needed. PMID- 16681739 TI - Hyperbaric oxygen therapy for delayed radiation injuries in gynecological cancers. AB - Delayed radiation-induced injuries are difficult to treat. The treatment of delayed radiation injuries with hyperbaric oxygen therapy (HBOT) is reported in small case series and case reports. This study reports the experience of a single institution with HBOT in delayed radiation injuries in patients with gynecological cancers. At least 20 sessions of 100% oxygen inhalation at 2.4 Atmospheric Absolutes (ATA) for 90 min in a hyperbaric chamber were carried out. Of the 14 patients included in the study, 10 patients have healed or showed improvement of more than 50%, resulting in a success rate of 71%. Mean follow-up was 31.6 months (range 6-70 months). The adverse events were acceptable. HBOT should be considered for patients with delayed radiation injuries, not responding to other treatments. PMID- 16681740 TI - Micrometastases detected by cytokeratin 19 expression in sentinel lymph nodes of patients with early-stage cervical cancer. AB - The purpose of this study was to detect micrometastases in sentinel lymph nodes (SLNs) by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) analyses for cytokeratin 19 (CK19) expression in early stage cervical cancer. One hundred twenty-five SLNs were collected from 46 patients with early-stage cervical cancer. Conventional histopathologic techniques revealed 14 metastatic SLNs from 11 out of 46 patients. CK19 expression was detected by RT-PCR and IHC in all the 125 SLNs. Cervical cancer tissues from nine patients and five pelvic lymph nodes from the patients without tumor were utilized as positive and negative controls, respectively. All the metastastic SLNs on conventional histopathologic techniques were positive by either RT-PCR or IHC analyses, while all the positive controls were positive and all the negative controls were negative as expected. Of 35 patients without metastatic SLNs on conventional histopathologic techniques, the detection rate of micrometastases was 42.85% by RT-PCR and 20% by IHC analyses. RT-PCR and IHC were more sensitive to identify micrometastases in SLNs of patients with early-stage cervical cancer than routine pathology. These findings demonstrated that micrometastasis could be identified by molecular technique such as RT-PCR and IHC analyses for CK19 expression. RT-PCR was more sensitive to detect micrometastases in SLNs than IHC in patients with early-stage cervical cancer. Therefore, molecular assessment of the SLNs may be a valuable tool to complement routine histologic examination of cervical cancer. The importance of micrometastases in SLNs is under close clinical observation to determine whether it can be used as a predicting factor to help us make decision whether to proceed with whole-pelvic lymph node dissection or as a prognostic factor for clinical outcome. PMID- 16681741 TI - Sentinel node identification in cervical cancer patients undergoing transperitoneal radical hysterectomy: a study of 100 cases. AB - We investigated the feasibility of sentinel lymph node (SN) identification using radioisotopic lymphatic mapping with technetium-99m-labeled nanocolloid and blue dye injection in 100 patients with early cervical cancer (FIGO stage IB1 in 58, IB2 in 18, and IIA in 24) undergoing radical hysterectomy with pelvic lymphadenectomy. At least one SN was found in 84% on one side and in 66% on both sides. The sentinel detection rates according to the stages were as follows: 96.6% in IB1, 66.7% in IB2, and 62.5% in IIA with at least one SN on one side, and 86.2% in IB1, 38.9% in IB2, and 37.5% in IIA with at least one SN on both sides. Successful identification of at least one SN was less likely in patients with tumors >2 cm (54% of SN) compared with those with tumors 1 mm (P= 0.014). Cox proportional hazards analysis revealed positive cone margins for invasive carcinoma as significant predictor of residual invasive disease (hazard ratio, 3.22; 95% CI 1.21-8.60, P= 0.019) In summary, patients with MIC and positive cone margins for high-grade lesions or invasive carcinoma are at high risk of residual neoplasia. Repeat cone biopsy should be performed to determine exactly the severity of lesion before planning treatment. PMID- 16681743 TI - Human immunodeficiency virus testing in patients with invasive cervical carcinoma: a prospective trial of the gynecologic oncology group. AB - To determine the frequency of positive human immunodeficiency virus (HIV) serostatus among North American women 50 years of age or younger with invasive cervical cancer and to define their tolerance to treatment. Consenting patients with newly diagnosed invasive cervical cancer, age 50 or younger were tested by enzyme-linked immunosorbent assay. The study design anticipated that approximately 3% of patients would be HIV positive. After the accrual of 913 eligible and evaluable patients, interim analysis revealed that only 9/913 ( approximately 1%) patients were HIV seropositive, indicating that it would not be feasible to achieve the study objective. The study was closed to further accrual. Between 1994 and 1997, the frequency of positive HIV serostatus among North American women with newly diagnosed cervical cancer was quite low. As a consequence, no evaluation of response to treatment or treatment tolerance can be made. PMID- 16681744 TI - Analyses of atypical squamous cells refined by the 2001 Bethesda System: the distribution and clinical significance of follow-up management. AB - This study was conducted to analyze the clinical significance of follow-up diagnostic methods of atypical squamous cells (ASC) (the 2001 Bethesda System) cases according to age. A computerized search of the cytology database was performed to retrieve all cases diagnosed as ASC from 2001 to 2003. The pathologic reports for all follow-up diagnoses were reviewed. We divided the patients into two groups according to their age, younger than 50 years of age and 50 years and older, and follow-up diagnoses were compared between the two groups. ASC was identified in 1035 (2.0%) of 49,882 women screened, and a total of 914 patients were eligible. In atypical squamous cells of undetermined significance (ASC-US) cases, colposcopically directed biopsy showed CIN I (CIN is cervical intraepithelial neoplasia) or higher grade lesions in 34.9% of cases younger than 50 years of age and in 17.4% of cases 50 years and older (P= 0.000). However, repeat Pap smears and human papillomavirus DNA testing showed no differences between the two groups. In contrast, the three methods did not exhibit significant difference between the two groups in patients with atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) (P= 0.743). Colposcopically directed biopsy for the ASC-US was more useful in patients younger than 50 years of age than in those who were 50 years and older. It is suggested that age should be considered in deciding follow-up diagnostic methods in patients with ASC-US. PMID- 16681745 TI - Radiation-induced benign glandular cells in posthysterectomy smears: a cytomorphologic and clinical analysis. AB - In this retrospective study, we aimed to investigate the frequency and cytomorphologic characteristics of benign glandular cells (BGCs) in hysterectomized individuals. We also discussed the possible effect of radiation therapy on these cells. We reviewed our cytopathology archive material through a 5.5-year period and found 1460 posthysterectomy vaginal smears. Of these, 508 smears were from patients who had undergone hysterectomy for a gynecological malignancy. Review of this vaginal cytology material revealed 17 posthysterectomy patients whose smears contained BGCs. We obtained detailed clinical information in 16 of these. In addition to routine Papanicolaou staining, mucicarmine stain was also used to demonstrate cytoplasmic mucin in some cases. All the patients had a history of gynecological malignancy and had radiation therapy. Glandular cells appeared singly or in rows and honeycomb groups and did not show cytologic atypia. We concluded that radiation might give rise to a metaplastic process in which basal cells of squamous epithelium of the vagina transform into glandular cells. Most probably this process is independent of radiation dosage and period and is irreversible. We also propose that the possibility of encountering glandular cells in posthysterectomy smears is higher than expected, if the mucin stains have been used for the microscopic examination. PMID- 16681746 TI - Female circumcision (female genital mutilation): a problem for brachytherapy in cervical cancer. AB - Female circumcision is a traditional practice common in African countries. It involves partial or total removal of external female genitalia. It has led to many complications, in particular, the scarring of the external genitalia. The consequence is a very narrow introitus making the intracavitary brachytherapy treatment component difficult when these women develop cancer of cervix. We present two such cases from our institution. Our aim is to make the radiation and gynecological oncologists, both in developed and developing countries, aware of this practice and the problems they can encounter in the management of such cases. Intracavitary brachytherapy is an important component in the potentially curative role of radiation therapy for cervical cancer. Every effort should be made to ensure that the sequelae of genital mutilation does not deprive these women of the same standard of care as the general population. PMID- 16681747 TI - Acceptability of human immunodeficiency virus testing in patients with invasive cervical cancer in Kenya. AB - Invasive cervical cancer (ICC) is common in areas where human immunodeficiency virus (HIV) is also prevalent. Currently, HIV seroprevalence as well as acceptability of HIV testing in ICC patients in Kenya is unknown. The objective of this study was to determine the acceptability of HIV testing among patients with ICC. Women with histologically verified ICC at Kenyatta National Hospital participated in the study. A structured questionnaire was administered to patients who gave informed consent. HIV pre- and posttesting counseling was done. Blood was tested for HIV using enzyme-linked immunosorbent assay. Overall, 11% of ICC patients were HIV seropositive. The acceptance rate of HIV testing was 99%; yet, 5% of the patients did not want to know their HIV results. Patients less than 35 years old were two times more likely to refuse the result of the HIV test (odds ratio [OR] 2.2). Patients who did not want to know their HIV results were three times more likely to be HIV seropositive (OR 3.1). Eighty four percent of the patients were unaware of their HIV seropositive status. The HIV-1 seroprevalence in ICC patients was comparable to the overall seroprevalence in Kenya. ICC patients were interested in HIV testing following pretest counseling. Offering routine HIV testing is recommended in ICC patients. PMID- 16681748 TI - Multinucleation of koilocytes is actually multilobation. AB - In order to clarify the complete cytomorphology of cytopathic changes as a consequence of human papillomavirus (HPV) infection, we performed three dimensional (3D) reconstruction from confocal fluorescent images. After confirming 22 HPV types using a DNA chip, we performed 3D confocal image restoration in human uterine cervical swab samples and corresponding tumor tissues. On restoration of 3D confocal images, the multinucleated feature of koilocytes was revealed to be multilobation of a single nucleus, as opposed to true multinucleation. PMID- 16681749 TI - Analysis of differential protein expression by cisplatin treatment in cervical carcinoma cells. AB - Cisplatin (cis-diaminedichloroplatinum), a DNA-damaging agent, which readily induces apoptosis in vitro, is one of the widely used anticancer drug in the treatment of human malignancies. Cisplatin has played an important role in cervical cancer management for effective chemotherapeutic regimen, but the underlying mechanisms inducing cell death at protein level are unknown. Using proteome analysis, an investigation aimed at a better understanding of the antiproliferative mechanisms by cisplatin was carried out in HeLa cervical carcinoma cells. In total, 21 protein spots were found to be differentially expressed following cisplatin treatment, of which 12 were upregulated (eg, regulator of G-protein signaling, TRAF:TNF (tumor necrosis factor) receptor associated factor-interacting protein [I-TRAF], and cyclin-dependent kinase inhibitor p27 [p27(kip1)]) and 9 were downregulated (eg, myc proto-oncoprotein [c myc] and proliferating cell nuclear antigen). Interestingly, we found the upregulation of proliferating cell nuclear antigen, which used molecular marker in cervical cancer screening. On the basis of proteomic data, we showed that cisplatin induced TRAF2-mediated NF-kappaB downregulation. In addition, our study demonstrated that cisplatin induced membrane death receptor-mediated and mitochondria-mediated apoptosis pathway. Our findings may offer new insights into the antiproliferative mechanism by cisplatin and its mode of action in cervical carcinoma cells. PMID- 16681750 TI - Distinctive cell cycle regulatory protein profiles by adenovirus delivery of p53 in human papillomavirus-associated cancer cells. AB - In this study, microarray analyses were performed to determine the time course of gene expression profiles in SiHa cells after infection with an adenovirus expressing p53 (Adp53). We then investigated the consequences of Adp53 gene transfer on the expression level of six genes associated with cell cycle control and on apoptosis and cell cycle arrest in SiHa cells and compared these results with those from CaSki and HeLa cells. Gene expression profiling of the p53 targeted genes in SiHa cells revealed that p21, p53, and mdm2 protein expression was significantly upregulated at 24 and 48 h. Western blot results revealed that p21 and p53 expression levels had significantly increased after Adp53 infection. Cyclin-dependent kinase 4 levels were decreased 48 h after treatment in SiHa and CaSki cells. Proliferating cell nuclear antigen levels were unchanged after Adp53 infection. Only SiHa cells exhibited significant cell death. Cell cycle arrest at the G1 phase was induced in the SiHa and HeLa cells but was not induced at the G2/M and S phases in the CaSki cells. These data support the notion that the understanding of p53-dependent apoptosis and cell growth arrest could be applicable to advanced strategies in the development of preferential tumor cell specific delivery. PMID- 16681751 TI - Phenotypic features with p53 alterations related to human papillomavirus and prognostic evaluation in cervical cancer. AB - Cervical cancer is one of the most common tumor affecting women worldwide. Human papillomavirus (HPV) was found to have a causal relationship with cervical cancer and its precursors. The interaction between HPV E6 protein and p53 was identified in in vitro studies. The aim of the study was to evaluate the prevalence of p53 alterations related to HPV infection and the prognostic significance of p53 alterations in cervical cancer. Studies were identified by a MEDLINE search, and all relevant articles were retrieved from 1991 to March 2004. The prevalence of p53 mutations is a rare event in cervical cancer. The correlation between p53 mutations and HPV or prognosis is controversial. Loss of heterozygosity (LOH) of p53 is more commonly found in cervical cancer and is related with the prognosis of this disease. There is no significant correlation between p53 polymorphism and development of cervical cancer. The p53 mutations were not commonly found in cervical cancer. LOH of p53 may contribute to the progression of this malignancy. p53 polymorphism failed to be an independent prognostic factor in predicting the outcome of patients with cervical cancer. Further, epidemiologic surveys should be undertaken in larger populations and in different geographical regions. PMID- 16681752 TI - Extended effects of human papillomavirus 16 E6-specific short hairpin RNA on cervical carcinoma cells. AB - Most cervical carcinomas express high-risk human papillomavirus (HPV) E6 and E7 oncogenes. Small interfering RNA can mediate sequence-specific inhibition of gene expression in mammalian cells. To find a most effective short hairpin RNA (shRNA) for HPV16 E6 messenger RNA (mRNA) and investigate the extended effects of the HPV16 E6 shRNA on cervical carcinoma cells, we stably transfected SiHa cells with four shRNA expression vectors (E6A-D). HPV16 E6A shRNA was found to be the most efficient in our study, which caused the reduction of HPV16 E6 mRNA to 10% in SiHa cells but did not reduce HPV18 E6 mRNA expression in HeLa cells. We subsequently demonstrated that E6A could stably express shRNA and effectively reduce HPV16 E6 and E7 viral genes expression in SiHa cells for more than 4 months. After E6 and E7 repression, there was a dramatic accumulation of p53, p21, and hypophosphorylated pRb proteins in cells. Furthermore, cell proliferation, colony formation ability, tumorigenicity, and in vitro cell invasive capability were suppressed substantially in E6A-transfected cells. These results suggest that the use of shRNA expression vector may be a potential approach for the treatment of persistent HPV infection and HPV-positive cervical carcinoma. PMID- 16681753 TI - Differential viral loads of human papillomavirus 16 and 58 infections in the spectrum of cervical carcinogenesis. AB - Human papillomavirus (HPV) load was reported to be related to the severity of cervical neoplasia but with controversy. The viral load-disease severity relationship was showed in HPV 16, but no study was made in HPV 58, the second most prevalent HPV in cervical cancer in East Asia. We studied cervical HPV loads in HPV 16- and HPV 58-infected cases of normal, low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and invasive cervical cancer (CC) by using quantitative polymerase chain reaction (Q-PCR) with type-specific primers in defined cell number. With the exception of HPV 16 infection in normal, viral loads varied greatly in each disease regardless of genotypes. The load of HPV 16 differed significantly among disease severities, with a dramatic increase from normal (1.14 +/- 2.25 copies/cell) to LSIL, HSIL, and CC (1599 +/- 2301, 7489 +/- 24,087 and 1878 +/- 2979 copies/cell, respectively) (P < 0.01). No significant difference was noted among different HPV 58 infections, with loads in normal, LSIL, HSIL, and CC of 503 +/- 641, 7951 +/- 27,557, 353 +/- 744, and 1139 +/- 2895 copies/cell, respectively. In comparison with HPV 16, HPV 58 subclinical infection confers a significant higher load (P < 0.01). Different HPV types behave differentially in the spectrum of cervical carcinogenesis. Unlike HPV 16, the infection load of HPV 58 does not correlate to the clinical severity. The wide variation of HPV loads among different HPV types and among squamous intraepithelial lesions and CC makes the viral load test unrealistic in differentiating different severities of cervical neoplasia. PMID- 16681754 TI - Molecular variants of human papillomavirus type 16 and risk for cervical neoplasia in South Africa. AB - Non-European variants of human papillomavirus (HPV) type 16 are generally associated with a greater risk of cervical neoplasia than European prototype variants. We investigated whether this association would persist in a population in which non-European HPV 16 variants were more common. We sequenced HPV 16 isolates in cervical samples collected from 93 Black South African women enrolled in a cervical cancer screening study and examined associations between cervical neoplasia identified though colposcopy with cervical biopsy and the specific HPV 16 variant identified. The European prototype variant (EP) was the most commonly identified variant in this population (47% of all isolates), but African variants (Af-1 and Af-2) were also quite common (41% of all isolates). In contrast to previous studies, we found no evidence that non-European variants were associated with an increased risk of neoplasia. Rather, most of the HPV 16-associated cancers were found in association with EP (71% of 14 cases). In this setting where African HPV 16 variants were common, no increased risk for cervical neoplasia was found among women with these variants compared with other HPV 16 variants. PMID- 16681755 TI - Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo. AB - Substantial studies have demonstrated that the initiation and progression of cervical cancer were closely associated with human papillomavirus (HPV) E6 and E7 oncogenes. The therapeutic strategy with ribozyme or antisense oligonucleotides to inhibit the expression of HPV E6 or E7 oncogenes showed effect to some degree, but problems such as low efficiency, short-period maintenance, and high cost still remain. The aim of this study was to investigate in vitro and in vivo the effect of HPV 16 E6 small interfering RNA (HPV 16 E6 siRNA) on cervical cancer cell line CaSki cells. The specific siRNA of HPV 16 E6 was synthesized and transfected into CaSki cells by liposome. The number of apoptotic cells, HPV 16 E6 messenger RNA (mRNA) level, and E6 protein expression were measured before and after the transfection by flow cytometry, reverse transcriptase-polymerase chain reaction, and Western blot, respectively. Cervical cancer in nude mice was established, and siRNA was injected directly into the nude mice peritoneal cavity or subcutaneous tumor. The efficiency of siRNA was evaluated by tumor volume change, HPV 16 E6 protein expression, and apoptosis of tumor cells. Apoptosis rate of CaSki cells at days 1, 2, 5, and 9 after siRNA transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV 16 E6 at the same time points were reduced by 77%, 83%, 59%, and 41%, respectively. But the mRNA level of beta-actin, as an internal control, showed no significant change. The inhibition rates of E6 protein synthesis at days 1, 2, 5, and 9 after the transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, whereas the protein levels of Lamin A/C, as internal control, had no change. In vivo, E6 siRNA administration groups showed a dramatic effect in inhibiting tumor growth, suppressing expression of E6 protein, and inducing tumor necrosis and apoptosis as compared with the control group. Direct injection of siRNA into subcutaneous tumor resulted in tumor suppression effect similar to that via the peritoneal cavity, and with additional injection better results could be achieved in cervical cancer CaSki cells. RNA interference exists, and the interference to HPV 16 E6 is specific and highly efficient both in vitro and in vivo. PMID- 16681756 TI - Erythropoietin receptor expression in biopsy specimens from patients with uterine cervix squamous cell carcinoma. AB - Hypoxia, an important mechanism of radioresistance, is a strong stimulus for erythropoietin (EPO) production. The stimulatory effects of EPO are mediated through the activation of its receptors, EPO receptors (EPORs). The objective of this study is to determine whether EPORs are expressed in biopsy specimens of patients with squamous cell carcinoma of the cervix. Eighteen biopsy specimens were studied after obtaining Institutional Review Board-approved consent. Standard immunohistochemistry techniques were utilized. Expression of EPORs was present in 16 out of 18 (88.9%) specimens. The intensity (qualitative) and the frequency (semiquantitative) of EPORs expression showed a statistically significant correlation (P= 0.00379). Statistical analysis was performed to determine whether EPORs expression is related to other parameters such as age, FIGO stage, histologic grade, and hemoglobin levels. Only age showed a statistically significant correlation with EPORs frequency of expression (P= 0.00878). Currently, work is in progress in our laboratory to study the radiobiologic effects of EPO on the radiation response of cultured cancer cell lines in vitro. PMID- 16681757 TI - Association between CD31 expression and histopathologic features in stage IB squamous cell carcinoma of the cervix. AB - This study was undertaken to evaluate the association between the expression of CD31 in the tumor and the histopathologic findings in patients with carcinoma of the cervix. This study included prospectively 30 women, aged 46.6 +/- 10.7 years, with stage IB squamous cell carcinoma of the cervix submitted to radical hysterectomy from November 2001 to September 2002. Samples from the tumor were taken and immunohistochemically evaluated by a monoclonal antibody for CD31. Clinicopathologic characteristics such as stage, tumor size, grade of differentiation, lymphatic vascular space invasion (LVSI), parametrial involvement, and status of pelvic lymph nodes were also recorded. The clinical stage (FIGO) was IB1 in 22 patients (73.3%) and IB2 in 8 patients (26.7%). The expression of CD31 was significantly associated with tumor size and the presence of LVSI, but not with grade of differentiation and vaginal or parametrial involvement (P= 0.03, P= 0.032, P= 0.352, P= 0.208, and P= 0.242, respectively). On univariate analysis, the presence of pelvic lymph node metastasis was influenced by LVSI (P= 0.003) and CD31 expression (P= 0.032). However, on multivariate analysis, the presence of LVSI (P= 0.007) was the only independent predictor of pelvic lymph node metastasis. The CD31 expression in tumor is significantly associated with LVSI and tumor size in patients with early-stage squamous cell carcinoma of the cervix. PMID- 16681759 TI - Increased cyclooxygenase-2 expression is correlated with suppressed antitumor immunity in cervical adenocarcinomas. AB - Cyclooxygenase-2 (COX-2) inhibition suppressed the growth of various tumors. The augmentation of antitumor immunity by increasing cytotoxic lymphocytes may be an important mechanism for COX-2 inhibition. Among cervical cancers, adenocarcinomas present more aggressive behavior and overexpressed COX-2. The expression of COX-2 and the CD8+ lymphocyte infiltrations were evaluated in this study by immunohistochemistry. We studied COX-2 expression and CD8+ lymphocyte infiltration in 55 women with cervical adenocarcinomas. COX-2 expression and tumor stromal CD8+ lymphocytes were evaluated by semiquantified methods. Tumor intraepithelial lymphocytes were counted under microscopic field of x200. Correlations between these data and other clinicopathologic features were investigated. Thirty-seven out of 55 (67.3%) cervical adenocarcinomas significantly expressed COX-2. Patients who died within 5 years showed higher percentage of COX-2 expression than survivors (100% vs 58.1%, P < 0.05). Victims also showed lesser intraepithelial CD8+ lymphocyte counts than survived patients (3.4 vs 26.4, P < 0.05). COX-2 expression and tumor intraepithelial lymphocyte count were reversely correlated with each other (correlation index: -0.38, P < 0.01). Up-regulated COX-2 expression and lesser tumor intraepithelial CD8+ lymphocyte count are poor prognostic indicators for cervical adenocarcinoma patients. COX-2 may play an important role in the suppression of host antitumor immunity in cervical adenocarcinomas. PMID- 16681758 TI - Inactivation of Crk SH3 domain-binding guanine nucleotide-releasing factor (C3G) in cervical squamous cell carcinoma. AB - C3G, a Crk SH3 domain-binding guanine nucleotide-releasing factor functions as a guanine nucleotide exchange factor for Rap1. It is activated via the Crk adaptor protein and plays an important role in transducing signals from receptors on the cell surface to the nucleus via the Ras/Raf/MAPK signal transduction pathway. However, since the experimental data result in pleiotropic effects in the cascade manner, its precise function remains unclear. Here we examined the C3G expression in cervical squamous cell carcinomas and found a marked decrease in the expression of C3G in a high incidence of said samples. In addition, we also demonstrated frequent hypermethylation of C3G, which resulted in an inactivation mechanism of the gene. Clinical and pathologic data failed to show any relationship between the human papillomavirus infection and the down-regulation of C3G. These results indicate that inactivation of C3G by de novo methylation plays an important role in the development of cervical squamous cell carcinoma. PMID- 16681760 TI - the management of vulval intraepithelial neoplasia in Northern Ireland. AB - In this study, we present the findings of a regional retrospective audit of all cases of vulval intraepithelial neoplasia (VIN) diagnosed in Northern Ireland (NI) over an 11-year period (1989-1999). During the period of the study, there were 97 cases of VIN. Cases of VIN were treated by many different clinicians at 14 hospitals. The commonest symptom at presentation was pruritus vulvae. The most common histopathologic diagnosis was VIN III (73%). In 52% of the cases, there was multifocal VIN, and in 43%, there was involvement of other sites such as the cervix, vagina, or anal region. The most common initial treatment was surgical excision, but a multitude of different treatments were performed initially. During the study period, 18 of 90 patients (20%) for whom follow-up was available developed invasive vulval squamous carcinoma. Most of the vulval cancers were superficially invasive, but three patients died of vulval cancer during the study period. This study illustrates that in NI, VIN is treated at many institutions by a multitude of clinicians. Management seems largely dependent on personal clinician preference and has been haphazard with little central coordination and organized strategy. VIN should be managed by clinicians with expertise in this field and who are treating significant numbers of patients according to evidence based protocols. PMID- 16681761 TI - A randomized phase II trial of indole-3-carbinol in the treatment of vulvar intraepithelial neoplasia. AB - The aim of this study was to determine the potential therapeutic benefits of indole-3-carbinol (I3C) in the management of vulvar intraepithelial neoplasia (VIN). Women with histologically confirmed high-grade VIN were randomized to receive 200 and 400 mg/day of I3C. Symptomatology by visual analog scale and vulvoscopic appearance were assessed at recruitment, 6 weeks, 3 months, and 6 months. Tissue biopsy to determine histologic response was obtained at completion of the study period. Urine samples were obtained at each visit to determine 2 hydroxyestrone to 16-alpha-hydroxyestrone ratios. Data from 12 women were suitable for analysis. There was a significant improvement in symptomatology with the introduction of I3C (itch, P= 0.018; pain, P= 0.028). Lesion size and severity were also significantly reduced (size, P= 0.005; appearance, P= 0.046). In addition, there was a significant increase in 2-hydroxyestrone to 16-alpha hydroxyestrone ratio following commencement of I3C, P= 0.05. However, tissue biopsy from the worst-affected vulval areas revealed no improvement in grade of VIN during the 6-month period, P= 0.317. There were no significant differences in results between those women taking 200 mg/day of I3C and those on 400 mg/day. This study has shown significant clinical improvement in symptomatology and vulvoscopic appearance of VIN with I3C therapy. Further clinical and scientific investigations are required to support these preliminary findings. PMID- 16681762 TI - Genital human papillomavirus infection in the male sexual partners of women with isolated vulvar lesions. AB - The purpose of this study was to detect human papillomavirus (HPV) positivity in the male sexual partners of women with isolated vulvar lesions. The male counterparts of the females were subjected to detailed physical and peniscopic evaluations. After the gross inspection, 5% acetic acid was applied and the whole genital organs were evaluated using a colposcope. Overall, 47 male sexual partners with isolated vulvar HPV lesions were included. None of the patients had condylomata acuminata. Twenty-six patients (55%) had papular or papillary lesions, and 21 (45%) had flat acetowhite lesions. Biopsies were taken from all these suspicious areas for histopathologic examination. Overall, 41 patients (87%) had the exact characteristics of HPV on biopsy specimens. Ninety-two percent of patients (24/26) having papular and/or papillary lesions on peniscopy had HPV on biopsy, while 81% of patients (17/21) who had acetowhite flat lesions had biopsy-confirmed HPV lesions. In conclusion, it is important to assess the male sexual partners of women with HPV-related lower genital tract disease. Peniscopy is valuable for detecting lesions, and histopathologic confirmation is mandatory. PMID- 16681763 TI - The role of omentectomy and appendectomy during the surgical staging of clinical stage I endometrial cancer. AB - Assessment of extrauterine spread is the most important objective of surgical staging in the endometrioid adenocarcinoma of uterine corpus. The role of omentectomy and appendectomy in the staging procedure is unclear. In this study, our objective was to determine whether omentectomy and appendectomy should be a part of the surgical staging in endometrioid adenocarcinoma of uterine corpus. Fifty-one patients who were diagnosed as clinical stage I endometrioid adenocarcinoma of corpus uteri were reviewed. Demographic, clinicopathologic, and surveillance data were collected from hospital charts. Grade, myometrial invasion, cervical and adnexal involvement and positive peritoneal cytology, lymph node and omentum and appendix involvement were recorded. The median age of 51 women was 60.45 years. Median follow-up period was 46.19 months. Microscopic omental involvement was detected in three cases (6%). Two cases (3.9%) of metastasis to appendix were seen. Omentum metastasis was more common in the patient with adnexal involvement, lymph node metastasis, and deep myometrial invasion (P= 0. 014, P= 0. 046, and P= 0. 033, respectively). Median survival was 53 months. We conclude that omentectomy gives additional information about extrauterine spread of tumor without increased operational morbidity and should be performed as a component of surgical staging in the presence of normal appeared omentum. PMID- 16681764 TI - Analysis of clinicopathologic factors predicting para-aortic lymph node metastasis in endometrial cancer. AB - The purposes of this study were to compare the relationships between para-aortic lymph node metastasis and various clinicopathologic factors to evaluate whether para-aortic lymph node dissection is necessary when treating endometrial cancer. A retrospective study was performed on 841 patients with endometrial cancer, who underwent the initial surgery at the Keio University Hospital. Clinicopathologic factors related to para-aortic lymph node metastasis significant on a univariate analysis were analyzed in a multivariate fashion using a logistic model. According to the multivariate analysis, the clinicopathologic factor most strongly related to the existence of para-aortic lymph node metastasis was positive pelvic lymph node metastasis (P < 0.01). Among the 155 patients who underwent pelvic and para-aortic lymph node dissection, the difference of 5-year overall survival by the presence of retroperitoneal lymph node metastasis was examined by Kaplan-Meier method. The prognosis was poor even if para-aortic lymph node dissection was performed in cases of positive para-aortic lymph node metastasis. In conclusion, when deciding whether to perform para-aortic lymph node dissection in patients with endometrial cancer, it is necessary to consider the pelvic lymph nodal status. If there is no pelvic lymph node metastasis, it could not be necessary to perform para-aortic lymph node dissection. PMID- 16681765 TI - Does immediate hormone replacement therapy affect the oncologic outcome in endometrial cancer survivors? AB - The purpose of this study was to evaluate the effect of immediate hormone replacement therapy (HRT) on oncologic outcome of patients with endometrial cancer. The patients were recruited prospectively after extensive discussion of risks and benefits of HRT. Continuous daily regimen of 0.625 mg conjugated equine estrogen plus 2.5 mg medroxyprogesterone acetate was initiated 4-8 weeks after surgery at first postoperative visit. The patients who had the same characteristics with the HRT group were assigned as a control group. Overall, 50 patients received HRT. There was no significant difference with respect to prognosticators between the HRT users and the control group. Seven patients (14%) stopped the use of HRT. Only two patients stopped the therapy before 24 months, and all the remaining patients used HRT for at least 24 months, with a mean value of 49.1 months. Neither the patients who used HRT nor the ones who left the therapy had recurrence at the time of writing of this article. This prospective case-control study showed that immediate postoperative use of HRT did not increase the recurrence or death rates in endometrial cancer survivors. PMID- 16681766 TI - The effect of long-term use of progesterone therapy on proliferation and apoptosis in simple endometrial hyperplasia without atypia. AB - The aim of this study was to evaluate the effect of long-term use of progesterone treatment on proliferation and apoptosis in simple endometrial hyperplasia without atypia. In this prospective control study, endometrial tissue samples of 19 patients with simple endometrial hyperplasia without atypia (group 1), posttreatment biopsy materials of the patients after 3 months of cyclic progesterone treatment with noretisterone for 10 days (group 2), and 18 endometrial biopsy materials of the control group (group 3) were examined for proliferative and apoptotic activities. There was a statistically significant difference between the median values of the proliferative index of the three groups (P = 0.000). The proliferative index was significantly higher in the endometrial hyperplasia group than in posttreatment group (P = 0.000). But there was no significant difference between posttreatment group and control group. The median value of apoptotic activity was significantly different between three groups (P = 0.000). Apoptotic index was highest in hyperplasia group. A significant decrease in apoptosis was observed after the progesterone treatment (P = 0.002). The lowest apoptotic activity was detected in the control group. In conclusion, 3 months of cyclic progesterone treatment reduces both proliferative and apoptotic activities in endometrial tissue with simple hyperplasia. PMID- 16681767 TI - Uterine effects of tamoxifen: a prospective study. AB - The purpose of the study was to evaluate tamoxifen-associated changes in the vagina and uterus in postmenopausal breast cancer patients. Between June 1994 and December 1998, 45 patients enrolled in a prospective study before commencing tamoxifen therapy. Patients with endometrial thickness >5 mm or neoplasia were excluded. Transvaginal ultrasonography, vaginal maturation indexes (VMI), and endometrial biopsy were performed at baseline and repeated at 6 months (n= 42), 1 year (n= 39), 2 years (n= 32), 3 years (n= 26), 4 years (n= 19), and 5 years (n= 15). For the 39 patients followed for 1 year, VMI (% parabasal/intermediate/superficial) was 21/71/8 at baseline compared with 1/90/9 at 1 year (P value = 0.0008/0.001/0.78). At baseline, mean endometrial thickness and uterine volume were 2.6 mm and 64 cm(3), respectively, compared with 5.8 mm and 84 cm(3) at 1 year (P= 0.0002, 0.002). At baseline, 80% of patients had atrophic endometrium and 9% proliferative endometrium compared with 61% and 26% at 1 year, respectively (P= 0.04). No cases of endometrial hyperplasia or adenocarcinoma were detected. Findings observed at 6 months persisted through 5 years of follow-up. Tamoxifen exerts a weak estrogenic effect on the vagina and uterus in highly prescreened postmenopausal women without preexisting endometrial pathology. PMID- 16681768 TI - Neuroendocrine features in poorly differentiated endometrioid adenocarcinomas of the endometrium. AB - This study aimed to clarify neuroendocrine features (NEF) in poorly differentiated (G3) endometrioid adenocarcinoma of the endometrium and to evaluate its prognostic significance. Forty cases with G3 carcinoma were investigated for NEF immunohistochemically. The histopathologic specimens were immunostained with chromogranin A, synaptophysin, and leu-7, using the labeled streptavidin-biotin method. The staining pattern was classified into diffuse, partial, or focal. The NEF was compared with clinicopathologic variables, including patients' survival. Chromogranin A was positive diffusely in 1 patient (2.5%), partially in 8 (20%), and focally in 13 (32.5%). Synaptophysin was positive diffusely in one (2.5%), partially in three (7.5%), and focally in nine (22.5%). Leu-7 was focally positive in 10 (25%) patients. The overall positive rate of the three neuroendocrine markers was 62.5%. A patient with diffusely positive staining for both chromogranin A and synaptophysin was diagnosed with neuroendocrine carcinoma. One or more neuroendocrine markers were positive in 25 cases (62.5%). Positive NEF was correlated with clinicopathologic parameters such as stage and myometrial invasion. The survival of the patients with positive NEF, especially with positive leu-7, was significantly lower than that without NEF. NEF was detected immunohistochemically in approximately 63% of the G3 carcinomas, and these patients had a poor prognosis. PMID- 16681769 TI - Expression of estrogen receptor-related receptor isoforms and clinical significance in endometrial adenocarcinoma. AB - Estrogen receptor-related receptors (ERRs) are members of orphan nuclear receptors closely related to the estrogen receptors (ERs). Researches showed that the ERRs bound to estrogen response elements and interfered in the ER signal pathway. Therefore, it might be associated with estrogen-dependent diseases. The purpose of this study was to explore whether ERRs were involved in the tumorigenesis of endometrial adenocarcinoma. We examined the expression of ERRs in endometrial adenocarcinoma and normal endometrium using semiquantitative reverse transcription-polymerase chain reaction and immunohistochemistry. Clinicopathologic features including FIGO stage, histologic grade, myometrial invasion, and nodal metastasis were reviewed. Results showed that messenger RNA (mRNA) levels of ERRalpha, ERRbeta, and ERRgamma were positively associated with the immunoreactivities (P= 0.009, P= 0.014, and P= 0.001, respectively). The expression rate and relative level of ERRalpha mRNA in ERalpha-positive endometrial adenocarcinomas were lower than in normal endometriums (P= 0.049 and P= 0.023), whereas the relative level of ERRgamma mRNA in ERalpha-positive endometrial adenocarcinomas was higher than in normal endometriums (P= 0.014). Expression of ERRalpha mRNA was positively correlated with FIGO stage (P= 0.019) and myometrial invasion (P= 0.043). A negative correlation was observed between expression of ERRgamma mRNA and nodal metastasis (P= 0.021). Results suggested that ERRalpha and ERRgamma might participate in the tumorigenesis of endometrial adenocarcinoma. ERRalpha and ERRgamma are promising to be new prognostic factors in endometrial adenocarcinoma. PMID- 16681770 TI - Genome-wide analysis of deoxyribonucleic acid in endometrial cancer using comparative genomic hybridization microarrays. AB - The aim of this study was to identify amplified oncogenes in endometrial cancer using array-based comparative genomic hybridization (array CGH). Despite its prevalence, the molecular mechanisms of endometrial carcinogenesis are still poorly understood. The selected array CGH allows the simultaneous examination of 58 oncogenes commonly amplified in human cancers and is capable of achieving increased mapping resolution compared with conventional CGH. A subset of 8 specimens from a bank of 60 malignant and normal specimens was selected for array analysis to identify potential genes of interest. TaqMan polymerase chain reaction was carried out on the 60 specimens to examine if aberrations at the genomic level correlated with gene expression and to compare expression in normal and malignant samples. Oncogenes amplified in the endometrial cancers included AR, PIK3CA, MET, HRAS, NRAS, D17S1670, FGFR1, CTSB, RPS6KB1, LAMC2, MYC, PDGFRA, FGF4/FGF3, PAKI, and FGR. Three genes were examined at the messenger RNA level. AR and PIK3CA were higher in normal specimens, and MET was higher in malignant samples, suggesting a role for MET in endometrial cancer. Newer arrays examining more genes and larger sample numbers are necessary to elucidate the carcinogenic pathway in endometrial cancer. PMID- 16681771 TI - Euonymus alatus (Thunb.) Sieb induces apoptosis via mitochondrial pathway as prooxidant in human uterine leiomyomal smooth muscle cells. AB - Euonymus alatus (Thunb.) Sieb (EA) is a traditional Korean herbal medicine, commonly used to treat tumors in Korea and China for centuries. Our earlier studies have indicated that EA exhibits antitumor properties, but its mechanism remains to be elucidated. In this study, we evaluated the molecular mechanism of EA in a human uterine leiomyomal smooth muscle cell (ULSMC) line. Water extracts of EA have been reported to not only function as antioxidants but also cause cytotoxic effect. We investigated the mechanism of EA-induced cytotoxicity in human ULSMC. When cells were cultured with 20-200 microg/mL EA for 6 h, caspase-3 was activated and then cells fell into apoptosis. Induction of apoptosis by EA was accompanied with increase of the cytosolic fractions of cytochrome c prior to the activation of caspase-3. The preculture with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, facilitated EA-induced induction of apoptosis. The preculture with N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone, a pan-caspase inhibitor, partially suppressed the induction of apoptosis. EA showed little toxic effect on peripheral blood mononuclear cells from healthy volunteers. These results indicate that EA acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway. PMID- 16681772 TI - p53, epidermal growth factor, and platelet-derived growth factor in uterine leiomyosarcoma and leiomyomas. AB - Uterine leiomyosarcoma (ULMS) is an aggressive gynecological disease. Although ULMS are often found in association with benign leiomyoma (LMA), little is known regarding the relationship between these benign and malignant smooth muscle neoplasms. The objective of this study was to evaluate the expression of epidermal growth factor (EGFR), platelet-derived growth factor (PDGFR), and p53 in ULMS specimens, their prognostic relevance, and the expression of these molecular markers when compared to benign LMA specimens. Between 1991 and 2001, 25 patients were identified with high-grade primary ULMS and for whom tissue was available. Tissue microarray was created with three representative cores from each of the ULMS cases as well as from 19 patients with benign uterine leiomyomata. Immunohistochemical (IHC) staining was performed for EGFR, PDGFR, and p53. Negative and positive IHC staining was scored for each marker. Outcome analysis was performed only for ULMS. Survival was determined from the time of initial diagnosis to last follow-up. Twelve (48%) ULMS expressed p53 compared to none of the LMA (P < 0.001), and 15 (60%) ULMS cases showed PDGFR expression compared to 32% of LMA samples (P= 0.08). EGFR expression did not differ between ULMS and LMA groups. ULMS patients with p53 expression had a poorer survival compared to ULMS patients with negative expression (P= 0.07). ULMS tumor stage had the strongest association with overall survival (P= 0.05). Our study supports previous investigations indicating that p53 expression may serve as a prognostic marker for ULMS patients. The difference in PDGFR expression between ULMS and LMA demonstrated a trend toward significance. EGFR was not commonly expressed in ULMS. These uniquely expressed markers may assist in stratifying patients by survival and identify novel therapeutic markers. Clearly, further investigation is needed. PMID- 16681773 TI - Whole-body positron emission tomography with F-18 fluorodeoxyglucose for the detection of recurrence in uterine sarcomas. AB - We evaluated the usefulness of whole-body positron emission tomography (PET) using F-18 fluorodeoxyglucose (FDG-PET) for the detection of recurrence in follow up patients after primary treatment of uterine sarcoma. Eight patients with pathologically proven uterine sarcoma underwent FDG-PET, computed tomography (CT), and ultrasonography (US). Final diagnoses of recurrence were established in five cases (three carcinosarcomas and two leiomyosarcomas). PET revealed recurrent sites in the intraperitoneum, liver, lung, bone, and retroperitoneal lymph nodes. However, the minimum size of the tumor detected by PET depended on the sites of recurrence. CT and US images showed two false-negative cases of intraperitoneal tumors. PET was able to detect a solitary small intraperitoneal tumor, which was very difficult to detect by CT and US. Positive PET findings did not affect the prognosis in three of the five recurrent patients; however, the remaining two patients consequently underwent the combination therapy consisting of surgery and chemotherapy and survived for more than 1 year after the positive FDG-PET results. Application of PET imaging for the early detection of recurrent sites was useful for the decision of treatment strategy for patients with recurrent uterine sarcoma. PMID- 16681774 TI - Uterine sarcomas in breast cancer patients treated with tamoxifen. AB - Tamoxifen (TMX) has been related with the development of uterine sarcomas. Since the first reported case in 1988, 65 TMX-related cases have been referred to. Here we present three new cases of uterine sarcomas in patients with breast cancer treated with TMX and we comment on the outcome of the cases described in the literature. In the past 25 years, 60 uterine sarcomas have been diagnosed and treated in Hospital Clinic. Three patients have previously received TMX 20 mg/day for 3, 5, and 7 years for breast cancer. Uterine sarcoma appeared 5, 5, and 7 years, respectively, after the start of TMX treatment, and all of them had stage I (FIGO) disease. Two patients had a carcinosarcoma and one patient had an adenosarcoma. After treatment, the disease progressed in two patients and the third patient is alive having a follow-up of 42 months. The low incidence of uterine sarcomas makes it difficult to establish a relationship with TMX. Nevertheless, looking at the literature data, 20 mg/day of TMX over 1 year could be enough to develop uterine sarcoma; the sarcoma appears mainly during the first 8 years and seem to behave more aggressively. Although only 65 cases have been reported in the past 14 years, a strict follow-up is necessary in patients with breast cancer receiving TMX therapy. PMID- 16681775 TI - Evolution of treatment of high-risk metastatic gestational trophoblastic tumors: Ain Shams University experience. AB - The aim of the current study is to evaluate the different treatment modalities used in the management of high-risk metastatic gestational trophoblastic tumors (GTT) between June 1992 and December 2004 at the Gynecologic Oncology Unit, Ain Shams University. Out of 261 patients diagnosed and treated for GTT, 70 (26.8%) were high risk metastatic patients based on the National Institutes of Health clinical classification. The mean age was 29.39 +/- 9.38 years (16-55 years), with six patients (8.6%) being older than 39 years, and the mean duration of follow-up was 79.74 +/- 40.44 months (6-157 months). Forty patients (57.14%) were diagnosed after molar pregnancy, 22 (31.43%) after abortion, and 8 (11.43%) after term pregnancy. Forty-two patients (60%) were diagnosed within 4 months of the occurrence of the disease, and 28 (40%) were diagnosed after more than 4 months. Sixty-seven patients were treated using different regimens according to the protocol of treatment at that time. The MAC regimen was used initially but has been subsequently abandoned in favor of EMA-CO (etoposide, methotrexate, dactinomycin, cyclophosphamide, and vincristine [Oncovin]) regimen, which was later modified by omitting the CO arm to decrease its toxicity. If resistance developed, platinum-based therapy was given in the form of EMA-EP. Recently, our unit incorporated paclitaxel in the third-line treatment. Surgical intervention was used selectively. Fifty-seven (81.4%) patients could be cured; 43 by initial chemotherapy, with a mean of 7 +/- 0.46 courses (6-15), and 14 were salvaged by second- or third-line chemotherapy. Fourteen patients (20%) died during the study period; one was unrelated to GTT, while three died of acute respiratory distress syndrome before instituting proper therapy and two died of treatment complications. Using univariate and multivariate Cox regression analyses, the presence of brain and/or liver metastases was found to be the worst prognostic variable affecting the survival, followed by resistance to combination chemotherapy and then the type of antecedent pregnancy. The projected 5-year survival as estimated by Kaplan-Meier method was 78%. PMID- 16681776 TI - Gestational trophoblastic tumor in pregnancy: a case report and review of the literature. AB - A case of gestational trophoblastic tumor (GTT) concurrent with an intrauterine pregnancy is reported in a 21-year-old gravida 2 para 0 (0010) who presented with dyspnea, orthopnea, headache, and blurring of vision at 33 weeks age of gestation. She had a history of hydatidiform mole for which curettage was done. Chest radiograph showed pulmonary metastases, with pleural effusion on both lungs. Serum beta subunit of human chorionic gonadotropin was abnormally elevated for age of gestation. Due to worsening maternal pulmonary condition, the patient underwent primary, low segment cesarean section and was subsequently started on multidrug chemotherapy. This is the first reported case of GTT in pregnancy in the Philippines in which both the mother and the infant survived. Several hypotheses regarding its origin, its diagnosis, and its management and prognosis are presented. This case report emphasizes the importance of early diagnosis and treatment to improve the prognosis of both the mother and the infant. PMID- 16681777 TI - The role of hysterotomy in the management of gestational trophoblastic neoplasia. AB - The management of late gestational trophoblastic disease recurrence is challenging. We present a case of a 16-year-old woman who was diagnosed with a gestational trophoblastic neoplasia 14 months after her hydatidiform mole pregnancy. A staging was performed revealing only an intramural lesion, which resembled a myoma, in the fundus of the uterus. Despite two course of methotrexate, the human chorionic gonadotropin (hCG) level increased slowly. The presentation was highly suggestive for a placental site trophoblastic tumor. A local resection of the tumor by hysterotomy was performed. Pathologic examination revealed a choriocarcinoma with tumor-free surgical margins. Subsequently, the patient received three cycles of EMA-CO (etoposide, methotrexate, actinomycin, cyclophosphamide, and vincristine). At present, 89 months after the hysterotomy, the patient is well, with no sings of recurrence. This report illustrates that it is mandatory to have a histologic diagnosis of chemoresistant gestational trophoblastic neoplasia before performing definitive surgery. PMID- 16681778 TI - Leiomyosarcoma of the vagina: case report and literature review. AB - Leiomyosarcoma of the vagina is quite rare. Extensive search of the literature discloses 137 case reports published over the past 40 years. Collectively, these reports give only cursory guidelines for diagnosis, management, and prognosis. We add another case to the sparse literature on leiomyosarcoma of the vagina. Treatment consisted of surgical excision. Tumor was found to extend to the margin of the excised specimen. The malignancy quickly recurred with lung metastases and the patient died. The very poor prognosis associated with this lesion emphasizes the critical need for early diagnosis and expeditious therapy. In the interest of improving salvage by prompt recognition and intervention, it is important for health-care professionals to be knowledgeable about and alert to this condition. PMID- 16681779 TI - Skeletal carcinomatosis in endometrial clear cell carcinoma at initial presentation: a case report. AB - Bone metastasis is frequently seen in different solid tissue malignancies. However, it is a very rare entity in endometrial cancers and most of the previous reports were localized to a single bone. To the best of our knowledge, this is the first report of a case with disseminated bone metastasis from endometrial clear cell carcinoma. A 69-year-old, multiparous woman presented with the complaint of postmenopausal bleeding. Diagnostic workup revealed endometrial clear cell carcinoma. After comprehensive surgical staging, the patient was found to be at FIGO stage IIIC. Three weeks after the operation, an intractable back pain developed. Direct graphics of the lumbosacral region were suspicious of metastasis, and further evaluation of the patient revealed metastasis in multiple bones including calvarium, thoracolumbal vertebrae, pelvic bones, costae, collum of the right femur, and trochanter major of the left femur. Bone metastasis in endometrial clear cell carcinoma may be seen at initial presentation and may involve multiple bones. Whole-body scanning with scintigraphic evaluation is a reasonable approach to evaluate the extension of the bone involvement. Further studies are needed to elucidate the true incidence and management of bone metastasis in endometrial clear cell carcinoma. PMID- 16681780 TI - A case of high-grade endometrial stromal sarcoma arising from endometriosis in the cul-de-sac. AB - This report describes a rare case of high-grade endometrial stromal sarcoma (ESS) arising from pathologically confirmed endometriosis in the cul-de-sac. A 37-year old woman presented with irregular menstruation, pelvic pain, and diarrhea. Magnetic resonance imaging and colon biopsy suggested endometriotic nodule of the cul-de-sac. The tumor size was reduced with hormonal therapy, and the residual tumor was excised, resulting in the pathologic diagnosis of endometriosis. Two years later, a soft mass reappeared with rapid growth. Tumor extraction was performed, and the histopathologic diagnosis was high-grade ESS. Neither hormonal therapy nor chemotherapy was effective, and the patient died 6 months postoperatively. ESS should be included in the differential diagnosis of malignant transformation of endometriosis. PMID- 16681781 TI - Recurrence of carcinoma cervix in the scar of previous cesarean section: a case report. AB - Although rare, reports of carcinoma cervix recurring in abdominal incision, episiotomy scars, laparoscopic port sites, and drain sites are available. However, recurrence in the scar of a previous cesarean section (CS) is unreported. A 49-year-old female with a diagnosis of keratinizing squamous cell carcinoma (SCC) of cervix, stage IIB, was treated by radical radiotherapy. She had undergone CS, through midline abdominal incision, for three previous deliveries. Twenty months later, she presented with a nodule of size 2.5 x 2 cm(2) on the 26-year-old abdominal CS scar. Infiltration of the skin and extension into the deeper structures of the abdominal wall was present in an area of 5 x 4 cm(2). There was no evidence of disease on the cervix. A fine-needle aspiration cytologic examination from the nodule revealed keratinizing SCC. Ultrasonography and computerized tomography scan revealed a mass lesion along the abdominal CS scar with subcutaneous nodule and peritoneal extension. Multiple metastatic lesions were present in the liver. She was treated with chemotherapy and radiotherapy. The patient has lived for more than 12.5 months. The first report of recurrence of carcinoma cervix in a CS scar is presented. Literature on surgical scar recurrence in carcinoma cervix is reviewed. PMID- 16681782 TI - Neoadjuvant chemotherapy followed by radical hysterectomy for invasive cervical cancer diagnosed during pregnancy: report of a case and review of the literature. AB - Although cervical carcinoma is among the most frequently encountered malignancies during pregnancy, only four cases of neoadjuvant chemotherapy during pregnancy have been reported. A 28-year-old A0P1G2M0 was diagnosed at 15 weeks with stage Ib1 invasive squamous cervical cancer. Because she strongly desired the continuation of this pregnancy, after extensive counseling she was treated with 75 mg/m(2) cisplatin every 10 days starting at 17 weeks. After six cycles, clinically and radiologically stable disease with normalization of the squamous cell carcinoma tumor marker was obtained. An elective cesarean delivery followed by radical hysterectomy and lymphadenectomy was performed at 32 weeks gestation. The pathology report revealed a moderately differentiated squamous cell carcinoma of 3.5 cm, and all 33 lymph nodes were free of disease. Neonatal examination of the baby could not reveal any abnormalities, and this was confirmed at 6 months. The use of neoadjuvant chemotherapy enabled us to continue this pregnancy until the fetus was viable. Cisplatin did not influence the short-term outcome, but only a long-term follow-up will inform us on its safety during pregnancy. PMID- 16681783 TI - Spontaneous bacterial peritonitis following treatment for cervical carcinoma. AB - Spontaneous bacterial peritonitis (SBP) is an acute bacterial infection usually associated with ascites and cirrhosis or is a complication of peritoneal dialysis. There are very few case reports of cancer patients who developed this disease. Furthermore, there have been no published case reports of successfully treated gynecological cancer patients who later developed SBP. We present a case involving a 41-year-old woman who was treated for cervical carcinoma in 1992. She underwent radical surgery and adjuvant chemoradiation therapy. Two years later, the patient presented with streptococcal group B cellulitis associated with left leg lymphedema. She recovered following antibiotic treatment but had recurrent episodes of streptococcal cellulitis in her leg over the past 10 years. In 2003, the patient was admitted to the hospital because of sepsis, acute renal failure, and SBP. She was treated and recovered following treatment. SBP is usually associated with cirrhosis. Although SBP is rarely seen in successfully treated gynecological cancer patients, oncologists should be aware of this clinical entity. Timely treatment is essential to maximize chances of survival. PMID- 16681784 TI - Splenic metastases from cervical carcinoma: a case report. AB - Splenic metastasis from squamous cell carcinoma of the uterine cervix is an unusual event in the natural history of the disease. The authors report one such uncommon occurrence in a 41-year-old female who presented initially with cervical carcinoma (stage IIB) and was treated with radical radiotherapy with concurrent weekly chemotherapy. Following a disease-free interval of less than a year, she developed hepatosplenic metastases despite being locally controlled. The literature relevant to the report is also discussed. This report reaffirms the notion that splenic metastases from cervical carcinoma are rare events but can occur as part of widespread dissemination. PMID- 16681785 TI - Metastatic tumor extending through the inferior vena cava into the right atrium: a case report of carcinoma of the uterine cervix with para-aortic lymph node metastases. AB - Carcinoma of the uterine cervix with cardiac metastasis is not uncommon in autopsy cases. However, an intraatrial tumor extending through the inferior vena cava (IVC) from the site of para-aortic lymph node metastasis has never been reported. A 57-year-old Japanese woman was admitted to an emergency care unit complaining of mild chest pain and shortness of breath. She had progressive multiple lymphatic metastases of stage IIIB squamous cell carcinoma of the uterine cervix that had initially been treated with concurrent chemoradiation. Echocardiogram showed pedunculated tumor in the right atrium (RA), and computed tomography demonstrated multiple pulmonary tumor embolism. Surgical specimen from the RA showed squamous cell carcinoma resembling the primary cervical tumor, and the peduncle appeared to originate from within the IVC. Postoperative ultrasonography showed severe stenosis of the abdominal IVC due to the invasive growth of para-aortic lymph node metastases. The stalk of the tumor originated from this lesion. We present an extremely unusual case of intraatrial metastatic tumor originating from the para-aortic lymph nodes of cervical cancer. PMID- 16681786 TI - Synchronous cervical and vulvar malign melanomas: metastasis or multifocality of the disease? A case report and review of the literature. AB - Vulvar melanoma is rare and has a worse prognosis and higher recurrence rate than cutaneous melanoma. Multifocality is also more common in vulvar melanomas. A case having synchronous cervix and vulvar malign melanoma is presented and discussed in the light of the literature whether it is a metastasis of vulvar malign melanoma to cervix or multifocal originated disease. In conclusion, it is important to evaluate the whole genital system in vulvar melanomas as it is in squamous cancers. PMID- 16681787 TI - Pleomorphic adenoma of the vulva: a review illustrated by a clinical case. AB - The pleomorphic adenoma of the vulva is an extremely scarce diagnosis. This form of neoplasm is more frequently observed in other topographic sites of the human body. Treatment modalities largely derive from experiences with the pleomorphic adenoma located in these other sites. As a result of similarity with other tumors and scarcity of the neoplasm, the diagnosis of pleomorphic adenoma of the vulva is easily missed in daily routine practice. Recurrences of this neoplasm are frequently observed with an estimated incidence of malignant transformation of 25%. Addressing this previously mentioned issue, an extensive surgical excision and long-term follow-up is advocated. This report is illustrated by the case of a 66-year-old woman who was admitted to our hospital with a pleomorphic adenoma of the vulva, diagnosed by pathologic assessments following surgical excision. As a result of the rarity of this diagnosis, characteristics and treatment of extravulvar in addition to vulvar localizations of the pleomorphic adenoma are reviewed. PMID- 16681788 TI - Primary malignant lymphoma of uterine cervix. AB - Primary malignant lymphoma of the uterine cervix is a rare disease. Malignant lymphoma can be clinically and histopathologically misdiagnosed for the infrequent presentation in this are. A case of 56-year-old woman with uterine cervical tumor with infiltration to both parametria is presented. A biopsy was performed and histopathological studies reported a large cell B lymphoma. After the diagnosis CT abdominal, pelvic and thoracic scan was performed and shows infiltration to posterior bladder without evidence of disease in lymph nodes or another organ. The patient was treated with chemotherapy and radiotherapy. Six month after finish the treatment is well and free of disease. PMID- 16681789 TI - Preoperative needle biopsy and immunohistochemical analysis for gastrointestinal stromal tumor of the rectum mimicking vaginal leiomyoma. AB - Accurate preoperative diagnosis for gastrointestinal stromal tumor (GIST) was obtained using the endoscopic approach in about 50% of the cases. Preoperative assessment is quite important in reproductive women for the treatment of pelvic tumor. We report the first case of GIST of the rectum that was accurately diagnosed with preoperative needle biopsy from the vaginal wall. The case was a 38-year-old woman who presented with a history of genital bleeding. Internal examination revealed a round and elastic mass (7 x 7 cm) in the left pelvic cavity. The biopsy specimen from the left sidewall of the vagina using a manually manipulated biopsy needle showed strong reaction for CD34 and CD117 (c-KIT). The patient underwent low anterior resection of the rectum, tumor resection, and partial resection of the posterior vagina. Preoperative biopsy with vaginal approach might be helpful to make a diagnosis for pelvic tumor of unknown origin and unknown malignant potential. PMID- 16681790 TI - Recurrent vulval congenital lymphangioma circumscriptum--a case report and literature review. AB - Lymphangioma circumscriptum (LC) is a defect of lymphatics in deep dermis. This abnormal saccular dilatation of lymphatics occurs in dermal papillae visible as vesicles clinically. The entity may be acquired or congenital. The occurrence of congenital variety of LC in vulva is extremely rare, with only few cases reported in the literature. We communicate one such case presenting as recurrent vulval growth along with a review of literature and treatment modalities. PMID- 16681791 TI - Is early postradiation dysplasia almost associated with poor prognosis? A case report and review of the literature. AB - Following radiotherapy for cervical carcinoma, abnormal cytologic changes have been reported in a number of publications. These reactions occur at varying periods of time following irradiation and do not necessarily reflect the presence of invasive cancer. On the basis of cytologic and histopathologic features, these reactions were determined as postradiation dysplasia (PRD). PRD has been reported to occur in 18.7-26% of patients treated by radiotherapy for cervical cancer. In the literature, it was reported that patients diagnosed with PRD less than 3 years after the initial diagnosis of cervical cancer had a mean survival rate of 33.8% as compared with a 100% 5-year survival rate in patients with a delayed (>3 years) onset of period. We present a case of stage IIIB cervical squamous cell carcinoma with PRD detected 6 months after radiotherapy. The patient is still tumor free 8 years after radiotherapy. In the light of this patient, we review the literature and discuss the relationship of PRD with survival in the cervical carcinomas. PMID- 16681792 TI - Successful treatment of two patients with gestational trophoblastic neoplasm presenting with emergent neurologic symptoms. AB - The objective of this study is to present the successful treatment of two young patients with gestational trophoblastic neoplasms (GTN) presenting with emergent neurologic symptoms without any gynecological problems. Case 1, a 22-year-old patient, was admitted to an infectious disease ward, with admitting diagnosis of encephalitis due to neurologic symptoms. Case 2, a 33-year-old patient, underwent craniotomy due to hemorrhagic brain tumor in the neurosurgery department. The diagnosis of GTN should be considered in any woman of reproductive age who has neurologic symptoms. It seems that multiagent chemotherapy in conjunction with whole-brain irradiation results in acceptable survival rate in brain metastatic GTN patients. Craniotomy is often necessary in fulminant cases. PMID- 16681793 TI - Recurrent partial hydatidiform mole: a report of a patient with three consecutive molar pregnancies. AB - Hydatidiform mole (HM) is the most common form of gestational trophoblastic neoplasia and is characterized by atypical hyperplastic trophoblasts and hydropic villi. Recurrence of HM is extremely rare. Here, we report the case of a patient with three consecutive partial HMs without normal pregnancy. A 28-year-old woman with gravida 3, para 0, was referred to our hospital with a diagnosis of an invasive mole in December 2003. She had three consecutive molar pregnancies in 2000, 2001, and 2003. All three molar pregnancies were evacuated by suction curettage and the patient was followed by serial beta-human chorionic gonadotropin levels. All three moles were histologically confirmed as partial moles. In the first two molar events no additional treatment after evacuation was required, but in the last event, the beta-human chorionic gonadotropin levels increased and an invasive mole was suspected. Diagnostic workup ruled out an invasive mole and choriocarcinoma. Karyotypic analysis of the patient and her husband was normal. The patient required chemotherapy for treatment of persistent disease. Recurrent partial HM is a very rare clinical disorder. Repetitive molar pregnancy is not an indication for chemotherapy, but persistent disease does require chemotherapy. PMID- 16681794 TI - The impact of obesity on surgery in gynecological oncology: a review. AB - Surgery represents a mainstay in the treatment of gynecological cancers. It is a common belief that operating on obese patients causes more peri- and postoperative complications than operating on nonobese patients. The surgical outcome in gynecological oncology can be evaluated by comparing intra- and postoperative complications, extent of lymphadenectomy, negativity of the specimens' margins, and percentage of optimal debulking between obese and nonobese patients affected by malignancies at the same stage. In this review, we analyze how obesity affects the feasibility of a correct oncologic procedure in case of cervical, endometrial, and ovarian cancer. We also describe the techniques that have been suggested in the literature to improve the surgical outcome on obese patients. PMID- 16681796 TI - From genes to neurons to brain. PMID- 16681797 TI - A hitchhiker's guide to behavioral analysis in laboratory rodents. AB - Genes and environment are both essential and interdependent determinants of behavioral responses. Behavioral genetics focuses on the role of genes on behavior. In this article, we aim to provide a succinct, but comprehensive, overview of the different means through which behavioral analysis may be performed in rodents. We give general recommendations for planning and performing behavioral experiments in rats and mice, followed by brief descriptions of experimental paradigms most commonly used for the analysis of reflexes, sensory function, motor function and exploratory, social, emotional and cognitive behavior. We end with a discussion of some of the shortcomings of current concepts of genetic determinism and argue that the genetic basis of behavior should be analyzed in the context of environmental factors. PMID- 16681798 TI - Genetic analysis of psychiatric disorders in humans. AB - Psychiatric disorders place a large burden not only on affected individuals and their families but also on societies and health services. Current treatment is only effective in a proportion of the patients, so considerable effort has been put into the development of new medications. The susceptibility to all major psychiatric disorders is, at least in part, genetic. Knowledge of the genes that underlie this susceptibility may lead to the identification of new drug targets and the development of more effective treatments. Therefore, numerous genetic studies in search for the genes involved in psychiatric disorders have been performed. Although results of both linkage and association studies have been inconsistent, several promising gene regions and candidate genes have been identified recently. In this article, we will review the strategies that proved to be successful in detecting genes for psychiatric disorders and we will provide some recommendations to increase the probability of detecting susceptibility genes in genetic studies of different designs. PMID- 16681799 TI - Genes and neurons: molecular insights to fear and anxiety. AB - Experimental animal models provide an important tool for the identification of inheritable components of fear and anxiety. 'Pavlovian' fear conditioning has been tremendously successful to characterize the neuronal circuitry and cellular mechanisms of the formation, consolidation and extinction of fear memories. Here we summarize recent progress that has led to the identification of gene products contributing to such experience-dependent changes in fear and anxiety and may guide the search for genetic factors involved in the development and treatment of human anxiety disorders. PMID- 16681800 TI - Dendritic pathology in mental retardation: from molecular genetics to neurobiology. AB - Mental retardation (MR) is a developmental brain disorder characterized by impaired cognitive performance and adaptive skills that affects 1-2% of the population. During the last decade, a large number of genes have been cloned that cause MR upon mutation in humans. The causal role of these genes provides an excellent starting point to investigate the cellular, neurobiological and behavioral alterations and mechanisms responsible for the cognitive impairment in mentally retarded persons. However, studies on Down syndrome (DS) reveal that overexpression of a cluster of genes and various forms of MR that are caused by single-gene mutations, such as fragile X (FraX), Rett, Coffin-Lowry, Rubinstein Taybi syndrome and non-syndromic forms of MR, causes similar phenotypes. In spite of the many differences in the manifestation of these forms of MR, evidence converges on the proposal that MR is primarily due to deficiencies in neuronal network connectivity in the major cognitive centers in the brain, which secondarily results in impaired information processing. Although MR has been largely regarded as a brain disorder that cannot be cured, our increased understanding of the abnormalities and mechanisms underlying MR may provide an avenue for the development of therapies for MR. In this review, we discuss the neurobiology underlying MR, with a focus on FraX and DS. PMID- 16681801 TI - Mitogen-activated protein kinase/extracellular regulated kinase signalling and memory stabilization: a review. AB - The function of mitogen-activated protein kinase (MAPK) in neurons has been the subject of considerable scrunity of late, and recent studies have given new insights into how this signalling cascade can regulate gene expression following cell-surface receptor activation. At the same time, a wealth of experimental data has demonstrated that the MAPK cascade is critically involved in the mechanisms underlying the type of enduring modification of neural networks required for the stability of memories, emphasizing the high level of interest in this signalling molecule. In this review, we briefly outline the main molecular events and mechanisms of the regulation of the MAPK cascade leading to transcriptional activation and summarize recent advances in our understanding of the functional role of this molecular signalling cascade in regulating brain plasticity, memory consolidation and memory reconsolidation. PMID- 16681802 TI - The genetic basis of circadian behavior. AB - In most species, an endogenous timing system synchronizes physiology and behavior to the rhythmic succession of day and night. The mammalian circadian pacemaker residing in the suprachiasmatic nuclei (SCN) of the hypothalamus controls peripheral clocks throughout the brain and the body via humoral and neuronal transmission. On the cellular level, these clockworks consist of a set of interwoven transcriptional/translational feedback loops. Recent work emphasizes the tissue specificity of some components of these molecular clockworks and the differential regulation of their rhythmicity by the SCN. PMID- 16681803 TI - Chromatin remodeling and neuronal function: exciting links. AB - Regulation of gene expression occurs at different levels, from DNA to protein, and through various mechanisms. One of them is modification of the chromatin structure, which is involved in the definition of transcriptional active and inactive regions of the chromosomes. These phenomena are associated with reversible chemical modifications of the genetic material rather than with variability within the DNA sequences inherited by the individual and are therefore called 'epigenetic' modifications. Ablation of the molecular players responsible for epigenetic modifications often gives rise to neurological and behavioral phenotypes in humans and in mouse models, suggesting a relevant function for chromatin remodeling in central nervous system function, particularly in the adaptive response of the brain to stimuli. We will discuss several human disorders that are due to altered epigenetic mechanisms, with special focus on Rett syndrome. PMID- 16681804 TI - Genetic basis of Alzheimer's dementia: role of mtDNA mutations. AB - Alzheimer's disease (AD) is the most common neurodegenerative disorder associated to dementia in late adulthood. Amyloid precursor protein, presenilin 1 and presenilin 2 genes have been identified as causative genes for familial AD, whereas apolipoprotein E epsilon4 allele has been associated to the risk for late onset AD. However, mutations on these genes do not explain the majority of cases. Mitochondrial respiratory chain (MRC) impairment has been detected in brain, muscle, fibroblasts and platelets of Alzheimer's patients, indicating a possible involvement of mitochondrial DNA (mtDNA) in the aetiology of the disease. Several reports have identified mtDNA mutations in Alzheimer's patients, suggesting the existence of related causal factors probably of mtDNA origin, thus pointing to the involvement of mtDNA in the risk contributing to dementia, but there is no consensual opinion in finding the cause for impairment. However, mtDNA mutations might modify age of onset, contributing to the neurodegenerative process, probably due to an impairment of MRC and/or translation mechanisms. PMID- 16681805 TI - In vitro investigations on the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone on Candida albicans. AB - Rilopirox and piroctone belong to the class of hydroxypyridone antimycotics. This class is not related to other antimycotics. In contrast to azole antimycotics and polyene antimycotics the mode of action of hydroxypyridone antimycotics is not fully understood. Inhibition of cellular uptake of essential compounds as well as loss of other compounds seems to be only a secondary effect of a primary not known action of these drugs. The antifungal effect in vitro depends on the medium used. The hyphal induction of Candida albicans is inhibited by hydroxypyridone antimycotics, but this effect is compensated by iron ions. A damage of the cell membrane and a direct influence on adenosine triphosphate synthesis, respectively, do not seem to be part of the mode of action. But there are clear hints that reactive oxygen species (ROS) and available metabolic activity are important parts of the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone. PMID- 16681806 TI - Antifungal effects of aminosulphoxide and disulphide derivatives. AB - 2-Benzenesulphinyl-(1,4)-naphtoquinone and 14 derivatives were synthesised and were used to evaluate their cytotoxicity against a human myelomonocyte cell line and their antifungal activity against two yeast, i.e. Candida albicans and C. tropicalis and against two filamentous fungi such as Aspergillus niger and Fusarium oxysporum and against one dermatophyte, namely Trichophyton tonsurans. The cytotoxicity and antifungal activities were investigated in comparison with amphotericin B as reference drug. No compound was significantly more toxic than amphotericin B at 0.2 microg ml(-1). The best results of antifungal activity were obtained with GFL 10, GFL 13 and GFL 30 on C. tropicalis, F. oxysporum and T. tonsurans. For C. albicans and A. niger, there was no difference between amphotericin B and the other molecules. The sterol quantitation, the time-kill curves were carried out for these three compounds in order to confirm their action in ergosterol synthesis. Time-kill curves showed a fungistatic activity. For C. tropicalis GFL 10, GFL 13 and GFL 30 increased the growth delay better than amphotericin B, in contrast to F. oxysporum. As for T. tonsurans, GFL10 and GFL13 gave a delay, but the effect of GFL 30 was a bit less marked. PMID- 16681807 TI - Characterisation of the anticryptococcal effect of the FC-1 toxin produced by Filobasidium capsuligenum. AB - The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC 1) which is highly effective against the opportunistic fungal pathogen Cryptococcus neoformans. The aim of this work was to study the effect of the toxin on C. neoformans cells. The sensitivities of strains representing eight molecular subtypes (VNI-IV and VGI-IV) of the C. neoformans species complex, and of an additional 50 clinical and environmental isolates were determined. Analysis of cellular DNA by laser scanning cytometry and fluorescein isothiocyanate (FITC) staining of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it may act as an ionophoric protein that disrupts the cytoplasmic membrane function. The competition assay results suggest that beta-1,6-glucan in the cell wall may provide the binding site for the killer protein. This anticryptococcal toxin has the potential to be applied as a therapeutic agent for the treatment of cryptococcosis. PMID- 16681808 TI - Paracoccidioides brasiliensis: attenuation of yeast cells by gamma irradiation. AB - Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America, and currently there is no effective vaccine. The aim of this study was to attenuate the yeast form of P. brasiliensis by gamma irradiation for further studies on vaccine research. Paracoccidioides brasiliensis (strain Pb 18) cultures were irradiated at doses between 0.5 and 8.0 kGy. After each dose the viability, reproductive ability and protein metabolism were evaluated. The comparison between the antigenic profile of irradiated and control yeast was made by Western blot and the virulence evaluated by the inoculation in C(57)Bl/J6 mice. At 6.5 kGy the yeast lost its reproductive capacity. The viability and the incorporation of [L-(35)S]-methionine were the same in control and up to 6.5 kGy irradiated cells, but 6.5 kGy-irradiated yeast secreted 40% less proteins. The Western blot profile was clearly similar in control and 6.5 kGy-irradiated yeast. No colony-forming unit (CFU) could be recovered from the tissues of the mice infected with the radioattenuated yeast. We concluded that for P. brasiliensis yeast it is possible to find a dose in which the pathogen loses its reproductive ability and virulence, while retaining its viability, metabolic activity and the antigenic profile. PMID- 16681809 TI - Epidemiological and aetiological study on tinea pedis and onychomycosis in Algeria. AB - Epidemiological studies on tinea pedis and onychomycosis, common fungal infections, have been conducted in many parts of the world. There are currently no studies of tinea pedis and/or onychomycosis in Algeria. The aim of this paper was to study the epidemiology of foot diseases, including tinea pedis and onychomycosis and to identify the aetiological factors of these infections in outpatients attending the Department of Dermatology of the Central Hospital of Army in Algiers, Algeria. A total of 1300 male subjects, mean age 35.9 +/- 16 years (range: 16-80) were recruited during the period November 2003 to November 2004 and were clinically examined. A complete dermatological examination was performed on all subjects, and skin and nail specimens of the feet were taken from those patients presenting signs of tinea pedis and/or onychomycosis for microscopy and fungal culture. Clinical diagnosis for tinea pedis and onychomycosis was suspected in 249 and 72 subjects, respectively, and confirmed in 197 and 60 cases, respectively, by positive cultures, resulting in a global prevalence of tinea pedis and toenail onychomycosis of 15% and 4.6% respectively. The age groups most commonly infected were 50-59 and 20-29 years. The yeast species Candida parapsilosis and the dermatophytic species Trichophyton rubrum were shown to be the most common pathogens in both tinea pedis (C. parapsilosis 20.4%; T. rubrum 17%) and onychomycosis (T. rubrum 35%; C. parapsilosis 28.3%). This is the first investigation dealing with fungal foot diseases in Algeria. The clinical and epidemiological data collected would serve as reference for future research and may be useful in the development of preventive and educational strategies. PMID- 16681810 TI - Duration and outcome of persistent candidaemia in newborn infants. AB - The aims of the study were to define the duration of candidaemia in newborn infants and to determine the incidence of persistent hospital-acquired candidaemia and its associated morbidity and mortality compared with non persistent candidaemia. This retrospective study, included 56 neonates admitted to the neonatal intensive care unit from 1996 to 2000 who had one or more positive blood cultures for Candida spp. The most prevalent pathogen was Candida albicans (64.3%). Mean disease duration was 6.9 +/- 5.5 days (median 6 days). Twenty-nine patients (52%) had a positive blood culture for >5 days (persistent candidaemia). There were no statistically significant differences between the infants with persistent and non-persistent candidaemia in background or predisposing clinical factors. Fungal endocarditis was present in four patients (13.7%) with persistent disease and one patient (3.7%) with non-persistent disease (odds ratio 4.19), and uveitis developed in one patient. Ten patients (17.8%) died--five with persistent and five with non-persistent disease (P = 1.0). These findings indicate that persistent neonatal candidaemia may be associated with an increased risk of Candida endocarditis, but not with other complications or increased mortality. PMID- 16681811 TI - Stress as a cause of chronic recurrent vulvovaginal candidosis and the effectiveness of the conventional antimycotic therapy. AB - Chronic Candida vulvovaginitis cannot, at present, be treated effectively with local or oral antimycotic medication. Gottlicher & Meyer [Vulvovaginalmykose. Klinische Ergebnisse einer epidemiologischen Langzeitstudie, Thieme, Stuttgart (1998); Mycoses41 (1998) 49] formulated a hypothesis to explain this fact. They say that stress is the main cause of vulvovaginal mycosis, and that frequently cited predisposing somatic risk factors are not causing the illness. Two research projects were carried out to test this hypothesis. The first project was conceived to provide direct evidence for two empirical statements derived from the Gottlicher-Meyer hypothesis: (i) stress as a predictor of vulvovaginitis is least valid as the commonly assumed somatic factors and (ii) combinations of factors that reliably discriminate index women from symptom-free women point to aspects of psychosocial development, particularly stress, and not to somatic factors. Between March and November 1999, 309 successive patients were randomly chosen from those successively treated in one in-patient and two out-patient settings. Of those chosen, 117 had had at least one episode of vaginal candidosis within the 2 years prior to enlistment in the study. The remaining 192 patients had experienced no such illness. For the 117 index patients both of the above empirical statements were confirmed by statistical analyses. The second project was designed to test predictions concerning the ineffectiveness of traditional antimycotic treatment derived from the Gottlicher-Meyer hypothesis. Three different empirical statements were derived from the hypothesis--each based on the assumption of a Poisson distribution of relapses that remains uninfluenced by treatment attempts. Each of the statements was tested and confirmed in independent samples--the first in the sample used in project one, the second in a sample of 206 women insured by public Healths Management Organizations (HMOs) and the third in a sample of 179 women insured by private HMOs. Each patient had evidenced Candida albicans vulvovaginitis at least once in the time interval between 1996 and 2000. Statistical analyses confirmed each of the empirical statements, thereby substantiating the Gottlicher-Meyer hypothesis. The authors conclude that (i) psychosocial factors, particularly stress, are the primary causes of Candida albicans vulvovaginitis. Accompanying somatic factors are of little statistical significance in explaining occurrence and relapse. (ii) Traditional antimycotic treatment influences only the symptoms of the illness, not its causes, and has no effect on the probability of a relapse. Increased treatment effectiveness can be achieved only at the cost of a complete re orientation concerning the causes of vaginal mycosis. Interventions designed to strengthen the patients' immune response are among the most promising. PMID- 16681812 TI - Diagnosis of aspergilloma in a pleural cavity (persistent pneumothorax) using classic imaging methods. AB - The diagnosis of pulmonary aspergillosis is based on serum-analysis, as well as histological and microbiological analysis of bronchial lavage and transbronchial biopsies. When Aspergillus develops within a preformed cavity, however, these tests are likely to be negative. In this situation, classic imaging techniques such as chest X-ray and high resolution-computed tomography (HR-CT) can be of great diagnostic use. We here describe the case of a 62-year-old woman with a history of breast cancer and subsequent ablation of the left breast and radiotherapy. The case demonstrates an example of a pleuropulmonary aspergilloma, in which sero- and micro-biological detection failed. Thorax HR-CT exhibited the cavity, a small persistent pneumothorax, partially filled by an oval density. This density clearly dislocated according to gravity following a positional change of the patient from supine to prone. The density thus revealed mobility which was typical of aspergilloma. Following excision, this diagnosis was confirmed. A density within a cavity may be differentiated by its mobility from differential diagnoses such as lung cancer which would not be expected to exhibit mobility. PMID- 16681813 TI - Onychomycosis in Lebanon: a mycological survey of 772 patients. AB - Accurate diagnosis of onychomycosis is based on clinical findings, direct microscopic investigation and mycological culture. If the diagnosis is not confirmed by culture and improvement does not occur, it is impossible to tell whether this represents treatment failure or an initial incorrect diagnosis. The aim of this study was to identify the major organisms involved in onychomycosis with emphasis on the importance of culture in treating onychomycosis. The study was performed at the Lebanese University, Beirut, Lebanon over a 5-year period (2000-2004). Clinically suspected patients were referred to our mycology laboratory for KOH test and culture. The study included 772 patients (520 women, 252 men). Cultures were positive in 54.3% of cases (predominantly male). The ratio of onychomycosis in toenails/fingernails was 1.9. In toenails, dermatophytes were found in 77.1% of cases, Candida in 18.9% and moulds in 4%. In fingernails, Candida was found in 81% of cases, dermatophytes in 18.1% and moulds in 0.9%. The most commonly isolated dermatophytes were Trichophyton mentagrophytes (36%), T. rubrum (27.5%) and T. tonsurans (26%). Pathogens involved in onychomycosis change according to each geographical area. Therefore, treatments should be based on studies carried out in the same region. PMID- 16681814 TI - In vitro activities of new and conventional antimycotics against fluconazole susceptible and non-susceptible Brazilian Candida spp. isolates. AB - The substantial increase in the rate of azole resistant Candida spp. yeast infections has become a serious treatment problem requiring new and more active antifungal agents. In this study, the in vitro activities of ravuconazole and albaconazole were compared with those of amphotericin B, flucytosine, itraconazole and fluconazole against 162 Brazilian isolates of Candida spp. from which 48 isolates had previously shown lower susceptibility or resistance to fluconazole. Ravuconazole susceptibility ranged from 84.6% (Candida albicans) to 100% for other species and albaconazole MIC(90) was < or =1.0 microg ml(-1) for all the species emphasising the potent activity of these triazoles. To our knowledge this is the first study evaluating the susceptibility of C. dubliniensis to albaconazole. PMID- 16681815 TI - Evaluation of antifungals in the surgical intensive care unit: a multi institutional study. AB - In the USA, >50% of candidemia episodes occur in medical or surgical intensive care units (SICU). However, studies focused on patterns and rationale for antifungal use are lacking. The objective of this study was to evaluate systemic antifungal usage in SICU patients. Retrospective audit of SICU patients receiving antifungal therapy from four American hospitals. Medical records were reviewed for demographics, hospital variables, microbiology results, antifungal regimens and indications for therapy. A total of 2411 patient-days of antifungal use were evaluated in 225 patients. Fluconazole was the most frequently prescribed antifungal (1846 patient-days) followed by amphotericin B deoxycholate (251 patient-days), lipid formulations of amphotericin B (201 patient-days), itraconazole (71 patient-days), and caspofungin (42 patient-days). Antifungals were prescribed empirically (44%), for preemptive therapy in critically ill patients colonised with Candida (43%), or for candidiasis (12%). Candida species were recovered from 98% of patients with positive fungal cultures most commonly from pulmonary (53%) or urinary sources (17%). Fluconazole is the most frequently prescribed antifungal agent in SICUs and is most often prescribed for empiric or preemptive indications. Research efforts to identify patients who warrant preemptive antifungal therapy for invasive candidiasis could dramatically change antifungal prescribing patterns in the SICU. PMID- 16681816 TI - Efficacy of topical griseofulvin in treatment of tinea corporis. AB - Tinea infections are among the most common dermatological conditions throughout the world. Griseofulvin is a classical oral fungistatic antibiotic, active against Epidermophyton floccosum, Trichophyton and Microsporum species, the causative fungi of tinea corporis. To evaluate the efficacy of topical griseofulvin in the treatment of tinea circinata using three different vehicles for drug delivery. Sixteen patients with tinea circinata were instructed to apply either griseofulvin gel form in group A or a similar placebo gel for control group; a niosomal gel formulation of griseofulvin for group B or; a liposomal gel formulation of griseofulvin for group C. Patients were evaluated both clinically and mycologically after 3 weeks. Marked improvement was seen for groups A, B and C both clinically and mycologically while no improvement was observed in the placebo group. Mild and transient irritation was reported in four patients. Our results show that topical griseofulvin preparations may be effective and safe in treating tinea circinata and that further large-scale studies may establish the high efficacy of the niosomal gel formulation. PMID- 16681817 TI - A comparative clinical study between 2 weeks of luliconazole 1% cream treatment and 4 weeks of bifonazole 1% cream treatment for tinea pedis. AB - The aim of the study was to compare the efficacy and safety of luliconazole 1% cream and bifonazole 1% cream as applied in the treatment of tinea pedis (interdigital-type and plantar-type). A multi-clinic, randomised single-blind, parallel group study with 34 hospitals and 11 clinics formed the study design. Five hundred and eleven patients with mycologically confirmed tinea pedis were included. Of the 489 evaluable patients, 247 were randomised to luliconazole, and 242 to bifonazole. Luliconazole 1% cream applied once a day for 2 weeks, followed by a placebo cream for 2 weeks, thereafter. Bifonazole 1% cream applied once a day for 4 weeks. Mycological effect (negative result on microscopy) and improvement of skin lesions were measured at weeks 1, 2, 3 and 4. Safety frequency and severity of adverse reactions were also measured. The improvement of skin lesions after 4 weeks was comparably good with rates of 91.5% vs. 91.7% (luliconazole vs. bifonazole). The mycological effect was characterised by high negative rates of 76.1% vs. 75.9% (luliconazole vs. bifonazole). The progression of tinea-related signs and symptom scores differed insignificantly between evaluated luliconazole and bifonazole treatment groups comprising a total of 500 patients. Both substances appeared to be comparably safe and well-tolerated. PMID- 16681818 TI - Successful caspofungin treatment of multidrug resistant Candida parapsilosis septicaemia in an extremely low birth weight neonate. AB - Candida septicaemia with multidrug resistance is an uncommon event in preterm neonates. We present an extremely low birth weight infant (gestational age of 27 weeks, birth weight of 980 g) who developed congenital Candida parapsilosis septicaemia. Because the fungus was resistant both to amphotericin B and fluconazole, caspofungin was chosen for therapy. The fungus was successfully eradicated without any clinical or laboratory adverse effects. PMID- 16681819 TI - First case report of empyema caused by Beauveria bassiana. AB - Infections with Beauveria bassiana are extremely rare in humans. A 51-year-old man was admitted to hospital with the diagnosis of lung adenocarcinoma. Thoracic wall resection with lobectomy was applied and empyema has developed after prolonged air leakage. B. bassiana was isolated in pleural fluid. The patient improved without antifungal therapy after thoracotomy with securing of air leakage. PMID- 16681820 TI - A case of bullous tinea pedis with dermatophytid reaction caused by Trichophyton violaceum. AB - Summary The authors report a case of bullous tinea pedis caused by Trichophyton violaceum with dermatophytid reaction in a 26-year-old woman who had been in Africa. Diagnosis was based on mycological examination (direct microscope observation and culture). Recovery was achieved after systemic antimycotic therapy with itraconazole and topical imidazole for a month. PMID- 16681821 TI - Successful treatment of Trichosporon mucoides infection with lipid complex amphotericin B and 5-fluorocytosine. AB - Infections in immunocompromised children can stem from bacteria, fungi, viruses, or protozoa, but most importantly, from the host's endogenous bacterial flora. Disseminated infection caused by Trichosporon species is one of the emerging mycoses in neutropenic patients, particularly when they are treated for haematological malignancy with cytotoxic and immunosuppressive chemotherapy. We report a 15-year-old boy with acute lymphoblastic leukaemia, whose Trichosporon mucoides infection was successfully treated with lipid complex amphotericin B plus 5-fluorocytosine. PMID- 16681822 TI - Onychomycosis in a diabetic patient due to Trichophyton gallinae. AB - A 67-year-old female suffering from diabetes mellitus type I revealed brownish yellow discolouration, subungual hyperkeratosis, and onychodystrophy of several finger nails. Upon culturing of nail specimens, Trichophyton gallinae could be repeatedly identified on Sabouraud glucose agar. To our knowledge, this is the first report of a human infection caused by this species in Germany. T. gallinae is a zoophilic dermatophyte that primarily infects higher animals, but can also be transmitted to humans sporadically. Herein, clinical, diagnostic, and epidemiological aspects of this zoophilic dermatophyte are briefly reviewed. PMID- 16681825 TI - Adoptive T-cell transfer in cancer immunotherapy. AB - Adoptive T-cell therapy has definite clinical benefit in relapsed leukaemia after allogeneic transplant and in Epstein-Barr virus-associated post-transplant lymphoproliferative disease. However, the majority of tumour targets are weakly immunogenic self-antigens and success has been limited in part by inadequate persistence and expansion of transferred T cells and by tumour-evasion strategies. Adoptive immunotherapy presents the opportunity to activate, expand and genetically modify T cells outside the tolerising environment of the host and a number of strategies to optimize the cellular product, including gene modification and modulation of the host environment, in particular by lymphodepletion, have been developed. PMID- 16681826 TI - Some considerations on the use of peptides and mRNA for therapeutic vaccination against cancer. AB - Active immunization against existing cancer is a field that is currently in development and is associated with a number of problems. The potential use of peptides as minimal essential T-cell antigens and of mRNA as a novel form of antigen with advantages is discussed, with special consideration of practical aspects. PMID- 16681827 TI - Immunological characteristics correlating with clinical response to immunotherapy in patients with advanced metastatic melanoma. AB - Current treatment options for advanced metastatic melanoma are limited to experimental regimen that provide poor survival outcomes. Immunotherapy is a promising alternative and we recently reported a clinical trial in which 6 out of 19 patients enrolled had objective clinical responses to a fully autologous melanoma/dendritic cell vaccine. The mechanism of the vaccine is not well understood, but we hypothesized that general immunocompetence may be a determinant of clinical response. We therefore examined the immune status of an expanded series of 21 patients who displayed varying clinical responses to the melanoma/dendritic cell vaccine. Immunocompetence was assessed using in vitro assays of lymphocyte function: survival, proliferation and cytokine responses to mitogen stimulation as well as T-cell receptor zeta expression and lymphocyte subset analysis. Although lymphocytes from patients mostly performed comparably to age-matched and sex-matched controls, in some assays we identified significant differences between complete clinical responders and other patients, both before and following vaccination. Surprisingly, before vaccination, only lymphocytes from clinical responder patients showed impaired in vitro survival. Following vaccination, T lymphocyte survival improved and cells recovered their ability to produce the Th1-associated cytokines TNF and IFN-gamma in response to anti-CD3 stimulation in vitro. No increase in Th1 cytokine production was observed in lymphocytes from patients who experienced partial clinical responses or progressive disease. We conclude that, before vaccination, patients who go on to have complete responses have immune characteristics suggestive of high cell turnover and low Th1-associated cytokine production, and that these can be reversed with vaccination. These results have potential implications for future immunotherapeutic strategies. PMID- 16681828 TI - Directions in the immune targeting of cancer: lessons learned from the cancer testis Ag NY-ESO-1. AB - Since the early 1990s, numerous cancer Ag have been defined and for a handful of these there is now some clinical experience, which has made it possible to assess their value as targets for cancer immunotherapy. The cancer-testis Ag have been particularly attractive because their expression is limited to cancer and virtually no non-malignant cells apart from germ cells and trophoblast. Among these, NY-ESO-1 has been the focus of our attention. The exceptional immunogenicity of this Ag coupled with its widespread distribution among many cancer types make it a very good vaccine candidate, with the potential to be used in vaccines against many types of malignancies. This article reviews emerging knowledge about the biology of NY-ESO-1 and experience with the early clinical development of vaccines directed against NY-ESO-1. These early studies have yielded a wealth of information about the immunology of NY-ESO-1 and set the scene for future clinical strategies for immune targeting of cancer. PMID- 16681829 TI - Reverse immunology approach for the identification of CD8 T-cell-defined antigens: advantages and hurdles. AB - One of the challenges of tumour immunology remains the identification of strongly immunogenic tumour antigens for vaccination. Reverse immunology, that is, the procedure to predict and identify immunogenic peptides from the sequence of a gene product of interest, has been postulated to be a particularly efficient, high-throughput approach for tumour antigen discovery. Over one decade after this concept was born, we discuss the reverse immunology approach in terms of costs and efficacy: data mining with bioinformatic algorithms, molecular methods to identify tumour-specific transcripts, prediction and determination of proteasomal cleavage sites, peptide-binding prediction to HLA molecules and experimental validation, assessment of the in vitro and in vivo immunogenic potential of selected peptide antigens, isolation of specific cytolytic T lymphocyte clones and final validation in functional assays of tumour cell recognition. We conclude that the overall low sensitivity and yield of every prediction step often requires a compensatory up-scaling of the initial number of candidate sequences to be screened, rendering reverse immunology an unexpectedly complex approach. PMID- 16681832 TI - Autophagy and antigen presentation. AB - CD4(+) T cells co-ordinate adaptive immunity and are required for immunological memory establishment and maintenance. They are thought to primarily recognize extracellular antigens, which are endocytosed, processed by lysosomal proteases and then presented on major histocompatibility complex (MHC) class II. However, recent studies have demonstrated that viral, tumour and autoantigens can gain access to this antigen presentation pathway from within cells by autophagy. This review will discuss the autophagic pathways that contribute to endogenous MHC class II antigen processing. Furthermore, potential characteristics of autophagy substrates, qualifying them to access these pathways, and regulation of autophagy will be considered. Finally, I will suggest how antigen presentation after autophagy might contribute to immune surveillance of infected and transformed cells. PMID- 16681833 TI - Autophagy and plant innate immunity. AB - Plant innate immunity is often associated with specialized programmed cell death at or near the site of pathogen infection. Despite the isolation of several lesion mimic mutants, the molecular mechanisms that regulate cell death during an immune response remain obscure. Recently, autophagy, an evolutionarily conserved process of bulk protein and organelle turnover, was shown to play an important role in limiting cell death initiated during plant innate immune responses. Consistent with its role in plants, several studies in animals also demonstrate that the autophagic machinery is involved in innate as well as adaptive immunities. Here, we review the role of autophagy in plant innate immunity. Because autophagy is observed in healthy and dying plant cells, we will also examine whether autophagy plays a protective or a destructive role during an immune response. PMID- 16681834 TI - Interferon signalling network in innate defence. AB - Interferons (IFNs) elicit multifaceted effects in host innate defence. Accumulating evidence revealed that not only the first identified Jak-Stat pathway but also other newly found signalling pathways are required for the induction of versatile responses by IFNs. In particular, type I IFNs are inducible by viral infection through the recognition of pathogen-associated molecules by pattern recognition receptors, and the induction of multiple IFN stimulated genes through the activation of type I IFN signalling confers antiviral and immunomodulatory activities. Any step in this process is often targeted by viruses for their immuno-evasion. The regulatory function of constitutive IFN-alpha/beta signalling has been recognized in terms of its boosting effect on cellular responsiveness in host defence systems. Further comprehensive understanding of IFN signalling may offer a better direction to unravelling the complex signalling networks in the host defence system, and may contribute to their more effective therapeutic applications. PMID- 16681836 TI - Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages. AB - After internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago-lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic'Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process. PMID- 16681835 TI - Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci. AB - A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Deltaorf1 and Deltaorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Deltaorf1 mutant using aspartic N peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Deltaorf1, Deltaorf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule. PMID- 16681837 TI - Niche-specific regulation of central metabolic pathways in a fungal pathogen. AB - To establish an infection, the pathogen Candida albicans must assimilate carbon and grow in its mammalian host. This fungus assimilates six-carbon compounds via the glycolytic pathway, and two-carbon compounds via the glyoxylate cycle and gluconeogenesis. We address a paradox regarding the roles of these central metabolic pathways in C. albicans pathogenesis: the glyoxylate cycle is apparently required for virulence although glyoxylate cycle genes are repressed by glucose at concentrations present in the bloodstream. Using GFP fusions, we confirm that glyoxylate cycle and gluconeogenic genes in C. albicans are repressed by physiologically relevant concentrations of glucose, and show that these genes are inactive in the majority of fungal cells infecting the mouse kidney. However, these pathways are induced following phagocytosis by macrophages or neutrophils. In contrast, glycolytic genes are not induced following phagocytosis and are expressed in infected kidney. Mutations in all three pathways attenuate the virulence of this fungus, highlighting the importance of central carbon metabolism for the establishment of C. albicans infections. We conclude that C. albicans displays a metabolic program whereby the glyoxylate cycle and gluconeogenesis are activated early, when the pathogen is phagocytosed by host cells, while the subsequent progression of systemic disease is dependent upon glycolysis. PMID- 16681838 TI - The enteropathogenic Escherichia coli EspF effector molecule inhibits PI-3 kinase mediated uptake independently of mitochondrial targeting. AB - Delivery of effector molecules into LMme(v) macrophages by enteropathogenic Escherichia coli, via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol-3 (PI-3) kinase-dependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE- and non-LEE-encoded effector proteins into host cells. Previous studies discounted essential roles for the LEE-encoded Map, EspF, Tir or Intimin proteins in this process but correlated it with loss of phosphorylation of the PI 3 kinase substrate, Akt (Celli et al., 2001, EMBO J 20: 1245-1258). Given the more recent finding that these bacterial proteins are multifunctional and can act together to subvert host cellular processes, we generated a quadruple deletion mutant (Map, Tir, EspF and Intimin deficient) to unearth any cooperativity in inhibiting uptake. The quadruple mutant was as defective as the T3SS-defective strain at preventing bacterial uptake with further studies revealing a surprising dependence on EspF but not Map, Tir or Intimin. Subversive activities previously associated with EspF are disruption of epithelial barrier function and programmed cell death, with the latter linked to EspF targeting mitochondria. Interestingly, the C-terminal domain possesses a polyproline motif associated with protein protein interactions. We demonstrate that EspF-mediated inhibition of PI-3 kinase dependent uptake: (i) is independent of mitochondrial targeting, (ii) requires the N-terminal domain with and (iii) the C-terminal domain is sufficient to disrupt barrier function but not inhibition of bacterial uptake. Moreover, loss of PI-3 kinase-dependent phosphorylation of Akt and gross changes in host phosphotyrosine protein profiles could not be linked to inhibition of the PI-3 kinase-dependent uptake process. PMID- 16681839 TI - Bacterial-associated cholera toxin and GM1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system. AB - To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae, we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM1. Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre incubation with GM1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells. PMID- 16681840 TI - A novel category of enteropathogenic Escherichia coli simultaneously utilizes the Nck and TccP pathways to induce actin remodelling. AB - Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) induce drastic reorganization of the microfilament cytoskeleton. EHEC and EPEC translocate Tir (translocated intimin receptor) which, once inserted into the host plasma membrane, binds the bacterial outer membrane adhesin intimin. Tir(EPEC) then becomes tyrosine phosphorylated facilitating the recruitment and site-specific binding of the eukaryotic adaptor Nck, which in turn binds and activates the Wiskott-Aldrich syndrome protein (N-WASP), leading to actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. In contrast, Tir(EHEC) has no Nck binding site; instead, EHEC utilizes the translocated effector TccP (Tir-cytoskeleton coupling protein) to bind and activate N-WASP. Here we report a novel class of EPEC that translocates both TccP and Tir(EPEC) like effector molecules. Consistent with these characteristics, we show that both the Tir-Nck and Tir:TccP actin remodelling pathways function simultaneously during infection, making this a novel and versatile EPEC category. PMID- 16681841 TI - Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. AB - Localized cell wall modification and accumulation of antimicrobial compounds beneath sites of fungal attack are common mechanisms for plant resistance to fungal penetration. In barley (Hordeum vulgare) leaves, light-microscopically visible vesicle-like bodies (VLBs) containing H(2)O(2) or phenolics frequently accumulate around cell wall appositions (syn. papillae), in which the penetration attempt of the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) is halted. By ultrastructural analyses, we demonstrated that the Bgh induced VLBs represent different structures. VLBs intensively stained by H(2)O(2) reactive dyes were actually small papillae instead of cytoplasmic vesicles. Other VLBs were identified as osmiophilic bodies or multivesicular compartments, designated paramural bodies (PMBs) and multivesicular bodies (MVBs). MVBs seemingly followed two distinct pathways: either they were engulfed by the tonoplast for degradation in the vacuole or they fused with the plasma membrane to release their internal vesicles into the paramural space and hence could be the origin of PMBs. MVBs and PMBs appeared to be multicomponent kits possibly containing building blocks to be readily assembled into papilla and antimicrobial compounds to be discharged against fungal penetration. Finally, we propose that released paramural vesicles might be similar to exosomes in animal cells. PMID- 16681842 TI - Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis. AB - YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence. PMID- 16681843 TI - Critical roles for excretory-secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae. AB - Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue-invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory-secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans-epoxysuccinyl-L-leuciloamido-(4-guanidino) butane (E 64) inhibited this process dose-dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion-secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host's tissues, suggesting their relevant function to tissue invasion/migration in the definitive host. PMID- 16681844 TI - Stimulation of the cytosolic receptor for peptidoglycan, Nod1, by infection with Chlamydia trachomatis or Chlamydia muridarum. AB - Infection of epithelial cells by the intracellular pathogen, Chlamydia trachomatis, leads to activation of NF-kappaB and secretion of pro-inflammatory cytokines. We find that overexpression of a dominant-negative Nod1 or depletion of Nod1 by RNA interference inhibits partially the activation of NF-kappaB during chlamydial infection in vitro, suggesting that Nod1 can detect the presence of Chlamydia. In parallel, there is a larger increase in the expression of pro inflammatory genes following Chlamydia infection when primary fibroblasts are isolated from wild-type mice than from Nod1-deficient mice. The Chlamydia genome encodes all the putative enzymes required for proteoglycan synthesis, but proteoglycan from Chlamydia has never been detected biochemically. Since Nod1 is a ubiquitous cytosolic receptor for peptidoglycan from Gram-negative bacteria, our results suggest that C. trachomatis and C. muridarum do in fact produce at least the rudimentary proteoglycan motif recognized by Nod1. Nonetheless, Nod1 deficiency has no effect on the efficiency of infection, the intensity of cytokine secretion, or pathology in vaginally infected mice, compared with wild type controls. Similarly, Rip2, a downstream mediator of Nod1, Toll-like receptor (TLR)-2, and TLR4, increases only slightly the intensity of chlamydial infection in vivo and has a very mild effect on the immune response and pathology. Thus, Chlamydia may not produce sufficient peptidoglycan to stimulate Nod1-dependent pathways efficiently in infected animals, or other receptors of the innate immune system may compensate for the absence of Nod1 during Chlamydia infection in vivo. PMID- 16681845 TI - Analysing ethnobotanical and fishery-related importance of mangroves of the East Godavari Delta (Andhra Pradesh, India) for conservation and management purposes. AB - Mangrove forests, though essentially common and wide-spread, are highly threatened. Local societies along with their knowledge about the mangrove also are endangered, while they are still underrepresented as scientific research topics. With the present study we document local utilization patterns, and perception of ecosystem change. We illustrate how information generated by ethnobiological research can be used to strengthen the management of the ecosystem. This study was conducted in the Godavari mangrove forest located in the East-Godavari District of the state Andhra Pradesh in India, where mangroves have been degrading due to over-exploitation, extensive development of aquaculture, and pollution from rural and urbanized areas (Kakinada).One hundred interviews were carried out among the fisherfolk population present in two mangrove zones in the study area, a wildlife sanctuary with strong conservation status and an adjacent zone. Results from the interviews indicated that Avicennia marina (Forsk.) Vierh., a dominant species in the Godavari mangroves, is used most frequently as firewood and for construction. Multiple products of the mangrove included the bark of Ceriops decandra (Griff.) Ding Hou to dye the fishing nets and improve their durability, the bark of Aegiceras corniculatum (L.) Blanco to poison and catch fish, and the leaves of Avicennia spp. and Excoecaria agallocha L. as fodder for cattle. No medicinal uses of true mangrove species were reported, but there were a few traditional uses for mangrove associates. Utilization patterns varied in the two zones that we investigated, most likely due to differences in their ecology and legal status. The findings are discussed in relation with the demographic and socio-economic traits of the fisherfolk communities of the Godavari mangroves and indicate a clear dependency of their livelihood on the mangrove forest.Reported changes in the Godavari mangrove cover also differed in the two zones, with significantly less perceptions of a decrease in the protected area, as compared to the adjacent non protected area. A posteriori comparisons between sequential satellite imagery (retrospective till 1977) and respondents that were at least 15 years back then, revealed a mangrove decrease which was however perceived to different extents depending on the area with which the fishermen were familiar. While local needs had not been incorporated in the existing policy, we created a framework on how data on ethnobotanical traditions, fishery-related activities and local people's perceptions of change can be incorporated into management strategies. PMID- 16681846 TI - Satisfaction survey with DNA cards method to collect genetic samples for pharmacogenetics studies. AB - BACKGROUND: Pharmacogenetic studies are essential in understanding the interindividual variability of drug responses. DNA sample collection for genotyping is a critical step in genetic studies. A method using dried blood samples from finger-puncture, collected on DNA-cards, has been described as an alternative to the usual venepuncture technique. The purpose of this study is to evaluate the implementation of the DNA cards method in a multicentre clinical trial, and to assess the degree of investigators' satisfaction and the acceptance of the patients perceived by the investigators. METHODS: Blood samples were collected on DNA-cards. The quality and quantity of DNA recovered were analyzed. Investigators were questioned regarding their general interest, previous experience, safety issues, preferences and perceived patient satisfaction. RESULTS: 151 patients' blood samples were collected. Genotyping of GST polymorphisms was achieved in all samples (100%). 28 investigators completed the survey. Investigators perceived patient satisfaction as very good (60.7%) or good (39.3%), without reluctance to finger puncture. Investigators preferred this method, which was considered safer and better than the usual methods. All investigators would recommend using it in future genetic studies. CONCLUSION: Within the clinical trial setting, the DNA-cards method was very well accepted by investigators and patients (in perception of investigators), and was preferred to conventional methods due to its ease of use and safety. PMID- 16681847 TI - Applying dynamic Bayesian networks to perturbed gene expression data. AB - BACKGROUND: A central goal of molecular biology is to understand the regulatory mechanisms of gene transcription and protein synthesis. Because of their solid basis in statistics, allowing to deal with the stochastic aspects of gene expressions and noisy measurements in a natural way, Bayesian networks appear attractive in the field of inferring gene interactions structure from microarray experiments data. However, the basic formalism has some disadvantages, e.g. it is sometimes hard to distinguish between the origin and the target of an interaction. Two kinds of microarray experiments yield data particularly rich in information regarding the direction of interactions: time series and perturbation experiments. In order to correctly handle them, the basic formalism must be modified. For example, dynamic Bayesian networks (DBN) apply to time series microarray data. To our knowledge the DBN technique has not been applied in the context of perturbation experiments. RESULTS: We extend the framework of dynamic Bayesian networks in order to incorporate perturbations. Moreover, an exact algorithm for inferring an optimal network is proposed and a discretization method specialized for time series data from perturbation experiments is introduced. We apply our procedure to realistic simulations data. The results are compared with those obtained by standard DBN learning techniques. Moreover, the advantages of using exact learning algorithm instead of heuristic methods are analyzed. CONCLUSION: We show that the quality of inferred networks dramatically improves when using data from perturbation experiments. We also conclude that the exact algorithm should be used when it is possible, i.e. when considered set of genes is small enough. PMID- 16681848 TI - Treatment of non-small cell lung cancer with intensity-modulated radiation therapy in combination with cetuximab: the NEAR protocol (NCT00115518). AB - BACKGROUND: Even today, treatment of Stage III NSCLC still poses a serious challenge. So far, surgical resection is the treatment of choice. Patients whose tumour is not resectable or who are unfit to undergo surgery are usually referred to a combined radio-chemotherapy. However, combined radio-chemotherapeutic treatment is also associated with sometimes marked side effects but has been shown to be more efficient than radiation therapy alone. Nevertheless, there is a significant subset of patients whose overall condition does not permit administration of chemotherapy in a combined-modality treatment. It could be demonstrated though, that NSCLCs often exhibit over-expression of EGF-receptors hence providing an excellent target for the monoclonal EGFR-antagonist cetuximab (Erbitux) which has already been shown to be effective in colorectal as well as head-and-neck tumours with comparatively mild side-effects. METHODS/DESIGN: The NEAR trial is a prospective phase II feasibility study combining a monoclonal EGF receptor antibody with loco-regional irradiation in patients with stage III NSCLC. This trial aims at testing the combination's efficacy and rate of development of distant metastases with an accrual of 30 patients. Patients receive weekly infusions of cetuximab (Erbitux) plus loco-regional radiation therapy as intensity-modulated radiation therapy. After conclusion of radiation treatment patients continue to receive weekly cetuximab for 13 more cycles. DISCUSSION: The primary objective of the NEAR trial is to evaluate toxicities and feasibility of the combined treatment with cetuximab (Erbitux) and IMRT loco regional irradiation. Secondary objectives are remission rates, 3-year-survival and local/systemic progression-free survival. PMID- 16681849 TI - The health of people classified as lesbian, gay and bisexual attending family practitioners in London: a controlled study. AB - BACKGROUND: The morbidity of gay, lesbian or bisexual people attending family practice has not been previously assessed. We compared health measures of family practice attendees classified as lesbian, gay and bisexual. METHODS: We conducted a cross-sectional, controlled study conducted in 13 London family practices and compared the responses of 26 lesbian and 85 bisexual classified women, with that of 934 heterosexual classified women and 38 gay and 23 bisexual classified men with that of 373 heterosexual classified men. Our outcomes of interest were: General health questionnaire; CAGE questionnaire; short form12; smoking status; sexual experiences during childhood; number of sexual partners and sexual function and satisfaction. RESULTS: In comparison to people classified as heterosexuals: men classified as gay reported higher levels of psychological symptoms (OR 2.48, CI 1.05-5.90); women classified as bisexual were more likely to misuse alcohol (OR 2.73, 1.70-4.40); women classified as bisexual (OR 2.53, 1.60-4.00) and lesbian (OR 3.13, 1.41-6.97) and men classified as bisexual (OR 2.48, 1,04, 5.86) were more likely to be smokers and women classified as bisexual (OR 3.27, 1.97-5.43) and men classified as gay (OR 4.86, 2.28-10.34) were much more likely to report childhood sexual experiences in childhood. Psychological distress was associated with reporting sexual experiences in childhood in men classified as gay and bisexual and women classified as heterosexual. Men classified as bisexual (OR 5.00, 1.73-14.51) and women classified as bisexual (OR 2.88, 1.24- 6.56) were more likely than heterosexuals to report more than one sexual partner in the preceding four weeks. Lesbian, gay and bisexual classified people encountered no more sexual function problems than heterosexuals but men classified as bisexual (OR 2.74, 1.12-6.70) were more dissatisfied with their sex lives. CONCLUSION: Bisexual and lesbian classified people attending London general practices were more likely to be smokers and gay classified men were at increased risk of psychological distress in comparison to heterosexual classified people. Increased awareness of the sexuality of people seen in primary care can provide opportunities for health promotion. PMID- 16681850 TI - Evidence of the exploitation of marine resource by the terrestrial insect Scapteriscus didactylus through stable isotope analyzes of its cuticle. AB - BACKGROUND: About 4 x 10(5) eggs in more than 5000 marine turtle nests are deposited every year on a 3.6 km long beach in French Guiana (South America). The dry biomass of eggs is estimated to be 5 x 10(3) kg, yet only 25% of this organic matter will return to the ocean in the form of hatchlings. Such amounts of organic matter are supposed to drive the functioning of the beach ecosystem. Previous studies have shown that egg predators and detritivorous organisms dominate the trophic relationships and the dynamics of the system. The role of a terrestrial insect Scapteriscus didactylus (Latreille), which damages up to 40% of the eggs of the marine turtle (Dermochelys coriacea), was unexpected. However it was impossible from direct observations to prove that the mole cricket consumed a significant amount of these eggs. Therefore, the precise place of the mole cricket in the nitrogen and carbon cycles of the beach ecosystem could not be determined. In order to answer this question, we looked for a marine signature of carbon and nitrogen source metabolized by the mole cricket. RESULTS: This study estimated the individual variability of delta13C and delta15N in the cuticle of Scapteriscus didactylus. The isotopic signature was compared between individuals collected at two sites: a village where mole crickets fed on human food scraps and the nearby Awala-Yalimapo beach, where food availability depends seasonally on the nesting sea turtles. The mole crickets collected near the habitations garbage showed no significant variations in the stable isotopic signature, within-and between age groups. On the contrary, isotopic values shifted from a signature of a terrestrial herbivorous diet in the mole crickets during early developmental stages, to isotopic values in adults in accordance with the exploitation of marine animal resources. CONCLUSION: The heterogeneity of individual signatures during the year is due to a selective exploitation of the food sources, differing in space and time. Some individuals, from the beach sample consumed a sufficient quantity of turtle eggs to induce the increase of isotopic enrichment observed in the cuticle. Scapteriscus didactylus is an opportunist feeder and plays a role in the turn over of the beach organic matter. PMID- 16681851 TI - Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model. AB - BACKGROUND: To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation. METHODS: Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system. RESULTS: Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium (p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group. CONCLUSION: Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research. PMID- 16681852 TI - Polyp in concha bullosa: a case report and review of the literature. AB - Polyp originating within a concha bullosa is uncommon; we report only the third such case in the English literature. A 45-year-old man presented with nasal obstruction and headache. Examination of the nose revealed right septal deviation and a hypertrophic left middle concha. Computed tomography confirmed right septal deviation and identified left concha bullosa with thickening of the mucosa covering this lesion. The lateral lamella of the affected turbinate was removed and a mass was excised. Histopathologic examination of the excised mass revealed polypoid hyperplasia. The rare finding of polyp in concha bullosa is discussed with a review of the literature. In any case of concha bullosa, computed tomography images should be carefully evaluated before surgery to check for other pathologies that might have arisen within the lesion. PMID- 16681854 TI - Identifying systematic reviews of the adverse effects of health care interventions. AB - BACKGROUND: In order to carry out a methodological research survey of systematic reviews of adverse effects we needed to retrieve a sample of systematic reviews in which the primary outcome is an adverse effect or effects. METHODS: We carried out searches of the Database of Abstracts of Reviews of Effects (DARE) and the Cochrane Database of Systematic Reviews (CDSR) for systematic reviews of adverse effects published between 1994 to 2005. The search strategies used a combination of text words in the title and abstract, Medical Subject Headings (MeSH) and subheadings/qualifiers. In addition, DARE records in progress were hand searched. No language restrictions were placed on any of the searches. The performance, in terms of sensitivity and precision, of the search strategies and their combinations were tested in DARE and CDSR. RESULTS: In total 3635 records were screened of which 257 met our inclusion criteria. The precision of the searches in CDSR was low (0% to 3%), and no one search strategy could retrieve all the relevant records in either DARE or CDSR. Hand searching the records from DARE and CDSR not retrieved by our searches indicated that we had missed relevant systematic reviews in both DARE and CDSR. The sensitivities of many of the search combinations were comparable to those found when searching for primary studies in which adverse effects are secondary outcomes. CONCLUSION: Searching major databases of systematic reviews, for systematic reviews of adverse effects, proved more difficult than anticipated due to a lack of standard terminology used by the authors, inadequate indexing and the variations in the search interfaces of the databases. At present hand searching all records in DARE and CDSR seems to be the only way to ensure retrieval of all systematic reviews of adverse effects in these databases. PMID- 16681853 TI - HER 2/neu protein expression in colorectal cancer. AB - BACKGROUND: Conflicting data exist about the prevalence of HER-2/neu overexpression in colorectal cancer ranging from 0 to 83 %. In our study we tried to clarify the extent of expression and its relationship to clinicopathological parameters. METHODS: This study involved 77 specimens of malignant colorectal cancer lesions of surgically resected patients. HER-2/neu immunohistochemistry was performed using the Hercep-Test Kit. RESULTS: Out of 77 specimens, 56 were Her-2/neu negative (70%), 20 (26%) showed a barely immunostaining (1+), only 1 (1%) was moderately (2+) and 2 (3%) were strongly positive (3+). Her-2/neu staining (moderately and strongly positive) was only detected in primary tumours of patients with confirmed metastases. No relationship was found between membranous HER-2 expression and patients' gender or differentiation. The median survival time of patients with positive HER-2/neu immunostaining was 21 versus 39 months in patients without HER-2/neu expression (p = 0.088). CONCLUSION: The c erbB protein expression was observed in colorectal cancer but rarely in the therapeutic range (2+ and 3+). There was no significant association with tumour grade, gender, localization of the primary tumour or survival. These data indicate that c-erbB-2 is unlikely to play a major role in the therapeutic management of colorectal cancer. PMID- 16681855 TI - Characterization of three different sensory fibers by use of neonatal capsaicin treatment, spinal antagonism and a novel electrical stimulation-induced paw flexion test. AB - In the present study, we first report an in vivo characterization of flexor responses induced by three distinct sine-wave stimuli in the electrical stimulation-induced paw flexion (EPF) test in mice. The fixed sine-wave electric stimulations of 5 Hz (C-fiber), 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber) to the hind paw of mice induced a paw-flexion response and vocalization. The average threshold for paw flexor responses by sine-wave stimulations was much lower than that for vocalization. Neonatally (P3) pretreatment with capsaicin to degenerate polymodal substance P-ergic C-fiber neurons increased the threshold to 5 Hz (C-fiber) stimuli, but not to 250 Hz (Adelta-fiber) and 2000 Hz (Abeta fiber). The flexor responses to 5 Hz stimuli were significantly blocked by intrathecal (i.t.) pretreatment with both CP-99994 and MK-801, an NK1 and NMDA receptor antagonist, respectively, but not by CNQX, an AMPA/kainate receptor antagonist. On the other hand, the flexor responses induced by 250 Hz stimuli were blocked by MK-801 (i.t.) but not by CP-99994 or CNQX. In contrast, flexor responses induced by 2000 Hz stimuli were only blocked by CNQX treatment. These data suggest that we have identified three pharmacologically different categories of responses mediated through different primary afferent fibers. Furthermore, we also carried out characterization of the in vivo functional sensitivity of each of the sensory fiber types in nerve-injured mice using the EPF test, and found that the threshold to both 250 Hz and 2000 Hz stimulations were markedly decreased, whereas the threshold to 5 Hz stimulations was significantly increased. Thus we found opposing effects on specific sensory fiber-mediated responses as a result of nerve injury in mice. These results also suggest that the EPF analysis is useful for the evaluation of plasticity in sensory functions in animal disease models. PMID- 16681856 TI - Costing the distribution of insecticide-treated nets: a review of cost and cost effectiveness studies to provide guidance on standardization of costing methodology. AB - BACKGROUND: Insecticide-treated nets (ITNs) are an effective and cost-effective means of malaria control. Scaling-up coverage of ITNs is challenging. It requires substantial resources and there are a number of strategies to choose from. Information on the cost of different strategies is still scarce. To guide the choice of a delivery strategy (or combination of strategies), reliable and standardized cost information for the different options is required. METHODS: The electronic online database PubMed was used for a systematic search of the published English literature on costing and economic evaluations of ITN distribution programmes. The keywords used were: net, bednet, insecticide, treated, ITN, cost, effectiveness, economic and evaluation. Identified papers were analysed to determine and evaluate the costing methods used. Methods were judged against existing standards of cost analysis to arrive at proposed standards for undertaking and presenting cost analyses. RESULTS: Cost estimates were often not readily comparable or could not be adjusted to a different context. This resulted from the wide range of methods applied and measures of output chosen. Most common shortcomings were the omission of certain costs and failure to adjust financial costs to generate economic costs. Generalisability was hampered by authors not reporting quantities and prices of resources separately and not examining the sensitivity of their results to variations in underlying assumptions. CONCLUSION: The observed shortcomings have arisen despite the abundance of literature and guidelines on costing of health care interventions. This paper provides ITN specific recommendations in the hope that these will help to standardize future cost estimates. PMID- 16681857 TI - Characterization of sensory neuron subpopulations selectively expressing green fluorescent protein in phosphodiesterase 1C BAC transgenic mice. AB - BACKGROUND: The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord. RESULTS: Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X3. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn. CONCLUSION: The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons. PMID- 16681858 TI - T-cell lymphoproliferative disorder of hand-mirror cell morphology presenting in an eosinophilic loculated peritoneal effusion, with omental "caking". AB - BACKGROUND: Cells with "hand mirror" morphology have not, to the best of our knowledge, been described in a primary effusion sample. This paper describes a case of T-cell lymphoma with eosinophilia in a patient with suspected peritoneal carcinomatosis. Rarely, a T-cell lymphoproliferative process may mimic primary peritoneal carcinomatosis, clinically suggested by a presentation in CT imaging of omental caking with bilateral massive loculated effusions in a patient without lymphadenopathy or splenomegaly. METHODS: A 60 year old caucasian male presented with vague abdominal discomfort and increasing abdominal girth. Computed tomography showed a two centimeter thick omental cake and a small loculated effusion. The clinical presentation and imaging findings were most consistent with peritoneal carcinomatosis. Cytologic evaluation of the effusion was undertaken for diagnostic study. RESULTS: Rapid intraprocedural interpretation of the effusion sample showed a monomorphic population of cells with "hand-mirror" cell morphology exhibiting cytoplasmic extensions (uropodia) with 3-5 course dark cytoplasmic granules and a rim of vacuolated cytoplasm capping the opposing "mirror head" side. These cells were seen within a background of mature eosinophils. Flow cytometric evaluation of the ascites fluid demonstrated an atypical T-cell population with the following immunophenotype: CD2-, CD3+, CD4-, CD5-, CD7-, CD8+, CD56+. T-cell receptor (TCR) gene rearrangement was positive for clonal TCR-gamma gene rearrangement, supporting the diagnosis of a T lymphoproliferative disorder. CONCLUSION: A T-cell lymphoproliferative process may present with "hand mirror" morphology in an effusion sample. These cells may show polar cytoplasmic vacuolization and 3-5 course granules within the "handle" of these unique cells. Cytoplasm shows peripheral constriction around the nucleus. PMID- 16681859 TI - Enhanced group II mGluR-mediated inhibition of pain-related synaptic plasticity in the amygdala. AB - BACKGROUND: The latero-capsular part of the central nucleus of the amygdala (CeLC) is the target of the spino-parabrachio-amygdaloid pain pathway. Our previous studies showed that CeLC neurons develop synaptic plasticity and increased neuronal excitability in the kaolin/carrageenan model of arthritic pain. These pain-related changes involve presynaptic group I metabotropic glutamate receptors (mGluRs) and postsynaptic NMDA and calcitonin gene-related peptide (CGRP1) receptors. Here we address the role of group II mGluRs. RESULTS: Whole-cell current- and voltage-clamp recordings were made from CeLC neurons in brain slices from control rats and arthritic rats (>6 h postinjection of kaolin/carrageenan into the knee). Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of afferents from the pontine parabrachial (PB) area. A selective group II mGluR agonist (LY354740) decreased the amplitude of EPSCs more potently in CeLC neurons from arthritic rats (IC50 = 0.59 nM) than in control animals (IC50 = 15.0 nM). The inhibitory effect of LY354740 was reversed by a group II mGluR antagonist (EGLU) but not a GABAA receptor antagonist (bicuculline). LY354740 decreased frequency, but not amplitude, of miniature EPSCs in the presence of TTX. No significant changes of neuronal excitability measures (membrane slope conductance and action potential firing rate) were detected. CONCLUSION: Our data suggest that group II mGluRs act presynaptically to modulate synaptic plasticity in the amygdala in a model of arthritic pain. PMID- 16681861 TI - An adult cystic fibrosis patient presenting with persistent dyspnea: case report. AB - BACKGROUND: Persistent dyspnea is a common finding in the cystic fibrosis patient that typically leads to further work up of an alternative pulmonary etiology. Adult cystic fibrosis patients; however, are growing in numbers and they are living into the ages in which coronary artery disease becomes prevalent. Coronary disease should be included in the consideration of diagnostic possibilities. CASE PRESENTATION: A 52-year-old white male with cystic fibrosis was evaluated for exertional dyspnea associated with vague chest discomfort. Diagnostic testing revealed normal white blood cell, hemoglobin and platelet count, basic metabolic panel, fasting lipid profile, HbA1c, with chest radiograph confirming chronic cystic findings unchanged from prior radiographs and an electrocardiogram that revealed sinus rhythm with left anterior fascicular block. Stress thallium testing demonstrated a reversible anteroseptal perfusion defect with a 55% left ventricular ejection fraction. Heart catheterization found a 99% occlusion of the left anterior descending artery extending into the two diagonal branches, with 100% obstruction of the left anterior descending artery at the trifurcation and 70% lesion affecting the first posterior lateral branch of the circumflex artery. CONCLUSION: This case report represents the first description in the medical literature of a cystic fibrosis patient diagnosed with symptomatic coronary artery disease. Applying a standard clinical practice guide proved useful toward evaluating a differential diagnosis for a cystic fibrosis patient presenting with dyspnea and chest discomfort. PMID- 16681860 TI - Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span. AB - BACKGROUND: The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words. RESULTS: An LDA model estimated from CGC items had better predictive performance than two standard models (unigram and mixture of unigrams) trained using the same data. To illustrate the practical utility of LDA models of biomedical corpora, a trained CGC LDA model was used for a retrospective study of nematode genes known to be associated with life span modification. Corpus-, document-, and word-level LDA parameters were combined with terms from the Gene Ontology to enhance the explanatory value of the CGC LDA model, and to suggest additional candidates for age-related genes. A novel, pairwise document similarity measure based on the posterior distribution on the topic simplex was formulated and used to search the CGC database for "homologs" of a "query" document discussing the life span modifying clk-2 gene. Inspection of these document homologs enabled and facilitated the production of hypotheses about the function and role of clk-2. CONCLUSION: Like other graphical models for genetic, genomic and other types of biological data, LDA provides a method for extracting unanticipated insights and generating predictions amenable to subsequent experimental validation. PMID- 16681862 TI - Adherence to the screening program for HBV infection in pregnant women delivering in Greece. AB - BACKGROUND: Hepatitis B infection (HBV) is a major Public Health Problem. Perinatal transmission can be prevented with the identification of HBsAg(+) women and administration of immunoprophylaxis to their newborns. A national prevention programme for HBV with universal screening of pregnant women and vaccination of infants is in effect since 1998 in Greece. METHODS: To evaluate adherence to the national guidelines, all women delivering in Greece between 17-30/03/03 were included in the study. Trained health professionals completed a questionnaire on demographic data, prenatal or perinatal screening for HBsAg and the implementation of appropriate immunoprophylaxis. RESULTS: During the study period 3,760 women delivered. Prenatal screening for HBsAg was documented in 91.3%. Greek women were more likely to have had prenatal testing. HBsAg prevalence was 2.89% (95%CI 2.3-3.4%). Higher prevalence of HBV-infection was noted in immigrant women, especially those born in Albania (9.8%). Other risk factors associated with maternal HBsAg (+) included young maternal age and absence of prenatal testing. No prenatal or perinatal HBsAg testing was performed in 3.2% women. Delivering in public hospital and illiteracy were identifiable risk factors for never being tested. All newborns of identified HBsAg (+) mothers received appropriate immunoprophylaxis. CONCLUSION: The prevalence of HBsAg in Greek pregnant women is low and comparable to other European countries. However, immigrant women composing almost 20% of our childbearing population, have significant higher prevalence rates. There are still women who never get tested. Universal vaccination against HBV at birth and reinforcement of perinatal testing of all women not prenatally tested should be discussed with Public Health Authorities. PMID- 16681863 TI - Lack of association between mannose-binding lectin gene polymorphisms and juvenile idiopathic arthritis in a Han population from the Hubei province of China. AB - Many studies have reported that polymorphisms of the mannose-binding lectin (MBL) gene are associated with autoimmune disease. Here, we investigate the relationship between MBL gene polymorphisms and susceptibility to juvenile idiopathic arthritis (JIA) in a Han-nationality population from the Hubei province of China. PCR-restriction fragment length polymorphism was used to investigate polymorphisms of codons 54 and 57 in exon 1 of the MBL gene in 93 patients with JIA and 48 control children. Neither group showed codon 57 polymorphisms. There was no significant difference in the genotypic frequencies of codon 54 between patients with JIA and healthy controls (wild type, 71.0% versus 75.0%, respectively; heterozygous type, 25.8% versus 25.0%, respectively; and homozygous type, 3.2% versus 0.0%, respectively). In addition, no association was found between the subgroups of patients with JIA and control individuals. Our results provide no evidence for a relationship between MBL gene mutation and susceptibility to JIA. PMID- 16681864 TI - Foreign body sensation in the throat due to displacement of the superior cornu of the thyroid cartilage: two cases and a literature review. AB - The sensation of a foreign body in the throat when swallowing is a common and very discomforting symptom for those who experience it. Common causes are gastroesophageal reflux disease and possibly upper oesophageal dysmotility. This complaint may be dismissed as psychogenic when a cause for the problem is not readily apparent. We present two cases in which the patient had a foreign body sensation in the throat, associated with a dry swallow. The most helpful diagnostic procedure was careful examination of the hypopharynx and neck and flexible fibre-optic rhinolaryngoscopy. One case was treated with surgery of the neck and larynx to trim the medially projecting superior cornu of the thyroid cartilage. Surgery was successful in eliminating the symptoms. Although an uncommon finding, our experience is that symptoms due to a medially displaced thyroid cornu should be considered in the differential diagnosis as an important and surgically correctable cause of persistent foreign body sensation at the hyoid level in the neck. PMID- 16681865 TI - The endoscopic treatment of gastrointestinal symptoms. PMID- 16681866 TI - [Intraoperative quick parathyroid hormone assay in thyroid surgery: preliminary experience on 36 cases]. AB - Through the analysis of our preliminary experience on 36 consecutive cases of thyroid surgery, we assessed the feasibility of the rapid intraoperative assay of the intact parathyroid hormone (iPTH) as predictive risk factor of hypocalcemia versus seric calcium level on the first post-operative day to select the patients eligible to an early discharge. Furthermore, we managed to determine if iPTH level during thyroid surgery could point out the cases in which parathyroid autotransplantation is necessary, as the macroscopic evaluation of the parathyroid gland's viability is inadequate. PMID- 16681867 TI - [Gastric lipoma presenting as intestinal obstruction]. AB - Gastric lipoma is a rare benign tumor. The symptoms are correlated with the size and the dimensions of neoplasm. It can be the cause of bleeding, gastroduodenal intussusception and intestinal obstruction, as in case reported and surgically treated. The Authors make a literature review to define the better diagnostic and surgical approach. PMID- 16681869 TI - [Appendiceal mucocele due to mucinous cystadenoma: case report and review of the literature]. AB - Mucinous cystadenomas of the appendix are rare tumours. In this article we describe the case of a young woman who presented with abdominal pain and a clinical pattern likely to a chronic appendicitis. At laparotomy we found a dilatation of the appendix which contained an abundant quantity of mucus. Histological examination showed a mucinous cystadenoma of the appendix. This neoplasm requires a surgical treatment, usually only appendectomy and right hemicolectomy in case of involvement of the caecum. During operation, the surgeon must take care in handling the appendix because of the risk of rupture with consequent dissemination of epithelial cells in the peritoneal cavity (pseudomyxoma peritonei). PMID- 16681868 TI - [Modern indications on the medical treatment of symptomatic diverticular disease]. AB - Authors refer about their experience on treatment of symptomatic diverticular disease both with antibiotic and anti-inflammatory drugs. They confirm the observation of other Authors on effectiveness of this new therapeutic approach versus traditional antibiotic therapy. PMID- 16681870 TI - [Double compression of the ulnar nerve at the elbow and at the wrist (double crush syndrome). Case report and review of the literature]. AB - Double compression of a peripheral nerve is not rare in medical practice. This article describes an ulnar neuropathy along the elbow and the wrist segments with electro-diagnostic examination (EDX). The proximal compression was an ulnar entrapment at the olecranon-epitrochlear semi-canal; the distal one was after the canal of Guyon, due to an arthro-synovial cyst arising from the pisohamatum joint. There aren't analogous clinical reports in the literature. PMID- 16681871 TI - Recurrent plexiform schwannoma in vestibular mucosa. AB - Schwannoma, also called neurilemmoma, is a benign neoplasm of peripheral nerve sheath. An infrequent location of a multiple intraoral plexiform schwannoma arising on the branches of the facial nerve in the vestibular mucosa of a young male patient is here discussed. Surgical treatment has been discussed on the base of histological findings and diagnosis. PMID- 16681872 TI - Breast surgery and sentinel node biopsy. Our experience. AB - Massive campaigns of screening of breast pathologies improved early diagnosis of breast cancers. Most of these cancers are small-sized (T1) and seldom show intraoperative nodal involvement. Sentinel node biopsy is the elective choice in the above mentioned cases because, if negative, it avoids axillary dissection. International literature reports rates of false negative sentinel node biopsy ranging among 4.5 and 12%; results in our experience account for almost 6%. As a consequence, there is the definite risk of potentially positive axilla that will not be dissected with subsequent risk of axillary recurrence within 24 months. The reason of that could be related to the fact that in Referral Centers this technique is performed in strictly selected patients, so as to gain a diagnostic accuracy of 98%. The rate of axillary recurrences does not justify the routine axillary dissection, since this is just a staging, not a therapeutic procedure. In case of doubt, it can be recommended a close and careful follow-up of the axilla. PMID- 16681873 TI - [Percutaneous radio frequency thermal ablation of non-resectable lung cancer: state of the art]. AB - BACKGROUND: Unfortunately, as of yet, most lung cancers are not operable as soon as diagnosis is available; in these situations chemo- and radio-therapy still play a key role, albeit palliative, improving survival rate moderately, but are not lacking in toxic effects, especially in case of concurrent pathology, reduced cardio-respiratory functionality or being advanced in years. Therefore thermal ablation mini-invasive techniques, already employed as ancillary treatments of hepatic cancer or in place of surgery, have been performed for these pathologies. AIM: Aim of this work is to define the current state of the art for Radio Frequency Ablation (RFA) to be performed on non-resectable lung cancer, also by means of a thorough review of international literature, from which to infer purposes, suggestions, methodologies, effectiveness, safety, complications and achievements, also in terms of the possible improvement of life quality and/or survival expectancy. PATIENTS AND METHODS: Patients have been carefully selected. Pulmonary nodules have been treated with TC or echo-guided percutaneous thermal ablation and, afterwards, evaluated by radiological and clinical (sometimes histopathological) follow-up. RESULTS: The size of the RFA-treated nodules is necessary in order to evaluate full or partial necrosis extent and, therefore, average survival rate. CONCLUSIONS: Availability of more extensive and homogeneous case histories, as well as standard follow-up (TC and/or histopathological sampling) methodologies, is required. Nevertheless several authors agree that RFA is a safe and effective technique within the framework of a substitutive or complementary treatment of non-operable lung cancer. The best results can be achieved for cancers less than 3 cm wide; RFA, performed before chemo- and/or radio-therapy, plays a neoadjuvant role for larger cancers, decreasing cancer volume and weakening the symptoms. PMID- 16681874 TI - Management of acute lower limb ischemia following percutaneous arterial closure device application: our experience. AB - INTRODUCTION: The Authors report their experience in the management of acute lower limb ischemia following percutaneous arterial closure device application. PATIENT AND METHODS: Five patients required an emergency vascular operations for acute lower limb ischemia. The symptoms onset was < 1 hour in 1 case, 4-12 hours in 2 cases and > 24-36 hours in 2 cases. A preoperative angiography was performed in all the cases. A transfemoral embolectomy was carried out. Direct suture repair were performed in three cases, vein patch angioplasty was carried out in two cases. In one case, a common femoral artery endarterectomy was performed. RESULTS: No post-operative mortality and limb loss occurred. CONCLUSIONS: Acute lower limb ischemia due to closure devices required an extensive approach with reconstruction in high risk septic area. Angiography is mandatory for surgical strategies. We prefer direct suture repair and vein path angioplasty for vascular reconstruction. PMID- 16681875 TI - [Nanotechnology in applied researches of medicine]. PMID- 16681876 TI - [Fabrication in science sounds alarm bell in academia circles]. PMID- 16681877 TI - [Effect of nanoparticle with vascular endothelial growth factor gene transferred into ischemic myocardium: experiment with rabbits]. AB - OBJECTIVE: To evaluate the possibility and efficiency of nanoparticle as a new vector in vascular endothelial growth factor (VEGF) gene transference, and investigate the efficacy of direct gene transfer of nanoparticle with VEGF(165) gene into ischemic myocardium. METHODS: Nanoparticle-VEGF (Np/VEGF) complex was prepared with poly (D, L-lactide-co-glycolide) (PLGA) loading VEGF(165) gene and the envelopment efficiency and size of the complex were determined. The Np/VEGF was transfected into the cultured myocardial cells, RT-PCR and ELISA were used to evaluate the transfection of VEGF. Suspension of Np/VEGF was injected into the myocardial tissue of 4 rabbits. 96 hours after operation myocardial tissue was obtained, made into sections, and observed with electron microscope. New Zealand White rabbits underwent thoracotomy followed by ligation of left anterior descending coronary artery to establish ischemic models. The New Zealand White rabbits were divided into 3 groups: Np/VEGF group (n = 12, nanoparticle with VEGF(165) were injected into the cordial myocardium), blank plasmid group (n = 12, injected with blank VEGF(165) plasmid), and control group (n = 8, injected with normal saline). Ultrasonography and immunohistochemistry with factor VIII related antigen were conducted to evaluate the cardiac function and the collateral circulation of the occluded artery. One month later the rabbits were killed to observe the vascularization of capillaries in the ischemic myocardium. RESULTS: The envelopment efficiency of the Np/VEGF complex thus prepared, 50 - 300 nm in size, were 1.87% y. RT-PCR and ELISA showed that VEGF gene had been successfully transfected into myocardial cells by the nanoparticles. A great number of nanoparticles were observed in the myocardial cytoplasm and nuclei. One month after operation, the ventricular wall motor amplitude of the Np/VEGF group was 1.87 mm +/- 0.32 mm, significantly larger than those of the blank plasmid group (1.59 mm +/- 0.24 mm, P < 0.05) and control group (0.93 mm +/- 0.40 mm, P < 0.05); and the left ventricular ejection fraction of the Np/VEGF group was 60% +/ 10%, significantly higher than those of the blank plasmid group (50% +/- 6%, P < 0.05) and control group (40% +/- 8%, P < 0.05). The capillary density at low power field (x 100) of the Np/VEGF group was 57 +/- 12, significantly higher than those of the VEGF group (41 +/- 14) and control group (24 +/- 8). CONCLUSION: Nanoparticle can act as a vector to transfect specific gene in vitro and in vivo. Direct gene transfer of nanoparticle with DNA encoding VEGF into the ischemic rabbit myocardium can increase capillary number; therefore it may be a novel therapeutic approach for myocardial ischemia. PMID- 16681878 TI - [Nanomechanical properties of vertebral trabeculae in ovariectomized rats]. AB - OBJECTIVE: To study the nanomechanical properties of the vertebral trabeculae of ovariectomized rat using nanoindentation. METHODS: Twenty 10-month-old SD rats were randomly divided into 2 equal groups: ovariectomized (OVX) group and Sham operation (SHAM) group. Fifteen weeks post-operationally dual energy X-ray absorptiometry was used to measure the bone mineral density (BMD) of the total body and of the sixth lumbar vertebra. Then the rats were killed. The BMD values of the sixth lumbar vertebrae were measured by DXA. Bone histomorphometry was performed on the proximal metaphysis of the right tibia. Three of the sixth lumbar vertebrae were randomly selected from each group and embedded in methyl methacrylate. Each vertebra was cut into two parts along the transverse direction in the middle point of longitudinal axis so as to expose the trabeculae on the cross section. The lower part was polished, trabeculae were randomly selected from 4 places, and 5 points from each place were randomly selected to undergo nanoindentation so as to measure the nanomechanical properties. RESULTS: Compared with the SHAM rats, the BMD of the sixth lumbar vertebra of the OVX rats was reduced significantly (P < 0.05). The histomorphometry of the tibia showed an increase in trabecular separation and a decrease in trabecular bone area fraction (both P < 0.05); the trabecular number and thickness decreased in these 2 groups, however, without significant difference between them. Nanoindentation tests showed that the values of hardness and elastic modulus of the trabeculae of the OVX rats were 0.91 GPa +/- 0.13 GPa and 21.01 GPa +/- 2.48 GPa respectively, not significantly different from those of the SHAM rats, 0.90 GPa +/- 0.09 GPa and 22.03 GPa +/- 2.44 GPa respectively. CONCLUSION: A novel technique, nanoindentation is able to directly measure the nanomechanical properties of trabeculae. Estrogen deficiency after ovariectomy induces significant osteoporotic change, but has no significant influence on the trabecular nanomechanical properties. PMID- 16681879 TI - [Nanomanipulation of sections of human tongue squamous cell carcinoma by atomic force microscopy]. AB - OBJECTIVE: To investigate the appropriate nanomanipulation of slides of human tongue squamous cell carcinoma tissue and cultured cells by atomic force microscopy (AFM), and develop methods to improve the resolution and contrast of AFM images. METHODS: Human tongue squamous carcinoma cells of the line Tca8113 were cultured. Cell masses were isolated and collected, and sectioned. Then the white lead and silvery white sections were transferred on mica slides, dried. Eighty slides were used and divided into 2 groups to be immersed into hydrogen dioxide of the concentrations 20% and 30% for 0, 5, 10, 15, 20, 25, 30, and 40 min respectively. Samples of human tongue squamous carcinoma were collected from 15 cases. Every sample was divided into 2 halves. Half of every sample underwent routine paraffin preparation of slide and adhered onto glass, dissolving of paraffin with dimethylbenzene for 0, 10, 15, 30, 45, or 60 min; and half of the sample underwent routine preparation of slides for electron microscopy, the white lead and silvery white sections were transferred on mica slides, dried. The slides were observed by contact mode AFM in air. RESULTS: In the ultrathin sections the metastructure of the cell such as the cell membrane, nuclear membrane and nucleolus could be distinct from each other clearly. The profile of the cell and the nucleus inside could be revealed by AFM after the paraffin sections were treated in dimethylbenzene for 15-30 min. AFM manipulation could be achieved within nanometer range by precise modulation of scanning force, area, angle and so on. The appropriate needle point should contain elastic coefficient > 5.0 N/m. The force exerted on the needle point should be within the range 50 300 nN. After location the scanning direction should be within the range of 20 nm 1 microm. CONCLUSION: A series of modified techniques of preparing and treating AFM samples, such as adhering the sections onto mica slide, dissolving of epoxy resin by hydrogen dioxide and removal of paraffin by dimethylbenzene, were developed, thus realizing high resolving power AFM imaging, especially for nuclear membrane, and nucleolus, and providing a base of AFM location, dissection, isolation and obtainment at nanometer scale. PMID- 16681880 TI - [Clinical management of adenomatous polyposis in patients with hereditary non polyposis colorectal cancer and familial adenomatous polyposis]. AB - OBJECTIVE: To investigate the validity and safety of different doses of non steroidal anti-inflammatory drugs (NSAID) in attempting to maintain the regression of colorectal adenomas in patients with familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). METHODS: Twenty-two FAP patients who were willing to receive celecoxib were randomly divided into 2 groups 400 mg/d group (n = 8, taking celecoxib 400 mg/d) and 200 mg/d group (n = 10, taking celecoxib 200 mg/d). Four FAP patients who refused celecoxib and selected aspirin 80 mg/d instead. Six HNPCC patients were given celecoxib 400 mg daily. The treatment lasted for 24 months in all groups. The efficacy was evaluated respectively by the number and grade of polyps by coloscopy every 3 months in the first year and every 6 months in the second year. RESULTS: Either dose of celecoxib could reduce polyps in the FAP patients, with a polyps reduction rate of 86.6% (280/323) in the 400 mg group, significantly higher than that in the 200 mg group [51.81% (129/249) of the aspirin group]. In 5 of the 6 HNPCC patients the polyps completely vanished after 9 months of treatment. Side effects, such as arrhythmia, angina pectoris, and nervous headache, were observed in the celecoxib 400 mg/d group. The side effects could be reversed by decreasing the dose of celecoxib or using aspirin instead. Only one patient in the celecoxib 200 mg/d group showed side effects. CONCLUSION: Celecoxib 400 mg daily is more effective but has more side effects. At first the patients should be treated with celecoxib 200 mg daily for a long time, or 400 mg/d in the first 6 months and then with a daily dose of 200 mg/d to maintain the treatment effects. Soluble aspirin has similar effects. PMID- 16681881 TI - [The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro]. AB - OBJECTIVE: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines. METHODS: Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells. RESULTS: Real time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference. CONCLUSION: Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis. PMID- 16681882 TI - [Difference in methylation of genomic DNA between gastric primary cancer and lymph nodes with metastatic gastric cancer]. AB - OBJECTIVE: To investigate the difference in methylation of genomic DNA between gastric primary cancer and lymph nodes with metastatic gastric cancer. METHODS: Methylation CpG island amplification (MCA) was used to enrich the methylated DNA sequences in the cancer tissues and lymph nodes with metastatic gastric cancer resected during operation from 5 patients. Representational difference analysis (RDA) was conducted with the MCA products from the lymph nodes with metastatic gastric cancer as testers and the MCA products of the gastric primary cancer as drivers. The differentially methylated DNA fragments were cloned and sequenced, and underwent similarity analysis by BLAST system. The relationship between the clone sequence and related gene was analyzed by GenBank. Hybridization analysis was performed, using the No 1-3 round RDA products and the MCA products of the tissues of gastric primary cancer and lymph nodes with metastatic gastric cancer with digoxin-labeled KL22 fragment as probe. RESULTS: Nineteen differentially methylated DNA sequences were obtained, distributed at the 5'-end, exon, intron, and 3'-end. KL59 fragment was located in the 9q21, the first exon of p16 gene. KL12 fragment was located in the promoter region of phosphotyrosine phosphatase receptor G (PTPRG) gene. Hybridization signals were obtained for all No 1-3 round RDA products and all testers, but not for the drivers. CONCLUSION: There is a difference in DNA methylation between the tissues of primary cancer and lymph nodes with metastatic gastric cancer. MCA-RNA is an effective method to study the gene methylation. PTPRG gene may be a candidate gene for metastasis of gastric cancer. PMID- 16681883 TI - [Inhibitory effect of antisense oligodeoxynucleotides targeting HER-2 mRNA on growth of breast cancer: experiment in vivo with mice]. AB - OBJECTIVE: To investigate the inhibitory effect of antisense oligodeoxynucleotides targeting HER-2 mRNA on growth of breast cancer. METHODS: Human breast cancer cells of the line SK-BR-3 that overexpresses HER-2 were injected subcutaneously into BALB/c nude mice. Seven to ten days after tumors were collected and made into homogenate. Antisense oligodeoxynucleotide HA6722 targeting HER-2 mRNA and its control sequence Scramble were synthesized. Forty nine BALB/c nude mice were injected with homogenate of SK-BR-3 cells and then randomly divided into 7 equal groups: Group 1, injected intravenously with docetaxel (TXT) 7.5 mg.kg(-1).d(-1) once a week twice; Group 2, injected intravenously with TXT 15 mg.kg(-1).d(-1) once a week twice; Group 3, injected intravenously with TXT 7.5 mg.kg(-1).d(-1) once a week twice + HA6722 5 mg.kg( 1).d(-1) for 12 days; Group 4, injected intravenously with HA6722 5 mg.kg(-1).d( 1) for 12 days; Group 5, injected intravenously with TXT 7.5 mg.kg(-1).d(-1) once a week twice + Scramble6722 5 mg.kg(-1).d(-1) for 12 days; and Group 7, injected with normal saline. After the drug administration the mice were observed for additional 3-4 weeks to measure the size of tumor every other day. Then the mice were killed and the tumors were taken out to be examined. The inhibition rate was calculated. RESULTS: The inhibition rate of tumor in Group 3 was 88.3%, not significantly different from that in Group 2 (88.7%, P > 0.05). The inhibition rate in Group 4 was 76.3%. The inhibition rates of tumor of Groups 2, 3, and 4 were all significantly higher than that in Group 7. However, the inhibition rate of tumor of Group 6 was not significantly different from that in Group 7. CONCLUSION: Antisense oligodeoxynucleotides targeting HER-2 mRNA inhibits the growth of breast cancer in a sequence specific and dose-dependent manner. PMID- 16681885 TI - [Transfection of recombinant bone morphogenetic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-beta]. AB - OBJECTIVE: To investigate the effects of bone morphogenetic protein (BMP)-7 on the extracellular matrix (ECM) accumulation induced by transforming growth factor (TGF)-beta. METHODS: Mouse full length BMP-7 cDNA was ligated into a eukaryotic expression vector pcDNA3.1. Restriction enzymatic analyses and DNA sequencing were used to confirm the accuracy of the BMP-7 expressing plasmid thus constructed. The recombinant expression plasmid pcDNA3.1-BMP-7 was transfected into cultured human renal tubular epithelial cells of the line HK-2 mediated by liposome. Positive clones were selected so as to obtain the human renal epithelial cells with stable transfection. These HK-2 cells were cultured and divided into 5 groups to be treated with 5 ng/ml TGF-beta, blank plasmid pcDNA3.1, blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta, pcDNA3.1-BMP-7, pcDNA3.1-BMP 7 + 5 ng/ml TGF-beta, and an additional grin the cells and the supernatant of the cell culture fluid were collected. The expression level of BMP-7 protein was determined by Western blotting. RT-PCR and ELISA were used to determine the mRNA and protein expression of collagen (Col) I and III, and fibronectin (FN) in the human renal tubular epithelial cells and supernatant of different groups. RESULTS: The recombinant plasmid pcDNA3.1-BMP-7 was successfully constructed. The cell mRNA expression levels of Col I and III and FN of the 5 ng/ml TGF-beta group and blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta group were all significantly higher than those of the blank plasmid pcDNA3.1 group, pcDNA3.1-BMP-7 group, and control group (all P < 0.05). The cell mRNA expression levels of Col I and FN of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta were all significantly lower than those of the TGF-beta group (all P < 0.05). The cell mRNA expression level of Col III of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta was lower, however, not significantly, than that of the TGF-beta group. The supernatant FN levels of the 5 ng/ml TGF-beta group and pcDNA3.1 + 5 ng/ml TGF-beta group were both significantly higher than that of the control group (both P < 0.05), and the supernatant FN level of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta group was significantly lower that that of the 5 ng/ml TGF-beta group (P < 0.05). CONCLUSION: Over-expression of BMP-7 significantly inhibits the increased syntheses of collagen I and III, and fibronectin induced by TGF-beta. BMP-7 exerts its antifibrotic effect partially through blocking the TGF-beta-induced accumulation of extracellular matrix in human renal tubular epithelial cells. PMID- 16681886 TI - [Non-infectious pulmonary complications after allogeneic hematopoietic stem cell transplantation: analysis of 17 cases]. AB - OBJECTIVE: To analyze the clinical feature, cause, treatment and outcome of late onset non-infectious pulmonary complications (LONIPC) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The clinical data of 17 patients with malignant hematological diseases who survived for at least 3 months and suffered from LONIPC after allo-HSCT were retrospectively analyzed. RESULTS: The incidence of LONIPC was 17.7% in allo-HSCT recipients. The median of onset of respiratory symptoms was 6.5 months (3-13.5 months). Seven patients came down with LONIPC during fast tapering of cyclosporine A prophylaxis and 15 patients had already chronic graft versus host disease (cGVHD) before the occurrence of respiratory symptoms. The initial symptoms were non-productive cough and dyspnoea in all patients; five of them had low grade or moderate fever. CT scans revealed patchy ground-glass opacities, irregular patchy consolidation, band-like opacities, and micronodular densities. Histological examination of transbronchial biopsy showed infiltration of lymphocyte and monocytes in interstitium, peribronchiolar fibrosis and alveoli pulmonis obliteration. The response rate of corticosteroids in addition to cyclosporine therapy was 70.6%. Treatment beginning at the early stage was more effective than that beginning late. The mortality rate of LONIPC was 35.3%. Chest CT scanning showed lung fibrosis in the patients with protracted LONIPC. CONCLUSION: The clinical manifestations and radiological changes of LONIPC are non-specific. The diagnosis is made by combination of functional and histological examinations and exclusion of pathogen infection. Examination of transbronchial biopsy is of significance for the diagnosis. LONIPC may be considered as pathognomonic of cGVHD in the lung of patients after allo-HSCT; and cGVHD should be regarded as a useful diagnostic proof for LONIPC. Earlier treatment with corticosteroids and maintenance treatment may result in improved survival and decrease of the fibrotic residue. PMID- 16681888 TI - [Polymorphisms of myocilin and optineurin in primary open angle glaucoma patients]. AB - OBJECTIVE: To detect the single nucleotide polymorphisms (SNPs) of the myocilin (MYOC) and optineurin (OPTN) genes, and to investigate their associations with high tension glaucoma (HTG) and normal tension glaucoma (NTG). METHODS: SNPs were detected using polymerase chain reaction (PCR), followed by conformation sensitive gel electrophoresis (CSGE) and fluorescent labeling automated DNA sequencing among 94 unrelated patients with HTG, 48 unrelated patients with NTG, and 77 unrelated control subjects. RESULTS: Fourteen MYOC sequence alterations were identified, five of them: V53A, I304I, T347T, 1-126T > C, and IVS2 + 172C > A, were novel. Among them, V53A was for the first time found in primary open angle glaucoma (POAG) patient. R76K usually occurred with the promoter polymorphism 1-83G > A. No sequence alterations in the MYOC gene showed significant differences among the HTG, NTG and control subjects (all P > 0.05). A total of 12 sequence alterations were identified in the OPTN gene, and three of them: V161M, I407T and L211L, were novel. Among them, I407T and L211L were found only in the HTG patients. The allele and genotype frequencies of T34T in the NTG patients were significantly higher than those of the controls (P = 0.001 and 0.004 respectively). In HTG, only the allele frequency of T34T was 24% (23/96), significantly higher than those of the NTG group (16.5%, 31/188) and the control group (9.1%, 14/154) (both P < 0.05). In addition, IVS8 + 20G > A was found only in the HTG (3.1%, 3/96) and NTG patients (3.7%, 7/188), and had significantly higher frequencies in the HTG and NTG patients when compared with the controls (P = 0.016 and 0.014, and P = 0.027 and 0.026). CONCLUSION: Polymorphisms in the MYOC and OPTN genes are associated with POAG in Chinese people. Moreover, sequence alterations not causing amino acid changes may play a role in the pathogenesis of POAG. PMID- 16681889 TI - [Spiral computed tomography images of extraluminal type gastric stromal tumors]. AB - OBJECTIVE: To evaluate the diagnostic value of spiral CT and its reconstruction techniques in the diagnosis of extraluminal type gastric stromal tumors. METHODS: The data of spiral CT performed 1 week before operation of 17 extraluminal type cases, 11 males and 6 females, aged 35.5 (13-67), from 33 patients of gastric stromal tumors proved by pathology and immunohistochemistry, were analyzed retrospectively. RESULTS: The accuracy rates of level diagnosis and qualitative diagnosis of extraluminal type gastric stromal tumor by spiral CT were 88.2% and 82.4% respectively. The main CT manifestations included: (1) extraluminal mass connected with the gastric wall, with round or lobulated margin and characteristic horn-like appearance and direct invasion, more than 5.0 cm in diameter; (2) inhomogeneous density of mass, including mild to moderate uneven enhancement in artery phase and obvious enhancement in vein and delay phase in 15 cases, and obvious enhancement in artery phase and decrease of enhancement in portal vein phases in 2 cases. (3) Irregular niches and sinus in the margins of the intraluminal masses (n = 11) and unclear border of the masses and invasion into nearby structures (n = 9); (4) connection of the tumor and stomach wall by broad boundary or failure to distinguish the tumor and stomach wall; and (5) invasion to the surrounding tissues and organs as a sign of malignant tumors. CONCLUSION: Spiral CT and its reconstruction techniques have important value in level diagnosis and qualitative diagnosis of extraluminal type gastric stromal tumors. PMID- 16681897 TI - [To enhance the research of antibiotic resistance and infection caused by drug resistance bacteria]. PMID- 16681896 TI - [To develop the therapy strategy of lower respiratory tract infection suited to the special situation in China]. PMID- 16681898 TI - [The role of clinical microbiology in infection control]. PMID- 16681899 TI - [Pay attention to the antibiotic resistance of the pathogenic bacteria causing community-acquire respiratory infections in childhood]. PMID- 16681900 TI - [The differential diagnosis and morphologic feature of bone marrow among a group of misdiagnosed infectious disorders]. PMID- 16681901 TI - [Strengthen the basic and clinical research of computer-assisted orthopaedic surgery and advance the clinical application thereof steadily]. PMID- 16681902 TI - [Molecular epidemiological study of colistin-only-sensitive strains of multi-drug resistant Acinetobacter baumannii]. AB - OBJECTIVE: To genetically analyze the epidemiology of the colistin-only-sensitive (COS) Acinetobacter baumannii (COS-AB) so as to provide experimental data for controlling COS-AB infection. METHODS: The drug resistance to 14 antibiotics of 136 COS-AB isolates collected in Ruijin Hospital from June 2004 to May 2005 were analyzed. 66 A. baumannii isolates were collected from May to December 2004, of which 33 strains were COS-AB and the other 33 non-COS-AB. Random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to analyze the homology of these strains. RESULTS: RAPD analysis (with the primers ERIC2 and 272) showed all COS-AB strains as identical, while PFGE analysis showed that these COS-AB were of two closely related genotypes distinctly different from that of the non-COS-AB. The COS-AB strains obtained from the Burns Ward were PFGE B type causing inter-ward outbreak of single clone. And the COS-AB strains obtained from another wards were PFGE BA type causing inter-departmental outbreak, mainly among the departments of surgical system. CONCLUSION: The cross-infection of COS-AB is severe. Strict disinfection and sterilization should be implemented. The epidemiology of COS-AB in environment and patients should be closely monitored. PFGE analysis is a more accurate method in typing A. baumannii. PMID- 16681903 TI - [A novel plasmid-mediated aminoglycosides-modifying enzyme gene, aph (2'')-Ie, in a strain of high-level gentamicin resistant Enterococcus casseliflavus]. AB - OBJECTIVE: To identify the aminoglycoside resistance gene in the high-level gentamicin resistant (HLGR) enterococcus and its transmission mechanism. METHODS: A HLGR strain, HZ95, of Enterococcus casseliflavus was screened by agar dilution method. The aminoglycoside activation enzyme genes were screened by PCR. The PCR products underwent purification, cloned, and sequenced. Plasmids were extracted and underwent Southern hybridization. Plasmid-mediated aminoglycoside modifying enzymes were cloned. The resistance of the HZ05 strain to different antimicrobial agents was determined by K-B method or agar dilution method. RESULTS: A new aminoglycosides-modifying enzyme, APH (2'')-Ie, leading to HLGR, was found in the plasmid of the HZ95 bacteria. The aph (2'')-Ie gene and partial sequence of a streptomycin adenylyltransferase gene (str) were contained in the 8.7-kb cloned fragment, and the transposase gene (tnpA) was on the upper stream. The aph (2'') Ie gene was located on the 16-kb plasmid with the amino acid sequence 96.1% homologous with chromosome-mediated APH (2'')-Id aminoglycoside-modifying enzyme. CONCLUSION: HLGR can be caused by a new plasmid-mediated aminoglycosides modifying enzyme, APH (2'')-Ie, which can be transmitted among plasmids or chromosome with other resistant genes through transposase. PMID- 16681904 TI - [Expression of high-level cephalosporinase due to mutation in the AmpC attenuator of a clinical Escherichia coli strain]. AB - OBJECTIVE: To study the resistant phenotype of a clinical strain of Escherichia coli and to explore the effect of its attenuator mutation on AmpC expression. METHODS: A clinical strain of Escherichia coli 20022 (ECO20022) resistant to cefoxitin was isolated clinically. The phenotype was examined by three dimensional methods, isoelectric focusing (IEF), and microdilution method. The regulator genes of ECO20022 were amplified and sequenced, and the difference between them was analyzed by BLAST method. Then the regulator genes were cloned into pCAT3-basic vector (a promoterless reporter gene vector). Microdilution method was used to detect the minimal inhibitory concentration (MIC) of chloramphenicol and ampicillin to this strain with E. coli ATCC25922 as quality control bacterium. ELISA was used to detect the content of chloramphenicol acetyl transferase (CAT). RESULTS: Compared to the standard E. coli K-12, there were four base substitutions, i.e., 22C-T, 26, 27TA-GT, and 32G-A in the attenuator region of ECO20022. Three-dimensional method showed that this strain was high AmpC-producing. IEF found that it produced three beta-lactamases with the values of PI of 5.4, 8.2, and 9.0 respectively. The beta-lactamase with the PI of 9.0 could be inhibited by cloxacillin but not by clavulanate. The strain was resistant to not only most of third generation cephalosporins, but also to cefepime; however it was still susceptible to carbapenem. The secondary structure of the attenuator RNA of ECO20022 was different from the traditional structure of E. coli K-12. The regulator gene was successfully cloned into pCAT3-basic vector and direct and indirect tests indicated that this regulator gene enhanced the CAT expressing level as much as 10 times that of Escherichia coli K-12. CONCLUSION: AmpC attenuator mutation leads to high AmpC expression in Escherichia coli, resulting in a significant rise of resistance level to beta-lactamase and a great menace to clinical antibiotic therapy. PMID- 16681906 TI - [Intra-operative stereotactic accuracy of computer-assisted robot orthopaedic trauma surgery planning system]. AB - OBJECTIVE: To investigate the feasibility and accuracy of the software computer assisted robot orthopaedic trauma surgery planning system (CAOTS). METHODS: The software CAOTS was developed and used on 85 cases of stereotactic operation, 24 model bones, 21 cadaveric extremity bones, and 40 patients. 307 groups of directional targets in these cases were randomly sampled. The distribution of error sources for evaluating system performance was embodied using Monte-Carlo method in order to derive the theoretic guarantees for further optimizing and enhancing the system performance, then the software SPSS 2.0 was used to analyze the errors. RESULTS: The statistical area of deviation number was 0.0408 +/- 0.4578 mm, corresponding to the result by Monte-Carlo method. Punching succeeded at the first run for all 307 cases without wrong locking and other clinical complications. CONCLUSION: Accurate and reliable, CAOTS improves the intra operative navigation techniques and facilitates the orthopedists to perform operation. PMID- 16681905 TI - [Characterization of macrolide-resistance mediated by transposable elements Tn1545 and Tn917 in clinical Isolates of Streptococcus pneumoniae]. AB - OBJECTIVE: To investigate the characterization of ermB gene expression and dissemination in macrolide-resistant Streptococcus pneumoniae (Sp) in Shanghai. METHODS: Eighty-six erythromycin-resistant isolates of Sp were isolated from 3 hospitals in Shanghai. E-test or K-B disk diffusion test were used to determine the susceptibility to 12 antibiotics according to the National Committee for Clinical Laboratory Standards. Macrolide resistant genes ermB and mefE, and transposable elements Tn1545 and Tn917 were amplified by PCR. The isolates were divided into Tn1545 and Tn917 groups according to the transposable elements thereof. Double disc test with erythromycin and clindamycin discs divided the isolates into 2 macrolide resistant phenotypes: cMLS(B) (inducible) phenotype and M phenotype (resistant to erythromycin and sensitive to clindamycin). BOX-PCR was used to analyze the homology of the S. pneumoniae. RESULTS: (1) Of the 86 erythromycin-resistant isolates, the positive rates of ermB, mefE, Tn1545, and Tn917 were 94%, 46%, 87% and 7% respectively. Tn1545 and Tn917 were not detected in 5 ermB-mdfE + strains. (2) Most strains in the Tn1545 and Tn917 groups were highly resistant to erythromycin with a MIC50 of 256 microg/ml. The Tn917 group had a lower MIC to beta-lactam antibiotics and lower resistance to tetracycline, levofloxacin, and compound sulfonamide in comparison with the Tn1545 group. (3) The most common macrolide resistance phenotype of the Tn1545 group was cMLS(B) phenotype. Three strains in the Tn917 group had a 194 bp deletion in the promoter region of ermB and an insertion of TAAA motif in the N end of leader peptide, resulting in the change of the ermB gene from inducible to constitutive expression. (4) BOX-PCR showed that Tn1545 and Tn917 might spread horizontally. CONCLUSION: In Shanghai ermB-mediated cMLS(B) is the most prevalent phenotype in macrolide-resistant Streptococcus pneumoniae isolates. Primarily, the ermB gene was carried and spread horizontally by Tn1545. PMID- 16681907 TI - [Effects of medical robot-assisted surgical navigation system in distal locking of femoral intramedullary nails: an experimental study]. AB - OBJECTIVE: To investigate the effects of medical robot-assisted surgical navigation system based on fluoroscopic images in distal locking of femoral intramedullary nails. METHODS: Using a robot-assisted computer-guided system designed based on modularization and minimization that permitted C-arm alignment assistance and real-time navigation control, provided constant feedback without the need for radiologic updates, thus avoiding constant X-ray exposure. The C-arm was used to collect the orthotopic and lateral X-ray images into the computer so as to calculate the locations of the target points. Nails were locked into 5 plastic femurs (Swiss Sybone, 35 holes), 2 dry human femoral specimens (12 holes), and one leg of fresh human cadaver (6 holes). Radiographs were taken to confirm that screws were positioned correctly, and fluoroscopic time associated with the locking procedure was recorded. RESULTS: All distal holes were locked successfully. In 6 (11.1%) of the 53 holes the drill bit touched the canal of the locking hole, albeit with no damage to the nail. The fluoroscopy time of per screw was 1.83 +/- 0.31 seconds. CONCLUSION: The medical robot-assisted surgical navigation system enables the physicians to precisely navigate surgical instruments throughout the procedure using just a few computer-calibrated radiographic images. The total radiation time per procedure can be significantly reduced because additional X-ray exposure is not required for tool navigation. The idea of a robot-assisted surgical navigation system is practicable. PMID- 16681908 TI - [Specific inhibition of HER-2 expression in ovarian carcinoma cells by siRNA targeting HER-2]. AB - OBJECTIVE: To investigate the effects of RNA interference (RNAi) targeting HER-2 gene on the biological traits of human ovarian carcinoma. METHODS: siRNA specific to HER-2 gene was synthesized according to the sequence in the GenBank. Human ovarian carcinoma cells of the line SKOV-3 were cultured and divided into 3 groups: control group; non-specific group, transfected with non-specific siRNA; and specific group, transfected with specific HER-2 siRNA. On the 5th day after transfection cisplatin was added into the culture fluid. The expression of HER-2 mRNA and the expression of protein both before and after transfection were detected by RT-PCR and Western blotting. The cell apoptosis was assessed by flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT method. RESULTS: Since the 3rd day after transfection the expression of HER-2 mRNA of the HER-2 siRNA group was suppressed till 10 days later. On the 7th day after transfection the expression rate of HER-2 protein of the HER-2 siRNA group was (25.5 +/- 0.8)%, significantly lower than those of the nonspecific siRNA group and control group, (95.7 +/- 0.8)% and (96.6 +/- 1.2)% respectively (both P < 0.001). On the 9th day after transfection no expression of HER-2 protein was found in the HER-2 siRNA group. The apoptosis rate of SKOV-3 cells increased time-dependently after transfection in the HER-2 siRNA group and reached the peak, (53.2 +/- 1.0)% on the 6th day, significantly higher than those of the non-specific siRNA group and control group, (4.1 +/- 0.3)% and (4.1 +/- 0.3)% respectively (both P < 0.001). After exposure to cisplatin for 24 hours, the survival rate of the HER-2 siRNA group was (58.4 +/- 0.8)%, significantly higher than those of the nonspecific siRNA group and control group, (68.0 +/- 0.6)% and (67.0 +/- 0.3)% respectively (both P < 0.001). CONCLUSION: siRNA targeting HER-2 synthesized in vitro and transfected into human ovarian carcinoma cells effectively suppresses the HER-2 expression, induces cell apoptosis, and increases the sensitivity to cisplatin of the cells. The successful application of HER-2 siRNA extends the list of available therapeutic modalities in the treatment of human ovarian cancer. PMID- 16681909 TI - [Clinical analysis of pneumomediastinum complicated in polymyositis and dermatomyositis]. AB - OBJECTIVE: To analyze the clinical features of pneumomediastinum complicated in polymyositis and dermatomyositis (PM/DM) and to study the pathogenesis thereof. METHODS: The clinical data of 4 patients with pneumomediastinum complicated in dermatomyositis out of 447 PM/DM patients hospitalized in Peking Union Medical College (PUMC) Hospital Jan 1989 to June 2005, were analyzed. The records of patients with PM/DM available in English throughout the world were reviewed to collect those with pneumomediastinum as a complication. And the data of these patients were analyzed, focusing mainly on the age, gender, peak of serum creatine kinase (CK), presence of pneumomediastinum, cutaneous vasculopathy, chest radiographic changes, tracheal cannula, management, and outcome. RESULTS: Among the 447 patients with PM/DM hospitalized in PUMC Hospital, 134 males and 313 females, aged 42 +/- 17, pneumomediastinum was observed as a complication in four patients, 3 males and 1 female, aged 12 - 43, with a prevalence rate of 0.9%. Together with 17 cases reported in the English literatures there were 21 patients with pneumomediastinum complicated in polymyositis and dermatomyositis (PM/DM in all. Only one of the literatures reported a prevalence rate as high as 8.3% (4/48), and other literatures were merely case reports. Compared with the PM/DM patients without pneumomediastinum the mean age of the PM/DM patients with pneumomediastinum was significantly younger (34:42, P < 0.01), the male: female ratio significantly higher (13:8 to 132:311, P < 0.01), the morbidity rates of interstitial lung disease and of cutaneous vasculopathy significantly higher (18/21 to 134/443, and 12/21 to 44/443, both P < 0.01). Although statistic analysis could not be undertaken because of the peak of CK not being provided in details in the literatures, the CK levels of the patients with pneumomediastinum were mostly normal or mildly higher with a peak lower than 500 U/L Three of the 4 patients with pneumomediastinum hospitalized in PUMC Hospital and 5 of the 443 patients (1.1%) without this complication received tracheal cannula. There was a significant association of pneumomediastinum with tracheal cannula (P = 0.000). CONCLUSION: Vasculopathy is strongly suspected as being responsible for the pneumomediastinum in DM, and male gender, younger age, interstitial lung disease, and tracheal cannula may be the risk factors of this pneumomediastinum complicated in PM/DM. PMID- 16681910 TI - [An unusual recombination event occurring between HLA-B and -Cw loci within a Chinese Han family]. AB - OBJECTIVE: To investigate the recombination event occurring between HLA-B and -Cw loci discovered in a family of Chinese Han nationality with an acute myeloid leukemia (AML) patient. METHODS: Peripheral blood samples were collected from a Chinese man with M5 type AML, aged 39, and his healthy wife and daughter, all of Han nationality. HLA class I (-A, -B, and -Cw) and II (-DRB1 and -DQB1) alleles were typed by both low and high resolution PCR with sequence specific primers (PCR-SSP) and sequence-based typing (SBT). Then the recombination sites were analyzed by family study. RESULTS: The 2 haplotypes of the patient, his daughter, and his wife were A*2402101-Cw*030401/0402-B*1301-DRB1*0406-DQR1*030302/0303 and A*02011-Cw*150201/0202-B*4002-DRB1*1405-DQB1*05031, A*02011-Cw*150201/0202-B*1301 DRB1*0406-DQB1*030302/0303 and A*2406-Cw*0602-B*1302_DRB1*070101/0102-DQB1*0202, and A*330301/0302-Cw*030201/0202-B*58-1-DRB1*17-DQB1*0202 and A*2406-Cw*0602 B*1302-DRB1*070101/0102-B*5801-DRB1*0202 respectively. Family study demonstrated that A*02011-Cw*150201/0202 recombination and B*1301-DRB1*030302/0303 recombination carried by the daughter came from the 2 isolated chromosomes of her father, indicating that the recombination event occurred between HLA-B and -Cw loci during meiosis of the father and resulted in a new HLA haplotype that was inherited by the daughter. CONCLUSION: An unusual HLA-B/Cw recombination event occurring between HLA-B and -Cw loci has been found in a Han family, which helps further study the mechanisms of HLA recombination. PMID- 16681911 TI - [Analysis of the causes of surgical complications of medial sphenoidal ridge meningioma]. AB - OBJECTIVE: To find out the causes of severe surgical complications of sphenoidal ridge meningioma of and the methods to avoid them. METHODS: The clinical data of 56 cases of sphenoidal ridge meningioma, 37 being medial (clinoidal) type, 5 being middle (alar) type, and 14 being lateral (pterional) type, with the diameters of 3.5 - 7 cm, 3.8 - 5.2 cm, and 3.8 - 7.5 cm respectively, who underwent operation. RESULTS: The tumors of 19 cases of lateral and middle types were resected completely without surgical complication. Facial nerve injury occurred in 2 cases of medial type because of failure to know well the course of facial nerve and over-stretching of skin flap. Oculomotor nerve injury occurred in 7 cases, 5 of which were transient, and trochlear nerve injury occurred in 1 case, all caused by inappropriate manipulations. Too early resection of tumor related vessel caused middle cerebral artery injury in one case. Resection of the tumor infiltrating the carotid artery inadequately caused injury of the carotid artery. Lenticulostriate artery injury was caused in 2 cases by over-separation of the lateral fissure. CONCLUSION: Severe surgical complications can be avoided if the surgeons, with great skills, are able to predict different topographic changes and difficulties encountered during operation in the light of the specific conditions in operation. PMID- 16681917 TI - [The first "World Kidney Day" is coming--paying more attention to the early diagnosis, treatment and prevention of chronic kidney disease]. PMID- 16681918 TI - [A DNA duplication at chromosome 10q24.3 is associated with split-hand split-foot malformation in a Chinese family]. AB - OBJECTIVE: To identify the disease-causing genetic alteration of split-hand/split foot malformation (SHFM) in a Chinese family. METHODS: Three of the 5 affected individuals from a four-generation Chinese SHFM family were examined physically and radiologically. Peripheral blood samples were collected from Digital photographs of the malformed hands and feet were taken. Peripheral blood samples were collected from 2 affected individuals, and lymphocytes were isolated to undergo high resolution G-banding. Genomic DNA was extracted from the whole blood samples of 4 available family members, including the 3 affected individuals. All 16 exons and their flanking intronic sequences of the TP63 gene were amplified using polymerase chain reaction (PCR) and sequenced directly. Microsatellite markers from the five SHFM loci were analyzed in the available family members by PCR, polyacrylamide gel electrophoresis and silver staining. For semi quantitative determination of the allele copy number, the polymorphic PCR amplified fragments representing genetic markers from the SHFM3 locus at chromosome 10q24.3 were sequenced in the affected individuals using normal individuals with identical genotypes as controls. RESULTS: All 3 existing affected individuals showed absence of 3 radial fingers, 2 affected individuals had a deep central cleft and central ray deficiency in the feet, and 1 affected individual had a fibular monodactyli, all limb malformations being bilateral and consistent with the phenotype of typical SHFM. G-banding showed normal karyotypes in the 3 affected individuals and no visible cytogenetic abnormality was found. Moreover, no mutation was identified in the TP63 gene. While no haplotype sharing was observed in the markers from loci SHFM1, SHFM4 and SHFM5, potential haplotype sharing was detected in the markers from two loci, SHFM2 and SHFM3, indicating possible causative mutation at SHFM2 or SHFM3. Furthermore, obviously biased silver density toward the allele fragments shared by the 3 affected individuals was observed in the markers from the SHFM3 locus. Comparative sequencing showed roughly one-fold increase of fluorescent signal of the shared fragments in the affected individuals. These results suggested a large-scale DNA duplication within the SHFM3 locus. CONCLUSION: A large-scale DNA duplication within the SHFM3 locus at chromosome 10q24.3 has been identified as the pathogenic genetic change in Chinese patients with SHFM. PMID- 16681920 TI - [Preliminary screening and identification of a peptide that binds specifically to gastric cancers cells with high metastasis to peritoneum]. AB - OBJECTIVE: To screen and identify peptides that binds specifically to gastric cancers cells with high metastasis to peritoneum so as to find appropriate vectors for targeting therapy for cancer. METHODS: Human gastric cancer cells of the line GC9811 and those with high metastasis to peritoneum of the line GC9811-P were co-incubated with the 12-mer bacteriophage random peptide library. After 3 round of repeated screening, phage clones were collected. Forty internalized phage single-stranded DNA that specifically binding to the GC98112-P cells were sequenced. GC9811 and GC9811-P cells were co-inoculated with 5 peptides with the N end marked with fluorescein isothiocyanate (FITC) and 1 un-related peptide not binding to GC9811 and GC9811-P cells. Fluorescence microscopy, ELISA, and flow cytometry were used to detect the binding activity. BALB/cnu/nu mice were inoculated intraperitoneally with GC9811 and GC9811-P cells and then randomly divided into. 2 equal groups: experimental group, inoculated with the peptide PIII-FITC and control group (inoculated with un-related peptide PC-FITC. Forty eight hours later the mice were killed and the peritoneum and tumor masses in different organs were collected and under fluorescence microscopy. RESULTS: DNA sequencing showed that 45% (18/45) of the isolated phages displayed repeated sequence SMSIASPYIALE, and SMSI was defined as a conservative motif. Obvious fluorescence was seen in the GC9811-P cells co-incubated with PIII-FITC and weak fluorescence was seen in the GC9811 cells co-incubated with PIII-FITC. Un-marked un-related peptide PC and PIII-FITC did not influenced the fluorescence staining of the GC9811-P cells, however, no fluorescence could be seen in the GC9811-P cells co-incubated with un-marked PIII and PIII-FITC. The fluorescence positive cell rate was 5.9% in the GC9811 cells co-incubated with PIII-FITC, and was 90.2% in the GC9811-P cells co-incubated with PIII-FITC. The fluorescence positive cell rates of the GC9811 cells and GC9811-P cells co-incubated with PC-FITC were 10.1% and 9.9% respectively 10.1% and 9.9% respectively. The fluorescence strength of the GC9811-P cells co-incubated with PIII-FITC was significantly greater than that of the GC9811-P cells co-incubated with PC-FTIC at any time-point and dose (all P < 0.01), and increased along with the increase of co-incubation time and dose of PIII-FITC. The peritoneal tumor tissues caused by the GC9811-P cells of the mice showed strong fluorescence and those caused by GC9811 cells only showed very weak fluorescence. Weak fluorescence could be seen in the tumor masses in the lymph nodes, liver, and muscle of the mice inoculated with GC9811-P cells and was not seen in the tissues of the mice inoculated with GC9811 cells. CONCLUSION: The sequence SMSIASPYIALE that specifically binds to human gastric cancer cells with high metastasis has been screened that has the potential to be used as a marker and targeting vector in diagnosis and treatment of gastric cancer. PMID- 16681921 TI - [Infection pathogen analysis of 2388 patients in an open hematology ward from 1993 to 2004]. AB - OBJECTIVE: To investigate the incidence of infection and pathogens in hematology ward. METHODS: The data of incidence, pathogen, and outcome of infection of 2388 hospitalized patients in an open hematology ward of Peking Union Medical College Hospital from 1993 to 2004 were analyzed retrospectively. RESULTS: The overall incidence of infection was 34.3% according to the person-times of hospitalization, 24.4% for nosocomial infection and 9.9% for community-acquired infection. Most of the pathogenic bacteria of the nosocomial infection were Gram negative. The most common bacteria in the sputum samples included Enterobacter (27%), Pseudomonas aeruginosa (16%) and coagulase negative Staphylococcus (MRSCoN, 12%), the most common bacteria in the blood samples included Escherichia coli (43%), Enterobacter cloacae (11%), and Klebsiella (11%). Whereas in the community-acquired infection the most common bacteria in the sputum samples were Haemophilus parainfluenzae (15%), MRSCoN (28%), and Staphylococcus epidermidis (10%), and the most common bacteria in the blood samples were MRSCoN (28%), E. coli (28%), and Klebsiella (9.4%). Fungi were more often found in nosocomial infection, especially in the sputum samples. 12-year follow up showed that in nosocomial infection Pseudomonas aeruginosa remained the most common bacteria in the sputum samples, whereas E. coli and Enterobacter cloacae became the major bacteria in the blood samples. In community-acquired infection, the proportion of Staphylococcus aureus, that of Klebsiella in blood samples, and that of E. coli in throat swab samples increased in recent years. The incidence of fungi infection had increased in both nosocomial infection and community acquired infection. The mortality of nosocomial infection was 6.1%, higher than that of the community-acquired infection, however, not statistically significant (P = 0.17). There was a trend of decrease in the mortality of community-acquired infection but did not in the nosocomial infection. CONCLUSION: The patients in hematology ward are susceptible to infection, especially nosocomial infection that has a higher mortality rate in comparison with the community-acquired infection, however, not statistically significant. The pathogens of nosocomial infection are most likely G- bacteria, fungi and other bacteria resistant to most antibiotics. The mortality rate of nosocomial infection remains almost unchanged in the 12-year follow up. PMID- 16681922 TI - [Profiles of type 1 and type 2 T cells in chronic idiopathic thrombocytopenic purpura]. AB - OBJECTIVE: To explore the profiles of type 1 and type 2 T cells in chronic idiopathic thrombocytopenic purpura (ITP) patients. METHODS: Samples of peripheral blood were collected from 30 chronic ITP patients, 8 males and 22 females, aged 34, 20 being in the active stage, and 10 in the remission stage, and 20 healthy persons, 7 males and 13 females, aged 31. Peripheral blood mononuclear cells (PBMCs) were isolated, cultured, and activated with PMA/ionomycin. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-gamma and interleukin (IL)-4 in the PBMCs so as to identify the Th1 cells (IFN-gamma(+) IL-4(+) CD4(+) cells), Th2 cells (IFN-gamma(-) IL-4(+) CD4(+) cells), Tc1 cells (IFN-gamma+ IL-4(-) CD8(+) cells), and Tc2 cells (IFN gamma(-) IL-4(+) CD8(+) cells). The ratios of Th2/Tc2, Th1/Th2, and Tc1/Tc2 were calculated. Samples of spleen tissue were collected from 8 patients with chronic ITP, 3 males and 5 females, aged 30, and 6 patients with hereditary spherocytosis (HS), 3 males and 3 females, aged 35, who underwent splenectomy. Splenocytes were isolated, cultured, and activated with PMA/ionomycin. Real-time PCR was used to detect the mRNA expression of T-bet and GATA-3 in the PBMCs and splenocytes. RESULTS: The Th1/Th2 ratio of the patients in active stage was 25.62, significantly higher than those of the patients in remission stage (9.86) and the control (8.29, both P < 0.01), and the Tc1/Tc2 ratio of the patients in active stage was 30.23, significantly higher than those of the patients in remission stage (10.10) and the controls (12.58, both P < 0.01). The Th1/Th2 ratio of the splenocytes of the patients in active stage was 41.46, significantly higher than that of the controls (16.30, P < 0.01), and the Tc1/Tc2 ratio of the splenocytes of the active ITP patients was 35.80, not significantly higher than that of the controls (16.30, P = 0.082). The GATA-3 mRNA expression level of the PBMCs of the active ITP patients was one-fifth of that of the controls (P < 0.05) and the GATA 3 mRNA expression level of the splenocytes of the ITP patients was 0.34 of that of the HS patients (P < 0.05), however, there was no difference in the T-bet expression among the 3 groups. The T-bet/GATA-3 ratio of the PBMCs of the active ITP patients was 5.85 times that of the controls and the T-bet/GATA-3 ratio of the splenocytes of the active ITP patients was 2.68 times that of the controls (both P < 0.01). CONCLUSION: ITP is a T1 cells (Th1 and Tc1) predominant disease. The T-bet/GATA-3 ratio may provide a surrogate marker of T1/T2 cytokine balance. Shifting the cytokine patterns from T1 to T2 may be a potential immunotherapy for ITP. PMID- 16681924 TI - [Characteristics of the daily blood glucose profiles of impaired glucose tolerance and type 2 diabetes mellitus subjects by continuous glucose monitoring system]. AB - OBJECTIVE: To investigate the characteristics of daily glucose profiles of the impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) subjects by continuous glucose monitoring system (CGMS) so as to instruct the development of rational programs of treatment. METHODS: The CGMS data of 6 normal glucose tolerance (NGT), 10 IGT, and 20 newly diagnosed T2DM subjects were analyzed. RESULTS: (1) The values of average blood glucose, standard deviation of blood glucose, total time of plasma glucose > or = 7.8 mmol/L (T(PG) > or = 7.8 mmol/L) of the T2DM subjects were all significantly higher than those of IGT subjects (all P < 0.05), and these values of the IGI subjects were all significantly higher than those of the NGT subjects (all P < 0.05). The CGMS blood glucose profile excursion were also more and more fluctuant with the deterioration of glucose tolerance. (2) In the IGT group, the time of PG reaching peak was 109 minutes +/- 32 minutes and the daily time of PG > or = 7.8 mmol/L was 3.0 hours +/- 0.8 hours. In the newly diagnosed T2DM group, the time of PG reaching peak was 92 minutes +/- 22 minutes, and the daily time of PG > or = 11.1 mmol/L was 12.6 hours +/- 1.3 hours. The blood glucose increased more quickly and more sharply, and continued for more time after breakfast. CONCLUSIONS: (1) CGMS data accurately display the characteristics of daily 24 h blood glucose profiles of the IGT and newly diagnosed T2DM subjects, and CGMS examination helps instruct the development of rational treatment program. (2) In daily life, the time of PG > or = 7.8 mmol/L in IGT subjects was nearly 3 hours. (3) With the deterioration of glucose tolerance, the postprandial glucose peak is elevated higher and more rapidly, and the daily blood glucose profile excursion becomes more and more fluctuant. PMID- 16681926 TI - [The predictive value of serum advanced fibrinogen and uric acid for acute coronary event risk]. AB - OBJECTIVE: To study the serum advanced fibrinogen (FIB) and uric acid (UA) in predicting acute coronary events risk. METHODS: 264 patients hospitalized with the suspected diagnosis of coronary heart disease (CHD) were divided into 2 groups according to the results of coronary arteriography: acute coronary event group (n = 195, 141 males and 54 females, aged 62 +/- 12, with laboratory and electrocardiogram proven acute myocardial infarction or unstable angina pectoris and obvious stricture of main coronary artery > 75%) and control group (n = 69, 31 males and 38 females, aged 65 +/- 9, without acute coronary event and with normal coronary artery or stricture of branches of coronary artery less than 50%). A week before the coronary arteriography fast peripheral venous blood samples were collected. The blood sugar, blood-lipid, FIB, and UA were determined. RESULTS: The FIB level of the acute coronary event group was 4.09 g/L +/- 1.36 g/L, significantly higher than that of the control group (3.52 g/L +/- 1.37 g/L, P < 0.05). The UA level of the acute coronary event group was 320 micromol/L +/- 91 micromol/L, not significantly different from that of the control group (302 micromol/L +/- 104 micromol/L, P > 0.05). Excluding the influence of age, sex, blood pressure, blood sugar, etc. multivariate analysis showed that FIB was a significant variable associated with acute coronary events, but UA, not entering the regression equation, was not an independent risk factor. When FIB and UA were both at high levels, acute coronary events risk was much higher than when any single index was at a high level. CONCLUSION: High FIB and UA jointly accelerate the development of atherosclerosis. It may be important to use the index of FIB associated with UA to predict acute coronary risk. PMID- 16681928 TI - [Application of homologous conduits with valves in the treatment of complex congenital heart disease with insufficient pulmonary blood flow in children]. AB - OBJECTIVE: To investigate the effects of homologous conduits with valves in the treatment of complex congenital heart disease with insufficient pulmonary blood flow in children. METHODS: Homologous conduits with valves were used in the right ventricular outflow tract reconstruction among 212 pediatric patients with complex congenital heart disease with insufficient pulmonary blood flow, aged 2.6 (0.5 - 15). The patients were followed up for 2.6 years (6 months to 7 years) and underwent echocardiography to assess the degree of pulmonary insufficiency and calcification of blood vessel. RESULTS: The success rate of operation was 96.5% (204/212). The ratio of right ventricle to aortic pressure was less then 0.55. During the follow-up, echocardiography showed valvular regurgitation and calcification of the blood vessel in 34 patients, 23 (11.2%) being mild, 7 (3.42%) being moderate, and 4 (1.96%) being sever. 170 patients did not show obvious pulmonary insufficiency. CONCLUSION: Application of homologous conduits with valves in the treatment of complex congenital heart disease with insufficient pulmonary blood flow in children shows excellent early results in preventing pulmonary insufficiency. PMID- 16681927 TI - [Identification of hepatitis B virus genotypes in patients with chronic hepatitis B from different nationalities in ethnic minority areas in Yunnan Province, China]. AB - OBJECTIVE: To determine whether there is evolutionary difference in hepatitis B virus (HBV) genotypes among the patients with chronic hepatitis B (CHB) of different nationalities and its clinical significance. METHODS: Peripheral blood samples were collected from 50 CHB patients, 25 of diverse nationalities and 25 of Han nationality from the ethnic minority regions in Yunnan Province, China, The HBV preS2/S (pre S2/S) and C genes were amplified by PCR. The PCR products were inserted into the vector pBluescriptIISK (pBS). The cloned preS2/S and C genes were sequenced. RESULTS: The sequences of HBV preS2/S and C genes from the 50 patients were 846 (with 49.1% of GC) and 552 (with 46.1% of GC) nucleotides (nt) in length, and encoded 281 and 183 amino acids (aa) respectively. These findings were registered in GenBank Accession Numbers: AY517619, AY517620, AY517488, AY517489, AY517598, AY517599. Compared with the HBV and subtype sequences in the GenBank database, the HBV preS2/S and C genes among all the subjects were homologous to ayw1 in sequence by 97.5% - 98.6% and 94.5% - 97.8% respectively. The "a" determinant region of S genes in all cases were found to be Arginine (AGA) and Lysine (AAA) at corresponding aa 122nd and 160th respectively. HBV genotype B was identified in all patients with CHB (ayw1 subtype). Genotypes A, C, D, E, F, G, and H were not detected in any of them. The quasi-species nature of the HBV in the sera was observed in 2 of the 50 samples examined (4%). There was not a significant difference in the prevalence of HBV genotype B between the 25 diverse nationality patients and the 25 control Han nationality patients (P > 0.05). In the 50 CHB patients, the preS2/S genes were identified to have aa substitutions at the positions R124K (1.1%), L172P (1.3%), M306T (1.5%), and I361M (1.6%), with a frequency of more than 1%. In all subjects, the frequency of aa G145R (0.4%) substitutions was less than 1%. In all subjects, nt variations of C genes caused aa substitutions among aa 27 - 63, 80 - 110, and 135 - 153 involved in T and B cell epitopes. In 45 CHB patients, C genes was identified to have aa substitutions at the positions V 27, N38, V63, Q135, and A147, due to nt variations of 1979A to G, 2012T to A, 2088G to T, 2304C to A, and 2339A to G transitions respectively. The frequency of aa substitutions of C genes was more than 1%. Whereas as for the other 5 severe CHB patients, the C gene variations of A to G, and A to C transitions at nt positions 2159 and 2189 led to aa substitutions of S to G and I to L at positions G87 and L97. No insertion or deletion was found in preS2/S and C regions. HBV genotype B was not relevant to different nationalities (all P > 0.05). CONCLUSION: It is the first time that the genotype of the HBV epidemic strains in the ethnic minority areas of Yunnan Province has been identified as genotype B subtype ayw1. The HBV genotype B is not related with nationality. A novel genotyping method by using PCR, gene cloning, followed by DNA sequencing that can identify all major genotypes has been developed. HBV genotype B is the geographic original strain in this area and is correlated with the severity of liver diseases and curative effect. HBV viral is the only significant variable associated with the CHB patients' prognosis. PMID- 16681929 TI - [Spleen-preserving distal pancreatectomy in treatment of solid-pseudopapillary neoplasm]. AB - OBJECTIVE: To investigate the feasibility and safety of spleen-preserving distal pancreatectomy with conservation of the splenic arteries and veins in treatment of benign neoplasms of distal pancreas. METHODS: Four patients with solid pseudopapillary neoplasm, 1 males and 3 females, aged 30 (17 - 37), underwent laparotomy. The distal pancreas with tumor was cut. The perforating branches between the splenic artery and vein and the distal pancreas were isolated, ligated, and cut. The splenic artery and vein and the short and left gastric vessels were all preserved. The spleen remained intact. RESULTS: The mean operation time was 208 minutes +/- 52 minutes, and the mean blood loss was 475 ml +/- 96 ml. Blood transfusion was not necessary. The mean post-operative hospital stay was 18 days +/- 13 days. No post-operative complications were found except for pancreatic leakage occurring in one patient that was cured by conservative treatment. No metastasis and recurrence was found. CONCLUSION: Safe and feasible, spleen-preserving distal pancreatectomy with conservation of the splenic arteries and veins is one of the best choices for treatment of benign and borderline tumors of pancreas. PMID- 16681930 TI - [Effects of norcantharidin on angiogenesis of human gallbladder carcinoma and its anti-angiogenic mechanisms]. AB - OBJECTIVE: To investigate the effects of norcantharidin (NCTD) on angiogenesis of human gallbladder carcinoma and its anti-angiogenic mechanisms. METHODS: Human gallbladder carcinoma cells of the line GBC-SD were cultured. BALB/c nude mice were inoculated subcutaneously with the GBC-SD cells and then randomly divided into 6 groups: NCTD group, injected intraperitoneally with 1/5 of the LD(50) of NCTD twice a week for 6 weeks; 5-fluorouracil (5-FU) group, injected intraperitoneally with 1/5 of the LD(50) of 5-FU twice a week for 6 weeks; endostatin (ES) group, intraperitoneally with ES; NCTD + 5-FU group, injected intraperitoneally with 1/5 of the LD(50) of NCTD and 1/5 of the LD(50) of 5-FU twice a week for 6 weeks; NCTD + ES group, injected intraperitoneally with 1/5 of the LD(50) of NCTD and ES twice a week for 6 weeks; and normal saline (NS) group (control group), injected with NS. The mice were killed in the 7th week. The tumors were taken out to measure their volumes and undergo microscopy. SABC method of immunohistochemistry was used to measure the microvessel density (MVD) and the protein expression of the angiogenesis-related factors: proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), angiopoietin (Ang)-2, thrombospondin (TSP), and tissue inhibitor of metalloprotease (TIMP)(2). Suspension of single tumor cell was prepared to examine the cell apoptosis by flow cytometry. RT-PCR was used to examine the mRNA expression of PCNA, VEGF, Ang-2, TSP, and TIMP2. RESULTS: (1) The MVD of the NCTD group was 4.12 +/- 1.4, significantly lower than those of the 5-FU group (15.8 +/ 5.9) and control group (17.6 +/- 3.2) (both P < 0.01), but not significantly different from those of the NCTD + 5-FU group (3.8 +/- 1.7), ES group (4.5 +/- 2.1), and NCTD + ES group (2.9 +/- 1.5) (all P > 0.05). The mice treated with NCTD showed significantly smaller tumor volume, lower PCNA protein expression, higher apoptotic rate, and higher PCNA/apoptosis ratio (P < 0.05 or P < 0.01), and significant correlation between MVD and tumor volume and between MVD and PCNA/apoptosis ratio (both P < 0.05). (2) The protein expression of VEGF and of Ang-2 of the NCTD group were both significantly lower than those of the control and 5-FU groups (all P < 0.01), however, not significantly different from those of the ES, NCTD + 5-FU, and NCTD + ES groups; and the protein expression of TSP and of TIMP2 of the NCTD group were both significantly higher than those of the control and 5-FU groups (all P < 0.01), however, not significantly different from those of the ES, NCTD + 5-FU, and NCTD + ES groups. MVD was positively correlated with VEGF and Ang-2 expression and negatively correlated with the expression of TSP and TIMP2 (all P < 0.05). (3) In comparison with the control group, the mRNA expression of VEGF and of Ang-2 of the tumor cells of the NCTD group were both significantly lower and the mRNA expression of TIMP2 was significantly higher. CONCLUSION: NCTD down-regulates the expression of the angiogenic factors, such as VEF|GF and Ang-2, and up-regulates the expression of the anti-angiogenic factors, such as TDP and TIMP2, thus inhibiting the angiogenesis in tumor, such as human gallbladder carcinoma, and further inhibiting the growth of tumor. PMID- 16681932 TI - [Comparative study of eukaryotic cell expression of wild-type and Pro370Leu mutation type myocilin gene]. AB - OBJECTIVE: To compare the eukaryotic cell expression of wild-type myocilin (MYOC) (wMYOC) gene and Pro370Leu mutation type MYOC (mMYOC) gene so as to understand the mechanism of primary open angle glaucoma (POAG). METHODS: HeLa cells were cultured and then transfected with the vector pEGFP-N3-wMYOC, recombinant plasmid with wild type MYOC gene and enhanced green fluorescein gene, or the vector pDsRed2-N1-mMYOC, recombinant plasmid with mutation MYOC gene and red fluorescein gene, respectively, or co-transfected with these 2 plasmids. Corresponding blank vectors pEGFP-N3 and pDsRed2-N1 were used as markers. Fluorescence microscopy was used to observe the localization of red fluorescence and green fluorescence. Laser co-focusing microscopy was used to observe the effect of co-transfection. The green and red fluorescein antibodies were examined by Western blotting. RESULTS: Green fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pEGFP-N3, in an even distribution; and red fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pDsRed2-N1, in an even distribution too. The cells transfected with pEGFP-N3-wMYOC showed evenly-distributed green fluorescence in the cytoplasm, and the cells transfected with pDsRed2-N1-mMYOC showed red fluorescence in the cytoplasm in a state of aggregation. Both red and green fluorescence could be seen in the cells co-transfected with pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC, both in a state of aggregation and co-localization. Laser co-focusing microscopy showed the same results. Protein with the relative molecular weight of 83,000, identical to that of the recombinant protein of wMYOC protein and green fluorescein, could be found in the culture fluid and cell lysate of the pEGFP-N3 wMYOC-transfected cells; however, could be found in the lysate only but not in the culture fluid of the co-transfected cells. Protein with the relative molecular weight of 81,000, identical to that of the recombinant protein of mMYOC protein and red fluorescein, could be found in the cell lysates of the pDsRed2-N1 mMYOC-transfected cells and the pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC co transfected cells, but not in the culture fluid of both cells. CONCLUSION: Both the WMYOC and mMYOC genes can be expressed and localized in the cytoplasm. mMYOC protein shoes a tendency of aggregation and disorder in secretion, and affects the expression and secretion of wMYOC, thus influencing the factions of the trabecular meshwork and causing POAG. PMID- 16681940 TI - [Deep research and practice on programming and foundation of emergency medical service system]. PMID- 16681942 TI - [The mutation R672H in SCN4A gene exists in Chinese patients with hypokalaemic periodic paralysis]. AB - OBJECTIVE: Mutation screening was performed on two Chinese families with HOKPP to locat the corresponding mutations and to specify the clinical features associated with the mutation. METHODS: Target-exon PCR and direct sequencing were used to screen mutation in the CACNA1S and SCN4A gene of all numbers of the two families. The clinical features of patients were summary. RESULTS: A heterozygous point mutation 2015G-->A causing R672H in the SCN4A was found in five patients and five normal relatives of the two families. Features of R672H mutation are incomplete penetrance, especially non-penetrance of phenotype in women and potassium is effective, but acetazolamide is not. CONCLUSION: The SCN4A R672H mutation exists in the Chinese family with HOKPP. PMID- 16681943 TI - [The study on the prevalence and associated risk factors of female urinary incontinence in Beijing women]. AB - OBJECTIVES: To evaluate the prevalence, the clinical characteristics and associated risk factors of urinary incontinence in Beijing women. METHODS: In the cross-sectional study, 5300 Beijing women aged over 20 years were interviewed through a questionnaire including International Consultation on Incontinence Questionnaire Female Lower Urinary Tract Symptoms Module (ICIQ-FLUTS). Aged from 20 to 99 years old (mean 46 +/- 17). Divided into 8 groups every 10 years. 4812 cases are married. RESULTS: The overall prevalence of UI was 38.5% (2008/5221), among theses women, 22.9% (1197/5221), had stress incontinence, 2.8% (147/5221) urge incontinence and 12.4% (649/5221) mixed incontinence. In multiple logistic models, age, delivery mode, smoking, hypertension, constipation, body mass index, chronic pelvic pain (CPP) were relation factors for stress incontinence. CONCLUSIONS: The prevalence of urinary incontinence is high (38.5%). Urinary incontinence is major disease that affects female quality of life. 22.9% (1197/5221) of the incontinence women had stress urinary incontinence, 2.8% (147/5221) urge urinary incontinence and 12.4% (649/5221) mixed urinary incontinence. Old, vaginal delivery, smoking, hypertension, constipation, CPP and higher BMI are potential risk factors for stress urinary incontinence. Cesarean section are protective factors for stress urinary incontinence. PMID- 16681944 TI - [Inhibition of HLA-DRB1*0405 gene expression by siRNA]. AB - OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) of HLA DRB1(*)0405 HLA-DRB1(*)0405 gene expression with plasmid-based siRNAs. METHOD: Plasmid expressing HLA-DRB1(*)0405-renilla fusion protein-siCHECK-2/HLA DRB1(*)0405, 6 different short hairpin RNAs (shRNAs) targeting 6 19 bp nucleotide sequences of HLA-DRB1(*)0405 (siRNA1 approximately 6), and one shRNA targeting the control non-specific sequence (siRNAC) were designed and constructed. Human embryonic kidney cells of the line 293 were cultured and co-transfected by lipids some with the plasmid siCHECK-2/HLA-DRB1(*)0405 and one specific shRNA expressing vector transiently, and cells without shRNA-transfection were used as negative controls. The impact of RNAi on HLA-DRB1(*)0405 expression was analyzed by real time fluorescence quantification RT-PCR and luciferase test. RESULTS: The expression of HLA-DRB1(*)0405 gene RNA of the 293 cells transfected with siRNA1, siRNA2, siRNA3, siRNA, and siRNA6 were down-regulated to 10.75%, 83.22%, 30.63%, 48.54%, and 89.92% that of the control group with the inhibition rates of 89.25%, 16.78%, 69.37%, 51.46%, and 10.08% respectively. However, no significant downregulation was showed in the cells transfected with siRNA4 and siRNAC. The 293 cells transfected with siRNA1 and siRNA 3 showed a significant downregulation of the protein expression of HLA-DRB1(*)0405 gene with the inhibitory rates of 6.70% and 36.85% respectively; however, the cells transfected with siRNA2, siRNA4, siRNA5, and siRNA6 did not show a significant downregulation. CONCLUSION: The significant inhibition of HLA-DRB1(*)0405 gene expression by siRNA suggests a therapeutic approach in rheumatoid arthritis: to use RNA interference (RNAi) to inhibit the abnormal immune reaction mediated by HLA-DRB1 in rheumatoid arthritis. PMID- 16681945 TI - [Anatomic study on the design of far-lateral transcondylar transtubercular keyhole approach assisted by neuro-navigation]. AB - OBJECTIVE: To design a new far-lateral transcondylar transtubercular keyhole approach assisted by neuro-navigation system according to the keyhole idea, and to explore the possibility of removing the occipital condyle and jugular tubercle precisely. METHODS: Navigation data were established on 8 cadaveric heads fixed by formalin and with their intracranial vessels perfused with colored silicone. Before the operation, circumscriptions of the occipital condyle and jugular tubercle were outlined with different colors in the navigation system in order to aid drilling them in operation. A 7 cm longitudinal "S" shaped skin incision was performed with its superior border 2 cm behind the middle point of mastoid and inferior margin at the level of C(2). After inverting the suboccipital muscles and exposing the far lateral part of the occipital bone, occipital condyle, hemilamina of C(1), vertebral artery and posterolateral portion of foramen magnum, a retro-condylar bone flap 3 cm in diameter was cut. Assisted by neuro navigation, not only were the maximal angle of visual field measured before and after the 1/3 and 1/2 posteromedial occipital condyle removal respectively, but also the anatomic structures were observed and measured. RESULTS: The incision of the retro-condylar keyhole approach fully met the needs of the far-lateral transcondylar transtubercular keyhole approach; partial occipital condyle and jugular tubercle could be precisely drilled with the aid of neuro-navigation, thus avoiding the bewilder in drilling process; the maximal angles of visual field were 39.2 degrees +/- 3.29 degrees (before condyle drilled), 51.5 degrees +/- 2.45 degrees (1/3 condyle drilled) and 57.5 degrees +/- 2.66 degrees (1/2 condyle drilled) respectively, and there were significant difference among them (P < 0.01). CONCLUSION: It is feasible to perform the far-lateral trascondylar transtubercular keyhole approach; the maximal angle of visual field is obviously increased by drilling partial occipital condyle, and the middle clivus can be increasingly exposed through removal of jugular tubercle. PMID- 16681946 TI - [Effects of peroxisome proliferators-activated receptor gamma agonists on transforming growth factor-beta1 and Smads signal pathway: experiment with rat renal fibroblasts]. AB - OBJECTIVE: To study the effects of peroxisome proliferators-activated receptor gamma (PPARgamma) agonists on transforming growth factor (TGF)-beta1-induced fibrotic responses in renal fibroblasts, so as to investigate its effects in prevention of tubulointerstitial fibrosis. METHODS: Rat renal fibroblasts of the line NRK/49F were cultured and divided into groups. In group 1 TGFbeta1 of the concentrations of 0, 1, 2, 5, and 10 ng/ml was added and co-cultured for 24 h. In group 2 TGFbeta1 of the concentration of 5 ng/ml was added and co-cultured for 0, 6, 12, and 24 h respectively. Groups 3, 4, and 5 were pretreated with 10 micromol/L15d-PGJ2, PPARgamma ligand, 10 micromol/L troglitazone, agonist of, and 10 micromol/L ciglitazone, both PPARgamma agonists, respectively for 2 h, then treated with 5 ng/ml TGFbeta1. A blank control group was set up. The cultured cells were collected. RT-PCR was used to detect the mRNA expression of TGF-beta1 indued fibronectin (FN). Western blotting was used to detect the expression of TGF-beta1-induced FN, Smad, and phosphorylated Smad (p-Smad) proteins. RESULTS: TGF-beta1 enhanced the FN mRNA expression in a dose- and time-dependent manner. The FN mRNA expression of the 5 ng/ml TGF-beta1 group was 3.7 times that of the control group (P < 0.05). The FN mRNA expression of the 15d-PGJ2, troglitazone-, and ciglitazone-pretreated groups were lower than that of the 5 ng/ml TGF-beta1 group by 37.3%, 41.5%, and 22.7% respectively (all P < 0.05). The p-Smad2/3 protein expression levels of the TGF-beta1 group began to increase 15 minutes after stimulation, increased in a time-dependent manner, peaked 1 hour after, and began to decrease 2 hours later. However, the levels of protein expression of total Smad2 and Smad3 did not change significantly in all groups. Both 2 ng/ml TGFbeta1 and 5 ng/ml TGFbeta1 significantly induced the increase of protein expression of p-Smad2/3 (all P < 0.05). The levels of protein expression of p Smad2 and p-Smad3 of the 5 ng/ml TGFbeta1 group were 3.42 and 0.97 times those of the 2 ng/ml TGFbeta1 (both P < 0.05). The levels of protein expression of p Smad2/3 of the 15d-PGJ2, troglitazone-, and ciglitazone-pretreated groups were all significantly lower than that of the 5 ng/ml TGFbeta1 group by 61.2%, 53.0%, and 59.5% (all P < 0.05), However, there was no significant difference among different drug-treated groups (all P > 0.05). CONCLUSION: Possibly through abrogating TGF-beta1/Smads signaling pathway, PPARgamma agonists inhibit TGF beta1-induced renal fibroblast extracellular matrix synthesis and may play a potential role in preventing tubulointerstitial fibrosis as a novel approach to prevent the progress of end stage renal dysfunction. PMID- 16681947 TI - [Effects of injecting urokinase via carotid artery in treatment of cerebral venous sinus thrombosis: an experiment with rabbit models]. AB - OBJECTIVE: To study the effects of injection of urokinase via carotid artery in treatment of cerebral venous sinus thrombosis. METHODS: Thirty-nine New Zealand white rabbits were made into models of cerebral venous sinus thrombosis with the superior sagittal sinus ligated and injected with suspension of cephalin kaolin and cephalin, and then were randomly divided into three groups: control group (n = 15), anticoagulation group (n = 12, infused with heparin 2 mgxkg(-1)xd(-1) via external carotid venous for 5 days), and thrombolysis group (n = 12, injected with urokinase 4000 Uxkg(-1)xd(-1) via carotid artery for 5 days). Laser Doppler scanning was used to determine the local cerebral blood flow before ligation, 20 and 90 min after ligation, and at the end of experiment. Five rabbits of the control group per each of the 3 time points (before ligation, 12 h after ligation, and at the end of experiment) were killed to collect the tissues of cerebral cortex in front of the ligated part to determine the brain water content. All the remaining rabbits were killed within 24 hours after the end of experiment to determine the brain water content and undergo microscopy. RESULTS: The local cerebral blood flow of the thrombolysis group was 94.8% +/- 0.3%, significantly higher than those of the anticoagulation group (88.6% +/- 0.3%) and control group (80.9% +/- 0.3%) (both P < 0.01). The brain water content of the thrombolysis group was 78.9% +/- 0.3%, significantly lower than those of the anticoagulation group (81.8% +/- 0.7%) and control group (82.1% +/- 1.0%) (both P < 0.05). CONCLUSION: Improving the local blood flow and reducing the brain water content, injection of urokinase via carotid artery is effective in treatment of cerebral venous sinus thrombosis. PMID- 16681948 TI - [The effect of VEGF antisense oligonucleotides combined with low molecular weight heparin on the growth and metastasis of mice Lewis lung cancer]. AB - OBJECTIVE: To investigate the inhibitory effect of vascular endothelial growth factor (VEGF) antisense oligonucleotides or/and dalteparin sodium (fragmin) on tumor growth and metastasis of mice Lewis lung cancer. METHODS: 40 mice with Lewis lung cancer were randomizedly divided into five groups: control group, VEGF antisense oligonucleotides (ASODN) group, VEGF mismatch sense oligonucleotides (MSODN) group, low molecular weight heparin (LMWH) group, and combined group. Sodium chloride, VEGF-ASODN, VEGF -MSODN, fragmin, and VEGF-ASODN plus fragmin were given respectively (once every two days, 15 times altogether). The volume and weight of subcutaneous tumors were measured, and the rates of lung metastasis were detected by HE staining. The microvessel density (MVD) in tumor mass were measured by immunohistochemistry staining. VEGF protein level in tumor tissue were detected by Western blot assay. RESULTS: After treatment, the tumor growth inhibitory rates were 47.34%, 27.31% and 59.03%, and the rates of lung metastasis were 37.5%, 37.5% and 25% in ASODN, LMWH, and the combined group, respectively. Tumor MVD and VEGF protein expression of the above three groups were lower than those of the control group. There was a significant difference in regard to the tumor growth inhibitory rates and MVD between the above three treated groups and the control group as well as the MSODN group (P < 0.05). CONCLUSIONS: VEGF-ASODN and fragmin may down-regulate VEGF gene expression and inhibit angiogenesis, Combined use of fragmin can enhance anti- tumor effect of VEGF-ASODN. PMID- 16681949 TI - [Risk factors affecting the survival rate in serious multiple trauma patients associated with acute lung injure/acute respiratory distress syndrome]. AB - OBJECTIVE: To investigate risk factors affecting the survival rate in serious multiple traumatic patients associated with acute lung injure (ALI)/acute respiratory distress syndrome (ARDS). METHODS: The clinical data of 76 serious multiple trauma patients associated with ALI and 95 cases in ARDS, totally 171 patients associated with ALI/ARDS, were retrospectively studied, and stepwise logistic regression analysis was used to analyze 20 possible risk factors affecting survival rate. RESULTS: The risk factors affecting survival rate in the ALI group: included smoking (B = -5.235, OR = 0.005, P = 0.001), sepsis secondary to trauma (B = -2.753, OR = 0.064, P = 0.031), and gastrointestinal hemorrhage (B = -2.876, OR = 0.056, P = 0.033). The risk factors affecting survival rate in the ARDS group included the time of induction factor persisting to attacking (B = 3.524, OR = 33.933, P = 0.008), sepsis secondary to trauma (B = -5.183, OR = 0.006, P = 0.004); renal insufficiency(B = -4.745, OR = 0.009, P = 0.009), and gastrointestinal hemorrhage (B = -6.335, OR = 0.002, P = 0.007). CONCLUSION: Different from the results of study of traditional risk factors affecting survival rate in ALI/ARDS, this study reveals that smoking may be an independent risk factor; the earlier ALI/ARDS appears, the lower the predictable survival rate in condition of serious multiple trauma; MODS is easily induced in the course of complicating renal insufficiency during the time of lung injury; sepsis and gastrointestinal hemorrhage are still the infective factors or infection inducing factors affecting the survival rate after trauma; and the primary disease causing infection must be treated actively. PMID- 16681951 TI - [Evaluation of stability of distal radius fracture after conservative treatment]. AB - OBJECTIVES: To evaluate the effect of conservative treatment on stability of different types of distal radius fracture so as to help select the optimal treatment of different distal radius fracture and verify the exact time when the patient need careful. METHODS: During the period of one year 103 consecutive patients of distal radius fracture were treated by closed reduction and below elbow cast immobilization and standard PA in the emergency room and were followed up 1, 2, and 4 weeks after the initial visit until the casts were removed. Lateral radiographic examination was conducted before and after reduction and during the reexamination. All the images were saved in a working server in hospital. The patients were classified according to the Cooney classification system, then the displacement, palmar tilt, radial inclination and radial shortening in every film were measured accurately and involvement of radiocarpal joint surface was noted. Combined with the assessment by the Lindstrom grading system, the radiographic results were divided into 2 groups: accepted and unaccepted. Finally, Pearson Chi-square test, likelihood ratio test, and Kendall correlation analysis were used to testify the radiographic results of different Cooney types. RESULTS: Fractures of Cooney type 1, 2 and 3 achieved good results after non-operative treatment with the rates of accepted results of 90%, 88.5%, and 88.9% respectively. Only 28.1% of the type 4 fractures reached the accepted criteria. The general rate of accepted results among the 103 fracture patients was 69.9%. The general rate of accepted results of the 103 patients was 69.9%. The rates of accepted results of the Cooney type 1, 2, 3, and 4 fractures were 90.0%, 88.5%, 88.9%, and 28.1% respectively. The radiographic result was significantly correlated with the Cooney classification with a large Kendall coefficient. Displacement after closed reduction was found in 36.5% of the type 2 fractures and 65.6% of the type 4 fractures and a large majority of them displayed instability within a week after the closed reduction and cast immobilization. CONCLUSION: A large majority of the distal radius fractures can achieve good results after treatment by closed reduction and cast immobilization, especially those of type 1, 2, and 3, for which conservative treatment should be the first choice. However, type 4 fracture is extremely unstable and a large percentage of it fails to get satisfactory result by non-operative treatment, so operative treatment is better for it. Reexamination 1 - 2 weeks after the manipulation should be emphasized so as to adopt effective treatment in time. PMID- 16681953 TI - [Experiences in surgical treatment of cardiac tumors]. AB - OBJECTIVE: To summarize the experiences in surgical treatment of cardiac tumors. METHODS: The results of surgical treatment of 494 patients with cardiac tumors hospitalized from 1975 to 2004, aged 43 +/- 16 (4 months to 78 years), 432 with myxoma, 27 with benign nonmyxoma, and 35 with malignant tumors, were analyzed. RESULTS: Complete resection of malignant tumor was achieved in 10 cases (28.6%), and subtotal resection was achieved in 15 cases (42.9%). or partial resection was performed based on the intra-operative condition. Only biopsy was performed in 10 patients (28.6%) because of extensive metastasis of tumor. Hospital death occurred in 11 myxoma patients (2.5%), and 3 malignant tumor patients (8.6%), and none of the benign non-myxoma tumor patients. The patients were followed up for 70 +/- 36 months with a follow-up rate of 92.9%. Late follow-up was performed for the 241 patients with myxoma who had underwent resection after 1990. Seventeen patients were lost to follow-up. There were 10 late deaths, 5 resulting from recurrence and 5 from unknown causes. Follow-up for malignant tumors showed 18 (51.4%) late deaths because of recurrence or metastasis, occurring 2 months to 2 years after operation. CONCLUSION: Excellent immediate and long-term operative results can be obtained for benign cardiac tumors. However, for the patients with malignant tumors the survival chances are dismal. PMID- 16681952 TI - [Clinical character and treatment of coexistence of cervical spine disease and lumbar spine degenerative disease]. AB - OBJECTIVE: To investigate the clinical character and surgical management in coexistence of cervical and lumbar spine disease. METHODS: 53 cases with coexistence of cervical and lumbar spine disease were analyzed for clinical character and follow-up outcomes. 19 single cervical operation cases, 16 single lumbar operation cases and 7 combined cervical lumbar operation cases, all patients were recorded with JOA score. RESULTS: The cases with cervical and lumbar intervertebral disc showed at least 2 segments. After 6 months follow-up, 35/44 cases symptom were relieved obviously, of which 24/35 cases were single operation, relief rate is 75.9%, satisfactory rate is 80.0%; of which 11/35 cases were continuous cervical lumbar operation, relief rate is 78.4%, satisfactory rate is 78.5%. 6/44 cases' symptom with only cervical or only lumbar operation were relieved incompletely, 3/44 cases' symptom with second operation relieved incompletely. 5 cases missed diagnosis on the first operation. 29 cases with responsible focus, of which 25 cases were followed up, relieved obviously, relief rate is 76.5%, satisfactory rate is 96.0%. CONCLUSION: The cases with cervical and lumbar intervertebral disc showed at least 2 segments, the same basis of the spinal canal stenosis and disc herniation is degeneration of spine. certain responsible focus and correct surgical management can get good treatment results. PMID- 16681962 TI - [Thermal ablation of tumor: present and prospect]. PMID- 16681954 TI - [Neuropsychiatric manifestations in systemic lupus erythematosus and the treatment of intrathecal methotrexate plus dexamethasone]. AB - OBJECTIVE: To study the neuropsychiatric (NP) manifestations of systemic lupus erythematosus (SLE) and evaluate treatment with intrathecal (IT) methotrexate (MTX) and dexamethasone (DXM). METHODS: 240 patients with NP syndromes of SLE (NPSLE) from 1990 to 2004 were retrospectively reviewed and IT injection group and non-IT injection group were compared. The side effects of IT injection were also discussed. 130 patients were followed up after discharge. RESULTS: Fifteen of the 19 ACR NP syndromes were identified. 86 (35.8%) patients presented one NP syndrome and 154 (64.2%) presented with more than one. The most frequent manifestations were headache and seizure disorder. 109 patients received IT injections. After IT injection, the CSF index had significantly improved. The mean SLEDAI score, mean duration of hospitalization and mortality rate of patients with IT injection were lower than those patients without IT injection (P < 0.05 - 0.001). The side effects of IT injection were found in 11.0% patients. 23 of 130 patients had recurrent NP events during follow up. CONCLUSION: There was heterogeneity of NPSLE in our study group. The most common NP features were headache and seizure. IT injection of MTX and DXM is an effective and safe alternative to traditional treatment of NPSLE. PMID- 16681963 TI - [Long-term results of percutaneous sonographically-guided microwave ablation therapy of early-stage hepatocellular carcinoma]. AB - OBJECTIVE: To evaluate the long-term results of percutaneous microwave coagulation therapy in the treatment of early hepatocellular carcinoma. METHODS: From May 1994 to June 2004, a total of 216 patients with 275 nodules of hepatocellular carcinoma whose diameter 3.0 cm in diameter. Forty-seven patients had single nodule, 44 patients had 2 nodules, and 93 patients had 3 or more nodules. Fifty-seven patients had tumor of low differentiation, 53 had tumor of middle differentiation, and 18 had highly differentiated tumors. Kaplan-Meier method was used to calculate the cumulative survival rate. Statistical comparison of the effects of potential predictive factors on survival rate was performed using log-rank analysis. Multivariate analysis of the survival rates was performed by using Cox's proportional hazard model. RESULTS: The 1, 2, 3, 4, and 5-year cumulative survival rates of all 128 patients were 90.88%, 73.84%, 51.09%, 36.14%, and 31.89% respectively. Univariate analysis showed that tumor size (P = 0.028), tumor differentiation (P = 0.026) and local recurrence or new metastases (P < 0.001) significantly affected the survival. Multivariate analysis revealed that tumor size (P = 0.035), recurrence or new metastases (P = 0.001) and tumor differentiation (P = 0.038) each had a significant effect on survival. CONCLUSION: There is a significantly higher probability of long-term survival for patients with well-differentiated tumors 3.0 cm or less in diameter and without recurrence or new metastasis after percutaneous microwave ablation. PMID- 16681968 TI - [The prevalence of disc hemorrhage and papillary atrophy in Beijing Eye Study]. AB - OBJECTIVE: To evaluate the prevalence of disc hemorrhage and papillary atrophy in Beijing defined area. METHODS: To review 4163 right eyes consecutive plain color fundus photographs of 4163 subjects (male/female = 1832/2331) who attended the eye screening. RESULTS: The prevalence of glaucoma is 3.0% (135/4439). Disc hemorrhage was found in 42 eyes of 42 subjects (1.0%), and more frequently in women. The prevalences of papillary atrophy (alpha zone and beta zone) were 70.98% (2955/4163) and 20.95% (872/4163), respectively. The prevalence of beta zone was greater in glaucoma group than in non-glaucomatous group (Fisher's test, P < 0.01). CONCLUSION: The prevalence of disc hemorrhage is low in whole population and women are easier to have disc hemorrhage with aging. Disc hemorrhage and papillary atrophy have different prevalences in defined-population study and might have close association on the early onset of glaucoma. PMID- 16681969 TI - [A survey of diabetes mellitus among inpatients in Guangdong province]. AB - OBJECTIVES: To investigate the prevalence of diabetes and impaired glucose regulation (IGR) among the inpatients and their clinical features. METHODS: A cross-sectional survey was conducted among the inpatients registered in ten university hospitals of Guangdong, China during the week before the Diabetes Day of 2004. The fasting plasma glucose (FPG) levels of first screen when the patients were hospitalized were recorded. Those with the FPG levels from 5.6 to 6.9 mmol/l. who had not been previously diagnosed as with diabetes (P-DM) underwent oral glucose tolerance test (OGTT). The patients were classified into 5 groups according to the OGTT results: normal glucose tolerance (NGT), DM, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), and IGT accompanied with IFG, the last 3 groups being called by a joint name of impaired glucose regulation (IGR). Those with FPG >or= 7.0 mmol/L who had not been diagnosed as with DM previously were diagnosed as newly-diagnosed DM (New-DM). RESULTS: Of the 8753 inpatients investigated 8.3% were with P-DM, 6.8% were diagnosed as with New-DM, and 4.9% were with IGR. OGTT was carried out in 433 inpatients. 27.9% of which were classified as with New-DM, and 49.9% with IGR. In the New-DM patients, 41.8% were accompanied with hypertension, 43.2% with proteinuria, and 67.6% with dyslipidemia. CONCLUSION: The prevalence rates of diabetes and IGR were 15.1% and 4.9% respectively among the inpatients investigated. The rate of diabetes was 27.9% among the patients underwent OGTT. Proteinuria, dyslipidemia, and hypertension were the concomitant disorders frequently found in New-DM. PMID- 16681970 TI - [Clinical value of the sixth edition TNM stages analysing prognosis of colorectal cancer]. AB - OBJECTIVES: To study the clinical value analyzing of colorectal cancer prognosis by the Sixth Edition TNM Stages. METHODS: 5481 cases with colorectal cancer and treated by operational methods, were collected. All the cases were separately staged by the Fifth Edition or Sixth Edition TNM Stages standards. The 5-year survival rates were analyzed by the life table method. RESULTS: The 5-year survival rates of the Fifth Edition TNM Stages of I, II, III and IV were 80.1%, 68.0%, 40.5% and 9.8% respectively. The 5-year survival rates of the Sixth Edition TNM Stages of II(A) and II(B) were 71.6% and 66.4% respectively, and of the stages III(A), III(B) and III(C) were 46.2%, 40.1% and 28.3% respectively. There were statistical differences among the sub-stages II and III, P < 0.05. CONCLUSION: The Sixth edition TNM Stages laid more stress on effect of the local infiltration depths and lymphatic metastasis in the prognosis of colorectal cancer, therefore, the stages were more fine, to analyze prognosis of the colorectal cancer were more precise. It is high clinical value for the individual complex treatment with every sub-stages. PMID- 16681972 TI - [Application of single stapler combined with prolapsing technique in anterior resection of ultra-low rectal cancer]. AB - OBJECTIVE: Evaluation of single stapler combined with prolapsing technique for anus-preserving of ultra-low rectal cancer and its indication as well as surgical procedure. METHODS: Forty-three patients with ultra-low low rectal cancer suitable for anterior resection were divided into two groups, single stapler combined with prolapsing technique was applied for experiment group, conventional double stapler technique was applied for control group. To compare the distal margin, local recurrence rate, complications, anal continence function and expenses. RESULTS: The distal margin of experimental group is significantly longer than that of control group (2.2 cm +/- 0.2 cm VS 1.9 cm +/- 0.4 cm, P = 0.006). The distance between dentate line and distal incision line of control group is much longer than experimental group (1.9 cm +/- 0.5 cm VS 1.3 cm +/- 0.3 cm, P < 0.001). There is no recurrence in experimental group but 3 cases recurrence within 1 year in control group. The anastomose fistula rate, instrument expenses of experimental group are less than those of control group. There is no distinct in anal continence between two groups. Occasional minor soiling is present in 1 case of experimental group. CONCLUSION: Single stapler combined with prolapsing technique is superior to double stapler technique for anus-preserving of ultra-low rectal cancer. PMID- 16681973 TI - [Bone marrow stem cells-based SERCA2a gene therapy for heart failure after acute myocardium infarction]. AB - OBJECTIVE: Explore the possibility of MSC to be used to target delivery of therapeutic gene and evaluate the therapeutic effects among gene therapy, MSC transplantation and MSC-based gene therapy. METHODS: MSC were infected with an adenoviral expression vector carrying SERCA2a. SD female rats were used to make animal model with heart failure after AMI and divided into 4 groups randomly. Group I (n = 7) received SERCA2a gene therapy, group II (n = 7) received MSC transplantation, group III (n = 8) received MSC infected with SERCA2a gene transplantation, and group IV (n = 7) received empty adenoviral vector. Cardiac function was evaluated by echocardiography and physiological recorder. SERCA2a gene and protein expression were evaluated by RT-PCR and Western blot respectively. RESULTS: Compared to group IV, EF and FS of group I, group II and group III were elevated significantly on 14 days after therapy (EF: 67.7 +/- 3.9, 62.6 +/- 4.0, 67.9 +/- 3.7 versus 45.0 +/- 2.2; FS: 33.9 +/- 1.9, 31.1 +/- 2.0, 33.9 +/- 1.9 versus 22.5 +/- 1.1, P < 0.05). While the elevation values of EF and FS began to reduce in group I 14 days after, it continued to increase in both group II and group III. Absolute value of LVEDP at 21 days after treatment was increased in group I, group II and group III compared to group IV (5.3 mm Hg +/- 1.2 mm Hg, 6.0 +/- 1.3 mm Hg, 6.2 mm Hg +/- 1.2 mm Hg versus 1.5 mm Hg +/- 0.2 mm Hg, P < 0.05), as well as absolute value of DP/dtmin (4756 mm Hg/s +/- 270 mm Hg/s, 5028 mm Hg/s +/- 253 mm Hg/s, 5283 mm Hg/s +/- 363 mm Hg/s versus 3201 mm Hg/s +/- 211 mm Hg/s, P < 0.05). DP/dtmax at 21 days after treatment increased in group I, group II and group III compared to group IV (6026 mm Hg/s +/- 281 mm Hg/s, 6278 mm Hg/s +/- 319 mm Hg/s, 7057 mm Hg/s +/- 389 mm Hg/s versus 5293 mm Hg/s +/- 360 mm Hg/s, P < 0.05). SERCA2a expressions and enzyme activity were significantly stronger in group I and group III than in group II and group IV. CONCLUSION: It showed that all MSC transplantation, SERCA2a gene therapy and MSC based gene therapy could enhance cardiac function. The recovered heart function continued to improve in MSC transplantation group and MSC-based gene therapy group up to 21 days, however slowed down in single gene therapy group in 21 days. Such therapeutic tendency of MSC-based gene therapy was stronger than that of MSC transplantation. Thus, MSC proved an effective platform for the targeted delivery of therapeutic gene. PMID- 16681975 TI - [Somatostatin levels and polymorphonuclear neutrophil life in intestinal ischemia reperfusion of macaque]. AB - OBJECTIVE: To investigate the association of somatostatin (SST) levels in plasma or jejunum tissue and polymorphonuclear neutrophil (PMN) life in intestinal ischemia reperfusion of macaque. METHODS: MODS model in macaque was established by clamping superior mesenteric artery. The levels of SST in plasma and jejunum tissue were measured by radioimmunoassay. After incubated with SST, the apoptotic PMN were determined by the morphological studies, flow cytometer and DNA agarose electrophoresis. The expression of SSTR on PMN was detected by biosensor. RESULTS: SST levels in macaque plasma or jejunum tissue were significantly decreased after intestinal ischemia reperfusion (IR) injury. The apoptotic rate of PMN was obviously reduced (15.4% +/- 1.4% to 3.5% +/- 0.5%, P < 0.05). However, the number of apoptotic macrophage from abdominal cavity was clearly increased. The apoptotic rate of PMN was significantly increased (20.0% +/- 2.2% to 50.2% +/- 1.8%, P < 0.01) after incubated with SST in vitro. Specific DNA ladder bands from PMN were visualized with agarose electrophoresis. Specific bindings between PMN and the antibodies for SSTR1 (548 RU/ml +/- 20 RU/ml) or SSTR2 (28 RU/ml +/- 21 RU/ml) were detected. CONCLUSION: The physiological apoptosis of PMN in macaque could be induced by SST through SSTRs on PMN. The decreased SST levels in macaque plasma or jejunum tissue in the state of intestinal ischemia reperfusion might extend PMN life and promote the occurrence of MODS. PMID- 16681976 TI - [pCKM-mPTH recombinant plasmid therapy for hypoparathyroidism by polycationic liposome-mediated transfection]. AB - OBJECTIVE: To investigate the optimal ratio of polycationic liposome mediated pCKM-mPTH recombinant gene transfer to skeleton cells in the gene therapy of hypoparathyroidism (HPT). METHODS: The protective roles of polycationic liposome to DNA were observed by the DNaseIsensitive test. Liposome-plasmid complex (lipoplexes) with various liposome to plasmid ratio and the naked plasmid were transferred both in vivo and in vitro. The supernatants and serums were collected for measuring parathyroid hormone (PTH) with radioimmunoassay. RESULTS: When the ratio of lipoplexes reached 2:1, there were obviously protective roles to DNA. While it was 3:1, mPTH transgene expressing reached peak which the PTH amount was (16.4 +/- 4.8) pg/10,000 cells/24 h. Furthermore, the amount serum PTH of the HPT model SD rats reached the highest (35.9 pg/ml +/- 2.2 pg/ml) till the concentration of pCKM-mPTH is 20 microg/100 microl with 3:1 ratio of lipoplexes. CONCLUSION: Polycationic liposomes are more effective than naked plasmids for pCKM-mPTH recombinant gene transfer to skeleton cells. The results also indicated that the effect of pCKM-mPTH transferring into muscles might be depended on the optimal ratio and dosage of lipoplexes. PMID- 16681977 TI - [Correlation of limb and myocardial ischemia postconditioning with acute myocardial reperfusion injury]. AB - OBJECTIVE: We tested the hypothesis that remote postconditioning, which is induced by a single 5-min episode of femoral artery occlusion and reperfusion applied just before the onset of coronary artery reperfusion, protects the myocardium from reperfusion injury. METHODS: In anesthetized open-chest rabbits, the left anterior descending artery (LAD) was occluded for 30 min and reperfused for 3 hrs. All rabbits were randomly divided into four groups (n = 10 in each group): (1) CONTROL: LAD occlusion and reperfusion only, with no other intervention; (2) Myocardial ischemic preconditioning (Pre-con): Three cycles of myocardial ischemia (5 min) and reperfusion (5 min) preceded the index ischemia/reperfusion protocol; (3) Myocardial ischemic postconditionng (Post con): After 30 min of LAD occlusion, reperfusion was initiated for 30 s followed by 30 s reocclusion. Three cycles of myocardial ischemia (30 s) and reperfusion (30 s) followed the index ischemia/reperfusion protocol. (4) Remote postconditioning (Re Post-con): After 24 min of LAD occlusion, the femoral artery was occluded for 5 min and released for 1 min before 3 hrs of LAD reperfusion. Myocardial infarct size and tissue myeloperoxidase (MPO) activity were determined at the end of the experiment. Plasma creatine kinase (CK) activity and malondialdehyde (MDA) activity were measured at baseline, the end of ischemia, and after 3 hrs of reperfusion respectively. RESULTS: Myocardial infarct size was significantly reduced in Pre-con (15.5% +/- 1.7%, P < 0.01), Post-con (16.15% +/- 2.05%, P < 0.01) and Re Post-con (17.11% +/- 1.70%, P < 0.01) groups as compared to CONTROL (31.46% +/- 1.28%). Results were confirmed by plasma CK activity (in Re Post-con 18.0 IU/g +/- 1.6 IU/g vs. CONTROL 45.6 IU/g +/- 5.5 IU/g). Plasma MDA was significantly less at 3 hrs of reperfusion in Pre-con (2.12 micromol/ml +/- 0.30 micromol/ml, P < 0.01), Post-con (2.17 micromol/ml +/- 0.24 micromol/ml, P < 0.01) and Re Post-con (2.16 micromol/ml +/- 0.33 micromol/ml, P < 0.01) than that in CONTROL (3.49 micromol/ml +/- 0.32 micromol/ml). Neutrophil accumulation (MPO activity) in the area at risk was less in Pre-con (1.43 U/100 g +/- 0.32 U/100 g, P < 0.01), Post-con (2.26 U/100 g +/- 0.28 U/100 g, P < 0.01) and Re Post-con (2.45 U/100 g +/- 0.28 U/100 g, P < 0.01) than that in CONTROL (5.44 U/100 g +/- 0.46 U/100 g). CONCLUSION: Remote limb postconditioing applied just before the onset of coronary artery reperfusion provides potent myocardial infarct size reduction, which is similar to the cardioprotective effect of myocardial postconditioning exerted during the first minutes of coronary reperfusion. The potential mechanism of this inter-organ remote postconditioning phenomenon might be associated with decreasing the injury caused by oxygen free radicals and strengthening the action of antioxidation. PMID- 16681978 TI - [Antisense to CDK4 inhibits the growth of human colon cancer cells HT29]. AB - OBJECTIVE: Antisense CDK4 was transfected into human colorectal cancer cells HT29 to examine the effect of it on the growth and proliferation of HT29 cells. METHODS: Antisense CDK4 transfected the HT29 cells with lipofectamine. The transfected effects were verified by the Northern blot, Western blot, morphological method, flow cell measure, etc. RESULTS: The transformant cells showed the expression of exogenous antisense CDK4 mRNA, and downmodulation of endogenous CDK4 mRNA expression and inhibition of its protein synthesis. The transformant cells exhibited the partial reversion of the malignant phenotype and behavior, and that the cell growth and the ability of colony formation in soft agar were inhibited. The arrest of G1 phase was also revealed. the apoptosis rate is higher in HT29-asCDK4. CONCLUSION: The antisense CDK4 gene can inhibited the cell growth and proliferation, and induce the cell apoptosis. It might provide a new way to gene therapy of the colorectal carcinoma. PMID- 16681979 TI - [Induction of chemical hepatocarcinogenesis and establishment of orthotopic liver transplantation model with hepatocarcinoma in rats]. AB - OBJECTIVE: To explore the inducing method for hepatic carcinoma in rats and the operative technique in establishment of orthotopic liver transplantation (OLT) model in rats with induced hepatic tumor. METHODS: Hepatic carcinoma was induced by diethylnitrosamine (DENA) in rats. Then OLT was performed, using a two-cuff vessel anastomosis method modified from that introduced by Kamada, with reconstruction of hepatic artery. RESULTS: At the observation time point (18 weeks) after the initiation of carcinogenesis, the one-month survival rate in OLT group was higher than that in non-OLT group, 56.3% and 21.4% respectively, P < 0.05. The operative successful rate in tumor rats group and control group was 87.5% and 90.0%, respectively. In OLT rats with hepatic carcinoma group, metastasis mainly occurred in lung and abdomen, while only 2 cases of intrahepatic recurrence were observed. There was significant difference between the tumor rats group and control group in cum survival rate. CONCLUSION: DENA can wholesale induce stable rat model for hepatic carcinoma. OLT can markedly elevate survival rate in rats with liver cancer. In rats with induced hepatic tumor, OLT model, suggested by the author, was established with hepatic arterial reconstruction and it offers a satisfied method with more similar pattern of physiological setting and carcinoma development situation. PMID- 16681980 TI - [Side effects of adjuvant chemotherapy and radiotherapy concurrently after conservative surgery for breast cancer]. AB - OBJECTIVE: Observe the side effects of adjuvant chemotherapy and radiotherapy concurrently after breast conservative surgery and investigate it feasible. METHODS: 40 breast cancer patients of conservative surgery were divided into two groups randomizedly, 20 patients of the study group were given adjuvant chemotherapy and radiotherapy concurrently, 20 patients of the control group were given adjuvant thermotherapy and radiotherapy sequently. Observe the side effects of the patients in the two groups and follow them 6 months. RESULTS: The leukopenia within the third chemotherapeutic cycle and the oral mucitis within the fourth chemotherapeutic cycle in the study group were more severe than those in the control group (P < 0.05). The other side effects within every chemotherapeutic cycles were similar in both groups, including skin reaction in radiotherapeutic area, gastroenteric reaction, alopecia, arthralgia, neurotoxicity and abnormal hepatic function. Following the patients of the two groups in 6 months, there were no significance in hemogram, hepatic and renal function, electrocardiogram and chest x-ray. CONCLUSION: It is feasible and safe to give the adjuvant chemotherapy and radiotherapy concurrently to the breast cancer patients after conservative surgery. PMID- 16681984 TI - Postsurgical ovarian failure after laparoscopic excision of bilateral endometriomas. AB - OBJECTIVE: This study was undertaken to determine the frequency of postsurgical ovarian failure in patients undergoing laparoscopic excision of bilateral endometriomas. STUDY DESIGN: Patients who had been operated on for bilateral ovarian endometriosis between January 1995 and December 2003 and who were younger than 40 years at the time of surgery were contacted by telephone and interviewed. RESULTS: A total of 126 patients were recruited. Mean +/- SD age of patients at the time of surgery was 30.4 +/- 4.3 years. Postsurgical ovarian failure was documented in 3 cases, corresponding to a rate of 2.4% (95% CI 0.5%-6.8%). In all cases, this complication occurred immediately after surgery. CONCLUSION: Patients who had been operated on for bilateral endometriomas have a low but definite risk of premature ovarian failure occurring immediately after surgery. PMID- 16681985 TI - Surgical therapy of recurrent vulvar cancer. AB - OBJECTIVE: The success of various surgical interventions in 201 cases with recurrent vulvar carcinoma was examined in the light of patients' pretreatment, surgical therapy, plastic reconstruction, and postoperative disease course. STUDY DESIGN: A databank of standardized clinical data was analyzed using statistical procedures. RESULTS: Therapy was selected on an individual basis according to tumor status. Recurrence at a site distant from the primary tumor, particularly in the inguinal region, indicated a markedly unfavorable prognosis. In contrast, tumors recurring locally did not exhibit any significant differences. Plastic surgery reconstruction led to improvements with respect to operability, wound healing, and survival. CONCLUSION: Individualized reconstructive surgery of the vulva leads to good results in patients with recurrent vulvar cancer. To an even greater extent than is the case for primary therapy, plastic surgery enlarges the spectrum of feasible surgical alternatives so that a more favorable oncological outcome and excellent cosmetic results may be expected. PMID- 16681987 TI - Pregnancy and estradiol modulate myometrial G-protein pathways in the guinea pig. AB - OBJECTIVE: Coupled to hundreds of receptors, G-proteins modulate signal transduction pathways and are important hormonal targets. The first objective was to determine the effect of pregnancy and estradiol on myometrial guanosine triphosphatase activity. The second objective was to begin dissecting the molecular mechanism(s) underlying alterations in guanosine triphosphatase activity. STUDY DESIGN: Myometrial tissue was obtained from pregnant, nonpregnant, and ovariectomized untreated and estradiol-treated guinea pigs. Myometrial membranes were prepared by homogenization and differential centrifugation. Basal high-affinity specific guanosine triphosphatase activity was quantitated by enzymatic assay and expressed in rhomol 32Pi per milligram protein per minute. Guanosine triphosphatase activity was stimulated using oxytocin, isoproterenol, and prostaglandin F2alpha. Specific G-protein subunits were quantitated using Western blots. G-protein associated gene expression was semiquantitated using HGU133A gene array chips from Affymetrix. RESULTS: Basal myometrial guanosine triphosphatase activity was increased in pregnant compared with nonpregnant animals. Estradiol increased basal myometrial guanosine triphosphatase activity, compared with untreated controls. The effect of estradiol on stimulated activity was agonist dependent. Both Galphas and Galphai isoform 1 protein levels were increased in myometrium from late pregnant compared with nonpregnant animals. By late gestation, the messenger ribonucleic acid levels of those genes were unaltered, compared with the nonpregnant animal. In general, the impact of pregnancy on G-protein family member gene messenger ribonucleic acid expression was modest. Only the small guanosine triphosphatase Rap1b demonstrated altered expression more than 2-fold during either myometrial quiescence (midpregnancy) or activation (term pregnancy) (up 3-fold during quiescence). Genomic network analyses revealed that the expression of another small guanosine triphosphatase, Rab7, was exclusively up-regulated (80%) during quiescence. During late pregnancy, network analysis showed that only G-protein beta was exclusively altered (up-regulated). Estradiol mimicked the pregnancy effect on both transcription and translation of G-protein family members for some but not all potentially relevant genes. CONCLUSION: The increase in functional myometrial guanosine triphosphatase activity during pregnancy may reflect increased synthesis of 1 or more small guanosine triphosphatase. PMID- 16681986 TI - Fetal membrane healing after spontaneous and iatrogenic membrane rupture: a review of current evidence. AB - In view of the important protective role of the fetal membranes, wound sealing, tissue regeneration, or wound healing could be life saving in cases of preterm premature rupture of the membranes. Although many investigators are studying the causes of preterm premature rupture of membranes, the emphasis has not been on the wound healing capacity of the fetal membranes. In this review, the relevant literature on the pathophysiologic condition that leads to preterm premature rupture of membranes will be summarized to emphasize a continuum of events between rupture and repair. We will present the current knowledge on fetal membrane wound healing and discuss the clinical implications of these findings. We will critically discuss recent experimental interventions in women to seal or heal the fetal membranes after preterm premature rupture of membranes. PMID- 16681989 TI - Can we screen for pelvic organ prolapse without a physical examination in epidemiologic studies? AB - OBJECTIVE: Large population-based epidemiologic studies of pelvic organ prolapse are rare. One barrier is the need for physical examination in order to confirm disease status. The objectives of this study were to develop a simple screening question for pelvic organ prolapse (POP) and to evaluate its test characteristics in high and low prevalence populations. STUDY DESIGN: Data from 100 women enrolled in the validation study of the Pelvic Floor Distress Inventory (PFDI) were used to identify the question or questions that most accurately identified women with advanced pelvic organ prolapse. After identifying an accurate and reliable screening question from this original group, its test characteristics were evaluated prospectively in 2 additional distinct populations: a group of 120 women presenting to a tertiary care urogynecology clinic (High prior probability of POP) and 448 women presenting to a nurse practitioner for annual gynecologic examination (Low prior probability of POP). Subjects in these 2 groups each completed the screening question and underwent a POPQ examination by a blinded examiner. RESULTS: A single question was identified from the original study population that most accurately and reliably identified those women with POP "Do you usually have a bulge or something falling out that you can see or feel in your vaginal area?" An affirmative answer to this question was 96% sensitive (95%CI 92-100) and 79% specific (95%CI 77-92) for prolapse beyond the hymen. The 1-week test-retest reliability was good (kappa .84). The prevalence of POP in this group was 29%. No other single question or group of questions had better test characteristics. When prospectively evaluated in the second High probability population (prevalence 39%), similar test characteristics were noted: sensitivity 85% (95%CI 71-93), specificity 86% (95%CI 75-92). However, when evaluated in the Low prior probability group (POP prevalence 3.8%) the specificity improved to 99% (95%CI 98-99), while the sensitivity decreased dramatically to 35% (95%CI 15-61). CONCLUSION: Screening for POP without a physical examination is subject to spectrum bias. Spectrum bias occurs when a diagnostic test performs differently in different groups of patients. In groups with a high prior probability of POP, a simple screening question can accurately screen for advanced POP without a physical exam. However, in groups with a low prior probability of POP such as might be seen in a population-based epidemiologic study, this question has poor sensitivity. PMID- 16681988 TI - Long-term neuroprotective effects of carbon dioxide on neonatal rat hypoxic ischemic brain injury: an experimental study of skilled motor tasks. AB - OBJECTIVE: This study was undertaken to investigate the long-term effect of hypercapnia on neonatal hypoxic-ischemic brain injury, we tested its effect in a neonatal rat hypoxia-ischemia model. STUDY DESIGN: The rats were subjected to unilateral carotid artery ligation and exposure to 8% oxygen for 30 minutes. Six percent carbon dioxide was administered to the neonatal rats during unilateral hypoxia-ischemia, and the motor function and neurologic outcomes were determined 3 months later. RESULTS: Significant motor functional improvement was observed in the hypercapnic animals, as judged by the Montoya staircase test. The unilateral brain injury was significantly ameliorated in the hypercapnic animals, and this amelioration was well correlated with the motor functional performance. Cerebral blood flow during hypoxia-ischemia, monitored by laser Doppler flowmetry, was better preserved in the hypercapnic animals. CONCLUSION: Our results suggest that mild hypercapnia during hypoxia-ischemia may provide long-lasting motor functional as well as neurologic protection for immature brains, possibly by increasing cerebral blood flow during hypoxia. PMID- 16681990 TI - Analysis of circulating annexin A5 parameters during pregnancy: absence of differences between women with recurrent spontaneous pregnancy losses and controls. AB - OBJECTIVE: We investigated whether levels of annexin A5 (AnxA5), AnxA5 anticoagulant activity and binding, and anti-AnxA5 antibodies might be altered in women with previous histories of recurrent spontaneous pregnancy losses (RSPL) when tested during pregnancy. STUDY DESIGN: Ninety pregnant women with histories of 3 or more RSPL (cases) and 150 women without adverse pregnancy histories (controls) were assayed for these AnxA5 parameters. RESULTS: There were no significant differences between cases and controls in AnxA5 levels (median, 23.1 ng/mL [range, 2.1-201.1 ng/mL] vs 19.7 ng/mL [2.1-151.5 ng/mL]; P = .20), AnxA5 anticoagulant activity (183% [101%-236%] vs 168% [128%-256%]; P = .39) and binding (6.0 ng/aliquot PL [2.1-19.5 ng/aliquot PL) vs 6.1 ng/aliquot PL [1.6 16.8 ng/aliquot PL]; P = .72), and anti-AnxA5 antibody levels. CONCLUSIONS: AnxA5 parameters do not distinguish between cases and controls when tested during pregnancy. The pregnant state itself appears to be associated with altered levels of AnxA5 parameters. PMID- 16681993 TI - Dipalmitoylation of a cellular uptake-mediating apolipoprotein E-derived peptide as a promising modification for stable anchorage in liposomal drug carriers. AB - Liposomes equipped with cellular uptake-mediating peptidic vector compounds have attracted much attention as target-specific drug delivery systems. Aside from the development of the target recognition motif itself, vector coupling to liposomes while conserving the active conformation constitutes an important element in carrier development. To elucidate the most efficient way for adsorptive peptide binding to liposomes, we synthesized and characterized two-domain peptides comprising a cationic sequence derived from the binding domain of apolipoprotein E (apoE) for the low-density lipoprotein receptor and different lipid-binding motifs, that is, an amphipathic helix, a transmembrane helix, single fatty acids or two palmitoyl chains. Peptide properties considered relevant for peptide liposome complexes to initiate an endocytotic cellular uptake such as lipid binding, helicity, stability of anchorage, bilayer-disturbing activity, and toxicity showed that the dipalmitoyl derivative was the most suitable to associate the apoE peptide to the surface of liposomes. The peptide showed pronounced lipid affinity and was stably anchored within the lipid bilayer on a time scale of at least 30 min. The helicity of about 40% in the lipid-bound state and the location of the amphipathic helix on the liposomal surface provided the prerequisites for interaction of the complex with the cell surface-located receptor. The concentration of the dipalmitoylated peptide to permeabilize neutral lipid bilayers (lipid concentration 25 microM) was 0.06 microM and a 2 microM concentration reduced cell viability to about 80%. Efficient internalization of liposomes bearing about 180 peptide derivatives on the surface into brain capillary endothelial cells was monitored by confocal laser scanning microscopy. The concept of complexation using dipalmitoylated peptides may offer an efficient substitute to covalent vector coupling and a prospective way to optimize the capacity of liposomes as drug delivery systems also for different targets. PMID- 16681992 TI - Correlated variation between the lateral basicranium and the face: a geometric morphometric study in different human groups. AB - OBJECTIVES: The significance of the cranial base in the contribution of normal and pathologic facial patterns is uncertain. The aim of this study was to explore and compare patterns of morphological covariation between the face and the lateral basicranium (anterior and middle cranial fossae) with covariation patterns between the face and the midline cranial base. DESIGN: We employed 2D geometric morphometrics and partial least squares analysis to quantify and compare correlation coefficient between principal variation patterns between basicranial and facial structures in lateral radiographs from 143 humans from Japan (N=45), Europe (N=65) and Africa (N=34). RESULTS: In both comparisons basicranial patterns co-varied with dolichofacial and brachyfacial morphologies. The findings showed that the midline cranial base is not significantly correlated of common facial patterns (r=0.37, P=0.09). Instead, the lateral basicranium is significantly (P=0.000), and significantly higher (r=0.6) correlated with facial variation than is the midline cranial base. CONCLUSIONS: The lateral basicranium may be of higher morphogenetic relevance for influencing facial variation than previously thought. This influence may be of clinical importance for the generation of skeletal class II or class III malocclusions. Further research should expand on geometric morphometric analysis of ontogenetic and clinical 3D data in order to advance in the understanding of these complex developmental relationships. PMID- 16681991 TI - Prolylcarboxypeptidase gene, chronic hypertension, and risk of preeclampsia. AB - OBJECTIVE: Renin-angiotensin System is essential for the homeostasis of blood pressure in humans. The roles of renin-angiotensin system gene polymorphisms including angiotensinogen, angiotensin-converting enzyme, renin and angiotensin II receptor, type 1 genes in the pathogenesis of preeclampsia have been extensively studied, but most association studies produced either negative or inconsistent results. Prolylcarboxypeptidase encodes a lysosomal enzyme and is a regulator for both renin-angiotensin system and the kallikrein-kinin system. There is no published study on prolylcarboxypeptidase gene and preeclampsia. STUDY DESIGN: We investigated the independent and joint association of five polymorphisms in angiotensinogen, angiotensin-converting enzyme, and prolylcarboxypeptidase gene and chronic hypertension with the risk of preeclampsia in 125 preeclamptic and 1040 non-preeclamptic black women enrolled at the Boston Medical Center. We used logistic regression models to estimate the odds ratios of risk for preeclampsia associated with each gene polymorphism and its joint association with chronic hypertension. RESULTS: No association was found in four polymorphisms in angiotensinogen and angiotensin-converting enzyme. Prolylcarboxypeptidase E112D (rs2298668) D allele along and jointly with chronic hypertension were associated with a significantly increased risk of preeclampsia. Compared to women with homozygous EE genotype and without chronic hypertension, higher risks of preeclampsia were observed in DD women without chronic hypertension (OR = 3.7, 95% CI, 1.2 - 12.4) and EE women with chronic hypertension (OR = 9.1, 95% CI: 4.7 - 17.6). Women with both D allele and chronic hypertension had the highest risk (OR = 158, 95% CI, 25-infinite). This finding was validated in an independent sample of 1,015 non-black women. We further compared the prolylcarboxypeptidase transcript levels in peripheral blood cells of 23 preeclamptic (30% with chronic hypertension) and 51 non-preeclamptic (6% with chronic hypertension) women 2 - 3 days after delivery. The PRCP transcript levels were lower in ED/DD women than in EE woman (P = .03) and lower in preeclamptic women than in non-preeclamptic women (P = .007). CONCLUSION: Our data showed that prolylcarboxypeptidase D allele coupled with chronic hypertension was associated with a significantly increased risk of preeclampsia in both black and non-black women. Gene expression assays lent further support for the functional significance of prolylcarboxypeptidase in the etiology of preeclampsia. PMID- 16681994 TI - Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna. AB - Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH(2)-terminus [Kato et al., Gene 334 (2004) 157-165]. Both genes had a 17 exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH(2)-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids. PMID- 16681995 TI - Substrate recognition ability differs among various prokaryotic tRNase Zs. AB - There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z. PMID- 16681996 TI - RhoA and Rac1 contribute to type III group B streptococcal invasion of human brain microvascular endothelial cells. AB - Type III group B streptococcus (GBS) has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying mechanisms remain incompletely understood. In the present study, we showed that the geranylgeranyl transferase I inhibitor, GGTI 298, not the farnesyltransferase inhibitor, FTI-277 inhibited type III GBS invasion of HBMEC. The substrates for GGTI-298 include Rho family GTPases, and we showed that RhoA and Rac1 are involved in type III GBS invasion of HBMEC. This was shown by the demonstration that infection with type III GBS strain K79 increased the levels of activated RhoA and Rac1 and GBS invasion was inhibited in HBMEC expressing dominant-negative RhoA and Rac1. Of interest, the level of activated Rac1 in response to type III GBS was decreased in HBMEC expressing dominant-negative RhoA, while the level of activated RhoA was not affected by dominant-negative Rac1. These findings indicate for the first time that activation of geranylgeranylated proteins including RhoA and Rac1 is involved in type III GBS invasion of HBMEC and RhoA is upstream of Rac1 in GBS invasion of HBMEC. PMID- 16681997 TI - HCV E2 may induce apoptosis of Huh-7 cells via a mitochondrial-related caspase pathway. AB - INTRODUCTION: One unusual characteristic of HCV is to establish chronic infection and the precise mechanisms remain unclear. MATERIALS AND METHODS: Huh-7 cells were transiently transfected with E2 and subjected to MTT assay, DNA fragmentation assay, and Western blotting to see the impact of E2 protein on apoptosis. RESULTS AND DISCUSSION: E2 may inhibit cell proliferation by inducing apoptosis and pro-caspases 3, 8, and 9 were cleaved and activated to result in the presence of active forms in a time-dependent fashion, which suggest that E2 induced apoptosis is caspase-dependent. Furthermore, the cytosolic level of cytochrome c was increased together with a gradually down-regulated Bcl-2 and up regulated Bax protein expression. The continuing reduction of Bid protein and the gradual increase of tBid protein also indicated that a time-dependent increased turn-over of Bid protein into tBid. Taken together, our data suggested that HCV E2 may induce apoptosis through a mitochondrial damage-mediated caspase pathway. PMID- 16681998 TI - Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells. AB - The anti-inflammatory activity of the predominant procyanidin dimer in cocoa, dimer B2, was investigated in this study. Pretreatment of the procyanidin dimer B2 reduced COX-2 expression induced by the endotoxin lipopolysaccharide (LPS) in differentiated human monocytic cells (THP-1) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by procyanidin, we examined their effects on the activation of extracellular signal-regulated protein kinase (ERK), Jun-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with procyanidin dimer B2 decreased the activation of ERK, JNK, and p38 MAPK. In addition, procyanidin dimer B2 suppressed the NF-kappaB activation through stabilization of IkappaB proteins, suggesting that these signal transducing enzymes could be potential targets for procyanidin dimer B2. By affecting the expression rather than the activity of COX-2, these in vitro data reported herein give further evidence on the anti-inflammatory protection by procyanidins. PMID- 16681999 TI - Molecular characteristics of IgA and IgM Fc binding to the Fcalpha/muR. AB - Fcalpha/mu receptor (Fcalpha/muR), a novel Fc receptor for IgA and IgM, is a type I transmembrane protein with an immunoglobulin (Ig)-like domain in the extracellular portion. Although IgA and IgM bind to Fcalpha/muR, the molecular and structural characteristics of the ligand-receptor interactions have been undetermined. Here, we developed twelve monoclonal antibodies (mAbs) against murine Fcalpha/muR by immunizing mice deficient in Fcalpha/muR gene. Eight mAbs totally or partially blocked IgA and IgM bindings to Fcalpha/muR. These blocking mAbs bound to a peptide derived from the Ig-like domain of murine Fcalpha/muR, which is conserved not only in human and rat Fcalpha/muR but also in polymeric Ig receptor (poly-IgR), another Fc receptor for IgA and IgM. These results suggest that IgA and IgM bind to an epitope in the conserved amino acids in the Ig-like domain of Fcalpha/muR as well as poly-IgR. PMID- 16682000 TI - Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration. AB - Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells. PMID- 16682001 TI - Modulation of angiogenesis by tissue inhibitor of metalloproteinase-4. AB - Despite the importance of MMP activity in the regulation of angiogenesis, relatively little is known about the role of TIMP-4, the most recently discovered endogenous MMP inhibitor, in modulating neovascularization. It has largely been assumed that all TIMPs are capable of inhibiting angiogenesis in vivo. However, it is now widely appreciated that TIMPs-1, -2, and -3 differ significantly in their ability to modulate angiogenic processes in vitro and angiogenesis in vivo. In order to study the effect of TIMP-4 in controlling angiogenesis, we have cloned and expressed TIMP-4 in a Pichia pastoris expression system, purified it to homogeneity, and tested its ability to regulate angiogenesis in vivo and in vitro. Our studies demonstrate that TIMP-4 is an inhibitor of capillary endothelial cell migration, but not of proliferation or of angiogenesis in vivo. PMID- 16682002 TI - F1F0-ATP synthase functions as a co-chaperone of Hsp90-substrate protein complexes. AB - Inhibition of heat shock protein 90 (Hsp90) has emerged as a novel intervention for the treatment of solid tumors and leukemias. Here, we report that F(1)F(0) ATP synthase, the enzyme responsible for the mitochondrial production of ATP, is a co-chaperone of Hsp90. F(1)F(0)-ATP synthase co-immunoprecipitates with Hsp90 and Hsp90-client proteins in cell lysates of MCF-7, T47D, MDA-MB-453, and HT-29 cancer cells. Inhibition of F(1)F(0)-ATP synthase by efrapeptins results in the disruption of the Hsp90 complexing with its substrate proteins and, in most cases, in the degradation of the latter. Hsp90-client proteins affected by the inhibition of F(1)F(0)-ATP synthase included ERalpha, mutated p53 (m.p53), Hsp70, Hsp27, and caspase-3 but not Raf-1. This is the first report identifying caspase 3 as a substrate protein of Hsp90. Unlike typical Hsp90 inhibitors, efrapeptin treatment triggers Hsp70 downregulation in parallel with depletion of Hsp90. This suggests that suppression of Hsp90 chaperone function through inhibition of F(1)F(0)-ATP synthase does not result in activation of transcription factor HSF 1, a generally unfavorable consequence of anti-cancer treatments based on Hsp90 inhibition. PMID- 16682003 TI - Hrt and Hes negatively regulate Notch signaling through interactions with RBP Jkappa. AB - Notch signaling is central to cell differentiation, organ development, and apoptosis. Upon ligand binding, the Notch intracellular domain (NotchIC) translocates to the nucleus to interact with its DNA-binding partner, RBP-Jkappa. The NotchIC-RBP-Jkappa complex activates target genes, such as those encoding the Hrt and Hes families of basic-helix-loop-helix (bHLH) transcriptional repressors. Hrt transcripts are enriched in the developing cardiovascular system, and mice lacking Hrt2 have cardiac malformations. Here we show that Hrt2 and Hes1 interact with RBP-Jkappa to negatively regulate Notch-dependent activation of Hrt and Hes expression. The bHLH domain of Hrt2 was necessary for this interaction, and disrupting the protein complex abrogated the negative autoregulation. The interaction did not interfere with the formation or DNA-binding of the NotchIC RBP-Jkappa complex, indicating direct inhibition by Hrt and Hes as co-repressors. These findings suggest a novel mechanism for negative feedback on Notch signaling that requires RBP-Jkappa to interact physically with Hrt and Hes. PMID- 16682004 TI - Serum non-transferrin-bound iron and low-density lipoprotein oxidation in heterozygous hemochromatosis. AB - Non-transferrin-bound iron (NTBI) is implicated in lipid peroxidation but the relation with oxidative modification of low-density lipoprotein (LDL) is not known. We assessed variables reflecting in vitro and in vivo LDL oxidation in two age- and sex-matched groups (n=23) of hereditary hemochromatosis heterozygotes (C282Y), characterized by a clear difference in mean serum NTBI (1.55+/-0.57 micromol/L vs 3.70+/-0.96 micromol/L). Plasma level of oxidized LDL (absolute and relative to plasma apolipoprotein B), and IgG and IgM antibodies to oxidized LDL, markers of in vivo LDL oxidation, did not differ between the groups with low and high serum NTBI. Mean lag-phase of in vitro LDL oxidation was also not significantly different between both study groups. CONCLUSION: these findings do not support the hypothesis that NTBI promotes oxidative modification of plasma LDL. PMID- 16682006 TI - Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis. AB - Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Our previous studies show that microtubule motor CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signaling cascade. However, the molecular function of CENP-E at the midbody had remained elusive. Here we show that CENP-E interacts with Skp1 at the midbody and participates in cytokinesis. CENP-E interacts with Skp1 in vitro and in vivo via its coiled-coil domain. Our yeast two-hybrid assays mapped the binding interfaces to the central stalk region of CENP-E (955-1571 aa) and the C-terminal 33 amino acids of Skp1, respectively. Our immunocytochemical studies revealed that CENP-E targets to the midbody prior to Skp1 and the midbody localization of CENP-E becomes diminished as Skp1 arrives at the midbody. Suppression of Skp1 in mitotic HeLa cells by siRNA resulted in accumulation of telophase cells with elongated inter-cell bridges and with midbodies stretched 2-3 times longer than that of normal cells. These Skp1-eliminated or -suppressed cells accumulate higher level of CENP-E, suggesting that spatiotemporal regulation of CENP-E degradation at the midbody is essential for cytokinesis. Over-expression of Skp1 lacking the CENP-E-binding domain confirmed that Skp1-CENP-E interaction is essential for faithful cytokinesis. We hypothesize that CENP-E degradation is essential for faithful mitotic exit and the proteolysis of CENP-E is mediated by SCF via a direct Skp1 link. PMID- 16682005 TI - Endothelin-1 inhibits adiponectin secretion through a phosphatidylinositol 4,5 bisphosphate/actin-dependent mechanism. AB - Adiponectin is an adipokine with profound insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties. Plasma levels of adiponectin are reduced in insulin resistant states such as obesity, type 2 diabetes and cardiovascular disease. However, the mechanism(s) by which adiponectin concentrations are decreased during disease development is unclear. Studies have shown that endothelin-1 (ET-1), a vasoconstrictor peptide, affects adipocyte glucose metabolism and secretion of adipokines such as leptin, resistin, and adiponectin. The goal of our study was to determine the mechanism by which ET-1 decreases adiponectin secretion. 3T3-L1 adipocytes were treated for 24h with ET-1 (10nM) and then stimulated with vehicle or insulin (100 nM) for a period of 1-2h. Chronic ET-1 (24h) treatment significantly decreased basal and insulin-stimulated adiponectin secretion by 66% and 47%, respectively. Inhibition of phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis by the PLCbeta inhibitor, U73122, or exogenous addition of PIP(2):histone carrier complex (1.25:0.625 microM) ameliorated the decrease in basal and insulin-stimulated adiponectin secretion observed with ET-1. However, treatment with exogenous PIP(2):histone carrier complex and the actin depolymerizing agent latrunculin B (20 microM) did not reverse the ET-1-mediated decrease in adiponectin secretion. In conclusion, we demonstrate that ET-1 inhibits basal and insulin-stimulated adiponectin secretion through PIP(2) modulation of the actin cytoskeleton. PMID- 16682007 TI - Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2. AB - Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti angiogenesis agent in both cancer and angiogenic retinopathies. PMID- 16682009 TI - 3D modeling of the activated states of constitutively active mutants of rhodopsin. AB - The activated (R*) states in constitutively active mutants (CAMs) of G-protein coupled receptors (GPCRs) are presumably characterized by lower energies than the resting (R) states. If specific configurations of TM helices differing by rotations along the long transmembrane axes possess energies lower than that in the R state for pronounced CAMs, but not for non-CAMs, these particular configurations of TM helices are candidate 3D models for the R* state. The hypothesis was studied in the case of rhodopsin, the only GPCR for which experimentally determined 3D models of the R and R* states are currently available. Indeed, relative energies of the R* state were significantly lower than that of the R state for the rhodopsin mutants G90D/M257Y and E113Q/M257Y (strong CAMs), but not for G90D, E113Q, and M257Y (not CAMs). Next, the developed build-up procedure successfully identified few similar configurations of the TM helical bundle of G90D/M257Y and E113Q/M257Y as possible candidates for the 3D model of the R* state of rhodopsin, all of them being in good agreement with the model suggested by experiment. Since constitutively active mutants are known for many of GPCRs belonging to the large rhodopsin-like family, this approach provides a way for predicting possible 3D structures corresponding to the activated states of the TM regions of many GPCRs for which CAMs have been identified. PMID- 16682008 TI - Interplay of Sps and poly(C) binding protein 1 on the mu-opioid receptor gene expression. AB - The proximal promoter of mouse mu-opioid receptor (MOR) gene is the dominant promoter for directing MOR-1 gene expression in brain. Sp1/Sp3 (Sps) and poly(C) binding protein 1 (PCBP) bind to a cis-element of MOR proximal promoter. Functional interaction between Sps and PCBP and their individual roles on MOR proximal core promoter were investigated using SL2 cells, devoid of Sps and PCBP. Each factor contributed differentially to the promoter, with a rank order of activity Sp1>Sp3>PCBP. Functional analysis suggested the interplay of Sps and PCBP in an additive manner. The in vivo binding of individual Sps or PCBP to MOR proximal promoter was demonstrated using chromatin immunoprecipitation (ChIP). Re ChIP assays further suggested simultaneous bindings of Sps and PCBP to the proximal promoter, indicating physiologically relevant communication between Sps and PCBP. Collectively, results documented that a functional coordination between Sps and PCBP contributed to cell-specific MOR gene expression. PMID- 16682010 TI - Characterization of a novel WHSC1-associated SET domain protein with H3K4 and H3K27 methyltransferase activity. AB - Evolutionary conserved SET domains were originally identified in three Drosophila proteins: suppressor of variegation (Su (var) 3-9), enhancer of zeste (E(z)), and the trithorax. Some of the SET-domain containing proteins have been known to elicit methylation of histone lysine residues. Based on a search for SET-domain containing proteins using bioinformatic tools, we identified and subsequently named a novel SET domain as WHISTLE, that has histone methyltransferase (HMTase) activity. To characterize WHISTLE, we performed an HMTase assay, mass spectrometric analysis, lysine specificity, and transfection assays. Mass spectrometric and immunoblot analysis revealed that WHISTLE di-methylates H3K4 and di-, and tri-methylates H3K27 of histones. Overexpression of WHISTLE repressed transcription of the SV40 promoter. Our results suggest that WHISTLE is a novel SET domain containing a protein with specific H3K4 and H3K27 HMTase activity. PMID- 16682011 TI - Structural and functional analogs of the novel mammalian neuropeptide, neuromedin S (NmS), in the dermal venoms of Eurasian bombinid toads. AB - We report the isolation and structural characterization of two neuromedin S (NmS) analogs, (NmS-17 and NmS-33), from the dermal venoms of Eurasian bombinid toads. NmS is a novel neuromedin U (NmU)-related peptide with potent anorexigenic and circadian rhythm-modulating properties recently discovered in mammals. Cloning of NmS precursor-encoding cDNAs from skin venom-derived libraries revealed the presence of a high degree of transcript splice variation comparable to that found previously for NmU in both amphibian skin and mammalian brain. Synthetic replicates of both amphibian NmS peptides evoked robust and dose-dependent transient increases in intracellular calcium ion concentrations in CHO cells that had been stably transfected with either FM-3/GPR66 or FM-4/TGR-1 human NmU receptors. The potency and efficacy of these amphibian skin peptides at such receptors were comparable to those observed with human NmS and rat NmS. These data show that NmS and NmU genes had already diverged at the level of the Amphibia and that differential splicing of their transcribed mRNAs has been highly conserved throughout tetrapod vertebrate evolution indicative of fundamental biological function. NmS is additionally a novel neuropeptide homolog that can be added to the biologically active peptide arsenal of amphibian venom/defensive skin secretions. PMID- 16682012 TI - Adenosine modulates vascular endothelial growth factor expression via hypoxia inducible factor-1 in human glioblastoma cells. AB - Hypoxia appears to induce a program which shifts the cellular phenotype toward an increase in extracellular adenosine. Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. Since in gliomas there is a strong correlation between HIF-1alpha expression, tumor grade and tumor vascularization, the aim of this study was to investigate whether adenosine may regulate HIF-1 in human glioblastoma cell lines. The results indicate that in the human hypoxic A172 and U87MG glioblastoma cell lines adenosine up-regulates HIF-1alpha protein expression via the A(3) receptor subtype. In particular, we investigated the effect of A(3) receptor antagonists on HIF-1 and vascular endothelial growth factor (VEGF) expression. We found that A(3) antagonists inhibit adenosine-induced HIF-1alpha and VEGF protein accumulation in the hypoxic cells. Investigations in the molecular mechanism showed that A(3) receptor stimulation activates p44/p42 and p38 MAPKs that are required for A(3)-induced increase of HIF-1alpha and VEGF. Further studies are required to demonstrate the in vivo relevance of these observations with regard to the proposed role for adenosine as a key element in hypoxia and in tumors. PMID- 16682013 TI - The role of vertebral column muscles in level versus upslope treadmill walking-an electromyographic and kinematic study. AB - To gain insight into the neural mechanisms controlling vertebral column movement and its role in walking, we performed kinematic and electromyographic (EMG) studies on cats during level and upslope treadmill walking. Kinematic data of the limbs and vertebral column were obtained with a high-speed camera synchronized with EMG recordings from levels T10, L1, and L5 of m. longissimus dorsi (Long). During a single-step cycle at all upslope angles, vertebral movement in the lateral (left-right), cranial-caudal (forward-backward), and dorsal-ventral (upward-downward) directions was observed. Lateral movements were produced by forelimb take-off and hindlimb landing, and forward and upward movements were produced by hindlimb extension. During the single-step cycle, each of the three epaxial muscles, m. multifidus, m. iliocostalis, and Long, showed two bilateral EMG bursts. The onset of the EMG bursts coincided with the left-right movements, suggesting that epaxial muscle activity depresses lateral movement. The termination of the EMG bursts correlated with the forward and downward phase of the step cycle, suggesting that contraction of the epaxial muscles produces forward and downward movements. EMG bursts of the epaxial muscles increase the stiffness and produce inwardly movements to decrease the lateral movements of the vertebral column and the termination of EMG bursts control the movements into cranial and ventral direction of the vertebral column. The results suggest that the rhythmic EMG bursts in the epaxial muscles are produced by pattern generators, and the timing of EMG bursts among the different levels of the epaxial muscles are altered by walking condition input via peripheral afferents and descending pathways. PMID- 16682014 TI - Structural analysis of water-soluble glucans from the root of Angelica sinensis (Oliv.) Diels. AB - Two water-soluble glucans (designated APS-1cI and APS-1cII) were extracted from the roots of Angelica sinensis (Oliv.) Diels and further purified by anion exchange and gel-filtration chromatography. Their molecular weights were determined to be 1.7 x 10(5) and 3.9 x 10(4)Da, respectively. The structures of the purified glucans were investigated by a combination of chemical and instrumental analysis, such as methylation analysis, periodate oxidation, GC-MS, as well as FTIR and NMR spectroscopy ((1)H, (13)C, H-H COSY, HSQC, HMBC, TOCSY and NOESY). The data obtained indicated that APS-1cI was a linear alpha-glucan composed of only (1-->6)-alpha-D-Glcp, and APS-1cII had a repeating unit consisting of (1-->4)-alpha-D-Glcp and (1-->6)-alpha-D-Glcp in a molar ratio of 4:1. Such glucans isolated from A. sinensis (Oliv.) Diels have not been previously reported. PMID- 16682016 TI - Dual level of interactions between calcineurin and PKC-epsilon in cardiomyocyte stretch. AB - OBJECTIVES: Myocardial stretch activates a number of interconnected pathways including the protein kinase C (PKC) pathway that in turn activates mitogen activated protein kinases (MAPK), leading to gene expression stimulation and ventricular hypertrophy. A role of calcineurin has also been shown during hypertrophy. The goal of our study was to look for a possible interconnection between PKC and calcineurin in myocardial stretch. METHODS: Neonatal rat cardiomyocytes were cultured for 5 days and a 15% stretch was applied. Expression of MAPK and PKC-epsilon was evaluated by Western blot analysis. The specific role of PKC-epsilon was evaluated by transfection of cardiomyocytes with a specific inhibitor peptide. Calcineurin and PKC-epsilon complex formation and co localization were evaluated by co-immunoprecipitation and by immunolocalization. RESULTS: The PKC isoform involved in stretch-induced ERK and JNK activations was PKC-epsilon. We show here that calcineurin is also found to be involved in the stretch response and that calcineurin and PKC-epsilon co-operate at 2 levels during stretch. First, stretch-induced translocation of PKC-epsilon from the cytosolic to the membrane fraction was inhibited by calcineurin inhibitors, indicating that calcineurin was necessary for PKC-epsilon activation induced by stretch. A second level of interaction was the formation of a calcineurin-PKC epsilon complex, which increased during stretch. Immunofluorescent studies indicated that, after stretch, calcineurin and PKC-epsilon were co-localized at the level of the perinuclear membrane. These results may have a major relevance in vivo since we also found similar PKC-epsilon-calcineurin complexes in the phase of thoracic aortic stenosis in rats during which heart failure develops. CONCLUSION: Calcineurin appears to be necessary for stretch-induced PKC-epsilon activation after which the phosphatase and the kinase are co-localized in a complex at the level of the perinuclear membrane where they may finely regulate the phosphorylation of their target proteins. PMID- 16682017 TI - Expression and regulation of SLC39A family zinc transporters in the developing mouse intestine. AB - Several ZIP genes (SLC39A family of metal transporters) play roles in zinc homeostasis. Herein, the temporal and spatial patterns of expression of the mouse ZIP1, 3, 4, and 5 genes in the developing intestine and the effects of maternal dietary zinc deficiency on these patterns of expression were examined. ZIP1 and ZIP3 genes, conserved members of the ZIP subfamily II, were found to be coexpressed during development. Expression of these genes was detected on day 14 of gestation in smooth muscle and the pseudostratified endoderm. By 5 days post partum, prominent expression became restricted to muscle and connective stroma. In contrast, expression of ZIP4 and ZIP5 genes, members of the ZIP subfamily called LIV-1, coincided with epithelial morphogenesis. ZIP5 expression was detected on d16 of gestation and localized to the basolateral membranes of the single-layered epithelium. ZIP4 expression was detected on d18 of gestation and localized to the apical membrane of villus epithelial cells. When dams were fed a zinc-deficient diet beginning at parturition, ZIP4 expression in the nursing neonate was greatly induced. In contrast, neonatal ZIP5 expression remained unchanged, but this protein was removed from the basolateral membrane of the enterocyte. These responses to dietary zinc deficiency mimic those found in the adult intestine. These studies reveal cell-type-specific expression of ZIP genes during development of the intestine, and suggest that the mouse intestine can elicit an adaptive response to dietary zinc availability at birth. PMID- 16682018 TI - Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. AB - Lifelong neurogenesis in vertebrates relies on stem cells producing proliferation zones that contain neuronal precursors with distinct fates. Proliferation zones in the adult zebrafish brain are located in distinct regions along its entire anterior-posterior axis. We show a previously unappreciated degree of conservation of brain proliferation patterns among teleosts, suggestive of a teleost ground plan. Pulse chase labeling of proliferating populations reveals a centrifugal movement of cells away from their places of birth into the surrounding mantle zone. We observe tangential migration of cells born in the ventral telencephalon, but only a minor rostral migratory stream to the olfactory bulb. In contrast, the lateral telencephalic area, a domain considered homologous to the mammalian dentate gyrus, shows production of interneurons and migration as in mammals. After a 46-day chase, newborn highly mobile cells have moved into nuclear areas surrounding the proliferation zones. They often show HuC/D immunoreactivity but importantly also more specific neuronal identities as indicated by immunoreactivity for tyrosine hydroxylase, serotonin and parvalbumin. Application of a second proliferation marker allows us to recognize label-retaining, actively cycling cells that remain in the proliferation zones. The latter population meets two key criteria of neural stem cells: label retention and self renewal. PMID- 16682019 TI - Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos. AB - The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation. PMID- 16682020 TI - Epithelial cell proliferation is promoted by the histamine H(3) receptor agonist (R)-alpha-methylhistamine throughout the rat gastrointestinal tract. AB - The temporal effect of (R)-alpha-methylhistamine on epithelial cell proliferation throughout the rat gastrointestinal tract was investigated. (R)-alpha methylhistamine was administered at 100 mg/kg orally and the rats were sacrificed 1, 24, 48, 72 and 144 h later. All the animals received 5-bromo-2'-deoxyuridine, (BrdU), 200 mg/kg i.p., 2 h before sacrifice. Gastrointestinal tissue was processed for histology and immunohistochemistry. (R)-alpha-methylhistamine caused a progressive increase in mucosal thickness of gastric fundus, distal small intestine and distal colon. Statistically significant differences from control values were found between 48 and 72 h after (R)-alpha-methylhistamine. (R)-alpha-methylhistamine significantly increased the number of BrdU-positive cells in the gastric fundus and antrum, intermediate and distal small intestine and distal colon. Peak effects were observed between 1 and 24 h after (R)-alpha methylhistamine administration. Proliferating cell number and mucosal thickness were comparable to those of control rats at 144 h. (R)-alpha-methylhistamine exerts a long lasting growth-promoting effect on the stomach, distal small intestine and distal colon. Present data support a role of histamine H(3) receptors in the normal regulation of cell cycle in epithelial tissue. PMID- 16682021 TI - Effect of scopolamine on the hippocampal theta rhythm during an eight-arm radial maze task in rats. AB - The changes in the hippocampal theta rhythm during an impairment of reference and working memory of radial maze task induced by scopolamine administration were studied. Intraperitoneal injection of scopolamine at doses of 0.5 and 1.0 mg/kg caused a significant increase in the number of total, reference memory and working memory errors. On the other hand, scopolamine significantly increased the hippocampal theta power (5-12 Hz) at doses (0.5 and 1.0 mg/kg) that caused an impairment of reference and working memory. A significant increase in the peak frequency of the hippocampal theta rhythm was also observed with scopolamine, even at a dose of 0.2 mg/kg. At doses of 0.2, 0.5 and 1.0 mg/kg, scopolamine caused a decrease in the locomotor activity during the radial maze task. From these results, it may be concluded that an increase in amplitude of the hippocampal theta rhythm induced by scopolamine is closely associated with memory/learning function of the eight-arm radial maze. PMID- 16682022 TI - Antinociceptive efficacy of lacosamide in a rat model for painful diabetic neuropathy. AB - Lacosamide was tested in the streptozotocin rat model of diabetic neuropathic pain in comparison to drugs which are commonly used in the treatment of diabetic neuropathic pain, i.e. antidepressants and anticonvulsants. In diabetic rats, lacosamide attenuated cold (10, 30 mg/kg, i.p.), warm (3, 10, 30 mg/kg, i.p.) and mechanical allodynia (30 mg/kg, i.p.). Streptozotocin-induced thermal and mechanical hyperalgesia were reduced by lacosamide at doses of 10 and 30 mg/kg, i.p. Morphine (3 mg/kg) showed similar efficacy on allodynia and hyperalgesia. Amitriptyline (10 mg/kg), venlafaxine (15 mg/kg), levetiracetam (180 mg/kg) and pregabalin (100 mg/kg) exhibited significant effects on thermal allodynia and mechanical hyperalgesia. Only treatment with amitriptyline (30 mg/kg, i.p.) produced full reversal of thermal allodynia comparable to lacosamide. Lamotrigine (45 mg/kg, i.p.) had no effect on both behavioral readouts. Lacosamide's potency and efficacy in reversing pain behavior might be due to its new, yet unknown mechanism of action. PMID- 16682023 TI - Topographical resolution of jaw movements mediated by cyclase- vs. non-cyclase coupled dopamine D(1)-like receptors: studies with SK&F 83822. AB - This study examined the effects on orofacial movement topography of SK&F 83822 ([R/S]-6-chloro-7,8-dihydroxy-3-allyl-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3 benzazepine), which stimulates dopamine D(1)-like receptors coupled to stimulation of adenylyl cyclase (AC) but not phosphoinositide (PI) hydrolysis, in comparison with SK&F 83959 ([R/S]-3-methyl-6-chloro-7,8-dihydroxy-1-[3-methyl phenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), which stimulates PI hydrolysis but not AC. SK&F 83822 alone induced chattering, while SK&F 83959 alone exerted little effect. SK&F 83822 and SK&F 83959 each in combination with the dopamine D(2)-like agonist quinpirole resulted in synergistic induction of non-chattering movements with tongue protrusions. These effects were blocked by the dopamine D(1)-like receptor antagonist SCH 23390 ([R]-3-methyl-7-chloro-8-hydroxy-1-phenyl 2,3,4,5-tetrahydro-1H-3-benzazepine). However, the dopamine D(2)-like receptor antagonist YM 09151-2 (cis-N-[1-benzyl-2-methyl-pyrrolidin-3-yl]-5-chloro-2 methoxy-4-methylaminobenzamide) exerted a biphasic effect on synergism with SK&F 83822: chattering was initially released but antagonised thereafter. Only antagonism was seen for synergism with SK&F 83959. While both AC- and PI-coupled dopamine D(1)-like receptors participate in synergistic dopamine D(1)-like:D(2) like receptor interactions, topographically specific synergistic and oppositional dopamine D(1)-like:D(2)-like interactions evident with SK&F 83822 reflect the involvement primarily of D(1)-like receptors coupled to AC rather than PI. PMID- 16682024 TI - In vitro and in vivo pharmacological characterization of the nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-ol. AB - It was recently reported that the hexapeptide Ac-RYYRIK-ol binds with high affinity nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors and competitively antagonizes N/OFQ actions in the mouse vas deferens assay. Here we further describe the in vitro and in vivo pharmacological features of this NOP receptor ligand. In mouse brain homogenate the degradation half life of Ac-RYYRIK-ol (2.48 min) was significantly higher than that of the parent compound Ac-RYYRIK-NH2 (1.20 min). In the electrically stimulated mouse vas deferens, Ac-RYYRIK-ol (10 1000 nM) competitively antagonized the inhibitory effect of N/OFQ (pA2=8.46), while in the isolated mouse colon the hexapeptide mimicked N/OFQ contractile effects thus behaving as a NOP receptor agonist (pEC50=9.09). This latter effect was no longer evident in colon tissues taken from mice knock out for the NOP receptor gene (NOP-/-). In vivo in mice, similarly to N/OFQ, Ac-RYYRIK-ol (dose range 0.001-1 nmol) produced: i) pronociceptive effects after intracerebroventricular (i.c.v.) administration and antinociceptive actions when given intrathecally (i.t.) in the tail withdrawal assay; ii) inhibition of locomotor activity and iii) stimulation of food intake after supraspinal administration. Finally, in the forced swimming test, Ac-RYYRIK-ol was inactive per se, but reversed the antidepressant-like effects elicited by the NOP receptor selective antagonist UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH2). Thus, in all these in vivo assays Ac-RYYRIK-ol mimicked the actions of N/OFQ showing however higher potency. In conclusion, Ac-RYYRIK-ol displayed a complex pharmacological profile which is likely due to the low efficacy agonist nature of this novel ligand of the NOP receptor. The high potency, selectivity of action, and in vivo effectiveness make Ac-RYYRIK-ol a useful pharmacological tool for future studies in the field of N/OFQ and its NOP receptor. PMID- 16682025 TI - Spectral attenuation of the mouse, rat, pig and human lenses from wavelengths 360 nm to 1020 nm. AB - The transmission spectrum of the lens is essential to calculate irradiance at the surface of the retina. Although the mouse is a common model for ophthalmic research, there is little information on the transmission of the mouse lens. Attenuation by the lens is partly dependent on lens thickness; however, the attenuation coefficient is independent of lens thickness. In this study, we measured the attenuation coefficients of the albino and pigmented mouse lens as well as those of the rat, pig, and human lenses, and provide a composite equation that describes the attenuation coefficients in the ultraviolet and visible bandwidths (360-700 nm). In the near infrared region (700-1020 nm), water absorption must be included to explain the measurements. PMID- 16682026 TI - Different death stimuli evoke apoptosis via multiple pathways in retinal pigment epithelial cells. AB - Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death. PMID- 16682027 TI - Three experimental glaucoma models in rats: comparison of the effects of intraocular pressure elevation on retinal ganglion cell size and death. AB - Glaucoma is a chronic and progressive optic nerve neuropathy involving the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is considered to be the major risk factor associated with the development of this neuropathy. The objective of the present study was to compare the effects on RGC survival of three different experimental methods to induce chronic elevation of IOP in rats. These methods were: (i) injections of latex microspheres into the eye anterior chamber; (ii) injections into the anterior chamber of a mixture of microspheres plus hydroxypropylmethylcellulose (HPM) and (iii) cauterization of three episcleral veins. The IOP of right (control) and left (glaucomatous) eyes was measured with an applanation tonometer in awake animals. Thirteen to 30 weeks later, RGCs were retrogradely labeled with 3% fluorogold. Subsequently, we analyzed the density of RGCs, as well as the major axis length and area of RGC soma resulting from the application of each method. A significant increase in IOP was found following application of each of the three methods. Cell death was evident in the glaucomatous eyes as compared to controls. However, no statistical differences were found between the extent of cell death associated with each of the three methods. IOP increase also induced a significant increase in the size of the soma of the remaining RGCs. In conclusion, the three methods used to increase IOP induce a similar degree of RGC death. Moreover, the extent of cell death was similar when the retinas were maintained under conditions of elevated IOP for 24 weeks in comparison to 13 weeks. PMID- 16682028 TI - Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice. AB - In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection. PMID- 16682029 TI - Uncinaria stenocephala: assessment of antigens for the immunodiagnosis of canine uncinariosis. AB - Although Uncinaria stenocephala is the most frequent hookworm in the intestine of dogs from Northern, Central and Southern Europe, little is known about its host parasite relationship. Three groups of sera from dogs (Group 1: dogs naturally infected only by U. stenocephala; Group 2: helminth-free dogs at necropsy, and Group 3: dogs parasitized by other helminths) were analyzed by ELISA using U. stenocephala antigens from adult worms (somatic and excretory-secretory antigens) and from L3 larvae (somatic antigens). All three sources of antigens were found to be suitable for immunodiagnosis of canine uncinariosis with up to 90% efficacy. However, an analysis to assess the diagnostic value of the different antigens demonstrated that the adult excretory-secretory antigens had a higher diagnostic efficacy (96.7%), indicating that this is the best antigen source for the diagnosis of Uncinaria infection. PMID- 16682030 TI - Schistosoma mansoni: biochemical characterization of lactate transporters or similar proteins. AB - While in medium containing glucose, schistosomes exhibit homolactic fermentation. Accumulation of lactate acid in tissue fluid causes lowering of pH and a resultant inhibition of metabolic pathways. This requires lactate transporter protein in homolactic fermentors to facilitate the translocation of lactate(-) and [H(+)] across their plasma membrane. The ex-vivo experiment assessed lactic acid secretion by adult worms in absence and the presence of lactic acid transporter protein inhibitors. Phloretin and alpha-cyano-4-hydroxycinnamate caused a combined 25-35% inhibition of lactic acid secretion and probenecid increased this inhibition to 65% of control values. The removal of inhibitors resulted in 80% recovery of lactic acid secretion. In the in-vitro studies using vesicles isolated from adult worms and from schistosomula, the effects of phloretin and alpha-cyano-4-hydroxycinnamate were greater, each causing approximately 80% inhibition independently. The data obtained in this study demonstrate the presence of lactic acid transporters or similar proteins in Schistosoma mansoni. PMID- 16682031 TI - Predictive value of the hemizona assay for pregnancy outcome in patients undergoing controlled ovarian hyperstimulation with intrauterine insemination. AB - OBJECTIVE: The hemizona assay (HZA) is an established functional test that examines in vitro sperm-zona pellucida binding capacity with high predictive power for fertilization outcome in IVF. The objective of this study was to evaluate the value of the HZA as a predictor of pregnancy in patients undergoing controlled ovarian hyperstimulation (COH) and intrauterine insemination (IUI). DESIGN: Prospective clinical study. SETTING: Academic center. PATIENT(S): Eighty two couples with unexplained or male factor infertility that underwent 313 IUI cycles. INTERVENTION(S): Basic semen analysis and HZA were performed within three months of starting COH/IUI therapy. MAIN OUTCOME MEASURE(S): Hemizona index (HZI) and clinical pregnancy. RESULT(S): Overall, patients with an HZI of <30 had a significantly lower pregnancy rate compared to patients with an HZI of > or =30 (11.1% vs. 40.6%, respectively; P<.05; relative risk for failure to conceive: 1.5 (confidence interval 1.2-1.9)). In all patients combined, and in the range of HZI 0-60, the duration of infertility (P=.000) and the HZI (P=.004) were significant determinants of conception (receiver operating characteristics (ROC) analysis). In couples with male infertility, the average path velocity and HZI were significant predictors of conception (P=.001 and P=.005, respectively, ROC analysis). The negative and positive predictive values of the HZA for pregnancy were 93% and 69%, respectively. Logistic regression analysis provided models of HZI (P=.021) and duration of infertility (P=.037) with highest predictability of conception in male factor and unexplained infertility groups, respectively. CONCLUSION(S): The HZA predicted pregnancy in the IUI setting with high sensitivity and negative predictive value in couples with male infertility. Results of this sperm function test are useful in counseling couples before allocating them into COH/IUI therapy. PMID- 16682032 TI - Individual differences in salivary cortisol: associations with common over-the counter and prescription medication status in infants and their mothers. AB - The purpose of the present study was to describe associations between the use of common over-the-counter (OTC) and prescription medications with individual differences in salivary cortisol in infants and their mothers. Participants were 1020 mothers and 852 infants (52.5% boys; ages 5.03-13.44 months) from economically disadvantaged and ethnically diverse families (38.4% African American) who donated saliva samples before, 20 and 40 min after infants participated in a series of challenging tasks. Samples (N=5616) were later assayed for cortisol. Medication information was content analyzed separately for infants (e.g., teething gels, nonsteroidal anti-inflammatory drugs, acetaminophen, decongestants) and mothers (e.g., narcotics, antidepressants, antipsychotics, contraceptives, glucocorticoids). A large percentage of infants (44%) and the majority of mothers (57.5%) had used at least one medication (range 0-4) in the previous 48 h. Most frequent were acetaminophen (e.g., Tylenol) and cold medications (e.g., decongestants) for infants and contraceptives and acetaminophen for mothers. Compared to infants not taking any medications, cortisol reactivity to the challenge tasks was less pronounced for infants taking acetaminophen. Cortisol levels were higher for mothers taking oral or transdermal contraceptives and acetylsalicylic acid (e.g., Aspirin) but lower for mothers taking pure agonist opioids (e.g., Oxycontin) compared to mothers not taking any medications. These medication-related differences remained significant after controlling for sampling time, fever, maternal anxiety and depression, infant temperament, ethnicity, SES, and health status. Recommendations are provided to steer investigators clear of these potential sources of unsystematic error variance in salivary cortisol. PMID- 16682033 TI - Masculinized otoacoustic emissions in female spotted hyenas (Crocuta crocuta). AB - In humans and rhesus monkeys, click-evoked otoacoustic emissions (CEOAEs) are stronger in females than in males, and there is considerable circumstantial evidence that this sex difference is attributable to the greater exposure to androgens prenatally in males. Because female spotted hyenas are highly androgenized beginning early in prenatal development, we expected an absence of sexual dimorphism in the CEOAEs of this species. The CEOAEs obtained from 9 male and 7 female spotted hyenas confirmed that expectation. The implication is that the marked androgenization to which female spotted hyenas are exposed masculinizes the cochlear mechanism responsible for CEOAEs. The CEOAEs measured in 3 male and 3 female hyenas that had been treated with anti-androgenic agents during prenatal development were stronger than the CEOAEs of the untreated animals, in accord with the implied inverse relationship between prenatal androgen exposure and the strength of the cochlear mechanisms producing CEOAEs. The CEOAEs of three ovariectomized females and two castrated males were essentially the same as those for the untreated females and males, suggesting that there is little or no activational effect of hormones on CEOAE strength in spotted hyenas. Distortion product OAEs (DPOAEs) also were measured. Those sex differences also were generally small (as they are in humans), and the effects of the anti-androgen agents were inconsistent. Thus, prenatal androgen exposure apparently does affect OAEs, but the effects appear to be greater for the reflection-based cochlear mechanism that underlies CEOAEs than for the nonlinear cochlear mechanism underlying DPOAEs. PMID- 16682034 TI - Delivery of the posterior arm reduces shoulder dimensions in shoulder dystocia. AB - OBJECTIVE: To evaluate a possible reduction in shoulder diameter and circumference by extending the posterior arm during delivery, for an easier birth in cases of shoulder dystocia. METHODS: In this study of 33 neonates the bisacromial diameter and axilloacromial circumference were measured within 72 h of birth, first with the neonate's arms by its chest and then with 1 arm extended above its head. Reductions in diameter and circumference were evaluated. RESULTS: The mean +/- SD differences in bisacromial diameter and axilloacromial circumference were 1.9 +/- 0.69 cm and 2.52 +/- 1.18 cm, respectively. A greater reduction was observed in neonates with a greater shoulder diameter. CONCLUSION: In cases of shoulder dystocia, delivery of the posterior arm should significantly reduce shoulder dimensions, especially in larger fetuses, and prevent a need for excessive traction. PMID- 16682035 TI - Cesarean delivery after successful external cephalic version of breech presentation at term. PMID- 16682036 TI - Measuring progress towards the MDG for maternal health: including a measure of the health system's capacity to treat obstetric complications. AB - OBJECTIVE: This paper argues for an additional indicator for measuring progress of the Millennium Development Goal for maternal health-the availability of emergency obstetric care. METHODS: MDG monitoring will be based on two indicators: the maternal mortality ratio and the proportion of births attended by skilled personnel. Strengths and weaknesses of a third indicator are discussed RESULTS: The availability of EmOC measures the capacity of the health system to respond to direct obstetric complications. Benefits to using this additional indicator are its usefulness in determining an adequate distribution of services and showing management at all levels what life-saving interventions are not being provided, and stimulate thought as to why. It can reflect programmatic changes over a relatively short period of time and data requirements are not onerous. CONCLUSION: A measure of strength of the health system is important since many interventions depend on the health system for their implementation. PMID- 16682037 TI - Indicators of prolonged hospital stay in hyperemesis gravidarum. PMID- 16682038 TI - Can the process indicators for emergency obstetric care assess the progress of maternal mortality reduction programs? An examination of UNFPA Projects 2000 2004. AB - BACKGROUND: In view of the disappointing progress made in the last 20 years in reducing maternal mortality in low-income countries and before going to scale in implementing the new evidence-based strategies, it is crucial to review and assess the progress made in pilot countries where maternal mortality reduction programs focused on emergency obstetric care. OBJECTIVE: To review the process indicators recommended for monitoring emergency obstetric care and their application in field situations, examining the conditions under which they can be used to assess the progress of maternal mortality reduction programs. METHODS: Five of the six UN recommended process indicators were monitored annually for 5 years in selected districts of Morocco, Mozambique, India and Nicaragua. Trends are presented and discussed. RESULTS: With specific variations due to different local situations in the four countries and in spite of variations in quality of data collection, all indicators showed a consistent positive trend, in response to the inputs of the programs. CONCLUSIONS: The UN process indicators for emergency obstetric care should continue to be promoted, but with two important conditions: (1) data collection is carefully checked for quality and coverage; (2) efforts are made to match process and outcome indicators (maternal and perinatal mortality, incidence of complications). PMID- 16682039 TI - Global patterns in availability of emergency obstetric care. AB - OBJECTIVE: This paper examines the availability of basic and comprehensive emergency obstetric care (EmOC), interventions used to treat direct obstetric complications. Determining what interventions are provided in health facilities is the first priority in analyzing a country's capabilities to treat obstetric emergencies. There are eight key interventions, six constitute basic EmOC and all eight comprehensive EmOC. METHODS AND RESULTS: Based on data from 24 needs assessments, the following global patterns emerge: comprehensive EmOC facilities are usually available to meet the recommended minimum number for the size of the population, basic EmOC facilities are consistently not available in sufficient numbers, both in countries with high and moderate levels of maternal mortality, and the majority of facilities offering maternity services provide only some interventions indicating an unrealized potential. CONCLUSION: Upgrading maternities, health centers and hospitals to at least basic EmOC status would be a major contributing step towards maternal mortality reduction in resource-poor countries. PMID- 16682040 TI - Monitoring of aortic and coronary atherosclerosis in native and non-native males of Yakutsk over 40 years. AB - We have analyzed the development of aortic and coronary atherosclerosis over 40 years in native and non-native males of Yakutsk by comparing the data obtained in 1963-1965, 1985-1987 and 2001-2003. The research was carried out in compliance with the WHO program for the atherosclerosis epidemiology studies using autopsy material. Subjects (361 natives and 692 non-natives) included in the research died of external causes at the age of 20-59 years and had no history of chronic diseases, which was confirmed by post-mortem examination. The prevalence and the extent of raised atherosclerotic lesions (fibrous plaques, complicated lesions and calcinosis) in the aorta and coronary arteries were determined by visual planimetry after staining with Sudan IV. An accelerated development of aortic and coronary atherosclerosis was revealed in both populations throughout the entire period of 40 years with more severe manifestations in non-native males. The severity of atherosclerosis increased considerably in native and non-native males aged over 40 years. The rate of the increase in the extent of raised atherosclerotic lesions tended to be greater in native than in non-native males. PMID- 16682041 TI - The -1131 T>C and S19W APOA5 gene polymorphisms are associated with high levels of triglycerides and apolipoprotein C-III, but not with coronary artery disease: an angiographic study. AB - High plasma concentrations of triglycerides (TG) and apolipoprotein C-III (ApoC III) are well-known risk factors for cardiovascular disease. Two variants of the recently discovered APOA5, 1131 C>T and S19W, have been associated with hypertriglyceridemia, whereas their relation with coronary artery disease (CAD) remains controversial. Nine hundred and thirteen angiografically defined patients (669 CAD and 244 CAD-free) were genotyped for APOA5 -1131 C>T and S19W polymorphisms. Carriership of the APOA5 -1131 C allele was identified, by multiple linear regression models, as a significant independent predictor for both TG (standardized beta-coefficient=0.112; p=0.010) and ApoC-III variability (standardized beta-coefficient=0.113; p=0.013). Similarly, APOA5 19W allele carriership was a significant independent predictor for both TG (standardized beta-coefficient=0.113; p=0.007) and ApoC-III variability (standardized beta coefficient=0.088; p=0.045). Despite the association with at-risk lipid profile, no significant difference was detected in the distribution of both APOA5 gene polymorphisms between subjects with or without CAD. Moreover, homozygous carriers of the APOC3 -455 C, another TG- and ApoC-III raising variant, showed a significant increased risk for CAD (OR 1.90 with 95% CI 1.002-3.62; p=0.049; by multiple logistic regression). Different genotypes, i.e., APOA5 and APOC3 variants, may lead to similar biochemical phenotypes, namely hypertriglyceridemia, but to contrasting clinical phenotypes such as the presence of angiographically proven CAD. PMID- 16682042 TI - Measurement of carotid artery intima-media thickness in dyslipidemic patients increases the power of traditional risk factors to predict cardiovascular events. AB - A longitudinal observational study investigated whether the measurement, in clinical practice, of carotid maximum intima-media thickness (Max-IMT) could be combined with the Framingham risk score (FRS) to improve the predictability of cardiovascular events in dyslipidemic patients who are at low or intermediate risk. Max-IMT was measured by ultrasound in 1969 patients attending a lipid clinic. The "best threshold values" (BTVs) above which we considered the Max-IMT to be abnormally high were calculated for our dyslipdemic population for each 10 year age interval in men and women. Two hundred and forty-two patients (age 54+/ 10 years; 43.8% women) with an FRS <20%, i.e. at low or intermediate risk, were monitored for more than 5 years. Twenty-four of these patients suffered a cardiovascular event within 5.1+/-2.3 years. Both FRS and Max-IMT proved to be independent outcome predictors (p<0.04, both), with a hazard ratio (HR) of 6.7 (95% CI 1.43, 31.04; p=0.015) in patients in whom FRS was 10-20% and Max-IMT was above the BTV (60th percentile of Max-IMT distribution for men or 80th for women). In Kaplan-Meier analysis, the Max-IMT significantly improved the predictive value of the FRS (chi(2)=8.13, p=0.04). Patients with FRS 10-20% (currently considered intermediate-risk) and also elevated Max-IMT values came into the same high-risk category as patients with FRS 20-30%. The combination of FRS with Max-IMT measurement can be used in routine clinical practice to greatly enhance the predictability of cardiovascular events in the large number of patients who fall into the intermediate-risk category, which currently does not call for aggressive preventive measures. PMID- 16682043 TI - Alanine aminotransferase predicts coronary heart disease events: a 10-year follow up of the Hoorn Study. AB - Alanine aminotransferase (ALT) is a marker of non-alcoholic fatty liver disease (NAFLD) and predicts incident type 2 diabetes mellitus (DM2). Recently, ALT was shown to be also associated with endothelial dysfunction and carotid atherosclerosis. We studied the predictive value of ALT for all-cause mortality, incident cardiovascular disease (CVD) and coronary heart disease (CHD) events in a population-based cohort of Caucasian men and women aged 50-75 years, at baseline. The 10-year risk of all-cause mortality, fatal and non-fatal CVD and CHD events in relation to ALT was assessed in 1439 subjects participating in the Hoorn Study, using Cox survival analysis. Subjects with prevalent CVD/CHD and missing data were excluded. As compared with the first tertile, the age- and sex adjusted hazard ratios (95% confidence intervals) for all-cause mortality, CVD events and CHD events were 1.30 (0.92-1.83), 1.40 (1.09-1.81) and 2.04 (1.35 3.10), respectively, for subjects in the upper tertile of ALT. After adjustment for components of the metabolic syndrome and traditional risk factors, the association of ALT and CHD events remained significant for subjects in the third relative to those in the first tertile, with a hazard ratio of 1.88 (1.21-2.92) and 1.75 (1.12-2.73), respectively. In conclusion, the predictive value of ALT for coronary events, seems independent of traditional risk factors and the features of the metabolic syndrome in a population-based cohort. Further studies should confirm these findings and elucidate the pathophysiological mechanisms. PMID- 16682044 TI - Comparison of mechanical behavior among the extrapulmonary arteries from rats. AB - Results of comparative tests on pulmonary arteries from untreated Long-Evans rats are presented from three sections of the artery: the trunk, and the right and left main extrapulmonary arteries. Analyses were conducted looking for mechanical differences between the flow (longitudinal) and circumferential directions, between the right and left main arteries, and between each of the mains and the trunk. The mechanical properties of rat pulmonary arteries were obtained with a bubble inflation technique. A flat disk of rat pulmonary artery was constrained at the periphery and inflated, and the geometry of the resulting bubble of material recorded from six different angles. To analyze the data, the area under the stress-strain curve was calculated for each test and orientation. This area, related to the strain-energy density, was calculated at stress equal to 200kPa, for the purpose of statistical comparison. The mean values for the area show that the trunk is less compliant than the main arteries; this difference is supported by histological evidence. When comparing the circumferential and longitudinal properties of the arteries, differences are found for the trunk and left main arteries, but with opposite orientations being more compliant. The mean values for the two orientations for the right main artery are statistically identical. There was indication of significant difference in mechanical properties between the trunk and the main arteries. The left main artery in the circumferential orientation is highly compliant and appears to strongly influence the likelihood that significant differences will exist when included in a statistical population. These data show that each section of the extrapulmonary arterial system should not be expected to behave identically, and they provide the baseline mechanical behavior of the pulmonary artery from normotensive rats. PMID- 16682045 TI - In vivo biomechanics of the fingerpad skin under local tangential traction. AB - Small patches of fingerpad glabrous skin in human subjects were tested in vivo for their biomechanical properties under tangential loading and for large deformations. These conditions included stretching and shearing the skin at a length scale of 0.3mm using an apparatus comprising a pair of piezoelectric benders arranged to increase the stiffness/free deflection tradeoff when compared to ordinary cantilevered benders. It was then possible to test the skin with up to 80% of tangential strain. With feedback control, it was also possible to create isotonic and isometric testing conditions. The results showed much variability across subjects and it was seen that the glabrous skin exhibited nonlinear stiffening in tangential traction. The skin was consistently more elastic across the ridges than along the ridges regardless of the location of the sample on the fingerpad. The skin behaved visco-elastically but relaxed about twice as fast than it crept. Finally, it was found that under large deformation, there was consistently 80% of hysteretic loss for a wide range of loading conditions. PMID- 16682046 TI - The effect of muscle fatigue on in vivo tibial strains. AB - Stress fracture is a common musculoskeletal problem affecting athletes and soldiers. Repetitive high bone strains and strain rates are considered to be its etiology. The strain level necessary to cause fatigue failure of bone ex vivo is higher than the strains recorded in humans during vigorous physical activity. We hypothesized that during fatiguing exercises, bone strains may increase and reach levels exceeding those measured in the non-fatigued state. To test this hypothesis, we measured in vivo tibial strains, the maximum gastrocnemius isokinetic torque and ground reaction forces in four subjects before and after two fatiguing levels of exercise: a 2km run and a 30km desert march. Strains were measured using strain-gauged staples inserted percutaneously in the medial aspect of their mid-tibial diaphysis. There was a decrease in the peak gastrocnemius isokinetic torque of all four subjects' post-march as compared to pre-run (p=0.0001), indicating the presence of gastrocnemius muscle fatigue. Tension strains increased 26% post-run (p=0.002, 95 % confidence interval (CI) and 29% post-march (p=0.0002, 95% CI) as compared to the pre-run phase. Tension strain rates increased 13% post-run (p=0.001, 95% CI) and 11% post-march (p=0.009, 95% CI) and the compression strain rates increased 9% post-run (p=0.0004, 95% CI) and 17% post-march (p=0.0001, 95% CI). The fatigue state increases bone strains well above those recorded in rested individuals and may be a major factor in the stress fracture etiology. PMID- 16682047 TI - Separation and determination of cations in beverage products by capillary zone electrophoresis. AB - Cation determination is important for quality control of beverage products. To determine a large group simultaneously, a capillary electrophoresis procedure is developed with indirect UV at 214 nm in a three-complex buffer system (10 mM N,N dimethylbenzylamine (DBA), 8 mM lactic acid and 2 mM 18-crown-6) with good mobility matching with desired cations. Under optimized conditions with pH adjusted to 4.65, a baseline separation is achieved for 14 cations (Rb(+), NH(4)(+), K(+), Ca(2+), Na(+), Mg(2+), Mn(2+), Co(2+), Fe(2+), Cd(2+), Cr(3+), Ni(2+), Zn(2+) and Cu(2+)) within 7 min using an uncoated silica column. To cover ng/l to mug/l range, both hydrostatic and electrokinetic sampling are studied, showing working ranges within (0.05-50)/(0.005-2) microg/l and detection limits (13-78)/(1.4-10) ng/l, respectively with satisfactory repeatability (RSD 0.31 0.47% for migration time, and 3.0-4.0% for peak height measurement). Agreeable results with established inductively coupled plasma-atomic emission spectrometry method have been obtained for orange juice and tea samples. PMID- 16682048 TI - Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis. AB - It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies. PMID- 16682049 TI - Liquid chromatography-UV diode-array detection method for multi-residue determination of macrolide antibiotics in sheep's milk. AB - A rapid, simple and sensitive liquid chromatography-UV diode-array detection method was developed for the simultaneous determination of seven macrolides (erythromycin, oleandomycin, roxithromycin, josamycin, spiramycin, tylosin and ivermectin) in sheep's milk. The column, mobile phase, temperature and flow rate were optimised to provide the best resolution of these analytes. The extraction of the antibiotic residues involves the treatment of protein-free samples with a combination of concentrated sodium hydroxide and ethyl acetate. Necessary defatting is achieved by alkaline hydrolysis. The recovery of each antibiotic was between 55% and 77%, with relative standard deviations ranging from 1% to 6.5%. The limit of quantification was 72.4 microg/kg for ivermectin, 48.3 microg/kg for roxithromycin, and 24.1 microg/kg for erythromycin, oleandomycin, spiramycin, josamycin and tylosin. The procedure was successfully used in the multi-residue determination of these macrolides at levels below the maximum concentrations legally allowed in milk samples. PMID- 16682050 TI - Simultaneous analysis of oxygenated and nitrated polycyclic aromatic hydrocarbons on standard reference material 1649a (urban dust) and on natural ambient air samples by gas chromatography-mass spectrometry with negative ion chemical ionisation. AB - This study deals with the development of a routine analytical method using gas chromatography-mass spectrometry with negative ion chemical ionisation (GC/NICI MS) for the determination of 17 nitrated polycyclic aromatic hydrocarbons (NPAHs) and 9 oxygenated polycyclic aromatic hydrocarbons (OPAHs) present at low concentrations in the atmosphere. This method includes a liquid chromatography purification procedure on solid-phase extraction (SPE) cartridge. Application of this analytical procedure has been performed on standard reference material (SRM 1649a: urban dust), giving results in good agreement with the few data available in the literature. The analytical method was also applied on ambient air samples (on both gas and particulate phases) from the French POVA program (POllution des Vallees Alpines). NPAHs concentrations observed for a rural site during the Winter period are about 0.2-100.0pgm(-3) in the particulate phase and about 0.0 20.0pgm(-3) in the gas phase. OPAHs present concentrations 10-100 times higher (0.1-2.0ngm(-3) and 0.0-1.4ngm(-3) for the particulate and the gas phases, respectively). These preliminary results show a good correlation between the characteristics of the sampling site and the compound origins (primary or secondary). PMID- 16682051 TI - Optimization of a microwave-assisted extraction method for the analysis of polycyclic aromatic hydrocarbons from fish samples. AB - A microwave-assisted extraction (MAE) procedure has been developed and optimized for the extraction of six regulated polycyclic aromatic hydrocarbons (PAHs) from muscle samples of polluted fish. The procedure involves the simultaneous microwave-assisted extraction of PAHs with n-hexane and the lipids hydrolysis with potassium hydroxide. Experimental design methodology allows a quick and robust optimization of operational parameters such as the extraction time, temperature, and solvent volumes. In these final optimized conditions, the procedure can be applied to a vast range of fat containing fish samples without significant changes, thus enabling its routine use. Recoveries around 90% for the studied compounds benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, and indene[1,2,3-cd]pyrene and quantification limits (between 0.07 and 0.53 ng/g dry weight) far below the regulated limits, have been obtained. The procedure is applied to several different fish samples. Further, accuracy validation using NIST SRM 2977 reference material was carried out. PMID- 16682052 TI - Validation of a tentative microsatellite marker for the dopamine D4 receptor gene by capillary gel electrophoresis. AB - Two to four-basepair-short tandem repeats (i.e. microsatellites) are broadly utilized as genetic markers for mapping disease loci in whole genome search analyses. Based on their close vicinity on chromosome 11, the D11S1984 microsatellite was anticipated as a tentative marker for the dopamine D4 receptor gene. A capillary gel electrophoresis based genotype analysis method and an in house made computational tool was developed for the analysis of the D11S1984 microsatellite marker to examine a healthy Hungarian population of n=106. The data obtained did not suggest significant linkage between the D11S1984 marker and the DRD4 gene. PMID- 16682053 TI - On-line hyphenation of flow injection, miniaturized capillary electrophoresis and atomic fluorescence spectrometry for high-throughput speciation analysis. AB - A hyphenated technique was developed for high-throughput speciation analysis by on-line coupling of flow injection (FI), miniaturized capillary electrophoresis (CE) and atomic fluorescence spectrometry (AFS). Two interfaces were used to couple all three systems: the first to couple FI and CE and the second to couple miniaturized CE and AFS. The first interface was a modified flow through chamber, connected to the FI valve with a piece of PTFE tube (0.1mm i.d.x 20 cm long). The capillary outlet was coupled to the AFS by using the second concentric "tube-in tube" interface. Split sampling was achieved in the electrokinetic mode. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were taken as model analytes to demonstrate the performance of the developed hyphenated technique. A volatile species generation (VSG) technique was employed to convert the analytes from the CE effluent into their respective volatile species. Baseline separation of Hg(II) and MeHg(I) was achieved by CE in a 50 microm i.d.x 8 cm long capillary at 3.0 kV within 60s. The precisions (RSD, n=12) were in the range of 0.7-0.9% for migration time, 3.8-4.2% for peak area, and 2.1-3.5% for peak height. The detection limits were 0.1 and 0.2 microgmL(-1) (as Hg) for Hg(II) and MeHg(I) with a sample throughput of 60 samples h(-1). The recoveries of both mercury species in the water samples studied were in the range of 93-106%. PMID- 16682054 TI - Improved preparation of class I HLA tetramers and their use in detecting CMV specific CTL. AB - Different methods were used to prepare HLA tetramers and the yields of each method were compared. Our results indicate that preliminary refolding of the heavy chain (Hc) and light chain (beta 2m) yields more monomer than the typical conventional method with urea-solubilized Hc and beta 2m. We then used the corresponding tetramers to detect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL). Increasing data suggest that the adoptive transfer of CMV specific CTL constitutes an effective strategy against CMV infections. We designed a method that efficiently induces CMV-specific CTL to a higher frequency in vitro than is currently achieved. This method increased the percentage of CMV specific CTL from below 1% to 20% of PBL, accounting for more than 40% of CD8+ T cells. Successful HLA tetramer preparation provides the basis for the subsequent detection of CMV-specific CTL in clinical applications. PMID- 16682055 TI - Structural basis of affinity maturation of the TEPC15/Vkappa45.1 anti-2-phenyl-5 oxazolone antibodies. AB - Affinity maturation is a process that leads to the emergence of more efficient antibodies following initial antigen encounter and represents a key strategy of the adaptive immunity of vertebrate organisms. Earlier and detailed sequence studies of the antibody response to a model antigen, the hapten 2-phenyl-5 oxazolone (phOx), define three different classes of antibodies. Class I antibodies use the V(H)Ox1/V(kappa)Ox1 gene pair and dominate the early stages of the anti-phOx response, class II antibodies use the V(kappa)Ox1 gene but a different V(H) segment and are common in the intermediate stages, and class III antibodies use the TEPC15/V(kappa)45.1 genes and play the greatest role in the late stages. Only the crystal structure of one anti-phOx antibody, the class II NQ10/12.5 Fab fragment, has been described. Here we report the crystal structures of the scFv form of the low and high affinity anti-phOx class III antibodies NQ10/1.12 and NQ16/113.8 complexed with the hapten. The two antibodies differ by nine amino acid substitutions, all located in the V(H) domain. Analysis of the two structures shows that affinity maturation results from an increase in surface complementarity, as a consequence of a finely tuned and highly concerted process chaperoned by the somatic mutations, and implies a more efficient hapten-induced fit in the mature antibody. The data also demonstrate that class III antibodies respond in a completely different way to the architectural problem of binding phOx compared to the class II antibody NQ10/12.5. PMID- 16682056 TI - Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines. AB - The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis. PMID- 16682057 TI - Nogo in multiple sclerosis: growing roles of a growth inhibitor. AB - In recent years, knowledge about the physiological functions of the Nogo-A protein has grown considerably, and this molecule has evolved from being one of the most important axonal regrowth inhibitors present in central nervous system (CNS) myelin, to several other potentially important roles in different areas such as nervous system development, epilepsy, vascular physiology, muscle pathology and CNS tumors. Therapeutically, targeting the Nogo-A protein by means of the immune response has been tried in an attempt to block neurite growth inhibition and promote regeneration in spinal cord injury models; the immune response to Nogo-A, however, has not been extensively studied. We propose to review recent evidence that Nogo-A may also play an important role in autoimmune demyelinating diseases such as experimental autoimmune encephalomyelitis and multiple sclerosis, including that Nogo-66 derived epitopes are encephalitogenic antigens in susceptible mouse strains, and that the immune response to Nogo-66 antigens includes both strong T cell and B cell activation, with epitope spreading of the antibody response to other myelin molecules. In CNS immunotherapy, careful targeting of neural self-antigens is a prerequisite in order to avoid unexpected deleterious effects, and increasing knowledge about the immune response to Nogo-A may provide a safe basis for the development of relevant therapeutic alternatives for several neurological conditions. PMID- 16682058 TI - Prevalence of Chiari I malformation and cerebellar ectopia in patients with pseudotumor cerebri. AB - BACKGROUND: Chiari I malformation (CM) may be present pre-surgically in pseudotumor cerebri (PTC) patients. Whether inferior tonsillar displacement (ITD) is coincidental or linked to increased intracranial pressure is unclear. This study aimed to identify the prevalence of both CM and cerebellar ectopia (CE) (ITD below the foramen magnum > or = 5 mm and 2-4 mm, respectively) in PTC patients. METHODS: Retrospective review combined with prospective assessment of 68 PTC patients with available brain magnetic resonance imaging (MRI) scans and reports. Data collected included patient demographics, height, weight, co-morbid conditions, and medications. MRIs were analyzed for cerebellar tonsillar position, and results were compared with original reports. RESULTS: Of 68 PTC patients, 60 (88%) had normal position of the cerebellar tonsils and 8 (12%) had ITD by report. Of the latter group, 4 were identified as CM and 4 as CE. On review of MRIs, however, 16 patients (24%) had ITD, 7 having CM and 9 having CE. All patients with ITD were female, most were overweight or obese, and most had presumed idiopathic intracranial hypertension (IIH). CONCLUSION: ITD exists pre surgically in a significant percentage of PTC patients. ITD is most common in obese or overweight women with presumed IIH. In fact, this subset of patients may actually represent a secondary form of PTC and may benefit from correction of ITD to restore normal intracranial pressure. PMID- 16682059 TI - Measuring sustained attention after traumatic brain injury: differences in key findings from the sustained attention to response task (SART). AB - Clinical reports after traumatic brain injury (TBI) suggest frequent difficulties with sustained attention, but their objective measurement has proved difficult. In 1997, Robertson and colleagues reported on a new sustained attention assessment tool, the sustained attention to response task (SART). Individuals with TBI were reported to produce more errors of commission on the SART than control participants, and both groups showed a relationship between SART errors and everyday lapses of attention as measured by the cognitive failures questionnaire (CFQ). Although few direct replications of these findings have been reported, the SART has been used widely as a measure of sustained attention in TBI, in normal controls, and in various other clinical samples. As part of a program of research on attention in TBI, we administered the SART and the CFQ to a sample of 34 survivors of moderate to severe TBI and to 35 control participants. CFQ scores reported by significant others showed clear group differences in everyday lapses of attention. Despite this, group differences in SART errors of commission were small and non-significant, and the correlations between SART errors and CFQ scores were small within both groups. Further analyses excluding participants with invalid score profiles, or restricting the analysis to the first performance of the SART failed to alter the results. These findings suggest that more research is needed to establish the validity of the SART as a measure of sustained attention after TBI, and to determine under what circumstances the original findings hold. PMID- 16682060 TI - Flavour-specific anticipatory hunger reinforced by either carbohydrate or protein. AB - Recent evidence indicates that rats can learn to avoid aversive consequences of several hours of food deprivation by eating more of food having orosensory characteristics that predict a protracted fast. Two new studies tested if macronutrient composition of a flavoured meal before the reinforcing fast influences this acquisition of anticipatory hunger/satiety in a smooth-brained mammal. In one study, female Sprague-Dawley rats were trained for 11 cycles of 4 days with experimental meals of either carbohydrate or protein flavoured with either grape or cherry, with one odour followed by a 3-h fast and the other by 10 h of food deprivation. Both nutrient groups acquired anticipatory hunger. Then that learnt increase in meal size started to extinguish the hunger-avoidance response to the longer fast. The other study tested if this finding extended to experimental meals that combined carbohydrate and protein, and had vanilla or chicken flavours, to fasts of 4 and 12 h and to male rats as well as females within a training period of 6 cycles of 4 days. Evidence of anticipatory hunger early in training was clear only in the males. The combined results from the two experiments indicate that either carbohydrate or protein is sufficient for negative reinforcement of flavour-specific anticipatory hunger when the shorter fast lasts for 3 h. This food-discriminative anticipatory adjustment of meal size could be an automatically learnt part of hunger management for socially scheduled eating in human beings. PMID- 16682061 TI - Locomotor, feeding and melatonin daily rhythms in sharpsnout seabream (Diplodus puntazzo). AB - Sharpsnout seabream is a marine teleost of increasing interest for Mediterranean aquaculture, but there is still a lack of information regarding its circadian organization. In this study, we have investigated sharpsnout seabream locomotor activity, feeding and plasma melatonin daily rhythms under a 12:12-h LD cycle, as well as the persistence of locomotor activity circadian rhythmicity under constant light (LL) conditions. When submitted to an LD cycle, most sharpsnout seabream displayed a diurnal locomotor pattern, with an average 74% of activity recorded during daytime. However, along the experiment 40% of fish spontaneously changed their locomotor rhythm phasing and became nocturnal. Feeding behaviour, nevertheless, remained strictly diurnal in all cases, with 97% of food demands being made during the light period. Free-running locomotor rhythms were recorded in one third of the fish kept under LL. Daily plasma melatonin levels displayed a rhythmic profile, with low daytime values (111 pg/ml) and high nighttime concentrations (791 pg/ml). Taken together, these results evidence a high degree of plasticity for sharpsnout seabream activity patterns, as well as phasing independence of locomotor and feeding rhythms. Finally, the existence of a well defined daily rhythm of plasma melatonin was found. PMID- 16682062 TI - First case of ivermectin-induced severe hepatitis. AB - Loiasis, caused by the filarial parasite Loa loa, is endemic in West and Central Africa. Ivermectin has been shown to be an effective treatment of loiasis. We report the case of a 20-year-old woman originally from Cameroon who was infected by the L. loa parasite and developed severe hepatitis, identified 1 month after a single dose of ivermectin. Liver biopsy showed intralobular inflammatory infiltrates, confluent necrosis and apoptosis, compatible with drug-induced liver disease. To our knowledge, this is the first case of ivermectin-induced severe liver disease published in the literature. PMID- 16682063 TI - TTSV1, a new virus-like particle isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax. AB - A new virus-like particle TTSV1 was isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax sampled at a hot spring region in Indonesia. TTSV1 had a spherical shape with a diameter of approximately 70 nm and was morphologically similar to the PSV isolated from a strain of Pyrobaculum. The 21.6 kb linear double-stranded DNA genome of TTSV1 had 38 open reading frames (ORFs), of which 15 ORFs were most similar to those of PSV. The remaining 23 ORFs showed little similarity to proteins in the public databases. Southern blot analysis demonstrated that the viral genome is not integrated into the host chromosome. TTSV1 consisted of three putative structural proteins of 10, 20, and 35 kDa in size, and the 10-kDa major protein was identified by mass spectrometry as a TTSV1 gene product. TTSV1 could be assigned as a new member of the newly emerged Globuloviridae family that includes so far only one recently characterized virus PSV. PMID- 16682065 TI - AFM study on the sorbed NOM and its fractions isolated from River Songhua. AB - Natural organic matter (NOM) and its fractions from River Songhua were concentrated and fractionated with the reverse osmosis technology and XAD resin. With the purpose of exploring the adsorbed morphology of NOM and its fractions from a specific water source, the tapping-mode atomic force microscopy (TMAFM) was employed. The effect of solution properties (pH, Ca(2+)) on NOM microtopography was also verified. The results showed that NOM and its fractions displayed obviously diverse morphologies due to their different characteristics. The macromolecular compounds with large SUVA value were prone to adsorb on the mica surface. The particle-shaped NOM macromolecules were loosely adsorbed on the mica surface, and the molecular aggregation phenomena were proved to be ubiquitous in this experimental condition. With the increase of pH value, the surface coverage of mica decreased evidently. Spherical conformation with relatively smaller diameters was observed in the acidic condition compared with the neutral and basic condition. In the presence of 0.1 mol L(-1) NaCl, aggregation phenomena were observed in all pH conditions and obvious aggregates were presented with the addition of only 5 mmol L(-1) CaCl(2). PMID- 16682064 TI - Evolution of virus-derived sequences for high-level replication of a subviral RNA. AB - Turnip crinkle virus (TCV) and its 356-nt satellite RNA satC share 151 nt of 3' terminal sequence, which contain 8 positional differences and are predicted to fold into virtually identical structures, including a series of four phylogenetically inferred hairpins. SatC and TCV containing reciprocal exchanges of this region accumulate to only 15% or 1% of wild-type levels, respectively. Step-wise conversion of satC and TCV 3'-terminal sequences into the counterpart's sequence revealed the importance of having the cognate core promoter (Pr), which is composed of a single hairpin that differs in both sequence and stability, and an adjacent short 3'-terminal segment. The negative impact of the more stable TCV Pr on satC could not be attributed to lack of formation of a known tertiary interaction involving the 3'-terminal bases, nor an effect of coat protein, which binds specifically to TCV-like Pr and not the satC Pr. The satC Pr was a substantially better promoter than the TCV Pr when assayed in vitro using purified recombinant TCV RdRp, either in the context of satC or when assayed downstream of non-TCV-related sequence. Poor activity of the TCV Pr in vitro occurred despite solution structure probing indicating that its conformation in the context of satC is similar to the active form of the satC Pr, which is thought to form following a required conformational switch. These results suggest that evolution of satC following its initial formation generated a Pr that can function more efficiently in the absence of additional TCV sequence that may be required for full functionality of the TCV Pr. PMID- 16682066 TI - The role of activated carbon on the removal of p-nitrophenol in an integrated three-phase electrochemical reactor. AB - Three-phase electrochemical reactor is still far from concerned in wastewater treatment in order to improve electrochemical treatment efficiency especially when the concentrations of organic pollutants are relatively low. This paper presents a novel process integrated electrocatalysis and activated carbon (AC) adsorption in a fluidization mode for p-nitrophenol (PNP) abatement, with special attention on probing the role of AC. Sparged by external gas (e.g., O(2)), the electrochemical reactor is actually a three-phase (gas, liquid, solid) reactor. By this one-step integrated process, the treatment efficiency was significantly promoted where PNP of initial concentration 150 mg l(-1) could be completely removed in no more than 30 min and it kept good performance for five consecutive runs, showing potential application for environmental remediation. In the integrated process, AC is in a dynamic state of adsorption and in situ electrochemical regeneration by the attack of electrogenerated hydroxyl radical on organic pollutants. When oxygen is sparged into the process, hydrogen peroxide can be formed by cathodic reduction and then decomposed by catalytic reaction on AC, which further promotes organic pollutants degradation. PMID- 16682067 TI - Effect of farmyard manure application on boron adsorption-desorption characteristics of some soils. AB - Boron (B) availability to crop plants depends on soil properties as well as management practices like liming, fertilization and use of organic manures. To assess the effect of farmyard manure (FYM) application on availability of added B, adsorption-desorption of B was investigated in five different soils receiving varying doses of FYM (0, 5 and 10 g FYM kg(-1) soil). Two surfaces Freundlich model was found best to account for B adsorption-desorption data of all soils. Application of FYM increased B adsorption capacities pertaining to low (K1) and high (K2) concentration ranges in all soils, except Soil C (Alfisol) having a pH of 9.8, in which the higher rate of FYM decreased the value of K2. Application of FYM did not change B desorption capacities of soils corresponding to low B concentration range (K(1)(1)) significantly, however, it increased B desorption capacity pertaining to high B concentration (K(1)(2)) in all soils, except Soils C (Alfisol) and E (Entisol) having pH of 9.8 and 5.1, respectively. Application of FYM increased the desorption slope factor applicable to low concentration range (1/n(1)(1)) in Soil A (Inceptisol), but decreased it in Soil E (Entisol). The 1/n(1)(2) (desorption slope factor applicable to high concentration range) decreased with FYM application in all soils except Soil E (Entisol), where it was increased. Boron desorption index (slope(ads)/slope(des)) decreased with FYM application in low B concentration range, but increased in high concentration range for all soils except soil E (Entisol, pH 5.1), in which a reverse trend was observed. Application of FYM increased the retention of added B in soils and may help reducing the leaching losses. PMID- 16682068 TI - Changes in amino acid profile and metal content in seeds of Cicer arietinum L. (chickpea) grown under various fly-ash amendments. AB - Seeds of Cicer arietinum L. plants are edible and a valuable source of protein. Accumulation of toxic metals in the edible part of the plant, grown in fields close to fly-ash (FA) landfills, may pose a threat to human health. In the present study, the effects of FA and its amendments with different ameliorants viz., garden soil (GS), press mud (PM) and saw dust (SD), on total soluble protein contents, amino acid composition and metal accumulation in seeds were investigated in var. CSG-8962 and var. C-235 of C. arietinum. Plants accumulated adequate amounts of essential metals viz. Fe, Cu, Zn in seeds, while the toxic metals such as Cd and Cr were taken up in smaller quantities. The accumulation of Cr and Cd was less in var. C-235 than var. CSG-8962. Amendment of FA with PM enhanced the amount of soluble protein and amino acids in both varieties and was found to be superior among all tested ameliorants. Both quantitative and qualitative analysis of amino acids showed better response in var. C-235 as compared to var. CSG-8962. Thus var. C-235 seems to be suitable for cultivation in FA contaminated areas due to more accumulation of essential metals and less accumulation of toxic metals in seeds. Application of PM may further improve the growth of plants and nutritional quality of seeds. PMID- 16682069 TI - Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation. AB - Coontail (Ceratophyllum demersum L.) plants when exposed to various concentrations of Pb (1-100microM) for 1-7days, exhibited both phytotoxic and tolerance responses. The specific responses were function of concentration and duration. Plants accumulated 1748mugPbg(-1) dw after 7d which reflected its metal accumulation ability, however most of the metal (1222microgg(-1) dw, 70%) was accumulated after 1d exposure only. The toxic effect and oxidative stress caused by Pb were evident by the reduction in biomass and photosynthetic pigments and increase in malondialddehyde (MDA) content and electrical conductivity with increase in metal concentration and exposure duration. Morphological symptoms of senescence phenomena such as chlorosis and fragmentation of leaves were observed after 7d. The metal tolerance and detoxification strategy adopted by the plant was investigated with reference to antioxidant system and synthesis of phytochelatins. Protein and antioxidant enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) showed induction at lower concentration and duration followed by decline. All enzymes except GPX showed maximum activity after 1d. An increase in cysteine, non-protein thiols (NP-SH) and glutathione (GSH) content was observed at moderate exposure conditions followed by decline. Phytochelatins (PC(2) and PC(3)) were synthesized to significant levels at 10 and 50microM Pb with concomitant decrease in GSH levels. Thus production of PCs seems important for the detoxification of metal, however it may lead to depletion of GSH and consequently oxidative stress. Results suggest that plants responded positively to moderate Pb concentrations and accumulated high amount of metal. Due to metal accumulation coupled with detoxification potential, the plant appears to have potential for its use as phytoremediator species in aquatic environments having moderate pollution of Pb. PMID- 16682070 TI - Acid-base properties of water-soluble organic matter of forest soils, studied by the pK-spectroscopy method. AB - Using the potentiometric titration and pK spectroscopy method, acid-base properties of water-soluble organic matter of forest soils have been studied. Five acidic classes composed of different substances with pK(a) values around 3.6; 4.8; 6.7; 8.7 and 9.7 have been identified. Testing the properties of soluble soil fraction, it is to be taken into account that when it is isolated from non-soluble soil matter, some water-soluble substances remain in soil and do not pass into the solution. Most firmly adsorbed in soil are water-soluble components with pK(a) 9.6-9.8. PMID- 16682072 TI - Is a differentiated advice by season and region necessary? AB - The ultraviolet radiation component in the solar spectrum varies greatly with season. In the summer, the significant short-wavelength (UV-B) radiation at midday can produce erythema in sensitive skin in less than 20 min in middle latitudes, and yet in winter months, the same midday exposure dose would require hours of exposure. The challenge for public health authorities is to provide simple, understandable messages for sensitive individuals to limit excessive exposure at appropriate times of the day during spring and summer months and yet not to take needless precautions or limit exposure during fall and winter months at mid and circumpolar latitudes. The appropriate exposure for beneficial effects is not possible to achieve at many latitudes during winter months, but is readily achieved in summer months. Simple messages should be tailored to the local times of day, reflecting the locale and season. One simple means to communicate the relative UV-B exposure relates to the length of one's shadow (the "Shadow Rule"). Further challenges are presented when apparently mixed messages would be justified for different skin phototypes. PMID- 16682071 TI - Biomarkers responses of the earthworm Eisenia fetida to acetochlor exposure in OECD soil. AB - To examine the potential of a suite of biomarkers as early warning indicators of environmental pollution, sperm count, neutral red retention time (NRRT) and DNA damage were measured in earthworm Eisenia fetida exposed to increasing concentrations of acetochlor in OECD soil. The neutral red retention time of earthworms coelomocytes was sensitive to acetochlor pollution, and decreased significantly when the concentration was more than 10mgkg(-1) after 30 and 60 days of exposure (P<0.05). The reduced neutral red retention time correlated with the soil acetochlor residual. Sperm count decreased significantly at the concentrations of 40 and 80mgkg(-1) after 15 days of exposure (P<0.05). The DNA damage of earthworms coelomocytes increased significantly after 30 days of exposure at the highest concentration (80mgkg(-1); P<0.05). Earthworms were under physiological stress at field dose of acetochlor (10mgkg(-1)). Higher concentrations of acetochlor caused sperm count decrease and DNA damage of earthworms. Such a suite of biomarkers could serve as indicators of the health of the soil environment and to evaluate the toxicity of acetochlor on earthworms or as a means of monitoring soil acetochlor pollution. PMID- 16682073 TI - Reinstatement of conditioned reinforcing properties of cocaine-conditioned stimuli. AB - The aim of the present experiment was to investigate the effects of cocaine primes and exposure to foot shock stress on reinstatement of operant responding maintained by a cocaine-conditioned stimulus in rats never trained to actively self-administer cocaine. Following a baseline session of responding for a light buzzer compound stimulus, rats underwent classical conditioning whereby the compound stimulus was paired with passive intravenous infusions of cocaine (vehicle, 0.5 or 1.0 mg/kg/inf). On subsequent test sessions, operant responding for the compound stimulus was re-assessed in the absence of cocaine. Finally, rats received a cocaine prime (20 mg/kg, i.p.) and foot shock stress prior to two separate test sessions assessing lever pressing for the cocaine-conditioned stimulus. It was found that the animals conditioned with cocaine displayed sustained responding on the lever activating the cocaine-conditioned stimulus. In addition, priming injections of cocaine reinstated responding for the light buzzer compound stimulus, and this effect was proportional to the dose of cocaine received during classical conditioning. Foot shock stress also reinstated responding, but its effect was smaller and observed only in animals conditioned with the highest dose of cocaine. These findings suggest that cocaine primes and stress can induce reinstatement by reactivating the motivational value of cocaine conditioned cues. PMID- 16682074 TI - Stepwise modification of lysine residues of glucose oxidase with citraconic anhydride. AB - Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213-216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure. PMID- 16682075 TI - Nanocomposites of hydroxyapatite with aspartic acid and glutamic acid and their interaction with osteoblast-like cells. AB - The direct synthesis of hydroxyapatite (HA)-aspartic acid (ASP) and HA-glutamic acid (GLU) nanocrystals was carried out in presence of different amounts of the amino acids in solution. ASP and GLU incorporation into HA crystals reduces the coherent length of the perfect crystalline domains along the long dimension (002) and, even more, along the cross section (310) of the apatite crystals, suggesting a specific interaction of the amino acids with the HA structure. FTIR analysis indicates that the carboxylic groups of the acidic amino acids interact with the calcium ions of HA. The relative amount of ASP incorporation into HA nanocrystals is greater than that of GLU, suggesting a greater affinity of ASP for HA. Osteoblast-like, MG63, cells cultured on the composite nanocrystals display good proliferation and increased values of ALP activity, collagen type I, TGF-betaI and osteocalcin production, indicating that the presence of the acidic amino acids enhances osteoblast activation and extra-cellular matrix mineralization processes. PMID- 16682076 TI - Comparative analysis of hypermethylation of cell cycle control and DNA-mismatch repair genes in low-density and CD34+ bone marrow cells from patients with myelodysplastic syndrome. AB - Hypermethylation of CpG islands within the promoter region is one of the mechanisms by which genes are inactivated and may be one of the reason for silencing of cell cycle control or DNA-mismatch repair genes in myelodysplastic syndrome (MDS). Since the function of cell cycle control genes including the cyclin-dependent kinase inhibitors known as p15(INK4b) and p16(INK4a), as well as p14(ARF) which blocks MDM-2 (an inhibitor of p53), the retinoblastoma (RB1) protein and the mismatch repair gene MGMT is critical for hematopoietic proliferation and differentiation, we performed methylation specific polymerase chain reaction (MSP) in low-density, non-adherent bone marrow cells from 49 patients with MDS. In addition, expression of p15(INK4b) and RB1 was analysed by quantitative real-time PCR. From selected patients, we analyzed the methylation pattern of cell cycle control genes in CD34+ bone marrow cells. Thirty-nine of 49 cases (80%) had at least one of five genes methylated in our MDS samples by analysing low-density non-adherent bone marrow cells. The frequency of p15(INK4b) methylation was 34 of 49 samples (69%). The incidence of methylation of both p14(ARF) and p16(INK4a) was four of 49 (8%). RB1 gene was methylated in seven samples (14%) and each patient had RA. Interestingly, none of these genes were methylated in the purified CD34+ hematopoietic stem cells from the MDS patients. Furthermore, all our RARS patients had a methylated p15(INK4b) promoter correlating with non-detectable expression of this gene in bone marrow cells from those patients. These results indicate that hypermethylation of cell cycle control genes in MDS may occur late during the differentiation of myelodysplastic stem cells. PMID- 16682077 TI - Activity of AMN107, a novel aminopyrimidine tyrosine kinase inhibitor, against human FIP1L1-PDGFR-alpha-expressing cells. AB - Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by tissue involvement and organ dysfunction due to abnormal eosinophil proliferation. In a subset of patients, this is caused by the FIP1L1 PDGFR-alpha fusion tyrosine kinase. Cumulative evidence indicates that the Bcr Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec) is active for the treatment of patients with HES, particularly those expressing the FIP1L1-PDGFR alpha oncoprotein. The novel tyrosine kinase inhibitor AMN107 was initially developed as a potent Bcr-Abl inhibitor based on the molecular structure of imatinib. We tested the in vitro efficacy of imatinib and AMN107 in the EOL-1 cell line and in cells from a patient with HES harboring the FIP1L1-PDGFR-alpha fusion kinase. AMN107 was as potent as imatinib in inducing apoptosis and inhibiting proliferation of EOL-1 cells, with IC(50) values of 0.54 and 0.20 nM, respectively. In addition, both drugs inhibited the phosphorylation of PDGFR alpha tyrosine kinase with equivalent efficacy. We conclude that AMN107 and imatinib are active and equipotent against cells expressing the FIP1L1-PDGFR alpha fusion gene. PMID- 16682078 TI - Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella. AB - A novel link between development and immunity in insects is introduced. Transiently enhanced expression of lysozyme, gallerimycin and the insect metalloproteinase inhibitor was discovered at the onset of metamorphosis of the greater wax moth, Galleria mellonella. Relative quantification of mRNAs encoding for these antimicrobial peptides using real-time PCR documents their induced expression during transformation of last instar larvae into prepupae and upon injection of either recombinant interstitial collagenase (MMP-1) or small-sized fragments of collagen type IV. The latter were also found to stimulate both nuclear import of c-Rel-proteins in the fat body, implicating activation of Toll or Imd-related signaling pathways, and subsequent synthesis of antimicrobial peptides. Obtained results implicate that degradation of collagen-IV by either microbial metalloproteinases associated with invading pathogens or endogenous matrix metalloproteinases contributing to degradation of extracellular matrix during metamorphosis stimulate innate immune responses. PMID- 16682079 TI - Thigh abscess caused by Eikenella corrodens and Streptococcus intermedius: a case report. AB - A 35-year-old Chinese female has a large thigh abscess without a clear source. No underlying diseases were revealed in the patient. The combination of clindamycin and cefazolin or vancomycin was administered but without a clinical response. Eikenella corrodens and Streptococcus intermedius were isolated from puncture drainage. Then, ceftriaxone was administered and a surgery of incision and further draining was carried. Eventually, the patient recovered. To our knowledge, this is the first case of thigh abscess in a previous healthy adult. PMID- 16682080 TI - Registration of in vivo magnetic resonance T1-weighted brain images to triphenyltetrazolium chloride stained sections in small animals. AB - Signal changes observed in high-resolution in vivo magnetic resonance (MR) images acquired during cerebral ischemia in small animal models must be correlated to molecular indicators of tissue damage obtained from digitized histological brain sections. An effective image registration technique that incorporates both a linear and non-linear thin plate spline transform was developed to compensate the distortions that occur in the brain during the extraction, fixation, and staining process. Features in different layers of the brain were utilized in conjunction with a radial guideline-assisted landmark selection method to register tissue layers with few distinguishing characteristics. Quantitative analysis using simulated data demonstrated average registration error of 400 microm (corresponding to approximately 2.5 pixels in the MR images) when > or =50 landmark points are used. Visual agreement was obtained between T(1)-weighted MR images and 2,3,5-triphenyltetrazolium chloride stained histology. These methods will allow accurate registration of in vivo images with histology to correlate in vivo surrogate markers of tissue damage with specific histological indicators of disease. PMID- 16682081 TI - Digit tapping model of functional activation in the rat somatosensory cortex. AB - To establish a non-invasive model for functional activation of the rat somatosensory cortex, the forepaw digits of halothane-anesthetized rats were tapped while the blood flow (laser-Doppler flow, LDF) and somatosensory evoked potential (SSEP) responses in the forelimb area of the somatosensory cortex (S1FL) were measured. The distal phalanges of the forepaw digits were lightly tapped for 10s with an aluminum bar at frequencies between 1 and 40 Hz, with 0.4 cm total bar displacement. The LDF signal was normalized to the baseline preceding each stimulus block and averaged. The LDF response to digit tapping in the contralateral, but not ipsilateral S1FL, commenced within 1s, peaked at 11+/ 0.5% (S.E.M.) above baseline within 2-3s, decreased to a plateau of 5+/-0.3% for the duration of the stimulation, and returned to baseline within 5-10s following tapping cessation. The LDF peak and plateau were not significantly different at different tapping frequencies. In the contralateral, but not ipsilateral, S1FLs, tapping produced an SSEP with positive (P1) and negative (N1) peaks at 27+/-0.5 and 47+/-0.2m s, respectively, after onset of the tap stimulation. As the tapping frequency increased from 1 to 20 Hz, the P1-N1 peak-to-peak amplitude decreased. At 30 and 40 Hz, the shortened interstimulus interval entrained the individual SSEPs into a steady-state evoked response. This study demonstrates that a robust functional activation of the forelimb region of primary somatosensory cortex of halothane-anesthetized rats can be produced by non-invasively tapping the forepaw digits and quantified with LDF and SSEP. PMID- 16682082 TI - Isolation of neural stem cells from the spinal cords of low temperature preserved abortuses. AB - In the present study, we show that neural stem cells can be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C for 2 h, 6 h and 12 h before use. Neural stem cells were isolated from cervical cord, thoracic cord and lumbar/sacral cord separately and induced to differentiate with fetal bovine serum. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Fluorescent immunocytochemistry was used to examine the expression of neural stem cell marker (nestin), neuronal marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation was prolonged to 12 h, the number of primary neurospheres decreased sharply. Neurospheres in all cultures showed nestin positive immunoreactivity and could yield astrocytes and neurons including cholinergic neurons in differential cultures. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for both experimentation and potential therapeutic uses. PMID- 16682083 TI - Preparation of a tissue-like cortical primary culture from embryonic rats using Matrigel and serum free Start V Medium. AB - To address the scientific quest for unravelling signalling pathways crucial in CNS development and function, cell culture systems have to be developed that are mimicking the physiological state of brain cells more efficiently. Here, we describe a method for cultivation of a virtual three-dimensional structure consisting of neural stem cell-derived cell types by using Matrigel as surface substrate and Start V as a serum free medium. We demonstrate that free floating dissociated cells form attached neurospheres from which cells start migration to surrounding areas and develop a virtual three-dimensional cell structure composed of neurons, glia and neural stem cells. Neuronal precursor cells differentiate into cholinergic and GABAergic cells and express vesicle proteins. Further, neuronal cells are interwoven with Nestin positive stem cells and GFAP positive astrocytes. Additionally, oligodendrocytes and microglia can also be detected in this neural tissue-like structure. As an example for studying cell migration we added externally microglial cells (BV2) and performed a confocal time lapse study. It revealed, that co-cultivated microglial cells migrated towards neurospheres within 14 h. Thus, the described method provides a serum free, tissue-like primary cell culture system of neural cells useful for the investigations of basic cell-cell interactions under in vitro conditions. PMID- 16682084 TI - Vaccinal control of Marek's disease: current challenges, and future strategies to maximize protection. AB - Marek's disease is an economically important lymphoid neoplasm of chickens, caused by oncogenic strains of Marek's disease herpesvirus. The disease can be successfully controlled by vaccination with attenuated or non-pathogenic MDV strains. However, vaccine failures do occur as field strains continue to evolve towards pathotypes of greater virulence, and this evolution is likely to be driven by the vaccines themselves. Two general strategies can be considered to improve protection by vaccination. Firstly by the development of novel vaccines, and secondly by maximizing the potential of existing vaccines. This second goal requires investigation of optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. Accurate quantitation of Marek's disease vaccine virus in vaccinated chicks will contribute significantly to our understanding of vaccinal protection. We recently developed a real-time polymerase chain reaction (PCR) assay for quantitation of CVI988 vaccine virus in the feather tips, a rich source of viral DNA which can easily be sampled in a non invasive manner. This PCR test is now used commercially to confirm the successful vaccination of chicks. We have also used the PCR to examine various aspects of vaccination in experimental chicks and commercial chicks with a view to determining how vaccine level in feathers correlates with protection against challenge, and for identifying optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. In this article we review some aspects of the current vaccinal control of Marek's disease, before highlighting some of the problems associated with current vaccines and vaccination strategies, and the challenges for the future. We go on to discuss the development and use of our real-time PCR feather test, its current applications and potential opportunities in Marek's disease vaccine research. PMID- 16682085 TI - Development of cattle TB vaccines in the UK. AB - In 1996, an independent scientific committee chaired by Professor John Krebs, tasked to review the problem of bovine tuberculosis (TB) in GB, concluded that vaccination of cattle offered the best long-term solution for controlling the disease in the National Herd. This view has been re-affirmed recently in the House of Commons Environment, Food and Rural Affairs Committee's report on Bovine TB (2004) and by the findings of the Independent Scientific Group Vaccine Scoping Sub-committee. Significant progress in developing TB vaccines for cattle has been made over the last 5 years. Specifically: (i) DNA or protein subunit vaccines used in combination with BCG have been shown to give superior protection against experimental challenge in cattle than BCG (heterologous prime-boost); (ii) prototype reagents that allow discrimination between vaccinated and infected animals have been developed; and (iii) and correlates of disease severity have been identified that can predict the success or failure of vaccination. These significant advances are detailed in this review with a summary of future directions that TB vaccine development for cattle is likely to take. PMID- 16682086 TI - A minor groove binder probe real-time PCR assay for discrimination between type 2 based vaccines and field strains of canine parvovirus. AB - A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries. PMID- 16682087 TI - Characterisation of the discrepancy between PCR and virus isolation in relation to classical swine fever virus detection. AB - In order to confirm and characterise further the discrepancies observed between diagnostic RT-nPCR and virus isolation results for the detection of classical swine fever virus (CSFV), a test panel of three new RT-PCRs was designed, amplifying parts of the NS2, NS3 and NS5A regions. Screening of negative samples by virus isolation with the new panel not only confirmed the discrepancies previously observed but also indicated that these were not associated with a specific genomic region. However, none of the PCR-positive samples were positive on all the different PCRs and preferential amplification was not obtained even when a more sensitive real-time RT-PCR was used. Furthermore, the primer dependent amplification, most likely caused by the presence of viral fragments, demonstrates the necessity of confirming a single positive PCR result, certainly in the presence of contradictory virus isolation results. The new PCR panel, in combination with sequencing, can be used as a tool to provide additional information on the nature of the viral RNA present in the sample. PMID- 16682088 TI - Energy metabolism of trypanosomatids: adaptation to available carbon sources. AB - Some development stages of the trypanosomatid protozoan parasites are well adapted to in vitro culture. They can be maintained in rich medium containing large excess of glucose and amino acids, which they use as carbon sources for ATP production. Under these growth conditions, carbon sources are converted into partially oxidized end products by so-called aerobic fermentation. Surprisingly, some species, such as the Trypanosoma brucei, Trypanosoma cruzi and Crithidia insect stages, prefer consuming glucose to amino acids, although their natural habitat is L-proline-rich. This review focuses on recent progress in understanding glucose and l-proline metabolism of insect stages, how these metabolic processes are regulated, and the rationale of the aerobic fermentation strategies developed by these parasites. PMID- 16682089 TI - The applicability of the Asian modified criteria of the metabolic syndrome in the Korean population. AB - BACKGROUND: We compared the metabolic profiles and risk of coronary artery disease (CAD) in Koreans with non-diabetic metabolic syndrome (MetS). [We applied four criteria of MetS: the NCEP criteria, the Asian modified NCEP (a-NCEP) criteria, the WHO criteria and the Asian modified WHO (a-WHO).] METHODS: The study group composed of 2724 subjects enrolled in the cardiovascular genome center. There were 728 patients with significant CAD. The different criteria of the MetS were applied for the study population. RESULTS: Among the 2724 participants, 522 (19.2%) met the NCEP criteria, 796 (29.2%) met the a-NCEP criteria, 361 (13.3%) met the WHO criteria and 576 (21.1%) met the a-WHO criteria. The clinical parameters, lipid profile, apoA1 and apoB level were not different between the participants classified as MetS by using the different criteria. The odds ratio for CAD prediction were not significantly different according to the metabolic criteria (odd ratio: 1.755 [95% CI: 1.423-2.163] in NCEP criteria, 2.120 [1.763-2.549] in a-NCEP criteria, 1.854 [1.466-2.343] in WHO criteria, 2.205 [1.810-2.687] in a-WHO criteria). The serum level of apoA1 and apoB showed strong correlations with MetS classified by all criteria and the HOMA index and insulin level showed better correlations with WHO-MetS criteria. CONCLUSIONS: All the MetS criteria showed similar metabolic profiles and all four criteria had similar predictive value for CAD. Conventional MetS criteria, applied to the non-diabetic Asian population, may underestimate the population at risk. Our data suggests that the Asian modified criteria will decrease the risk for underdiagnosis while demonstrating similar metabolic profiles and CAD risk compared to the conventional criteria. PMID- 16682090 TI - Genetic engineering of wheat--current challenges and opportunities. AB - Wheat is one of the major staple food crops grown worldwide; however, productivity in cereal crops has not kept pace with the world population growth. A significant increase in wheat production (>40% by 2020) is needed simply to keep up with the growing demand. This increase is unlikely to be achieved by conventional plant breeding methods because of the limited gene pool available. The application of recombinant techniques to improve wheat quality and yield is not only desirable but also has potential to open up new opportunities. Although there has been significant progress in developing gene-transformation technologies for improving these traits, this remains an important challenge for plant biotechnology. Obstacles to translate the full potential of the genomic era to wheat breeding include the need to develop elite wheat varieties without selectable markers, introducing minimal or nil intergenic DNA and social and market issues concerning genetically engineered food products. PMID- 16682091 TI - AFLP characterization of Southern Europe population of Aspergillus Section Nigri from grapes. AB - Members of Aspergillus belonging to Section Nigri are distributed worldwide and are mainly responsible for the ochratoxin A accumulation in grapes and wine, particularly in Southern Europe. Limited information is available on the species composition and genetic variability of black Aspergilli strains occurring on grapes. We analyzed 283 representative strains from the main wine producing European countries collected in 2001-2002 (Italy, France, Spain, Portugal, Greece and Israel) using amplified fragment length polymorphisms (AFLP) technique. Four main groups were obtained by AFLP clustering analysis of these strains and three of them showed a well defined homogeneous population/species with intraspecific homology higher than 48%: Aspergillus carbonarius (105 strains), Aspergillus tubingensis (69 strains), and Aspergillus "uniseriate" (56 strains) with a similarity less than 20% to the Aspergillus japonicus type strain. The fourth cluster, that we called "A. niger like" (44 strains), showed low homology with A. niger type strain (35%) and high internal heterogeneity. Finally, nine strains could not be assigned readily to any of the type strain of the A. nigri Section. These findings indicate that the Aspergillus Section Nigri strains occurring on grapes in Southern Europe represent a complex of species, and some of these are peculiar to grapes. PMID- 16682092 TI - Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure. AB - Escherichia coli ATCC 25922 in phosphate buffered saline solution (PBS, pH 7.1, 10(8) CFU/ml) was inactivated by high hydrostatic pressure (HHP, 400 to 600 MPa) treatment at 25 degrees C for 10 min. Colonies of E. coli were not detected on non-selective plate count agar immediately after a HHP-treatment of at least 550 MPa. E. coli subjected to at least 500 MPa in PBS were incubated at 4, 25, and 37 degrees C for 120 h. No colonies were detected on plate count agar throughout the 120-h incubation period at 4 or 37 degrees C. In contrast, the number of E. coli during storage at 25 degrees C increased from an undetectable level (< 1 CFU/ml) to the level of initial cell counts regardless of the treatment pressure level. The recovery in PBS required a maximum time of 48 h, while the period during which cell numbers remained at an undetectable level increased from 24 to 72 h as the treatment pressure increased. E. coli treated at 550 and 600 MPa in PBS were inoculated into trypticase soy broth (TSB) and stored at 4, 25, and 37 degrees C for 120 h. No recovery was recorded in TSB during the 120-h storage at 37 degrees C. In contrast, the number of E.coli during storage at 25 degrees C in TSB increased beyond the level of initial cell counts regardless of the treatment pressure level. The recovery of cell numbers observed in TSB was faster than that in PBS samples, as bacterial growth in TSB assisted faster recovery. When the incubation temperature in PBS was shifted to 25 degrees C after 120-h at 4 or 37 degrees C, recovery of E. coli was observed in samples shifted from 4 to 25 degrees C regardless of the treatment pressure. However, the time during which cell numbers remained at an undetectable level was extended by increasing the level of treatment pressure, and recovery required a maximum time of 48 h. On the other hand, no recovery was observed with HHP-treated E. coli subjected to an incubation temperature shift from 37 to 25 degrees C. This study indicates that an appropriate incubation temperature after HHP-treatment is needed to optimize the recovery of HHP-injured bacteria and thus prevent overestimation of the lethal effect of HHP-treatment. PMID- 16682093 TI - Prevalence and characteristics of Escherichia coli O26 and O111 from cattle in Korea. AB - Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome worldwide. E. coli O26 and O111 are the serotypes most frequently isolated from human EHEC infections in Korea. Cattle are considered to be the major sources of E. coli O26 and O111. This study investigated the prevalence of E. coli O26 and O111 in fecal samples from cattle in Korea from April 2002 to March 2004. Out of 809 samples, 54 (6.67%), 37 (4.57%), and 16 (1.98%) tested positive for O26, O111, and both O26 and O111, respectively. Most of the E. coli O26 and O111 strains were isolated from May to October of each year. PCR analysis of the EHEC virulence markers revealed that most of the E. coli O26 and O111 isolates were positive for ehxA, eaeA and stx1 and/or stx2. These results suggest that the majority of Korean E. coli O26 and O111 isolates from cattle can cause serious diseases in humans. PMID- 16682094 TI - Occurrence and preliminary study of antimicrobial resistance of enterococci isolated from dairy goats in Spain. AB - The present study performed in Spain was designed to investigate the occurrence and antimicrobial resistance of enterococci in faecal and bulk tank milk samples from 222 healthy Murciano-Granadina dairy goats reared on 12 farms. Enterococci were isolated in 54.5% and 63.6% of the faecal and bulk tank milk samples, respectively. Enterococci were detected more frequently from goat kids (70%) than from replacement animals (44.4%) and adults (56.4%). Seven species were found in the faecal samples but the most common species detected were Enterococcus faecium (32.3%), Enterococcus faecalis (27.6%) and Enterococcus hiriae (22%). In contrast, only E. faecium and E. faecalis were found in the bulk tank milk samples. According to NCCLS (2002), of the 134 strains studied, 17 (12.7%) were resistant to at least 7 antimicrobials and 14 of these strains were resistant to vancomycin. PMID- 16682095 TI - Regulated readthrough: a new method for the alternative tagging and targeting of recombinant proteins. AB - We report here a new method for the alternative peptide tagging of recombinant proteins from mammalian cell lines. This method, which we called regulated readthrough, exploits the property of aminoglycoside antibiotics to promote translational readthrough of nonsense codons. The basic expression cassette includes a translational fusion between a gene of interest and a membrane targeting peptide, which are separated by a nonsense codon. In the presence of an aminoglycoside antibiotic, translational readthrough is promoted and results in the targeting of the fusion protein to the cell membrane, thus allowing the efficient flow cytometry-based isolation of cells expressing very high levels of recombinant protein. For downstream applications requiring the production of soluble recombinant protein, the cells are cultured in the absence of aminoglycoside, leading to an efficient translational termination. By combining different translation termination signals that exhibit various susceptibilities to aminoglycoside-mediated translational readthrough with flow cytometry capabilities, it is possible to use this technology for other applications such as functional library screening or monitoring the stability of recombinant protein production. PMID- 16682096 TI - FEM simulation of the eye structure with bio-heat analysis. AB - Computer simulation on medical sciences has gain increasing popularity as computational technology advances. Successful thermal modeling of the human eye will assist in enabling early detections of eye abnormalities such as inflammatory. However, validity of every computer simulated results must be benchmarked with experimental measurement and this can be a daunting task especially in biomedical fields where experimental data is not in abundance. This paper presents a 2D finite element (FE) human eye model developed to simulate its thermal steady state conditions based on the properties and parameters reported in the open literatures. The results are verified with experimental and computational results obtained by previous studies on human as well as animal eyes. Results show discrepancy of only 0.33% when compared to images from infrared (IR) screening and a difference of only 0.127% compared to another finite element model. The sensitivity analysis also provides good agreement with results by previous studies. This promising simulation allows new possibility in computational methods for eye health care. PMID- 16682097 TI - Relationship of mother and child food purchases as a function of price: a pilot study. AB - To our knowledge, there are no data on parental influences on child purchasing behavior of healthy or unhealthy foods. Mothers and children in ten families were given 5.00 US dollars to purchase portions of preferred fruits/vegetables and high energy-dense snack foods for each of ten trials of price manipulations. For five of the trials the price of the fruit/vegetable increased in price from 0.50 US dollars to 2.50 US dollars (in 0.50 US dollar increments), while the price of the energy-dense snack food remained constant at 1.00 US dollar. For the remaining five trials, the commodity that previously rose in price remained constant at 1.00 US dollars and the other commodity varied from 0.50 US dollars to 2.50 US dollars. Same-price elasticity was shown for both the child and parent purchases, and parent purchases were significantly related to child purchases of both healthy (regression estimate = 0.46, p < 0.001) and unhealthy (regression estimate = 0.12, p = 0.036) foods. Children's purchases of unhealthy snack food items were positively related to family socioeconomic status, and negatively related to child age. These results indicate that parental food choice and purchasing behaviors may play a role in the development of children's purchasing of both healthy and unhealthy foods. PMID- 16682098 TI - Expanding the concept of parental control: a role for overt and covert control in children's snacking behaviour? AB - The existing literature on parental control and children's diets is confusing. The present paper reports two studies to explore an expanded conceptualisation of parental control with a focus on overt control which 'can be detected by the child' and covert control which 'cannot be detected by the child'. In study 1, 297 parents of children aged between 4 and 11 completed a measure of overt control and covert control alongside ratings of their child's snacking behaviour as a means to assess who uses either overt or covert control and how these aspects of parental control relate to a child's snacking behaviour. The results showed that lighter parents and those with children perceived as heavier were more likely to use covert control and those from a higher social class were more likely to use overt control. Further, whilst greater covert control predicted a decreased intake of unhealthy snacks, greater overt control predicted an increased intake of healthy snacks. In study 2, 61 parents completed the same measure of overt and covert control alongside the three control subscales of the Child Feeding Questionnaire [Birch, L.L., Fisher, J.O., Grimm-Thomas, Markey, C.N., Sawyer, R. (2001). Confirmatory factor analysis of the Child Feeding Questionnaire: A measure of parental attitudes, beliefs and practices about child feeding and obesity proneness. Appetite, 36, 201-210] to assess degrees of overlap between these measures. The results showed that although these five measures of control were all positively correlated, the correlations between the new and existing measures indicated a maximum of 21% shared variance suggesting that covert and overt control are conceptually and statistically separate from existing measures of control. To conclude, overt and covert control may be a useful expansion of existing ways to measure and conceptualise parental control. Further, these constructs may differentially relate to snacking behaviour which may help to explain some of the confusion in the literature. PMID- 16682099 TI - Ethical and legal aspects of enteral nutrition. AB - European ethical and legal positions with regard to EN vary slightly from country to country but are based on a common tradition derived from Graeco Roman ideas, religious thought and events of the 20th century. The Hippocratic tradition is based on 'beneficience' (do good) and 'non-maleficience' (do no harm). Religious thinking is based upon the presumption of providing food and drink by whatever means unless burden outweighs benefit. The concept of 'autonomy' (the patients right to decide) arose following in the decades after the Second World War and is enshrined in Human Rights law. The competent patient has the right to participate in decision making and to refuse treatment although the doctor is not obliged to give treatment which he or she considers futile or against the patient's interests. The incompetent patient is protected by law. The fourth principle is that of 'justice' i.e. equal access to healthcare for all. The law regards withholding and withdrawing treatment as the same. It also defines the provision of food and drink by mouth as basic care and feeding by artificial means as a medical treatment. It requires doctors to act in the best interests of the patient. PMID- 16682100 TI - Pathological consequences to reticuloendothelial system organs following infusion of unstable all-in-one mixtures in rats. AB - BACKGROUND: Recent evidence of lung injury from the infusion of unstable lipid injectable emulsions as all-in-one mixtures (AIOs) was shown in a guinea pig infusion model. METHODS: We extended this research to a Sprague-Dawley rat infusion model, focusing our analyses on the dose of large-diameter fat globules (expressed as the volume-weighted percent of fat larger than 5 or 10 microm, PFAT5 or PFAT10) from the dispersed phase of emulsion mixtures of varying levels of stability, and the potential injurious effects on major tissues of the reticuloendothelial system (i.e., lungs and liver). RESULTS: Two identical infusion experiments (n=13 rats/study), involving stable (s-AIO) vs. unstable (u AIO) mixtures were separately conducted, and differed in two respects: (1) duration of AIO infusion (24 h vs. 72 h) and, (2) starting PFAT5 levels for the u AIO (24- vs. 72-h PFAT5: 0.682+/-0.055% vs. 0.117+/-0.024%, respectively). In both experiments, s-AIOs vs. u-AIOs were infused, and evidence of hepatic oxidative stress was noted by significantly higher tissue concentrations of malondialdehyde (MDA) during infusion of u-AIOs. The higher concentrations of MDA in the livers of animals receiving the u-AIOs were also accompanied by significantly higher plasma concentrations of AST in both infusion experiments suggesting injury. Levels of cytokines (IL-1beta, TNFalpha) in the lungs and livers in both infusion studies were variable. CONCLUSIONS: These results demonstrate the infusion of u-AIOs with starting PFAT5 levels of approximately 0.1% show evidence of pathological consequences to the liver and lungs, and therefore, such unstable AIO mixtures should probably be avoided in the clinical setting. PMID- 16682101 TI - Proteomic analysis of Neisseria lactamica and N eisseria meningitidis outer membrane vesicle vaccine antigens. AB - Vaccines to prevent meningococcal disease have been developed from the outer membrane vesicles (OMVs) of Neisseria meningitidis and the related commensal organism Neisseria lactamica. In addition to lipopolysaccharide and the major porins, these vaccines contain a large number of proteins that are incompletely characterised. Here we describe comparative proteomic analyses of the N. lactamica OMV vaccine and OMVs from a serogroup B strain of N. meningitidis. Tandem mass-spectrometry data for trypsinised N. lactamica OMV vaccine were matched to an incompletely assembled genome sequence from the same strain to give 65 robust protein identifications and a further 122 single- or two-peptide matches. Fifty-seven N. meningitidis K454 proteins were identified robustly (and a further 68 from single- or two-peptide matches) by inference from the N. meningitidis MC58 genome. The results suggest that OMVs have a hitherto unappreciated complexity and pinpoint novel candidate antigens for further characterisation. PMID- 16682102 TI - Executive function deficits in autism spectrum disorders and attention deficit/hyperactivity disorder: examining profiles across domains and ages. AB - Deficits in 'executive function' (EF) are characteristic of several clinical disorders, most notably Autism Spectrum Disorders (ASD) and Attention Deficit/Hyperactivity Disorder (ADHD). In this study, age- and IQ-matched groups with ASD, ADHD, or typical development (TD) were compared on a battery of EF tasks tapping three core domains: response selection/inhibition, flexibility, and planning/working memory. Relations between EF, age and everyday difficulties (rated by parents and teachers) were also examined. Both clinical groups showed significant EF impairments compared with TD peers. The ADHD group showed greater inhibitory problems on a Go-no-Go task, while the ASD group was significantly worse on response selection/monitoring in a cognitive estimates task. Age-related improvements were clearer in ASD and TD than in ADHD. At older (but not younger) ages, the ASD group outperformed the ADHD group, performing as well as the TD group on many EF measures. EF scores were related to specific aspects of communicative and social adaptation, and negatively correlated with hyperactivity in ASD and TD. Within the present groups, the overall findings suggested less severe and persistent EF deficits in ASD (including Asperger Syndrome) than in ADHD. PMID- 16682103 TI - Simultaneous masking in a dichotic emotion detection task. AB - The present study investigated the possible role of ceiling effects in producing laterality effects of small magnitude in dichotic emotion detection. Twenty two right-handed undergraduate students participated in the present experiment. They were required to detect the presence of a target emotion in the expressions tones of happiness, sadness, anger, and neutrality presented dichotically. Stimuli were adjusted to 70 dB and occurred simultaneously with a white noise mask that had an intensity of 65, 70, 80, or 85 dB. Results showed a left ear advantage (LEA) for the 65 dB mask and a right ear advantage for the 85 dB mask, but only after two testing sessions. The possible existence of a generalized right ear bias that might affect the observed LEA for non-verbal tasks is discussed. Alternative explanations and limitations of the present experiment are also presented. PMID- 16682104 TI - A continuity between bipolar II depression and major depressive disorder? AB - BACKGROUND: A recent series of studies has questioned the current categorical split of mood disorders into bipolar and depressive disorders. Mixed states, especially mixed depression (i.e., depression plus co-occurring, noneuphoric, hypomanic symptoms) might support a continuity between bipolar II (BP-II) depression and major depressive disorder (MDD). The aim of the study was to assess the distribution of intradepressive hypomanic symptoms rating between BP II and MDD depressions. A bi-modal distribution would support a categorical distinction, and no bi-modality would support continuity. METHODS: Consecutive 389 BP-II and 261 MDD major depressive episode (MDE) outpatients were interviewed (off psychoactive drugs) with the Structured Clinical Interview for DSM-IV, the Hypomania Interview Guide (HIG, to assess intradepressive hypomanic symptoms), and the Family History Screen, by a mood specialist psychiatrist in a private practice. Mixed depression was defined as MDE plus 3 or more intradepressive, noneuphoric hypomanic symptoms, a definition validated by Akiskal and Benazzi. The distribution of intradepressive hypomanic symptoms rating was studied by Kernel density estimate and by histogram. RESULTS: BP-II depression, versus MDD depression, had significantly lower age at onset, was significantly more likely to be atypical and mixed, had more depression recurrences, and a higher bipolar family history loading. BP-II depression, versus MDD depression, had significantly more irritability, racing/crowded thoughts, distractibility, psychomotor agitation, talkativeness, increased goal-directed activity, and excessive risky activities. HIG scores were significantly higher in BP-II. The distribution of intradepressive hypomanic symptoms rating showed no bi-modality in the entire depression sample. CONCLUSIONS: Interpretation of study findings relies on the method used to define a categorical disorder. By using classic diagnostic validators (such as family history and age at onset), BP-II and MDD depressions would seem to be distinct disorders. Instead, by using the 'bi modality' approach, a continuity would seem to be supported. Which of these methods for classification is the best has yet to be shown. PMID- 16682105 TI - Examination of free radical metabolism and antioxidant defence system elements in patients with obsessive-compulsive disorder. AB - Free radicals and oxidative stress are involved in the pathogenesis of various diseases. Malondialdehyde (MDA) is a natural product of lipid peroxidation in all mammalian cells. Vitamins C and E are nonenzymatic antioxidant structures. Our study investigated the role of free radicals in the obsessive-compulsive disorder (OCD). The participants were 30 patients with OCD that were drug-free at least for a month and a control group of 30 healthy subjects, matched with respect to age and sex. In both groups, the levels of erythrocyte malondialdehyde and the plasma vitamin C and E concentrations were measured. The levels of malondialdehyde were significantly higher in the patient group than in the control group (p<.01). The levels of plasma vitamin E were significantly lower in the patient than in the control group (p<.02). Although our patient group had slightly lower concentrations of plasma vitamin C compared to our control group, the difference between these two groups was not statistically significant. There was a significant correlation between increasing malondialdehyde levels and decreasing vitamin E concentrations. This study shows the presence of a significant relationship of OCD and oxidative stress, and consequently, an involvement of free radicals and of the antioxidant defence. Biochemical studies may contribute to the understanding of OCD and its treatment. PMID- 16682106 TI - Can rodents be used to model interferon-alpha-induced depressive symptoms? PMID- 16682107 TI - Quetiapine-induced neuroleptic malignant syndrome in dementia with Lewy bodies: a case report. AB - Patients with dementia with Lewy bodies (DLB) are particularly vulnerable to adverse effects of neuroleptics; this sensitivity is included among the clinical diagnostic criteria for DLB. Recently atypical neuroleptics, which carry less risk of extrapyramidal side effects than typical agents, have come into increasing use in treating psychotic symptoms and behavioral disturbances related to DLB. The present report is the first to describe a DLB patient who developed neuroleptic malignant syndrome (NMS) induced by quetiapine, an atypical neuroleptic known to have relatively infrequent extrapyramidal side effects in DLB patients. Physicians should be aware of the possibility of the occurrence of NMS in DLB even when atypical neuroleptics are administered. PMID- 16682108 TI - Protection of renal tubular cells against the cytotoxicity of cadmium by glycine. AB - Glycine treatment is reported to protect against the nephrotoxicity of cadmium (Cd) in rats. The purpose of the present study was to explore the mechanism of this protection using a renal epithelial cell line, LLC-PK(1). The cells were incubated with 10-30 microM Cd in serum-free DMEM and cytotoxicity was evaluated by LDH leakage into the incubation medium. Under these conditions, 20 and 30 microM Cd concentrations were cytotoxic. As compared to the non-Cd-exposed cells, the LDH release was elevated more than six-fold in cells exposed to 30 microM Cd for 24h. Co-treatment with 5-50mM glycine was cytoprotective in a concentration dependent manner. Prior treatment with 50 mM glycine for 16 h, or co-treatment for 24h, reduced LDH leakage due to 30 microM Cd exposure by 60 and 66%, respectively. Co-incubation with 50 mM alanine was also protective but only about half as effective as with glycine. During the first 4h, prior to the onset of any significant cell membrane damage, the Cd-exposed cells accumulated 0.55 microg Cd/mg protein. Glycine pre-treatment or co-treatment reduced Cd accumulation by about one-quarter or one-half, respectively. To delineate the mechanism of glycine's effect on Cd accumulation, the efflux of Cd was studied after a 30 min pulse exposure. The results suggested that pre-treatment reduced Cd accumulation by increasing its efflux from the cells. In contrast, co-treatment reduced Cd efflux, suggesting that the co-treatment lowered Cd accumulation by suppressing its uptake. When co-incubated, Cd and glycine formed a complex that was apparently responsible for the marked reduction in Cd uptake. It is concluded that, regardless of the mode of treatment, glycine is cytoprotective against Cd and that it may do so by lowering the intracellular Cd burden. PMID- 16682109 TI - Amyloid beta and neuromelanin--toxic or protective molecules? The cellular context makes the difference. AB - Alzheimer's disease (AD) and Parkinson's disease (PD) share several pathological mechanisms. The parallels between amyloid beta (Abeta) in AD and alpha-synuclein in PD have been discussed in several reports. However, studies of the last few years show that Abeta also shares several important characteristics with neuromelanin (NM), whose role in PD is emerging. First, both molecules accumulate with aging, the greatest risk factor for AD and PD. Second, in spite of their different structures, Abeta and NM have similar characteristics that could also lead to neuroprotection. Metals are required to catalyze their formation and they can bind large amounts of these metals, generating stable complexes and thus playing a protective role against metal toxicity. Moreover, they may be able to remove toxic species such as oligopeptides and excess cytosolic dopamine. Third, both Abeta and NM have been implicated in parallel aspects of the neuronal death that underlies AD and PD, respectively. For example, both molecules can activate microglia, inducing release of toxic factors such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO). A careful analysis of these parallel effects of Abeta and NM, including their seemingly paradoxical ability to participate in both cell death and protection, may lead to an improved understanding of the roles of these molecules in neurodegeneration and also provide insights into possible parallels in the pathological mechanisms underlying AD and PD. PMID- 16682110 TI - A population-based study of 518 very preterm neonates from high-risk pregnancies: prognostic value of umbilical and cerebral artery Doppler velocimetry for mortality before discharge and severe neurological morbidity. AB - OBJECTIVE: To evaluate in everyday practice the predictiveness of fetal umbilical artery and cerebral artery Doppler examination for mortality before discharge and for severe neurological morbidity among very preterm neonates from high-risk pregnancies. METHODS: Data came from a population-based study (EPIPAGE) of all births before 33 weeks' gestation during 1 year in nine French regions. We examined the prognostic value of Doppler findings among the liveborn singletons delivered after pregnancies with maternal hypertension or antenatal suspicion of small-for-gestational-age status. RESULTS: This study included 518 fetuses. Predischarge mortality for infants with abnormal umbilical artery Doppler findings was not significantly higher than for those with normal findings. Mortality for infants with abnormal cerebral artery Doppler findings was significantly higher in the bivariate analysis (crude OR: 3.5 (1.6-7.4)). After adjustment, the association between mortality and abnormal cerebral artery Doppler findings remained significant in the subgroup with an abnormal umbilical artery Doppler assessment (OR: 5.1 (1.1-23)). There was no significant association between neurological morbidity and Doppler findings. CONCLUSION: The prognostic value of Doppler examinations appears lower in this study than in previous hospital series. This suggests the need for quality control and improvements in these examinations. PMID- 16682111 TI - Ultrasonographic cervical length measurement at 10-14 and 20-24 weeks gestation and the risk of preterm delivery. AB - OBJECTIVE: To compare cervical length measurements at 10-14 and 20-24 weeks gestation in asymptomatic women with singleton pregnancies and to assess the measurements as a predictor of preterm delivery. STUDY DESIGN: In this prospective study, cervical length was measured in 152 asymptomatic women with singleton pregnancies using transvaginal ultrasonography at 10-14 and 20-24 weeks gestation. The primary outcome measure was spontaneous preterm delivery before 35 weeks of gestation. The mean cervical length was calculated at both stages, and lengths were compared between the term and preterm groups. RESULTS: The rate of spontaneous preterm deliveries was 10.5%. The mean cervical length at 10-14 and 20-24 weeks was 40.5 and 37.1mm, respectively. The cervical length at 10-14 weeks was not significantly different between those who delivered at term (40.9 mm) and those who delivered preterm (38.6 mm). By contrast, the cervical length at 20-24 weeks was significantly shorter in the group that had preterm deliveries (28.4 mm) than in those who had term deliveries (37.8 mm) (P < 0.001). The cervical shortening was more apparent in the group that delivered prematurely (from 38.6 to 28.4 mm) than in that which delivered at term (from 40.9 to 37.8 mm). CONCLUSION: Cervical length measurement used to predict preterm delivery was found to be more predictive at 20-24 weeks. Cervical length measurement at 10-14 weeks was not reliable for predicting preterm delivery. The mean cervical length tapered gradually from the first to the second scan, and the more rapid cervical shortening was found to be associated with increased risk for preterm delivery. PMID- 16682112 TI - Atherosclerosis in women with endometriosis. AB - OBJECTIVE: This study aims to determine whether women with endometriosis have greater subclinical atherosclerosis than the general population. STUDY DESIGN: This case-control study included 66 women with endometriosis and 66 controls matched for age and body mass index. All subjects were >or=35 years old. Exclusion criteria were obesity, diabetes, hypertension, hyperlipidemia, renal or metabolic diseases. Before laparoscopy, all patients underwent a measurement of intima-media thickness (IMT) and distensibility coefficient (DC) on the common carotid artery. In addition, blood samples were taken to determine the levels of lipids, fibrinogen, C-reactive protein, homocysteine, fasting glycemia, antithrombin III, plasminogen, protein C, protein S, and activated protein C resistance. RESULTS: All the biochemical parameters evaluated had similar levels in the two study groups. IMT was similar in women with endometriosis and in controls both on left (p=0.330) and right (p=0.648) carotid artery. Similarly, no significant difference was observed in the DC between women with endometriosis and controls both on left (p=0.539) and right (p=0.178) carotid artery. No significant difference was observed in IMT and DC between women with mild and severe endometriosis. CONCLUSION: Women with endometriosis do not have more subclinical atherosclerosis than the general population. PMID- 16682113 TI - Elements of a cost-benefit analysis for improving salmonid spawning habitat in the River Wye. AB - Contingent valuation methods were used to derive an economic value for salmonid spawning habitat restoration. Anglers' willingness to pay for such restoration and farmers' willingness to accept compensation for lost productivity due to the restoration were investigated using questionnaire and face-to-face surveys, respectively. These elements were combined with the Wye Habitat Improvement Project (WHIP) budget into a cost-benefit analysis (CBA) based upon direct use values only. The CBA assumed that the WHIP will improve salmonid stock. Different levels of investment were considered and the CBA gave a positive net present value indicating an economically viable project and benefit-to-cost ratios greater than one for minor projects. However, the number of anglers using the Wye would need to double or triple in order to justify the full WHIP budget. Management of an environmental resource must consider users' and stakeholders' needs and opinions. It was found that anglers on the Wye value habitat/scenery in its own right and that this played an important part in the activity. Farmers' agreement regarding involvement in such projects was more likely to be determined by their political and ethical views than receiving an economic compensation. PMID- 16682114 TI - Influence of alkalinity, hardness and dissolved solids on drinking water taste: A case study of consumer satisfaction. AB - Two surveys of consumer satisfaction with drinking water conducted by Taiwan Water Supply Corp. are presented in this study. The study results show that although a lot of money was invested to modify traditional treatment processes, over 60% of local residents still avoided drinking tap water. Over half of the respondents felt that sample TT (from the traditional treatment process) was not a good drinking water, whether in the first or second survey, whereas almost 60% of respondents felt that samples PA, PB, CCL and CT (from advanced treatment processes) were good to drink. For all drinking water samples, respondent satisfaction with a sample primarily depended on it having no unpleasant flavors. Taiwan Environmental Protection Administration plans to revise the drinking water quality standards for TH and TDS in the near future. The new standards require a lower TH concentration (from currently 400mg/L (as CaCO(3)) to 150mg/L (as CaCO(3))), and a lower TDS maximum admissible concentration from the current guideline of 600 to 250mg/L. Therefore, this study also evaluated the impacts on drinking water tastes caused by variations in TH and TDS concentrations, and assessed the need to issue more strict drinking water quality standards for TH and TDS. The research results showed that most respondents could not tell the difference in water taste among water samples with different TDS, TH and alkalinity. Furthermore, hardness was found to be inversely associated with cardiovascular diseases and cancers, and complying with more strict standards would lead most water facilities to invest billions of dollars to upgrade their treatment processes. Consequently, in terms of drinking water tastes alone, this study suggested that Taiwan Environmental Protection Administration should conduct more thorough reviews of the scientific literature that provides the rationale for setting standards and reconsider if it is necessary to revise drinking water quality standards for TH and TDS. PMID- 16682115 TI - Activation of mitogen-activated protein kinase (MAPK) by GnRH is cell-context dependent. AB - The interaction of GnRH with its cognate receptor (GnRHR) in pituitary gonadotropes includes activation of Gq/G11 and phospholipase Cbeta (PLCbeta), which generates the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which are required for Ca2+ mobilization and PKC isoforms activation. Activation of PKC in pituitary gonadotropes leads to the activation of the major members of the mitogen-activated protein kinase superfamily (MAPK), namely: extracellular signal-regulated kinase (ERK), jun-N-terminal Kinase (JNK) and p38MAPK. The above pathways mediate GnRH-induced gonadotropin release and synthesis. Here we summarise the diverse mechanisms utilized by GnRH to activate the MAPK members and show that they depend on "cell-context". PMID- 16682116 TI - Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein beta confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models. AB - Steroidogenic acute regulatory protein (StAR) mediates translocation of cholesterol to the inner membranes of steroidogenic mitochondria, where it serves as a substrate for steroid synthesis. Transcription of StAR in the gonads and adrenal cells is upregulated by trophic hormones, involves downstream signaling pathways and a cohort of trans-factors acting as activators or suppressors of StAR transcription. This study suggests that a 21 basepair long sequence positioned at -81/-61 of the murine StAR promoter is sufficient to confer a robust hormonal activation of transcription in ovarian granulosa cells treated with FSH. We show that recombinant GATA-4 and CCAAT/enhancer-binding protein beta (C/EBPbeta) bind to the promoter at -66/-61 and -81/-70 and activate transcription of a reporter gene when co-expressed in heterologous human embryonic kidney 293 (HEK293) cells. In this cell model, C/EBPbeta and GATA-4 synergize in a sequence dependent manner and p300/CBP further maximizes their joint activities. Inhibitors of the transcriptional activators, such as liver enriched inhibiting protein (C/EBPbeta-LIP), Friend of GATA-4 (FOG-2) protein and the viral E1A protein abolished the respective factor-dependent activities in HEK293 cells. Binding assays suggest that a dual binding of C/EBPbeta and GATA-4 to the promoter depends on the molar ratio of the factors present while demonstrating GATA-4 predominant association with the promoter DNA. This pattern may reflect on StAR expression at the time of corpus luteum formation when C/EBPbeta levels peak, as does StAR expression. PMID- 16682117 TI - Synthesis of a quaternary amine anion exchange resin and study [of] its adsorption behaviour for chromate oxyanions. AB - Glycidyl methacrylate/N,N'-methylene bis-acrylamide (GMA/MBA) was prepared and allowed to react with tetraethylenepentamine (TEP) to give glycidyl methacrylate amine resin (RPA) followed by treatment with glycidyl trimethylammonium chloride (GTA) to give glycidyl methacrylate resin bearing quaternary ammonium chloride moieties (RQA). Zeta potential measurements showed that RQA particles are positively charged over pH 2-10 indicating the strong basic nature of the quaternary amine sites. The effect of pH on the recovery of chromate by RPA and RQA was examined. The results indicated that RQA is an efficient sorbent for chromate from both acidic and basic media. The repeated use of RQA was tested through stripping the adsorbed chromate using a mixture of 0.05 NaOH and 2 M NaCl in the case of the uptake from acidic media and using 2 M NaCl solution in the case of alkaline solutions. PMID- 16682118 TI - No association of 5-HT2C, 5-HT6, and tryptophan hydroxylase-1 gene polymorphisms with personality traits in the Japanese population. AB - Serotonin 2C receptor (5-HT(2C)), serotonin 6 receptor (5-HT(6)), and tryptophan hydroxylase-1 (TPH1) genes could be candidates for personality-related genes considering the role of serotonin in various mental functions and behavior. However, a limited number of studies have investigated the association between these genes and personality traits. In the present study, we investigated the three serotonin-related genes, 5-HT(2C), 5-HT(6), and TPH1 genes, in relation to personality traits in the Japanese population. The Cys23Ser polymorphisms in the 5-HT(2C) gene, the 267T/C polymorphism of the 5-HT(6) gene, and the 779A/C polymorphisms in the TPH1 gene were genotyped in 253 healthy Japanese subjects. Personality traits were evaluated by using the Revised NEO Personality Inventory (NEO PI-R) and the State-Trait Anxiety Inventory (STAI). As a result, no significant association was observed between the polymorphisms and the NEO PI-R or the STAI scores. The present results did not provide evidence for the association between the three serotonin-related genes and personality traits. The genes might not have major role in the development of personality traits, although further investigation with larger sample size may be recommended for conclusion. PMID- 16682119 TI - Individual differences in sucrose intake and preference in the rat: circadian variation and association with dopamine D2 receptor function in striatum and nucleus accumbens. AB - Measurement of sucrose intake or preference is currently in widespread use in preclinical psychopharmacology, and used for predicting sensitivity to rewards, but limited information is available about the consistency of individual sucrose intake or preference. In the present study, individual differences in sucrose intake and preference in free-feeding rats were studied during the dark and light phases, and associations of these measures with the function of D(2) receptors in the striatum and nucleus accumbens were characterized. Altogether eight two bottle tests were carried out intermittently during light and dark phase. Ten days after the last test, animals were sacrificed. Intake, and to a lesser degree preference of sucrose during the dark phase were higher as compared to the light phase, and sucrose intake, but not preference was individually very consistent across different tests, especially during the dark phase. The average dark phase sucrose intake and preference correlated positively with dopamine-dependent [(35)S]GTPgammaS binding in nucleus accumbens. Dopamine-dependent [(35)S]GTPgammaS binding in striatum correlated negatively with sucrose preference in the first test. This study has demonstrated that sucrose intake is an individually stable trait, especially when measured during the dark phase, and persistent individual differences in sucrose consumption and possibly reward sensitivity in general are related to dopamine D(2) receptor function in the nucleus accumbens. Individual differences in D(2) receptor function in the striatum may influence behaviour of rats in novel situations. PMID- 16682120 TI - Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM. AB - Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria. PMID- 16682121 TI - The natural hosts of larvae and nymphs of Amblyomma tigrinum Koch, 1844 (Acari: Ixodidae). AB - The hosts of larvae and nymphs of Amblyomma tigrinum, a tick whose adults feed on wild and domestic Canidae in South America, are uncertain. A 17 months survey was carried out trapping wild vertebrates in north-western Cordoba, Argentina, to evaluate their parasitism with A. tigrinum subadults. Larvae and nymphs of this tick species were identified conventionally and by comparison of 16S rDNA sequences with GenBank deposited sequences. A total of 207 small and medium-sized rodents and 182 birds were captured and examined for ticks. Most ticks on birds were from ground forest feeding birds (BB) with a minimal contribution of birds feeding in open pastures. All ticks from rodents were obtained from representatives of the families Cricetidae (SR) and Caviidae (MR). Percent of larvae infestation was higher (P<0.01, Chi-square distribution) in BB (55.2%) and SR (46.4%) than in MR (17.4%) and the same trend was found for number of larvae on these hosts (test of Kruskal-Wallis). Caviidae (only representative Galea musteloides) rodents were extremely prone to be infested with nymphs of A. tigrinum (94.2%) followed by BB (50.6%) and SR (3.6%) (P<0.01) and the same tendency was found for number of nymphs (P<0.01). The index of aggregation for nymphs on MR was the lowest (0.409) followed by nymphs on BB (0.706) which may be a consequence of higher and homogenous exposure of G. musteloides to host-seeking nymphs. Several BB are food source for both larvae and nymphs of A. tigrinum while for rodents larvae were common only on SR (mainly on the Sigmodontinae Akodon dolores and Graomys sp.) and nymphs feed almost exclusively on MR. Therefore, both birds and rodents are of importance for the survival strategy of A. tigrinum subadults. The plasticity of A. tigrinum to colonize areas with different climates plus the capacity of their subadults to feed on hosts widely distributed indicates that this tick has the potential to become a widespread parasite but this does not seem to be the actual situation. Several proposals are presented to further understand its ecology. PMID- 16682122 TI - PCR-based detection of Theileria ovis in Rhipicephalus bursa adult ticks. AB - Tick-borne diseases in ruminants are common in tropical and subtropical regions and lead to meat and milk production losses. In this study, polymerase chain reaction (PCR) was used to assess the presence of Theileria ovis in Rhipicephalus bursa ticks. We have demonstrated that the PCR enabled detection of T. ovis in field isolates of R. bursa collected from naturally infested sheep and goats in eastern Turkey. The sampling was done in spring season (between May and June 2004). A total of 420 R. bursa were collected and randomly selected 192 number of them (97 female and 95 male) were dissected. Primers specific for 520 bp fragments small subunit ribosomal RNA (ssu rRNA) gene of T. ovis amplified products from 37 of the 192 (19.27%) samples. The parasite was detected in 17 (17.52%) female and in 20 (21.05%) male ticks. Two T. ovis amplicons from the tick samples were purified and sequenced. The resulting sequences were identical to the nucleotide sequence of the Turkish sheep strain of T. ovis. These results showed that R. bursa might play an important role in the field as a natural vector of T. ovis. PMID- 16682123 TI - Cyathostomes in horses in Canada resistant to pyrantel salts and effectively removed by moxidectin. AB - Clinical trials using fecal egg count reduction tests and coproculture were conducted with yearlings and mares on a farm in 1997. Fecal samples were taken from each horse to estimate the number of strongyle eggs/g feces with Cornell Wisconsin centrifugal flotation and Cornell-McMaster dilution techniques. Eleven of 15 yearlings, which had been on a daily feeding of grain with pyrantel tartrate for 66 d were found with strongyle eggs in feces. This was the first time the in-feed medication had been used on the farm. Nine yearlings were randomised into three groups; continuation of daily pyrantel tartrate or one treatment with pyrantel pamoate or moxidectin. Two of three yearlings given pyrantel tartrate or pamoate had no reduction in the eggs/g feces. These six yearlings were then given moxidectin and in all yearlings the eggs/g feces was reduced to zero. The 66 d of pyrantel tartrate use was an inadequate time for development of resistant cyathostomes and a hypothesis was the resistance was due to extensive use on the farm over many years of pyrantel pamoate at twice the label dose for control of tapeworms. That hypothesis was tested with 12 mares with strongyle eggs in the feces randomised into two treatment groups: pyrantel pamoate at label dose or moxidectin. Five of six mares given pyrantel had <80% reduction in egg/g feces. These mares were then given moxidectin and in all mares the eggs/g feces was reduced to zero. Only cyathostomes were found on culture and apparently there was side resistance among the pyrantel salts. PMID- 16682124 TI - Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs. AB - Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (> or = 1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR-RFLP can be used to identify Leishmania species in dog tissue imprint stained slides. PMID- 16682125 TI - The establishment rate of a sheep nematode: revisiting classics using a meta analysis of 87 experiments. AB - Strongyle nematode establishment rate in their host is a highly variable life history trait, which makes it difficult to estimate. A meta-analysis was applied to the nematode Teladorsagia circumcincta of sheep in order to acquire a general framework of the factors modulating this life trait. A linear model was built with individual data on 540 infected lambs extracted from 13 articles. Lambs breed and age, time lag between last infection and the interaction between infection mode, infective dose and the number of repeated infective doses were significantly related with the establishment rate. The influence of infection mode on nematode establishment rate was also evaluated by comparing nematode establishment rate distributions within lamb populations infected under different conditions. Natural and repeated experimental infections lead to similar distribution of establishment rate. Conversely, these infection conditions resulted in different parasite establishment rates in average (12.7 and 23.4%, respectively). Three hypotheses are discussed to explain this result: immune protective response, host avoidance behaviour and parasite virulence. PMID- 16682127 TI - Classic Kaposi's sarcoma presenting first with gastrointestinal tract involvement in a HIV-negative Inuit male--a case report and review of the literature. AB - Kaposi's sarcoma (KS) is a multicentric low-grade vascular malignancy. In North America, it is usually seen in AIDS and solid organ transplant populations. Classic KS is a subtype that traditionally occurs in elderly HIV-negative males of Mediterranean, Eastern European, and Jewish descent. Patients with classic KS characteristically present with skin lesions in the distal extremities. Involvement of the viscera is uncommon in classic KS, but may occur in the late stages of the disease. We report the first case of classic KS presenting in the gastrointestinal tract of an elderly HIV-negative Inuit male from Northern Quebec, Canada. PMID- 16682126 TI - The dynamic range and domain-specific signals of intracellular calcium in photoreceptors. AB - Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca(2+) signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca(2+) gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca(2+) concentration within synaptic terminals of approximately 2 microM and lower (approximately 750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca(2+) enters the two compartments via both local L-type Ca(2+) channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca(2+) channels and plasma membrane Ca(2+) ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca(2+) homeostasis is controlled in a region-specific manner by direct Ca(2+) entry and diffusion as well as Ca(2+) extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca(2+) signaling in photoreceptors is approximately 40 fold, from approximately 50 nM in the light to approximately 2 microM in darkness. PMID- 16682128 TI - Adenomyoma with goblet and Paneth cells of the ileum. AB - A case of ileal adenomyoma with goblet and Paneth cells is reported. A 75-year old man died of ruptured hepatocellular carcinoma. As an incidental finding at autopsy, a 9 x 7 x 6 mm(3)-sized nodule was found in the ileal wall. Histologically, the lesion occupied the submucosa and muscularis propria, and consisted of glandular structures of various sizes and interlacing smooth muscle bundles surrounding the glandular elements. Goblet cells and Paneth cells were interspersed in the glandular element. Immunohistochemically, the glandular element was positive for cytokeratin (CK) 7 and negative for CK 20. This is the first reported case of adenomyoma of the gastrointestinal tract that contained Paneth cells. The result of the immunohistochemical staining favored the heterotopic pancreas theory concerning its pathogenesis. The appearance of goblet and Paneth cells might be the result of metaplasia. PMID- 16682129 TI - Impact of conventional radiotherapy on health-related quality of life and critical functions of the head and neck. AB - PURPOSE: Head-and-neck radiotherapy is associated with significant morbidities. Our purpose was to document impact of morbidities by use of multiple objective measures and health-related quality of life (HR-QOL). METHODS AND MATERIALS: Ten head-and-neck cancer patients were evaluated before receiving conventional head and-neck radiotherapy and at 1 month and 6 months after treatment. We evaluated weight, saliva production, diet, swallow function, auditory function, and HR-QOL. RESULTS: After radiotherapy, weight was reduced in 89% of subjects. Salivary function was significantly reduced and did not resolve by 6 months. Diet impairment and abnormalities in swallowing function persisted at 6 months. Perception of physical functioning was reduced after treatment, and swallowing, coughing, and dry-mouth symptoms increased. Very few changes were observed in auditory function. CONCLUSIONS: Conventional head-and-neck radiotherapy is associated with substantial functional deficits and diminished HR-QOL. Deficits reported here can serve as a baseline for comparison with results derived from new radiotherapy-treatment techniques. PMID- 16682130 TI - The challenging role of radiation therapy for very young children with rhabdomyosarcoma. AB - PURPOSE: To evaluate local control and toxicity for very young children treated with multimodality therapy for rhabdomyosarcoma (RMS). METHODS AND MATERIALS: From 1990 to 2004, 20 patientsor=1 year after diagnosis (15) in terms of mild, moderate, or severe deficits. RESULTS: Median follow-up was 33 months for survivors and 23 months for all patients. Two-year actuarial local control, event free survival, disease-specific survival, and overall survival were 84%, 52%, 74%, and 62%, respectively. All patients who began EBRT60 years, 25% with severe neurologic deficits, 44% after gross total resection, 52% with high-dose radiotherapy, and 16% with chemotherapy). Variations in the clinical-therapeutic features in three subsequent periods (1985 through 2001) were evaluated, along with overall survival for the different subgroups. RESULTS: The proportion of women, of older patients, of those with worse neurologic performance status (NPS), with WHO Grade 4, and with smaller tumors increased with time, as did the proportion of those treated with radical surgery, hypofractionated radiotherapy, and more sophisticated radiotherapy techniques, after staging procedures progressively became more accurate. The main prognostic factors for overall survival were age, sex, neurologic performance status, WHO grade, extent of surgery, and radiation dose. CONCLUSIONS: Recently, broader selection criteria for radiotherapy were adopted, together with simpler techniques, smaller total doses, and larger fraction sizes for the worse prognostic categories. Younger, fit patients are treated more aggressively, more often in association with chemotherapy. Survival did not change over time. The accurate evaluation of neurologic status is therefore of utmost importance before the best treatment option for the individual patient is chosen. PMID- 16682132 TI - Renal dysfunction after total body irradiation: dose-effect relationship. AB - PURPOSE: Late complications related to total body irradiation (TBI) as part of the conditioning regimen for hematopoietic stem cell transplantation have been increasingly noted. We reviewed and compared the results of treatments with various TBI regimens and tried to derive a dose-effect relationship for the endpoint of late renal dysfunction. The aim was to find the tolerance dose for the kidney when TBI is performed. METHODS AND MATERIALS: A literature search was performed using PubMed for articles reporting late renal dysfunction. For intercomparison, the various TBI regimens were normalized using the linear quadratic model, and biologically effective doses (BEDs) were calculated. RESULTS: Eleven reports were found describing the frequency of renal dysfunction after TBI. The frequency of renal dysfunction as a function of the BED was obtained. For BED>16 Gy an increase in the frequency of dysfunction was observed. CONCLUSIONS: The tolerance BED for kidney tissue undergoing TBI is about 16 Gy. This BED can be realized with highly fractionated TBI (e.g., 6x1.7 Gy or 9x1.2 Gy at dose rates>5 cGy/min). To prevent late renal dysfunction, the TBI regimens with BED values>16 Gy (almost all found in published reports) should be applied with appropriate shielding of the kidneys. PMID- 16682133 TI - Highly effective local control and palliation of mantle cell lymphoma with involved-field radiation therapy (IFRT). AB - PURPOSE: Although radiosensitivity of mantle cell lymphoma (MCL) has been demonstrated in vitro, radiotherapy is rarely employed in treatment of MCL. We studied clinical responses of MCL patients treated with involved-field radiation therapy (IFRT) predominantly for local control and/or palliation. METHODS AND MATERIALS: A total of 21 consecutive patients (38 sites) treated with IFRT for MCL were retrospectively analyzed. Median age was 68. Seventeen patients had Stage IV/relapsed disease, 1 had Stage II, and 3 had Stage I disease. Most patients received prior chemotherapy, with an average of two combinations per patient. Mean number of sites treated per patient was two. Mean total dose was 30 Gy. RESULTS: Mean follow-up was 13 months. Overall local response rate was 100%. Complete response was obtained in 64% of the sites and partial response in 36%. Average time to response was 20 days. Twenty-eight sites had a response before radiation therapy was complete. Of 16 sites associated with pre-IFRT pain or discomfort, 15 exhibited post-IFRT relief. Thirteen sites (34%) exhibited local progression, with a median time to progression of 10 months, and an average response duration of 9 months. Five patients experienced Grade II radiation related toxicity. No Grade III toxicity was reported. Twelve-month overall survival for patients receiving IFRT was 55%. CONCLUSIONS: Radiotherapy provided effective and lasting local responses in MCL patients and was associated with minimal toxicity. Radiation doses required for most lesions were relatively low and responses were noticed early in the course of treatment. Radiation therapy should be considered early in the course of relapsing, refractory, or localized MCL. PMID- 16682134 TI - Concurrent weekly docetaxel and concomitant boost radiation therapy in the treatment of locally advanced squamous cell cancer of the head and neck. AB - PURPOSE: In a Phase I/II trial, we investigated concurrent weekly docetaxel and concomitant boost radiation in patients with locally advanced squamous cell cancer of the head and neck (SCCHN) after induction chemotherapy. PATIENTS AND METHODS: Patients presented with American Joint Committee on Cancer Stage III/IV and were treated initially with induction chemotherapy using cisplatinum/5 fluorouracil (PF), carboplatinum-5-FU, or docetaxel-PF. Patients then received docetaxel four times weekly with concomitant boost (CB) radiation (1.8 Gy once daily X20, 1.8/1.5 Gy twice a day). Fifteen patients each received 20 mg/M2 and 25 mg/M2. RESULTS: Thirty-one patients were enrolled and 30 were evaluable for response and toxicity. Median follow-up was 42 months (range, 27-63 months). Primary sites were: oropharynx 19, oral cavity 2, larynx/hypopharynx 5, and unknown primary 4. Eighty-seven percent of patients had N2/N3 disease; 60% had T3/T4 disease. Twenty percent of patients had a complete response (CR) to induction chemotherapy. After chemoradiotherapy, 21 of 30 patients had a CR, 2 had progressive disease, and 7 had partial response (PR). Nineteen of 26 patients presenting with neck disease had neck dissections, and 7 of 19 were positive. Ninety-three percent of all patients were rendered disease-free after all planned therapy. Treatment failed in 8 patients, and 7 have died of disease. An additional patient died with no evidence of disease. Twenty-one patients (70%) are currently alive with no evidence of disease. No acute dose-limiting toxicity was observed at either dose level. CONCLUSIONS: This intensive treatment regimen of concurrent docetaxel/concomitant boost radiation and surgery after induction chemotherapy in poor prognosis patients yields good local regional control and survival. Docetaxel/CB chemoradiotherapy represents an aggressive alternative regimen to platinum-based chemoradiotherapy or surgery in patients who have a poor response to induction chemotherapy. PMID- 16682136 TI - Patterns of failure after resection of non-small-cell lung cancer: implications for postoperative radiation therapy volumes. AB - PURPOSE: To analyze local-regional patterns of failure after surgical resection of non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: This retrospective analysis included 61 patients who underwent resection of NSCLC at Duke University Medical Center. Inclusion into the study required the following: margin-negative resection, no neoadjuvant/adjuvant radiation therapy (RT), first recurrence involving a local-regional site, and imaging studies available for review. Sites of intrathoracic disease recurrence were documented. Diagrams were constructed that illustrated sites of failure on the basis of lobe of primary tumor. Failure rates were compared by application of a two-tailed Fisher's exact test. RESULTS: All patients had CT imaging for review, and 54% also had PET imaging. The median number of local-regional recurrent sites was two (range, 1-6). For all patients, the most common site of failure was the bronchial stump/staple line (44%), which was present more often in those who had a wedge resection than in those who had a more radical procedure (79% vs. 34%, p=0.005). Patients with initial nodal involvement (pN1-2) were not more likely to have involvement of the mediastinum than were patients with pN0 disease (64% vs. 72%, p=0.72), but were more likely to have involvement of the supraclavicular fossa (27% vs. 4%, p=0.04). Mediastinal involvement, without overt evidence of hilar involvement, occurred in 59% of patients. Left-sided tumors tended to involve the contralateral mediastinum more frequently than did right-sided tumors. Patterns of failure after resection are diagrammed and follow a fairly predictable pattern on the basis of involved lobe. CONCLUSIONS: These data may help clinicians construct postoperative RT volumes that are smaller than ones traditionally utilized, which may improve the therapeutic ratio. PMID- 16682135 TI - Hemoglobin levels do not predict biochemical outcome for localized prostate cancer treated with neoadjuvant androgen-suppression therapy and external-beam radiotherapy. AB - PURPOSE: To investigate whether hemoglobin (Hb) levels affect outcome in men with localized prostate adenocarcinoma (LPA) treated with neoadjuvant androgen suppression therapy (NAST) and external-beam radiotherapy (EBRT). METHODS AND MATERIALS: A total of 563 men with LPA treated with NAST (median: 5.3 months) and EBRT who had Hb levels during treatment were retrospectively reviewed. Patient, tumor, and treatment variables, including the following Hb variables, were subjected to univariate and multivariable analyses to identify factors that predict biochemical control (bNED) and overall survival (OS): pre-EBRT Hb, Hb nadir during EBRT, and change in Hb from pre-EBRT to nadir during EBRT. RESULTS: Median PSA follow-up was 4.25 years. Forty-nine percent of men were anemic during EBRT, with a median Hb of 13.4 g/dL, and 68% experienced a decline in Hb from pre EBRT to during EBRT of median 0.6 g/dL. Five-year Nadir+2 bNED and OS rates were similar for anemic and nonanemic patients during EBRT. High percent-positive biopsies, PSA and Gleason score, and use of AA monotherapy predicted worse bNED. High stage and age predicted worse OS. Hb variables were not predictive of bNED or OS. CONCLUSIONS: Anemia is a common side effect of NAST and is usually mild. Hb levels, however, do not predict biochemical control or survival. PMID- 16682137 TI - Computerized tomographic simulation compared with clinical mark-up in palliative radiotherapy: a prospective study. AB - PURPOSE: To evaluate the impact of computed tomographic (CT) planning in comparison to clinical mark-up (CM) for palliative radiation of chest wall metastases. METHODS AND MATERIALS: In patients treated with CM for chest wall bone metastases (without conventional simulation/fluoroscopy), two consecutive planning CT scans were acquired with and without an external marker to delineate the CM treatment field. The two sets of scans were fused for evaluation of clinical tumor volume (CTV) coverage by the CM technique. Under-coverage was defined as the proportion of CTV not covered by the CM 80% isodose. RESULTS: Twenty-one treatments (ribs 17, sternum 2, and scapula 2) formed the basis of our study. Due to technical reasons, comparable data between CM and CT plans were available for 19 treatments only. CM resulted in a mean CTV under-coverage of 36%. Eleven sites (58%) had an under-coverage of >20%. Mean volume of normal tissues receiving >/=80% of the dose was 5.4% in CM and 9.3% in CT plans (p = 0.017). Based on dose-volume histogram comparisons, CT planning resulted in a change of treatment technique from direct apposition to a tangential pair in 7 of 19 cases. CONCLUSIONS: CT planning demonstrated a 36% under-coverage of CTV with CM of ribs and chest wall metastases. PMID- 16682138 TI - Gamma-Knife radiosurgery in the management of melanoma patients with brain metastases: a series of 106 patients without whole-brain radiotherapy. AB - PURPOSE: To assess retrospectively a strategy that uses Gamma-Knife radiosurgery (GKR) in the management of patients with brain metastases (BMs) of malignant melanoma (MM). METHODS: GKR without whole-brain radiotherapy (WBRT) was performed for patients with Karnofsky Performance Status (KPS) of 60 or above who harbored 1 to 4 BMs of 30 mm or less and was repeated as often as needed. Survival was assessed in the whole population, whereas local-control rates were assessed for patients with follow-up longer than 3 months. RESULTS: A total of 221 BMs were treated in 106 patients; 61.3% had a single BM. Median survival from the time of GKR was 5.09 months. Control rate of treated BMs was 83.7%, with 14% of complete response (14 BMs), 42% of partial response (41 BMs), and 43% of stabilization (43 BMs). In multivariate analysis, survival prognosis factors retained were KPS greater than 80, cortical or subcortical location, and Score Index for Radiosurgery (SIR) greater than 6. On the basis of KPS, BM location, and age, a score called MM-GKR, predictive of survival in our population, was defined. CONCLUSION: Gamma-Knife radiosurgery provides a surgery-like ability to obtain control of a solitary BM and could be consider as an alternative treatment to the combination of GKR+WBRT as a palliative strategy. MM-GKR classification is more adapted to MM patients than are SIR, RPA and Brain Score for Brain Metastasis. PMID- 16682139 TI - Beta-ray brachytherapy with 106Ru plaques for retinoblastoma. AB - PURPOSE: A retrospective analysis of 134 patients who received (106)Ru brachytherapy for retinoblastomas (175 tumors in 140 eyes). Treatment and follow up were analyzed with special emphasis on tumor control organ, preservation, and late complications. RESULTS: Treated tumors had a mean height and diameter of 3.7+/-1.4 mm and 5.0+/-2.8 disk diameters, respectively. The radiation dose values were recalculated according to the calibration standard recently introduced by the National Institute of Standards and Technology. The recalculation revealed a mean applied dose of 419 Gy at the sclera (SD, 207 Gy) and 138 Gy (SD, 67 Gy) at the tumor apex. The 5-year tumor control rate was 94.4%. Tumor recurrence was more frequent in eyes with vitreous tumor cell seeding or fish-flesh regression. The estimated 5-year eye preservation rate was 86.5%. Previous treatment by brachytherapy or external beam radiotherapy, as well as a large tumor diameter, were significant factors for enucleation. The radiotherapy-induced complications after 5 years of follow-up were retinopathy (22%), optic neuropathy (21%), and cataract (17%). These complications were significantly more frequent after prior brachytherapy or external beam radiotherapy. CONCLUSION: Brachytherapy using (106)Ru plaques is a highly efficient therapy with excellent local tumor control and an acceptable incidence of side effects. PMID- 16682140 TI - Pediatric cerebral arteriovenous malformations: the role of stereotactic linac based radiosurgery. AB - PURPOSE: To evaluate retrospectively clinical outcome and obliteration rates after linac-based radiosurgery (RS) in children with cerebral arteriovenous malformations (AVM). METHODS AND MATERIALS: Between 1996 and 2002, 22 children with cerebral AVM were treated at our institution. Mean age at treatment was 11.8 years (range, 4.4-16.4 years). Classification according to Spetzler-Martin was 1 child grade I (4%), 7 grade II (32%), 12 grade III (56%), 1 grade IV (4%), and 1 grade V (4%). Median single dose was 18 Gy/80%-isodose. Median AVM volume was 4.2 mL (range, 0.4-26.5 mL). Median RS-based AVM-score was 1.07 (range, 0.61-3.55). Fifty-nine percent of children experienced intracranial hemorrhage before RS. Median follow-up was 3.1 years (range, 1.7-7.3 years). RESULTS: Actuarial complete obliteration rate (CO) was 54% after 3 years and 65% after 4 years, respectively. Median time interval to CO was 27.1 months. Intracranial hemorrhage after RS was seen in five children after median 13.9 months. Annual bleeding risk was 9.1% after 1 year and 13.6% after 2 years. Maximum diameter>or=3 cm and AVM volume>or=6 mL were significant predictors for intracranial hemorrhage. Neurologic deficits were improved/completely dissolved in 58% of children and remained stable in 42%. No new onset of neurologic dysfunction was seen after RS. CONCLUSIONS: RS is safe and effective in pediatric cerebral AVM with high obliteration rates. Size and volume of AVM are significant predictors for intracranial bleeding. The same treatment guidelines as in adults should be applied. Careful long-term follow-up observation is required after RS from long life expectation. PMID- 16682141 TI - Feasibility of radiotherapy after high-dose dense chemotherapy with epirubicin, preceded by dexrazoxane, and paclitaxel for patients with high-risk Stage II-III breast cancer. AB - PURPOSE: To verify the feasibility of, and quantify the risk of, pneumonitis from locoregional radiotherapy (RT) after high-dose dense chemotherapy with epirubicin and paclitaxel with peripheral blood progenitor cell support in patients with high-risk Stage II-III breast cancer. METHODS AND MATERIALS: Treatment consisted of a mobilizing course of epirubicin 150 mg/m2, preceded by dexrazoxane (Day 1), paclitaxel 175 mg/m2 (Day 2), and filgrastim; followed by three courses of epirubicin 150 mg/m2, preceded by dexrazoxane (Day 1), paclitaxel 400 mg/m2 (Day 2), and peripheral blood progenitor cell support and filgrastim, every 16-19 days. After chemotherapy, patients were treated with locoregional RT, which included the whole breast or the chest wall, axilla, and supraclavicular area. RESULTS: Overall, 64 of 69 patients were evaluable. The interval between the end of chemotherapy and the initiation of RT was at least 1.5-2 months (mean 2). No treatment-related death was reported. After a median follow-up of 27 months from RT (range 5-77 months), neither clinically relevant radiation pneumonitis nor congestive heart failure had been reported. Minor and transitory lung and cardiac toxicities were observed. CONCLUSION: Sequential high doses of epirubicin, preceded by dexrazoxane, and paclitaxel did not adversely affect the tolerability of locoregional RT in breast cancer patients. The risk of pneumonitis was not affected by the use of sequential paclitaxel with an interval of at least 1.5-2 months between the end of chemotherapy and the initiation of RT. Long-term follow up is needed to define the risk of cardiotoxicity in these patients. PMID- 16682143 TI - Change in volume of lumpectomy cavity during external-beam irradiation of the intact breast. AB - PURPOSE: Definition of the lumpectomy cavity is an important component of irradiation of the breast. We use computed tomography (CT)-based planning and contour the lumpectomy volume on the planning CT. We obtained a second CT in the 4th or 5th week of treatment for boost planning and compared the volume change with the first planning-CT scan. METHODS AND MATERIALS: This retrospective study reviewed the planning-CT data for 20 patients. In the first CT, images were obtained from the mandible to 2 cm below the breast in 3-mm slices. In the second CT, for the boost, images were obtained from the top to the bottom of the clinically defined breast, in 3-mm slices. Lumpectomy cavities were contoured on both CT scans and volumes compared. RESULTS: Sixteen of the 20 patients (80%) had more than a 20% decrease from the first to the second volume, with a corresponding 95% confidence interval. The mean decrease was 16.13 cc, with a standard deviation of 14.05. The Spearman correlation coefficient of 0.18 did not show a significant correlation between the initial volume and the percent change. CONCLUSIONS: During external breast irradiation, many patients will have significant volume reduction in the lumpectomy cavity. Because CT-based definition of the lumpectomy cavity can influence the planning of a boost technique, further study appears warranted. PMID- 16682142 TI - Multi-institutional analysis of early squamous cell carcinoma of the hypopharynx treated with radical radiotherapy. AB - PURPOSE: To analyze the outcomes of patients with early hypopharyngeal cancer treated with radical radiotherapy (RT). METHODS AND MATERIALS: Ten institutions combined the data from 115 patients with Stage I-II hypopharyngeal squamous cell carcinoma treated with definitive RT between 1990 and 2001. The median patient age was 67 years; 99 patients were men and 16 were women. Of the 115 patients, 39 had Stage I and 76 had Stage II disease. Conventional fractionation was used in 98 patients and twice-daily RT in 17 patients; chemotherapy was combined with RT in 57 patients. The median follow-up period was 47 months. RESULTS: The overall and disease-specific 5-year survival rate for 95 patients without synchronous malignancies was 66.0% and 77.4%, respectively. The 5-year disease-specific survival rate by T stage was 95.8% for patients with T1 disease and 70.1% for patients with T2 disease (p=0.02). Of the 115 patients, local control with laryngeal voice preservation was achieved in 34 of 39 patients with T1 lesions, including 7 patients successfully salvaged, and in 56 of 76 patients with T2 lesions. Sixty-five patients (56.5%) had synchronous or metachronous cancers. Of the 115 patients, 19 died of hypopharyngeal cancer, 10 died of second primary cancers, and 14 died of other causes during the study and follow-up periods. CONCLUSIONS: Patients with early hypopharyngeal cancer tended to have a good prognosis after RT. However, second malignancies had an adverse effect on the overall outcomes of patients with early hypopharyngeal cancer. PMID- 16682144 TI - Is there a role for postimplant dosimetry after real-time dynamic permanent prostate brachytherapy? AB - PURPOSE: To evaluate the correlation of real-time dynamic prostate brachytherapy (RTDPB) dosimetry and traditional postimplant dosimetry for permanent prostate brachytherapy. METHODS AND MATERIALS: A total of 164 patients underwent RTDPB for clinically confined prostate cancer. Of these 164 patients, 45 were implanted with 103Pd and 119 with 125I. Additionally, 44 patients underwent combined external beam radiotherapy and brachytherapy and 120 patients underwent brachytherapy alone. The postimplant dosimetry with computed tomography was performed at 4 weeks and compared with the RTDPB dose plan using the intraclass correlation coefficient. The millicurie/gram of the prostate volume and the percentage of the minimal dose to 90% of the prostate relative to the prescribed implant dose (D90%) of the RTDPB patients was compared with 400 patients treated with a free-seed technique. RESULTS: The mean D90% achieved in the operating room and on the 3-week dose plan was 109% (range, 93-139%) and 105% (range, 88-140), respectively. The mean percentage of prostate volume receiving 100% of the prescribed minimal peripheral dose (V100) achieved in the operating room and on the 3-week dose plan was 93% (range, 78-98%) and 91% (range, 64-98%), respectively. The intraclass correlation coefficient for each calculated relationship was 0.586 for D90 (p<0.001), 1.19 for V100 (p=0.135), 0.692 for the urethral D90 (p<0.001), 0.602 for the maximal rectal dose (p<0.001), 0.546 for D90 with 125I (p<0.001), and 0.565 for D90 with 103Pd (p<0.001). A 12% decrease was noted in the millicurie/gram of the isotope, with a 2.5% increase in the D90 comparing RTDPB and the free-seed technique. CONCLUSION: The results of this study demonstrated a correlation between the dose assessment obtained intraoperatively and postoperatively at 3 weeks. With reliable dose data available in the operating room, our results question the need for routine postimplant dose studies. Furthermore, patients treated with RTDPB received less radioactivity per gram of the prostate with a corresponding small increase in the D90. Future analyses will assess variations in the inverse dose planning rules and the clinical correlation of patients undergoing RTDPB vs. older techniques for toxicity and biochemical outcomes. PMID- 16682145 TI - Significant reduction of normal tissue dose by proton radiotherapy compared with three-dimensional conformal or intensity-modulated radiation therapy in Stage I or Stage III non-small-cell lung cancer. AB - PURPOSE: To compare dose-volume histograms (DVH) in patients with non-small-cell lung cancer (NSCLC) treated by photon or proton radiotherapy. METHODS AND MATERIALS: Dose-volume histograms were compared between photon, including three dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), and proton plans at doses of 66 Gy, 87.5 Gy in Stage I (n=10) and 60-63 Gy, and 74 Gy in Stage III (n=15). RESULTS: For Stage I, the mean total lung V5, V10, and V20 were 31.8%, 24.6%, and 15.8%, respectively, for photon 3D CRT with 66 Gy, whereas they were 13.4%, 12.3%, and 10.9%, respectively, with proton with dose escalation to 87.5 cobalt Gray equivalents (CGE) (p=0.002). For Stage III, the mean total lung V5, V10, and V20 were 54.1%, 46.9%, and 34.8%, respectively, for photon 3D-CRT with 63 Gy, whereas they were 39.7%, 36.6%, and 31.6%, respectively, for proton with dose escalation to 74 CGE (p=0.002). In all cases, the doses to lung, spinal cord, heart, esophagus, and integral dose were lower with proton therapy even compared with IMRT. CONCLUSIONS: Proton treatment appears to reduce dose to normal tissues significantly, even with dose escalation, compared with standard-dose photon therapy, either 3D-CRT or IMRT. PMID- 16682146 TI - Effect of beam channel plugging on the outcome of gamma knife radiosurgery for trigeminal neuralgia. AB - PURPOSE: We studied the influence of using plugs for brainstem protection during gamma knife radiosurgery (GKR) of trigeminal neuralgia (TN), with special emphasis on irradiation doses delivered to the trigeminal nerve, pain outcomes, and incidence of trigeminal dysfunction. METHODS AND MATERIALS: A GKR procedure for TN using an anterior cisternal target and a maximum dose of 90 Gy was performed in 109 patients. For 49 patients, customized beam channel blocking (plugs) were used to reduce the dose delivered to the brainstem. We measured the mean and integrated radiation doses delivered to the trigeminal nerve and the clinical course of patients treated with and without plugs. RESULTS: We found that blocking increases the length of trigeminal nerve exposed to high-dose radiation, resulting in a significantly higher mean dose to the trigeminal nerve. Significantly more of the patients with blocking achieved excellent pain outcomes (84% vs. 62%), but with higher incidences of moderate and bothersome trigeminal nerve dysfunction (37% mild/10% bothersome with plugs vs. 30% mild/2% bothersome without). CONCLUSIONS: The use of plugs to protect the brainstem during GKR treatment for TN increases the dose of irradiation delivered to the intracisternal trigeminal nerve root and is associated with an important increase in the incidence of trigeminal nerve dysfunction. Therefore, beam channel blocking should be avoided for 90 Gy-GKR of TN. PMID- 16682147 TI - Predictors of mortality after prostate-specific antigen failure. AB - PURPOSE: We identified factors associated with the length of survival after prostate-specific antigen (PSA) failure. METHODS AND MATERIALS: The study cohort comprised 81 of 206 men enrolled on a randomized trial evaluating external-beam radiation therapy (RT) with or without androgen suppression therapy (AST) and who experienced PSA failure. Salvage AST was administered at a PSA level of approximately 10 ng/mL as per protocol. Cox regression was used to determine factors associated with length of survival after PSA failure. RESULTS: A PSA DT (doubling time) <6 months (p = 0.04) and age at the time of PSA failure (p = 0.009) were significantly associated with length of survival. By 5 years, 35% and 65% of all-cause mortality was from prostate cancer in men whose age at PSA failure was 75 or higher vs. <75, respectively. Across all ages, 0%, 4%, as compared with 63% of men, were estimated to die of prostate cancer within 5 years after PSA failure if their PSA DT was >12, 6-12, or <6 months, respectively. CONCLUSIONS: Advanced age and a PSA DT <6 months at the time of PSA failure are associated with a significantly shorter survival. PMID- 16682148 TI - Pretreatment prognostic factors in patients with early-stage (I/II) non-small cell lung cancer treated with hyperfractionated radiation therapy alone. AB - PURPOSE: To investigate influence of various pretreatment prognostic factors in patients with early stage (I/II) non-small-cell lung cancer (NSCLC) treated with hyperfractionated radiation therapy alone. PATIENTS AND METHODS: One hundred and sixteen patients were treated with tumor doses of 69.6 Gy, 1.2-Gy, twice-daily fractionation. There were 49 patients with Stage I and 67 patients with Stage II. Eighty patients had Karnofsky performance status (KPS) 90-100 and 95 patients had <5% weight loss. Peripheral tumors were observed in 57 patients. Squamous histology was observed in 70 patients and the majority of patients had concomitant disease (n=72). RESULTS: The median survival time for all patients was 29 months; 5-year survival was 29%. The median time to local progression and the distant metastasis were not achieved, whereas 5-year local progression-free and distant metastasis-free survivals were 50% and 72%, respectively. Multivariate analysis identified KPS, weight loss, location, histology, and the reason for not undergoing surgery as prognostic factors for survival. KPS, location, and histology influenced local progression-free survival, whereas only KPS and weight loss influenced distant metastasis-free survival. CONCLUSIONS: This retrospective analysis identified KPS and weight loss as the most important prognostic factors of outcome in patients with early-stage NSCLC treated with hyperfractionation radiation therapy. PMID- 16682149 TI - Androgen-deprivation therapy does not impact cause-specific or overall survival after permanent prostate brachytherapy. AB - PURPOSE: To determine if androgen-deprivation therapy (ADT) has an impact on cause-specific, biochemical progression-free, or overall survival after prostate brachytherapy. METHODS AND MATERIALS: From April 1995 through June 2002, 938 consecutive patients underwent brachytherapy for clinical Stage T1b to T3a (2002 AJCC) prostate cancer. All patients underwent brachytherapy more than 3 years before analysis. A total of 382 patients (40.7%) received ADT with a duration of 6 months or less in 277 and more than 6 months in 105. The median follow-up was 5.4 years. Multiple clinical, treatment, and dosimetric parameters were evaluated as predictors of cause-specific, biochemical progression-free, and overall survival. RESULTS: The 10-year cause-specific, biochemical progression-free, and overall survival rates for the entire cohort were 96.4%, 95.9%, and 78.1%, respectively. Except for biochemical progression-free survival in high-risk patients, ADT did not statistically impact any of the three survival categories. A Cox linear-regression analysis demonstrated that Gleason score was the best predictor of cause-specific survival, whereas percent-positive biopsies, prostate volume, and risk group predicted for biochemical progression-free survival. Patient age and tobacco use were the strongest predictors of overall survival. One hundred two patients have died, with 80 of the deaths a result of cardiovascular disease (54) and second malignancies (26). To date, only 12 patients have died of metastatic prostate cancer. CONCLUSIONS: After brachytherapy, androgen-deprivation therapy did not have an impact on cause specific or overall survival for any risk group; however, ADT had a beneficial effect on biochemical progression-free survival in high-risk patients. Cardiovascular disease and second malignancies far outweighed prostate cancer as competing causes of death. PMID- 16682150 TI - Prevertebral muscle involvement in nasopharyngeal carcinoma. AB - PURPOSE: The purpose of this study is to evaluate the prevalence and prognostic significance of prevertebral muscle involvement in patients with nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS: Between July 1990 and December 2001, 521 newly diagnosed patients with NPC treated at Koo Foundation Sun Yat-Sen Cancer Center (KF-SYSCC) were examined with magnetic resonance imaging (MRI) for evidence of prevertebral muscle involvement before treatment. Patients were staged according to the 1997 American Joint Committee on Cancer staging classification of NPC based on the physical exams and MRI findings. All patients received radiotherapy with or without chemotherapy. The association between clinical prevertebral muscle involvement and posttreatment outcomes (overall survival, locoregional recurrence, and distant metastasis) were evaluated using Cox regression model to adjust for other prognostic factors. RESULTS: Of 521 patients treated at KF-SYSCC, 181 (35%) patients were found to have prevertebral muscle involvement, one-third in those with Stage II/III tumors and two-thirds in those with Stage IV tumor. In multivariate analysis accounting for all previously known prognostic factors, prevertebral muscle invasion was associated with an increased risk for any recurrence (adjusted relative risk, 2.01; p<0.001), locoregional recurrence (adjusted relative risk, 2.69; p<0.001), and distant metastasis (adjusted relative risk, 2.25; p<0.001), and with a borderline significant increased risk for overall survival (adjusted relative risk, 1.44; p=0.10). CONCLUSIONS: Prevertebral muscle involvement is an independent prognostic factor for NPC recurrence. PMID- 16682151 TI - Impact of viral E2-gene status on outcome after radiotherapy for patients with human papillomavirus 16-positive cancer of the uterine cervix. AB - PURPOSE: Integration of high-risk papillomavirus DNA has been considered an important step in oncogenic progression to cervical carcinoma. Disruption of the human papillomavirus (HPV) genome within the E2 gene is frequently a consequence. This study investigated the influence of episomal viral DNA on outcome in patients with advanced cervical cancer treated with primary radiotherapy. METHODS AND MATERIALS: Paraffin-embedded biopsies of 82 women with locally advanced cervical cancer could be analyzed for HPV infection by multiplex polymerase chain reaction (PCR) by use of SPF1/2 primers. E2-gene intactness of HPV-16-positive samples was analyzed in 3 separate amplification reactions by use of the E2A, E2B, E2C primers. Statistical analyses (Kaplan-Meier method; log-rank test) were performed for overall survival (OS), disease-free survival (DFS), local progression-free survival (LPFS), and distant metastases-free survival (DMFS). RESULTS: Sixty-one (75%) of 82 carcinomas were HPV positive, 44 of them for HPV 16 (72%). Seventeen of the 44 HPV-16-positive tumors (39%) had an intact E2 gene. Patients with a HPV-16-positive tumor and an intact E2 gene showed a trend for a better DFS (58% vs. 38%, p = 0.06) compared with those with a disrupted E2 gene. A nonsignificant difference occurred regarding OS (87% vs. 66%, p = 0.16) and DMFS (57% vs. 48%, p = 0.15). CONCLUSION: E2-gene status may be a promising new target, but more studies are required to elucidate the effect of the viral E2 gene on outcome after radiotherapy in HPV-positive tumors. PMID- 16682152 TI - Intraoperative electron-beam therapy for primary and recurrent retroperitoneal soft-tissue sarcoma. AB - PURPOSE: This study assesses the long-term outcome of patients with retroperitoneal soft-tissue sarcomas treated by maximal resection in combination with intraoperative electron-beam therapy (IOERT) and postoperative external-beam radiotherapy. METHODS AND MATERIALS: From 1991 to 2004, 67 patients were treated with curative intent for primary (n = 26) or recurrent (n = 41) retroperitoneal soft-tissue sarcoma. All patients underwent maximal resection in combination with IOERT (mean dose, 15 Gy), 45 patients underwent additional postoperative EBRT, and 20 patients were previously irradiated. RESULTS: The 5-year actuarial overall survival (OS), disease-free survival, local control (LC), and freedom from metastatic disease of all patients was 64%, 28%, 40%, and 50%, respectively. The 5-year LC inside the IOERT field was 72%. For patients who completed IOERT and EBRT after R0-resection 5-year and 10-year OS was 80%, and 5-year and 10-year LC was 100%. Only 1 of the 21 patients after R0-resection and only 8 of 34 patients after R1-resection compared with 9 of 12 patients after R2-resection experienced inside IOERT-field relapse. Grade II or higher late complications were seen in 21% of the patients, but only 2 patients required surgical intervention because of late complications. CONCLUSION: In selected patients, IOERT results in excellent local control and survival, with acceptable morbidity. PMID- 16682153 TI - Long-term results of irradiation for paraganglioma. AB - PURPOSE: The management of paragangliomas is controversial. Observation, surgery, external-beam radiotherapy (EBRT), and stereotactic radiosurgery (SRS) may, alone or in combination, be appropriate, depending on the size and extent of the tumor, previous treatment, and patient age, general health, and neurologic condition. Few data exist regarding long-term tumor control and late effects after EBRT or SRS. METHODS AND MATERIALS: We performed a retrospective review of all patients treated with EBRT or SRS for paraganglioma at our institution between 1967 and 1994. The endpoints of the study were tumor control and late complications. RESULTS: The 33 patients in this study had a median follow-up of 13 years (range, 4 months to 36 years). The 10-year tumor control rate was 92% (95% confidence interval, 75-98%). At the last follow-up visit, no patient had developed a radiation-induced malignancy. CONCLUSION: External-beam RT and SRS are safe and effective for enlarging and/or symptomatic paragangliomas. The risk of developing a delayed radiation-induced malignancy after EBRT or SRS is low. This risk must be weighed against the significant immediate and permanent risk of cranial nerve deficits if the tumor is untreated or is surgically resected. This risk must also be weighed against the immediate but low risk of surgical mortality. PMID- 16682154 TI - Novel object exploration in ravens (Corvus corax): effects of social relationships. AB - Social context has been shown to encourage, or to delay object exploration and learning. This ambiguity might be due to factors such as social relationships and personality of the individuals involved. Here, we investigated in ravens (Corvus corax) individuals' consistency in response to novel objects over development and across contexts: alone versus social. In the social setting we focussed on the effects of social relationships on social facilitation during the approach to novel objects. We tested 11 hand-raised ravens with novel objects individually at three and six months of age and in dyadic combinations at six months of age. Individuals were consistent over development and contexts in their response to different novel objects. Birds joined siblings faster to approach novel objects than non-siblings. They also spent more time sitting close to siblings than to non-siblings. In male-male dyads but not in female-female dyads, subordinates approached the novel objects significantly faster than dominant birds. In contrast, dominant males were the first to approach the novel objects in mixed sex combinations. Hence, the effect of social context seems to depend on the social relationships towards the companions and on the combination of the sexes. PMID- 16682155 TI - Lateralised swimming behaviour in the California sea lion. AB - Lateralised motor behaviour in the pinnipeds has been subject to little investigation. This study examined the swimming behaviour of seven zoo-housed California sea lions to determine whether they exhibited a directional bias in their motor behaviour. Data were collected on the direction of the animals' swimming patterns from the point of entering a pool of water from dry land. Each animal was studied for 100 episodes of swimming. All seven of the sea lions showed significant (P<0.001) bias in the direction of their swimming, although unidirectional bias was not observed at the level of the population. The direction of the sea lions' swimming patterns varied significantly according to the animals' sex. Males showed a preference at the level of the population for swimming in a clockwise direction, while females showed a population-level counterclockwise swimming preference. Overall, the findings appear to suggest that California sea lions, like other marine mammals, exhibit motor bias in the direction of their swimming patterns, although further work using larger sample sizes is needed before more firm conclusions regarding motor laterality in this species can be reached. PMID- 16682156 TI - Serum and liver tissue biotinidase enzyme activity in rats which were administrated to valproic acid. AB - Valproic acid (VPA) is an antiepileptic drug widely used and well-tolerated by most of patients. Its non-dose-dependent side effects seen mostly are the temporary gastrointestinal disturbances including anorexia and nausea, and hepatoxicity. As to its dose-dependent side effects are the weight loss, tremor, skin eruption and the alopecia. In this study we aimed to put forward the biotinidase deficiency considered as a possible cause of alopecia in the rats administered with valproic acid, and the correlation between liver and serum biotinidase enzyme activities (BEA) and transaminases, albumin and serum valproic acid levels. In our study, 4 groups of which one of them was a control group, each consisting of 15 male Wistar rats was organized. 200, 400, and 600 mg/kg/day of VPA, and distilled water, two divided doses per day, were administered per orally to VPA-1, VPA-2, VPA-3, and control group, respectively, in 60 days. Their serum and liver biotinidase enzyme activities, serum AST, ALT, albumin, and valproic acid levels were measured. Alopecia was seen in the subjects of 6.6% of VPA-1, 13.3% of VPA-2, and 26.6% of VPA-3. Significant difference in the liver tissues BEA was noted only between VPA-3 and the control group. Reductions were observed both in the liver tissues BEA and the serum BEA levels, which are inversely proportional to the VPA doses. A positive correlation between the liver biotinidase enzyme activities and the serum valproic acid levels, and the negative correlation between the liver tissues biotinidase activities and the serum valproic acid levels were noted, respectively. As a conclusion, the partial alopecia which is an initial symptom of reduced biotinidase activity may also be created depending on the reduction of biotinidase activity during valproic acid therapy. The alopecia which may further be observed in the patients receiving valproic acid therapy may be prevented by means of administration of biotin in a dose of 10 mg/day. PMID- 16682157 TI - Abnormal movement-related cortical potential in patients with subcortical heterotopia. AB - Movement-related cortical potential (MRCP) is recorded to investigate the cerebral motor preparation in two patients with subcortical heterotopia. Magnetic resonance imaging (MRI) studies were conducted in the two patients. Scalp MRCP elicited by self-paced voluntary extension of the wrist was recorded. The MRCP waveforms and the potential maps were compared to those of an age-matched normal subject. MRI reveals subcortical heterotopia in the right frontal area and bilateral centrum semiovale in one patient and in the left parietal lobe in the other. Both of the patients had history of early onset epilepsy and were hypersensitive to unexpected loud noises. Distinctly different from the normal MRCP, the waveforms of the patients are flattened in all scalp electrodes by moving either the right or the left hand. And the topographic potential map detects no evidence of preponderance of negativity on the hemisphere contralateral to the moving side in the patients. The ectopic neurons of the subcortical heterotopia may produce chaotic discharges, which can interrupt or cancel the well oriented dipole of the MRCP activities. PMID- 16682158 TI - The validity of post-concussion syndrome in children: a controlled historical cohort study. AB - The aim of this controlled historical cohort study was to assess the validity of post-concussion syndrome in children. We identified 301 children aged 4-15 years who had sustained an isolated brain concussion, and another group of 301 children who sustained any other mild body injury excluding the head. Parents from both groups filled in standardized questionnaires containing questions about the health condition of the children: headache, neck pain, dizziness, malaise, fatigability, exercise or noise intolerance, irritability, weepiness, sadness, anxiety, nocturnal enuresis, tics, sleep disorders, memory or learning difficulties, hyperactivity, seizures, attention disorder, buzzing in the ears, subjective parental concerns about the child's health condition, and parental concerns about their child having a brain disorder. The severity of the complaints was rated on the Visual Analogue Scale. After the final exclusion, 102 pairs strictly matched by sex, age, and the date of trauma were analyzed. The differences of parental complaints about the health condition of their children between case and control groups were statistically insignificant for all symptoms, except parental concerns about their child having brain damage which were significantly higher in the case group. The likelihood of parental concerns about the possibility of their child having brain damage was 2.7 times higher in the case group. Headache, learning difficulties, and sleep disorders were significant variables predicting the concerns. These results question the validity of the post-concussion syndrome in children. PMID- 16682159 TI - Column-switching high-performance liquid chromatographic analysis of fluvastatin in rat plasma by direct injection. AB - A column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of fluvastatin in rat plasma. Plasma samples were diluted with an equal volume of mobile phase, i.e. acetonitrile-5 mM potassium phosphate buffer (pH 6.8) (15:85, v/v), and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then back-flushed onto an analytical column and quantified with fluorescence detection (lambdaex=305 nm; lambdaem=390 nm). The standard curve for the drug was linear in the range 0.5-100 ng mL(-1) in rat plasma. The limit of quantitation for plasma was found to be 0.5 ng mL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple and rapid because of a minimized sample preparation and appears to be useful for the pharmacokinetic study of fluvastatin. PMID- 16682160 TI - Effect of calcination cycles on the preparation of tin oxide based traditional drug: studies on its formation and characterization. AB - The preparation method of metal based Indian traditional drugs involves conversion of a pure metal into its oxide by repeated high temperature calcination cycles. In this work, the effect of number of calcination cycles followed in the preparation of tin oxide based Ayurvedic drug, 'vanga bhasma' was studied by a systematic characterization of the drug samples after various calcination stages. It was found that tin was in the form of Sn4+ state and that the formation of SnO2 proceeded step-wise through Sn(OH)4. PMID- 16682161 TI - A feasibility study on direct assay of an aqueous formulation by chiral supercritical fluid chromatography (SFC). AB - Supercritical fluid chromatography (SFC) has gained considerable importance in the area of Separation Science in pharmaceutical analysis over the past few years. The synthesis of chiral compounds is of particular significance in the pursuit of new drug entities. SFC is rapidly replacing high performance liquid chromatography (HPLC) in many pharmaceutical and biotechnological companies as the standard screening and method development tool for chiral compounds. Analysis of pharmaceutical formulations of research compounds is an area where SFC is recently being explored as a possible alternate or complementary technique to HPLC in limited scope. A feasibility study was carried out to perform direct assay of a chiral drug compound AZM in 100% aqueous formulations by SFC. The results indicated that this approach has the potential to significantly reduce the typical sample processing time prior to analysis. The method was reproducible, linear over a wide dynamic range, and sensitive enough to detect the minor enantiomeric impurity in the chiral drug compound investigated here. Further application will be pursued for other research compounds in the future to illustrate the broader applicability of this approach. PMID- 16682163 TI - Development an ion-pair liquid chromatographic method for determination of sotalol in plasma using a monolithic column. AB - A rapid and sensitive ion-pair HPLC method using a monolithic column and fluorescence detection has been developed for quantification of sotalol in plasma. The assay enables the measurement of sotalol for therapeutic drug monitoring with a minimum quantification limit of 10 ng ml(-1). The analytical method involves simple, one-step protein precipitation and no extraction procedure is needed. Sample preparation is fast and the analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column at ambient temperature. The mobile phase was 10% acetonitrile, 0.001 M heptane sulfonic acid, 0.02 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to pH 5.5 at a flow rate of 1.8 ml/min. The excitation wavelength was set at 235 nm, emission at 300 nm. The calibration curve was linear over the concentration range 20-1500 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 7%. The method has been applied to the determination of sotalol in plasma from 12 subjects dosed with racemic sotalol. PMID- 16682162 TI - Gas chromatography nitrogen phosphorous detection (GC-NPD) assay of tofisopam in human plasma for pharmacokinetic evaluation. AB - Tofisopam (1-(3,4-dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3 benzodiazepine) has been shown to be an effective anxiolytic agent in the wide ranging clinical practice. A high sensitive gas chromatography nitrogen phosphorous detection (GC-NPD) bioanalytical method was developed and validated for the purpose of pharmacokinetic study of tofisopam. A liquid-liquid extraction method was used for the sample preparation. The mean recovery for tofisopam was 69.8% and the inter- and intra-day precision values were well below the 15% limit established for bioanalytical methods. A similar compound, girizopam was used as internal standard. The assay was linear in the 5-500 ng/ml range corresponding to therapeutically relevant plasma levels. The concentrations of the compound were measured in the plasma samples of 12 healthy male volunteers and the pharmacokinetic parameters were determined from the plasma concentration-time data. A rapid absorption and distribution, relatively short biological half-life and considerable inter-individual variation in the plasma concentration levels of parent compound were the main characteristics of the pharmacokinetics of tofisopam. According to these results, the new (GC-NPD) bioanalytical method proved to be capable of measuring concentration of tofisopam in human plasma and was successfully applied in a single dose pharmacokinetic study. PMID- 16682164 TI - Flow-injection chemiluminescent determination of cefprozil using Tris (2,2' bipyridyl) ruthenium (II)-permanganate system. AB - A rapid and sensitive chemiluminescence (CL) method using flow-injection (FI) has been developed for the determination of a second generation cephalosporin, cefprozil. The method is based on the CL reaction of cefprozil with acidic potassium permanganate and tris (2,2'-bipyridyl) ruthenium (II), Ru (bipy)3(2+). The CL intensity is greatly enhanced when quinine sulfate is used as a sensitizer. After optimization of the different experimental parameters, a calibration graph was obtained over a concentration range of 0.1-3.0 microg ml( 1) with minimum detectability of 0.005 microgml(-1) (S/N=3). The correlation coefficient was 0.9998 (n=6) with a relative standard deviation (%R.S.D.) of 1.63% for 2.0 microgml(-1). The proposed method was successfully applied to commercial tablets. The average percentage recovery (n=6) was 99.9+/-1.40. PMID- 16682165 TI - Clinical presentation and vascular imaging in giant cell arteritis of the femoropopliteal and tibioperoneal arteries. Analysis of four cases. AB - We present four patients with rapidly progressive claudication of the lower limbs due to extracranial giant cell arteritis. Additional findings suggestive of giant cell arteritis were involvement of the axillary or brachial arteries in two patients, symptoms of polymyalgia rheumatica or temporal arteritis in three, and all patients had severely elevated erythrocyte sedimentation rate and C-reactive protein level. Lower limb involvement affected preferentially the femoropopliteal, deep femoral, and tibioperoneal arteries. Hypoechogenic, concentric mural thickening suggestive of vasculitis was readily visible in all involved arterial segments by duplex ultrasound imaging, whereas angiography was rather unspecific. Typical changes for large-vessel vasculitis were also detectable by magnetic resonance imaging and fluorine-18-desoxyglucose positron emission tomography. More widespread use of these vascular imaging techniques may show that giant cell arteritis of the lower limbs is more frequent than previously assumed. PMID- 16682166 TI - Outcomes of carotid endarterectomy for asymptomatic stenosis in Sweden are improving: Results from a population-based registry. AB - OBJECTIVE: In large randomized trials, carotid endarterectomy (CEA) for asymptomatic stenosis has shown a net benefit compared with best medical treatment. To justify an increased number of procedures for this indication, the perioperative risk of stroke or death must not exceed that of the trials. The aim of this study was to evaluate the outcome in routine clinical practice in Sweden in a population-based study. METHODS: The Swedish Vascular Registry (Swedvasc) covers all centers performing CEA. Data on all registered CEAs during 1994 to 2003 were analyzed both for the whole time period and for two 5-year periods to study alterations over time. Four validation procedures of the registry were performed. Medical records were reviewed for both a random sample and a target sample (a total of 12% of the CEAs for asymptomatic stenosis). Swedvasc data were cross-matched with the In-Patient-Registry (used for reimbursement) and the Population-registry (death). RESULTS: A total of 6182 CEAs were registered, 671 being for asymptomatic stenosis. In the validation process, no missed registration of major stroke or death was found. Patients with asymptomatic stenosis had, when the whole time-period was analyzed, a perioperative combined stroke or death rate of 2.1%. Outcome improved over time; the combined stroke or death rate decreased from 3.3% (11/330) from 1994 to 1998 to 0.9% (3/341) from 1999 to 2003 (P = .026). During the second time period, no patient with a perioperative major stroke or death was reported. CONCLUSIONS: This extensively validated national audit of CEA for patients with asymptomatic carotid artery stenosis showed results well comparable with those of the randomized trials. The results improved over time. PMID- 16682167 TI - Chylous ascites after pelvic lymph node dissection for gynecologic cancer. PMID- 16682169 TI - Management of peritoneal surface malignancy with cytoreductive surgery and perioperative intraperitoneal chemotherapy. AB - AIMS: A new treatment strategy combining maximal cytoreductive surgery for treatment of macroscopic disease and maximal perioperative intraperitoneal chemotherapy for residual microscopic disease, suggests that in a selected group of patients benefit is possible. The purpose of this study was to report our experience with this combined treatment and to identify the principal prognostic factors. METHODS: The study included 266 patients from 9 institutions operated on between July 1990 and July 2004. The median age was 55 years. RESULTS: The mortality rate was 7.8% and the morbidity rate 37.5%. The overall median survival was 13.7 months. Positive independent prognostic factors by multivariate analysis were gender, perioperative intraperitoneal chemotherapy and treatment by the second-look procedure. CONCLUSIONS: The therapeutic approach combining cytoreductive surgery with perioperative intraperitoneal chemotherapy achieved long-term survival in a selected group of patients with an acceptable morbidity and mortality. PMID- 16682170 TI - Propolis-induced genotoxicity and antigenotoxicity in Chinese hamster ovary cells. AB - Propolis has been used in folk medicine since ancient times and is known for its antimicrobial, antiparasitic, antiviral, anti-inflammatory, antitumoral and antioxidant properties. In view of the great therapeutic interest in propolis and the small number of studies regarding its mechanism of action, the aim of the present study was to evaluate the mutagenic and antimutagenic effects of propolis using Chinese hamster ovary cells. Parameters such as the frequency of chromosome aberrations and mitotic index were analyzed. The results showed that, on one hand, the highest propolis tested concentration displayed a small but significant increase in the frequency of chromosome aberrations, and on the other hand, it was observed that the lowest tested concentration significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that propolis shows the characteristic of a "Janus" compound, i.e., propolis is genotoxic at higher concentrations, while at lower concentrations it display a chemopreventive effect on doxorubicin-induced mutagenicity. Flavonoids may be the components of propolis responsible for its both mutagenic and antimutagenic effects, once these compounds may act either as pro-oxidant or as free radicals scavenger, depending on its concentration. PMID- 16682171 TI - Determining treatment outcome in early intervention programs for autism spectrum disorders: a critical analysis of measurement issues in learning based interventions. AB - One of the areas receiving the greatest attention from researchers studying autism spectrum disorders in recent years involves psychologically based early intervention programs. Various claims of cure, marked improvement in social and communication skills, and improved I.Q. are among the conclusions that have been drawn by various researchers. However, little has been done to analyze the dependent variables used in these studies and their impact on the conclusions reached regarding treatment effectiveness. Obviously, this set of measures is crucial since these methods define which behaviors "improved" and to what extent. The present review analyzes the current status, strengths, and weaknesses of these measurements. PMID- 16682172 TI - The Src homology 2-containing inositol 5-phosphatase 1 (SHIP1) is involved in CD32a signaling in human neutrophils. AB - Phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P(3)) plays important signaling roles in immune cells, particularly in the control of activating pathways and of survival. It is formed by a family of phosphatidylinositol 3' kinases (PI3Ks) which phosphorylate PtdIns(4,5)P(2) in vivo. In human neutrophils, the levels of PtdIns(3,4,5)P(3) increase rapidly at the leading edge of locomoting cells and at the base of the phagocytic cup during FcgammaR mediated particle ingestion. Even though these, and other, data indicate that PtdIns(3,4,5)P(3) is involved in the control of chemotaxis and phagocytosis in human neutrophils, the mechanisms that regulate its levels have yet to be fully elucidated in these cells. We evaluated the potential implication of SHIP1 and PTEN, two lipid phosphatases that utilize PtdIns(3,4,5)P(3) as substrate, in the signaling pathways called upon in response to CD32a cross-linking. We observed that the cross-linking of CD32a resulted in a transient accumulation of PtdIns(3,4,5)P(3). CD32a cross-linking also induced the tyrosine phosphorylation of SHIP1, its translocation to the plasma membrane and its co-immunoprecipitation with CD32a. CD32a cross-linking had no effect on the level of serine/threonine phosphorylation of PTEN and did not stimulate its translocation to the plasma membrane. PP2, a Src kinase inhibitor, inhibited the tyrosine phosphorylation of SHIP1 as well as its translocation to the plasma membrane. Wortmannin, a PI3K inhibitor, had no effect on either of these two indices of activation of SHIP1. Our results indicate that SHIP1 is involved, in a Src kinase-dependent manner, in the early signaling events observed upon the cross-linking of CD32a in human neutrophils. PMID- 16682174 TI - Subcutaneous delivery of insulin loaded poly(fumaric-co-sebacic anhydride) microspheres to type 1 diabetic rats. AB - The method of phase inversion nanoencapsulation (PIN) and microencapsulation was used to produce biodegradable poly(fumaric-co-sebacic anhydride) (p(FASA)) microspheres that contain insulin. Microspheres were characterized by SEM and a laser light scattering technique to determine particle size distribution. Insulin stability was determined by RP and SEC HPLC. Release rate studies were conducted and microspheres were administered subcutaneously (SQ) to type 1 diabetic rats to determine the bioactivity of insulin at three different dosages. Pharmacokinetic parameters for SQ experiments were measured using the trapezoidal rule by plotting average plasma insulin level (PIL) vs. time and determining peak concentration (CP), the time of peak concentration (TP), duration of PIL curve (D), and relative bioavailability (RB). When our insulin containing formulation was analyzed by HPLC, there was no evidence of high molecular weight transformation (HMWT) or deamidated products. In addition, we effectively altered the onset, peak, and duration of insulin action after SQ injection. PMID- 16682173 TI - Independent roles of Drosophila Moesin in imaginal disc morphogenesis and hedgehog signalling. AB - The three ERM proteins (Ezrin, Radixin and Moesin) form a conserved family required in many developmental processes involving regulation of the cytoskeleton. In general, the molecular function of ERM proteins is to link specific membrane proteins to the actin cytoskeleton. In Drosophila, loss of moesin (moe) activity causes incorrect localisation of maternal determinants during oogenesis, failures in rhabdomere differentiation in the eye and alterations of epithelial integrity in the wing imaginal disc. Some aspects of Drosophila Moe are related to the activity of the small GTPase RhoA, because the reduction of RhoA activity corrects many phenotypes of moe mutant embryos and imaginal discs. We have analysed the phenotype of moesin loss-of-function alleles in the wing disc and adult wing, and studied the effects of reduced Moesin activity on signalling mediated by the Notch, Decapentaplegic, Wingless and Hedgehog pathways. We found that reductions in Moesin levels in the wing disc cause the formation of wing-tissue vesicles and large thickenings of the vein L3, corresponding to breakdowns of epithelial continuity in the wing base and modifications of Hedgehog signalling in the wing blade, respectively. We did not observe any effect on signalling pathways other than Hedgehog, indicating that the moe defects in epithelial integrity have not generalised effects on cell signalling. The effects of moe mutants on Hedgehog signalling depend on the correct gene-dose of rhoA, suggesting that the requirements for Moesin in disc morphogenesis and Hh signalling in the wing disc are mediated by its regulation of RhoA activity. The mechanism linking Moesin activity with RhoA function and Hedgehog signalling remains to be elucidated. PMID- 16682175 TI - Determination of Pulmozyme (dornase alpha) stability using a kinetic colorimetric DNase I activity assay. AB - An enzymatic activity assay was developed for the determination of dornase alpha human recombinant desoxyribonuclease (DNase I) stability. The method was adapted from a colorimetric endpoint enzyme activity assay for DNase I based on the degradation of a DNA/methyl green complex. With the described modifications the kinetic measurement of enzyme activity is feasible on an automated analyzer system within a rather short time. The development of this assay was based on the need for reliable detection of a possible loss of enzyme activity after transferring the commercial therapeutic agent into sealed glass vials required for a placebo-controlled study. The measuring range of this stability test was from 0 to 3000 U/L corresponding to 0-120% of the original enzyme activity; CV values of control solutions inside the measuring range were between 3% and 5%. The enzyme activity decreased less than 15% during the observation period of 180 days. In conclusion the current kinetic assay is a reliable method for a simple time-saving determination of DNase I activity to test Pulmozyme stability as required for quality control. As dornase alpha is used for inhalation, this method also proved its reliability in testing DNase stability during aerosolization with new inhalation devices (e-flow). PMID- 16682176 TI - Quantification of the compactibility of pharmaceutical powders. AB - The purpose of this study is to investigate and to quantify the compactibility of pharmaceutical powders by a simple linear relationship between the diametral compressive strength of tablets and the applied compaction pressure. The mechanical strength of the tablets is characterized as the crushing force normalized with the dimension of the tablet and termed the specific crushing strength, SCS. The proposed model: SCS=Cp*P+b estimates the slope of the regression line Cp as a dimensionless compactibility parameter and is reported with the corresponding standard deviation SCp. The linear region of the compactibility profile is selected using the 95% predictability limits bordering the regression line. Eleven different materials were tested and acceptable fits to the linear model were observed in all cases. The ability of the model to discriminate between the investigated materials is excellent, in cases where the difference may be difficult to show a simple t-test is used as an inference tool. No difference was found between lactose tablets of different masses (500 and 1000 mg). A relationship between the compactibility parameter and the compressibility characterized by the Walker coefficient is demonstrated. PMID- 16682177 TI - In vitro phototoxicity of 5-aminolevulinic acid and its methyl ester and the influence of barrier properties on their release from a bioadhesive patch. AB - Topical administration of excess exogenous 5-aminolevulinic acid (ALA) leads to selective accumulation of the potent photosensitiser protoporphyrin IX (PpIX) in neoplastic cells, which can then be destroyed by irradiation with visible light. Due to its hydrophilicity, ALA penetrates deep lesions, such as nodular basal cell carcinomas (BCCs) poorly. As a result, more lipophilic esters of ALA have been employed to improve tissue penetration. In this study, the in vitro release of ALA and M-ALA from proprietary creams and novel patch-based systems across normal stratum corneum and a model membrane designed to mimic the abnormal stratum corneum overlying neoplastic skin lesions were investigated. Receiver compartment drug concentrations were compared with the concentrations of each drug producing high levels of PpIX production and subsequent light-induced kill in a model neoplastic cell line (LOX). LOX cells were found to be quite resistant to ALA- and M-ALA-induced phototoxicity. However, drug concentrations achieved in receiver compartments were comparable to those required to induce high levels of cell death upon irradiation in cell lines reported in the literature. Patches released significantly less drug across normal stratum corneum and significantly more across the model membrane. This is of major significance since the selectivity of PDT for neoplastic lesions will be further enhanced by the delivery system. ALA/M-ALA will only be delivered in significant amounts to the abnormal tissue. PpIX will only then accumulate in the neoplastic cells and the normal surrounding tissue will be unharmed upon irradiation. PMID- 16682178 TI - Guest editorial letter: Molecular machines in the matrix? PMID- 16682179 TI - Adenosine cardioprotection study in clinical setting of paroxysmal supraventricular tachycardia. AB - PSVT attack of >20min and frequency >160 is well-recognized model of myocardial dysfunction. We measured 6-keto-PGF1alpha and TXB(2) before and after adenosine administration to assess its cardioprotective potential. A total of 64 patients were randomly assigned as having acute episode of PSVT to adenosine or verapamil group. A bolus of 6mg of adenosine up to the maximum dose of 12 or 5mg of verapamil up to the maximum dose of 10mg were given, until the sinus rhythm was restored. The levels of PGI(2), TXA(2) and TAS were measured in three different time intervals. In adenosine group all parameters were normalized after 20min of conversion to sinus rhythm. The ratio of PGI(2)/TXA(2) increased after 5min of conversion to SR (P<0.01). Also, the ratio of TXA(2)/TAS was decreased for ADO (P<0.01). This is the first study to demonstrate that adenosine exerts cardioprotective effect. PMID- 16682180 TI - The maintenance and masking of chromosome termini. AB - Telomeres, the natural termini of eukaryotic chromosomes, have been the subject of intense interest during the last decade because of the roles that these chromosome termini perform in both cancer and aging. As we become more cognizant of the consequences of telomere dysfunction on several aspects of human health, significant attention is focused on understanding at a molecular level how the many telomere-associated factors perform their activities. PMID- 16682183 TI - Delivering cancer stem cell therapies - a role for nanomedicines? AB - Cancer stem cells (CSCs), i.e. cancer cells that can self-renew, constitute only a minority of the cells of a tumour, but, because of their ability to initiate and repopulate tumours, failure to control CSCs can potentially lead to tumour re growth, even though the bulk tumour may have been treated successfully. Nanomedicines improve spatio-temporal control over drug kinetics and distribution, thus opening the prospect of safer and more specific therapies to address the challenges posed by CSCs. In particular, these systems have the potential to facilitate CSC-aware therapy by overcoming resistance to conventional cytotoxic drugs and by targeting novel therapies to the tumour and CSC-marker positive cells. This review examines the implications of the CSC paradigm specifically for the development of nanomedicines, i.e. therapies based on macromolecules or supramolecular aggregates. PMID- 16682182 TI - Transport of messenger RNA from the nucleus to the cytoplasm. AB - All movement of molecules and macromolecules between the cytoplasm and the nucleus takes place through nuclear pore complexes (NPCs), very large macromolecular complexes that are the only channels connecting these compartments. mRNA export is mediated by multiple, highly conserved protein factors that couple steps of nuclear pre-mRNA biogenesis to mRNA transport. Mature messenger ribonucleoproteins (mRNPs) diffuse from sites of transcription to NPCs, although some active genes are positioned at the nuclear periphery where they interact physically with components of NPCs. As properly processed mRNPs translocate through the pore, certain mRNP proteins are removed, probably through the enzymatic action of the DEAD-box helicase Dbp5, which binds to Nup159 and Gle1, components of the cytoplasmic filaments of the NPC. Gle1 and the phosphoinositide IP6 activate Dbp5's ATPase activity in vitro and this could provide critical spatial regulation of Dbp5 activity in vivo. PMID- 16682184 TI - Identification of potent 5-pyrimidinyl-2-aminothiazole CDK4, 6 inhibitors with significant selectivity over CDK1, 2, 5, 7, and 9. AB - 5-Pyrimidinyl-2-aminothiazole 1 was identified as an inhibitor of cyclin dependent kinases (CDKs) by a screening of the Merck sample repository. The introduction of a methyl group at the C-5 or C-6 position on the pyrimidine ring, directed toward the gate keeper residue of CDK4 (Phe93), led to significant enhancement of selectivity for CDK4 over other CDKs. Compound 3 exhibited more than 300-fold selectivity for CDK4 over CDK1, 2, 5, 7, and 9. Subsequent improvements in aqueous solubility afforded compound 4, which is available for further in vivo studies and this compound inhibited pRb phosphorylation and BrdU incorporation in tumor models. PMID- 16682185 TI - Highly selective and potent agonists of sphingosine-1-phosphate 1 (S1P1) receptor. AB - Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4>10,000 fold, S1P1/S1P5>600-fold, while EC50 (S1P1) <0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-lowering effect. PMID- 16682186 TI - Synthesis and evaluation of N-acyl sulfonamides as potential prodrugs of cyclin dependent kinase inhibitor JNJ-7706621. AB - A novel prodrug strategy for cyclin-dependent kinase inhibitor JNJ-7706621 has been explored. Through N-acylation of a sulfonamide substituent, tails containing different solubilizing groups (amino, carboxyl, alkoxyl, and hydroxyl) were attached to JNJ-7706621. Most of the prodrugs exhibited good aqueous solubility and the N-acyl groups on the sulfonamide were metabolically cleaved to generate active drug in rat PK study. PMID- 16682187 TI - Novel class of cyclophosphamide prodrug: cyclophosphamide spiropiperaziniums (CPSP). AB - A novel class of cyclophosphamide spiropiperaziniums was synthesized and evaluated for their in vivo anti-cancer activities against S180 and H22. Most of them exhibited definite activities. Especially, compounds 8b and 8k showed good anti-cancer activities, meanwhile, 8k also showed much lower toxicity than CP. Several interesting structure-activity relationships were revealed. PMID- 16682188 TI - Critical modifications of the ISO-1 scaffold improve its potent inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity. AB - Based on the scaffold of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of the proinflammatory cytokine MIF, two critical modifications and chiral resolution have significantly improved the potency of the inhibition. Compound (R)-17 is 20-fold more potent than ISO-1 and inhibits MIF tautomerase activity with an IC(50) of 550 nM. PMID- 16682189 TI - QSAR analysis of some 5-amino-2-mercapto-1,3,4-thiadiazole based inhibitors of matrix metalloproteinases and bacterial collagenase. AB - A quantitative structure-activity relationship (QSAR) study has been performed on 5-amino-2-mercapto-1,3,4-thiadiazole based inhibitors of matrix metalloproteinases (MMPs) and a bacterial collagenase known as Clostridium histolyticum collagenase (ChC) to understand the structural features influencing the affinity of these inhibitors towards the enzyme. The compounds in the selected series were characterized by topological and fragmental descriptors calculated using QuaSAR module of molecular operating environment (MOE). An indicator variable was also assigned to account for the presence of amide function in vicinity of sulfonamide group in the parent structure. Correlations between different inhibitory activities and calculated predictor variables were established through stepwise multiple regression employing the method of least squares. The results of the study indicates that MMP inhibitory activity of 5 amino-2-mercapto-1,3,4-thiadiazoles can be successfully explained in terms of topology of the molecule. The obtained correlations also suggest that increase in the number of fluorine atoms in the aromatic ring will augment inhibitory activity of these molecules against all the MMPs probably by virtue of hydrogen bond interaction with some complementary groups in the active site of the enzymes. One prime requirement for better inhibition of MMPs (except for MMP-1) and ChC identified from the present study is the presence of amide function in vicinity of sulfonamide group in the parent structure as suggested by the presence of indicator variable in almost all correlations. While MMP-1 and ChC inhibitory activity of the compounds studied is shown to be dependent on Kier's first order carbon valence molecular connectivity index indicating that increase in branching and presence of heteroatoms in the molecule will improve the MMP-1 and ChC inhibitory potency of 5-amino-2-mercapto-1,3,4-thiadiazoles, correlations derived for other enzymes (MMP-2, MMP-8, MMP-9) are quite similar. In addition to the number of fluorine atoms and presence of indicator variable, MMP-2, MMP-8 and MMP-9 inhibitory activity of 5-amino-2-mercapto-1,3,4-thiadiazoles is found to be dependent on Kier's alpha modified index of third order in such a way that infer, terminally branched functions will increase the affinity of these molecules to the MMPs. PMID- 16682190 TI - Small-molecule microarrays: development of novel linkers and an efficient detection method for bound proteins. AB - Novel isocyanate and diazoketone linkers possessing polyoxypropylenediamine as a spacer for small-molecule microrray are developed. White light interferometry is introduced to detect bound proteins on the glass slides without using chemically modified proteins. PMID- 16682191 TI - Opioid receptor binding and antinociceptive activity of the analogues of endomorphin-2 and morphiceptin with phenylalanine mimics in the position 3 or 4. AB - Endomorphin-2 (EM-2) and morphiceptin are the same class of putative mu-opioid receptor ligands. To investigate the effectiveness of phenylglycine (Phg, L or D) and homophenylalanine (Hfe) as the surrogates of phenylalanine in the position 3 and/or 4 of them, a series of their analogues were synthesized. Opioid receptor binding affinities were determined. Two analogues, [Hfe3]EM-2 and [Phg4] (EM 2/morphiceptin), showed different but potent antinociceptive activity in mouse hot-plate test, the results combined with their half-lives of degradation by mouse brain homogenate could also present some evidence to the in vivo degradative mechanism of EM-2. PMID- 16682192 TI - DNA sequence recognition by Hoechst 33258 conjugates of hairpin pyrrole/imidazole polyamides. AB - A series of hairpin pyrrole/imidazole polyamides linked to a Hoechst 33258 (Ht) analogue (5-7) were synthesized on solid-phase by adopting an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. The dsDNA binding properties of Ht-polyamides 5-7 were determined by thermal denaturation experiments. Hairpin Ht-polyamides 5-7 bound to dsDNA sequences 16 and 18 show DeltaTm values that are 14-18 degrees higher than linear Ht-polyamides bound to the same sequences. All three Ht-polyamides were found to be selective for their 9-bp match dsDNA sequences, supporting a relative stronger interaction of an Im/Py anti-parallel dimer with an appropriately positioned G/Cbp rather than sequences containing only A/Tbps. In addition, Ht-polyamides 5 and 7 showed a 20 fold preference for a properly placed G/Cbp over a C/Gbp, while 6 showed a 10 fold preference. PMID- 16682193 TI - Discovery and SAR of 2-amino-5-(thioaryl)thiazoles as potent and selective Itk inhibitors. AB - A series of structurally novel aminothiazole based small molecule inhibitors of Itk were prepared to elucidate their structure-activity relationships (SARs), selectivity, and cell activity in inhibiting IL-2 secretion in a Jurkat T-cell assay. Compound 3 is identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo and was previously reported to reduce lung inflammation in a mouse model of ovalbumin induced allergy/asthma. PMID- 16682194 TI - Synthesis and in vitro anti-tumor activity of N-{1-[(3-thioxo-5,6 dihydroimidazo[2,1-c][1,2,4]thiadiazol-7-ylthio)thiocarbonyl]-2 imidazolidene}arylsulfonamides. AB - A series of N-{1-[(3-thioxo-5,6-dihydroimidazo[2,1-c][1,2,4]thiadiazol-7 ylthio)thiocarbonyl]-2-imidazolidene}arylsulfonamides (2a-z) was obtained by reacting 6,7-dihydro-1H-imidazo[2,1-c][1,2,4]thiadiazol-3-thione (1) with arylsulfonyl chlorides. The relationships between structure and anti-tumor activity revealed that compound 2o with p-Cl substituent at the phenyl ring was most active (-log GI50>8.00, -log TGI=7.66) and was found to exhibit high selectivity toward the leukemia CCRF-CEM cell line (Deltaf=3.08 and 3.31, respectively). PMID- 16682195 TI - Novel gamma-secretase inhibitors discovered by library screening of in-house synthetic natural product intermediates. AB - Screening of our in-house compound library comprised of intermediates of natural product synthesis projects resulted in discovering two novel gamma-secretase inhibitors, which coincidently had similar moieties, that is, cyclohexenone and two aryl groups arranged on the core six-membered ring. Structure-activity relationship studies of these compounds were also developed. PMID- 16682196 TI - Cyclobutane derivatives as potent NK1 selective antagonists. AB - A series of novel cyclobutane derivatives as potent and selective NK1 receptor antagonists is described. Several compounds in this series exhibited high in vitro binding affinity (Ki 50 microM), an effect that was unrelated to the inhibition of ABA biosynthesis, seedling growth was not affected by 100 microM abamineSG. These results suggest that abamineSG is a more potent and specific inhibitor of ABA biosynthesis than abamine. PMID- 16682206 TI - 4'-Fluorinated carbocyclic nucleosides: synthesis and inhibitory activity against S-adenosyl-L-homocysteine hydrolase. AB - 4'-Fluorinated analogue of 9-[(1'R,2'S,3'R)-2',3'-dihydroxy-cyclopentan-1' yl]adenine (DHCaA) and their related analogues were systematically synthesized under the Mitsunobu and palladium(0) coupling conditions followed by fluorination with inversion of the configuration by using diethylaminosulfur trifluoride, respectively. 4'-beta-Fluoro DHCaA and 2-amino-4'-alpha-fluoro DHCaA demonstrated slight inhibitory activity against human and Plasmodium falciparum S-adenosyl-L homocysteine hydrolase, respectively. PMID- 16682207 TI - An easy preparation of 'monolithic type' hydrophilic solid phase: capability for affinity resin to isolate target proteins. AB - An easy preparation method of monolithic type hydrophilic solid phase was discussed. Newly invented functional monomer with a hydrophilic cross-linking agent was co-polymerized to realize well-controlled monolithic co-continuous structure by use of diethylene glycol as porogenic solvent. We were able to control the content of the functional monomer up to 40 vol% without loss of monolithic structure. Those prepared were utilized as affinity resins after immobilization of FK506, an immunosuppressive drug as a ligand. It was found that the affinity resins prepared were hydrophilic enough to eliminate non-specific adsorption of proteins, while two of the target proteins of FK506 tested were successfully captured. PMID- 16682208 TI - Synthesis and evaluation of glycosidase inhibitory activity of N-butyl 1-deoxy-D gluco-homonojirimycin and N-butyl 1-deoxy-L-ido-homonojirimycin. AB - Conjugate addition of n-butyl amine to d-glucose derived alpha,beta-unsaturated ester 4 afforded beta-amino esters 5a,b that on reduction of ester group, 1,2 acetonide deprotection, and reductive amination led to the formation of corresponding N-butyl 1-deoxy-D-gluco-homonojirimycin 2c and N-butyl 1-deoxy-L ido-homonojirimycin 2d which were found to be selective beta-glucosidase inhibitors with an IC(50) value in millimolar range. PMID- 16682210 TI - The new models of the human telomere d[AGGG(TTAGGG)3] in K+ solution. AB - The human telomeric sequence d[AGGG(TTAGGG)(3)] has been found to form different types of G-quadruplex structures. NMR revealed that in Na(+) solution this 22 nucleotide (nt) sequence exhibits an antiparallel structure, whereas crystallographic studies in the presence of K(+) showed a dramatically different parallel structure. The structure of this 22 nt sequence in the presence of K(+) has drawn intense interest as the intracellular K(+) concentration is greater than that of Na(+). However, the question of the type of structure for the 22 nt telomeric sequence in K(+) solution remains open. In this study, we substituted the Gs in the sequence with 8-bromoguanine and examined the resultant structures and thermal stabilities by circular dichroism (CD) spectroscopy. The results suggest that the 22 nt in K(+) solution exists as a mixture of mixed parallel/antiparallel and chair-type G-quadruplex. To date, the exact structure of human telomeric G-quadruplex in K(+) solution is extremely controversial. The present study provides valuable information for understanding the discrepancies between the crystal and solution studies. We discuss the possible implications of the structure in understanding higher-order telomeric DNA structure and T-loop formation. PMID- 16682209 TI - Synthesis and biological evaluation of novel heterocyclic ionone-like derivatives as anti-inflammatory agents. AB - Five- and six-membered heterocyclic ionone-like derivatives 4-6 have been synthesised in one step and with good yield from the key intermediate 3a and appropriate bifunctional reagents. Four were active as inhibitors of the respiratory burst of human neutrophils without affecting cell viability. The two most active compounds (5a,d) tested in neutrophil migration assays, were also found to be potent inhibitors of neutrophil chemotactic responsiveness. These two molecules could be considered as lead compounds of new drugs which can be an effective tool to treat psoriasis and related neutrophilic dermatoses. PMID- 16682211 TI - One novel quinoxaline derivative as a potent human cyclophilin A inhibitor shows highly inhibitory activity against mouse spleen cell proliferation. AB - Cyclophilin A (CypA) is a ubiquitous cellular enzyme playing critical roles in many biological processes, and its inhibitor has been reported to have potential immunosuppressive activity. In this work, we reported a novel quinoxaline derivative, 2,3-di(furan-2-yl)-6-(3-N,N-diethylcarbamoyl-piperidino)carbonylamino quinoxaline (DC838, 3), which was confirmed to be a potent inhibitor against human CypA. By using the surface plasmon resonance (SPR) and fluorescence titration techniques, the kinetic analysis of CypA/DC838 interaction was quantitatively performed. CypA peptidyl prolyl cis-trans isomerase (PPIase) activity inhibition assay showed that DC838 demonstrated highly CypA PPIase inhibitory activity. In vivo assay results showed that DC838 could inhibit mouse spleen cell proliferation induced by concanavalin A (Con A). Molecular docking simulation further elucidated the specific DC838 binding to CypA at the atomic level. The current work should provide useful information in the discovery of immunosuppressor based on CypA inhibitor. PMID- 16682212 TI - Pedunculopontine neurons are involved in network changes in the kindling model of temporal lobe epilepsy. AB - It is well known that epileptogenesis is associated with widespread neuronal network changes in brain regions adjacent to the seizure focus but also in remote structures including basal ganglia. Besides the superior colliculus, the pedunculopontine tegmental nucleus (PPN) is one of three main target regions of basal ganglia output activity and is reciprocally connected with the substantia nigra pars reticulata (SNr), which is critically involved in seizure propagation and manipulation. We here tested the hypothesis if, apart from the traditional view that the superior colliculus mediates seizure-gating mechanisms of the SNr, the PPN is involved in kindling-induced network changes. Rats were electrically kindled via the ipsilateral basolateral amygdala. In vivo extracellular single unit recordings of right PPN neurons were performed in kindled rats 1 day after a generalized seizure in order to examine kindling-associated rather than seizure associated activity changes. The main findings of the study were (1) a seizure outlasting drastically reduced firing rate of PPN neurons and (2) an increase in burst and irregular firing pattern in kindled rats compared with sham-kindled and naive controls. These changes are likely to be caused by an altered inhibitory input from the SNr. Furthermore, kindling caused (3) the oscillation frequency of PPN neurons to shift towards lower frequencies. The kindling-induced activity changes were found to be anatomically restricted to the PPN, indicating that network changes follow distinct anatomical routes. We demonstrated that the PPN is strongly affected by the functional reorganization of neurocircuitry associated with kindling. The underlying mechanisms are discussed. The findings are relevant for a better understanding of kindling-associated network changes and might provide new targets for therapeutic manipulations in epilepsies. PMID- 16682213 TI - Preoperative C-reactive protein is predictive of long-term outcome after coronary artery bypass surgery. AB - BACKGROUND: Increased levels of C-reactive protein (CRP) are associated with the presence and severity of atherosclerosis, and with increased risk of coronary events as well as of cardiac events after coronary percutaneous intervention. METHODS: We have investigated whether preoperative CRP had an impact on the long term outcome of 843 patients who underwent on-pump coronary artery bypass surgery (CABG). RESULTS: Among operative survivors, patients with preoperative CRP < 1.0 mg/dL had significantly better 12-year overall survival rate (74.1% vs 63.0%, p = 0.004) and survival freedom from fatal cardiac event (86.7% vs 78.1%). Multivariate analysis including patients' age, extracardiac arteriopathy, urgent/emergent operation, recent myocardial infarction, congestive heart failure, left ventricular ejection fraction, atrial fibrillation, transient ischemic attack/stroke, number of distal anastomoses, diabetes, and preoperative CRP > or = 1.0 mg/dL or <1.0 mg/dL, showed that the latter was an independent predictor of late all-cause mortality (p = 0.017, RR 1.60, 95% CI 1.09-2.35). Its impact on overall survival was particularly evident in patients with left ventricular ejection fraction <50% (CRP < 1.0 mg/dL: 58.7% vs CRP > or = 1.0 mg/dL: 43.7%, p < 0.00001). CONCLUSIONS: Increased preoperative levels of CRP are associated with significantly decreased overall survival after primary on-pump CABG. PMID- 16682214 TI - Epigenetic and proteolytic inactivation of 14-3-3sigma in breast and prostate cancers. AB - 14-3-3sigma is an epithelial marker whose expression is induced by DNA damage through a p53-dependent pathway. 14-3-3sigma functions sequesters cyclin B1-CDC2 complexes outside the nucleus and thereby contributes to a G2 arrest. Down regulation or lack of 14-3-3sigma is a frequent event in breast and prostate cancers. Epigenetic silencing by CpG methylation, p53 inactivation, and proteasome-dependent proteolysis leads to loss of 14-3-3sigma. Hypermethylation of the 14-3-3sigma gene is often observed in precancerous lesions and likely to be causally linked to the onset of cancer. Proteolytic inactivation of 14-3 3sigma has been recently found in breast and prostate cancers. In breast cancer, the estrogen-responsive E3 ubiquitin ligase Efp specifically targets 14-3-3sigma for degradation. The E2 ubiquitin conjugating enzyme UBC8 and Efp also mediates ISG15 modification of 14-3-3sigma. Detection of 14-3-3sigma inactivation on the protein or DNA methylation level may be used for cancer prognosis. Furthermore, 14-3-3sigma may be a potential therapeutic target in breast and prostate cancer. PMID- 16682215 TI - The IgLON protein Lachesin is required for the blood-brain barrier in Drosophila. AB - In the mammalian peripheral nervous system, nerve insulation depends on the integrity of paranodal junctions between axons and their ensheathing glia. Ultrastructurally, these junctions are similar to the septate junctions (SJ) of invertebrates. In Drosophila, SJ are found in epithelia and in the glia that form the blood-brain barrier (BBB). Drosophila NeurexinIV and Gliotactin, two components of SJ, play an important role in nerve ensheathment and insulation. Here, we report that Drosophila Lachesin (Lac), another SJ component, is also required for a functional BBB. In the developing nervous system, Lac is expressed in a dynamic pattern by surface glia and a subset of neurons. Ultrastructural analysis of Lac mutant embryos shows poorly developed SJ in surface glia and epithelia where Lac is expressed. Mutant embryos undergo a phase of hyperactivity, with unpatterned muscle contractions, and subsequently become paralyzed and fail to hatch. We propose that this phenotype reflects a failure in BBB function. PMID- 16682216 TI - GSK3beta and PKCzeta function in centrosome localization and process stabilization during Slit-mediated neuronal repolarization. AB - In comparison with other migratory cells, neurons exhibit a unique, highly polarized morphology and a distinctive pattern of movement. This migration consists of a repeating of three distinct phases: neurite outgrowth, movement of the centrosome into the leading process, and translocation of the nucleus towards the centrosome. The direction of movement is under the control of extracellular guidance cues, but mechanisms by which these determine neuronal polarity, centrosome position, and neuronal movement are not well understood. We found that in primary olfactory bulb neuronal precursors, Slit-mediated repolarization consisted of growth of a new process from the previous trailing edge, then reorientation of the centrosome followed by nuclear translocation in the reverse direction. Inhibition of cell polarity factors GSK3beta or PKCzeta resulted in impaired centrosome reorientation and process stabilization. Our findings suggest that activation of cell polarity signaling and positioning of the centrosome ahead of the nucleus are important steps in repolarization in response to guidance cues. PMID- 16682217 TI - Manufacturing and supply of monovalent oral polio vaccines. AB - A new polio vaccine was developed, produced and licensed by sanofi pasteur at the request of the World Health Organization (WHO) for mass immunization campaigns in endemic countries such as Egypt. The new vaccine, monovalent oral polio vaccine 1 or mOPV1, is currently used in Egypt as a critical part of a new WHO strategy to end polio type 1 transmission by the end of the year 2005 (types 2 and 3 polioviruses have already been eliminated from Egypt). To answer this specific need, an urgent program was mounted by Sanofi pasteur to manufacture 50million doses for Egypt, in close collaboration with WHO and National Regulatory Agencies (France and Egypt). The joint efforts between manufacturer, regulators and the WHO resulted in the quickest ever vaccine development and licensure and WHO pre qualification. The production of mOPV was based on existing tOPV but with appropriate "change control" procedures to assure the quality of the product, and to distinguish mOPV from tOPV. Key success factors included clear and careful definition of the project; close collaboration between manufacturer, regulators and WHO; and commitment and motivation of staff. As a result, development and production of mOPV1 vaccine were carried out in a drastically reduced time period, leading to the release and delivery of the first 15 million doses of mOPV1 in April 2005. PMID- 16682218 TI - Production, testing and perspectives of IPV and IPV combination vaccines: GSK biologicals' view. AB - GSK Biologicals has been involved in the production of Polio vaccine since the early start of Polio vaccination, beginning with the first generation of Inactivated Polio Vaccine (IPV). Over time, the company has developed solid industrial experience and knowledge that significantly contributes today to the quality of our Polio vaccines. GSK Biologicals' current IPV is now routinely produced according to the process defined by Van Wezel (RIVM) in the late seventies, using Vero cells and micro-carrier technology in bioreactors. In addition to compliance with current requirements (World Health Organization, European Pharmacopoeia, Code of Federal Regulations USA), the quality of the routine vaccine is guaranteed by numerous additional data related to the characterization, to the consistency, and to the validation of the process and the testing. This supplementary data package will allow, for instance, for the application of the in vitro potency testing for routine release instead of the in vivo testing. The present views on the Polio vaccine strategy for the post eradication era have portrayed a very limited role for the current IPV. The main reasons relate to post-eradication bio-containment needs and to production capacity and costs. A reevaluation of the classic approach taken to the use of the current IPV produced from wild type polio strains positions this vaccine as a real alternative to other strategies, allowing us to take advantage of the excellent performance of IPV over many years. PMID- 16682219 TI - First decade (1950-1960) of studies and trials with the polio vaccine. AB - The chronology of the development of polio vaccines following the first human trials of attenuated poliovirus vaccine in 1950 is described by me as a witness to the first decade of trials and tribulations following my discovery of polio vaccine in 1950. Mass vaccination trials are considered to be the most important phase of the discovery of oral polio vaccine (OPV). These took place in the Belgian Congo, Poland, Croatia, Switzerland, and finally in the former Soviet Union. By 1960, approximately 13 million individuals had been vaccinated with the Koprowski oral polio vaccine and over 11 million with the Sabin vaccine. PMID- 16682220 TI - Interrupting poliovirus transmission -- new solutions to an old problem. AB - Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, knowledge as to the nature of circulating polioviruses and the challenges to their interruption has increased tremendously, particularly during the period 2000-2005. By January 2006, however, the systematic application of the standard polio eradication strategies, combined with recent refinements, had reduced the number of countries with ongoing transmission of indigenous wild polioviruses to just four (Nigeria, India, Pakistan, and Afghanistan), the lowest ever in history. In addition, only 8 of the 22 areas that had been re-infected by wild poliovirus in 2003-2005 still required large-scale 'mop-up' activities and circulating vaccine-derived poliovirus (cVDPV) outbreaks were being readily addressed. This progress, despite new challenges late in the GPEI, was greatly facilitated by a range of solutions that included two new monovalent oral polio vaccines (mOPVs), new and robust international standards for polio outbreak response, and renewed political commitment across the remaining infected countries. PMID- 16682221 TI - Regulation and standardization of IPV and IPV combination vaccines. AB - Inactivated poliovirus vaccine (IPV) is not only increasingly used on a global basis but also is a component of many combination vaccines. Standardization and control of IPV continues to be a challenge for manufacturers and regulators. A rat immunogenicity assay is currently recommended by many authorities, including WHO, as the definitive in vivo potency. Alternative in vitro assays to determine D-antigen content have been developed and are routinely used in some countries to assess IPV potency assay. However, the other less reliable in vivo immunogenicity assays are also used (e.g. monkey, chick). We review some of the regulatory challenges facing current and future IPV assessment, with a focus on the relevance of in vivo and in vitro tests, considerations for Sabin derived IPV and discussion of future efforts for standardization. PMID- 16682222 TI - No evidence for prolonged excretion of polioviruses in persons with residual paralytic poliomyelitis in Ethiopia, Pakistan and Guatemala. AB - Persons who have developed acute flaccid paralysis following infection with wild type polioviruses or vaccine-associated paralytic poliomyelitis usually excrete polioviruses for only a few weeks. However, some patients with paralytic poliomyelitis have had prolonged excretion of polioviruses for periods of up to 10 years after onset of disease. Most prolonged excretors have been identified in industrialized countries. We studied 348 patients 2-28 years old in Ethiopia, Pakistan and Guatemala with residual paralytic poliomyelitis to determine if they had IgA or IgG deficiency or persistent poliomyelitis excretion at least 1 year after onset of disease. None of the 348 affected individuals had IgG deficiency or persistent poliovirus excretion. One child had borderline low serum IgA concentration. Since we did not study children under 2 years of age, persons born with IgG deficiency disorders may have died in developing countries where replacement immunoglobulin therapy is not readily available. Nevertheless, persistent poliovirus excretion among persons 2 years of age and older with residual paralytic poliomyelitis is uncommon in developing countries. PMID- 16682223 TI - Poliovirus excretion in Guatemalan adults and children with HIV infection and children with cancer. AB - More than 20 patients with persistent poliovirus infections have been identified and reported to WHO. To date, almost all of these patients have had B-cell immune deficiency disorders. Since there are limited data on patients with HIV infection who have received oral poliovirus vaccine (OPV), we studied adults and children to determine if persons with acquired immunodeficiency due to HIV infection or cancer chemotherapy in a developing country setting had prolonged excretion of polioviruses. Stool samples from 94 HIV-infected children and 101 adults and 50 children surviving cancer in Guatemala City were cultured for polioviruses. No polioviruses were detected in any of the 195 persons with HIV infection or the 50 with cancer. The evidence from this and other studies indicates that the persistent poliovirus excretion in HIV-infected individuals is an unlikely event. PMID- 16682224 TI - A vision of a world without polio: the OPV cessation strategy. AB - Once the eradication of wild poliovirus has been confirmed, the public health benefits of routine immunization with OPV will no longer outweigh the burden of disease either due to paralysis caused by OPV (vaccine associated paralytic polio), or by outbreaks caused by circulating vaccine-derived polioviruses. The eventual cessation of OPV use in routine immunization programmes worldwide will become necessary to assure a lasting eradication of polio. As the world moves towards polio eradication and its certification, preparations are therefore being intensified for OPV cessation, and the risk management framework for safe OPV cessation is being put in place. The framework includes bio-containment of all known poliovirus and potentially infected substances, development of an international stockpile of oral polio vaccine, ensuring a mechanism for continued global surveillance and response for polio after eradication has been certified, and national policies if countries decide to continue vaccinating with inactivated polio vaccine (IPV). It is ironic that the vaccine on which the world has depended for polio eradication will itself become a risk to eradication once the transmission of wild poliovirus has been interrupted. Final preparations for the eventual global and simultaneous cessation of OPV will require the same level of international cooperation and coordination that has brought the world to the verge of polio eradication. PMID- 16682225 TI - Post-eradication poliovirus facility-associated community risks. AB - Minimizing the risk of poliovirus transmission from the poliovirus facility to an increasingly susceptible community is crucial when global poliovirus transmission and OPV use stops. Community risks of exposure to wild poliovirus as well as Sabin strains are highest from facility personnel who are unknowingly contaminated or infected. Immunization with OPV or IPV prevents poliomyelitis, but neither vaccine fully inhibits silent infection of the gut. Facility environments maintained at low relative humidity (<50%) may reduce poliovirus survival and inhalation risk. Circulating antibodies reduce personnel infection risks from injection or virus entry through breaks in skin or mucous membranes. Community exposure risk through inhalation of contaminated air effluent is likely low in most modern facilities. Community risks through ingestion of liquid effluents are facility-specific and may range from high to low. This assessment of community risks, when combined with assessments of facility-specific hazards and the consequences of wild or Sabin poliovirus transmission, provides the foundation for effective risk management. PMID- 16682226 TI - Purification of GFP fusion proteins from transgenic plant cell cultures. AB - Green fluorescence protein (GFP) has become a widely used reporter in many areas of life science. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. GFP itself has been purified from recombinant organisms by several methods, often involving unfavorable conditions (e.g., use of organic solvents and/or low pH) that may be destabilizing to some proteins. In this study, we have developed a general recovery scheme that entails a simple three-step purification procedure for GFP fusion proteins produced in tobacco suspension cells, with the intent of maximizing purity and yield under gentle conditions so as to maintain the integrity of the fusion partner. Ammonium sulfate treatment at 30% (v/v) precipitated particulate matter and removed aggregated material while simultaneously maintaining GFP solubility and increasing hydrophobicity. Hydrophobic interaction chromatography was then performed to eliminate the majority of background proteins while eluting GFP and fusions in a low ionic buffer suitable to be directly applied to an ion-exchange column as the final step. Three intracellular proteins, secreted alkaline phosphatase (SEAP), and granulocyte-macrophage colony-stimulating factor (GMCSF), each fused to GFP, as well as GFP itself, were recovered with yields exceeding 70% and purity levels over 80%. This purification scheme exploits the hydrophobic nature of GFP while maintaining a gentle environment for labile fusion partners. Although some optimization may be required, we believe this scheme may serve as a benchmark for purifying other GFP fusion proteins. PMID- 16682227 TI - Overexpression, refolding, and purification of polyhistidine-tagged human retinoic acid related orphan receptor RORalpha4. AB - RORalpha4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORalpha4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6M guanidinium-HCl and renatured by gradual removal of guanidinium-HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20mg of pure and native RORalpha4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORalpha in mice with high sensitivity and specificity. PMID- 16682228 TI - Heterologous expression of lipoprotein-associated phospholipase A2 in different expression systems. AB - Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a key enzyme involved in atherosclerosis, and has been considered as a new target for drug discovery. The major difficulty for high-throughput screening of Lp-PLA(2) inhibitors and for functional studies was their fast and efficient production. Purification of native Lp-PLA(2) from human plasma was complicated and produced a very low yield. We herein examined the feasibility of expressing and purifying recombinant Lp PLA(2) in different heterologous expression systems. The fusion Lp-PLA(2) was expressed at high levels and exhibited strong enzyme activity in insect cell baculovirus expression system. The functional enzyme could also be produced in Pichia pastoris. The inclusion of a Kozak sequence increased greatly the expression level of recombinant Lp-PLA(2) in insect cells, but had little effect on the expression of recombinant Lp-PLA(2) in P. pastoris and Escherichia coli. P. pastoris-produced Lp-PLA(2) could be purified rapidly and conveniently through a one-step procedure, while baculovirus-produced Lp-PLA(2) could be efficiently purified through a two-step procedure. This ability to readily produce recombinant Lp-PLA(2) could provide a screening model for Lp-PLA(2) inhibitors and will facilitate further studies on this enzyme. PMID- 16682229 TI - Crystal structure at 1.9A of E. coli ClpP with a peptide covalently bound at the active site. AB - ClpP, the proteolytic component of the ATP-dependent ClpAP and ClpXP chaperone/protease complexes, has 14 identical subunits organized in two stacked heptameric rings. The active sites are in an interior aqueous chamber accessible through axial channels. We have determined a 1.9 A crystal structure of Escherichia coli ClpP with benzyloxycarbonyl-leucyltyrosine chloromethyl ketone (Z-LY-CMK) bound at each active site. The complex mimics a tetrahedral intermediate during peptide cleavage, with the inhibitor covalently linked to the active site residues, Ser97 and His122. Binding is further stabilized by six hydrogen bonds between backbone atoms of the peptide and ClpP as well as by hydrophobic binding of the phenolic ring of tyrosine in the S1 pocket. The peptide portion of Z-LY-CMK displaces three water molecules in the native enzyme resulting in little change in the conformation of the peptide binding groove. The heptameric rings of ClpP-CMK are slightly more compact than in native ClpP, but overall structural changes were minimal (rmsd approximately 0.5 A). The side chain of Ser97 is rotated approximately 90 degrees in forming the covalent adduct with Z-LY-CMK, indicating that rearrangement of the active site residues to a active configuration occurs upon substrate binding. The N-terminal peptide of ClpP-CMK is stabilized in a beta-hairpin conformation with the proximal N terminal residues lining the axial channel and the loop extending beyond the apical surface of the heptameric ring. The lack of major substrate-induced conformational changes suggests that changes in ClpP structure needed to facilitate substrate entry or product release must be limited to rigid body motions affecting subunit packing or contacts between ClpP rings. PMID- 16682230 TI - Mechanical properties of cardiac titin's N2B-region by single-molecule atomic force spectroscopy. AB - Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91-98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at approximately 95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the approximately 100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes. PMID- 16682231 TI - Anisotropic lattice distortions in biogenic calcite induced by intra-crystalline organic molecules. AB - We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2.10( 3)) was detected along the c-axis. Under heat treatment above 200 degrees C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells. PMID- 16682232 TI - Quantitative investigation of antigen and immune response in nervous and lymphoid tissues of Atlantic halibut (Hippoglossus hippoglossus) challenged with nodavirus. AB - The present study reports the quantitative analysis of the spatio-temporal development of nodavirus infection and corresponding immune response in juvenile Atlantic halibut (Hippoglossus hippoglossus) challenged by intramuscular injection of nodavirus. Novel quantitative real-time RT-PCR protocols were applied to evaluate the absolute copy numbers of nodavirus RNA2 (RNA2) and secretory-IgM mRNA (sec-igmicro) in the eye, brain, mid/posterior kidney and spleen sampled over a period of 81 days. In the eye and brain, levels of both RNA2 and sec-igmicro increased significantly early in the infection. In the spleen and mid/posterior kidney, both RNA2 and sec-igmicro were detected but the levels remained unchanged during the experimental period. The levels of RNA2 and sec-igmicro in the eye and brain were strongly correlated (P<0.0001). Nodavirus antigen was demonstrated by immunohistochemistry (IHC) in the retina of eyes from a relatively few fish from day 34 post challenge (brain not examined), but not at any time in the spleen and anterior kidney. By IHC, IgM+ cells were observed in conjunction with nodavirus positive IHC labelling in the retina. In both the spleen and anterior kidney, the number of IgM+ cells increased from day 3 post challenge. By conventional real-time RT-PCR, RNA2 was only sporadically demonstrated in the posterior intestine, heart and gills. ELISA analysis revealed a nodavirus specific antibody response in serum that was significant from day 18 post challenge. No clinical signs or mortality related to nodavirus infection were observed in the challenged halibut. The results suggest that the nodavirus infection induced a significant antibody response through activation of B-cells in the kidney and spleen, and involved a substantial migration of antibody secreting cells to infected peripheral tissues. PMID- 16682233 TI - Quantification of mechanical vibration during diffusion tensor imaging at 3 T. AB - Subjects sense clear mechanical vibrations during diffusion tensor imaging (DTI). These vibrations, likely resulting from diffusion-sensitizing gradients, have been assumed to be of the same strength and phase in all parts of the magnetic resonance imaging (MRI) scanner so that they could be ignored. However, our measurements, carried out from several parts of the MRI scanner and its surroundings using an optical laser-based interferometer, demonstrate an uneven distribution of mechanical vibrations within the scanner. The measurements were performed during DT scanning at 3 T, with various diffusion-weighting parameters, by positioning a phantom in the head coil and/or a human subject on the patient bed. The vibration-related movement was caused by the diffusion-sensitizing gradients and was maximally 0.5 mm with typical settings used in brain imaging. The compensation for eddy currents, done with gradients in our DTI sequence, increased the vibration level by a factor of 1.5 or more with diffusion-weighting parameter b = 1000 s/mm(2) and by a factor of 3 or more with b = 3000 s/mm(2). Mechanical vibrations stayed at an acceptable level with b < or = 1000 s/mm(2), resulting in additional signal losses of 5-17%. Vibration levels might be reduced by adjusting imaging parameters, by modifying the gradient waveforms in the DTI sequence, and by redesigning the mechanics of patient bed to effectively dampen the movements. PMID- 16682234 TI - Cerebral perfusion mapping using a robust and efficient method for deconvolution analysis of dynamic contrast-enhanced images. AB - Dynamic contrast-enhanced (DCE) imaging using MRI or CT is emerging as a promising tool for diagnostic imaging of cerebral disorders and the monitoring of tumor response to treatment. In this study, we present a robust and efficient deconvolution method based on a linearized model of the impulse residue function, which allows for the mapping of functional cerebral parameters such as cerebral blood flow, volume, mean transit time, and permeability. Monte Carlo simulation studies were performed to study the accuracy and stability of the proposed method, before applying it to clinical study cases of patients with cerebral tumors imaged using DCE CT. Functional parameter maps generated using the proposed method revealed the locations of the cerebral tumors and were found to be of sufficiently good clarity for marked regional differences in tissue vascularity and permeability to be assessed. In particular, tumor visualization and delineation were found to be better on the parameter maps that were indicative of the breakdown of the blood-brain barrier. PMID- 16682235 TI - The neural basis of social tactics: An fMRI study. AB - One of the most powerful ways of succeeding in complex social interactions is to read the minds of companions and stay a step ahead of them. In order to assess neural responses to reciprocal mind reading in socially strained human relationships, we used a 3-T scanner to perform an event-related functional magnetic resonance imaging study in 16 healthy subjects who participated in the game of Chicken. Statistical parametric mapping showed that the counterpart effect (human minus computer) exclusively activated the medial frontal area corresponding to the anterior paracingulate cortex (PCC) and the supramarginal gyrus neighboring the posterior superior temporal sulcus (STS). Furthermore, when we analyzed the data to evaluate whether the subjects made risky/aggressive or safe/reconciliatory choices, the posterior STS showed that the counterpart had a reliable effect regardless of risky or safe decisions. In contrast, a significant opponent x selection interaction was revealed in the anterior PCC. Based on our findings, it could be inferred that the posterior STS and the anterior PCC play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. PMID- 16682236 TI - Roles for the interleukin-4 receptor and associated JAK/STAT proteins in human articular chondrocyte mechanotransduction. AB - OBJECTIVE: To identify functional interleukin-4 (IL4) receptor (IL4R) subtypes and associated Janus kinase/signal transducers and activators of transcription (JAK/STAT) molecules in human articular chondrocytes and assess the role of JAK/STAT proteins in chondrocyte mechanotransduction. METHODS: Expression of IL4R subunits and associated molecules was assessed by immunohistochemistry and western blotting. Functional IL4R were identified by chemical crosslinking of IL4 stimulated chondrocytes and western blotting. JAK and STAT phosphorylation was assessed by western blotting. RESULTS: Chondrocytes from normal and osteoarthritic (OA) cartilage express IL4Ralpha, gammac and IL13Ralpha1 subunits (components of the Type I and Type II IL4R). In the presence of IL4 only functional Type II IL4Rs were identified in normal or OA chondrocytes. With the exception of STAT2, no differences in JAK/STAT expression were detected between normal and OA cartilage. STAT2 was expressed in OA but not normal chondrocytes. Mechanical stimulation (MS) resulted in an IL4R-dependent increase in phosphorylated Tyk2 in normal chondrocytes, which could be abolished by IL1beta preincubation. No phosphorylation of STAT5 or STAT6 was detected in either normal or OA chondrocytes following mechanical stimulation (MS) IL4 stimulation resulted in a decrease in Tyk2 phosphorylation and an increase in phosphorylation of STAT6 in both normal and OA chondrocytes. CONCLUSION: Chondrocytes from normal and OA cartilage signal through a Type II IL4R. This signalling is via a STAT6 independent pathway. Differences in IL4 signalling are likely due to crosstalk between integrin and cytokine signalling pathways, and not differences in IL4R expression. PMID- 16682237 TI - PEG-hirudin/iloprost coating of small diameter ePTFE grafts effectively prevents pseudointima and intimal hyperplasia development. AB - OBJECTIVES: Small diameter PTFE grafts are prone to thrombosis and intimal hyperplasia development. Heparin graft coating has beneficial effects but also potential drawbacks. The purpose of this study was to evaluate the experimental efficacy of PEG-hirudin/iloprost coated small caliber PTFE grafts. METHODS: Thirty-six femoro-popliteal ePTFE grafts (expanded polytetrafluoroethylene, diameter 4 mm) were inserted into 18 pigs. Grafts were randomised individually for each leg and grouped for 3 groups. Group I consisted of native ePTFE grafts, group II were grafts coated with a polylactide polymer (PLA) without drugs and group III grafts were coated with PLA containing a polyethylene glycol (PEG) hirudin/iloprost combination. The follow-up period was 6 weeks. Patency rates were calculated and development of pseudointima inside the grafts was noted. Thickness of intimal hyperplasia at the distal anastomoses was measured using light microscopy. RESULTS: Patency rates for group I were 6/9 (67%), for group II 9/10 (90%) and 12/12 (100%) for group III. In groups I and II there was a significant reduction of blood flow proximal to the graft at graft harvest, to 29+/-12 and 28+/-20 ml/min respectively (both p<0.01 versus preoperative value), whilst in group III blood flow, 99+/-21 ml/min, remained at the preoperative level. Subtotal stenosis due to development of pseudointima was noted in each of the native and PLA coated grafts but not in group III grafts. Intimal hyperplasia at the distal anastomosis was lowest in group III. CONCLUSIONS: The PEG hirudin/iloprost coating of ePTFE prostheses effectively reduced pseudointima and intimal hyperplasia development and led to superior graft patency. PMID- 16682238 TI - The impact of lesion length on angiographic restenosis after vertebral artery origin stenting. AB - OBJECTIVE: To evaluate the effect of lesion length on in-stent restenosis (ISR) of vertebral artery (VA) origin stenting. METHODS: We retrospectively analyzed the medical and radiological records of patients receiving VA origin stenting from March 1999 to June 2005. They were subdivided according to lesion length. ISR was defined as >50% diameter narrowing in stent. RESULTS: Eighty symptomatic patients (64 male, mean age 72 years) with 90 lesions treated with balloon expandable tubular coronary stents were enrolled. There were 34 patients with 38 short lesions (length5 mm, <10 mm, group 2) and 9 patients with 10 long lesions (length>or=10 mm, group 3). Eighty seven bare-metal stents and 3 drug-eluting stents were implanted. Repeat angiography was done in 40 lesions (44%) at 11.7+/-9.6 months. The ISR rate in group 1, 2, 3 is 21%, 29%, and 50% (p=0.486). Multivariable Cox regression analysis showed lesion length was the only significant independent predictor of ISR (hazard ratio: 1.19, p=0.039). CONCLUSION: ISR of VA origin stenting is common. Lesion length is an important predictor of ISR in VA origin stenting. PMID- 16682239 TI - Arterial consequences of recreational drug use. AB - BACKGROUND: Recreational drug use is becoming an increasing problem throughout the world. Many of the drugs used and their routes of administration have the potential to cause damage to the vascular system. Intravenous drug administration with the risk of arterial puncture predisposes to the formation of infected pseudoaneurysms. Inadvertent intra-arterial injection predisposes to distal limb ischaemia. Cocaine has numerous effects not only on the heart, but also potentially on any vascular bed. METHODS: A systematic review of published literature with regard to the arterial consequences of recreational drug abuse was undertaken by undergoing an electronic search. RESULTS: Most of the available literature is in the form of case reports and case series. Pseudoaneurysm formation is a serious consequence for intravenous drug users. For femoral aneurysms vessel ligation is the safest option with a low amputation risk. A high proportion of patients do have symptoms of claudication in the long term. Intra arterial injections can result in limb threatening ischaemia. Systemic anticoagulation forms the mainstay of treatment. Cocaine has significant effects upon both the myocardium and the arterial tree in general. Arterial problems must always be considered in cocaine users who present acutely. CONCLUSION: The consequences of recreational drug use will result in an increased exposure of vascular surgeons to its associated complications posing unique and challenging problems. PMID- 16682240 TI - Short-term efficacy of pharmacotherapeutic interventions in osteoarthritic knee pain: A meta-analysis of randomised placebo-controlled trials. AB - BACKGROUND: Pain is the most debilitating symptom in osteoarthritis of the knee (OAK). AIM AND METHODS: To determine the short-term pain-relieving effects of seven commonly used pharmacological agents for OAK pain by performing a systematic review of randomised placebo-controlled trials. RESULTS: In total, 14,060 patients in 63 trials were evaluated. Opioids and oral NSAIDs therapy in patients with moderate to severe pain (mean baseline 64.3 and 72.8 mm on VAS respectively) had maximum efficacies compared to placebo at 2-4 weeks of 10.5 mm [95% CI: 7.4-13.7] and 10.2 mm [95% CI: 8.8-11.2] respectively. The efficacy of opioids may be inflated by high withdrawal rates (24-50%) and "best-case" scenarios reported in intention-to-treat analyses. In patients with moderate pain scores on VAS (mean range from 51 to 57 mm), intra-articular steroid injections and topical NSAIDs had maximum efficacies at 1-3 weeks of 14.5mm [95% CI: 9.7 19.2] and 11.6 mm [95% CI: 7.4-15.7], respectively. Paracetamol, glucosamin sulphate and chondroitin sulphate had maximum mean efficacies at 1-4 weeks of only 4.7 mm or lower. Heterogeneity tests revealed that best efficacy values of topical NSAIDs may be slightly deflated, while data for oral NSAIDs may be slightly inflated due to probable patient selection bias. CONCLUSION: Clinical effects from pharmacological interventions in OAK are small and limited to the first 2-3 weeks after start of treatment. The pain-relieving effects over placebo in OAK are smaller than the patient-reported thresholds for relevant improvement. PMID- 16682241 TI - A theoretical study of a family of new quinoxaline derivatives. AB - Hybrid density functional calculations are performed on a series of 21 new quinoxaline derivatives, which would likely exhibit important biological activities. Optimized geometries, harmonic vibrational frequencies, and (1)H chemical shifts are reported and compared with experimental data when available. In addition, melting points of 75 derivatives are predicted resorting to the Quantitative Structure-Property Relationship (QSPR) Theory, doing the variable selection by means of the Replacement Method and using 875 theoretical descriptors obtained from Dragon 5 software. The best relationship found has seven descriptors with R=0.8818 and R(l-10%-o)=0.7705. PMID- 16682242 TI - Roles of neuraminidase in the initial stage of influenza virus infection. AB - We propose a concept that neuraminidase (NA) promotes virus entry into target cells during the initial stage of viral infection, in addition to the generally accepted concept that influenza virus NA promotes the release of progeny virus from a host cell at the final stage of viral replication. When NA activity was inhibited with specific inhibitors such as zanamivir and oseltamivir carboxylate, infection efficiency of the virus to MDCK and A549 cells was reduced to approximately 1/4 and 1/8, respectively. NA inhibitors did not significantly affect virus binding and envelope fusion activities, when assessed using an erythrocyte and virus system. Since the initial stage of viral infection involves binding of the virus to the target cell, virus entry into an endosome and envelope fusion with the endosomal membrane, our results indicated that NA inhibitors interfered with the virus entry step. Thus, NA is thought to promote virus entry, and thereby enhances infection efficiency. PMID- 16682243 TI - Non-invasive bioluminescence imaging for monitoring herpes simplex virus type 1 hematogenous infection. AB - Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the replication and tropism of KOS strain HSV-1 viruses expressing the firefly luciferase reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro light emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection. PMID- 16682244 TI - Toward direct biocontrol using surface EMG signals: control of finger and wrist joint models. AB - Increased interest in virtual reality (VR) and telemanipulation has created a growing need for the development of new interfacing devices for measuring controlling actions of the human hand. The objective of the present study was to determine if surface electromyography signals (SEMG) from the flexor digitorum superficialis (FDS), and flexor carpi ulnaris (FCU) generated during flexion extension of the human index finger and wrist can be used for controlling the flexion-extension of the finger and wrist of a simple geometric computer model. A simple geometric computer model of finger and wrist joints was developed. Eighteen subjects controlled the computer model using the SEMG signals from their FDS and FCU. The results indicate that the SEMG signals from the FDS and FCU muscles can be used as a direct biocontrol technique for controlling the finger and wrist models. This study establishes the proof of concept for direct biological control of the dynamic motion of the finger and wrist models for use in virtual reality environments and telemanipulation. PMID- 16682245 TI - Advanced lung ventilation system (ALVS) with linear respiratory mechanics assumption for waveform optimization of dual-controlled ventilation. AB - The present paper describes the functional features of an advanced lung ventilation system (ALVS) properly designed for the optimization of conventional dual-controlled ventilation (DCV), i.e. with pressure-controlled ventilation with ensured tidal or minute volume. Considering the particular clinical conditions of patients treated with controlled ventilation the analysis and synthesis of ALVS control have been performed assuming a linear respiratory mechanics. Moreover, new airways pressure waveforms with more physiological shape can be tested on simulators of respiratory system in order to evaluate their clinical application. This is obtained through the implementation of a compensation procedure making the desired airways pressure waveform independent on patient airways resistance and lung compliance variations along with a complete real-time monitoring of respiratory system parameters leading the ventilator setting. The experimental results obtained with a lung simulator agree with the theoretical ones and show that ALVS performance is useful for the research activity aiming at the improvement of both diagnostic evaluation and therapeutic outcome relative to mechanical ventilation treatments. PMID- 16682246 TI - Ovarian cancer: homeobox genes, autocrine/paracrine growth, and kinase signaling. AB - Epithelial ovarian cancer, the fourth leading cause of cancer deaths in American women, is currently classified by surgical and histologic appearance. However, the predictive value of this classification is limited. The risk of epithelial ovarian cancer increases with the number of ovulatory events. It is now thought that different ovarian tumors are derived from a single ovarian surface epithelial precursor cell with the degree and pattern of differentiation determined by combinatorial expression of homeobox genes normally involved in differentiation of the female genital tract. This aberrant differentiation occurs in association with histology-specific genomic aberrations, genomic instability, and resultant chromosomal changes, and may be triggered by prolonged abnormal or excessive exposure of surface epithelial cells to autocrine/paracrine stimulation by sex steroids and other growth factors. As the disease progresses, activation of kinase pathways and continued abnormal autocrine/paracrine stimulation contribute to genomic instability but also identify potential targets for novel therapeutic intervention. PMID- 16682247 TI - Hitting the SPOT: small-molecule macroarrays advance combinatorial synthesis. AB - The small-molecule macroarray represents a new tool to accelerate combinational library synthesis and screening. This array platform originates from the SPOT synthesis technique, or the spatially addressed synthesis of peptides on cellulose supports. Recent advances in the field have expanded this technique beyond peptidic systems into the realm of complex small-molecule synthesis. Small molecule macroarrays offer some significant advantages over traditional combinatorial synthesis platforms--these focused, 50-200 compound arrays are straightforward to synthesize, inexpensive, and amenable to numerous screening applications where the array compounds are either bound to or cleaved from the planar support. Critical advances in the small-molecule macroarray technique are highlighted herein, including the use of microwave-assisted organic reactions, multicomponent reactions, and automated spotting methods to further accelerate and broaden macroarray technology. PMID- 16682248 TI - Small molecule screening by imaging. AB - Useful small molecule tools can be discovered in imaging screens that measure phenotype in single cells or small organisms. Recent examples include identification of small molecule inhibitors of processes such as cell migration, cytokinesis, mitotic spindle length determination, melanogenesis, aggresome formation, membrane transport and nuclear export. Imaging screens are currently limited by challenges in the areas of image analysis and target identification. We discuss the use of model organisms such as zebrafish in screens and review different methods of target identification. The emerging field of automated image analysis is also introduced. PMID- 16682249 TI - Vibrational spectral analysis of DL-valine DL-valinium and DL-methionine DL methioninium picrates. AB - A comparative study of infrared and Raman spectra of DL-valine DL-valinium picrate (DL-VVP) and DL-methionine DL-methioninium picrate (DL-MMP) at the room temperature in 4000-50 cm(-1) range helps to determine the effect of hydrogen bonds in these crystals. The existence of the zwitterion and the protonated form in both the crystals has been observed. In DL-MMP, the methionine and the methioninium residues form a dimer through a strong hydrogen bond between them in the crystal. The characteristic bands due to gem-methyl group are observed in the case of DL-VVP. Factor group analysis has been made and the numbers of vibrational modes have been calculated. The tentative assignments of the observed bands are given. The picrate group forms the anion in both the crystals and it is unaffected by the presence of the cations. PMID- 16682250 TI - The molecular structure and vibrational spectra of 1-amino-5-benzoyl-4 phenylpyrimidin-2(1H) by Hartree-Fock and density functional methods. AB - The molecular structure and vibrational spectra of 1-amino-5-benzoyl-4 phenylpyrimidin-2(1H) (C(17)H(13)N(3)O(2)) have been investigated by Hartree-Fock and density functional method using standard B3LYP with 6-31G(d) basis set. The calculated results of the geometric bond lengths and bond angles obtained by using HF and DFT (B3LYP) are in very good agreement with the experimental values. Comparison of the observed fundamental vibrational frequencies of 1-amino-5 benzoyl-4-phenylpyrimidin-2(1H) (C(17)H(13)N(3)O(2)) and calculated results by density functional B3LYP and Hartree-Fock methods indicate that B3LYP is superior to the scaled Hartree-Fock approach for molecular vibrational problems. PMID- 16682251 TI - Ventilatory and gas-exchange responses to incremental exercise performed with reduced muscle glycogen content. AB - This study examined the relationship between minute ventilation (VE), CO2 production (VCO2), and blood lactate concentration ([La-]) during incremental exercise performed with reduced muscle glycogen stores. Nine untrained female subjects (25.3+/-4.2 year) performed incremental cycling in a normal glycogen (NG) state and under conditions of reduced muscle glycogen (RG) content. To reduce muscle glycogen stores, subjects cycled to exhaustion (124+/-33 min) at a power output corresponding to their gas-exchange anaerobic threshold. Peak oxygen uptake (VO2peak) was unchanged with glycogen reduction, even though subjects achieved a significantly lower maximal power output in the RG state (p<0.05). Peak blood [La-] decreased significantly by 37% in the RG state (p<0.001). At any percentage of VO2peak, O2 uptake and VE were similar for both treatment conditions, whereas VCO2 and respiratory exchange ratio values were lower during the RG trial than under NG conditions. Therefore, VE/VCO2 tended to be higher and end-tidal CO2 partial pressure tended to be lower during exercise performed in the RG state. VE was significantly correlated with VCO2 under both treatment conditions (NG: r=0.99, p<0.01; RG: r=0.99, p<0.01). However, the slope of the VE VCO2 relationship was significantly elevated during the RG trial (p<0.01). VE during exercise was similar under both treatment conditions, even though VCO2 and blood [La-] were lower during the RG trial compared to the NG trial. This suggests that factors other than CO2 delivery to the lung and metabolic acidosis play an important role in regulating VE during exercise. PMID- 16682252 TI - Targeting the stromal microenvironment in chronic inflammation. AB - A characteristic feature of chronic inflammatory reactions is their persistence and predilection for certain sites. The molecular basis for such tissue tropism (as, for example, seen with metastatic spread) has until recently remained obscure, but recent studies have strongly implicated tissue-resident, stromal cells, such as macrophages, endothelial cells and fibroblasts. These cell types make attractive therapeutic targets as they help define the three-dimensional structure of tissues and are key orchestrators of the inflammatory infiltrate. Most current anti-inflammatory therapies target immune cells in an attempt to inhibit the production of pro-inflammatory mediators; however, an equally important target is the active induction of anti-inflammatory mediators involved in the resolution of inflammation. Recent work suggests that stromal cells are an important source of these mediators. Targeting of multiple signals may be required to inhibit tissue damage associated with inflammatory disease. Cells of the monocyte lineage are present as tissue-resident cells and interact closely with other stromal populations. These cells form an ideal target for modulation of the inflammatory environment as, in some cases, they appear to induce tissue repair. Therapeutic manipulation of the stromal microenvironment has been particularly effective in treating cancer and is likely to provide a novel method to achieve improved control of chronic inflammatory disease. PMID- 16682253 TI - Partial interferon-gamma receptor deficiency and non-tuberculous mycobacterial lung disease. AB - Interferon-gamma (IFN-gamma) plays a key role in the host defense response against mycobacterial disease, and a complete or partial deficiency in IFN-gamma receptor 1 (IFN-gammaR1) or IFN-gamma receptor 2 (IFN-gammaR2) has been reported to contribute to susceptibility to disseminated infection with non-tuberculous mycobacteria (NTM). However, IFN-gammaR1 and IFN-gammaR2 deficiencies have not yet been studied in adult patients with isolated NTM lung disease. The purpose of the present study was to evaluate whether partial IFN-gammaR1 and IFN-gammaR2 deficiency are associated with human susceptibility to NTM lung disease. We studied 40 patients with NTM lung disease (Mycobacterium avium complex infection, 20 patients; Mycobacterium abscessus infection, 20 patients) for partial IFN gammaR1 and IFN-gammaR2 deficiency. Genomic DNA was amplified by polymerase chain reaction and sequenced for revealing mutations of the IFN-gammaR1 and IFN-gammaR2 gene. None of the patients had previously reported homozygous recessive missense mutation causing an amino-acid substitution in the extracellular domain of the receptor (I87T) and hotspot for small deletions (818delT, 818del4) of the IFN gammaR1 or homozygous missense mutation (R114C) of the IFN-gammaR2. In conclusion, in adult patients with isolated NTM lung disease, there is no evidence for previously known genetic defects of partial deficiencies of IFN gammaR1 and IFN-gammaR2 to correlate with disease. PMID- 16682254 TI - Analysis of tumors arising in male B6C3F1 mice with and without AAV vector delivery to liver. AB - The present study reports on the frequency of liver tumors observed in a gene therapy study with AAV vectors in male mice of the B6C3F1 hybrid background, which are known to have a high frequency of spontaneous liver tumors. Male mice with mutations in their Otc gene and their wild-type siblings received AAV vectors expressing either the murine Otc or the LacZ gene. Untreated control animals were included in the study. All experimental groups, including wild-type and OTC-deficient animals not treated with vector, developed liver nodules, which in some cases were due to hepatocellular carcinoma. Vector DNA was lower in tumors than in adjacent normal liver. A statistical analysis of the data did not show an association between treatment with Otc vectors and formation of tumors in OTC-deficient mice. However, mice treated with LacZ vectors showed increased risks of tumor formation and hepatocellular carcinoma relative to untreated animals or animals that had received vectors with Otc as the transgene. It appears that AAV vectors alone do not contribute to the formation of tumors in these strains of mice although the expression of LacZ alone or in combination with vector may be problematic. PMID- 16682255 TI - Relationship of smoking and fibrosis in patients with chronic hepatitis C. AB - BACKGROUND & AIMS: Preliminary studies have suggested that in patients with chronic hepatitis C (CHC), cigarette smoking increases the risk for developing liver fibrosis. Hypoxia caused by smoking may induce expression of the cytokines' vascular endothelial growth factor (VEGF) and VEGF-D and their corresponding soluble tyrosine kinase receptors fms-like tyrosine kinase receptor (s-Flt) and kinase insert domain receptor (s-KDR). These cytokine levels are increased in animals with cirrhosis and in human beings with CHC. We studied whether the concentrations of VEGF, VEGF-D, s-Flt, and s-KDR were increased in CHC smokers with and without hepatic fibrosis. METHODS: A total of 170 CHC patients were identified retrospectively from a single center's database. In 59 patients, serum levels of VEGF, VEGF-D, s-Flt, and s-KDR were measured using an enzyme-linked immunosorbent assay. RESULTS: All 170 patients were hepatitis C virus RNA positive, 117 (69%) were men, 43 (25%) were smokers, and their mean (+/-SD) age was 47 (+/-6) years. Overall, 21% of smokers had Metavir fibrosis scores of 3 and 4 compared with 14% of nonsmokers (P < .01). In an age-weighted multivariate model using step-wise logistic regression, smoking, infection with hepatitis C virus genotype 1, male sex, and increased VEGF-D concentration all were significant independent predictors of more severe liver fibrosis (P < .05 for all observations). CONCLUSIONS: These data suggest that CHC patients who smoke may have more hepatic fibrosis. The data also suggest that increased VEGF and VEGF-D concentrations are associated with smoking and may be involved in the molecular mechanisms of fibrogenesis. PMID- 16682256 TI - Endoscopic ultrasound and positron emission tomography for lung cancer staging. AB - BACKGROUND AND AIMS: Accurate assessment of mediastinal lymph nodes is vital for optimum treatment allocation in lung cancer patients. Currently available strategies fail to identify many patients with advanced mediastinal disease, resulting in unnecessary surgery. We prospectively compared 2 promising new modalities, positron emission tomography (PET) and endoscopic ultrasound (EUS), for staging mediastinal lymph nodes. METHODS: Consenting patients with lung cancer who also were suitable candidates for surgery were enrolled in the study. Patients underwent both PET and EUS. Outcomes were analyzed by surgery results or follow-up with serial imaging. RESULTS: Seventy-two eligible patients were enrolled, and adequate data were available for 65 patients. The final diagnosis was based on tissue analysis in 59 patients and 1-year radiologic follow-up evaluation in 6 patients. PET correctly diagnosed mediastinal lymph node status in 77% of patients, and EUS fine-needle aspiration was correct in 94% of patients (P = .012). The overall sensitivity, specificity, and accuracy of PET were 61%, 91%, and 77% compared with 87%, 100%, and 94% for EUS. We estimated that EUS obviated a surgical procedure in 55% (95% confidence interval, 40%-69%) of patients with radiologic evidence of mediastinal metastasis, and in 22% (95% confidence interval, 10%-41%) of patients without radiologic evidence of mediastinal metastasis. CONCLUSIONS: EUS fine-needle aspiration was more accurate than PET in staging mediastinal lymph nodes in lung cancer patients, and resulted in a substantial reduction in mediastinoscopy and thoracotomy. PMID- 16682257 TI - Gastric varix associated with autoimmune pancreatitis. PMID- 16682258 TI - Polymeric diet alone versus corticosteroids in the treatment of active pediatric Crohn's disease: a randomized controlled open-label trial. AB - BACKGROUND & AIMS: Nutritional therapy has been reported to have an almost equivalent efficacy of corticosteroids in achieving clinical remission in active Crohn's disease (CD). However, the effects of both treatments on intestinal mucosal inflammation rarely are reported. In a randomized controlled trial in children with active CD we compared the efficacy of nutritional therapy alone or corticosteroids on clinical variables and intestinal mucosal healing. METHODS: In a prospective, 10-week open-label trial, children with active, naive CD were randomized to orally polymeric formula alone or oral corticosteroids. The clinical activity index and nutritional and activity serum variables were evaluated at week 0 and then every 2 weeks; intestinal mucosal inflammation was assessed through endoscopy and histology at weeks 0 and 10. Primary efficacy outcomes were clinical remission and mucosal healing. RESULTS: Of the 37 children randomized, 19 received polymeric formula and 18 received corticosteroids. At week 10, on an intention-to-treat basis, the proportion of patients achieving clinical remission was comparable between the 2 groups (polymeric formula: 15/19 [79%; 95% confidence interval (CI), 56%-92%]; corticosteroid group: 12/18 [67%; 95% CI, 44%-84%]; P = .4; not significant). On the contrary, the proportion of children showing mucosa healing was significantly higher in the polymeric (14/19; 74%; 95% CI, 51%-89%) than the corticosteroid group (6/18 [33%; 95% CI, 16%-57%]; P < .05). At week 10 both endoscopic and histologic scores significantly decreased only in the polymeric group (P < .001). CONCLUSIONS: In children with active and recently diagnosed CD, a short course of polymeric diet is more effective than corticosteroids in inducing healing of gut inflammatory lesions. PMID- 16682259 TI - Screening for early pancreatic neoplasia in high-risk individuals: a prospective controlled study. AB - BACKGROUND & AIMS: Individuals with a strong family history of pancreatic cancer and persons with Peutz-Jeghers syndrome (PJS) have an increased risk for pancreatic cancer. This study screened for early pancreatic neoplasia and compared the pancreatic abnormalities in high-risk individuals and control subjects. METHODS: High-risk individuals with PJS or a strong family history of pancreatic cancer were prospectively evaluated with baseline and 12-month computed tomography (CT) scan and endoscopic ultrasonography (EUS). If EUS was abnormal, EUS-fine-needle aspiration and endoscopic retrograde cholangiopancreatography (ERCP) were performed. Surgery was offered to patients with potentially neoplastic lesions. Radiologic findings and pathologic diagnoses were compared. Patients undergoing EUS and/or ERCP for benign non-pancreatic indications were concurrently enrolled as control subjects. RESULTS: Seventy eight high-risk patients (72 from familial pancreatic cancer kindreds, 6 PJS) and 149 control patients were studied. To date, 8 patients with pancreatic neoplasia have been confirmed by surgery or fine-needle aspiration (10% yield of screening); 6 patients had 8 benign intraductal papillary mucinous neoplasms (IPMNs), 1 had an IPMN that progressed to invasive ductal adenocarcinoma, and 1 had pancreatic intraepithelial neoplasia. EUS and CT also diagnosed 3 patients with 5 extrapancreatic neoplasms. At EUS and ERCP abnormalities suggestive of chronic pancreatitis were more common in high-risk patients than in control subjects. CONCLUSIONS: Screening EUS and CT diagnosed significant asymptomatic pancreatic and extrapancreatic neoplasms in high-risk individuals. IPMN should be considered a part of the phenotype of familial pancreatic cancer. Abnormalities suggestive of chronic pancreatitis are identified more commonly at EUS and ERCP in high-risk individuals. PMID- 16682260 TI - Maintenance of healed erosive esophagitis: a randomized six-month comparison of esomeprazole twenty milligrams with lansoprazole fifteen milligrams. AB - BACKGROUND AND AIMS: The aim was to compare esomeprazole with lansoprazole for the maintenance of healed erosive esophagitis and resolution of gastroesophageal reflux disease-related symptoms in a United States population. METHODS: Patients who entered this double-blind, randomized, parallel-group, multicenter, maintenance trial had been treated and healed (no endoscopic evidence of erosive esophagitis) with esomeprazole 40 mg or lansoprazole 30 mg once daily (patients with Los Angeles grades C and D erosive esophagitis at baseline) or esomeprazole 40 mg (patients with Los Angeles grades A and B erosive esophagitis at baseline) and had no heartburn or acid regurgitation symptoms during the previous week. Patients were randomized to maintenance once-daily therapy with esomeprazole 20 mg (n = 512) or lansoprazole 15 mg (n = 514) for up to 6 months. Esophago gastroduodenoscopies were done at months 3 and 6, and investigators assessed symptom severity at months 1, 3, and 6. Endoscopic/symptomatic remission was defined as no erosive esophagitis and no study withdrawal as a result of reflux symptoms. RESULTS: The estimated endoscopic/symptomatic remission rate during a period of 6 months was significantly higher (P = .0007) for patients who received esomeprazole 20 mg once daily (84.8%) compared with those who received lansoprazole 15 mg (75.9%). Most patients had no heartburn (383/462 and 369/466) or acid regurgitation (401/462 and 400/466) symptoms at 6 months, and there were no significant differences between treatments. Both treatments were well tolerated. CONCLUSION: Esomeprazole 20 mg is more effective than lansoprazole 15 mg in maintaining endoscopic/symptomatic remission in patients with healed erosive esophagitis. PMID- 16682261 TI - Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo. AB - Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the delta-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins. PMID- 16682262 TI - Hormesis/preconditioning mechanisms, the nervous system and aging. AB - Throughout life, organisms and their cells are subjected to various stressors which they must respond to adaptively in order to avoid disease and death. Accordingly, cells possess a variety of stress-responsive signaling pathways that are coupled to kinase cascades and transcription factors that induce the expression of genes that encode cytoprotective proteins such as protein chaperones (PC), growth factors and antioxidant enzymes. Emerging findings suggest that many of the environmental factors that improve health and so prolong lifespan (for example, dietary restriction, exercise and cognitive stimulation) exert their beneficial effects through a hormesis-like mechanism. Here we describe data supporting the hormesis hypothesis of disease resistance and longevity, with a focus on findings from studies of the nervous system in this laboratory. PMID- 16682263 TI - Catheter-related thrombosis associated with elevated factor VIII levels in cystic fibrosis. AB - We report a cystic fibrosis (CF) subject with extensive, central venous catheter associated thrombosis and sustained elevation of factor VIII to levels normally associated with significantly increased risks of deep venous thrombosis. To determine the potential significance of this finding, the prevalence of elevated factor VIII levels in 22 adults with CF was investigated. Mean (S.D.) factor VIII level was 177 (43) U/dl, with 77% of subjects having levels >150 U/dl. The high prevalence of elevated factor VIII levels questions the significance of this finding in CF subjects with catheter-related thrombosis. PMID- 16682264 TI - Analysis of liposomes by capillary electrophoresis and their use as carrier in electrokinetic chromatography. AB - This contribution reviews work about liposomes in the context of electrically driven separation methods in the capillary format. The discussion covers four topics. The one broaches the application of liposomes as pseudo-stationary phases or carriers in vesicle or liposome electrokinetic chromatography (EKC) in the way as microemulsions and micelles are used; it includes the chromatographic use of liposomal bilayers as stationary phases attached to the wall for capillary electrochromatography (CEC). The second topic is the characterization and separation of liposomes as analytes by capillary electrophoresis (CE). Then the determination of distribution coefficients and binding constants between liposomes and ligands is discussed, and finally work dealing with peptides and proteins are reviewed with lipid bilayers as constituents of the electrically driven separation system. PMID- 16682265 TI - Inflammation and miscarriage. AB - Most relevant studies in animals and humans indicate that some degree of systemic or uterine inflammation is necessary both for normal implantation and pregnancy. However, if inflammation becomes too excessive it might cause pregnancy complications such as fetal resorption/miscarriage. The main regulator of the correct level of inflammation at the feto-maternal interface seems to be the uterine CD16(-) CD56(bright) natural killer (NK) cells. Trophoblast debris, apoptotic cells and progesterone probably stimulate/regulate the production of inflammatory cytokines from these cells. Miscarriage of karyotypically normal embryos may occur when the level of inflammation at the feto-maternal interface falls outside the optimal range. This may be caused by an insufficient influx of CD56(bright) NK cells into the decidua, too little soluble histocompatibility leukocyte antigen (HLA)-G secretion from the trophoblast, hypersecretion of inflammatory cytokines due to the presence of high-production polymorphisms, presence of maternal HLA-DR alleles associated with high tumor necrosis factor (TNF)-alpha production, or maternal mannose-binding lectin deficiency. PMID- 16682266 TI - Mandibular phenotype of p20C/EBPbeta transgenic mice: Reduced alveolar bone mass and site-specific dentin dysplasia. AB - CCAAT enhancer binding proteins (C/EBP) comprise a family of basic-leucine zipper transcription factors that regulate cellular differentiation and function. To determine the role of C/EBP transcription factors in osteoblasts and odontoblasts, we generated a transgenic (TG) mouse model with Co1a1 (pOBCol3.6) promoter-targeted expression of a FLAG-tagged dominant negative C/EBP isoform, p20C/EBPbeta (previously LIP). Two of the four transgenic lines presented with abnormalities in the developing incisors, including breakage, overgrowth, and malocclusion. Histological examination revealed that the amount of alveolar bone was reduced in TG compared to wild-type (WT) mice. By microcomputed tomography (microCT), the bone volume fraction of the mandible was reduced at the level of the first and third molars, demonstrating a severe mandibular osteopenia. The lingual dentin morphology of TG incisors differed dramatically from WT. Labial dentin (enamel side) showed normal thickness and tubular dentin structure, whereas the lingual dentin was thinner (25-30% of WT at the alveolar crest) with an amorphous globular structure characteristic of dentin dysplasia. FLAG immunostaining was seen in both lingual and labial odontoblasts, indicating that the site-specific defect was not due to a lack of labial transgene expression. Northern blot analysis demonstrated reduced osteocalcin expression in TG mandibles, while bone sialoprotein was increased, consistent with prior results in calvariae and long bones. Dental sialophosphoprotein, a marker of the odontoblast lineage whose absence causes dentin dysplasia, was modestly reduced in TG mice by Northern blot and in situ hybridization analysis. By fluorescence microscopy, pOBCol2.3-GFP, a marker of the odontoblast lineage, was expressed in both labial and lingual odontoblasts, although GFP-marked lingual odontoblasts were more flattened than WT cells. Moreover, GFP-positive processes in the lingual dentin tubules were truncated and less organized than those in WT dentin. MicroCT analysis showed reduced tissue density in the lingual dentin. These data suggest that C/EBP transcription factors may be involved in the regulation of odontoblast polarization and dentin matrix production. PMID- 16682267 TI - Low lumbar spine bone mineral density in both male and female endurance runners. AB - There have been many reports of low bone mineral density (BMD) in female endurance runners. Although there have been several reports of low BMD in male runners, it is unclear how comparable the problem is to that in females. We compared BMD between male and female endurance runners and with a reference population. One hundred and nine endurance runners (65 females, 44 males) aged 19 50 years participated and had been training regularly for at least 3 years (32 187.2 km week(-1)) in events from 3 km to the marathon. BMD was measured at the lumbar spine (L2-L4) and hip by DXA. A questionnaire assessed training and menstrual status. Lumbar spine T scores were similar in male and female runners ( 0.8 (0.8) versus -0.8 (0.7); f = 0.015; P = 0.904) as were total hip T scores (0.6 (7.9) versus 0.5 (9.2); f = 0.192; P = 0.662). The proportion of male runners with low lumbar spine BMD (<-1.0) (n = 16 from 44) compared to that of females (n = 27 from 65) (P = 0.675). Males had lower spine T scores than eumenorrhoeic females (-0.8 (0.7) versus -0.4 (0.7); f = 5.169; P = 0.03). There were moderate negative correlations between weekly running distance and lumbar spine BMD in males and females (r(2) = 0.267; 0.189; P < 0.001), independent of menstrual status in females (r(2) = 0.192; P < 0.001). Lumbar spine but not hip T scores were greater in runners who participated in resistance training at least twice-a-week (male: -0.4 versus -1.1; female: -0.5 versus -1.1; P < 0.01). Using multiple regression, running distance (-) and BMI (+) together best predicted lumbar spine T scores (r(2) = 0.402; P < 0.01) in females. Although weak, BMI (+) best predicted hip T scores (r(2) = 0.167; P < 0.05). In males, running distance and training years (-) together best predicted lumbar spine T scores (r(2) = 0.400; P < 0.01). Training years (-) best predicted hip T scores (r(2) = 0.361; P < 0.01). To conclude, our findings suggest that male runners face the same bone threat at the spine, as female runners. Further research in male athletes is required. Incorporation of regular resistance training into an athlete's training programme may be a useful preventative strategy. PMID- 16682268 TI - Molecular imaging of the dopaminergic system and its association with human cognitive function. AB - Molecular imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT) has recently been used to examine dopamine (DA) function and its relationship with cognition in human subjects. This article will review PET and SPECT studies that have explored the relationship between cognitive processes and components of the DA system (pre-, intra-, and postsynaptic) in healthy and patient populations such as Parkinson's disease (PD), schizophrenia, Huntington's disease, and aging. It is demonstrated that DA activity modulates a range of frontal executive-type cognitive processes such as working memory, attentional functioning, and sequential organization, and alterations of DA within the fronto-striato-thalamic circuits might contribute to the cognitive impairments observed in PD, schizophrenia, and normal aging. Although associations between DA and cognitive measures need to be considered within the context of fronto-striato-thalamic circuitry, it is suggested that striatal (especially caudate) DA activity, particularly via D2 receptors, might be important for response inhibition, temporal organization of material, and motor performance, whereas cortical DA transmission via D1 receptors might be important for maintaining and representing on-going behavior. PMID- 16682269 TI - The role of extrastriatal dopamine D2 receptors in schizophrenia. AB - Despite numerous studies on extrastriatal regions involved in schizophrenia, studies on the functional implications of dopamine (DA) D2 receptors in the extrastriatal regions, including the cortex and thalamus, are limited. We review postmortem and in vivo human imaging studies as well as animal studies, focusing on the function of extrastriatal DA D2 receptors and their role in the pathophysiology of schizophrenia. Based on recent findings, cortical DA D2 receptors may interact with the gamma-aminobutyric acid system to modulate DA transmission, and thalamic DA D2 receptors are likely to participate in sensory gating function into the prefrontal cortex. We have found decreased DA D2 receptors in the anterior cingulate cortex and thalamic subregions of patients with schizophrenia. These observations may suggest that alterations of extrastriatal DA D2 receptors are involved in dysregulation of DA transmission and sensory signals from the thalamus to the cortex. Excessive excitatory signals from the thalamus might flow into the cortical neurotransmission system, aggravating dysregulation of DA transmission in both the striatal and extrastriatal regions in schizophrenia. These notions suggest the need for future investigations of extrastriatal DA D2 receptor function to gain important clues regarding the pathogenesis and of possible treatments for schizophrenia. PMID- 16682270 TI - Britain gives up smoking. PMID- 16682271 TI - Posttranscriptional mechanisms regulating the inflammatory response. AB - The inflammatory response is a complex physiologic process that requires the coordinate induction of cytokines, chemokines, angiogenic factors, effector enzymes, and proteases. Although transcriptional activation is required to turn on the inflammatory response, recent studies have revealed that posttranscriptional mechanisms play an important role by determining the rate at which mRNAs encoding inflammatory effector proteins are translated and degraded. Most posttranscriptional control mechanisms function to dampen the expression of pro-inflammatory proteins to ensure that potentially injurious proteins are not overexpressed during an inflammatory response. Here we discuss the factors that regulate the stability and translation of mRNAs encoding pro-inflammatory proteins. PMID- 16682272 TI - Negative signaling in Fc receptor complexes. AB - Cell activation results from the transient displacement of an active balance between positive and negative signaling. This displacement depends in part on the engagement of cell surface receptors by extracellular ligands. Among these are receptors for the Fc portion of immunoglobulins (FcRs). FcRs are widely expressed by cells of hematopoietic origin. When binding antibodies, FcRs provide these cells with immunoreceptors capable of triggering numerous biological responses in response to a specific antigen. FcR-dependent cell activation is regulated by negative signals which are generated together with positive signals within signalosomes that form upon FcR engagement. Many molecules involved in positive signaling, including the FcRbeta subunit, the src kinase lyn, the cytosolic adapter Grb2, and the transmembrane adapters LAT and NTAL, are indeed also involved in negative signaling. A major player in negative regulation of FcR signaling is the inositol 5-phosphatase SHIP1. Several layers of negative regulation operate sequentially as FcRs are engaged by extracellular ligands with an increasing valency. A background protein tyrosine phosphatase-dependent negative regulation maintains cells in a "resting" state. SHIP1-dependent negative regulation can be detected as soon as high-affinity FcRs are occupied by antibodies in the absence of antigen. It increases when activating FcRs are engaged by multivalent ligands and, further, when FcR aggregation increases, accounting for the bell-shaped dose-response curve observed in excess of ligand. Finally, F-actin skeleton-associated high-molecular weight SHIP1, recruited to phosphorylated ITIMs, concentrates in signaling complexes when activating FcRs are coengaged with inhibitory FcRs by immune complexes. Based on these data, activating and inhibitory FcRs could be used for new therapeutic approaches to immune disorders. PMID- 16682273 TI - The surprising diversity of lipid antigens for CD1-restricted T cells. AB - CD1 proteins have been conserved throughout mammalian evolution and function to present lipid antigens to T cells. Crystal structures of CD1-lipid complexes show that CD1 antigen-binding grooves are composed of four pockets and two antigen entry portals. This structural information now provides a detailed understanding of how CD1-binding grooves capture a surprisingly diverse array of lipid ligands. CD1-expressing APCs are able to acquire lipid antigens from their own pool of lipids and from exogenous sources, including microbial pathogens, bystander cells, or even the systemic circulation. CD1 proteins bind to certain antigens using high stringency loading reactions within endosomes that involve low pH, glycosidases, and lipid transfer proteins. Other antigens can directly load onto CD1 proteins using low stringency mechanisms that are independent of cellular factors. New evidence from in vivo systems shows that CD1-restricted T cells influence outcomes in infectious, autoimmune, and allergic diseases. These studies lead to a broader view of the natural function of alphabeta T cells, which involves recognition of both cellular proteins and lipids. PMID- 16682274 TI - Lysophospholipids as mediators of immunity. AB - Lysophospholipids (LPLs) are lipid-derived signaling molecules exemplified by lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Originally identified as serum-associated growth factors, these mediators now are known to signal through a family of diverse G protein-coupled receptors (GPCRs). Virtually all cells that participate in the immune response express multiple receptors for LPLs. The development of antibody reagents that recognize the receptors for each LPL and the derivation of receptor-selective agonists and receptor-null mouse strains have provided insights into the widely diverse functions of LPLs in immune responses, particularly the role of S1P in lymphocyte trafficking. This review focuses on the biology of the LPLs as these molecules relate to functional regulation of immune cells in vitro and to the regulation of integrated immune responses in vivo. PMID- 16682275 TI - Systemic mastocytosis. AB - Systemic mastocytosis is a fascinating disease with diverse clinical features. There have been numerous advances in understanding the basis of clinical manifestations of this disease and of its molecular pathogenesis in the last several decades. The development of methods to study mast cell biology using cell culture and murine models has proven invaluable in this regard. Clarification of the roles of mast cells in various biological processes has expanded our understanding of their importance in innate immunity, as well as allergy. New diagnostic methods have allowed the design of detailed criteria to assist in distinguishing reactive mast cell hyperplasia from systemic mastocytosis. Variants and subvariants of systemic mastocytosis have been defined to assist in determining prognosis and in management of the disease. Elucidation of the roles of the Kit receptor tyrosine kinase and signal transduction pathway activation has contributed to development of potential targeted therapeutic approaches that may prove useful in the future. PMID- 16682276 TI - Regulation of fibrosis by the immune system. AB - Inflammation and fibrosis are two inter-related conditions with many overlapping mechanisms. Three specific cell types, macrophages, T helper cells, and myofibroblasts, each play important roles in regulating both processes. Following tissue injury, an inflammatory stimulus is often necessary to initiate tissue repair, where cytokines released from resident and infiltrating leukocytes stimulate proliferation and activation of myofibroblasts. However, in many cases this drive stimulates an inappropriate pro-fibrotic response. In addition, activated myofibroblasts can take on the role of traditional APCs, secrete pro inflammatory cytokines, and recruit inflammatory cells to fibrotic foci, amplifying the fibrotic response in a vicious cycle. Moreover, inflammatory cells have been shown to play contradictory roles in initiation, amplification, and resolution of fibrotic disease processes. The central role of the macrophage in contributing to the fibrotic response and fibrotic resolution is only beginning to be fully appreciated. In the following review, we discuss the fibrotic disease process from the context of the immune response to injury. We review the major cellular and soluble factors controlling these responses and suggest ways in which more specific and, hopefully, more effective therapies may be derived. PMID- 16682277 TI - Immunity and acquired alterations in cognition and emotion: lessons from SLE. AB - Classic immunologic teaching describes the brain as an immunologically privileged site. Studies of neuroimmunology have focused for many years almost exclusively on multiple sclerosis, a disease in which inflammatory cells actually infiltrate brain tissue, and the rodent model of this disease, experimental allergic encephalitis. Over the past decade, however, increasingly, brain-reactive antibodies have been demonstrated in the serum of patients with numerous neurological diseases. The contribution these antibodies make to neuronal dysfunction has, in general, not been determined. Here, we describe recent studies showing that serum antibodies to the N-methyl-D-aspartate receptor occur frequently in patients with systemic lupus erythematosus and can cause alterations in cognition and behavior following a breach in the blood-brain barrier. PMID- 16682278 TI - Immunodeficiencies with autoimmune consequences. AB - Far from being mutually exclusive, immunodeficiency and autoimmunity may occur simultaneously. During the last years, analysis of Autoimmune Polyendocrinopathy- Candidiasis--Ectodermal Dystrophy (APECED) and Immunodysregulation- Polyendocrinopathy--Enteropathy--X-linked (IPEX), two rare monogenic forms of immunodeficiency associated with autoimmunity, has led to the identification of Auto Immune Regulator (AIRE) and Forkhead Box P3 (FOXP3), essential transcriptional regulators, involved in central tolerance and peripheral immune homeostasis, respectively. Characterization of the molecular and cellular mechanisms involved in APECED, and recognition that AIRE expression is sustained by effective thymopoiesis, has recently allowed to define that the autoimmunity of Omenn syndrome, a combined immunodeficiency due to defects of V(D)J recombination, also results from defective expression of AIRE. The implications of identification of the basis of autoimmunity in these rare forms of immunodeficiency have important implications for a better understanding of more common autoimmune disorders, and for development of novel therapeutic approaches. PMID- 16682279 TI - Allele-specific loss of heterozygosity in multiple colorectal adenomas: toward an integrated molecular cytogenetic map II. AB - Colorectal cancer (CRC) remains a significant public health challenge despite our increased understanding of the genetic defects underlying the pathogenesis of this common disease. It has been thought that multiple mechanisms lead to the malignant phenotype, with familial predisposition syndromes accounting for only a small proportion of all CRC cases. To identify additional loci likely involved in CRC and to test the hypothesis of allele-specific loss of heterozygosity (LOH) for the localization of CRC susceptibility genes, we initially conducted a genome wide allelotyping analysis of 48 adenomas from a patient with familial adenomatous polyposis coli (FAP) and 63 adenomas from 7 patients with sporadic CRC using 79 fluorescently tagged oligonucleotide primers amplifying microsatellite loci covering the human genome. Frequent allelic losses were identified at D17S802 (41%), D7S518 (40%), D18S53 (38%), D10S249 (32%), D2S391 (29%), D16S419 (27%), D15S1005 and D15S120 (24%), D9S274 and D11S1318 (23%), D14S65 (20%), D14S274 and D17S953 (19%), D19S424 (18%), D5S346 and D1S397 (15%), and D6S468 (13%) in multiple FAP adenomas. Common LOH was also detected at D4S1584 (42%), D11S968 (31%), D17S953 (28%), D5S394, D9S286 and D10S249 (24%), D8S511 (23%), D13S158 (21%), D7S669 (20%), D18S58 (19%), D2S162 and D16S432 (16%), D2S206 (15%), D7S496 and D17S946 (14%), D6S292 (13%), D4S1586 and D8S283 (11%), and D1S2766 (10%) in multiple CRC adenomas. In addition, allele-specific LOH at D5S346, D15S1005, and D15S120 was observed in multiple FAP adenomas (P < 0.01) and at D2S206 and D16S423 in multiple CRC (P < 0.05). To compare our data to previous reports, we determined the band-specific frequency of chromosomal imbalances in CRC karyotypes reported in the Mitelman database, and from the CGH results of cases accessible through the PROGENETIX website. Furthermore, published genome-wide allelotyping analysis of CRC and other allele-specific LOH studies were compiled and collated with our LOH data. The combined results not only provide a comprehensive view of genetic losses in CRC, indicating the comparability of these different techniques, but they also reveal different novel loci in multiple adenomas from FAP and sporadic CRC patients, suggesting that they represent a distinct subtype of CRC in terms of allelic losses. Allele specific LOH is an alternative approach for cancer gene mapping. PMID- 16682280 TI - Transitional cell bladder tumor: predicting recurrence and progression by analysis of microsatellite loss of heterozygosity in urine sediment and tumor tissue. AB - Transitional cell bladder tumors (TCT) is prone to recurrence (60-80%) after tumor resection. Up to 25% of these patients will progress, so it is important to find reliable predictive markers. We analyzed for loss of heterozygosity (LOH) with respect to 13 microsatellites located on 10 different chromosomal arms. This analysis was performed on the urine sediment and tumor tissue from 59 patients with bladder TCT and on the urine and normal-looking mucosa from 25 patients with a history of bladder TCT but no evidence of disease at the time of the study inclusion. The median follow-up period was 23.1 months (range, 2-48 months) for the 59 patients with bladder TCT and 25 months (range, 4-57 months) for the 25 patients without evidence of ongoing active disease. Correlation between LOH and eventual recurrence, progression, and mortality was investigated. In patients with noninvasive TCT, correlation between 11p tumor tissue LOH and recurrence was found. Similarly, 8p LOH in both urine sediment and tumor tissue correlated with progression. Finally, in the group of patients with a history of bladder TCT, normal tissue 8p and/or 11p LOH correlated with recurrence. PMID- 16682281 TI - Chromosomal aberrations in 130 patients with multiple myeloma studied by interphase FISH: diagnostic and prognostic relevance. AB - The study described the molecular cytogenetic characterization of myeloma cells in 130 patients via interphase fluorescence in situ hybridization. Nine repetitive DNA probes (for chromosomes 3, 7, 9, 11, 15, 17, 18, X, and Y) as well as seven single-copy DNA probes (for chromosomes 13, 17, 21, and two each for chromosomes 5 and 22) were used for the hybridizations. Using this panel of probes, we were able to show aberrations in 86% of patients. Most of them had one to three aberrations. There was a distinct correlation between the number of aberrations per patient and the tumor stage. Thus, the proportion of patients with 8-12 aberrations increased from 16% in stage II to 26% in stage III. There were marked differences among the chromosomes with respect to the prevalence of genomic losses and gains and deletions of gene loci. Chromosomes 3, 5, 7, 9, 11, 15, and 21 showed a preference for genomic gains. Losses were most often found for chromosomes 13 and 17 (locus specific) as well as for the X and Y chromosomes. The frequency of monosomies and trisomies were approximately the same for chromosomes 15 and 18, which indicates a skewed pattern of distribution. We found two specific aberrations that caused distinct changes in the survival rates of the patients: deletion 13q14 (28% of patients) and translocation of the IGH locus 14q32 (79% of 39 patients who were analyzed separately). The results obtained in this study yielded data of extremely relevant prognostic value. PMID- 16682282 TI - Evaluation of microsatellite amplifications at chromosomal locus 3q26 as surrogate marker for premalignant changes in mucosa surrounding head and neck squamous cell carcinoma. AB - We analyzed tumor and surrounding mucosal samples of head and neck squamous cell carcinoma by fragment analysis for gain of genomic material as a potential indicator for oncogenic transformation. Our aim was to evaluate the potential value of this fast and sensitive polymerase chain reaction (PCR)-based method for intra-operative detection of chromosomal aberrations as a surrogate marker for incomplete tumor resection. Biopsies of the primary tumors and adjacent macroscopically nonmalignant mucosa 1 and 2 cm away from the tumor margins were collected from 20 patients. DNA were isolated, and 11 microsatellite markers at loci 3q25.31 approximately 3q28 were amplified by PCR. Allelic losses or gains were analyzed by capillary electrophoresis. Imbalanced alleles were common in the samples evaluated. In the median gains of the informative loci were detected in 67% of the primary tumors, 22% of the samples taken at 1 cm and 15% of the samples taken at 2 cm distance. We observed gains of 3q at least in one microsatellite in all primary tumors, in 15 (1 cm) and 12 (2 cm) nonmalignant mucosa samples. Gain of genetic material is frequent in tumor-surrounding mucosa. Detection of small chromosomal aberrations is possible using the PCR-based, highly sensitive, and fast-to-perform fragment analysis technique. The value for the diagnosis and prognosis will have to be proven in follow-up studies. PMID- 16682283 TI - Allelic imbalance of HER2 variant in sporadic breast and ovarian cancer. AB - Both breast and ovarian cancers are associated with HER2 receptor activation, which usually results from receptor overexpression and/or gene amplification. The HER-2 gene harbors a polymorphism at codon 655 (GTC/valine to ATC/isoleucine) in the transmembrane domain region, which has been associated with an elevated risk of breast cancer. The objective of this study was to determine whether the polymorphism is under a selection pressure during breast and ovarian carcinogenesis. The Ile/Val genotype was present in 41% (9/22) of the normal DNA of breast cancer patients. An allelic imbalance in the tumor tissue was found in three breast tumors, with overrepresentation of the Val allele. HER-2 was amplified and overexpressed in these tumors. Half of the eight ovarian tumor patients carried heterozygous Ile/Val genotypes. In contrast to breast tumors, all these ovarian cancer specimens showed the presence of the Ile allele. In our selected set of tumors, the Val allele was overrepresented in the subset of HER2 positive breast cancers and the Ile allele in serous ovarian cancer. Further analyses of tumors with known gene amplifications and overexpression may reveal novel associations between germline polymorphisms and development of sporadic tumors. PMID- 16682284 TI - Detection of abnormalities of PRV-1, TPO, and c-MPL genes detected by fluorescence in situ hybridization in essential thrombocythemia. AB - No specific diagnostic markers have been described in essential thrombocythemia (ET). PRV-1 (polycythemia rubra vera-1), TPO (thrombopoietin), and c-MPL (myeloproliferative leukemia virus oncogene) genes are candidate ET molecular markers because of their implication in the pathogenesis of ET. We have studied the status of PRV-1, TPO, and c-MPL genes in 30 ET patients by a fluorescence in situ hybridization (FISH) technique using three noncommercial, locus-specific probes for PRV-1 (BAC RP11-160A19, located at 19q13.2), TPO (BAC RP11-45NP16, located at 3q27), and c-MPL (BAC RP11-297L5, located at 1p34). FISH study showed no PRV-1, TPO, and c-MPL cytogenetic abnormalities in any of the analyzed cases. Our results suggest a lack of structural and numerical rearrangements (deletions, translocations, or amplifications) of PRV-1, TPO, and c-MPL genes in ET patients. PMID- 16682285 TI - A molecular study of first and second RB1 mutational hits in retinoblastoma patients. AB - RB1 mutations accountable for biallelic inactivation are crucial events in the development of retinoblastoma because a first mutation (M1) predisposes to retinoblastoma while a second mutation (M2) is required for tumor development. Mutational analyses of this gene showed a wide spectrum of genetic alterations (single base substitutions, insertions, or deletions, as well as small and large deletions). The most frequent second hit in retinoblastoma patients is loss of heterozygosity (LOH) followed by promoter methylation. Molecular analyses of RB1 mutations were conducted in 36 patients (20 unilateral and 16 bilateral) using polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP) analysis, sequencing, and LOH analysis. Sixty-four amplified fragments showing abnormal SSCP patterns were sequenced, and mutations were confirmed in five patients (13.89%). Four mutations were located at coding regions, and a fifth one was found at an exon-intron junction. Two mutations were C-->T transitions, two were small-length deletions, and one was a G-->A transition. A total of 47.05% patients showed LOH. In one patient, the parental origin of the mutated allele was detected: the allele retained in the tumor was the paternal one. This work helps to characterize the spectrum of mutations in the Brazilian population, and to confirm that formaldehyde-fixed paraffin tissue can provide valuable information on the RB1 status in retinoblastoma patients. PMID- 16682286 TI - Myelodysplastic syndromes with isolated deletion of the long arm of the chromosome X as a sole cytogenetic change. AB - Deletions of Xq are extremely rare events in myelodysplastic syndromes (MDS) patients and were previously described in five patients, in two of them as a sole chromosome abnormality. We found isolated del(Xq) in 3 of 127 MDS patients with clonal chromosome changes. Detailed analysis of clinical and morphological data of presented and previously published cases indicates the following: (1) del(X)(q24) and del(X)(q13) are nonrandom chromosomal abnormalities in MDS; (2) MDS with deletions of Xq affect exclusively females ages 46-65; and (3) deletions of Xq are associated with refractory anemia with excess blasts (RAEB) and indicate an unfavorable prognosis. PMID- 16682287 TI - Favorable outcome of triploid neuroblastomas: a contribution to the special oncogenesis of neuroblastoma. AB - There is a well-known association between patient outcome and tumor ploidy in neuroblastoma. To date, however, most clinical trials have not used this parameter for therapy stratification. Using conventional cytogenetics and fluorescence in situ hybridization (FISH), we investigated 36 tumors in terms of ploidy and chromosome 1 copy number (polysomy). In addition, interphase FISH for polysomy was performed on a second cohort of 440 neuroblastomas, together with the status of 1p, MYCN, and 11q. The main goals were as follows: (1) to assess the reliability of FISH to determine ploidy; (2) to illustrate associations between somy 1 and clinical/biologic factors; and (3) to investigate the role of somy 1 for predicting outcome. The comparison between karyotyping and FISH in the smaller cohort revealed 86% consistency between ploidy and polysomy (31/36). According to FISH, trisomic tumors in the second cohort showed structural chromosomal aberrations less frequently compared to di-/tetrasomic tumors (15 vs. 60%, P < 0.001). The portion of trisomic neuroblastomas was higher in stages 1, 2, and 4S versus stages 3 and 4 (55 vs. 24%, P < 0.001) and in children 18 months or younger versus those older than 18 months (55 vs. 19%, P < 0.001). Prognosis was significantly better for trisomic tumors versus di-/tetrasomic in the whole cohort [event-free (EFS) and overall survival (OS), P < 0.001]. In the subgroup without abnormalities of other molecular markers, EFS of trisomic neuroblastomas was better (P = 0.048), but was most likely due to an unequal stage distribution. In further subgroups, in terms of age and stage, significance between the somy groups was not reached, neither for EFS nor OS. The multivariate analyses including age, stage, chromosomal markers, and somy 1 confirmed the lack of independent prognostic power for the copy number of chromosome 1. This study demonstrates the following: (1) FISH is a practical alternative to other more labor-intensive techniques for determining ploidy; (2) trisomic tumors correlate with younger age at diagnosis, localized stage, and the lack of structural alterations; and (3) polysomy is not an independent prognostic marker. The sharp decline of trisomic tumors after the age of 18 months supports the idea of different genetic tumor entities. PMID- 16682288 TI - Genome-wide semiquantitative microsatellite analysis of human hepatocellular carcinoma: discrete mapping of smallest region of overlap of recurrent chromosomal gains and losses. AB - Recurrent chromosomal gains at 1q, 6p, 8q, and 17q, or losses at 1p, 4q, 6q, 8p, 9p, 13q, 16q, and 17p are common features of human hepatocellular carcinoma (HCC). For precise determination of the shortest region of overlap (SRO), 49 HCC obtained at the time of surgery or autopsy were subjected to comprehensive microsatellite analysis by using 400 markers distributed at almost equal distances throughout the 22 autosomes and X chromosomes. Each allele showing imbalance was subjected to comparative duplex polymerase chain reaction using a retained allele as an internal control to determine whether the imbalance was the result of chromosomal gain or loss. The following SRO of recurrent chromosomal gains and losses were determined: -1p36.22 approximately p36.33, D1S450-D1S2893, 5.0 mega-base pairs (Mbp); +1q23.3 approximately q25.3, D1S2878-D1S2619, 16.9 Mbp; -4q21.2 approximately q24, D4S2964-D4S1572, 23.0 Mbp; -6q23.3 approximately qter, D6S292-qter, 34.7 Mb; -8p22 approximately p23.1, D8S549-D8S550, 4.8 Mbp; +8q12.2 approximately q24.13, D8S260-D8S514, 61.8 Mbp; -13q13.3 approximately q22.1, D13S218-D13S156, 35.6 Mbp; -16q22.1 approximately qter, D16S503-qter, 26.7 Mbp; and -17p12 approximately pter, D17S921-pter, 14.2 Mbp. Contrary to our initial expectations, many HCC showed major deletions or additions of chromosome arms, so that a number of genes were included in the SRO. Although some putative oncogenes or tumor suppressor genes mapped in these SRO may be important, relative copy number changes of numerous other genes may affect pathogenesis of HCC. PMID- 16682289 TI - Delineation by molecular cytogenetics of 5q deletion breakpoints in myelodyplastic syndromes and acute myeloid leukemia. AB - Deletions of 5q in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are of different extents and the majority map to sub-bands 5q13.3 and 5q33.1. To further pinpoint these deletions, we have performed a detailed interphase fluorescence in situ hybridization (I-FISH) analysis with precisely mapped BAC probes. Eleven MDS and two AML patients with a sole cytogenetically visible del(5q) were studied. The proximal deletion endpoints were localized between 75 and 86 megabases (Mb) (5q13, five times), 86 and 96 Mb (5q14 approximately q15, four times), and at various sites in the other four. The distal breakpoints mapped between 153 and 155 Mb (5q33.2, five times), 156 and 158 Mb (5q33.3, three times), 158 and 164 Mb (5q34, two times), and 164 and 181 Mb (telomere) in three. The largest deletion was approximately 70 Mb and the smallest was 43 Mb. These studies show that cytogenetically similar appearing deletions in 5q are highly variable in molecular terms. We also found that in MDS cases with a blast count between 0 and 13%, cells with a del(5q) were present in 24-90% of interphase (nondividing) cells and in 30-100% of metaphase (dividing) cells. In the two AML patients with a blast count of 30 and 80%, del(5q) was found in 35 and 95% interphase cells and 95 and 100% of metaphase cells, respectively. This demonstrates that a low blast count can be associated with a high proportion of 5q- cells in the bone marrow. PMID- 16682290 TI - Juvenile myelomonocytic leukemia in a child with Crohn disease. AB - Intestinal adenocarcinoma is a well-known complication of inflammatory bowel disease. Hematologic malignancies, most commonly lymphoma or acute myeloid leukemia, represent a much less well-recognized complication of these disorders; these typically occur in adults with ulcerative colitis. We report a fatal case of juvenile myelomonocytic leukemia associated with monosomy 7 in a young child with a clinical history of Crohn disease. Neither the leukemia nor the cytogenetic aberration has been previously reported in a patient with inflammatory bowel disease. The aggressive disease course emphasizes the need for proper recognition and further study of this unusual complication. PMID- 16682292 TI - A diminutive chromosome 21 centromere in acute lymphoblastic leukemia. AB - A chance observation of a tiny constitutional variant for the centromere of chromosome 21 in two patients with acute lymphoblastic leukemia (ALL), suggested a possible correlation with the cytogenetic findings in their leukemic cells. Interphase FISH revealed three 13/21 centromeric signals and a single MLL signal in the blast cells of each patient. Metaphase FISH with dual-color application of whole-chromosome paint (wcp) and centromeric probes for chromosome 21 showed two copies of chromosome 21, one with a tiny centromeric signal which corresponded to the invisible centromere in the interphase cells. Patient 2700 had a normal karyotype in his bone marrow at diagnosis. All metaphases from his stimulated peripheral blood also had the tiny chromosome 21 centromere, proving it to be a constitutional variant. Patient 3314 showed the abnormal karyotype 46,XY,inv(1)(p?q?),del(11)(q?),del(12)(p?),inc in his bone marrow. Interphase FISH revealed only one copy each of the ABL and ETV6 genes, in addition to the loss of the MLL signal. The question arises, is there an association between the diminutive centromeric signals for chromosome 21 and the chromosomal instability demonstrated by the deletions of key genes from the leukemic blasts of these two patients? PMID- 16682291 TI - Emergence of chronic myelogenous leukemia in a patient with primary thrombocythemia and absence of BCR/ABL rearrangement. AB - A 65-year-old woman presented with clinical features of primary thrombocythemia (PT), and absence of the BCR/ABL fusion gene. She responded to hydroxyurea treatment, although after 1 year she required progressive increases in the dose. Six years later, she maintained a high platelet count despite hydroxyurea at 2 g/day and treatment was changed to anagrelide. After 3 weeks, both platelet and leukocyte counts increased. A karyotype study detected the Philadelphia chromosome in all of the 24 metaphases studied. Fluorescent in situ hybridization (FISH) analysis revealed the BCR/ABL rearrangement. The patient was treated with imatinib mesylate and achieved a normal platelet and leukocyte count in 3 weeks. Patients presenting clinical features of PT expressing the Ph chromosome or the BCR/ABL fusion gene have been well documented but, to our knowledge, this is the first report of evolution from typical PT to chronic myeloid leukemia. PMID- 16682293 TI - A variant of the SYT-SSX2 fusion gene in a case of synovial sarcoma. AB - Synovial sarcoma is a malignant soft tissue tumor harboring a tumor-specific fusion gene, SYT-SSX, of which exon 10 of the SYT gene is fused to exon 6 of the SSX gene is the common form. Here we report a case of synovial sarcoma with a novel form of the SYT-SSX2 fusion transcript, in which 75 bases were inserted at the common fusion junction. Computer analyses revealed that 15 bases were from intron 10 of the SYT gene, and 10 from the end of intron 4, and 50 from exon 5 of the SSX2 gene. Precise analyses of genomic breakpoints in SYT and SSX2 loci revealed that the reciprocal translocation creating the fusion gene was associated with a large deletion in both loci. The structure of SYT-SSX2 suggests that the fusion transcript in this case was created using a cryptic splicing acceptor site 15 bases upstream of the genomic fusion point, incorporating intronic sequences in mature mRNA. Reexamination of two variant SYT-SSX2 genes reported previously revealed that unknown sequences inserted at the common junction points were derived from intron sequences, as in the present case. PMID- 16682294 TI - A suggested protocol for obtaining high-quality skin metaphases from primary cutaneous T-cell lymphoma. PMID- 16682295 TI - Translocations of 17q21 approximately qter in neuroblastoma cell lines infrequently include the topoisomerase IIalpha gene. PMID- 16682296 TI - Nitrate-dependent [Fe(II)EDTA]2- oxidation by Paracoccus ferrooxidans sp. nov., isolated from a denitrifying bioreactor. AB - Enrichments with [Fe(II)EDTA]2- as electron donor and nitrate or nitrite as electron acceptor were established using an inoculum from a bioreactor performing denitrification. A nitrate-reducing, [Fe(II)EDTA]2- oxidizing strain was isolated and named strain BDN-1. The G + C content of strain BDN-1 was 67%, and the organism was closely affiliated to Paracoccus denitrificans, P. pantotrophus and P. versutus by 16S rRNA sequence comparison. Results from DNA-DNA hybridization, rep-PCR, and whole cell protein analysis gave congruent results confirming the genotypic and phenotypic differences between strain BDN-1 and the other species of Paracoccus. From these results, we considered strain BDN-1 as a novel species for which we propose the name Paracoccus ferrooxidans. Apart from [Fe(II)EDTA]2-, BDN-1 could also use thiosulfate and thiocyanate as inorganic electron donors. Nitrate, nitrite, N2O, [Fe(II)EDTA.NO]2- and oxygen could be used by strain BDN-1 as electron acceptors. Repeated transfer on a culture medium with bicarbonate as the sole carbon source confirmed that strain BDN-1 was a facultative autotroph. [Fe(II)EDTA]2- oxidation dependent denitrification was also performed by other Paracoccus species, that were closely affiliated to P. ferrooxidans. PMID- 16682297 TI - Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica). AB - A new thermophilic spore-forming strain MR3CT was isolated from geothermal soil located on Mount Rittmann in Antarctica. Strain MR3CT was Gram-positive, rod shaped, occurring in pairs or filamentous. Growth was observed between 45 and 65 degrees C (optimum 61 degrees C) and at pH 5.0-6.5 (optimum pH 5.6). It was capable of utilizing galactose, trehalose, maltose and sucrose. The microorganism produced an exopolysaccharide and synthesized an extracellular constitutive amylolytic activity. The G + C content of DNA was 43.5 mol%. On the basis of 16S rRNA gene sequence similarity, strain MR3CT was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain MR3C1T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MR3CT from the validly published Anoxybacillus species. MR3CT therefore represents a new species, for which the name Anoxybacillus amylolyticus sp. nov., is proposed, with the type strain MR3CT (= ATCC BAA-872T = DSM 15939T = CIP 108338T). PMID- 16682298 TI - Hormone replacement therapy and the cardiovascular system lessons learned and unanswered questions. AB - Cardiovascular disease is the leading cause of death among women in the U.S., exceeding breast cancer mortality in women of all ages. Women present with cardiovascular disease a decade after men, and this has been attributed to the protective effect of female ovarian sex hormones that is lost after menopause. Animal and observational studies have shown beneficial effects of hormone therapy when it is initiated early in the perimenopausal period or before the development of significant atherosclerosis. However, randomized, placebo-controlled trials in older women have not shown any benefit in either primary prevention or secondary prevention of cardiovascular events, with a concerning trend toward harm. This review outlines the lessons learned from the basic science, animal, observational, and randomized trials, and then summarizes yet-unanswered questions of hormone therapy and cardiovascular risk. PMID- 16682299 TI - Fourth annual American College of Cardiology international lecture: a journey in the interventional field. PMID- 16682300 TI - Cardiac regeneration. AB - The role and even the existence of new myocyte formation in the adult heart remain controversial. Documentation of cell cycle regulators, deoxyribonucleic acid synthesis, and mitotic images has only in part modified the view that myocardial growth can be accomplished exclusively from hypertrophy of an irreplaceable population of differentiated myocytes. However, myocyte regeneration and death occur physiologically, and these cellular processes are enhanced in pathologic states. These observations have challenged the view of the heart as a postmitotic organ and have proposed a new paradigm in which parenchymal and non-parenchymal cells are continuously replaced by newly formed younger populations of myocytes as well as by vascular smooth muscle and endothelial cells. Heart homeostasis is regulated by a stem cell compartment characterized by multipotent cardiac stem cells that possess the ability to acquire the distinct cell lineages of the myocardium. Similarly, adult bone marrow cells are able to differentiate into cells beyond their own tissue boundary and create cardiomyocytes and coronary vessels. This process has been termed developmental plasticity or transdifferentiation. Because of these properties, bone marrow cells and cardiac stem cells have been employed experimentally in the reconstitution of dead myocardium after infarction. These cell classes hold promise for the treatment of heart failure in humans. PMID- 16682301 TI - Regeneration gaps: observations on stem cells and cardiac repair. AB - Substantial evidence indicates that cell transplantation can improve function of the infarcted heart. A surprisingly wide range of non-myogenic cell types improves ventricular function, suggesting that benefit may result in part from mechanisms that are distinct from true myocardial regeneration. While clinical trials explore cells derived from skeletal muscle and bone marrow, basic researchers are investigating sources of new cardiomyocytes, such as resident myocardial progenitors and embryonic stem cells. In this commentary, we briefly review the evolution of cell-based cardiac repair, discuss the current state of clinical research, and offer some thoughts on how newcomers can critically evaluate this emerging field. PMID- 16682302 TI - A novel drug-eluting stent coated with an integrin-binding cyclic Arg-Gly-Asp peptide inhibits neointimal hyperplasia by recruiting endothelial progenitor cells. AB - OBJECTIVES: Novel stents loaded with an integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) were analyzed for their potential to limit coronary neointima formation and to accelerate endothelialization by attracting endothelial progenitor cells (EPCs). BACKGROUND: Re-endothelialization is important for healing after arterial injury. METHODS: Effects of cRGD on EPC number, recruitment in flow, and invasion were analyzed in vitro. A durable polymer coating containing 67 microg cRGD per stent was developed for Guidant Tetra stents. Twelve cRGD-loaded polymer, 12 unloaded polymer, and 12 bare metal stents were deployed in porcine coronary arteries. Quantification of cRGD in peri-stent tissue was established by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Histomorphometry and immunostaining were performed after 4 and 12 weeks. Recruitment of labeled porcine EPCs was assessed 7 days after intracoronary infusion. RESULTS: The cRGD clearly supported the outgrowth, recruitment, and migration of EPCs in vitro. At 4 weeks, there was no difference for mean neointimal area and percent area stenosis in the cRGD-loaded, polymer, or bare metal stent group. At 12 weeks, neointimal area (2.2 +/- 0.3 mm2) and percent area stenosis (33 +/- 5%) were significantly reduced compared with polymer stents (3.8 +/- 0.4 mm2, 54 +/- 6%; p = 0.010) or bare metal stents (3.8 +/- 0.3 mm2, 53 +/- 3%; p < 0.001). The HPLC/MS confirmed cRGD tissue levels of 1 to 3 mug/stent at 4 weeks, whereas cRGD was not detectable at 12 weeks. Staining for CD34 and scanning electron microscopy indicated enhanced endothelial coverage on cRGD-loaded stents at 4 weeks associated with a significant increase in the early recruitment of infused EPCs. CONCLUSIONS: Stent coating with cRGD may be useful for reducing in-stent restenosis by accelerating endothelialization. PMID- 16682303 TI - Learning curves and reliability measures for virtual reality simulation in the performance assessment of carotid angiography. AB - OBJECTIVES: Improvement in performance as measured by metric-based procedural errors must be demonstrated if virtual reality (VR) simulation is to be used as a valid means of proficiency assessment and improvement in procedural-based medical skills. BACKGROUND: The Food and Drug Administration requires completion of VR simulation training for physicians learning to perform carotid stenting. METHODS: Interventional cardiologists (n = 20) participating in the Emory NeuroAnatomy Carotid Training program underwent an instructional course on carotid angiography and then performed five serial simulated carotid angiograms on the Vascular Interventional System Trainer (VIST) VR simulator (Mentice AB, Gothenburg, Sweden). Of the subjects, 90% completed the full assessment. Procedure time (PT), fluoroscopy time (FT), contrast volume, and composite catheter handling errors (CE) were recorded by the simulator. RESULTS: An improvement was noted in PT, contrast volume, FT, and CE when comparing the subjects' first and last simulations (all p < 0.05). The internal consistency of the VIST VR simulator as assessed with standardized coefficient alpha was high (range 0.81 to 0.93), except for FT (alpha = 0.36). Test-retest reliability was high for CE (r = 0.9, p = 0.0001). CONCLUSIONS: A learning curve with improved performance was demonstrated on the VIST simulator. This study represents the largest collection of such data to date in carotid VR simulation and is the first report to establish the internal consistency of the VIST simulator and its test-retest reliability across several metrics. These metrics are fundamental benchmarks in the validation of any measurement device. Composite catheter handling errors represent measurable dynamic metrics with high test-retest reliability that are required for the high-stakes assessment of procedural skills. PMID- 16682304 TI - Oxidized low-density lipoprotein in children with familial hypercholesterolemia and unaffected siblings: effect of pravastatin. AB - OBJECTIVES: To assess the role of oxidized phospholipids (OxPLs) in children with familial hypercholesterolemia (FH) and the effect of pravastatin. BACKGROUND: Oxidized phospholipids are a major component of oxidized low-density lipoprotein (OxLDL) and are bound to lipoprotein (a) [Lp(a)]. The significance of OxPL markers in children is unknown. METHODS: Children with FH were randomized to placebo (n = 88) or pravastatin (n = 90) after instruction on American Heart Association step II diet. Unaffected siblings (n = 78) served as controls. The OxPL content on apolipoprotein B-100 (apoB) detected by antibody E06 (OxPL/apoB ratio), immunoglobulin (Ig)G and IgM immune complexes per apoB (IC/apoB) and on all apoB particles (total apoB-IC = IC/apoB multiplied by plasma apoB levels), autoantibodies to malondialdehyde (MDA)-low-density lipoprotein (LDL), Lp(a), and apoB levels were measured at baseline and after two years of treatment. RESULTS: Compared with unaffected siblings, children with FH had significantly lower levels of OxPL/apoB but higher levels of IgG and IgM total apoB-IC and IgM MDA LDL autoantibodies. From baseline to two-year follow-up, compared with placebo pravastatin treatment resulted in a greater mean percentage change in apoB ( 18.7% vs. 0.3%; p = 0.001), total IgG apoB-IC (-31.9% vs. -12.2%; p < 0.001), and total IgM apoB-IC (-25.5% vs. 13.2%; p = 0.001). Interestingly, pravastatin also resulted in higher OxPL/apoB (48.7% vs. 29.3%; p = 0.028) and Lp(a) levels (21.9% vs. 10.7%; p = 0.044). CONCLUSIONS: Compared with unaffected siblings, children with FH are characterized by elevated levels of apoB-IC and IgM MDA-LDL autoantibodies. Compared with placebo, pravastatin led to a greater reduction in apoB-IC but also to a greater increase in OxPL/apoB and Lp(a), which may represent a novel mechanism of mobilization and clearance of OxPL. PMID- 16682305 TI - Time dependence of defibrillator benefit after coronary revascularization in the Multicenter Automatic Defibrillator Implantation Trial (MADIT)-II. AB - OBJECTIVES: The study was designed to assess the effect of elapsed time from coronary revascularization (CR) on the benefit of the implantable cardioverter defibrillator (ICD) and the risk of sudden cardiac death (SCD) in patients with ischemic left ventricular dysfunction. BACKGROUND: The ICD improves survival in appropriately selected high-risk cardiac patients by 30% to 54%. However, in the Coronary Artery Bypass Graft (CABG)-Patch trial no evidence of improved survival was shown among a similar population of patients in whom an ICD was implanted prophylactically at the time of elective CABG. METHODS: The outcome by time from CR was analyzed in 951 patients in whom a revascularization procedure was performed before enrollment in the Multicenter Automatic Defibrillator Implantation Trial (MADIT)-II. RESULTS: The adjusted hazard ratio (HR) of ICD versus conventional therapy was 0.64 (p = 0.01) among patients enrolled more than six months after CR, whereas no survival benefit with ICD therapy was shown among patients enrolled six months or earlier after CR (HR = 1.19; p = 0.76). In the conventional therapy group, the risk of cardiac death increased significantly with increasing time from CR (p for trend = 0.009), corresponding mainly to a six fold increase in the risk of SCD among patients enrolled more than six months after CR. CONCLUSIONS: In patients with ischemic left ventricular dysfunction, the efficacy of ICD therapy after CR is time dependent, with a significant life saving benefit in patients receiving device implantation more than six months after CR. The lack of ICD benefit when implanted early after CR may be related to a relatively low risk of SCD during this time period. PMID- 16682306 TI - Tincture of time: when to implant a prophylactic cardioverter-defibrillator following coronary revascularization? PMID- 16682307 TI - Prognostic utility of microvolt T-wave alternans in risk stratification of patients with ischemic cardiomyopathy. AB - OBJECTIVES: The purpose of this study was to assess if microvolt T-wave alternans (MTWA) is an independent predictor of mortality in patients with ischemic cardiomyopathy. BACKGROUND: Microvolt T-wave alternans has been proposed as an effective tool for identifying high-risk patients with ischemic cardiomyopathy who are likely to benefit from implantable cardioverter-defibrillator (ICD) therapy. However, earlier studies have been limited in their ability to control for baseline differences between MTWA-negative and -non-negative (positive and indeterminate) patients. METHODS: We enrolled 768 consecutive patients with ischemic cardiomyopathy (left ventricular ejection fraction < or =35%) and no prior history of ventricular arrhythmia. All patients underwent baseline MTWA testing and were classified as MTWA negative or non-negative. Multivariable Cox regression analyses, stratified by ICD status, were used to determine the association between MTWA testing and mortality after adjusting for demographic, clinical, and treatment differences between MTWA-negative and -non-negative patients. RESULTS: We identified 514 (67%) patients with a non-negative MTWA test. After multivariable adjustment, a non-negative MTWA test was associated with a significantly higher risk for all-cause (stratified hazard ratio [HR] = 2.24 [95% confidence interval 1.34 to 3.75]; p = 0.002) and arrhythmic mortality (stratified HR = 2.29 [1.00 to 5.24]; p = 0.049) but not for nonarrhythmic mortality (stratified HR = 1.77 [0.84 to 3.74]; p = 0.13). In subgroup analyses, a non-negative MTWA test was also associated with a higher risk for all-cause mortality in patients with ejection fractions < or =30% (stratified HR = 2.10 [1.18 to 3.73]; p = 0.01) and after excluding those with indeterminate MTWA tests (stratified HR = 2.08 [1.18 to 3.66]; p = 0.01). CONCLUSIONS: Microvolt T-wave alternans is a strong and independent predictor of all-cause and arrhythmic mortality in patients with ischemic cardiomyopathy. PMID- 16682308 TI - Tpeak-Tend and Tpeak-Tend dispersion as risk factors for ventricular tachycardia/ventricular fibrillation in patients with the Brugada syndrome. AB - OBJECTIVES: Our objective in this study was to evaluate Tpeak-Tend interval (Tp e) and other electrocardiographic parameters as risk factors for recurrence of life-threatening cardiac events in patients with the Brugada syndrome (BS). BACKGROUND: The Tp-e interval in the electrocardiogram (ECG) has been reported to predict life-threatening arrhythmias in the long QT syndrome. METHODS: Twenty nine patients with the ECG pattern of BS and 29 healthy age- and gender-matched controls were studied. The follow-up period was 42.65 +/- 24.42 months (range 11 to 108 months). RESULTS: Upon presentation, five patients had suffered aborted sudden death, five syncope, and two presyncope. Eleven patients with the ECG pattern of BS had a prolonged (>460 ms) QTc in V2 but usually not in inferior or left leads. No patient had abnormally prolonged QT dispersion. Programmed electrical stimulation induced ventricular tachycardia/fibrillation in 5 out of 26 patients. Inducibility did not predict recurrence of events. Cardioverter defibrillators were implanted in 14 patients (all symptomatic and two asymptomatic). During follow-up, nine symptomatic patients experienced recurrences. Previous cardiac events and a QTc >460 ms in V2 were significant risk factors (p = 0.00002 and p = 0.03, respectively). Tp-e and Tp-e dispersion were significantly prolonged in patients with recurrences versus patients without events (104.4 and 35.6 ms vs. 87.4 and 23.2 ms; p = 0.006 and p = 0.03, respectively) or controls (90.7 and 17.9 ms; p = 0.02 and p = 0.001, respectively). CONCLUSIONS: Our study demonstrates significant correlation between previous events, QTc >460 ms in V2, Tp-e, and Tp-e dispersion and occurrence of life-threatening arrhythmic events, suggesting that these parameters may be useful in risk stratification of patients with the Brugada syndrome. PMID- 16682309 TI - Aerobic training decreases B-type natriuretic peptide expression and adrenergic activation in patients with heart failure. AB - OBJECTIVES: We sought to evaluate the effect of physical training on neurohormonal activation in patients with heart failure (HF). BACKGROUND: Patients with HF benefit from physical training. Chronic neurohormonal activation has detrimental effects on ventricular remodeling and prognosis of patients with HF. METHODS: A total of 95 patients with HF were assigned randomly into two groups: 47 patients (group T) underwent a nine-month training program at 60% of the maximal oxygen uptake (VO2), whereas 48 patients did not (group C). The exercise load was adjusted during follow-up to achieve a progressive training effect. Plasma assay of B-type natriuretic peptide (BNP), amino-terminal pro brain natriuretic peptide (NT-proBNP), norepinephrine, plasma renin activity, and aldosterone; quality-of-life questionnaire; echocardiogram; and cardiopulmonary stress test were performed upon enrollment and at the third and ninth month. RESULTS: A total of 85 patients completed the protocol (44 in group T, left ventricular ejection fraction [EF] 35 +/- 2%, mean +/- SEM; and 41 in group C, EF 32 +/- 2%, p = NS). At the ninth month, patients who underwent training showed an improvement in workload (+14%, p < 0.001), peak VO2 (+13%, p < 0.001), systolic function (EF +9%, p < 0.01), and quality of life. We noted that BNP, NT-proBNP, and norepinephrine values decreased after training (-34%, p < 0.01; -32%, p < 0.05; -26%, p < 0.01, respectively). Increase in peak VO2 with training correlated significantly with the decrease in both BNP/NT-proBNP level (p < 0.001 and p < 0.01, respectively). Patients who did not undergo training showed no changes. CONCLUSIONS: Clinical benefits after physical training in patients with HF are associated with blunting of adrenergic overactivity and of natriuretic peptide overexpression. PMID- 16682310 TI - Comparison of radiation doses from multislice computed tomography coronary angiography and conventional diagnostic angiography. AB - OBJECTIVES: The aim of this study was to quantify and compare effective doses from conventional angiography and multislice computed tomography (MSCT) coronary angiography using a 16-slice scanner. BACKGROUND: Multislice computed tomography is now a viable modality for cardiac imaging. However, for any diagnostic use of ionizing radiation, the risk to the patient must be considered and justified. METHODS: Multislice computed tomography angiography and conventional angiography were used to assess 180 patients with suspected coronary artery disease. Estimates of effective dose were derived from exposure data recorded for each patient examination. For each modality, a comparable calculation technique was used, based on Monte Carlo modeling of the standard Cristy phantom. RESULTS: In a subset of 91 directly comparable patients the mean effective dose for MSCT coronary angiography was 14.7 mSv (SD 2.2) and that for conventional angiography was 5.6 mSv (SD 3.6). A significant difference in effective dose was seen between the two protocols. CONCLUSIONS: The mean effective dose for MSCT coronary angiography was significantly higher than that for conventional angiography. As MSCT cardiac scanners become increasingly available, operators must be aware of the radiation dose and the factors that affect it. PMID- 16682311 TI - Radiation exposure of computed tomography and direct intracoronary angiography: risk has its reward. AB - A hallmark of noninvasive testing has been the identification of patients with coronary artery disease. Now, with multislice computed tomography (MSCT), information about coronary anatomy can be obtained without the need for catheterization. A major concern with the application of MSCT coronary angiography is the radiation exposure to the patient. Both MSCT and selective coronary angiography share the risks of procedure-related complications, such as allergic contrast reactions, and stochastic risks (i.e., cancer induction) of low level radiation. There is a substantially higher radiation dose for MSCT angiography (effective dose [ED] 14 mSv) than for CCA (ED 6 mSv). These exposures yield lifetimes risks of 0.07% and 0.02%, respectively, of inducing a fatal cancer in the general (i.e., age- and gender-averaged) population. However, CCA poses additional serious risks associated with cardiac catheterization, yielding a non-radiogenic risk of mortality--excluding contrast reactions--of 0.11%. Combining the radiogenic and non-radiogenic risks (0.02% and 0.11%, respectively) yields a 0.13% overall risk of mortality from CCA--nearly two-fold higher than that for MSCT angiography (0.07%). If one were to use the lower, more age appropriate risk factors for the older patient population in question, the radiogenic risks of both CCA and MSCT would be reduced by about one-half, further widening the overall safety ratio of MSCT relative to CCA. When weighing the relative risks of alternative medical procedures, therefore, it is imperative that one consider the overall risk of the respective procedures. PMID- 16682312 TI - The relationship between plasma osteoprotegerin levels and coronary artery calcification in uncomplicated type 2 diabetic subjects. AB - OBJECTIVES: This study sought to prospectively evaluate the relationship between plasma osteoprotegerin (OPG), inflammatory biomarkers (high-sensitivity C reactive protein [hs-CRP], interleukin-6 [IL-6], coronary artery calcification (CAC), and cardiovascular events in patients with type 2 diabetes. BACKGROUND: Arterial calcification is a prominent feature of atherosclerosis and is associated with an increased risk of cardiovascular events. Osteoprotegerin is a cytokine that has recently been implicated in the regulation of vascular calcification. METHODS: A total of 510 type 2 diabetic patients (53 +/- 8 years; 61% male) free of symptoms of cardiovascular disease were evaluated by CAC imaging. Risk factors, hs-CRP, IL-6, and OPG levels were measured. Patients were followed up for cardiovascular events (cardiac death, myocardial infarction, acute coronary syndrome, late revascularization, and nonhemorrhagic stroke). RESULTS: Significant CAC (>10 Agatston units) was seen in 236 patients (46.3%); OPG was significantly elevated in patients with increased CAC. In multivariable analyses, OPG retained a strong association with elevated CAC scores after adjustment for age, gender, and other risk factors (odds ratio = 2.84, 95% confidence interval 2.2 to 3.67; p < 0.01). Sixteen cardiovascular events occurred during a mean follow-up of 18 +/- 5 months. The waist-to-hip ratio, United Kingdom Prospective Diabetes Study (UKPDS) risk score, OPG level, and CAC score were significant predictors of time to cardiovascular events in a univariate Cox proportional hazards model. In the multivariate model, the CAC score was the only independent predictor of adverse events. Levels of hs-CRP and IL-6 were related to neither the extent of CAC nor short-term events. CONCLUSIONS: A high proportion of asymptomatic diabetic patients have significant subclinical atherosclerosis. Of the biomarkers studied, only OPG predicted both subclinical disease and near-term cardiovascular events. Therefore, measurement of OPG merits further investigation as a simple test for identifying high-risk type 2 diabetic patients. PMID- 16682313 TI - Validation and re-evaluation of a discriminant model predicting anatomic suitability for biventricular repair in neonates with aortic stenosis. AB - OBJECTIVES: The purpose of this study was to validate and re-evaluate our previously reported scoring systems for predicting optimal management in neonates with aortic stenosis (AS). BACKGROUND: In 1991, we reported a multivariate discriminant equation and an ordinal scoring system for predicting which neonates with AS are suitable for biventricular repair and which are better served by single ventricle management. METHODS: Retrospective analysis was performed to: 1) validate our scoring systems in 89 additional neonates with AS and normal mitral valve area, 2) assess the effects of 5% measurement variation on predictive scores, 3) evaluate our cohort with the Congenital Heart Surgeons' Society scoring system, and 4) repeat the discriminant analysis on the basis of all 126 patients. RESULTS: The original scores each predicted outcome accurately in 68 patients (77%). Minor (5%) measurement variation changed the outcome predicted by the discriminant equation in 8 patients (9%) and by the threshold system in 13 patients (15%). The most accurate model for predicting survival with a biventricular circulation among the full cohort is: 10.98 (body surface area) + 0.56 (aortic annulus z-score) + 5.89 (left ventricular to heart long-axis ratio) 0.79 (grade 2 or 3 endocardial fibroelastosis) - 6.78. With a cutoff of -0.65, outcome was predicted accurately in 90% of patients. CONCLUSIONS: Both of our original scoring systems are less accurate at predicting outcome than in our original analysis. Revised discriminant analysis yielded a model similar to our original equation that was 90% accurate at predicting survival with a biventricular circulation among neonates with AS and a mitral valve area z-score >-2. PMID- 16682314 TI - A novel contrast removal system from the coronary sinus using an adsorbing column during coronary angiography in a porcine model. AB - OBJECTIVES: This study examined the efficacy of a novel system to remove contrast from the coronary sinus (CS) using an adsorbing column during coronary angiography. BACKGROUND: Contrast-induced nephropathy (CIN) has become a serious problem for patients with pre-existing renal insufficiency undergoing percutaneous coronary intervention (PCI). METHODS: Eight swine were studied to evaluate the efficacy of the contrast removal system. A newly developed 8-F blood suction catheter was inserted into the CS via the right femoral vein. The venous blood from the CS was transferred into the 500-ml contrast-adsorbing column using an extracorporeal system. A total of 155 ml of contrast medium was infused selectively into the coronary artery. Five swine were treated extracorporeally for 90 min using adsorbing columns, and three were treated without columns as a control group. RESULTS: The mean calculated iodine removal rate was 49.4%. The area under the curve of the iodine concentration in the group treated with the column was significantly smaller, by 60%, than that of the group treated without the column (p = 0.0003). No significant adverse effects were observed in the subjects' vital signs throughout the experiments. CONCLUSIONS: The contrast removal system from CS is effective and safe during coronary angiography in swine. This technique may be useful for preventing CIN in patients with renal insufficiency undergoing PCI. PMID- 16682315 TI - The effects of combined versus selective adrenergic blockade on left ventricular and systemic hemodynamics, myocardial substrate preference, and regional perfusion in conscious dogs with dilated cardiomyopathy. AB - OBJECTIVES: Given that adverse effects of chronic sympathetic activation are mediated by all three adrenergic receptor subtypes (beta1, beta2, alpha1), we examined the effects of standard doses of carvedilol and metoprolol succinate (metoprolol controlled release/extended release [CR/XL]) on hemodynamics, myocardial metabolism, and regional organ perfusion. BACKGROUND: Both beta1 selective and combined adrenergic blockade reduce morbidity and mortality in heart failure. Whether there are advantages of one class over the other remains controversial, even in the wake of the Carvedilol Or Metoprolol European Trial (COMET). Similarly, the mechanistic basis for the relative differences is incompletely understood. METHODS: Thirty-three conscious, chronically instrumented dogs with pacing-induced (240 min(-1) for 4 weeks) dilated cardiomyopathy (DCM) were randomized to carvedilol (25 mg twice daily, Coreg, Glaxo Smith Kline, Research Triangle, North Carolina) or metoprolol succinate (100 mg qd, Toprol XL, Astra Zeneca, Wilmington, Delaware). Left ventricular and systemic hemodynamics, myocardial substrate uptake, and norepinephrine spillover were measured before and after three days of treatment. Regional (renal, hepatic, skeletal muscle) blood flows were measured using neutron-activated microspheres. RESULTS: Both agents had comparable heart rate effects. However, carvedilol treated dogs showed significantly greater increases in stroke volume and cardiac output and decreases in left ventricular end-diastolic pressure and systemic vascular resistance. Carvedilol increased renal, hepatic, and skeletal muscle blood flow. Carvedilol increased myocardial glucose uptake and suppressed norepinephrine and glucagon. Carvedilol antagonized the response to exogenous norepinephrine to a greater extent than metoprolol CR/XL. CONCLUSIONS: At doses inducing comparable heart rate reductions, short-term treatment with carvedilol had superior hemodynamic and metabolic effects compared with metoprolol CR/XL. These data suggest important advantages of blocking all three adrenergic receptor subtypes in DCM. PMID- 16682316 TI - Serofendic acid, a novel substance extracted from fetal calf serum, protects against oxidative stress in neonatal rat cardiac myocytes. AB - OBJECTIVES: We examined whether serofendic acid (SFA) has protective effects against oxidative stress in cardiac myocytes. BACKGROUND: We previously identified a novel endogenous substance, SFA, from a lipophilic extract of fetal calf serum. Serofendic acid protects cultured neurons against the cytotoxicity of glutamate, nitric oxide, and oxidative stress. METHODS: Primary cultures of neonatal rat cardiac myocytes were exposed to oxidative stress (H2O2, 100 micromol/l) to induce cell death. Effects of SFA were evaluated with a number of markers of cell death. RESULTS: Pretreatment with SFA (100 micromol/l) significantly suppressed markers of cell death, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining and cell viability assay. Loss of mitochondrial membrane potential (DeltaPsi(m)) is a critical step of the death pathway, which is triggered by matrix calcium overload and reactive oxygen species. Serofendic acid prevented the DeltaPsi(m) loss induced by H2O2 in a concentration-dependent manner (with saturation by 100 micromol/l). Serofendic acid remarkably suppressed the H2O2-induced matrix calcium overload and intracellular accumulation of reactive oxygen species. The protective effect of SFA was comparable to that of a mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channel opener, diazoxide. Furthermore, mitoK(ATP) channel blocker, 5-hydroxydecanoate (500 micromol/l), abolished the protective effect of SFA. Co-application of SFA (100 micromol/l) and diazoxide (100 micromol/l) did not show an additive effect. Thus, SFA inhibited the oxidant-induced mitochondrial death pathway, presumably through activation of the mitoK(ATP) channel. CONCLUSIONS: Serofendic acid protects cardiac myocytes against oxidant-induced cell death by preserving the functional integrity of mitochondria. PMID- 16682317 TI - Diastolic stress echocardiography: hemodynamic validation and clinical significance of estimation of ventricular filling pressure with exercise. AB - OBJECTIVES: Our study attempted to validate a Doppler index of diastolic filling (E/E') during exercise with simultaneously measured left ventricular diastolic pressure (LVDP), investigate its association with exercise capacity, and understand which patients to select for testing. BACKGROUND: The ratio of early diastolic transmitral velocity to early diastolic tissue velocity approximates LVDP at rest, but there is limited validation of exercise E/E' with invasive hemodynamic measurement, and its clinical implications are unclear. METHODS: The ratio of early diastolic transmitral velocity to early diastolic tissue velocity was measured at rest and during supine cycle ergometry in 37 patients undergoing left heart catheterization. In addition to correlation between invasive and estimated LVDP, the accuracy of different cutoffs for identification of elevated LVDP (>15 mm Hg) was determined at both rest and exercise. Doppler index of diastolic filling was also measured at rest and immediately after maximal treadmill exercise in 166 patients to investigate the association between exercise E/E' and exercise capacity (<8 metabolic equivalents [METs]). RESULTS: In patients undergoing invasive measurement, nine (24%) had elevation of LVDP only during exercise. There was a good correlation between E/E' and LVDP at rest (r = 0.67) and during exercise (r = 0.59), and the regressions at rest and exercise corresponded closely. Receiver-operator curve analysis indicated that a cutoff value of 13 for exercise E/E' identified patients with an elevated LVDP during exercise. A post-exercise E/E' >13 was highly specific (90%) for reduced exercise capacity, and even after classification of resting E/E', exercise E/E' permitted classification of patients with exercise capacity <8 METs or > or =8 METs. CONCLUSIONS: The ratio of early diastolic transmitral velocity to early diastolic tissue velocity correlates with invasively measured LVDP during exercise. It can be used to reliably identify patients with elevated LVDP during exercise and reduced exercise capacity. PMID- 16682318 TI - President's page: American College of Cardiology fellowship: where ideals and personal responsibility intersect. PMID- 16682319 TI - Early versus delayed enalapril in patients with left ventricular systolic dysfunction: impact on morbidity and mortality 15 years after the SOLVD trial. PMID- 16682320 TI - Short QT interval and atrial fibrillation in patients without structural heart disease. PMID- 16682321 TI - Association of pulsus paradoxus with obesity in normal volunteers. PMID- 16682322 TI - Diabetes lowers six-minute walk test performance in heart failure. PMID- 16682323 TI - Life-threatening coronary artery spasm following sirolimus-eluting stent deployment. PMID- 16682324 TI - Endothelial dysfunction after drug-eluting stent was never predicted in preclinical studies. PMID- 16682326 TI - Preventing type 2 diabetes mellitus with angiotensin blockade: is it clinically relevant? PMID- 16682327 TI - Long-term bosentan treatment in children with pulmonary arterial hypertension. PMID- 16682329 TI - Limitations of crush technique. PMID- 16682332 TI - Autapses. PMID- 16682333 TI - Insect flight. PMID- 16682334 TI - An ancient Fox gene cluster in bilaterian animals. PMID- 16682335 TI - Nanosecond-scale kinetics of nematocyst discharge. PMID- 16682336 TI - Molecular evidence for dim-light vision in the last common ancestor of the vertebrates. PMID- 16682338 TI - Nuclear actin: a lack of export allows formation of filaments. AB - Actin has been found in nuclei of many cell types, but little is known about its form and function. A recent study has shown that a lack of specific export allows actin to accumulate in the nucleus, where it forms a network of actin filaments that may be required to stabilize the giant nucleus of the Xenopus oocyte. PMID- 16682339 TI - Social learning: ants and the meaning of teaching. AB - Recent research on ants shows that running in tandem might serve the function of teaching naive ants about the path to a target. Although these new experiments represent perhaps the most highly controlled study of teaching in animals to date, the findings prompt the question of how teaching formally differs from other forms of communication. PMID- 16682340 TI - Cell mechanics: filaminA leads the way. AB - Actin filaments are thought to be the major structural components of most eukaryotic cells, but reconstituted actin networks have yet to account for the remarkable strength exhibited by cellular networks. A new study has found that reconstituted networks that include the cross-linker filaminA can replicate many of the mechanical properties of cells if they are stressed prior to mechanical measurement. PMID- 16682341 TI - Comparative neurophysiology: an electric convergence in fish. AB - Two lineages of fishes convergently evolved electric organs; recent research has shown that they independently took advantage of an extra copy of a sodium channel gene to alter channel kinetics for the electric organ discharge. PMID- 16682342 TI - Visual physiology: perceived size looms large. AB - Visual illusions tell us that size perception depends heavily upon complex contextual cues, often thought to be extracted by brain areas high in the visual hierarchy. Now, a new study shows that perceived size is reflected in activity as early as the primary visual cortex. PMID- 16682343 TI - Sympatric speciation: why the controversy? AB - Two recent studies provide convincing evidence that speciation without geographic isolation--sympatric speciation--has occurred within isolated island habitats, but we remain ignorant of how frequent the process really is. PMID- 16682344 TI - Conservation: losing biodiversity by reverse speciation. AB - A large fraction of the world's species diversity is of recent evolutionary origin, and has evolved as a by-product of divergent adaptation in heterogeneous environments. Recent research provides a dire warning that homogenizing environments may cause the rapid loss of such species through a reversal of the speciation process. PMID- 16682345 TI - Cell migration: neurons go with the flow. AB - Cilia lining the surfaces of the brain ventricles may be responsible for the graded distribution of chemorepellents that drive the directed migration of neurons. PMID- 16682346 TI - Integrin alpha5beta1 and fibronectin regulate polarized cell protrusions required for Xenopus convergence and extension. AB - BACKGROUND: Integrin recognition of fibronectin is required for normal gastrulation including the mediolateral cell intercalation behaviors that drive convergent extension and the elongation of the frog dorsal axis; however, the cellular and molecular mechanisms involved are unclear. RESULTS: We report that depletion of fibronectin with antisense morpholinos blocks both convergent extension and mediolateral protrusive behaviors in explant preparations. Both chronic depletion of fibronectin and acute disruptions of integrin alpha5beta1 binding to fibronectin increases the frequency and randomizes the orientation of polarized cellular protrusions, suggesting that integrin-fibronectin interactions normally repress frequent random protrusions in favor of fewer mediolaterally oriented ones. In the absence of integrin alpha5beta1 binding to fibronectin, convergence movements still occur but result in convergent thickening instead of convergent extension. CONCLUSIONS: These findings support a role for integrin signaling in regulating the protrusive activity that drives axial extension. We hypothesize that the planar spatial arrangement of the fibrillar fibronectin matrix, which delineates tissue compartments within the embryo, is critical for promoting productive oriented protrusions in intercalating cells. PMID- 16682347 TI - Human Scc4 is required for cohesin binding to chromatin, sister-chromatid cohesion, and mitotic progression. AB - BACKGROUND: Sister-chromatid cohesion depends on the cohesin complex whose association with chromatin is mediated by Scc2 and Scc4 in budding yeast. Both cohesin and Scc2 have been conserved from yeast to humans, but no Scc4 orthologs have been identified. Mutation of Scc2 orthologs causes defects in cohesion, transcription, and development, resulting in Cornelia de Lange syndrome in humans. RESULTS: We have identified a family of tetratricopeptide repeat proteins that share weak sequence similarities with yeast Scc4. This family includes MAU 2, which is required for development of the nervous system in Caenorhabditis elegans. We show that the human member of this family is associated with Scc2, is bound to chromatin from telophase until prophase, and is required for association of cohesin with chromatin during interphase. Cells lacking Scc4 lose sister chromatid cohesion precociously and arrest in prometaphase. Mitotic chromosomes in Scc4-depleted cells lack cohesin, even though the cohesin-protecting proteins Sgo1 and Bub1 are normally enriched at centromeres and separase does not seem to be active. CONCLUSION: Our data indicate that human Scc4 is required for the association of cohesin with chromatin, which is a prerequisite for the establishment of sister-chromatid cohesion and for chromosome biorientation in mitosis. The proteinaceous machinery that is required for loading of cohesin onto chromatin is therefore conserved from yeast to humans. The finding that Caenorhabditis elegans MAU-2 is an ortholog of Scc4 further supports the notion that the Scc2-Scc4 complex is required for developmental processes in metazoans. PMID- 16682348 TI - A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4. AB - Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes. PMID- 16682349 TI - The identification of genes involved in the stomatal response to reduced atmospheric relative humidity. AB - Stomatal pores of higher plants close in response to decreases in atmospheric relative humidity (RH). This is believed to be a mechanism that prevents the plant from losing excess water when exposed to a dry atmosphere and as such is likely to have been of evolutionary significance during the colonization of terrestrial environments by the embryophytes. We have conducted a genetic screen, based on infrared thermal imaging, to identify Arabidopsis genes involved in the stomatal response to reduced RH. Here we report the characterization of two genes, identified during this screen, which are involved in the guard cell reduced RH signaling pathway. Both genes encode proteins known to be involved in guard cell ABA signaling. OST1 encodes a protein kinase involved in ABA-mediated stomatal closure while ABA2 encodes an enzyme involved in ABA biosynthesis. These results suggest, in contrast to previously published work, that ABA plays a role in the signal transduction pathway connecting decreases in RH to reductions in stomatal aperture. The identification of OST1 as a component required in stomatal RH and ABA signal transduction supports the proposition that guard cell signaling is organized as a network in which some intracellular signaling proteins are shared among different stimuli. PMID- 16682350 TI - Two kinesins are involved in the spatial control of cytokinesis in Arabidopsis thaliana. AB - In plant cells, the plane of division is anticipated at the onset of mitosis by the presence of a preprophase band (PPB) of microtubules and F-actin at a cortical site that circumscribes the nucleus. During cytokinesis, the microtubule and F-actin-based phragmoplast facilitates construction of a new cell wall and is guided to the forecast division site. Proper execution of this process is essential for establishing the cellular framework of plant tissues. The microtubule binding protein TANGLED1 (TAN1) of maize is a key player in the determination of division planes . Lack of TAN1 leads to misguided phragmoplasts and mispositioned cell walls in maize. In a yeast two-hybrid screen for TAN1 interacting proteins, a pair of related kinesins was identified that shares significant sequence homology with two kinesin-12 genes in Arabidopsis thaliana (A. thaliana): PHRAGMOPLAST ORIENTING KINESIN 1 and 2 (POK1, POK2). POK1 and POK2 are expressed in tissues enriched for dividing cells. The phenotype of pok1;pok2 double mutants strongly resembles that of maize tan1 mutants, characterized by misoriented mitotic cytoskeletal arrays and misplaced cell walls. We propose that POK1 and POK2 participate in the spatial control of cytokinesis, perhaps via an interaction with the A. thaliana TAN1 homolog, ATN. PMID- 16682351 TI - Sister-chromatid telomere cohesion is nonredundant and resists both spindle forces and telomere motility. AB - It is well documented that inactivation of essential cohesion proteins results in precocious sister-chromatid separation. On average, however, only approximately 55% of cohesin-deficient budding yeast cells arrested prior to anaphase contain separated sister chromatids , suggesting that cohesin-independent factors also contribute to sister-chromatid pairing. Recently, redundant pairing mechanisms were found to occur at both rDNA and centromeres . Here, we tested whether redundant mechanisms also function to pair telomeres or whether cohesins provide sole pairing activity. Results from both mcd1 and ctf7 mutant cells show that nearly 100% of telomeres separate prior to anaphase, twice the cohesion defect reported for centromeres. Such complete loci separation reveals that cohesins are singularly responsible for maintaining telomere cohesion, in contrast to other loci. We also found that sister telomeres moved 141% farther apart than centromeres. Telomere separation occurred in the absence of spindle microtubules and an actin cytoskeleton and persisted in cells abrogated for Mps3p function-an integral nuclear envelope protein previously shown to function in cohesion . These findings are consistent with numerous studies that telomeres translocate along the nuclear periphery and provide new evidence that telomere dynamics can contribute to sister-chromatid separation, independent of centromere motility. PMID- 16682352 TI - Conscious awareness of flicker in humans involves frontal and parietal cortex. AB - Even when confined to the same spatial location, flickering and steady light evoke very different conscious experiences because of their distinct temporal patterns. The neural basis of such differences in subjective experience remains uncertain . Here, we used functional MRI in humans to examine the neural structures involved in awareness of flicker. Participants viewed a single point source of light that flickered at the critical flicker fusion (CFF) threshold, where the same stimulus is sometimes perceived as flickering and sometimes as steady (fused) . We were thus able to compare brain activity for conscious percepts that differed qualitatively (flickering or fused) but were evoked by identical physical stimuli. Greater brain activation was observed on flicker (versus fused) trials in regions of frontal and parietal cortex previously associated with visual awareness in tasks that did not require detection of temporal patterns . In contrast, greater activation was observed on fused (versus flicker) trials in occipital extrastriate cortex. Our findings indicate that activity of higher-level cortical areas is important for awareness of temporally distinct visual events in the context of a nonspatial task, and they thus suggest that frontal and parietal regions may play a general role in visual awareness. PMID- 16682353 TI - Phenotypic variation and natural selection at catsup, a pleiotropic quantitative trait gene in Drosophila. AB - Quantitative traits are shaped by networks of pleiotropic genes . To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the level of quantitative trait nucleotides (QTNs). Catecholamines up (Catsup), which encodes a negative regulator of tyrosine hydroxylase , the rate-limiting step in the synthesis of the neurotransmitter dopamine, is a pleiotropic quantitative trait gene in Drosophila melanogaster. We used association mapping to determine whether the same or different QTNs at Catsup are associated with naturally occurring variation in multiple quantitative traits. We sequenced 169 Catsup alleles from a single population and detected 33 polymorphisms with little linkage disequilibrium (LD). Different molecular polymorphisms in Catsup are independently associated with variation in longevity, locomotor behavior, and sensory bristle number. Most of these polymorphisms are potentially functional variants in protein coding regions, have large effects, and are not common. Thus, Catsup is a pleiotropic quantitative trait gene, but individual QTNs do not have pleiotropic effects. Molecular population genetic analyses of Catsup sequences are consistent with balancing selection maintaining multiple functional polymorphisms. PMID- 16682354 TI - DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. AB - trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY. PMID- 16682355 TI - Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway. AB - Plants leaves develop proximodistal, dorsoventral (adaxial-abaxial), and mediolateral patterns following initiation. The Myb domain gene PHANTASTICA (PHAN) is required for adaxial fate in many plants , but the Arabidopsis ortholog ASYMMETRIC LEAVES1 (AS1) has milder effects, suggesting that alternate or redundant pathways exist . We describe enhancers of as1 with more elongate and dissected leaves. As well as RDR6, an RNA-dependent RNA polymerase previously proposed to influence as1 through microRNA , these enhancers disrupt ARGONAUTE7 (AGO7)/ZIPPY, SUPPRESSOR OF GENE SILENCING3 (SGS3), and DICER-LIKE4 (DCL4), which instead regulate trans-acting small interfering RNA (ta-siRNA) . Microarray analysis revealed that the AUXIN RESPONSE FACTOR genes ETTIN (ETT)/ARF3 and ARF4 were upregulated in ago7, whereas FILAMENTOUS FLOWER (FIL) was upregulated only in as1 ago7 double mutants. RDR6 and SGS3 likewise repress these genes, which specify abaxial fate . We show that the trans-acting siRNA gene TAS3, which targets ETT and ARF4, is expressed in the adaxial domain, and ett as1 ago7 triple mutants resemble as1. Thus FIL is downregulated redundantly by AS1 and by TAS3, acting through ETT, revealing a role for ta-siRNA in leaf polarity. RDR6 and DCL4 are required for systemic silencing, perhaps implicating ta-siRNA as a mobile signal. PMID- 16682356 TI - Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. AB - MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) in plants form through distinct pathways, although they function as negative regulators of mRNA targets by similar mechanisms . Three ta-siRNA gene families (TAS1, TAS2, and TAS3) are known in Arabidopsis thaliana. Biogenesis of TAS3 ta-siRNAs, which target mRNAs encoding several AUXIN RESPONSE FACTORs (including ARF3/ETTIN and ARF4 ) involves miR390-guided processing of primary transcripts, conversion of a precursor to dsRNA through RNA-DEPENDENT RNA POLYMERASE6 (RDR6) activity, and sequential DICER LIKE4 (DCL4)-mediated cleavage events. We show that the juvenile-to-adult phase transition is normally suppressed by TAS3 ta-siRNAs, in an ARGONAUTE7-dependent manner, through negative regulation of ARF3 mRNA. Expression of a nontargeted ARF3 mutant (ARF3mut) in a wild-type background reproduced the phase-change phenotypes detected in rdr6-15 and dcl4-2 mutants, which lose all ta-siRNAs. Expression of either ARF3 or ARF3mut in rdr6-15 plants, in which both endogenous and transgenic copies of ARF3 were derepressed, resulted in further acceleration of phase change and severe morphological and patterning defects of leaves and floral organs. In light of the functions of ARF3 and ARF4 in organ asymmetry, these data reveal multiple roles for TAS3 ta-siRNA-mediated regulation of ARF genes in developmental timing and patterning. PMID- 16682365 TI - A fresh approach to an old issue. PMID- 16682366 TI - Conceptual model for nursing and health policy: what role for history? PMID- 16682367 TI - Staffing ratios in New York: a decade of debate. AB - The effectiveness and desirability of mandated nurse staffing ratios have been discussed for more than 10 years. The author summarizes this debate as it unfolded in New York State. Although staffing ratios were always supported within the context of labor contracts negotiated by the New York State Nurses Association, there was a more gradual acceptance of legislation mandating staffing radios statewide. Current legislative proposals have focused on the need to base ratios in current research and include provisions that would allow adjustment of ratios with input from nurses in direct care. PMID- 16682368 TI - Assessing California's nursing home staffing standards. PMID- 16682369 TI - Nurse-to-patient ratios: what do we know? PMID- 16682370 TI - Nursing organizations call for phase-out of agricultural practices that promote antibiotic resistance. AB - The use of antibiotics in agriculture is considered a contributing factor to the problem of antibiotic resistance. A majority of antibiotics and related drugs produced in the United States are not used to treat the infirm, but rather are used as feed additives for agricultural animals to promote growth and compensate for stressful and crowded growing conditions. Significant efforts must be made to decrease inappropriate overuse in animals and agriculture. Several leading health and political organizations have begun to address the issue. The American Nurses Association has established policies that call on Congress, the U.S. Food and Drug Administration, and meat producers to promptly phase out the agricultural practices that promote antibiotic resistance. PMID- 16682371 TI - Women who did not succeed in the work-based welfare program. AB - Welfare reform, enacted more than 5 years ago, created dramatic changes in the lives of single mothers living in poverty. The purpose of this study was to describe the lives of women who were unable to sustain involvement with work based welfare. A multimethodological design and snowball sampling were used to gather qualitative and quantitative data from 31 urban women. Instruments were a demographic form, an interview guide, and the General Well-Being Schedule from the U.S. Health and Nutrition Examination Survey. Interviews were taped, transcribed, and entered into software to facilitate analysis. Thematic coding and narrative analysis were done. The study population had higher levels of severe and moderate distress than the reference standards for the general population. Barriers within the Temporary Assistance for Needy Families (TANF) system and personal barriers were identified by the women. Policy changes that address these obstacles would serve as blueprints for achieving the Healthy People 2010 goals for the nation. PMID- 16682372 TI - Practice patterns of underrepresented minority nurse practitioners in New York state, 2000. AB - Using data collected from a sample of 1,319 nurse practitioners (NPs) in New York state in 2000, differences in practice patterns by race and ethnicity were identified. NPs from underrepresented minority (URM) groups were more likely than non-URM NPs to work in hospitals, community health centers, and schools but less likely to work in physician offices and NP practices. URM NPs were also more likely to report a specialty certification in women's health. Several different measures of primary care provision were examined, with URM NPs substantially more likely to meet all four measures of primary care practice than non-URM NPs. Finally, URM NPs were more likely than non-URM NPs to practice in federally designated health profession shortage areas. PMID- 16682373 TI - Innovation in nursing education: a statewide grant initiative. AB - Meeting the challenges of the nursing shortage appears to be daunting for schools of nursing across the nation as they struggle to meet educational capacity needs with limited resources. A statewide grant program was developed and implemented through legislative initiatives providing opportunities for schools of nursing to respond to the need for innovation in nursing education. This article shares some of the successes and challenges identified through the process of implementing and evaluating this grant program. Insights into the political and regulatory process are provided as a model for other states to consider. Examples of educational strategies that were successful in improving the recruitment and retention of students and faculty are discussed. Nursing and other health care leaders across the nation should look to policy initiatives such as grant programs to infuse innovation into educational programs and to address educational capacity issues compounding the nursing shortage. PMID- 16682374 TI - Nursing recruitment and retention in New York state: a qualitative workforce needs assessment. AB - This study's objective is to determine major barriers to nursing recruitment and retention as a basis for the New York State Area Health Education Center (NYS AHEC) System's strategic plan for its nursing recruitment and retention initiative, using qualitative assessment with maximum variation purposeful sample. Nine focus groups were conducted in eight rural and urban regions. Three analysts evaluated data following a theory-driven immersion-crystallization approach. Fifty-six practicing nurses, nurse managers, nursing educators, and health care administrators participated. Participants identified workforce recruitment challenges, including labor market dynamics, professional image in the media, and workforce education and training. Workforce retention challenges were also identified, including working conditions, workforce compensation, and barriers to continuing education. Findings confirm documented national and state nursing workforce issues, identify rural-urban distinctions not evidenced in previous research, and provide priorities for the NYS AHEC System pertaining to access to formal and continuing education, faculty shortages, work-place best practices, and recruitment. PMID- 16682375 TI - Mandatory reporting of health care-associated infections: Kingdon's multiple streams approach. PMID- 16682376 TI - Adolescent homelessness, nursing, and public health policy. AB - Homelessness among American adolescents is not a new phenomenon. The United States has a long history of youth who are runaways and/or homeless. The majority of adolescents who are homeless believe that they have no acceptable housing placement options. The foster care system often fails to help youth deal with problems. The impact of homelessness on youth's social functioning and health status should be raised at the community level, particularly by community nurses and other health professions who interact with adolescents who are homeless. By using Kingdon's multiple streams model, nurses can advocate for homeless youth by accurately defining and reframing adolescent homelessness for policy makers and including provision of shelter as a policy priority. PMID- 16682377 TI - Patent foramen ovale with a riding vermicular thrombus causing paradoxical and massive pulmonary embolism. PMID- 16682378 TI - Intravascular ultrasound predictors of angiographic restenosis after sirolimus eluting stent implantation. AB - AIMS: In many countries, drug-eluting stent implantation is the dominant interventional strategy. We evaluated the clinical, angiographic, procedural, and intravascular ultrasound (IVUS) predictors of angiographic restenosis after sirolimus-eluting stent (SES) implantation. METHODS AND RESULTS: SES implantation was successfully performed in 550 patients with 670 native coronary lesions. Six month follow-up angiography was performed in 449 patients (81.6%) with 543 lesions (81.1%). Clinical, angiographic, procedural, and IVUS predictors of restenosis were determined. Using multivariable logistic regression analysis, the only independent predictors of angiographic restenosis were post-procedural final minimum stent area by IVUS [odds ratio (OR)=0.586, 95% confidence interval (CI) 0.387-0.888, P=0.012] and IVUS-measured stent length (OR=1.029, 95% CI 1.002 1.056, P=0.035). Final minimum stent area by IVUS and IVUS-measured stent length that best separated restenosis from non-restenosis were 5.5 mm2 and 40 mm, respectively. Lesions with final minimum stent area<5.5 mm2 and stent length>40 mm had the highest rate of angiographic restenosis [17.7% (11/62)], P<0.001 compared with other groups. CONCLUSION: Independent predictors of angiographic restenosis after SES implantation were post-procedural final minimum stent area by IVUS and IVUS-measured stent length. The angiographic restenosis rate was highest in lesions with stent area<5.5 mm2 and stent length>40 mm. PMID- 16682379 TI - Role of N-terminal brain natriuretic peptide (N-TproBNP) in scleroderma associated pulmonary arterial hypertension. AB - AIMS: The aims of this study were to evaluate the diagnostic value and to explore the prognostic value of N-terminal brain natriuretic peptide (N-TproBNP) in patients with systemic sclerosis (SSc) both with and without pulmonary arterial hypertension (PAH). METHODS AND RESULTS: N-TproBNP, six-minute walk distance (SMWD), haemodynamics (at right heart catheterization) or tricuspid gradient (by echocardiography), and survival were assessed in 109 patients with SSc. The study population included 68 individuals with PAH [mean pulmonary artery pressure (PAP) >25 mmHg and pulmonary capillary wedge pressure <15 mmHg] and 41 individuals without PAH. In patients with PAH, the prognostic value of baseline and change in WHO functional class, N-TproBNP levels, and SMWD were compared using Kaplan-Meier survival curves and Cox proportional hazard analysis. The mean duration of follow up was 10 months (range 1-18 months). One year survival in patients with normal PAP was 100% when compared with 83.5% in those with SSc-PAH (P < 0.05). The patients without PAH had a mean N-TproBNP level of 139 pg/mL (SD 151); those with SSc-PAH had a significantly higher mean N-TproBNP level of 1474 pg/mL (SD 2642) (P = 0.0002). Among patients with PAH for every order of magnitude increase in N TproBNP level there was a four-fold increased risk of death (P = 0.002 for baseline level and P = 0.006 for follow-up level). Baseline N-TproBNP levels were correlated positively with mean PAP (r = 0.62; P < 0.0001), pulmonary vascular resistance (PVR) (r = 0.81; P < 0.0001), and inversely with SMWD (r = -0.46; P < 0.0001). Among patients with SSc-PAH, 13 patients (19%) were in WHO functional classes II and had mean N-TproBNP levels of 325 pg/mL (SD 388). Fifty-three patients (78%) were in WHO classes III and IV and had significantly higher mean N TproBNP levels of 1677 pg/mL (SD 2835) (P = 0.02). At an N-TproBNP level of 395 pg/mL, the sensitivity and specificity for predicting the presence of SSc-PAH were 56 and 95% respectively. CONCLUSION: Raised N-TproBNP levels are directly related to the severity of PAH. In screening programs, SSc patients with an N TproBNP in excess of 395 pg/mL have a very high probability of having pulmonary hypertension. Baseline and serial changes in N-TproBNP levels are highly predictive of survival. A 10-fold increase in N-TproBNP level on therapy is associated with a greater than three-fold increase in mortality, and may indicate therapeutic failure. PMID- 16682380 TI - Differential change in left ventricular mass and regional wall thickness after cardiac resynchronization therapy for heart failure. AB - AIMS: LV reverse remodelling has been shown to be a favourable response after cardiac resynchronization therapy (CRT) in many clinical trials. This study investigated whether left ventricular (LV) reverse remodelling after CRT has any structural benefit, which include the improvement of LV mass or regional wall thickness. METHODS AND RESULTS: Fifty patients (66 +/- 11 years) receiving CRT were followed up for at least 3 months. Echocardiography with tissue Doppler imaging was performed serially before and at day 1 and 3 months after CRT. Although LV end-systolic volume (LVESV) was decreased at day 1 after CRT (141 +/- 74 vs. 129 +/- 71 cm(3), P < 0.001), further LV reverse remodelling was observed at 3 months (110 +/- 67 cm(3), P < 0.001 vs. day 1). LV ejection fraction increased at day 1 (26.5 +/- 9.3 vs. 28.5 +/- 9.1%, P < 0.005) and was further improved at 3 months (34.2 +/- 10.5%, P < 0.001 vs. day 1). However, reduction of LV mass (231 +/- 67 vs. 213 +/- 59 g, P < 0.001) and regional wall thickness was only observed at 3 months, but not at day 1. The improvement of LV mass correlated with the change in LVESV (r = 0.66, P < 0.001) and the baseline systolic asynchrony index (Ts-SD) (r = -0.52, P < 0.001). LV mass was only decreased significantly in responders of LV reverse remodelling (245 +/- 66 vs. 207 +/- 61 g, P < 0.001), but increased in non-responders (209 +/- 64 vs. 223 +/- 56 g, P = 0.02). Responders had significant decrease in thickness of all the four walls for -6 to -11% (all P < or =0.02), whereas non-responders had increased thickness in septal and lateral walls for +11% (both P < 0.05). CONCLUSION: The acute reduction in LV volume after CRT is mediated by haemodynamic and geometric benefits without actual changes in LV mass. However, at 3-month follow-up, reduction in LV mass and regional wall thickness was demonstrated, which represents structural reverse remodelling. Such benefit was only observed in volumetric responders but was worsened in non-responders. PMID- 16682381 TI - Evidence-based use of levosimendan in different clinical settings. AB - Levosimendan is a new calcium sensitizer and K-ATP channel opener. Compared with other inodilators, it improves myocardial contractility without increasing oxygen requirements and induces peripheral and coronary vasodilation with a potential anti-stunning, anti-ischaemic effect. The documentation regarding levosimendan is one of the largest ever on the safety and efficacy of a new pharmacological agent in acute heart failure syndromes. Recent experiences in small-scale studies and randomized clinical trials have led to greater interest in the use of this drug for the support of impaired cardiac function also in patients with ischaemic heart disease and cardiogenic or septic shock. It is also demonstrated that this drug could be used as bridge therapy for the peri-operative phase of cardiac surgery in both adult and paediatric populations. This review summarizes the evidence from published scientific literature regarding the use of levosimendan in various clinical settings. PMID- 16682382 TI - Prognosis of coronary flow reserve: a new therapeutic target? PMID- 16682383 TI - C-reactive protein gene haplotypes and risk of coronary heart disease: the Rotterdam Study. AB - AIMS: C-reactive protein is associated with risk of cardiovascular disease. However, whether C-reactive protein is a marker of severity of cardiovascular disease or actually is involved in its pathogenesis remains unknown. We investigated the relation between C-reactive protein haplotypes, representing the comprehensive variation of the C-reactive protein gene, and coronary heart disease. METHODS AND RESULTS: The Rotterdam Study is a prospective population based study among men and women aged 55 years and older. C-reactive protein was associated with risk of coronary heart disease, with a multivariable adjusted hazard ratio of 1.9 (95% CI 1.5-2.4) for the highest vs. the lowest quartile. Four C-reactive protein haplotypes were present with overall frequencies of 32.8, 31.7, 29.5, and 5.9%. C-reactive protein serum levels were significantly different according to C-reactive protein haplotypes. C-reactive protein haplotypes were not associated with coronary heart disease. CONCLUSION: Steady state C-reactive protein serum level is influenced by C-reactive protein gene haplotypes. Although elevated C-reactive protein level has lately been found to be a consistent and relatively strong risk factor for cardiovascular disease, our study does not support that the common variation in the C-reactive protein gene has a large effect on the occurrence of coronary heart disease. PMID- 16682384 TI - A comparison of dual vs. triple antiplatelet therapy in patients with non-ST segment elevation acute coronary syndrome: results of the ELISA-2 trial. AB - AIMS: To compare dual vs. triple antiplatelet pre-treatment in patients with non ST-elevation acute coronary syndrome (NSTE ACS) who were planned for early catheterization. METHODS AND RESULTS: A total of 328 consecutive patients with NSTE ACS were included and were randomized to pre-treatment with dual (n = 166, aspirin, clopidogrel 600 mg) or triple antiplatelet therapy (n = 162, aspirin, clopidogrel 300 mg, and Tirofiban). The primary endpoint was enzymatic infarct size, defined as cumulative LDH release (LDHQ(48)). Initial TIMI flow of the culprit vessel was a pre-specified secondary endpoint. Angiography was performed in 98% of patients at a median of 23 h after admission. Enzymatic infarct size (median, 25-75%) was 166 (60-349) IU/L in the triple group compared with 193 (75 466) IU/L in the dual group (P = 0.2). Initial TIMI 3 flow of the culprit vessel was significantly more often observed after triple antiplatelet therapy (67 vs. 47%, P = 0.002). At 30 days follow-up, myocardial infarction (MI) occurred in 46% of patients in the triple antiplatelet group, compared with 57% in the dual antiplatelet group, P = 0.052. No significant difference in bleeding was present. CONCLUSION: This study showed that in patients with NSTE ACS, triple antiplatelet pre-treatment was associated with a non-significant reduction in enzymatic infarct size, a significantly better initial perfusion of the culprit vessel, and a trend towards a better survival without death or MI. Further, large-scale studies should be performed to find whether the beneficial trend in favour of triple antiplatelet pre-treatment can be reproduced. PMID- 16682386 TI - Progress in intellectual disabilities services in Greece: diagnostic, educational and vocational services. PMID- 16682387 TI - Attitudes of direct care staff towards external professionals, team climate and psychological wellbeing: a pilot study. AB - Relationships with external professionals may be an important factor in the psychological wellbeing of care staff. A questionnaire to measure team climate and staff wellbeing was developed, tested and revised. Seventy-two direct care staff completed the revised questionnaire, which had acceptable levels of internal consistency. Significant correlations were found between care staff attitudes towards professionals and levels of team climate and psychological wellbeing. The pattern of relationships was explored by path analysis. Further work is needed on the reliability and validity of the questionnaire. There is a clear association between attitudes to professionals and team climate and between some aspects of climate and psychological wellbeing. Professionals should adopt collaborative ways of working based on problem-solving approaches with staff teams, with the aim of delivering improved quality of care to residents. PMID- 16682385 TI - Placental-related diseases of pregnancy: Involvement of oxidative stress and implications in human evolution. AB - Miscarriage and pre-eclampsia are the most common disorders of human pregnancy. Both are placental-related and exceptional in other mammalian species. Ultrasound imaging has enabled events during early pregnancy to be visualized in vivo for the first time. As a result, a new understanding of the early materno-fetal relationship has emerged and, with it, new insight into the pathogenesis of these disorders. Unifying the two is the concept of placental oxidative stress, with associated necrosis and apoptosis of the trophoblastic epithelium of the placental villous tree. In normal pregnancies, the earliest stages of development take place in a low oxygen (O2) environment. This physiological hypoxia of the early gestational sac protects the developing fetus against the deleterious and teratogenic effects of O2 free radicals (OFRs). In miscarriage, development of the placento-decidual interface is severely impaired leading to early and widespread onset of maternal blood flow and major oxidative degeneration. This mechanism is common to all miscarriages, with the time at which it occurs in the first trimester depending on the aetiology. In contrast, in pre-eclampsia the trophoblastic invasion is sufficient to allow early pregnancy phases of placentation but too shallow for complete transformation of the arterial utero placental circulation, predisposing to a repetitive ischaemia-reperfusion (I/R) phenomenon. We suggest that pre-eclampsia is a three-stage disorder with the primary pathology being an excessive or atypical maternal immune response. This would impair the placentation process leading to chronic oxidative stress in the placenta and finally to diffuse maternal endothelial cell dysfunction. PMID- 16682388 TI - Growing pains: a personal development program for students with intellectual and developmental disabilities in a specialist school. AB - A personal development program, entitled 'Growing Pains', was developed for 11-to 15-year-old students with intellectual and developmental disabilities in a specialist school. Seven topics were identified as important in supporting students through the physical and emotional changes of adolescence. A single group, non-experimental outcome evaluation was conducted on a 20 week trial program. Teacher-developed checklists of observable skills were completed pre-and post-program for 68 participants yielding individual 'personal development' scores and whole group 'topic' scores, graphed to observe pre-post differences. Parents and teachers completed feedback questionnaires. Graphed data showed positive trends in individual personal development and most topic scores. The topic 'personal hygiene' showed almost no change across the group but parent and teacher feedback identified it as highly important. Causality cannot be established using this design; however, positive trends justified persisting with the program. Future studies might investigate effective means of teaching personal hygiene skills. PMID- 16682389 TI - Successful experience of people with Down syndrome. AB - The aim of this study was to investigate the successful experiences of people with Down syndrome to provide insights into the potential of people with intellectual disabilities. A qualitative approach was adopted, and 10 recipients of the Stevie Award for Outstanding Persons with Down syndrome were interviewed. Interviews with the participants' parents and other significant persons were also conducted. Content analysis and constant comparative method were adopted for data analysis. All 10 participants achieved in life when they demonstrated the motivation and perseverance to try new things and active involvement in extra curricular activities and volunteer service. Role modeling and support from parents and teachers helped participants to develop talents in sports and arts. Effective family support and ongoing collaboration among family, school and community facilities are vital in enabling people with intellectual disabilities to develop their potential for a life of quality. PMID- 16682390 TI - Age at death of people with an intellectual disability in Ireland. AB - This study examined the age at death of people with an intellectual disability in Ireland. Using data from the National Intellectual Disability Database, the average age at death of 1120 people who died between 1996 and 2001 was 45.68 years, with no difference in lifespan between men and women. A more severe level of intellectual disability predicted shorter lifespan. In general, those requiring lower levels of day-to-day support and supervision had a longer lifespan. Those living in the family home had the shortest lifespan while those in hospital residential environments had the longest lifespan. Those not attending any day service had a longer lifespan than those attending a range of day services. There were also differences in age at death across different health board regions. These findings are discussed in light of their implications for service planning. PMID- 16682391 TI - Effects of an education programme on family participation in the rehabilitation of children with developmental disability. AB - The growth and development of children with developmental disability depend very much on the parents. The present study was designed to examine whether family focused education could enhance parental skills, knowledge and competency. The enhancement would in turn result in greater parental participation in the rehabilitation process of these children. The parental needs of Chinese parents were reviewed for the formulation of the Family-Focused Education Programme, which was then implemented, and evaluated using a pre-test/post-test control group design. Forty parents were invited to participate in the study using a convenience sampling technique. The mean scores of these variables were in the direction of increasing after the programme, though repeated measures ANOVA did not indicate any statistically significant changes in parenting knowledge, attitude and stress. It was apparent that the Family-Focused Education Programme enhanced parental competence. Undoubtedly, the family-focused approach is crucial for enhancing parenting competency, and education is fundamental to enabling parents. PMID- 16682392 TI - Old issues and new directions: perceptions of advocacy, its extent and effectiveness from a qualitative study of stakeholder views. AB - This article describes and discusses 27 interviews that explored perceptions of the effectiveness of advocacy services for people with learning disabilities in the UK. The views of stakeholders on the current position of advocacy services, how services are evaluated and recommendations for change are central themes in the analysis of the interviews. The discussion suggests that although evaluation of effectiveness is increasingly important in the light of policy change and funding constraints, it is not universally in place and available tools are considered to be inadequate for the task. The explanation of this may lie in the continuing debates over the meaning and purpose of evaluation and fears that it is one-dimensional. Future investment and commitment to advocacy schemes will need to address these issues. PMID- 16682394 TI - Natural radionuclides in zircon and related radiological impacts in mineral separation plants. AB - The activity concentration of uranium and thorium present in zircon obtained from mineral sand industries are presented. External gamma radiation levels and inhalation of airborne dust are found to be the significant routes of radiation exposure to occupational workers. The annual average dose attributed to zircon processing is estimated to be 2.3 mSv in the plants under study. This paper presents the results of external gamma measurements, estimation of airborne radioactivity in zircon process locations and radon and thoron in the occupational environment of two mineral separation plants in India. Analyses of the solid wastes and liquid effluent generated and resultant environmental impacts are indicated. PMID- 16682395 TI - Modelling the effect of non-uniform radon progeny activities on transformation frequencies in human bronchial airways. AB - The effects of radiological and morphological source heterogeneities in straight and Y-shaped bronchial airways on hit frequencies and microdosimetric quantities in epithelial cells have been investigated previously. The goal of the present study is to relate these physical quantities to transformation frequencies in sensitive target cells and to radon-induced lung cancer risk. Based on an effect specific track length model, computed linear energy transfer (LET) spectra were converted to corresponding transformation frequencies for different activity distributions and source-target configurations. Average transformation probabilities were considerably enhanced for radon progeny accumulations and target cells at the carinal ridge, relative to uniform activity distributions and target cells located along the curved and straight airway portions at the same exposure level. Although uncorrelated transformation probabilities produce a linear dose-effect relationship, correlated transformations first increase depending on the LET, but then decrease significantly when exceeding a defined number of hits or cumulative exposure level. PMID- 16682396 TI - Screening for autism: agreement with diagnosis. AB - Screening measures to identify very young children at risk for autism spectrum disorders include the Modified Checklist for Autism in Toddlers (M-CHAT) and the Social Communication Questionnaire (SCQ). To examine the validity of these written questionnaires, parents completed them prior to their child's diagnostic assessment at a tertiary autism clinic. The M-CHAT was given to 84 parents of 2- to 3-year-olds and the SCQ to 94 parents of 4- to 6-year-olds. On both measures sensitivity was higher than specificity with positive predictive values 0.63 0.68. False negatives, or children with autism who were missed by screening, were somewhat higher functioning than true positives. Results were better for parents who spoke English as a second language, contrary to expectations. At this stage of development these tools would be recommended as part of more comprehensive surveillance programmes to identify children in need of further assessment but not to 'screen out' the possibility of autism. PMID- 16682397 TI - Follow-up of children with autism spectrum disorders from age 2 to age 9. AB - The purpose of the present study was to examine the developmental outcomes of children 7 years after their initial diagnosis. Children diagnosed with autism or PDD-NOS at age 2 received follow-up evaluations at age 9. Diagnostic stability was high, with 88 percent of the sample obtaining autism spectrum diagnoses at age 9. Cognitive scores improved considerably for a large segment of the sample, with over 50 percent obtaining scores in the average range at follow-up. Language outcomes were also positive at follow-up; 88 percent of the sample demonstrated at least some functional language, and 32 percent were able to engage in conversational exchanges. Early characteristics that predicted outcome status were: age of diagnosis, age 2 cognitive and language scores, and total hours of speech-language therapy between ages 2 and 3. These findings highlight the potential long-term benefits of both early identification and early intervention, and provide additional evidence for the importance of promoting public awareness of the early signs of autism. PMID- 16682398 TI - Can the Children's Communication Checklist differentiate autism spectrum subtypes? AB - The study explored whether children with high functioning autism (HFA), Asperger syndrome (AS), and pervasive developmental disorder not otherwise specified (PDD NOS) can be differentiated on the Children's Communication Checklist (CCC). The study also investigated whether empirically derived autistic subgroups can be identified with a cluster analytic method based on the Autism Diagnostic Interview-Revised. Fifty-seven children with HFA, 47 with AS, 31 with PDD-NOS, and a normal control group of 47 children between 6 and 13 years participated. Children with HFA, AS, and PDD-NOS showed pragmatic communication deficits in comparison to the controls. Little difference was found between the three subtypes with respect to their CCC profile. A three-cluster solution explained the data best. The HFA cluster showed most autism characteristics, followed by the combined HFA + AS cluster, and then the PDD-NOS cluster. The findings support the autism spectrum concept based on severity of symptom impairment rather than distinct categories. PMID- 16682399 TI - Labels increase attention to novel objects in children with autism and comprehension-matched children with typical development. AB - This study used an intact group comparison to examine attention following in 34 children aged 2 years diagnosed with autism spectrum disorder (ASD) matched pairwise for vocabulary comprehension with a group of typically developing toddlers. For both groups of children, the presence of verbal labels during a referential task increased attention to a novel object over and above the attention-facilitating effect of child-directed talking without labeling. The typically developing children displayed more attention following than comprehension matched children with ASD across experimental conditions and there was no significant difference between the groups in the facilitative effect of hearing verbal labels. Implications for word-learning theory, intervention strategies and future research are considered. PMID- 16682400 TI - Risperidone and late onset tics. PMID- 16682401 TI - Risperidone induced oedema in a child with learning disability and autism. PMID- 16682403 TI - Hormonal characteristics of follicular fluid from women receiving either GnRH agonist or hCG for ovulation induction. AB - BACKGROUND: A recent prospective randomized study from our group compared GnRH agonist (0.5 mg buserelin) and hCG (10,000 IU) for triggering of ovulation following a flexible antagonist protocol. The agonist group showed a poor reproductive outcome despite luteal phase support with progesterone and estradiol (E(2)). In the present prospective observational study, the health status of follicles from the above study was monitored by analysing the hormonal content of frozen/thawed follicular fluid samples. The aim was to test whether the poor reproductive outcome could be related to a defective pre-ovulatory follicular maturation resulting in oocytes with a compromised developmental competence. METHODS: Hormone concentrations were measured in two individual follicular fluid samples from each of 32 women receiving buserelin and 37 receiving hCG, thus representing a subset of the follicles retrieved. RESULTS: Follicular fluid levels of LH in the agonist group as compared with the hCG group was 11.1 +/- 0.5 versus 3.6 +/- 0.3 IU/l (mean +/- SEM; P < 0.001); FSH, 6.3 +/- 0.6 versus 3.3 +/ 0.2 IU/l (P < 0.001); hCG, not determined versus 139+/-8 IU/l; E(2), 1.9 +/- 0.2 versus 1.8 +/- 0.2 micromol/l (P > 0.10); progesterone, 70 +/- 4 versus 93 +/- 6 micromol/l (P < 0.001); inhibin-A, 36.9 +/- 3.1 versus 37.1 +/- 2.5 ng/ml (P > 0.10) and inhibin-B, 35.6 +/- 2.8 versus 40.1 +/- 3.1 ng/ml (P > 0.10). Thus, pronounced hormonal differences exist in follicular fluid, and the collective concentration of all three gonadotropins and the follicular fluid concentration of progesterone were much higher in the group of women receiving hCG for ovulation induction. CONCLUSION: The study suggests that GnRH agonist results in proper pre-ovulatory follicular maturation, but the ovulatory signal--probably in synergy with the resulting pituitary down-regulation--is too low to support appropriate corpus luteum (CL) function. PMID- 16682404 TI - A novel route for F-box protein-mediated ubiquitination links CHIP to glycoprotein quality control. AB - In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif. PMID- 16682405 TI - Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases. AB - SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L methionine to the lysine epsilon-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product epsilon N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the epsilon-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine epsilon-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation. PMID- 16682406 TI - Widely divergent biochemical properties of the complete set of mouse DC-SIGN related proteins. AB - The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors. PMID- 16682407 TI - Crystal structure of 2-nitropropane dioxygenase complexed with FMN and substrate. Identification of the catalytic base. AB - Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2 nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His(152) as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism. PMID- 16682408 TI - The repeat domain of the melanosomal matrix protein PMEL17/GP100 is required for the formation of organellar fibers. AB - Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles. PMID- 16682409 TI - Genetic deletion of NAD(P)H:quinone oxidoreductase 1 abrogates activation of nuclear factor-kappaB, IkappaBalpha kinase, c-Jun N-terminal kinase, Akt, p38, and p44/42 mitogen-activated protein kinases and potentiates apoptosis. AB - The NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II enzyme that reduces and detoxifies quinones and their derivatives. Although overexpressed in tumor cells, the NQO1 has been linked with the suppression of carcinogenesis, and the effect of NQO1 on tumor necrosis factor (TNF), a cytokine that mediates tumorigenesis through proliferation, invasion, angiogenesis, and metastasis of tumors, is currently unknown. The purpose of our study was to determine the role of NQO1 in TNF cell signaling by using keratinocytes derived from wild-type and NQO1 gene deleted mice. TNF induced nuclear factor (NF)-kappaB activation in wild-type but not in NQO1-deleted cells. The treatment of wild-type cells with dicoumarol, a known inhibitor of NQO1, also abolished TNF-induced NF-kappaB activation. NF kappaB activation induced by lipopolysaccharide, phorbol ester, and cigarette smoke, was also abolished in NQO1-deleted cells. The suppression of NF-kappaB activation was mediated through the inhibition of IkappaBalpha kinase activation, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Further, the deletion of NQO1 abolished TNF-induced c-Jun N-terminal kinase, Akt, p38, and p44/p42 mitogen-activated protein kinase activation. TNF also induced the expression of various NF-kappaB-regulated gene products involved in cell proliferation, antiapoptosis, and invasion in wild-type NQO1 keratinocytes but not in NQO1 deleted cells. The suppression of these antiapoptotic gene products increased TNF induced apoptosis in NQO1-deleted cells. We also found that TNF activated NQO1, and NQO1-specific small interfering RNA abolished the TNF-induced NQO1 activity and NF-kappaB activation. Overall, our results indicate that NQO1 plays a pivotal role in signaling activated by TNF and other inflammatory stimuli and that its suppression is a potential therapeutic strategy to inhibit the proliferation, survival, invasion, and metastasis of tumor cells. PMID- 16682410 TI - The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin. AB - Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate. PMID- 16682411 TI - Analysis of the oligomeric structure of the motor protein prestin. AB - Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed approximately 11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an approximately 11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestin-prey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the approximately 11-nM particles in the outer hair cell's basolateral membrane. PMID- 16682412 TI - The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells. AB - DNA CpG methylation can cooperate with histone H3 lysine 9 (H3-K9) methylation in heterochromatin formation and gene silencing. Trimethylation of H3-K9 by the recently identified euchromatic histone methyltransferase SETDB1/ESET may be responsible for transcriptional repression of certain promoters. Here, we show that SETDB1 associates with endogenous DNA methyltransferase activity. SETDB1 interacts with the de novo DNA methyltransferases DNMT3A and DNMT3B but not with the maintenance methyltransferase DNMT1. The interaction of SETDB1 with DNMT3A was further characterized and confirmed by in vivo and in vitro interaction studies. A direct interaction of the two proteins occurs through the N terminus of SETDB1 and the plant homeodomain of DNMT3A. Co-expression of SETDB1 and DNMT3A was essential for repression of reporter gene expression in a Gal4-based tethering assay and resulted in their recruitment to the artificial promoter. We further demonstrate that the CpG-methylated promoters of the endogenous p53BP2 gene in HeLa cells and the RASSF1A gene in MDA-MB-231 cells are simultaneously occupied by both SETDB1 and DNMT3A proteins, which provides evidence for SETDB1 being at least partly responsible for H3-K9 trimethylation at the promoter of RASSF1A, a gene frequently silenced in human cancers. In summary, our data demonstrate the direct physical interaction and functional connection between the H3-K9 trimethylase SETDB1 and the DNA methyltransferase DNMT3A and thus contribute to a better understanding of the complexity of the self-reinforcing heterochromatin machinery operating at silenced promoters. PMID- 16682413 TI - Orchiectomy attenuates post-ischemic oxidative stress and ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase. AB - Males are much more susceptible to ischemia/reperfusion (I/R)-induced kidney injury when compared with females. Recently we reported that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney susceptibility to I/R injury in mice. Although reactive oxygen species and antioxidant defenses have been implicated in I/R injury, their roles remain to be defined. Here we report that the orchiectomized animal had significantly less lipid peroxidation and lower hydrogen peroxide levels in the kidney 4 and 24 h after 30 min of bilateral renal ischemia when compared with intact or dihydrotestosterone-treated orchiectomized males. The post-ischemic kidney expression and activity of manganese superoxide dismutase (MnSOD) in orchiectomized mice was much greater than in intact or dihydrotestosterone-administered orchiectomized mice. Four hours after 30 min of bilateral ischemia, superoxide formation was significantly lower in orchiectomized mice than in intact mice. In Madin-Darby canine kidney cells, a kidney epithelial cell line, 1 mm H(2)O(2) decreased MnSOD activity, an effect that was potentiated by pretreatment with dihydrotestosterone. Orchiectomy prevented the post-ischemic decrease of catalase activity. Treatment of male mice with manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a SOD mimetic, reduced the post-ischemic increase of plasma creatinine, lipid peroxidation, and tissue hydrogen peroxide. These results suggest that orchiectomy accelerates the post-ischemic activation of MnSOD and reduces reactive oxygen species and lipid peroxidation, resulting in reduced kidney susceptibility to I/R injury. PMID- 16682414 TI - N-glycosylation affects the molecular organization and stability of E-cadherin junctions. AB - Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with gamma-catenin and vinculin compared with the endogenous E cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization. PMID- 16682415 TI - Hypothyroidism reduces tricarboxylate carrier activity and expression in rat liver mitochondria by reducing nuclear transcription rate and splicing efficiency. AB - The tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by approximately 40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3'-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels. PMID- 16682416 TI - Reversible post-translational modification of proteins by nitrated fatty acids in vivo. AB - Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3 phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys 149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking. PMID- 16682417 TI - Pregnane X receptor is a target of farnesoid X receptor. AB - The pregnane X receptor (PXR) is an essential component of the body's detoxification system. PXR is activated by a broad spectrum of xenobiotics and endobiotics, including bile acids and their precursors. Bile acids in high concentrations are toxic; therefore, their synthesis is tightly regulated by the farnesoid X receptor, and their catabolism involves several enzymes regulated by PXR. Here we demonstrate that the expression of PXR is regulated by farnesoid X receptor. Feeding mice with cholic acid or the synthetic farnesoid X receptor (FXR) agonist GW4064 resulted in a robust PXR induction. This effect was abolished in FXR knock-out mice. Long time bile acid treatment resulted in an increase of PXR target genes in wild type mice. A region containing four FXR binding sites (IR1) was identified in the mouse Pxr gene. This region was able to trigger an 8-fold induction after GW4064 treatment in transactivation studies. Deletion or mutation of single IR1 sites caused a weakened response. The importance of each individual IR1 element was assessed by cloning a triple or a single copy and was tested in transactivation studies. Two elements were able to trigger a strong response, one a moderate response, and one no response to GW4064 treatment. Mobility shift assays demonstrated that the two stronger responding elements were able to bind FXR protein. This result was confirmed by chromatin immunoprecipitation. These results strongly suggest that PXR is regulated by FXR. Bile acids activate FXR, which blocks synthesis of bile acids and also leads to the transcriptional activation of PXR, promoting breakdown of bile acids. The combination of the two mechanisms leads to an efficient protection of the liver against bile acid induced toxicity. PMID- 16682418 TI - Transforming growth factor beta (TGF-beta) signaling is regulated by electrical activity in skeletal muscle cells. TGF-beta type I receptor is transcriptionally regulated by myotube excitability. AB - Transforming growth factor (TGF-beta) is involved in several cellular processes such as cell proliferation, differentiation, and apoptosis. At the cell surface, TGF-beta binds to serine-threonine kinase transmembrane receptors (type II and type I) to initiate Smad-dependent intracellular signaling cascades. During the early stages of skeletal muscle differentiation, myotubes start to evoke spontaneous electrical activity in association with contractions that arise following the maturation of the excitation-contraction apparatus. In this work, we report that TGF-beta-dependent signaling is regulated by electrical activity in developing rat primary myotubes, as determined by Smad2 phosphorylation, Smad4 nuclear translocation, and p3TPLux reporter activity. This electrical activity dependent regulation is associated with changes in TGF-beta type I receptor (TbetaRI) levels, correlated with changes in transducing receptors at the cell membrane (measured through radiolabeling binding assays). The inhibition of electrical activity with tetrodotoxin, a voltage-dependent sodium channel blocker, increases TbetaRI levels via a transcription-dependent mechanism. In contrast, the promotion of electrical activity in myotube cultures, induced by the up-regulation of voltage-dependent sodium channels or by direct stimulation with extracellular electrodes, causes TbetaRI levels to decrease. Similar results were obtained in denervated adult muscles, suggesting that electrical activity dependent regulation of TbetaRI also occurs in vivo. Additional results suggest that this activity-dependent regulation is mediated by myogenin. Altogether, these findings support the possibility for a novel regulatory mechanism acting on TGF-beta signaling cascade in skeletal muscle cells. PMID- 16682419 TI - Feedback of the kinesin-1 neck-linker position on the catalytic site. AB - Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state. PMID- 16682420 TI - HIV prevention in Mexican schools: prospective randomised evaluation of intervention. AB - OBJECTIVE: To assess effects on condom use and other sexual behaviour of an HIV prevention programme at school that promotes the use of condoms with and without emergency contraception. DESIGN: Cluster randomised controlled trial. SETTING: 40 public high schools in the state of Morelos, Mexico. PARTICIPANTS: 10 954 first year high school students. INTERVENTION: Schools were randomised to one of three arms: an HIV prevention course that promoted condom use, the same course with emergency contraception as back-up, or the existing sex education course. Self administered anonymous questionnaires were completed at baseline, four months, and 16 months. Students at intervention schools received a 30 hour course (over 15 weeks) on HIV prevention and life skills, designed in accordance with guidelines of the joint United Nations programme on HIV/AIDS. Two extra hours of education on emergency contraception were given to students in the condom promotion with contraception arm. MAIN OUTCOME MEASURES: Primary outcome measure was reported condom use. Other outcomes were reported sexual activity; knowledge and attitudes about HIV and emergency contraception; and attitudes and confidence about condom use. RESULTS: Intervention did not affect reported condom use. Knowledge of HIV improved in both intervention arms and knowledge of emergency contraception improved in the condom promotion with contraception arm. Reported sexual behaviour was similar in the intervention arms and the control group. CONCLUSION: A rigorously designed, implemented, and evaluated HIV education course based in public high schools did not reduce risk behaviour, so such courses need to be redesigned and evaluated. Addition of emergency contraception did not decrease reported condom use or increase risky sexual behaviour but did increase reported use of emergency contraception. PMID- 16682421 TI - Observing local and global properties of metabolic pathways: 'load points' and 'choke points' in the metabolic networks. AB - MOTIVATION: The local and global aspects of metabolic network analyses allow us to identify enzymes or reactions that are crucial for the survival of the organism(s), therefore directing us towards the discovery of potential drug targets. RESULTS: We demonstrate a new method ('load points') to rank the enzymes/metabolites in the metabolic network and propose a model to determine and rank the biochemical lethality in metabolic networks (enzymes/metabolites) through 'choke points'. Based on an extended form of the graph theory model of metabolic networks, metabolite structural information was used to calculate the k shortest paths between metabolites (the presence of more than one competing path between substrate and product). On the basis of these paths and connectivity information, load points were calculated and used to empirically rank the importance of metabolites/enzymes in the metabolic network. The load point analysis emphasizes the role that the biochemical structure of a metabolite, rather than its connectivity (hubs), plays in the conversion pathway. In order to identify potential drug targets (based on the biochemical lethality of metabolic networks), the concept of choke points and load points was used to find enzymes (edges) which uniquely consume or produce a particular metabolite (nodes). A non pathogenic bacterial strain Bacillus subtilis 168 (lactic acid producing bacteria) and a related pathogenic bacterial strain Bacillus anthracis Sterne (avirulent but toxigenic strain, producing the toxin Anthrax) were selected as model organisms. The choke point strategy was implemented on the pathogen bacterial network of B.anthracis Sterne. Potential drug targets are proposed based on the analysis of the top 10 choke points in the bacterial network. A comparative study between the reported top 10 bacterial choke points and the human metabolic network was performed. Further biological inferences were made on results obtained by performing a homology search against the human genome. AVAILABILITY: The load and choke point modules are introduced in the Pathway Hunter Tool (PHT), the basic version of which is available on http://www.pht.uni koeln.de. PMID- 16682422 TI - HoSeqI: automated homologous sequence identification in gene family databases. AB - We present a web service allowing to automatically assign sequences to homologous gene families from a set of databases. After identification of the most similar gene family to the query sequence, this sequence is added to the whole alignment and the phylogenetic tree of the family is rebuilt. Thus, the phylogenetic position of the query sequence in its gene family can be easily identified. AVAILABILITY: http://pbil.univ-lyon1.fr/software/HoSeqI/. PMID- 16682423 TI - Prelude and Fugue, predicting local protein structure, early folding regions and structural weaknesses. AB - Prelude&Fugue are bioinformatics tools aiming at predicting the local 3D structure of a protein from its amino acid sequence in terms of seven backbone torsion angle domains, using database-derived potentials. Prelude(&Fugue) computes all lowest free energy conformations of a protein or protein region, ranked by increasing energy, and possibly satisfying some interresidue distance constraints specified by the user. (Prelude&)Fugue detects sequence regions whose predicted structure is significantly preferred relative to other conformations in the absence of tertiary interactions. These programs can be used for predicting secondary structure, tertiary structure of short peptides, flickering early folding sequences and peptides that adopt a preferred conformation in solution. They can also be used for detecting structural weaknesses, i.e. sequence regions that are not optimal with respect to the tertiary fold. AVAILABILITY: http://babylone.ulb.ac.be/Prelude_and_Fugue. PMID- 16682424 TI - PACK: Profile Analysis using Clustering and Kurtosis to find molecular classifiers in cancer. AB - MOTIVATION: Elucidating the molecular taxonomy of cancers and finding biological and clinical markers from microarray experiments is problematic due to the large number of variables being measured. Feature selection methods that can identify relevant classifiers or that can remove likely false positives prior to supervised analysis are therefore desirable. RESULTS: We present a novel feature selection procedure based on a mixture model and a non-gaussianity measure of a gene's expression profile. The method can be used to find genes that define either small outlier subgroups or major subdivisions, depending on the sign of kurtosis. The method can also be used as a filtering step, prior to supervised analysis, in order to reduce the false discovery rate. We validate our methodology using six independent datasets by rediscovering major classifiers in ER negative and ER positive breast cancer and in prostate cancer. Furthermore, our method finds two novel subtypes within the basal subgroup of ER negative breast tumours, associated with apoptotic and immune response functions respectively, and with statistically different clinical outcome. AVAILABILITY: An R-function pack that implements the methods used here has been added to vabayelMix, available from (www.cran.r-project.org). CONTACT: aet21@cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online. PMID- 16682425 TI - Rational use of antimicrobials in infants in primary care of Bahrain. AB - This nationwide prescription-based study was undertaken to evaluate antimicrobial prescribing for infants, and to identify prescribing errors in infants in 20 primary care health centres of Bahrain. Data was collected on a daily basis by pharmacists in May 2004. Antimicrobials ranked the 5th most common drugs prescribed in infants; beta-lactams, notably amoxycillin and cephalexin, comprised 81.6% of overall prescribed antimicrobials. Antimicrobials were prescribed to approximately one out of four infants (23.8%), prescribed more often to infants aged 9-12 months of age. Approximately one-fifth (22%) of infants received antimicrobials at subtherapeutic daily doses. The prescribing errors were related to dosage (39.2%), strength/doses (26.4%), frequency of dosing (19.2%), and duration of therapy (4.5%). Antimicrobial dosage prescribed in relation to infants' age revealed a positive correlation for amoxycillin (r = 0.264; p < 0.0001), cephalexin (r = 0.223; p = 0.029), erythromycin (r = 0.127; p = 0.284), and a negative correlation for metronidazole (r = -0.183; p = 0.415). Overt use of antimicrobials in infants, and prescription-writing errors (in dosages and frequency of dosing) were common in primary care. Dosage adjustment in relation to developmental stages of infants has received inadequate attention. Effective strategies to improve prescription writing skills are urgently required. PMID- 16682426 TI - Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21. AB - BACKGROUND: T cell-mediated immunity plays a central part in the pathogenesis of tissue damage in inflammatory bowel disease (IBD). The mechanism by which T cells mediate tissue damage during IBD remains unclear, but evidence indicates that T cell-derived cytokines stimulate fibroblasts to synthesise matrix metalloproteinases (MMPs), which then mediate mucosal degradation. We have previously shown that, in IBD, there is high production of interleukin (IL) 21, a T cell-derived cytokine, which enhances Th1 activity. AIM: To investigate whether IL21 controls MMP production by intestinal fibroblasts. METHODS: IL21 receptor (IL21R) was evaluated in intestinal fibroblasts by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Fibroblasts were stimulated with IL21 and MMPs were evaluated by RT-PCR and western blotting. The effect of a neutralising IL21R fusion protein (IL21R/Fc) on the induction of MMPs in fibroblasts stimulated with IBD lamina propria mononuclear cell (LPMC) supernatants was also evaluated. RESULTS: Intestinal fibroblasts constitutively express both IL21R and the common gamma chain receptor, which are necessary for IL21-driven signalling. IL21 enhances fibroblast production of MMP-1, MMP-2, MMP 3 and MMP-9, but not tissue inhibitors of MMP-1 and MMP-2. Moreover, IL21 synergises with tumour necrosis factor alpha to increase synthesis of MMP synthesis. IL21 enhances MMP secretion without affecting gene transcription and protein synthesis. IBD LPMC supernatants stimulate MMP secretion by intestinal fibroblasts, and this effect is partly inhibited by IL21R/Fc. CONCLUSIONS: These results suggest that fibroblasts are a potential target of IL21 in the gut and that IL21 controls MMP secretion by fibroblasts. PMID- 16682427 TI - Endoscopic indications for endoscopic mucosal resection of laterally spreading tumours in the colorectum. AB - BACKGROUND: Laterally spreading tumours (LSTs) in the colorectum are usually removed by endoscopic mucosal resection (EMR) even when large in size. LSTs with deeper submucosal (sm) invasion, however, should not be treated by EMR because of the higher risk of lymph node metastasis. AIMS: To determine which endoscopic criteria, including high magnification pit pattern analysis, are associated with sm invasion in LSTs and clarify indications for EMR. METHODS: Eight endoscopic criteria from 511 colorectal LSTs (granular type (LST-G type); non-granular type (LST-NG type)) were evaluated retrospectively for association with sm invasion, and compared with histopathological findings. RESULTS: LST-NG type had a significantly higher frequency of sm invasion than LST-G type (14% v 7%; p<0.01). Presence of a large nodule in LST-G type was associated with higher sm invasion while pit pattern (invasive pattern), sclerous wall change, and larger tumour size were significantly associated with higher sm invasion in LST-NG type. In 19 LST-G type with sm invasion, sm penetration determined histopathologically occurred under the largest nodules (84%; 16/19) and depressed areas (16%; 3/19). Deepest sm penetration in 32 LST-NG type was either under depressed areas (72%; 23/32) or lymph follicular or multifocal sm invasion (28%; 1/32 and 8/32, respectively). CONCLUSIONS: When considering the most suitable therapeutic strategy for LST-G type, we recommend endoscopic piecemeal resection with the area including the large nodule resected first. In contrast, LST-NG type should be removed en bloc because of the higher potential for malignancy and greater difficulty in diagnosing sm depth and extent of invasion compared with LST-G type. PMID- 16682428 TI - Pit patterns in rectal mucosa assessed by magnifying colonoscope are predictive of relapse in patients with quiescent ulcerative colitis. AB - BACKGROUND: Relapse of ulcerative colitis is difficult to predict by routine colonoscopy. A high-resolution video-magnifying colonoscope with chromoscopy enables the observation of colorectal mucosal pit patterns. AIMS: To investigate the association of pit patterns as assessed by magnifying colonoscopy (MCS) with histological inflammation and mucosal chemokine activity in patients with quiescent ulcerative colitis, and to prospectively analyse the prognostic factors that may predict exacerbations. METHODS: MCS was performed in 113 patients with ulcerative colitis in remission. Pit patterns in the rectal mucosa were classified into four MCS grades on the basis of size, shape and arrangement. Mucosal interleukin (IL) 8 activity was measured in biopsy specimens of rectal mucosa and the specimens were assessed for histological disease activity. The patients were then followed until relapse or for a maximum of 12 months. Multivariate survival analysis was carried out to determine the independent predictors of clinical relapse. RESULTS: A positive correlation was identified between MCS grade, histological grade (p = 0.001) and mucosal IL8 activity (p<0.001). Multivariate proportional hazard model analysis showed that MCS grade was a significant predictor of relapse (relative risk 2.06, p = 0.001). Kaplan Meier estimate of relapse during 12 months of follow-up was found to increase with increasing MCS grade, with values of 0% for grade 1, 21% for grade 2, 43% for grade 3 and 60% for grade 4. CONCLUSION: MCS grading is associated with the degree of histological inflammation and mucosal IL8 activity in patients with quiescent ulcerative colitis, and may predict the probability of subsequent disease relapse in patients with ulcerative colitis in remission. PMID- 16682429 TI - TP53 and progression from Barrett's metaplasia to oesophageal adenocarcinoma in a UK population cohort. AB - BACKGROUND AND AIMS: Oesophageal adenocarcinoma frequently develops on a background of metaplastic Barrett's epithelium. The development of malignancy is accompanied by genetic alterations, which may be promising biomarkers of disease progression. METHODS: A case control study was conducted nested within a large unselected population based cohort of Barrett's patients. Incident oesophageal malignancies and high grade dysplasias were identified. For each case up to five controls were matched on age, sex, and year of diagnosis. Biopsies from the time of diagnosis of Barrett's epithelium were stained immunohistochemically for TP53, cyclin D1, cyclooxygenase 2 (COX-2), and beta-catenin proteins. RESULTS: Twenty nine incident oesophageal malignancies and six cases of high grade dysplasia were identified. The odds of diffuse or intense TP53 staining were substantially elevated in biopsies from patients who developed oesophageal adenocarcinoma compared with controls (odds ratio (OR) 11.7 (95% confidence interval (CI) 1.93, 71.4)). This difference was also present when all cases were considered (OR 8.42 (95% CI 2.37, 30.0). Despite the association with TP53 staining, only 32.4% of cases had an initial biopsy showing diffuse/intense TP53 staining. There were no significant associations between cyclin D1, COX-2, or beta-catenin staining and case control status. The OR for positive staining for both TP53 and COX-2 was markedly increased in cases compared with controls (OR 27.3 (95% CI 2.89, 257.0)) although only 15% of cases had positive staining for both markers. CONCLUSIONS: Immunohistochemical detection of TP53 expression is a biomarker of malignant progression in Barrett's oesophagus but sensitivity is too low to act as a criterion to inform endoscopic surveillance strategies. Additional biomarkers are required which when combined with TP53 will identify, with adequate sensitivity and specificity, Barrett's patients who are at risk of developing cancer. PMID- 16682430 TI - Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins. AB - BACKGROUND: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. AIMS: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. PATIENTS: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. METHODS: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. RESULTS: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; p = 0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5-5.3), p = 0.0009; and HR 2.8 (95% CI 1.4-5.5), p = 0.004, respectively). CONCLUSIONS: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma. PMID- 16682431 TI - Monoaminergic neurotransmission is altered in hepatitis C virus infected patients with chronic fatigue and cognitive impairment. AB - BACKGROUND: The majority of patients with hepatitis C virus (HCV) infection suffer from disabling fatigue, cognitive dysfunction, and quality of life reduction. Meanwhile, there is increasing evidence that HCV infection can affect brain function. Recent studies have shown that fatigue and psychomotor slowing may resolve in patients with hepatitis C after treatment with ondansetron. This observation indicates alteration of serotonergic neurotransmission in HCV infected patients with chronic fatigue. METHODS: Data from 20 HCV infected patients who were referred to our clinic because of disabling fatigue and cognitive decline of unknown cause were analysed retrospectively. Patients had undergone a diagnostic programme, including clinical and psychometric examination, electroencephalogram (EEG), magnetic resonance imaging of the brain, cerebrospinal fluid analysis, and I-123-beta-CIT (2beta-carbomethoxy-3-beta-(4 [(123)I]iodophenyl)tropane) single photon emission computerised tomography (SPECT) studies of serotonin and dopamine transporter binding capacity. RESULTS: All patients had pathological results on the fatigue impact scale. Two thirds of patients showed pathological attention test results. EEG, magnetic resonance imaging, and cerebrospinal fluid analysis were normal. Pathological dopamine transporter binding was present in 12/20 (60%) patients and pathological serotonin transporter binding in 8/19 (50%) patients. Patients with normal SPECT results did not significantly differ from controls with regard to psychometric test results. Interestingly, patients with both decreased serotonin and dopamine transporter binding showed significantly impaired performance in most of the tests applied. Comorbidity that could have impaired cerebral function was excluded in all patients. CONCLUSION: Our findings indicate alteration of serotonergic and dopaminergic neurotransmission in HCV infected patients with chronic fatigue and cognitive impairment. PMID- 16682433 TI - The cost-effectiveness of improving malaria home management: shopkeeper training in rural Kenya. AB - Home management is a very common approach to the treatment of illnesses such as malaria, acute respiratory infections, tuberculosis, diarrhoea and sexually transmitted infections, frequently through over-the-counter purchase of drugs from shops. Inappropriate drugs and doses are often obtained, but interventions to improve treatment quality are rare. An educational programme for general shopkeepers and communities in Kilifi District, rural Kenya was associated with major improvements in the use of over-the-counter anti-malarial drugs for childhood fevers. The two main components were workshop training for drug retailers and community information activities, with impact maintained through on going refresher training, monitoring and community mobilization. This paper presents the cost and cost-effectiveness of the programme in terms of additional appropriately treated cases, evaluating both its measured cost-effectiveness in the first area of implementation (early implementation phase) and the estimated cost-effectiveness of the programme recommended for district-level implementation (recommended district programme). The proportion of shop-treated childhood fevers receiving an adequate amount of a recommended antimalarial rose from 2% to 15% in the early implementation phase, at an economic cost of 4.00 US dollars per additional appropriately treated case (2000 US dollars). If the same impact were achieved through the recommended district programme, the economic cost per additional appropriately treated case would be 0.84 US dollars, varying between 0.37 US dollars and 1.36 US dollars in the sensitivity analysis. As with most educational approaches, the programme carries a relatively high initial financial cost, of 11,477 US dollars (0.02 per capita US dollars) for the development phase and 81,450 US dollars (0.17 per capita US dollars) for the set up year, which would be particularly suitable for donor funding, while the annual costs of 18,129 US dollars (0.04 per capita US dollars) thereafter could be contained within the budget of a typical District. To reach the Abuja target of 60% of those suffering from malaria in sub-Saharan Africa having access to affordable and appropriate treatment within 24 hours, improvements in community-based malaria treatment are urgently required. From these results, policymakers can estimate costs for district-scale shopkeeper training programmes, and will be able to assess their relative cost-effectiveness as comparable evaluations become available from home management interventions in the future. Extrapolation of the results using a simple decision tree model to estimate the cost per DALY averted indicates that the intervention is likely to be considered highly cost-effective in comparison with standard benchmarks for interventions in low-income countries. PMID- 16682432 TI - STAT3 activation via interleukin 6 trans-signalling contributes to ileitis in SAMP1/Yit mice. AB - BACKGROUND AND AIMS: SAMP1/Yit mice spontaneously develops intestinal inflammation. Previously, we demonstrated that the signal transducer and activator of transcription (STAT)-3/suppressor of cytokine signalling (SOCS)-3 pathway is pivotal in human inflammatory bowel disease. In our studies in SAMP1/Yit mice, the aim was to investigate whether STAT3 activation contributes to ileitis and to examine the therapeutic effects of this signal blockade. METHODS: Intestinal expression of phospho-STAT3 in SAMP1/Yit mice and control AKR/J mice was examined by western blotting and immunohistochemistry. SOCS3 and interleukin 6 (IL-6) mRNA were determined by northern blotting and reverse transcription-polymerase chain reaction, respectively. We also examined the effects of intravenously injected hyper-IL-6, an IL-6/soluble IL-6 receptor fusion protein, and of soluble gp130-Fc, a specific inhibitor of soluble IL-6 receptor signalling, on STAT3 phosphorylation and disease severity in SAMP1/Yit mice. RESULTS: Phospho-STAT3 was expressed strongly during the disease course in SAMP1/Yit mice but only transiently in AKR/J mice. Phospho-STAT3 was localised to epithelial and mononuclear cells in the diseased intestine of SAMP1/Yit mice. SOCS3 as well as IL-6 mRNAs were expressed in affected intestine. Administration of hyper-IL-6 caused disease exacerbation and enhancement of STAT3 phosphorylation. In contrast, soluble gp130-Fc administration ameliorated the disease and suppressed STAT3 phosphorylation. CONCLUSION: STAT3 signalling is critical in the development of intestinal inflammation in SAMP1/Yit mice. Blockade of this signalling pathway by soluble gp130-Fc may have therapeutic effects in inflammatory bowel disease. PMID- 16682434 TI - Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody. AB - Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP 1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody. PMID- 16682437 TI - Equity of access to dialysis facilities in Wales. AB - BACKGROUND: Demand for dialysis, particularly, in-centre haemodialysis (HD), is growing, and more units will be needed. Travel time to treatment is consistently a major area of concern for patients. AIM: To analyse access to current dialysis facilities in Wales, and use the data to help plan for new dialysis units. METHODS: We analysed a combination of UK Renal Registry, Welsh population census data, the Welsh Index of Multiple Deprivation 2005 (WIMD), travel time and geographical information systems. RESULTS: Prevalence of HD fell significantly with increasing travel time from units. This was not influenced by the WIMD. Prior to the opening of a new HD unit in Aberystwyth, prevalence in the surrounding area was significantly lower than for Wales as whole, but within 2 years, prevalence had risen to approximate national levels. In Haverfordwest, an area >30 min drive from any current facility, prevalence is consistently and significantly lower than for Wales as a whole, and has not shown the growth seen elsewhere in the country. DISCUSSION: The ability to combine data has enabled modelling of the likely immediate impact of opening a new unit in Haverfordwest, and also provided an estimate of its required capacity. This multidisciplinary approach to demand analysis should help to highlight areas of under-provision, and facilitate the planning of the sites and sizes of new dialysis units in Wales. PMID- 16682435 TI - Inhibitors of differentiation (ID1, ID2, ID3 and ID4) genes are neuronal targets of MeCP2 that are elevated in Rett syndrome. AB - Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2, encoding methyl-CpG-binding protein 2. MeCP2 is a transcriptional repressor elevated in mature neurons and is predicted to be required for neuronal maturation by regulating multiple target genes. Identifying primary gene targets in either Mecp2-deficient mice or human RTT brain has proven to be difficult, perhaps because of the transient requirement for MeCP2 during neuronal maturation. In order to experimentally control the timing of MeCP2 expression and deficiency during neuronal maturation, human SH-SY5Y cells undergoing mature neuronal differentiation were transfected with methylated MeCP2 oligonucleotide decoy to disrupt the binding of MeCP2 to endogenous targets. Genome-wide expression microarray analysis identified all four known members of the inhibitors of differentiation or inhibitors of DNA-binding (ID1, ID2, ID3 and ID4) subfamily of helix-loop-helix genes as novel neuronal targets of MeCP2. Chromatin immunoprecipitation analysis confirmed binding of MeCP2 near or within the promoters of ID1, ID2 and ID3, and quantitative RT-PCR confirmed increased expression of all four Id genes in Mecp2-deficient mouse brain. All four ID proteins were significantly increased in Mecp2-deficient mouse and human RTT brain using immunofluorescence and laser scanning cytometric analyses. Because of their involvement in cell differentiation and neural development, ID genes are ideal primary targets for MeCP2 regulation of neuronal maturation that may explain the molecular pathogenesis of RTT. PMID- 16682438 TI - Colonoscopy first for iron-deficiency anaemia: a Numbers Needed to Investigate approach. AB - BACKGROUND: British Society of Gastroenterology guidelines recommend that gastrointestinal investigations should be considered in males and post-menopausal women presenting with iron-deficiency anaemia (IDA). AIM: To compare the diagnostic yields and clinical effectiveness of upper and lower gastrointestinal (GI) investigation in detecting malignancy among patients presenting with IDA. DESIGN: Retrospective review of case notes, endoscopy records and radiology reports. METHODS: We reviewed the results of 3798 investigations in 2600 patients presenting to our hospital with IDA from October 1995 to December 2003. The findings of the 2318 gastroscopies were compared with those of the 896 colonoscopies and the 584 barium enemas. Patients diagnosed with GI malignancy were identified and their outcomes determined. RESULTS: Gastroscopy identified 44 patients with newly-diagnosed upper GI cancer (18 oesophageal, 26 gastric). Thus for patients being gastroscoped for IDA, the Numbers Needed to Investigate (NNI) to detect each cancer was 53. Five-year survival for these 44 patients was 10%, so the NNI to identify each curable upper GI malignancy was 527. Colonoscopy or barium enema identified 111 (7.5%) patients with newly diagnosed colorectal cancer, giving a NNI of 13. Their 5-year survival was 35%, giving a NNI to identify each curable colorectal cancer patient of 38. DISCUSSION: Potentially curable gastrointestinal malignancy was diagnosed over 13 times more commonly using colonoscopy or barium enema vs. gastroscopy. For patients presenting with IDA, our findings favour investigating the lower GI tract first, or performing both gastroscopy and colonoscopy during the same endoscopy list. PMID- 16682439 TI - Chronic kidney disease: evolving strategies for detection and management of impaired renal function. AB - Nephrologists have long been concerned about late referral of patients with severe kidney disease, and resultant poor outcomes on dialysis. But there is an increasing realisation that mild to moderate chronic kidney disease is far more common than previously appreciated. Furthermore, the main consequence of chronic kidney disease is not progression to dialysis, but increased risk of cardiovascular disease. Chronic kidney disease is at least as common and important a risk factor for cardiovascular disease as diabetes mellitus. The MDRD formula is a well-validated formula to estimate glomerular filtration rate, which is now being widely implemented by clinical chemistry laboratories, and should increase the recognition of chronic kidney disease. The K/DOQI classification of chronic kidney disease has gained international acceptance and provides the structure to guide referral and management. This classification, and associated guidelines, also focus attention on areas where evidence is lacking, and which urgently require research. These current developments will substantially change and improve how chronic kidney disease is identified and managed. PMID- 16682440 TI - Prospective drug utilization evaluation of three broad-spectrum antimicrobials: cefepime, piperacillin-tazobactam and meropenem. AB - BACKGROUND: Cefepime, piperacillin-tazobactam and meropenem are among the broadest-spectrum and most expensive antimicrobials. AIM: To evaluate guidelines for appropriate use of these drugs. METHODS: We developed guidelines for use of these antibiotics, and conducted a two-phase drug utilization evaluation. We included all patients who received one of the study drugs during two 3-month periods, with an educational intervention in the intervening period. Appropriateness was determined for initiation of treatment, and for adaptation or continuation of established treatment. RESULTS: Overall, 205 patients received 271 courses with one of these antibiotics, for a total of 709 defined daily doses (DDD) of cefepime, 543 of piperacillin-tazobactam, and 680 of meropenem (8.3, 6.3 and 7.9 DDD/1000 admission days, respectively). Of these 271 courses, 234 were appropriate (86%). Treatment was continued for > or =5 days in 60%, of which 88% were appropriate (NS). Of the 271 courses, 210 (77%) were empirical (83% appropriate), while 61 (23%) were based on a relevant culture result (97% appropriate) (p < 0.001). Appropriateness differed significantly between departments (p < 0.001), and between the two phases (p < 0.001). The major difference between the two surveys was a decrease in meropenem usage (p < 0.05). DISCUSSION: The vast majority of courses with cefepime, piperacillin-tazobactam and meropenem are empirically selected and continued, underlying the importance of an optimal initial choice. Antibiotic guidelines, in conjunction with formal infectious disease consultation, can contribute to more appropriate use of these drugs. PMID- 16682441 TI - Phage display mediated immuno-PCR. AB - Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10,000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications. PMID- 16682442 TI - Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. AB - Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3' to 5' exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing. PMID- 16682443 TI - Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation. AB - We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA-protein complexes with nucleotide resolution in living cells. PMID- 16682444 TI - Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells. AB - We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3'-phosphoadenosine 5'-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans. PMID- 16682445 TI - Alu RNP and Alu RNA regulate translation initiation in vitro. AB - Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation. PMID- 16682446 TI - Kinetics of double-chain reversals bridging contiguous quartets in tetramolecular quadruplexes. AB - Repetitive 5'GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5'GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallel-stranded quadruplex formed by TG4T. We demonstrate large differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG4T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG4T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data. PMID- 16682447 TI - Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation. AB - Chromatin-modifying factors play key roles in transcription, DNA replication and DNA repair. Post-translational modification of these proteins is largely responsible for regulating their activity. The FACT (facilitates chromatin transcription) complex, a heterodimer of hSpt16 and SSRP1, is a chromatin structure modulator whose involvement in transcription and DNA replication has been reported. Here we show that nucleosome binding activity of FACT complex is regulated by poly(ADP-ribosyl)ation. hSpt16, the large subunit of FACT, is poly(ADP-ribosyl)ated by poly(ADP-ribose) polymerase-1 (PARP-1) resulting from physical interaction between these two proteins. The level of hSpt16 poly(ADP ribosyl)ation is elevated after genotoxic treatment and coincides with the activation of PARP-1. The enhanced hSpt16 poly(ADP-ribosyl)ation level correlates with the dissociation of FACT from chromatin in response to DNA damage. Our findings suggest that poly(ADP-ribosyl)ation of hSpt16 by PARP-1 play regulatory roles for FACT-mediated chromatin remodeling. PMID- 16682448 TI - Telomeres, chromosome instability and cancer. AB - Telomeres are composed of repetitive G-rich sequence and an abundance of associated proteins that together form a dynamic cap that protects chromosome ends and allows them to be distinguished from deleterious DSBs. Telomere associated proteins also function to regulate telomerase, the ribonucleoprtotein responsible for addition of the species-specific terminal repeat sequence. Loss of telomere function is an important mechanism for the chromosome instability commonly found in cancer. Dysfunctional telomeres can result either from alterations in the telomere-associated proteins required for end-capping function, or from alterations that promote the gradual or sudden loss of sufficient repeat sequence necessary to maintain proper telomere structure. Regardless of the mechanism, loss of telomere function can result in sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles, leading to extensive DNA amplification and large terminal deletions. B/F/B cycles terminate primarily when the unstable chromosome acquires a new telomere, most often by translocation of the ends of other chromosomes, thereby providing a mechanism for transfer of instability from one chromosome to another. Thus, the loss of a single telomere can result in on-going instability, affect multiple chromosomes, and generate many of the types of rearrangements commonly associated with human cancer. PMID- 16682449 TI - Detecting uber-operons in prokaryotic genomes. AB - We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: http://csbl.bmb.uga.edu/uber, the first of its kind. PMID- 16682450 TI - A periodic pattern of mRNA secondary structure created by the genetic code. AB - Single-stranded mRNA molecules form secondary structures through complementary self-interactions. Several hypotheses have been proposed on the relationship between the nucleotide sequence, encoded amino acid sequence and mRNA secondary structure. We performed the first transcriptome-wide in silico analysis of the human and mouse mRNA foldings and found a pronounced periodic pattern of nucleotide involvement in mRNA secondary structure. We show that this pattern is created by the structure of the genetic code, and the dinucleotide relative abundances are important for the maintenance of mRNA secondary structure. Although synonymous codon usage contributes to this pattern, it is intrinsic to the structure of the genetic code and manifests itself even in the absence of synonymous codon usage bias at the 4-fold degenerate sites. While all codon sites are important for the maintenance of mRNA secondary structure, degeneracy of the code allows regulation of stability and periodicity of mRNA secondary structure. We demonstrate that the third degenerate codon sites contribute most strongly to mRNA stability. These results convincingly support the hypothesis that redundancies in the genetic code allow transcripts to satisfy requirements for both protein structure and RNA structure. Our data show that selection may be operating on synonymous codons to maintain a more stable and ordered mRNA secondary structure, which is likely to be important for transcript stability and translation. We also demonstrate that functional domains of the mRNA [5' untranslated region (5'-UTR), CDS and 3'-UTR] preferentially fold onto themselves, while the start codon and stop codon regions are characterized by relaxed secondary structures, which may facilitate initiation and termination of translation. PMID- 16682451 TI - Evidence supporting predicted metabolic pathways for Vibrio cholerae: gene expression data and clinical tests. AB - Vibrio cholerae, the etiological agent of the diarrheal illness cholera, can kill an infected adult in 24 h. V.cholerae lives as an autochthonous microbe in estuaries, rivers and coastal waters. A better understanding of its metabolic pathways will assist the development of more effective treatments and will provide a deeper understanding of how this bacterium persists in natural aquatic habitats. Using the completed V.cholerae genome sequence and PathoLogic software, we created VchoCyc, a pathway-genome database that predicted 171 likely metabolic pathways in the bacterium. We report here experimental evidence supporting the computationally predicted pathways. The evidence comes from microarray gene expression studies of V.cholerae in the stools of three cholera patients [D. S. Merrell, S. M. Butler, F. Qadri, N. A. Dolganov, A. Alam, M. B. Cohen, S. B. Calderwood, G. K. Schoolnik and A. Camilli (2002) Nature, 417, 642-645.], from gene expression studies in minimal growth conditions and LB rich medium, and from clinical tests that identify V.cholerae. Expression data provide evidence supporting 92 (53%) of the 171 pathways. The clinical tests provide evidence supporting seven pathways, with six pathways supported by both methods. VchoCyc provides biologists with a useful tool for analyzing this organism's metabolic and genomic information, which could lead to potential insights into new anti bacterial agents. VchoCyc is available in the BioCyc database collection (http://BioCyc.org). PMID- 16682452 TI - Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection. AB - We previously conducted an in vitro selection experiment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nucleotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short14-15 nt catalytic DNA motif commonly known as 8 17. Herein, we report extensive truncation experiments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8-17 and to determine the effect of excess sequence elements on the activity of this motif and the outcome of selection. Although we observed beneficial, detrimental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2 fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activity, called G15-30, was approximately 2.5-fold larger and experienced a approximately 4-fold greater inhibitory effect from excess sequence elements than the average 8-17 motif. Our results suggest that 8-17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a previously unappreciated selective advantage that may contribute to its widespread recurrence. PMID- 16682453 TI - Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion. AB - Hypoxia-induced stress plays a central role in retinal vascular disease and cancer. Increased hypoxia-inducible factor-1 alpha (Hif-1 alpha) expression leads to HIF-1 formation and the production of vascular endothelial growth factor (VEGF). Cytokines, including insulin-like growth factor-1 (IGF-1), also stimulate VEGF secretion. In this study, we examined the relationship between IGF-1 signaling, HIF-1 alpha protein turnover and VEGF secretion in the ARPE-19 retinal pigment epithelial cell line. Northern analysis revealed that IGF-1 stimulated Hif-1 alpha message expression, whereas the hypoxia-mimetic CoCl2 did not. CoCl2 treatment increased Hif-1 alpha protein accumulation to a greater extent than IGF 1 treatment. However, IGF-1 stimulated a more significant increase in VEGF secretion. IGF-1-stimulated VEGF promoter activity was phosphatidylinositol 3 kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent, whereas VEGF secretion was only partially reduced by inhibition of PI3K/Akt/mTOR and HIF-1 activities. Analysis of VEGF promoter truncation mutants indicated that sensitivity to CoCl2 was hypoxia response element (HRE)-dependent with the region upstream of the HRE conferring IGF-1 sensitivity. In conclusion, IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways. PMID- 16682454 TI - A selective peroxisome proliferator-activated receptor gamma modulator with distinct fat cell regulation properties. AB - Adipogenesis is an important process for the improvement of insulin resistance by peroxisome proliferator-activated receptor (PPAR) gamma agonists, such as rosiglitazone and pioglitazone. FK614 [3-(2,4-dichlorobenzyl)-2-methyl-N (pentylsulfonyl)-3-Hbenzimidazole-5-carboxamide] is a structurally novel class of PPARgamma agonist that improves insulin sensitivity in animal models of type 2 diabetes. Herein, we characterize FK614, a selective PPARgamma modulator (SPPARM) with differential properties affecting the regulation of fat cell function. FK614 behaves as a partial agonist in inducing the interaction of PPARgamma with both transcriptional coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, but as a full agonist with both PPAR binding protein and PPAR-interacting protein, which are required for PPARgamma mediated adipogenesis. In the differentiating 3T3-L1 adipocytes, the levels of adipose fatty acid-binding protein (aP2) mRNA expression and triglyceride accumulation induced by FK614 were as efficacious as those of rosiglitazone and pioglitazone. In contrast, the effect of FK614 on aP2 gene expression in mature adipocytes was less than that of the other PPARgamma agonists. Furthermore, the long-term treatment of mature adipocytes with rosiglitazone and pioglitazone reduced the expression of phosphodiesterase 3B, the down-regulation of which has an important role in the development of insulin resistance; however, FK614 had no such effect in mature adipocytes. Thus, FK614 behaves as an SPPARM with differential effects on the activation of PPARgamma at each stage of adipocyte differentiation. The stage-dependent selectivity of FK614 may contribute to its enhanced insulin sensitization in differentiating adipocytes and to reduced insulin resistance at the stage of adipocyte hypertrophy. PMID- 16682455 TI - The pharmacology of two novel long-acting phosphodiesterase 3/4 inhibitors, RPL554 [9,10-dimethoxy-2(2,4,6-trimethylphenylimino)-3-(n-carbamoyl-2-aminoethyl) 3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one] and RPL565 [6,7-dihydro-2 (2,6-diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido[6,1-a]isoquinolin-4-one]. AB - The pharmacology of two novel, trequinsin-like PDE3/4 inhibitors, RPL554 [9,10 dimethoxy-2(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7 tetrahydro-2H-pyrimido-[6,1-a]isoquinolin-4-one] and RPL565 [6,7-dihydro-2-(2,6 diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido[6,1-a]isoquinolin-4-one], has been investigated in a number of in vitro and in vivo assays. Electrical field stimulation-induced contraction of guinea pig superfused isolated tracheal preparations was significantly inhibited by RPL554 (10 microM) and RPL565 (10 microM) (percentage control; 93 +/- 1.2 and 84.4 +/- 2.7, respectively). Contractile responses were suppressed for up to 12 h after termination of superfusion with RPL554 demonstrating a long duration of action. RPL554 and RPL565 inhibited, in a concentration-dependent manner, lipopolysaccharide-induced tumor necrosis factor alpha release from human monocytes [IC50; 0.52 microM (0.38 0.69) and 0.25 microM (0.18-0.35), respectively] and proliferation of human mononuclear cells to phytohemagglutinin [IC50; 0.46 microM (0.24-0.9) and 2.90 microM (1.6-5.4), respectively]. The inhibitory effect of these drugs in vitro was translated into anti-inflammatory activity in vivo. RPL554 (10 mg/kg) and RPL565 (10 mg/kg) administered orally significantly inhibited eosinophil recruitment following antigen challenge in ovalbumin-sensitized guinea pigs. Likewise, inhalation of dry powder containing RPL554 by conscious guinea pigs (25% in micronized lactose) 1.5 h before antigen exposure significantly inhibited the recruitment of eosinophils to the airways. Exposure of conscious guinea pigs to inhalation of dry powder containing RPL554 (2.5%) and RPL565 (25%) in micronized lactose significantly inhibited histamine-induced plasma protein extravasation in the trachea and histamine-induced bronchoconstriction over a 5.5 h period. Thus, RPL554 and RPL565 are novel, long-acting PDE 3/4 inhibitors exhibiting a broad range of both bronchoprotective and anti-inflammatory activities. PMID- 16682456 TI - Evidence of mRNA-mediated intron loss in the human-pathogenic fungus Cryptococcus neoformans. AB - Introns are a defining feature of eukaryotic genomes, though the mechanism of intron gain or loss is not well understood. Reverse transcription of mRNA followed by homologous recombination with the genome has been posited as a mechanism of intron loss, though little direct evidence of recent loss events has been described to support this model. We find supporting evidence for an mRNA mediated mechanism of loss through comparative genome analyses that revealed a recent loss of 10 adjacent introns in a 22-exon gene in the human-pathogenic fungus Cryptococcus neoformans. We surveyed the gene structures of the entire genomes of Cryptococcus gattii, which diverged from the C. neoformans lineage 37 million years ago (Mya), and C. neoformans var. grubii and var. neoformans, which diverged 18 Mya. Our comparison revealed greater than 99.9% intron conservation, with evidence from 20 genes showing evidence of intron loss, but no convincing evidence of intron gain. Our findings confirm that Cryptococcus introns have been quite stable over recent evolutionary time, with occasional mRNA-mediated intron loss events. PMID- 16682457 TI - Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans. AB - The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5' region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae. PMID- 16682458 TI - The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia. AB - Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling. PMID- 16682460 TI - Expression of the Candida albicans morphogenesis regulator gene CZF1 and its regulation by Efg1p and Czf1p. AB - The ability of Candida albicans to transit between different cellular morphologies is believed to be important for virulence. Morphological transitions occur in response to a variety of environmental signals. One such signal is encountered when cells are grown in a semisolid matrix. An important regulator of cellular morphology is the putative transcription factor CZF1. Here we demonstrate that transcription of CZF1 is responsive to growth parameters such as the temperature, carbon source, growth phase of cells, and the physical environment. In wild-type cells, a CZF1 transcript of about 4 kb was expressed when cells were grown embedded in semisolid agar medium, as well as in late exponential phase when cells were grown in liquid medium. Deletion of EFG1, a key regulator of morphogenesis, abolished CZF1 expression. Overexpression of CZF1 revealed that this gene also autoregulates its expression. Efg1p and Czf1p were shown by chromatin immunoprecipitation to act by binding to the promoter of CZF1. The coupling of environmental cues to the expression of a morphogenetic transcription factor may allow C. albicans to coordinate morphogenesis in response to specific conditions encountered in the human host. PMID- 16682459 TI - Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo. AB - Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate, pyruvate, and fatty acids. Using mutants unable to either transport or utilize these compounds, we demonstrated that no individual nitrogen source was essential, while glucose was the most significant primary carbon source for yeast survival in vivo. Hydrolysis of the storage carbohydrate glycogen made a slight contribution for in vivo survival compared with a substantial requirement for trehalose hydrolysis. The ability to sense and respond to low glucose concentrations was also important for survival. In contrast, there was little or no requirement in vivo in this assay for any of the nitrogen-sensing pathways, nitrogen catabolite repression, the ammonium- or amino acid-sensing pathways, or general control. By using auxotrophic mutants, we found that some nitrogenous compounds (polyamines, methionine, and lysine) can be acquired from the host, while others (threonine, aromatic amino acids, isoleucine, and valine) must be synthesized by the pathogen. Our studies provide insights into the yeast-host environment interaction and identify potential antifungal drug targets. PMID- 16682461 TI - Unique posttranslational modifications of chitin-binding lectins of Entamoeba invadens cyst walls. AB - Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an approximately 30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans. PMID- 16682462 TI - Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains. AB - The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking. PMID- 16682463 TI - Expression of a mitochondrial peroxiredoxin prevents programmed cell death in Leishmania donovani. AB - Leishmania promastigote cells transmitted by the insect vector get phagocytosed by macrophages and convert into the amastigote form. During development and transformation, the parasites are exposed to various concentrations of reactive oxygen species, which can induce programmed cell death (PCD). We show that a mitochondrial peroxiredoxin (LdmPrx) protects Leishmania donovani from PCD. Whereas this peroxiredoxin is restricted to the kinetoplast area in promastigotes, it covers the entire mitochondrion in amastigotes, accompanied by dramatically increased expression. A similar change in the expression pattern was observed during the growth of Leishmania from the early to the late logarithmic phase. Recombinant LdmPrx shows typical peroxiredoxin-like enzyme activity. It is able to detoxify organic and inorganic peroxides and prevents DNA from hydroxyl radical-induced damage. Most notably, Leishmania parasites overexpressing this peroxiredoxin are protected from hydrogen peroxide-induced PCD. This protection is also seen in promastigotes grown to the late logarithmic phase, also characterized by high expression of this peroxiredoxin. Apparently, the physiological role of this peroxiredoxin is stabilization of the mitochondrial membrane potential and, as a consequence, inhibition of PCD through removal of peroxides. PMID- 16682464 TI - Stress by heat shock induces massive down regulation of genes and allows differential allelic expression of the Gal/GalNAc lectin in Entamoeba histolytica. AB - Gene expression analysis by microarray assay revealed that when exposed to stress, Entamoeba histolytica exhibits a specific heat shock response, together with a dramatic overall reduction in gene transcription as well as differential allelic expression of key genes participating in virulence, such as the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin. PMID- 16682465 TI - Characterization and differential nuclear localization of Nopp140 and a novel Nopp140-like protein in trypanosomes. AB - Trypanosomatids possess two homologues of Nopp140: a canonical Nopp140 and a Nopp140-like protein (TbNoLP) in which a GAR domain replaces the C-terminal SRP40 domain. Both are phosphorylated and coimmunoprecipitate with RNA polymerase I. Each paralogue has a distinct subnuclear localization, and depletion of TbNoLP produces an enlarged nucleolus in which TbNopp140-containing regions disperse. The restricted occurrence pattern of NoLP proteins reflects an intriguing convergence in evolution, suggestive of a function in nucleoplasmic small nucleolar ribonucleoprotein shuttling. PMID- 16682466 TI - Unraveling the basic principles. PMID- 16682467 TI - Stress and the colon: central-vagal or direct peripheral effect of CRF? PMID- 16682468 TI - CCK- and leptin-induced vagal afferent activation: a model for organ-specific endocrine modulation of visceral sensory information. PMID- 16682469 TI - Inward rectifier currents in pericytes. PMID- 16682470 TI - Activation of NADPH-diaphorase-positive projections to the rostral ventrolateral medulla following cardiac mechanoreceptor stimulation in the conscious rat. AB - Stimulation of cardiac mechanoreceptors during volume expansion elicits reflex compensatory changes in sympathetic nerve activity (SNA). The hypothalamic paraventricular nucleus (PVN) and nucleus of the tractus solitarius (NTS) are autonomic regions known to contribute to this reflex. Both of these nuclei project to the rostral ventrolateral medulla (RVLM), critical in the tonic generation of SNA. Recent reports from our laboratory show that these pathways 1) are activated following cardiac mechanoreceptor stimulation, and 2) produce nitric oxide, known to influence SNA. The aims of the present study were to determine whether 1) the activated neurons within the PVN and NTS were nitrergic and 2) these neurons projected to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retrogradely transported tracer into the pressor region of the RVLM and the placement of a balloon at the right venoatrial junction. In conscious rats, the balloon was inflated to stimulate the cardiac mechanoreceptors or was left uninflated. Balloon inflation elicited a significant increase in Fos-positive neurons in the parvocellular PVN (sevenfold) and NTS (fivefold). In the PVN, 51% of nitrergic neurons and 61% of RVLM-projecting nitrergic neurons were activated. In the NTS, these proportions were 8 and 18%, respectively. The data suggest that nitrergic neurons within the PVN and, to a lesser extent, in the NTS, some of which project to the RVLM, may contribute to the central pathways influencing SNA elicited by cardiac mechanoreceptor stimulation. PMID- 16682471 TI - Tachycardia during fever: is it neural or humoral? PMID- 16682472 TI - A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle. AB - Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD. PMID- 16682473 TI - Rapid, sensitive, and specific lateral-flow immunochromatographic device to measure anti-anthrax protective antigen immunoglobulin g in serum and whole blood. AB - Evidence from animals suggests that anti-anthrax protective antigen (PA) immunoglobulin G (IgG) from vaccination with anthrax vaccine adsorbed (AVA) is protective against Bacillus anthracis infection. Measurement of anti-PA IgG in human sera can be performed using either enzyme-linked immunosorbent assay or fluorescent covalent microsphere immunoassay (ELISA) (R. E. Biagini, D. L. Sammons, J. P. Smith, B. A. MacKenzie, C. A. Striley, V. Semenova, E. Steward Clark, K. Stamey, A. E. Freeman, C. P. Quinn, and J. E. Snawder, Clin. Diagn. Lab. Immunol. 11:50-55, 2004). Both these methods are laboratory based. We describe the development of a rapid lateral-flow immunochromatographic assay (LFIA) test kit for the measurement of anti-PA IgG in serum or whole-blood samples (30-microl samples) using colloidal gold nanoparticles as the detection reagent and an internal control. Using sera from 19 anthrax AVA vaccinees (anti PA IgG range, 2.4 to 340 microg/ml) and 10 controls and PA-supplemented whole blood samples, we demonstrated that the LFIA had a sensitivity of approximately 3 microg/ml anti-PA IgG in serum and approximately 14 microg/ml anti-PA IgG in whole blood. Preabsorption of sera with PA yielded negative anti-PA LFIAs. The diagnostic sensitivity and specificity of the assay were 100% using ELISA measured anti-PA IgG as the standard. This kit has utility in determining anti-PA antibody reactivity in the sera of individuals vaccinated with AVA or individuals with clinical anthrax. PMID- 16682474 TI - Peripheral blood mononuclear cells from individuals infected with human T-cell lymphotropic virus type 1 have a reduced capacity to respond to recall antigens. AB - Evidence indicates that human T-cell lymphotropic virus type 1 (HTLV-1) infection leads to chronic immunosuppression and a greater susceptibility to infectious diseases. Spontaneous in vitro proliferation of peripheral blood mononuclear cells (PBMC) is an important immunological feature of HTLV-1-infected individuals. However, the association between spontaneous proliferation and immunosuppression is not clear. In this study, we evaluated the cellular immune responses of PBMC from 58 asymptomatic HTLV-1-infected individuals with PBMC showing or not showing spontaneous proliferation. Individuals with PBMC that spontaneously proliferated had increased proportions of CD4 T cells expressing CD45RO and dramatically reduced responses to recall antigens. In addition, frequencies of positive responses to recall antigens were also decreased in HTLV infected individuals without spontaneous proliferation of PBMC. There was a polyclonal expansion of multiple T-cell receptor Vbeta families of CD4+ T lymphocytes in patients with spontaneous proliferation. We observed that HTLV-1 induced an immunosuppression characterized by a decrease in the stimulation index to a recall antigen, even in individuals who did not present spontaneous proliferation. On the other hand, only patients with PBMC presenting spontaneous proliferation showed polyclonal activation and increased proportion of CD4 T cells expressing CD45RO. PMID- 16682475 TI - Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses. AB - An immunochromatographic test for the simultaneous detection of Babesia caballi- and B. equi-specific antibodies (BceICT) was developed using a recombinant B. caballi 48-kDa rhoptry protein (rBc48) and a recombinant truncated B. equi merozoite antigen 2 (rEMA-2t). An evaluation of the ability of the BceICT to detect antibodies in sera from uninfected horses and experimentally infected horses showed high sensitivities and specificities of 83.3% (10/12 sera) and 92.9% (52/56 sera), respectively, for the anti-B. caballi antibody and 94.1% (16/17 sera) and 88.2% (45/51 sera), respectively, for the anti-B. equi antibody. Results from the detection of antibodies in field-collected sera indicated that the BceICT results corresponded with those of enzyme-linked immunosorbent assays (ELISA), showing 91.8% correspondence (67/73 sera) for B. caballi and 95.9% correspondence (70/73 sera) for B. equi, and that the BceICT results also corresponded with the ICT for B. caballi and for B. equi, both of which were 98.2% (55/56 sera). The comparable results of the ICT and ELISA and the simplicity and rapidity of the performance of the ICT suggest that the BceICT would be a feasible test for the simultaneous serodiagnosis of both agents of equine babesiosis in the field. PMID- 16682476 TI - Lymphatic tissue fibrosis is associated with reduced numbers of naive CD4+ T cells in human immunodeficiency virus type 1 infection. AB - The organized structure of lymphatic tissues (LTs) constitutes a microenvironment referred to as a niche that plays a critical role in immune system homeostasis by promoting cellular interactions and providing access to cytokines and growth factors on which cells are dependent for survival, proliferation, and differentiation. In chronic human immunodeficiency virus type 1 (HIV-1) infection, immune activation and inflammation result in collagen deposition and disruption of this LT niche. We have previously shown that these fibrotic changes correlate with a reduction in the size of the total population of CD4+ T cells. We now show that this reduction is most substantial within the naive CD4+ T-cell population and is in proportion to the extent of LT collagen deposition in HIV-1 infection. Thus, the previously documented depletion of naive CD4+ T cells in LTs in HIV-1 infection may be a consequence not only of a decreased supply of thymic emigrants or chronic immune activation but also of the decreased ability of those cells to survive in a scarred LT niche. We speculate that LT collagen deposition might therefore limit repopulation of naive CD4+ T cells with highly active antiretroviral therapy, and thus, additional treatments directed to limiting or reversing inflammatory damage to the LT niche could potentially improve immune reconstitution. PMID- 16682477 TI - Use of miniaturized protein arrays for Escherichia coli O serotyping. AB - Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O serotyping methods. PMID- 16682478 TI - Evaluation of various methods of maternal placental blood collection for immunology studies. AB - The collection of maternal placental intervillous blood (IVB), without contamination of fetal blood and with an accurate mononuclear cell profile, is essential for immunological studies of placental malaria and other infectious diseases of the placenta. We have compared five documented methods of IVB collection: perfusion, incision, biopsy, tissue grinding, and puncture (prick) for fetal blood contamination and mononuclear cell profiles using flow cytometry. Twenty-five placentas were obtained from Plasmodium falciparum and human immunodeficiency virus-negative primigravid and secundigravid women delivering at Nyanza Provincial Hospital in Kisumu, western Kenya. Each of the five methods was performed on the same placenta. Fetal red blood cell contamination was significantly lower for the prick and perfusion methods (4.1% and 8.3%, respectively) than for incision (59.5%), biopsy (42.6%), and tissue grinding (19.9%). Significant variation was noted among the five methods in the percentages of monocytes, total T cells, CD4+ and CD8+ T cells, B cells, and NK cells. Further, a pairwise comparison of prick and perfusion, the two methods with low fetal blood contamination, showed that they were not different for fetal red blood cell contamination levels; however, prick yielded significantly higher percentages of CD4 T cells and CD4 memory T cells than perfusion. Collection by prick was determined to be the best method of intervillous blood collection for immunology studies, and perfusion represented the next best method of choice due to high sample volume yield. Overall, in considering the advantages/disadvantages of the two methods with low fetal cell contamination, we conclude that a combination of prick and perfusion is most suitable for immunology studies. PMID- 16682479 TI - Changes in activation states of murine polymorphonuclear leukocytes (PMN) during inflammation: a comparison of bone marrow and peritoneal exudate PMN. AB - To study different activation states in polymorphonuclear leukocytes (PMN) in mice, we compared the function of murine PMN obtained from the bone marrow (BMPMN) with those of PMN obtained by intraperitoneal induction with thioglycolate (TGPMN) or uric acid (UAPMN). When stimulated with chemotactic peptides, e.g., formyl-methionyl-leucyl-phenylalanine (fMLF), WKYMVM, or WKYMVm, the TGPMN and UAPMN showed greatly enhanced generation of reactive oxygen species (ROS) compared with BMPMN, which suggests that exudation to the peritoneum per se induces a primed state in the cells. The WKYMVm peptide was the most potent stimulant of ROS generation, and it desensitized for subsequent stimulation with fMLF or WKYMVM. This desensitization was broken by the addition of cytochalasin B. The TGPMN and UAPMN appeared to be fully primed, since no increase in response was induced by pretreatment with tumor necrosis factor alpha (TNF-alpha). In contrast, the BMPMN response was increased 2.5- to 3-fold. The differences in oxidative responses were supported by degranulation studies. Preincubation with TNF-alpha promoted CR3 expression on BMPMN, and this level of expression was also enhanced by WKYMVm. In contrast, CR3 expression on untreated TGPMN and UAPMN was already similar to that on TNF-alpha-primed BMPMN and could be only slightly enhanced by TNF-alpha treatment. Taken together, these results indicate that BMPMN are in a resting state and have the capacity to become primed, while peritoneal exudate PMN are already fully primed upon isolation. These results have major implications for murine neutrophil research and show the importance of defining which PMN subsets to use when investigating murine models. PMID- 16682480 TI - Eosinophil activity in Schistosoma mansoni infections in vivo and in vitro in relation to plasma cytokine profile pre- and posttreatment with praziquantel. AB - Eosinophil activity in vivo and in vitro was studied in relation to infection intensities and plasma cytokine profiles of 51 Schistosoma mansoni-infected Ugandan fishermen before treatment and 24 h and 3 weeks posttreatment. Blood eosinophil numbers significantly declined 24 h posttreatment, but significant eosinophilia had developed by 3 weeks posttreatment. Cellular eosinophil cationic protein (ECP) content increased significantly during the transient eosinopenia but was significantly reduced 3 weeks later. No similar reduction in cellular eosinophil protein X (EPX) content was seen. Before treatment, S. mansoni infection intensity was positively correlated with 24-h boosts in plasma interleukin-5 (IL-5) and IL-6 levels, which were in turn negatively correlated with the posttreatment fall in eosinophil numbers. Significant correlations were observed between pretreatment infection intensities and plasma IL-10 and eotaxin levels. Treatment induced significant fluctuations in plasma IL-5, IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), and eotaxin levels. Optimal relative release of ECP and EPX in vitro was detected in S. mansoni soluble egg antigen stimulated cultures during transient eosinopenia. Our data suggest that blood eosinophils are activated during S. mansoni infection and that treatment induces a burst in released antigens, causing increased production of IL-5, IL-6, IL-10, and eotaxin; a drop in TNF-alpha levels; and a transient sequestration of eosinophils, which leaves fewer degranulated eosinophils in the circulation 24 h posttreatment, followed by the development of eosinophilia 3 weeks later. During these events, it appears that preferential release of ECP occurs in vivo. Moreover, it is possible that infection intensity-dependent levels of plasma IL 10 may be involved in the prevention of treatment-induced anaphylactic reactions. PMID- 16682481 TI - Production of an anti-severe acute respiratory syndrome (SARS) coronavirus human monoclonal antibody Fab fragment by using a combinatorial immunoglobulin gene library derived from patients who recovered from SARS. AB - A combinatorial human immunoglobulin gene library was constructed from the peripheral lymphocytes of two patients who recovered from severe acute respiratory syndrome (SARS). The library was screened for the production of Fab antibody fragments to a recombinant spike protein of SARS-associated coronavirus (SARS-CoV). One Fab clone, AS3-3, reacted with the spike protein in an enzyme linked immunosorbent assay. The dissociation constant of AS3-3 was 1.98 x 10(-8) M. Immunofluorescent microscopy revealed that it reacted with SARS-CoV-infected cells. The library seems to be a potent tool for the production of human antibodies to SARS-CoV. PMID- 16682482 TI - A simple manual rosetting method for absolute CD4+ lymphocyte counting in resource-limited countries. AB - A CD4 monoclonal antibody which reacts with CD4+ lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries. PMID- 16682483 TI - Regulation of thick ascending limb transport: role of nitric oxide. AB - Nitric oxide (NO) plays a role in many physiological and pathophysiological processes. In the kidney, NO reduces renal vascular resistance, increases glomerular filtration rate, alters renin release, and inhibits transport along the nephron. The thick ascending limb is responsible for absorbing 20-30% of the filtered load of NaCl, much of the bicarbonate that escapes the proximal nephron, and a significant fraction of the divalent cations reclaimed from the forming urine. Additionally, this nephron segment plays a role in K+ homeostasis. This article will review recent advances in our understanding of the role NO plays in regulating the transport processes of the thick ascending limb. NO has been shown to inhibit NaCl absorption primarily by reducing Na+-K+-2Cl- cotransport activity. NO also inhibits bicarbonate absorption by reducing Na+/H+ exchange activity. It has also been reported to enhance luminal K+ channel activity and thus is likely to alter K+ secretion. The source of NO may be vascular structures such as the afferent arteriole or vasa recta, or the thick ascending limb itself. NO is produced by NO synthase 3 in this segment, and several factors that regulate its activity both acutely and chronically have recently been identified. Although the effects of NO on thick ascending limb transport have received a great deal of attention recently, its effects on divalent ion absorption and many other issues remain unexplored. PMID- 16682484 TI - Regulation of epithelial sodium channels by the ubiquitin-proteasome proteolytic pathway. AB - Amiloride-sensitive epithelial Na+ channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends on a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits: alpha, beta, and gamma. The COOH-terminal domain of all three subunits is intracellular and contains a proline-rich motif (PPxY). Mutations or deletion of this PPxY motif in the beta- and gamma-subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell-expressed, developmentally downregulated protein (Nedd4-2), to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin proteasome system, but when Madin-Darby canine kidney cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected renal cells (A6) expressing endogenous ENaC, ENaC is indeed degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by Nedd4-2 activity. In this review, we discuss the role of the ubiquitin conjugation and the alternative degradative pathways (lysosomal or proteasomal) in regulating the rate of ENaC turnover in untransfected renal cells and compare this regulation to that of transfected cell systems. PMID- 16682485 TI - Regulation of TRPV5 and TRPV6 by associated proteins. AB - The epithelial Ca2+ channels TRPV5 and TRPV6 are the most Ca2+-selective members of the TRP channel superfamily. These channels are the prime target for hormonal control of the active Ca2+ flux from the urine space or intestinal lumen to the blood compartment. Insight into their regulation is, therefore, pivotal in our understanding of the (patho)physiology of Ca2+ homeostasis. The recent elucidation of TRPV5/6-associated proteins has provided new insight into the molecular mechanisms underlying the regulation of these channels. In this review, we describe the various means of TRPV5/6 regulation, the role of channel associated proteins herein, and the relationship between both processes. PMID- 16682486 TI - Dietary fiber decreases colonic epithelial cell proliferation and protein synthetic rates in human subjects. AB - Although it has been proposed that high fiber consumption can prevent proliferative diseases of the colon, the clinical data to support this hypothesis have been inconsistent. To provide a more robust measure of the effects of fiber on colonic mucosal growth than previous studies, we evaluated both cell proliferation and colonic mucosal protein synthesis in nine healthy volunteers after they consumed a typical Western diet (<20 g fiber/day) or a Western diet supplemented with wheat bran (24 g/day) in a randomized crossover design. Biopsies taken from the sigmoid colon were used to assess mucosal proliferation by determining proliferating cell nuclear antigen (PCNA) in crypt cells and to assess mucosal protein synthetic rate using stable isotopically labeled leucine infusion. Fiber supplementation produced a 12% decrease in labeling index (%crypt cells stained with PCNA) (P < 0.001) and an 11% decrease in mucosal protein fractional synthetic rate (FSR; P < 0.05). Moreover, mucosal protein FSR correlated directly with labeling index (r2= 0.22, P < 0.05). These data demonstrate that increased wheat bran consumption decreases colonic mucosal proliferation and support the potential importance of dietary fiber in preventing proliferative diseases of the colon. PMID- 16682487 TI - Interleukin-6 release from human abdominal adipose cells is regulated by thyroid stimulating hormone: effect of adipocyte differentiation and anatomic depot. AB - Adipose cells are extrathyroidal targets of thyroid-stimulating hormone (TSH). TSH stimulates interleukin-6 (IL-6) release from adipocytes. We examined TSH responsiveness as a function of stage of differentiation or adipose tissue depot in cultured adipose cells and determined the effect of TSH on extrathyroidal IL-6 production in vivo. Stromal preadipocytes, isolated from human abdominal subcutaneous or omental adipose tissue, and their differentiated counterparts were studied. IL-6 protein concentration in the medium was measured after TSH stimulation. Basal IL-6 release was greater for preadipocytes than differentiated adipocytes, whether derived from subcutaneous or omental fat depots. A depot dependent effect (omental > subcutaneous) on basal IL-6 release was observed for preadipocytes (1.6-fold, P < 0.05); a similar trend for differentiated adipocytes was not significant (6.2-fold, P > 0.05). IL-6 responsiveness to TSH was observed upon differentiation, but only for subcutaneous adipocytes (1.9-fold over basal, P < 0.001). To determine if TSH could stimulate IL-6 release from extrathyroidal tissues in vivo, we measured serum IL-6 levels from five thyroidectomized patients who received recombinant human (rh) TSH and found that levels increased by threefold on days 3 and 4 (P < 0.05) after its administration. Our data demonstrate that stage of differentiation and fat depot origin affect basal and TSH-stimulated IL-6 release from adipose cells in culture. Furthermore, rhTSH elevates serum IL-6 response in thyroidectomized patients, indicating an extrathyroidal site of TSH action. PMID- 16682488 TI - Mixed meal and light exercise each recruit muscle capillaries in healthy humans. AB - Intense exercise and insulin each increases total limb blood flow and recruits muscle capillaries, presumably to facilitate nutrient exchange. Whether mixed meals or light exercise likewise recruits capillaries is unknown. We fed 18 (9 M, 9 F) healthy volunteers a 480-kcal liquid mixed meal. Plasma glucose, insulin, brachial artery flow, and forearm muscle microvascular blood volume were measured before and after the meal. Brachial artery flow and microvascular volume were also examined with light (25% max), moderate (50%), and heavy (80%) forearm contraction every 20 s in 5 (4 M, 1 F) healthy adults. After the meal, glucose and insulin rose modestly (to approximately 7 mM and approximately 270 pM) and peaked by 30 min, whereas brachial artery blood flow (P < 0.05) and the microvascular volume (P < 0.01) each increased significantly by 60 min, and microvascular flow velocity did not change. For exercise, both 50 and 80%, but not 25% maximal handgrip, increased average forearm and brachial artery blood flow (P < 0.01). Flow increased immediately after each contraction and declined toward basal over 15 s. Exercise at 25% max increased microvascular volume threefold (P < 0.01) without affecting microvascular flow velocity or total forearm blood flow. Forearm exercise at 80% maximal grip increased both microvascular volume and microvascular flow velocity (P < 0.05 each). We conclude that light exercise and simple meals each markedly increases muscle microvascular volume, thereby expanding the endothelial surface for nutrient exchange, and that capillary recruitment is an important physiological response to facilitate nutrient/hormone delivery in healthy humans. PMID- 16682489 TI - Effect of adipocyte beta3-adrenergic receptor activation on the type 2 diabetic MKR mice. AB - The antiobesity and antidiabetic effects of the beta3-adrenergic agonists were investigated on nonobese type 2 diabetic MKR mice after injection with a beta3 adrenergic agonist, CL-316243. An intact response to acute CL-316243 treatment was observed in MKR mice. Chronic intraperitoneal CL-316243 treatment of MKR mice reduced blood glucose and serum insulin levels. Hyperinsulinemic euglycemic clamps exhibited improvement of the whole body insulin sensitivity and glucose homeostasis concurrently with enhanced insulin action in liver and adipose tissue. Treating MKR mice with CL-316243 significantly lowered serum and hepatic lipid levels, in part due to increased whole body triglyceride clearance and fatty acid oxidation in adipocytes. A significant reduction in total body fat content and epididymal fat weight was observed along with enhanced metabolic rate in both wild-type and MKR mice after treatment. These data demonstrate that beta3 adrenergic activation improves the diabetic state of nonobese diabetic MKR mice by potentiation of free fatty acid oxidation by adipose tissue, suggesting a potential therapeutic role for beta3-adrenergic agonists in nonobese diabetic subjects. PMID- 16682490 TI - Risk-stratified seroprevalence of severe acute respiratory syndrome coronavirus among children in Hong Kong. AB - BACKGROUND: Severe acute respiratory syndrome was relatively mild in children, and the incidence was significantly lower when compared with adults. Although previous seroepidemiological studies demonstrated that asymptomatic infection was uncommon among health care workers and adult contacts of patients with severe acute respiratory syndrome, it is unclear whether this would extend to the pediatric population. OBJECTIVE: The purpose of this study was to determine the seroprevalence of severe acute respiratory syndrome coronavirus among asymptomatic children living near Amoy Gardens (site of largest community outbreak of severe acute respiratory syndrome in Hong Kong) compared with a low risk region where no community transmission occurred. METHODS: The study was conducted from September to October 2003. Target subjects living in the defined high-risk and low-risk areas were approached through the schools within the respective localities. We recruited 353 and 361 children, respectively, from the high-risk and low-risk areas and collected 3 to 5 mL of blood for severe acute respiratory syndrome coronavirus IgG antibody testing by immunofluorescence antibody assay and confirmation by neutralization test. Parents of all of the subjects who joined the study were contacted by telephone, and a standardized questionnaire was administered by a research nurse to collect information including sociodemographic data, history of severe acute respiratory syndrome coronavirus infection in the subjects and members of the household, history of contact with known cases of severe acute respiratory syndrome, presence of severe acute respiratory syndrome-like symptoms since onset of the severe acute respiratory syndrome epidemic, travel history of the child and his/her relatives within the 15 days before any such symptom onset, use of health service as a result of such symptoms, and whether there were deaths of relatives as a result of severe acute respiratory syndrome. RESULTS: Two (0.57%) of 353 asymptomatic children from the high-risk area were tested positive for severe acute respiratory syndrome coronavirus antibody compared with 0 of 361 in the low-risk region. None of the 14 children who lived in the high-risk area and had known contacts with severe acute respiratory syndrome patients were seropositive. CONCLUSIONS: As in adults, subclinical severe acute respiratory syndrome coronavirus infection was rare in children in the 2003 epidemic. The very low seroprevalence implies little or no population herd immunity to protect against future resurgence of severe acute respiratory syndrome. PMID- 16682491 TI - Improvement in risk factors for metabolic syndrome and insulin resistance in overweight youth who are treated with lifestyle intervention. AB - OBJECTIVE: To evaluate the prevalence of risk factors that are associated with the metabolic syndrome and insulin resistance in overweight youth and to determine the effect of a short-term, family-centered, lifestyle intervention on various associated anthropometric and metabolic measures. METHODS: Overweight youth who were between 8 and 16 years of age participated in a 12-week, family centered, lifestyle intervention program. Anthropometric and metabolic measures were assessed before the program in all participants (n = 109) and after the program in a subset of the participants (n = 43). RESULTS: At baseline, 49.5% of youth had multiple risk factors associated with the metabolic syndrome, based on a modified definition of the National Cholesterol Education Program, and 10% had impaired fasting glucose and/or impaired glucose tolerance. Measures of insulin resistance correlated significantly with the risk factors of the metabolic syndrome. Forty-three youth had pre- and postintervention evaluations that showed statistically significant improvements in body mass index, systolic blood pressure, lipids (total, low-density lipoprotein cholesterol, and triglycerides), postprandial glucose, and leptin levels. CONCLUSION: Overweight youth have multiple risk factors associated with the metabolic syndrome. A 12-week lifestyle program may have a positive effect on reducing risk factors for the metabolic syndrome and insulin resistance in overweight youth. PMID- 16682492 TI - Incidence and clinical presentation of invasive neonatal group B streptococcal infections in Germany. AB - OBJECTIVE: Streptococcus agalactiae (group B Streptococcus) is an important cause of morbidity and mortality in newborn infants. So far, there have been no published data on the incidence, morbidity, and mortality of invasive neonatal group B Streptococcus infections in Germany. METHODS: A prospective active surveillance study involving all of the pediatric hospitals, which reported their cases to the German Pediatric Surveillance Unit, and all of the microbiological laboratories serving pediatric hospitals, which reported their cases to the Laboratory Sentinel Group at Robert Koch Institute Berlin, was conducted between 2001 and 2003. Capture-recapture analysis was used to evaluate the completeness of reported neonatal invasive group B Streptococcus infections. RESULTS: We collected and analyzed data from 347 and 360 infants with invasive group B Streptococcus infection during the first 3 months of life, as reported by pediatricians to the German Pediatric Surveillance Unit and microbiologists to the Robert Koch Institute Berlin, respectively. Using capture-recapture analysis, we calculated an incidence of 0.47 per 1000 live births. Nearly 60% of the infants suffered from early-onset disease, and 16% of these presented with meningitis. In contrast, 61.8% of infants with late-onset disease presented with meningitis. Prematurity was present in 22.4% of early-onset disease and 39.7% of late-onset disease cases, respectively. A high proportion of infants suffered from sequelae because of group B Streptococcus infection at the time of discharge from the hospital. Most common sequelae were hydrocephalus and cerebral seizure. Case fatality rate was 4.3%. CONCLUSIONS: This study, which is the first to provide information on the current national incidence and morbidity of invasive group B Streptococcus infection in Germany, demonstrates remarkable country specific variation in comparison with other European countries, which gather data in a similar fashion. Therefore, the importance of country-specific prevention guidelines has to be stressed. PMID- 16682493 TI - The c-FLIP-NH2 terminus (p22-FLIP) induces NF-kappaB activation. AB - c-FLIP proteins (isoforms: c-FLIP(L), c-FLIP(S), and c-FLIP(R)) play an essential role in the regulation of death receptor-induced apoptosis. Here, we demonstrate that the cytoplasmic NH2-terminal procaspase-8 cleavage product of c-FLIP (p22 FLIP) found in nonapoptotic malignant cells, primary T and B cells, and mature dendritic cells (DCs) strongly induces nuclear factor kappaB (NF-kappaB) activity by interacting with the IkappaB kinase (IKK) complex via the IKKgamma subunit. Thus, in addition to inhibiting apoptosis by binding to the death-inducing signaling complex, our data demonstrate a novel mechanism by which c-FLIP controls NF-kappaB activation and life/death decisions in lymphocytes and DCs. PMID- 16682494 TI - RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation. AB - Hematopoietic stem cells (HSCs) sustain lifelong production of all blood cell types through finely balanced divisions leading to self-renewal and differentiation. Although several genes influencing HSC self-renewal have been identified, to date no gene has been described that, when activated, enhances HSC self-renewal and, when inactivated [corrected] promotes HSC differentiation. We observe that the retinoic acid receptor (RAR)gamma is selectively expressed in primitive hematopoietic precursors and that the bone marrow of RARgamma knockout mice exhibit markedly reduced numbers of HSCs associated with increased numbers of more mature progenitor cells compared with wild-type mice. In contrast, RARalpha is widely expressed in hematopoietic cells, but RARalpha knockout mice do not exhibit any HSC or progenitor abnormalities. Primitive hematopoietic precursors overexpressing RARalpha differentiate predominantly to granulocytes in short-term culture, whereas those overexpressing RARgamma exhibit a much more undifferentiated phenotype. Furthermore, loss of RARgamma abrogated the potentiating effects of all-trans retinoic acid on the maintenance of HSCs in ex vivo culture. Finally, pharmacological activation of RARgamma ex vivo promotes HSC self-renewal, as demonstrated by serial transplant studies. We conclude that the RARs have distinct roles in hematopoiesis and that RARgamma is a critical physiological and pharmacological regulator of the balance between HSC self renewal and differentiation. PMID- 16682495 TI - Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. AB - Atherosclerosis is an inflammatory disease of large arteries. Flow cytometry of aortic cell suspensions showed that B and T lymphocytes and some macrophages and dendritic cells are already present in the adventitia of normal/noninflamed mouse aortas. Adoptively transferred lymphocytes constitutively homed to the aorta and resided within the adventitia up to 7 d after transfer. Lymphocyte trafficking into normal/noninflamed or atherosclerosis-prone aortas was partially L-selectin dependent. Antigen-activated dendritic cells induced increased T lymphocyte proliferation within the aorta 72 h after adoptive transfer. During progression of atherosclerosis in apolipoprotein-E-deficient mice, the total number of macrophages, T cells, and dendritic cells, but not B cells, increased significantly. This alteration in immune cell composition was accompanied by the formation of tertiary lymphoid tissue in the adventitia of atherosclerotic aortas. These results demonstrate that lymphocytes already reside within the normal/noninflamed aorta before the onset atherosclerosis as a consequence of constitutive trafficking. Atherosclerosis induces the recruitment of macrophages and dendritic cells that support antigen presentation. PMID- 16682496 TI - Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations. AB - Rare human immunodeficiency virus 1-infected individuals, termed elite suppressors (ES), maintain plasma virus levels of <50 copies/ml and normal CD4 counts without therapy. The major histocompatibility complex class I allele group human histocompatibility leukocyte antigen (HLA)-B*57 is overrepresented in this population. Mutations in HLA-B*57-restricted epitopes have been observed in ES, but their significance has remained unclear. Here we investigate the extent and impact of cytotoxic T lymphocyte (CTL) escape mutations in HLA-B*57+ ES. We provide the first direct evidence that most ES experience chronic low level viremia. Sequencing revealed a striking discordance between the genotypes of plasma virus and archived provirus in resting CD4+ T cells. Mutations in HLA-B*57 restricted Gag epitopes were present in all viruses from plasma but were rare in proviruses, suggesting powerful selective pressure acting at these epitopes. Surprisingly, strong CD8+ T cell interferon-gamma responses were detected against some mutant epitopes found in plasma virus, suggesting the development of de novo responses to viral variants. In some individuals, relative CD8+ T cell interleukin-2 responses showed better correlation with the selection observed in vivo. Thus, analysis of low level viremia reveals an unexpectedly high level of CTL escape mutations reflecting selective pressure acting at HLA-B*57-restricted epitopes in ES. Continued viral suppression probably reflects CTL responses against unmutated epitopes and residual or de novo responses against epitopes with escape mutations. PMID- 16682497 TI - Plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus. AB - Respiratory syncytial virus (RSV) infection is widely spread and is a major cause of bronchiolitis in infants and high-risk adults, often leading to hospitalization. RSV infection leads to obstruction and inflammation of the airways and induction of innate and acquired immune responses. Because dendritic cells (DCs) are essential in the elicitation of these immune responses, we investigated the presence and the role of dendritic cell subtypes upon RSV infection in the lung. Here, we report that RSV infection increased the number of both conventional and plasmacytoid dendritic cells in the lung and the lung draining lymph nodes. In particular, the increase in plasmacytoid dendritic cell numbers was sustained and lasted until 30 d after infection. Depletion of plasmacytoid dendritic cells resulted in decreased RSV clearance. In addition, depletion of plasmacytoid dendritic cells resulted in an exacerbation of all manifestations of immune-mediated pathology caused by RSV infection. In conclusion, this study demonstrates that both conventional and plasmacytoid dendritic cells are attracted to the site of RSV infection. It is demonstrated that plasmacytoid dendritic cells play a protective role during RSV infection by modulation of local immune responses. PMID- 16682499 TI - The impact of DM on MHC class II-restricted antigen presentation can be altered by manipulation of MHC-peptide kinetic stability. AB - DM edits the peptide repertoire presented by major histocompatibility complex class II molecules by professional antigen-presenting cells (APCs), favoring presentation of some peptides over others. Despite considerable research by many laboratories, there is still significant uncertainty regarding the biochemical attributes of class II-peptide complexes that govern their susceptibility to DM editing. Here, using APCs that either do or do not express DM and a set of unrelated antigens, we found that the intrinsic kinetic stability of class II peptide complexes is tightly correlated with the effects of DM editing within APCs. Furthermore, through the use of kinetic stability variants of three independent peptides, we demonstrate that increasing or decreasing the kinetic stability of class II-peptide complexes causes a corresponding alteration in DM editing. Finally, we show that the spontaneous kinetic stability of class II complexes correlates directly with the efficiency of presentation by DM+ APCs and the immunodominance of that class II-peptide complex during an immune response. Collectively, these results suggest that the pattern of DM editing in APCs can be intentionally changed by modifying class II-peptide interactions, leading to the desired hierarchy of presentation on APCs, thereby promoting recruitment of CD4 T cells specific for the preferred peptides during an immune response. PMID- 16682498 TI - Reprogramming of CTLs into natural killer-like cells in celiac disease. AB - Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon gamma-producing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP 70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases. PMID- 16682500 TI - E proteins and Notch signaling cooperate to promote T cell lineage specification and commitment. AB - The helix-loop-helix protein, E47, is essential for both B- and T-lineage development. Here we demonstrate that in vitro E47 and Notch signaling act in concert to promote T cell development from fetal hematopoietic progenitors and to restrain development into the natural killer and myeloid cell lineages. The expression of an ensemble of genes associated with Notch signaling is activated by E47, and additionally, Notch signaling and E47 act in parallel pathways to induce a T lineage-specific program of gene expression. Enforced expression of the intracellular domain of Notch rescues the developmental arrest at the T cell commitment stage in E2A-deficient fetal thymocytes. Finally, we demonstrate that regulation of Hes1 expression by Notch signaling and E47 is strikingly similar to that observed during Drosophila melanogaster sensory development. Based on these observations, we propose that in developing fetal thymocytes E47 acts to induce the expression of an ensemble of genes involved in Notch signaling, and that subsequently E47 acts in parallel with Notch signaling to promote T-lineage maturation. PMID- 16682501 TI - Telomerase flies the coop: the telomerase RNA component as a viral-encoded oncogene. AB - Telomerase, the enzyme that elongates our telomeres, is crucial for cancer development based on extensive analyses of human cells, human cancers, and mouse models. New data now suggest that a viral telomerase RNA gene encoded by Marek's disease virus (MDV), an oncogenic herpesvirus of chickens, promotes tumor formation. These findings highlight the importance of telomerase in cancer and raise new questions regarding the mechanisms by which the telomerase RNA component supports tumorigenesis. PMID- 16682502 TI - Jekyll and hyde: two faces of cannabinoid signaling in male and female fertility. AB - Mammalian reproduction is a complicated process designed to diversify and strengthen the genetic complement of the offspring and to safeguard regulatory systems at various steps for propagating procreation. An emerging concept in mammalian reproduction is the role of endocannabinoids, a group of endogenously produced lipid mediators, that bind to and activate cannabinoid receptors. Although adverse effects of cannabinoids on fertility have been implicated for years, the mechanisms by which they exert these effects were not clearly understood. With the identification of cannabinoid receptors, endocannabinoid ligands, their key synthetic and hydrolytic pathways, and the generation of mouse models missing cannabinoid receptors, a wealth of information on the significance of cannabinoid/endocannabinoid signaling in spermatogenesis, fertilization, preimplantation embryo development, implantation, and postimplantation embryonic growth has been generated. This review focuses on various aspects of the endocannabinoid system in male and female fertility. It is hoped that a deeper insight would lead to potential clinical applications of the endocannabinoid signaling as a target for correcting infertility and improving reproductive health in humans. PMID- 16682503 TI - Lithium inhibits ceramide- and etoposide-induced protein phosphatase 2A methylation, Bcl-2 dephosphorylation, caspase-2 activation, and apoptosis. AB - Lithium confers cell protection against stress and toxic stimuli. Although lithium inhibits a number of enzymes, the antiapoptotic mechanisms of lithium remain unresolved. Here, we report a novel role of lithium on the blockage of ceramide- and etoposide-induced apoptosis via inhibition of protein phosphatase 2A (PP2A) activity. Overexpression of PP2A resulted in caspase-2 activation, mitochondrial damage, and cell apoptosis that were inhibited by okadaic acid (OA) and lithium. Lithium and OA abrogated ceramide- and etoposide-induced Bcl-2 dephosphorylation at serine 70. Furthermore, ceramide- and etoposide-induced PP2A activation involved methylation of PP2A C subunit, which lithium suppressed. Lithium caused dissociation of PP2A B subunit from the PP2A core enzyme, whereas ceramide caused recruitment of the B subunit. Taken together, lithium exhibited an antiapoptotic effect by inhibiting Bcl-2 dephosphorylation and caspase-2 activation, which involved, at least in part, a mechanism of down-regulating PP2A methylation and PP2A activity. PMID- 16682504 TI - Simultaneous activation of the delta opioid receptor (deltaOR)/sensory neuron specific receptor-4 (SNSR-4) hetero-oligomer by the mixed bivalent agonist bovine adrenal medulla peptide 22 activates SNSR-4 but inhibits deltaOR signaling. AB - Hetero-oligomerization among G protein-coupled receptors has been proposed to contribute to signal integration. Because sensory neuron-specific receptors (SNSRs) and the opioid receptors (OR) share a common ligand, the bovine adrenal medulla peptide (BAM) 22, and have opposite effects on pain modulation, we investigated the possible consequences of deltaOR/SNSR-4 hetero-oligomerization on the signaling properties of both receptor subtypes. Bioluminescence resonance energy transfer revealed that the human deltaOR has similar propensity to homo oligomerize and to form hetero-oligomers with human SNSR-4 when coexpressed in human embryonic kidney 293 cells. The hetero-oligomerization leads to a receptor form displaying unique functional properties. Individual activation of either deltaOR or SNSR-4 in cells coexpressing the two receptors led to the modulation of their respective signaling pathways; inhibition of adenylyl cyclase and activation of phospholipase C, respectively. In contrast, the deltaOR/SNSR-4 bivalent agonist BAM22, which could activate each receptor expressed individually, fully activated the SNSR-4-dependent phospholipase C but did not promote deltaOR-mediated inhibition of adenylyl cyclase in deltaOR/SNSR-4 coexpressing cells. Likewise, concomitant activation of the deltaOR/SNSR-4 hetero oligomer by selective deltaOR and SNSR-4 agonists promoted SNSR-4 but not deltaOR signaling, revealing an agonist-dependent dominant-negative effect of SNSR-4 on deltaOR signaling. Furthermore, the deltaOR selective antagonist naltrexone trans inhibited the SNSR-4-promoted phospholipase C activation mediated by BAM22 but not by the SNSR-4-selective agonists, suggesting a bivalent binding mode of BAM22 to the deltaOR/SNSR-4 hetero-oligomer. The observation that BAM22 inhibited the Leu-enkephalin-promoted cAMP inhibition in rat dorsal root ganglia neurons supports the potential physiological implication of such regulatory mechanism. PMID- 16682505 TI - Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells. AB - The ability of two opioid agonists, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and morphine, to induce mu-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2 dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes. PMID- 16682506 TI - Gene expression in head hair follicles plucked from men and women. AB - Characterizing gene expression in hair follicles can help to elucidate the hair growth cycle by delineating the genes and pathways involved in follicular growth and degeneration. The objectives of this study were to determine whether intact RNA could be extracted from a small number of plucked, unstaged hair follicles in sufficient quantity to conduct gene expression profiling, and to conduct global gene expression profiling. To this end, RNA was extracted from 1 to 3 unstaged follicles plucked from the scalp of 36 volunteers. The average quantifiable yield of RNA/follicle was 112.5 ng. Ribosomal ratios were lower than normally expected, but investigation indicated the RNA was intact. Ten of the samples were amplified and hybridized to Affymetrix genechips. On average, 2,567 of the total probe sets (8,500) were expressed in each sample; 1,422 were expressed in all 10 samples; 97 were significantly changed in one gender compared to the other, and 41 had high levels of interindividual variability. This study demonstrates that RNA of sufficient quantity and quality to use in microarray hybridizations can be obtained from as little as a single plucked human hair follicle. Genes expressed in all individuals are probably related to follicular growth and could form a starting set for developing signatures of toxicant exposure. The differentially expressed genes could be involved in producing gender and interindividual differences in hair growth. PMID- 16682507 TI - Human bone marrow mesenchymal stem cells transfected with human insulin genes can secrete insulin stably. AB - Beta-cell replacement therapy by pancreatic islet transplantation has become a promising treatment for type 1 diabetes. However, the limited supply of human islet tissue prevents this therapy from being widely used to treat patients with type 1 diabetes. In order to obtain insulin-secreting cells, retrovirus vector pLNCX was used to transfer the human insulin gene into human bone marrow mesenchymal stem cells (hMSCs). The hMSCs were isolated from bone marrow of healthy volunteers and were expanded in vitro. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify the insulin DNA fragment from a healthy pancreas sample. The recombinant vector pLNCX-Ins was constructed by cloning the insulin DNA fragment into retrovirus vector pLNCX. After being packaged by BD RetroPack PT67 packaging cells, the virus that contained the insulin gene was used to infect hMSCs. Transcription and expression of the insulin gene in transfected hMSCs were examined by RT-PCR and immunofluorescence. The transfected hMSCs stably secreted insulin into culture media for >3 weeks. Thus, insulin gene-transfected hMSCs can secrete insulin and provide a new way to cope with the shortage of beta cells for therapy of type 1 diabetes. PMID- 16682508 TI - Expression patterns of ER-alpha, PR, HER-2/neu, and EGFR in different cell origin subtypes of high grade and non-high grade ductal carcinoma in situ. AB - We have previously reported that high grade and non-high grade ductal carcinoma in situ (DCIS) of the breast can be subdivided into 3 cell origin subtypes (luminal, basal/stem, and null), and that high grade DCIS is more frequently associated with basal/stem cell subtypes compared to non-high grade DCIS. Here we refine the relationships between these 3 subtypes and the expression patterns of estrogen receptor-alpha (ER-alpha), progesterone receptor (PR), HER-2/neu, and epidermal growth factor receptor (ERFR) in 53 cases of non-high grade and 46 cases of high nuclear grade DCIS. Using a panel of antibodies to ER-alpha, PR, HER-2/neu, and EGFR, along with cytokeratin (CK) markers (CK5/6, CK8, CK14, CK17, and CK18), we found that all 3 cell origin subtypes can express ER-alpha and PR, and their expression is higher in non-high grade DCIS than in high grade DCIS; the expression of HER-2/neu is associated with luminal subtype only in non-high grade DCIS, but can be seen in all 3 subtypes in high grade DCIS; the expression of EGFR is low and is present only in luminal cell subtypes in both high and non high grade DCIS. Basal/ stem cell and null cell subtypes occur in younger patients in non-high grade DCIS compared to high grade DCIS. In conclusion, the expression patterns of ER-alpha, PR, HER-2/neu, and EGFR are markedly different in different cell origin subtypes of both high grade and non-high grade DCIS, suggesting that cell origin subtypes as well as nuclear grade contribute to the biological and molecular heterogeneity of DCIS. PMID- 16682509 TI - Cytokine patterns correlate with liver damage in patients with chronic hepatitis B and C. AB - T-cell immunoregulatory cytokines influence the persistence of hepatitis C virus (HCV) chronic infection and the extent of liver damage. Th1 cytokines positively correlate with hepatic inflammation in chronic hepatitis B virus (HBV) infection. The pro-inflammatory, cytokines IL-6 and IL-18, are involved in viral clearance and in metabolic and viral hepatic diseases, respectively. The aim of this study was to evaluate the profile of Th1/Th2 cytokines in HCV and HBV hepatitis. HBV infected patients showed higher plasma IFN-gamma levels than the HCV+ patients or the control group (p <0.0001). Plasma TNF-alpha and IL-2 were higher in HBV+ in comparison to HCV+ patients (p <0.001) or the control group (p <0.005). Plasma IL 6 and IL-18 were higher in both groups of patients compared to the control group (p <0.04). In HCV+ and HBV+ groups, IL-6 was positively correlated with the duration of the illness (p <0.01 and <0.001, respectively) and viral load (p <0.001 and <0.001, respectively), while IL-18 was positively correlated with serum ALT activity (p <0.01 and <0.001, respectively) and serum AST activity (p <0.01 and <0.001, respectively). We found that in HCV+ and HBV+ patients there are higher levels of Th1 cytokines, particularly in the course of chronic hepatitis B, and that IL-18 and IL-6 levels may have important roles as markers of both inflammation and hepatic injury, particularly in the course of hepatitis C. PMID- 16682510 TI - Increased ALZ-50 immunoreactivity in CSF of pseudotumor cerebri patients. AB - Pseudotumor cerebri (PTC) is characterized by increased intracranial pressure and papilledema without a mass lesion. PTC predominantly affects obese women. Currently, the pathogenesis of PTC is obscure. Since cytoskeletal abnormalities are found in many neurodegenerative diseases, we hypothesized that some cytoskeletal protein might be involved in the pathophysiology of PTC. Western blotting with specific antibody probes was employed to evaluate ALZ-50 immunoreactive protein, cytoskeletal microtubule-associated protein (MAP), and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) samples from 8 PTC patients and 6 controls. Immunoblotting of ALZ-50 in CSF revealed intense staining of 50 kDa protein bands in 7 of 8 PTC patients, while weak staining was found in 4 of 6 controls. Moderate staining of ALZ-50 was seen in 1 of 8 PTC patients and in 2 of 6 controls. CSF blots with anti-ALZ-50 antibody also showed intense staining of a 65 kDa protein band in 3 of the 8 patients but in none of the controls. In anti-MAP CSF blots of the PTC patients and controls, weak staining of the MAP 60 kDa and 50 kDa protein bands was observed. Weak staining of 60 kDa bands was also observed in anti-GFAP CSF blots of all PTC patients and controls. In CSF blots reacted with anti-GFAP antibody, 65 kDa and 32 kDa bands were evident in some PTC patients, but in none of the controls. This study indicates that ALZ-50 immunoreactivity is elevated in CSF of PTC patients. The ALZ-50 immunoreactive protein, either normal tau protein or its phosphorylated variant, may be useful as a biomarker for the diagnosis of PTC. Since the ALZ-50 monoclonal antibody was generated against brain homogenate from Alzheimer's disease (AD) patients, this study suggests a possible link between PTC and AD. PMID- 16682511 TI - Assessment of serum free light chain assays for plasma cell disorder screening in a Veterans Affairs population. AB - This study evaluated serum kappa and lambda free light chain (FLC) concentrations in a Veterans Affairs (VA) population. We hypothesized that our older, mostly male, population should not differ in serum FLC ranges from levels previously established for younger male and female populations and that the assay would improve our screening protocol for plasma cell dyscrasias (PCD). Serum kappa and lambdaFLC were assayed in 312 consecutive serum samples collected during a 16 week period from veterans whose clinical presentation indicated a need for serum protein electrophoresis (SPEP) analysis. We reviewed our laboratory information system (LIS) files to evaluate the patients' diagnoses and treatment status in conjunction with serum FLC levels. All assays and validation studies were conducted using an immunoturbidimetric method with a Roche/Hitachi 911 modular analytical system. The intra-assay variability (CV) was <5%, based on 13 replicate assays of 4 control samples and 1 blank sample. Of the 312 patients, the SPEP results were normal in 235 and abnormal in 77. Of the 235 patients with normal SPEP results, 37 had abnormal FLC values and 20 of these were diagnosed as PCD. Of the 77 patients with abnormal SPEP results, only 9 had diagnoses unrelated to PCD. Using the FLC assay in conjunction with retrospective reviews of medical records, we obtained an 86% detection rate of PCD. This detection rate increased to 100% when both SPEP and FLC results were considered. In conclusion, this study documents an important role for serum FLC assays in diagnosing and monitoring PCD in a VA population. Our results support previously established serum FLC reference ranges that were obtained in younger, male and female populations. Using the serum FLC results in conjunction with SPEP results improves the sensitivity and specificity for managing VA patients whose clinical presentation indicates the need to evaluate PCD. PMID- 16682512 TI - Serum C-reactive protein as a marker for wellness assessment. AB - C-reactive protein (CRP), a nonspecific inflammatory marker, is widely used to monitor treatment of cardiovascular diseases (high serum CRP levels indicate poor outcome of heart disease). A healthy lifestyle decreases serum CRP levels, while obesity, physical inactivity, and smoking increase them. CRP, a stable pentameric protein, has a half-life of 19 hr, is not subject to diurnal variation, and can serve as a marker of wellness and a candidate for future direct access testing for people monitoring their health after adopting a healthier lifestyle. The CRP level may be influenced more by lifestyle than by genetics. Monozygotic twins may not have the same CRP level; within each twin pair, the one with higher adiposity generally has a higher CRP level than the one with low adiposity. Chronic diseases generally have a lower prevalence among Asians than among Westerners. Asians also have lower CRP levels than Westerners. In large population studies, the median CRP level of Asians is only one-tenth that of Westerners. Is there a factor in the lifestyle or diet of Asians that accounts for lower CRP levels? For example, a statin inhibitor of cholesterol synthesis occurs in red yeast rice, an important component of the Asian diet. In summary, CRP is a marker for monitoring cardiovascular therapy and assessing the wellness of the general population. Through improving health and preventing disease, CRP testing may help lower a nation's health costs. PMID- 16682513 TI - Is nasal polyposis a determinant of bronchial hyperresponsiveness and altered quality of life in asthmatic subjects? A case-control study. AB - Asthmatic patients with nasal polyposis (NP) have been reported to have a high prevalence of bronchial hyperresponsiveness (BHR) and a worsening of quality of life (QoL). The aim of this case-control study was to evaluate if NP is a determinant of BHR and is responsible for modifying the QoL in asthmatic subjects. Eighty-nine asthmatic subjects, including 24 patients with NP and 65 patients without NP (controls), underwent spirometry, methacholine challenge test (MCHt), skin prick tests, and were evaluated with the Asthma Quality of Life Questionnaire (AQLQ). Results of the MCHt test are expressed as the provocative concentration of methacholine that causes 20% (PC20) fall of forced expiratory volume at 1 sec (FEV1). The PC20 (mean +/- SD) in NP cases was 1149 +/- 668 microg/ml vs 894 +/- 691 microg/ml in controls (p <0.001). This demonstrates that BHR was not enhanced by the presence of NP in asthmatic subjects. No significant differences were found between the NP cases and controls for overall QoL or for single QoL domains. This study shows that the presence of NP did not impair the QoL of asthmatic patients, as indicated by the items included in the AQLQ questionnaire. PMID- 16682514 TI - Effects of diabetes duration and glycemic control on free radicals in children with type 1 diabetes mellitus. AB - Parameters of lipid peroxidation, protein oxidation, and antioxidant defense systems were measured in blood samples from 47 children with type 1 diabetes mellitus and from 51 healthy controls, matched for age and sex. In the diabetic children, chemiluminescent assay of plasma superoxide anion gave photoemission (counts x 10(3), mean +/- SD) of 674 +/- 412, which were significantly higher than those in the controls (452 +/- 185; p <0.05). Plasma vitamin A levels in the diabetic children (243 +/- 90 microg/dl) were also higher than those in the controls (207 +/- 59 microg/dl, p <0.05). In a subgroup of 24 diabetic children with blood HbA1C levels >or=8.5%, plasma lipoperoxide (LPO) and vitamin E levels were higher (p <0.05) than those in 23 diabetic children with blood HbA1C levels <8.5%. In a subgroup of 26 children with diabetes duration >or=5 yr, plasma LPO levels were higher (p <0.05) than those in 21 children with diabetes duration <5 yr. These findings confirm the presence of oxidant stress in children with type 1 diabetes mellitus and demonstrate that certain indices of oxidant stress are influenced by the duration of diabetes and by the efficacy of glycemic control. These observations suggest that supportive therapy aimed at oxidative stress may help to prevent clinical complications in children with type 1 diabetes mellitus. PMID- 16682515 TI - Murine cytomegalovirus infection markedly reduces serum MCP-1 levels in MCP-1 transgenic mice. AB - Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory chemokine believed to play a major role in atherogenesis. Injured endothelial cells express MCP-1, which attracts monocytes to the blood vessel wall and leads to the formation of atheromas. Cytomegalovirus infection may also play a role in atherogenesis and accelerates inflammation in tissues that overexpress MCP-1. To examine the relationship of cytomegalovirus infection and MCP-1, we infected MCP 1 transgenic mice with murine cytomegalovirus (MCMV) and collected serum 6 days post-infection to evaluate TH1-related cytokine levels by ELISA. Serum levels of IL-10, IL-12 and IFN-gamma were increased in MCP-1 transgenic mice on day 6 following MCMV infection, while levels of IL-1beta and TNF-alpha were undetectable. However, MCP-1 serum levels were reduced >50% in MCP-1 transgenic mice following MCMV infection compared to uninfected transgenic mice. This effect was not as dramatic when an M33 null MCMV was administered to MCP-1 transgenic mice. The mechanism by which MCMV lowers serum MCP-1 levels is unknown, but this effect may enhance the survival of the virus and thus allow CMV to contribute to the chronic inflammation of atherogenesis. PMID- 16682516 TI - Fatty acid synthase and its mRNA concentrations are decreased at different times following Hoechst 33342-induced apoptosis in BC3H-1 myocytes. AB - Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 microg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not. Hoechst 33342 at 20 microg/ml reduced the concentration of FAS protein, which was followed more than 6 hr later by a reduction in FAS mRNA. In conclusion, the inhibition of fatty acid synthesis induced by 20 microg/ml of Hoechst 33342 is attributed to the degradation of FAS protein by activated caspases rather than by inhibition of FAS enzyme activity or FAS mRNA synthesis. PMID- 16682517 TI - Computation of decision levels from differentiated logistic regression probability curves. AB - The determination of clinical decision levels (DL) or "cut-offs" for laboratory parameters involves the analysis of sensitivity and specificity at varying levels of the predictor variable (PV). Commonly, receiver-operator characteristic (ROC) curves are used for this purpose. However, the association between a binary outcome choice and a continuous PV is often tested for statistical significance by logistic regression (LoRe), which also provides estimates of outcome probability (P) at various levels of the PV. Utilizing a graphical procedure based on the 1st [f'(P)] and 2nd [f"(P)] derivatives of the probability curve, DL were computed for simulated data sets (sims) and for actual data from a case control study and compared with those obtained from ROC curves. Sims were constructed for 5 sets of two outcomes (n = 50, each outcome) of normally distributed data with progressive overlap and for 2 sets of fewer data (n = 15 and 9 per outcome, respectively). Additionally, data from a study of the relationship between serum Mg+2 concentration and outcomes in chronic obstructive pulmonary disease (COPD) were analyzed. DL from LoRe was taken to be the point where f"(P) = 0. For sims, the DL from LoRe correlated well with the optimum DL from ROC analysis (n = 7; r2 = 0.93; p = 0.0004). DL for Mg+2 in COPD data from LoRe was 0.83 mmol/L compared to mean of 0.82 mmol/L by ROC. These data suggest that, when the strength of association between outcomes and PV is analyzed by LoRe, DL can be determined from the probability curves. Moreover, LoRe may provide a useful method to determine DL with less ambiguity than those obtained from ROC curves, as well as provide measures of dispersion for the DL. PMID- 16682518 TI - Erythrocyte antioxidant enzymes in patients with cataract. AB - The pathogenesis of cataract has been found to be influenced by a number of factors including oxidative stress. Catalase, glutathione peroxidase (GPX), and superoxide dismutase (SOD) are some of the antioxidant enzymes that protect the body from oxidative damage. The present study investigates the activities of erythrocyte catalase, GPX, and SOD with respect to senile cataract (non-diabetic cataract) and osmotic cataract (diabetic cataract) in a Sri Lankan population. One hundred and two non-diabetic subjects (50 with cataract and 52 non-cataract) and 106 diabetic subjects (56 with cataract and 50 non-cataract) were recruited into the study. Erythrocyte catalase, GPX, and SOD activities were assayed and the data were analysed by t-test (p <0.05 for significance). In the non-diabetic group, significantly low levels of catalase, GPX, and SOD activities were associated with cataract when compared with non-cataract. No significant changes in catalase, GPX, and SOD activities were observed in the diabetic group between cataract and non-cataract. Senile cataract (non-diabetic cataract) was associated with significantly low levels of erythrocyte catalase, GPX, and SOD when compared with osmotic cataract (diabetic cataract). Positive correlations were observed between catalase and SOD (r = 0.75), catalase and GPX (r = 0.63), and SOD and GPX (r = 0.59) in subjects with senile cataracts. Our results indicate that erythrocyte antioxidant enzyme levels are decreased in senile cataract as opposed to osmotic cataract. Assays of these erythrocyte enzyme activities could provide a marker to identify individuals predisposed to senile cataract. PMID- 16682519 TI - Severe hemolytic disease of the newborn in a group B African-American infant delivered by a group O mother. AB - Maternal-fetal ABO incompatibility is a common hematological problem affecting the newborn. In general, hemolysis is minimal and the clinical course is relatively benign, rarely causing the escalating levels of hyperbilirubinemia and significant anemia commonly associated with Rh hemolytic disease of the newborn (HDN). The incidence of HDN ranges from one in 150 births to 1:3000 births, depending on the degree of anemia and level of serum bilirubin. The etiology of ABO hemolytic disease of the newborn (ABO-HDN) is complex because anti-A and anti B antibodies are composed mainly of IgM. Since only IgG antibodies cross the placenta, those pregnant women with high levels of IgG anti-A,B, anti-A, or anti B with an ABO incompatible fetus will be the ones to give birth to an infant with ABO-HDN. We describe a case of a B/Rh positive term newborn born to an O/Rh negative African-American mother demonstrating aggressive hemolysis and a robust response of the bone marrow. This case was successfully managed with phototherapy and simple RBC transfusion without the need for exchange transfusion. PMID- 16682520 TI - A rare case of a multifocal extra-adrenal myelolipoma with markedly hypocellular bone marrow. AB - Extra-adrenal myelolipomas are rare, generally solitary, non-functioning tumors consisting of adipose tissue and hematopoetic elements. These lesions are not known to be associated with underlying hematologic disorders or documented bone marrow abnormalities. We describe the extremely rare occurrence of multifocal, extra-adrenal myelolipomas in a 35-yr-old male. A markedly hypocellular bone marrow is noted, despite normal peripheral blood counts. To our knowledge, this is the first case report of a markedly abnormal marrow examination in a patient with extra-adrenal myelolipoma, and implicates the extra-osseous masses as the driving force behind his normal hematopoesis. PMID- 16682521 TI - Blastic natural killer (NK) cell leukemia (agranular CD4+CD56+ leukemia). AB - Blastic NK cell lymphoma is a rare hematolymphoid neoplasm. This report illustrates an unusual presentation of this entity, namely as a primary leukemia, but without skin lesions. PMID- 16682522 TI - A unique pancreatic tumor with exclusive hepatocytic differentiation. AB - Only 7 cases of pancreatic tumor with hepatocytic differentiation have been reported in the literature, including 6 cases of hepatoid carcinoma and one case of hepatoid adenoma. Diagnosis of hepatoid carcinoma depends on recognition of characteristic histological features, supported by other evidence linked to hepatic lineage including alpha-fetoprotein production, positive immunoreactivity to liver synthesized proteins, and in situ hybridization detection of albumin mRNA. In addition, a synchronous focus of carcinoma arising in pancreatic ducts, islet cells, or acinar cells is essential. We report a unique case of pancreatic tumor with exclusive hepatocytic differentiation. In this tumor, we were unable to find a synchronous focus of carcinoma arising in pancreatic ducts, islet cells, or acinar cells, ruling out the possibility of its being hepatoid carcinoma. Long term follow-up can help to determine whether this tumor is benign or malignant. The patterns of reticulin staining and immunohistochemical staining are suggestive of malignancy, but mitotic activity is low and nuclear pleomorphism is minimal. PMID- 16682523 TI - A note from history: the first biochemical test for detection of cancer. PMID- 16682524 TI - Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling. AB - The Epstein-Barr virus (EBV) noncoding RNAs, EBV-encoded RNA 1 (EBER1) and EBER2, are the most abundant viral transcripts in all types of latently infected human B cells, but their function remains unknown. We carried out heterokaryon assays using cells that endogenously produce EBERs to address their trafficking, as well as that of the La protein, because EBERs are quantitatively bound by La in vivo. Both in this assay and in oocyte microinjection assays, EBERs are confined to the nucleus, suggesting that their contribution to viral latency is purely nuclear. EBER1 does not bind exportin 5; therefore, it is unlikely to act by interfering with microRNA biogenesis. In contrast, La, which is a nuclear phosphoprotein, undergoes nucleocytoplasmic shuttling independent of the nuclear export protein Crm1. To ensure that small RNA shuttling can be detected in cells that are negative for EBER shuttling, we demonstrate the shuttling of U1 small nuclear RNA. PMID- 16682525 TI - Focal adhesions are hotspots for keratin filament precursor formation. AB - Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement. PMID- 16682526 TI - The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function. AB - The nuclear pore complex (NPC) is a large channel that spans the two lipid bilayers of the nuclear envelope and mediates transport events between the cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins, and the role of these proteins in NPC function and assembly remains poorly understood. We investigate the function of the three integral membrane nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the correct localization of nuclear transport cargoes and of components of the NPC. However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p, as cells lacking both of these proteins show enhanced NPC disruption. Electron microscopy studies reveal that the absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to an increased diffusion between the cytoplasm and the nucleus. PMID- 16682527 TI - Direct evidence for coherent low velocity axonal transport of mitochondria. AB - Axonal growth depends on axonal transport. We report the first global analysis of mitochondrial transport during axonal growth and pauses. In the proximal axon, we found that docked mitochondria attached to the cytoskeletal framework that were stationary relative to the substrate and fast axonal transport fully accounted for mitochondrial transport. In the distal axon, we found both fast mitochondrial transport and a coherent slow transport of the mitochondria docked to the axonal framework (low velocity transport [LVT]). LVT was distinct from previously described transport processes; it was coupled with stretching of the axonal framework and, surprisingly, was independent of growth cone advance. Fast mitochondrial transport decreased and LVT increased in a proximodistal gradient along the axon, but together they generated a constant mitochondrial flux. These findings suggest that the viscoelastic stretching/creep of axons caused by tension exerted by the growth cone, with or without advance, is seen as LVT that is followed by compensatory intercalated addition of new mitochondria by fast axonal transport. PMID- 16682528 TI - The Fat1 cadherin integrates vascular smooth muscle cell growth and migration signals. AB - The significance of cadherin superfamily proteins in vascular smooth muscle cell (VSMC) biology is undefined. Here we describe recent studies of the Fat1 protocadherin. Fat1 expression in VSMCs increases significantly after arterial injury or growth factor stimulation. Fat1 knockdown decreases VSMC migration in vitro, but surprisingly, enhances cyclin D1 expression and proliferation. Despite limited similarity to classical cadherins, the Fat1 intracellular domain (Fat1(IC)) interacts with beta-catenin, inhibiting both its nuclear localization and transcriptional activity. Fat1 undergoes cleavage and Fat1(IC) species localize to the nucleus; however, inhibition of the cyclin D1 promoter by truncated Fat1(IC) proteins corresponds to their presence outside the nucleus, which argues against repression of beta-catenin-dependent transcription by nuclear Fat1(IC). These findings extend recent observations about Fat1 and migration in other cell types, and demonstrate for the first time its anti proliferative activity and interaction with beta-catenin. Because it is induced after arterial injury, Fat1 may control VSMC functions central to vascular remodeling by facilitating migration and limiting proliferation. PMID- 16682529 TI - E-cadherin engagement stimulates proliferation via Rac1. AB - E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK-52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120-catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell-cell contact may trigger or inhibit epithelial cell proliferation in different settings. PMID- 16682530 TI - Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway. AB - We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain-containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal sending cells needs to proceed past coat disassembly to activate Notch. PMID- 16682531 TI - Improvement of postherpetic neuralgia after treatment with intravenous acyclovir followed by oral valacyclovir. AB - BACKGROUND: Postherpetic neuralgia (PHN) is a complication of shingles (herpes zoster), a painful rash due to varicella-zoster virus reactivation. Studies of patients with PHN and zoster sine herpete (radicular pain without rash) support the notion that low-grade viral ganglionitis contributes to pain. If chronic pain reflects active infection, then antiviral therapy may help patients with PHN. OBJECTIVE: To determine whether antiviral treatment helps reduce PHN-associated pain. DESIGN: Prospective, open-label phase I/II clinical trial. SETTING: Tertiary care university hospital. PATIENTS: Fifteen patients with moderate to severe PHN. INTERVENTIONS: Intravenous acyclovir at a dosage of 10 mg/kg every 8 hours for 14 days followed by oral valacyclovir at a dosage of 1000 mg 3 times per day for 1 month. MAIN OUTCOME MEASURE: Numeric Rating Scale for Pain score. RESULTS: As defined by a decrease of 2 or more points on the Numeric Rating Scale for Pain, 8 (53%) of 15 patients reported improvement. CONCLUSION: Clinical improvement reported by most of our patients warrants further investigation in a larger, randomized, double-blind, placebo-controlled trial. PMID- 16682532 TI - Neuropathology of amnestic mild cognitive impairment. PMID- 16682533 TI - The neuropathology of aging, mild cognitive impairment, and Alzheimer disease. PMID- 16682534 TI - Mitochondria in Parkinson disease: back in fashion with a little help from genetics. AB - Parkinson disease is a devastating neurodegenerative disorder with no known cure. Impairment in mitochondrial dysfunction is thought to play a major role in the pathogenesis. Recent genetic advances suggest that mitochondrial dysfunction may be the primary defect. Drugs that target the mitochondria may therefore represent the best hope for disease-modifying therapies in Parkinson disease. PMID- 16682535 TI - Reasons why few patients with acute stroke receive tissue plasminogen activator. AB - Despite the US Food and Drug Administration's approval in 1996, tissue plasminogen activator (tPA) therapy for acute ischemic stroke remains substantially underused. We reviewed 3 potential reasons for low rates of tPA use: poor patient education, physicians' perceived risk of legal liability from negative patient outcomes, and insufficient reimbursement. The recent addition of diagnosis-related grouping code 559 will provide higher payment for stroke patients treated with tPA, creating a natural experiment to examine our third reason. PMID- 16682536 TI - Neuropathologic features of amnestic mild cognitive impairment. AB - BACKGROUND: The neuropathologic substrate of amnestic mild cognitive impairment (aMCI) is not known. OBJECTIVE: To determine the neuropathologic features of patients who died while their clinical classification was aMCI. DESIGN: Cohort study. SETTING: Community based. PARTICIPANTS: Sixty-six individuals, including 15 who had memory impairment beyond that allowed for aging but who were not demented, were studied along with 28 clinically healthy individuals and 23 patients with probable Alzheimer disease (AD) for comparison. MAIN OUTCOME MEASURES: Standard neuropathologic techniques and classification according to Khachaturian, Consortium to Establish a Registry for Alzheimer Disease, and National Institute on Aging-Reagan criteria were used to analyze autopsy tissue from 15 individuals who died while their clinical diagnosis was aMCI. For comparison, autopsy data on age-matched groups of clinically healthy individuals and patients with probable AD were analyzed. RESULTS: Most patients with aMCI did not meet the neuropathologic criteria for AD, but their pathologic findings suggest a transitional state of evolving AD. All the patients with aMCI had pathologic findings involving medial temporal lobe structures, likely accounting for their memory impairment. In addition, there were many concomitant pathologic abnormalities, including argyrophilic grain disease, hippocampal sclerosis, and vascular lesions. CONCLUSIONS: The neuropathologic features of aMCI matched the clinical features and seemed to be intermediate between the neurofibrillary changes of aging and the pathologic features of very early AD. PMID- 16682537 TI - Neuropathologic outcome of mild cognitive impairment following progression to clinical dementia. AB - BACKGROUND: The pathologic outcome of patients diagnosed with mild cognitive impairment (MCI) following progression to dementia is poorly understood. OBJECTIVE: To determine the pathologic substrates of dementia in cases with prior diagnosis of amnestic MCI. DESIGN AND SETTING: Community-based cohort. PATIENTS: Thirty-four subjects followed up prospectively as part of a community-based study who were diagnosed with amnestic MCI, progressed to clinical dementia, and underwent subsequent postmortem brain analysis. MAIN OUTCOME MEASURES: Neuropathologic analyses resulted in assignment of a primary pathologic diagnosis and included staging of Alzheimer pathologic abnormalities and identification of contributing vascular disease, Lewy bodies, and argyrophilic grains. RESULTS: Although the majority of subjects progressed both clinically and pathologically to Alzheimer disease (AD), 10 (29%) of them developed non-AD primary pathologic abnormalities. All of the cases were found to have sufficient pathologic abnormalities in mesial temporal lobe structures to account for their amnestic symptoms regardless of the cause. Most subjects were found to have secondary contributing pathologic abnormalities in addition to primary pathologic diagnoses. No significant differences between subjects with and without neuropathologically proven AD were detected in demographic variables, apolipoprotein E genotype, or cognitive test measures at onset of MCI, onset of dementia, or last clinical evaluation. CONCLUSIONS: The neuropathologic outcome of amnestic MCI following progression to dementia is heterogeneous, and it includes AD at a high frequency. Complex neuropathologic findings including 2 or more distinct pathologic entities contributing to dementia may be common in community-based cohorts. Neither demographic variables nor cognitive measures had predictive value in determining which patients diagnosed with MCI will develop the neuropathologic features of AD. PMID- 16682539 TI - Distinguishing sleep disorders from seizures: diagnosing bumps in the night. AB - BACKGROUND: Abnormal paroxysmal events in sleep may be parasomnias or epileptic seizures. In nocturnal frontal lobe epilepsy (NFLE), the unusual seizure features often lead to diagnostic confusion with nonepileptic parasomnias; video electroencephalography monitoring is usually required to make the diagnosis. OBJECTIVE: To examine the reliability of the clinical history in diagnosing NFLE, using the Frontal Lobe Epilepsy and Parasomnias (FLEP) scale. DESIGN: The FLEP scale, comprising specific questions reflecting the diagnostic features of NFLE and parasomnias, was developed by an expert panel following review of the literature. It was then validated in a sample of individuals with firmly diagnosed nocturnal events. SETTING: Patients were recruited after appropriate diagnostic workup in tertiary sleep and epilepsy referral centers in Melbourne, Australia. PARTICIPANTS: Sixty-two patients (45 males) [corrected] with paroxysmal nocturnal events. Intervention Two independent interviews were conducted in each case, with the patient and a witness, by researchers blinded to the diagnosis. MAIN OUTCOME MEASURE: The diagnosis obtained from scores on the FLEP scale was compared with the confirmed diagnosis in each patient. RESULTS: Nocturnal frontal lobe epilepsy was correctly diagnosed from the FLEP score in 31 of 31 patients, with a sensitivity of 1.0 (95% confidence interval [CI], 0.85 1.00), specificity of 0.90 (95% CI, 0.73-0.97), positive predictive value of 0.91 (95% CI, 0.75-0.97), and negative predictive value of 1.00 (95% CI, 0.85-1.00). CONCLUSIONS: A diagnosis of NFLE can be made reliably using the clinical features identified in the FLEP scale. This may reduce the requirement for tertiary referral and extensive inpatient monitoring. PMID- 16682538 TI - Conversion of mild cognitive impairment to Alzheimer disease predicted by hippocampal atrophy maps. AB - BACKGROUND: While most patients with mild cognitive impairment (MCI) transition to Alzheimer disease (AD), others develop non-AD dementia, remain in the MCI state, or improve. OBJECTIVE: To test the following hypotheses: smaller hippocampal volumes predict conversion of MCI to AD, whereas larger hippocampal volumes predict cognitive stability and/or improvement; and patients with MCI who convert to AD have greater atrophy in the CA1 hippocampal subfield and subiculum. DESIGN: Prospective longitudinal cohort study. SETTING: University of California Los Angeles Alzheimer's Disease Research Center. PATIENTS: We followed up 20 MCI subjects clinically and neuropsychologically for 3 years. MAIN OUTCOME MEASURE: Baseline regional hippocampal atrophy was analyzed with region-of-interest and 3 dimensional hippocampal mapping techniques. RESULTS: During the 3-year study, 6 patients developed AD (MCI-c), 7 remained stable (MCI-nc), and 7 improved (MCI i). Patients with MCI-c had 9% smaller left and 13% smaller right mean hippocampal volumes compared with MCI-nc patients. Radial atrophy maps showed greater atrophy of the CA1 subregion in MCI-c. Patients with MCI-c had significantly smaller hippocampi than MCI-i patients (left, 24%; right, 27%). Volumetric analyses showed a trend for greater hippocampal atrophy in MCI-nc relative to MCI-i patients (eg, 16% volume loss). After permutation tests corrected for multiple comparison, the atrophy maps showed a significant difference on the right. Subicular differences were seen between MCI-c and MCI-i patients, and MCI-nc and MCI-i patients. Multiple linear regression analysis confirmed the group effect to be highly significant and independent of age, hemisphere, and Mini-Mental State Examination scores at baseline. CONCLUSIONS: Smaller hippocampi and specifically CA1 and subicular involvement are associated with increased risk for conversion from MCI to AD. Patients with MCI-i tend to have larger hippocampal volumes and relative preservation of both the subiculum and CA1. PMID- 16682540 TI - The malignant course of "benign hallucinations" in Parkinson disease. AB - OBJECTIVE: To monitor progression of "benign hallucinations" in Parkinson disease (PD). METHODS: We searched our data repository for subjects with PD with 3 sets of neuropsychological testing during 3 years and Unified Parkinson's Disease Rating Scale thought disorder scores taken at 4- to 12-month intervals during this period. We found 48 patients with benign hallucinations, defined as a thought disorder score of 2 (benign hallucinations, insight retained), receiving no treatment for hallucinations. We followed up thought disorder scores under best medical management for at least 3 years or until a thought disorder score of 3 (loss of insight) or 4 (delusions) occurred. In subjects whose thought disorder scores remained at 2, we assessed neuroleptic use and decreases in PD medications to abate hallucinations. RESULTS: Most subjects (81%) progressed to thought disorder scores of 3 or 4. In 7 (78%) of 9 subjects who retained a thought disorder score of 2, PD medications were reduced to treat hallucinations, and 3 subjects (33%) also received neuroleptics. If the composite end point (any of the criteria) was used, 96% of subjects progressed, with only 2 subjects continuing with stable, untreated benign hallucinations. CONCLUSIONS: Because hallucinations progress, the concept of benign hallucinations is prognostically misleading. Though hallucinations with retained insight may be "benign" for the moment, they portend serious consequences. The term benign hallucinations of PD should be considered generally unsound and dropped from operative vocabulary. PMID- 16682541 TI - History of vascular disease and mild parkinsonian signs in community-dwelling elderly individuals. AB - BACKGROUND: Mild parkinsonian signs (MPS) are commonly found during the clinical examination of older people. These signs could reflect the accumulation of vascular pathological changes in the brain. If the etiology were vascular, one could hypothesize that individuals with MPS would be more likely to have systemic vascular disease than would their counterparts without these signs. OBJECTIVES: To examine whether MPS are associated with a history of vascular disease and whether certain MPS (rigidity, changes in axial function) are more strongly associated with a history of vascular disease than are other MPS (tremor). METHODS: Evaluation of older people without dementia in Washington Heights Inwood, northern Manhattan, NY. The presence of vascular diseases was assessed with a structured health interview. RESULTS: Mild parkinsonian signs were present in 375 (16.4%) of 2286 participants without dementia. Diabetes mellitus, heart disease, peripheral vascular disease, and stroke were more prevalent in participants with MPS than without MPS, whereas nonvascular diseases (cancer, thyroid disease) were not. In a logistic regression analysis that adjusted for age, sex, education, and depressive symptoms, the number of vascular diseases was associated with MPS (odds ratio, 1.31 [95% confidence interval, 1.18-1.46]; P<.001). The combination of diabetes mellitus and heart disease increased the odds of MPS by 70% and the combination of these with stroke increased the odds by 332%. CONCLUSIONS: The presence of MPS in elderly individuals might reflect, in part, the accumulation of vascular pathological changes in the basal ganglia or white matter regions caused by preventable vascular diseases. Prospective and pathological studies are needed to further explore this finding. PMID- 16682542 TI - Testosterone therapy in men with Parkinson disease: results of the TEST-PD Study. AB - BACKGROUND: Testosterone deficiency has been reported in patients with Parkinson disease (PD), Alzheimer disease, and Huntington disease. It is not known whether testosterone therapy (TT) in men with borderline hypogonadism and neurodegenerative diseases will be of substantial benefit. Previously, we reported that testosterone deficiency is more common in patients with PD compared with age-matched control subjects, and we also reported in 2 small open-label studies that some nonmotor symptoms responded favorably to TT. OBJECTIVE: To define the effects of TT on nonmotor and motor symptoms in men with PD and probable testosterone deficiency. DESIGN: Double-masked, placebo-controlled, parallel-group, single-center trial. PATIENTS: Two experimental groups: patients with PD who were receiving either TT or placebo. INTERVENTIONS: Participants received either the study drug by intramuscular injection (200 mg/mL of testosterone enanthate every 2 weeks for 8 weeks) or placebo (isotonic sodium chloride solution injections). In patients in each group, the testosterone serum concentration was obtained at each study visit. During 2 study visits, testosterone levels were blindly evaluated and the intramuscular testosterone dose was increased by 200 mg/mL if the free testosterone value failed to double from the baseline value. MAIN OUTCOME MEASURES: The primary outcome variable was the St Louis Testosterone Deficiency Questionnaire, and secondary outcome measures included measures of mood, cognition, fatigue, motor function, and frequency of adverse events. At the end of the double-blind phase, all patients were offered open-label TT and were followed up after 3 and 6 months. RESULTS: Fifteen patients in the placebo group (mean age, 69.9 years), receiving a mean total levodopa equivalent dose of 924 mg/d, had a baseline free testosterone level of 47.91 pg/mL, compared with 15 patients in the TT group (mean age, 66.7 years), receiving an average total levodopa equivalent dose of 734 mg/d, who had a baseline free testosterone level of 63.49 pg/mL. Testosterone was generally well tolerated. More subjects in the TT group experienced lower extremity edema (40% vs 20%). In 2 patients, 1 in each group, prostate-specific antigen levels were elevated from baseline. The improvement in the TT group compared with the placebo group (1.7 vs 1.1) on the St Louis Testosterone Deficiency Scale was not statistically significant. In addition, there were no significant differences in motor and nonmotor features of PD between the 2 groups, although a few subscales showed improvements (Hopkins Verbal Learning Test, P<.04; and Backward Visual Span subtrial, P<.03). However, long-term open-label TT resulted in delayed but sustained improvement in subjects in the TT group who continued to receive treatment (n = 6) compared with subjects in the placebo group who elected not to receive TT (n = 3). CONCLUSIONS: Testosterone therapy was generally well tolerated in elderly men with PD and probable testosterone deficiency. While there was no significant difference in the motor and nonmotor scales between the TT and placebo groups at the end of 8 weeks compared with baseline, this may be due to several study limitations, including small sample size, a strong placebo effect with intramuscular therapy, and short follow-up that did not allow measurement of delayed effects of TT in some subjects. Until more definitive studies are reported, practitioners should be particularly cautious in treatment of low testosterone concentrations in men with PD and borderline testosterone deficiency, and careful consideration should be given to the risks vs the benefits of TT. PMID- 16682543 TI - Magnetization transfer magnetic resonance imaging and clinical changes in patients with relapsing-remitting multiple sclerosis. AB - BACKGROUND: Magnetization transfer (MT) magnetic resonance imaging (MRI) can provide in vivo quantitative estimates of microscopic tissue damage in normal appearing white matter (NAWM) and gray matter (GM) from patients with multiple sclerosis (MS). OBJECTIVE: To determine whether a one-time MT MRI can provide markers of short-term disease evolution in patients with relapsing-remitting MS. DESIGN: Eighteen-month observational study. SETTING: Neuroimaging Research Unit, Scientific Institute and University Ospedale San Raffaele. PATIENTS: Twenty-two patients with untreated relapsing-remitting MS. MAIN OUTCOME MEASURES: Relapse rate; disability according to the Expanded Disability Status Scale (EDSS); dual echo, 2-dimensional gradient echo with and without a saturation MT pulse and T1 weighted MRIs of the brain; and MT ratio (MTR) histograms for NAWM and GM. RESULTS: During the study period, 13 patients (59%) experienced 25 relapses. The median EDSS score was 1.25 (range, 0-3.5) at study entry and 1.75 (range, 0-3) at study exit. Significant, although moderate, correlations were found between average GM MTR values at baseline and EDSS changes during the study period (r = 0.44; P = .04). A trend was observed for the correlation between NAWM MTR values at baseline and the EDSS changes throughout 18 months (r = -0.42; P = .05). For the relation between EDSS changes and baseline GM MTR, the slope of the regression line was -0.5 (95% confidence interval, -1.0 to 0.0), indicating that a decrease in the baseline GM MTR of 1% predicted an increase in the EDSS score of 0.5 point throughout the 18 months. CONCLUSION: This study indicates that a "snapshot" MT MRI assessment detects subtle brain tissue changes that are associated with short-term disability accumulation in patients with relapsing remitting MS. PMID- 16682544 TI - Venturi mask adjuvant oxygen therapy in severe acute ischemic stroke. AB - BACKGROUND: The effect of oxygen therapy in acute ischemic stroke remains undetermined. OBJECTIVE: To investigate the feasibility of eubaric hyperoxia therapy by Venturi mask (VM) in a group of patients who experienced a severe acute ischemic stroke. DESIGN: Patients experiencing a first-ever large middle cerebral artery infarction were recruited within 48 hours after stroke. Patients were subdivided to undergo therapy with a VM with a fraction of inspired oxygen of 40% or with a nasal cannula. A large middle cerebral artery infarction was defined as a large low-attenuation area of more than one third of the middle cerebral artery territory on brain images. Stroke severity was evaluated by the National Institutes of Health Stroke Scale. RESULTS: Seventeen patients were enrolled in the VM group and 29 in the nasal cannula group. All the demographic and clinical characteristics were equally distributed initially. The mean initial National Institutes of Health Stroke Scale score was 20.5 and 18.9 in the VM and nasal cannula groups, respectively. Atrial fibrillation was found in 11 (65%) patients in the VM and 17 (59%) patients in the nasal cannula groups. The VM therapy was initiated within 13.7 (range, 3.0-41.5) hours after stroke and the duration was 132.9 (range, 48.0-168.5) hours. In-hospital mortality was 1 (6%) in the VM group and 7 (24%) in the nasal cannula group (P=.12). In the VM group, there were fewer incidences of fever (4 [24%] vs 15 [52%]; P=.06), pneumonia (1 [6%] vs 6 [21%]; P=.18), and respiratory failure (3 [18%] vs 8 [28%]; P=.45), but a higher incidence of bedsores (3 [18%] vs 2 [7%]; P=.29). CONCLUSIONS: By using VM therapy with a fraction of inspired oxygen of 40%, there might be less mortality and comorbidities in treated patients who experienced a severe acute ischemic stroke. Further randomized confirmatory studies should explore the decreased mortality in patients who experience a severe acute ischemic stroke, especially in those with a large middle cerebral artery infarction who undergo VM therapy with a fraction of inspired oxygen of 40%. PMID- 16682545 TI - Nerve conduction abnormalities in patients with MELAS and the A3243G mutation. AB - BACKGROUND: Mitochondrial DNA point mutations are especially deleterious to tissues with high energy demand, including the peripheral nervous system. Neuropathy has been associated with several mitochondrial diseases, including MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes). OBJECTIVE: To evaluate nerve conduction in a genotypically and phenotypically homogeneous group of patients with MELAS and the A3243G mutation. DESIGN: We studied 30 patients with MELAS and the A3243G mutation using neurophysiological techniques, medical history questionnaires, laboratory tests, and a standardized neurological examination. RESULTS: Twenty-three subjects (77%) had abnormal nerve conduction measures. Symptoms suggestive of neuropathy were present in only half of the patients, but almost all had decreased reflexes or distal sensory findings on examination. Nerve conduction abnormalities were predominantly axonal and sensory and mainly present in the legs. Patients with nerve conduction abnormalities tended to be older and were more likely male. CONCLUSIONS: Peripheral nerve impairment is common in those with MELAS and the A3243G mutation, and may be subclinical. Male sex and older age may add to the genetic disposition to develop neuropathy. PMID- 16682546 TI - Eight novel mutations in SPG4 in a large sample of patients with hereditary spastic paraplegia. AB - BACKGROUND: Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous disorders characterized by progressive spasticity of the lower limbs. Mutations in the SPG4 gene, which encodes spastin protein, are responsible for up to 45% of autosomal dominant cases. OBJECTIVE: To search for disease causing mutations in a large series of Italian patients with HSP. DESIGN: Samples of DNA were analyzed by direct sequencing of all exons in SPG4. Samples from a subset of patients were also analyzed by direct sequencing of all exons in SPG3A, SPG6, SPG10, and SPG13. SETTING: Molecular testing facility in Italy. PATIENTS: Sixty unrelated Italian patients with pure (n = 50) and complicated (n = 10) HSP. MAIN OUTCOME MEASURES: Mutations in SPG4, SPG3A, SPG6, SPG10, and SPG13. RESULTS: We identified 12 different mutations, 8 of which were novel, in 13 patients. No mutations of any of the other HSP genes tested were found in 15 patients with sporadic pure HSP who did not have mutations in the SPG4 gene. CONCLUSIONS: The overall rate of mutation in the SPG4 gene within our sample was 22%, rising to 26% when only patients with pure HSP were considered. The negative result obtained in 15 patients without mutations in SPG4 in whom 4 other genes were analyzed (SPG3A, SPG6, SPG10, and SPG13) indicate that these genes are not frequently mutated in sporadic pure HSP. PMID- 16682547 TI - Hereditary spastic paraplegia with thin corpus callosum: reduction of the SPG11 interval and evidence for further genetic heterogeneity. AB - BACKGROUND: Hereditary spastic paraplegia (HSP) with thin corpus callosum (TCC) is an autosomal recessive form of complicated HSP mainly characterized by slowly progressive spastic paraparesis and mental deterioration beginning in the second decade of life. The locus for HSP-TCC, designated SPG11, was mapped to chromosome 15q13-15 in some of the affected families from Japan, Europe, and North America, spanning an interval of 17.5 megabases (Mb). OBJECTIVE: To perform a clinical and genetic study of HSP-TCC. DESIGN AND SETTING: Case series; multi-institutional study. PATIENTS: Seven patients with HSP-TCC who belong to 3 consanguineous families of Arab origin residing in Israel. RESULTS: The 7 patients manifested a relatively similar combination of adolescence-onset cognitive decline and spastic paraparesis with TCC on brain magnetic resonance imaging. After excluding the SPG7 locus, we tested the 3 families for linkage to the SPG11, SPG21/MAST, and ACCPN loci associated with autosomal recessive disorders with TCC. Two families showed evidence for linkage to SPG11 (Z(max) = 5.55) and reduced the candidate region to 13 Mb. CONCLUSIONS: Our findings in HSP-TCC further confirm its worldwide distribution and genetic heterogeneity, and they significantly reduce the candidate SPG11 interval. PMID- 16682548 TI - Nontuberculous mycobacterial infection of a metastatic brain neoplasm in an immunocompromised patient. AB - BACKGROUND: Nontuberculous mycobacterial infections occur in immunocompromised patients but so rarely involve the central nervous system (CNS) that they may not be included in a differential diagnosis of CNS lesions in such patients. OBJECTIVE: To illustrate a putative mechanism for nontuberculous mycobacterial infection of the CNS via breakdown of the blood-brain barrier by metastatic neoplasm. RESULTS: A 56-year-old man who had undergone renal transplantation in February 2003 and was taking an immunosuppressive regimen of mycophenolate mofetil and cyclosporine was seen in the emergency department after a syncopal episode. Head computed tomography revealed a single focal occipital lesion with vasogenic edema. Hospital admission and further workup led to diagnosis of metastatic carcinoma infected with nontuberculous mycobacteria in the setting of a disseminated nontuberculous mycobacterial infection. CONCLUSION: This case illustrates that breakdown of the blood-brain barrier by metastatic neoplasm may provide a route of access for a pathogen that is not normally seen in the CNS. PMID- 16682549 TI - Nonvasculitic autoimmune inflammatory meningoencephalitis imitating Creutzfeldt Jakob disease. AB - BACKGROUND: Nonvasculitic autoimmune inflammatory meningoencephalitis and Creutzfeldt-Jakob disease can present as rapidly progressive encephalopathies with similar clinical features. Slowing of background rhythm is an electroencephalographic characteristic shown by both, but persistent periodic sharp waves are more specific for Creutzfeldt-Jakob disease and have not been reported in nonvasculitic autoimmune inflammatory meningoencephalitis or related autoimmune meningoencephalitides. OBJECTIVE: To describe a patient with clinical (rapidly progressive myoclonus, dementia, and Parkinsonism) and electroencephalographic findings (persistent periodic sharp waves) that diagnostically suggest Creutzfeldt-Jakob disease. DESIGN AND SETTING: A case report at the Mayo Clinic Arizona, Scottsdale. RESULTS: The patient made a dramatic recovery with resolution of the periodic sharp wave complexes after treatment with high-dose corticosteroids. Our case is the first reported case of a patient with probable nonvasculitic autoimmune inflammatory meningoencephalitis and electroencephalographic periodic complexes suggestive of Creutzfeldt-Jakob disease. CONCLUSION: Rapidly progressive encephalopathy with periodic sharp wave complexes can be associated with a reversible autoimmune syndrome. PMID- 16682550 TI - Superior divisional third cranial nerve paresis: clinical and anatomical observations of 2 unique cases. AB - BACKGROUND: Within the midbrain, the third nerve nucleus is composed of a complex of subnuclei. The fascicular portion of the nerve courses through the red nucleus and exists in the midbrain just medial to the cerebral peduncle. The cisternal portion of the nerve is a single structure that divides into a superior branch and an inferior branch in the region of the cavernous sinus and superior orbital fissure. OBJECTIVE: To describe 2 patients with superior divisional third cranial nerve paresis resulting from a lesion involving the cisternal portion of the nerve prior to its anatomical bifurcation. PATIENTS: Case 1 was a 77-year-old man with a superior divisional third nerve palsy as the presenting manifestation of a posterior communicating artery aneurysm. Case 2 was a 41-year-old woman who developed a superior divisional third nerve palsy following anterior temporal lobectomy for epilepsy. RESULTS: In both cases, the presumed location of the lesion was the cisternal portion of the third cranial nerve. CONCLUSIONS: Although the anatomical division of the third cranial nerve occurs in the region of the anterior cavernous sinus or superior orbital fissure, there is a topographical arrangement of the motor fibers within the cisternal portion of the nerve. The clinical evaluation of a patient with a third cranial nerve paresis requires an understanding of the regional neuroanatomy and topographical organization of the nerve. PMID- 16682551 TI - Magnetic resonance imaging in poliomyelitis. PMID- 16682552 TI - Cerebral vasculitis in ulcerative colitis. PMID- 16682553 TI - Suprascapular nerve entrapment in a canoeist. PMID- 16682554 TI - Early descriptions of Parkinson disease in ancient China. PMID- 16682555 TI - Characterizing the mechanisms and progression in multiple sclerosis: evidence and new hypotheses for future directions. PMID- 16682556 TI - Pure monoparesis: what makes it different. PMID- 16682557 TI - 30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15. AB - Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized. PMID- 16682558 TI - Functional conformations of the L11-ribosomal RNA complex revealed by correlative analysis of cryo-EM and molecular dynamics simulations. AB - The interaction between the GTPase-associated center (GAC) and the aminoacyl tRNA.EF-Tu.GTP ternary complex is of crucial importance in the dynamic process of decoding and tRNA accommodation. The GAC includes protein L11 and helices 43-44 of 23S rRNA (referred to as L11-rRNA complex). In this study, a method of fitting based on a systematic comparison between cryo-electron microscopy (cryo-EM) density maps and structures obtained by molecular dynamics simulations has been developed. This method has led to the finding of atomic models of the GAC that fit the EM maps with much improved cross-correlation coefficients compared with the fitting of the X-ray structure. Two types of conformations of the L11-rRNA complex, produced by the simulations, match the cryo-EM maps representing the states either bound or unbound to the aa-tRNA.EF-Tu.GTP ternary complex. In the bound state, the N-terminal domain of L11 is extended from its position in the crystal structure, and the base of nucleotide A1067 in the 23S ribosomal RNA is flipped out. This position of the base allows the RNA to reach the elbow region of the aminoacyl-tRNA when the latter is bound in the A/T site. In the unbound state, the N-terminal domain of L11 is rotated only slightly, and A1067 of the RNA is flipped back into the less-solvent-exposed position, as in the crystal structure. By matching our experimental cryo-EM maps with much improved cross correlation coefficients compared to the crystal structure, these two conformations prove to be strong candidates of the two functional states. PMID- 16682559 TI - Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain. AB - Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings. PMID- 16682560 TI - Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity. AB - Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended ("off-target") consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3' UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with dependence on siRNA concentration similar to that of silencing of the target transcript. Thus, short stretches of sequence complementarity to the siRNA or shRNA seed region are key to the silencing of unintended transcripts. PMID- 16682561 TI - Off-target effects by siRNA can induce toxic phenotype. AB - Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA. PMID- 16682562 TI - Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. AB - Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts. PMID- 16682563 TI - Identification of small Hfq-binding RNAs in Listeria monocytogenes. AB - The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Gram-positive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes. PMID- 16682564 TI - Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA. AB - TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG206 codon was dependent on its closeness to the upstream AUG69 codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides. PMID- 16682565 TI - Analysis of a 3'-translation enhancer in a tombusvirus: a dynamic model for RNA RNA interactions of mRNA termini. AB - Tomato bushy stunt virus is a (+)-strand RNA virus that is neither 5'-capped nor 3'-polyadenylated. Translation of viral proteins is instead mediated by an RNA element, the 3'-cap-independent translational enhancer (3'CITE), which is located in its 3' untranslated region (UTR). The 3'CITE is proposed to recruit the translational machinery to the viral message, while a long-distance RNA-RNA interaction between the 3'CITE and 5' UTR is thought to deliver the 43S ribosomal subunit to the 5' end of the viral mRNA. Here we provide the first evidence that the 5' UTR and 3'CITE interact physically. Mutational analysis showed that formation of this RNA-RNA interaction in vitro correlates well with efficient translation in vivo, thus supporting its functional relevance. Other analyses of the 3'CITE confirmed an overall Y-shaped RNA secondary structure and demonstrated the importance of numerous minor structural features for efficient translation of viral mRNAs. Functional studies on the role of the 5' UTR revealed that despite the absence of a cap structure, 43S subunits load at the very 5' end and scan in a 3' direction. These results indicate that the 5'-3' RNA-RNA interaction is likely disrupted by scanning ribosomal subunits and suggest a dynamic model for the interaction of mRNA termini during active translation. PMID- 16682566 TI - An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation. AB - Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy. PMID- 16682567 TI - Patient preferences can be misleading as explanations for racial disparities in health care. AB - Addressing racial differences in US health care is difficult, in part because the origins of these differences are complex and poorly understood and also because only some differences may represent legitimate agendas for reform. For example, racial differences that result from differences in access or from clinician prejudices are symptoms of a problem that must be addressed; in contrast, racial differences that arise from different preferences for health care might be seen as acceptable consequences of variation in personal or cultural values that are worth respecting and preserving. However, we believe that this distinction is sometimes overstated. We present several fictitious clinical stories to illustrate how what appear to be individual or cultural preferences are often substantially shaped by modifiable practices of the health care system. True differences in preference that are worthy of respect surely exist between individuals or demographic groups, but some apparent differences in preference may actually reflect problems with the health care system that are worthy of remediation. Uncovering the hidden constraints that we impose on health care choice may help us reduce unwanted disparities in health care. PMID- 16682568 TI - Clinical pharmacists and inpatient medical care: a systematic review. AB - BACKGROUND: The role of clinical pharmacists in the care of hospitalized patients has evolved over time, with increased emphasis on collaborative care and patient interaction. The purpose of this review was to evaluate the published literature on the effects of interventions by clinical pharmacists on processes and outcomes of care in hospitalized adults. METHODS: Peer-reviewed, English-language articles were identified from January 1, 1985, through April 30, 2005. Three independent assessors evaluated 343 citations. Inpatient pharmacist interventions were selected if they included a control group and objective patient-specific health outcomes; type of intervention, study design, and outcomes such as adverse drug events, medication appropriateness, and resource use were abstracted. RESULTS: Thirty-six studies met inclusion criteria, including 10 evaluating pharmacists' participation on rounds, 11 medication reconciliation studies, and 15 on drug specific pharmacist services. Adverse drug events, adverse drug reactions, or medication errors were reduced in 7 of 12 trials that included these outcomes. Medication adherence, knowledge, and appropriateness improved in 7 of 11 studies, while there was shortened hospital length of stay in 9 of 17 trials. No intervention led to worse clinical outcomes and only 1 reported higher health care use. Improvements in both inpatient and outpatient outcome measurements were observed. CONCLUSIONS: The addition of clinical pharmacist services in the care of inpatients generally resulted in improved care, with no evidence of harm. Interacting with the health care team on patient rounds, interviewing patients, reconciling medications, and providing patient discharge counseling and follow-up all resulted in improved outcomes. Future studies should include multiple sites, larger sample sizes, reproducible interventions, and identification of patient specific factors that lead to improved outcomes. PMID- 16682569 TI - Are lifestyle measures effective in patients with gastroesophageal reflux disease? An evidence-based approach. AB - Lifestyle modifications are first-line therapy for patients with gastroesophageal reflux disease (GERD). We applied an evidence-based approach to determine the efficacy of lifestyle measures for GERD management. We used PubMed and Ovid to perform a search of the literature published between 1975 and 2004 using the key words heartburn, GERD, smoking, alcohol, obesity, weight loss, caffeine or coffee, citrus, chocolate, spicy food, head of bed elevation, and late-evening meal. Each study was reviewed by 2 reviewers who assigned one of the following ratings: evidence A, randomized clinical trials; evidence B, cohort or case control studies; evidence C, case reports or flawed clinical trials; evidence D, investigator experience; or evidence E, insufficient information. We screened 2039 studies and identified 100 that were relevant. Only 16 clinical trials examined the impact on GERD (by change in symptoms, esophageal pH variables, or lower esophageal sphincter pressure) of the lifestyle measure. Although there was physiologic evidence that exposure to tobacco, alcohol, chocolate, and high-fat meals decreases lower esophageal sphincter pressure, there was no published evidence of the efficacy of dietary measures. Neither tobacco nor alcohol cessation was associated with improvement in esophageal pH profiles or symptoms (evidence B). Head of bed elevation and left lateral decubitus position improved the overall time that the esophageal pH was less than 4.0 (evidence B). Weight loss improved pH profiles and symptoms (evidence B). Weight loss and head of bed elevation are effective lifestyle interventions for GERD. There is no evidence supporting an improvement in GERD measures after cessation of tobacco, alcohol, or other dietary interventions. PMID- 16682570 TI - Cost-effectiveness of continuous positive airway pressure therapy for moderate to severe obstructive sleep apnea/hypopnea. AB - BACKGROUND: Obstructive sleep apnea/hypopnea (OSAH) is a common disorder characterized by recurrent collapse of the upper airway during sleep, and is associated with an increased risk of motor vehicle crashes (MVCs). Common first line therapy for OSAH is continuous positive airway pressure (CPAP). We assessed the cost-effectiveness of CPAP therapy vs none for the treatment of OSAH. METHODS: We used a 5-year Markov model that considers the costs and quality-of life improvements of CPAP therapy, accounting for the gains from reduced MVC rates. Utility values were obtained from published studies. The MVC rates under the CPAP and no-CPAP scenarios were calculated from National Highway Traffic Safety Administration data and a systematic review of published studies. Costs of MVCs, equipment, and physicians were obtained from US Medicare and the National Highway Traffic Safety Administration. The target population included male and female patients aged 25 to 54 years and newly diagnosed as having moderate to severe OSAH. We examined the findings from the perspectives of a third-party payer and society. RESULTS: From a third-party payer or a societal perspective, CPAP therapy was more effective but more costly than no CPAP, with incremental cost-effectiveness ratios of $3354 or $314 per quality-adjusted life-year gained, respectively. The incremental cost-effectiveness ratio estimate was most dependent on viewpoint (varying more than 10-fold between societal and third party payer perspectives) and choice of utility measurement method (varying more than 5-fold between the use of standard gamble and EuroQol 5D utility assessment values). CONCLUSION: When quality of life, costs of therapy, and MVC outcomes are considered, CPAP therapy for patients with OSAH is economically attractive. PMID- 16682571 TI - Lifestyle-related risk factors and risk of future nursing home admission. AB - BACKGROUND: While risks of disease, hospitalization, and death attributable to lifestyle-related factors such as smoking, inactivity, and obesity have been well studied, their associations with nursing home admission are less well known. These risk factors are usually established by middle age, but nothing is known about how they relate to long-term risk of nursing home admission in this age group. METHODS: Cox proportional hazards regressions were used to analyze risk of nursing home admission over 2 decades of follow-up (1971-1975 to 1992) in a nationally representative, longitudinal survey of community-dwelling adults aged 45 to 74 years at baseline. Middle-aged (45-64 years at baseline) and elderly persons (aged 65-74 years at baseline) were analyzed separately: 230 (6.5%) of 3526 middle-aged respondents and 728 (24.7%) of 2936 elderly ones had 1 or more nursing home admissions. Baseline risk factors included smoking, inactivity, obesity, elevated blood pressure, elevated total cholesterol level, and diabetes mellitus, which were defined according to national guidelines. RESULTS: All lifestyle-related factors, except total cholesterol level, were associated with higher risk of nursing home admission during follow-up in one or both age groups. Risk ratios were higher in middle-aged than in elderly persons. In those aged 45 to 64 years at baseline, diabetes more than tripled the risk of nursing home admission (relative risk, 3.25; 95% confidence interval, 2.04-5.19); smoking, inactivity, and elevated systolic blood pressure had relative risks of 1.56, 1.40, and 1.35, respectively. Obesity was a risk factor for those aged 65 to 74 years at baseline, but not for the middle-aged subjects. Persons with 2 lifestyle related factors were at greatly increased risk, especially if 1 was diabetes. CONCLUSIONS: Lifestyle factors are important contributors to the long-term risk of nursing home admission. Modifying lifestyle, especially in middle age, may reduce the risk of admission. PMID- 16682572 TI - Depression outcome in inpatients with congestive heart failure. AB - BACKGROUND: High rates of depression are found among hospitalized patients with congestive heart failure. Little is known about the outcome of depression in these patients or factors that influence that outcome. METHODS: To assess baseline patient characteristics as predictors of time to remission in depressed inpatients with congestive heart failure, consecutive patients older than 50 years admitted with congestive heart failure were screened for major and minor depression using a structured clinical interview. Patients with minor depression were reevaluated at 6 and 12 weeks, and those with major depression at 6, 12, 18, and 24 weeks using the Longitudinal Interview Follow-up Evaluation. RESULTS: Of a total of 473 depressed patients, 404 (247 with minor depression and 157 with major depression) were identified and followed up. Patients with minor depression were followed up for an average of 11.3 weeks, during which 64.0% went into remission; those with major depression were followed up for 20.2 weeks, during which 47.8% went into remission. Baseline predictors of shorter time to remission for minor depression were less severe depression (hazard ratio [HR], 0.95; 95% confidence interval [CI], 0.92-0.98) and fewer comorbid illnesses (HR, 0.92; 95% CI, 0.87-0.98); patients who were younger and had better physical functioning and those not treated with antidepressants also tended to go into remission faster. For major depression, less severe depression was the primary predictor (HR, 0.92; 95% CI, 0.88-0.96), although patients who were younger, male, without a history of depression, and with fewer comorbid medical disorders also tended to go into remission faster. Fewer than 50% of patients with major depression received treatment, and only 12% had psychiatric consultations. CONCLUSIONS: The outcome of minor depression may be more dependent on physical stressors, whereas major depression seems more affected by intrinsic vulnerability. Many patients with major depression were not treated, and few had psychiatric consultations. PMID- 16682573 TI - Comparison of outcomes using 2 delivery models of anticoagulation care. AB - BACKGROUND: Studies demonstrate the effectiveness of anticoagulation management service (AMS) in providing antithrombotic therapy for eligible patients. We sought to extend this concept by determining whether an interim telephone model (IT) is comparable to our current AMS model at achieving optimal therapeutic outcomes. METHODS: The 36-month trial (24-month study plus 12-month extension) enrolled 192 eligible patients receiving long-term warfarin therapy at a Veterans Affairs hospital. Consenting participants were randomly assigned to either our current face-to-face clinic model (AMS), or our IT model. The primary outcome was the percentage of time individuals' international normalized ratios (INRs) were maintained within their target INR range (2.0-3.0 or 2.5-3.5). Secondary outcomes included the number of adverse events (eg, thromboembolism or hemorrhage) experienced during the study. RESULTS: We found no statistically significant difference between the 2 groups in the percentage of time maintained within INR target range overall (55.1% for AMS; 57.8% for IT; P = .28) nor over the course of the study. There were no statistically significant differences in the rate of thromboembolic or serious bleeding events between IT and AMS participants. Nevertheless, we did note differences related to intensity of anticoagulation. The IT group receiving treatment at a higher intensity (INR, 2.5-3.5) experienced greater anticoagulation control (P = .04) and fewer complications than the AMS group. The IT participants, however, reported a significantly higher rate of minor bleeding events, experienced mainly by those at an INR range of 2.0 to 3.0. CONCLUSION: Our IT model is a viable modification of our AMS model for the management of patients undergoing chronic anticoagulant therapy. PMID- 16682574 TI - Heart failure and risk of dementia and Alzheimer disease: a population-based cohort study. AB - BACKGROUND: Heart failure has been linked to cognitive impairment in several previous studies, but to our knowledge, no investigations have explored the relationship between heart failure and the risk of dementia. We sought to examine the hypothesis that heart failure is a risk factor for dementia and Alzheimer disease. METHODS: A community-based cohort of 1301 individuals 75 years or older and without dementia in Stockholm, Sweden, was examined 3 times over a 9-year period to detect patients with dementia and Alzheimer disease using the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition. Heart failure was defined according to the guidelines of the Task Force on Heart Failure of the European Society of Cardiology by integrating clinical symptoms and signs with inpatient register entries and use of cardiac medications. Data were analyzed using Cox proportional hazards models with adjustment for major potential confounders. RESULTS: During the 6534 person years of follow-up (mean, 5.02 years per person), 440 subjects were diagnosed as having dementia, including 333 with Alzheimer disease. At baseline, heart failure was identified in 205 subjects. Heart failure was associated with a multi adjusted hazard ratio (HR) of 1.84 (95% confidence interval [CI], 1.35-2.51) for dementia and 1.80 (95% CI, 1.25-2.61) for Alzheimer disease. Use of antihypertensive drugs (83% of which are diuretics) seemed to reduce dementia risk due to heart failure (HR, 1.38; 95% CI, 0.99-1.94). Heart failure and low diastolic pressure (< 70 mm Hg) had an additive effect on the risk for dementia (HR, 3.07; 95% CI, 1.67-5.61). CONCLUSIONS: Heart failure is associated with an increased risk of dementia and Alzheimer disease in older adults. Antihypertensive drug therapy may partially counteract the risk effect of heart failure on dementia disorders. PMID- 16682575 TI - Reducing warfarin medication interactions: an interrupted time series evaluation. AB - BACKGROUND: Computerized decision support reduces medication errors in inpatients, but limited evidence supports its effectiveness in reducing the coprescribing of interacting medications, especially in the outpatient setting. The usefulness of academic detailing to enhance the effectiveness of medication interaction alerts also is uncertain. METHODS: This study used an interrupted time series design. In a health maintenance organization with an electronic medical record, we evaluated the effectiveness of electronic medical record alerts and group academic detailing to reduce the coprescribing of warfarin and interacting medications. Participants were 239 primary care providers at 15 primary care clinics and 9910 patients taking warfarin. All 15 clinics received electronic medical record alerts for the coprescription of warfarin and 5 interacting medications: acetaminophen, nonsteroidal anti-inflammatory medications, fluconazole, metronidazole, and sulfamethoxazole. Seven clinics were randomly assigned to receive group academic detailing. The primary outcome, the interacting prescription rate (ie, the number of coprescriptions of warfarin interacting medications per 10 000 warfarin users per month), was analyzed with segmented regression models, controlling for preintervention trends. RESULTS: At baseline, nearly a third of patients had an interacting prescription. Coinciding with the alerts, there was an immediate and continued reduction in the warfarin interacting medication prescription rate (from 3294.0 to 2804.2), resulting in a 14.9% relative reduction (95% confidence interval, -19.5 to -10.2) at 12 months. Group academic detailing did not enhance alert effectiveness. CONCLUSIONS: This study, using a strong and quasi-experimental design in ambulatory care, found that medication interaction alerts modestly reduced the frequency of coprescribing of interacting medications. Additional efforts will be required to further reduce rates of inappropriate prescribing of warfarin with interacting drugs. PMID- 16682576 TI - Corticosteroids and the risk of atrial fibrillation. AB - BACKGROUND: High-dose (pulse) corticosteroid therapy has been associated with the development of atrial fibrillation. This association, however, is mainly based on case reports. METHODS: To test the hypothesis that high-dose corticosteroid exposure increases the risk of new-onset atrial fibrillation, we performed a nested case-control study within the Rotterdam Study, a population-based cohort study among 7983 older adults. Cases were defined as persons with incident atrial fibrillation between July 1, 1991, and January 1, 2000. Their date of diagnosis was defined as the index date. All noncases within the Rotterdam Study who were alive and eligible on this index date were used as controls. Subsequently, we compared the proportion of cases and controls that received a corticosteroid prescription within 1 month preceding the index date. Corticosteroid exposure was categorized into high-dose exposure (oral or parenteral steroid at a daily dose > or =7.5 mg of prednisone equivalents) and low-intermediate-dose exposure (<7.5 mg of prednisone equivalents or inhaled corticosteroids). RESULTS: There were 385 eligible cases of new-onset atrial fibrillation during the study period. The risk of new-onset atrial fibrillation was significantly higher for persons who received a corticosteroid prescription within 1 month before the index date than for those without (odds ratio [OR], 3.75; 95% confidence interval [CI], 2.38 5.87). However, only high-dose corticosteroid use was associated with an increased risk (OR, 6.07; 95% CI, 3.90-9.42), whereas low-intermediate-dose use was not (OR, 1.42; 95% CI, 0.72-2.82). The association of atrial fibrillation with high-dose corticosteroid use was largely independent of the indication for corticosteroid therapy, since the risk of new-onset atrial fibrillation was not only increased in patients with asthma or chronic obstructive pulmonary disease (OR, 4.02; 95% CI, 2.07-7.81) but also in patients with rheumatic, allergic, or malignant hematologic diseases (OR, 7.90; 95% CI, 4.47-13.98). CONCLUSION: Our findings strongly suggest that patients receiving high-dose corticosteroid therapy are at increased risk of developing atrial fibrillation. PMID- 16682577 TI - Off-label prescribing among office-based physicians. AB - BACKGROUND: Unlike medicines prescribed for Food and Drug Administration-approved indications, off-label uses may lack rigorous scientific scrutiny. Despite concerns about patient safety and costs to the health care system, little is known about the frequency of off-label drug use or the degree of scientific evidence supporting this practice. METHODS: We used nationally representative data from the 2001 IMS Health National Disease and Therapeutic Index (NDTI) to define prescribing patterns by diagnosis for 160 commonly prescribed drugs. Each reported drug-diagnosis combination was identified as Food and Drug Administration-approved, off-label with strong scientific support, or off-label with limited or no scientific support. Outcome measures included (1) the proportion of uses that were off-label and (2) the proportion of off-label uses supported by strong scientific evidence. Multivariate analyses were used to identify drug-specific characteristics predictive of increased off-label use. RESULTS: In 2001, there were an estimated 150 million (95% confidence interval, 127-173 million) off-label mentions (21% of overall use) among the sampled medications. Off-label use was most common among cardiac medications (46%, excluding antihyperlipidemic and antihypertensive agents) and anticonvulsants (46%), whereas gabapentin (83%) and amitriptyline hydrochloride (81%) had the greatest proportion of off-label use among specific medications. Most off-label drug mentions (73%; 95% confidence interval, 61%-84%) had little or no scientific support. Although several functional classes were associated with increased off label use (P<.05), few other drug characteristics predicted off-label prescription. CONCLUSIONS: Off-label medication use is common in outpatient care, and most occurs without scientific support. Efforts should be made to scrutinize underevaluated off-label prescribing that compromises patient safety or represents wasteful medication use. PMID- 16682578 TI - Unopposed estrogen therapy and the risk of invasive breast cancer. AB - BACKGROUND: Although short-term unopposed estrogen use does not seem to increase breast cancer risk, the effect of longer-term estrogen use remains unclear. We sought to assess the relationship between longer-term use of unopposed estrogen and the risk of invasive breast cancer over an extended follow-up period. METHODS: Within the Nurses' Health Study, a prospective cohort study, we observed 11 508 postmenopausal women who had a hysterectomy and reported information on estrogen use at baseline (1980). The study population was expanded every 2 years to include women who subsequently became postmenopausal and had a hysterectomy, so that 28 835 women were included in the final follow-up period (2000-2002). Estrogen use was assessed from self-reported data on biennial questionnaires. The main outcome was invasive breast cancer. RESULTS: A total of 934 invasive breast cancers were included in the analysis. Breast cancer risk increased with duration of unopposed estrogen use among longer-term users with the highest risk seen in cancers positive for estrogen receptor (ER+) and progesterone receptor (PR+). The multivariate relative risks (RRs) and 95% confidence intervals (CIs) for breast cancer with current use of unopposed estrogen for less than 5 years, 5 to 9.9 years, 10 to 14.9 years, 15 to 19.9 years, and 20 years or longer were, respectively, 0.96 (95% CI, 0.75-1.22), 0.90 (95% CI, 0.73-1.12), 1.06 (95% CI, 0.87-1.30), 1.18 (95% CI, 0.95-1.48), and 1.42 (95% CI, 1.13-1.77) (P for trend <.001). The risk of ER+/PR+ breast cancers was noted to be statistically significant after 15 years of current use (RR, 1.48; 95% CI, 1.05-2.07). CONCLUSION: Users of unopposed estrogen were at increased risk of breast cancer but only after longer-term use. PMID- 16682579 TI - Improving the management of pain in hospitalized adults. AB - BACKGROUND: Pain is a major quality issue. The objective of this study was to evaluate the effectiveness of a series of interventions on pain management. METHODS: This controlled clinical trial (April 1, 2002, to February 28, 2003) involved the staggered implementation of 3 interventions into 2 blocks of matched hospital units. The setting was an 1171-bed hospital. A total of 3964 adults were studied. Interventions included education, standardized pain assessment using a 1 or 4-item (enhanced) pain scale, audit and feedback of pain scores to nursing staff, and a computerized decision support system. The main outcome measures were pain assessment and severity and analgesic prescribing. RESULTS: Units using enhanced pain scales had significantly higher pain assessment rates than units using 1-item pain scales (64% vs 32%; P<.001), audit and feedback of pain results was associated with increases in pain assessment rates compared with units in which audit and feedback was not used (85% vs 64%; P<.001), and the addition of the computerized decision support system was associated with significant increases in pain assessment only when compared with units without audit and feedback (79% vs 64%; P<.001). The enhanced pain scale was associated with significant increases in prescribing of World Health Organization step 2 or 3 analgesic for patients with moderate or severe pain compared with the 1-item scale (83% vs 66%; P=.01). The interventions did not improve pain scores. CONCLUSIONS: A clinically meaningful pain assessment instrument combined with either audit and feedback or a computerized decision support system improved pain documentation to more than 80%. The enhanced pain scale was associated with improved analgesic prescribing. Future interventions should be directed toward altering physician behavior related to titration of opioid analgesics. PMID- 16682580 TI - The role of inflammation for heart disease risk cannot be determined by correlations between C-reactive protein and risk factors. PMID- 16682581 TI - Statins and fracture: are all variables accounted for? PMID- 16682582 TI - Diabetes and cancer screening: a win-win situation or a zero game? PMID- 16682583 TI - Thiazolidinediones and the risk of nontraumatic fractures in patients with diabetes: in reply. PMID- 16682584 TI - Effect of moderate intraocular pressure changes on topographic measurements with confocal scanning laser tomography in patients with glaucoma. AB - OBJECTIVE: To evaluate optic disc topography changes after intraocular pressure (IOP) modulation in patients with glaucoma. METHODS: Twenty-three patients with glaucoma were studied. Three mean optic disc topography images were obtained with the Heidelberg Retina Tomograph II at baseline and weeks 1, 2, 4, and 8 (visits 1, 2, 3, 4, and 5, respectively). Topical medications were discontinued in the study eye after visit 1 and resumed after visit 4 but maintained in the contralateral control eye. Central corneal thickness was measured at the last visit. Topographic changes were determined by stereometric parameters (rim area and mean cup depth) and at discrete topographic locations using the Topographic Change Analysis program (from the Heidelberg Retina Tomograph II). RESULTS: In the study eyes, IOP increased significantly (5.4 mm Hg at visit 4; P<.001) after withdrawal of topical medications but returned to baseline levels after resuming medications; no statistically significant topographic changes, however, were observed. Moreover, no relationship between change in IOP and stereometric parameters was observed. Central corneal thickness was not associated with changes in optic disc topography induced by IOP modulation. CONCLUSION: In patients with glaucoma, significant but relatively moderate IOP increases and decreases on the order of 5 mm Hg did not appear to have an effect on optic disc topography. PMID- 16682585 TI - Triamcinolone as adjunctive treatment to laser panretinal photocoagulation for proliferative diabetic retinopathy. AB - OBJECTIVE: To evaluate intravitreal injection of triamcinolone acetonide before laser panretinal photocoagulation (PRP) in the treatment of proliferative diabetic retinopathy. METHODS: This interventional case series included 9 patients with bilateral proliferative diabetic retinopathy. One eye received intravitreal triamcinolone before PRP (injected eye) and the other, PRP alone (control eye). The main outcome measures were the change in planimetric area of fluorescein leakage from retinal neovascularization and in central macular thickness on optical coherence tomography at 3, 6, 9, and 12 months. Secondary end points were change in vision, intraocular pressure, and cataract progression. RESULTS: All patients completed 9 months and 5 patients, 12 months of follow-up. Initial mean (SD) planimetric area of fluorescein leakage and central macular thickness were 7.22 (5.70) mm(2) and 372.11 (91.88) microm in injected eyes and 9.08 (6.17) mm(2) and 355.33 (115.23) microm in control eyes, respectively. At the 9- and 12-month intervals, the planimetric area of fluorescein leakage decreased by 86% and 88% in injected eyes and 33% and 50% in controls, respectively. Central macular thickness significantly decreased in injected eyes and increased in control eyes. Vision slightly improved in injected eyes and worsened in control eyes. CONCLUSION: Intravitreal injection of triamcinolone before PRP may be useful in improving the effects of PRP in eyes with proliferative diabetic retinopathy by reducing neovascularization and macular thickening. PMID- 16682586 TI - Macular grid photocoagulation after intravitreal triamcinolone acetonide for diffuse diabetic macular edema. AB - OBJECTIVE: To evaluate the clinical outcomes of macular laser photocoagulation after the intravitreal injection of 4 mg of triamcinolone acetonide (IVTA) for diffuse diabetic macular edema (DME). METHODS: Eighty-six eyes of 74 patients with diffuse DME were randomized into 2 groups. The laser group eyes (n = 48) were subjected to a macular grid laser photocoagulation 3 weeks after IVTA. The control group eyes (n = 38) underwent only IVTA. Both groups were compared with regard to the changes in visual acuity and central macular thickness at 3 weeks, 3 months, and 6 months after IVTA. RESULTS: The mean central macular thickness before, 3 weeks after, and 3 and 6 months after IVTA were 538, 250, 295, and 301 mum in the laser group vs 510, 227, 302, and 437 mum in the control group, respectively. The logMAR visual acuities were not significantly different between the 2 groups at baseline and at 3 weeks after IVTA but were significantly better in the laser group at 3 (P = .02) and 6 months (P<.001) after IVTA. CONCLUSIONS: Macular laser coagulation effectively maintains improved visual acuity after IVTA for diffuse DME and is believed to reduce recurrent DME after IVTA. PMID- 16682587 TI - Occult with no classic subfoveal choroidal neovascular lesions in age-related macular degeneration: clinically relevant natural history information in larger lesions with good vision from the Verteporfin in Photodynamic Therapy (VIP) Trial: VIP Report No. 4. AB - OBJECTIVE: To determine whether data from patients with age-related macular degeneration (AMD) assigned to the placebo group in the Verteporfin in Photodynamic Therapy (VIP) Trial provide a rationale for continuation or cessation of follow-up of individuals with subfoveal occult choroidal neovascularization (CNV) with no classic lesions, presumed recent disease progression, larger lesion size (>4 disc areas), and a higher level of visual acuity (approximate Snellen equivalent, > or =20/50 in the affected eye) in whom no treatment is given at initial examination. METHODS: In a prospective, noncomparative case series, angiograms of participants assigned to a placebo group who had occult with no classic lesion composition at baseline were reviewed to identify conversion to minimally classic (area of classic CNV >0% but <50% of the entire lesion area) or predominantly classic (area of classic CNV > or =50% of the entire lesion area) composition. RESULTS: Of the 114 patients with AMD assigned to the placebo group, 89 were judged to have occult with no classic lesion composition at baseline in the study eye when fluorescein angiograms were reviewed in late 2001 for this report. By 24 months, 7 (8%) of the 89 patients had lesions that converted to predominantly classic composition, and 41 (46%) had minimally classic composition. Among the 24 patients with a baseline visual acuity better than 20/50(-1) and lesion size greater than 4 disc areas whose lesions did not convert to predominantly classic composition, the visual acuity of 18 (75%) dropped below 20/50. Six of these 18 continued to have occult with no classic CNV with a visual acuity of 20/100 or better and had a lesion size no greater than 9 disc areas at the time that visual acuity dropped below 20/50. CONCLUSIONS: Continued monitoring, rather than cessation of follow-up, is recommended for patients with occult with no classic lesions, similar to those patients enrolled in the VIP Trial who did not initially receive treatment when they had relatively large lesions with good visual acuity. In these cases, if visual acuity decreases or predominantly classic features develop, photodynamic therapy with verteporfin or pegaptanib sodium injections may be considered. PMID- 16682588 TI - Effect of systemic administration of simvastatin on retinal circulation. AB - OBJECTIVE: To investigate the effect of systemic administration of simvastatin on the retinal circulation. METHODS: The effects of systemic administration of simvastatin on the retinal circulation after 90 minutes and after 7 days were studied in a placebo-controlled, double-masked, clinical trial among 12 healthy men. We used laser Doppler velocimetry to measure vessel diameter and blood velocity and calculated the blood flow in retinal arteries and veins. We also measured the intraocular pressure and the plasma nitrite/nitrate levels, the stable end products of nitric oxide metabolism. RESULTS: There were no significant changes in any retinal circulatory parameters at 90 minutes after administration of simvastatin. Daily administration of simvastatin for 7 days significantly increased blood velocity and blood flow in retinal arteries and veins but did not significantly change vessel diameter. The intraocular pressure significantly decreased at 90 minutes and at 7 days after administration of simvastatin. Simvastatin also significantly increased the plasma nitrite/nitrate levels. CONCLUSION: Simvastatin induced an increase in blood velocity and blood flow in retinal arteries and veins, increased the plasma nitrite/nitrate levels, and decreased the intraocular pressure, probably through the increase in nitric oxide. PMID- 16682589 TI - Visual outcomes after 3-port lens-sparing vitrectomy in stage 4 retinopathy of prematurity. AB - OBJECTIVE: To assess the visual acuity of eyes successfully treated with 3-port lens-sparing vitrectomy for stage 4 retinopathy of prematurity. METHODS: Of 102 consecutive eyes achieving at least posterior pole reattachment, 30 eyes of 26 patients were tested by Teller or Allen acuity measurements and were subsequently converted to logarithm of the minimum angle of resolution (logMAR). Visual outcomes were also examined as either favorable or unfavorable (Snellen equivalent >20/200). RESULTS: Seventy-two eyes were not tested because of either inability to perform testing (age or neurologic sequelae related to prematurity) or loss of follow-up. Of those tested, mean +/- SD logMAR visual acuity for the stage 4A and stage 4B groups was 0.51 +/- 0.09 (Snellen approximate 20/62) and 1.03 +/- 0.19 (Snellen approximate 20/200), respectively (odds ratio, 0.39; 95% confidence interval, 0.24-0.64; P = .001). Of those eyes assessed by Teller measurements, 10 of 10 stage 4A eyes and 3 (37.5%) of 8 stage 4B eyes had favorable outcomes; among eyes assessed with Allen measurements, 4 of 4 stage 4A eyes and 0 of 8 stage 4B eyes had favorable outcomes. CONCLUSIONS: The majority of eyes were not tested. Among eyes tested after successful 3-port lens-sparing vitrectomy, some eyes treated prior to macular detachment may be associated with a more favorable outcome and improved maintenance of functional visual acuity. PMID- 16682590 TI - Multiple mechanisms of extraocular muscle "overaction". AB - OBJECTIVE: To assign more specific pathophysiologic processes to the protean patterns of extraocular muscle "overaction" that we see in clinical practice. METHODS: By extrapolating from known principles of striated muscle physiology, a cohesive theory about extraocular muscle behavior is derived. RESULTS: The key to understanding apparent extraocular muscle overaction is to differentiate between a muscle that has decreased elasticity and one that is strengthened. Primary inferior oblique muscle overaction has the characteristics of a muscle that primarily has decreased elasticity, the superior rectus overaction/contraction syndrome appears to represent a muscle that is strengthened, and inferior oblique overaction secondary to ipsilateral superior oblique palsy has elements of both decreased elasticity and strengthening. Many motility patterns that appear to be due to an overacting muscle may in fact be caused by other muscles than the suspected one. CONCLUSION: Apparent extraocular muscle overaction can be caused by many different factors. PMID- 16682591 TI - Radial optic neurotomy for management of hemicentral retinal vein occlusion. AB - OBJECTIVE: To evaluate the effect of radial optic neurotomy on visual acuity (VA) and foveal thickness in patients with hemicentral retinal vein occlusion. METHODS: A prospective noncomparative case series of 13 eyes in 13 patients with hemicentral retinal vein occlusion and a preoperative VA of 20/60 or less from a total of 232 retinal vein occlusions diagnosed. All patients underwent pars plana vitrectomy, posterior hyaloid dissection, and radial optic neurotomy at the nasal border of the optic disc. RESULTS: Visual acuity and macular thickness were measured with optical coherence tomography. Nine patients (69.2%) gained 2 or more Snellen lines of vision, and in 4 patients (30.8%) VA improved by 4 or more Snellen lines (median visual acuity, 20/50; mean VA, 20/45; P<.01) (average gain, 2.7 Snellen lines). The decrease in foveal thickness was statistically significant (P<.01) (median decrease, 297 microm). Final VA was statistically related to decreased macular thickness at optical coherence tomography (P = .03; rho = -0.62). Retinochoroidal shunts developed in 6 patients (46.1%) at the radial optic neurotomy site. No surgical complications were observed. CONCLUSIONS: Radial optic neurotomy seems to be a potential treatment in selected patients with hemicentral retinal vein occlusion, probably because of the more rapid appearance of retinochorioretinal collateral vessels, which promote faster resolution of macular edema. PMID- 16682592 TI - Optic neuropathy in patients using amiodarone. AB - OBJECTIVE: To refine the criteria for the diagnosis of amiodarone-related optic neuropathy by including a broader spectrum of clinical features, thus helping to differentiate this entity from nonarteritic anterior ischemic optic neuropathy coincidentally affecting a patient taking amiodarone. METHODS: A retrospective case review of 22 patients who developed optic neuropathy while taking amiodarone, in whom other systemic causes were excluded. RESULTS: We identified 3 groups of patients: those in whom a diagnosis of amiodarone-induced optic neuropathy seems probable (n = 14), those in whom an association with amiodarone optic neuropathy is indeterminate (n = 5), and those in whom the occurrence of nonarteritic anterior ischemic optic neuropathy seems to be coincidental (ie, unrelated to amiodarone) (n = 3). We formulated specific diagnostic criteria for each of these categories. CONCLUSIONS: We recommend a systematic approach that includes assessment of bilaterality, mode of onset, degree of optic nerve dysfunction, structure of the uninvolved optic disc in unilateral cases, and systemic toxic effects. Such well-defined diagnostic criteria can help the clinician in the treatment of patients with this disorder. PMID- 16682593 TI - Basement membrane and collagen deposition after laser subepithelial keratomileusis and photorefractive keratectomy in the leghorn chick eye. AB - OBJECTIVE: To evaluate corneal scar formation and new collagen deposition after laser subepithelial keratomileusis (LASEK) compared with photorefractive keratectomy (PRK) in the leghorn chick corneal model. METHODS: Leghorn chick corneas treated with LASEK surgery (using 20% ethanol for 30 seconds) or PRK were evaluated by indirect confocal immunofluorescence and transmission electron microscopy at 1, 2, 7, 14, and 28 days after surgery. New collagen deposition was determined by dichlorotriazinylaminofluorescein staining 2 and 4 weeks after surgery. RESULTS: Laminin was detected around the basal layers during the immediate postoperative period and 4 weeks after LASEK surgery, and from day 2 onwards after PRK. Collagen III deposition in the cornea was about 3 times greater with PRK than with LASEK. The thickness of new collagen deposition at 4 weeks was 34 microm +/- 2.5 microm in the PRK group, which was significantly greater than that of the LASEK group (11 microm +/- 1 microm; P<.001). CONCLUSIONS: Reduced subepithelial stromal tissue deposition was observed in LASEK-treated eyes compared with PRK-treated eyes. Postoperative preservation of the epithelial basement membrane and survival of epithelial cells in LASEK and possibly in epithelial laser in situ keratomileusis may contribute to this phenomenon. CLINICAL RELEVANCE: An advantage of LASEK over PRK is the reduction of postoperative haze. PMID- 16682594 TI - Chemokine receptor CCR5 expression in conjunctival epithelium of patients with dry eye syndrome. AB - OBJECTIVE: To characterize chemokine receptor CCR5 expression on the conjunctival epithelium in dry eye syndromes. METHODS: Conjunctival impression cytology samples were obtained from normal subjects (n = 15) and patients with dry eye syndrome (n = 45). Cells were harvested from impression cytology samples, and flow cytometry was performed to quantitatively analyze the cell surface expression of chemokine receptor CCR5. Characterization of CCR5-positive cells was done by 2-color flow cytometry using fluorescein-conjugated anti-CCR5 and phycoerythrin-conjugated anti-CD45 antibodies (where CD45 is a marker for bone marrow-derived cells). To study CCR5 messenger RNA transcripts, real-time polymerase chain reaction was done on RNA isolated from the impression cytology samples of normal subjects (n = 5) and patients with dry eye syndrome (n = 14). RESULTS: We found significant up-regulation in cell surface expression of CCR5 in patients with both aqueous tear-deficient and evaporative forms of dry eye syndrome (P<.001). The real-time polymerase chain reaction results (for messenger RNA) corroborated the flow cytometry data (for protein). The majority of the cells expressing CCR5 were non-bone marrow-derived resident epithelial cells of the conjunctiva. CONCLUSION: Our findings suggest that CCR5 up-regulation is significantly associated with dry eye syndrome-associated ocular surface disease. Clinical Relevance Chemokine receptor CCR5 or its ligands may serve as useful targets for modulation of tissue immunoinflammatory responses in dry eye syndromes. PMID- 16682595 TI - Endothelin B receptor in human glaucoma and experimentally induced optic nerve damage. AB - OBJECTIVE: To assess endothelin B receptor (ETbR) expression in human glaucomatous optic nerves and the spatial relationship between ETbR and astrocytes. METHODS: Twenty-six eyes from 16 glaucoma patients and 10 normal control subjects were immunohistochemically labeled with antibodies to ETbR. The immunoreactivity was quantified and compared between normal and glaucomatous eyes with an image analysis system. Tissues were also double-labeled for ETbR and astrocytes. In addition, the optic nerve of a monkey with regional degeneration induced by laser coagulation was examined with the same techniques. RESULTS: The frequency of positive ETbR immunoreactivity was higher in human glaucomatous optic nerves as compared with age-matched controls (9/16 vs 1/10, P = .02). The ETbR immunoreactivity colocalized with astrocytic processes and was quantitatively higher in the glaucomatous eyes (P = .02). In the monkey, the regions of degeneration showed increased ETbR associated with reactive astrocytes and was highest at the borders between normal areas and degeneration. CONCLUSION: Increased ETbR immunoreactivity in diseased optic nerves and its association with astrocytes suggest that the glia-endothelin system may be involved in the pathologic mechanisms of neuronal degeneration. Clinical Relevance The study supports the clinical observation of endothelin involvement in glaucoma and provides direct evidence that the endothelin system is associated with glaucomatous pathologic abnormalities. PMID- 16682596 TI - Ten-year incidence of retinal vein occlusion in an older population: the Blue Mountains Eye Study. AB - OBJECTIVE: To assess the 10-year incidence of retinal vein occlusion (RVO) and its predictors in an older population. METHODS: The Blue Mountains Eye Study examined 3654 residents aged 49 years and older (82.4% response) from 1992 to 1994, reexamined 2335 residents (75.1% of survivors) from 1997 to 1999, and reexamined 1952 residents (75.6% of survivors) from 2002 to 2004. Incident RVO was assessed from stereoscopic retinal photographs. Kaplan-Meier cumulative 10 year incidence was calculated. RESULTS: After excluding 47 residents with RVO at baseline and 171 residents with no photographs at either follow-up examination, 2346 residents were considered at risk of developing RVO. The cumulative 10-year incidence of RVO was 1.6%. Age was significantly associated with the incidence of RVO (P = .03, Mantel-Haenszel chi(2) test for trend). Factors predicting the incidence of RVO included mean arterial blood pressure (age-adjusted odds ratio [OR], 1.41 per 10-mm Hg increase), ocular perfusion pressure (OR, 1.71 per 10-mm Hg increase), obesity (OR, 2.16), and presence of retinal arteriolar wall signs (focal narrowing: OR, 3.37; arteriovenous nicking: OR, 4.09; and opacification: OR, 4.89). CONCLUSIONS: Older age (> or =70 years), increasing mean arterial blood pressure, and atherosclerotic retinal vessel signs were significant predictors of incident RVO. PMID- 16682597 TI - Nonarteritic anterior ischemic optic neuropathy and sildenafil. PMID- 16682598 TI - Report of a conjunctival myxoma case and review of the literature. PMID- 16682599 TI - Corneal decompensation following bleb revision with absolute alcohol: clinical pathological correlation. PMID- 16682600 TI - Verteporfin photodynamic therapy of choroidal neovascularization secondary to ocular toxoplasmosis. PMID- 16682601 TI - Visual impairment and deafness in young children: consider the diagnosis of congenital infection with cytomegalovirus, even years after birth. PMID- 16682602 TI - Case of Stargardt disease caused by uniparental isodisomy. PMID- 16682603 TI - Optical coherence tomography demonstration of macular infarction in sickle cell retinopathy. PMID- 16682604 TI - Necrobiotic xanthogranuloma with posterior scleritis. PMID- 16682605 TI - Phakic status affects vitreous penetration of topical moxifloxacin. PMID- 16682606 TI - NO break-ins at water gate. PMID- 16682607 TI - Aquaporin-1 transports NO across cell membranes. AB - NO plays a role in the regulation of blood pressure through its effects on renal, cardiovascular, and central nervous system function. It is generally thought to freely diffuse through cell membranes without need for a specific transporter. The water channel aquaporin-1 transports low molecular weight gases in addition to water and is expressed in cells that produce or are the targets of NO. Consequently, we tested the hypothesis that aquaporin-1 transports NO. In cells expressing aquaporin-1, NO permeability correlated with water permeability. NO transport was reduced by 71% by HgCl2, an inhibitor of aquaporin-1. Transport of NO by aquaporin-1 saturated at 3 micromol/L NO and displayed a K(1/2) (the concentration of NO that produces half of the maximum transport rate) of 0.54 micromol/L. Reconstitution of purified aquaporin-1 into lipid vesicles increased NO influx by 316%. In endothelial cells, lowering aquaporin-1 expression with a small interfering RNA (siRNA) blunted aquaporin-1 expression by 54% and NO release by 44%. We conclude that NO transport by aquaporin-1 may allow cells to control intracellular NO levels and effects. NO transport by aquaporin-1 may play a role in central nervous system, vascular and renal function, and consequently blood pressure. Disruption of NO transport by aquaporin-1 offers an alternate cause for diseases currently explained by inadequate NO bioavailability. PMID- 16682608 TI - Nephron number, uric acid, and renal microvascular disease in the pathogenesis of essential hypertension. PMID- 16682609 TI - Effects of low birth weight in 8- to 13-year-old children: implications in endothelial function and uric acid levels. AB - Low birth weight has been associated with an increased incidence of adult cardiovascular disease. Endothelial dysfunction and high levels of serum uric acid are associated with hypertension. In this study, we have determined whether uric acid is related to blood pressure and vascular function in children with low birth weight. We evaluated vascular function using high-resolution ultrasound, blood pressure, and uric acid levels in 78 children (35 girls, 43 boys, aged 8 to 13 years). Increasing levels of uric acid and systolic blood pressure were observed in children with low birth weight. Birth weight was inversely associated with both systolic blood pressure and uric acid; on the other hand, uric acid levels were directly correlated with systolic blood pressure in children of the entire cohort. Low birth weight was associated with reduced flow-mediated dilation (r=0.427, P<0.001). Because the children with low birth weight had elevated uric acid as well as higher systolic blood pressure levels, we evaluated the correlation between these variables. In the low birth weight group, multiple regression analysis revealed that uric acid (beta=-2.886; SE=1.393; P=0.040) had a graded inverse relationship with flow-mediated dilation, which was not affected in a model adjusting for race and gender. We conclude that children with a history of low birth weight show impaired endothelial function and increased blood pressure and uric acid levels. These findings may be early expressions of vascular compromise, contributing to susceptibility to disease in adult life. PMID- 16682610 TI - Treating hypertension in acute stroke: a better arrow for the quiver. PMID- 16682611 TI - Transdermal glyceryl trinitrate lowers blood pressure and maintains cerebral blood flow in recent stroke. AB - High blood pressure (BP) is common in acute stroke and is independently associated with a poor outcome. Lowering BP might improve outcome if it did not adversely affect cerebral blood flow (CBF) or cerebral perfusion pressure. We investigated the effect of glyceryl trinitrate ([GTN] an NO donor) on quantitative CBF, BP, and cerebral perfusion pressure in patients with recent stroke. Eighteen patients with recent (<5 days) ischemic (n=16) or hemorrhagic (n=2) stroke were randomly assigned (2:1) to transdermal GTN (5 mg) or control. CBF (global, hemispheric, arterial territory, and lesion, using xenon computed tomography) and BP (peripheral and central) were measured before and 1 hour after treatment with GTN. The effects of GTN on CBF and BP were adjusted for baseline measurements (ANCOVA). GTN lowered peripheral systolic BP by (mean) 23 mm Hg (95% CI, 2 to 45; P=0.03) and central systolic BP by 22 mm Hg (95% CI, 0 to 44; P=0.048). In contrast, GTN did not alter CBF (mL/min per 100 g): global -1.2 (95% CI, -6.5 to 4.2; P=0.66) and ipsilateral hemisphere -1.4 (95% CI, -7.6 to 4.9; P=0.65) or area of stroke oligemia, penumbra, or core (as defined by critical CBF limits). Contralateral CBF did not change: hemisphere 0 (95% CI, -7 to 6; P=0.96). GTN did not alter cerebral perfusion pressure or zero-filling pressure. Significant reductions in BP after transdermal GTN are not associated with changes in CBF or cerebral perfusion pressure or cerebral steal in patients with recent stroke. Trials need to assess the effect of lowering BP on functional outcome. PMID- 16682612 TI - Platelet expression profiling and clinical validation of myeloid-related protein 14 as a novel determinant of cardiovascular events. AB - BACKGROUND: Platelets participate in events that immediately precede acute myocardial infarction. Because platelets lack nuclear DNA but retain megakaryocyte-derived mRNAs, the platelet transcriptome provides a novel window on gene expression preceding acute coronary events. METHODS AND RESULTS: We profiled platelet mRNA from patients with acute ST-segment-elevation myocardial infarction (STEMI, n=16) or stable coronary artery disease (n=44). The platelet transcriptomes were analyzed and single-gene models constructed to identify candidate genes with differential expression. We validated 1 candidate gene product by performing a prospective, nested case-control study (n=255 case control pairs) among apparently healthy women to assess the risk of future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) associated with baseline plasma levels of the candidate protein. Platelets isolated from STEMI and coronary artery disease patients contained 54 differentially expressed transcripts. The strongest discriminators of STEMI in the microarrays were CD69 (odds ratio 6.2, P<0.001) and myeloid related protein-14 (MRP-14; odds ratio 3.3, P=0.002). Plasma levels of MRP-8/14 heterodimer were higher in STEMI patients (17.0 versus 8.0 microg/mL, P<0.001). In the validation study, the risk of a first cardiovascular event increased with each increasing quartile of MRP-8/14 (Ptrend<0.001) such that women with the highest levels had a 3.8-fold increase in risk of any vascular event (P<0.001). Risks were independent of standard risk factors and C-reactive protein. CONCLUSIONS: The platelet transcriptome reveals quantitative differences between acute and stable coronary artery disease. MRP-14 expression increases before STEMI, and increasing plasma concentrations of MRP-8/14 among healthy individuals predict the risk of future cardiovascular events. PMID- 16682613 TI - Chemokine CXCL10 promotes atherogenesis by modulating the local balance of effector and regulatory T cells. AB - BACKGROUND: Studies to define the overall contribution of lymphocytes to lesion formation in atherosclerosis-susceptible mice have demonstrated relatively subtle effects; the use of lymphocyte-deficient mice, however, compromises both the effector and regulatory arms of the immune system. Here, we tested the hypothesis that deletion of CXCL10 (IP-10), a chemokine specific for effector T cells that has been localized within atherosclerotic lesions, would significantly inhibit atherogenesis. METHODS AND RESULTS: Compound deficient Apoe(-/-)/Cxcl10(-/-) mice fed a Western-style diet for either 6 or 12 weeks demonstrated significant reductions in atherogenesis as compared with Apoe(-/-) controls, as assessed by both aortic en face and cross-sectional analyses. Immunohistochemical studies revealed a decrease in the accumulation of CD4+ T cells, whereas quantitative polymerase chain reaction analysis of lesion-rich aortic arches demonstrated a marked reduction in mRNA for CXCR3, the CXCL10 chemokine receptor. Although overall T-cell accumulation was diminished significantly, we found evidence to suggest that regulatory T-cell (Treg) numbers and activity were enhanced, as assessed by increased message for the Treg-specific marker Foxp3, as well as increases in immunostaining for the Treg-associated cytokines interleukin-10 and transforming growth factor-beta1. We also documented naturally occurring Treg cells in human atherosclerotic lesions. CONCLUSIONS: We provide novel evidence for a functional role for the effector T-cell chemoattractant CXCL10 in atherosclerotic lesion formation by modulating the local balance of the effector and regulatory arms of the immune system. PMID- 16682614 TI - Predictive factors of restenosis after coronary implantation of sirolimus- or paclitaxel-eluting stents. AB - BACKGROUND: The efficacy of drug-eluting stents in reducing restenosis risk has not been uniform across patient subsets. Identifying predictive factors of restenosis may help improve outcomes after percutaneous coronary interventions. METHODS AND RESULTS: All patients who underwent successful implantation of sirolimus- or paclitaxel-eluting stents in native vessels for de novo lesions between August 2002 and December 2004 were eligible for this study. All data were prospectively collected. Angiographic restenosis was defined as diameter stenosis > or =50% at follow-up in the in-segment area. Target lesion revascularization was defined as any revascularization procedure involving the target lesion. Included in this study were 1845 patients with 2093 target lesions. Multivariable analysis showed that vessel size, final diameter stenosis, and drug-eluting stent type were the strongest predictors of restenosis. A 0.5-mm decrease in vessel size was associated with adjusted odds ratios (ORs) of 1.74 (95% CI, 1.31 to 2.32) for angiographic restenosis and 1.65 (95% CI, 1.22 to 2.23) for target lesion revascularization. A 5% increase in final diameter stenosis was associated with adjusted ORs of 1.30 (95% CI, 1.15 to 1.47) for angiographic restenosis and 1.18 (95% CI, 1.03 to 1.35) for target lesion revascularization. Compared with paclitaxel-eluting stent, sirolimus-eluting stent was associated with adjusted ORs of 0.60 (95% CI, 0.44 to 0.81) for angiographic restenosis and 0.67 (95% CI, 0.49 to 0.91) for target lesion revascularization. CONCLUSIONS: Vessel size and drug-eluting stent type are the most important predictors of angiographic and clinical restenosis, with drug-eluting stent type having a particular impact on restenosis of small coronary vessels. PMID- 16682615 TI - Percutaneous septal sinus shortening: a novel procedure for the treatment of functional mitral regurgitation. AB - BACKGROUND: The septal-to-lateral (SL) mitral annular diameter is increased in functional mitral regurgitation (MR). We describe a novel percutaneous technique (the percutaneous septal sinus shortening system) that ameliorates functional MR in an ovine model. METHODS AND RESULTS: Sheep underwent rapid right ventricular pacing to obtain moderate to severe functional MR with SL enlargement. The percutaneous septal sinus shortening system was placed via standard interventional techniques consisting of a bridge (suture) element between interatrial septal wall and great cardiac vein anchors. Through progressive tensioning of the bridge element, direct SL shortening was achieved. Sheep underwent short-term (n=19) and long-term (n=4) evaluation after device implantation. In short-term studies, SL diameter decreased an average of 24% (32.5+/-3.5 to 24.6+/-2.4 mm; P<0.001), and MR grade significantly improved (2.1+/-0.6 to 0.4+/-0.4; P<0.001). Despite continued rapid pacing, chronic device implantation resulted in durable SL shortening (30.4+/-1.9 mm before implantation to 25.3+/-0.8 mm at 30 days; P=0.01) and MR reduction (1.8+/-0.5 before implantation to 0.2+/-0.1 at 30 days; P=0.01). Increased cardiac output, decreased wedge pressure, and decreased brain natriuretic peptide levels were observed in animals undergoing long-term device implantation. CONCLUSIONS: The percutaneous septal sinus shortening system is effective in ameliorating functional MR in an ovine tachycardia model. The procedure, which uses standard catheter techniques, can be deployed largely under fluoroscopic guidance. The unique bridge element appears durable and allows direct and precise SL shortening to a diameter optimal for MR reduction. PMID- 16682616 TI - Secular trends in deaths from cardiovascular diseases: a 25-year community study. AB - BACKGROUND: Although age-adjusted cardiovascular disease (CVD) mortality has declined over the past decades, controversies remain about whether this trend was similar across locations of death and disease categories and about the existence of age and sex disparities. METHODS AND RESULTS: We examined CVD mortality trends in Olmsted County, Minnesota, between 1979 and 2003 using the categories defined by the American Heart Association, including coronary heart disease (CHD), non CHD diseases of the heart, and noncardiac circulatory diseases. Data on demographics, cause, and location of death of all 6378 residents who died of CVD were analyzed. Although decreases in the age-adjusted rates occurred in all groups, the magnitude of the decline varied widely. Lesser annual declines were noted in out-of-hospital than in-hospital deaths (1.8% versus 4.8%; P<0.001), in older than in younger persons (1.5% at age > or =85 years versus 3.9% for those < or =74 years of age; P<0.001), and in women relative to men (2.5% versus 3.3%; P=0.007). Furthermore, although CHD showed a marked annual decrease (3.3%), more modest decrements were found for non-CHD diseases of the heart (2.1%) and noncardiac circulatory diseases (2.4%) (P=0.02 and P=0.04 for the comparison with CHD decline, respectively). CONCLUSIONS: Over the past 25 years, CVD mortality declined markedly in the community, but there were large disparities in the magnitude of the decline, resulting in a shift in the distribution toward out-of hospital and non-CHD deaths. Further reduction in CVD mortality will require strategies directed at elderly persons and women, in whom out-of-hospital rates have improved only minimally. PMID- 16682617 TI - Modulation of CD4(+)CD28null T lymphocytes by tumor necrosis factor-alpha blockade in patients with unstable angina. AB - BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that favors the expansion of CD4(+)CD28null T cells, an aggressive and unusual proinflammatory lymphocyte subset frequently observed in patients with unstable angina (UA). The purpose of the present ex vivo study was to evaluate whether inflammation in patients with UA may be modulated by selective blockade of TNF alpha. METHODS AND RESULTS: Peripheral blood samples were collected from 17 patients with UA (Braunwald's class IIIB). CD4(+)CD28null T cells were assessed by flow cytometry and expressed as a percentage of all CD4+ T cells after 24 hours of incubation of whole blood with and without increasing doses (0.1, 1, 10, and 100 microg/mL) of infliximab, an anti-TNF-alpha monoclonal antibody. In addition, CD28 expression was assessed and expressed as mean fluorescence intensity (geometric mean of the CD28 fluorescence value on all CD4+ T cells). CD4(+)CD28null T-cell percentage decreased from a median of 6.2% (range, 1.2% to 23.9%) to 4.9% (range, 1.1% to 21.9%), 4.5% (range, 1.1% to 21.6%), and 4.1% (range, 0.4% to 21.4%) after incubation with 1, 10, and 100 microg/mL of infliximab (P for trend=0.043). Analysis of CD28 mean fluorescence intensity showed that the expression of CD28 on cell surface significantly increased after incubation with increasing doses of infliximab (P for trend=0.03). CONCLUSIONS: The findings of this ex vivo study show that CD4(+)CD28null T-cell expansion in patients with UA may be reduced by selective TNF-alpha blockade. Further studies are warranted to evaluate the clinical benefit of CD4(+)CD28null T-cell modulation. PMID- 16682618 TI - Multiorgan engraftment and differentiation of human cord blood CD34+ Lin- cells in goats assessed by gene expression profiling. AB - To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+ Lin- cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45-55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2-36% of all cells examined). We identified human beta2 microglobulin positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions. PMID- 16682619 TI - Highly conserved proteins that modify plant ethylene responses. PMID- 16682620 TI - A common allosteric site and mechanism in caspases. AB - We present a common allosteric mechanism for control of inflammatory and apoptotic caspases. Highly specific thiol-containing inhibitors of the human inflammatory caspase-1 were identified by using disulfide trapping, a method for site-directed small-molecule discovery. These compounds became trapped by forming a disulfide bond with a cysteine residue in the cavity at the dimer interface approximately 15 A away from the active site. Mutational and structural analysis uncovered a linear circuit of functional residues that runs from one active site through the allosteric cavity and into the second active site. Kinetic analysis revealed robust positive cooperativity not seen in other endopeptidases. Recently, disulfide trapping identified a similar small-molecule site and allosteric transition in the apoptotic caspase-7 that shares only a 23% sequence identity with caspase-1. Together, these studies show a general small-molecule binding site for functionally reversing the zymogen activation of caspases and suggest a common regulatory site for the allosteric control of inflammation and apoptosis. PMID- 16682621 TI - Progression of prostate cancer by synergy of AKT with genotropic and nongenotropic actions of the androgen receptor. AB - Classic work by Huggins and Hodges demonstrated that human prostate cancer regresses dramatically during antihormonal therapy but recurs frequently with androgen independence. Perturbations in the androgen receptor (AR) and PTEN-AKT signaling axes are significantly correlated with the progression of prostate cancer. Genetic alterations of the AR cause receptor hypersensitivity, promiscuity, and androgen-independent receptor transactivation. Prostate cancers maintain an elevated AKT activity through the loss of PTEN function or the establishment of autocrine signaling by growth factors and cytokines. We used an in vivo prostate regeneration system to investigate the biological potency of the potential crosstalk between these two signal transduction pathways. We demonstrate a direct synergy between AKT and AR signaling that is sufficient to initiate and progress naive adult murine prostatic epithelium to frank carcinoma and override the effect of androgen ablation. Both genotropic and nongenotropic signals mediated by AR are essential for this synergistic effect. However, phosphorylation of AR by AKT at Ser-213 and Ser-791 is not critical for this synergy. These results suggest that more efficient therapeutics for advanced prostate cancer may need to target simultaneously AR signaling and AKT or the growth factor receptor tyrosine kinases that activate AKT. PMID- 16682622 TI - A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer. AB - The development of acquired resistance to ErbB2 tyrosine kinase inhibitors limits the clinical efficacy of this class of cancer therapeutics. Little is known about the mechanism(s) of acquired resistance to these agents. Here we establish a model of acquired resistance to N-{3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5 ({[2 (methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (lapatinib), an inhibitor of ErbB2 and ErbB1 tyrosine kinases by chronically exposing lapatinib-sensitive ErbB2-overexpressing breast cancer cells to lapatinib, simulating the clinic where lapatinib is administered on a daily chronic basis. Analysis of baseline gene expression in acquired lapatinib-resistant and parental cells indicates estrogen receptor (ER) signaling involvement in the development of resistance. Using gene interference, we confirm that acquired resistance to lapatinib is mediated by a switch in cell survival dependence and regulation of a key antiapoptotic mediator from ErbB2 alone to codependence upon ER and ErbB2 rather than loss of ErbB2 expression or insensitivity of ErbB2 signaling to lapatinib. Increased ER signaling in response to lapatinib is enhanced by the activation of factors facilitating the transcriptional activity of ER, notably FOXO3a and caveolin-1. Importantly, we confirm that lapatinib induces ER signaling in tumor biopsies from patients with ErbB2-overexpressing breast cancers receiving lapatinib therapy. These findings provided the rationale for preventing the development of acquired resistance by simultaneously inhibiting both ER and ErbB2 signaling pathways. Establishing clinically relevant models of acquired resistance to ErbB2 kinase inhibitors will enhance therapeutic strategies to improve clinical outcomes for patients with ErbB2-overexpressing breast cancers. PMID- 16682624 TI - The real estate of cardiac signaling: location, location, location. PMID- 16682623 TI - Neurons produce type I interferon during viral encephalitis. AB - Type I interferons, also referred to as IFN-alpha/beta, form the first line of defense against viral infections. Major IFN-alpha/beta producers in the periphery are the plasmacytoid dendritic cells (pDCs). Constitutive expression of the IFN regulatory factor (IRF)-7 enables pDCs to rapidly synthesize large amounts of IFN alpha/beta after viral infection. In the central nervous system (CNS), pDCs are considered to be absent from the parenchyma, and little is known about the cells producing IFN-alpha/beta. The study presented here aimed to identify the cells producing IFN-alpha/beta in the CNS in vivo after infection by neurotropic viruses such as Theiler's virus and La Crosse virus. No cells with high constitutive expression of IRF-7 were detected in the CNS of uninfected mice, suggesting the absence of cells equivalent to pDCs. Upon viral infection, IFN beta and some subtypes of IFN-alpha, but not IFN-epsilon or IFN-kappa, were transcriptionally up-regulated. IFN-alpha/beta was predominantly produced by scattered parenchymal cells and much less by cells of inflammatory foci. Interestingly, in addition to some macrophages and ependymal cells, neurons turned out to be important producers of both IFN-alpha and IFN-beta. However, only 3% of the infected neurons produced IFN-alpha/beta, suggesting that some restriction to IFN-alpha/beta production existed in these cells. All CNS cell types analyzed, including neurons, were able to respond to type I IFN by producing Mx or IRF-7. Our data show that, in vivo, neurons take an active part to the antiviral defense by being both IFN-alpha/beta producers and responders. PMID- 16682625 TI - Covalent chemistry on distended proteins. PMID- 16682626 TI - Stable time filtering of strongly unstable spatially extended systems. AB - Many contemporary problems in science involve making predictions based on partial observation of extremely complicated spatially extended systems with many degrees of freedom and with physical instabilities on both large and small scale. Various new ensemble filtering strategies have been developed recently for these applications, and new mathematical issues arise. Because ensembles are extremely expensive to generate, one such issue is whether it is possible under appropriate circumstances to take long time steps in an explicit difference scheme and violate the classical Courant-Friedrichs-Lewy (CFL)-stability condition yet obtain stable accurate filtering by using the observations. These issues are explored here both through elementary mathematical theory, which provides simple guidelines, and the detailed study of a prototype model. The prototype model involves an unstable finite difference scheme for a convection-diffusion equation, and it is demonstrated below that appropriate observations can result in stable accurate filtering of this strongly unstable spatially extended system. PMID- 16682627 TI - Profile of David W. McLaughlin. PMID- 16682628 TI - Corticotropin-releasing hormone induces skin vascular permeability through a neurotensin-dependent process. AB - Many skin disorders are associated with increased numbers of activated mast cells and are worsened by stress; however, the mechanism underlying these processes is not understood. Corticotropin-releasing hormone (CRH) is secreted under stress from the hypothalamus, but also in the skin, where it induces mast cell activation and vascular permeability. We investigated the effect of CRH in a number of animal models by using i.v. Evans blue extravasation as a marker of vascular permeability. Intradermal CRH is among the most potent peptides at 100 nM, its effect being nearly comparable to that of neurotensin (NT). Pretreatment of skin injection sites with the NT receptor antagonist SR48692 blocks CRH induced vascular permeability, which is diminished in NT-/- mice, implying that NT is necessary for the effect of CRH. CRH and NT precursor mRNA are shown to be expressed in both dorsal root ganglia and skin, whereas the latter also expresses mRNA for prohormone convertase 5, an enzyme that cleaves pro-NT into its active form. We also show that the effect of both CRH and NT is absent in W/W(v) mast cell-deficient mice; however, only a fraction of skin mast cells express CRH receptors, as shown by FACS analysis of CRH receptor (CRHR) and c-kit double positive disaggregated mouse skin mast cells. These findings suggest that CRH induces skin vascular permeability through NT acting on mast cells and that both peptides should be considered in the pathogenesis of skin disorders exacerbated by stress. PMID- 16682629 TI - Medicare prescription drug coverage: consumer information and preferences. AB - We investigate prescription drug use, and information and enrollment intentions for the new Medicare Part D drug insurance program, using a sample of Medicare eligible subjects surveyed before open enrollment began for this program. We find that, despite the complexity of competing plans offered by private insurers under Part D, a majority of the Medicare population had information on this program and a substantial majority planned to enroll. We find that virtually all elderly, even those with no current prescription drug use, can expect to benefit from enrollment in a Part D Standard plan at the low premiums available in the current market. However, there is a significant risk that many eligible seniors, particularly low-income elderly with poor health or cognitive impairment, will make poor enrollment and plan choices. PMID- 16682630 TI - The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences. AB - We investigated whether genes escape X chromosome inactivation by positioning outside of the territory defined by XIST RNA. Results reveal an unanticipated higher order organization of genes and noncoding sequences. All 15 X-linked genes, regardless of activity, position on the border of the XIST RNA territory, which resides outside of the DAPI-dense Barr body. Although more strictly delineated on the inactive X chromosome (Xi), all genes localized predominantly to the outer rim of the Xi and active X chromosome. This outer rim is decorated only by X chromosome DNA paints and is excluded from both the XIST RNA and dense DAPI staining. The only DNA found well within the Barr body and XIST RNA territory was centromeric and Cot-1 DNA; hence, the core of the X chromosome essentially excludes genes and is composed primarily of noncoding repeat-rich DNA. Moreover, we show that this core of repetitive sequences is expressed throughout the nucleus yet is silenced throughout Xi, providing direct evidence for chromosome-wide regulation of "junk" DNA transcription. Collective results suggest that the Barr body, long presumed to be the physical manifestation of silenced genes, is in fact composed of a core of silenced noncoding DNA. Instead of acting at a local gene level, XIST RNA appears to interact with and silence core architectural elements to effectively condense and shut down the Xi. PMID- 16682631 TI - V1a vasopressin receptors maintain normal blood pressure by regulating circulating blood volume and baroreflex sensitivity. AB - Arginine-vasopressin (AVP) is a hormone that is essential for both osmotic and cardiovascular homeostasis, and exerts important physiological regulation through three distinct receptors, V1a, V1b, and V2. Although AVP is used clinically as a potent vasoconstrictor (V1a receptor-mediated) in patients with circulatory shock, the physiological role of vasopressin V1a receptors in blood pressure (BP) homeostasis is ill-defined. In this study, we investigated the functional roles of the V1a receptor in cardiovascular homeostasis using gene targeting. The basal BP of conscious mutant mice lacking the V1a receptor gene (V1a-/-) was significantly (P < 0.001) lower compared to the wild-type mice (V1a+/+) without a notable change in heart rate. There was no significant alteration in cardiac functions as assessed by echocardiogram in the mutant mice. AVP-induced vasopressor responses were abolished in the mutant mice; rather, AVP caused a decrease in BP, which occurred in part through V2 receptor-mediated release of nitric oxide from the vascular endothelium. Arterial baroreceptor reflexes were markedly impaired in mutant mice, consistent with a loss of V1a receptors in the central area of baroreflex control. Notably, mutant mice showed a significant 9% reduction in circulating blood volume. Furthermore, mutant mice had normal plasma AVP levels and a normal AVP secretory response, but had significantly lower adrenocortical responsiveness to adrenocorticotropic hormone. Taken together, these results indicate that the V1a receptor plays an important role in normal resting arterial BP regulation mainly by its regulation of circulating blood volume and baroreflex sensitivity. PMID- 16682633 TI - A likelihood approach to analysis of network data. AB - Biological, sociological, and technological network data are often analyzed by using simple summary statistics, such as the observed degree distribution, and nonparametric bootstrap procedures to provide an adequate null distribution for testing hypotheses about the network. In this article we present a full likelihood approach that allows us to estimate parameters for general models of network growth that can be expressed in terms of recursion relations. To handle larger networks we have developed an importance sampling scheme that allows us to approximate the likelihood and draw inference about the network and how it has been generated, estimate the parameters in the model, and perform parametric bootstrap analysis of network data. We illustrate the power of this approach by estimating growth parameters for the Caenorhabditis elegans protein interaction network. PMID- 16682632 TI - Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers. AB - Mu opioid receptors are critical for heroin dependence, and A118G SNP of the mu opioid receptor gene (OPRM1) has been linked with heroin abuse. In our population of European Caucasians (n = 118), approximately 90% of 118G allelic carriers were heroin users. Postmortem brain analyses showed the OPRM1 genotype associated with transcription, translation, and processing of the human striatal opioid neuropeptide system. Whereas down-regulation of preproenkephalin and preprodynorphin genes was evident in all heroin users, the effects were exaggerated in 118G subjects and were most prominent for preproenkephalin in the nucleus accumbens shell. Reduced opioid neuropeptide transcription was accompanied by increased dynorphin and enkephalin peptide concentrations exclusively in 118G heroin subjects, suggesting that the peptide processing is associated with the OPRM1 genotype. Abnormal gene expression related to peptide convertase and ubiquitin/proteosome regulation was also evident in heroin users. Taken together, alterations in opioid neuropeptide systems might underlie enhanced opiate abuse vulnerability apparent in 118G individuals. PMID- 16682634 TI - The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. AB - NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects. PMID- 16682635 TI - Loss of Siglec expression on T lymphocytes during human evolution. AB - We report here that human T cells give much stronger proliferative responses to specific activation via the T cell receptor (TCR) than those from chimpanzees, our closest evolutionary relatives. Nonspecific activation using phytohemagglutinin was robust in chimpanzee T cells, indicating that the much lower response to TCR simulation is not due to any intrinsic inability to respond to an activating stimulus. CD33-related Siglecs are inhibitory signaling molecules expressed on most immune cells and are thought to down-regulate cellular activation pathways via cytosolic immunoreceptor tyrosine-based inhibitory motifs. Among human immune cells, T lymphocytes are a striking exception, expressing little to none of these molecules. In stark contrast, we find that T lymphocytes from chimpanzees as well as the other closely related "great apes" (bonobos, gorillas, and orangutans) express several CD33-related Siglecs on their surfaces. Thus, human-specific loss of T cell Siglec expression occurred after our last common ancestor with great apes, potentially resulting in an evolutionary difference with regard to inhibitory signaling. We confirmed this by studying Siglec-5, which is prominently expressed on chimpanzee lymphocytes, including CD4 T cells. Ab-mediated clearance of Siglec-5 from chimpanzee T cells enhanced TCR-mediated activation. Conversely, primary human T cells and Jurkat cells transfected with Siglec-5 become less responsive; i.e., they behave more like chimpanzee T cells. This human-specific loss of T cell Siglec expression associated with T cell hyperactivity may help explain the strikingly disparate prevalence and severity of T cell-mediated diseases such as AIDS and chronic active hepatitis between humans and chimpanzees. PMID- 16682636 TI - Low-frequency normal modes that describe allosteric transitions in biological nanomachines are robust to sequence variations. AB - By representing the high-resolution crystal structures of a number of enzymes using the elastic network model, it has been shown that only a few low-frequency normal modes are needed to describe the large-scale domain movements that are triggered by ligand binding. Here we explore a link between the nearly invariant nature of the modes that describe functional dynamics at the mesoscopic level and the large evolutionary sequence variations at the residue level. By using a structural perturbation method (SPM), which probes the residue-specific response to perturbations (or mutations), we identify a sparse network of strongly conserved residues that transmit allosteric signals in three structurally unrelated biological nanomachines, namely, DNA polymerase, myosin motor, and the Escherichia coli chaperonin. Based on the response of every mode to perturbations, which are generated by interchanging specific sequence pairs in a multiple sequence alignment, we show that the functionally relevant low-frequency modes are most robust to sequence variations. Our work shows that robustness of dynamical modes at the mesoscopic level is encoded in the structure through a sparse network of residues that transmit allosteric signals. PMID- 16682637 TI - Motor cortex maps articulatory features of speech sounds. AB - The processing of spoken language has been attributed to areas in the superior temporal lobe, where speech stimuli elicit the greatest activation. However, neurobiological and psycholinguistic models have long postulated that knowledge about the articulatory features of individual phonemes has an important role in their perception and in speech comprehension. To probe the possible involvement of specific motor circuits in the speech-perception process, we used event related functional MRI and presented experimental subjects with spoken syllables, including [p] and [t] sounds, which are produced by movements of the lips or tongue, respectively. Physically similar nonlinguistic signal-correlated noise patterns were used as control stimuli. In localizer experiments, subjects had to silently articulate the same syllables and, in a second task, move their lips or tongue. Speech perception most strongly activated superior temporal cortex. Crucially, however, distinct motor regions in the precentral gyrus sparked by articulatory movements of the lips and tongue were also differentially activated in a somatotopic manner when subjects listened to the lip- or tongue-related phonemes. This sound-related somatotopic activation in precentral gyrus shows that, during speech perception, specific motor circuits are recruited that reflect phonetic distinctive features of the speech sounds encountered, thus providing direct neuroimaging support for specific links between the phonological mechanisms for speech perception and production. PMID- 16682638 TI - Light chain inclusion permits terminal B cell differentiation and does not necessarily result in autoreactivity. AB - Mice in which the Jkappa cluster was replaced with a VkappaJkappa rearranged gene were studied. More than 90% of B cells from homozygous mutant mice expressed the transgenic kappa chain but showed a slightly reduced level of kappa transcripts compared with WT B lymphocytes. Light chain inclusion was apparent in 10% of B cells from these mice and raised 25% in hemizygous mice with a still lower expression of the knockin kappa chain. Beyond the rules of clonal selection, peripheral B cells developed in such animals, with included cells being activated and differentiating into class-switched or antibody-secreting cells. The high amount of included mature B cells was associated with an increase of hybrid kappa/lambda immunoglobulins but not with the increased prevalence of autoantibodies. Altogether, these data suggest that light chain exclusion prevalent in normal B cells mostly results from ordered rearrangements and stochastic mechanisms but is neither tightly ensured by a stringent cell selection process nor absolutely required for normal B cell function. PMID- 16682639 TI - Suppressor of cytokine signaling 3 limits protection of leukemia inhibitory factor receptor signaling against central demyelination. AB - Enhancement of oligodendrocyte survival through activation of leukemia inhibitory factor receptor (LIFR) signaling is a candidate therapeutic strategy for demyelinating disease. However, in other cell types, LIFR signaling is under tight negative regulation by the intracellular protein suppressor of cytokine signaling 3 (SOCS3). We, therefore, postulated that deletion of the SOCS3 gene in oligodendrocytes would promote the beneficial effects of LIFR signaling in limiting demyelination. By studying wild-type and LIF-knockout mice, we established that SOCS3 expression by oligodendrocytes was induced by the demyelinative insult, that this induction depended on LIF, and that endogenously produced LIF was likely to be a key determinant of the CNS response to oligodendrocyte loss. Compared with wild-type controls, oligodendrocyte-specific SOCS3 conditional-knockout mice displayed enhanced c-fos activation and exogenous LIF-induced phosphorylation of signal transducer and activator of transcription 3. Moreover, these SOCS3-deficient mice were protected against cuprizone-induced oligodendrocyte loss relative to wild-type animals. These results indicate that modulation of SOCS3 expression could facilitate the endogenous response to CNS injury. PMID- 16682640 TI - Transferable anticancer innate immunity in spontaneous regression/complete resistance mice. AB - Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites. PMID- 16682641 TI - Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling. AB - To achieve full ripening, climacteric fruits, such as tomato require synthesis, perception and signal transduction of the plant hormone ethylene. The nonripening phenotype of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato is the result of reduced ethylene responsiveness in fruit tissues. In addition, a subset of ethylene responses associated with floral senescence, abscission, and root elongation are also impacted in mutant plants, but to a lesser extent. Using positional cloning, we have identified an identical 334-bp deletion in a gene of unknown biochemical function at the Gr/Nr-2 locus. Consistent with a dominant gain of function mutation, this deletion causes ectopic expression of Gr/Nr-2, which in turn leads to ripening inhibition. A CaMV35::GR transgene recreates the Gr/Nr-2 mutant phenotype but does not lead to a global reduction in ethylene responsiveness, suggesting tissue-specific modulation of ethylene responses in tomato. Gr/Nr-2 encodes an evolutionary conserved protein of unknown biochemical function that we associate here with ethylene signaling. Because Gr/Nr-2 has no sequence homology with the previously described Nr (Never-ripe) ethylene receptor of tomato we now refer to this gene only as GR. Identification of GR expands the current repertoire of ethylene signaling components in plants and provides a tool for further elucidation of ethylene response mechanisms and for controlling ethylene signal specificity in crop plants. PMID- 16682642 TI - REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis. AB - Arabidopsis thaliana has five ethylene hormone receptors, which bind ethylene and elicit responses critical for plant growth and development. Here we describe a negative regulator of ethylene responses, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which regulates the function of at least one of the receptors, ETR1, in Arabidopsis. RTE1 was identified based on the ability of rte1 mutations to suppress ethylene insensitivity of the dominant gain-of-function allele etr1-2. rte1 loss-of-function mutants have an enhanced ethylene response that closely resembles the etr1 null phenotype. The etr1 rte1 double null mutant is identical to the etr1 and rte1 single null mutants, suggesting that the two genes act in the same pathway. rte1 is unable to suppress the etr1-1 gain-of-function allele, placing RTE1 at or upstream of ETR1. rte1 also fails to suppress gain-of-function mutations in each of the four other ethylene receptor genes. RTE1 encodes a previously undescribed predicted membrane protein, which is highly conserved in plants, animals [corrected] and protists but absent in fungi and prokaryotes. Ethylene treatment induces RTE1 expression, and overexpression of RTE1 confers reduced ethylene sensitivity that partially depends on ETR1. These findings demonstrate that RTE1 is a negative regulator of ethylene signaling and suggest that RTE1 plays an important role in ETR1 function. PMID- 16682643 TI - Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP. AB - The Wingless pathway plays an essential role during synapse development. Recent studies at Drosophila glutamatergic synapses suggest that Wingless is secreted by motor neuron terminals and binds to postsynaptic Drosophila Frizzled-2 (DFz2) receptors. DFz2 is, in turn, endocytosed and transported to the muscle perinuclear area, where it is cleaved, and the C-terminal fragment is imported into the nucleus, presumably to regulate transcription during synapse growth. Alterations in this pathway interfere with the formation of new synaptic boutons and lead to aberrant synaptic structures. Here, we show that the 7 PDZ protein dGRIP is necessary for the trafficking of DFz2 to the nucleus. dGRIP is localized to Golgi and trafficking vesicles, and dgrip mutants mimic the synaptic phenotypes observed in wg and dfz2 mutants. DFz2 and dGRIP colocalize in trafficking vesicles, and a severe decrease in dGRIP levels prevents the transport of endocytosed DFz2 receptors to the nucleus. Moreover, coimmunoprecipitation experiments in transfected cells and yeast two-hybrid assays suggest that the C terminus of DFz2 interacts directly with the PDZ domains 4 and 5. These results provide a mechanism by which DFz2 is transported from the postsynaptic membrane to the postsynaptic nucleus during synapse formation and implicate dGRIP as an essential molecule in the transport of this signal. PMID- 16682644 TI - Development of NG2 neural progenitor cells requires Olig gene function. AB - In the adult central nervous system, two distinct populations of glial cells expressing the chondroitin sulfate proteoglycan NG2 have been described: bipolar progenitor cells and more differentiated "synantocytes." These cells have diverse neurological functions, including critical roles in synaptic transmission, repair, and regeneration. Despite their potential importance, the genetic factors that regulate NG2 cell development are poorly understood, and the relationship of synantocytes to the oligodendroglial lineage, in particular, remains controversial. Here, we show that >90% of embryonic and adult NG2 cells express Olig2, a basic helix-loop-helix transcription factor required for oligodendrocyte lineage specification. Analysis of mice lacking Olig function demonstrates a failure of NG2 cell development at embryonic and perinatal stages that can be rescued by addition of a transgene containing the human OLIG2 locus. These findings show a general requirement for Olig function in NG2 cell development and highlight further roles for Olig transcription factors in neural progenitor cells. PMID- 16682645 TI - Simvastatin promotes Th2-type responses through the induction of the chitinase family member Ym1 in dendritic cells. AB - Statins, best known for their lipid-lowering actions, also possess immunomodulatory properties. Recent studies have shown a Th2-biasing effect of statins, although the underlying mechanism has not been identified. In this study, we investigated whether simvastatin can exercise a Th2-promoting effect through modulation of function of dendritic cells (DCs) without direct interaction with CD4+ T cells. Exposure of DCs to simvastatin induced the differentiation of a distinct subset of DCs characterized by a high expression of B220. These simvastatin-conditioned DCs up-regulated GATA-3 expression and down regulated T-bet expression in cocultured CD4+ T cells in the absence of additional simvastatin added to the coculture. The Th2-biased transcription factor profile induced by simvastatin-treated DCs also was accompanied by increased Th2 (IL-4, IL-5, and IL-13) and decreased Th1 (IFN-gamma) cytokine secretion from the T cells. The Th2-promoting effect of simvastatin was found to depend on the chitinase family member Ym1, known to be a lectin. Anti-Ym1 antibody abolished the Th2-promoting effect of simvastatin-treated DCs. Also, simvastatin was unable to augment Ym1 expression in DCs developed from STAT6-/- or IL-4R alpha-/- mice. Thus, modulation of Ym1 production by DCs identifies a previously undescribed mechanism of Th2 polarization by statin. PMID- 16682646 TI - Evidence that feedback inhibition of NAD kinase controls responses to oxidative stress. AB - Formation of NADP+ from NAD+ is catalyzed by NAD kinase (NadK; EC 2.7.1.23). Evidence is presented that NadK is the only NAD kinase of Salmonella enterica and is essential for growth. NadK is inhibited allosterically by NADPH and NADH. Without effectors, NadK exists as an equilibrium mixture of dimers and tetramers (KD = 1.0 +/- 0.8 mM) but is converted entirely to tetramers in the presence of the inhibitor NADPH. Comparison of NadK kinetic parameters with pool sizes of NADH and NADPH suggests that NadK is substantially inhibited during normal growth and, thus, can increase its activity greatly in response to temporary drops in the pools of inhibitory NADH and NADPH. The primary inhibitor is NADPH during aerobic growth and NADH during anaerobic growth. A model is proposed in which variation of NadK activity is central to the adjustment of pyridine nucleotide pools in response to changes in aeration, oxidative stress, and UV irradiation. It is suggested that each of these environmental factors causes a decrease in the level of reduced pyridine nucleotides, activates NadK, and increases production of NADP(H) at the expense of NAD(H). Activation of NadK may constitute a defensive response that resists loss of protective NADPH. PMID- 16682647 TI - Purkinje neuron degeneration in nervous (nr) mutant mice is mediated by a metabolic pathway involving excess tissue plasminogen activator. AB - Purkinje neurons (PNs), the central cells in cerebellar circuitry and function, constitute a vulnerable population in many human genetic, malignant, hypoxic, and toxic diseases. In the nervous (nr) mutant mouse, the majority of PNs die in the fourth to fifth postnatal weeks, but the responsible molecules are unknown. We first disclose a remarkable increase in mRNA expression and protein concentration in the nr cerebellum of tissue plasminogen activator (tPA), a gene closely linked to the mapped but as-yet-uncloned nr locus. Evidence that excessive tPA triggers nr PN death was obtained with organotypic slice cultures expressing the nr PN phenotype, in which an inhibitor of tPA led to increased nr PN survival. An antagonist of protein kinase C, a downstream component in the tPA pathway, also increased nr PN survival. Additional downstream targets in the tPA pathway (the mitochondrial voltage-dependent anion channel, brain-derived neurotrophic factor, and neurotrophin 3) were also abnormal, in parallel with the alterations in PN mitochondrial morphology, dendritic growth, and synaptogenesis that culminate in nr PN death and motor incoordination. We thus propose a molecular pathway by which the excessive tPA in nr cerebellum mediates PN degeneration. PMID- 16682648 TI - Pannexin 1 in erythrocytes: function without a gap. AB - ATP is a widely used extracellular signaling molecule. The mechanism of ATP release from cells is presently unresolved and may be either vesicular or channel mediated. Erythrocytes release ATP in response to low oxygen or to shear stress. In the absence of vesicles, the release has to be through channels. Erythrocytes do not form gap junctions. Yet, here we show with immunohistochemical and electrophysiological data that erythrocytes express the gap junction protein pannexin 1. This protein, in addition to forming gap junction channels in paired oocytes, can also form a mechanosensitive and ATP-permeable channel in the nonjunctional plasma membrane. Consistent with a role of pannexin 1 as an ATP release channel, ATP release by erythrocytes was attenuated by the gap junction blocker carbenoxolone. Furthermore, under conditions of ATP release, erythrocytes took up fluorescent tracer molecules permeant to gap junction channels. PMID- 16682649 TI - Sex-specific influences of vasopressin on human social communication. AB - Arginine vasopressin (AVP) and related peptides affect social behaviors in numerous species, but AVP influences on human social functions have not yet been established. Here, we describe how intranasal AVP administration differentially affects social communication in men and women, and we propose a mechanism through which it may exert those influences. In men, AVP stimulates agonistic facial motor patterns in response to the faces of unfamiliar men and decreases perceptions of the friendliness of those faces. In contrast, in women, AVP stimulates affiliative facial motor patterns in response to the faces of unfamiliar women and increases perceptions of the friendliness of those faces. AVP also affected autonomic responsiveness to threatening faces and increased anxiety, which may underlie both communication patterns by promoting different social strategies in stressful contexts in men and women. PMID- 16682650 TI - Targeted disruption of growth hormone receptor interferes with the beneficial actions of calorie restriction. AB - Reduced intake of nutrients [calorie restriction (CR)] extends longevity in organisms ranging from yeast to mammals. Mutations affecting somatotropic, insulin, or homologous signaling pathways can increase life span in worms, flies, and mice, and there is considerable evidence that reduced secretion of insulin like growth factor I and insulin are among the mechanisms that mediate the effects of CR on aging and longevity in mammals. In the present study, mice with targeted disruption of the growth hormone (GH) receptor [GH receptor/GH-binding protein knockout (GHRKO) mice] and their normal siblings were fed ad libitum (AL) or subjected to 30% CR starting at 2 months of age. In normal females and males, CR produced the expected increases in overall, average, median, and maximal life span. Longevity of normal mice subjected to CR resembles that of GHRKO animals fed AL. In sharp contrast to its effects in normal mice, CR failed to increase overall, median, or average life span in GHRKO mice and increased maximal life span only in females. In a separate group of animals, CR for 1 year improved insulin sensitivity in normal mice but failed to further enhance the remarkable insulin sensitivity in GHRKO mutants. These data imply that somatotropic signaling is critically important not only in the control of aging and longevity under conditions of unlimited food supply but also in mediating the effects of CR on life span. The present findings also support the notion that enhanced sensitivity to insulin plays a prominent role in the actions of CR and GH resistance on longevity. PMID- 16682652 TI - Leukoencephalopathy with cerebral calcifications and cysts. PMID- 16682651 TI - Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated. AB - Gametes rely heavily on posttranscriptional control mechanisms to regulate their differentiation. In eggs, maternal mRNAs are stored and selectively activated during development. In the male, transcription ceases during spermiogenesis, necessitating the posttranscriptional regulation of many paternal mRNAs required for spermatozoan assembly and function. To date, most of the testicular mRNAs known to be translationally regulated are initially transcribed in postmeiotic cells. Because protein synthesis occurs on polysomes and translationally inactive mRNAs are sequestered as ribonucleoproteins (RNPs), movement of mRNAs between these fractions is indicative of translational up- and down-regulation. Here, we use microarrays to analyze mRNAs in RNPs and polysomes from testis extracts of prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds. Consistent with published reports, many of the translationally delayed postmeiotic mRNAs shift from the RNPs into the polysomes, establishing the validity of this approach. In addition, we detect another 742 mouse testicular transcripts that show dramatic shifts between RNPs and polysomes. One subgroup of 35 genes containing the known, translationally delayed phosphoglycerate kinase 2 (Pgk2) is initially transcribed during meiosis and is translated in later-stage cells. Another subgroup of 82 meiotically expressed genes is translationally down-regulated late in spermatogenesis. This high throughput approach defines the changing translation patterns of populations of genes as male germ cells differentiate and identifies groups of meiotic transcripts that are translationally up- and down-regulated. PMID- 16682653 TI - Clinical findings of the phakomatoses: von Hippel-Lindau disease. PMID- 16682654 TI - The away neurology rotation: is the grass greener on the other side? AB - Medical students and residents often struggle with the pros and cons of pursuing an away rotation. The purpose of this article is to share personal perspectives on the benefits and costs of an away rotation. PMID- 16682655 TI - Active control trials for epilepsy: avoiding bias in head-to-head trials. PMID- 16682656 TI - The insular cortex and cardiac response to stroke. PMID- 16682657 TI - Appointing a proxy for research consent after one develops dementia: the need for further study. PMID- 16682658 TI - The portrayal of coma in contemporary motion pictures. AB - BACKGROUND: Coma has been a theme of screenplays in motion pictures, but there is no information about its accuracy. METHODS: The authors reviewed 30 movies from 1970 to 2004 with actors depicting prolonged coma. Accurate depiction of comatose patients was defined by appearance, the complexity of care, accurate cause of coma and probability of awakening, and appropriate compassionate discussion between the physician and family members. Twenty-two key scenes from 17 movies were rated for accuracy by a panel of neurointensivists and neuroscience nurses and then were shown to 72 nonmedical viewers. Accuracy of the scenes was assessed using a Likert Scale. RESULTS: Coma was most often caused by motor vehicle accidents or violence (63%). The time in a comatose state varied from days to 10 years. Awakening occurred in 18 of 30 motion pictures (60%). Awakening was sudden with cognition intact, even after prolonged time in a coma. Actors personified "Sleeping Beauty" (eyes closed, beautifully groomed). Physicians appeared as caricatures. Only two movies had a reasonable accurate representation (Dream Life of Angels and Reversal of Fortune). The majority of the surveyed viewers identified inaccuracy of representation of coma, awakenings, and conversations on the experience of being in a coma, except in 8 of the 22 scenes (36%). Twenty eight of the 72 viewers (39%) could potentially allow these scenes to influence decisions in real life. CONCLUSIONS: Misrepresentation of coma and awakening was common in motion pictures and impacted on the public perception of coma. Neurologic advice regarding prolonged coma is needed. PMID- 16682659 TI - Diffusion MRI abnormalities after prolonged febrile seizures with encephalopathy. AB - BACKGROUND: Patients with encephalopathy heralded by a prolonged seizure as the initial symptom often have abnormal subcortical white matter on diffusion weighted MRI (DWI). OBJECTIVE: To determine if these patients share other common features. METHODS: Patients with encephalopathy heralded by a prolonged seizure and followed by the identification of abnormal subcortical white matter on MRI were collected retrospectively. Their clinical, laboratory, and radiologic data were reviewed. RESULTS: Seventeen patients were identified, ages 10 months to 4 years. All had a prolonged febrile seizure (longer than 1 hour in 12 patients) as their initial symptom. Subsequent seizures, most often in clusters of complex partial seizures, were seen 4 to 6 days after the initial seizure in 16 patients. Outcome ranged from almost normal to severe mental retardation. MRI performed within 2 days of presentation showed no abnormality. Subcortical white matter lesions were observed on DWI between 3 and 9 days in all 17 patients. T2-weighted images showed linear high intensity of subcortical U fibers in 13 patients. The lesions were predominantly frontal or frontoparietal in location with sparing of the perirolandic region. The diffusion abnormality disappeared between days 9 and 25, and cerebral atrophy was detected later than 2 weeks. Three patients having only frontal lesions had relatively good clinical outcome. CONCLUSIONS: Although the pathophysiologic mechanism remains unknown, these patients seem to have a distinctive encephalopathy syndrome. MRI is helpful in establishing the diagnosis of this encephalopathy. PMID- 16682660 TI - A meta-analysis of individual patient responses to lamotrigine or carbamazepine monotherapy. AB - OBJECTIVE: To compare the effects of carbamazepine and lamotrigine monotherapy for people with partial onset seizures or generalized onset tonic-clonic seizures. METHODS: A systematic review and meta-analysis using data on individual patients from randomized trials comparing lamotrigine with carbamazepine monotherapy. The review draws on the search strategy developed for the Cochrane Epilepsy Group, which searches MEDLINE and the Cochrane Controlled Trials register, and hand searches relevant journals. The outcomes considered were time to antiepileptic drug withdrawal, which would usually be for either inadequate seizure control or unacceptable adverse effects, time to first seizure, and 6 month remission. RESULTS: Five randomized trials were identified containing data for 1,384 participants. Time to treatment withdrawal significantly improved with lamotrigine compared to carbamazepine (hazard ratio 0.55, 95% CI 0.35 to 0.84, random effects), while time to first seizure (hazard ratio 1.14, 95% CI 0.92 to 1.43, fixed effects) and seizure freedom at 6 months (relative risk 0.92, 95% CI 0.81 to 1.04, fixed effects) favor carbamazepine although the results are not significant. CONCLUSIONS: Lamotrigine is significantly less likely to be withdrawn than carbamazepine, but results for time to first seizure suggest a nonsignificant trend that carbamazepine may be superior in terms of seizure control. Trials were of too short a duration to measure clinically important efficacy outcomes such as time to 12-month remission. Current industry-sponsored trials fail to adequately inform clinical practice and further more clinically relevant trials are needed in which longer-term outcomes are assessed before the place of lamotrigine in the treatment of epilepsy is defined. PMID- 16682661 TI - Use of antiepileptic drugs and risk of fractures: case-control study among patients with epilepsy. AB - OBJECTIVE: To study the association between use of antiepileptic drugs (AEDs) and risk of fractures. METHODS: The authors obtained data from the General Practice Research Database (GPRD). A case-control study was nested within a cohort of patients with active epilepsy. Cases were patients with a first fracture after cohort entry. Up to four controls were matched to each case by practice, sex, year of birth, timing of first epilepsy diagnosis, index date, and duration of GPRD history. Cumulative exposure to AEDs was assessed by summing the duration of all AED prescriptions. A distinction was made between AEDs that induce the hepatic cytochrome P-450 enzyme system and AEDs that do not. Medical conditions and drugs known to be associated with bone metabolism or falls were evaluated as potential confounders. Conditional logistic regression analysis was used to calculate odds ratios (ORs) and 95% CIs. RESULTS: The study population comprised 1,018 cases and 1,842 matched controls. The risk of fractures increased with cumulative duration of exposure (p for trend < 0.001), with the strongest association for greater than 12 years of use: adjusted OR 4.15 (95% CI 2.71 to 6.34). Risk estimates were higher in women than in men. There was no difference between users of AEDs that induce and AEDs that do not induce the hepatic cytochrome P-450 system. CONCLUSIONS: Long-term use of AEDs was associated with an increased risk of fractures, especially in women. More research on mechanisms of AED-induced bone breakdown and female vulnerability to the effects of AEDs on bone health is warranted. PMID- 16682662 TI - Elevated troponin levels are associated with higher mortality following intracerebral hemorrhage. AB - BACKGROUND: Elevated troponin levels are an independent indicator of poor outcome in ischemic stroke. The authors performed a retrospective study to ascertain whether elevated cardiac troponin I (cTnI) influences outcome from intracerebral hemorrhage (ICH). METHODS: Patients were included if they had a cTnI level measured and a head CT performed within 24 hours of presentation with a spontaneous ICH. Those with recent stroke, angina, or myocardial ischemia were excluded. CT scans were reviewed to determine the hematoma size, location, presence of intraventricular or subarachnoid hemorrhage, hydrocephalus, and midline shift. RESULTS: Of the 729 ICH patients admitted over 4 years, 235 were included in the analysis. Most exclusions were for medical reasons or because of lack of a CT. Mortality was higher in the 18% with a peak cTnI level > 0.4 ng/mL (58 vs 34%, p = 0.009) and having elevated cTnI was an independent predictor of in-hospital mortality (Exp [beta] 3.68, 95% CI 1.2 to 11.2, p = 0.023). Three patients (1.2%) died due to cardiac events, all of whom had an elevated cTnI level on admission. CONCLUSIONS: Elevated cardiac troponin I (cTnI) values occur frequently in ICH and are independently associated with higher in-hospital mortality. Although cardiac causes of death were higher in those with elevated cTnI levels, due to its very low frequency (1.2%) this finding remains preliminary. PMID- 16682663 TI - A population-based study of the incidence and prognosis of lacunar stroke. AB - OBJECTIVE: To evaluate incidence and prognosis of lacunar stroke in a prospective, population-based patient registry. METHODS: The authors included first-ever strokes occurring between 1994 and 1998. They assessed incidence, risk factors, mortality, and recurrence in patients with lacunar stroke. RESULTS: The authors identified 491 patients (15.3%) with lacunar stroke (252 men and 239 women) and 2,153 patients (67.3%) with nonlacunar stroke (998 men and 1,155 women). Crude annual incidence rate for a first-ever lacunar stroke was 33.0/100,000 (95% CI 30.2 to 36.0). At the univariate logistic regression analysis among patients with lacunar stroke there was a higher proportion of cigarette smoking and hypercholesterolemia and a lower proportion of chronic atrial fibrillation than in patients with nonlacunar stroke. For lacunar stroke, the 30-day case-fatality rate was 4.3% (95% CI 2.5 to 6.1) and the 1-year case fatality rate was 13.0% (95% CI 10.0 to 16.0). During the first year of follow-up the average annual stroke recurrence rate was lower in patients with lacunar (2.83%; 95% CI 1.36 to 4.30) than in those with nonlacunar stroke (5.10%; 95% CI 4.17 to 6.03) while from the second year onward, rates were similar in both groups. CONCLUSION: In the short term, patients with nonlacunar stroke had more vascular events, but in the long term, the risk of death and of stroke recurrence was similar. PMID- 16682664 TI - Retinal vessel diameters and risk of stroke: the Rotterdam Study. AB - BACKGROUND: Retinal vessels may provide information on cerebral vascular pathology, because they share many features with cerebral vessels. A smaller ratio of the retinal arteriolar-to-venular diameters reportedly predicts the risk of stroke. It is unclear if this is due to arteriolar narrowing or venular dilation. OBJECTIVE: To investigate whether smaller arteriolar or larger venular diameters are related to the risk of stroke and cerebral infarction. METHODS: This study was based on the prospective population-based Rotterdam Study and included 5,540 participants of 55 years or over, who had gradable fundus transparencies and were free of stroke at baseline (1990 to 1993). For each participant, retinal arteriolar and venular diameters were measured on digitized images of one eye. Follow-up for first-ever stroke was complete until January 1, 2002. RESULTS: After a mean follow-up of 8.5 years, 411 participants had a stroke, of whom 259 had cerebral infarction. Larger venular diameters were associated with an increased risk of stroke (hazard ratio [HR] adjusted for age and sex per SD increase: 1.12 [95% CI: 1.02 to 1.24]) and cerebral infarction (HR: 1.15 [95% CI: 1.02 to 1.29]). Smaller arteriolar diameters were neither related to the risk of stroke (HR per SD decrease: 1.02 [95% CI: 0.93 to 1.13]) nor to the risk of cerebral infarction (HR: 1.02 [95% CI: 0.90 to 1.15]). After additional adjustment for other cardiovascular risk factors, the results did not change. CONCLUSIONS: Larger retinal venular diameters are associated with an increased risk of stroke and cerebral infarction. The role of venules in cerebrovascular disease warrants further exploration. PMID- 16682665 TI - Impact of metabolic syndrome on prognosis of symptomatic intracranial atherostenosis. AB - BACKGROUND: The metabolic syndrome (MetS) is a cluster of risk factors linked to insulin resistance that increase an individual's risk of atherosclerotic vascular disease. The authors evaluated the prevalence and prognosis of the MetS among individuals with symptomatic intracranial arterial stenosis. METHODS: Patients enrolled in the Warfarin-Aspirin Symptomatic Intracranial Disease trial were evaluated in this post-hoc analysis. Baseline characteristics and outcome were compared in patients with the MetS vs patients without the MetS. RESULTS: Among 476 patients, the prevalence of the MetS was 43%. MetS patients were more likely to be younger, female, and white. During a mean follow-up period of 1.8 years, time to the first of ischemic stroke, myocardial infarction, or vascular death was shorter among patients with the MetS with a hazard ratio (syndrome/no syndrome) of 1.6 (95% CI = 1.1 to 2.4, p = 0.0097). Time to ischemic stroke alone was also shorter among patients with the MetS with a hazard ratio (syndrome/no syndrome) of 1.7 (95% CI = 1.1 to 2.6, p = 0.012). When controlling for individual factors of the definition, MetS was not significant (combined outcome: p = 0.14; ischemic stroke: p = 0.074). CONCLUSIONS: The metabolic syndrome is present in about half of individuals with symptomatic intracranial atherosclerotic disease and is associated with a substantially higher risk of major vascular events. The metabolic syndrome may not provide additional ability to predict outcomes beyond the individual factors for patients with intracranial stenosis. PMID- 16682666 TI - Predictors of hemorrhage in patients with untreated brain arteriovenous malformation. AB - BACKGROUND: Intracranial hemorrhage is a serious possible complication in patients with brain arteriovenous malformation (AVM). Several morphologic factors associated with hemorrhagic AVM presentation have been established, but their relevance for the risk of subsequent AVM hemorrhage remains unclear. METHODS: The authors analyzed follow-up data on 622 consecutive patients from the prospective Columbia AVM database, limited to the period between initial AVM diagnosis and the start of treatment (i.e., any endovascular, surgical, or radiation therapy). Univariate and multivariate logistic regression and Cox proportional hazard models were applied to analyze the effect of patient age, gender, AVM size, anatomic location, venous drainage pattern, and associated arterial aneurysms on the risk of intracranial hemorrhage at initial presentation and during follow-up. RESULTS: The mean pretreatment follow-up was 829 days (median: 102 days), during which 39 (6%) patients experienced AVM hemorrhage. Increasing age (hazard ratio [HR] 1.05, 95% CI 1.03 to 1.08), initial hemorrhagic AVM presentation (HR 5.38, 95% CI 2.64 to 10.96), deep brain location (HR 3.25, 95% CI 1.30 to 8.16), and exclusive deep venous drainage (HR 3.25, 95% CI 1.01 to 5.67) were independent predictors of subsequent hemorrhage. Annual hemorrhage rates on follow-up ranged from 0.9% for patients without hemorrhagic AVM presentation, deep AVM location, or deep venous drainage to as high as 34.4% for those harboring all three risk factors. CONCLUSIONS: Hemorrhagic arteriovenous malformation (AVM) presentation, increasing age, deep brain location, and exclusive deep venous drainage appear to be independent predictors for AVM hemorrhage during natural history follow-up. The risk of spontaneous hemorrhage may be low in AVMs without these risk factors. PMID- 16682667 TI - Prevalence and severity of microbleeds in a memory clinic setting. AB - OBJECTIVE: To determine prevalence and severity of microbleeds (MBs) in a large cohort of patients attending a memory clinic. METHODS: The authors consecutively included patients attending their memory clinic between January 2002 and April 2005. They analyzed prevalence and number of MBs according to demographic, diagnostic, and MRI data. RESULTS: The authors included 772 patients (53% men, age 66 +/- 11). One hundred twenty-seven patients (17%) exhibited at least one MB. The prevalence differed according to diagnostic groups (p < 0.0001): Sixty five percent of patients with vascular dementia exhibited MBs vs 18% of Alzheimer disease patients, 20% of mild cognitive impairment patients, and 10% of patients with subjective complaints. The presence of MBs was associated with age, white matter hyperintensities, lacunar infarcts, and infarcts. CONCLUSION: The prevalence of microbleeds (MBs) in a large cohort of patients attending a memory clinic is higher than previously described in community samples and lower than reported in stroke patients. This finding of a relatively high proportion of MBs in Alzheimer disease and mild cognitive impairment provides further evidence for the involvement of vascular factors in neurodegenerative diseases such as Alzheimer disease. PMID- 16682668 TI - Speaking of research advance directives: planning for future research participation. AB - OBJECTIVE: To examine one model of research advance directive as a possible way to reduce the mismatch between patient and proxy choices and also to learn more about how patients with mild to moderate dementia may want to keep decision making or cede it to their proxies in the future. METHODS: Separate interviews were conducted with 149 dyads of dementia patients and family proxies about future enrollment in five types of research. Subsequent joint interviews were conducted with 69 of those dyads to discuss their separately articulated decisions and ask whether the patient prefers future enrollment decisions to be made as he or she directs today or as the proxy deems best in the future. RESULTS: Patients chose to cede future decision making to their proxies in 82.9% of the trials. Patients ceded decisions to their proxies in 80.7% of those trials about which the dyad had given opposite answers (n = 74, 49.7%). Patients who had expressed discomfort about the prospect of the proxy making an enrollment decision in a trial (n = 49, 32.9%) ceded decision making to their proxies in 45.7% of those trials. CONCLUSIONS: Both patients and proxies were willing to discuss future research enrollment in the context of an advance directive for research. Such a document may be helpful to proxies and researchers in the future to judge the types of research and associated risks patients are willing to enroll in. Although most patients willingly cede future decisions to their proxies, a sizeable minority do not wish to do so. PMID- 16682669 TI - Cognitive performance predicts treatment decisional abilities in mild to moderate dementia. AB - OBJECTIVE: To examine the contribution of neuropsychological test performance to treatment decision-making capacity in community volunteers with mild to moderate dementia. METHODS: The authors recruited volunteers (44 men, 44 women) with mild to moderate dementia from the community. Subjects completed a battery of 11 neuropsychological tests that assessed auditory and visual attention, logical memory, language, and executive function. To measure decision making capacity, the authors administered the Capacity to Consent to Treatment Interview, the Hopemont Capacity Assessment Interview, and the MacCarthur Competence Assessment Tool--Treatment. Each of these instruments individually scores four decisional abilities serving capacity: understanding, appreciation, reasoning, and expression of choice. The authors used principal components analysis to generate component scores for each ability across instruments, and to extract principal components for neuropsychological performance. RESULTS: Multiple linear regression analyses demonstrated that neuropsychological performance significantly predicted all four abilities. Specifically, it predicted 77.8% of the common variance for understanding, 39.4% for reasoning, 24.6% for appreciation, and 10.2% for expression of choice. Except for reasoning and appreciation, neuropsychological predictor (beta) profiles were unique for each ability. CONCLUSIONS: Neuropsychological performance substantially and differentially predicted capacity for treatment decisions in individuals with mild to moderate dementia. Relationships between elemental cognitive function and decisional capacity may differ in individuals whose decisional capacity is impaired by other disorders, such as mental illness. PMID- 16682670 TI - APOE epsilon variation in multiple sclerosis susceptibility and disease severity: some answers. AB - BACKGROUND: Previous studies have examined the role of APOE variation in multiple sclerosis (MS), but have lacked the statistical power to detect modest genetic influences on risk and disease severity. The meta- and pooled analyses presented here utilize the largest collection, to date, of MS cases, controls, and families genotyped for the APOE epsilon polymorphism. METHODS: Studies of MS and APOE were identified by searches of PubMed, Biosis, Web of Science, Cochrane Review, and Embase. When possible, authors were contacted for individual genotype data. Meta analyses of MS case-control data and family-based analyses were performed to assess the association of APOE epsilon genotype with disease risk. Pooled analyses of MS cases were also performed to assess the influence of APOE epsilon genotype on disease severity. RESULTS: A total of 22 studies (3,299 MS cases and 2,532 controls) were available for meta-analysis. No effect of epsilon2 or epsilon4 status on MS risk was observed (summary OR 1.14, 95% CI 0.96-1.34 and OR 0.89, 95% CI 0.78-1.01). Results obtained from analyses of APOE genotypes in 1,279 MS families were also negative (p = 0.61). Finally, results from pooled analyses of 4,048 MS cases also argue strongly that APOE epsilon status does not distinguish a relapsing-remitting from primary progressive disease course, or influence disease severity, as measured by the Expanded Disability Status Scale and disease duration. CONCLUSION: Overall, these findings do not support a role for APOE in multiple sclerosis, and underscore the importance of using large sample sizes to detect modest genetic effects, particularly in studies of genotype-phenotype relationships. PMID- 16682671 TI - MRI T2 lesion burden in multiple sclerosis: a plateauing relationship with clinical disability. AB - BACKGROUND: Previous studies have shown only modest correlation between multiple sclerosis (MS) lesions on MRI and clinical disability. OBJECTIVE: To investigate the relationship between proton density/T2-weighted (T2) burden of disease (BOD) quantitatively measured on MRI scans and clinical determinants including disability. METHODS: Using the Sylvia Lawry Centre for Multiple Sclerosis Research (SLCMSR) database, the authors studied baseline T2 BOD data from a pooled subsample of 1,312 placebo MS patients from 11 randomized controlled trials. Univariate comparisons guided development of multiple regression models incorporating the most important clinical predictors. RESULTS: Significant, although weak to moderate, correlations were found between T2 BOD and age at disease onset, disease duration, disease course, disability (as measured by the Expanded Disability Status Scale [EDSS]), relapse rate, certain presenting symptoms, and gadolinium enhancement. An unexpected but key finding that persisted in the multiple regression analyses was a plateauing relationship between T2 BOD and disability for EDSS values above 4.5. CONCLUSIONS: This study confirmed the limited correlation between clinical manifestations and T2 burden of disease (BOD) but revealed an important plateauing relationship between T2 BOD and disability. PMID- 16682672 TI - Saccade dynamics in peripheral vs central sixth nerve palsies. AB - OBJECTIVE: To investigate differences between peripheral idiopathic and central sixth nerve palsies from brainstem damage by comparing peak velocities and durations of horizontal saccades. METHODS: Fourteen patients with unilateral incomplete sixth nerve palsies caused by idiopathic, presumed ischemic, peripheral damage, 5 with incomplete central (fascicular) palsy caused by brainstem lesions, and 10 controls were studied. Palsies under 1 month in duration were designated as acute and those of longer duration were chronic. Among peripheral palsies, five were acute, nine were chronic. Among central palsies, two were acute, three were chronic. Subjects made +/- 10 deg horizontal saccades while wearing search coils. Serial recordings were made in seven patients with acute palsy (five peripheral, two central). RESULTS: Centrifugal abducting saccadic velocities in the paretic eye were subnormal in both central and peripheral acute palsies, as anticipated from lateral rectus weakness. In chronic central palsies, abducting velocities in the paretic eye remained reduced. However, in chronic peripheral palsies, velocities became normal in the tested range of excursion, within 2 months of onset, despite persisting abduction deficit. CONCLUSIONS: Saccade peak velocities are reduced and their durations are prolonged in the field of action of acutely palsied peripheral and central nerves. Speeds remain reduced in chronic central (fascicular) palsies, consistent with limited regeneration within the brain. Saccade speeds are repaired in chronic peripheral palsies, probably by remyelination and axonal regeneration, and perhaps also by central monocular adaptation of innervation selectively to the paretic eye, in order to drive both eyes rapidly and simultaneously into the paretic field of motion. PMID- 16682673 TI - Coronary artery disease is associated with Alzheimer disease neuropathology in APOE4 carriers. AB - OBJECTIVE: To examine the associations between postmortem Alzheimer disease (AD) neuropathology and autopsy-verified cardiovascular disease. METHODS: The authors examined 99 subjects (mean age at death = 87.6; SD = 8.7) from the Mount Sinai School of Medicine Department of Psychiatry Brain Bank who were devoid of cerebrovascular disease-associated lesions or of non-AD-related neuropathology. Density of neuritic plaques (NPs) and neurofibrillary tangles (NFTs) as well as coronary artery and aortic atherosclerosis, left ventricular wall thickness, and heart weight were measured. Partial correlations were used to assess the associations of the four cardiovascular variables with NPs and NFTs in the hippocampus, entorhinal cortex, and multiple regions of the cerebral cortex after controlling for age at death, sex, dementia severity, body mass index, and ApoE genotype. These analyses were also repeated separately for ApoE4 carriers and noncarriers. RESULTS: The extent of coronary artery disease and to a lesser extent atherosclerosis were significantly associated with the density of cardinal neuropathologic lesions of AD in this autopsy sample (significant correlations between 0.22 and 0.29). These associations were more pronounced for the ApoE4 allele carriers (n = 42; significant correlations between 0.34 and 0.47). CONCLUSIONS: The degree of coronary artery disease is independently associated with the cardinal neuropathological lesions of Alzheimer disease. These associations are primarily attributable to individuals with the ApoE4 allele. PMID- 16682674 TI - Multi-detector CT angiography in intracranial dural AV fistula at the foramen magnum. PMID- 16682675 TI - Trying to tell a tale: discourse impairments in progressive aphasia and frontotemporal dementia. AB - OBJECTIVE: To assess discourse in patients with frontotemporal dementia (FTD). METHODS: The authors asked patients with progressive nonfluent aphasia (PNFA), patients with semantic dementia (SemD), and nonaphasic patients with a disorder of social comportment and executive functioning (SOC/EXEC) to narrate the story of a wordless children's picture book. RESULTS: The authors found significant discourse impairments in all three groups of patients. Moreover, there were qualitatively important differences between the groups. Patients with PNFA had the sparsest output, producing narratives with the fewest words per minute. Patients with SemD had difficulty retrieving words needed to tell their narratives. Though not aphasic, patients with SOC/EXEC had profound difficulty organizing their narratives, and they could not effectively express the point of the story. This deficit correlated with poor performance on a measure of executive resources requiring an organized mental search. In addition, a correlation of narrative organization with cortical atrophy in patients with SOC/EXEC was significant in right frontal and anterior temporal brain regions. CONCLUSIONS: Impaired day-to-day communication in nonaphasic frontotemporal dementia patients with a disorder of social comportment and executive functioning is due in part to a striking deficit in discourse organization associated with right frontotemporal disease. Difficulty with discourse in progressive aphasia is due largely to the language impairments of these patients. PMID- 16682676 TI - Double dissociation between representational personal and extrapersonal neglect. AB - BACKGROUND: Although perceptual and representational neglect are frequently associated, the demonstration of a double dissociation between both neglect forms suggests that both rely on different central mechanisms. In addition, perceptual neglect can be selectively observed within personal space or extrapersonal space. However, it is not known whether the latter dissociation also exists in representational neglect. METHODS: The authors investigated this question in two brain-damaged patients with anatomically different lesions sites, using neuropsychological tests specifically designed to assess perceptual and representational neglect in both personal and extrapersonal space. RESULTS: Patients presented a double dissociation with respect to personal and extrapersonal space in representational neglect. CONCLUSIONS: These data suggest that the cerebral networks that process mental space representation use similar principles of space compartmentalization as those used by cerebral networks processing perceived space. PMID- 16682677 TI - Sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease. AB - BACKGROUND: The prevalence and characteristics of sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease (sCJD) are poorly understood. METHODS: Seven consecutive patients with definite sCJD underwent a systematic assessment of sleep-wake disturbances, including clinical history, video-polysomnography, and actigraphy. Extent and distribution of neurodegeneration was estimated by brain autopsy in six patients. Western blot analyses enabling classification and quantification of the protease-resistant isoform of the prion protein, PrPSc, in thalamus and occipital cortex was available in four patients. RESULTS: Sleep-wake symptoms were observed in all patients, and were prominent in four of them. All patients had severe sleep EEG abnormalities with loss of sleep spindles, very low sleep efficiency, and virtual absence of REM sleep. The correlation between different methods to assess sleep-wake functions (history, polysomnography, actigraphy, videography) was generally poor. Brain autopsy revealed prominent changes in cortical areas, but only mild changes in the thalamus. No mutation of the PRNP gene was found. CONCLUSIONS: This study demonstrates in sporadic Creutzfeldt-Jakob disease, first, the existence of sleep-wake disturbances similar to those reported in fatal familial insomnia in the absence of prominent and isolated thalamic neuronal loss, and second, the need of a multimodal approach for the unambiguous assessment of sleep-wake functions in these patients. PMID- 16682678 TI - Initial pharmacotherapy in a population of veterans with Parkinson disease. AB - The authors analyzed patient and prescribing provider characteristics for 530 veterans identified from VA pharmacy records with Parkinson disease (PD) and initial pharmacotherapy. Neurologists prescribed 29% of initial therapy. While a patient being younger and seeing a movement disorder specialist predicted receiving dopamine agonists, only 20% of patients younger than age 65 years received dopamine agonists. Initial pharmacotherapy is strongly influenced by the provider's specialty but mostly initiated by providers without PD expertise. PMID- 16682679 TI - Safety of rasagiline in elderly patients with Parkinson disease. AB - The authors examined age effects on adverse events from two randomized, controlled trials of rasagiline, comparing younger (younger than 70 years) and older (70 years and older) subjects. Older patients were more prone to serious adverse effects than younger patients, but there was no statistical interaction between age and rasagiline exposure. This absence of an age-rasagiline interaction suggests that rasagiline does not require special safety precautions for elderly subjects with Parkinson disease. PMID- 16682680 TI - Evaluation of sleep and daytime somnolence in spinocerebellar ataxia type 6 (SCA6). AB - To determine the frequency of subjective sleep problems in spinocerebellar ataxia type 6 (SCA6), the authors surveyed 25 patients and 25 age-matched controls with the Epworth Sleepiness Scale (ESS) and Pittsburgh Sleep Quality Index (PSQI). The ESS was higher in patients with SCA6 (9.12 +/- 5.80; CI: 2.28) than in controls (4.96 +/- 3.01; CI: 1.18) (p = 0.003, t test). The PSQI was higher in patients with SCA6 (7.96 +/- 4.65; CI: 1.86) than in controls (5.08 +/- 3.39; CI: 1.36) (p = 0.018, t test). PMID- 16682681 TI - Voglibose inhibits postprandial hypotension in neurologic disorders and elderly people. AB - The authors examined blood pressure, glucose, insulin, and neurotensin before and after intake of 75 g glucose with or without voglibose in 28 neurologic patients and 20 healthy controls. Voglibose significantly prevented hypotension and neurotensin increment after glucose intake and had no influence on glucose or insulin increment. These results suggest that voglibose benefits postprandial hypotension. PMID- 16682682 TI - MATILDE regimen followed by radiotherapy is an active strategy against primary CNS lymphomas. AB - The authors assessed MATILDE chemotherapy followed by response-tailored radiation therapy in 41 patients aged 70 years or younger with primary CNS lymphoma in a Phase II trial. With response rates of 76% after MATILDE and 83% after chemotherapy with or without radiation therapy, this was an active strategy, particularly in low- to intermediate-risk patients (International Extranodal Lymphoma Study Group [IELSG] score). Myelosuppression was the dose-limiting toxicity, with 9.5% of lethal complications. After a median follow-up of 49 months, a plateau in the survival curve (5-year overall survival: 41 +/- 7%) was obtained. PMID- 16682683 TI - POLG1, C10ORF2, and ANT1 mutations are uncommon in sporadic progressive external ophthalmoplegia with multiple mitochondrial DNA deletions. AB - The authors sequenced POLG1, C10ORF2, and ANT1 in 38 sporadic progressive external ophthalmoplegia patients with multiple mitochondrial DNA (mtDNA) deletions. Causative mutations were identified in approximately 10% of cases, with two unrelated individuals harboring a novel premature stop codon mutation (1356T>G). None had a mutation in C10ORF2 or ANT1. In the majority of patients, the primary nuclear genetic defect is likely to affect other unknown genes important for mtDNA maintenance. PMID- 16682684 TI - Lunar phases and seizure occurrence: just an ancient legend? AB - The authors retrospectively reviewed all neurologic records of an emergency unit from 1999 to 2003 to identify a potential association between lunar phases and seizure occurrence. Overall 859 patients admitted for seizure occurrence were divided into the four quarters of the synodic month according to moon phases. A significant clustering of seizures around the full moon period was observed, supporting the ancient belief of periodic increased seizure frequency during full moon days. PMID- 16682685 TI - Perception of motor strength and stimulus magnitude are correlated in stroke patients. AB - The authors examined whether perception of contralateral limb strength is altered and whether perception of strength correlates with perception of stimulus intensity (magnitude) in a prospective sample of patients with unilateral right (RHL: n = 13) and left (LHL: n = 6) hemisphere lesions due to stroke. Patients with RHL tended to overestimate strength and patients with LHL tended to underestimate strength; both patterns were highly correlated with altered perception of stimulus magnitude. PMID- 16682687 TI - Eyelid apraxia associated with subthalamic nucleus deep brain stimulation. PMID- 16682686 TI - Variable benefit in neuropsychological function in HIV-infected HAART-treated patients. AB - The authors examined cognitive performance change in 101 individuals with advanced HIV infection on highly active antiretroviral therapy (HAART), using standard neuropsychological testing in three visits, over a 27-month-period. Cognitive performance stabilized in a majority of HIV+ participants over time. A neuroactive HAART regimen was associated with neuropsychological improvement. Decline occurred in a minority with lower nadir CD4. The current CD4 count and plasma viral load were not associated with cognitive change. PMID- 16682688 TI - Neuropathy progressing to myeloneuropathy 20 years after partial gastrectomy. PMID- 16682689 TI - What's the use? PMID- 16682690 TI - Sildenafil increases cerebrovascular reactivity: a transcranial Doppler study. PMID- 16682691 TI - Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium. PMID- 16682692 TI - Stiff child syndrome with mutation of DYT1 gene. PMID- 16682693 TI - Does pain change the brain? PMID- 16682694 TI - Effect of mitoxantrone on MRI in progressive MS: results of the MIMS trial. PMID- 16682695 TI - Concurrent dolichoectasia of basilar and coronary arteries. PMID- 16682696 TI - An open study of botulinum-A toxin treatment of trigeminal neuralgia. PMID- 16682698 TI - Uncharted territory: Hippocratic ethics and health systems. PMID- 16682700 TI - Former director general: Health Canada needs broader drug safety powers. PMID- 16682701 TI - Alberta leads country in e-health records. PMID- 16682702 TI - Dispute over Vioxx study plays out in New England journal. PMID- 16682703 TI - Marijuana use by tobacco smokers and nonsmokers: who is smoking what? PMID- 16682704 TI - The Smoking Regulatory Index: a new way to measure public health performance. PMID- 16682705 TI - Strategies for preventing calcium oxalate stones. PMID- 16682706 TI - Raccoon roundworm. PMID- 16682707 TI - Ecchymotic groin plaques in an immunocompromised man. PMID- 16682708 TI - Effect of neighbourhood income and maternal education on birth outcomes: a population-based study. AB - BACKGROUND: Maternal socioeconomic status (SES) is an important determinant of inequity in maternal and fetal health. We sought to determine the extent to which associations between adverse birth outcomes and SES can be identified using individual-level measures (maternal level of education) and community-level measures (neighbourhood income). METHODS: In Quebec, the birth registration form includes a field for the mother's years of education. Using data from birth registration certificates, we identified all births from 1991 to 2000. Using maternal postal codes that can be linked to census enumeration areas, we determined neighbourhood income levels that reflect SES. RESULTS: Lower levels of both maternal education and neighbourhood income were associated with elevated crude risks of preterm birth, small-for-gestational-age (SGA) birth, stillbirth and neonatal and postneonatal death. The effects of maternal education were stronger than, and independent of, those of neighbourhood income. Compared with women in the highest neighbourhood income quintile, women in the lowest quintile were significantly more likely to have a preterm birth (adjusted odds ratio [OR] 1.14, 95% confidence interval [CI] 1.10-1.17), SGA birth (OR 1.18, 95% CI 1.15 1.21) or stillbirth (OR 1.30, 95% CI 1.13-1.48); compared with mothers who had completed community college or at least some university, mothers who had not completed high school were significantly more likely to have a preterm birth (adjusted OR 1.48, 95% CI 1.44-1.52), SGA birth (OR 1.86, 95% CI 1.82-1.91) or stillbirth (OR 1.54, 95% CI 1.36-1.74). INTERPRETATION: Individual and, to a lesser extent, neighbourhood-level SES measures are independent indicators for subpopulations at risk of adverse birth outcomes. Women with lower education levels and those living in poorer neighbourhoods are more vulnerable to adverse birth outcomes and may benefit from heightened clinical vigilance and counselling. PMID- 16682709 TI - Efficacy and safety of unfractionated heparin versus enoxaparin: a pooled analysis of ASSENT-3 and -3 PLUS data. AB - BACKGROUND: The optimal antithrombotic therapy to accompany tenecteplase in cases of acute ST-segment elevation myocardial infarction (STEMI) remains unclear. We undertook a prespecified pooled analysis of data from the ASSENT-3 and ASSENT-3 PLUS trials. METHODS: We created a combined database of the 2040 and 818 patients who received enoxaparin in ASSENT-3 and ASSENT-3 PLUS, respectively, and compared them with the 2038 and 821 patients who received unfractionated heparin. RESULTS: The efficacy end point (a composite of 30-day mortality, reinfarction or refractory ischemia) was 12.2% with enoxaparin versus 16.0% with unfractionated heparin (p < 0.001); the combined end point of efficacy plus safety (a composite of 30-day mortality, reinfarction, refractory ischemia, intracranial hemorrhage [ICH] or major systemic bleeding) was 15.0% versus 18.0%, respectively (p = 0.003) [corrected] The 1049 patients urgently revascularized had greater benefit from enoxaparin (15.4% v. 10.1%, p = 0.013), yet the excess in major systemic bleeding evident with enoxaparin (3.3% v. 2.4%, p = 0.01) was largely confined to the 3492 patients without or before revascularization. Although ICH rates in the groups were similar (1.3% v. 0.9%, p = 0.26), an excess of ICH occurred among those administered enoxaparin during the ASSENT-3 PLUS trial (6.7% v. 0.8%, p = 0.013), especially among women over 75 years of age. INTERPRETATION: These data demonstrated the benefit of enoxaparin used in conjunction with tenecteplase, but raised caution about its prehospital use to treat STEMI in elderly women. PMID- 16682710 TI - Socioeconomic inequality in birth outcomes: what do the indicators tell us, and where do we find the data? PMID- 16682711 TI - Low-molecular-weight heparins as adjunctive therapy to thrombolytics: extracting the best data. PMID- 16682713 TI - Diabetes and the proactive laboratory. PMID- 16682714 TI - Maternal mortality: an important priority. PMID- 16682712 TI - Current management of the complications of portal hypertension: variceal bleeding and ascites. AB - Portal hypertension is one of the main consequences of cirrhosis. It results from a combination of increased intrahepatic vascular resistance and increased blood flow through the portal venous system. The condition leads to the formation of portosystemic collateral veins. Esophagogastric varices have the greatest clinical impact, with a risk of bleeding as high as 30% within 2 years of medium or large varices developing. Ascites, another important complication of advanced cirrhosis and severe portal hypertension, is sometimes refractory to treatment and is complicated by spontaneous bacterial peritonitis and hepatorenal syndrome. We describe the pathophysiology of portal hypertension and the current management of its complications, with emphasis on the prophylaxis and treatment of variceal bleeding and ascites. PMID- 16682717 TI - Yes to "febrile," no to "flu-like". PMID- 16682718 TI - Sex ratio for medical residencies. PMID- 16682719 TI - 2006 update of recommendations for the use of white blood cell growth factors: an evidence-based clinical practice guideline. AB - PURPOSE: To update the 2000 American Society of Clinical Oncology guideline on the use of hematopoietic colony-stimulating factors (CSF). UPDATE METHODOLOGY: The Update Committee completed a review and analysis of pertinent data published from 1999 through September 2005. Guided by the 1996 ASCO clinical outcomes criteria, the Update Committee formulated recommendations based on improvements in survival, quality of life, toxicity reduction and cost-effectiveness. RECOMMENDATIONS: The 2005 Update Committee agreed unanimously that reduction in febrile neutropenia (FN) is an important clinical outcome that justifies the use of CSFs, regardless of impact on other factors, when the risk of FN is approximately 20% and no other equally effective regimen that does not require CSFs is available. Primary prophylaxis is recommended for the prevention of FN in patients who are at high risk based on age, medical history, disease characteristics, and myelotoxicity of the chemotherapy regimen. CSF use allows a modest to moderate increase in dose-density and/or dose-intensity of chemotherapy regimens. Dose-dense regimens should only be used within an appropriately designed clinical trial or if supported by convincing efficacy data. Prophylactic CSF for patients with diffuse aggressive lymphoma aged 65 years and older treated with curative chemotherapy (CHOP or more aggressive regimens) should be given to reduce the incidence of FN and infections. Current recommendations for the management of patients exposed to lethal doses of total body radiotherapy, but not doses high enough to lead to certain death due to injury to other organs, includes the prompt administration of CSF or pegylated G-CSF. PMID- 16682720 TI - Oral second-line chemotherapy for advanced non-small-cell lung cancer: the bottom line. PMID- 16682721 TI - Topoisomerase IIalpha gene amplification and response to anthracycline-containing adjuvant chemotherapy in breast cancer. PMID- 16682722 TI - When the risk of febrile neutropenia is 20%, prophylactic colony-stimulating factor use is clinically effective, but is it cost-effective? PMID- 16682723 TI - FCGR2A polymorphism is correlated with clinical outcome after immunotherapy of neuroblastoma with anti-GD2 antibody and granulocyte macrophage colony stimulating factor. AB - PURPOSE: Anti-GD2 murine IgG3 antibody 3F8 kills neuroblastoma cells by antibody dependent cell-mediated cytotoxicity (ADCC). Granulocyte macrophage colony stimulating factor (GM-CSF) enhances phagocyte-mediated ADCC. The differential affinity of the human FCGR polymorphic alleles for 3F8 may influence the effectiveness of antibody immunotherapy. PATIENTS AND METHODS: The entire cohort of high risk neuroblastoma patients (N = 136) treated on protocol using 3F8 and GM-CSF were the subjects of this analysis. Tumor response was measured by standard clinical tools plus sensitive molecular monitoring using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Polymorphic alleles of FCGR2A and FCGR3A were determined by PCR plus direct sequencing using genomic DNA samples obtained from marrow or blood of patients. RESULTS: FCGR2A (R/R) genotype correlated with progression-free survival for the entire cohort (P = .049) and for the subset of patients with no history of prior relapse (P = .023). FCGR2A (R/R) also correlated with marrow remission 2.5 months after treatment initiation: by histology (P = .021 and P = .036, for the entire cohort and the subset, respectively) and by qRT-PCR (P = .052 and P = .033, respectively). CONCLUSION: The favorable outcome associated with FCGR2A (R/R) genotype is consistent with the proposed role of FCGR2A and phagocyte-mediated ADCC in 3F8 plus GM-CSF immunotherapy. PMID- 16682724 TI - Quantitative fluoroestradiol positron emission tomography imaging predicts response to endocrine treatment in breast cancer. AB - PURPOSE: In breast cancer, [(18)F]fluoroestradiol (FES) positron emission tomography (PET) correlates with estrogen receptors (ER) expression and predicts response to tamoxifen. We tested the ability of FES-PET imaging to predict response to salvage hormonal treatment in heavily pretreated metastatic breast cancer patients, predominantly treated with aromatase inhibitors. PATIENTS AND METHODS: Initial FES uptake measurements in 47 patients with ER-positive tumors were correlated with subsequent tumor response to 6 months of hormonal treatment. Most patients had bone dominant disease and prior tamoxifen exposure. Response was compared to initial FES-PET uptake, measured qualitatively and quantitatively using standardized uptake value (SUV) and estradiol-binding flux. RESULTS: Eleven of 47 patients (23%) had an objective response. While no patients with absent FES uptake had a response to treatment, the association between qualitative FES-PET results and response was not significant (P = .14). However, quantitative FES uptake and response were significantly associated; zero of 15 patients with initial SUV less than 1.5 responded to hormonal therapy, compared with 11 of 32 patients (34%) with SUV higher than 1.5 (P < .01). In the subset of patients whose tumors did not overexpress HER2/neu, 11 of 24 patients (46%) with SUV higher than 1.5 responded. CONCLUSION: Quantitative FES-PET can predict response to hormonal therapy and may help guide treatment selection. Treatment selection using quantitative FES-PET in our patient series would have increased the rate of response from 23% to 34% overall, and from 29% to 46% in the subset of patients lacking HER2/neu overexpression. A multi-institutional collaborative trial would permit definitive assessment of the value of FES-PET for therapeutic decision making. PMID- 16682725 TI - Cost-effectiveness of adding granulocyte colony-stimulating factor to primary prophylaxis with antibiotics in small-cell lung cancer. AB - PURPOSE: Recently, a Dutch, randomized, phase III trial demonstrated that, in small-cell lung cancer patients at risk of chemotherapy-induced febrile neutropenia (FN), the addition of granulocyte colony-stimulating factor (GCSF) to prophylactic antibiotics significantly reduced the incidence of FN in cycle 1 (24% v 10%; P = .01). We hypothesized that selecting patients at risk of FN might increase the cost-effectiveness of GCSF prophylaxis. METHODS: Economic analysis was conducted alongside the clinical trial and was focused on the health care perspective. Primary outcome was the difference in mean total costs per patient in cycle 1 between both prophylactic strategies. Cost-effectiveness was expressed as costs per percent-FN-prevented. RESULTS: For the first cycle, the mean incremental costs of adding GCSF amounted to 681 euro (95% CI, -36 to 1,397 euro) per patient. For the entire treatment period, the mean incremental costs were substantial (5,123 euro; 95% CI, 3,908 to 6,337 euro), despite a significant reduction in the incidence of FN and related savings in medical care consumption. The incremental cost-effectiveness ratio was 50 euro per percent decrease of the probability of FN (95% CI, -2 to 433 euro) in cycle 1, and the acceptability for this willingness to pay was approximately 50%. CONCLUSION: Despite the selection of patients at risk of FN, the addition of GCSF to primary antibiotic prophylaxis did not result in cost savings. If policy makers are willing to pay 240 euro for each percent gain in effect (ie, 3,360 euro for a 14% reduction in FN), the addition of GCSF can be considered cost effective. PMID- 16682726 TI - Pegylated liposomal doxorubicin and trastuzumab in HER-2 overexpressing metastatic breast cancer: a multicenter phase II trial. AB - PURPOSE: Cardiotoxicity precludes the concurrent use of doxorubicin and trastuzumab. Because pegylated liposomal doxorubicin (PLD) has equal efficacy but significantly less cardiotoxicity than conventional doxorubicin, this phase II study assessed the rate of cardiotoxicity and efficacy of first-line PLD plus trastuzumab in HER-2-positive metastatic breast cancer (MBC). PATIENTS AND METHODS: Women with HER-2-positive, measurable MBC, and a baseline left ventricular ejection fraction (LVEF) > or = 55% were treated with PLD 50 mg/m2 every 4 weeks for six cycles and weekly trastuzumab (4 mg/kg loading dose then 2 mg/kg thereafter). Cardiotoxicity was defined as symptomatic congestive heart failure (CHF) with > or = 10% decline in LVEF to below lower limits of normal, > or = 15% decline in LVEF without symptomatic CHF, or less than 10% LVEF decline to less than 45%. RESULTS: Thirty women were enrolled, 13 had received prior adjuvant anthracyclines. A median 5.5 cycles of PLD were administered. Mean baseline LVEF was 62.8%, 59.5% after cycle four, and 58.3% after cycle six. Three patients (10%) developed protocol-defined cardiotoxicity. No patients developed symptomatic CHF. Response rate was 52%, with an additional 38% stable disease rate. At a median follow-up of 13.9 months, the median progression-free survival was 12.0 months; median overall survival has not yet been reached. The most common adverse events were grade 3 hand-foot syndrome (30%) and grade 3/4 neutropenia (27%). CONCLUSION: The combination of PLD and trastuzumab is a well tolerated and active regimen in HER-2-positive MBC. Cardiotoxicity was observed, but limited to asymptomatic declines in LVEF. Further evaluation of this combination is warranted. PMID- 16682727 TI - Phase III study comparing oral topotecan to intravenous docetaxel in patients with pretreated advanced non-small-cell lung cancer. AB - PURPOSE: This open-label, randomized, multicenter, phase III study compared oral topotecan versus intravenous (IV) docetaxel in patients with previously treated non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with stage III or IV NSCLC, performance status < or = 2, who had received only one prior chemotherapy regimen, were randomly assigned to treatment with oral topotecan 2.3 mg/m2/d on days 1 to 5 or IV docetaxel 75 mg/m2 day 1 every 21 days. RESULTS: A total of 829 patients were randomly assigned. In intent-to-treat analysis, 1-year survival rates were 25.1% with topotecan and 28.7% with docetaxel. The difference of -3.6% (95% CI, -9.59% to 2.48%) met the predefined criteria for noninferiority of topotecan relative to docetaxel because the lower limit of the 95% CI was above -10%. Median survival was 27.9 weeks with topotecan and 30.7 weeks with docetaxel. Although not statistically significant (log-rank P = .057), the higher survival rate with docetaxel was maintained across the entire treatment period. The median time to progression was 11.3 weeks with topotecan versus 13.1 weeks with docetaxel (log-rank P = .02). The overall response rate was 5% in each group. Grade 3/4 neutropenia occurred more frequently with docetaxel (60% v 50%). Grade 3/4 anemia and thrombocytopenia occurred more frequently with topotecan (26% v 10% and 26% v 7%, respectively). CONCLUSION: Oral topotecan provides activity in the treatment of relapsed, locally advanced, unresectable NSCLC. Both regimens were well tolerated with differing safety profiles. Topotecan may provide an option for patients who desire an orally available treatment after relapse. PMID- 16682728 TI - Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER 2/neu-amplified breast cancer: Scandinavian Breast Group Trial 9401. AB - PURPOSE: Amplification of the HER-2/neu and topoisomerase IIalpha (TOP2A) genes has been linked to the effects of anthracyclines. Their role in predicting the outcome of anthracycline-based adjuvant chemotherapy for breast cancer patients has remained controversial. PATIENTS AND METHODS: The present substudy of the Scandinavian Breast Group trial 9401, in which an epirubicin-based regimen (nine courses of tailored and dose-escalated fluorouracil, epirubicin, and cyclophosphamide [FEC]) was compared with three or four courses of standard FEC followed by bone marrow-supported high-dose chemotherapy (cyclophosphamide, thiotepa, and carboplatin), included high-risk breast cancer patients (with eight or more positive axillary lymph nodes or at least five nodes with additional poor prognostic indicators). Amplification of HER-2/neu was determined retrospectively in paraffin-embedded tumor tissue sections by chromogenic in situ hybridization. TOP2A was tested only in HER-2/neu-amplified tumors. RESULTS: HER-2/neu amplification alone, which was present in 32.7% of the tumors, was a strong prognostic factor for short relapse-free (P = .0034) and overall survival (P = .0008) but showed no direct association with treatment assignment. TOP2A coamplification, which was present in 37% of HER-2/neu-amplified tumors, was associated with better relapse-free survival in patients treated with tailored and dose-escalated FEC (hazard ratio [HR] = 0.45; P = .049). A statistical multivariate Cox's regression analysis confirmed the predictive significance of TOP2A coamplification (HR = 0.30; P = .020) in HER-2/neu-amplified tumors. There was no such association in patients with HER-2/neu-amplified tumors without TOP2A gene amplification. CONCLUSION: Coamplification of HER-2/neu and TOP2A may define a subgroup of high-risk breast cancer patients who benefit from individually tailored and dose-escalated adjuvant anthracyclines. PMID- 16682729 TI - Long-term follow-up after reduced-intensity conditioning allogeneic transplantation for acute myeloid leukemia/myelodysplastic syndrome: late CNS relapses despite graft-versus-host disease. PMID- 16682730 TI - Don't deny liver transplantation to HIV patients with hepatocellular carcinoma in the highly active antiretroviral therapy era. PMID- 16682731 TI - Disparities in cancer care: a worldwide perspective and roadmap for change. PMID- 16682732 TI - Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. AB - Efforts to reduce global cancer disparities begin with an understanding of geographic patterns in cancer incidence, mortality, and prevalence. Using the GLOBOCAN (2002) and Cancer Incidence in Five Continents databases, we describe overall cancer incidence, mortality, and prevalence, age-adjusted temporal trends, and age-specific incidence patterns in selected geographic regions of the world. For the eight most common malignancies-cancers of lung, breast, colon and rectum, stomach, prostate, liver, cervix, and esophagus-the most important risk factors, cancer prevention and control measures are briefly reviewed. In 2002, an estimated 11 million new cancer cases and 7 million cancer deaths were reported worldwide; nearly 25 million persons were living with cancer. Among the eight most common cancers, global disparities in cancer incidence, mortality, and prevalence are evident, likely due to complex interactions of nonmodifiable (ie, genetic susceptibility and aging) and modifiable risk factors (ie, tobacco, infectious agents, diet, and physical activity). Indeed, when risk factors among populations are intertwined with differences in individual behaviors, cultural beliefs and practices, socioeconomic conditions, and health care systems, global cancer disparities are inevitable. For the eight most common cancers, priorities for reducing cancer disparities are discussed. PMID- 16682733 TI - Interpreting disparate responses to cancer therapy: the role of human population genetics. AB - Increasingly, investigators are recognizing differences in tumor biology, drug metabolism, toxicity, and therapeutic response among different patient populations receiving anticancer agents. These observations provide exciting opportunities to identify the factors most important for predicting individual variability in pharmacologically relevant phenotypes and consequently for personalizing the delivery of cancer therapy. Although pharmacogenomic differences may explain some of these disparities, rigorous investigation of both genetic and nongenetic differences is important to identify the variables most important for optimal selection and dosing of treatment for an individual patient. For example, pharmacogenetic tests currently used in cancer therapy, such as genotyping UGT1A1 to reduce the incidence of severe toxicity of irinotecan and sequencing epidermal growth factor receptor from tumors to identify somatic mutations conferring sensitivity to tyrosine kinase inhibitors, were developed without initial identification of interpopulation differences. Although interpopulation variability in toxicity and efficacy of these agents has been observed, the basis for these population differences remains only partially explained. Here, we review concepts of human population genetics to inform interpretations of disparate drug effects of cancer therapy across patient populations. Understanding these principles will help investigators better design clinical trials to identify the variables most relevant to subsequent individualization of a cancer therapy. PMID- 16682734 TI - Ethnic differences in response to epidermal growth factor receptor tyrosine kinase inhibitors. AB - The identification of somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) and their correlation with response to EGFR inhibitors has become an important event in the fields of cancer genetics and therapeutics. The initial observation of a higher response to gefitinib and erlotinib in patients of Asian origin was followed by the discovery that they harbor more frequent EGFR mutations in NSCLC; this raises the issue of ethnic diversity in the pathogenesis of given tumors. In a similar fashion, amplification of the closely related HER2 gene, which could also have implications for the treatment of NSCLC, is also more frequent in East Asian patients. On the other hand, EGFR gene amplification may be more prevalent in Western populations. The implication of these findings is that ethnicity may indicate different genetic backgrounds in common tumors that may influence clinical outcome and response to therapy. Therefore, in clinical trials with tyrosine kinase inhibitors and other molecular-targeted therapies, the inclusion of a global population appears to be required. PMID- 16682735 TI - Genetics, epidemiology, and cancer disparities: is it black and white? AB - Epidemiologic studies attempt to understand the distribution and determinants of human disease. Epidemiologic research often incorporates information about race, ethnicity, or ancestry, usually as a self-identified race or ethnicity (SIRE) variable. Differences in the distribution and determinants of disease on the basis of SIRE may be identified in these studies. In addition, genetic and other biologic differences according to SIRE are frequently reported. If these differences are real and meaningful, they may have value in identifying disease causative or -preventive factors, and thus may be beneficial to human health. However, the concepts of race, ethnicity, or ancestry are often poorly considered or crudely applied, particularly in genetic studies of disease etiology or outcome. Consequently, results suggesting genetic differences with respect to disease etiology or outcome across SIRE groups may not be meaningful; in fact, these differences may prove harmful if they propagate stereotypes or spurious differences. Therefore, it is critical to properly consider the meaning, definitions, and use of race, ethnicity, or ancestry in molecular epidemiologic studies. PMID- 16682736 TI - Racial and ethnic disparities in breast cancer mortality: are we doing enough to address the root causes? AB - Breast cancer is the most common noncutaneous malignancy and the second most lethal form of cancer among women in the United States. Mortality from breast cancer has declined since the late 1980s, but this decline has been steeper among white women compared with black women. As a result, the black:white mortality rate ratio has increased over the last two decades. Other ethnic minorities also suffer from disproportionately high breast cancer mortality rates. This review discusses the causes of racial and ethnic disparities in breast cancer mortality and describes the most common approaches to reducing these disparities. The literature suggests that outcome disparities are related to patient-, provider-, and health system-level factors. Lack of insurance, fear of testing, delay in seeking care, and unfavorable tumor characteristics all contribute to disparities at the patient level. At the provider level, insufficient screening, poor follow up of abnormal screening tests, and nonadherence to guideline-based treatments add to outcome disparities. High copayment requirements, lack of a usual source of care, fragmentation of care, and uneven distribution of screening and treatment resources exacerbate disparities at the health system level. Although pilot programs have increased breast cancer screening among select populations, persistent disparities in mortality suggest that changes are needed at the policy level to address the root causes of these disparities. PMID- 16682737 TI - Colorectal cancer model of health disparities: understanding mortality differences in minority populations. AB - African Americans are more likely to be diagnosed with and die as a result of colorectal cancer than white patients. This review briefly documents these differences and explores the factors that may contribute to advanced stage at diagnosis and reduced survival once African Americans are diagnosed with colorectal cancer. Attention is focused on what is known about the role of socioeconomic status, cancer screening, comorbidities and lifestyle factors, tumor biology and genetics, and the differences in the receipt of and benefit of appropriate therapy. Finally, areas of ongoing and future research and policy initiatives aimed at reducing disparities are discussed. PMID- 16682738 TI - Disparities in gastric cancer chemotherapy between the East and West. AB - There are still remarkable disparities in the treatment of gastric cancer between the East and West. Treatment outcomes for this disease have improved in Japan due to early detection and surgical resection with systematic node dissections, such as D2, whereas gastric cancer remains a virulent disease in Western countries. Differences in the types of surgery and their outcomes affect how adjuvant trials are conducted and interpreted. Recent Western randomized trials demonstrated the significant survival benefit of adjuvant chemoradiotherapy or intensive combination chemotherapy. However, baseline surgical quality and outcomes were quite different from those in Japan, and Japanese surgical/medical oncologists have not accepted the Western results. Several disparities are also evident in the results of chemotherapy trials for advanced gastric cancer. Although similar results were obtained with randomized studies using older regimens, the interpretation of the results differed between Japan and other countries. A combination of cisplatin and fluorouracil was used as the reference arm in ongoing randomized trials in most countries, whereas single-agent fluorouracil or S-1 alone was used in Japanese trials. Two triplet regimens have already demonstrated significant prolongation of survival in Western studies. However, these benefits seem to be marginal and these regimens may be replaced by newer regimens, which will soon be available in Europe and Asia, where a total of 2,600 patients have been accrued. Although these disparities between regions must be overcome, it is time for both Eastern and Western investigators to pursue further benefits by incorporating new agents into treatment regimens. PMID- 16682739 TI - Disparities in genetic testing: thinking outside the BRCA box. AB - The impact of predictive genetic testing on cancer care can be measured by the increased demand for and utilization of genetic services as well as in the progress made in reducing cancer risks in known mutation carriers. Nonetheless, differential access to and utilization of genetic counseling and cancer predisposition testing among underserved racial and ethnic minorities compared with the white population has led to growing health care disparities in clinical cancer genetics that are only beginning to be addressed. Furthermore, deficiencies in the utility of genetic testing in underserved populations as a result of limited testing experience and in the effectiveness of risk-reducing interventions compound access and knowledge-base disparities. The recent literature on racial/ethnic health care disparities is briefly reviewed, and is followed by a discussion of the current limitations of risk assessment and genetic testing outside of white populations. The importance of expanded testing in underserved populations is emphasized. PMID- 16682740 TI - Between and within: international perspectives on cancer and health disparities. AB - The purpose of this article is to compare reasons for cancer health disparities in developing and developed countries. By 2010, approximately 60% of new cancer cases will occur in the developing world, higher than rates developed countries. However, disparities exist not only between countries but also within countries. Cancer epidemiology in developing countries is paradoxical: Increased incidence is partially due to increased development resulting in longer life expectancy and unhealthy lifestyle behaviors. Reduced mortality from infectious diseases results in relatively greater mortality from chronic diseases. However, infectious diseases are also risk factors for the leading causes of cancer mortality in these countries. While health disparities in developing versus developed countries are quantitatively worlds apart, they are qualitatively rather similar. They share common causes, such as environmental pollution, the need for social justice, large gaps between the rich and the poor, lack of access to cancer resources, and health services that are available to some but not to all. While industrialization and urbanization elevate a country's economic base while contributing to cancer incidence and mortality. Strategies to reduce international cancer disparities include country- and regional-level interventions, utilizing nongovernmental organizations, and developing long-term inter-institutional partnerships. Although economic aid is undoubtedly necessary, it is not sufficient to control cancer in the developing world. To address these problems, it will be necessary to focus attention on what can be done locally within countries, not only between countries. PMID- 16682741 TI - Reducing cancer disparities for minorities: a multidisciplinary research agenda to improve patient access to health systems, clinical trials, and effective cancer therapy. AB - Ethnic diversity in recruitment is a vital prerequisite to eliminating health disparities in cancer treatment, control and prevention programs. Much anecdotal reporting, but little scientific rigor, has been applied to the study of methods to improve the recruitment of minorities into cancer control or treatment trials. Even the most innovative research is stymied by the lack of representative samples of the populations that the research is designed to serve. The goals of this article are to describe a theory-driven framework for improving minority recruitment to clinical and cancer control trials, to explain organizational prerequisites to improving minority recruitment, and to provide empirical evidence of success in initial efforts to recruit to cancer control studies. These programs are offered as models for improving minority recruitment to cancer control and -treatment trials, and minority access to cancer treatment in general. PMID- 16682742 TI - Post-traumatic stress symptoms during treatment in mothers of children with leukemia. PMID- 16682743 TI - Neoadjuvant chemotherapy for metastatic colon cancer: too much caution and still too much to be assessed. PMID- 16682744 TI - ASEtrap: a biological method for speeding up the exploration of spliceomes. AB - Alternative splicing (AS) of pre-messenger RNA is a major mechanism for generating protein diversity from a limited number of genes in higher eukaryotes, and it constitutes a central mode of genetic regulation. Thus, efficient methods are needed to systematically identify new AS events at a genomic scale across different tissues, stages of development, and physiological or pathological conditions in order to better understand gene expression. To fulfill this goal, we have designed the ASEtrap, which is a cloning procedure for producing AS libraries that is based on a single-stranded trap consisting of an ssDNA-binding protein. In this paper, we have applied our approach to the construction of an AS library and a Control library from human placenta. By analyzing 9226 and 9999 sequences of the AS and Control libraries, respectively, we show that internal AS events (events that can be identified by the sole resources provided by either the AS or the Control library) and the discovery rate of new AS events measured at early stages of sequencing were nine to 10 times higher in the former than in the latter. Moreover, by performing a search for new AS events within a group of 162 known drug target genes, we identified six new events in six genes, and we observed that they all were discovered exclusively through the AS library. Thus, it appears that ASEtrap has the potential to greatly facilitate the determination of the total complement of splice variants expressed in human, as well as other organisms. PMID- 16682745 TI - Isolation and characterization of human apolipoprotein M-containing lipoproteins. AB - Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDL(apoM+)) contained significantly more free cholesterol than HDL lacking apoM (HDL(apoM-)) (5.9 +/- 0.7% vs. 3.2 +/- 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDL(apoM+) inhibited Cu(2+)-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDL(apoM-). In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM. PMID- 16682746 TI - Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras. AB - The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain. PMID- 16682748 TI - Microsatellite mapping of the genes for brittle rachis on homoeologous group 3 chromosomes in tetraploid and hexaploid wheats. AB - The brittle rachis character, which causes spontaneous shattering of spikelets, has an adaptive value in wild grass species. The loci Br1 and Br2 in durum wheat (Triticum durum Desf.) and Br3 in hexaploid wheat (T. aestivum L.) determine disarticulation of rachides above the junction of the rachilla with the rachis such that a fragment of rachis is attached below each spikelet. Using microsatellite markers, the loci Br1, Br2 and Br3 were mapped on the homoeologous group 3 chromosomes. The Br2 locus was located on the short arm of chromosome 3A and linked with the centromeric marker, Xgwm32, at a distance of 13.3 cM. The Br3 locus was located on the short arm of chromosome 3B and linked with the centromeric marker, Xgwm72 (at a distance of 14.2 cM). The Br1 locus was located on the short arm of chromosome 3D. The distance of Br1 from the centromeric marker Xgdm72 was 25.3 cM. Mapping the Br1, Br2 and Br3 loci of the brittle rachis suggests the homoeologous origin of these 3 loci for brittle rachides. Since the genes for brittle rachis have been retained in the gene pool of durum wheat, the more closely linked markers with the brittle rachis locus are required to select against brittle rachis genotypes and then to avoid yield loss in improved cultivars. PMID- 16682747 TI - Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach. AB - Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO(3)](-)) in the negative ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/mul concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies. PMID- 16682749 TI - Effects of heavy metal pollution on genetic variation and cytological disturbances in the Pinus sylvestris L. population. AB - This isoenzymatic and cytogenetic study has shown significant differences in genetic composition between two groups of Pinus sylvestris trees: tolerant and sensitive to heavy metal pollution. Total and mean numbers of alleles and genotypes per locus were higher in the pollution-sensitive group of trees, but heterozygosity (Ho) was lower in this group. Fixation index (F) indicates that trees tolerant for pollution were in the Hardy-Weinberg equilibrium, while the sensitive group had a significant excess of homozygosity. Cytological analyses demonstrated numerous aberrations of chromosomes in meristematic root tissue of seedlings developed from seeds collected from trees in the polluted area. The aberrations included chromosome bridges and stickiness, laggards, retarded and forward chromosomes, and their fragments. The mitotic index was markedly lower in this group of seedlings, as compared to the control. Both isoenzymatic and cytological analyses showed a significant influence of heavy metal ions on the genetic structure of the Pinus sylvestris population. PMID- 16682750 TI - Screening for mutations affecting sexual reproduction after activation tagging in Arabidopsis thaliana. AB - In this work, a seed-set-based screening was performed on 70 lines of Arabidopsis thaliana after activation tagging mutagenesis to identify mutations in reproductive mechanisms. Five mutants showed significantly lower seed set than the wild type and confirmed the phenotype in the progeny. This phenotype was linked with the marker gene bar carried by T-DNA conferring glufosinate resistance. Genetic analysis revealed that the mutation inheritance was sporophytic in 3 mutants and gametophytic in 2 mutants. In addition, 2 mutants had an extra T-DNA copy. Thus activation tagging can be an effective strategy to identify new mutations affecting sporogenesis or gametogenesis. PMID- 16682752 TI - A novel mitochondrial intergenic spacer reflecting population structure of Pacific oyster. AB - Nucleotide sequence divergence in a novel major mitochondrial DNA intergenic spacer (IGS) of Pacific oyster Crassostrea gigas was analyzed for 29 cultured individuals within the Goseong population (Korea). A total of 7 variable sites were detected within the IGS, and the relative frequency of nucleotide alteration was determined to be 1.16%;. All alterations were due to a single nucleotide substitution, and 5 transitions and 2 transversions were observed. Among 29 specimens, only 8 haplotypes could be identified, and 6 of the haplotypes were unique to particular specimens. Pairwise genetic diversity of all 8 haplotypes was calculated to be 0.412+/-0.134 from multiple sequence substitutions based on the two-parameter model. The phylogenetic tree obtained for these haplotypes according to the neighbor-joining method illustrated a single cluster of linkages, which comprised 5 haplotypes associated with 23 specimens, while the other 3 haplotypes associated with 6 specimens were scattered. The results indicate that the IGS is higher polymorphic and thus more suitable as a genetic marker for population structure analysis of Pacific oyster than the mtDNA coding regions, such as cytochrome c oxidase I and 16S ribosomal RNA genes. PMID- 16682751 TI - Effect of selected factors on the effectiveness of Capsicum annuum L. anther culture. AB - The primary aim of the study was to establish the effectiveness of induced androgenesis in in vitro anther culture of two pepper (Capsicum annuum L.) breeding lines--ATZ1 and PO, and a hybrid between these two lines (ATZ1 x PO)F1. Anther culture was maintained according to the method developed by Dumas de Vaulx et al. (1981) with some modifications. The experiment revealed that the effectiveness of androgenesis ranged from 4 %; for the ATZ1 line to 1.5 %; for the (ATZ1 x PO)F1 and strongly depended on the developmental stage of flower buds, as well as the conditions for anther culture maintenance. The development of androgenic embryos was successfully induced only in anthers which originated from the flower buds with petals equal or slightly longer than sepals and there was a clear relationship between the length of the period of anther induction on CP medium and the level of kinetin in R1 regeneration medium. PMID- 16682753 TI - Adjustments for heterogeneous herd-year variances in a random regression model for genetic evaluations of Polish Black-and-White cattle. AB - The study investigated the existence of heterogeneous variance in first-lactation daily milk yield of Polish Black-and-White cows across herds in different years. Bayesian Information Criterion was used to show that the model with unequal residual variances for different herd-years was more plausible than the model assuming equal variances. A method of adjusting phenotypic records was developed to account for unequal variability in herd-years. Factors used for the data adjustment considered variation of general residuals and residuals for specific herd-years. The size of herd-year was also taken into account. Varied power of corrections was used to analyze the effect of adjustment on estimated breeding values. The method was applied to daily milk records of 817,165 primiparous cows. The effectiveness of the data adjustment was evaluated by the analysis of differences between each bull's breeding value and its parental index. Data correction reduced the average difference and variance of differences between breeding values and parental indices. Accounting for the size of herd-year classes in correction factors improved the efficiency of heterogeneous variance adjustment. PMID- 16682754 TI - Polymorphisms in coding and regulatory regions of the porcine MYF6 and MYOG genes and expression of the MYF6 gene in m. longissimus dorsi versus productive traits in pigs. AB - MYOG and MYF6 belong to the MyoD gene family. They code for the bHLH transcription factors playing a key role in later stages of myogenesis: differentiation and maturation of myotubes. Three SNPs in porcine MYF6 and two in porcine MYOG were analysed in order to establish associations with chosen carcass quality and growth rate traits in Polish Landrace, Polish Large White and line 990 sows. No statistically significant effect of SNP in the promoter region of the MYF6 gene on its expression measured on mRNA level was found. Associations between the genotype at the MYF6 locus and carcass quality traits appeared to be breed-dependent. The C allele in the case of SNP in the promoter region and GC haplotype in exon 1 were advantageous for right carcass side weight in Polish Landrace sows and disadvantageous for this trait in Polish Large White sows. These gene variants were also the most advantageous for loin and ham weight in sows of line 990. The mutation in exon 1 of the MYOG gene had no statistically significant association with carcass quality traits and the mutation in the 3' flanking region had the breed-dependent effect as well. These results suggest that SNPs analysed in this study are not causative mutations, but can be considered as markers of some other, still unrevealed genetic polymorphism that influences the physiological processes and phenotypic traits considered in this study. PMID- 16682755 TI - Associations between the prolactin receptor gene polymorphism and reproductive traits of boars. AB - The prolactin receptor gene (PRLR), located on chromosome 16 in pigs, is a candidate gene for reproductive traits. The experiment was aimed to detect the DNA mutations in this gene and to find probable relations between the genotype and some reproductive traits in boars. The polymorphism in the PRLR gene was identified by PCR-RFLP method using specific primers and the restriction enzyme AluI. In total 229 boars of various breeds were genotyped. The frequency of allele A was estimated at 0.62 and allele B at 0.38. Genotype AA was found at a frequency of 0.45, AB at 0.35 and BB at 0.20. We found associations between PRLR genotype and ejaculate volume, sperm concentration, percentage of live sperm, and number of live sperm in the ejaculate (P < 0.01). PMID- 16682756 TI - Birth defects surveillance in Ukraine: a process. AB - Birth defects (BD) surveillance using international standards was introduced in Ukraine by a network of five BD centers located in northwestern, central and southern regions. BD centers provide resources to access current and comprehensive information and to nurture partnerships with physicians, administrators, parental support groups, educators, and humanitarian assistance organizations. One outcome was the vigorous and popular website International BD Information Systems (IBIS). The network is now incorporated as OMNI-Net Ukraine. The program has documented high prevalence rates of neural tube defects (NTD); fetal alcohol effects (FAE); and idiopathic developmental retardation among orphans that prompted prevention and amelioration initiatives. Further program objectives include: universal folic acid flour fortification, as recommended by the Ukrainian Academy of Medicine; continued research on methods to reduce FAE in collaboration with partners from California; opening other early infant stimulation centers funded by local authorities, modeled on those in Rivne and Lutsk; and linking BD prevention with bioethical considerations, which is a topic of interest in Ukraine in part enhanced by the effects of Chornobyl. PMID- 16682757 TI - The impact of prenatal diagnosis on neural tube defect (NTD) pregnancy versus birth incidence in British Columbia. AB - The birth incidence of neural tube defect (NTD) cases in British Columbia (B.C.), and elsewhere in North America, is reported to be declining. This decline is being attributed to folic acid (FA) supplementation and food fortification, but 2nd trimester prenatal screening of pregnancies for NTDs and other congenital anomalies has increased during this timeframe, as well. This descriptive, population-based study evaluates the impact of prenatal screening of NTD-affected pregnancies on (1) pregnancy outcome and (2) reporting of NTD births to the provincial Health Status Registry (B.C.H.S.R.); and it assesses (3) the use of periconceptional FA supplementation. NTD cases were ascertained from medical records of health centres providing care to families with NTD-affected pregnancies and newborns; and from NTD cases reported to the B.C.H.S.R. In 1997 1999, the B.C.H.S.R. published a NTD incidence of 0.77/1000. In this study, 151 NTD-affected pregnancies were identified, with an incidence of 1.16/1000. Partial Reporting of induced abortions in a NTD incidence 45.5% low than the actual incidence. Medical records were available for review on 144/151 pregnancies. Prenatal screening identified 86.1% (124/144) of NTD-affected pregnancies, with 72.6% (90/124) resulting in pregnancy termination, and 27.4% (34/124) continuing to term. Use of FA supplementation in the periconceptional period was recorded in 36.4% of pregnancies (39/107). Thus in B.C. the decline in the NTD incidence is due predominantly to pregnancy terminations following prenatal diagnosis, which reduces the NTD incidence by 60%, from 1.16/1000 to 0.47/1000. Continued efforts for primary and the option of secondary prevention of NTDs are recommended in order to improve newborn health in B.C. and elsewhere. These interventions need to be monitored, however, for optimal health care planning. PMID- 16682758 TI - A novel c.581C>T transition localized in a highly conserved homeobox sequence of MSX1: is it responsible for oligodontia? AB - Even though selective tooth agenesis is the most common developmental anomaly of human dentition, its genetic background still remains poorly understood. To date, familial as well as sporadic forms of both hypodontia and oligodontia have been associated with mutations or polymorphisms of MSX1, PAX9, AXIN2 and TGFa, whose protein products play a crucial role in odontogenesis. In the present report we described a novel mutation of MSX1, which might be responsible for the lack of 14 permanent teeth in our proband. However, this c.581C>T transition, localized in a highly conserved homeobox sequence of MSX1, was identified also in 2 healthy individuals from the proband's family. Our finding suggests that this transition might be the first described mutation of MSX1 that might be responsible for oligodontia and showing incomplete penetrance. It may also support the view that this common anomaly of human dentition might be an oligogenic trait caused by simultaneous mutations of different genes. PMID- 16682759 TI - Trisomy 18 in neonates: prenatal diagnosis, clinical features, therapeutic dilemmas and outcome. AB - The study aimed to analyse the clinical courses of aggressively treated neonates with cytogenetically confirmed trisomy 18, with special attention focused on the efficiency of prenatal diagnostics, associated malformations, therapeutic dilemmas and outcomes. We investigated retrospectively the data concerning 20 neonates with trisomy 18, admitted to the Neonatal Intensive Care Unit (NICU) in Katowice between January 2000 and February 2005. Their birth weights ranged from 650 g to 2400 g, mean 1812 g; gestational age ranged from 27 to 42 weeks, median 38 weeks. Intrauterine growth retardation was noticed in 90% of neonates. Trisomy 18 was suspected prenatally in 40% of cases. Most (80%) of newborns were delivered by caesarean section (92% of neonates with prenatally unrecognized chromosomal defects, 62% of neonates with trisomy 18 suspicion) and 70% of infants needed respiratory support immediately after birth. Cardiac defects were present in 95%, central nervous system malformations in 65%, severe anomalies of digestive system or abdominal wall in 25% of patients. Nine surgical operations were performed during hospitalization (4 were palliative cardiac surgeries). Six patients (30%) survived the neonatal period and were discharged from the NICU. The median survival of the neonates who died was 20 days. In 4 cases cardiac problems implicated their death; in others, deaths were attributed to multiorgan failure, prematurity and/or infection. Further improvement of efficiency of prenatal ultrasound screening for diagnosis of trisomy 18 in the fetus is necessary. A lack of prenatal diagnosis of trisomy 18 in the fetus results in a high rate of unnecessary caesarean sections in these pregnancies. Despite the aggressive treatment most neonates with trisomy 18 died during the neonatal period. The majority of deaths were attributed to cardiorespiratory and multiorgan failure. Concerning the poor prognosis, prompt karyotyping (using FISH) of clinically suspected trisomy 18 is very important, because many invasive procedures and surgeries may then be avoided. PMID- 16682760 TI - Urological anomalies in children with renal agenesis or multicystic dysplastic kidney. AB - This study aimed to determine the frequency of associated urological abnormalities in children with unilateral renal agenesis (RA) or multicystic dysplastic kidney (MCDK). In total, 38 children (10 girls, 28 boys) were studied: 21 with RA and 17 with MCDK. In 14 children (37%) anomalies of the urinary tract were suspected prenatally in ultrasound studies. In the remaining 24 children the diagnosis of RA/MCDK was made postnatally: in 13 (34%) in the first 7 days of life, in 11 (29%) at the age of 8 days to 34 months, mean 10.6+/-8.05 months. Voiding cystourethrography was done in 36 (95%) children, the isotopic 99mTc EC/DMSA scan of the kidney in 29 (67%), and urography in 8. Urological anomalies were present in 11 (29%) children: in 7 (33%) with RA and in 4 (24%) with MCDK. Vesicoureteral reflux was diagnosed in 8 children: grade II in 4, III in 3, and IV in 1 (in 1 child to duplicated, in 1 to ectopic kidney); ureterovesical junction obstruction in 2 (9.5%); and ureteropelvic junction obstruction in 1 (4.8%). Among them, 2 children demanded surgery on the contralateral urinary tract: pyeloplasty in 1, antireflux procedure in 1; while 9 children were treated conservatively. Compensatory hypertrophy of the contralateral kidney was found in 90% of children. Thus due to an increased risk of pathological changes in the single functioning kidney, lifelong nephrological care is recommended in patients with unilateral RA/MCDK. PMID- 16682761 TI - Ultrasound diagnostic schema for the determination of increased risk for chromosomal fetal aneuploidies in the first half of pregnancy. AB - The aim of the study was to develop an early ultrasound diagnostic schema for the determination of increased risk for fetal chromosomal aneuploidies. The study was conducted on a population of 1318 pregnant women divided into 2 groups: 1255 women with the normal course of pregnancy and 63 women with diagnosed fetal abnormalities. There were 34 cases of chromosomal abnormalities (trisomy 21, 18, 13; triploidy; unbalanced inversion 9; deletion 16) and 29 cases of structural malformations. The estimation of the range of normal values was performed for the nuchal translucency (NT) measurement between 11 and 13 weeks and the nasal bone length (NB) measurement between 12 and 20 week. The results obtained in the collective set of normal pregnancies constituted the basis for the calculation of the range of normal values. The measurements of NB and NT showed a linear value increase with the pregnancy course. The following test characteristics (correlation to CRL) were recorded: NB - sensitivity 60%, specificity 98%, positive predictive value (PPV+) 43%, negative predictive value (NPV-) 98.9%. For the assumption that the test outcome means the presence or absence of the nasal bone in the ultrasound scan the sensitivity was 40%, but specificity 100%; NT - sensitivity 63.6%, specificity 98.2%, PPV+ 38.9%, NPV - 98.2%; NT + NB - presents similar characteristic to the NB or NT alone - sensitivity 55.6%, specificity 98.6%, PPV+ 50%, NPV - 98.9%. The following test characteristics for chromosomal aberration markers (correlation to BPD) were observed: NB - sensitivity 68.4%, specificity 97.4%, PPV+ 56.5%, NPV - 98.4%; NT - sensitivity 73.9%, specificity 97.9%, PPV+ 54.8%, NPV- 99.2%; NT + NB - sensitivity 94.7%, specificity 98.9%, PPV+ 90%, NPV - 99.7%, respectively. The "genetic sonogram" protocol for the structural defect detection was analysed: sensitivity was 80%, specificity 100%, PPV+ 100%, NPV - 99.7%. It is concluded that the new biometric parameter--nasal bone length (NB) and the corrected one--nuchal translucency thickness (NT) are useful markers for fetal abnormalities, especially for chromosomal aberrations. High predictive values of the diagnostic schema for the detection of aneuploidies and structural defects indicate that its application in correlation with the biparietal diameter (BPD) is highly recommended. The proposed schema is an effective algorithm for prenatal diagnostics characterised by high prognostic values. The possible introduction of the schema could result in a decrease of the invasive procedure rates, which could minimise the rate of miscarriages as a complication of amniocenteses. PMID- 16682762 TI - Knowledge among young people about folic acid and its importance during pregnancy: a survey in the Federal State of Saxony-Anhalt (Germany). AB - Periconceptional folic acid supplementation is recommended to prevent congenital malformations, mainly neural tube defects, but only 7% of pregnant women in Saxony-Anhalt (Germany) take folic acid at least 4 weeks before conception and in the first 3 months of pregnancy. From March to June 2004, we sent standardized questionnaires about folic acid and its importance during pregnancy to 33 schools in the Federal State of Saxony-Anhalt. A total of 4332 young people aged 1521 years completed the questionnaire, of which 2632 were girls (61%) and 1685 were boys (39%). The majority of them (61%) had heard about folic acid, but only 5% knew that it is a vitamin and 0.7% were aware of the physiological functions of folic acid. Only 22% of the young people answered that folic acid should be taken before and during pregnancy, whereas almost all respondents knew other precautions during pregnancy, e.g. "no smoking" and "no alcohol". Our survey shows that the level of awareness of the importance of folic acid at schools is very low. We suggest that the problem of folic acid should be included in the curricula of biology classes at schools to spread the knowledge of this subject among young people. PMID- 16682763 TI - Trends in diagnosis and prevalence of critical congenital heart defects in the Podkarpacie province in 2002-2004, based on data from the Polish Registry of Congenital Malformations. AB - This paper presents results of data collection regarding congenital heart defects (CHD) for the Polish Registry of Congenital Malformations (PRCM) from the Podkarpacie province in 2002-2004. Routine methods (fetal echocardiography, clinical examination) and recently also unique methods (screening echocardiography and pulse oximetry) were used for early detection of critical cardiac malformations. Critical CHD were detected there in 107 newborns in 2002 2004, so the mean prevalence reached 1.71/1000. Within this group, death rate decreased from 8 deaths in 2002 to 2 deaths in 2004, mainly thanks to improved detection and treatment of CHD. PMID- 16682764 TI - Harlequin ichthyosis--difficulties in prenatal diagnosis. AB - Ichthyoses belong to the group of genodermatoses, characterized by hyperkeratosis and desquamation of the epidermis. Clinical manifestation is heterogeneous and depends on the type of the disease. Harlequin foetus is the most severe form of congenital ichtyosis, inherited as an autosomal recessive trait. The disfunction of the epidermis begins prenatally. Neonates are often born prematurely, in severe condition. At present better care and treatment prolong the length and quality of children's life. We report a case of harlequin ichthyosis. Parents were healthy and there was no history of ichthyosis or other congenital anomalies in the family. Sonography at the 26th week of gestation revealed anomalies of the fetal face; however, the diagnosis of harlequin ichthyosis was not established prenatally. The male child was born alive at the 37th week of the third pregnancy, with birth weight of 2900 g. Typical features of harlequin ichthyosis were present at birth. Intensive neonatological care was necessary. The child survived and at the time of the report was 6 months old and in good condition. PMID- 16682765 TI - Dental treatment strategies in a 40-year-old patient with cleidocranial dysplasia. AB - Oral anomalies and dental treatment in a patient with cleidocranial dysplasia (referred to the dental clinic at the age of 40 years) are presented. Five supernumerary teeth were found in the patient: three in the maxilla in the area of molars and two in the mandibula in the area of premolars. Therapy included surgical exposure of impacted teeth in combination with removal of supernumerary teeth. PMID- 16682766 TI - Structure of myotoxin II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom. AB - Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 angstroms resolution and the anion-binding site has been characterized. PMID- 16682768 TI - Expression, purification, crystallization and preliminary X-ray diffraction analysis of human phosphoribosyl pyrophosphate synthetase 1 (PRS1). AB - Phosphoribosyl pyrophosphate synthetase (PRS; EC 2.7.6.1) catalyzes the reaction of ribose-5-phosphate (R5P) with ATP to yield AMP and PRPP (5-phosphoribosyl-1 pyrophosphate), which is necessary for the de novo and salvage pathways of purine , pyrimidine- and pyridine-nucleotide biosynthesis. PRPP is a metabolite that is required at all times in the cell and is thus central to life. In this study, human PRS1 was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals in complex with Mg2+, inorganic phosphate (P(i)) and ATP were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.6 A resolution. The crystal belongs to space group R3, with unit cell parameters a = b = 168.846, c = 61.857 angstroms, assuming two molecules in the asymmetric unit and a volume-to-weight ratio of 2.4 angstroms3 Da(-1), which was consistent with the result calculated from the self-rotation function. PMID- 16682767 TI - Structure of ribose 5-phosphate isomerase from Plasmodium falciparum. AB - The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 angstroms resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway. The P. falciparum enzyme belongs to the ribose 5 phosphate isomerase A family, Pfam family PF06562 (DUF1124), and is structurally similar to other members of the family. PMID- 16682769 TI - Cloning, purification and preliminary crystallographic analysis of the Bacillus subtilis GTPase YphC-GDP complex. AB - The Bacillus subtilis YphC gene encodes an essential GTPase thought to be involved in ribosome binding and whose protein product may represent a target for the development of a novel antibacterial agent. Sequence analysis reveals that YphC belongs to the EngA family of GTPases, which uniquely contain two adjacent GTP-binding domains. Crystals of a selenomethionine-incorporated YphC-GDP complex have been grown using the hanging-drop vapour-diffusion method and polyethylene glycol as a precipitating agent. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.71, b = 65.05, c = 110.61 angstroms, and have one molecule in the asymmetric unit. Data sets at three different wavelengths were collected on a single crystal to 2.5 angstroms resolution at the Daresbury SRS in order to solve the structure by MAD. Ultimately, analysis of YphC in complex with GDP may allow a greater understanding of the EngA family of essential GTPases. PMID- 16682770 TI - Crystallization and preliminary X-ray analysis of the rhamnogalacturonan lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide lyase family 11. AB - Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a beta-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P2(1) and diffract to 2.40 angstroms resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 A, beta = 94.9 degrees. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase. PMID- 16682771 TI - Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved. AB - Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ(gamma) subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 angstroms, beta = 104.5 degrees. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described. PMID- 16682772 TI - Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the alpha crystallin family. AB - The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 angstroms resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 angstroms. The crystal structure was solved by molecular replacement methods. Structure refinement is in progress. PMID- 16682773 TI - Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre. AB - Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579 793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10,000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kalpha radiation to at least 1.9 angstroms resolution and a complete data set was collected from a single crystal to 2.2 angstroms resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 angstroms, beta = 101.3 degrees, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre head structures. Structure solution is in progress. PMID- 16682774 TI - Synthesis, capillary crystallization and preliminary joint X-ray and neutron crystallographic study of Z-DNA without polyamine at low pH. AB - In order to crystallographically study the hydration of the major groove (convex surface) of Z-DNA, the oligonucleotide d(CGCGCG) has been synthesized. Single crystals were grown by vapor diffusion using the hanging-drop and sitting-drop methods for X-ray studies and by batch crystallization and evaporation within silicon tubes for neutron studies. Hexagonal crystals were obtained without the use of duplex-stabilizing polyamines and at an acid pH. X-ray data collected at room temperature (1.5 angstroms resolution; unit-cell parameters a = 17.90, b = 30.59, c = 44.61 angstroms) and at 100 K (1 angstroms resolution; a = 17.99, b = 30.98, c = 44.07 angstroms) and neutron data collected at room temperature (1.6 angstroms resolution; a = 18.00, b = 31.16, c = 44.88 angstroms) indicate that the DNA is in the Z-form packing in space group P2(1)2(1)2(1). PMID- 16682775 TI - Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering. AB - The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF65) binds the polypyrimidine tract near the 3' splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein-nucleic acid complex. Cocrystals of a U2AF65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 angstroms resolution and contain one complex per asymmetric unit. PMID- 16682776 TI - Preliminary X-ray investigations of several crystal forms of the ferripyoverdine FpvA outer membrane receptor from Pseudomonas aeruginosa bound to ferripyoverdine. AB - Ferripyoverdine transport across the outer membrane of Pseudomonas aeruginosa by the pyoverdine receptor FpvA and the transcriptional regulation of FpvA involve interactions of the FpvA N-terminal TonB box and signalling domain with proteins from the inner membrane. Several crystallization conditions of FpvA-Pvd-Fe solubilized in C8E4 detergent were obtained and X-ray data were collected from three crystal forms. The resolution limits range from 3.15 to 2.7 angstroms depending on the crystal form. From preliminary analysis of the electron-density maps, the first full-length structure of an outer membrane receptor including a signalling domain should be determined. PMID- 16682777 TI - Crystallization of recombinant Haemophilus influenzae e (P4) acid phosphatase. AB - Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host-pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 angstroms resolution native X-ray diffraction data set. The space group is P4(2)2(1)2, with unit-cell parameters a = 65.6, c = 101.4 angstroms, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase. PMID- 16682778 TI - Purification, crystallization and preliminary X-ray diffraction of human S100A15. AB - Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7. PMID- 16682779 TI - Crystallization and preliminary X-ray analysis of CooA from Carboxydothermus hydrogenoformans. AB - CooA, a homodimeric haem-containing protein, is responsible for transcriptional regulation in response to carbon monoxide (CO). It has a b-type haem as a CO sensor. Upon binding CO to the haem, CooA binds promoter DNA and activates expression of genes for CO metabolism. CooA from Carboxydothermus hydrogenoformans has been overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal belongs to space group P2(1), with unit-cell parameters a = 61.8, b = 94.7, c = 92.8 angstroms, beta = 104.8 degrees. The native and anomalous difference Patterson maps indicated that two CooA dimers are contained in the asymmetric unit and are related by a translational symmetry almost parallel to the c axis. PMID- 16682780 TI - Expression, purification, crystallization and preliminary X-ray characterization of the GRP carbohydrate-recognition domain from Homo sapiens. AB - Galectins are a family of animal lectins which share similar carbohydrate recognition domains (CRDs) and an affinity for beta-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38-172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging drop vapour-diffusion method from a solution containing 2% PEG 400 and 2M ammonium sulfate in 100 mM Tris-HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 angstroms at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 angstroms, beta = 118.72 degrees. The estimated Matthews coefficient was 2.6 angstroms3 Da(-1), corresponding to 51.8% solvent content. PMID- 16682781 TI - Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH. AB - Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an approximately 220 angstroms extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell's outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 angstroms resolution and belong to space group P2(1), with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high throughput crystallization robot at the Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 angstroms resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress. PMID- 16682782 TI - Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the glutamate-1-semialdehyde aminotransferase from Bacillus subtilis. AB - 5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole-biosynthesis pathway. Plants, algae and many other bacteria synthesize ALA from glutamate by a C5 pathway in which the carbon skeleton of glutamate is converted into ALA by a series of enzymes. Glutamate-1-semialdehyde aminotransferase (GSAT) is the last enzyme in this pathway. The gene that codes for GSAT was amplified from the cDNA library of Bacillus subtilis and overexpressed in Escherichia coli strain BL21(DE3). The protein was purified and crystallized. Well diffracting single crystals were obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray diffraction studies yielded excellent diffraction data to a resolution of 2.0 angstroms. PMID- 16682784 TI - Differential expression of platelet glycoprotein Ia/IIa in Taiwan Chinese corresponds to glycoprotein ia gene polymorphisms. AB - Glycoprotein (GP) Ia/IIa plays a key role in platelet function by acting as a primary receptor for subendothelial collagen, thereby contributing to arterial thrombosis. In this study, we found that the expression level of platelet GPIa/IIa among Taiwan Chinese varies over sixfold and this difference relates either to the GPIa gene 807 C/T dimorphisms or the appearance of BglII restriction sequence within intron G. This is the first study to demonstrate the wide variation of platelet GPIa/IIa density in an Oriental population and its correlation to the GPIa gene polymorphisms. These results provide useful tools for predicting platelet GPIa/IIa density of Chinese. PMID- 16682783 TI - Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15. AB - Almost all alginate lyases depolymerize alginate in an endolytical fashion via a beta-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2(1) and diffracted to 2.8 angstroms resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 angstroms, beta = 91.5 degrees. PMID- 16682785 TI - The mechanism of endothelium-independent relaxation induced by the wine polyphenol resveratrol in human internal mammary artery. AB - Resveratrol, a stilbene polyphenol found in grapes and red wine, produces vasorelaxation in both endothelium-dependent and endothelium-independent manners. The mechanisms by which resveratrol causes vasodilatation are uncertain. The aim of this study was to investigate the mechanism(s) of endothelium-independent resveratrol-induced vasorelaxation in human internal mammary artery (HIMA) obtained from male patients undergoing coronary artery bypass surgery and to clarify the contribution of different K+ channel subtypes in resveratrol action in this blood vessel. HIMA rings without endothelium were precontracted with phenylephrine. Resveratrol induced a concentration-dependent relaxation of the HIMA. A highly selective blocker of ATP-sensitive K+ channels, glibenclamide, as well as nonselective blockers of Ca2+-sensitive K+ channels, tetraethylammonium and charybdotoxin, did not block resveratrol induced relaxation of HIMA rings. 4 Aminopyridine (4-AP), non selective blocker of voltage gated K+ (KV) channels, and margatoxin that inhibits KV1.2, KV1.3, and KV1.6 channels abolished relaxation of HIMA rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed HIMA rings with denuded endothelium. It seems that 4-AP- and margatoxin-sensitive K+ channels located in smooth muscle of HIMA mediated this relaxation. PMID- 16682786 TI - Glucocorticoid is involved in food-entrainable rhythm of mu-opioid receptor expression in mouse brainstem and analgesic effect of morphine. AB - The repeated manipulation of feeding schedule has a marked influence on the chronopharmaological aspects of many drugs. In this study, we investigated the role of endogenous glucocorticoid in the mechanism by which restricting the feeding time modulates the analgesic effect of morphine. Male ICR mice were housed under a light-dark cycle (light on from 07:00 to 19:00) with food and water ad libitum or under repeated time-restricted feeding (feeding time from 09:00 to 17:00) for 2 weeks before the experiment. Under the ad libitum feeding, mRNA levels of mu-opioid receptor and its binding capacity in mouse brainstem increased around the early dark phase, following the 24-h variation in circulating glucocorticoid levels. As a consequence, potent analgesic effects of morphine were observed in mice injected with the drug during the dark phase. Daily restricted feeding modulated the time-dependency of mu-opioid receptor function, accompanied by the alteration of the rhythm in circulating glucocorticoid levels. Under the time-restricted feeding, potent analgesic effects of morphine were found in mice injected with the drug during the light phase. Because the manipulation of feeding schedule was unable to produce the food-entrainable rhythm in the expression of mu-opioid receptor in the brainstem of adrenalectomized mice, endogenous rhythm of glucocorticoid secretion seems to be involved in the mechanism by which the time-restricted feeding modulates the analgesic effects of morphine. PMID- 16682787 TI - Development and evaluation of a direct sandwich enzyme-linked immunosorbent assay for the quantification of guinea-pig immunoglobulin e. AB - The purpose of this study was to develop and evaluate a direct sandwich enzyme linked immunosorbent assay (ELISA) for the immunoglobulin E (IgE) in serum and plasma from guinea pig using mouse monoclonal antibodies specific for guinea-pig IgE. Mouse monoclonal antibodies were raised against purified IgE protein. The ELISA was performed using a combination of two anti-IgE monoclonal antibodies. One antibody was labeled with horseradish peroxidase (HRP), and the other was coated on polystyrene wells. Purified guinea-pig IgE was used as the standard material. The validity of the ELISA was confirmed by precision, dilution, recovery, and interference tests. The range of detection was 3.1-800 ng of IgE mass per mL of serum and plasma. The intra- and inter-assay coefficients of variation were 4.6% and 5.7%, respectively, or less. The recovery test showed variation only between 92.1% and 111.8%, and the anticoagulants showed noninterference with the IgE assay. The mean serum IgE mass concentration in OVA sensitized guinea pigs was 29438 ng/mL, and it was 48.6 ng/mL in normal guinea pigs. The present ELISA is useful and practical for specific measurement of the guinea-pig IgE, and it is surmised that it would be suitable for use in allergological and pharmacological research. PMID- 16682788 TI - Brain cytokines and chemokines: roles in ischemic injury and pain. AB - Cytokines and chemokines were originally identified as essential mediators for inflammatory and immune responses. Enhanced production and release of cytokines/chemokines are observed also in the central nervous system (CNS) under diverse pathological conditions. There is growing evidence showing that brain cytokines/chemokines play crucial roles in the neuro-glio-vascular interaction underlying the pathology of various brain disorders and therefore are potential targets for development of novel and effective therapeutics for CNS diseases. Here the evidence of the involvement of cytokines/chemokines in ischemic brain injury and pain is reviewed. PMID- 16682789 TI - Ca2+-dependent inositol 1,4,5-trisphosphate and nitric oxide signaling in cerebellar neurons. AB - Intracellular Ca(2+) signals are important for the regulation of synaptic functions in the central nervous system. In this review, I summarize findings of our recent studies on upstream and downstream Ca(2+) signaling mechanisms in cerebellar synapses using novel molecular imaging methods. Inositol 1,4,5 trisphosphate (IP(3))-induced Ca(2+) release plays a pivotal role in central synapses. The visualization of IP(3) at fine dendrites of Purkinje cells (PCs) using a fluorescent IP(3) indicator showed that intracellular Ca(2+) concentration has a stimulatory effect on phospholipase C activity, which catalyzes IP(3) production. This indicates that metabotropic and ionotropic glutamate receptors collaborate to generate IP(3) signals. Using a novel nitric oxide (NO) indicator, the spatial distribution of NO signals originating from parallel fiber (PF) terminals was visualized. Our results show that the NO signal decays steeply with distance from the site of production in the cerebellum and is dependent on PF stimulation frequency in a biphasic manner. NO released from PF terminals generated a synapse-specific long-term potentiation of PF-PC synapse when PF was stimulated at certain frequencies. These imaging studies clarified new aspects of the regulatory mechanisms of synaptic functions. PMID- 16682790 TI - Pharmacological studies on fullerene (C60), a novel carbon allotrope, and its derivatives. AB - Fullerene (C(60)), a condensed ring aromatic compound with extended pi systems, is a novel carbon allotrope. Because of its poor solubility in polar solvents, investigation of the biological and pharmacological properties of fullerene has been difficult. Recently, water-soluble fullerene derivatives have been synthesized, and we and others have found that they have potent and selective pharmacological effects on organs, cells, enzymes, and nucleic acids. In the presence of fullerene C(60) derivative (10(-5) M), endothelium-dependent relaxations induced by agonists in the vascular system were eliminated and acetylcholine-induced contractile response of smooth muscle was observed. Some investigators have reported free radical-scavenging activity and direct nitric oxide-quenching activity of fullerene derivatives. Knowledge of the chemical modifications, biological significance, and materials applications of functionalized fullerenes is growing rapidly; and these compounds are emerging as new tools in the field. The focus of this review is to introduce several pharmacological effects of fullerenes and to discuss the possible mechanisms of the pharmacological actions caused by previously synthesized fullerenes. PMID- 16682791 TI - Measuring heart rate variability with wavelet thresholds and energy components in healthy subjects and patients with congestive heart failure. AB - BACKGROUND: Analysis of wavelet thresholds and energy components can be used to differentiate patients with congestive heart failure (CHF) from healthy subjects and to demonstrate the improvement of heart rate variability after treatment with beta-blockers in the patient group. METHODS: Nine patients with CHF without concurrent beta-blocker therapy and 15 healthy volunteers were recruited and received 24-hour Holter recording. Wavelet thresholds and energy components were calculated. The patients with CHF were then treated with atenolol for 3 months. Another Holter recording was repeated and associated wavelet parameters were estimated. RESULTS: Wavelet thresholds were statistically higher in normal subjects than those derived from patients with CHF (62.40 +/- 20.65 vs. 16.90 +/- 9.72, p < 0.001). The difference was still significant after control of heart rate and the total number of heart beats (74.85 +/- 22.68 vs. 32.51 +/- 18.73, p < 0.001). The energy component of HRV in the finest wavelet domain was also significantly higher in the normal subjects (243.01 +/- 237.69 x 10(-6) vs. 40.65 +/- 39.65 x 10(-6), p = 0.001). These two measures increased after treatment with beta-blockers in the patients with CHF, but not to the extent of those of normal subjects. CONCLUSION: Analysis of wavelet thresholds and energy components has provided robust and accurate measurements for background HRV. PMID- 16682792 TI - Frequency of dementia etiologies in four ethnic groups. AB - BACKGROUND/AIMS: Research on dementia among ethnically diverse populations in the USA has focused primarily on Alzheimer disease and vascular dementia, but there are limited data on other neurodegenerative causes of dementia. METHODS: To determine the frequency of neurodegenerative disorders in four ethnic groups, data collected at Alzheimer's Disease Research Centers of California for assessments between 1992 and 2002 were analyzed retrospectively. Cases of Alzheimer disease, vascular dementia, dementia with Lewy bodies, frontotemporal lobar degeneration, Parkinson disease and progressive supranuclear palsy were identified for 452 Asian and Pacific Islander, 472 Black, 675 Latino and 4,926 White patients. RESULTS: The percentage of non-Whites diagnosed as having dementia with Lewy bodies was lower than that of Whites. Frontotemporal lobar degeneration was as common in Asians and Pacific Islanders compared to Whites, but less common in Blacks and Latinos. Progressive supranuclear palsy was more frequent in Asians and Pacific Islanders compared to Whites, but equally common in Blacks and Latinos. CONCLUSION: Additional study is needed on the social and biological factors that influence the diagnosis and prevalence of non-Alzheimer and nonvascular dementias among diverse ethnic groups. PMID- 16682793 TI - Stability of CSF beta-amyloid(1-42) and tau levels by APOE genotype in Alzheimer patients. AB - BACKGROUND: Cerebrospinal fluid (CSF) measures of beta-amyloid(1-42 )and tau differ between patients with Alzheimer's Disease (AD) and elderly normal controls. The effect of time and APOE genotype on these biomarkers continues to be elucidated. METHODS: We assessed CSF beta-amyloid(1-42) and tau in 20 mild-to moderate AD patients, 11 APOE epsilon4+ and 9 APOE epsilon4-, over a mean time of 3.8 years (range 1-11.1 years). RESULTS: Over the period measured, CSF beta amyloid(1-42) levels were lower in APOE epsilon4+ compared to APOE epsilon4- patients, and the levels decreased over time. Tau levels were stable over time and did not show an effect of APOE allele. CONCLUSIONS: While this is a limited clinical sample, the further decrease in CSF beta-amyloid(1-42 )(i.e., more abnormal) combined with the CSF tau stability over a mean period of almost 4 years suggests that beta-amyloid(1-42 )and tau maintain their potential usefulness as diagnostic biomarkers over time. These findings should be taken into account if CSF beta-amyloid(1-42) and tau are used as measures of treatment response. PMID- 16682794 TI - Heterogeneity in executive impairment in patients with very mild Alzheimer's disease. AB - BACKGROUND/AIMS: The presence of executive impairment in mild Alzheimer's disease (AD) has primarily been demonstrated by means of group comparison. Whether executive dysfunction is a common feature of mild AD or only present in a subgroup of patients remains unclear. The aim of this study was to describe the frequency of impairment on a set of internationally well-known executive tests in patients with very mild AD. METHODS: Thirty-six patients with very mild AD (MMSE scores above 23) and 32 healthy control subjects were administered a battery of 7 executive tests: Trail Making part B, Stroop Interference Test, modified Wisconsin Card Sorting Test (WCST), category- and letter-based verbal fluency, a design fluency task and the Similarities subtest from WAIS. Impairment was defined as a score of 2 SD or more below control means. RESULTS: Executive impairment on at least 1 measure was seen in 76% of the patients, and 50% were impaired on 2 or more tests. Trail Making B and Stroop Interference Test were impaired in more than 40%, whereas only few patients were impaired on Similarities, WCST and design fluency. A wide variation of executive test profiles was seen among the patients. CONCLUSION: Executive impairments are common in early AD and not just a feature characteristic of a subgroup of patients. Complex attentional skills are more frequently affected than other executive functions. There is, however, considerable heterogeneity among AD patients in the pattern of executive dysfunction. PMID- 16682795 TI - The effect of apolipoprotein polymorphism on brain in mild cognitive impairment: a voxel-based morphometric study. AB - We investigated the effect of apolipoprotein E (ApoE) on the whole brain in 51 individuals with mild cognitive impairment using voxel-based morphometry. Between cases heterozygous for the ApoE epsilon4 (n = 15) and those who were ApoE epsilon4 noncarriers (n = 28), only the right parahippocampal gyrus, with the entorhinal cortex included, reached the level of statistical significance. In cases homozygous for the epsilon4 allele (n = 8) versus noncarriers, the greatest atrophy was located in the right amygdala followed by the right parahippocampal gyrus, the left amygdala and the left medial dorsal thalamic nucleus. PMID- 16682796 TI - Blood pressure management in acute stroke: a long-standing debate. AB - Although elevated blood pressure (BP) levels are a common complication of acute stroke, whether of ischaemic or haemorrhagic type, a long-standing debate exists regarding the management of post-stroke hypertension. In the absence of solid, randomised data from controlled trials, the current observational evidence allows different approaches, since theoretical arguments exist for both lowering BP in the setting of acute stroke (reduce the risk of stroke recurrence, of subsequent oedema formation, of rebleeding and haematoma expansion in patients with cerebral bleeding) as well as leaving raised BP levels untreated (avoid reduction in cerebral perfusion pressure and blood flow to viable ischaemic tissue in the absence of normal autoregulation). The present review will summarize the evidence for and against the therapeutic manipulation of BP in acute stroke provided by the currently available observational studies and randomised trials, consider the ongoing clinical trials in this area and address the present recommendations regarding this conflicting issue. PMID- 16682797 TI - Chronic deep brain stimulation of the subthalamic nucleus for Parkinson's disease: effects on cognition, mood, anxiety and personality traits. AB - OBJECTIVE: To evaluate modifications occurring in cognitive functions and behavioural aspects in a group of 72 consecutive patients with Parkinson's disease (PD) 15 months after bilateral deep brain stimulation (DBS) of the subthalamic nucleus (STN). METHODS: 72 consecutive PD patients bilaterally implanted for DBS of the STN were evaluated before and after surgery with a mean follow-up of 15 months. A neuropsychological assessment was performed to evaluate reasoning (Raven Colour Matrices), memory (Bisyllabic Word Repetition Test, Corsi's Block-Tapping Test, Paired-Associate Learning) and frontal executive functions (Trail Making Test Part B, Nelson Modified Card Sorting Test, phonemic and category verbal fluency tasks). Mood and suicidal ideation were evaluated using the Beck Depression Inventory (BDI). Anxiety was measured by means of the State-Trait Anxiety Inventory and personality traits were evaluated with the Structured Clinical Interview for the DSM-III-R Axis II Disorders (SCID-II). Assessment of thought disorders and apathy was based on subitems of the Unified Parkinson's Disease Rating Scale. RESULTS: The comparisons between pre- and postoperative neuropsychological test scores showed a significant worsening only in phonemic and semantic verbal fluency tasks, while fewer errors were found in the Nelson Modified Card Sorting Test. Globally, behavioural assessment evidenced a small improvement in mood, as assessed by the BDI, in obsessive-compulsive and paranoid personality traits (SCID-II). Thought disorders worsened while suicidal ideation, anxiety and apathy showed no postoperative modifications. The analysis of individual outcomes (+/-1 SD criterion) evidenced a relevant postoperative cognitive decline in 3 patients out of 65 (4.5%). Moreover, following implantation, 1 patients exhibited psychosis (1.5%), 2 patients experienced a clinically relevant worsening of depressive symptoms (3%), 7 patients showed an increase in anxiety (12%) and 3 patients a worsening in depression and anxiety symptoms (3%). On the contrary, 12 patients (20%) showed a relevant improvement in mood and 14 patients (23%) a relevant reduction of anxiety symptoms after the surgery. CONCLUSIONS: The present study confirms that STN DBS is cognitively safe since the only relevant change observed was a mild decrease in verbal fluency tasks. Globally, a small postoperative improvement was found in the BDI, and in two SCID-II subscales concerning obsessive-compulsive and paranoid personality traits, even though postoperative behavioural disturbances can occur in individual patients. PMID- 16682798 TI - Neuropsychological changes in patients with carotid stenosis after carotid endarterectomy. AB - We investigated changes in neuropsychological function in patients with carotid stenosis following carotid endarterectomy (CEA) in relation to cerebral hemodynamics. The subjects were 24 patients who underwent CEA and 17 healthy controls matched by age, educational level, gender and handedness. Cerebral angiography, single-photon emission computed tomography (SPECT) and neuropsychological tests were performed approximately 1 week before and 3 weeks after CEA in all patients. In the patient group, the categories achieved in the new modified Wisconsin Card Sorting Test (WCST) improved significantly after CEA (4.0 +/- 2.0; before CEA: 2.3 +/- 2.1, p < 0.01). The perseverative errors of Nelson in the WCST improved significantly (before CEA: 9.1 +/- 6.9, after CEA: 3.3 +/- 3.4, p < 0.01). The difficulty maintaining set in the WCST improved significantly (before CEA: 2.3 +/- 1.9, after CEA: 1.3 +/- 1.7, p < 0.05). In the control group, none of the test scores showed significant improvement between the first and second tests. Moreover, frontal lobe function improved significantly in those patients with baseline carotid artery diameter of more than 70% of normal, or whose preoperative cerebral perfusion reserve was reduced by less than 15% of a response to acetazolamide. Our results suggest that CEA improves frontal lobe function in patients with severe carotid stenosis or reduced cerebral perfusion reserve. PMID- 16682799 TI - Perception of emotional facial expressions at different intensities in early symptomatic Huntington's disease. AB - BACKGROUND: While there is abundant evidence that patients with Huntington's disease (HD) have an impairment in the recognition of the emotional facial expression of disgust, previous studies have only examined emotion perception using full-blown facial expressions. OBJECTIVE: The current study examines the perception of facial emotional expressions in HD at different levels of intensity to investigate whether more subtle deficits can be detected, possible also in other emotions. METHOD: We compared early symptomatic HD patients with healthy matched controls on emotion perception, presenting short video clips of a neutral face changing into one of the six basic emotions (happiness, anger, fear, surprise, disgust and sadness) with increasing intensity. Overall face perception ability as well as depressive symptoms were taken into account. RESULTS: A specific impairment in recognizing the emotions disgust and anger was found, which was present even at low emotion intensities. CONCLUSION: These results extend previous findings and support the use of more sensitive emotion perception paradigms, which enable the detection of subtle neurobehavioral deficits even in the pre- and early symptomatic stages of the disease. PMID- 16682800 TI - Intravenous immunoglobulin: striving for appropriate use. AB - BACKGROUND: Intravenous immunoglobulin (IVIG) is the mainstay therapy in human immune deficiency states characterized by qualitative and quantitative reductions in B cells. In addition, however, there is widespread use of IVIG in a number of other areas, including neuroimmunologic, infectious, dermatologic, hematologic, autoimmune, inflammatory and idiopathic disorders. In many of these cases, there are little objective data to support the use. METHODS: We performed a review of more than 400 publications in PubMed using the key words 'intravenous immunoglobulin' and excluded publications that focused on immune deficiency, for which the indication for IVIG is already clear. RESULTS: For a number of off label indications, there is significant evidence of efficacy and IVIG has become the standard of care for many clinical syndromes other than immune deficiency. In some conditions, however, the data have not been well controlled or randomized and are often limited to case reports that are difficult to interpret. Although the critical shortage of IVIG of the last decade is no longer an issue, IVIG is expensive and not without risk. The use of IVIG should be based not only on clinical data, but also, and especially, on the biological rationale for its use. CONCLUSIONS: The appropriate use of IVIG is an important issue that is difficult to resolve, and will continue to challenge clinicians based on expense and potentially limited supply, including the intrinsic limitations of donor plasma. The establishment of national and international voluntary registries to report use of IVIG in disorders for which evidence is lacking would be a first step toward facilitating randomized, controlled clinical trials. PMID- 16682801 TI - A rare intestinal manifestation in a patient with common variable immunodeficiency and strongyloidiasis. AB - We report an immunodeficient patient with a rare gastrointestinal manifestation. A 26-year-old male with common variable immunodeficiency (CVID) and bronchiolitis obliterans, who was on intravenous gamma-globulin and prednisone, presented diffuse abdominal pain, nausea, vomiting and constipation of 3 days' duration. He reported 5 years of recurrent respiratory infections and diarrhea with negative stool tests, including tests for Strongyloides stercoralis. A physical exam revealed a poor general condition, anemia, dehydration and a distended painful abdomen with guarding, without abdominal sounds. The radiological study showed marked dilation of the small bowel that was edematous. Resection of the affected loop was performed and the histopathologic study showed transmural infection with S. stercoralis and hemorrhagic necrosis of the muscular layer, without mucosal destruction. The patient developed malabsorption syndrome and septic shock; he was treated with antibiotics and thiabendazole and was finally discharged in a good general condition. CVID is a rare disease and its association with systemic strongyloidiasis is very uncommon, but it has been reported in patients on corticosteroids. Hemorrhagic necrosis of the muscular layer without mucosal destruction was not found in the literature studied. PMID- 16682802 TI - Endothelial and epithelial expression of eotaxin-2 (CCL24) in nasal polyps. AB - BACKGROUND: Nasal polyposis is mostly associated with eosinophilia of mucosal tissue. This points to the implication of CC chemokines in nasal eosinophilia. Recently the CC chemokine eotaxin-2 (CCL24) was identified. This study was initiated to localize the cellular source, analyze expression of mRNA, and quantify protein synthesis of CCL24. METHODS: Specimens of nasal inferior turbinates from controls and polypous tissue from patients suffering from chronic polypous sinusitis were collected. Furthermore, fibroblasts and epithelial cells were cultured. CCL24 protein was analyzed by immunohistochemistry and ELISA, expression of mRNA by SQ-RT-PCR. RESULTS: CCL24 was observed in endothelial and epithelial cells. Specimens from patients expressed significantly (>2fold) more CCL24 mRNA than controls. Fibroblasts and unstimulated cells did not express CCL24 mRNA. Upon stimulation with TNF-alpha, INF-gamma, IL-4, or costimulation with TNF-alpha and INF-gamma CCL24 mRNA was significantly enhanced (3.2-19.6%). In controls, fibroblast, and unstimulated cells CCL24 protein was below detection limit. Most polyps comprised significant amounts of CCL24 (mean 0.24 ng/mg). TNF alpha, INF-gamma or IL-4 induced CCL24 protein (0.1-0.3 ng/ml) in epithelial cells. Costimulation with TNF-alpha and IL-4 (0.1-30 and 1-30 ng/ml, respectively) synergistically induced synthesis of CCL24 protein (0.18-0.31 ng/ml). CONCLUSION: In nasal polyps endothelial and epithelial cells are obviously the main source of CCL24, which was shown for transcription (mRNA) and production (protein) levels and was associated with diseases. Results gave evidence of CLL24- directed migration of cells from inside (the bloodstream) to the epithelial side (mucosa) in eosinophilic inflammatory diseases, e.g. nasal polyposis. PMID- 16682803 TI - Correction of endothelial dysfunction in diabetic female rats by tetrahydrobiopterin and chronic insulin. AB - Diabetes-induced vascular dysfunction has mainly been studied in males. However, the mechanisms involved may not correspond to those in females. Here we analyzed the effects of tetrahydrobiopterin (BH(4)) and chronic insulin on the physiology of mesenteric arterioles of alloxan-diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy), nitric oxide synthase (NOS) activity (conversion of L-arginine to L-citrulline), eNOS gene expression (RT-PCR), NO production (diaminofluorescein), reactive oxygen species (ROS) generation (intravital fluorescence microscopy) and Cu/Zn superoxide dismutase (SOD) activity (spectrophotometry) and gene expression (RT-PCR). The reduced endothelium-dependent vasodilation of diabetic females was corrected by both BH(4) and insulin. NOS activity was decreased by diabetes, but insulin did not correct it. However, NOS expression was not modified by either diabetes or insulin. Arterioles of diabetic rats exhibited lower NO production, which was fully corrected by BH(4) and only partially by insulin. ROS generation was increased in diabetic rats, and both BH(4) and insulin normalized it. Diabetes did not change SOD activity and gene expression. However, insulin increased SOD activity but not its expression. Our data suggest that, similarly to males, endothelial dysfunction in female diabetic rats involves an altered ROS/NO imbalance. In contrast to males, however, insulin does not regulate NOS in the microcirculation of diabetic females. PMID- 16682804 TI - Postural effects on interstitial fluid pressure in humans. AB - BACKGROUND: Direct assessment of the effect of postural changes on interstitial fluid pressure (IFP) in the human skin under physiological conditions is important for the understanding of mechanisms involved in diseases resulting in lower limb edema. Previous techniques to measure IFP had limitations of being invasive, and acute measurements were not possible. Here we describe the effect of postural changes on IFP in the skin of the foot using the minimally invasive servonulling technique. RESULTS: Measurements were performed in 12 healthy subjects. IFP (means +/- SD) was significantly higher in the sitting (5.1 +/- 2.9 mm Hg) than in the supine position (-0.3 +/- 3.6 mm Hg, p = 0.04) when measured in the sitting position first. The difference between the sitting and the supine position was not significant when measurements were taken in the supine position first [from 1.0 +/- 4.3 (supine) to 3.6 +/- 6.7 mm Hg (sitting), p = 0.46]. Spontaneous low-frequency pressure fluctuations occurred in 58% of the recordings during sitting, which was almost twice as frequent as in the supine position (33%; p = 0.001), while no effects on lymphatic capillary network extension were observed (p = 0.12). CONCLUSION: Using the servonulling micropressure system, postural effects on IFP can be directly assessed. IFP is higher in the sitting position, but differences are influenced by the time in the upright position. PMID- 16682805 TI - Transcription factor Ets-1 regulates fibroblast growth factor-1-mediated angiogenesis in vivo: role of Ets-1 in the regulation of the PI3K/AKT/MMP-1 pathway. AB - We previously demonstrated that a modified secreted form of fibroblast growth factor 1 (FGF-1), a prototypic member of the FGF family, has the ability to stimulate angiogenesis in an in vivo model of angiogenesis, the so-called chick chorioallantoic membrane assay or CAM. We recently defined the importance of the phosphatidylinositol 3-kinase/AKT pathway in FGF-1-mediated angiogenesis in this model using specific pharmacological inhibitors. In our continuing efforts to define the molecular signaling pathway regulating FGF-1-induced angiogenesis in vivo, we utilized a transcription factor activity assay and identified transcription factor Ets-1 as a critical effector of FGF-1-induced angiogenesis. Both activity and mRNA expression levels of the Ets-1 molecule were increased in response to FGF-1 overexpression in CAMs, as documented by electrophoretic mobility shift assay (gel shift) and reverse transcription real-time PCR techniques, respectively. Furthermore, the delivery of Ets-1 antisense (AS) into CAM tissues effectively reduced angiogenesis in the CAM assay. In addition, both Ets-1 AS-treated chicken CAMs and cultured endothelial cells exhibited a reduction in matrix metalloproteinase 1 gene expression levels. The Ets-1 AS treated endothelial cells also demonstrated a reduction in migration. These data suggest that Ets-1 activation is a requisite for FGF-1-mediated angiogenesis in vivo. Therefore, Ets-1 might be a potential target for the generation of inhibitor drugs for the treatment of FGF-dependent pathological angiogenesis such as metastatic tumors, rheumatoid arthritis and diabetic retinopathy. PMID- 16682806 TI - GABA(B1) knockout mice reveal alterations in prolactin levels, gonadotropic axis, and reproductive function. AB - gamma-Aminobutyric acid (GABA) has been implicated in the control of hypophyseal functions. We evaluated whether the constitutive loss of functional GABA(B) receptors in GABA(B1) knockout (GABA(B1)(-/-)) mice alters hormonal levels, under basal and stimulated conditions, and reproductive function. The serum hormone levels were measured by radioimmunoassay, the estrous cyclicity was evaluated by vaginal lavages, and the mating behavior was determined by the presence of vaginal plugs. A moderate hyperprolactinemic condition was observed, in which prolactin increase and thyroid-stimulating hormone decrease were similar between genotypes. Basal luteinizing hormone (LH), follicle-stimulating hormone, thyroid stimulating hormone, and growth hormone levels were similar between genotypes in each sex. Analysis of the gonadotropin axis revealed no differences in puberty onset between female genotypes. In con trast, the estrous cyclicity was significantly disrupted in GABA(B1)(-/-) female mice, showing significantly extended periods in estrus and shortened periods in proestrus. Reproduction was significantly compromised in GABA(B1)(-/-) females, with a significantly lower proportion of mice (37.5%) getting pregnant during the first 30 days of mating as compared with wild-type controls (87.5%). Moreover, only 14% of vaginal plug positive GABA(B1)(-/-) females had successful pregnancies as compared with 75% in the controls. In addition, the postovariectomy LH rise was significantly advanced in GABA(B1)(-/-) mice, while the response to estradiol feedback was similar in both genotypes. In conclusion, our endocrine analysis of GABA(B1)(-/-) mice reveals that GABA(B) receptors are involved in the regulation of basal prolactin titers. Moreover, the hypothalamic-hypophyseal-ovarian axis is seriously disturbed, with alterations in cyclicity, postcastration LH increase, and fertility indexes. The molecular mechanism underlying these hormonal disturbances remains to be addressed. PMID- 16682807 TI - Differential modulation of ovine fetal ACTH secretion by PGHS-1 and PGHS-2. AB - BACKGROUND/AIMS: We have previously demonstrated that prostaglandin generation within the fetal brain augments or partially mediates fetal reflex responsiveness to hypotension. The present study was performed to test the relative roles of prostaglandin endoperoxide synthases-1 and -2 (PGHS-1 and 2, or COX-1 and 2) as potential mediators of this interaction. METHODS: Chronically catheterized and instrumented fetal sheep were subjected to transient brachiocephalic occlusion (BCO) after intracerebroventricular injection of resveratrol (PGHS-1 or COX-1 inhibitor), nimesulide (PGHS-2 or COX-2 inhibitor), or vehicle. RESULTS: BCO decreased arterial pressure perfusing the fetal brain and stimulated increases in systemic blood pressure and heart rate as well as in circulating concentrations of ACTH. Inhibition of PGHS-1 and PGHS-2 had differential effects on fetal ACTH secretion. Pre-BCO concentrations of plasma ACTH increased in response to nimesulide, while the fetal ACTH response to BCO was delayed by resveratrol. Prior to the BCO, nimesulide also increased fetal blood pressure and decreased fetal heart rate. The injections of resveratrol and nimesulide did not alter placental biosynthesis of prostaglandins and therefore acted within the fetal brain. CONCLUSION: We conclude that prostaglandin generated in the fetal brain by the action of PGHS-1 augments fetal ACTH reflex responses to BCO but that, in contrast, the action of PGHS-2 is inhibitory to ACTH secretion. PMID- 16682808 TI - Management dilemmas in the treatment and follow-up of advanced juvenile nasopharyngeal angiofibroma. AB - Advanced juvenile nasopharyngeal angiofibroma (JNA) (i.e. disease with intracranial extension) is reportedly uncommon. The optimum management form of such a clinical situation currently remains controversial. This review, of the recent English published literature, discusses the common JNA classification systems advocated, shows the advantages and disadvantages of surgical approaches used (both open, endoscopic or combined), and re-evaluates the current role of radiotherapy (for initial management or treatment of residual/recurrent disease). We also discuss the typical presentation of these patients, the role of pre operative embolization, as well as surgical techniques to minimize recurrence, and advocate a post-treatment follow-up protocol. PMID- 16682809 TI - JNK regulation of oncogenesis. AB - The literature provides strong precedent for both pro-tumorigenic and tumor suppressor roles for the c-Jun N-terminal kinases (JNKs) in the setting of oncogenesis. Clearly, JNKs are activated by numerous oncogenes and growth factors and the literature documents a role for these MAP kinases in cell proliferation and transformation. By contrast, JNKs mediate signals from diverse stimuli that result in cell death or differentiation and a role for JNKs as tumor suppressors has emerged. This enigmatic nature of the JNKs in the setting of oncogenesis is considered herein. Further illumination of the complex and context-dependent functions of the JNKs in cancer cells is of obvious importance for the rational use of small molecule JNK inhibitors as therapeutics. PMID- 16682810 TI - The modulation of inflammatory gene expression by lipids: mediation through Toll like receptors. AB - Toll-like receptors (TLRs) were evolved to detect invading pathogens and to induce innate immune responses in order to mount host defense mechanisms. It becomes apparent that the activation of certain TLRs is also modulated by endogenous molecules including lipid components, fatty acids. Results from epidemiological and animal studies demonstrated that saturated and polyunsaturated dietary fatty acids can differentially modify the risk of development of many chronic diseases. Inflammation is now recognized as an important underlying etiologic condition for the pathogenesis of many chronic diseases. Therefore, if the activation of TLRs and consequent inflammatory and immune responses are differentially modulated by types of lipids in vivo, this would suggest that the risk of the development of chronic inflammatory diseases and the host defense against microbial infection may be modified by the types of dietary fat consumed. PMID- 16682811 TI - Mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein. AB - Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a distant member of the Group 2 coronaviruses, has recently been identified as the etiological agent of severe acute respiratory syndrome (SARS). The genome of SARS CoV contains four structural genes that are homologous to genes found in other coronaviruses, as well as six subgroup-specific open reading frames (ORFs). ORF3 encodes a predicted 154-amino-acid protein that lacks similarity to any known protein, and is designated 3b in this article. We reported previously that SARS CoV 3b is predominantly localized in the nucleolus, and induces G0/G1 arrest and apoptosis in transfected cells. In this study, we show that SARS-CoV 3b fused with EGFP at its N- or C- terminus co-localized with a mitochondria-specific marker in some transfected cells. Mutation analysis of SARS-CoV 3b revealed that the domain spanning amino acids 80 to 138 was essential for its mitochondria localization. These results provide new directions for studies of the role of SARS-CoV 3b protein in SARS pathogenesis. PMID- 16682812 TI - Mapping of quantitative trait loci for salt tolerance at the seedling stage in rice. AB - Salt tolerance was evaluated at the young seedling stage of rice (Oryza sativa L.) using recombinant inbred lines (MG RILs) from a cross between Milyang 23 (japonica/indica) and Gihobyeo (japonica). 22 of 164 MG RILs were classified as tolerant with visual scores of 3.5-5.0 in 0.7% NaCl. Interval mapping of QTLs related to salt tolerance was conducted on the basis of the visual scores at the young seedling stage. Two QTLs, qST1 and qST3, conferring salt tolerance, were detected on chromosome 1 and 3, respectively, and the total phenotypic variance explained by the two QTLs was 36.9% in the MG RIL population. qST1 was the major QTL explaining 27.8% of the total phenotypic variation. qST1 was flanked by Est12 RZ569A, and qST3 was flanked by RG179-RZ596. The detection of new QTLs associated with salt tolerance will provide important information for the functional analysis of rice salt tolerance. PMID- 16682813 TI - The ozone stress transcriptome of pepper (Capsicum annuum L.). AB - We used cDNA microarrays to monitor the transcriptome of ozone stress-regulated genes (ORGs) in two pepper cultivars [Capsicum annuum cv. Dabotop (ozone sensitive) and Capsicum annuum cv. Buchon (ozone-tolerant)]. Ozone stress up- or down-regulated 180 genes more than three-fold. Transcripts of 84 of these ORGs increased, transcripts of 88 others diminished, and those of eight either accumulated or diminished at different time points in the two cultivars or changed in only one of the cultivars. 67% (120) of the ORGs were regulated differently in ozone-sensitive and ozone-tolerant peppers, most being specifically up-regulated in the ozone-sensitive cultivar. Many were also represented in the plant defense transcriptome against non-host pathogen infection, and some in the transcriptomes for cold, drought, and salinity stresses. PMID- 16682814 TI - Myogenic differentiation of p53- and Rb-deficient immortalized and transformed bovine fibroblasts in response to MyoD. AB - We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and p16(INK4a) functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and p16(INK4a) pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions. PMID- 16682815 TI - Antitumor activity of the novel human cytokine AIMP1 in an in vivo tumor model. AB - Although AIMP1 (previously known as p43) is one of three auxiliary proteins bound to a macromolecular aminoacyl tRNA complex, it is also secreted as a cytokine controlling both angiogenesis and immune responses. Here we show that systemically administered purified recombinant human AIMP1 had anti-tumor activity in mouse xenograft models. In Meth A-bearing Balb/c mice, tumor volume increased about 28 fold in the vehicle treatment group, while an increase of about 16.7 fold was observed in the AIMP1-treated group. We also evaluated the anti-tumor activity of AIMP1 in combination with a sub-clinical dose of the cytotoxic anti-tumor drug, paclitaxel. The growth of NUGC-3 human stomach cancer cells was suppressed by 84% and 94% by the combinations of 5 mg/kg paclitaxel + 25 mg/kg AIMP1 (p = 0.03), and 5 mg/kg paclitaxel + 50 mg/kg AIMP1 (p = 0.02), respectively, while 5 mg/kg paclitaxel alone suppressed growth by only 54% (p = 0.02). A similar cooperative effect of AIMP1 and paclitaxel was observed in a lung cancer xenograft model. These results suggest that AIMP1 may be useful as a novel anti-tumor agent. PMID- 16682816 TI - Dose-dependent UV stabilization of p53 in cultured human cells undergoing apoptosis is mediated by poly(ADP-ribosyl)ation. AB - The effect of poly(ADP-ribosyl)ation on the stability of p53 in SK-HEP1 cells treated with UV light was examined. Intracellular levels of p53 increased in cells treated with a low dose of UV light (20 J/m2), whereas they increased but then declined after a higher dose of UV (100 J/m2). Intracellular levels of p53 in the UV treated SK-HEP1 cells were dependent on the UV dose. Use of proteasome inhibitors revealed that p53 is degraded by proteasomal proteolysis after high doses of UV light. We present evidence that, at low doses, poly(ADP ribose)polymerase (PARP) poly(ADP-ribosyl)ates p53 and protects it from proteasomal degradation before caspase-3 is activated, whereas at high doses the cells undergo UV induced apoptosis and PARP is cleaved by caspase-3 before it can protect p53 from degradation. Destabilization of p53 by cleavage of PARP may be important in cell fate decision favoring apoptosis. PMID- 16682817 TI - Cellular changes resulting from forced expression of glypican-3 in hepatocellular carcinoma cells. AB - Glypican-3 (GPC3) is a member of the glypican family, which encodes cell-surface heparan-sulfate proteoglycans, and is frequently upregulated in hepatocellular carcinoma (HCC). We have recently reported that blocking endogenous GPC3 expression promotes the growth of HCC cell lines, suggesting that GPC3 plays a negative role in HCC cell proliferation. Here, we report that forced expression of GPC3 reduced the growth of HCC cells. We also found that FGF2-mediated cell proliferation was inhibited by GPC3. In addition, we observed that the adhesion of HCC cells to collagen type I and fibronectin was decreased by GPC3, whereas cellular migration and invasiveness were stimulated. Collectively, these results suggest that progression of hepatocellular carcinoma is associated with upregulation of GPC3. PMID- 16682818 TI - A helix-induced oligomeric transition of gaegurin 4, an antimicrobial peptide isolated from a Korean frog. AB - Gaegurin 4 (GGN4), a novel peptide isolated from the skin of a Korean frog, Rana rugosa, has broad spectrum antimicrobial activity. A number of amphipathic peptides closely related to GGN4 undergo a coil to helix transition with concomitant oligomerization in lipid membranes or membrane-mimicking environments. Despite intensive study of their secondary structures, the oligomeric states of the peptides before and after the transition are not well understood. To clarify the structural basis of its antibiotic action, we used analytical ultracentrifugation to define the aggregation state of GGN4 in water, ethyl alcohol, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The maximum size of GGN4 in 15% HFIP corresponded to a decamer, whereas it was monomeric in buffer. The oligomeric transition is accompanied by a cooperative 9 nm blue-shift of maximum fluorescence emission and a large secondary structure change from an almost random coil to an alpha-helical structure. GGN4 induces pores in lipid membranes and, using electrophysiological methods, we estimated the diameter of the pores to be exceed 7.3 A, which suggests that the minimal oligomer structure responsible is a pentamer. PMID- 16682819 TI - Nerve growth factor activates brain-derived neurotrophic factor promoter IV via extracellular signal-regulated protein kinase 1/2 in PC12 cells. AB - Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC. PMID- 16682820 TI - Identification of a peptide enhancing mucosal and systemic immune responses against EGFP after oral administration in mice. AB - Gangliosides are receptors for various peptides and proteins including neuropeptides, beta-amyloid proteins, and prions. Recently, the role of gangliosides in mucosal immunization has attracted attention due to the emerging interest in oral vaccination. Ganglioside GM1 exists in abundance on the surface of the M cells of Peyer's patch, a well-known mucosal immunity induction site. In the present study we identified a peptide ligand for GM1 and tested whether it played a role in immune induction. GM1-binding peptides were selected from a phage-displayed dodecapeptide library and one peptide motif, GWKERLSSWNRF, was fused to the C-terminus of enhanced green fluorescent protein (EGFP). The fusion protein, but not EGFP fused with a control peptide, was concentrated around Peyer's patch after incubation in the lumen of the intestine ex vivo. Furthermore, oral feeding of the fusion protein but not control EGFP induced mucosal and systemic immune responses against EGFP resembling Th2-type immune responses. PMID- 16682821 TI - The study of Bfa1p(E438K) suggests that Bfa1 control the mitotic exit network in different mechanisms depending on different checkpoint-activating signals. AB - During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit via mitotic checkpoints. In budding yeast, mitotic exit is controlled by a regulatory cascade called the mitotic exit network (MEN). The MEN is regulated by a small GTPase, Tem1p, which in turn is controlled by a two-component GAP, Bfa1p-Bub2p. Recent results suggested that phosphorylation of Bfa1p by the polo-related kinase Cdc5p is also required for triggering mitotic exit, since it decreases the GAP activity of Bfa1p-Bub2p. However, the dispensability of GEF Lte1p for mitotic exit has raised questions about regulation of the MEN by the GTPase activity of Tem1p. We isolated a Bfa1p mutant, Bfa1p(E438K), whose overexpression only partially induced anaphase arrest. The molecular and biochemical functions of Bfa1p(E438K) are similar to those of wild type Bfa1p, except for decreased GAP activity. Interestingly, in BFA1(E438K) cells, the MEN could be regulated with nearly wild type kinetics at physiological temperature, as well as in response to various checkpoint activating signals, but the cells were more sensitive to spindle damage than wild type. These results suggest that the GAP activity of Bfa1p-Bub2p is responsible for the mitotic arrest caused by spindle damage and Bfa1p overproduction. In addition, the viability of cdc5-2 delta bfa1 cells was not reduced by BFA1(E438K), suggesting that Cdc5p also regulates Bfa1p to activate mitotic exit by other mechanism(s), besides phosphorylation. PMID- 16682822 TI - Transcriptional regulation of the methuselah gene by dorsal protein in Drosophila melanogaster. AB - The Drosophila methuselah (mth) mutant has an approximately 35 percent increase in average lifespan, and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat. To examine the transcriptional regulation of mth, we used luciferase assays employing Drosophila S2 cells. Two positive control elements were found at -542 to -272 (PE1) and +28 to +217 (PE2), where putative binding sites for transcription factors including Dorsal (Dl) were identified. Cotransfection of a Dl expression plasmid with a mth luciferase reporter plasmid resulted in decreased reporter activity. PE1 and PE2, the minimal elements for strong promoter activity, were required for maximal repression by Dl protein. The N-terminal Rel homology domain (RHD) of Dl was not sufficient for repression of mth. We demonstrated by chromatin affinity precipitation (ChAP) assays in S2 cells that Dl bound to the putative PE1 binding site. Unexpectedly, semi-quantitative RT-PCR analysis revealed that the level of mth transcripts was reduced in dl flies. However, the in vivo result support the view that mth expression is regulated by dl, since it is well known that Dl functions as both a transcriptional activator and repressor depending on what other transcription factors are present. These findings suggest that both innate immunity and resistance to stress are controlled by Dl protein. PMID- 16682823 TI - Functional annotation and analysis of Korean patented biological sequences using bioinformatics. AB - A recent report of the Korean Intellectual Property Office (KIPO) showed that the number of biological sequence-based patents is rapidly increasing in Korea. We present biological features of Korean patented sequences though bioinformatic analysis. The analysis is divided into two steps. The first is an annotation step in which the patented sequences were annotated with the Reference Sequence (RefSeq) database. The second is an association step in which the patented sequences were linked to genes, diseases, pathway, and biological functions. We used Entrez Gene, Online Mendelian Inheritance in Man (OMIM), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Through the association analysis, we found that nearly 2.6% of human genes were associated with Korean patenting, compared to 20% of human genes in the U.S. patent. The association between the biological functions and the patented sequences indicated that genes whose products act as hormones on defense responses in the extra cellular environments were the most highly targeted for patenting. The analysis data are available at http://www.patome.net. PMID- 16682824 TI - Enhanced expression of plasma glutathione peroxidase in the thymus of mice treated with TCDD and its implication for TCDD-induced thymic atrophy. AB - The potent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces thymus atrophy in experimental animals. However, its mechanism of action is not fully understood. To gain insight into its immunosuppressive effect, Balb/c mice were intraperitoneally injected with TCDD (30 microg/kg body weight) and genes regulated by TCDD were identified using cDNA arrays [Park and Lee (2002)]. One of the regulated genes was that for plasma glutathione peroxidase (pGPx). Upon TCDD injection, pGPx mRNA levels in the thymus increased, in parallel with increases in GPx activity and the frequency of anti-human pGPx antibody-reactive cells. pGPX mRNA levels were also moderately up-regulated in the testis and spleen. This is the first report that a particular isotype of the glutathione peroxidase family is regulated by TCDD at both mRNA and protein levels. pGPx is expressed in various tissues in contact with body fluids, and detoxifies hydrogen peroxides and lipid hydroperoxides. It will be of interest to assess the role of pGPx in TCDD-induced thymic atrophy. PMID- 16682825 TI - Analysis of intragenic Ds transpositions and excision events generating novel allelic variation in rice. AB - Even though Ac/Ds gene-tagging systems have been established in many higher plants, maize is the only major plant in which short-distance transposition of Ac/Ds has been utilized to probe gene function. This study was performed to evaluate the efficiency of obtaining new alleles and functional revertants from Ds insertion loci in rice. By analyzing 1,580 plants and the progeny of selected lines, the insertion sites and orientations of Ds elements within 16 new heritable alleles of three rice loci were identified and characterized. Intragenic transposition was detected in both directions from the original insertion sites. The closest interval was 35 bp. Three of the alleles had two Ds elements in cis configuration in the same transcription units. We also analyzed the excision footprints of intragenic and extragenic transpositions in Ds inserted alleles at 5 loci. The 134 footprints obtained from different plants revealed predominant patterns. Ds excision at each locus left a predominant footprint at frequencies of 30-75%. Overall, 66% of the footprints were 7-bp additions. In addition, 16% of the excisions left 0-, 3-, 6-, and 9-bp additions with the potential of conserving reading frame. PMID- 16682826 TI - Phosphorylation of eukaryotic elongation factor 2 can be regulated by phosphoinositide 3-kinase in the early stages of myoblast differentiation. AB - We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3 kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in G2/M and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in G0/G1 while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation. PMID- 16682827 TI - Identification of two types of naturally-occurring intertypic recombinants of Epstein-Barr virus. AB - Two Epstein-Barr virus (EBV) types, type 1 and type 2, maintain the same allelic specificity at four genomic loci encoding the EBNA2, -3A, -3B, and -3C proteins. We have previously described 16 EBV-transformed B-lymphoblastoid cell lines derived from Korean cancer patients, and the EBNA2 types of the EBV isolates therein. In this study, the allelic types of the EBNA2, -3A, -3B, and -3C genes of these EBV isolates were determined. We report the identification of two distinct types of naturally occurring intertypic recombinants, one with genotype EBNA2 type 1/EBN3A, -3B, -3C type 2 and the other with genotype EBNA2, -3A type 1/EBNA3B, -3C type 2. The existence of these intertypic recombinants indicates that various intertypic EBV strains may be circulating in the human population, in addition to typical EBV-1 and EBV-2 strains. PMID- 16682828 TI - Detection of abnormally high amygdalin content in food by an enzyme immunoassay. AB - Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput. PMID- 16682829 TI - Clinical effects of early treatment with insulin glargine in patients with cystic fibrosis and impaired glucose tolerance. AB - Diabetes mellitus is an increasing complication of cystic fibrosis (CF), as a result of the improved life expectancy. There is clear association between diabetes and increased morbidity and mortality. Lung function and clinical status deteriorate up to 2-4 yr before the diagnosis of CF-related diabetes (CFRD). The aim of our study was to evaluate the effects, on glucose homeostasis and clinical status, of the early treatment with insulin glargine in CF patients with impaired glucose tolerance (IGT). We selected six subjects with IGT diagnosed at oral glucose tolerance test (OGTT). Median age was 18.12 yr (range 9.2-27.8). Insulin glargine was administered at the median dosage of 0.3 U/kg/day (range 0.2-0.5). After the initial adjustment of the dosage, no patient manifested hypoglycemia during treatment. Median glycosylated hemoglobin (HbA1c) did not show any significant variation during treatment: it was 5.9% at baseline (range 5.5-6.2) and 6.1% (range 5.0-6.7) at the end of follow-up (p=0.496). Median body mass index (BMI) z-score significantly increased during treatment, from -0.95 (range 3.2-+0.6) at baseline to -0.5. (range -3.0-+0.9) at the end of follow-up (p=0.026). Lung function, measured by median forced expiratory volume in the first second (FEV1%), showed a mild but significant improvement during insulin treatment. It was 72.7% at baseline (range 41.5-98.4) and 76.7% (range 42.0 106.8) at the end of follow-up (p=0.027). No significant variation was found between the number of hospitalizations for clinical exacerbation (no./patient/yr) in the last 2 yr before treatment and during follow-up. Median number at baseline was 1.95/patient/yr (range 1-3) and 2.0/patient/yr (range 1-3) at follow-up (p=0.715). Our data seem to indicate that early insulin therapy can be safe, no patient manifested hypoglycemia or other adverse effects during treatment. Insulin is an anabolic hormone implicated in both lipid and protein metabolism. The appearance of IGT out of infections can indicate an early insulin deficiency, with a potential impact on the nutritional and clinical status of the patient, even before the appearance of overt diabetes. Larger controlled trials are necessary to verify if early insulin therapy is able to reduce the deterioration of nutritional status and lung function associated with the onset of IGT. PMID- 16682830 TI - Endothelial dysfunction in subjects with subclinical hyperthyroidism. AB - Numerous clinical and experimental reports suggest that a high von Willebrand factor (vWf) level reflects endothelial damage or endothelial dysfunction. The present study was designed to evaluate vWf in subclinical hyperthyroidic subjects compared with euthyroidic subjects. We selected 20 subclinical hyperthyroidic subjects and 20 euthyroidic control subjects matched for age, gender and body mass index. The level of vWf was significantly higher in the subclinical hyperthyroidic group than in the euthyroidic group (42.9+/-9.6 vs 37.6+/-6.4%, p=0.026). In conclusion, our results suggest that subjects with subclinical hyperthyroidism tend to have an endothelial dysfunction. Endothelial dysfunction could contribute to increasing the cardiovascular risk in subclinical hyperthyroidism. PMID- 16682831 TI - Mild hyperhomocysteinemia and the common C677T polymorphism of methylene tetrahydrofolate reductase gene are not associated with the metabolic syndrome in Type 2 diabetes. AB - A moderate increase of total homocysteine (tHcy) plasma levels seems to increase cardiovascular disease (CVD) risk in Type 2 diabetic subjects, but its relationship with diabetes and insulin-resistance is still controversial. We examined whether mild hyperhomocysteinemia and its major genetic determinant would cluster with the metabolic syndrome (MS) in Type 2 diabetes. One hundred Type 2 diabetic subjects with and without MS were enrolled in the study. Fasting tHcy, vitamin B12, and folate plasma levels, insulin-resistance [assessed by homeostasis model assessment, (HOMAIR)] and the methylene tetrahydrofolate reductase (MTHFR) C677T genotype were assessed in all the participants. Geometric mean tHcy concentration and the prevalence of mild hyperhomocysteinemia, as commonly defined by tHcy >/=15 micromol/l, were comparable in diabetic subjects with and without MS, even after adjustment for age, sex, vitamin B12, folate and creatinine levels. In both groups, the MTHFR C677T genotype distribution was not significantly different from the Hardy-Weinberg equilibrium, with a TT homozygous frequency of 21% in subjects with and 18% in those without the syndrome (p=ns). tHcy plasma levels and the degree of insulin-resistance did not differ across MTHFR genotypes in both groups, even after multivariable adjustment. Overall, tHcy significantly correlated with creatinine (r=0.25; p=0.009) and trygliceride concentrations (r=0.24; p=0.02), but not with HOMAIR. At multivariate analysis, only creatinine was significantly correlated with tHcy levels (beta=0.42; p=0.001). In conclusion, hyperhomocysteinemia and the common C677T variant of MTHFR gene are not associated with MS in Type 2 diabetic subjects. PMID- 16682832 TI - Congenital adenohypophysis aplasia: clinical features and analysis of the transcriptional factors for embryonic pituitary development. AB - Anterior pituitary agenesis (APA) has very rarely been reported. Therefore, its phenotypical and genotypical features are not well known. The aim of this study was to ascertain whether the clinical picture in 4 subjects with APA and multiple pituitary hormone deficiencies (MPHD) was different compared to the one observed in a selected control group consisting of 7 MPHD individuals with hypoplastic (and not aplastic) adenohypophysis and pituitary stalk interruption syndrome. Another goal was to investigate genetic basis of APA by analyzing for the first time in this condition many of the transcriptional factors which are required for both structural development and cellular differentiation of hypophysis. Age at diagnosis was significantly lower in APA children than in controls (1.5+/-2.3 vs 11.1+/-7.6 yr, p<0.0005). Microphallus and neonatal cholestasis were observed only in APA subjects (chi-squared=4.3, p<0.05) and also neonatal hypoglycemia was more frequent in APA patients than in controls (X2=4.05, p<0.05). Molecular analyses of the genes of the transcriptional factors POU1F1, PROP1, LHX3, LHX4, ISL1 and HESX1 detected no mutations in APA patients. CONCLUSIONS: a) if compared with a selected cohort of MPHD patients with both adenohypophysis hypoplasia and pituitary stalk interruption syndrome, the ones with APA show an earlier and more severe picture of hypopituitarism; b) mutations in several transcription factors that are known to be essential for the development of Rathke's pouch are not necessarily found in humans with APA. PMID- 16682834 TI - Calcaneus ultrasound in males: normative data in the Croatian population (ECUM study). AB - Limited number of dual X-ray absorptiometry (DXA) devices in Croatia makes this diagnostic technique unavailable to the majority of the population. Quantitative ultrasound (QUS) of the calcaneus could be an alternative tool for assessing fracture risk. However, age-specific normative data should be obtained before using the QUS in everyday clinical practice. The aim of our Epidemiology of Calcaneus Ultrasound in Males (ECUM) study is to establish the normative QUS data in a healthy sample of Croatian males. A total of 1002 male participants, aged 20 99, recruited in different Croatian counties, were included in the study. In each subject broadband ultrasound attenuation (BUA), speed of sound (SOS) and quantitative ultrasound index (QUI) of the left calcaneus were measured using the Sahara ultrasound device (Hologic). The coefficients of variation were 2.85 for BUA, 0.37 for SOS and 2.49 for QUI. Significant declining with age was found for all three parameters, BUA (p<0.001), SOS (p<0.001) and QUI (p<0.001), with respective r values 0.14, 0.27 and 0.23. The peak SOS (1,562.8+/-28.5 m/sec) and QUI (103.6+/-16.5) values were observed in the third decade, whereas the peak BUA value (86.2+/-19.2 db/MHz) was observed in the fourth decade. A subgroup of 103 participants, aged 20-29, was used to estimate young adult mean and SD for QUI and calculate the T-scores. Using the World Health Organization diagnostic criteria the rates of osteoporosis (T-score<-2.5) in the males aged 50 and older was 5.8%. However, when we used the cut-off value of the T-score<-1.8, as previously suggested, prevalence of osteoporosis in Croatian males >50 yr was 16.2%. Although further studies might improve our understanding of the QUS role in the fracture prediction, we hope that the results presented here will improve the clinical management of osteoporosis in males. PMID- 16682833 TI - The relationship between blood pressure and serum parathyroid hormone with special reference to urinary calcium excretion: the Tromso study. AB - The aim of the present cross-sectional epidemiological study from Tromso, Northern Norway, was to evaluate the relation between blood pressure and serum PTH, and to examine whether this relation can be explained by a blood pressure induced increase in urinary calcium. Ten thousand-four hundred and nineteen subjects were invited to participate and 8,128 attended. Those with serum calcium outside the reference range (2.20-2.60 mmol/l), with increased serum creatinine (upper limit 120 micromol/l for men and 100 micromol/l for women) and those using antihypertensive medication were excluded. Three thousand- six hundred and twenty subjects had complete data on outcome measures. Height, weight, blood pressure, serum calcium, PTH, and creatinine were measured and smoking status recorded. A morning urine sample was collected and urinary calcium, sodium and creatinine measured. The urinary calcium/urinary creatinine ratio (Uca/Ucr) and urinary sodium/urinary creatinine ratio (Una/Ucr) were calculated. There was a significant association between both systolic and diastolic blood pressure and serum PTH. The Uca/Ucr increased with increasing blood pressure. However, the Uca/Ucr did not affect the association between blood pressure and serum PTH in a multiple linear regression model. The relationship between blood pressure and serum PTH was also seen in subjects with similar Uca/Ucr, and a negative association between serum PTH and the Uca/Ucr was found. In conclusion, blood pressure and serum PTH are associated. This association cannot be explained by the urinary calcium excretion alone. PMID- 16682835 TI - Weight loss in obese children carrying the proopiomelanocortin R236G variant. AB - To acquire more information relative to the course of obesity in conditions of food restriction in subjects carrying mutations in the melanocortin signaling pathway, 710 obese children (mean age: 9.5+/-2.1 yr; mean z-score body mass index: 3.63+/-1.6) were genotyped for the proopiomelanocortin (POMC) R236G substitution, a variant which has been associated to early onset obesity, by restriction fragment length polymorphism (RFLP) analysis. Three children were heterozygotes for the R236G variant (0.4%). One of them had the metabolic syndrome. This variant was not found in 400 controls. The 3 probands followed a hypocaloric balanced diet and, after about 12 months, normalized their weight as well as fat mass and insulin resistance. The patient with the metabolic syndrome reversed this condition. These results show that a) the R236G substitution of POMC gene, although not a major cause of obesity among Italian obese children and adolescents, is associated with early onset obesity, and that b) inherited alterations of the melanocortin signaling pathway, independently of the degree of obesity, do not preclude the possibility to lose weight in mutated individuals following a hypocaloric diet. PMID- 16682836 TI - Increase in adiponectin levels during pioglitazone therapy in relation to glucose control, insulin resistance as well as ghrelin and resistin levels. AB - Glitazones increase the secretion of the adipocyte-derived hormone adiponectin. Furthermore, the gastric signal peptide ghrelin is known to suppress adiponectin expression in adipocyte cell culture models. It is not known whether the increase in adiponectin during glitazone therapy is due to a suppression of ghrelin levels, a decrease of resistin concentrations or an amelioration of glucose control. In 10 patients (age 71+/-9 yr, body mass index 29.9+/-3.6 kg/m(2), HbA1c 6.9+/-0.5%) with Type 2 diabetes, who had already been treated with sulfonylureas, we additionally initiated a pioglitazone therapy (30 mg/day) for 12 weeks. To investigate the pioglitazone effect independently of blood glucose, glycosylated hemoglobin (HbA1c) was kept unchanged by reducing the daily dose of sulfonylurea if necessary. Ghrelin concentration [radioimmunoassay (RIA), Phoenix Pharmaceuticals, Mountain View, CA, USA], adiponectin levels [enzyme-linked immunosorbent assay (ELISA), Biovendor, Heidelberg, Germany] as well as resistin concentrations (ELISA, Linco Research, St. Charles, MO, USA) were measured before and after pioglitazone. Glucose control remained unchanged within the 12-week pioglitazone therapy (HbA1c 6.9+/-0.5% before vs 6.8+/-0.6% after pioglitazone) while body weight increased from 86.6+/-9.2 to 88.0+/-9.4 kg (p<0.05), and insulin concentration decreased from 19.6+/-5.7 to 10.1+/-1.6 microU/ml (p<0.05). Adiponectin concentration increased in all patients from 7.70+/-2.47 to 23.33+/ 8.28 microg/ml (p<0.01), while resistin concentrations tended to decrease (by 15%; p=0.059). However, ghrelin remained unchanged during therapy. No correlations were observed either between ghrelin, resistin, insulin and adiponectin, or between body weight and hormone plasma levels. The increase in adiponectin levels during pioglitazone therapy seems to be at least partly independent of blood glucose and insulin concentration as well as of ghrelin levels, and it was not associated with a decrease in resistin concentrations. PMID- 16682837 TI - A follow-up of GH-dependent biomarkers during a 6-month period of the sporting season of male and female athletes. AB - In order to verify the effects of the sporting season (entailing periods of training, competition, recovery, resting) on GH-dependent parameters in male and female athletes from different sporting disciplines, 47 male and female athletes (3 rowers, 5 swimmers, 7 alpine skiers, 3 soccer players, 7 middle distance runners, 14 sprinters, 4 triathletes, 1 road walker, 3 cyclists) were followed-up for a period of 6 months. Blood samples were taken every two months for the evaluation of IGF-I, N-terminal propeptide of type III procollagen (PIIINP) and C terminal cross-linked telopeptide of type I collagen (ICTP). Abnormal IGF-I, PIIINP and ICTP levels were observed during the follow-up period in 7/100 (7%), 9/100 (9.0%) and 8/100 (8%) samples of the male group, respectively, and in 9/88 (10.2%), 1/88 (1.1%) and 0/88 (0%) samples of the female group, respectively. Abnormal levels appeared to be randomly distributed over the different periods of the sporting season and within male and female subjects, with the large majority of abnormal values being found in the younger athletes. Taking into account all the tests done during the 6-month period (no. 564), individual markers falling outside the normal range (for age) were observed in a small number of instances (34/564 tests done, 24/300 for males and 10/264 for females). When our method for the detection of exogenous recombinant GH (rhGH) administration, based on the concomitant determination of these three peripheral GH-dependent markers and on the attribution of specific scores, was applied in the same athlete at a given time point of the 6-month period, the prevalence of a positive score was extremely low (ie, 3/188 samples or 1.6%). Total positive scores were actually recorded in only three male athletes (2 swimmers and 1 skier, aged <21 yr) at one occasion during the 6-month period considered. In contrast, no total positive scores were found in female athletes (ie, 0/88 samples). In conclusion, the concentrations of IGF-I, PIIINP and ICTP were stable and not significantly modified during 6 months of a sporting season (entailing periods of training, competition, recovery, resting) in athletes from different sporting disciplines. Therefore our method, based on the concomitant determination of three peripheral GH-dependent biomarkers appears safe, acceptable, relatively inexpensive and repeatable (in case of positive or suspected values) immediately or at different intervals of the sporting season. Further additional studies are requested to precise the cut-off values for narrower age-class subdivisions in both genders in order to improve the proposed method. PMID- 16682838 TI - Treatment of post-partum thyrotoxicosis. AB - Thyrotoxicosis occurs more frequently during the post-partum period than at other times in women of childbearing age. Graves' disease and post-partum thyroiditis are two major causes of thyrotoxicosis in this period. The major task lies in differentiation of these two diseases in the post-partum period; since throtoxicosis caused by post-partum thyroiditis usually does not require treatment. The radioiodine uptake is elevated or normal in Graves' disease and low in post-partum thyroiditis, and TSH-receptor antibodies are positive in Graves' and negative in post-partum thyroiditis. Post-partum thyrotoxicosis due to Graves' disease may be treated with radioiodine but it requires radiation safety measurements for infant and is contraindicated if the mother is breast feeding. Antithyroid drugs are the mainstay of the treatment of post-partum thyrotoxicosis. Recent investigations conclude that neither propylthiouracil nor methimazole cause any alterations in thyroid function and physical and mental development of infants breast-fed by lactating thyrotoxic mothers, and both can be safely administered in moderately high doses during lactation. PMID- 16682839 TI - Association between breast cancer and autoimmune thyroid disorders: no increase of lymphocytic infiltrates in breast malignant tissues. AB - An association between thyroid autoimmunity and breast cancer (BC) has been consistently reported, but the cause of this association is still unknown. The role of lymphocytic infiltration (LI) in breast tumorigenesis is controversial and several data suggest that in BC an increase of lymphoid cell infiltrates or a dysfunctional local immune response may be detected very early during tumor development. Chronic autoimmune thyroiditis is characterized by different degrees of LI in thyroid gland and BC cells share some antigenic properties similar to those detected in thyroid tissue, such as sodium iodide symporter (NIS) and peroxidase activity. The aim of this study was to evaluate the frequency and amount of LI in malignant and in normal peritumoral breast tissues, as expression of autoimmune morphological changes, in a group of BC patients with thyroid autoimmunity. We suppose that an increased LI in breast tissues of this group of patients may help explain the association between BC and thyroid autoimmunity. The study group included 26 BC patients with thyroperoxidase antibodies positivity (TPOAb+), 14 of them (53.8%) with Hashimoto's thyroiditis (HT), and 30 BC patients with no evidence of thyroid autoimmune disorders. Malignant and surrounding normal breast tissues were assessed for LI. The amount of LI was scored as very scanty or scanty (LI S) and moderate or marked (LI M), independently by two expert pathologists. LI S was detected in 19/26 (73.1%) BC tissues from patients with TPOAb positivity and LI M in 7 (26.9%). All BC patients with HT had LI S. LI S was detected in 25/30 (83%) and LI M in 5/30 (17%) of BC tissue from patients with no thyroid autoimmunity. The difference in the amount of LI of BC tissues in patient with or without autoimmune thyroid disorders was not significant. The LI was generally absent or very scanty in remote breast tissue in all cases. In conclusion, in breast malignancies the presence of humoral and/or clinical evidence of thyroid autoimmunity is not associated to autoimmune morphological changes of cancer and peritumoral normal tissue. The LI does not seem to have any role in tumorigenesis in patients with BC and thyroid autoimmunity. PMID- 16682840 TI - A novel heterozygous missense mutation in the vasopressin moiety is identified in a Japanese person with neurohypophyseal diabetes insipidus. AB - The autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by diverse mutations in one allele of the gene that encodes the arginine vasopressin (AVP) precursor protein, AVP-neurophysin II (AVP-NP II). Most of the mutations identified so far are located in either the signal peptide or NP II moiety. Two recently published mutations in the AVP gene identified in kindreds with adFNDI predict a substitution of histidine for tyrosine at position 2 and a deletion of phenylalanine at position 3 in AVP. They are unique among adFNDI mutations in that they are the only adFNDI mutations that affect amino acid residues in the AVP moiety of the pro-hormone. Here, we report a novel heterozygous missense mutation in the AVP moiety of the AVP-NP II gene in a Japanese person with neurohypophyseal diabetes insipidus (DI). This mutation occurs at position 2 in AVP and predicts a substitution of serine for tyrosine (Y21S). It is expected to interfere with normal binding of AVP with NP II, and thus result in misfolding of the precursor proteins. The data of this study support the notion that mutations affecting the AVP moiety can result in the initiation of the pathological processes. PMID- 16682841 TI - Akinetic mutism followed by a manic reaction on introduction of steroid replacement for Addison's disease. AB - Neuropsychiatric changes during exogenous corticosteroid administration are well recognized. However, reports of neuropsychiatric reactions to corticosteroid replacement for Addison's disease are distinctively rare. We report on a patient with primary adrenocortical insufficiency, initially presenting with depressive symptoms, who developed akinetic mutism followed by acute manic illness shortly after the initiation of steroid replacement. Both disorders occurred with physiological doses of hydrocortisone and resolved spontaneously. The pathogenesis of the above neuropsychiatric reactions is discussed in the context of glucocorticoid receptor-related brain effects of glucocorticoids. In addition, this report points to the need for accurate psychiatric assessment of patients with Addison's disease upon introduction of replacement therapy. PMID- 16682842 TI - The IGF system in a case of Costello syndrome. AB - Costello syndrome is characterized by facial dysmorphia, hyperpigmented skin, palmar and plantar hyperkeratosis, curly hair, perioral and nasal papillomata (more rarely localized anally and on vocal cords), short stature, mental retardation and sociable personality. Although growth retardation is typical of Costello syndrome, its cause is not defined. We report on a 10-yr-old Caucasian girl affected by Costello syndrome with fasting hypoglycemia and short stature, associated low circulating levels of acid-labile subunit (ALS), relatively low levels of IGF-I and IGFBP-3, and normal IGF-II, mostly circulating in a binary complex with IGFBP-2 and -6 instead of in a 150 kDa ternary complex. The reduced ALS concentration and the consequent impaired formation of the circulating 150 kDa ternary complex can induce an accelerated clearance rate of IGF peptides and of IGFBP-3, contributing to the decreased IGF-I growth promoting activity in our patient. Moreover, the presence of IGF-II in the binary complex, which has been postulated to increase the insulin-like effects of these peptides, can explain, at least in part, the patient's asymptomatic fasting hypoglycemia. PMID- 16682843 TI - Central diabetes insipidus in a patient with malaria tropica. AB - Up to 21% of severe cases of malaria tropica are associated with polyuria and are life-threatening. We describe a 39-yr-old man with malaria tropica who developed disseminated intravascular coagulation, polyuria, and a pituitary lesion. Empiric treatment with vasopressin improved the polyuria. This is the first case of malaria tropica in which central diabetes insipidus has been documented. PMID- 16682844 TI - Beyond semantics: defining hyponatremia in secondary adrenal insufficiency. PMID- 16682845 TI - The metabolic syndrome in polycystic ovary syndrome. AB - Much overlap is present between the polycystic ovary syndrome (PCOS) and the metabolic syndrome. This article reviews the existing data regarding the prevalence, characteristics, and treatment of the metabolic syndrome in women with PCOS. The prevalence of the metabolic syndrome in PCOS is approximately 43 47%, a rate 2-fold higher than that for women in the general population. High body mass index and low serum HDL cholesterol are the most frequently occurring components of the metabolic syndrome in PCOS. The pathogenic link between the metabolic syndrome and PCOS is most likely insulin resistance. Therefore, the presence of the metabolic syndrome in PCOS suggests a greater degree of insulin resistance compared to PCOS without the metabolic syndrome. Obesity, atherogenic dyslipidemia, hypertension, impaired fasting glucose/impaired glucose tolerance, and vascular abnormalities are all common metabolic abnormalities present in PCOS. Lifestyle modification has proven benefit and pharmacological therapy with insulin-sensitizing agents has potential benefit in the treatment of the metabolic syndrome in women with PCOS. PMID- 16682847 TI - Development of Graves' hyperthyroidism during the early phase of pregnancy in a patient with pre-existing and long-standing Hashimoto's hypothyroidism. PMID- 16682846 TI - Melatonin and nitric oxide. AB - Melatonin is a product of the amino acid tryptophan in the pineal gland. Once synthesized, the specific mechanisms governing the release of melatonin from the pineal gland and its functions are largely unknown. Besides its regulatory role in circadian rhythms in mammals, because of its widespread subcellular distribution, melatonin contributes to the reduction of oxidative damage in both the lipid and the aqueous environments of the cell. This postulate is widely supported by the experimental observations showing that melatonin protects lipids in membranes, proteins in the cytosol, and DNA in the nucleus and mitochondria from free radical damage. Melatonin thus reduces the severity of disease conditions where free radicals are implicated. The direct free radical scavenging effects of melatonin are receptor independent. It has recently been shown that it has an ability to scavenge free radicals, including hydroxyl radicals, hydrogen peroxide, peroxyl radicals, singlet oxygen and nitric oxide (NO) and peroxynitrite anion. An excessive amount of NO, a free radical which is generated by the inducible form of NO synthase, is known to cause cytotoxic changes in cells. Hence, NO synthase is considered a pro-oxidative enzyme, and any factor that reduces its activity would be considered an antioxidant. Recent studies have shown that melatonin inhibits the activity of NO synthase, beside its NO and peroxynitrite scavenging activity. Thus, inhibition of NO production may be another means whereby melatonin reduces oxidative damage under conditions, such as ischemia-reperfusion, sepsis, etc, where NO seems to be important in terms of the resulting damage. PMID- 16682848 TI - A pilot study evaluating a minimal contact telephone and mail weight management intervention for primary care patients. AB - OBJECTIVE: This study examined an 8-week telephone and mail weight management intervention for Veterans who are unable to attend frequent clinical visits. METHODS: A mail and phone based intervention was compared to a "usual care" intervention in a Hospital clinic. 26 participants (80% male; mean wt=246.2 lbs, BMI=34.7) completed the study. All participants received weight management counseling at weeks 1 and 8. Participants in the intervention group received phone calls and mailings during weeks 2-7. RESULTS: The intervention group lost more weight, although it was not statistically significant. Multivariate analyses showed stage of change was associated with weight loss and attendance. Treatment group and role limitations due to emotional problems were related to weight loss. The intervention group significantly increased their intake of fruits and vegetables. CONCLUSION: This pilot study suggests that minimal interventions may be a method to reach populations that do not have access to more intensive treatments for weight management. PMID- 16682849 TI - Symptoms of attention deficit hyperactivity disorder in severely obese women. AB - OBJECTIVE: Past and current symptoms of Attention Deficit Hyperactivity Disorder (ADHD) were assessed in a clinical sample of severely obese females. METHOD: Core symptoms of ADHD were examined in 75 consecutive, severely obese (BMI > or = 35) women referred to a medical specialist for the non-surgical treatment of obesity. Subjects completed both a retrospective report of childhood symptoms of ADHD (Wender Utah Scale) and two standardized adult ADHD symptom scales. RESULTS: The frequency of clinically suggestive elevations in ADHD scores was substantially and significantly higher than the normative samples in 9 out of 11 symptom subscales. Inattentive symptoms, but not hyperactive symptoms of ADHD, were frequently reported. Overall, 26.7% of the sample reported significant symptoms of ADHD in both childhood and adulthood. CONCLUSIONS: This preliminary study suggests that severely obese women report significant symptomatology related to both childhood and adult ADHD. PMID- 16682850 TI - Bulimic Investigatory Test, Edinburgh (BITE). A validation study of the Italian version. AB - OBJECTIVE: To explore the psychometric properties of the Italian version of the Bulimic Investigatory Test, Edinburgh (BITE). SUBJECTS: A general population sample of 995 subjects (621 females), a clinical sample of 388 eating disordered females and a clinical sample of 710 patients with obesity (575 females). RESULTS: Internal consistency was satisfactory. The factor analysis confirmed that the Symptom Scale is mono-factorial. Patients with bulimia nervosa (BN) purging showed the highest scores and those with anorexia nervosa (AN) restricting type the lowest. Obese patients showed higher BITE scores than controls in both sexes (P<0.01) and those with binge eating disorder (BED) showed higher scores than the rest of the sample (P<0.05). In patients with obesity with the threshold of 10 the Symptom Scale had a sensitivity for BED of 93% and a specificity of 55%; with the threshold of 20, sensitivity and specificity were, respectively, 44% and 92%. Normative values for BITE total and sub-scale scores in clinical and non-clinical samples are reported. CONCLUSION: The Italian version of BITE is psychometrically sound and it can be a useful screening tool. Our data suggest that Symptom Scale, with a threshold of 20, has a good sensitivity for BN, although it does not effectively discriminate patients with BN from those affected by other eating disorders with binge-eating (BED, AN bingeing-purging type). PMID- 16682851 TI - Caloric requirement for refeeding inpatients with anorexia nervosa: the contribution of anxiety exercise, and cigarette smoking. AB - BACKGROUND: Refeeding inpatients with anorexia nervosa (AN) is costly, stressful, and can precipitate the refeeding syndrome. Caloric intake is usually increased gradually from a low starting point until a steady weight gain is achieved. There is no reliable equation that predicts the number of calories required for a weight gain. It was our clinical suspicion that anxiety, exercise, and cigarette smoking might increase the caloric need for refeeding. METHOD: We conducted an observational cohort study of 17 females with AN admitted to an inpatient eating disorder unit for refeeding. We estimated the energy intake by observation, the caloric expenditure due to exercise with a triaxial accelerometer, the number of cigarettes smoked by history, and the anxiety by the Beck Anxiety Inventory (BAI). RESULTS: Neither anxiety, exercise, or cigarette smoking predicted the caloric requirement for refeeding, individually or in combination. DISCUSSION: Our data suggest that the caloric requirement for weight gain during refeeding is not predicted by the patient's anxiety, exercise or smoking habits. The standard methods of estimating caloric requirements for refeeding remain indirect calorimetry and previous history. PMID- 16682852 TI - For an anthropology of eating disorders. A pornographic vision of the self. AB - In reading the considerations of the leading contemporary sociologists, attentive observers of the metamorphosis of identity in the post-modern age, a game came to my mind: "what if I mixed up quotes from these scholars' papers with extracts from patients' clinical reports, especially people suffering from so-called "eating disorders"? The objective of this game is to show how difficult it is to pick out the clinical fragments from the sociologists' descriptions of the "never ending task of assembling our Self". The game is to try to separate psychopathology from the Late Modern physiology of identity. The great psychopathologists from the last century offered us two major meaning organizers of identity and its disorders: the Freudian and the Jaspersian. Freud's notion of the "discontents with civilization" served to explain that widespread sense of malaise that characterizes the modern condition. As civilization has been built on our restraining our drives, civilized man ended up "trading in a part of his chances for happiness for a bit of security". Security is guaranteed by our submitting to norms of civilized living together. Neurosis, that feeling that permeates modern human beings who neither belong to themselves nor to others, is the agonizing result. The Jaspersian concept of the Ego consciousness is mainly based on the Kantian Self, the identity pole of subjectivity, standing above the stream of changing experience. It is a necessary condition for coherent experience. Also the Jaspersian Self presupposes coherence, since its main features include identity through time, a sense of unity and one of demarcation from the external world, and a feeling of being actively involved in one's own experiences and performances. In late modernity, identity is a task. Post-modern people have the task and necessity to be perpetually constructing themselves. Being, Self, are organized in a reflexive way. Individuals are forced to choose their own life style among a multitude of alternatives. Are the Freudian and the Kantian/Jaspersian models of the Self now still proving to be suitable for an adequate psychopathological analysis? PMID- 16682853 TI - Orthorexia nervosa: validation of a diagnosis questionnaire. AB - AIM: To validate a questionnaire for the diagnosis of orhorexia oervosa, an eating disorder defined as "maniacal obsession for healthy food". MATERIALS AND METHODS: 525 subjects were enrolled. Then they were randomized into two samples (sample of 404 subjects for the construction of the test for the diagnosis of orthorexia ORTO-15; sample of 121 subjects for the validation of the test). The ORTO-15 questionnaire, validated for the diagnosis of orthorexia, is made-up of 15 multiple-choice items. RESULTS AND CONCLUSION: The test we proposed for the diagnosis of orthorexia (ORTO 15) showed a good predictive capability at a threshold value of 40 (efficacy 73.8%, sensitivity 55.6% and specificity 75.8%) also on verification with a control sample. However, it has a limit in identifying the obsessive disorder. For this reason we maintain that further investigation is necessary and that new questions useful for the evaluation of the obsessive-compulsive behavior should be added to the ORTO-15 questionnaire. PMID- 16682854 TI - Temperament and character inventory and pharmacotherapeutic outcome in bulimia nervosa. AB - OBJECTIVE: To assess the relationship between the personality dimensions measured by the temperament and character inventory (TCI) and pharmacotherapeutic outcome in bulimia nervosa (BN). METHODS: Thirty female BN patients aged 19.5+/-2.9 years were enrolled to receive 12 weeks' treatment with fluoxetine or buspirone and assessed using the Polish version of the TCI. The personality dimensions of the patients with good and poor treatment responses were compared. RESULTS: The subjects with a good outcome had a higher self-directedness and lower harm avoidance score; this difference was more pronounced in the fluoxetine-treated subjects. At multiple regression analysis, only self-directedness predicted a good outcome. DISCUSSION: The results indicate that self-directedness is associated with a good pharmacotherapeutic outcome in BN. This seems to confirm the results of previous studies of the pharmacotherapy of depression and cognitive-behavioural therapy (CBT) in BN. PMID- 16682855 TI - Health and appearance concerns in young Caucasian and African-American women. AB - OBJECTIVE: The primary aim of this study was to examine the health- and appearance-related concerns of young African-American and Caucasian females. RESEARCH METHODS: Participants were twenty Caucasian and twenty African-American college women, who were matched on age and BMI. All participants were administered the Multidimensional Body-Self Relations Questionnaire (MBSRQ), a demographics instrument, and a short inventory to assess concerns about health, appearance, and weight management, in a single testing session; height and weight data were self-reported. RESULTS: African-American women and Caucasian women were not significantly different in their level of health consciousness or perception of health; however, African-American women were significantly more satisfied with their physical appearance in comparison to Caucasian women. Among African American women, perception of health and level of health consciousness were highly related; this relationship was not found in Caucasian women. In addition, perception of health was related to satisfaction with appearance in both groups, although the relationship was stronger among African-Americans. Among Caucasian women, satisfaction with physical appearance was highly correlated with perception of weight status (from underweight to very overweight); this relationship was not found among African-American females. An inverse relationship between body mass index (BMI) and appearance evaluation was found only for Caucasian females. DISCUSSION: These findings suggest that, on the whole, young African-American and Caucasian college females report similar evaluations of health and values placed on appearance and health, and differ primarily in terms of satisfaction with physical appearance. PMID- 16682856 TI - Reliability of the AccuSens Taste Kit(c) in patients with eating disorders. AB - Zinc deficiency is a putative risk factor for anorexia nervosa (AN). Detecting zinc deficiency may therefore be important in treatment. However, serum zinc is not a good measure of total body zinc. An alternative test for zinc deficiency is taste testing because zinc deficiency is known to impair taste (dysgeusia). To determine whether taste testing could be used in this way, we measured the reliability of the only commercially available taste test in 16 patients with eating disorders. The results were analyzed graphically and with the kappa statistic (K). The taste test was found to be unreliable and should not be used to determine zinc status. PMID- 16682857 TI - Satiety and hunger: some considerations from feast and famine aboriginal (stone age men) experiences. PMID- 16682858 TI - Recovery of menstrual cycle after therapy for anorexia nervosa. AB - OBJECTIVES: Although the relation between sexual dysfunction, undernourishment and weight loss is clear, amenorrhea sometimes precedes weight loss and may persist despite later weight gain. We therefore studied the variations in anthropometric variables in women with restrictive anorexia nervosa (AN) at the time they recovered their menstrual cycle. METHOD: We undertook a longitudinal study of 40 women with AN. All were in secondary amenorrhea at the start of the outpatient treatment program. Leptin concentrations were measured during the early follicular phase after the patients had recovered their menstrual cycle and had had two regular cycles. Body composition was measured by multifrequency bioelectric impedentiometry and the lean mass, fat mass and percentage fat were recorded. RESULTS: The menstrual cycle returned in 25% of the patients with a BMI of 17 or lower, in 50% of the patients with fat percentages of 20% or lower and in 25% with fat percentages of 17% or lower. Over 20% of the patients recovered their menstrual cycle without having leptin figures above 1.85 ng/ml. DISCUSSION: Prediction of recovery of menstruation in women with AN is difficult, as anthropometric variables alone are not sufficient; other factors have also to be taken into account. PMID- 16682859 TI - Quality of life assessment for patients with eating disorders. AB - The purpose of this study was to examine the construct validity of Visual Analogue Scale (VAS)-quality of life for patients with eating disorders in relation to the following instruments: Body Awareness Scale Health, (BAS-H) and its Interview Scale for Body Ego (ISBE), Eating Disturbances Scale (EDS-5), Body Attitude Test (BAT) and Comprehensive Body Examination (CBE), part of respiration, subscale 1. In total 87 individuals were examined: 26 patients with anorexia nervosa (AN), 20 patients with bulimia nervosa (BN) and 41 patients with eating disorders not otherwise specified (EDNOS). The highest significant correlations (0,43-0,61) were found between VAS-quality of life and BAS-H (subscale grounding), variables of EDS-5 (feeling of control and guilt), BAT (subscale 2) and CBE (breathing variables). In conclusion, this study indicated that VAS-quality of life seemed to more accurately capture mental and psychosomatic factors than physical and health-related factors. PMID- 16682860 TI - Measuring change in parotid gland size: test-retest reliability of a novel method. AB - BACKGROUND: There is currently no convenient method for measuring parotid gland hypertrophy, a common condition among patients with bulimia nervosa (BN) and anorexia nervosa (AN). OBJECTIVE: To develop a technique for reliably estimating change in parotid gland size. METHODS: A method for measuring facial width as a surrogate marker of parotid gland size was developed using calipers to measure between defined reference points located on the parotid gland region. The method was tested for reliability when performed by a single operator and used to determine face width measurements of 15 control subjects. RESULTS: Face width measurements were reliable when performed by a single operator. Face width measurements of control subjects ranged from 9.1 cm to 15.3 cm. DISCUSSION: The caliper method of measuring changes in parotid size is a novel method of measurement of parotid hypertrophy. It is quick, non-invasive and inexpensive and is highly reliable in the hands of a single operator. PMID- 16682861 TI - The relationship between self-rated health, stress, health care, overall quality of life and weight in a rural population. AB - OBJECTIVE: To determine if there is a relationship between self-rated health, stress, health care, satisfaction, overall quality of life scores and weight. DESIGN: A mailed survey and retrospective chart review of people living in the Bella Coola Valley who attend the Bella Coola Medical Clinic. STUDY POPULATION: Adults living in the Bella Coola Valley who are registered with the Bella Coola Medical Clinic. MAIN OUTCOME MEASURES: Self-rated health, stress, health care received, satisfaction with health, happiness, overall quality of life scores and weight (body mass index). RESULTS: An estimated 1734 residents live in the Bella Coola and are registered with the clinic. A total of 968 useable surveys were returned for a response rate of 56% (968/1734). Nine hundred and eighteen survey respondents had a recent weight in kilograms documented; 803 survey respondents had a height documented. A higher weight was associated with poorer self-rated health, higher stress levels, and lower satisfaction with health. It was also associated with lower self-esteem and satisfaction scores, particularly in younger obese people. A higher weight was not correlated with spirituality, overall quality of life, health care rating, or happiness scores. CONCLUSION: Increasing weight may contribute to poorer health, higher stress, lower satisfaction with health and poorer self-esteem. However, we found no evidence that increased weight impairs happiness or overall quality of life. This may be one reason for the lack of success of weight loss strategies that focus on happiness and overall quality of life to increase readiness and motivation. Alternatively, focussing on secondary medical benefits and self-esteem may be useful. PMID- 16682862 TI - Differences in diet composition of Brazilian adolescent girls with positive or negative score in the Eating Attitudes Test. AB - Dieting, a behavioral phenomenon which is becoming more frequent among adolescents, is certainly involved in the pathogenesis of eating disorders, especially anorexia nervosa (AN), and may result in an unbalanced nutrition. Our study aimed at identifying girls with symptoms of AN and describing their diets. We assessed girls with symptoms by using the Eating Attitudes Test and considered girls with a score > or = 20 as having symptoms of AN. To evaluate their diets, a "modified" 24-hour recall was applied on three alternate days. Twenty-one percent of the 279 girls studied showed symptoms of AN. Analysis of the food intake showed that calories, calcium and iron were below the recommendations of the Standing Committee on the Specific Evaluation of Dietary Reference Intakes (DRI Committee) and the National Research Council, while protein and carbohydrate were within the recommended range and fat was above the percentage of recommended total energy intake. Compared to girls without symptoms, they had a significantly smaller intake of calories and iron, and a higher intake of protein. Findings shown in this paper point out the need for intervention in adolescents because their dieting behavior can predispose to an eating disorder. PMID- 16682863 TI - Gender comparisons in psychological characteristics of obese, binge eaters. AB - OBJECTIVE: To investigate differences between male and female, obese binge eaters in levels of depression, anxiety and self-esteem. In addition, to make comparisons in these psychological characteristics, for both genders, between obese, binge eaters and obese nonbingers. METHOD: Participants consisted of 48 female (26 binge eaters and 22 nonbingers) and 13 male (4 binge eaters and 9 nonbingers) outpatients in a hospital weight-loss program. Participants completed the following: Questionnaire on Eating and Weight Patterns--Revised, Beck Depression Inventory, Beck Anxiety Inventory and the Coopersmith Self-Esteem Inventory. RESULTS: Females had a significantly higher level of depression (p<0.003) than males. No other significant gender differences were found. However significant differences between binge eaters and nonbingers were found, for both genders, in depression (p<0.0001) and anxiety (p<0.008) with the binge eaters having higher scores than the nonbingers, and in self-esteem (p<0.0001) with the binge eaters having lower scores than the nonbingers. CONCLUSION: Females have higher levels of depression compared to males, but no gender differences were found in anxiety or self-esteem. In both genders, binge eaters have higher levels of depression and anxiety and lower levels of self-esteem compared to nonbingers. PMID- 16682864 TI - Pilot treatment program for shoplifting in eating disorders. AB - OBJECTIVE: There are no established treatment programs for shoplifting in eating disorder patients. Our objective was to observe the effect of an established behavioural treatment program in a series of eating disorder patients. METHODS: Patients with eating disorders who shoplift voluntarily took part in an 8-week behavioral treatment program at the Elizabeth Fry Society of Greater Vancouver, British Columbia. They completed assessments at the first session, last session, and at 1 and 6 months post intervention. RESULTS: Six patients enrolled in the study and three patients completed the treatment program. Only one patient reported a decrease in shoplifting frequency. All subjects reported an increase in self-esteem and ability to control shoplifting impulses. DISCUSSION: Our findings suggest that behavioral therapy may be effective in treating shoplifting in eating disorders, but that longer sessions and follow-up may be necessary to show benefit. A randomized control trial with longer-term follow-up is needed to determine whether there is a benefit. PMID- 16682865 TI - Psychometric properties of the eating attitudes test and children's eating attitudes test in Croatia. AB - The factor structure of the children's version of the Eating Attitudes Test (ChEAT) and Eating Attitudes Test (EAT-26) were examined in Croatian girls of different ages. A self-report survey was given to 225 girls (Grades 5 to 8), 525 high school girls (Grades 9 to 12), and 646 female university students. Factor analysis revealed the existence of four factors for ChEAT, and three interpretable factors for EAT-26. Internal consistency of both instruments was satisfactory. 10.3% of school girls scored 20 or higher on ChEAT, when 7.6% of high school girls and 11.3% of university students had elevated EAT-26 scores. The ChEAT and EAT-26 were useful for screening large non-clinical groups and measuring disturbed eating behaviours. Those with elevated ChEAT and EAT-26 scores were more likely than those with lower scores to be engaged in extreme weight control methods (e.g. vomiting, binging). PMID- 16682866 TI - The agreement between the MedGem indirect calorimeter and a standard indirect calorimeter in anorexia nervosa. AB - OBJECTIVES: Measurement of the basal metabolic rate (BMR) can be used to estimate the calories required for weight gain during refeeding in anorexia nervosa (AN). The reference method for measuring the BMR is indirect calorimetry. MedGem has developed a new indirect calorimeter that calculates the metabolic rate much more quickly than standard indirect calorimeters. This study compared the BMR measured by the MedGem and standard indirect calorimetry in an AN population. METHODS: We measured the BMR using the Deltatrac metabolic cart followed immediately by the MedGem indirect calorimeter in 27 subjects (12 patients and 15 controls). RESULTS: Bland-Altman plots show that there is poor agreement between the BMR reported by the MedGem compared to the Deltatrac. DISCUSSION: Until better agreement with standard indirect calorimetry can be shown the MedGem should not be used for calorimetry in AN. Possible factors that may limit the MedGem's reliability include patient discomfort with the mouthpiece, use of a fixed RQ, and the short sampling period. PMID- 16682867 TI - Obesity, self-complexity, and compartmentalization: on the implications of obesity for self-concept organization. AB - OBJECTIVE: The relationship between obesity and structural aspects of the self concept was examined in adult women. Participants were 119 adult women [age range: 18-73, M=26.9; body mass index (BMI) range: 16.2-54.7, M=27.3] who completed measures of self-esteem, self-complexity, and the spontaneous self concept. BMI was associated with less complex and more compartmentalized self knowledge and more frequent mention of weight-stereotypic traits as self descriptive. The findings are discussed in the context of research on obesity- related stigma. PMID- 16682868 TI - An open trial of reboxetine in obese patients with binge eating disorder. AB - BACKGROUND: The objective of this study was to evaluate the effectiveness and safety of reboxetine, a selective noradrenaline reuptake inhibitor, in the treatment of obese patients with binge eating disorder (BED). METHOD: Nine outpatients with BED and obesity received reboxetine 8 mg/day for 12 weeks. The number of days with binge eating episodes per week was considered the primary outcome measure. Body weight, body mass index (BMI), Binge Eating Scale (BES), Clinical Global Impression Severity (CGI-S) and the World Health Organization Quality of Life Assessment Scale (WHOQOL-BREF) scores were used as secondary outcome measures. Paired t-test with the last observation carried forward was used to analyze changes from baseline to endpoint. RESULTS: Five patients completed the study and showed a complete remission of BED. The mean binge days per week was significantly reduced from 4.6+/-1.8 to 0.2+/-0.6, at the end of the study (p=0.0002). Mean BES scores were decreased from 32.3+/-6.3 to 9.3+/-6.8 (p=0.0003). There was also a statistically significant decrease in BMI (from 36.5+/-4.48 to 35.06+/-4.49; p=0.01), and in CGI-S (from 5.1+/-0.9 to 1.4+/-1.01; p=0.0002). Quality of life, assessed using the WHOQOL-BREF was significantly improved on overall quality of life and general health (p=0.02) and on psychological (p=0.03) domain. No serious adverse events were observed. CONCLUSION: Reboxetine may be an effective and well-tolerated agent in the treatment of BED in obese patients. PMID- 16682869 TI - Socio-cultural and cognitive predictors of eating disorder symptoms in young girls. AB - There is some evidence for a relationship between socio-cultural variables and the development of disordered eating or concerns. However, the role of individual cognition in adding to this relationship has not yet been investigated. The current study therefore had two main questions. Firstly, which of the socio cultural factors investigated (parental, peers and the media) predict girls' eating disorder related symptoms? Secondly, do individuals' cognitions add to this prediction? Thirty-eight girls participated in the study. They completed the Eating Attitudes Test (EAT), Eating Disorder Belief Questionnaire (EDBQ), measures of parental, peer and media influence, and a measure of their awareness and internalisation of societal standards of attractiveness. The results indicated that several of the socio-cultural factors were related to girls' EAT score. The belief that being thinner would make boys like them more was the most significant predictor in the whole sample (and in younger girls). Individual cognitions added significantly to this prediction in the whole sample but not in the younger girls. For older girls, the importance of magazines as a source of information about beauty and ideals was the strongest predictor of EAT score, and their cognitions added significantly to this prediction. It is concluded that peer and media influences are important determinants of girls' eating disorder related symptoms. However, individual cognitions add to this relationship, particularly in older girls. The implications and limitations of the study are discussed. PMID- 16682870 TI - Predicting caregiver-reported behavior problems in cocaine-exposed children at 3 years. AB - Predictors of caregiver-reported behavior problems for 3-year-olds with prenatal cocaine exposure (PCE) and matched controls were examined using structural equation modeling. We tested whether PCE had a direct effect on child behavior problems in a model that included other prenatal drug exposure, child sex, caregiver depression, and the quality of the child's home environment. The sample (N = 256) was drawn from a longitudinal, prospective study of children of (predominantly crack) cocaine-using women and controls matched on race, socioeconomic status, parity, and pregnancy risk. Child Behavior Problems was modeled as a latent variable composed of the 48-item Conners' Parent Report Scale Conduct Problem and Impulsive-Hyperactive scales and the Eyberg Child Behavior Inventory Intensity scale. Caregiver depression was the only significant predictor of Child Behavior Problems. Mean levels of caregiver self-reported depression and reported child behavior problems did not differ between groups. Mean depression scores were well above the recommended clinical cutoff while mean child behavior problems scores were within normal limits. The model explained 21% of the variance in caregiver-reported child behavior problems in our sample of rural African American, low SES youngsters. Non-maternal caregivers of cocaine exposed children had significantly lower mean depression scores and mean child behavior problems ratings for 2 of 3 scales used in the study compared to biological mothers of children with PCE and controls. For all groups, much larger proportions of children were rated as having clinically significant behavior problems than would be expected based on the prevalence of behavior problems in the general population. PMID- 16682871 TI - Effects of breastfeeding on weight changes in family-based pediatric obesity treatment. AB - Research indicates that breastfeeding may provide protective effects against the development of obesity; however, breastfed children may still become obese because of the obesogenic environment. This study is designed to examine the effects of retrospective recall of breastfeeding on weight changes in children participating in a 6-month behavioral treatment program for childhood obesity. The independent variable of breastfeeding was defined as children who were exclusively breastfed for 4 weeks (excluding water or medication) versus those who were never breastfed. Child percent overweight and body mass index changes during 6 and 12 months were evaluated for 94 families based on mother report of breastfeeding status using analysis of covariance, controlling for socioeconomic status and initial child weight status. Data were compiled for secondary analysis from pediatric obesity randomized controlled outcome studies evaluating core components of family-based treatments. Results showed that, compared with nonbreastfed (formula) children (n = 28), breastfed children (n = 66) showed significantly larger reductions in (mean +/- SEM) percent overweight at 6 months (-15.2 +/- 1.1 vs -10.2 +/- 1.7, p <.05) and 1 year (-10.3 +/- 1.3 vs -5.9 +/- 1.8, p <.05). Similarly, breastfed children showed greater reductions in body mass index at 6 months (-2.1 +/- 0.19 vs -1.1 +/- 0.28) and 1 year (-0.8 +/- 0.23 vs +0.1 +/- 0.32). Findings suggest the beneficial effects of breastfeeding may extend beyond obesity prevention to include improved outcome in family-based pediatric obesity treatment. Potential mechanisms relating breastfeeding, obesity prevention, and enhanced outcome in pediatric obesity treatment are discussed. PMID- 16682872 TI - Self-efficacy, outcome expectations, and diabetes self-management in adolescents with type 1 diabetes. AB - The goal of this research was to develop and evaluate measures of adolescent diabetes management self-efficacy and outcome expectations that reflect developmentally relevant, situation-specific challenges to current diabetes regimens. Self-efficacy for diabetes management, expected outcomes of adherence, adherence to the diabetes regimen, and glycemic control were assessed in 168 adolescents (ages 10-16 years) with type 1 diabetes. Factor analyses indicated a single scale for self-efficacy and two distinct factors representing positive and negative outcome expectations. Reliability and predictive validity of the new scales were supported. In regression analyses, self-efficacy and the interaction of self-efficacy with expectations of positive outcomes were significantly associated with diabetes self-management adherence and glycemic control in older adolescents. The effect of self-efficacy was greatest when adolescents had stronger beliefs in the beneficial outcomes of adherence. These brief measures can be used to identify youths at risk of poor diabetes self-management. Interventions targeting self-efficacy may lead to improved diabetes self management. PMID- 16682873 TI - Does "stubbornness" have a role in pediatric constipation? AB - The objective of this study was to determine if children with constipation are more stubborn, both in general and specifically regarding toileting behaviors, than children without constipation. A secondary objective was to determine if constipated children who are more stubborn are less likely to respond to routine therapeutic interventions than less stubborn constipated children. One hundred one children aged 2 to 6 years, who were first-time presenters (never received treatment) to their primary care physician (PCP) with constipation, were compared with 84 nonconstipated control children of similar age range. Comparison measures included general stubbornness and toilet-specific stubbornness (active resistance to participating in appropriate toileting behaviors). Measures of stubbornness were generated from retrospective questionnaires, prospective toileting diaries completed by the parents, and direct experimenter observations. The constipated children were treated by their PCP for 2 months and then reassessed. Constipated children were perceived by their parents to be significantly more stubborn than control children generally and specifically in terms of toileting. Some study evidence suggested that constipated children who continued to have difficulties after 2 months of treatment by their PCPs were perceived by their parents to have significantly more general stubbornness than constipated children who responded to treatment. Parent-perceived toilet-specific stubbornness significantly improved after successful treatment of the constipation by their PCP. The finding that constipated children had more parent-perceived stubbornness than children without constipation is notable because it may play a role in the development and/or maintenance of this bowel dysfunction as well as being an obstacle in treatment compliance. PMID- 16682874 TI - School achievement in a regional cohort of children born very low birthweight. AB - Children born very low birthweight (VLBW, <=1500 g) have historically had lower average school achievement than their normal birthweight peers. However, perinatal care and survival have changed dramatically since prior cohorts accrued, prompting reassessment. Surfactant therapy became generally available 8/1/1990, and the use of ante- and postnatal steroids increased substantially around this time. Standardized test scores and teacher ratings in math, reading, science, and social studies were obtained at age 10 for a cohort of children admitted to six regional NICUs in Wisconsin and Iowa, 8/1/1988 - 6/30/1991. We compared achievement between the VLBW cohort and controls from the same school districts. Among VLBW children, we determined neonatal and early childhood factors associated with achievement on standardized tests (ordinal logistic regression) and teacher ratings (linear regression) and evaluated whether achievement differed by birth year. Compared to population controls, VLBW children's greatest deficits occurred in mathematics. Scores on the standardized math exam and teacher ratings of overall achievement were positively associated with birthweight, social function measured at age five, and socioeconomic status. VLBW children born in the post-surfactant era (after 8/1/1990) had lower achievement on the standardized math exam than children born during the pre surfactant era of the prior two years. Lower achievement in the post-surfactant era may be due to greater survival among less healthy neonates or increased exposure to postnatal steroids. VLBW children more likely to struggle academically could be identified by early childhood measures, allowing for targeted interventions to mitigate their difficulties. PMID- 16682875 TI - Mothers' views on hyperactivity: a cross-cultural perspective. AB - To examine cultural variation in mothers' perceptions of hyperactive behaviors in school-age boys, we surveyed 135 mothers in 3 ethnic groups: Puerto Rican, Central and South American, and Anglo. Mothers read or heard 8 taped vignettes of boys with behaviors related to DSM-IV hyperactivity criteria. In 50% of the vignettes, Spanish-responding mothers were less likely to consider the boys' behaviors expected than were the English-responding mothers. In 62.5% of the vignettes, Latina mothers expressed more interest in discussing behaviors with their physicians than did the Anglo mothers, and in 62.5% of the vignettes, Spanish-responding mothers expressed more interest in discussing behaviors with their physicians than did the English-responding mothers. We found no association of the 2 scores by the bicultural scale. Mothers' perception of hyperactivity boys varies both with ethnicity and language of response. Latina mothers, especially Spanish-responding mothers, seem interested in discussing children's behavior with physicians. PMID- 16682876 TI - The New WISC-IV. PMID- 16682877 TI - Female monozygotic twins with selective mutism--a case report. AB - Selective mutism is a rare social anxiety disorder characterized by a total lack of speech in certain specific situations despite the ability to speak in others. Both genetic and psychosocial factors are thought to be involved in its presentation, persistence, and response to treatment. This case report describes a case of young female monozygotic twins who presented with selective mutism and their treatment spanning a 2-year period. It highlights the strong genetic association along with environmental factors such as social isolation and consequences of maternal social phobia, all contributing to treatment resistance, despite an intensive multimodal biopsychosocial approach. General issues related to the difficulties in treating monozygotic twins are also addressed. PMID- 16682879 TI - Bowlby and Robertson revisited: the impact of isolation on hospitalized children during SARS. AB - Psychosocial issues related to pediatric isolation remain relevant to both clinical practice and research. The early theories of Bowlby and Robertson are central to this discussion for it was predominantly their work that elicited several policy changes aimed at improving psychosocial care in pediatric settings. Recognizing the significant effects of isolating hospitalized children is just as relevant today where serious infectious diseases can lead to separation of family members. An ethnographic, qualitative study examined the experiences and perspectives of children hospitalized because of SARS (severe acute respiratory syndrome), their parents, and pediatric health care providers. The sample included 23 participants: five children, 10 parents, and eight health care providers. Data analyses illuminated a range of perceived experiences for this triadic sample. Themes related to the effects of social isolation on children were predominant. They included emotional upheaval, changes in parental and professional roles, and familial experiences following hospital discharge. Finally, the paper draws on the findings of the present study and other recent work in offering recommendations for effective clinical approaches in the event of future outbreaks. PMID- 16682880 TI - ADHD, medication, and the military service: a pediatrician's dilemma. PMID- 16682882 TI - Caring for migrant and refugee children: challenges associated with mental health care in pediatrics. AB - This article reviews aspects of the mental health care of migrant and refugee children. It highlights the challenges of access to care for these children and of considering the role of pediatricians in their mental health care. It also looks at the sources of differences in presentation of mental health issues of migrant youth when compared with dominant culture youth, examining the contributions of culture, context, and the families' own views. Models of care will be described that have tried to elicit a better understanding of the difficulties migrant and refugee children may encounter. Some avenues through which we may expand our current psychiatric models of care will also be addressed. These avenues include the use of interpreters and cultural brokers, addressing the debate around ethnic matching between therapists and patients, promoting a sensitivity to otherness and mediation, and looking at the importance of time issues. PMID- 16682883 TI - Emotion regulation in children and adolescents. AB - Within the past two decades, an "affect revolution" [Fischer and Tangney, Self conscious Emotions: The Psychology of Shame, Guilt, Embarrassment, and Pride 1995:3-22] in research has revolutionized the ways in which emotion processes have been conceptualized and subsequently studied. This review discusses the literature on emotion regulation (ER) in childhood and adolescence by first summarizing the trajectory of emotional development from infancy through adolescence, followed by a discussion of the biological and environmental influences on ER, and then a review of the literature linking ER to psychosocial functioning. The penultimate section offers practical suggestions for identifying ER difficulties in children and strategies for intervention efforts. Potential areas for future research conclude the review. PMID- 16682885 TI - Stimulant medications and their adverse events and the food and drug administration: what can we conclude? PMID- 16682886 TI - With the recent Food and Drug Administration (FDA) Advisory Committee's recommendation to put a black box warning on stimulants, the government has joined the media in raising alarm about the medical treatment of attention deficit disorder. PMID- 16682888 TI - How to ENHANCE our knowledge of activated protein C during pediatric sepsis trials: pediatric versus adult trials. PMID- 16682889 TI - The authors reply: academic and industry partnerships. PMID- 16682890 TI - Planning for the unexpected: extracorporeal membrane oxygenation in the catheterization laboratory and beyond. PMID- 16682891 TI - Vasopressin in sepsis: a world of complexity. PMID- 16682892 TI - CPOE revisited: a computerized calculator for continuous medication infusions. PMID- 16682893 TI - Indirect Fick principle: great idea, but can we use it in critical care?. PMID- 16682894 TI - The impact of recent surgical innovation on the postoperative care of stage I palliation for hypoplastic left heart syndrome. PMID- 16682895 TI - Opioids causing a back up in the pediatric intensive care unit? No solution yet. PMID- 16682896 TI - Sepsis-induced pulmonary edema: what do we know? PMID- 16682900 TI - Hyponatremia, hypernatremia: a physiological approach. AB - Natremia belongs to the toolbox of the practicing intensivist. It is an indicator of the hydration status, which is an item that must be continuously monitored in critically ill patients. Hyponatremia is not rare (1% to 2% of hospitalised patients), and hypernatremia is about 10 times less frequent while hypernatremia always indicates hypertonicity, hyponatremia is not equivalent to hypotonicity. Diagnosis depends on the history, clinical examination and basic biochemical data. It should be kept in mind that obtaining urine samples is as important as plasma samples in this respect. The first step consists in confirming that hyponatremia is hypotonic. The second step is to assess the renal response to hypotonicity. Hypotonic hyponatremia will be considered in association with hypovolemia, euvolemia or hypervolemia. The constitution of a hyperosmolar state requires an inadequate water intake The main goal of the treatment is not to normalize numbers (they must always be checked first), but to treat symptoms. Tolerance must always be appreciated. The mathematical formulas proposed are of interest for a better understanding, but should not be followed strictly. PMID- 16682898 TI - Severe diabetic ketoacidosis: hyperventilation or relative hypoventilation. PMID- 16682901 TI - Low vs high positive end-expiratory pressure in the ventilatory management of acute lung injury. AB - Positive end-expiratory pressure (PEEP) has become an essential component of the care of many critically ill patients who require ventilatory support. The application of PEEP is expected to improve lung mechanics and gas exchange as it recruits lung volume. In the last 3 decades, research of the effects of PEEP in animal models of lung injury and in patients with acute respiratory failure has produced a plethora of information. Support for the use of PEEP comes from historical comparisons and a few randomized controlled studies. Although the data from those animal studies and clinical trials could be seen as very convincing, there are insufficient data to propose an universal approach for the use of PEEP in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In this article I will review the basic mechanisms of PEEP and the current knowledge of the effects of PEEP on the evolution and outcome of ALI/ARDS. PMID- 16682902 TI - Positron emission tomography imaging of regional lung function. AB - Regional pulmonary perfusion and ventilation can be assessed by imaging, with positron emission tomography (PET), the pulmonary kinetics of [13N]nitrogen (13N2). Because of its low solubility in blood and tissues, 13N2 infused intravenously in saline solution evolves into the alveolar airspace at first pass, where it accumulates in proportion to regional perfusion during a short apnea. In contrast, infused 13N2 is not retained in non-aerated regions, which do not exchange gas. Robust estimates of regional perfusion and shunt are obtained by modeling the pulmonary kinetics of 13N2 infused as a bolus during a short apnea. Regional ventilation is measured by modeling the washout of 13N2 after breathing is resumed. Regional gas content and dead space ventilation can be measured with inhalation of 13N2. Application of this novel functional imaging technique can further the understanding of the pathophysiology of a variety of pulmonary processes. This review briefly describes the methodological aspects of PET imaging of regional perfusion and ventilation and then focuses on insights in the pathophysiology of acute lung injury and asthma that have been gained by imaging the pulmonary kinetics of 13N2 with PET. PMID- 16682903 TI - Progenitor cells in acute lung injury. AB - AIM: Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are common and serious pulmonary illnesses associated with a high morbidity and mortality. A specific therapy that improves outcomes in these disorders has not been forthcoming. There is growing interest to explore novel therapies, namely stem cells, to repair damage to the lung that is observed in these disorders. In this manuscript, stem cell investigations in the field of pulmonary medicine are delineated, with a focus on endothelial progenitor cells (EPCs) in the setting of ALI. METHODS: A review of the pertinent literature in this field is presented, including recent investigations in animal models. Work related to the assessment of EPCs in human subjects after transplantation and in patients with pneumonia is described. The measurement of circulating EPCs in those with ALI is presented in detail. RESULTS: Patients with ALI have an increased number of circulating EPCs than do healthy controls. Further, a greater number of circulating EPCs is associated with improved outcomes in a multivariable analysis correcting for the effects of age, gender, and severity of illness scores. CONCLUSIONS: EPCs represent a rapidly expanding interest in critical care medicine. However, their specific properties and utilities with regards to lung injury need to be further delineated prior to their use in the therapy of ALI. PMID- 16682904 TI - The effect of alcohol abuse on ARDS and multiple organ dysfunction. AB - A history of alcohol abuse is very common and many times unrecognized in critically ill patients. The consequences of alcohol abuse are multifactorial, and it is associated with excessive morbidity and increased mortality. Alcohol causes acute and chronic dysfunction in multiple organ systems, and the underlying mechanisms responsible for organ injury are complex. In adults with septic shock, a history of alcohol abuse is associated with an increased incidence of Acute Respiratory Distress Syndrome (ARDS) and the development of more severe organ dysfunction. This increased susceptibility to developing acute lung injury has been evaluated by many investigators, and the common variable appears to be oxidative stress. In this article, we review the epidemiology of alcohol abuse and its association with ARDS. In addition, we provide an overview of the mechanisms thought to contribute to ARDS and multiple organ dysfunction. PMID- 16682905 TI - Treatment of critical bleeding in trauma patients. AB - AIM: Massive haemorrhage after trauma is a big challenge for care-givers, being a leading cause of early in-hospital mortality. Surgical bleeding may be easily controlled with several techniques. Otherwise, consumptive coagulopathy is often extremely difficult to stop. An adjunctive strategy to treat traumatic coagulopathic bleeding is recombinant activated factor VII (rFVIIa) (NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark). METHODS: All major trauma victims haemodinamically unstable (systolic blood pressure < 90 mmHg or > 90 mmHg with massive infusions or vasopressors) admitted to the Emergency Department of the Niguarda Ca' Granda Hospital in Milan from October 2002 to September 2005 were reviewed. Mechanical bleeding was controlled with interventional techniques when indicated. Blood derivatives were administrated to maintain haemoglobin> 7 g/dL, INR < 1.5, fibrinogen > 1 and platelet count > 50 x 109. Off-label administration of rFVIIa was performed in the last year in any coagulopathic salvageable patient when all other strategies failed to control bleeding. RESULTS: Major trauma were 942, mean age 32.49+/-18.44 years, 94% blunt trauma, 25.13% haemodynamically unstable. Deaths occurred in 17.02% of cases before any procedure. Emergency invasive treatments were performed in 72.34% of cases. Infusions restored haemodynamic stability in 10.63% of patients. In average 9.4+/-4.1 units of red blood cells were transfused in unstable patients. rFVIIa (dosing 60-100 mg/kg) was administrated in 12 patients. Mortality occurred in 33.33% of cases. The principal cause of death was brain injury. A femoral artery thrombosis was observed in a mangled leg. No other adverse effects due to rFVIIa were documented. CONCLUSIONS: Off-label administration of rFVIIa was able to reverse life-threatening bleeding not manageable with standard strategies in our series of major trauma patients without systemic adverse effects. PMID- 16682906 TI - Critical bleeding in pregnancy: a novel therapeutic approach to bleeding. AB - AIM: In the developed countries the frequency of life threatening post-partum hemorrhages (PPH) is 1 in 1,000 deliveries with a risk of death of 1-2/100,000 deliveries. Hysterectomies for intractable bleeding are carried out in approximately 50% of the cases. The majority of PPH have obstetrical causes, most frequently atony of the uterus. Hereditary and acquired hemostatic defects are very rare. Guidelines of standard surgical and medical measures are available. In this paper we focus on the use of activated recombinant factor VII (rFVIIa) in PPH. METHODS: A computerized literature search was carried out in PubMed and Ovid for papers published between 2001 and 2005 in the English literature reporting on life-threatening PPH treated with rFVIIa after failure of conventional therapy, including hysterectomy. RESULTS: We identified 11 papers including 39 patients; in 18 of them the laboratory data were indicative for disseminated intravascular coagulation and in 24 hysterectomy was carried out. Controlled or reduced bleeding was reported in 38 out of 39 treated patients. CONCLUSIONS: The bleeding can occur in a series of events conductive to metabolic complications, hypoxia, disseminate intravascular coagulation, organ damage and multiorgan failure, progressively exhaustive. The therapeutic intervention must be instituted as early as possible before successive complications ensue. These preliminary reports in PPH after failure of conventional standard therapy suggest that rFVIIa is an active agent but should be administered as early as possible before the consequences of severe and intractable bleeding. PMID- 16682907 TI - Metabolic and endocrine effects of sedative agents. AB - Metabolically critical illness can be divided in two phases, acute and prolonged. Whereas the acute or hypermetabolic phase is characterized by elevated circulating concentration of catabolic hormones and substrate utilization to provide energy to vital organs, the prolonged or catabolic phase of critical illness is marked by reduced endocrine stimulation and severe loss of body cell mass. The most common analgesic and sedative agents used in the intensive care unit, if used in small or moderate doses, do not interfere significantly with the metabolic milieu; however, prolonged infusions, and in high doses, without adequate monitoring of level of sedation and quality of analgesia, can precipitate morbid events. Further research is needed in the metabolic aspects of analgesia and sedation in the intensive care unit, particularly if a multimodal pharmacologic strategy is used whereby multiple interventions aim at minimizing the risk of overdosing and contributing to attenuation of the stress response associated with critical illness. PMID- 16682908 TI - Acquired weakness in the ICU: critical illness myopathy and polyneuropathy. AB - Illnesses commonly encountered in the ICU, such as sepsis, have frequently been associated with neuromuscular weakness and may play a role in the development of CIM and CIP, whose incidence in the critically ill is greater than initially reported. Although difficult to diagnose from history and clinical/laboratory findings alone, the use of electromyographic and nerve conduction testing is helpful in establishing these diagnoses. Information regarding prognosis of these disorders is limited, and there are no specific therapies that improve outcome. Acquired neuromuscular weakness in the ICU affects a significant number of patients and may continue to affect their quality of life long after discharge. Although diagnostic techniques are readily available, additional research is necessary to obtain adequate prognostic information and therapeutic options for these patients. PMID- 16682909 TI - How to improve ischemic stroke treatment in the fibrinolysis era. AB - In the last 15 years new therapeutic approaches have influenced the treatment of ischemic stroke victims. Aim of this review is to point out the elements of a modern approach to the acute stroke patient. The likelihood of saving ischemic cerebral tissue is time-dependent and the treatment goal is to minimise brain damage. The NINDS trial has documented a higher likelihood of better outcome if the fibrinolytic therapy is administered within 3 h of onset of symptoms. To reach this target several interventions are necessary. First of all, education is needed to diffuse public awareness of stroke warning signs. Moreover, out-of hospital treatment should be optimised with rapid triage and transport to an hospital with a comprehensive stroke approach. The early hospital phase should comprehend a rapid evaluation and an urgent CT scan. After the verification of the inclusion/exclusion criteria, in a authorised SITS-MOST centre, the patient should receive fibrinolytic therapy. The diagnostic and the therapeutic phase should include rapid identification and treatment of secondary insults, as hyperthermia and hyperglycaemia, that have a negative influence on outcome. Despite advances in diagnosis and monitoring, fibrinolytic therapy is the only treatment with a proven efficacy in achieving a higher functional outcome. The narrow time-window is the reason for the need of rapid and well-organised out-of hospital and in-hospital systems. PMID- 16682910 TI - Postoperative metabolic stress: interventional strategies. AB - More and more evidence accumulates suggesting that the reversal of the catabolic responses to surgery is associated with better outcome. The potential for anesthesiologists to be involved in altering outcome by simply preserving a normal blood glucose, providing optimal pain control and perioperative feeding is tremendous. PMID- 16682912 TI - Substrate metabolism during anesthesia. PMID- 16682913 TI - Interactions between general and regional anesthesia. AB - Neuraxial blockade is commonly used to abolish sensations elicited by noxious stimuli during surgical procedures. Proven advantages of combined anesthesia include early recovery from general anesthesia and postoperative analgesia, together with likely decreases in blood loss, cardiac dysrhythmias, or ischemic events and postoperative deep vein thrombosis. The side effects of the technique are related to the dose or site of local anesthetic administration and to light general anesthesia, which can result in awareness during surgery. Varying degrees of synergistic interactions have been reported among the drugs used to achieve the anesthetic state. Spinal anaesthesia appears to have sedative effects, and local anesthetics used for neuraxial blockade have been found to reduce the induction and maintenance dosage of midazolam, thiopental, propofol and inhaled anesthetics. The growing interest in combining local and general anesthesia has led to studies investigating possible interactions between general anesthesia and local anesthetics administered via spinal or epidural routes. Neuraxial blockade reduces sedative and anesthetic requirements by decreasing ascending sensory input into the brain. This has important clinical implications, as anesthetists should expect to reduce anesthetic and sedative drug doses during neuraxial blockade, unless the blockade involves lower dermatomes alone. Clinical practice of anesthesia is a polypharmacy, wherein the anesthetic state is the net result of the action of different drugs and their interaction in the presence of a surgical stimulus. PMID- 16682914 TI - Perioperative platelet transfusion. Recommendations of the French Health Products Safety Agency (AFSSAPS) 2003. AB - The aim of this paper is to present the recommendations of the Agence Francaise de Securite Sanitaire des Produits de Sante (AFSSaPS; French Safety Agency for Health Products). A panel of experts reviewed and graded the literature on platelet transfusions; recommendations were formulated. Threshold platelet counts (PC) for transfusions in the perioperative context have not been clearly defined and should be determined by the existence of hemorrhagic risk factors. In the case of commonly practiced invasive procedures, the recommendation is to transfuse in order to achieve PC > 50,000 x microL-1. In the absence of platelet dysfunction, regardless of the type of surgery, the standard hemorrhagic risk threshold for surgery is 50,000 x microL-1. It has not been proven that the risk threshold is different according to the type of surgery. For neurosurgery and ophthalmologic surgery involving the posterior segment of the eye, a PC of 100,000 x microL-11 is required. For axial regional anesthesia, a PC of 50,000 x microL-11 is sufficient for spinal anesthesia; a PC of 80,000 x microL-11 has been proposed for epidurals. During massive transfusion, prophylactic platelet infusion cannot be recommended beyond a loss of two blood volumes in less than 24 h (Professional Consensus). As for the therapeutic transfusion of plasma and/or platelets, as much as possible, platelet deficit should be documented with test results (PC and fibrinogen) before transfusing. In the event of bleeding, platelet transfusion may precede plasma infusion. However, although this recommendation has been the subject of several professional consensus agreements, it is not based on any randomized studies. Threshold PC for perioperative transfusions have not been clearly defined and most recommendations are the result of a professional consensus. PMID- 16682916 TI - Preoperative parental information and parents' presence at induction of anaesthesia. AB - Preoperative preparation of paediatric patients and their environment in order to prevent anxiety is an important issue in paediatric anaesthesia. Anxiety in paediatric patients may lead to immediate negative postoperative responses. When a child undergoes surgery, information about the child's anaesthesia must be provided to parents who are responsible for making informed choices about healthcare on their child's behalf. A combination of written, pictorial, and verbal information would improve the process of informed consent. The issue of parental presence during induction of anaesthesia has been a controversial topic for many years. Potential benefits from parental presence at induction include reducing or avoiding the fear and anxiety that might occur in both the child and its parents, reducing the need for preoperative sedatives, and improving the child's compliance even if other studies showed no effects on the anxiety and satisfaction level. The presence of other figures such as clowns in the operating room, together with one of the child's parents, is an effective intervention for managing child and parent anxiety during the preoperative period. PMID- 16682915 TI - Caudal anesthesia in pediatrics: an update. AB - AIM: Caudal anesthesia is one of the most used-popular regional blocks in children. This technique is a useful adjunct during general anesthesia and for providing postoperative analgesia after infraumbilical operations. The quality and level of the caudal blockade is dependent on the dose, volume, and concentration of the injected drug. Although it is a versatile block, one of the major limitations of the single-injection technique is the relatively short duration of postoperative analgesia. The most frequently used method to further prolong postoperative analgesia following caudal block is to add different adjunct drugs to the local anesthetics solution. Only few studies evaluated quality and duration of caudal block against the volume of the local anaesthetic applied. After reviewing recent scientific literature, the authors compare the duration of postoperative analgesia in children scheduled for hypospadia repair when 2two different volumes and concentrations of a fixed dose of ropivacaine are used. METHODS: After informed parental consent, 30 children (ASA I, 1-5 years old) were enrolled in a multicentre, perspective, not randomized, observational study conducted in two 2 children hospitals. After premedication with midazolam, anesthesia was induced with thiopental and maintained with sevoflurane in oxygen/air. After induction, patients received a caudal blockade either with ropivacaine 0.375% at 0.5 mL/kg (Low Volume High Concentration Group, LVHC; n = 15), or ropivacaine 0.1% at 1.8 mLl/kg (High Volume Low Concentration Group, HVLC; n = 15). Surgery was allowed to begin 10ten minutes after performing the block. MAC-hour was calculated. In the recovery room, pain was assessed using the Children's Hospital of Eastern Ontario Pain Scale (CHEOPS). In addition, the motor block was scored. After transferral to the ward, the patients were observed for 24 hours for signs of postoperative pain. The time period to first supplemental analgesic demand, i.e., from establishment of the block until the first registration of a CHEOPS score = or > 9, was considered the primary endpoint of the study. The time periods were compared using analysis of variance adjusted for age, weight and duration of surgical procedure as covariates. RESULTS: All patients were judged to have sufficient intraoperative analgesia, and none of them received additional analgesics intraoperatively. Patients' characteristics were similar, besides the age (32+/-10 vs 24 +/- 9 months; P < 0.05) and weigh (15.13 +/- 3.92 vs 11.93 +/- 1.83; P = 0.08). Analgesics were needed after 520 +/- 480 min in the LVHC and 952 +/- 506 min in the HVLC group (P < 0.05). Motor block was less in the HVLC group. CONCLUSIONS: In children undergoing hypospadia repair, caudal block with a ''high volume, low concentration'' regimen produces prolonged analgesia and less motor block, compared to a ''low volume, high concentration'' regimen. PMID- 16682917 TI - Using the nerve stimulator for peripheral or plexus nerve blocks. AB - Conventional methodology for nerve location utilizes anatomical landmarks followed by invasive exploration with a needle to a suitable endpoint. An appropriate endpoint can be either anatomical in nature (e.g. transaterial technique) or functional (paresthesia or motor response to electrical stimulation). Ability to electrically stimulate a peripheral nerve or plexus depends upon many variables, including; 1) conductive area at the electrode, 2) electrical impedance, 3) electrode-to-nerve distance, 4) current flow (amperage), and 5) pulse duration. Electrode conductive area follows the equation R = rhoL/A, where R = electrical resistance, p = tissue resistivity, L = electrode-to-nerve distance, and A = electrode conductive area. Therefore resistance varies to the inverse of the electrode's conductive area. Tissue electrical impedance varies as a function of the tissue composition. In general, tissues with higher lipid content have higher impedances. Modern electrical nerve stimulators are designed to keep current constant, in spite of varying impedance. The electrode-to-nerve distance has the most influence on the ability to elicit a motor response to electrical stimulation. This is governed by Coulomb's law: E = K(Q/r2) where E = required stimulating charge, K= constant, Q = minimal required stimulating current, and r = electrode-to-nerve distance. Therefore, ability to stimulate the nerve at low amperage (e.g. < 0.5 mA), indicates an extremely close position to the nerve. Similarly, increasing current flow (amperage) increases the ability to stimulate the nerve at a distance. Increasing pulse duration increases the flow of electrons during a current pulse at any given amperage. Therefore, reducing pulse duration to very short times (e.g. 0.1 or 0.05 ms) diminishes current dispersion, requiring the needle tip to be extremely close to the nerve to elicit a motor response. The above parameters can be varied optimally to enhance successful nerve location and subsequent blockade. Unlike imaging modalities such as ultrasonography, electrical nerve stimulation depends upon nerve conduction. Similarly, percutaneous electrode guidance (PEG) makes use of the above variables to allow prelocation of the nerve by transcutaneous stimulation. PMID- 16682918 TI - Epidemiology of ARDS. AB - For decades the incidence of the acute respiratory distress syndrome (ARDS) has been unclear. A first effort to tackle the problem was undertaken by the National Heart and Lung Task Force who, in 1972, provided the scientific community with an arguable estimate of 75 cases per 100,000 inhabitants/year for the USA. Nearly 20 years later, the first population based studies yielded figures of 1.5-4.5 cases per 100 000 inhabitants/year in Europe. Epidemiologic research became much more focused when in 1992 the new ARDS and acute lung injury (ALI) definitions of the American-European Consensus Conference became available. In subsequent studies in which these refined definitions were used, incidence figures ranging from 13-23 cases per 100,000 inhabitants/year for ARDS and 18 cases per 100,000 inhabitants/year for ALI were reported. Latest results from a high-class epidemiological study conducted in Seattle, suggested ARDS/ALI incidence figures of 59 and 79 cases per 100,000 inhabitants/year, respectively. These new figures, similar to the one proposed in 1972, led to a paradigm shift: ARDS and ALI are no longer viewed as rare syndromes but as widespread diseases with a massive socio economic impact that is comparable with the burden from breast cancer, AIDS, asthma or myocardial infarction. PMID- 16682911 TI - Continuous quality improvement in intensive care medicine. The GiViTI Margherita Project - Report 2005. AB - AIM: The assessment of the quality of intensive care medicine is mandatory in the modern healthcare system. In Italy, the GiViTI (Gruppo Italiano per la Valutazione degli Interventi in Terapia Intensiva) network is working in this field since 1991 and it now involves 295 out of the about 450 Italian intensive care units (ICU). In 2002 GiViTI launched a project for the continuous quality assessment and improvement that is now joined by 180 ICUs. Data collected in 2005 are analyzed and presented. METHODS: All admitted patients were entered in a validated software, which performs a multitude of validity checks during the data entry. Data were further reviewed by the co-ordinating center; patients admitted in months with more than 10% of incomplete or inconsistent records in each ICU were excluded from the analysis. Each year, a multivariate logistic regression model is fitted to identify predictors of hospital mortality. Starting from the SAPS 2 and the 2004 GiViTI model predictions of hospital mortality, two calibration tables and curves are presented. RESULTS: In 2005, 180 Italian ICUs collected data on 55 246 patients. After excluding those admitted in months with an unjustified lower recruitment rate or with less than 90% of complete and consistent data, we had 52 816 (95.6%) valid cases. Although the rough hospital mortality in 2005 was 1% higher than in 2004 (22.6% vs 21.5%), the adjusted mortality shows a statistically significant 4% reduction (obser-ved-to-expected ratio: 0.96; 95% CI: 0.94-0.97). CONCLUSIONS: Italian ICUs in 2005 performed better than in 2004, at a parity of patient severity. PMID- 16682919 TI - Quality of life after intensive care in trauma patients. AB - Published studies and our own preliminary results show that Intensive Care Unit (ICU) patients admitted after severe trauma, all report reduced quality of life (QOL) after discharge. In contrast to other ICU patients, this reduction is decreased by time, but does not seem to reach pre-ICU levels. Interventions to improve this situation have not been documented. This should be a high priority aspect for all health personnel caring for patients surviving severe trauma. PMID- 16682920 TI - Hemodynamic monitoring in ICU. AB - Oxygen supply to all tissues is possible only in a condition of adequate blood circulation. Oxygen demand is the driving force that is responsive of hemodynamic adjustment. The human body acts on four modulators (intravascular volume, inotropy, vasoactivity, chrono-tropy) in order to adjust the hemodynamic state. Hemodynamic monitoring consists of techniques able to assess the hemodynamic status and to understand the mechanism of patient decompensation; its goal is to maintain adequate tissue perfusion through appropriate therapeutic interventions. An early diagnosis of hemodynamic alteration is crucial for an early treatment; several reports have explored the effectiveness of hemodynamic manipulations and results are conflicting: too many variables can, in fact, modify the results: timing and lenght of the treatment, drugs used, etc. However, at least, in some specific settings, as sepsis, early intervention has a positive impact on mortality. In this presentation it will be briefly analyzed the most common parameters used in the ICU. Arterial pressure, central venous pressure, pulmonary artery catheter derived parameters, SvO2 and their relation with organ perfusion are considered and positive and negative aspects of this type of monitoring is reviewed. Starting from these considerations we would like to underline the importance of understanding the physiological basis of monitoring and the correct interpretation of data in order to have improvement on patient outcome. PMID- 16682921 TI - Intraoperative use of recombinant activated factor VII (rFVIIa). AB - Recombinant activated factor VII (rFVIIa, Novoseven, Novo Nordisk, Denmark) was introduced as a prohemostatic agent in the early 80s: the only indication approved in USA by Food and Drug Administration (FDA) is the spontaneous bleeding in congenital hemophilia patients who developed inhibitors to FVIII and FIX. Recently, EMEA approved the use of rFVIIa in congenital hemophilia patients with inhibitors undergoing surgery, in subjects with congenital FVII deficiency undergoing surgical or invasive procedures, in patients with acquired hemophilia and in case of Glanzmann's thromboasthenia. Out of these approved indications, the off label use of rFVIIa is rapidly expanding, particularly in surgical patients with acquired coagulation disorders in order to manage severe, uncontrolled bleeding nonresponsive to conventional therapeutic measures or to reduce blood loss and transfusion requirements in potentially bleeding surgical procedures (major liver surgery, liver transplantation, major abdominal or obstetric surgery, trauma surgery). This paper reviews the more recent data coming from retrospective or prospective studies performed in different surgical settings: so far, the major point to be addressed is the place for rFVIIa as an adjunctive but sometimes lifesaving treatment to control haemostasis and critical bleeding in surgery and critically ill patients. PMID- 16682922 TI - Echocardiographic assessment of cardiovascular failure. AB - In the last few years echocardiography has gained an increasing role in critical care settings as invaluable tool for hemodynamic assessment of the unstable patient, due to its unique features of dynamic bedside imaging technique which can yield both morphologic and functional information. The main characteristics of cardiovascular function can be thoroughly explored, and a practical clinically oriented approach can lead to answer the crucial questions of patient management, integrating (and often substituting) invasive monitoring, and allowing invasive monitoring pitfalls correction. Therapeutic impact of transesophageal echocardiography (TEE) has proven to be substantial in intensive care unit (ICU) population, although large randomized controlled studies are currently missing. Echocardiography requires specific training, but short training focused on the key hemodynamic information obtained from the shocked patient is proving to be effective. Echocardiographic hemodynamic evaluation should become part of routine assessment in the ICU soon, and critical care teams could achieve a hierarchic organization with respect to echocardiographic skills, with all members being at least able to perform a basic ultrasound examination of the heart, and a fewer who have gone through higher level formal training and board certification. PMID- 16682923 TI - The endothelial function in cardiac surgery. AB - Cardiac operations with cardiopulmonary bypass exerts many different actions which modify the natural function of endothelial cells. The main determinant is the activation of the coagulation system both through the intrinsic and extrinsic pathways, leading to an overwhelming thrombin formation. To counteract the coagulant effects of thrombin, heparin is used in large doses. As a result, the endothelium is asked to promote all its anticoagulant properties, basically through the AT release from the surface, the tissue factor pathway inhibitor release, and the activation of the protein C protein S system. At the end of cardiac operations, all these systems are depleted, and low levels of antithrombin, tissue factor pathway inhibitor, protein C are available for further anticoagulant effects. There is the evidence that levels of antithrombin activity below 50% at the end of cardiac operations with cardiopulmonary bypass are associated to bad outcomes in terms of surgical revision rate, thromboembolic events, and neurological events. Exogenous antithrombin administration has a well defined action in limiting thrombin formation during cardiac operations; however, we are still lacking an evidence-based information about the clinical impact of this and others possible preventive strategies based on exogenous administration of antithrombin before or during cardiac operations. PMID- 16682924 TI - Microcirculatory recruitment maneuvers correct tissue CO2 abnormalities in sepsis. AB - The rises in tissue partial pressure of carbon dioxide have been observed in critically ill patients with shock and sepsis for a long time and have been proposed to be an earlier and more reliable marker of tissue hypoxia than traditional markers. However, the mechanisms leading to those increases, especially in sepsis and endotoxemia, are not well understood. Recent studies provided further data, supporting the idea that the origin of those increases in partial pressure of CO2 in sepsis as being caused by microcirculatory perfusion deficit resulting in mitochondrial depression by time. Previously, we have termed this condition where despite correction of systemic oxygen delivery variables, regional hypoxia and oxygen extraction deficit persist as microcirculatory and mitochondrial distress syndrome (MMDS). Recent findings support the idea that the progression from early to severe sepsis is accompanied or possibly even caused by microcirculatory dysfunction, which leads to mitochondrial dysfunction by time. Therefore early identification of microcirculatory dysfunction and correction with microcirculatory recruitment maneuvers are needed to ensure adequate microcirculatory perfusion and tissue oxygenation. Microcirculatory imaging, such as SDF imaging technique, appears to be a very useful tool for this task and its combination together with other systemic and regional tissue oxygenation measurements may provide more information regarding the tissue oxygenation and will be a very promising tool for microcirculatory researchers and the management of critically ill patients at the bedside. PMID- 16682925 TI - Assessing dead space. A meaningful variable? AB - The recording of dead space will give information on how much of total ventilation that reaches both ventilated and perfused alveoli and thus allows gas exchange between alveoli and pulmonary blood. Realising that CO2 retention can be an effect not only of low total ventilation but also of increased dead space is one important information. Moreover, dead space will give insight into the matching of ventilation and perfusion. This is because dead space is affected by a number of factors: 1) tubings and valves that the subject has to rebreath through (apparatus dead space), 2/ Airways (anatomical dead space), 3/ Non perfused but ventilated alveoli, e.g. pulmonary embolus (alveolar dead space), 4/ Excessive ventilation of alveoli in relation to their perfusion that can be seen in chronic obstructive lung disease (another form of alveolar dead space), and 5/ So called "shunt dead space" that is an erroneous description of right to left lung shunt that brings the higher CO2 concentration in venous blood to the arterial side thereby producing an arterial-to-end-tidal CO2 difference. The dead spaces 2-5 are called physiological dead space. The recording of dead spaces can be done according to the Riley three-compartment model or by analysis of the expired CO2 curve. However, both are subjected to potential errors that have to be considered to make a dead space recording meaningful. A correct measurement and calculation of the dead space will give valuable information on the ventilatory support of the critically ill patient and can also be a valuable diagnostic tool. It should therefore not be forgotten in the intensive care setting. PMID- 16682926 TI - Gastric tonometry. AB - Gastric tonometry was originally proposed to assess the splanchnic perfusion. Several technological improvements have been introduced over the years and, to date, the preferred way to estimate the splanchnic perfusion is to rely on the arterial-gastric PCO2 gap. In this brief review we will discuss the value of the gastric tonometry, its physiological background and the clinical results observed so far. PMID- 16682927 TI - Metabolic treatment of critically ill patients: energy balance and substrate disposal. AB - Oxidation of substrates is the main biochemical process used by the human body to produce energy. Different substrates (carbohydrates, lipids, and proteins) have different effects on oxygen consumption and carbon dioxide production: during the critical phase of pathologies it could be relevant pay attention to the use of various nutrients, that have some altered effect respect to the normal subjects metabolism, and during the length of metabolic treatment, too. Generally, nutrition lead to replenish body stores, while endogenous substrates are used to be oxidized. Critically ill patients show a preference for prompt energy availability (i.e. glucose) to avoid endogenous protein catabolism; lipids are shown to have a more pronounced storage effect. Adequate amount of energy intake in carbohydrates determine an increase of RQ, that means a shift from a more lipid-based to a more glucose-based oxidation. Composition of dietary intake can be usefully different for each pathology, and also for different periods of the same pathology, because critically ill patients have a variety of metabolic needs during their stay in ICU. PMID- 16682928 TI - The ratio between arterio-venous PCO2 difference and arterio-jugular oxygen difference as estimator of critical cerebral hypoperfusion. AB - AIM: The aim of this study was to evaluate the arterio-venous difference in carbon dioxide tension (DPCO2) and the ratio between DPCO2 and arterio-jugular oxygen difference (AJDO2) as indicators of compensated or uncompensated cerebral hypoperfusion. METHODS: Cerebral blood flow (CBF) was reduced stepwise in 6 pigs by inducing intracranial hypertension with consequently cerebral perfusion pressure (CPP) reduction: CBF 100%, 50-60 % of baseline, 20-30% of baseline. Intracranial pressure (ICP), mean arterial pressure (MAP), CPP and CBF (laser Doppler method) were continuously recorded. Superior sagittal sinus was punctured for the determination of AJDO2 and DPCO2. RESULTS: CBF impairment was accompanied by changes in AJDO2 from 6.03 +/- 1.21 vol% to 7.32 +/- 1.30 vol%, up to 8.07 +/- 1.32 vol% (P < 0.01), in DPCO2 from 12.17 +/- 3.25 mmHg to 16 +/- 4.12 mmHg, up to 26.5 +/- 6.41 mmHg (P < 0.01), and DPCO2/AJDO2 ratio from 2.05 +/- 0.39 to 2.06 +/- 0.72 up to 3.41 +/- 1.09 in the 3 phases (P < 0.05). CONCLUSIONS: When CBF declines AJDO2 increases, indicating greater extraction of O2 to satisfy aerobic metabolism. However, this mechanism can no longer compensate once a critical CBF threshold is reached. DPCO2 rises slowly during moderate CBF reduction because of defective washout; the rise is steeper during marked CBF impairment when anaerobic metabolism takes place. During cerebral hypoperfusion the venous blood gases and acid base variables mirror the degree of cerebral perfusion. In particular the DPCO2, and the DPCO2/ AJDO2 ratio may be useful markers of critical brain hypoperfusion. PMID- 16682929 TI - Respiratory acidosis: is the correction with bicarbonate worth? AB - Bicarbonate infusion is traditionally used to increase pH during metabolic acidosis, but it has been also suggested to increase the pH during permissive hypercapnia. In this paper we will discuss the physicochemical effect of adding (Na+ HCO3-), first in a closed system (venous blood) and then in an open system (the blood after the lung). According to Stewart model, in the closed system two independent variables are changed (CO2 and strong ion difference). As a first result changes in pH are negligible. If the CO2 is cleared by the lung and the PCO2 is maintained as before the infusion, the rise in pH is due to the SID increase caused by the (Na+) rise. The effect is independent on (HCO3-) infusion and equivalent to adding (Na+ OH-) instead of (Na+ HCO3-). PMID- 16682930 TI - Metabolic treatment of critically ill patients: energy expenditure and energy supply. AB - Nutrition in critically ill patients should be considered as therapy: assessing the energy expenditure and the termogenic effect of food, and knowing the differences among composition and amount of given substrates, it is possible restore, maintain, or at least limit the derangement of energy equilibrium. Energy metabolism comprehends assumption, storage and oxidation of nutrients: all these factors could be discriminant in critical clinical conditions, particularly cardiac and respiratory failure. Then, this review would lead the decision making process beginning from biochemistry and bioenergetics, until the metabolic strategy practically usable at the bedside of patients during the whole critical phase of their pathology. PMID- 16682931 TI - Hypercapnia: permissive and therapeutic. AB - Hypercapnia has traditionally been avoided in the quest to keep parameters normal. Recent understanding of the role of excessive tidal stretch has prompted clinicians to avoid high tidal volumes or plateau pressures, and the resulting permissive hypercapnia has been increasingly tolerated by clinicians. Newer data point to the potential for elevated CO2 to be protective, and in some experimental situations, to cause harm. The protective effects of so called therapeutic hypercapnia' remain experimental at present, but promising laboratory studies suggest potential roles for the eventual selective application at the bedside. PMID- 16682932 TI - Volumetric capnography in the mechanically ventilated patient. AB - Expiratory capnogram provides qualitative information on the waveform patterns associated with mechanical ventilation and quantitative estimation of expired CO2. Volumetric capnography simultaneously measures expired CO2 and tidal volume and allows identification of CO2 from 3 sequential lung compartments: apparatus and anatomic dead space, from progressive emptying of alveoli and alveolar gas. Lung heterogeneity creates regional differences in CO2 concentration and sequential emptying contributes to the rise of the alveolar plateau and to the steeper the expired CO2 slope. The concept of dead space accounts for those lung areas that are ventilated but not perfused. In patients with sudden pulmonary vascular occlusion due to pulmonary embolism, the resultant high V/Q mismatch produces an increase in alveolar dead space. Calculations derived from volumetric capnography are useful to suspect pulmonary embolism at the bedside. Alveolar dead space is large in acute lung injury and when the effect of positive end expiratory pressure (PEEP) is to recruit collapsed lung units resulting in an improvement of oxygenation, alveolar dead space may decrease, whereas PEEP induced overdistension tends to increase alveolar dead space. Finally, measurement of physiologic dead space and alveolar ejection volume at admission or the trend during the first 48 hours of mechanical ventilation might provide useful information on outcome of critically ill patients with acute lung injury or acute respiratory distress syndrome. PMID- 16682933 TI - Role of extracorporeal lung assist in the treatment of acute respiratory failure. AB - For patients with most severe acute respiratory distress syndrome (ARDS) conservative treatment with lung protective ventilation is often not sufficient to prevent life-threatening hypoxemia and additional strategies are necessary. Extracorporeal lung assist (ECLA) or extracorporeal membrane oxygenation (ECMO) using capillary membrane oxygenators can provide sufficient gas exchange and lung rest. In 2 randomized trials mortality was unchanged for ECMO. Today an technically enhanced ECMO is used for most severe ARDS using clinical algorithm and different case studies demonstrated a survival rate about 56%. Today miniaturized ECMO with optimized blood pumps and oxygenators are available and could enhance safety and clinical management. Another approach is an arterio venous pumpless interventional lung assist (ILA) with a low resistance oxygenator. Advantages seem a simplified clinical management and less blood trauma. At present new devices are developed for chronic respiratory failure or bridge to lung transplant. Oxygenators with even less flow resistance could be implanted paracorporeal using the right ventricle as driving force. An intravascular oxygenator has been developed using the combination of a miniaturized blood pump and an oxygenator for implantation in the vena cava. Well designed clinical trials are necessary to demonstrate a clinical benefit for these experimental devices. PMID- 16682935 TI - From cradle to grave: pancreatic beta-cell mass and glucagon-like peptide-1. AB - Type 2 diabetes mellitus and its clinical correlates, including impaired fasting blood glucose, obesity and insulin resistance, represent a significant public health issue worldwide, with the prevalence of these metabolic conditions increasing exponentially. Given the staggering financial costs and human suffering incurred by diabetes and its co-morbid conditions, any safe new therapeutic interventions that prove to have a beneficial effect in reducing the incidence of diabetes in susceptible individuals or in preventing progression of the disease would have major public health benefits. Studies on the regulation of beta-cell mass have demonstrated a remarkable plasticity, from fetal through adult life, as well as in response to a variety of stresses. These findings are considered in this review in the context of newer studies on the intestinal hormone, glucagon-like peptide-1, which not only enhances beta-cell function, but also stimulates beta-cell growth, neogenesis and survival. PMID- 16682934 TI - Meaning of arterio-venous PCO2 difference in circulatory shock. AB - The arterovenous difference in carbon dioxide tension (DeltaPCO2) can be calculated after simultaneous sampling of arterial blood (PaCO2) and of mixed venous blood from the distal of a pulmonary artery catheter (PvCO2). Under physiological conditions, DeltaPCO2 ranges from 2 to 5 mmHg. The DeltaPCO2 depends on carbon dioxide and cardiac output by a complex fashion. In this article, we detail the influence of these factors on DeltaPCO2 in normoxic conditions and in hypoxic conditions. We bring evidence that DeltaPCO2 cannot serve as a marker of tissue hypoxia contrary to what was initially thought. However, DeltaPCO2 can be considered as a marker of the adequacy of venous blood flow (i.e. cardiac output) to remove the total CO2 produced by the peripheral tissues. In this regard, the knowledge of DeltaPCO2 should help the clinicians for the decision of giving therapy aimed at increasing cardiac output. PMID- 16682936 TI - The clinical utility of leptin therapy in metabolic dysfunction. AB - Obesity and diabetes are major public health threats worldwide. Insulin resistance appears to be a significant factor in this global epidemic. In this present review, we have focused on a human model of insulin resistance which embodies many of the metabolic abnormalities that are associated with the morbidity of diabetes and obesity. Lipodystrophy in rodents and humans is a severe model of insulin resistance, and we use a novel therapeutic approach with the administration of the newly discovered leptin to ameliorate many of these metabolic abnormalities. The ability to study the administration of leptin in this setting of severe insulin resistance allows us perform a coveted look into a human condition where metabolic dysfunction can be reversed or controlled. PMID- 16682937 TI - Therapies for the treatment of type 2 diabetes mellitus based on incretin action. AB - Orally ingested glucose leads to a much higher insulin response than intravenous glucose leading to identical postprandial plasma glucose excursions. This phenomenon, termed ''incretin effect'' comprises up to 60% of the postprandial insulin secretion and is diminished in type 2 diabetes. The gastrointestinal hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) promote the incretin effect. Type 2 diabetes is characterized by an incretin defect: while GIP does not stimulate insulin secretion, GLP-1 action is still preserved under supraphysiological concentrations. GLP-1 stimulates insulin secretion only under hyperglycaemic conditions, therefore it does not cause hypoglycaemia. Furthermore, GLP-1 inhibits glucagon secretion and delays gastric emptying. In vitro and animal data demonstrated that GLP-1 increases beta cell mass by stimulating islet cell neogenesis and by inhibiting apoptosis of islets. The improvement of beta cell function can be indirectly observed from the increased insulin secretory capacity of humans receiving GLP-1. In contrast to GIP, GLP-1 may represent an attractive therapeutic method for type 2 diabetes due to its multiple effects also including the simulation of satiety in the central nervous system by acting as transmitter or by crossing the blood brain barrier. Native GLP-1 is degraded rapidly upon intravenous or subcutaneous administration and is therefore not feasible for routine therapy. Long-acting GLP-1 analogs (e.g. Liraglutide) and exendin-4 (Exenatide, Byetta) that are resistant to degradation, called ''incretin mimetics'' are approved (Exenatide, Byetta) or in clinical trials. DPP-4-inhibitors (e.g. Vildagliptin), Sitagliptin and Saxagliptin) that inhibit the enzyme DPP-4 responsible for incretin degradation are also under study. PMID- 16682938 TI - Regulation of cerebral glucose metabolism. AB - The brain uses glucose as a primary fuel for energy generation. Glucose gains entry into the brain by facilitated diffusion across the blood-brain barrier. Glucose transport may adapt during changes in cerebral glucose metabolism, neural activation and changes in plasma glucose levels. Within the brain, glucose is either oxidized to produce ATP or used to synthesize glycogen. To ensure the delivery of a continuous supply of glucose to maintain normal cellular function, the brain has developed a complex regulatory system to preserve its supply. Gluco sensing neurons have been demonstrated in various regions of the brain and they appear to play an important role in not only detecting changes in brain glucose levels but also in initiating responses to maintain constant brain glucose levels. In this review, we will discuss the regulation of brain glucose metabolism (CMR(gluc)) and how it adapts to chronic changes in glycemia, like that seen in hyperglycemic patients with diabetes mellitus or patients with type 1 diabetes, recurrent hypoglycemia, and hypoglycemia unawareness. We will also consider the role of brain glycogen in providing fuel for energy under conditions of stress. PMID- 16682939 TI - The rational use of fine needle aspiration biopsy (FNAB) in diagnosing thyroid nodules. AB - AIM: Fine needle aspiration biopsy (FNAB) plays a crucial role in the diagnosis of thyroid nodules and enables the number of surgical operations to be reduced. Theoretically, FNAB should be carried out on all nodules, though currently only those displaying certain characteristics are biopsied. Indeed, to perform FNAB on all nodules may be regarded as an excess of zeal. Therefore, it seems advisable that the endocrinologist should be able to confirm on the spot the necessity and utility of FNAB. METHODS: We evaluated on a sample of 263 consecutive requests (209 female, 57 male; age 56.7+/-13.7 years) for FNAB in 2004: 1) the appropriateness of the investigation, 2) expected efficacy, 3) practical efficacy, 4) efficiency. FNAB was performed under echo-guidance in accordance with the standard technique. In 50%, 36%, 6%, 3%, 2% and 1% of cases, the echographic diagnosis was of MNG, UNG, pseudo-nodular lesion in ATD, lymph-node, neck cyst, suspected parathyroid lesion and tumefaction of the salivary glands, respectively. A pre-FNAB clinical risk score was assigned to each case on the basis of clinical and echographic data, with a maximum possible score of 11. The results of FNAB were subdivided into 5 categories according to the criteria of the BTA (Thy1-Thy5). After FNAB, a decisional category was assigned, ranging from ''observation'' to ''surgery''; this was subsequently (7-18 months) compared with the management strategy adopted by the attending physician. Information was gathered by means of telephone enquiry. RESULTS: 1) Appropriateness: on the basis of clinical and echographic findings, FNAB was not judged appropriate in 24% of cases because of either the lack of confirmation of a significant target (34%) or a low pre-FNAB risk score (range 0-2) (66%). The decisional category was ''observation'' in 87% of cases and ''further investigation'' in 13%. 2) Expected efficacy: FNAB was performed in 76% of cases. The biopsies (3%) performed on swollen lymph-nodes and extra-thyroid neck tumefactions, in which biochemical evaluation was positive, proved to be diagnostic but not classifiable according to the BTA. In 82% of the remaining cases, the result was Thy2 (observation) or Thy 4-5 (surgery). Thy3 results (surgery) were rare (1%). Thy1 results (16%) were yielded by the aspiration of colloid cysts (29%), solid lesions (10%) characterised by means of PTH-FNAB and Tg-FNAB, nodules (9%) no longer detectable on repetition of FNAB, nodules (16%) in which FNAB was already a repetition of a non-diagnostic investigation (2003), and nodules (9%) in which the presence of normal thyrocytes, ''hot'' scintigraphic image and prior decision of the surgeon advised against repeating FNAB. Of the patients with Thy1 results, 26% refused to repeat FNAB. In all, 95% of FNAB supported by biochemical evaluation yielded results that usefully contributed to patient management. The correlation between pre-FNAB clinical risk and cytological score according to the BTA proved significant (P<0.001). No difference in diameter was recorded between nodules with adequate cytology (23.3+/-0.9 mm) and those with inadequate cytology (25.2+/ 1.6 mm). 3) Practical efficacy: 75% of patients were reached by telephone. In most cases, observation was the most frequent clinical choice, after echography and/or FNAB. The decisional category assigned after FNAB correlated significantly (P<0.001) with the approach adopted by the attending physician. d) Efficiency: following FNAB, 11 patients were assigned to surgery. DTC was detected in 100% of these cases (1 follicular carcinoma, 1 insular carcinoma, 9 papillary carcinoma). The success of FNAB (9/11) in detecting lesions that proved malignant on histological examination (11/11) was significant (P<0.05). Of the 2 Thy 3 cases, 1 was follicular carcinoma and 1 was follicular adenoma with adjacent papillary carcinoma. The incidence of thyroid carcinomas in the population studied was 5.5%. CONCLUSIONS: 1) Together with clinical-biochemical evaluation, echo-guided FNAB re-mains the first-line diagnostic test in the management of thyroid nodules; 2) a pre-FNAB clinical risk score is useful in limiting the number of probably inappropriate investigations; 3) efficacy, in terms of cytology results that are useful for patient management after FNAB (and after biochemical evaluation, when indicated) is high, enabling patients to be stratified in classes with different subsequent pathways; 4) in the vast majority of cases, FNAB influences subsequent clinical decisions; 5) false negatives cannot be excluded, while false positives are practically nil; 6) further indications may be yielded by studies on larger populations, and new prospects may emerge from the application of other techniques associated to FNAB. PMID- 16682940 TI - Effects of thyroxine withdrawal in biochemical parameters and cardiac function and structure in patients with differentiated thyroid cancer. AB - AIM: Patients with differentiated thyroid carcinoma (DTC) are closely monitored during the first decade after diagnosis. At intervals of 1-2 years withdrawal of suppressive doses of T(4) is recommended in order to check thyroglobulin (Tg) levels under increased TSH. T(4) therapy is usually withdrawn for 5 weeks (during the first 3 weeks patients receive treatment with T(3) instead of T(4), and the last 2 weeks stop all medication). There are a few reported studies looking into the effects of T(4) withdrawal in athyreotic patients in terms of biochemical parameters and ultrasound indices. We studied patients with DTC at two time points: during suppressive T(4) treatment and at the end of the T(4) withdrawal protocol in order to identify acute changes that become apparent after 5 weeks of treatment modification. METHODS: Hormonal and biochemical parameters were measured as well as ultrasound indices of cardiac function and structure. RESULTS: Statistically significant increases were found in total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol and triglycerides with T(4) withdrawal. Creatine phosphokinase showed a striking increase with treatment withdrawal. In addition, liver enzymes, total protein and albumin concentrations increased. Creatinine levels increased significantly and sodium decreased on stopping T(4) treatment. The ultrasound indices of cardiac function and structure did not show significant changes. CONCLUSIONS: Acute hypothyroidism following T(4) withdrawal in DTC patients leads to important biochemical changes without significant alterations in cardiac function and structure. These changes may adversely affect patients, especially older patients or those with other chronic diseases. PMID- 16682941 TI - Fine needle aspiration biopsy-RT-PCR molecular analysis of thyroid nodules: a useful preoperative diagnostic tool. AB - Fine needle aspiration biopsy (FNAB)-based cytology is an accurate preoperative diagnostic method for the evaluation of thyroid nodules. Despite its high specificity and sensitivity, the results are non-diagnostic in a significant number of cases, commonly due to inadequacy of the samples or the presence of undetermined cellular morphology. Overlap-ping cytologic features among follicular-derived lesions are the most common causes of cyto-histopathologic discordances. The management of thyroid nodules with a non-diagnostic FNAB remains controversial. The significant probability of malignancy imposes either the repeat of the procedure or direct surgery. Recent studies have demonstrated the important role of molecular-based techniques such as RT-PCR in the diagnosis of thyroid lesions. Follicular cells obtained from the remaining material within the needle can be used for RNA extraction and then for RT-PCR amplification of specific mRNAs that are expressed in benign or malignant thyroid nodules. Furthermore, FNAB-RT-PCR in combination with molecular scanning techniques or direct sequencing provides a valuable screening tool for mutations. The present review highlights the applications of FNAB-RT-PCR method in the identification of new diagnostic molecular markers. The parallel molecular and cytological analysis of FNABs may contribute to a more accurate diagnosis and, thus, to a more specific clinical management of thyroid nodules. PMID- 16682942 TI - Pheochromocytoma associated with adrenocortical tumor in the same gland. Two case reports and literature review. AB - Pheochromocytomas are catecholamine-producing neuroendocrine tumours arising from chromaffin cells of the adrenal medulla or extra-adrenal paraganglionic system that show 2 distinctive features, rarity and clinical variability. Pheochromocytoma occasionally is associated with pathological lesions of the adrenal cortex. We present 2 cases of patients referred to our hospital with a finding of clinical suspected pheochromocytoma. Both of them were hypertensive; the first patient with typical symptoms of pheochromocytoma and the second patient with chest pain and hypertension resistant to pharmacological treatment. The diagnosis of pheochromocytoma was confirmed in both cases with laboratory analysis and the lesion was achieved by employing 3 imaging techniques: computed tomography (CT), magnetic resonance imaging (MRI) and scintigraphy with (123)I metaiodobenzilguanidine (MIBG). The patients underwent adrenalectomy and in the same adrenal gland we found a pheochromocytoma associated with a nonfunctioning cortical adenoma. As far as we know few cases with this association are available in the literature. PMID- 16682943 TI - MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2. AB - Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway. PMID- 16682944 TI - MADD/DENN splice variant of the IG20 gene is necessary and sufficient for cancer cell survival. AB - The IG20 gene is overexpressed in human tumors and cancer cell lines, and encodes at least four splice variants (SVs) namely, IG20pa, MADD, IG20-SV2 and DENN-SV. Earlier, gain-of-function studies showed that IG20-SVs can exhibit diverse functions and play a critical role in cell proliferation and apoptosis. Expression of exogenous IG20pa or DENN-SV rendered cells either susceptible or resistant to induced apoptosis, respectively, whereas MADD and IG20-SV2 had no apparent effect. In order to understand the contrasting effects of the IG20-SVs in a physiologically more relevant system, we expressed exon-specific small hairpin RNAs (shRNAs) to selectively knockdown specific IG20-SVs. Consistent with an earlier study, knockdown of all IG20-SVs resulted in spontaneous apoptosis of HeLa and PA-1 cells. In addition, we unambiguously demonstrated that knockdown of MADD can render cells susceptible to spontaneous apoptosis but had no discernible effect on cell proliferation, colony size or cell cycle progression. Moreover, expression of MADD alone, and not DENN-SV, in the absence of endogenous IG20-SVs was sufficient to prevent spontaneous apoptosis. Our results show the utility of shRNAs for selective knockdown of particular IG20-SVs and their potential therapeutic value in cancer. Further, they demonstrate that MADD alone is sufficient and necessary for cancer cell survival. PMID- 16682945 TI - DEP-1 protein tyrosine phosphatase inhibits proliferation and migration of colon carcinoma cells and is upregulated by protective nutrients. AB - The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients. PMID- 16682946 TI - Hypoxia-inducible factor-1alpha expression requires PI 3-kinase activity and correlates with Akt1 phosphorylation in invasive breast carcinomas. AB - Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1alpha protein is associated with poor patient survival and therapy resistance in many types of solid tumors. Insight into HIF-1alpha regulation in solid tumors is important for therapeutic strategies. In this study, we determined the pathophysiological relevance of HIF 1alpha regulation by the oncogenic phosphatidylinositol 3'-kinase (PI 3 kinase)/Akt signaling pathway. We modeled the physiology of hypoxic tumor regions by culturing carcinoma cells under low oxygen tension in the absence of serum. We observed that hypoxic induction of HIF-1alpha protein was decreased by serum deprivation. Overexpression of dominant-active Akt1 restored HIF-1alpha expression, whereas inhibition of PI 3-kinase activity reduced hypoxic HIF-1alpha protein levels to a similar extent as serum deprivation. Immunohistochemical analysis of 95 human breast cancers revealed that lack of Akt1 phosphorylation correlates with low HIF-1alpha levels. To our knowledge, this is the first reported comparison between HIF-1alpha expression and Akt phosphorylation in human carcinomas. We conclude that Akt activity is physiologically relevant for HIF-1alpha expression in breast cancer. This implies that HIF-1alpha function might be therapeutically targeted by inhibition of the PI 3-kinase/Akt pathway. PMID- 16682947 TI - Mice with skin-specific DNA repair gene (Ercc1) inactivation are hypersensitive to ultraviolet irradiation-induced skin cancer and show more rapid actinic progression. AB - Ercc1 has an essential role in the nucleotide excision repair (NER) pathway that protects against ultraviolet (UV)-induced DNA damage and is also involved in additional repair pathways. The premature death of simple Ercc1 mouse knockouts meant that we were unable to study the role of Ercc1 in the skin. To do this, we have used the Cre-lox system to generate a skin-specific Ercc1 knockout. With a Cre transgene under control of the bovine keratin 5 promoter we achieved 100% recombination of the Ercc1 gene in the epidermis. Hairless mice with Ercc1 deficient skin were hypersensitive to the short-term effects of UV irradiation, showing a very low minimal erythemal dose and a dramatic hyperproliferative response. Ultraviolet-irradiated mice with Ercc1-deficient skin developed epidermal skin tumours much more rapidly than controls. These tumours appeared to arise earlier in actinic progression and grew more rapidly than tumours on control mice. These responses are more pronounced than have been reported for other NER-deficient mice, demonstrating that Ercc1 has a key role in protecting against UV-induced skin cancer. PMID- 16682948 TI - Mitochondrially targeted wild-type p53 suppresses growth of mutant p53 lymphomas in vivo. AB - The complex apoptotic functions of the p53 tumor suppressor are central to its antineoplastic activity in vivo. Besides its well-known action as a transcriptional activator of apoptotic genes, p53 exerts a direct proapoptotic role at the mitochondria via protein-protein interactions with Bcl2 family members, thus executing the shortest known circuitry of p53 death signaling. We recently reported that exclusive delivery of p53 to mitochondria exerts a significant in vivo tumor suppressor activity in p53-null lymphomas. However, it was unknown whether mitochondrially targeted p53 has suppressor activities in tumors harboring missense mutants, which constitute the vast majority of p53 alterations in human tumors. Here, we show that targeting p53 to mitochondria does confer a significant growth disadvantage in B-lymphomas expressing various point mutants of p53, resulting in efficient apoptosis induction in vitro and in vivo in mice. PMID- 16682949 TI - Centrosomal proteins Nde1 and Su48 form a complex regulated by phosphorylation. AB - The centrosome modulates spindle formation and plays a critical role in guiding proper segregation of chromosomes during cell division. Centrosome aberrations, frequently seen in human tumors, may cause abnormal chromosome segregation and contribute to malignant transformation. To explore the components of the centrosomes, we previously identified a novel centrosomal protein called Su48. To further characterize the Su48-containing protein ensemble in the centrosome, we performed yeast two-hybrid screens and isolated a number of Su48-interacting molecules, including the centrosomal protein Nde1. Here, we demonstrate that Su48 can associate with Nde1. Moreover, we found that Nde1 is subjected to phosphorylation in vivo. In particular, we identified six putative Cdc2 phosphorylation sites in Nde1 and found that alteration of these sites diminishes phosphorylation by Cdc2 in vitro and affects the stability of Su48-Nde1 interactions and the centrosomal localization of Nde1. Ablation of Nde1 by gene specific small interfering RNA causes mitotic delay and cell death, coupled with a modest decrease in the incidence of the cells that harbor excessive centrosomes. Collectively, our findings indicate that Nde1 can form a protein complex with Su48 in the centrosome and plays an important role for successful mitosis. PMID- 16682950 TI - Involvement of programmed cell death 4 in transforming growth factor-beta1 induced apoptosis in human hepatocellular carcinoma. AB - The programmed cell death 4 (PDCD4) gene was originally identified as a tumor related gene in humans and acts as a tumor-suppressor in mouse epidermal carcinoma cells. However, its function and regulatory mechanisms of expression in human cancer remain to be elucidated. We therefore investigated the expression of PDCD4 in human hepatocellular carcinoma (HCC) and the role of PDCD4 in human HCC cells. Downregulation of PDCD4 protein was observed in all HCC tissues tested compared with corresponding noncancerous liver, as revealed by Western blotting or immunohistochemical staining. Human HCC cell line, Huh7, transfected with PDCD4 cDNA showed nuclear fragmentation and DNA laddering characteristic of apoptotic cells associated with mitochondrial changes and caspase activation. Transforming growth factor-beta1 (TGF-beta1) treatment of Huh7 cells resulted in increased PDCD4 expression and occurrence of apoptosis, also concomitant with mitochondrial events and caspase activation. Transfection of Smad7, a known antagonist to TGF-beta1 signaling, protected cells from TGF-beta1-mediated apoptosis and suppressed TGF-beta1-induced PDCD4 expression. Moreover, antisense PDCD4 transfectants were resistant to apoptosis induced by TGF-beta1. In conclusion, these data suggest that PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, and a possible tumor suppressor in hepatocarcinogenesis. PMID- 16682951 TI - SEPT2 is a new fusion partner of MLL in acute myeloid leukemia with t(2;11)(q37;q23). AB - We have identified a new mixed lineage leukemia (MLL) gene fusion partner in a patient with treatment-related acute myeloid leukemia (AML) presenting a t(2;11)(q37;q23) as the only cytogenetic abnormality. Fluorescence in situ hybridization demonstrated a rearrangement of the MLL gene and molecular genetic analyses identified a septin family gene, SEPT2, located on chromosome 2q37, as the fusion partner of MLL. RNA and DNA analyses showed the existence of an in frame fusion of MLL exon 7 with SEPT2 exon 3, with the genomic breakpoints located in intron 7 and 2 of MLL and SEPT2, respectively. Search for DNA sequence motifs revealed the existence of two sequences with 94.4% homology with the topoisomerase II consensus cleavage site in MLL intron 7 and SEPT2 intron 2. SEPT2 is the fifth septin family gene fused with MLL, making this gene family the most frequently involved in MLL-related AML (about 10% of all known fusion partners). The protein encoded by SEPT2 is highly homologous to septins 1, 4 and 5 and is involved in the coordination of several key steps of mitosis. Further studies are warranted to understand why the septin protein family is particularly involved in the pathogenesis of MLL-associated leukemia. PMID- 16682952 TI - MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors. AB - To determine whether integration of human papillomavirus (HPV) DNA sequences could lead to the deregulation of genes implied in oncogenesis, we analysed the HPV integration sites in a series of nine cell lines derived from invasive genital carcinomas. Using in situ hybridization, HPV16 or 18 sequences were found at chromosome band 8q24, the localization of MYC, in IC1, IC2, IC3, IC6 and CAC-1 cells and at other sites in IC4, IC5, IC7 and IC8 cells. We then localized viral sequences at the molecular level and searched for alterations of MYC structure and expression in these cells. MYC genomic status and viral integration sites were also analysed in primary tumors from which IC1, IC2, IC3 and IC6 cells were derived. In IC1, IC2 and CAC-1 cells, HPV DNA was located within 58 kb of MYC, downstream, upstream, or within MYC. In IC3 and IC6 cells, HPV DNA was located 400-500 kb upstream of MYC. Amplification studies showed that, in IC1, IC2 and IC3, viral and MYC sequences were co-amplified in an amplicon between less than 50 and 800 kb in size. MYC amplification was also observed in primary tumors, indicating that this genetic alteration, together with viral insertion at the MYC locus, had already taken place in vivo. MYC was not amplified in the other cell lines. MYC mRNA and protein overexpression was observed in the five cell lines in which the HPV DNA was inserted close to the MYC locus, but in none of the lines where the insertion had occurred at other sites. MYC activation, triggered by the insertion of HPV DNA sequences, can be an important genetic event in cervical oncogenesis. PMID- 16682953 TI - Suppression of replication fork progression in low-dose-specific p53-dependent S phase DNA damage checkpoint. AB - The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53-/- MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53-/- MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53-/- MEFs. These results demonstrate that the novel low-dose-specific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA. PMID- 16682954 TI - 15-Lipoxygenase-2 gene regulation by its product 15-(S)-hydroxyeicosatetraenoic acid through a negative feedback mechanism that involves peroxisome proliferator activated receptor gamma. AB - An inverse relationship exists between the expression of 15-lipoxygenase-2 (15 LOX-2) and peroxisome proliferator-activated receptor gamma (PPARgamma) in normal prostate epithelial cells (PrECs) compared with their expression in prostate carcinoma cells (PC-3). The reason for this difference, however, is unknown. We hypothesized that this inverse expression partly involves the 15-LOX-2 promoter and 15-S-hydroxyeicosatetraenoic acid (15-(S)-HETE), a product of 15-LOX-2 that binds to PPARgamma. We identified an active steroid nuclear receptor half-site present in the 15-LOX-2 promoter fragment F-5 (-618/+177) that can interact with PPARgamma. After forced expression of wild-type PPARgamma, 15-(S)-HETE (1 microM) decreased F-5 reporter activity in PrECs whereas forced expression of 15-LOX-2 resulted in 15-(S)-HETE production which enhanced F-5 activity in PC-3. In contrast, the expression of dominant-negative PPARgamma reversed the transcriptional activation of F-5 by enhancing it 202-fold in PrEC or suppressing it in PC-3; the effect in PC-3 was positively increased 150-fold in the presence of 15-(S)-HETE (1 microM). Peroxisome proliferator-activated receptor gamma interacted with 15-LOX-2 promoter sequences in pulldown experiments using biotinylated 15-LOX-2 (-560/-596 bp) oligonucleotides. In gelshift analyses PPARgamma and orphan receptor RORalpha were shown to interact with the F-5 fragment in PC-3 cells. These data suggest that crosstalk mechanisms exist between the 15-LOX-2 gene and PPARgamma to counterbalance expression and help explain the inverse relationship of these genes in normal versus cancer cells. PMID- 16682955 TI - Expression of the SNT-1/FRS2 phosphotyrosine binding domain inhibits activation of MAP kinase and PI3-kinase pathways and antiestrogen resistant growth induced by FGF-1 in human breast carcinoma cells. AB - Fibroblast growth factor (FGF) signaling can bypass the requirement for estrogen receptor (ER) activation in the growth of ER-positive (ER+) breast cancer cells. Fibroblast growth factor-1 stimulation leads to phosphorylation of the adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT-1) on C-terminal tyrosine residues, whereas it is constitutively bound through its N-terminal phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). By expressing the PTB domain of SNT-1 (SNT-1 PTB) in an inducible manner in an ER+ breast carcinoma line, ML20, we asked whether we could uncouple FGFR activation from its downstream signaling components and abrogate FGF-1-induced antiestrogen-resistant growth. Induction of SNT-1 PTB resulted in a significant decrease of FGF-1-dependent tyrosine phosphorylation of endogenous SNT-1, strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1, and reduced activation of Ras, mitogen-activated protein kinase (MAP kinase), and Akt. SNT-1 PTB also inhibited the phosphorylation of p70S6K on Thr421/Ser424 and Ser411, which may result from the abrogation of MAP kinase activity. Moreover, we also observed a decreased phosphorylation of the MAP kinase-independent site Thr389. This may reflect both inhibition of PI-3 kinase pathways and mammalian target of rapamycin (mTOR)-dependent signaling, as the phosphorylation of Thr389 site was sensitive to treatment with the PI3-K and mTOR inhibitors, LY294002 and rapamycin, respectively. Collectively these results suggest that SNT-1 plays a pivotal role in FGF-dependent activation of the Ras-MAP kinase, PI-3 kinase, and mTOR pathways in these cells. Fibroblast growth factor-1 dependent colony formation of ML20 cells in media containing the pure antiestrogen ICI 182,780 was also markedly inhibited upon induction of SNT-1 PTB, suggesting that blockade of FGFR-SNT-1 interactions might abrogate FGF-mediated antiestrogen resistance in breast cancers. PMID- 16682956 TI - Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors. AB - Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression. PMID- 16682957 TI - Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB. AB - Overexpression of cyclooxygenase-2 (Cox-2) is thought to exert antiapoptotic effects in cancer. Here we show that the tumor suppressor p53 upregulated Cox-2 in esophageal and colon cancer cell lines by inducing the binding of nuclear factor-kappaB (NF-kappaB) to its response element in the COX-2 promoter. Inhibition of NF-kappaB prevented p53 induction of Cox-2 expression. Cooperation between p53 and NF-kappaB was required for activation of COX-2 promoter in response to daunomycin, a DNA-damaging agent. Pharmacological inhibition of Cox-2 enhanced apoptosis in response to daunomycin, in particular in cells containing active p53. In esophageal cancer, there was a correlation between Cox-2 expression and wild-type TP53 in Barrett's esophagus (BE) and in adenocarcinoma, but not in squamous cell carcinoma (P<0.01). These results suggest that p53 and NF-kappaB cooperate in upregulating Cox-2 expression, promoting cell survival in inflammatory precursor lesions such as BE. PMID- 16682958 TI - Chronic UVA irradiation of human HaCaT keratinocytes induces malignant transformation associated with acquired apoptotic resistance. AB - Ultraviolet A (UVA, 315-400 nm), constituting about 95% of ultraviolet irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. It has proven difficult to show direct actions of UVA as a carcinogen in human cells. Here, we demonstrate that chronic UVA exposures at environmentally relevant doses in vitro can induce malignant transformation of human keratinocytes associated with acquired apoptotic resistance. As evidence of carcinogenic transformation, UVA long-treated (24 J/cm(2) once/week for 18 weeks) HaCaT (ULTH) cells showed increased secretion of matrix metalloproteinase (MMP-9), overexpression of keratin 13, altered morphology and anchorage-independent growth. Malignant transformation was established by the production of aggressive squamous cell carcinomas after inoculation of ULTH cells into nude mice (NC(r)-nu). ULTH cells were resistant to apoptosis induced not only by UVA but also by UVB and arsenite, two other human skin carcinogens. ULTH cells also became resistant to apoptosis induced by etoposide, staurosporine and doxorubicin hydrochloride. Elevated phosphorylation of protein kinase B (PKB, also called AKT) and reduced expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were detected in ULTH cells. The resistance of ULTH cells to UVA-induced apoptosis was reversed by either inhibition of phosphatidylinositol 3-kinase (PI-3K) or adenovirus expression of PTEN or dominant negative AKT. These data indicate that UVA has carcinogenic potential in human keratinocytes and that the increased AKT signaling and decreased PTEN expression may contribute to this malignant transformation. Further comparisons between the transformed ULTH and control cells should lead to a better understanding of the mechanism of UVA carcinogenesis and may help identify biomarkers for UVA-induced skin malignancies. PMID- 16682959 TI - Involvement of serine/threonine protein phosphatases sensitive to okadaic acid in restraint stress-induced hyperlocomotion in cocaine-sensitized mice. AB - We used okadaic acid (OA), a potent preferential inhibitor of PP2A and PP5 but not PP1 (PP subfamilies), to examine the involvement of serine/threonine protein phosphatase (PP) in behavioral sensitization stimulated by treatment with cocaine in mice. Repeated administration of cocaine (10 mg kg(-1)) once a day for five consecutive days produced a progressive increase in locomotor activity that was maintained after the cessation of cocaine treatment, as revealed by the fact that a challenge dose of cocaine given on day 7 of withdrawal reproduced an enhanced stimulant effect. On the seventh day of withdrawal, OA-sensitive PP activity and expression of PP2A and PP5, but not PP1gamma, were increased in whole-cell extract of the nucleus accumbens and the ventral tegmental area in cocaine sensitized mice, compared to saline-treated mice. Restraint stress increased locomotor activity in cocaine-sensitized mice on day 7 after drug administration was ceased. The locomotor activity was more susceptible to restraint-elicited enhancement in cocaine-sensitized mice than in saline-treated mice. The restraint induced hyperlocomotion was suppressed by a single intracerebroventricular injection of OA immediately before restraint in cocaine-sensitized mice, but this suppression did not occur in saline-treated mice. The membrane fraction of the whole brain in cocaine-sensitized mice showed that OA-sensitive activity levels rise after mice are subjected to restraint, and this is concomitant with an increase in expression levels of PP2A and PP5, but not PP1gamma. These results suggest that the upregulated OA-sensitive PPs are involved in stress-induced hyperlocomotion in cocaine-sensitized mice. There may be intracellular mechanisms mediating psychostimulant cross-sensitization to stress underlying the spontaneous recurrence of its psychosis. PMID- 16682960 TI - Intrathecal treatment with sigma1 receptor antagonists reduces formalin-induced phosphorylation of NMDA receptor subunit 1 and the second phase of formalin test in mice. AB - Although previous reports have suggested that the sigma 1 (sigma(1)) receptor may be involved in pain sensation, its specific site of action has not been elucidated. The aim of present study was to determine the role of the spinal sigma(1) receptor in formalin-induced pain behavior, spinal cord Fos expression and phosphorylation of N-methyl-D-aspartate receptor subunit 1 (pNR1). Intrathecal (i.t.) pretreatment with the selective sigma(1) receptor antagonist, BD-1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide) (10-100 nmol) dose dependently reduced formalin-induced pain behaviors in second phase, but not first phase, of the formalin test. I.t. injection of BD-1047 also reduced formalin-evoked Fos expression and pNR1 at the protein kinase C-dependent site, serine-896 (Ser896) and the protein kinase A dependent site, serine-897 (Ser897) in spinal dorsal horn.i.t. BMY-14802 ((alpha (4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazinebutanol hydrochloride) (10-100 nmol, sigma(1) receptor antagonist and 5-HT(1A) receptor agonist) dose dependently reduced formalin-induced pain behaviors in both phases. However, the 5-HT(1A) receptor might not be involved in the antinociceptive effect of BMY 14802 on the second phase, since i.t. pretreatment with the 5-HT(1A) receptor antagonist propranolol ((S)-1-isopropylamino-3-(1-naphthyloxy)-2-propanol hydrochloride) (injected 10 min prior to i.t. BMY-14802) partially blocked the effect of BMY-14802 on the first phase of the formalin test but did not affect the inhibitory effect of BMY-14802 on the second phase. In addition, i.t. BMY 14802 significantly reduced formalin-evoked Fos expression and pNR1 (Ser896 and Ser897) expression in spinal dorsal horn. The results of this study suggest that selective blockage of spinal sigma(1) receptors can reduce pain behaviors, spinal cord Fos expression and pNR1 (Ser896 and Ser897) expression associated with the second phase of the formalin test. PMID- 16682961 TI - Activation of D2-like receptors induces sympathetic climactic-like responses in male and female anaesthetised rats. AB - In anaesthetised male rats an intravenous (i.v.) injection of p-chloroamphetamine (PCA) produced a specific patterned bursting response in the sympathetic vas deferens nerve (VDN) that corresponds to ejaculation. In the present, study selective dopamine agonists and antagonists were used to investigate whether dopaminergic mechanisms influence the generation of this ejaculatory-related response. Administration of a mixed D(1/2) receptor agonist (0.1-1.0 mg kg(-1) apomorphine i.v.) also evoked the characteristic bursting pattern responses in the VDN. Similar, but fewer, burst pattern responses could also be evoked by a selective D(2/3) receptor agonist (0.1-2.0 mg kg(-1) piribedil). Responses to 1.0 mg kg(-1) apomorphine were blocked by pretreatments with either 0.5 mg kg(-1) remoxipride (D(2) receptor antagonist) or 0.5 mg kg(-1) nafadotride (D(3) receptor antagonist), suggesting that D(2)-like receptors were involved. Responses could not be evoked by i.v. injections of apomorphine (1.0 mg kg(-1)) in anaesthetised male rats with a midthoracic spinal section, indicating that activation of D(2)-like receptors at supraspinal sites leads to an increase in the excitability of the lumbosacral pattern generator for ejaculation. In anaesthetised female rats a similar patterned bursting response occurred in the uterine nerve (UN) in response to apomorphine (0.5-2.0 mg kg(-1) i.v.). Thus a common neural mechanism may regulate sexual climactic reflexes in both sexes. PMID- 16682962 TI - AT2 receptor-mediated vasodilation in the mouse heart depends on AT1A receptor activation. AB - Angiotensin (Ang) II type 2 (AT(2)) receptors are believed to counteract Ang II type 1 (AT(1)) receptor-mediated effects. Here, we investigated AT(2) receptor mediated effects on coronary and cardiac contractility in C57BL/6 mice. Hearts were perfused according to Langendorff. Baseline coronary flow (CF) and left ventricular systolic pressure (LVSP) were 2.7 +/- 0.1 ml min(-1) and 111 +/- 3 mmHg (n = 50), respectively. Ang II (n = 14) concentration dependently decreased CF and LVSP, by maximally 41 +/- 4 and 25 +/- 3%, respectively (pEC(50)s 7.41 +/- 0.12 and 7.65 +/- 0.12). The AT(1) receptor antagonist irbesartan (n = 4) abolished all Ang II-induced changes, whereas the AT(2) receptor antagonist PD123319 (n = 6) enhanced (P < 0.05) the effect of Ang II on CF (to 59 +/- 1%) and LVSP (to 44 +/- 2%), without altering its potency. A similar enhancement was observed in the presence of nitric oxide (NO) synthase inhibitor N(omega)-nitro-L arginine methyl ester HCl (L-NAME; n = 4). On top of L-NAME, PD123319 no longer affected the response to Ang II (n = 4). The AT(2) receptor agonist CGP42112A (n = 4) did not affect CF or LVSP, nor did CGP42112A (n = 4) alter the constrictor response to the alpha(1)-adrenoceptor agonist phenylephrine. Furthermore, Ang II exerted no effects in hearts of AT(1A)(-/-) mice (n = 5), whereas its effects in hearts of AT(1A)(+/+) wild-type control mice (n = 7) were indistinguishable from those in hearts of C57BL/6 mice. In conclusion, Ang II exerts opposite effects on coronary and cardiac contractility in the mouse heart via activation of AT(1A) and AT(2) receptors. AT(2) receptor-mediated effects depend on NO and occur only in conjunction with AT(1A) receptor activation. PMID- 16682963 TI - Loss of nitrergic neurotransmission to mouse corpus cavernosum in the absence of neurturin is accompanied by increased response to acetylcholine. AB - The neurotrophic factor, neurturin (NTN), plays an important role in parasympathetic neural development. In the penis, parasympathetic nitrergic/cholinergic nerves mediate the erectile response. However, despite reduced parasympathetic penile innervation in mice lacking the NTN receptor, glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)2, they are capable of erection and reproduction. Our aim was to assess neural regulation of erectile tissues from mice lacking NTN. Responses of cavernosal smooth muscle were studied in vitro, monitoring agonist- and nerve-evoked changes in tension. Frequency-dependent nerve-evoked relaxations in the presence of guanethidine were markedly reduced in the mutant mice compared to wild types (19 vs 72% of phenylephrine pre-contraction). Atropine reduced the amplitude in wild type mice to 61%, but abolished relaxations in knockout mice. In wild-type and knockout animals, nitric oxide synthase inhibition abolished neurogenic relaxations. In NTN knockout animals, EC(50) values for nitric oxide-dependent relaxations to acetylcholine and muscarine were increased approximately 0.5 log units. In contrast, contractions to electrical stimulation or phenylephrine, and relaxations to bradykinin or the nitric oxide donor, sodium nitroprusside, were unaltered. Immunohistochemistry confirmed that nerves immunoreactive for nitric oxide synthase, vesicular acetylcholine transporter and vasoactive intestinal polypeptide were substantially reduced in cavernosum of NTN knockout mice. Parallel immunohistochemical and pharmacological studies in GFRalpha2 knockout animals showed the same changes from their wild types as the NTN knockout animals. The data demonstrate that NTN is essential for normal development of penile erection-inducing nerves and that its absence leads to increased responsiveness to muscarinic agonists, possibly as a compensatory mechanism. PMID- 16682964 TI - mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis. AB - Several studies have described functional interactions between opioid and cannabinoid receptors; the underlying mechanism(s) have not been well explored. One possible mechanism is direct receptor-receptor interactions, as has been demonstrated for a number of G-protein-coupled receptors. In order to investigate interactions between opioid and cannabinoid receptors, we epitope tagged mu, delta and kappa opioid receptors with Renilla luciferase and CB1 cannabinoid or CCR5 chemokine receptors with yellow fluorescent protein and examined the extent of substrate hydrolysis induced bioluminescence resonance energy transfer (BRET) signal. We find that coexpression of opioid receptors with cannabinoid receptors, but not with chemokine receptors, leads to a significant increase in the level of BRET signal, suggesting that the opioid-cannabinoid interactions are receptor specific. In order to examine the implications of these interactions to signaling, we used GTPgammaS binding and mitogen-activated protein kinase (MAPK) phosphorylation assays and examined the effect of receptor activation on signaling. We find that the mu receptor-mediated signaling is attenuated by the CB1 receptor agonist; this effect is reciprocal and is seen in heterologous cells and endogenous tissue expressing both receptors. In order to explore the physiological consequences of this interaction, we examined the effect of receptor activation on the extent of Src and STAT3 phosphorylation and neuritogenesis in Neuro-2A cells. We find that the simultaneous activation of mu opioid and CB1 cannabinoid receptors leads to a significant attenuation of the response seen upon activation of individual receptors, implicating a role for receptor-receptor interactions in modulating neuritogenesis. PMID- 16682965 TI - Opioid and cannabinoid receptors: friends with benefits or just close friends? AB - mu-Opioid (MOP) and cannabinoid CB1 receptors mediate overlapping pharmacological responses in clinically important areas such as drug abuse and pain management, and functional interactions between agonists at these receptors have long been recognized. In the present issue of this Journal, Rios and co-workers have provided the first strong evidence that the two receptors interact directly when coexpressed in the same cells. The authors report a close physical association between MOP and CB1 receptors and novel pharmacological interactions of MOP and CB1 agonists. They argue that MOP/CB1 heterodimer formation explains these interactions. If correct, the direct interaction of MOP and CB1 pharmacophores in a quaternary complex would provide real benefits by opening the potential for development of novel MOP/CB1 small molecules or new strategies for use of current ligands. However, a lot more evidence will be required before the heterodimer interpretation can be accepted. If it turns out that MOP and CB1 receptors do not readily form hetero-oligomers, the study by Rios and co-workers shows that they are still friends but there may be few benefits. PMID- 16682966 TI - Endocannabinoids potently protect the newborn brain against AMPA-kainate receptor mediated excitotoxic damage. AB - Brain lesions induced in newborn mice or rats by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) or S-bromowillardiine (acting on AMPA-kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage associated with cerebral palsy. Exogenous and endogenous cannabinoids have received increasing attention as potential neuroprotective agents in a number of neurodegenerative disorders of the adult. One recent study showed neuroprotection by the cannabinoid agonist WIN-55212 in a newborn rat model of acute severe asphyxia. The present study was designed to assess the neuroprotective effects of the endogenous cannabinoid anandamide using a well-defined rodent model of neonatal excitotoxic brain lesions. In this model, anandamide provided dose-dependent and long-lasting protection of developing white matter and cortical plate reducing the size of lesions induced by S-bromowillardiine. Anandamide had only marginal neuroprotective effect against ibotenate-induced cortical grey matter lesions. Anandamide-induced neuroprotection against AMPA-kainate receptor-mediated brain lesions were blocked by a CB1 antagonist but not by a CB2 antagonist. Furthermore, anandamide effects were mimicked by a CB1 agonist but not by a CB2 agonist. Real-time PCR confirmed the expression of CB1 receptors, but not CB2 receptors, in the untreated newborn neocortex. Finally, neuroprotective effects of anandamide in white matter involved increased survival of preoligodendrocytes and better preservation of myelination. The present study provides experimental support for the role of endocannabinoids as a candidate therapy for excitotoxic perinatal brain lesions. PMID- 16682967 TI - Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats. AB - We have examined the relative contributions of small- and intermediate conductance Ca(2+)-activated K(+) channels (SK(Ca) and IK(Ca)) to the endothelium derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 microM indomethacin plus 100 microM N(omega)-nitro-L-arginine. SK(Ca) was selectively inhibited with 100 nM apamin, whereas IK(Ca) was blocked with 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17- to 20-week-old control and diabetic rats (approximately -54 mV). Responses elicited by 1 and 10 microM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to 69.5 +/- 0.8 mV (ZDF; n = 12) and -73.2 +/- 0.6 mV (Lean; n = 12; P < 0.05) evoked by 10 microM ACh). The IK(Ca)-mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately -65 mV on exposure to 10 microM ACh). However, SK(Ca)-mediated responses were significantly reduced in the diabetic group (hyperpolarizations to -63.1 +/- 1.0 mV (ZDF; n = 6) and -71.5 +/- 1.2 mV (Lean; n = 6; P < 0.05) on exposure to 10 microM ACh. Impaired ACh responses were not observed in arteries from 5- to 6-week-old (pre diabetic) animals. SK(Ca) subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SK(Ca)-mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SK(Ca) and IK(Ca) pathways is discussed. PMID- 16682968 TI - Correlation between in vitro and in vivo concentration-effect relationships of naproxen in rats and healthy volunteers. AB - Understanding the mechanisms underlying the analgesic effect of new cyclooxygenase inhibitors is essential to identify dosing requirements in early stages of drug development. Accurate extrapolation to humans of in vitro and in vivo findings in preclinical species is needed to optimise dosing regimen in inflammatory conditions. The current investigation characterises the inhibition of prostaglandin E2 (PGE(2)) and thromboxane B2 (TXB(2)) by naproxen in vitro and in vivo in rat and human blood. The inhibition of PGE(2) in the absence or presence of increasing concentrations of naproxen (10(-8)-10(-1) M) was measured by ex vivo whole blood stimulation with LPS, whereas inhibition of TXB(2) was measured in serum following blood clotting. In further experiments, inhibition of PGE(2) and TXB(2) levels was also assessed ex vivo in animals treated with naproxen (2.5, 10, 25 mg kg(-1)). Subsequently, pharmacokinetic (PK)/pharmacodynamics (PD) modelling of in vitro and in vivo data was performed using nonlinear mixed effects in NONMEM (V). Inhibition of PGE(2) and TXB(2) was characterised by a sigmoid E(max) model. The exposure-response relationships in vitro and in vivo were of the same order of magnitude in both species. IC(80) estimates obtained in vitro were similar for PGE(2) inhibition (130.8 +/- 11 and 131.9 +/- 19 10(-6) M, mean +/- s.d. for humans and rats, respectively), but slightly different for TXB(2) inhibition (103.9+/-15 and 151.4 +/- 40 10(-6) M, mean +/- s.d. for humans and rats, respectively, P < 0.05). These differences, however, may not be biologically relevant. The results confirm the value of exposure-effect relationships determined in vitro as a means to predict the pharmacological activity in vivo. This analysis also highlights the need to parameterise concentration-effect relationships in early drug development, as indicated by the estimates of IC(80) for PGE(2) and TXB(2) inhibition. PMID- 16682969 TI - A common variant associated with prostate cancer in European and African populations. AB - With the increasing incidence of prostate cancer, identifying common genetic variants that confer risk of the disease is important. Here we report such a variant on chromosome 8q24, a region initially identified through a study of Icelandic families. Allele -8 of the microsatellite DG8S737 was associated with prostate cancer in three case-control series of European ancestry from Iceland, Sweden and the US. The estimated odds ratio (OR) of the allele is 1.62 (P = 2.7 x 10(-11)). About 19% of affected men and 13% of the general population carry at least one copy, yielding a population attributable risk (PAR) of approximately 8%. The association was also replicated in an African American case-control group with a similar OR, in which 41% of affected individuals and 30% of the population are carriers. This leads to a greater estimated PAR (16%) that may contribute to higher incidence of prostate cancer in African American men than in men of European ancestry. PMID- 16682970 TI - Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. AB - Joubert syndrome-related disorders (JSRD) are a group of syndromes sharing the neuroradiological features of cerebellar vermis hypoplasia and a peculiar brainstem malformation known as the 'molar tooth sign'. We identified mutations in the CEP290 gene in five families with variable neurological, retinal and renal manifestations. CEP290 expression was detected mostly in proliferating cerebellar granule neuron populations and showed centrosome and ciliary localization, linking JSRDs to other human ciliopathies. PMID- 16682971 TI - Positional cloning of Sorcs1, a type 2 diabetes quantitative trait locus. AB - We previously mapped the type 2 diabetes mellitus-2 locus (T2dm2), which affects fasting insulin levels, to distal chromosome 19 in a leptin-deficient obese F2 intercross derived from C57BL/6 (B6) and BTBR T+ tf/J (BTBR) mice. Introgression of a 7-Mb segment of the B6 chromosome 19 into the BTBR background (strain 1339A) replicated the reduced insulin linked to T2dm2. The 1339A mice have markedly impaired insulin secretion in vivo and disrupted islet morphology. We used subcongenic strains derived from 1339A to localize the T2dm2 quantitative trait locus (QTL) to a 242-kb segment comprising the promoter, first exon and most of the first intron of the Sorcs1 gene. This was the only gene in the 1339A strain for which we detected amino acid substitutions and expression level differences between mice carrying B6 and BTBR alleles of this insert, thereby identifying variation within the Sorcs1 gene as underlying the phenotype associated with the T2dm2 locus. SorCS1 binds platelet-derived growth factor, a growth factor crucial for pericyte recruitment to the microvasculature, and may thus have a role in expanding or maintaining the islet vasculature. Our identification of the Sorcs1 gene provides insight into the pathway underlying the pathophysiology of obesity induced type 2 diabetes mellitus. PMID- 16682972 TI - Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1. AB - Vernalization is the process by which sensing a prolonged exposure to winter cold leads to competence to flower in the spring. In winter annual Arabidopsis thaliana accessions, flowering is suppressed in the fall by expression of the potent floral repressor FLOWERING LOCUS C (FLC). Vernalization promotes flowering via epigenetic repression of FLC. Repression is accompanied by a series of histone modifications of FLC chromatin that include dimethylation of histone H3 at Lys9 (H3K9) and Lys27 (H3K27). Here, we report that A. thaliana LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) is necessary to maintain the epigenetically repressed state of FLC upon return to warm conditions typical of spring. LHP1 is enriched at FLC chromatin after prolonged exposure to cold, and LHP1 activity is needed to maintain the increased levels of H3K9 dimethylation at FLC chromatin that are characteristic of the vernalized state. PMID- 16682973 TI - The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. AB - The molecular basis of nephronophthisis, the most frequent genetic cause of renal failure in children and young adults, and its association with retinal degeneration and cerebellar vermis aplasia in Joubert syndrome are poorly understood. Using positional cloning, we here identify mutations in the gene CEP290 as causing nephronophthisis. It encodes a protein with several domains also present in CENPF, a protein involved in chromosome segregation. CEP290 (also known as NPHP6) interacts with and modulates the activity of ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation. NPHP6 is found at centrosomes and in the nucleus of renal epithelial cells in a cell cycle dependent manner and in connecting cilia of photoreceptors. Abrogation of its function in zebrafish recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome. Our findings help establish the link between centrosome function, tissue architecture and transcriptional control in the pathogenesis of cystic kidney disease, retinal degeneration, and central nervous system development. PMID- 16682974 TI - Function-blocking autoantibodies to the melanin-concentrating hormone receptor in vitiligo patients. AB - Vitiligo is a common depigmenting skin disorder resulting from the loss of melanocytes in the cutaneous epidermis. Although the cause of the disease remains obscure, autoimmune mechanisms are thought to be involved. Recently, melanin concentrating hormone receptor (MCHR)-binding autoantibodies have been identified in vitiligo patients. In the present study, we aimed to determine if MCHR autoantibodies could also affect receptor function either by direct activation or by blocking its response to melanin-concentrating hormone. The results indicated that 10/18 (56%) vitiligo patient IgG samples inhibited the function of MCHR expressed in a Chinese hamster ovary cell line. In contrast, neither control (n=20) nor SLE patient (n=10) IgG samples blocked receptor function. Compared with healthy controls, MCHR function-blocking autoantibodies were found at a significantly increased frequency in the vitiligo patient group (P=0.0004). No MCHR-activating autoantibodies were detected in any of the vitiligo patient, SLE patient or control IgG samples that were analysed. In addition, vitiligo patient IgGs were tested for MCHR autoantibodies that could mediate antibody-dependent cell-mediated cytotoxicity via the receptor. However, this could only be demonstrated in two vitiligo patient sera. Overall, this work has provided additional evidence that MCHR is a B-cell autoantigen in vitiligo and has demonstrated the existence of MCHR function-blocking autoantibodies further to the receptor-binding autoantibodies previously reported. PMID- 16682975 TI - The antifibrogenic effect of (-)-epigallocatechin gallate results from the induction of de novo synthesis of glutathione in passaged rat hepatic stellate cells. AB - Hepatic stellate cells (HSC) are the major players during hepatic fibrogenesis. Overproduction of extracellular matrix (ECM) is a characteristic of activated HSC. Transforming growth factor-beta (TGF-beta) is the most potent fibrogenic cytokine while connective tissue growth factor (CTGF) mediates the production of TGF-beta-induced ECM in activated HSC. HSC activation and hepatic fibrogenesis are stimulated by oxidative stress. Glutathione (GSH) is the most important intracellular antioxidant. The aim of this study is to explore the mechanisms of (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, in the inhibition of ECM gene expression in activated HSC. It is hypothesized that EGCG inhibits ECM gene expression in activated HSC by interrupting TGF-beta signaling through attenuating oxidative stress. It is found that EGCG interrupts TGF-beta signaling in activated HSC by suppressing gene expression of type I and II TGF-beta receptors. EGCG inhibits CTGF gene expression, leading to the reduction in the abundance of ECM, including alphaI(I) procollagen. Exogenous CTGF dose dependently eliminates the antifibrogenic effect. EGCG attenuates oxidative stress in passaged HSC by scavenging reactive oxygen species and reducing lipid peroxidation. De novo synthesis of GSH is a prerequisite for EGCG to interrupt TGF-beta signaling and to reduce the abundance of alphaI(I) procollagen in activated HSC in vitro. Taken together, our results demonstrate that the interruption of TGF-beta signaling by EGCG results in the suppression of gene expression of CTGF and ECM in activated HSC in vitro. In addition, our results, for the first time, demonstrate that the antioxidant property of EGCG derived from de novo synthesis of intracellular GSH plays a critical role in its antifibrogenic effect. These results provide novel insights into the mechanisms of EGCG as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. PMID- 16682976 TI - The relationship between body mass index and health-related quality of life: comparing the EQ-5D, EuroQol VAS and SF-6D. AB - BACKGROUND: No other studies have compared the relationship between body mass index (BMI) and health-related quality of life (HRQL) on more than one utility measure. Estimating the HRQL effects of obesity on a (common) utility scale enables the relative cost-effectiveness of interventions designed to alleviate obesity to be estimated. OBJECTIVE: To examine the relationship between BMI and HRQL according to the EQ-5D, EuroQol visual analogue scale (EQ-VAS) and SF-6D. METHODS: Patients aged >/=45 years at one UK general practice were asked to complete the EQ-5D, EQ-VAS, SF-36 questionnaire (used to derive the SF-6D), and information on their characteristics and co-morbidity. Body mass index was categorized according to the World Health Organization (WHO) recommendations. Regression analysis was used to compare the HRQL of normal BMI patients to the HRQL of patients in other BMI categories, while controlling for patient characteristics and co-morbidity. RESULTS: A total of 1865 patients responded (67%), mean BMI 26.0 kg/m(2), 16% obese (BMI>/=30). Patients with back pain, hip pain, knee pain, asthma, diabetes or osteoarthritis were also significantly more likely to be obese. After controlling for other factors, compared to normal BMI patients, obese patients had a lower HRQL according to the EQ-5D (P<0.01), EQ-VAS (P<0.001) and SF-6D (P<0.001). Pre-obese patients were not estimated to have a significantly lower HRQL, and underweight patients were only estimated to have a significantly lower HRQL according to the SF-6D. These results arose because, on the EQ-5D, obese patients were found to have significantly more problems with mobility and pain, compared to physical functioning, social functioning and role limitations on the SF-6D. Whereas, according to the SF-6D, underweight patients had significantly more problems on the dimension of role limitation. CONCLUSION: The EQ-5D, EQ-VAS and SF-6D were in agreement that, relative to a normal BMI, obesity is associated with a lower HRQL, even after controlling for patient characteristics and co-morbidity. These three measures are thereby sensitive to the HRQL effects of obesity and can be used to estimate the cost-effectiveness of interventions designed to alleviate obesity. PMID- 16682977 TI - Altered insulin-mediated and insulin-like growth factor-1-mediated vasorelaxation in aortas of obese Zucker rats. AB - OBJECTIVE: Insulin and insulin-like growth factor-1 (IGF-1) have vasorelaxant effects in vivo, which is dependent on nitric oxide (NO) production. The aim of this study was to investigate the vasorelaxant responses mediated by insulin and/or IGF-1 in aortas of obese Zucker rats. METHODS: The thoracic aortas of eight lean and eight obese Zucker rats (6 months old) were isolated for vasorelaxation analysis. Insulin-induced and IGF-1-induced vasorelaxant responses were evaluated by the isometric tension of aortic rings in the organ bathes. The roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS) in vasorelaxant responses were examined by treating selective inhibitors, such as wortmannin (an inhibitor of PI3K) and N (omega)-nitro-L-arginine methyl ester (L NAME, a NOS inhibitor). In addition, the vascular responses to sodium nitroprusside (SNP), a direct vasodilator of vascular smooth muscle, were examined. RESULTS: The insulin-induced vasorelaxation in aortas of obese rats was significantly decreased, whereas the IGF-1-induced vasorelaxation was significantly increased, compared with that in lean rats. After the pre administration of wortmannin or L-NAME, the altered insulin-induced or IGF-1 induced vasorelaxation was abolished. There was no significant difference in the SNP-induced vasorelaxation between lean and obese rats. CONCLUSION: Our findings suggested that the decreased insulin-mediated vasorelaxation in obese rats appeared to be counteracted by the increased IGF-1-mediated vasorelaxation. Furthermore, the NO-dependent pathway was involved in the altered vasorelaxant responses. However, the SNP-induced vasorelaxation was not changed in obese rats. PMID- 16682978 TI - Reducing weight increases postural stability in obese and morbid obese men. AB - OBJECTIVE: To investigate the effect of weight loss on balance control in obese and morbid obese men. METHODS: In a longitudinal and clinical intervention study, postural stability was measured with a force platform before and after weight loss in men. Weight loss was obtained in obese men (mean body mass index (BMI)=33.0 kg/m(2)) by hypocaloric diet until resistance and in morbid obese men (mean BMI=50.5 kg/m(2)) by bariatric surgery. Morbid obese men were tested before surgery, and 3 and 12 months after surgery when they had lost 20 and nearly 50% of initial body weight, respectively. Normal weight individuals (mean BMI=22.7 kg/m(2)) were tested twice within a 6- to 12-month period to serve as control. Body fatness and fat distribution measures, and posturographic parameters of the center of foot pressure (CP) along the antero-posterior and medio-lateral axes for conditions with and without vision were performed in all subjects. RESULTS: Weight loss averaged 12.3 kg after dieting and 71.3 kg after surgery. Body weight remained unchanged in the control group. After weight loss, nearly all measures of postural stability were improved with and without vision (i.e., CP speed and range in antero-posterior and medio-lateral axes). A strong linear relationship was observed between weight loss and improvement in balance control measured from CP speed (adjusted R (2)=0.65, P<0.001). CONCLUSION: Weight loss improves balance control in obese men and the extent of the improvement is directly related to the amount of weight loss. This should decrease the habitual greater risk of falling observed in obese individuals. PMID- 16682979 TI - Differential association of C-reactive protein with adiposity in men and women in an Aboriginal community in northeast Arnhem Land of Australia. AB - OBJECTIVE: To examine the relationship between C-reactive protein (CRP), adiposity and other metabolic abnormalities in an Aboriginal community in Northern Australia. DESIGN: Cross-sectional analysis of data obtained between 2001 and 2003 from 379 Aboriginal people residing in a geographically isolated community. RESULTS: Mean (95% CI) CRP in women and men was 4.06 cholesterol (3.53, 4.66) mg/l and 3.42 (2.94, 3.97) mg/l, respectively (P=NS). The prevalence of the metabolic syndrome (US National Cholesterol Education [corrected] Program (NCEP) definition) was significantly higher for women than men (41 vs 18%, chi (2)=20.94, P<0.001). C-reactive protein correlated strongly with adiposity in women (waist circumference, waist to hip ratio and body mass index; r>/=0.514, P<0.01) but much less strongly in men (r25% decrease in total NIH-CPSI compared with 78.9% (15 of 19) of group 2 (P=0.041). Also, more patients in group 2 (78.9%) were rated as responders with a 6-point decrease in NIH-CPSI compared with group 1 (47.6%) (P=0.041). The early results suggest that ExMI combined with alpha-blocker therapy has better effect than alpha-blocker monotherapy for the treatment of CP/CPPS. PMID- 16683010 TI - Histopathological findings after radiofrequency (RITA) treatment for prostate cancer. AB - Radiofrequency interstitial tumor ablation (RITA) is a thermal ablation method that uses needles and low radiofrequency (RF) energy. The aim of our study was to evaluate the histopathology of thermal lesions induced by RF energy delivered interstitially in prostate cancer patients who subsequently underwent prostatectomy, and to determine the feasibility, effectiveness and safety of this new method in a pilot study. PMID- 16683009 TI - Fatty acid oxidation is a dominant bioenergetic pathway in prostate cancer. AB - Most malignancies have increased glycolysis for energy requirement of rapid cell proliferation, which is the basis for tumor imaging through glucose analog FDG (2 deoxy-2-fluoro-D-glucose) with positron emission tomography. One of significant characteristics of prostate cancer is slow glycolysis and low FDG avidity. Recent studies showed that prostate cancer is associated with changes of fatty acid metabolism. Several enzymes involved in the metabolism of fatty acids have been determined to be altered in prostate cancer relative to normal prostate, which is indicative of an enhanced beta-oxidation pathway in prostate cancer. Increased fatty acid utilization in prostate cancer provides both ATP and acetyl-coenzyme A (CoA); subsequently, increased availability of acetyl-CoA makes acceleration of citrate oxidation possible, which is an important energy source as well. Dominant fatty acid metabolism rather than glycolysis has the potential to be the basis for imaging diagnosis and targeted treatment of prostate cancer. PMID- 16683011 TI - Prostate cancer incidence varies among males from different Y-chromosome lineages. AB - The incidence rate of prostate cancer in African-American males is two times higher than Caucasian men and ten times higher than Japanese men. The geographical specificity of Y haplogroups implies that males from different ethnic groups undoubtedly have various Y lineages with different Y-chromosomal characteristics that may affect their susceptibility or resistance to such a male specific cancer. To confirm this hypothesis we studied the Y-chromosomal haplogroups of 92 Japanese prostate cancer patients comparing them with randomly selected 109 unrelated healthy Japanese male controls who were confirmed to be residents of the same geographical area. Males could be classified using three binary Y-chromosome markers (sex-determining region Y (SRY), YAP, 47z) into four haplogroups DE, O2b(*), O2b1, and untagged group. Our results confirmed that prostate cancer incidence varies among males from different Y-chromosome lineages. Males from DE and the untagged haplogroups are at a significantly higher risk to develop prostate cancer than O2b(*) and O2b1 haplogroups (P=0.01), odds ratio 2.17 and 95% confidence interval (1.16-4.07). Males from haplogroup DE are over-represented in the patient group showing a percentage of 41.3%. The underlying possible causes of susceptibility variations of different Y lineages for such a male-specific cancer tumorigenesis are discussed. These findings explain the lower incidence of prostate cancer in Japanese and other South East Asian males than other populations. To our knowledge, this is the first reliable study examining the association between prostate cancer and Y-chromosomal haplogroups, comparing prostate cancer patients with carefully selected matched controls. PMID- 16683012 TI - De novo calcification of liver and nodal metastases in prostate carcinoma. AB - Prostate cancer has a distinctly recognized pattern of metastases: multifocal and osteoblastic lesions involving the axial skeleton and non-calcified lymph nodes in the pelvic and lumbar aortic groups. Most adenocarcinomas are capable of producing macrocalcification. We report a case of prostate cancer with de novo calcified metastases to the liver and retroperitoneal lymph nodes mimicking the pattern usually seen in mucin-producing adenocarcinomas arising from the gastrointestinal tract. To our knowledge, this is the first such case to be reported in the literature. We propose a multifactorial mechanism that supports dystrophic calcification in this case. The knowledge of atypical presentation of metastatic disease can prevent diagnostic delay and prompt initiation of therapy. PMID- 16683013 TI - Permanent prostate brachytherapy: a century of technical evolution. AB - PURPOSE: To summarise the practical aspects of the development of techniques of interstitial permanent prostate brachytherapy (PPB) implantation. Prostate brachytherapy dates back to Pasteau's publication in 1913 describing the insertion of a radium capsule into the prostatic urethra to treat carcinoma of the prostate. Various implantation methods were employed but with unsatisfactory results until the development of the transrectal ultrasound in the 1980s. The subsequent two-stage Seattle technique allowed for a planned homogenous distribution of radioactive sources throughout the gland resulting in biochemical control comparable to surgical and external beam radiotherapy series. With the advent of advanced computer software and improved imaging, the technique has developed accordingly to a single stage procedure with on-table dosimetric assessment. The principles of targeting dose to the prostate while avoiding surrounding organs at risk remain as relevant today as nearly a century ago. There is an array of techniques to consider for the novice PPB provider. Whether the evolution of PPB techniques will translate into improved biochemical control is yet to be seen. PMID- 16683014 TI - Evidence of a novel biomarker, alphas1-Casein, a milk protein, in benign prostate hyperplasia. AB - Benign prostate hyperplasia (BPH) is a common disease in elderly men. Although it is a non-malignant disease, it has a significant detrimental impact on the quality of life in patients with late-stage disease. Owing to the lack of specific markers, diagnosis of early-stage BPH has been proven unsuccessful. Recently, using two-dimensional electrophoresis, we identified a group of prostatic secretory proteins that are specifically produced by BPH cells (Xu et al., Electrophoresis 2003; 24: 1311). In this study, we investigated the potential diagnostic value of one of the secretory proteins, alphas1-Casein, in BPH by inmmunohistological staining of normal, BPH and prostate cancer tissues. We found that 90% (20 out of 22) of BPH tissues showed moderate to strong alphas1 Casein protein expression whereas none of the normal tissues (0 out of 10) and less than 10% of the prostate cancer tissues (3 out of 30) showed similar staining intensity. Our results suggest that alphas1-Casein may be a potential biomarker for early identification of BPH patients. PMID- 16683015 TI - SAGE2Splice: unmapped SAGE tags reveal novel splice junctions. AB - Serial analysis of gene expression (SAGE) not only is a method for profiling the global expression of genes, but also offers the opportunity for the discovery of novel transcripts. SAGE tags are mapped to known transcripts to determine the gene of origin. Tags that map neither to a known transcript nor to the genome were hypothesized to span a splice junction, for which the exon combination or exon(s) are unknown. To test this hypothesis, we have developed an algorithm, SAGE2Splice, to efficiently map SAGE tags to potential splice junctions in a genome. The algorithm consists of three search levels. A scoring scheme was designed based on position weight matrices to assess the quality of candidates. Using optimized parameters for SAGE2Splice analysis and two sets of SAGE data, candidate junctions were discovered for 5%-6% of unmapped tags. Candidates were classified into three categories, reflecting the previous annotations of the putative splice junctions. Analysis of predicted tags extracted from EST sequences demonstrated that candidate junctions having the splice junction located closer to the center of the tags are more reliable. Nine of these 12 candidates were validated by RT-PCR and sequencing, and among these, four revealed previously uncharacterized exons. Thus, SAGE2Splice provides a new functionality for the identification of novel transcripts and exons. SAGE2Splice is available online at http://www.cisreg.ca. PMID- 16683016 TI - The signal in the genomes. PMID- 16683017 TI - Practical strategies for discovering regulatory DNA sequence motifs. PMID- 16683018 TI - Codon usage domains over bacterial chromosomes. AB - The geography of codon bias distributions over prokaryotic genomes and its impact upon chromosomal organization are analyzed. To this aim, we introduce a clustering method based on information theory, specifically designed to cluster genes according to their codon usage and apply it to the coding sequences of Escherichia coli and Bacillus subtilis. One of the clusters identified in each of the organisms is found to be related to expression levels, as expected, but other groups feature an over-representation of genes belonging to different functional groups, namely horizontally transferred genes, motility, and intermediary metabolism. Furthermore, we show that genes with a similar bias tend to be close to each other on the chromosome and organized in coherent domains, more extended than operons, demonstrating a role of translation in structuring bacterial chromosomes. It is argued that a sizeable contribution to this effect comes from the dynamical compartimentalization induced by the recycling of tRNAs, leading to gene expression rates dependent on their genomic and expression context. PMID- 16683019 TI - Positive selection, relaxation, and acceleration in the evolution of the human and chimp genome. AB - For years evolutionary biologists have been interested in searching for the genetic bases underlying humanness. Recent efforts at a large or a complete genomic scale have been conducted to search for positively selected genes in human and in chimp. However, recently developed methods allowing for a more sensitive and controlled approach in the detection of positive selection can be employed. Here, using 13,198 genes, we have deduced the sets of genes involved in rate acceleration, positive selection, and relaxation of selective constraints in human, in chimp, and in their ancestral lineage since the divergence from murids. Significant deviations from the strict molecular clock were observed in 469 human and in 651 chimp genes. The more stringent branch-site test of positive selection detected 108 human and 577 chimp positively selected genes. An important proportion of the positively selected genes did not show a significant acceleration in rates, and similarly, many of the accelerated genes did not show significant signals of positive selection. Functional differentiation of genes under rate acceleration, positive selection, and relaxation was not statistically significant between human and chimp with the exception of terms related to G protein coupled receptors and sensory perception. Both of these were over represented under relaxation in human in relation to chimp. Comparing differences between derived and ancestral lineages, a more conspicuous change in trends seems to have favored positive selection in the human lineage. Since most of the positively selected genes are different under the same functional categories between these species, we suggest that the individual roles of the alternative positively selected genes may be an important factor underlying biological differences between these species. PMID- 16683020 TI - Changing cellular location of CheZ predicted by molecular simulations. AB - In the chemotaxis pathway of the bacterium Escherichia coli, signals are carried from a cluster of receptors to the flagellar motors by the diffusion of the protein CheY-phosphate (CheYp) through the cytoplasm. A second protein, CheZ, which promotes dephosphorylation of CheYp, partially colocalizes with receptors in the plasma membrane. CheZ is normally dimeric in solution but has been suggested to associate into highly active oligomers in the presence of CheYp. A model is presented here and supported by Brownian dynamics simulations, which accounts for these and other experimental data: A minority component of the receptor cluster (dimers of CheA(short)) nucleates CheZ oligomerization and CheZ molecules move from the cytoplasm to a bound state at the receptor cluster depending on the current level of cellular stimulation. The corresponding simulations suggest that dynamic CheZ localization will sharpen cellular responses to chemoeffectors, increase the range of detectable ligand concentrations, and make adaptation more precise and robust. The localization and activation of CheZ constitute a negative feedback loop that provides a second tier of adaptation to the system. Subtle adjustments of this kind are likely to be found in many other signaling pathways. PMID- 16683021 TI - Designing a nanotube using naturally occurring protein building blocks. AB - Here our goal is to carry out nanotube design using naturally occurring protein building blocks. Inspection of the protein structural database reveals the richness of the conformations of proteins, their parts, and their chemistry. Given target functional protein nanotube geometry, our strategy involves scanning a library of candidate building blocks, combinatorially assembling them into the shape and testing its stability. Since self-assembly takes place on time scales not affordable for computations, here we propose a strategy for the very first step in protein nanotube design: we map the candidate building blocks onto a planar sheet and wrap the sheet around a cylinder with the target dimensions. We provide examples of three nanotubes, two peptide and one protein, in atomistic model detail for which there are experimental data. The nanotube models can be used to verify a nanostructure observed by low-resolution experiments, and to study the mechanism of tube formation. PMID- 16683022 TI - Pseudo-messenger RNA: phantoms of the transcriptome. AB - The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo-messenger RNA to be RNA molecules that resemble protein-coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo-messenger RNAs (approximately half of which are transposon-associated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein-coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense-mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non-standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level. PMID- 16683023 TI - SPA: a probabilistic algorithm for spliced alignment. AB - Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5' and 3' ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice site that we also find in the mouse dataset. The SPA software package is available at http://www.biozentrum.unibas.ch/personal/nimwegen/cgi-bin/spa.cgi. PMID- 16683024 TI - Distinguishing protein-coding from non-coding RNAs through support vector machines. AB - RIKEN's FANTOM project has revealed many previously unknown coding sequences, as well as an unexpected degree of variation in transcripts resulting from alternative promoter usage and splicing. Ever more transcripts that do not code for proteins have been identified by transcriptome studies, in general. Increasing evidence points to the important cellular roles of such non-coding RNAs (ncRNAs). The distinction of protein-coding RNA transcripts from ncRNA transcripts is therefore an important problem in understanding the transcriptome and carrying out its annotation. Very few in silico methods have specifically addressed this problem. Here, we introduce CONC (for "coding or non-coding"), a novel method based on support vector machines that classifies transcripts according to features they would have if they were coding for proteins. These features include peptide length, amino acid composition, predicted secondary structure content, predicted percentage of exposed residues, compositional entropy, number of homologs from database searches, and alignment entropy. Nucleotide frequencies are also incorporated into the method. Confirmed coding cDNAs for eukaryotic proteins from the Swiss-Prot database constituted the set of true positives, ncRNAs from RNAdb and NONCODE the true negatives. Ten-fold cross validation suggested that CONC distinguished coding RNAs from ncRNAs at about 97% specificity and 98% sensitivity. Applied to 102,801 mouse cDNAs from the FANTOM3 dataset, our method reliably identified over 14,000 ncRNAs and estimated the total number of ncRNAs to be about 28,000. PMID- 16683025 TI - Heterotachy in mammalian promoter evolution. AB - We have surveyed the evolutionary trends of mammalian promoters and upstream sequences, utilising large sets of experimentally supported transcription start sites (TSSs). With 30,969 well-defined TSSs from mouse and 26,341 from human, there are sufficient numbers to draw statistically meaningful conclusions and to consider differences between promoter types. Unlike previous smaller studies, we have considered the effects of insertions, deletions, and transposable elements as well as nucleotide substitutions. The rate of promoter evolution relative to that of control sequences has not been consistent between lineages nor within lineages over time. The most pronounced manifestation of this heterotachy is the increased rate of evolution in primate promoters. This increase is seen across different classes of mutation, including substitutions and micro-indel events. We investigated the relationship between promoter and coding sequence selective constraint and suggest that they are generally uncorrelated. This analysis also identified a small number of mouse promoters associated with the immune response that are under positive selection in rodents. We demonstrate significant differences in divergence between functional promoter categories and identify a category of promoters, not associated with conventional protein-coding genes, that has the highest rates of divergence across mammals. We find that evolutionary rates vary both on a fine scale within mammalian promoters and also between different functional classes of promoters. The discovery of heterotachy in promoter evolution, in particular the accelerated evolution of primate promoters, has important implications for our understanding of human evolution and for strategies to detect primate-specific regulatory elements. PMID- 16683026 TI - Clusters of internally primed transcripts reveal novel long noncoding RNAs. AB - Non-protein-coding RNAs (ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22 to 25-nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases (Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full-length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine-rich regions within longer parental transcripts. We therefore conducted a genome-wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty-six regions were identified, each of which mapped outside known protein-coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT-PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air. PMID- 16683027 TI - A method for similarity search of genomic positional expression using CAGE. AB - With the advancement of genome research, it is becoming clear that genes are not distributed on the genome in random order. Clusters of genes distributed at localized genome positions have been reported in several eukaryotes. Various correlations have been observed between the expressions of genes in adjacent or nearby positions along the chromosomes depending on tissue type and developmental stage. Moreover, in several cases, their transcripts, which control epigenetic transcription via processes such as transcriptional interference and genomic imprinting, occur in clusters. It is reasonable that genomic regions that have similar mechanisms show similar expression patterns and that the characteristics of expression in the same genomic regions differ depending on tissue type and developmental stage. In this study, we analyzed gene expression patterns using the cap analysis gene expression (CAGE) method for exploring systematic views of the mouse transcriptome. Counting the number of mapped CAGE tags for fixed-length regions allowed us to determine genomic expression levels. These expression levels were normalized, quantified, and converted into four types of descriptors, allowing the expression patterns along the genome to be represented by character strings. We analyzed them using dynamic programming in the same manner as for sequence analysis. We have developed a novel algorithm that provides a novel view of the genome from the perspective of genomic positional expression. In a similarity search of expression patterns across chromosomes and tissues, we found regions that had clusters of genes that showed expression patterns similar to each other depending on tissue type. Our results suggest the possibility that the regions that have sense-antisense transcription show similar expression patterns between forward and reverse strands. PMID- 16683028 TI - A simple physical model predicts small exon length variations. AB - One of the most common splice variations are small exon length variations caused by the use of alternative donor or acceptor splice sites that are in very close proximity on the pre-mRNA. Among these, three-nucleotide variations at so-called NAGNAG tandem acceptor sites have recently attracted considerable attention, and it has been suggested that these variations are regulated and serve to fine-tune protein forms by the addition or removal of a single amino acid. In this paper we first show that in-frame exon length variations are generally overrepresented and that this overrepresentation can be quantitatively explained by the effect of nonsense-mediated decay. Our analysis allows us to estimate that about 50% of frame-shifted coding transcripts are targeted by nonsense-mediated decay. Second, we show that a simple physical model that assumes that the splicing machinery stochastically binds to nearby splice sites in proportion to the affinities of the sites correctly predicts the relative abundances of different small length variations at both boundaries. Finally, using the same simple physical model, we show that for NAGNAG sites, the difference in affinities of the neighboring sites for the splicing machinery accurately predicts whether splicing will occur only at the first site, splicing will occur only at the second site, or three nucleotide splice variants are likely to occur. Our analysis thus suggests that small exon length variations are the result of stochastic binding of the spliceosome at neighboring splice sites. Small exon length variations occur when there are nearby alternative splice sites that have similar affinity for the splicing machinery. PMID- 16683029 TI - Differential use of signal peptides and membrane domains is a common occurrence in the protein output of transcriptional units. AB - Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome. PMID- 16683030 TI - Complex Loci in human and mouse genomes. AB - Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes. PMID- 16683031 TI - The abundance of short proteins in the mammalian proteome. AB - Short proteins play key roles in cell signalling and other processes, but their abundance in the mammalian proteome is unknown. Current catalogues of mammalian proteins exhibit an artefactual discontinuity at a length of 100 aa, so that protein abundance peaks just above this length and falls off sharply below it. To clarify the abundance of short proteins, we identify proteins in the FANTOM collection of mouse cDNAs by analysing synonymous and non-synonymous substitutions with the computer program CRITICA. This analysis confirms that there is no real discontinuity at length 100. Roughly 10% of mouse proteins are shorter than 100 aa, although the majority of these are variants of proteins longer than 100 aa. We identify many novel short proteins, including a "dark matter" subset containing ones that lack detectable homology to other known proteins. Translation assays confirm that some of these novel proteins can be translated and localised to the secretory pathway. PMID- 16683032 TI - Mice and men: their promoter properties. AB - Using the two largest collections of Mus musculus and Homo sapiens transcription start sites (TSSs) determined based on CAGE tags, ditags, full-length cDNAs, and other transcript data, we describe the compositional landscape surrounding TSSs with the aim of gaining better insight into the properties of mammalian promoters. We classified TSSs into four types based on compositional properties of regions immediately surrounding them. These properties highlighted distinctive features in the extended core promoters that helped us delineate boundaries of the transcription initiation domain space for both species. The TSS types were analyzed for associations with initiating dinucleotides, CpG islands, TATA boxes, and an extensive collection of statistically significant cis-elements in mouse and human. We found that different TSS types show preferences for different sets of initiating dinucleotides and cis-elements. Through Gene Ontology and eVOC categories and tissue expression libraries we linked TSS characteristics to expression. Moreover, we show a link of TSS characteristics to very specific genomic organization in an example of immune-response-related genes (GO:0006955). Our results shed light on the global properties of the two transcriptomes not revealed before and therefore provide the framework for better understanding of the transcriptional mechanisms in the two species, as well as a framework for development of new and more efficient promoter- and gene-finding tools. PMID- 16683033 TI - Multiple mechanisms promote the retained expression of gene duplicates in the tetraploid frog Xenopus laevis. AB - Gene duplication provides a window of opportunity for biological variants to persist under the protection of a co-expressed copy with similar or redundant function. Duplication catalyzes innovation (neofunctionalization), subfunction degeneration (subfunctionalization), and genetic buffering (redundancy), and the genetic survival of each paralog is triggered by mechanisms that add, compromise, or do not alter protein function. We tested the applicability of three types of mechanisms for promoting the retained expression of duplicated genes in 290 expressed paralogs of the tetraploid clawed frog, Xenopus laevis. Tests were based on explicit expectations concerning the ka/ks ratio, and the number and location of nonsynonymous substitutions after duplication. Functional constraints on the majority of paralogs are not significantly different from a singleton ortholog. However, we recover strong support that some of them have an asymmetric rate of nonsynonymous substitution: 6% match predictions of the neofunctionalization hypothesis in that (1) each paralog accumulated nonsynonymous substitutions at a significantly different rate and (2) the one that evolves faster has a higher ka/ks ratio than the other paralog and than a singleton ortholog. Fewer paralogs (3%) exhibit a complementary pattern of substitution at the protein level that is predicted by enhancement or degradation of different functional domains, and the remaining 13% have a higher average ka/ks ratio in both paralogs that is consistent with altered functional constraints, diversifying selection, or activity-reducing mutations after duplication. We estimate that these paralogs have been retained since they originated by genome duplication between 21 and 41 million years ago. Multiple mechanisms operate to promote the retained expression of duplicates in the same genome, in genes in the same functional class, over the same period of time following duplication, and sometimes in the same pair of paralogs. None of these paralogs are superfluous; degradation or enhancement of different protein subfunctions and neofunctionalization are plausible hypotheses for the retained expression of some of them. Evolution of most X. laevis paralogs, however, is consistent with retained expression via mechanisms that do not radically alter functional constraints, such as selection to preserve post-duplication stoichiometry or temporal, quantitative, or spatial subfunctionalization. PMID- 16683034 TI - Chemical genetics reveals an RGS/G-protein role in the action of a compound. AB - We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation. PMID- 16683035 TI - Virtual histology of transgenic mouse embryos for high-throughput phenotyping. AB - A bold new effort to disrupt every gene in the mouse genome necessitates systematic, interdisciplinary approaches to analyzing patterning defects in the mouse embryo. We present a novel, rapid, and inexpensive method for obtaining high-resolution virtual histology for phenotypic assessment of mouse embryos. Using osmium tetroxide to differentially stain tissues followed by volumetric X ray computed tomography to image whole embryos, isometric resolutions of 27 mum or 8 mum were achieved with scan times of 2 h or 12 h, respectively, using mid gestation E9.5-E12.5 embryos. The datasets generated by this method are immediately amenable to state-of-the-art computational methods of organ patterning analysis. This technique to assess embryo anatomy represents a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developmental patterning defects attributed to genetically engineered mutations and chemically induced embryotoxicity. PMID- 16683036 TI - Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs. AB - The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species. PMID- 16683037 TI - Genome Network and FANTOM3: assessing the complexity of the transcriptome. PMID- 16683038 TI - Balancing selection and its effects on sequences in nearby genome regions. AB - Our understanding of balancing selection is currently becoming greatly clarified by new sequence data being gathered from genes in which polymorphisms are known to be maintained by selection. The data can be interpreted in conjunction with results from population genetics models that include recombination between selected sites and nearby neutral marker variants. This understanding is making possible tests for balancing selection using molecular evolutionary approaches. Such tests do not necessarily require knowledge of the functional types of the different alleles at a locus, but such information, as well as information about the geographic distribution of alleles and markers near the genes, can potentially help towards understanding what form of balancing selection is acting, and how long alleles have been maintained. PMID- 16683040 TI - More than 25 years of urogenital Chlamydia trachomatis research in the Netherlands. AB - This article provides an overview of the Dutch work performed on urogenital Chlamydia trachomatis infections which started over 30 years ago. We will review past PhD research, 50% of which involved C. trachomatis as the main focus of the thesis, as well as research by current PhD fellows investigating (partially) C. trachomatis, and publications from Dutch authors or co-authors and the main discussion forums. PMID- 16683039 TI - DNA methylation supports intrinsic epigenetic memory in mammalian cells. AB - We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation. PMID- 16683041 TI - Prevalence of urogenital Chlamydia trachomatis infections in the Netherlands suggests selective screening approaches. Results from the PILOT CT Population Study. AB - Chlamydia trachomatis screening is being considered in the Netherlands, but policy recommendations are hampered by the lack of population-based data. We studied the prevalence of chlamydia infection in 15-29-year-old women and men in a national representative sample of 21,000 inhabitants of rural and urban areas in the Netherlands. Of this sample, 41% responded by sending in urine and an answered questionnaire, while 11% returned a refusal card. The overall prevalence of chlamydia infection was 2.0% (CI: 1.7-2.3); 2.5% (CI: 2.0-3.0) in women and 1.5% (1.1-1.8) in men. Chlamydia prevalence was significantly greater in very highly urbanized areas (3.2%, CI: 2.4-4.0) compared to rural areas (0.6%, CI: 0.1 1.1). In very highly urbanized areas the greatest prevalence was found among 15 19-year-old women (4.3%) and among 25-29-year-old men (4.2%). A risk profile could be determined and a prediction rule was developed. These data suggest that nationwide systematic screening is not indicated in the Netherlands and that targeted approaches are a better option. Roll-out of selective screening is recommended. PMID- 16683042 TI - Chlamydia antibody testing in subfertile women. AB - In the majority of women, Chlamydia trachomatis infections remain asymptomatic. These unrecognized and untreated infections may increase the risk for tubal factor subfertility at a later age. Since the association between C. trachomatis IgG antibodies in serum and tubal pathology was noticed, C. trachomatis IgG antibody testing has been used as a screening test for tubal factor subfertility. The diagnostic accuracy of C. trachomatis IgG antibody testing is limited, however. Since women who have persistent chlamydia infections are considered to be at the highest risk for developing late sequelae, the predictive value of markers of persistent infections have been studied in subfertile women. Patients who had C. trachomatis IgG antibodies (as markers of a previous infection), and an elevated C-reactive protein within the normal range (as a marker of a persisting infection) had the highest risk for having tubal pathology. PMID- 16683043 TI - Lymphogranuloma venereum proctitis: An emerging sexually transmitted disease in HIV-positive men in the Netherlands. AB - A recent outbreak of lymphogranuloma venereum (LVG) proctitis caused by Chlamydia trachomatis serovar L2 has been detected in HIV-positive men in the Netherlands and Belgium. This sexually transmitted disease (STD), which is well known and frequently occurring in tropical countries, was quite unusual in Europe until 2003. STDs with ulcerative lesions, such as LGV, facilitate transmission of other microorganisms, including HIV and hepatitis C. This in combination with risky sexual behavior, such as unprotected anal sexual intercourse or use of sex toys, increases the risk of blood-blood contact and hence the risk of contracting multiple STDs. Two cases of patients who in a short time period contracted multiple STDs, including LGV proctitis, is presented. PMID- 16683044 TI - Chlamydia trachomatis infections in Nicaragua: preliminary results from a competitive voucher scheme to prevent and treat sexually transmitted infections and HIV/AIDS among sex workers. AB - A donor-supported competitive voucher scheme in Nicaragua provides prevention and treatment services for sexually transmitted infections (STIs) and HIV/AIDS to high-risk populations such as sex workers and their partners and clients. Beyond detecting and treating STIs, HIV and AIDS, these health services can also raise awareness of risks and promote safer behavior, leading to widespread benefits. This review describes the voucher scheme, summarizes published data on Chlamydia trachomatis infection in Nicaragua and provides preliminary prevalence data on C. trachomatis obtained in 2003 through the voucher scheme. PMID- 16683045 TI - Animal models for the study of Chlamydia trachomatis infections in the female genital infection. AB - Despite intensive research on chlamydial pathogenesis and host immune responses, vaccine development has been hampered by incomplete understanding of the virulence factors and the critical factors governing protective immunity. It is unknown why certain Chlamydia trachomatis serovars cause asymptomatic or symptomatic infection or ascend to the upper genital tract. Former studies have used nonprimate monkey, mice or guinea-pig infection models. However, pigs are genetically and physiologically related to man and are susceptible to chlamydial infections. The main objective of the present study was the validation of the pig as an alternative animal model for C. trachomatis female genital tract infection. PMID- 16683046 TI - Inclusion proteins of Chlamydiaceae. AB - Chlamydiaceae are obligate intracellular pathogens with family members among the etiological agents of several human diseases, such as blinding trachoma, sexually transmitted disease (Chlamydia trachomatis) and pneumonia (Chlamydophila pneumoniae, Chlamydophila psittaci). The bacteria replicate intracellularly in a membrane-bound vacuole termed inclusion. The chlamydial inclusion is effectively separated from eukaryotic endocytic pathways. More than two decades ago it was already speculated that Chlamydiae might modify the inclusion membrane through the insertion of chlamydial-derived components. However, because the classical genetic approaches cannot be applied to manipulate these bacteria, it took more than 10 years before definitive proof was obtained that Chlamydiae indeed actively modify the inclusion membrane by the insertion of proteins of chlamydial origin, first observed by Rockey et al. in 1995. This review will focus on the structural and functional aspects of inclusion proteins of Chlamydiaceae, thereby summarizing data obtained by in vitro studies and comparative genomics. PMID- 16683047 TI - Innate immunity in defense against Chlamydia trachomatis infections. AB - Innate immunity is of key importance in primary recognition of invading pathogens. Infected epithelial cells respond in similar, but not identical ways to different invading pathogens and the pathogens are capable of modifying the host cell response. Chlamydia trachomatis is a major cause of preventable blindness in underdeveloped countries and of sexually transmitted infections with sequelae such as infertility, pelvic inflammatory disease, and extrauterine gravidity throughout the world. Limited knowledge about molecular mediators and effectors, immunocompetent cells, and host response in chlamydial mucosal infections will be described. Recent findings of a differential response to invasive and noninvasive chlamydial infections are highlighted. PMID- 16683048 TI - An integrated approach to the study of Chlamydia trachomatis infection of the female genital tract. AB - The two major aims of this thesis were firstly, to provide a basis for an integrated approach to study the oculogenital biovar of Chlamydia trachomatis in order to explain the complex and varied course of female genital tract infection; and secondly, to use the understanding gained through this approach to suggest possible intervention strategies. To accomplish this end, the projects conducted and reported upon in the thesis fall into four categorical sections. Part I of the thesis addresses the bacterial factors that contribute to the pathogenesis of infection using both in vitro observations and an in vivo experimental murine model. In Part II, host immunogenetic factors are analyzed using gene knockout mice and single nucleotide polymorphism (SNP) analysis of human genes. In Part III, the efficacy of prophylactic intervention on the susceptibility and course of female genital tract infection with C. trachomatis in the mouse is evaluated. Finally, in Part IV, the natural immune response to the pathogen and the relationship that these responses have on conferring protection and/or contributing to the pathology of disease are investigated to bring us closer to the ultimate protective intervention strategy: a vaccine against C. trachomatis. This article summarizes the aims and results of the thesis, followed by a general discussion and conclusions. PMID- 16683049 TI - Recognition of pathogenic microorganisms by Toll-like receptors. AB - Toll-like receptors (TLRs) have been identified as a major class of pattern recognition receptors. Recognition of pathogen-associated molecular patterns (PAMPs) by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of innate immune response. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of all the major classes of microbial pathogens. By studying fungal infection in knock-out mice deficient in either TLRs or TLR-associated adaptor molecules, it appeared that specific TLRs play differential roles in the activation of the various arms of the innate immune response. Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of antiinflammatory cytokines. These new data have substantially increased our knowledge of the recognition of microbial pathogens, and this remains one of the most active areas of research in the field of infectious diseases. PMID- 16683050 TI - Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection. AB - The use of an integrated approach to the study of Chlamydia trachomatis infection of the female genital tract, presented at the mini-symposium "Chlamydia trachomatis infections" and described in the thesis of Joseph M. Lyons, has resulted in the creation of the ICTI consortium. The ICTI consortium is based on strong interaction and collaboration between basic scientists, clinicians, epidemiologists, and health care policy makers. This translational approach will help to further the valuable insight into the immunopathogenesis of this sexually transmitted infection (STI) and the development of new intervention strategies, including the vaccines and screening programs necessary to effectively diagnose, treat and prevent C. trachomatis infection. A background of the need for this integrated approach is presented and the goals and participants of the consortium are described. PMID- 16683051 TI - Nitric oxide in children with persistent asthma. AB - OBJECTIVE: To assess the difference in exhaled nitric oxide levels in atopic and nonatopic asthmatic patients treated with anti-inflammatory drugs, and to compare exhaled nitric oxide measurement with lung function tests. METHODS: Cross sectional study with 45 consecutively selected patients with moderate and severe persistent asthma, aged between 6 and 17 years, and treated with anti inflammatory drugs for at least 1 year. The patients were split into two groups: atopic ones (with positive skin tests) and nonatopic ones. The clinical and functional assessments and the measurement of exhaled nitric oxide were carried out concomitantly. RESULTS: There was a male predominance (62.5%), with an age range between 6 and 13 years (mean of 10.4 years) in 85% of the patients. Neither the symptoms associated with asthma (p = 0.07), allergic rhinitis (p = 0.17), food allergy (p = 0.09), necessity of systemic corticosteroids (p = 0.10), antileukotrienes (p = 0.20) and antihistamines (p = 0.70), nor the three parameters used to assess lung function (FEV(1), FEV(1)/FVC and FEF(25-75%), p > or = 0.14) were statistically significant. The frequency of eczema (p < 0.005) and exhaled nitric oxide levels (p < 0.001) were higher among atopic patients. CONCLUSION: Results suggest that clinical and functional stability of asthma among atopic patients does not necessarily reflect an efficient control over the inflammatory process and a higher probability for recurrence after discontinuation of anti-inflammatory therapy. PMID- 16683052 TI - Vaccination in special situations. AB - OBJECTIVE: To review the indications, contraindications and efficacy of vaccination in some special situations: immunosuppression, prematurity, pregnancy and post-exposure situations. SOURCES OF DATA: Systematic review of articles published during the two last decades, found in MEDLINE, SciELO and Lilacs databases; guidelines of Programa Nacional de Imunizacoes (Brazilian National Immunization Program), 2001 to 2004, and of Programa Nacional de DST/AIDS (Brazilian National STD/AIDS Program), 2004. Abstracts published in national and international pediatric and infectious disease congress annals during the last five years were also consulted. SUMMARY OF THE FINDINGS: Some special situations, such as immunosuppression, prematurity, pregnancy and exposure to infectious diseases increased the risk of diseases or adverse post-vaccination events. In these situations, special vaccines or special vaccination schedules are indicated, or vaccines should be postponed or even forbidden. In general, toxoid or inactivated vaccines can be used, considering the possibility of insufficient immune response. For immunosuppressed patients, in accordance with the type of immunosuppression, live virus or bacterial vaccines should be avoided, because of the risk of vaccine agent spread. Immunization should include not only the patient, but his/her home and day-care contacts as well. CONCLUSIONS: Knowledge about the schedule indicated for each situation improves the chances of better vaccine protection and decreases the risk of adverse events. Immunosuppressed or immunodeficient patients whose post-vaccine antibody titers are not available should be considered susceptible when exposed to infectious disease, and all the available prophylactic measures should be implemented, even when the vaccination schedule is correct. PMID- 16683054 TI - Molecular cytogenetic characteristics of Down syndrome newborns. AB - Down syndrome (DS) is a multifactorial disorder with a high predisposition to leukemia and other malignancies. A change in the replication pattern from synchronous in normal genes to asynchronous in DS amniocytes has previously been reported. The objective of this study was to evaluate additional molecular cytogenetic factors which could re-emphasize the high correlation between DS cells and genetic instability. We found a higher rate of random aneuploidy in chromosomes 9 and 18 and a higher rate of asynchronous replication in the subtelomeric region or DS leukocytes than in cells from normal newborns. In addition, the telomere capture phenomenon was observed in the DS leukocytes but not in normal controls. The molecular cytogenetic factors observed in the DS individuals are known to correlate with genomic instability and with predisposition to cancer. PMID- 16683055 TI - Polymorphic alleles of the human MEI1 gene are associated with human azoospermia by meiotic arrest. AB - Genetic mechanisms are implicated as a cause of some male infertility, yet are poorly understood. Mouse meiotic mutant mei1 (meiosis defective 1) was isolated by a screening of infertile mice. Male mei1 mice have azoospermia due to meiotic arrest, and the mouse Mei1 gene is responsible for the mei1 phenotype. To investigate whether human MEI1 gene defects are associated with azoospermia by meiotic arrest, we isolated the human MEI1 cDNA based on the mouse Mei1 amino acid sequence. MEI1 is expressed specifically in the testis. Mutational analysis by direct sequencing of all MEI1 coding regions was performed in 27 men (13 European Americans, 13 Israeli and 1 Japanese) having azoospermia due to complete early meiotic arrest. This identified four novel, coding single-nucleotide polymorphisms (cSNPs), i.e., SNP1 (T909G), SNP2 (A1582G), SNP3 (C1791A) and SNP4 (C2397T) in exons 4, 8, 9 and 14, respectively. Using these cSNPs, an association study was carried out between 26 non-Japanese patients with azoospermia and two sets of normal control men (61 normal European Americans and 60 Israelis). Consequently, SNP3 and SNP4 were shown to be associated with azoospermia among European Americans (P =0.0289 and P =0.0299 for genotype and allele frequencies at both the polymorphic sites, respectively), although no such association was observed among Israelis (P >0.05). Haplotype estimation revealed that the frequencies of SNP3-SNP4 (C-T), SNP3-SNP4 (A-C) and SNP3-SNP4 (A-T) were higher in the European American patients, and the frequency of SNP3-SNP4 (A-T) was also higher than in both control groups. These results suggest that MEI1 may play a role in meiosis during spermatogenesis, especially in European Americans. PMID- 16683056 TI - Comparing infant and juvenile behavior in bonobos (Pan paniscus) and chimpanzees (Pan troglodytes): a preliminary study. AB - The dichotomy between the two Pan species, the bonobo (Pan paniscus) and chimpanzee (Pan troglodytes) has been strongly emphasized until very recently. Given that most studies were primarily based on adult individuals, we shifted the "continuity versus discontinuity" discussion to the infant and juvenile stage. Our aim was to test quantitatively, some conflicting statements made in literature considering species differences between immature bonobos and chimpanzees. On one hand it is suggested that infant bonobos show retardation in motor and social development when compared with chimpanzees. Additionally it is expected that the weaning process is more traumatic to chimpanzee than bonobo infants. But on the other hand the development of behaviors is expected to be very similar in both species. We observed eight mother-infant pairs of each species in several European zoos. Our preliminary research partially confirms that immature chimpanzees seem spatially more independent, spending more time at a larger distance from their mother than immature bonobos. However, the other data do not seem to support the hypothesis that bonobo infants show retardation of motor or social development. The development of solitary play, environmental exploration, social play, non-copulatory mounts and aggressive interactions do not differ between the species. Bonobo infants in general even groom other group members more than chimpanzee infants. We also found that older bonobo infants have more nipple contact than same aged chimpanzees and that the weaning process seems to end later for bonobos than for immature chimpanzee. Additionally, although immature bonobos show in general more signs of distress, our data suggest that the weaning period itself is more traumatic for chimpanzees. PMID- 16683060 TI - Heat stress increases protein antigen transport across the intestinal epithelium via a mechanism of impairing proteolytic enzymatic activity. AB - It has not been fully understood how intact protein antigens escape digestion in the course of absorption. The present study was designed to investigate the mechanism that heat stress induced an increase in intact protein antigen absorption. Human colonic cell line Caco-2 cells were treated with high temperature (37 to 43 degrees C) for 60 min. Epithelial permeability was evaluated by horseradish peroxidase (HRP) flux and dextran flux. Activity of the major intracellular proteolytic enzyme, acid phosphatase, in Caco-2 cells was determined. HRP products in Caco-2 cells were observed by electron microscopy (EM) and analyzed with a computerized image processing system. Heat stress significantly increased intact protein HRP transport across Caco-2 monolayers, decreased acid phosphatase activity of the cells, and significantly reduced transepithelial electric resistance of Caco-2 cells. EM results showed that HRP transport across Caco-2 monolayers occurred mainly via the intracellular pathway. PMID- 16683059 TI - Influence of hydrocortisone, prednisolone, and NO association on the evolution of acute pancreatitis. AB - Leukocyte activation, inflammatory up-regulation, and microcirculatory disruption associated with ischemia-reperfusion injury are hallmarks in the pathogenesis of acute pancreatitis (AP). NO donors ensure microvascular integrity, while glucocorticoids act as anti-inflammatory and immune modulator drugs. AP was induced by the biliopancreatic duct outlet exclusion-closed duodenal loops (BPDOE CDLs) model. Treatment with hydrocortisone (6 mg/kg) or prednisolone (0.5 mg/kg) alone or together with DETA-NO (0.5 mg/kg) was done (a)1 hr pre or (b)1 hr post, or (c) 1 hr pre and 4 hr post ,or (d) 4 hr post triggering AP. NOS inhibition by L-NAME (15 mg/kg) and glucocorticoid receptor blockage by mifepristone (3 mg/kg) was considered. AP severity was assessed by biochemical and histopathological analyses. Treatment with glucocorticoids together with DETA-NO 1 hr pre and 4 hr post BPDOE-CDLs reduced serum amylase, lipase, C-reactive protein, IL-6, IL-10, hsp72, and 8-isoprostane as well as pancreatic and lung myeloperoxidase. Acinar and fat necrosis, hemorrhage, and neutrophil infiltrate were also decreased. Hydrocortisone together with DETA-NO rendered the best results. We conclude that AP severity was significantly diminished by glucocorticoids associated with DETA NO, with the optimal dose and time point of administration being crucial to provide adequate protection against AP. PMID- 16683061 TI - Acute treatment with the antidepressants bupropion and sertraline do not influence memory retrieval in man. AB - OBJECTIVE: Several evidences implicate that monoamines play a modulatory role in the brain mechanisms underlying encoding and retrieval of emotional memories. Recent experiments demonstrate that acute monoaminergic potentiation with the antidepressants bupropion or sertraline enhance the retrieval of longterm emotional memory in rodents. In the present study, we tested the hypothesis that acute monoaminergic re-uptake inhibition with these antidepressants might enhance retrieval of emotional memory in man. METHODS: The central monoaminergic system was stimulated with either bupropion or sertraline in a double-blind, randomized, placebo-controlled design with 105 healthy adult subjects divided in three groups (placebo, 150 mg-bupropion and 50 mg-sertraline). Memory was evaluated with a 'surprise' memory test 7 days after the presentation of an emotional story and with a word-cued autobiographical memory test. RESULTS: A total of 99 volunteers completed the experimental procedures. Contrasting to our prediction, we found no memory enhancing effect for either drug in both memory tests. All groups showed the expected heightened memory performance to the middle 'emotive' phase of the story. CONCLUSION: Stimulation of the central monoaminergic system with the antidepressants bupropion and sertraline did not enhance the retrieval of long term emotional memories in man. PMID- 16683062 TI - The clinical impact of mood disorder comorbidity on obsessive-compulsive disorder. AB - The present study examines the effect of concomitant major depressive or bipolar disorder on clinical symptoms of patients with obsessive-compulsive disorder (OCD). Forty-nine patients classified as OCD without a mood disorder, 26 classified as OCD with bipolar disorder (OCD-BD) and 42 classified as OCD with major depressive disorder (OCD-MDD) according to DSM-IV diagnostic criteria were included in the study. The groups were compared with respect to demographic variables and scores obtained on various scales. The OCD-BD group had more symmetry/exactness obsessions and ordering/arranging compulsions, and a more episodic course of illness and had better insight compared to the other two groups. Levels of anxiety, depression, disability and obsessive-compulsive symptom severity were significantly higher in the OCDMDD group. The rate of social phobia was higher in OCD-BD patients, whereas the rates of generalized anxiety disorder and simple phobias were higher in OCDMDD group. These findings suggest that comorbidity of major depressive disorder may increase the severity of OCD symptoms. On the other hand, bipolar disorder comorbidity may constitute a subgroup which is characterized by a higher rate of episodic course and better insight. PMID- 16683063 TI - Levetiracetam for cerebellar tremor in multiple sclerosis: an open-label pilot tolerability and efficacy study. AB - PURPOSE: Disabling tremor is frequent in multiple sclerosis (MS) and its treatment remains challenging. We conducted an open-label trial to evaluate the effect of levetiracetam (LEV) to treat cerebellar tremor in MS patients. PATIENTS AND METHODS: Fourteen MS patients, aged 27 to 57 years, with cerebellar tremor. Tremor duration ranged from 3 to 14 years. The tremor clinical rating scale, the spiral drawings scale, and ataxia clinical scale were used to assess the severity of tremor. Data about the tremor-induced disability were obtained by using the specific Activities of Daily Living Questionnaire (ADL). LEV was orally administered at a starting dose of 500 mg twice daily for one week followed by increments of 500 mg twice daily each week up to the target dose of 50 mg/Kg/day. Patients were evaluated at baseline and two weeks after the end of titration phase. The Wilcoxon matched-pairs test was used for statistical analysis. RESULTS: Eleven patients completed the trial. LEV administration was associated with subjective and objective improvement of the tremor, with significant lowering of all tremor measurements' sum of scores as well as of ADL mean score between the baseline and follow-up. No correlation was found between the degree of improvement of the tremor and the disease duration or progression. LEV was well tolerated by subjects who completed the study. CONCLUSIONS: LEV could be useful for the management of cerebellar tremor in MS and the good tolerability makes it easy to test. LEV long-term efficacy should be confirmed in extended studies. PMID- 16683066 TI - Autonomics and cognition. PMID- 16683067 TI - A world without pain or tears. AB - The world of the child with familial dysautonomia (FD), a genetic disorder affecting development of the sensory and autonomic nervous system, is not idyllic. However, over the last 35 years advances in supportive treatments have improved morbidity and mortality. Recent genetic breakthroughs have further expanded thinking about this disorder and suggested innovative approaches to modifying genetic expression. This article reviews the current supportive treatment modalities and their rationale, as well as the suggested new treatments that may alter the function and prognosis of an individual affected with FD. PMID- 16683068 TI - Phase of the menstrual cycle does not affect orthostatic tolerance in healthy women. AB - Women of child-bearing age have a lower orthostatic tolerance (OT) than older women or men, and women suffering from frequent syncopal episodes often comment that their symptoms occur at certain times of the menstrual cycle. However, it is not known whether, in asymptomatic women, OT varies at different phases of the menstrual cycle. We studied 8 healthy asymptomatic women aged 26.8 +/- 3.4 years. We determined OT using a test of combined head-up tilting and lower body suction. We continuously monitored beat-to-beat blood pressure (Finapres), heart rate (ECG), and cerebral and forearm blood flow velocities (Doppler ultrasound). On each test day we assessed carotid baroreceptor sensitivity from suction/pressure applied to a neck chamber. We also determined estradiol and progesterone levels from a venous blood sample. Tests were performed in early follicular and late luteal phases, and during ovulation. Serum concentrations of estradiol (pmol x l( 1)) and progesterone (nmol x l(-1)) were in follicular phase 464.1 +/- 63 and 6.3 +/- 2.8; ovulation 941.6 +/- 298 and 5.8 +/- 1.2; luteal phase 698 +/- 188 and 32.3 +/- 9.6. Progesterone levels were significantly higher in the luteal phase (p < 0.001). OT was not different on any test day: follicular 31.9 +/- 1.6 min, ovulation 31.3 +/- 0.7 min; luteal 31.1 +/- 2.2 min. Supine and tilted heart rates and blood pressures, the maximum heart rate, and the cerebral autoregulatory and forearm vascular resistance responses to the orthostatic stress were similar during all studies. Both cardiac and vascular resistance carotid baroreceptor sensitivities were also similar on all test days. These results suggest that there is no difference in either OT or cardiovascular control at the tested phases of the menstrual cycle in healthy women. PMID- 16683069 TI - Beta-2 adrenergic receptor polymorphisms and the forearm blood flow response to mental stress. AB - Circulating epinephrine plays an important role in skeletal muscle vasodilation during mental stress. Normotensive adults homozygous for glycine (Gly) of the Arg16/Gly beta2-adrenergic receptor polymorphism have a greater forearm beta2 receptor mediated vasodilation and a higher cardiac output response to isometric handgrip than arginine (Arg) homozygotes. To test the hypothesis that the Arg16/Gly beta2-adrenergic receptor polymorphism affects the forearm blood flow (FBF) and hemodynamic response to mental stress, and whether venous catecholamine concentrations predicted these responses, we measured venous epinephrine, norepinephrine, heart rate (HR), arterial pressure (Finapres), and FBF during mental stress in healthy subjects homozygous for Gly16 (n = 30; mean age +/- SE: 30 +/- 1.2, 13 women) and Arg16 (n = 17, age 30 +/- 1.6, 11 women). Resting HR, blood pressure, and FBF responses to mental stress were similar between genotype groups. There were positive correlations between epinephrine and peak FBF (r = 0.694, P < 0.001), peak forearm vascular conductance (r = 0.677, P < 0.001) and the change in epinephrine to the change in HR (r = 0.456, P = 0.002) in all subjects. These correlations were not significantly different in the Gly16 and Arg16 groups. We conclude that venous epinephrine predicts the FBF response to mental stress, and the increase in epinephrine is also correlated with the increase in HR. Furthermore, the Arg16/Gly beta2-receptor polymorphism has no significant influence on the FBF or cardiovascular responses to mental stress. PMID- 16683070 TI - Cognitive functioning in orthostatic hypotension due to pure autonomic failure. AB - Psychophysiological science proposes close interactions between cognitive processes and autonomic responses, yet the consequences of autonomic failure on cognitive functioning have not been documented. This pilot study investigates, for the first time, the cognitive profile of 14 patients with Pure Autonomic Failure (PAF). Each patient was administered a comprehensive battery of neuropsychological tests and neuroimaging investigation. A number of patients (n = 6) presented with cognitive impairment. The two most frequent types of impairment were: deficits of speed and attention, and executive functioning. Impairments of free recall memory, intellectual functioning, nominal and calculation functions were also documented, albeit in a much lower frequency. These cognitive changes were not always associated with white matter abnormalities. We speculate that the cognitive impairments associated with PAF represent consequences of systemic hypotension with cerebral underperfusion. However, a failure in integrated bodily arousal responses during cognitive behaviours may also contribute to some of the observed deficits. PMID- 16683074 TI - A phase I and pharmacokinetic study of irofulven and cisplatin administered in a 30-min infusion every two weeks to patients with advanced solid tumors. AB - BACKGROUND: To determine maximum tolerated dose (MTD), recommended dose, safety and pharmacokinetics of irofulven combined with cisplatin in advanced solid tumor patients. PATIENTS AND METHODS: Cisplatin and irofulven were given sequentially i.v. over 30 min on day 1 and 15 every 4 weeks. Four dose levels (DL) were explored: irofulven (mg/kg)/cisplatin (mg/m2): DL1: 0.3/30; DL2: 0.4/30; DL3: 0.4/40; DL4: 0.5/40. Dose-limiting toxicity (DLT) included dosing omission and delay > 1 week. MTD was the DL with DLT in 2/2 or > or = 2/6 patients during cycle 1-2. RESULTS: Between March 2002 and April 2003, 33 patients were treated. DLT occurred in 1/6 patients in DL1 (hypomagnesemia, hypocalcemia); 1/6 in DL2 (thrombocytopenia); 2 heavily pretreated patients out of 6 patients in DL3 (neutropenic infection, thrombocytopenia, stomatitis); 2/3 in DL4 (asthenia, blurred vision). Three DLT occurred in 12 additional patients treated at DL2. No toxic deaths occurred; grade 4 toxicity and grade 3 non-hematological toxicity were infrequent. Six patients reported grade 1-2 visual events. Antitumor activity was observed over a broad spectrum of tumor types in all DLs: 1 partial response in bulky sarcoma (DL1); 1 clinical response in endometrial carcinoma (DL1); 2 partial responses not confirmed due to discontinuation (ovarian DL2, renal DL4); 8 stabilizations > 3 months; PSA response: 3/9 prostate cancer patients. Irofulven showed rapid elimination and high interpatient variability. Platinum and irofulven pharmacokinetics did not suggest drug-drug interactions. CONCLUSION: Irofulven with cisplatin was adequately tolerated and substantial evidence of antitumor activity was observed. The recommended dose is irofulven 0.4 mg/kg and cisplatin 30 mg/m2. PMID- 16683072 TI - The Wnt-dependent signaling pathways as target in oncology drug discovery. AB - Our current understanding of the Wnt-dependent signaling pathways is mainly based on studies performed in a number of model organisms including, Xenopus, Drosophila melanogaster, Caenorhabditis elegans and mammals. These studies clearly indicate that the Wnt-dependent signaling pathways are conserved through evolution and control many events during embryonic development. Wnt pathways have been shown to regulate cell proliferation, morphology, motility as well as cell fate. The increasing interest of the scientific community, over the last decade, in the Wnt-dependent signaling pathways is supported by the documented importance of these pathways in a broad range of physiological conditions and disease states. For instance, it has been shown that inappropriate regulation and activation of these pathways is associated with several pathological disorders including cancer, retinopathy, tetra-amelia and bone and cartilage disease such as arthritis. In addition, several components of the Wnt-dependent signaling pathways appear to play important roles in diseases such as Alzheimer's disease, schizophrenia, bipolar disorder and in the emerging field of stem cell research. In this review, we wish to present a focused overview of the function of the Wnt dependent signaling pathways and their role in oncogenesis and cancer development. We also want to provide information on a selection of potential drug targets within these pathways for oncology drug discovery, and summarize current data on approaches, including the development of small-molecule inhibitors, that have shown relevant effects on the Wnt-dependent signaling pathways. PMID- 16683073 TI - A phase I study of flavopiridol and docetaxel. AB - BACKGROUND: Flavopiridol is a cyclin dependent kinase inhibitor. Preclinical models suggest a sequence dependent synergy between flavopiridol and taxanes. The primary objective of this study was to determine the maximum tolerated dose (MTD) of flavopiridol and docetaxel and the influence of flavopiridol on the pharmacokinetics of docetaxel. METHODS: The major eligibility criteria included: a diagnosis of non-hematologic cancer with no conventional effective therapy, normal organ function, and ECOG performance status of 0-2. Patients were treated with docetaxel followed 24 h later by flavopiridol given via continuous intravenous infusion over a 24-h period. The starting doses of docetaxel and flavopiridol were 60 and 60 mg/m2, respectively. Cycles were repeated every 21 days. All patients received diarrhea prophylaxis consisting of bismuth subsalicylate. RESULTS: Ten patients (M:F 4:6; median age 56 years) were treated. The median number of cycles per patient was 2 (range 1-6). Two of the three patients on dose level 1 developed dose-limiting toxicities consisting of neutropenia and fever. Seven patients were subsequently enrolled on dose level -1 (docetaxel 60 mg/m2, flavopiridol 50 mg/m2). One episode of grade 3 diarrhea was reported at dose level -1. CONCLUSIONS: Neutropenia complicated by infection was the major dose-limiting toxicity. The recommended doses of flavopiridol and docetaxel for phase II trials are 50 and 60 mg/m2 every three weeks, respectively. PMID- 16683075 TI - Phase I trial of thalidomide and interleukin-2 in patients with metastatic renal cell carcinoma. AB - BACKGROUND: The treatment of advanced renal cell cancer remains unsatisfactory, therefore new combination regimens such as thalidomide and IL-2 are of interest. A phase I trial of SC IL-2 and oral thalidomide was performed to identify the toxicity, maximum tolerated dose (MTD) and preliminary clinical activity of this regimen. METHODS: 33 patients with advanced/metastatic RCC were enrolled. An established 8-week outpatient schedule of subcutaneously administered IL-2 in escalating doses, days 1-5, for 6 weeks with a 2 week rest was utilized with daily oral thalidomide. Cohorts of 4-6 patients were treated at 4 dose levels. RESULTS: Toxicity was moderate to severe and related to dose level. All patients developed fever, chills and fatigue. 29/33 patients developed < or = Grade 2 desquamation of hands and feet and/or rash. Dose limiting toxicity (DLT) included Grade 3 neutropenia and pulmonary embolus. The maximum tolerated dose (MTD) of IL 2 and thalidomide was 9.0 MIU/m2 s.c. days 1-5, weeks 1 to 6 and 100 mg p.o. daily, respectively. A median of 2 cycles of therapy was administered (range 1 9). 2/33 patients responded (1 CR--prior IL-2 therapy, 1 PR--no prior therapy) with an overall response of 6% (95% CI, 1-20%). One minimal response was converted to a surgical CR (remains disease free at 24 + months). CONCLUSION: Outpatient administration of IL-2 and thalidomide is possible with acceptable toxicity. Further evaluation of this regimen is underway. PMID- 16683076 TI - Phase II trial of R115777 (NSC #70818) in patients with advanced colorectal cancer: a Southwest Oncology Group study. AB - PURPOSE: The purpose of this Phase II multi-institutional trial was to determine the efficacy and toxicity of R115777 in previously untreated patients with metastatic colorectal carcinoma. PATIENTS AND METHODS: Patients were required to have histologically confirmed colorectal cancer with distant metastatic disease that was not surgically curable. They could not have received prior chemotherapy for metastatic disease. R115777 was given at a dose of 300 mg p.o. twice a day for 21 days every 28 days until tumor progression or toxicity or other reason for discontinuation occurred. The primary endpoint was to determine the confirmed response probability with this treatment. RESULTS: There were 55 eligible patients accrued to the study. There were no complete responses, but one confirmed partial response for a confirmed response probability of 2% (95%CI 0 10%). Three additional patients had an unconfirmed partial response for an overall response probability of 7%. The time to treatment failure was 1.7 months and the estimated median survival was 8.1 months. One patient died of treatment related infection and there were 7 other patients with grade 4 toxicities consisting of neutropenia, leukopenia, febrile neutropenia and thrombocytopenia, depression, increased bilirubin, anemia, and pneumonitis/infiltrates. CONCLUSION: R115777 given as a single agent by this dose and schedule is ineffective in patients with metastatic colorectal cancer. PMID- 16683077 TI - A multi-center phase II study of sequential paclitaxel and bryostatin-1 (NSC 339555) in patients with untreated, advanced gastric or gastroesophageal junction adenocarcinoma. AB - PURPOSE: Protein Kinase C (PKC), involved in transmembrane signaling of cell surface receptors, promotes carcinogenesis and tumor progression. Bryostatin-1 competes with PKC for phorbol esters (tumor promoters), thus inhibiting tumor progression. Bryostatin-1 also increases cytotoxicity of paclitaxel in a sequential fashion. We studied sequential paclitaxel and bryostatin-1 in patients with untreated, advanced gastric adenocarcinoma. METHODS: Patients with histologic proof of gastric or gastroesophageal junction adenocarcinoma with advanced, measurable cancers were eligible. Patients were required to have near normal organ function and ECOG performance status of 0 or 1. All patients gave an informed consent. Patients received paclitaxel 80 mg/m2 in 2 h intravenously on day 1 and bryostatin-1 40 mcg/m2 in 1 h intravenously on day 2 each week for 3 consecutive weeks out of 4. Primary objective was to assess the objective response rate. RESULTS: In a multi-center setting, 37 patients were enrolled and 35 were assessable for response. A confirmed partial response rate was 29%. The median time-to-progression was 4.25 months and the median survival time was 8 months. Grade 3 cumulative myalgias occurred in 55% of patients. Twelve patients discontinued therapy due to myalgias, including 6 patients who had not progressed after achieving a partial response. Other toxic effects were uncommon. CONCLUSIONS: Sequential paclitaxel plus bryostatin-1 resulted in a superior response rate than would be expected of paclitaxel alone in patients with untreated, advanced gastric or gastroesophageal junction adenocarcinoma. Further development of this combination is warranted once an effective method to ameliorate or prevent myalgias can be established. PMID- 16683078 TI - Quetiapine-fluvoxamine combination during pregnancy and while breastfeeding. PMID- 16683080 TI - The anterior mediastinum: anatomy and imaging procedures. AB - The mediastinum is divided into compartments (anterior, middle, posterior) on the basis of lateral chest radiographs. Several anatomical and radiological classifications of the mediastinum are reported in the literature. Most mediastinal abnormalities are initially suspected following chest radiography; the need for further investigation and the most appropriate imaging modality are largely dictated by the tentative diagnosis made on this examination. Although routine chest radiography initiates the evaluation of mediastinal disorders, it is rarely diagnostic: notable exceptions are teeth or bones within a mass, which are diagnostic of a teratoma; air/fluid levels suggest an oesophageal origin, hernia, cyst, or abscess. Chest radiography is followed by spiral computed tomography (sCT). However, even sCT with contrast material is occasionally diagnostic (a confident diagnosis can be made of some lesions such as mature teratoma and mediastinal goiter) but is usually sufficient for preoperative evaluation before mediastinotomy or mediastinoscopy: it is instrumental in planning further diagnostic workup. In certain cases, magnetic resonance imaging (MRI) may be complementary to sCT, but its use is not considered routine. Besides, although the anterior mediastinum is suitable for sonographic examination, the diagnostic value of ultrasonography has not been fully exploited. Thyroid scanning with radioactive iodine is useful in identifying and evaluating masses of suspected thyroid origin. The role of fluorodeoxyglucose positron emission tomography (FDG-PET) in mediastinal diseases continues to be evaluated: it has potential for differentiating between benign and malignant disease and is expected to play a more extensive role in the imaging of mediastinal neoplasms in the future. In this paper, the radiological features of masses located in the anterior mediastinum are discussed, with particular reference to radiographic and CT patterns useful to the clinician's everyday practice. PMID- 16683081 TI - The anterior mediastinum: diseases. AB - Mediastinal tumours are frequently asymptomatic and first noted on routine chest radiograph. In most cases, evaluation should proceed to spiral computed tomography (sCT) of the chest with iodinated contrast material. The specific location and appearance of tumours on sCT is instrumental in planning further diagnostic and treatment strategies. Primary tumours in the anterior mediastinum account for half of all mediastinal masses. They comprise various benign and malignant neoplasms, but a wide variety of nonneoplastic lesions (developmental, inflammatory) can present as a localised mass in this compartment. The most common primary anterior mediastinal tumours are thymoma, teratoma and lymphoma; all other lesions are rare. Nonneoplastic conditions include thymic cysts, lymphangioma and intrathoracic goitre. Understanding the pathology, clinical presentation, imaging and diagnosis of the major tumour types is instrumental in the safe and efficient work-up of a mediastinal mass. Patients with primary mediastinal masses and cysts will usually undergo surgical resection; radiological and clinical features should prompt limited biopsy specimens followed by oncologic consultation, and chemotherapy or radiotherapy when appropriate. The objective of this review was to examine the role of diagnostic imaging in the management of masses of the anterior mediastinum. PMID- 16683082 TI - Three-dimensional analysis of pulmonary nodules by MSCT with Advanced Lung Analysis (ALA1) software. AB - PURPOSE: The purpose of this study was to test the reproducibility of the three dimensional (3D) Advanced Lung Analysis software (3D-ALA, GE Healthcare) in the estimation of pulmonary nodule volume. MATERIALS AND METHODS: We retrospectively reviewed the unenhanced multislice CT scans (Lightspeed Pro 16 GE) of 77 patients with a solitary pulmonary nodule (n=71) or metastatic pulmonary disease (n=6). A total of 103 pulmonary nodules (19 well-circumscribed, 45 juxtavascular and 39 juxtapleural) were analysed grouped into five classes based on diameter: <5 mm, 10 nodules (9.7%); >or=5 to <10 mm, 25 nodules (24.2%); >or=10 mm to <15 mm, 41 nodules (39.8%); >or=5 to <18 mm, 14 nodules (13.6% ); >or=8 to <30 mm, 13 nodules (12.62%). The following acquisition parameters were used: slice thickness 0.625 mm, reconstruction interval 0.4 mm, pitch 0.562:1, 140 kV, 300 mAs, field of view 13 cm, bone kernel. For each of the 103 nodules three, 3D volume measurements were obtained by the 3D-ALA software. The reproducibility of nodule segmentation was evaluated according to a visual score (1=optimal, >or=95%; 2=fair, 90-95%; 3=poor, 0.05). CONCLUSIONS: Three-dimensional volume measurement with ALARiassunto 1 software is reproducible for all nodules as regards dimension and site. ALA-1 software provided a good and reproducible volume measurement in well circumscribed and most juxtavascular nodules. Volumetric evaluation and reproducibility of volume estimation in juxtapleural pulmonary nodules, particularly those adjacent to diaphragmatic pleura, is inadequate, and software improvement is needed. PMID- 16683083 TI - Imaging before and after multimodal treatment for malignant pleural mesothelioma. AB - PURPOSE: Computed tomography (CT), magnetic resonance (MR) and positron emission tomography (PET) have a very important role in the diagnosis of malignant pleural mesothelioma (MPM) in the choice of chemoradiotherapy alone or in combination with surgery and in evaluating possible recurrence. It is also essential for assessing the possible benefits of radical surgery (pleuropneumonectomy) in terms of patient survival. MATERIALS AND METHODS: We considered 28 patients suffering from MPM whose mean survival after diagnosis was 15-18 months. Sixteen of these patients had radiotherapy or chemoradiotherapy alone, according to standard protocols, while 12 also underwent surgery. The CT features of MPM were thoroughly examined, as was the role of PET and CT-PET in achieving accurate disease staging and consequent selection of candidates for surgery. RESULTS: Nine of the 12 patients who underwent pleuropneumonectomy had no significant survival advantage over the mean survival in the 16 who were not operated whereas the other three lived 1-3 years longer. Two patients underwent surgery after an optimal response to chemoradiotherapy, but both survived less than a year due to particularly aggressive recurrences. CONCLUSIONS: CT, PET and CT-PET are indicated for diagnosis and, above all, for staging of MPM, in the selection of patients who might benefit from surgery after neoadjuvant therapy and also in identifying small recurrences and/or remote metastases. Being highly specific, PET is essential in the follow-up of patients undergoing chemoradiotherapy alone and/or surgery. Each imaging modality has its advantages and limitations, but their combined use is crucial in determining the most appropriate treatment options for patients with MPM. PMID- 16683084 TI - Volumetric evaluation of therapy response in patients with lung metastases. Preliminary results with a computer system (CAD) and comparison with unidimensional measurements. AB - PURPOSE: The aim of this study was to assess the efficacy of a Computer-Aided Detection (CAD) system in the identification of lung metastases and to compare the volumetric CAD measurements with unidimensional observer measurements in the evaluation of treatment response in oncology patients. MATERIALS AND METHODS: Two observers (A and B) evaluated nine patients undergoing lung computed tomography (CT) just before and immediately after treatment with chemotherapy. Multislice CT scans were performed before and after the injection of contrast material with a high-resolution protocol (collimation 4x1 mm, 100 mAs, 120 kV). Response Evaluation Criteria in Solid Tumours (RECIST) criteria were used to consider the disease as stable, increased or decreased. Subsequently, target lesions (most significant lesions identified before and after chemotherapy) were evaluated with a computerised system (CAD) to establish volumetric measurements. Observers' unidimensional measurements and CAD volumetric measurements were analysed for comparison. RESULTS: Twenty-four nodules (diameter: 5-18 mm in the first study and 4-20 mm in the follow-up study) were included. Observers agreed in the assessment of therapy response in 21 nodules: eight were considered to have increased in size, and 13 were judged stable. Observer and CAD measurements disagreed in three nodules: two were considered stable by radiologists and increased by CAD; one was considered increased by radiologists and stable by CAD.As regards patient response, radiologists disagreed in two cases. CAD and observers did not agree in one case. CONCLUSIONS: Our preliminary data suggest that volumetric measurements can modify the diagnostic and therapeutic evaluation of oncology patients under chemotherapy.CAD volumetric measurements allow an easy and objective evaluation, reducing interobserver variability in the evaluation of chemotherapy response. PMID- 16683085 TI - Coronary artery anomalies: incidence, pathophysiology, clinical relevance and role of diagnostic imaging. AB - Conventional coronary angiography is the gold standard for the diagnosis of coronary artery anomalies. Coronary anomalies are relatively rare findings in patients undergoing conventional coronary angiography for suspected obstructive coronary artery disease. Recently, the increasing performance of diagnostic techniques, such as electron beam tomography (EBT), magnetic resonance (MR) and, more recently, multislice computed tomography (MSCT), has enabled their application to cardiac imaging. MSCT, in particular, has a prominent role in coronary imaging due to its spatial and temporal resolution and three-dimensional capabilities. We report the incidence and pathophysiology of coronary artery anomalies based on the capabilities of recent diagnostic tools with the aim of improving an accurate and noninvasive diagnostic approach. PMID- 16683086 TI - Magnetic resonance diffusion-weighted imaging: extraneurological applications. AB - Diffusion-weighted (Dw) imaging has for a number of years been a diagnostic tool in the field of neuroradiology, yet only since the end of the 1990s, with the introduction of echoplanar imaging (EPI) and the use of sequences capable of performing diffusion studies during a single breath hold, has it found diagnostic applications at the level of the abdomen. The inherent sensitivity to motion and the magnetic susceptibility of Dw sequences nonetheless still create problems in the study of the abdomen due to artefacts caused by the heartbeat and intestinal peristalsis, as well as the presence of various parenchymal-gas interfaces. With regard to focal liver lesions, a review of the literature reveals that Dw imaging is able to differentiate lesions with high water content (cysts and angiomas) from solid lesions. With regard to the latter, although there are differences between benign forms [focal nodular hyperplasia (FNH), adenoma] and malignant forms [metastasis, hepatocellular carcinoma (HCC)] in their apparent diffusion coefficient (ADC) in the average values for histological type, there is a significant overlap in values when lesions are assessed individually, with the consequent problem of their correct identification. One promising aspect is the possibility of quantifying the degree of fibrosis in patients with chronic liver disease and cirrhosis given that the deposit of collagen fibres "restricts" the motion of water molecules and therefore reduces ADC values. However, even in this field, studies can only be considered preliminary and far from real clinical applications. The retroperitoneum is less affected by motion artefacts and similarly deserves the attention of Dw imaging. Here it is possible to differentiate mucin-producing tumours of the pancreas from pseudocystic forms on the basis of ADC values even though the limited spatial resolution of Dw imaging does not enable the identification of small lesions. Dw imaging may be applied to the study of the kidney to differentiate hydronephrosis from pyonephrosis and with regard to tumours, solid from pseudocystic forms. In addition, given that renal parenchyma has significantly variable ADC values on the basis of the anatomic section and physiological conditions, the possibility of assessing functional alterations is currently being studied. Indeed, a good correlation has been found between ADC values and glomerular filtration rate. With regard to musculoskeletal applications, the absence of motion artefacts in the regions studied has enabled the development of sequences less sensitive to magnetic susceptibility and with greater spatial resolution than EPI. Attempts have therefore been made to use Dw imaging in the characterization of soft-tissue tumours although the findings so far have been disputed. Greater agreement has been found regarding sensitivity of the technique in assessing response of these tumours to chemotherapy: tumour necrosis is thought to increase ADC whereas the persistence of vital neoplastic tissue tends to lower it. One of the most promising applications of Dw imaging is without doubt the assessment of vertebral collapse where a high ADC has been shown to be associated with an osteoporotic cause and a low ADC with a neoplastic cause. Nonetheless, even here, a moderate overlap between ADC values of the two types has been encountered. Dw imaging has also been used in the assessment of bone marrow cellularity: areas of tightly packed cells show a higher ADC value than hypocellular areas. In particular, no significant difference in ADC is noted between normal hypercellular bone marrow and hypercellular bone marrow secondary to lymphomatous infiltration whereas this difference is significant between hypocellular, normocellular and haematopoietic hypercellular bone marrow. With regard to the study of joints, the limited structure dimensions, particularly cartilage, creates technical difficulties related to spatial resolution and an adequate signal-to-noise ratio, problems that can only be solved by further technological developments. Lastly, a significant difference in ADC values between degenerative and inflammatory effusion has been found, a fact that may be explained as the result of the activity of hyaluronidase present in inflammatory forms, which causes a reduction in the concentration of hyaluronic acid with a consequent decrease in viscosity. PMID- 16683087 TI - Estimation of the phosphocreatine T1 time constant using a clinical NMR scanner. AB - AIM: The aim of this study was to determine the "spin-lattice relaxation" or "T1" time constant of phosphocreatine in the human gastrocnemius muscle, both at rest and during aerobic exercise, when a clinical nuclear magnetic resonance (NMR) scanner is available. MATERIALS AND METHODS: Using the multipoint saturation recovery technique, we tested four acquisition protocols differing in number and duration of repetition times. Moreover, two mathematical models describing the phenomenon were also evaluated. Protocols and models were tested on a phantom containing 1 kg of distilled water in which were dissolved 5.88 g sodium chloride, 1 cm3 phosphoric acid and 7.58 g sodium phosphate dodecahydrate. One protocol only was used on a group of four healthy volunteers both at rest and during exercise. Each volunteer repeated the rest-exercise sequence three times. RESULTS AND CONCLUSIONS: On the phantom, the average T1 values for the various protocols and mathematical models used proved to differ widely, ranging from 0.61 s to 7.20 s. On volunteers, the T1 values obtained at rest and during exercise were not significantly different--0.91 s on average. Correction of this T1 value with the results obtained using the phantom provides a T1 value of 5.73 s, which is comparable with the value reported in the literature for resting conditions only. PMID- 16683088 TI - Clinical and radiological findings in Kirner's deformity. A report of nine cases. AB - PURPOSE: The purpose of this study is to report radiological findings and evaluate the frequency with which Kirner's deformity is found in a large paediatric population. MATERIALS AND METHODS: We reviewed X-ray films of the hands taken over the past 23.5 years in order to assess not only bone age but all sorts of bone abnormalities (trauma, haematological disorders, bone syndromes and dysplasias, dysmorphic features, rheumatic disorders, etc.) to retrospectively evaluate all those presenting Kirner's deformity. RESULTS: Among the 16,326 patients who underwent X-ray study of the hands, nine (six males and three females, 0.055% of the total) presented the typical radiological features of Kirner's deformity; no patient had a family history of the disorder except for one, in whom the suspicion of familiarity could not be verified radiologically. The diagnosis of Kirner's deformity was straightforward in most cases. In one case, a girl affected by Turner's syndrome, the abnormality was suspected, and its appearance, development and evolution was followed over 10 years. CONCLUSIONS: Although rare, as also shown by our study, Kirner's deformity must be recognised and properly diagnosed in order to spare the subject unnecessary surgical procedures. PMID- 16683089 TI - Role of ultrasonography in detecting mammographically occult breast carcinoma in women with dense breasts. AB - PURPOSE: The purpose of this study was to assess the usefulness of routine ultrasonography in women with negative mammography and dense breasts [Breast Imaging Reporting and Data System (BIRADS D3-4)]. MATERIALS AND METHODS: We applied a protocol involving routine ultrasonography in a consecutive series of subjects with negative mammography and dense breasts. After evaluation by internal and external reviewers of cancers detected by ultrasonography performed to confirm negative mammography, we determined the additional cancer detection rate of ultrasonography and the cost of the protocol. RESULTS: Out of 17,883 total mammographies, 167 cancers were diagnosed (detection rate: 0.93%). Out of 257 suspicious mammographies, 138 cancers were detected. Out of 17,626 negative mammographies, 6,449 (36.5%) were classified as "dense breast" and underwent ultrasonography: 29 cancers were detected (detection rate: 0.44%, or 17.3% of total cancers). Out of 25 cancer cases reviewed, negative mammography and asymptomatic status was confirmed in 15 (detection rate 0.23%, or 8.9% of total cancers). The cancer detection rate was 0.11%, 0.22%, 0.32% and 0.14% for age groups <40, 40-49, 50-59 and >59, respectively. The cost per additional carcinoma detected by ultrasonography alone was euro 25,847.85 whereas that per examined woman was euro 21.68. CONCLUSIONS: The study confirms the possibility that ultrasonography can detect mammographically occult breast carcinoma in dense breasts. The evidence is insufficient to recommend this policy in routine screening practice but suggests that, at least in current clinical practice, adding ultrasonography in dense breasts may be useful despite the substantial costs. PMID- 16683090 TI - Radiological treatment of male varicocele: technical, clinical, seminal and dosimetric aspects. AB - PURPOSE: The purpose of this study was to present our experience with percutaneous treatment of male varicocele considering technical, clinical, seminal and dosimetric aspects. MATERIALS AND METHODS: At baseline and at 6 months' follow-up, 290 male patients aged between 18 and 37 (average age 27.3 years) with left (266 cases) or bilateral (24 cases) varicocele underwent clinical assessment, Doppler ultrasonography (US), laboratory testing of free and total serum testosterone, leutenising hormone (LH) and follicle stimulating hormone (FSH) gonadotropins, inhibin B and spermiogram. In 223 cases, selective catheterisation of the spermatic vein was performed with a right transfemoral approach. Two hundred and six out of 223 underwent radiological treatment of varicocele; in 194, hydroxy-poliethoxydocanol (Aetoxysclerol) was used only whereas in 12 cases (5.8%), 5 ml of absolute alcohol and a Gianturco coil (0.038 in. Cook coil, 10 mmx50 mm) were also used. In 17/223 patients (7.6%), sclerotherapy was contraindicated or not technically feasible. Sixty-seven patients refused radiological treatment and were used as a control group. In 20 patients, the following parameters were measured: dose area product, entrance surface dose, effective dose and gonad dose. RESULTS: Technical success was achieved in 206/223 cases; two phlebographic examinations (immediately following administration of the sclerosing agent and after 15-20 min) showed prethrombotic endoluminal alterations of the internal spermatic vein. At 6 months' follow-up, 172/206 patients (83.49%) showed complete resolution of the varicocele whereas 34/206 (16.5%) had only partial disengorgement of the pampiniform plexus. In these 206 patients, the spermogram showed a significant increase in sperm concentration (52.1+/-4.1 vs. 44.2+/-3.6 million/ml, p=0.002) and motility (40.5+/-2.2 vs. 33.3+/-2.0%, p=0.0001), with negligible morphological changes. In the control group and in the other 17 untreated patients, no variations in seminal parameters were observed. The following minor procedural complications were recorded: two cases of acute abdominal pain, three of vagal crisis during administration of sclerosing agent that resolved spontaneously and two of spermatic cord inflammation that resolved within days after medical therapy. We recorded no statistically significant differences with regard to testicular volume or serum hormone levels between the treated and untreated groups. Maximum effective dose and maximum gonad dose equivalent were 6.9 mSv and 0.69 mSv, respectively. DISCUSSION AND CONCLUSIONS: Percutaneous radiological treatment of varicocele is a minimally invasive technique, which is well tolerated by patients and able to significantly improve seminal parameters. The principal technical limitation to percutaneous treatment is related to difficult selective catheterisation of the spermatic vein due to anatomic alterations, spasms and intimal dissection of the vein. Moreover, when the cremasteric vein is incontinent, inguinal surgical ligation provides better results. In the majority of cases, administration of at least 3 ml sclerosing agent at 3% ensures occlusion of the gonadic vein above the abdominal collaterals, which are responsible for long-term recurrence if not treated. In the remaining cases, absolute alcohol and metallic coils can be used to complete the treatment. The positive results in seminal parameters do not, however, allow for reliable assessment of patients' fertility. Finally, we believe that radiological procedures are not indicated or justified when prolonged catheterisation with elevated gonadic irradiation is needed. PMID- 16683091 TI - Multidetector-row CT angiography of renal artery stenosis in 50 consecutive patients: prospective interobserver comparison with DSA. AB - PURPOSE: The purpose of this study was to establish the diagnostic value of multidetector-row computed tomography (MDCT) angiography compared with digital subtraction angiography (DSA) for detection and quantification of both main and accessory renal artery stenosis in patients with secondary hypertension. MATERIALS AND METHODS: Fifty consecutive patients scheduled for DSA were considered candidates for MDCT angiography. In all patients, MDCT angiography of the abdominal aorta was performed before DSA. For the purpose of interpretation, the arteries were separately interpreted either with DSA or MDCT angiography in order to provide qualitative and quantitative information. For qualitative evaluation, one experienced reader graded the opacification of renal arteries as excellent, good or poor; for quantitative evaluation, MDCT and DSA were independently evaluated for the number of renal arteries and the presence, location and degree of stenosis in random order by three readers. On the basis of consensus readings, calculations of sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) for detection of degree of stenosis were made by using DSA findings as the standard of reference. Interobserver variability was also assessed. RESULTS: With regard to qualitative analysis, arterial enhancement was considered excellent in 39 patients and good in 11. For quantitative analysis, 73 arteries were classified as normal with DSA. Although 72 of these were also classified as normal with CT angiography, one was overestimated by one grade; at DSA, 16 arteries were classified as moderately stenotic; in two arteries, there was an overestimation of one grade. Perfect correlation was achieved for the diagnosis of occlusion. In two patients, all three readers detected multiple severe stenoses on both modalities, with a "string-of-beads" appearance typical of fibromuscular dysplasia. Accessory arteries were correctly identified as such by all three readers on either DSA or MDCT. Levels of sensitivity, specificity and accuracy regarding degree of stenosis were 100%, 98.6% and 96.9%, respectively, with PPV and NPV of 97.6% and 100%, respectively. When we considered significant arterial stenosis (50%-100% luminal narrowing), sensitivity, specificity and accuracy were 100%, 97.3% and 97.8%, respectively, with a PPV and NPV of 98.2% and 97.8%, respectively. For all observers, interobserver agreement was almost perfect (k=0.81-1) for both MDCT and DSA, with a k value between 0.82 and 0.95. CONCLUSIONS: MDCT angiography is very accurate and robust, even for the assessment of renal artery stenosis, and has the potential to become a viable substitute, in most cases, for diagnostic catheter-based DSA. PMID- 16683092 TI - Monitoring of the medical radiological exposures of the population of the Emilia Romagna Region. AB - PURPOSE: The Italian Decree of Law 187/2000 provides for many fulfilments relevant to justification and optimisation of medical exposures that can complicate the daily work of radiology departments if considered as mere legal requirements. On the contrary, this law should be regarded as a good opportunity to analyse and optimise working practices. To this end, the Emilia- Romagna Region carried out an initial assessment of medical exposures to its population in 2001 followed by a second survey taking into account new dosimetric evaluations. This paper illustrates the results of this second survey and analyses the most significant parameters in comparison with similar studies reported in the literature. MATERIALS AND METHODS: We first determined the examinations to be considered: 12 easily identifiable examinations divided into macroaggregates were selected for conventional radiography and computed tomography (CT). Hospitals of the Emilia-Romagna Region were directly asked to provide the number of examinations performed subdivided by type and grouped by nomenclature code, some technical parameters related to both examination protocol and equipment and the value of dose quantities as measured by local medical physicists. RESULTS: Study of distribution of the entrance skin dose for different examinations in single hospitals showed no systematic differences in kilovoltage settings versus dose whereas the number of examinations tended to be inversely proportional to dose. These trends could be explained by the fact that in hospitals where many examinations of the same type are performed, operators, equipment and procedures are well integrated, leading to a level of specialisation that allows efficient interaction in order to deliver an "optimal dose". Analysis of the "entrance skin dosemax"/"entrance skin dosemin" ratios for various projections and comparison with literature data seem to show that a "scale factor" has a fundamental role in the variability of entrance skin dose values amongst hospitals and that "chest" examinations are the most critical, with the greatest differences in entrance skin doses. CONCLUSIONS: The evaluations performed in this study show that this type of analysis heavily relies not only on the cooperation of all professionals responsible for patient radiation protection but also on the experience gained during previous surveys because data collection is a very critical process that can invalidate, if not carefully performed, all subsequent processing. PMID- 16683093 TI - Interrupted aortic arch in a adult: diagnosis with MSCT. AB - Interrupted aortic arch (IAA) is a rare severe congenital heart defect defined as a complete loss of luminal and anatomic continuity between ascending and descending aorta. It is important that we made a good diagnosis and accurate evaluation of their morphologic conditions before a heart operation. We presented an 18-year-old girl of interrupted IAA diagnosed by multi-slice computed tomography angiography. PMID- 16683094 TI - The current and future applications of microorganism in the bioremediation of cyanide contamination. AB - Inorganic cyanide and nitrile compounds are distributed widely in the environment, chiefly as a result of anthropogenic activity but also through cyanide synthesis by a range of organisms including higher plants, fungi and bacteria. The major source of cyanide in soil and water is through the discharge of effluents containing a variety of inorganic cyanide and nitriles. Here the fate of cyanide compounds in soil and water is reviewed, identifying those factors that affect their persistence and which determine whether they are amenable to biological degradation. The exploitation of cyanides by a variety of taxa, as a mechanism to avoid predation or to inhibit competitors has led to the evolution in many organisms of enzymes that catalyse degradation of a range of cyanide compounds. Microorganisms expressing pathways involved in cyanide degradation are briefly reviewed and the current applications of bacteria and fungi in the biodegradation of cyanide contamination in the field are discussed. Finally, recent advances that offer an insight into the potential of microbial systems for the bioremediation of cyanide compounds under a range of environmental conditions are identified, and the future potential of these technologies for the treatment of cyanide pollution is discussed. PMID- 16683104 TI - Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) of the small bowel: a rare cause of intestinal obstruction. PMID- 16683108 TI - Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein-2 (rhBMP-2). A pilot study in dogs. AB - The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2 biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm(2)), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm(2))] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependent manner. PMID- 16683109 TI - Retrograde femoral nailing in elderly patients with supracondylar fracture femur; is it the answer for a clinical problem? AB - BACKGROUND: Supracondylar fractures of femur constitute 7% of all femoral fractures. In elderly patients, they are invariably low-energy fractures predisposed to by osteoporosis. Treatment of these fractures in the elderly is a challenging task for most orthopaedic surgeons. There is no consensus on what would be the ideal treatment for such cases. This study looks at the results of retrograde femoral nailing as a treatment option for this vulnerable group of patients. PATIENTS AND METHODS: This retrospective study looks at 23 elderly patients with supracondylar fractures of the femur treated by retrograde femoral nailing. Patients had an average age of 75 years (range between 65 years and 97 years). All patients were assessed with regard to operative time, blood loss, hospital stay, and postoperative complications. All patients were assessed clinically and radiologically every 6 weeks for average period of 14 months (range 12 to 18 months). RESULTS: two patients died a few weeks postoperatively. Average operative time 70 minutes, average blood loss 350ml. Radiologically all cases united, 39.2% had angular malalignment. There were no cases of implant or fixation failure. CONCLUSION: Retrograde femoral nailing is a surgically limited and reliable procedure for elderly patients with supracondylar fractures of the femur without intra-articular extension. Although it has a high incidence of angular malalignment, the overall functional demands of this age group are perhaps not affected much by that particular complication. PMID- 16683110 TI - Intramedullary knee arthrodesis as a salvage procedure after failed total knee replacement. AB - Septic and aseptic loosening with or without extensive bone loss after total knee replacement are the most common indications for knee fusion. Both external fixation and intramedullary nailing can be used for the treatment, though the latter appears to be the method of choice for most patients. Nine patients were treated after a total knee replacement failure using intramedullary nailing. A long intramedullary nail with a proximal interlocking screw was used in five cases, and a customised nail was used in four cases. Successful fusion occurred in eight of nine patients (89%). Average time for the joint union was 6.5 months, and average operative blood loss was 860 ml. In two patients, iliac crest and patellar bone graft were also used. In conclusion, intramedullary nailing can give excellent results in achieving knee fusion after a failed knee replacement as it allows early weight bearing and at the same time offers stability, pain relief, and a high rate of union, even though the surgical technique is demanding. PMID- 16683112 TI - Bone tunnel enlargement after ACL reconstruction using autologous hamstring tendons: a CT study. AB - PURPOSE: To evaluate prospectively the increase in the size of the tibial and femoral bone tunnel following arthroscopic anterior cruciate ligament (ACL) reconstruction with quadrupled-hamstring autograft. METHODS: Twenty-five consecutive patients underwent arthroscopic ACL reconstruction with quadrupled hamstring autograft. Preoperative clinical evaluation was performed using the Lysholm knee score, Tegner activity level, and International Knee Documentation Committee forms and a KT-1000 arthrometer (side to side). Computed tomography (CT) of the femoral and tibial tunnel was performed on the day after operation in all cases and at mean follow-up of 10 months (range 9-11 months). RESULTS: All of the clinical evaluation scales performed showed an overall improvement. The postoperative anterior laxity difference was <3 mm in 16 patients (70%) and 3-5 mm in seven patients (30%). The mean average femoral tunnel diameter increased significantly (3%) from 9.04+/-0.05 mm postoperatively to 9.3+/-0.8 mm at 10 months; tibial tunnel increased significantly (11%) from 9.03+/-0.04 mm to 10+/ 0.8 mm. There were no statistically significant differences between tunnel enlargement, clinical results, and arthrometer evaluation. CONCLUSIONS: The rate of tunnel widening observed in this study seems to be lower than that reported in previous studies that used different techniques. We conclude that an anatomical surgical technique and a less aggressive rehabilitation process influenced the amount of tunnel enlargement after ACL reconstruction with doubled hamstrings. PMID- 16683114 TI - Estimation of dose to the unborn child at diagnostic X-ray examinations based on data registered in RIS/PACS. AB - The aim of this work was to determine mean absorbed doses to the unborn child in common conventional X-ray and computed tomography (CT) examinations and to find an approach for estimating foetal dose based on data registered in the Radiological Information System/Picture Archive and Communication System (RIS/PACS). The kerma-area product (KAP) and CT dose index (CTDI(vol)) in common examinations were registered using a human-shaped female dosimetry phantom. Foetal doses, D(f), were measured using thermoluminescent dosimeters placed inside the phantom and compared with calculated values. Measured foetal doses were given in relation to the KAP and the CTDI(vol) values, respectively. Conversion factor D(f)/KAP varies between 0.01 and 3.8 mGy/Gycm(2), depending on primary beam position, foetus age and beam quality (tube voltage and filtration). Conversion factors D(f)/CTDI(vol) are in the range 0.02 - 1.2 mGy/mGy, in which the foetus is outside or within the primary beam. We conclude that dose conversion factors based on KAP or CTDI(vol) values automatically generated by the RIS/PACS system can be used for rapid estimations of foetal dose for common examination techniques. PMID- 16683113 TI - High failure rate of a modern, proximally roughened, cemented stem for total hip arthroplasty. AB - The role of surface finish on the survivorship of cemented femoral stems continues to be debated. A total of 34 proximally roughened cemented stems were implanted in 33 consecutive patients undergoing total hip arthroplasty by a single surgeon. An alarmingly high failure rate was observed, prompting a retrospective chart review, analysis of radiographs, and evaluation of retrieved stems and pathological specimens. Nineteen patients were available with more than two years follow-up. Of these 19 patients, nine stems had failed (47%) due to severe osteolysis and stem loosening. Failures were significantly more common in the male gender (p<0.005), and young (p=0.05), tall (p<0.002), and heavy patients (p<0.004). All failed revised hips showed severe metallosis, with both gross and microscopic evidence of metallic shedding from the stems. Our findings suggest that this proximally roughened stem is susceptible to early failure. Failure is characterized by stem debonding, subsidence within the cement mantle, shedding of metallic and cement particles due to fretting, and rapidly progressive osteolysis. These findings have been observed with other rough surface finish cemented stems. PMID- 16683115 TI - Additional value of PET-CT in the staging of lung cancer: comparison with CT alone, PET alone and visual correlation of PET and CT. AB - Integrated positron emission tomography (PET) and computed tomography (CT) is a new imaging modality offering anatomic and metabolic information. The purpose was to evaluate retrospectively the accuracy of integrated PET-CT in the staging of a suggestive lung lesion, comparing this with the accuracy of CT alone, PET alone and visually correlated PET-CT. Fifty patients undergoing integrated PET-CT for staging of a suggestive lung lesion were studied. Their tumor, node, metastasis (TNM) statuses were determined with CT, PET, visually correlated PET-CT and integrated PET-CT. These TNM stages were compared with the surgical TNM status. Integrated PET-CT was the most accurate imaging technique in the assessment of the TNM status. Integrated PET-CT predicted correctly the T status, N status, M status and TNM status in, respectively, 86%, 80%, 98%, 70% versus 68%, 66%,88%, 46% with CT, 46%, 70%, 96%, 30% with PET and 72%, 68%, 96%, 54% with visually correlated PET-CT. T status and N status were overstaged, respectively, in 8% and 16% with integrated PET-CT, in 20% and 28% with CT, in 16% and 20% with PET, in 12% and 20% with visually correlated PET-CT and understaged in 6% and 4% with integrated PET-CT, versus 12% and 6% with CT, 38% and 10% with PET and 12% with visually correlated PET-CT. Integrated PET-CT improves the staging of lung cancer through a better anatomic localization and characterization of lesions and is superior to CT alone and PET alone. If this technique is not available, visual correlation of PET and CT can be a valuable alternative. PMID- 16683116 TI - Transjugular liver biopsy: prospective evaluation of the angle formed between the hepatic veins and the vena cava main axis and modification of a semi-automated biopsy device in cases of an unfavorable angle. AB - In cases of transjugular liver biopsies, the venous angle formed between the chosen hepatic vein and the vena cava main axis in a frontal plane can be large, leading to technical difficulties. In a prospective study including 139 consecutive patients who underwent transjugular liver biopsy using the Quick-Core biopsy set, the mean venous angle was equal to 49.6 degrees. For 21.1% of the patients, two attempts at hepatic venous catheterization failed because the venous angle was too large, with a mean of 69.7 degrees. In all of these patients, manual reshaping of the distal curvature of the stiffening metallic cannula, by forming a new mean angle equal to 48 degrees , allowed successful completion of the procedure in less than 10 min. PMID- 16683117 TI - MRI-guided needle localization of suspicious breast lesions: results of a freehand technique. AB - Magnetic resonance imaging (MRI) can detect clinically and mammographically occult breast lesions. In this study we report the results of MRI-guided needle localization of suspicious breast lesions by using a freehand technique. Preoperative MRI-guided single-needle localization was performed in 220 patients with 304 MRI-only breast lesions at our hospital between January 1997 and July 2004. Procedures were performed in an open 0.5-T Signa-SP imager allowing real time monitoring, with patient in prone position, by using a dedicated breast coil. MRI-compatible hookwires were placed in a noncompressed breast by using a freehand technique. MRI findings were correlated with pathology and follow-up. MRI-guided needle localization was performed for a single lesion in 150 patients, for two lesions in 56 patients, and for three lesions in 14 patients. Histopathologic analysis of these 304 lesions showed 104 (34%) malignant lesions, 51 (17%) high-risk lesions, and 149 (49%) benign lesions. The overall lesion size ranged from 2.0-65.0 mm (mean 11.2 mm). No direct complications occurred. Follow up MRI in 54 patients showed that two (3.7%) lesions were missed by surgical biopsy. MRI-guided freehand needle localization is accurate and allows localization of lesions anterior in the breast, the axillary region, and near the chest wall. PMID- 16683118 TI - Evaluating a three dimensional model of diffuse photosynthetically active radiation in maize canopies. AB - Diffuse photosynthetically active radiation (DPAR) is important during overcast days and for plant parts shaded from the direct beam radiation. Simulation of DPAR interception by individual plant parts of a canopy, separately from direct beam photosynthetically active radiation (PAR), may give important insights into plant ecology. This paper presents a model to simulate the interception of DPAR in plant canopies. A sub-model of a virtual maize canopy was reconstructed. Plant surfaces were represented as small triangular facets positioned according to three-dimensionally (3D) digitized data collected in the field. Then a second sub model to simulate the 3D DPAR distribution in the canopy was developed by dividing the sky hemisphere into a grid of fine cells that allowed for the anisotropic distribution of DPAR over the sky hemisphere. This model, DSHP (Dividing Sky Hemisphere with Projecting), simulates which DSH (Divided Sky Hemisphere) cells are directly visible from a facet in the virtual canopy, i.e. not obscured by other facets. The DPAR reaching the center of a facet was calculated by summing the amounts of DPAR present in every DSH cell. The distribution of DPAR in a canopy was obtained from the calculated DPARs intercepted by all facets in the canopy. This DSHP model was validated against DPAR measurements made in an actual maize (Zea mays L.) canopy over selected days during the early filling stage. The simulated and measured DPAR at different canopy depths showed a good agreement with a R (2) equaling 0.78 (n=120). PMID- 16683119 TI - Immunohistochemistry of whole-organ sections of advanced human laryngeal cancer. AB - Whole-mount sections have been in the interest of laryngologists for long time. The aim of this study was to demonstrate the technical aspects of processing horizontal whole-mount sections of advanced laryngeal cancer specimens after total laryngectomy. Those sections may provide new insights in the biology of laryngeal cancer. Six excised human larynges were block-embedded in paraffine. Serial sections were obtained as thin as 9 mum. Sections were stained by Giemsa and standard immunohistochemistry protocols with commercial antibodies against Cytokeratine5/6, Ki-67, Topoisomerase IIalpha, and p53. Four high-power fields were selected randomly in each section of a surface grid and the percentage of positive tumor cells was noted for each antibody in the respective field. Morphometric surface maps of protein expression were generated for each parameter. The tissues remained intact without major artifacts. Specific characteristics of the tumors were identified after evaluation of the whole-mount sections. Staining of cytokeratine was homogenous, whereas nuclear markers showed a distinct heterogeneity in the respective staining patterns. By analyzation of color-coded fusion images the spatial expression of the respective antibodies could be visualized. PMID- 16683120 TI - Atypical haemangioma of the internal auditory meatus: a case report. AB - Haemangiomas involving the internal auditory meatus (IAM) are rare and can mimic other frequent lesions of the IAM such as schwannomas by their clinical and imaging aspects. The case of a patient with an atypical IAM haemangioma fluctuating in size is reported to highlight this diagnostic possibility. A 36 year-old female presented with a sudden and recurrent left sensorineural hearing loss (SNHL). Three consecutive MRIs were performed in a 10-month period of preoperative observation. They showed a fluctuation of the tumour signal and size. The surgical removal was performed via a translabyrinthine approach. Pathological findings were consistent with the diagnosis of a heamangioma. MRI and CT findings suggested the diagnosis of IAM heamangioma, but the rapid variation in size and signal was misleading. This phenomenon may be due to haemorrhage or oedema, and can be accompanied by a deterioration of the hearing function. Early surgical resection is the treatment of choice and allows to confirm the diagnosis. PMID- 16683121 TI - The cost-utility of lumbar disc herniation surgery. AB - The cost and utility of surgery for a herniated lumbar disc has not been determined simultaneously in a single cohort. The aim of this study is to perform a cost-utility analysis of surgical and nonsurgical treatment of patients with lumbar disc herniation. Ninety-two individuals in a cohort of 1,146 Swedish subjects underwent lumbar disc herniation surgery during a 2-year study. Each person operated on was individually matched with one treated conservatively. The effects and costs of the treatments were determined individually. By estimating quality of life before and after the treatment, the number of quality adjusted life years (QALY) gained with and without surgery was calculated. The medical costs were much higher for surgical treatment; however, the total costs, including disability costs, were lower among those treated surgically. Surgery meant fewer recurrences and less permanent disability benefits. The gain in QALY was ten times higher among those operated. Lower total costs and better utility resulted in a better cost utility for surgical treatment. Surgery for lumbar disc herniation was cost-effective. The total costs for surgery were lower due to lower recurrence rates and fewer disability benefits, and surgery improved quality of life much more than nonsurgical treatments. PMID- 16683122 TI - Assessment of CAOS as a training model in spinal surgery: a randomised study. AB - The objectives of this study were (1) to quantify the benefit of computer assisted orthopaedic surgery (CAOS) pedicle screw insertion in a porcine cadaver model evaluated by dissection and computed tomography (CT); (2) to compare the effect on performance of four surgeons with no experience of CAOS, and varying experience of pedicle screw insertion; (3) to see if CT with extended windows was an acceptable method to evaluate the position of the pedicle screws in the porcine cadaver model, compared to dissection. This was a prospective, randomised, controlled and blinded porcine cadaver study. Twelve 6-month-old porcine (white skinned Landrace) lumbar spines were scanned pre-operatively by spiral CT, as required for the CAOS computer data set. Computer randomisation allocated the specimens to one of four surgeons, all new to CAOS but with different levels of experience in spinal surgery. The usual anatomical landmarks for the freehand technique were known to all four surgeons. Two pedicles at each vertebral level were randomly allocated between conventional free hand insertion and an electromagnetic image guided surgery (NAVITRAK) and 6.5 mm cancellous AO screws inserted. Post-operatively, spiral CT was blindly evaluated by an independent radiologist and the spine fellow to assess the accuracy of pedicle screw placement, by each method. The inter- and intra-observer reliability of CT was evaluated compared to dissection. The pedicle screw placement was assessed as perfect if within the pedicle along its central axis, or acceptable (within < 2 mm from perfect), and measured in millimetres from perfect thereafter. One hundred and sixty-six of 168 pedicles in 12 porcine spines were operated on. Complete data were present for 163 pedicles (81 CAOS, 82 freehand). In the CAOS group 84% of screws were deemed acceptable or perfect, compared to 75.6% with the freehand technique. Screw misplacement was significantly reduced using CAOS (P = 0.049). Seventy-nine percent of CAOS screws were ideally placed compared with 64% with a conventional freehand technique (P = 0.05). A logistic linear regression model showed that the miss placed pedicle screw rate was significantly reduced using CAOS (P = 0.047). CAOS benefited the least experienced surgeons most (the research registrars acceptable rate increased from 70 to 90% and the spine fellow from 76 to 86%). CAOS did not have a statistically significant effect on the experienced consultant spine surgeon increasing from 70 to 79% (P = 0.39). The experienced general orthopaedic surgeon did not benefit from CAOS (P = 0.5). CT compared to dissection showed an intra-observer reliability of 99.4% and inter observer reliability of 92.6%. The conclusions of this study were as follows: (1) an increased number of pedicle screws were ideally placed using the CAOS electromagnetic guidance system compared to the conventional freehand technique; (2) junior surgeons benefited most from CAOS; (3) we believe CAOS (Navitrak) with porcine lumbar spines evaluated by post operative CT, represents a useful model for training junior surgeons in pedicle screw placement; (4) experienced spine surgeons, who have never used CAOS, may find CAOS less helpful than previously reported. PMID- 16683124 TI - Influence of different transition metals in phthalocyanines on their interaction energies with volatile organic compounds: an experimental and computational study. AB - Experimental partition coefficients were determined for a series of volatile organic compounds (VOCs) (acetonitrile, n-butylamine, n-octane tetrachloroethene, and toluene) for the interaction with 2,3,9,10,16,17,23,24-octakis(octyloxy) phthalocyaninato complexes, PcM(OR)(8), with varying central metal atoms [M=H(2) (metal-free), Ni, Pd, Cu, Zn]. Large partition coefficients for toluene were observed in the case of the nickel and palladium phthalocyanines, whereas for the corresponding zinc-containing compound, interaction with n-butylamine resulted in a high value for the partition coefficient. Interaction energies for model coordination complexes were obtained at the ab initio LMP2/ LACVP* level of theory. The interaction of various small volatiles with the various PcM(OR)(8) compounds was studied using the PM3 semiempirical Hamiltonian. Large values for interaction energies correspond to particularly strong partition coefficients, suggesting that coordination of the volatiles to the central metal dominates over the often discussed pi-system stacking at the PcM(OR)(8)'s. PMID- 16683125 TI - Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides. AB - The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C18 chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product's structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides. PMID- 16683126 TI - Biodegradation of 8-anilino-1-naphthalenesulfonic acid by Pseudomonas aeruginosa. AB - Pseudomonas aeruginosa, isolated from soil near tannery effluent was able to degrade 8-anilino-1-naphthalenesulfonic acid (ANSA), a sulfonated aromatic amine. The organism degraded this amine up to a concentration of 1,200 mg l(-1) using glucose and ammonium nitrate as carbon and nitrogen sources respectively. The degradation started when the organism reached its late exponential growth phase. Salicylic acid and beta-ketoadipic acid were identified as intermediate compounds using HPLC and GC-MS and provide evidence for ortho pathway reactions. Further proof for the pathway is obtained from the dioxygenase activity of the strain growing exponentially in medium with ANSA and glucose. PMID- 16683128 TI - Biodegradability of end-groups of the biocide polyhexamethylene biguanide (PHMB) assessed using model compounds. AB - Polyhexamethylene biguanide (PHMB), a biocide used in a wide variety of disinfection and preservation applications, is a polydisperse mixture in which the end-groups may be any combination of amine, guanidine and cyanoguanidine. Using PHMB model compounds (1,6-diaminohexane; 1,6-diguanidinohexane; 1,6 di(cyanoguanidino)hexane; 4-guanidinobutyric acid), we have determined the biodegradation characteristics of each end-group in several strains of bacteria isolated for their ability to utilise PHMB as a sole source of nitrogen. Bacteria were screened for growth at the expense of each model compound (at non-inhibitory concentrations) as sole nitrogen source. None of the isolated bacteria was capable of utilising a cyanoguanidine end-group as growth substrate, whereas several bacteria were shown to utilise amine or guanidine end-groups. In particular, a strain of Pseudomonas putida was capable of extensive growth with 1,6-diguanidinohexane as a sole nitrogen source, with complete removal of guanidine groups from culture medium within 2 days, and with concomitant formation of unsubstituted urea, which in turn was also utilised by the organism. We conclude that whilst amine and guanidine end-groups in PHMB are likely to be susceptible to biodegradation, cyanoguanidine end-groups are likely to be recalcitrant. PMID- 16683129 TI - [The medial approach for total hip replacement]. AB - BACKGROUND: In an anatomical study we investigated the medial approach to the hip joint and developed the surgical technique for patients with hip joint replacement. MATERIAL AND METHODS: The temporary detachment of the adductor longus tendon allows a perfect exposure of the surgical site without risk of damaging important structures. RESULTS: The operation is possible with normal instruments. The blood loss is little. CONCLUSION: The abductor apparatus (gluteal muscles and fascia lata) and the superior gluteal nerve remain intact. Immediate lateral positioning is allowed after surgery. The scar is unremarkable. PMID- 16683130 TI - [Conventional vs minimally invasive total hip arthroplasty. A prospective study of rehabilitation and complications]. AB - BACKGROUND: In a prospective, nonrandomized study the outcome in terms of rehabilitation and complications of total hip arthroplasty (THA) through a superior capsulotomy exposure (study group) was compared to THA performed through a direct lateral exposure (control group). PATIENTS AND METHODS: The study group (106 THA) and the control group (107 THA) were controlled for complexity and had no significant differences in age, sex, diagnosis, or body mass index. RESULTS: The study group had improved recovery at 6 weeks after surgery which was statistically significant (p<0.001). In addition, the study group had a lower incidence of perioperative complications. CONCLUSION: The current study demonstrates the potential that less-invasive surgical techniques with the philosophy of maximally preserving the abductors, posterior capsule, and short rotators may result in a safer operation with an accelerated recovery. PMID- 16683131 TI - Association of a polymorphism in the betacellulin gene with type 1 diabetes mellitus in two populations. AB - Betacellulin, a member of the epidermal growth factor family, is expressed in fetal and adult pancreas. In vitro and in vivo studies suggest a role for betacellulin in islet neogenesis and regeneration. Therefore, a mutation in the betacellulin gene might lead to fewer beta cells. With reduced beta cell reserve, beta cells may not be able to compensate for an autoimmune attack, and in turn, susceptibility to type 1 diabetes mellitus (T1DM) would increase. Previous mutational analysis identified seven polymorphisms in the betacellulin gene [5' UT (-233G>C, -226A>G), exon 1 (TGC19GGC, Cys7Gly), exon 2 (CTC130TTC, Leu44Phe), exon 4 (TTG370ATG, Leu124Met), intron 2 (-31T>C), and intron 4 (-4C>T)]. An association study of these variants with T1DM was first carried out in 100 Caucasian subjects with T1DM and 282 Caucasian subjects without diabetes recruited at the University of Maryland. The frequency of the intron 4 T-4 allele was significantly higher among nondiabetic controls than that among diabetic cases (0.29 vs 0.21, p=0.04). Allele frequencies for the other polymorphisms did not differ significantly between cases and controls. The intron 4 T-4 association was then replicated by transmission disequilibrium testing in a separate population of Caucasian parent/offspring with T1DM trios (n=168 trios, 113 informative) recruited at the Medical College of Wisconsin (p=0.024). An interaction of the intron 4 T-4 allele and human leukocyte antigen (HLA) was also detected with undertransmission of the T allele in those T1DM subjects with susceptible HLA types as compared to those T1DM subjects without susceptible HLA types (p=0.018). RNA studies of the intron T-4 variant showed similar RNA levels for intron 4 T-4 and intron 4 C-4 alleles. Additionally, there was no evidence for an effect of this variant on exon-intron splicing. We conclude that the intron 4 T-4 allele in the betacellulin gene is associated with lower risk of T1DM and may interact with HLA. Further studies will be necessary to establish the significance of this association. PMID- 16683133 TI - Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains. AB - We improved the hydrogen yield from glucose using a genetically modified Escherichia coli. E. coli strain SR15 (DeltaldhA, DeltafrdBC), in which glucose metabolism was directed to pyruvate formate lyase (PFL), was constructed. The hydrogen yield of wild-type strain of 1.08 mol/mol glucose, was enhanced to 1.82 mol/mol glucose in strain SR15. This figure is greater than 90 % of the theoretical hydrogen yield of facultative anaerobes (2.0 mol/mol glucose). Moreover, the specific hydrogen production rate of strain SR15 (13.4 mmol h(-1) g(-1) dry cell) was 1.4-fold higher than that of wild-type strain. In addition, the volumetric hydrogen production rate increased using the process where cells behaved as an effective catalyst. At 94.3 g dry cell/l, a productivity of 793 mmol h(-1) l(-1) (20.2 l h(-1) l(-1) at 37 degrees C) was achieved using SR15. The reported productivity substantially surpasses that of conventional biological hydrogen production processes and can be a trigger for practical applications. PMID- 16683134 TI - Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2. AB - We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO(3) in the reaction mixture. Only 5 or 48 % of 1,3 dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO(3), respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction. PMID- 16683135 TI - Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. AB - Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone. Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability. They have become a general and versatile tool for DNA and RNA recognition. LNA is a general and versatile tool for specific, high-affinity recognition of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). LNA can be used for designing LNA oligoes for hybridization studies or as real time polymerase chain reaction probes in the form of Taqman probes. LNA also has therapeutic and diagnostic applications. PNA is another type of DNA analog with neutral charge. The extreme stability of PNA makes it an ideal candidate for the antisense and antigene application. PNA is used as probe for gene cloning, mutation detection, and in homologous recombination studies. It was also used to design transcription factor decoy molecules for target gene induction. Morpholino, another structural type, was devised to circumvent cost problems associated with DNA analogs. It has become the premier knockdown tool in developmental biology due to its cytosolic delivery in the embryos by microinjection. Thus, the nucleic acid analogs provide an advantage to design and implementation, therapies, and research assays, which were not implemented due to limitations associated with standard nucleic acids chemistry. PMID- 16683136 TI - Spatial coding capacity of central otolith neurons. AB - This review focuses on recent approaches to unravel the capacity of otolith related brainstem neurons for coding head orientations. In the first section, the spatiotemporal features of central vestibular neurons in response to natural otolithic stimulation are reviewed. Experiments that reveal convergent inputs from bilateral vestibular end organs bear important implications on the processing of spatiotemporal signals and integration of head orientational signals within central otolith neurons. Another section covers the maturation profile of central otolith neurons in the recognition of spatial information. Postnatal changes in the distribution pattern of neuronal subpopulations that subserve the horizontal and vertical otolith systems are highlighted. Lastly, the expression pattern of glutamate receptor subunits and neurotrophin receptors in otolith-related neurons within the vestibular nuclear complex are reviewed in relation to the potential roles of these receptors in the development of vestibular function. PMID- 16683137 TI - Visual motion detection in hierarchical spatial frames of reference. AB - Neurophysiological and neuroimaging work has uncovered modulatory influence of long-range lateral connections from outside of the classical receptive field on neuronal and behavioral responses to localized targets. We report two psychophysical experiments investigating visual detection of real and apparent motion in central vision with and without remote and immediate stationary references. At a particular temporal frequency (0.1-12.8 Hz), participants adjusted the amplitude of either triangle-wave (real) or square-wave (stroboscopic/apparent) oscillatory motion of a vertical bar along a straight, horizontal trajectory for the first impression of the target's stationarity/nonstationarity (the displacement threshold). In the relative motion conditions, a stationary reference bar was positioned 23' apart from the target; in the absolute motion conditions, the bar was absent. The thresholds were measured with a dimly-lit uniform background (13 x 13 degrees ) and either in the darkness (experiment 1) or moving-background conditions (experiment 2). For both real and apparent motion, varying the observation conditions yields three sensitivity levels: irrespective of the background, the lowest thresholds occur in the presence of an immediate reference, followed by the moderately increased thresholds obtained with a dimly-lit background alone. The equally high thresholds occur in the darkness and moving-background conditions without any visible stationary references. The results suggest that the spatial frames of reference for visual motion detection are hierarchically nested, yet independent. The findings provide support for the view that absolute motion perception should be considered relative, extending neurophysiological evidence for the existence of long-range lateral connections across the visual field. PMID- 16683138 TI - The effects of repetitive transcranial magnetic stimulation on cortical inhibition in healthy human subjects. AB - It has been suggested that the therapeutic effects of repetitive transcranial magnetic stimulation (rTMS) are mediated through changes in cortical inhibition (CI). However, in healthy human subjects the effects of rTMS on CI have been inconsistent. Therefore, this study sought to improve on the methodological limitations of previous studies by exploring several different rTMS-stimulus conditions on inhibition in the human motor cortex. In the first experiment, 12 healthy control subjects were randomly assigned to receive regular 1, 10 or 20 Hz rTMS in a counterbalanced order with sessions separated by at least 1 week. In the second experiment, 10 of these 12 subjects received priming rTMS (600 stimuli at 6 Hz followed by 600 stimuli at 1 Hz). Cortical inhibition was indexed using short-interval intracortical inhibition (SICI) and cortical silent period (CSP). Corticospinal excitability was indexed using motor threshold and MEP amplitude. We found no significant overall change in SICI, although there was a significant correlation between changes in SICI with baseline SICI. Subjects with greater SICI at baseline tended to have reduction in SICI post-rTMS, whereas subjects with less SICI tended to have increase in SICI post-rTMS. There was also a significant lengthening of the CSP with higher stimulation frequencies compared to lower stimulation frequencies. These findings suggest that rTMS increases CI, particularly in subjects with reduced baseline inhibition, a finding consistent with the concept of homeostatic plasticity. Baseline physiological characteristics may be further explored as a method to select patients who may benefit from rTMS treatment. PMID- 16683139 TI - Individual variation in feeding habitat use by adult female green sea turtles (Chelonia mydas): are they obligately neritic herbivores? AB - Satellite telemetry and stable isotope analysis were used to confirm that oceanic areas (where water depths are >200 m) are alternative feeding habitats for adult female green sea turtles (Chelonia mydas), which have been thought to be obligate herbivores in neritic areas (where depths are <200 m). Four females were tagged with satellite transmitters and tracked during post-nesting periods from Ogasawara Islands, Japan. Three females migrated to neritic habitats, while transmissions from another female ceased in an oceanic habitat. The overall mean nighttime dive depths during oceanic swimming periods in two females were <20 m, implying that the main function of their nighttime dives were resting with neutral buoyancy, whereas the means in two other females were >20 m, implying that they not only rested, but also foraged on macroplankton that exhibit diel vertical migration. Comparisons of stable carbon and nitrogen isotope ratios between 89 females and the prey items in a three-source mixing model estimated that 69% of the females nesting on Ogasawara Islands mainly used neritic habitats and 31% mainly used oceanic habitats. Out of four females tracked by satellite, two females were inferred from isotope ratios to be neritic herbivores and the two others oceanic planktivores. Although post-nesting movements for four females were not completely consistent with the inferences from isotope ratios, possibly due to short tracking periods (28-42 days), their diving behaviors were consistent with the inferences. There were no relationships between body size and the two isotope ratios, indicating a lack of size-related differences in feeding habitat use by adult female green turtles, which was in contrast with loggerhead sea turtles (Caretta caretta). These results and previous findings suggest that ontogenetic habitat shifts by sea turtles are facultative, and consequently, their life histories are polymorphic. PMID- 16683140 TI - DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine. AB - Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5 azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 microM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G1 phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 microM 5-azaC may have enhanced potential for porcine NT. PMID- 16683146 TI - Short-term preoperative radiotherapy in rectal cancer patients leads to a reduction of the detectable number of lymph nodes in resection specimens. AB - BACKGROUND AND AIMS: The Union Internationale Contre le Cancer and American Joint Committee on Cancer classification propose that pN(0)-classified colorectal lymphadenectomy specimens will ordinarily include 12 or more tumor-negative lymph nodes. We performed a clinical trial to investigate whether a short-term preoperative radiotherapy (5x5 Gy) leads to a reduction of the number of lymph nodes in rectal cancer specimens after total and partial mesorectal excision (TME and PME, respectively). MATERIALS AND METHODS: Within a 5-year period, 28 (15%) of 148 rectal cancer patients underwent hypofractionated preoperative radiotherapy in this monocenter study, whereas 120 patients (85%) underwent TME/PME surgery alone. The main criterion was the number of lymph nodes in TME/PME specimens. We used a stratified one-sided Wilcoxon-Mann-Whitney test to test for a significant difference in the number of lymph nodes, stratifying for tumor location and postoperative tumor stage. Patients who were suspected of having any alterations in the number of pelvic lymph nodes were excluded from the study. RESULTS: Fewer lymph nodes were detected in the TME/PME specimens of patients who received hypofractionated preoperative radiotherapy compared to patients who underwent TME/PME surgery alone (12 detectable lymph nodes vs 15; p=0.0005). Tumor location (p=0.095) and tumor stage (p=0.093) did not significantly influence the number of lymph nodes in this study. CONCLUSIONS: We conclude that a 5x5 Gy short-term preoperative radiotherapy leads to a reduction in the number of lymph nodes in TME/PME specimens. Because neoadjuvant therapy in rectal cancer for T(2) and T(3) tumors has advanced a new therapeutic standard procedure, in the future, less lymph nodes will be detected in TME/PME specimens. This might influence the required number of lymph nodes in current staging systems for rectal cancer in the future. PMID- 16683147 TI - Successful doxycycline treatment of lymphatic fistulas: report of five cases and review of the literature. AB - BACKGROUND AND AIMS: Lymphatic fistulas are commonly known complications after lymph node or soft tissue resection, requiring long and sometimes difficult therapy. Doxycycline is an antibiotic agent, which was used in the treatment of pneumothoraces and pleural effusions. The aim of the study was to evaluate the effect of local doxycycline instillation on postoperative lymphatic effusions. MATERIALS AND METHODS: Five patients with postoperative lymphatic fistulas of different locations (inguinal, gluteal, and thoracic) were included. Patients received local doxycycline instillation after a postoperative observation period of 19-27 days without spontaneous closure of the fistulas. RESULTS: Postoperative lymphatic fistulas closed after a single injection of doxycycline in four out of five patients within 2 days, in one patient, injection had to be repeated to achieve closure of the fistula. Drain removal was possible within 4 days after instillation in all patients; no complications occurred. CONCLUSIONS: Doxycycline can be used for treatment of persistent postoperative lymphatic fistulas in different locations. In further studies, this therapeutic concept should be evaluated with larger numbers of patients with regard to the ideal point of time, frequency, and mode of application. PMID- 16683151 TI - [Implementing knowledge. Tasks and activities of Working Group 3 on "Healthy Aging" of the German Platform for Prevention and Health Promotion]. AB - It is indisputable that preventive and health-promoting interventions are also effective for older people. In order to put this knowledge into practice, several important questions have to be answered first. Which preventive goals have priority? Which contents have to be conveyed in the context of "healthy aging"? Which strategies of intervention have proven effective so far? How can older people be addressed in their surroundings? Working Group 3 on "Healthy Aging" of the German Forum/Platform for Prevention and Health Promotion is currently discussing these questions. The following article presents the proceedings as well as the results of Working Group 3 up to now. PMID- 16683155 TI - [Long-term experience with Cohen ureteral reimplantation in bilateral vesicoureteral reflux in childhood]. AB - INTRODUCTION: In bilateral VUR, Cohen cross-trigonal ureteric reimplantation is a popular but also controversial surgical approach. We present our own experience in a retrospective analysis. PATIENTS AND METHODS: Between 1990 and 2005, 41 children (26 girls, 15 boys) with bilateral reflux [92 renal units (RU)] underwent ureteric reimplantation. The mean age was 4.5 (0.3-12) years. Eight patients had ureteral duplication (six unilateral, two bilateral); 12 of 41 patients had no intraoperative ureteral stenting. Seven patients had prior surgery for VUR. A successful result was defined as absence of VUR, significant UVJ obstruction, or voiding dysfunction throughout the follow-up. RESULTS: The mean follow-up was 7.8 (0.5-15) years. Eight patients (19.5%) had 13 complications. One patient had an intraoperative small bowel lesion (2%). Six patients (14.6%) had UTI. Four patients (9.8%) showed transient UVJ obstruction. Three required a temporary percutaneous nephrostomy. Two of these patients had no intraoperative ureteral stenting. Recurrence of VUR was found in 2 patients (4.8%) and 2/92 RU (2.2%), respectively. Complications were more frequent in high grade VUR, ureter duplex, or unstented ureteral reimplantation. Prior surgery for VUR did not influence the postoperative outcome. Postoperative voiding disorders were not observed. CONCLUSIONS: Two unilateral recurrences of VUR were observed, requiring a reoperation in one patient. A reoperation for UVJ obstruction was not necessary. Related to 92 RU the surgical success rate was 97.8%. Intraoperative ureteral stenting has to be considered with respect to the current discussion of shortening inpatient procedures. In our experience, the perioperative risk was elevated in patients with high-grade VUR or ureteral duplication. PMID- 16683157 TI - Three-dimensional volume rendering of tibiofibular joint space and quantitative analysis of change in volume due to tibiofibular syndesmosis diastases. AB - OBJECTIVE: The diagnosis of ankle syndesmosis injuries is made by various imaging techniques. The present study was undertaken to examine whether the three dimensional reconstruction of axial CT images and calculation of the volume of tibiofibular joint space enhances the sensitivity of diastases diagnoses or not. DESIGN: Six adult cadaveric ankle specimens were used for spiral CT-scan assessment of tibiofibular syndesmosis. After the specimens were dissected, external fixation was performed and diastases of 1, 2, and 3 mm was simulated by a precalibrated device. Helical CT scans were obtained with 1.0-mm slice thickness. The data was transferred to the computer software AcquariusNET. Then the contours of the tibiofibular syndesmosis joint space were outlined on each axial CT slice and the collection of these slices were stacked using the computer software AutoCAD 2005, according to the spatial arrangement and geometrical coordinates between each slice, to produce a three-dimensional reconstruction of the joint space. The area of each slice and the volume of the entire tibiofibular joint space were calculated. The tibiofibular joint space at the 10th-mm slice level was also measured on axial CT scan images at normal, 1, 2 and 3-mm joint space diastases. RESULTS: The three-dimensional volume-rendering of the tibiofibular syndesmosis joint space from the spiral CT data demonstrated the shape of the joint space and has been found to be a sensitive method for calculating joint space volume. We found that, from normal to 1 mm, a 1-mm diastasis increases approximately 43% of the joint space volume, while from 1 to 3 mm, there is about a 20% increase for each 1-mm increase. CONCLUSIONS: Volume calculation using this method can be performed in cases of syndesmotic instability after ankle injuries and for preoperative and postoperative evaluation of the integrity of the tibiofibular syndesmosis. PMID- 16683160 TI - Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simple to compound. AB - Seed plants with compound leaves constitute a polyphyletic group, but studies of diverse taxa show that genes of the class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) family are often involved in compound leaf development. This suggests that knox1 genes have been recruited on multiple occasions during angiosperm evolution (Bharathan et al. in Science 296:1858-1860, 2002). In agreement with this, we demonstrate that the simple leaf of dandelion (Taraxacum officinale Web.) can be converted into a compound leaf by the constitutive expression of heterologous knox1 genes. Dandelion is a rosette plant of the family Asteraceae, characterised by simple leaves with deeply lobed margins and endogenous knox1 gene expression. Transgenic dandelion plants constitutively expressing the barley (Hordeum vulgare L.) hooded gene (bkn3, barley knox3) or the related bkn1 gene, developed compound leaves featuring epiphyllous rosettes. We discuss these results in the context of two current models of compound leaf formation. PMID- 16683161 TI - Strawberry fruit protein with a novel indole-acyl modification. AB - Achenes and receptacle tissue of Fragaria vesca, L. cultivar Yellow Wonder were shown to contain conjugated indole-3-acetic acid (IAA) that was not soluble in organic solvents and yielded IAA after strong alkaline hydrolysis, suggestive of IAA attached to plant proteins. This solvent insoluble conjugated IAA accounted for between 0.4 and 4 ng of IAA per gram fresh weight of tissue in both achenes and receptacles. To investigate this strawberry conjugate class further, a polyclonal antibody was produced to IAA-glycine attached to BSA that detected neutral indole acid esters, monocarboxylic-amino acid IAA conjugates and IAA proteins. Using immunoblotting, both achenes and receptacles of strawberry were shown to have primarily an immuno-detectable band at 76 kDa. Two-dimensional polyacrylamide gel electrophoresis yielded a wide band that was analyzed by LC MS/MS analysis following in-gel trypsin digestion. Peptides derived from the immuno-detectable band were tentatively identified by peptide fragment analysis as being from either a chaperonin related to the hsp60 class of proteins or, alternatively, an ATP synthase. This is one of the first reports of an IAA modified protein in fruit tissue. PMID- 16683165 TI - [Prof. Dr. medicine em. Mogens Schou (1918-2005)]. PMID- 16683163 TI - Role of with-no-lysine [K] kinases in the pathogenesis of Gordon's syndrome. AB - Gordon's syndrome, also known as pseudohypoaldosteronism type II (PHA II) or familial hypertension with hyperkalemia, is an autosomal-dominant disease characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis, and normal glomerular filtration rate. Recent positional cloning has linked mutations of WNK1 and WNK4 to Gordon's syndrome. With-no-lysine [K] (WNK) kinases are a new family of large serine-threonine protein kinases with an atypical placement of the catalytic lysine. Here, we review the pathogenesis of PHA II based on current understanding of the actions of WNK1 and WNK4 on Na+ and K+ handling in the renal distal tubule. PMID- 16683168 TI - [Morphology, family history, and age at diagnosis of 26 patients with Axenfeld Rieger syndrome and glaucoma or ocular hypertension]. AB - PURPOSE: The Axenfeld-Rieger syndrome (ARS) shows genetic and morphologic heterogeneity and is associated with glaucoma in 50% of the patients. METHODS: Ocular, dental, and systemic anomalies, maximum intraocular pressure (IOPmax), frequency of ARS or glaucoma in the family history (FH), and age at diagnosis (AAD) of 26 consecutively examined patients with ARS and glaucoma or elevated IOP were evaluated retrospectively. RESULTS: In 65.4% of the patients hypoplasia of the iris was found. Almost 50% of the patients had systemic anomalies, dental anomalies being the most frequent. Of 26 patients, 12 (46.2%) had an ARS in the FH; 57.7% of the patients had a FH of glaucoma with or without ARS. No significant differences in IOPmax and frequency of iris hypoplasia and glaucoma surgery were found when patients with and without ARS in their FH and patients with and without iris hypoplasia were compared. CONCLUSION: Patients with iris hypoplasia and patients with ARS and/or glaucoma in the FH do not show a worse glaucoma prognosis than patients without iris hypoplasia or without a FH of glaucoma or ARS. ARS was diagnosed within the 1st year of life in approximately half of the patients. Therefore, children of ARS patients should be screened as soon as possible to improve early diagnosis of ARS and glaucoma and to improve glaucoma prognosis. As the dental and facial anomalies may require treatment in the first dentition, patients with ARS should be referred to a dentist or orthodontist. PMID- 16683169 TI - [In vivo imaging of the conjunctival epithelium using confocal laser scanning microscopy]. AB - BACKGROUND: In various ocular diseases, cytomorphological findings of the ocular surface are an essential component of clinical diagnostics. When evaluating the conjunctival epithelium, minimally invasive acquisition of biomaterial is necessary for lab and technical processing and in vitro histological examination. To examine corneal structures in vivo, confocal laser scanning microscopy is a successful standard method. Our aim was to employ in vivo confocal laser scanning microscopy also for examining the conjunctival epithelium. MATERIAL AND METHOD: Results were analyzed and compared with cytomorphological findings of impression cytology. Accordingly, the basic features of conjunctival in vivo examination using RLSM were described and defined. In vivo images were analyzed and compared with impression cytological slide preparations (n=110) of 23 healthy test persons. Examination was standardized. Finally, the confocal laser scan images were compared to the impression cytological patterns. RESULTS: Due to the distribution of reflectors (pixel brightness), diagnostic analysis of important morphological structures (cell nucleus, cytoplasm, nucleus/plasma relation) of the conjunctiva is possible. Secretory cells of the epithelium (goblet cells) can be easily recognized by their size. Highly reflective pixels depict cell walls or wide intercellular spaces with high contrast. CONCLUSIONS: The in vivo investigation of important anatomical and morphological structures of the conjunctival epithelium is possible using RLSM. The distribution pattern of goblet cell pixel brightness may correlate with various secretion contents or suggest distinct, recognizable, functional conditions (hypo- or hypersecretion). PMID- 16683170 TI - The role of the feedforward paradigm in cognitive psychology. AB - Feedforward control is a process adjusting behaviour in a continuative way. Feedforward takes place when an equilibrium state is disrupted and the system has to automatically retrieve the homeostatic stable state. It also occurs when a perturbation is previewed and must be eliminated in order to achieve a desired goal. According to the most general definition, a feedforward process operates by fixing the future representation of the desired state, the achieving of which stops the process. Then, feedforward works by means of the refinement determined by successive comparisons between the actual and target products. In its applications, a feedforward process is thought to be modulated by the subject's purpose and the environmental state. Over the years, the feedforward process has assumed different connotations in several contests of cognitive psychology. An overview of the research fields in psychology that significantly progressed with the introduction of a feedforward paradigm is provided by: (a) reviewing models in which the feedforward concept plays a fundamental role in the system control; (b) examining critical experiments related to the interaction of feedforward and feedback processes; (c) evidencing practical applications for some of the presented feedforward-based architectures. PMID- 16683171 TI - The supplementary motor area in motor and perceptual time processing: fMRI studies. AB - The neural bases of timing mechanisms in the second-to-minute range are currently investigated using multidisciplinary approaches. This paper documents the involvement of the supplementary motor area (SMA) in the encoding of target durations by reporting convergent fMRI data from motor and perceptual timing tasks. Event-related fMRI was used in two temporal procedures, involving (1) the production of an accurate interval as compared to an accurate force, and (2) a dual-task of time and colour discrimination with parametric manipulation of the level of attention attributed to each parameter. The first study revealed greater activation of the SMA proper in skilful control of time compared to force. The second showed that increasing attentional allocation to time increased activity in a cortico-striatal network including the pre-SMA (in contrast with the occipital cortex for increasing attention to colour). Further, the SMA proper was sensitive to the attentional modulation cued prior to the time processing period. Taken together, these data and related literature suggest that the SMA plays a key role in time processing as part of the striato-cortical pathway previously identified by animal studies, human neuropsychology and neuroimaging. PMID- 16683172 TI - Conditional visuo-motor learning and dimension reduction. AB - Conditional visuo-motor learning consists in learning by trial and error to associate visual cues with correct motor responses, that have no direct link. Converging evidence supports the role of a large brain network in this type of learning, including the prefrontal and the premotor cortex, the basal ganglia BG and the hippocampus. In this paper we focus on the role of a major structure of the BG, the striatum. We first present behavioral results and electrophysiological data recorded from this structure in monkeys engaged in learning new visuo-motor associations. Visual stimuli were presented on a video screen and the animals had to learn, by trial and error, to select the correct movement of a joystick, in order to receive a liquid reward. Behavioral results revealed that the monkeys used a sequential strategy, whereby they learned the associations one by one although they were presented randomly. Human subjects, tested on the same task, also used a sequential strategy. Neuronal recordings in monkeys revealed learning-related modulations of neural activity in the striatum. We then present a mathematical model inspired by viability theory developed to implement the use of strategies during learning. This model complements existing models of the BG based on reinforcement learning RL, which do not take into account the use of strategies to reduce the dimension of the learning space. PMID- 16683173 TI - Inertia and memory in ambiguous visual perception. AB - Perceptual multistability during ambiguous visual perception is an important clue to neural dynamics. We examined perceptual switching during ambiguous depth perception using a Necker cube stimulus, and also during binocular rivalry. Analysis of perceptual switching time series using variance-sample size analysis, spectral analysis and time series shuffling shows that switching times behave as a 1/f noise and possess very long range correlations. The long memory feature contrasts sharply with the traditional satiation models of multistability, where the memory is not incorporated, as well as with recently published models of multistability and neural processing, where memory is excluded. On the other hand, the long memory feature favors the concept of "dynamic core" or coalition of neurons, where neurons form transient coalitions. Perceptual switching then corresponds to replacement of one coalition of neurons by another. The inertia and memory measures the stability of a coalition: a strong and stable coalition has to be won over by another similarly strong and stable coalition, resulting in long switching times. The complicated transient dynamics of competing coalitions of neurons may be addressable using a combination of functional imaging, measurement of frequency-tagged magnetoencephalography and frequency-tagged encephalography, simultaneous recordings of groups of neurons in many areas of the brain, and concepts from statistical mechanics and nonlinear dynamics theory. PMID- 16683174 TI - The Torino Inter-University Center for Cognitive Science. PMID- 16683175 TI - Visual interaction: models, systems, prototypes. The Pictorial Computing Laboratory at the University of Rome La Sapienza. AB - This paper reports on the research activities performed by the Pictorial Computing Laboratory at the University of Rome, La Sapienza, during the last 5 years. Such work, essentially is based on the study of humancomputer interaction, spans from metamodels of interaction down to prototypes of interactive systems for both synchronous multimedia communication and groupwork, annotation systems for web pages, also encompassing theoretical and practical issues of visual languages and environments also including pattern recognition algorithms. Some applications are also considered like e-learning and collaborative work. PMID- 16683176 TI - Peripheral neuropathy in two patients with rheumatoid arthritis receiving infliximab treatment. AB - Antitumor necrosis alpha agents have been successfully used for the treatment of rheumatoid and seronegative arthritis, Crohn's disease, psoriasis, and severe cases of vasculitis. Several side effects have been observed in patients receiving these agents including hypersensitivity reactions, infections, drug induced lupus, or demyelinating syndromes. The presence of peripheral neuropathy has been reported only in isolated cases. We describe two cases of peripheral neuropathy which occurred in patients with rheumatoid arthritis receiving infliximab treatment, one with multifocal motor neuropathy with conduction block and another with axonal sensory polyneuropathy, reversed upon discontinuation of infliximab and intravenous gammaglobulin treatment. PMID- 16683177 TI - Nocardia brain abscesses in a male patient with SLE: successful outcome despite delay in diagnosis. AB - We report a 37-year-old African-American man with systemic lupus erythematosus (SLE) diagnosed in May 2001 when he presented with biopsy-proven nephritis. He had been treated intermittently from May 2001 to November 2004 with intravenously (i.v.) administered cyclophosphamide and high doses of prednisone due to unrelenting proteinuria. In November 2004, he was admitted to the hospital because of deterioration of renal function and massive proteinuria (21 g dl(-1) 24 h(-1)) and treated with pulses of methylprednisolone and two courses of i.v. administered cyclophosphamide. His hospital course was complicated by cellulitis and bacteremia with Pseudomonas spp. and Streptococcus bovis. He was discharged on prednisone 60 mg daily, ciprofloxacin, augmentin, and hemodialysis. He was readmitted a week later with new onset of seizure activity, slurred speech, and left-sided hemiparesis. Magnetic resonance imaging of the brain revealed multiple ringlike enhancing foci in the frontal and occipital lobes. Brain biopsy was performed, and Gram stain and initial cultures were negative. Empiric tobramycin, cefepime, and metronidazole were administered. Diagnosis was delayed for several months, but culture eventually grew Nocardia asteroides. Trimethoprim sulfomethoxazole and linezolid therapy was begun. This was followed by slow, but steady, clinical improvement. Risk factors, diagnostic clues, and treatment are reviewed. PMID- 16683178 TI - Body composition: gender-specific risk factor of reduced quantitative ultrasound measures in older people. AB - INTRODUCTION: Body composition has been reported as a significant determinant of bone mineral density. However, data regarding the relationship between the body composition and quantitative ultrasound (QUS) are scarce. METHODS: This cross sectional study examined risk factors of reduced QUS measures in older men and women. A total of 699 subjects aged 60 years old and over-458 women and 241 men who lived in rural communities of Thailand were randomly recruited. RESULTS: By using broadband ultrasound attenuation at the cutoff point of mean -1.0 SD based on young Thai people, the independent factors associated with reduced QUS measures in men were: poor activity of daily living, older age, and low fat mass. As for older women, the independent factors were the number of years since menopause, lack of regular exercise, lower height, and low fat mass. When using the stiffness index at the cutoff point of mean -1.6 SD based on young Thai people, the independent factors associated with reduced QUS measures in men were older age and low fat mass. Meanwhile, the number of years since menopause and low lean body mass were independent risk factors in women. CONCLUSIONS: Poor activity of daily living in men or lack of regular exercise in women was found to be a modifiable risk factor of reduced QUS measures. Years since menopause and low lean body mass were significant factors determining reduced QUS measures in women. Fat mass was an independent factor associated with reduced QUS measures both in men and women. PMID- 16683179 TI - Accuracy of pQCT for evaluating the aged human radius: an ashing, histomorphometry and failure load investigation. AB - INTRODUCTION: Quantifying the determinants of bone strength is essential to understanding if or how the structure will fail under load. Determining failure requires knowledge of material and geometric properties. However, characterizing the relative contributions of geometric parameters of bone to overall bone strength has been difficult to date because of limitations in imaging technology. Peripheral quantitative computed tomography (pQCT) uses digital images to derive estimates of bone strength in the peripheral skeleton and is a relatively safe technique to differentiate cortical from trabecular bone and assess bone geometry and density. However, in a compromised osteoporotic bone, thin cortices and low scan resolution can limit accurate analysis. METHODS: Therefore, in this two-part investigation we scanned ten pairs (n=20) of fresh-frozen radial specimens [female, mean (SD) age 79(6) years] using pQCT (XCT 2000) at the 4 and 30% sites of the distal radius. We investigated the accuracy of four different acquisition resolutions (200, 300, 400, 500 microm) and several analysis modes and thresholds. We evaluated (1) the accuracy of the Norland/Stratec XCT 2000 pQCT in assessing low-density bones by comparing pQCT outcomes to ashing and histomorphometry and (2) the association of geometric parameters by pQCT and areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry (DXA) to failure load at the distal radius. RESULTS: Using histomorphometry and ashing as reference standards, we found that pQCT scans varied systematically and underestimated or overestimated total area and mineral content at the radial midshaft depending on the analysis algorithm and selected threshold. Overall, most pQCT analysis modes were accurate. In the mechanical testing studies, bone mineral content and cortical bone content at the midshaft were strongly associated with failure load. The pQCT parameters that best accounted for failure load were total content at the 4% site and cortical thickness at the 30% site and they accounted for up to 81% of the variance. The best DXA predictor of failure load was total density at the distal third site and it explained 75% of the variance. CONCLUSIONS: In summary, analysis mode, resolution and thresholding affected pQCT outputs at the radial midshaft. This study extends our understanding of pQCT analysis and provides important data regarding determinants of bone strength at the distal radius. PMID- 16683180 TI - No effect of vitamin K1 intake on bone mineral density and fracture risk in perimenopausal women. AB - INTRODUCTION: Vitamin K functions as a co-factor in the post-translational carboxylation of several bone proteins, including osteocalcin. AIM: The aim of this study was to investigate the relationship between vitamin K(1) intake and bone mineral density (BMD) and fracture risk in a perimenopausal Danish population. DESIGN: The study was performed within the Danish Osteoporosis Prevention Study (DOPS), including a population-based cohort of 2,016 perimenopausal women. During the study approximately 50% of the women received hormone replacement therapy (HRT). Associations between vitamin K(1) intake and BMD were assessed at baseline and after 5-years of follow-up (cross-sectional design). Moreover, associations between vitamin K(1) intake and 5-year and 10 year changes in BMD were studied (follow-up design). Finally, fracture risk was assessed in relation to vitamin K(1) intake (nested case-control design). RESULTS: In our cohort, dietary vitamin K(1) intake (60 mug/day) was close to the daily intake recommended by the Food and Agriculture Organization (FAO). Cross sectional and longitudinal analyses showed no associations between intake of vitamin K(1) and BMD of the femoral neck or lumbar spine. Neither did BMD differ between those 5% that had the highest vitamin K(1) intake and those 5% that had the lowest. During the 10-years of follow-up, 360 subjects sustained a fracture (cases). In a comparison between the cases and 1,440 controls, logistic regression analyses revealed no difference in vitamin K(1) intake between cases and controls. CONCLUSION: In a group of perimenopausal and early postmenopausal women, vitamin K(1) intake was not associated with effects on BMD or fracture risk. PMID- 16683181 TI - Effect of different LLLT on pituitrin-induced bradycardia in the rabbit. AB - The objective of this paper was to observe the effect of low-level combined- or single-laser irradiation on bradycardia produced by pituitrin in rabbits. A combined-laser apparatus was made. A 10.6-microm CO2 laser and a 650-nm semiconductor laser, transmitted by different optical fibers, converged to output and irradiate on the Neiguan (PC6) acupuncture point in rabbits with bradycardia produced by pituitrin. Thirty minutes after the model was set, the heart rates of the combined-laser Neiguan group made quicker recoveries than those of the model control group, the laser-control group, or the single-laser Neiguan group (P<0.05), and the heart rates of the single-CO2-laser Neiguan group were similar to those of the normal group (P>0.05). However, there were significant differences between the 650-nm-laser Neiguan group and the normal control group (P<0.05). The combined-laser irradiation certainly has a curative effect on bradycardia produced by pituitrin. A single CO2 laser could accelerate the recovery from bradycardia, while single 650-nm-laser irradiation on the Neiguan acupoint does not produce such an effect. PMID- 16683182 TI - Copper-1,10-phenanthroline-induced apoptosis in liver carcinoma Bel-7402 cells associates with copper overload, reactive oxygen species production, glutathione depletion and oxidative DNA damage. AB - The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper 1,10-phenanthroline, Cu(OP)2, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)2 or Cu2+ alone. It was found that the apoptosis induced by Cu(OP)2 could not be induced by Cu2+ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2',7' dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)2 in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)2. PMID- 16683184 TI - Perceived impact by administrators of psychiatric emergency services after changes in a state's mental health system. AB - As a safety net, psychiatric emergency services are sensitive to system changes. To determine the impact of a state's changes in its mental health system, administrators of publicly funded psychiatric emergency services were surveyed. They reported few (M=0.8) negative changes in coordination of care but 77% endorsed change in administrative burden (54% saying it negatively affected quality of services). Reporting negative effect of administrative burden was associated with treating more persons with substance abuse problems and greater challenge posed by distance to local providers. These results suggest that impact of state-level changes was not uniform but associated with local characteristics. PMID- 16683187 TI - Editorial to the second issue dedicated to Lewis Sheiner. PMID- 16683189 TI - Physical quality and carotene content of solar-dried green leafy and yellow succulent vegetables. AB - The effects of vegetable type, vegetable dimensions, and solar drier load on dehydration rate; and texture, color, water activity, and carotene content of solar-dried carrots, sweet potatoes, and collard greens were studied. Mean dehydration rates (moisture loss,%/hr) for solar dried loads of 430 g/m2 and 715 g/m2 were 3.3 +/- 0.30% and 3.8 +/- 0.20% for carrots and sweet potatoes, respectively. Loads of 360 g/m2 and 465 g/m2 of collard greens had dehydration rates of 6.3 +/- 0.10% moisture loss per hr. The results showed that vegetable type accounted for significant differences (p < 0.01) in dehydration rate, and beta-carotene content. Vegetable dimensions affected (p < 0.05) water activity. Solar drier load affected water activity (p < 0.01), and hue angle (p < 0.05). Beta-carotene contents (dry basis) of dehydrated carrot, sweet potato, and collard treatments were 10.9-17.4%, 7.6-9.8%, and 11.9-21.5%, respectively. Among the carrot treatments, the 5-mm thick slices packed at a load of 715 g/m2 contained the highest beta-carotene (17.4%, dry basis) and vitamin A activity (362 IU/g, dry basis), and good physical properties. For collard greens, the 2-cm and 3-cm wide strips packed at 360 g/m2 loads had the best combinations of high beta-carotene (21.5% and 17.2%, dry basis, respectively), vitamin A activity (357.2 and 293.1 IU/g, dry basis, respectively), and optimal color, texture, and water activity. Beta-carotene losses due to solar dehydration were 48.9-67.5%, 4.0-5.8%, and 1.9-19.8% (dry basis) in carrots, sweet potato and collard greens, respectively. PMID- 16683188 TI - The zinc containing pro-apoptotic protein siva interacts with the peroxisomal membrane protein pmp22. AB - Host answers to pathogen attacks define the course of pathogenic events and decide about the fate of the host organism. Infection with coxsackievirus B3 (CVB3) can induce severe myocarditis and pancreatitis. The interplay between host factors and virus components is crucial for the fate of the infected host. As we have shown before, expression of the pro-apoptotic host protein Siva is significantly increased after CVB3 infection, and infected cells are removed by programmed cell death. Analysis of Siva expressed in Escherichia coli revealed that this protein binds three zinc ions, suggesting a rather complex three dimensional structure. By screening a human heart cDNA library we found a new interaction partner of Siva. The peroxisomal membrane protein PMP22 may be involved in the host response against CVB3. Previous investigations showed that Siva interacts with the cytoplasmic C-terminus of CD27, a member of the tumor necrosis factor receptor group, and transmits an apoptotic signal. With the help of directed two-hybrid assays we determined the N-terminal part of Siva as the binding region for CD27. PMID- 16683202 TI - The mandibular gland secretions of the leaf-cutting ants Atta sexdens sexdens and Atta opaciceps exhibit intercaste and intercolony variations. AB - The mandibular gland secretions of worker castes from wild colonies of the leaf cutting ants Atta sexdens sexdens and Atta opaciceps were analyzed quantitatively by gas chromatography-mass spectrometry. The secretions comprised a complex mixture of volatile, mainly oxygenated compounds, and their profiles exhibited considerable qualitative and quantitative variations among species and castes. The known alarm pheromone 4-methyl-3-heptanone was common to both species. The elevated relative proportions of this ketone found in the secretions of gardeners and generalists suggest that such castes are primarily responsible for the production and release of the alarm pheromone. Quantitative variations (but no qualitative differences) in the profiles of secretions of soldiers from different colonies of A. sexdens sexdens were detected, supporting the view that intraspecific colony recognition is mediated through mandibular gland secretions. Subsequent laboratory assays showed that, among the compounds identified by GC MS, 4-methyl-3-heptanone elicited a strong alarm response in workers of A. sexdens sexdens and A. opaciceps. PMID- 16683203 TI - Female sex pheromone of a carpenter moth, Cossus insularis (Lepidoptera: Cossidae). AB - This study describes the identification of a sex pheromone component of a cossid moth, Cossus insularis. Coupled gas chromatographic-electroantennographic detection (GC-EAD) analysis of solid-phase microextraction (SPME) collections of volatiles released by live female moths showed that two compounds elicited EAG responses from the antennae of male moths. These compounds were identified as (E) 3-tetradecenyl acetate (E3-14:Ac) and (Z)-3-tetradecenyl acetate (Z3-14:Ac) by mass spectral analysis and retention index comparisons with synthetic standards. The ratio of E3-14:Ac and Z3-14:Ac was 95:5 in the effluvia of a female. In field bioassays, sticky traps baited with blends of E3-14:Ac and Z3-14:Ac showed that E3-14:Ac is an essential component of the pheromone. However, the role of Z3 14:Ac is unclear, because E3-14:Ac as a single component was as attractive to male moths as blends of E3-14:Ac and Z3-14:Ac, including the 95:5 blend released by live female moths. PMID- 16683204 TI - Phase I study of gefitinib, oxaliplatin, 5-fluorouracil, and leucovorin (IFOX) in patients with advanced solid malignancies. AB - PURPOSE: Aphase 1 study of gefitinib in combination with oxaliplatin, 5 fluorouracil and leucovorin (IFOX)was conducted to evaluate the safety and feasibility of this regimen. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with escalating doses of gefitinib (250 mg or 500 mg once daily) in combination with FOLFOX (oxaliplatin, 5-fluorouracil, and leucovorin). The initial dose of oxaliplatin was 70 mg/m2 with sequential dose escalation to 85 mg/m2. RESULTS: Sixteen patients received a total of 138 14-day courses of daily gefitinib in combination with FOLFOX. Escalation of gefitinib from 250 mg/d to 500 mg/d with FOLFOX was well-tolerated. In addition, no severe toxicities precluded subsequent dose escalation of oxaliplatin from 70 mg/m2 to 85 mg/m2 at which no dose-limiting toxicity was seen. No further dose escalation was performed as this represented the oxaliplatin dose administered in the standard FOLFOX-4 regimen. The most predominant toxicity was diarrhea, which was well controlled with oral antidiarrheal agents. Four partial remissions occurred in patients with metastatic colorectal cancer. CONCLUSIONS: Gefitinib as a 500 mg daily continuous dose was well tolerated in combination with full doses of FOLFOX 4. PMID- 16683205 TI - A phase I safety and dose escalation trial of docetaxel combined with GEM231, a second generation antisense oligonucleotide targeting protein kinase A R1alpha in patients with advanced solid cancers. AB - PURPOSE: GEM231 is a second-generation antisense oligonucleotide targeting the mRNA of the R1alpha regulatory subunit of cAMP dependent protein kinase A. Preclinical studies have demonstrated synergistic antitumor activity when GEM231 is combined with docetaxel. This trial assesses the safety of this combination. EXPERIMENTAL DESIGN: Docetaxel was administered once every three weeks (one cycle) at doses between 50-75 mg/m2. GEM231 was administered twice weekly at 220 mg/m2 for 3 (schedule-A), or 2 (schedule-B) weeks. RESULTS: Twenty patients with chemotherapy-refractory advanced cancer received a total of 39 cycles of therapy. Six patients in schedule-A received docetaxel 50 mg/m2, and 14 patients in schedule-B received docetaxel 50-75 mg/m2. In schedule-A, 2 of 6 patients developed cycle-1 dose limiting toxicity (DLT)-grade-3 fatigue or grade-3 serum transaminase elevation. In schedule-B, 1 of 4 patients developed cycle-1 DLT at the highest dose of docetaxel tested (75 mg/m2)--grade-3 febrile neutropenia. Subsequent dose escalations were not pursued since the overall incidence of grade 3 toxicities (including those that occurred after cycle 1) was 75%, and this dose was close to the single agent MTD of docetaxel. Grade-3 toxicities included fatigue (2 patients), transaminase elevation (4 patients), and altered mentation (1 patient). The mean post-infusion aPTT was significantly higher than the pre infusion value [14.8 seconds; p<0.001]; however, there were no hemorrhagic episodes. CONCLUSIONS: The recommended dose for further development of the combination of docetaxel and GEM231 is 75 mg/m2 and 220 mg/m2, respectively. It is important to administer GEM231 twice weekly for 2 consecutive weeks followed by a one-week break. PMID- 16683206 TI - Attentional modulation of firing rate and synchrony in a model cortical network. AB - The response of a neuron in the visual cortex to stimuli of different contrast placed in its receptive field is commonly characterized using the contrast response curve. When attention is directed into the receptive field of a V4 neuron, its contrast response curve is shifted to lower contrast values (Reynolds et al., 2000). The neuron will thus be able to respond to weaker stimuli than it responded to without attention. Attention also increases the coherence between neurons responding to the same stimulus (Fries et al., 2001). We studied how the firing rate and synchrony of a densely interconnected cortical network varied with contrast and how they were modulated by attention. The changes in contrast and attention were modeled as changes in driving current to the network neurons. We found that an increased driving current to the excitatory neurons increased the overall firing rate of the network, whereas variation of the driving current to inhibitory neurons modulated the synchrony of the network. We explain the synchrony modulation in terms of a locking phenomenon during which the ratio of excitatory to inhibitory firing rates is approximately constant for a range of driving current values. We explored the hypothesis that contrast is represented primarily as a drive to the excitatory neurons, whereas attention corresponds to a reduction in driving current to the inhibitory neurons. Using this hypothesis, the model reproduces the following experimental observations: (1) the firing rate of the excitatory neurons increases with contrast; (2) for high contrast stimuli, the firing rate saturates and the network synchronizes; (3) attention shifts the contrast response curve to lower contrast values; (4) attention leads to stronger synchronization that starts at a lower value of the contrast compared with the attend-away condition. In addition, it predicts that attention increases the delay between the inhibitory and excitatory synchronous volleys produced by the network, allowing the stimulus to recruit more downstream neurons. PMID- 16683207 TI - Effects of stimulus transformations on estimates of sensory neuron selectivity. AB - Stimulus selectivity of sensory systems is often characterized by analyzing response-conditioned stimulus ensembles. However, in many cases these response triggered stimulus sets have structure that is more complex than assumed. If not taken into account, when present it will bias the estimates of many simple statistics, and distort the estimated stimulus selectivity of a neural sensory system. We present an approach that mitigates these problems by modeling some of the response-conditioned stimulus structure as being generated by a set of transformations acting on a simple stimulus distribution. This approach corrects the estimates of key statistics and counters biases introduced by the transformations. In cases involving temporal spike jitter or spatial jitter of images, the main observed effects of transformations are blurring of the conditional mean and introduction of artefacts in the spectral decomposition of the conditional covariance matrix. We illustrate this approach by analyzing and correcting a set of model stimuli perturbed by temporal and spatial jitter. We apply the approach to neurophysiological data from the cricket cercal sensory system to correct the effects of temporal jitter. PMID- 16683208 TI - The influence of cortical feature maps on the encoding of the orientation of a short line. AB - The inhomogeneous distribution of the receptive fields of cortical neurons influences the cortical representation of the orientation of short lines seen in visual images. We construct a model of the response of populations of neurons in the human primary visual cortex by combining realistic response properties of individual neurons and cortical maps of orientation and location preferences. The encoding error, which characterizes the difference between the parameters of a visual stimulus and their cortical representation, is calculated using Fisher information as the square root of the variance of a statistically efficient estimator. The error of encoding orientation varies considerably with the location and orientation of the short line stimulus as modulated by the underlying orientation preference map. The average encoding error depends only weakly on the structure of the orientation preference map and is much smaller than the human error of estimating orientation measured psychophysically. From this comparison we conclude that the actual mechanism of orientation perception does not make efficient use of all the information available in the neuronal responses and that it is the decoding of visual information from neuronal responses that limits psychophysical performance. PMID- 16683210 TI - On the sensitive dependence on initial conditions of the dynamics of networks of spiking neurons. AB - We have previously formulated an abstract dynamical system for networks of spiking neurons and derived a formal result that identifies the criterion for its dynamics, without inputs, to be "sensitive to initial conditions". Since formal results are applicable only to the extent to which their assumptions are valid, we begin this article by demonstrating that the assumptions are indeed reasonable for a wide range of networks, particularly those that lack overarching structure. A notable aspect of the criterion is the finding that sensitivity does not necessarily arise from randomness of connectivity or of connection strengths, in networks. The criterion guides us to cases that decouple these aspects: we present two instructive examples of networks, one with random connectivity and connection strengths, yet whose dynamics is insensitive, and another with structured connectivity and connection strengths, yet whose dynamics is sensitive. We then argue based on the criterion and the gross electrophysiology of the cortex that the dynamics of cortical networks ought to be almost surely sensitive under conditions typically found there. We supplement this with two examples of networks modeling cortical columns with widely differing qualitative dynamics, yet with both exhibiting sensitive dependence. Next, we use the criterion to construct a network that undergoes bifurcation from sensitive dynamics to insensitive dynamics when the value of a control parameter is varied. Finally, we extend the formal result to networks driven by stationary input spike trains, deriving a superior criterion than previously reported. PMID- 16683209 TI - A computational model of how an interaction between the thalamocortical and thalamic reticular neurons transforms the low-frequency oscillations of the globus pallidus. AB - In Parkinson's disease, neurons of the internal segment of the globus pallidus (GPi) display the low-frequency tremor-related oscillations. These oscillatory activities are transmitted to the thalamic relay nuclei. Computer models of the interacting thalamocortical (TC) and thalamic reticular (RE) neurons were used to explore how the TC-RE network processes the low-frequency oscillations of the GPi neurons. The simulation results show that, by an interaction between the TC and RE neurons, the TC-RE network transforms a low-frequency oscillatory activity of the GPi neurons to a higher frequency of oscillatory activity of the TC neurons (the superharmonic frequency transformation). In addition to the interaction between the TC and RE neurons, the low-threshold calcium current in the RE and TC neurons and the hyperpolarization-activated cation current (I (h)) in the TC neurons have significant roles in the superharmonic frequency transformation property of the TC-RE network. The external globus pallidus (GPe) oscillatory activity, which is directly transmitted to the RE nucleus also displays a significant modulatory effect on the superharmonic frequency transformation property of the TC-RE network. PMID- 16683211 TI - Fitting experimental data to models that use morphological data from public databases. AB - Ideally detailed neuron models should make use of morphological and electrophysiological data from the same cell. However, this rarely happens. Typically a modeler will choose a cell morphology from a public database, assign standard values for Ra, Cm, and other parameters and then do the modeling study. The assumption is that the model will produce results representative of what might be obtained experimentally. To test this assumption we developed models of CA1 hippocampal pyramidal neurons using 4 different morphologies obtained from 3 public databases. The multiple run fitter in NEURON was used to fit parameter values in each of the 4 morphological models to match experimental data recorded from 19 CA1 pyramidal cells. Fits with fixed standard parameter values produced results that were generally not representative of our experimental data. However, when parameter values were allowed to vary, excellent fits were obtained in almost all cases, but the fitted parameter values were very different among the 4 reconstructions and did not match standard values. The differences in fitted values can be explained by very different diameters, total lengths, membrane areas and volumes among the reconstructed cells, reflecting either cell heterogeneity or issues with the reconstruction data. The fitted values compensated for these differences to make the database cells and experimental cells more similar electrotonically. We conclude that models using fully reconstructed morphologies need to be calibrated with experimental data (even when morphological and electrophysiological data come from the same cell), model results should be generated with multiple reconstructions, morphological and experimental cells should come from the same strain of animal at the same age, and blind use of standard parameter values in models that use reconstruction data may not produce representative experimental results. PMID- 16683212 TI - Low-molecular-weight heparin compared with unfractionated heparin for patients with non-ST-segment elevation acute coronary syndromes treated with glycoprotein IIb/IIIa inhibitors: results from the CRUSADE initiative. AB - BACKGROUND: Both heparin and glycoprotein (GP) IIb/IIIa inhibitor therapy and early invasive management strategies are recommended by the American College of Cardiology (ACC)/American Heart Association (AHA) guidelines for the treatment of patients with non-ST-segment elevation acute coronary syndromes (NSTE ACS). However, controversy exists about which form of heparin-unfractionated (UF) or low-molecular-weight (LMW)-is preferable. We sought to compare the efficacy and safety of these treatment strategies in a large contemporary population of patients with NSTE ACS. METHODS: Using data from the CRUSADE Initiative, we evaluated LMWH and UFH in high-risk NSTE ACS patients (positive cardiac markers and/or ischemic ST-segment changes) who had received early (< 24 hours) GP IIb/IIIa inhibitor therapy and underwent early invasive management. In-hospital outcomes were compared among treatment groups. RESULTS: From a total of 11,358 patients treated at 407 hospitals in the US from January 2002-June 2003, 6881 (60.6%) received UFH and 4477 (39.4%) received LMWH. Patients treated with UFH were more often admitted to a cardiology inpatient service (73.6% vs. 65.5%, P < 0.0001) and more frequently underwent diagnostic catheterization (91.8% vs. 85.9%, P < 0.0001) and percutaneous coronary intervention (PCI) (69.7% vs. 56.9%, P < 0.0001) than patients treated with LMWH. The point estimate of the adjusted risk of in-hospital death or reinfarction was slightly lower among patients treated with LMWH (odds ratio [OR] 0.81, 95% confidence interval [CI] 0.67-0.99) and the risk of red blood cell transfusion was similar (OR 1.01, 95% CI 0.89 1.15). Among patients who underwent PCI within 48 hours, adjusted rates of death (OR 1.14, 95% CI 0.71-1.85), death or reinfarction (OR 0.93, 0.67-1.31), and transfusion (OR 1.16, 0.89-1.50) were similar. Patients who underwent PCI more than 48 hours into hospitalization had reduced rates of death (OR 0.64, 0.46 0.88), death or reinfarction (OR 0.57, 0.44-0.73), and transfusion (OR 0.66, 0.52 0.84). CONCLUSIONS: In routine clinical practice, patients treated with GP IIb/IIIa inhibitors have slightly improved outcomes and similar bleeding risks with LMWH than with UFH. These findings are consistent with current ACC/AHA guidelines but raise important questions about the safety and effectiveness of antithrombotic therapy in real-world clinical practice. Using data from the CRUSADE Initiative, we evaluated low-molecular-weight heparin (LMWH) and unfractionated heparin (UFH) in high-risk patients with non-ST-segment elevation acute coronary syndromes (NSTE ACS) who received early (<24 hours) glycoprotein (GP) IIb/IIIa inhibitors and early invasive management. In-hospital outcomes were compared among treatment groups. LMWH was associated with slightly improved clinical outcomes and similar rates of transfusion compared with UFH. Our results support the current ACC/AHA guidelines recommendations but raise concerns about the safety and efficacy of UFH in the setting of background use of upstream GP IIb/IIIa inhibitors for patients with NSTE ACS in routine clinical practice. PMID- 16683213 TI - Enhanced spontaneous thrombolysis: a new therapeutic challenge. AB - Spontaneous thrombolysis is an endogenous protective mechanism against lasting arterial thrombotic occlusion, which is implicated in the pathogenesis of myocardial infarction and acute coronary events. Novel therapies for coronary heart disease (CHD) targeting atherosclerosis and thrombosis, together with cardiovascular prevention programs targeting risk-factors and lifestyle provide evidence that CHD is preventable. Although reduced fibrinolytic activity is a recognized risk-factor for ischemic cardiovascular events, it has so far been neglected. Our knowledge of the fibrinolytic effect of drugs commonly used for CHD such as antiplatelet agents (aspirin, ticlopidine, clopidogrel), anti diabetic biguanides (phenformin, metformin) or anti-hypertensive drugs is scanty and conflicting. This is mainly due to the lack of a global test of spontaneous thrombolysis, as opposed to fibrinolysis of plasma or whole blood, i.e. the assessment of various activators and inhibitors of the fibrinolytic system. A recently described technique allows the measurement of spontaneous thrombolysis, that is, lysis of an autologous platelet-rich thrombus in the absence of added plasminogen activators. Early results suggest that this test may have significant clinical potential both in identifying those at risk of fatal cardiac events and in finding new therapeutic avenues or lifestyles to improve spontaneous thrombolytic activity. PMID- 16683214 TI - Association of glycoprotein IIb/IIIa inhibitors and long-term survival following administration during percutaneous coronary intervention for acute myocardial infarction. AB - OBJECTIVES: We sought to evaluate the impact of GP IIb/IIIa receptor blockers on long-term mortality in patients undergoing PCI for AMI. BACKGROUND: Glycoprotein (GP) IIb/IIIa inhibitors are potent suppressors of platelet aggregation and when used during percutaneous coronary intervention (PCI) for the treatment of acute myocardial infarction (AMI) may improve short-term clinical outcomes, including survival. However, the impact of GP IIb/IIIa treatment during PCI for AMI on long term survival is unknown. METHODS: Patients undergoing primary or rescue PCI for AMI within 24 hours of symptom onset with or without GP IIb/IIIa inhibitor treatment were identified from a multicenter PCI database. All cause mortality at a mean follow-up of 3 years was the primary end point. RESULTS: Of the 269 patients treated with primary or rescue PCI for AMI, 107 (40%) received a GP IIb/IIIa antagonist. Patients treated with GP inhibitors were more likely to present with or develop heart failure (13% vs. 6.2%, P = 0.052). Left ventricular ejection fraction was reduced in those treated with GP IIb/IIIa antagonists (44% vs. 48%, P = 0.051). The extent of coronary artery disease did not differ between groups. Stent use was 80% in both groups. Procedural success was high and did not differ between groups. In-hospital mortality was low and did not differ between groups. The mortality at a mean follow-up of 3 years was 1.9% among patients treated with a GP IIb/IIIa antagonist and 15% for those who were not treated (log rank P = 0.0005). Treatment with a GP IIb/IIIa antagonist was independently associated with a significant reduction in the hazard of long-term mortality (Hazard Ratio, 0.159; 95% Confidence Interval, 0.034-0.729; P = 0.018). CONCLUSIONS: Treatment of patients undergoing PCI for AMI with GP IIb/IIIa antagonists appears to be associated with a profound reduction in late mortality. PMID- 16683215 TI - The effect of high plasma levels of angiotensin-converting enzyme (ACE) and plasminogen activator inhibitor (PAI-1) on the reperfusion after thrombolytic therapy in patients presented with acute myocardial infarction. AB - The resistance to thrombolytic agents and delays in reperfusion occur in more than 30% after acute myocardial infarction. This may play an important role in the unsuccessful recanalization after thrombolytic therapy. The aim of this study is to assess the clinical and biochemical markers of reperfusion after different types of thrombolytic therapy and to find out the relationship between PAI-1 and ACE serum levels and the short-term outcome. Pretreatment ACE and PAI-1 plasma levels of 184 patients with acute myocardial infarction, treated with thrombolytic therapy were determined. Failure of thrombolysis was considered when reperfusion was delayed as assessed by noninvasive reperfusion criteria, reinfarction, and impaired left ventricular function. High plasma level of ACE (> 50 U/L), PAI-1 (> 43 ng/ml) and both was found in 57, 108 and 32 patients respectively. Subjects with high ACE plasma levels were characterized by impaired LV systolic function (79.0% vs. 75.0%), new Q-wave (88.4% vs. 74.2%), less reperfusion arrhythmia (19.3% vs. 22.8%) and prolonged hospitalization (70% vs. 66%) but no statistical significance was observed. High enzymes levels of PAI-1 were observed with higher incidence of anterior myocardial infarction (50.0% vs. 41.0%), lesser ST segment resolution (65.6% vs. 58.8%), reinfarction (6.3% vs. 5.9%), and impaired LV systolic function (90.6% vs. 76.0%), and prolonged hospitalization (70.4% vs. 63.4). There was a statistically significant difference between thrombolytic agents in the presence of high ACE regarding hospital overstay (p = 0.02). While the presence of high PAI-1 was significantly affect the degree of ST-segment resolution (p = 0.03). CONCLUSION: High plasma ACE and/or PAI-1 plays a considerable role in the higher incidence of unsuccessful reperfusion and impaired left ventricular function after thrombolytic therapy. A rapid diagnostic tool that enables physician of detecting those enzymes before giving thrombolytic therapy may change the strategy of treatment to offer another effective revascularization method. PMID- 16683216 TI - Evaluation of platelet function in aspirin treated patients with CAD. AB - BACKGROUND: Studies of platelet function in the acute coronary syndrome (ACS) have revealed both increased and unchanged platelet activation. To obtain a better understanding of platelet function in coronary artery disease in the setting of aspirin therapy, we performed platelet functional testing in patients with ACS and compared results to patients without CAD. METHODS: We measured platelet aggregation and activation in response to ADP and epinephrine in 80 age and gender matched hospitalized patients (40 with ACS, 40 with non-cardiac chest pain (NCCP)). All subjects received ASA (81-325 mg). Platelet aggregation was performed using standard light transmission in platelet-rich plasma and activation was measured via flow cytometric analyses. We also studied platelet function under high shear rates measured by the platelet function analyzer (PFA 100). RESULTS: ASA effect was found to be present in all subjects by blunted platelet aggregation in response to arachidonic acid. Patients with ACS showed significantly higher levels of platelet aggregation to epinephrine compared to patients with NCCP (p = 0.001). Other measures of platelet function including ADP aggregation, Pselectin, activated glycoprotein IIb/IIIa expression, and PFA-100 were unchanged between the two groups. CONCLUSIONS: We have found conflicting results of platelet functional testing in ACS. Specifically aspirin therapy in patients with ACS is effective in suppressing the platelet release response and is effective in the partial suppression of platelet aggregation; however, it appears that ACS patients have increased platelet aggregation to adrenergic stimuli when compared to age and gender matched controls without CAD despite the use of aspirin. In some studies, because ACS patients have an accentuated response to adrenergic stimuli this might be interpreted as aspirin resistance. Our study suggests that depending on the assay used to determine aspirin resistance, not all patients with this label are resistant to the biological effects of aspirin but they may have higher than normal baseline platelet sensitivity to adrenergic stimuli. PMID- 16683217 TI - Symptomatic intracerebral hematomas in posterior circulation stroke patients anticoagulated with heparin. AB - BACKGROUND: A lot of clinicians use heparin in patients with posterior circulation stroke. Frequency and risk factors of symptomatic intracerebral hematoma (ICH) in posterior circulation infarct patients anticoagulated with unfractionated heparin are not known. METHODS: To determine the incidence and the risk factors of the heparin-related ICH in posterior circulation infarct patients, we retrospectively reviewed the clinical features of 37 patients who had acute posterior circulation infarct and received intravenous heparin after they underwent brain computed tomographic scans and diffusion-weighted imaging (DWI). Follow-up brain scans were obtained at any time if clinical symptoms worsened. Volumes of acute posterior circulation infarction were calculated on DWI. RESULTS: Of 37 patients, four (10.8%) developed symptomatic ICH during heparin infusion. The location of ICH was cerebellum in all the hemorrhagic worsening patients. We found that the size of an acute infarction calculated on DWI is the risk factor of symptomatic ICH during intravenous heparin therapy in patients with posterior circulation infarct. CONCLUSIONS: Until a large prospective study is performed, it may be prudent to avoid heparin infusion in patients with large posterior circulation infarct documented on DWI. PMID- 16683218 TI - What are the barriers to warfarin use in atrial fibrillation?: Development of a questionnaire. AB - BACKGROUND: Despite evidence of their benefit and efforts to increase usage, anticoagulation for stroke prophylaxis in atrial fibrillation (AF) patients remains underutilized. Previous surveys have assessed reasons for underuse of anticoagulation but have limitations including non-structured approach for eliciting barriers and use of clinical vignettes and not patient-level data. The objectives of this study were to develop a questionnaire to assess barriers to anticoagulation use for stroke prophylaxis in AF patients at a patient- and physician-level and to conduct a preliminary field-test of the instrument. METHODS: Barriers to warfarin use were identified from a literature review, input from clinical experts, and a physician focus group. A sample of US physicians who treat AF patients completed the questionnaire. Physicians ranked their reluctance on a 1-10 scale (10 = very reluctant) in general to prescribe warfarin if a specific barrier was present in a patient and then indicated critical barriers to prescribing warfarin in a sample of their own AF patients not receiving warfarin. RESULTS: Forty-one barriers to warfarin use were identified and classified into 4 groups: patient medical characteristics (n = 17), health care system factors (n = 7), patient capability (n = 12), and patient preference (n = 5). Several new items were developed (e.g., difficulty in obtaining venous access), existing items were revised (e.g., timeframe for bleeding episodes subdivided into > or or = 70 years presenting with localized prostate cancer. METHODS: A matched pair analysis compared patients age < or = 55 years (Group 1) who were treated with 3-dimension conformal radiation without androgen deprivation to men age > or = 60 years and < 70 years (Group 2), and men age > or = 70 years (Group 3) who were treated at the Fox Chase Cancer Center between November 1989 and October 2001. The groups were matched for disease stage (T1/T2b vs. T2C/T3), Gleason grade (2-6 vs. 7-10), radiation dose (< 70 Gray [Gy] vs. > or = 70-76 Gy vs. > or = 76 Gy), and pretreatment prostate-specific antigen (PSA) level. Estimates of outcome were accomplished using Kaplan-Meier methodology and compared by age group using the log-rank test. RESULTS: Eighty-four men were identified according to the selection criteria. No statistically significant difference was found in the 5 year overall survival rates for Group 1, Group 2, and Group 3 (94%, 95%, and 87%, respectively) or the 5-year rate of freedom from biochemical failure in Group 1, Group 2, and Group 3 (82%, 76%, and 70%, respectively), or freedom from distant metastases (96%, 97%, and 98%, respectively). CONCLUSIONS: Men age < or = 55 years who present with localized prostate cancer do not appear to have a worse prognosis. External beam radiation therapy appears to be a viable treatment alternative and should be offered to men age < or = 55 years who present with organ-confined prostate cancer. PMID- 16683225 TI - Selecting patients for breast-conserving therapy: the importance of lobular histology. AB - BACKGROUND: Concerns exist regarding the ability to select patients with lobular carcinoma for breast-conserving therapy (BCT) using mammography. The purpose of this study was to compare the conversion rate from BCT to mastectomy among patients with lobular (ILC) and ductal carcinoma (IDC), and to compare the number of resections needed to obtain negative margins. METHODS: In all, 349 patients with pure or mixed ILC were identified from a database and 318 were successfully matched to 2 controls with IDC on the basis of year of diagnosis, menopausal status, and stage of disease. Conditional logistic regression was used to control for matching. RESULTS: Patients with ILC were older, had larger tumors (2.6 vs. 2.1 cm; P<.001), and were more likely to have contraindications to BCT (25% vs. 20%). In patients thought to be candidates for BCT, no difference in success rate was noted on the basis of histologic type after controlling for age and stage. In those undergoing BCT, no difference in the number of procedures was seen on the bases of histology after adjustment for age and stage. CONCLUSIONS: Patients with ILC are not more likely to fail BCT than those with IDC, nor do they require more operations to obtain negative margins. These results do not support the use of more extensive preoperative evaluation such as magnetic resonance imaging in patients with ILC. PMID- 16683226 TI - Origin of dental occlusion in tetrapods: signal for terrestrial vertebrate evolution? AB - Evolutionary changes of the dentition in tetrapods can be associated with major events in the history of terrestrial vertebrates. Dental occlusion, the process by which teeth from the upper jaw come in contact with those in the lower jaw, appears first in the fossil record in amniotes and their close relatives near the Permo-Carboniferous boundary approximately 300 million years ago. This evolutionary innovation permitted a dramatic increase in the level of oral processing of food in these early tetrapods, and has been generally associated with herbivory. Whereas herbivory in extinct vertebrates is based on circumstantial evidence, dental occlusion provides direct evidence about feeding strategies because jaw movements can be reconstructed from the wear patterns of the teeth. Examination of the evolution of dental occlusion in Paleozoic tetrapods within a phylogenetic framework reveals that this innovation developed independently in several lineages of amniotes, and is represented by a wide range of dental and mandibular morphologies. Dental occlusion also developed within diadectomorphs, the sister taxon of amniotes. The independent, multiple acquisition of this feeding strategy represents an important signal in the evolution of complex terrestrial vertebrate communities, and the first steps in the profound changes in the pattern of trophic interactions in terrestrial ecosystems. PMID- 16683227 TI - Evidence for disease-regulated transgene expression in the brain with use of lentiviral vectors. AB - In this study we have developed and validated a novel approach of transgene regulation in the brain. By using lentiviral vectors that incorporate promoters of genes that are up-regulated during different pathological states, we were able to regulate transgene expression in accordance with the disease process. When using a glial fibrillary acidic protein promoter, efficient disease regulation in glial cells was achieved after an excitotoxic lesion or a 6-hydroxydopamine (6 OHDA) lesion. Transgene expression was physiologically regulated and displayed a dose-dependent increase depending on the severity of lesion. Efficient regulation was also achieved in neurons when using a preproenkephlin promoter in 6-OHDA lesioned rats, allowing combined regulation and targeting. This disease-regulated approach allows control of transgene expression in the brain without the use of inducer molecules and without overexpression of transactivator proteins. PMID- 16683228 TI - Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP-induced morphologic changes. AB - Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP-treated and non-treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP treated astrocytes after stimulation with ATP by high-performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1-43 imaging, appeared in cyclic AMP-treated astrocytes in a punctiform fashion, but not in non-treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP-induced morphologic changes, and in particular, a v-SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP-induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP-treated astrocytes in response to ATP released from neighboring neurons and astrocytes. PMID- 16683229 TI - Gas-chromatographic separation of stereoisomers of dipeptides. AB - Synthetic dipeptides comprising mixtures of enantiomers, diastereomers, or sequential isomers were converted into their N-perfluoroacetyl dipeptide esters (perfluoroacetyl: trifluoroacetyl, pentafluoroacetyl, heptafluorobutyryl; ester: methyl, 1-propyl, 2-propyl, 2,2,2-trifluoroethyl) and analyzed by GC-MS on the chiral stationary phases Chirasil-L-Val and Lipodex-E using helium as carrier gas. Further, dipeptides were converted into their N-trifluoroacetyl dipeptide S (+)-2-butyl esters and separated on achiral phenylmethyl polysiloxane column (HP 5 MS). Derivatization of dipeptides was performed at ambient temperature in order to avoid formation of the corresponding diketopiperazines. The best separation of stereoisomers was achieved with TFA and PFP methyl esters on Chirasil-L-Val. PMID- 16683230 TI - Spontaneous resolution of 2,3-bis-fluoren-9-ylidenesuccinic acid induced by achiral guest inclusion. AB - The racemic title host compound (1) formed homochiral inclusion crystals (conglomerate) of 1 with acetone, while heterochiral inclusion crystals (racemate) were obtained when complexed with ethyl acetate. Upon treatment of the homochiral inclusion crystals of 1 with gaseous acetyl chloride in the solid state, optically active 2,3-bis(fluorenylidene)succinic anhydride (3) was obtained in high optical purity. PMID- 16683231 TI - Improved organotypic cell culture model for analysis of the neuronal circuit involved in the monosynaptic stretch reflex. AB - Knowledge regarding neuronal circuit formation is central for the understanding of the vast network making up the brain. It is therefore necessary to find novel ways to analyze the mechanisms involved in well-defined neural circuits. We present an improved in vitro model of the monosynaptic stretch reflex circuit, based on primary organotypic cell cultures. By using limb tissue as a source of muscle fibers instead of circumspinal tissue we could make the in vitro system more in vivo like in the sense that it focuses on the stretch reflex involving limb muscles. Furthermore, our analyses showed that this procedure allows muscle fibers to follow the normal developmental pattern. Particularly interesting was the finding of slow tonic myosin heavy chain expressing muscle fibers, a developmental marker for muscle spindles, in the cultures showing that this system has the potential to contain the complete reflex circuits. PMID- 16683232 TI - The posterior deep temporal nerve: its relationship with the human cranial base. AB - In an earlier dry-skull study (Dias et al. [2001] Int J Osteoarchaeol 11:241 248), a hitherto undescribed groove was observed in the infratemporal region of the greater wing of the sphenoid bone in the cranial base, passing laterally from the foramen ovale onto the squamous temporal bone. We found a very high incidence of this groove, 81% on one side at least, in modern South-Asian Indian adult crania, but much less so, 50%, in a Polynesian sample of both New Zealand Maori and Moriori adult crania. This article reports our finding of the groove, at least on one side, in 85% of a modern Portuguese skeletal population, and our dissection study on twenty European cadavers confirms that the groove, present in all, is formed by the posterior deep temporal nerve. PMID- 16683233 TI - Medical students speak: a two-voice comment on learning professionalism in medicine. AB - We are two medical students. For one of us, medical school is just beginning; for the other, it is coming to an end. Our experiences are different, but our message is the same. Professionalism is a vital component in the field of medicine. Characteristics such as trustworthiness, compassion, integrity, honesty, leadership, and social responsibility must be embraced by the next generation of doctors so the future healthcare system will be one that patients and physicians admire and respect. To reach this goal, it is important to understand how medical students today view professionalism and how such a construct is integrated into medical education. We hope to provide insight into this area by reflecting on the lessons we have learned regarding professionalism in medical school. Professionalism, like the medical field itself, is a life-long learning process. By encouraging this process early in medical training, future doctors will be able to provide their patients with highest quality care. PMID- 16683234 TI - AUF-1 mediates inhibition by nitric oxide of lipopolysaccharide-induced matrix metalloproteinase-9 expression in cultured astrocytes. AB - Neuroinflammatory diseases are associated with increased production of matrix metalloproteinase-9 (MMP-9) and excessive generation of nitric oxide (NO). NO has been reported to have variable effects on MMP-9 gene expression and activation in various cell types. In the present study, we investigated the effect of NOon MMP 9 expression in primary cortical astrocytes. Zymography and real-time PCR showed that lipopolysaccharide (LPS) dramatically increased latent MMP-9 gelatinolytic activity and MMP-9 mRNA expression. By using the NO donor DETA NONOate, we observed a dose-dependent inhibition of MMP-9 induction by LPS. Active forms of MMP-9 were not found by zymography after NO treatment. The MEK1/2 inhibitor U0126 completely inhibited LPS-induced MMP-9, which was partially inhibited by the p38 MAPK inhibitor SB203580. NO had no effect on LPS-stimulated ERK1/2 and p38 MAPK activation, suggesting that the inhibitory action of NO occurs downstream of MAPK cascades. Real-time PCR analysis showed that NO accelerated the degradation of MMP-9 mRNA after LPS induction. Western blotting and pull-down assay demonstrated that NO increased AUF-1 expression as well as its specific binding to the MMP-9 gene 3'-untranslated region. Knockdown of AUF-1 with siRNA partially reversed the inhibitory action of NO on LPS-stimulated MMP-9 induction. We conclude that NO does not activate MMP-9 in astrocyte cultures but reduces LPS-induced MMP-9 expression via accelerating MMP-9 mRNA degradation, which is partially mediated by AUF-1. Our results suggest that elevated NO concentrations may suppress MMP-9 and restrict the inflammatory response in neurodegenerative diseases. PMID- 16683235 TI - Distribution and expression of tissue inhibitors of metalloproteinase in dorsal root entry zone and dorsal column after dorsal root injury. AB - To understand whether tissue inhibitors of metalloproteinase (TIMPs) contribute to the failure of regenerating sensory axons to enter the spinal cord, we used in situ hybridization and immunocytochemistry to examine the expression of TIMP1, TIMP2, and TIMP3 in the dorsal root, dorsal root entry zone (DREZ), and dorsal column after dorsal root injury in adult rats. We found that the three TIMPs and their mRNAs were up-regulated in a time-, region-, and cell-type-specific manner. Strong up-regulation of all three TIMPs was seen in the injured dorsal roots. TIMP2 was also significantly up-regulated in the DREZ and degenerating dorsal column, where TIMP1 and TIMP3 showed only moderate up-regulation. Most cells up regulating the TIMPs in the DREZ and degenerating dorsal column were reactive astrocytes, but TIMP2 was also up-regulated by microglia/macrophages, especially at long postoperative survival times. These results suggest that TIMPs may be involved in controlling tissue remodelling following dorsal root injury and that manipulation of the expression of TIMPs may provide a means of promoting axonal regeneration into and within the injured spinal cord. PMID- 16683236 TI - Correlation between the four types of acromion and the existence of enthesophytes: a study on 423 dried scapulas and review of the literature. AB - The purpose of this study was to correlate the four types of acromial shape with the existence of enthesophytes, which together comprise two important parameters for subacromial impingement syndrome and rotator cuff tears. In addition, a review of the literature was carried out. Four hundred twenty-three dried scapulas were studied at the Department of Anatomy in the University of Cologne, Germany. Four types of acromion were found: the three classical ones as described by Bigliani et al. ([1986] Orthop Trans 10:216) and a fourth one, where the middle third of the undersurface of acromion was convex (Gagey et al. [1993] Surg Radiol Anat 15:63-70). The correlation between the four types of acromion and the presence of enthesophytes at its anterior undersurface was also recorded. The distribution of acromial types was as follows: type I, flat, 51 (12.1%); type II, curved, 239 (56.5%); type III, hooked, 122 (28.8%); and type IV, convex, 11 (2.6%). Enthesophytes were found in 1 of type I (2%), in 19 of type II (7.9%), in 46 of type III (37.7%), and in 0 (0%) of type IV acromions. Overall, 66 (15.6%) out of 423 scapulas had enthesophytes. In all cases, they were localized at the site of the coracoacromial ligament insertion on the acromion. Enthesophytes were significantly (P < 0.05) more common in type III acromions and this combination is particularly associated with subacromial impingement syndrome and rotator cuff tears. In type I and in type IV acromions, the incidence of enthesophytes is very small and, according to other studies, with these two acromial types rotator cuff tears are also rare. PMID- 16683237 TI - Professionalism and anatomy: How do these two terms define our role? PMID- 16683238 TI - Using gross anatomy to teach and assess professionalism in the first year of medical school. AB - The public is uneasy about the lack of professionalism in physicians chronicled in the different news media. Since professionalism is a set of values developed over a period of time, it is imperative that the evaluation of medical students in regard to these values begins early in their medical school education. In the Gross Anatomy laboratory there are opportunities for students to display such aspects of professionalism as responsibility/accountability for actions, working with others (teamwork), respect for patients, and social responsibility. Because students spend a significant time in the Gross Anatomy laboratory with the faculty, this setting can provide the forum for faculty to assess the expression of principles of professionalism by the students. This requires faculty who are committed to evaluating the progress of students in this regard. Faculty can monitor work ethic, interpersonal relationships, and overall attitude of these students throughout the course. Thus, aspects of professionalism can be introduced, monitored, and evaluated as early as the first semester of medical school. PMID- 16683239 TI - Overcoming the tension between scientific and religious views in teaching anatomical dissection: the Israeli experience. AB - More than three thousand years of Jewish historical and scholarly writings have addressed the problematic relationship between anatomical dissection for the purpose of medical education and Judaism, which values the wholeness and sanctity of the human body. The Department of Anatomy at Tel Aviv University's Sackler Faculty of Medicine has sought to bridge the gap between science and Jewish religious-cultural values. The Department requires students to conduct laboratory dissections on cadavers in an ethical and respectful manner. Student emotions are also addressed by the Department as students are encouraged to share their apprehensions and concerns about participating in dissections in discussion groups. At the same time, the high academic standards of the medical school are strictly upheld, ensuring that each student has a thorough knowledge of human anatomy. Teaching anatomy in Israeli medical school involves reconciling two conflicting approaches to dissection: (1) The scientific-medical approach, which views the human body as inert material and anatomical dissection as a means of studying anatomy and gaining medical knowledge. (2) A Jewish religious point of view, which perceives anatomical dissection as a threat to the sanctity of a human body and leads to the defilement of those participating in the dissection. In this article, the views of major Jewish scholars regarding dissection are presented and discussed in relation to their implementation in the dissection theater. These views are examined in an anthropological light based on observations in the dissection room and interviews with students and faculty members. The findings reflect the emotions and concerns of Israeli medical students at the Sackler Faculty of Medicine in particular as well as those of the Israeli-Jewish population as a whole. In the dissection theater, medical students must gain a comprehensive understanding of human anatomy while dealing with their own personal ethical, cultural, and religious views on death and dying. Confronting these issues enhances both personal growth as individuals and professional behavior as future physicians. PMID- 16683240 TI - Patient confidentiality: implications for teaching in undergraduate medical education. AB - Respect for patient confidentiality is one of the core concepts of professionalism and should have high priority in medical education. Confidentiality should be introduced early in the curriculum so that students understand their ethical, professional, and legal obligations throughout their medical studies and later professional career. Anatomists have important opportunities for teaching professional values including confidentiality and should be major contributors to a multidisciplinary teaching on professionalism. Students should make a formal commitment to and be assessed on patient confidentiality in the context of professionalism. A Faculty development program on confidentiality and data protection will inform and support teachers in delivering these objectives. It is recommended that medical schools have a policy on patient confidentiality and a disciplinary procedure for the management of students who breach patient confidentiality. PMID- 16683241 TI - Integrating professionalism in early medical education: the theory and application of reflective practice in the anatomy curriculum. AB - Renewed emphases on teaching professionalism require physicians to develop the ability to critically reflect upon their own decisions. Innovative programs that address teaching professionalism within medical curricula have been implemented in almost all medical schools. The foundation for many of these programs is "reflection," which is regarded as a core skill in professional competence. In order to achieve the desired outcomes and meet the demands of a required curriculum, an understanding of educational concepts in the designing of medical curricula is essential. Educators recognize that, for most medical students, professional growth is initiated during the first year of the medical curriculum and, therefore, traditionally pure content delivery courses such as first year anatomy course are being utilized now in order to explore issues related to critical thinking and professionalism. As a result, learning strategies such as "reflective practice" are beginning to play an important role in curriculum design. This article provides an overview of the theory of reflective practice, and demonstrates how reflective practice may be integrated into the anatomy curriculum. In order to incorporate reflective exercises into a curriculum, the basic elements of a reflective process are defined, strategies to implement reflective exercises within the course are described, and the benefits of reflective practice are highlighted. Therefore, in creating an environment that fosters reflective learning, the gap between theory and practice may be consolidated, which in the context of anatomy promotes the issue of teaching for relevance and clinical application. PMID- 16683242 TI - The role of gross anatomy in promoting professionalism: a neglected opportunity! AB - Professionalism is one of the six areas of competency defined by the Accreditation Council for Graduate Medical Education (ACGME). Unprofessional behavior is the single most common cause for disciplinary action against medical students in their clinical rotations, residents, and clinical practitioners. The Association of American Medical Colleges (AAMC) and the National Board of Medical Examiners (NBME) will like to see professionalism taught and evaluated across medical school. Gross Anatomy, with cadaver laboratory dissection, is in a unique position to preside over a rich number of activities where behaviors of professionalism can be taught, practiced, and rewarded. Such activities will be comparable to the behaviors of professionalism taught in clinical rotations. This article highlights the essential involvement and tools that can be used to teach, evaluate, and promote behaviors of professionalism accessible in the laboratory with cadaver dissection. PMID- 16683243 TI - Frank H. Netter, M.D. (1906-1991): the artist and his legacy. AB - The renowned medical illustrator Frank H. Netter, M.D., was born on 25 April 1906 in Brooklyn, NY, and died on 17 September 1991 in New York, NY, at the age of 85. In his professional career, Dr. Netter painted over 4,000 medical illustrations, many of which became the center pieces of over 250 issues of Clinical Symposia and were compiled in the 13 volume set of The CIBA Collection of Medical Illustrations. Two years prior to his death, the Atlas of Human Anatomy was published, considered by many to be Dr. Netter's crowning achievement. The Atlas rapidly became the most widely used atlas of anatomy in American medical schools and currently is published in 16 languages. Netter's impressive career as an illustrator and his unique style and legacy to medical education are summarized briefly in this review. PMID- 16683244 TI - The oblique cord of the forearm in man. AB - There is minimal and often conflicting data in the literature regarding the oblique cord of the forearm. The current study seeks to elucidate further the anatomy of this structure of the upper extremity. In adult cadavers, the oblique cord was observed for and, when found, measurements were made of it. Ranges of motion were carried out while observation of the oblique cord was made. An oblique cord was found on 52.6% of sides. Gantzer's muscle was found on 55% of sides and, when present, had attachment into the oblique cord on five sides. The oblique cord was present on 13 sides with a Gantzer's muscle. Of the 20 sides with an oblique cord, no Gantzer's muscle was found on 10. The mean length of the oblique cord was 3.4 cm. In the majority of specimens, this cord tapered from proximal to distal. The proximal, middle, and distal widths of this structure had means 9, 7, and 4 mm, respectively. The oblique cord was found to travel approximately 45 degrees from a line drawn through the ulna and more or less traveled perpendicular to the insertion site of the bicipital tendon. This ligament was lax in the neutral position and with pronation became lax in all specimens. The oblique cord progressively became taut with increased supination from the neutral position and was maximally taut with the forearm fully supinated. Tautness of this cord was also found with distal distraction of the radius. Following the transection of the oblique cord, no discernable difference was observed in regard to maximal supination of the forearm or distal distraction of the radius. No obvious instability of the proximal forearm was found following transection of the oblique cord. Functionally, although the oblique cord may resist supination, it is unlikely that this structure affords significant stability to the proximal forearm, as it was often absent, of a very small caliber, and based on our observations, following its transection, the amount of supination of the forearm did not increase. Moreover, one would expect that this structure would never resist supination alone, as the larger overlying muscles would become taut prior to calling upon the action of this cord. Based on our findings, the function of the oblique cord appears insignificant in providing significant stability to the proximal forearm; however, further investigative studies are now necessary to confirm these data. PMID- 16683245 TI - Promoter methylation in circadian genes of endometrial cancers detected by methylation-specific PCR. AB - Methylation of CpG dinucleotides in the promoter sequence of a gene can lead to deregulated and suppressed gene expression. In this study, we have developed procedures for methylation-specific polymerase chain reaction (MSP) and sequencing analysis to determine CpG methylation status of the promoter sequences of nine circadian genes in 35 endometrial cancers (EC) and paired noncancerous endometrial tissues. DNA methylation was found in the promoter sequences of PER1, PER2, and CRY1, but not of other six circadian genes in the ECs and normal tissues examined. Eleven of the 35 EC tissues showed CpG methylation in the promoter sequences of PER1, PER2, or CRY1. Of these 11 cases, 1 had promoter methylation in all the three genes, 1 in PER1 and PER2, 3 in PER1 and CRY1, and 6 in PER1, respectively. In comparison, promoter CpG methylation of PER1, PER2, or CRY1 was found in only 7 of 35 paired noncancerous tissues including 2 in PER1 and PER2, 2 in PER1, and 3 in CRY1. In summary, promoter methylation in the PER1, PER2, or CRY1 circadian genes was detected in about one-third of EC and one-fifth of noncancerous endometrial tissues of 35 paired specimens indicating possible disruption of the circadian clock in the development of EC. PMID- 16683246 TI - The anatomic relationships of the axillary nerve and surgical landmarks for its localization from the anterior aspect of the shoulder. AB - The axillary nerve has long been known to be one of the nerves vulnerable to damage during shoulder arthroscopic and open surgical procedures. The relationship of the axillary nerve to the shoulder capsule and the subscapularis muscle has not been well defined in orthopedic literature. This descriptive anatomical study aimed to present the course and the relations of the axillary nerve with neighboring neurovascular structures and the shoulder capsule and to define anatomical landmarks and regions that can be used practically in anterior surgical approaches to the shoulder region. To investigate the course of the axillary nerve and its relationship with neighboring structures, 30 shoulders of 15 fixed adult cadavers were dissected under the microscope through an anterior approach. A triangle-shaped anatomic area containing the axillary neurovascular bundle was defined. The closest distance between the axillary nerve and the anteromedial aspect of the coracoid tip and the glenoid labrum was measured as 3.7 cm and 1.1 cm on average, respectively. The distance between the anteromedial aspect of the coracoid tip and the point where the nerve passes through the medial edge of the subscapularis was measured as 2.5 cm on average. The results of this study demonstrate the anatomic pattern and the course of the axillary nerve and its relations with the shoulder capsule. Knowing the exact localization of the axillary nerve under the guidance of the defined anatomic triangle may provide a safer surgery. PMID- 16683247 TI - Lingual foramina on the mandibular midline revisited: a macroanatomical study. AB - The purpose of the present study was to determine the incidence, size, location, course, and content of the foramina and bony canals located on the lingual side of the mandibular midline. Fifty dry human mandibles were morphometrically analyzed by measuring the distances of these midline foramina from the mandibular base and the dimensions of these foramina and their bony canals. In addition, macro- and microanatomical dissection was performed on 12 intact cadaver mandibles. The macroanatomic midline foramina were classified into superior and inferior genial spinal foramina according to their vertical location with respect to the genial spines. This study showed that out of 50 dry mandibles, 49 (98%) had at least one midline lingual foramen; only one lacked a true midline foramen. Evaluation of the microanatomical dissections indicated a clear neurovascular bundle in both superior and inferior genial spinal foramina and canals. For the superior canal, the content was found to derive from the lingual artery and the lingual nerve. For the inferior canal, however, the arterial origin was submental and/or sublingual, while the innervation derived from a branch of the mylohyoid nerve. In conclusion, different kinds of lingual foramina have been identified according to their location. The superior and inferior genial spinal foramina have different neurovascular contents, determined by their anatomical location above or below the genial spines. PMID- 16683248 TI - An aberrant anatomic variation along the course of the ulnar nerve above the elbow with coexistent cubital tunnel syndrome. AB - We report on a patient with an unusual anatomic variation along the course of ulnar nerve above the elbow who had cubital tunnel syndrome. The variation consisted of a cutaneous neural branch that was originating at a distance of approximately 40 mm proximal to the medial epicondyle, and from the radial aspect of the main trunk of ulnar nerve. The branch had a superficial course and it was passing distally, anterior to the medial epicondyle without penetrating the fascia of the flexor muscles origin. Anterior intramuscular transposition of the ulnar nerve was performed leaving the newly found branch over the fascia between the muscles and the adipose subcutaneous tissue. PMID- 16683249 TI - Assessment of carcinogenic potential of repeated fish fried oil in mice. AB - Our prior studies have shown that single topical treatment of repeated fish fried oil extract (RFFE), containing various polycyclic aromatic hydrocarbons (PAHs), to the dorsal epidermis of mice caused enhancement of DNA damage along with higher expression of p53 and p21WAF1 proteins and cell-cycle arrest. In the present study carcinogenic potential of repeated fish fried oil (RFFO) and RFFE was assessed. Single topical application of RFFO (100 microL/animal) and RFFE (100-500 microg/animal) to Swiss albino female mice resulted in significant induction (1.8- to 7.4-fold) of ornithine decarboxylase activity. Twice weekly topical application of methylcholanthrene (MCA) for 24 wk or single topical application of 7,12-dimethylbenzanthracene (DMBA) or RFFO or RFFE, as initiator followed by twice weekly application of 12-O-tetradecanoyl phorbol myristate acetate (TPA) as promoter for 24 wk, resulted in development of skin papillomas after 6, 7, 18, and 9 wk, respectively. The cumulative number of tumors in MCA, DMBA/TPA, RFFE (200 microg)/TPA, and RFFE (500 microg)/TPA groups were 276, 168, 34, and 58 after 24 wk while negligible or minimal initiating activity was noticed in RFFO/TPA group. No tumors were found in animals either given twice weekly topical application of RFFO or a single initiating dose of DMBA followed by twice weekly application of RFFO. Histopathology of skin of animals treated with RFFE/TPA showed marked proliferation of epidermal layers along with abnormal mitosis and multinucleated tumor appearance. Skin of animals in groups RFFO/TPA and DMBA/RFFO showed sloughing and regeneration of epidermal layers, oedema along with proliferation of fibroblasts. Histochemical localization of gamma-glutamyl transpeptidase was found to be substantially higher in skin of mice treated with RFFO/TPA and RFFE/TPA. Animals treated with RFFO/TPA, DMBA/RFFO, and RFFE/TPA resulted in significant induction of cutaneous aryl hydrocarbon hydroxylase (AHH) (421-432%), ethoxyresorufin-O-deethylase (252-316%), and glutathione S transferase (133-245%) activities. Animals treated with RFFO/TPA, DMBA/RFFO, and RFFE/TPA led to significant reduction in glutathione content (39-44%) with a concomitant increase in lipid peroxidation (254-492%). Animals treated with RFFO/TPA and RFFE/TPA led a significant decrease in catalase (43-69%) and superoxide dismutase (20-31%) activities while glutathione reductase activity was found to be diminished (23-51%) in RFFO, RFFO/TPA, DMBA/RFFO, and RFFE/TPA treated groups. These results suggest that RFFE possess skin tumor initiating activity and that it may have weak promoting activity as well, which may involve free radicals. PMID- 16683251 TI - Correlation of visinin-like-protein-1 expression with clinicopathological features in squamous cell carcinoma of the esophagus. AB - EF-hand Ca(2+)-sensor proteins are key molecules for transducing Ca(2+) signals into physiological answers and changes in cytosolic Ca(2+) concentration control a variety of cellular responses, including proliferation, migration, and differentiation, which are relevant for tumor progression. The Ca(2+)-sensor visinin-like protein-1 (VILIP-1) has recently attracted major interest due to its putative tumor suppressor function. Whereas VILIP-1 is expressed in normal skin, it is downregulated in skin tumors in a murine tumor model. The aim of this study was to investigate the expression of the Ca(2+)-sensor VILIP-1 in squamous cell carcinoma of the esophagus and to correlate expression levels with clinicopathological features of the tumor. We examined VILIP-1 expression in 54 specimens of esophageal squamous cell carcinomas and 24 normal esophagus tissues, with immunohistochemical staining and immunofluorescence co-staining techniques. VILIP-1 expression was completely lost or significantly reduced in esophageal tumor tissue compared with normal squamous epithelium. Correlation with clinicopathological features indicated that there was significantly less VILIP-1 expression in lymph node positive (N = 1) versus lymph node negative (N = 0) tumors (P = 0.002). Although there was no significant difference between highly (G(1)), moderately (G(2)) and poorly differentiated (G(3)) tumors (P = 0.177), VILIP-1 expression in tumors is significantly correlated with the depth of tumor invasion (P = 0.028 between T1, T2, T3, and T4). In contrast, co-staining with the proliferation marker Ki-67 indicated no significant correlation with proliferation rates in tumors (Ki-67 index of the tumor). In summary, the expression of the Ca(2+)-sensor VILIP-1 was found to be lost during development of squamous cell carcinoma of the esophagus. The protein expression level significantly correlates with invasive features, such as depth of tumor invasion and local lymph node metastasis, but not with proliferation rate of tumor cells. PMID- 16683250 TI - Chronic thrombin exposure results in an increase in apolipoprotein-E levels. AB - Studies have shown that individuals with both a history of traumatic brain injury and inheritance of apolipoprotein E-4 (ApoE4) allele are associated with a poor neurologic outcome and an increased risk for Alzheimer's disease. We assessed the hypothesis that thrombin released during brain injury causes an increase in apolipoprotein-E levels and such increase in the levels of apolipoprotein-E4 isoform may have amyloidogenic effects. Rats received either thrombin (100 nm, 0.25 microl/hr, 28 days) or vehicle via intracerebroventricular (i.c.v.) infusion. Thrombin treatment increased apolipoprotein-E levels in hippocampus as compared to vehicle treatment (P < 0.001). Infusion of human apolipoprotein-E4 (0.6 ng/hr, i.c.v., 56 days) into rats resulted in beta-amyloid deposition and increased the number of GFAP-positive astrocytes. ApoE4 infusion also resulted in significant spatial memory deficits. These findings suggest that thrombin released during brain injury may contribute to an increase in apolipoprotein-E levels. Such increase in Apolipoprotein-E4 isoform facilitates beta-amyloid deposition and cognitive deficits. PMID- 16683252 TI - Mice with genetically altered GABA transporter subtype I (GAT1) expression show altered behavioral responses to ethanol. AB - It is widely accepted that the GABAergic system plays an important role in the action of ethanol in vivo. GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites in the CNS and regulates GABAergic transmissions. In this study, mice lacking the GAT1 were developed by homologous recombination. Both hetero- and homozygous GAT1 mutant mice were tested for ethanol, saccharin or quinine consumption, ethanol-conditioned place preference, ethanol-conditioned taste aversion, ethanol-simulated motor activity, and ethanol-induced sedation/hypnosis. The GAT1(-/-) mice showed decreased ethanol aversion and ethanol reward, and insensitivity to both the sedative/hypnotic and the motor stimulant effects of ethanol, along with increased avoidance of quinine preference and consumption. GAT1(+/-) mice showed significantly increased consumption of ethanol and saccharin, however, enhanced the rewarding and preference effect of ethanol, increased avoidance of quinine, and higher sensitivity to the motor stimulant effect of ethanol. These results demonstrate that GAT1, perhaps in a bi-directional way, modulates some behavioral effects of ethanol. The GAT1 mutant mice provided us a very useful model to investigate the mechanisms of ethanol action in vivo. PMID- 16683253 TI - Protein kinase Cepsilon and development of squamous cell carcinoma, the nonmelanoma human skin cancer. AB - Protein kinase C (PKC) represents a large family of phosphatidylserine (PS) dependent serine/threonine protein kinases. At least five PKC isoforms (alpha, delta, epsilon, eta, and zeta) are expressed in epidermal keratinocytes. PKC isoforms are differentially expressed in proliferative (basal layer) and nonproliferative compartments (spinous, granular, cornified layers), which exhibit divergence in their roles in the regulation of epidermal cell proliferation, differentiation, and apoptosis. Immunocytochemical localization of PKC isoforms indicate that PKCalpha is found in the membranes of suprabasal cells in the spinous and granular layers. PKCepsilon is mostly localized in the proliferative basal layers. PKCeta is localized exclusively in the granular layer. PKCdelta is detected throughout the epidermis. PKC isozymes exhibit specificities in their signals to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform mediates the induction of squamous cell carcinoma (SCC) elicited either by the DMBA-TPA protocol or by repeated exposures to ultraviolet radiation (UVR). PKCepsilon overexpression, which sensitizes skin to UVR-induced carcinogenesis, suppresses UVR-induced sunburn (apoptotic) cell formation, and enhances both UVR-induced levels of TNFalpha and hyperplasia. UVR induced sunburn cell formation is mediated by Fas/Fas-L and TNFalpha NFR1 extrinsic apoptotic pathways. The death adaptor protein termed Fas-associated death domain (FADD) is a common adaptor protein for both of these apoptotic pathways. PKCepsilon inhibits UVR-induced expression of FADD leading to the inhibition of both apoptotic pathways. It appears that PKCepsilon sensitizes skin to the development of SCC by UVR by transducting signals, which inhibit apoptosis on one hand, and enhances proliferation of preneoplastic cells on the other hand. PMID- 16683254 TI - A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting. AB - Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population. PMID- 16683255 TI - TGFBI gene mutations in corneal dystrophies. AB - The lattice corneal dystrophies (LCD) and granular corneal dystrophies (GCD) are autosomal dominant disorders of the corneal stroma. They are bilateral, progressive conditions characterized by the formation of opacities arising due to the deposition of insoluble material in the corneal stroma leading to visual impairment. The LCDs and GCDs are distinguished from each other and are divided into subtypes on the basis of the clinical appearance of the opacities, clinical features of the disease, and on histopathological staining properties of the deposits. The GCDs and most types of LCD arise from mutations in the transforming growth factor beta-induced (TGFBI) gene on chromosome 5q31. Over 30 mutations causing LCD and GCD have been identified so far in the TGFBI. There are two mutation hotspots corresponding to arginine residues at positions 124 and 555 of the transforming growth factor beta induced protein (TGFBIp) and they are the most frequent sites of mutation in various populations. Mutations at either of these two hotspots result in specific types of LCD or GCD. The majority of identified mutations involve residues in the fourth fasciclin-like domain of TGFBIp. PMID- 16683256 TI - DSC perfusion MRI-Quantification and reduction of systematic errors arising in areas of reduced cerebral blood flow. AB - Dynamic susceptibility contrast (DSC)-MRI is commonly used to measure cerebral perfusion in acute ischemic stroke. Quantification of perfusion parameters involves deconvolution of the tissue concentration-time curves with an arterial input function (AIF), typically with the use of singular value decomposition (SVD). To mitigate the effects of noise on the estimated cerebral blood flow (CBF), a regularization parameter or threshold is used. Often a single global threshold is applied to every voxel, and its value has a dramatic effect on the CBF values obtained. When a single global threshold was applied to simulated concentration-time curves produced using exponential, triangular, and boxcar residue functions, significant systematic errors were found in the measured perfusion parameters. We estimate the errors obtained for different sampling intervals and signal-to-noise ratios (SNRs), and discuss the source of the systematic error. We present a method that partially corrects for the systematic error in the presence of an exponential residue function by applying a linear fit, which removes underestimates of long mean transit time (MTT) and overestimates of short MTT. For example, the correction reduced the error at a temporal resolution of 2.5 s and an SNR of 30 from 29.1% to 11.7%. However, the error is largest in the presence of noise and at MTTs that are likely to be encountered in areas of hypoperfusion; furthermore, even though it is reduced, it cannot be corrected for exactly. PMID- 16683257 TI - Rapid MR elastography using selective excitations. AB - Rapid MR elastography (MRE) techniques using spatially-selective excitations to reduce acquisition times to a few seconds or less were devised and tested. The techniques included reduced field of view (rFOV) MRE and 1D MRE (beam MRE) using 2D spatially selective RF excitations for gradient-echo (GRE) applications and intersecting 90 degrees and 180 degrees slice-selective excitations for spin-echo (SE) applications. It was shown that scan times could be reduced by a factor of 8 using rFOV MRE, and by 64 using beam MRE, while still obtaining stiffness estimates comparable to full-FOV MRE. Results were shown in gel phantom experiments as well as in the case of a preserved postmortem breast tissue specimen with a stiff lesion. These methods can be used to more rapidly interrogate regions of interest (ROIs) in tissue to quickly obtain information about the viscoelastic properties of that tissue. PMID- 16683258 TI - Quantitative microanatomy of jaw muscle attachment in extant diapsids. AB - Muscular reconstructions in vertebrate paleontology have often relied heavily on the presence of "muscle scars" and similar osteological correlates of muscle attachment, a practice complicated by the fact that approximately half of tendinous muscle attachments to bone in extant vertebrates do not leave readily interpretable scars. Microanatomical and histological correlates of tendinous muscle attachment are much less ambiguous. This study examines the microanatomical correlates of muscle attachment for the mandibular adductors in six species of diapsids. Most prominent tendinous or aponeurotic muscle attachments display a high density of extrinsic fibers (similar to Sharpey's fibers). There is also some indication that the density of extrinsic fibers at an attachment may be directly related to the amount of stress exerted on that attachment. The presence of comparable densities of extrinsic fibers in fossil tissue constitutes strong and readily interpretable positive evidence for the presence of adjacent fibrous connective tissue in life. Microanatomy and histology provide reliable data about muscle attachments that cannot be gleaned from gross observation alone. These additional data, when coupled with existing muscular reconstruction techniques, may be essential to the resolution of ambiguous character states, and will provide more severe tests for long-standing hypotheses of musculature in extinct diapsids. Increasing the accuracy and precision of muscular reconstructions lends greater strength to any phylogenetic, paleobiological, or paleoecological inferences that draw upon these reconstructions as important lines of evidence. PMID- 16683260 TI - Saturated double-angle method for rapid B1+ mapping. AB - For in vivo magnetic resonance imaging at high field (> or =3 T) it is essential to consider the homogeneity of the active B(1) field (B(1)+), particularly if surface coils are used for RF transmission. A new method is presented for highly rapid B(1)+ magnitude mapping. It combines the double angle method with a B(1) insensitive magnetization-reset sequence such that the choice of repetition time (TR) is independent of T(1) and with a multislice segmented (spiral) acquisition to achieve volumetric coverage with adequate spatial resolution in a few seconds. Phantom experiments confirmed the accuracy of this technique even when TR << T(1), with the side effect being lowered SNR. The speed of this method enabled B(1)+ mapping in the chest and abdomen within a single breath-hold. In human cardiac imaging, the method enabled whole-heart coverage within a single 16-s breath-hold. Results from phantoms and healthy volunteers at 1.5 T and 3 T are presented. PMID- 16683261 TI - Active catheter tracking using parallel MRI and real-time image reconstruction. AB - In this work active MR catheter tracking with automatic slice alignment was combined with an autocalibrated parallel imaging technique. Using an optimized generalized autocalibrating partially parallel acquisitions (GRAPPA) algorithm with an acceleration factor of 2, we were able to reduce the acquisition time per image by 34%. To accelerate real-time GRAPPA image reconstruction, the coil sensitivities were updated only after slice reorientation. For a 2D trueFISP acquisition (160 x 256 matrix, 80% phase matrix, half Fourier acquisition, TR = 3.7 ms, GRAPPA factor = 2) real-time image reconstruction was achieved with up to six imaging coils. In a single animal experiment the method was used to steer a catheter from the vena cava through the beating heart into the pulmonary vasculature at an image update rate of about five images per second. Under all slice orientations, parallel image reconstruction was accomplished with only minor image artifacts, and the increased temporal resolution provided a sharp delineation of intracardial structures, such as the papillary muscle. PMID- 16683262 TI - Computational design of proteins stereochemically optimized in size, stability, and folding speed. AB - Artificial proteins potentially barrier-free in the folding kinetics are approached computationally under the guidance of protein-folding theories. The smallest and fastest folding globular protein triple-helix-bundle (THB) is so modified as to minimize or eliminate its presumed barriers in folding speed. As the barriers may reside in the ordering of either secondary or tertiary structure, the elements of both secondary and tertiary structure in the protein are targeted for prenucleation with suitable stereochemically constrained amino acid residues. The required elements of topology and sequence for the THB are optimized independently; first the topology is optimized with simulated annealing in polypeptides of highly simplified alphabet; next, the sequence in side chains is optimized using the standard inverse design methods. The resultant three best adapted THBs, variable in topology and distinctive in sequences, are assessed by comparing them with a few benchmark proteins. The results of mainly molecular dynamics (MD) comparisons, undertaken in explicit water at different temperatures, show that the designed sequences are favorably placed against the chosen benchmarks as THB proteins potentially thermostable in the native folds. Folding simulation experiments with MD establish that the designed sequences are rapid in the folding of individual helices, but not in the evolution of tertiary structure; energetic cum topological frustrations remain but could be the artifacts of the starting conformations that were chosen in the THBs in the folding simulations. Overall, a practical high-throughput approach for de novo protein design has been developed that may have fruitful application for any type of tertiary structure. PMID- 16683263 TI - Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe. AB - BACKGROUND: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. METHODS: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. RESULTS: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase-3-deficient MCF7-Fas cells and a caspase-6-specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase-3 activation consistently preceded caspase-6 by approximately 30 min following induction of apoptosis. CONCLUSIONS: We have developed a novel FRET-based probe for simultaneous detection of two caspase activities in living cells using flow cytometry. Simultaneous detection of two caspase activities using this probe has clearly provided information of the ordering of caspase-3 and -6 in the apoptotic pathway. PMID- 16683264 TI - Ligand binding by estrogen receptor beta attached to nanospheres measured by fluorescence correlation spectroscopy. AB - Although many indirect methods have been chosen to study the system of estrogen receptor ligand binding, an ideal method is fluorescence correlation spectroscopy (FCS). FCS is nondestructive to the sample, uses very small sample volumes, and operates well within physiological concentration ranges. The methodology was developed to biotinylate the estrogen receptor beta-ligand binding domain (ERbeta LBD) using biotin with a very short spacer and to then attach this protein to a 40 nm neutravidin-coated bead (nanosphere). Diffusional FCS data were obtained for a fluorescently labeled coactivator peptide, steroid receptor coactivator peptide-1 (A-SRC-1(2)), in the absence and presence of bead-bound ERbeta-LBD. Data were also acquired in the presence of one of the endogenous ligands for ERbeta, 17beta-estradiol, and with tamoxifen. The bead strategy resulted in a decreased receptor diffusion coefficient and consequent increase in the decay time of the FCS autocorrelation functions for receptor-bound, labeled SRC-1(2). Thus, free and bound coactivators were much more readily distinguished by FCS. Discrimination between the fluorescently labeled unbound and bound species could be determined in autocorrelation functions obtained in as few as 30 s. The advantage of using FCS with the ERbeta-LBD: bead methodology is the ability to obtain reliable and reproducible data in a short time frame. PMID- 16683265 TI - Progressive increase of frontostriatal brain activation from childhood to adulthood during event-related tasks of cognitive control. AB - Higher cognitive inhibitory and attention functions have been shown to develop throughout adolescence, presumably concurrent with anatomical brain maturational changes. The relatively scarce developmental functional imaging literature on cognitive control, however, has been inconsistent with respect to the neurofunctional substrates of this cognitive development, finding either increased or decreased executive prefrontal function in the progression from childhood to adulthood. Such inconsistencies may be due to small subject numbers or confounds from age-related performance differences in block design functional MRI (fMRI). In this study, rapid, randomized, mixed-trial event-related fMRI was used to investigate developmental differences of the neural networks mediating a range of motor and cognitive inhibition functions in a sizeable number of adolescents and adults. Functional brain activation was compared between adolescents and adults during three different executive tasks measuring selective motor response inhibition (Go/no-go task), cognitive interference inhibition (Simon task), and attentional set shifting (Switch task). Adults compared with children showed increased brain activation in task-specific frontostriatal networks, including right orbital and mesial prefrontal cortex and caudate during the Go/no-go task, right mesial and inferior prefrontal cortex, parietal lobe, and putamen during the Switch task and left dorsolateral and inferior frontotemporoparietal regions and putamen during the Simon task. Whole-brain regression analyses with age across all subjects showed progressive age-related changes in similar and extended clusters of task-specific frontostriatal, frontotemporal, and frontoparietal networks. The findings suggest progressive maturation of task-specific frontostriatal and frontocortical networks for cognitive control functions in the transition from childhood to mid-adulthood. PMID- 16683266 TI - High growth rate and substrate exhaustion results in rapid cell death and lysis in the thermophilic bacterium Geobacillus thermoleovorans. AB - Batch cultures of the thermophilic bacterium Geobacillus thermoleovorans T80 attained extremely high-specific glucose utilization rates leading to high specific growth rates, followed by extensive cell death and lysis with the onset of substrate exhaustion. The dramatic decrease in live cell numbers, as determined by flow cytometry, was accompanied by the release of soluble protein. Once the growth phase reached the point of commitment to lysis created by the impending exhaustion of substrate, the addition of extra carbon substrate did not halt the rapid death rate and lysis, although, towards the end of the exponential growth phase, the substrate was utilized producing only a small additional biomass concentration as a result of the net effect of cell growth and death. This lytic phenomenon was observed when a range of different carbon substrates (glucose, pyruvate, acetate, n-hexadecane, nutrient broth), as well as ammonium (the nitrogen source) in the presence of excess carbon source, reached near exhaustion. The rate and extent of cell death and the ensuing lysis depend on the culture growth rate. Cultures batch grown with a lower initial substrate concentration, or at a lower temperature, or at lower dilution rates for continuous-flow cultures, exhibited a lower rate and extent of cell death and lysis. Batch re-culture of the persister cells resulted in a behavior identical to that of the original culture indicating that these cells were not genetically modified. The glucose utilization, cell growth and death rates were mathematically described based on Monod kinetics and estimated values of pertinent biokinetic constants are reported. PMID- 16683267 TI - A fluid dynamics approach to bioreactor design for cell and tissue culture. AB - The problem of controlling cylindrical tank bioreactor conditions for cell and tissue culture purposes has been considered from a flow dynamics perspective. Simple laminar flows in the vortex breakdown region are proposed as being a suitable alternative to turbulent spinner flask flows and horizontally oriented rotational flows. Vortex breakdown flows have been measured using three dimensional Stereoscopic particle image velocimetry, and non-dimensionalized velocity and stress distributions are presented. Regions of locally high principal stress occur in the vicinity of the impeller and the lower sidewall. Topological changes in the vortex breakdown region caused by an increase in Reynolds number are reflected in a redistribution of the peak stress regions. The inclusion of submerged scaffold models adds complexity to the flow, although vortex breakdown may still occur. Relatively large stresses occur along the edge of disks jutting into the boundary of the vortex breakdown region. PMID- 16683268 TI - Activation of innate immunity by prostate specific antigen (PSA). AB - BACKGROUND: Prostate specific antigen (PSA) is a serine protease secreted by the prostatic epithelium. The only known function of the protein is to cleave seminogelin. We wished to determine if PSA activated peripheral blood mononuclear cells (PBMC). METHODS: PBMC and selected sub-populations were cultured with purified PSA. Secretion of IFNgamma was measured by cytokine capture flow cytometry and enzyme-linked immunosorbent assay. RESULTS: We observed secretion of IFNgamma and a proliferative response in PBMC cultured with PSA. We found that NK cells were the source of the IFNgamma but NK cells were not directly stimulated by PSA. Rather, a soluble factor secreted primarily by CD14 monocytes in response to PSA stimulated NK cells to secrete IFNgamma. DISCUSSION: PSA induces a pro-inflammatory response that results in the secretion of INFgamma by NK cells. The presence of large amounts of PSA could contribute to the common finding of inflammatory infiltrates in the prostate. PMID- 16683269 TI - New bone formation and osteolysis by a metastatic, highly invasive canine prostate carcinoma xenograft. AB - BACKGROUND: Osteoblastic metastases are commonly induced by prostate cancer. A canine prostate carcinoma xenograft (Ace-1) was developed and used to evaluate neoplastic prostate cell growth, metastasis, and effects on bone formation in nude mice. METHODS: Characteristics of the Ace-1 cells were evaluated with histopathology, radiography, and bioluminescent imaging (BLI). Immunohistochemistry and quantitative RT-PCR were used to evaluate the expression of factors important in the development of osteoblastic metastases. RESULTS: The Ace-1 cells were invasive and induced bone formation and destruction. Radiographs demonstrated a mixed osteoblastic/osteolytic reaction. Lung and lymph node metastases occurred in 30% of mice. The tumor cells expressed parathyroid hormone related protein (PTHrP-141 isoform), cathepsin K, keratins 8/18, and vimentin, but not keratins 5/14, and were androgen receptor negative. Intracardiac (IC) injections resulted in metastases in vertebrae and long bones. CONCLUSIONS: The Ace-1 xenograft is a useful model for investigating the pathogenesis of prostate cancer invasion and mixed osteoblastic/osteolytic bone metastases. PMID- 16683270 TI - NF-kappaB activation upregulates fibroblast growth factor 8 expression in prostate cancer cells. AB - BACKGROUND: Fibroblast growth factor 8 (FGF8) is over-expressed in prostate cancer (CaP) correlating with high-grade disease and reduced survival. The role of acetylation in transcriptional regulation of FGF8 was investigated using the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). METHODS: FGF8 transcriptional response to TSA was investigated by gene reporter assays, RT-PCR, and Western blotting. Chromatin immunoprecipitation (ChIP) assays were also performed. RESULTS: FGF8 is upregulated in response to TSA treatment along with NF-kappaB transcriptional activity. Over-expression of p65 activated FGF8 transcription. ChIP assays revealed p65 recruitment to the fgf8 promoter, containing putative NF-kappaB binding sites, post TSA stimulation. PI-3K activity is required for TSA mediated FGF8 upregulation. CONCLUSION: Using TSA treatment in prostate cancer cells, a requirement of PI-3K activity in mediating TSA function is demonstrated and a novel role for NF-kappaB in the regulation of FGF8 expression is uncovered. PMID- 16683271 TI - Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases. AB - BACKGROUND: The present study was undertaken to evaluate anti-proliferative and apoptotic activities of mahanine, a plant derived carbazole alkaloid, in prostate cancer cells and to determine its molecular mechanism by which it induces apoptotic cell death. METHODS: The growth inhibitory and apoptotic inductive effect of mahanine on prostate cancer cells were examined by measuring cell proliferation and BrdU labeling, caspase activity, DNA fragmentation, and Western blot analyses. RESULTS: Mahanine inhibited growth of PC3 and LNCaP prostate cancer cells in a dose and time-dependent manner. Mechanistically, mahanine inhibited cell-survival pathway by dephosphorylation of PIP3 dependent kinase 1 (PDK1) thereby deactivation of Akt and downregulation of Bcl-xL. In addition, mahanine activated caspase pathway (caspases 9 and 3) and eventually cleavage of DNA repair enzyme, PARP resulting DNA fragmentation and apoptosis. CONCLUSIONS: Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway. PMID- 16683272 TI - A pilot study with a new, rapid-exchange, thrombus-aspirating device in patients with thrombus-containing lesions: the Diver C.E. study. AB - BACKGROUND: In patients with acute coronary syndromes (ACS), distal embolization of thrombotic material is more likely to play a key role in the pathogenesis of myocardial no-reflow during percutaneous coronary intervention (PCI). Thus, interventional techniques able to reduce thrombus burden at the culprit vessel might improve final myocardial reperfusion. OBJECTIVE: To evaluate a new rapid exchange thrombus-aspirating catheter, the Diver C.E., in patients with thrombotic coronary lesions undergoing PCI. METHODS: Fifty patients with acute myocardial infarction (n = 44) or with non-ST-elevation ACS and angiographic evidence of coronary thrombus (n = 6) undergoing urgent PCI were prospectively enrolled. The Diver C.E. was used to aspirate coronary thrombus from the culprit lesion after placement of the guidewire. Adjunctive balloon inflations and stent implantation were used to achieve good angiographic result. Angiographic coronary flow (by means of TIMI score and corrected TIMI frame count, cTFC), thrombus score (TS), and myocardial perfusion (by means of postintervention myocardial blush grade, MBG) were assessed in all patients. RESULTS: The device could be successfully employed in 96% of the cases (48/50) and yielded significant (P < 0.0001) acute reduction in thrombus burden (TS: predevice 3.5 +/- 0.8, postdevice 2.5 +/- 0.9) and improvement in coronary flow (TIMI grade: predevice 1.0 +/- 0.9, postdevice 2.0 +/- 0.9; CTFC predevice 71 +/- 31, postdevice 39 +/- 26). Final TIMI grade 0-1 was observed in one patient only (2%). A significant (P = 0.02) correlation was found between preintervention TS and efficacy of thrombus aspiration. A more pronounced acute reduction of thrombus burden after thrombus aspiration (TS reduction > or = 2) was associated with a better postintervention angiographic myocardial perfusion (MBG 2.3 +/- 0.9 vs 1.7 +/- 0.8; P = 0.05). CONCLUSIONS: This new, easy-to-use, device is able to reduce thrombus burden and to improve coronary flow in patients with thrombus-containing lesions. The improvement in myocardial perfusion associated to greater thrombus removal highlights the importance of thrombus aspiration in the management of thrombus burdened coronary lesions. PMID- 16683273 TI - "Head-to-head comparison between sirolimus-eluting and paclitaxel-eluting stents in patients with complex coronary artery disease: an intravascular ultrasound study". AB - BACKGROUND: The aim of this study was to assess neointimal hyperplasia following sirolimus-eluting (SES) and paclitaxel-eluting stents (PES) implantation in a patients with complex coronary disease. METHOD: Between January to December 2004, 70 patients were enrolled in this study (SES = 37; PES = 33. The primary objective was to assess the efficacy of SES and PES on neointimal proliferation inhibition in patients with complex coronary lesions by volumetric 3D intravascular ultrasound (IVUS) assessment at six-month follow-up. RESULTS: Baseline clinical, demographic or angiographic characteristics were well balanced in both groups. All procedures as well as hospitalisation were uneventful. The percentage of B2/C lesions in our study was > 90% in both groups. The IVUS assessed in-stent mean neointimal hyperplasia volume was significantly lower in lesions treated with SES compared to PES (4.1 +/- 11 mm3 vs. 17.4 +/- 23 mm3, p < 0.002) at 6 month follow-up. No difference in both MACE (3.0 versus 6.0%, p = NS) and restenosis (5.4 versus 9.1%, p = NS) were found. The in-segment late loss at six month was 0.26 mm in the SES and 0.48 mm in the PES group (p = NS). CONCLUSIONS: The present study showed reduced neointimal proliferation after sirolimuseluting as compared to paclitaxel-eluting stents in patients with complex coronary artery disease. Both SES and PES were associated with low rate of angiographic restenosis or major adverse cardiovascular events. PMID- 16683274 TI - Prenatal diagnosis of a de novo complex chromosome rearrangement (CCR) mediated by six breakpoints, and a review of 20 prenatally ascertained CCRs. AB - OBJECTIVES: To describe the cytogenetic and FISH characterization of a prenatally diagnosed de novo complex chromosome rearrangement (CCR), showing the involvement of four chromosomes and six breakpoints, and review the literature concerning prenatally detected CCRs in order to obtain insights into addressing karyotype phenotype correlations in prenatal genetic counseling. METHODS: Conventional protocols were used to set up cultures and chromosome preparations. Commercial and homemade probes were used for the FISH analyses. RESULTS: An apparently balanced de novo t(4;10;20) was prenatally identified by means of cytogenetic analysis. FISH revealed a rearrangement mediated by six breakpoints and the insertion of chromosome 8 material within the 4q region. The pregnancy was interrupted. The fetus showed malformations and anomalous cortical neuron migration. The assembled list of 20 prenatally detected CCRs points to the preferential involvement of chromosomes 4, 6 and 14. The involvement of chromosome 20 is described here for the first time. CONCLUSIONS: FISH analysis is essential for the accurate definition of a complex rearrangement. Phenotype description of fetuses carrying CCRs investigated by means of molecular cytogenetic techniques may contribute to improving and personalizing genetic counseling in prenatal diagnosis. PMID- 16683276 TI - Trisomy 15 mosaicism owing to familial reciprocal translocation t(1;15): implication for prenatal diagnosis. AB - We describe a 4-year-old female child with severe global mental retardation, myoclonic epilepsy, proximal hypotonia and dysmorphisms, whose prenatal diagnosis following amniocentesis revealed a constitutional female karyotype carrying a t(1;15)(q10;p11) familial reciprocal translocation. Post-natal high-resolution karyotype, Fluorescence in situ hybridization (FISH) screening for subtelomeric rearrangements, VNTR search for UPD15 in the blood and fibroblast, and WCP1 and 15 in the mother, failed to provide an explanation for the complex clinical phenotype of the proband. Since the pachytene configuration of the translocated chromosomes defines a high probability of 3:1 segregation, an extensive workup was undertaken to look for a possibly cryptic mosaicism. Four percent of the cells with trisomy 15 was found in the peripheral blood lymphocytes examined by classical cytogenetic technique and interphase FISH analysis. The clinical features associated with cryptic trisomy 15 mosaicism and the problems concerning prenatal diagnosis and genetic counselling for carriers of translocations at high risk of 3:1 segregation are discussed. PMID- 16683277 TI - Photoelectrochemical kinetics of eosin y-sensitized zinc oxide films investigated by scanning electrochemical microscopy. AB - Eosin Y is used as a sensitizer for nanoporous zinc oxide films for prospective applications in photoelectrochemical solar cells. The kinetics of the reduction of the intermittently formed photo-oxidized dye molecules by iodide ions in the electrolyte phase was investigated by using the feedback mode of scanning electrochemical microscopy (SECM). The bulk solution phase contained triiodide as electron transfer mediator, from which the ultramicroelectrode-generated iodide ions acted as electron donors for photo-oxidized Eosin Y molecules (D(+) (ads)) at the zinc oxide sample. Effective rate constants for the dye regeneration could be extracted from the SECM approach curves. The effective rate constants at different triiodide concentrations could be related to the rate constant for the reaction of the dissolved donor with photo-oxidized Eosin Y bound to ZnO, as well as to the overall rate of the photosensitization process. For the reaction D(+) (ads) + 1.5 I(-)-->D(ads) + 0.5 I(3) (-) a rate constant of k(ox) = (1.4+/ 0.8)x10(8) cm(9/2) mol(-3/2) s(-1) was determined. PMID- 16683278 TI - Sandwich complexes based on the "all-metal" Al4 2- aromatic ring. AB - We report on novel sandwichlike structures [Al(4)MAl(4)](q-) (q=0-2 and M=Ti, V, Cr, Zr, Nb, Mo, Hf, Ta and W) based on the recently synthesized all-metal aromatic Al(4)(2-) square ring. The sandwichlike structures have two aromatic tetraaluminum square rings which trap a transition-metal cation from either the first, second, or third row. The stability of the anionic sandwichlike complexes towards electron detachment is discussed, and addition of alkali cations is found to stabilize the 2- charged complexes, preventing spontaneous electron detachment. Once the sandwichlike complexes are formed, the Al(4)(2-) square properties remain nearly unchanged; this fact strongly supports the hypothesis that in these complexes the Al(4)(2-) square rings remain aromatic. PMID- 16683279 TI - Competing magnetic interactions in Na10Co4O10, studied by neutron diffraction. AB - Na(10)Co(4)O(10) was investigated by neutron powder diffraction at 230, 70, and 4 K. The crystal structure, determined previously by X-ray diffraction on single crystals, was confirmed. Na(10)Co(4)O(10) orders magnetically below 37 K. All observed magnetic reflections could be indexed by integers (hkl) with respect to the chemical unit cell and the magnetic propagation vector q=0. The refinement was performed in the Shubnikov space group C2/c and indicated a collinear antiferromagnetic spin structure. The determined spin arrangement is consistent with the magnetic intratetramer interactions suggested previously from the analysis of magnetic susceptibility data: the magnetic moments of the central Co(III) ions of the Co(4)O(10) tetramer lie parallel to each other and couple in an antiparallel fashion to the terminal Co(II) moments. The Rietveld analysis shows that the net moments of 0.64 mu(B) per tetramer form ferromagnetic layers parallel to the ab plane. Adjacent layers are coupled antiferromagnetically along c. The spins are aligned in the ac plane along the line connecting adjacent Co(II) and Co(III) ions of the tetramer. We have determined unusually low values for the ordered magnetic moments of 2.43(5) mu(B) and 2.11(6) mu(B) for Co(III) and Co(II), respectively. The occurrence of spontaneous magnetization below 37 K indicates a slight canting of 2.2 degrees of the antiferromagnetic structure. A representation analysis shows that a weak ferromagnetic component along b is compatible with the determined antiferromagnetic structure. PMID- 16683280 TI - Asymmetric rhodium-catalyzed hydrogenation meets gold-catalyzed cyclization: Enantioselective synthesis of 8-hydroxytetrahydroisoquinolines. AB - Different furyl-substituted (Z)-dehydroamino acid derivatives were hydrogenated with the rhodium/Mandyphos(OMe)-system to give enantiomeric excesses between 80 and 98 %. The absolute configuration of the newly formed stereogenic center was determined by anomalous diffraction to be R. These chiral furyl alanines were transferred into 8-hydroxytetrahydroisoquinolines by employing gold-catalyzed arene synthesis as the key step. During the latter reaction sequence, also including either a propargylation or a reduction, a protection of the hydroxy group, and a subsequent propargylation, no racemization of the stereogenic center was observed. With very electron-rich furans, instead of the 8 hydroxytetrahydroquinolines as products, furans anellated to seven-membered rings with exocyclic C-C double bonds are formed under the same reaction conditions. PMID- 16683281 TI - Spin-density distribution in the copper site of azurin. AB - A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur. PMID- 16683282 TI - Ab initio molecular dynamics study of the keto-enol tautomerism of acetone in solution. AB - We have studied the keto-enol interconversion of acetone to understand the mechanism of tautomerism relevant to numerous organic and biochemical processes. Applying the ab initio metadynamics method, we simulated the keto-enol isomerism both in the gas phase and in the presence of water. For the gas-phase intramolecular mechanism we show that no other hydrogen-transfer reactions can compete with the simple keto-enol tautomerism. We obtain an intermolecular mechanism and remarkable participation of water when acetone is solvated by neutral water. The simulations reveal that C deprotonation is the kinetic bottleneck of the keto-enol transformation, in agreement with experimental observations. The most interesting finding is the formation of short H-bonded chains of water molecules that provide the route for proton transfer from the carbon to the oxygen atom of acetone. The mechanistic picture that emerged from the present study involves proton migration and emphasizes the importance of active solvent participation in tautomeric interconversion. PMID- 16683283 TI - Proton transfer in ionic water clusters. PMID- 16683284 TI - A metal-ion-releasing probe for DNA detection by catalytic signal amplification. PMID- 16683285 TI - Charge transfer between metal nanoparticles interconnected with a functionalized molecule probed by surface-enhanced Raman spectroscopy. PMID- 16683286 TI - A general and versatile method for C-C cross-coupling synthesis of conjugated enynes: one-pot sequence starting from carbonyl compounds. PMID- 16683287 TI - Inorganic molecular capsules: from structure to function. PMID- 16683288 TI - Microwave-enhanced ionothermal synthesis of aluminophosphate molecular sieves. PMID- 16683289 TI - Glycerol as a source for fuels and chemicals by low-temperature catalytic processing. PMID- 16683290 TI - Synthesis and reactions of highly strained 2,3-bridged 2H-azirines. PMID- 16683291 TI - Coupling of electrochemistry and fluorescence microscopy at indium tin oxide microelectrodes for the analysis of single exocytotic events. PMID- 16683292 TI - Diastereoselective synthesis of methylenecyclopropanes from chiral cyclopropene derivatives. PMID- 16683293 TI - Total synthesis of the light-harvesting carotenoid peridinin. PMID- 16683294 TI - Modular asymmetric synthesis of aigialomycin D, a kinase-inhibitory scaffold. PMID- 16683295 TI - RhI-catalyzed C-C bond activation: seven-membered ring synthesis by a [6+1] carbonylative ring-expansion reaction of allenylcyclobutanes. PMID- 16683296 TI - A quadruply azulene-fused porphyrin with intense near-IR absorption and a large two-photon absorption cross section. PMID- 16683297 TI - Maternal anxiety at amniocentesis and plasma cortisol. AB - OBJECTIVES: To assess whether anticipation of amniocentesis is linked with maternal anxiety, and whether this anxiety is associated with increased maternal plasma cortisol. METHODS: Two hundred and fifty-four women awaiting a morning amniocentesis for karyotyping (gestation range 15-37 weeks, median 17 weeks) completed Spielberger state and trait anxiety inventory (STAI) questionnaires, and provided blood samples immediately before the procedure for cortisol assay. Six hundred and five women at mean gestation of 20 weeks, attending the same hospital for routine ultrasound but not for amniocentesis, also completed Spielberger STAI questionnaires and served as a comparison group for the anxiety ratings. RESULTS: Mean state and trait anxiety scores (+/- SD) in the comparison group of 605 women at mean gestation of 20 weeks were 36.1 +/- 10.2 (range 20-70) and 35.6 +/- 8.9 (range 20-73), respectively. The mean state anxiety score (+/ SD) of 49.8 +/- 14.0 (range 20-77) of the amniocentesis group was considerably higher than the comparison group (p < 0.001), although the mean trait anxiety score in the amniocentesis group was similar at 36.4 +/- 8.6 (range 21-60). The state, but not trait, anxiety correlated with plasma cortisol (r = 0.176, p = 0.005). Maternal cortisol in the amniocentesis group increased with gestational age (r = 0.310, p < 0.001), whereas state anxiety scores showed no significant change with increase in gestational age (r = - 0.042, ns). Multivariate analysis demonstrated that maternal state anxiety was positively correlated with plasma cortisol independent of gestation and time of collection. CONCLUSION: Women awaiting amniocentesis experience a high state anxiety associated with modestly increased plasma cortisol. PMID- 16683298 TI - Postnatal follow-up of prenatally diagnosed trisomy 16 mosaicism. AB - OBJECTIVE: To determine the long-term outcome of pregnancies prenatally diagnosed with trisomy 16 and identify variables associated with the outcome. METHODS: We reviewed all published and our unpublished data from trisomy 16 pregnancies for which outcomes were available for children of greater than 1 year of age. RESULTS: Nineteen cases were diagnosed with trisomy 16 on chorionic villus sampling (CVS) and 17 cases at amniocentesis. Age at last follow-up ranges from 1 to 13 years. Among the CVS group, four out of five patients, with a birth weight and/or length below -2 SD and postnatal growth information, showed catch-up growth (80%). Among the amniotic fluid (AF) group, the birth weight was available in 13 cases. Eleven of the 13 cases had a birth weight less than -2 SD. In eight cases, the length was also below -2 SD (length data unavailable in one case). Nine out of ten cases (90%) and seven out of eight (87.5%) showed catch-up growth for weight and length, respectively. In terms of development, no cases of CVS mosaicism had global developmental delay. One child had a history of delay in speech development. Among the AF-detected cases, 4/17 cases had global developmental delay. All four children with global developmental delay had more than one major malformation compared to 6 out of 32 children in the group with normal development (p = 0.004). The finding of uniparental disomy (UPD) was not associated with developmental delay. CONCLUSIONS: The majority of prenatally diagnosed trisomy 16 mosaic cases have a good postnatal outcome. However, the finding of mosaicism on AF and the presence of major congenital anomalies are associated with an increased risk of developmental delay. PMID- 16683299 TI - Severe lupus responds to stem cell therapy. PMID- 16683300 TI - By the way, doctor. A recent test showed I have some hip osteopenia. Now every day I take two Tums Ultra (800 mg of calcium) plus a multivitamin (200 mg of calcium). I also get a total of 800 International Units (IU) of vitamin D daily. I've been advised that Tums aren't sufficient. Do I need to switch to a more expensive calcium supplement? PMID- 16683301 TI - Niger welcomes largest bednet distribution in history. PMID- 16683302 TI - Millions in Niger facing food shortages once again. PMID- 16683303 TI - Septic shock; current pathogenetic concepts from a clinical perspective. PMID- 16683304 TI - Seeing the big picture. PMID- 16683305 TI - Finding your north. PMID- 16683306 TI - Tweaking stem cells can repair joint damage in mice. PMID- 16683307 TI - The dark side of "heartburn". Acid reflux, often known as "heartburn", is more than an annoyance; it may raise your risk for esophageal cancer. PMID- 16683308 TI - Watchful waiting for hernias. PMID- 16683309 TI - Healthy brain in a healthy, social body. Physical exercise is as beneficial to mental alertness as learning new things and staying socially connected. PMID- 16683310 TI - Certain drugs might slow progression of Parkinson's disease. PMID- 16683311 TI - Statins may aid arthritis. PMID- 16683312 TI - Vertebrae treatments may cause new fractures. PMID- 16683313 TI - Eat less, live longer. PMID- 16683315 TI - Sprained ankles then, arthritis now? PMID- 16683314 TI - Meniscus health linked to knee osteoarthritis. Think twice before you get rid of that old, worn-out meniscus. You may need it, after all. PMID- 16683316 TI - Thyroid problems can impact heart health. Subtle abnormalities may contribute to heart conditions, two studies suggest. PMID- 16683317 TI - Researchers affirm more lifestyle links to cancer. Several recent studies take a novel approach to unraveling the mystery of the cancer-diet link. PMID- 16683318 TI - Radiation seed implants for early breast cancer. PMID- 16683319 TI - I lead a stressful life taking care of my husband, who had a stroke two years ago, and managing our household, our finances and our medical care. I am 67 and seem to be bearing up well physically, except for high blood pressure. But I'm worried the stress may be taking a toll on my mind. Lately I have had a difficulty concentrating and remembering. I have forgotten important things, like doctors' instructions and paying my bills, which is very unusual for me. Could stress be causing my memory lapses? If this is the case, what can I do about it? PMID- 16683320 TI - [Tuberculosis throughout the times]. AB - This paper outlines a picture of the representations of tuberculosis in different decades and how they have been handled by clinical medicine. The history of the disease, from the early days of the XIX century hygienist policies to the current control and prevention programs, displays a continual change of focus: social views were sometimes highlighted (the environment, work, housing), and at times the focus shifted to individual viewpoints (body fluids, lifestyle) regarding forms of prevention and cure of the disease. Such diverse representations and concepts about tuberculosis have influenced the way in which medicine and sick individuals perceived the disease and dealt with it. PMID- 16683321 TI - ["Nursing research--what we owe our patients." Interview by Urs Luthi]. PMID- 16683322 TI - Bioinformatics: from molecules to systems. Proceedings of a discussion meeting. April 4-5, 2005. London, United Kingdom. PMID- 16683323 TI - What is genetic information, and why is it significant? A contextual, contrastive, approach. AB - Is genetic information of special ethical significance? Does it require special regulation? There is considerable contemporary debate about this question (the 'genetic exceptionalism' debate). 'Genetic information' is an ambiguous term and, as an aid to avoiding conflation in the genetic exceptionalism debate, a detailed account is given of just how and why 'genetic information' is ambiguous. Whilst ambiguity is a ubiquitous problem of communication, it is suggested that 'genetic information' is ambiguous in a particular way, one that gives rise to the problem of 'significance creep' (i.e., where claims about the significance of certain kinds of genetic information in one context influence our thinking about the significance of other kinds of genetic information in other contexts). A contextual and contrastive methodology is proposed: evaluating the significance of genetic information requires us to be sensitive to the polysemy of 'genetic information' across contexts and then examine the contrast in significance (if any) of genetic, as opposed to nongenetic, information within contexts. This, in turn, suggests that a proper solution to the regulatory question requires us to pay more attention to how and why information, and its acquisition, possession and use, come to be of ethical significance. PMID- 16683324 TI - Killing for knowledge. AB - I distinguish between four arguments commonly used to justify experimentation on animals (I). After delineating the autonomy of the question of experiments from other topics within animal ethics (II), I examine and reject each of these justifications (III-VI). I then explore two arguments according to which animal dependent experimentation should continue even if it is immoral (VII). I close with the way in which liberationists' strategic considerations modify the moral conclusions of my analysis. PMID- 16683325 TI - Organ sales and moral distress. AB - The possibility that organ sales by living adults might be made legal is morally distressing to many of us. However, powerful arguments have been provided recently supporting legalisation (I consider two of those arguments: the Consequentialist Argument and the Autonomy Argument). Is our instinctive reaction against a market of organs irrational then? The aim of this paper is not to prove that legalization would be immoral, all things considered, but rather to show, first, that there are some kinds of arguments, offered in favour of legalisation, that are, in an important sense, illegitimate, and second, that even if legalisation might not be wrong all things considered, there are good reasons for our negative moral intuitions. Moreover, identifying these reasons will help highlight some features of moral decisions in non-ideal situations, which in turn might be relevant to some other moral or policy choices. PMID- 16683326 TI - The detection of minimal alcoholic liver disease by three methods. AB - The serum aspartate transaminase, 2-h post-prandial bile acids and the aminopyrine breath test were measured in 14 alcoholics with histologically proved minimal liver damage. A raised aspartate transaminase value was found in 64% (9/14) of the patients and was the commonest abnormality found. In three patients all three tests were normal. Six patients stopped abusing alcohol and, when reassessed a mean of 33 days later, showed significant changes in the mean aspartate transaminase value and the mean value for the aminopyrine breath test. There was no significant change in the mean post-prandial bile acids value. The remaining eight patients continued to drink alcohol and, when reassessed at a mean of 118 days, showed no significant change in any of these indices. Of the methods assessed, the serum aspartate transaminase appeared to be the most useful for detecting and monitoring patients with minimal alcoholic liver disease. However, all three tests failed to detect an unacceptably high percentage of the patients, and liver biopsy therefore remains a more certain diagnostic method. PMID- 16683327 TI - [Transgenic plants: the future of sustainable agriculture]. AB - There has been a daily increase of evidence of the advantages of the transgenic plants which have been licensed so far. Every new product offers greater advantages as regards productivity, reduction of environmental impact, vitamin value and so on. The secret of the transgenic method lies in the fact that it enables strict selection in every phase in the laboratory and during field tests. Though it is still not possible to be risk-free, we can certainly trust transgenics better than plants classified as "organic". PMID- 16683328 TI - Retraction statement. PMID- 16683329 TI - [The critical scientists' voice]. AB - The intricate debate over genetically modified organisms (GMOs) involves powerful economic interests, as well as ethical, legal, emotional and scientific aspects, some of which are dealt with in this paper.(It is possible to identify two main groups of scientists across the GMOs divide: the triumphalist and the critical group.) Scientists in the triumphalist group state that GMOs and their derivatives are safe for the environment and do not offer health hazards any more than similar, non-genetically modified, products. This view is disputed by the critical scientists, who are prompted by the scarcity of studies on the environmental impacts and toxicity of GMOs, and who point out flaws in tests performed by the same companies which hold the patents. They are also critical of the current state of the process of gene transference, lacking accuracy, a fact which, coupled with the scant knowledge available about 97% of the genome functions, may produce unforseeable effects with risks for the environment and public health yet to be assessed. Examples of such effects are: the transference of alien genes [??] to other species, the emergence of toxins, the creation of new viruses, the impacts on beneficial insects and on biodiversity in general. PMID- 16683330 TI - [Transgenics and productivity in the Brazilian agriculture]. PMID- 16683331 TI - [Concerns over transgenic soybean]. PMID- 16683332 TI - [A change in the scientific paradigm]. PMID- 16683333 TI - [Public opinion on transgenics]. PMID- 16683334 TI - [A chronology from newspaper clippings]. PMID- 16683335 TI - [Preserved stegomyia and vinegared microbes: Brazil's triumph in Berlin]. AB - The article investigates the circumstances leading to Brazil being awarded the first prize at the XIV International Conference on Demography and Hygiene, held in Berlin in 1907. An examination of the unpublished correspondence between Henrique da Rocha Lima and Oswaldo Cruz prior to the conference/exhibition reveals the peculiarities of a Brazilian-style event, where the outcome seems to be determined by haphazard incidents rather than by meticulously planned strategies. Their letters can also be read as a narrative celebrating individual qualities, as they attribute the victory in Berlin much more to Rocha Lima's than Oswaldo Cruz's personal merits. In light of the Raizes do Brasil, a classic by Sergio Buarque de Holanda, this article also addresses the issue regarding the dispute between the Iberian and Anglo-Saxon worlds over the Brazilian victory. PMID- 16683336 TI - [Life in Manguinhos: the career trajectory of a group of scientists at the Instituto Oswaldo Cruz]. AB - This article aims to analyze the career trajectory of a group of biomedical scientists of Manguinhos and it focuses on the process of construction of their professional careers between the 20s and the 50s. It is centered on case studies, with a view to evaluating the sociability and consolidation means employed by the group, the recruiting strategies, as well as the values which shaped their identity in the daily life inside the Instituto Oswaldo Cruz, deemed to be one of the leading loci of biomedical sciences in Brazil. PMID- 16683337 TI - [Public health and chemical-pharmaceutical companies]. AB - This article is concerned with the establishment of the Brazilian pharmaceutical industry. It addresses some issues of relevance to social-economic history, such as the emergence of the public health system, procedures for combating infectious disease, the relations between disinfection campaigns and the chemical industry, serotherapy and the production of serums and vaccines by public research institutions and private pharmaceutical companies. Focusing on the private pharmaceutical industry in Brazil, with special reference to the Instituto Pinheiros - Produtos Terapeuticos S.A., this article highlights the relations between scientists technology and product development. It also considers the debate which involved the scientific community and some governmental research institutions for public health policy development in the state of Sao Paulo. PMID- 16683338 TI - [Scientific diffusion in Rio de Janeiro: some considerations about the 20s]. AB - In this article, we collate data about the activities aimed at the diffusion of scientific knowledge, which were developed in Rio de Janeiro in the 20s, and we discuss their main characteristics. We also point out the significant involvement of members of the scientific and academic communities in such activities. The main research questions guiding the investigation are concerned with the identification of the main characteristics of scientific diffusion at the time, the main actors in such a process, their form of organization, the research means and instruments used. We also provide a general analysis of the motivations, interests, and the philosophical and political perspectives of science in the 20s, as reflected in the type of scientific diffusion produced at the time. PMID- 16683339 TI - [On the founders of the Institute of Mathematics and Physics, University of Bahia]. AB - The reduced number of female students of mathematics at the University of Bahia School of Philosophy (Faculdade de Filosofia, Universidade da Bahia - FF/UBa) is quite surprising. To date, they are concentrated in areas traditionally viewed as feminine whereas men predominate in the mathematical fields. I have examined interview data from a few women who graduated in mathematics and went on to teach at the University of Bahia School of Mathematics (Faculdade de Filosofia - FF) and at the Institute of Mathematics and Physics (Instituto de Matematica e Fisica - IMF), where they were soon to outnumber men and constitute the majority of the mathematics teaching staff. In this study, I have investigated the course of their careers over time: from their early student days, through their time as teaching assistants and professors, and finally as founders of the Institute of Mathematics and Physics, in 1960. Special reference is made to Martha Maria de Souza Dantas, organizer of the I Brazilian Conference on Mathematics Teaching, an event which has provided the groundwork for what was to become the Institute (IMF); and to Arlete Cerqueira Lima, the mastermind behind its creation. PMID- 16683342 TI - [Researchers' virtual libraries: its role in science and technology]. PMID- 16683343 TI - [The scientific and technological policy depicted in the annual addresses of heads of state]. PMID- 16683344 TI - Health care expenditures in the OECD. PMID- 16683345 TI - Will the rising Social Security full retirement age affect the disability insurance rolls? PMID- 16683346 TI - Do advertisements for smoking cessation products help smokers quit? PMID- 16683347 TI - [Factors involved in learning efficacy of patients with mild cognition disorders]. PMID- 16683349 TI - [Automobile driving by old demented patients--an issue that has to be addressed immediately]. PMID- 16683348 TI - [Automobile driving and protection of the rights of aged patients with dementia]. PMID- 16683350 TI - [Status of automobile driving by demented patients and the response by physicians]. PMID- 16683351 TI - [Traffic safety management for the elderly--with special reference to automobile driving by aged patients with cognition disorders]. PMID- 16683353 TI - [Consensus on demented patients giving up automobile driving and the role of physicians]. PMID- 16683352 TI - [Process in which aged patients with dementia give up automobile driving--dealing with family members and dilemma that patients face]. PMID- 16683354 TI - [From the study on Alzheimer disease to that of cognition disorders seen in psychiatric diseases]. PMID- 16683355 TI - [Rehabilitation of cognition disorders: an overview]. PMID- 16683356 TI - [Concept of cognition rehabilitation in Alzheimer disease and a preliminary trial]. PMID- 16683358 TI - [Not Available]. PMID- 16683357 TI - [Not Available]. PMID- 16683360 TI - [Not Available]. PMID- 16683359 TI - [Not Available]. PMID- 16683361 TI - [Not Available]. PMID- 16683362 TI - [Not Available]. PMID- 16683363 TI - [Not Available]. PMID- 16683364 TI - [Not Available]. PMID- 16683365 TI - [Not Available]. PMID- 16683366 TI - [Not Available]. PMID- 16683367 TI - [Not Available]. PMID- 16683369 TI - [Not Available]. PMID- 16683368 TI - [Not Available]. PMID- 16683371 TI - [Not Available]. PMID- 16683370 TI - [Not Available]. PMID- 16683372 TI - [Not Available]. PMID- 16683373 TI - [Not Available]. PMID- 16683374 TI - [Not Available]. PMID- 16683375 TI - [Not Available]. PMID- 16683376 TI - Quality control of mammography screening in the Veneto Region. Evaluation of four programs at a local health unit level--analysis of the frequency and diagnostic pattern of interval cancers. AB - AIMS AND BACKGROUND: Service mammography screening has been reported to have suboptimal performance compared to controlled trials. The aim of this study was to evaluate the sensitivity of the mammography screening program in four Local Health Units (ASL) and the possible causes of diagnostic error in cases further surfacing as interval cancers. MATERIAL AND METHODS: Interval cancers were identified by cross checking of screened women databases with hospital discharge records reporting breast cancer. Proportional interval cancer incidence (observed interval cancers/expected invasive cancers) was determined by matching the database of women screened during 1999-2002 to the hospital discharge records databases during 1999-2003. The ratio of observed interval cancer rate to underlying incidence was compared to international standards and with literature data. Screening mammograms reported as negative and followed by interval cancers were randomly mixed with true-negative controls, and the resulting set underwent blind review by an external radiologist who applied the conventional criteria recommended for the classification of the type of diagnostic error (occult, minimal signs, screening error). RESULTS: Matching of screening archives with the hospital discharge records databases allowed for the identification of 154 invasive interval cancers compared to 480 expected. The proportional observed/expected interval cancer incidence in the first and second year of the interval was 21% and 46%, respectively (ASL 1 = 14% or 38%, ASL 2 = 19% or 48%, ASL 3 = 30% or 53%, ASL 4 = 25% or 49%). Radiological review included 38 further interval cancer cases, identified after the time limits defined for proportional interval cancer incidence assessment, and could not include 18 interval cancers, not retrieved from ASL 4 archives: overall, 174 interval cancers were reviewed, of which 135 were classified as occult (77.3%) (ASL 1 = 83.3%, ASL 2 = 71.1%, ASL 3 = 78.6%, ASL 4 = 75%), 12 (6.9%) as minimal signs (ASL 1 = 6.6%, ASL 2 = 11.5%, ASL 3 = 2.4%, ASL 4 = 5%), and 27 (15.5%) as screening error (ASL 1 = 8.3%, ASL 2 = 17.3%, ASL 3 = 19.0%, ASL 4 = 25%). CONCLUSIONS: Observed proportional interval cancer incidence was lower than commonly reported for service screening programs and currently recommended (< 30% in the first, < 50% in the second year of the interval). The analysis of interval cancer causes showed a screening error rate below the maximum acceptable standard (< 20% of interval cancers should be classified as screening error) in three of four programs and in average figures. Substantial differences observed among single programs (one did not comply to recommended standards) suggest that space is available for the improvement of overall performance by optimizing program organization and by further training of radiologists. Overall, the analysis showed a good sensitivity of the screening program in the Veneto Region, although the performance was inferior to that of excellence centers, and further action to improve it is possible. Assessment and review of interval cancers is an early indicator of screening efficacy which has not yet been fully adopted in Italian screening programs. Although using hospital discharge records to identify interval cancers may be affected by limited errors, such a procedure is particularly convenient, as data from hospital discharge records are available much in advance compared to cancer registries and are the most reliable source of information for areas uncovered by a cancer registry. Hospital discharge records-based procedures for interval cancers assessment should be employed routinely in screening programs. PMID- 16683377 TI - Weekly docetaxel and gemcitabine following docetaxel plus epirubicin or vinorelbine as first-line treatment of metastatic breast cancer: results of a multicenter phase II study. AB - AIMS AND BACKGROUND: Sequential docetaxel and gemcitabine following initial docetaxel plus epirubicin or vinorelbine association could be worthwhile as first line treatment of metastatic breast cancer. METHODS: Fifty-eight patients entered a phase II study that included two sequential phases. In the first phase, 36 and 22 patients previously unexposed or exposed to adjuvant anthracyclines received the association of docetaxel (75 mg/m2, day 1) with epirubicin (75 mg/m2, day 1) or vinorelbine (20 mg/m2, days 1 and 5), respectively, every 21 days for 4 courses. In the second phase, patients who had a response (R) or stable disease (SD) received docetaxel (35 mg/m2) and gemcitabine (800 mg/m2) on days 1, 8 and 15 every 28 days for 4 courses. RESULTS: In the first phase, grade > or = III neutropenia occurred in 51% and 37% of patients during docetaxel-epirubicin and docetaxel-vinorelbine, respectively. In the second phase, it occurred in the 27% and 15% of patients initially treated with docetaxel-epirubicin and docetaxel vinorelbine, respectively. On an intention to treat basis, the complete (CR) + partial response (PR) rate to the first phase was 71%, and 22% of patients had SD, without a significant difference between the docetaxel-epirubicin and docetaxel-vinorelbine arms. After the second phase, the CR + PR rate was 65%, and 14% of patients had SD. Median time to progression and survival were 12.1 and 22.0 months, respectively, without a significant difference between patients initially treated with docetaxel-epirubicin and docetaxel-vinorelbine. CONCLUSIONS: Following an initial docetaxel-based treatment, weekly docetaxel and gemcitabine maintains high percentages of R and SD, with improved toxicity. Survival was similar in patients previously untreated and treated with adjuvant anthracyclines. PMID- 16683378 TI - Third-line hormonal treatment with exemestane in postmenopausal patients with advanced breast cancer progressing on letrozole or anastrozole. A phase II trial conducted by the Hellenic Group of Oncology (HELGO). AB - AIMS AND BACKGROUND: The understanding of hormonal therapies in postmenopausal women with metastatic breast cancer has advanced greatly in the past several decades. With the introduction of orally active, potent and selective third generation aromatase inhibitors (anastrozole, letrozole and exemestane), approaches to the treatment of hormone-sensitive advanced breast cancer are undergoing reevaluation. For treatment of advanced or metastatic disease that has progressed on tamoxifen, all three agents are active. The purpose of the study was to assess the antitumor efficacy and tolerance of exemestane administered as third-line hormonal therapy to postmenopausal women with metastatic breast cancer refractory to letrozole and anastrozole. STUDY DESIGN: Sixty postmenopausal women with stage IV hormone receptor-positive carcinoma of the breast were enrolled in the study. All patients had received two prior hormonal manipulations and had measurable or assessable disease. All adverse events were monitored. RESULTS: Objective tumor response was achieved in 12 (20%) patients (95% CI, 9.6-30.4). The overall clinical benefit was 38.3% (95% CI, 21.2-49.3), and the median duration of objective tumor response was 20 months (range, 9-26). The median time to death was 17.4 months (95% CI, 16.14-18.66). CONCLUSIONS: Exemestane represents an active and well-tolerated treatment option in pretreated patients with advanced breast cancer who have received standard first- and second line hormonal therapies. By extending the sequence of hormonal therapy, disease progression and the need for chemotherapy may be significantly delayed. PMID- 16683379 TI - Significance of the relative size of a breast tumor in deciding the method of surgical treatment. AB - AIM: In the surgical treatment of breast cancer there are basically two options: radical and breast-sparing surgery. The main criterion in deciding the mode of treatment is the absolute size of the primary tumor. The purpose of this study was to analyze the relative size of a breast tumor as a prognostic factor and to establish boundary values of the relative tumor size for breast-sparing or radical surgery. METHODS: The relative volume of the breast tumor was analyzed in 200 female patients treated at the Zagreb University Hospital for Tumors. All patients were diagnosed with ductal invasive carcinoma and underwent radical surgery. RESULTS: The study found the correlation between the relative volume of the tumor and tumor grade (r = 0.805, P = 0.24) and between the relative volume of the tumor and the involvement of regional lymph nodes (r = 0.493, P = 0.14). These correlations confirmed the possible prognostic value of the relative size of a breast tumor. CONCLUSIONS: Three percent is the boundary value of the relative volume of a tumor at which its biological potential changes and the pathohistological level of a tumor and the number of affected lymph nodes increases. The relative size of 3% could therefore be considered a boundary value in making a decision about the method of surgical treatment (breast-sparing or radical surgery). For tumors with a relative size of less than 3%, breast conserving surgery is recommended for its lesser malignant potential and the possibility of performing wide extirpation (extirpation 1-2 cm from the tumor margin), regardless of the size of the breast. PMID- 16683380 TI - The safe association of intraoperative evaluation of surgical margins and neoadjuvant chemotherapy in breast cancer larger than 3 centimeters. AB - OBJECTIVES AND BACKGROUND: The objective of the present study was to determine whether neoadjuvant chemotherapy in women with breast cancer larger than 3 cm associated with breast-conserving surgery plus intraoperative evaluation of surgical margins is safe. METHODS: A total of 164 patients with breast cancer larger than 3 cm in diameter were submitted to neoadjuvant chemotherapy between 1992 and 1998 and followed until 2003. We used neoadjuvant chemotherapy in pulses at 21-day intervals with 5-fluorouracil (500 mg/m2), epirubicin (50 mg/m2) and cyclophosphamide (500 mg/m2) (FEC). Initially, 3 cycles of chemotherapy were administered, followed by surgery and 6 additional chemotherhapy cycles. During surgery, an evaluation of the surgical margins was performed. RESULTS: Quadrantectomy was performed in 102 patients (62.2%) and mastectomy in 62 (37.8%). Local recurrence in 8 patients, metastasis in 37, and 21 deaths were observed. The curves of local recurrence for quadrantectomy and mastectomy were similar (P = 0.654 and P = 0.841, respectively), and so were the numbers of local recurrence (P = 0.4438). The curves of disease-free survival for quadrantectomy and mastectomy were different (P = 0.034 and P = 0.033, respectively). However, no statistically significant difference was observed in the number of events (P = 0.1283). A statistically significant difference was observed for the curves (P = 0.001 and P = 0.000) and the number (P = 0.0034) of deaths between patients undergoing quadrantectomy or mastectomy. CONCLUSIONS: Neoadjuvant chemotherapy can reduce surgery complexity and is safe when associated with intraoperative evaluation of the surgical margins, without changing the local recurrence rate, disease-free survival, and overall survival. PMID- 16683381 TI - Long-term consequences of total gastrectomy: quality of life, nutritional status, bacterial overgrowth and adaptive changes in esophagojejunostomic mucosa. AB - OBJECTIVES: The aim of the study was to evaluate long-term quality of life and adaptive changes in the mucosa of the proximal section of the small intestine used for esophagojejunostomy reconstruction in stomach cancer patients after total gastrectomy. MATERIAL AND METHODS: Thirty-one patients who had undergone stomach cancer-related total gastrectomy were included in the study, which spanned a period of 48 to 127 months (79.6 months on the average) after the surgery. The analysis included: a) evaluation of selected biochemical parameters; b) microbiological evaluation of esophagojejunostomic area; c) evaluation of adaptive changes in esophagojejunostomic mucosa using light and electron microscopy; d) quality of life evaluation with a Troidl questionnaire. RESULTS: Quality of life was subjectively rated as good or very good by almost all subjects. The analyzed biochemical parameters were within the range of normal values in all the subjects with the exception of mild abnormalities in alkaline phosphatase and vitamin B12 levels in some patients. Microbiological examination of mucosal specimens from below the esophagojejunostomy revealed significant bacterial flora overgrowth in all the patients, with streptococci being the most abundant species. Light and electron microscopy examination of the epithelium confirmed it was normal and characteristic of a healthy small intestine. CONCLUSIONS: Long-term quality of life in patients after complete stomach resection is considered good or very good, irrespective of the reconstruction method used, and the esophagojejunostomic mucosa of the reconstructed area is normal and typical for a healthy small intestine. PMID- 16683382 TI - Kidney-sparing radiotherapy by multiple-field three-dimensional technique in the postoperative management of patients with gastric cancer: comparison with standard two-field conformal technique. AB - AIMS AND BACKGROUND: The opposed two-field technique is the standard approach for delivering adjuvant radiotherapy to patients with resected gastric cancer. Since a considerable radiation dose may reach both kidneys with this beam arrangement, with a potential risk of late effects, we investigated whether the CT-based multiple-field (M-F) approach was superior in terms of sparing critical organs at risk. METHODS: From March 2001 to March 2004, 19 patients with radically resected gastric cancer entered the study. They were treated with adjuvant chemoradiotherapy according to the INT 0116 protocol. For each patient dose volume histograms were calculated and the volume values of both kidneys and liver receiving 25 Gy (V25), 30 Gy (V30) and 40 Gy (V40) individually estimated with the M-F and two-field techniques were compared in detail. RESULTS: Right kidney median V25, V30 and V40 values for the two-field and M-F techniques were 1.50%, 0%, 0% and 0%, 0% and 0%, respectively (P < 0.005, P < 0.01, P < 0.05). Left kidney median V25, V30 and V40 values for the M-F and two-field technique were 16%, 9.80%, 0.90% and 33.20%, 30.20%, 21.40% (P < 0.001, P < 0.0005, P < 0.0005). Liver median V25, V30 and V40 values for the M-F and two-field techniques were 51.30%, 22.30%, 8.90% and 13.30%, 11.60%, 8.10%, respectively (P < 0.0001, P < 0.0005 and P = 0.18). CONCLUSIONS: Our comparison revealed that with the multiple field technique the right kidney may be largely spared from irradiation; with respect to the two-field technique, the left kidney may receive a significantly reduced dose; however, the liver receives an increased dose that warrants careful long-term monitoring of hepatic function. PMID- 16683383 TI - Long-term results of conventional radiotherapy versus accelerated hyperfractionated radiotherapy versus concomitant radiotherapy and chemotherapy in locoregionally advanced carcinoma of the oropharynx. AB - AIMS AND BACKGROUND: To compare conventional fractionation (CF) radiation therapy (RT), arm A, versus a split-course accelerated hyperfractionated schedule (S AHF), arm B, versus CFRT plus concomitant chemotherapy (CT), arm C, in terms of five-year survival and toxicity for squamous cell tumors of the oropharynx. METHODS AND STUDY DESIGN: Between January 1993 and June 1998, 192 previously untreated patients with stage III and IV oropharyngeal carcinoma (excluding T1N1 and T2N1) were enrolled in a multicenter randomized phase III trial (ORO 93-01). In arms A and C, 66 to 70 Gy in 33 to 35 fractions was administered five days a week for six and a half to seven weeks. In arm B, the dose delivered was 64 to 67.2 Gy in two fractions of 1.6 Gy every day, five days a week, with a planned two-week split at 38.4 Gy. In arm C the CT regimen consisted of three cycles of carboplatin and 5-fluorouracil (CBDCA 75 mg/m2 on days 1 to 4 and 5-FU 1000 mg/m2 i.v. on days 1 to 4 every 28 days). RESULTS: No statistically significant difference was found in five-year overall survival (P = 0.39): 21% for arm A, 21% for arm B, and 40% for arm C. Similarly, there was no statistically significant difference in terms of five-year relapse-free survival: 15% for arm A, 17% for arm B, and 36% for arm C. There was a slight trend towards better five-year locoregional control (P = 0.07) for the combined arm: patients without locoregional relapse were 48% in arm C, 21% in arm A and 18% in arm B. Locoregional control was significantly better when arm C was compared with arms A and B combined (P = 0.02; arm A+B 20%; arm C 48%). Distant metastases were fairly balanced in the three arms (A: 14; B: 9; C: 11), with a tendency towards more frequent isolated distant metastasis development in arm C (8 of 11 [72%] versus 7 of 23 [30%] in arms A+B). Five-year second-tumor-free survival was 85%. The 13 second tumors were equally distributed and were mainly correlated with tobacco and alcohol consumption (five lung, two esophagus, two oral cavity, one larynx, one pancreas, one hepatocarcinoma, one myeloma). Arm C showed slightly more G3+ late side effects involving subcutaneous tissues and mucosa, although significant late sequelae were relatively uncommon and the mucosal side effects were mostly transient. The occurrence of persistent G3 xerostomia was comparable in the three treatment arms. CONCLUSIONS: The results obtained with the combination of CT and RT compared with RT alone did not reach statistical significance, but combined treatment almost doubled the five-year overall survival, relapse-free survival and locoregional control rate. Patients with advanced squamous cell carcinomas of the oropharynx who are medically suitable for the combined approach should be treated with a combination of radiotherapy and chemotherapy. The occurrence of second tumors is relatively common in these patients and may contribute substantially to the causes of death. PMID- 16683384 TI - Two exercise schemes in postoperative breast cancer: comparison of effects on shoulder movement and lymphatic disturbance. AB - AIMS AND BACKGROUND: We attempted to determine the best method of performing the exercises in rehabilitation after breast cancer surgery and their influence on postoperative complications. Currently, there are no guidelines for these exercises. The scope of this study was to evaluate the effect of two schemes of physiotherapy exercises (directed or free) on shoulder function and lymphatic disturbance in postoperative rehabilitation. SUBJECTS: Sixty women who underwent a modified radical mastectomy or quadrantectomy with axillary dissection were randomized into two groups. METHODS: The physiotherapy technique used was kinesiotherapy. The directed group performed physiotherapy following a regimen of 19 exercises. The free group performed the exercises following the biomechanical physiological movements of the shoulder without a previously defined sequence or number of repetitions. RESULTS: The averages of the flexion, abduction and external rotational movements of the shoulder showed better recovery with statistical significance in the directed group compared to the free group. There was no significant difference between the groups in lymphatic disturbance. CONCLUSION: At the end of 42 days of follow-up, the movements of flexion, extension, abduction and external rotation of the shoulder were better rehabilitated in the directed group. The two schemes of exercises were not different with regard to lymphatic disturbance. PMID- 16683385 TI - Disclosure of cancer diagnosis to patients and their relatives in Turkey: views of accompanying persons and influential factors in reaching those views. AB - AIMS AND BACKGROUND: In Turkey, it is a common belief that most family members of patients with cancer would not want them to be informed of a diagnosis of cancer. Our aim was to evaluate the attitudes and opinions of people accompanying cancer patients, regarding cancer diagnosis disclosure. METHODS: In a cross-sectional study 270 caregivers accompanying cancer patients during outpatient chemotherapy sessions were asked to fill in a questionnaire to determine their opinions regarding whether the diagnosis of cancer should be disclosed to the patients and their relatives or not. Timing of telling the diagnosis and from whom it should be learned were queried as well. Possible influential factors for the answers were analyzed with the chi-square test. RESULTS: Of the 270 accompanying persons, 130 (48.2%) said that the patients should be informed of the diagnosis, whereas a greater number (236, 87.4%) believed that the patient's relatives should be informed. Being younger than 40 years old (P = 0.0005), being unmarried (P = 0.002), having a higher educational status (P = 0.0001) and having passed less than four months since the diagnosis (P = 0.005) positively affected opinions regarding telling the truth to the patient. Higher education (P = 0.012) and high monthly income (P = 0.002) positively affected opinions regarding disclosing a diagnosis of cancer to the patient's relatives. CONCLUSION: As a result, in a survey of caregivers' points of view, more than half of the accompanying persons did not agree with disclosing a cancer diagnosis to patients, whereas the majority agreed with disclosing it to the relatives, and educational level seemed to be the major influential factor. PMID- 16683386 TI - Levels of VE-cadherin increase independently of VEGF in preoperative sera of patients with colorectal cancer. AB - AIMS AND BACKGROUND: Vascular endothelial cadherin (VE-cadherin) preserves the tightness of the mature vascular network as a component of endothelial adherens junctions. Vascular endothelial growth factor (VEGF) makes VE-cadherin dissociate from complexes with beta-catenin, so that endothelial cells can loosely proliferate and migrate. We searched for relationships between VEGF and VE cadherin levels in preoperative sera of patients with colorectal cancer (CRC). We also compared VE-cadherin levels of control and preoperative CRC sera in relation to clinicopathological features. METHODS: We measured with an ELISA kit the serum levels of the proteins in preoperative samples from 125 CRC patients and in samples from 16 healthy volunteers. RESULTS: Serum VE-cadherin was about fourfold higher in CRC patients than in controls (P < 0.00001), with similar results being found in subgroups with different clinicopathological features versus controls. VE-cadherin was not correlated with VEGF in the entire group of CRC patients nor in the subgroups of node-positive and node-negative patients, different grades of histological differentiation (G2 or G3), extent of tumor growth (pT1+pT2 or pT3+pT4), histopathological type (adenocarcinoma or mucinous carcinoma), sex, age, and tumor site (colon or rectum). However, the serum levels of VE-cadherin and VEGF in CRC patients, which were higher than the mean values of controls, tended towards a negative correlation in node-positive patients (P = 0.078, r = 0.279). CONCLUSIONS: VEGF and VE-cadherin seem to be independent markers of angiogenesis in CRC with no significant correlation between their serum levels. PMID- 16683387 TI - Neuroendocrine tumors of the larynx: a clinical report and literature review. AB - Neuroendocrine tumors of the larynx represent a rare group of neoplasms characterized by pathological and biological heterogeneity. The histological diagnosis is the most important step in the appropriate management of these tumors and the prognosis varies according to histological types. Here we report on a case of a laryngeal neuroendocrine tumor occurring in a 67-year-old man who underwent several locoregional and systemic treatments. Because of the very low incidence of neuroendocrine tumors in the larynx, a review of the literature has also been performed. PMID- 16683388 TI - Cardiac metastases in malignant fibrous histiocytoma. A case report. AB - Malignant fibrous histiocytoma metastasizing to the left ventricle is an uncommon form of cardiac malignancy. This report describes a rare case of left ventricular metastases from a malignant fibrous histiocytoma of the posterior compartment of the right thigh, recurring five years after treatment with surgery, hyperthermic perfusion of the limb and radiotherapy. As the patient presented symptoms of cardiac tamponade, open heart surgery was performed through a median sternotomy; however, the tumor was not resectable and only a biopsy was performed. A partial response was obtained with standard and high-dose chemotherapy with peripheral blood progenitor cell transplantation. The response continued to improve with immunotherapy. The patient returned to normal physical activity. He died four years later due to a ventricular arrhythmia. PMID- 16683390 TI - Ileal carcinoid with liver metastasis presenting after ten years with abdominal mass and right heart failure: report of a case. AB - A case of ileal carcinoid metastatic to the liver is reported. The diagnosis was made and treatment given ten years after the detection of a left hypervascular liver mass, which was first confounded with a hemangioma. The onset of right heart failure led to surgical replacement of the tricuspid and pulmonary valves. After cardiac surgery the patient underwent an ileal resection and left hepatectomy for a cystic left liver metastasis. Isolated right heart failure and cystic degeneration of a liver metastasis are uncommon features of metastatic carcinoid tumors; only a few cases have been described in the literature. Cardiac surgery is recommended before liver surgery to reduce venous pressure and consequent bleeding during hepatectomy. Surgical treatment of liver metastases may relieve endocrine symptoms and result in an overall five-year survival rate of 47%. PMID- 16683389 TI - HPV 6-positive giant keratoacanthoma in an immunocompetent patient. AB - Keratoacanthoma (KA) is a clinically distinct, rapidly growing lesion that generally presents as a solitary crateriform nodule in sun-exposed areas in elderly, fair-skinned individuals. A KA larger than 20-30 mm is referred to as giant keratoacanthoma, a relatively rare lesion especially in young patients. Such lesions grow rapidly with possible destruction of underlying tissues. In addition to ultraviolet exposure, KAs have also been associated with chemical carcinogens, chemical peels, genetic factors, chronic skin conditions that produce scarring, trauma and thermal burns. Immunosuppressed patients, especially after transplantation, also develop KAs. A viral etiology has been suggested but not confirmed. We encountered a case of giant keratoacanthoma (greater than 50 mm in diameter) with induration of underlying structures on the upper lip of a 39 year-old male sailor. The patient reported sudden appearance and rapid enlargement of the lesion in only three weeks. Biopsy of the cutaneous lesion and the characteristic clinical history suggested the diagnosis of keratoacanthoma. Total excision with primary closure of the defect by a nasolabial advancement flap was performed. Histological examination of the tumor mass confirmed the diagnosis of KA with infiltrative growth and perineural invasion. Immunosuppression was excluded by blood analyses, as were HIV, syphilis and hepatitis infections. Only low-risk genital HPV type 6 was detected in the lesion, suggesting a possible cocarcinogenic effect of HPV and UV light in a chronically sun-exposed patient. PMID- 16683391 TI - [Novel methods of treatment for colorectal carcinoma: from oral chemotherapy to angiogenesis inhibitors]. PMID- 16683392 TI - [Erythropoietin today? Contrary opinions]. PMID- 16683393 TI - [ECCO 2005. Advances in the prevention of chemotherapy-induced nausea and vomiting]. PMID- 16683394 TI - Consensus report: First European Workshop on Tobacco Use Prevention and Cessation for Oral Health Professionals. PMID- 16683395 TI - Public health aspects of tobacco control: setting the agenda for action by oral health professions across Europe. AB - Tobacco use is a significant public health problem across Europe. Each year over half a million Europeans die prematurely due to a smoking-related disease. Tobacco use is a primary cause of many oral diseases and adverse oral conditions. The prevalence of tobacco use varies considerably across Europe, although in many countries overall rates of use have declined in recent years. However, tobacco use among women and young people is rising in several European countries. Tobacco behaviour is influenced by an array of factors, and quitting is a major challenge for many tobacco users. Tobacco use is now considered a chronic progressive relapsing condition requiring very specific support and assistance. To reduce tobacco use across Europe, a range of complementary actions and policies are required at an international, national and local level. The WHO Framework Convention on Tobacco Control (FCTC) outlines an array of evidence-based policies that can be implemented to prevent tobacco use and promote cessation. National dental associations and professional groups across Europe have an important role to play in supporting the ratification and implementation of the FCTC. The aim of this paper is to outline the public health aspects of tobacco control and highlight how the oral health professions across Europe can become actively engaged in this important and relevant area of prevention. PMID- 16683396 TI - Evaluation of tobacco use cessation (TUC) counselling in the dental office. AB - Tobacco use cessation (TUC) in dentistry is critical to reducing the effect of a major risk factor for both oral and systematic diseases. The effect of TUC interventions has been widely reported. The data show that the success of TUC without professional support is negligible but that behavioural and pharmacological interventions are effective. Furthermore, the greater the intensity of support, the greater the quit rate and success rates are similar comparing different health care professionals including dental professionals. Although few studies have been performed in dental practice, it is clear that TUC should be embedded in routine oral health care. In addition to evaluating the effect of TUC, several studies have investigated barriers to implementing TUC in dental settings. A large number of barriers have been reported. These studies highlight the importance of further training for dental professionals but also identify the need for major cultural and policy changes to facilitate the provision of TUC. Research on barriers to TUC in dental care could be facilitated by employing qualitative or mixed methods designs and studies that evaluate the impact of changing such barriers on TUC provision. Such an approach will help to close the gap between research findings and implementation. Regarding the measurement of outcomes from TUC, no gold standards exist currently. Therefore both self-reported and biochemical measures of tobacco use should be reported in evaluation studies. It is also clear that feedback from biochemical testing of tobacco use can increase success rates in tobacco use cessation. PMID- 16683397 TI - Tobacco use prevention and cessation in dental and dental hygiene undergraduate education. AB - Oral health care professionals are aware of their responsibility to advise patients to stop using tobacco. However, they do not feel sufficiently prepared to help their patients to quit, and consequently are not confident in providing these preventive measures. This fact reflects the lack of emphasis on tobacco cessation in both dental and dental hygiene undergraduate education. It may therefore be assumed that improvement of dental and dental hygiene education in tobacco use cessation counselling may result in increased self-confidence and frequency of its provision. The importance of making space in the curriculum for tobacco use prevention and cessation has to be emphasised. Dental schools and dental hygiene programmes have to be reminded of the key role the dental profession has in tobacco control. Next to the public health aspect of tobacco control, such involvement may be both an ethical and a legal responsibility. The implementation of effective tobacco use prevention and cessation in a dental educational setting requires a multidisciplinary approach involving the school's entire teaching personnel and external experts. In general, a knowledge base attained through lecture, Problem-Based Learning (PBL), or E-Learning, and clinical skills attained through clinical instructions and practices is required. It is suggested that curriculum content should include (1) the biological effects of tobacco use, (2) the history of tobacco culture and psychosocial aspects of tobacco use, (3) prevention and treatment of tobacco use and dependence, and (4) development of clinical skills for tobacco use prevention and cessation. PMID- 16683398 TI - Continuing education of tobacco use cessation (TUC) for dentists and dental hygienists. AB - Tobacco use prevention (TUC) guidelines are mostly designed for undergraduate education. Most practising dental professionals have not been trained in TUC and so current and future guidelines need to be adapted for continuing education. It is important to motivate dental professionals to be involved in TUC. 'The 3 Ts' is one suggested method of stimulating this motivation. Two levels of TUC are recommended, and both brief advice and enhanced interventions can be incorporated into routine practice. It is recommended that TUC continuing education on these interventions should be provided by a team of dental and trans- disciplinary experts. The maintenance of TUC involvement can be divided in individual and collective strategies. The international dental professional organisations can provide important 'benchmarks' for minimum clinical standards and for the involvement of both national dental organisations and individual dental health professionals in TUC continuing education. PMID- 16683399 TI - Assessing behavioural change support abilities of the oral healthcare team. AB - Competent behavioural change intervention can be learned, practised and developed. Therefore, the teaching and assessment of this ability should be within the scope of both the undergraduate and post-graduate curriculum. Assessment should target knowledge base and skills in the areas of counselling, communication and behaviour. Assessment of the knowledge base should ideally be conducted in a comprehensive, multidisciplinary, centrally based manner in the pre-clinical curriculum. Assessment of skills in the areas of communication, counselling and behaviour change is a wider aim that should be integral throughout the curriculum. In continuing education (CE) environments, an initial 'screening' assessment would help educators to adjust the course to the participants' background and needs. Furthermore, three major assessment schemes are proposed: (1) assessment of knowledge and skills, (2) evaluation of the whole course by the participants, and (3) assessment of the implementation process, four to six months after completion. PMID- 16683400 TI - [Detection of free cancer cells in peritoneal cavity in patients surgically treated for gastric adenocarcinoma]. PMID- 16683401 TI - [Detection of free cancer cells in peritoneal cavity in patients surgically treated for gastric adenocarcinoma]. AB - BACKGROUND/AIM: Peritoneal metastasis is a leading cause of therapeutic failure after an operative treatment of patients with gastric adenocarcinoma. Free cancer cells might induce or indicate an early peritoneal seeding with a subsequent peritoneal metastasis. The aim of this study was to determine the frequency of the presence of free cancer cells in the peritoneal cavity in the patients surgically treated for gastric adenocarcinoma, and its relation to certain clinical, operative and pathohistological paramethers. METHODS: Inside a period from April 2000, and April 2004, the total of 100 patients underwent intraoperative peritoneal lavage for cytological examination. Immediately after the laparotomy, 200 ml physiologic saline, heated to 37 degrees C, was introduced into the abdominal cavity, mannualy dispersed and collected from the region around the gastric tumor and the pouch of Douglas. The nucleated cell layer was smeared on four glass slides for every patient and dyed with May-Grunwald-Giemsa stain. The cytological findings were defined as positive or negative according to the presence of cancer cells. The frequency of positive cytological findings was compared to the location and the diameter of the cancer, pathohistological type of carcinoma, pathohistological stage of the disease, lymph node and the liver and/or peritoneal metastases and the type of surgical procedure. RESULTS: Free cancer cells were found in 24 (24%) of the patients, while in 76 (76%) of them cytological findings were negative. A statistically highly significant difference (p < or = 0.001) in the frequency of positive cytological finding was found between the groups of patients with and without cancer invasion of serosa, with cancer diameters > 5 cm and < or = 5 cm, in the stage of disease I, II and III, IV, with macroscopically present and without metastases, with re section and D2 lymphadenectomy and palliative procedure. Free cancer cells were statistically more frequently (p < or = 0.05) detected in the patients with lymph nodes metastases comparing to the patients with out lymph nodes involvement. The results of the univariate analysis showed that the cancer diameter > 5 cm, tumor invasion of serosa, pathohistological stage of the disease III and IV and macroscopically visible metastases were the most important risk factors for the free cancer cells detection. CONCLUSION: Peritoneal lavage cytology was shown to be a useful tool for the detection of the group of patients with greatest risk of peritoneal dissemination. The frequency of positive cytological findings was highly associated with the diameter of the tumor and the cancer invasion of serosa. Cytological examination of peritoneal lavage fluid improved the accuracy of staging and selection of patients who might have benefit from neoadjuvant chemotherapy. PMID- 16683402 TI - Autopsy and postmortem examination case study on genetic risk factors for cardiac death: polymorphisms of endothelial nitric oxide synthase gene Glu298Asp variant and T-786C mutation, human paraoxonase 1 (PON1) gene and alpha2beta-adrenergic receptor gene. AB - BACKGROUND/AIM: The Glu298Asp variant in exon 7 and T-786C mutation in the 5' flanking region of the endothelial nitric oxide synthase (eNOS) gene, paraoxonase I gene (PON1), and alpha2beta-adrenergic receptor gene (alpha2beta-AR) have been reported to be genetic risk factors for coronary heart disease (CHD). The aim of this study was to investige the effects of these four genetic polymorphisms on the probability of death due to CHD, using data obtained from medico-legal autopsies. METHODS: Blood samples from three groups: healthy controls, dead cases with CHD and without CHD (the latter as a control for dead cases) were used. After DNA extraction, genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) test. RESULTS: The frequency of the T allele in Glu298Asp variant in the dead cases with CHD was significantly higher than that in the healthy control (p < 0.001, OR = 4.47) and that in the dead cases without CHD (p < 0.001, OR = 7.62). The gene frequency of PON1 was significandy different (p = 0.007) between dead cases with and without CHD, and was also significantly different (p = 0.025) between the healthy control and dead cases without CHD. The gene frequency of PON1 was not significantly different (p = 0.401) between the healthy controls and dead cases with CHD. Hence this gene was not associated with death due to CHD. The other polymorphisms (T-786C mutation, alpha2beta-AR) also showed no effect on death due to CHD. CONCLUSION: The polymorphism of Glu298Asp eNOS gene in dead cases may be useful for determining the cause of death in CHD cases in the Japanese population. PMID- 16683403 TI - [Expression of Bcl-2 protein and the amplification of c-myc gene in patients with chronic myeloid leukemia]. AB - BACKGROUND/AIM: Chronic myeloid leukemia (CML) represents a malignant myeloproliferative disease developed out of pluripotent hematopoietic stem cell that contains the fusion bcr-abl gene. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about the disease progress. The aim of our study was to carry out the analysis of the presence of the amplification of the c-myc oncogene, as well as the analysis of the changes in the expression of Bcl-2 in the patients with CML. METHODS: Our study included 25 patients with CML (18 in chronic phase, 7 in blast transformation). Using an immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) method, we analyzed the expression of cell death protein in the mononuclear bone marrow cells of 25 CML patients. By a differential PCR (polymerase chain reaction) method, we followed the presence of amplified c-myc gene in mononuclear peripheral blood cells. RESULTS: The level of the expression of Bcl-2 protein was considerably higher in the bone marrow samples of the patients undergoing blast transformation of the disease. The amplification of c myc gene was detected in 30% of the patients in blast transformation of the disease. CONCLUSION: The expression of Bcl-2 protein and the amplification of c myc gene are in correlation with the disease progression. PMID- 16683404 TI - Pattern of utilization of benzodiazepines in patients with hypertension: a pilot study. AB - BACKGROUND/AIM: The analysis of drug prescribing in general practice in Serbia showed that the use of benzodiazepines is most frequently associated with hypertension. The aim of this study was to establish the correlation of the characteristics of patients with hypertension to antihypertensive drug therapy, and the intake of benzodiazepines. METHODS: A special questionnaire was used for interviewing the patients (n = 171) chronically treated for hypertenson. Statistical tests used were chi2-test and Student's t-test. RESULTS: No differences were noted in terms of age, gender, education, body weight, smoking habits and blood pressure (155 +/- 4.9/100 +/- 2.7 mmHg vs. 160 +/- 2.2/105 +/- 3.7 mmHg), between the group I (antihypertensive drugs+benzodiazepines: n = 79), and the group II (antihypertensives only: n = 92). The patients taking benzodiazepines received a lower number of different antihypertensive drugs (2.3 +/- 0.09 vs. 2.7 +/- 0.10; p < 0.01), but the total antihypertensive drug load was significantly greater than in the group II (2.6 +/- 0.10 vs. 1.9 +/- 0.15 defined daily doses (DDD)/patient/day; p < 0.01). Benzodiazepines were taken for anxiety (62%) and hypertension (21%), rarely for insomnia, mostly once a day, at bedtime. About half the patients took benzodiazepines regularly for months or years aware of the risk for addiction. Diazepam was used by 82% of the patients. The average daily exposure to benzodiazepines was 0.45 +/- 0.05 DDD/patient/day. The drug was bought without prescription in 25% of the patients, and without consulting a physician in 12% of them. CONCLUSION: The study confirmed a close association of hypertension with the use of benzodiazepines. The frequent use of benzodiazepines in the patients with hypertension might be caused by an inadequate response to antihypertensive drug therapy, besides anxiety and insomnia. The therapeutic efficacy of a long-term use of low doses of benzodiazepines in hypertension requires further investigation. PMID- 16683405 TI - [Gynecology and obstetrics in the Vojnosanitetski Pregled]. PMID- 16683406 TI - [Pregnancy outcome during the bombing of Yugoslavia from March 24 to June 9, 1999]. AB - BACKGROUND/AIM: The aim of this study was to evaluate pregnancy outcome during the bombing of Yugoslavia in the period from March 24 to June 9, 1999. METHODS: A retrospective study included a total of 81 spontaneous abortion following XII gestation week, and 1448 deliveries, hospitalized in the regional hospital. The analyzed were: the incidence of spontaneous abortion, Cesarean section, post-term delivery, vaginal delivery following the previous Cesarean section within the period from March 24 to June 9, 1999, and compared to the same periods in 1998 and 2000 by the use of chi2 and Kolgomorov-Smirnov tests. RESULTS: Under the conditions of a three-month stress imposed by the bombing, significantly increased were the incidence of spontaneous abortion and vaginal delivery following the previous Cesarean section, while the incidence of Cesarean section and post-term delivery were decreased, but the incidence of perinatal outcome was paradoxically improved. The analysis of findings on admittance revealed that iterative Cesarean section was performed electively, close to the expected term of delivery, and vaginal delivery following the previous Cesarean section mainly two weeks before that term with the admittance finding confirming a high active stage pregnancy. CONCLUSIONS: Within the bombing, statistically significantly was increased the percentage of abortions after XII gestation week, and the biological duration of pregnancy was reduced. The reduced duration of pregnancy complete with the accelerated fetal mutation (also caused by the stress) resulted in better perinatal outcome, and statistically significantly lower percentage of Cesarean section. PMID- 16683407 TI - [Tumor marker CA-125 in adnexal inflammatory tumors]. AB - BACKGROUND/AIM: The glycoprotein of a high molecular weight CA-125, which is not a specific tumor marker of ovarian cancer, is secreted by the endothelial cells of most pelvic organs. Endometriosis, inflammatory processes in the pelvic cavity, as well as some nongynecoligical malignant diseases, could be followed by the increased values of CA-125. Serial assessment of the values of CA-125 makes it possible to avoid surgical treatment, and, by means of the used conservative treatment, to avoid malignant diseases not to be noticed. METHODS: The study included 57 female patients hospitalized due to inflammable adnexal tumors. Besides following the values of serum CA-125 during and after the therapy, also performed were the transvaginal Doppler ultrasonography and the determination of the values of resistance index (RI). RESULTS: In 27 patients (55.1%) the CA-125 values ranged from 38.8 U/ml to 794 U/ml, while in 30 of the patients they were within the range of normal. In this group of the patients, besides the increased values of CA-125, also increased were the values of leucocytes (11(9)/1-20(9)/1), as well as the sedimentation rates (65-120) within the first hour. In all the 57 patients, transvaginal Doppler ultrasonography revealed the presence of adnexal tumor of inflammatory kind. The measured values of RI were within the range of 0.539-0.681. Eight of the patients were treated by the conservative--triple antibiotic therapy, while in 49 patients explorative laparotomy was performed. Hystorectomy was done in 12 of the patients, and one-side adnexectomy in 37 of them. CONCLUSION: The method for the assessment of CA-125 is simple and available which facilitates the monitoring of surgical, conservative or the combined therapy that is particularly significant in younger patients with inflammable adnexal tumor developed on the basis of endometrosis. PMID- 16683408 TI - [Effects of tibolone on the risk factors for cardiovascular diseases in postmenopausal women]. AB - BACKGROUND/AIM: Tibolone is a preparation that belongs to the group of steroidal substances. The effects of the use of the use of tibolone are the consequence of the activities of its metabolities, considering that their hormonal activity depends on the type of tissue in which they develop. The aim of this study was to evaluate the influence of the use of tibolone on risk factors for the development of cardiovascular diseases in postmenopausal women. METHODS: A prospective observational stady included 94 patients who had the concentration of 17beta estradiol < 50 pg/ml and who was in menopause more than a year. Out of the total number of patients, 63 accepted to receive tibolone 2.5 mg daily (tibolone group), while 31 of the patients refused to take tibolone (control group). We measured the concentration of lipids (cholesterol, LDL cholesterol, HDL cholesterol, triglicerides), antitrombin III, fibrinogen, and C-reactive protein, before and after the treatment within a 6-month period. Then, we compared the difference between the values of concentrations and tested the statistical significance of the difference. We also evaluated the changes of values in the concentrations of the examinated parametars inside a 6-month period in the control group. RESULTS: In 31 patients of the control group, from the control group there were no significant changes in the values of the defined parametars as compared to their initial values after six months. But there were changes of statistical significance (p < 0.001) in values of the concentrations of the exeminated parametars before and after the treatment in the tibolone group. In fact, we recorded decreases in the total cholesterol by 17.8%, HDL cholesterol by 27%, LDL cholesterol by 4% (without statistical significance p > 0.05) and triglicerids by 35%. There were no statistical differences in the concentrations of antitrombin III, fibrionogen, and C-reactive proteine in the tibolone group before and after the treatment CONCLUSION: The use of tibolone dose decrease the concentration of the total cholesterol, triglicerides, HDL cholesterol, without a significant decrease of LDL cholesterol. Also, the use of tibolone does not have any significant effect on the concentrations of antitrombin III, fibrinogen and C reactive proteine. The number of serum parameters measured in this study was limited, thus that was the reason to discuss only about the metabolism of lipids in the patients from the tibolone group. The final condusion about the risk for cardiovascular diseases in the patients on tibolone, howerer, reqnires were extensive further clinical exeminations. PMID- 16683409 TI - [Liver damage caused by hepatitis C viral infection and ethyl alcohol consumption]. AB - BACKGROUND/AIM: Hepatitis C virus infection (HCV) is a complex disease, most commonly chronicle (80-85%). The aim of this research was to determinate the level of the liver damage in the patients cansed by HCV in conjunction with consuming ethyl alcohol. METHODS: The research included 15 patients with chronic HCV infection supported by the misuse of ethyl alcohol, as well. The diagnosis of C infection hepatitis was proved using the ELISA test and PCR method. RESULTS: The results of the study showed the liver damage by both HCV infection and ethyl alcohol, which was verified by the presence of biochemical changes and patohystological processing of the patients (liver biopsy and prosection). Patohystological changes were at the level of liver cirrhosis and carcinoma (2 patients). There was a signficant difference between the two subgroups (p < 0.001) regarding the examined values gamma-GT, PLT and PTV. The basic therapeutic procedure was to introduce this category of patients into alcohol abstinence, and, in a few patients, to apply the antivirus therapy, as well. CONCLUSION: Based on the number of the examined patients (n = 15), we could conclude that a prolonged ethyl alcohol misuse with the presence of HCV infection was in a correlation with the liver disease progression. PMID- 16683410 TI - [Thanatophobia in the patients on dialysis]. AB - BACKGROUND/AIM: Thanatophobia is an exaggerated, specific, structured fear of death. It appears in childhood and continues to grow over the years, and in the old age it is accompanied with nosophobia and other mental disorders. The aim of this study was to analyze thanatophobia in dialysed patients which was in the direct connection with a basic disorder, and the influence of this disorder on functioning and the quality of life of the patients on dialysis. METHODS: In the study we examined 753 patients from the chronic program of haemodialysis in a period from 1999 to 2004. The patiens were classified in two groups: 348 randomized patients with Balkan Endemic Nephropathy (BEN), and the control group (N18) of patients with terminal renal insufficiency, and other diagnoses (n = 405). Since the study was a comparative, cross-sectional one, the patients were tested by the appropriate questionnaires for anxiety, depression and general mental functioning. Statistical analysis was done by the standard descriptive and analytic statistic methods. RESULTS: Based on socio-demographics data we revealed a highly significant difference regarding the place of living between the groups BEN and N18 (chi2 = 23.970; p < 0.01), the frequency of occurrence of renal comorbidity (chi2 = 23.970; p < 0.01), the frequency of familly renal comorbidity in siblings (chi2 = 23.970; p < 0.01), and the frequency of migrationes (chi2 = 4.874; p < 0.01). According to psychiatry scales, the patiens from the BEN group were significantly more anxious and depressive than those from the control group. CONCLUSION: The signs of thanatophobia were revealed in both examined groups, but significantly more in the patients with BEN than in those with other nephrologic diseases. Thanatophobia starts before dialysis, and dialysis structures it into fear of death which is in a direct connection with the basic disorder. This intensive fear may be connected with dementia and depression, but also with other mental disorders. PMID- 16683411 TI - [Human ehrlichiosis]. AB - BACKGROUND: Human ehrlichiosis is a newly recognized disease. It is a tick-borne disease caused by several bacterial species of the genhus Erlichia. These are small gram-negative pleomorphic cocci, that are obligatory intracellular bacteria. Tick Ixodes is the principle vector in Europe, and Amblyomma amenicanum in the United States. Bacterial organisms replicate in a tick, and are transmited from infected cells in a vector to the blood cells of animals or humans. Human ehrlichiosis is a name for a group of diseases caused by different species of Ehrlichia. One of them is the disease named human monocytic ehrlichiosis, caused by Ehrlichia chaffeensis, and the other is a human granulocytic ehrlichiosis caused by Anaplasma phagocytophilia. CASE REPORT: We reported a 23-year-old patient admitted for the clinical treatment with the symptoms of high febrility (above 40 degrees C), headache, vomiting, general weakness and exhaustion, but without data on a tick bite. The patient was treated with trimetoprim sulfamethoxazole for a week when Ehrlichia chaffeensis was confirmed by the immunofluoroscence test, and the therapy contimed with doxacyclin. CONCLUSION: Human ehrlichiosis is also present in our country, so this disease should be considered everyday, especially in infectology practice. PMID- 16683412 TI - [Influence of metal-ceramic fixed dental restorations on the occurrence of discoloration of gingiva]. AB - BACKGROUND: Adverse effects of dental cast alloys on the health of patients are the problem in clinical practice. The aim of this paper was to describe the case of a patient with discolorated gingivae in the presence of fixed dental restorations and used diagnostic and therapeutic procedures. CASE REPORT: A 30 year old pacient, presented with the complaints about unsatisfactory esthetic of his fixed dental restorations. He complained about the greyish discoloration of gingivae, inappropriate color of the crowns, and a larger space between the central incisors. Both discolorated and normal gingivae around the fixed dental restorations were taken by excision and the samples of gingivae were examined histopathologically, and by using the Atomic Absorption Spectrophotometry test (AAS). Histopathological examination of the discolorated gingivae showed the presence of non-specific inflammation with a foreign body. AAS revealed the presence of various metals in the samples and the higher metal contents in the samples of discolorated gingivae as compared with the samples of normal gingivae. New metal-ceramic crowns were made for the patient. CONCLUSION: A main cause of greyish discoloration of gingivae was presence of a metal in gingival tissue. After the excision of discolorated gingivae old metal-ceramic crowns should be replaced with new crowns. PMID- 16683413 TI - [Historical review of the development of military medical corps--part 1]. PMID- 16683414 TI - Management of ovarian cancer. An impressive history of improvement in survival and quality of life. AB - Over the past 2 decades, we have seen major progress in the management of women with ovarian cancer, with improvements in both overall survival and quality of life. To truly appreciate this progress, it is important to understand the state of affairs regarding the treatment of ovarian cancer in the early 1980s. This paper will discuss that historical background, describe the increasingly favorable impact of evolving treatment paradigms in ovarian cancer, and note future directions for clinical research in this complex disease process. PMID- 16683415 TI - Treatment of stage I-III non-small-cell lung cancer in the elderly. AB - Elderly patients with stage I-III non-small-cell lung cancer (NSCLC) constitute a peculiar patient population and need specific therapeutic approaches. Limited resections are an attractive alternative for elderly patients with resectable NSCLC because of the potential reduction in postoperative complications. Curative radiation therapy is an acceptable alternative for elderly patients who are unfit for or refuse surgery. Hypofractionated stereotactic body radiation therapy is of particular interest for this population because of its favorable tolerance. Elderly patients may tolerate chemotherapy poorly because of comorbidity and organ failure. The survival benefit obtained with adjuvant platinum-based chemotherapy in the younger population may vanish or decrease in the elderly because of a potential higher toxic death rate or lower compliance to treatment. The efficacy and feasibility of adjuvant chemotherapy for elderly patients need to be investigated in specific trials. Neoadjuvant chemotherapy remains an experimental approach under investigation in the general patient population, and consequently should not be considered in clinical practice in the elderly. Retrospective analyses on chemoradiation in elderly patients should be considered globally ambiguous and at risk of selection bias. Only specifically designed prospective studies will elucidate the real role and feasibility of this combined approach in the treatment of unselected elderly patients. PMID- 16683416 TI - Identification and treatment of aggressive thyroid cancers. Part 2: risk assessment and treatment. AB - Most thyroid cancers are slow-growing, easily treatable tumors with an excellent prognosis after surgical resection and targeted medical therapy. Unfortunately, 10% to 15% of thyroid cancers exhibit aggressive behavior and do not follow an indolent course. Approximately one-third of patients with differentiated thyroid cancers will have tumor recurrences. Distant metastases are present in about 20% of patients with recurrent cancer. Approximately half of patients with distant metastases die within 5 years. The loss of the ability to concentrate radio iodine and produce thyroglobulin is a sign of dedifferentiation, which occurs in about 30% of patients with persistent or recurrent thyroid cancer. Dedifferentiation is associated with poorer responses to conventional therapy and difficulty monitoring tumor burden. Clinicians must identify tumors with more aggressive biology and treat them accordingly with more aggressive regimens. Part 1 of this two-part article, which appeared in March, described in detail the distinct types of thyroid cancer, as well as risk factors, outcomes, treatment, and prognostic factors, with a focus on thyroid cancers of follicular cell origin. Part 2 covers risk assessment and staging, findings that suggest the presence of aggressive tumors, recurrent/metastatic disease, and treatment with chemotherapy and external-beam radiotherapy. Experimental treatments utilizing molecular targets, redifferentiation agents, and gene therapy are covered briefly as well. PMID- 16683417 TI - Polypoid lesions of the lower female genital tract. PMID- 16683418 TI - The FDA and the person with cancer: give PROs a chance. PMID- 16683419 TI - [The past, present and future of the reparative and reconstructive surgery]. AB - OBJECTIVE: To expound the progress of the reparative and reconstructive surgery (RRS), especially in the recent 20 years, and the developmental direction in the future. METHODS: We reviewed the information from many books, journals, and conference compilations concerned with RRS. RESULTS: RRS emerged with modern surgery. After its development for 20 years, distinguished achievements were obtained in technologies, e.g., biological fixation of bone fracture, bioactive materials, stem cells, tissue engineering, bioactive factor, and induction/derivation regeneration of tissues and organs. Moreover, ideas were continuously refreshed in RRS, such as emphasis on the equal importance of donor and recipient in autologous transplantation, and of structural and functional restoration in dentofacial surgery. CONCLUSION: Further research programs should be conducted, especially in the fields of creative theories, innovative techniques, and rehabilitation engineering. PMID- 16683420 TI - [Development of the tissue-autografting]. AB - OBJECTIVE: To introduce the current situation and prospect of the tissue autografting, such as the flaps, muscle flaps, and bone(periosteum) flaps, and its application in reparative and reconstructive surgery. METHODS: Based on our own experiences and combined with the review of the literature at home and abroad, the latest development of the tissue-autografting was analyzed. RESULTS: The femoral anterolateral flap, latissimus dorsi muscle flap, upper arm lateral flap, scap flap, temporal fascial flap and perforator flap are the frequently used in clinic. Of all the perforator flap had such advantages as better repair of the recipient sites and less damage of the donor site. Because of more advantages of the free myocutaneous flap transplantation, it substituted the free muscle transplantation. The latissimus dorsi muscle myocutaneous flap was the most frequently used in the transplant of the vessels, with preserved function of the thoracodorsal nerve or with repair of the defected tissues by the bridge. The most common donor sites of the bone were ribs, iliac bone, fibula and scapula, so the severe bone defects or the bone non-union, femoral head ischemic necrosis, and the bone graft from the tumor removal could be managed with the bones from those sites. CONCLUSION: The autografting in repairing the tissue defect has become one of the most important surgical techniques in reparative and reconstructive surgery. PMID- 16683421 TI - [Advance in management of skeletal trauma of the extremities]. AB - OBJECTIVE: To investigate the advance in the management of skeletal trauma of the extremities. METHODS: The literature at home and abroad was reviewed, and the research findigs with clinical experience in the therapeutic methods for fracture of the extremities were summarized. RESULTS: The concept on fracture management was renewed, the minimally invasive surgery (MIS) was developed and popularized, the implantation was improved, the navigation technique with computer-assisted surgery was applied, and the tissue engineering was developed. The fracture management was changed from tubular bone, the boanatomical reduction with absolutely rigid fixation to the biological osteosynthesis with protection of the fracture environment. The minimally invasive surgical techniques included the minimally invasive plate osteosynthesis, intramedullary nailing, external fixation, arthroscopic surgery, and computer-assisted surgery. In concordance with the MIS principles, the new implants, such as the locking compression plate, and the less invasive stabilization system were well designed and put into clinical practice so as to provide effective therapeutic results in treating osteoporotic fractures and complicated articular and/or metaphyseal fractures. In treatment of the delayed union or non-union of fractures, more effective techniques were employed, including the application of bone substitutes, which are degradable and have properties of bone conduction and induction. In the repair of segmental defects of the long tubular bone, the bone transport and the vascularized bone grafts could work well. The investigation of the bone engineering revealed its great potentiality. CONCLUSION: Fracture of the extremities is a common problem and its management should emphasize the recovery of the extremity function of the patient in addition to emphasis on the replacement and fixation of the biological structures. The combination of bone engineering and microsurgery represents the development tendency in this field. PMID- 16683422 TI - [Recent progress in diagnosis and treatment of the injury to the peripheral nerve]. AB - OBJECTIVE: To investigate the latest development in diagnosis and treatment of the injury to the peripheral nerve. METHODS: The literature at home and abroad was reviewed, and the research findings with clinical experience in diagnosis and treatment for the injury to the peripheral nerve were summarized. RESULTS: The treatment for the total brachial plexus avulsion injury was successfully performed by the extra-plexus nerve transfer. The avulsion of the brachial plexus could be directly repaired by the healthy C7 nerve root transfer through the anterior spinal approach. The forearm flexors could be reinforced by the neurovascularized gracilis transplantation. MRI and CTM were the best methods of early diagnosis for the brachial plexus injury. The pure upper or lower root avulsion of the brachial plexus injuries could be repaired by the intra-plexus nerve transfer, which involved a transfer of part of the ulnar nerve in the arm to the motor nerve of the biceps for C5-C6 avulsion of the brachial plexus,and a transfer of selective fascicles of the healthy C7 nerve root or brachial muscle branch to the flexors muscle fascicles of the median nerve or anterior interosseous nerve. The thoracic outlet syndrome always occurred in this position when the neck muscle fatigue occurred in the typists or the game-players after their longstanding looking forwards. The C5 and C6 roots were rolled by the tendenofibrotic tissue at the origin of the scalenus. After a procaine block, the symptom subsided. Radiation neuropathy was a series of pathological changes caused by overdoses of the radiation therapy. The pathologic findings consisted of a series of vessel damages and final coagulation necrosis, which induced clusters of abnormally-dilated, thin-walled telangiectasias. Radiation neuropathy could be detected by MRI and could be differentiated from tumor. Once the diagnosis was established, the therapy should be begun early, including the systemic use of steroids, anticoagulation, and hyperbaric oxygen. CONCLUSION: Great progress has been made in diagnosis and treatment of the injury to the peripheral nerve, but a further study should be performed to promote regeneration of the nerves and reconstruction of the related functions. PMID- 16683423 TI - [Advancement in the repair and reconstruction of the injured spine and spinal cord]. AB - OBJECTIVE: To investigate the latest research and the therapeutic development in the injuries to the spine and spinal cord. METHODS: Literature concerned was reviewed, combined with our own research and clinical experience, to summarize the trend of the researches and their clinical application in the treatment of the injured spine and spinal cord. RESULTS: The posterior approach atlantoaxial stabilization technique changed the conventional wiring technique into the transarticular screw fixation to the plate and pedicle or the lateral mass screw fixation technique. The clinical application of the transoralpharyngeal atlantoaxial reduction plate fixation technique showed a good effect on the reduction of atlantoaxial dislocation. However, there were no unified criteria for selection of the surgical approach, fixation level, and fusion mode in the treatment of thoracolumbar spinal fractures. Under optimal conditions, both the anterior and the posterior approaches could achieve good clinical effects on decompression and spinal reconstruction. The single level fixation technique showed some advantages in treating certain types of thoracolumbar spinal fractures when compared with the traditional cross-sectional fixation. The endoscopy-assistant and image-guiding spinal intervention techniques were evolved in China during these years. In the treatment of the obstinate painful osteoporotic vertebral compressive fracture, percutaneous vertebroplasty and kyphoplasty achieved good results in the pain relief and spinal reconstruction. Numerous basic and clinical researches have given us a further understanding of the medical protection of acute spinal cord injury, and biological treatments have given us new ideas on neural reparation and regeneration. Cell transplantation and gene therapy have become the most promising treatment strategies in this field. CONCLUSION: With the rapid development of spine surgery, the repair and reconstruction of the injured spine and spinal cord made a great stride in the recent years. PMID- 16683424 TI - [Effects of the nerve, endocrine, and immune factors on the skin repair and regeneration]. AB - OBJECTIVE: To investigate the effects of the nerve, endocrine, and immune factors on the process of wound healing and regeneration of the skin after injury and to review the research work in the past years in this area. METHODS: The prospective study was made to explore the relationship among the nerve, endocrine, immune factors, and skin tissue repair and regeneration, and to summarize the recent advance in this area. RESULTS: The nerve, endocrine, and immune factors played an important role in the repair and generation of the skin after injury. CONCLUSION: As there has been fewer studies made in this field, we should reinforce the related basic research in this field. PMID- 16683425 TI - [Reparation and reconstruction of the postburn deformity and functional disability]. AB - OBJECTIVE: To investigate the development made in the reparation and reconstruction of the postburn deformity and functional disability in the advanced-stage patients. METHODS: Based on the reviewed literature at home and abroad and combined with our clinical experience, the new reparative and reconstructive techniques for the patients with advanced-stage deformity and functional disability were evaluated. RESULTS: The reparative and reconstructive microsurgical techniques achieved a significant development in treating the following pathologic changes after burn: deformity due to proliferation and contracture of the scars, severe hand deformity, defects occurring in the muscle tendons and nerves due to electricity burn, and defects occurring in the long tubular bones of the extremities. CONCLUSION: Although there has been a great achievement in this field, there is still a hard task of finding out newer therapeutic approaches and achieving more effective results in the future. PMID- 16683426 TI - [Recent development of reparative and reconstructive surgery on oral and maxillofacial regions]. AB - OBJECTIVE: To summarize the latest development of the oral and maxillofacial reparative and reconstructive surgery. METHODS: The literature at home and abroad was reviewed and the main creative concepts in this field were summarized and analyzed. RESULTS: In the terms of the oral and maxillofacial reparative and reconstructive surgery, the significant achievements were made in the following aspects: transplantation of revascularized tissues, artificial materials, platelet-rich plasma, distraction osteogenesis, microsurgery on fracture, arthroplasty, dynamic repair, and computer-assisted design. CONCLUSION: Based on the previous achievements, the oral and maxillofacial reparative and reconstructive surgery will have a further development, especially in the individualized treatment. PMID- 16683427 TI - [Stem cells: ideal seed cells for reconstruction of tissues and organs]. AB - OBJECTIVE: To investigate an important role of the stem cells in reconstructing the tissues and organs. METHODS: Based on our own researches and combined with the review of the literature at home and abroad, the latest development of the cell therapy with the stem cells and the application of the seed cells in the tissue engineering were analyzed. RESULTS: As the stem cells are the origin of the human tissues and organs and have a higher self-renewal ability and extensive characteristics of proliferation in vitro, their imbedding and multi-differential potentialities were illustrated. Both the embryonic stem cells and the adult stem cells had a wide prospect as ideal seed cells for reparation and reconstruction of the impaired human tissues and organs. CONCLUSION: The stem cells can play an important role in repairing and reconstructing the injured tissues and organs and they have a promising prospect in clinical application. The further research and wide application of the stems cells will significantly improve the therapeutic effects on the injured tissues and organs. PMID- 16683428 TI - [Biomaterials used for reparative and reconstructive surgery]. AB - OBJECTIVE: To investigate the currently-used biomaterials in reparative and reconstructive surgery and to clarify the relationship between the development of biomaterials and the progress of reparative and reconstructive surgery. METHODS: Based on the author's many years' scientific researches and combined with the literature available at home and abroad, the biomaterials used in the clinical practice, and their kinds and application fields were summarized. RESULTS: Based on the sufficient knowledge of the component structure of biomaterials and the patient's pathological status, the matching biomaterials could be designed and developed. According to the analysis on some common defects occurring in the skin, bone, cartilage, vocal cord, nerve, and drum membrane, the methods of repairing the defects with biomaterials that we had developed, such as collagen, chitosan, and hyaluronate, achieved good results. CONCLUSION: The rapid development of biomaterials can greatly promote progress of reparative and reconstructive surgery and there exists a dependence relationship between the two. The related histological responses and the importance of biological estimation after implantation of biomaterials should be emphasized. PMID- 16683429 TI - [Development in the field of plastic surgery]. AB - OBJECTIVE: To summarize the recent development in the plastic and reconstructive surgery. METHODS: The related literature was reviewed and the main achievements in the field of plastic surgery were summarized. RESULTS: Plastic and reconstructive surgery was one of the quickly-developed fields in the world medicine, especially aesthetic plastic surgery. Reconstructive plastic surgery and aesthetic plastic surgery would be bind in the future. The unaesthetic hand surgery" was a very important branch of the hand surgery, so not only the function recovery of the hand deformities but also the aesthetic sculpture for the deformed hand should be emphasized. Evaluation on measurement of the facial paralysis reconstruction was a main part of the facial examination. The allotransplantation of the face became more developed. The anatomical research on the cutaneous perforator arteries, vascularisation of the biceps femorismuscle, and the gracilis perforator flap was well performed by the plastic surgeons. CONCLUSION: The quick development has happened in the reparative and reconstuctive surgery and the aesthetic plastic surgery. We must not only pay attention to the organ defect reconstruction and tissue deformity repair but also to the aesthetic sculpture for the deformed parts of the body. PMID- 16683430 TI - [Rapid development of cosmetic medicine in China]. AB - OBJECTIVE: To review and summarize the development during the last 20 years and the current status of cosmetic medicine, i.e., cosmetic surgery, in China, for the healthier development of this specialty in the future. METHODS: Literature concerned was reviewed, including conference abstracts, papers, and publications, and the present status and problems were analyzed. RESULTS: Cosmetic medicine was recognized as an independent specialty and gained its clear definition. The development of cosmetic medicine is an inevitable trend of the changing medical modules and the developing science and civilization. This trend fulfilled the need of the people. The related problems consisted of a high complication rate, confusion of management, and insufficient specific knowledge in part of the providers. CONCLUSION: The development of cosmetic medicine is an inevitable trend of the civilization development. For the healthy development of this specialty, scientific management and systemic education for the providers are crucial. Only those who have the plastic surgery background are able to participate in this practice. PMID- 16683431 TI - [A comparative research of multilevel cervical spondylotic myelopathy treated by two different anterior operative methods]. AB - OBJECTIVE: To compare the outcomes of two operative methods, the anterior decompression in subsection and the anterior decompression in one section, which were used to treat multilevel cervical spondylotic myelopathy (CSM). METHODS: Data of multilevel CSM undergoing the anterior decompression in subsection (33 cases, the subsection group) and the anterior decompression in one section (19 cases, the one section group) from July 1999 to January 2004 were retrospectively analyzed. The incidence of perioperative complications and the rate of fusion were evaluated by the postoperative X-ray and MRI examinations, and improvement of the neurological function was evaluated by the JOA score. RESULTS: The incidence of perioperative complications was 36.8% in the one section group, mainly including immigration of the plate and grafts, which was settled by the revision surgery; while the incidence of perioperative compilcations was 12.1% in the subsection group, mainly including the immigration of the titanium mesh. There was a significant difference between the two groups (P < 0.05). 84.2% of the patients in the one section group and 81.8% of the patients in the subsection group developed bony fusion by the end of the follow-up (9-31 mon, averaged 11.2 mon), and there was no significant difference between the two groups (P > 0.05). According to the JOA score, the ratio of the improvement in the neurological function was 70.4% in the subsection group and 64.4% in the one section group. There was no significant difference between the two groups (P > 0.05). CONCLUSION: The anterior decompression in subsection is more rational for the surgical treatment on the multilevel CSM than the anterior decompression in one section. It can provide an equal decompressive effect but a more stable local mechanical environment right after the surgery and can maintain it well, which is critical for the bony fusion. PMID- 16683432 TI - [Comparing loss of anterior column height and cervical Cobb angle with three different types of anterior cervical discectomy and fusion]. AB - OBJECTIVE: To compare the loss of anterior column height and cervical Cobb angle with three different types of anterior cervical discectomy and fusion (ACDF). METHODS: A prospective randomized study was performed on 60 patients who had undergone ACDF with the autologous iliac crest graft (group A, n=20), the autogenous bone and the anterior cervical locking plates (group B, n=20), and Syncage-C filled with the local autograft reamings (group C, n=20) from January 1998 to January 2003. The patients diagnosed as having cervical radiculopathy (RP) and cervical spondylotic myelopathy (CSM) were indicated for ACDF. Of the patients, 41 were male and 19 female with a mean age of 57 years (range, 36-68) and their average course of disease was 6.2 months (range, 1-36). There were 36 one-level and 24 two-level fusions from C(3,4) to C7, T1. Radiological measurements were performed on the cervical radiographs taken before operation, 7 days and 3 months after operation, and on the last day of the follow-up; then, the height and Cobb angle of the fused segment, functional restoration, and clinical outcome were evaluated in the three groups. RESULTS: The follow-up of more than 2 years (range, 2-7) showed that the average loss of anterior column height and Cobb angle of the fused segments in groups A and B, which had not preserved the endplate, had a greater increase than that in group C, which had preserved the endplate. Of the patients, 12 had autograft collapse, 3 autograft displacement, and 10 postural abnormality between the fused segments, most of which happened in groups A and B. The fusion rate was 93.3% (56 cases) according the strict arthrodesis criteria; their excellent and good rate in the functional assessment was 83.3% (RP 90.4%, CSM 79.5%); the overall satisfactory (excellent and good) rates in groups A, B and C were 75%, 85% and 90%, respectively. CONCLUSION: To increase the resistance to graft subsidence, which is a major reason for narrowness of the fused segments, and to maintain normal cervical curvature, we should improve our skills of bone grafting performance, preserve the endplate, carefully evaluate the degree of osteoporosis before operation, and use anterior cervical locking plate and/or fusion with Syncage-C when necessary. PMID- 16683433 TI - [Comparison of restoring and maintaining the cervical curvature and height by using three different anterior cervical plate systems]. AB - OBJECTIVE: To investigate and compare the effects of restoring and maintaining the cervical curvature and height of the fused segment by using three different anterior cervical plate systems. METHODS: From January 2002 to June 2004, 122 patients underwent anterior cervical decompression, autogenous iliac bone graft and plate fixation. Of the 122 patients (85 males, 37 females, aged 14-70), 37 underwent surgery involving the fixation with the Orion plate system, 39 with the Zephir plate system, and 46 with the Codman plate system. The cervical curvature and height of the fused segment were measured on the lateral X-ray films so as to compare the changes of the conditions preoperatively, 1 week after surgery, and during the follow-up, and also to compare the difference among the three groups. RESULTS: The follow-up of the patients for 6-35 months (average 17.3 months) showed that all the patients developed the bone fusion 6 months after operation. There was a significant improvement in the cervical curvature and height of the fused segment before operation versus 1 week after operation (P < 0.05); however, there were no significant changes 1 week after operation versus during the follow up in each group(P > 0.05); there was no significant difference among the three groups (P > 0.05). CONCLUSION: The three plate systems can effectively reconstruct and maintain the cervical curvature and height of the fused segment, with a satisfactory effect in a short term. PMID- 16683434 TI - [Comparison between solis fixation fusion and titanium plate fixation by cervical anterior approach]. AB - OBJECTIVE: To investigate the effect difference between the Solis fixation fusion and the titanium plate fixation by the cervical anterior approach after decompression and bone graft implantation. METHODS: Of the 104 patients with cervical disease from September 2001 to March 2004, 36 were treated with the Solis implantation after decompression by the cervical anterior approach, and 68 were treated with the titanium plate fixation after decompression and bone graft implantation. The recovery of the neurological function in all the patients were assessed with the JOA Scoring at 6 weeks, 3, 6, 12, 24 and 36 months. The fragment fusion and its stability as well as the changes in the intervertebral height were assessed with X-ray examination. RESULTS: According to the JOA Scoring, the excellent and good outcomes accounted for 94.4% in the Solis group and 94.1% in the titanium plate group. In all the patients, the fragment fusion was achieved in 3 months. The change in the Cobb angle of the fused fragment was less than 5 degrees at the flexion-extension posture, 3.6 +/- 0.8 degrees in the Solis group, 2.4 +/- 0.7 degrees in the titanium plate group. There was significant differences between the two groups (P < 0.05). The intervertebral height of the operation fragment in the Solis group increased 1.6 +/- 0.7 mm, which was higher than that in the titanium plate group (P < 0.05). CONCLUSION: Clinical effects of the two internal fixation operations are good; however, the Solis fixation has more advantages because of its simpler performance, less trauma, and fewer complications. PMID- 16683435 TI - [Preliminary application of anterior uncovertebral joint resection in cervical spondylotic treatment]. AB - OBJECTIVE: To investigate the effect and safety of the anterior uncovertebral joint resection in treatment of cervical spondylotic myelopathy accompanied by radiculopathy. METHODS: From March 2002 to July 2004, 9 patients (5 males, 4 females; aged 38-66) with cervical spondylotic myelopathy accompanied by radiculopathy underwent the anterior uncovertebral joint resection and discectomy using the titanium cage implantation. There were 6 unilateral and 3 bilateral compressed nerve roots. RESULTS: During the follow-up for 3-16 months, CT scanning or X-ray films revealed that all the patients improved with an enlargement of the intervertebral foramen, and MRI demonstrated that compression of the spinal cord and nerve roots vanished. Seven patients had a relief in the radicular pain although the other 2 still had a residual pain. All the patients improved to some extent in symptoms of myelopathy. No patient suffered vertebral artery injury during the operation. CONCLUSION: The uncovertebral joint resection can directly decompress the nerve roots. The key to avoidance of an injury to the vertebral artery is a good understanding of the regional anatomy and a good performance of the operation. PMID- 16683436 TI - [Two-level cervical artificial disc replacement combined with nearby segments fused for multi-level disc herniations]. AB - OBJECTIVE: To evaluate the therapeutic effect of the two-level cervical artificial disc replacements combined with the nearby segments fused for the multi-level disc herniations and to investigate their interaction during the treatment. METHODS: The patient undergoing surgery was diagnosed as having intervertebral disc herniations in the C3 7 intervertebral discs. During the operation, the C(4-6) discs were replaced with two Bryan artificial discs while the C(3,1) and C(6,7) discs were removed, followed by the bone grafting and the Zephir instrumentation. The range of motion (ROM) was assessed before and immediately after operation by the flexion-extension radiography. The patient's functional exercise began as early as 2 days after operation and lasted for 3 weeks, followed by immobilization for 3 months. RESULTS: According to the follow up for 1 year and 2 months, the patient could move his neck in all directions without pain. The spinal and neural symptoms were significantly alleviated. ROM was close to the normal level postoperatively. CONCLUSION: Artificial disc replacement combined with bone grafting and fusion is a new way to treat multi level disc herniations surgically, with good clinical results. Early cervical functional exercise followed by restriction is an effective way to deal with the contradiction of motion and immobilization. PMID- 16683437 TI - [Selection of low instrumentation vertebrae with CD hybrid technique in king types III and IV scoliosis]. AB - OBJECTIVE: To review the King-types III and IV patients treated by the CD hybrid technique and evaluate clinical results on the shorter fusion levels. METHODS: Fifty-eight patients with idiopathic scoliosis were treated by the CD hybrid method from March 2000 to January 2003, among whom 40 were grouped as King-type I and 18 as King-type N; 41 were female and 17 were male. The Cobb angle of the thoracic curve was averaged 64 degrees (range 50-83 degrees), and the curve flexibility was 62%. The compensative lumbar curve was averaged 37 degrees (range 16-48 degrees), and the curve flexibility was 105%. With the neutral rotational vertebrae as a basis to select the low instrumentation vertebrae, the neutral rotational vertebrae or the vertebrae at 1 or 2 levels proximal to the neutral rotational vertebrae were selected as the low instrumentation vertebrae in all the patients. Standing AP and lateral radiographs were taken respectively at the discharge, during the follow-up after discharge, and at the final follow-up. RESULTS: The patients were followed up for an average of 2. 4 years (range 1.8 3.2). The corrected curves lost an average of 3.1 degrees (range--1-5 degrees)and the correction rate of the thoracic curve was 68% at the final follow-up. The plumb line from C7 was parallel to the sacral midline in 56 patients. The lumbar curves were corrected to an average of 8 degrees (2-13 degrees) automatically. The lumbosacral angle was corrected automatically by 53% and the low instrumentation vertebrae in 48 patients turned into stable vertebrae. The low instrumentation vertebrae lost 1.4 segments on average compared with the Harrington principle. No spinal imbalance was clinically observed in all the patients. CONCLUSION: The choice of the low instrumentation vertebrae as the neutral rotational vertebrae can have a good result in the clinical practice. It can be applied in the CD hybrid technique in treatment of idiopathic thoracic curves. PMID- 16683438 TI - [A long-term follow-up of lumbar laminotomy and replantation]. AB - OBJECTIVE: To evaluate lumbar laminotomy and replantation in prevention of spinal unstability and peridural adhesion after laminectomy. METHODS: From February 1995 to March 2001, a total of 169 patients (96 males, 73 females, aged 22-63) with lesions in the lumbar vertebral canals underwent surgery, in which the lesions were removed after laminectomy and then the excised laminae were replanted. RESULTS: The follow-up for 5-9 years showed that all the patients had no complications after the lesions were removed. According to the evaluation criteria formulated by WANG Yongti, 81 patients had an excellent result, 67 had a good result, 19 had a fair result, and 2 had a poor result. 87.6% of the patients obtained quite satisfactory results. The X-ray films demonstrated that the replanted laminae obtained bony healing and the spine was stable. The CT scanning demonstrated that the canals were enlarged with a smooth and glossy interior. CONCLUSION: Lumbar laminotomy and replantation is reasonable in design and convenient in performance, which can be promoted as a basic operation in spinal surgery. PMID- 16683439 TI - [Allogenous bone plate reconstructing spinal channel and grafting in treatment of thoracolumbar burst fracture with paraplegia]. AB - OBJECTIVE: To evaluate the method of the allogenous bone plate reconstructing the spinal channel and grafting in treatment of thoracolumbar burst fracture with paraplegia. METHODS: Thirty-six patients with thoracolumbar burst fracture with paraplegia were included in this study. Their ages ranged from 18 to 56 (average, 38). The vertebral injury involved T11 in 3 patients, T12 in 10 patients, L1 in 14 patients, L2 in 7 patients, and L3 in 2 patients. Neurological deficits were classified by the Frankel grading. There were 9 patients in grade A, 11 patients in grade B, 13 patients in grade C, and 3 patients in grade D. All the patients were treated with the anterior approach, decompression of the spinal channel, interbody graft, and internal fixation. The grafting materials consisted of the allogenous femoral bone plate that was decreased in advance and implanted in the intervertebral posterior region, with cut ribs and bone mills during the decompression. RESULTS: Postoperative CT scanning showed clearance of the spinal cord compression and expansion of the spine channel. During the follow-up period averaged 2 years, almost all the patients showed an improvement in the neurological function. Spinal fusion occurred in 32 patients. There was no screw loosened or broken. Only 1 patient failed to achieve the fusion. CONCLUSION: The anterior approach, allograft bone plate reconstructing the spine channel is a safe and effective method in treatment of the thoracolumbar burst fracture with paraplegia, which may be a replacement of the autogenous iliac bone graft. PMID- 16683440 TI - [Anterior decompression and reconstruction with internal fixation for severe thoracolumbar burst fracture]. AB - OBJECTIVE: To explore the injury mechanism of the severe thoracolumbar burst fracture and the necessity of anterior decompression and reconstruction with internal fixation. METHODS: From January 1999 to January 2004, 21 patients were treated with anterior decompression and reconstruction. The fractures were located at T12 in 6 patients, L1 in 12, L2 in 4, L3 in 3,and L4 in 1. Four patients were treated with the "anterior approach" and "posterior approach" surgeries for severe column fractures. RESULTS: All the patients were restored to the normal physiological radian, and the spinal canal was decompressed completely. They were followed up for 1-6 years, and the bony fusion was observed radiologically. The spinal cord function was improved to the 1-3 Frankel grade in all the patients except 2. There were no such complications as leakage of the cerebrospinal fluid, plate-screw loosening or breaking, or segment instability. The clinical effects were satisfactory. CONCLUSION: The operation of the anterior decompression and reconstruction with internal fixation for severe thoracolumbar burst fracture has advantages of complete decompression, full bone-grafting, and firm internal fixation. It can restore the spinal height and improve the spinal cord function. PMID- 16683441 TI - [The finite element analysis of lumbar fusions]. AB - OBJECTIVE: To investigate the stability and the stress distributions of L(3-5) fused with three different approaches (interbody, posterolateral and circumferential fusions) and to investigate degeneration of the segment adjacent to the fused functional spinal unit. METHODS: A detailed L(3-5) three-dimensional nonlinear finite element model of a normal man aged 32 was established and validated. Based on the model, the destabilized model, the interbody, posterolateral and circumferential fusions models of L45 were established. After the loadings were placed on all the models, we recorded the angular motions of the fused segment and the Von Mises stress of the adjacent intervertebral disc. RESULTS: The circumferential fusion was most stable than the others, and the interbody fusion was more stable than the posterolateral fusion. The maximal Von Mises stress of the adjacent L3.4 intervertebral finite all the models was ranked descendingly as flexion, lateral bending, torsion and extension. For the three kinds of fusions, the stress increment of the L34 intervertebral disc was ranked ascendingly as interbody fusion, posterolateral fusion and circumferential fusion. CONCLUSION: After destabilization of the L(4.5) segment, the stability of the circumferential fusion is better than that of the others, particularly under the fluxional or extensional loading. The stability of the interbody fusion is better than that of the posterolateral fusion, except for under the fluxional loading. The feasibility of adjacent segment degeneration can be ranked descendingly as: circumferential fusion, posterolateral fusion and interbody fusion. PMID- 16683442 TI - [Diagnosis and treatment of far-lateral lumbar disc herniation]. AB - OBJECTIVE: To investigate diagnosis and treatment of far-lateral lumbar disc herniations. METHODS: The clinical data from 16 patients with far-lateral lumbar disc herniations from January 1999 to January 2004 were retrospectively analyzed. The CT scanning showed that the shadow density of the CT scanning values in the corresponding intra-foramen, extra-foramen and all-foramen was as the same as that of the intervertebral disc. Of the 16 patients, 10 were operated on by the interlaminar approach, 3 were operated on by the laterolaminar approach, 3 were operated on by the combined interlaminar and laterolaminal approach. RESULTS: According to the follow-up for 6 months to 5 years, excellent results were obtained in 8 patients, good results in 5, and fair results in 3. The postoperative CT examination showed that the space occupying in the foramen or extra-foramen of the corresponding segment vanished and the nerve root compression of the identical segment also vanished. CONCLUSION: The lamellar high resolution CT is a better way to diagnose lumbar disc herniation. The operative approach should be chosen according to the position of the intervertebral disc protrusion, pathologic type, and presence or absence of the lesions in the vertebral canal. PMID- 16683443 TI - [Treatment of multi-segmental lumbar intervertebral disc protrusion with limited recessive decompression]. AB - OBJECTIVE: To retrospectively analyze the clinical and imaging features of multi segmental lumbar intervertebral disc protrusion and its treatment with the limited recessive decompression operation. METHODS: Twenty-two patients (14 males and 8 females, aged 49-68 years) were admitted to hospital from March 1999 to March 2004. They suffered from multi-segmental lumbar intervertebral disc protrusion that involved L1-S1 and were treated with the limited recessive decompression operation. RESULTS: The follow-up for 4-21 months showed that 16 of the patients had an excellent outcome, 5 had a good outcome, and 1 had fair outcome. There were no such operative complications as nerve root lesions and putamen lesions. CONCLUSION: The limited recessive decompression operation is one of the available good treatments for multi-segmental lumbar intervertebral disc protrusion. It solves problems of herniation and stenosis and maintains stability of the spine. PMID- 16683444 TI - [Canal laminoplasty with titanium miniplate re-implantation of vertebral lamina]. AB - OBJECTIVE: To evaluate the reliability and the advantage of canal laminoplasty with laminoplasty over laminectomy for treatment of lumbar spinal stenosis. METHODS: From June 2000 to September 2004, the titanium miniplate fixation technique was applied to re-implantation of the vertebral lamina in the lumbar spine. The vertebral lamina was made with a specially-made osteotome and a special technique in 18 patients with lumbar spinal stenosis. RESULTS: The patients were followed up for 1 yr and 8 mon on average (range, 3 mon-4 yr and 3 mon) and were observed to have a bony fusion of the re-implanted lamina 3-9 months postoperatively. There was no nonunion, displacement of the re-implanted lamina, overgrowth of the anterior bone edge of osteotomy, recompression of the nerves or instability of the lumbar spine. CONCLUSION: The result demonstrates that canal laminoplasty with the titanium miniplate re-implantation of the vertebral lamina in lumbar spine can restore the normal anatomy, keep stability of the spine, and avoid adhesion and scar in the canal. PMID- 16683445 TI - [Surgical treatment for spinal canal stenosis of retrograde degeneration]. AB - OBJECTIVE: To investigate the surgical effect on different types of spinal canal stenosis of retrograde degeneration. METHODS: The retrospective analysis was performed on 96 patients (85 males, 11 females; average age 54, range 39-71) admitted from September 2001 to January 2004 for spinal canal stenosis of retrograde degeneration. The patients were divided into five groups according to their clinical symptoms and signs and the imaging of the spinal cord. In group A, 39 patients had one segment of the spinal canal stenosis and they underwent the recessively expanding operation through the intervertebral canal by opening a window between the vertebral plates. In group B, 21 patients had stenosis of the central vertebral canal of one segment and they underwent excision of the intervertebral disc through the window opened between the vertebral plates and the recessively expanding operation. In group C, 18 patients had degenerative spinal derangement of one segment and they underwent decompression through the window opened between the bilateral vertebral plates and the recessively expanding operation, and then underwent the internal fixation and bone grafting. In group D, 11 patients had the mixed stenosis of the vertebral canal of more than 2 segments and they underwent the recessively expanding operation through the window opened bilaterally on the diseased segment and excision of the yellow ligament and the intervertebral disc. In group E, 7 patients had degenerative lateral curvature of the spine and they underwent the combined surgical procedures including decompression, internal fixation, and bone grafting. All the patients were followed up after operation. RESULTS: The follow-up of the 96 patients for 6-36 months with an average of 12.2 months showed that they had no postoperative complications. According to the JOA Scoring, 85 patients had an excellent result, 9 had a good result, 1 had a fair result, and only 1 had a poor result. The X-ray films revealed no mistaken placing of the nails on the vertebral arch, broken nails or loosened nails. CONCLUSION: The limited surgery and effective decompression can improve curative effects and reduce complications of spinal canal stenosis of retrograde degeneration. The imaging of the spinal cord has an important value in the choice of surgical protocols for spinal canal stenosis of retrograde. PMID- 16683446 TI - [Perioperative treatment for senile lumbar disease accompanied by internal disease]. AB - OBJECTIVE: To investigate the surgical method and perioperative treatment for senile lumbar disease accompanied by internal disease. METHODS: From June 2000 to December 2003, the complete neurological and physical examinations were performed on the patients before operation, as treatment of internal diseases could improve the patients' conditions. Lumbar operations were performed on 125 patients, among whom 23 had simple lumbar disc herniation, 13 had lumbar spine stenosis, 81 had lumbar disc herniation with lumbar spine stenosis, and 8 had spondylolisthesis. The JOA score was 11.6 +/- 2.5. There were 3 patients undergoing fenestration + discectomy, 16 undergoing semi-laminectomy + discectomy, 82 undergoing total laminectomy + discectomy, 5 undergoing total-laminectomy+discectomy+pedicle fixation, 11 undergoing lamina decompression+nerve-root pathway decompression, and 8 undergoing pedicle screw fixation+bone graft and fusion in spondylolisthesis. RESULTS: With an effective medical treatment, the internal disease produced little effect on the operation. Improved functions and bone fusions were observed after operation. According to the JOA standards, the average alleviation rate was 87.9%. CONCLUSION: Early neurological examination and proper treatment of internal diseases are the keys to the successful operation on the senile patients with lumbar disease. Radiological data are important in avoidance of mistaken diagnosis. PMID- 16683447 TI - [Development and application of artificial vertebral body in spinal surgery]. AB - OBJECTIVE: To study the development, investigation, and application of the artificial vertebral body so as to provide an essential reference for the future research and clinical application. METHODS: The recent articles on materials, types, and clinical applications of the artificial vertebral body were reviewed. RESULTS: The materials used for the artificial vertebral body were porcelain, alloy, variant bone, and composite. But each of them had its own advantages and disadvantages. The types of the artificial vertebral body were grouped as expandable and non-expandable ones; however, the expandable type was much better. The artificial vertebral body had been applied to the treatments of spinal tumor, tuberculosis, fracture, and infection, with better effects. CONCLUSION: The artificial vertebral body can be extensively applied. However, the materials and types need to be improved. PMID- 16683448 TI - [Construction and identification of eukaryotic expression plasmid pcDNA3. 1-BACE and its transient expression in COS-7 cells]. AB - OBJECTIVE: To generate eukaryotic expression vector of pcDNA3. 1-beta-site amyloid precursor protein cleaving enzyme (BACE) and obtain its transient expression in COS-7 cells. METHODS: A 1.5 kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells by the RT-PCR method and was cloned into the plasmid pcDNA3.1. The vector was identified by the double digestion with restriction enzymes BamHI and XhoI and was sequenced by the Sanger-dideoxy mediated chain termination. The expression of the BACE gene was detected by immunocytochemistry. RESULTS: The results showed that the cDNA fragment included 1.5 kb total coding region. The recombinant eukaryotic cell expression vector of pcDNA3. 1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells transfected with the pcDNA3. 1-BACE plasmid expressed a high level of the BACE protein in the cytoplasm. CONCLUSION: The recombinant plasmid pcDNA3. 1-BACE can provide a very useful tool for the research on the cause of Alzheimer's disease and lay an important foundation for preventing Alzheimer's disease. PMID- 16683449 TI - [Effect of pilose antler polypeptides on the apoptosis of rabbit marrow mesenchymal stem cells differentiated into chondrogenic phenotype in vitro]. AB - OBJECTIVE: To observe the effect of pilose antler polypeptides (PAP) on the apoptosis of rabbit marrow mesenchymal stem cells (MSCs) differentiated into chondrogenic phenotype by interleukin 1beta (IL-1beta) so as to optimize the seeding cells in cartilage tissue engineering. METHODS: The MSCs were separated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into chondrogenic phenotype by transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). According to different medias, the MSCs were randomly divided into four groups: group A as black control group, group B (100 ng IL 1beta), group C (10 microg/ml PAP + 100 ng IL-1beta) and group D (100 ng/ml TGF beta1 + 100 ng IL-1beta). The samples were harvested and observed by morphology, flow cytometry analysis, RT-PCR and ELISA at 24, 48 and 72 hours. RESULTS: The intranuclear chromatin agglutinated into lump and located under nuclear membranes which changed into irregular shape at 24 hours. The intranuclear chromatin agglutinated intensified at 48 hours. Then the nuclear fragments agglutinated into apoptosis corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosis rate of cells, the mRNA expression of Caspase 3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A, C and D (P < 0.01). CONCLUSION: Caspase-3 is involved in apoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3. PMID- 16683450 TI - [Effects of different durations and times of ischemic preconditioning on ischemia reperfusion injury to tram flaps in rats]. AB - OBJECTIVE: To determine whether the different durations and times of the ischemic preconditioning affect the effectiveness of the ischemic preconditioning. METHODS: Ninety male Wistar rats were randomly divided into the control group and the eight preconditioned groups of 10 rats each. A transverse rectus abdominis musculocutaneous flap (TRAM) was elevated in each rat. The flaps were preconditioned by clamping the pedicle and reperfusing for 5 or 10 minutes per cycle. This was repeated for one or two cycles. The controls were simply perfused for 30 minutes. Each flap was then subjected to 4 hours of the global ischemia. Three rats in each group were killed for an estimate of the water content in the muscle and for observation on the muscular structure under microscope. The flap surface survival areas of the other rats were calculated on the 7th postoperative day by the computerized video planimetry. RESULTS: The water content in the muscle was evidently reduced. The mean survival area of the flap in every preconditioned group increased by 2-3 times compared with that of the controls (P < 0.001). The different procedures of the ischemic preconditioning produced different protective effects. CONCLUSION: The ischemic preconditioning is an available means to alleviate an ischemia-reperfusion injury to the transverse rectus abdominis musculocutaneous flap in rats. The effect of the ischemic preconditioning is affected by the duration and time of the ischemic preconditioning. PMID- 16683452 TI - Safety of fetal olfactory ensheathing cell transplantation in patients with chronic spinal cord injury. A 38-month follow-up with MRI. AB - OBJECTIVE: To determine the safety of the fetal olfactory ensheathing cell (OEC) transplantation in patients with chronic spinal cord injury (SCI) by examination of the magnetic resonance imaging (MRI). METHODS: A prospective clinical study involving 16 patients with chronic SCI was designed to investigate the feasibility and biological safety of the fetal OEC transplantation in treatment of SCI. The olfactory bulbs from the 3-4-month-old aborted human fetuses following the strict ethical guidelines were harvested and trypsinized down to single fetal OEC. These cells were then cultured for 12-17 days and were prepared for a clinical use. From November 2001 to December 2002, 16 patients with chronic SCI were randomly enrolled. The patients suffered from SCI for 1.5-8 years (average 4.3 years) after the injury. The suspension (50 microl) containing about 1 x 10(6) fetal OECs was transplanted by an injection into the patients' spinal cords above and below the injury site. All the patients were assessed before the transplantation and were followed up with MRI for 29-42 months (average 38 mon) after the transplantation. RESULTS: No cell-related adverse effects were observed in any patient during the follow-up period. The follow-up with MRI did not reveal any development of optic glial tumor, tumor-like mass, new hemorrhage, edema, expanding cyst, new cyst formation, infection or disruption of the neural structure in the transplant site of all the patients. CONCLUSION: This is the first clinical study demonstrating the long-term safety of the OEC therapy for SCI. The results indicate that our protocol is feasible and safe in treatment of patients with chronic SCI within 38 months after the injury. Although the size of the samples for our study was not big enough, the positive results of the study have encouraged us to make a further research in this field. PMID- 16683451 TI - Influence factors for functional improvement after olfactory ensheathing cell transplantation for chronic spinal cord injury. AB - OBJECTIVE: To explore the influence factors for the functional improvement after the fetal olfactory ensheathing cell (OEC) transplantation for chronic spinal cord injury(SCI). METHODS: The olfactory bulbs were harvested and trypsinized down to single fetal OEC. They were cultured for 12-17 days prepared for use. From November 2001 to December 2003, a total of 300 patients volunteered for the fetal OEC transplantation, among whom 222 suffered from complete chronic SCI and 78 suffered from incomplete chronic SCI. The procedures were performed on the patients with a disease course ranging from 6 months to 31 years (average 3.1 years) after their injuries. The fetal OEC was transplanted by the form of injections into the spinal cord at the upper and lower ends of the injury site. All the patients were assessed by the ASIA standard before the transplantation and 2-8 weeks after the transplantation. The influence factors including age, sex, duration after the injury, and injury degrees and levels were compared with those in the functional improvement after fetal OEC transplantation. RESULTS: The partially-improved neurological functions assessed by the ASIA standard were indicated by the motor scores increasing from 39.1 +/- 20.6 to 45.9 +/- 20.3 (P < 0.001), the light touch scores from 51.7 +/- 24.9 to 63.4 +/- 23.0 (P < 0.001), and the pin prick scores from 53.0 +/- 24.2 to 65.3 +/- 22.7 (P < 0.001). There was no significant difference in the functional improvement of the motor, light touch, and pin brick when compared with the age, sex, duration after the injury, and the injury degrees and levels. The motor scores and light touch scores at the cervical level were higher than the scores at the thoracic level. CONCLUSION: The fetal OEC transplantation can partially improve the neurological functions quickly in treatment of the chronic spinal cord injury. All the influence factors except the motor scores and light touch scores, which were higher at the cervical level than at thoracic level, have no impact on the functional improvement after the fetal OEC transplantation. PMID- 16683453 TI - [Degradation of antigen-extracted meniscus in vitro]. AB - OBJECTIVE: To study degradation of the antigen-extracted meniscus in PBS solution with no enzyme or with different enzymes. METHODS: Four types of enzymes (collagenase, hyaluronidase, trypsin, papain) were used to enzymolyze the antigen extracted meniscus and the fresh meniscus for 3, 7, 15 and 30 days (37 degrees C). The antigen-extracted meniscus and the fresh meniscus were immersed in PBS solution (37 degrees C) for 30 days. Weight loss measurement, UV spectrophotometry, and scanning electron microscopy (SEM) were used to characterize the degraded materials. RESULTS: The two types of the materials were remarkably digested under the enzymes, especially under trypsin. The degradation curves showed that the antigen-extracted meniscus was enzymolyzed less than the fresh meniscus. The degradation products were grouped as amino, peptide, and polycose by the analysis. Both of the materials could hardly be hydrolyzed in PBS solution without the enzymes. The four different enzymes had different surface morphologies under the examination of SEM. CONCLUSION: The antigen-extracted meniscus is enzymolyzed more slowly than the fresh meniscus in vitro, and the result can be used as a guideline to the further research. PMID- 16683454 TI - [Effect of morphological changes in proximal femur on prosthesis selection of total hip arthroplasty in patients with ankylosing spondylitis]. AB - OBJECTIVE: To investigate the effect of the morphological changes in the proximal femur on the prosthesis selection in the total hip arthroplasty in the patients with ankylosing spondylitis. METHODS: The experimental group was composed of 13 patients (16 hips) with ankylosing spondylitis, which was treated with the total hip arthroplasty, and the control group was composed of 16 patients (19 hips) with non-ankylosing spondylitis, which was also treated with the total hip arthroplasty. In the two groups, the measurements of Singh index, canal flare index, morphological index of the cortex and cortical index were performed in the two groups. RESULTS: The results of the statistical analysis on Singh index, canal flare index, morphological index of the cortex and cortical index in the experimental group were 3.81 +/- 0.54, 2.63 +/- 0.41, 2.02 +/- 0.38 and 1.69 +/- 0.69, respectively, but 4.63 +/- 0.62, 3.03 +/- 0.27, 2.76 +/- 0.28 and 2.12 +/- 0.24, respectively in the control group. There was a significant difference in Singh index, canal flare index, and morphological index of the cortex between the two groups (P < 0.05), while there was no statistical difference in cortical index between the two groups (P > 0.05). The patients with ankylosing spondylitis had more serious osteoporosis in their proximal femur. CONCLUSION: Cemented femoral prosthesis should be used in the total hip replacement in patients with ankylosing spondylitis, and the revision total hip arthroplasty should be performed on patients with more serious osteoporosis. PMID- 16683455 TI - [A kind of improved operative correction of severe inverted nipples]. AB - OBJECTIVE: To introduce a kind of improved operative method for correction of severe inverted nipples. METHODS: Sixteen patients (aged 28-38 years) with 31 inborn inverted nipples were operated with the improved method from January 2002 to March 2005. The nipple was incised horizontally through the middle, the fibrotic band was released, and the tissue below the nipple was contracted. Then the defective space with mammary gland flap. Finally, a Z-shaped suture was employed at each base of nipple. RESULTS: The shapes of these nipples were satisfactory. No infection and necrosis of the nipples occurred. The follow-up showed that the results were perfect in the sensory and elective functions of nipples. All the corrected nipples did not have recurrence except one nipple. The scars in the local site were limited and inconspicuous. CONCLUSION: Severe inverted nipples can be corrected effectively by the combined improved incisions and Z-shaped sutures. This method is suitable for the female patients with no requirement for feeding. PMID- 16683456 TI - [Expression of nerve growth factor and its receptor during osteoinduction of recombined human bone morphogenetic protein 2]. AB - OBJECTIVE: To identify the expression of nerve growth factor (NGF) and its high affinity receptor (tyrosine kinase receptor A, TrkA) during bone induction by recombined human bone morphogenetic protein 2 (rhBMP-2) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) and to discuss the role of NGF on the bone induction of the BMP. METHODS: Thirty-six ICR mice were divided into the experimental group and the control group at random. rhBMP-2 /collagen sponge and collagen sponge were implanted into the right thigh muscle pouches of the mice in the experimental group and the control group, respectively. The tissues in the implanted site of the two groups were removed on the 7th, 14th and 21st day after the implantation. Histological, immunohistochemical and RT-PCR analyses were performed to detect osteoinductive effects of rhBMP-2 and the expression of NGF and TrkA. RESULTS: Gross observation showed that a solid lump was found in the right thigh in the experimental group on the 7th day and became harder on the 14th and 21st day, which was not found in the control group. rhBMP-2 /collagen sponge displayed a potent ability to induce bone formation, while immunostaining for NGF and TrkA was observed in the course of osteoinduction by rhBMP-2. On the 7th day in the experimental group, NGF positive immunostaining reached the peak in the stage of chondrogenesis and there were a large number of cells expressing NGF, including fibroblasts, chondroblasts, chondrocytes, and osteoblasts; then, there was a decrease in the number of the positive cells and in the intensity of immunostaining on the 14th and 21st day. Staining of TrkA was similar to that of NGF. The expression level of the mRNA of NGF during the course of bone induction peaked 7 days rhBMP 2/collagen is a kind of satisfactory osteoinductive after the implantation and then decreased. CONCLUSION: material, and many different kinds of cells induced by rhBMP-2 can express NGF and TrkA, which suggests that NGF may play an important role in the osteogenesis initiated by exogenous BMP through direct and indirect pathways. PMID- 16683457 TI - [Human embryonic esophagus squamous epithelial cells cultured through microscopic stripping technique and digestion in vitro]. AB - OBJECTIVE: To discuss the application possibility of the microscopic stripping technique used in the primary culture of human embryonic esophagus squamous epithelial cells, and of the methods for the isolation, depuration and subculture of the esophagus epithelial cells in vitro. METHODS: The squamous epithelial cells were obtained from the esophagus mucous membrane of the 20-week abortion fetus through the microscopic stripping technique, and were digested with trypsin. Then, the morphological, immunohistochemical observation and the growth curve of the isolated cells were studied. RESULTS: The isolated cells were spherical in the cell suspension and spherical-like or polygon-like after attachment to the culture flask. The squamous epithelial specialized cytokeratin staining was strongly positive. And the morphological studies by the transmission electron microscopy indicated that the cultured cells were squamous epithelial cells. The squamous epithelial cells reached the peak level 3-4 days after the transfer of the culture. The absorbance at 3 and 4 days was significantly higher than that at 1, 2, 5 and 6 days (P < 0.05). CONCLUSION: A large number of squamous epithelial cells can be available with the microscopic stripping technique and the digestion method. The cultured squamous epithelial cells can be proliferated quickly, and fit for the tissue engineering study. PMID- 16683458 TI - [Comparison of two sorts of bio-derived materials after being explanted in the repaired rabbit corneal defect]. AB - OBJECTIVE: To compare the effects of the denuded freeze-dried-amniotic-membrane and the denuded freeze-dried bovine corneal stroma when they were explanted to repair the corneal defect of rabbits. METHODS: The amenia from healthy human placentae were prepared with the method reported by LUO Jingcong, which were freeze-dried and sterilized. The bovine cornea was also denuded by trypsin, rinsed, freeze-dried, and sterilized. Twenty Japan rabbits were divided into group A (the amniotic group) and group B (the bovine-corneal-stroma group) at random. The defect was made, which was 7.5 mm in diameter and 1/3 of the thickness of the cornea, and the two kinds of materials were explanted to repair the defect. The vascularization and the changes of the operated eye were observed. The samples were taken at 2, 4 and 8 weeks for histological examination. RESULTS: The explanted materials were not melted or excluded. There were visible neovessels in both groups, yet there was no significant difference between them. According to the histological observation, there was severe inflammation in both groups 2 weeks after operation, the fibroblasts were proliferated, and the collagen fibers were disorganized; however,the reactions became milder from 4 weeks after operations, and the neovessels could be seen in groups A and B; at 8 weeks, the collagen fibers were more organized in groups A and B; however,there was still a small area of disorganized fibers left. CONCLUSION: The two materials can lead to rejection to some extent, and so they need to be improved. PMID- 16683459 TI - [The experimental study on culture of human oral keratinocyte and epithelium using autologous serum and fetal bovine serum]. AB - OBJECTIVE: To investigate the possibility of culturing human oral keratinocyte using autologous serum in order to provide theoretical and technical foundation for clinical application of tissue engineering oral mucosa epithelium. METHODS: The human oral keratinocytes were cultured by the medium containing different concentrations of autologous serum (10%, 20%, 30%)and fetal bovine serum (10%), respectively. The growth conditions for the cell and the mucosa epithelium in the groups were observed, the cell growth curves were drawn, and the population doubling time (PDT) was counted. RESULTS: The results showed that the human oral keratinocyte could proliferate well in the medium containing autologous serum or fetal bovine serum. The differences in the 24-hour clone rate and PDT were not significant. Both the area and the thickness of the cultured oral epithelium increased with the increase of the autologous serum concentration, and the difference between autologous serum and fetal bovine serum was significant, especially with the medium containing 20% autologous serum (P < 0.05). The human nature of the cultured epithelium was demonstrated by the immunofluorescent mouse anti-HLA antigen. CONCLUSION: The autologous serum can replace the fetal bovine serum to culture the oral keratinocyte well, and the cultured oral mucosa epithelium can be better differentiated in the autologous serum than in the fetal bovine serum. PMID- 16683460 TI - [Optimizing culture system of mouse marrow mesenchymal stem cells in vitro and investigating effect of alcohol and acetaldehyde on the cells]. AB - OBJECTIVE: To optimize the in vitro culture system of C57/BL6 marrow mesenchymal stem cells (MSCs) and to investigate the effect of alcohol and acetaldehyde on MSCs. METHODS: The MSCs were isolated from the femur marrow of C57/BL6 mice and were cultured in the optimized system, so that highly-purified MSCs were harvested and identified by immunohistochemistry. Then, MSCs were cultured in the medium containing alcohol or its metabolic product acetaldehyde, with the following concentration groups: alcohol 5.7, 17.0, 50.0, 100.0 and 150.0 mmol/L; acetaldehyde 4.5, 0.9, 0.18, 0.036, 0.0072, 0.00144, 0.000288 mmol/L. MSCs were cultured with alpha-MEM as the control group. After 3 days, their proliferation activity was measured by the MTT method. RESULTS: MSCs within 6 passages had a good stability and a high proliferation activity. They were identified to express CD90 but no CD34. The MTT assay showed that alcohol at the concentration greater than 100.0 mmol/L and acetaldehyde at the concentration greater than 4.5 mmol/L could inhibit proliferation of MSCs (P < 0.05). But the proliferation activity might rise with an increase in the acetaldehyde concentration smaller than 0.18 mmol/L (P < 0.05). CONCLUSION: The optimized culture system can effectively isolate and culture MSCs. Both alcohol and acetaldehyde can inhibit proliferation of MSCs but toxicity of acetaldehyde is more serious. PMID- 16683461 TI - [Study on cell senescence induced by D-galactose in cultured rat mesenchymal stem cells]. AB - OBJECTIVE: To study morphological and biological senescence changes induced by D galactose in the cultured rat mesenchymal stem cells. METHODS: After 3rd generations cultured in the DMEM-F12, MSCs were changed into DMEM-F12 medium containing 8 g/L D-galactose and cultured to the 6th generations as the inducement group. The comparison were the 6th generations which was cultured in the DMEM-F12 medium all along, and then identified by surface wave. Using flow cytometer to check the comparisons cell cycle change after swing in with 8 g/L D galactose within the 4 days. In the first 7 days to draw the growth curve to the two groups. Optical and electronic microscope were used to identify the influences of characteristic morphological of mesenchymal stem cells of the two groups, the influences of biological markers were identified by single cell gel electrophoresis and beta-galactose dye. Results After treatment with D-galactose, the mesenchymal stem cells displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers; 85% of the cells were X-gal dye masculine, and the signal cell gel electrophoresis showed DNA damnification. The flow cytometry showed that 90% of the cells stayed in G0/G1, but the cells in S and G2/M almost disappeared. However, the cells in the control group had no such DNA damages. CONCLUSION: D-galactose can induce senescence of the mesenchymal stem cells, and 8 g/L is the best concentration to do so. This study has provided a good model for the research of the mesenchymal stem cells senescence. PMID- 16683462 TI - Public interest responsibilities of professionals. PMID- 16683463 TI - An audit of a specialist old age psychiatry liaison service to a medium and a high secure forensic psychiatry unit. AB - The elderly prison population is increasing and there is a significant amount of unidentified psychiatric morbidity among elderly prisoners. A sizeable number of elderly subjects are referred to regional forensic units. These units are able to provide advice but are reluctant to admit frail, physically ill and vulnerable elderly into their unit because the ward environment is considered inappropriate for them. A sizeable number of inpatients in medium and high secure units are elderly. This paper is an exploratory audit of referrals from a medium and high secure forensic psychiatry unit to a specialist consultation-only liaison old age psychiatry service, which was specifically developed to service the forensic unit. The demographic and clinical characteristics of the patients seen by this specialist service were similar to elderly inpatients in medium and high secure units. All referrals were judged to be appropriate and new management advice was provided in all cases. Main reasons for referral included diagnostic advice, placement advice and treatment advice. Establishing a diagnosis of dementia was considered important because Alzheimer's disease and Lewy body dementia can be treated with cholinesterase inhibitors. Placement advice was the most difficult to provide because of paucity of residential and nursing homes able and willing to accept patients with a forensic history. There is a need for a comprehensive model of specialist forensic old age psychiatry service at a regional or supraregional level. A consensus needs to be reached on the exact configuration of such a service. PMID- 16683464 TI - Alleged dowry death: a study of homicidal burns. AB - Homicidal burning of married women in India is a major concern for the Government, law-enforcing authorities, the judiciary, the police and medico-legal experts all over the country who are associated with dowry disputes. Dowry death, a heinous crime, is gradually engulfing and polluting the entire society. Sections 304(B) and 498(A) of the Indian Penal Code (IPC) have been introduced and Sections 174 Criminal Procedure Code (Cr PC) and 176 Cr PC have been modified but, instead of deterring dowry deaths, the occurrences are increasing day by day and the number of cases coming for post-mortem examination is rising. Both parties, that is, the parents and in-laws of the deceased, are becoming adept at manufacturing circumstantial evidence to serve their own interests. Therefore, to establish the truth and for the smooth administration of justice, reliable unbiased medical evidence has to be corroborated. This study presents retrospective research carried out in the tertiary care teaching hospital of MKCG Medical College, Berhampur, Eastern India, for a five-year period between January 1998 to December 2002. From a total of 58 burned wives, 32 (55.1%) were homicidal burns. These cases were analyzed in view of epidemiological and medico-legal aspects. The majority of the affected women (90.63%) were in the 18 to 26 years of age group. Death occurred within the first few years of marriage and most of them had no children. All the victims belonged to the Hindu religion and 62.5% of incidents occurred during the daytime. In 71.87% cases the incident occurred in rural areas. Eighty-three per cent of cases occurred in a joint family within the house. In most cases, kerosene was the accelerant and flame was the causative agent. In more than 68.75% of cases the total body surface area involved was more than 80%. Most victims died at the time of the incident, or within the subsequent 24 hours, due to hypovolumeic shock. The education status in 70.21% cases was either illiterate or under matriculation. More stringent laws and female independence both mentally and economically will reduce the homicidal burns in young brides. PMID- 16683465 TI - The mental health needs of people with a learning disability detained in police custody. AB - Few research studies examine the prevalence or mental health needs of people with a Learning Disability (LD) detained in police custody. This paper describes the population of detainees with an LD who presented to an inner city inter-agency police liaison service during a three-year period. Two forensically trained Community Mental Health Nurses (CMHNs) screened all custody record forms (n=9014) for evidence of a mental health problem or LD. The CMHNs interviewed positively screened detainees (n=1089) using a battery of measures designed to assess mental health status, risk-related behaviour and alcohol or drug abuse. Almost one-in ten of those interviewed (95/1089) were judged to have a possible or definite LD. Fifty-two per cent were cases on the General Health Questionnaire (GHQ) whilst 61% attained 'above threshold' Brief Psychiatric Rating Scale (BPRS) scores. The majority (63%) had a history of causing harm to others while 56 per cent had a history of self-harm. More than half (56%) regularly consumed harmful levels of alcohol while one-in-four (27%) reported abusing drugs. Higher than expected numbers of detainees have a learning disability and most have complex mental health needs. A police liaison service offers a way of identifying people with LD and connecting them with appropriate health and social care agencies. PMID- 16683466 TI - Using the inflammatory cell infiltrate to estimate the age of human burn wounds: A review and immunohistochemical study. AB - The burn wound healing process, which is reviewed in this paper, has features that differ from the healing of incised cutaneous wounds. This study used immunohistochemical staining and cell counting to examine the inflammatory cell response in biopsy samples of burn wounds from live human subjects obtained at six hours until 23 days after injury in order to determine how the age of a burn could be estimated. Acute inflammatory cells predominated in samples taken six hours to two days after injury. However, neutrophils were often minimal in early samples or could be present late. Elevated numbers of macrophages tended to be encountered from days 2 to 20, but it was not uncommon to observe a minimal or absent macrophage response. Unexpectedly, there was no trend in the number of lymphocytes. A small study was also made of burn wound samples that had been obtained at post-mortem examination of subjects that died in a fire or up to 77 days after injury from fire. This revealed a similar trend of neutrophil and macrophage accumulation. Additionally, it appeared that an increase in the number of lymphocytes occurred late, from 35 days. IN CONCLUSION: If neutrophils predominate, the wound is probably less than a couple of days old. When macrophages are abundant the wound is probably a few days to weeks old. However, as expected from the review of the literature, the inflammatory cell infiltrate may be low or absent in burn wounds, which can render determination of the age of burn wounds difficult. PMID- 16683467 TI - Prison suicides and self-harming behaviours in Italy, 1990-2002. AB - Data on suicides, attempted suicides and other self-harming behaviours that occurred in Italian prisons from 1990 to 2002 was studied on official records, as documented by the Ministry of Justice. Over the study interval, completed suicide rates in Italian prisons were constantly about ten times higher than among the general population, but over time they did not increase significantly despite nearly a doubling in the absolute number of inmates. The rate of completed suicides in overcrowded prisons was about ten times higher than in non overcrowded ones. Attempted suicides were about ten times higher than completed suicides, with females being significantly more likely to attempt to take their own lives and males being more likely to complete suicide. Self-injuring acts without the intent to die involve about one in every ten individuals, with foreigners (non-EU citizens mostly) being twice as likely to self-harm than residents. A better identification of the people suffering from mental disorders and a reduction in prison overcrowding are two key issues that need to be implemented to reduce the impact of suicide and self-harming behaviours among convicts. PMID- 16683468 TI - Homicidal sharp force injuries inflicted by family members or relatives. AB - We retrospectively reviewed 35 autopsy cases where death had resulted from homicidal sharp force injuries and compared cases where the injuries had been inflicted by family members or relatives (relative group) with cases where the injuries had been inflicted by an unrelated person (stranger group). We reviewed the age and sex of the victims, the number of stab wounds, the site of the stab wounds, the presence of defence wounds, the detection of alcohol and other drugs and the mental status of the victims and perpetrators. We found the following tendencies: (a) a female victim was more frequently killed by a relative than by a stranger; (b) the percentage of cases receiving a single stab wound and the percentage of cases receiving more than ten stab wounds were both unexpectedly higher in the relative group than in the stranger group, and (c) in the stranger group, when there were no defence wounds, the victim had usually consumed alcohol, whereas when there were neither defence wounds nor alcohol intake, the case usually fell into the relative group. These tendencies will contribute towards our forensic appraisement in autopsy cases resulting from sharp force injuries. PMID- 16683469 TI - Inter- and intra-individual variation in applied force when listening at a surface, and resulting variation in earprints. AB - We aimed to explore the level of inter- and intra-individual variation in applied force when listening at a surface, and assess the resulting variation in earprints. We further intended to identify possible sources of this variation. Forty subjects each listened twenty-four times at a surface while applied force was recorded. In between efforts the level and frequency of the target sound, and the level of ambient noise were varied. Each listening effort was characterized by two values: the mean of a series of force recordings ('functional force') and the highest force reading of the effort ('peak value'). A mixed model analysis of variance revealed that repetition during multiple efforts of listening and the level of the target sound significantly affected both values for applied force. The frequency of the target sound affected the peak value, but we assume this was due to confounding effects. The level of ambient noise did not affect applied force. To explore the correlation between values for applied force of various efforts by single ear, the intra-class correlation coefficient was calculated. For functional force it was 0.80; for the peak value it was 0.79. To study intra individual variation in earprints, five prints from each ear were lifted and studied. Variation in prints is discussed. PMID- 16683470 TI - Historical and medico-legal aspects of infanticide: an overview. AB - In practice, all cases of abandoned newborn deaths are reported to the police/coroner, who asks for a post-mortem examination because only this can establish the viability of the infant, proof of separate existence, and the cause and manner of death in such cases. The pathologist who conducts the autopsy will have to use his skills and experience to assess a wide variety of marks and injuries so as to give his opinion fairly and frankly, not allowing any personal feelings to influence his findings. In some cases, he may be unable to give a firm opinion and should not shirk from stating this clearly. This paper examines some historical, legal and pathological aspects of infanticide. PMID- 16683471 TI - The Abbreviated Injury Scale and its correlation with preventable traumatic accidental deaths: a study from South Delhi. AB - Anatomic trauma scoring systems are fundamental to trauma research. The Abbreviated Injury Scale (AIS) and its derivative, the Injury Severity Score (ISS), are the most frequently used scales. In a prospective study, 400 autopsies of road traffic accident victims performed between January 2002 and December 2003 were coded according to the AIS and ISS methods. All the cases were classified into different injury groups according to the Injury Severity Scale. Fifty-eight cases (14.5%) were assigned an ISS value of <25; 244 (61%) cases were valued between 25-49; 38 cases (9.5%) were valued between 50-74 and 60 (15%) cases had a value of 75. On analysis of medical care, in cases with ISS<50, about 96% of the victims did not receive optimal care quickly enough with a lack of pre-hospital resuscitation measures and lengthy transportation time to hospital being of major importance. PMID- 16683472 TI - Fatal electrocution in adults--a 30-year study. AB - A retrospective study was undertaken of all cases of death in adults (>16 years) due to electrocution which were autopsied at Forensic Science SA, Adelaide, Australia, over a 30-year period from 1973 to 2002. A total of 96 cases were identified with 87 males (91%) (mean age=41.6 years; range 17 to 86 years) and nine females (9%) (mean age=49.2 years; range 20 to 76 years). Deaths were due to accidents in 66 cases (69%; M:F = 63:3), suicides in 28 cases (29%; M:F=24:4) and homicides in two cases (2%). Both homicide victims were females, with females accounting for only 5% of the accidents and 14% of suicides. The number of accidental deaths increased until the early 1990s and then declined, whereas suicidal electrocutions were only found in the last 20 years of the study. Most deaths (N = 80; 83%) were due to low voltage circuits (<1000 volts), with deaths due to high voltages occurring in 15 cases (16%). One death was due to lightning (1%). While suicides were evenly distributed throughout the year, most accidental deaths occurred in late spring and summer (N = 42; 64%) with the lowest number of accidental deaths occurring in winter and early spring. This report demonstrates a significantly higher rate of electrocution deaths among males, with a summer predominance of accidental deaths, most likely due to increased outdoor activities in better weather. The reduction in cases over the last decade of the study may be a reflection of the success of workplace and domestic safety campaigns. Female electrocution suicides and electrocution homicides were rare events. PMID- 16683473 TI - Fatal neglect of the elderly by a spouse: a case report. AB - Neglect is one of the most common forms of elder abuse, along with physical and psychological abuse. It is defined as an elderly person alone who is not able to provide for him or herself the services necessary to maintain physical and mental health, or who is not receiving those services from a responsible carer. We present an unusual case of neglect where a 69-year-old diabetic man was left lying on the ground by his spouse after a sudden fall from a cerebral stroke. The man was not able to move or to get up and his spouse, unable to lift him up, did not seek help. Although the victim's spouse reported having taking care of him, the man died four days later from a fatal hyperglycaemia. PMID- 16683474 TI - Air gun--a deadly toy?: A case report. AB - Air guns (air rifles) are used throughout the world as instruments of amusement such as toys in funfairs, for bird hunting and firearms training. In India and in many other countries, this instrument neither comes under the purview of the Arms Act, nor is there is any restriction on the user's age. This enables a person to gain access to this instrument quite easily. Sometimes serious and fatal injuries result when it is used by an ignorant person or by a criminal. There are reports which suggest that these 'toys' can cause painful injuries but only a few cases of death have been reported. There is no literature about the features of injuries that help to establish the range of fire by an air gun. Here we report a case where a boy was accidentally shot to death while watching bird shooting. We attempt to correlate the injury with the range of fire. PMID- 16683475 TI - HIV/AIDS, psychiatric disorder and sexual assault in Transkei: a case report. AB - HIV infection and psychiatric disorders have a complex relationship. HIV infection could lead to psychiatric disorders, and psychiatric patients are more vulnerable to HIV infection. HIV is not only an illness which is associated with stigmatization and discrimination, but also has several risks attached including physical and sexual abuse. There is a scarcity of literature on HIV, mental illness and sexual assault although they are a common trio. The author describes an HIV-positive woman with a psychiatric disorder. She suffered from AIDS and periodic psychotic epidsodes. On a day when she had such an episode she became a rape victim. The history, and physical and genital examination has been described. The need for further research in the area of HIV/AIDS and mental health has been discussed. Preventive strategies have been recommended for HIV infected individuals in poor health resource settings. PMID- 16683476 TI - Smooth the elective OR schedule? A large hospital makes it happen. PMID- 16683477 TI - Medicare to cover more obesity surgery. PMID- 16683478 TI - Crucial conversations: a key for safety. Interviewed by Leslie Flowers. PMID- 16683479 TI - What does JCAHO expect for handoffs? PMID- 16683480 TI - Tips for introducing SBAR in the OR. PMID- 16683481 TI - Passing the baton' for smooth handoffs. PMID- 16683482 TI - A SHARED tool strengthens handoffs. PMID- 16683484 TI - ASC safety begins with patient selection. PMID- 16683483 TI - The right antibiotic at the right time. PMID- 16683485 TI - Synaesthesia: an overview of contemporary findings and controversies. AB - Research on synaesthesia is undergoing something of a renaissance, having initially been a hot topic in psychology and philosophy in the late 19th and early 20th centuries. One hundred years ago investigators failed to define an objective framework within which to characterise the phenomenon, and so interest in the topic waned. With the cognitive revolution and the rapid rise of new experimental techniques in human neuroscience, interest in synaesthesia as a legitimate topic for scientific investigation has been rekindled. Since the 1980s there has been rapid growth in scientific and media interest in synaesthesia, and there is now a sense that real progress is being made. This Special Issue of Cortex brings together the largest collection of empirical papers on the topic of synaesthesia to date. The 21 papers collected herein showcase the many significant advances that have been made in understanding the behavioural and neural bases of synaesthesia. The contributions reflect the work of more than 60 investigators from nine different countries. We are confident that the papers presented in this Special Issue will set the agenda for synaesthesia research for many years to come. PMID- 16683486 TI - Diagnosing and phenotyping visual synaesthesia: a preliminary evaluation of the revised test of genuineness (TOG-R). AB - Synaesthesia, a neurological condition affecting approximately .05% of the population, is characterised by anomalous sensory perception: a stimulus in one sensory modality triggers an automatic, instantaneous, consistent response in another modality (e.g., sound evokes colour) or in a different aspect of the same modality (e.g., black text evokes colour). As evidence was limited to case studies based on self-report, the existence of synaesthesia was regarded with scepticism until the development of the Test of Genuineness (TOG) in 1987, which measures the consistency of stimulus-response linkage: synaesthetes typically score between 70-90% range, whereas controls typically score between 20-38%. However, the TOG had only limited ability to quantify the characteristics of visual synaesthesia. In this study, the revised Test of Genuineness (TOG-R), utilising the Pantone-based Cambridge Synaesthesia Charts, was given to 26 synaesthetes and 23 controls. Results confirmed that the TOG-R is equally accurate in the diagnosis of synaesthesia; synaesthetes scored significantly (t47 = 16.01, p < .001) higher (mean = 71.3%, SEM = 1.4%) than controls (mean = 33%, SEM = 2.0%). The TOG-R provides greater precision in quantifying the closeness of colour matches and enables a more detailed analysis of visual synaesthesia. Synaesthetes were phenotyped into broad- and narrowband based on their overall responsiveness to auditory stimuli, with bandwidth determined primarily by responsiveness to non-word stimuli. They were further sub-phenotyped based on responses to sub-groups of stimuli into word-colour (WC) and music-colour (MC). Development of this instrument has important implications for the diagnosis and phenotyping of visual synaesthesia. PMID- 16683487 TI - Monozygotic twins' colour-number association: a case study. AB - A case study of a pair of monozygotic twins, both of whom show a strong and enduring colour-number association, is reported. The origin of the colours, in a jigsaw puzzle, is known. Neither reports conscious photisms typical of synaesthesia, but a Stroop task of naming the colours of digits shows an interference effect with incongruent colours. PMID- 16683488 TI - Mitempfindung in synaesthetes: co-incidence or meaningful association? AB - Mitempfindung is the referral of a tactile sensation to a location far away from the stimulation site. This brief note reports a 40% incidence of Mitempfindung in a sample of 20 digit-color synaesthetes which contrasts with a 10% incidence in two control groups of non-synaesthetes. Phenomenological similarities between Mitempfindung and synaesthesia are discussed, and we propose that an erratic neural connectivity underlies both phenomena. To better characterize the nature of the purported neurophysiological peculiarities, prospective studies are needed that characterize Mitempfindung in synaesthetes and nonsynaesthetes. PMID- 16683489 TI - Effect of a synesthete's photisms on name recall. AB - A multilingual, colored-letter synesthete professor (MLS), 9 nonsynesthete multilingual professors and 4 nonsynesthete art professors learned 30 names of individuals (first and last name pairs) in three trials. They recalled the names after each trial and six months later, as well as performed cued recall trials initially and after six months. As hypothesized, MLS recalled significantly more names than control groups on all free recall tests (except after the first trial) and on cued recall tests. In addition, MLS gave qualitatively different reasons for remembering names than any individual control participant. MLS gave mostly color reasons for remembering the names, whereas nonsynesthetes gave reasons based on familiarity or language or art knowledge. Results on standardized memory tests showed that MLS had average performance on non-language visual memory tests (the Benton Visual Retention Test-Revised--BURT-R, and the Rey-Osterrieth Complex Figure Test--CFT), but had superior memory performance on a verbal test consisting of lists of nouns (Rey Auditory-Verbal Learning Test--RAVLT). MLS's synesthesia seems to aid memory for visually or auditorily presented language stimuli (names and nouns), but not for non-language visual stimuli (simple and complex figures). PMID- 16683490 TI - Synaesthesia: pitch-colour isomorphism in RGB-space? AB - A new experimental technique found coloured-hearing synaesthetes to possess localised isomorphism between pitch and colour. We found significantly more consistency in synaesthetes' pitch-colour matches than controls, with matches unaffected by musical experience, not facilitated by absolute pitch (AP) and responded to on the basis of pitch alone, without interference from note-name information. Synaesthetes also placed their colour responses to quartertones (notes falling between adjacent semitones) significantly closer to the RGB midpoint of their responses to the semitones lying either side. This is partial evidence for a direct, localised pitch-colour correspondence. Criteria for possible between-synaesthete similarities in patterning are also discussed, with limited evidence for localised patterning presented. PMID- 16683491 TI - Synesthetic colors determined by having colored refrigerator magnets in childhood. AB - Synesthesia is a condition in which percepts in one modality reliably elicit secondary perceptions in the same or a different modality that are not in the stimulus. In a common manifestation, synesthetes see colors in response to spoken or written letters, words and numbers. In this paper we demonstrate that the particular colors seen by a grapheme-color synesthete AED were learned from a set of refrigerator magnets and that the synesthesia later transferred to Cyrillic in a systematic way, with the colors induced by the Cyrillic letters determined by their visual or phonetic similarity to English letters. Closer examination of the data reveals that letters of either language that are more visually similar to the English capitals in the magnet set are also more saturated. In order to differentiate AED's synesthesia from ordinary memory, we use a novel psychophysical method to show that AED's synesthetic colors are subject to ordinary lightness constancy mechanisms. This suggests that the level of representation at which her synesthesia arises is early in the stream of visual processing. PMID- 16683493 TI - Perceptual interaction between real and synesthetic colors. AB - People with color-graphemic synesthesia experience vivid, reliable color upon viewing achromatic alphanumeric characters. Recent evidence indicates that synesthetic color experiences are as perceptually real as actual colors are for non-synesthetic observers. To investigate possible interactions between real and synesthetic colors, we tested two adult color-graphemic synesthetes on a pair of perceptual grouping tasks. In Experiment 1, we employed a well-known phenomenon of motion perception, bistable apparent motion, to explore whether synesthetic colors interact with real colors in grouping over time. Two-frame apparent motion sequences were presented with both path lengths and colors systematically manipulated. Results showed that synesthetic colors of motion tokens interacted with matching real colors of the corresponding motion tokens, which could subsequently bias perceived direction of motion. In Experiment 2, we exploited binocular rivalry, a condition under which two dissimilar monocular images compete with each other and result in perceptual switches, to explore whether synesthetic colors interact with real colors in grouping over space. Pairs of rival images with two different characters were presented dichoptically with colors of characters manipulated. Results showed that synesthetic and real colors of characters tended to group together, which, in turn, promoted the perceived global dominance during binocular rivalry. Therefore, the present results identify substantial interaction between synesthetic colors and real colors in perceptual grouping. PMID- 16683492 TI - Contrast affects the strength of synesthetic colors. AB - Grapheme-color synesthesia is an automatic, involuntary experience of seeing colors when viewing numbers, letters or words on a printed page. Previous research has demonstrated that synesthesia is a genuine perceptual phenomenon, but crucially, all of these experiments have used high-contrast letters and numbers. Our synesthete, JC, anecdotally reported that the strength of his synesthetic colors varied depending on whether the graphemes were presented in high or low contrast. To test this, we asked JC to rate the strength of his experiences to letters of different contrasts on three different dates. JC's ratings of the strength of his synesthetic colors consistently declined monotonically with contrast, suggesting that his synesthetic colors were reduced or absent at low contrasts. To more precisely quantify the impact of this, we then tested JC on modified versions of our embedded figures task (Ramachandran and Hubbard, 2001a) and crowding task (Ramachandran and Hubbard, 2001b) by presenting displays with varying contrast between the graphemes and the background. Behavioral data in the contrast variant of our embedded figures task showed that JC performed significantly better than controls at high contrast, replicating our previous findings. However, at low contrast this advantage was eliminated, consistent with his reports of weaker or absent colors. A similar, but weaker pattern of results was found in the modified version of our crowding task. These results suggest that JC's synesthetic colors may be elicited at contrast dependent stages of visual processing. We propose that regions of the fusiform gyrus specialized for letter and number grapheme recognition that have been shown to respond in a contrast dependent manner mediate JC's synesthetic colors. However, whether this is true for all grapheme-color synesthetes or is only true of the group we refer to as lower synesthetes, remains to be seen. PMID- 16683494 TI - Automatic perception and synaesthesia: evidence from colour and photism naming in a stroop-negative priming task. AB - It is widely assumed that synaesthetic perception is highly automatic, as shown by Stroop test. Furthermore, it has been shown that, although automatic, it can be suppressed leading to Negative Priming (NP). However, these assumptions have not been consistently investigated, as not many papers have measured Stroop in synaesthesia, and only one used a NP procedure. Two experiments were carried out in a female synaesthete (MA), and 13 control participants, in which numbers and letters were displayed in colours either congruent or incongruent with MA's photisms. In contrast to control participants, MA showed significant Stroop effect both when naming the colours and when naming the photisms (slower RT when naming a colour or photism that was incongruently coloured versus congruently coloured). For comparison, we also report a control experiment in which the first letters of colour names were displayed in either congruent (e.g., B in blue) or incongruent (e.g., B in red) colours. Significant Stroop and NP effects were found when a control group named the displayed colour of these letters. The synaesthesic Stroop effect shown by MA was greater than that observed in the Control Experiment when MA was to name the displayed colour, but smaller when she was to name the photism of the stimuli. Regarding NP, MA showed an effect similar to that observed in the Control Experiment, but only when she was to name the photisms of the stimuli. Altogether, these results show that synaesthetic perception is highly automatic and difficult to inhibit. PMID- 16683495 TI - Attentional load attenuates synaesthetic priming effects in grapheme-colour synaesthesia. AB - One of the hallmarks of grapheme-colour synaesthesia is that colours induced by letters, digits and words tend to interfere with the identification of coloured targets when the two colours are different, i.e., when they are incongruent. In a previous investigation (Mattingley et al., 2001) we found that this synaesthetic congruency effect occurs when an achromatic-letter prime precedes a coloured target, but that the effect disappears when the letter is pattern masked to prevent conscious recognition of its identity. Here we investigated whether selective attention modulates the synaesthetic congruency effect in a letter priming task. Fourteen grapheme-colour synaesthetes and 14 matched, non synaesthetic controls participated. The amount of selective attention available to process the letter-prime was limited by having participants perform a secondary visual task that involved discriminating pairs of gaps in adjacent limbs of a diamond surrounding the prime. In separate blocks of trials the attentional load of the secondary task was systematically varied to yield 'low load' and 'high load' conditions. We found a significant congruency effect for synaesthetes, but not for controls, when they performed a secondary attention demanding task during presentation of the letter prime. Crucially, however, the magnitude of this priming was significantly reduced under conditions of high-load relative to low-load, indicating that attention plays an important role in modulating synaesthesia. Our findings help to explain the observation that synaesthetic colour experiences are often weak or absent during attention demanding tasks. PMID- 16683496 TI - Do synaesthetic colours act as unique features in visual search? AB - For individuals with grapheme-colour synaesthesia, letters, numbers and words elicit vivid and highly consistent colour experiences. A critical question in determining the mechanisms underlying the phenomenon is whether synaesthetic colours arise early in visual processing, prior to the allocation of focused attention, or at some later stage following explicit recognition of the inducing form. If the synaesthetic colour elicited by an achromatic target emerges early in visual processing, then the target should be relatively easy to find in an array of achromatic distractor items, provided the target and distractors elicit different synaesthetic colours. Here we present data from 14 grapheme-colour synaesthetes and 14 matched non-synaesthetic controls, each of whom performed a visual search task in which a target digit was distinguished from surrounding distractors either by its unique synaesthetic colour or by its unique display colour. Participants searched displays of 8, 16 or 24 items for a specific target. In the chromatic condition, target and distractor digits were presented in different colours (e.g., a yellow '2' amongst blue '5's). In the achromatic condition, all digits in the display were black, but targets elicited a different synaesthetic colour from that induced by the distractors. Both synaesthetes and controls showed the expected efficient (pop-out) search slopes when the target was defined by a unique display colour. In contrast, search slopes for both groups were equally inefficient when the target and distractors were achromatic, despite eliciting distinct colours for the synaesthetes under normal viewing conditions. These results indicate that, at least for the majority of individuals, synaesthetic colours do not arise early enough in visual processing to guide or attract focal attention. Our findings are consistent with the hypothesis that graphemic inducers must be selectively attended to elicit their synaesthetic colours. PMID- 16683497 TI - Does binding of synesthetic color to the evoking grapheme require attention? AB - The neural mechanisms involved in binding features such as shape and color are a matter of some debate. Does accurate binding rely on spatial attention functions of the parietal lobe or can it occur without attentional input? One extraordinary phenomenon that may shed light on this question is that of chromatic-graphemic synesthesia, a rare condition in which letter shapes evoke color perceptions. A popular suggestion is that synesthesia results from cross-activation between different functional regions (e.g., between shape and color areas of the ventral pathway). Under such conditions binding may not require parietal involvement and could occur preattentively. We tested this hypothesis in two synesthetes who perceived grayscale letters and digits in color. We found no evidence for preattentive binding using a visual search paradigm in which the target was a synesthetic inducer. In another experiment involving color judgments, we show that the congruency of target color and the synesthetic color of irrelevant digits modulates performance more when the digits are included within the attended region of space. We propose that the mechanisms giving rise to this type of synesthesia appear to follow at least some principles of normal binding, and even synesthetic binding seems to require attention. PMID- 16683498 TI - The role of meaning in grapheme-colour synaesthesia. AB - When the synaesthete, J, is shown black graphemes, in addition to perceiving the black digits or letters she also experiences highly specific colours that overlay the graphemes (e.g., 5 is pink, S is green). We used ambiguous graphemes in a Stroop-type task to show that the exact same forms (e.g., 5) can elicit different synaesthetic colours depending on whether they are interpreted as digits or letters. J was shown strings of black digits (e.g., 3 4 5 6 7) or words (e.g., M U S I C for 1 sec. All but one of the graphemes then disappeared and the remaining grapheme changed to a colour that J had to name as quickly as possible. The key trials involved coloured graphemes that were ambiguous (e.g., the 5 in the strings above could be interpreted either as a digit or as a letter). On congruent trials, the colour of the ambiguous target grapheme was the same as J's photism for the digit or letter interpretations of the grapheme. On incongruent trials, the colours of the ambiguous target graphemes were different than the colours of J's photisms for the digit or letter interpretations of the graphemes. On digit-context incongruent trials, the ambiguous graphemes were presented in J's colour for the letter-interpretations of the graphemes, whereas on letter context incongruent trials, the ambiguous graphemes were presented in J's colours for the digit-interpretations of the graphemes. Thus the same ambiguous grapheme (e.g., a pink 5) served as a congruent stimulus in one context and an incongruent stimulus in another context. J's response times showed that ambiguous graphemes elicited different photisms depending on whether they were interpreted as digits or letters. This finding suggests that it is not the form but the meaning of graphemes (whether they are interpreted as digits or letters) that determines the colours of synaesthetic photisms. PMID- 16683499 TI - Number synaesthesia: when hearing "four plus five" looks like gold. AB - Whilst lexical-colour synaesthesia has been widely studied, the number-colour pairing has received relatively less attention. Since one very strong variable is the structure and "ordinality" within a set of inducers (e.g., letters of the alphabet, days of the week and months of the year), the inherent hierarchy of numbers suggests that number-synaesthesia should be found in most synaesthetes. Three synaesthetes were recruited who showed between 80 and 100% consistency of synaesthetic responses to a set of stimuli across a three month interval. Their synaesthesia for numbers was investigated using an extension of Dixon et al.'s (2000) "2 plus 5 equals yellow" paradigm. Participants saw single digits and operators for an arithmetic process followed by a coloured square which was either congruent or incongruent with their individual colour for the answer; the task was to name the colour followed by stating the solution to the arithmetic sum. To see whether number-processing synaesthesia extends beyond the visual domain into the auditory domain the same procedure was used with the digits and operators being presented aurally. Relative to age-matched controls congruency effects were found for the synaesthetes. These findings add to the suggestion that synaesthesia can occur at a conceptual level where physical experience of an inducer is not necessary. Additionally, the findings showed that for one synaesthete there was a statistical difference in the auditory but not visual condition whilst for the remaining two synaesthetes, the reverse pattern was observed. This variable pattern adds to the body of literature that further illustrates the heterogeneity of synaesthesia. The findings are incorporated into Rich and Mattingley's (2002) neurocognitive model of synaesthesia and observations which cannot yet be explained are highlighted as future directions for research. PMID- 16683500 TI - Color congruity effect: where do colors and numbers interact in synesthesia? AB - The traditional size congruity paradigm is a Stroop-like situation where participants are asked to compare the values of two digits and ignore the irrelevant physical sizes of the digits (e.g., 3 5). Here a color congruity paradigm was employed and the irrelevant physical sizes were replaced by irrelevant colors. MM, a digit-color synesthete, yielded the classical congruity effect. Namely, she was slower to identify numerically larger numbers when they deviated from her synesthetic experience than when they matched it. In addition, the effect of color on her comparative judgments was modulated by numerical distance. In contrast, performance of non-synesthetes was not affected by the colors. On the basis of neurophysiological studies of magnitude comparison and interference between numerical and physical information, it is proposed that the interaction between colors and digits in MM occurs at the conceptual level. Moreover, by using the current paradigm it is possible to determine the stage at which color-digit binding in synesthesia occurs. PMID- 16683501 TI - Sound-colour synaesthesia: to what extent does it use cross-modal mechanisms common to us all? AB - This study examines a group of synaesthetes who report colour sensations in response to music and other sounds. Experiment 1 shows that synaesthetes choose more precise colours and are more internally consistent in their choice of colours given a set of sounds of varying pitch, timbre and composition (single notes or dyads) relative to a group of controls. In spite of this difference, both controls and synaesthetes appear to use the same heuristics for matching between auditory and visual domains (e.g., pitch to lightness). We take this as evidence that synaesthesia may recruit some of the mechanisms used in normal cross-modal perception. Experiment 2 establishes that synaesthetic colours are automatically elicited insofar as they give rise to cross-modal Stroop interference. Experiment 3 uses a variant of the cross-modal Posner paradigm in which detection of a lateralised target is enhanced when combined with a non informative but synaesthetically congruent sound-colour pairing. This suggests that synaesthesia uses the same (or an analogous) mechanism of exogenous cross modal orienting as normal perception. Overall, the results support the conclusion that this form of synaesthesia recruits some of the same mechanisms used in normal cross-modal perception rather than using direct, privileged pathways between unimodal auditory and unimodal visual areas that are absent in most other adults. PMID- 16683502 TI - Linguistic determinants of word colouring in grapheme-colour synaesthesia. AB - Previous studies of grapheme-colour synaesthesia have suggested that words tend to be coloured by their initial letter or initial vowel (e.g., Baron-Cohen et al., 1993; Ward et al., 2005). We examine this assumption in two ways. First, we show that letter position and syllable stress have been confounded, such that the initial letters of a word are often in stressed position (e.g., 'wo-man, 'ta-ble, 'ha-ppy). With participant JW, we separate these factors (e.g., with stress homographs such as 'con-vict vs. con-'vict) and show that the primary determinant of word colour is syllable stress, with only a secondary influence of letter position. We show that this effect derives from conceptual rather than perceptual stress, and that the effect is more prominent for synaesthetes whose words are coloured by vowels than by consonants. We examine, too, the time course of word colour generation. Slower colour naming occurs for spoken versus written stimuli, as we might expect from the additional requirement of grapheme conversion in the former. Reaction time data provide evidence, too, of incremental processing, since word colour is generated faster when the dominant grapheme is flagged early rather than late in the spoken word. Finally, we examine the role of non-dominant graphemes in word colouring and show faster colour naming when later graphemes match the dominant grapheme (e.g., ether) compared to when they do not (e.g., ethos). Taken together, our findings suggest that words are coloured incrementally by a process of competition between constituent graphemes, in which stressed graphemes and word-initial graphemes are disproportionately weighted. PMID- 16683503 TI - Pupillometry of grapheme-color synaesthesia. AB - Pupil diameters of color-grapheme synaesthetes were measured with an infrared eye tracker while Stroop-like alphanumeric symbols were passively viewed. Pupils dilated more when synaesthetes viewed incongruently-colored symbols than congruently-colored symbols or symbols printed in the standard black ink. The results show that the physiological measure of pupillary diameter can be used as a marker of the synaesthetic experience. PMID- 16683504 TI - Neuronal correlates of colour-graphemic synaesthesia: a fMRI study. AB - Synaesthesia is a perceptual phenomenon in which specific events in one sensory modality induce experiences in another. In colour-graphemic synaesthesia, subjects report colour experiences induced by written letters. Our subjects displayed this type of synaesthesia, as verified by a test of the consistency of the perceptual associations over time, and had no history of neurological or psychiatric disorders. We investigated the hypothesis that the synaesthetic colour experience is accompanied by an activation of the human colour area (V4/V8) using functional magnetic resonance imaging (fMRI). With retinotopic and colour mapping we could confirm that colour stimuli specifically activate area V4/V8. For the study of colour-graphemic synaesthesia we used an AB boxcar design with blocks of letters that elicited a synaesthetic colour experience (condition A) alternating with blocks of letters that did not (condition B). In both hemispheres condition A led to a significantly higher activation of V4/V8 than condition B. These findings support the hypothesis that the grapheme-induced colour perception in synaesthesia is caused by an activation of the colour areas of the human visual cortex. PMID- 16683505 TI - Activation of color-selective areas of the visual cortex in a blind synesthete. AB - Many areas of the visual cortex are activated when blind people are stimulated naturally through other sensory modalities (e.g., haptically; Sadato et al., 1996). While this extraneous activation of visual areas via other senses in normal blind people might have functional value (Kauffman et al., 2002; Lessard et al., 1998), it does not lead to conscious visual experiences. On the other hand, electrical stimulation of the primary visual cortex in the blind does produce illusory visual phosphenes (Brindley and Lewin, 1968). Here we provide the first evidence that high-level visual areas not only retain their specificity for particular visual characteristics in people who have been blind for long periods, but that activation of these areas can lead to visual sensations. We used fMRI to demonstrate activity in visual cortical areas specifically related to illusory colored and spatially located visual percepts in a synesthetic man who has been completely blind for 10 years. No such differential activations were seen in late-blind or sighted non-synesthetic controls; neither were these areas activated during color-imagery in the late-blind synesthete, implying that this subject's synesthesia is truly a perceptual experience. PMID- 16683506 TI - Evidence against functionalism from neuroimaging of the alien colour effect in synaesthesia. AB - Coloured hearing synaesthetes experience colours to heard words, as confirmed by reliability of self-report, psychophysical testing and functional neuroimaging data. Some also describe the 'alien colour effect' (ACE): in response to colour names, they experience colours different from those named. We have previously reported that the ACE slows colour naming in a Stroop task, reflecting cognitive interference from synaesthetically induced colours, which depends upon their being consciously experienced. It has been proposed that the hippocampus mediates such consciously experienced conflict. Consistent with this hypothesis, we now report that, in functional magnetic resonance imaging of the Stroop task, hippocampal activation differentiates synaesthetes with the ACE from those without it and from non-synaesthete controls. These findings confirm the reality of coloured hearing synaesthesia and the ACE, phenomena which pose major challenges to the dominant contemporary account of mental states, functionalism. Reductive functionalism identifies types of mental states with causal roles: relations to inputs, outputs and other states. However, conscious mental states, such as experiences of colour, are distinguished by their qualitative properties or qualia. If functionalism is applied to conscious mental states, it identifies the qualitative type of an experience with its causal role or function. This entails both that experiences with disparate qualitative properties cannot have the same functional properties, and that experiences with disparate functional properties cannot have the same qualitative properties. Challenges to functionalism have often denied the first entailment. Here, we challenge the second entailment on empirical grounds. In coloured hearing synaesthesia, colour qualia are associated with both hearing words and seeing surfaces; and, in the ACE, these two functions act in opposition to one another. Whatever its merits as an account of other mental states, reductive functionalism cannot be the correct account of conscious experiences. PMID- 16683507 TI - The rule of reason. PMID- 16683508 TI - The state of Massachusetts health insurance--Spring 2006. PMID- 16683509 TI - What is all this complaining? PMID- 16683511 TI - Ten under 10. PMID- 16683510 TI - Massachusetts fluoridation update 2006. AB - Massachusetts has a long history of activity with community water fluoridation. Although the state has 3.8 million people living in 137 fluoridated communities, there are more than 2 million people who do not have these benefits. The Bay State is ranked 35th in the country regarding the percent of people on public water supplies with fluoridation. We can do better than that. We have more than 60 years of experience receiving the health and economic benefits of fluoridation in our country; however, there is still a lot of misinformation about fluoridation, and the unreliable nature of information posted on the Internet exacerbates much of this misinformation. Dental professionals, their patients, and decision-makers must be continuously educated about the safety, health, and economic benefits of community water fluoridation. Patients from 6 months to 16 years of age living in nonfluoridated communities should be prescribed supplemental fluoride. Dental professionals in nonfluoridated communities should assist them to become fluoridated. All dental professionals need to become more involved in the leadership of their communities. PMID- 16683512 TI - Financial pitfalls of expansion projects (and how to avoid them). PMID- 16683513 TI - American Heart Association changes CPR guidelines. PMID- 16683514 TI - Saving teeth through nonsurgical endodontic retreatment. PMID- 16683515 TI - Bisphosphonate-associated Osteonecrosis of the jaws and endodontic treatment: two case reports. AB - Bisphosphonates are commonly used in the management of bone diseases, such as osteoporosis and Paget's disease, and to prevent bone complications and treat malignant hypercalcemia in certain types of cancer. Although this class of drugs has clear evidence of medical efficacy, there are an, increasing number of reports of bisphosphonate-associated osteonecrosis of the jaws that have substantial implications for the patient and for the treating dentist. This article reviews proposed possible mechanisms of bisphosphonate-associated osteonecrosis of the jaws and describes two case reports where non-surgical and surgical root canal treatment were precipitating factors. Recommendations for prevention and treatment of the disease follow. Thorough history-taking and timely consultation with the patient's oral surgeon and oncologist are emphasized. PMID- 16683516 TI - A clinico-pathologic correlation. PMID- 16683517 TI - Management of reactive gingival nodules. PMID- 16683518 TI - Dentistry: the early years. PMID- 16683519 TI - [The induction of CYP3A regulated by pregnane X receptor and its significance in drug metabolism]. PMID- 16683520 TI - [Advances in the research of anti-hepatic fibrosis drugs]. PMID- 16683521 TI - Preparation and characterization of PLGA microspheres containing a staphylokinase variant (K35R). AB - AIM: To produce poly (lactic-co-glycolic acid) (PLGA) microspheres, containing a staphylokinase variant (K35R, DGR) with reduced immunogenecity and antiplatelet aggregation activities, which allowed the preservation of protein stability during both particle processing and drug release. METHODS: DGR-loaded microspheres were fabricated using a double emulsion-solvent evaporation technique. The effects of preparative parameters, such as stirring rate, polymer concentration, and the excipients of both internal and external aqueous phase (W2), on DGR encapsulation efficiency and microsphere characteristics were investigated. In vitro and in vivo release of DGR were conducted and the cause for instability of DGR during release was also investigated. RESULTS: Moderate ultrasonic treatment of aqueous DGR/dichloromethane mixtures caused approximately. Eighty four per cent DGR denaturation. However, the activity recovery of DGR almost amounted to 100% when 2% polyvinyl alcohol (PVA) was addled into the aqueous phase. It was found that NaCl in the external water phase significantly increased DGR encapsulation efficiency. Furthermore, NaCl in the external water phase played a role in determining size and surface morphology of microsphere. In vitro release test showed a burst release of DGR from microspheres, followed by sustained release of 50% total activity over 15 days. In vivo experiments showed that DGR released from microspheres sustained 5 days. Denaturation of DGR within microspheres might be resulted from acidic microclimate. CONCLUSION: The stability of DGR was effectively protected during microencapsulation and a relatively high encapsulation efficiency of DGR was obtained. PLGA microspheres could be an effective carrier for DGR. PMID- 16683522 TI - [Liver targeting of cationic liposomes modified with soybean-derived sterylglucoside in vitro]. AB - AIM: To construct a liposomal liver targeting delivery system by adding soybean derived sterylglucoside (SG) to the cationic liposomes. METHODS: The physico chemical properties of SG modified cationic lipsomes were investigated using fluorescein sodium (FS) as a model drug, as well as the interaction of SG modified liposomes with HepG2 2. 2. 15 cells in the point of involvement of asialoglycoprotein receptor (ASGP-R) mediated transfection. Liver targeting of modified cationic liposomes were also investigated using liver perfusing technique, and hepatocytes and non-hepatocytes were separated and examined after perfusing. RESULTS: All the formula yielded high incorporation efficiency (83.12% - 91.74%), small particle size (93.0 - 124.4 nm). The zeta potential of blank liposomes all showed positive values. The transfection efficiency of FS entrapped in SG-liposomes with HepG2 2.2. 15 was significantly higher than that of liposomes without modification. The transfection of SG-liposomes were reduced significantly by the 20/30 mmol galactose as a competitor of ASGP-R. All the cationic liposomes showed high level of liver uptake of FS. Compared with the uptake of non-hepatocytes of each respectively, only SG/Brij-35 liposomes showed difference in FS uptake by hepatocytes (P < 0.05). CONCLUSION: It showed that SG/Brij-35 modified cationic liposomes are potentially useful drug carrier to liver but may be affected by different modification. PMID- 16683523 TI - [Pharmacokinetics of breviscapine liposomes following intravenous injection in Beagle dogs]. AB - AIM: To prepare the breviscapine liposomes and study the pharmacokinetics of breviscapine liposomes in Beagle dogs. METHODS: The cross-over design (two periods) was employed. Six Beagle dogs were administrated a single intravenous dosage of 28 mg of breviscapine liposomes and reference preparation, respectively, scutellarin in plasma of 6 dogs at different sampling time was determined by RP-HPLC. The pharmacokinetic parameters were calculated by 3P97 program and compared by statistic analysis. RESULTS: The mean concentration-time curves of breviscapine liposomes and reference preparation were both fitted to two-compartment model with the main pharmacokinetic parameters as follows: T 1/2 alpha were (4.4 +/- 0.7) min and (1.8 +/- 1.3) min respectively; T 1/2 beta were (55 +/- 27) min and (28 +/- 23) min respectively; V(c) were (1 580 +/- 265) mL and (2 460 +/- 2 200) mL respectively; CL(s) were (88 +/- 10) mL x min(-1) and (324 +/- 69) mL x min(-1) respectively; and AUC(0-720) were (363 +/- 42) microg x min x mL(-1) and (102 +/- 19) microg x min x mL(-1) respectively. The T 1/2 alpha, CL(s) and AUC(0-720) of breviscapine liposomes all had significant difference from those of reference preparation, after the data were examined by a one-way analysis of variance (ANOVA). CONCLUSION: Compared with the reference preparation, breviscapine liposomes had a much more higher concentration in plasma and contained characteristic of sustained-release, which ameliorated the pharmacokinetic properties of scutellarin. PMID- 16683524 TI - [Effect of self-microemulsifying system on cell tight junctions]. AB - AIM: To study the effect of negatively charged and positively charged self microemulsifying systems (SMES) on the cellular tight junction complex was to be investigated at molecular cell level. METHODS: Human intestinal epithelial Caco-2 cell model was established. Effect of formulations on the transepithelial electrical resistance (TEER) and permeability of the paracellular transport marker mannitol were measured to evaluate the cell integrity. Changes in subcellular localization of the tight junction protein zona occludens 1 (ZO-1) and cytoskeleton protein actin by immunofluorescence were also assessed after treatment of two SMESs in different dilutions. RESULTS: The TEER of cell monolayers was not markedly affected by negatively charged SMES in different dilutions. The positively charged SMES could significantly decrease the TEER (P < 0.05) in three dilutions. The full recovery of TEER was found after the treatment of lower dilution for 2 h, then cultured for 48 h, while the recovery of TEER was 81.3% of control in 1 : 50 dilution. Two SMESs could enhance the apparent permeability coefficient of mannitol (2.9 - 64.6 folds), which depended on the dilution times. The immunofluorescent results indicated that the distribution of ZO-1 and actin were discrete in cell membrane after the treatment of formulation. Since the positively charged microemulsion could bind to the epithelial cell membrane by electrostatic interaction, the actin of the cells undergone some kind of stress stimulated by the higher concentration of microemulsion was more markedly affected than the negatively charged SMES. Effect of formulations on ZO 1 and actin relied on the dilution. CONCLUSION: SMES is able to enhance the paracellular transport marker mannitol. The mechanism of opening of tight junctions by SMES might be the change of distribution of ZO-1 and actin. PMID- 16683525 TI - [Effect of ginkgolide B on the function of rat aorta smooth cells and U937 cells stimulated by oxLDL]. AB - AIM: To investigate the effect of ginkgolide B on the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL or PAF. In addition, to analyze whether the effect of oxLDL is mediated through PAF receptor. METHODS: Using 3H-Tdr incorporation assay, the proliferation of VSMC was measured. The protein and mRNA level of MCP-1 and IL-8 in U937 cells were determined by RT-PCR and ELISA. Using Western blotting the p65 and IkappaB was quantified. The binding of oxLDL to U937 cell was measured by a radio-ligand binding assay of 3H-PAF. RESULTS: Ginkgolide B inhibited, in dose-dependent manner, the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL, and inhibited the oxLDL-induced p65 activation and depletion of IKappaB. oxLDL inhibited PAF binding to U937 cells. CONCLUSION: Ginkgolide B, as a PAF antagonist, possesses the effect of inhibiting the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL in vitro. The effect of oxLDL is, at least in part, mediated through PAF receptor. PMID- 16683526 TI - [Antitumor effect of the novel immunoconjugate composed of pingyangmycin and anti type IV collagenase monoclonal antibody]. AB - AIM: To study the antitumor effect of an immunoconjugate composed of pingyangmycin (PYM) and anti-type IV collagenase monoclonal antibody (mAb) 3G11. METHODS: 3G11-PLG-PYM immunoconjugate was prepared by linking 2-iminothiolane (2 IT) modified mAb to PYM via N-succinimidyl-3-(2-pyridyldithiol) -propionate (SPDP) derived poly-alpha-L-glutamic acid (PLG) backbone as the intermediate drug carrier. Characterization of the conjugate was performed by SDS-PAGE and spectrophotometry. Immunoreactivity of the conjugate against type IV collagenase was determined by ELISA. The cytotoxicity of the conjugate to hepatoma 22 (H22) and KB cells was examined by MTT assay. Antitumor effect of the conjugate in vivo was evaluated in mice bearing subcutaneously implanted H22 tumor, the candidate drugs were administered intravenously by "q2d x 6" regimen. RESULTS: The molecular weight of the conjugate was approximately 170 kD. The molecular ratio of 3G11-PLG-PYM was 1 : 2. 4 : 10. The conjugate retained part of the immunoreactivity of mAb 3G11 against the antigen. The cytotoxicity of the conjugate to H22 and KB cells was moderate comparing with free PYM. In vivo however, free PYM inhibited the growth of H22 by 60.6% on day 22 at the dose of 10 mg x kg(-1), while the equivalent dose of 3G11-PLG-PYM conjugate reached 90.8%. The median survival time of the mice treated with the conjugate was prolonged by 71.7% as compared with that of the untreated group, whereas that of free PYM prolonged only 10.9%. 3G11-PLG-PYM conjugate was notably more effective than free PYM in tumor suppression and life span prolongation. CONCLUSION: 3G11 PLG-PYM displayed more marked antitumor efficacy than free PYM in vivo and might be a novel candidate for cancer treatment. PMID- 16683527 TI - [Application of new method for data processing in metabonomic studies]. AB - AIM: To search for and application of new method for data processing in metabonomic studies. METHODS: The paper proposed that in the processing of metabonomic data, robust PCA method can be used to diagnose outliers; and unstable variables judged by comparison between difference within class and difference among classes should be excluded before data analysis; moreover, the data should be properly scaled before further processing. The proposed methods were used to preprocess metabolomic data of four genotypes of the Arabidopsis thaliana plants. RESULTS AND CONCLUSION: The outcome demonstrated that the application of these methods can obviously improve clustering and biomarker identifying results. PMID- 16683528 TI - [Effect of Angelica sinensis polysaccharide fraction AP-3 on IL-2 and IFN-gamma induction]. AB - AIM: To investigate the effects of Angelica sinensis polysaccharide fraction AP-3 on IL-2 and IFN-gamma induction and its further immunomodulatory feature. METHODS: The percentage of CD4+ lymphocyte was detected by flow cytometric method, the production of IL-2 and IFN-gamma in cell culture supernatant were determined by ELISA, mRNA expressions of IL-2 and IFN-gamma cytokines were detected by RT-PCR. RESULTS: At the range of 0. 6 - 2 micromol x L(-1), AP-3 significantly enhanced the percentage of CD4+ lymphocytes in total splenocytes. At the range of 2 - 6 micromol x L(-1), the treatment of AP-3 augmented both productions of IL-2 in cell culture supernatant and cell IL-2 mRNA transcription level in a time and dose dependent manner. While in the case of IFN-gamma, AP-3 stimulated at early time after exposure but down-regulated thereafter. CONCLUSION: Angelica sinensis polysaccharide could regulate the immune response through upregulating IL-2, IFN-gamma expression and activating Th1 cell. PMID- 16683529 TI - [Synthesis and antibacterial activity of dl-7-(4,4-dimethyl-3 aminomethylpyrrolidinyl) -quinolones]. AB - AIM: To explore new agents of quinolone derivatives with high activity against Gram-positive organisms. METHODS: dl-7-(4,4-Dimethyl-3- aminomethylpyrrolidinyl) quinolones were designed and synthesized, and their activity against Gram positive organisms was tested in vitro. RESULTS: Ten target compounds were obtained. The structures of these compounds were confirmed by 1H NMR, MS. The target compounds with dl-4,4-dimethyl-3-( methyl) aminomethylpyrrolidine side chains had high activity against Gram-positive organisms. Especially the MIC values of compound 22 for 4 strains of Gram-positive resistant bacteria (two strains of MRSA and two of MRSE) were 0.015 -0.5 mg x L(-), which exhibited more potent activities than gatifloxacin (4 - 128 times). Its MIC value for Pseudomonas aeruginosa 03-5 (0.008 mg x L(-1)) was 4 times as that of gatifloxacin (0.03 mg x L(-1)). CONCLUSION: The compound 22 showed high activity against Gram-positive organisms in vitro and it is worth of more investigation. PMID- 16683530 TI - [Synthesis and immunosuppressive activity of new artemisinin derivatives containing polyethylene glycol group]. AB - AIM: To search for new artemisinin derivatives with higher immunosuppressive activity. METHODS: Two kinds of new artemisinin derivatives containing polyethylene glycol group were synthesized from dihydroartemisinin via condensation and esterification. These compounds were assayed for their inhibitory activity on ConA-induced T cell proliferation and LPS-induced B cell proliferation. RESULTS: Twenty three new compounds (2a - 2f, 3a - 3d, 4a - 4f, 6a, 6b and 7a - 7g) were synthesized and identified by 1H NMR and elemental analysis. CONCLUSION: These compounds had immunosuppressive activity in vitro. Among them, the symmetrical substituted compound 2 and 6 had higher activity than mono-substituted compound 3, 4 and 7. Especially, compounds 2a - 2f remarkably exhibited higher inhibition in comparison with artemisinin and artesunate. PMID- 16683531 TI - [Synthesis and vascular relaxing activity of arecoline derivatives coupled with nitric oxide donors]. AB - AIM: To search for potential anti-atherosclerosis drugs with vascular relaxation activity, a series of agonists of endothelial targets were designed and synthesized. METHODS: Coupling N-methyl-1,2, 3,6-tetrahydrapyridine ring system with 3,4-dibenzenesulfonyl-1,2,5-oxadiazole-2-oxide through esterification or amidation, a series of arecoline derivatives containing NO donors were designed and synthesised. RESULTS: A novel series of compounds structurally related to arecoline have been prepared, the proposed structures of eighteen new compounds were established by IR, 1H NMR, MS spectroscopy and elemental analysis. The effects of the target compounds on the vasodilation activity were tested in the isolated preparation of mice thoratic aorta. CONCLUSION: This preliminary pharmacological tests showed that the candidates have good vasodilation activities and were worthy to be intensively studied. PMID- 16683532 TI - [Isolation and structure identification of a new isoflavone from Psoralea corylifolias]. AB - AIM: To study the chemical constituents of the fruits of Psoralea corylifolia L. METHODS: The constituents were separated and purified by column chromatography with normal phase silica gel and polyamide. Their structures were identified on the basis of physical and spectral data (MS, NMR, HMBC and HMQC). RESULTS: Six compounds were isolated and identified as psoralen (1), isopsoralen (2), bavachalcone (3), psoralidin (4), daidzin (5) and bavadin (6). CONCLUSION: Compound 6 is a new compound. Compound 5 was isolated from this plant for the first time. PMID- 16683533 TI - [Metabolites of R, S-1-(2-methoxyphenyl)-4-[3-(naphtha-1-yl-oxy)-2-hydroxypropyl] piperazin in rat plasma]. AB - AIM: To study the metabolites of R, S-1-(2-methoxypheyl)-4-[3-(naphthal-yl-oxy)-2 hydroxypropyl] -piperazine, (naftopidil, NAF), a novel antihypertensive drug in rat plasma. METHODS: The rat plasma samples were analyzed by LC/MS after oral administration of NAF. According to MS relativity of metabolites and parent compound (NAF) and metabolic rule of compound with similar structure, the structure of potential metabolites were postulated. Phase I metabolites were identified by HPLC/MS and by comparison with authentic standards, phase II conjugates were indirectly identified with beta-D-glucuronidase in presence or absence of glucuronidase selective inhibitor D-saccharric acid beta-1,4-Lactone. RESULTS: Phase I metabolites desmethyl-naftopidil (DMN), (phenyl) hydroxynaftopidil (PHN), (naphthyl) -hydroxy-naftopidil (NHN) were separated and identified in rat plasma by comparison with reference substances, phase II conjugates, NAF and NHN glucuronide conjugates were separated and tentatively identified by hydrolysis with glucuronidase, the aglycones, NAF and NHN, were identified in rat plasma. CONCLUSION: The major metabolic pathway of NAF in rat plasma should be the hydroxylation of the phenyl or nephthyl moiety of NAF and demethylation of NAF. Therefore, (naphthyl) hydroxyl-metabolite and NAF followed by conjugation with beta-glueuronic acid. PMID- 16683534 TI - [In vitro metabolic interaction between diphenytriazol and steroid hormone drugs]. AB - AIM: To observe the metabolic interaction between diphenytriazol and steroid hormone drugs, and provide some useful information for clinical medication. METHODS: The steroid hormone drugs which may be co-administrated with diphenytriazol were selected, such as mifepriston, estradiol, medroxyprogesterone acetate, progesterone, norethisterone and so on. Diphenytriazol was incubated with each drug in rat liver microsome. The residual concentration of diphenytriazol or steroid hormone drugs in the microsomal incubates was determined by reversed-phase high-performance liquid chromatography, separately. The inhibition constants (K(i)) for each of them were calculated. RESULTS: The inhibition constant K(is) of diphenytriazol for the metabolism of mifepristone, estradiol, medroxyprogesterone acetate, progesterone and norethisterone were (201.3 +/- 1.0), (94 +/- 4), (128.7 +/- 2.2), (64 +/- 5) and (80 +/- 4) micromol x L(-1), respectively. The inhibition constants K(i) of steroid hormone drugs for the metabolism of diphenytriazol was (66.9 +/- 2.2) micromol x L(-1) for estradiol, (60.0 +/- 2.3) micromol x L(-1) for medroxyprogesterone acetate, (163 +/- 10) micromol x L(-1) for progesterone and (88 +/- 5) micromol x L(-1) for norethisterone, respectively. CONCLUSION: Diphenytriazol shows metabolism interaction with steroid hormone drugs such as estradiol, medroxyprogesterone acetate, progesterone and norethisterone. PMID- 16683535 TI - Analysis of intraspecific variation of Chinese Carthamus tinctorius L. using AFLP markers. AB - AIM: To investigate the intraspecific variation of Carthamus tinctorius L. (safflower) and establish foundation for further breeding of safflower germplasm resource and screening the quality correlation genes. METHODS: Amplified fragment length polymorphism (AFLP) was carried out to analyze genetic variation of 28 safflower populations collected in China. Unweighed pair-group method of with arithmetical averages (UPGMA) cluster analysis was used to construct a dendrogram and to estimate the genetic distances among the populations. RESULTS: All populations could be uniquely distinguished using 12 selected primer combinations. Similarity coefficients ranged from 0.48 to 0.96 among the populations. Dendrogram revealed distinct segregation of all the cultivars into three main groups and one midst group. CONCLUSION: Limited genetic diversity exists within the tested 28 collections at intra specific level and AFLP-based phyiogeny was not absolutely consistent with that based on morphological characters may be due to the interaction effect between genotype and environment. PMID- 16683536 TI - [Recent advances on the study between steroids and brain function]. AB - It is vital for steroid hormones synthesized in the gonad gland, adrenal gland and placenta to the development and physiological metabolism of the body. In particular, its regulatory effect on brain functions has attracted more and more attention, as nearly all the steroid hormones can be synthesized de novo in the brain. This means steroids derived from brain and peripheral gland can modulate the physiology and pathology of the brain reciprocally and importantly on many aspects such as learning and memory, synaptic transmission, neuroprotection, neurodegenerative diseases (especially Alzheimer's disease), emotion, stress, and menstrual-cycle-linked disorders. In the present review, some new advances are summarized. PMID- 16683537 TI - [Processing of spatial information by hippocampal place cells]. AB - Hippocampal place cells can process the environmental inputs and make up a cognitive map in the hippocampus, or strengthen the synaptic connections within an association cortical cell assembly,thus creating a permanent engram for a spatial site. Outputs from the hippocampus are then integrated with other inputs within the nucleus accumbens and finally initiate a goal-directed behavior through the motor circuit. PMID- 16683538 TI - [The progress in proteomics researches of Alzheimer's disease]. AB - Proteomics, an important discipline in genomic era, is the integrated study of protein properties on a large scale. Application of proteomics researches in Alzheimer's disease (AD), which is one of the most common and complex neurodegeneration disorders, we can not only elucidate the essential issues of disease in protein level, but give a whole view in the pathological mechanisms identification, diagnosis and therapeutic targets discovery as well. The three aspects about the progresses in proteomics researches of AD were reviewed in this article. PMID- 16683539 TI - [Progress in the research of germ cell from human embryonic stem cells]. AB - As the development of spontaneous differentiation of germ cells and gametogenesis from mouse embryonic stem cells (mES) in vitro, hES (human embryonic stem cells) also have potential to differentiated into germ cells in theory. This review focuses on the stem cells niches and genes regulating the hES differentiation toward germ cells, as well as the recent advance and application on the reproductive medicine and therapy of infertility. PMID- 16683540 TI - [Advances of nutrigenomics]. AB - With the development and application in the life science of genomics and bioinformatics, nutrigenomics has emerged and become the new frontier for nutrition science. The main contents of nutrigenomics include the mechanism of nutrients and phytochemicals on the transcription, translation and metabolism of human genes, especially the molecular mechanism and requirement of nutrients, the personal recipe and food safety, etc. In this article, the main research fields, advances and the trend in the future of nutrigenomics are discussed. PMID- 16683541 TI - [Roles of vasodilator-stimulated phosphoprotein in the regulation of cytoskeleton]. AB - Regulation of cytoskeletal dynamics is required to coordinate cell adhesion, morphology change and cell motility. Vasodilator-stimulated phosphoprotein (VASP) is an actin-binding protein which is composed of EVH1, EVH2 domains and PRR. It has been implicated in actin-based processes such as axon guidance, T cell polarization and fibroblast migrations. The regulation of VASP phosphorylation, which is controlled by PKG (cGMP-dependent protein kinase) and PKA (cAMP dependent protein kinase), could correspond to the "switch" process in its communication with cytoskeleton during modification of focal adhesion. PMID- 16683542 TI - [Surgical stress and immunosuppression]. AB - Perioperative psychological and physiological stress can suppress cell-mediated immunity, including the decrease of T lymphocytes and natural killer (NK) cells in the number and activities. A stronger immunosuppression is found in larger postoperative stress than in smaller one, which may interfere with the recovery from the surgical hurt. Generally, the surgical stress-induced immunosuppression can recover, which depends on the degree of surgical injury. The mechanisms through which surgical stress induces the immunosuppression are closely related to hypothalamus-pituitary-adrenocortical axis, sympathetic nervous system, cytokines, opioids and T cell signal molecule. Some strategies may be used to prevent surgical stress-induced immunosuppression, which includes analgesic drugs devoid of immunosuppressive effects and protease inhibitor. PMID- 16683543 TI - [Current progress of mitochondrial transcription factor A]. PMID- 16683544 TI - [A brief review on the mechanism and function of macropinocytosis]. PMID- 16683545 TI - [Macrophage migration inhibitory factor and its role in atherosclerosis]. PMID- 16683546 TI - [Progress in angiogenesis regulation of perlecan]. PMID- 16683547 TI - [The role of beta3-adrenergic receptors in the regulation of cardiac function]. PMID- 16683548 TI - [Application of single cell gene expression analysis techniques on neurosciences research]. PMID- 16683549 TI - [Recent advances in Nurr1 gene differentiating stem cells into dopaminergic neuron]. PMID- 16683550 TI - [The progress of Mash-1 gene associated with neural development and differentiation]. PMID- 16683551 TI - [The role of synaptotagmin in neurotransmitter release]. PMID- 16683552 TI - [Progress in the study of OPHN1 gene]. PMID- 16683553 TI - [The involvement of phasic bursting activation of midbrain dopamine neurons in reinforcement]. PMID- 16683554 TI - [Expression and regulation of angiogenesis-related factors during embryo implantation]. PMID- 16683555 TI - [Research advancement of beta-chemokine RANTES]. PMID- 16683556 TI - [Study of two electrophoresis procedures of seminal plasma proteins]. AB - OBJECTIVE: To analyse clinical feasibility of two electrophoresis procedures of seminal plasma proteins, agarose gel electrophoresis and SDS-agarose gel electrophoresis. METHODS: Sixty-nine semen samples were examined and classified into three groups: the asthenozoospermia (n = 22), the asthenoteratozoospermia (n = 19), and the relative normal group (n = 28) with normal routine and special test results, according to WHO routine and special test criterion. Then, the seminal plasma protenis were separated by two different electrophoresis, with SDS agarose and agarose support medium, the buffer pH 7.0 and 9.2 respectively. The agarose gel electrophoresis was done under various sample loading time, motion power and staining modules. The completed gels were scanned and compared the each other statistically. RESULTS: Seminal plasma proteins can be separated into 4 strips by SDS-agarose gel electrophoresis with acid crystal violet, and the strips were diffusion and with dark background. However, 6 clear strips named A, B, C, D, E, and F can be obtained by agarose gel electrophoresis with 6 min. After samples were loaded and stained by amidoblack, there showed appropriate spaces among strips, and it was very easy to scan the drying gel by a densitometer. Using agarose gel electrophoresis, the statistical difference in strip C and E was shown between the asthenozoospermia and the relative normal group, and between the asthenozoospermia and the asthenoteratozoospermia, however, not between the relative normal and the asthenoteratozoospermia group. Moreover, the samples in the relative normal group with normal routine and special test results were in different electrophoresis maps. CONCLUSION: Agarose gel electrophoresis of seminal plasma proteins with buffer pH 9.2, 6 min. sample loading and amidoblack stain was a simple, fast and fit technique for clinic. PMID- 16683557 TI - [Establishment of the 2-D synthetic map of total protein of normal human spermatozoa enriched with low abundance protein]. AB - OBJECTIVE: To separate the low abundance protein and establish the 2-DE synthetic map of total protein of human normal spermatozoa by using the 2-DE technology. METHODS: All the needed human spermatozoa were collected and mixed, and proteins were extracted at one time with the method of urea/thiourea and ultra-sound. 0.8 mg, 0.6 mg, 0.5 mg, 0.3 mg sperm protein extracts were separated with 2-DE. Analyzed with MALDI-TOF-MS, PI and MW of 2 spots were obtained. Then set the 2 spots as the referent spots, different maps were compared and analyzed. At last, a synthetic map enriched with low abundance protein was obtained. RESULTS: 1,080 +/- 23 protein spots have been separated on the 2-DE map with standard 0.5 mg loading amount and a synthetic map A was constructed which consist of 889 matched protein spots on the all maps with 0.5 mg loading amount. 381, 50 and 32 new spots were detected individually on the maps with 0.8 mg, 0.6 mg and 0.3 mg protein loading amount. A synthetic map with 1,352 protein spots was obtained. CONCLUSION: Low abundance protein was separated and a synthetic map enriched with low abundant protein was obtained by changing the protein loading amount. PMID- 16683558 TI - [Effect of nonylphenol on testosterone secretion of rat leydig cells in vitro]. AB - OBJECTIVE: To investigate the effect of nonylphenol (NP) on testosterone secretion of Leydig cells. METHODS: A primary culture system of Leydig cells was set up, followed by identification of Leydig cells with 3beta-HSD staining. After treatment with different concentrations of NP, testosterone secretion was detected and morphological examination was performed. RESULTS: Following treatment with NP, morphologic changes of Leydig cells were detected, with decreased cell density at high doses of NP. Testosterone increased at lower concentrations of NP while decreased at high concentrations of NP. CONCLUSION: Lower doses of NP can stimulate the secretion of testosterone, but increased exposure to NP will inhibit testosterone secretion of Leydig cells. Besides, high concentrations of NP can cause death of Leydig cells in vitro. PMID- 16683559 TI - [Construction and identification for cell strain of anti-human sperm protein 22 monoclonal antibodies]. AB - OBJECTIVE: To prepare monoclonal antibodies of mice anti-human sperm protein 22 (SP22) and to identify their specificities. METHODS: BALB/c mice were immunized with human SP22, monoclonal antibodies were prepared by hybridoma technique and the sensitivity and specificity of SP22 McAb were investigated by ELISA and Western-blot assay, respectively. The distribution of human SP22 in sperm were shown by immunohistochemistry. RESULTS: Three strains of hybridoma cells were obtained, with the affinity constant (K) of 1.0 x 10(7) L/mol and the titers were 1:10(3) and 1:3,200 in the mixed supernatant of cell cultures and abdominal dropsy, respectively. IgG isotype of the antibody was identified as IgG1. Western blot demonstrated that there was a specific recognition between human SP22 and the obtained monoclonal antibody. Immunohistochemistry displayed that human SP22 mainly distributed on the acrosome surface of human sperm. CONCLUSION: The monoclonal antibodies of anti-human 22 was prepared by the technique of hybridoma cell has higher titer and specificity, which can combine specially with the SP22 protein on the surface of human sperm. PMID- 16683560 TI - [Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21]. AB - OBJECTIVE: To produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21. METHODS: Bmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET 28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot. RESULTS: Mouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody. CONCLUSION: There was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly. PMID- 16683561 TI - [Influence of LY294002 on sperm motility in asthenozoospermia patients in vitro]. AB - OBJECTIVE: To investigate the effects of LY294002, a phosphatidylinositol 3 kinase inhibition, on sperm motility in asthenozoospermia patients in vitro, and further analyze the possible molecular mechanism. METHODS: Sperm aseptically obtained by masturbation and prepared by swim-up technique from 10 patients with asthenozoospermia and 10 healthy fertile men were incubated with different concentrations of LY294002. Measurements of motility were carried out at 10, 30 and 60 min in all specimens by CASA. RESULTS: The sperm in asthenozoospermia patients treated with LY294002 showed a significant increase in sperm progressive motility, the percentage of motile cells, VSL and VAP. CONCLUSION: LY294002 can enhance the motility of sperm in asthenozoospermia patients in vitro. PMID- 16683562 TI - [Plasma endothelin-1 in patients with prostate cancer and its clinical significance]. AB - OBJECTIVE: To investigate plasma endothelin-1 (ET-1) level in patients with prostate cancers and its clinical significance. METHODS: Plasma ET-1 level was measured by radioimmunoassay in 31 patients of prostate cancer (23 with non-HRPC, 8 with HRPC) and 26 patients of BPH. RESULTS: Compared with each other of the ET 1 level, there were no significant difference among the BPH group,non-HRPC group and HRPC group. No significant difference was found either between bone metastasis (BM) and non- BM, between high and middling differentiation prostate cancer group, as well as in different PSA level groups (P >0.05). But the ET-1 level in low differentiation prostate cancer was notably lower than those of the high and middle respectively (P < 0.05). CONCLUSION: To detect plasma endothelin 1 (ET-1) level is not a useful method to evaluate the development and the prognosis of prostate cancer. PMID- 16683563 TI - [Antagonistic effects of vitamin E on the testicular injury by cyclophosphamide in mice]. AB - OBJECTIVE: To observe the protective effects of vitamin E on the testicular injury by cyclophosphamide in mice, and the correlative mechanism. METHODS: Fifty sexually mature male mice were randomly divided into five groups: the cyclophosphamide group (the CP group), the low-dose vitamin E group (the low-dose group), the middle-dose vitamin E group (the middle-dose group), the high-dose vitamin E group (the high-dose group), the matched control group (the control group). The first four groups were given cyclophosphamide by gavage at a dose of 5 mg/(kg x d). The low-dose group, the middle-dose group and the high-dose group were given vitamin E by subcutaneous injection at doses of 30 mg/(kg x d), 50 mg/(kg x d) , 70 mg/(kg x d) after 4 h of cyclophosphamide treatment. The control group was gavaged with equivalent normal saline. The treatment period for all groups was 28 days. The level of plasma FSH, LH, T and the activity of testicular SOD, GSHPx, CAT and the level of testicular MDA were detected. The histological structure and the ultrastructure of the testis were examined by light microscope and electron microscope. RESULTS: As compared with the CP group, the plasma FSH, LH, T level and the SOD, GSHPx, CAT activity in the middle-dose group and the high-dose group were higher (P< 0.05, P< 0.01), MDA level significantly lower(P<0.01). The histological structure and the ultrastructure of the testis were in the normal range. CONCLUSION: Vitamin E has protective effects on the testicular injury by cyclophosphamide in mice. The possible mechanism of vitamin E may be its scavenging free radical and antioxidant effects, as well as it may have some stimulatory effects on gonadotrophin releasing of pituitary anterior lobe. PMID- 16683564 TI - [Protective effect of pentoxifylline on spermatogenesis following testicular torsion/detorsion in rats]. AB - OBJECTIVE: To investigate the protective effect of pentoxifylline on spermatogenesis following testicular torsion/detorsion in rats. METHODS: Healthy male Sprague-Dawley rats (n = 24) were divided into three groups randomly, each comprising 8 rats. In Group I, rats underwent a sham operation. In Group II and III, animals were submitted to unilateral 720 degrees testicular torsion, then detorsion in two hours. Infusion of isotonic saline and pentoxifylline into tail vein was initiated 15 minutes prior to relief of torsion in Group II and III respectively. Twenty four hours later, testes were examined for evidence of germ cell apoptosis by the flow cytometry and the level of total antioxidant capability (T-AOC) and malondialdehyde (MDA) by spectrophotometry. RESULTS: Compared with that of group II, the number of apoptotic germ cell and the level of MDA decreased remarkably in Group III, but T-AOC increased significantly (P <0.01). CONCLUSION: Pentoxifylline provided significant rescue of testicular function after acute experimental torsion. PMID- 16683565 TI - [Effect of tail-suspension on the reproduction of adult male rats]. AB - OBJECTIVE: To study the effects on the male reproduction in adult male rats and its mechanisms through simulated weightlessness using tail-suspension, in order to do a basic works of exploring the effects on human being's reproduction in outer space. METHODS: Forty Spraque-Dawley adult male rats were randomly divided into four groups, two experimental groups and two control groups. Rats in the two experimental groups were tail-suspended for 14 d and 28 d respectively, then we examined the weight and morphology of testis, the quality and amount of sperm, also tested the serum hormone by radioimmunoassay and analyzed apoptosis rate of testicular cells by TUNEL in the experimental rats and control rats. RESULTS: After tail-suspension, the weight of testis, the sperm count and sperm motility significantly decreased (P <0.05), while the apoptosis rate of testicular cells and the amount of abnormal sperm markedly increased (P <0.05). The content of testosterone significantly decreased (P <0.05), but the contents of FSH and LH mildly increased (P > 0.05). These changes were not significant between two experimental groups (P > 0.05). In addition, the seminiferous tubules became atrophy with the reduction of the layers of seminiferous epithelium, and sperm amount in lumens of seminiferous tubules decreased in experimental groups. The above were more remarkable in the 28 d experimental group. CONCLUSION: Simulating weightlessness has a harmful effect on reproduction of adult male rats. These may be caused by inducing apoptosis. The blocking apoptosis of testicular cells may be useful in improving the harmful effect. PMID- 16683566 TI - [Detection of peripheral blood Th1/Th2 cell ratio in patients with chronic abacterial prostatitis/chronic pelvic pain syndrome]. AB - OBJECTIVE: To evaluate the change of peripheral Th1/Th2 cells in patients with CAP/CPPS and its significance in various clinical types of CAP/CPPS. METHODS: Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) secreted by CD3+ CD8(-)T cells were detected by flow cytometry in 35 patients with CAP/CPPS and 12 healthy volunteers, and significance of Th1/Th2 cells ratio in the etiology of CAP/CPPS was analyzed. RESULTS: Compared with control group, peripheral Th1 cells were significantly increased in patients with IIIA and III B CAP/CPPS, the Th1/Th2 cell ratio was apparently increased (P <0.05), while Th2 cells was not different from that in the control(P>0.05 ); Th1, Th2 cells and Th1/Th2 in patients with IIIA CPPS were not statistically different from those in III B CPPS respectively (P > 0.05) . CONCLUSION: There is a Th1/Th2 imbalance in CAP/CPPS patients with an increase of Th1 type cytokines. Th1 cell may play an important role in the pathogenesis of CAP/CPPS. PMID- 16683567 TI - [Study on fetal SRY gene in maternal plasma using nested polymerase chain reaction]. AB - OBJECTIVE: To develop a nested polymerase chain reaction (PCR) technique for fetal SRY gene identification using cell-free fetal DNA in maternal plasma. METHODS: Peripheral blood samples were obtained from 30 pregnant women and cell free DNA was extracted by the phenol/chloroform method from plasma. The nested PCR was carried out to amplify the fragment of SRY gene by two sets of PCR primer pairs. Direct sequencing analysis was then performed on the PCR product. RESULTS: Among the 17 women bearing male fetuses, SRY sequences were detected in 15 plasma samples after nested PCR amplification, while none of the 13 women bearing female fetuses had the positive results. The accuracy and sensitivity were 93.3% (28/30) and 88.2% (15/17), respectively. CONCLUSION: The phenol/chloroform extraction for fetal DNA in maternal plasma was effective and simple. And the nested PCR amplification of SRY sequence is a convenient and low-cost approach for the non invasive early prenatal diagnosis of sex-linked inheritant diseases. PMID- 16683568 TI - [Urethroplasty with oblique preputial island flap for the treating of hypospadia]. AB - OBJECTIVE: To Explore the effect of oblique preputial island flap for the treating of hypospadias. METHODS: Fifty-one patients were performed one-stage urethroplasty with oblique preputial island flap to repair hypospadias. RESULTS: All cases resulted in a good contour of the penis without any redundancy and a normal anatomic position of slit-shaped urethral meatus. The urination was perfect. Six patients occurred complications (3 cases of urinary fistula, 3 cases of meatal stenosis). CONCLUSION: With extensive scope of materials, reliable blood supply of skin flap, satisfactory appearance of shaping penis and few complications, one-stage urethroplasty with oblique preputial island flap is an effective method to repair hypospadias of penile type and penile-scrotal type. PMID- 16683569 TI - [Clinical value of CK34BE12 combining the expression of protein P53 gene and prostate specific antigen for the differential diagnosis of prostate carcinoma]. AB - OBJECTIVE: To improve the level of clinical diagnosis and differential diagnosis of benign and malignant prostate lesions. METHODS: One hundred and nine cases of prostate cancer and prostate hyperplasia were evaluated by the expression of high molecular weight cytokeratin (CK34BE12), prostate specific antigen (PSA) and protein P53 gene using the immunohistochemical technique. RESULTS: The basal cells in all of the benign lesions were stained with the CK34BE12 and PSA, while it had not immunoreactivity with P53. In contrast, the prostate carcinoma were not stained or partly stained with the CK34BE12 and PSA, but P53 show significant immunoreactivity with the tissue. CONCLUSION: Based on the routine histological studies with the expression of CK34BE12 and PSA together, they can indicate the existence of basal-cell distinctly and show indirectly whether the basal-cell is integrated. Combining the expression of P53 to determine the existence of cancer gene, it can help to distinguish benign and malignant prostate lesions. PMID- 16683570 TI - [An investigation on AIDS knowledge of Chinese peacekeepers in Liberia and effects of relevant health education]. AB - OBJECTIVE: To investigate and analyze AIDS knowledge of the Chinese peacekeepers in Liberia and to evaluate the effects of AIDS education in order to conduct more effective health education of AIDS and increase AIDS knowledge among the Chinese peacekeepers in the areas with a high incidence of AIDS. METHODS: A questionnaire was designed on the basis of that recommended by WHO to investigate the knowledge of AIDS among the 528 Chinese peacekeepers in Liberia respectively before and after relevant health education, and the results were compared. RESULTS: There was an obvious deficiency in AIDS knowledge among the subjects before the education and an evident increase after it, and the difference was significant (P <0.01). CONCLUSION: The Chinese peacekeepers are deficient in AIDS knowledge, and the deficiency can be effectively improved by relevant health education, which is essential for the health of the Chinese peacekeepers in the areas with a high incidence of AIDS. PMID- 16683571 TI - [Expression and significance of survivin protein, PTEN in prostatic cancer]. AB - OBJECTIVE: To study the expressions of survivin, PTEN and their relationships with tissue grade and pathology stage in prostatic carcinoma (PCa). METHODS: The immunohistological staining was used to evaluated the expressions of survivin protein and PTEN in 43 case of prostatic carcinoma (PCa) and 5 cases of benign prostatic hyperplasia (BPH). RESULTS: The positive rate of survivin protein was 81.40%. Expression of survivin protein in 5 case of BPH was negative. The positive rate of PTEN was 30.23%, and the higher the grade and the clinical stage of tumors were, the lower the expression of PTEN was, PTEN of 5 case of BPH was positive. CONCLUSION: The positively correlation was found between the abnormal expressions of survivin protein, PTEN and the biological behavior of prostatic carcinoma (PCa). Detection of survivin combined with PTEN is valuable for diagnosing PCa, and evaluating malignancy extent and prognosis. PMID- 16683572 TI - [Study of the changes of acrosomal enzyme and nitric oxide synthase, superoxide dismutase of infertile patients with positive antisperm antibody in seminal plasma]. AB - OBJECTIVE: To study the impacts of positive antisperm antibody (AsAb) in seminal plasma on acrosomal enzyme activity, nitric oxide synthase (NOS) and superoxide dismutase (SOD) levels of spermatozoa. METHODS: Swatch from 40 infertile patients with positive AsAb in seminal plasma as experimental group, and 40 fertile men as control group. Acrosomal enzyme activity was detected by the BAEE/ADH unitive method, NOS was detected by the redoxreaction assay, and SOD level was measured xanthine oxidase method. RESULTS: Compared with control group, acrosomal enzyme activity of spermatozoa of experimental group was significantly decreased (P <0.01), NOS activity was apparently increased (P < 0.01), and SOD level in seminal plasma was markedly decreased (P<0.01). CONCLUSION: It may be possible that the positive AsAb in seminal plasma beget infertility through the changes of acrosomal enzyme of spermatozoa, SOD and NOS activities in seminal plasma. PMID- 16683573 TI - [Expression of Bcl-2 and Bax in the impairment of contralateral testes of unilateral cryptorchidism in rats]. AB - OBJECTIVE: To investigate the relationship between germ cell apoptosis and expressions of Bcl-2 and Bax in contralateral testes of experimental unilateral cryptorchid rats. METHODS: Twenty male Sprague-Dawley rats were randomly divided into 2 groups (namely cryptorchid group and control group), with 10 rats in each group. Cryptorchid animal model was established, and contralateral testes were captured 90 days later. The evidence of germ cell apoptosis was detected by TUNEL assay. The expressions of Bcl-2 and Bax were detected by immunohistochemical method. RESULTS: In the contralateral testes of experimental unilateral cryptorchidism, apoptosis index of germ cell and Bax expression significantly increased compared with those in the control group, respectively (P < 0.01), while Bcl-2 expression and testis weight obviously decreased (P <0.01). The apoptotic cells were mostly pachytene spermatocytes and round spermatids. CONCLUSION: The germ cell apoptosis is highly correlated with expressions of Bcl 2 and Bax in contralateral testes of experimental unilateral cryptorchidism. Bcl 2/Bax plays an important role in germ cell apoptosis. PMID- 16683574 TI - [The application of quality control system in reproductive medicine center]. AB - Uniform standards are required for evaluating the quality of reproductive medicine center engaged in vitro-fertilization and embryo transfer. Clinic pregnancy rates in common use are not in evaluation focus. More internationalized standard methods should be used to assess the quality of IVF center. PMID- 16683575 TI - [Proteomics and its application in the research of prostatic carcinoma]. AB - With the development of the proteomics technology, more and more tumorous labeled substrates have been discovered and applied to clinical practice. This can provide the forepart diagnosis of tumor and prognosis monitoring with the reliable evidence. A summary is given of the study strategy of proteomics and its application in the prostatic carcinoma research. PMID- 16683576 TI - [Preliminary experience in treatment of chronic prostatitis/chronic pelvic pain syndrome with Longjintonglin capsule]. AB - OBJECTIVE: To explore the effectiveness of Longjintonglin capsule in treatment of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: One hundred and thirty-six patients accorded with the demands of chronic prostatitis type III of National Institute of Health (NIH), among them there were 84 cases of chronic prostatitis type IIIA and 52 cases of type IIIB. All of the patients accepted oral Longjintonglin capsule for 3 tablets, 3 times per day, for 3 months. NIH- chronic prostatitis symptom index (NIH-CPSI) and counting leukocytes in expressed prostatic secretions (EPS) were used as target to evaluate the effectiveness. RESULTS: One hundred and thirty-one cases accomplished treatment, in which there were 81 cases of type IIIA, and 50 cases of type IIIB. Overall scores of NIH-CPSI before and after treatment were(24.8 +/- 5.6) and (13.3 +/- 6.8) points respectively, which dropped significantly (average 11.5 points), (P < 0.01). The symptom scores before and after treatment were (15.4 +/- 4.3) and (8.7 +/- 3.9) respectively, which also decreased significantly (average 6.7 points), (P <0.01). The scores for quality of life before and after treatment were (9.4 +/- 3.7) and (4.6 +/- 2.7) respectively, which dropped significant (average 4.8 points) (P < 0.01). The leukocytes counts before and after treatment in patients with CP/CPPs type IIIA were (21.7 +/- 14.5)/HP and (8.8 +/- 12.6)/HP, respectively. Twenty four cases(18.3%) got cured, 41 cases (31.3%) notable effect, 40 cases (30.5%) some effect, 26 cases (19.8%) no effect, and total effective rate was 80.1%. No apparent side effect was observed in all patients accepted treatment, especially for their kidney and hepatic function. CONCLUSION: Longjintonglin capsule was a safe and effective medicine in treatment of CP/CPPS without significant side effect. PMID- 16683577 TI - [Efficacy and safety of vardenafil in difficult-to-treat erectile dysfunction men]. AB - This paper reviewed the efficacy and safety of vardenafil, a highly selective phosphodiesterase type 5 (PDE 5) inhibitor, in men with erectile dysfunction who were difficult to treat. Several large-scale studies indicated vardenafil was effective and safe in the treatment of these difficult-to-treat ED patients, including ED with depression or diabetes, ED after radical retropubic prostatectomy, ED caused by spinal cord injury, and sildenafil nonresponders. Vardenafil provides a rational treatment alternative. PMID- 16683578 TI - Dead Daphnia flag mystery water contaminants. PMID- 16683579 TI - A tiny but powerful microbial fuel cell. PMID- 16683580 TI - Perchlorate found in vitamins and elsewhere. PMID- 16683581 TI - Is EPA's Performance Track running off the rails? PMID- 16683582 TI - Bottled antimony. PMID- 16683583 TI - Will climate change worsen infectious diseases? PMID- 16683584 TI - Winter motor-vehicle emissions in Yellowstone National Park. PMID- 16683585 TI - Deriving site-specific 2,2-bis(chlorophenyl)-1,1-dichloroethylene quality criteria of water and sediment for protection of common tern populations in Bohai Bay, North China. AB - In this paper we first present a method of deriving site-specific water and sediment quality criteria of chemicals for protecting wildlife populations. The method has two steps: (1) identification of the threshold concentration in specific tissue corresponding to a population benchmark response and (2) extrapolating quality criteria on the basis of the bioaccumulation in tissue from the water or sediment (e.g., the bioaccumulation factor (BAF) and biota/ sediment accumulation factor (BSAF)). The method was applied to derive 2,2 bis(chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) quality criteria of water and sediment of Bohai Bay, China, for common tern populations. The benchmark concentration in eggs of the common tern was determined to be 663 ng/g wet weight from the relationship curve between concentration (biotic burden in eggs) and the intrinsic rate of population increase (r) calculated by an age-structured matrix approach. The p,p'-DDE concentrations (mean 58.79 and ranging from 13.14 to 326.60 ng/g wet weight) measured in common tern eggs (n = 35), collected from the Beidagang wetland on the coast of Bohai Bay in China, and those in surface water or sediment from literature values were combined to estimate the probability distribution of BAF and BSAF using the Monte Carlo method. Finally, the marine water and sediment quality criteria of p,p'-DDE in Bohai Bay were estimated to be 4 ng/L and 1.9 ng/g dry weight, respectively. PMID- 16683586 TI - Changes in uranium speciation through a depth sequence of contaminated Hanford sediments. AB - The disposal of basic sodium aluminate and acidic U(VI)-Cu(ll) wastes in the now dry North and South 300 A Process Ponds atthe Hanford site resulted in a groundwater plume of U(VI). To gain insight into the geochemical processes that occurred during waste disposal and those affecting the current and future fate and transport of this uranium plume, the solid-phase speciation of uranium in a depth sequence of sediments from the base of the North Process Pond through the vadose zone to groundwater was investigated using standard chemical and mineralogical analyses, electron and X-ray microprobe measurements, and X-ray absorption fine structure spectroscopy. Near-surface sediments contained uranium coprecipitated with calcite, which formed due to overneutralization of the waste ponds with base (NaOH). At intermediate depths in the vadose zone, metatorbernite [Cu(UO2PO4)2 x 8H2O] precipitated, likely during pond operations. Uranium occurred predominantly sorbed onto phyllosilicates in the deeper vadose zone and groundwater; sorbed uranium was also an important component at intermediate depths. Since the calcite-bearing pond sediments have been removed in remediation efforts, uranium fate and transport will be controlled primarily by desorption of the sorbed uranium and dissolution of metatorbernite. PMID- 16683587 TI - Tracing source pollution in soils using cadmium and lead isotopes. AB - Tracing the source of heavy metals in the environment is of key importance for our understanding of their pollution and natural cycles in the surface Earth reservoirs. Up to now, most exclusively Pb isotopes were used to effectively trace metal pollution sources in the environment. Here we report systematic variations of Cd isotope ratios measured in polluted topsoils surrounding a Pb-Zn refinery plant in northern France. Fractionated Cd was measured in soil samples surrounding the refinery, and this fractionation can be attributed to the refining processes. Despite the Cd isotopic ratios being precisely measured, the obtained uncertainties are still large compared to the total isotopic variation. Nevertheless, for the first time, Cd isotopically fractionated by industrial processes may be traced in the environment. On the same samples, Pb isotope systematics suggested that materials actually used by the refinery were not the major source of Pb in soils, probably because refined ore origins changed over the 100 years of operation. On the other hand, Cd isotopes and concentrations measured in topsoils allowed identification of three main origins (industrial dust and slag and agriculture), assuming that all Cd ores are not fractionated, as suggested by terrestrial rocks so far analyzed, and calculation of their relative contributions for each sampling point. Understanding that this refinery context was an ideal situation for such a study, our results lead to the possibility of tracing sources of anthropogenic Cd and better constrain mixing processes, fluxes, transport, and phasing out of industrial input in nature. PMID- 16683588 TI - Comparison of daytime and nighttime concentration profiles and size distributions of ultrafine particles near a major highway. AB - Previously we have conducted systematic measurements of the concentration and size distribution of ultrafine particles in the vicinity of major highways during daytime in Los Angeles. The present study compares these with similar measurements made at night. Particle number concentration was measured by a condensation particle counter (CPC) and size distributions in the size range from 7 to 300 nm were measured by a scanning mobility particle sizer (SMPS). Measurements were taken at 30, 60, 90, 150, and 300 m upwind and downwind from the center of the 1-405 freeway. Average traffic flow at night was about 25% of that observed during the day. Particle number concentration measured at 30 m downwind from the freeway was 80% of previous daytime measurements. This discrepancy between changes in traffic counts and particle number concentrations is apparently due to the decreased temperature, increased relative humidity, and lower wind speed at night. Particle size distributions do not change as dramatically as they did during the daytime. Particle number concentration decays exponentially downwind from the freeway similarly to what was observed during the day, but at a slower rate. No particle number concentration gradient has been observed for the upwind side of the freeway. No PM2.5 and very weak PM10 concentration gradients were observed downwind of thefreeway at night. Ultrafine particle number concentration measured at 300 m downwind from the freeway was still distinguishably higher than upwind background concentration at night. These data may be used to help estimate exposure to ultrafine particles in the vicinity of major highways for epidemiology studies. PMID- 16683589 TI - Metal(loid) diagenesis in mine-impacted sediments of Lake Coeur d'Alene, Idaho. AB - Mining activity along the South Fork of the Coeur d' Alene River in northern Idaho has resulted in fluvial mine tailings enriched in Pb, As, Ag, Sb, Hg, Cd, and Zn deposited on the lakebed of Lake Coeur d'Alene, thus serving as a potential benthic source of inorganic contaminants. Our objective was to characterize the dominant solid phase materials and diagenetic processes controlling metal(loid) solubilities, and thus their potential release to the overlying water column. Aqueous and solid concentrations of metal(loid) contaminants were examined along with distinct species of Fe and S within sediments and interstitial water. A gradient from oxic conditions at the sediment water interface to anoxic conditions below 15 cm exists at all sites, resulting in a dynamic redox environment that controls the partitioning of contaminants. Fluvial deposition from frequent seasonal flood events bury ferric oxides residing at the sediment-water interface leading to reductive dissolution as they transition to the anoxic zone, consequently releasing associated metal(loids) to the interstitial water. Insufficient sulfur limits the formation of sulfidic minerals, but high carbonate content of this mining region buffers pH and promotes formation of siderite. Diagenetic reactions create chemical gradients encouraging the diffusion of metal(loids) toward the sediment--water interface, thereby, increasing the potential for release into the overlying water. PMID- 16683590 TI - Bioavailability of iron sensed by a phytoplanktonic Fe-bioreporter. AB - This study describes a short-term (12 h) evaluation of iron (Fe) bioavailability to an Fe-dependent cyanobacterial bioreporter derived from Synechococcus PCC 7942. Several synthetic ligands with variable conditional stability constants for Fe(lll) (K* of 10(19.8) to 10(30.9)), in addition to several defined natural Fe binding ligands and a fulvic acid of aquatic origin (Suwannee River), were used to elucidate the forms of Fe that are discerned by this phytoplanktonic microbe: Fe-HEBD (log conditional stability constant, K*, = 28.1, HEBD = N,N'-di(2 hydroxybenzyl)ethylenediamine-N,N'-diacetic acid monohydrochloride hydrate), Fe HDFB (K* = 30.9, DFB = desferroxamine B), Fe-ferrichrome (K* = 23.2), Fe-DTPA (K* = 21.1, DTPA = diethylenetrinitrilopentaacetic acid), Fe-(8HQS)2 (K* = 20.4, 8HQS = 8-hydroxyquinoline-5-sulfonic acid), Fe-CDTA (K* = 19.8, CDTA = trans-1,2 cyclohexylenedinitrilotetraacetic acid), and Fe-EDTA (K* = 19.2). Iron bioavailability sensed by the bioreporter was related to diffusion limitation and activity of high-affinity transporters rather than by siderophore secretion. Iron complexed with a K* < 23.2 contributes to the bioavailable pool; bioavailability could be explained by disjunctive ligand exchange considerations and fully, partially, and nonbioavailable complexes could be distinguished according to their conditional stability constant. The use of Fe-bioreporters provides a relevant measurement of bioavailability to an important group of primary producers in freshwaters (cyanobacteria) and is thus a promising technique for understanding Fe cycling in aquatic systems. PMID- 16683591 TI - Partitioning CO2 effluxes from an Atlantic pine forest soil between endogenous soil organic matter and recently incorporated 13C-enriched plant material. AB - Soil CO2 effluxes from recently added 13C-labeled phytomass versus endogenous soil organic matter (SOM) were studied in an acid soil from Atlantic pine forests (NW Spain). After several cultures to incorporate fresh 13C-enriched Lolium perenne to a Humic Cambisol with predominance of humus--Al over humus--Fe complexes, potential soil C mineralization was determined by laboratory aerobic incubation (84 days). Isotopic 13C analyses of SOM fractions were assessed to know in which organic compartments the 13C was preferentially incorporated. Although in the 13C-labeled soil the C mineralization coefficient totalized less than 3% of soil C, the 13C mineralization coefficient exceeded 14%, indicating a greater lability of the newly incorporated organic matter. Organic compounds coming from added phytomass showed a higher lability and contributed considerably to the total soil CO2 effluxes (52% of total soil CO2 evolved during the first decomposition stages and 27% at the end), even though added-C comprised less than 4% of total soil C. Good determination coefficients, when values of CO2--C released were fitted to a first-order double exponential kinetic model, support the existence of two C pools of different lability. Kinetic parameters obtained with this model indicated that phytomass addition augmented the biodegradability of the labile pool (instantaneous mineralization rate k increased from 0.07 d(-1) to 0.12 d(-1)) but diminished that of the recalcitrant pool (instantaneous mineralization rate h decreased from 2.7 x 10(-4) d(-1) to 1.6 x 10(-4) d(-1)). Consequently, the differentiation between both SOM pools increased, showing the importance of SOM quality on CO2 emissions from this kind of soil to the atmosphere. PMID- 16683592 TI - Cyanogen bromide formation from the reactions of monobromamine and dibromamine with cyanide ion. AB - Cyanide ion (CN-) was found to reactwith monobromamine (NH2Br) and dibromamine (NHBr2) according to the reactions NH2Br + CN- + H20 --> NH3 + BrCN + OH- and NHBr2 + CN- + H20 --> NH2Br + BrCN + OH- with respective reaction rate constants of 2.63 x 10(4) M9-10 s(-1) and 1.31 x 10(8) M(-1) s(-1). These values were found to be 10(5)-10(6) times greater than those for the corresponding reactions between chloramine species and CN-. As a result, bromamines, even if present at relatively low concentrations, would tend to outcompete chloramines in reacting with CN-, and thus, the formation of BrCN would predominate that of ClCN through these reaction mechanisms. The NH2Br reaction was found to be general-acid catalyzed. The third-order catalysis rate constants for H+, H2PO4-, HPO4(2-), H3BO3, and NH4+ correlated linearly with their corresponding acid dissociation constants, consistent with the Bronsted-Pedersen relationship. The NHBr2 reaction did not undergo catalysis. A model was developed to predict the concentrations of bromamines over time on the basis ofthe above two reactions with CN- and bromamine formation/decomposition reactions previously reported. PMID- 16683593 TI - Iron-mediated oxidation of antimony(III) by oxygen and hydrogen peroxide compared to arsenic(III) oxidation. AB - Antimony is used in large quantities in a variety of products, though it has been declared as a pollutant of priority interest by the Environmental Protection Agency of the United States (USEPA). Oxidation processes critically affect the mobility of antimony in the environment since Sb(V) has a greater solubility than Sb(lll). In this study, the cooxidation reactions of Sb(lIl) with Fe(ll) and both O2 and H2O2 were investigated and compared to those of As(III). With increasing pH, the oxidation rate coefficients of Sb(lll) in the presence of Fe(ll) and O2 increased and followed a similar pH trend as the Fe(ll) oxidation by O2. Half lives of Sb(lll) were 35 and 1.4 h at pH 5.0 and pH 6.2, respectively. The co oxidation with Fe(ll) and H2O2 is about 7000 and 20 times faster than with Fe(ll) and O2 at pH 3 and pH 7, respectively. For both systems, *OH radicals appear to be the predominant oxidant below approximately pH 4, while at more neutral pH values, other unknown intermediates become important. The oxidation of As(lll) follows a similar pH trend as the Sb(lll) oxidation; however, As(lll) oxidation was roughly 10 times slower and only partly oxidized in most of the experiments. This study shows that the Fe(ll)-mediated oxidation of Sb(Ill) can be an important oxidation pathway at neutral pH values. PMID- 16683594 TI - Reexamination of hexafluorosilicate hydrolysis by 19F NMR and pH measurement. AB - The dissociation of hexafluorosilicate has been reinvestigated due to recent suggestions that fluorosilicate intermediates may be present in appreciable concentrations in drinking water. 19F NMR spectroscopy has been used to search for intermediates in the hydrolysis of hexafluorosilicate. No intermediates were observable at 10(-5) M concentrations under excess fluoride forcing conditions over the pH range of 3.5-5. A single intermediate species, assigned as SiF5(-) or its hydrate, was detected below pH 3.5. At moderate pH values of 4 and 5 silica oligomerization in the solutions studied made it difficult to directly determine the hexafluorosilicate equilibrium constant. Under more acidic conditions the average pKd, or negative log of the dissociation constant Kd, determined by 19F NMR measurements, was 30.6. We also investigated the behavior of hexafluorosilicate in common biological buffer reagents including phosphate/citrate, veronal/HCI buffers, and Ringer's solution. The buffer capacity of all of these systems was found to be insufficient to prevent acidic shifts in pH when hexafluorosilicate was added. The pH change is sufficient explanation for the observed inhibition of acetylcholinesterase that was previously attributed to hexafluorosilicate hydrolysis intermediates. PMID- 16683595 TI - The Prestige oil spill. 2. Enhanced biodegradation of a heavy fuel oil under field conditions by the use of an oleophilic fertilizer. AB - A field bioremediation assay using the oleophilic fertilizer S200 was carried out 10 months after the Prestige heavy fuel-oil spill on a beach of the Cantabrian coast (North Spain). The field survey showed that S200 significantly enhanced the biodegradation rate, particularly of high molecular weight n-alkanes, alkylcyclohexanes, and benzenes, and alkylated PAHs, paralleling the results previously found in vitro. The most significant molecular bioremediation indicators were the depletion of diasteranes and C-27 sterane components. Enhanced isomeric selectivity was also observed within the C1-phenanthrenes and dibenzothiophenes. Through the analysis of some target aliphatic and aromatic hydrocarbons a number of chemical indicators for assessing the efficiency of field bioremediation as well as identifying the source of highly weathered samples collected in the area after the spill are defined. PMID- 16683596 TI - On the relative significance of bacteria for the distribution of polychlorinated biphenyls in Arctic Ocean surface waters. AB - This study presents the first field observations of polychlorinated biphenyls (PCB) in bacteria in oceanic waters. To contribute to the limited knowledge of what role bacteria play in the dynamics of hydrophobic organic contaminants (HOCs) in surface seawater, PCB concentrations were measured in bacteria (0.2-2 microm) collected at seven stations in the northern Barents Sea marginal ice zone (MIZ) and the central Arctic Ocean. Concentrations of individual PCB congeners in bacteria were 0.5-5 ng/g oc (organic carbon), which was as high as or higher than PCB concentrations in bulk particulate organic carbon (POC, "phytoplankton"; > 0.7 microm). Considering the relative biomasses of phytoplankton and bacteria, the amount of PCB in bacteria was generally 5-20% of that in phytoplankton, but at two stations the bacterial biomass contained more PCBs than the phytoplankton pool. This study further showed that efficient PCB uptake in bacteria may be described by an apparent equilibrium partitioning model with linear regressions between the organic-carbon-normalized partition coefficient and the octanol-water partition coefficient (log K(bact-oc)-log K(ow)). PMID- 16683597 TI - Assessment of the reproductive-endocrine disrupting potential of chlorine dioxide oxidation products of plant sterols. AB - This study examined the hypothesis that chlorine dioxide bleaching used in pulp and paper production causes the formation of reproductive-endocrine disrupting compounds from plant sterols. This was tested by conducting a laboratory simulation of the chlorine dioxide oxidation of two plant sterols, beta sitosterol and stigmasterol. Oxidation products of the plant sterol beta sitosterol were purified and identified and found to be cholestan-24-ethyl-3-one, 4-cholestene-24-ethyl-3-one, and 4-cholestene-24-ethyl-3,6-dione. The first two compounds were found in a number of pulp and paper effluents and biosolids. The sterols and their oxidation products were tested in vitro using bioassays for androgenicity and estrogenicity. A 28 d in vivo bioassay was employed to examine masculinization in female mosquitofish. In vitro bioassays revealed little estrogenic activity in the parent sterols or in mixtures of their oxidation products. Androgenic activity as measured by the androgen receptor binding bioassay was in the order of 19-96 microg/g testosterone equivalents but with no increase or decrease with chlorine dioxide oxidation. The mosquitofish bioassay did not show significant masculinization for any of the preparations tested. A number of androstane steroids were identified in the sterols tested, however, those compounds could only account for a small fraction of the androgenic activity in the sterols. It was clear that the parent sterols were not themselves acting as androgens in the bioassays used. This study indicated that chlorine dioxide oxidation of sterols produced predominantly oxidized sterols that were not likely to act through androgenic or estrogenic mechanisms. PMID- 16683598 TI - Phylogenetic and functional biomakers as indicators of bacterial community responses to mixed-waste contamination. AB - Few studies have demonstrated changes in community structure along a contaminant plume in terms of phylogenetic, functional, and geochemical changes, and such studies are essential to understand how a microbial ecosystem responds to perturbations. Clonal libraries of multiple genes (SSU rDNA, nirK, nirS, amoA, pmoA, and dsrAB) were analyzed from groundwater samples (n = 6) that varied in contaminant levels, and 107 geochemical parameters were measured. Principal components analyses (PCA) were used to compare the relationships among the sites with respect to the biomarker (n = 785 for all sequences) distributions and the geochemical variables. A major portion of the geochemical variance measured among the samples could be accounted for by tetrachloroethene, 99Tc, No3, SO4, Al, and Th. The PCA based on the distribution of unique biomarkers resulted in different groupings compared to the geochemical analysis, but when the SSU rRNA gene libraries were directly compared (deltaC(xy) values) the sites were clustered in a similar fashion compared to geochemical measures. The PCA based upon functional gene distributions each predicted different relationships among the sites, and comparisons of Euclidean distances based upon diversity indices for all functional genes (n = 432) grouped the sites by extreme or intermediate contaminant levels. The data suggested that the sites with low and high perturbations were functionally more similar than sites with intermediate conditions, and perhaps captured the overall community structure better than a single phylogenetic biomarker. Moreover, even though the background site was phylogenetically and geochemically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site. An understanding of microbial community-level responses within an ecological framework would provide better insight for restoration strategies at contaminated field sites. PMID- 16683599 TI - Strontium speciation during reaction of kaolinite with simulated tank-waste leachate: bulk and microfocused EXAFS analysis. AB - Radioactive strontium (90Sr) is an important constituent of the complex wastes from past nuclear weapons production and has been stored in underground tanks at U.S. DOE sites (e.g., Hanford, WA). Using bulk and microfocused EXAFS spectroscopy, we examined temporal changes in solid-phase Sr speciation in kaolinite samples reacted for 1-369 d with high-pH, high ionic strength synthetic tank-waste leachate containing Sr(2+) and Cs(+) at 10(-3) mol kg(-1). Analyses of bulk EXAFS spectra showed that Sr initially forms a precipitate by 7 d with a local structure similar to SrCO(3-) (s). At 33 d, microfocused EXAFS of individual particles in one sample revealed a mixture of hydrated and dehydrated Sr associated with neoformed sodalite-type phases. At aging times of 93 d and longer, bulk EXAFS spectra and supporting characterizations indicated nonexchangeable Sr with a local structure consistent with incorporation into increasingly crystalline aluminosilicate particles, particularly sodalite. These experimental studies suggest that irreversible trapping of radionuclides occurs if they are present during the formation and aging of feldspathoid alteration products of local Si-bearing sediment minerals. This may serve as an effective contaminant sequestration mechanism at sites such as Hanford. PMID- 16683600 TI - Triclosan reactivity in chloraminated waters. AB - Triclosan, widely employed as an antimicrobial additive in many household personal care products, has recently been detected in wastewater treatment plant effluents and in source waters used for drinking water supplies. Chloramines used either as alternative disinfectants in drinking water treatment or formed during chlorination of nonnitrified wastewater effluents have the potential to react with triclosan. This study examined triclosan reactivity in chloraminated waters over the pH range of 6.5-10.5. Experimental and modeling results show that monochloramine directly reacts with the phenolate form of triclosan; however, the reaction is relatively slow as evinced by the second-order rate constant k(ArO) NH2Cl = 0.025 M(-1) s(-1). Kinetic modeling indicates that for pH values less than 9.5, reactions between triclosan and two monochloramine autodecomposition intermediates, hypochlorous acid (k(ArO)-HOC = 5.4 x 10(3) M(-1) s(-1)) and dichloramine (k(ArO)-NHCl2 = 60 M(-1) s(-1)), are responsible for a significant percentage of the observed triclosan decay. The products of these reactions include three chlorinated triclosan byproducts as well as 2,4-dichlorophenol and 2,4,6-trichlorophenol. Low levels of chloroform were detected after 1 week at pH values of 6.5 and 7.5. The slow reactivity of triclosan in the presence of chloramines explains the recalcitrance of this species in nonnitrified wastewater effluents. PMID- 16683601 TI - Complexation of copper(ll) in organic soils and in dissolved organic matter- EXAFS evidence for chelate ring structures. AB - Associations with functional groups of natural organic matter (NOM) are of great importance for bioavailability, toxicity, and mobility of trace metals in soils and waters. In this study, the coordination chemistry of copper, Cu(ll), in organic soils and dissolved organic matter (DOM) from soils and streams was investigated by extended X-ray absorption fine structure (EXAFS) spectroscopy. In both soil organic matter (SOM) and DOM (990-11 000 microg Cu g(-1) dryweight, pH 2.8-6.3), Cu(ll) was coordinated by 4 oxygen/ nitrogen (O/N) atoms at a distance of 1.92-1.95 angstroms in the first coordination shell. These four atoms are positioned in the equatorial plane of a Jahn-Teller distorted octahedron. In samples with a pH of 4.8-6.3, a second coordination shell with 2.0-3.8 C atoms was located at a distance of 2.76-2.86 angstroms. A significant improvement (19 39%) of the fit was obtained by including a third coordination shell with 2.0-3.8 O/C atoms involved in single scattering at an average distance of 3.69 angstroms and multiple scattering at an average distance of 4.19 angstroms. Our results provide evidence for inner-sphere complexation of Cu(ll) in NOM and suggest that Cu(ll) is complexed by either one or two five-membered chelate rings involving possible combinations of amino, carboxyl, or carbonyl functional groups. Ion activity measurements showed that less than 0.2% of total Cu was in the form of free Cu2+ in our samples at pH 4.8-6.3. PMID- 16683602 TI - High power density from a miniature microbial fuel cell using Shewanella oneidensis DSP10. AB - A miniature microbial fuel cell (mini-MFC) is described that demonstrates high output power per device cross-section (2.0 cm2) and volume (1.2 cm3). Shewanella oneidensis DSP10 in growth medium with lactate and buffered ferricyanide solutions were used as the anolyte and catholyte, respectively. Maximum power densities of 24 and 10 mW/m2 were measured using the true surface areas of reticulated vitreous carbon (RVC) and graphite felt (GF) electrodes without the addition of exogenous mediators in the anolyte. Current densities at maximum power were measured as 44 and 20 mA/m2 for RVC and GF, while short circuit current densities reached 32 mA/m2 for GF anodes and 100 mA/m2 for RVC. When the power density for GF was calculated using the cross sectional area of the device or the volume of the anode chamber, we found values (3 W/m2, 500 W/m3) similar to the maxima reported in the literature. The addition of electron mediators resulted in current and power increases of 30-100%. These power densities were surprisingly high considering a pure S. oneidensis culture was used. We found that the short diffusion lengths and high surface-area-to-chamber volume ratio utilized in the mini-MFC enhanced power density when compared to output from similar macroscopic MFCs. PMID- 16683603 TI - Coupled partitioning, dilution, and chemical aging of semivolatile organics. AB - A unified framework of semi-volatile partitioning permits models to efficiently treat both semi-volatile primary emissions and secondary organic aerosol production (SOA), and then to treat the chemical evolution (aging) of the aggregate distribution of semi-volatile material. This framework also reveals critical deficiencies in current emissions and SOA formation measurements. The key feature of this treatment is a uniform basis set of saturation vapor pressures spanning the range of ambient organic saturation concentrations, from effectively nonvolatile material at 0.01 microg m(-3) to vapor-phase effluents at 100 mg m(-3). Chemical evolution can be treated by a transformation matrix coupling the various basis vectors. Using this framework, we show that semi volatile partitioning can be described in a self-consistent way, with realistic behavior with respect to temperature and varying organic aerosol loading. The time evolution strongly suggests that neglected oxidation of numerous "intermediate volatility" vapors (IVOCs, with saturation concentrations above approximately 1 mg m(-3)) may contribute significantly to ambient SOA formation. PMID- 16683604 TI - A dynamic model to study the exchange of gas-phase persistent organic pollutants between air and a seasonal snowpack. AB - An arctic snow model was developed to predict the exchange of vapor-phase persistent organic pollutants between the atmosphere and the snowpack over a winter season. Using modeled meteorological data simulating conditions in the Canadian High Arctic, a single-layer snowpack was created on the basis of the precipitation rate, with the snow depth, snow specific surface area, density, and total surface area (TSA) evolving throughout the annual time series. TSA, an important parameter affecting the vapor-sorbed quantity of chemicals in snow, was within a factor of 5 of measured values. Net fluxes for fluorene, phenanthrene, PCB-28 and -52, and alpha- and gamma-HCH (hexachlorocyclohexane) were predicted on the basis of their wet deposition (snowfall) and vapor exchange between the snow and atmosphere. Chemical fluxes were found to be highly dynamic, whereby deposition was rapidly offset by evaporative loss due to snow settling (i.e., changes in TSA). Differences in chemical behavior over the course of the season (i.e., fluxes, snow concentrations) were largely dependent on the snow/air partition coefficients (K(sa)). Chemicals with relatively higher K(sa) values such as alpha- and gamma-HCH were efficiently retained within the snowpack until later in the season compared to fluorene, phenathrene, and PCB-28 and -52. Average snow and air concentrations predicted by the model were within a factor of 5-10 of values measured from arctic field studies, but tended to be overpredicted for those chemicals with higher K(sa) values (i.e., HCHs). Sensitivity analysis revealed that snow concentrations were more strongly influenced by K(sa) than either inclusion of wind ventilation of the snowpack or other changes in physical parameters. Importantly, the model highlighted the relevance of the arctic snowpack in influencing atmospheric concentrations. For the HCHs, evaporative fluxes from snow were more pronounced in April and May, toward the end of the winter, providing evidence that the snowpack plays an important role in influencing the seasonal increase in air concentrations for these compounds at this time of year. PMID- 16683605 TI - Modeling flow around bluff bodies and predicting urban dispersion using large eddy simulation. AB - Modeling air pollutant transport and dispersion in urban environments is especially challenging due to complex ground topography. In this study, we describe a large eddy simulation (LES) tool including a new dynamic subgrid closure and boundary treatment to model urban dispersion problems. The numerical model is developed, validated, and extended to a realistic urban layout. In such applications fairly coarse grids must be used in which each building can be represented using relatively few grid-points only. By carrying out LES of flow around a square cylinder and of flow over surface-mounted cubes, the coarsest resolution required to resolve the bluff body's cross section while still producing meaningful results is established. Specifically, we perform grid refinement studies showing that at least 6-8 grid points across the bluff body are required for reasonable results. The performance of several subgrid models is also compared. Although effects of the subgrid models on the mean flow are found to be small, dynamic Lagrangian models give a physically more realistic subgrid scale (SGS) viscosity field. When scale-dependence is taken into consideration, these models lead to more realistic resolved fluctuating velocities and spectra. These results set the minimum grid resolution and subgrid model requirements needed to apply LES in simulations of neutral atmospheric boundary layer flow and scalar transport over a realistic urban geometry. The results also illustrate the advantages of LES over traditional modeling approaches, particularly its ability to take into account the complex boundary details and the unsteady nature of atmospheric boundary layer flow. Thus LES can be used to evaluate probabilities of extreme events (such as probabilities of exceeding threshold pollutant concentrations). Some comments about computer resources required for LES are also included. PMID- 16683606 TI - Adjoint sensitivity analysis for a three-dimensional photochemical model: implementation and method comparison. AB - Photochemical air pollution forms when emissions of nitrogen oxides (NO(x)) and volatile organic compounds (VOC) react in the atmosphere in the presence of sunlight. The goal of applying three-dimensional photochemical air quality models is usually to conduct sensitivity analysis: for example, to predict changes in an ozone response due to changes in NO(x) and VOC emissions or other model data. Forward sensitivity analysis methods are best suited to investigating sensitivities of many model responses to changes in a few inputs or parameters. Here we develop a continuous adjoint model and demonstrate an adjoint sensitivity analysis procedure that is well-suited to the complementary case of determining sensitivity of a small number of model responses to many parameters. Sensitivities generated using the adjoint method agree with those generated using other methods. Compared to the forward method, the adjoint method had large disk storage requirements but was more efficient in terms of computer processor time for receptor-based investigations focused on a single response at a specified site and time. The adjoint method also generates sensitivity apportionment fields, which reveal when and where model data are important to the target response. PMID- 16683607 TI - Modeling semivolatile organic aerosol mass emissions from combustion systems. AB - Experimental measurements of gas-particle partitioning and organic aerosol mass in diluted diesel and wood combustion exhaust are interpreted using a two component absorptive-partitioning model. The model parameters are determined by fitting the experimental data. The changes in partitioning with dilution of both wood smoke and diesel exhaust can be described by two lumped compounds in roughly equal abundance with effective saturation concentrations of approximately 1600 microg m(-3) and approximately 20 microg m(-3). The model is used to investigate gas-particle partitioning of emissions across a wide range of atmospheric conditions. Under the highly dilute conditions found in the atmosphere, the partitioning of the emissions is strongly influenced by the ambient temperature and the background organic aerosol concentration. The model predicts large changes in primary organic aerosol mass with varying atmospheric conditions, indicating that it is not possible to specify a single value for the organic aerosol emissions. Since atmospheric conditions vary in both space and time, air quality models need to treat primary organic aerosol emissions as semivolatile. Dilution samplers provide useful information about organic aerosol emissions; however, the measurements can be biased relative to atmospheric conditions and constraining predictions of absorptive-partitioning models requires emissions data across the entire range of atmospherically relevant concentrations. PMID- 16683608 TI - HPLC-DAD and Q-TOF MS techniques identify cause of Daphnia biomonitor alarms in the River Meuse. AB - Several water companies in The Netherlands use a combination of specifically targeted compound analysis (HPLC-UV and GC-MS) and effect monitoring (continuous biotests) to monitor source water quality and to screen for unknown compounds. In spring 2004, the Daphnia biomonitor at Keizersveer monitoring station alongside the River Meuse recorded several alarms. In this study, the combination of HPLC DAD and Q-TOF MS techniques was used to identify the so-far unknown microcontaminant related to this Daphnia alarm as 3-cyclohexyl-1,1-dimethylurea. The maximum concentration of this compound in the River Meuse at the time of the alarm was estimated to be 5 microg/L. The response of the waterfleas to this compound was confirmed with a short-term and a long-term verification test. The origin of the pollutant is still unknown. This paper shows that the combined application of on-line continuous biotests and advanced chemical analysis is an effective tool for the detection and identification of unknown, potentially hazardous compounds for surface water quality monitoring. Biological effect monitoring and specific compound analysis complement each other and together provide the best possible insight in rapid surface water quality changes. PMID- 16683609 TI - Fractionation mechanisms of rare earth elements (REEs) in hydroponic wheat: an application for metal accumulation by plants. AB - Fractionations of rare earth elements (REEs) in wheat (Triticum aestivum L.) were observed through application of exogenous mixed REEs under hydroponic conditions. Middle REE (MREE), light REE (LREE), and heavy REE (HREE) enrichments were found in roots, stems, and leaves, respectively, accompanied by the tetrad effect (an effect that can cause a split of REE patterns into four consecutive segments) in these organs. Investigations into REE speciation in roots and in the xylem sap with X-ray absorption spectroscopy (XAS) and nanometer-sized TiO2 adsorption techniques, associated with other controlled experiments, demonstrated that REE fractionations in wheat were caused by the combined effects of chemical precipitation, cell wall absorption, and solution complexation by organic ligands in the xylem vessels. REE fractionations in wheat, which were derived from the small differences of chemical properties across REE series, may reflect a sensitive internal chemical environment that influences plant accumulation for REEs and their analogues actinide radionuclides. PMID- 16683610 TI - Web-accessible scientific workflow system for performance monitoring. AB - We describe the design and implementation of a web-accessible scientific workflow system for environmental performance monitoring. This workflow environment integrates distributed automated data acquisition with server side data management and information visualization through flexible browser-based data access tools. Component technologies include a rich browser-based client, a back end server for methodical data processing, user management, and result delivery, and third party applications which are invoked by the back-end using web services. This environment allows for reproducible, transparent result generation by a diverse user base, and provides a seamless integration between data selection, analysis applications, and result delivery. This workflow system has been implemented for several sites and monitoring systems with different degrees of complexity. PMID- 16683611 TI - Permeability of collapsed cakes formed by deposition of fractal aggregates upon membrane filtration. AB - We have investigated, theoretically, the physical properties of cake layers formed from aggregates to obtain a better understanding of membrane systems used in conjunction with coagulation/flocculation pretreatment. We developed a model based on fractal theory and incorporated a cake collapse effect to predict the porosity and permeability of the cake layers. The floc size, fractal dimension, and transmembrane pressure were main parameters that we used in these model calculations. We performed experiments using a batch cell device and a confocal laser-scanning microscope to verify the predicted specific cake resistances and porosities under various conditions. Based on the results of the model, the reduction in inter-aggregate porosity is more important than that in intra aggregate porosity during the cake collapsing process. The specific cake resistance decreases upon increasing the aggregate size and decreasing the fractal dimensions. The modeled porosities and specific cake resistances of the collapsed cake layer agreed reasonably well with those obtained experimentally. PMID- 16683612 TI - Membrane fouling of submerged membrane bioreactors: impact of mean cell residence time and the contributing factors. AB - In this study, four bench-scale pre-anoxic submerged membrane bioreactors (MBR) were operated simultaneously at different mean cell residence times (MCRTs) (3, 5, 10, and 20 days) to systematically elucidate the contributing factors of membrane fouling. Severe membrane fouling was first observed in the 3-day followed by the 5-day MCRT MBRs. Minimal membrane fouling was detected in the 10 and 20-day MCRT MBRs. The fouling of microfiltration membrane was not controlled by mixed liquor suspended solids concentration or zeta potential of biomass. Instead, membrane fouling rate increased with increasing soluble microbial products and extracellular polymeric substances concentrations, which both increased with decreasing MCRT. Total organic carbon, protein, carbohydrate, and UV254 absorbance in the mixed liquor supernatant increased with decreasing MCRT and were consistently higher than those of the effluent. Accumulation of carbohydrates rather than proteins in the mixed liquor supernatant was found to decrease with increasing MCRT. Normalized capilliary suction time value rather than the capilliary suction time value would indicate membrane fouling potential of a mixed liquor. Image analysis of the fouled membrane using scanning electron microscope and confocal laser scanning microscope showed that biofilm formation was the cause of membrane fouling. PMID- 16683613 TI - Development of cost-effective noncarbon sorbents for Hg(0) removal from coal fired power plants. AB - Noncarbonaceous materials or mineral oxides (silica gel, alumina, molecular sieves, zeolites, and montmorillonite) were modified with various functional groups such as amine, amide, thiol, urea, and active additives such as elemental sulfur, sodium sulfide, and sodium polysulfide to examine their potential as sorbents for the removal of elemental mercury (Hg(0)) vapor at coal-fired utility power plants. A number of sorbent candidates such as amine- silica gel, urea- silica gel, thiol- silica gel, amide-silica gel, sulfur-alumina, sulfur-molecular sieve, sulfur-montmorillonite, sodium sulfide-montmorillonite, and sodium polysulfide-montmorillonite, were synthesized and tested in a lab-scale fixed-bed system under an argon flow for screening purposes at 70 degrees C and/or 140 degrees C. Several functionalized silica materials reported in previous studies to effectively control heavy metals in the aqueous phase showed insignificant adsorption capacities for Hg(0) control in the gas phase, suggesting that mercury removal mechanisms in both phases are different. Among elemental sulfur-, sodium sulfide-, and sodium polysulfide-impregnated inorganic samples, sodium polysulfide-impregnated montmorillonite K 10 showed a moderate adsorption capacity at 70 degrees C, which can be used for sorbent injection prior to the wet FGD system. PMID- 16683614 TI - RCRA toxicity characterization of discarded electronic devices. AB - The potential for discarded electronic devices to be classified as toxicity characteristic (TC) hazardous waste under provisions of the Resource Conservation and Recovery Act (RCRA) using the toxicity characteristic leaching procedure (TCLP) was examined. The regulatory TCLP method and two modified TCLP methods (in which devices were disassembled and leached in or near entirety) were utilized. Lead was the only element found to leach at concentrations greater than its TC limit (5 mg/L). Thirteen different types of electronic devices were tested using either the standard TCLP or modified versions. Every device type leached lead above 5 mg/L in at least one test and most devices leached lead above the TC limit in a majority of cases. Smaller devices that contained larger amounts of plastic and smaller amounts of ferrous metal (e.g., cellular phones, remote controls) tended to leach lead above the TC limit at a greater frequency than devices with more ferrous metal (e.g., computer CPUs, printers). PMID- 16683615 TI - Platinum particle size and support effects in NO(x) mediated carbon oxidation over platinum catalysts. AB - Platinum metal was dispersed on microporous, mesoporous, and nonporous support materials including the zeolites Na-Y, Ba-Y, Ferrierite, ZSM-22, ETS-10, and AIPO 11, alumina, and titania. The oxidation of carbon black loosely mixed with catalyst powder was monitored gravimetrically in a gas stream containing nitric oxide, oxygen, and water. The carbon oxidation activity of the catalysts was found to be uniquely related to the Pt dispersion and little influenced by support type. The optimum dispersion is around 3-4% corresponding to relatively large Pt particle sizes of 20-40 nm. The carbon oxidation activity reflects the NO oxidation activity of the platinum catalyst, which reaches an optimum in the 20-40 nm Pt particle size range. The lowest carbon oxidation temperatures were achieved with platinum loaded ZSM-22 and AIPO-11 zeolite crystallites bearing platinum of optimum dispersion on their external surfaces. PMID- 16683616 TI - Treatment of volatile organic chemicals on the EPA Contaminant Candidate List using ozonation and the O3/H2O2 advanced oxidation process. AB - Seven volatile organic chemicals (VOCs) on the EPA Contaminant Candidate List together with 1,1-dichloropropane were studied for their reaction kinetics and mechanisms with ozone and OH radicals during ozonation and the ozone/ hydrogen peroxide advanced oxidation process (O3/H2O2 AOP) using batch reactors. The three aromatic VOCs demonstrated high reactivity during ozonation and were eliminated within minutes after ozone addition. The high reactivity is attributed to their fast, indirect OH radical reactions with k(OH,M) of (5.3-6.6) x 10(9) M(-1) s( 1). Rates of aromatic VOC degradation are in the order 1,2,4-trimethylbenzene > p cymene > bromobenzene. This order is caused by the selectivity of the direct ozone reactions (k(O3,M) ranges from 0.16 to 304 M(-1) s(-1)) and appears to be related to the electron-donating or -withdrawing ability of the substituent groups on the aromatic ring. The removal rates for the five aliphatic VOCs are much lower and are in the order 1,1-dichloropropane > 1,3-dichloropropane > 1,1 dichloroethane > 2,2-dichloropropane > 1,1,2,2-tetrachloroethane. The second order indirect rate constants for the aliphatic VOCs range from 0.52 x 10(8) to 5.5 x 10(8) M(-1) s(-1). The relative stability of the carbon-centered intermediates seems to be related to the relative reactivity of the aliphatic VOCs with OH radicals. Except for 1,3-dichloropropane, ozonation and the O3/H2O2 AOP are not effective for the removal of other aliphatic VOCs. Bromide formation during the ozonation of bromobenzene indicates that bromate can be formed, and thus, ozonation and O3/H2O2 AOP may not be suitable for the treatment of bromobenzene. PMID- 16683617 TI - Phosphate removal from wastewaters using a weak anion exchanger prepared from a lignocellulosic residue. AB - Surface modifications of lignocellulosic residues has become increasingly important for improving their applications as adsorbents. In this study a new adsorbent system (BS-DMAHP) containing dimethylaminohydroxypropyl (DMAHP) weak base groups was prepared by the reaction of banana stem (BS), a lignocellulosic residue with epichlorohydrin and dimethylamine followed by treatment of hydrochloric acid. The original BS and BS-DMAHP were characterized with the help of surface area analyzer, infrared spectroscopy (IR) and scanning electron microscopy (SEM). Surface charge density of the samples as a function of pH was investigated using potentiometric titrations. Adsorbent exhibits very high adsorption potential for phosphate and more than 99.0% removal was achieved in the pH range of 5.0-7.0. Adsorption has been found to be concentration dependent and endothermic and follows a reversible second-order kinetics. The Langmuir and Freundlich isotherm models were applied to describe the equilibrium data. Equilibrium data agreed very well with the Langmuir model. Adsorption experiments were conducted using a commercial chloride form Duolite A-7, a weak base anion exchanger. The removal efficiency was tested using fertilizer industry wastewater. Adsorbed phosphate on BS-DMAHP can be recovered by treating with 0.1 M NaOH solution. A stability test operated for four cycles indicate a capacity loss of < 12.0%. PMID- 16683618 TI - Micellar enhanced ultrafiltration for arsenic(V) removal: effect of main operating conditions and dynamic modelling. AB - In this work arsenic removal by micellar enhanced ultrafiltration (MEUF) was investigated using cetylpyridinium chloride (CPC) in ceramic membrane apparatus. Permeability tests and discontinuous diafiltration tests were performed in different operating conditions to evaluate the effect of membrane pore size (20 and 50 nm), transmembrane pressure, pH, surfactant concentration (1-3 mM), and arsenic concentration (10-40 mg/L) on permeate flux decline, arsenic, and CPC rejections. These preliminary experimental results showed that a ceramic membrane with large pore size allows treament of high fluxes of concentrated arsenic bearing solutions even by using low surfactant concentrations. Arsenic concentration in the permeate was at the 1 ppm level, with feed As concentrations (10 ppm) that are larger than those generally used in MEUF studies and with CPC amounts that are lower than the usual ones. In addition, operating conditions adopted in these tests obtained CPC concentrations in the permeate always lower than its critical micellar concentration (0.9 mM). Dynamic simulations of discontinuous two-step diafiltration tests allowed a simple and adequate representation of the performance of the process especially for 1 mM CPC, while discrepancies for 2.5 mM CPC level denoted complex interactions between CPC and As. PMID- 16683619 TI - Uptake of metals during chelant-assisted phytoextraction with EDDS related to the solubilized metal concentration. AB - The use of chelants to enhance phytoextraction is one method being tested to make phytoextraction efficient enough to be used as a remediation technique for heavy metal pollution in the field. We performed pot experiments with sunflowers in order to investigate the use of the biodegradable chelating agent SS-EDDS for this purpose. We used singly and combined contaminated soils (Cu, Zn) and multimetal contaminated field soils (Cu, Zn, Cd, Pb). EDDS (10 mmol kg(-10 soil) increased soil solution metals greatly for Cu (factor 840-4260) and Pb (factor 100-315), and to a lesser extent for Zn (factor 23-50). It was found that Zn (when present as the sole metal), Cu, and Pb uptake by sunflowers was increased by EDDS, butin multimetal contaminated soil Zn and Cd were not. EDDS was observed in the sunflower roots and shoots at concentrations equal to metal uptake. The different metal uptake in the various soils can be related to a linear relationship between Cu and Zn in soil solution in the presence of EDDS and plant uptake, indicating the great importance of measuring and reporting soil solution metal concentrations in phytoextraction studies. PMID- 16683620 TI - Inactivation of biological agents using neutral oxone-chloride solutions. AB - Bleach solutions containing the active ingredient hypochlorite (OCl-) serve as powerful biological disinfectants but are highly caustic and present a significant compatibility issue when applied to contaminated equipment or terrain. A neutral, bicarbonate-buffered aqueous solution of Oxone (2K2HSO5.KHSO4.K2SO4) and sodium chloride that rapidly generates hypochlorite and hypochlorous acid (HOCl) in situ was evaluated as a new alternative to bleach for the inactivation of biological agents. The solution produced a free chlorine (HOCl + OCl-) concentration of 3.3 g/L and achieved > or =5.8-log inactivation of spores of Bacillus atrophaeus, Bacillus thuringiensis, Aspergillus niger, and Escherichia coli vegetative cells in 1 min at 22 degrees C. Seawaterwas an effective substitute for solid sodium chloride and inactivated 5 to 8 logs of each organism in 10 min over temperatures ranging from -5 degrees C to 55 degrees C. Sporicidal effectiveness increased as free chlorine concentrations shifted from OCl- to HOCl. Neutrally buffered Oxone-chloride and Oxone-seawater solutions are mitigation alternatives for biologically contaminated equipment and environments that would otherwise be decontaminated using caustic bleach solutions. PMID- 16683621 TI - Reduction of acrolein by elemental iron: kinetics, pH effect, and detoxification. AB - Acrolein is a highly toxic alpha,beta-unsaturated aldehyde that is widely used as a biocide, a cross-linking agent, and an intermediate in the chemical industry, among other applications. In this study we investigated the reductive transformation of acrolein by elemental iron and evaluated the feasibility of using iron to detoxify acrolein. At acidic and neutral pH, acrolein was transformed by iron through reduction of the C=C double bond to propionaldehyde. The reduction appeared to involve the chemisorption of acrolein to the iron surface followed by reduction of adsorbed acrolein. Both the adsorption and reduction rate constants decreased with increasing pH. Between pH 7.0 and 7.4, the acrolein adsorption rate constant decreased precipitously, resulting in a sharp decline in its removal rate. At higher pH, acrolein disappeared rapidly in control without iron, presumably due to reversible, base-catalyzed hydration. At equilibrium, approximately 93% of acrolein was hydrated, corresponding to an equilibrium constant of 13. Acrolein at 25 mg/L completely inhibited aerobic respiration; in contrast, its reduction product propionaldehyde was biodegradable. This suggests that elemental iron may be used to pretreat acrolein containing wastes prior to aerobic biodegradation. To our knowledge, this is the first report of reduction and detoxification of an alpha,beta-unsaturated aldehyde by elemental iron. PMID- 16683622 TI - Organic/inorganic hybrid filters based on dendritic and cyclodextrin "nanosponges" for the removal of organic pollutants from water. AB - Long-alkyl chain functionalized poly(propylene imine) dendrimer, poly(ethylene imine) hyperbranched polymer, and beta-cyclodextrin derivatives, which are completely insoluble in water, have the property of encapsulating organic pollutants from water. Ceramic porous filters can be impregnated with these compounds resulting in hybrid organic/ inorganic filter modules. These hybrid filter modules were tested for the effective purification of water, by continuous filtration experiments, employing a variety of water pollutants. It has been established that polycyclic aromatic hydrocarbons (PAHs) can be removed very efficiently (more than 95%), and final concentrations of several ppb (microg/ L) are easily obtained. Representatives of the pollutant group of trihalogen methanes (THMs), monoaromatic hydrocarbons (BTX), and pesticides (simazine) can also be removed (>80%), although the filters are saturated considerably faster in these cases. PMID- 16683623 TI - Effect of Fenton reagent dose on coexisting chemical and microbial oxidation in soil. AB - The ability of modified Fenton reactions to promote simultaneous chemical and biological oxidation in an artificially contaminated soil was studied in batch laboratory slurry reactors. Tetrachloroethene (PCE) and oxalate (OA) were used to distinguish chemical oxidation from aerobic heterotrophic metabolism. PCE was mineralized by Fenton reactions, but OA was not oxidized. Indigenous soil microorganisms did not degrade added PCE aerobically but readily assimilated OA. Fenton reactions were promoted at the natural soil pH (7.6) by adding H2O2 and Fe(III), with nitrilotriacetic acid (NTA) as a chelator, at a constant molar ratio of H2O2/Fe(III)/NTA of 50:1:1. The *OH-mediated mineralization of PCE was demonstrated by adding 2-propanol (an *OH scavenger), which inhibited PCE oxidation. In subsequent dosing studies, PCE oxidation served as an indicator of Fenton reactions, while OA assimilation, dissolved oxygen (DO) concentration, and heterotrophic plate counts were indicators of aerobic microbial activity. Increasing Fenton doses to 20 times that required to achieve 95% PCE oxidation only delayed OA assimilation by 500 min and reduced plate counts by 1.5 log units g(-1) soil. Results show that aerobic metabolism can coexist with Fenton oxidation in soils. PMID- 16683624 TI - New Orleans soil lead (Pb) cleanup using Mississippi River alluvium: need, feasibility, and cost. AB - In New Orleans, LA prior to hurricane Katrina 20-30% of inner-city children had elevated blood Pb levels > or =10 microg/ dL and 10 census tracts had a median surface soil level of Pb >1000 mg/kg (2.5 times the U.S. standard). This project tests the feasibility of transporting and grading contaminated properties (n = 25) with 15 cm (6 in.) of clean Mississippi River alluvium from the Bonnet Carre Spillway (BCS) (median soil Pb content 4.7 mg/kg; range 1.7-22.8). The initial median surface soil Pb was 1051 mg/kg (maximum 19 627). After 680 metric tons (750 tons) of clean soil cover was emplaced on 6424 m2 (69 153 ft2), the median surface soil Pb decreased to 6 mg/kg (range 3-18). Interior entrance wipe samples were collected at 10 homes before and after soil treatment and showed a decreasing trend of Pb (p value = 0.048) from a median of 52 microg/ft2 to a median of 36 microg/ft2 (25th and 75th percentiles are 22 and 142 microg/ft2 and 12 and 61 microg/ft2, respectively). Average direct costs for properties with homes were $3,377 ($1.95 per square foot), with a range of $1,910-7,020, vs $2,622 ($0.61 per square foot), with a range of $2,400-3,040 for vacant lots. Approximately 40% (86,000) of properties in New Orleans are in areas of >400 mg Pb/kg soil and estimated direct costs for treatment are between $225.5 and $290.4 million. Annual costs of Pb poisoning in New Orleans are estimated at approximately $76 million in health, education, and societal harm. Urban accumulation of Pb is an international problem; for example, the new Government of Norway established a policy precedence for an isolated soil cleanup program at daycare centers, school playgrounds, and parks to protect children. New Orleans requires a community-wide soil cleanup program because of the extent and quantity of accumulated soil Pb. The post-Katrina benefits of reducing soil Pb are expected to outweigh the foreseeable costs of Pb poisoning to children returning to New Orleans. PMID- 16683625 TI - Carbonation of steel slag for CO2 sequestration: leaching of products and reaction mechanisms. AB - Carbonation of industrial alkaline residues can be used as a CO2 sequestration technology to reduce carbon dioxide emissions. In this study, steel slag samples were carbonated to a varying extent. Leaching experiments and geochemical modeling were used to identify solubility-controlling processes of major and trace elements, both with regard to carbonation mechanisms and the environmental properties of the (carbonated) steel slag. Carbonation was shown to reduce the leaching of alkaline earth metals (except Mg) by conversion of Ca-phases, such as portlandite, ettringite, and Ca-(Fe)-silicates into calcite, possibly containing traces of Ba and Sr. The leaching of vanadium increased substantially upon carbonation, probably due to the dissolution of a Ca-vanadate. The reactive surface area of Al- and Fe-(hydr)oxides increased with the carbonation degree, which tends to reduce the leaching of sorption-controlled trace elements. Sorption on Mn- (hydr)oxides was found to be required to adequately model the leaching of divalent cations, but was not influenced by carbonation. Consideration of these three distinct reactive surfaces and possible (surface) precipitation reactions resulted in adequate modeling predictions of oxyanion and trace metal leaching from (carbonated) steel slag. Hence, these surfaces exert a major influence on the environmental properties of both fresh and carbonated steel slag. PMID- 16683626 TI - Hybrid life-cycle assessment of natural gas based fuel chains for transportation. AB - This research compares the use of natural gas, methanol, and hydrogen as transportation fuels. These three fuel chains start with the extraction and processing of natural gas in the Norwegian North Sea and end with final use in Central Europe. The end use is passenger transportation with a sub-compact car that has an internal combustion engine for the natural gas case and a fuel cell for the methanol and hydrogen cases. The life cycle assessment is performed by combining a process based life-cycle inventory with economic input-output data. The analysis shows that the potential climate impacts are lowest for the hydrogen fuel scenario with CO2 deposition. The hydrogen fuel chain scenario has no significant environmental disadvantage compared to the other fuel chains. Detailed analysis shows that the construction of the car contributes significantly to most impact categories. Finally, it is shown how the application of a hybrid inventory model ensures a more complete inventory description compared to standard process-based life-cycle assessment. This is particularly significant for car construction which would have been significantly underestimated in this study using standard process life-cycle assessment alone. PMID- 16683627 TI - Ambient particulate matter exhibits direct inhibitory effects on oxidative stress enzymes. AB - A primary mechanistic hypothesis by which ambient air particles have a significant negative impact on human health is via the induction of pulmonary inflammatory responses mediated through the generation of reactive oxygen species (ROS). Development of a biosensor for the assessment of particulate ROS activity would be a significant advance in air pollution monitoring. The objective of this study was to evaluate whether air particulates interact directly with protective enzymes involved in oxidative stress responses. We performed enzyme activity assays on four enzymes involved in oxidative stress responses (Cu/Zn superoxide dismutase, Mn superoxide dismutase, glutathione peroxidase, and glutathione reductase) in the presence of particles of varying toxicities and found distinctive inhibition patterns. On the basis of these findings, we suggest a strategy for an enzyme bioassay that could be used to assess the potential of particles to generate ROS-induced responses. PMID- 16683628 TI - Comparison of soil Pb in vitro bioaccessibility and in vivo bioavailability with Pb pools from a sequential soil extraction. AB - In this study, 15 soils ranging in Pb content from 32 to 6330 mg kg(-1) were subjected to in vitro gastrointestinal extractions with and without added powdered milk. Before and after treatment, Pb in the soils was fractionated according to a 7-step sequential extraction procedure. A subset of five soils and Pb acetate was used for a minipig dosing study. The amount of bioaccessible Pb determined with the in vitro system ranged from 3 to 20% without powdered milk and from 11 to 56% with powdered milk. The higher bioaccessibility of Pb in the in vitro model with addition of powdered milk was related to a depletion of Pb in the organic Pb pool and indicates that soluble milk constituents compete with soil organic ligands for Pb. The absolute and relative bioavailabilities of Pb in the minipig dosing experiment were not related to bioaccessible Pb determined in any of the two in vitro systems. However, relative bioavailabilities in liver, kidney, and total uptake were highly correlated to Pb in the third fraction of the sequential extraction that is attributed to easily reducible Mn oxides. These results indicate that reductive processes in the intestine may be more relevant for Pb absorption than the initial solubilization in the acidic stomach. PMID- 16683629 TI - Estrogenic activity of polychlorinated biphenyls present in human tissue and the environment. AB - This study evaluated the estrogenicity of polychlorinated biphenyls (PCBs) present in environmental media and human tissue and assessed exposure pathways for PCB-derived estrogenic potency in air, soil, and dust from New Bedford, MA, an area with a PCB-contaminated Superfund site. Thirty-four PCB congeners were assayed for estrogenic potency using E-SCREEN, an assay based on the estrogen dependent proliferation of MCF-7 cells in vitro. Childhood exposure to estradiol equivalents via PCBs in environmental media was estimated byweighting previously reported New Bedford congener-specific concentrations by their relative estrogenic potency and published inhalation and soil ingestion rates. Thirteen congeners were weakly estrogenic in E-SCREEN: PCBs 17, 18, 30, 44, 49, 66, 74, 82, 99, 103, 110, 128, and 179. These PCBs were typically 6 orders of magnitude less potent than 17beta-estradiol, with proliferative potencies ranging from 0.0007% to 0.0040%. Of the environmental media assessed, air (inhalation) had the highest PCB-derived estradiol-equivalent exposure. PCB estrogenic potency information from this study provides an important resource both for preliminary estimation of routes of human exposure to xenoestrogens and for application to human health studies focused on estrogen-responsive health outcomes, such as reproductive development and related malignancies. PMID- 16683630 TI - Organochlorines in antarctic and arctic avian top predators: a comparison between the South Polar Skua and two species of northern hemisphere gulls. AB - Different organochlorine compounds (OCs) were measured in the blood of breeding south polar skuas (Catharacta maccormicki) at Svarthamaren, Dronning Maud Land (Antarctica) and compared to those in two species of northern hemisphere gulls: the Arctic glaucous gull (Larus hyperboreus) and the subarctic great black-backed gull (Larus marinus). The skuas had 8% and 29% of the SigmaOC levels (45 ng/g, wet weight) of glaucous gulls (591 ng/g) and great black-backed gulls (158 ng/g), respectively. Polychlorinated biphenyls (PCBs) and p,p' dichlorodiphenyldichloroethylene (p,p'-DDE) were very low in skuas compared to northern gulls, but the mean hexachlorobenzene (HCB) level was 1.7 times higher than in great black-backed gulls and one-third of the glaucous gull level. Mirex levels in skuas were among the highest reported in birds, the mean level being 3 and 26 times higher than those in glaucous gull and great black-backed gulls, respectively. In skuas, the mean levels of HCB, oxychlordane, p,p'-DDE, and PCBs increased by about 30% during a 2-week period, and mirex increased by nearly 60%. In glacuous gulls, HCB, p,p'-DDE, and PCBs increased by 10-20%. For HCB, mirex, and oxychlordane, only a relatively small proportion of the increase in skuas could be explained by changes in lipid pools and the levels at first sampling, compared to glaucous gulls. Thus, skuas were probably accumulating these compounds when present in Antarctica. p,p'-DDE and PCB levels, in contrast, seemed much more stable in the skuas. Relatively high levels of mirex and HCB in south polar skuas are concerning with regard to potential adverse effects. PMID- 16683631 TI - A new method using a health index (HI) to screen low level toxic organic substances in consumer products. AB - To ensure selection of appropriate materials for industrial use in terms of toxicity, a procedure to estimate a health index (HI) that can be used for prioritizing less hazardous components of consumer products is described. The HI is based on the occupational exposure limits (OELs) of organic substances that compose the products. To calculate the HI of a product, it is often necessary to predict OEL values for the substances for which an OEL value has not yet been promulgated. We developed a method to estimate the OEL values from median lethal dose (LD50) data of rodent. A good correlation between known OEL values and the LD50 data was obtained by a multivariate regression analysis by introducing organic compensation coefficients, which were caluculated as 10 to the Bth power where B is the regression coefficient of dummy variables denoting the characteristics of the organic compounds such as functional groups, molecular structures, and so on. We believe that the use of the present method should be limited to predicting unknown OEL values for the HI and used for material prioritization. It should not be extended to determinations of regulatory OELs. PMID- 16683632 TI - Using yeast RNA as a probe for generation of hydroxyl radicals by earth materials. AB - Inhalation of certain types of particulate matter can lead to lung disease. The reactivity of these particles and, in part, the pathologic responses that result are dictated by their physicochemical properties. The ability of particles to induce the generation of reactive oxygen species (ROS), especially hydroxyl radicals in vivo, is one property that has been correlated to the development of lung disease. Several minerals, such as quartz and asbestos, are known to generate hydroxyl radicals and cause lung disease, but many other minerals have never been tested. Here, we describe a technique employing yeast RNA as a probe to screen for mineral-generated hydroxyl radicals. The stability of RNA in the presence of hydrogen peroxide, ferrous iron, hydroxyl radicals, and several common minerals (quartz, albite, forsterite, fayalite, hematite, magnetite, coal, and pyrite) was examined. 3'-(p-Aminophenyl) fluorescein (APF) was used to verify mineral generation of ROS. RNA is stable in the presence of hydrogen peroxide, quartz, and albite; while it degrades in the presence of ferrous iron, hydroxyl radicals, and the other minerals. Coal and pyrite are the most reactive both in RNA degradation and hydroxyl radical generation. This noncellular technique provides a straightforward way to compare many different particles simultaneously. Those particles showing reactivity toward RNA using this method are high-priority candidates for further in vitro and possibly in vivo tests. PMID- 16683633 TI - Cloning and real-time PCR testing of 14 potential biomarkers in Eisenia fetida following cadmium exposure. AB - Important biological activities could be affected in metal exposed species, and amongthe main physiological functions, immunity may provide one (or more) effector(s) which expression can be directly affected by a metal exposure in various macroinvertebrates. As many proteinic effectors showed a high degree of homology between species, we have developed a PCR approach to characterize partial mRNA sequences of selected effectors in the laboratory model, Eisenia fetida. After cloning, levels of expression of each gene were analyzed following exposures (80 and 800 mg/kg) to cadmium spiked soils using real-time PCR. An implemented approach was allowed to test quickly potential biomarkers in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, trancriptionally controlled tumor protein, protein kinase C, and ubiquitin. Most of the selected effectors did not show variations of expression level after exposure. Others expressed weak changes of expression as heat shock proteins. At lastfor catalase and metallothionein, early suitable variations of expression were observed. PMID- 16683634 TI - Humic substances and crude oil induce cytochrome P450 1A expression in the Amazonian fish species Colossoma macropomum (Tambaqui). AB - Cytochrome P450 1A (CYP1A) induction is used widely as a biomarker of exposure to pollutants, such as petroleum hydrocarbons, yet CYP1A inducibility has been characterized in few tropical fish. Using Western blot analysis, catalytic assay, and immunohistochemistry, we evaluated CYP1A induction in an Amazonian fish (tambaqui; Colossoma macropomum) acclimated to humic substances (HS) and acutely exposed to crude oil. HS are ubiquitous in Amazonian waters, and they are known to affect the bioavailability of pollutants. CYP1A activity was also measured in fish exposed for 10 days to a range of concentrations of HS from both natural and commercial sources. Crude oil induced CYP1A expression in tambaqui, as expected. Exposure to both HS and crude oil resulted in greater levels of CYP1A expression relative to that in fish exposed to petroleum alone. Interestingly, CYP1A induction was also observed in fish exposed to HS alone. Induction by HS was concentration-dependent, and activity was higher in fish exposed to HS from the commercial source than in fish exposed to the HS from the natural source. The use of CYP1A as a biomarker of exposure to pollutants such as petroleum hydrocarbons in fish living in environments rich in humic substances should be considered with caution given that HS themselves induce CYP1A expression. Our results suggest that there may be as yet unknown CYP1A inducing components (aryl hydrocarbon receptor agonists) in humic substances. PMID- 16683635 TI - [Evaluation and management of pressure ulcers in the elderly]. PMID- 16683636 TI - [Molecular mechanism tissue-specific actions of selective estrogen receptor modulators]. PMID- 16683637 TI - [Cerebrovascular disease in the elderly people]. PMID- 16683638 TI - [Trends of infectious diseases in the elderly]. PMID- 16683639 TI - [Alzheimer's disease and diet]. PMID- 16683640 TI - [Support of leaving hospital from viewpoint of health promotion]. PMID- 16683641 TI - [Social work intervention at the time of discharge from the hospital]. PMID- 16683642 TI - [The present status and the future prospects of the medical network--from the standpoint of a community hospital]. AB - Our hospital receives elderly patients who are disabled and have dementia, dysphagia, infectious diseases, and/or decubitus from acute hospitals. We treat, rehabilitate, and return those patients to their home. For this purpose we have many rehabilitation staff and several teams such as a nutrition support team, an infection control team, and a decubitus control team. Hospitals treating acute conditions do not have enough rehabilitation staff, and frail elderly patients tend to suffer a decrease in their activities of daily living. Therefore a close relationship between hospitals treating acute conditions and rehabilitation hospitals is important to avoid disuse syndrome. We also treat in-home patients. Those patients are often more disabled and financially burdened than in previous years. Therefore they need more help from the medical and social welfare system of Japan. PMID- 16683643 TI - [Diastolic heart failure in the elderly]. PMID- 16683644 TI - [Management of atrial fibrillation associated with heart failure in the elderly]. PMID- 16683645 TI - [Impact of PCI on heart failure complicating acute myocardial infarction in elderly patients]. PMID- 16683646 TI - [Multidisciplinary approach to elderly patients with congestive heart failure prevents re-admission]. PMID- 16683647 TI - [Aging society and social security of Japan]. PMID- 16683648 TI - [Long-term care insurance and medical insurance]. PMID- 16683650 TI - [Considering medical care and long-term care insurance scheme for the elderly- from the perspective of health economics and policy]. PMID- 16683649 TI - [The medical and care insurance system for the aged]. PMID- 16683651 TI - [Association of high blood pressure to lacunae and ischemic white-matter change]. PMID- 16683652 TI - [Guideline for the treatment of diabetes, hyperlipidemia, and ischemic heart disease in elderly patients]. PMID- 16683653 TI - [Clinical guidelines for elderly patients: special reference to respiratory system and aspiration pneumonia]. PMID- 16683654 TI - [Background and problems in constructing disability prevention guidelines]. AB - AIM: This study aims to describe the factors that must be considered for the construction of guidelines to prevent disability of the elderly. METHOD: All community-dwelling residents aged 65 and over and without functional decline (n = 1,560) were assessed in 1996 and were followed annually until 2002. The prevalence and incidence of low to severe disability, and their association with chronic conditions present at the commencement of the study, was analyzed. RESULT: An increase of mild functional decline was more prevalent than severe functional decline. The accumulation of mild disability was more prominent in women. The major chronic conditions associated with mild disability were chronic arthritis and diabetes in women, and cerebrovascular accident and malignancy in men. CONCLUSIONS: The process of functional decline, gender and the endpoint outcome level must be taken into account on the development of preventive guideline for elderly disability. PMID- 16683655 TI - [Behavioral and psychological symptoms, cognitive impairment and caregiver burden related to Alzheimer's disease patients treated in an outpatient memory clinic]. AB - AIM: The relationships among behavioral and psychological symptoms of dementia (BPSD), cognitive impairment of Alzheimer's disease (AD) patients and the caregiver burden of their caregivers were investigated in an outpatient memory clinic. METHODS: Forty-six pairs of AD patients and their family caregivers were involved in this study. Neuropsychiatry Inventory (NPI) was used to estimate BPSD, to which memory symptoms were added as a subcategory of BPSD. MMSE, word fluency, clock drawing test and category-cued memory test were used for cognitive measurement. Zarit burden interview (ZBI) and CES-D were used to assess caregiver burden. RESULTS: Among 11 BPSD subcategories, memory symptoms, apathy, depression, delusion, aggression and anxiety were prevalent BPSD was a strong determinant of caregiver burden. Among BPSD symptoms, anxiety, aggression and aberrant motor behavior were significantly related to ZBL In terms of the relationship between BPSD and cognitive impairment, the scores for delusion and apathy were significantly related to the cognitive decline. On the other hand, patients who showed symptoms related to memory and depression had higher cognitive function than those who did not. CONCLUSION: These analyses will contribute to better assessment of AD patients and their caregivers, hopefully resulting in better support for them. PMID- 16683656 TI - [Effects of age on the relationship between cardio-ankle vascular index and atherosclerotic progression in patients with type 2 diabetes mellitus]. AB - AIM: A new indicator of arterial stiffness, cardio-ankle vascular index (CAVI), has recently been developed, instead of conventional pulse wave velocity. CAVI is proposed as a blood pressure-independent indicator of atherosclerosis, however, there have been few studies on the association of CAVI with atherosclerosis. The purpose of this study was to investigate effects of age on the relationship between CAVI and atherosclerotic risk in patients with diabetes mellitus. METHODS: The relationship between CAVI and atherosclerotic risk was investigated in 105 subjects with type 2 diabetes mellitus (mean age, 65.1 years old). The mean intima-media thickness (IMT) of the carotid artery was used as an indicator of atherosclerotic progression. RESULTS: In 55.2% of the subjects, CAVI was abnormally high (> or =9.0). In simple regression analysis, CAVI showed significant correlations with age, duration of diabetes and IMT. In logistic regression analysis, crude odds of abnormally high values of CAVI were significant for highest versus lowest tertile groups of mean arterial pressure (MAP) and IMT. Age-and sex-adjusted odds of abnormally high values of CAVI were significant for highest versus lowest tertile groups of mean arterial pressure (MAP) and serum total cholesterol. In multiple regression analysis, CAVI showed significant correlation with IMT independently of MAP but not independently of age, while the significant correlation of ankle-brachial pressure index (ABPI) with IMT was independent of age, sex and MAP. CONCLUSIONS: CAVI reflects atherosclerotic risk and the relationship between CAVI and atherosclerotic progression is strongly confounded by age. Thus, age should be taken into account when CAVI is used as an indicator of atherosclerotic progression. PMID- 16683657 TI - [Investigation of the nutritional status of the elderly for the development of guidelines for nutritional management in the ultra-aged society]. AB - AIM: This study was conducted to evaluate the nutrient intake and the nutritional state of elderly people who live independently in a region with an aging rate (percentage of the elderly 65 or above to the entire population) of 50% and to develop guidelines for nutritional improvement to extend the healthy lifespan in the upcoming ultra-aged society in which, by definition, aging rate exceeds 21%. METHODS: The study investigated the diet, physical constitution and blood biochemistry of 38 elderly people living in a region with an aging rate of 50% and compared the results with 23 elderly people living in a region with an aging rate of 18%. RESULTS: In the ultra-aged region the elderly people had a tendency for serum, triglyceride to be high and calcium to be low. The rate of the elderly living alone was high and the solitary elderly tended to have poor intake of vegetable fats, albumin, iron and zinc. The elderly men in the ultra-aged region had the lowest rate of recommended dietary intakes of calcium and vitamin B1. CONCLUSIONS: The results indicated that extending a healthy lifespan in an ultra aged society requires less intake of animal fats and more intake of vitamin B1 and calcium and a close assessment of the nutritional state of zinc, iron, and selenium. In order to prevent lifestyle-related diseases and to extend healthy life expectancy, the nutrition management of the elderly living alone is particularly important PMID- 16683658 TI - [Collaboration between university hospitals and local medical communities in Japan evaluated by a questionnaire among doctors]. AB - AIM AND METHODS: To provide quality medical service in a society with many elderly, university hospitals should clearly define their roles and contribute to the establishment of an integrated, comprehensive medical system. In this study, we conducted a questionnaire survey of doctors working at Kyoto University Hospital and at private practices in Kyoto about their attitude toward collaboration between the university hospital and private practices and asked their opinions on the role of university hospitals in the local medical community. RESULTS: The survey showed that doctors both at the university hospital and in private practice regarded close collaboration as important, but also highlighted obstacles and problems that block collaboration. CONCLUSION: Overcoming these problems and constructing collaboration models will be a key for university hospitals to fully contribute to the establishment of a quality medical system. PMID- 16683660 TI - [Relationship between activity of daily living and motor function in the community-dwelling elderly after hip fracture]. AB - AIM: To clarify the relationship between ADL and motor functions in 44 community dwelling elderly patients with hip fracture. METHODS: The questionnaire comprised items on BADL, IADL, ability to walk, and certification for long-term care. The assessment of motor functions included grip strength, knee extension strength, 10 m walking time, TUG, FRT, and Romberg rate and shift length of the center of gravity measured by a stabilometer. Based on these tests, relationships between the motor functions and ADL were evaluated in elderly patients after hip fracture. RESULTS: Significant correlations were found between BADL and grip strength, knee extension strength, and FRT. As for IADL, significant correlations were found with grip strength, knee extension strength, 10-m walking speed, TUG, FRT, and MMSE items. Similarly, regarding ability to walk and the certification of long-term care, significant correlations were shown with grip strength, knee extension strength, 10 m walking speed, and MMSE items. CONCLUSION: The relationships between ADL and motor functions were confirmed in community dwelling elderly patients with hip fracture. The parameters related to muscle strength and balance ability that were assessed in this study are those that can be improved through constant training after discharge from hospital. The validity of these parameters as the indices for future intervention studies to assess the maintenance and improvement of ADL after discharge and prognosis was indicated. PMID- 16683659 TI - [The efficacy and adverse effects of rituximab with CHOP or THP-COP in old-old and extremely old patients with diffuse large B cell lymphoma]. AB - AIM: We examined the efficacy and adverse effects of rituximab with CHOP (cyclophosphamide, doxorubicin, vincristin, prednisone) or THP-COP (pirarubicin, cyclophosphamide, vincristin, prednisone) in previously untreated old-old and extremely old patients with diffuse large B cell lymphoma (DLBCL). METHODS: Subjects were 13 initial DLBCL patients consisting of 7 men and 8 women with a median age of 79 years (range 75-91 years). These patients received CHOP or THP COP plus 375mg/m2 rituximab intravenously given on the day before each cycle. The dose was adjusted depending on the patient's age and associated complications. Administration was performed for 6 to 8 cycles, whenever possible. RESULTS: Seven patients (54%) achieved a complete response (CR), 4 (31%) achieved a partial response (PR), 2 (15%) failed to respond. The 2-year survival rates were 62%. The CR rate and survival rate were higher than in patients previously treated with CHOP alone in our hospital, but there was no statistically significant difference. The most frequent adverse effect was bone marrow suppression, observed in 9 patients (69.2%). Adverse effects that seemed to be infusion reaction occurred in 4 patients (30.7%). These adverse effects related to infusion reaction disappeared by reducing or stopping rituximab. Four (30.7%) patients died and 3 of those died due to disease progression. One of those died 1.5 month after he began chemotherapy and the cause of death was unknown. CONCLUSION: This result suggested that both CHOP and THP-COP combined with rituximab were safe and effective for old-old and extremely old patients. PMID- 16683661 TI - [A ninety-eight-year-old woman with ruptured pancreatic anaplastic mucinous cystadenocarcinoma]. AB - A 98-year-old woman was admitted to our hospital complaining of anorexia, epigastralgia, and vomiting. An elastic hard tumor was palpable in her epigastric region. CT and US examination revealed a huge cystic lesion adjacent to the left lobe of the liver and the stomach. Her serum levels of CEA (13.6 ng/ml), CA19-9 (95 U/ml) and CA125 (99 U/ml) were high. She suffered from aspiration pneumonia on the 10th day of admission, which progressed to acute respiratory distress syndrome. On the 20th day of admission, the epigastric tumor suddenly disappeared. She passed away on the 31st day due to respiratory failure. Autopsy revealed that she had a ruptured pancreatic anaplastic mucinous cystadenocarcinoma. To the best of our knowledge, this is the oldest reported case of ruptured pancreatic cystadenocarcinoma in the world. PMID- 16683662 TI - Bleaching non vital primary teeth: case report. AB - Trauma and pulpal infections in primary dentition are part of the routine of the pediatric dentist. Common consequences in these cases are alterations in dental color, compromising patient's esthetics and his interaction in social environment. Bleaching intends to preserve dental structure already weakened and to show immediate esthetic results. This clinical case shows a bleaching technique in devitalized primary teeth using bleaching agent with 35% hydrogen peroxide activated by photo polymerizer. This technique is simple and shows immediate satisfactory results. PMID- 16683663 TI - Behavior and orofacial characteristics of children with attention-deficit hyperactivity disorder during a dental visit. AB - ADHD is a neuropsychological disorder, affecting attention, impulsiveness and activeness. The study included 36 children with ADHD, 47 without, and two silent observers. A dental form, SNAP-IV and ADHDT symptom checklists were used. Statistically significant differences were observed in hospitalization histories, oral habits, tongue characteristics, and facial biotype. Differences in orofacial characteristics and behavior between the groups were confirmed. PMID- 16683664 TI - Early childhood caries (ECC): a preventive-conservative treatment mode during a 12-month period. AB - PURPOSE: To evaluate a preventive treatment mode for early childhood caries (ECC). POPULATION AND METHODS: The population to be studied included 30 children who, over a 12-month period, presented with ECC to a private dental clinic. Parents preferred non-invasive, preventive treatment over restorations. Parents were given hygiene and proper feeding instructions. Mesial slicing was performed where proximal caries was observed. Children were examined once every two months. They received supervised professional topical fluoride treatment. Plaque level, brushing, stopping the bottle, eating sweets, appearance of new lesions or exacerbation of existing condition were observed and recorded. RESULTS: In the vast majority of patients, the progression of ECC was arrested after the preventive regimen. Three children required restorations. They had failed to limit sugar consumption and to comply with brushing instruction. General improvement was observed in plaque control, brushing habits and sweets consumption. CONCLUSION: Preventive measures may successfully arrest ECC and thereby avoid invasive procedures as well as the need of anesthesia. PMID- 16683665 TI - Spontaneous eruption of an occult incisor fragment from the lip after eight months: report of a case. AB - Large majorities of dental traumas are associated with soft-tissue injury and tooth fragments. They are often found embedded in the oral soft tissues, most commonly the lip. Early detection and complete removal of such fragments is critical in order to prevent infection and scarring. Adequate communication between medical and dental professionals involved is of considerable value in the management of such cases. This report presents a case of spontaneous extrusion of an occult incisor fragment from the lower lip, eight months after trauma. PMID- 16683666 TI - Eruption cyst treated with a laser powered hydrokinetic system. AB - Eruption cysts are benign cysts that appear on the mucosa of a tooth shortly before its eruption. The majority disappear on their own. If they hurt, bleed or are infected they may require surgical treatment to expose the tooth and drain the content. Here we present a clinical case of a six-year-old child with an eruption cyst on the permanent maxillary central left incisor, which was handled using treatment with an Er,Cr-YSGG laser. It did not require suture, there was no haemorrhage, swelling, infection or postoperative pain. The treatment of eruption cysts with this technique facilitates obtaining the cooperation of pediatric patients and makes behavior management by the pediatric dentist easier. PMID- 16683668 TI - Reattachment and orthodontic extrusion in the management of an incisor crown-root fracture: a case report. AB - This article describes the management of a case of oblique crown root fracture of a central incisor where the fractured fragment was used as a temporary replacement crown in order to maintain esthetics during the period of orthodontic extrusion of the remaining root. Placement of the final restoration after orthodontic extrusion resulted in good esthetics and periodontal health postoperatively. The merits and demerits of this treatment modality are discussed. PMID- 16683667 TI - Human pulpal response to mineral trioxide aggregate (MTA): a histologic study. AB - The purpose of this study was to study the histologic changes in the dental pulp following pulpotomy with Mineral Trioxide Aggregate (MTA) and Calcium hydroxide. Pulpotomies were performed on premolar teeth that were to be extracted for orthodontic reasons. The radicular pulp was capped with either MTA or Calcium hydroxide and restored with IRM. The teeth were extracted at 4 and 8 week intervals, fixed in 10% formalin and then kept in 5% nitric acid for 28 days for demineralization. Longitudinal sections were then prepared and viewed under light microscope. The pulps capped with MTA (at the end of 4 weeks and 8 weeks) showed dentin bridge formation which was more homogenous and continuous with the original dentin when compared to the pulps capped with calcium hydroxide. The pulpal inflammation was also less in the MTA group as compared to the calcium hydroxide group at the end of 4 and 8 weeks. PMID- 16683669 TI - Resin composite sealant vs. polyacid-modified resin composite applied to post eruptive mature and immature molars: two year clinical study. AB - The aim of this study was to evaluate the effect of the post eruptive enamel maturation on the durability of polyacid-modified resin composite (PMRC; Dyract Seal) and resin composite (Fissurit F) sealant and understanding the effect of posteruptive enamel maturation on the sealant retention. To determine the possible effect of the enamel maturation to the sealant retention, the success of the sealants applied to the newly erupted mandibular first molars of the 6 to 7 years old children, who were in the fourth or fifth eruption stage according to Dennison's classification were compared with the sealants applied to first mandibular molars which were in the mouth at least for two years of the 9 years old children. The molars of the younger children were accepted as immature and the elders were posteruptively mature. The sealant retention was evaluated for two years. At the end of two years the retention rates of Dyract Seal were significantly lower. Maturation has no effect on the sealant retention. Dyract Seal application after NRC conditioning is not recommended. PMID- 16683670 TI - Black stains in the mixed dentition: a PCR microbiological study of the etiopathogenic bacteria. AB - The aim of this work is to emphasize that particular stains on the third cervical of the buccal and lingual surfaces in mixed dentition, called "black stain." Previous research showed the microbiological etiology of this discoloration by chromogen bacterias. Our study shows bacteria spp involved in stains by means of PCR process and electrophoresis gel on the agarose medium. Sample was formed by 100 subject with black stain and 100 control subjects stain-free. A statistical analysis (SPSS 10.0) using X2 was performed in this study. Porphyromonas gingivalis and Prevotella melaninogenica, were not involved in both in black stain subjects and in the control. On the contrary, Actinomyces could be involved in the pigmentation process. PMID- 16683671 TI - A retrospective evaluation of the eruption of impacted permanent incisors after extraction of supernumerary teeth. AB - AIMS: Delayed eruption of teeth may be caused by the presence of one or more supernumerary teeth. The purpose of this study was to evaluate findings, predisposing factors and differentiate different techniques used that affect the outcome following removal of supernumerary teeth. A comprehensive literature review was also undertaken. METHODS: A longitudinal retrospective study was carried out at the Royal Liverpool Children's Hospital. A total of 120 patients were identified from the general anesthesia records that had supernumeraries extracted. Only 43 cases had delayed eruption of teeth caused by supernumeraries. The pre and post extraction record data collected were the gender, radiographic assessment, position of the supernumerary, age at time of referral and extraction of the supernumerary, age at time of eruption of the impacted tooth and the orthodontic and surgical management. RESULTS: The mean age of referral was 9.1 years with a male to female ratio of 4.4:1. There was a greater predilection for supernumeraries to be on the left side and be positioned palatally. Tuberculate type supernumeraries were the most frequent followed by the conical type. Spontaneous eruption of the impacted tooth occurred in 49% of cases. Eruption of the impacted tooth within eighteen months following removal of the supernumerary was observed in 91% of cases. The chronological age and space availability were the two factors that were critical in determining if eruption was spontaneous following removal of the supernumerary. CONCLUSIONS: The findings of this study reiterates the fact that given early referral, sufficient space and time, the majority of teeth prevented from erupting by a supernumerary tooth would erupt spontaneously following removal of the supernumerary alone. Randomized multi centre prospective studies are suggested. PMID- 16683672 TI - Efficacy of various intracanal medicaments against Enterococcus faecalis in primary teeth: an in vivo study. AB - The aim of this study was to evaluate the in vivo efficacy of three intracanal medicaments (Ca(OH)2, 1% chlorhexidine gel and 1% chlorhexidine gel with Ca(OH)2 against Enterococcus faecalis in necrotic primary teeth. As a conclusion, chlorhexidine gel with or without Ca(OH)2 was more effective than Ca(OH)2 alone against Enterococcus faecalis. PMID- 16683673 TI - Evaluation of fluoride release from teeth after topical application of NaF, SnF2 and APF and antimicrobial activity on mutans streptococci. AB - The objectives of this study were to evaluate and compare the amount and pattern of fluoride release from teeth after topical application of 2% NaF, 8% SnF2 and 1.23% APF at different time intervals. The growth inhibitory effects of this released fluoride ion was assessed on mutans streptococci (MS) and correlated with the fluoride release. Forty premolars divided into four groups were subjected to different topical fluoride treatments. All the teeth were immersed individually in deionized water and were transferred to containers at 1 hour, 1 day and 1 week time intervals. 240 samples in total were used for fluoride estimation by ion selective electrode method and the samples from the other subgroup were used for evaluation of antimicrobial activity on mutans streptococci (MS) by bacterial inhibition assay method. The results showed that the highest fluoride release (7.83 +/- 0.55 ppm) was seen in SnF2 treated specimens, as compared to that of NaF (3.71 +/- 0.60ppm) and APF (3.30 +/- 0.51ppm), the difference being statistically significant (P<0.01). This was observed immediately after 1 hour, followed by a drastic reduction thereafter. No zones of inhibition were observed at the released fluoride concentrations at different time intervals in the different groups. IN CONCLUSION: 8% SnF2 is expected to have greater anticaries property from the high fluoride releasing property for prolonged period of time. PMID- 16683674 TI - Scanning electron microscopy evaluation of the effect of etching agents on human enamel surface. AB - Acid etching promotes microporosities on enamel surface, which provide a better bonding surface to adhesive materials. The purpose of this study was to comparatively analyze the microstructure of enamel surface after etching with 37% phosphoric acid or with two self-etching primers, Non-rinse conditioner (NRC) and Clearfil SE Bond (CSEB) using scanning electron microscopy. Thirty sound premolars were divided into 3 groups with ten teeth each: Group 1: the buccal surface was etched with 37% phosphoric acid for 15 seconds; Group 2: the buccal surface was etched with NRC for 20 seconds; Group 3: the buccal surface was etched with CSEB for 20 seconds. Teeth from Group 1 were rinsed with water; teeth from all groups were air-dried for 15 seconds. After that, all specimens were processed for scanning electron microscopy and analyzed in a Jeol 6100 SEM. The results showed deeper etching when the enamel surface was etched with 37% phosphoric acid, followed by NRC and CSEB. It is concluded that 37% phosphoric acid is still the best agent for a most effective enamel etching. PMID- 16683675 TI - The detection of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in tooth, tongue and buccal mucosa plaques in children, using immunoslot blot assay (IBA). AB - The present study was to investigate the distribution of typical periodontpathic bacteria (Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans) in tooth, tongue and buccal mucosa plaques in 3 to 17 Year old children. Clinical parameters (Rates of df, d, DMF, and D; plaque and gingival index) for each subject were determined prior to the collection of each site plaque. Three periodontopathic bacteria on each site samples were detected using IBA. The frequency of three bacteria for tooth plaque was higher than that for tongue or buccal mucosa plaque. The frequency of Porphyromonas gingivalis and Prevotella intermedia in supragingival plaques was significantly higher than that of corresponding ones in tongue or buccal mucosa plaques. The three bacteria also occurred more frequently in subjects aged between 10 and 14 years. Periodontopathic bacteria may be enhanced in circumpubertal children. PMID- 16683676 TI - Pallister-Killian syndrome (PKS): clinical case report. AB - Pallister-Killian Syndrome is a rare dysmorphic condition characterized by specific clinical manifestations and tetrasomy 12p. This paper focuses on the general and orofacial clinical manifestations. PMID- 16683677 TI - Giant cell fibroma: a case report. AB - This article describes a case of giant cell fibroma in a 3-year-old boy, which is an uncommon age for this lesion. The cause of this pathology has not been determined. The lesion was excised by electro surgery and submitted to a histological exam, which confirmed the diagnosis. No recurrence was observed. Pediatric dentists should be capable to diagnose and treat this kind of lesion despite this lesion being an unusual pathology among children. PMID- 16683678 TI - Skeletal maturation analysis by morphological evaluation of the cervical vertebrae. AB - The determination of skeletal maturation by morphological evaluation of the cervical vertebrae was evaluated in a 100 cephalograms. The analysis showed that this method was reproducible for assessing the individual's growth curve. PMID- 16683679 TI - Home to the masters. PMID- 16683680 TI - Single-session treatment of a major complication of dens invaginatus: a case report. AB - Dens invaginatus is a dental malformation that may give rise to several complications. Caries of the invagination can severely weaken the whole tooth, making it susceptible to fracture. Subgingival fractures are major complications threatening tooth survival and usually require periodontal/orthodontic/prosthetic treatment if long-term viability is to be ensured. This article describes a case of single-session restoration of a fractured invaginated tooth by means of endodontic treatment followed by fragment reattachment. PMID- 16683681 TI - The pier abutment: a review of the literature and a suggested mathematical model. AB - Many clinicians consider the prosthetic restoration of missing posterior teeth by means of a 5-unit fixed partial denture with a pier abutment a non-favorable situation. The present article assesses the validity of the pier abutment by means of a critical review of the literature and mathematical analysis of the forces generated within a fixed partial denture of this design. Few studies are available, and none substantiate the theory that the pier abutment serves as a fulcrum. A mathematical model suggests that debonding may occur in the anterior abutment, but not due to the teetering of the fixed partial denture around the pier abutment. PMID- 16683682 TI - The use of ozone in dentistry and maxillofacial surgery: a review. AB - Ozone has been successfully used in medicine because of its microbiologic properties for more than 100 years. Its bactericide, virucide, and fungicide effects are based on its strong oxidation effect with the formation of free radicals as well as its direct destruction of almost all microorganisms. In addition, ozone has a therapeutic effect that facilitates wound healing and improves the supply of blood. For medical purposes, ozone may be applied as a gas or dissolved in water. Despite the advantages that the therapeutic use of ozone offers, reservations remain in terms of its application in the oral and maxillofacial area. Particularly, the gaseous application of ozone is critically evaluated because of its possible side effects on the respiratory system. The objective of this article is to provide an overview of the current applications of ozone in dentistry and oral surgery. Research was based on peer-reviewed sources found through a Medline/PubMed search and other textbooks, reviews, and journals. PMID- 16683683 TI - Performance of 2 packable composites at 12 months. AB - OBJECTIVE: The purpose of this randomized, double-blind, clinical trial was to compare the marginal seal of 2 packable resin composite materials in moderate to large lesions on molars. METHOD AND MATERIALS: Fifty participants in need of a moderate to large Class 2 or complex Class 1 molar restoration were randomly distributed into 4 groups, to receive either Alert (Jeneric/Pentron) or SureFil (Dentsply/Caulk) resin composite with or without a surface sealer. Each participant received one restoration. With the exception that study protocol limited increments to no more than 4 mm, teeth were restored according to the manufacturers' instructions, and surface sealer was applied after finishing in the designated groups. Use of Alert includes routine placement of a flowable composite liner. Clinical performance of the restorations was evaluated in 8 categories at baseline, 6 months, and 12 months. The 2 materials were compared to determine if a difference in marginal seal existed between groups. The number of restorations exhibiting marginal staining was compared using Fischer's exact test at a significance level of 5%. RESULTS: Six participants did not present for the 12-month recall. At 12 months, 19 (90.5%) Alert restorations and 15 (68.2%) SureFil restorations did not exhibit marginal staining. There was no statistically significant difference between the 2 restorative materials for marginal staining. Overall, 3 restorations were rated as failures. CONCLUSION: At 12 months, materials placed with a flowable liner were not associated with a significant reduction in marginal staining. PMID- 16683685 TI - Restoration of maxillary incisors with a zirconia all-ceramic system: a case report. AB - The increase in patient demands for enhanced esthetics has prompted the development of several all-ceramic restorative systems. Despite their esthetic advantage, unsatisfactory mechanical properties have restricted the clinical indications of all-ceramic restorations. Recently, densely sintered zirconia based restorations have become available. This material appears to have adequate strength and toughness to allow for the fabrication of anterior and posterior crowns and fixed partial dentures. This clinical report describes the procedures used for the restoration of maxillary incisors with the zirconia-based Lava system. PMID- 16683684 TI - Root canal transportation with a Ni-Ti rotary file system and stainless steel hand files in simulated root canals. AB - OBJECTIVE: The purpose of this study was to compare the root canal transportation of the crown-down technique performed with the Ni-Ti rotary ProFile system (Dentsply/Maillefer), with the step-back technique using stainless steel K Flexofiles (Dentsply/Maillefer). METHOD AND MATERIALS: Thirty simulated root canals in resin blocks were equally divided into 2 groups. The first group was instrumented with the ProFile system in a crown-down technique and the second group with hand K-Flexofiles in a step-back technique. After instrumentation, resin blocks of both groups were scanned by a transparency scanner, and the derived images were superimposed with the scanned image of an uninstrumented block. Transportation was digitally calculated by a computer software, and results were statistically evaluated by Student t test. RESULTS: Statistically significant differences (P < .05) were found among the 2 groups at 1, 2, 4, 5, and 6 mm from the apical foramen. No statistically significant difference was detected at 3 mm from the apical foramen. CONCLUSION: ProFile caused less transportation at 1 and 2 mm from the apex, whereas at 3 mm, both instrumentation techniques caused the same transportation. Standard deviation was less in the ProFile group than in the hand file group, indicating a more standardized preparation. PMID- 16683686 TI - Pain-related impairment and health care utilization in children and adolescents: a comparison of orofacial pain with abdominal pain, back pain, and headache. AB - OBJECTIVE: The purpose was to compare orofacial (temporomandibular) pain with other pain complaints regarding impairment and health care utilization in a sample of 1,011 children and adolescents from a metropolitan area in Germany. METHOD AND MATERIALS: Individuals aged 10 to 18 years were sampled in schools using a 2-stage cluster technique. Orofacial pain in the previous month was assessed according to the Research Diagnostic Criteria for Temporomandibular Disorders. Additionally, participants were questioned about headache, back pain, abdominal pain, pain-related impairment, and health care utilization (visits to doctors, analgesic consumption) in the previous month. RESULTS: Headache was reported by 50% (95% CI: 45% to 56%) of participants, stomach pain by 36% (95% CI: 32% to 41%), back pain by 31% (95% CI: 25% to 36%), and orofacial pain by 15% (95% CI: 12% to 18%). Girls were more affected than boys. The range of "severe" and "very severe" impairment lay between 8% (orofacial pain) and 22% (headache). The range of treatment demand was between 10% and 17% (orofacial pain: 15%), and the range for analgesic consumption between 18% and 24% (orofacial pain: 22%). The more pain experienced, the more impairment, doctor consultations, and analgesic consumption were reported (Chi2test: P < 0.05). Risk of orofacial pain was 60% higher for subjects with head, back, and/or abdominal pain (odds ratio: 1.6; 95% CI: 1.3 to 1.9). CONCLUSION: In children and adolescents, orofacial pain occurs about half as often as other pain complaints. However, relative to their prevalence the different pain complaints are similar regarding impairment and health care utilization. PMID- 16683687 TI - In vitro antibacterial efficacy of a new chlorhexidine slow-release device. AB - OBJECTIVE: The aim of this study was to evaluate and compare the antibacterial effect of chlorhexidine and calcium hydroxide slow-release devices on oral bacteria. METHOD AND MATERIALS: The agar diffusion test was used to evaluate the antibacterial effect of the slow-release devices Activ Point (chlorhexidine; Roeko) and Calcium Hydroxide Plus Point (Roeko) on 8 anaerobic and 2 facultative oral bacterial strains, as well as 2 bacterial cultures randomly sampled from necrotic root canals and incubated in anaerobic and aerobic broth for 48 hours. The efficiency of the medicaments against the various strains was evaluated using a nonparametric test (Friedman type) and a second nonparametric test (Wilcoxon's signed rank test). RESULTS: Activ Point produced significantly larger inhibition zones (P < .001) than the calcium hydroxide slow-release device around all tested bacterial strains. CONCLUSION: The chlorhexidine slow-release device (Activ Point) exhibited significant antibacterial activity in the agar diffusion test and merits study as an intracanal medicament. PMID- 16683688 TI - Evaluation of 2 thione primers and 3 resin adhesives for silver-palladium-copper gold alloy bonding. AB - OBJECTIVE: The purpose of this study was to evaluate 2 thione primers and 3 resin adhesives for enhancement of bonding strength to a silver-palladium-copper-gold alloy. METHOD AND MATERIALS: Two different sized disk specimens (10- and 8-mm diameter by 2.5-mm thick) were prepared from a silver-palladium-copper-gold alloy (Castwell M.C. 12, GC). The specimens were airborne-particle abraded with 50 microm-grain alumina, conditioned either with a thiouracil primer (Metaltite, Tokuyama Dental) or with a triazine dithione primer (V-Primer, Sun Medical), and then bonded with 1 of 3 acrylic resins: a benzoyl peroxide-amine redox-initiated resin adhesive (Multi-Bond, Tokuyama Dental) or a tri-nbutylborane-initiated resin adhesive (Super-Bond C&B and Super-Bond Quick, Sun Medical). For each adhesive, unprimed specimens were prepared as experimental controls. Shear bond strength was determined after thermocycling (100,000 cycles). RESULTS: Use of primers significantly (P < .05) enhanced the bond strength of specimens in all adhesives. Irrespective of the type of primer, the strength of Multi-Bond adhesive was significantly (P < .05) lower than that of Super-Bond C&B and Super Bond Quick adhesives. The strength of the 2 tri-n-butylborane-initiated adhesives did not differ significantly (P > .05). The mean strength of the Super-Bond C&B adhesive was 40.4 MPa with Metaltite and 37.8 MPa with V-Primer; that of Super Bond Quick adhesive was 40.9 MPa with Metaltite and 36.5 MPa with V-Primer. CONCLUSION: Use of thione primers effectively enhanced the strength of the bond to the silver-palladium-copper-gold alloy. Furthermore, the combinations of primers and tri-n-butylborane-initiated adhesives were found to be more efficient for bonding. PMID- 16683689 TI - Aggressive periodontitis in a patient with chronic cutaneous lupus erythematosus: a case report. AB - Lupus erythematosus is considered to be a high risk factor for periodontitis. As an autoimmune disease of unknown origin, cutaneous lupus erythematosus (CLE) is subdivided into 3 categories: chronic (CCLE), subacute (SCLE), and acute (ACLE). While the ACLE has a high prevalence of conjunctive periodontal lesions, aggressive periodontitis in patients with CCLE has been rarely reported. This article describes the case of a patient diagnosed with aggressive periodontitis. Three months after the diagnosis of periodontitis, the patient experienced advancing hair loss (alopecia), pale fingers and toes, as well as edema in the legs and around the eyes. Skin biopsy showed follicular hyperkeratosis with perivascular mononuclear cell infiltrate. Colliquation of the basal cells, thickening of the basal lamina, and vacuolar degeneration of basal keratinocytes were also found. A lupus band test was positive, and diagnosis of CCLE was established. Three months following the treatment of lupus with antimalarial agents, the periodontal condition became stable with no further exacerbation or progression of the existing periodontitis. An 11-month postsurgical follow-up revealed stable periodontal and general medical conditions. A patient's medical history should be re-evaluated in the event of recurrence of periodontal lesions refractory to periodontal treatment. The control of systemic conditions like lupus erythematosus is essential for a good prognosis in the treatment of periodontitis as well as for the general health of the patient. PMID- 16683690 TI - A tribute to professor Autar Singh Paintal (1925-2004). PMID- 16683691 TI - Immunolocalization of tandem pore domain K+ channels in the rat carotid body. PMID- 16683692 TI - Neuroglobin, a new oxygen binding protein is present in the carotid body and increases after chronic intermittent hypoxia. PMID- 16683693 TI - Hypoxia-inducible factor (HIF)-1alpha and endothelin-1 expression in the rat carotid body during intermittent hypoxia. PMID- 16683694 TI - Expression of HIF-2alpha and HIF-3alpha in the rat carotid body in chronic hypoxia. PMID- 16683695 TI - Modulation of gene expression in subfamilies of TASK K+ channels by chronic hyperoxia exposure in rat carotid body. PMID- 16683696 TI - Postnatal changes in gene expression of subfamilies of TASK K+ channels in rat carotid body. PMID- 16683697 TI - Morphological changes in the rat carotid body in acclimatization and deacclimatization to hypoxia. PMID- 16683698 TI - Effect of carbon dioxide on the structure of the carotid body: a comparison between normoxic and hypoxic conditions. PMID- 16683699 TI - S-nitrosoglutathione (SNOG) accumulates hypoxia inducible factor-l alpha in main pulmonary artery endothelial cells but not in micro pulmonary vessel endothelial cells. PMID- 16683700 TI - Changes in antioxidant protein SP-22 of chipmunk carotid bodies during the hibernation season. PMID- 16683701 TI - Potential role of mitochondria in hypoxia sensing by adrenomedullary chromaffin cells. PMID- 16683702 TI - Localization of Ca/calmodulin-dependent protein kinase I in the carotid body chief cells and the ganglionic small intensely fluorescent (SIF) cells of adult rats. PMID- 16683703 TI - Dual origins of the mouse carotid body revealed by targeted disruption of Hoxa3 and Mash1. PMID- 16683704 TI - Genetic regulation of chemoreceptor development in DBA/2J and A/J strains of mice. PMID- 16683705 TI - Genetic influence on carotid body structure in DBA/2J and A/J strains of mice. PMID- 16683706 TI - The effect of hyperoxia on reactive oxygen species (ROS) in petrosal and nodose ganglion neurons during development (using organotypic slices). PMID- 16683707 TI - Carotid body volume in three-weeks-old rats having an episode of neonatal anoxia. PMID- 16683708 TI - The effect of development on the pattern of A1 and A2a-adenosine receptor gene and protein expression in rat peripheral arterial chemoreceptors. PMID- 16683709 TI - A comparative study of the hypoxic secretory response between neonatal adrenal medulla and adult carotid body from the rat. PMID- 16683710 TI - In search of the acute oxygen sensor: functional proteomics and acute regulation of large-conductance, calcium-activated potassium channels by hemeoxygenase-2. PMID- 16683711 TI - Does AMP-activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to pulmonary artery constriction? PMID- 16683712 TI - Function of NADPH oxidase and signaling by reactive oxygen species in rat carotid body type I cells. PMID- 16683713 TI - Hypoxemia and attenuated hypoxic ventilatory responses in mice lacking heme oxygenase-2: evidence for a novel role of heme oxygenase-2 as an oxygen sensor. PMID- 16683714 TI - Regulation of a TASK-like potassium channel in rat carotid body type I cells by ATP. PMID- 16683715 TI - Accumulation of radiolabeled N-oleoyl-dopamine in the rat carotid body. PMID- 16683716 TI - Profiles for ATP and adenosine release at the carotid body in response to O2 concentrations. PMID- 16683717 TI - Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells. PMID- 16683718 TI - Does AMP-activated protein kinase couple hypoxic inhibition of oxidative phosphorylation to carotid body excitation? PMID- 16683719 TI - Mitochondrial ROS production initiates Abeta1-40-mediated up-regulation of L-type Ca2+ channels during chronic hypoxia. PMID- 16683720 TI - Acute hypoxic regulation of recombinant THIK-1 stably expressed in HEK293 cells. PMID- 16683721 TI - Differential expression of oxygen sensitivity in voltage-dependent K channels in inbred strains of mice. PMID- 16683722 TI - An overview on the homeostasis of Ca2+ in chemoreceptor cells of the rabbit and rat carotid bodies. PMID- 16683723 TI - Midbrain neurotransmitters in acute hypoxic ventilatory response. PMID- 16683724 TI - Chronic intermittent hypoxia enhances carotid body chemosensory responses to acute hypoxia. PMID- 16683725 TI - The cell-vessel architecture model for the central respiratory chemoreceptor. PMID- 16683726 TI - Loop gain of respiratory control upon reduced activity of carbonic anhydrase or Na+/H+ exchange. PMID- 16683727 TI - Adrenaline increases carotid body CO2 sensitivity: an in vivo study. PMID- 16683729 TI - Effects of low-dose methazolamide on the control of breathing in cats. PMID- 16683728 TI - Peripheral chemoreceptor activity on exercise-induced hyperpnea in human. PMID- 16683730 TI - Stimulus interaction between hypoxia and hypercapnia in the human peripheral chemoreceptors. PMID- 16683731 TI - Gene expression and signaling pathways by extracellular acidification. PMID- 16683732 TI - Hypoxic modulation of the cholinergic system in the cat carotid glomus cell. PMID- 16683733 TI - Are there "CO2 sensors" in the lung? PMID- 16683734 TI - Nitric oxide in brain glucose retention after carotid body receptors stimulation with cyanide in rats. PMID- 16683735 TI - Pulmonary nociceptors are potentially connected with neuroepithelial bodies. PMID- 16683736 TI - Modulators of cat carotid body chemotransduction. PMID- 16683737 TI - Identification and characterization of hypoxia sensitive Kvalpha subunits in pulmonary neuroepithelial bodies. PMID- 16683738 TI - Voltage-dependent K channels in mouse glomus cells are modulated by acetylcholine. PMID- 16683739 TI - Modification of the glutathione redox environment and chemoreceptor cell responses. PMID- 16683740 TI - Carotid body transmitters actions on rabbit petrosal ganglion in vitro. PMID- 16683741 TI - Potassium channels in the central control of breathing. PMID- 16683742 TI - Role of endothelin-1 on the enhanced carotid body activity induced by chronic intermittent hypoxia. PMID- 16683744 TI - A statistical thermodynamic model of the protein ensemble. PMID- 16683748 TI - Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences. PMID- 16683746 TI - Molecular dynamics: survey of methods for simulating the activity of proteins. PMID- 16683747 TI - Protein-solvent interactions. PMID- 16683745 TI - Protein folding thermodynamics and dynamics: where physics, chemistry, and biology meet. PMID- 16683749 TI - Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution. PMID- 16683750 TI - Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. PMID- 16683751 TI - NMR residual dipolar couplings as probes of biomolecular dynamics. PMID- 16683752 TI - Dynamical contributions to enzyme catalysis: critical tests of a popular hypothesis. PMID- 16683753 TI - Protein folding pathways studied by pulsed- and native-state hydrogen exchange. PMID- 16683754 TI - Probing protein folding and conformational transitions with fluorescence. PMID- 16683756 TI - Probing conformational fluctuation of proteins by pressure perturbation. PMID- 16683755 TI - Single-molecule fluorescence studies of protein folding and conformational dynamics. PMID- 16683758 TI - Characterizing the protein folding transition state using psi analysis. PMID- 16683759 TI - Conformation of the backbone in unfolded proteins. PMID- 16683760 TI - Protein folding-simulation. PMID- 16683761 TI - GroEL-GroES-mediated protein folding. PMID- 16683757 TI - Early events in protein folding explored by rapid mixing methods. PMID- 16683762 TI - Folding and stability of alpha-helical integral membrane proteins. PMID- 16683763 TI - Photoconversion in the red fluorescent protein from the sea anemone Entacmaea quadricolor: is cis-trans isomerization involved? AB - Proteins from the family of the green fluorescent protein (GFP) are presently extensively used in molecular and cellular biology. Recent studies suggest that isomerization of the chromophore occurs upon excitation and is involved in nonradiative deactivation. Using Raman spectroscopy, we report on photoinduced cis-trans isomerization in the red fluorescent protein eqFP611 from the sea anemone Entacmaea quadricolor. The crystal structure of eqFP611 shows that the chemical structure of the chromophore, p-hydroxybenzylidene-imidazolinone with an extended -conjugated system, is nearly identical to the chromophore of other red fluorescent proteins such as DsRed and HcRed. However, the chromophore of eqFP611 has a trans configuration whereas the chromophore of DsRed has a cis configuration. Upon irradiation with 532-nm light, the absorption of eqFP611 peaking at 559 nm diminished, and concomitantly a drastic decrease in the quantum yield of fluorescence as well as more complex decay kinetics was observed. Upon irradiation, changes in the Raman spectrum of eqFP611 were observed, and the relative intensities and peak positions of the irradiated eqFP611 showed striking similarity with the peaks in the Raman spectrum of DsRed. These observations are tentatively interpreted as trans-to-cis isomerization of the chromophore taking place upon irradiation together with the opening of new, nonradiative pathways. PMID- 16683764 TI - An unusual cationic [2 + 2] cycloaddition in a divergent total synthesis of hongoquercin A and rhododaurichromanic acid A. AB - An unusual polyene cyclization pathway that led to a divergent total synthesis of hongoquercin A and rhododaurichromanic acid A is described here. This work uncovered a unique cationic cyclobutane formation that could be relevant to the biosynthetic pathway for the formation of cyclobutane containing terpenoids in addition to rhododaurichromanic acids. PMID- 16683765 TI - Negative differential resistance and hysteresis through an organometallic molecule from molecular-level crossing. AB - Analogous to a quantum double-dot system, diblock structured molecules could also show negative differential resistance (NDR). Using combined density functional theory and nonequilibrium Green function technique, we show that molecular-level crossing in a molecular double-dot system containing cobaltocene and ferrocene leads to NDR and hysteresis. PMID- 16683766 TI - Captured and cross-linked palladium nanoparticles. AB - Polystyrene-poly(ethylene glycol) resin-captured cross-linked palladium nanopaticles were prepared via a straightforward route, and their heterogeneous behavior was truly confirmed by various tests. They were applied to aqueous Suzuki cross-coupling reactions with various aryl bromides and recycled up to six times without loss of activity. PMID- 16683768 TI - Redox-assisted asymmetric ostwald ripening of CdSe dots to rods. AB - The redox-assisted asymmetric Ostwald ripening of dots to rods was observed upon annealing a concentrated dispersion of CdSe nanocrystals (NCs) containing 0.1 M CdCl2 in 3-amino-1-propanol (APOL)/H2O (v/v = 9/1) at 135 degrees C. Transmission electron microscopic investigation, along with UV-vis and photoluminescence results, revealed that, while the length of these NCs increased upon annealing, their diameter remained constant. The surface oxidation of NC Se atoms to SeO2 and its subsequent dissolution into the basic APOL/H2O mixture as SeO32- was found instrumental for such dot-to-rod transformation. The amine-assisted SeO2 reduction to Se0 (as confirmed by X-ray photoelectron spectroscopy and X-ray diffraction results) provides the Se source for further NC growth. The preferential growth along the c-axis leads to the formation of rods with zinc blende CdSe structure at its growing ends, due to the low-temperature growth conditions. PMID- 16683767 TI - Singlet oxygen generation from water-soluble quantum dot-organic dye nanocomposites. AB - Water-soluble quantum dot-organic dye nanocomposites have been prepared via electrostatic interaction. We used CdTe quantum dots with diameters up to 3.4 nm, 2-aminoethanethiol as a stabilizer, and meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP) as an organic dye. The photophysical properties of the nanocomposite have been investigated. The fluorescence of the parent CdTe quantum dot is largely suppressed. Instead, indirect excitation of the TSPP moiety leads to production of singlet oxygen with a quantum yield of 0.43. The nanocomposite is sufficiently photostable for biological applications. PMID- 16683769 TI - Water activated by atomic oxygen on Au(111) to oxidize CO at low temperatures. AB - The Au(111) surface was populated with atomic oxygen [16O] followed by oxygen labeled water [H218O] at surface temperatures as low as 77 K. When a CO beam was impinged on this surface, both [C16O16O] and [C16O18O] were produced. The results strongly suggest the direct involvement and promoting role of water in CO oxidation on oxygen covered Au(111) at low temperatures. PMID- 16683770 TI - Dynamic control of racemization rate through E-Z photoisomerization of azobenzene and subsequent partial photoresolution under circular polarized light. AB - We have synthesized bicyclic azobenzene dimers that possess enantiomers whose racemization rates could be controlled reversibly through E-Z photoisomerization of the azobenzene units. Upon alternating the exposure to r- and l-CPL, we were able to repeatedly perform partial enrichment of (S)- and (R)- enantiomers, respectively. PMID- 16683771 TI - Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays. AB - In this communication, single molecules of beta-galactosidase were captured on a 1 mm femtoliter array using biotin-streptavidin binding. The femtoliter arrays, containing 24 000 individual reaction chambers, permit digital concentration readout as the percentage of reaction vessels that successfully capture a target molecule is correlated to the bulk target concentration. This capture and readout approach should prove useful for DNA and antibody assays that utilize an enzyme label to catalyze the generation of a fluorescent signal. PMID- 16683772 TI - Nanopore formation and phosphatidylserine externalization in a phospholipid bilayer at high transmembrane potential. AB - Atomic-resolution molecular dynamics simulations of lipid bilayers containing 7% phosphatidylserine (PS) on one leaflet are consistent with experimental observations of membrane poration and PS externalization in living cells exposed to nanosecond, megavolt-per-meter electric pulses. Nanometer-diameter aqueous pores develop within nanoseconds after application of an electric field of 450 mV/nm, and electrophoretic transport of the anionic PS headgroup along the newly constructed hydrophilic pore surface commences even while pore formation is still in progress. PMID- 16683773 TI - Orientation of deoxyhemoglobin at high magnetic fields: structural insights from RDCs in solution. AB - Human normal adult hemoglobin (Hb A) is a tetrameric protein molecule of ~64 kDa consisting of two identical -chains and two identical -chains of 141 and 146 amino acid residues each and four bound heme moieties. In the oxygen-free form of Hb A, also known as deoxyhemoglobin A (deoxy-Hb A), the hemes are paramagnetic with S = 2. We have measured the one-bond spin-spin couplings (1JNH + 1DNH) on (15N,2H)-labeled deoxy-Hb A in solution as a function of magnetic field strengths from 11.7 to 21.1 T and found that these couplings are linearly proportional to the square of the magnetic field. This field dependence provides an opportunity to extract the residual dipolar couplings (RDCs, 1DNH) and, thus, to compare predictions about the solution structure of deoxy-Hb A to crystal structures for this molecule. Such comparison is essential for our understanding of the structure, dynamics, and function of this allosteric protein under conditions close to the physiological state. This report illustrates the usefulness of using the magnetic-field dependent RDCs to determine the solution structure of a large paramagnetic protein. This method is especially valuable for those proteins whose structures must be determined in an oxygen-free environment. PMID- 16683774 TI - Interfacing carbon nanotubes with living cells. AB - We developed a polymer coating for carbon nanotubes (CNTs) that mimics the mucin glycoprotein coating of mammalian cells. CNTs coated with these mucin mimic polymers have two novel properties: they can bind to carbohydrate receptors, providing a means for biomimetic interactions with cell surfaces, and, importantly, they are rendered nontoxic to cells. PMID- 16683775 TI - Internal water molecules of the proton-pumping halorhodopsin in the presence of azide. AB - In the FTIR study of rhodopsins, we have so far found that strongly hydrogen bonded water molecules (O-D stretch at <2400 cm-1) are only present in the proteins exhibiting proton-pumping activity. Halorhodopsin (HR) is a light-driven chloride pump in haloarchaea, which does not possess such water molecules. On the other hand, it is known that addition of azide converts HR into a proton pump. Although the mechanism has not been understood, we observed strongly hydrogen bonded water molecules in the azide-bound HR of Natronobacterium pharaonis (pHR). This finding is consistent with the previous results, implying that the presence of strongly hydrogen-bonded water molecules is requested for the proton-pumping function of rhodopsins. PMID- 16683776 TI - Self-induced 1-D molecular chain growth of thiophene on Ge(100). AB - The adsorption of thiophene on Ge(100) has been studied using scanning tunneling microscopy (STM), high-resolution core-level photoemission spectroscopy (HRPES), and density functional theory (DFT) calculations. Until now, thiophene is known to react with the Ge(100) dimer through a [4 + 2] cycloaddition reaction at room temperature, similar to the case of thiophene on Si(100). However, we found that thiophene has two adsorption geometries on Ge(100) at room temperature, such as a kinetically favorable Ge-S dative bonding configuration and a thermodynamically stable [4 + 2] cycloaddition adduct. Moreover, our STM results show that under 0.25 ML thiophene molecules preferentially produce one-dimensional molecular chain structures on Ge(100) via the Ge-S dative bonding configuration. PMID- 16683777 TI - From monomeric nanofibers to PbS nanoparticles/polymer composite nanofibers through the combined use of gamma-irradiation and gas/solid reaction. AB - Organic metal-salt (lead dimethacrylate (Pb(MA)2)) nanofibers are prepared, and these Pb(MA)2 monomeric nanofibers are successfully converted into PbS nanoparticles/polymer composite nanofibers through the combined use of gamma irradiated polymerization and gas/solid reaction. The resulting composite nanofibers have excellent thermal and chemical stability, and the PbS nanoparticles (with diameters of about 4 nm) are well dispersed in the polymer fiber matrices. This approach could also be extended to methacrylates containing other metal ions. We anticipate that this method would provide a platform for the fabrication of diverse and multifunctional polymer nanocomposite fibers, which would have potential applications in fabricating devices with optical, electric, and magnetic properties. PMID- 16683778 TI - Glycosynthase-mediated synthesis of glycosphingolipids. AB - Glycosphingolipids play crucial roles in virtually every stage of the cell cycle, and their clinical administration has been proposed as a treatment for Alzheimer's, Parkinson's, stroke, and a range of other conditions. However, lack of supply has severely hindered testing of this potential. A novel glycosynthase based synthetic strategy is demonstrated, involving a mutant of an endoglycoceramidase in which the catalytic nucleophile has been ablated. This mutant efficiently couples a range of glycosyl fluoride donors with a range of sphingosine-based acceptors in yields around 95%. This technology opens the door to large-scale production of glycosphingolipids and, thus, to clinical testing. PMID- 16683779 TI - Asymmetric catalysis of the [5 + 2] cycloaddition reaction of vinylcyclopropanes and pi-systems. AB - As part of our studies of metal-catalyzed [m + n (+...o)] cycloadditions, we have previously reported the rhodium-catalyzed [5 + 2] cycloaddition of vinylcyclopropanes (VCPs) and pi-systems. These studies have led to Rh(I) complexes that catalyze these reactions in minutes at room temperature or in water without organic solvents. We describe a comparative evaluation of several chiral catalysts for the [5 + 2] reaction, evaluation of a preferred catalyst, [((R)-BINAP)Rh]+SbF6-, with substrates differing in substitution and tether types producing enantiomeric excesses >/=95% for several systems. A predictive model for the selectivity is also presented. PMID- 16683780 TI - Asymmetric synthesis of protected arylglycines by rhodium-catalyzed addition of arylboronic acids to N-tert-butanesulfinyl imino esters. AB - A new method for the Rh(I)-catalyzed addition of arylboronic acids to N-tert butanesulfinyl imino esters has been developed for the asymmetric synthesis of arylglycine derivatives. This method provides high yields (61-90%) and diastereoselectivities (>98:2) for a variety of functionalized arylboronic acids. The N-sulfinyl arylglycine ester products are versatile intermediates for further transformations, including selective protecting group removal, conversion to beta amino alcohols, and direct incorporation into peptides. PMID- 16683781 TI - PtCl2-catalyzed rearrangement of methylenecyclopropanes. AB - Alkylidenecyclopropanes readily convert into cyclobutene derivatives on treatment with catalytic amounts of PtCl2. The reaction is strongly accelerated when performed under an atmosphere of CO (1 atm). The resulting cyclobutenes are isolated in good to excellent yields for substrates bearing aliphatic as well as aromatic substituents R on their olefinic site. If the substituent R, however, is a very electron-rich arene, the cyclobutenes initially formed react further to give dimeric products with a previously unknown 1,2,2a,7a tetrahydrospiro[cyclobuta[a]indene-7,1'-cyclobutane skeleton. A mechanism accounting for these experimental observations as well as for a deuterium labeling experiment is proposed which implies reactive intermediates at the nonclassical cation/carbene interface. Furthermore it is shown that the PtCl2 catalyzed cyclobutene formation can be geared with subsequent ring-opening/ring closing metathesis (ROM/RCM) events. Finally, a convenient "one pot" method for the preparation of the alkylidenecyclopropane substrates used in this study is presented, which is based on a modified Julia-Kocienski olefination of aldehydes with readily available 1-tert-butyl-1H-tetrazol-5-yl-cyclopropyl sulfone under Barbier conditions. PMID- 16683782 TI - Highly enantioselective aerobic oxidation of alpha-hydroxyphosphonates catalyzed by chiral vanadyl(V) methoxides bearing N-salicylidene-alpha-aminocarboxylates. AB - An unprecedented vanadyl(V) methoxide complex 4 derived from 3,5-dibromo-N salicylidene-l-tert-leucinate enables highly enantioselective aerobic oxidations of alpha-hydroxyphosphonates at ambient temperature with selectivity factors ranging from 3 to >99. PMID- 16683783 TI - A short enantioselective pathway for the synthesis of the anti-influenza neuramidase inhibitor oseltamivir from 1,3-butadiene and acrylic acid. AB - A short synthetic pathway has been developed for the synthesis of oseltamivir (1) or the enantiomer (ent-1). The intermediates and conditions for this process are summarized in Scheme 1. The synthesis provides a number of advantages: (1) use of inexpensive and abundant starting materials; (2) complete enantio-, regio-, and diastereocontrol; (3) avoidance of explosive, azide-type intermediates; (4) good overall yield (ca. 30%, still not completely optimized); and (5) scalability. PMID- 16683784 TI - De novo synthesis of Tamiflu via a catalytic asymmetric ring-opening of meso aziridines with TMSN3. AB - An asymmetric ring-opening reaction of meso-aziridines with TMSN3 was developed using a catalyst prepared from Y(OiPr)3 and chiral ligand 2 in a 1:2 ratio. Excellent enantioselectivity was realized from a wide range of substrates with a practical catalyst loading. The products were efficiently converted to enantiomerically enriched 1,2-diamines, which are versatile chiral building blocks for pharmaceuticals and chiral ligands. This reaction was applied to a catalytic asymmetric synthesis of Tamiflu, a very important anti-influenza drug containing a chiral 1,2-diamino functionality. PMID- 16683786 TI - Nanotube confinement denatures protein helices. AB - In striking contrast to simple polymer physics theory, which does not account for solvent effects, we find that physical confinement of solvated biopolymers decreases solvent entropy, which in turn leads to a reduction in the organized structural content of the polymer. Since our theory is based on a fundamental property of water-protein statistical mechanics, we expect it to have broad implications in many biological and material science contexts. PMID- 16683785 TI - Palladium-catalyzed enantioselective C-3 allylation of 3-substituted-1H-indoles using trialkylboranes. AB - We have developed a new enantioselective C-3 allylation of 3-substituted indoles using allyl alcohol and trialkylboranes. Asymmetric syntheses of 3,3 disubstituted indolines and indolenines in enantiomeric excesses up to 90% have been achieved using the bulky borane 9-BBN-C6H13 as the promoter of the reaction. The dependence of the selectivity on the nature of the borane suggests that the boron reagent has a role beyond promoting ionization of the allyl alcohol. A protocol for oxidation of indolenines to oxindoles has also been developed and led to a formal synthesis of (-)-phenserine. PMID- 16683787 TI - The triplet energy of thymine in DNA. AB - Norfloxacin (NFX) photosensitizes formation of thymine dimers (T<>T) in DNA, while its N(4') acetyl derivative (ANFX) does not. This is evident from the observation of single-strand breaks after enzymatic treatment with T4 endonuclease V and subsequent gel electrophoresis. The triplet energies of NFX and ANFX are estimated at 273 and 268 kJ/mol, respectively, on the basis of triplet-triplet energy transfer quenching by a set of biphenyl and naphthalene derivatives. Hence, the triplet energy of thymine in DNA (i.e., the value for a photosensitizer to produce T<>T) can be estimated at 270 kJ/mol. PMID- 16683788 TI - Periodic mesoporous organosilica hollow spheres with tunable wall thickness. AB - Periodic mesoporous organosilica (PMO) hollow spheres with tunable wall thickness have been successfully synthesized by a new vesicle and a liquid crystal "dual templating" mechanism, which may be applicable for drug and DNA delivery systems, biomolecular encapsulation, as well as nanoreactors for conducting biological reactions at the molecular levels. PMID- 16683789 TI - Dearomatization and functionalization of terpyridine by lutetium(III) alkyl complexes. AB - Lutetium(III)-bis(alkyl) and -tris(alkyl) fragments supported by either 2,2':6',2' '-terpyridine or 4,4',4' '-tri-tert-butyl-2,2':6',2' '-terpyridine are not stable and undergo facile 1,3-alkyl migration under ambient conditions resulting in dearomatization and ortho (2' or 6') functionalization of the terpyridyl ligand, clearly demonstrating that the terpyridyl ligand framework is not as innocent as previously thought. PMID- 16683790 TI - Single quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracers. AB - The present study describes a stabilization of single quantum dot (QD) micelles by hydrophobic silica precursors and an extension of the silica layer to form a silica shell around the micelle. The obtained product consists of up to 92% of single nanocrystals (CdSe, CdSe/ZnS, or CdSe/ZnSe/ZnS quantum dots) in the silica micelles, coated with silica shell. The thickness of silica shell could vary, starting from 3 to 4 nm. Increasing the shell thickness increases the photoluminescent characteristics of QDs in aqueous solution. The silica-shelled single CdSe/ZnS QD micelles possess a high quantum yield in aqueous solution, a controlled small size, sharp photoluminescence spectra (fwhm approximately 30 nm), an absence of aggregation, and a high transparency. The presence of a hydrophobic layer between the QD and silica shell ensures an incorporation of other hydrophobic molecules (with interesting properties) in the close proximity of nanocrystal. Thus, it is possible to combine the characteristics of hybrid material with the priority of small size. The nanoparticles are amino functionalized and ready for conjugation. A comparatively good biocompatibility is demonstrated. The nanoparticles show ability for intracellular delivery and are noncytotoxic during long-term incubation with viable cells in the absence of light exposure, which makes them appropriate for cell tracing and drug delivery. PMID- 16683791 TI - A high-resolution solid-state NMR approach for the structural studies of bicelles. AB - Bicelles are increasingly being used as membrane mimicking systems in NMR experiments to investigate the structure of membrane proteins. In this study, we demonstrate the effectiveness of a 2D solid-state NMR approach that can be used to measure the structural constraints, such as heteronuclear dipolar couplings between 1H, 13C, and 31P nuclei, in bicelles without the need for isotopic enrichment. This method does not require a high radio frequency power unlike the presently used rotating-frame separated-local-field (SLF) techniques, such as PISEMA. In addition, multiple dipolar couplings can be measured accurately, and the presence of a strong dipolar coupling does not suppress the weak couplings. High-resolution spectra obtained from magnetically aligned DMPC:DHPC bicelles even in the presence of peptides suggest that this approach will be useful in understanding lipid-protein interactions that play a vital role in shaping up the function of membrane proteins. PMID- 16683792 TI - Perfectly straight nanowires of fullerenes bearing long alkyl chains on graphite. AB - Fullerene derivatives bearing long alkyl chains epitaxially adsorb on the basal plane of graphite forming well-ordered one-dimensional lamellae. Within the lamellae, the C60 moieties are organized in a zigzag-type fashion. The ordering is mainly governed by the substitution pattern and chain length of the alkyl chains. The electrochemical activity of the C60 groups is fully maintained in the surface-confined assemblies. PMID- 16683793 TI - Palladium-catalyzed [3 + 3] cycloaddition of trimethylenemethane with azomethine imines. AB - A palladium-catalyzed [3 + 3] cycloaddition of trimethylenemethane (TMM) with azomethine imines has been developed to produce hexahydropyridazine derivatives under simple and mild conditions. The use of substituted TMM precursors highlights the difference of this system from previously reported [3 + 2] cycloaddition of TMMs under palladium catalysis. The present [3 + 3] cycloaddition reactions are also applicable to couplings with nitrones. PMID- 16683794 TI - Nickel-catalyzed multicomponent connection of dimethylzinc, alkynes, 1,3 butadiene, aldehydes, and amines. AB - A nickel(0) species catalyzes the five-component connection reaction of dimethylzinc, alkynes, 1,3-butadiene, aldehydes, and amines, furnishing either 1 or 2 selectively in good yields depending on the kinds of amines, aromatic or aliphatic amines, respectively. PMID- 16683795 TI - Xenon biosensor amplification via dendrimer-cage supramolecular constructs. AB - Polyamidoamine dendrimers were synthesized with a single biotin moiety and used with cryptophane-A cages to form supramolecular biosensor constructs. These new biosensors amplified the NMR signals obtained from polarized xenon 8 times more than the original Xe biosensor. PMID- 16683796 TI - Rhodium-catalyzed cycloisomerization of N-propargyl enamine derivatives. AB - A novel rhodium-catalyzed cycloisomerization has been developed which converts N propargyl enamine derivatives to their isomeric six-membered azacycles in good yields under mild reaction conditions. The [Rh(C2H2)2Cl]2/P(4-F-C6H4)3 catalyst, in combination with DABCO as a base and DMF as a solvent, was found to be effective in promoting the cyclization process. The reaction is proposed to proceed through an intramolecular nucleophilic attack of the enamine to the rhodium vinylidene followed by a base-mediated proton-shunting process. The base was found to play a crucial role in the transformation, indicating the proton migration to be an integral component of the catalytic cycle. An aprotic polar solvent was required for high conversion, which points to the intermediacy of a zwitterionic intermediate. The present reaction should prove useful for the efficient synthesis of various six-membered azacycles that are important structural motifs of many natural isolates and medicinal agents. PMID- 16683797 TI - Ultrafast T-jump in water: studies of conformation and reaction dynamics at the thermal limit. AB - We report the development of ultrafast T-jump with time resolution reaching the fundamental time scale of water thermalization time ( approximately 5 ps). The T jump heats the bulk water up to 20 degrees C via the overtone absorption of the OH- stretch at 1.5 mum. The example given here shows the application of the methodology, for the first time, and the results demonstrate distinct time scales for solvation, conformational change, and thermal reaction. PMID- 16683798 TI - On the origin of the unique properties of supported Au nanoparticles. AB - The unique catalytic activity of supported Au nanoparticles has been ascribed to various effects including thickness/shape, the metal oxidation state, and support effects. Previously, we reported the synthesis of ordered Au monolayers and bilayers on TiO(x), with the latter being significantly more active for CO oxidation than the former. In the present study, the electronic and chemical properties of ordered monolayer and bilayer Au films have been characterized by infrared reflection adsorption spectroscopy using CO as a probe and ultraviolet photoemission spectroscopy. The Au overlayers are found to be electron-rich and to have significantly different electronic properties compared with bulk Au. The common structural features of ordered Au bilayers and Au bilayer nanoparticles on TiO2(110) are described, and the exceptionally high catalytic activity of the Au bilayer structure related to its unique electronic properties. PMID- 16683799 TI - Chiroptical and computational studies of a bridled chiroporphyrin and of its nickel(II), copper(II), and zinc(II) complexes. AB - Circular dichroism (CD) spectra and density functional theory (DFT) calculations are reported for a series of conformationally bistable chiroporphyrins with 8 methylene bridles MBCP-8, which can display either an alphaalphaalphaalpha or an alphabetaalphabeta orientation of their meso substituents. From DFT geometry optimizations, the most stable form of ZnBCP-8 is found to be the alphaalphaalphaalpha conformer. By passing to NiBCP-8, there is a strong stabilization of the alphabetaalphabeta conformation with respect to the alphaalphaalphaalpha conformation, consistent with the X-ray structures of alphaalphaalphaalpha-ZnBCP-8 and alphabetaalphabeta-NiBCP-8. A correlation between the sign of the CD signal in the Soret region and the conformation of the BCP-8 compounds is reported: the alphaalphaalphaalpha conformers H2BCP-8 and ZnBCP-8 show a positive CD signal, whereas the alphabetaalphabeta conformers NiBCP-8 and CuBCP-8 exhibit a negative signal. The possible contributions to the rotational strengths of alphabetaalphabeta-NiBCP-8 and alphaalphaalphaalpha-ZnBCP 8, calculated on the basis of their crystal structures, have been analyzed. The CD signals are found to result from a combination of both the inherent chirality of the porphyrin and of extrinsic contributions due to the chiral bridles. These results may have a broad significance for understanding the chiroptical properties of chiral porphyrins and hemoproteins and for monitoring stimuli responsive, conformationally bistable chiroporphyrin compounds. PMID- 16683800 TI - Effect of hydration on coordination properties of uranyl(VI) complexes. A first principles molecular dynamics study. AB - Results from Car-Parrinello molecular dynamics simulations are reported for [UO2(OH2)5]2+, UO2(NO3)2(OH2)2, and UO2(NO3)2(eta2-tmma) (tmma = tetramethylmalonamide) in the gas phase and in aqueous solution. The distances between uranyl and neutral ligands such as water and tmma are decreased by up to 0.2 angstroms upon hydration, whereas those between uranyl and the nitrate ion are increased by up to 0.08 angstroms. According to pointwise thermodynamic integration involving constrained molecular dynamics simulations, solvation facilitates the transition of the chelating nitrate ligand to a eta1-bonding mode: the free energy of UO2(eta2-NO3)(eta1-NO3)(OH2)2 relative to the bis chelating minimum drops from 3.9 kcal/mol in vacuo to 1.4 kcal/mol in water. Optimizations in a polarizable continuum (specifically, the conductor-like screening model in conjunction with the zero-order regular approximation and triple-zeta Slater basis sets) can qualitatively reproduce the geometrical changes from explicit hydration. PMID- 16683801 TI - Optimization of unnatural base pair packing for polymerase recognition. AB - As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet. PMID- 16683802 TI - Experimental and theoretical investigations of new dinuclear palladium complexes as precatalysts for the amination of aryl chlorides. AB - A series of new palladium dinuclear species with general formula [Pd2X(mu-X)[mu P(t)Bu2(Bph-R)]] (X = Cl, Br; Bph = biphenyl; R = H, Me, NMe2) have been prepared. The two palladium centers in these species are bridged by one of the aromatic rings of the biphenyl group present in the corresponding phosphine. The X-ray crystal structure of one of these complexes has been obtained, providing a clear picture of the bonding pattern. The stability of these dimers in solution is shown to be highly dependent on the nature of the phosphine R group and also on the bridging halide. When R = NMe2, the dimers dissociate, yielding the palladium(II) compounds PdX2[P(t)Bu2(BPh-NMe2)] (X = Cl, Br), and the X-ray crystal structure of one of them (X = Br) has shown that the biphenyl group from the phosphine interacts directly with the metal center. This interaction seems to play an important role in stabilizing the otherwise coordinatively unsaturated palladium(II) complex. In contrast, when R = H or Me, the analogous monomeric palladium(II) complexes are unstable and undergo cyclometalation to generate a palladium(II) dinuclear species in which each of the two phosphines cyclometalates with the palladium centers forming a strained four-membered ring. In addition to their unusual structures, these aryl-bridged dimers have also proven to be excellent precatalysts for the amination of aryl chlorides. To rationalize some of the experimental results, a detailed DFT computational study has been carried out and is presented herein. PMID- 16683804 TI - Photoreversible DNA condensation using light-responsive surfactants. AB - A means to control DNA compaction with light illumination has been developed using the interaction of DNA with a photoresponsive cationic surfactant. The surfactant undergoes a reversible photoisomerization upon exposure to visible (trans isomer, more hydrophobic) or UV (cis isomer, more hydrophilic) light. As a result, surfactant binding to DNA and the resulting DNA condensation can be tuned with light. Dynamic light scattering (DLS) measurements were used to follow lambda-DNA compaction from the elongated-coil to the compact globular form as a function of surfactant addition and light illumination. The results reveal that compaction occurs at a surfactant-to-DNA base pair ratio of approximately 7 under visible light, while no compaction is observed up to a ratio of 31 under UV light. Upon compaction, the measured diffusion coefficient increases from a value of 0.6 x 10(-8) cm2/s (elongated coil with an end-to-end distance of 1.27 microm) to a value of 1.7 x 10(-8) cm2/s (compact globule with a hydrodynamic radius of 120 nm). Moreover, the light-scattering results demonstrate that the compaction process is completely photoreversible. Fluorescence microscopy with T4-DNA was used to further confirm the light-scattering results, allowing single-molecule detection of the light-controlled coil-to-globule transition. These structural studies were combined with absorbance and fluorescence spectroscopy of crystal violet in order to elucidate the binding mechanism of the photosurfactant to DNA. The results indicate that both electrostatic and hydrophobic forces are important in the compaction process. Finally, a DNA-photosurfactant-water phase diagram was constructed to examine the effects of both DNA and surfactant concentration on DNA compaction. The results reveal that precipitation, which occurs during the latter stages of condensation, can also be reversibly controlled with light illumination. The combined results clearly show the ability to control the interaction between DNA and the complexing agent and, therefore, DNA condensation with light. PMID- 16683803 TI - Modulation of the axial water hydrogen-bonding properties by chemical modification of the substrate in resting state, substrate-bound heme oxygenase from Neisseria meningitidis; coupling to the distal H-bond network via ordered water molecules. AB - The hydrogen bonding of ligated water in ferric, high-spin, resting-state substrate complexes of heme oxygenase from Neisseria meningitidis has been systematically perturbed by variable electron-withdrawing substituents on the hemin periphery. The pattern of 1H NMR-detected dipolar shifts due to the paramagnetic anisotropy is strongly conserved among the four complexes, with the magnitude of dipolar shifts or anisotropy increasing in the order of substituent formyl < vinyl < methyl. The magnetic anisotropy is axial and oriented by the axial Fe-His23 bond, and while individual anisotropies have uncertainties of approximately 5%, the relative values of deltachi (and the zero-field splitting constant, D proportional, variant deltachi(ax)) are defined to 1%. The unique changes in the axial field strength implied by the variable zero-field splitting are in accord with expectations for the axial water serving as a stronger H-bond donor in the order of hemin substituents formyl > vinyl > methyl. These results establish the axial anisotropy (and D) as a sensitive probe of the H-bonding properties of a ligated water in resting-state, substrate complexes of heme oxygenase. Correction of observed labile proton chemical shifts for paramagnetic influences indicates that Gln49 and His53, some approximately 10 angstroms from the iron, sense the change in the ligated water H-bonding to the three nonligated ordered water molecules that link the two side chains to the iron ligand. The present results augur well for detecting and characterizing changes in distal water H-bonding upon mutagenesis of residues in the distal network of ordered water molecules and strong H-bonds. PMID- 16683805 TI - High resolution spectral differentiation of enantiomers: benzo[a]pyrene tetrols complexed with a promiscuous antibody. AB - The interaction of the highly cross-reactive anti-PAH monoclonal antibody with four diastereomeric benzo[a]pyrene tetrols (BPTs) is studied by means of fluorescence line-narrowing spectroscopy. It is shown that the interaction of enantiomers of cis-BPT and trans-BPT with the antibody involves different complex geometries. These spatially different ligand-protein interactions alter the relative intensities of the excited-state vibrational frequencies of immunocomplexed molecules allowing for unambiguous spectroscopic resolution of all four enantiomeric isomers. This study represents the first example of a high resolution, fluorescence-based spectroscopic method capable of enantiospecific differentiation. PMID- 16683806 TI - Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions. AB - Site-saturated mutagenesis experiments were carried out on the His234 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) to characterize its functional role in ERG7 activity and to determine its effect on the oxidosqualene cyclization/rearrangement reaction. Two novel intermediates, (13alphaH)-isomalabarica-14(26),17E,21-trien-3beta-ol and protosta-20,24-dien 3beta-ol, isolated from ERG7(H234X) mutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikov cation and protosteryl cation that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. In addition, we obtained mutants that showed a complete change in product specificity from lanosterol formation to either protosta-12,24 dien-3beta-ol or parkeol production. Finally, the repeated observation of multiple abortive and/or alternative cyclization/arrangement products from various ERG7(H234X) mutants demonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affect both the stability of the cation-pi interaction and generate product diversity. PMID- 16683807 TI - Dynamical valence fluctuation at the charge-density-wave phase boundary in iodide bridged Pt compound [Pt(chxn)2I]I2. AB - We synthesized a novel iodo-bridged linear chain platinum compound, having the quasi-two-dimensional charge-density-wave (CDW) ground state and the smallest band gap. In this compound, we discovered an anomalous valence state in the boundary region at which the CDW phase alternates in the crystal by means of ESR, X-ray diffuse scattering, STM, and electrical resistivity. This anomalous state can be explained by the fast fluctuation between Pt(IV)-I...Pt(II) and Pt(II)...I Pt(IV) in the double well potential. This is the first observation of the dynamical fluctuation of the CDW phase among the quasi one-dimensional halogen bridged complexes. PMID- 16683808 TI - High-quality sodium rare-earth fluoride nanocrystals: controlled synthesis and optical properties. AB - We report a general synthesis of high-quality cubic (alpha-phase) and hexagonal (beta-phase) NaREF4 (RE: Pr to Lu, Y) nanocrystals (nanopolyhedra, nanorods, nanoplates, and nanospheres) and NaYF(4):Yb,Er/Tm nanocrystals (nanopolyhedra and nanoplates) via the co-thermolysis of Na(CF3COO) and RE(CF3COO)3 in oleic acid/oleylamine/1-octadecene. By tuning the ratio of Na/RE, solvent composition, reaction temperature and time, we can manipulate phase, shape, and size of the nanocrystals. On the basis of its alpha --> beta phase transition behavior, along the rare-earth series, NaREF4 can be divided into three groups (I: Pr and Nd; II: Sm to Tb; III: Dy to Lu, Y). The whole controlled-synthesis mechanism can be explained from the point of view of free energy. Photoluminescent measurements indicate that the value of I610/I590 and the overall emission intensity of the NaEuF4 nanocrystals are highly correlative with the symmetries of the Eu3+ ions in both the lattice and the surface. PMID- 16683809 TI - 17O solid-state NMR spectroscopic studies of the involvement of water vapor in molecular sieve formation by dry-gel conversion. AB - Dry-gel conversion is a relatively new approach for molecular sieve synthesis. This method potentially has several advantages over the traditional hydrothermal synthesis and can be used to prepare molecular sieves with certain unique properties. The technique involves treating the predried reactive gel powder with water vapor at elevated temperature and pressure. The role of water vapor in this apparent solid transformation is, however, not clear. In this work, we directly monitored the involvement of 17O-enriched water vapor in crystallization of AlPO4 11 (an aluminophosphate-based molecular sieve) by 17O solid-state NMR spectroscopy. In addition to 17O magic-angle spinning technique, several dipolar coupling based double-resonance methods including 17O[27Al], 17O[31P] rotational echo double-resonance, 17O --> 31P and 1H --> 17O cross polarization techniques were used for spectral editing to select different 17O species. The results show that water from the vapor phase slowly exchanges with water molecules strongly bound to the AlPO intermediates first. Then 17O atoms are gradually incorporated in both P-O-H and P-O-Al units in the layered intermediate. There are three different P sites in AlPO4-11. Interestingly, during the transformation from the layered intermediate to AlPO4-11, the 17O atoms prefer to bond to the P2 and P3, but not to P1. The absence of 17O atoms in the first coordination sphere of P1 site suggests that some building units such as joint four- and six-membered rings involving hydrogen bonding with structure-directing agents are common in both layered intermediate and AlPO4-11 and they are not affected by the transformation from the layered phase to the AlPO4-11 framework. PMID- 16683810 TI - A new class of silica cross-linked micellar core-shell nanoparticles. AB - Micellar nanoparticles made of surfactants and polymers have attracted wide attention in the materials and biomedical community for controlled drug delivery, molecular imaging, and sensing; however, their long-term stability remains a topic of intense study. Here we report a new class of robust, ultrafine silica core-shell nanoparticles formed from silica cross-linked, individual block copolymer micelles. Compared with pure polymeric micelles, the main advantage of the new core-shell nanoparticles is that they have significantly improved stability and do not break down during dilution. We also studied the drug loading and release properties of the silica cross-linked micellar particles, and we found that the new core-shell nanoparticles have a slower release rate which allows the entrapped molecules to be slowly released over a much longer period of time under the same experimental conditions. A range of functional groups can be easily incorporated through co-condensation with the silica matrix. The potential to deliver hydrophobic agents into cancer cells has been demonstrated. Because of their unique structures and properties, these novel core-shell nanoparticles could potentially provide a new nanomedicine platform for imaging, detection, and treatment, as well as novel colloidal particles and building blocks for mutlifunctional materials. PMID- 16683811 TI - Synthesis of bis(indenyl)zirconium dihydrides and subsequent rearrangement to eta5,eta3-4,5-dihydroindenediyl ligands: evidence for intermediates during the hydrogenation to tetrahydroindenyl derivatives. AB - Exposure of eta9,eta5-bis(indenyl)zirconium sandwich complexes to 4 atm of H2 resulted in facile oxidative addition to furnish the corresponding zirconocene dihydrides, (eta5-C9H5-1,3-R2)2ZrH2 (R = SiMe3, SiMe2Ph, CHMe2). Continued hydrogenation completed conversion to the tetrahydroindenyl derivatives, (eta5 C9H9-1,3-R2)2ZrH2. Deuterium labeling studies established that dihydrogen (dideuterium) addition to the benzo rings is intramolecular and stereospecific, occurring solely from the endo face of the ligand, proximal to the zirconium. In the absence of dihydrogen, the bis(indenyl)zirconium dihydrides rearranged to new zirconium monohydride complexes containing an unusual eta5,eta3-4,5 dihydroindenediyl ligand, arising from metal-to-benzo ring hydrogen transfer. Mechanistic studies, including a normal, primary kinetic isotope effect measured at 23 degrees C, are consistent with a pathway involving regio- and stereoselective insertion of a benzo C=C bond into a zirconium hydride. The stereochemistry of the insertion reaction, and hence the eta5,eta3-4,5 dihydroindenediyl product, is influenced by the presence of donor ligands and controlled by the preferred conformation of the indenyl rings. Exposure of the zirconium hydrides containing the eta5,eta3-4,5-dihydroindenediyl rings to 1 atm of dihydrogen afforded the tetrahydroindenyl zirconium dihydride complexes, establishing the intermediacy of this unusual coordination environment during benzo ring hydrogenation. PMID- 16683812 TI - Efficient light harvesting in dye-endcapped conjugated polymers probed by single molecule spectroscopy. AB - The development of sophisticated microscopic models of energy transfer in linear multichromophoric systems such as conjugated polymers is rarely matched by suitable experimental studies on the microscopic level. To assess the roles of structural, temporal, and energetic disorder in energy transfer, single molecule spectroscopic investigations of the elementary processes leading to energetic relaxation in conjugated polymers are desirable. We present a detailed study of energy transfer processes occurring in dye-endcapped conjugated polymer molecules on the single molecule level. These processes are mostly masked in ensemble investigations. Highly efficient intramolecular energy transfer along a single polyindenofluorene chain to a perylene endcap occurs in many instances and is resolved in real time. We further consider the spectral emission characteristics of the single molecule, the polarization anisotropy which reveals the chain conformation, the fluorescence intermittency, and the temperature dependence and conclude that the efficiency of energy transfer in the ensemble is controlled by the statistics of the individual molecules. The weak thermal activation of energy transfer indicates the involvement of vibrational modes in interchromophoric coupling. Whereas backbone-endcap coupling is strong, the rate-limiting step for intramolecular energy transfer is the migration along the backbone. The results are particularly relevant to understanding undesired exciton trapping on fluorenone defects in polyfluorenes. PMID- 16683813 TI - Unexpected chemical and electrochemical properties of M3N@C80 (M = Sc, Y, Er). AB - The unexpected isomerization of N-ethyl [6,6]-pyrrolidino-Y3N@C80 to the [5,6] regioisomer is reported, as well as the synthesis, characterization, and electrochemical analysis of Er3N@C80 derivatives. A complete electrochemical study of the M3N@C80 species (M = Sc, Y, Er) and their derivatives is presented. We introduce electrochemistry as a new tool in the characterization of the [5,6] and [6,6] regioisomers of trimetallic nitride endohedral metallofullerenes. PMID- 16683814 TI - A pirouette on a metallofullerene sphere: interconversion of isomers of N tritylpyrrolidino I(h) Sc3N@C80. AB - The pure I(h) isomer of Sc3N@C80 was allowed to react with N-triphenylmethyl-5 oxazolidinone via the corresponding azomethine ylide. The reaction results in the formation of two monoadducts; one (1b) is the kinetic product, and the other (1a) is thermodynamically more stable. Small amounts of the bisadducts were also formed. The structure of the thermodynamic monoadduct 1a was shown conclusively by NMR spectroscopy and X-ray crystallography to result from addition across the 5,6-ring junction. The kinetic product 1b was demonstrated to be the 6,6-ring juncture adduct on the basis of NMR experiments and X-ray crystallography. In refluxing chlorobenzene pure 1b was converted to the more thermodynamically stable 1a isomer. These N-tritylpyrrolidino derivatives are potentially useful precursor compounds for further derivatization for various applications. PMID- 16683815 TI - A nickel tripeptide as a metallodithiolate ligand anchor for resin-bound organometallics. AB - The molecular structure of the acetyl CoA synthase enzyme has clarified the role of individual nickel atoms in the dinickel active site which mediates C-C and C-S coupling reactions. The NiN2S2 portion of the biocatalyst (N2S2 = a cysteine glycine-cysteine or CGC4- tripeptide ligand) serves as an S-donor ligand comparable to classical bidentate ligands operative in organometallic chemistry, ligating the second nickel which is redox and catalytically active. Inspired by this biological catalyst, the synthesis of NiN2S2 metalloligands, including the solid-phase synthesis of resin-bound Ni(CGC)2-, and sulfur-based derivatization with W(CO)5 and Rh(CO)2+ have been carried out. Through comparison to analogous well-characterized, solution-phase complexes, Attenuated Total Reflectance FTIR spectroscopy establishes the presence of unique heterobimetallic complexes, of the form [Ni(CGC)]M(CO)x, both in solution and immobilized on resin beads. This work provides the initial step toward exploitation of such an evolutionarily optimized nickel peptide as a solid support anchor for hybrid bioinorganic organometallic catalysts. PMID- 16683816 TI - Tandem nucleophilic addition/fragmentation reactions and synthetic versatility of vinylogous acyl triflates. AB - A thorough analysis of the chemistry of vinylogous acyl triflates provides insight into important chemical processes and opens new directions in synthetic technology. Tandem nucleophilic addition/C-C bond cleaving fragmentation reactions of cyclic vinylogous acyl triflates 1 yield a variety of acyclic acetylenic compounds. Full details are disclosed herein. A wide array of nucleophiles, such as organolithium and Grignard reagents, lithium enolates and their analogues, hydride reagents, and lithium amides, are applied. The respective reactions produce ketones 2, 1,3-diketones and their analogues 3, alcohols 4, and amides 5. The present reactions are proposed to proceed through a 1,2-addition of the nucleophile to the carbonyl group of starting triflates 1 to form tetrahedral alkoxide intermediates C, followed by Grob-type fragmentation, which effects C-C bond cleavage to yield acyclic acetylenic compounds 2-5 and 7. The potent nucleofugacity of the triflate moiety is channeled through the sigma bond framework of 1, providing direct access to the fragmentation pathway without denying other typical reactions of cyclic vinylogous esters. The synthetic versatility of vinylogous acyl triflates, including functionalization reactions of the cyclic enone core (1 --> 6 or 8), is also illustrated. PMID- 16683817 TI - New palladium(II)-(eta(3/5)- or eta1-indenyl) and dipalladium(I)-(mu,eta3 indenyl) complexes. AB - Reaction of the dimeric species [(eta3-Ind)Pd(mu-Cl)]2 (1) (Ind = indenyl) with NEt3 gives the complex (eta(3-5)-Ind)Pd(NEt3)Cl (3), whereas the analogous reactions with BnNH2 (Bn = PhCH2) or pyridine (py) afford the complexes trans L2Pd(eta1-Ind)Cl (L = BnNH2 (4), py (5)). Similarly, the one-pot reaction of 1 with a mixture of BnNH2 and the phosphine ligands PR3 gives the mixed-ligand, amino and phosphine species (PR3)(BnNH2)Pd(eta1-Ind)Cl (R = Cy (6a), Ph (6b)); the latter complexes can also be prepared by addition of BnNH2 to (eta(3-5) Ind)Pd(PR3)Cl (R = Cy (2a), Ph (2b)). Complexes 6 undergo a gradual decomposition in solution to generate the dinuclear Pd(I) compounds (mu,eta3-Ind)(mu Cl)Pd2(PR3)2 (R = Cy (7a), Ph (7b)) and the Pd(II) compounds (BnNH2)(PR3)PdCl2 (R = Cy (8a), Ph (8b)), along with 1,1'-biindene. The formation of 7 is proposed to proceed by a comproportionation reaction between in situ-generated Pd(II) and Pd0 intermediates. Interestingly, the reverse of this reaction, disproportionation, also occurs spontaneously to give 2. All new compounds have been characterized by NMR spectroscopy and, in the case of 3, 4, 5, 6a, 7a, 7b, and 8a, by X-ray crystallography. PMID- 16683819 TI - Imaging nanoscale spatio-temporal thermal fluctuations. AB - Spatial and temporal fluctuations of the electric polarization were imaged in polymer thin films near the glass transition using electric force microscopy. Below the glass transition the fluctuations are quasi-static, and spatial fluctuations were found to quantitatively agree with predictions for thermal fluctuations. Temporal fluctuations appear near the glass transition. Images of the space-time nanoscale dynamics near the glass transition are produced and analyzed. PMID- 16683820 TI - Nitrogen violation of the isolated pentagon rule. AB - We examine a range of fullerene isomers with and without the presence of nitrogen using density-functional-based calculations. We show that nitrogen stabilizes those with neighboring pentagons, allowing sp(2) bonded carbon networks to overcome the conventional rule that paired pentagons are not allowed through energetic constraints. This implies a new family of azafullerenes containing nitrogen-stabilized pentagon pairs, many of which will be considerably smaller than C(60). We discuss the relevance for nitrogen-doped nanotube structures and the topological importance for nanotube distortion and closure. PMID- 16683822 TI - Self-avoiding flexible polymers under spherical confinement. AB - We compute the free energy of confinement for a flexible self-avoiding polymer inside a spherical cavity. Accurate numerical results allow us to arbitrate between three competing scaling predictions. For moderate confinement, the free energy exhibits a power-law dependence on cavity size that is different from what is observed for planar and cylindrical confinement. At high monomer concentrations, crossover to a different scaling regime is observed, consistent with the screening of the excluded-volume interactions. We demonstrate how our findings lead to a revised prediction for the escape time of a polymer from a spherical confinement. PMID- 16683823 TI - Field-effect transistors assembled from functionalized carbon nanotubes. AB - We have fabricated field-effect transistors from carbon nanotubes using a novel selective placement scheme. We use carbon nanotubes that are covalently bound to molecules containing a hydroxamic acid functionality. The functionalized nanotubes bind strongly to basic metal oxide surfaces, but not to silicon dioxide. Upon annealing, the functionalization is removed, restoring the electronic properties of the nanotubes. The devices thus fabricated show excellent electrical characteristics. PMID- 16683824 TI - Tunable single-walled carbon nanotube microstructure in the liquid and solid states using poly(acrylic acid). AB - Poly(acrylic acid) is shown to control the level of SWNT dispersion in aqueous mixtures and the state of dispersion in a solid composite. At low pH, PAA stabilized suspensions containing 0.1 wt % SWNT have a waterlike viscosity, but this mixture thickens as the pH is raised. This behavior is reversed when pH is again lowered. Changing pH varies the SWNT microstructure between aggregated and well-exfoliated states, as evidenced by electron microscopy and electrical conductivity measurements. PMID- 16683825 TI - Effects of sidewall functionalization on conducting properties of single wall carbon nanotubes. AB - We investigated the conducting properties of functionalized single wall nanotubes (SWNTs) with a finite addend concentration. Robust differences are found between monovalent and divalent additions. For the former a small number of addends can significantly disrupt the ballistic conductance of nanotubes near the Fermi level. As the concentration increases the conductance decreases rapidly and approaches zero at addend to C ratio around 25%. In contrast, divalent functionalizations have weak effects, and the nanotube quantum conductance remains above 50% of that of a perfect tube even for an addend concentration as large as 25%. These differences can be attributed to the formation of impurity states near the Fermi level for monovalent additions, while divalent addends create impurity states far away from the Fermi level. PMID- 16683827 TI - Scanning tunneling microscope excited cathodoluminescence from ZnS nanowires. AB - The electronic excitation spectrum of ZnS nanowires (NWs) is characterized by cathodoluminescence (CL) excited using the tip of a scanning tunneling microscope as a highly localized and bright source of low-energy (150-350 eV) electrons with impinging power densities in the range of up to 60 kW/cm(2) or more. The CL spectra reveal significant differences when compared with the photoluminescence spectra of ZnS NWs. The differences can be associated with the properties of the surface region of the NWs, which are preferentially emphasized in the CL spectra owing to the small probing depth of low-energy electrons. PMID- 16683828 TI - Low-frequency current fluctuations in individual semiconducting single-wall carbon nanotubes. AB - We present a systematic study on low-frequency current fluctuations of nanodevices consisting of one single semiconducting nanotube, which exhibit significant 1/f-type noise. By examining devices with different switching mechanisms, carrier types (electrons vs holes), and channel lengths, we show that the 1/f fluctuation level in semiconducting nanotubes is correlated to the total number of transport carriers present in the system. However, the 1/f noise level per carrier is not larger than that of most bulk conventional semiconductors, e.g., Si. The pronounced noise level observed in nanotube devices simply reflects on the small number of carriers involved in transport. These results not only provide the basis to quantify the noise behavior in a one-dimensional transport system but also suggest a valuable way to characterize low-dimensional nanostructures based on the 1/f fluctuation phenomenon. PMID- 16683829 TI - A single electron transistor on an atomic force microscope probe. AB - We report fabrication as well as proof-of-concept experiments of a noninvasive sensor of weak nanoscale electric fields. The sensor is a single electron transistor (SET) placed at the tip of a noncontact atomic force microscope (AFM). This is a general technology to make any nanometer-sized lithography pattern at edges or tips of a cantilever. The height control of the AFM allows the SET to hover a few nanometers above the substrate, improving both the electric field sensitivity and lateral resolution of the electrometer. Our AFM-SET sensor is prepared by a scalable technology. It means that the probe can be routinely fabricated and replaced, if broken. PMID- 16683830 TI - Electromechanical carbon nanotube switches for high-frequency applications. AB - We describe the fabrication and characterization of a nanoelectromechanical (NEM) switch based on carbon nanotubes. Our NEM structure consists of single-walled nanotubes (SWNTs) suspended over shallow trenches in a SiO(2) layer, with a Nb pull electrode beneath. The nanotube growth is done on-chip using a patterned Fe catalyst and a methane chemical vapor deposition (CVD) process at 850 degrees C. Electrical measurements of these devices show well-defined ON and OFF states as a dc bias up to a few volts is applied between the CNT and the Nb pull electrode. The CNT switches were measured to have speeds that are 3 orders of magnitude higher than MEMS-based electrostatically driven switches, with switching times down to a few nanoseconds, while at the same time requiring pull voltages less than 5 V. PMID- 16683832 TI - Measurement of the adhesion force between carbon nanotubes and a silicon dioxide substrate. AB - Carbon nanotube adhesion force measurements were performed on single-walled nanotubes grown over lithographically defined trenches. An applied vertical force from an atomic force microscope (AFM), in force distance mode, caused the tubes to slip across the 250-nm-wide silicon dioxide trench tops at an axial tension of 8 nN. The nanotubes slipped at an axial tension of 10 nN after being selectively coated with a silicon dioxide layer. PMID- 16683835 TI - Ejection dynamics of a simple liquid from individual carbon nanotube nozzles. AB - Molecular dynamics simulations show that the flow of a high pressurized atomic liquid inside carbon nanotube "pipets" occurs in one-atom-thick well-defined laminae. Fluxes and velocities at ejection are a function of the inlet diameter and the type of outlet. In the conditions investigated here, the force of the ejected liquid is similar in value to that of biomotors, while the output per second is of the order of picoliters. PMID- 16683837 TI - Addressable molecular tweezers for DNA-templated coupling reactions. AB - Here we report the construction of fully addressable DNA-based molecular tweezers to actuate coupling reactions in a programmable fashion. Three tweezers, each bearing two coupling reactants, are self-assembled on a linear DNA track. A fourth tweezer floating freely in solution can be brought to any one of the tweezers and close them by the addition of a unique pair of "fuel" DNA strands. The coupling reactions happen when the tweezers are closed, and this can be controlled sequentially from one tweezer to another. A molecular device of this kind would not only enable programmable chemical reactions but also allow distance-dependent control of biomolecular interactions. PMID- 16683839 TI - Differential ion transport induced electroosmosis and internal recirculation in heterogeneous osmosis membranes. AB - Water and ion transport through a heterogeneous membrane separating two electrolyte solutions at different concentrations is investigated by using molecular dynamics simulations. The membrane features pairs of oppositely charged pores with identical diameters. Simulation results indicate that the differential transport of K(+) and Cl(-) ions through the membrane pores creates an electrical potential difference across the membrane, which then induces an electroosmotic water flux. The induced electroosmosis creates an internal recirculation loop of water between adjacent pores. The implications of these new observations are discussed. PMID- 16683841 TI - Patterning polyethylene oligomers on carbon nanotubes using physical vapor deposition. AB - Periodic patterning on one-dimensional (1D) carbon nanotubes (CNTs) is of great interest from both scientific and technological points of view. In this letter, we report using a facile physical vapor deposition method to achieve periodic polyethylene (PE) oligomer patterning on individual CNTs. Upon heating under vacuum, PE degraded into oligomers and crystallized into rod-shaped single crystals. These PE rods periodically decorate on CNTs with their long axes perpendicular to the CNT axes. The formation mechanism was attributed to "soft epitaxy" growth of PE oligomer crystals on CNTs. Both SWNTs and MWNTs were decorated successfully with PE rods. The intermediate state of this hybrid structure, MWNTs absorbed with a thin layer of PE, was captured successfully by depositing PE vapor on MWNTs detached from the solid substrate, and was observed using high-resolution transmission electron microscopy. Furthermore, this hybrid structure formation depends critically on CNT surface chemistry: alkane modification of the MWNT surface prohibited the PE single-crystal growth on the CNTs. We anticipate that this work could open a gateway for creating complex CNT based nanoarchitectures for nanodevice applications. PMID- 16683842 TI - Control of nanopore wetting by a photochromic spiropyran: a light-controlled valve and electrical switch. AB - By modifying the surface of nanoporous alumina membranes using mixtures of a photochromic spiropyran and hydrophobic molecules, it is possible to control the admission of water into the membrane using light. When the spiropyran is in the thermally stable, relatively hydrophobic closed form, the membrane is not wet by an aqueous solution. Upon exposure to UV light, the spiropyran photoisomerizes to the more polar merocyanine form, allowing water to enter the pores and cross the membrane. Thus, the photosensitive membrane acts as a burst valve, allowing the transport of water and ions across the membrane. If the aqueous solution contains ions, then the membrane acts as an electrical switch; photoisomerization leads to a two-order-of-magnitude increase in ionic conductance, allowing a current to flow across the membrane. Exposure to visible light causes photoisomerization of the merocyanine back to the closed, spiro form, but dewetting of the membrane does not occur spontaneously, due to a high activation barrier. PMID- 16683843 TI - Adsorption of polyelectrolyte molecules to a nanostructured monolayer of amphiphiles. AB - We investigate the conformation of atactic poly(sodium 4-styrene sulfonate) (PSS) molecules both in water-ethanol solutions and adsorbed to a template provided by a nanostructured monolayer of flat-lying octadecylamines on graphite. The conformations of the PSS molecules in these solutions undergo two conformational transitions, a re-entry transition at 40 vol % and a coil-globule conformational transition at 80 vol % ethanol. On the surface the conformations are characterized by two different length scales: on a short length scale they are defined by the template, whereas the large-scale structures reflect the conformations in solution. We conclude that the conformational transitions are discontinuous on the level of single molecules and appear continuous in the ensemble. PMID- 16683848 TI - In situ encapsulation of horseradish peroxidase in electrospun porous silica fibers for potential biosensor applications. AB - Nanoporous silica nanofibers have been employed as a matrix to encapsulate horseradish peroxide enzymes via a simple electrospinning method. A viscous solution of prehydrolyzed tetramethyl orthosilicate, beta-d-glucose, poly(vinyl alcohol), and enzymes were employed as spinning solution to generate porous fibers in the form of nonwoven mats. The silica fiber mats thus produced have a high surface area because of the small diameter (100 to 200 nm) of the fibers as well as the extreme porosity (2 to 4 nm) of individual fibers caused by the glucose template present in them. The high surface area, mechanical flexibility, thermal stability, reusability, and freedom of encapsulating various enzymes make porous silica nanofibers excellent biosensors. PMID- 16683850 TI - Anchoring of nematic liquid crystals on viruses with different envelope structures. AB - The ordering of synthetic liquid crystals near surfaces is known to be dependent on the nanoscopic structure and chemical functionality of surfaces. In this letter, we report that the orientational ordering of synthetic liquid crystals on surfaces decorated with viruses is also dependent on the structures of the viruses. Each of the four virions investigated had diameters of approximately 100 nm, but three of the viruses (influenza virus, La Crosse virus, and vesicular stomatitis virus) were enveloped in a lipid bilayer, whereas one virus (adenovirus) was not. We observed that lipid bilayer-enveloped viruses induce homeotropic (perpendicular) ordering of a nematic liquid crystal upon contact with the liquid crystal. In contrast, nonenveloped virus (adenovirus)-treated surfaces caused a near-planar orientation of the liquid crystal. We conclude that the homeotropic ordering of liquid crystals is a signature of the presence of enveloped viruses present on surfaces. These results suggest new approaches to the design of nanostructured materials that incorporate viruses as well as suggest methods that can be used to amplify the presence of nanoscopic virions into micrometer-sized domains of liquid crystal that can be optically probed. PMID- 16683851 TI - Qdot nanobarcodes for multiplexed gene expression analysis. AB - We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4 fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods. PMID- 16683856 TI - The nature of the discontinuation syndrome associated with antidepressant drugs. AB - A common phenomenon accompanying treatment with nearly every major class of antidepressant is the emergence of the discontinuation syndrome in some patients. It is seen most frequently after the abrupt cessation of agents with shorter half lives. The term withdrawal has been used in the past; however, the distinctions between discontinuation symptoms and drug withdrawal are clear. Thus, the use of proper terminology when discussing this phenomenon with patients will help to alleviate concerns and stop the spread of common misperceptions. In addition, awareness of the unique nature of discontinuation effects and a grasp of the typical time frame of their emergence can assist in distinguishing between discontinuation syndrome and relapse. As a result, it is vital that both patients and their relatives, especially caregivers, be provided with adequate education and a realistic and objective appraisal of expected outcomes upon initiation of antidepressant treatment. PMID- 16683857 TI - Physiologic mechanisms underlying the antidepressant discontinuation syndrome. AB - The rate at which serotonin reuptake inhibitor (SRI) treatment is terminated and the duration of treatment appear to be key factors in predicting discontinuation symptoms. The development of animal models to explain the mechanisms of this clinical problem has proved challenging, because less than half of all patients experience any discontinuation symptoms, many of which are subjective in nature. One explanation is that SRI discontinuation symptoms may arise from the rapid decrease in serotonin (5-HT) availability when treatment ends abruptly. Yet, it would appear that discontinuation discomforts may not be mediated exclusively through 5-HT receptors, given the major regulatory role 5-HT exerts on a number of specific chemical receptor systems in the brain. As a result, attempts to explain the determinants of this phenomenon rely on a certain level of speculation. This article examines the possible physiologic bases for the antidepressant discontinuation syndrome and briefly describes these adaptations. It discusses the 3 systems most likely to account for at least part of the symptomatology--the 5-HT, the norepinephrine, and the cholinergic systems--and the possible interactions among them. It also attempts to explain their implications in the therapeutic actions of antidepressants in patients with affective and anxiety disorders. PMID- 16683858 TI - Prospective studies of adverse events related to antidepressant discontinuation. AB - The value of a prospective assessment of discontinuation-emergent symptoms proximal to the termination of antidepressant treatment cannot be overstated. Though varying in frequency and intensity, nearly all classes of antidepressants have been linked with discontinuation reactions and the associated psychological, physical, and somatic discomfort. Spontaneous reports have been typically used to gauge the risks of discontinuation reactions. Judging from a number of prospective studies, spontaneous reports very likely underestimate the occurrence of discontinuation reactions. This probability suggests that systematic inquiry must urgently become a part of the assessment in antidepressant discontinuation studies. Insight into the number and type of events that may occur following antidepressant discontinuation may be gleaned from instruments such as the Discontinuation-Emergent Signs and Symptoms Scale. This article takes a comprehensive view of a number of studies dealing with discontinuation-related adverse events. It discusses key issues in the analysis of incidence rates of antidepressant discontinuation-emergent adverse events such as the obvious bias of both clinicians' and patients' being aware of the treatment discontinuation. This article also looks at early prospective studies of antidepressant discontinuation reactions based on spontaneous reports and discusses, while making the case for, prospective studies based on systematic inquiry. PMID- 16683859 TI - Monoamine depletion studies: implications for antidepressant discontinuation syndrome. AB - The likelihood of a connection between serotonin reuptake inhibitor (SRI) discontinuation and an acute reduction in synaptic serotonin (5-HT) has ignited interest in the similarities between SRI discontinuation syndrome and the symptoms observed after acute tryptophan depletion, which reduces synaptic 5-HT levels. An open question is whether these 2 phenomena have shared characteristics because of a similar underlying mechanism. The evidence in support of a similar underlying mechanism includes the observation that comparable proportions of SRI treated patients experience depressive symptoms following tryptophan depletion and SRI discontinuation. Furthermore, the proportion of people who have emotional changes with rapid antidepressant discontinuation may be parallel to the proportion of people who experience those changes with rapid tryptophan depletion. PMID- 16683860 TI - Antidepressant discontinuation syndrome: consensus panel recommendations for clinical management and additional research. AB - OBJECTIVE: Currently, no evidence-based guidelines exist for the management of serotonin reuptake inhibitor (SRI) discontinuation syndrome. This article summarizes recommendations with respect to future research as well as clinical management recommendations for SRI discontinuation syndrome. PARTICIPANTS: In April 2004, a panel of physicians convened in New York City to discuss recommendations for clinical management of and additional research on SRI discontinuation syndrome. EVIDENCE: Previous guidance for management of SRI discontinuation syndrome was proposed in 1997 in a consensus meeting also chaired by Alan F. Schatzberg. A literature search of the PubMed database was conducted to identify articles on SRI discontinuation syndrome that have been published since 1997. CONSENSUS PROCESS: The 2004 panel reviewed important preclinical and clinical studies, discussed prospective investigation of this syndrome in clinical trials, and suggested the establishment of a research network to collect data in naturalistic settings. The panel also reviewed the management recommendations published in 1997 and subsequently updated the recommendations, taking into account the latest clinical data as well as the personal experience of its members with patients. CONCLUSIONS: Additional preclinical and clinical studies are necessary to further elucidate the underlying biological mechanisms of SRI discontinuation syndrome and to identify the patient populations and agents that are most affected by this phenomenon. Management strategies include gradual tapering of doses and should emphasize clinical monitoring and patient education. PMID- 16683861 TI - The clinical applications of a systems approach. PMID- 16683863 TI - Model selection and the molecular clock. PMID- 16683862 TI - Relaxed phylogenetics and dating with confidence. AB - In phylogenetics, the unrooted model of phylogeny and the strict molecular clock model are two extremes of a continuum. Despite their dominance in phylogenetic inference, it is evident that both are biologically unrealistic and that the real evolutionary process lies between these two extremes. Fortunately, intermediate models employing relaxed molecular clocks have been described. These models open the gate to a new field of "relaxed phylogenetics." Here we introduce a new approach to performing relaxed phylogenetic analysis. We describe how it can be used to estimate phylogenies and divergence times in the face of uncertainty in evolutionary rates and calibration times. Our approach also provides a means for measuring the clocklikeness of datasets and comparing this measure between different genes and phylogenies. We find no significant rate autocorrelation among branches in three large datasets, suggesting that autocorrelated models are not necessarily suitable for these data. In addition, we place these datasets on the continuum of clocklikeness between a strict molecular clock and the alternative unrooted extreme. Finally, we present analyses of 102 bacterial, 106 yeast, 61 plant, 99 metazoan, and 500 primate alignments. From these we conclude that our method is phylogenetically more accurate and precise than the traditional unrooted model while adding the ability to infer a timescale to evolution. PMID- 16683864 TI - Chromosomal organization: mingling with the neighbors. PMID- 16683865 TI - Citation advantage of open access articles. AB - Open access (OA) to the research literature has the potential to accelerate recognition and dissemination of research findings, but its actual effects are controversial. This was a longitudinal bibliometric analysis of a cohort of OA and non-OA articles published between June 8, 2004, and December 20, 2004, in the same journal (PNAS: Proceedings of the National Academy of Sciences). Article characteristics were extracted, and citation data were compared between the two groups at three different points in time: at "quasi-baseline" (December 2004, 0-6 mo after publication), in April 2005 (4-10 mo after publication), and in October 2005 (10-16 mo after publication). Potentially confounding variables, including number of authors, authors' lifetime publication count and impact, submission track, country of corresponding author, funding organization, and discipline, were adjusted for in logistic and linear multiple regression models. A total of 1,492 original research articles were analyzed: 212 (14.2% of all articles) were OA articles paid by the author, and 1,280 (85.8%) were non-OA articles. In April 2005 (mean 206 d after publication), 627 (49.0%) of the non-OA articles versus 78 (36.8%) of the OA articles were not cited (relative risk = 1.3 [95% Confidence Interval: 1.1-1.6]; p = 0.001). 6 mo later (mean 288 d after publication), non-OA articles were still more likely to be uncited (non-OA: 172 [13.6%], OA: 11 [5.2%]; relative risk = 2.6 [1.4-4.7]; p < 0.001). The average number of citations of OA articles was higher compared to non-OA articles (April 2005: 1.5 [SD = 2.5] versus 1.2 [SD = 2.0]; Z = 3.123; p = 0.002; October 2005: 6.4 [SD = 10.4] versus 4.5 [SD = 4.9]; Z = 4.058; p < 0.001). In a logistic regression model, controlling for potential confounders, OA articles compared to non-OA articles remained twice as likely to be cited (odds ratio = 2.1 [1.5-2.9]) in the first 4-10 mo after publication (April 2005), with the odds ratio increasing to 2.9 (1.5-5.5) 10-16 mo after publication (October 2005). Articles published as an immediate OA article on the journal site have higher impact than self-archived or otherwise openly accessible OA articles. We found strong evidence that, even in a journal that is widely available in research libraries, OA articles are more immediately recognized and cited by peers than non-OA articles published in the same journal. OA is likely to benefit science by accelerating dissemination and uptake of research findings. PMID- 16683866 TI - Open access increases citation rate. PMID- 16683867 TI - Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs. AB - BACKGROUND: Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. METHODS AND FINDINGS: To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10) particles, two logs lower than that used previously. CONCLUSIONS: Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10) rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate. PMID- 16683868 TI - Laboratory productivity and the rate of manual peripheral blood smear review: a College of American Pathologists Q-Probes study of 95,141 complete blood count determinations performed in 263 institutions. AB - CONTEXT: Automated laboratory hematology analyzers are capable of performing differential counts on peripheral blood smears with greater precision and more accurate detection of distributional and morphologic abnormalities than those performed by manual examinations of blood smears. Manual determinations of blood morphology and leukocyte differential counts are time-consuming, expensive, and may not always be necessary. The frequency with which hematology laboratory workers perform manual screens despite the availability of labor-saving features of automated analyzers is unknown. OBJECTIVE: To determine the normative rates with which manual peripheral blood smears were performed in clinical laboratories, to examine laboratory practices associated with higher or lower manual review rates, and to measure the effects of manual smear review on the efficiency of generating complete blood count (CBC) determinations. DESIGN: From each of 3 traditional shifts per day, participants were asked to select serially, 10 automated CBC specimens, and to indicate whether manual scans and/or reviews with complete differential counts were performed on blood smears prepared from those specimens. Sampling continued until a total of 60 peripheral smears were reviewed manually. For each specimen on which a manual review was performed, participants indicated the patient's age, hemoglobin value, white blood cell count, platelet count, and the primary reason why the manual review was performed. Participants also submitted data concerning their institutions' demographic profiles and their laboratories' staffing, work volume, and practices regarding CBC determinations. The rates of manual reviews and estimations of efficiency in performing CBC determinations were obtained from the data. SETTING: A total of 263 hospitals and independent laboratories, predominantly located in the United States, participating in the College of American Pathologists Q-Probes Program. RESULTS: There were 95,141 CBC determinations examined in this study; participants reviewed 15,423 (16.2%) peripheral blood smears manually. In the median institution (50th percentile), manual reviews of peripheral smears were performed on 26.7% of specimens. Manual differential count review rates were inversely associated with the magnitude of platelet counts that were required by laboratory policy to trigger smear reviews and with the efficiency of generating CBC reports. Lower manual differential count review rates were associated with laboratory policies that allowed manual reviews solely on the basis of abnormal automated red cell parameters and that precluded performing repeat manual reviews within designated time intervals. The manual scan rate elevated with increased number of hospital beds. In more than one third (35.7%) of the peripheral smears reviewed manually, participants claimed to have learned additional information beyond what was available on automated hematology analyzer printouts alone. CONCLUSION: By adopting certain laboratory practices, it may be possible to reduce the rates of manual reviews of peripheral blood smears and increase the efficiency of generating CBC results. PMID- 16683870 TI - Defining error in anatomic pathology. AB - CONTEXT: Although much has been said and written about medical error and about error in pathology since the publication of the Institute of Medicine's report on medical error in 1999, precise definitions of what constitutes error in anatomic pathology do not exist for the specialty. Without better definitions, it is impossible to accurately judge errors in pathology. The lack of standardized definitions has implications for patient care and for the legal judgment of malpractice. OBJECTIVES: To review the goals of anatomic pathology, to discuss the problems inherent in applying these goals to the judgment of error in pathology, to offer definitions of major and minor errors in pathology, and to discuss error in anatomic pathology in relation to the classic laboratory test cycle. DATA SOURCES: Existing literature. CONCLUSION: Definitions for major and minor error in anatomic pathology are proffered, and anatomic pathology error is characterized in the classic test cycle. PMID- 16683871 TI - Errors and error rates in surgical pathology: an Association of Directors of Anatomic and Surgical Pathology survey. AB - This survey on errors in surgical pathology was commissioned by the Association of Directors of Anatomic and Surgical Pathology Council to explore broad perceptions and definitions of error in surgical pathology among its membership and to get some estimate of the perceived frequency of such errors. Overall, 41 laboratories were surveyed, with 34 responding to a confidential questionnaire. Six small, 13 medium, and 10 large laboratories (based on specimen volume), predominantly located in the United States, were surveyed (the remaining 5 laboratories did not provide this particular information). The survey questions, responses, and associated comments are presented. It is clear from this survey that we lack uniformity and consistency with respect to terminology, definitions, and the identification/documentation of errors in surgical pathology. An appeal is made for the urgent need to reach some consensus in order to address these discrepancies as we prepare to combat the issue of errors in surgical pathology. PMID- 16683872 TI - Pediatric surgical pathology: pitfalls and strategies for error prevention. AB - CONTEXT: Few data exist regarding quality measures for pediatric surgical pathology, types of errors, or how error-prone situations and diagnostic pitfalls can be minimized. OBJECTIVE: This review reports on survey findings regarding methodology for quality assurance and error detection measurement and classification in pediatric surgical pathology. It presents information regarding, and quality aspects of, intraoperative consultations in pediatric surgical pathology. General strategies for identifying diagnostic pitfalls in pediatric surgical pathology are briefly discussed. DATA SOURCES: A survey of children's hospitals based on a survey created by the Association of Directors of Anatomic and Surgical Pathology, literature review, and institutional quality assurance records provided information for this review. CONCLUSIONS: Approaches to quality assurance and error reduction in pediatric surgical pathology are similar to those used in general surgical pathology. The children's hospitals that were surveyed used a variety of standard quality assurance measures. Because of differences in data collection, classification, and reporting, it is not possible to provide a detailed analysis of the types of diagnostic error across institutions at this time. Intraoperative consultations are a potential source of error. Pediatric neoplasms and Hirschsprung disease account for the majority of intraoperative consultations in the pediatric setting. Further considerations include the unique aspects of pediatric medical and neoplastic disorders and special diagnostic criteria, classification, grading, and staging requirements. PMID- 16683873 TI - Errors in surgical neuropathology and the influence of cognitive biases: the psychology of intelligence analysis. AB - CONTEXT: A significant difficulty that pathologists encounter in arriving at a correct diagnosis is related to the way information from various sources is processed and assimilated in context. OBJECTIVE: These issues are addressed by the science of cognitive psychology. Although cognitive biases are the focus of a number of studies on medical decision making, few if any focus on the visual sciences. DATA SOURCES: A recent publication authored by Richards Heuer, Jr, The Psychology of Intelligence Analysis, directly addresses many of the cognitive biases faced by neuropathologists and anatomic pathologists in general. These biases include visual anticipation, first impression, and established mindsets and subconsciously influence our critical decision-making processes. CONCLUSIONS: The book points out that while biases are an inherent property of cognition, the influence of such biases can be recognized and the effects blunted. PMID- 16683874 TI - Medicolegal aspects of error in pathology. AB - OBJECTIVE: To discuss the various ways error is defined in surgical pathology. To identify errors in pathology practice identified by an analysis of pathology malpractice claims. DESIGN: Three hundred seventy-eight pathology malpractice claims were reviewed. Nuisance claims and autopsy claims were excluded; 335 pathology claims remained and were analyzed to identify repetitive patterns of specimen type and diagnostic category. SETTING: All pathology malpractice claims reported to The Doctors Company of Napa, Calif, between 1998 and 2003. RESULTS: Fifty-seven percent of malpractice claims involved just 5 categories of specimen type and/or diagnostic error, namely, breast specimens, melanoma, cervical Papanicolaou tests, gynecologic specimens, and system (operational) errors. Sixty three percent of claims involved failure to diagnose cancer, resulting in delay in diagnosis or inappropriate treatment. CONCLUSION: A false-negative diagnosis of melanoma was the single most common reason for filing a malpractice claim against a pathologist. Nearly one third involved melanoma misdiagnosed as Spitz nevus, "dysplastic" nevus, spindle cell squamous carcinoma, atypical fibroxanthoma, and dermatofibroma. While breast biopsy claims were a close second to melanoma, when combined with breast fine-needle aspiration and breast frozen section claims, breast specimens were the most common cause of pathology malpractice claims. Cervical Papanicolaou test claims were third in frequency behind melanoma and breast; 98% involved false-negative Papanicolaou tests. Forty two percent of gynecologic surgical pathology claims involved misdiagnosed ovarian tumors, and 85% of these were false-negative diagnoses of malignancy. The most common cause of system errors was specimen "mix-ups" involving breast or prostate needle biopsies. PMID- 16683875 TI - Surgical pathology--second reviews, institutional reviews, audits, and correlations: what's out there? Error or diagnostic variation? AB - CONTEXT: A variety of methodologies have been used to report error rates in surgical pathology within the peer-reviewed medical literature. The media has selectively and superficially reported these error rates creating a climate of disinformation between physicians and the public. OBJECTIVES: To review the medical literature on diagnostic error in surgical pathology and summarize and compare these data with selected reports in the print and broadcast media. DESIGN: A search of the medical literature from the National Library of Medicine database using the heading "Error and Pathology Diagnosis." RESULTS: Three thousand nine hundred ninety-two citations were found, of which 83 directly measured in some manner errors in surgical and cytopathology. Major error rates ranged from 1.5% to 5.7% globally for institutional consults. Error rates were less, 0.26% to 1.2% for global in-house prospective review and 4.0% for in-house and retrospective blinded review. Error rates also varied by anatomic site: skin, institutional consult, 1.4%; prostate, institutional consult, 0.5%; and thyroid, institutional consult, 7.0%. Error rates reported in citations used by the Wall Street Journal were as follows: prostate, Gleason score changed by 1 point, 44% and resultant change in treatment for prostate cancer, 10%; for breast, altered lumpectomy or mastectomy plan, 8%; and diagnosis changed for thyroid lesions, 18%. Errors in second opinion on breast lesions (single pathologist author for the study) fall within the range of global reviews. Errors for second opinions on prostate cancer were principally 81% upgrades in Gleason score for prostate core needle biopsies. However, this resulted in an upgrade of patient risk category in only 10.8% of patients. Data for the article on change in diagnosis of thyroid lesions were incomplete. There appeared to be 3 significant diagnostic errors (4.5%). CONCLUSIONS: Pathology is not immune to the power of the media to create concern about accuracy of diagnosis in surgical pathology and cytopathology. Detailed analysis of the medical literature cited by the media determines that painting the big picture and hitting the highlights can be profoundly misleading. PMID- 16683876 TI - Comparing methods to measure error in gynecologic cytology and surgical pathology. AB - CONTEXT: Both gynecologic cytology and surgical pathology use similar methods to measure diagnostic error, but differences exist between how these methods have been applied in the 2 fields. OBJECTIVE: To compare the application of methods of error detection in gynecologic cytology and surgical pathology. DATA SOURCES: Review of the literature. CONCLUSIONS: There are several different approaches to measuring error, all of which have limitations. Measuring error using reproducibility as the gold standard is a common method to determine error. While error rates in gynecologic cytology are well characterized and methods for objectively assessing error in the legal setting have been developed, meaningful methods to measure error rates in clinical practice are not commonly used and little is known about the error rates in this setting. In contrast, in surgical pathology the error rates are not as well characterized and methods for assessing error in the legal setting are not as well defined, but methods to measure error in actual clinical practice have been characterized and preliminary data from these methods are now available concerning the error rates in this setting. PMID- 16683877 TI - Error reduction in surgical pathology. AB - CONTEXT: Because of its complex nature, surgical pathology practice is inherently error prone. Currently, there is pressure to reduce errors in medicine, including pathology. OBJECTIVE: To review factors that contribute to errors and to discuss error-reduction strategies. DESIGN: Literature review. RESULTS: Multiple factors contribute to errors in medicine, including variable input, complexity, inconsistency, tight coupling, human intervention, time constraints, and a hierarchical culture. Strategies that may reduce errors include reducing reliance on memory, improving information access, error-proofing processes, decreasing reliance on vigilance, standardizing tasks and language, reducing the number of handoffs, simplifying processes, adjusting work schedules and environment, providing adequate training, and placing the correct people in the correct jobs. CONCLUSIONS: Surgical pathology is a complex system with ample opportunity for error. Significant error reduction is unlikely to occur without a sustained comprehensive program of quality control and quality assurance. Incremental adoption of information technology and automation along with improved training in patient safety and quality management can help reduce errors. PMID- 16683878 TI - Improving patient safety through quality assurance. AB - CONTEXT: Anatomic pathology laboratories use several quality assurance tools to detect errors and to improve patient safety. OBJECTIVE: To review some of the anatomic pathology laboratory patient safety quality assurance practices. DESIGN: Different standards and measures in anatomic pathology quality assurance and patient safety were reviewed. MAIN OUTCOME MEASURES: Frequency of anatomic pathology laboratory error, variability in the use of specific quality assurance practices, and use of data for error reduction initiatives. RESULTS: Anatomic pathology error frequencies vary according to the detection method used. Based on secondary review, a College of American Pathologists Q-Probes study showed that the mean laboratory error frequency was 6.7%. A College of American Pathologists Q-Tracks study measuring frozen section discrepancy found that laboratories improved the longer they monitored and shared data. There is a lack of standardization across laboratories even for governmentally mandated quality assurance practices, such as cytologic-histologic correlation. The National Institutes of Health funded a consortium of laboratories to benchmark laboratory error frequencies, perform root cause analysis, and design error reduction initiatives, using quality assurance data. Based on the cytologic-histologic correlation process, these laboratories found an aggregate nongynecologic error frequency of 10.8%. Based on gynecologic error data, the laboratory at my institution used Toyota production system processes to lower gynecologic error frequencies and to improve Papanicolaou test metrics. CONCLUSION: Laboratory quality assurance practices have been used to track error rates, and laboratories are starting to use these data for error reduction initiatives. PMID- 16683879 TI - Critical values in anatomic pathology. AB - Similar to critical values (CVs) in clinical pathology, occasional diagnoses in surgical pathology and cytology could require immediate notification of the physician to rapidly initiate treatment. However, there are no established CV guidelines in anatomic pathology. A retrospective review of surgical pathology reports was recently conducted to study the incidence of CVs in surgical pathology and to survey the perceptions of pathologists and clinicians about CVs in surgical pathology, with a similar analysis of CVs performed in cytology. The results indicated that CVs in surgical pathology and cytology are uncommon but not rare and that there is a wide range of opinion among pathologists and between pathologists and clinicians about the need for an immediate telephone call and about the degree of urgency. It was obvious from the study that there is a lack of consensus in identifying what constitutes surgical pathology and cytology CV cases. Since the Institute of Medicine's report on medical errors, there has been an increasing number of initiatives to improve patient safety. Having guidelines for anatomic pathology CVs could enhance patient safety, in contrast to the current practice in which CV cases are managed based on common sense and on personal experience. Therefore, a discussion involving the pathology community might prove useful in an attempt to establish anatomic pathology CV guidelines that could represent a practice improvement. PMID- 16683880 TI - Communicating critical values in anatomic pathology. AB - Critical values in anatomic pathology are usually information sensitive, whereas most such values in laboratory medicine are time sensitive. However, there is an important time element in anatomic pathology as well. Pathologists should be aware that many medicolegal actions against radiologists are based on failure to communicate "abnormal" results in a timely manner. Are pathologists the next group that will be targeted? Pathologists can spend much time trying to communicate important data that will affect patient care to someone who will accept the information. This is not an efficient use of pathologists' professional time. Most important, what are our obligations to patients to communicate "critical" abnormal results to the treating physician? What results need to be so communicated? Are pathologists obliged to contact the patient directly if there is a failure to communicate the critical results to a clinician? We explore these questions to promote discussion of these important issues as they relate to pathologists' liability and to patient care. PMID- 16683881 TI - Determining customer satisfaction in anatomic pathology. AB - CONTEXT: Measurement of physicians' and patients' satisfaction with laboratory services has become a standard practice in the United States, prompted by national accreditation requirements. Unlike other surveys of hospital-, outpatient care-, or physician-related activities, no ongoing, comprehensive customer satisfaction survey of anatomic pathology services is available for subscription that would allow continual benchmarking against peer laboratories. Pathologists, therefore, must often design their own local assessment tools to determine physician satisfaction in anatomic pathology. OBJECTIVE: To describe satisfaction survey design that would elicit specific information from physician customers about key elements of anatomic pathology services. DESIGN: The author shares his experience in biannually assessing customer satisfaction in anatomic pathology with survey tools designed at the Henry Ford Hospital, Detroit, Mich. Benchmarks for physician satisfaction, opportunities for improvement, and characteristics that correlated with a high level of physician satisfaction were identified nationally from a standardized survey tool used by 94 laboratories in the 2001 College of American Pathologists Q-Probes quality improvement program. RESULTS: In general, physicians are most satisfied with professional diagnostic services and least satisfied with pathology services related to poor communication. CONCLUSIONS: A well-designed and conducted customer satisfaction survey is an opportunity for pathologists to periodically educate physician customers about services offered, manage unrealistic expectations, and understand the evolving needs of the physician customer. Armed with current information from physician customers, the pathologist is better able to strategically plan for resources that facilitate performance improvements in anatomic pathology laboratory services that align with evolving clinical needs in health care delivery. PMID- 16683882 TI - Molecular pathology: future issues. AB - CONTEXT: The field of molecular pathology is expanding in complexity. To achieve competency, vigilance is required. OBJECTIVE: To review the advances in clinically useful molecular biologic techniques and to identify their applications in clinical practice, as presented at the 13th Annual William Beaumont Hospital DNA Symposium. DATA SOURCES: The 4 manuscripts submitted were reviewed and their major findings were compared with the literature on the same or related topics. STUDY SELECTION: Manuscripts address the use of molecular or immunophenotyping by flow cytometry to evaluate the origin or presence of sepsis, respectively; the use of imatinib mesylate to treat chronic myeloid leukemia and the nature of resistance to imatinib; and the use of 9 and 10 fluorochromes during clinical flow cytometric studies. DATA SYNTHESIS: The epidemiologic evaluation of a septic outbreak may be monitored using molecular techniques that track the relatedness of isolates. A potential biomarker for the presence of early sepsis is CD64. Intracellular signal transduction pathways are altered in malignancy. Imatinib mesylate inhibits the BCR-ABL kinase created by translocation of the long arms of chromosomes 9 and 22 in chronic myeloid leukemia. Resistance to imatinib may be secondary to mutation in the BCR-ABL kinase domain or residual leukemic stem cells that imatinib does not kill. The use of 9 or 10 fluorochromes simultaneously during flow cytometry has many clinical advantages; however, software for data analysis is needed. CONCLUSION: The current postgenomic era will continue to emphasize the use of microarrays and database software for genomic, transcriptomic, proteomic, nutrigenomic, and pharmacogenomics screening to search for a useful clinical assay. The number of molecular pathologic techniques will expand as additional disease-associated mutations are defined. PMID- 16683883 TI - Neutrophil CD64 is an improved indicator of infection or sepsis in emergency department patients. AB - CONTEXT: Sepsis, affecting millions of individuals annually with an associated high mortality rate, is among the top 10 causes of death. In addition, improvements in diagnostic tests for detecting and monitoring sepsis and infection have been limited in the last 25 years. Neutrophil CD64 expression has been proposed as an improved diagnostic test for the evaluation of infection and sepsis. OBJECTIVE: To evaluate the diagnostic performance of a quantitative flow cytometric assay for leukocyte CD64 expression in comparison with the standard tests for infection/sepsis in an ambulatory care setting. DESIGN: Prospective analysis of 100 blood samples from patients from an emergency department setting in a 965-bed tertiary care suburban community hospital was performed for neutrophil CD64 expression, C-reactive protein, erythrocyte sedimentation rate, and complete blood count. The laboratory findings were compared with a clinical score for the likelihood of infection/sepsis, which was obtained by a blinded retrospective chart review. RESULTS: The diagnostic performance, as gauged by the clinical score, varied with neutrophil CD64 (sensitivity 87.9%, specificity 71.2%, efficiency 76.8%) and outperformed C-reactive protein (sensitivity 88.2%, specificity 59.4%, efficiency 69.4%), absolute neutrophil count (sensitivity 60.0%, specificity 50.8%, efficiency 53.8%), myeloid left shift (sensitivity 68.2%, specificity 76.3%, efficiency 73.3%), and sedimentation rate (sensitivity 50.0%, specificity 65.5%, efficiency 61.0%). CONCLUSION: Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice. PMID- 16683884 TI - The application of molecular techniques to the study of hospital infection. AB - CONTEXT: Nosocomial infections represent an important cause of morbidity and mortality in hospital settings, resulting in high health care costs. The roles of an epidemic investigation are to recognize that a problem exists, to compare characteristics of affected persons with those of similar but unaffected persons (case-control study), and to identify risk factors. Integrating typing methods as part of conventional epidemiologic surveillance is cost-effective and results in a reduction in rates of nosocomial infections. During the past 10 years, there has been unprecedented progress in molecular biology and in the application of nucleic acid technology to the study of the epidemiology of human infections. OBJECTIVES: To summarize the available molecular tests for determination of the relatedness of microorganisms causing nosocomial infections, emphasizing the most useful applications of the tests to the study of the epidemiology of hospital acquired infection; and to discuss the appropriate use of these tests in the prevention and control of hospital-associated infection. DATA SOURCE: Published English-language literature from 1980 to the present. CONCLUSIONS: Pulsed-field gel electrophoresis is the method of choice for strain delineation. The newest techniques include polymerase chain reaction and multilocus sequence typing, in which various housekeeping genes that are stable markers of strain identity are sequenced. Molecular techniques are broadly applicable to the study of diverse pathogens. Typing data obtained by DNA analysis should always be considered together with epidemiologic information, because only this combination will enable the most accurate epidemiologic evaluation. PMID- 16683885 TI - Imatinib resistance: obstacles and opportunities. AB - OBJECTIVE: To review the current status of resistance to imatinib mesylate (IM) in patients with chronic myelogenous leukemia, and the obstacles and opportunities presented by the development of this resistance. DATA SOURCES AND STUDY SELECTION: Review of selected studies obtained from a MEDLINE search encompassing the years 1950 to 2004. DATA EXTRACTION AND DATA SYNTHESIS: Relevant information from the selected studies was abstracted and summarized. CONCLUSIONS: The identification of the Philadelphia chromosome and the subsequent discovery that it represents a translocation between the long arms of chromosomes 9 and 22 producing an aberrant tyrosine kinase, known as BCR-ABL1, has catalyzed our understanding and treatment of this hematologic malignancy. An extensive search for molecules to block the aberrant BCR-ABL1 protein resulted in the development of IM as an orally bioavailable agent with remarkable efficacy in producing hematologic, cytogenetic, and molecular remissions. However, follow-up of patients treated with IM has demonstrated that some patients can develop resistance to IM with progression of their leukemia. Multiple mechanisms of resistance have been identified. The dominant mechanism appears to be mutations in the kinase domain of BCR-ABL1, which result in altered affinity of IM for the BCR-ABL1 protein. Recently, small-molecule, combined SRC and ABL1 inhibitors have been developed and entered into clinical trials. These inhibitors appear effective in inhibiting most of the mutant BCR-ABL1 molecules that are resistant to IM. The rapid development of new therapies for treatment of chronic myelogenous leukemia brings the promise that this disorder can be cured or controlled in many patients with oral drugs that have a low toxicity profile. PMID- 16683886 TI - 9-color and 10-color flow cytometry in the clinical laboratory. AB - CONTEXT: The development of commercial flow cytometers capable of detecting more than 10 simultaneous fluorescent signals presents opportunities for improved diagnosis and monitoring of patients with leukemia and lymphoma. OBJECTIVE: To describe instrument and reagent characteristics necessary for successful 9-color and 10-color flow cytometry in a clinical setting. DESIGN: Systematic review of issues related to instrument settings, reagent performance, and general principles of panel construction. RESULTS: Nine-color and 10-color flow cytometry offers the possibility for increased accuracy in population identification, the ability to obtain detailed information from paucicellular specimens, improved laboratory efficiency, and the means to consistently detect abnormal populations at low levels. Careful attention to details of instrument and reagent performance allows for the development of panels suitable for screening of samples for leukemia and lymphoma in a clinical setting. CONCLUSIONS: The characteristics of this technique are particularly well suited to the analysis of leukemia and lymphoma and have the potential to revolutionize and standardize this type of analysis in the clinical laboratory. PMID- 16683887 TI - Evaluation of Her-2/neu gene status in osteosarcoma by fluorescence in situ hybridization and multiplex and monoplex polymerase chain reactions. AB - CONTEXT: Previous reports suggest that the human epidermal growth factor 2 (HER 2/neu) receptor may be overexpressed in osteosarcoma. OBJECTIVE: To determine whether osteosarcomas have amplifications of the HER-2/neu gene. DESIGN: We studied a series of osteosarcomas by fluorescence in situ hybridization (FISH) and by 2 real-time polymerase chain reaction assays that measure the amount of HER-2/neu DNA relative to a control gene. The HER-2/ neu monoplex and multiplex assays were capable of identifying those cases of breast cancer that were known to overexpress HER-2/neu as assessed by FISH. We initially studied 21 cases of osteosarcoma by FISH analysis (using a technique that included a probe for chromosome 17), 11 of which had their HER-2/neu gene amplification status previously reported. RESULTS: None of these osteosarcoma cases showed HER-2/neu amplification by our FISH analysis and subsequent quantitative (multiplex) polymerase chain reaction. Apparent expression of HER-2/neu protein was observed in several of the cases but the immunoreactivity was localized to the cytoplasm and was not membranous in character. An additional 35 osteosarcoma specimens were subjected to monoplex polymerase chain reaction analysis, and amplifiable DNA was recovered from 19 specimens (54%). None of these samples had HER-2/neu amplification by monoplex PCR analysis and only one case had membranous immunoreactivity graded as 1+. CONCLUSION: Although a small subset of osteosarcomas had weak noncircumferential membranous immunoreactivity for HER 2/neu protein, no osteosarcomas demonstrated positive (2+ or 3+) immunoreactivity for HER-2/ neu protein and none showed HER-2/neu gene amplification by either FISH or polymerase chain reaction. PMID- 16683888 TI - Thin glomerular basement membrane nephropathy: incidence in 3471 consecutive renal biopsies examined by electron microscopy. AB - CONTEXT: Thin glomerular basement membrane (GBM) nephropathy is often equated with benign familial hematuria, although it may occur sporadically and may not always be benign. Thin GBM nephropathy is reported to occur in at least 1% of the population, although its reported incidence varies considerably in different studies because there are presently no uniform criteria for its diagnosis by electron microscopy (EM). OBJECTIVE: To determine the incidence of thin GBM nephropathy in a large sample of renal biopsies using a basic methodology that can easily be applied in any diagnostic EM laboratory. DESIGN: Direct measurements of GBM thickness were made from electron micrographs at 3 specified points along each of 10 randomly selected glomerular capillaries to determine an average GBM thickness for each of 50 males and 50 females, ages 9 to 80 years, with minimal-change nephropathy or acute interstitial nephritis, without hematuria. The means of the average GBM thickness values were 330 +/- 50 (SD) nm in the males and 305 +/- 45 nm in the females; normal ranges for each sex were defined as being within 2 SD of these means. The average GBM thickness was then similarly determined for renal biopsies with suspected thin GBMs examined from January 2000 to December 2004; a total of 3471 renal biopsies were examined by EM during this period. SETTING: Academic medical center renal pathology/EM laboratory. RESULTS: Excluding biopsies with immunoglobulin A nephropathy, which is known to be frequently associated with thin GBMs, and biopsies with Alport syndrome, 67 biopsies (1.9% of total) had an average GBM thickness below the sex specific normal range. Of these, 37 biopsies were performed specifically because of hematuria and had an average GBM thickness of 121 to 215 nm (mean, 185 +/- 20 nm). The remaining 30 (0.9%) biopsies, with average GBM thicknesses of 143 to 227 nm (mean, 190 +/- 20 nm), represent cases of incidentally discovered thin GBM nephropathy. CONCLUSIONS: Based on the frequency of incidentally discovered cases and taking into account excluded cases and biopsies (eg, with diabetic nephropathy) in which diagnosis of incidental thin GBM nephropathy by EM is not possible, the incidence of thin GBM nephropathy in our population is estimated to be between 1% and 2%. Diseases most often associated with incidental thin GBM nephropathy were focal segmental glomerulosclerosis (10 cases) and minimal-change nephropathy (5 cases). PMID- 16683889 TI - Juxtaglomerular cell tumor of kidney with CD34 and CD117 immunoreactivity: report of 5 cases. AB - CONTEXT: Juxtaglomerular cell tumor is a rare renal neoplasm. Renin immunohistochemistry and electron microscopic documentation of rhomboid crystals are the primary methods of diagnosing this benign tumor. OBJECTIVES: In this retrospective study, we evaluated the morphologic, immunohistochemical, and ultrastructural features of 5 cases of juxtaglomerular cell tumor to determine the effectiveness of CD34 and CD117 immunohistochemistry for the diagnosis of this tumor. DESIGN: We reviewed 5 cases with clinical, histologic, immunohistochemical, and ultrastructural aspects. RESULTS: Three women and 2 men with a mean age of 37.8 years (range, 16-60 years) were included in this study. All patients presented with severe hypertension. All tumors were well circumscribed and ranged from 1.5 cm to 8.5 cm (mean, 4.4 cm). On light microscopic examination, we found solid sheets and nests of tumor cells with oval to-round nuclei and eosinophilic cytoplasm. Low-power microscopic examination disclosed a hemangiopericytic vascular pattern. Immunohistochemistry results were as follows: vimentin (positive), renin (weakly positive), smooth muscle actin (focal immunoreactivity), and cytokeratin (negative). All 5 tumors were immunoreactive for CD34 and CD117. Electron microscopy revealed rhomboid crystals in the cytoplasm. Postoperatively, 4 patients were normotensive and 1 patient experienced persistent mild hypertension. CONCLUSIONS: Our findings indicate that immunohistochemistry for CD34 and CD117 are effective at diagnosing juxtaglomerular cell tumor. Juxtaglomerular cell tumor should be considered in the diagnosis of any renal tumors with epithelioid cells and negative initial cytokeratin immunohistochemistry. PMID- 16683890 TI - An overview of inflicted head injury in infants and young children, with a review of beta-amyloid precursor protein immunohistochemistry. AB - CONTEXT: Inflicted traumatic brain injury of infants and young children results in a complex array of autopsy findings. In many cases, immunostains for beta amyloid precursor protein are used to detect axonal injury. Interpretation of the gross, microscopic, and immunostaining results requires the integration of the many facets of the individual case. OBJECTIVE: In this article we review the gross and microscopic findings associated with inflicted traumatic brain injury. The application and interpretation of beta-amyloid precursor protein immunostains are discussed and photomicrographs are used to illustrate immunostaining patterns. DATA SOURCES: The pertinent literature is integrated into a review of the subject. CONCLUSIONS: Inflicted traumatic brain injury often results in subdural, subarachnoid, retinal, and optic nerve sheath hemorrhage. These findings must be interpreted within the entire context of the case. Beta-amyloid precursor protein immunostains may be helpful in illustrating the traumatic nature of the injuries in some cases. PMID- 16683891 TI - Iatrogenic Trichuris suis infection in a patient with Crohn disease. AB - We report a case of biopsy-proven iatrogenic infection by the pig whipworm Trichuris suis in a patient with Crohn disease. The deliberate therapeutic ingestion of T suis ova has been adopted as an experimental approach to the treatment of Crohn disease in an effort to promote a switch from the T helper subtype 1 to T helper subtype 2 inflammatory phenotype in vivo. This report examines the morphology of the immature and adult T suis, the effects of this intervention on the immunophenotype of the bowel mucosa, and it also raises the possibility of persistent active infection in man. PMID- 16683892 TI - Synchronous pulmonary carcinoma and pleural diffuse malignant mesothelioma. AB - Synchronous pulmonary carcinoma and pleural diffuse malignant mesothelioma is rare. Cases from the archives of 2 large referral centers were reviewed to identify cases of synchronously occurring pulmonary carcinoma and pleural diffuse malignant mesothelioma. Three cases of synchronous pulmonary carcinoma and pleural diffuse malignant mesothelioma were identified from more than 16,000 pleuropulmonary cases and were reviewed for demographic, clinical, radiographic, histologic, and immunohistochemical findings. The patients were men who were 63, 67, and 77 years old. Two had positive smoking histories; the smoking history of the other patient is unknown. One patient had a positive history of asbestos exposure; one patient had no history of asbestos exposure; and one patient's history of asbestos exposure is unknown. The patients underwent surgery for treatment of adenocarcinoma that was diagnosed preoperatively. Two of the adenocarcinomas were of a predominantly bronchioloalveolar pattern. No diffuse malignant mesothelioma was identified preoperatively. Diffuse malignant mesothelioma was suspected on the basis of pleural involvement by tumor with histology differing from that of the adenocarcinoma. Tumor immunostaining supported the diagnoses. The average survival after diagnosis was 6 weeks or less. In summary, the paucity of cases at 2 large referral centers and the paucity of cases reported in the English language literature highlights the rarity of synchronous pulmonary carcinoma and pleural diffuse malignant mesothelioma. These synchronous neoplasms occur in patients who have risk factors for both neoplasms independently. Length of survival following diagnosis is bleak. PMID- 16683893 TI - Asphyxiation caused by giant fibrovascular polyp of the esophagus. AB - Giant fibrovascular polyps of the esophagus are rare, benign, "tumorlike" lesions that typically present as large pedunculated growths arising in the cervical esophagus. The predominant histologic component of these lesions is variable, often resulting in misdiagnosis. Clinically, these polyps present with nonspecific symptoms and are often undiagnosed or misdiagnosed until they are significant in size. Diagnosis is best made by upper endoscopic evaluation; surgical excision is the definitive treatment. Although rare, asphyxia resulting from obstruction of the glottis is the most serious complication. We describe a case of asphyxiation caused by laryngeal occlusion by a giant esophageal polyp and we provide a review of the literature. PMID- 16683894 TI - Desmoplastic small round cell tumors: cytologic, histologic, and immunohistochemical features. AB - Desmoplastic small round cell tumor (DSRCT) is a recently recognized clinicopathologic entity that has a predilection for adolescent males and usually affects the abdominal cavity. Due to its uncommon nature, many pathologists lack experience with this tumor. The literature regarding DSRCT is reviewed with special attention to its histologic and cytologic diagnosis. Morphologic features of DSRCT and its immunohistochemical and cytogenetic profile are summarized and differential diagnosis with other small round cell tumors is discussed. As observed by both histologic and cytologic examinations, small round blue cells and fibrosclerotic stroma are the striking morphologic features of DSRCT. The typical immunohistochemical profile is characterized by coexpression of epithelial, mesenchymal, myogenic, and neural markers. Cytogenetically, this tumor harbors a specific karyotypic abnormality, namely t(11;22)(p13;q12). These features distinguish DSRCT from other members of the family of small round cell tumors. PMID- 16683895 TI - Premature neonate with apnea and trace amounts of hemoglobin S and A detected by high-performance liquid chromatography. Sickle cell trait/disease in a prematurely born baby with hampered switch from gamma-globin to beta-globin chain synthesis. PMID- 16683896 TI - Left facial mass in an elderly man. Metastasizing atypical fibroxanthoma of the skin. PMID- 16683897 TI - Skin clues to a systemic illness. Secondary syphilis. PMID- 16683899 TI - A 40-year-old woman with facial papules and flank pain. Birt-Hogg-Dube syndrome. PMID- 16683900 TI - A 67-year-old woman with abdominal distention, vaginal bleeding, and elevated CA 125 level. Pure Sertoli cell tumor of the ovary with differentiation varying from well-differentiated tubules, to intermediate foci, to sarcomatoid spindle cell areas. PMID- 16683901 TI - A 63-year-old woman with chronic pelvic pain. Uterine tumor resembling ovarian sex cord tumor. PMID- 16683902 TI - A 37-year-old woman with a presacral mass. Tailgut cyst (retrorectal cystic hamartoma). PMID- 16683903 TI - A patient with shortness of breath and a left hilar mass. Biphasic pulmonary blastoma. PMID- 16683904 TI - A 59-year-old woman with a spindle cell lesion of the breast. Low-grade (fibromatosis-like) spindle cell carcinoma of the breast. PMID- 16683905 TI - Scotopic and photopic visual thresholds and spatial and temporal discrimination evaluated by behavior of mice in a water maze. AB - Methods that allow specific manipulations of the mouse genome have made it possible to alter specific aspects of photoreceptor function within the mouse retina. Mice with photoreceptors that have altered photosensitivities and altered photoresponse kinetics are now available. Methods are needed that can show how those perturbations in photoreceptor response characteristics translate into perturbations in visual sensitivity and perception. We have adapted a previously described method to evaluate visual threshold, spatial discrimination and temporal discrimination in mice swimming in a water maze. In this report we describe the sensitivities of rod-mediated and cone-mediated vision using GNAT1-/ and GNAT2-/- mice. Cone-mediated vision is approximately 10,000 times less sensitive than rod-mediated vision in mice. We also demonstrate that mice can distinguish striped from solid objects in the water maze and that they can distinguish flickering from continuous illumination. PMID- 16683906 TI - Photosensitized generation of singlet oxygen. AB - This work gives an overview of what is currently known about the mechanisms of the photosensitized production of singlet oxygen. Quenching of pi pi* excited triplet states by O2 proceeds via internal conversion of excited encounter complexes and exciplexes of sensitizer and O2. Both deactivation channels lead with different efficiencies to singlet oxygen generation. The balance between the deactivation channels depends on the triplet-state energy and oxidation potential of the sensitizer, and on the solvent polarity. A model has been developed that reproduces rate constants and efficiencies of the competing processes quantitatively. Sensitization by excited singlet states is much more complex and hence only qualitative rules could be elaborated, despite serious efforts of many groups. However, the most important deactivation paths of fluorescence quenching by O2 are again directed by excess energies and charge-transfer interactions similar to triplet-state quenching by O2. Finally, two recent developments in photosensitization of singlet oxygen are reviewed: Two-photon sensitizers with particular application potential for photodynamic therapy and fluorescence imaging of biological samples and singlet oxygen sensitization by nanocrystalline porous silicon, a material with very different photophysics compared to molecular sensitizers. PMID- 16683907 TI - Issues with prescribed medications in Aboriginal communities: Aboriginal health workers' perspectives. AB - INTRODUCTION: The health of Indigenous Australians remains appalling. The causes of this situation are multi-factorial, however one contributing factor is poor medication compliance within Aboriginal populations. Anecdotal evidence provided by Aboriginal health workers in western New South Wales (NSW), Australia, has suggested that there are problems associated with the use of prescribed medications within the Aboriginal community. Aboriginal health workers form a core component of the Aboriginal health service sector and they have an in-depth knowledge of the community and its healthcare provision, as well as a familiarity with clinic patients and families. As such they are an important group whose opinions and beliefs about medication use in the Aboriginal population should be investigated. While there have been studies on the issues of prescribing in Aboriginal communities and access to medications, limited investigation into the use of prescribed medicines in Aboriginal communities and the role of the pharmacist in that process, has taken place. Therefore, this research aimed to identify the type of and reasons for inappropriate use of prescribed medications within Aboriginal communities serviced by the Mid Western Area Health Service (since incorporated into the Greater West Area Health Service) as perceived by the Aboriginal health workers in the area, and to explore strategies in conjunction with those Aboriginal health workers to address identified issues. METHODS: Qualitative, in-depth interviews were held with 11 Aboriginal health workers employed in Community Health Centres and hospitals in the Mid Western Area Health service of NSW. The interviews were audiotaped and transcribed verbatim. The transcripts were content analysed for emerging themes. The interviews explored the beliefs, perceptions and experiences of the Aboriginal health workers regarding prescribed medication use, the role of the pharmacist, and identification of future strategies to improve medication use in local Aboriginal communities. RESULTS: The Aboriginal health workers reported a general lack of access to medications and frequent inappropriate use of medications due to limited understanding, literacy and information all of which lead to non compliance with instructions. Medication sharing was common in their communities. They reported that many Aboriginal people were uncomfortable seeking medicines advice, and the consumer medicine information provided was often difficult to understand, culturally inappropriate and unlikely to be utilised. Strategies suggested to improve pharmacist services and access to the services were a more 'Aboriginal friendly' environment, relationship development between pharmacists and Aboriginal health workers, cultural awareness programs for pharmacists and their staff, provision of disease state management services and medicine education programs by pharmacists for Aboriginal health workers. CONCLUSION: Medication misunderstandings and non-compliance within the Aboriginal community frequently occur. Suggestions to improve access, understanding and compliance, along with the education and training of Aboriginal health workers may provide tools for self-determination. Pharmacists may be well positioned to provide Aboriginal health workers with medicines information and patient education skills, and encourage the effective use of medicines within the Aboriginal community. PMID- 16683913 TI - Setting SNAREs in a different wood. AB - Vesicle traffic is essential for cell homeostasis, growth and development in plants, as it is in other eukaryotes, and is facilitated by a superfamily of proteins known as soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs). Although SNAREs are well-conserved across phylla, genomic analysis for two model angiosperm species available to date, rice and Arabidopsis, highlights common patterns of divergence from other eukaryotes. These patterns are associated with the expansion of some gene subfamilies of SNAREs, the absence of others and the appearance of new proteins that show no significant homologies to SNAREs of mammals, yeast or Drosophila. Recent findings indicate that the functions of these plant SNAREs also extend beyond the conventional 'housekeeping' activities associated with vesicle trafficking. A number of SNAREs have been implicated in environmental responses as diverse as stomata movements and gravisensing as well as sensitivity to salt and drought. These proteins are essential for signal transduction and response and, in most cases, appear also to maintain additional roles in membrane trafficking. One common theme to this added functionality lies in control of non-SNARE proteins, notably ion channels. Other examples include interactions between the SNAREs and scaffolding or other structural components within the plant cell. PMID- 16683914 TI - Morphogenesis of the endoplasmic reticulum: beyond active membrane expansion. AB - The endoplasmic reticulum (ER) of higher eukaryotic cells is a dynamic network of interconnected membrane tubules that pervades almost the entire cytoplasm. On the basis of the morphological changes induced by the disruption of the cytoskeleton or molecular motor proteins, the commonly accepted model has emerged that microtubules and conventional kinesin (kinesin-1) are essential determinants in establishing and maintaining the structure of the ER by active membrane expansion. Surprisingly, very similar ER phenotypes have now been observed when the cytoskeleton-linking ER membrane protein of 63 kDa (CLIMP-63) is mutated, revealing stable attachment of ER membranes to the microtubular cytoskeleton as a novel requirement for ER maintenance. Additional recent findings suggest that ER maintenance also requires ongoing homotypic membrane fusion, possibly controlled by the p97/p47/VICP135 protein complex. Work on other proteins proposed to regulate ER structure, including huntingtin, the EF-hand Ca(2+)-binding protein p22, the vesicle-associated membrane protein-associated protein B and kinectin isoforms further contribute to the new emerging concept that ER shape is not only determined by motor driven processes but by a variety of different mechanisms. PMID- 16683915 TI - P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies. AB - The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC). PMID- 16683912 TI - Calcium-dependent and -independent interactions of the S100 protein family. AB - The S100 proteins comprise at least 25 members, forming the largest group of EF hand signalling proteins in humans. Although the proteins are expressed in many tissues, each S100 protein has generally been shown to have a preference for expression in one particular tissue or cell type. Three-dimensional structures of several S100 family members have shown that the proteins assume a dimeric structure consisting of two EF-hand motifs per monomer. Calcium binding to these S100 proteins, with the exception of S100A10, results in an approx. 40 degrees alteration in the position of helix III, exposing a broad hydrophobic surface that enables the S100 proteins to interact with a variety of target proteins. More than 90 potential target proteins have been documented for the S100 proteins, including the cytoskeletal proteins tubulin, glial fibrillary acidic protein and F-actin, which have been identified mostly from in vitro experiments. In the last 5 years, efforts have concentrated on quantifying the protein interactions of the S100 proteins, identifying in vivo protein partners and understanding the molecular specificity for target protein interactions. Furthermore, the S100 proteins are the only EF-hand proteins that are known to form both homo- and hetero-dimers, and efforts are underway to determine the stabilities of these complexes and structural rationales for their formation and potential differences in their biological roles. This review highlights both the calcium-dependent and -independent interactions of the S100 proteins, with a focus on the structures of the complexes, differences and similarities in the strengths of the interactions, and preferences for homo- compared with hetero dimeric S100 protein assembly. PMID- 16683916 TI - Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'. AB - Ricin is transported from early endosomes and/or the recycling compartment to the trans-Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo- and vector-based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A' mRNA was simultaneously up-regulated, the inhibition of ricin sulfation was abolished, indicating that the up-regulation of Rab6A' may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A' gave approximately 40% reduction in ricin sulfation. The up-regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A' function. The depletion of both Rab6A and Rab6A' gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A' seem to be involved in the transport of ricin from endosomes to the Golgi apparatus. PMID- 16683918 TI - Identification of an intracellular trafficking and assembly pathway for HIV-1 gag. AB - Retroviral Gag proteins are membrane-bound polyproteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle production is determined. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV-1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)-like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amounts of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV-1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag association with MVB-like compartments regulates the site of HIV-1 budding and particle formation. PMID- 16683917 TI - Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization. AB - Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. PMID- 16683920 TI - Haptically linked dyads: are two motor-control systems better than one? PMID- 16683919 TI - Rab23: what exactly does it traffic? AB - Rab23 is the product of the gene mutated in the mouse open brain1 phenotype, which displays neural tube defects. It appears to antagonize sonic hedgehog (Shh) mediated signaling during mouse development, presumably by regulating the intracellular trafficking of one or more of Shh's-signaling components. The Shh receptor Patched1 (Ptch1) and its downstream effector Smoothened (Smo) were initial prime suspects as they are membrane proteins whose cellular dynamics are modulated by the Shh signal. However, Rab23 mutants do not appear to affect the localization and dynamics of either protein. Genetic analyses have now shown that Rab23 functions downstream of Smo and affects the function of the Shh-regulated Gli family of transcription factors in a more direct manner than previously thought. A plethora of proteins that influence Shh signaling and whose cellular trafficking could potentially be regulated by Rab23 has also emerged. These include members of the intraflagellar transport complex, as well as motor proteins responsible for their assembly at the cilia. Rab23 is also expressed in adult mouse neurons and may thus have functions beyond embryonic developmental stages and Shh signaling. We discuss these new findings and explore the myriad of possibilities whereby Rab23 may function. PMID- 16683921 TI - Conditions under which function information attenuates name extension via shape. AB - Children often extend names to novel artifacts on the basis of overall shape rather than core properties (e.g., function). This bias is claimed to reflect the fact that nonrandom structure is a reliable cue to an object having a specific designed function. In this article, we show that information about an object's design (i.e., about its creator's intentions) is neither necessary nor sufficient for children to override the shape bias. Children extend names on the basis of any information specifying the artifact's function (e.g., information about design, current use, or possible use), especially when this information is made salient when candidate objects for extension are introduced. Possible mechanisms via which children come to rely less on easily observable cues (e.g., shape) and more on core properties (e.g., function) are discussed. PMID- 16683922 TI - Prosodic knowledge affects the recognition of newly acquired words. AB - An eye-tracking study examined the involvement of prosodic knowledge- specifically, the knowledge that monosyllabic words tend to have longer durations than the first syllables of polysyllabic words--in the recognition of newly learned words. Participants learned new spoken words (by associating them to novel shapes): bisyllables and onset-embedded monosyllabic competitors (e.g., baptoe and bap). In the learning phase, the duration of the ambiguous sequence (e.g., bap) was held constant. In the test phase, its duration was longer than, shorter than, or equal to its learning-phase duration. Listeners' fixations indicated that short syllables tended to be interpreted as the first syllables of the bisyllables, whereas long syllables generated more monosyllabic-word interpretations. Recognition of newly acquired words is influenced by prior prosodic knowledge and is therefore not determined solely on the basis of stored episodes of those words. PMID- 16683923 TI - An examination of sources of peer-review bias. AB - Peer-review ratings of 1,983 posters submitted for three annual conferences of a professional society were examined for evidence of bias. Hypotheses derived from the literature on the better-than-average effect were tested by analyzing 7,383 sets of ratings. Reviewers who authored posters gave lower average ratings than reviewers who did not author posters. Posters having authorship that included at least one reviewer received higher ratings than those having only nonreviewing authors. Reviewers' experience and professional role were also explored as biasing factors. The ratings were converted into z scores, and differences in reliability and acceptance decisions were examined. Implications for current peer review practices are discussed. PMID- 16683924 TI - Looking deathworthy: perceived stereotypicality of Black defendants predicts capital-sentencing outcomes. AB - Researchers previously have investigated the role of race in capital sentencing, and in particular, whether the race of the defendant or victim influences the likelihood of a death sentence. In the present study, we examined whether the likelihood of being sentenced to death is influenced by the degree to which a Black defendant is perceived to have a stereotypically Black appearance. Controlling for a wide array of factors, we found that in cases involving a White victim, the more stereotypically Black a defendant is perceived to be, the more likely that person is to be sentenced to death. PMID- 16683925 TI - Priming race in biracial observers affects visual search for Black and White faces. AB - We examined whether or not priming racial identity would influence Black-White biracial individuals' ability to visually search for White and Black faces. Black, White, and biracial participants performed a visual search task in which the targets were Black or White faces. Before the task, the biracial participants were primed with either their Black or their White racial identity. All participant groups detected Black faces faster than White faces. Critically, the results also showed a racial-prime effect in biracial individuals: The magnitude of the search asymmetry was significantly different for those primed with their White identity and those primed with their Black identity. These findings suggest that top-down factors such as one's racial identity can influence mechanisms underlying the visual search for faces of different races. PMID- 16683926 TI - Ethnic diversity and perceptions of safety in urban middle schools. AB - Students' perceptions of their safety and vulnerability were investigated in 11 public middle schools (more than 70 sixth-grade classrooms) that varied in ethnic diversity. Results of hierarchical linear modeling analyses indicate that higher classroom diversity is associated with feelings of safety and social satisfaction. African American (n= 511) and Latino (n= 910) students felt safer in school, were less harassed by peers, felt less lonely, and had higher self worth the more ethnically diverse their classrooms were, even when controlling for classroom differences in academic engagement. Results at the school level were similar to those at the classroom level; higher ethnic diversity was associated with lower levels of self-reported vulnerability (but no difference in self-worth) in both fall and spring of sixth grade. In the spirit of Brown v. Board of Education, the current findings offer new empirical evidence for the psychological benefits of multiethnic schools. PMID- 16683927 TI - Shared system for ordering small and large numbers in monkeys and humans. AB - There is increasing evidence that animals share with adult humans and perhaps human infants a system for representing objective number as psychological magnitudes that are an analogue of the quantities they represent. Here we show that rhesus monkeys can extend a numerical rule learned with the values 1 through 9 to the values 10, 15, 20, and 30, which suggests that there is no upper limit on a monkey's numerical capacity. Instead, throughout the numerical range tested, both accuracy and latency in ordering two numerical values were systematically controlled by the ratio of the values compared. In a second experiment, we directly compared humans' and monkeys' performance in the same ordinal comparison task. The qualitative and quantitative similarity in their performance provides the strongest evidence to date of a single nonverbal, evolutionarily primitive mechanism for representing and comparing numerical values. PMID- 16683928 TI - Numeracy and decision making. AB - A series of four studies explored how the ability to comprehend and transform probability numbers relates to performance on judgment and decision tasks. On the surface, the tasks in the four studies appear to be widely different; at a conceptual level, however, they all involve processing numbers and the potential to show an influence of affect. Findings were consistent with highly numerate individuals being more likely to retrieve and use appropriate numerical principles, thus making themselves less susceptible to framing effects, compared with less numerate individuals. In addition, the highly numerate tended to draw different (generally stronger or more precise) affective meaning from numbers and numerical comparisons, and their affective responses were more precise. Although generally helpful, this tendency may sometimes lead to worse decisions. The less numerate were influenced more by competing, irrelevant affective considerations. Analyses showed that the effect of numeracy was not due to general intelligence. Numerical ability appears to matter to judgments and decisions in important ways. PMID- 16683929 TI - Recommendations implicit in policy defaults. AB - Should people be considered organ donors after their death unless they request not to be, or should they not be considered donors unless they request to be? Because people tend to stay with the default in a variety of domains, policymakers' choice of default has large and often important effects. In the United States, where the organ-donation policy default is "not a donor," about 5,000 people die every year because there are too few donors. Four experiments examined two domains-being an organ donor and saving for retirement-where default effects occur and have important implications. The results indicate that default effects occur in part because policymakers' attitudes can be revealed through their choice of default, and people perceive the default as indicating the recommended course of action. Policymakers need to be aware of the implicit messages conveyed by their choice of default. PMID- 16683930 TI - Acting intentionally and the side-effect effect. AB - The concept of acting intentionally is an important nexus where theory of mind and moral judgment meet. Preschool children's judgments of intentional action show a valence-driven asymmetry. Children say that a foreseen but disavowed side effect is brought about "on purpose" when the side effect itself is morally bad, but not when it is morally good. This is the first demonstration in preschoolers that moral judgment influences judgments of whether something was done on purpose (as opposed to judgments of purpose influencing moral judgment). Judgments of intentionality are usually assumed to be purely factual. That these judgments are sometimes partly normative-even in preschoolers-challenges current understanding. Young children's judgments regarding foreseen side effects depend on whether the children process the idea that the character does not care about the side effect. As soon as preschoolers effectively process the theory-of-mind concept "not care that P," children show the side-effect effect. PMID- 16683931 TI - Dual processing in reasoning: two systems but one reasoner. AB - Human reasoning has been characterized as an interplay between an automatic belief-based system and a demanding logic-based reasoning system. The present study tested a fundamental claim about the nature of individual differences in reasoning and the processing demands of both systems. Participants varying in working memory capacity performed a reasoning task while their executive resources were burdened with a secondary task. Results were consistent with the dual-process claim: The executive burden hampered correct reasoning when the believability of a conclusion conflicted with its logical validity, but not when beliefs cued the correct response. However, although participants with high working memory spans performed better than those with lower spans in cases of a conflict, all reasoners showed similar effects of load. The findings support the idea that there are two reasoning systems with differential processing demands, but constitute evidence against qualitative individual differences in the human reasoning machinery. PMID- 16683932 TI - The effect of personal relevance and approach-related action expectation on relative left frontal cortical activity. AB - Past research using a variety of methods has suggested that the frontal cortex is asymmetrically involved in the experience and expression of positive (or approach motivational) and negative (or withdrawal motivational) affects, with the left frontal region being involved in positive affects (or approach) and the right frontal region being involved in negative affects (or withdrawal). However, some studies have failed to replicate these effects, leaving many scientists questioning the meaning of the past supportive findings. To examine these inconsistencies in results, we tested the hypothesis that increasing the personal relevance of the stimuli and approach motivational intensity would increase relative left frontal activation. Results supported the predictions. Moreover, by showing the predicted effects with anger-inducing stimuli, the results demonstrated that motivational direction, rather than affective valence, accounts for asymmetrical frontal cortical activity. PMID- 16683933 TI - Suppression of emotional and nonemotional content in memory: effects of repetition on cognitive control. AB - Two experiments utilized a think/no-think paradigm to examine whether cognitive control of memories differs depending on whether they contain information with negative or neutral emotional content. During a training phase, participants learned face-word pairs (Experiment 1) or face-picture pairs (Experiment 2). In a subsequent experimental phase, participants were shown faces and told to think of the items paired with some of the faces and to try not to think of the items paired with other faces. Finally, in a test phase, participants were again shown each face and asked to recall the item with which it had been paired previously. Results for both verbal (Experiment 1) and nonverbal (Experiment 2) items indicated that the facilitatory and inhibitory influences of cognitive control were larger for negative than neutral items. PMID- 16683934 TI - Controlling the integration of emotion and cognition: the role of frontal cortex in distinguishing helpful from hurtful emotional information. AB - Emotion has been both lauded and vilified for its role in decision making. How are people able to ensure that helpful emotions guide decision making and irrelevant emotions are kept out of decision making? The orbitofrontal cortex has been identified as a neural area involved in incorporating emotion into decision making. Is this area's function specific to the integration of emotion and cognition, or does it more broadly govern whether emotional information should be integrated into cognition? The present research examined the role of orbitofrontal cortex when it was appropriate to control (i.e., prevent) the influence of emotion in decision making (Experiment 1) and to incorporate the influence of emotion in decision making (Experiment 2). Together, the two studies suggest that activity in lateral orbitofrontal cortex is associated with evaluating the contextual relevance of emotional information for decision making. PMID- 16683935 TI - Indoor exposures are not the same as outdoor exposures! PMID- 16683936 TI - Influence of ozone-limonene reactions on perceived air quality. AB - This study conducted short-term assessments of perceived air quality (PAQ) for six different realistic concentrations of ozone and limonene, separately or together, in room air. The impact of filtration and the influence of the ozone generation method were also examined. The evaluations were made in four identical 40 m3 low-polluting test offices ventilated at 1.4 h(-1) or in two identical 30 m3 stainless-steel chambers ventilated at 1.9 h(-1). Concentrations of ozone, total volatile organic compounds and size-fractionated particles were continuously monitored in each experiment. The results indicate that, for each of the six conditions, the PAQ was poorer when ozone and limonene were present together compared with when only ozone or only limonene was present. In the test offices a correlation was observed between the number of secondary organic aerosols produced by a given ozone/limonene condition and the sensory pollution load for that condition. The particles themselves do not appear to be the primary causative agents, but instead are co-varying surrogates for sensory offending gas phase species. PRACTICAL IMPLICATIONS: Although the health consequences of long term exposures to the products of ozone-initiated indoor chemistry remain to be determined, we judge that the sensory offending nature of selected products provides an additional reason to limit indoor ozone levels. Devices that emit ozone at significant rates should not be used indoors. Ozone-filtration of make up air should also be beneficial in mechanically ventilated buildings located in regions that repeatedly violate outdoor ozone standards. Additionally, the use of limonene containing products should be curtailed during periods when indoor ozone levels are elevated. PMID- 16683937 TI - Cleaning products and air fresheners: emissions and resulting concentrations of glycol ethers and terpenoids. AB - Experiments were conducted to quantify emissions and concentrations of glycol ethers and terpenoids from cleaning product and air freshener use in a 50-m3 room ventilated at approximately 0.5/h. Five cleaning products were applied full strength (FS); three were additionally used in dilute solution. FS application of pine-oil cleaner (POC) yielded 1-h concentrations of 10-1300 microg/m3 for individual terpenoids, including alpha-terpinene (90-120), d-limonene (1000 1100), terpinolene (900-1300), and alpha-terpineol (260-700). One-hour concentrations of 2-butoxyethanol and/or d-limonene were 300-6000 microg/m3 after FS use of other products. During FS application including rinsing with sponge and wiping with towels, fractional emissions (mass volatilized/dispensed) of 2 butoxyethanol and d-limonene were 50-100% with towels retained, and approximately 25-50% when towels were removed after cleaning. Lower fractions (2-11%) resulted from dilute use. Fractional emissions of terpenes from FS use of POC were approximately 35-70% with towels retained, and 20-50% with towels removed. During floor cleaning with dilute solution of POC, 7-12% of dispensed terpenes were emitted. Terpene alcohols were emitted at lower fractions: 7-30% (FS, towels retained), 2-9% (FS, towels removed), and 2-5% (dilute). During air-freshener use, d-limonene, dihydromyrcenol, linalool, linalyl acetate, and beta citronellol) were emitted at 35-180 mg/day over 3 days while air concentrations averaged 30-160 microg/m3. PRACTICAL IMPLICATIONS: While effective cleaning can improve the healthfulness of indoor environments, this work shows that use of some consumer cleaning agents can yield high levels of volatile organic compounds, including glycol ethers--which are regulated toxic air contaminants- and terpenes that can react with ozone to form a variety of secondary pollutants including formaldehyde and ultrafine particles. Persons involved in cleaning, especially those who clean occupationally or often, might encounter excessive exposures to these pollutants owing to cleaning product emissions. Mitigation options include screening of product ingredients and increased ventilation during and after cleaning. Certain practices, such as the use of some products in dilute solution vs. full-strength and the prompt removal of cleaning supplies from occupied spaces, can reduce emissions and exposures to 2-butoxyethanol and other volatile constituents. Also, it may be prudent to limit use of products containing ozone-reactive constituents when indoor ozone concentrations are elevated either because of high ambient ozone levels or because of the indoor use of ozone-generating equipment. PMID- 16683938 TI - Fungal and endotoxin measurements in dust associated with respiratory symptoms in a water-damaged office building. AB - We investigated the associations of fungal and endotoxin levels in office dust with respiratory health in 888 (67% participation) occupants of a water-damaged building. We analyzed floor and chair dusts from 338 workstations for culturable fungi and endotoxin. Based on averages, we ranked each floor of the building as low, medium, or high for occupants' exposure to each of these agents. Multivariate logistic regression models for building-related symptoms included this ranking of fungi and endotoxin, age, gender, race, smoking status, and duration of occupancy. Using floor dust measures, we found significantly increased odds for lower respiratory symptoms [wheeze, chest tightness, attacks of shortness of breath, and attacks of cough: odds ratios (OR) = 1.7 (95% confidence interval (CI): 1.02-2.77) to 2.4 (95% CI: 1.29-4.59)], throat irritation [OR = 1.7, (95% CI: 1.06-2.82)], and rash/itchy skin [OR = 3.0, (95% CI: 1.47-6.19)] in the highest fungal exposure group compared to the lowest, with generally linear exposure-response relationships. Nonlinear relationships were observed for many of these symptoms and endotoxin in floor dust. Interaction models showed that endotoxin modified effects of fungi on respiratory symptoms. Our findings of exposure interactions and exposure-response relationships of fungal and endotoxin with increased risk of building-related symptoms contribute to an understanding of the role of microbial agents in building-related asthma and respiratory and systemic symptoms. PRACTICAL IMPLICATIONS: Our demonstration of exposure-response relationships between measurements of fungi and/or endotoxin in floor dusts and building-related symptoms implies that microbial agents in floor dust may be a good surrogate measure for dampness-related bioaerosol exposure, considering that measurements of microbial agents in air often fail to demonstrate the associations between exposure and health. In addition, our finding that endotoxin exposure may change the effect of fungal exposure (and vice versa) on respiratory heath suggests that exposure to both fungi and endotoxin should be assessed in epidemiological investigations examining the effect of fungal or endotoxin exposure on respiratory health in indoor environments. PMID- 16683939 TI - Analysis of indoor particle size distributions in an occupied townhouse using positive matrix factorization. AB - From late 1999 to early March 2000, measurements of particle number (particles 0.01-20 microm in aerodynamic diameter) concentrations were made inside of a townhouse occupied by two non-smoking adults and located in Reston, VA (approximately 25 miles northwest of Washington, DC). The particle size measurements were made using an SMPS and an APS as well as a Climet optical scattering instrument. In this study, positive matrix factorization (PMF) was used to study the indoor particle size distributions. The size distributions or profiles obtained were identified by relating the obtained source contributions to the source information provided by the occupants. Nine particle sources were identified, including two sources associated with gas burner use: boiling water and frying tortillas. Boiling water for tea or coffee was found to be associated only with the smallest particles, with a number mode close to the detection limit of the SMPS (i.e., 0.01 microm). Frying tortillas produced particles with a number mode at about 0.09 microm while broiling fish produced particles with a number mode at about 0.05 microm. A citronella candle was often burned during the study period, and this practice was found to produce a 0.2-microm modal number distribution. Other indoor particle sources identified included sweeping/vacuuming (volume mode at 2 microm); use of the electric toaster oven (number mode at 0.03 microm); and pouring of kitty litter (volume mode over 10 microm). Two outdoor sources were also resolved: traffic (number mode at about 0.15 microm) and wood smoke (major number mode at about 0.07 microm). The volume distributions showed presence of coarse particles in most of the resolved indoor sources probably caused by personal cloud emissions as the residents performed the various indoor activities. PRACTICAL IMPLICATIONS: This study has shown that continuous measurements of indoor particle number and volume concentrations together with records of personal activities are useful for indoor source apportionment models. Each of the particle sources identified in this study produces distinct size distributions that may be useful in studying the mortality and morbidity effects of airborne particulate matter because they will have different penetrability and deposition patterns. PMID- 16683940 TI - Particle size distributions and concentrations of airborne endotoxin using novel collection methods in homes during the winter and summer seasons. AB - A comparison study of novel collection methods for airborne bacteria and endotoxin was performed in an environmentally controlled chamber and in pilot field studies. Airborne particulate matter was collected in swirling liquid impingers, air-monitoring filter cassettes, and with a micro-orifice uniform deposit impactor (MOUDI) to evaluate aerodynamic particle size distributions. Environmentally controlled chamber studies showed that impingers and MOUDI recovered significantly more airborne bacteria than filter cassettes, whereas collection methods for airborne endotoxin were not significantly different. In addition, total airborne bacteria and endotoxin concentrations were measured indoors and outdoors at three homes in Boulder, CO during winter and summer seasons. Indoor concentrations collected with the three different samplers were significantly different for airborne endotoxin, but not for airborne bacteria. Total airborne bacteria indoors and outdoors significantly varied with seasons. Outdoor airborne endotoxin significantly varied with season; no seasonal variation was seen for indoor airborne endotoxin. Indoor and outdoor levels were not significantly different for both airborne bacteria and endotoxin. The largest proportion of endotoxin was associated with airborne particulate matter <1 microm. PRACTICAL IMPLICATIONS: This study compared sampling methods for airborne endotoxin, a potent and nonspecific immune system stimulant which can induce negative health responses. The data from this study showed that swirling liquid impingers and the micro-orifice uniform deposit impactor (MOUDI) recovered significantly more airborne endotoxin than the more widely adapted method of collecting airborne endotoxin on membrane filters, when collection methods were applied in realistic settings (homes). The MOUDI measured the particle size distribution of airborne endotoxin, which can be useful for determining endotoxin respiratory toxicity and its health effects. PMID- 16683941 TI - A nationwide study of indoor and outdoor environments in allergen avoidance and conventional daycare centers in Sweden. AB - Sweden has had specialized 'allergen avoidance daycare centers' (AADC) since 1979. The aim was to compare AADC with ordinary daycare centers (ODC). Through contacts with municipalities and hospitals, 72 AADC were identified. For each AADC, the two nearest ODCs served as controls. A questionnaire was sent to the local directors of the 216 daycare centers (531 sections), 83% responded. A total of 39% of the ODCs had ever had dampness/molds, 12% were near (<500 m) stables/farms, 23% were closer than 50 m to a heavy trafficked road, and 13% were exposed to wood smoke in winter. Only 11% of the AADCs had any child with furred pets at home, while 97% of the ODCs had children with furred pets. Bans on smoking at home and on perfumes were more common at the AADCs (P = 0.001). Fewer AADCs had PVC floors (OR = 0.53; P = 0.01), dampness/molds (OR = 0.55; P = 0.04), shelves (OR 3.03; P = 0.001), curtains (OR = 1.67; P = 0.047), and flowers (OR = 0.03; P < 0.001), while more had daily floor cleaning (OR = 19.9; P = 0.004), weekly wiping of furniture (OR = 11.0; P = 0.001), and washing of pillows/mattresses (OR = 2.74; P = 0.005) and curtains (OR = 9.07; P = 0.001). In conclusion, allergy avoidance daycare centers differ from other daycare centers, and may have better indoor environments. PRACTICAL IMPLICATIONS: There is a need to improve the indoor environments of daycare centers, including reduction of building dampness and molds. Allergen avoidance daycare centers (AADC) in Sweden differ from ordinary daycare centers in many respects, with fewer indoor and building factors related to dust, allergens and irritants. This shows that the indoor environments of daycare centers can be improved. Data suggest that AADC may have lower levels of pet allergens, and this is beneficial for children with pet allergy. The effects of these improvements on indoor exposures and health of the children need to be further evaluated. PMID- 16683942 TI - Volatile organic compound (VOC) adsorption on material: influence of gas phase concentration, relative humidity and VOC type. AB - This paper presents the results of a factorial experiment design analysis to investigate volatile organic compounds (VOC) adsorption on a ceiling tile. The impacts of three factors, VOC gas phase concentration, relative humidity, and VOC type, as single parameters and as a combination, on adsorption have been investigated. Cyclohexane, toluene, ethyl acetate, isopropyl alcohol and methanol were the five VOCs used in this study. A factor significant level was determined through evaluating its F value and comparing it with the critical value of F distribution at 95% confidence level. It was found that: (i) neither the relative humidity and gas phase concentration nor any interaction effect between them had significant impacts on toluene adsorption on the ceiling tile; (ii) the adsorption isotherm appeared to be linear for the non-polar compounds and non linear for the semi-polar and polar compounds; (iii) no significant impact of relative humidity on adsorption was observed for most VOC compounds except for methanol; and (iv) the ceiling tile had the highest adsorption capacity toward the polar compounds, followed by the aromatic compounds and aliphatic compounds. In addition, the statistical analysis regarding the experimental results of toluene as a single compound or as a part of a mixture showed that toluene adsorption capacity on the ceiling tile as a single compound was higher than as a part of a mixture. PRACTICAL IMPLICATIONS: Building materials and furnishings may act as source and sink of VOCs in the indoor environment. In this study, a factorial experiment design analysis technique was used to show the impact of three factors, VOC gas phase concentration, relative humidity, and VOC type, as single parameters and as a combination, on the adsorption process (sink effect). The aim was to better understand the interaction between these parameters and to verify the common assumptions made in the model development and measurement of indoor air quality. PMID- 16683945 TI - Abstracts of The Royal Australasian College of Surgeons Annual Scientific Congress, Sydney, Australia, 15-19 May 2006. PMID- 16683943 TI - Children's homes--determinants of moisture damage and asthma in Finnish residences. AB - Certain housing characteristics increase the risk for moisture damage, which has been associated with increased risk for asthma in children. Modeling moisture damage as a function of these characteristics could therefore provide a simple tool to estimate building-related risk for asthma. This study aimed to find out specific associations between asthma case-control status of children and moisture damage and housing characteristics. The data consisted of information on 121 asthmatic children and predominately two age-, gender- and place of residence matched control children for every case, and information on moisture damage and housing characteristics in the homes of the children. In a previous study, we found a statistically significant association between moisture damage observations in main living areas and asthma in children. Using logistic regression, five models were formulated to predict moisture damage status of the homes and moisture damage status of living areas. The models were able to classify the damage status correctly in 65.0-87.7% of the homes (kappa values 0.10-0.47) as functions of housing characteristics. None of the models qualified as a significant determinant of the case-control status of the children. PRACTICAL IMPLICATIONS: It can be hypothesized that building-related risk for asthma could be roughly estimated using models predicting moisture damage status of buildings as a function of easily obtainable housing characteristics. The results of this study indicated that, with a moderate certainty, it is possible to model moisture damage status of buildings using housing characteristics. However, the models developed did not associate with asthma in children. In conclusion, it was not possible to estimate the risk for asthma by studying housing characteristics only, but detailed information on moisture damage (e.g. location of damage) was crucial for such estimation. PMID- 16683946 TI - Abstracts from the 41st Congress of the RANZCP, 28 May-1 June 2006. PMID- 16683947 TI - Progress of rotary blood pumps. PMID- 16683948 TI - Living with the Jarvik 2000: a five-plus year experience. PMID- 16683949 TI - Third-generation blood pumps with mechanical noncontact magnetic bearings. AB - This article reviews third-generation blood pumps, focusing on the magnetic levitation (maglev) system. The maglev system can be categorized into three types: (i) external motor-driven system, (ii) direct-drive motor-driven system, and (iii) self-bearing or bearingless motor system. In the external motor-driven system, Terumo (Ann Arbor, MI, U.S.A.) DuraHeart is an example where the impeller is levitated in the axial or z-direction. The disadvantage of this system is the mechanical wear in the mechanical bearings of the external motor. In the second system, the impeller is made into the rotor of the motor, and the magnetic flux, through the external stator, rotates the impeller, while the impeller levitation is maintained through another electromagnetic system. The Berlin Heart (Berlin, Germany) INCOR is the best example of this principle where one-axis control combination with hydrodynamic force achieves high performance. In the third system, the stator core is shared by the levitation and drive coil to make it as if the bearing does not exist. Levitronix CentriMag (Zurich, Switzerland), which appeared recently, employs this concept to achieve stable and safe operation of the extracorporeal system that can last for a duration of 14 days. Experimental systems including HeartMate III (Thoratec, Woburn, MA, U.S.A.), HeartQuest (WorldHeart, Ottawa, ON, Canada), MagneVAD (Gold Medical Technologies, Valhalla, NY, U.S.A.), MiTiHeart (MiTi Heart, Albany, NY, U.S.A.), Ibaraki University's Heart (Hitachi, Japan) and Tokyo Medical and Dental University/Tokyo Institute of Technology's disposable and implantable maglev blood pumps are also reviewed. In reference to second-generation blood pumps, such as the Jarvik 2000 (Jarvik Heart, New York, NY, U.S.A.), which is showing remarkable achievement, a question is raised whether a complicated system such as the maglev system is really needed. We should pay careful attention to future clinical outcomes of the ongoing clinical trials of the second-generation devices before making any further remarks. What is best for patients is the best for everyone. We should not waste any efforts unless they are actually needed to improve the quality of life of heart-failure patients. PMID- 16683950 TI - HeartQuest ventricular assist device magnetically levitated centrifugal blood pump. AB - Improvements in implantable ventricular assist device (VAD) performance will be required to obtain patient outcomes that are comparable with those of heart transplantation. The HeartQuest VAD (WorldHeart, Oakland, CA, U.S.A.) is an advanced device, with full magnetic suspension of the rotor, designed to address specific clinical shortcomings in existing devices and to maximize margins of safety and performance for an implantable assist device. The device dimensions are 35 x 75 mm, with a total weight of 440 g. The system was designed using extensive computer modeling of device function; a total of two iterations of device prototypes were built before building the clinical version. Animal study results have been very promising, with over 30 calf studies completed. Plasma free hemoglobin levels returned to preoperative levels, and other hematology results were in the normal ranges. Highlights include clean surfaces seen in a 116-day experiment with no anticoagulation after day 43. Feasibility clinical trials are planned to start in 2006. PMID- 16683951 TI - Development of Lorentz force-type self-bearing motor for an alternative axial flow blood pump design. AB - A Lorentz force-type self-bearing motor was developed to provide delivery of both motoring torque and levitation force for an alternative axial flow blood pump design with an enclosed impeller. The axial flow pumps currently available introduce electromagnetic coupling from the motor's stator to the impeller by means of permanent magnets (PMs) embedded in the tips of the pump's blades. This design has distinct disadvantages, for example, pumping efficiency and electromagnetic coupling transmission are compromised by the constrained or poor geometry of the blades and limited pole width of the PMs, respectively. In this research, a Lorentz force-type self-bearing motor was developed. It is composed of (i) an eight-pole PM hollow-cylindrical rotor assembly supposedly to house and enclose the impeller of an axial flow blood pump, and (ii) a six-pole stator with two sets of copper wire and different winding configurations to provide the motoring torque and levitating force for the rotor assembly. MATLAB's xPC Target interface hardware was used as the rapid prototyping tool for the development of the controller for the self-bearing motor. Experimental results on a free/simply supported rotor assembly validated the design feasibility and control algorithm effectiveness in providing both the motoring torque and levitation force for the rotor. When levitated, a maximum orbital displacement of 0.3 mm corresponding to 1050 rpm of the rotor was measured by two eddy current probes placed in the orthogonal direction. This design has the advantage of eliminating the trade-off between motoring torques, levitating force, and pumping efficiency of previous studies. It also indicated the benefits of enclosed-impeller design as having good dynamic response, linearity, and better reliability. The nonmechanical contact feature between rotating and stationary parts will further reduce hemolysis and thromboembolitic tendencies in a typical blood pump application. PMID- 16683952 TI - A review of leakage flow in centrifugal blood pumps. AB - This article presents a new approach in determining the functional relationship between the leakage flow in a centrifugal blood pump and various parameters that affect it. While high leakage flow in a blood pump is essential for good washout and can help prevent thrombus formation, excessive leakage flow will result in higher fluid shear stress that may lead to hemolysis. Dimensional analysis is employed to provide a functional relationship between leakage flow rate and other important parameters governing the operation of a centrifugal blood pump. Results showed that pump performance with a smaller gap clearance is clearly superior compared to those of two other similar pumps with larger gap clearances. It was also observed that the nondimensional leakage flow rate varies almost linearly with dimensionless pump head. It also decreases with increasing volume flow rate. A smaller gap clearance will also increase the flow resistance and hence, decrease the nondimensional leakage flow rate. Increasing surface roughness, length of the gap clearance passage, or loss coefficient of the gap geometry will increase losses and hence, decrease the leakage flow rate. It is also interesting to note that for a given pump and gap clearance geometry, the nondimensional leakage flow rate is almost independent of the Reynolds number when specific speed is constant. PMID- 16683953 TI - Estimation of early-stage malfunction using implantable artificial heart sound in animal experiments. AB - We have developed an automatic diagnosis system of an artificial heart in order to ensure the safety of the patient implanted with the artificial heart. The automatic diagnosis system is composed of an electro-stethoscope system, adaptive noise canceller (ANC), and artificial neural network (ANN). The ANC effectively eliminates ambient noise from the sound signal of the artificial heart detected by the electro-stethoscope, and a filtered sound signal is separated into each frequency components by fast Fourier transformation. Each frequency component of an artificial heart's acoustic signal is fed into the ANN in order to make a diagnosis of pump condition. The automatic diagnosis system was evaluated in mock circulatory tests and a long-term animal experiment using a goat implanted with an undulation pump ventricular assist device (UPVAD). In mock circulatory tests, the ANN was able to detect pump failing conditions, which were occlusion of inflow and outflow cannula and deterioration of the ball bearing. In a long-term animal experiment, after training the ANN using UPVAD's sound signal in normal condition, the diagnosis system continuously monitored UPVAD's sound signal detected by the electro-stethoscope placed on the surface of the left thoracic cavity of the goat. The UPVAD was stopped by rupture of a diaphragm in the pump on the ninth day of operation. We were able to identify initial signs of malfunction of the pump on the eighth day, while the UPVAD was able to operate normally. In conclusion, the automatic diagnosis system for malfunction of the artificial heart has enough performance to detect early stages of malfunction of the artificial heart, and it contributes to ensure the patient's safety. PMID- 16683954 TI - Hemolysis resulting from surface roughness under shear flow conditions using a rotational shear stressor. AB - The degree of hemolysis as a function of surface roughness value and roughened area under shear flow conditions was investigated using a rotational shear stressor. The shearing portion of the stressor is cone shaped in its upper and lower positions, with a cylindrical central section. Surface roughness was applied to the cylindrical section. Bovine blood was sheared for 30 min over a set of roughened surfaces of between arithmetic mean roughness (Ra) 0.1 and 0.8 mm covering 10% of the surface area of the cylindrical section (equivalent to 1.8% of the whole blood contact area) at a shear rate of 3750/s. The threshold value thus obtained for rapid increase in hemolysis was between Ra 0.6 and 0.8 mm. When sheared with a roughened surface of Ra 0.8 mm applied to the cylindrical surface at areas between 0 and 100% (equivalent to between 0 and 18% of the whole blood-contacting area), the hemolysis level did not increase from 10 to 100%, but a significant difference was obtained between 0 and 10%. This suggests that red blood cells were destroyed not by fatigue failure caused by rolling on the roughened surface, but due to the high shear stress generated by surface roughness. Moreover, it appears that the shear stress was generated over the entire cylindrical section, regardless of the area of surface roughness. PMID- 16683955 TI - In vitro study to estimate particle release from a centrifugal blood pump. AB - Centrifugal pumps have been increasingly used in clinical settings. Like roller pumps, centrifugal pumps can cause debris release due to mechanical stress. The objectives of this study were to evaluate in vitro the particle release from a centrifugal pump, Gyro Pump (Japan Medical Materials Co., Osaka, Japan), which is a pivot-bearing supported pump clinically used in Japan, and to identify the released particles. In the clean room Class 10,000, the pump was operated for 24 h at 4000 rpm and 6 L/min in a mock loop filled with lactated Ringer's solution. After 24 h, the sample fluid and a blank were filtered with a 0.45-microm membrane filter for microscopic counting, followed by observation with a scanning electron microscope and element analysis with an X-ray spectrometer. Microscopic countings were 128 +/- 42 in the test samples (n = 10) of the Gyro Pump and 98 +/ 42 in the blank samples (n = 10) (P = 0.12). The oxygen/carbon atomic ratio of the particles in the test samples was 0.32 +/- 0.06, which was similar to the ratio of the particles in the blank sample (0.34 +/- 0.06). The profiles of elements with an X-ray spectrometer showed that the released particles from the Gyro Pump were not derived from the pump materials. In conclusion, an in vitro test system has been established for estimation of particle release from a centrifugal pump. Based upon the results with the system, the Gyro Pump with a pivot-bearing system has little risk to release debris particles even in a severe condition. PMID- 16683956 TI - Detection of left ventricle function from a magnetically levitated impeller behavior. AB - The magnetically levitated (Mag-Lev) centrifugal rotary blood pump (CRBP) with two-degrees-of-freedom active control is promising for safe and long-term support of circulation. In this study, Mag-Lev CRBP controllability and impeller behavior were studied in the simulated heart failure circulatory model. A pneumatically driven pulsatile blood pump (Medos VAD [ventricular assist device]-54 mL) was used to simulate the left ventricle (LV). The Mag-Lev CRBP was placed between the LV apex and aortic compliance tank simulating LV assistance. The impeller behavior in five axes (x, y, z, theta, and phi) was continuously monitored using five eddy current sensors. The signals of the x- and y-axes were used for feedback active control, while the behaviors of the other three axes were passively controlled by the permanent magnets. In the static mock circuit, the impeller movement was controlled to within +/-10-+/-20 microm in the x- and y axes, while in the pulsatile circuit, LV pulsation was modulated in the impeller movement with the amplitude being 2-22 microm. The amplitude of impeller movement measured at 1800 rpm with the simulated failing heart (peak LV pressure [LVP] = 70 mm Hg, mean aortic pressure [AoP(mean)] = 55 +/- 20 mm Hg, aortic flow = 2.7 L/min) was 12.6 microm, while it increased to 19.2 microm with the recovered heart (peak LVP = 122 mm Hg, AoP(mean) = 100 +/- 20 mm Hg, aortic flow = 3.9 L/min). The impeller repeated the reciprocating movement from the center of the pump toward the outlet port with LV pulsation. Angular rotation (theta, phi) was around +/-0.002 rad without z-axis displacement. Power requirements ranged from 0.6 to 0.9 W. Five-axis impeller behavior and Mag-Lev controller stability were demonstrated in the pulsatile mock circuit. Noncontact drive and low power requirements were shown despite the effects of LV pulsation. The impeller position signals in the x- and y-axes reflected LV function. The Mag-Lev CRBP is effective not only for noncontact low power control of the impeller, but also for diagnosis of cardiac function noninvasively. PMID- 16683957 TI - Computational fluid dynamics and digital particle image velocimetry study of the flow through an optimized micro-axial blood pump. AB - A detailed knowledge of the flow field in a blood pump is indispensable in order to increase the efficiency of the pump and to reduce the shear-induced hemolysis. Thus, three different impeller designs were developed and tested by means of computational fluid dynamics (CFD) and digital particle image velocimetry (DPIV). The results show a good agreement of CFD and DPIV data. An optimization of the impeller could be achieved by following the concept of turbulent drag reduction for the axisymmetric center body. PMID- 16683958 TI - Computational fluid dynamics analysis of the pediatric tiny centrifugal blood pump (TinyPump). AB - We have developed a tiny rotary centrifugal blood pump for the purpose of supporting circulation of children and infants. The pump is designed to provide a flow of 0.1-4.0 L/min against a head pressure of 50-120 mm Hg. The diameter of the impeller is 30 mm with six straight vanes. The impeller is supported by a hydrodynamic bearing at its center and rotated with a radial coupled magnetic driver. The bearing that supports rotation of the impeller of the tiny centrifugal blood pump is very critical to achieve durability, and clot-free and antihemolytic performance. In this study, computational fluid dynamics (CFD) analysis was performed to quantify the secondary flow through the hydrodynamic bearing at the center of the impeller and investigated the effects of bearing clearance on shear stress to optimize hemolytic performance of the pump. Two types of bearing clearance (0.1 and 0.2 mm) were studied. The wall shear stress of the 0.1-mm bearing clearance was lower than that of 0.2-mm bearing clearance at 2 L/min and 3000 rpm. This was because the axial component of the shear rate significantly decreased due to the narrower clearance even though the circumferential component of the shear rate increased. Hemolysis tests showed that the normalized index of hemolysis was reduced to 0.0076 g/100 L when the bearing clearance was reduced to 0.1 mm. It was found that the CFD prediction supported the experimental trend. The CFD is a useful tool for optimization of the hydrodynamic bearing design of the centrifugal rotary blood pump to optimize the performance of the pump in terms of mechanical effect on blood cell elements, durability of the bearing, and antithrombogenic performance. PMID- 16683959 TI - Optimization of an axial flow heart pump with active and passive magnetic bearings. AB - Optimization of a magnetically suspended left ventricular assist device (LVAD) is crucial. We desire a totally implantable, long-life LVAD that delivers the necessary flow rate, pressure rise, and blood compatibility. By using a novel combination of passive and active magnetic bearings (AMBs), we have developed an axial flow LVAD prototype, the LEV-VAD, which provides an unobstructed blood flow path, preventing stagnation regions for the blood. Our current effort is focused on the optimization of the magnetic suspension system to allow for control of the AMB, minimizing its size and power consumption. The properties of the passive magnetic bearings and AMBs serve as parameter space, over which a cost function is minimized, subject to constraints such as suspension stability and sufficient disturbance rejection capabilities. The design process is expected to lead to the construction of a small prototype pump along with the necessary robust controller for the AMB. Sensitivity of the LVAD performance with respect to various design parameters is examined in-depth and an optimized, more compact LVAD prototype is designed. PMID- 16683960 TI - Development of integrated electronics unit for drive and control of undulation pump-left ventricular assist device. AB - In this study, we have developed an implantable electronics unit (IEU) for driving an undulation pump-left ventricular assist device (UP-LVAD). The IEU consists of a pump driver, three series-connected lithium ion batteries (1800 mAh), a charger, and a transcutaneous information transmission system. These electronic subunits were encapsulated in a case (110 x approximately 80 x approximately 30 mm) made of epoxy resin. The IEU was evaluated in two animal experiments using goats implanted with the UP-LVAD. The lithium ion batteries in the IEU provided 30-min energy supply daily to the UP-LVAD. The transcutaneous information transmission system transmitted data bidirectionally between the IEU and the personal computer at the data transmission ratio of 56 kbps without any transmission error. We could obtain survival days of 27 and 28 days supporting cardiac function with the UP-LVAD system. The temperature inside the IEU case was maintained under 45 degrees C, and there was evidence of a burn on the surrounding tissue in autopsies in each experiment. Based on the results, the IEU is promising to be suitable for drive and control of an implantable UP-LVAD. PMID- 16683961 TI - Advances in the treatment of affective disorders. PMID- 16683962 TI - Suicide and antidepressants: what is the evidence? PMID- 16683963 TI - Anticonvulsants in bipolar disorder. AB - OBJECTIVE: To review the literature on the effectiveness of anticonvulsant drugs in the management of bipolar disorder. METHODS: A selective review of the literature. RESULTS: Valproate is an effective drug, alone or in combination, for mania, but has limited benefit in bipolar depression. Although valproate is widely used in maintenance therapy of bipolar disorder, and secondary analyses suggest benefit, in the largest randomised maintenance trial there was only a trend favouring valproate. Lamotrigine has benefit in bipolar depression and maintenance, but not in mania. Carbamazepine is effective in mania. Other anticonvulsant drugs have been tried in mania, but with mixed results. CONCLUSIONS: Valproate, lamotrigine and carbamazepine have a valuable place in the management of bipolar disorder. PMID- 16683964 TI - Prospects for the treatment of depression. AB - Antidepressant drugs represent the principal form of treatment for major depressive disorder. While there are a plethora of medications available for this task, current drugs have many shortcomings. In the face of these deficiencies there is an ongoing search for new agents. The search has been guided, in part, by drug design based on existing agents and their putative mechanism of action. This has been less than fruitful in addressing inadequacies of existing medications as it has not produced compounds which are novel in terms of pharmacological mechanisms. Recent insights from molecular biological approaches hold promise for the discovery of novel compounds, in particular the so-called neurogenesis hypothesis suggests novel therapeutic approaches. Although significantly modified over the years, the monoamine hypothesis of depression and antidepressant drug action still remains an important driving force behind the development of new compounds. Several recently marketed agents and some in early phase development tend to conform to these existing mechanistic hypotheses. Clearly the place of these agents in the treatment of depression is dependent on issues such as short- and long-term safety and efficacy. Duloxetine has been developed as a dual monoamine re-uptake inhibitor. Agomelatine is a compound with major effects on the circadian system as well as effects on subtypes of the serotonin receptor system. While the mechanism of action of this compound is not certain, recent evidence would suggest that the drug exerts its effects through antagonist actions at serotonin receptors. Compounds based on the hypothalamic pituitary adrenal axis, substance P antagonism and other neuropeptides have potential application for the treatment of depression but require further development before that potential is realized. PMID- 16683965 TI - Antiglucocoticoid treatments for depression. AB - OBJECTIVE: To selectively review the literature germane to antiglucocoticoid treatments for depression. METHOD: Selective review of the relevant literature. RESULTS: Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis has been well-described in both bipolar and unipolar depression. Hypercortisolaemia, possibly secondary to breakdown in glucocorticoid-receptor-mediated negative feedback mechanisms within the HPA axis, may be central to the pathogenesis of both depressive symptoms and the neurocognitive deficits which characterize these disorders. Strategies to counteract the effects of elevated cortisol, which may potentially restore HPA axis integrity, have been the focus of recent research. CONCLUSIONS: Both preclinical and clinical studies report encouraging results which suggest that lowering circulating cortisol levels or blocking the effects of elevated cortisol with antagonists, which may up-regulate glucocorticoid receptors, has therapeutic benefits in terms of improvements in depressive symptoms and some domains of neurocognitive function. PMID- 16683966 TI - Transcranial magnetic stimulation for depression. AB - OBJECTIVE: To review the accumulated literature on the efficacy, safety and predictors of response for repetitive transcranial magnetic stimulation (rTMS) in the treatment of depression. METHODS: A descriptive review of the more than 25 published sham-controlled rTMS studies in depression was undertaken, focusing on reported meta-analyses as well as individual trial reports. Potential determinants of efficacy were examined, including the form of shams employed, stimulation parameters and clinical features. RESULTS: There is now clear evidence for the statistical superiority of left-prefrontal high frequency rTMS compared with sham therapy. However, the clinical benefits are marginal in the majority of reports. There is also still considerable uncertainty concerning the optimal stimulation parameters. Those clinical features which appear to be associated with greater response include younger age, lack of refractoriness to antidepressants and no psychotic features. CONCLUSIONS: Current studies confirm statistical efficacy, but insubstantive clinical benefit. Large multicentre studies currently underway should clarify if rTMS should be approved by regulatory agencies for widespread clinical availability. Furthermore, there is a need to clarify the preferred parameters for administering this treatment. PMID- 16683967 TI - Transition to psychosis: 6-month follow-up of a Chinese high-risk group in Hong Kong. AB - OBJECTIVES: The identification of individuals at high risk of becoming psychotic within the near future creates opportunities for early intervention before the onset of psychosis. This study sets out to identify a group of symptomatic young people in a Chinese population with the high likelihood of transition to psychosis within a follow-up period of 6 months, and to determine the rate of transition to psychosis in this group. METHOD: Symptomatic individuals with a family history of psychotic disorder, sub-threshold psychotic symptoms or brief transient psychotic symptoms were identified using the operationalized criteria of an 'At Risk Mental State'. The individuals were prospectively assessed monthly on a measure of psychopathology for 6 months. RESULTS: Eighteen out of 62 individuals (29%) made the transition to frank psychosis within a 6 month follow up period, with the majority occurring within 3 months. In addition, significant differences were found in the intake Positive and Negative Syndrome Scale, Comprehensive Assessment of 'At Risk Mental State' and Global Assessment of Functioning scores between the group that ultimately became psychotic and the group that did not. CONCLUSION: The period of the highest risk of transition to psychosis was within the 3 months after the study began. Thus, distressed youths in our outpatient clinic, who meet the high-risk criteria should be monitored most closely in the initial 3 months, particularly those individuals with high levels of psychopathology and functional decline. PMID- 16683968 TI - Pathways to care in a New Zealand first-episode of psychosis cohort. AB - OBJECTIVES: To present the clinical and sociodemographic characteristics of patients referred to a New Zealand early intervention for psychosis service and to describe the pathways to care for these patients. METHOD: Clinical and sociodemographic characteristics were examined in 200 patients consecutively admitted to an early psychosis service over a 5-year period. Assessment measures included DSM-IV diagnosis, the Quality of Life Scale, Positive and Negative Syndrome Scale and information on pathways to care. RESULTS: The majority of patients were young (mean age = 22.35), single (90.1%), male (72.5%), of New Zealand European descent (63.7%), living in the parental home (60.2%) and on a government benefit (60.2%). Overall, 40.8% of patients were initially diagnosed with a schizophrenia spectrum disorder, 36.4% with a mood disorder and 22.8% were classified as 'other psychoses' (substance-induced psychosis, brief psychotic disorder, etc.). Those with a schizophrenia spectrum disorder had a median duration of untreated psychosis of 120 days compared with 29 days for other diagnoses. Overall, 64.1% of referrals came from the Inpatient Service, 36.3% of patients had a compulsory admission under the Mental Health Act, and 41.8% had contact with police in 6 months prior to referral to the service. No differences in inpatient admission rates, police involvement or compulsory admission were evident for ethnicity or diagnosis. CONCLUSIONS: The pathways to care, clinical and sociodemographic descriptors of the present sample are somewhat different to those previously reported in international cohorts. This reflects the inclusion of patients with affective psychoses, the age range of 18-30 years and New Zealand's distinctive healthcare system. Examination of the sociodemographic characteristics and pathways to care in the present cohort suggests that service reconfigurations are required to optimize the experience of inpatient admission, given the substantial proportion of clients with non-schizophrenia spectrum disorders that present acutely for treatment. PMID- 16683969 TI - Pattern and correlates of inpatient admission during the initial acute phase of first-episode psychosis. AB - OBJECTIVES: The first aim of this study was to examine the rate, pattern and correlates of inpatient admission during the first 3 months of treatment for first-episode psychosis (FEP). The second aim was to determine whether the pattern of inpatient admission during this period was associated with remission of psychotic symptoms or inpatient service use at 15-month follow-up. METHOD: One hundred and four consecutive patients with FEP at a specialist treatment service were approached to participate in a follow-up study. Patients were grouped on the basis of the pattern of inpatient admission (none, one, or multiple) during the first 3 months of treatment. Clinical ratings at baseline and 3-month follow-up, and ratings of remission of psychotic symptoms at 3 and 15-month follow-up, were available for two-thirds of the patients. Inpatient data for the 15-month follow up period were derived from an electronic database for most patients (n = 98). RESULTS: Eighty (76.9%) of the 104 patients were admitted to an inpatient unit during the first 3 months of treatment. Fifty-nine (56.7%) patients had a single admission and 21 (20.2%) had multiple admissions. At baseline, inpatient admission was associated with a diagnosis of affective psychosis and more severe behavioural and functional disturbance but not positive psychotic symptoms. Multiple admissions were associated with risks to self or others at baseline and 3-month follow-up, and lack of remission of positive symptoms at 3 and 15-month follow-up. There was no association between the pattern of inpatient admission during the initial 3-month period and inpatient service use during the following 12-month period. CONCLUSIONS: The substantial proportion of young patients with FEP admitted to hospital emphasizes the need for youth-friendly treatment environments and practices. Although patients with multiple admissions during the initial treatment period are less likely to achieve remission, these patients are no more likely to establish a pattern of revolving-door hospitalizations compared with other patients. PMID- 16683970 TI - Treatment with olanzapine, risperidone or typical antipsychotic drugs in Asian patients with schizophrenia. AB - OBJECTIVE: To examine clinical outcomes in Asian patients with schizophrenia receiving monotherapy with olanzapine, risperidone or typical antipsychotics in naturalistic settings. METHOD: In this report, data from the first 12 months of the prospective, observational, 3-year Intercontinental Schizophrenia Outpatient Health Outcomes study are presented for patients from participating Asian countries (Korea, Taiwan and Malaysia) who were started on, or switched to, monotherapy with olanzapine (n = 484), risperidone (n = 287) or a typical antipsychotic drug (n = 127) at baseline. RESULTS: At 12 months, overall reduction in the score of Clinical Global Impressions-Severity of Illness rating scale was greatest with olanzapine (p < 0.001 vs typical agents), followed by risperidone (p = 0.007 vs typical agents) treatment. Olanzapine treatment was found to have significantly better effects than typical agents on negative and depressive symptom scores, and significantly greater improvements than risperidone on negative and cognitive symptoms. The occurrence of extrapyramidal symptoms was least likely with olanzapine (p < 0.001 vs typical agents, and p = 0.012 vs risperidone), while the estimated odds of tardive dyskinesia were greatest in the typical treatment group (p = 0.046 vs olanzapine, and p = 0.082 vs risperidone). Mean weight increase was greater for olanzapine-treated patients compared with the other agents (p = 0.030 vs typical agents and p < 0.001 vs risperidone). The risk of menstrual disturbance was relatively high with risperidone when compared with olanzapine treatment (p < 0.001). CONCLUSIONS: The results of this observational study indicate that, in Asian patients with schizophrenia, olanzapine may offer benefits when compared with typical agents or risperidone. However, the significantly greater odds of weight gain should be considered in the clinical management of olanzapine-treated patients. PMID- 16683971 TI - Detection of non-adherent behaviour in early psychosis. AB - OBJECTIVE: Adherence to antipsychotic treatment is an important aspect of the long-term management of schizophrenia. The evaluation of adherence is often difficult in the clinical setting. This study compared patient self-reporting and clinician judgment of adherence behaviour in patients with early and chronic schizophrenia. METHOD: Clinician-rated questionnaires and parallel patient self rated questionnaires were administered to 229 patients with early schizophrenia (illness duration < 5 years) and 255 patients with chronic schizophrenia. Items in the questionnaires addressed two forms of adherence behaviour (forgetting to take medication and deciding to stop medication) as well as attitudes toward medication. RESULTS: Significant non-adherent behaviour was reported by patients, particularly in the early schizophrenia group. Non-adherent behaviour was related to feelings of embarrassment about taking medication. Both non-adherence and embarrassment were under-recognized by clinicians. Starting from a prior probability of 0.24, knowledge of the patients' attitudes increased the posterior probability to 0.33, whereas clinicians' detection of non-adherence (deciding to stop medication) improved the posterior probability to 0.65. When both clinicians' evaluations and patients' attitudes were known, the posterior probability improved to 0.68. CONCLUSION: Non-adherence is a widespread phenomenon in early schizophrenia. Increasing clinicians' sensitivity to patients' feelings of embarrassment may be an important factor in the detection of non-adherence. When taking a base rate of non-adherence into consideration, clinicians' evaluations appeared to be more effective in detecting non-adherence than simple information obtained from patients on their attitudes toward medication. Pragmatic real-life estimation of non-adherence has important implications for the possibility of intervention. PMID- 16683972 TI - Community-based vocational rehabilitation: effectiveness and cost impact of a proposed program model. AB - OBJECTIVE: To study the vocational, service use and relative cost impact for schizophrenia or schizoaffective disorder of an innovative community-based vocational rehabilitation program. METHOD: Participants were consecutive enrollees in a community-based vocational rehabilitation program who had remained in the program for at least 1 year. Lifetime vocational history and mental health service utilization for 2 years prior and up to 4 years subsequent to program enrollment were studied. Retrospective data were collected using a questionnaire completed by the patient and available family and case workers, patient interview and chart review. RESULTS: Months in paid work increased after enrollment, while earned income did not (most work was low wage and/or part-time). Annual inpatient days decreased precipitously, a change which could not be explained by hospitalization trends during the same period. Average relative cost units, based on charges for mental health services used, dropped over 70% following enrollment. CONCLUSIONS: Community-based vocational rehabilitation may be cost effective in this population, largely as a result of its impact on hospitalizations and utilization of the most costly services. Such programs represent an important alternative to supported employment that may be particularly suited to cognitively or functionally impaired patients unwilling or unable to work in a competitive environment. PMID- 16683973 TI - Deinstitutionalization of long-stay patients with schizophrenia: the 2-year social and clinical outcome of a comprehensive intervention program in Japan. AB - OBJECTIVES: The Sasagawa Project aimed to investigate the effects of deinstitutionalization and evidence-based strategies for the treatment of mental disorders among long-stay patients after their discharge from a mental hospital using a quasi-experimental longitudinal study design and to assess the patients' social and clinical outcomes over a 2-year post-discharge period. METHOD: Seventy eight patients with schizophrenia were transferred to a community facility (Sasagawa Village) following the closure of Sasagawa Hospital in Koriyama in March 2002. The patients had undergone psychosocial training following the protocol outlined by the Optimal Treatment Project. All evaluations were performed prior to the patients' discharge and were repeated 12 and 24 months after discharge using the Positive and Negative Syndrome Scales, the Global Assessment for Functioning, the Schedule for Assessment of Insight, the Rehabilitation Evaluation Hall and Baker Scale, the Social Functioning Scale, the Drug Attitude Inventory, and the Mini-Mental State Examination. RESULTS: During the 24-month study period, 18 residents had incidents that made their continued stay at Sasagawa Village impossible. Only four (5.1%) of these residents were readmitted to psychiatric wards because of exacerbations of their conditions. Twelve residents were admitted to hospital because of serious physical illnesses. The 60 residents who remained in the community facility for 2 years demonstrated significant improvements in not only their psychiatric symptoms, but also their social functioning, as evidenced by their scores for Social Activity, Speech Skills, Disturbed Speech, Self-Care and General Behaviour on the Rehabilitation Evaluation Hall and Baker Scale and Withdrawal, Independence (Performance), Independence (Competence), and Employment on the Social Functioning Scale. CONCLUSIONS: Careful planning that minimized social and clinical dislocation may have contributed to the successful transition from mental hospital to community facility assessed in this study. Patients with a long history of illness showed favourable outcomes with little clinical deterioration and various improvements in their psychiatric symptoms and social functioning. PMID- 16683974 TI - Enhancing employment services for people with severe mental illness: the challenge of the Australian service environment. AB - OBJECTIVES: Comparatively few people with severe mental illness are employed despite evidence that many people within this group wish to obtain, can obtain and sustain employment, and that employment can contribute to recovery. This investigation aimed to: (i) describe the current policy and service environment within which people with severe mental illness receive employment services; (ii) identify evidence-based practices that improve employment outcomes for people with severe mental illness; (iii) determine the extent to which the current Australian policy environment is consistent with the implementation of evidence based employment services for people with severe mental illness; and (iv) identify methods and priorities for enhancing employment services for Australians with severe mental illness through implementation of evidence-based practices. METHOD: Current Australian practices were identified, having reference to policy and legal documents, funding body requirements and anecdotal reports. Evidence based employment services for people with severe mental illness were identified through examination of published reviews and the results of recent controlled trials. RESULTS: Current policy settings support the provision of employment services for people with severe mental illness separate from clinical services. Recent studies have identified integration of clinical and employment services as a major factor in the effectiveness of employment services. This is usually achieved through co-location of employment and mental health services. CONCLUSIONS: Optimal evidence-based employment services are needed by Australians with severe mental illness. Providing optimal services is a challenge in the current policy environment. Service integration may be achieved through enhanced intersectoral links between employment and mental health service providers as well as by co-locating employment specialists within a mental health care setting. PMID- 16683975 TI - Association between childhood trauma and dissociation among patients with borderline personality disorder. AB - OBJECTIVE: To examine the relationship between childhood trauma and dissociative experience in adulthood in patients with borderline personality disorder. METHOD: Dissociative experiences scale scores and subscale scores for the Childhood Trauma Questionnaire were correlated in 139 patients. Patients were dichotomized into high or low dissociators using the Median Dissociative Experiences Scale score as the cut-off. RESULTS: Childhood Trauma Questionnaire Subscale scores for emotional and physical abuse and emotional neglect but not sexual abuse correlated significantly with Dissociative Experiences Scale scores. High dissociators reported significantly greater levels of emotional abuse, physical abuse, emotional neglect and physical neglect but not sexual abuse than low dissociators. CONCLUSION: Patients with borderline personality disorder therefore demonstrated levels of dissociation that increased with levels of childhood trauma, supporting the hypothesis that traumatic childhood experiences engender dissociative symptoms later in life. Emotional abuse and neglect may be at least as important as physical and sexual abuse in the development of dissociative symptoms. PMID- 16683976 TI - Childhood adversity and adult personality. AB - OBJECTIVES: To explore how recalled childhood adversity affects trait measures of personality in three age cohorts of an Australian adult population and to examine the effects of particular adversities on adult personality traits. METHOD: A total of 7485 randomly selected subjects in the age bands of 20-24, 40-44 and 60 64 years were interviewed at the outset of a longitudinal community study of psychological health in the Canberra region of Australia. In the initial interview, subjects answered 17 questions about domestic adversity and three questions on positive aspects of upbringing to age 16 years. Personality traits were measured by Eysenck Personality Questionnaire, Behavioural Activation and Inhibition Scales, Positive and Negative Affect Scales and a measure of dissocial behaviours. RESULTS: Higher levels of childhood adversity substantially increase the risk of high neuroticism (OR = 2.6) and negative affect (OR = 2.6), less for behavioural inhibition (OR = 1.7) and for dissocial behaviour (OR = 1.7). No significant effect is seen for extraversion, psychoticism or behavioural activation. Age and gender had little effect on the pattern of risk. Maternal depression has significant and substantial independent effects on measures of neuroticism and negative affect as well as most other measures of personality. CONCLUSION: Childhood domestic adversity has substantial associations with clinically important aspects of personality: neuroticism and negative affect. Only small effects are seen on behavioural inhibition and dissocial behaviour, and no significant effect on extraversion and behavioural activation. These unexpected findings contradict clinical belief. Maternal psychological ill-health is pre-eminent among adversities predicting later disadvantageous traits, even for those traits that had only the slightest association with childhood adversity. Consequences of childhood adversity prevail throughout the lifespan in men and women equally. The study underlines the importance of childhood domestic adversity and especially maternal psychological ill-health as a target for preventive intervention for psychological difficulties at all ages. PMID- 16683977 TI - Examining geographical and household variation in mental health in Australia. AB - OBJECTIVE: International research has failed to demonstrate area effects in the distribution of common mental disorders. In contrast, strong and robust household effects are evident, though relatively rarely examined. This study investigated household and area effects in the distribution of mental health scores using Australian data. METHOD: Analysis of data from the first wave of the Household, Income and Labour Dynamics in Australia Survey: a large representative survey of 13 969 Australian adults. Multilevel regression methods were used to model variance in the mental health scale and mental component summary scale of the Short-Form 36 at the individual, household and area (Census Collection District) levels. A number of risk factors at various levels of the model were also examined. RESULTS: Very little variance in mental health scores occurred at the area level (1.5%), whereas significant and substantial variance occurred at the household level (23.0%). The variance at the household level remained highly significant following the inclusion of a range of risk factors at the individual, household and area levels. CONCLUSIONS: The results confirm the absence of substantial area-level variation in mental health using Australian data. The findings highlight the importance of focusing on household-level characteristics in future research. PMID- 16683978 TI - Successful treatment with lamotrigine in bipolar depression: a study from Turkey. PMID- 16683979 TI - Stuporous catatonia in an elderly bipolar patient: response to olanzapine. PMID- 16683984 TI - A three-dimensional model of differentiation of immortalized human bronchial epithelial cells. AB - A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration. PMID- 16683985 TI - Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. AB - Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5 azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes. PMID- 16683986 TI - An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells. AB - Human embryonic stem (ES) cells are usually co-cultivated with supporting cells consisting of short-term cultures of fibroblasts (not an immortalized line) in a medium lacking serum. This method has promoted important progress in the field, but suffers from certain disadvantages. By serial cultivation for 27 consecutive transfers and about 63 cell generations, we have evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports the multiplication of H9 cells better than the 3T3 line. It supports the growth of H9 cells as well as do available short-term fibroblast cultures, but maintains more effectively the stem cell character of the H9 cells, judging by their better retention of Oct4. We have made MMM cells resistant to blasticidin and zeocin, the most efficient antibiotics for selection of stable transformants. In the presence of zeocin, the resistant MMM were able to support multiplication and selection of ES cells transfected with an exogenous gene encoding zeocin resistance. PMID- 16683987 TI - Gene expression analysis identifies novel genes participating in early murine liver development and adult liver regeneration. AB - Adult liver tissue regeneration may recapitulate molecular events of liver organogenesis. As gaps in our understanding of the fundamental processes that govern development and regeneration of the liver still exist, we studied gene expression in the developing liver at embryonic day 9.5 post coitum (E d9.5 p.c.). Microarray data from E d9.5 p.c. as well as previously published data from embryonic day 11.5 post coitum (E d11.5 p.c.) and embryonic day 13.5 post coitum (E d13.5 p.c.) were subjected to cluster analysis. This led to the identification of 130 genes which were characterized by continuous expression at all stages of liver development with peak expression of 44 genes at E d9.5 p.c. Five of these genes, previously not known to be associated with early liver development or with adult liver regeneration were selected for further analysis. The expression of the genes was studied by real-time polymerase chain reaction at 0, 2, 4, 6, 12, 24 and 48 hr after partial hepatectomy in the adult liver. Two of the genes, growth arrest protein 43 (GAP43) and paired-like homeodomain transcription factor 2 (Pitx2) were exclusively detected at 24 hr, whereas the genes Twist1, Midkine, and zinc finger protein of cerebellum 1 (Zic1) each showed a specific expression profile in the regenerating liver with peak expressions at 4, 24, and 6 hr, respectively. In summary, we were able to identify novel genes, that may act as regulators during liver formation as well as in the regeneration phase of adult liver. This information may contribute to the development of new targets for the treatment of liver diseases in the future. PMID- 16683988 TI - Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing. AB - In addition to their well-known antinociceptive action, opioids can modulate non neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing. PMID- 16683989 TI - Epidermis-restricted expression of zebrafish cytokeratin II is controlled by a 141/+85 minimal promoter, and cassette -141/-111 is essential for driving the tissue specificity. AB - We isolated a 2.3 kb DNA segment from the upstream region of the zebrafish cytokeratin II (zfCKII) gene. Transgenic embryos, produced by using a series of 5' deletions linked to the red fluorescent protein (RFP) reporter, showed that the -141/+85 segment of zfCKII directed RFP expression in epidermal cells, whereas the -111/+85 segment did not. When -141/-111 was deleted from -355/+85 and microinjected into one-celled embryos, no fluorescence was observed at later stages, indicating that the -141/-111 segment is required for green fluorescent protein expression in epidermal cells. Furthermore, when a putative KLF-binding site at -119/-117 was mutated, RFP expression rates and intensities were reduced dramatically, although still observed, suggesting that -119/-117 within -141/-111 is a key cis-element for controlling epidermis-specific expression of the zfCKII gene. Finally, we generated a zebrafish transgenic line, Tg(zfCKII(2.3):RFP), which carries an upstream 2.3 kb regulatory region of the zfCKII gene fused with RFP. The expression pattern in the epidermal cells of Tg(zfCKII(2.3):RFP) fish recapitulated that of the endogenous gene. F2 embryos derived from Tg(zfCKII(2.3):RFP) males crossed with wild-type females revealed that the earliest onset of RFP expression was at the sphere stage, indicating that this transgenic approach can be used for studying zygotic expression of maternally inherited genes. PMID- 16683990 TI - The pharmacokinetics of coagulation factors. AB - In this article, we provide a summary of the generally accepted approaches to the design and analysis of studies examining the pharmacokinetic (PK) profile of an infused coagulation factor in patients with a deficiency of one or more of these factors. Furthermore, we briefly review the known PK results for various commercially available coagulation factor preparations under single and continuous infusion. PMID- 16683991 TI - Haemophilia and ageing. AB - Men with haemophilia have not only the challenges of living with HIV and/or HCV infection and premature arthritis as complications of their disorder, but they also confront the other ails of ageing. These include genitourinary problems such as prostatic hypertrophy, prostatic cancer and renal stone disease, and arterial disease for which haemophilia is not protective. Progressive arthritis and declining fitness may lead to loss of independence which causes great concern. Associated with the physical aspects of ageing, many patients also suffer from psychological symptoms which may be precipitated by changes in work such as early retirement and altered family dynamics. Many older men with haemophilia may never have consulted primary care physicians because of the rarity and complexity of their disorder. Haemophilia centre staff often assume responsibility for the identification and management of all health problems of their patients. Even when other clinicians are involved, patients require their centre's involvement in the investigation and support of many procedures such as coronary artery surgery and urological surgery. This paper addresses falls in the older man with haemophilia, their causes and consequences and cardiovascular problems in particular. Very little literature has been published about these common problems. We need to be aware of the ageing issues in haemophilia and develop 'wellness' programs which are directed to the early identification of disease as well as preventative strategies to reduce the physical and psychological impacts of ageing. PMID- 16683992 TI - The natural evolution of haemophilia care: developing and sustaining comprehensive care globally. AB - Comprehensive care is vital for patients with haemophilia to prevent early death and free patients from the complications that inhibit living normal lives. Experience has shown that once introduced in a country, there is a progressive restoration of normal healthy lives to the haemophilia community. Accompanying this progress is a gradual decreased dependency on the haemophilia comprehensive centre - except during brief periods when expertise contained within the comprehensive centre is mandatory for life-saving clinical management or to prevent severe morbidity. During each stage of the natural evolution of comprehensive haemophilia care in a country, challenges to the existence of the centre occur, which threaten the comprehensive treatment concept. The haemophilia community must understand this natural evolution and be prepared to work collaboratively with governments, physicians and other patients to ensure that centres retain the expertise to meet the emergent needs when they arise. PMID- 16683993 TI - Quality of life assessment in clinical practice in haemophilia treatment. AB - The modern management of haemophilia has greatly influenced not only survival of patients, their clinical symptoms and orthopaedic outcome but also their perceived quality of life (QoL). QoL measures recently became an essential part of clinical trials being one of the most important patient-rated outcomes (PROs). Moreover, QoL assessment is essential in pharmacoeconomics. In clinical trials, not only clinical outcomes but also the so-called PROs and health-economic outcomes are included. Different types of economic evaluations may be conducted in order to describe the economic burden of a disease condition, to determine the cost of care and to assess and evaluate alternative treatments in terms of both costs and effects as well as benefits and outcomes with the aim of optimizing the use of resources. For these evaluations, the following analyses are performed: descriptive cost of illness study, incremental cost-effectiveness analysis, incremental cost-utility analysis and incremental cost-benefit analysis. By contrast, PROs are derived from direct patient reports and they allow to evaluate the impact of a disease and its treatment on patients' well-being and functioning. PROs include health-related quality of life, patient preferences/utilities, treatment satisfaction and other PROs such as functional assessment, etc. Choosing a QoL measure, study- and instrument-related aspects have to be taken into account. Finally, QoL assessment will become a part of the regular clinical assessment of persons with haemophilia, in order to provide trustworthy data of perceived well-being to be compared over time and in order to assess treatment efficacy and quality of care. PMID- 16683994 TI - Ethical issues in haemophilia. AB - Ethical issues surrounding both the lack of global access to care as well as the implementation of advancing technologies, continue to challenge the international haemophilia community. Haemophilia is not given the priority it deserves in most developing countries. Given the heavy burdens of sickness and disease and severe resource constraints, it may not be possible to provide effective treatment to all who suffer from the various 'orphan' diseases. Nevertheless, through joint efforts, some package of effective interventions can be deployed for a significant number of those who are afflicted with 'orphan' diseases. With cost effective utilization of limited resources, a national standard of care is possible and affordable. Gene-based diagnosis carries attendant ethical concerns whether for clinical testing or for research purposes, even as the list of its potential benefits to the haemophilia community grows rapidly. As large-scale genetic sequencing becomes quicker and cheaper, moving from the research to the clinic, we will face decisions about the implementation of prenatal, neonatal and other screening programs. Such debates will require input from not just the health care professionals but from all stakeholders in the haemophilia community. Finally, long-term therapeutic success gene transfer in small and large animal models raises the question of when and in which patient population the novel therapeutic approach should first be studied in humans with haemophilia. Although gene therapy represents a worthy goal, the central question for the haemophilia community should be whether it wishes to volunteer itself as a model for a much broader set of innovations. PMID- 16683995 TI - Cellular and genetic therapies for haemophilia. AB - Haemophilia continues to be a prime target for a variety of gene and cell-based therapies. Pre-clinical successes in both mouse and dog models of the disease have been documented with a variety of approaches over the past decade, and there have now been six small clinical trials of gene transfer in haemophilia. To date, the only significant adverse events documented in these trials have been related to host immune responses, indicating that immunologic barriers continue to represent the major obstacle to achieving success of gene transfer in humans. Despite these challenges, new strategies are being explored with novel serotypes of viral vectors and with the use of transient periods of immunosuppression to attenuate the immune response to the vector and transgene product following gene delivery. Two new clinical trials, both using AAV vectors, will likely start within the next year, and additional large animal pre-clinical studies using other viral vector-mediated approaches for gene transfer are expected in the near future. PMID- 16683996 TI - Strategies towards a longer acting factor VIII. AB - The reduced mortality, improved joint outcomes and enhanced quality of life, which have been witnessed in the developed world for patients with haemophilia, have been an outstanding achievement. Advancements in biotechnology contributed significantly through the development of improved pathogen screening, viral inactivation techniques and the development of recombinant clotting factors. These were partnered with enhanced delivery of care through comprehensive haemophilia centres, adoption of home therapy and most recently effective prophylaxis. This came at great costs to governments, medical insurers and patients' families. In addition, barriers persist limiting the adoption and adherence of effective prophylactic therapy. Biotechnology has been successful at overcoming similar barriers in other disease states. Long-acting biological therapeutics are an incremental advance towards overcoming some of these barriers. Strategies that have been successful for other therapeutic proteins are now being applied to factor VIII (FVIII) and include modifications such as the addition of polyethylene glycol (PEG) polymers and polysialic acids and alternative formulation with PEG-modified liposomes. In addition, insight into FVIII structure and function has allowed targeted modifications of the protein to increase the duration of its cofactor activity and reduce its clearance in vivo. The potential advantages and disadvantages of these approaches will be discussed. PMID- 16683997 TI - Why do inhibitors develop? Principles of and factors influencing the risk for inhibitor development in haemophilia. AB - The formation of inhibitory alloantibodies is, in the postinfectious era, the most severe and costly complication of replacement therapy in patients with haemophilia. The complexity of the immune response to the infused factor becomes more and more obvious as knowledge in the area increases. Antibodies develop as a result of a complex multi-factorial interaction between antigen-presenting cells, T- and B-lymphocytes. Genetic susceptibility of cell surface molecules, such as the major histocompatibility complex, the T-cell receptor and cytokine receptors, as well as various immunomodulatory molecules have a major impact on the outcome. In addition, environmental factors probably influence the risk of inhibitor development. The current concept of inhibitor development is reviewed. A better understanding of the pathophysiological mechanisms involved will facilitate improvement of therapy in the future, and hopefully provide an opportunity to prevent this complication of treatment. PMID- 16683998 TI - Achieving and maintaining quality in the laboratory. AB - In order to ensure the delivery of a service of the highest possible quality, it is an essential requirement that laboratories undertake strict internal quality control (QC) measures as well as participate in external quality assessment (EQA) schemes. For any given test, a critical part of the internal QC process involves the establishment of reference intervals using samples taken from normal individuals, and then calculating limits representing the 95% range. This forms the basis for assessment of abnormal test results, which will in turn impact on laboratory performance in proficiency testing exercises in EQA programmes. Whereas for plasma-based assay systems, variability in performance in EQA exercises is usually determined by measurement of a coefficient of variation (CV), results of genetic testing is usually measured in absolute terms. Despite this, results of genetic EQA programmes confirm that errors in testing do occur, as much because of inadvertent sample switching and transcription errors as to analytical mistakes. EQA programmes involving identification of mutations by DNA sequencing, such as haemophilia, is made difficult by the high information content of sequence data. Nevertheless, results show that errors are usually made in the naming of the mutations, indicating that this is an evolving and poorly standardized area. Developing countries face particular challenges in the encouragement of laboratories to participate in local EQA programmes, as well as in relation to the logistical issues of sample provision, distribution and result collation in an effective and affordable manner. PMID- 16683999 TI - Laboratory issues in bleeding disorders. AB - The clinical history of the patient and of his/her relatives is the most important tool for making correct diagnosis of inherited or acquired bleeding disorders. Several attempts have been made by clinicians to evaluate the sensitivity and specificity of bleeding symptoms. Specific and detailed questionnaires have been designed to quantify the bleeding tendency of patients with von Willebrand's disease (VWD) and a bleeding score has been calculated. VWD is considered the most frequent inherited bleeding disorder according to population studies: however, due to the complexity of its diagnosis, the number of patients with correct diagnosis of VWD in many developing countries is relatively low and most cases remain still under- or misdiagnosed. Once bleeding history is carefully evaluated by means of a bleeding score, the laboratory workout should be organized to find out the specific defect of haemostasis responsible for bleeding. Since factors involved in haemostasis are many, the correct approach must include first level screening tests with the aim to identify the abnormal phase of haemostasis involved: then, second level tests should be focused on the specific factors within the abnormal step of haemostasis. Among many other acquired bleeding disorders related to clinical conditions or to the use of drugs, the acquired inhibitors of haemostasis are rare but should be immediately characterized by appropriate laboratory tests because they can be often life-threatening for the patients. PMID- 16684000 TI - New approaches for measuring coagulation. AB - Although specific assays of coagulation factors are essential for diagnostic purposes they only give partial information about an individual's haemostatic state. This can be better assessed by various global tests, and recent developments and evaluations of five such tests are described in this symposium: the PFA-100; waveform analysis; thrombin generation; overall haemostasis potential; thrombelastography. Each test has advantages in various applications, but the thrombin generation test and waveform analysis have been found most useful in haemophilia, whilst the PFA-100 is helpful in von Willebrand's disease. PMID- 16684002 TI - HIV and HCV coinfected haemophilia patients: what are the best options of orthopaedic treatment? AB - In the era of contemporary orthopaedics, haematology and internal medicine, it is obvious that surgery can be indicated in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) coinfected haemophilia patients suffering from severe and painful musculoskeletal problems. However, the expected high risk of infection and other postoperative complications is a concern due to the poor health status that many of these patients present. In a HIV and HCV coinfected haemophilia patient, the orthopaedic surgeon together with the multidisciplinary haemophilia team should weigh the risks and benefits carefully. Clinical and immunological status should be considered before suggesting a surgical procedure (specially a joint arthroplasty) in this group of patients. If a surgical procedure is contraindicated, conservative treatment could be an alternative, although many times with inferior results. Usually, surgical procedures can better relieve pain for several years and improve the quality of life in this cohort of patients. Regarding anaesthetics, drugs metabolized by the liver or the kidney should be avoided depending on every particular case. Rachianaesthesia is more recommendable than epidural anaesthesia in elderly patients in whom general anaesthesia sometimes could be dangerous, although in all ages we prefer general anaesthesia. The size of the needle should be small (size G-27), and we never use spinal catheters. PMID- 16684001 TI - Genetic diagnosis of haemophilia and other inherited bleeding disorders. AB - Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders. PMID- 16684003 TI - Recent developments in clinimetric instruments. AB - Assessment of impairment and function is essential in order to monitor joint status and evaluate therapeutic interventions in patients with haemophilia. The improvements in the treatment of haemophilia have required the development of more sensitive tools to detect the more minor dysfunctions that may now be apparent. This paper outlines some of the recent developments in this field. The Haemophilia Joint Health Score (HJHS) provides a systematic and robust measure of joint impairment. The MRI Scoring System has been designed to provide a comprehensive scoring system combining both progressive and additive scales. The Functional Independence Score for Haemophilia (FISH) has been developed to assess performance of functional activities and can be used in conjunction with the Haemophilia Activities List (HAL) which provides a self report measure of function. It is recommended that both measures are evaluated as these tools measure different constructs. Further refinement and testing of the psychometric properties of all of these tools is in progress. More widespread use of these tools will enable the sharing of data across the world so promoting best practice and ultimately enhancing patient care. PMID- 16684004 TI - Physiotherapy following elective orthopaedic procedures. AB - As haemophilic arthropathy and chronic synovitis are still the most important clinical features in people with haemophilia, different kinds of invasive and orthopaedic procedures have become more common during the last decades. The availability of clotting factor has made arthroplasty of one, or even multiple joints possible. This article highlights the role of physiotherapy before and after such procedures. Synovectomies are sometimes advocated in people with haemophilia to stop repetitive cycles of intra-articular bleeds and/or chronic synovitis. The synovectomy itself, however, does not solve the muscle atrophy, loss of range of motion (ROM), instability and poor propriocepsis, often developed during many years. The key is in taking advantage of the subsequent, relatively safe, bleed-free period to address these important issues. Although the preoperative ROM is the most important variable influencing the postoperative ROM after total knee arthroplasty, there are a few key points that should be considered to improve the outcome. Early mobilization, either manual or by means of a continuous passive mobilization machine, can be an optimal solution during the very first postoperative days. Muscle isometric contractions and light open kinetic chain exercises should also be started in order to restore the quadriceps control. Partial weight bearing can be started shortly after, because of quadriceps inhibition and to avoid excessive swelling. The use of continuous clotting factor replacement permits earlier and intensive rehabilitation during the postoperative period. During the rehabilitation of shoulder arthroplasty restoring the function of the rotator cuff is of utmost importance. Often the rotator cuff muscles are inhibited in the presence of pain and loss of ROM. Physiotherapy also assists in improving pain and maintaining ROM and strength. Functional weight-bearing tasks, such as using the upper limbs to sit and stand, are often discouraged during the first 6 weeks postoperatively. This may be influenced by the condition of the joints of the lower limbs. Attention should be given to the total chain of motion, of which the shoulder itself is only a part. We conclude that physiotherapy management is of major importance in any invasive or orthopaedic procedure, regardless of which joints are involved. Both pre- and postoperative physiotherapy, as part of comprehensive care is needed to achieve optimal functional outcome and therefore optimal quality of life for people with haemophilia. PMID- 16684005 TI - Total joint replacement in patients with inhibitors. AB - Today, total joint replacement is the treatment of choice for chronic haemophilic arthropathy of the knee and hip in developed and developing countries. After the last World Haemophilia Congress and Musculoskeletal Congress, we cannot say the same for haemophilic patients with inhibitors because elective surgery today is still limited to few centres and extremely few patients. This is because until the first half of the 1990s, performing surgery in haemophilic patients with inhibitors was associated with a high risk of bleeding. With the availability of activated recombinant factor VII, the first surgical procedures were performed, but they still remain limited because of the elevated costs of replacement therapy. Our goal for the future must be to ensure the same possibility of surgical intervention in haemophilic patients both with and without inhibitors. This will be possible, thanks to the experience of some centres with an increased number of patients, where today this kind of surgery is routinely performed. PMID- 16684006 TI - Pathogenesis of haemophilic arthropathy. AB - The pathogenetic mechanism of haemophilic arthropathy is multifactorial and includes degenerative cartilage-mediated and inflammatory synovium-mediated components. Intra-articular blood first has a direct effect on cartilage, as a result of the iron-catalysed formation of destructive oxygen metabolites (resulting in chondrocyte apoptosis), and subsequently affects the synovium, in addition to haemosiderin-induced synovial triggering. Both processes occur in parallel, and while they influence each other they probably do not depend on each other. This concept resembles degenerative joint damage as found in osteoarthritis as well as inflammatory processes in rheumatoid arthritis. These processes finally result in a fibrotic and destroyed joint. PMID- 16684007 TI - Tissue engineering in musculoskeletal problems related to haemophilia. AB - This article is a review of how advances in tissue engineering can be applied to the musculoskeletal pathology of patients with haemophilia. This article will also explain the theory that the deterioration of joints in patients with haemophilia is due to biological and mechanical causes. Current concepts of tissue engineering would be to replace the degenerated and damaged tissue by live cells, using them as a biological implant. However, before these new technologies are applied, an appropriate control of their indication and results is required. PMID- 16684008 TI - Congenital platelet disorders: overview of their mechanisms, diagnostic evaluation and treatment. AB - The bleeding problems associated with common and rare inherited platelet disorders illustrate the importance of platelets to normal haemostasis. At sites of injury, platelets normally adhere, undergo activation, secretion and aggregate formation, and they provide the membrane surface for the assembly of coagulation to generate thrombin. The causes of inherited disorders that alter platelet haemostatic functions are quite diverse, ranging from defects in receptors critical to platelet adhesion and aggregation, to defects in signalling molecules or in transcription factors important for production of functional platelets. The mechanisms of impaired platelet function are largely unknown for the more common disorders that alter platelet activation, secretion and the secondary wave of platelet aggregation. The diagnostic evaluation of congenital platelet disorders has been challenging as some 'platelet-type' bleeding symptoms, such as bruising, are quite common in the general population. Moreover, the diagnostic tests used by clinical laboratories to evaluate disorders of platelet function have not been standardized. In individuals recognized to have an inherited defect in platelet function, therapy is important for controlling and preventing bleeding episodes. Presently, there are a number of choices to consider for the management of bleeding symptoms, including menorrhagia. This paper reviews the causes, diagnostic evaluation and therapies for common and rare congenital platelet disorders. PMID- 16684009 TI - Rare bleeding disorders. AB - Deficiencies of coagulation factors other than factor VIII and factor IX (afibrinogenemia, FII, FV, FV+FVIII, FVII, FX, FXI, FXIII) that cause bleeding disorders (RBDs) are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500,000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. The study of the genetic basis of these disorders could represent an important tool for prevention through prenatal diagnosis. Treatment of patients with RBDs during bleeding episodes or surgery is a challenge because of the lack of experience and the paucity of data. For some deficiency factor concentrates are still non available and severe complications can occur. These complications can be minimized by assessment of risks of bleeding and thrombosis, use of haemostatic means other than blood components or no therapy at all. The RBDs pose a problem for guideline writers because there are no suitable clinical trials to supply good evidence for how these people are best treated. The lack of adequate information on clinical manifestations, treatment and genetic basis of RBDs could be improved by the collection of data in an International Database (http://www.rbdd.org), linkable to others previously published. This could be a useful tool to fill the gap between clinical data and clinical practice. This article reviews the genetic basis of RBDs, problems and complications of treatment, problems in the preparation of suitable guidelines for treatment and the future perspectives of the International Registry on RBDs. PMID- 16684010 TI - Current understanding of von Willebrand's disease in women - some answers, more questions. AB - Considerable progress has been made in the past decade in describing the obstetrical and gynaecological aspects of von Willebrand's disease (VWD). In addition, epidemiological studies have established an approximately 11-16% prevalence of the laboratory diagnosis of VWD in women presenting with menorrhagia. However, it is not established presently whether an upfront VWD screening should be a part of the standard evaluation of menorrhagia. This is because it is presently not known whether therapy in the VWD patient tailored specifically for VWD will appreciably alter the natural history of menorrhagia compared with the non-VWD menorrhagia patient. There are also subtleties involved in securing the diagnosis of VWD in women presenting with menorrhagia in terms of fluctuation of von Willebrand factor (VWF) levels vis-a-vis the menstrual cycle and the potential impact of oral contraceptive on VWF levels. Regarding management of VWD-related menorrhagia, pending ongoing comparative trials of intranasal desmopressin (DDAVP), tranexamic acid, oral contraceptive and the levonorgestrel intrauterine device, specific recommendations cannot be made presently regarding the superiority of one intervention compared with the other. The management of VWD-related postpartum haemorrhage is also an area of active debate in terms of 'best practice' in type 1 (? prophylactic DDAVP), type 2 [? expectant management if factor VIII:C (FVIII:C) level normalizes] and type 3 patients (? intensity and duration of infusional therapy with a VWF-containing plasma-derived FVIII concentrate). This review summarizes the present state of knowledge and highlights numerous questions for future study based on our present understanding of VWD in women. PMID- 16684011 TI - Von Willebrand's disease: clinical management. AB - The aim of treatment of von Willebrand's disease (VWD) is to correct the dual defect of haemostasis, i.e. the abnormal platelet adhesion due to reduced and/or dysfunctional von Willebrand factor (VWF) and the abnormal coagulation expressed by low levels of factor VIII (FVIII). Desmopressin (DDAVP) is the treatment of choice for type 1 VWD because it can induce release of normal VWF from cellular compartments. Prospective studies on biological response versus clinical efficacy of DDAVP in VWD type 1 and 2 are in progress to further explore its benefits and limits as therapeutic option. In type 3 and in severe forms of type 1 and 2 VWD, DDAVP is not effective and for these patients plasma virally inactivated concentrates containing VWF and FVIII are the mainstay of treatment. Several intermediate- and high-purity VWF/FVIII concentrates are available and have been shown to be effective in clinical practice (bleeding and surgery). New VWF products almost devoid of FVIII are now under evaluation in clinical practice. Although thrombotic events are rare in VWD patients receiving repeated infusions of concentrates, there is some concern that sustained high FVIII levels may increase risk of postoperative venous thromboembolism. Dosage and timing of VWF/FVIII administrations should be planned to keep FVIII level between 50 and 150 U/dL. Appropriate dosage and timing in repeated infusions are also very important in patients exposed to secondary long term prophylaxis for recurrent bleedings. PMID- 16684012 TI - Prevention and treatment of musculoskeletal disease in the haemophilia population: role of prophylaxis and synovectomy. AB - Prophylaxis is defined as primary (started before the onset of joint damage) or secondary (started after the onset of joint damage). The aim of primary prophylaxis is to prevent recurrent bleeding into joints and the development of chronic arthropathy in later life. When started early, and at most after two joint bleeds, the result is predictably excellent if there is compliance with the primary prophylaxis regimen. In order to decrease the need for central venous access devices to assure reliable venous access, a number of centres start primary prophylaxis with once weekly infusions with dose-escalation based on frequency of joint bleeding. A major unanswered question is whether primary prophylaxis can be safely discontinued in adolescents/young adults and if so, when. A promising predictor for the milder bleeding phenotype in persons with severe haemophilia is a later onset of joint bleeding. Once joint damage has occurred as a result of recurrent bleeding, secondary prophylaxis can only retard, but not prevent, ongoing joint damage. Other strategies to decrease recurrent bleeding from target joints include surgical synovectomy (ideally performed using an arthroscopic technique), radionuclide synovectomy and chemical synovectomy. These interventions have very good outcomes when performed by an experienced team. Given the very high cost of factor concentrates required for programmes of prophylaxis prospective studies that document benefits to the child and family, e.g. quality of life are to be encouraged. PMID- 16684013 TI - Treatment for all: a vision for the future. AB - The World Federation of Hemophilia (WFH) has defined a new strategic plan which maps out where the organization is going and what it can do for our members. The plan embraces the vision of Treatment for All; i.e. one day, treatment will be available for all those with inherited bleeding disorders, regardless of where they live. Treatment for All means proper diagnosis, management, and care by a multidisciplinary team of trained specialists. It means safe, effective treatment products are available for all people with inherited bleeding disorders. It means expanding services beyond haemophilia, to those with von Willebrand's disease, rare factor deficiencies, and inherited platelet disorders. Today, more than 75% of the global bleeding disorders community receive either inadequate or no treatment whatsoever. Our mission is to improve treatment where it is limited or does not exist. At the same time, we must sustain the many gains we have achieved thus far. The challenge is immense. Making our vision a reality requires us to be focused and deliberate about the programmes we undertake and the commitments we make. Building on past strategic plans, this plan presents a vision for the continued success of the WFH over the next 3-5 years. PMID- 16684014 TI - Initial experience in the use of integrated electroanatomic mapping with three dimensional MR/CT images to guide catheter ablation of atrial fibrillation. AB - INTRODUCTION: No prior studies have reported the use of integrated electroanatomic mapping with preacquired magnetic resonance/computed tomographic (MR/CT) images to guide catheter ablation of atrial fibrillation (AF) in a series of patients. METHODS AND RESULTS: Sixteen consecutive patients with drug refractory AF underwent catheter ablation under the guidance of a three dimensional (3D) electroanatomic mapping system (Carto, Biosense Webster, Inc., Diamond Bar, CA, USA). Gadolinium-enhanced MR (n = 8) or contrast-enhanced high resolution CT (n = 8) imaging was performed within 1 day prior to the ablation procedures. Using a novel software package (CartoMerge, Biosense Webster, Inc.), the left atrium (LA) with pulmonary veins (PVs) was segmented and extracted for image registration. The segmented 3D MR/CT LA reconstruction was accurately registered to the real-time mapping space with a combination of landmark registration and surface registration. The registered 3D MR/CT LA reconstruction was successfully used to guide deployment of RF applications encircling the PVs. Upon completion of the circumferential lesions around the PVs, 32% of the PVs were electrically isolated. Guided by a circular mapping catheter, the remaining PVs were disconnected from the LA using a segmental approach. The distance between the surface of the registered 3D MR/CT LA reconstruction and multiple electroanatomic map points was 3.05 +/- 0.41 mm. No complications were observed. CONCLUSIONS: Three-dimensional MR/CT images can be successfully extracted and registered to anatomically guided clinical AF ablations. The display of detailed and accurate anatomic information during the procedure enables tailored RF ablation to individual PV and LA anatomy. PMID- 16684015 TI - Three-dimensional image integration: a first experience with guidance of atrial fibrillation ablations. PMID- 16684016 TI - Catheter ablation of stable and unstable ventricular tachycardias in patients with arrhythmogenic right ventricular dysplasia. AB - INTRODUCTION: A reentrant circuit within an area of abnormal myocardium is suspected as the origin of ventricular tachycardia (VT) in patients with arrhythmogenic right ventricular dysplasia (ARVD). OBJECTIVES: To examine the relationship between the reentrant circuits of VT and the abnormal electrograms in ARVD, and to assess the feasibility of a block line formation in the reentrant circuit isthmus utilizing electroanatomical mapping system (CARTO) guidance. METHODS AND RESULTS: An electrophysiological study and catheter ablation (CA) were performed in 17 ARVD patients (13 men, 47 +/- 17 year) using CARTO. Endocardial mapping during sinus rhythm demonstrated electrogram abnormalities extended from the tricuspid annulus (TA) or the right ventricular outflow tract in 16 of 17 patients. In 13 hemodynamically stable VTs, the reentrant circuits and critical slow conduction sites for the CA were investigated during VTs. The entire macro-reentrant pathway was identified in 6/13 stable VTs (figure-of-8 in 4, single loop in 2). In the remaining seven VTs, a focal activation pattern was found in four and an unidentifiable pattern in three. CA successfully abolished all the macro-reentrant and focal tachycardias, however, not effective in three unidentifiable VTs. In the 13 cases with unstable VT, the linear conduction block zone was produced between the sites with abnormal electrograms and the TA. Ultimately, 23/26 VTs (88%) became noninducible after the CA. During follow-up (26 +/- 15 months), 13/17 patients remained free from any VT episodes. CONCLUSIONS: CARTO is useful for characterizing the anatomical and electrophysiological substrates, and for identifying the optimal ablation sites for VT associated with ARVD. PMID- 16684017 TI - Catheter ablation of ventricular tachycardia in ARVC: is curative treatment at the horizon? PMID- 16684018 TI - SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias. AB - INTRODUCTION: We aimed at identifying the molecular defect underlying the clinical phenotype of a Finnish family with a cardiac conduction defect and atrial arrhythmias. METHODS AND RESULTS: A large Finnish family was clinically evaluated (ECG, 24-hour ambulatory ECG, echocardiography). We performed linkage analysis with markers flanking the SCN5A gene and subsequently sequenced the SCN5A gene. Five family members had atrial arrhythmias and intracardiac conduction defects, and due to bradycardia needed a pacemaker when adolescents. No heart failure or sudden cardiac death was observed. Left ventricle dilatation was seen in one individual and three individuals had a slightly enlarged right ventricle. Premature death due to stroke occurred in one subject during the study, and two other members had suffered from stroke at young age. Linkage analysis favored the role of the SCN5A gene in disease pathogenesis, and direct sequencing disclosed D1275N mutation. This alteration was present not only in all six affected individuals, but also in two young individuals lacking clinical symptoms. CONCLUSIONS: Cardiac conduction defect and atrial arrhythmias in a large Finnish family appear to result from the SCN5A D1275N mutation. Although no sudden cardiac death was recorded in the family, at least three affected members had encountered brain infarction at the age of 30 or younger. PMID- 16684019 TI - Predictors of appropriate defibrillator therapy among patients with an implantable defibrillator that delivers cardiac resynchronization therapy. AB - INTRODUCTION: The purpose of this study was to determine predictors of appropriate implantable defibrillator (ICD) therapy among patients with heart failure who are treated with a cardiac resynchronization therapy-defibrillator (CRT-D). METHODS AND RESULTS: Patients enrolled in the Ventak CHF/Contak CD study were treated with a CRT-D device and were required to have NYHA class II-IV CHF, QRS duration > or = 120 msec, and a class I or II indication for an ICD. The study database was retrospectively analyzed during the 6-month postimplant period to identify predictors of appropriate ICD therapy. Five hundred and one of the 581 patients enrolled in the trial had successful device implantation and were included in this analysis. Patients were mostly male (83%), 66 +/- 11 years old, and had coronary artery disease (69%), a mean left ventricular ejection fraction (EF) = 0.22 +/- 0.07, and NYHA class II (33%), III (58%), or IV (9%) CHF symptoms. During 6 months of follow-up, 73 of 501 (14%) patients received an appropriate ICD therapy. Two independent predictors of appropriate therapy were identified: a history of a spontaneous, sustained ventricular arrhythmia (HR = 2.05; 95% CI = 1.31-3.20; P = 0.002) and NYHA class IV CHF (HR = 1.81; 95% CI = 1.10-2.96; P = 0.019). When patients with NYHA class II were excluded from analysis, a history of a sustained ventricular arrhythmia and the presence of NYHA class IV CHF symptoms remained as independent predictors of appropriate ICD therapy. CONCLUSIONS: In a select population of advanced heart failure patients receiving a CRT-D, NYHA class IV CHF was a powerful independent predictor of appropriate ICD therapy. Approximately one-quarter of the patients with NYHA class IV CHF who received a CRT-D device received an appropriate ICD therapy within 3 months after implant. Additional studies are needed to confirm an association between class IV CHF symptoms and an increased frequency of ICD shocks. PMID- 16684020 TI - CRT-D therapy in heart failure: how much do NYHA class IV patients benefit? PMID- 16684021 TI - Transcranial Doppler detection of microembolic signals during pulmonary vein antrum isolation: implications for titration of radiofrequency energy. AB - BACKGROUND: Cerebrovascular events are an important complication during pulmonary vein antrum isolation (PVAI). Microembolic signals (MES) have been associated with stroke and neurological impairment. However, the incidence of MES during PVAI, and their relationship to microbubble formation and radiofrequency (RF) parameters are unknown. OBJECTIVES: We sought to assess the relationship between MES, microbubble detection, and neurological outcome and the impact of RF titration strategy on these parameters. METHODS: We studied 202 patients in two groups undergoing PVAI using an intracardiac echocardiography (ICE)-guided technique. MES were detected by transcranial Doppler (TCD) using insonation of the middle cerebral arteries. The number of microbubbles on ICE were qualitatively labeled as FEW, MODERATE, and SHOWER. In group I (n = 107), RF output was titrated to avoid microbubble formation and in group II (n = 95), standard power-limited RF output was used. RESULTS: TCD detected MES in all 202 patients during PVAI with an average of 1,793 +/- 547 per patient; 90% were detected during left atrial ablation. Over 85% of MES occurred after microbubbles. Group I patients had significantly lower numbers of MES (1,015 +/- 438 per patient) compared to group II patients (2,250 +/- 864 per patient) (P < 0.05). Group II also had a 3.1% incidence of acute neurological complications versus 0.9% in group I (P = 0.10). Patients with clinical events had significantly higher numbers of MES. There were no significant correlations between RF power, temperature, or impedence and MES number. CONCLUSIONS: MES directly correlate to the amount of microbubble formation on ICE, and may result in cerebroembolic complications. Titration of RF according to microbubble formation by ICE during PVAI may be important for minimizing the occurrence of MES and possibly acute neurological complications. PMID- 16684022 TI - Cerebral microembolism during AF ablation: an innocent bystander or an accessory to brain injury? PMID- 16684023 TI - Right ventricular versus biventricular antitachycardia pacing in the termination of ventricular tachyarrhythmia in patients receiving cardiac resynchronization therapy: the ADVANCE CRT-D trial. AB - BACKGROUND: The purpose of this investigation is to compare the efficacy of antitachycardia pacing (ATP) delivered via the right ventricular (RV) lead versus ATP delivered simultaneously via the right and left ventricular leads (biventricular [BiV]) in the termination of ventricular tachyarrhythmia (VT) in patients receiving cardiac resynchronization therapy (CRT) with ICD capabilities. METHODS AND RESULTS: The ADVANCE CRT is a prospective, multicenter, randomized, parallel trial evaluating RV versus BiV ATP in the termination of VT in CRT patients. The study will test the hypothesis that BiV ATP is superior to RV ATP in the termination of VT and fast VT. All patients with class I and IIa indications for an ICD implantation and CRT are included. The sample size has been estimated to 400 participants followed for 12 months to show a 10% benefit of BiV versus RV ATP. The efficacy of BiV ATP to terminate all VT presents the primary endpoint. The investigation is expected to be completed in 2007. CONCLUSIONS: The ADVANCE CRT trial is the first large randomized clinical investigation evaluating the efficacy of BiV ATP in patients under CRT and ICD therapy. PMID- 16684024 TI - Narrow, slow-conducting isthmus dependent left atrial reentry developing after ablation for atrial fibrillation: ECG characterization and elimination by focal RF ablation. AB - INTRODUCTION: The complete circuit of reentrant left atrial tachycardias (LATs) occurring after ablation for atrial fibrillation (AF) has not been well described. Identifying discrete isthmuses critical to these LATs may simplify their elimination by catheter ablation. METHODS AND RESULTS: Fifteen patients (all male, 56 +/- 8 years) with 15 reentrant LATs following AF ablation underwent activation and entrainment mapping. Eleven patients (11 LATs) had a single localized site with low amplitude (0.16 +/- 0.05 mV), fractionated long duration (131 +/- 23 msec) electrograms coinciding with an isoelectric interval of 106 +/- 24 msec between flutter waves on all 12 ECG leads. Three-dimensional mapping and entrainment revealed this site to be a narrow markedly slowly conducting isthmus adjacent to ablated left (n = 8) or right (n = 3) pulmonary vein (PV) ostia, and critical to nine small diameter (15 +/- 3 mm) and two large diameter (49 +/- 2 mm) circuits. One radiofrequency (RF) application on this isthmus eliminated LAT in all 11 patients. Four patients (four LATs) with large circuits around the mitral annulus and/or PV ostia lacked isoelectric ECG intervals and slow conducting isthmuses and required multiple RF applications across anatomically wide, rapidly conducting isthmuses. CONCLUSION: Focally ablatable narrow isthmuses of slow conduction are critical for the majority of reentrant LAT occurring after ablation for AF. The role and presence of these isthmuses can be anticipated by observing significant isoelectric intervals between flutter waves on all 12-surface ECG leads. Their distinctive electrophysiological characteristics allow their identification and elimination by simple RF ablation. PMID- 16684025 TI - Slow conduction and flutter following atrial fibrillation ablation: proarrhythmia or unmasking effect of radiofrequency application? PMID- 16684026 TI - Results of the multicenter RENEWAL 3 AVT clinical study of cardiac resynchronization defibrillator therapy in patients with paroxysmal atrial fibrillation. AB - INTRODUCTION: Atrial fibrillation impacts the clinical course of up to 50% of patients with advanced heart failure (HF) who are eligible for cardiac resynchronization therapy with a defibrillator (CRT-D). While RV-based defibrillators are available with advanced atrial diagnostics and therapies that provide rapid diagnosis and treatment of spontaneously occurring atrial tachycardia/fibrillation (AT/AF) episodes, there is no CRT-D device that combines atrial/ventricular and CRT therapies. PURPOSE: The purpose of the prospective multicenter RENEWAL 3 AVT study is to assess the performance of atrial diagnostics and therapies used in combination with a CRT-D device. METHODS: Enrolled patients were required to have indications for a CRT-D device and a documented episode of AT/AF within 12 months of enrollment. A total of 170 patients were enrolled over 9 months (85% male; mean age 72 +/- 10 years; NYHA classification: 88% III, 12% IV; left ventricular ejection fraction [LVEF] mean 23 +/- 6%; mean QRS duration 150 +/- 25 msec; 78% ischemic etiology). The documented atrial arrhythmia was AF in 77% of patients. A total of 60% of patients had the CRT-D device placed for primary prevention of sudden death and 40% of patients had a history of ventricular arrhythmia in addition to HF. The device operates in the biventricular (BiV) triggered mode for sensed ventricular events associated with AF. RESULTS: A total of 159 patients (95%) had a successful CRT-D implant. Over a mean follow-up of 5.7 +/- 2.3 months, there were a total of 152 atrial shocks delivered in 108 patients for induced (93%) or spontaneous (7%) occurring episodes of AF. Spontaneously occurring AF was observed in 40 patients (25%). The rate of first shock conversion was 118/152 (78%, mean energy 11.6 +/- 5.9 J). Overall shock therapy conversion rate was 138/152 (91%). The number of shock conversions resulting in sinus rhythm maintained for at least 2 minutes postshock was 87% for induced episodes. Therapy was delivered for spontaneous ventricular tachycardia/fibrillation in nine patients (6%). There was no instance of ventricular proarrhythmia associated with atrial shock therapies, undersensing of ventricular arrhythmias, or interruption of CRT therapy associated with the combined device. CONCLUSIONS: In CRT-D candidates with a history of AF, 25% experience recurrent AF within 6 months of implant. Atrial detection and ventricular detection, shock, and resynchronization therapies are not compromised by the addition of atrial therapies to a CRT-D device. PMID- 16684027 TI - Comparison of the effects of VVI versus DDD pacing on cardiac baroreflex function. AB - INTRODUCTION: Conventional baroreceptor-heart rate (HR) reflex sensitivity cannot be examined in chronotropically incompetent patients or in pacemaker recipients. However, cardiac baroreceptor reflex sensitivity (BRS)-stroke volume (SV), which is closely and linearly correlated with BRS-HR, may be an alternative in that population. The aim of this study was to compare the BRS-SV in pacemaker recipients with a fixed HR paced in VVI versus DDD modes in the supine and upright positions. METHODS: The pacing mode was set randomly to DDD or VVI with complete atrial and/or ventricular capture, then crossed over to the alternate mode in 9 recipients of dual-chamber pacemakers with atrioventricular (AV) block. Beat-to-beat mean blood pressure and SV were measured in the supine and upright positions, using a tilt table. The BRS-SV, expressed in %/mmHg, was the ratio of low-frequency (LF) power to total power (TP) of SV variability, measured by spectral analysis of spontaneous variations in mean blood pressure and SV. RESULTS: BRS-SV was significantly lower in the VVI than in the DDD mode in the supine (37.2 +/- 26.7 vs 14.5 +/- 7.7%/mmHg) and upright (22.9 +/- 16.9 vs 10.6 +/- 6.6%/mmHg) positions (P < 0.05 for both comparisons). CONCLUSIONS: VVI pacing is adverse from the standpoint of cardiac autonomic baroreflex function. A decreased BRS-SV may be one of the factors involved in the hemodynamic intolerance associated with VVI pacing. PMID- 16684028 TI - Cardiac reflexes during pacing: are we getting to the heart of the matter? PMID- 16684029 TI - Selective aldosterone blockade suppresses atrial tachyarrhythmias in heart failure. AB - INTRODUCTION: Renin-angiotensin-aldosterone system activation may be involved in the pathogenesis of atrial arrhythmias in congestive heart failure (CHF). The effects of aldosterone blockade on atrial tachyarrhythmias have not been evaluated. This study's aim was to determine whether selective aldosterone blockade suppresses atrial tachyarrhythmia inducibility and modifies atrial electrical and/or structural remodeling in a canine model of rapid ventricular pacing (RVP)-induced CHF. METHODS AND RESULTS: Dogs were assigned randomly to treatment with oral placebo or eplerenone (50 mg/day) and divided into four groups: two sham-operated (no RVP) and two RVP groups. After 5 weeks of no RVP or RVP at 230 beats/min along with concurrent placebo or eplerenone treatment, dogs underwent electrophysiologic and echocardiographic studies. Sustained atrial tachyarrhythmia inducibility (>10-minute duration), atrial effective refractory periods (ERPs), systolic and diastolic function, and left atrial and left ventricular (LV) chamber sizes were assessed. Placebo-treated RVP dogs developed CHF with LV systolic and diastolic dysfunction, left atrial and LV enlargement, increased atrial ERPs, and inducible sustained atrial tachyarrhythmias. Eplerenone treatment in RVP dogs significantly suppressed sustained atrial tachyarrhythmia inducibility, nonuniformly prolonged atrial ERPs and attenuated LV diastolic dysfunction without modifying left atrial or LV dilation or ejection fractions in CHF. Isoproterenol (2-4 microg/min) reversed eplerenone's atrial antiarrhythmic and ERP prolonging effects in CHF. Eplerenone did not alter atrial ERPs in sham (no RVP) dogs without CHF. CONCLUSIONS: Eplerenone suppresses inducibility of sustained atrial tachyarrhythmias, selectively prolongs atrial ERPs, and attenuates LV diastolic remodeling in RVP-induced CHF. Aldosterone blockade may be a promising new approach for atrial tachyarrhythmia prevention in CHF. PMID- 16684030 TI - Aldosterone antagonism as an antiarrhythmic approach for atrial arrhythmias in heart failure. PMID- 16684031 TI - Protection of the coronary arteries during epicardial radiofrequency ablation with intracoronary chilled saline irrigation: assessment in an in vitro model. AB - INTRODUCTION: The coronary arteries can be damaged during epicardial radiofrequency ablation (RFA) procedures. We hypothesized that intracoronary irrigation with chilled saline may be a useful technique for minimizing heat induced damage to the coronary artery endothelium during this procedure. METHODS AND RESULTS: Twenty-nine ablation procedures were performed on 17 freshly excised ovine hearts. Radiofrequency current was delivered through an internally cooled, 4-mm-tip ablation catheter placed directly over the coronary artery (24 applications) and over noncoronary epicardium (5 applications). An Amplatz coronary catheter was used to internally irrigate the coronary artery with either 37 degrees C or 5 degrees C 0.9% saline (12 ablations each group). Fluroptic temperature probes were placed within the artery lumen under the ablation site and 15 mm distal from the ablation site. The peak intracoronary temperature directly under the ablation catheter was significantly lower (P = 0.001) in the chilled than in the nonchilled saline irrigation group (23.6 degrees C, interquartile range [IQR] 15.7-39.8 vs 54.6 degrees C, IQR 48.9-58.6). Blue tetrazolium stained lesion sections showed that the median distance between the ablation lesion and the artery wall was significantly higher (P = 0.004) for the chilled versus the nonchilled saline irrigation group (0.42 mm, IQR 0.25-0.70 vs 0.00 mm, IQR 0.00-0.28). CONCLUSIONS: Intracoronary irrigation with chilled saline may protect the coronary artery endothelium from heat-induced damage during epicardial RFA. PMID- 16684032 TI - Epicardial ablation: reducing the risks. PMID- 16684033 TI - T-wave oversensing in implantable cardiac defibrillators is due to technical failure of device sensing. AB - The expanded indications for the use of implantable cardioverter defibrillators (ICDs) for primary prevention of sudden cardiac death have increased concerns about inappropriate delivery of therapies. Dual-chamber systems have improved the capacity of ICDs to differentiate ventricular arrhythmias from supraventricular arrhythmias. Still, T-wave oversensing is a major source of inappropriate therapy. It is likely the true incidence of T-wave oversensing is greater than reported as documented events reflect only those stored in a device memory. Reviewing cases of T-wave oversensing that failed noninvasive correction; we found successful resolution resulted from generator replacement. We conclude that T-wave oversensing is due to inadequate signal processing by some ICD generators. PMID- 16684034 TI - Narrow complex tachycardia after slow pathway ablation: continue ablating? PMID- 16684035 TI - Simultaneous wide and narrow QRS complex tachycardias in a child: what is the mechanism? PMID- 16684036 TI - Electrocardiographic hallmark of arrhythmogenic right ventricular cardiomyopathy. PMID- 16684037 TI - Brugada-type ECG pattern and extreme QRS complex widening with propafenone overdose. PMID- 16684038 TI - Continuous and discontinuous propagation in heart muscle. AB - Although cardiac muscle has been considered as an electrical syncytium for a long time, several aspects of conduction of the electrical impulse in the heart could not be explained by a continuous approach. Even the simple anisotropic nature of cardiac muscle gives rise to a number of conduction characteristics that contradicts with results from continuous models. It is, however, evident that cardiac conduction must be discontinuous in nature owing to recurrent resistive discontinuities caused by the cellular interconnections. In this review, the role of discontinuities at the cellular and macroscopic level on cardiac propagation will be discussed. PMID- 16684040 TI - Catheter ablation of long-lasting persistent atrial fibrillation: clinical outcome and mechanisms of subsequent arrhythmias. PMID- 16684041 TI - Risk factors for requirement of permanent pacemaker implantation after aortic valve replacement. AB - BACKGROUND: The aim of this study was to evaluate the frequency of requirement for permanent pacing and related risk factors after aortic valve replacement. METHODS: Among 465 patients operated between 1994 and 2004, 19(4.1%) patients with a mean age 49.9 +/- 17.2 years required the implantation of a permanent pacemaker. Eleven of them were female (57.9%). The main indication was aortic stenosis (89.5%). Severe annular calcification was documented in 78.9% of them, and the aortic valve was bicuspid in 57.9%. RESULTS: Risk factors for permanent pacing after aortic valve replacement (AVR) identified by univariate analysis were female sex, hypertension, preoperative ejection fraction, aortic stenosis, annular calcification, bicuspid aorta, presence of right bundle branch block (RBBB) or left bundle branch block (LBBB), prolonged aortic cross-clamp and perfusion times, and preoperative use of calcium channel blockers. Multivariate analysis showed that female sex (p = 0.01, OR; 5.21, 95% CI: 1.48-18.34), annular calcification (p < 0.001, OR; 0.05, 95% CI: 0.01-0.24), bicuspid aortic valve (p = 0.02, OR; 0.24, 95% CI: 0.07-0.84), presence of RBBB (p = 0.009, OR; 0.03, 95% CI: 0.003-0.44) or LBBB (p = 0.01, OR; 0.13, 95% CI: 0.02-0.69), hypertension (p = 0.03, OR; 0.22, 95%CI: 0.05-0.89), and total perfusion time (p = 0.002, OR; 1.05, 95% CI: 1.01-1.08) were associated risk factors. CONCLUSION: Irreversible atrioventricular block requiring a permanent pacemaker implantation is an uncommon complication after AVR. Risk factors are annular calcification, bicuspid aorta, female sex, presence of RBBB or LBBB, prolonged total perfusion time, and hypertension. PMID- 16684044 TI - Different hemodynamic stress of the ascending aorta wall in patients with bicuspid and tricuspid aortic valve. AB - It is unclear whether ascending aorta dilation in patients with bicuspid aortic valve is caused by abnormal hemodynamics or by a common developmental defect of the aortic valve and aortic wall. We performed an echocardiographic study to examine the differences in hemodynamic stress at the ascending aorta in patients with bicuspid and tricuspid aortic valve. We studied prospectively 58 consecutive patients referred for preoperative echocardiographic examination with aortic valve stenosis and either bicuspid or tricuspid valve and an ascending aortic diameter of 30% (HEF). To further evaluate the LVD patients, they were divided into three subgroups base on LVEF: 0% to 10%, 11% to 20%, and 21% to 30%. Data were collected prospectively and entered into the departmental database of the Society of Thoracic Surgeons. RESULTS: Patients in the LVD group had increased incidence of diabetes, chronic obstructive pulmonary disease (COPD), peripheral vascular disease, prior myocardial infarction (MI), congestive heart failure, and less elective procedures compared to the HEF group. Despite this greater risk profile, operative mortality (LVD 2.6% vs. HEF 1.2%, p = 0.19), the incidence of stroke (2.6% vs. 1.0%, p = 0.13), and perioperative MI (0.9% vs. 0.7%) were not statistically different between the groups. The incidence of respiratory (14.8% vs. 1.9%, p < 0.001), renal (5.2% vs. 1.0%, p < 0.001), and vascular (5.2% vs. 0.5%, p < 0.001) complications was significantly higher in the LVD group, resulting in a longer hospital length of stay (8 +/- 8 vs. 6 +/- 4 days, p < 0.0001). In a multivariate analysis, advanced age was as an independent predictor of hospital mortality. Average follow-up in 108 (94%) LVD patients was 36 +/- 22 months (range 2 to 78 months). Twenty-one patients expired during the follow-up, for nine the causes were cardiac-related. Three- and 5-year survival rates were 91 +/- 3% and 76 +/- 6%, respectively. Independent predictors of mid-term mortality in the LVD group by a multivariate analysis included female gender, renal failure, respiratory complications, and grade I/II mitral regurgitation (MR). At the time of follow-up, 72% of LVD patients were in functional class I/II. There were no statistically significant differences in short- and mid-term outcomes among the LVD subgroups. CONCLUSION: CABG in patients with severe LVD can be performed with a low mortality, albeit with higher morbidity and longer length of hospital stay, than patients with LVEF >30%. Low ejection fraction per se was not a predictor of hospital mortality. CABG should be considered a safe and effective therapy for low ejection fraction patients with ischemic heart disease. Mitral valve repair/replacement in the presence of moderate degree of MR should be considered at the time of the initial operation. PMID- 16684047 TI - Severe left ventricular dysfunction: a continuous surgical challenge. PMID- 16684048 TI - Determinants of outcomes for postcardiotomy VAD placement: an 11-year, two institution study. AB - OBJECTIVE: Ventricular assist device (VAD) placement after postcardiotomy failure is associated with a high mortality. We sought to determine prognostic factors in order to help better select patients who may benefit from VAD placement. METHODS: From January 1992 to January 2003, 63 patients out of approximately 30,000 cardiac surgery patients (0.21%) developed postcardiotomy failure requiring VADs. Planned VAD for bridge to transplant or bridge to recovery were excluded. Multivariate logistic regression analysis, based on pre-VAD placement parameters, was used to determine prognostic factors for in-hospital 30-day mortality. RESULTS: Overall operative mortality was 73% (46 of 63). Multivariate logistic regression analysis identified median age /=0 mEq/L (each additional mEq/L) prior to VAD placement (OR = 0.60, 95% CI = 0.48 to 0.91, p = 0.012) to be independent predictors of improved 30-day survival. CONCLUSIONS: Postcardiotomy failure is a rare event but is associated with a very poor prognosis despite salvage therapies utilizing VADs. Age /=0 (mEq/L) prior to VAD placement are associated with improved 30-day survival. PMID- 16684049 TI - Condition critical: can mechanical support prevent death due to postcardiotomy shock? PMID- 16684050 TI - Minimally invasive reoperative isolated valve surgery: early and mid-term results. AB - OBJECTIVE: Minimally invasive, nonsternotomy approaches for valve procedures may reduce the risks associated with cardiac surgery after prior sternotomy and may improve outcomes. We analyzed our institutional experience to test this hypothesis. METHODS: Between 1995 and 2002, 498 patients with previous cardiac operations via sternotomy underwent isolated valve surgery: 337 via median sternotomy (aortic = 160; mitral = 177) and 161 via mini-thoracotomy (aortic = 61; mitral = 100). Data were collected prospectively using the New York State Cardiac Surgery Report Form. RESULTS: Preoperative incidences of congestive heart failure, renal disease, and nonelective procedures were higher in the sternotomy group. Hospital mortality was significantly lower with the minimally invasive approach, 5.6% (9/161) versus 11.3% (38/337) (univariate, p = 0.04). However, multivariate analysis (odds ratio: 95% confidence intervals, p value) revealed that chronic obstructive pulmonary disease (6.6: 1.4 to 3.1, p = 0.001), renal disease (4.1: 1.52 to 11.2, p = 0.01), cerebrovascular disease (2.2: 1.03 to 4.78, p = 0.04), and ejection faction <30% (1.5: 0.96 to 5.5, p = 0.06) were associated with increased mortality. While mean bypass time, cross-clamp times, and stroke rates were comparable between groups, patients undergoing minimally invasive valve surgery had no deep wound infections (0% vs 2.4%, p = 0.05), less need for blood products (p = 0.02), and shorter hospital stays (p = 0.009). Five year survival was higher with minimally invasive techniques as compared to a sternotomy approach (92.4 +/- 2% and 86.0 +/- 2%, respectively, p = 0.08). CONCLUSIONS: Reoperative valve surgery can be safely performed using a nonsternotomy, minimally invasive approach, with at least equal mortality, less hospital morbidity, decreased hospital length of stay, and slightly favorable mid term survival as compared to sternotomy. PMID- 16684051 TI - Morbidity and mortality of cardiac surgery following renal transplantation. AB - BACKGROUND: With improved survival following renal transplantation, the number of patients undergoing cardiac surgery has increased. The purpose of this study was to review the morbidity, mortality, and allograft function in renal transplant patients undergoing major cardiac surgery. METHODS: Retrospective database review of consecutive renal transplant patients undergoing cardiac surgery from 1987 to 2002. Patients requiring dialysis (D) before cardiac surgery versus those with stable renal transplants (ND) were compared. RESULTS: Cardiac surgery was performed in 46 patients during the study period. Twenty patients (42%) required dialysis (D) before surgery while 26 (58%) had stable allograft function (ND). Among patients who had stable allograft function prior to surgery, there was no allograft loss. In the ND group, preoperative and discharge creatinine levels were 2.17 +/- 1.0 and 2.4 +/- 1.5 mg/dL, respectively. All operative deaths occurred in the dialysis dependent group. The 30-day and 3-year survival, respectively was 80% and 20% in the D group compared to 100% and 69% amongst the ND group (p or = 50 days ('Late'), and 110 had RDI < 50 days ('Early'). In the Late group there were only 2 deaths from cancer and 93.7% cancer-specific five year survival (c5ys), compared with 22 and 65.3%, respectively, in the Early one (P = 0.007). There were more Duke's A cases in the Late group (38.6%vs 15.2%, P = 0.006), but this did not fully explain the improved survival. Comparisons for each Duke's Stage showed improved c5ys for Late Duke's B ones (100% of 16 vs 60.3% of 54, P = 0.039). Late patients had more low risk symptoms than Early ones, both overall (31.8%vs 13.7%, P = 0.013) and in Duke's B cases (56%vs 15.3%, P = 0.003). Tumours were smaller in the Late group (length 35.3 vs 41.6 mm, P= 0.04); this difference was confined to the Duke's A patients and sigmoid tumours. Late sigmoid tumours were not only shorter (32.4 vs 45.9 mm, P = 0.02) but also were all cured (c5ys 100% of 18 vs 60.3% of 23, P = 0.011). There were no differences between Late and Early groups in: age (mean 69.9 years), sex (male 57.7%), date of diagnosis (mean December 1998), ASA comorbidity index (mean 1.9), number of lymph nodes found in the operative specimen (mean 8.6), or histological grading (moderate differentiation 94.4%). CONCLUSION: In the context of modern rapid access clinics, symptomatic CRC patients with delay between referral and diagnosis (even if this is several months or occasionally more than a year) have less aggressive tumours and markedly better long-term cure rate than their earlier diagnosed counterparts. Attempts to speed up further the diagnosis would be a waste of time and resources, being unlikely to make an appreciable difference to the overall cure rate. PMID- 16684088 TI - Negative influence of delayed surgery on survival after preoperative radiotherapy in rectal cancer. AB - OBJECTIVE: In Europe, until recently the standard treatment for locally advanced rectal cancer was preoperative radiotherapy (RT). The objective of this study was to evaluate the influence on survival of intervals between diagnosis and treatment. PATIENTS AND METHODS: The influence on survival of intervals between diagnosis and surgery (Dg-Surg), diagnosis and initiation of RT (Dg-Rad), and completion of RT and surgery (Rad-Surg) was evaluated in a retrospective series of patients treated with preoperative RT. Between 1991 and 1998, 102 patients received treatment with preoperative RT without concomitant chemotherapy at the Rene Gauducheau Cancer Center. Patients generally received 45 Gy (80%) in 25 fractions over 35 days for T2-T3-T4 N0-N1 M0 rectal adenocarcinoma located mainly (62.7%) in the lower third of the rectum (< or = 5 cm from anal margin). Thirty five pN1 patients were treated with postoperative chemotherapy. Differences between survival were assessed by the log-rank test, and prognostic factors by the Cox test. RESULTS: Median time was 14.7 weeks for Dg-Surg, 4.6 weeks for Dg Rad and 5.1 weeks for Rad-Surg. Median follow-up from diagnosis was 57.4 months. Five-year local relapse-free survival was 83.9%, metastasis-free survival 64% and overall survival 60.8%. No factor was predictive of tumour response to RT. Log rank and multivariate analysis showed that overall survival was significantly influenced by lower-third tumours, pT, pN and Dg-Surg (poorer survival when > or = 16 weeks: OR = 2.59, P = 0.005). Metastasis-free survival correlated significantly with Dg-Surg (> or = 16 weeks: OR = 2.05, P = 0.05). CONCLUSION: An interval of more than 16 weeks between diagnosis and surgery may reduce overall survival of patients treated with preoperative RT for locally advanced rectal cancer. Surgery should be performed shortly after completion of RT for patients with no possibility of sphincter preservation, or a minimal risk of morbidity from an abdominoperineal excision. PMID- 16684089 TI - Prognostic significance of CEA levels and positive cytology in peritoneal washings in patients with colorectal cancer. AB - OBJECTIVE: The aims of this prospective study were to determine carcinoembryonic antigen (CEA) levels and incidence of cytology in peritoneal washings of patients with colorectal cancer, correlate the results with various histopathological factors and determine their significance as prognostic factors of the disease. METHODS: From 1992 to 1999, 98 patients with adenocarcinoma of the colon or intraperitoneal rectum underwent curative surgery and enrolled in this study. RESULTS: Overall, 25 (26.3%) of 95 patients were found to have positive cytology. The proportion of patients with positive cytology was higher in the recurrence group (36.4%) than in the groups of 5-year survival and hepatic metastases (24.6% and 26.3%, respectively), but this difference was not significant. The 5-year survival group had the lowest peritoneal CEA levels compared with the other groups, but this difference was not significant. Peritoneal cytology and CEA level alone were not sensitive, specific or accurate enough indicators in predicting survival, hepatic metastases or local recurrence. The analysis of patients with positive cytology and high peritoneal CEA level revealed that their combination can predict local recurrence with accuracy of 85%. CONCLUSIONS: The presence of free malignant cells, as detected by cytology and CEA level, in the peritoneal cavity of patients with resectable colorectal cancer had no detectable impact on survival, hepatic metastases or local recurrence rate. However, local recurrence can be predicted with accuracy of 85% in patients who have positive cytology and high peritoneal CEA level at the same time. PMID- 16684090 TI - Laparoscopic vs open subtotal colectomy for benign and malignant disease. AB - AIM: The present meta-analysis aims to compare short-term and long-term outcomes in patients undergoing laparoscopic or open subtotal colectomy for benign and malignant disease. METHODS: A literature search of Medline, Ovid, Embase and Cochrane databases was performed to identify studies published between 1992 and 2005, comparing laparoscopic (LSC) and open (OSC) subtotal colectomy. A random effect meta-analytical technique was used and sensitivity analysis performed on studies published since the beginning of 2000, higher quality papers, those reporting on more than 40 patients, and those studies reporting on adult cases or acute colitis. RESULTS: A total of eight studies satisfied the criteria for inclusion. These included outcomes on 336 patients, 143 (42.6%) of whom had undergone laparoscopic resection, with an overall conversion rate to open surgery of 5% (range 0-11.8%). Operative time was significantly longer in the laparoscopic group by 86.2 min (P < 0.001) and throughout subgroup analysis, although it was only in patients with acute colitis that this finding was without significant heterogeneity. Operative blood loss was less in the laparoscopic group by 57.5 millilitres in high quality and studies published since 2000, and 65.3 millilitres in those reporting on more than 40 patients. There was no significant difference in early or long-term complications between the groups. A statistically significant reduction in length of postoperative stay was observed in the laparoscopic groups by 2.9 days (P < 0.001). CONCLUSION: Laparoscopic subtotal colectomy was associated with longer operating times but a reduced length of stay compared to open surgery. Although short-term outcomes were equivalent in both groups, the suggested benefits in terms of reduced long-term obstructive complications were not supported by this meta-analysis. PMID- 16684091 TI - Necrotizing fasciitis--a rare complication of 'un-bridged' ileostomy. PMID- 16684092 TI - A second case of long-term incontinence following botulinum injection for anal fissure. PMID- 16684093 TI - Mortality after acute surgery for complications of diverticular disease of the sigmoid colon is almost exclusively due to patient-related factors. PMID- 16684094 TI - New models for circadian systems in microorganisms. AB - Microorganisms provide important model systems for studying circadian rhythms, and they are overturning established ideas about the molecular mechanisms of rhythmicity. The transcription/translation feedback model that has been accepted as the basis of circadian clock mechanisms in eukaryotes does not account for old data from the alga Acetabularia demonstrating that transcription is not required for rhythmicity. Moreover, new results showing in vitro rhythmicity of KaiC protein phosphorylation in the cyanobacterium Synechococcus, and rhythmicity in strains of the fungus Neurospora carrying clock gene null mutations, require new ways of looking at circadian systems. PMID- 16684095 TI - Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl deoxynivalenol chemotypes in Fusarium. AB - The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product. PMID- 16684096 TI - Spherical body formation in the spirochaete Brachyspira hyodysenteriae. AB - When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6 12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera. PMID- 16684097 TI - Characterization of Helicobacter pylori sigma54 promoter-binding activity. AB - Several Helicobacter pylori flagellar genes require sigma(54) for their transcription. Predicted H. pylori sigma(54)-dependent promoters display a preference for A at position -23 instead of C or T as occurs in promoters from most other bacteria. Substitution of the A at position -23 of the H. pylori flaB promoter with a C did not effect expression of a flaB'-'xylE reporter gene in H. pylori, whereas T or G substitutions at this position drastically reduced expression. Results of gel mobility shift assays that used DNA probes corresponding to core promoter sequences and a H. pylori sigma(54) protein fused to the Escherichia coli maltose-binding protein suggested that H. pylori sigma(54) has a higher affinity for promoters with an A at the -23 position. The failure to observe an effect on expression for the flaB mutant promoter with the A to C substitution at the -23 position indicates that sequences flanking the core promoter region may assist binding of H. pylori sigma(54) to the mutant flaB promoter. Alternatively, H. pylori RNA polymerase or the sigma(54)-dependent activator FlgR may compensate for the reduced affinity of sigma(54) for the mutant flaB promoter. PMID- 16684098 TI - Identification and characterization of the loci encoding the competence associated alternative sigma factor of Streptococcus gordonii. AB - In naturally-competent streptococci such as Streptococcus pneumoniae, expression of the late competence operons is regulated by ComX (sigma(X)), the competence specific alternative sigma factor. In this study, duplicate genes (comR1 and comR2) encoding the putative ComX homologue of the oral bacterium Streptococcus gordonii were identified. Like the identical twin comX loci of S. pneumoniae, both comR determinants are independently functional as well as responsive to the ComDE signal transduction system activated by competence-stimulating peptide. However, in contrast to the comX system, nucleotide sequence analyses in combination with in trans complementation studies with a comR null mutant demonstrate that the identical 83 bp tracts (Region I) located immediately upstream of the comR structural genes are insufficient to confer wild-type competence levels. Wild-type transformation levels required additional distal nonhomologous DNA segments (Region II). Our findings suggest that alternative regulatory elements, under overall control of the ComDE pathway, may influence expression of the comR loci. PMID- 16684099 TI - A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence. AB - Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification. PMID- 16684100 TI - Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts. AB - We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria. PMID- 16684101 TI - Formation of water-soluble metal cyanide complexes from solid minerals by Pseudomonas plecoglossicida. AB - A few Pseudomonas species are able to form hydrocyanic acid (HCN), particularly when grown under glycine-rich conditions. In the presence of metals, cyanide can form water-soluble metal complexes of high chemical stability. We studied the possibility to mobilize metals as cyanide complexes from solid minerals using HCN forming microorganisms. Pseudomonas plecoglossicida was cultivated in the presence of copper- and nickel-containing solid minerals. On powdered elemental nickel, fast HCN generation within the first 12 h of incubation was observed and water-soluble tetracyanaonickelate was formed. Cuprite, tenorite, chrysocolla, malachite, bornite, turquoise, millerite, pentlandite as well as shredded electronic scrap was also subjected to a biological treatment. Maximum concentrations of cyanide-complexed copper corresponded to a solubilization of 42% and 27% when P. plecoglossicida was grown in the presence of cuprite or tenorite, respectively. Crystal system, metal oxidation state and mineral hydrophobicity might have a significant influence on metal mobilization. However, it was not possible to allocate metal mobilization to a single mineral property. Cyanide-complexed gold was detected during growth on manually cut circuit boards. Maximum dicyanoaurate concentration corresponded to a 68.5% dissolution of the total gold added. These findings represent a novel type of microbial mobilization of nickel and copper from solid minerals based on the ability of certain microbes to form HCN. PMID- 16684102 TI - S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus. AB - S-adenosylmethionine (SAM), the major methyl donor in diverse biological processes, was previously documented as a regulator for secondary metabolism in Streptomyces. The present study demonstrates that exogenous SAM, in a quantity as low as 10 muM, enhanced streptomycin production and adpA transcription in both Streptomyces griseus wild-type strain and mutant HO1, which displays no ArpA repression on the adpA promoter. SAM also enhanced xylE expression driven by the promoter of adpA or strR in a heterologous host, S. lividans. This report substantiates that exogenous SAM promotes adpA transcription in S. griseus, which is involved in the SAM-mediated promotion of streptomycin, and that the mechanism underlying this event is shared in S. lividans. PMID- 16684103 TI - Comparative fatty acid profiling of Mucor rouxii under different stress conditions. AB - To understand the relationship between fatty acid metabolism and the growth morphology of Mucor rouxii, fatty acid profiling was studied comparatively in cells grown under conditions which included different atmospheric conditions or the addition of phenethyl alcohol (PEA). The significant difference in fatty acid profiles from M. rouxii grown under aerobic or anaerobic conditions was not found to be directly related to morphological growth. Oxygen limitation, which induced the formation of pure multipolar budding yeasts, led to a decrease in long-chain fatty acids-- particularly unsaturated fatty acids-- and an increase in medium chain saturated fatty acids, a finding which contrasted with the aerobic cultures, including mycelia and PEA-induced bipolar budding cells. High levels of C18 : 1Delta(9) were found in aerobic yeast cultures with additional PEA when compared to that in the aerobically grown mycelia. The identification of unusual fatty acids in Mucor in response to alcoholic and hypoxic stresses - including odd-numbered fatty acids and 7-hydroxy dodecanoic acid (7-OH C12 : 0) in addition to the more common fatty acids - implied that an important role existed for these unusual fatty acids. PMID- 16684104 TI - Factors affecting the attachment of rhizospheric bacteria to bean and soybean roots. AB - The plant rhizosphere is an important soil ecological environment for plant microorganism interactions, which include colonization by a variety of microorganisms in and around the roots that may result in symbiotic, endophytic, associative, or parasitic relationships within the plant, depending on the type of microorganisms, soil nutrient status, and soil environment. Rhizosphere competence may be attributable to the differences in the extent of bacterial attachment to the root surface. We present results of the effect of various factors on the attachment to bean (Phaseolus vulgaris) and soybean (Glycine max) roots of some bacterial species of agronomic importance, such as Rhizobium tropici, Rhizobium etli, Ensifer fredii (homotypic synonym Sinorhizobium fredii), and Azospirillum brasilense; as well as the attachment capability of the plant growth promoting rhizobacteria Pseudomonas fluorescens and Chryseobacterium balustinum. Additionally, we have studied various bacterial traits, such as autoaggregation and flagella movements, which have been postulated to be important properties for bacterial adhesion to surfaces. The lack of mutual incompatibility between rhizobial strains and C. balustinum has been demonstrated in coinoculation assays. PMID- 16684105 TI - Rapid discrimination of cytK-1 and cytK-2 genes in Bacillus cereus strains by a novel duplex PCR system. AB - Bacillus cereus is the causative agent of gastrointestinal diarrhoea. At least three known enterotoxins may be involved in this syndrome: nonhaemolytic (Nhe) enterotoxin, Hbl enterotoxin and cytotoxin K. Two different forms were recently described for cytotoxin K, encoded by cytK-1 and cytK-2 genes. The CytK-1 toxin appeared to carry a high toxicity, but there is currently no method available to rapidly detect and discriminate the B. cereus strains able to produce this CytK-1 form. In this study, a duplex PCR assay was developed and validated on 162 known cytotoxin-containing strains. This PCR method is the first molecular tool to provide rapid detection and discrimination of cytK-1- and cytK-2-carrying B. cereus strains. PMID- 16684106 TI - Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function. AB - Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F(+) and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F(+). The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics. PMID- 16684107 TI - Strong antimicrobial activity of Lactobacillus rhamnosus GG against Salmonella typhimurium is due to accumulation of lactic acid. AB - Spent culture supernatant (SCS) of the probiotic Lactobacillus rhamnosus GG had been reported to exert antibacterial activity against Salmonella typhimurium. However, the chemical identity of the antimicrobial compound(s) responsible remained unknown. A survey of the antimicrobial compounds produced by L. rhamnosus GG was performed. Lactobacillus rhamnosus GG produced a low-molecular weight, heat-stable, non-proteinaceous bactericidal substance, active at acidic pH against a wide range of bacterial species. SCS of L. rhamnosus GG grown in MRS medium contained five compounds that could meet the above description, if present at the appropriate concentration. Based on different experimental approaches, it could be concluded that under the growth conditions tested, the strong antimicrobial activity of L. rhamnosus GG against Salmonella was mediated by lactic acid. PMID- 16684108 TI - Functional characterization of AAA family FtsH protease of Mycobacterium tuberculosis. AB - FtsH is a membrane-bound ATP-dependent zinc-metalloprotease which proteolytically regulates the levels of specific membrane and cytoplasmic proteins that participate in diverse cellular functions, and which therefore might be of critical importance to a human pathogen such as Mycobacterium tuberculosis. As the substrates of MtFtsH in mycobacteria are not known, we examined whether recombinant MtFtsH could complement the lethality of a DeltaftsH3::kan mutation in Escherichia coli and elicit proteolytic activity against the known substrates of E. coli FtsH, namely heat shock transcription factor sigma(32) protein, protein translocation subunit SecY and bacteriophage lambdaCII repressor protein. The MtFtsH protein could not only efficiently complement lethality of DeltaftsH3::kan mutation in E. coli, but could also degrade all three heterologous substrates with specificity when expressed in ftsH-null cells of E. coli. These observations probably reveal the degree of conservation in the mechanisms of substrate recognition and cellular processes involving FtsH protease of M. tuberculosis and E. coli. PMID- 16684109 TI - Involvement of quorum sensing and RpoS in rice seedling blight caused by Burkholderia plantarii. AB - Burkholderia plantarii is a plant pathogen responsible for causing rice seedling blight. The molecular mechanisms responsible for this pathogenicity are currently unknown. In this study, we report the identification and characterization of N acyl homoserine lactone quorum sensing and the stationary phase RpoS sigma factor of B. plantarii. Both global regulatory systems are involved in causing rice seedling blight. This is the first report of gene regulators of B. plantarii implicated in the disease. PMID- 16684110 TI - Characterization of glycerate kinase (2-phosphoglycerate forming), a key enzyme of the nonphosphorylative Entner-Doudoroff pathway, from the thermoacidophilic euryarchaeon Picrophilus torridus. AB - Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Entner-Doudoroff (ED) pathway. Here we report the characterization of a key enzyme of this pathway, glycerate kinase (2-phosphoglycerate forming). The enzyme was purified 5,100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to 2 phosphoglycerate. The enzyme showed highest activity at 60 degrees C and pH 7.3, with ATP as phosphoryl donor and Mg(2+) as divalent cation. By MALDI-TOF analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus tenax, for which the operation of nonphosphorylative ED pathways, involving 2 phosphoglycerate forming glycerate kinases, has been proposed. PMID- 16684111 TI - Brucella abortusd-alanyl-D-alanine carboxypeptidase contributes to its intracellular replication and resistance against nitric oxide. AB - Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes, and cause abortion in domestic animals and undulant fever in humans. However, the mechanism and factors of virulence are not fully understood. In the present study, a D alanyl-D-alanine carboxypeptidase (DAP) mutant of Brucella abortus failed to replicate in mouse macrophages and HeLa cells, and showed less virulence than the wild type in mice. Under nitric oxide (NO) stress, the growth of the DAP mutant in vitro decreased and it also had less capability to reduce NO than the wild type. Intracellular replication of the DAP mutant was partially restored by pretreatment of macrophages with the NO synthase inhibitor, 1-phenyl-imidazole, and the level of expression of the NO reductase gene, norB, in the DAP mutant was lower than that in the wild type. These results suggest that DAP contributes to resistance against NO and that it is required for the intracellular growth of the bacterium. PMID- 16684112 TI - Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species. AB - Burkholderia species are widely distributed in the natural environment. We evaluated the use of the recA gene in a cultivation-independent approach to examine the Burkholderia diversity associated with the maize rhizosphere. Two types of recA gene library were constructed, one with broad-specificity recA primers (BUR1 and BUR2) and a second from the products of nested PCRs using Burkholderia-specific primers (BUR3 and BUR4). The broad-specificity primer set provided near full-length recA sequences (869 bp) suitable for the creation of robust environmental sequence data sets; however, the nested PCR approach demonstrated the greatest specificity (84%) for detection of Burkholderia species recA genes. In addition, the screening approach was able to identify recA phylotypes matching Burkholderia cepacia complex species previously cultivated from the maize samples and discriminate these from other Burkholderia. The ecological benefit of Burkholderia species cultivated from maize rhizosphere is well documented, however, the fact that the majority of Burkholderia recA genes detected in this study (90%) were suggestive of novel taxa indicates that a wealth of potentially important interactions with uncultivated Burkholderia species remain unstudied in this habitat. PMID- 16684113 TI - Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae. AB - A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes. PMID- 16684114 TI - Pyruvate-converting activity in the spores of the microsporidian genus Paranosema (Antonospora). AB - Microsporidia, a large group of fungi-related protozoa with an obligate intracellular lifestyle, are characterized by a drastically reduced cell machinery and a unique metabolism. These parasites possess genes encoding glycolysis components and glycerol-phosphate shuttle, but lack typical mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except for two subunits of the E(1) enzyme of the pyruvate dehydrogenase complex. This study demonstrates that in spite of the above, destroyed spores of the microsporidian Paranosema (Antonospora) grylli and P. locustae deplete pyruvate content in the incubation medium. This activity is sensitive to heat, proportionally distributed between the soluble and the insoluble fractions and does not depend on additional ions or cofactors. PMID- 16684115 TI - Regulated expression of HPrK/P does not affect carbon catabolite repression of the xyn operon and of rocG in Bacillus subtilis. AB - HPr kinase/phosphorylase (HPrK/P), a central metabolic regulator in many Gram positive bacteria, reversibly phosphorylates HPr and Crh, thus controlling their activities as effectors of CcpA predominantly in carbon catabolite repression (CCR). We have placed the constitutively expressed hprK in its native chromosomal locus under anhydrotetracycline-dependent transcriptional control to establish the correlation between HPrK/P amounts and the efficiency of CCR in Bacillus subtilis. This resulted in about eightfold repression of HPrK/P expression but had no effect on CCR as monitored by xynP'-lacZ reporter gene expression and by analysis of RocG protein amounts. These results suggest that very small amounts of HPrK/P are sufficient for complete CCR and that control of HPrK/P activity depends only on the presence of effectors and not on the abundance of the enzyme. PMID- 16684116 TI - Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis. AB - Flavonoids, polyphenolic compounds found in plants, have demonstrated activity against several parasites and can augment the efficacy of other drugs by either increasing the uptake or decreasing the efflux of these drugs. We evaluated 11 of these compounds alone or in combination in order to test the hypothesis that flavonoids are effective against Cryptosporidium parvum and Encephalitozoon intestinalis. Using in vitro cell culture assays, HCT-8 cells or E6 cells were infected with C. parvum and E. intestinalis, respectively, and treated with compounds at doses ranging from 1 to 200 microM. We found that six compounds were active against C. parvum. Naringenin and genistein had the greatest activities with EC(50) of 15 and 25 microM, respectively. Two compounds, quercetin and apigenin, had activity against E. intestinalis at EC(50) of 15 and 50 microM, respectively. The EC(50) of trifluralin, a dinitroaniline compound, was decreased significantly when combined with genistein in an in vitro assay, suggesting that compounds may be used alone on in combination with other moderately active drugs to increase efficacy. In addition, induction of apoptosis by these compounds was studied but not observed to be a significant mechanism of action. PMID- 16684117 TI - The effects of alpha-lactalbumin and glycomacropeptide on the association of CaCo 2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri. AB - Two milk components, alpha-lactalbumin (alpha-La) and glycomacropeptide (GMP) may inhibit intestinal infection/intoxification. (3)[H] thymidine-labeled enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium (ATCC 6994) or Shigella flexneri (ATCC 9199) were introduced to CaCo-2 cultures and their association with CaCo-2 cells was assessed. Undigested, pepsin-digested and pepsin- and pancreatin-digested alpha-lactalbumin and glycomacropeptide inhibited association. Thus, milk supplemented with alpha-lactalbumin and glycomacropeptide might be effective in inhibiting associations of the pathogens EPEC, Salmonella typhimurium, and Shigella flexneri to intestinal cells. PMID- 16684119 TI - Elevated levels of C-reactive protein are associated with impaired coronary collateral development. AB - BACKGROUND: In vitro studies have shown that C-reactive protein (CRP) attenuates nitric oxide production and inhibits angiogenesis, which may result in impaired collateral development. The aim of this study was to investigate the association between high sensitivity CRP (hsCRP) levels and the extent of coronary collaterals. MATERIALS AND METHODS: We investigated the association between hsCRP levels and the extent of coronary collaterals according to the Rentrop classification in a cohort of 185 patients who had high-grade coronary stenosis or occlusion on their angiograms. RESULTS: Mean age was 62 years and 80% were males. Subjects with a higher grade of collaterals were significantly less likely to have diabetes mellitus (OR; 0.48, 95% and CI; 0.28, 0.83) or acute coronary syndrome (OR; 0.58, 95% and CI; 0.33, 0.99), but they were more likely to have higher number of vessels with significant stenosis (OR; 1.41, 95% and CI; 1.03, 1.93) and to have received statins (OR; 1.84, 1.09, 3.13). The mean hsCRP values reduced significantly as the Rentrop grades increased (trend, P = 0.0006). After adjusting for age, gender, statin use, clinical presentation with acute coronary syndrome, diabetes mellitus and the number of vessels with significant stenosis, each 10-unit increase in hsCRP values corresponded to a 31% reduced odds of having a higher collateral score (OR; 0.69, 95% and CI; 0.53, 0.90). CONCLUSIONS: Our findings indicate that elevated hsCRP levels are associated with a significant impairment in coronary collateralization. These data suggest a previously unrecognized mechanism through which inflammation may worsen cardiovascular outcomes. PMID- 16684120 TI - Endothelial dysfunction in Buerger's disease and its relation to markers of inflammation. AB - BACKGROUND: Buerger's disease (BD) is a segmental occlusive vascular disease. The aim of this study was to detect functional changes in brachial artery and asymptomatic morphological changes in extra-cranial carotid arteries not affected by the disease process and to assess markers of inflammation and endothelial damage. MATERIALS AND METHODS: Fourteen patients in the remission phase of BD and the same number of age- and sex-matched healthy controls were included in the study. The capability of endothelium-dependent (flow-mediated) and endothelium independent dilation of the brachial artery and intima-media thickness of the carotid arteries were measured using high-resolution ultrasound. Laboratory parameters of endogenous fibrinolytic activity, inflammation and endothelial dysfunction were also measured. RESULTS: Patients with BD had a diminished capability of endothelium-dependent vasodilation and higher levels of some circulating markers of inflammation, such as leukocytes, C-reactive protein, intercellular adhesion molecule-1 and E-selectin. Intercellular adhesion molecule 1 levels were related to some of the inflammatory markers (sedimentation rate, C reactive protein, alpha2-globulins and fibrinogen), while E-selectin was correlated with decreased endogenous blood fibrinolytic activity. Endothelium dependent vasodilation was in negative correlation with the relative share of neutrophil granulocytes. There were no significant differences in intima-media thickness between patients with BD and controls. CONCLUSIONS: Our study has expressed generalized functional arterial disorder in patients with BD not accompanied by any measurable morphological changes of the carotid arterial wall. Functional deterioration of brachial artery could be related to increased levels of various inflammatory markers--the process which is most probably the basic pathogenetic mechanism of the disease. PMID- 16684121 TI - Postprandial interstitial insulin concentrations in type 2 diabetes relatives. AB - BACKGROUND: An endothelial barrier for the insulin transport from the circulation to the target tissues of insulin has previously been suggested to contribute to insulin resistance. The interstitial insulin concentration (I-insulin) and insulin kinetics following a mixed meal have, however, previously not been characterized in human adipose tissue. SUBJECTS AND METHODS: Eight nondiabetic first-degree relatives (FDR) of type 2 diabetes patients were recruited. Their I insulin was measured by microdialysis after a test meal with or without oral administration of the insulin secretagogue nateglinide (120 mg). In parallel, adipose tissue blood flow and lipolysis were measured by xenon-clearance and microdialysis, respectively. RESULTS: The I-insulin increased after the test meal, and this response was more prominent on the day the subjects received the nateglinide tablet when compared with the day the subjects received the placebo tablet [I-insulin incremental area under the curve (IAUC) nateglinide 7612 +/- 3032 vs. Plac 4682 +/- 2613 pmol L(-1) min; P < 0.05, mean +/- SE]. However, the postprandial I-insulin(max)/P-insulin(max) ratio was similar on the two test days (nateglinide: 213 +/- 62 vs. 501 +/- 92 pmol L(-1), I/P-ratio: 0.38 +/- 0.06 and placebo: 159 +/- 39 vs. 410 +/- 74 pmol L(-1), I/P-ratio: 0.36 +/- 0.05). There was no difference in time of onset of insulin action in situ, or responsiveness, when comparing placebo and nateglinide. CONCLUSIONS: Microdialysis can now be used to measure the I-insulin in human adipose tissue following a mixed meal. The data also showed that the transendothelial delivery of insulin occurs rapidly, supporting the concept that transcapillary insulin transfer is a nonsaturable process in nondiabetic first-degree relatives of type 2 diabetes patients. PMID- 16684122 TI - Leptin and ghrelin expression in adipose tissues and serum levels in gastric banding patients. AB - BACKGROUND: To determine how leptin and ghrelin are expressed in the adipose tissues of obese adults undergoing gastric banding (LAGB), and to correlate tissue expression with serum concentrations and parameters of the metabolic syndrome. MATERIALS AND METHODS: A cross-sectional analysis of 92 patients: 61 obese patients with a body mass index (BMI) 49.2 +/- 1 kg m(-2) received LAGB, 20 patients underwent band exchange (BMI, 36.6 +/- 1.4 kg m(-2)) and 11 adult patients (BMI, 24.3 +/- 0.6 kg m(-2)) with fundoplication served as controls. Clinical data such as BMI and blood pressure were evaluated along with subcutaneous and visceral adipose tissue gene expression and fasting levels of leptin and ghrelin. Tissue transcripts were measured using real-time PCR, serum protein concentrations radio-immunologically. RESULTS: Leptin gene expression was highest in the primary LAGB group and more pronounced in subcutaneous fat in both sexes (P < 0.0001). Serum leptin concentrations were highest in the LAGB group (P < 0.001), whereby women exhibited higher serum levels than men. Leptin concentrations correlated positively to expression in subcutaneous fat (P < 0.0001), and leptin expression was also correlated to BMI and systolic blood pressure. We detected ghrelin gene expression in both types of fat. The ghrelin mRNA amounts in adipose tissues were similar in both sexes and comparable within groups; serum concentrations were lower in patients with primary LAGB than in controls (P < 0.05). CONCLUSIONS: Human adipose tissue expression of leptin is weight-course dependent and ghrelin is constitutional. Serum levels of leptin, but not of ghrelin, are indicative of an adaptive pattern of local gene expression in obese subjects undergoing weight reduction. PMID- 16684123 TI - Soluble CD40L in patients with morbid obesity: significant reduction after bariatric surgery. AB - BACKGROUND: Morbid obesity is associated with increased cardiovascular morbidity and mortality. Recent studies suggest that soluble CD40 Ligand (sCD40L) may play a pathogenetic role in atherothrombotic complications in cardiovascular disease as well as in inflammation and thrombosis. As morbid obesity is closely associated with chronic inflammation and insulin resistance (IR), it was of interest to study sCD40L in patients with morbid obesity before and after massive weight loss induced by bariatric surgery. PATIENTS AND METHODS: A total of 34 patients (mean age 40 +/- 12 years) with morbid obesity were studied before and 27.2 months after bariatric surgery. High sensitivity assays were used to measure concentrations of fasting sCD40L, monocyte-chemoattractant-protein-1 (MCP-1) and high-sensitive C-reactive protein (hsCRP). To investigate the associations of concentration changes of the parameters studied, differences between pre- and post-operative data were assessed and tested by univariate and multivariate linear regression analysis. RESULTS: After a mean weight loss of 33.1 +/- 18.4 kg, circulating sCD40L decreased significantly from (3.7 +/- 1.5) ng mL(-1) to (2.2 +/- 0.7) ng mL(-1), (P < 0.001). The decline in sCD40L after weight loss correlated significantly with the decrease in fasting insulin, 2-h insulin, HOMA insulin resistance (HOMA-IR), triglycerides, and the inflammatory biomarkers MCP 1 and hsCRP. CONCLUSIONS: We have shown a marked decrease in circulating sCD40L in association with an improvement of both insulin resistance and chronic inflammation in morbidly obese patients after bariatric surgery. As high sCD40L was shown to predict cardiovascular death and myocardial infarction in several prospective studies, the observed marked lowering of sCD40L might be of clinical relevance in morbidly obese patients. PMID- 16684124 TI - In vivo effects of imatinib mesylate on human haematopoietic progenitor cells. AB - BACKGROUND: Imatinib mesylate has considerable antineoplastic activity in patients with chronic myeloid leukaemia (CML) and some solid tumours. Although originally regarded as nontoxic for normal haematopoiesis, mild to moderate myelosuppression is a commonly observed side-effect of this treatment. Recently, this molecule has been shown to suppress normal haematopoietic progenitor cells in vitro. This is the first study that has investigated the effect of imatinib on haematopoietic progenitor cells in vivo. MATERIALS AND METHODS: We investigated the number of circulating haematopoietic progenitor cells in 79 patients with CML and five patients with solid tumours who were treated with imatinib for at least 3 months. Bone marrow progenitor cells were assessed in a subgroup of 18 patients with CML after 12 months of imatinib treatment. Results were compared with haematopoietic progenitor cell numbers of normal controls. RESULTS: Circulating progenitors of all classes were significantly decreased in CML up to 24 months of imatinib therapy compared with healthy controls (median progenitor cells in CML after 12 months: CFU-GM 62, range 0-2543; BFU-E 216, range 0-3259; CFU-GEMM 0, range 0-139; versus controls: CFU-GM 208, range 50-936; BFU-E 690, range 120 1862; CFU-GEMM 20, range 4-77; P < 0.001). Similar reductions in the number of progenitor cells derived from bone marrow were found in a subgroup of 18 patients with CML. In patients with solid tumours the number of circulating progenitor cells was significantly lower under treatment with imatinib when compared with the controls. Withdrawal of imatinib in a patient with a malignant brain tumour resulted in a prompt normalization of circulating progenitors. CONCLUSIONS: This study suggests that imatinib exerts myelosuppressive effects through inhibition of haematopoietic progenitor cells. PMID- 16684126 TI - Lipid-laden macrophage index in healthy canines. AB - BACKGROUND: The quantity of lipids in alveolar macrophages is used clinically as an indicator of aspiration, which is associated with increased lung inflammation. This is determined in the macrophages obtained from bronchoalveolar lavage (BAL) and is expressed as lipid-laden macrophage index (LLMI). Although there is ample data on LLMI in human subjects, there is no published data pertaining to the baseline measures of the LLMI in canines, which are extensively used for experimental studies on gastroesophageal reflex (GER) and airway diseases. Primary aim of the present study was to collect data pertaining to the cytology and LLMI in BAL fluids obtained from healthy dogs. MATERIALS AND METHODS: Eight dogs underwent a bronchoscopy with BAL collection, and esophageal pH monitoring to determine the reflux index (RI). The BAL fluid was processed and reviewed under a microscope to determine the proportions of the various cell types and the LLMI. RESULTS: The median RI among the subjects was found to be 0.6 (0.0, 1.2). The BAL cytology analysis showed 77.5% (71.0, 83.5) macrophages, 21.0 (13.0, 24.5) lymphocytes and 2.5 (1.5, 5.0) neutrophils. The median LLMI was found to be 156 (111, 208). CONCLUSIONS: Although the differential cell counts in the dogs' BAL fluid was comparable to that of other experimental animals and humans, the LLMI was distinctly higher than the corresponding value reported for other species. As LLMI is a valuable modality for evaluation of intrapulmonary pathophysiology, these data on LLMI can be used as a species-specific standard for canine subjects used for experimental studies on GER and airway diseases. PMID- 16684125 TI - Effect of stealthy liposomal topotecan plus amlodipine on the multidrug-resistant leukaemia cells in vitro and xenograft in mice. AB - BACKGROUND: Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy as the over-expressed MDR protein acts as an efflux pump, which leads to a reduction in the uptake of the anticancer agent by tumour cells. We combined topotecan and amlodipine together into the stealthy liposomes, in which amlodipine was applied as a MDR reversing agent to overcome the resistance. MATERIALS AND METHODS: Cytotoxicity, apoptosis and the signalling pathway assays were performed on human chronic myelogenous leukaemia K562, promyelocytic leukaemia HL-60 and MDR HL-60 cells, respectively. Pharmacokinetics and antitumour activity studies were performed on normal Kunming mice and female BALB/c nude mice with MDR HL-60 xenografts, respectively. RESULTS: Topotecan alone was effective in inhibiting the growth of non-resistant leukaemia cells, K562 and HL-60 cells but not the growth of MDR HL-60 cells. The resistance of topotecan in MDR HL-60 cells was potently reversed by the addition of amlodipine. Moreover, amlodipine enhanced the apoptosis-inducing effect of topotecan synergistically. Apoptosis was through activating caspases in a cascade: first, the initiator caspase 8 and then effectors caspase 3/7 (total activity of caspases 3 and 7) were activated. Being encapsulated into the stealthy liposomes with an acidic internal medium, topotecan existed dominantly in an active lactone species, which was reversibly changed from an inactive carboxylate form via a pH dependent reaction. After administration of stealthy liposomes to mice, the blood exposure of the lactone form was evidently increased and extended. The antitumour effects in the MDR HL-60 xenografted tumour were stealthy liposomal topotecan (SLT) plus amlodipine > SLT > un-encapsulated topotecan > blank control. CONCLUSIONS: The enhanced antitumour activity in the MDR HL-60 cells by the SLT plus amlodipine could be owing to multiple reasons: (a) synergistic apoptosis inducing effect, (b) reversing MDR by amlodipine and (c) increasing the availability of active lactone of topotecan by the stealthy liposomes. The apoptosis induced by amlodipine is through caspase 8 and then the 3/7 signalling pathway. PMID- 16684127 TI - Surfactant protein D in human lung diseases. AB - The lung is continuously exposed to inhaled pollutants, microbes and allergens. Therefore, the pulmonary immune system has to defend against harmful pathogens, while an inappropriate inflammatory response to harmless particles must be avoided. In the bronchoalveolar space this critical balance is maintained by innate immune proteins, termed surfactant proteins. Among these, surfactant protein D (SP-D) plays a central role in the pulmonary host defence and the modulation of allergic responses. Several human lung diseases are characterized by decreased levels of bronchoalveolar SP-D. Thus, recombinant SP-D has been proposed as a therapeutical option for cystic fibrosis, neonatal lung disease and smoking-induced emphysema. Furthermore, SP-D serum levels can be used as disease activity markers for interstitial lung diseases. This review illustrates the emerging role of SP-D translated from in vitro studies to human lung diseases. PMID- 16684128 TI - Hyperproinsulinaemia in normoglycaemic lipodystrophic HIV-infected patients. AB - BACKGROUND: We aimed to investigate whether the insulin precursors, intact (IP) and 32-33 split proinsulin (SP), which are elevated in states of insulin resistance and predict type 2 diabetes, would be elevated in human immunodeficiency virus (HIV)-infected patients with lipodystrophy (LIPO). MATERIALS AND METHODS: Forty-three normoglycaemic HIV-infected patients [18 LIPO and 18 without lipodystrophy (NONLIPO) receiving antiretroviral drugs, and seven patients naive to antiretroviral drugs (NAIVE)] were examined. Insulin precursors were measured during fasting, during an intravenous glucose tolerance test and during a hyperinsulinaemic-euglycaemic clamp, respectively. Insulin secretion rates (ISR) were determined by deconvolution of C-peptide concentrations. Disposition index (DI) was calculated as insulin sensitivity (Si(RD)) multiplied by the first-phase insulin response to intravenous glucose. RESULTS: LIPO exhibited increased fasting IP and SP (P < 0.05), a higher proportion of elevated fasting IP (3.1 pmol L(-1), 66% vs. 33% and 28%, P < 0.05) and SP (7.2 pmol L( 1), 50%, 11% and 0%, P < 0.01), reduced Si(RD) (> 50%, P < 0.001) and increased ISR (P < 0.001) compared with NONLIPO and NAIVE. Fasting SP and IP correlated positively with ISR (P < 0.001) and inversely and hyperbolically with Si(RD) (P < 0.001). Fasting SP/insulin ratio correlated inversely with Si(RD) (P < 0.05). Incremental IP + SP/insulin ratio after an intravenous glucose bolus correlated inversely with DI (P < 0.01), but did not differ between study groups. CONCLUSIONS: Proinsulin appeared to be increased in HIV-lipodystrophy, but no more than caused by the increased ISR. Nevertheless, the inverse correlations between SP/insulin ratio versus Si(RD) and incremental total proinsulin/insulin ratio versus DI may argue for a subtle beta-cell dysfunction in those patients with insulin resistance and low DI. PMID- 16684129 TI - Jacques Benoit Lecture. Information processing in the hypothalamus: peptides and analogue computation. AB - Peptides in the hypothalamus are not like conventional neurotransmitters; their release is not particularly associated with synapses, and their long half-lives mean that they can diffuse to distant targets. Peptides can act on their cells of origin to facilitate the development of patterned electrical activity, they can act on their neighbours to bind the collective activity of a neural population into a coherent signalling entity, and the co-ordinated population output can transmit waves of peptide secretion that act as a patterned hormonal analogue signal within the brain. At their distant targets, peptides can re-programme neural networks, by effects on gene expression, synaptogenesis, and by functionally rewiring connections by priming activity-dependent release. PMID- 16684130 TI - Do corticosteroids damage the brain? AB - Corticosteroids are an essential component of the body's homeostatic system. In common with other such systems, this implies that corticosteroid levels in blood and, more importantly, in the tissues remain within an optimal range. It also implies that this range may vary according to circumstance. Lack of corticosteroids, such as untreated Addison's disease, can be fatal in humans. In this review, we are principally concerned with excess or disturbed patterns of circulating corticosteroids in the longer or shorter term, and the effects they have on the brain. PMID- 16684131 TI - PC1/3 and PC2 gene expression and post-translational endoproteolytic pro opiomelanocortin processing is regulated by photoperiod in the seasonal Siberian hamster (Phodopus sungorus). AB - A remarkable feature of the seasonal adaptation displayed by the Siberian hamster (Phodopus sungorus) is the ability to decrease food intake and body weight (by up to 40%) in response to shortening photoperiod. The regulating neuroendocrine systems involved in this adaptation and their neuroanatomical and molecular bases are poorly understood. We investigated the effect of photoperiod on the expression of prohormone convertases 1 (PC1/3) and 2 (PC2) and the endoproteolytic processing of the neuropeptide precursor pro-opiomelanocortin (POMC) within key energy balance regulating centres of the hypothalamus. We compared mRNA levels and protein distribution of PC1/3, PC2, POMC, adrenocorticotrophic hormone (ACTH), alpha-melanocyte-stimulating hormone (MSH), beta-endorphin and orexin-A in selected hypothalamic areas of long day (LD, 16:8 h light:dark), short day (SD, 8:16 h light:dark) and natural-day (ND, photoperiod depending on time of the year) acclimated Siberian hamsters. The gene expression of PC2 was significantly higher within the arcuate nucleus (ARC, P < 0.01) in SD and in ND (versus LD), and is reflected in the day length profile between October and April in the latter. PC1/3 gene expression in the ARC and lateral hypothalamus was higher in ND but not in SD compared to the respective LD controls. The immunoreactivity of PC1/3 cleaved neuropeptide ACTH in the ARC and PC1/3-colocalised orexin-A in the lateral hypothalamus were not affected by photoperiod changes. However, increased levels of PC2 mRNA and protein were associated with higher abundance of the mature neuropeptides alpha-MSH and beta endorphin (P < 0.01) in SD. This study provides a possible explanation for previous paradoxical findings showing lower food intake in SD associated with decreased POMC mRNA levels. Our results suggest that a major part of neuroendocrine body weight control in seasonal adaptation may be effected by post translational processing mediated by the prohormone convertases PC1/3 and PC2, in addition to regulation of gene expression of neuropeptide precursors. PMID- 16684132 TI - Pituitary cocaine- and amphetamine-regulated transcript expression depends on the strain, sex and oestrous cycle in the rat. AB - Cocaine- and amphetamine-regulated transcript (CART) mRNA and peptides are abundant in the adenohypophysis, but their role in pituitary function has not yet been elucidated. CART peptides were recently shown to colocalise with luteinising hormone (LH) or prolactin in rat anterior pituitary, and contradictory results concerning the peptide effects on pituitary hormonal secretions were obtained in vitro from pituitary cell cultures. Thus, we reinvestigated the expression of CART mRNA within the pituitary. Immunohistochemistry for pituitary hormones was performed on sections from adult male Wistar rats followed by in situ hybridisation using CART mRNA antisense 35S-labelled probes. The most represented CART-expressing cells were lactotrophs (42 +/- 1% of CART cells) and gonadotrophs (32 +/- 3%), followed by thyrotrophs (10 +/- 2%), corticotrophs (7 +/- 2%) and somatotrophs (6 +/- 1%). In the pars tuberalis, CART mRNA was easily detectable in gonadotrophs and lactotrophs and, to a lesser extent, in corticotrophs and thyrotrophs. CART peptide was quickly and potently released from perifused pituitary by depolarisation (K+ 30 mM for 15 min; 465 +/- 37% over basal release, n = 5). Gonadotrophin-releasing hormone and thyrotrophin-releasing hormone (0.1 microM) were also active to a lesser extent (138 +/- 11% and 71 +/- 17, n = 7, respectively). CART (0.1 microM) did not modify basal LH or prolactin release but selectively inhibited K+-induced LH release without affecting K+-induced prolactin secretion. Pituitary CART mRNA and content were sex dependent and varied during the oestrous cycle, being lower in dioestrous 2. Pituitary CART content also varied widely amongst rat strains being five to six-fold higher in Wistar and Fischer rats compared to Brown Norway and Lou C rats. Ageing differentially affected pituitary CART mRNA and content, resulting in a marked decrease in Lou C and an increase in Wistar and Sprague-Dawley rats. Taken together, these results suggest that pituitary CART expression is dependent of the sex steroid environment and may be physiologically involved in LH secretion. PMID- 16684133 TI - Dynamic changes in dopaminergic neurotransmission induced by a low concentration of bisphenol-A in neurones and astrocytes. AB - One of the most common chemicals that behaves as an endocrine disruptor is the compound 4,4'-isopronylidenediphenol, called bisphenol-A (BPA). We previously reported that prenatal and postnatal exposure to BPA potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant induced pharmacological actions. Many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. The present study aimed to investigate the role of neurone-astrocyte communication in the enhancement of dopaminergic neurotransmission induced by BPA. We found that treatment of mouse purified astrocytes and neurone/glia cocultures with BPA in vitro caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of glial fibrillary acidic protein. A low concentration of BPA significantly enhanced the Ca2+ responses to dopamine in both neurones and astrocytes. Furthermore, a high concentration of BPA markedly induced the activation of caspase-3, which is a marker of neuronal apoptotic cell death in mouse midbrain neurone/glia cocultures. By contrast, treatment with 17beta oestradiol (E2) had no such effects. Prenatal and neonatal exposure to BPA led to an enhancement of the dopamine-dependent rewarding effect induced by morphine. These findings provide evidence that BPA alters dopamine responsiveness in neurones and astrocytes and that, at least in part, it may contribute to potentiate the development of psychological dependence on drugs of abuse. PMID- 16684134 TI - Regulation of salmon gonadotrophin-releasing hormone gene expression by sex steroids in rainbow trout brain. AB - Salmon gonadotrophin-releasing hormone (sGnRH) is the major form of gonadotrophin releasing hormone in the brain of Salmonids and is encoded by two different genes: sGnRH1 and sGnRH2. In the present study, we examined the expression patterns of these two genes during development and throughout the reproductive cycle of the female rainbow trout (Oncorhynchus mykiss), and also investigated the feedback action of sex steroids on brain mRNA levels. Both genes are expressed as early as 14 days postfertilisation and show a similar expression pattern during early life stages. In the adult female, sGnRH1 and sGnRH2 mRNAs are both present in neurones located in the ventral forebrain. This gene expression in the brain appears to be low during early vitellogenesis, and increases during oocyte maturation to reach a maximum after ovulation. The expression of sGnRH1 was not modified by in vivo steroid treatments in any experiment; however, testosterone and 5alpha-dihydrotestosterone down-regulate brain sGnRH2 gene in immature and adult ovariectomised females. Oestradiol treatment decreases sGnRH2 mRNA levels in the brain of adult ovariectomised females only. In the triploid fish brain, none of the steroids affect brain sGnRH mRNA levels. Our results suggest that, unlike sGnRH1, the sGnRH2 gene is under a strongly androgenic inhibitory control in the immature and adult female rainbow trout. PMID- 16684135 TI - Brain-derived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin. AB - Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, occurs abundantly in the brain, where it exerts a variety of neural functions. We previously demonstrated that BDNF also exists in the endocrine melanotroph cells in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis, suggesting that BDNF, in addition to its neural actions within the brain, can act as a hormone. In the present study, we tested whether BDNF, in addition to its neural and hormonal roles, can be released as a neurohormone from the neural pituitary lobe of X. laevis. By light immunocytochemistry, we show that BDNF is present in perikarya, in ventrolaterally projecting axons of the hypothalamic magnocellular nucleus and in the neural lobe of the pituitary gland, and that it coexists in these structures with the amphibian neurohormone, mesotocin. The neural lobe was studied in detail at the ultrastructural level. Two types of neurohaemal axon terminals were observed, occurring intermingled and in similar numbers. Type A is filled with round, moderately electron-dense secretory granules with a mean diameter of approximately 145 nm. Type B terminals contain electron-dense and smaller, ellipsoid granules (long and short diameter approximately 140 and 100 nm, respectively). BDNF is exclusively present in secretory granules of type A axon terminals. Double gold-immunolabelling revealed that BDNF coexists in these granules with mesotocin. Furthermore, we demonstrate in an superfusion study performed in vitro that mesotocin stimulates peptide release from the endocrine melanotroph cells. On the basis of these data, we propose that BDNF can act on these cells as a neurohormone. PMID- 16684136 TI - Abnormalities of the hippocampus are similar in deoxycorticosterone acetate-salt hypertensive rats and spontaneously hypertensive rats. AB - Hippocampal neuropathology is a recognised feature of the brain in spontaneously hypertensive rats (SHR), but similar studies are lacking in another model of hypertension, the mineralocorticoid-salt-treated rat. The present study aimed to compare changes in hippocampal parameters in 16-week-old male SHR (blood pressure approximately 190 mmHg) and their normotensive Wistar-Kyoto controls, with those of male Sprague-Dawley rats receiving (i) 10 mg deoxycorticosterone acetate (DOCA) every other day during 3 weeks and drinking 1% NaCl solution (blood pressure approximately 160 mmHg) and normotensive controls treated with (ii) DOCA and drinking water, (iii) drinking water only or (iv) 1% NaCl only. In these experimental groups, we determined: (i) cell proliferation in the dentate gyrus (DG) using the 5-bromo-2'-deoxyuridine-labelling technique; (ii) the number of glial fibrillary acidic protein (GFAP) positive astrocytes under the CA1, CA3 and DG; (iii) the number of apolipoprotein E (ApoE) positive astrocytes as a marker of potential neuronal damage; and (iv) the number of neurones in the hilus of the DG, taken as representative of neuronal density in other hippocampal subfields. Changes were remarkably similar in both models, indicating a decreased cell proliferation in DG, an increased number of astrocytes immunopositive for GFAP and ApoE and a reduced number of hilar neurones. Although hypertension may be a leading factor for these abnormalities, endocrine mechanisms may be involved, because hypothalamic-pituitary function, mineralocorticoid receptors and sensitivity to mineralocorticoid treatment are stimulated in SHR, whereas high exogenous mineralocorticoid levels circulate in DOCA-treated rats. Thus, in addition to the deleterious effects of hypertension, endocrine factors may contribute to the abnormalities of hippocampus in SHR and DOCA-treated rats. PMID- 16684137 TI - Serum from heifer calves treated with bovine growth hormone affects the rate of proliferation of C2C12 myogenic cells dependent on the plane of nutrition: the role of insulin-like growth factor-I and IGF-binding proteins-2 and -3. AB - The present in vitro experiments were carried out in order to study whether variations in the bovine growth hormone (bGH)/insulin-like growth factor (IGF)-I axis induced by plane of nutrition and bGH treatment of heifer calves caused variations in serum-induced proliferation of C2C12 myoblasts. Serum was obtained from two groups each of six heifer calves (195 +/- 8 kg) before (d -1) and after treatment with 15 mg/day of bGH for 6 days (d 6) fed either a low (GHL) or a high plane (GHH) of nutrition. Preceding the experiment all 12 heifer calves were fed at the low plane of nutrition. At d 6, serum concentrations of insulin and IGF-I were increased while that of IGF-binding proteins (IGFBP)-2 was decreased in GHH, but unchanged in GHL calves. Serum-induced proliferation of C2C12 myoblasts, was elevated at d 6 by GHH treatment. Especially human IGFBP-3 but also bovine IGFBP 2 added to cell cultures inhibited the rate of proliferation of C2C12 myoblasts stimulated by human IGF-I. The present results showed that GH treatment causes changes in the GH/IGF axis, which leads to changes in serum-induced growth of C2C12 muscle cells dependent on the plane of nutrition that mimic in vivo effects of GH treatment, which indicate an endocrine contribution of the IGF system. However, drawbacks of this suggestion are discussed. PMID- 16684138 TI - Resting energy expenditure and body composition of Labrador Retrievers fed high fat and low fat diets. AB - A high dietary fat intake may be an important environmental factor leading to obesity in some animals. The mechanism could be either an increase in caloric intake and/or a decrease in energy expenditure. To test the hypothesis that high fat diets result in decreased resting energy expenditure (REE), we measured REE using indirect calorimetry in 10-adult intact male Labrador Retrievers, eating weight-maintenance high-fat (HF, 41% energy, average daily intake: 8018 +/- 1247 kJ/day, mean +/- SD) and low-fat (LF, 14% energy, average daily intake: 7331 +/- 771 kJ/day) diets for a 30-day period. At the end of each dietary treatment, body composition measurements were performed using dual-energy X-ray absorptiometry. The mean +/- SD REE was not different between diets (4940 +/- 361 vs. 4861 +/- 413 kJ/day on HF and LF diets respectively). Measurements of fat-free mass (FFM) and fat mass (FM) also did not differ between diets (FFM: 26.8 +/- 2.3 kg vs. 26.3 +/- 2.5 kg; FM: 3.0 +/- 2.3 vs. 3.1 +/- 1.5 kg on HF and LF diets respectively). In summary, using a whole body calorimeter, we found no evidence of a decrease in REE or a change in body composition on a HF diet compared with LF diet. PMID- 16684139 TI - Protein profiling of organic stone matrix and urine from dogs with urolithiasis. AB - Two-thirds of the organic matrix in urinary stones consists of proteins. Their relationship to calculogenesis remains controversial with regard to their effect as inhibitors or promoters during stone formation. The purpose of the present study was to determine the differences in peptide and protein pattern between the urine of stone formers (n = 23) and control dogs (n = 12), as well as between organic matrix of different urinary stones (struvite n = 11, calcium oxalate n = 8, uric acid n = 4) using surface-enhanced laser desorption/ionization time-of flight mass spectrometry. Specific differences in protein and peptide profiles were found in the organic matrix of different mineral compositions. Characteristic differences were also found in urinary peptide and protein pattern especially in molecular masses below 20 kDa between affected and healthy dogs. Based on the obtained molecular masses they were in some cases tentatively identified as proteins that are known to be involved in stone formation in humans. The study shows that in dogs, specific-urinary peptides and proteins might be associated with urolithiasis. It indicates the importance to further characterize those proteins for possible diagnostic purposes in prognosis and therapy. PMID- 16684140 TI - Effect of chicory products with different inulin content on rat caecum physiology. AB - A 4-week experiment carried out on rats addressed the analysis of physiological properties of model casein diets supplemented with 5% cellulose or different preparations of chicory roots: flour, pulp, high- and low-molecular inulin (IN(HM) and IN(LM) respectively). Of all preparations, only pulp did not induce the hypertrophy of caecum walls or an increase in the bulk of caecal digesta. Chicory preparations lowered caecal pH and ammonia concentration (except IN(LM) preparation), evoked increased hydration of caecal digesta and protein content. The highest concentration of volatile fatty acids (VFA) was observed in rats fed a diet containing chicory pulp and IN(HM), whereas the lowest VFA concentration was reported after the administration of an IN(LM)-containing diet. The VFA pool was the highest in rats receiving a diet supplemented with chicory flour. All preparations examined diminished the coefficient of protein digestibility but not decrease nitrogen retention. The lowest levels of glucose and total cholesterol, at concurrently the highest level of HDL fraction, were recorded in serum of rats fed IN(LM). A distinct increase in intestinal wall and intestinal digesta, as well as the highest production of VFA and the lowest pH of caecal digesta indicate that chicory flour ingested to a semi-synthetic diet had the most profitable effect on the functioning of rat caecum, compared with other chicory products. Compared with the IN(HM), the preparation of IN(LM) increased caecal wall hypertrophy without increasing VFA production, still simultaneously increasing ammonia content and pH of caecal digesta, decreasing the total cholesterol concentration and increasing HDL content in serum. PMID- 16684141 TI - Weight loss effect of dietary diacylglycerol in obese dogs. AB - Obesity in dogs and cats have been increasingly recognized in recent years. Because obesity underlies various diseases, pet owners and veterinarians have an important responsibility to help animals lose weight and maintain their health. Diet therapy, however, is typically based on limited calorie intake and animals may suffer stress from hunger and this is also a concern to animal owners. For this reason, many clients drop out of weight control programmes. In the present study, we focused on dietary diacylglycerol (DAG) as a potentially effective ingredient for canine weight control without caloric restriction. We replaced a portion of the fat in dog food with either DAG or triacylglycerol (TAG), referred to as DAG or TAG diets here, and fed overweight beagle dogs (body condition score of 4 or higher) with either the DAG or TAG diet for a 6-week period. Results indicated that, even though the food composition other than fat type were identical, dogs fed the DAG diet showed a statistically significant reduction in body weight averaging a 2.3% reduction within 6 weeks while the TAG-fed dogs maintained their obese body weights. In addition, the DAG group also showed a reduction in body fat content, serum triglyceride and total cholesterol concentrations. These results suggest the possibility of developing a pet food using DAG to control weight and serum lipid levels without compromising caloric intake. PMID- 16684142 TI - Bioavailability of zinc glycinate in comparison with zinc sulphate in the presence of dietary phytate in an animal model with Zn labelled rats. AB - The objective of this study was to quantify the bioavailability of zinc (Zn) from sulphate and glycinate as representatives of inorganic and organic zinc sources. The semi-synthetic basal diet contained 2 microg/g of native Zn and was fortified with pure sodium-phytate (8 g/kg) in order to simulate conditions of common cereal-based meals. The basal diet was supplemented with either 53 microg/g of Zn from sulphate (control) or 10 microg/g of Zn from either sulphate (ZnSulphate) or glycinate (ZnGly). Twenty-four (65)Zn-labelled, growing rats weighing 133 g were allotted to the three diets (eight animals per treatment) and were kept pair-fed to ZnSulphate for 15 days. Zn contents in blood plasma, femur and whole body, as well as, plasma alkaline phosphatase activities were reduced compared with control indicating a zinc deficiency in ZnSulphate and ZnGly treatment. This allowed their differentiation in zinc bioavailability. True absorption of dietary Zn was significantly higher in ZnGly than in ZnSulphate (51% vs. 44%) while losses of endogenous faecal Zn and urinary Zn were not affected to a quantitatively relevant extent (mean: 17% and 2% of intake). This resulted in a +30% significantly improved Zn retention for ZnGly (33% vs. 25%) and a lower severity on Zn deficiency symptoms compared with ZnSulphate. Metabolic utilization accounted for 95% of absorbed dietary Zn for both Zn sources. Overall, the bioavailability of zinc glycinate was significantly superior by 16% to zinc sulphate (49% vs. 42%), mainly because of a higher absorptive potential at presence of a strong anti-nutritive component (phytate) in the diet. PMID- 16684143 TI - Nutritional evaluation of commercial dry dog foods by near infrared reflectance spectroscopy. AB - Near infrared reflectance spectroscopy (NIRS) was used to predict the nutritional value of dog foods sold in Chile. Fifty-nine dry foods for adult and growing dogs were collected, ground and scanned across the visible/NIR range and subsequently analysed for dry matter (DM), crude protein (CP), crude fibre (CF), total fat, linoleic acid, gross energy (GE), estimated metabolizable energy (ME) and several amino acids and minerals. Calibration equations were developed by modified partial least squares regression, and tested by cross-validation. Standard error of cross validation (SE(CV)) and coefficient of determination of cross validation (SE(CV)) were used to select best equations. Equations with good predicting accuracy were obtained for DM, CF, CP, GE and fat. Corresponding values for and SE(CV) were 0.96 and 1.7 g/kg, 0.91 and 3.1 g/kg, 0.99 and 5.0 g/kg, 0.93 and 0.26 MJ/kg, 0.89 and 12.4 g/kg. Several amino acids were also well predicted, such as arginine, leucine, isoleucine, phenylalanine-tyrosine (combined), threonine and valine, with values for and SE(CV) (g/kg) of 0.89 and 0.9, 0.94 and 1.3, 0.91 and 0.5, 0.95 and 0.9, 0.91 and 0.5, 0.93 and 0.5. Intermediate values, appropriate for ranking purposes, were obtained for ME, histidine, lysine and methionine-cysteine. Tryptophan, minerals or linoleic acid were not acceptably predicted, irrespective of the mathematical treatment applied. It is concluded that NIR can be successfully used to predict important nutritional characteristics of commercial dog foods. PMID- 16684144 TI - Digestibility and digestive organ development in indigenous and improved chickens and ducks fed diets with increasing inclusion levels of cassava leaf meal. AB - Growing indigenous Cambodian chickens and ducks, and broiler chickens and White Pekin ducks were fed diets containing 0%, 7%, 14% and 20% of cassava leaf meal (CLM) to study the effects of CLM level on diet digestibility and gastrointestinal tract (GIT) and organ development. The coefficient of total tract apparent digestibility (CTTAD) of dry matter (DM) and intake of digestible DM decreased with increased dietary CLM. DM and digestible DM intake was higher for local breeds than for the corresponding exotic breeds, and higher for ducks than for chickens (p < 0.001), although there were no species or breed effects on CTTAD of DM (p > 0.05). Weight of small intestine, caeca, gizzard and pancreas, expressed as per kg body weight, increased with increased CLM in the diet (p < 0.001). There was no consistent diet effect on liver weight. Length of small intestine and caeca, expressed on a mass-specific basis, increased with dietary CLM content (p < 0.001). When expressed as per kg body weight small intestine, proventriculus, gizzard, pancreas and liver weights, and small intestine length, were higher in ducks than in chickens (p < 0.001), and were higher in the indigenous than in the improved breeds (p < 0.01), except for small intestine weights, which were similar. However chickens had higher weight of caeca (p < 0.001) and colon (p < 0.01) in absolute units and per kg body weight. PMID- 16684145 TI - Olaquindox and cyadox stimulate growth and decrease intestinal mucosal immunity of piglets orally inoculated with Escherichia coli. AB - One hundred and fifty piglets were randomly allotted to one of six treatments to determine the effects of olaquindox and cyadox on growth and intestinal immune response including the number of intraepithelial lymphocytes and immunoglobulin A secreting cells (ASCs) during the three-week period. A 2 x 3 factorial arrangement of treatments was employed with the following factors: (1) Escherichia coli (O(139):K(88), 10(10) CFU) inoculation or control and (2) no antimicrobials, 100 mg/kg olaquindox and 100 mg/kg cyadox in the basal diet respectively. The antimicrobial supplementations improved (p < 0.01) average daily gain and feed conversion ratio (FCR) during the experiment. Average daily gain and FCR in the cyadox-supplemented pigs were higher (p < 0.05) than those in the olaquindox-supplemented pigs. Intraepithelial lymphocytes and ASCs decreased (p < 0.05) when the diets were supplemented. Jejunal ASCs in the cyadox supplemented pigs were lower (p < 0.05) than those in the olaquindox-supplemented pigs. Olaquindox and cyadox suppressed E. coli-induced intestinal immune activation, which may be involved in the observed growth promotion. PMID- 16684146 TI - Recovery of age-dependent immunological deterioration in old mice by thyroxine treatment. AB - On the basis that multiple interactions exist between thyroid hormones and immune system, and ageing is accompanied by changes in thyroid hormone secretion, it seems possible that thyroid hormones may be involved in the age-related immune dysfunction. The present study was conducted to evaluate in vivo and in vitro effects of thyroxine (T(4)) treatment on both cell-mediated and humoral immune responses of aged mice. In a trial to improve age-associated immune dysfunction, T(4) (0.2, 1.0 and 5.0 microg) was subcutaneously supplemented to BALB/c mice (over 18 months old) for 30 consecutive days. The present results showed that exogenous treatment of aged mice with T(4) was associated with a marked increase in serum T(4) level, and the total number of peripheral blood leukocytes as well as the total cellularity of thymus, spleen, peripheral lymph nodes (PLNs), mesenteric lymph nodes (MLNs) and bone marrow (BM). T(4) treatment also caused a significant increase in the total and differential numbers of peritoneal exudate cells (PECs), while it caused a slight increase in macrophages' phagocytic activity of PEC. Moreover, T(4) treatment elicited a statistically significant increase in both plaque-forming cell and rosette-forming cell responses. In vitro results showed that the addition of T(4) at concentrations of 0.001, 0.005 and 0.025 microg/well substantially potentiated the ability of splenocytes from aged mice to proliferate in the presence of concanavalin-A mitogen. Histological examination of thymuses from T(4)-treated aged mice revealed that the cortex was preferentially enlarged and repopulated with immature thymocytes. The present study postulates that thyroid hormones may be involved in the observed decrease in the immune responsiveness during ageing, and that T(4) treatment to aged mice is able to restore the age-related decline of the immune efficiency. PMID- 16684148 TI - Guest editorial: Shedding new light on the twilight zone. PMID- 16684147 TI - Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. AB - Three hundred and thirty-six 1-day-old male Hubbard HI-Ye broiler hybrids, kept in battery cages, were fed with diets based on maize (groups I and II) or wheat and barley (groups III and IV) and supplemented with or without plant extract (XT* 100 mg/kg) containing 5% carvacrol, 3% cinnamaldehyde and 2% of capsicum oleoresin. The morphological and histochemical examinations were carried out on days 21 and 42 of bird's age. The middle part of glandular part of the stomach and 30 mm long segment from the central part of the small intestine (jejunum) were taken out and then prepared for morphometrical and histochemical assays. Mobilization of mucocytes in superficial epithelium of the glandular stomach and increased secretion of neutral mucopolysaccharides and small amounts of sialomucins with or without local cell disruption with releasing of large amounts of mucus were observed in both 'grain' groups of 21-day-old birds fed with extract. In some animals, particularly those fed mixtures with plant extract, the folds of the proventriculum mucosa were fused into large, unshaped structures. In groups fed with plant extract the mucus secretion intensity and accumulation inside cells of the gastrointestinal mucosa were slightly higher. Morphological changes on gastrointestinal mucosa observed in young chickens fed XT were reduced in older animals. The results of this study showed that the increased releasing of large amounts of mucus and the creation of a thick layer of mucus on glandular stomach and wall of jejunum in chickens fed diets with plant extract could suggest villi-related protective properties of the use of the carvacrol, cinnamaldehyde and capsaicin mixture. This can explain the reduced possibility of adhesion to epithelium and number of Escherichia coli, Clostridium perfringes and fungi in the intestinal content of bird fed with XT supplemented diet. In morphometrical parameters of depth of jejunum crypt and height of villi, the influence of kind of grain and extract supplementation was observed in 21-day-old chickens only. The significant interaction between higher jejunum wall villi layer was observed only in chickens fed on maize diet supplemented with plant extract. PMID- 16684149 TI - Into the twilight zone: the complexities of mesopic vision and luminous efficiency. AB - Of all the functions that define visual performance, the mesopic luminous efficiency function is probably the most complex and hardest to standardise or model. Complexities arise because of the substantial and often rapid visual changes that accompany the transition from scotopic to photopic vision. These are caused not only by the switch from rod to cone photoreceptors, but also by switches between different post-receptoral pathways through which the rod and cone signals are transmitted. In this review, we list several of the complexities of mesopic vision, such as rod-cone interactions, rod saturation, mixed photoreceptor spectral sensitivities, different rod and cone retinal distributions, and the changes in the spatial properties of the visual system as it changes from rod- to cone-mediated. Our main focus, however, is the enormous and often neglected temporal changes that occur in the mesopic range and their effect on luminous efficiency. Even before the transition from rod to cone vision is complete, a transition occurs within the rod system itself from a sluggish, sensitive post-receptoral pathway to a faster, less sensitive pathway. As a consequence of these complexities, any measure of mesopic performance will depend not only on the illumination level, but also on the spectral content of the stimuli used to probe performance, their retinal location, their spatial frequency content, and their temporal frequency content. All these should be considered when attempting to derive (or to apply) a luminous efficiency function for mesopic vision. PMID- 16684150 TI - Toward a CIE supplementary system of photometry: brightness at any level including mesopic vision. AB - Photometry for brightness at any level including mesopic vision is described on the basis of the work conducted towards the development of a Commission Internationale de l'Eclairage (CIE) supplementary system of photometry in CIE technical committees. Several critical items in developing such a system are discussed: (1) how to scale brightness using the concept of equivalent luminance; (2) the basic vision model of brightness perception; (3) a description of the chromatic contribution to brightness at the photopic as well as at the mesopic level; (4) formulation of an adaptation coefficient for rod-cone interaction. A possible photometric model for brightness is described based on these considerations, together with a quantitative evaluation of the model using experimental data. PMID- 16684151 TI - Mesopic spectral sensitivity functions based on visibility and recognition contrast thresholds. AB - Human vision works in a complex way in the mesopic luminance range which is not yet clearly understood, although several important visual tasks are performed at these luminance levels. In this study, spectral data for detection visibility and recognition contrast sensitivity were obtained in visual experiments. From our data it appears that chromatic channels also influence mesopic contrast sensitivity. An achromatic model of spectral sensitivity is fitted to our experimental data and the remaining chromatic components are discussed. PMID- 16684152 TI - Visual performance in night-time driving conditions. AB - This paper introduces an experimental multitechnique method which was developed to establish a basis for a task performance-based mesopic photometry. This approach considers night-time driving by dividing visual performance into three visual tasks, of which achromatic threshold and reaction time are presented. The performance of both visual tasks decreased with decreasing luminance level from 1 to 0.01 cd m(-2), showing the strong effect of light level on visual performance in driving. The behaviour of the achromatic contrast threshold and reaction time for low-contrast targets was similar in terms of spectral effects, the strongest effects occurring at the lower mesopic levels. Both measures showed the Purkinje shift with decreasing luminance levels. The experimental data were used to calculate mesopic performance measures with the new mesopic model. The results imply that compared with V(lambda), spectral sensitivity in night-time driving can be better described with a mesopic model based on visual performance measures. PMID- 16684153 TI - Target detection and driving behaviour measurements in a driving simulator at mesopic light levels. AB - During night-time driving hazardous objects often appear at mesopic light levels, which are typically measured using light meters with a spectral sensitivity that is only valid for photopic light levels. In order to develop suitable mesopic models a target detection experiment was performed in a driving simulator. While subjects drove along a winding road they had to respond to randomly presented circular targets at various eccentricities. The background luminance ranged between 0.01 and 10 cd m(-2), and was either white, yellow, red or blue. The RT, number of missed targets, and driving behaviour were measured. The results show that target detection and driving performance get poorer with decreasing background luminance and increasing eccentricity of the target, in particular for the red colour. At high driving speeds and low luminances subjects tend to neglect left off-axis targets. Luminance calculated with existing reaction-time based mesopic models fits better to RT data than the widely-used photopic luminance. PMID- 16684155 TI - Changes in reaction time and search time with background luminance in the mesopic range. AB - Vision relies on both rod and cone signals over a large range of ambient illumination that encompasses a number of common situations. It is important, therefore, to understand how performance changes with light level in functional visual tasks. We measured reaction times and search times using achromatic targets to examine the relationship between latency and luminance contrast as a function of background luminance. Visual search was more robust to changes in luminance than reaction time; search performance could be made invariant by scaling the effects of contrast, but the range of reaction time changed significantly over the mesopic range. We also investigated the extent to which two mesopic visual performance models described the dependence of reaction time and search time on stimulus spectra, using coloured stimuli. The 'effective contrast' model that we examined described the spectral dependence of both reaction time and search time well. A model for mesopic luminous efficiency based on reaction times described the spectral dependence of each response only in conditions where there was little influence of chromatic signals. PMID- 16684154 TI - Characterizing luminous efficiency functions for a simulated mesopic night driving task based on reaction time. AB - The objective of this study is to test the luminous efficiency functions V(lambda), V'(lambda), V(10)(lambda) and their linear combinations on the basis of a data set gained from a simulated mesopic night-time driving experiment. Another aim is to provide 'real-world' data for the 'X framework' or 'linear combination model', and to find out its limits in a practical situation. Human performance was measured by the reaction time method. Results show that the single parameter of the linear combination of photopic and scotopic luminous efficiency functions can be determined analytically with little variation for a given mesopic background luminance level and a given visual target colour, but the computation leads to considerable deviations comparing all three target colours (red, green and blue) used in the experiment. The conclusion for the given experimental conditions is that the single parameter of the linear combination model has an increasing deviation for lower background luminance levels. PMID- 16684156 TI - Brightness contrast perception in the mesopic region. AB - Subjective evaluations of brightness contrast were obtained for Munsell chips of varying hues, lightness values and sizes against a white background illuminated in turn by four white illuminants at four background luminances in the mesopic region. Chip lightness was the strongest variable, resulting in a family of monotonic functions relating brightness contrast ratings to chip lightness. At the lowest light levels a nearly linear relationship was found between chip lightness and subjective ratings. As light levels increased, the relationship became curvilinear such that at the lowest contrast, chromatic brightness became important for subjective ratings. Within the level of precision for this experiment, chip hue and size as well as the spectral power distribution of the illuminants did not significantly impact the overall brightness contrast ratings. The subjective ratings of brightness contrast were analysed as a function of the luminance contrast of the stimuli as determined by conventional photometry and by three models of mesopic vision. The response functions relating subjective rating to the stimulus characterized in terms of these models were similar to those obtained with conventional photometry. PMID- 16684157 TI - Spectral discomfort glare sensitivity under low photopic conditions. AB - Car headlamp manufacturers have to optimise the headlamp spectrum to produce maximum visibility and minimal glare. There are very few investigations on the best spectrum for mesopic visibility and even less data on the spectrum of discomfort glare. We have performed pilot measurements to determine the spectral discomfort glare sensitivity under situations similar to night-driving conditions and conclude that it is not a simple additivity of the V(lambda) and the V' (lambda) functions. PMID- 16684158 TI - The dazzle reflex: electrophysiological signals from ocular muscles reveal strong binocular summation effects. AB - Under dark adapted or dim conditions the mammalian visual system is carefully programmed to respond rapidly to the sudden onset of bright lights. This response, called the dazzle reflex, is controlled from sub-cortical structures of the brain. It is known anecdotally that exposure to a bright light when dark adapted induces an instinctive closure of one eye to reduce the pain associated with dazzle. This binocular summation of the dazzle response has not previously been reported. The dazzle reflex can be measured in human subjects by recording the electrical activity from surface electrodes located near the muscles around the eye. In this paper we report an investigation of the apparent binocular summation of the dazzle reflex using this technique. The data reveal a clear difference between monocular and binocular stimulation, with the binocular response being much larger than the monocular response. Furthermore this monocular/binocular difference arises only if the stimulus duration is longer than approximately 1 s. These observations are interpreted in terms of the known physiology of blink mechanisms. PMID- 16684159 TI - Clinical disorders affecting mesopic vision. AB - Vision in the mesopic range is affected by a number of inherited and acquired clinical disorders. We review these conditions and summarize the historical background, describing the clinical characteristics alongside the genetic basis and molecular biological mechanisms giving rise to rod and cone dysfunction relevant to twilight vision. The current diagnostic gold standards for each disease are discussed and curative and symptomatic treatment strategies are summarized. PMID- 16684160 TI - Editorial: Stem cells: regeneration in the clinic. PMID- 16684161 TI - An evaluation of nursing practice models in the context of the severe acute respiratory syndrome epidemic in Hong Kong: a preliminary study. AB - AIM AND OBJECTIVE: Like other health-care workers, Hong Kong nurses had their professional knowledge and skills seriously challenged during the SARS outbreak. Could current nursing practices support the care of SARS or SARS-like patients in the future? If not, alternative practices would be needed. Providing a preliminary understanding, this paper compares the conventional with different nursing delivery models in a simulated SARS ward and focuses on nurses' efficiency, infection control practices and views of the two models. DESIGN AND METHODS: This study was conducted in three phases. First, a baseline understanding of nursing practices was achieved through four workflow observations. In an eight-hour day, four research assistants observed nursing activities in the medical and fever wards. These data were used in the second phase to construct two sets of clinical vignettes, pertaining to SARS patient care in both conventional and alternative practice models. These scripts were discussed with nine nurses of various ranks from the hospital under study for their expert validation and input. In the third phase, nurse participants and patient actors enacted the vignettes in a simulated setting. Video-taped observations and four nurse participant interviews were employed. Observational data were analysed through descriptive statistics and independent t-tests. Textual data were coded and categorized for common meanings. RESULTS: Conventional practice from the findings consisted of cubicle and named nurse nursing. While the former reflected modified team and functional nursing, it did not confine patient care within a cubicle as suggested by its name. The latter depicted a modified primary nursing approach in a team, with delegation of care. Preliminary findings concerning infection control and nurse satisfaction revealed that the alternative model had an advantage over the conventional. RELEVANCE TO CLINICAL PRACTICE: This study findings lay the foundation for clinical trials, which would evaluate the significance of patient-care quality, cost-effectiveness and better human resource management by restructuring current nursing practices. PMID- 16684162 TI - Nurse-related factors in the delivery of preoperative patient education. AB - AIMS AND OBJECTIVES: This article aimed to explore the factors relating to nurses themselves that influence the delivery of preoperative patient education in everyday surgical clinical contexts at one large general adult teaching hospital in Ireland. BACKGROUND: As landmark studies of preoperative education undertaken in the UK in the 1970s identified the superiority of structured programmes of patient education over and above 'regular' preoperative care, there have been many intervention/outcome experimental studies carried out in this area. However, there has been little interpretative work conducted that explores the regular, or 'usual' preoperative education given to patients in everyday surgical units. Methods. A sample of 12 experienced surgical nurses was selected and each participant was interviewed in depth. Data were analysed using a qualitative strategy resembling grounded theory. RESULTS: Findings indicate that preoperative education was variously interpreted by participants, and participants' accounts suggested that different understandings and practices by nurses resulted in patients receiving different levels of care. In addition, diverse levels of knowledge and experience of individual nurses resulted in unevenness in the type of preoperative education that patients received. A number of participants advocated a more formal method of preparation for nurses in the area of preoperative education. Finally, in some surgical areas, specialist nurses worked side-by-side with regular ward nurses and their input in preoperative education was largely seen by participants in a positive way, particularly in view of the structural constraints that ward nurses faced. CONCLUSIONS: We conclude that nurse-related factors in preoperative education do not arise in a vaccum, but rather are related to the wider organizational practices and culture. The difficulties with preoperative education identified in data may be addressed through organizational investment in preoperative education. RELEVANCE TO CLINICAL PRACTICE: This paper produces evidence for nurses and clinical nurse managers about how nurse-related factors impede preoperative education, and may provide a starting point for how to begin to address obstacles to better preoperative care. PMID- 16684163 TI - Effect of nurse-led gut-directed hypnotherapy upon health-related quality of life in patients with irritable bowel syndrome. AB - AIMS AND OBJECTIVES: This study quantified health-related quality of life in a group of irritable bowel syndrome patients and measures changes following a treatment programme of nurse-led gut-directed hypnotherapy. BACKGROUND: It is well recognized that health-related quality of life can be severely impaired in patients suffering form the irritable bowel syndrome. Current conventional treatment for irritable bowel syndrome is often unsatisfactory. In contrast it has been shown that gut-directed hypnotherapy is an effective treatment of irritable bowel syndrome with up to three-quarters of patients reporting symptomatic improvement. DESIGN/METHOD: Seventy-five patients (55 females/20 males, median age 37.1 years, age range 18-64) comprised the study group. Physical symptoms of irritable bowel syndrome were recorded using seven-day diary cards. On presentation the predominant symptoms were abdominal pain (61%), altered bowel habit (32.5%), and abdominal distension/bloating (6.5%) in the patient group. An irritable bowel syndrome quality of life questionnaire was used to define health-related quality of life. Psychological well-being was measured using the Hospital Anxiety and Depression Scale. Data analysis was carried out using MINITAB, Release 12 for Windows. RESULTS: Physical symptoms statistically improved after hypnotherapy. There were also significant statistical improvements (P < 0.001) in six of the eight health-related quality of life domains measured (emotional, mental health, sleep, physical function, energy and social role). These improvements were most marked in female patients who reported abdominal pain as their predominant physical symptom. Anxiety and depression improved following treatment. CONCLUSION: Gut-directed hypnotherapy has a very positive impact on health-related quality of life with improvements in psychological well being and physical symptoms. It appears most effective in patients with abdominal pain and distension. Relevance to clinical practice. This study demonstrates that by integrating complementary therapies into conventional care that gastrointestinal nurses have a potential role in the management of irritable bowel syndrome. PMID- 16684164 TI - Patients' experiences of living with oesophageal cancer. AB - AIMS: The aim of this study is to describe patients' experiences of living with oesophageal cancer and how they seek information. BACKGROUND: Oesophageal cancer is a devastating disease with poor prognosis. Nursing care for individuals with oesophageal cancer requires increased knowledge of how they experience illness and how it affects them. METHOD: Data were collected by semi-structured, qualitative interviews with 13 participants. Content analysis was used to analyse data. RESULTS: Four themes were identified: (i) Experiences of becoming a patient diagnosed with oesophageal cancer is distinguished by the participants' experiences of vague symptoms, of receiving the diagnosis and of existential concerns evoked by the illness. (ii) Experiences of undergoing investigations and treatment consist of the participants' experiences of extreme tiredness in relation to investigations and treatment. (iii) Experiences of intrusions in daily life is conceptualized by the participants' experiences of how the illness influenced their daily life. (iv) Managing a life-threatening illness consists of a variety of strategies, which the participants employed to manage their life threatening illness. CONCLUSIONS: The participants were unprepared of receiving a diagnosis of oesophageal cancer. Dysphagia, fatigue and uncertainty influenced the participants' everyday life. To manage the illness one of their strategies was seeking for information. The physicians were considered the main source of information, but family as well as friends with medical knowledge were also acknowledged as valuable sources. RELEVANCE TO CLINICAL PRACTICE: Understanding patients' experiences of living with oesophageal cancer is important to improve nursing care. When caring for these patients, focus ought to be on the whole family. In nursing care, it is important to be aware of the effects of dysphagia and fatigue. Health-care professionals ought to organize meetings with fellow patients and recommend literature and websites that provide patients with high quality information. PMID- 16684165 TI - Evidence for early oral feeding of patients after elective open colorectal surgery: a literature review. AB - AIM: To review research on early oral feeding following elective, open colorectal surgery. BACKGROUND: Fasting following gastrointestinal surgery is a traditional surgical practice, based on fears of causing postoperative complications if oral intake begins before bowel function returns, but fasting following elective surgery is questionable as a best practice. METHODS: Searches in Journals@Ovid CINAHL, MEDLINE, PubMed, Web of Science and The Cochrane Library for primary studies, published during 1995-2004, used the keywords: 'surgery', 'postoperative', 'elective, 'colorectal', 'bowel, 'colon', 'oral', 'enteral', 'feeding', 'early', 'traditional'. Studies of adults undergoing elective, open colorectal surgery who were allowed fluids and food before bowel function returned (early feeding) were included. Outcomes of interest were safety, tolerability, duration of gastrointestinal ileus and length of hospital stay. Critical appraisal of randomized and controlled studies was undertaken following inclusion. RESULTS: Fifteen studies comprising 1352 patients were reviewed. All studies concluded early feeding was safe, based on complications rates. Total complications were 12.5% (range 0-25%) for 935 early feeding patients, with no increased risk of anastomotic leak, aspiration pneumonia, or bowel obstruction. For all studies an average of 86% patients (range 73-100%) tolerated early feeding. Studies demonstrating faster resolution of postoperative ileus or shorter hospitalization were associated with multimodal perioperative care, including early mobilization, epidural analgesia and comprehensive patient education. Appraisal of five randomized trials revealed no blinding and inadequate randomization. CONCLUSIONS: This review supports early oral feeding after elective, open colorectal surgery and challenges the traditional practice of fasting patients until return of bowel function. Early feeding was safe, well tolerated and easy to implement. Reduced length of ileus and shorter hospitalization may occur with multimodal protocols. RELEVANCE TO CLINICAL PRACTICE: Nurses can highlight this new evidence for other health professionals, advocate development of clinical protocols featuring early feeding and participate in multi-disciplinary, multi-method research regarding benefits of early feeding. PMID- 16684166 TI - The qualitative experience of Chinese parents with children diagnosed of cancer. AB - AIM: The present study aimed to describe the coping experiences of Chinese parents with children diagnosed as having cancer during the treatment stage. BACKGROUND: Cancer is the second major cause of death among children in Hong Kong, it claims the lives of 60-70 children per year. Childhood cancer has tremendous impact on the family, especially the parents. It is, therefore, important to understand parents' psychological functioning and coping experience. METHODS: A phenomenological approach was used. Data were collected by qualitative interviews and analysed following Colaizzi's phenomenological methodology. A purposive sample of nine parents whose children were diagnosed of having childhood cancer was recruited from a regional hospital in Hong Kong. RESULTS: Four themes emerged describing parents' coping experiences: shock and denial, establishing the meaning or the situation, confronting the reality and establishing a new perspective. The initial reactions of the parents to the diagnosis were shock, denial and worry. However, they quickly accepted the reality and regarded their child's illness as their 'fate' that they had to accept. They were committed to the care of the sick child and seek informational and emotional support to cope with the situation. All of them were able to identify positive aspects from the illness experience and establish hope for the future. Chinese cultural beliefs might help the parents cope positively and avoid negative emotions. CONCLUSION: This study found some commonalities of coping experience in both Western and Chinese culture. It adds knowledge to the coping experience of Chinese parents at the treatment phase of their children's illness and highlighted the need for emotional and information support for parents. RELEVANCE TO CLINICAL PRACTICE: Education programme and mutual support group would be helpful to parents. Nurses have to learn how different cultural groups and subcultural groups in the society cope to provide competent cultural care. PMID- 16684167 TI - Revalidation of the postpartum stress scale. AB - AIMS AND OBJECTIVES: The purpose of this study was to test further and validate the postpartum stress scale developed for Taiwanese women. BACKGROUND: The postpartum stress scale was developed to measure postpartum stress in Taiwanese women. However, over the last decade, the social context in Taiwan has changed and several items in the scale needed to be re-examined. DESIGN: Non-experimental quantitative research with repeated measures at the first and fifth week of the postpartum period was conducted for this study. METHODS: A proportional stratified quota was used to sample from the 10 hospitals and six clinics with the highest birth rates in Kaohsiung, Taiwan. Participants were 505 and 518 postpartum women at each time point, respectively. RESULTS: Factor analysis at two points in time identified three attributes of postpartum stress: (a) maternal role attainment, (b) lack of social support, and (c) negative body changes. The Cronbach's alphas at each time point were 0.94 and 0.92, respectively. CONCLUSIONS: The results support the postpartum stress scale as a validated instrument that has been conceptualized, created, and tested with Taiwanese postpartum women. RELEVANCE TO CLINICAL PRACTICE: This study was done in the hope that women experiencing specific postpartum stressors would be detected and subsequently helped by supportive nursing intervention that provides stressor specific coping resources. PMID- 16684168 TI - Nurses as providers of support for mothers of premature infants. AB - AIM: The study aims to explore the supportive behaviour of nurses as experienced by mothers of premature infants in Hong Kong. BACKGROUND: The sudden, unexpected delivery of a premature infant is an extreme shock to parents. Studies have confirmed the importance of nursing support to the relief of stress of parents. DESIGN: A descriptive study design. METHODS: A convenience sampling of 37 mothers in a neonatal Intensive Care Unit were recruited from a regional hospital to complete the 'Nurses Parent Support Tool' (NPST) which consists of four aspects: communication information support, emotional support, parental esteem support and quality care-giving support. Six mothers were interviewed after the completion of the tool to give examples of supportive and non-supportive behaviour. RESULTS: The results showed that all of the mothers rated all nursing support items as important. However, there was a significant mean difference between perceived and received nursing support. These findings demonstrated that parents desired more nursing support than they received, particularly in the area of supportive communication and the giving of information. Comparatively, emotional support was rated less important as well as having been less often received than the other three domains of support. Interviews with six mothers further gave examples and described the types, attributes and the phases of support in the Chinese setting. CONCLUSION: The findings indicate that Chinese parents are desirous of more nursing support than they received. RELEVANCE TO CLINICAL PRACTICE: Nurses should be aware of the importance of tailoring nursing support to meet the needs of parents of premature infants. PMID- 16684169 TI - A comparison of HIV positive and negative pregnant women at a public sector hospital in South Africa. AB - AIM: The aim of the study was to compare HIV positive and negative pregnant women with respect to maternal and neonatal outcome to inform the development of clinical practice guidelines. BACKGROUND: HIV infection in pregnancy places an added burden on the physical ability of the woman's body to cope with pregnancy. As a result HIV causes an exaggeration of the problems related to pregnancy. METHOD: Data were collected by means of a retrospective record review conducted on 212 stratified randomly selected HIV positive and 101 matched HIV negative pregnant women. The two sample t-test and Fisher exact test were used to compare the maternal and neonatal outcomes of HIV positive and negative pregnant women. RESULTS: HIV positive pregnant women had a significantly lower haemoglobin (10.85 vs. 11.48 g/dl; P = 0.001), attended significantly fewer antenatal clinic appointments (4.03 vs. 4.63; P = 0.04), weighed significantly less (72.07 vs. 76.69 kg; P = 0.02) and were significantly more likely to present with an abnormal vaginal discharge (32.55 vs. 24.75%; P = 0.02) than HIV negative pregnant women. The difference in the prevalence in HIV positive pregnant women of pregnancy induced hypertension (16.98 vs. 9.90%; P = 0.06), syphilis infection (5.95 vs. 0.99%; P = 0.062) and urinary tract infection (15.53 vs. 7.92%; P = 0.06) approached significance when compared with HIV negative pregnant women. HIV positive pregnant women were significantly more likely to present with intrauterine growth retardation (4.72 vs. 0%; P = 0.03), significantly more likely to deliver earlier (37.92 vs. 38.51 weeks; P = 0.03) and significantly more likely to deliver neonates weighing less (2969.98 vs. 3138.43 g; P = 0.01) than HIV negative pregnant women. CONCLUSION: The Department of Health attributes the high rate of HIV and AIDS related maternal morbidity and mortality in South Africa to the absence of accepted and practical guidelines for midwives' antenatal assessment and management of HIV positive pregnant women. Relevance to clinical practice. This study identifies maternal and neonatal outcomes related to HIV infection in pregnancy and provides evidence required to inform the development of clinical practice guidelines. PMID- 16684170 TI - A comparison of written materials vs. materials and counselling for women with sexual dysfunction and multiple sclerosis. AB - AIM: Evaluate whether symptoms of vaginal dryness, low libido, less intense or delayed orgasm could be improved in women with multiple sclerosis who took part in an education or education plus counselling programme. BACKGROUND: Sexual dysfunction, a prevalent symptom in women with multiple sclerosis, can negatively affect quality-of-life. METHODS: Women attending a large multiple sclerosis clinic were invited and 62 were randomized into one of two groups. Group 1 received written materials on primary, secondary and tertiary sexual dysfunction in multiple sclerosis as well as additional resources (books, websites, list of local psychologists specializing in sexual counselling). Group 2 received the same written materials as well as three counselling sessions from the clinic nurse, the latter two by telephone. The primary outcome measures were the expanded disability status scale and the multiple sclerosis intimacy and sexuality questionnaire-19. Repeated-measures analysis of variance was used to evaluate sexual dysfunction score over time and to compare two groups. RESULTS: At baseline, total expanded disability status scale scores were not correlated with primary, secondary or tertiary sexual dysfunction. Total multiple sclerosis intimacy and sexuality questionnaire-19 score was correlated with use of anti cholinergic medications [r (54) = 0.28, P < 0.05], but no other medications, alcohol or tobacco use. Both groups had equivalent and significant reductions in primary sexual dysfunction [F (1) = 14.79, P < 0.001] postintervention. There was a trend towards an interaction effect for tertiary sexual dysfunction [F (1) = 2.88, P = 0.096], in the direction of group 2 (education and counselling). Subjectively, women welcomed the opportunity to discuss sexual concerns and noted that the written information allowed a framework for initiating discussion with their spouses. CONCLUSION: Relatively straightforward interventions provided by a clinic nurse may help women cope with the symptoms of sexual dysfunction associated with multiple sclerosis. Women who do not benefit from basic interventions could then be referred to an expert sexual dysfunction practitioner. RELEVANCE TO CLINICAL PRACTICE: Women with multiple sclerosis experience many disease-related physical and emotional challenges of which sexuality is only one. Sensitivity to sexual dysfunction and being willing to approach the topic is appreciated by women with multiple sclerosis. Nurses do not require in-depth expertise to offer some basic suggestions which may significantly improve life quality and assist the woman with multiple sclerosis to talk about or cope with sexuality issues. PMID- 16684171 TI - Children's and young people's experiences of chronic renal disease: a review of the literature, methodological commentary and an alternative proposal. AB - AIM: The aims of this paper were to review and critique existing research literature on children's and young people's experiences of chronic renal disease and to propose alternative approaches that may be more fruitful in addressing existing research shortcomings. BACKGROUND: Chronic renal disease, which results in approximately 1.6-4 new cases per year per million population in the 0-15 years age group, is a serious illness that causes severe and irreversible reduction in kidney function. Despite modern medical advances, its significance and implications for the lives of the children and young people concerned are profound. METHOD: Salient literature for this review was obtained using the major health and social science electronic databases such as Medline, CINAHL, Psyclit and Sociofile. Manual searching of relevant books, journals and 'grey literature', combined with the genealogy approach, extended and strengthened the search. CONCLUSIONS: Research in this area focuses mainly on two areas, namely psychological adjustment and adaptation to end-stage renal disease. This research is grounded within a framework of empirical psychology that values objectivity, measurement and quantification. This predominantly psychometric approach is critiqued for simplifying the complex experience of end-stage renal disease and for pathologizing children and young people with this disease. We identify a significant gap in the research literature, namely the lack of research that takes into account these children's and young peoples'own perspectives of their experiences. RELEVANCE TO CLINICAL PRACTICE: Chronic renal disease has a significant impact on children's and young people's lives. Understanding the experiences of these children is important for the provision of effective healthcare. Conducting child-centred qualitative research in this area would allow us to explore vital questions of meaning, perception and understanding. If health and social care organizations claim to provide 'consumer-focused' services, it behoves us to develop first a clearer understanding of the lives and experiences of children and families who seek our help and to use this knowledge and understanding to plan and provide more grounded and responsive services. PMID- 16684172 TI - Adolescents with Type 1 Diabetes: transition between diabetes services. AB - AIM: The research aimed to develop a sustainable and coordinated approach to facilitating the transition between diabetes services for adolescents. The objectives were to: (1) involve key diabetes health delivery stakeholders in expressing their concerns and issues about current service delivery and ways to improve same, and (2) reveal from the perspective of the adolescents living with Type 1 Diabetes their experiences surrounding the process of transition. BACKGROUND: This paper presents research that sought to identify the major concerns and issues that stakeholders had about transition and to reveal the experience of transition for the adolescent with Type 1 Diabetes. Key representatives from seven public diabetes services in Adelaide, South Australia worked collaboratively to answer the objectives of this inquiry. Approach. Interview data were generated and analysed using a response focus framework provided by fourth generation evaluation research. In this study, the focus was on common concerns, claims and issues raised by health care professionals (n = 21) and adolescents (n = 10) aged between 15 and 18 years about transferring from children's to adult diabetes services. FINDINGS: Data revealed education and dietetic advice was reactive rather than proactive and that the paediatric model of care is philosophically and practically different to the adult model of diabetes care. Three phases of transition were identified: preparation, formal transition and evaluation. Our findings indicated that these stages of transition were not being fully implemented in health units. CONCLUSION: The project findings have set the scene to establish a multidisciplinary working party to work collaboratively across agencies to develop effective transition pathways. RELEVANCE TO CLINICAL PRACTICE: The role of diabetes nurse educators and dietitians in South Australia is under-used throughout the transition process. Diabetes nurse educators are in an ideal position to prepare, coordinate and evaluate transitional processes. PMID- 16684173 TI - The effectiveness of a diabetes nurse clinic in treating older patients with type 2 diabetes for their glycaemic control. AB - AIMS: The aim of this study is to determine the effectiveness of a diabetes nurse clinic intervention in controlling the poor glycaemia of older patients with type 2 diabetes. RESEARCH METHOD: This is a quasi-experimental design with pre- and follow-up tests. The study was conducted in a regional acute hospital in Hong Kong. A total of 150 (75 controls, 75 cases) poor glycaemic control older patients with diabetes were recruited in the study. OUTCOME MEASURES: Biomedical and psychological data were collected at pre- and follow-up period and compared between groups. RESULTS: The study results show an effective intervention of diabetes nurse clinic in giving consultation and education to the type 2 diabetic patients. Subjects in the nurse follow-up group showed an improvement in the HbA1c, and systolic blood pressure and reduction on healthcare utilization. CONCLUSION: The study provided evidence to support the diabetes nurse clinic in treating the older patients with diabetes. This study has provided confidence to the diabetes nurse to treat the unstable older patients with diabetes. RELEVANCE TO CLINICAL PRACTICE: Diabetes mellitus is a serious health concern that most commonly affects older people. As indicated by the results of this study, this educational programme can act as an effective nursing intervention to the type 2 diabetic patients. PMID- 16684174 TI - A pilot study of the effects of a specialist nurse-led cognitive-behavioural therapy service on coping, respiratory function and quality of life for patients with bronchiectasis. PMID- 16684175 TI - Commentary on Woodward VA, Webb C & Prowse M (2005) Nurse consultants: their characteristics and achievements. Journal of Clinical Nursing 14, 845-854. PMID- 16684177 TI - Commentary on Dunnion ME & Kelly HW (2005) From the emergency department to home: discharge planning and communication of information between an emergency department and primary care sector following discharge of older people from an emergency department of a rural general hospital. Journal of Clinical Nursing 14, 776-785. PMID- 16684178 TI - Commentary on Coyne I (2006) Consultation with children in hospital: children, parents' and nurses' perspectives. Journal of Clinical Nursing 15, 61-71. PMID- 16684181 TI - Commentary on Wright D, Fenwick J, Stephenson P & Monterosso L (2005) Speculum 'self insertion': a pilot study. Journal of Clinical Nursing 14, 1098-1111. PMID- 16684182 TI - Commentary on Lauder W, Mummery K & Sharkey S (2006) Social capital, age and religiosity in people who are lonely. Journal of Clinical Nursing15, 334-340. PMID- 16684183 TI - The publication of studies without ethical permission. PMID- 16684184 TI - PHN competencies. PMID- 16684185 TI - Comparison of the dissemination and implementation of standardized public health nursing competencies in academic and practice settings. AB - OBJECTIVES: To assess the use of the "Core Competencies for Public Health Professionals" standards in practice and academic work settings by public health nurses (PHNs), and to determine differences between practitioners and faculty. DESIGN: Nonexperimental, descriptive study using a cross-sectional survey. SAMPLE: Three public health nursing (PHN) organizations sent invitations to all members. A total of 334 (18.7%) from an estimated 1,786 members completed the survey. MEASUREMENTS: The investigators developed a 17-item web-based survey with open- and closed-ended responses, using Rogers' diffusion of innovations as a theoretical framework. RESULTS: Respondents are equally familiar with the competencies for public health professionals disseminated by the Council on Linkages and for PHNs by the Quad Council of Public Health Nursing Organizations (Quad Council). Two thirds of PHNs are aware of the competencies after only 2 years, primarily from professional PHN organizations. Faculty are adopting and using the competencies at a significantly faster rate than practitioners. CONCLUSIONS: Faculty and practitioners who use the competencies value them, and rarely discontinued their use after adoption. Efforts to promote diffusion among faculty and especially practitioners need to continue. Professional organizations can actively provide and share examples of useable formats and best practices associated with the competencies. PMID- 16684186 TI - Development of the public health nursing competency instrument. AB - This paper describes the development and initial testing of an instrument to measure population-based public health nursing competencies. Although multiple lists of public health competencies exist, literature review did not elicit a valid instrument that could measure changes in public health nursing competency over time. The public health nursing competency instrument, consisting of 195 measurable activities organized in the framework of the nursing process, was developed. Competency scores of practicing public health nurses significantly increased after a continuing education series, and the instrument was confirmed by experts to be a valid reflection of public health nursing practice. The time required for instrument development exceeded expectations because of the multiple stages of delineating competencies and validating data with national experts. PMID- 16684187 TI - A competency-based approach to public health nursing performance appraisal. AB - OBJECTIVES: To integrate public health nursing (PHN) competencies into a comprehensive performance review instrument for nurses at multiple practice levels in an urban public health department. DESIGN: Based on thorough review of PHN competency literature, the tool evaluates performance for 5 nursing practice classifications (Staff RN, Public Health Nurse, Nurse Practitioner, Clinical Nurse Specialist, Nursing Supervisor) in eight PHN domains (assessment, policy development/program planning, evaluation, communication, cultural competency, partnership/collaboration, disease prevention/health promotion, leadership/systems thinking). SAMPLE: Tool was piloted with over 50 nurses from PHN workforce (n>400) of Public Health-Seattle & King County (Washington). METHOD: Pilot testing includes all components of the performance appraisal system: Public Health Competency Grid, statement of general workplace expectations, Nursing Performance Appraisal Tool, and supporting documents defining performance elements by job classification. RESULTS: Supervisors find the tool easy to use and report that it provides opportunity for real communication between employee and supervisor. Nurses at all practice levels report that it effectively describes/evaluates their practice. CONCLUSIONS: This tool is an efficient performance appraisal instrument providing meaningful feedback to nursing employees within a framework of PHN competencies. Adopting such tools in PHN practice can help nurses to better understand their role in population-based public health efforts. PMID- 16684188 TI - Use of public health nurse competencies to develop a childcare health consultant workforce. AB - The purpose of this article is to describe the efforts in the state of Georgia to train public health nurse-childcare health consultants (PHN-CCHCs) using the framework of the "Core competencies for public health practice." OBJECTIVES: The goal of the training was twofold: (1) to prepare a statewide cadre of PHNs as the primary workforce for Georgia's emerging childcare health consultation (CCHC) system and (2) to prepare their district nurse directors to lead and support CCHCs. DESIGN: Administrators attended a 2-day workshop followed by access to executive coaching for their management teams. PHNs participated in a three-phase training program, with phases 1 and 3 offered as 3-day workshops with field experiences, and phase 2 offered online and as a practicum. SAMPLE: Forty-four administrators and over 85 PHN-CCHCs completed the training. RESULTS: Graduates of the program reported satisfaction with training and reported the use of PHN core competencies in CCHC. Graduates also found enhanced skills in using core competencies to be applicable to a variety of population-based practices. Beyond CCHC being instituted in selected health districts, interest in CCHC has occurred statewide. CONCLUSIONS: The PHN-CCHC program enhanced the knowledge and use of core competencies and heightened interest in CCHC statewide. PMID- 16684189 TI - Use of PHN competencies and ACHNE essentials to develop teaching-learning strategies for generalist C/PHN curricula. AB - This paper provides a useful tool for the undergraduate community/public health nursing (C/PHN) faculty member to design courses and learning activities, and to interpret C/PHN education needs to undergraduate curriculum committees and administrators. Specifically, this paper provides a tangible bridge between the Public Health Nursing Competencies (Quad Council of Public Health Nursing Organizations, 2004) and the Association of Community Health Nursing Educators (ACHNE) Essentials of Baccalaureate Education (2000) for both didactic and clinical learning experiences. The tables may be used in multiple ways, including curriculum monitoring and improvement, course development and instructional design, clinical practice planning, and as a foundation for evaluation of conceptual learning and practice competence for the C/PHN generalist. Because C/PHN experiences in undergraduate education are unique and context based, the tables exemplify how two key guiding documents mutually frame the C/PHN educational experience supported by specific learning activities. Further, at a minimum, MSN preparation as a C/PHN specialist is clearly necessary for the teaching and learning of baccalaureate curricular components of C/PHN. PMID- 16684190 TI - Developing population health competencies among public health nurses in Georgia. AB - As part of a new vision for public health in Georgia, the role of the public health nurse (PHN) is shifting from a predominantly individual and clinic-based care model to a population health practice model. Based on focus groups conducted with management and frontline PHNs in 2001, nurses in Georgia were unprepared for this transition and lacked a strong understanding of population health concepts and competencies. To prepare nurses for their new and expanded responsibilities, an online population health course was developed specifically for currently employed PHNs. The Quad Council PHN Competencies (2003) provided the roadmap in identifying the population health online course and continuing education training program priority competencies for PHNs in Georgia. Along with incorporating the competencies into case studies and didactic learning, the required projects served as evidence of competency application. The population health online course was offered from fall 2000 to spring 2005 in collaboration with Georgia schools of nursing and has enabled a cohort of nurses to lead the way as Georgia transitions into a new public health model. Nurses who completed the course reported greater experience with and an understanding of population health competencies. PMID- 16684191 TI - Self-reported competency of public health nurses and faculty in Illinois. AB - Having a public health workforce with a high level of competency is a prerequisite for having an effective public health system. The purpose of these two studies was to assess the competency level of practicing public health nurses (PHNs; n=168) from 50 local health agencies and public health nursing faculty (n=46) from 31 nursing programs in Illinois. The questionnaire consisted of nine reliable scales using self-reported levels of competence in each PHN competency domain. Overall, PHNs reported only feeling competent in one domain: "linking people to services." Although PHN faculty felt competent across the nine domains, they did not report feeling competent to teach any of the domains. Thus, PHNs and public health nursing faculty need education and training to meet the professionally established level of competence. PMID- 16684192 TI - Developing public health competence among RN-to-BSN students in a rural community. AB - A new RN/BSN nursing program offers rural students in a western state the opportunity to address significant health care needs on a local level by developing public health leadership competencies in their home communities. The innovative program, funded by a grant from the Health Resources and Services Administration, makes it possible for RNs to complete their BSN degrees without travel, as they position themselves to provide critically needed health care leadership in their local areas. Partnerships between the university, community colleges, and local health agencies allow students in the RN-to-BSN program to benefit from a streamlined BSN admission process, onsite mentoring, and newly developed courses that lead students to reflect on health needs in their home communities. On the basis of Public Health Nursing Competencies as defined by the Nursing Quad Council (2004), the re-designed curriculum prepares students for public health leadership by encouraging application of competencies while participating in the delivery of essential public health services in their communities. Initial response to this new opportunity indicates that students can develop as leaders by developing public health competencies, and facets of the program may encourage more students to commit to completing the BSN while increasing capacity among PHNs. PMID- 16684193 TI - Use of health fairs to develop public health nursing competencies. AB - This paper describes a community-campus partnership for health education established through the effort of faculty, students, and the community. Health fairs designed to address issues of concern to a community agency were originally conducted with nursing students. This partnership between nursing and a community was expanded to include social work, physical therapy, and medical students. A shared learning experience was structured through the presentation of health fairs in collaboration with agencies in Burlington, Vermont. One goal of nursing education is to produce professionals who have the beginning competencies of public health nursing. Reflection on the process, relationships, and outcomes of the health fairs revealed that students attained these beginning competencies in all eight public health nursing competency domains. Combining community-campus partnerships and an interdisciplinary focus enabled nursing students to become more responsive to community needs and to learn to work collaboratively toward creating healthier communities, thus building skills required for public health nursing. PMID- 16684194 TI - Matching a graduate curriculum in public/community health nursing to practice competencies: the Rush University experience. AB - An evidence-based approach to Public/Community Health Nursing (P/CHN) requires that P/CHN educators prepare practitioners with the relevant skills, attitudes, and knowledge. Such education should be competency-based and have measurable outcomes to demonstrate student preparation. In 2003, the Quad Council competencies were developed to be applied at two levels of public health nursing practice: the staff nurse/generalist role and the manager/specialist/consultant role. This paper describes a process for evaluation and revision of a graduate curriculum to prepare Advanced Practice Clinical Nurse Specialists (CNS) in P/CHN, to ensure that the educational program addresses and develops knowledge and proficiency in all relevant competencies. This paper documents the process of integrating the competencies throughout the P/CHN graduate curriculum at varying levels, guiding students to achieve proficiency in each competency by the end of the program. Measurement of achievement in these competencies will be discussed, and examples provided. Advanced Practice Public Health Nurses educated via this competency-based approach will be prepared to sit for national certification as a CNS in Public/Community Health, and to assume leadership roles in public health nursing. PMID- 16684195 TI - Development of public health nursing competencies: an oral history. AB - This paper identifies the external and internal forces that led to the initiation and completion of a set of Public Health Nursing (PHN) competencies by nursing representatives from the Quad Council (QC) organizations: the Association of Community Health Nursing Educators, the American Public Health Association/Public Health Nursing Section, the Association of State and Territorial Directors of Nursing, and the American Nurses Association Congress on Nursing Practice and Economics. Discussion on the need for competencies began in 1988 with the Institute of Medicine report, The Future of Public Health, which cited a widening gap between the education and practice of public health (PH). PH leaders promptly responded by initiating many interactions to improve academic programs and enhance workforce development, including the development of competencies for PH professionals. PHN responded through the Quad Council of Public Health Nursing Organizations, which completed development of a set of national PHN competencies in 2003. The unfolding of that process is reported from the content-specific oral histories of five PHN leaders who served on the QC and participated in developing the PHN competencies. PMID- 16684197 TI - Elementary, my dear readers. PMID- 16684198 TI - Guiding mothers' management of health problems of very low birth-weight infants. AB - OBJECTIVE: Explore the feasibility, usefulness, and outcomes of a pilot program to support mothers in developing competencies for managing health problems of their very low birth-weight (VLBW) infants in partnership with the primary care clinician (PCC). DESIGN: In a randomized study, mothers who received guided participation (GP) and printed guidelines for managing VLBW infant health problems were compared with mothers who received only the guidelines and standard care (GL group). SAMPLE: All mothers (GP = 20; GL = 11) were at least 18 years old and English speaking. Infants were all VLBW (< or =1,500 g). INTERVENTION: GP began during the infant's neonatal intensive care unit stay and continued with public health nurses (PHNs) and a family service clinician through the infant's first 4 postterm months. MEASUREMENTS: Intervention feasibility and usefulness were assessed with maternal and clinician feedback. Outcomes included maternal and clinician appraisal of mothers' use of clinical resources and mothers' perceptions of primary-care quality and the family-PCC relationship. RESULTS: Intervention feasibility and usefulness were supported. GP and GL groups did not differ significantly on outcomes. CONCLUSIONS: Findings indicate a longer intervention period, GP organized by infant problem episodes, and enhancement of the PHN role in the context of interdisciplinary and interagency collaboration. PMID- 16684199 TI - Assisting women to learn myocardial infarction symptoms. AB - OBJECTIVE: The purpose of this study was to test how teaching format (factual versus storytelling) and restructuring the social norm of caring for others to caring for self affects how women learn to identify and respond to myocardial infarction (MI) symptoms. DESIGN: The study was a randomized pretest posttest full factorial experiment. SAMPLE: One hundred and thirteen women participated. MEASURES: Before and after reading the intervention pamphlet, the women wrote all the MI symptoms that they knew and rated their intention to call 911 if symptoms occurred. INTERVENTION: The women read one of the four MI pamphlets corresponding to the four conditions. RESULTS: No significant effects for learning MI symptoms resulted from teaching format or social norms. Women learned three additional MI symptoms. All responded with high intention to call 911 if MI symptoms occurred. CONCLUSIONS: Women can learn additional MI symptoms from reading a brief pamphlet about MI symptoms. Use of a storytelling format and the social norm of caring for self might not impact how many MI symptoms women learn. Studies using audiovisuals and larger samples are needed to clarify whether storytelling format and the social norm of caring for self-impact learning MI symptoms. PMID- 16684200 TI - Key informants' perceptions of health care for elders at the U.S.-Mexico border. AB - OBJECTIVE: This study was part of a systematic assessment to gain an understanding of aging in a rural community along the U.S.-Mexico border in Arizona. DESIGN: It used a participatory action research design. Interviews were conducted among key informants (n = 29), including health and social services providers and community leaders. Content analysis focused on identifying common themes and concerns related to elders. RESULTS: Major themes identified were (1) social infrastructure issues, (2) community awareness of health problems, (3) socioeconomic problems, (4) cultural influences on health beliefs, practice, and use of services, (5) image of aging, (6) border issues, and (7) educational needs for service providers. CONCLUSION: Elders living at the border are a particularly vulnerable population with strong cultural affiliations and health disparities. Although the population of elders has been growing at the border, the traditional focus on the health care needs of youth and children has contributed to the neglect of issues facing elders. This study provided community key informants' perspective of aging to community members, policy makers, service agencies, and health care providers for community development. A conceptual framework was developed for educational programs and research projects for health professionals who care for elders at the border. PMID- 16684201 TI - Evaluation of a community-based outreach worker program for children with asthma. AB - OBJECTIVE: An asthma outreach worker (AOW) can provide home-based education about asthma management and methods to minimize home environmental asthma triggers. A theory-based AOW program was implemented by a community partnership and its effectiveness was evaluated. DESIGN: Baseline and follow-up surveys were used to evaluate the effectiveness of the AOW. SAMPLE: The convenience sample consisted of 60 caregivers whose children received AOW services. MEASURES: Quality of life, use of asthma management plans, medication use, health care utilization, home environmental behavior changes to reduce triggers, and satisfaction with AOW services were self-reported by caregivers. RESULTS: Caregivers reported significantly higher quality of life at follow-up than at baseline. At follow-up, 93% of the children had asthma management plans as compared with 31% at baseline. Self-reported hospitalizations were significantly reduced. All of the families made changes to minimize household asthma triggers. Caregivers reported high satisfaction with the AOW and 90% of them felt that the home environmental assessment conducted by the AOW helped improve their child's asthma. CONCLUSION: Short-term community-based AOW services for children can be effective in enhancing self-management capabilities, improving the quality of life, increasing the use of asthma management plans, and helping families reduce asthma triggers in the home environment. PMID- 16684202 TI - Perceptions of effectiveness of nurse-managed clinics: A cross-cultural study. AB - OBJECTIVE: To study perceptions of the effectiveness of two nurse-managed clinics (NMCs), one in the United States and the other in New Zealand. DESIGN: Cross cultural evaluation study utilizing qualitative methodology; two rounds of in depth interviews were tape recorded, transcribed verbatim, and analyzed for themes. SAMPLE: Data were initially collected from 21 participants. Sixteen of the original participants participated in a second interview. INTERVENTION: The first round of interviews and provisional data analysis were conducted in the respective NMCs; joint data analysis to identify themes and develop a preliminary joint report of findings followed. Sixteen participants responded to the provisional report; additional data analysis resulted in the final report of findings. RESULTS: Analysis of the data yielded three categories: the NMC milieu affects perceived effectiveness; perceptions are shaped by contrasting with past experiences; and the level of care affects personal health care decisions. CONCLUSIONS: The milieu created in the NMC enhances perceptions of effectiveness and responsibility for personal health care. Further research related to effectiveness of NMC models of care and client outcomes is needed. Qualitative methodology is recommended for cross-cultural research. PMID- 16684203 TI - Canadian public health nurses' job satisfaction. AB - OBJECTIVES: This study was undertaken to test the applicability of using a standardized questionnaire for measuring public health nurse (PHN) job satisfaction and to determine whether or not scores changed over 30 months. The importance of establishing a method for ongoing measurement of PHN job satisfaction was underscored by changing directions in practice and an emphasis on building public health capacity. METHOD: A 30-month interval, repeated measures descriptive survey design was used. SAMPLE: A randomly selected sample of 87 PHNs employed within 1 Canadian regional health authority participated. MEASUREMENT: The survey questionnaire, the Index of Worklife Satisfaction, was designed to measure the importance of and satisfaction with 6 components of job satisfaction. RESULTS: Pay and autonomy were the most important components; the order of the 4 remaining components changed from first to second surveys. Professional status, autonomy, and interaction were the most satisfying components; PHN satisfaction with professional status and interaction improved significantly over 30 months. A majority of subjects reported that direct client care/client response/making a difference were worklife aspects providing them with most satisfaction. CONCLUSIONS: A valid, reliable questionnaire suitable for ongoing measurement was tested with PHNs, and baseline levels of their job satisfaction were established. PMID- 16684204 TI - Outbreak investigations: Community participation and role of community and public health nurses. AB - Community and public health nurses (C/PHNs) may play a vital role in the investigation of disease outbreaks. C/PHNs possess skills in conducting interviews on sensitive subjects and in collaborating with communities. C/PHNs maintain key links to community providers, symptomatic clients, their families and associates, as well as community institutions where outbreaks occur. This combination of skills makes C/PHNs ideally suited to perform outbreak investigations. There are, however, pressing questions about whether C/PHNs are adequately prepared to contribute to investigation outcomes, to foster participation of affected communities, and to fully apply nursing skills to outbreak investigations to stop the spread of disease. Using one case study, the authors explore investigation outcomes, community participation issues, educational preparation, and public health funding and workforce policies required to achieve these ends successfully. One model of community participation in the steps of outbreak investigation and several Quad Council domains and competencies are proposed for use in practice. Questions regarding the use of emergency preparedness funding and employment of C/PHNs in epidemiology roles are raised. PMID- 16684205 TI - Fit or fat? A review of the debate on deaths attributable to obesity. AB - Scientific debate surrounding the burden of obesity on the population's health has escalated in recent months following the release of differing reports on the population attributable fraction of obesity mortality. Differing views on statistical methodology for estimating the number of deaths attributable to being overweight and obese were widely reported in the popular media. There was concern that the public was left unsure of how to perceive the health information about obesity. Furthermore, food industry groups exploited this moment of scientific development to generate support in corporate interests in the name of "consumer freedom." This review examines the state of the debate on deaths attributable to obesity. Public health nurses can use this information to work collaboratively with their service populations to ensure that accurate public health information is disseminated regarding obesity and mortality. This paper also highlights areas of obesity-related public health research that have been emphasized by this debate. Nurses should be actively engaged in such research in an effort to understand the epidemic of obesity and develop programs and influence policy to curb the determinants of obesity. PMID- 16684206 TI - Social exchange as a framework for client-nurse interaction during public health nursing maternal-child home visits. AB - The purpose of this paper was to develop a nursing-focused use of social exchange theory within the context of maternal-child home visiting. The nature of social exchange theory, its application to client-nurse interaction, and its fit with an existing data set from a field research investigation were examined. Resources exchanged between the nurse and clients were categorized and compared across the patterns of home visiting, nursing strategies based on exchange notions were identified, and variations in exchange were linked with client outcomes. The nurse provided resources within the categories of information, status, service, and goods. Clients provided time, access to the home, space within the home to conduct the visit, opportunities to observe maternal-child interaction, access to the infant, and information. The ease and breadth of resource exchange varied across the patterns of home visiting. The social exchange perspective was useful in categorizing resources, specifying and uncovering new resource categories, understanding nursing strategies to initiate and maintain the client-nurse relationship, and linking client-nurse interactive phenomena with client outcomes. Social exchange theory is potentially useful for understanding client nurse interaction in the context of maternal-child home visits. PMID- 16684207 TI - Utility of qualitative research findings in evidence-based public health practice. AB - Epidemiological data, derived from quantitative studies, provide important information about the causes, prevalence, risk correlates, treatment and prevention of diseases, and health issues at a population level. However, public health issues are complex in nature and quantitative research findings are insufficient to support practitioners and administrators in making evidence informed decisions. Upshur's Synthetic Model of Evidence (2001) situates qualitative research findings as a credible source of evidence for public health practice. This article answers the following questions: (1) where does qualitative research fit within the paradigm of evidence-based practice and (2) how can qualitative research be used by public health professionals? Strategies for using qualitative research findings instrumentally, conceptually, and symbolically are identified by applying Estabrooks' (1999) conceptual structure of research utilization. Different research utilization strategies are illustrated through the use of research examples from the field of work on intimate partner violence against women. Recommendations for qualitative researchers disseminating findings and for public health practitioners/policy makers considering the use of qualitative findings as evidence to inform decisions are provided. PMID- 16684209 TI - The nurse midwives' story, an allegory. 1952. AB - This is an excerpt from a fictional account of a nurse midwifery student on the verge of graduation from the Maternity Center Association (MCA) School of Nurse Midwifery. The original article was written by 6 students at MCA about nurse midwifery and published in the original Public Health Nursing in 1952. After graduation, 4 of the authors served in mission groups in Saudi Arabia, Formosa, and the Philippines, and the others were employed in California and New York. The story conveys the hopes and limitations perceived by the midwifery students and serves as an allegory for those who push the boundaries of accepted practice. PMID- 16684208 TI - Instrument development of the Confidence in Home Care Services Questionnaire for use with elders and caregivers of Mexican descent. AB - Mexican American elders use home care services less than non-Hispanic white elders, and a larger study is testing whether lack of confidence in home care services, measured by the Community Service Attitude Inventory, is a factor. In order to further develop the questionnaire for use with Mexican American elders and caregivers, qualitative interviews were conducted with Mexican American elders (n = 5), Mexican American caregivers (n = 5), and home care providers (n = 5). Content analysis of interviews supported 2 dimensions: confidence and fear/worry. The research team developed 29 items from the dimensions. Testing of the items (n = 15) suggested content validity and two additional items. The revised questionnaire was translated and tested for language equivalence in Spanish and English, assisted by a local community advisory council (n = 9). Through collaboration, bicultural/bilingual teams and community partners refined 1 instrument that can be used to measure one of many barriers to equity in health care services with vulnerable populations. Thematic findings may be incorporated into nurses' interventions as they offer home care services to families. PMID- 16684210 TI - Cell-free artificial liver support. PMID- 16684211 TI - Albumin regeneration in liver support-comparison of different methods. AB - Albumin is the most abundant human plasma protein. Among many other functions it is an important transporter of hydrophobic internal and external substances such as intermediate and end products of metabolism and drugs. In liver failure the albumin binding capacity is decreased because of a disproportion between available albumin molecules caused by decreased hepatic synthesis and hydrophobic toxins because of decreased hepatic clearance. The resulting increase in plasma and tissue concentrations of these substances is associated with multiple organ dysfunctions frequently seen in severe liver failure. The scope of the present article is to compare different liver support strategies with regard to their ability to regenerate the patients albumin pool by removing albumin-bound toxins. Most prominent technique in this group is the molecular adsorbent recirculating system (MARS). It will be compared with single pass albumin dialysis (SPAD), fractionated plasma separation and adsorption system (FPSA, Prometheus), and plasma perfusion/bilirubin adsorption with special regard to efficacy and selectivity. PMID- 16684212 TI - Principles of bound solute dialysis. AB - Toxins that bind to albumin in the bloodstream and are associated with progressing liver failure have proven refractory to removal by conventional hemodialysis. Such toxins can, however, be removed by adding a binder to the dialysate that serves to capture the toxin as it is dialyzed across the membrane. Several approaches based upon this concept are in various stages of clinical evaluation. The thermodynamic basis common to these approaches has been used to develop an engineering description of 'bound solute dialysis' which has further been used to define the clinical expectations and limitations of the approach. Three dimensionless, independently controllable, operating parameters emerged from this analysis (i): kappa, the dialyzer mass transfer/blood flow rate ratio (clinical range: 0.5-2.5); (ii) alpha, the dialysate/blood flow rate ratio (clinical range: 0.1-2.0); and (iii) beta, the dialysate/blood binder concentration ratio (clinical range: 0.02-5.0). In the absence of binder in the dialysate, bound toxin removal is sensitive to kappa and alpha, with greater removal associated with greater kappa and/or alpha. Bound toxin removal, however, is dependent primarily upon kappa and independent of alpha and beta once a small amount of binder, beta > 0.02, is added to the dialysate. The improvement in bound toxin removal over conventional hemodialysis is dependent upon how tightly the toxin binds albumin ranging from a 6-fold increase for a relatively tightly bound solute such as unconjugated bilirubin, to 1.5-fold increase for a less tightly bound drug such as warfarin at 24 h perfusion time. Clinically, bound solute dialysis can be practiced in single-pass mode with as little as 1-2 g albumin/L dialysate. Because of the constraints imposed by the thermodynamic nature of the process, intervention should be made as early in the disease progression as feasible. PMID- 16684213 TI - How can liver toxins be removed? Filtration and adsorption with the Prometheus system. AB - The application of extracorporeal blood circuits in liver failure therapy has its roots in the two functions of the liver, first as a detoxifying and second as a synthetizing organ. In contrast to hydrophilic uremic toxins, most liver toxins are hydrophobic and bind preferentially to blood proteins. Consequently, the majority of these compounds cannot be removed by hemodialysis or similar dialytic procedures. Current systems use albumin as a transport vehicle for hydrophobic compounds across high flux membranes (e.g. albumin-dialysis, molecular adsorbent recirculating system (MARS)). In contrast to these devices, the Prometheus system (Fresenius Medical Care, Bad Homburg, Germany) applies filtration across highly permeable membranes with a molecular weight cut-off of >300.000. These membranes facilitate a direct filtration of most of the toxin-bearing proteins. In a secondary circuit these toxins are then removed by adsorber beads assembled in specially designed cartridges. The protein-containing toxin-free solution returns to the primary circuit. Clinical testing of the Prometheus system's safety and efficacy parameters showed that cell counts and coagulation factors were not significantly affected. Total bilirubin-, bile acid- and plasma ammonia-levels were reduced in vivo by -21%, -43% and -40%, respectively. First successful therapeutic results have been obtained for patients treated for drug abuse and for patients waiting for transplantation. Thus, a combination of plasma fractionation with highly permeable membranes followed by a secondary circuit with adsorber cartridges proves to be the most effective method of removing toxic waste in liver failure. Further investigations will follow in order to extend the application of the Prometheus system to larger cohorts of patients. PMID- 16684214 TI - Review article: clinical experience with Prometheus. AB - Prometheus is a new extracorporeal liver support device which facilitates the combined removal of both albumin-bound and water-soluble toxins based upon the method of fractionated plasma separation and adsorption (FPSA). The pilot trial included 11 patients with acute-on-chronic liver failure and concomitant renal failure. Prometheus therapy was found to be safe except for a reversible decrease of blood pressure. In three patients, clotting of the secondary system occurred. Prometheus treatment significantly improved blood levels of protein-bound (conjugated bilirubin, bile acids, ammonia) and water-soluble (creatinine, urea) substances. Thus, Prometheus might be a new therapeutic option in patients with severe hepatorenal syndrome. Furthermore, there is some preliminary experience with Prometheus in the treatment of refractory cholestatic pruritus and in successful bridging to liver transplantation. In order to compare extraction capacities of Prometheus and the molecular adsorbent recirculating system (MARS), five patients were crossover-treated with both systems. Prometheus resulted in significantly higher reduction ratios of bilirubin, ammonia and urea. Another study closely monitored whether the device causes an unselective removal. Neither important cytokines nor coagulation factors were found to be removed. In conclusion, Prometheus seems to be a new therapeutic option in artificial liver support. A significant improvement of the biochemical milieu was already observed after two treatments. The potential to remove protein-bound and water-soluble substances has been shown without signs of a significant unselective removal. PMID- 16684215 TI - A novel plasma filtration therapy for hepatic failure: preclinical studies. AB - There is a need to develop artificial means of liver replacement and/or assistance with the aim of either supporting patients with borderline functional liver cell mass until their liver regenerates, or until a donor liver becomes available for transplantation. Selective plasma filtration is a novel approach to blood purification therapy designed to reduce the level of circulating toxins of hepatic and renal failure, mediators of inflammation and inhibitors of hepatic regeneration. The results of preclinical studies indicate that treatment of pigs with experimentally-induced fulminant hepatic failure is safe and effective in extending survival time and arresting brain swelling. In addition, the amount of ammonia, aromatic amino acids, IL6, TNFalpha and C3a removed during the 6-h treatment in the present study was higher by 34% to 175% than the total plasma content of those substances at the start of therapy. PMID- 16684216 TI - Sorbent suspensions vs. sorbent columns for extracorporeal detoxification in hepatic failure. AB - Hepatic failure is a significant medical problem which has been unsuccessfully treated by hemodialysis. However, similar therapies using recirculated dialysate regenerated by sorbents in place of single-pass dialysate have been beneficial in treating acute-on-chronic liver failure. The advantages of sorbent-based treatments include some selectivity of toxin removal and improved removal of protein-bound toxins. Activated carbon has been extensively used in detoxification systems, but has often had insufficient toxin capacity. Powdered activated carbon, because of its large surface area, can provide greater binding capacity for bilirubin and other toxins than granular carbon commonly used in detoxifying columns. Methods of using powdered carbon in extracorporeal blood treatment devices are reviewed in the present paper, including liver dialysis and a new sorbent suspension reactor (SSR); and the abilities and limitations of the SSR and columns to process protein solutions are discussed. PMID- 16684217 TI - Fluidized bed adsorbent systems for extracorporeal liver support. AB - Acute liver failure based on acute-on-chronic liver failure (AoCLF) or on acute severe damage of the liver caused by different etiologies includes complex mechanisms resulting in severe disturbances of principle liver functions. In order to compensate the liver's function of detoxification as efficiently as possible, fluidized bed absorbent systems have been designed. In these systems, small particles with specific adsorption properties for toxins related to acute liver failure are applied. A special technology based on adsorbents in suspension has been developed under the guidance of our group and is prepared for clinical application during the coming year. This technology is called microspheres-based detoxification system (MDS) and is based on microadsorbents with a diameter of 1 10 microm which are recirculated in suspension. The safety of the MDS is guaranteed by the use of fluorescently labeled magnetic microparticles, which in case of a membrane-leakage are detected in the blood circuit by an optical system equipped with a magnetic trap. In vitro tests with two kinds of microadsorbents (a combination of a hydrophobic neutral resin and an anion exchange resin) showed excellent efficiency of the system with respect to adsorption capacity as well as to the kinetics of elimination of albumin-bound substances (e.g. unconjugated bilirubin or cholic acid) and of non-protein-bound substances (e.g. phenol). Moreover, using a plasma filter or the Albuflow filter as membrane filters in the blood circuit, the MDS technology offers the possibility to remove inflammatory mediators such as tumor necrosis factor-alpha (TNF) by additional use of specific adsorbents. PMID- 16684219 TI - Bioartificial liver: its pros and cons. AB - Both the large variety of liver functions for maintaining body homeostasis and the proven effectivity of whole liver transplantation in the therapy of acute liver failure (ALF), are important reasons to presume that cell-free liver support systems will not be able to adequately support the failing liver. Accordingly, bioartificial liver (BAL) systems have shown their efficacy in experimental ALF models in small and large animals, and have shown to be suitable and safe in phase 1 studies in humans with ALF. However, the optimal BAL system is still under development. Important issues are the source of the cellular component and the configuration of the BAL system with regard to cell attachment, mass transfer characteristics and oxygenation at site. The deficiency of all BAL systems to excrete bile effectively is another important topic for improvement. The great challenge for the future is to develop a well-functioning and safe human hepatic cell line which can replace the widely used porcine (xenogeneic) hepatocytes. Theoretically, a combination of a cell-free liver support system and a BAL system might be optimal for the treatment of ALF patients in the near future. PMID- 16684218 TI - Artificial liver support system in China: a review over the last 30 years. AB - Severe viral hepatitis with high mortality is the most common cause of liver failure in China. Treatment of severe viral hepatitis by hemoperfusion was initially adopted in the late 1970s and early 1980s. Following 10 years of development in China, a plasma exchange (PE)-centered artificial liver support system (ALSS), principally dependent on PE technology was developed. Based on the condition and symptoms of each patient, PE was given alone, or combined with hemodialysis, hemofiltration, hemodiafiltration, hemoperfusion, or plasma perfusion. In the late 1990s, training courses for ALSS were developed, and ALSS began to be carried out across China. Guidelines for artificial liver therapy were formulated and published by the Artificial Liver and Liver Failure Group of the Chinese Society of Infection. In recent years, new methods have been attempted, including small pore-size plasma separators, a molecular adsorbent based recirculating system (MARS), and a continuous albumin purification system (CAPS). According to a retrospective analysis published in 2004, ALSS therapy significantly (P < 0.001) improved the survival rate of patients with severe hepatitis compared with patients who received only medicines (43.4%, 157/362 vs. 15.4%, 55/358 in chronic patients and 78.9%, 30/38 vs. 11.9%, 5/42 in acute and subacute patients). Furthermore, ALSS has also proved valuable as a bridge to liver transplantation in the treatment of patients with end-stage severe hepatitis in China. More recently, ALSS has been used in the treatment of drug induced liver failure, acute fatty liver during pregnancy, and other difficult-to treat disorders in China. PMID- 16684220 TI - Cell-free artificial liver support: design of appropriate clinical studies. AB - The clinical use of cell-free liver support devices is dependent upon proof of safety and efficacy in clinical trials. The current published data to support their use is limited in both quantity and quality. In most studies, the methodology is such that the effects of these devices are difficult to establish with certainty. Bias might be introduced by the use of uncontrolled studies, and in randomized controlled trials limited size, poorly matched control groups or medical therapy or the use of clinically inappropriate endpoints might be important. Guidelines are now available to provide a framework for the design and execution of such trials, specifically to minimize the systematic errors that are frequently present and that might result in biased estimates of treatment effects. In the present review, the limitations of current studies are discussed and suggestions made as to the design and conduct of future clinical trials. PMID- 16684221 TI - rHuG-CSF increases the platelet-neutrophil complex formation and neutrophil adhesion molecule expression in volunteer granulocyte and stem cell apheresis donors. AB - Several reports have shown that granulocyte colony-stimulating factor (G-CSF) administration induces a transient, mild hypercoagulable state, which might predispose certain donors to thrombotic complications. In the present study, changes in the expression of neutrophil adhesion molecules (CD11b/CD18, CD62L) and platelet-neutrophil complex formation following rHuG-CSF administration were investigated in normal granulocyte and stem cell donors. For granulocyte apheresis (N = 10), rHuG-CSF (5 microg/kg) was given subcutaneously every 12 h three times and apheresis was carried out two hours after the last dose. For stem cell apheresis (N = 8), rHuG-CSF (10 microg/kg/day) was given subcutaneously for 5 days and apheresis was carried out when peripheral CD34+ cell counts exceeded 20 cell/microL. Expression of neutrophil adhesion molecules (CD11b/CD18, CD62L) and platelet-neutrophil complex formation following rHuG-CSF administration were investigated in donors by a flow cytometric method. A significant increase in neutrophil counts (P < 0.001), and decreases in platelet counts (P < 0.01) and hemoglobin levels (P < 0.01) occurred following G-CSF administration. The expression of CD11b/CD18 significantly increased (P < 0.001) over pretreatment values with G-CSF administration and returned to baseline 1 week after stopping the drug. In contrast, CD62L expression was decreased (P < 0.01) with G-CSF and returned to normal after cessation of the drug. rHuG-CSF caused more than a two fold increase (from 0.3 to 7.0 x 10(9)/L) in circulating platelet-neutrophil complexes (P < 0.01), which returned to normal after 1 week. Although clinical significance of these laboratory changes is not clear, the occurrence of neutrophil activation and increased platelet-neutrophil complex formation might predispose certain donors or patients to thrombotic complications following G-CSF administration. PMID- 16684222 TI - Immunoadsorption plasmapheresis using a phenylalanine column as an effective treatment for lupus nephritis. AB - Immunoadsorption plasmapheresis (IAPP) is effective for eliminating pathogenic molecules such as anti-DNA antibody (anti-DNA Ab) and immune complexes from the serum of patients with systemic autoimmune diseases. The purpose of this study was to assess patients with lupus nephritis (LN) treated by IAPP using a phenylalanine column and determine its efficacy with respect to conventional therapies. Six patients (M = 1, F = 5) with histologically proven LN associated with proteinuria and abnormal sedimentation on urinalysis were the subjects for this study. All were treated with oral corticosteroid (prednisolone 1 mg/kg/day) and IAPP (Immusorba PH - 350; 2 L of plasma twice weekly for 2 weeks). Serum anti DNA Ab and complement, urinary protein, and creatinine clearance were measured over 6 weeks (pretreatment, before and after each of 2 IAPP sessions, and 1 and 4 weeks after the second IAPP session). Clinical efficacy of IAPP was compared with conventional pharmacotherapy regimes by conducting a retrospective review of 23 LN patients treated at our hospital using corticosteroid pulse therapy (CSPT, N = 7, intravenous methylprednisolone 500 mg/day for 3 days), intravenous cyclophosphamide pulse therapy (IVCY, N = 7), or corticosteroid monotherapy (CSMT, N = 9, oral prednisone 1 mg/kg body weight daily, for 4 weeks). Immunosuppressants and anticoagulants were not used. With IAPP, mean urinary protein excretion decreased from 2.2 +/- 1.7 g/day pretreatment to 0.4 +/- 0.6 g/day post-treatment (P < 0.001). Mean serum anti-DNA Ab also decreased from 84.0 +/- 88.1 U/mL pretreatment to 5.8 +/- 5.5 U/mL post-treatment (P < 0.05). In combination with corticosteroid therapy, IAPP would appear to be an effective and safe treatment for LN. PMID- 16684223 TI - Prevalence of hepatitis E virus infection in regular hemodialysis patients. AB - The percentage of patients infected with blood-borne diseases, including hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, is high in patients undergoing hemodialysis regularly. Hepatitis E virus (HEV) is transmitted via the fecal-oral route, and blood-borne HEV infection has also been reported recently. On the basis of these findings, we investigated the actual status of HEV infection in regular hemodialysis patients. Out of 1077 patients undergoing hemodialysis at two key hospitals and three outpatient hemodialysis clinics, 300 were randomly selected as the subjects. Among these 300 hemodialysis patients, 19.0% were IgG-type anti-HEV antibody-positive. The percentage of HEV infected patients increased with patient age and it was particularly high in patients 40 years of age or older. The percent IgG-type anti-HEV antibody positivity was not significantly different (P = 0.14) between anti-HCV antibody positive patients (27.8%) and anti-HCV antibody-negative patients (17.8%). The percentage of HEV-infected patients among the hemodialysis patients was higher than that previously reported among patients with healthy kidneys. No correlation was observed between the percentage of HEV-infected patients and HCV infection incidence or a history of blood transfusion. The percent IgG-type anti-HEV antibody positivities were significantly different among the facilities. It was impossible to specify the route of infection, and the correlation between the incidence of infection and hemodialysis therapy was not clear. Because more routes of infection are possible for patients undergoing dialysis than for persons with normal kidney function, it seems necessary to analyze the significance of infection incidence, the route of infection and infection prevention measures. PMID- 16684224 TI - Is the volume of the parathyroid gland a predictor of Maxacalcitol response in advanced secondary hyperparathyroidism? AB - We evaluated the relationship between the volume of parathyroid glands estimated by ultrasonography (US) and response of 22-oxa calcitriol (Maxacalcitol, OCT) in patients with secondary hyperparathyroidism (2HPT) to evaluate whether the volume can be a predictor of the OCT response. Eleven institutes participated in this study. Ninety-four patients with advanced 2HPT were enrolled. The volume of the parathyroid glands were estimated by US before and 6 months after OCT treatment. The response of OCT treatment was classified into three groups (Group A: i-PTH < 300 pg/mL; Group B: 300 pg/mL < or = i-PTH < 500 pg/mL; Group C: i-PTH > or = 500 pg/mL). Forty-eight patients were in Group A, 28 patients in Group B, and 18 patients in Group C. The PTH levels at the beginning and 6 months were 458.3 199.1 pg/mL (P < 0.0001) in Group A, 524.6-403.2 pg/mL (P = 0.007) in Group B and 736.7-613.6 pg/mL (ns) in Group C, respectively. The volume of the largest gland in Group B was significantly larger than that in Group A (96.2 vs. 343.2 mm3: P < 0.001). Clinical factors affecting response of OCT was evaluated by logistic regression analysis and only the volume of the largest gland was a significant factor. In the patients whose volume was less than 300 mm3, the OCT response was significantly effective. We conclude that the glandular volume of the largest parathyroid gland estimated by US can be a useful factor to predict the OCT response in patients with moderate or severe renal HPT. PMID- 16684225 TI - Producing patterns in plants. PMID- 16684226 TI - Common symbiosis genes of Lotus japonicus are not required for intracellular accommodation of the rust fungus Uromyces loti. PMID- 16684227 TI - Unravelling phenotypic plasticity -- why should we bother? AB - The ability of a genotype to change its phenotype was once considered rather a nuisance -- making it difficult to define a genotype. This led to the idea that there was a problem called 'instability'. But quite early it was recognized that stability was under genetic control, and was a character like other attributes of an individual. From this realization came the idea that there were two sides to the character of 'instability', and that the ability to change could be important. This ability was thus given the title of 'plasticity'. Once recognized, it became clear from surveys of different species and populations that plasticity can (i) be a complex character, and (ii) be selected to fit species to the particular demands of different environments. For plants, which cannot meet variations in environment like animals by behavioural responses, phenotypic plasticity can be very important. Plants should therefore be valuable tools for unravelling the mechanisms of plasticity whilst also demonstrating its contribution to fitness experimentally. We ought also to be able to demonstrate that appropriate genetic variability is available through which complex responses can be built up by selection. Genes must exist not only to determine character means, but also to determine character response, which adds interesting complexity to our ideas about evolution. PMID- 16684228 TI - Mind the gap: the emerging synthesis of plant 'eco-devo'. PMID- 16684229 TI - Plant-microbe interactions in Yucatan: hurricanes didn't curb the whirlwind of discovery. PMID- 16684230 TI - Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. AB - Insects select their hosts, but trees cannot select which herbivores will feed upon them. Thus, as long-lived stationary organisms, conifers must resist the onslaught of varying and multiple attackers over their lifetime. Arguably, the greatest threats to conifers are herbivorous insects and their associated pathogens. Insects such as bark beetles, stem- and wood-boring insects, shoot feeding weevils, and foliage-feeding budworms and sawflies are among the most devastating pests of conifer forests. Conifer trees produce a great diversity of compounds, such as an enormous array of terpenoids and phenolics, that may impart resistance to a variety of herbivores and microorganisms. Insects have evolved to specialize in resistance to these chemicals -- choosing, feeding upon, and colonizing hosts they perceive to be best suited to reproduction. This review focuses on the plant-insect interactions mediated by conifer-produced terpenoids. To understand the role of terpenoids in conifer-insect interactions, we must understand how conifers produce the wide diversity of terpenoids, as well as understand how these specific compounds affect insect behaviour and physiology. This review examines what chemicals are produced, the genes and proteins involved in their biosynthesis, how they work, and how they are regulated. It also examines how insects and their associated pathogens interact with, elicit, and are affected by conifer-produced terpenoids. PMID- 16684231 TI - Seduced by the dark side: integrating molecular and ecological perspectives on the influence of light on plant defence against pests and pathogens. AB - Plants frequently suffer attack from herbivores and microbial pathogens, and have evolved a complex array of defence mechanisms to resist defoliation and disease. These include both preformed defences, ranging from structural features to stores of toxic secondary metabolites, and inducible defences, which are activated only after an attack is detected. It is well known that plant defences against pests and pathogens are commonly affected by environmental conditions, but the mechanisms by which responses to the biotic and abiotic environments interact are only poorly understood. In this review, we consider the impact of light on plant defence, in terms of both plant life histories and rapid scale molecular responses to biotic attack. We bring together evidence that illustrates that light not only modulates defence responses via its influence on biochemistry and plant development but, in some cases, is essential for the development of resistance. We suggest that the interaction between the light environment and plant defence is multifaceted, and extends across different temporal and biological scales. PMID- 16684232 TI - Genetic interactions in the control of flowering time and reproductive structure development in tomato (Solanum lycopersicum). AB - Different tomato (Solanum lycopersicum) mutants, affected in flowering time, reproductive structure or plant architecture, were crossed to produce double mutants in order to investigate gene interactions in flowering regulation in this autonomous species with a sympodial growth habit. The compound inflorescence: uniflora, uniflora: self pruning, uniflora: blind, and jointless: uniflora double mutants all produced solitary flowers like their uniflora parent, instead of inflorescences. All double mutants were late flowering. uniflora: blind and uniflora: self pruning had flowering times intermediate between those of their two parents. jointless: uniflora and compound inflorescence: uniflora flowered later than uniflora, the mutant with the most delayed flowering. All double mutants developed strong lateral shoots at node levels approximately corresponding to the level at which their parent cultivars initiated their first reproductive structure, which is a typical trait of uniflora. These results suggest that the UNIFLORA gene acts upstream of the other investigated genes in controlling flowering in tomato, and that floral transition of the primary shoot and floral transition of sympodial segments are regulated differently. PMID- 16684233 TI - How does the meristem of sunflower capitulum cope with tissue expansion and floret initiation? A quantitative analysis. AB - The coordination between floret initiation and tissue expansion has been studied and quantified in the apical meristem of sunflower (Helianthus annuus) plants grown under different light availability. A method was developed to quantify tissue expansion in the meristem during floret initiation from measurements of meristem area, number of florets and primordium size. Initially, floret initiation and tissue expansion occurred simultaneously at the meristem surface. The duration of this phase remained unchanged across environments, whereas the rate of tissue expansion varied greatly. Floret initiation rate depended on meristem initial size and tissue-expansion rate. Thereafter, floret initiation continued without tissue expansion in the meristem, resulting in a rapid decrease of meristem area. A set of equations was proposed to predict floret initiation rate and floret number as a function of the rates of tissue expansion in the meristem before and during floret initiation. This formalism demonstrated the role of tissue expansion in determining the final number of florets, and provided a framework to analyse the response of floret initiation to genotype and environment. PMID- 16684235 TI - Transcript profiling of a xylem vs phloem cDNA subtractive library identifies new genes expressed during xylogenesis in Eucalyptus. AB - Eucalyptus is one of the world's main sources of biomass. The genus includes species representing the principle hardwood trees used for pulp and paper. Here, we aimed to identify genes specifically expressed in differentiating secondary xylem compared with phloem. We constructed a xylem vs phloem subtractive library (Xp) that generated 263 unique sequences. By transcript profiling of xylem, phloem, vascular cambium and leaves using macroarrays, we classified the 263 unigenes into distinct tissue-specific groups. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the differential expression of representative expressed sequence tags (ESTs). A total of 87 unigenes were preferentially expressed in xylem. They were involved in functional categories known to play roles in xylogenesis, such as hormone signaling and metabolism, secondary cell wall thickening and proteolysis. Some of these genes, including unknown genes, may be considered xylem-specific and they are likely to control important functions in xylogenesis. These data shed light on the cellular functions of xylem cells and, importantly, provide us with a portfolio of Eucalyptus xylem genes that may be major players in the control of wood formation and quality. PMID- 16684236 TI - The heavy metal hyperaccumulator Thlaspi caerulescens expresses many species specific genes, as identified by comparative expressed sequence tag analysis. AB - Thlaspi caerulescens is a natural zinc (Zn), cadmium (Cd) and nickel (Ni) hyperaccumulator and an emerging plant model species to study heavy metal hyperaccumulation and tolerance. This paper describes the analysis of the first expressed sequence tag (EST) collection from T. caerulescens. This collection is a new resource to unravel the molecular basis of plant metal homeostasis, tolerance and hyperaccumulation. In total, 4289 ESTs were generated originating from Zn-exposed root and shoot tissues, leading to 3709 T. caerulescens assembled partial cDNA sequences (unigenes). In comparison to Arabidopsis or other publicly available plant sequences, a fraction of c. 8% of the T. caerulescens unigenes (TcUGs) had no significant similarity with any known DNA sequence and, so far, these sequences are T. caerulescens specific. Three per cent of the TcUGs correspond to Arabidopsis thaliana orthologues that, as yet, have not been found to be expressed. The T. caerulescens transcriptome generally relates very well to the A. thaliana transcriptome, although, compared with other closely related species, a relatively large number of T. caerulescens-specific transcripts were found. T. caerulescens also expresses a relatively large number of genes which are expressed at a very low level in A. thaliana. PMID- 16684237 TI - Root aeration in rice (Oryza sativa): evaluation of oxygen, carbon dioxide, and ethylene as possible regulators of root acclimatizations. AB - Adventitious roots of rice (Oryza sativa) acclimatize to root-zone O(2) deficiency by increasing porosity, and induction of a barrier to radial O(2) loss (ROL) in basal zones, to enhance longitudinal O(2) diffusion towards the root tip. Changes in root-zone gas composition that might induce these acclimatizations, namely low O(2), elevated ethylene, ethylene-low O(2) interactions, and high CO(2), were evaluated in hydroponic experiments. Neither low O(2) (0 or 0.028 mol m(-3) O(2)), ethylene (0.2 or 2.0 microl l(-1)), or combinations of these treatments, induced the barrier to ROL. This lack of induction of the barrier to ROL was despite a positive response of aerenchyma formation to low O(2) and elevated ethylene. Carbon dioxide at 10 kPa had no effect on root porosity, the barrier to ROL, or on growth. Our findings that ethylene does not induce the barrier to ROL in roots of rice, even though it can enhance aerenchyma formation, shows that these two acclimatizations for improved root aeration are differentially regulated. PMID- 16684234 TI - Transcriptome analysis of bud burst in sessile oak (Quercus petraea). AB - Expression patterns of hundreds of transcripts in apical buds were monitored during bud flushing in sessile oak (Quercus petraea), in order to identify genes differentially expressed between the quiescent and active stage of bud development. Different transcriptomic techniques combining the construction of suppression subtractive hybridization (SSH) libraries and the monitoring of gene expression using macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were performed to dissect bud burst, with a special emphasis on the onset of the process. We generated 801 expressed sequence tags (ESTs) derived from six developmental stages of bud burst. Macroarray experiment revealed a total of 233 unique transcripts exhibiting differential expression during the process, and a putative function was assigned to 65% of them. Cell rescue/defense , metabolism-, protein synthesis-, cell cycle- and transcription-related transcripts were among the most regulated genes. Macroarray and real-time RT-PCR showed that several genes exhibited contrasted expressions between quiescent and swelling buds, such as a putative homologue of the transcription factor DAG2 (Dof Affecting Germination 2), previously reported to be involved in the control of seed germination in Arabidopsis thaliana. These differentially expressed genes constitute relevant candidates for signaling pathway of bud burst in trees. PMID- 16684238 TI - Morpho-anatomical, physiological and biochemical adjustments in response to root zone salinity stress and high solar radiation in two Mediterranean evergreen shrubs, Myrtus communis and Pistacia lentiscus. AB - Salt- and light-induced changes in morpho-anatomical, physiological and biochemical traits were analysed in Myrtus communis and Pistacia lentiscus with a view to explaining their ecological distribution in the Mediterranean basin. In plants exposed to 20 or 100% solar radiation and supplied with 0 or 200 mm NaCl, measurements were conducted for ionic and water relations and photosynthetic performance, leaf morpho-anatomical and optical properties and tissue-specific accumulation of tannins and flavonoids. Net carbon gain and photosystem II (PSII) efficiency decreased less in P. lentiscus than in M. communis when exposed to salinity stress, the former having a superior ability to use Na(+) and Cl(-) for osmotic adjustment. Morpho-anatomical traits also allowed P. lentiscus to protect sensitive targets in the leaf from the combined action of salinity stress and high solar radiation to a greater degree than M. communis. Salt and light-induced increases in carbon allocated to polyphenols, particularly to flavonoids, were greater in M. communis than in P. lentiscus, and appeared to be related to leaf oxidative damage. Our data may conclusively explain the negligible distribution of M. communis in open Mediterranean areas suffering from salinity stress, and suggest a key antioxidant function of flavonoids in response to different stressful conditions. PMID- 16684239 TI - Performance of seedlings of Mediterranean woody species under experimental gradients of irradiance and water availability: trade-offs and evidence for niche differentiation. AB - The aim of the study was to assess the potential importance for Mediterranean plants of trade-offs in the response to irradiance and water availability at the regeneration stage. Survival and growth patterns across an experimentally imposed irradiance gradient (1, 6, 20 and 100% sunlight) were studied in seedlings of eight Mediterranean woody species, together with the impact of a simulated summer drought. We found evidence of some of the trade-offs previously reported for non Mediterranean plant communities, such as between survival in the shade and relative growth rate (RGR) at high light, but no evidence for others, such as between shade and drought tolerances. The impact of drought on survival and RGR was stronger in high light than in deep shade. The observed species-specific differences in performance provide a mechanistic basis for niche differentiation at the regeneration stage, contributing to possible explanations of species coexistence in Mediterranean ecosystems. PMID- 16684240 TI - Wood density and vessel traits as distinct correlates of ecological strategy in 51 California coast range angiosperms. AB - Wood density and vessel characteristics are functionally interrelated, yet they may have distinct ecological associations. In a comparative study of 51 angiosperm species ranging from chaparral shrubs to riparian trees, we examined relationships among wood density and vessel traits and their ecological correlates. Mean vessel lumen area and vessel density (number mm(-2)) varied widely (7- to 10-fold). In multivariate analyses, both vessel traits were negatively correlated with wood density, which varied more narrowly (< 2-fold). Vessel density and lumen area were inversely related across species, allowing a broad range of vessel traits within a narrow range of wood density. Phylogenetic independent contrasts indicated correlated inverse evolutionary change in vessel traits. Each trait had a distinct pattern of ecological correlation -- wood density was most strongly associated with soil water, and vessel traits showed contrasting relationships with plant height. Within a narrow range of wood density, there was significant variation in vessel traits. Given their particular ecological associations, the results suggest that wood density and vessel traits describe two distinct ecological axes. PMID- 16684241 TI - Interactions of drought and shade effects on seedlings of four Quercus species: physiological and structural leaf responses. AB - Here, we investigated the physiological and structural leaf responses of seedlings of two evergreen and two deciduous Quercus species, grown in a glasshouse and subjected to contrasted conditions of light (low, medium and high irradiance) and water (continuous watering vs 2-months drought). The impact of drought on photosynthetic rate was strongest in high irradiance, while the impact of shade on photosynthetic rate was strongest with high water supply, contradicting the hypothesis of allocation trade-off. Multivariate causal models were evaluated using d-sep method. The model that best fitted the dataset proposed that the variation in specific leaf area affects photosynthetic rate and leaf nitrogen concentration, and this trait determines stomatal conductance, which also affects photosynthetic rate. Shade conditions seemed to ameliorate, or at least not aggravate, the drought impact on oak seedlings, therefore, the drought response on leaf performance depended on the light environment. PMID- 16684242 TI - A test of the generality of leaf trait relationships on the Tibetan Plateau. AB - Leaf mass per area (LMA), nitrogen concentration (on mass and area bases, N(mass) and N(area), respectively), photosynthetic capacity (A(mass) and A(area)) and photosynthetic nitrogen use efficiency (PNUE) are key foliar traits, but few data are available from cold, high-altitude environments. Here, we systematically measured these leaf traits in 74 species at 49 research sites on the Tibetan Plateau to examine how these traits, measured near the extremes of plant tolerance, compare with global patterns. Overall, Tibetan species had higher leaf nitrogen concentrations and photosynthetic capacities compared with a global dataset, but they had a slightly lower A(mass) at a given N(mass). These leaf trait relationships were consistent with those reported from the global dataset, with slopes of the standardized major axes A(mass)-LMA, N(mass)-LMA and A(mass) N(mass) identical to those from the global dataset. Climate only weakly modulated leaf traits. Our data indicate that covarying sets of leaf traits are consistent across environments and biogeographic regions. Our results demonstrate functional convergence of leaf trait relationships in an extreme environment. PMID- 16684243 TI - Antecedent moisture and seasonal precipitation influence the response of canopy scale carbon and water exchange to rainfall pulses in a semi-arid grassland. AB - The influences of prior monsoon-season drought (PMSD) and the seasonal timing of episodic rainfall ('pulses') on carbon and water exchange in water-limited ecosystems are poorly quantified. *In the present study, we estimated net ecosystem exchange of CO(2) (NEE) and evapotranspiration (ET) before, and for 15 d following, experimental irrigation in a semi-arid grassland during June and August 2003. Rainout shelters near Tucson, Arizona, USA, were positioned on contrasting soils (clay and sand) and planted with native (Heteropogon contortus) or non-native invasive (Eragrostis lehmanniana) C4 bunchgrasses. Plots received increased ('wet') or decreased ('dry') monsoon-season (July-September) rainfall during 2002 and 2003. Following a June 2003 39-mm pulse, species treatments had similar NEE and ET dynamics including 15-d integrated NEE (NEE(pulse)). Contrary to predictions, PMSD increased net C uptake during June in plots of both species. Greater flux rates after an August 2003 39-mm pulse reflected biotic activity associated with the North American Monsoon. Furthermore, August NEE(pulse) and ecosystem pulse-use efficiency (PUE(e) = NEE(pulse)/ET(pulse)) was greatest in Heteropogon plots. PMSD and rainfall seasonal timing may interact with bunchgrass invasions to alter NEE and ET dynamics with consequences for PUE(e) in water limited ecosystems. PMID- 16684244 TI - Long-term change in the sensitivity of tree-ring growth to climate forcing in Larix decidua. AB - Tree rings are widely used long-term proxy data which, if combined with long-term instrumental climate records, can provide excellent information on global climate variability. This research aimed to determine whether interannual climate-growth responses in Alpine treeline forests are stationary over time. We used tree-ring width chronologies of Larix decidua (European larch) from 17 sites and monthly temperatures and precipitation data for the period 1800-1999. Climate-growth relationships were assessed with correlation and response functions, and their stationarity and consistency over time were measured using moving correlation. Tree-ring chronologies showed similar interannual variations over the last two centuries, suggesting that the same climatic factors synchronously limited growth at most sites. The most sensitive variables showed significant transient responses varying within the time period, indicating a possible deviation from the uniformitarian principle applied to dendroclimatology. If these findings are confirmed in future studies on other species and in other regions, we suggest that time-dependent variables should be taken into account to avoid overestimation of treeline advance, future forest carbon storage in temperature limited environments and inaccurate reconstruction of past climate variability. PMID- 16684245 TI - Species composition of an ectomycorrhizal fungal community along a local nutrient gradient in a boreal forest. AB - Soil abiotic factors are considered to be important in determining the distribution of ectomycorrhizal (ECM) fungal species; however, there are few field data to support this. Here, we relate ECM species distributions to changes in soil chemistry along a short (90-m), natural nutrient gradient. The ECM community was characterized, using morphological and molecular techniques, in soil samples collected at 10-m intervals. There were pronounced changes in ECM fungal community structure along the transect, with many taxa showing discrete distributions. Although there was a change of host from Pinus to Picea along the gradient, host-specific fungi did not account for the observed change in community structure. Ordination analyses showed that community structure was strongly correlated with soil characteristics, in particular extractable ammonium and base saturation. However, autocorrelation among soil parameters makes it difficult to isolate the effects of individual parameters. The distinctive changes in soil and vegetation along the transect used in this study provided an exceptional opportunity to examine the local-scale impact of natural spatial heterogeneity on an ECM fungal community. PMID- 16684246 TI - Evolution of unisexual flowers in grasses (Poaceae) and the putative sex determination gene, TASSELSEED2 (TS2). AB - Unisexuality has evolved repeatedly in flowering plants, but its genetic control is not understood in most cases. In maize (Zea mays), unisexual flower development is regulated by a short-chain dehydrogenase/reductase protein, TASSELSEED2 (TS2), but its role in other grass lineages is unknown. TS2 was cloned and sequenced from a broad range of grasses and compared to available sequences from other flowering plants using phylogenetic analysis and tests for selection. Gene expression was investigated using reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. TS2 orthologs appear to be restricted to monocots. The TS2 protein sequence was found to be generally under purifying selection in bisexual and unisexual lineages alike. Only one site, in unisexual herbaceous bamboos, is potentially under positive selection. TS2 was expressed broadly in all sampled tissues of unisexual and bisexual grasses, and was also expressed in rice flowers in floral organs that do not abort. TS2 may have a more general developmental role in most grasses than programmed cell death of the developing gynoecium, but has been co-opted to this role within a subset of Poaceae, probably as a result of alterations in the activity or regulation of other genes in the gynoecial pathway. PMID- 16684248 TI - Self-monitoring: individual differences in orientations to the social world. AB - In their articles in this special section of the Journal of Personality, the authors have focused their attention on the role of individual differences in self-monitoring for a variety of interpersonal phenomena. In so doing, the authors have provided an overview of the theoretical and empirical contributions of the psychology of self-monitoring to the domains of interest: close relationships, consumer behavior, behavior in the workplace, and social interaction. As each of the contributing authors to this special section suggests, much more theoretical and empirical work is in order if the impact of individual differences in self-monitoring for the phenomena reviewed here is to be fully appreciated. Moreover, the four domains of interest represented in this special section by no means exhaust the areas to which theorists and researchers have applied or can apply the psychology of individual differences in self monitoring. Given the large nomological network that currently exists involving the self-monitoring construct, it is anticipated that the breadth and depth of applications of the psychology of self-monitoring will only continue to expand as it has in the last 30 years since the appearance of the construct in the literature. PMID- 16684249 TI - Self-monitoring in social interaction: the centrality of self-affect. AB - In this review, we examine the role of self-monitoring in social interaction. We first note that the presumed ease with which self-monitors adapt to new social contexts is more apparent than real, being the self-conscious outcome of (1) high self-monitors' preference for clearly defined situations, (2) their use of scripts regarding typical situations, (3) their ability to formulate effective plans of action before social encounters, and (4) their ability to use other people's behavior as a guide. We then examine the strong motive of high self monitors to express and evoke high levels of positive affect in their interpersonal relationships. Two recent unstructured dyadic interaction studies lead us to argue that the primary concern of high self-monitors during social interaction is to regulate their own self-affect through effective impression management. In this sense, it really is the self that is closely monitored whenever self-monitoring processes influence social interaction. PMID- 16684250 TI - Self-monitoring at work: a motive-based perspective. AB - It is argued that the recent study of personality in industrial and organizational (I-O) psychology is characterized by two limitations: (a) almost complete reliance on the Big Five to the exclusion of other personality variables (most notably, self-monitoring) and (b) insufficient theoretical attention paid to the criteria in work-related personality research. In an attempt to overcome both of these limitations, we review theoretical and empirical evidence for the relevance of self-monitoring in organizational life, with particular attention paid to the criteria grounded in socioanalytic theory of getting along, getting ahead, and making sense. Extant research indicates that high self-monitors are particularly good at getting along (e.g., meeting others' social expectations) and getting ahead (e.g., job performance and leadership emergence), but the evidence is more mixed with regard to making sense. We conclude with a discussion of practical concerns in considering the use of self-monitoring for managerial selection and a research agenda for the future to further elaborate a theory of self-monitoring at work. PMID- 16684251 TI - Self-monitoring and consumer psychology. AB - Research on the relations between self-monitoring differences and two important areas of consumer behavior, reactions to specific advertising approaches and product evaluation strategies, is reviewed and evaluated. First, research on the responsiveness of high and low self-monitors to image-oriented and product quality-based advertising attempts is addressed. Although findings generally indicate that high self-monitors are more responsive to image-oriented ads and that low self-monitors are more responsive to quality-based appeals, these results have not been robust. Possible reasons for this inconsistency are explored. Second, research on the means by which high and low self-monitors evaluate consumer products is examined. Findings suggest that when judging product quality, low self-monitors tend to rely on product performance and high self-monitors more likely use the product's image-enhancing capabilities. Additionally, research indicates that self-image/product-image congruency is generally a more important concern for low self-monitors, but high and low self monitors' differential reliance on this congruency might be moderated by the situations in which the product is typically used. Additional possible delimiting conditions of these general findings are discussed. PMID- 16684252 TI - Self-monitoring and close relationships. AB - Three types of close relationships have received attention from theorists and researchers interested in self-monitoring: friendships, romantic relationships, and marriage. Our review of this literature was organized around three phases of relationships: initiation, maintenance, and dissolution. Across the three types of relationships, consistent differences between high self-monitors and low self monitors emerged concerning the structure of their social relationships (segmented vs. integrated), the basis for choosing friends and romantic partners (activity-based vs. person-based), and the orientation taken to romantic and marital partners (uncommitted vs. committed). Across all three types of relationships, however, little is known about the processes and consequences involved in the dissolution of close relationships for high self-monitors and low self-monitors. Relatively little is also known about the processes used by high self-monitors and low self-monitors to maintain their friendships and marriages. In addition to addressing these deficiencies in the literature, theorists and researchers interested in self-monitoring and close relationships need to develop sophisticated, causal models that can account for (a) interaction exchanges in the relationships, (b) dyadic as well as individual levels of analysis, and (c) temporal and situational changes in the course of close relationships. PMID- 16684253 TI - Cross-sectional and longitudinal tests of the Personality and Role Identity Structural Model (PRISM). AB - A conceptual hierarchy termed the Personality and Role Identity Structural Model, or PRISM, is offered as a framework for incorporating situational information into trait models. PRISM assumes that personality is structured hierarchically with general dispositions subsuming context-specific dispositions (role identities), which, in turn, subsume role-based thoughts, feelings, and behaviors. At Wave 1 (N = 149), we found that general traits account for commonalities across traits measured within role identities and that role identities mediate the relationship between general traits and role criteria. In a longitudinal follow-up of a subsample of the original participants (Wave 2; N = 62), we found that general traits and role-identity traits were more stable than role experiences over time. Also, changes in role experiences were related to changes in role-identity traits, and, in turn, changes in role-identity traits were related to changes in general traits. The potential of PRISM for use in understanding the development of personality traits is discussed. PMID- 16684254 TI - Subjective impact, meaning making, and current and recalled emotions for self defining memories. AB - Two studies examined the impact of self-defining events on individuals (i.e., subjective impact), meaning making with regard to these events, and how subjective impact may account for the pattern of current and recalled emotions for these self-defining memories (Singer & Moffitt, 1991-1992). In Study 1, participants recalled self-defining memories, indicating how much impact the recalled events have had on them and described meaning making for these events. Subjective impact was shown to be a good marker for meaning making. Participants in Study 2 each recalled five self-defining memories, reporting their current emotions about the events, the emotions they recalled feeling at the time, and the impact the events have had on them. As expected, for negative memories, people reported less negative emotion (e.g., sadness) and more positive emotion (e.g., pride) compared to how they recalled feeling at the time. For positive memories, people reported equally intense positive emotion (e.g., love) and less negative emotion (e.g., fear) compared to how they recalled feeling at the time. These patterns of current and recalled emotions were accounted for by impact ratings. PMID- 16684255 TI - Generativity and successful parenting: an analysis of young adult outcomes. AB - Generativity scores were assessed in parents and correlated with offspring outcomes. The offspring were participants in a longitudinal study spanning their first and senior years of college. Generativity of parents was positively related to offspring agreeableness and conscientiousness. Parental generativity was also related to offspring scores on future time orientation and positive affect. In addition, generative parents seemed to model their political interests to offspring, and that modeling was related to children's higher scores on generativity and greater interest in politics. Parental generativity was also related to offspring religiosity. Most of these relationships remained significant after controlling for offspring scores on generativity. Generativity of parents appears to be related to successful offspring outcomes. PMID- 16684256 TI - Individual differences in anger and sadness: in pursuit of active situational features and psychological processes. AB - In the cognitive-affective personality system (CAPS) theory of Mischel and Shoda (1995, 1998), personality is conceived as a system of cognitions and affects that mediates between active situational features and behavior. Two major tasks for this approach to personality are the search for active situational features and for mediating psychological processes within a behavioral domain of interest. We report two studies to address these tasks for the domain of anger and sadness. To design these studies and to analyze the obtained data, novel extensions of our previous developed Triple Typology Model (TTM; Vansteelandt & Van Mechelen, 1998) are proposed. These extensions allow the researcher to test hypotheses concerning potentially relevant active situational features in a systematic and confirmatory way and to examine psychological processes as they occur in concrete situations. PMID- 16684257 TI - Locus of control predicts appraisals and cardiovascular reactivity to a novel active coping task. AB - These two studies investigated the influence of dispositional locus of control (LOC) on subjective and physiological responses to a novel laboratory stressor task. Across two studies, 64 healthy undergraduate students, ages 18-22, completed Levenson's (LOC) scales for internal, powerful others, and chance prior to performing a video-game task. Participants rated pretask and posttask stressfulness and coping ability (i.e., measures of primary and secondary appraisal). Cardiovascular measures (heart period, HP; preejection period, PEP; respiratory sinus arrhythmia, RSA; cardiac output, CO; systolic blood pressure, SBP; diastolic blood pressure, DBP & total peripheral resistance, TPR) were recorded during 4-minute baseline and 4-minute stressor task periods. The internal LOC factor predicted pretask reports of coping ability as well as posttask reports of stressfulness. In contrast, the powerful-others LOC factor predicted cardiac changes (HP, PEP, and RSA) during the task but not cardiac output or any other vascular change measure. These results underscore the importance of using the three subscales of the Levenson LOC to assess relationships between dispositional LOC and the response to stressors because self-reported appraisals of a task are predicted by a different component of dispositional LOC than are task-related cardiovascular functions. PMID- 16684259 TI - Natural history of gastric cancer after Helicobacter pylori eradication in Japan: after endoscopic resection, after treatment of the general population, and naturally. PMID- 16684260 TI - VacA-associated inhibition of T-cell function: reviewed and reconsidered. AB - Chronic Helicobacter pylori infection is characterized by dense infiltration of the mucosa with neutrophilic granulocytes, lymphocytes, and monocytes/macrophages. Among these different cell types, T-lymphocytes are the most intriguing and crucial cells for the elimination of the bacteria. Previous studies have elucidated possible mechanisms on how bacteria could interfere with the human immune response and claimed that especially the secreted vacuolating toxin VacA may be responsible for the chronic persistence of the bacteria. Some of these results have to be interpreted with caution and may just describe in vitro phenomena; others may reveal promising facts. PMID- 16684261 TI - Design and planned analyses of an ongoing randomized trial assessing the preventive effect of Helicobacter pylori eradication on occurrence of new gastric carcinomas after endoscopic resection. AB - BACKGROUND: A causal relationship between Helicobacter pylori infection and gastric cancer has been established. A nonrandomized study has shown eradication of H. pylori after endoscopic resection (ER) of early gastric cancer inhibits development of new carcinomas. SUBJECTS AND METHODS: Eligible subjects are patients with H. pylori infection who are newly diagnosed with early gastric cancer and plan to have ER or who are in the post-resection follow-up phase after ER time of enrollment. Patients are randomly allocated to the eradication or the control arms (no eradication and standard of care). Patients will be evaluated by endoscopy at 0.5, 1, 2, and 3 years after randomization. Diagnosis of a new carcinoma at another site of the stomach is defined as primary endpoint, and recurrence of tumors at the resection site as a secondary endpoint. In addition to intention-to-treat and per-protocol analyses using proportional hazards models, time to recurrence will be compared between treatment and control using multiple logistic regression analyses. In the latter two situations, the models will be adjusted for the factors exerting significant influences on the results. RESULTS: Five hundred and forty-two subjects have been enrolled into the study and are being followed-up. CONCLUSIONS: This study will have the statistical power to demonstrate whether H. pylori eradication therapy exerts any clinically relevant inhibitory effects on occurrence or recurrence of gastric cancer. In addition, it will be able to test the hypothesis that H. pylori infection is a promoter in gastric carcinogenesis. PMID- 16684262 TI - Efficacy of metronidazole as second-line drug for the treatment of Helicobacter pylori Infection in the Japanese population: a multicenter study in the Tokyo Metropolitan Area. AB - BACKGROUND: With the increase in the frequency of clarithromycin-resistant Helicobacter pylori (H. pylori), there is rising concern about the decline of the eradication rate of this infection following treatment. The Tokyo Hp Study Group examined the eradication rate in response to a second-line regimen consisting of proton pump inhibitor (PPI), amoxicillin, and metronidazole by conducting a multicenter study in the Tokyo Metropolitan Area. MATERIALS AND METHODS: Two hundred and twenty-eight patients with H. pylori infection, in whom the first line therapy with a PPI, amoxicillin, and clarithromycin administered for 1 week had failed to eradicate the infection, were enrolled in this study. These cases were randomly assigned to one of the two second-line regimens containing metronidazole (PPI/AM500 or PPI/AM750) administered for 1 week. 13C-urea breath test was performed as a diagnostic method test for H. pylori infection not earlier than 8 weeks after the second-line therapy. RESULTS: Intention-to-treat (ITT) and per-protocol (PP) analyses revealed an eradication rate of 87.6 and 90.6%, respectively, following PPI/AM500 treatment, and 86.9 and 88.6%, respectively, following PPI/AM750 treatment. Neither analysis revealed any significant difference in the eradication rate between PPI/AM500 and PPI/AM750 (p = .876 and .621, respectively). According to ITT and PP analyses, the eradication rates following treatment with PPI/AM500 were 85.2 and 88.5% with the use of lansoprazole, 62.5 and 62.5% with the use of omeprazole, and 93.2 and 96.5% with the use of rabeprazole, respectively. There was a significant difference in the eradication rates between PPI (omeprazole)/AM500 and PPI (rabeprazole)/AM500. In the case of PPI/AM750, the corresponding eradication rates were 84.8 and 87.0% with the use of lansoprazole, 92.9 and 92.9% with the use of omeprazole, and 92.9 and 92.9% with the use of rabeprazole, respectively. There were no significant differences in the eradication rates obtained with the use of the three PPIs. CONCLUSIONS: Both PPI/AM500 and PPI/AM750 administered for 1 week appeared to be highly effective second-line regimens for the treatment of H. pylori infection in Japanese patients. From the viewpoint of adverse events, PPI/AM500 appeared to be safe compared with PPI/AM750. PMID- 16684263 TI - Asymptomatic Helicobacter pylori infection and iron deficiency are not associated with decreased growth among Alaska Native children aged 7-11 years. AB - INTRODUCTION: Alaska Native children have high Helicobacter pylori infection and iron deficiency prevalences, and their average height-for-age is lower than US reference populations. During a clinical trial to determine the impact of H. pylori treatment on iron deficiency, we evaluated the effects of H. pylori infection and treatment on growth. MATERIALS AND METHODS: We measured height and weight for children aged 7-11 years in western Alaska using village-based measuring devices. H. pylori infection was determined by urea breath test and iron deficiency using serum ferritin. Children with H. pylori infection and iron deficiency entered the treatment phase and received iron alone or iron plus triple therapy for H. pylori. Follow-up evaluations occurred at 2, 8, and 14 months. We evaluated the association between baseline H. pylori infection and growth; among children in the treatment phase, we also assessed the effect of H. pylori resolution on growth. RESULTS: At baseline, 566 (87.1%) of 650 children were infected with H. pylori. Neither height and weight, nor body mass index differed by H. pylori infection status. Of 189 children in the treatment phase, 20 (10.6%) were uninfected at all three follow-up periods, and 54 (28.6%) were uninfected for one or two periods. Compared with continuously infected children, children in these two groups had little evidence of improvements in any of the measured growth outcomes. CONCLUSIONS: H. pylori infection is not related to growth among Alaska Native children aged 7-11 years. Growth deficiency should not be considered an indication for H. pylori therapy. PMID- 16684264 TI - High rate of Helicobacter pylori reinfection in children and adolescents. AB - AIMS: Primary Helicobacter pylori infection occurs predominantly in childhood. The aims of this study were to establish the rate of H. pylori reinfection after successful eradication in children and adolescents and to determine the risk factors associated with reinfection. PATIENTS AND METHODS: This retrospective study involved 45 children (20 girls, 25 boys) who met the following criteria: eradication of H. pylori confirmed at least 4 weeks after the completion of therapy, and the search for reinfection at least one year after control of eradication of H. pylori. Demographic data, socioeconomic status and living conditions were recorded. RESULTS: Forty-five children aged 1.2-17.6 years (median, 10.9 years) at the time of H. pylori treatment were reviewed 1 to 9 years after H. pylori eradication. Eight children (18%) had been reinfected (5.4% to 6% per patient-year). Six of 25 (24%) children older than 10 years at the time of diagnosis became reinfected. None of the studied risk factors was associated with reinfection. However, having a sibling younger than 5 years was found in four of seven (57%) reinfected children versus five of 24 (21%) nonreinfected children (p = .08). CONCLUSION: Children become reinfected more frequently than adults. Adolescents become reinfected, whereas acquisition of primary H. pylori infection occurs predominantly in early childhood. Close contact with young children, especially siblings, younger than 5 years could be a more important risk factor than the age of the patient at the time of treatment for the high rate of reinfection in childhood. PMID- 16684265 TI - Cardiovascular autonomic response to food ingestion in patients with gastritis: a comparison between Helicobacter pylori-positive and -negative patients. AB - BACKGROUND: Feeding evokes a cardiovascular response associated to an increased sympathetic drive. The role of the parasympathetic component in this regard is less clear. Improvement of postprandial vasovagal complaints after Helicobacter pylori eradication in three cases led us to assess autonomic response to feeding in H. pylori-positive patients in search for an exacerbated parasympathetic response. MATERIAL AND METHODS: Patients with mild or moderate chronic histologic gastritis were studied. Subjects with the same diagnosis but testing negative for H. pylori were used as controls. Noninvasive cardiovascular tests were applied before and after feeding. RESULTS: On sympathetic tests, standing postprandial blood pressure was lower than preprandially in 5/12 H. pylori-positive patients and in 0/9 controls, p = .045. Resting postprandial BP on handgrip test was significantly lower than preprandially only in H. pylori-positive patients (71 +/ 8 versus 76 +/- 6 mmHg, p = .0068). Regarding parasympathetic tests, the 4-s unloaded exercise revealed greater initial heart rate response to unloaded cycling after feeding than preprandially again only in H. pylori-positive patients (1.40 +/- 0.20 versus 1.33 +/- 0.17, p = .0195). On tests influenced by both branches of the autonomic system, a difference was seen in the chronotropic response to handgrip. Postprandial heart rate on effort of H. pylori-positive patients was not higher than preprandially in contrast to controls [intervals between 2 consecutive R waves on electrocardiogram (R-R intervals) of 666 +/- 39 versus 685 +/- 62 ms, p = .0195], suggesting blunted sympathetic activation in the former. CONCLUSION: Supporting the observations that motivate the study, our findings indicate blunted sympathetic reactivity and exacerbated vagal response to feeding in H. pylori-positive patients. PMID- 16684266 TI - Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori. AB - BACKGROUND: Amoxicillin-based therapies are highly effective for the treatment of Helicobacter pylori infections, but the efficacy may decrease as the incidence of amoxicillin resistance is increasing. So far, the molecular mechanism underlying stable amoxicillin resistance has only been identified for a few naturally occurring amoxicillin-resistant (Amx) H. pylori isolates, and is mediated by mutations in penicillin-binding protein 1A (PBP1A). In this study the molecular mechanism underlying amoxicillin resistance of seven additional Amx H. pylori isolates has been established. METHODS: H. pylori strain 26695 (minimal inhibitory concentration (MIC) 0.125 mg/l) was naturally transformed with total DNA and pbp1A polymerase chain reaction (PCR) products from the seven Amx H. pylori isolates, and the MIC of amoxicillin and pbp1A gene sequence of the obtained Amx transformants were determined. RESULTS: Replacement of the wild-type pbp1A gene of H. pylori reference strain 26695 by the pbp1A gene of the Amx H. pylori isolates resulted in an increased MIC (0.5-1.0 mg/l). Sequence analysis of the smallest PBP1A fragments able to transfer the resistance indicated that several amino acid substitutions in or adjacent to the second (SKN402-404) and third (KTG555-557) conserved penicillin-binding protein motifs (PBP-motifs) mediate amoxicillin resistance in H. pylori. This was confirmed by site-directed mutagenesis using oligonucleotides that contained defined mutations in or adjacent to these PBP-motifs. CONCLUSION: In naturally occurring Amx H. pylori isolates, amoxicillin resistance is mediated by various mutational changes located in or adjacent to the second and third PBP-motifs of the PBP1A. Although we cannot exclude the role of the other genes in amoxicillin resistance, it is likely that multiple mutational changes in the PBP1A gene are the predominant cause of amoxicillin resistance in H. pylori. The findings of this study currently preclude the rapid detection of amoxicillin resistance in H. pylori by molecular tests. PMID- 16684267 TI - Assessing the therapeutic use of Lafoensia pacari St. Hil. extract (mangava brava) in the eradication of Helicobacter pylori: double-blind randomized clinical trial. AB - BACKGROUND: The eradication of Helicobacter pylori is easily achieved by combining antisecretory agents and antibiotics; however, the cost of these associations is very high for the population of Third World countries, where the prevalence of the infection is even higher and leads to markedly reduced treatment effectiveness. We tested a plant (Lafoensia pacari) that is used in the central region of Brazil. According to previous studies, this plant has high concentrations of ellagic acid, which presents gastric antisecretory and antibacterial actions. MATERIAL AND METHODS: One hundred dyspeptic, urease positive patients were randomized to receive 500 mg of methanolic extract of L. pacari (n = 55) or placebo (n = 45), for 14 days, in a double-blind clinical trial. The main variables assessed were the eradication of H. pylori 8 weeks after the intervention and complete symptom relief at the end of the treatment. RESULTS: The examinations (urease and histology) showed persistence of H. pylori in 100% of participants. Complete symptom relief was experienced by 42.5% of patients (95% CI: 29.4-55.8) in the intervention group and by 21% (95% CI: 8.8 33.1) in the control group, p = .020. The side-effects were minimal and similar in both groups. CONCLUSIONS: The extract of L. pacari as a single agent was not effective to eradicate H. pylori. However, it was well tolerated and many participants reported relief of symptoms. Future studies may test the agent using larger doses and longer periods, in monotherapy or in combination with antibiotics. PMID- 16684268 TI - Interleukin-6 induction by Helicobacter pylori in human macrophages is dependent on phagocytosis. AB - BACKGROUND: The colonization of the gastric mucosa with Helicobacter pylori is accompanied by elevated levels of proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and IL-8. The aim of our study was to determine the mechanisms of IL-6 stimulation in phagocytes upon H. pylori infection. MATERIALS AND METHODS: We investigated the secretion of IL-6 by different professional phagocytes from murine and human origin, including granulocyte- and monocyte-like cells and macrophages derived from human peripheral blood monocytes (PBMCs). The influence of viability, phagocytosis, and the impact of different subcellular fractions of H. pylori bacteria were evaluated. RESULTS: IL-6 levels induced by H. pylori were low in cell lines derived from murine and human monocytes and in human granulocyte-like cells. By contrast, macrophages derived from human PBMCs were highly responsive to both H. pylori and Escherichia coli. IL-6 induction was blocked by inhibition of actin-dependent processes prior to infection with H. pylori, but not with E. coli or E. coli lipopolysaccharide (LPS). Using cell fractionation, the most activity was found in the H. pylori membrane. H. pylori LPS exhibited a 10(3)- to 10(4)-fold lower biologic activity than E. coli LPS, suggesting a minor role for toll-like receptor 4 (TLR4)-mediated signalling from the exterior. CONCLUSIONS: From these data, we conclude that macrophages may be a major source of IL-6 in the gastric mucosa upon H. pylori infection. The IL-6 induction by H. pylori in these cells is a multifactorial process, which requires the uptake and presumably degradation of H. pylori bacteria. PMID- 16684269 TI - Evolution of the management of Helicobacter pylori infection in general practice. PMID- 16684270 TI - Helicobacter pylori antimicrobial resistance and risk factors in Denmark 1998 2004: no need for concern? PMID- 16684274 TI - Calciphylaxis--a topical overview. AB - 'Calciphylaxis', a calcification syndrome associated with ischaemic cutaneous necrosis, is acquired naturally in humans in disease states. It is a life and limb-threatening complication, usually observed in patients with renal disease and secondary hyperparathyroidism, but known to occur in the absence of renal or parathyroid disease. The reported mortality rate, which ranges from 60-80%, relates to wound infection, sepsis and organ failure. It is a small-vessel vasculopathy, which is estimated to occur in about 4% of haemodialysis patients. Clinically, violaceous, reticulate areas of cutaneous necrosis and eschar may be evident, particularly in the extremities. In addition to the clinical picture, a raised calcium phosphorous product, an elevated parathyroid hormone level, radiographic evidence of vessel and soft-tissue calcification and the finding of mural calcification affecting small arteries and arterioles on histopathology help to confirm the diagnosis of this entity which generally has a poor prognosis. A high index of suspicion and an active multidisciplinary management approach, with rigorous attention to wound care and prevention of sepsis, are vital in the management of these patients. In this overview, we discuss the pathophysiology, clinical features and associations, risk factors, diagnosis and management issues relating to calciphylaxis. PMID- 16684275 TI - Atopic eczema: what's new? AB - Atopic eczema (AE) is a chronic inflammatory skin disease characterized by recurrent intense pruritus and a typical age-related distribution of skin lesions. Several new aspects with regard to the pathogenetic background as well as strategies for prevention, diagnosis and treatment of AE have emerged. There are ongoing studies on genetic susceptibility loci, as well as environmental and nutritional factors associated with an increase or a decrease of AE lesions. The atopy patch test is now available for identification of allergens in aeroallergen triggered AE. New topical therapies, such as the calcineurin inhibitors, have broadened the therapeutic armamentarium substantially. In order to increase knowledge and coping strategies, patient education programs have been launched. Learning objective Upon completing this paper, the reader should be aware of new developments in AE, especially on nomenclature, prevention strategies, diagnostic tests, as well as therapeutic options. PMID- 16684276 TI - Horizontal advancement flap for lateral dorsal nasal defects. AB - BACKGROUND: Repairing dorsal nasal defects is a frequent challenge for dermatologic surgeons, mainly due to the high frequency of basal cell carcinomas on this site. Obvious scars, mismatched skin and distortion of the nasal contour are the surgical hazards that must be avoided in these cases. AIM: Our aim was to perform surgery involving a simple flap in order to repair medium to large defects on the dorsal side of the nose. METHODS: The dorsal horizontal advancement flap was studied in 12 patients, in order to evaluate the benefits and limits of this surgical procedure. RESULTS: The resulting scars on most of our patients were well-camouflaged among their natural skin lines, and there was neither distortion of the alar contour nor the nostril. CONCLUSIONS: This flap is easy to perform and, in selected cases, provides an outstanding alternative to second-intention healing, full-thickness skin grafts, transposition, rotation and pedicle flaps. PMID- 16684277 TI - Comparison of various insulin sensitivity indices in psoriatic patients and their relationship with type of psoriasis. AB - AIM: We aimed to identify insulin resistance and its possible association with types, duration and severity of psoriasis, and to evaluate various simple insulin sensitivity indices and beta-cell function in psoriasis. METHODS: A cross sectional study was performed in 110 non-obese adults (18-50 years old): 70 with psoriasis (53 type I, 17 type II psoriasis) and 40 healthy individuals. Blood glucose, insulin and C-peptide levels were measured. Oral glucose tolerance test (OGTT); insulin sensitivity and beta-cell function indices derived from a single sample and OGTT were determined and compared in three groups. RESULTS: Total, type I and type II psoriatics had IGT rates of 18.6%, 13.2% and 40%, respectively. In the control group IGT was only 2.5%. Homeostasis Model Assessment (HOMA) beta cell index, fasting insulin, Raynaud index, HOMA-IR and FIRI results were higher in total, type I and type II psoriatics than in controls (P < 0.05, for all). Fasting Belfiore index, QUICKY index, ISI HOMA and FIRI(-1) results were lower in total, type I and type II psoriatics than in controls (P < 0.05, for all), and type I psoriatics had higher levels of these indices than type II psoriatics (P < 0.05, for all). CONCLUSION: Our study showed that psoriatic patients were more insulin resistant than healthy subjects and type II psoriatics were more susceptible than type I psoriatics to develop IGT. We suggest that beta-cell function and insulin sensitivity indices are useful methods for measuring insulin resistance in psoriatics. We propose that OGTT should be applied especially in type II psoriatics because of increased rate of IGT in this group. PMID- 16684278 TI - A 308-nm monochromatic excimer light in the treatment of palmoplantar psoriasis. AB - BACKGROUND: Various reports have shown the efficacy of narrow-band UVB (311-313 nm) and excimer laser (308 nm) in the treatment of psoriasis. OBJECTIVE: To prove the efficacy of light produced by xenon-chloride excimer at 308 nm (monochromatic excimer light, MEL) in the treatment of palmoplantar psoriasis (PP). METHODS: Fifty-four patients (29 male and 25 female) affected by PP were treated with MEL every 7-14 days. A mean number of 10 sessions was performed with an increase of the dose depending on patient's skin type and response. RESULTS: All 54 patients completed the treatment. After 4 months of MEL we observed a complete remission in 31 patients, a partial remission in 13 patients, and a moderate improvement in 10 patients. CONCLUSIONS: These results suggest that MEL can be considered as a valid therapeutic option for treatment of selected forms of PP. PMID- 16684279 TI - The effects of calcipotriol and methylprednisolone aseponate on bcl-2, p53 and ki 67 expression in psoriasis. AB - OBJECTIVE: The decrease of physiological apoptosis in the psoriatic lesions is thought to be involved in the pathogenesis of psoriasis, and induction of apoptosis was shown to contribute to the regression of psoriatic hyperplasia. In the present study, we compared the effects of calcipotriol and methylprednisolone aseponate (MPA) treatments on bcl-2, p53 and ki-67 expressions in psoriatic patients in order to define a relationship between regulation of apoptosis and healing process in psoriasis. METHODS: Thirty psoriatic patients with stable and moderate chronic plaque psoriasis applied either calcipotriol or MPA ointment for 6 weeks twice daily. Evaluation of bcl-2, p53 and ki-67 positivity was performed at baseline and was repeated at sixth week for each therapy. RESULTS: The mean percentage of positive keratinocytes was 8.63 +/- 7.15% for p53, 20.66 +/- 14.45% for ki-67, and 3.74 +/- 2.83% for bcl-2 in psoriatic skin at baseline. Normal skin values were 3.27 +/- 3.21% for p53, 4.93 +/- 4.77% for ki-67, and 1.80 +/- 0.41% for bcl-2. The psoriatic skin showed higher ki-67 (P < 0.05) and bcl-2 (P < 0.05) expression rates when compared to normal skin. The p53 positivity observed in psoriatic skin and normal skin was not significantly different (P > 0.05). Following calcipotriol and MPA treatments, there was a significant reduction in p53 and ki-67 positivity accompanied by an increase in bcl-2 positivity (P < 0.05 each). No significant differences were found at sixth week between calcipotriol and MPA groups with respect to p53, ki-67 and bcl-2 positivity (P > 0.05). The post-treatment psoriatic skin showed lower expression of p53, higher expressions of ki-67 and bcl-2 when compared to normal skin (P < 0.05 each). CONCLUSION: The results of this study provide evidence that both calcipotriol and MPA decrease the p53 and ki-67 expression and increase bcl-2 expression. However, it should further be elucidated if these changes were the common behaviour of psoriatic keratinocytes to any antipsoriatic medication. PMID- 16684280 TI - Efficacy and functionality of silver-coated textiles in patients with atopic eczema. AB - BACKGROUND: Microbial skin colonization with Staphylococcus aureus is known to play an important role in atopic eczema (AE). Recently, an antibacterial effect of silver-coated textiles on S. aureus colonization has been demonstrated. OBJECTIVES: To investigate clinical efficacy and functionality of silver-coated textiles in AE, a multicentre, double-blind, placebo-controlled trial was conducted. PATIENTS/METHODS: From November 2001 to August 2002, 68 consecutive outpatients clinically diagnosed with generalized AE were included in the study. Inclusion criteria were the clinical diagnosis of AE with a moderate severity as measured by the scoring of atopic dermatitis (SCORAD) index with at least 20. Patients were instructed to wear either silver-coated (verum, 35 patients + 2 dropouts) or cotton garments (placebo, 22 patients + 9 dropouts) directly on the skin for 2 weeks. Only basic skin care and ongoing therapy with topical steroids or oral antihistamines was permitted. Clinical severity was assessed using the 'SCORAD' before, during and at the end of study. Quality of life (QOL), wearing comfort (WC) and functionality (FU) of study clothes were measured in parallel. Patients documented their subjective and objective symptoms daily. RESULTS: In the verum group, eczema improved significantly after 1 week with further enhancement until the end of study (P = 0.03 and P < 0.001). Silver-coated textiles were comparable to cotton in WC and FU. Pruritus and self-assigned skin condition improved significantly more than with placebo (P < 0.001 and P = 0.003). CONCLUSIONS: In conclusion, silver-coated textiles are able to improve objective and subjective symptoms of AE significantly within 2 weeks, showing a good wearing comfort and functionality comparable to cotton. PMID- 16684281 TI - UVB therapy of pityriasis lichenoides--our experience with 29 patients. AB - BACKGROUND: Pityriasis lichenoides comprises a clinical and pathological spectrum of disorders. So far no highly effective treatment has been reported. Previous small studies have suggested that ultraviolet B (UVB) is a good alternative. METHODS: This is a retrospective analysis of 29 pityriasis lichenoides patients treated in our institution with broad- or narrow-band UVB during the period 1996 2002. Twenty-one of these patients had one or more previous unsuccessful treatments. RESULTS: Complete response was achieved in 93.1% in both treatment groups, with 73% of them still relapse free after a mean follow-up of 58 and 38 months in broad- and narrow-band UVB treatment groups, respectively. Mild side effects were observed in about one-third of the patients. CONCLUSION: We believe both forms of UVB are a good option for pityriasis lichenoides and should be considered as the first line in generalized cases interested in treatment. PMID- 16684282 TI - The role of IVIg treatment in severe pemphigus vulgaris. AB - BACKGROUND: High-dose intravenous immunoglobulin (IVIg) has become a part of the treatment armentarium in pemphigus vulgaris (PV). Some consider IVIg as an adjuvant steroid sparing agent in PV, while others as disease modifying that can be used as monotherapy. METHODS: We report our experience with a series of 12 PV patients with severe disease treated with IVIg as an adjuvant therapy. RESULTS: Ten of 12 patients (83%) showed response to six cycles of IVIg, six (50%) having complete remission and four (33%) having a partial response. This response rate is concordant with previous reports. The therapy was well tolerated. In all 12 patients, treatment with IVIg allowed a gradual reduction of prednisone dose compared with baseline levels. CONCLUSION: IVIg treatment was beneficial as a steroid sparing agent in our series of patients with severe PV. PMID- 16684283 TI - Combination of narrow band UVB and topical calcipotriol for the treatment of vitiligo. AB - BACKGROUND: Narrow band ultraviolet B (NB-UVB) phototherapy has been used successfully for the treatment of vitiligo. Recently, topical calcipotriol has also been claimed to be effective, either as monotherapy or as a part of combination therapies. OBJECTIVE: The aim of the present study was to compare the clinical efficacy of NB-UVB and NB-UVB plus topical calcipotriol in the treatment of vitiligo. METHODS: NB-UVB treatment was given to 24 patients with generalized vitiligo three times weekly. Topical calcipotriol cream was only applied to the lesions located on the right side of the body. Treatment was continued for 6 months. Treatment efficacy was evaluated by determining the average response rates of the lesions at 3-month intervals. RESULTS: The average response rates of patients receiving combination of NB-UVB plus calcipotriol and NB-UVB alone were 51 +/- 19.6% and 39 +/- 18.9%, respectively. The median cumulative UVB dose and number of UVB exposures for initial repigmentation were 6345 mj/cm(2) (range; 2930-30980) and 18 (range; 12-67) for the combination therapy, and 8867.5 mj/cm(2) (range; 2500-30980) and 24 (range; 15-67) for the narrow band UVB therapy, respectively. CONCLUSIONS: These findings indicate that concurrent topical calcipotriol potentates the efficacy of NB-UVB in the treatment of vitiligo. This combination not only provides earlier pigmentation with lower total UVB dosage and less adverse UVB effects, but also reduces the duration and cost of treatment as well. PMID- 16684284 TI - The efficacy of excimer laser (308 nm) for vitiligo at different body sites. AB - BACKGROUND: The treatment with XeCl-excimer laser generated 308-nm UVB radiation has shown promising results in patients with vitiligo. OBJECTIVE: In this controlled, prospective trial we studied the primary efficacy (start and grade of repigmentation) and patient's satisfaction of XeCl-excimer laser for treatment of vitiligo patches at different body sites and re-evaluated the achieved repigmentation 12 months after the end of therapy. METHODS: Twenty-five patients with generalized or localized vitiligo with a total of 85 lesions at different body sites were enrolled in this study. Vitiligo patches were treated with 308-nm XeCl-excimer laser 3 times a week for 6 to 10 weeks. The overall repigmentation grade of each treated lesion was evaluated once a week on a 5 point scale rating from 0 (no repigmentation), 1 (1-5%), 2 (6-25%), 3 (26-50%), 4 (51-75%), to 5 (76 100%). RESULTS: Twenty-four patients completed the study. Within 6 to 10 weeks of treatment 67% of the patients (16/24) developed follicular repigmentation of at least one of their vitiligo lesions. Lesion repigmentation started after a mean of 13 treatments in lesions located on the face, trunk, arm, and/or leg (high responder location), and after a mean of 22 treatments in lesions located on the elbow, wrist, dorsum of the hand, knee, and/or dorsum of the foot (low-responder location). Untreated control lesions and lesions located on the fingers did not achieve any repigmentation. After 10 weeks of treatment repigmentation of more than 75% was found in 25% (7/28) of lesions of the high-responder location group versus 2% (1/43) of lesions of the low-responder location group. In most cases, laser-induced repigmentation was persistent, as determined 12 months after the end of treatment. CONCLUSIONS: 308-nm excimer laser is an effective modality for the treatment of vitiligo. However, similar to other non-surgical treatment modalities, the therapeutic effect is mainly dependent on the location of vitiligo lesions. PMID- 16684285 TI - UVB in the management of early stage mycosis fungoides. AB - BACKGROUND: Several options for treatment of early mycosis fungoides (MF) offer similar success rates. Previous small studies have shown UVB to be at least as effective as PUVA. OBJECTIVE: To summarize our experience with UVB treatment of early MF. METHODS: A retrospective analysis of early-stage MF patients treated by narrow band (NB) or broad band (BB) UVB in our institution between 1996 and 2002. Most patients achieving complete response (CR) were put on maintenance until natural sun exposure was possible and followed up every 3-6 months. The results were compared to those previously reported regarding PUVA. RESULTS: Sixty-eight and 43 patients were treated by NB and BB UVB, respectively. Eighty-six per cent (84 and 89% in NB and BB UVB groups, respectively) of IA and 71% (78 and 44% in NB and BB UVB groups, respectively) of IB patients achieved CR within a mean of 12.8 and 10.6 weeks, respectively. When maintenance was stopped, 65 and 30% had not relapsed after an average follow up of 27 and 222 weeks, respectively. Non relapse rate was 33 and 48% for those having had vs. those not having had maintenance, respectively. CONCLUSIONS: Our results are comparable to all previously reported for skin-targeted treatments, including PUVA and, to our belief, reflect the nature of early MF, in which CR can probably be achieved in most of the patients. Among the responding patients there is no relapse during prolonged follow-up in about one third of the cases. Thus, we believe treatment should be stopped completely following first CR induction and maintenance treatment should be considered for relapsing patients only. Both broad and narrow UVB options are good and future choices should be made on the basis of short- and long-term side-effects. PMID- 16684286 TI - Narrowband UVB phototherapy for small plaque parapsoriasis. AB - BACKGROUND: Narrowband UVB (NB-UVB) phototherapy has been shown to be effective for the treatment of various dermatoses. OBJECTIVE: To analyze the effects of NB UVB phototherapy for small plaque parapsoriasis (SPP). METHODS: The response of 45 patients (24 females, 21 males, age range 20-58 years) with histologically confirmed SPP were assessed. NB-UVB therapy was given 3-4 times weekly. The initial treatment dose was 70% of the minimal erythema dose. The doses were increased gradually with a standard increment of 20/10/0. Clinical response was determined as follows: complete response (CR), at least 90% clearing of skin lesions; partial response (PR), at least 50% but less than 90% clearing and no response (NR), less than 50% clearing. The follow-up period was 6-24 months after the treatment. RESULTS: NB-UVB treatment led to CR in 33 of 45 patients (73.3%) with a mean cumulative dose of 14.3 J/cm(2) (range 3.2-24.1 J/cm(2)) after a mean number of 29 exposures (range 16-51 sessions); PR in 12 of 45 (26.6%) with a cumulative dose of 15.6 J/cm(2) (range 10.4-23.3 J/cm(2)) after a mean number of 29.4 exposures (range 25-50 sessions). Nineteen patients with CR had skin phototype II, 13 had type III and 1 had type I. Among the patients with PR, 7 had skin phototype II and 5 had type III. Postinflammatory hyperpigmentation was observed in 51% of the patients. Relapses occurred in six patients within a mean time of 7.5 months (2-12 months). CONCLUSION: NB-UVB phototherapy has several advantages over treatment with broadband UVB and PUVA. NB-UVB therapy for patients with SPP is an effective, safe and practical alternative treatment modality. Further larger studies with longer follow-up periods are necessary to determine the proper clinical response and long-term complications of NB-UVB therapy in this disease. PMID- 16684287 TI - Critical study of hair growth analysis with computer-assisted methods. AB - BACKGROUND: Computer-assisted image analysis has been proposed for human hair growth studies. METHODS: The performances of Trichoscan, a commercially available automated system combining epiluminiscence microscopy with digital image analysis, developed for office-based hair growth measurements, have been evaluated comparatively on the same skin sites using standardized photographic equipment and calibrated processing for contrast-enhanced phototrichogram (CE PTG) analysis. This reference method has been validated with scalp biopsies and histological examination of serial sectioning. RESULTS: Besides edge effects, hair fibres escaped the Trichoscan analysis for various reasons including, but not limited to, thickness, pigmentation, closeness and crossing. CONCLUSION: Most of these problems have been identified in the late 1980s and remain largely unsolved by the processing software that was evaluated in 2004. Therefore claims promoting the Trichoscan method for accurate hair measurements in clinical trials on scalp and body hair are not supported by the present investigation. The speed at which the analysis is performed is outweighed by the errors in signal detection. Therefore we suggest that improvements must be clearly documented before Trichoscan is established for quantified diagnostic purposes and detailed hair cycle monitoring during hair trials. PMID- 16684288 TI - Cutaneous hyperpigmentation induced by omeprazole mimicking ashy dermatosis. AB - BACKGROUND: Omeprazole has been associated with multiple adverse effects including skin reactions but, to date, cutaneous hyperpigmentation has not been described as an adverse effect of this drug. OBSERVATIONS: We describe a case of a 52-year-old Caucasian woman who developed skin hyperpigmentation in the upper trunk, mimicking ashy dermatosis, 2 months after initiating omeprazole treatment. Histopathologic examination of a skin biopsy taken from a pigmented macule showed dermal macrophages containing golden-brown granules, which also displayed a sulphur peak on energy-dispersive X-ray microanalysis. High-performance liquid chromatography (HPLC) and mass spectrometry were also performed on the drug and on a biopsy specimen revealing the same chromatograms as well as the same mass spectra. CONCLUSIONS: According to our results, omeprazole itself may induce cutaneous pigmentation and, to our knowledge, this is the first report of this finding. PMID- 16684289 TI - Stevens-Johnson syndrome in two patients treated with cranial irradiation and phenytoin. AB - Stevens-Johnson syndrome (SJS) is a severe cutaneous eruption that most often appears as an adverse reaction to medication. In this report, we present two patients diagnosed with SJS, which developed in patients with brain tumour, after receiving cranial irradiation and phenytoin. Concomitant application of these two therapies may play an important role in the occurrence of the disease. PMID- 16684290 TI - Oculocutaneous tyrosinaemia or tyrosinaemia type 2: a case report. AB - Oculo-cutaneous tyrosinaemia type II is an autosomal recessive disease due to an abnormality of tyrosine metabolism, probably because of a deficiency of cytoplasmic tyrosine aminotransferase. It presents as a varying association of focal palmoplantar keratosis, bilateral keratitis and mental retardation. Herein, we report an 8-year-old boy with palmoplantar hyperkeratosis with peripheral oozing and dendritic keratitis appearing after the skin lesions. There was no mental deterioration despite the long delay in diagnosis of the disorder. The diagnosis was confirmed by the presence of hypertyrosinaemia and the absence of hepatorenal lesion. The child exhibited a remarkable degree of improvement in the hyperkeratotic lesions and keratitis after the dietary modifications were instituted. In conclusion, chronic focal bullous palmoplantar hyperkeratosis along with keratitis should alert the clinician to screen for abnormal serum and/or urine tyrosine level. Awareness of the presenting signs and symptoms may speed up the diagnosis and initiation of a tyrosine and phenylalanine-restricted diet that is most efficient in improving the symptoms and preventing visual and cognitive impairment. PMID- 16684291 TI - Bilateral inguinal hernia with dislocation of great saphenous vein as complication of long-standing granulomatous slack skin: a case report. AB - Granulomatous slack skin (GSS) represents a rare variant of mycosis fungoides, histologically characterized by a variably deep T helper lymphocytes infiltrate with alteration of the dermal elastic tissue and consequent elastolysis, elastophagocytosis and numerous giant cells. Clinically, a development of unelastic, slack skin, especially on flexural areas, is observed. Hereby, we describe a man with a 12-year history of GSS. In 2002, for practical (limitation of movement, deambulation) and cosmetic reasons, he underwent the surgical excision of loose and sagging skinfold over inguinal area, and, afterwards, of the opposite affected inguinal skin. The surgical treatment of bilateral inguinal hernia with reposition of inguinal dislocated vasculature is also reported. In both cases the excised material confirmed the former diagnosis of GSS and revealed a very deep, muscular infiltrate of neoplastic lymphocytes. One year later, a new excision of GSS on the axillae was made. Now, after 2 years, deambulation keeps improving, although an initial relapse of the inguinal slack skin has been observed. PMID- 16684292 TI - Calciphylaxis associated with alcoholic cirrhosis. AB - Calciphylaxis is an uncommon disease characterized by calcification of dermal vessels that determines skin necrosis. Calciphylaxis has been almost exclusively reported in association with renal failure and altered phosphor-calcium metabolism. Only a few cases have been described in hyperparathyroidism, malignancies, and, recently, cirrhosis. We report a patient that developed calciphylaxis related to end-stage alcoholic cirrhosis, without any alteration in the phosphocalcic and parathyroid hormone metabolisms. Possible contributing factors were repeated albumin infusions and low levels of protein C and S. PMID- 16684293 TI - Malignant atrophic papulosis: endocardial involvement and positive anticardiolipin antibodies. PMID- 16684294 TI - Rhinophyma in an adolescent. PMID- 16684295 TI - Partial unilateral lentiginosis with mixed lentigo/jentigo histological pattern. PMID- 16684296 TI - Primary cutaneous MALT-type lymphoma and Helicobacter pylori: a possible relationship. PMID- 16684297 TI - Eccrine squamous syringometaplasia in a patient with systemic lupus erythematosus. PMID- 16684298 TI - Nodular erythema as early sign of systemic aspergillosis. PMID- 16684299 TI - Orf followed by erythema multiforme. PMID- 16684300 TI - Etanercept-induced dermatitis in a patient with rheumatoid arthritis. PMID- 16684302 TI - Aspirin-induced unilateral angioedema of the tongue. PMID- 16684301 TI - Pachyonychia congenita: treatment of the thickened nails and palmoplantar circumscribed callosities with urea 40% paste. PMID- 16684303 TI - Parvovirus B19 primary infection with vasculitis: DNA identification in cutaneous lesions and sera. PMID- 16684304 TI - Cutaneous melanoma cases observed in Antalya from 1994 to 2003: clinical and demographical properties. PMID- 16684305 TI - Coexistence of three variants of porokeratosis with multiple squamous cell carcinomas arising from lesions of giant hyperkeratotic porokeratosis. PMID- 16684306 TI - Cutaneous sclerosis associated with left ventricular noncompaction, myopathy, polyneuropathy and pancytopenia with eosinophilia. PMID- 16684307 TI - Sex and age distribution of patients with lichen planus. PMID- 16684308 TI - Irritated seborrhoeic keratosis presenting as a cutaneous horn. PMID- 16684309 TI - Dyschromatosis universalis hereditaria as an autosomal recessive disease in five members of one family. PMID- 16684310 TI - The identification of primary human herpesvirus 7 infection in young adults with pityriasis rosea by investigating avidity of antibodies. PMID- 16684311 TI - Kaposiform hemangioendothelioma in an adult with an unusual presentation. PMID- 16684312 TI - Acute severe form of lichenoid graft-versus-host disease after donor lymphocyte infusions. PMID- 16684313 TI - Does stereotactic core needle biopsy increase the risk of local recurrence of invasive breast cancer? PMID- 16684314 TI - Local recurrence of breast cancer after skin-sparing mastectomy following core needle biopsy: case reports and review of the literature. AB - The latest advances in diagnostic and therapeutic procedures for breast cancer have provided valuable technological breakthroughs. Yet the long-term consequences of these modern methods are still quite unclear. Such is the case for stereotactic or ultrasound-guided histologic needle biopsy and skin-sparing mastectomy. We report on three patients who presented with multicentric breast cancer diagnosed by stereotactic needle biopsy and treated by skin-sparing mastectomy. All three patients developed recurrence at the core needle entry site. Records of 58 patients with breast cancer who were treated by skin-sparing mastectomy followed by immediate reconstruction (with transverse rectus abdominis muscle [TRAM] flap or tissue expander) at the Breast Diseases Division of Buenos Aires British Hospital between December 1999 and December 2003 were reviewed retrospectively. Eleven of these patients were diagnosed by histologic needle biopsy. The mean follow-up was 28 months (range 5-60 months). Three (skin or subcutaneous) local recurrences at the needle entry site, diagnosed in a mean time of 23.6 months (16, 22, and 23 months), were reported. The three patients underwent complete resection with clear margins, radiation therapy to the "neobreast," and tamoxifen. All three patients are disease free with a mean postrecurrence follow-up of 24.3 months (30, 23, and 22 months). Based on the evidence of displacement of tumor cells and the potential nonresection of such tumor seeding at the time of skin-sparing mastectomy, as well as the poor probability of postoperative radiation therapy, we recommend surgical resection of the needle biopsy tract, including the dermal entry site, at the time of mastectomy. PMID- 16684315 TI - Intraoperative palpation for clinically suspicious axillary sentinel lymph nodes reduces the false-negative rate of sentinel lymph node biopsy in breast cancer. AB - Axillary sentinel lymph node biopsy (SLNB) is widely used to identify the first lymph node draining breast tumors. When the sentinel lymph node is free of metastasis, axillary dissection is avoided because the rest of the nodes are expected to be negative as well. A false-negative rate of 5% is considered acceptable. In the case of a false-negative SLNB, adjuvant local and systemic treatments might be suboptimal. We assessed the effect of intraoperative axillary palpation for clinically suspicious lymph nodes that are not otherwise detected by radioactive tracer or blue dye on the false-negative rate of SLNB in breast cancer patients. Our prospective database of patients having surgery for primary invasive breast cancer and who had a SLNB from 2000 to 2004 was reviewed. Only patients with clinically negative nodes preoperatively were included. The procedure included preoperative injection of radiotracer, with dye injection as backup, and intraoperative palpation of the axilla for suspicious lymph nodes that were not radioactive or blue. Of the 290 patients, 89 (30.7%) had sentinel node involvement by tumor. Seven patients had clinically suspicious nodes identified solely by palpation and not by tracer, in addition to sentinel lymph nodes detected by tracer. In five of the seven patients, the nodes harbored metastasis. In four of these five patients (4.5% of the 89 patients with axillary involvement), the palpable nodes were the only ones involved. A generous axillary incision and systematic palpation of the axilla reduces the false-negative rate and should be a part of the SLNB procedure. PMID- 16684316 TI - Radioguided surgery using intravenous 99mTc sestamibi associated with breast magnetic resonance imaging for guidance of breast cancer resection. AB - The purpose of this study was to evaluate the feasibility of surgery radioguided with intravenous (99m)Tc sestamibi associated with magnetic resonance imaging (MRI) of the breast to detect tumor extent and guide complete tumor resection. A descriptive experimental study was developed with 10 breast cancer patients (stage IIA-IIB) who underwent mastectomy. From 2 to 10 days before surgery, the patients underwent breast MRI with 0.1 mmol/kg of gadolinium and scintimammography with a 740 MBq of (99m)Tc sestamibi. A region of interest was drawn around the tumor image and an uptake curve as a function of time was plotted to calculate the optimal time to perform radioguided surgery. In the perioperative period, the same dose of (99m)Tc sestamibi was intravenously injected into the patients. Tumor resection was performed under the guidance of a gamma probe. MRI was used to evaluate the skin and deep fascia involvement and to detect occult tumor foci which were also excised. Subsequently a modified radical mastectomy was performed. Tumor and residual breast were histopathologically examined. In a series of 10 women, all demonstrated (99m)Tc sestamibi uptake in tumor cells. Eight patients showed no disease in the residual breast, one presented with one foci of invasive ductal carcinoma measuring 0.5 cm in diameter located 5 cm from the tumor bed, and one presented with one foci of ductal carcinoma in situ measuring 0.8 cm at the resection margin. The mean tumor size in the histopathologic assessment was 3.3 cm and in MRI was 5.0 cm. Radioguided surgery using (99m)Tc sestamibi associated with MRI is a feasible technique that can be employed in tumor resection. PMID- 16684317 TI - Oncologic and aesthetic results following breast-conserving therapy with 0.5 cm margins in 100 consecutive patients. AB - The purpose of this study was to evaluate the oncologic and aesthetic results of patients undergoing breast-conserving therapy with 0.5 cm surgical margins and determine the factors that influence the need for reconstruction. One hundred consecutive patients who underwent breast-conserving surgery with 0.5 cm surgical margins followed by radiotherapy for invasive cancer and ductal carcinoma in situ (DCIS) were followed prospectively and evaluated for recurrence and aesthetic result. Thirteen patients underwent reexcision to achieve a 0.5 cm margin. Factors including breast size, location of the tumor, specimen size and volume, tumor size and volume, and TNM stage, if axillary dissection or reexcision were required, were included in the analysis. Aesthetic evaluation consisted of both patient rating and an independent observer rating on a 10-point scale that assessed volume, shape, symmetry, areola/nipple, and scar. Of the 100 patients that underwent breast-conserving therapy, the overall aesthetic results revealed that 8% of the patients scored themselves seven or less, another 8% were scored seven or less by the independent observer, and another 7% were scored seven or less by both the patient and the observer. Of these patients, only one proceeded to have a reconstructive procedure. Analysis of variance revealed a significant correlation between tumor size (cm(2)) and an aesthetic score of seven or less (p = 0.023), and specimen volume (cm(3)) and an aesthetic score of seven or less (p = 0.039). Chi-squared analysis revealed a significant difference (chi(2) = 4.39, p < 0.5) in the aesthetic result in patients with stage IIA disease. Other independent factors such as age, breast size, location of the tumor, axillary dissection, and reexcision did not influence the overall aesthetic result. A Pearson correlation of patient and independent observer ratings showed a positive correlation (r = 0.4; 95% confidence interval [CI] 0.19-0.57) between the two groups. There were zero local recurrences of breast cancer during the study period. Our results demonstrate that following breast-conserving therapy with a minimum of 0.5 cm resection margins, it is possible to achieve excellent oncologic and aesthetic results. Patients with large tumors that require a large volume of resection or with stage IIA disease should be considered for reconstructive evaluation. PMID- 16684319 TI - Double heterozygotes for non-Caucasian families with mutations in BRCA-1 and BRCA 2 genes. AB - The recent discovery of the BRCA-1 and BRCA-2 genes as contributing factors to hereditary breast cancer has significantly improved our understanding of familial breast cancer. Deleterious mutations in both BRCA-1 and BRCA-2 in an individual patient is exceedingly rare, with few case reports in the literature, particularly among non-Caucasian, non-Jewish families. We describe here two cases of deleterious mutations in both genes in a cohort of Korean women with early onset breast cancer. PMID- 16684318 TI - The role of ultrasound in the surgical management of patients diagnosed with ductal carcinoma in situ of the breast. AB - The purpose of this study was to determine the usefulness of ultrasound (US) as an adjunct to mammography (MMG) in the surgical treatment planning for patients with ductal carcinoma in situ (DCIS) of the breast. A total of 119 patients diagnosed with DCIS, who were treated between 1999 and 2002, were identified from the institutional database. US and MMG size of suspicious abnormalities, pathologic tumor size, and findings of axillary US and surgical axillary evaluation were analyzed. The median size difference of US versus pathologic tumor size and MMG versus pathologic tumor size was 1.0 and 2.0 cm, respectively. Correlation coefficients for US versus pathologic tumor size and MMG versus pathologic tumor size were 0.53 and -0.09, respectively. The negative and positive predictive values of axillary US to predict nodal disease were 93% and 27%, respectively. US evaluation appears to be a useful tool in conjunction with MMG in determining the extent of disease in patients diagnosed with DCIS. However, the low positive predictive value of US for evaluating metastases to axillary lymph nodes does not justify the routine use of this modality for axillary evaluation in patients with DCIS. Therefore the use of US for patients diagnosed with DCIS needs further investigation. PMID- 16684320 TI - Reliability and validity of the body image after breast cancer questionnaire. AB - The purpose of this study was to determine the reliability and validity of the Body Image After Breast Cancer Questionnaire (BIBCQ) in a series of outpatients with breast cancer. One hundred sixty-four breast cancer patients attending outpatient clinics completed questionnaires at baseline. The patients' BIBCQ scores were compared with their scores on related psychological measures including depression, self-esteem, quality of life, and sexual functioning. Scores on the BIBCQ for women after mastectomy and breast conservation were compared. Select items of the BIBCQ were compared between women with and without breast cancer. Patients received a second questionnaire after a 2 week interval to assess test-retest reliability. Good reliability was found for the six scales (ranging from 0.77 to 0.87). The BIBCQ correlated with similar measures as predicted, but not with a measure of social desirability. The BIBCQ distinguished between women treated with lumpectomy and mastectomy, and between women with breast cancer and a control group, supporting the validity of the BIBCQ. The BIBCQ provides a reliable and valid assessment of the long-term impact of breast cancer on body image. It is suitable for use in research focusing on this issue. PMID- 16684321 TI - Paget's disease of the breast areola mimicking cutaneous melanoma. AB - Paget's disease of the breast nipple and areola complex represents a cutaneous manifestation of an underlying breast malignancy. The typical skin changes characteristic of breast Paget's disease are usually easily distinguished from those found of malignant melanoma. The rare finding of a pigmented cutaneous breast lesion mimicking melanoma presents a diagnostic dilemma. This case presentation describes a pigmented mammary lesion involving the areola that had undergone changes in diameter and pigmentation. The lesion was not associated with nipple drainage, nipple excoriation, dermal ulceration, or a palpable breast mass. The areola lesion was highly suggestive of a dermatologic malignancy such as malignant melanoma. As such, a biopsy was performed with initial findings suggestive of cutaneous melanoma. Upon more detailed analysis of a larger excision specimen, immunohistochemical staining was highly supportive of the diagnosis of breast Paget's disease. A total mastectomy with sentinel lymph node evaluation was performed along with immediate reconstruction. The final pathology revealed a small focus of ductal carcinoma in situ of the breast associated with the areola changes. Histologic and immunohistochemical analysis of a biopsy specimen were paramount in differentiating these two lesions. PMID- 16684322 TI - Papillary lesions of the breast. AB - Papillary lesions of the breast include a variety of benign, atypical, and malignant lesions whose hallmark is the formation of either papillary tufts composed only of epithelium, or true papillary structures with fibrovascular support and epithelium. The precise diagnosis has significance because of the variability in treatment modalities. This article attempts to describe the basic features seen in papillary-type lesions and their correlation with imaging studies. PMID- 16684323 TI - Ultrasound for accurate measurement of invasive breast cancer tumor size. AB - Accurate presurgical assessment of tumor size is important for choosing appropriate treatment, especially with the increasing use of neoadjuvant and minimally invasive therapy. Breast sonography is increasingly used by breast surgeons as a part of their basic clinical evaluation. We undertook this study to compare clinical evaluation, mammography, and breast sonography for evaluating breast tumor size. A prospective analysis of 124 consecutive patients with palpable breast cancer was performed. Tumor masses belonging to T1 and small T2 were selectively selected. All women had clinical, mammographic, and sonographic assessment of tumor size. Measurements were compared to the pathologic tumor size of the surgical specimen. Both mammographic and sonographic measurements tend to underestimate tumor size, while clinical assessment tends to overestimate it. Ultrasound was significantly more accurate in determining tumor size. The maximal tumor diameter measured was within 2 mm of the pathologic tumor size in 45.2% of cases measured by breast ultrasound, 28.2% of cases measured by mammography, and 14.5% of cases measured clinically. These data suggest that ultrasound is more accurate than clinical breast examination and mammography in assessing breast cancer size. Ultrasound assessment should be used by surgeons as an accurate adjunct to clinical examination in outpatient breast clinics. PMID- 16684324 TI - Tubular adenoma of the breast in an 84-year-old woman: report of a case simulating breast cancer. AB - Tubular adenomas of the breast are rare and they mainly occur in young women. They must be differentiated from other benign lesions and from malignant breast cancer. Preoperative diagnosis is difficult because both radiologic and cytohistologic examinations performed before surgery are often not diagnostic; therefore, in most cases, surgical excision is the only way to reach a precise diagnosis and a definitive treatment. This article describes a case of an 84-year old woman suffering from tubular adenoma of the breast. The authors underline the difficulty in preoperative differentiation between benign and malignant breast lesions. PMID- 16684325 TI - Male breast hemangioma--a rare entity: a case report and review of the literature. AB - We report the case of a 77-year-old man with a very rare breast tumor: hemangioma. The clinical examination found a large (6 cm diameter) lump and mammography revealed a well-defined high-density lesion with lobulated contour. Simple mastectomy was performed. The histology confirmed the diagnosis of benign hemangioma. There has been no local recurrence after 9 years of follow-up. There is very little literature on this type of tumor in men. Generally hemangiomas are large and ultrasound and magnetic resonance imaging (MRI) are useful for the diagnosis. The important differential diagnosis with angiosarcoma is based on specific histologic patterns. Moreover, malignant transformation of hemangioma is rare, but possible. PMID- 16684326 TI - Mammary gouty tophus: a case report and review of the literature. AB - We present an uncommon presentation of a common disease in a completely asymptomatic woman. She presented with a solitary breast nodule incidentally found on her first mammography. A step-by-step evaluation of the breast nodule, including fine-needle aspiration and excisional biopsy, was done which revealed mammary gouty tophus. PMID- 16684327 TI - Necrotizing infection of the breast mimicking carcinoma. AB - Necrotizing fasciitis is a polymicrobial infection of the skin, subcutaneous tissue, and fascia with a fulminant course and a high morbidity and mortality. It rarely affects the breast. We report a postmenopausal woman presenting with necrotizing infection of the breast requiring mastectomy. PMID- 16684328 TI - Wegener's granulomatosis of the breast. AB - A 60-year-old woman presented with a painless self-detected breast lump. After wide local excision, the histology of this radiologically suspicious lump turned out to be an inflammatory lesion with granulomatous foci, suggesting a diagnosis of granulomatous mastitis. Prior to the excision biopsy, this woman had suffered from and been treated for anterior uveitis, secretory otitis media, and proximal myopathy. Four weeks later, a skin biopsy of a rapidly developing widespread petechial rash, mainly affecting the lower limbs, confirmed the diagnosis as Wegener's granulomatosis. Treatment with intravenous steroids and later cyclophosphamide produced rapid remission. PMID- 16684329 TI - Hemangioma of the breast: clinical, mammographic, and ultrasound features. PMID- 16684330 TI - Lupus mastitis in the male breast mimicking inflammatory carcinoma. PMID- 16684331 TI - Hematoma mimicking local recurrence of breast cancer. PMID- 16684332 TI - Metaplastic ossification in mammary fibroadenoma. PMID- 16684333 TI - Mimicry of breast carcinoma. PMID- 16684334 TI - Genetic counseling and management of newly diagnosed breast cancer patients at genetic risk for BRCA germline mutations. PMID- 16684337 TI - The use of nonionized contrast media with surgical biopsies for nonpalpable microcalcified breast lesions. PMID- 16684338 TI - Possible association between nanobacteria and malignant microcalcifications in breast cancer. PMID- 16684339 TI - Neutrophil elastase predicts trastuzumab responsiveness in metastatic breast cancer. PMID- 16684341 TI - Viral particles of the endogenous retrovirus ZAM from Drosophila melanogaster use a pre-existing endosome/exosome pathway for transfer to the oocyte. AB - BACKGROUND: Retroviruses have evolved various mechanisms to optimize their transfer to new target cells via late endosomes. Here, we analyzed the transfer of ZAM, a retroelement from Drosophila melanogaster, from ovarian follicle cells to the oocyte at stage 9-10 of oogenesis, when an active yolk transfer is occurring between these two cell types. RESULTS: Combining genetic and microscopic approaches, we show that a functional secretory apparatus is required to tether ZAM to endosomal vesicles and to direct its transport to the apical side of follicle cells. There, ZAM egress requires an intact follicular epithelium communicating with the oocyte. When gap junctions are inhibited or yolk receptors mutated, ZAM particles fail to sort out the follicle cells. CONCLUSION: Overall, our results indicate that retrotransposons do not exclusively perform intracellular replication cycles but may usurp exosomal/endosomal traffic to be routed from one cell to another. PMID- 16684342 TI - A preliminary DTI study showing no brain structural change associated with adolescent cannabis use. AB - Analyses were performed on brain MRI scans from individuals who were frequent cannabis users (N = 10; 9 males, 1 female, mean age 21.1 +/- 2.9, range: 18-27) in adolescence and similar age and sex matched young adults who never used cannabis (N = 10; 9 males, 1 female, mean age of 23.0 +/- 4.4, range: 17-30). Cerebral atrophy and white matter integrity were determined using diffusion tensor imaging (DTI) to quantify the apparent diffusion coefficient (ADC) and the fractional anisotropy (FA). Whole brain volumes, lateral ventricular volumes, and gray matter volumes of the amygdala-hippocampal complex, superior temporal gyrus, and entire temporal lobes (excluding the amygdala-hippocampal complex) were also measured. While differences existed between groups, no pattern consistent with evidence of cerebral atrophy or loss of white matter integrity was detected. It is concluded that frequent cannabis use is unlikely to be neurotoxic to the normal developing adolescent brain. PMID- 16684343 TI - Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter. AB - BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs 111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl. PMID- 16684345 TI - Correction of scaling mismatches in oligonucleotide microarray data. AB - BACKGROUND: Gene expression microarray data is notoriously subject to high signal variability. Moreover, unavoidable variation in the concentration of transcripts applied to microarrays may result in poor scaling of the summarized data which can hamper analytical interpretations. This is especially relevant in a systems biology context, where systematic biases in the signals of particular genes can have severe effects on subsequent analyses. Conventionally it would be necessary to replace the mismatched arrays, but individual time points cannot be rerun and inserted because of experimental variability. It would therefore be necessary to repeat the whole time series experiment, which is both impractical and expensive. RESULTS: We explain how scaling mismatches occur in data summarized by the popular MAS5 (GCOS; Affymetrix) algorithm, and propose a simple recursive algorithm to correct them. Its principle is to identify a set of constant genes and to use this set to rescale the microarray signals. We study the properties of the algorithm using artificially generated data and apply it to experimental data. We show that the set of constant genes it generates can be used to rescale data from other experiments, provided that the underlying system is similar to the original. We also demonstrate, using a simple example, that the method can successfully correct existing imbalances in the data. CONCLUSION: The set of constant genes obtained for a given experiment can be applied to other experiments, provided the systems studied are sufficiently similar. This type of rescaling is especially relevant in systems biology applications using microarray data. PMID- 16684344 TI - Calcium ion currents mediating oocyte maturation events. AB - During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. PMID- 16684346 TI - Perceived quality of life in obsessive-compulsive disorder: related factors. AB - BACKGROUND: Obsessive-compulsive disorder (OCD) affects young adults and has great impact on the social, emotional and work spheres. METHODS: We measured perceived quality of life (QOL) in OCD patients, in order to analyse socio demographic and clinical factors that may be associated with QOL perception. 64 OCD outpatients were assessed with the Mini International Neuropsychiatric Interview for DSM-IV, the Yale-Brown Obsessions and Compulsions scale (Y-BOCS), Hamilton's depression scale and the SF-36 self-administered global QOL perception scale. RESULTS: We found a correlation among Hamilton's scale scores and all SF 36 subscales. The severity of the obsessive-compulsive disorder was correlated with all SF-36 subscales and with the highest scores in Hamilton's scale. The obsessions subscale was correlated to all SF-36 subscales, while the compulsions subscale was correlated only to social functioning, emotional role, mental health and vitality. Compulsions were not related to general health perception. There were significant differences between OCD patients and the Spanish general population in all SF-36 subscales except those related to physical health and pain. Gender, age, age of onset of the disorder, years of evolution and marital status of the patients did not significantly affect quality of life perception. Being employed was related to better scores in the subscale of physical role. Patients with medical comorbidity scored lower in the subscales of general health, social functioning and mental health. Patients with comorbid psychiatric disorders had worse scores in the subscales of pain, general health, social functioning and mental health. CONCLUSION: Quality of life perception was different in OCD patients and the general population. Quality of life perception was related to severity of the disorder, physical and psychiatric comorbidity and employment status. PMID- 16684347 TI - A survey of the use of electronic scientific information resources among medical and dental students. AB - BACKGROUND: To evaluate medical and dental students' utilization of electronic information resources. METHODS: A web survey sent to 837 students (49.9% responded). RESULTS: Twenty-four per cent of medical students and nineteen per cent of dental students searched MEDLINE 2+ times/month for study purposes, and thiry-two per cent and twenty-four per cent respectively for research. Full-text articles were used 2+ times/month by thirty-three per cent of medical and ten per cent of dental students. Twelve per cent of respondents never utilized either MEDLINE or full-text articles. In multivariate models, the information-searching skills among students were significantly associated with use of MEDLINE and full text articles. CONCLUSION: Use of electronic resources differs among students. Forty percent were non-users of full-text articles. Information-searching skills are correlated with the use of electronic resources, but the level of basic PC skills plays not a major role in using these resources. The student data shows that adequate training in information-searching skills will increase the use of electronic information resources. PMID- 16684348 TI - Synaptic depression and short-term habituation are located in the sensory part of the mammalian startle pathway. AB - BACKGROUND: Short-term habituation of the startle response represents an elementary form of learning in mammals. The underlying mechanism is located within the primary startle pathway, presumably at sensory synapses on giant neurons in the caudal pontine reticular nucleus (PnC). Short trains of action potentials in sensory afferent fibers induce depression of synaptic responses in PnC giant neurons, a phenomenon that has been proposed to be the cellular correlate for short-term habituation. We address here the question whether both this synaptic depression and the short-term habituation of the startle response are localized at the presynaptic terminals of sensory afferents. If this is confirmed, it would imply that these processes take place prior to multimodal signal integration, rather than occurring at postsynaptic sites on PnC giant neurons that directly drive motor neurons. RESULTS: Patch-clamp recordings in vitro were combined with behavioral experiments; synaptic depression was specific for the input pathway stimulated and did not affect signals elicited by other sensory afferents. Concordant with this, short-term habituation of the acoustic startle response in behavioral experiments did not influence tactile startle response amplitudes and vice versa. Further electrophysiological analysis showed that the passive properties of the postsynaptic neuron were unchanged but revealed some alterations in short-term plasticity during depression. Moreover, depression was induced only by trains of presynaptic action potentials and not by single pulses. There was no evidence for transmitter receptor desensitization. In summary, the data indicates that the synaptic depression mechanism is located presynaptically. CONCLUSION: Our electrophysiological and behavioral data strongly indicate that synaptic depression in the PnC as well as short-term habituation are located in the sensory part of the startle pathway, namely at the axon terminals of sensory afferents in the PnC. Our results further corroborate the link between synaptic depression and short-term habituation of the startle response. PMID- 16684349 TI - Pim-1 kinase phosphorylates RUNX family transcription factors and enhances their activity. AB - BACKGROUND: The pim family genes encode oncogenic serine/threonine kinases which in hematopoietic cells have been implicated in cytokine-dependent signaling as well as in lymphomagenesis, especially in cooperation with other oncogenes such as myc, bcl-2 or Runx family genes. The Runx genes encode alpha-subunits of heterodimeric transcription factors which regulate cell proliferation and differentiation in various tissues during development and which can become leukemogenic upon aberrant expression. RESULTS: Here we have identified novel protein-protein interactions between the Pim-1 kinase and the RUNX family transcription factors. Using the yeast two-hybrid system, we were able to show that the C-terminal part of human RUNX3 associates with Pim-1. This result was confirmed in cell culture, where full-length murine Runx1 and Runx3 both coprecipitated and colocalized with Pim-1. Furthermore, catalytically active Pim 1 kinase was able to phosphorylate Runx1 and Runx3 proteins and enhance the transactivation activity of Runx1 in a dose-dependent fashion. CONCLUSION: Altogether, our results suggest that mammalian RUNX family transcription factors are novel binding partners and substrates for the Pim-1 kinase, which may be able to regulate their activities during normal hematopoiesis as well as in leukemogenesis. PMID- 16684350 TI - Phylophenetic properties of metabolic pathway topologies as revealed by global analysis. AB - BACKGROUND: As phenotypic features derived from heritable characters, the topologies of metabolic pathways contain both phylogenetic and phenetic components. In the post-genomic era, it is possible to measure the "phylophenetic" contents of different pathways topologies from a global perspective. RESULTS: We reconstructed phylophenetic trees for all available metabolic pathways based on topological similarities, and compared them to the corresponding 16S rRNA-based trees. Similarity values for each pair of trees ranged from 0.044 to 0.297. Using the quartet method, single pathways trees were merged into a comprehensive tree containing information from a large part of the entire metabolic networks. This tree showed considerably higher similarity (0.386) to the corresponding 16S rRNA-based tree than any tree based on a single pathway, but was, on the other hand, sufficiently distinct to preserve unique phylogenetic information not reflected by the 16S rRNA tree. CONCLUSION: We observed that the topology of different metabolic pathways provided different phylogenetic and phenetic information, depicting the compromise between phylogenetic information and varying evolutionary pressures forming metabolic pathway topologies in different organisms. The phylogenetic information content of the comprehensive tree is substantially higher than that of any tree based on a single pathway, which also gave clues to constraints working on the topology of the global metabolic networks, information that is only partly reflected by the topologies of individual metabolic pathways. PMID- 16684351 TI - A long-term investigation of the anti-hepatocarcinogenic potential of an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra. AB - BACKGROUND: A decoction comprised of Nigella sativa seeds, Hemidesmus indicus root bark and Smilax glabra rhizome is being recommended for cancer patients by a family of traditional medical practitioners of Sri Lanka. Previous investigations have demonstrated that a short term (10 weeks) treatment with the decoction can significantly inhibit diethylnitrosamine (DEN) mediated expression of Glutathione S-transferase P form (GST-P) in rat liver. The objective of the present investigation was to determine whether long term (16 months) treatment with the decoction would be successful in inhibiting in rat livers, not only DEN- mediated expression of GST-P, but also the carcinogen mediated development of overt tumours (OT) or histopathological changes leading to tumour development (HT). METHODS: Thirty-six male Wistar rats were divided into 3 groups of 12 each. Groups 1 and 2 were injected intraperitoneally (i.p) with DEN (200 mg/kg) while group 3 was injected normal saline (NS). Twenty-four hours later, decoction (DC; 6 g/kg body weight/day) was orally administered to group 1 rats, while groups 2 and 3 (DEN-control and normal control) were given distilled water (DW). Treatment with DC or DW continued for 16 months. At the end of the 9th month and 16th months (study 1 and study 2 respectively), six rats from each group were sacrificed, and livers observed for OT or HT, both visually and by subjecting liver sections to staining with Haemotoxylin and Eosin (H & E), Sweet's Silver stain (for reticulin fibers), Periodic Acid Schiff (PAS) staining (for glycogen), and immunohistochemical staining (for GST-P). RESULTS: At the end of 9 months (study 1) a hepatocellular adenoma (HA) developed in one of the rats in the DEN + DW treated group (group 2). At the end of 16 months (study 2), livers of all rats of group 2 developed OT and HT. Large areas of GST-P positive foci were also observed. No OT, HT or GST-P positive foci were detected in any of the other groups. CONCLUSION: Protection against DEN-mediated carcinogenic changes in rat liver can be achieved by long term treatment with the DC comprised of N. sativa seeds, S. glabra rhizome and H. indicus root bark. PMID- 16684352 TI - JMATING: a software for the analysis of sexual selection and sexual isolation effects from mating frequency data. AB - BACKGROUND: Many different sexual isolation and sexual selection statistics have been proposed in the past. However, there is no available software that implements all these statistical estimators and their corresponding tests for the study of mating behaviour. RESULTS: JMATING is an easy-to-use program developed in Java for the analysis of mating frequency data to study sexual selection and sexual isolation effects from laboratory experiments as well as descriptive studies accomplished in the wild. The software allows the re-organization of the data previous to the analysis, the estimation of the most important estimators, and a battery of complementary statistical tests. CONCLUSION: JMATING is the first complete and versatile software for the analyses of mating frequency data. It is available at http://www.uvigo.es/webs/c03/webc03/XENETICA/XB2/JMsoft.htm and requires the Java runtime environment. PMID- 16684354 TI - Spatially targeting Culex quinquefasciatus aquatic habitats on modified land cover for implementing an Integrated Vector Management (IVM) program in three villages within the Mwea Rice Scheme, Kenya. AB - BACKGROUND: Continuous land cover modification is an important part of spatial epidemiology because it can help identify environmental factors and Culex mosquitoes associated with arbovirus transmission and thus guide control intervention. The aim of this study was to determine whether remotely sensed data could be used to identify rice-related Culex quinquefasciatus breeding habitats in three rice-villages within the Mwea Rice Scheme, Kenya. We examined whether a land use land cover (LULC) classification based on two scenes, IKONOS at 4 m and Landsat Thematic Mapper at 30 m could be used to map different land uses and rice planted at different times (cohorts), and to infer which LULC change were correlated to high density Cx. quinquefasciatus aquatic habitats. We performed a maximum likelihood unsupervised classification in Erdas Imagine V8.7 and generated three land cover classifications, rice field, fallow and built environment. Differentially corrected global positioning systems (DGPS) ground coordinates of Cx. quinquefasciatus aquatic habitats were overlaid onto the LULC maps generated in ArcInfo 9.1. Grid cells were stratified by levels of irrigation (well-irrigated and poorly-irrigated) and varied according to size of the paddy. RESULTS: Total LULC change between 1988-2005 was 42.1 % in Kangichiri, 52.8 % in Kiuria and and 50.6 % Rurumi. The most frequent LULC changes was rice field to fallow and fallow to rice field. The proportion of aquatic habitats positive for Culex larvae in LULC change sites was 77.5% in Kangichiri, 72.9% in Kiuria and 73.7% in Rurumi. Poorly - irrigated grid cells displayed 63.3% of aquatic habitats among all LULC change sites. CONCLUSION: We demonstrate that optical remote sensing can identify rice cultivation LULC sites associated with high Culex oviposition. We argue that the regions of higher Culex abundance based on oviposition surveillance sites reflect underlying differences in abundance of larval habitats which is where limited control resources could be concentrated to reduce vector larval abundance. PMID- 16684353 TI - Muscarinic receptor signaling in the pathophysiology of asthma and COPD. AB - Anticholinergics are widely used for the treatment of COPD, and to a lesser extent for asthma. Primarily used as bronchodilators, they reverse the action of vagally derived acetylcholine on airway smooth muscle contraction. Recent novel studies suggest that the effects of anticholinergics likely extend far beyond inducing bronchodilation, as the novel anticholinergic drug tiotropium bromide can effectively inhibit accelerated decline of lung function in COPD patients. Vagal tone is increased in airway inflammation associated with asthma and COPD; this results from exaggerated acetylcholine release and enhanced expression of downstream signaling components in airway smooth muscle. Vagally derived acetylcholine also regulates mucus production in the airways. A number of recent research papers also indicate that acetylcholine, acting through muscarinic receptors, may in part regulate pathological changes associated with airway remodeling. Muscarinic receptor signalling regulates airway smooth muscle thickening and differentiation, both in vitro and in vivo. Furthermore, acetylcholine and its synthesizing enzyme, choline acetyl transferase (ChAT), are ubiquitously expressed throughout the airways. Most notably epithelial cells and inflammatory cells generate acetylcholine, and express functional muscarinic receptors. Interestingly, recent work indicates the expression and function of muscarinic receptors on neutrophils is increased in COPD. Considering the potential broad role for endogenous acetylcholine in airway biology, this review summarizes established and novel aspects of muscarinic receptor signaling in relation to the pathophysiology and treatment of asthma and COPD. PMID- 16684355 TI - Simple tandem repeat (TTTA)n polymorphism in CYP19 (aromatase) gene and breast cancer risk in Nigerian women. AB - BACKGROUND: Breast cancer is the most common cancer and the leading cause of cancer related deaths in women worldwide. The incidence of the disease is increasing globally and this increase is occurring at a faster rate in population groups that hirtherto enjoyed low incidence. This study was designed to evaluate the role of a simple tandem repeat polymorphism (STRP) in the aromatase (CYP19) gene in breast cancer susceptibility in Nigerian women, a population of indigenous sub-Saharan African ancestry. METHODS: A case-control study recruiting 250 women with breast cancer and 250 women without the disease from four University Teaching Hospitals in Southern Nigeria was carried out between September 2002 and April 2004. Participants were recruited from the surgical outpatient clinics and surgical wards of the Nigerian institutions. A polymerase chain reaction (PCR)-based assay was employed for genotyping and product sizes were detected with an ABI 3730 DNA Analyzer. RESULTS: Conditional logistic regression analysis revealed that harboring the putative high risk genotypes conferred a 29% increased risk of breast cancer when all women in the study were considered (Odds ratio [OR] = 1.29, 95% confidence interval [CI] 0.83-2.00), although this association was not statistically significant. Subgroup analysis based on menopausal status showed similar results among premenopausal women (OR = 1.35, 95% CI 0.76-2.41 and postmenopausal women (OR = 1.27, 95% CI 0.64-2.49). The data also demonstrated marked differences in the distribution of (TTTA)n repeats in Nigerian women compared with other populations. CONCLUSION: This study has shown that harboring 10 or more repeats of the microsatellite (TTTA)n repeats of the CYY19 gene is associated with a modest increased risk of breast cancer in Nigerian women. PMID- 16684356 TI - Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays. AB - BACKGROUND: Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB) macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia. RESULTS: RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4%) from 12 farms, including 9 farms (14 samples) located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species. CONCLUSION: This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial tests like RLB that clearly identify the parasite and can detect subclinical infections are essential to establish good control measures. PMID- 16684357 TI - Regularized binormal ROC method in disease classification using microarray data. AB - BACKGROUND: An important application of microarrays is to discover genomic biomarkers, among tens of thousands of genes assayed, for disease diagnosis and prognosis. Thus it is of interest to develop efficient statistical methods that can simultaneously identify important biomarkers from such high-throughput genomic data and construct appropriate classification rules. It is also of interest to develop methods for evaluation of classification performance and ranking of identified biomarkers. RESULTS: The ROC (receiver operating characteristic) technique has been widely used in disease classification with low dimensional biomarkers. Compared with the empirical ROC approach, the binormal ROC is computationally more affordable and robust in small sample size cases. We propose using the binormal AUC (area under the ROC curve) as the objective function for two-sample classification, and the scaled threshold gradient directed regularization method for regularized estimation and biomarker selection. Tuning parameter selection is based on V-fold cross validation. We develop Monte Carlo based methods for evaluating the stability of individual biomarkers and overall prediction performance. Extensive simulation studies show that the proposed approach can generate parsimonious models with excellent classification and prediction performance, under most simulated scenarios including model mis-specification. Application of the method to two cancer studies shows that the identified genes are reasonably stable with satisfactory prediction performance and biologically sound implications. The overall classification performance is satisfactory, with small classification errors and large AUCs. CONCLUSION: In comparison to existing methods, the proposed approach is computationally more affordable without losing the optimality possessed by the standard ROC method. PMID- 16684358 TI - Do patients receive recommended treatment of osteoporosis following hip fracture in primary care? AB - BACKGROUND: Osteoporosis results in fractures and treatment of osteoporosis has been shown to reduce risk of fracture particularly in those who have had a history of fracture. METHODS: A prospective study was conducted using patients admitted to a hip fracture rehabilitation program at a large referral center to evaluate the use of treatments recommended for secondary prevention of osteoporotic fracture between September 1, 2001 and September 30, 2003. The frequency of medication use for the treatment of osteoporosis including estrogen replacement therapy, bisphosponates, calcitonin, calcium and vitamin D therapy was determined on admission, at 6 weeks post discharge and one year following discharge. All patients were discharged to the care of their family physician. All family physicians in the referral region received a copy of the Canadian Consensus recommendations for osteoporosis management 1-3 months prior to the study. RESULTS: During the study period, 174 patients were enrolled and 121 completed all assessments. Fifty-seven family physicians were identified as caring for 1 or more of the study patients. Only 7 patients had previous BMD, only 5 patients had previously been prescribed a bisphosponate and 14 patients were taking calcium and/or vitamin D. All patients were prescribed 2500 mg calcium, 400 IU vitamin D and 5 mg residronate daily during rehabilitation and at discharge.Following discharge, a significant improvement was seen in all clinical indices of functional mobility, including the functional independence measure (FIM), walking distance, fear of falling score (FFS), and the Berg balance score (BBS). At six weeks a significant (p < 0.01) decrease in calcium and vitamin D use was observed. All patients remained compliant with residronate therapy. At twelve months 71 patients remained on residronate (p < 0.01), 10 were now taking alternate bisphosphonate therapy and few were taking calcium and/or vitamin D (p < 0.001). FIM, FFS and Berg scores were significantly decreased from discharge (p < 0.001) while walking distance was unchanged. CONCLUSION: Few patients admitted for hip fracture had previously taken recommended osteoporosis therapy including bisphosphonates. While compliance with Canadian Consensus recommendations was observed at six weeks, this was not the case at twelve months post hip fracture rehabilitation. Interventions to improve not only the detection and treatment of osteoporosis but also the ongoing treatment and management post-fracture need to be developed and implemented. PMID- 16684359 TI - Optimizing search strategies to identify randomized controlled trials in MEDLINE. AB - BACKGROUND: The Cochrane Highly Sensitive Search Strategy (HSSS), which contains three phases, is widely used to identify Randomized Controlled Trials (RCTs) in MEDLINE. Lefebvre and Clarke suggest that reviewers might consider using four revisions of the HSSS. The objective of this study is to validate these four revisions: combining the free text terms volunteer, crossover, versus, and the Medical Subject Heading CROSS-OVER STUDIES with the top two phases of the HSSS, respectively. METHODS: We replicated the subject search for 61 Cochrane reviews. The included studies of each review that were indexed in MEDLINE were pooled together by review and then combined with the subject search and each of the four proposed search strategies, the top two phases of the HSSS, and all three phases of the HSSS. These retrievals were used to calculate the sensitivity and precision of each of the six search strategies for each review. RESULTS: Across the 61 reviews, the search term versus combined with the top two phases of the HSSS was able to find 3 more included studies than the top two phases of the HSSS alone, or in combination with any of the other proposed search terms, but at the expense of missing 56 relevant articles that would be found if all three phases of the HSSS were used. The estimated time needed to finish a review is 1086 hours for all three phases of the HSSS, 823 hours for the strategy versus, 818 hours for the first two phases of the HSSS or any of the other three proposed strategies. CONCLUSION: This study shows that compared to the first two phases of the HSSS, adding the term versus to the top two phases of the HSSS balances the sensitivity and precision in the reviews studied here to some extent but the differences are very small. It is well known that missing relevant studies may result in bias in systematic reviews. Reviewers need to weigh the trade-offs when selecting the search strategies for identifying RCTs in MEDLINE. PMID- 16684360 TI - Reversible hypothyroidism and Whipple's disease. AB - BACKGROUND: The major cause of primary hypothyroidism is autoimmune mediated with progressive and permanent destruction of the thyroid gland resulting in life-long replacement therapy. Treatable and reversible hypothyroidism is unusual and here forth is such a case due to infection of the thyroid gland with Tropheryma whippleii, Whipple disease. CASE PRESENTATION: A 45 year-old female presented with symptoms and signs consistent with primary hypothyroidism, which was also confirmed biochemically. Her response to thyroxine replacement therapy was poor however, requiring a significantly elevated amount. Further investigation revealed the presence of Whipple's disease involving the gastrointestinal trace and possibly the thyroid gland. Her thyroxine requirement decreased drastically following appropriate antimicrobial therapy for Whipple's disease to the extent that it was ceased. Thyrotropin releasing hormone testing in the steady state suggested there was diminished thyroid reserve due to Whipple's disease. CONCLUSION: This is the first ante-mortem case report studying the possible involvement of the thyroid gland by Whipple's disease. Despite the normalization of her thyroid function test biochemically after antibiotic therapy, there is diminished thyroid reserve thus requiring close and regular monitoring. PMID- 16684361 TI - Nucleotide sequence analyses of the MRP1 gene in four populations suggest negative selection on its coding region. AB - BACKGROUND: The MRP1 gene encodes the 190 kDa multidrug resistance-associated protein 1 (MRP1/ABCC1) and effluxes diverse drugs and xenobiotics. Sequence variations within this gene might account for differences in drug response in different individuals. To facilitate association studies of this gene with diseases and/or drug response, exons and flanking introns of MRP1 were screened for polymorphisms in 142 DNA samples from four different populations. RESULTS: Seventy-one polymorphisms, including 60 biallelic single nucleotide polymorphisms (SNPs), ten insertions/deletions (indel) and one short tandem repeat (STR) were identified. Thirty-four of these polymorphisms have not been previously reported. Interestingly, the STR polymorphism at the 5' untranslated region (5'UTR) occurs at high but different frequencies in the different populations. Frequencies of common polymorphisms in our populations were comparable to those of similar populations in HAPMAP or Perlegen. Nucleotide diversity indices indicated that the coding region of MRP1 may have undergone negative selection or recent population expansion. SNPs E10/1299 G>T (R433S) and E16/2012 G>T (G671V) which occur at low frequency in only one or two of four populations examined were predicted to be functionally deleterious and hence are likely to be under negative selection. CONCLUSION: Through in silico approaches, we identified two rare SNPs that are potentially negatively selected. These SNPs may be useful for studies associating this gene with rare events including adverse drug reactions. PMID- 16684362 TI - The Australian experiment: the use of evidence based medicine for the reimbursement of surgical and diagnostic procedures (1998-2004). AB - BACKGROUND: In 1998 a formal process using the criteria of safety, effectiveness and cost-effectiveness (evidence based medicine) on the introduction and use of new medical procedures was implemented in Australia. As part of this process an expert panel, the Medical Services Advisory Committee (MSAC) was set up. This paper examines the effectiveness of this process based on the original criteria, that is, evidence based medicine. METHOD: The data for this analysis was sourced primarily from that made available in the public domain. The MSAC web site provided Minutes from MSAC meetings; Annual Reports; Assessment and Review reports; Progress status; and Archived material. RESULTS: The total number of applications submitted to the MSAC has been relatively low averaging approximately only fourteen per year. Additionally, the source of applications has quickly shifted to the medical devices, equipment and diagnostic industry as being the major source of applications. An overall average time for the processing of an application is eighteen months. Negative recommendations were in most cases based on insufficient clinical evidence rather than clinical evidence that clearly demonstrated a lack of clinical effectiveness. It was rare for a recommendation, either positive or negative, to be based on cost-effectiveness. CONCLUSION: New medical procedures are often the result of a process of experimentation rather than formally conducted research. Affordability and the question of who should pay for the generation, collection and analysis of the clinical evidence is perhaps the most difficult to answer. This is especially the case where the new procedure is the result of a process of experimentation with an old procedure. A cost-effective way needs to be found to collect acceptable levels of evidence proving the clinical effectiveness of these new procedures, otherwise the formal processes of evaluation such as that used by the Australian MSAC since 1998 will continue to run the risk of committing Type II errors, that is, denying access to medical procedures that are beneficial and efficient. PMID- 16684364 TI - Use of an integrated clinical trial database to evaluate the effect of timing of drotrecogin alfa (activated) treatment in severe sepsis. AB - INTRODUCTION: Several studies have indicated that early identification and treatment of patients with severe sepsis using standard supportive care improves outcomes. Earlier treatment with drotrecogin alfa (activated) (DrotAA) may also improve outcomes in severe sepsis. Using a recently constructed integrated severe sepsis database, our objectives in this study were to describe the influence of baseline clinical characteristics on timing of DrotAA treatment in patients with severe sepsis, to evaluate the efficacy of DrotAA with respect to timing of administration, and to examine the association between early intervention with DrotAA and patient outcomes, using adjustments for imbalances. METHODS: The database comprises data from 4,459 patients with severe sepsis (DrotAA, n = 3,228; placebo, n = 1,231) included in five clinical trials conducted in tertiary care institutions in 28 countries. Placebo data came only from randomized trials, whereas data for the DrotAA group came from randomized (PROWESS) and open label/observational (ENHANCE) trials. RESULTS: Increased time-to-treatment with DrotAA was significantly associated with more organ dysfunction, greater need of mechanical ventilation, vasopressor use, or recent surgery. Earlier treatment was associated with higher baseline Acute Physiology and Chronic Health Evaluation (APACHE II) scores. Adjusted and unadjusted survival analyses suggested that compared with placebo, DrotAA treatment provided a potential survival benefit, regardless of time to treatment. Survival curves of DrotAA patients treated early compared with those treated late began to separate at 14 days. By 28 days, patients treated earlier had higher survival than those treated later (76.4% versus 73.5%, p = 0.03). Sepsis-induced multiorgan dysfunction was the most common cause of death followed by refractory shock and respiratory failure. Modeling of the treatment effect, as a function of time to treatment, suggested increased benefit with earlier treatment. CONCLUSION: Using an integrated database of five severe sepsis trials and appropriate statistical adjustments to reduce sources of potential bias, earlier treatment with DrotAA seemed to be associated with a lower risk-adjusted mortality than later treatment. These data suggest that earlier treatment with DrotAA may provide most benefit for appropriate patients. PMID- 16684363 TI - Conserved genes in a path from commensalism to pathogenicity: comparative phylogenetic profiles of Staphylococcus epidermidis RP62A and ATCC12228. AB - BACKGROUND: Staphylococcus epidermidis, long regarded as an innocuous commensal bacterium of the human skin, is the most frequent cause of nosocomial infections associated with implanted medical devices. This conditional pathogen provides a model of choice to study genome landmarks correlated with the transition between commensalism and pathogenicity. Traditional investigations stress differences in gene content. We focused on conserved genes that have accumulated small mutation differences during the transition. RESULTS: A comparison of strain ATCC12228, a non-biofilm forming, non-infection associated strain and strain RP62A, a methicillin-resistant biofilm clinical isolate, revealed consistent variation, mostly single-nucleotide polymorphisms (SNPs), in orthologous genes in addition to the previously investigated global changes in gene clusters. This polymorphism, scattered throughout the genome, may reveal genes that contribute to adaptation of the bacteria to different environmental stimuli, allowing them to shift from commensalism to pathogenicity. SNPs were detected in 931 pairs of orthologs with identical gene length, accounting for approximately 45% of the total pairs of orthologs. Assuming that non-synonymous mutations would mark recent evolution, and hence be associated to the onset of the pathogenic process, analysis of ratios of non-synonymous SNPs vs synonymous SNPs suggested hypotheses about possible pathogenicity determinants. The N/S ratios for virulence factors and surface proteins differed significantly from that of average SNPs. Of those gene pairs, 40 showed a disproportionate distribution of dN vs dS. Among those, the presence of the gene encoding methionine sulfoxide reductase suggested a possible involvement of reactive oxygen species. This led us to uncover that the infection associated strain was significantly more resistant to hydrogen peroxide and paraquat than the environmental strain. Some 16 genes of the list were of unknown function. We could suggest however that they were likely to belong to surface proteins or considered in priority as important for pathogenicity. CONCLUSION: Our study proposed a novel approach to identify genes involved in pathogenic processes and provided some insight about the molecular mechanisms leading a commensal inhabitant to become an invasive pathogen. PMID- 16684365 TI - Kinetic bed therapy to prevent nosocomial pneumonia in mechanically ventilated patients: a systematic review and meta-analysis. AB - INTRODUCTION: Nosocomial pneumonia is the most important infectious complication in patients admitted to intensive care units. Kinetic bed therapy may reduce the incidence of nosocomial pneumonia in mechanically ventilated patients. The objective of this study was to investigate whether kinetic bed therapy reduces the incidence of nosocomial pneumonia and improves outcomes in critically ill mechanically ventilated patients. METHODS: We searched Medline, EMBASE, CINAHL, CENTRAL, and AMED for studies, as well as reviewed abstracts of conference proceedings, bibliographies of included studies and review articles and contacted the manufacturers of medical beds. Studies included were randomized or pseudo randomized clinical trials of kinetic bed therapy compared to standard manual turning in critically ill mechanically ventilated adult patients. Two reviewers independently applied the study selection criteria and extracted data regarding study validity, type of bed used, intensity of kinetic therapy, and population under investigation. Outcomes assessed included the incidence of nosocomial pneumonia, mortality, duration of ventilation, and intensive care unit and hospital length of stay. RESULTS: Fifteen prospective clinical trials were identified, which included a total of 1,169 participants. No trial met all the validity criteria. There was a significant reduction in the incidence of nosocomial pneumonia (pooled odds ratio (OR) 0.38, 95% confidence interval (CI) 0.28 to 0.53), but no reduction in mortality (pooled OR 0.96, 95%CI 0.66 to 1.14), duration of mechanical ventilation (pooled standardized mean difference (SMD) -0.14 days, 95%CI, -0.29 to 0.02), duration of intensive care unit stay (pooled SMD -0.064 days, 95% CI, -0.21 to 0.086) or duration of hospital stay (pooled SMD 0.05 days, 95% CI -0.18 to 0.27). CONCLUSION: While kinetic bed therapy has been purported to reduce the incidence of nosocomial pneumonia in mechanically ventilated patients, the overall body of evidence is insufficient to support this conclusion. There appears to be a reduction in the incidence of nosocomial pneumonia, but no effect on mortality, duration of mechanical ventilation, or intensive care or hospital length of stay. Given the lack of consistent benefit and the poor methodological quality of the trials included in this analysis, definitive recommendations regarding the use of this therapy cannot be made at this time. PMID- 16684366 TI - Autoimmune targeting of key components of RNA interference. AB - RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production. PMID- 16684367 TI - Local treatment with the selective IkappaB kinase beta inhibitor NEMO-binding domain peptide ameliorates synovial inflammation. AB - Nuclear factor (NF)-kappaB is a key regulator of synovial inflammation. We investigated the effect of local NF-kappaB inhibition in rat adjuvant arthritis (AA), using the specific IkappaB kinase (IKK)-beta blocking NF-kappaB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-beta-induced IkappaB alpha phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-alpha and IL-1-beta in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-beta-induced TNF-alpha production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-alpha-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-kappaB blockade using a small peptide inhibitor of IKK-beta has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-beta-targeted NF-kappaB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis. PMID- 16684368 TI - Interleukin-15 and interferon-gamma participate in the cross-talk between natural killer and monocytic cells required for tumour necrosis factor production. AB - We have characterized the lymphocyte subset and the receptor molecules involved in inducing the secretion of TNF by monocytic cells in vitro. The TNF secreted by monocytic cells was measured when they were co-cultured with either resting or IL 15-stimulated lymphocytes, T cells, B cells or natural killer (NK) cells isolated from the peripheral blood of healthy subjects and from the synovial fluid from patients with inflammatory arthropathies. Co-culture with IL-15-activated peripheral blood or synovial fluid lymphocytes induced TNF production by monocytic cells within 24 hours, an effect that was mainly mediated by NK cells. In turn, monocytic cells induced CD69 expression and IFN-gamma production in NK cells, an effect that was mediated mainly by beta2 integrins and membrane-bound IL-15. Furthermore, IFN-gamma increased the production of membrane-bound IL-15 in monocytic cells. Blockade of beta2 integrins and membrane-bound IL-15 inhibited TNF production, whereas TNF synthesis increased in the presence of anti-CD48 and anti-CD244 (2B4) monoclonal antibodies. All these findings suggest that the cross talk between NK cells and monocytes results in the sustained stimulation of TNF production. This phenomenon might be important in the pathogenesis of conditions such as rheumatoid arthritis in which the synthesis of TNF is enhanced. PMID- 16684369 TI - RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals. AB - BACKGROUND: Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions. RESULTS: We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1alpha as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi. CONCLUSION: Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions. PMID- 16684370 TI - Influence of metabolic network structure and function on enzyme evolution. AB - BACKGROUND: Most studies of molecular evolution are focused on individual genes and proteins. However, understanding the design principles and evolutionary properties of molecular networks requires a system-wide perspective. In the present work we connect molecular evolution on the gene level with system properties of a cellular metabolic network. In contrast to protein interaction networks, where several previous studies investigated the molecular evolution of proteins, metabolic networks have a relatively well-defined global function. The ability to consider fluxes in a metabolic network allows us to relate the functional role of each enzyme in a network to its rate of evolution. RESULTS: Our results, based on the yeast metabolic network, demonstrate that important evolutionary processes, such as the fixation of single nucleotide mutations, gene duplications, and gene deletions, are influenced by the structure and function of the network. Specifically, central and highly connected enzymes evolve more slowly than less connected enzymes. Also, enzymes carrying high metabolic fluxes under natural biological conditions experience higher evolutionary constraints. Genes encoding enzymes with high connectivity and high metabolic flux have higher chances to retain duplicates in evolution. In contrast to protein interaction networks, highly connected enzymes are no more likely to be essential compared to less connected enzymes. CONCLUSION: The presented analysis of evolutionary constraints, gene duplication, and essentiality demonstrates that the structure and function of a metabolic network shapes the evolution of its enzymes. Our results underscore the need for systems-based approaches in studies of molecular evolution. PMID- 16684371 TI - Validation of the International Classification of Functioning, Disability and Health (ICF) Core Set for rheumatoid arthritis from the patient perspective using focus groups. AB - Functioning is recognized as an important study outcome in rheumatoid arthritis (RA). The Comprehensive ICF Core Set for RA is an application of the International Classification of Functioning, Disability and Health (ICF) of the World Health Organisation with the purpose of representing the typical spectrum of functioning of patients with RA. To strengthen the patient perspective, persons with RA were explicitly involved in the validation of the Comprehensive ICF Core Set for RA using qualitative methodology. The objective of the study was twofold: to come forward with a proposal for the most appropriate methodology to validate Comprehensive ICF Core Sets from the patient perspective; and to add evidence to the validation of the Comprehensive ICF Core Set for RA from the perspective of patients. The specific aims were to explore the aspects of functioning and health important to patients with RA using two different focus group approaches (open approach and ICF-based approach) and to examine to what extent these aspects are represented by the current version of the Comprehensive ICF Core Set for RA. The sampling of patients followed the maximum variation strategy. Sample size was determined by saturation. The focus groups were digitally recorded and transcribed verbatim. The meaning condensation procedure was used for the data analysis. After qualitative data analysis, the resulting concepts were linked to ICF categories according to established linking rules. Forty-nine patients participated in ten focus groups (five in each approach). Of the 76 ICF categories contained in the Comprehensive ICF Core Set for RA, 65 were reported by the patients based on the open approach and 71 based on the ICF-based approach. Sixty-six additional categories (open approach, 41; ICF-based approach, 57) that are not covered in the Comprehensive ICF Core Set for RA were raised. The existing version of the Comprehensive ICF Core Set for RA could be confirmed almost entirely by the two different focus group approaches applied. Focus groups are a highly useful qualitative method to validate the Comprehensive ICF Core Set for RA from the patient perspective. The ICF-based approach seems to be the most appropriate technique. PMID- 16684372 TI - Building bridges with astrocytes for spinal cord repair. AB - Simultaneous suppression of glial scarring and a general enhancement of axonal outgrowth has now been accomplished in an adult rat model of spinal cord transection. Transplantation of a novel astrocyte cell type derived from glial restricted precursors in vitro raise the eventual possibility of cellular therapy for spinal cord injury. PMID- 16684373 TI - Erroneous measurement of haemodynamic parameters by PiCCO monitor in a critically ill patient with renal replacement therapy: a case report. PMID- 16684374 TI - Time-point measurement is critical in hormone characterization. PMID- 16684375 TI - Early venovenous haemodiafiltration for sepsis-related multiple organ failure. PMID- 16684377 TI - Food purchasing patterns in purchase-driven societies. PMID- 16684376 TI - Biology and therapy of fibromyalgia: pain in fibromyalgia syndrome. AB - Fibromyalgia (FM) pain is frequent in the general population but its pathogenesis is only poorly understood. Many recent studies have emphasized the role of central nervous system pain processing abnormalities in FM, including central sensitization and inadequate pain inhibition. However, increasing evidence points towards peripheral tissues as relevant contributors of painful impulse input that might either initiate or maintain central sensitization, or both. It is well known that persistent or intense nociception can lead to neuroplastic changes in the spinal cord and brain, resulting in central sensitization and pain. This mechanism represents a hallmark of FM and many other chronic pain syndromes, including irritable bowel syndrome, temporomandibular disorder, migraine, and low back pain. Importantly, after central sensitization has been established only minimal nociceptive input is required for the maintenance of the chronic pain state. Additional factors, including pain related negative affect and poor sleep have been shown to significantly contribute to clinical FM pain. Better understanding of these mechanisms and their relationship to central sensitization and clinical pain will provide new approaches for the prevention and treatment of FM and other chronic pain syndromes. PMID- 16684378 TI - Out of the box. PMID- 16684379 TI - Sixth Boyd Orr Memorial Lecture. PMID- 16684380 TI - The impact of knowledge and social influences on adolescents' breast-feeding beliefs and intentions. AB - OBJECTIVES: Many health promotion educational interventions assume that increasing knowledge directly influences beliefs, intentions and behaviour, whereas research suggests that knowledge alone is insufficient for behavioural change. Social cognition frameworks such as the Theory of Reasoned Action propose a central role for beliefs and social normative influences. This Scottish study evaluates the role of knowledge and social influences (subjective norms, exposure to breast-feeding, social barriers) on beliefs and future intentions to breast feed or bottle-feed. Social influences from family and peers are investigated. DESIGN: A cross-sectional between-subjects observational design was used. A questionnaire was administered to a sample of 229 (46%) male and 267 (54%) female adolescents aged 11-18 years. SETTING: Participants completed questionnaires during lessons at three secondary schools in Central Scotland. RESULTS: Knowledge about health benefits of breast-feeding was generally poor. Analyses found that perceived social barriers to breast-feeding moderated the relationship between knowledge and beliefs. More knowledge, positive beliefs and supportive subjective norms also predicted future intentions to breast-feed. Parental norms exerted greater influence than peer norms on adolescents' breast-feeding beliefs. CONCLUSIONS: Knowledge and social influences are important predictors of positive breast-feeding beliefs and future intentions to breast-feed in adolescents. This has important implications for breast-feeding health promotion interventions in young people. PMID- 16684381 TI - Unintended pregnancies are associated with less likelihood of prolonged breast feeding: an analysis of 18 Demographic and Health Surveys. AB - OBJECTIVE: To examine the relationship between unintended pregnancies and prolonged breast-feeding among 18 countries on an individual and aggregated level. DESIGN AND SETTING: Regional multivariate logistic regression analyses were conducted to examine this association based on 18 Demographic and Health Surveys conducted between 1995 and 2000. SUBJECTS: Women who had a live child between 13 and 36 months old were included in these analyses (n=41 353). RESULTS: Regression models were adjusted for 10 covariates including child age, maternal age, pregnancy status of mother at the time of interview and parity. In 11 out of the 18 countries the odds ratio (OR) of the association between unintended pregnancies and prolonged breast-feeding was <1.0, reaching statistical significance in three countries. Pooled analyses of all 18 countries detected a significant association between unintended pregnancies and less likelihood of prolonged breast-feeding (OR=0.90, 95% confidence interval=0.85-0.96). CONCLUSIONS: Prospective studies are needed to further understand if and how pregnancy intentions influence breast-feeding outcomes in different settings. PMID- 16684382 TI - Breast-feeding and feeding practices of infants in a developing country: a national survey in Lebanon. AB - OBJECTIVE: Breast-feeding (BF) provides the ideal food for the healthy growth and development of infants. The prevalence of BF in Lebanon shows mixed results. The present study was the first large-scale, extensive survey on BF parameters in Lebanon that aimed to explore demographic, socio-economic and other fundamental issues associated with the initiation and duration of BF by Lebanese mothers. DESIGN: The survey was cross-sectional in design and administered over 10 months. SETTING: Information on all variables was collected from mothers at health centres. SUBJECTS: Two-stage sampling was conducted to select participants. A total of 1,000 participants were randomly selected. A consent form was provided to each participant. Data were collected from 830 of these. RESULTS: Almost all mothers were Lebanese, married and had given birth in a hospital. About a third stated that breast milk was the first food introduced after birth. Although 55.9% started breast-feeding their newborns within a few hours after birth, and 18.3% within half an hour, 21.2% replied that they initiated BF a few days after birth. Only 4.6% of the mothers replied that they never breast-fed their infant. Timing of initiation of BF was associated with the type of delivery (vaginal/Caesarean section) and hospital-related factors (rooming-in, night feedings and frequency of mother-infant interaction). Of the mothers who breast-fed exclusively beyond 6 months, 86.7% had initiated BF a few hours following delivery, while only 13.3% had initiated BF a few days later. Compared with the exceptionally high proportion of BF initiation, exclusivity of BF was low, dropping to 52.4% at 1 month. Exclusivity of BF was also associated with place of residence (urban/rural) and negatively associated with educational level of the mother. Duration of BF was inversely associated with the use of pain killers during delivery and maternal education. Rural mothers and those who practised exclusive BF maintained BF for a longer duration. CONCLUSION: Initiation rates of BF are very high in Lebanon but rates of exclusive BF are low and duration of BF is short. Future research targeting the factors associated with BF, with particular emphasis on exclusivity, is needed. For the 95.4% of mothers who initiated BF, an ecological perspective on intervention aimed at women and their social support system is required to improve duration and exclusivity. PMID- 16684383 TI - Predictors of poor anthropometric status among children under 2 years of age in rural Uganda. AB - OBJECTIVE: To assess predictors of poor anthropometric status among infants and young children. DESIGN: Cross-sectional survey. SETTING: The rural subsistence agricultural district of Hoima, western Uganda. SUBJECTS: Seven hundred and twenty children aged 0-23 months with their mothers/carers. METHODS: Participants were recruited in September 2002, using a two-stage cluster sampling methodology. A structured questionnaire was administered to mothers in their home settings. Information on health, household socio-economic status, child feeding practices and anthropometric measurement was gathered. Conditional logistic regression analysis was applied taking into account the hierarchical relationships between potential determinants of poor anthropometric status. RESULTS: The mean Z-score for weight-for-height was -0.2 (95% confidence interval (CI) -0.1, -0.7), for height-for-age was -1.1 (95% CI -1.2, -0.9) and for weight-for-age was -0.7 (95% CI -0.8, -0.6). Wasting was independently associated only with a history of fever in the 2 weeks prior to the survey (odds ratio (OR)=4.4, 95% CI 1.5, 13), while underweight was associated with a history of fever (OR=2.4, 95% CI 1.3, 4.4) and cough (OR=3.0, 95% CI 1.3, 6.8). Stunting was positively associated with a wider range of factors, including: history of a fever episode (OR=1.7, 95% CI 1.0, 2.9), lack of a latrine in the household (OR=2.7, 95% CI 1.5, 4.9), failure to de worm children 12 months or older (OR=1.7, 95% CI 1.1, 2.8), and being born to a non-formally educated mother compared with mothers educated above primary school (OR=2.1, 95% CI 1.1, 4.0). CONCLUSIONS: In analyses guided by the hierarchical interrelationships of potential determinants of malnutrition, wasting and underweight turned out to be independently predicted by morbidity (proximal) factors. Stunting, however, was predicted by socio-economic (distal), environmental and health-care (intermediate) factors in addition to morbidity. Strategies aimed at improving the growth of infants and young children in rural communities should address morbidity due to common childhood illness coupled with environmental and socio-economically oriented measures. PMID- 16684384 TI - Nutritional status of Amerindian children from the Beni River (lowland Bolivia) as related to environmental, maternal and dietary factors. AB - OBJECTIVES: To report the nutritional status of children aged up to 15 years and their mothers living in a remote Amazonian area of Bolivia, and to study its main social, familial and maternal determinants. SETTING: Fifteen Beni River communities located at the foot of the Andes. DESIGN: Cross-sectional survey of riverside populations. All childbearing mothers and their children in the 15 communities were examined. METHODS: Information on household production, dietary habits and demography was collected. Individual clinical, anthropometric and parasitological examinations were carried out. SUBJECTS: A total of 631 persons were examined: 171 mothers and 460 children and adolescents aged 0 to 15 years. RESULTS: There were no cases of severe wasting, but 41% of 0-5-year-olds and 36% of 5-10-year-olds were stunted. Among 346 stool specimens examined, 75% were positive for at least one helminth. Diversity of food and quality of diet were satisfactory in 54% of households, but 27% had low diversity scores. Mothers were lighter and shorter than those observed at the national level: 20% had height below 145 cm. Prevalence of anaemia (42%) was also higher. In pre-school children, multivariate analysis indicated a relationship between growth retardation and household factors such as dietary quality, ethnic group and clinical state, but not maternal anthropometry. In contrast, in school-age children and adolescents, growth retardation was related to maternal characteristics. CONCLUSIONS: Growth retardation appeared mainly during the weaning period and did not seem to improve thereafter. To ameliorate this situation, an effort should be made to prevent common parasitic and infectious diseases in young children. Follow-up of pregnant mothers during pregnancy and delivery also needs to be reinforced. PMID- 16684385 TI - Short-term daily or weekly administration of micronutrient Sprinkles has high compliance and does not cause iron overload in Chinese schoolchildren: a cluster randomised trial. AB - OBJECTIVES: To examine consumption rates and serum ferritin (SF) concentrations (as a marker of safety) among schoolchildren (3-6 years) provided with daily and weekly micronutrients. DESIGN AND METHODS: Micronutrients were provided for one school term (13 weeks) to a kindergarten in northern China as single-dose Sprinkles sachets containing 30 mg of iron as encapsulated ferrous fumarate, 5 mg zinc gluconate, 50 mg vitamin C, 300 microg vitamin A, 7.5 microg vitamin D3 and 150 microg folic acid. Sixteen classrooms were randomly assigned to: (1) daily supplements for 5 days a week (daily group); (2) weekly supplements (weekly group); or (3) no supplements (control group). Consumption of sachets was monitored for each child and SF concentrations were measured at the end of study. Random effects general linear models and graphs were used to compare the groups. RESULTS: A total of 415 children from 16 classrooms entered the study. At the end of the study, mean consumption rates per child were 86% (daily group; standard deviation (SD) 12%) and 87% (weekly group; SD 16%). Median SF concentrations were 71 microg l(-1) (range 27-292 microg l(-1); daily group), 55 microg l(-1) (range 11-299 microg l(-1); weekly group) and 54 microg l(-1) (range 7-327 microg l(-1); control group); the overall difference was not significant (P=0.06). However, the daily group was significantly different from the control (P=0.02); daily and weekly groups had higher SF at lower percentiles and similar SF at higher percentiles compared with the control group. CONCLUSION: The high consumption rates and appropriate SF concentrations in the supplemented groups suggest that a short-term school programme with Sprinkles is an efficient and safe way to provide micronutrients (including iron). PMID- 16684386 TI - Anaemia and vitamin A status among adolescent schoolboys in Dhaka City, Bangladesh. AB - OBJECTIVES: To investigate the prevalence of anaemia and subclinical vitamin A deficiency among adolescent schoolboys in Dhaka City, Bangladesh, and to identify factors related to anaemia and vitamin A status. DESIGN: A cross-sectional study. SETTING: Government high schools in Dhaka City, Bangladesh. SUBJECTS AND METHODS: A total of 381 boys, aged 11-16 years, from 10 schools in Dhaka City participated in the study. Socio-economic, anthropometric and dietary data were collected. Haemoglobin and serum retinol (vitamin A) concentrations were determined. RESULTS: Seven per cent of the boys were anaemic and 22% had serum vitamin A levels below the adequate level of 1.05 micromol l(-1), with only 1.5% having subclinical vitamin A deficiency (<0.70 micromol l(-1)). Food frequency data revealed poor dietary habits. Multiple regression analysis showed that age, body mass index (BMI), parents' occupation, serum vitamin A level and frequency of intakes of meat and fruit were significantly independently related to haemoglobin level. The overall F-ratio (13.1) was highly significant (P<0.000) and the adjusted R(2) was 0.192. For serum vitamin A, BMI, father's education, per capita expenditure on food, haemoglobin concentration and frequency of intake of vitamin A-rich fruit were found to be significantly independently related. The overall F ratio (14.5) was highly significant (P<0.000) and the adjusted R(2) was 0.186. CONCLUSION: The data show that adolescent schoolboys in Dhaka City have anaemia and inadequate vitamin A status, although the extent of the problems is lower than in other population groups in the country. Sociodemographic and dietary factors appear to have important relationships with anaemia and vitamin A status of these boys. PMID- 16684387 TI - The determinants of overweight and obesity among 10- to 15-year-old schoolchildren in the North West Province, South Africa - the THUSA BANA (Transition and Health during Urbanisation of South Africans; BANA, children) study. AB - AIM: To investigate the determinants of overweight and obesity among 10- to 15 year-old schoolchildren in a population in the transitional phase in the North West Province of South Africa. METHODS: A cross-sectional survey was used to investigate weight status (anthropometric indicators) and determinants of overweight/obesity including dietary intake, physical activity and socio-economic status. A single, random sample (n=1257), stratified for gender, type of school and ethnic group, was used. Data were collected on demographics, family circumstances, habitual physical activity, dietary intake and anthropometry to evaluate weight status and body fat content. One-way analysis of variance, the generalised linear models procedure of SAS and the Tukey post hoc honest significant difference test were used to analyse the data. RESULTS: Few children were overweight or obese (7.8%) according to International Obesity Task Force (IOTF) standards (body mass index (BMI)-for-age). These standards were compared with other accepted standard values. Both Cole's IOTF/BMI-for-age standard and the sum of skinfold thicknesses standard classified normal-weight status similarly at a level of 92% (P<0.01) and were found to be useful in determining overweight/obesity. The prevalence rate was higher in females and white children, and was more apparent in urban areas, smaller households and children of parents with low- or high-income occupations. Boys and pre-menarcheal girls had mean body fat percentage in the normal/optimal range, whereas that of post-menarcheal girls was moderately high. Few variables showed a significant association with high body fat percentage: in boys, only the number of members in the household and physical activity levels over the weekend; in girls, only age. The overweight/obese boys mostly lived in smaller households, and the overweight/obese post-menarcheal girls were most inactive on both weekdays and weekends, and more overweight with increasing age. CONCLUSION: Smaller households, inactivity and increasing age for girls were found to be determinants that influence the development of overweight/obesity, while female gender and age post-menarche were identified as determinants of higher body fat content. For overweight/obesity prevention, the focus should be on pre-menarcheal girls, aged 10-13 years, using these determinants to identify overweight/obesity risk. Preventive programmes should aim to increase the physical activity of children to improve their current and future weight status. PMID- 16684388 TI - Milk as a food for growth? The insulin-like growth factors link. AB - OBJECTIVE: The mechanisms underlying the association of insulin-like growth factor-I (IGF-I) and leg length (a marker of prepubertal growth) with cancer risk are uncertain. One hypothesis is that diet in early childhood might provide the link. The aim of the present study was to examine the association between early diet - in particular, the intakes of cows' milk and dairy products - and height, leg length and IGF-I levels at age 7-8 years. SUBJECTS: Children participating in the Avon Longitudinal Study of Parents and Children. DESIGN: Diet was assessed using a 3-day unweighed food record. Anthropometry, IGF-I and insulin-like growth factor-binding protein-3 (IGFBP-3) were measured by standard methods. RESULTS: Data on both diet and height were available for 744 children (404 boys) and on diet and IGF for 538 (295 boys). After adjusting for energy, both cows' milk and dairy product intakes were positively associated with IGF-I (P=0.040 and 0.027, respectively) and IGFBP-3 levels (P=0.082 and 0.067, respectively). These associations persisted on adjustment for potential confounders, but were abolished on controlling for protein intake. In energy-adjusted models there was only weak evidence of associations of milk and dairy product intakes with anthropometry. In boys only, dairy product intake was positively associated with leg length (equivalent to a 0.058 (0.002, 0.114) standard deviation score increase in leg length per 100 g increase in daily intake). CONCLUSIONS: These data provide some evidence that variation in childhood milk and dairy product intakes underlies associations of leg length, IGF-I and cancer risk. The association appears to be due to the protein content of milk. PMID- 16684389 TI - Intake of specific flavonoids and risk of acute myocardial infarction in Italy. AB - OBJECTIVE: As intake of flavonoids has been associated with reduced risk of coronary heart disease but data on the relation with specific classes of flavonoids are scarce, we assessed the relation between dietary intake of specific classes of flavonoids and the risk of acute myocardial infarction (AMI) in an Italian population. DESIGN: Case-control study. Dietary information was collected by interviewers on a questionnaire tested for validity and reproducibility. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were obtained by multiple logistic regression models including terms for energy and alcohol intake, as well as sociodemographic factors, tobacco and other major recognised risk factors for AMI. SETTING: Milan, Italy, between 1995 and 2003. SUBJECTS: Cases were 760 patients, below age 79 years, with a first episode of non-fatal AMI, and controls were 682 patients admitted to hospital for acute conditions unrelated to diet. RESULTS: A reduced risk of AMI was found for increasing intake of anthocyanidins (OR=0.45, 95% CI 0.26-0.78 for the highest vs. the lowest quintile, Ptrend=0.003) and flavonols (OR=0.65, 95% CI 0.41-1.02, Ptrend=0.02). A tendency towards reduced risks, although not significant, was observed for flavan-3-ols (OR=0.73, 95% CI 0.48-1.10) and total flavonoids (OR=0.74, 95% CI 0.49-1.14). No meaningful heterogeneity was found between the sexes. No association emerged for other flavonoids, including isoflavones, flavanones and flavones. CONCLUSIONS: High intake of anthocyanidins reduced the risk of AMI even after allowance for alcohol, fruit and vegetables, supporting a real inverse association between this class of flavonoids and AMI risk. PMID- 16684390 TI - Socio-economic pathways to diet: modelling the association between socio-economic position and food purchasing behaviour. AB - OBJECTIVES: To examine the association between education level and food purchasing behaviour and the contribution of dietary knowledge to this relationship; and the association between household income and purchasing behaviour and the contribution made by subjective perceptions about the cost of healthy food. DESIGN AND SETTING: The study was conducted in Brisbane City (Australia) in 2000. The sample was selected using a stratified two-stage cluster design. Data were collected by face-to-face interview from residents of private dwellings (n=1003), and the response rate was 66.4%. Dietary knowledge was measured using a 20-item index that assessed general knowledge about food, nutrition, health and their interrelationships. Food-cost concern was measured using a three-item scale derived from principal components analysis (alpha=0.647). Food purchasing was measured using a 16-item index that reflected a household's purchase of grocery items that were consistent (or otherwise) with dietary guideline recommendations. Associations among the variables were analysed using linear regression with adjustment for age and sex. RESULTS: Significant associations were found between education, household income and food purchasing behaviour. Food shoppers with low levels of education, and those residing in low income households, were least likely to purchase foods that were comparatively high in fibre and low in fat, salt and sugar. Socio-economic differences in dietary knowledge represented part of the pathway through which educational attainment exerts an influence on diet; and food purchasing differences by household income were related to diet in part via food-cost concern. CONCLUSIONS: Our findings suggest that socio-economic differences in food purchasing behaviour may contribute to the relationship between socio-economic position and food and nutrient intakes, and, by extension, to socio-economic health inequalities for diet-related disease. Further, socio-economic differences in dietary knowledge and concerns about the cost of healthy food play an important role in these relationships and hence should form the focus of future health promotion efforts directed at reducing health inequalities and encouraging the general population to improve their diets. PMID- 16684391 TI - Food-purchasing patterns for home: a grocery store-intercept survey. AB - OBJECTIVES: To identify the most common frequency of food-purchasing patterns and relate this pattern to characteristics of individuals and families. DESIGN: A customer-intercept survey was conducted in the greater Houston area, Texas, USA, in 2002. The frequency of food shopping at supermarkets, convenience stores and restaurants to buy food for eating at home was assessed. SUBJECTS: A total of 823 adults (78.5% female; mean age 37.4 years) who went to any of several grocery or convenience stores, including European, Hispanic and African Americans, and Asian/Pacific Islanders. RESULTS: Major food-shopping patterns were a weekly big trip with a few small trips (34.9%), biweekly big trips with a few small trips (21.9%), no big shopping trips (15.4%), a weekly big trip without small trips (13.9%), a monthly big trip (8.3%), and biweekly big trips without small trips (6.4%). While 61.1% of participants never went to convenience stores to buy fruit and vegetables (F&V) for eating at home, 67% went to restaurants for F&V. African American families shopped for food least frequently, while Asian American families shopped for food most frequently. Educational level was negatively associated with the use of convenience stores and positively associated with take away from restaurants. CONCLUSIONS: There is substantial variability in the frequency of food shopping. Future research on food shopping should incorporate this variable. PMID- 16684392 TI - The effect of personal characteristics on the validity of nutrient intake estimates using a food-frequency questionnaire. AB - OBJECTIVE: To assess validity of the Nambour food-frequency questionnaire (FFQ) relative to weighed food records (WFRs), and the extent to which selected demographic, anthropometric and social characteristics explain differences between the two dietary methods. DESIGN: Inter-method validity study; 129-item FFQ vs. 12 days of WFR over 12 months. SETTING: Community-based Nambour Skin Cancer Prevention Trial. SUBJECTS: One hundred and fifteen of 168 randomly selected participants in the trial (68% acceptance rate) aged 25-75 years. RESULTS: Spearman correlations between intakes from the two methods ranged from 0.18 to 0.71 for energy-adjusted values. Differences between FFQ and WFR regressed on personal characteristics were significantly associated with at least one characteristic for 16 of the 21 nutrients. Sex was significantly associated with differences for nine nutrients; body mass index (BMI), presence of any medical condition and age were each significantly associated with differences for three to six nutrients; use of dietary supplements and occupation were associated with differences for one nutrient each. There was no consistency in the direction of the significant associations. Regression models explained from 7% (riboflavin) to 27% (saturated fat) of variation in differences in intakes. CONCLUSIONS: The relative validity of FFQ estimates for many nutrients is quite different for males than for females. Age, BMI, medical condition and level of intake were also associated with relative validity for some nutrients, resulting in the need to adjust intakes estimates for these in modelling diet-disease relationships. Estimates for cholesterol, beta-carotene equivalents, retinol equivalents, thiamine, riboflavin and calcium would not benefit from this. PMID- 16684393 TI - News and information: the future of nutrition in Africa is bright. PMID- 16684395 TI - Pathophysiology and therapy for haemoglobinopathies. Part II: thalassaemias. AB - Thalassaemias result from mutations of the globin genes that cause reduced or absent haemoglobin production and thus interfere with the critical function of oxygen delivery. They represent the most common single-gene disorders, with 4.83% of the world population carrying globin gene variants. Reduced or absent alpha globin (alpha-thalassaemia) or beta-globin (beta-thalassaemia) leads to anaemia and multifaceted clinical syndromes. In this second of two reviews on the pathophysiology of haemoglobinopathies, we describe the clinical features, pathophysiology and molecular basis of alpha- and beta-thalassaemias. We then discuss current targeted therapies, including the new oral iron chelators, which, along with chronic transfusions, constitute the mainstay of symptomatic therapy for the majority of patients. Finally, we describe potentially curative therapies, such as bone marrow transplant, and discuss some of the outstanding research studies and questions, including the upcoming field of gene therapy for beta-thalassaemia. An accompanying article on haemoglobinopathies (Part I) focuses on sickle cell disease. PMID- 16684396 TI - Alternate forms of logical memory and verbal fluency tasks for repeated testing in early cognitive changes. AB - BACKGROUND: Repeat cognitive testing is an essential diagnostic strategy to measure changes in cognition over time when following people with memory problems. Alternate forms may avert practice effects that can mimic improvements in cognition. We evaluated alternate forms of verbal fluency and logical memory (paragraph recall) tasks to evaluate their equivalence for clinical use. METHODS: Participants with mild cognitive impairment (MCI) and dementia were recruited from five outpatient memory clinics and one nursing home. Participants with normal cognition (NC) were recruited from family members or friends. Verbal fluency categories of animals, cities & towns, fruits & vegetables and first names were used. Scores were recorded for 0-30 seconds, 31-60 seconds and errors. For the logical memory task, participants were read one of three different paragraphs and then were asked to recall the story. Immediate recall and delayed recall scores were recorded. The Standardized Mini-mental State Examination, the AB Cognitive Screen and the 15-point Geriatric Depression Scale were administered as part of the assessment. Analyses were performed using means, frequency distributions, t-tests, receiver-operating characteristic curves and effect sizes. RESULTS: There were 46 NC participants, 45 with MCI and 55 with dementia. For verbal fluency, the mean number of animals, cities & towns, names or fruits & vegetables named in 60 seconds did not differ significantly within each cognitive group. First names was an easier category than the others: NC named 16.9-22.3 items, MCI named 11.6-14.4 items and dementia named 8.1-11.4 items. The mean number of items immediately recalled in logical memory was not significantly different for the three paragraphs. The verbal fluency task (in 60 seconds) and logical memory immediate recall were highly sensitive and specific to differences between NC and MCI (areas under the curves 0.87 and 0.76, respectively). CONCLUSIONS: Alternate forms allow serial testing without learning bias. Verbal fluency and logical memory tasks are sensitive to early cognitive changes. PMID- 16684397 TI - Evaluation of a psychiatric day hospital program for elderly patients with mood disorders. AB - BACKGROUND: Very little is known about the utility of psychiatric day hospitals for elderly adults with mood disorders. The objectives of this study were to evaluate a long-standing day-hospital program and to explore whether demographic and non-demographic patient characteristics were associated with treatment outcomes. METHOD: We used t-tests to compare retrospective admission and discharge data for 708 patients over a 16-year period, and multiple regression to examine predictors of improvement. RESULTS: Depressed patients showed statistically and clinically significant improvements on the Geriatric Depression Scale and the Hamilton Depression Rating Scale. The number and severity of depressive symptoms at admission were strongly related to treatment outcomes. After controlling for initial levels of depression, demographic characteristics did not predict improvement, and axis I and II diagnoses modestly and inconsistently predicted improvement. CONCLUSIONS: A biopsychosocially-focused day-hospital treatment program was associated with improvements in depression in a large sample of elderly adults with mood disorders. Except for depression severity at admission, patient characteristics had very little impact on treatment outcomes, suggesting that day hospital programs are beneficial for a wide range of depressed elderly adults. PMID- 16684398 TI - The association between congestive heart failure and cognitive performance in a primary care population of elderly adults: the Steel Valley Seniors Survey. AB - BACKGROUND: Evidence suggests an association between congestive heart failure (CHF) and cognitive function, particularly in heart transplant patients and patients hospitalized for CHF. We examined the association between CHF and cognitive performance in stable outpatients recruited from primary care. METHODS: This is a cross-sectional secondary data analysis of the Steel Valley Seniors Survey, an epidemiological study of elderly primary care outpatients. Participants aged >/= 65 years were recruited in primary care clinics. The study cohort (n = 354) is a subgroup, composed of subjects with Mini-mental State Examination score < 25, and a random sample of the remaining, who underwent a baseline assessment in the home. The assessment included demographics, comorbid illnesses, depressive symptoms, functional status, a neurological examination and a neuropsychological battery. CHF is defined by self-report and/or chart review, and stable CHF as not being hospitalized in the year prior to the assessment. The associations between CHF and specific cognitive tests were examined by bivariate analysis and logistic regression, controlling for demographic variables. RESULTS: Subjects with CHF performed worse on tests of visual memory [10.1 (S.D. 5.4) vs. 12.7 (S.D. 5.2), p = 0.007], Trailmaking B [0.1 (0.1) vs. 0.2 (0.1), p = 0.002], category fluency [11.1 (4.4) vs. 13.4 (4.5), p = 0.008], and clock drawing [5.6 (1.9) vs. 6.7 (1.4), p < 0.001] compared to subjects without CHF, after adjustment for relevant demographic variables. CONCLUSION: CHF is associated with lower cognitive functioning in a population of patients with stable heart failure in primary care settings. PMID- 16684399 TI - Increased risk of dementia following mild head injury for carriers but not for non-carriers of the APOE epsilon4 allele. AB - BACKGROUND: The epsilon4 allele of apolipoprotein E (APOE) and head injury are risk factors for dementia diseases, and may act synergistically to further increase the risk. The aim of this study was to examine the association between mild head injury, APOE and dementia. METHODS: Data were obtained from the Betula prospective population-based study of aging, memory, and health. The study included 543 participants in the age range 40-85 years, free of dementia at baseline, who were followed up within a 5-year interval. Dementia was classified using DSM-IV criteria. Information on previous head injury was obtained through screening of the participants' answers to health questionnaires at baseline and at follow-up. RESULTS: Subjects with head injury but without APOE epsilon4 had no increased risk of dementia. Subjects with APOE epsilon4 had an increased risk and those with both APOE epsilon4 and head injury had the highest risk of dementia (odds ratio = 5.2). CONCLUSIONS: APOE epsilon4 constitutes a risk factor for dementia, mild injury in isolation does not increase the risk, but head injury in combination with the APOE epsilon4 leads to increased risk of dementia. PMID- 16684400 TI - On the need for combining complementary analyses to assess the effect of a candidate gene and the evolution of its polymorphism: the example of the Major Histocompatibility Complex in chicken. AB - The aim of this paper is to combine different but complementary approaches to check the neutrality of a given locus in a selected population. Analysis was undertaken through the polymorphism's evolution compared with that predicted under the effect of drift and through the analysis of the variance components of the measured traits, considering the effect of the locus as either a fixed or a random effect. This study deals with the case of the MHC locus, using both data from experimental lines of chicken selected for three different criteria of immune response, and frequencies of the genotyped haplotypes over time. Both the evolution of the polymorphism and the variance components approach have led to the conclusion that the MHC locus has an effect on the trait affecting antibody production against the Newcastle disease virus. Results have also highlighted the interest in using various methods in the case of low allelic frequencies. However, none of the common hypotheses, overdominance or frequency-dependent selection, was sufficient to explain the observed variation of the MHC polymorphism, which was displayed by the temporal variation of the allelic frequencies. PMID- 16684401 TI - The emerging strains of Shigella dysenteriae type 2 in Bangladesh are clonal. AB - A total of 113 strains of Shigella dysenteriae type 2 isolated from patients attending the Dhaka diarrhoea treatment centre of ICDDR,B: Centre for Health and Population Research during the period 1999-2004 were studied. Serotype of the isolates was confirmed using commercially available antisera. Except for arabinose fermentation, all the strains had similar biochemical reactions. More than 60% of the strains were sensitive to commonly used antibiotics; only 6% (n=7) of the strains were resistant to nalidixic acid, and none of the strains were resistant to mecillinam and ciprofloxacin. All strains were invasive as demonstrated by the presence of a 140 MDa plasmid, ial, sen and ipaH genes, Congo Red absorption ability and by the Sereny test performed on representative strains. Plasmid patterns were heterogeneous but more than 50% of strains were confined to a single pattern. All strains possessed a 1.6 MDa plasmid and 87% of the strains contained a 4 MDa plasmid. Middle-range plasmids (90 MDa to 30 MDa) present in 36% of the strains were not associated with antibiotic resistance. All the strains were clustered within a single type with four subtypes by pulsed field gel electrophoresis while ribotyping patterns of all the strains were identical. PMID- 16684402 TI - Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus isolated from the nasopharynx of healthy children attending day-care centres in the Czech Republic. AB - Nasopharyngeal carriage of potential pathogens was studied in 425 healthy 3- to 6 year-old children attending 16 day-care centres (DCCs) in nine Czech cities during the winter 2004-2005. The overall carriage of pathogens was 62.8% (Streptococcus pneumoniae, 38.1%; Haemophilus influenzae, 24.9%; Moraxella catarrhalis, 22.1%; Staphylococcus aureus, 16%). An age-related downward trend was observed for colonization with respiratory pathogens in contrast to Staph. aureus whose carriage was significantly higher among older children. The following serotypes of colonizing S. pneumoniae were the most predominant: 23F (20.6%), 6A (15.1%), 6B (12.7%), 18C (7.8%), 15B and 19F (6% each). The majority (94.3%) of H. influenzae isolates were non-typable; among capsulated isolates, serotype b was not found. Decreased susceptibility to penicillin was determined in 3% of pneumococci; 4.6% of H. influenzae strains and 85.1% of M. catarrhalis strains produced beta-lactamase. As for non-beta-lactam antibiotics, pneumococci resistant to trimethoprim-sulphamethoxazole were the most common (15.7%) among the attendees. PMID- 16684404 TI - Hepatitis A surveillance in England--how many cases are not reported and does it really matter? AB - Underreporting of hepatitis A infection in England may be high and a number of outbreaks have occurred undetected by routine surveillance. We evaluated surveillance of hepatitis A cases by employing capture-recapture analysis on data from two distinct outbreaks of hepatitis A. The overall reporting of cases of hepatitis A was 81.7% (95% CI 55.3-95) in the first outbreak in North East England and reporting through Lab Base was 65.7% (95% CI 42.8-76.4). In the second outbreak in the East Midlands the overall reporting of hepatitis A cases was 27.8% (95% CI 19-38.7) and through Lab Base 16.6% (95% CI 11.4-23.1). Underreporting of hepatitis A cases is high. Public health interventions exist to prevent and control outbreaks of hepatitis A. The lack of reliable data on incidence and prevalence hampers effective public health management of this disease. PMID- 16684403 TI - Incubation period as a clinical predictor of botulism: analysis of previous izushi-borne outbreaks in Hokkaido, Japan, from 1951 to 1965. AB - This study investigated 21 foodborne type-E botulism outbreaks, without antitoxin administration, from 1951 to 1965 in Hokkaido, Japan, to characterize the descriptive epidemiology and evaluate the relationship between case fatality and incubation period. The median (25-75% quartile) attack rate and case fatality, which were evaluated by outbreak, were 58.3% (38.0-73.2) and 25.7% (0.1-50.0) respectively. Individual records of 64 diagnoses, including 31 deaths, were also examined using logistic regression analysis, revealing that a shorter incubation period is likely to result in a significantly higher risk of death (P=0.01). The observed case fatality was more than 50% for those who developed symptoms within the first 18 h after exposure, possibly reflecting underlying dose-dependent mechanisms. In the event of intentional contamination of food with botulinum toxin, rapidly determining the incubation periods may be critical for guiding public health response efforts. PMID- 16684422 TI - Breast cancer follow-up: could primary care be the right venue? AB - AIMS: To identify the elements of a follow-up protocol for treated breast cancer patients in primary care with reference to key stakeholders in one region of the UK. METHODS: Stage 1: a survey of 100 consecutive hospital records relating to patients treated for primary breast cancer. The most common problems managed at follow-up and the type and frequency of resources used were identified. Stage 2: focus groups with stakeholders identifying potential barriers to follow-up of breast cancer patients in primary care after successful therapy. Stage 3: a nominal group outlined the elements of a follow-up protocol in primary care. RESULTS: The most frequently recorded problems in 702 patient years of follow-up were anxiety, unrelated medical problems and joint pain. Anxiety and depression tend to present relatively soon and are often enduring whereas concomitant medical problems also present later. Health care professionals considered patients difficult to manage because symptoms of recurrence require investigation for absolute reassurance of the symptomatic patient. However, investigations other than mammograms were seldom necessary. CONCLUSIONS: A multidisciplinary panel identified attention to the psychosocial sequelea of breast cancer as a vital aspect of follow-up. Patients and their partners are preoccupied with a fear of recurrence. This may manifest in a variety of guises including mental health problems. These can be addressed in primary care especially with the support of counsellors, with teamwork and agreed protocols for referral back to specialists when indicated. PMID- 16684423 TI - Drug utilization of ezetimibe in rehabilitation centres: registry analysis of factors influencing prescription and effectiveness of treatment. AB - BACKGROUND: While randomized controlled trials (RCTs) generate informative data about clinical outcomes, by their nature they cannot provide information about drug utilization and factors influencing prescribing decisions. In the secondary prevention of patients with cardiac events, lipid lowering therapy with statins and other agents, such as cholesterol absorption inhibitors (CAI, e.g. ezetimibe) plays a pivotal role and is often initiated or modified in rehabilitation centres. The aims of the present study were to analyse factors that influence the prescribing decisions of physicians, and to investigate success rates of lipid lowering therapy with ezetimibe after adjustment for covariates. METHODS: Ninety three rehabilitation centres throughout Germany documented a total of 17029 patients in cardiac rehabilitation, of which 6976 (41.6%) were prescribed a CAI. A logistic regression model with forward selection based on 31 potential regressors for ezetimibe prescription (demographics, diagnosis, risk factors etc.) was used to construct a propensity score, which reflects the inclination of physicians to prescribe CAI. This score was subsequently used for bias reduction in the comparison of co-medications and success rates. RESULTS: Nineteen variables were associated with ezetimibe prescriptions, the most important ones being total cholesterol, level of education, unstable angina pectoris and arterial hypertension. Ezetimibe was more frequently prescribed together with simvastatin and pravastatin than with other statins, and frequently together with aspirin or beta blockers, respectively. After adjustment for baseline lipid values and covariates, the probability of target level achievement appears to be substantially higher for patients on ezetimibe than for those without ezetimibe. CONCLUSIONS: Other factors than conventional risk factors contribute to the CAI prescription habits of physicians. Additional lipid level reductions due to ezetimibe are seen in routine health care corresponding to findings from randomized studies. PMID- 16684424 TI - Rationale and design for the CARDIOVIT Study (Cardiovit, Atherosclerotic vascular disease and hypeRhomocysteinemia: an epiDemiological study in Indians, additionally evaluating the effect of Oral VITamin supplementation). AB - BACKGROUND AND RATIONALE: Atherosclerotic vascular disease (AVD), including coronary heart disease (CHD), is on the rise in India. Mortality due to CHD is estimated to be 40% higher in Indian Asians compared with Europeans. Hyperhomocyst(e)inaemia (hypertHcy) is emerging as a novel independent risk factor for AVD. Moreover, there is increasing evidence of its association with CHD in subjects of Indian origin possibly because of inadequate folic acid and B(12) status. However, there is a lack of epidemiological data on its prevalence in Indians. A need to evaluate the optimum dose of folic acid required to reduce plasma homocyst(e)ine (tHcy) also exists. OBJECTIVES AND METHODS: The CARDIOVIT study, divided into two stages, is designed to determine the prevalence and relevance of hypertHcy in Indian subjects and also evaluate the tHcy-lowering effect of a marketed multivitamin formulation (Cardiovit). Stage I is a cross sectional epidemiological study to estimate the prevalence of hypertHcy in a randomly selected population in South India. We planned to screen 2000 subjects between 25 and 64 years of age for established risk factors; plasma tHcy levels will be estimated and the correlation of AVD with established risk factors, plasma tHcy and nutritional factors was evaluated. Subjects having tHcy levels > 12 micromol/L are recruited in Stage II; a randomized, three-arm, double-blind, double-dummy, placebo-controlled, parallel-group, comparative study designed to evaluate the tHcy-lowering effect of a multivitamin formulation containing 300 microg folic acid, 1 microg vitamin B(12) and 1.5 mg vitamin B(6), along with Vitamins A, C, E and selenium. Subjects will be randomized into three groups (n = 64 completed subjects each) and receive either two placebos, or one placebo and multivitamin, or two multivitamin tablets for 6 weeks. Serum vitamin B(12), serum and red cell folate are estimated at baseline and at 6 weeks. Descriptive, univariate analysis, multiple logistic regression model and appropriate statistical tests will be used to analyse the prevalence of hypertHcy, association with risk factors of AVD, efficacy and safety of the multivitamin formulation and other data per protocol. CONCLUSIONS: CARDIOVIT is one of the first and largest studies in India to provide insight into the prevalence of hypertHcy, its association with AVD and conventional risk factors and the role of multivitamins in reducing it. PMID- 16684425 TI - Effect of a new leuprorelin formulation on testosterone levels in patients with advanced prostate cancer. AB - BACKGROUND AND SCOPE: Leuprorelin is a well known luteinising hormone releasing hormone (LHRH) agonist. The drug is effective in the treatment of advanced prostate cancer and is well tolerated. This article reviews published literature (based on a search of PubMed, EMBASE and Biosis databases to the end of 2005) and other sources of data on a new formulation of leuprorelin acetate (Eligard) for use in the treatment of hormone-dependent advanced prostate cancer. This product takes advantage of a novel delivery system (Atrigel) which forms an implant in situ that is capable of delivering double doses of leuprorelin consistently to provide better, more sustained testosterone suppression compared with a microsphere leuprolide acetate formulation. Two formulations, 7.5 mg and 22.5 mg, are currently available with duration of action of 1 and 3 months, respectively. The 2-week stability at room temperature prior to mixing facilitates its use and reduces the potential for waste. FINDINGS: In clinical studies of the new leuprorelin acetate formulation reviewed here, all patients achieved testosterone levels < or = 50 ng/dL and up to 98% of patients showed levels comparable to those resulting from surgical bilateral orchidectomy (< or = 20 ng/dL). Both formulations showed minimal breakthroughs, defined as a rise in testosterone levels after reaching levels of 50 ng/dL. The safety profile is typical of LHRH agonists, with mild to moderately severe 'hot flushes' being the most common adverse event. The higher dose of 22.5 mg, with a volume of 0.375 mL is administered subcutaneously via a small 20G needle, causing little local discomfort. CONCLUSION: Prostate cancer remains a major cause of morbidity and mortality in older men. In the majority of cases, suppression of serum testosterone levels is very effective. The level of testosterone suppression is currently under debate, with ideal suppression levels ranging from 20 to 50 ng/dL. Not all LHRH agonist therapy achieves the same degree of testosterone suppression as bilateral orchidectomy. The new leuprorelin acetate (Eligard) appears to achieve a testosterone suppression of 20 ng/dL in 98% of patients, while maintaining a side effect profile comparable to other products in its class. PMID- 16684426 TI - Does the treatment of gastroesophageal reflux disease (GERD) meet patients' needs? A survey-based study. AB - OBJECTIVE: Symptoms of gastroesophageal reflux disease (GERD) affect approximately 20% of people on a weekly basis. A number of different therapies are prescribed to control the disease. This survey-based study was carried out to assess patients' and physicians' perceptions of GERD and its treatment. METHODS: Randomly selected general practitioners (GPs) from five countries (USA, UK, Japan, Germany and France) took part in a faceto-face interview, using a standard questionnaire, concerning the last four GERD patients (those taking GERD medication) who had consulted them and who consented to be interviewed. Those patients were then interviewed via telephone, also using a standard questionnaire. RESULTS: Completed questionnaires were available for 927 of the 1044 patients who were identified. The mean length of time that patients suffered GERD symptoms prior to consultation was more than 1.5 years, with 52.3% of those consulting a GP stating the reason they sought medical attention was that 'symptoms were too uncomfortable to bear'. Only 36% of patients receiving prescription therapy reported that they were currently asymptomatic; 20.5% of patients were also taking at least one over-the-counter (OTC) medication. CONCLUSIONS: In the primary care setting, many patients receiving GERD therapy do not have fully controlled symptoms. It is recommended that GPs question patients routinely about persistent symptoms on therapy, and OTC use, in order that effective treatment choices are made in the management of GERD. PMID- 16684427 TI - Costs of detection and treatment of cervical cancer, cervical dysplasia and genital warts in the UK. AB - OBJECTIVE: Infection with human Papillomavirus (HPV) is a necessary cause of cervical cancer (CC) and genital warts (GW). HPV vaccination studies have shown excellent efficacy against HPV-induced lesions. To assess the cost-effectiveness of a HPV quadrivalent (6, 11, 16 and 18) vaccine it is necessary to estimate the costs of managing current levels of HPV-related diseases. This study estimates the annual 2003 expenditures in the UK for CC screening, follow-up of abnormal findings, CC treatment and GW treatment. DESIGN AND METHODS: CC screening programmes provided the annual number of screening tests, their results and use of colposcopy procedures in women with abnormal findings. Incident CC cases and hospital admissions for CC in 2003 were used to estimate CC costs. Health Protection Agency data provided the annual number of new, recurrent or persistent cases of GW treated in Genitourinary Medicine (GUM) clinics. Treatment patterns for managing GW were estimated by GUM clinicians. The annual physician visits, tests, procedures, hospital admissions and topical genital wart medications were costed to estimate the total annual expenditures for CC and GW. RESULTS: There were 4.8 million screening tests and 230 303 colposcopy procedures. Estimated costs for screening, management of abnormal and inadequate findings were 138.5 million pounds sterlings. Annual management costs for incident and prevalent CC cases were 46.8 million pounds sterlings. There were an estimated 76 457 incident and 55 657 recurrent/persistent GW cases in 2003. The costs for managing these cases were approximately 22.4 million pounds sterlings. Total annual estimated costs for CC screening, management and treatment of GW were 208 million pounds sterlings and ranged from 186.9 pounds sterlings to 214 million pounds sterlings based upon sensitivity analyses. CONCLUSIONS: The direct medical costs for the NHS associated with detection and management of CC, cervical dysplasia and treatment of GW in the UK are substantial. These medical costs are invaluable for future cost-effectiveness analyses of a quadrivalent HPV vaccine programme. PMID- 16684428 TI - Self-monitoring of glucose in type 2 diabetes mellitus: a Bayesian meta-analysis of direct and indirect comparisons. AB - OBJECTIVE: To evaluate the relative effectiveness of interventions with self monitoring blood glucose and self-monitoring of urine glucose, versus interventions without self-monitoring, in terms of HbA(1c) reductions in type 2 diabetes mellitus. METHODS: Thirteen published full reports on randomised controlled trials investigating the effects of self-monitoring glucose were identified by a systematic search of Medline, Embase, the Cochrane Library (1966 Nov 2005) and previous reviews. Three types of studies were included: self monitoring of blood glucose versus no self-monitoring, self-monitoring of blood glucose versus self-monitoring of urine glucose and self-monitoring of blood glucose with regular feedback versus monitoring without feedback. The internal validity of studies was assessed systematically by two reviewers, using 13 criteria of a validated list. Results from the three types of studies were analysed simultaneously with a Bayesian metaanalysis of direct and indirect comparisons. RESULTS: Adjusted for baseline HbA(1c) level and internal validity, interventions with self-monitoring of blood glucose showed a reduction in HbA(1c) of 0.40 percentage-points (%) (95% credible interval [CrI] 0.07 to 0.70%) in comparison to interventions without self-monitoring. Regular feedback more than doubled the HbA(1c) reduction. Self-monitoring of urine glucose showed comparable results to interventions without self-monitoring (0.02% decrease in HbA(1c); 95% CrI -0.62 to 0.70%). There is a 88% probability that interventions with self monitoring blood glucose are more effective than interventions with urine glucose monitoring (relative reduction in HbA(1c) is 0.38%, 95% CrI -0.30 to 1.00%). CONCLUSION: The randomized clinical trials performed to date provided positive results on the effectiveness of interventions with self-monitoring of blood glucose in type 2 diabetes mellitus. Regular medical feedback of the monitored HbA(1c) levels is important. Furthermore, self-monitoring of blood glucose is likely to be more effective than self-monitoring of urine glucose. PMID- 16684429 TI - Direct costs of warfarin treatment among patients with atrial fibrillation in a Finnish health care setting. AB - OBJECTIVE: The main objective was to estimate the mean direct costs of warfarin treatment for atrial fibrillation (AF) patients. Secondly, the costs of initiating warfarin treatment during a 60-day period and the impact of International Normalized Ratio (INR) and co-morbidities on costs were estimated. DESIGN AND DATA: The study was performed as a retrospective cohort study over a 12-month period in a Finnish communal health care setting. All AF patients aged 65 years or older (n = 250) with warfarin treatment were identified from the database of the health service district of an urban area. Patient specific information related to comorbidities, INR-control, complications and health care resource use were collected. Cost information was obtained from the Finnish national health care unit cost list. METHODS: The effect of treatment balance and other background variables on treatment costs were evaluated using ordinary least squares regression (OLS), log-transformed OLS and generalized linear model (GLM). The mean costs were calculated on the basis of the different models and bias corrected and accelerated (BCa) bootstrap confidence intervals (CIs) were calculated for the mean costs. RESULTS: The best fitting cost model was log transformed OLS. The costs of warfarin treatment on the basis of the log transformed model were 589.82 Euros (BCa 95% CI: 586.68-591.99) per patient compared to 616.00 Euros (BCa 95% CI: 579.98-652.96) obtained with the OLS-model. For the treatment initiation period, the mean costs were 263 Euros (BCa 95% CI: 218.90-314.71). Depending on the way that INR-control was defined, the mean costs were 95.27 Euros or 166.92 Euros higher for patients who were not in the defined INR-balance. CONCLUSIONS: The INR-control has a significant impact on the warfarin treatment costs. The choice of model influences the estimated mean costs. In addition, different models identify statistically significant effects between different background variables and costs. PMID- 16684430 TI - Efficacy and tolerability of adjunctive lamotrigine for refractory epilepsy in institutional or community residents with mental retardation. AB - OBJECTIVE: To determine whether the efficacy and tolerability of adjunctive lamotrigine differ as a function of whether persons with mental retardation and refractory epilepsy resided in institutions or the community. RESEARCH DESIGN AND METHODS: The analysis was retrospective and employed data from a previously published, open-label study. To be eligible for enrollment, patients needed to be receiving stable regimens of up to three antiepileptic medications at enrollment and to have experienced at least two seizures per month in the 3 months prior to enrollment. The study included an Escalation Phase during which lamotrigine was titrated to a target dose, an 8-week Maintenance Phase during which doses of lamotrigine and concomitant antiepileptic drugs were maintained, and a 12-week Optimization Phase during which doses of antiepileptic drugs could be adjusted. Outcomes of interest included baseline characteristics; the percentage of persons with 25%, 50%, and 75% reductions in seizure frequency and with zero seizures; investigator-rated clinical status; and mean scores on the Habilitative Improvement Scale and the Aberrant Behavior Checklist. The small sample size did not allow for sufficient power for statistical comparisons between groups. RESULTS: Demographics and baseline clinical characteristics were similar between groups (31 institutional and 36 community residents) except institutional residents were more likely to have severe or profound mental retardation and complex partial and partial secondarily generalized seizures. The percentages of persons with 25%, 50%, and 75% reductions in seizure frequency and zero seizures were 64%, 50%, 29%, and 18% respectively, for community residents and 42%, 38%, 23%, and 12% for institutional residents. Investigator-rated clinical status at the end of the Maintenance Phase was improved versus baseline in 68% of community residents and 62% of institutional residents. Most individuals (81% of institutionalized persons, 69% of community-dwelling persons) were considered to have experienced no change in adverse events with the addition of lamotrigine. The mean Habilitative Improvement Scale score was significantly improved (p < or = 0.05) versus baseline at the end of the Escalation, Maintenance, and Optimization Phases in both groups. CONCLUSION: The efficacy and tolerability of lamotrigine were similar between institutional residents and community residents despite the numerically higher prevalence of severe or profound mental retardation among institutional residents. PMID- 16684431 TI - Initial experience with teriparatide in the United States. AB - Teriparatide has been commercially available in the United States (US) for over 3 years. This summary spans the early experience with this therapy. As of December 31, 2005, over 235 000 patients had filled a prescription for teriparatide world wide. Data collected from July to December 2004, from 15,000 retail pharmacies in the US, indicated that the mean age of patients was 67.5 years, and more recent data collected from January through October 2005 indicated that 90% of patients were female. According to market research conducted with prescribing physicians from February through March of 2005, it is estimated that over 80% of patients receiving prescriptions for teriparatide had already experienced one or more prior fractures. Since teriparatide is administered subcutaneously, it is important that patients receive training on the use of the teriparatide injection device (i.e., the pen device). Educational programs are available for those who have been prescribed teriparatide therapy. Patients may also contact a customer care program regarding a variety of topics, including pen device use. Based on patient feedback, design changes have been implemented in the pen device to facilitate optimal use. Updates have also been made to the prescribing information to reflect the post-marketing surveillance experience. Adverse experiences reported to date have been consistent with the current product label and with cumulative teriparatide clinical trial experience. As of December 31, 2005 no reports of pathology-confirmed osteosarcoma have been received for individuals who have been treated with teriparatide, either with the commercially available drug or in clinical trials. We are unaware of any reports of osteosarcoma in association with other preparations of teriparatide, or other peptides of parathyroid hormone, either in the setting of clinical trials or from marketed drug experience. PMID- 16684432 TI - Are generic formulations of carvedilol of inferior pharmaceutical quality compared with the branded formulation? AB - INTRODUCTION: Carvedilol is a comprehensive beta(1)-, beta(2)-, and alpha(1) adrenoreceptor blocker marketed as Dilatrend by F. Hoffmann-La Roche Ltd. (Roche) and as Coreg by GlaxoSmithKline for the treatment of hypertension, stable angina pectoris, post myocardial infarction with left ventricular dysfunction and all degrees of symptomatic chronic heart failure. OBJECTIVES: In this report, the pharmaceutical qualities of Dilatrend 6.25 mg, 12.5 mg and 25 mg tablets and 35 randomly selected carvedilol generic products from 20 manufacturers in 19 countries have been assessed according to the European Pharmacopoeia and the Roche specifications. METHODS: The generic products were subjected to four key tests: carvedilol content, tablet hardness, tablet dissolution and purity. RESULTS: All three Dilatrend strengths conformed to specifications. At least 17/35 (48.6%) generic products failed the specifications due to: incorrect mean carvedilol content (outside 95-105%) - three products; excess impurities (> 0.3%) - one product; incorrect tablet hardness (outside 30-70 N) - 11 products; inadequate dissolution (< 75% in 30 min) - nine products. Seven products (20%) failed two tests, generally hardness and dissolution. CONCLUSION: The dose-for dose substitution of the original formulation of carvedilol (Dilatrend) with a pharmaceutically different, and possibly inferior, generic copy may conceivably result in a change in the efficacy of the treatment, because of an unanticipated change in pharmacokinetics or bioequivalence, and/or in a change in tolerability due to impurities. PMID- 16684433 TI - Efficacy and safety of tamsulosin for benign prostatic hyperplasia: clinical experience in the primary care setting. AB - OBJECTIVE: This study evaluated the efficacy and safety of the alpha(1A)/alpha(1D) subtype-selective blocker tamsulosin for the increasingly common treatment of benign prostatic hyperplasia (BPH) in the primary care setting. METHODS: A total of 493 men (age > or = 45 years), 99.6% of whom had moderate or severe BPH at baseline, were given tamsulosin 0.4 mg/day in a multicenter, open-label study conducted over 45 days by 42 primary care physicians and two urologists. RESULTS: Mean American Urological Association (AUA) Symptom Score decreased by 7.5 from a baseline of 20.0 on day 4, representing a 37.5% improvement over baseline (p < 0.001). AUA Obstructive and Irritative Scores declined significantly by day 4 (-4.7 and -2.7, respectively), as did AUA Bother Score (-5.4, p < 0.001) and mean BPH Impact Score (-2.5, p < 0.001). The Investigator's Global Assessment showed slight or greater improvement in 77.2% of patients (13.7% markedly improved). Effects were maintained from day 4 through day 45. CONCLUSIONS: Overall, patients treated with tamsulosin in a primary care setting experienced rapid, significant improvement in their signs and symptoms of BPH, based upon the change in the AUA Symptom Score. Tamsulosin was well tolerated; no new safety concerns were observed. Tamsulosin was not associated with significant effects on blood pressure or first-dose hypotension. PMID- 16684434 TI - Iron and the anaemia of chronic disease: a review and strategic recommendations. AB - BACKGROUND: The incidence of anaemia is high in many chronic conditions, yet it often receives little attention. SCOPE/METHODS: A panel of international experts with experience in haematology, nephrology, oncology, rheumatology and pharmacy was convened to prepare strategic guidelines. A focused literature search was conducted after key issues had been identified. A series of recommendations was agreed, backed, wherever possible, by published evidence which is included in the annotations. RECOMMENDATIONS: Anaemia is a critical issue for patients with chronic diseases. Healthcare professionals need to recognise that anaemia is a frequent companion of cancer and chronic conditions such as rheumatoid arthritis and heart failure. It reduces patients' quality of life and can increase morbidity and mortality. Anaemia should be considered as a disordered process in which the rate of red cell production fails to match the rate of destruction which leads eventually to a reduction in haemoglobin concentration; this process is common to all chronic anaemias. The aim of anaemia management should be to restore patient functionality and quality of life by restoring effective red cell production. Blood transfusion can elevate haemoglobin concentration in the short term but does nothing to address the underlying disorder; red cell transfusion is, therefore, not an appropriate treatment for chronic anaemia. Patients with anaemia of chronic disease may benefit from iron therapy and/or erythropoiesis stimulating agents (ESAs). Intravenous iron should be considered since this can be given safely to patients with chronic diseases while intramuscular iron causes unacceptable adverse effects and oral iron has limited efficacy in chronic anaemia. CONCLUSION: The management of anaemia calls for the development of a specialist service together with education of all healthcare professionals and transfer of skills from areas of good practice. Improvement in the management of anaemia requires a fundamental change of attitude from healthcare professionals. PMID- 16684435 TI - Efficacy and safety of silver textile in the treatment of atopic dermatitis (AD). AB - BACKGROUND: Patients with atopic dermatitis (AD) have an increased tendency to develop bacterial skin infections. Colonization with Staphylococcus aureus is known to be a major trigger and might also play a pathophysiological role. Because of their antiseptic action, silver-coated textiles suppress S. aureus colonization and toxin formation, thus damping the inflammatory reaction. OBJECTIVES: To evaluate the clinical effectiveness and safety of a special silver textile in the treatment of patients suffering from acute AD. METHODS: In a randomized phase II monocenter parallel-group comparative study 30 patients were recruited (average age 25.5 years, min. 4 years, max. 70 years) who were affected by AD in an acute phase. During the first study phase from Day 1 to Day 14, 10 patients received a silver textile (Group 1), 10 a silver-free textile (Group 2), and 10 prednicarbate ointment (Group 3). In the second phase from Day 15 to Day 28 all patients wore the silver textile, and during the follow-up period from Day 28 to Day 56 no textiles were used. Prednicarbate ointment was allowed as emergency medication, but ointment consumption was measured. The overall severity of the disease was evaluated using the SCORAD index as the primary efficacy parameter. Secondary parameters included severity of pruritus and the patients' assessment of their disease control (uncontrolled, limited, good or complete). Safety tests included hematology, blood chemistry, urinalysis for silver, and physical examination for silver deposits in the skin and mucous membranes. RESULTS: The initial SCORAD was 61.6 (IQR 26.6, min. 30.6, max. 99.9). At the end of the Study Phase 1 the SCORAD had improved significantly in the patients of Groups 1 (74.6-29.9, p = 0.005) and 3 (57.8-24.0, p = 0.009). During Study Phase 2 healing of eczema continued in Group 1 (SCORAD 29.9-18.1, p = 0.037), was observed in Group 2 (48.2-24.1, p = 0.015), and remained at an improved level in Group 3 (SCORAD 24-23.5). Consumption of prednicarbate ointment (Phase 1, Phase 2, follow-up period, medians are given): Group 1: 135 g, 10 g, 45 g; Group 2: 13 g, 0 g, 0 g; Group 3: 145 g, 30 g, 90 g. Silver textiles reduced the severity of the pruritus (p = 0.031); silver-free textiles (n.s.) and prednicarbate (n.s.) were less effective. No undesired events were observed. CONCLUSION: The elastic silver textile worn directly against the skin led to an impressive improvement of AD and a reduction in the use of prednicarbate ointment. PMID- 16684436 TI - Glimepiride versus pioglitazone combination therapy in subjects with type 2 diabetes inadequately controlled on metformin monotherapy: results of a randomized clinical trial. AB - OBJECTIVE: To compare the effect of add-on glimepiride or pioglitazone in subjects with type 2 diabetes inadequately controlled on metformin monotherapy. RESEARCH DESIGN AND METHODS: Multicenter, randomized, parallel-group, open-label, forcedtitration study involving 203 adults with poorly controlled type 2 diabetes (A1C 7.5-10%) on metformin monotherapy. Subjects were randomized to receive glimepiride or pioglitazone, titrated to the maximum dose for 26 weeks. Subjects were evaluated for A1C changes, fasting plasma glucose (FPG), insulin, C-peptide, and lipid levels. Safety outcomes and diabetes-related healthcare resource utilization were also evaluated. RESULTS: Both treatment groups achieved similar and significant mean decreases from baseline to endpoint (week 26) in A1C (p = 0.0001) and FPG (p < 0.05). Glimepiride therapy, however, resulted in a more rapid decline in A1C levels at weeks 6, 12, and 20 vs. pioglitazone (p < 0.05). A mean A1C < or = 7% was reached faster in the glimepiride group (median, 80-90 days vs. 140-150 days [p = 0.024]). Total and LDL cholesterol were significantly higher with pioglitazone treatment than with glimepiride at endpoint (p < 0.05). Glimepiride treatment was associated with an increased risk of hypoglycemia and pioglitazone with higher rate of peripheral edema. Healthcare resource utilization was similar between groups, but total healthcare costs were significantly lower for glimepiride versus pioglitazone over the course of the study, driven largely by drug costs. The use of fasting C-peptide concentration > or = 0.27 nmol/L in the inclusion criteria was a potential limitation as it may have included those patients with an improved probability for glimepiride or pioglitazone response. In addition, a larger patient population would have provided a greater degree of data applicability. CONCLUSIONS: In patients with type 2 diabetes inadequately controlled on metformin monotherapy, add-on glimepiride or pioglitazone results in similar overall improvements in glycemic control. Compared with pioglitazone, glimepiride is associated with faster glycemic control, lower total and LDL cholesterol levels and reduced short-term healthcare costs. PMID- 16684437 TI - The efficacy and safety of armodafinil as treatment for adults with excessive sleepiness associated with narcolepsy. AB - OBJECTIVE: This study assessed the efficacy and safety of armodafinil, the longer half-life enantiomer of modafinil, for the treatment of excessive sleepiness in patients with narcolepsy. RESEARCH DESIGN AND METHODS: This was a multicenter double-blind study with 196 patients (aged 18-65 years) randomized to receive armodafinil 150 mg (n = 65), armodafinil 250 mg (n = 67), or placebo (n = 64) once daily for 12 weeks. MAIN OUTCOME MEASURES: Efficacy was assessed using the Maintenance of Wakefulness Test (MWT) (six 20-min subtests across the day), the Clinical Global Impression of Change (CGI-C), subjective measures of sleepiness (Epworth Sleepiness Scale), patient diaries, and evaluations of cognitive performance (Cognitive Drug Research) and fatigue (Brief Fatigue Inventory). RESULTS: Armodafinil significantly increased MWT mean sleep latency (at 0900 1500) compared with placebo. The mean change from baseline at final visit for armodafinil was an increase of 1.3, 2.6, and 1.9 min in the 150-mg, 250-mg, and combined groups, respectively, compared with a decrease of 1.9 min for placebo (p < 0.01 for all three comparisons). Mean late-day MWT latency (1500-1900) was also significantly improved (difference of armodafinil combined group relative to placebo at final visit: 2.8 min, p = 0.0358). The proportions of patients who showed at least minimal improvement in the CGIC rating from baseline to final visit in the armodafinil 150-mg, 250-mg, and combined groups were 69%, 73%, and 71%, respectively, compared with 33% for placebo (p < 0.0001). Both doses were associated with statistically significant improvements in memory, attention, and fatigue (p < 0.05). The most common adverse events in patients receiving armodafinil were headache, nausea, and dizziness. CONCLUSIONS: Armodafinil significantly improved ability to sustain wakefulness throughout the day in patients with narcolepsy. Armodafinil also significantly improved overall clinical condition, memory, attention, and fatigue when compared with placebo. PMID- 16684438 TI - Public acceptance of drug use for non-disease conditions. AB - OBJECTIVE: This article deals with the issue of ordinary healthy people using drugs to improve or enhance non-disease conditions. The objective is to illuminate the extent of public acceptance of this practice. RESEARCH DESIGN AND METHODS: The results are based on two studies: a classically structured telephone interview with 961 Danes in 1999 and an Internet questionnaire survey of 2735 Danes in 2003. The two studies cannot be compared due to differences in recruitment and methodology. RESULTS: Based on basic descriptive statistics, the studies show substantial public acceptance of the use of drugs for non-disease conditions. Men in particular look favourably on the use of drugs by healthy individuals. People with less education find this type of drug use unacceptable to a greater extent than those with more education, who are more positive. If we look at political affiliation, a pattern emerges. People who did not vote or voted for one of the left-wing parties are less likely to accept this type of drug use. The extent to which people work with drugs professionally does not seem to influence the extent to which they are positive or negative about this type of drug use. CONCLUSION: The implications of this development encompass a wide range of concerns, from the individual risk of side effects to general issues concerning the prioritisation of health care resources. The results need more attention, including further studies, professional consideration and health policy decisions. PMID- 16684439 TI - The VA Ostomy Health-Related Quality of Life Study: objectives, methods, and patient sample. AB - OBJECTIVE: To present the design and methods of a multisite study of health related quality of life (HR-QOL) in veterans living with ostomies. RESEARCH DESIGN AND METHODS: Veterans from Tucson, Indianapolis, and Los Angeles VA Medical Centers were surveyed using the validated City of Hope ostomy-specific tool (mCOH-QOL-Ostomy) and the SF-36V. Cases (ostomates) had a major gastrointestinal procedure that required an intestinal stoma, while controls had similar procedures for which an ostomy was not required. Ostomy subjects were recruited for four focus groups in each of two sites divided by ostomy type (colostomy versus ileostomy) and overall mCOH-QOL-Ostomy HR-QOL score (highest versus lowest quartile). The focus groups further evaluated barriers, concerns, and adaptation methods and skills. MAIN OUTCOME MEASURES: This report presents recruitment results, reliability of survey instruments, and demographic characteristics of the sample. RESULTS: The overall response (i.e., recruitment) rate across all sites was 48% and by site was 53%, 57%, and 37%, respectively (p < 0.001). Internal consistency reliability estimates indicated that both instruments remain reliable in this population (Cronbach's alpha for HR-QOL domains/scales: 0.71-0.96). Cases and controls were similar in demographic characteristics. Proportions of minority subjects matched projections from the site patient populations. Subjects with ostomies had significantly longer time since surgery than controls (p < 0.001). Focus groups were comprised of two to six subjects per group and demonstrated racial diversity at the Los Angeles site. CONCLUSIONS: The unique design of our study of VA patients with ostomies is an illustration of a successful mixed methods approach to HR-QOL research. We collected meaningful quantitative and qualitative data that will be used in the development of new approaches to care that will lead to improved functioning and well-being in persons living with ostomies. Subsequent reports will provide the results of this research project. PMID- 16684440 TI - Therapeutic angiogenesis by bone marrow implantation for critical hand ischemia in patients with peripheral arterial disease: a pilot study. AB - OBJECTIVE: Implantation of bone marrow mononuclear cells (BM-MNCs), including endothelial progenitor cells, into ischemic lower limbs has been shown to improve symptoms in patients with peripheral arterial diseases (PAD). This study investigated whether BM-MNC implantation (BMI) is also effective for the ischemic hands of these patients. METHODS: Seven PAD patients with hand ischemia were enrolled: six patients had thromboangiitis obliterans and one had collagen disease. All seven had symptoms involving either resting pain or non-healing ischemic ulcers of the hand. Approximately 600 mL of MNCs were separated from BM and concentrated to a final volume of 40-50 mL, which were injected into ischemic hands. Ischemic status was evaluated by measuring the digital/brachial pressure index (DBI), visual analog pain scale, and the healing of ulcers before and 6 months after BMI. RESULTS: The mean number of implanted MNCs, CD34-positive cells, and CD34,133-positive cells was 3.67 +/- 0.53 x 10(9), 4.94 +/- 2.45 x 10(7), and 2.52 +/- 1.57 x 10(7), respectively. Mean DBI in those patients was 0.15 +/- 0.30 before BMI and significantly increased to 0.67 +/- 0.19 at 6 months after BMI (p = 0.004). All patients also showed improvement of pain scale and ischemic ulcers. There was no significant correlation between the number of implanted cells and improvement in the degree of DBI or the pain scale. CONCLUSION: Autologous BMI could be a promising and safe method of therapeutic angiogenesis for critical hand ischemia in PAD patients. PMID- 16684472 TI - Why do we need an oral health care policy in Canada? AB - Although health care is a right of citizenship, severe inequities in oral health and access to care persist. This paper provides information on the financing, organization and delivery of oral health services in Canada. It concludes that dental care has largely fallen out of consideration as health care. The increasing costs of dental insurance and disparities in oral health and access to care threaten the system"s sustainability. The legislation that allows the insured to receive tax-free care and requires all taxpayers to subsidize that expenditure is socially unjust. Unless an alternative direction is taken, dentistry will lose its relevance as a profession working for the public good and this will be followed by further erosion of public support for dental education and research. However, never before have we had the opportunity presented by high levels of oral health, the extensive resources already allocated to oral health care, plus the support of other organizations to allow us to consider what else we might do. One of the first steps would be to establish new models for the delivery of preventive measures and care that reach out to those who do not now enjoy access. PMID- 16684441 TI - Guidelines for selecting among different types of bootstraps. AB - BACKGROUND: The bootstrap has become very popular in health economics. Its success lies in the ease of estimating sampling distribution, standard error and confidence intervals with few or no assumptions about the distribution of the underlying population. OBJECTIVE: The purpose of this paper is three-fold: (1) to provide an overview of four common bootstrap techniques for readers who have little or no statistical background; (2) to suggest a guideline for selecting the most applicable bootstrap technique for your data; and (3) to connect guidelines with a real world example, to illustrate how different bootstraps behave in one model, or in different models. RESULTS: The assumptions of homoscedasticity and normality are key to selecting the best bootstrapping technique. These assumptions should be tested before applying any bootstrapping technique. If homoscedasticity and normality hold, then parametric bootstrapping is consistent and efficient. Paired and wild bootstrapping are consistent under heteroscedasticity and non-normality assumptions. CONCLUSION: Selecting the correct type of bootstrapping is crucial for arriving at efficient estimators. Our example illustrates that if we selected an inconsistent bootstrapping technique, results could be misleading. An insignificant effect of controller treatment on total health expenditures among asthma patients would have been found significant and negative by an improperly chosen bootstrapping technique, regardless of the type of model chosen. PMID- 16684473 TI - Oral health care in Canada--a view from the trenches. AB - PURPOSE: Concern is increasing over the effect of lack of access to oral health care on the oral health, and hence general health, of disadvantaged groups. In preparation for a national symposium on this issue, key informants across Canada were canvassed for their perceptions of oral health services and their recommendations for improving oral health care delivery. This paper reports the results of that survey. METHOD: A questionnaire was constructed to address problems facing agencies with responsibility for meeting the oral health care needs of people receiving government assistance, the underhoused and the working poor. The survey was sent to 200 agencies, government and professional organizations. Data from the returned questionnaires were entered into a Statistical Package for the Social Sciences database and analyzed. Responses from Ontario were compared with those from the rest of Canada, those from government organizations were compared with others and results were examined by cultural nature of clients and by type of organization. RESULTS: In assessing the positive aspects of oral health care, 84% of respondents agreed that public programs were useful and 81% felt that dentists offer good care. However, 77% disagreed that preventive care is accessible and that access to dentists and dental specialists is easy. More Ontarians than others thought that there are few alternative settings for care delivery (95% vs. 83%) and that the poor feel unwelcome in dental offices (83% vs. 70%). The issues most commonly identified were the need for alternative delivery sites, such as community health centres where service delivery could be affordable, accountable and sustainable; the need for oral health to be recognized as part of general health; regulatory issues (e.g., expanding practice opportunities for non-dentist oral health care providers and removing restrictions on other dental health professionals in providing basic care to the financially challenged); and training. DISCUSSION: The survey helped to identify access and care issues across the country. There was considerable agreement that lack of access to dental care services is an important detriment to the oral and general health of many Canadians. Respondents believe that dental health is isolated from general health. PMID- 16684474 TI - Teaching the use of resin composites in Canadian dental schools: how do current educational practices compare with North American trends? AB - The placement of resin composites in posterior teeth is now a common procedure in dental practice. The aim of this study was to investigate current teaching of this procedure in Canadian dental schools and to compare trends in teaching with those in the United States. This study complements other investigations in which we examined teaching of the use of posterior resin composites in dental schools in the United States, Ireland and the United Kingdom. A questionnaire was distributed by email to the faculty member in each of the 10 dental schools in Canada with responsibility for teaching the operative dentistry curriculum, including the placement of posterior resin composites. The response rate was 100%. More teaching of posterior resin composites was noted since the time of a survey in the late 1990s. The amount of teaching and clinical experience in the use of posterior resin composites in Canadian dental schools seems to be higher than in dental schools in the United States. As noted in surveys of other countries, variation among Canadian teaching programs was found to persist in relation to techniques and technologies used. PMID- 16684475 TI - The Oral-B CrossAction manual toothbrush: a 5-year literature review. AB - The design of the modern conventional manual toothbrush can be attributed to Dr. Robert Hutson, a Californian periodontist, who in the early 1950s developed the multitufted, flattrimmed, end-rounded nylon filament brush that became known as the Oral-B manual toothbrush. The trademark Oral-B emphasized that this was an oral brush, designed to clean all parts of the oral cavity, not merely a toothbrush. Flat-trimmed conventional toothbrushes based on the original Oral-B design have good plaque-removing capability when used carefully. However, limitations in terms of patients" brushing technique and brushing time necessitated a radical change in bristle pattern to improve performance, especially at approximal sites and along the gumline. RATIONALE FOR PRODUCT DEVELOPMENT: Detailed studies of the tooth-brushing process, using advanced scientific and ergonomic research methods, led to new toothbrush designs intended to maximize the efficacy of brushing efforts. These studies showed that the point of greatest interproximal penetration occurs when the direction of brushing changes; bristles angle back into the interproximal space, moving down and back up the adjoining approximal surface. These mechanics were further optimized on the basis of standardized evaluations of brush-design characteristics, including combinations of tuft lengths, insertion angles and tuft layout. With conventional vertical bristles these improvements yield limited benefits because only a few bristles are correctly positioned at the interproximal junction when the brush changes direction. Ultimately, a design with bristle tufts arranged at 16 masculine from vertical along the horizontal brush head axis was identified, in which the maximum number of bristles operated at the optimum angle throughout the brushing cycle. This design was significantly more effective (p < 0.001) than others in terms of penetration (by 9.6%) and cleaning effectiveness per brush stroke (by 15.5%). EFFECTIVENESS: This discovery paved the way for a new toothbrush design with a unique patented array of tufts, which became known as the Oral-B CrossAction brush. This design was selected for extensive independent studies designed to evaluate plaque removal at the gingival margins and in the approximal areas and longer-term control of gingivitis, relative to current standard designs. In a series of studies (published in 2000), 14 single-brushing comparisons and 2 longer-term studies demonstrated the consistent superiority of the Oral-B CrossAction brush over the equivalent commercial standards. Since then, several additional studies have contributed further positive performance data for the CrossAction brush. Two of the studies demonstrated that plaque removal by this brush was superior to that of 15 other manual toothbrushes, and further investigations contributed similarly positive data. Longer-term data have confirmed superior CrossAction performance and the long-term benefits of improved efficacy, particularly for gingivitis. DISCUSSION: Novel approaches to toothbrush design have produced a toothbrush that, when tested in a large number of clinical studies, has consistently met or exceeded established standards of efficacy. The literature contains a wealth of performance data on various toothbrush designs, but none of these designs shows the year-on-year consistency and reproducibility of the Oral-B CrossAction. PMID- 16684476 TI - Orofacial granulomatosis: 2 case reports and literature review. AB - Orofacial granulomatosis comprises a group of diseases characterized by noncaseating granulomatous inflammation affecting the soft tissues of the oral and maxillofacial region. The most common clinical presentation is persistent swelling of one or both lips. It is important to establish the diagnosis accurately because this condition is sometimes a manifestation of Crohn's disease or sarcoidosis. This article describes 2 cases of orofacial granulomatosis, in one of which the condition was a manifestation of Crohn"s disease. The diagnostic approach to and the treatment of orofacial granulomatosis are reviewed. PMID- 16684477 TI - Extensive papillomatosis of the palate exhibiting epithelial dysplasia and HPV 16 gene expression in a renal transplant recipient. AB - We report a unique case of extensive papillomatosis of the palate in a renal transplant recipient. The condition resembled inflammatory papillary hyperplasia; it exhibited severe epithelial dysplasia and concurred with generalized gingival hyperplasia. We document and discuss the probable multifactorial etiology of the lesions, including evidence for human papillomavirus (HPV) type 16 expression, as detected by in situ reverse transcription polymerase chain reaction. This report illustrates the need for careful clinical investigation and follow-up of immunosuppressed individuals presenting with apparently benign, common oral lesions. PMID- 16684478 TI - Cleidocranial dysplasia: 2 generations of management. AB - Patients with cleidocranial dysplasia (CCD) commonly present with significant dental problems, such as retention of multiple deciduous teeth, impaction or delay in eruption of permanent teeth and, often, the presence of supernumerary teeth. Several approaches have been described for the management of such patients. We report 2 cases illustrating the shift in the management paradigm from edentulation and prosthetic replacement to orthodontically assisted forced eruption and fixed appliance orthodontic treatment combined with orthognathic surgery. PMID- 16684479 TI - [Diagnosis and treatment of solitary thyroid nodule--study and evaluation in Portugal]. AB - INTRODUCTION: The best diagnostic and treatment strategy for an approach to the nodular thyroid disease continues to be a controversial issue. OBJECTIVES: The aim of this study was to characterise medical practice in the diagnosis and treatment of nodular thyroid disease by endocrinologists and surgeons in Portugal in 2002. METHODS: A questionnaire based on that used by the European Thyroid Association and the American Thyroid Association was drawn up. The questionnaire, based on a well-defined index case, was circulated by the Portuguese Endocrinology Society to endocrinologists and surgeons: 42 year-old woman with solitary thyroid nodule measuring 2 x 3 cm, with no history of malfunction or painful symptoms. Each doctor was asked to reply as to the adopted diagnosis and therapy procedures for the index case. Eleven variations to the original case were proposed in order to evaluate the alterations for each variation. RESULTS: 1492 questionnaires were sent out, 163 to endocrinologists and 1329 to surgeons. A total of 104 were returned. The global response rate was 7%. The response rate for endocrinologists was 27% and 4.5% for surgeons. Of the 104 questionnaires returned, 42% were from endocrinologists and 58% from surgeons. Concerning tests prescribed, surgeons would use more tests than endocrinologists for the index case. The main differences in laboratory terms were the higher number of prescriptions for total T4 and T3 and thyroglobulin by surgeons and more prescriptions for AATPO by endocrinologists. The average number of tests was 4.6, 4.1 for endocrinologists and 5.1 for surgeons. Relative to imaging and cytology, 32% of doctors advocated a scintigraphy to diagnose the index case, with no significant differences between endocrinologists and surgeons. Ultrasonography was used by over 85% of respondents. 90% prescribed a cytology, 83% guided by palpation and 18% ultrasonography-guided. Concerning treatment, 33% of doctors advocated levothyroxin treatment; surgery was advocated by 16.3% of endocrinologists and 36.6% of surgeons. Meanwhile, the majority of doctors (68%) would opt for no treatment and simply maintain the patient under surveillance. CONCLUSIONS: There are important differences in the approach to nodular thyroid disease among the various doctors and specialists, which highlight the difficulty in achieving a diagnostic and therapeutic consensus. PMID- 16684480 TI - [Newborns from assisted reproductive technology at the Hospital de Sao Joao]. AB - Over the last 20 years, assisted reproductive technology has been increasingly used. Ever since the first newborn resulting from artificial insemination, over two centuries ago, thousands more children were born resulting from these techniques. In Portugal, assisted reproductive technology began in 1985 at the Hospital de Sao Joao (HSJ). Although the success of these techniques is often judge by the pregnancy rate, occasionally by the birth rate, the most important issue of this evaluation should be the neonatal health. A retrospective study to evaluate the use of assisted reproductive technology at the HSJ, as well as the occurrence of complications during pregnancy and the evolution of the newborns in the neonatal period, during the period between 1999 and 2003, was undertaken. A hundred and thirty-eight pregnant women were included, which stands for about 1% of the total number of pregnant women at the HSJ in the period in question, with an average of age 32.2 +/- 4.1 and of infertility 6.2 +/- 3.8 years. The number of attempts for a successful pregnancy was 2.7 +/- 2.1. The most used technique was intracytoplasmatic sperm injection. Eighteen percent of the pregnancies resulted in abortion. The incidence of multiple pregnancies was 30%. A hundred and fifty-six infants were born, which stands for about 1% of the total number of births at the HSJ in the period in question, with a gestational age of 36.4 +/- 3 weeks and weight at birth of 2674.5 +/- 761.4 g. The incidence of preterm birth was 41%. Seventy-eight percent of the newborns had a good neonatal outcome, whereas the other 22% were admitted to a neonatal intensive care unit for an average period of 17 days. The most frequent neonatal morbidity was respiratory. One newborn died. Despite the need for several attempts of assisted reproductive technology, pregnancy complications, neonatal morbidity and mortality, the use of these techniques allowed for most couples to overcome their infertility problem. PMID- 16684481 TI - [Determination of HER2 by fluorescence in situ hybridization (FISH) in breast cancer. Experience of the Reference Laboratory of Lisbon]. AB - The knowledgement of HER2/c-erbB-2 status in breast cancer is essential for the eligibility of the patients for therapy with monoclonal antibody anti-HER 2/trastuzumab. From the various existent techniques for the determination of HER 2, the most widely used are: immunohistochemistry (IHC), that estimates the protein expression, and in situ hybridization (FISH), that evaluates its amplification. FISH is essential as complementary of IHQ in tumors with equivocal or non interpreted staining, and also in those with complete weak to moderate membrane staining in >10 % of neoplastic cells (score 2+) because, of these, only cases with gene amplification (FISH positive) respond to therapy. Recent studies also support that FISH should be performed in cases with score 3+, because 10-12% of them don't have amplification. The expensive equipment and reagents for standard FISH method and the need of extensive training and expertise for both the technique and the evaluation, justify a centralized testing in Reference Laboratories. Our aim was to describe the work of the Department of Pathology of the Portuguese Institute of Oncology of Lisbon as a Reference Laboratory in the execution of FISH technique and to present the results obtained from May 2001 to August 2004. FISH was performed with Kit INFORM HER2/neu Gene Detection System, using the BenckMark system of Ventana. We evaluated a series of 4499 invasive carcinoma primary of the breast, that included 587 cases of the Portuguese Institute of Oncology of Lisboa and 3912 cases from different Hospitals of Lisboa, Cascais, Almada, Barreiro, Santarem, Evora, Faro, Portimao, Guimaraes, Funchal and Angra do Heroismo. We verified that 591 cases (13.5%) had HER2 gene amplification, being those patients eligible for therapy with trastuzumab. PMID- 16684482 TI - [Paraquat intoxication and hemocarboperfusion]. AB - In the northeast of Portugal, paraquat intoxication is common. We report 31 patients, admitted at our institution from April 1997 to June 2004, with suspected paraquat intoxication. Thirty cases had suicidal intention and all have ingested the toxic by mouth. Treatment consisted of skin and digestive decontamination with gastric washing and activated charcoal or Fuller's Earth. The first medical care was performed after 1 h 14 min at median [9 min-11 h 26 min]. By laboratory measurement (n=13) and/or clinical observation (n=11) it was possible to confirm the gastrointestinal absorption of paraquat for 24 patients (17 male-7 female; 49 +/- 17 years). The estimated ingested volume was higher for 15 non-survivors relatively to nine survivors (170 +/- 60 mL vs 25 +/- 10 mL; P<0,001). Initial serum paraquat of seven non-survivors was also higher than the serum level of six survivors (8.3 +/- 6.9 mg/L vs 0.4 +/- 0.3 mg/L; P < 0.01). Remarkably, all patients with serum paraquat higher than 1.7 ml/l did not survived. The paraquat urine level, simultaneous to the first serum determination, was higher for six survivors (38 +/- 37 mg/L) comparing with four non-survivors patients (465 +/- 536 mg/L; P<0.04). Hemoperfusion with activated charcoal was performed after December 1997 until June 2004 for 25 patients. Twenty of the 24 patients confirmed for paraquat intoxication were treated with this technique (12/15 of the non-survivors and 8/9 of the survivors; p=NS). Total number of sessions was identical (2.0 +/- 1.0 vs 1.5 +/- 1.2; P=NS), total time of hemoperfusion was higher (7 h 58 min vs 5 h 37 min; P=0.07) but the beginning of the first session was later (7 h 44 min vs 4 h 18 min; P=0.04) for the survivors. Clinical signs and laboratory analysis were collected at admission trying to detect markers of prognostic survival value. Hypernatremia, hypokaliemia, hyperglycemia and acute renal failure were more frequent for non survivors (P<0.05) but the variable timing of the first laboratorial determination jeopardize our analysis, perhaps excluding acute renal failure. Rapid evolution to shock, lead to death for seven patients at the first day of admission and to another four deaths at the next day. Median survival of non survivors was 1.2 days [0.1-13.2]. Evolution to pulmonary fibrosis for the nine survivors was not investigated (median follow-up: 14 days [2-1053]). Paraquat intoxication was highly lethal, leading to a 63% mortality rate of our patients. Hemoperfusion did not reveal any survival advantage for our patients. PMID- 16684483 TI - [Medicine, doctors and persons. Understanding stress to prevent burnout]. AB - This review is focused on doctors' occupational stress. A synthesis of the broad negative impact of distress is presented, as well as its spreading importance and magnitude according to recent research. Contemporary definitions of stress and burnout models are underlined with a remembering of their historical evolution. A synthesis of stress physiopathology is presented. The core risk factors of occupational medical stress are detailed and discussed, with more emphasis in those related to doctors vulnerabilities. Great need actually unmeeted regards the public recognition of the professional medical risks, the acceptance by the doctors of their normal fragility as humans, and their insight about their own vulnerabilities as a group with specifics professional representation. The conclusion is that there is a major and urgent need for the doctors to develop and practice competences of stress management. PMID- 16684484 TI - [Hymenoptera venom allergy--new diagnostic methods]. AB - During the past years, the number of patients with both anaphylaxis to hymenoptera sting and negative IgE detected by conventional methods has been increasing. To obviate this problem new assays have been tried, such as Western blot and in vitro challenge tests involving basophil activation, with either the measurement of histamine and sulphidoleukotrien released (CAST--Cellular Antigen Stimulation Test) or the detection of activation markers on the cell surface with flow cytometric analysis (Flow CAST). In this paper the principles of these assays as well as their diagnostic value in hymenoptera allergy are briefly reviewed. PMID- 16684485 TI - [The victim as object of the medico-legal intervention]. AB - We present a reflection about the victim as the object of the medico-legal intervention, that opens perspectives not only to the evidence research (as a techno-scientific contribute to support Justice in what concerns bio-psycho social questions), but also to the support of victims which, in a last analysis, is no more than another aspect of this the social contribution of this science to ensure the best interest of the victim. This reflection aims to contribute to a better knowledge of the victim in his/her various perspectives. Accordingly, during the exposition, we refer some contributes of Victimology towards the understanding of the victims' behaviour's. Afterwards, we focus on the problems related to the aetiologies (with a particular reference to trauma), levels and consequences (physical, psychological and socio-economical) of victimization, as well as the methodologies of victims approach, medico-legal evaluation and reparation. Considering victims as being not only those who suffer directly the consequences of the victimizing phenomena, but also those that suffer from it indirectly and even secondarily, we shall approach the topic regarding both perspectives. We highlight the importance of understanding the victim as a person, not just understanding him/her confined to the organic aspect (as it used to be until recently in certain fields of medico-legal intervention, and as it still happens due to some legal obligations, for instance in the Labour Law), but considering the person in a global way (body, capacities, life situations and subjectivity). In conclusion, we highlight the importance of this topic to the medico-legal mission, while taking part in various multidisciplinary interventions and being involved in strategies and measures which purpose is to prevent violence, promote safety, avoid secondary victimization and revictimization as well as guarantee the victims' protection and reintegration, in a work attitude that should happen, even more and more, as a real network. PMID- 16684486 TI - [Xerostomia in palliative care]. AB - Xerostomia is the subjective feeling of mouth dryness, caused or not by function lowering of salivary glands, with decrease of saliva quality or quantity. It's a frequent symptom in palliative care patients and its prevalence is referred to be 60% to 88% in advanced and progressive oncological disease patients. Xerostomia has physical, social and psychological consequences. Saliva plays an important role in maintaining the best physiological conditions of mouth. Besides humidifying the oral cavity tissues, its lubricating properties help swallowing, talking and prevents other damages caused by mechanical and noxious microbiological agents. Xerostomia is caused by three basic mechanisms: factors that compromise the salivary centre, factors related to the autonomic stimulation or factors related to salivary glands themselves. The diagnosis is mainly clinical. Mouth condition must be thoroughly evaluated. If justified, a quantitative evaluation of saliva secretion, in rest and under stimulation, might be done. The treatment must be oriented by aetiology and directed towards the disease effects in patient comfort and quality of life. During treatment, the use of xerogenic drugs should be controlled, hydration should be promoted and other symptom control measures improved. The symptomatic treatment has three pathways: the increasing of saliva production by mechanical, gustatory or pharmacological stimulation; the using of saliva substitutes and the improving of active mouth care. Mechanical stimulation is obtained by chewing gum and gustatory stimulation may be reached by sucking Vitamin C tablets. Pilocarpine is the available drug to improve salivation. A soft diet must be advised, hard and dry food, tobacco and alcoholic beverages should be avoided. It's important that health workers teach patients with xerostomia the best way to get relief and the measures to prevent its complications that could, even more, compromise their quality of life. PMID- 16684487 TI - [Heat waves: health impacts]. AB - During the summer of 2003, record high temperatures were reported across Europe, causing thousands of casualties. Heat waves are sporadic recurrent events, characterised by intense and prolonged heat, associated with excess mortality and morbidity. The most frequent cause of death directly attributable to heat is heat stroke but heat waves are known to cause increases in all-cause mortality, specially circulatory and respiratory mortality. Epidemiological studies demonstrate excess casualties cluster in specific risk groups. The elderly, those with chronic medical conditions and the socially isolated are particularly vulnerable. Air conditioning is the strongest protective factor against heat related disorders. Heat waves cause disease indirectly, by aggravating chronic disorders, and directly, by causing heat-related illnesses (HRI). Classic HRI include skin eruptions, heat cramps, heat syncope, heat exhaustion and heat stroke. Heat stroke is a medical emergency characterised by hyperthermia and central nervous system dysfunction. Treatment includes immediate cooling and support of organ-system function. Despite aggressive treatment, heat stroke is often fatal and permanent neurological damage is frequent in those who survive. Heat related illness and death are preventable through behavioural adaptations, such as use of air conditioning and increased fluid intake. Other adaptation measures include heat emergency warning systems and intervention plans and environmental heat stress reduction. Heat related mortality is expected to rise as a consequence of the increasing proportion of elderly persons, the growing urban population, and the anticipated increase in number and intensity of heat waves associated with global warming. Improvements in surveillance and response capability may limit the adverse health conditions of future heat waves. It is crucial that health professionals are prepared to recognise, prevent and treat HRI and learn to cooperate with local health agencies. PMID- 16684488 TI - [Cutaneous leishmaniosis in Portugal due to Leishmania infantum MON-1]. AB - Leishmania infantum zymodeme MON-1 was isolated from a skin lesion in a child in Lisboa area (Portugal) who had never travelled abroad. Succefull therapy was achieved by excisional biopsy of the lesion. This is the first evidence that this agent is responsible by cutaneous leishmaniasis in the country. PMID- 16684489 TI - [Friedreich ataxia and diabetes mellitus--family study]. AB - Friedreich's ataxia (FA) is one of the genetic syndromes sometimes associated with diabetes and the most common hereditary ataxia. It is a autosomal recessive neurodegenerative disease, caused by a mutation in the FRDA gene, which originates decreased expression of frataxin, a mitochondrial protein involved in iron metabolism. The disorder is usually manifest in childhood and is characterised by ataxia, dysarthria, scoliosis and feet deformity. About two thirds of patients have hypertrophic cardiomyopathy, 10% have diabetes and 20% have another glucose homeostasis disorder. Both insulin resistance and beta-cell dysfunction are implicated in this patients' diabetes pathophysiology. The mean half-life is 35 years. Cause of death is usually related to cardiomyopathy or diabetes' complications. We report the case study of two twin sisters with 28 years old, in whom FA was diagnosed in the first decade, both of them with diabetes since their early twenties. A third sister with FA is reported, with no glucose homeostasis disorder. They also have two healthy male brothers. Based in this cases, the FA associated diabetes pathophysiology is discussed, concerning the therapeutic approach to these patients and to their diabetic relatives without neurologic symptoms. The role of molecular genetic testing and genetic counselling are also debated. PMID- 16684490 TI - [Microlithiasis and testicular tumour]. AB - Testicular microlithiasis is a rare entity, usually asymptomatic and bilateral. There are however reports that until 40% of the cases may be related with testicular tumours. We report an 11-year-old boy, with a four-month history of left testicular mass. Sonography showed increased volume of left testis and bilateral microlithiasis. Testicular biopsy revealed Sertoli cell tumour and he was submitted to left radical orquidectomy. Testicular cancer is often curable, especially if diagnosed and treated early. The association of malignancy justifies long term clinical and ultrasound follow-up of testicular microlithiasis. PMID- 16684492 TI - Recent insights into cardiac hypertrophy and left ventricular remodeling. AB - Cardiac hypertrophy is a compensatory mechanism of the heart to maintain cardiac output under stresses that compromise cardiac function. Mechanical stretch and neurohumoral factors induce changes in intracellular signaling pathways resulting in increased protein synthesis and activation of specific genes promoting cardiac growth, eventually leading to left ventricular remodeling and cardiac dysfunction. The remodeling process results from alterations in cardiac myocytes as well as the extracellular matrix. PMID- 16684491 TI - Screening for ventricular remodeling. AB - Current guidelines emphasize the importance of preventing heart failure (HF) by targeting people with preclinical forms of the disease. Accordingly, there is considerable interest in identifying left ventricular (LV) remodeling, the fundamental substrate for HF, in asymptomatic individuals in the community. Increased LV mass and asymptomatic LV systolic and LV diastolic dysfunction are the remodeling phenotypes that could be potentially considered for population wide screening. Plasma levels of natriuretic peptides (NP) have been extensively investigated for such screening purposes. However, a majority of investigations suggest that their performance characteristics are suboptimal for identifying LV remodeling phenotypes unless high-risk individuals (eg, older men with hypertension) are targeted. In general, the prevalence of LV systolic dysfunction in women is too low to justify screening. In recent reports, sequential screening strategies combining urine and plasma NP or plasma NP and hand-held portable echocardiography have been advocated as potential approaches to identify asymptomatic LV systolic dysfunction in a cost-effective manner. Additional studies are warranted to confirm these findings. PMID- 16684493 TI - Treating heart failure with cardiac contractility modulation electrical signals. AB - Major advances have been made over the past two decades in the pharmacologic treatment of chronic heart failure (HF). Angiotensin-converting enzyme inhibitors, beta-blockers, and aldosterone antagonists have had a substantial impact on reducing mortality and morbidity in patients with HF and low left ventricular ejection fraction. These treatments delayed the progression toward advanced intractable HF but did not arrest progressive worsening of the disease. Patients on optimal medical therapy continued to deteriorate, albeit at a much slower pace, ultimately requiring further intervention. This gave rise to a host of device-based therapies that emerged in recent years to address this unmet need. Device therapies such as cardiac resynchronization, the CorCap cardiac support device (Acorn Cardiovascular, Inc., St. Paul, MN), and the OPTIMIZER System (Impulse Dynamics USA, Inc., Orangeburg, NY) are a few examples. This review addresses the progress made to date in the development and implementation of cardiac contractility modulation (CCM) as a device-based therapy for the treatment of patients with advanced HF. Treatment of patients with HF using CCM electrical signals is at present an investigational form of therapy. PMID- 16684494 TI - Mechanisms and evidence for the role of enhanced external counterpulsation in heart failure management. AB - Balloon counterpulsation has gained widespread acceptance as a therapy for cardiogenic shock. However, over the past four decades a parallel method of noninvasive counterpulsation, enhanced external counterpulsation (EECP), has been defined and developed. Mechanisms of benefit for this technology continue to emerge and include enhanced coronary and other key target organ perfusion beds. Other mechanisms include angiogenesis and enhanced cellular metabolism. Beyond putative mechanisms there is ample evidence for improved and sustained outcomes in patients with and without left ventricular dysfunction. This evidence comes from long-term registry reports and randomized clinical trials. With respect to heart failure (HF), there is registry, pilot trial, and randomized clinical trial evidence of safety and efficacy. This paper summarizes some of the mechanisms and outcomes of EECP in HF patients and helps to elucidate the role of EECP in the management of patients with chronic HF. PMID- 16684495 TI - Exercise training in heart failure. AB - The reduction of exercise capacity with early occurrence of fatigue and dyspnea is a hallmark of heart failure syndrome. There are objective similarities between heart failure and muscular deconditioning. Deficiencies in peripheral blood flow and skeletal muscle function, morphology, metabolism, and function are present. The protective effects of physical activity have been elucidated in many recent studies: training improves ventilatory control, skeletal muscle metabolism, autonomic nervous system, central and peripheral circulation, and heart function. These provide the physiologic basis to explain the benefits in terms of survival and freedom from hospitalization demonstrated by physical training also in heart failure. PMID- 16684496 TI - Diuretic resistance in heart failure. AB - The use of diuretics for the treatment of heart failure (HF) is ubiquitous in any basic HF medical regimen. Although initially these drugs clearly show benefit by relieving symptomatic episodes of decompensated HF, long-term use of these drugs can lead to a "diuretic-resistant" state and is associated with an increased risk of morbidity and mortality. A number of factors may be responsible for this, including dietary noncompliance, inadequate diuretic dosing or methods of administration, and concomitant use of certain medications. Diuretics themselves may set in motion an iatrogenic cardiorenal syndrome leading to worsening renal function and diuretic resistance. The methods for overcoming this resistance are varied and require a focused approach with emphasis on relieving the congestive symptoms related to HF while attempting to preserve renal function and minimize any untoward systemic effects. PMID- 16684497 TI - Haptic and visual perception of roughness. AB - In this study, we are interested in the following two questions: (1) how does perceived roughness correlate with physical roughness, and (2) how do visually and haptically perceived roughness compare? We used 96 samples of everyday materials, such as wood, paper, glass, sandpaper, ceramics, foams, textiles, etc. The samples were characterized by various different physical roughness measures, all determined from accurately measured roughness profiles. These measures consisted of spectral densities measured at different spatial scales and industrial roughness standards (R(a), R(q) and R(z)). In separate haptic and visual conditions, 12 naive subjects were instructed to order the 96 samples according to perceived roughness. The rank orders of both conditions were correlated with the various physical roughness measures. With most physical roughness measures, haptic and visual correspondence with the physical ordering was about equal. With others, haptic correspondence was slightly better. It turned out that different subjects ordered the samples using different criteria; for some subjects the correlation was better with roughness measures that were based on higher spatial frequencies, while others seemed to be paying more attention to the lower spatial frequencies. Also, physical roughness was not found to be the same as perceived roughness. PMID- 16684498 TI - Surface geometry influences the shape of illusory contours. AB - Geometric and neural models of illusory-contour (IC) synthesis currently use only local contour geometry to derive the shape of ICs. Work on the visual representation of shape, by contrast, points to the importance of both contour and surface geometry. We investigated the influence of surface-based geometric factors on IC shape. The local geometry of inducing-contour pairs was equated in stereoscopic IC displays, and the shape of the enclosed surface was varied by manipulating sign of curvature, cross-axial shape width, and medial-axis geometry. IC shapes were measured using a parametric shape-adjustment task (Experiment 1) and a dot-adjustment task (Experiment 2). Both methods revealed large influences of surface geometry. ICs enclosing locally concave regions were perceived to be systematically more angular than those enclosing locally convex regions. Importantly, the influence of sign of curvature was modulated significantly by shape width and medial-axis geometry: IC shape difference between convex and concave inducers was greater for narrow shapes than wider ones, and greater for shapes with straight axis and symmetric contours (diamond versus bowtie), than those with curved axis and parallel contours (bent tubes). Even at the level of illusory "contours," there is a contribution of region-based geometry which is sensitive to nonlocal shape properties involving medial geometry and part decomposition. Models of IC synthesis must incorporate the role of nonlocal region-based geometric factors in a way that parallels their role in organizing visual shape representation more generally. PMID- 16684499 TI - Soybean ATP sulfurylase, a homodimeric enzyme involved in sulfur assimilation, is abundantly expressed in roots and induced by cold treatment. AB - Soybeans are a rich source of protein and a key feed ingredient in livestock production, but lack sufficient levels of cysteine and methionine to meet the nutritional demands of swine or poultry as feed components. Although engineering the sulfur assimilatory pathway could lead to increased sulfur-containing amino acid content, little is known about this pathway in legumes. Here, we describe the cloning and characterization of soybean ATP sulfurylase (ATPS), which acts as the metabolic entry point into the sulfur assimilation pathway. Analysis of the ATPS clone isolated from a soybean seedling cDNA library revealed an open-reading frame, encoding a 52 kDa polypeptide with an N-terminal chloroplast/plastid transit peptide, which was related to the enzymes from Arabidopsis, potato, human, and yeast. Soybean ATP sulfurylase was expressed in Escherichia coli and purified to apparent homogeneity. Based on gel-filtration chromatography, the enzyme functions as a 100 kDa homodimer. Analysis of genomic DNA by Southern blotting revealed that multiple genes encode ATP sulfurylase in soybean. Analysis of the transcript profiles retrieved from a soybean EST database indicated that ATP sulfurylase mRNA was most abundant in root tissue. Cold treatment induced mRNA accumulation and enhanced the specific activity of ATP sulfurylase in root tissue. Northern blot analysis indicated a decline in the ATP sulfurylase transcript levels during seed development. Likewise, ATP sulfurylase specific activity also declined in the later stages of seed development. Increasing the expression levels of this key enzyme during soybean seed development could lead to an increase in the availability of sulfur amino acids, thereby enhancing the nutritional value of the crop. PMID- 16684502 TI - Cooperative unfolding of Escherichia coli ribosome recycling factor originating from its domain-domain interaction and its implication for function. AB - Cooperative unfolding of Escherichia coli ribosome recycling factor (RRF) and its implication for function were investigated by comparing the in vitro unfolding and the in vivo activity of wild-type E. coli RRF and its temperature-sensitive mutant RRF(V117D). The experiments show that mutation V117D at domain I could perturb the domain II structure as evidenced in the near-UV CD and tyrosine fluorescence spectra though no significant globular conformation change occurred. Both equilibrium unfolding induced by heat or denaturant and kinetic unfolding induced by denaturant obey the two-state transition model, indicating V117D mutation does not perturb the efficient interdomain interaction, which results in cooperative unfolding of the RRF protein. However, the mutation significantly destabilizes the E. coli RRF protein, moving the thermal unfolding transition temperature range from 50-65 to 35-50 degrees C, which spans the non-permissive temperature for the growth of E. coli LJ14 strain (frr(ts)). The in vivo activity assays showed that although V117D mutation results in a temperature sensitive phenotype of E. coli LJ14 strain (frr(ts)), over-expression of mutant RRF(V117D) can eliminate the temperature sensitive phenotype at the non-permissive temperature (42 degrees C). Taking all the results into consideration, it can be suggested that the mechanism of the temperature sensitive phenotype of the E. coli LJ14 cells is due to inactivation of mutant RRF(V117D) caused by unfolding at the non-permissive temperatures. PMID- 16684501 TI - Kinetic analysis of the effects of monovalent cations and divalent metals on the activity of Mycobacterium tuberculosis alpha-isopropylmalate synthase. AB - Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) is a member of the family of enzymes that catalyze a Claisen-type condensation. In this work we characterized the monovalent and divalent specificity of MtIPMS using steady state kinetics. The monovalent cation dependence of the kinetic parameters of substrates and divalent metals indicates that K+ is the likely physiological activator. K+ acts most likely as an allosteric activator, and exerts part of its effect through the catalytic divalent metal. The divalent metal specificity of MtIPMS is broad, and Mg2+ and Mn2+ are the metals that cause the highest activation. Interestingly, Zn2+, first assigned as the catalytic metal, inhibits the enzyme with submicromolar affinity. The features of monovalent cation and divalent metal activation, as well as the inhibition by Zn2+ and Cd2+, are discussed in light of the kinetic and structural information available for MtIPMS and other relevant enzymes. PMID- 16684500 TI - Thermodynamics of the reactions of carbamoyl phosphate. AB - Two measurements of equilibrium constants by Marshall and Cohen make it possible to calculate standard Gibbs energies of formation of the species of carbamate and carbamoyl phosphate. Carbamate formation from carbon dioxide and ammonia does not require an enzyme, and the equilibrium concentrations of carbamate in ammonium bicarbonate are calculated. Knowing the values of standard Gibbs energies of formation of species of carbamate and carbamoyl phosphate make it possible to calculate the dependencies of the standard transformed Gibbs energies of formation of these reactants on pH and ionic strength and to calculate apparent equilibrium constants for several enzyme-catalyzed reactions and several chemical reactions. These calculations are sufficiently complicated that computer programs in Mathematica are used to make tables and plots. The dependences of apparent equilibrium constants on pH are consequences of the production or consumption of hydrogen ions, which are shown in plots. As usual the increase in the number of enzyme-catalyzed reactions for which apparent equilibrium constants can be calculated is larger than the number of reactions required to obtain the thermodynamic properties of the species involved. PMID- 16684503 TI - An experimental investigation of the impact of biological and psychological causal explanations on anxious and depressed patients' perception of a person with panic disorder. AB - It is often suggested that biological accounts of the cause of mental health problems are beneficial in health education initiatives. However, an alternative view is that the idea of a diseased brain may result in stigma and therapeutic pessimism in sufferers, professionals and the public with implications for the perception of unpredictability and risk. Anxious and depressed patients (n=49) were randomly allocated to three experimental conditions. Prior to watching a video of a person suffering from panic disorder, participants were told either that research indicated that panic was caused by biological factors, by psychological factors or the cause was unclear (control condition). Those in the biological condition were significantly more pessimistic about the patient's prospects for recovery and rated risks as higher compared to those in the psychological condition. The results call into question the widely accepted practice of promoting biological/disease explanations of mental health problems. PMID- 16684504 TI - A chitinase structurally related to the glycoside hydrolase family 48 is indispensable for the hormonally induced diapause termination in a beetle. AB - Two proteins (APAP I and II) of the glycoside hydrolase family 48 (Family GH48) were isolated from the active adults of the leaf beetle Gastrophysa atrocyanea. Full-length and cDNAs were sequenced. APAP I expression and function were examined in detail. The protein has a chitinase but not a glucanase and cellobiohydrolase activity. It is expressed in the feeding stages, including beetles whose diapause was terminated with a juvenile hormone agonist. Suppression of the APAP I expression by means of RNA interference prevented the hormonal termination of diapause. PMID- 16684505 TI - Regulation of integrin alpha10 expression in chondrocytes by the transcription factors AP-2epsilon and Ets-1. AB - Expression of integrin alpha10 is initiated at the beginning of chondrogenesis and continues throughout cartilage development in adult cartilage. In our study, we aim to identify regulatory sequences that control the cell-type specific expression of the human integrin alpha10 gene. Therefore, promoter constructs harboring 1139bp 5' of the transcriptional start site of the human integrin alpha10 gene were analyzed. Our experiments localized a promoter region that directs high levels of expression specifically in chondrocytes. A sequence analysis detected three consensus AP-2 binding sites within this functional domain. Functionality of these sites was tested and confirmed by cotransfection of AP-2 in a luciferase reporter assay. Interestingly, EMSA identified AP 2epsilon as the major AP-2 protein binding to the AP-2 consensus sequences. Additionally, Ets-1 was shown to be a positive regulator of the integrin alpha10 expression whereas Sox9 was irrelevant. Taken together, these results suggest that AP-2epsilon and Ets-1 are involved in the regulation of integrin alpha10 transcription in chondrocytes. PMID- 16684506 TI - Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability. AB - Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression. PMID- 16684507 TI - Expression and characteristics of vanilloid receptor 1 in the rabbit submandibular gland. AB - Vanilloid receptor 1 (VR1) is a polymodal receptor originally found in sensory neurons of the central nervous system. Recent evidence indicates that VR1 is also expressed in non-neuronal tissues. We report here endogenous expression of VR1 in rabbit submandibular gland (SMG) and its possible role in regulating saliva secretion based on: (i) the expression of VR1 mRNA and protein detected in SMG; (ii) VR1 was mainly localized in the basolateral membrane of duct cells and the cytoplasm of acinar cells and also in cytoplasm of primary cultured neonatal rabbit SMG cells; (iii) stimulation of neonatal rabbit SMG cells with capsaicin induced a significant increase in intracellular calcium, and capsazepine, a VR1 antagonist, abolished this increase; (iv) infusion of capsaicin via the external carotid artery to isolated SMG increased saliva secretion of the gland. These findings indicated that VR1 was expressed in SMG and appeared to play an important role in regulating saliva secretion. PMID- 16684508 TI - Epidermal-type fatty acid binding protein as a negative regulator of IL-12 production in dendritic cells. AB - Fatty acids and their metabolites have recently been shown to modulate various functions of dendritic cells (DCs) including their differentiation and cytokine production, although the mechanisms underlying their cellular functions are not fully understood. In view of our previous finding that epidermal-type fatty acid binding protein (E-FABP) was exclusively expressed in splenic DCs among FABP family, we examined the phenotype of E-FABP-null mutant mice in order to elucidate the functional significance of E-FABP expression in DCs. Although E FABP-null mutant mice showed no apparent abnormalities in the population density and subset distribution of DCs as well as the microscopic morphology in the spleen, DCs isolated from E-FABP-null spleen showed enhanced production of IL 12p70, a key cytokine for innate immune responses, in response to appropriate stimuli as compared with wild-type. In real-time PCR, the expression level of IL 12p35 mRNA after LPS stimuli was much higher in mutant DCs when compared with wild-type, while no apparent change of IL-12p40 mRNA level was detected. Phosphorylated forms of p38 mitogen-activated protein kinase (p38MAPK) and IkappaB-alpha, molecules critical for IL-12 production, were detected at higher levels in E-FABP-null-mutant DCs after LPS stimuli when compared with wild-type counterparts. Collectively, it is suggested that E-FABP may be a novel negative regulator of IL-12 production in DCs, and this regulation may be exerted via its involvement in the p38MAPK-mediated transcription of IL-12p35. PMID- 16684509 TI - Paradoxical effects of the phage display-derived peptide antagonist IGF-F1-1 on insulin-like growth factor-1 receptor signaling. AB - The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1, (125)I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [(3)H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [(3)H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor. PMID- 16684510 TI - The spatio-temporal brain dynamics of processing and integrating sound localization cues in humans. AB - Interaural intensity and time differences (IID and ITD) are two binaural auditory cues for localizing sounds in space. This study investigated the spatio-temporal brain mechanisms for processing and integrating IID and ITD cues in humans. Auditory-evoked potentials were recorded, while subjects passively listened to noise bursts lateralized with IID, ITD or both cues simultaneously, as well as a more frequent centrally presented noise. In a separate psychophysical experiment, subjects actively discriminated lateralized from centrally presented stimuli. IID and ITD cues elicited different electric field topographies starting at approximately 75 ms post-stimulus onset, indicative of the engagement of distinct cortical networks. By contrast, no performance differences were observed between IID and ITD cues during the psychophysical experiment. Subjects did, however, respond significantly faster and more accurately when both cues were presented simultaneously. This performance facilitation exceeded predictions from probability summation, suggestive of interactions in neural processing of IID and ITD cues. Supra-additive neural response interactions as well as topographic modulations were indeed observed approximately 200 ms post-stimulus for the comparison of responses to the simultaneous presentation of both cues with the mean of those to separate IID and ITD cues. Source estimations revealed differential processing of IID and ITD cues initially within superior temporal cortices and also at later stages within temporo-parietal and inferior frontal cortices. Differences were principally in terms of hemispheric lateralization. The collective psychophysical and electrophysiological results support the hypothesis that IID and ITD cues are processed by distinct, but interacting, cortical networks that can in turn facilitate auditory localization. PMID- 16684511 TI - Increased isoform-specific membrane translocation of conventional and novel protein kinase C in human neuroblastoma SH-SY5Y cells following prolonged hypoxia. AB - Several studies have suggested that protein kinase C (PKC) plays a key role in the mechanism of cerebral ischemic/hypoxic preconditioning (I/HPC). However, detailed information regarding PKC isoforms in response to brain ischemia/hypoxia and their potential role in neuroprotection is unclear. Previous studies in our laboratory have demonstrated that the levels in membrane translocation of conventional PKC (cPKC) betaII, gamma, and novel PKCepsilon (nPKC), but not cPKCalpha, betaI, nPKCdelta, eta, mu, theta, and atypical PKC (aPKC) zeta and iota/lambda, were increased significantly in the hippocampus and cortex of intact mice with hypoxic preconditioning. To further detect cPKC and nPKC isoforms activation following prolonged hypoxia in vitro, we tested the membrane translocation (an indicator of PKC activation) of cPKCalpha, betaI, betaII, and gamma, and nPKCdelta, epsilon, eta, mu, and theta in a human neuroblastoma SH SY5Y cell line following sustained hypoxic exposure (1% O(2)/5% CO(2)/94% N(2)). Using Western blot and immunocytochemistry methods, we found that the levels of cPKCalpha, betaI, betaII, and nPKCepsilon, but not nPKCdelta, eta, mu, and theta, membrane translocation were increased significantly (P < 0.05, n = 8) in a time dependent manner (from 0.5 to 24 h) following sustained hypoxic exposure. Similarly, the immunostaining experiment also showed a noticeable translocation of cPKCalpha, betaI, betaII, and nPKCepsilon from the cytosol to the perinuclear or membrane-related areas after 6 h posthypoxic exposure. In addition, no cPKCgamma was detected in this cell line under either a normoxic or hypoxic condition. These results suggested that prolonged hypoxia may induce the activation of cPKCalpha, betaI, betaII, and nPKCepsilon by triggering their membrane translocation in SH-SY5Y cells. PMID- 16684512 TI - Prolonged bihemispheric alterations in unfolded protein response related gene expression after experimental stroke. AB - After ischemia, endoplasmic reticulum (ER) stress pathways are activated that include unfolded protein response (UPR) and protein synthesis inhibition (PSI). Both of these mechanisms aim to restore ER functioning mainly by inhibition of translation and increased processing of excess proteins in ER. We were interested in the role of these pathways during spontaneous recovery after transient middle cerebral artery occlusion (MCAO) in rats. The spontaneous recovery of rats was assessed with a limb-placing test. The expression of ER-stress-related genes (IRE1, ATF6, GRP78, eif2alpha, ATF4, PERK) was studied by using in situ hybridization in different brain areas on post-operative days 2, 7, 14 and 28. Elevated signals were detected in striatum contralateral to the lesion on days 2 (PERK and IRE1) and 14 post-ischemia (IRE1). Gene expression was elevated on day 7 in the striatum ipsilateral to the lesion (ATF6 and GRP78) and on day 14 (GRP78) post-ischemia. Furthermore, elevated levels of GRP78 were detected on day 14 after ischemia in the ipsilateral sensorimotor cortex. These results suggest that altered gene expression related to unfolded protein response may be more long lasting than expected following focal cerebral ischemia. In addition, these results show that the response to ER stress differs ipsi- and contralaterally after MCAO in rats. Since these differences are detected in both hemispheres only in areas adjacent to the lesion, UPR may contribute to spontaneous recovery after MCAO in rats. PMID- 16684513 TI - CB1 knockout mice display significant changes in striatal opioid peptide and D4 dopamine receptor gene expression. AB - Antagonism of the CB(1) cannabinoid receptor (CB(1) receptor) by rimonabant (SR141716) reduces self-administration of alcohol and other drugs of abuse in animal models. These findings suggest that the CB(1) receptor may be a target for genetic differences that modify the salient features of rewarding drugs. In the present study, wild-type (CB(1) (+/+)) are compared to transgenic mice deficient in CB(1) receptors (CB(1) (-/-)). The goal was to investigate the influences of the cannabinoid receptor system on opioid peptide gene expression and on dopamine receptor gene expression which is commonly influenced by substances of abuse. We demonstrate using reverse transcription and real-time polymerase chain reaction (PCR) that striatal mRNA for preproenkephalin (PPENK) and preprodynorphin (PPDYN) in the CB(1) (-/-) striatum increases when compared to CB(1) (+/+). Real-time PCR analyses to evaluate D(2) and D(4) dopamine receptor gene expression in striatum isolated from CB(1) (+/+) and CB(1) (-/-) revealed a nearly 2-fold increase in D(4) receptor mRNA in the striatum from CB(1) (-/-) mice and no significant change in D(2) expression. In contrast, treatment of C57BL/6 mice with the CB(1) receptor antagonist, rimonabant, produced a reduction of both D(2) and D(4) dopamine receptor expression in the striatum. These data suggest that genetic differences in CB(1) receptor may exert a modulatory effect on D(4) dopamine receptor and opioid peptide gene expression. PMID- 16684514 TI - Human MT/V5 activity on viewing eye gaze changes in others: A magnetoencephalographic study. AB - The present study used magnetoencephalography (MEG) to investigate human MT/V5 activity when observing changes in eye gaze. Subjects viewed a face in which the eyes changed to look either directly at (BACK) or away from (AWAY) the subject in a series of apparent motion conditions. BACK involved 2 directions, from left to center (LC) and from right to center (RC). Likewise, AWAY involved 2 directions, from center to left (CL) and from center to right (CR). A clear MEG component, 1M, was elicited with all eye gaze changes. Mean peak latency was 157 ms and was unaffected by stimulus condition. The equivalent current dipole (ECD) was localized to human MT/V5. Two main effects were noted: (1) ECD moment was significantly larger for BACK than for AWAY; and (2) 1M ECD locations were more posterior for AWAY than for BACK. Gaze direction, with LEFT involving CL and RC and RIGHT involving CR and LC, showed no significant effects. These data indicate that MT/V5 responds to gaze direction rather than eye position, and that eye movements directed at the viewer elicit the strongest effects. Processing of gaze change is NOT sensitive to eye direction per se but rather is modulated by eye gaze relative to the viewer. PMID- 16684515 TI - The supragenual nucleus: a putative relay station for ascending vestibular signs to head direction cells. AB - Head direction (HD) cells located in several regions of the brain, including the postsubiculum, retrosplenial cortex, lateral dorsal thalamic nucleus, anterior dorsal thalamic nucleus, and lateral mammillary nucleus, provide a signal of the rat's momentary directional heading. Experimental evidence suggests that vestibular inputs are critical for the maintenance these cells' directional sensitivity. However, it is still unclear how vestibular information is conveyed to the HD cell-related circuitry. In a recent study, the supragenual nucleus (SG) was suggested as a putative relay of vestibular inputs to this circuitry. In the present study, using anterograde and retrograde tract-tracing methods, we first investigated whether the SG is in a position to convey vestibular inputs. Next, we examined the projections of the SG with the Phaseolus vulgaris leucoagglutinin method. Our results indicate that the SG receives direct inputs from the medial vestibular nucleus and projects to elements of the HD cell-related circuitry, providing a massive input to the contralateral dorsal tegmental nucleus and a moderately dense projection to the shell region of the lateral mammillary nucleus. Overall, the present findings serve to clarify how vestibular inputs reach the HD cell-related circuit and point out the SG as an important interface to this end. PMID- 16684516 TI - Theiler's virus induces the MIP-2 chemokine (CXCL2) in astrocytes from genetically susceptible but not from resistant mouse strains. AB - The murine encephalomyelitis virus of Theiler (TMEV) induces demyelination in susceptible strains of mice by a CD4(+) Th1 T cell mediated immunopathologic process. We focused on the production of one chemokine, the macrophage inflammatory protein-2 (MIP-2 or CXCL2), by cultured mouse astrocytes infected with the BeAn strain of TMEV. Analysis of a murine genome DNA hybridized with cRNA from mock- and TMEV-infected astrocytes, revealed up-regulation of three sequences encoding MIP-2. Northern blot analysis indicated increased MIP-2 mRNA expression. Levels of MIP-2 in the supernatants of infected cells as detected by ELISA, varied directly with the multiplicity of infection used. This secreted CXCL2 was biologically active inducing chemoattraction of neutrophils but not of lymphocytes. CXCL2 was specifically induced by TMEV infection, since induction was inhibited by anti TMEV antibodies. The inflammatory cytokines, IL-1alpha and TNF-alpha, which are also induced in astrocytes by TMEV, were very potent inducers of CXCL2. Nevertheless, both mechanisms of induction follows different pathways as antibodies to both cytokines fails to inhibit TMEV-induced CXCL2 up regulation. Sera from TMEV-infected SJL/J mice with chronic demyelination, but not from BALB/c TMEV-resistant mice, revealed CXCL2 at the peak of clinical disease. Our main novel finding is the strain-dependent differences in CXCL2 expression both in vitro and in vivo. This suggest an role for this chemokine in attracting immune cells within the CNS, which in turn, might trigger demyelination in this experimental model of MS. PMID- 16684517 TI - Inhibitor-resistant type I receptors reveal specific requirements for TGF-beta signaling in vivo. AB - Activin/nodal-like TGF-beta superfamily ligands signal through the type I receptors Alk4, Alk5, and Alk7, and are responsible for mediating a number of essential processes in development. SB-431542, a chemical inhibitor of activin/nodal signaling, acts by specifically interfering with type I receptors. Here, we use inhibitor-resistant mutant receptors to examine the efficacy and specificity of SB-431542 in Xenopus and zebrafish embryos. Treatment with SB 431542 eliminates Smad2 phosphorylation in vivo and generates a phenotype very similar to those observed in genetic mutants in the nodal signaling pathway. Inhibitor-resistant Alk4 efficiently rescues Smad2 signaling, developmental phenotype, and marker gene expression after inhibitor treatment. This system was used to examine type I receptor specificity for several activin/nodal ligands. We find that Alk4 can efficiently rescue signaling by a wide range of ligands, while Alk7 can only weakly rescue signaling by the same ligands. In whole embryos, nodal signaling during gastrulation can be rescued with Alk4, but not Alk7, while Alk5 can only mediate signaling by ligands expressed later in development. The combination of the ALK inhibitor SB-431542 with inhibitor-resistant ALKs provides a powerful set of tools for examining nodal/activin signaling during embryogenesis. PMID- 16684518 TI - Mosaic analysis of extended auricle1 (eta1) suggests that a two-way signaling pathway is involved in positioning the blade/sheath boundary in Zea mays. AB - The maize leaf develops in a simple, stereotypical manner; therefore, it serves as a basic model to understand the processes involved in forming developmental boundaries. extended auricle1 (eta1) is a pleiotropic maize mutant that affects proximodistal leaf development. Mutant eta1 individuals display basipetal displacement of the blade/sheath boundary and the boundary between auricle and blade is not clearly delineated, leading to an undulating auricle. SEM analysis shows that eta1 is required for proper placement of the blade/sheath boundary on the adaxial leaf surface. Examination of vascular and cellular organization indicates that eta1 affects not only placement of the blade/sheath boundary, but also differentiation of cell types within the blade/sheath boundary. Genetic mosaic analysis was used to determine the effect of eta1 mutant tissue on wild type leaf development and to resolve the site and timing of the Eta1+ gene product. Interestingly, sectors of eta1 tissue affect the placement of the blade/sheath boundary even in wild-type tissue. These results suggest that a two way signaling pathway may be involved in the positioning of the blade/sheath boundary. Based on these data, we propose a model for Eta1+ function in the maize leaf. PMID- 16684519 TI - Low activity by the calpain system in primate lenses causes resistance to calcium induced proteolysis. AB - The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin. PMID- 16684520 TI - Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells. AB - The aim of the study was to evaluate the antiproliferative and cytotoxic properties of triamcinolone acetonide (TA) on human retinal pigment epithelium cells (ARPE19) and the role of epicellular crystalline deposits. Monolayer cultures of ARPE19 cells were used. Purified or unpurified crystalline TA suspension (0.01-1.0 mg/ml) or the vehicle alone (benzyl alcohol, 0.025% 0.00025%), diluted in culture medium, were added to the cells that were either grown on cell culture dishes covered by a protecting membrane filter insert or without a filter. After 1, 3, 5 and 7 days mitochondrial activity was measured using the MTT assay and the morphology assessed microscopically. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays ARPE19 cells were grown to confluence and then cultured in a serum-deficient medium to ensure a static milieu. Annexin V-FITC and propidium iodide co-staining was performed and analyzed by flow cytometry. Exposure to TA without direct cellular contact showed a moderate antiproliferative activity resulting in a dose-dependent suppression of DNA synthesis (maximum 42.7%), but not a cytotoxic effect. In contrast, adherent deposits of crystalline TA particles on top of the cell layer caused a rapid-progressive and dose-dependent cell death preceded by an early phosphatidylserine externalization to the outer leaflet of the plasma membrane. Within a healthy, confluent cell layer the number of viable cells decreased by 14.2, 20.8 and 68.8%, respectively, after one day of direct exposure. Exposure to the vehicle alone caused only a slight growth inhibitory effect in a proliferating cell layer, but early signs of cell death were detected even at the lowest concentration tested. In conclusion, the effect of the vehicle is less pronounced than formerly assumed, but not negligible, thus indicating a beneficial effect of purification. While non-adherent TA, if purified, appears to be safe in clinically used concentrations, direct physical contact with crystalline particles might cause a local, rapid-progressive cytotoxicity that involves the induction of the apoptotic cascade. Therefore, epiretinal deposits after intravitreal TA administration might be critical in terms of long-term biocompatibility. PMID- 16684521 TI - Aqueous humour- and growth factor-induced lens cell proliferation is dependent on MAPK/ERK1/2 and Akt/PI3-K signalling. AB - The aqueous humour of the eye is a rich source of growth factors, many of which have been shown to be lens cell mitogens; however, the identity of the endogenous mitogen(s) for lens cells is still unknown. As a first approach to identify the mechanisms by which these aqueous humour-derived growth factors induce lens cell proliferation, the present study set out to examine MAPK/ERK1/2 and PI3-K/Akt signalling associated with lens cell proliferation. Using a lens explant system, we examined the effects of different lens mitogens (aqueous humour, FGF, PDGF, IGF and EGF) using 5'-2'-bromo-deoxyuridine incorporation. In addition, we adopted immunolabelling techniques to compare the roles that the ERK1/2 and PI3-K signalling pathways play in regulating lens cell proliferation. We showed that the aqueous humour, and all the other growth factors examined, could activate ERK1/2 and PI3-K/Akt signalling. By targeting these pathways using specific pharmacological inhibitors, we were able to show that both ERK1/2 and PI3-K signalling are required for growth factor-induced lens cell proliferation, and that there was a strong correlation between the spatial distribution of proliferating cells in lens explants with ERK1/2 labelling. Furthermore, our blocking studies confirmed that PI3-K/Akt signalling can act upstream of ERK1/2, potentiating ERK1/2 phosphorylation in growth factor-induced lens cell proliferation. A better understanding of the signalling pathways required for aqueous humour-induced lens cell proliferation may ultimately allow us to identify the mitogen(s) that are important for regulating lens cell proliferation in situ. PMID- 16684522 TI - Role of nuclear factor-kappaB in interleukin-1-induced collagen degradation by corneal fibroblasts. AB - The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration. The role of nuclear factor (NF)-kappaB in the IL-1-induced degradation of collagen by corneal fibroblasts that underlies corneal ulceration was investigated. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen with or without IL-1 and sulfasalazine, an inhibitor of NF-kappaB activation. Collagen degradation was assessed from the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) into culture supernatants was examined by immunoblot analysis and gelatin zymography, and the cellular abundance of MMP and TIMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation and degradation of the NF-kappaB-inhibitory protein IkappaB-alpha were examined by immunoblot analysis. The subcellular localization and DNA binding activity of the p65 subunit of NF-kappaB were evaluated by immunofluorescence analysis and with a colorimetric assay, respectively. The transactivation activity of NF-kappaB was assessed with a reporter gene assay. Sulfasalazine inhibited IL-1-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also inhibited the stimulatory effects of IL-1 on the synthesis or activation of various MMPs in a concentration-dependent manner. IL-1 induced the phosphorylation and degradation of IkappaB-alpha, the nuclear translocation and up-regulation of the DNA binding activity of the p65 subunit of NF-kappaB, and the activation of NF-kappaB in a manner sensitive to sulfasalazine. These results suggest that NF-kappaB contributes to the IL-1 induced degradation of collagen by corneal fibroblasts and is therefore a potential therapeutic target for treatment of corneal ulcers. PMID- 16684523 TI - Multiplex fluorescence in situ hybridization identifies novel rearrangements of chromosomes 6, 15, and 18 in primary uveal melanoma. AB - Uveal melanomas are the commonest ocular tumour of adults and are characterized by reproducible alterations of chromosomes 1, 3, 6 and 8. These alterations are of prognostic relevance and have also be shown to correlate to high risk and low risk metastatic categories of uveal melanoma as defined by micro-array analysis. It is, however, possible that a catalogue of relevant genetic alterations, involving gene rearrangement rather than amplification, have as yet eluded identification. To address this point we examined 14 primary uveal melanomas, using 24 colour multiplex fluorescence in situ hybridization (M-FISH). All tumours were karyotyped following G-Banding, and M-FISH was performed to confirm and clarify the identity of abnormal chromosomes. M-FISH data were obtained from all tumours and was able to establish the nature of most abnormalities not fully characterized by cytogenetics. Abnormalities of chromosome 6 were far more frequent than previously indicated, in approximately 70% of cases, indicating they have been substantially underrepresented in past studies of uveal melanoma. Spindle melanomas were found to have novel rearrangements affecting in particular chromosomes 6, 15 and 18, suggesting that juxtaposition of genes through translocational events may play a role in the development of some uveal melanomas. In conclusion, this study is the largest of primary uveal melanoma analysed by M-FISH and indicates that alterations of chromosome 6 have previously been underestimated. Furthermore spindle melanomas are prone to rearrangements affecting chromosomes 6, 15 and 18, which may relate to early changes in uveal melanoma development or associate with those melanomas of a more differentiated status. PMID- 16684524 TI - Regulation of cysteine cathepsin expression by oxidative stress in the retinal pigment epithelium/choroid of the mouse. AB - Cystatin C is the major inhibitor of the cysteine cathepsins. Polymorphisms in the cystatin C gene have recently been associated with the risk of developing Age related Macular Degeneration (AMD). Oxidative stress is also thought to play a key role in the pathogenesis of AMD. We surveyed the retinal pigment epithelium (RPE) and choroid of the C57BL/6J mouse for the expression of the cysteine cathepsins under normoxic and hyperoxic (75% O(2)) conditions. Microarray analysis of RPE/choroid mRNA revealed the expression of cathepsins B and L, as well as cystatin C under all experimental conditions. The microarray results were confirmed by real-time quantitative polymerase chain reaction (PCR). Localization of the mRNA species for cystatin C and cathepsin B, as well as, localization of protein species for cystatin C, cathepsins B and L were performed to evaluate the tissue distribution of these species. Our results indicate that cystatin C is largely synthesized in the RPE and secreted from the basal side. Cathepsin B is the major cysteine protease in the RPE and choroid. The expression of all mRNAs and proteins was elevated by exposure to oxidative stress. PMID- 16684525 TI - Constitutive expression of abiotic stress-inducible hot pepper CaXTH3, which encodes a xyloglucan endotransglucosylase/hydrolase homolog, improves drought and salt tolerance in transgenic Arabidopsis plants. AB - Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants. PMID- 16684526 TI - Redox-responsive in vitro modulation of the signalling state of the isolated PrrB sensor kinase of Rhodobacter sphaeroides NCIB 8253. AB - Prr is a global regulatory system that controls a large and diverse range of genes in Rhodobacter sphaeroides in response to changing conditions of environmental redox potential. PrrB is the membrane-bound sensor kinase and previously we showed that the purified, detergent-solubilised intact membrane protein is functional in autophosphorylation, phosphotransfer and phosphatase activities. Here we confirm that it also senses and responds directly to its environmental signal, redox potential; strong autophosphorylation of PrrB occurred in response to dithiothreitol (DTT)-induced reducing conditions (and levels increased in response to a wide 0.1-100 mM DTT range), whilst under oxidising conditions, PrrB exhibited low, just detectable levels of autophosphorylation. The clear response of PrrB to changes in reducing conditions confirmed its suitability for in vitro studies to identify modulators of its phosphorylation signalling state, and was used here to investigate whether PrrB might sense more than one redox-related signal, such as signals of cell energy status. NADH, ATP and AMP were found to exert no detectable effect on maintenance of the PrrB-P signalling state. By contrast, adenosine diphosphate produced a very strong increase in PrrB-P dephosphorylation rate, presumably through the back-conversion of PrrB-P to PrrB. PMID- 16684527 TI - PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3. AB - Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy. PMID- 16684528 TI - Featuring... Theresia Stradal. Interview by Tine Walma. PMID- 16684530 TI - MRI-guided gene therapy. AB - MRI has the ability to generate high-contrast and high-resolution images, to obtain multiple diagnostic evaluations of organ function and morphology, and to provide multiple image planes with no risk of ionizing radiation. Recent efforts have focused on using MR technology to monitor gene delivery, to enhance gene transfection/transduction, and to track gene expression. This review summarizes the current status of MRI-guided gene therapy. PMID- 16684529 TI - Akt1 is dynamically modified with O-GlcNAc following treatments with PUGNAc and insulin-like growth factor-1. AB - The Ser/Thr kinase Akt1 is activated by growth factors subsequent to its phosphorylation on Thr308 and Ser473. In the present study, Akt1 was found to be constitutively modified with O-GlcNAc. Treatment of SH-SY5Y cells with O(2 acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which inhibits the enzymatic removal of O-GlcNAc from proteins, increased cytosolic O GlcNAc-Akt1 levels. Treatment of cells with insulin-like growth factor-1 (IGF-1) also increased O-GlcNAc-Akt1 levels and increased Akt1 phosphorylation. PUGNAc treatment did not attenuate IGF-1 induced Akt1 phosphorylation. These results indicate that Akt1 can be simultaneously modified with O-GlcNAc and phosphorylated. However, PUGNAc induced the nuclear accumulation of Akt1 suggesting that the O-GlcNAc-modification on Akt1 may play a role in Akt1 nuclear localization. PMID- 16684531 TI - The transition metal chelator nicotianamine is synthesized by filamentous fungi. AB - Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+-hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray-ionization-quadrupole-time-of-flight mass spectrometry we prove the formation of nicotianamine in N. crassa. Transcript level is strongly upregulated under Zn deficiency as shown by real time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions. PMID- 16684532 TI - A component of BRAF-HDAC complex, BHC80, is required for neonatal survival in mice. AB - BHC80 is a component of BRAF-HDAC complex (BHC) involved in transcriptional repression of neuron-specific genes in non-neuronal cells. However, BHC80 is present in both neuronal and non-neuronal cells. To explore the physiological importance of BHC80 in vivo, and the precise mechanism underlying neuron-specific gene repression by BHC80, we have produced mutant mice lacking Bhc80. The loss of Bhc80 resulted in neonatal lethality without sucking mother's breast milk sufficiently. Although Bhc80-deficient mice showed no developmental defect in the neuronal and non-neuronal tissues, Bhc80 is indispensable for the survival of neonatal pups. PMID- 16684533 TI - Caspase-8 acts as a key upstream executor of mitochondria during justicidin A induced apoptosis in human hepatoma cells. AB - Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption. PMID- 16684534 TI - VHA-8, the E subunit of V-ATPase, is essential for pH homeostasis and larval development in C. elegans. AB - Vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans. PMID- 16684535 TI - Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase. AB - Three methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6 methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10(3) m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine methyltransferase activity was detected. PMID- 16684538 TI - Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells. AB - Coronavirus envelope (E) protein is a small integral membrane protein with multi functions in virion assembly, morphogenesis and virus-host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS-CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N- and C-termini of the SARS-CoV E protein are exposed to the cytoplasmic side of the membranes (N(cyto)C(cyto)). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N terminus exposed luminally and the C-terminus exposed cytoplasmically (N(exo(lum) )C(cyto)). Intriguingly, a minor proportion of the SARS-CoV E protein was found to be modified by N-linked glycosylation on Asn 66 and inserted into the membranes once with the C-terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS-CoV E protein may provide a useful clue to the pathogenesis of SARS-CoV. PMID- 16684536 TI - Erythropoietin production: Molecular mechanisms of the antagonistic actions of cyclic adenosine monophosphate and interleukin-1. AB - Erythropoietin (Epo) mRNA expression is suppressed by interleukin 1 (IL-1). Cyclic adenosine monophosphate (cAMP) can increase Epo mRNA and Epo protein levels in IL-1 treated HepG2 cells to some extent. To identify molecular mechanisms of this reaction we investigated three transcription factors (NF kappaB, GATA-2 and HIF-1) that control the Epo gene. Western blot analyses and electrophoretic mobility shift assays (EMSAs) revealed that IL-1 strongly activated NF-kappaB, which is a likely suppressor of the Epo promoter. Treatment of the cells with dibutyryl-cAMP (Bt2-cAMP) inhibited the activation of NF-kappaB by IL-1. Bt2-cAMP increased GATA-2 DNA binding. Since GATA-2 is a suppressor of the Epo promoter, GATA-2 activation was unlikely to cause the increase of Epo mRNA expression in IL-1 treated cells. Furthermore, Western blots, EMSAs and reporter gene studies showed that Bt2-cAMP was without effect on the hypoxia inducible transcription factor HIF-1. Thus, NF-kappaB is probably the primary transcription factor by which cAMP counteracts the inhibition of Epo gene expression by IL-1. PMID- 16684539 TI - para- and ortho-Pyridinium aldoximes in reaction with acetylthiocholine. AB - In the oximolysis reaction para-aldoximes K027 and TMB-4 react faster with ATCh than ortho-aldoximes HI-6 and K033. The reaction rate constants at 25 degrees C were 22 M(-1) min(-1) for HI-6 and K033, 230 M(-1) min(-1) for TMB-4 and 306 M( 1) min(-1) for K027. Semi-empirical calculations showed that differences in rates do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance of the oxime group within the molecule. Thermodynamic parameters, DeltaG#, DeltaH# and DeltaS#, were also determined for oximolysis reaction. PMID- 16684540 TI - Platelet-activating factor-induced NF-kappaB activation enhances VEGF expression through a decrease in p53 activity. AB - We investigated the role of p53 in nuclear factor (NF)-kappaB dependent, platelet activating factor (PAF)-induced vascular endothelial growth factor (VEGF) expression. Transfected NF-kappaB subunits in ECV304 cells increased the tumor necrosis factor-alpha promoter activity, which was completely inhibited by p53. Transfected p53 increased p53RE promoter activity, which was completely inhibited by NF-kappaB subunits, indicating that cross-regulation occurs between NF-kappaB and p53. PAF-induced increase in VEGF expression was correlated with decreased p53 activity. These data suggest that NF-kappaB-dependency of the PAF-induced increase in VEGF expression is due to decreased p53 activity, which is reciprocally regulated by increased NF-kappaB activity. PMID- 16684541 TI - The amino acid sensitive TOR pathway from yeast to mammals. AB - The target of rapamycin (TOR) is an ancient effector of cell growth that integrates signals from growth factors and nutrients. Two downstream effectors of mammalian TOR, the translational components S6K1 and 4EBP1, are commonly used as reporters of mTOR activity. The conical signaling cascade initiated by growth factors is mediated by PI3K, PKB, TSC1/2 and Rheb. However, the process through which nutrients, i.e., amino acids, activate mTOR remains largely unknown. Evidence exists for both an intracellular and/or a membrane bound sensor for amino acid mediated mTOR activation. Research in eukaryotic models, has implicated amino acid transporters as nutrient sensors. This review describes recent advances in nutrient signaling that impinge on mTOR and its targets including hVps34, class III PI3K, a transducer of nutrient availability to mTOR. PMID- 16684542 TI - Cloning of stanniocalcin (STC) cDNAs of divergent teleost species: Monomeric STC supports monophyly of the ancient teleosts, the osteoglossomorphs. AB - Molecular cloning of teleost stanniocalcin (STC) cDNAs was undertaken in two species of order Osteoglossiformes of subdivision Osteoglossomorpha and one species of each of orders Cypriniformes and Perciformes within the subdivision Euteleostei. The elephantnose (Gnathonemus petersii) and the butterflyfish (Pantadon buchholzi) are basal teleosts in different osteoglossiforme suborders yet their 218 amino acid (aa) mature hormones, from prehormones of 249 and 251aa, respectively, have only 10 cysteine residues. A substitution for cysteine at the intermonomeric disulfide linkage site, implies that their STCs exist as monomeric peptides, as is the case with STC from another osteoglossormorph, arawana [Amemiya, Y., Marra, L.E., Reyhani, N., Youson, J.H., 2002. Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution. Mol. Cell. Endocrinol. 188, 141-150]. The STC cDNA of the generalized teleost and cyprinid, the white sucker (Catostomus commersoni), encodes a prehormone of 249aa with a signal peptide of 31aa and a mature protein of 218aa that possesses 11 cysteine residues. The latter feature is consistent with a previous analysis that white sucker mature STC is a glycosylated, homodimeric peptide [Amemiya, Y., Marra, L.E., Reyhani, N., Youson, J.H., 2002. Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution. Mol. Cell. Endocrinol. 188, 141-150]. An open reading frame of the STC cDNA of the derived teleost and perciforme, the smallmouth bass (Micropterus dolomieui), encodes a prehormone of 255aa with a signal peptide of 33aa and a mature protein of 222aa. The position of the 11 cysteines in smallmouth bass STC suggests that it exists as a homodimeric peptide. A phylogenetic analysis, using the new STC-1 amino acid sequences and those in the gene data base provided strong support for monophyly of the Osteoglossomorpha and indicated, with positioning of white sucker and smallmouth bass, that this molecule has some utility as a taxonomic marker. This analysis also suggested that two STC-1 gene sequences exist in multiple fish genomes, and that they may be a product of the fish-specific genome duplication. The mutation in the osteoglossomorph STC likely occurred after the appearance of the first teleosts and before movement of the tectonic plates. PMID- 16684543 TI - Assessment of paraoxonase and arylesterase activities in patients with iron deficiency anemia. AB - Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL) associated enzyme with three activities which are paraoxonase, arylesterase and dyazoxonase. We aimed to determine serum (a) paraoxonase and arylesterase activities and, lipid hydroperoxide (LOOH) levels in patients with iron deficiency anemia (IDA) (b) whether there is an association between the development of atherosclerosis and paraoxonase/arylesterase activities in patients with IDA. Twenty-five female with IDA and 22 healthy female as control were enrolled in the study. Serum basal/salt stimulated paraoxonase and arylesterase activities were measured spectrophotometrically. LOOH levels were measured by ferrous oxidation with xylenol orange assay. Basal/salt-stimulated paraoxonase and arylesterase activities were significantly lower in patients with IDA than controls (p<0.001; for all), while LOOH levels were significantly higher (p<0.001). Our results show that paraoxonase and arylesterase activities, which have antiatherogenic capability, are decreased in patients with IDA. Reduced paraoxonase and arylesterase activities may play a role in pathogenesis of atherosclerosis through increased susceptibility to lipid peroxidation in patients with IDA. PMID- 16684545 TI - Antioxidant activity of coumarins and flavonols from the resinous exudate of Haplopappus multifolius. AB - The antioxidant activity of eight coumarins and two flavonols isolated from Haplopappus multifolius was studied with the DPPH radical method. Results show that a high concentration of phenolic coumarins and the presence of quercetin and rhamnetin in the exudates could account for the protection of the plant against oxidative stress. Structures for the coumarins 6-hydroxy-7-[(E,E)-3',7'-dimethyl 2',4',7'-octatrienyloxy] coumarin and 7-[(E)-3'-methyl-4'-hydroxy-2'-butenyloxy] coumarin are proposed on the basis of spectroscopic evidence. PMID- 16684544 TI - Brain metabolic changes associated with predispotion to onset of major depressive disorder and adjustment disorder in cancer patients--a preliminary PET study. AB - OBJECTIVES: To explore neurobiological risk factors for major depressive disorder (MDD) and adjustment disorder in cancer patients by examining regional brain metabolism before psychiatric manifestation using positron emission tomography and by prospectively observing depressive and anxiety symptoms. METHOD: Cancer patients who showed no psychiatric symptoms when they underwent 18F fluorodeoxyglucose positron emission tomography (18F-FDG PET) were followed up for one year using the Hospital Anxiety and Depression Scale (HADS). Fourteen patients who showed high HADS scores and 14 patients who showed low HADS scores were assessed by a psychiatrist 2 years after the PET scan and grouped into the deterioration group (n=10) and the no-change group (n=9). 18F-FDG PET images were analyzed to examine the difference in local brain glucose metabolism between the two groups. RESULTS: The deterioration group showed a decreased glucose metabolism in the right medial frontal gyrus (BA6) and an increased glucose metabolism in the right posterior cingulate (BA29), right anterior cingulate (BA25), left subcallosal gyrus (BA25), and left caudate compared with the no change group. CONCLUSION: Cancer patients who later developed MDD or adjustment disorder showed regional brain metabolic changes. These regions may be associated with vulnerability to the onset of MDD or adjustment disorder in cancer patients. PMID- 16684546 TI - What sort of networks are public health networks? AB - BACKGROUND: Re-organization of the English National Health Service (NHS) has fragmented the public health workforce, relocating teams from about 100 health authorities into over 300 primary care trusts (PCTs). The UK Government announced the setting up of public health networks (PHNs) as a solution to the problems created by fragmentation. METHODS: Fifty-seven semi-structured telephone interviews were held with key players in PHNs in all strategic health authority areas in England in early 2003. RESULTS: PHNs appeared to be primarily networks of public health professionals rather than of organizations. Informants were unsure about PCTs' commitment to public health. Predominantly, members were those NHS personnel with a clear and explicit public health role. Most PHNs intended to include others later (e.g. health visitors, environmental health officers), although a few thought that inclusivity was essential from the start. Continuing professional development for public health personnel dominated the work being undertaken, with some collaborative work across PCTs. PHNs were seen as a compulsory reconfiguration of existing networks, and informants doubted that they were appropriate for the many levels of networking that public health work requires. CONCLUSION: The formation of PHNs does not appear to have been either necessary or sufficient. However, the public health community has a well established tradition of networking, and therefore has the skills to use PHNs advantageously. PMID- 16684547 TI - Rural and urban differences in blood pressure and hypertension in Ghana, West Africa. AB - BACKGROUND: Hypertension, once rare in traditional African societies, is rapidly becoming a major public health problem. OBJECTIVE: To assess urban and rural differences in blood pressure (BP) and hypertension, and to determine factors associated with BP in this sub-Saharan Africa population. STUDY DESIGN: Cross sectional survey. SETTING: Ashanti region of Ghana, West Africa. PARTICIPANTS: There were 1431 participants (644 males and 787 females). Of these, 578 were from the rural setting (237 males and 341 females) and 853 from the urban setting (407 males and 446 females). RESULTS: Age-adjusted mean systolic and diastolic BP levels were lower in rural men than in urban men (129/75 versus 133/78, P<0.001). The mean systolic and diastolic BP levels were also lower in rural women than in urban women (126/76 versus 131/80, P<0.001). After adjustments for age, the odds ratios (95% CI) for being hypertensive were 1.9 (1.3-2.9; P<0.01) for urban men and 1.9 (1.3-2.8; P<0.0001) for urban women. Urban women were more likely than rural women to be aware of their hypertensive condition (odds ratio 2.3, 95% CI, 1.2-4.2; P<0.001). Treatment and control of hypertension did not differ between the groups in either men or women. In multiple linear regression analysis, age, urban dwelling, BMI and heart rate were independently associated with systolic and diastolic BP in both men and women. Smoking and alcohol consumption were independently associated with systolic and diastolic BP but only in men. CONCLUSION: The findings of this study demonstrate that high BP (hypertension) is an important public health burden in both urban and rural settings in this sub Saharan African population. Cost-effective public health measures are urgently needed to prevent high BP from becoming another public health burden. PMID- 16684548 TI - A study on the prevalence of and risk factors for neck pain in secondary school teachers. PMID- 16684549 TI - Mental health and sexual risk behaviours in a South African township: a community based cross-sectional study. AB - OBJECTIVES: Despite the high prevalence of both mental illness and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) in developing countries, there are few data on the association between different forms of mental illness and sexual risk behaviours in resource-poor settings. The objective of this study was to examine the association between mental illness and HIV risk behaviours in a South African township. STUDY DESIGN: A cross-sectional study was performed among 645 individuals living in households selected at random. METHODS: A self-administered translated questionnaire investigated sexual risk behaviours [including sexual partners, condom use, casual sexual contacts, and sex in exchange for money, drugs or a place to stay (transactional sex)], depression (measured using the Center for Epidemiological Studies Depression Scale), alcohol abuse (from the Alcohol Use Disorders Identification Test), and post-traumatic stress disorder (based on the Life Event Checklist). RESULTS: Of the 645 individuals who completed the survey, 33% reported depression, 17% reported alcohol abuse, and 15% reported post-traumatic stress disorder. After adjusting for demographic characteristics, the presence of any of these three conditions was strongly associated with experiences of forced sex [adjusted odds ratio (AOR) 2.53; 95% confidence intervals (CI) 1.60-4.02], transactional sex (AOR 2.88; 95% CI 1.29-6.48) and increased condom use (AOR 2.07; 95% CI 1.32 3.25). CONCLUSIONS: These findings emphasize the substantial burden of mental illness in this setting, and its association with forced and transactional sex. The temporal nature of these associations is not always clear from this cross sectional study, and additional prospective research is required. Public health interventions are needed to address the dual burden of HIV/AIDS and mental illness in this and similar settings. PMID- 16684550 TI - Validity of the single-item question on self-rated health status in first generation Turkish and Moroccans versus native Dutch in the Netherlands. AB - BACKGROUND: The single-item question on self-rated health has been used in many studies as a global measure of general health. It is unclear whether ethnic minority groups in the Netherlands attach the same meaning to the single-item question as the native Dutch people do. OBJECTIVE: To assess the validity of using the single-item question on self-rated health in comparing health status in native Dutch with first generation Turkish and Moroccan ethnic groups in the Netherlands. METHODS: The associations between self-reported chronic illnesses and self-rated health were used to examine convergent validity, and self-rated health and health care use for predictive validity using logistic regression analysis. RESULTS: In general, chronic illnesses were associated with fair health and poor health ratings in all the ethnic groups but there were important differences in associations between the groups. There were significant interactions between ethnicity and chronic illnesses on fair health, and poor health, independent of socio-demographical factors. There was also significant interaction between ethnicity and self-rated health on health care uses. These findings indicate that the meaning(s) attached to the single-item question differ between these ethnic groups. CONCLUSION: The study findings suggest that the use of the single-item question on self-rated health to compare native Dutch with the first generation Turkish and Moroccan ethnic groups is not valid. These findings imply that researchers need to be cautious about the interpretation of self-rated health ratings when comparing different ethnic groups. A qualitative research is needed to find out more about how these single-item ratings are being interpreted by Turkish and Moroccan elderly in the Netherlands. PMID- 16684551 TI - Comparison of protein phosphatase inhibition activities and mouse toxicities of microcystins. AB - Eight naturally purified microcystins (MCs), including MC-LR, -FR, -WR, -RR, [d Asp(3)]MC-FR, -WR, -RR, and [Dha(7)]MC-RR were utilized to determine the effects of amino acid substitutions and modifications on MC-induced protein phosphatase inhibition activity and mouse toxicity. Catalytic subunits of protein phosphatase 1 (PP-1) and 2A (PP-2A) were purified and subjected to the inhibition assays, and intraperitoneal injection was used to administer MCs into mice for the toxicity assay. It is found that the replacement of the non-polar amino acid l-leucine at the second position of these heptacyclic peptide toxins by a polar l-arginine reduces their mouse toxicities and inhibitory activities against PP-1 and PP-2A to different extends. Demethylation of methyldehydroalanine (Mdha) at the seventh amino acid of MC-RR exhibits the least mouse toxicity and phosphatase inhibition. The loss of a methyl group on the common methylaspartic acid (MeAsp) at the third position of MC-FR, -WR, and -RR does not alter their toxicity levels, but dominantly reduces their activities in PP-1 inhibition compared to other substitutions or modifications. This suggests that the methyl group on MeAsp is also important for MCs inhibition. However, such a tendency is not observed for PP-2A. By comparing the LD(50) values of the mouse toxicity assay and IC(50) values of the PP-1 and PP-2A inhibition assay of eight MCs using linear regression, it is evident that the MC-induced toxicity is much more related to the inhibition of PP-2A than PP-1, which suggests that PP-2A inhibition may play a major role in the MC-induced mouse toxicity. PMID- 16684552 TI - Functional characterization of HIV-1 Nef mutants in the context of viral infection. AB - Nef is an important pathogenesis factor of HIV-1 with a multitude of effector functions. We have designed a broad panel of isogenic viruses encoding defined mutants of HIV-1(SF2) Nef and analyzed their biological activity in the context of productive HIV-1 infection. Analysis of subcellular localization, virion incorporation, downregulation of cell surface CD4 and MHC-I, enhancement of virion infectivity and facilitation of HIV replication in primary human T lymphocytes mostly confirmed the mapping of Nef determinants previously reported upon isolated expression of Nef. However, reduced activity in downregulation of CD4, infectivity enhancement and virion incorporation of a Nef variant (Delta12 39) lacking an amphipatic helix required for binding of a cellular kinase complex and the association of Nef with MHC-I/AP-1 suggested a novel role of this N terminal motif. The SH3 binding motif of Nef was partially required for infectivity enhancement and replication but not for receptor downmodulation. In contrast to previous results obtained using other Nef alleles, non-myristoylated SF2-Nef was only partly defective when expressed during HIV infection and was present in HIV-1 particles. Importantly, incorporation of Nef into HIV-1 virions was not required for any of the tested Nef activities. Altogether, this study provides a broad characterization and mapping of multiple Nef activities in HIV infected cells. The results emphasize that multiple activities govern Nef's effects on HIV replication and argue against a role of virion incorporation for Nef's activity as pathogenicity factor. PMID- 16684553 TI - Test stimulus characteristics determine the perceived speed of the dynamic motion aftereffect. AB - Using a speed-matching task, we measured the speed tuning of the dynamic motion aftereffect (MAE). The results of our first experiment, in which we co-varied dot speed in the adaptation and test stimuli, revealed a speed tuning function. We sought to tease apart what contribution, if any, the test stimulus makes towards the observed speed tuning. This was examined by independently manipulating dot speed in the adaptation and test stimuli, and measuring the effect this had on the perceived speed of the dynamic MAE. The results revealed that the speed tuning of the dynamic MAE is determined, not by the speed of the adaptation stimulus, but by the local motion characteristics of the dynamic test stimulus. The role of the test stimulus in determining the perceived speed of the dynamic MAE was confirmed by showing that, if one uses a test stimulus containing two sources of local speed information, observers report seeing a transparent MAE; this is despite the fact that adaptation is induced using a single-speed stimulus. Thus while the adaptation stimulus necessarily determines perceived direction of the dynamic MAE, its perceived speed is determined by the test stimulus. This dissociation of speed and direction supports the notion that the processing of these two visual attributes may be partially independent. PMID- 16684554 TI - Age related changes in accommodative dynamics in humans. PMID- 16684555 TI - Evidence for two interacting temporal channels in human visual processing. AB - Previous studies have generally estimated that two independent channels underlie human temporal vision: one broad and low-pass, the other high, and band-pass. We confirm this with iso-oriented targets and masks. With orthogonal masks, the same high-frequency channel emerges but no low-pass channel is observed, indicating the high-frequency channel is orientation invariant, and possibly pre-cortical in origin. In contrast, orientation dependence for low frequencies suggests a cortical origin. Subsequent masking experiments using unoriented spatiotemporal filtered noise demonstrated that high-frequency masks (>8Hz) suppress low frequency targets (1 and 4Hz), but low frequencies do not suppress high frequencies. This asymmetry challenges the traditional assumption of channel independence. To explain this, we propose a two-channel model in which a non orientation-selective high-frequency channel suppresses an orientation-tuned low frequency channel. This asymmetry may: (i) equalise the over-representation of low temporal-frequency energy in natural stimuli (1/f power spectrum); (ii) contribute to motion deblurring. PMID- 16684556 TI - Endogenous influences on perceptual bistability depend on exogenous stimulus characteristics. AB - We investigated the influence of changing physical parameters and task on bistable perception of an ambiguously rotating sphere (SFM). Increasing dot density and velocity decreased the duration of perceptual phases during both passive viewing and voluntary control exertion. Our main finding is that voluntary control of perception depends on the physical parameters constituting the stimulus. This dependency places important constraints on the mechanisms mediating voluntary control as these mechanisms cannot operate independently of stimulus characteristics. In addition, local asymmetries in dot-densities can trigger alternations towards the most salient direction, which is not necessarily associated with largest number of dots: competition between perceptual interpretations during SFM appears to occur between surface-based representations rather than between individual elements. Finally, we show that voluntary control remains effective, even when attentive tracking of individual stimulus elements is no longer possible. PMID- 16684557 TI - Modeling the crystallization of magnesium ammonium phosphate for phosphorus recovery. AB - The crystallization of magnesium ammonium phosphate (MAP) is one of the main processes for recovering P and N from wastewater. Chemically defined solution systems were designed; the saturation indices (SIs) of the solution systems with respect to MAP were derived by using a geochemical aqueous model Program, PHREEQC 2.11; the effects of the solution conditions were evaluated using thermodynamic theories. The concentrations of P and Mg in the tested solutions were 10-600 mg l(-1) and 24-720 mg l(-1), respectively, the molar ratios of N/P and pH values of the solutions varied in the ranges of 1-40 and 6.0-12.0, respectively. The temperature of all the tests was set at 25 degrees C. The test results show that the SI value of MAP is the logarithmic functions of the concentrations of P, ammonium-N and Mg, and increases with the increase of the concentration of each element. The SI value of MAP is a polynomial function of pH value of the solution, and the optimum pH value for the crystallization of MAP is 9.0 but increases slightly with the increase of the N/P. Moreover, the SI value of MAP is a power law function of the ionic strength of solutions but decreases with its increase. The adjustment of the Mg concentration and the control of solution pH are two effective methods for the control of the crystallization of MAP. The results obtained from the research can be used to guide the design and control of MAP crystallization process for the removal and recovery of P. PMID- 16684558 TI - Embryonal rhabdomyosarcoma of the uterus in a postmenopausal woman. Case report and review of the literature. AB - BACKGROUND: Embryonal rhabdomyosarcoma is a rare sarcoma which characteristically occurs in non genitourinary sites in children. CASE: We present a case of uterine embryonal rhabdomyosarcoma in a postmenopausal patient who presented with increasing abdominal girth, early satiety, weight loss, and pelvic pain. CONCLUSION: Embryonal rhabdomyosarcoma does not commonly originate from the uterine corpus, and it is rarely seen in postmenopausal patients. A review of the literature confirms the unique nature of this case. PMID- 16684559 TI - Determinants of influenza vaccination in hard-to-reach urban populations. AB - OBJECTIVE: Influenza vaccination rates among disadvantaged minority and hard-to reach populations are lower than in other groups. We assessed the barriers to influenza vaccination in disadvantaged urban areas. METHODS: We conducted a cross sectional study, using venue-based sampling, collecting data on residents of eight neighborhoods throughout East Harlem and the Bronx, New York City. RESULTS: Of 760 total respondents, 461 (61.6%) had received influenza vaccination at some point in their life. In multivariable models, having access to routine medical care, receipt of health or social services, having tested positive for HIV, and current interest in receiving influenza vaccination were significantly associated with having received influenza vaccination in the previous year. Of participants surveyed, 79.6% were interested in receiving an influenza vaccination at the time of survey. Among participants who had never previously received influenza vaccination in the past, 73.4% were interested in being vaccinated; factors significantly associated with an interest in being vaccinated were minority race, lower annual income, history of being homeless, being uninsured/underinsured, and not having access to routine medical care. CONCLUSIONS: Participants who are unconnected to health or social services or government health insurance are less likely to have been vaccinated in the past although these persons are willing to receive vaccine if it were available. PMID- 16684560 TI - Physical activity among Dutch workers--differences between occupations. AB - OBJECTIVE: This study describes the total physical activity and the contribution of work to total physical activity among Dutch workers. The study was conducted in the Netherlands in the years 2000-2002. METHODS: 2,417 respondents of a cross sectional survey representing the Dutch adult working population were included. Physical activity was measured by the proportion meeting the current public health physical activity recommendations and by total physical activity (in minutes per week). Seven occupational groups and 28 sectors were distinguished. Unadjusted analyses were carried out using chi-square test and one-way analysis of variance. RESULTS: On average, 53.7% was sufficiently active according to the recommended levels. Work contributed for 30% to total physical activity. There were significant differences between the occupations and sectors in total physical activity levels as well as in the amount of work-related physical activity. Those in policy and higher executive functions were least active at work with work contributing for 19.5% to total physical activity, whereas those working in agricultural occupations were most active at work with work contributing for 55.1% to total physical activity. CONCLUSIONS: Work is still an important source of total physical activity among workers. Based on the differences observed, branch-specific strategies to enhance work-related physical activity are recommended. PMID- 16684561 TI - Redox proteomics in the mussel, Mytilus edulis. AB - Pollutants (e.g. PAHs, metals) cause oxidative stress (OS) by forming reactive oxygen species. Redox proteomics provides a means for identifying protein specific OS effects in Mytilus edulis. Groups of mussels were sampled from a clean site in Cork Harbour, Ireland and exposed to 1 mM H2O2 in holding tanks. Protein extracts of gill and digestive gland were separated by two dimensional electrophoresis and similar protein expression profiles were found. Effects of OS on disulphide bridge patterns were investigated in diagonal gels by separating proteins in non-reducing conditions followed by a second reducing dimension. Immunoprecipitation selected carbonylated and glutathionylated proteins. These methodologies can contribute to redox proteomic studies of pollutant responses in marine organisms. PMID- 16684562 TI - Lead contamination of small cetaceans in European waters--the use of stable isotopes for identifying the sources of lead exposure. AB - Lead concentrations and isotopic composition have been measured in bone and teeth of small cetaceans belonging to three species (Delphinus delphis, Phocoena phocoena and Stenella coeruleoalba), to evaluate the toxicological risk and to determine sources of lead in the European waters. Lead concentrations, far lower than threshold value inducing toxic effects in human, were higher in teeth than in bones, but highly correlated between the two tissues (r=0.92, p<0.001). Large variations of 206Pb/207Pb values in bone tissue showed that cetaceans must be submitted to various atmospheric influences. No geographical differences appeared which is consistent with studies on their distribution indicating seasonal movements between Brittany waters and the Bay of Biscay. The negative correlation between 206Pb/207Pb ratios and age of the individuals reflected the decrease in the production of alkyl lead in Europe, i.e., the increasing use of unleaded gasoline. PMID- 16684563 TI - Approaches to extrapolating animal toxicity data on organic solvents to public health. AB - Synthesizing information about the acute neurotoxicity of organic solvents into predictive relationships between exposure and effect in humans is difficult because (1) data are usually derived from experimental animals whose sensitivity to the chemical relative to humans is unknown; (2) the specific endpoints measured in laboratory animals seldom translate into effects of concern in humans; and (3) the mode of action of the chemical is rarely understood. We sought to develop approaches to estimate the hazard and cost of exposure to organic solvents, focusing on the acute behavioral effects of toluene in rats and humans. Available published data include studies of shock avoidance behavior in rats and choice reaction time in humans. A meta-analysis of these data suggested that a 10% change in rat avoidance behavior occurs at a blood concentration of toluene 25 times higher than the concentration at which a 10% change in human choice reaction time occurs. In contrast, our in vitro studies of nicotinic acetylcholine receptors indicated that human and rat receptors do not differ in sensitivity to toluene. Analysis of other dose-response relationships for visual and cognitive functions in rats suggests that the apparent difference between rats and humans may be driven by the specific endpoints measured in the two species rather than by inherent differences in sensitivity to toluene. We also explored the hypothesis that dose-equivalence relationships may be used to compare the societal costs of two chemicals. For example, ethanol-induced changes in choice reaction time, for which societal costs are estimatable, may be used as a benchmark effect for estimating the monetary benefits of controlling exposure to organic solvents. This dose-equivalence method is applicable for solvents because this set of data fulfills three important assumptions about equivalence relationships based on a single effect: (1) a common dose metric (concentration of the chemical in the brain); (2) a common effect to provide a linking variable (choice reaction time); and (3) a common mode of action (interference with neuronal ion channel function). PMID- 16684564 TI - Hippocampal cell alterations induced by the inhalation of vanadium pentoxide (V(2)O(5)) promote memory deterioration. AB - Spatial memory may be severely impaired as a consequence of ageing and neurodegenerative diseases, conditions that include neuronal damage. Vanadium (V) is a metalloid widely distributed in the environment and exerts severe toxic effects on a wide variety of biological systems. Reports about V inhalation toxicity on the CNS are limited, thus the purpose of this study is to determine the effects of Vanadium pentoxide (V(2)O(5)) inhalation (0.02M) on the memory and its correlation with the cytology of the hippocampus CA1. Forty eight CD-1 male mice were trained in spatial memory tasks and inhaled 1h twice a week; after each inhalation animals were evaluated and sacrificed from 1 to 4 weeks, perfused and processed for Golgi method and for ultrastructure evaluation. The cytological analysis consisted in counting the number of dendritic spines of 20 pyramidal neurons of hippocampus CA1, as well as ultrastructural characteristics. Results show that V inhalation produces a time dependent loss of dendritic spines, necrotic-like cell death, and notorious alterations of the hippocampus CA1 neuropile, which correlate with spatial memory impairment. Our data suggest that V induces important cellular and functional alterations, fact that deserves special attention since the concentration's trend of this element in the atmosphere is increasing. PMID- 16684566 TI - Structure-toxicity relationships for different types of dinuclear platinum complexes. AB - Nine structurally distinct dinuclear platinum complexes have been evaluated in a novel model system for the investigation of renal epithelial toxicity of platinum drugs. The results showed that these compounds are toxic when applied at the basolateral side of renal epithelia, whereas their toxic effects on the apical side are negligible. Such a difference in toxicity of the complexes has been found to result from their poor uptake through the apical membrane, as compared to the basolateral membrane. Toxicity of the compounds on the basolateral side varies depending on their structure. Structure-toxicity relationships for the group of complexes with rigid ligands and for the group of complexes with flexible ligands are discussed. Among the dinuclear complexes with rigid ligands, sterically hindered complexes are less toxic, due to their poor uptake and low reactivity towards glutathione. Within the group of complexes with flexible ligands, cis-configured isomers are more toxic than their trans-counterparts. PMID- 16684565 TI - Direct binding of Cu(II)-complexes of oxicam NSAIDs with DNA backbone. AB - Drugs belonging to the non-steroidal anti-inflammatory drug group (NSAID) are not only used as anti-inflammatory and analgesic agents, but also exhibit chemopreventive and chemosuppressive effects on various cancer cell lines. They exert their anticancer effects by inhibiting both at the protein level and/or at the transcription level. Cu(II) complexes of these NSAIDs show better anti-cancer effects than the bare drugs. Considering the above aspects, it is of interest to see if Cu(II) complexes of these drugs can exert their effects directly at the DNA level. In this study, we have used UV-Vis spectroscopy to characterize the complexation between Cu(II) and two NSAIDs belonging to the oxicam group viz. piroxicam and meloxicam, both of which exhibit anticancer properties. For the first time, this study shows that, Cu(II)-NSAID complexes can directly bind with the DNA backbone, and the binding constants and the stoichiometry or the binding site sizes have been determined. Thermodynamic parameters from van't Hoff plots showed that the interaction of these Cu(II)-NSAID complexes with ctDNA is an entropically driven phenomenon. Circular dichroism (CD) spectroscopy showed that the binding of these Cu(II)-NSAIDs with ctDNA result in DNA backbone distortions which is similar for both Cu(II)-piroxicam and Cu(II)-meloxicam complexes. Competitive binding with a standard intercalator like ethidium bromide (EtBr) followed by CD as well as fluorescence measurements indicate that the Cu(II) NSAID complexes could intercalate in the DNA. PMID- 16684567 TI - Synthesis, crystal structure and DNA binding studies of a binuclear copper(II) complex with phenanthroline. AB - A new binuclear copper(II) complex, [Cu2Phen2Cl4] (Phen=1,10-phenanthroline), has been synthesized and characterized. Single crystal X-ray diffraction results suggest that this complex structure belongs to monoclinic crystal system, Cc (no. 9) with the cell dimensions: a=9.849(2)A, b=17.833(4)A, c=13.374(3)A, beta=106.61(3) degrees , V=2251.0(8)A(3), Dc=1.8569 Mgm(-3), F(000)=1256.0, Z=4. One Cu(II) central atom situated in a distorted square planar geometry is four coordinated. The other situated in a distorted square pyramidal geometry is five coordinated. Only one bridging Cl atom exists in the complex. Spectroscopic studies, including electronic absorption and fluorescence spectra, conductivity measurements and parallel factor analysis (PARAFAC) of fluorescence excitation emission three-way data array, were carried out on the DNA binding behavior of the complex. All the results suggested that the breakage of DNA secondary structure took place at low molar ratio of complex to DNA (0.3 at most) and intercalation into the base pair of DNA took place at high molar ratio. Additionally, the equilibrium concentration of EB-DNA and EB (EB: ethidium bromide) could be directly obtained by PARAFAC algorithm, proved to be a convincing method for studying the interaction of complexes with DNA. PMID- 16684568 TI - Characterization and crystal structure of cadmium(II) halide complexes with amino acids and their derivatives: VII. Crystal structures of aquadibromo(3 aminopropanoic acid)cadmium(II), dichloro(4-aminobutanoic acid)cadmium(II), diaquabis(aminohexanoic acid)cadmium(II) tetrachlorocadmium(II), and dibromo(azetidine-3-carboxylic acid)cadmium(II). AB - Seven cadmium complexes: [CdX2(Hapro)(H2O)n] (X: Cl(1), Br(2)), [CdX2(Hgaba)] (X: Cl(3), Br(4)), [Cd(Hahex)2(H2O)2][CdCl4] (5), and [CdX2(Haze-3)](H2O)n (X: Cl(6), Br(7)) have been prepared and investigated by means of IR and FT Raman spectra. The crystal and molecular structures of 2, 3, 5 and 7 were determined by a single crystal X-ray diffraction method. In complex 2, the cadmium atom is in a distorted octahedral geometry, ligated by two carboxyl oxygen atoms of Hapro, a water molecule, and three bromine atoms; one is terminal and each of the other two is bridging two cadmium atoms to make a polymer. The structure of 3 consists of one-dimensional polymers bridged by two chlorine atoms and a carboxyl group. The carboxyl oxygen atoms of Hgaba coordinate forkedly to two cadmium atoms. The cadmium atom of [Cd(Hahex)2(H2O)2]2+ in complex 5 is in a distorted octahedral geometry, ligated by four carboxyl oxygen atoms of two molecules of Hahex and by two water molecules. [Cd(Hahex)2(H2O)2]2+ exists between two layers which are formed of infinite [CdCl4]2- chains. The carboxyl oxygen atoms of Hahex coordinate to the same cadmium atom. In complex 7, the cadmium atom is ligated by two carboxyl oxygen atoms and four bridging bromine atoms to make a polymer. PMID- 16684569 TI - The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein. AB - The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein. PMID- 16684570 TI - 3D-QSAR study for DNA cleavage proteins with a potential anti-tumor ATCUN-like motif. AB - Genomics projects have elucidated several genes that encode protein sequences. Subsequently, the advent of the proteomics age has enabled the synthesis and 3D structure determination for these protein sequences. Some of these proteins incorporate metal atoms but it is often not known whether they are metal-binding proteins and the nature of the biological activity is not understood. Consequently, the development of methods to predict metal-mediated biological activity of proteins from the 3D structure of metal-unbound proteins is a goal of major importance. More specifically, the amino terminal Cu(II)- and Ni(II) binding (ATCUN) motif is a small metal-binding site found in the N-terminus of many naturally occurring proteins. The ATCUN motif participates in DNA cleavage and has anti-tumor activity. In this study, we calculated average 3D electrostatic potentials (xi(k)) for 265 different proteins including 133 potential ATCUN anti-tumor proteins. We also calculated xi(k) values for the total protein or for the following specific protein regions: the core, inner, middle, and outer orbits. A linear discriminant analysis model was subsequently developed to assign proteins into two groups called ATCUN DNA-cleavage proteins and non-active proteins. The best model found was: ATCUN=1.15.xi(1)(inner)+2.18.xi(5)(middle)+27.57.xi(0)(outer) 27.57.xi(0)(total)+0.09. The model correctly classified 182 out of 197 (91.4%) and 61 out of 66 (92.4%) proteins in training and external predicting series', respectively. Finally, desirability analysis was used to predict the values for the electrostatic potential in one single region and the combined values in two regions that are desirable for ATCUN-like proteins. To the best of our knowledge, the present work is the first study in which desirability analysis has been used in protein quantitative-structure-activity-relationship (QSAR). PMID- 16684571 TI - Cardiac arrest associated with febrile illness due to U.K. acquired Cyclospora cayetanensis. AB - This report describes a 43 yr old man diagnosed with U.K. acquired cyclospora cayetanensis infection resulting in fever and diarrhoea. In course of the febrile illness, he suffered an out of hospital cardiac arrest. Extensive cardiac investigation including a transthoracic echocardiogram, coronary angiogram, and cardiac electrophysiological studies failed to identify the cause. The possible links between cyclospora infection and cardiac arrest are explored. Fever has been reported as a precipitant for idiopathic ventricular fibrillation in patients with the Brugada syndrome but also rarely in individuals with normal hearts. Clinicians should be aware of a possible link between any febrile illness and potentially fatal ventricular dysrhythmia. PMID- 16684572 TI - Eosinophil and IgA responses in sheep infected with Teladorsagia circumcincta. AB - Infections of sheep with the gastrointestinal nematode Teladorsagia circumcincta are characterised by increased concentrations of IgA, eosinophilia and mastocytosis but the interactions between these immune responses are unclear. We investigated the kinetics of the parasite-specific IgA and eosinophil responses in controlled infections in lambs to determine if there were any associations with subsequent worm growth and survival. IgA and eosinophil responses had very similar kinetics and variation in both responses accounted for more of the variation in adult worm length than either trait alone suggesting that IgA and eosinophils interact in regulating the growth of T. circumcincta. Curiously, those animals with higher peak eosinophil responses had longer worms at slaughter emphasising the intriguing complexity of the immune responses to these parasitic infections. PMID- 16684573 TI - Cervical cancer: treatment challenges in the developing world. PMID- 16684574 TI - REMOVED: Boron neutron capture therapy in the treatment of glioblastoma: As effective, more effective or less effective than photon irradiation? AB - This article has been removed consistent with Elsevier Policy on Article Withdrawal. Please see .The Publisher apologizes for any inconvenience this may cause. PMID- 16684575 TI - Posture and motion variability in non-repetitive manual materials handling tasks. AB - In developing a motion prediction model it is important to initially consider the sources of variability that a model should reproduce. This initial step is followed by model evaluation, where the variability predicted by the model can be a useful test parameter. An existing lifting-motion dataset collected under controlled laboratory conditions was employed here to evaluate quantitatively some important sources of variability for lift motion modeling. The main source of variability was the segment being analyzed, which accounted for more than 20% of the overall variability. There was substantial left-right symmetry in individual segment variability estimates, which were largest for the upper arm segment and tended to be larger for the upper limbs than the lower limbs. Task related factors accounted for variability mainly as a function of the segment being considered. Within-participant variability contributions to the dataset were relatively small, whereas the contribution of between-participants variability was dependent on the segment (as large as 50%) and could indicate different lifting strategies across participants. Variability was found to remain relatively constant across the different stages of the lifting movements. Implications of these results for the development and evaluation of motion prediction models are presented. Specifically, while task characteristics may be important modifiers of the mean segment trajectory during a lifting movement, their influence on variability differs based on the segment that is being considered. The relevance of the findings is discussed in terms of their utility in the ergonomic design of tasks and work spaces. PMID- 16684576 TI - Medullary monoamines and NMDA-receptor regulation of cardiovascular responses during peripheral nociceptive stimuli. AB - We have previously reported that AMPA-receptor blockade within the rostral ventrolateral medulla (RVLM) attenuates cardiovascular responses and extracellular concentrations of glutamate during mechanical, but not during thermal stimulation [Gray, T., Lewis III, E., Maher, T.J., Ally, A., 2001. AMPA receptor blockade within the RVLM modulates cardiovascular responses via glutamate during peripheral stimuli. Pharmacol. Res. 43, 47-54]. In this study, we examined the role of NMDA-receptor blockade within the RVLM on cardiovascular responses and release of biogenic monoamines (serotonin [5HT], dopamine [DA], and norepinephrine [NE]) during both mechanical and thermal nociception using anesthetized Sprague-Dawley rats. Both mechanical and thermal stimulation have been shown to activate peripheral Adelta and C-fiber polymodal nociceptors. Noxious mechanical stimuli were induced by applying a pinch to alternate hindpaw for 5s while the noxious thermal stimuli involved immersion of the metatarsus of alternate hindpaw in a water bath at a temperature of 52 degrees C for 5 s. Mechanical stimulation increased mean arterial pressure (MAP), heart rate (HR), extracellular fluid 5HT, and DA concentrations (n=10). However, extracellular levels of NE were decreased within the RVLM. Furthermore, NMDA-receptor blockade with a competitive antagonist, AP-7 (200 nM), within the RVLM attenuated the cardiovascular responses and changes in 5HT and DA, but had no effect on NE levels. The thermal stimulation elicited similar increases in MAP and HR, however, extracellular levels of 5HT or DA did not change. Concentrations of NE were decreased during a thermal stimulation similar to the levels observed following mechanical stimuli. In contrast to mechanical stimuli, bilateral administration of AP-7 (200-1 mM) into the RVLM had no effect on cardiovascular responses, 5HT, DA or NE concentrations during a thermal stimulation. These results show that NMDA receptors within the RVLM most likely play a role in modulating cardiovascular responses by altering 5HT and DA concentrations within the RVLM during mechanical but not thermal nociception. Overall, the present study delineates the NMDA-receptor mediated central integrative mechanisms within the RVLM that coordinate processing of sensory impulses arising from peripheral noxious stimulation. PMID- 16684577 TI - Inhibition of aromatase improves nutritional status following portacaval anastomosis in male rats. AB - BACKGROUND/AIMS: The portacaval anastamosis (PCA) rat is a model of the nutritional and endocrine consequences of liver cirrhosis. We hypothesized that failure to gain body weight in the PCA rat was the consequence of low testosterone levels and will be reversed by 4-hydroxyandrostenedione, a specific aromatase inhibitor. METHODS: Male Sprague-Dawley rats subjected to an end-to side PCA or sham surgery were administered either 4-hydroxyandrostenedione or vehicle. Food intake, food efficiency, and body weight were measured, animals sacrificed 3 weeks after surgery, visceral organs harvested and plasma sex steroids measured. Hepatic RNA was extracted and dot blots done to quantify the expression of sex hormone dependent enzymes 16alpha hydroxylase and 15beta hydroxylase. RESULTS: Growth, food intake, food efficiency and plasma testosterone were lower and plasma estradiol higher in PCA than sham rats. Hepatic expression of testosterone driven 16alpha hydroxylase was lower and estradiol driven 15beta hydroxylase higher in PCA rats. These alterations were reversed by 4-hydroxyandrostenedione. CONCLUSIONS: These observations demonstrate that use of aromatase inhibitor reverses the nutritional and endocrine abnormalities in the PCA rat and suggest that this approach may be useful in cirrhotic patients. PMID- 16684578 TI - Co-infection of human parvovirus B19 in Vietnamese patients with hepatitis B virus infection. AB - BACKGROUND/AIMS: Human parvovirus B19 (B19) has been identified in the serum of hepatitis B virus (HBV) infected patients. However, the effect of B19-infection on the course of HBV-associated liver disease has not previously been investigated. We examined the prevalence of B19-DNA in HBV-infected Vietnamese patients and analysed the association between co-infection and the clinical outcome of HBV-infection. METHODS: Serum samples from 399 HBV-infected patients and 64 healthy individuals were analysed for the presence of B19-DNA by PCR and DNA-sequencing. RESULTS: B19-DNA was detected in 99/463 (21.4%) individuals. The proportion of HBV-infected patients who were also co-infected with B19 was higher than the healthy controls (P<0.001). B19-DNA was detected more frequently in patients with HBV-associated hepatocellular carcinoma compared to patients with acute and chronic HBV, HBV-associated liver cirrhosis and healthy subjects (P<0.006). A positive correlation was also found between B19-DNA loads and both serum HBV-DNA loads and alanine aminotransferase (rho>0.250 and P<0.05). CONCLUSIONS: Our findings demonstrate that B19-infection is frequent in HBV infected Vietnamese patients. Also, a significant correlation exists between HBV/B19 co-infection and a greater likelihood of progression to more severe hepatitis B-associated liver disease. Further studies are required to determine the role of B19-infection on HBV-associated pathogenesis. PMID- 16684579 TI - Cancer incidence in people with hepatitis B or C infection: a large community based linkage study. AB - BACKGROUND/AIMS: Risks of hepatocellular carcinoma (HCC) following hepatitis B and/or hepatitis C virus (HBV/HCV) infection are well known, those for other cancers are less well understood. The aim was to quantify the risk of cancers among persons diagnosed with HBV/HCV infections. METHODS: The data from a cohort of 39109 HBV, 75834 HCV, and 2604 HBV/HCV co-infected persons notified to the State health department, 1990-2002, were probabilistically linked to the Cancer Registry and standardised incidence ratios (SIRs) for cancer were calculated. RESULTS: The match rate for any cancer was 2.7%, 2.3% and 3.3% for HBV, HCV and HBV/HCV co-infected notifications. SIRs for HCC were 30.6 (95% CI 25.7-36.5), 22.7 (95% CI 19.1-26.5) and 30.3 (95% CI 13.6-67.5), respectively. Increased risk was detected for Burkitt's lymphoma and HBV (SIR 12.9, 95% CI 5.4-30.9) and immunoproliferative malignancies following HCV (SIR 5.6, 95% CI 1.8-17.5). CONCLUSIONS: The risk of HCC in the infected cohort was 20-30 times greater than in the uninfected population with SIRs two to three times greater than those for the other HBV/HCV infection associated cancers. The modest though significant risk of immunoproliferative malignancies associated with HCV infection is consistent with recent findings. PMID- 16684580 TI - Prioritisation by physicians in the Netherlands--the growth hormone example in drug reimbursement decisions. AB - Drug treatment and reimbursement is an area of ever growing complexity in health priority setting. This paper assesses the National Registry of Growth Hormone Treatment (LRG) responsible for making prioritisation decisions in the Dutch drug reimbursement system in the treatment of growth hormone, using the framework for fairness. We used qualitative research consisting of semi-structured interviews and focus group sessions combined with quantitative methods to audit the decisions of the forum. The rationing decisions of the forum demonstrate accountability for reasonableness by the conditions for transparency, relevance, and appeal. Most rationales for the decisions are public and transparent. The patients and paediatricians see decisions made by the LRG as clinical and therefore relevant decisions. They also refer to extensive appeal procedures. The case also raises important issues regarding the legitimacy of expert-based priority setting as the cyclic nature of guideline development conflicts with the need for maintaining strict rationing criteria. In 13% of the patients, the sick funds did cover treatment as the forum advised them to do, but according to guideline criteria it may be unlikely that these patients have growth hormone deficiency. According to the LRG, however, only 2% of the decisions are inconsistent with the guidelines, as some criteria on what to do in case of more uncertainty, shifted. For the forum, it seems rather unthinkable to go against the professional norms, in spite of formal national regulations. For the Health Care Insurance Board (CVZ), it was not considered possible to go against national regulations, especially as professional norms have shifted without informing policy makers and patient representatives. PMID- 16684581 TI - A new software tool for 3D motion analyses of the musculo-skeletal system. AB - BACKGROUND: Many clinical and biomechanical research studies, particularly in orthopaedics, nowadays involve forms of movement analysis. Gait analysis, video fluoroscopy of joint replacement, pre-operative planning, surgical navigation, and standard radiostereometry would require tools for easy access to three dimensional graphical representations of rigid segment motion. Relevant data from this variety of sources need to be organised in structured forms. Registration, integration, and synchronisation of segment position data are additional necessities. With this aim, the present work exploits the features of a software tool recently developed within a EU-funded project ('Multimod') in a series of different research studies. METHODS: Standard and advanced gait analysis on a normal subject, in vivo fluoroscopy-based three-dimensional motion of a replaced knee joint, patellar and ligament tracking on a knee specimen by a surgical navigation system, stem-to-femur migration pattern on a patient operated on total hip replacement, were analysed with standard techniques and all represented by this innovative software tool. Segment pose data were eventually obtained from these different techniques, and were successfully imported and organised in a hierarchical tree within the tool. FINDINGS: Skeletal bony segments, prosthesis component models and ligament links were registered successfully to corresponding marker position data for effective three-dimensional animations. These were shown in various combinations, in different views, from different perspectives, according to possible specific research interests. INTERPRETATION: Bioengineering and medical professionals would be much facilitated in the interpretation of the motion analysis measurements necessary in their research fields, and would benefit therefore from this software tool. PMID- 16684582 TI - Estimates of ozone AOT40 from passive sampling in forest sites in South-Western Europe. AB - Weekly-fortnightly ozone (O3) concentrations measured by passive sampling at 81 forest monitoring plots in France, Italy, Spain and Switzerland over the period 2000-2002 were used to estimate the cumulative exposure index AOT40. The estimation method is based on a deterministic model which describes the O3 daily profile as a function of relative altitude (the difference between the altitude of the site and the lowest altitude within a 5 km radius) and the time of the day. Estimated AOT40 values (AOT40(e)) were evaluated against co-located automatic measurement stations and with 14 independent automatic stations located throughout Italy whose weekly mean O3 values were used to simulate passive samplers. AOT40 can be predicted by modelling passive sampling data (R2: 0.90; P<0.0001, SE of estimates: 3271 ppb h), although considerable deviations can occur for individual sites. Estimated AOT40 shows a distinct, significant latitudinal and altitudinal gradient. Taking the 3-year average as a whole, exceedance of critical level of 5000 ppb h occurs at 77-100% of the monitored sites, respectively. PMID- 16684583 TI - A review of perchloroethylene and rat mononuclear cell leukemia. AB - Mononuclear cell leukemia (MNCL) is an extremely common spontaneous disease of ageing F344 rats accompanied by splenomegaly, anemia, thrombocytopenia, and leukemic infiltration (initially of the spleen, liver, and lung). Rare in other rat strains, incidence in F344 rats is variable, has been increasing, and can exceed 70% in controls. MNCL cells possess natural killer (NK) cell characteristics and apparently, the neoplastic cells derive from large granular lymphocytes (LGL), hence the alternative name of LGL leukemia. LGL leukemia is uncommon in man and occurs in two forms: T-LGL leukemia which has a chronic course, and the much rarer NK-LGL leukemia. In addition to cell type, the latter resembles F344 LGL leukemia being acute in course and involving more pronounced splenomegaly and thrombocytopenia. Chemically related increases in MNCL in F344 rats have not been associated with induction of human LGL leukemia. Carcinogenicity studies of perchloroethylene (PERC) in several rat strains have shown moderate, not clearly dose-related, increases in MNCL only in F344 rats (two studies). There was no consistent decrease in latency and the incidence in the PERC treated groups is within the overall control range. As a response in a rat strain highly predisposed to developing MNCL, these results are not considered predictive for human cancer risk. PMID- 16684584 TI - Rhythm deficits in 'tone deafness'. AB - It is commonly observed that 'tone deaf' individuals are unable to hear the beat of a tune, yet deficits on simple timing tests have not been found. In this study, we investigated rhythm processing in nine individuals with congenital amusia ('tone deafness') and nine controls. Participants were presented with pairs of 5-note sequences, and were required to detect the presence of a lengthened interval. In different conditions the sound sequences were presented isochronously or in an integer-ratio rhythm, and these were either monotonic or varied randomly in pitch. It was found that the 'tone deaf' participants exhibited inferior rhythm analysis for the sequences that varied in pitch compared to those that did not, whereas the controls obtained equivalent thresholds for these two conditions. These results suggest that the rhythm deficits in congenital amusia result from the pitch-variations in music. PMID- 16684585 TI - Learned irrelevance and response perseveration in a total change dimensional shift task. AB - Thirty-six healthy participants received a discrimination learning task requiring the identification of a relevant stimulus dimension. After successful learning, the relevant dimension was shifted unannounced. All exemplars of the two dimensions presented after the shift were novel, implying a 'total change' design. In three experimental conditions, participants could either make only errors reflecting perseveration of responding to the former relevant dimension, continued ignoring of the former irrelevant dimension, or both. After the shift, the participants in the perseveration condition made fewer errors than did those in the other two conditions, which did not differ. These results imply a predominance of the learned irrelevance mechanism even when any direct transfer of learning about exemplars in the pre-shift phase is precluded. PMID- 16684587 TI - A spreadsheet model for developing field indicators and grazing management tools to meet environmental and production targets for dairy farms. AB - Changes in livestock farm structure, such as increasing land area per animal, as well as developments in national and European agricultural policies may lead to changes in grazing and fertilizer management practices for environmental or economic reasons. To facilitate choices and the learning of new practices at the farm level, such as the amount of land to allocate for grazing or of fertilizer to apply, we propose to combine a simplified grass growth and N model with two sward indicators. One assesses the sward nitrogen status to evaluate animal excreted N; the other assesses the standing herbage mass to characterize the grazing management. Following a description of the model (first part), we use it as a research tool for highlighting grazing management (second part). First we analyze how stocking rate, N excreted, grazing and N use efficiency varied according to management (i.e., the time between two grazing events), sward (N status, leaf lifespan) and weather characteristics. Next we use it for determining field indicator thresholds at key periods that allow agricultural and environmental aims to be met; these thresholds being intended to give guidance to meet farmers' objectives. In the last part, we illustrate how to combine model and field indicators for planning and monitoring a management strategy suitable for the management of risks. PMID- 16684586 TI - Serum pro-hepcidin and iron markers during uncomplicated pregnancy. PMID- 16684588 TI - Induction of iron homeostasis genes during estrogen-induced uterine growth and differentiation. AB - We have previously used genome-wide transcript profiling to investigate the relationships between changes in gene expression and physiological alterations during the response of the immature mouse uterus to estrogens. Here we describe the identification of a functionally inter-related group of estrogen-responsive genes associated with iron homeostasis, including the iron-binding protein lactotransferrin, the ferroxidase ceruloplasmin, the iron delivery protein lipocalin 2 and the iron-exporter ferroportin. Quantitative real-time PCR revealed that the expression of these genes increases with time during the uterotrophic response, reaching maximal levels in the post-proliferative phase (between 48 and 72 h). In contrast, the heme biosynthesis genes aminolevulinic acid synthase 1 and 2 were maximally induced by estrogen at 2 and 4 h, respectively, prior to increased cell proliferation. Together, these data reveal that estrogen induces the temporally coordinated expression of iron homeostasis genes in the mouse uterus, and suggest an important role for iron metabolism during sex steroid hormone-induced uterine cell growth and differentiation. PMID- 16684589 TI - Ruptured hemangioma of the umbilical cord and intrauterine fetal death, with review data. AB - A case of intrauterine fetal death with rupture of a hemangioma in the umbilical cord is presented. Hemangiomas are uncommon tumors of the umbilical cord, and their clinical significance is not entirely clear, but associations with polyhydramnios, fetal disseminated intravascular coagulation, and fetal hydrops have been described. In a high proportion of the umbilical cord hemangiomas reported in the literature (32 cases), the fetus had died in utero as in the present case and only ten cases were vital and completely normal infants. Associated malformations and complications of the umbilical cord hemangioma are reviewed. PMID- 16684590 TI - Focal myointimal hyperplasia of mesenteric veins is associated with previous trauma in surgical specimens. AB - Idiopathic myointimal hyperplasia of mesenteric veins (IMHMV) is a rare and poorly understood disease that occurs in the rectosigmoid colon of predominantly young, previously healthy, male patients. This disease is often confused with chronic idiopathic inflammatory bowel disease clinically, and pathologists may miss the diagnosis unless elastin stains are performed because diseased veins may readily be mistaken for arteries. The etiology of IMHMV is unclear, but a traumatic pathomechanism resulting in arterialization of the veins has been proposed. Review of bowel resection specimens (n=68) for non-neoplastic disease within a 1-year period in patients younger than 50 years of age revealed 10 cases with focal mesenteric vein myointimal hyperplasia. Significantly more cases with focal myointimal hyperplasia of mesenteric veins (MHMV) were associated with pre resection trauma to the involved bowel segment (5/11 vs. 5/57; p=0.0016). A significant association of MHMV with pre-resection trauma supports the hypothesis that idiopathic myointimal hyperplasia of the mesenteric veins may be the result of trauma through torsion/stretching of the sigmoid colon and, subsequently, increased mesenteric venous pressure through arterialization. PMID- 16684591 TI - Canine leptospirosis--do we have a problem? AB - Acute canine leptospirosis is well known to vet surgeons. To protect dogs against this lethal disease, vaccination is widely used. However, chronic forms of leptospirosis have been noticed even in vaccinated animals, generally induced by bacteria from serogroups other than Icterohaemorrhagiae and Canicola such as Sejroe, Australis or Grippotyphosa. In a survey on 98 ill cats, 48% were positive in microagglutination test (MAT) to Leptospira spp., showing that this infection is also frequent in the feline species. PMID- 16684592 TI - Structure and locomotion of adult in vitro regenerated spiral ganglion growth cones-- a study using video microscopy and SEM. AB - Neuronal development and neurite regeneration depends on the locomotion and navigation of nerve growth cones (GCs). There are few detailed descriptions of the GC function and structure in the adult auditory system. In this study, GCs of adult dissociated and cultured spiral ganglion (SG) neurons were analyzed in vitro utilizing combined high resolution scanning electron microscopy (SEM) and time lapse video microscopy (TLVM). Axon kinesis was assessed on planar substratum with growth factors BDNF, NT-3 and GDNF. At the nano-scale level, lamellipodial abdomen of the expanding GC was found to be decorated with short surface specializations, which at TLVM were considered to be related to their crawling capacity. Filopodia were devoid of these surface structures, supporting its generally described sensory role. Microspikes appearing on lamellipodia and axons, showed circular adhesions, which at TLVM were found to provide anchorage of the navigating and turning axon. Neurons and GCs expressed the DCC-receptor for the guidance molecule netrin-1. Asymmetric ligand-based stimulation initiated turning responses suggest that this attractant cue influences steering of GC in adult regenerating auditory neurites. Hopefully, these findings may be used for ensuing tentative navigation of spiral ganglion neurons to induce regenerative processes in the human ear. PMID- 16684593 TI - Hypoglycemic and hypolipidemic action of Du-zhong (Eucommia ulmoides Oliver) leaves water extract in C57BL/KsJ-db/db mice. AB - The anti-diabetic efficacy of Du-zhong (Eucommia ulmoides Oliver) leaves water extract (WDZ) was investigated in type 2 diabetic animals. The WDZ was given to C57BL/KsJ-db/db mice as a dietary supplement based on 1% dried whole Du-zhong leaves (0.187 g WDZ/100 g standard diet) for 6 weeks. The WDZ supplementation significantly lowered the blood glucose level and enhanced the glucose disposal in an intraperitoneal glucose tolerance test. The plasma insulin and C-peptide levels were significantly higher in the WDZ group than in the control group, while the glucagon level was lower. The hepatic glucokinase activity was significantly higher in the WDZ group, whereas, the glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities were significantly lower. The WDZ supplementation also significantly lowered the hepatic fatty acid synthase, HMG CoA reductase and ACAT activities compared to the control group, while it elevated the lipoprotein lipase activity in the skeletal muscle. The WDZ also altered the plasma and hepatic lipid levels by lowering the cholesterol and triglyceride concentrations, while elevating the plasma HDL-cholesterol level. Therefore, these results suggest that WDZ may partly ameliorate hyperglycemia and hyperlipidemia with type 2 diabetes through increasing glycolysis, suppressing gluconeogenesis and the biosynthesis of fatty acid and cholesterol in the liver. PMID- 16684594 TI - Genetic variation of 13 STR loci in the four endogamous tribal populations of Eastern India. AB - We have analysed 13 autosomal STR loci in four endogamous tribal populations from two eastern states (Orissa and Nagaland) of India. The Gadaba, Kuvi Khond and Lotha Naga populations have not been analysed for microsatellite genetic variation previously. The allele frequencies for all loci are within the range observed in the geographical region and racial background, though some alleles showed greater variation. Departures from the Hardy-Weinberg equilibrium were tested by three methods and two loci (THO1 and TPOX) showed significant departures for all measures in Gadaba and Lotha Naga populations. The exclusion probability and discrimination probability were high for all analysed loci in all populations. There is no evidence for association of alleles among the STR loci studied. This allele frequency information will be useful for forensic, paternity and population genetic studies. PMID- 16684595 TI - The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes. AB - The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan. PMID- 16684597 TI - Screening for gynaecological cancers. AB - BACKGROUND: The role of screening in gynaecological cancers is under evaluation. With mass screening proven effective in significantly reducing cervical cancer mortality, there is an interest in developing other screening methods to detect gynaecological malignancies early. This review covers advances in cervical cancer screening, strategies being investigated in ovarian cancer screening and the lack of justification in screening for endometrial, vulval and vaginal cancers. METHODS: A Medline based literature search was performed for articles relating to screening for different gynaecological malignancies. Additional original papers cited in those identified by the initial search were also reviewed. RESULTS: Advances in cervical cancer screening include liquid-based cytology and HPV testing. Results of ongoing trials are awaited before these can be fully implemented. The results of the two large, multicentre, randomised controlled trials being conducted in the United Kingdom and United States (UKCTOCS and PLCO study, respectively) to assess impact of screening on ovarian cancer mortality will shed some light on the need to implement screening for ovarian cancer in the general population. Novel markers, serum proteomic profiles and Doppler are some of the other technologies being explored. Currently, screening for endometrial cancer is not advocated as most women present with symptoms in early disease with good survival outcomes. Vulval and vaginal cancers are too rare to justify mass screening. CONCLUSION: Methods to screen for various gynaecological malignancies need further evaluation before implementation in the general population. Results of large multicentred trials are awaited. Presently, screening for endometrial, vaginal and vulval cancers is not justified. PMID- 16684596 TI - Host-specific bacterial lineages in the taxon 2 and 3 complex of Pasteurellaceae. AB - The aim of the investigation was to determine the genetic relationship of a phenotypically diverse strain collection of epidemiologically unrelated strains from the taxon 2 and 3 complex of Bisgaard isolated from different hosts. A total of 325 isolates belonging to the taxon 2 and 3 complex of Bisgaard was characterized phenotypically in 82 characters. The genetic relationship among a subset of 60 isolates was investigated by amplified fragment length polymorphism (AFLP). The isolates were selected aiming at including the broadest diversity with regards to phenotype and host spectrum. The results suggested a statistically clear association between AFLP clusters and the host species families Columbidae (pigeon, dove), Anatidae (duck, goose) and Psittacidae (parrot, parakeet, budgerigar), respectively. This association was further supported by results from previous whole cell protein profiling and DNA:DNA hybridization studies. In conclusion, it appears that distinct genetic lineages within the taxon 2 and 3 complex of Bisgaard have evolved specificity for host bird species of different families. The observed host specificity of taxon 2 and 3 organisms may be used in future diagnostics and studies elucidating aspects of pathogenicity and epidemiology associated with the different lineages and their respective hosts. PMID- 16684598 TI - Novel spastin (SPG4) mutations in Italian patients with hereditary spastic paraplegia. AB - Spastic paraplegia type 4 is caused by mutations in the gene that encodes spastin (SPG4), a member of the AAA protein family. A cohort of 34 unrelated Italian patients with pure spastic paraplegia, of which 18 displayed autosomal dominant inheritance and 16 were apparently sporadic, were screened for mutations in the SPG4 gene by denaturing high performance liquid chromatography. We identified a previously reported mutation in a sporadic patient with pure hereditary spastic paraplegia. We also identified eight unrelated patients with pure autosomal dominant hereditary spastic paraplegia carrying five novel mutations in the SPG4 gene (one missense mutation, c.1304 C>T; one nonsense mutation, c.807C>A; two frameshift mutations, c.1281dupT, c.1514_1515insATA; and one splicing mutation, c.1322-2A>C). The frequency for SPG4 mutations detected in autosomal dominant hereditary spastic paraplegia was 44.4%. This study contributes to expand the spectrum of SPG4 mutations in Italian population. PMID- 16684599 TI - Is selection required for the accumulation of somatic mitochondrial DNA mutations in post-mitotic cells? AB - Mitochondrial DNA (mtDNA) mutations accumulate in the skeletal muscle of patients with mtDNA disease, and also as part of healthy ageing. Simulations of human muscle fibres suggest that, over many decades, the continuous destruction and copying of mtDNA (relaxed replication) can lead to dramatic changes in the percentage level of mutant mtDNA in non-dividing cells through random genetic drift. This process should apply to both pathogenic and neutral mutations. To test this hypothesis we sequenced the entire mitochondrial genome for 20 muscle fibres from a healthy elderly 85-year-old individual, chosen because of the low frequency of cytochrome c oxidase negative fibres. Phenotypically neutral single base substitutions were detected in 15% of the healthy fibres, supporting the hypothesis that positive selection is not essential for the clonal expansion of mtDNA point mutations during human life. Treatments that enhance mtDNA replication, such as vigorous excercise, could amplify this process, with potentially detrimental long-term consequences. PMID- 16684600 TI - 140th ENMC International Workshop: Myotonic Dystrophy DM2/PROMM and other myotonic dystrophies with guidelines on management. PMID- 16684601 TI - Novel slow-skeletal myosin (MYH7) mutation in the original myosin storage myopathy kindred. AB - Myosin storage myopathy (OMIM 608358), a congenital myopathy characterised by subsarcolemmal, hyaline-like accumulations of myosin in Type I muscle fibres, was first described by Cancilla and Colleagues in 1971 [Neurology 1971;21:579-585] in two siblings as 'familial myopathy with probable lysis of myofibrils in type I muscle fibres'. Two mutations in the slow skeletal myosin heavy chain gene (MYH7) have recently been associated with the disease in other families. We have identified a novel heterozygous Leu1793Pro mutation in MYH7 in DNA from paraffin sections of one of the original siblings. This historical molecular analysis confirms the original cases had myosin storage myopathy. PMID- 16684602 TI - Different early pathogenesis in myotilinopathy compared to primary desminopathy. AB - Mutations in the human myotilin gene may cause limb-girdle muscular dystrophy 1A and myofibrillar myopathy. Here, we describe a German patient with the clinically distinct disease phenotype of late adult onset distal anterior leg myopathy caused by a heterozygous S55F myotilin mutation. In addition to a thorough morphological and clinical analysis, we performed for the first time a protein chemical analysis and transient transfections. Morphological analysis revealed an inclusion body myopathy with myotilin- and desmin-positive aggregates. The clinical and pathological phenotype considerably overlaps with late onset distal anterior leg myopathy of the Markesbery-Griggs type. Interestingly, all three analyzed myotilin missense mutations (S55F, S60F and S60C) do not lead to gross changes in the total amount of myotilin or to aberrant posttranslational modifications in diseased muscle, as observed in a number of muscular dystrophies. Transiently transfected wild-type and S55F mutant myotilin similarly colocalised with actin-containing stress fibers in BHK-21 cells. Like the wild type protein, mutated myotilin did not disrupt the endogenous desmin cytoskeleton or lead to pathological protein aggregation in these cells. This lack of an obvious dominant negative effect sharply contrasts to transfections with, for instance, the disease-causing A357P desmin mutant. In conclusion our data indicate that the disorganization of the extrasarcomeric cytoskeleton and the presence of desmin-positive aggregates are in fact late secondary events in the pathogenesis of primary myotilinopathies, rather than directly related. These findings suggest that unrelated molecular pathways may result in seemingly similar disease phenotypes at late disease stages. PMID- 16684603 TI - 136th ENMC International Workshop: Charcot-Marie-Tooth disease type 1A (CMT1A)8 10 April 2005, Naarden, The Netherlands. PMID- 16684604 TI - Burkholderia cepacia complex species: health hazards and biotechnological potential. AB - The Burkholderia cepacia complex is a group of nine closely related bacterial species that have useful properties in the natural environment as plant pest antagonists, plant growth promoters and degradative agents of toxic substances. Because these species are human opportunistic pathogens, especially in cystic fibrosis patients, biotechnological applications that involve environmental releases have been severely restricted. Recent progress in understanding the taxonomy, epidemiology and ecology of the B. cepacia complex species has unravelled considerable variability in their pathogenicity and ecological properties, which has set the basis for a reassessment of the risk posed by individual species to human health. PMID- 16684605 TI - Mitochondria in DRG neurons undergo hyperglycemic mediated injury through Bim, Bax and the fission protein Drp1. AB - Dorsal root ganglia (DRG) neurons degenerate in diabetic neuropathy (DN) and exhibit mitochondrial damage. We studied mitochondria of cultured DRG neurons exposed to high glucose as an in vitro model of DN. High glucose sequentially increases the expression, activation and localization of the pro-apoptotic proteins Bim and Bax and the mitochondrial fission protein dynamin-regulated protein 1 (Drp1). High glucose causes association of Drp1/Bax, similar to other apoptotic stimuli. Collectively, these events promote mitochondrial fragmentation and reduce mitochondrial number, suggestive of apoptotic mitochondrial fission. Drp1 is also upregulated in DRG from experimentally diabetic rats, suggesting a role for mitochondrial fission in DN. Insulin-like growth factor-I (IGF-I) protects high glucose-treated DRG neurons by preventing mitochondrial accumulation of Bim and Bax but does not modulate Drp1 expression or localization. We propose that mitochondria are compromised by convergence of Bim/Bax proteins with Drp1, which contributes to high glucose-induced injury in DRG neurons. PMID- 16684606 TI - SIRT1 and endocrine signaling. AB - Sirtuins (Sir2-related enzymes) are a recently discovered class of NAD(+) dependent protein deacetylases that regulate gene expression in a variety of organisms by deacetylation of modified lysine residues on histones, transcription factors and other proteins. Conservation of sirtuin regulation of the insulin insulin-like growth factor I signaling pathway has been observed for Caenorhabditis elegans and mammals, indicating an ancient role for sirtuins in the modulation of organism adaptations to nutritional intake. The human sirtuin SIRT1 regulates a number of transcription factors that modulate endocrine signaling, including peroxisome proliferator-activated receptor gamma, peroxisome proliferator-activated receptor gamma coactivator 1alpha, forkhead-box transcription factors and p53. PMID- 16684607 TI - Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly. AB - The Ena/VASP and WASP family of proteins play distinct roles in actin cytoskeleton remodeling. Ena/VASP is linked to actin filament elongation, whereas WASP plays a role in filament nucleation and branching mediated by Arp2/3 complex. The molecular mechanisms controlling both processes are only emerging. Both Ena/VASP and WASP are multidomain proteins. They both present poly-Pro regions, which mediate the binding of profilin-actin, followed by G-actin-binding (GAB) domains of the WASP-homology 2 (WH2) type. However, the WH2 of Ena/VASP is somewhat different from that of WASP, and has been poorly characterized. Here we demonstrate that this WH2 binds profilin-actin with higher affinity than actin alone. The results are consistent with a model whereby allosteric modulation of affinity drives the transition of profilin-actin from the poly-Pro region to the WH2 and then to the barbed end of the filament during elongation. Therefore, the function of the WH2 in Ena/VASP appears to be to "process" profilin-actin for its incorporation at the barbed end of the growing filament. Conformational changes in the newly incorporated actin subunit, resulting either from nucleotide hydrolysis or from the G- to F-actin transition, may serve as a "sensor" for the processive stepping of Ena/VASP. Conserved domain architecture suggests that WASP may work similarly. PMID- 16684608 TI - Differential macrophage polarisation during parasitic infections in common carp (Cyprinus carpio L.). AB - In many parasitic infections both classically activated macrophages (caMF) and alternatively activated macrophages (aaMF) play a pivotal role. To investigate if both types of macrophages also play an important role during parasitic infections in fish, we infected carp with either Trypanoplasma borreli or Trypanosoma carassii and determined the activation state of the head kidney leukocytes (HKL). Nitrite production was used as read-out for caMF and arginase activity as read out for aaMF. Basal nitrite production and arginase activity of HKL were moderately different between the two infections. Differences were observed, however, after ex vivo re-stimulation of HKL. Re-stimulation with LPS and T. borreli lysates increased nitrite production by HKL of T. borreli-infected fish. Re-stimulation with cAMP increased arginase activity in HKL of T. carassii infected fish. Our results indicate that T. borreli-infected carp are more prone to increase nitrite production by caMF while T. carassii-infected fish are more prone to increase arginase activity by aaMF. PMID- 16684609 TI - The effect of sodium alginate on the immune response of tiger shrimp via dietary administration: activity and gene transcription. AB - The total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (release of superoxide anions), and superoxide dismutase (SOD) activity, as well as expressions of beta-1,3-glucan-binding protein (betaGBP), prophenoloxidase (proPO), peroxinectin (PE), cytosolic SOD (cyt-SOD), penaeidin-5 (PA-5), and a single whey acidic protein (WAP) domain protein (SWDP) gene were determined in the tiger shrimp Penaeus monodon (15.6-19.5g) which had individually been fed diets containing sodium alginate at 0, 1.0, or 2.0gkg(-1) for 5months. Results showed that shrimp fed a diet containing 1.0 and 2.0gkg(-1) sodium alginate had significantly increased SOD activity but decreased respiratory bursts. The expressions of betaGBP, PE, cyt-SOD, PA-5, and SWDP were significantly elevated in shrimp fed the T) and -188/-189 (T insertion). From further sequencing analysis, the promoter region of the 3.2-kb allele showed four normal transcriptional sequences positioned at nt -126C, -113G, -110T, and -103A. However, other nucleotides such as at nt -294T, -293A, and -282A were unchanged. We concluded that the 6.2-kb TaqI fragments of the CYP21A2 haplotype may lead to an incorrect result in the analysis between CYP21A2 and CYP21A1P. The formation of the 3.2-kb TaqI fragment allele which can be mistaken for the CYP21A1P gene may be caused by small-scale conversions of the CYP21A1P gene located between nt -126C and -282A. Therefore, the CYP21A2 haplotype not only presents a 3.7-kb TaqI fragment but also may possibly exist in multiple forms including both 6.2- and 3.2-kb fragments. PMID- 16684615 TI - Sonochemical preparation of carbon nanosheet from carbon black. AB - Preparation of carbon nanosheet via ultrasound irradiation of carbon black under ambient conditions was reported for the first time. The structure of resulting carbon nanosheet was characterized by TEM, HRTEM, EDX and AFM. The experimental results showed that the carbon nanosheet is composed of ordered graphite carbon layers with a thickness of several nanometers. PMID- 16684616 TI - An experimental and theoretical study on the interaction of PHBA ions and H2O molecules. AB - We studied the dependence of the Raman spectra on the concentration of PHBA aqueous solution under UV laser excitation. Through analyzing the spectra, we conclude that the interaction between PHBA ions and H(2)O molecules is weak. To further explore the problem, we studied the interaction between PHBA ions and H(2)O molecules by virtue of theoretical calculations, DFT-B3PW91/6-31+g(*) was employed. We draw a coincident conclusion with the experiments and dug out the reasonable interaction model that reflects the actual interaction configuration between PHBA ions and H(2)O molecules. We supply a new thinking for studying interactions between solute molecules and solvent molecules, which also can be applied to interactions between solute molecules and other solute molecules in solutions. PMID- 16684617 TI - A novel colorimetric sensor of dihydrogen-phosphate based on metal complex between 8-hydroxy quinoline-5-azo-4'-nitrobenzene and cobalt. AB - A novel colorimetric sensor based on 8-hydroxy quinoline-5-azo-4'-nitrobenzene (1) was prepared and used for recognizing anions. 1 and its metal complex (1.Co) were found to show response to anions such as CH(3)CO(2)(-), H(2)PO(4)(-), HSO(4)(-), F(-) and dramatic color changes were observed. The selectivity and sensitivity of 1 and 1.Co for sensing anions were different, which was in the order of CH(3)CO(2)(-)>F(-)>H(2)PO(4)(-)>>HSO(4)(-) for 1 and H(2)PO(4)( )>HSO(4)(-)>CH(3)CO(2)(-) approximately F(-) for 1.Co, respectively. In CH(3)CN, sensor 1.Co exhibited excellent specificity toward H(2)PO(4)(-), and the color variety was dependent on the concentration of H(2)PO(4)(-) which was attributed to anion structure and stability of anionic complex (1-anion), metal complex (1 Co) and inorganic complex (Co-anion). PMID- 16684618 TI - UV and fluorescence spectral changes induced by neodymium binding of N,N' ethylenebis[2-(o-hydroxyphenolic)glycine] and N,N'-di(2 hydroxybenzyl)ethylenediamine-N,N' diacetic acid. AB - In 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.4 and room temperature, the binding of neodymium to N,N'-ethylenebis[2-(o hydroxyphenolic)glycine] (EHPG), or N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N' diacetic acid (HBED) had been studied from 210 to 330 nm by means of difference UV spectra. Two peaks at 240 and 292 nm appear in difference UV spectra after neodymium binding to EHPG or HBED. The 1:1 stable complex can be confirmed from spectral titration curves. The molar extinction coefficient of Nd-EHPG and Nd HBED complexes are Deltaepsilon(Nd-EHPG)=(12.93+/-0.21) x 10(3)cm(-1)M(-1), Deltaepsilon(Nd-HBED)=(14.45+/-0.51) x 10(5)cm(-1)M(-1) at 240 nm, respectively. Using EDTA as a competitor, the conditional equilibrium constants of the complexes are logK(Nd-EHPG)=11.89+/-0.09 and logK(Nd-HBED)=12.19+/-0.15, respectively. At the same conditions, fluorescence measurements show that neodymium binding to EHPG leads to a quenching of the fluorescence of EHPG at near 310 nm. However, there is no obvious fluorescence change of HBED at 318 nm with the binding of neodymium to HBED. PMID- 16684619 TI - Automated detection of videotaped neonatal seizures based on motion segmentation methods. AB - OBJECTIVE: This study was aimed at the development of a seizure detection system by training neural networks using quantitative motion information extracted by motion segmentation methods from short video recordings of infants monitored for seizures. METHODS: The motion of the infants' body parts was quantified by temporal motion strength signals extracted from video recordings by motion segmentation methods based on optical flow computation. The area of each frame occupied by the infants' moving body parts was segmented by direct thresholding, by clustering of the pixel velocities, and by clustering the motion parameters obtained by fitting an affine model to the pixel velocities. The computational tools and procedures developed for automated seizure detection were tested and evaluated on 240 short video segments selected and labeled by physicians from a set of video recordings of 54 patients exhibiting myoclonic seizures (80 segments), focal clonic seizures (80 segments), and random infant movements (80 segments). RESULTS: The experimental study described in this paper provided the basis for selecting the most effective strategy for training neural networks to detect neonatal seizures as well as the decision scheme used for interpreting the responses of the trained neural networks. Depending on the decision scheme used for interpreting the responses of the trained neural networks, the best neural networks exhibited sensitivity above 90% or specificity above 90%. CONCLUSIONS: The best among the motion segmentation methods developed in this study produced quantitative features that constitute a reliable basis for detecting myoclonic and focal clonic neonatal seizures. The performance targets of this phase of the project may be achieved by combining the quantitative features described in this paper with those obtained by analyzing motion trajectory signals produced by motion tracking methods. SIGNIFICANCE: A video system based upon automated analysis potentially offers a number of advantages. Infants who are at risk for seizures could be monitored continuously using relatively inexpensive and non invasive video techniques that supplement direct observation by nursery personnel. This would represent a major advance in seizure surveillance and offers the possibility for earlier identification of potential neurological problems and subsequent intervention. PMID- 16684620 TI - Toward an objective quantification of impaired manual dexterity following stroke: the usefulness of precision grip measures. PMID- 16684621 TI - Committed to memory: lineage choices for activated T cells. AB - The mechanisms for the generation of memory T cells and their delineation into heterogeneous subsets remain unknown. The linear model for memory T-cell generation from differentiated effector cells has been favored, although there is evidence that memory T cells can emerge directly from naive T cells undergoing homeostatic expansion and from activated T cells lacking effector functions. Here, we discuss the evidence from diverse studies of memory generation that support a new 'intersecting pathway' model for memory T-cell generation in which antigen-driven effector differentiation and homeostasis-driven memory differentiation follow distinct but analogous pathways. Antigen withdrawal during effector differentiation enables intersection with the memory pathway through a pre-memory intermediate, and memory heterogeneity is influenced by homeostasis, migration and persistence in vivo. PMID- 16684622 TI - Gene therapy advances but struggles to interpret safety data in small animal models. PMID- 16684623 TI - Co-evolution of the outer surface protein C gene (ospC) and intraspecific lineages of Borrelia burgdorferi sensu stricto in the northeastern United States. AB - Clinical and tick isolates of Borrelia burgdorferi sensu stricto, the bacterial agent of Lyme disease, from the northeastern United States were sequenced at 12 loci located on the main chromosome and 7 plasmids (lp54, cp26, cp9, lp17, lp25, lp28-2, and lp38). The outer surface protein C gene (ospC) showed the highest number (12) of major alleles (defined as alleles differing by 5% or more in nucleotide sequence) while other ORFs had only two to four major alleles. A non recombining chromosomal marker, the rrs-rrlA ribosomal RNA spacer, was used to infer the intraspecific phylogeny among these B. burgdorferi isolates. We were thus able to analyze the multilocus genotypes in the context of a B. burgdorferi intraspecific phylogeny. Except for ospC, sequence variations at plasmid-borne loci showed broad inconsistency with the intraspecific phylogeny, supporting DNA exchanges mediated by plasmid transfers. The multilocus linkage frequently observed in B. burgdorferi populations is due more likely to a "founder effect" than to a lack of recombination. The exceptional phylogenetic consistency of ospC, in conjunction with its selectively maintained high intraspecific diversity, suggested a dominant role ospC plays in the initiation and maintenance of adaptive differentiation in B. burgdorferi. PMID- 16684625 TI - Determining factors associated with shoulder dystocia: a population-based study. AB - OBJECTIVE: The study was aimed to define obstetric factors associated with shoulder dystocia. METHODS: A population-based study comparing all singleton, vertex, term deliveries with shoulder dystocia with deliveries without shoulder dystocia was performed. Statistical analysis was done using multiple logistic regression analysis. RESULTS: Shoulder dystocia complicated 0.2% (n = 245) of all deliveries included in the study (n = 107965). Independent risk factors for shoulder dystocia in a multivariable analysis were birth-weight > or =4000 g (OR = 24.3; 95% CI 18.5-31.8), vacuum delivery (OR = 5.7, 95% CI 3.4-9.5), diabetes mellitus (OR = 1.7, 95% CI 1.2-2.5) and lack of prenatal care (OR = 1.5, 95% CI 1.1-2.3). A significant linear association was found between birth-weight and shoulder dystocia, using the Mantel-Haenszel procedure. Pregnancies complicated with shoulder dystocia had higher rates of third-degree perineal tears as compared to the comparison group (0.8% versus 0.1%; P < 0.001). Similarly, perinatal mortality was higher among newborns delivered after shoulder dystocia as compared to the comparison group (3.7% versus 0.5%; OR = 7.4, 95% CI 3.5-14.9, P < 0.001). In addition, these newborns had higher rates of Apgar scores lower than 7 at 1 and 5 min as compared to newborns delivered without shoulder dystocia (29.7% versus 3.0%; OR = 13.8, 95% CI 10.3-18.4, P < 0.001 and 2.1% versus 0.3%; OR = 7.2, 95% CI 2.8-18.1, P < 0.001, respectively). Combining risk factors such as large for gestational age, diabetes mellitus and vacuum delivery increased the risk for shoulder dystocia to 6.8% (OR = 32.6, 95% CI 10.1-105.8, P < 0.001). CONCLUSIONS: Independent factors associated with shoulder dystocia were birth weight > or =4000 g, vacuum delivery, diabetes mellitus and lack of prenatal care. PMID- 16684626 TI - Gore-Tex mesh pelvic occlusion and secondary colpopexy: A new surgical technique for posthysterectomy vaginal vault prolapse. AB - OBJECTIVE: This article presents and discusses a new surgical abdominal technique for the treatment of posthysterectomy vaginal vault prolapse. METHOD: It provides support of the peritoneal surface of the pelvic floor by means of a Gore-Tex mesh, which closes this space. The vaginal vault is fixed to the centre of the mesh. STUDY DESIGN: Descriptive study. RESULTS: Sixteen patients with vaginal vault prolapse were operated on and postoperative follow-up time ranged from 16 to 46 months. There was only one case in which the mesh had to be removed due to infection and posterior erosion of the vaginal wall, and no cases of recurrent vaginal vault prolapse. CONCLUSION: A Gore-Tex mesh, placed at the top of the vaginal vault and extending across the pelvic floor, can effectively treat posthysterectomy vault prolapse. PMID- 16684628 TI - Neurology case studies: cerebrovascular disease. AB - Data from randomized therapeutic trials often provide little relevant evidence for therapeutic decisions physicians make daily. By illustrating the nuances of these four complex cases involving cerebrovascular disease, the authors stress the importance of more time spent by specialists at the bed-side, exploring patients' symptoms and learning their thoughts, fears, biases, and wishes. PMID- 16684629 TI - Diagnosis and management of multiple sclerosis: case studies. AB - Although substantial capabilities have emerged in the ability to globally manage patients who have MS, clinicians continue to be confronted with formidable challenges. Reduction in disease activity and its impact on dis-ability progression remains the central objective of disease-modifying therapy and most current MS research initiatives. Nevertheless, the principal factors that determine the day-to-day limitations on functional capabilities(activities of daily living, work performance, quality of life, and so forth)are a derivative of the pathophysiology of the disease process itself. The substrate for these limitations is inherent in the pathology of demyelination and axonal dysfunction. Identifying measures that can optimize the performance and fidelity of axonal conduction mechanisms may translate into a reduction in MS-related symptoms. Chronic neurologic disease management (with MS representing a signature example) can be optimized when all members of the care team (including patients and their families) collaborate in the co-ordination of interdisciplinary care models that address all aspects of suffering. PMID- 16684630 TI - Syncope: case studies. AB - In this series of clinical vignettes, the authors have attempted to provide a "feel" for the varied causes of syncope. The neurologist should be able to diagnose most causes of syncope using a simple algorithmic approach. Initial evaluation includes detailed clinical history, physical examination, and 12-lead ECG. Following initial evaluation, the cause of syncope is usually immediately apparent (typical story for vasovagal syncope, clinically demonstrable autonomic failure, long QT), strongly suspected (syncope preceded by chest pain or palpitations), or uncertain. In the latter group of patients, further workup will depend on the suspicion or documented presence of heart disease. In those with a single episode of syncope and no evidence of heart disease, further workup may not be necessary. In patients over 60 years of age with recurrent episodes and no cardiac history or abnormal ECG, tilt-table testing and carotid sinus massage may be diagnostic. If no diagnosis is found, an implantable loop monitor may be needed. Patients with heart disease will need the most comprehensive evaluations, possibly including exercise testing, cardiac electrophysiology, and tilt-table testing. As better understanding of pathophysiology and epidemiology emerge, under-standing of the diagnosis and treatment of syncope will improve. In the meantime, there is no substitute for astute clinical acumen. PMID- 16684631 TI - Neuromuscular disorders in clinical practice: case studies. AB - Neuromuscular disorders represent a large group of highly varied and interesting clinical disorders, many of which have major general medical manifestations. These disorders can be diagnosed largely based on the patient's history and physical examination with a little help from modern technology. Despite the outdated belief that neurologic conditions are diagnosed but rarely treatable, all cases discussed herein represent disorders for which there are extensive options and opportunities for meaningful management. These 16 brief case overviews challenge and refresh diagnostic skills and provide the framework for selected comments regarding management options. PMID- 16684632 TI - Sleep disorders: case studies. AB - This article reviews nine case studies of patients presenting with sleep complaints. PMID- 16684633 TI - Epilepsy case studies. AB - Epilepsy is a common disorder affecting all age groups. Diagnosis depends on accurate eyewitness description and electroencephalography. Many genetic, metabolic, and structural perturbations of cortical function can cause seizures. MRI is the most important test for etiology. Medication selection is based on classification of seizure type and epilepsy syndrome, with consideration of patient age, gender, and comorbidities. Surgery is a good treatment for some patients who have medically refractory partial-onset seizures. PMID- 16684634 TI - Spine disorder case studies. AB - Spine and limb pain related to degenerative, wear-and-tear changes of the disks, joints, and soft tissues of the spine (spondylosis) is exceedingly common. Identical spine and limb pain can be caused by fracture, tumor, or infection. Patients who have a history of significant trauma, signs or symptoms of systemic disease, neurologic impairment, and profound pain require further evaluation, and some may require surgical intervention to decompress the spinal nerves, spinal cord, or cauda equina or to stabilize adjacent spinal segments. The discovery of red flags in the history or on examination can identify patients who should have imaging and possibly surgical intervention. Neurologic assessment is required to determine which patients could benefit from surgery. The natural history for most acute spondylotic conditions is favorable. Treatment for acute and chronic spine related pain syndromes is available. PMID- 16684635 TI - Case studies in neuro-ophthalmology for the neurologist. AB - In summary, neurologists should be aware of emergent neuro-ophthalmic conditions: (1) temporal arteritis (GCA), (2) IIH, (3) intracranial shunt malfunction, (4) pituitary apoplexy, and (5) pupil-involved TNP. Earlier recognition and treatment of these disorders makes a difference in final out-come. Appropriate evaluation and management may be vision or life saving. PMID- 16684636 TI - Case studies of uncommon headaches. AB - The following interesting and uncommon headache disorders are presented through case studies: exploding head syndrome, hypnic headache, neck-tongue syndrome, "Alice in Wonderland" syndrome, nummular headache, red ear syndrome, burning mouth syndrome, spontaneous intracranial hypotension syndrome, and cardiac cephalalgia. PMID- 16684637 TI - Psychosis in Parkinson's disease: case studies. AB - Psychosis is common in patients who have PD and leads to significant disability. Patients often can be managed with non-pharmacologic interventions or with decreasing doses of anti-parkinsonism medications. If these interventions are insufficient, then atypical antipsychotics should be considered. Clozapine is used in more refractory cases and requires stringent monitoring for agranulocytosis. PMID- 16684638 TI - Neuromuscular disorders in medical and surgical ICUs: case studies in critical care neurology. AB - The differential diagnosis of generalized weakness in ICU patients is quite broad. Although neuromuscular disorders are the most common causes of generalized weakness, a thorough evaluation is necessary to delineate the underlying cause of weakness. Biochemical studies, neuroimaging, and electrophysiologic studies help to delineate most of the common disorders associated with weakness. Prompt identification of a neurologic disorder and initiation of therapy speeds up recovery and reduces morbidity and mortality in these patients. PMID- 16684639 TI - Functional symptoms in neurology: case studies. AB - In this series of case vignettes, the authors have emphasized that the diagnosis of functional symptoms should normally rest on the presence of positive evidence of the problem being functional rather than the absence of evidence of organic disease. In addition, practitioners should be prepared to make a functional diagnosis in a patient who also has evidence of disease. Misdiagnosis of functional symptoms occurs no more than for other neuro-logical and psychiatric disorders. The neurologist has an important role in being able to transmit the diagnosis in a way that will not offend the patient but will also facilitate recovery. The key elements of this explanation are making the patient feel believed and emphasizing potential reversibility. A multidisciplinary approach involving concurrent physical and psychological treatments is often recommended, although further study is required to determine the best approaches to explain and treat these conditions. PMID- 16684640 TI - A Raman and infrared spectroscopic study of the uranyl silicates--weeksite, soddyite and haiweeite. AB - Raman spectroscopy has been used to study the molecular structure of a series of selected uranyl silicate minerals, including weeksite K2[(UO2)2(Si5O13)].H2O, soddyite [(UO2)2SiO4.2H2O] and haiweeite Ca[(UO2)2(Si5O12(OH)2](H2O)3 with UO2(2+)/SiO2 molar ratio 2:1 or 2:5. Raman spectra clearly show well resolved bands in the 750-800 cm-1 region and in the 950-1000 cm-1 region assigned to the nu1 modes of the (UO2)2+ units and to the (SiO4)4- tetrahedra. For example, soddyite is characterized by Raman bands at 828.0, 808.6 and 801.8 cm-1 (UO2)2+ (nu1), 909.6 and 898.0 cm-1 (UO2)2+ (nu3), 268.2, 257.8 and 246.9 cm-1 are assigned to the nu2 (delta) (UO2)2+. Coincidences of the nu1 (UO2)2+ and the nu1 (SiO4)4- is expected. Bands at 1082.2, 1071.2, 1036.3, 995.1 and 966.3 cm-1 are attributed to the nu3 (SiO4)4-. Sets of Raman bands in the 200-300 cm-1 region are assigned to nu2 (delta) (UO2)2+ and UO ligand vibrations. Multiple bands indicate the non-equivalence of the UO bonds and the lifting of the degeneracy of nu2 (delta) (UO2)2+ vibrations. The (SiO4)4- tetrahedral are characterized by bands in the 470-550 cm-1 and in the 390-420 cm-1 region. These bands are attributed to the nu4 and nu2 (SiO4)4- bending modes. The minerals show characteristic OH stretching bands in the 2900-3500 cm-1 and 3600-3700 cm-1. PMID- 16684641 TI - Cardiovagal autonomic dysfunction in relation to HIV-associated lipodystrophy. AB - PURPOSE: Human immunodeficiency virus (HIV)-associated lipodystrophy (LD) may be mediated by changes in the autonomic nervous system. We examined the autonomic function among HIV-infected patients with LD compared to HIV-infected patients without LD (non-LD) and HIV-negative controls (controls). METHOD: This cross sectional study examined cardiovagal autonomic function among the three groups. LD was defined in HIV-infected patients as increased visceral adipose accumulation and peripheral lipoatrophy. Cardiovagal autonomic testing was assessed by measuring heart rate variability during rest, paced breathing, and upright tilt and was analyzed in time and frequency domains. RESULTS: Cardiovagal testing was performed on 58 participants: 26 controls, 20 non-LD, and 12 LD. After adjustment for visceral fat, time domain analysis showed decreased heart rate variability in patients with LD compared to the other groups (p < .05). The frequency domain analysis showed decreased high-frequency power and increased low to high-frequency power ratio in the LD group compared to both groups during rest and to non-LD during tilt (p < .05). CONCLUSION: Patients with LD have altered cardiovagal modulation. Patients with LD had lower heart rate variability and higher sympathetic modulation compared to non-LD and controls. These alterations may be prognostic of increased cardiovascular disease morbidity and mortality. PMID- 16684642 TI - Randomized trials stopped early for harm in HIV/AIDS: a systematic survey. AB - PURPOSE: The decision to stop trials early because of the harmful effects of the intervention is complex and requires weighing statistical, logistical, and ethical considerations. We assessed the prevalence of randomized clinical trials (RCTs) stopped early for harm in HIV/AIDS and determined the quality of reporting of methods to inform the decision to stop the trial. METHOD: We searched 11 electronic databases and major conference abstract databases, contacted trialist and advocacy groups, and searched the Internet. We selected RCTs stopped early for harm. We extracted data on journal and year of publication, reporting of methods and funding, planned sample size, number and planning of interim analyses, stopping rules, and effect size of the harm outcomes. RESULTS: We found 10 RCTs stopped early for harm (median, n = 85; range, 7-1227). Most interventions (n = 9) were antiviral drugs; one trial studied vitamins to prevent vertical transmission of HIV. Five studies reported a priori defined adverse events, and only 1 trial reported planned stopping guidelines. The primary harm outcomes reported across trials included toxicity, death, and increased mother-to child transmission. Two trials were stopped due to sudden unanticipated adverse events (Stevens-Johnson syndrome, death, and encephalopathy). Relative risk point estimates for harm ranged from 1 to 6.18. Six studies reported the presence of a data safety and monitoring board. CONCLUSION: The reporting of methods to inform the decision to stop trials for harm in this population is deficient in a variety of ways, including lack of stopping guidelines. Clinicians should interpret RCTs stopped early for harm with caution and interpret the results in light of related evidence. Trialists should improve the transparency of their decision-making regarding early stopping for harmful effects. PMID- 16684644 TI - David Kupfer: a career retrospective. AB - David Kupfer's research career spanned 50 years and he authored or co-authored over 160 papers and book chapters. Although best known for his work centering on cytochrome P450 metabolism of prostaglandins, steroids, and proestrogenic compounds, David's research also contributed key advances in the areas of P450 induction and catalytic mechanism, breast cancer therapy, and analytical methodology. His research is reviewed here. PMID- 16684645 TI - David Kupfer, Ph.D. A mentor and a scientist. AB - I worked with the late Dr. David Kupfer for nearly nine years at the Worcester Foundation/University of Massachusetts Medical School, Worcester, MA. I was involved in the metabolism of methoxychlor and tamoxifen, the areas of research close to David's heart. We demonstrated the metabolic pathways of these compounds in rats and humans, and the covalent binding to microsomal proteins that could result in long-term toxic manifestations. I learned a lot from David, who was a mentor and friend/colleague. His death has left a void in my heart and he will be sorely missed. PMID- 16684646 TI - A photographic diary of travels with David Kupfer. AB - The present article focuses on David Kupfer as a colleague and fellow traveler who participated in numerous meetings on cytochrome P450 held at exotic venues. It was always a pleasure to renew a long-standing friendship with David by meeting him at these meetings. His inscrutable smile combined with residues of an accent derived from his earlier background in Poland and Israel characterized this warm and delightful man. A number of photos of David in various locales are presented with this article in an attempt to fully capture the outstanding qualities of this man. PMID- 16684647 TI - Transcriptional regulation of the human pregnane-X receptor. AB - This review addresses the general structure and function of nuclear receptors and places specific emphasis on their role in xenosensing, resulting in the activation of a battery of genes mediating drug metabolism, conjugation, and transport. The pregnane-X receptor is a nuclear receptor that functions to control a battery of genes predominantly involved in drug metabolism and we place emphasis on how this important cellular mediator is transcriptionally activated. We have identified both positive and negative regulatory elements in the PXR promoter, the balance of which dictates the steady state expression of the PXR gene. PMID- 16684648 TI - Synthetic drugs and natural products as modulators of constitutive androstane receptor (CAR) and pregnane X receptor (PXR). AB - Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are members of the nuclear receptor superfamily. These transcription factors are predominantly expressed in the liver, where they are activated by structurally diverse compounds, including many drugs and endogenous substances. CAR and PXR regulate the expression of a broad range of genes, which contribute to transcellular transport, bioactivation, and detoxification of numerous xenochemicals and endogenous substances. This article discusses the importance of these receptors for pharmacology and toxicology, emphasizing the role of individual drugs and natural products as agonists, indirect activators, inverse agonists, and antagonists of CAR and PXR. PMID- 16684649 TI - Phenobarbital confers its diverse effects by activating the orphan nuclear receptor car. AB - In the early 1960s, phenobarbital (PB) was shown to induce hepatic drug metabolism and the induction was implicated in the molecular mechanism of drug tolerance development. Since then, it has become evident that PB not only induces drug metabolism, but also triggers pleiotropic effects on liver function, such as cell growth and communication, proliferation of the endoplasmic reticulum, tumor promotion, glucose metabolism, steroid/thyroid hormone metabolism, and bile acid synthesis. Upon activation by PB and numerous PB-type inducers, the nuclear receptor CAR mediates those pleiotropic actions by regulating various hepatic genes, utilizing multiple regulatory mechanisms. PMID- 16684651 TI - Activated estrogens and antiestrogens: a 30-year journey with David Kupfer. AB - David Kupfer had a passion for drug metabolism and used his talents to understand the putative metabolic activation of the insecticides o, p'DDT and methoxychlor to estrogens. His research helped to create a scientific foundation for the current interest in endocrine disruption. With the increasing clinical significance of tamoxifen in the late 1980s, and the proposal to test tamoxifen as a breast cancer chemopreventive in healthy women, David initiated laboratory studies on the mechanisms of tamoxifen metabolism. He was the first to note that tamoxifen is metabolically activated to alkylating species. Tamoxifen and insecticides covalently bind to microsomal proteins. His contribution presaged worldwide studies of the induction of rat liver carcinogenesis by tamoxifen. PMID- 16684650 TI - The biological actions of dehydroepiandrosterone involves multiple receptors. AB - Dehydroepiandrosterone has been thought to have physiological functions other than as an androgen precursor. The previous studies performed have demonstrated a number of biological effects in rodents, such as amelioration of disease in diabetic, chemical carcinogenesis, and obesity models. To date, activation of the peroxisome proliferators activated receptor alpha, pregnane X receptor, and estrogen receptor by DHEA and its metabolites have been demonstrated. Several membrane-associated receptors have also been elucidated leading to additional mechanisms by which DHEA may exert its biological effects. This review will provide an overview of the receptor multiplicity involved in the biological activity of this sterol. PMID- 16684652 TI - David Kupfer and the metabolism connection: on aromatase inhibitors and tamoxifen. AB - This tribute to Dr. David Kupfer describes his contributions to understanding the actions and interactions of tamoxifen and aromatase inhibitors through their metabolites and their role in breast cancer treatment and prevention. PMID- 16684653 TI - The role of flavin-containing monooxygenase (FMO) in the metabolism of tamoxifen and other tertiary amines. AB - Tamoxifen is utilized in breast cancer therapy and in chemoprevention. Tamoxifen may enhance risk for other neoplasias, especially endometrial cancer. The risk:benefit depends on the rate of metabolic activation versus detoxication. Cytochrome P450-dependent alpha-hydroxylation, followed by sulfonation, represents a metabolic activation pathway, producing products capable of covalent DNA adduction. In contrast, tamoxifen N-oxygenation represents a detoxication pathway, with the caveat that N-oxides can be reduced back to the parent amines. The N-oxygenation pathway will be the focus for this review. Dr. David Kupfer pioneered studies on cytochrome P450 and flavin-containing monooxygenase (FMO) tamoxifen metabolism. We collaborated with Dr. Kupfer's laboratory and recently determined that the low level of tamoxifen N-oxide production in human liver microsomes may be explained by the kinetics of FMO1 versus FMO3. PMID- 16684654 TI - Organophosphorus chemicals: potent inhibitors of the human metabolism of steroid hormones and xenobiotics. AB - Although it has been known for some time that organophosphate chemicals containing the P = S moiety are irreversible inhibitors of cytochrome P450, this knowledge has not been generally applied to the human metabolism of xenobiotics. Recent studies have demonstrated that organophosphate insecticides containing this moiety are potent inhibitors of the metabolism of both xenobiotics and endogenous substrates by human liver microsomes and by specific human cytochrome P450 isoforms. PMID- 16684655 TI - David Kupfer at meetings and play. AB - David Kupfer was a very sociable person, someone with whom it was a pleasure and delight to attend meetings and discuss science. He had a highly developed sense of humor and enjoyed life fully. The following pictures, hopefully capture some of his essence. PMID- 16684656 TI - Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. AB - Mitochondrial P450 type enzymes catalyze central steps in steroid biosynthesis, including cholesterol conversion to pregnenolone, 11beta and 18 hydroxylation in glucocorticoid and mineralocorticoid synthesis, C-27 hydroxylation of bile acids, and 1alpha and 24 hydroxylation of 25-OH-vitamin D. These monooxygenase reactions depend on electron transfer from NADPH via FAD adrenodoxin reductase and 2Fe-2S adrenodoxin. These systems can function as a futile NADPH oxidase, oxidizing NADPH in absence of substrate, and leak electrons via adrenodoxin and P450 to O(2), producing superoxide and other reactive oxygen species (ROS). The degree of uncoupling depends on the P450 and steroid substrate. Studies with purified proteins and overexpression in cultured cells show consistently that adrenodoxin, but not reductase, is responsible for ROS production that can lead to apoptosis. In the ovary and corpus luteum, antioxidant enzyme activities superoxide dismutase, catalase, and glutathione peroxidase parallel steroidogenesis. Antioxidant beta-carotene, alpha-tocopherol, and ascorbate can protect against oxidative damages of P450 systems. In testis Leydig cells, steroidogenesis is associated with aging of the steroidogenic capacity. PMID- 16684657 TI - Sex and the liver - a journey through five decades. AB - Metabolism of steroids and drugs in rodents is sexually differentiated. The reason for this turned out to be the sexually differentiated growth hormone (GH) secretory pattern regulating the expression of a number of hepatic cytochrome P 450 genes. Although not fully resolved, it is clear that several signaling pathways and transcription factors are involved in mediating the effects of GH. It may be argued that such a well-controlled physiological system should have an important biological role and we speculate that the demands of a robust hepatic steroid metabolism during pregnancy has led to the development of this sexually differentiated hypothalamo-pituitary-liver axis. PMID- 16684658 TI - Cytochromes P450--a family of proteins and scientists-understanding their relationships. AB - The unifying thread of this review involves NADPH-cytochrome P450 reductase (CYPOR), the microsomal enzyme responsible for transferring electrons to cytochromes P450, as well as several other monooxygenase systems, a lifelong interest of the corresponding author. The intersection of her research with that of Dr. David Kupfer, their resulting collaboration, and the beginning of a long standing study of fatty acid- and eicosanoid-metabolizing cytochromes P450 (CYP4A gene subfamily), including the role of cytochrome b5, will be reported. The culmination of this interest now involves purification and characterization of the human mutants of CYPOR that have been implicated in pathologies, such as Antley-Bixler syndrome. PMID- 16684659 TI - The role of trace metals in cytochrome P4501 regulation. AB - Trace metals and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental co-contaminants and the trace metals could influence the carcinogenicity of the PAHs by altering their extent of induction of cytochromes P4501A1, 1A2, and 1B1 (CYP). Studies in cell lines from humans, rodents, chickens, and fish, and in cell culture generally indicate that trace metals diminish the inductive potency of PAHs for these CYPs. The extent of the effect is species-, metal-, PAH-, and metal dose-dependent. Both transcriptional and post-translational mechanisms are involved in the trace metal-mediated down regulation of the CYP1 forms. The latter mechanism incorporates induction of heme oxygenase-1 by the metals, with resultant heme catabolism. Thus, trace metals could diminish the carcinogenicity of PAHs. PMID- 16684660 TI - On the mechanism of hepatocarcinogenesis of benzodiazepines: evidence that diazepam and oxazepam are CYP2B inducers in rats, and both CYP2B and CYP4A inducers in mice. AB - The aim of this study was to evaluate diazepam and oxazepam as cytochrome P450 inducers at doses previously shown to cause liver tumors in mice but not rats. In rats, diazepam and oxazepam induced CYP2B, and were as effective as phenobarbital despite lacking phenobarbital's tumor-promoting effect in rats. In mice, diazepam and oxazepam induced both CYP2B and CYP4A at dietary doses associated with liver tumor formation. It remains to be determined why diazepam and oxazepam induce CYP4A in mice but not rats and whether this difference accounts for the apparent species difference in the tumor-promoting activity of diazepam and oxazepam. PMID- 16684661 TI - Molecular mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin cardiovascular embryotoxicity. AB - 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons are widespread environmental contaminants and potent developmental toxicants. Hallmarks of embryonic exposure include edema, hemorrhage, and mortality. Recent studies in zebrafish and chicken have revealed direct impairment of cardiac muscle growth that may underlie these overt symptoms. TCDD toxicity is mediated by the aryl hydrocarbon receptor, but downstream targets remain unclear. Oxidative stress and growth factor modulation have been implicated in TCDD cardiovascular toxicity. Gene expression profiling is elucidating additional pathways by which TCDD might act. We review our understanding of the mechanism of TCDD embryotoxicity at morphological and molecular levels. PMID- 16684662 TI - CYP1A in TCDD toxicity and in physiology-with particular reference to CYP dependent arachidonic acid metabolism and other endogenous substrates. AB - Toxicologic and physiologic roles of CYP1A enzyme induction, the major biochemical effect of aryl hydrocarbon receptor activation by TCDD and other receptor ligands, are unknown. Evidence is presented that CYP1A exerts biologic effects via metabolism of endogenous substrates (i.e., arachidonic acid, other eicosanoids, estrogens, bilirubin, and melatonin), production of reactive oxygen, and effects on K(+) and Ca(2+) channels. These interrelated pathways may connect CYP1A induction to TCDD toxicities, including cardiotoxicity, vascular dysfunction, and wasting. They may also underlie homeostatic roles for CYP1A, especially when transiently induced by common chemical exposures and environmental conditions (i.e., tryptophan photoproducts, dietary indoles, and changes in oxygen tension). PMID- 16684663 TI - Physiological significance and expression of P450s in the developing eye. AB - Expression of 10 CYP orthologs (Families 1-3) in developing mouse conceptus is constitutive. These forms have specific temporal and spatial expression. Studies on CYP1B1 indicate its requirement for normal eye development, both in human and mouse. The distribution of the enzyme in the mouse eye is in three regions, which may reflect three different, perhaps equally important, functions in this organ. Its presence in the inner ciliary and lens epithelia appears to be necessary for normal development of the trabecular meshwork and its function in regulating intraocular pressure. Its expression in the retinal ganglion and inner nuclear layers may reflect a role in maintenance of the visual cycle. Its expression in the corneal epithelium may indicate a function in metabolism of environmental xenobiotics. PMID- 16684664 TI - Peroral IMUNOR, a low-molecular-weight immunomodulator prepared from disintegrated and ultrafiltered leukocytes, enhances recovery from myelosuppression induced by cisplatin or 5-fluorouracil. AB - A single dose of IMUNOR, a low-molecular-weight immunodulator prepared from disintegrated and ultrafiltered pig leukocytes, was found to enhance recovery of murine pool of hemopoietic progenitor cells for granulocytes and macrophages damaged by a single injection of cytotoxic drugs 5-fluorouracil or cisplatin. The best results were obtained after the treatment with IMUNOR on days 3 or 4 after the injection of 5-fluorouracil or cisplatin. These results together with previous findings obtained in our laboratory suggest that IMUNOR has the potential to become a part of treatment schemes in oncological practice aimed at alleviation of myelosuppression evoked by cytotoxic anti-tumor therapy. PMID- 16684666 TI - Prophylactic dose of neem (Azadirachta indica) leaf preparation restricting murine tumor growth is nontoxic, hematostimulatory and immunostimulatory. AB - Significant restriction of growth of Ehrlich's carcinoma was observed following prophylactic treatment on Swiss albino mice with neem leaf preparation (NLP-1 unit) once weekly for four weeks. Toxic effects of this particular dose (1 unit), along with 0.5 unit and 2 units of NLP doses, were evaluated on different murine physiological systems. One hundred percent of mice could tolerate 4 injections of 0.5 and 1 unit NLP doses. Body weight, different organ-body weight ratios and physical behavior of treated mice remained completely unchanged during treatment with different NLP doses. All of these NLP doses were observed to stimulate hematological systems as evidenced by the increase in total count of RBC, WBC and platelets and hemoglobin percentage. As histological changes as well as elevation in serum alkaline phosphatase, SGOT, SGPT were not observed in mice treated with three different doses of NLP, the nonhepatotoxic nature of NLP was proved. The level of serum urea remained unaltered and normal architecture of the cortical and medullary parts of the kidney were also preserved after NLP treatment. Increased antibody production against B16 melanoma antigen was detected in mice immunized with 0.5 unit and 1 unit of NLP. Number of splenic T lymphocytes (CD4+ and CD8+) and NK cells were also observed to be increased in mice injected with 0.5 unit and 1 unit of NLP. However, NLP dose of 2 units could not exhibit such immunostimulatory changes; NLP mediated immunostimulation was correlated well with the growth restriction of murine carcinoma. In other words, tumor growth restriction was observed only when mice were injected with immunostimulatory doses of NLP (0.5 unit and 1 unit). PMID- 16684665 TI - Influenza virus host response of C57Bl/6 mice treated with TACI-Ig. AB - TACI-Ig is a soluble glycoprotein comprised of a human IgG1-Fc fused with the extracellular domain of the human TACI receptor. Chronic exposure to TACI-Ig is associated with reduced circulating B cells in mouse and non-human primates, and a concomitant decrease in circulating immunoglobulin. Because of these activities, TACI-Ig is in clinical evaluation for treatment of various autoimmune diseases and B cell malignancies. In this study, the effect of TACI-Ig treatment on the ability of C57Bl/6 mice to clear influenza virus was evaluated. C57Bl/6 mice were exposed to vehicle (negative control), dexamethasone (positive control), or TACI-Ig (0.05, 0.50, or 5.0 mg/kg, SC, thrice weekly) from within one week prior to viral exposure through 21 days thereafter. Dexamethasone treatment of influenza-infected mice prolonged the infection, and decreased survival, body weight, lymphoid organ weight, influenza-specific IgM and IgG, and viral clearance relative to control animals, consistent with its expected immunosuppressive activity. Animals treated with TACI-Ig (0.05, 0.50, and 5.0 mg/kg) demonstrated a dose-dependent decrease in spleen weight and influenza specific IgG and IgM in both lung and serum relative to control animals. In addition, flow cytometric analyses showed a decrease in B cells, but not T cells, in peripheral blood in animals treated with TACI-Ig. However, neither viral clearance nor survival was affected by TACI-Ig treatment. These data demonstrate the expected B cell-specific pharmacological effects of TACI-Ig in influenza challenged C57Bl/6 mice without apparent effect on influenza virus clearance. It is concluded that non-B cell related antiviral competence remains intact during TACI-Ig treatment. PMID- 16684667 TI - Effect of the traditional Korean immunomodulating formulation, Gamguntang (GGT), on experimental thyroiditis model. AB - The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells. PMID- 16684668 TI - In vivo heparan sulfate treatment alters the immune response of normal and LLC bearing mice. AB - Despite the large amount of research dedicated to the understanding and treatment of tumor growth, the majority of cancers continue to lack effective therapeutic options. As in the case of most solid tumors, growth requires evasion of the host immune system. Our previous work using the Lewis Lung Carcinoma (LLC) model of tumor bearing (TB)-mice has shown several tumor-induced immune suppressing effects to be present. These effects include a decreased T-cell proliferative response to Con A and altered cytokine secretion patterns that favor neither a Th1 nor a Th2 response. To address these immune alterations, immune modulating approaches have been a central area of study. Of the many potential immune modulating compounds, we believe promising therapeutic potential lies in the heparin family. Heparan sulfate (HS), in particular, has been shown to increase T cell proliferative response in non TB-mouse splenocytes as well as promotion of a beneficial Th1 response. In this paper, we studied the potential of HS to decrease tumor burden via in vivo treatment of TB-mice. Results showed both normal and TB-mice splenocytes had a dose response change in proliferation as a result of HS treatment. Furthermore, splenocytes from HS treated TB-mice showed a potentially beneficial decrease in basal level proliferation. On gross examination, HS treatment produced a decrease in tumor surface necrosis with a visible (2 +/- 1.8%) surface necrotic area in treated mice as opposed to a (43 +/ 16%) surface necrotic area in untreated mice. HS treatment decreased TB-mice splenomegaly when comparing mice spleen weights in treated (0.3 +/- 0.05 g) vs. untreated (0.14 +/- 0.02 g) groups. These results show a potential role of HS as an immune modulating agent with antitumor properties. PMID- 16684669 TI - Interleukin-6 and oxidative stress in plasma of alloxan-induced diabetic rabbits after pioglitazone treatment. AB - There is evidence that oxidative stress might be implicated in promoting a state of systemic inflammation in diabetic patients. Understanding the role of reactive oxygen species in the inflammatory response in diabetes becomes essential in finding preventive treatments. Pioglitazone is a new oral antidiabetic agent with potent antioxidant and anti-inflammatory properties. The drug is a high affinity ligand of peroxisome proliferator-activated receptor gamma. This receptor seems to be involved in the control of inflammation by modulating the production of inflammatory mediators. In the present study, the changes in some markers of enhanced oxidative stress and in the level of pro-inflammatory interleukin-6 (IL 6) were examined in plasma of diabetic rabbits after 4 and 8 weeks of pioglitazone treatment. Ascorbic acid (AA) concentration and total antioxidant status (TAS) in plasma of diabetic animals were diminished and significantly elevated after pioglitazone treatment (p < 0.05). Protein carbonyl groups (PCG) content and IL-6 concentration were elevated in plasma of diabetic animals and significantly diminished after pioglitazone treatment. The results obtained in the present study confirm the relations of cytokine systems with oxidative stress in plasma of diabetic subjects. They also suggest the antioxidative and antinflammatory properties of pioglitazone. PMID- 16684670 TI - The influence of human neutrophils on N-nitrosodimethylamine (NDMA) synthesis. AB - N-nitrozodimethyloamine (NDMA) is a carcinogenic compound that can be formed in vivo. NDMA is synthesized from precursors-amines and nitrosating agents. Nitrosating agents are formed through the reaction of oxide, reactive oxygen species and nitric oxide (NO). Human neutrophils (PMN) are an important source of the most reactive oxygen species as well as of the nitric oxide. The increase in oxygen metabolism of PMN can lead to the increase nitrosating agent and nitroso forms. Inflammatory process is associated with locally decreased pH that may favor nitrosation reaction. In the present study, we estimated the NDMA synthesis by LPS-stimulated PMN in the presence of the iNOS inhibitor--N-nitro-L-arginine methyl ester (L-NAME). In the nitrosation reaction dimethylamine (DMA) was used as substrat. The viability of the cells was measured by cytometric method. NDMA concentrations the culture media was measured by GCMS method. NO production was estimated by Griess's method. Expression of iNOS was determined by western blotting. Results obtained showed that DMA nitrosation is most effective in pH between 3-4.5. Nonstimulated PMN produced lower concentrations of NO than LPS stimulated cells (1.27 microg/cm3 and 1.57 microg/cm3, respectively). In the culture of nonstimulated PMN supplemented with DMA, there was NDMA (mean--0.99 ng/cm3). In the culture of LPS-stimulated PMN in the presence of DMA, the concentration of NDMA was higher than in the culture of nonstimulated PMN (median -1.45 ng/cm3). In the supernatants of cells incubated without DMA and with DMA, LPS and L-NAME, no NDMA was detected. These results indicate that PMN can be one of sources of nitrosating agents and can play a role in endogenous NDMA synthesis. Stimulation of PMN can lead to the increase of NDMA concentration following the increase of NO production. Different pathological conditions associated with PMN activation as well as the decreased pH may favor endogenous NDMA synthesis. PMID- 16684671 TI - Biological role of the N-formyl peptide receptors. AB - Ligation of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to its specific cell surface receptors triggers different cascades of biochemical events, eventually leading to cellular activation. The formyl peptide receptors (FPRs) are members of the seven-transmembrane, G-protein coupled receptors superfamily, expressed at high levels on polymorphonuclear and mononuclear phagocytes. The main responses elicited upon ligation of formylated peptides, referred to as cellular activation, are those of morphological polarization, locomotion, production of reactive-oxygen species and release of proteolytic enzymes. FPRs have in recent years been shown to be expressed also in several non myelocytic populations, suggesting other unidentified functions for this receptor family, independent of the inflammatory response. Finally, a number of ligands acting as exogenous or host-derived agonists for FPRs, as well as ligands acting as FPRs antagonists, have been described, indicating that these receptors may be differentially modulated by distinct molecules. PMID- 16684672 TI - Antioxidant and anti-inflammatory activities of the plant Andrographis paniculata Nees. AB - In this study, we explored the antioxidant and anti-inflammatory properties of the medicinal herb Andrographis paniculata using in vitro as well as in vivo systems. Methanolic extract of Andrographis paniculata was found to inhibit formation of oxygen derived free radicals such as superoxide (32%) hydroxyl radicals (80%) lipid peroxidation (80%) and nitric oxide (42.8%) in in vitro system. In vivo studies using BALB/c mice models also showed significant inhibition in PMA induced superoxide (32.4%) and nitric oxide (65.3%) formation. Interestingly we also found that, administration of Andrographis paniculata extract produced complete inhibition of carageenan induced inflammation compared with control models. PMID- 16684673 TI - Adjuvant effect of lemnan, pectic polysaccharide of callus culture of Lemna minor L. at oral administration. AB - A pectic polysaccharide, lemnan LMC, was extracted from the callus of duckweed Lemna minor L. and was tested for adjuvant properties at oral administration with protein antigen. Mice were orally immunized thrice with weekly interval with free hen's egg lysozyme or lysozyme with LMC. Lemnan LMC was shown to increase delayed type hypersensitivity and serum antilysozyme IgG responses. LMC was established to increase levels of both serum IgG1 and IgG2a subclasses. The concentration of malondialdehyde and myeloperoxidase activity were found to be higher in the tissue samples obtained from small intestine of mice immunized with mixture of lysozyme/LMC than those immunized with lysozyme only. Thus, lemnan appeared to be useful as the adjuvant for oral immunization. PMID- 16684674 TI - Polimorphonuclear cell-mediated oxidative stress: sink for reactive oxygen species and cell various type damage. AB - Reactive oxygen species (ROS) are produced in animals and humans under physiologic and pathologic conditions. Polymorphonuclear cells (PMNs) and other professional phagocytes are able to generate large amounts of ROS that have not only antimicrobial capacity but are also deleterious to mammalian cells and responsible for many chronic diseases. In particular, ROS produced in large amounts by the massively infiltrating leukocytes in inflammed tissues are believed to constitute a major tissue-destructive force and may contribute significantly to the pathogenesis of several inflammatory diseases. Inflammation can accelerate the development of cancer: in fact, it seems that a part of the predisposition to cancer may be attributed to the oxidants released by the phagocytes at inflammatory site and then to the effects of continuous damage over a life span by ROS. The focus of this study was to investigate the differential capacity of ROS capture and the relative cellular damage degree in gastric, intestinal and fibroblastic cell lines. These various cell types were in vitro used as sink for ROS released by co-cultured fMLP-stimulated human polymorphonuclear cells. Our data demonstrated that cell lines showed a differential capacity of ROS capture correlated to cellular damage, probably due to a different cell susceptibilty to the oxidative challenge produced by stimulated PMNs. PMID- 16684675 TI - Placenta hominis protects osteoporosis in ovariectomized rats. AB - In China, Japan, and Korea, placenta hominis extracts (PHEs) are used clinically for the treatment of osteoporosis. The anti-osteoporotic effect of PHEs was studied. The trabecular bone area and thickness in OVX rats decreased by 50% from those in sham-operated rats; these decreases were completely inhibited by administration of PHEs for 7 weeks. Osteoclast numbers and the osteoblast surface were enhanced in OVX rats, but PHEs had no effect on these phenomena. Serum phosphorus and alkaline phosphatase in OVX rats increased compared to those in sham-operated rats, but the increases were not affected by the administration of PHEs. Thyroxine (T4) level was stimulated in OVX rats. The extracts inhibited the T4 level in the OVX rats. These results strongly suggest that PHEs be effective in preventing the development of bone loss induced by OVX in rats. PMID- 16684676 TI - Iron-radiofrequency synergism in lymphomagenesis. AB - The parenteral iron administration effects on the acceleration of lymphomagenesis by radiofrequency exposure were investigated using an animal model that develops spontaneous lymphomas with ageing. Complementary studies of the in vivo uptake of 59Fe-labeled ferric gluconate and ferric-ATP complex showed differences ob absorption and excretion between both iron compounds. In vitro assays of their effects on calcium cellular uptake using a cell model and tissues homogenates showed a molecular structure-dependence. The current results (mortality, clinical and histopathological examinations) demonstrated a synergism between radiofrequency and ferric gluconate, and the increased risk of radiofrequency exposure when it is simultaneous to parenteral iron administration. PMID- 16684677 TI - Ethical aspects of publishing. PMID- 16684678 TI - Psychiatric symptoms associated with oculogyric crisis: a review of literature for the characterization of antipsychotic-induced episodes. AB - Antipsychotics have been found to induce recurrent psychotic episodes lasting minutes to hours, mostly accompanied by oculogyric crisis (OGC). To characterize this side effect, antipsychotic-induced and postencephalitic OGCs that were reported in the literature were compared to find out common characteristics of OGCs and their associated symptoms. Both postencephalitic and antipsychotic induced OGCs were found to occur late in the day and at regular intervals, and were associated with autonomic symptoms such as profuse sweating, facial flushing, transitory hypertension and difficulty in micturition. They were often associated also with transient psychiatric episodes: visual hallucinations and illusions, auditory hallucinations, delusions, catatonic phenomena, obsessive thoughts and panic attacks. These (OGC) characteristics will be useful in recognizing antipsychotic-induced psychiatric episodes. The associated psychiatric episodes were noted to recur occasionally also without OGC in a few postencephalic cases, and during gradual dose reduction or after a switch to a novel or low-potency antipsychotic in drug-induced cases. These findings suggest that episodes with the OGC characteristics but without OGC per se, may be less severe reactions to antipsychotic medication than those with OGC, and may represent manifestations of subclinical OGC. PMID- 16684679 TI - Effect of olanzapine treatment on platelet glutamine synthetase-like protein and glutamate dehydrogenase immunoreactivity in schizophrenia. AB - According to contemporary views, the glutamatergic system is implicated in the pathogenesis of schizophrenia, and atypical neuroleptics exert their effects (at least partially) through the glutamatergic system. Immunoreactive glutamate metabolising enzymes, such as glutamine synthetase-like protein (GSLP) and two glutamate dehydrogenase isoenzymes (GDH), have been discovered in human platelets. The amount of GSLP in the platelets of 40 chronic patients with schizophrenia was found to be significantly higher than in 33 controls (consistent with our previous finding of increased amounts of GSLP in the prefrontal cortex of chronic schizophrenia patients). Moreover, survival analysis of the group of patients treated with olanzapine for 28 weeks showed that the larger amount of GSLP measured in platelets before treatment, the shorter the treatment time needed to achieve a positive clinical response (defined a priori as > or = 20% reduction in PANSS total score from the initial level before the treatment). Hence, GSLP level may serve as a predictor of the treatment duration to achieve a positive outcome with olanzapine. Both GSLP and GDH were found significantly changed in the course of treatment; hence, treatment with olanzapine influences the amounts of glutamate-metabolising enzymes in the platelets of chronic schizophrenia patients. PMID- 16684680 TI - The influence of concomitant antidepressant medication on safety, tolerability and clinical effectiveness of electroconvulsive therapy. AB - BACKGROUND: A major problem in the treatment of severe depression is the onset latency until clinical improvement. So far, electroconvulsive therapy (ECT) is the most effective somatic treatment of depression. This holds especially true for treatment-refractory disturbances. However, not all patients respond to conventional unilateral ECT. In certain cases, subsequent clinical response can be achieved using bilateral or high-dose unilateral ECT. Also, a concomitant pharmacotherapy can be utilized to augment therapeutic effectiveness. Surprisingly, data in this field are widely lacking and only few studies showed advantages of an ECT/tricyclic antidepressant combination. METHOD: We retrospectively evaluated 5482 treatments in 455 patients to investigate possible therapeutic advantages in combination therapies versus ECT monotherapy. Main outcome criteria were clinical effectiveness and tolerability. Moreover, treatment modalities and ictal neurophysiological parameters that might influence treatment outcome were analysed. RESULTS: A total of 18.2% of our treatments were ECT monotherapy, 8.87% were done with one antidepressant. Seizure duration was unaffected by the most antidepressants. SSRI caused a lengthened seizure activity. Postictal suppression was lower in mirtazapine and higher in SSRI and SNRI treated patients. A significant enhancement of therapeutic effectiveness could be seen in the patient group receiving tricyclics, SSRI or mirtazapine. Serious adverse events were not recorded. CONCLUSION: Our study supports the hypothesis that mirtazapine can be used to enhance the therapeutic effectiveness of ECT. Controlled studies are necessary to further investigate the possible advantages of ECT and pharmacotherapy combinations, especially the use of modern dually acting antidepressants which have proven their good effectiveness in treatment-resistant depression. PMID- 16684681 TI - P300 differences exist between Tourette's syndrome with and without attention deficiency and hyperactivity disorder in children. AB - OBJECTIVE: To study the characteristics of P300 in Tourette's syndrome (TS) with and without attention deficiency and hyperactivity disorder (ADHD). METHOD: Auditory evoked P300 were recorded in 19 TS only (TS-ADHD) children, 15 TS with ADHD (TS + ADHD) children and 20 unaffected control subjects, and their waveforms, amplitudes, latencies and topographies were compared at Fz, Cz, C3, C4 and Pz. RESULTS: The TS + ADHD group showed shorter latencies than control subjects at all electrode sites (P<0.05 or 0.01), and the TS-ADHD group at CZ and PZ (P<0.05); however, there was no significant difference between control subjects and the TS-ADHD group. The TS-ADHD group showed smaller amplitudes than the control group at all electrode sites (P<0.05), and the TS + ADHD group at Cz (P<0.05); however, there were no significant differences between control subjects and the TS + ADHD group. There was no significant difference in the prevalence of abnormal waveforms between the control, TS, TS-ADHD and TS + ADHD groups, but there were significant differences in the variability of localization of P300 between the control and the TS group (P=0.003), control and TS + ADHD groups (P=0.000), and the TS-ADHD and TS + ADHD groups (P=0.039). P300 in the TS + ADHD group tended to spread out to the left and that of the TS-ADHD group tended to spread out to the right. CONCLUSIONS: P300 differences exist between TS-ADHD and TS + ADHD in children. These suggested that establishment different development defects or delay of communications between different structures rather than a delay in maturation of the structures themselves may be involved in TS + ADHD and TS-ADHD children and ADHD symptoms in TS patients are likely a trait rather than adventitious or acquired within the TS syndrome. PMID- 16684682 TI - Three-dimensional models of neurotransmitter transporters and their interactions with cocaine and S-citalopram. AB - Drugs that act on the human serotonin transporter (hSERT), human dopamine transporter (hDAT) and human noradrenaline transporter (hNET) are important in antidepressant treatment and well known in drug abuse. The investigation of their molecular mechanisms of action is very useful for designing new ligands with a therapeutic potential. The detailed three-dimensional molecular structure of any monoamine transporter is not known, but the three-dimensional electron density projection map of Escherichia coli Na+/H+ antiporter (NhaA) has provided structural basis for constructing models of such transporters using molecular modelling techniques. Three-dimensional models of these drug targets give insight into their structure, mechanisms and drug interactions. In these molecular modelling studies, an Escherichia coli NhaA model was first constructed based on its three-dimensional electron density projection map and experimental studies on NhaA and the Escherichia coli lactose permease symporter (Lac permease). Then three-dimensional models of the neurotransmitter transporters hDAT, hSERT and hNET were constructed based on the NhaA model and studies of ligand binding to mutated dopamine transporter (DAT) and serotonin transporter (SERT). The structural properties of these neurotransmitter transporter models have been examined, and their interactions with cocaine and S-citalopram have been investigated. PMID- 16684683 TI - The problem of delusional ugliness: is it really body dysmorphic disorder? AB - Currently, the DSM-IV allows individuals with dysmorphic delusions to be diagnosed with either delusional body dysmorphic disorder (BDD) and delusional disorder, somatic type (DDST). However, given the growing acceptance of a dimensional perspective in psychopathology, it is conceivable that future editions of the DSM may recommend the exclusion of the diagnosis of DDST whenever isolated dysmorphic delusions are present, arguing that the latter should be considered no more than a symptom of BDD. But is the concept of DDST condemned to extinction in favor of that of delusional BDD? While some studies suggest that non-delusional and delusional BDD/DDST may be indistinguishable from the clinical, neuroanatomical, and therapeutic perspectives, several facts support the utility of the DDST concept. Firstly, DDST is a wider construct than delusional BDD. Secondly, it is unclear whether DDST in general (as opposed to delusional BDD) belongs to the obsessive-compulsive spectrum. Thirdly, differential pharmacological response may not be an adequate criteria for blending non-delusional and delusional BDD/DDST. Fourthly, "delusional" BDD may not be delusional at all. Finally, there is more about delusion than just an "extreme" conviction. Future studies are urgently needed in order to substantiate our judgement regarding the existence of diagnostic limits between delusional BDD and DDST with dysmorphic delusions. PMID- 16684684 TI - Abdominal cutaneous nerve entrapment syndrome (ACNES) in a patient with a pain syndrome previously assumed to be of psychiatric origin. AB - A 21-year-old female patient with chronic abdominal pain was referred to a psychiatric outpatient clinic after gastroenterological and gynaecological pathogeneses had been excluded and a treatment with an antidepressant had had no beneficial effects. The mental state examination, however, revealed no psychopathology whatsoever. The patient was injected with a local anaesthetic loco dolenti which resulted in immediate pain relief. She was diagnosed with abdominal cutaneous nerve entrapment syndrome (ACNES); no psychiatric diagnosis was given. PMID- 16684685 TI - The treatment of recurring auditory hallucinations in schizophrenia with rTMS. AB - BACKGROUND: Auditory hallucinations are a common and disabling problem for many patients with schizophrenia and often fail to respond to optimal antipsychotic therapy. Repetitive transcranial magnetic stimulation (rTMS) has recently been trialled as an alternative treatment option for these patients. These studies have generally been positive, but treatment has only been provided for short periods of time and little is known about the longer-term impact of TMS on the course of hallucinations. METHOD: We describe two cases in which rTMS was provided to patients upon relapse of hallucinations following initial successful rTMS treatment in a clinical trial. RESULTS: A repeat course of rTMS resulted in a marked improvement in the symptoms experienced by these two patients. CONCLUSIONS: rTMS appears to have potential as a long-term treatment for patients with auditory hallucinations, but requires ongoing systematic investigation. PMID- 16684686 TI - Aripiprazole in patients with Tourette syndrome. AB - The treatment of the Gilles de la Tourette syndrome (GTS) is often challenging. One reason for this is the high neuropsychiatric cormorbidity in terms of ADHD or obsessive-compulsive symptoms. Dopaminergic modulation e.g. with antidopaminergic medication is an important part of the medical therapy aimed at motor and vocal tics. We report recent experiences with treatment with aripiprazole, a novel antipsychotic agent, which not only improved motor and vocal tics but also ameliorated some behavioural symptoms of the GTS cluster. Furthermore, we discuss possible pharmacological mechanisms for the observed effects. PMID- 16684687 TI - Addressing the limitations of the CATIE study. PMID- 16684688 TI - Short-term lower leg growth in asthmatic children treated with inhaled beta2 agonists. AB - BACKGROUND: Knemometry studies of growth suppressive effects of inhaled glucocorticoids in children with asthma usually allow participating children to use concomitant inhaled beta2-agonists. Systemic beta2-agonists, however, have been found to suppress growth hormone secretion and this has caused concern about a possible confounding effect of inhaled beta2-agonists on results of growth studies of exogenous glucocorticoids. AIM: The study evaluated whether inhaled salbutamol adversely affects short-term growth. SUBJECTS AND METHODS: Fifteen children aged 6-12 years with mild asthma were enrolled in a single-blind, randomized crossover study with two 2-week treatment periods and a 1-week run-in. During the active period treatment dry powder salbutamol (Ventoline Diskhaler) 200 microg was inhaled three times a day. During the comparative period no treatment was given. Knemometry of the right lower leg was performed on the first and the last day of each period. RESULTS: Mean lower leg growth rates (SEM) during no-treatment and salbutamol periods were 0.35 (0.06) and 0.42 (0.07) mm per week, respectively (P = 0.35, t = -0.98, 95% CI: 0.25-0.93 mm per week). CONCLUSIONS: Inhaled salbutamol 200 microg three times daily does not suppress short-term growth in asthmatic children. Inhaled beta2-agonists in equipotent doses and regimens can be safely used in short-term knemometric growth studies of exogenous glucocorticoids without any risk of confounding the results. PMID- 16684689 TI - Alu insertion polymorphisms in Native Americans and related Asian populations. AB - BACKGROUND: Alu insertions provide useful markers for the study of inter population affinities and historical processes, but data on these systems are not numerous in Native Americans and related populations. AIM: The study aimed to answer the following questions: (a) do the population relationships found agree with ethnic, historical and geographical data? and (b) what can heterozygote levels and associated results inform us about the events that led to the colonization of the New World? SUBJECTS AND METHODS: Twelve Alu insertion polymorphisms were studied in 330 individuals belonging to South American Native, Siberian and Mongolian populations. These data were integrated with those from 526 persons, to ascertain the relationships between Asian, Northern Arctic and Amerindian populations. RESULTS: A decreasing trend concerning heterozygosities and amount of gene flow was observed in the three sets, in the order indicated above. Most results indicated the validity of these subdivisions. However, no clear structure could be observed within South American Natives, indicating the importance of dispersive (genetic drift, founder effects) factors in their differentiation. CONCLUSIONS: The answers to the questions are: (a) yes; and (b) an initial moderate bottleneck, intensified by more recent historical events (isolation and inbreeding), can explain the current Amerindian pattern of diversity. PMID- 16684690 TI - Spatial parameters of walking gait and footedness. AB - OBJECTIVE: The present study was undertaken to assess whether footedness has effects on selected spatial and angular parameters of able-bodied gait by evaluating footprints of young adults. SUBJECTS AND METHODS: A total of 112 males and 93 females were selected from among students and staff members of the University of Malawi using a simple random sampling method. Footedness of subjects was assessed by the Waterloo Footedness Questionnaire Revised. Gait at natural speed was recorded using the footprint method. The following spatial parameters of gait were derived from the inked footprint sequences of subjects: step and stride lengths, gait angle and base of gait. The anthropometric measurements taken were weight, height, leg and foot length, foot breadth, shoulder width, and hip and waist circumferences. RESULTS: The prevalence of right-, left- and mix-footedness in the whole sample of young Malawian adults was 81%, 8.3% and 10.7%, respectively. One-way analysis of variance did not reveal a statistically significant difference between footedness categories in the mean values of anthropometric measurements (p > 0.05 for all variables). Gender differences in step and stride length values were not statistically significant. Correction of these variables for stature did not change the trend. Males had significantly broader steps than females. Normalized values of base of gait had similar gender difference. The group means of step length and normalized step length of the right and left feet were similar, for males and females. There was a significant side difference in the gait angle in both gender groups of volunteers with higher mean values on the left side compared to the right one (t = 2.64, p < 0.05 for males, and t = 2.78, p < 0.05 for females). One-way analysis of variance did not demonstrate significant difference between footedness categories in the mean values of step length, gait angle, bilateral differences in step length and gait angle, stride length, gait base and normalized gait variables of male and female volunteers (p > 0.05 for all variables). CONCLUSIONS: The present study demonstrated that footedness does not affect spatial and angular parameters of walking gait. PMID- 16684692 TI - Statistical validation of air-displacement plethysmography for body composition assessment in children. AB - BACKGROUND: Body composition assessment of children has been hindered by the absence of a safe, quick, and easily tolerated gold standard technique. Existing validation studies of air-displacement plethysmography (ADP) have been based on small, narrowly defined samples, using simple linear regression or Bland-Altman analyses. AIM: Correlations within a multitrait-multimethod matrix (MTMM) and factor analytic methodologies were used to evaluate ADP as a valid and reliable body composition technique for children. SUBJECTS AND METHODS: Fat mass (FM), fat free mass (FFM) and per cent body fat (%BF) were measured in 139 children, 7-10 years old, by ADP, dual energy X-ray absorptiometry (DXA) and anthropometry (ANTH). MTMM and factor analysis were used to compare assessment techniques. RESULTS: Reliability estimates were lower for ADP than for either ANTH or DXA. Convergent and discriminant correlations between ADP and ANTH or DXA were high for identical as well as non-identical measures. Two body composition factors (Fatness, Leanness) and two technique-related factors (Bod Pod, Anthropometry) were identified. CONCLUSION: ADP offers a valid and reliable means of assessing body composition in children but does not perform as well as ANTH or DXA. MTMM and factor analytic methodologies offer an effective alternative to assessing body composition. PMID- 16684691 TI - Familial gene analysis for Wilson disease from north-west Indian patients. AB - BACKGROUND: A number of polymerase chain reaction (PCR) based techniques like single-strand conformational polymorphism (SSCP), amplification refractory mutation system (ARMS)-PCR, semi-nested PCR and dinucleotide-repeat marker analysis have been used in the diagnosis of asymptomatic Wilson disease (WD) patients and the carrier status of WD families. In the present study, we explore the utility of mutation analysis in combination with restriction fragment length polymorphism (RFLP) in the genetic diagnosis of WD. AIM: The study was planned to provide a molecular diagnostic tool for the diagnosis of asymptomatic WD patients as well as assessment of the carrier status of WD families. SUBJECTS AND METHODS: Four WD families were analyzed in which parents and siblings showed no clinical manifestations or biochemical abnormalities. The parents of the WD patients were not consanguineous and had no family history of WD. Mutations in ATP7B were characterized using SSCP and DNA sequencing. Further, RFLP was developed for the analysis of characterized mutations in ATP7B from the WD patients, their parents and siblings. RESULTS: Three mutations, Q1256R, A1003T and I1102T, were characterized in WD patients, using SSCP and DNA sequencing. These mutations created/deleted restriction sites for AccII, Bsh1236I and EcoRI restriction enzymes respectively. Despite having no clinical manifestations nor any significant alteration in biochemical investigations, eight carriers and one asymptomatic WD patient were diagnosed in 13 members of the patients families by restriction digestion analysis. CONCLUSION: The report demonstrates that mutation analysis in combination with RFLP is useful for diagnosis of asymptomatic WD patients as well as for the elucidation of the carrier status of the patients' family members. It is noteworthy that this combinational methodology provides a positive diagnosis in siblings/parents where biochemical parameters are ambiguous. PMID- 16684694 TI - Short-term growth in healthy infants, schoolchildren and adolescent girls. AB - BACKGROUND: There is agreement amongst most investigators now on the fact that growth, when measured over short periods, is an irregular and non-linear process. However, many aspects related to this process still remain controversial, for example, the continuity or discontinuity of the daily changes and the degree of existence of age differences in growth patterns. AIM: In this study we analyse and compare growth in height based on daily measurements in three samples of healthy children: Eight infants (I), 16 schoolchildren (S) and 10 adolescent girls during their pubertal growth spurt (A). SUBJECTS AND METHODS: Standing height or supine length were measured five times a week during 128 days, with Harpenden instruments and standardized techniques. A jump-preserving smoothing technique, based on the technical error of measurement (TEM) with a hard rejection criterion was used to analyse our data. TEM varied between 0.08 and 0.20 cm. RESULTS: Three types of events were found: (1) stasis (changes in smoothed values indistinguishable from zero over 7 or more consecutive days), (2) steep changes (any daily increment greater than 0.3 cm over 1 day), and (3) continuous growth (small, and continued increments over time). The three types were present in all infants, adolescents and schoolchildren, with the exception of four schoolchildren who did not show steep changes. In the three samples, steep changes accounted for 32.7% (r = 15.3-54.5%) of the total growth during the study period. Mean numbers of 1-day steep changes were 4.4, 1.4 and 2.1 for samples I, S and A, respectively. Mean numbers of stasis periods were 3.0, 2.9 and 4.3 for samples I, S and A, respectively. The longest stasis period observed was 28 consecutive days in one infant and one school child. Mean number of steep changes per child was significantly greater in infants. CONCLUSION: Our findings support the concept of growth as a non-linear and irregular process with three phenomena: Stasis, steep changes and continuous growth present in all ages, including infancy, school age and adolescence. PMID- 16684693 TI - Ethnicity and lipoprotein(a) polymorphism in Native Mexican populations. AB - BACKGROUND: Lp(a) is a lipoparticle of unknown function mainly present in primates and humans. It consists of a low-density lipoprotein and apo(a), a polymorphic glycoprotein. Apo(a) shares sequence homology and fibrin binding with plasminogen, inhibiting its fibrinolytic properties. Lp(a) is considered a link between atherosclerosis and thrombosis. Marked inter-ethnic differences in Lp(a) concentration related to the genetic polymorphism of apo(a) have been reported in several populations. AIM: The study examined the structural and functional features of Lp(a) in three Native Mexican populations (Mayos, Mazahuas and Mayas) and in Mestizo subjects. METHODS: We determined the plasma concentration of Lp(a) by immunonephelometry, apo(a) isoforms by Western blot, Lp(a) fibrin binding by immuno-enzymatic assay and short tandem repeat (STR) polymorphic marker genetic analysis by capillary electrophoresis. RESULTS: Mestizos presented the less skewed distribution and the highest median Lp(a) concentration (13.25 mg dL(-1)) relative to Mazahuas (8.2 mg dL(-1)), Mayas (8.25 mg dL(-1)) and Mayos (6.5 mg dL(-1)). Phenotype distribution was different in Mayas and Mazahuas as compared with the Mestizo group. The higher Lp(a) fibrin-binding capacity was found in the Maya population. There was an inverse relationship between the size of apo(a) polymorphs and both Lp(a) levels and Lp(a) fibrin binding. CONCLUSION: There is evidence of significative differences in Lp(a) plasma concentration and phenotype distribution in the Native Mexican and the Mestizo group. PMID- 16684695 TI - An investigation of sequence deletions of amelogenin (AMELY), a Y-chromosome locus commonly used for gender determination. AB - BACKGROUND: Accurate gender determination is crucial in many scientific disciplines, but especially so in prenatal diagnosis of X-linked diseases and forensic investigations. Today, molecular techniques, especially typing for a length variation in the X-Y homologous amelogenin gene (AMELX and AMELY), are used for gender assignation. This amelogenin test is an integral part of most PCR multiplex kits that are used for DNA profiling, but in 1998 there was a report of two normal males being typed as female with this test. Subsequently, a small number of amelogenin negative (or AMELY null) males have been reported in various populations but little data are available characterising these deletions. AIMS: The study aims to determine the size of the deletion in five AMELY null males by typing DNA samples for markers surrounding this gender-determining locus. The possible relationships among the AMELY null samples are examined through analysis of their deletion size and associated Y-chromosome microsatellite haplotypes. We also attempt to determine the frequency of AMELY negative males in Australia. SUBJECTS AND METHODS: DNA samples from five AMELY null males, from different geographical regions, were made available for this study. The samples were typed for eight sites, all located on the short arm of the Y chromosome, using PCR and gel electrophoresis. Eleven Y-chromosome specific microsatellites were also typed on each sample in order to generate haplotypes for phylogenetic analysis. A questionnaire was sent to all Australian forensic centres requesting information on the frequency of AMELY negative males observed in their laboratories. RESULTS: Two different sized deletions were seen in the five AMELY null samples. One deletion (in two samples) has a size of between 304 and 731 kbp, whereas the other (in three samples) ranges between 712 and 1001 kbp. Y-microsatellite haplotypes indicate that the smaller deletion is probably identical in the two samples, but this is not the case with the larger deletion. The frequency of AMELY negative is rare in Australia, with an overall frequency of 0.02%. CONCLUSION: Comparisons of both deletion size and haplotypes with published data suggest that most AMELY nulls are the result of independent evolutionary events, even in those populations where the frequency is relatively high. Although AMELY null males are extremely rare in most populations, typing an additional gender determining locus should be considered in forensic investigations where the reference sample is of unknown gender. PMID- 16684696 TI - Eye measurements in 7-year-old black South African children. AB - BACKGROUND: Ethnic variation often renders anthropometric reference values obtained in one population unsuitable for use in others. Fetal alcohol syndrome (FAS) diagnosis relies in part on the evaluation of certain anthropometric facial features. Measurements of these facial features in South African children have not been compared with measurements obtained in other populations. AIM: The study seeks to determine the suitability of reference values obtained in other populations for the diagnosis of the facial phenotype associated with FAS in South African children. PARTICIPANTS AND METHODS: Palpebral fissure length (PFL), interpupillary distance (IPD), inner canthal distance (ICD) and outer canthal distance (OCD) measured in a group of black South African children were obtained from digital photographs using stereophotogrammetry, and compared with measurements published for other populations. The study population comprised 17 7 year-old boys and 17 7-year-old girls. The precision and reliability of measurements were examined with reference to published data. RESULTS: Eye distance measurements in the study population do not consistently reflect those in any one other population for which such measurements have been published. CONCLUSION: Population-specific reference values of eye distance measurements should be established for South African children. PMID- 16684697 TI - Overweight and obesity in affluent school children of Punjab. AB - In the present cross-sectional study, an attempt has been made to report the prevalence of overweight and obesity in school-going children of the affluent families of Punjab. A total of 1000 children (490 boys and 510 girls) were measured for height and weight. Overweight and obesity were assessed using age and sex-specific body mass index (BMI) cut-off points. In the present study, 12.24% boys and 14.31% girls were overweight, and 5.92% boys and 6.27% girls were obese. The prevalence of overweight and obesity among the affluent school children of Punjab was as high or higher as in some developed countries. PMID- 16684701 TI - Identification of binaural integration deficits in children with the Competing Words Subtest: standard score versus interaural asymmetry. AB - The Competing Words Subtest is a commonly used dichotic listening test for assessing binaural integration in children suspected of having an auditory processing disorder. In 124 children, standard scores from the subtest suggested a binaural integration deficit in 23% of the children tested. Because standard scores are derived from the combined scores of both ears during the test, children with normal performance in one ear and weak performance in the other ear may be overlooked. For these children, a measure of interaural asymmetry may be a more sensitive indicator of a binaural integration deficit. When an age appropriate criterion for interaural asymmetry from the Competing Words Subtest was used, the incidence of a binaural integration deficit increased to 51% of the children tested. Four typical patterns of dichotic listening performance were identified based on results from the two scoring techniques. PMID- 16684702 TI - Validity and accuracy of electric response audiometry using the auditory steady state response: evaluation in an empirical design. AB - The validity and accuracy of the application of the auditory steady-state response (ASSR) to electric response audiometry (ERA) was tested further in a study permitting subjects to be their own controls for hearing loss. Simulated sensorineural hearing loss (SSHL) of complex configuration and varying degrees was effected using filtered masking noise. Thresholds estimated via ASSR-ERA were compared to those measured via conventional pure-tone audiometry. Further, the slow vertex potential N1-P2 was recorded to permit a comparison with an evoked response test of common content validity and known accuracy. Results in a homogeneous subject sample demonstrated strong interest correlation and agreement within 10 dB at 1000 to 4000 Hz (on average), but not at 500 Hz. The configurations determined by ASSR-ERA followed behavioral audiometric patterns well, except for the mildest degree of SSHL tested. Consequently, limitations of ERA remain, although ASSR-ERA appears to be quite valid overall and promises (justifiably) broad clinical applicability. PMID- 16684703 TI - Differentiation of migrainous positional vertigo (MPV) from horizontal canal benign paroxysmal positional vertigo (HC-BPPV). AB - This article presents an approach to differentiation of migrainous positional vertigo (MPV) from horizontal canal benign paroxysmal positional vertigo (HC BPPV). Such an approach is essential because of the difference in intervention between the two disorders in question. Results from evaluation of the case study presented here revealed a persistent ageotropic positional nystagmus consistent with MPV or a cupulolithiasis variant of HC-BPPV. The patient was treated with liberatory maneuvers to remove possible otoconial debris from the horizontal canal in an attempt, in turn, to provide further diagnostic information. There was no change in symptoms following treatment for HC-BPPV. This case was diagnosed subsequently as MPV, and the patient was referred for medical intervention. Treatment has been successful for 22 months. Incorporation of HC BPPV treatment, therefore, may provide useful information in the differential diagnosis of MPV and the cupulolithiasis variant of HC-BPPV. PMID- 16684704 TI - A Swedish version of the Hearing In Noise Test (HINT) for measurement of speech recognition. AB - A Swedish Hearing In Noise Test (HINT), consisting of everyday sentences to be used in an adaptive procedure to estimate the speech recognition thresholds in noise and quiet, has been developed. The material consists of 250 sentences, with a length of five to nine syllables, normalized for naturalness, difficulty and reliability. The sentences were recorded with a female speaker. From the sentences, 25 phonemically balanced lists were created. All lists fluctuate less than 1 dB of the overall mean. The standard deviation of the test-retest difference is 0.94 dB when testing with one list, and decreases to 0.68 dB and 0.56 dB for two and three lists, respectively. The average speech recognition thresholds in noise for the Swedish sentences were -3.0 dB signal/noise ratio (SD=1.1 dB). The present study has resulted in a well-defined and internationally comparable set of sentences, which can be used in Swedish audiological rehabilitation and research to measure speech recognition in noise and quiet. PMID- 16684705 TI - Measuring outcomes of a communication program for older people with hearing impairment using the International Outcome Inventory. AB - The main objective of this study was to describe the outcomes of a communication education program for older people with hearing impairment using the International Outcome Inventory--Alternative Interventions (IOI-AI) and the version for significant others (IOI-AI-SO). Ninety-six people aged 58 to 94 years participated in an interactive group education program for two hours per week for five weeks. The IOI-AI was administered at one to two weeks after the last educational session and 29 significant others also completed the IOI-AI-SO at this time. Overall, positive results were obtained using both questionnaires, and satisfaction with the program was particularly high. Findings also compared favourably to reports of outcomes for other audiological interventions (i.e., another communication training program and hearing aid fitting). Principal components analysis of the IOI-AI revealed a somewhat different factor structure than the original IOI-HA. The two versions of the IOI applied in this study are recommended as simple and effective measures of the outcomes of alternative interventions. PMID- 16684706 TI - A study of hearing changes among military conscripts in the Swedish Army. AB - The aim of the study was to investigate the incidence and the relative risk of significant threshold shift (STS, >or=15 dB deterioration at any ear and audiometric frequency) during primary military service (7-9 months), and to investigate whether subjects with an initial slight hearing loss (thresholds>or=25 dB HL at any audiometric frequency and ear) were under increased risk. The investigation was made as a prospective audiometric study and included 747 men. An age-matched group of 138 individuals served as an unexposed control group, whose incidence of STS was 2.9%. In the exposed group the incidence was 7.9% and the relative risk 2.7 risk ratio (RR). In the subgroup of 95 persons, who already at reporting-for-training had a mild hearing loss, the incidence was 17%. The relative risk for STS in this group compared to the control group was 6.8 (RR), and compared to those with normal hearing at reporting was 3.1 (RR). In spite of hearing conservation efforts, hearing deterioration still occurs, above all in the artillery. Those who already at reporting-for-training had a mild hearing loss were at higher risk for STS compared to those with initially normal hearing. PMID- 16684707 TI - Factors that influence the use of assistance technologies by older adults who have a hearing loss. AB - The objective of this study was to describe and better understand the factors that influence the use of assistance technologies by older adults who have a hearing loss. We were interested in adopting a methodological approach that would provide an in-depth account of individual experiences related to the use of these technologies. A qualitative research design was therefore selected. Audio recorded interviews were conducted with ten individuals who were 65 years of age or older and were current successful assistance technology users. Thematic analysis was used to draw meaning from the interview transcripts. The results suggest that successful use of these assistance technologies involves the recognition of hearing difficulties, an awareness that technological solutions exist, consultation for and acquisition of devices, and adapting to device use and modified behaviour. These four landmarks seem to be crucial stages when people either move toward successful assistance technology use or are discouraged from assistance technology use. Based on these results, a representative model of assistance technology awareness, acquisition and utilization is proposed. PMID- 16684708 TI - Effect of natural analogues of trans-resveratrol on cytochromes P4501A2 and 2E1 catalytic activities. AB - The aim was to assess the inhibitory effect of a series of naturally occurring trans-resveratrol analogues on cytochromes P450, namely CYP1A2 and CYP2E1, in vitro in order to analyse any structure-activity relationships. 3,5-Dimethoxy-4' hydroxy-trans-stilbene (pterostilbene), 3,4',5-trimethoxy-trans-stilbene (TMS), 3,4'-dihydroxy-5-methoxy-trans-stilbene (3,4'-DH-5-MS) and 3,5-dihydroxy-4' methoxy-trans-stilbene (3,5-DH-4'-MS) inhibited the activity of CYP1A2, with K(i) = 0.39, 0.79, 0.94 and 1.04 microM, respectively. Piceatannol (3,3',4,5' tetrahydroxy-trans-stilbene) was the least potent inhibitor of CYP1A2 with a K(i) = 9.67 microM. Piceatannol and TMS in the concentration range 1-100 microM did not inhibit CYP2E1 activity. The activity of this enzyme likewise was not significantly influenced by pterostilbene and 3,5-DH-4'-MS with IC(50) > 100 microM, whereas 3,4'-DH-5-MS appeared to be a moderately potent, competitive inhibitor of CYP2E1 (K(i) = 42.6 microM). Structure-activity relationship analysis leads to the conclusion that the substitution of hydroxy groups of resveratrol with methoxy groups increases the inhibition of CYP1A2, yet the number and position of methylation are not essential. However, the 4'-hydroxy group in trans-resveratrol and its analogues may play an important role in the interaction with a binding site of CYP2E1. PMID- 16684709 TI - Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5. AB - To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL(int) estimates using CYP3A5 compared with CYP3A4 including midazolam (CL(int) = 3.4 versus 3.3 microl min(-1) pmol( 1)). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL(int) = 10.3 versus 2.4 microl min(-1) pmol(-1)), and ritonavir (CL(int) = 0.76 versus 0.47 microl min(-1) pmol(-1)). The majority of compounds studied showed a greater inhibitory potential (IC(50)) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4. PMID- 16684710 TI - Species differences in hydrolase activities toward OT-7100 responsible for different bioavailability in rats, dogs, monkeys and humans. AB - OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a] pyrimidine) is an amide moiety-bearing pyrazolopyrimidine derivative with a potential analgesic effect. To determine the factors responsible for observed species differences in the bioavailability of this drug, human and experimental animal samples were used to investigate in vitro microsomal and cytosolic hydrolase activities in the liver and small intestine vis-a-vis the pharmacokinetics of OT-7100. The AUC(0-t) values of OT-7100 after oral administration in rats, dogs and monkeys were 0.163, 0.0383 and 0.00147 microg h ml(-1) divided by mg kg(-1), respectively. The bioavailabilities of OT-7100 after oral administration in rats, dogs and monkeys were 36, 17 and 0.3%, respectively. The plasma concentration-time profiles of intravenously administrated OT-7100 in rats, dogs and monkeys were similar. The hydrolase activities toward OT-7100 in liver microsomes or cytosol were approximately similar in rats, dogs, monkeys and humans. In contrast, hydrolase activities of small intestinal microsomes from monkeys were higher (36.1 ng mg protein(-1) min(-1)) than those of rats, dogs and humans (5.4, 1.4 and 4.3 ng mg protein(-1) min(-1), respectively). These results suggest that the primary factor influencing first-pass metabolism for the OT-7100 is enzymatic hydrolysis in the small intestine. This information provides an important index for extrapolating the pharmacokinetics of drugs in humans using studies on monkeys. PMID- 16684711 TI - Aldehyde oxidase and its contribution to the metabolism of a structurally novel, functionally selective GABAA alpha5-subtype inverse agonist. AB - (3-Tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5 ylmethoxy)pyrazolo[1,5-d] [1,2,4]triazine was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5-containing receptors, which enhances performance in animal models of cognition. The routes of metabolism of this compound in rat, dog, rhesus monkey and human in vitro systems, and in vivo in rat, dog and rhesus monkey have been characterized. The current study demonstrates that both a cytosolic oxidative reaction and cytochrome P450 play important roles in the metabolism of the compound. Chemical inhibition studies showed the oxidation in human cytosol to be catalysed predominantly by aldehyde oxidase rather than the related enzyme, xanthine oxidase. The aldehyde oxidase-mediated metabolites were present in vitro and in vivo in both rat and rhesus monkey, and also in vitro in man. They were absent both in vitro and in vivo in dog. PMID- 16684712 TI - Marked species differences in the bioavailability of midazolam in cynomolgus monkeys and humans. AB - The bioavailability (F) of midazolam in cynomolgus monkeys (0.02) was markedly lower than that in humans (0.24-0.46) and the reason for this difference in F between the two species was investigated. Based on the area under the plasma concentration-time curve after intravenous and intraportal infusion to cynomolgus monkeys, the hepatic availability (F(h)) was estimated as 0.66. The fraction of dose absorbed (F(a)) estimated from the single-pass intestinal perfusion method was 1.0 in cynomolgus monkeys. The intestinal availability (F(g) = F/F(a)/F(h)) was calculated as 0.03 in cynomolgus monkeys. Since the F(a) of midazolam has been reported to be almost 1.0 in humans, F(h) and F(g) were calculated as 0.33 0.76 and 0.46-1.00 when the reference values for hepatic blood flow (1026-1530 ml h(-1) kg(-1)) were used. In conclusion, the main reason for low F in cynomolgus monkeys was the markedly higher first-pass intestinal metabolism seen in cynomolgus monkeys compared with humans. PMID- 16684713 TI - Pharmacokinetics and clearance processes of UK-279,276 (rNIF) in rat and dog: comparison with human data. AB - UK-279,276, or recombinant neutrophil inhibitory factor (rNIF), is a glycoprotein that binds to the human CD11b receptor (IC(50) = <0.5 nM) and inhibits neutrophil binding to vascular endothelial cells. In dogs, the pharmacokinetics of UK 279,276 are non-linear with clearance slowing as dose increases (apparent clearance 0.06 ml min(-1) kg(-1) at 0.l mg kg(-1) decreasing to 0.02 ml min(-1) kg(-1) at 2 mg kg(-1)). The observations are in keeping with human pharmacokinetic data. In rats, clearance is linear (0.07-0.13 ml min(-1) kg(-1)) across a similar dose range (0.006-2.25 mg kg(-1)) compared with dogs and humans. These data indicate that two clearance processes act on UK-279,276 in dogs and humans, but only one clearance process occurs in rats. A non-saturable, low affinity/high-capacity clearance process is present in all animal species studied as well as in human, and it is believed to be hepatic uptake mediated by the asialoglycoprotein receptor. A second, saturable, clearance process is present in dogs and humans that is not apparent in the rat. This high-affinity/low-capacity process is thought to be mediated by interaction with the CD11b receptor, which is the pharmacological target for this molecule. This hypothesis is supported by the marked species differences seen for the affinity of CD11b for UK-279,276, with dogs and humans having high-affinity (IC(50) = <0.5 nM), whilst the rat affinity is low (IC(50) = 134 nM). PMID- 16684714 TI - Dichotomous effect of aerosolized hyaluronan in a hamster model of endotoxin induced lung injury. AB - Inhalation of aerosolized low-molecular-weight (150-kDa) hyaluronan (HA) was previously shown by this laboratory to prevent experimentally induced pulmonary emphysema without associated toxicity. Nevertheless, other investigators have found that low-molecular-weight HA may be proinflammatory, prompting the authors to determine if aerosolized HA could possibly enhance pulmonary inflammation in a different model of lung injury involving intratracheal instillation of endotoxin to hamsters. Results indicate that exposure to HA following endotoxin administration significantly increased lung inflammation, whereas pretreatment with HA had the opposite effect. PMID- 16684715 TI - Acute bronchial obstruction in sheep: histopathology and gland cytokine expression. AB - An ovine model of smoke inhalation and burn (S+B) injury models the pathophysiology of these injuries in humans. This study examines the degree of airway obstruction, associated histopathology, and bronchial gland cell expression of cytokines during the first 24 hours after S+B injury in sheep. Changes in the mean degree of obstruction were limited to the bronchial airways, showing significant increases in obstruction with time, P<.05. At 4 hours after injury, the obstructive material was predominantly mucus, with neutrophils clustered around and within gland acini. At 8 to 24 hours, bronchial obstruction was characterized by increased inflammatory cell accumulation. Immunohistochemical results showed that gland cells constitutively express and secrete interleukin (IL)-1beta, and that after injury there is an increase in the percentage of gland cells staining for IL-1alpha, IL-8, and tumor necrosis factor (TNF)-alpha, P<.05. PMID- 16684716 TI - Heritable and pharmacological influences on pauses and apneas in inbred mice during anesthesia and emergence. AB - Inherited differences in response to hypercapnia are augmented by volatile anesthetics. Therefore, the authors tested the hypotheses that (1) the incidence of pauses and apneas also increase under anesthesia; (2) there is a difference in the incidence between mouse strains; and (3) there is a difference in the incidence of pauses and apneas depending on the volatile agent. The authors assessed respiratory pauses and apneas at rest; during anesthesia with isoflurane, sevoflurane, and desflurane; and at recovery in C3, B6, and F1 mice. The results are compared using analysis of variance (ANOVA) (P<.05). Awake, there was no difference between mouse strains (Bb, 0.3+/-0.7 P min(-1), C3, 0.4+/-0.7 P min(-1) and F1, 0.4+/-0.5 P min(-1)). In contrast, during anesthesia C3 mice showed a significantly higher incidence of pauses and apneas. There was no increase for B6 and F1 mice. There was no difference depending on the volatile agent. These results indicate an inheritance of a susceptibility to pauses and apneas under the influence of volatile anesthetics, albeit independent of the agent used. This response seems to be independent from the inherited response to hypercapnia. PMID- 16684717 TI - Rapid pulmonary expression of acute-phase reactants after local lipopolysaccharide exposure in mice is followed by an interleukin-6 mediated systemic acute-phase response. AB - This study investigated local and systemic innate immune responses in lipopolysaccharide (LPS)-induced lung inflammation in mice. Intratracheal LPS exposure resulted in increased pulmonary mRNA expression for acute-phase reactants (APRs) alpha(1)-antitrypsin (alpha(1)-AT), alpha(1)-acid glycoprotein (AGP), and LPS-binding protein (LBP) from 4 hours post exposure. Although pulmonary serum amyloid P component (SAP) mRNA was not increased, systemic levels of SAP, AGP, and LBP were elevated from 24 hours post exposure. Systemic APRs increase was associated with hepatic mRNA expression. As in vivo neutralization of interleukin (IL)-6, but not tumor necrosis factor (TNF)-alpha, fully ablated hepatic APR mRNA expression, IL-6 may act as signaling molecule between lung and liver. In conclusion, pulmonary LPS exposure induced rapid APR expression in lung, which precedes IL-6-mediated systemic elevation of APRs associated with hepatic APRs expression. PMID- 16684718 TI - Elastin defects in the lungs of avian and murine models of homocysteinemia. AB - Homocysteinemia in animals is associated with disruption of the elastic fiber component of the extracellular matrix, resulting in vascular complications. The authors have utilized both avian and murine models to investigate the effects of homocysteinemia on lung development and repair following injury. Days old chicks were fed a diet containing 2% methionine for 3 weeks. Pregnant mice were given 2% methionine in the diet and feeding continued for up to 6 weeks after birth. The lungs were removed and examined for defects in elastin fiber formation. Methionine levels were elevated 20-fold in the serum from chicks receiving the methionine and 10-fold in pregnant mice. The elastic fibers in the parabronchi and air capillaries of chicks receiving methionine were thin and clearly disrupted. In the 2% methionine neonatal pups, normal lung development was prevented and the alveoli were significantly enlarged. However, after the pups reached 10 days of age the 2% methionine lungs did not differ histologically from the normal controls. Fetal mice reflected the same serum methionine levels as the dams fed the 2% methionine diet, yet after birth the serum levels of the neonates returned to control levels within 3 days. The authors found that the high serum methionine levels of the dams were not transferred to the milk, allowing the pups to reverse the histopathology observed early and then develop normally. The ability of the lung to replace elastin following elastase injury was not different in mice raised on the 2% methionine diet compared to controls. The studies show that continuous exposure of the developing lung to high circulating levels of methionine/homocysteine can result in major disruptions of elastic fibers and lung architecture. However, young mammals such as the mouse are protected from extended lung pathology because toxic levels of methionine are not transferred through the mothers milk. PMID- 16684719 TI - Patterns of speciation in the Yucca moths: parallel species radiations within the Tegeticula yuccasella species complex. AB - The interaction between yuccas and yucca moths has been central to understanding the origin and loss of obligate mutualism and mutualism reversal. Previous systematic research using mtDNA sequence data and characters associated with genitalic morphology revealed that a widespread pollinator species in the genus Tegeticula was in fact a complex of pollinator species that differed in host use and the placement of eggs into yucca flowers. Within this mutualistic clade two nonpollinating "cheater" species evolved. Cheaters feed on yucca seeds but lack the tentacular mouthparts necessary for yucca pollination. Previous work suggested that the species complex formed via a rapid radiation within the last several million years. In this study, we use an expanded mtDNA sequence data set and AFLP markers to examine the phylogenetic relationships among this rapidly diverging clade of moths and compare these relationships to patterns in genitalic morphology. Topologies obtained from analyses of the mtDNA and AFLP data differed significantly. Both data sets, however, corroborated the hypothesis of a rapid species radiation and suggested that there were likely two independent species radiations. Morphological analyses based on oviposition habit produced species groupings more similar to the AFLP topology than the mtDNA topology and suggested the two radiations coincided with differences in oviposition habit. The evolution of cheating was reaffirmed to have evolved twice and the closest pollinating relative for one cheater species was identified by both mtDNA and AFLP markers. For the other cheater species, however, the closest pollinating relative remains ambiguous, and mtDNA, AFLP, and morphological data suggest this cheater species may be diverged based on host use. Much of the divergence in the species complex can be explained by geographic isolation associated with the evolution of two oviposition habits. PMID- 16684720 TI - Differentiating between hypotheses of lineage sorting and introgression in New Zealand alpine cicadas (Maoricicada Dugdale). AB - Lineage sorting and introgression can lead to incongruence among gene phylogenies, complicating the inference of species trees for large groups of taxa that have recently and rapidly radiated. In addition, it can be difficult to determine which of these processes is responsible for this incongruence. We explore these issues with the radiation of New Zealand alpine cicadas of the genus Maoricicada Dugdale. Gene trees were estimated from four putative independent loci: mitochondrial DNA (2274 nucleotides), elongation factor 1-alpha (1275 nucleotides), period (1709 nucleotides), and calmodulin (678 nucleotides). We reconstructed phylogenies using maximum likelihood and Bayesian methods from 44 individuals representing the 19 species and subspecies of Maoricicada and two outgroups. Species-level relationships were reconstructed using a novel extension of gene tree parsimony, whereby gene trees were weighted by their Bayesian posterior probabilities. The inferred gene trees show marked incongruence in the placement of some taxa, especially the enigmatic forest and scrub dwelling species, M. iolanthe. Using the species tree estimated by gene tree parsimony, we simulated coalescent gene trees in order to test the null hypothesis that the nonrandom placement of M. iolanthe among gene trees has arisen by chance. Under the assumptions of constant population size, known generation time, and panmixia, we were able to reject this null hypothesis. Furthermore, because the two alternative placements of M. iolanthe are in each case with species that share a similar song structure, we conclude that it is more likely that an ancient introgression event rather than lineage sorting has caused this incongruence. PMID- 16684723 TI - Changes in mental health financing since 1971: implications for policymakers and patients. AB - The aggregate share that total mental health spending claims of national income has been stable over the past thirty-five years. This stability is a consequence of immense change--new organizational technologies, new treatment technologies, and a growing supply of providers. Aggregate spending stability has been accompanied by major shifts in the composition of financing, which have tended to spread the costs of mental illness more broadly but also have led to fragmentation in public responsibility for people with mental illnesses. Recent developments suggest that financing could be further constrained in the future, even as fragmentation continues to increase. PMID- 16684724 TI - Rising mental health costs: what are we getting for our money? AB - This synthesis of recent epidemiological and service use data concludes that, in aggregate, rising mental health spending since the early 1990s appear to be purchasing improvements in access to care and to represent a good value for society. However, there is also evidence of continuing waste and quality deficits. Although mental health faces some unique challenges, these patterns are more similar to than different from those seen in all of health care. These parallels suggest the importance of learning from and working with the broader health care system in improving the returns on U.S. mental health spending. PMID- 16684725 TI - The costs of mental health parity: still an impediment? AB - Parity in mental health benefits rectifies unfairness in health insurance coverage and reduces financial risk for those with mental illness. However, increased coverage for mental illness has been seen as creating inefficiencies and increasing total spending, based largely on results from the RAND Health Insurance Experiment conducted in the 1970s. Newer evidence suggests that cost control techniques associated with managed care give health plans alternatives to discriminatory coverage for containing costs. We review both eras of research on mental health insurance and conclude that comprehensive parity implemented in the context of managed care would have little impact on total spending. PMID- 16684726 TI - Prices, profits, and innovation: examining criticisms of new psychotropic drugs' value. AB - High profits and high drug costs have brought increased scrutiny of the pharmaceutical industry over the issue of whether the drugs they produce are worth the costs. I examine several related complaints, including the proliferation of me-too drugs and product reformulations, which some argue have little value relative to their cost; the baseless promotion of newer drug classes as more effective than existing, less expensive drugs; legal strategies to extend market exclusivity that result in high brand-name drug prices for an extended period of time; and large promotional expenditures that result in higher prices. PMID- 16684728 TI - Substance abuse with mental disorders: specialized public systems and integrated care. AB - Separate public financing and regulation of substance (SA) abuse treatment distinct from mental health (MH) treatment preserves a focus on the special needs of those with substance abuse but creates challenges to providing appropriate care for the large number of people with co-occurring conditions. This paper reviews recent efforts to overcome these challenges through clinical and systems approaches that better integrate care. Although much progress has been made for some subgroups of people with co-occurring disorders, further efforts to develop and sustain clinically integrated service delivery approaches within separate systems, particularly in SA treatment settings, are needed. PMID- 16684729 TI - From silos to bridges: meeting the general health care needs of adults with severe mental illnesses. AB - Despite their higher burden of chronic general medical disorders, adults with severe mental illnesses have poorer access to and quality of general health care compared to people without mental illnesses. Because a key contributor to this situation is the separation between the mental health care sector (patients' health care home) and the general health care sector, bridges to connect these systems are needed. We discuss obstacles to clinical integration and propose strategies to address them. PMID- 16684730 TI - Beyond integration: challenges for children's mental health. AB - This paper reviews access, outcomes, and quality in the children's mental health system. We contend that a major focal point of future reforms should be at the organization level of care delivery. We identify five areas for intentional policy action to better infuse quality into the system. We also call for building upon the momentum of recent high-visibility reform proposals and for renewed advocacy to advance quality-linked perspectives into the children's mental health system beyond its focus on children with severe emotional disturbances. PMID- 16684731 TI - Ensuring quality and access in mental health services. PMID- 16684732 TI - What is necessary to transform the quality of mental health care. AB - Improving the quality of care is a national priority in the United States; however, it is not clear how to accelerate progress for mental health care. We recommend advances in three capacities: (1) developing quality improvement resources applicable to a diverse set of mental health disorders, clients, and service settings; (2) improving the infrastructure for providing evidence-based psychotherapy and psychosocial interventions; and (3) promoting innovation in financial incentives for quality improvement in mental health care. We also discuss the need to develop leadership among health care stakeholders and community engagement to promote public commitment to high-quality care in mental health. PMID- 16684733 TI - The struggle to provide community-based care to low-income people with serious mental illnesses. AB - This paper describes gaps in services for low-income people with serious mental illnesses as reported by mental health professionals and other observers in twelve U.S. communities. According to respondents, service gaps have grown in recent years--especially for uninsured people--as a result of state budget pressures and Medicaid cost containment policies. Growing service gaps contribute to the high prevalence of serious mental illness among the homeless and incarcerated populations, as well as crowding of emergency departments. Some states and communities are aggressively addressing these gaps, although funding for new programs remains scarce. PMID- 16684735 TI - Mental health in the Medicare Part D drug benefit: a new regulatory model? AB - The Medicare Prescription Drug, Improvement, and Modernization Act (MMA) of 2003 represents the most significant expansion of Medicare benefits since the program's inception and has important implications for mental health. Medicare will become a major payer for psychotropic medications through the new prescription drug benefit. The structure of the drug benefit's delivery system creates incentives for plans to underprovide medications, like psychotropic drugs, that are used persistently and are associated with high expected costs. Regulators have put policies in place to counteract these incentives. This paper examines these strategies' likely success and suggests additional approaches to be considered. PMID- 16684736 TI - The patient as a policy factor: a historical case study of the consumer/survivor movement in mental health. AB - This paper analyzes the history of the modern consumer/survivor movement and its impact on the policy-making climate in the mental health field. The growing attentiveness to consumers' perspectives is presented largely as a consequence, not a cause, of radical restructurings of the mental health system. Consumers' perspectives have entered policy discourse in the wake of policy failures and have flourished in a climate of perpetual crisis and tight budgets. Precisely because it has been such a contested arena for so long, the mental health field has produced some innovative responses to demands for patient empowerment. PMID- 16684737 TI - A seat at the table: trend or illusion? AB - Responding to Nancy Tomes's conclusion that mental health consumers are beneficiaries but not primary causes of a new attentiveness to consumers' concerns, this review finds various historical and contemporary reasons for the mental health consumer movement to remain vigilant: the system's shallow acceptance of consumer self-determination; continued reliance on expedient coercive tactics rather than therapeutic engagement; the currently narrow reach of initiatives in self-direction; and the fragmentation of authority in the delivery of public services and supports needed by mental health consumers. PMID- 16684738 TI - Psychiatry and the consumer movement. AB - We agree that consumers' perspectives have had a limited impact on major policy shifts but note that they have resulted in important changes in the role of patients and families in all aspects of mental health treatment. In addition, we take issue with the use of "consumer" and "survivor" as one combined term because of the words' very different connotations. The field of psychiatry and the consumer movement may still differ in their views on the need for involuntary treatment but are now generally allied around the need for more effective and better-funded psychiatric services. PMID- 16684739 TI - Defining 'mental illness' in mental health policy. AB - Mental health policy is shaped fundamentally by the definition of mental illness associated with the policy. Changing policies reflect changing definitions. At various times, the definition may be narrow or broad with respect to the scope of conditions covered by a specific policy. The priority accorded to impairment severity is the most crucial and enduring policy issue related to the definition of mental illness and the scope of that definition. This paper explores the role of definitions in framing mental health policy, using examples from the history of policy making over the past half-century. PMID- 16684740 TI - Malpractice premiums and physicians' income: perceptions of a crisis conflict with empirical evidence. AB - The conventional wisdom is that malpractice premiums have steadily risen and now constitute a crisis for medical practice. The best available data suggest otherwise. American Medical Association (AMA) surveys of self-employed physicians from 1970 to 2000 indicate that premiums rose until 1986, then declined until 1996, rose thereafter, but were lower in 2000 than in 1986. Other items represented a much greater share of total practice expenses in 1970 yet increased rapidly until 1996 and moderately thereafter, while spending on premiums fell during 1986-2000. National trends were reflected with variations in obstetrics/gynecology, surgery, and anesthesiology and in nine regions surveyed. PMID- 16684741 TI - The relationship between health plan advertising and market incentives: evidence of risk-selective behavior. AB - Medicare beneficiaries are now facing advertising from an unprecedented number of health plans that are offering prescription drug coverage. Previous Medicare managed care efforts have been undermined by risk selection, the practice of enrolling healthier and therefore less costly patients. In this study we explore how the content of health plan advertising is related to the competitiveness of the health plan market. We find that increased competition is associated with greater use of advertising that targets healthier patients. PMID- 16684742 TI - Consumer-directed health insurance products: local-market perspectives. AB - During the past few years, health plans have focused product development on consumer-driven health plans. This paper examines how these products are faring in twelve Community Tracking Study (CTS) communities. Although there has been a proliferation in the number and variety of consumer-directed plan options available, employers have taken a cautious approach. Given the increased financial stake and decision-making responsibility consumers hold when enrolled in these plans, respondents expressed frustration that the availability of information support has lagged behind the demands placed on consumers. PMID- 16684743 TI - Why employer-sponsored insurance coverage changed, 1997-2003. AB - Four and a half million Americans gained employer-sponsored health insurance coverage during 1997-2001, while nearly nine million lost coverage in the ensuing economic downturn (2001-2003), after population growth was accounted for. Macroeconomic trends affecting employment, job quality, and incomes drove most of the coverage changes, although key factors varied during the two periods. Take-up rates affected coverage, mostly reflecting the interaction of premium cost trends and labor-market tightness, but take-up also was influenced by the implementation of the State Children's Health Insurance Program (SCHIP) during 1997-2001. Coverage among low-income people was most affected by economic conditions and premium costs. PMID- 16684744 TI - Construction activity in U.S. hospitals. AB - Hospital construction activity is increasing, but little information exists on what types of hospital capacity are affected and what is motivating specific efforts. Our analysis of Round Five Community Tracking Study data revealed four general types of activity: new hospital construction or expansion of existing general hospital capacity; new or expanded capacity in specialty services; replacement of aged facilities; and expansion of capacity-constrained services. Some of these actions are responsive to community need, but others resemble a medical-arms-race response. Overall, current construction activity will provide more convenient access for some consumers but at high cost if excess capacity results. PMID- 16684745 TI - National trends in adult hospice use: 1991-1992 to 1999-2000. AB - This study examines hospice use among adult hospice patients based on the 1992 2000 National Home and Hospice Care Surveys, the 1997-1999 National Nursing Home Surveys, and the 1991-2000 annual Underlying and Multiple Cause-of-Death Files. The total number of adult hospice patients tripled between 1991-1992 and 1999 2000. The majority of inpatient hospice patients resided in nursing homes. The increased hospice utilization rates and increased percentage of adult hospice patients with short stays in hospice indicate changes in hospice enrollment patterns over time. Hospice is still in the process of growing toward a steady state. PMID- 16684746 TI - Influencing cancer policy. AB - This paper describes the Institute of Medicine's (IOM's) eight-year experiment in examining cancer policy issues through the National Cancer Policy Board and more recently through the early phases of the National Cancer Policy Forum. Both were primarily funded by the National Cancer Institute and Centers for Disease Control and Prevention. The forum identifies and explores issues but, unlike the board, does not author IOM advisory reports. We report on the events that led to the establishment of these activities and discuss the factors that could lead to success in influencing policy, using examples of actual reports and effects on public- and private-sector programs and policies. PMID- 16684747 TI - Doctoring across the language divide. PMID- 16684748 TI - Innocence abroad. PMID- 16684749 TI - Health care spending and use of information technology in OECD countries. AB - In 2003, the United States had fewer practicing physicians, practicing nurses, and acute care bed days per capita than the median country in the Organization for Economic Cooperation and Development (OECD). Nevertheless, U.S. health spending per capita was almost two and a half times the per capita health spending of the median OECD country. One proposal for both lowering health spending and improving quality is the adoption of health information technology (HIT). The United States lags as much as a dozen years behind other industrialized countries in HIT adoption--countries where national governments have played major roles in establishing the rule, and health insurers have paid most of the costs. PMID- 16684750 TI - Generosity and adjusted premiums in job-based insurance: Hawaii is up, Wyoming is down. AB - This paper reports national and state findings on the generosity or actuarial value of U.S. employer-based plans and adjusted premiums in 2002. The basis for our calculations is simulated bill paying for a large standardized population. After adjusting for the quality of benefits, we find from regression analysis that adjusted premiums are 18 percent higher in the nation's smallest firms than in firms with 1,000 or more workers. They are 25 percent higher in indemnity plans and 18 percent higher in preferred provider organizations than in health maintenance organizations. The generosity of coverage increased from 1997 to 2002. PMID- 16684751 TI - The predictive accuracy of the New York State coronary artery bypass surgery report-card system. AB - We examined the impact of New York State's public reporting system for coronary artery bypass surgery fifteen years after its launch. We found that users who picked a top-performing hospital or surgeon from the latest available report had approximately half the chance of dying as did those who picked a hospital or surgeon from the bottom quartile. Nevertheless, performance was not associated with a subsequent change in market share. Surgeons with the highest mortality rates were much more likely than other surgeons to retire or leave practice after the release of each report card. PMID- 16684752 TI - Strengthening a state's health advocacy infrastructure. AB - Although coordination among advocates may be a critical ingredient for achieving health policy change, opportunities for discussions that lead to cooperation among advocates are rare. For the past seven years, the California Wellness Foundation has funded an annual two-day retreat for California health advocates. These gatherings have created a reflective time period for them to consider their differing advocacy agendas as well as areas of overlap. The retreats have also helped to soften the fault lines that can be barriers to cooperation. An independent evaluation has provided some important points for foundations to consider when making grants for such a retreat. PMID- 16684754 TI - Defining a future for fee-for-service Medicare. AB - The Medicare Prescription Drug, Improvement, and Modernization Act (MMA) provides economic incentives that favor health plans over traditional fee-for-service (FFS) Medicare. This reflects an ideological preference for private plans rather than government-administered pricing and recognition that private plans can use tools effectively to improve quality. However, enrollment projections indicate that FFS will continue to attract the majority of beneficiaries for years to come. We argue that MMA's contractor reform provisions create the opportunity to build critical FFS infrastructure, and contractors have the potential to encourage quality and manage utilization to compete with private plans in a modernized Medicare. PMID- 16684756 TI - Specialty hospitals: pro and Kahn. PMID- 16684758 TI - Examining hospital costs. PMID- 16684759 TI - Nurse-to-patient ratios. PMID- 16684760 TI - Nurse staffing and quality. PMID- 16684763 TI - Managing consumerism. PMID- 16684762 TI - Cost sharing revisited. PMID- 16684764 TI - Establishment of a reliable method for direct proteome characterization of human articular cartilage. AB - Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma. PMID- 16684765 TI - Analysis of the Salmonella typhimurium proteome through environmental response toward infectious conditions. AB - Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis. PMID- 16684766 TI - Modifications of the lipoamide-containing mitochondrial subproteome in a yeast mutant defective in cysteine desulfurase. AB - Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis. PMID- 16684767 TI - High dynamic range characterization of the trauma patient plasma proteome. AB - Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses. PMID- 16684768 TI - ABIN-1 binds to NEMO/IKKgamma and co-operates with A20 in inhibiting NF-kappaB. AB - Nuclear factor kappaB (NF-kappaB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF kappaB is dependent on the phosphorylation of the inhibitory subunit IkappaBalpha that is mediated by a multimeric, high molecular weight complex, called IkappaB kinase (IKK) complex. This is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, NEMO/IKKgamma. The latter protein is essential for the activation of IKKs and NF-kappaB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-kappaB) as a NEMO/IKKgamma-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-kappaB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-kappaB at the level of the IKK complex and that A20 inhibits activation of NF-kappaB by de ubiquitination of NEMO/IKKgamma. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKgamma and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-kappaB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKgamma and facilitates A20-mediated de-ubiquitination of NEMO/IKKgamma, thus resulting in inhibition of NF-kappaB. PMID- 16684769 TI - Molecular identification and characterization of a family of kinases with homology to Ca2+/calmodulin-dependent protein kinases I/IV. AB - Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2. PMID- 16684770 TI - Differential actin-dependent localization modulates the evolutionarily conserved activity of Shroom family proteins. AB - Shroom is an actin-associated determinant of cell morphology that is required for neural tube closure in both mice and frogs. Shroom regulates this process by causing apical constriction of epithelial cells via a pathway involving myosin II. Here we report on characterization of the Shroom-related proteins Apxl and KIAA1202 and their role in cell architecture. Shroom, Apxl, and KIAA1202 exhibit differing abilities to interact with the actin cytoskeleton. In fibroblasts, Shroom readily associates with actin stress fibers and induces bundling, Apxl is found on cortical actin, and KIAA1202 is localized to a cytoplasmic population of F-actin. In epithelial cells, Apxl and KIAA1202 do not induce apical constriction as Shroom does, but have the capacity to do so if targeted to the apical junctional complex. To determine whether the activity of Shroom-like proteins is conserved in invertebrates, we have tested the ability of the lone Shroomrelated protein in Drosophila, CG8603, to activate the constriction pathway. A chimeric protein consisting of the Shroom targeting domain and the Drosophila protein elicits constriction. Finally, we show that Apxl is involved in regulating the cytoskeletal organization and architecture of endothelial cells. We predict that the ability of Shroom-like proteins to regulate cellular morphology is conserved in evolution and is regulated in part by subcellular localization. PMID- 16684771 TI - Functional characterization of the flagellar glycosylation locus in Campylobacter jejuni 81-176 using a focused metabolomics approach. AB - Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6 trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways. PMID- 16684772 TI - Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core. AB - Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells. PMID- 16684773 TI - Phosphorylation of Ime2 regulates meiotic progression in Saccharomyces cerevisiae. AB - Ime2p is a meiosis-specific protein kinase in Saccharomyces cerevisiae that controls multiple steps in meiosis. Although Ime2p is functionally related to the Cdc28p cyclin-dependent kinase (CDK), no cyclin binding partners that regulate its activities have been identified. The sequence of the Ime2p catalytic domain is similar to CDKs and mitogen-activated protein kinases (MAPKs). Ime2p is activated by phosphorylation of its activation loop in a Cak1p-dependent fashion and is subsequently phosphorylated on multiple residues as cells progress through meiosis. In this study, we show that Ime2p purified from meiotic cells is phosphorylated on Thr(242) and Tyr(244) in its activation loop and on Ser(520) and Ser(625) in its C terminus. Ime2p autophosphorylates on threonine in its activation loop in vitro consistent with autophosphorylation of Thr(242) playing a role in its activation. Moreover, autophosphorylation in cis is required for Ime2p to become hyperphosphorylated. Phosphorylation of the C-terminal serines is not essential to sporulation. However, Ime2p C-terminal phosphorylation site mutants genetically interact with components of the FEAR network that controls exit from meiosis I. These data suggest that Ime2p plays a role in controlling the exit from meiosis I and demonstrate that a phospho-modification pathway regulates Ime2p during the different phases of meiotic development. PMID- 16684774 TI - Elucidation of steps in the capture of a protein substrate for efficient encapsulation by GroE. AB - We have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T --> R(1)) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R(1) --> R(2)) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R(2) state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL.GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R(2).GroES --> R(3).GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL.GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES. PMID- 16684775 TI - The role of phospholipid hydroperoxide glutathione peroxidase isoforms in murine embryogenesis. AB - Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5 17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice. PMID- 16684776 TI - Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms. AB - The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 A resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR. PMID- 16684777 TI - Essential role of 3'-untranslated region-mediated mRNA decay in circadian oscillations of mouse Period3 mRNA. AB - Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, we focused on the 3'-untranslated region (3'-UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3'-UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3'-UTR-mediated mRNA decay on circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Interestingly, a stabilizing mutation of 3'-UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3'-UTR began to increase earlier than that in the presence of wild-type 3' UTR. Consequently, luciferase mRNA with mutant 3'-UTR displayed oscillation patterns with a prolonged rising phase. Our results indicate that mPer3 3'-UTR mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post-transcriptional control of circadian mRNA oscillations. PMID- 16684778 TI - Alteration of Escherichia coli topoisomerase IV conformation upon enzyme binding to positively supercoiled DNA. AB - Escherichia coli topoisomerase IV (topo IV) is an essential enzyme that unlinks the daughter chromosomes for proper segregation at cell division. In vitro, topo IV readily distinguishes between the two possible chiralities of crossing segments in a DNA substrate. The enzyme relaxes positive supercoils and left handed braids 20 times faster, and with greater processivity, than negative supercoils and right-handed braids. Here, we used chemical cross-linking of topo IV to demonstrate that enzyme bound to positively supercoiled DNA is in a different conformation from that bound to other forms of DNA. Using three different reagents, we observed novel cross-linked species of topo IV when positively supercoiled DNA was in the reaction. We show that the ParE subunits are in close enough proximity to be cross-linked only when the enzyme is bound to positively supercoiled DNA. We suggest that the altered conformation reflects efficient binding by topo IV of the two DNA segments that participate in the strand passage reaction. PMID- 16684780 TI - Linking observational and genetic approaches to determine the role of C-reactive protein in heart disease risk. PMID- 16684779 TI - Essential role of KIBRA in co-activator function of dynein light chain 1 in mammalian cells. AB - Recently dynein light chain 1 (DLC1), a cytoskeleton signaling component, has been shown to interact with and transactivate estrogen receptor-alpha (ER), leading to increased expression of ER target genes and growth stimulation of breast cancer cells. However, the molecular mechanism by which DLC1 regulates the ER pathway remains poorly understood. To gain insights into the putative mechanism, here we set out to identify novel DLC1-interacting proteins. We identified KIBRA, a WW domain- and a glutamic acid stretch-containing protein, as a DLC1-binding protein and showed that it interacts with DLC1 both in vitro and in vivo. We found that KIBRA-DLC1 complex is recruited to ER-responsive promoters. We also found that KIBRA-DLC1 interaction is mandatory for the recruitment and transactivation functions of ER or DLC1 to the target chromatin. Finally we found that KIBRA interacts with histone H3 via its glutamic acid-rich region and that such interaction might play a mechanistic role in conferring an optimal ER transactivation function as well as the proliferation of ligand stimulated breast cancer cells. Together these findings indicate that DLC1-KIBRA interaction is essential for ER transactivation in breast cancer cells. PMID- 16684781 TI - Angiotensin II receptor blockers and coronary artery disease: 'presumed innocents'. PMID- 16684782 TI - The health promoting sports club in Finland--a challenge for the settings-based approach. AB - The purpose of this article is, first, to compile a frame of reference for the health promoting sports club and, second, to develop standards for the concept. This concept is based on the settings-based health promotion approach. Sports clubs are a new setting for health promotion, which until now has been little examined from a settings point of view. Nevertheless, this concept has much potential. For example, sports clubs attract a large number of children and adolescents and their educational nature can be considered to be informal. The present standards were developed using the Delphi method. The researcher, in cooperation with a panel of experts (experts in health promotion, n = 11, and experts in sports clubs, n = 16), sought to create a consensus statement on the standards. At the preliminary stage of the study 64 original standards were created on the basis of existing literature and the principles of the Ottawa Charter. During the three rounds of the Delphi process 15 standards were evaluated as the most important. After the Delphi process, the researcher modified the standards by eliminating overlap, interpolating seven standards to involve all strategic areas of the Ottawa Charter and creating a preliminary typology of the standards. At the subsequent stages of the study, indicators for these standards will be drafted and tested in practice. Therefore, this study would provide tools for determining and evaluating how health promoting a particular sports club is. PMID- 16684783 TI - Eye diseases among elderly drivers in Finland. PMID- 16684784 TI - Respiratory symptoms and lung function in Bangkok school children. AB - BACKGROUND: Previous epidemiological studies have shown acute effects of ambient air pollutants in children with respiratory disorders. METHODS: The chronic effects of air pollution in Bangkok children were investigated. Children aged 10 15 years were examined for lung functions using spirometry tests and for respiratory symptoms by the American Thoracic Society's Division of Lung Diseases (ATS-DLD-78-C) questionnaire during May-August 2004. Effects of residential area were estimated by multiple logistic regression analysis. Of the 878 children, 722 (82%) had completed lung function test and ATS-DLD questionnaire. RESULTS: In children, who live in roadside (R) and general (G) areas with high (H) pollution, the prevalence of respiratory symptoms increased significantly [odds ratios (95% confidence interval) in HR and HG are 2.44 (1.21-4.93) and 2.60 (1.38-4.91), respectively]. Children with normal lung function were less observed in H- and M polluted roadside and general area [HR, OR = 1.41 (95% CI 0.89-2.22); HG, 1.08 (0.71-1.64); and MR, 0.99 (0.63-1.57)]. Residential locations and family members were associated with the prevalence of respiratory symptoms, whereas factors such as the responder of ATS-DLD, gender, age, residential years, home size, parental smoking habits, use of air conditioners, and domestic pets were not associated. Age was associated with the impaired lung function, whereas others factors were not associated. CONCLUSION: The prevalence of respiratory symptoms and impaired lung function were higher among children living in areas with high pollution than those in areas with low pollution. PMID- 16684785 TI - Socio-economic position and adolescents' health in Italy: the role of the quality of social relations. AB - BACKGROUND: The quality of social relations in adolescence is possibly one of the major determinants of habits that can influence the health of young people, and it may also be one of the mediators of the effect of social position on health. In this paper we propose to test these hypotheses for Italian adolescents, in order to suggest interventions aimed at improving their health. METHODS: The Italian data of the HBSC (Health Behaviour in School-Aged Children) survey 2001 02 have been analysed, and the distribution of the perceived quality of social relations has been described, stratified by age, gender, and economic well-being. Logistic models have been fitted using health behaviours as dependent variables and economic well-being and social relations as determinants. RESULTS: The quality of relations with adults seems to decrease consistently from age 11 through age 15, while the relation with peers improves. The relation with the father seems positively correlated with economic well-being. Difficult relations with adults are associated with higher probability of smoking, drinking alcohol and using cannabis; difficult relations with peers are associated with lower physical activity and lower probability of having used cannabis. CONCLUSIONS: Even if the relations with adults become less important in adolescence, they are still associated with health behaviours. Our results fit the framework of socialization theories and can be used for planning adequate health education interventions. PMID- 16684786 TI - Periventricular heterotopia: phenotypic heterogeneity and correlation with Filamin A mutations. AB - Periventricular heterotopia (PH) occurs when collections of neurons lay along the lateral ventricles or just beneath. Human Filamin A gene (FLNA) mutations are associated with classical X-linked bilateral periventricular nodular heterotopia (PNH), featuring contiguous heterotopic nodules, mega cisterna magna, cardiovascular malformations and epilepsy. FLNA encodes an F-actin-binding cytoplasmic phosphoprotein and is involved in early brain neurogenesis and neuronal migration. A rare, recessive form of bilateral PNH with microcephaly and severe delay is associated with mutations of the ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2) gene, required for vesicle and membrane trafficking from the trans-Golgi. However, PH is a heterogeneous disorder. We studied clinical and brain MRI of 182 patients with PH and, based on its anatomic distribution and associated birth defects, identified 15 subtypes. Classical bilateral PNH represented the largest group (98 patients: 54%). The 14 additional phenotypes (84 patients: 46%) included PNH with Ehlers-Danlos syndrome (EDS), temporo-occipital PNH with hippocampal malformation and cerebellar hypoplasia, PNH with fronto-perisylvian or temporo-occipital polymicrogyria, posterior PNH with hydrocephalus, PNH with microcephaly, PNH with frontonasal dysplasia, PNH with limb abnormalities, PNH with fragile-X syndrome, PNH with ambiguous genitalia, micronodular PH, unilateral PNH, laminar ribbon-like and linear PH. We performed mutation analysis of FLNA in 120 patients, of whom 72 (60%) had classical bilateral PNH and 48 (40%) other PH phenotypes, and identified 25 mutations in 40 individuals. Sixteen mutations had not been reported previously. Mutations were found in 35 patients with classical bilateral PNH, in three with PNH with EDS and in two with unilateral PNH. Twenty one mutations were nonsense and frame-shift and four missense. The high prevalence of mutations causing protein truncations confirms that loss of function is the major cause of the disorder. FLNA mutations were found in 100% of familial cases with X-linked PNH (10 families: 8 with classical bilateral PNH, 1 with EDS and 1 with unilateral PH) and in 26% of sporadic patients with classical bilateral PNH. Overall, mutations occurred in 49% of individuals with classical bilateral PNH irrespective of their being familial or sporadic. However, the chances of finding a mutation were exceedingly gender biased with 93% of mutations occurring in females and 7% in males. The probability of finding FLNA mutations in other phenotypes was 4% but was limited to the minor variants of PNH with EDS and unilateral PNH. Statistical analysis considering all 42 mutations described so far identifies a hotspot region for PNH in the actin-binding domain (P < 0.05). PMID- 16684787 TI - Local and global motion after-effects are both enhanced in migraine, and the underlying mechanisms differ across cortical areas. AB - Visual after-effects are illusions that occur after prolonged viewing of visual displays (pattern adaptation). The motion after-effect (MAE), for example, is an illusory impression of motion that is seen after viewing moving displays. After effects have been used extensively in basic vision research as well as in clinical settings, and have been reported to be enhanced in migraine. Pattern adaptation is a cortical phenomenon that reflects both cellular mechanisms acting within individual neurons and specific interactions between groups of neurons activated by the adapting display. A remarkable feature of the MAE is that its duration is only slightly reduced if a delay is inserted between the end of the adaptation and the test display ('storage'). The reduction is consistent with recovery of the cellular component, and the residual with network changes that are maintained during the delay. This study aimed (i) to assess explanations for prolonged MAEs in migraine by teasing apart the proposed cellular and network components of adaptation using storage; (ii) to determine the extent of cortical abnormality in migraine using local and global MAEs, which reflect adaptation at different stages of the visual system. Fifty migraine (22 with, 28 without aura) and 50 control participants adapted to motion before viewing a stationary or dynamic (random motion) test, which consequently appeared to move in the opposite direction (local and global MAEs, respectively). Half of the trials included a delay between the adapting and test displays. The results extend those reported previously, as both local and global MAEs lasted longer in migraine compared with the control group. Global MAEs survived delays almost completely for both groups, whereas local MAEs were reduced to a greater extent in migraine. There were no significant differences between migraine subgroups classified according to the presence or absence of visual aura. These results suggest that cellular recovery is slowed in migraine for early but not later visual cortical areas. Sustained network changes following adaptation are implicated across cortical areas. Differences between people with and without migraine on various measures of visual perception have been attributed to abnormal cortical processing in migraine, variously described by hyperexcitability, heightened responsiveness and/or a lack of intra-cortical inhibition. The results are not consistent with hyperexcitability resulting from a lack of inhibition in migraine, but are consistent with extended suppression of intra-cortical excitation. The implications of these results for alternative models of hyperexcitability are discussed. PMID- 16684788 TI - Slow oscillatory activity and levodopa-induced dyskinesias in Parkinson's disease. AB - The pathophysiology of levodopa-induced dyskinesias (LID) in Parkinson's disease is not well understood. We have recorded local field potentials (LFP) from macroelectrodes implanted in the subthalamic nucleus (STN) of 14 patients with Parkinson's disease following surgical treatment with deep brain stimulation. Patients were studied in the 'Off' medication state and in the 'On' motor state after administration of levodopa-carbidopa (po) or apomorphine (sc) that elicited dyskinesias in 11 patients. The logarithm of the power spectrum of the LFP in selected frequency bands (4-10, 11-30 and 60-80 Hz) was compared between the 'Off' and 'On' medication states. A peak in the 11-30 Hz band was recorded in the 'Off' medication state and reduced by 45.2% (P < 0.001) in the 'On' state. The 'On' was also associated with an increment of 77. 6% (P < 0.001) in the 4-10 Hz band in all patients who showed dyskinesias and of 17.8% (P < 0.001) in the 60-80 Hz band in the majority of patients. When dyskinesias were only present in one limb (n = 2), the 4-10 Hz peak was only recorded in the contralateral STN. These findings suggest that the 4-10 Hz oscillation is associated with the expression of LID in Parkinson's disease. PMID- 16684789 TI - Paclitaxel, 5-fluorouracil and hydroxyurea concurrent with radiation in locally advanced nasopharyngeal carcinoma. AB - BACKGROUND: Concurrent chemoradiotherapy (CRT) is the standard treatment for locally advanced nasopharyngeal carcinoma (NPC). We conducted a phase II trial using paclitaxel, 5-fluorouracil and hydroxyurea concurrent with radiation (TFHX). PATIENTS AND METHODS: Fifty-nine patients with locally advanced NPC were treated with CRT consisting of 4-day continuous infusions of paclitaxel (20 mg/m(2)/d) and 5-fluorouracil (600 mg/m(2)/d), and oral hydroxyurea 500 mg bid for nine doses, every 3 weeks concurrent with radiotherapy (RT). RT consisted of once daily 200cGy fractions 5 times per week to a total of 7000cGy. RESULTS: Complete response was seen in 86% and 71% of patients at 4 and 12 months after CRT. The median follow-up was 34 months. Twenty-three patients experienced relapse. Sixteen deaths occurred: 13 from progressive disease. Three-year overall survival and progression-free survival were 72% and 54% respectively, with locoregional and distant control rates of 83% and 64% at 3 years respectively. Grade 3 to 4 acute toxicities included oropharyngeal mucositis in 81% of patients treated, dermatitis in 63%, weight loss in 32%, and neutropenia in 22%. Neutropenic fever was seen in 14%. There were no treatment-related deaths from acute toxicity. CONCLUSIONS: TFHX is shown to be feasible in NPC. Non-cross resistant induction chemotherapy should be further studied with this regimen. PMID- 16684790 TI - Toxic epidermal necrolysis in patients with malignancies. PMID- 16684791 TI - Population-based screening for prostate cancer by measuring free and total serum prostate-specific antigen in Iran. AB - PURPOSE: To evaluate the natural background of prostate cancer in Iran a large population-based study of screening using total prostate-specific antigen (tPSA) and per cent free PSA (fPSA) as the initial test was performed. MATERIALS AND METHODS: For 9 years (1996 to 2004) in Tehran, Iran, 3670 Iranian men older than 40 years were mass checked by PSA-based screening. They were invited to have a digital rectal examination (DRE), serum PSA assay and transrectal ultrasonography (TRUS)-guided sextant prostate biopsy to see if the DRE was clinically suspicious of malignancy, the serum PSA was > or =2.1 ng/ml or free-to-total PSA (f/tPSA) ratio < or=15%. RESULTS: In 433 (11.8%) of screened males, tPSA levels exceeded the cut-off value of > or =2.1 ng/ml and 128 prostate cancers were diagnosed [positive predictive value (PPV) 29.6%] corresponding to an overall detection rate of 3.5%. Altogether 138 cancers were detected (detection rate 3.8%); none were stage M(1), three were stage N(+) and 4 stage T(3). A threshold tPSA of > or =2.1 ng/ml would have detected 128 cancers in 447 biopsied men (PPV 29%). There were 109 of 138 (79%) men with cancer who had an f/tPSA of < or =15%, while 152 of 305 (49.8%) with benign biopsies had a f/tPSA of < or =15%, which corresponds to a PPV of 30.8%. CONCLUSION: PSA-based screening with low PSA cut-off values increase the detection rate of clinically significant, organ confined and potentially curable prostate cancer. Further studies are warranted in order to determine the incidence and prevalence of prostate cancer in different ethnic groups. PMID- 16684792 TI - In response to: 'On prejudice and facts and choices', an editorial by Kohne and Folprecht. PMID- 16684793 TI - TAT 2006. Abstracts of the 4th International Symposium on Targeted Anticancer Therapies, 16-18 March, 2006, Amsterdam, The Netherlands. PMID- 16684795 TI - Review paper: principles and applications of surface analytical techniques at the vascular interface. AB - Surface properties have been found to be one of the key parameters which cause degradation and of thrombogenicity in all polymers used in biomedical devices, thus signifying the importance and the necessity for quantitative and accurate characterization of the polymer surface itself as used in the construction of the device. The characterization techniques employed generally involve thermal and spectroscopic measurements, in which class the electrochemical investigations and scanning probe microscopies can also be included. Current hypotheses on the correlations that exist between surface parameters and hemocompatibility and degradation of polymers are examined herein, but concentrating on the field of clinically utilized polymeric materials as used within medical devices themselves. Furthermore, this review provides a brief but complete synopsis of these techniques and other emerging ones, which have proven useful in the analysis of the surface properties of polymeric materials as used in the construction of cardiovascular devices. Statements and examples are given as to how specific information can be acquired from these differing methodologies and how it aids in the design and development of new polymers for usage in biomedical device construction. PMID- 16684796 TI - Construction of chitosan-gelatin-hyaluronic acid artificial skin in vitro. AB - To further enhance the properties of chitosan (Cs)-gelatin (Gel) scaffolds for skin tissue engineering, hyaluronic acid (HA) is introduced to the Cs-gel complex. Porous scaffolds composed of Cs, Gel, and HA are prepared using the freeze-drying method. The scaffold has an interconnected pore structure with two different pore size layers. The water uptake ability, flexibility, and biocompatibility of the scaffold are greatly increased with the incorporation of HA. To construct an artificial skin in vitro, fibroblasts and keratinocytes are co-cultured in Cs-Gel-HA scaffolds at an air-liquid interface. After 2 weeks of co-culture, the epithelial layer becomes progressively stratiform, including cubic perpendicularly oriented cells and a superficial layer of flattened cells. Immunohistochemical analyses confirmed the presence of laminin and type IV collagen, typical molecules of the basement membrane. The results of this study suggest that it is possible to construct a functional artificial skin in vitro and the Cs-Gel-HA scaffold is a promising matrix for skin tissue engineering. PMID- 16684797 TI - Long-term in vivo degradation of poly-L-lactide (PLLA) in bone. AB - The use of absorbable orthopedic implants has increased substantially during the last decade. Currently, most of them are fabricated from poly-L-lactide (PLLA), its co-polymers, or mixtures with other constituents. In vivo, PLLA persists for years after its surgical role has ended, which is confirmed by a long-term histological study of PLLA implanted in sheep either as functional interference screws or nonfunctional rods. The first tissue reaction is the sequestration of the implant within new bone during the initial 3 months. After a nonreactive period, a second tissue reaction is associated with the early signs of structural disintegration of the PLLA at 1 year. Subsequently, as the polymer mass reduces, it is replaced by a relatively avascular fibrous tissue containing macrophages and having an occasional multinucleated giant cell on the implant surface. After 3 years much of the polymer is still present, although as isolated fragments. The tissue reactions can be explained in terms of the physical chemistry of PLLA degradation. Though biocompatible, the excessive longevity of PLLA and the absence of its replacement by bone, indicates that despite being satisfactory clinically, it is not an ideal implant material, and that improved absorbable materials need to be developed. PMID- 16684798 TI - Dental composites based on amorphous calcium phosphate - resin composition/physicochemical properties study. AB - This study explores how the resin composition/structure affects the physicochemical properties of copolymers and their amorphous calcium phosphate (ACP)-filled composites. A series of photo-polymerizable binary and ternary matrices are formulated utilizing 2,2-bis[ p-(2(')-hydroxy 3(')methacryloxypropoxy)phenyl]propane, 2,2-bis[ p-(2(') methacryloxypropoxy)phenyl]-propane (EBPADMA), or a urethane dimethacrylate as base monomers, and triethylene glycol dimethacrylate or hexamethylene dimethacrylate (HmDMA) with or without 2-hydroxyethyl methacrylate (HEMA) as diluent monomer. Unfilled copolymers and composites filled with 40% by mass zirconia-hybridized ACP are evaluated for biaxial flexure strength (BFS), degree of conversion (DC), mineral ion release, polymerization shrinkage (PS), and water sorption (WS). The average DC values are 82-94% and 74-91% for copolymers and composites, respectively. Unrelated to the resin composition, the PS values of composites are up to 8.4 vol. % and the BFS values of wet composite specimens are on average 51 +/- 8 MPa. The maximum WS values attained in copolymers and composites reach 4.8 mass%. Inclusion of hydrophobic HmDMA monomer in the matrices significantly reduces the WS. The levels of Ca and PO(4) released from all types of composites are significantly above the minimum necessary for the re deposition of apatite to occur. Elevated Ca, and to a lesser extent PO(4) release, is observed in HEMA-containing, ternary EBPADMA formulations. Further resin reformulations may be needed to improve the PS of composite specimens. Poor dispersion of ;as-synthesized' ACP within the composite contributes to their inferior mechanical performance. PMID- 16684799 TI - Imbibition of Swietenia macrophylla (Meliaceae) seeds: the role of stomata. AB - BACKGROUND AND AIMS: The occurrence of stomata in seed coats is uncommon and there is limited information about their function(s). The aim of this study was to verify the distribution of stomata in seed coats of Swietenia macrophylla and to relate it to the imbibition process and aspects of the structure of the outer integument layers. METHODS: For the structural and ultrastructural studies, the seeds were processed using the usual techniques and studied under light and scanning electron microscopes. Histochemical tests were employed to identify the cell wall composition in the different seed coat portions. To assess the role of the stomata in the imbibition, non-impervious seeds were compared with partially impervious ones, in which only the embryo, median or hilar regions were left free. Further, the apoplastic pathway marker was employed to confirm the role of the stomata as sites of water passage during imbibition. KEY RESULTS: A positive relationship was observed between seed coat thickness and stomata density. The stomata were devoid of movement, with a large pore. They occurred in large numbers in the embryo region and extended with lower frequency towards the wing. Imbibition rates were related to stomata density, suggesting that the stomata act as preferential sites for water entry in the S. macrophylla seeds. CONCLUSIONS: At maturity, the stomata in the seed coat play a significant role in seed imbibition. The data may also infer that these permanently opened stomata have an important role in gas exchange during seed development, aiding embryo respiration. PMID- 16684800 TI - Emtricitabine intolerance in treatment-experienced patients switched from lamivudine: a method of assessing toxicity. PMID- 16684801 TI - Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs. AB - The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology. PMID- 16684802 TI - Increased maternal nutrition alters development of the appetite-regulating network in the brain. AB - Individuals exposed to an increased nutrient supply before birth have a high risk of becoming obese children and adults. It has been proposed that exposure of the fetus to high maternal nutrient intake results in permanent changes within the central appetite regulatory network. No studies, however, have investigated the impact of increased maternal nutrition on the appetite regulatory network in species in which this network develops before birth, as in the human. In the present study, pregnant ewes were fed a diet which provided 100% (control, n = 8) or approximately 160% (well-fed, n = 8) of metabolizable energy requirements. Ewes were allowed to lamb spontaneously, and lambs were sacrificed at 30 days of postnatal age. All fat depots were dissected and weighed, and expression of the appetite-regulating neuropeptides and the leptin receptor (OBRb) were determined by in situ hybridization. Lambs of well-fed ewes had higher glucose (Glc) concentrations during early postnatal life (F = 5.93, P<0.01) and a higher relative subcutaneous (s.c.) fat mass at 30 days of age (34.9+/-4.7 g/kg vs. 22.8+/-3.3 g/kg; P<0.05). The hypothalamic expression of pro-opiomelanocortin was higher in lambs of well-fed ewes (0.48+/-0.09 vs. 0.28+/-0.04, P<0.05). In lambs of overnourished mothers, but not in controls, the expression of OBRb was inversely related to total relative fat mass (r2 = 0.50, P = 0.05, n = 8), and the direct relationship between the expression of the central appetite inhibitor CART and fat mass was lost. The expression of neuropeptide Y and AGRP was inversely related to total relative fat mass (NPY, r2 = 0.28, P<0.05; agouti related peptide, r2 = 0.39, P<0.01). These findings suggest that exposure to increased nutrition before birth alters the responses of the central appetite regulatory system to signals of increased adiposity after birth. PMID- 16684803 TI - Detecting and profiling tissue-selective genes. AB - The widespread use of DNA microarray technologies has generated large amounts of data from various tissue and/or cell types. These data set the stage to answer the question of tissue specificity of human transcriptome in a comprehensive manner. Our focus is to uncover the tissue-gene relationship by identifying genes that are preferentially expressed in a small number of tissue types. The tissue selectivity would shed light on the potential physiological functions of these genes and provides an indispensable reference to compare against disease pathophysiology and to identify or validate tissue-specific drug targets. Here we describe a systematic computational and statistical approach to profile gene expression data to identify tissue-selective genes with the use of a more extensive data set and a well-established multiple-comparison procedure with error rate control. Expression data of 35,152 probe sets in 97 normal human tissue types were analyzed, and 3,919 genes were identified to be selective to one or a few tissue types. We presented results of these tissue-selective genes and compared them to those identified by other studies. PMID- 16684805 TI - Growth in clopidogrel-aspirin combination therapy. PMID- 16684804 TI - Diabetes-altered gene expression in rat skeletal muscle corrected by oral administration of vanadyl sulfate. AB - Treatment with vanadium, a representative of a class of antidiabetic compounds, alleviates diabetic hyperglycemia and hyperlipidemia. Oral administration of vanadium compounds in animal models and humans does not cause clinical symptoms of hypoglycemia, a common problem for diabetic patients with insulin treatment. Gene expression, using Affymetrix arrays, was examined in muscle from streptozotocin-induced diabetic and normal rats in the presence or absence of oral vanadyl sulfate treatment. This treatment affected normal rats differently from diabetic rats, as demonstrated by two-way ANOVA of the full array data. Diabetes altered the expression of 133 genes, and the expression of 30% of these genes dysregulated in diabetes was normalized by vanadyl sulfate treatment. For those genes, the ratio of expression in normal animals to the expression in diabetic animals showed a strong negative correlation with the ratio of expression in diabetic animals to the expression in diabetic animals treated with vanadyl sulfate (P = -0.85). The genes identified belong to six major metabolic functional groups: lipid metabolism, oxidative stress, muscle structure, protein breakdown and biosynthesis, the complement system, and signal transduction. The identification of oxidative stress genes, coupled with the known oxidative chemistry of vanadium, implicates reactive oxygen species in the action of this class of compounds. These results imply that early transition metals or compounds formed from their chemical interactions with other metabolites may act as general transcription modulators, a role not usually associated with this class of compounds. PMID- 16684806 TI - Prevalence of diarrhea at a university hospital and association with modifiable risk factors. AB - OBJECTIVE: To assess the prevalence of diarrhea at a university-affiliated medical center and the presence of modifiable risk factors. METHODS: A point prevalence survey was conducted. All patients hospitalized for more than 24 hours were asked if they were experiencing diarrhea. Stools of patients not previously tested were assessed for Clostridium difficile (CD) toxins A and B. Univariate analysis and multivariate logistic regression analyses were used to identify modifiable variables associated with diarrhea (significance defined as p < 0.05). RESULTS: Four hundred eighty-five hospitalized patients were interviewed, of whom 60 (12.4%) reported 2 or more loose, unformed stools in the last 24 hours. Six of 81 (7.4%) patients tested positive for CD toxin. Three (50%) of the CD toxin positive patients had not previously been tested during the current admission. Patients with diarrhea were more likely to have tested CD toxin-positive (OR 10.6; p = 0.01), received antibiotics (OR 1.79; p = 0.04), or been hospitalized for a longer period of time (p = 0.04). CONCLUSIONS: Diarrhea was prevalent in 12.4% of hospitalized patients at a large university hospital at one point in time. Patients with diarrhea were more likely to have CD infection, receive antibiotics, or experience a longer hospitalization. Half of the CD diarrhea cases occurring in the hospital had been previously unidentified. Hospitalized patients should be evaluated for diarrhea on an ongoing basis with appropriate interventions instituted. PMID- 16684807 TI - Symmetrical peripheral gangrene and neutropenia following propylthiouracil. PMID- 16684808 TI - Rash with both clopidogrel and ticlopidine in two patients following percutaneous coronary intervention with drug-eluting stents. AB - OBJECTIVE: To describe 2 cases of rash that occurred following oral administration of both clopidogrel and ticlopidine and to review previously published case reports. CASE SUMMARY: Two patients developed maculopapular pruritic rashes that began on the abdomen and spread to the back, neck, and face following clopidogrel administration after placement of a drug-eluting intracoronary stent. Following recurrence of the rash after ticlopidine was initiated, thienopyridines were discontinued, and the patients were treated for 3 6 months with aspirin, cilostazol, and enoxaparin or warfarin for prevention of stent thrombosis and reinfarction. One patient self-discontinued cilostazol, reinitiated clopidogrel, and redeveloped a rash. DISCUSSION: While there have been several published cases of thienopyridine-associated rash, there have been only 2 reported cases of cross-sensitivity between orally administered clopidogrel and ticlopidine. Preliminary reports suggest that clopidogrel desensitization may be accomplished in selected patients several months to years following thienopyridine rash using an allergy desensitization protocol, with close monitoring for anaphylaxis. An objective causality assessment using the Naranjo probability scale indicated that both ticlopidine and clopidogrel were probable causes of the rash in the first patient; in the second patient, clopidogrel was judged a definite cause and ticlopidine was a probable cause of the rash. CONCLUSIONS: In a patient who develops a rash following clopidogrel treatment after intracoronary stent placement, ticlopidine therapy should be attempted, provided the initial reaction did not include life-threatening symptoms. In a patient who experiences rash with both clopidogrel and ticlopidine and does not have a contraindication to cilostazol or anticoagulation, therapy with aspirin, cilostazol, and either enoxaparin or warfarin may be administered for 2-6 months following placement of a drug-eluting stent. PMID- 16684809 TI - The role of vitamin D in multiple sclerosis. AB - OBJECTIVE: To evaluate the literature about the role of vitamin D in the prevention and treatment of multiple sclerosis (MS). DATA SOURCES: MEDLINE (1966 April 2006) and International Pharmaceutical Abstracts (1970-April 2006) searches were performed. In addition, pertinent references from identified articles were obtained. Key search terms included vitamin D, 25-hydroxyvitamin D, vitamin D deficiency, and multiple sclerosis. DATA SYNTHESIS: Vitamin D supplementation prevented the development and progression of experimental autoimmune encephalitis, an animal model of MS, in mice. A large, prospective, cohort study found that vitamin D supplementation was associated with a 40% reduction in the risk of developing MS. Four small, noncontrolled studies suggested that vitamin D supplementation may decrease exacerbation of MS symptoms. CONCLUSIONS: Vitamin D supplementation may help prevent the development of MS and may be a useful addition to therapy. However, current studies are in small populations and are confounded by other variables, such as additional vitamin and mineral supplementation. PMID- 16684810 TI - The wild ride: cardiology and clinical pharmacy 1967-2005. PMID- 16684811 TI - Role of risperidone in children with autism spectrum disorder. AB - OBJECTIVE: To review the clinical trials investigating the efficacy and safety of risperidone in the treatment of children with autism spectrum disorder (ASD). DATA SOURCES: Searches of MEDLINE/PubMed (1992-February 2006) were conducted, as well as an extensive manual review of journals, using the key words autism and risperidone. STUDY SELECTION AND DATA EXTRACTION: Only double-blind, placebo controlled trials were included for review. DATA SYNTHESIS: ASD is the most common of the pervasive developmental disorders. The main characteristics (core symptoms) of autism are impairment in social skills, problems communicating, and stereotypical movements. Behavioral manifestations or maladaptive behaviors include aggression, irritability, hyperactivity, inattention, impulsivity, tantrums, and self-injurious behavior. CONCLUSIONS: Based on the data examined, risperidone appears efficacious and safe for treating certain behavioral aspects of autism including irritability, aggression, hyperactivity, and stereotypy. It does not appear to be as effective for the treatment of the core symptoms of autism. PMID- 16684812 TI - Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells. AB - Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense-core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense core vesicle exocytosis nor the functional relationship between them is clear. In this study, we expressed a number of different Rab proteins, from Rab1 to Rab41 in PC12 cells and systematically screened them for those that are specifically localized on dense-core vesicles. We found that four Rabs (Rab3A, Rab27A, Rab33A, Rab37) are predominantly targeted to dense-core vesicles in PC12 cells, and that three of them (Rab3A, Rab27A, Rab33A) are endogenously expressed on dense-core vesicles. We further investigated the effect of silencing each Rab with specific small interfering RNA on vesicle dynamics by total internal reflection fluorescence microscopy in a single PC12 cell. Silencing either Rab3A or Rab27A in PC12 cells significantly decreased the number of dense-core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step(s) of dense-core vesicles to the plasma membrane. PMID- 16684813 TI - Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin. AB - The dystrophin glycoprotein complex links laminin in the extracellular matrix to the cell cytoskeleton. Loss of dystrophin causes Duchenne muscular dystrophy, the most common human X-chromosome-linked genetic disease. The alpha7beta1 integrin is a second transmembrane laminin receptor expressed in skeletal muscle. Mutations in the alpha7 integrin gene cause congenital myopathy in humans and mice. The alpha7beta1 integrin is increased in the skeletal muscle of Duchenne muscular dystrophy patients and mdx mice. This observation has led to the suggestion that dystrophin and alpha7beta1 integrin have complementary functional and structural roles. To test this hypothesis, we generated mice lacking both dystrophin and alpha7 integrin (mdx/alpha7(-/-)). The mdx/alpha7(-/-) mice developed early-onset muscular dystrophy and died at 2-4 weeks of age. Muscle fibers from mdx/alpha7(-/-) mice exhibited extensive loss of membrane integrity, increased centrally located nuclei and inflammatory cell infiltrate, greater necrosis and increased muscle degeneration compared to mdx or alpha7-integrin null animals. In addition, loss of dystrophin and/or alpha7 integrin resulted in altered expression of laminin-alpha2 chain. These results point to complementary roles for dystrophin and alpha7beta1 integrin in maintaining the functional integrity of skeletal muscle. PMID- 16684814 TI - A MORN-repeat protein is a dynamic component of the Toxoplasma gondii cell division apparatus. AB - Apicomplexan parasites divide and replicate through a complex process of internal budding. Daughter cells are preformed within the mother on a cytoskeletal scaffold, endowed with a set of organelles whereby in the final stages the mother disintegrates and is recycled in the emerging daughters. How the cytoskeleton and the various endomembrane systems interact in this dynamic process remains poorly understood at the molecular level. Through a random YFP fusion screen we have identified two Toxoplasma gondii proteins carrying multiple membrane occupation and recognition nexus (MORN) motifs. MORN1 is highly conserved among apicomplexans. MORN1 specifically localizes to ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, a specialized nuclear structure that organizes the mitotic spindle. Time-lapse imaging of tagged MORN1 revealed that these structures are highly dynamic and appear to play a role in nuclear division and daughter cell budding. Overexpression of MORN1 resulted in severe but specific defects in nuclear segregation and daughter cell formation. We hypothesize that MORN1 functions as a linker protein between certain membrane regions and the parasite's cytoskeleton. Our initial biochemical analysis is consistent with this model. Whereas recombinant MORN1 produced in bacteria is soluble, in the parasite MORN1 was associated with the cytoskeleton after detergent extraction. PMID- 16684815 TI - A complex interaction pattern of CIS and SOCS2 with the leptin receptor. AB - Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity. PMID- 16684816 TI - The Drosophila phosphatidylinositol transfer protein encoded by vibrator is essential to maintain cleavage-furrow ingression in cytokinesis. AB - Cytokinesis requires the coordination of cytoskeletal and plasma membrane dynamics. A role for phosphatidylinositol lipids has been proposed for the successful completion of cytokinesis but this is still poorly characterised. Here, we show mutants of the gene vibrator, previously found to encode the Drosophila phosphatidylinositol transfer protein, produce multinucleate cells indicative of cytokinesis failure in male meiosis. Examination of fixed preparations of mutant spermatocytes showed contractile rings of anillin and actin that were of normal appearance at early stages but were larger and less well organised at later stages of cytokinesis than in wild-type cells. Time-lapse imaging revealed sequential defects in cytokinesis of vibrator spermatocytes. In cells that fail cytokinesis, central spindle formation occurred correctly, but furrow ingression was delayed and the central spindle did not become compressed to the extent seen in wild-type cells. Cells then stalled at this point before the apparent connection between the constricted cytoskeleton and the plasma membrane was lost; the furrow then underwent elastic regression. We discuss these defects in relation to multiple functions of phosphoinositol lipids in regulating actin dynamics and membrane synthesis. PMID- 16684817 TI - Mesenchymal stem cells reside in virtually all post-natal organs and tissues. AB - Mesenchymal stem cells (MSCs) are multipotent cells which can give rise to mesenchymal and non-mesenchymal tissues in vitro and in vivo. Whereas in vitro properties such as (trans)differentiation capabilities are well known, there is little information regarding natural distribution and biology in the living organism. To investigate the subject further, we generated long-term cultures of cells with mesenchymal stem cell characteristics from different organs and tissues from adult mice. These populations have morphology, immunophenotype and growth properties similar to bone marrow-derived MSCs. The differentiation potential was related to the tissue of origin. The results indicate that (1) cells with mesenchymal stem characteristics can be derived and propagated in vitro from different organs and tissues (brain, spleen, liver, kidney, lung, bone marrow, muscle, thymus, pancreas); (2) MSC long-term cultures can be generated from large blood vessels such as the aorta artery and the vena cava, as well as from small vessels such as those from kidney glomeruli; (3) MSCs are not detected in peripheral blood. Taken together, these results suggest that the distribution of MSCs throughout the post-natal organism is related to their existence in a perivascular niche. These findings have implications for understanding MSC biology, and for clinical and pharmacological purposes. PMID- 16684818 TI - Vitamin D status in relation to one-year risk of recurrent falling in older men and women. AB - BACKGROUND: Falls frequently occur in the elderly and are a major cause of morbidity and mortality. OBJECTIVE: The objective of the study was to prospectively investigate the association between serum 25-hydroxyvitamin D [25(OH)D] levels and risk of recurrent falling in older men and women. DESIGN: This was a prospective cohort study. SETTING: An age- and sex-stratified random sample of the Dutch older population was determined. SUBJECTS: Subjects included 1231 men and women (aged 65 yr and older) participating in the Longitudinal Aging Study Amsterdam. MEASUREMENTS: Baseline serum 25(OH)D was determined by a competitive protein binding assay. During 1 yr, falls were prospectively recorded by means of a fall calendar. RESULTS: Low 25(OH)D (<10 ng/ml) was associated with an increased risk of falling. After adjustment for age, sex, education level, region, season, physical activity, smoking, and alcohol intake, the odds ratios (95% confidence interval) were 1.78 (1.06-2.99) for subjects who experienced two falls or more as compared with those who did not fall or fell once and 2.23 (1.17 4.25) for subjects who fell three or more times as compared with those who fell two times or less. There was a statistically significant effect modification by age, and stratified analyses (<75 and > or = 75 yr) showed that the associations were particularly strong in the younger age group; the odds ratios (95% confidence interval) were 5.21 (2.03-13.40) for two falls or more and 4.96 (1.52 16.23) for three falls or more. CONCLUSIONS: Poor vitamin D status is independently associated with an increased risk of falling in the elderly, particularly in those aged 65-75 yr. PMID- 16684819 TI - The diagnosis of ectopic focal hyperinsulinism of infancy with [18F]-dopa positron emission tomography. AB - BACKGROUND: Congenital hyperinsulinism (CHI) is a cause of severe hypoglycemia in the neonatal and infancy period. Histologically, there are two subtypes with diffuse and focal disease. The preoperative differentiation of these two forms is very important because the surgical management is radically different. The focal form of the disease can be cured if the focal lesion can be localized accurately and completely resected with surgery. AIM: We report the case of a child who underwent three pancreatectomies with a choledochoduodenostomy and a cholecystectomy but continued to have severe hyperinsulinemic hypoglycemia. METHODS/RESULTS: Radiological investigations including imaging with (18)fluoro-L Dopa positron emission tomography scan showed a clear focus of increased (18)F fluoro-L-Dopa uptake in the vicinity of the former head of the pancreas. On the magnetic resonance imaging scan, this focal uptake appeared to localize adjacent or next to duodenum (in the wall or cavity of the duodenum). CONCLUSIONS: This unique case highlights the importance of correctly localizing and completely resecting the focal lesion in patients with CHI. (18)Fluoro-L-Dopa positron emission tomography scan can identify ectopic focal lesions in patients with CHI. PMID- 16684821 TI - Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AB - CONTEXT: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is known as a rare hereditary disease with classic triad of mucocutaneous candidiasis, hypoparathyroidism, and adrenocortical failure, two of which, diagnostic dyad, are required for the diagnosis. Evidently many patients suffer unrecognized because the condition is more variable and complex. OBJECTIVE: The objective of the study was to describe the variability of autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy for promoting recognition and adequate follow-up of patients. SETTING: The Finnish series of patients is the largest internationally. PATIENTS: The study population was all 91 known Finnish patients. RESULTS: Besides the classical triad, a dozen autoimmune endocrine and other components occurred variably, several of them dangerous. The initial manifestation appeared within the age range of 0.2-18 yr, mucocutaneous candidiasis being part of it in 60% of the patients, hypoparathyroidism in 32%, and adrenocortical failure in 5%. But 23% of the patients had one to six other components before the diagnostic dyad: hepatitis, keratoconjunctivitis, chronic diarrhea, periodic rash with fever. The dyad appeared 0.2-20 yr later. Prevalence of most components increased with age, diabetes mellitus, hypothyroidism, and testicular failure becoming common toward middle age. Tubulointerstitial nephritis occurred in 9% of the patients, apparent mineralocorticoid excess in 9%, asplenia in 19% of adults, and oral or esophageal squamous cell carcinoma in 10% of patients older than 25 yr. CONCLUSIONS: Any child or young adult with one of the many disease components should be examined for others and consideration of AIRE mutation assay. PMID- 16684820 TI - Regulation, function, and dysregulation of endocannabinoids in models of adipose and beta-pancreatic cells and in obesity and hyperglycemia. AB - CONTEXT: Cannabinoid CB(1) receptor blockade decreases weight and hyperinsulinemia in obese animals and humans in a way greatly independent from food intake. OBJECTIVE: The objective of this study was to investigate the regulation and function of the endocannabinoid system in adipocytes and pancreatic beta-cells. DESIGN, SETTING, AND PATIENTS: Mouse 3T3-F442A adipocytes and rat insulinoma RIN-m5F beta-cells, pancreas and fat from mice with diet induced obesity, visceral and sc fat from patients with body mass index equal to or greater than 30 kg/m(2), and serum from normoglycemic and type 2 diabetes patients were studied. MAIN OUTCOME MEASURE: Endocannabinoid enzyme and adipocyte protein expression, and endocannabinoid and insulin levels were measured. RESULTS: Endocannabinoids are present in adipocytes with levels peaking before differentiation, and in RIN-m5F beta-cells, where they are under the negative control of insulin. Chronic treatment of adipocytes with insulin is accompanied by permanently elevated endocannabinoid signaling, whereas culturing of RIN-m5F beta-cells in high glucose transforms insulin down-regulation of endocannabinoid levels into up-regulation. Epididymal fat and pancreas from mice with diet induced obesity contain higher endocannabinoid levels than lean mice. Patients with obesity or hyperglycemia caused by type 2 diabetes exhibit higher concentrations of endocannabinoids in visceral fat or serum, respectively, than the corresponding controls. CB(1) receptor stimulation increases lipid droplets and decreases adiponectin expression in adipocytes, and it increases intracellular calcium and insulin release in RIN-m5F beta-cells kept in high glucose. CONCLUSIONS: Peripheral endocannabinoid overactivity might explain why CB(1) blockers cause weight-loss independent reduction of lipogenesis, of hypoadiponectinemia, and of hyperinsulinemia in obese animals and humans. PMID- 16684823 TI - Metformin treatment to prevent early puberty in girls with precocious pubarche. AB - CONTEXT AND OBJECTIVE: Girls with precocious pubarche (PP, pubic hair at < 8 yr of age) are at high risk for early onset and rapid progression of puberty, in particular if their prenatal growth was restrained, i.e. low birth weight (LBW), and followed by rapid postnatal catch-up of weight gain. We postulated that insulin resistance contributes to early onset and rapid progression of puberty in LBW-PP girls and thus explored the puberty-delaying effects of insulin sensitization with metformin initiated shortly after PP diagnosis. SETTING, DESIGN, AND PATIENTS: The study population consisted of 38 prepubertal LBW girls with PP attributed to exaggerated adrenarche [mean body weight, 2.4 kg; age, 7.9 yr; body mass index (BMI), 18.4 kg/m(2)]. These girls were randomly assigned to remain untreated (n = 19) or to receive metformin (n = 19; 425 mg/d) for 2 yr. MAIN OUTCOME MEASURES: Pubertal staging, age at menarche, body composition by absorptiometry, fasting insulin, glucose, lipids, leptin, IGF-I, IGF-binding protein-1, testosterone, dehydroepiandrosterone sulfate, and SHBG were the main outcome measures. RESULTS: Metformin treatment was associated with a less adipose body composition (and lower serum leptin levels) and with a 0.4-yr delay in the clinical onset of puberty (Tanner B2; 9.5 vs. 9.1 yr; P < 0.01). These findings were corroborated by a delay of at least 1 yr in the puberty-associated rise of circulating IGF-I (P < 0.01). Available results also point to a metformin associated delay of menarche (P < 0.02). Gain in height and lean mass was not divergent between study subgroups. CONCLUSION: The efficacy of early metformin treatment in PP girls is here extended to include not only a less adipose body composition after 2 yr but also a less advanced onset of puberty, whereas height gain is maintained. These findings open the perspective that, ultimately, metformin treatment may also prove to heighten the short adult stature of LBW-PP girls. PMID- 16684822 TI - Analysis of DAX1 (NR0B1) and steroidogenic factor-1 (NR5A1) in children and adults with primary adrenal failure: ten years' experience. AB - CONTEXT: Primary adrenal failure is a life-threatening condition that can be caused by a range of etiologies, including autoimmune, metabolic, and developmental disorders. The nuclear receptors DAX1 (NR0B1) and steroidogenic factor-1 (SF1/Ad4BP, NR5A1) play an important role in adrenal development and function, and mutations in these transcription factors have been found in patients with adrenal hypoplasia. OBJECTIVE: Our objective was to investigate the prevalence of DAX1 and SF1 mutations in children and adults with primary adrenal failure of unknown etiology (i.e. not caused by congenital adrenal hyperplasia, adrenoleukodystrophy, or autoimmune disease). PATIENTS: One hundred seventeen patients were included. Eighty-eight individuals presented in infancy or childhood with adrenal hypoplasia or primary adrenal failure of unknown etiology (n = 64 46,XY phenotypic males; n = 17 46,XY gonadal dysgenesis/impaired androgenization; n = 7 46,XX females). Twenty-nine individuals presented in adulthood with Addison's disease of unknown etiology. METHODS: Mutational analysis of DAX1 (NR0B1) (including exon 2alpha/1A) and SF1 (NR5A1) was done by direct sequencing. RESULTS: DAX1 mutations were found in 58% (37 of 64) of 46,XY phenotypic boys referred with adrenal hypoplasia and in all boys (eight of eight) with hypogonadotropic hypogonadism and a family history suggestive of adrenal failure in males. SF1 mutations causing adrenal failure were found in only two patients with 46,XY gonadal dysgenesis. No DAX1 or SF1 mutations were identified in the adult-onset group. CONCLUSIONS: DAX1 mutations are a relatively frequent cause of adrenal failure in this group of boys. SF1 mutations causing adrenal failure in humans are rare and are more likely to be associated with significant underandrogenization and gonadal dysfunction in 46,XY individuals. PMID- 16684824 TI - Commentary: Polycystic ovary syndrome--increased or preserved ovarian sensitivity to insulin? PMID- 16684825 TI - Effects of teriparatide, alendronate, or both on bone turnover in osteoporotic men. AB - CONTEXT: We have previously demonstrated that alendronate reduces the ability of teriparatide to increase bone mineral density (BMD) in osteoporotic men. The underlying basis for this observation is poorly understood. OBJECTIVE: The primary aim of this study was to determine whether teriparatide increases osteoblast activity when the ability of teriparatide to increase osteoclast activity is suppressed by alendronate. DESIGN: This was a nonblinded, randomized, controlled trial. SETTING: The study was conducted at the General Clinical Research Center of a teaching hospital. PATIENTS: We studied 63 men, age 46-85, with low spine and/or hip BMD. INTERVENTIONS: Subjects received alendronate 10 mg daily (group 1), teriparatide 37 microg sc daily (group 2), or both (group 3) for 30 months. Teriparatide was begun at month 6. MAIN OUTCOME MEASURES: The primary endpoint was the change in serum N-telopeptide, osteocalcin, and amino-terminal propeptide of type 1 procollagen. RESULTS: In men receiving teriparatide monotherapy (group 2), levels of all bone turnover markers increased markedly during the first 6 months of teriparatide administration and then declined toward baseline during the next 18 months. In men who received combination therapy (group 3), bone turnover marker levels declined in the first 6 months (while receiving alendronate alone) and then returned to baseline levels (N-telopeptide) or above (osteocalcin and amino-terminal propeptide of type 1 procollagen) after teriparatide was added. Changes in each marker were significantly different between groups 1 and 2 (all P values < 0.001), groups 1 and 3 (all P values < 0.001), and groups 2 and 3 (all P values < 0.03). CONCLUSIONS: As with BMD, alendronate impairs the action of teriparatide to increase bone turnover in men. PMID- 16684826 TI - Goitrous congenital hypothyroidism and hearing impairment associated with mutations in the TPO and SLC26A4/PDS genes. AB - CONTEXT: Pendred syndrome (PS) and thyroid peroxidase (TPO) deficiency are autosomal-recessive disorders that result in thyroid dyshormonogenesis. They share congenital hypothyroidism, goiter, and an iodide organification defect as common features. Whereas the hallmark of PS is sensorineural deafness, other forms of congenital hypothyroidism may also lead to hearing impairment. Therefore, a definite diagnosis may be difficult and require molecular genetic analyses. CASE REPORT: The propositus presented at birth with primary hypothyroidism and goiter. He also had congenital bilateral moderate hearing loss, and PS was suspected. METHODS: We sequenced the SLC26A4/PDS and TPO genes in the propositus and tested familial segregation of mutations in all available family members who were phenotypically normal. The functional consequences of the identified pendrin mutation (p.R776C) were studied in vitro. RESULTS: Sequencing of the SLC26A4/PDS gene revealed a single monoallelic missense mutation in the propositus (p.R776C). This mutation, which was inherited from his unaffected mother, has previously been identified in an individual with deafness and an enlarged vestibular aqueduct. Sequencing of the TPO gene revealed compound heterozygosity for a novel nonsense mutation (p.Q235X) and a known missense mutation (p.Y453D). The mutant pendrin (p.R776C) retained its ability to transport iodide in vitro. CONCLUSIONS: These results show that the propositus carries three sequence variants in two genes: a monoallelic SLC26A4/PDS sequence variant and compound heterozygous TPO mutations. Our study illustrates that if only a single heterozygous SLC26A4/PDS mutation is found in a patient with goiter and deafness, other genetic explanations should be considered. PMID- 16684827 TI - Hyperthyrotropinemia in obese children is reversible after weight loss and is not related to lipids. AB - CONTEXT: There is some controversy whether T(4) treatment is indicated in obese humans with hyperthyrotropinemia. OBJECTIVE: The objective of this study was to examine whether hyperthyrotropinemia is a cause or a consequence of obesity. DESIGN: The study was designed as a cross-sectional comparison between obese and lean children and includes a 1-yr follow-up study. SETTING: The study was set in a primary care facility. PATIENTS: The patients were 246 obese and 71 lean children. INTERVENTION: The 1-yr intervention program was based on exercise, behavior therapy, and nutrition education. MAIN OUTCOME MEASURES: The main outcome measures were TSH, free T(3) (fT3), free T(4) (fT4), high-density lipoprotein, low-density lipoprotein, and total cholesterol at baseline and 1 yr later. RESULTS: TSH (P = 0.009) and fT3 (P = 0.003) concentrations were significantly higher in obese children than in normal weight children, whereas there was no difference in fT4 levels (P = 0.804). Lipids did not correlate significantly to thyroid hormones in cross-sectional and longitudinal analyses. fT3, fT4, and lipids did not differ significantly in the 43 (17%) children with TSH levels above the normal range from the children with TSH levels within the normal range. Substantial weight loss in 49 obese children led to a significant reduction of TSH (P = 0.035) and fT3 (P = 0.036). The 197 obese children without substantial weight loss demonstrated no significant changes of thyroid hormones. CONCLUSIONS: Because fT3 and TSH were moderately increased in obese children and weight loss led to a reduction, the elevation of these hormones seems to be rather a consequence of obesity than a cause of obesity. Because fT3 and TSH were both increased in obesity and thyroid hormones were not associated to lipids, we put forward the hypothesis that there is no necessity for thyroxine treatment. PMID- 16684828 TI - Adult height in patients with permanent growth hormone deficiency with and without multiple pituitary hormone deficiencies. AB - CONTEXT: It has been reported that patients with multiple pituitary hormone deficiencies (MPHDs) achieve a greater final height, compared with patients with isolated GH deficiency (IGHD). However, the outcome of patients with permanent GH deficiency (GHD) has not yet been reported. OBJECTIVES: The objectives of the study were to evaluate and compare adult height data and the effect of spontaneous or induced puberty after long-term treatment with GH in young adults with either permanent IGHD or MPHD. DESIGN AND SETTING: This was a retrospective multicenter study conducted in university research hospitals and a tertiary referral endocrine unit. PATIENTS AND METHODS: Thirty-nine patients with IGHD (26 males, 13 females) and 49 with MPHD (31 males, 18 females), diagnosed at a median age of 7.7 and 6.9 yr, respectively, were reevaluated for GH secretion after adult height achievement (median age 17.6 and 19.8 yr). The diagnosis of permanent GHD was based on peak GH levels less than 3 microg/liter after an insulin tolerance test or peak GH levels less than 5 microg/liter after two different tests. Fifteen subjects had idiopathic GHD and seventy-three had magnetic resonance imaging evidence of congenital hypothalamic-pituitary abnormalities. Height sd score (SDS) was analyzed at diagnosis, the onset of puberty (either spontaneous or induced), and the time of GH withdrawal. RESULTS: The subjects with IGHD entered puberty at a median age of 12.6 yr (females) and 13.4 yr (males). Puberty was induced at a median age of 13.5 and 14.0 yr, respectively, in males and females with MPHD. Median height SDS at the beginning of puberty was similar in the IGHD and MPHD subjects. Total pubertal height gain was similar between patients with IGHD or MPHD. Median adult height was also not significantly different between IGHD and MPHD patients (males, 168.5 vs. 170.3 cm; females, 160.0 vs. 157.3 cm). The adult height SDS of the IGHD subjects was positively correlated with height at the time of diagnosis and with total pubertal height gain. Conversely, the adult height SDS of the MPHD subjects was positively correlated with both the duration of GH treatment and height SDS at the time of GHD diagnosis. CONCLUSIONS: Adult height in patients with permanent IGHD and spontaneous puberty is similar to adult height in patients with MPHD and induced puberty. PMID- 16684829 TI - Changes induced by physical activity and weight loss in the morphology of intermyofibrillar mitochondria in obese men and women. AB - CONTEXT: In obesity, skeletal muscle insulin resistance may be associated with smaller mitochondria. OBJECTIVE: Our objective was to examine the effect of a lifestyle-modification intervention on the content and morphology of skeletal muscle mitochondria and its relationship to insulin sensitivity in obese, insulin resistant subjects. DESIGN: In this prospective interventional study, intermyofibrillar mitochondrial content and size were quantified by transmission electron microscopy with quantitative morphometric analysis of biopsy samples from vastus lateralis muscle. Systemic insulin sensitivity was measured with euglycemic hyperinsulinemic clamps. SETTING: The study took place at a university based clinical research center. PARTICIPANTS: Eleven sedentary, overweight/obese volunteers without diabetes participated in the study. INTERVENTION: Intervention included 16 wk of aerobic training with dietary restriction of 500-1000 kcal/d. MAIN OUTCOME MEASURES: We assessed changes in mitochondrial content and size and changes in insulin sensitivity. RESULTS: The percentage of myofiber volume occupied by mitochondria significantly increased from 3.70 +/- 0.31 to 4.87 +/- 0.33% after intervention (P = 0.01). The mean individual increase was 42.5 +/- 18.1%. There was also a change in the mean cross-sectional mitochondrial area, increasing from a baseline of 0.078 +/- 0.007 to 0.091 +/- 0.007 microm(2) (P < 0.01), a mean increase of 19.2 +/- 6.1% per subject. These changes in mitochondrial size and content highly correlated with improvements in insulin resistance (r = 0.68 and 0.72, respectively; P = 0.01). CONCLUSIONS: A combined intervention of weight loss and physical activity in previously sedentary obese adults is associated with enlargement of mitochondria and an increase in the mitochondrial content in skeletal muscle. These findings indicate that in obesity with insulin resistance, ultrastructural mitochondrial plasticity is substantially retained and, importantly, that changes in the morphology of mitochondria are associated with improvements in insulin resistance. PMID- 16684830 TI - Long-term outcome of 444 patients with distant metastases from papillary and follicular thyroid carcinoma: benefits and limits of radioiodine therapy. AB - AIM: The goal of this study was to estimate the cumulative activity of (131)I to be administered to patients with distant metastases from thyroid carcinoma. METHODS: A total of 444 patients were treated from 1953-1994 for distant metastases from papillary and follicular thyroid carcinoma: 223 had lung metastases only, 115 had bone metastases only, 82 had both lung and bone metastases, and 24 had metastases at other sites. Treatment consisted of the administration of 3.7 GBq (100 mCi) (131)I after withdrawal of thyroid hormone treatment, every 3-9 months during the first 2 yr and then once a year until the disappearance of any metastatic uptake. Thyroxine treatment was given at suppressive doses between (131)I treatment courses. RESULTS: Negative imaging studies (negative total body (131)I scans and conventional radiographs) were attained in 43% of the 295 patients with (131)I uptake; more frequently in those who were younger, had well-differentiated tumors, and had a limited extent of disease. Most negative studies (96%) were obtained after the administration of 3.7-22 GBq (100-600 mCi). Almost half of negative studies were obtained more than 5 yr after the initiation of the treatment of metastases. Among patients who achieved a negative study, only 7% experienced a subsequent tumor recurrence. Overall survival at 10 yr after initiation of (131)I treatment was 92% in patients who achieved a negative study and 19% in those who did not. CONCLUSION: (131)I treatment is highly effective in younger patients with (131)I uptake and with small metastases. They should be treated until the disappearance of any uptake or until a cumulative activity of 22 GBq has been administered. In the other patients, other treatment modalities should be used when tumor progression has been documented. PMID- 16684831 TI - Mechanisms of irregular bleeding with hormone therapy: the role of matrix metalloproteinases and their tissue inhibitors. AB - CONTEXT: Irregular bleeding is common in users of combined hormone therapy (HT) and often leads to invasive and expensive investigations to exclude underlying pathology. The mechanisms of HT-related bleeding are poorly understood. Endometrial matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to regulate bleeding during the normal menstrual cycle and are known to be altered in breakthrough bleeding with progestogen-only contraception. OBJECTIVE: The aim of this study was to determine how HT exposure alters endometrial production of MMP-1, -3, -9, and -14 and their tissue inhibitors TIMP 1, -2, -3, and -4 and to determine the relationship between MMP and TIMP production and bleeding patterns in HT users. Endometrial leukocytes regulating MMP production and activation were also assessed. DESIGN: A prospective observational study was conducted between 2003 and 2005. SETTING AND PATIENTS: The study occurred at a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia, and included 25 postmenopausal women not taking HT and 73 women taking combined HT. INTERVENTIONS: Endometrium was obtained during and outside bleeding episodes. MAIN OUTCOME MEASURES: We assessed production of MMP-1, -3, -9, and -14 and their tissue inhibitors TIMP-1, -2, -3, and -4 and their relationship to bleeding patterns in HT users. RESULTS: All MMPs studied, with the exception of MMP-9, were expressed at low levels in postmenopausal endometrium. Increases in both MMP-3 and -9 localization were seen in association with irregular bleeding, but these did not reach statistical significance. Endometrial production of TIMP-1 was significantly increased in association with bleeding. Endometrial leukocytes were not related to bleeding, with the exception of uterine natural killer cells, which were significantly increased during bleeding, as previously published. CONCLUSIONS: Irregular bleeding in HT users is associated with a distinct pattern of MMP and TIMP production that differs from that seen in normal menstrual bleeding and from that seen in contraceptive-related breakthrough bleeding. This suggests that the endometrial balance between MMP and TIMP contributes to vascular breakdown with HT but by a different mechanism than that seen in normal menstruation or in breakthrough bleeding. PMID- 16684832 TI - Activating mutations in the luteinizing hormone receptor gene: a human model of non-follicle-stimulating hormone-dependent inhibin production and germ cell maturation. AB - CONTEXT: Familial male-limited precocious puberty is a dominant autosomal genetic disease caused by activating LH receptor gene mutations, clinically expressed only in males. In preliminary studies, in addition to the expected testosterone increase, we found high inhibin B levels before the age of normal puberty. OBJECTIVES: The objective of the study was to assess the cellular origin of serum inhibin thanks to testis section immunostaining. MAIN OUTCOME MEASURES: Serum testosterone, gonadotropin, inhibin B, pan-alphaC-inhibin, and anti-Mullerian hormone levels were measured. Immunostaining was performed using specific anti alpha- and anti-beta-subunit antibodies. SUBJECTS AND METHODS: Five boys from three families (mutation M398T or I542L) were investigated at onset (2-6 yr), on ketoconazole treatment, and at adolescence. Testis biopsies were performed in three subjects before the disease was fully characterized. RESULTS: The high testosterone levels were suppressed by ketoconazole. Anti-Mullerian hormone levels were inversely related to testosterone: low at diagnosis, elevated after testosterone suppression. Despite FSH suppression, inhibin B and pan-alphaC inhibin levels were high from clinical onset to adolescence. Biopsy specimens showed normal Sertoli cell complement and germ cell maturation until the spermatocyte II stage. Sertoli and Leydig cells displayed positive inhibin alpha subunit immunostaining. Only Leydig cells and spermatogonia stained positively for the inhibin betaB-subunit. CONCLUSIONS: Familial male-limited precocious puberty is a unique model of inhibin B secretion, demonstrating that Leydig cells can produce significant amounts of the dimeric molecule. Our results also suggest that the pubertal FSH rise is not required for full expression of the two inhibin B genes and for the initiation of germ cell maturation. PMID- 16684833 TI - Pernicious anemia and widespread absence of gastrointestinal endocrine cells in a patient with autoimmune polyglandular syndrome type I and malabsorption. AB - CONTEXT: Autoimmune polyglandular syndrome type I (APS I) is characterized by multiple endocrine gland failures, with other manifestations such as gastrointestinal (GI) symptoms. OBJECTIVE: The objective of the study was to study the histopathological and immunological findings in the GI mucosa of a patient with typical features of APS I, malabsorption, and pernicious anemia. DESIGN AND PATIENT: Biopsies from the GI tract of a patient with APS I were immunostained with chromogranin for GI endocrine cells (GIECs). Blinded slides were graded for numbers of endocrine cells. Normal gastric mucosa was exposed to the patient's serum to test for circulating anti-GIEC and antiparietal cell antibodies using indirect immunofluorescence. SETTING: The study was conducted at the Departments of Pediatrics and Medical Gastroenterology in an academic medical center. RESULTS: The patient's GI mucosa demonstrated absence of GIECs throughout, including gastric gastrin-secreting cells, and her laboratory tests for serum gastrin levels were low normal. Both GIECs and parietal cells were absent in her gastric corpus. The patient's serum contained anti-GIEC antibody but no antiparietal cell antibody. CONCLUSIONS: These observations suggest that GIECs in APS I are subject to an autoimmune destruction that can cause widespread GIEC loss. This could explain the GI dysfunctions that are often noted in the syndrome including malabsorption and atrophic gastric changes with pernicious anemia. We also hypothesize that absence of gastric parietal cells may result mainly from hypogastrinemia that is mainly the loss of gastrin-secreting cells rather than from immune-mediated destruction of parietal cells like that seen in the atrophic gastritis associated with adult-onset pernicious anemia. PMID- 16684834 TI - Leptin interferes with adrenocorticotropin/3',5'-cyclic adenosine monophosphate (cAMP) signaling, possibly through a Janus kinase 2-phosphatidylinositol 3 kinase/Akt-phosphodiesterase 3-cAMP pathway, to down-regulate cholesterol side chain cleavage cytochrome P450 enzyme in human adrenocortical NCI-H295 cell line. AB - CONTEXT: Obesity has adverse effects on adrenocortical functions. Adipocyte derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. OBJECTIVE: We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI H295 tumor cell line. METHODS: Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system. RESULTS: Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP responsive region located between -1.7 and -1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3-kinase/Akt (wortmannin) as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including nonspecific 3-isobutyl-1-methylxanthine and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-nonhydrolyzable N(6)-monobutyryl-cAMP. CONCLUSIONS: Leptin interferes with ACTH/cAMP signaling, possibly through a cAMP-degrading mechanism involving activation of JAK2, phosphatidylinositol 3-kinase, and PDE3, to down regulate P450scc expression and consequent adrenal steroidogenesis. PMID- 16684835 TI - An increased coronary risk is paradoxically associated with common cholesteryl ester transfer protein gene variations that relate to higher high-density lipoprotein cholesterol: a population-based study. AB - BACKGROUND: Several cholesteryl ester transfer protein (CETP) polymorphisms affect high-density lipoprotein (HDL) cholesterol, but the impact of CETP gene variants on incident coronary disease in the general population is uncertain after correction for their effect on HDL cholesterol. DESIGN: We determined relationships between the CETP -629C-->A promoter (n = 8141), the TaqIB (n = 8289), and the I405V (n = 8265) polymorphisms, serum lipids, C-reactive protein, and clinical factors with incident coronary heart disease (defined as death from or hospitalization for myocardial infarction, ischemic heart disease, or coronary intervention) during a median of 4.94 yr follow-up. SUBJECTS: A predominantly Caucasian general population was studied. RESULTS: HDL cholesterol was 0.08 mmol/liter higher in -629A carriers than in -629CC homozygotes (P < 0.001). The unadjusted coronary hazard was 1.26 [95% confidence interval (CI), 0.95-1.68; P = 0.11] in A carriers compared with CC homozygotes and increased to 1.46 (95% CI, 1.10-1.95; P = 0.01) after adjustment for HDL cholesterol. This effect remained after additional adjustment for apolipoprotein A-I, triglycerides, C-reactive protein, age, and gender. Likewise, the HDL-cholesterol-adjusted hazard ratio was also higher in AA than in CC homozygotes (hazard ratio, 1.72; 95% CI, 1.22-2.42; P < 0.01). Similar findings were obtained with the TaqIB polymorphism. The 405V allele was weakly associated with incident coronary heart disease after HDL cholesterol adjustment (P = 0.09). CONCLUSIONS: A common CETP promoter polymorphism, which beneficially contributes to higher HDL cholesterol, is paradoxically associated with increased incidence of coronary disease in the general population. Thus, CETP gene variation may affect coronary risk apart from the level of HDL cholesterol. PMID- 16684836 TI - Glucocorticoid receptor polymorphism affects transrepression but not transactivation. AB - CONTEXT: Glucocorticoids (GCs) are extensively used in the treatment of inflammatory and autoimmune diseases. Their beneficial effects are thought to be mediated by GC transrepression on gene expression. However, their use is limited by serious adverse effects, presumably mediated by GC transactivation of gene expression. OBJECTIVE: The objective of the study was to investigate the effect of the GC receptor haplotype, characterized by the GR-9beta polymorphism, on GC transactivation and transrepression. DESIGN AND METHODS: This was a cross sectional study in 216 persons randomly selected from participants in The Rotterdam Study, a population-based cohort study in the elderly. Clinical and biochemical parameters of GC sensitivity were measured: weight, height, waist to hip ratio, glucose, insulin, total cholesterol, high-density lipoprotein, and C reactive protein. In a dexamethasone suppression test, the response of serum cortisol concentrations was studied. Genotyping for four GC receptor polymorphisms was performed. In addition, ex vivo experiments were performed with leukocytes of 10 healthy controls and two persons homozygous for the GR-9beta polymorphism, in which the expression of two GC-sensitive genes, GC-induced leucine zipper and IL-2, was measured. RESULTS: Persons carrying the GR-9beta haplotype without 22/23EK (n = 53) revealed no significant differences in their body mass index, waist to hip ratio, fat spectrum, and insulin sensitivity or in their cortisol response to dexamethasone and levels of C-reactive protein, compared with noncarriers (n = 113). Ex vivo, GC-induced up-regulation of GC induced leucine zipper mRNA via transactivation did not significantly differ in GR-9beta homozygotes, whereas the down-regulation of IL-2 expression via transrepression was decreased. CONCLUSION: Persons carrying the GR-9beta haplotype seem to have a decreased GC transrepression with normal transactivation. PMID- 16684837 TI - Frozen embryo transfers: implications of clinical and embryological factors on the pregnancy outcome. AB - BACKGROUND: Frozen embryo transfers are characterized by impaired pregnancy outcome and increased incidence of pregnancy loss as compared with fresh IVF/ICSI embryo transfers. In this study, we performed a retrospective analysis of clinical and embryological factors that potentially influence the outcome of frozen embryo transfer. METHODS: We reviewed the outcome of 1242 frozen embryo transfers with respect to the age of the woman, the method of fertilization, embryo quality before and after freezing and the number of embryos transferred. RESULTS AND CONCLUSIONS: The pregnancy (positive hCG) and clinical pregnancy rates were 25.8 and 21.1%, respectively. A total of 107 (33.3%) of the 321 pregnancies identified by a positive hCG test miscarried either before (18.4%) or after (15%) the clinical recognition of gestational sac(s). The delivery rate for the frozen embryo transfers analysed was 17.2%. Our data revealed that the delivery rate after frozen embryo transfer was dependent on both the woman's age and the quality of embryos transferred, at the same time being unaffected by IVF/ICSI treatment. In addition, the increased woman's age at IVF/ICSI treatment was identified as the only parameter elevating the biochemical pregnancy rate, whereas the clinical abortion rate was found to be unrelated to the clinical or embryological parameters studied. PMID- 16684838 TI - Abnormal glucose tolerance in Chinese women with polycystic ovary syndrome. AB - BACKGROUND: The aims of this study were to analyse the prevalence of impaired glucose tolerance (IGT) and diabetes mellitus (NIDDM) in Chinese polycystic ovary syndrome (PCOS) patients and to assess the ability of screening tests to predict these abnormalities within this population. METHODS: A total of 102 PCOS patients were evaluated. All patients underwent oral glucose tolerance tests (OGTTs) with blood samples taken at 0, 1 and 2 h. The 2-h plasma glucose level was used to categorize subjects as having IGT or NIDDM. RESULTS: The prevalence of IGT was 20.5% and that of NIDDM was 1.9%. There was no significant relationship between BMI and 2-h plasma glucose levels. The areas under the receiver operating characteristic (ROC) curve for glucose to insulin ratio (G:I), homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) were 0.702, 0.734 and 0.733 respectively. ROC analysis suggested a threshold value of 10.7 in G:I ratio (73.9% sensitivity and 59.5% specificity), a value of 2.14 in HOMA (73.9% sensitivity and 73.4% specificity) and a value of 0.34 in QUICKI (73.9% sensitivity and 73.4% specificity) for the prediction of abnormal glucose tolerance (IGT and NIDDM). CONCLUSIONS: Chinese women with PCOS are at increased risk of IGT and NIDDM. Even though G:I, HOMA and QUICKI are easier than OGTT, they could not replace the role of 2-h post-challenge plasma glucose level in the screening of IGT and NIDDM in PCOS women. PMID- 16684839 TI - Mifepristone-induced nitric oxide release and expression of nitric oxide synthases in the human cervix during early pregnancy. AB - BACKGROUND: Nitric oxide (NO) is a factor in cervical ripening, perhaps under the control of progesterone. We studied the effects of the antiprogesterone mifepristone on the release of NO and on the expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) in the uterine cervix of women in early pregnancy. METHODS: Thirteen women were treated with oral mifepristone (200 mg), and 15 women were studied as controls. Cervical fluid samples were collected before treatment then hourly up to 3 h, and the samples were assayed for the concentration of nitric oxide metabolites (NOx). In addition, cervical biopsy samples from six women treated with mifepristone and from six controls were assessed for iNOS and eNOS by immunohistochemistry and Western blotting. RESULTS: In 1-3 h, mifepristone induced 7.4- to 17.2-fold elevations in cervical fluid NOx concentrations; no change was seen in the controls. The expression of both iNOS and eNOS was detected in the cervical cells. The expression of cervical iNOS was strong in five of the six women treated with mifepristone but was not strong in any of the six control women. CONCLUSION: This is the first study to show that mifepristone stimulates the release of NO and the expression of iNOS in cervical cells of women in early pregnancy. This may be one mechanism by which mifepristone initiates cervical ripening. PMID- 16684840 TI - Biochemical pregnancy after fertilization of an oocyte aspirated from a heterotopic autotransplant of cryopreserved ovarian tissue: case report. AB - Autotransplantation of frozen/thawed ovarian tissue in women undergoing cancer therapy has so far led to the birth of two healthy babies. In both cases, it can be discussed whether the fertilized oocyte originated from the transplant or from the native ovary. We now present a biochemical pregnancy achieved after heterotopical autotransplantation of cryopreserved ovarian cortical tissue and hence the unquestionable proof that pregnancy can occur after transplantation of cryopreserved ovarian tissue. A woman diagnosed with Hodgkin's lymphoma had ovarian tissue cryopreserved at the age of 28, before receiving chemotherapy and radiation therapy that rendered her amenorrhoeic. After complete remission, she had autotransplantation of ovarian tissue to the remaining ovary, to the right pelvic wall and to a midline subperitoneal pocket on the lower abdominal wall. The transplanted tissue resumed hormone secretion and follicles developed in all three locations. Three times during 8 months, when follicles could not be visualized in other locations, oocytes were aspirated from the subperitoneal autotransplanted tissue on the lower abdominal wall. Twice, an oocyte was retrieved, fertilized by intracytoplasmatic sperm injection (ICSI) and transferred to the woman's uterus. One of the treatments resulted in a positive pregnancy test 14 days after transfer. Clinical pregnancy, however, was not achieved. In conclusion, heterotopic autotransplantation of cryopreserved ovarian tissue can sustain follicle development. The oocytes of aspirated mature follicles are capable of fertilization after ICSI, and the resulting embryo is competent of producing hCG at detectable levels. PMID- 16684841 TI - Control of estradiol secretion in reproductive ageing. AB - BACKGROUND: Estradiol (E(2)) concentration is preserved in older reproductive aged women despite a decrease in follicle number and androstenedione (AD) levels. We hypothesized that increased aromatase activity accounts for E(2) preservation in older women. METHODS: Older (36-46 years; n = 11) and younger (21-35 years; n = 10) women with 25- to 35-day menstrual cycles participated in a parallel design study. Daily blood samples were drawn starting at menses, and recombinant human FSH (rhFSH), 150 IU, was administered when the dominant follicle's diameter was > or =16 mm. FSH, LH, E(2), estrone (E(1)), AD and the AD/E(1) ratio were compared. RESULTS: E(2) and E(1) concentrations and the E(1)/E(2) ratio were similar across the follicular phase in older compared with younger women, whereas AD and the AD/E(1) ratio were lower. Older women had higher FSH concentrations in the early follicular phase and fewer small follicles. RhFSH-stimulated changes in E(1) were similar between older and younger subjects despite the smaller number of follicles. CONCLUSIONS: These findings suggest that E(2) secretion is maintained by increased aromatase function in older compared with younger reproductive-aged women, whereas there is no apparent difference in 17beta-hydroxysteroid dehydrogenase activity. The increased aromatase is probably driven by increased FSH in the early follicular phase and compensates for the decreased follicle number in older reproductive-aged women. PMID- 16684842 TI - Unexplained infertility: does it really exist? AB - Recent medical literature has quite extensively addressed the use of various terminologies within the field of reproductive medicine. This discussion has, however, so far overlooked the fact that one of the most frequently made diagnosis, so-called unexplained infertility (UI), not only didactically but, even more importantly, clinically, appears unsustainable as an independent diagnosis. The arguments in support of such a contention are manifold. The diagnosis of UI is highly subjective. It is dependent on which diagnostic tests have been performed (or have been omitted) and at what level of quality. Paradoxically, a diagnosis of UI will, therefore, be more often reached if the diagnostic workup is incomplete or of poor quality. Supported by evidence from the literature, the argument is made that the conditions, most frequently misdiagnosed as UI, are endometriosis, tubal infertility (especially distal and peritubal disease), premature ovarian ageing and immunological infertility. Because of the obvious unreliability of a diagnosis of UI and the widely reported unevenness in diagnostic criteria, we recommend the abandonment of UI as a formal infertility diagnosis. Better efforts to reach infertility diagnoses more accurately should improve the diagnostic accuracy of hitherto frequently missed diagnoses, which often falsely have led to a diagnosis of UI. PMID- 16684843 TI - Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications. AB - BACKGROUND: Sperm DNA integrity is an important factor in the prognosis of male fertility. In this study, we investigated intra-individual variation of sperm chromatin structure assay (SCSA) parameters in infertility patients undergoing assisted reproductive techniques (ARTs). METHODS: Retrospective study of 282 consecutive patients referred for ART [intrauterine insemination (IUI), IVF or ICSI] with repeated (between 2 and 5) SCSA measurements. RESULTS: Mean coefficient of variation (CV) of DNA Fragmentation Index (DFI) for repeated SCSA measurements was 29%. A high proportion [37%; 95% confidence interval (CI): 27%, 49%] of patients with DFI >30% in the first test had DFI <30% in the second test. Also, a considerable proportion (27%; 95% CI : 16%, 40%) of patients with 21-30% DFI values in the first test had DFI >30% in the second test. CONCLUSIONS: Intra individual variability in DFI is significant, therefore repeated SCSA measurements are recommended. The biological mechanisms behind these variations remain to be elucidated. PMID- 16684844 TI - Abnormal cervical cytology in women eligible for IVF. AB - BACKGROUND: Chlamydia trachomatis is more prevalent in subfertile women than in the general population and is the leading cause of tubal factor subfertility. As C. trachomatis infections are sexually transmitted, it can be expected that infections with human papillomavirus (HPV) are also more prevalent in this group of women. HPV is a necessary cause for the development of cervical (pre )malignancies. We therefore hypothesized that subfertile women are more likely to have HPV-induced cervical abnormalities compared to the general population. METHODS: In this retrospective case-control study, all cervical smears of women visiting the fertility clinic for IVF (cases) and of women attending the population-based screening programme for cervical cancer (controls) were retrieved from an electronic database and assessed. RESULTS: The cases (n = 669) showed significantly more abnormal cervical smears compared to the controls (77,055) (6.1 and 3.9%, respectively, P < 0.02). CONCLUSIONS: The probability that subfertile women eligible for IVF are diagnosed with a high-grade cervical lesion is almost twice as high compared to women in the general population. We therefore suggest to take a cervical smear from all women referred for fertility problems. PMID- 16684845 TI - High NK cell activity in recurrent miscarriage: what are we really measuring? AB - BACKGROUND: Several studies have shown that women with unexplained recurrent miscarriage (RM) have increased numbers and activity of peripheral blood NK cells and that elevated levels of these cells predict subsequent miscarriages in women with RM. Because catecholamines rapidly mobilize NK cells into the circulation, such increases may not reflect a steady state of overactive immunity but may result from a transient increase in the number of NK cells because of the stress associated with blood withdrawal. METHODS: Blood was drawn from 22 controls and 38 RM patients immediately after vein cannulation, and again 20 min later. The percentage of NK cells within lymphocytes, their concentration per microlitre of blood and their activity were assessed. RESULTS: All three indices of NK cells did not change in the controls across the two samples. However, women with RM had elevated levels in all three NK indices in the first blood sample, but these levels declined to values similar to those seen in the controls. This decline was mainly observed in primary aborters whose NK activity was highest in the first blood withdrawal. Accordingly, there was a high correlation between the magnitude of the decline and the initial NK cell indices in women with RM. The change in activity highly correlated with the change in the concentration of NK cells. CONCLUSION: The increased NK number and activity previously observed in RM patients may result from a transient stress response at the time of blood withdrawal. Patients with primary RM may be characterized by exaggerated acute stress responses in other circumstances. PMID- 16684846 TI - Progesterone regulates HLA-G gene expression through a novel progesterone response element. AB - BACKGROUND: We have previously demonstrated that progesterone has a stimulatory effect on HLA-G gene expression. Because this effect was abolished by the anti progestin, RU486, we hypothesize that this effect is through receptor-mediated up regulation of the HLA-G gene. The objective of this study was to explore the molecular mechanisms of this effect. METHODS: The transient transfection of a chloramphenicol acetyltransferase (CAT) construct containing a fragment of the HLA-G gene promoter into the JEG-3 choriocarcinoma cell line was performed. An electrophoretic mobility shift assay (EMSA) and a DNA fragment-binding enzyme linked immunosorbent assay (ELISA) were carried out to locate a specific progesterone response element (PRE) in the HLA-G gene promoter region. RESULTS: Progesterone treatment of JEG-3 cells transfected with the HLA-G gene promoter CAT construct resulted in an increase of CAT synthesis, whereas RU486 blocked this transcriptional activation. A novel PRE-binding site sequence, with 60% homology to that of wild-type mouse mammary tumour virus (MMTV) PRE, was discovered in this region. CONCLUSION: The effect of progesterone on HLA-G gene expression is through progesterone receptor (PR) activation, followed by binding to a novel PRE in the HLA-G promoter region. Therefore, one of the mechanisms of immunomodulation by progesterone during pregnancy may be through the regulation of HLA-G gene expression via this novel PRE. PMID- 16684847 TI - Effects of aripiprazole on alcohol intake in an animal model of high-alcohol drinking. AB - AIMS: This study examined the effects of aripiprazole, a novel atypical antipsychotic drug with partial agonist properties at dopamine D2 receptors, on the voluntary limited access alcohol drinking of alcohol-preferring AA (Alko, Alcohol) rats. METHODS: AA rats were taught to drink 10% alcohol in a 4 h limited access paradigm. Effects of acute aripiprazole (0, 0.3, 1.0, and 3.0 mg/kg) on the limited access alcohol drinking were studied. In repeated treatment experiment, aripiprazole (0, 1.0, and 6.0 mg/kg) was administered once daily over five successive days. To reveal any effect by aripiprazole not selective for alcohol drinking, 0.025% saccharin solution was substituted for alcohol during the 4 h limited access, and acute treatments were repeated. The effects of aripiprazole on ambulatory locomotor activity were tested with doses that were used in the acute experiments. RESULTS: Acute aripiprazole at the doses of 0.3, 1.0, and 3.0 mg/kg had no effect on alcohol drinking. Repeated treatment with the aripiprazole dose of 6.0 mg/kg significantly diminished alcohol drinking at the 1 h time point. This dose had no effect on saccharin drinking when given acutely. Acute aripiprazole at the doses of 1.0, 3.0, and 6.0 mg/kg significantly suppressed locomotor activity. CONCLUSIONS: Aripiprazole decreased limited access alcohol drinking in AA rats, but only at a high dose that also strongly suppressed locomotor activity. PMID- 16684848 TI - Route of administration (enteral or parenteral) affects the contribution of L glutamine to de novo L-arginine synthesis in mice: a stable-isotope study. AB - A pathway from enteral L-glutamine as substrate for L-arginine synthesis is suggested by previous studies. L-Glutamine and L-glutamine dipeptides exhibit numerous beneficial effects in experimental and clinical studies. In trauma patients, enteral L-glutamine supply increased plasma L-arginine. The present study was designed to quantify the contribution of L-glutamine to the de novo L citrulline and L-arginine synthesis in mice when L-glutamine is administered in a high dose of labeled L-glutamine or L-alanyl-L-glutamine by the enteral or parenteral route. For this purpose, male Swiss mice (n = 43) underwent a laparotomy, and catheters were inserted for sampling and infusion. A primed, constant, and continuous infusion of L-alanyl-L-[2-(15)N]glutamine (dipeptide groups) or L-[2-(15)N]glutamine (free L-glutamine groups), simultaneously with L [ureido-(13)C,(2)H(2)]citrulline and L-[guanidino-(15)N(2),(2)H(2)]arginine, was given (steady-state model). Mice received the L-glutamine tracers intravenously (jugular vein) or enterally (duodenum). Enrichments of metabolites were measured by LC-MS. Arterial L-glutamine concentrations were the highest in the intravenous dipeptide group. L-Glutamine was converted to L-citrulline and L-arginine when L [2-(15)N]glutamine and L-alanyl-L-[2-(15)N]glutamine were given by enteral or parenteral route. The contribution of L-glutamine to the de novo synthesis of L citrulline and L-arginine was higher in the enteral groups when compared with the intravenous groups (P < 0.005). Therefore, the route of administration (enteral or parenteral) affects the contribution of L-glutamine, provided as free molecule or dipeptide, to the de novo synthesis of L-arginine in mice. PMID- 16684849 TI - Body mass index, free insulin-like growth factor I, and physical function among older adults: results from the ilSIRENTE study. AB - The aim of the present study was to evaluate the mediating role played by obesity on the relationship of free insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) with muscle strength and physical performance. Data were from baseline evaluation of the ilSIRENTE Study. Muscle strength was measured by hand grip strength. Physical performance was assessed using the walking speed and the 0-3 Short Physical Performance Battery (SPPB) score. Based on its median value, free IGF-I was categorized in the following two groups: low IGF-I (IGF-I <0.65 ng/ml; n = 174) and high IGF-I (IGF-I > or =0.65 ng/ml; n = 175). Similarly, IGFBP-3 was categorized in the following two groups: low IGFBP-3 (IGFBP-3 <4,319.9 ng/ml; n = 174) and high IGFBP-3 (IGFBP-3 > or =4,319.9 ng/ml; n = 175). Body mass index (BMI) was categorized as follows: <25 kg/m(2) (n = 160), 25-29.9 kg/m(2) (n = 133), > or =30 kg/m(2) (n = 56). Mean age of the 349 participants was 85.8 yr, and 234 (67%) were women. After adjusting for potential confounders, no significant association of IGF-I and IGFBP-3 with study outcomes was observed. After the study sample was stratified by BMI groups, compared with participants with low IGF-I level, those with high IGF-I level had a significantly better grip strength [35.2 +/- 1.6 vs. 29.2 +/- 2.0 (SE) kg, P = 0.03], walking speed (0.55 +/- 0.04 vs. 0.40 +/- 0.04 m/s, P = 0.01), and SPPB score (1.9 +/- 0.1 vs. 1.5 +/ 0.1 m/s, P = 0.01) but only in the group with BMI > or =30 kg/m(2) and not in other BMI groups. A statistically significant interaction between BMI and IGF-I level was observed on all study outcomes. By contrast, no association was observed between IGFBP-3 and study outcomes, independently of BMI. In conclusion, high IGF-I level is associated with better physical function in older persons with obesity, but not in nonobese subjects. PMID- 16684850 TI - Current and future approaches using genetically modified mice in endocrine research. AB - Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre-loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism. PMID- 16684852 TI - The release of GLP-1 and ghrelin, but not GIP and CCK, by glucose is dependent upon the length of small intestine exposed. AB - Previous observations suggest that glucagon-like peptide-1 (GLP-1) is released into the bloodstream only when dietary carbohydrate enters the duodenum at rates that exceed the absorptive capacity of the proximal small intestine to contact GLP-1 bearing mucosa in more distal bowel. The aims of this study were to determine the effects of modifying the length of small intestine exposed to glucose on plasma concentrations of GLP-1 and also glucose-dependent insulinotropic peptide (GIP), insulin, cholecystokinin (CCK) and ghrelin, and antropyloric pressures. Glucose was infused at 3.5 kcal/min into the duodenum of eight healthy males (age 18-59 yr) over 60 min on the first day into an isolated 60-cm segment of the proximal small intestine ("short-segment infusion"); on the second day, the same amount of glucose was infused with access to the entire small intestine ("long-segment infusion"). Plasma GLP-1 increased and ghrelin decreased (P < 0.05 for both) during the long-, but not the short-, segment infusion. By contrast, increases in plasma CCK and GIP did not differ between days. The rises in blood glucose and plasma insulin were greater during the long- than during the short-segment infusion (P < 0.05). During the long- but not the short-segment infusion, antral pressure waves (PWs) were suppressed (P < 0.05). Isolated pyloric PWs and basal pyloric pressure were stimulated on both days. In conclusion, the release of GLP-1 and ghrelin, but not CCK and GIP, is dependent upon >60 cm of the intestine being exposed to glucose. PMID- 16684851 TI - Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction. AB - The most energy-requiring organ in the body, the cardiac muscle, relies primarily on lipoprotein-derived fatty acids. Prenatal loss of cardiac lipoprotein lipase (LPL) leads to hypertriglyceridemia, but no cardiac dysfunction, in young mice. Cardiac specific loss of LPL in 8-wk-old mice was produced by a 2-wk tamoxifen treatment of MerCreMer (MCM)/Lpl(flox/flox) mice. LPL gene deletion was confirmed by PCR analysis, and LPL mRNA expression was reduced by approximately 70%. One week after tamoxifen was completed, triglyceride was increased with LPL deletion, 162 +/- 53 vs. 91 +/- 21 mg/dl, P < 0.01. Tamoxifen treatment of Lpl(flox/flox) mice did not cause a significant increase in triglyceride levels. Four weeks after tamoxifen, MCM/Lpl(flox/flox) mice had triglyceride levels of 190 +/- 27 mg/dl, similar to those of mice with prenatal LPL deletion. One week after the tamoxifen, MCM/Lpl(flox/flox), but not Lpl(flox/flox), mice had decreases in carnitine palmitoyl transferase I mRNA (18%) and pyruvate dehydrogenase kinase 4 mRNA (38%). These changes in gene expression became more robust with time. Acute loss of LPL decreased ejection fraction and increased mRNA levels for atrial natriuretic factor. Our studies show that acute loss of LPL can be produced and leads to rapid alteration in gene expression and cardiac dysfunction. PMID- 16684853 TI - Fatty acid transport and FAT/CD36 are increased in red but not in white skeletal muscle of ZDF rats. AB - An increased rate of fatty acid transport into skeletal muscle has been has been linked to the accumulation of intramuscular lipids and insulin resistance, and red muscles are more susceptible than white muscles in developing fatty acid mediated insulin resistance. Therefore, we examined in Zucker diabetic fatty (ZDF) rats, relative to lean rats, 1) whether rates of fatty acid transport and transporters (FAT/CD36 and FABPpm) were upregulated in skeletal muscle during the transition from insulin resistance (week 6) to type 2 diabetes (weeks 12 and 24), 2) whether such changes occurred primarily in red skeletal muscle, and 3) whether changes in FAT/CD36 and GLUT4 were correlated. In red muscles of ZDF compared with lean rats, the rates of fatty acid transport were upregulated (+66%) early in life (week 6). Compared with the increase in fatty acid transport in lean red muscle from weeks 12-24 (+57%), the increase in fatty acid transport rate in ZDF red muscle was 50% greater during this same period. In contrast, no differences in fatty acid transport rates were observed in the white muscles of lean and ZDF rats at any time (weeks 6-24). In red muscle only, there was an inverse relationship between FAT/CD36 and GLUT4 protein expression as well as their plasmalemmal content. These studies have shown that, 1) before the onset of diabetes, as well as during diabetes, fatty acid transport and FAT/CD36 expression and plasmalemmal content are upregulated in ZDF rats, but importantly, 2) these changes occurred only in red, not white, muscles of ZDF rats. PMID- 16684854 TI - Elevated plasma free fatty acids decrease basal protein synthesis, but not the anabolic effect of leucine, in skeletal muscle. AB - Elevations in free fatty acids (FFAs) impair glucose uptake in skeletal muscle. However, there is no information pertaining to the effect of elevated circulating lipids on either basal protein synthesis or the anabolic effects of leucine and insulin-like growth factor I (IGF-I). In chronically catheterized conscious rats, the short-term elevation of plasma FFAs by the 5-h infusion of heparin plus Intralipid decreased muscle protein synthesis by approximately 25% under basal conditions. Lipid infusion was associated with a redistribution of eukaryotic initiation factor (eIF)4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex. This shift was associated with a decreased phosphorylation of eIF4G but not 4E-BP1. Lipid infusion did not significantly alter either the total amount or phosphorylation state of mTOR, TSC2, S6K1, or the ribosomal protein S6 under basal conditions. In control rats, oral leucine increased muscle protein synthesis. This anabolic response was not impaired by lipid infusion, and no defects in signal transduction pathways regulating translation initiation were detected. In separate rats that received a bolus injection of IGF-I, lipid infusion attenuated the normal redistribution of eIF4E from the active to inactive complex and largely prevented the increased phosphorylation of 4E-BP1, eIF4G, S6K1, and S6. This IGF-I resistance was associated with enhanced Ser(307) phosphorylation of insulin receptor substrate-1 (IRS-1). These data indicate that the short-term elevation of plasma FFAs impairs basal protein synthesis in muscle by altering eIF4E availability, and this defect may be related to impaired phosphorylation of eIF4G, not 4E-BP1. Moreover, hyperlipidemia impairs IGF-I action but does not produce leucine resistance in skeletal muscle. PMID- 16684855 TI - Role of ovarian hormones in the regulation of protein metabolism in women: effects of menopausal status and hormone replacement therapy. AB - The age-related decline in fat-free mass is accelerated in women after menopause, implying that ovarian hormone deficiency may have catabolic effects on lean tissue. Because fat-free tissue mass is largely determined by its protein content, alterations in ovarian hormones would likely exert regulatory control through effects on protein balance. To address the hypothesis that ovarian hormones regulate protein metabolism, we examined the effect of menopausal status and hormone replacement therapy (HRT) on protein turnover. Whole body protein breakdown, oxidation, and synthesis were measured under postabsorptive conditions using [(13)C]leucine in healthy premenopausal (n = 15, 49 +/- 1 yr) and postmenopausal (n = 18, 53 +/- 1 yr) women. In postmenopausal women, whole body protein turnover and plasma albumin synthesis rates (assessed using [(13)C]leucine and [(2)H]phenylalanine) were also measured following 2 mo of treatment with oral HRT (0.625 mg conjugated estrogens + 2.5 mg medroxyprogesterone acetate, n = 9) or placebo (n = 9). No differences in whole body protein breakdown, oxidation, or synthesis were found between premenopausal and postmenopausal women. Protein metabolism remained similar between groups after statistical adjustment for differences in adiposity and when subgroups of women matched for percent body fat were compared. In postmenopausal women, no effect of HRT was found on whole body protein breakdown, synthesis, or oxidation. In contrast, our results support a stimulatory effect of HRT on albumin fractional synthesis rate, although this did not translate into alterations in circulating albumin concentrations. In conclusion, our results suggest no detrimental effect of ovarian hormone deficiency coincident with the postmenopausal state, and no salutary effect of hormone repletion with HRT, on rates of whole body protein turnover, although oral HRT regimens may increase the synthesis rates of albumin. PMID- 16684856 TI - Endurance training has little effect on active muscle free fatty acid, lipoprotein cholesterol, or triglyceride net balances. AB - We evaluated the hypothesis that net leg total FFA, LDL-C, and TG uptake and HDL C release during moderate-intensity cycling exercise would be increased following endurance training. Eight sedentary men (26 +/- 1 yr, 77.4 +/- 3.7 kg) were studied in the postprandial state during 90 min of rest and 60 min of exercise twice before (45% and 65% V(O2 peak)) and twice after 9 wk of endurance training (55% and 65% posttraining V(O2 peak)). Measurements across an exercising leg were taken to be a surrogate for active skeletal muscle. To determine limb lipid exchange, femoral arterial and venous blood samples drawn simultaneously at rest and during exercise were analyzed for total and individual FFA (e.g., palmitate, oleate), LDL-C, HDL-C, and TG concentrations, and limb blood flow was determined by thermodilution. The transition from rest to exercise resulted in a shift from net leg total FFA release (-44 +/- 16 micromol/min) to uptake (193 +/- 49 micromol/min) that was unaffected by either exercise intensity or endurance training. The relative net leg release and uptake of individual FFA closely resembled their relative abundances in the plasma with approximately 21 and 41% of net leg total FFA uptake during exercise accounted for by palmitate and oleate, respectively. Endurance training resulted in significant changes in arterial concentrations of HDL-C (49 +/- 5 vs. 52 +/- 5 mg/dl, pre vs. post) and LDL-C (82 +/- 9 vs. 76 +/- 9 mg/dl, pre vs. post), but there was no net TG or LDL C uptake or HDL-C release across the resting or active leg before or after endurance training. In conclusion, endurance training favorably affects blood lipoprotein profiles, even in young, healthy normolipidemic men, but muscle contractions per se have little effect on net leg LDL-C, or TG uptake or HDL-C release during moderate-intensity cycling exercise. Therefore, the favorable effects of physical activity on the lipid profiles of young, healthy normolipidemic men in the postprandial state are not attributable to changes in HDL-C or LDL-C exchange across active skeletal muscle. PMID- 16684857 TI - Insulin resistance and whole body energy homeostasis in obese adolescents with fatty liver disease. AB - Obese adolescents are at risk of developing NAFLD and type 2 diabetes. We measured noninvasively the IHF content of obese adolescents to ascertain whether it is associated with insulin resistance and abnormal energy homeostasis. IHF content, whole body energy homeostasis, insulin sensitivity, and body composition were measured using localized hepatic (1)H-MRS, indirect calorimetry, fasting derived and 3-h-OGTT-derived surrogate indexes (HOMA2 and WBISI), and DEXA, respectively, in 54 obese adolescents (24 female and 30 male, age 13 +/- 2 yr, BMI >99th percentile for their age and sex). NAFLD (defined as IHF content >5% wet weight) was found in 16 individuals (30%) in association with higher ALT (P < 0.006), Hb A(1c) (P = 0.021), trunk fat content (P < 0.03), and lower HDL cholesterol (P < 0.05). Individuals with NAFLD had higher fasting plasma glucose (89 +/- 8 vs. 83 +/- 9 mg/dl, P = 0.01) and impaired insulin sensitivity (HOMA2 and WBISI, P < 0.05). Meanwhile, parameters of insulin secretion were unaffected. Their reliance on fat oxidation in the fasting state was lower (RQ 0.83 +/- 0.08 vs. 0.77 +/- 0.05, P < 0.01), and their ability to suppress it during the oral glucose challenge was impaired (P < 0.05) vs. those with normal IHF content. When controlling for trunk fat content, the correlation between IHF content and insulin sensitivity was weakened, whereas the correlation with fasting lipid oxidation was maintained. In conclusion, NAFLD is common in childhood obesity, and insulin resistance is present in association with increased trunk fat content. In contrast, the rearrangement of whole body substrate oxidation in these youngsters appeared to be an independent feature. PMID- 16684858 TI - Adjuvant therapy in rectal cancer: what is the truth? PMID- 16684859 TI - Evidence-based risk factors for seroma formation in breast surgery. AB - BACKGROUND: Seroma is a common problem in breast surgery. The aim of this systematic review was to identify risk factors for seroma formation. METHODS: Articles published in English were obtained from searches of Medline and additional references were found in the bibliographies of these articles. Risk factors were graded according to the quality and strength of evidence and to the direction of association. RESULTS: One meta-analysis, 51 randomized controlled trials, 7 prospective studies and 7 retrospective studies were identified. There was no risk factor supported by strong evidence, but there was moderate evidence to support a risk for seroma formation in individuals with heavier body weight, extended radical mastectomy as compared with simple mastectomy, and greater drainage volume in the initial 3 days. On the other hand, the following factors did not have a significant influence on seroma formation: duration of drainage; hormone receptor status; immobilization of the shoulder; intensity of negative suction pressure; lymph node status or lymph node positivity; number of drains; number of removed lymph nodes; previous biopsy; removal of drains on the fifth postoperative day versus when daily drainage volume fell to minimal; stage; type of drainage (closed suction versus static drainage); and use of fibrinolysis inhibitor. In contrast, sentinel lymph node biopsy reduced seroma formation. Evidence was weak, or unproven, for other factors that were commonly cited in the literature. CONCLUSIONS: Although a number of factors have been correlated with seroma formation, strong evidence is still scarce. However, there is evidence showing that sentinel lymph node biopsy reduces seroma formation. PMID- 16684860 TI - Phase I/II study of oxaliplatin with weekly bolus fluorouracil and high-dose leucovorin (ROX) as first-line therapy for patients with colorectal cancer. AB - BACKGROUND: Infusional fluorouracil (5-FU) and leucovorin (LV) with oxaliplatin is one of the current standard regimens for the treatment of patients with metastatic colorectal cancer. Weekly bolus 5-FU with high-dose LV (Roswell Park Memorial Institute Regimen: RPMI) is the most commonly used regimen in Japan. The objectives of this study were to determine the recommended dose (RD) of RPMI combined with oxaliplatin and to evaluate the toxicity and efficacy at the RD. METHODS: The subjects were 18 patients with metastatic colorectal cancer. Oxaliplatin (85 mg/m2) was given intravenously over 2 h on days 1 and 15 with l LV (250 mg/m2) given intravenously over 2 h and 5-FU as an intravenous bolus on days 1, 8, and 15. This treatment was repeated every 4 weeks. The dose of 5-FU was escalated from 400 mg/m2 (level 1) to 500 mg/m2 (level 2). RESULTS: A total of 14 patients received level 1, and 4 received level 2. Three of the patients had dose-limiting toxicity (DLT) in cycle 1 of level 2 (grade 3 thrombocytopenia, grade 4 neutropenia and grade 2 neutropenia in one patient each), requiring that treatment was delayed for longer than 7 days. None of the 14 patients given level 1 had DLT or grade 3 or 4 gastrointestinal toxicity. Sensory neuropathy occurred in all patients. Objective response rates were 61% in the 18 patients studied and 64% at level 1. The median time to progression was 171 days, and the median overall survival time was 603 days in the 18 patients studied. CONCLUSIONS: Oxaliplatin (85 mg/m2) with weekly bolus 5-FU (400 mg/m2) and high-dose l-LV (250 mg/m2) is recommended for further phase III studies in patients with metastatic colorectal cancer. PMID- 16684861 TI - Incidence and survival of mesothelioma in Osaka, Japan. AB - BACKGROUND: Mortality statistics show rapid increase in the number of deaths from mesothelioma. However, population-based study of the incidence and the survival has never been conducted. Time-trends and regional differences in the incidence of mesothelioma in Osaka were examined together with their 5-year survival. METHODS: Individual data for mesothelioma were retrieved from Osaka Cancer Registry during the period 1966-2001. Annual incidence rates were calculated for every 3 years from 1975 to 2001, and age-standardized rates were calculated with the Japanese model population of 1985. Standardized incidence ratios were also calculated by age-specific number of population of each municipality and the corresponding age-specific incidence rates of mesothelioma in Osaka Prefecture during the period 1981-2001. The survival analysis was performed with the Kaplan Meier method, based on the newly reported cases diagnosed during the period 1975 1997. RESULTS: Incidence rates of mesothelioma have increased rapidly both among males and females in Osaka during the past few decades. Geographical differences in the standardized incidence ratios were found to be remarkable in Osaka Prefecture. The result shows that the survival of malignant mesothelioma was very poor (5-year survival and median survival time: 5.1% and 6 months for males, 10.2% and 5 months for females). CONCLUSIONS: Incidence of mesothelioma has increased remarkably in Osaka, Japan, during past few decades. Geographical variations in the incidence were also suggested. Five-year survival of the patients was very poor. PMID- 16684862 TI - A case of malignant solitary fibrous tumor of the pleura. PMID- 16684863 TI - International comparisons of cumulative risk of liver cancer, from Cancer Incidence in Five Continents Vol. VIII. PMID- 16684864 TI - Images in cardiovascular medicine. Compression of the pulmonary vein after right sided pneumonectomy. PMID- 16684865 TI - Images in cardiovascular medicine. Balloon valvuloplasty of a stenosed bioprosthetic tricuspid valve. PMID- 16684866 TI - Images in cardiovascular medicine. Percutaneous closure of a left ventricular free-wall rupture site. PMID- 16684868 TI - Letter regarding article by Banki et al., "Acute neurocardiogenic injury after subarachnoid hemorrhage.". PMID- 16684870 TI - Viewpoint: stem cells in cardiology. PMID- 16684871 TI - Preventing vascular events due to elevated blood pressure. PMID- 16684872 TI - Timing of surgical intervention in chronic mitral regurgitation: is vigilance enough? PMID- 16684873 TI - Evaluating drug effects in the post-Vioxx world: there must be a better way. PMID- 16684874 TI - Neovascularization in human atherosclerosis. PMID- 16684875 TI - Clinical trials in the wake of Vioxx: requiring statistically extreme evidence of benefit to ensure the safety of new drugs. PMID- 16684877 TI - A biochemical rationale for the anticancer effects of Hsp90 inhibitors: slow, tight binding inhibition by geldanamycin and its analogues. AB - Heat shock protein (Hsp)90 is emerging as an important therapeutic target for the treatment of cancer. Two analogues of the Hsp90 inhibitor geldanamycin are currently in clinical trials. Geldanamycin (GA) and its analogues have been reported to bind purified Hsp90 with low micromolar potency, in stark contrast to their low nanomolar antiproliferative activity in cell culture and their potent antitumor activity in animal models. Several models have been proposed to account for the approximately 100-fold-greater potency in cell culture, including that GA analogues bind with greater affinity to a five-protein Hsp90 complex than to Hsp90 alone. We have determined that GA and the fluorescent analogue BODIPY-GA (BDGA) both demonstrate slow, tight binding to purified Hsp90. BDGA, used to characterize the kinetics of ligand-Hsp90 interactions, was found to bind Hsp90alpha with k(off) = 2.5 x 10(-3) min(-1), t(1/2) = 4.6 h, and Ki* = 10 nM. It was found that BDGA binds to a functional multiprotein Hsp90 complex with kinetics and affinity identical to that of Hsp90 alone. Also, BDGA binds to Hsp90 from multiple cell lysates in a time-dependent manner with similar kinetics. Therefore, our results indicate that the high potency of GA in cell culture and in vivo can be accounted for by its time-dependent, tight binding to Hsp90 alone. In the broader context, these studies highlight the essentiality of detailed biochemical characterization of drug-target interactions for the effective translation of in vitro pharmacology to cellular and in vivo efficacy. PMID- 16684878 TI - Probing a protein-protein interaction by in vitro evolution. AB - In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity driven stringency selections. Simultaneously, the antibody potency to inhibit IL 13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope. PMID- 16684876 TI - Adenosine A2a blockade prevents synergy between mu-opiate and cannabinoid CB1 receptors and eliminates heroin-seeking behavior in addicted rats. AB - Relapse is the most serious limitation of effective medical treatment of opiate addiction. Opiate-related behaviors appear to be modulated by cannabinoid CB1 receptors (CB1) through poorly understood cross-talk mechanisms. Opiate and CB1 receptors are coexpressed in the nucleus accumbens (NAc) and dorsal striatum. These regions also have the highest density of adenosine A2a receptors (A2a) in the brain. We have been investigating the postsynaptic signaling mechanisms of mu opiate receptors (MORs) and CB1 receptors in primary NAc/striatal neurons. In this article, we present evidence that MOR and CB1 act synergistically on cAMP/PKA signaling in NAc/striatal neurons. In addition, we find that synergy requires adenosine and A2a. Importantly, an A2a antagonist administered either directly into the NAc or indirectly by i.p. injection eliminates heroin-induced reinstatement in rats trained to self-administer heroin, a model of human craving and relapse. These findings suggest that A2a antagonists might be effective therapeutic agents in the management of abstinent heroin addicts. PMID- 16684879 TI - Sox10 regulates ciliary neurotrophic factor gene expression in Schwann cells. AB - Ciliary neurotrophic factor (Cntf) plays an essential role in postnatal maintenance of spinal motoneurons. Whereas the expression of this neurotrophic factor is low during embryonic development, it is highly up-regulated after birth in myelinating Schwann cells of rodents. To characterize the underlying transcriptional mechanisms, we have analyzed and compared the effects of various glial transcription factors. In contrast to Pit-1, Oct-1, Unc-86 homology region (POU) domain class 3, transcription factor 1 (Oct6/SCIP/Tst-1) and paired box gene 3 (Pax3), SRY-box-containing gene 10 (Sox10) induces Cntf expression in Schwann cells. Subsequent promoter analysis using luciferase reporter gene and EMSA identified the corresponding response elements within the Cntf promoter. Overexpression of Sox10 in primary sciatic nerve Schwann cells leads to a >100 fold up-regulation of Cntf protein, and suppression of Sox10 by RNA interference in the spontaneously immortalized Schwann cell line 32 reduces Cntf expression by >80%. Mice with heterozygous inactivation of the Sox10 gene show significantly reduced Cntf protein levels in sciatic nerves, indicating that Sox10 is necessary and sufficient for regulating Cntf expression in the peripheral nervous system. PMID- 16684880 TI - Genetic variation in putative regulatory loci controlling gene expression in breast cancer. AB - Candidate single-nucleotide polymorphisms (SNPs) were analyzed for associations to an unselected whole genome pool of tumor mRNA transcripts in 50 unrelated patients with breast cancer. SNPs were selected from 203 candidate genes of the reactive oxygen species pathway. We describe a general statistical framework for the simultaneous analysis of gene expression data and SNP genotype data measured for the same cohort, which revealed significant associations between subsets of SNPs and transcripts, shedding light on the underlying biology. We identified SNPs in EGF, IL1A, MAPK8, XPC, SOD2, and ALOX12 that are associated with the expression patterns of a significant number of transcripts, indicating the presence of regulatory SNPs in these genes. SNPs were found to act in trans in a total of 115 genes. SNPs in 43 of these 115 genes were found to act both in cis and in trans. Finally, subsets of SNPs that share significantly many common associations with a set of transcripts (biclusters) were identified. The subsets of transcripts that are significantly associated with the same set of SNPs or to a single SNP were shown to be functionally coherent in Gene Ontology and pathway analyses and coexpressed in other independent data sets, suggesting that many of the observed associations are within the same functional pathways. To our knowledge, this article is the first study to correlate SNP genotype data in the germ line with somatic gene expression data in breast tumors. It provides the statistical framework for further genotype expression correlation studies in cancer data sets. PMID- 16684881 TI - The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs. AB - Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme-inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs. PMID- 16684882 TI - The deprotonated guanine-cytosine base pair. AB - Awareness of the harmful effects of radiation has increased interest in finding the mechanisms of DNA damage. Radical and anion formation among the DNA base pairs are thought to be important steps in such damage [Collins, G. P. (2003) Sci. Am. 289 (3), 26-27]. Energetic properties and optimized geometries of 10 radicals and their respective anions derived through hydrogen abstraction from the Watson-Crick guanine-cytosine (G-C) base pair have been studied using reliable theoretical methods. The most favorable deprotonated structure (dissociation energy 42 kcal x mol(-1), vertical detachment energy 3.79 eV) ejects the proton analogous to the cytosine glycosidic bond in DNA. This structure is a surprisingly large 12 kcal x mol(-1) lower in energy than any of the other nine deprotonated G-C structures. This system retains the qualitative G C structure but with the H...O2 distance dramatically reduced from 1.88 to 1.58 A, an extremely short hydrogen bond. The most interesting deprotonated G-C structure is a "reverse wobble" incorporating two N-H...N hydrogen bonds. Three different types of relaxation energies (4.3-54 kcal x mol(-1)) are defined and reported to evaluate the energy released via different mechanisms for the preparation of the deprotonated species. Relative energies, adiabatic electron affinities (ranging from 1.93 to 3.65 eV), and pairing energies are determined to discern which radical will most alter the G-C properties. The most stable deprotonated base pair corresponds to the radical with the largest adiabatic electron affinity, 3.65 eV. This value is an enormous increase over the electron affinity (0.60 eV) of the closed-shell G-C base pair. PMID- 16684883 TI - Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-suppressor mechanism. AB - Genomic material from chromosome band 13q14.3 distal to the retinoblastoma locus is recurrently lost in a variety of human neoplasms, indicating an as-yet unidentified tumor-suppressor mechanism. No pathogenic mutations have been found in the minimally deleted region until now. However, in B cell chronic lymphocytic leukemia tumors with loss of one copy of the critical region, respective candidate tumor-suppressor genes are down-regulated by a factor >2, which would be expected by a normal gene-dosage effect. This finding points to an epigenetic pathomechanism. We find that the two copies of the critical region replicate asynchronously, suggesting differential chromatin packaging of the two copies of 13q14.3. Although we also detect monoallelic silencing of genes localized in the critical region, monoallelic expression originates from either the maternal or paternal copy, excluding an imprinting mechanism. DNA methylation analyses revealed one CpG island of the region to be methylated. DNA demethylation of this CpG island and global histone hyperacetylation induced biallelic expression, whereas replication timing was not affected. We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. Accordingly, deletion of the single active copy of 13q14.3 results in significant down-regulation of the candidate genes and loss of function, providing a model for the interaction of genetic lesions and epigenetic silencing at 13q14.3 in B cell chronic lymphocytic leukemia. PMID- 16684884 TI - Tbx1 haploinsufficiency is linked to behavioral disorders in mice and humans: implications for 22q11 deletion syndrome. AB - About 35% of patients with 22q11 deletion syndrome (22q11DS), which includes DiGeorge and velocardiofacial syndromes, develops psychiatric disorders, mainly schizophrenia and bipolar disorder. We previously reported that mice carrying a multigene deletion (Df1) that models 22q11DS have reduced prepulse inhibition (PPI), a behavioral abnormality and schizophrenia endophenotype. Impaired PPI is associated with several psychiatric disorders, including those that occur in 22q11DS, and recently, reduced PPI was reported in children with 22q11DS. Here, we have mapped PPI deficits in a panel of mouse mutants that carry deletions that partially overlap with Df1 and have defined a PPI critical region encompassing four genes. We then used single-gene mutants to identify the causative genes. We show that PPI deficits in Df1/+ mice are caused by haploinsufficiency of two genes, Tbx1 and Gnb1l. Mutation of either gene is sufficient to cause reduced PPI. Tbx1 is a transcription factor, the mutation of which is sufficient to cause most of the physical features of 22q11DS, but the gene had not been previously associated with the behavioral/psychiatric phenotype. A likely role for Tbx1 haploinsufficiency in psychiatric disease is further suggested by the identification of a family in which the phenotypic features of 22q11DS, including psychiatric disorders, segregate with an inactivating mutation of TBX1. One family member has Asperger syndrome, an autistic spectrum disorder that is associated with reduced PPI. Thus, Tbx1 and Gnb1l are strong candidates for psychiatric disease in 22q11DS patients and candidate susceptibility genes for psychiatric disease in the wider population. PMID- 16684885 TI - siRNA in human cells selectively localizes to target RNA sites. AB - Recent observations of RNA interference (RNAi) in the nuclei of human cells raise key questions about the extent to which nuclear and cytoplasmic RNAi pathways are shared. By directly visualizing the localization of small interfering RNA (siRNA) in live human cells, we show here that siRNA either selectively localizes in the cytoplasm or translocates into the nucleus, depending on where the silencing target RNA resides. Two siRNAs that target the small nuclear 7SK and U6 RNAs localize into the nucleus as duplexes. In contrast, an siRNA targeting the cytoplasmic hepatitis C virus replicon RNA dissociates, and only antisense strand distributes in the cytoplasm of the cells harboring the target RNA, whereas sense strand gets degraded. At the same time, both strands of the latter siRNA are distributed throughout the cytoplasm and nucleus in cells lacking the silencing target RNA. These results suggest the existence of a mechanism by which the RNAi machinery orchestrates a target-determined localization of the siRNA and the corresponding RNAi activity, and also provide evidence for formation of nuclear programmed active RNA induced silencing complexes directly in the nucleus. PMID- 16684886 TI - The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases. AB - Pseudomonas aeruginosa is both a ubiquitous environmental bacterium and an opportunistic human pathogen. A remarkable metabolic versatility allows it to occupy a multitude of ecological niches, including wastewater treatment plants and such hostile environments as the human respiratory tract. P. aeruginosa is able to degrade and metabolize biocidic SDS, the detergent of most commercial personal hygiene products. We identify SdsA1 of P. aeruginosa as a secreted SDS hydrolase that allows the bacterium to use primary sulfates such as SDS as a sole carbon or sulfur source. Homologues of SdsA1 are found in many pathogenic and some nonpathogenic bacteria. The crystal structure of SdsA1 reveals three distinct domains. The N-terminal catalytic domain with a binuclear Zn2+ cluster is a distinct member of the metallo-beta-lactamase fold family, the central dimerization domain ensures resistance to high concentrations of SDS, whereas the C-terminal domain provides a hydrophobic groove, presumably to recruit long aliphatic substrates. Crystal structures of apo-SdsA1 and complexes with substrate analog and products indicate an enzymatic mechanism involving a water molecule indirectly activated by the Zn2+ cluster. The enzyme SdsA1 thus represents a previously undescribed class of sulfatases that allows P. aeruginosa to survive and thrive under otherwise bacteriocidal conditions. PMID- 16684888 TI - All-trans retinoic acid-induced expression of bactericidal/permeability increasing protein (BPI) in human myeloid cells correlates to binding of C/EBPbeta and C/EBPepsilon to the BPI promoter. AB - Bactericidal/permeability-increasing protein (BPI) neutralizes the proinflammatory effects of lipopolysaccharide and is of potential clinical use in the treatment of fulminant Gram-negative infections. BPI is a cationic protein with antibacterial activity stored in azurophil (primary) granules of neutrophil granulocytes. However, the absence of BPI in patients with specific granule deficiency indicates a transcriptional control of BPI, which is distinct from that of other azurophil granule proteins. Accordingly, we demonstrate in vivo that the BPI mRNA level peaks, together with mRNA for specific granule proteins, during the myelocytic and metamyelocytic stage of granulocytic maturation. The human promyelocytic cell line NB4 expresses several azurophil granule proteins, but expression of BPI is undetectable. We show that treatment of NB4 cells with all-trans retinoic acid (ATRA) induces BPI expression at mRNA and at protein level. The induction is dependent on de novo protein synthesis, as judged by sensitivity to cycloheximide. Previous investigations have indicated a potential role of CCAAT/enhancer-binding protein (C/EBP) transcription factors in the regulation of BPI expression. Here, we show that induction of NB4 cells with ATRA correlates to direct binding of C/EBPbeta and C/EBPepsilon to the proximal BPI promoter, as determined by electrophoretic mobility shift analysis and chromatin immunoprecipitation. The dependency on C/EBPbeta and C/EBPepsilon provides an explanation for delayed BPI mRNA expression, as compared with mRNA of other azurophil granule proteins. PMID- 16684887 TI - Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin. AB - Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 micros after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition. PMID- 16684890 TI - Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells. AB - CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation, the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study, we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells, TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently, reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, which relays signals to downstream signaling components, also showed reduced phosphorylation, which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings, the defect is accompanied with impaired actin cap formation, implicating a failure of actin remodeling of the cells. Together, our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways, which could be critical for inducing the distinct behavior of these cells. PMID- 16684889 TI - IL-5-mediated eosinophil survival requires inhibition of GSK-3 and correlates with beta-catenin relocalization. AB - Interleukin (IL)-5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine-specific alpha-chain and a beta-chain shared with the receptors for IL-3 and the granulocyte macrophage-colony stimulating factor. The molecular mechanisms by which IL-5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL-5 withdrawal induces eosinophil apoptosis through a mitochondria-dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol-3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL-5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK-3). We analyzed expression of FOXO3a-dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl-2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK-3 is activated after IL-5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. beta-catenin, a direct GSK-3 substrate, is present in the nucleus of IL-5-stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK-3-dependent manner. This is the first report describing a potential role for GSK-3 and beta-catenin in regulating eosinophil survival and suggests a novel mechanism by which IL-5 inhibits the constitutive apoptotic program in these cells. PMID- 16684891 TI - The generation and modulation of antigen-specific memory CD8 T cell responses. AB - The immune system has adapted to effect different mechanisms to combat the multitude of potential pathogens in our environment. In particular, CD8 T cells are participants in the immune response to intracellular pathogens, which include viruses, certain types of bacteria, and protozoa. Classified as members of the adaptive immune system, antigen-specific CD8 T cells after activation eventually form a pool of memory. Memory cells have an enhanced ability to protect against subsequent infections. The generation of antigen-specific CD8 T cells, therefore, is a potential approach in the design of vaccines, especially for those pathogens in which the humoral response is insufficient to protect the host. PMID- 16684892 TI - Arteriogenesis depends on circulating monocytes and macrophage accumulation and is severely depressed in op/op mice. AB - It has been suggested that monocytes/macrophages represent the pivotal cell type during early adaptive growth of pre-existent arterial anastomoses toward functional collateral arteries (arteriogenesis) upon arterial occlusion. This hypothesis was supported by previous studies providing evidence that elevation of the peripheral monocyte count, increased monocyte survival (e.g., granulocyte macrophage-colony stimulating factor), as well as enhanced attraction or adhesion (e.g., monocyte chemoattractant protein 1; intercellular adhesion molecule 1) of the latter cells correlates directly with the arteriogenic response to restore tissue perfusion. However, the experimental proof of the essential role of monocytes/macrophages remains to be given. We therefore hypothesized that arteriogenesis is reduced in a genuine, nonpharmocologically induced monocyte/macrophage-deficient model of femoral artery occlusion in osteopetrotic (op/op) mice. Total leukocyte count did not differ between op/op mice and control (B6C3Fe a/a-Csf1(+/+)) mice. op/op mice show a significant monocytopenia (0.67%+/ 0.38% vs. 1.53%+/-0.32%), granulocytosis (33.66%+/-6.67% vs. 22.83+/-7.47%), and a concomitant, relative lymphopenia (65.67%+/-6.58% vs. 75.65%+/-7.31%). Microsphere-based perfusion measurement 7 days after femoral artery occlusion demonstrated a significantly reduced perfusion restoration upon femoral artery occlusion in op/op mice as compared with controls (28.19%+/-0.91% vs. 47.88%+/ 2.49%). The application of a novel method of high resolution (microfocus X-ray system) angiography revealed a reduction of proliferation and diameter of collateral arteries. Quantitative immunohistology showed significantly lower numbers of macrophages in the surrounding tissue of proliferating arteries. This study provides additional evidence for the preeminent role of monocytes/macrophages during arteriogenesis in a genuine model of monocyte deficiency, i.e., without pharmacological intervention. PMID- 16684893 TI - Enhanced early vascular permeability in gelatinase B (MMP-9)-deficient mice: putative contribution of COX-1-derived PGE2 of macrophage origin. AB - Increased vascular permeability leading to vascular leakage is a central feature of all inflammatory reactions and is critical for the formation of an inflammatory exudate. The leakage occurs because of gap formation between endothelial cells and breakdown of the basement membrane barriers. The present study aimed to investigate the role of gelatinase B [matrix metalloproteinase 9 (MMP-9)], known to be involved in neutrophil exudation, in changes of vascular permeability at the early stages of acute zymosan peritonitis. We show that although MMP-9 is being released already within the first minutes of peritonitis, its lack, induced pharmacologically or genetically, does not decrease but rather increases vasopermeability. In mice treated with an inhibitor of gelatinases (A and B), a tendency to increased vasopermeability existed, and in MMP-9-/- mice [knockout (KO)], the difference was statistically significant in comparison with their controls. Moreover, in intact KO mice, significantly augmented production of prostaglandin E(2) (PGE(2)) of cyclooxygenase 1 (COX-1) origin was detected, and depletion of peritoneal macrophages, but not mast cells, decreased vasopermeability in KO mice. Thus, the increase of vasopermeability observed on KO mice is a result of the increased production of COX-1-derived PGE(2) by peritoneal macrophages. We conclude that genetic deficiency in gelatinase B might lead to the development of a compensatory mechanism involving the COX pathway. PMID- 16684894 TI - Commentary: what explains widening geographic differences in life expectancy in New Zealand? PMID- 16684895 TI - Commentary: development of the Aberdeen children of the 1950s study. PMID- 16684896 TI - Epidemiology of a primary pneumonic plague in Kantoshu, Manchuria, from 1910 to 1911: statistical analysis of individual records collected by the Japanese Empire. AB - BACKGROUND: Among the potential uses of Yersinia pestis, intentional release of its aerosolized form, causing person-to-person transmission, is thought to be the most threatening. With the current rarity of pneumonic plague epidemics, our epidemiological knowledge remains insufficient for detailed characterization of effective control measures. METHODS: Temporal patterns and key biological parameters of a pneumonic plague epidemic in Manchuria from 1910 to 1911 were analysed based on historical records collected by Kanto Totokufu, the administration of the Japanese Empire in Manchuria at that time. The serial intervals were fitted to gamma distribution using the maximum likelihood method, and time-delay distributions from onset-to-admission, admission-to-death, and onset-to-death were investigated. RESULTS: Whereas a total of 228 cases were diagnosed with pneumonic plague in areas under direct control of the Japanese Empire, 4,781 cases were also recorded in surrounding areas. Although the epidemic grew exponentially in the early phase, the average doubling time steadily increased reflecting successful control efforts. The estimated mean serial interval (and standard deviation) was 5.7 (3.6) days. All cases with known dates of onset were admitted to hospital within 4 days after onset, and the mean time from onset to admission was 1.1 (0.4) days. CONCLUSIONS: The increase in doubling time demonstrates the efficient and rapid countermeasures employed. Since the short interval from onset to death implies the importance of rapid responses, the challenge in confronting a future bioterrorist attack is to implement rapid and appropriate integration of control measures both at the individual and community level to prevent further transmissions as well as lower case fatality. PMID- 16684898 TI - Commentary: bonding, bridging, and linking--but still not much going on. PMID- 16684899 TI - Widening socioeconomic inequalities in US life expectancy, 1980-2000. AB - BACKGROUND: This study examines changes in the extent of inequalities in life expectancy at birth and other ages in the United States between 1980 and 2000 by gender and socioeconomic deprivation levels. METHODS: A factor-based deprivation index consisting of 11 education, occupation, wealth, income distribution, unemployment, poverty, and housing quality indicators was used to define deprivation deciles, which were then linked to the US mortality data at the county-level. Life expectancy estimates were developed by age, gender, and deprivation levels for three 3 year time periods: 1980-82, 1989-91, and 1998 2000. Inequalities in life expectancy were measured by the absolute difference between the least-deprived group and each of the other deprivation deciles. Slope indices of inequality for each gender and time period were calculated by regressing life expectancy estimates on deprivation levels using weighted least squares models. RESULTS: Those in less-deprived groups experienced a longer life expectancy at each age than their counterparts in more-deprived groups. In 1980 82, the overall life expectancy at birth was 2.8 years longer for the least deprived group than for the most-deprived group (75.8 vs 73.0 years). By 1998 2000, the absolute difference in life expectancy at birth had increased to 4.5 years (79.2 vs 74.7 years). The inequality indices also showed a substantial widening of the deprivation gradient in life expectancy during the study period for both males and females. CONCLUSIONS: Between 1980 and 2000, those in higher socioeconomic groups experienced larger gains in life expectancy than those in more-deprived groups, contributing to the widening gap. PMID- 16684900 TI - Regulation of dopamine transporter trafficking by intracellular amphetamine. AB - The dopamine (DA) transporter (DAT) mediates the removal of released DA. DAT is the major molecular target responsible for the rewarding properties and abuse potential of the psychostimulant amphetamine (AMPH). AMPH has been shown to reduce the number of DATs at the cell surface, and this AMPH-induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT hDAT). However, AMPH did not induce cytosolic redistribution in an uptake impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A-hDAT. Furthermore, direct intracellular application of AMPH, via a whole-cell patch pipette, stimulated the trafficking of Y335A-hDAT. Taken together, these data suggest that the DAT transport cycle is not required for AMPH-induced down-regulation and that an increase of intracellular AMPH is an essential component of DAT redistribution. PMID- 16684901 TI - Role of adipose tissue as an inflammatory organ in human diseases. AB - Reviews on the inflammatory role of adipose tissue outside the field of metabolism are rare. There is increasing evidence provided by numerous basic research studies from nearly all internal medicine subspecializations that adipocytes and adipocytokines are involved in primary inflammatory processes and diseases. Therefore, it is the aim of the present review to discuss and to summarize the current knowledge on the inflammatory role of adipocytokines and special types of regional adipocytes such as retroorbital, synovial, visceral, subdermal, peritoneal, and bone marrow adipocytes in internal medicine diseases. Future clinical and therapeutic implications are discussed. PMID- 16684902 TI - Assessment of children's growth and development in the 21st century. PMID- 16684903 TI - Clinical skills at conferences. PMID- 16684904 TI - Transitions in breastfeeding: daily parent diaries provide evidence of behavior over time. AB - This study addressed a key question for assessing breastfeeding duration: at what point is an infant considered no longer exclusively breastfed or no longer breastfed at all? Mothers provided longitudinal infant feeding data via daily checklists. Transitions between exclusive to partial breastfeeding and partial to no breastfeeding were compared across 11 time periods for 10 age groups of infants. Daily transitions between exclusive and partial breastfeeding were common, especially for infants 6 months of age and younger, and transitions from partial to no breastfeeding occurred much more quickly than transitions from exclusive to partial breast-feeding. Ages at supplementation and weaning calculated in 1-day or 7-day spans correlated highly (intraclass correlation = .99). These results support the Breastfeeding Definitions and Data Collection Periods guideline recently developed by the Breastfeeding Committee for Canada and may bring the breastfeeding research and clinical communities closer to a consensus on the definition of breastfeeding over time. PMID- 16684905 TI - Evaluation of breastfeeding of very low birth weight infants: can we use the infant breastfeeding assessment tool? AB - The objective of this study was to evaluate the use of the Infant Breastfeeding Assessment Tool (IBFAT, Matthews, 1993) to assess breastfeeding in very low birth weight (VLBW, < 1.5 kg) infants. Thirty-four mothers of singleton VLBW infants completed the IBFAT and underwent a standardized feeding observation at 35 weeks corrected age. Median feeding duration was 14.5 minutes (range, 1-20), intake per feed was 6.0 mL (range, 0-60), and intake rate was 0.5 mL/minute (range, 0-3). IBFAT scores were correlated with feeding observation measures. The IBFAT summary score was significantly correlated with milk intake volume (r = 0.651, P < .001) and intake rate (r = 0.559, P = .001). The IBFAT sucking quality score was significantly associated with percent time sucking (r = 0.559, P = .001) and sucking bursts (r = 0.632, P< .001 on the feeding observation. The authors conclude that although IBFAT scores correlated with objective feeding measures, these data do not support use of the IBFAT to identify infants with adequate as compared to inadequate intakes. Further study is needed. PMID- 16684906 TI - Fatigue and breastfeeding: an inevitable partnership? AB - Postpartum fatigue is a normal condition that most women experience. Breastfeeding is often associated in women's minds as contributing to the feeling of overall perceived fatigue, and many women indicate that they have ceased breastfeeding because of fatigue. However, the relationship between feeding choice and perceived fatigue has never been established. Two hundred and fifty three women participated in a study examining whether perceived fatigue differed for bottle-feeding and breastfeeding women at 3 different times during the postpartum period (2-4 days, 6 weeks, and 12 weeks postpartum). Results showed no significant differences for these 2 groups, suggesting that perceived fatigue during the postpartum period is not dependent on feeding choice. Additional analyses examining other variables with a potential effect were nonsignificant. Because perceived physical fatigue does not appear to be dependent on feeding choice, women should be prepared for the feeling of perceived fatigue during the postpartum period while at the same time be reassured that feeding choice is not correlated. PMID- 16684907 TI - Health workers' support for breastfeeding in Ibadan, Nigeria. AB - Breastfeeding in Nigeria is universal, and exclusive breastfeeding was introduced in 1992, yet no study has assessed health workers' support for breastfeeding at the grassroots level. This study assessed health workers' tangible support for breastfeeding at primary care facilities in Ibadan and factors affecting it, including knowledge of and attitudes toward breastfeeding. Among the 386 workers, there was moderate support for breastfeeding (median score = 15.0, maximum = 20). Following multivariate analysis, young age of worker (20-29 years; odds ratio [OR] = 2.9, 95% confidence interval [CI]: 1.2-6.8), more than 5 years of post training experience (OR = 2.3, 95% CI: 1.2-4.4), senior profession (OR = 2.1, 95% CI: 1.0-4.4), high breastfeeding knowledge scores (OR = 2.5, 95% CI: 1.4-4.5), and sufficient opportunities to practice tangible breastfeeding support (OR = 4.3, 95% CI: 2.4-7.7) were found to predict tangible breastfeeding support. Deliberate efforts should be made to incorporate continuing education workshops to better prepare health professionals for their role in providing tangible breastfeeding support at the primary care level. PMID- 16684908 TI - An investigation of the field trip model as a method for teaching breastfeeding to pediatric residents. AB - Pediatricians in training are underexposed to breastfeeding issues and as a result are not fully prepared to promote breastfeeding and support the breastfeeding mother. This study is a pre-post evaluation of the effectiveness of a pilot breastfeeding curriculum. Using the "field trip model," pediatric residents participated in 4 half-day teaching sessions at community sites, including a visit to a La Leche League home meeting, a Kaiser lactation consultant clinic, hospital-based lactation rounds, and a children's hospital based referral clinic. The objective of this study was to evaluate the effectiveness of this curriculum using a modified version of a previously published questionnaire that assesses knowledge about (70 items), attitude toward (6 items), and experience with breastfeeding (11 items). Residents enrolled in the field trip model of breastfeeding instruction exhibited significant increases in attitude and experience scores and self-reported high levels of satisfaction compared to controls. PMID- 16684909 TI - Ethics and lactation consultants: developing knowledge, skills, and tools. AB - This article studies the role of ethics in the context of the work of International Board Certified Lactation Consultants. It provides an overview of some of the main ethical approaches with the goal of contributing to the knowledge, skills, and tools required by lactation consultants. Five main sections structure the article: background, current literature, ethical theory and principles, implications for lactation consultants, and decision-making frameworks. Background about the International Board of Lactation Consultant Examiners and International Board Certified Lactation Consultants and the significance of applied ethics in their work are described. Current literature regarding ethics and lactation consultants is reviewed. Because a computer-based literature search yielded a lack of articles, ethics in nursing literature is also presented. Three main ethical theories and 5 key ethical principles are explored with a view to their implications for lactation consultants. Finally, decision-making frameworks are considered as systematic tools for making ethical decisions. PMID- 16684910 TI - Milk oversupply. PMID- 16684911 TI - What to expect when you see a lactation consultant. PMID- 16684916 TI - Does maternal diet contribute to colic among breastfed infants? PMID- 16684917 TI - Focal liver lesions: SPIO-, gadolinium-, and ferucarbotran-enhanced dynamic T1 weighted and delayed T2-weighted MR imaging in rabbits. AB - PURPOSE: To compare a superparamagnetic iron oxide (SPIO), VSOP-C184, with a gadopentetate dimeglumine with regard to signal-enhancing effects on T1-weighted dynamic magnetic resonance (MR) images and with another SPIO contrast medium with regard to signal-reducing effects on delayed T2-weighted MR images. MATERIALS AND METHODS: All experiments were approved by the responsible Animal Care Committee. Twenty rabbits (five for each contrast agent and dose) implanted with VX-2 carcinoma were imaged at 1.5 T. VSOP-C184 at 0.015 and 0.025 mmol Fe/kg was compared with gadopentetate dimeglumine at 0.15 mmol Gd/kg and ferucarbotran at 0.015 mmol Fe/kg. The imaging protocol comprised a T1-weighted dynamic gradient echo (GRE) MR before injection and at 6-second intervals for up to 42 seconds after injection and a T2-weighted turbo spin-echo MR before and 5 minutes after injection. Images were evaluated quantitatively, and contrast media were compared by using nonparametric analysis of variance. RESULTS: At dynamic T1-weighted GRE MR imaging with 0.015-mmol Fe/kg VSOP-C184, 0.025-mmol Fe/kg VSOP-C184, gadopentetate dimeglumine, and ferucarbotran, the median peak contrast-to-noise ratio (CNR) was 20.7 (25th percentile, 16.3; 75th percentile, 22.6), 24.2 (25th percentile, 19.3; 75th percentile, 28.5), 16.4 (25th percentile, 13.7; 75th percentile, 20.3), and 14.0 (25th percentile, 11.4; 75th percentile, 16.8), respectively. Both doses of VSOP-C184 yielded significantly higher CNR (P < .05) than the other two agents. At T2-weighted turbo spin-echo imaging with 0.015-mmol Fe/kg VSOP-C184, 0.025-mmol Fe/kg VSOP-C184, gadopentetate dimeglumine, and ferucarbotran, the median CNR was 15.0 (25th percentile, 13.4; 75th percentile, 21.3), 15.7 (25th percentile, 14.5; 75th percentile, 19.8), 11.3 (25th percentile, 8.2; 75th percentile, 12.2), and 15.7 (25th percentile, 12.5; 75th percentile, 22.4), respectively. There was no significant difference between VSOP C184 and ferucarbotran; both had a significantly higher CNR than did gadopentetate dimeglumine. CONCLUSION: VSOP-C184 produces higher liver-to-tumor contrast at dynamic T1-weighted imaging than does gadopentetate dimeglumine; at delayed T2-weighted imaging, the contrast is comparable to that achieved with ferucarbotran. PMID- 16684918 TI - Hepatic macrosteatosis: predicting appropriateness of liver donation by using MR imaging--correlation with histopathologic findings. AB - PURPOSE: To retrospectively evaluate the diagnostic performance of magnetic resonance (MR) imaging in predicting the appropriateness of liver donation in potential living liver donors by using histopathologic results as the reference standard. MATERIALS AND METHODS: This study was approved by institutional review board; all patients gave informed consent for the use of MR data for future research. Fifty-seven potential liver donors (40 male, 17 female; age range, 17 57 years; mean age, 32 years) underwent dual-echo 1.5-T MR imaging. Two radiologists qualitatively graded each MR image, with consensus for disagreements. Livers were assigned one of three degrees of hepatic steatosis on the basis of changes in hepatic signal intensity (SI) between in-phase and opposed-phase images. For quantitative analysis, a third radiologist calculated mean hepatic and mean splenic SI by averaging 25 hepatic regions of interest and three splenic regions of interest. Relative SI decrease (RSID) in the liver on opposed-phase images compared with in-phase images was calculated. Linear regression analysis was used to correlate RSID with the degree of total steatosis, macrosteatosis, and microsteatosis. Diagnostic performance for predicting the appropriateness of liver donation was analyzed. RESULTS: Histologic findings of macrosteatosis resulted in 52 patients being categorized as appropriate donors, with the remaining five being categorized as inappropriate donors. RSID was correlated with total steatosis (r = 0.850). When the RSID criterion for inappropriateness of liver donation was set at 20%, the sensitivity, specificity, and accuracy were 100%, 92.3%, and 93%, respectively. When RSID was used, four livers that had been misclassified as inappropriate for transplantation were found to have microsteatosis of various degrees and a less than moderate degree of macrosteatosis at histologic analysis. Qualitative and quantitative analyses were comparably accurate. CONCLUSION: When an RSID criterion of less than 20% was used, dual-echo MR imaging facilitated the correct prediction of appropriateness of liver donation in 53 of 57 patients. PMID- 16684919 TI - Mild cognitive impairment: evaluation with 4-T functional MR imaging. AB - PURPOSE: To prospectively assess abnormalities in brain activation patterns during encoding and retrieval in subjects with mild cognitive impairment by using 4-T functional magnetic resonance (MR) imaging. MATERIALS AND METHODS: The institutional review board approved this HIPAA-compliant study; all subjects gave written informed consent. Twenty patients with mild cognitive impairment (12 men, eight women; mean age, 75.0 years +/- 7.6 [standard deviation]) and 20 elderly control subjects (nine men, 11 women; mean age, 71.2 years +/- 4.5) underwent functional MR imaging at 4 T during a novel-versus-familiar face-name encoding retrieval task. The magnitude of blood oxygen level-dependent brain responses across the entire brain were compared within and between subjects with mild cognitive impairment and control subjects by using a voxelwise random-effects model. A one-sample t test was used for within-group analysis; an analysis-of covariance model (with age as a covariate) was used for between-group analysis. RESULTS: Brain regions activated by the task (prefrontal, medial temporal, and parietal regions) during encoding were similar to those activated during retrieval, with larger areas activated during retrieval. Subjects with mild cognitive impairment showed decreased magnitude of activation in bilateral frontal cortex regions (during encoding and retrieval), the left hippocampus (during retrieval), and the left cerebellum (during encoding) compared with magnitude of activation in control subjects (P < .001). Patients with mild cognitive impairment showed increased activation in the posterior frontal lobes (during retrieval) (P < .001). Lower hippocampal activation during retrieval was the most significant correlate of clinical severity of memory loss in mild cognitive impairment (P < .001). CONCLUSION: A difference exists in the response of brain regions underlying encoding and retrieval in mild cognitive impairment. Memory deficits in mild cognitive impairment may be linked to functional alterations in several specific brain regions both inside and outside the medial temporal lobe. PMID- 16684920 TI - Differentiating benign from malignant solid breast masses: comparison of two dimensional and three-dimensional US. AB - PURPOSE: To compare prospectively obtained static two-dimensional (2D) and three dimensional (3D) ultrasonographic (US) images in the diagnostic performance of radiologists with respect to the differentiation of benign from malignant solid breast masses with histopathologic examination as the reference standard. MATERIALS AND METHODS: This study had institutional review board approval, and patient informed consent was obtained. Conventional 2D and 3D US images were obtained from 141 patients (age range, 25-71 years; mean age, 46 years) with 150 solid breast masses (60 cancers and 90 benign lesions) before excisonal or needle biopsy. Four radiologists who had not performed the examinations independently reviewed 2D US images and stored 3D US data and provided a level of suspicion concerning probability of malignancy. The sensitivity, specificity, and negative predictive values of 2D images were compared with those of 3D US images. RESULTS: For all readers, 3D US images were the same as or better than 2D US images in terms of sensitivity (100% vs 100% for reader 1; 100% vs 98% for reader 2; 98% vs 93% for reader 3; 93% vs 92% for reader 4), specificity (58% vs 56% for reader 1; 51% vs 46% for reader 2; 83% vs 72% for reader 3; 86% vs 84% for reader 4), and negative predictive values (100% vs 100% for reader 1; 100% vs 98% for reader 2; 99% vs 94% for reader 3; 95% vs 94% for reader 4). These differences, however, were not statistically significant (P > .05). CONCLUSION: The performance of the radiologists with respect to the characterization of solid breast masses with static 2D US images was similar to that with 3D US data. PMID- 16684921 TI - Incidental pulmonary emboli in oncology patients: prevalence, CT evaluation, and natural history. AB - PURPOSE: To retrospectively determine the prevalence and natural history of incidental pulmonary emboli in oncology patients, the number of such cases reported at initial thoracic computed tomographic (CT) image interpretation, and the factors that contribute to underdiagnosis. MATERIALS AND METHODS: Institutional review board approval, which included HIPAA-compliant access to protected health information and waived patient consent requirements, was obtained for this retrospective study. Four hundred three consecutive oncology patients (199 male, 204 female; age range, 14-87 years; mean age, 55 years) in whom adequate-quality multidetector thoracic CT was performed within a 10-day period for indications other than pulmonary emboli assessment were identified. There were 31 (7.7%) inpatients at the time of imaging. Each imaging case was reviewed by two independent radiologists, and all pulmonary emboli were confirmed by a panel of three thoracic radiologists. Clinical charts were reviewed for demographic data, embolus detection, and outcomes up to 2 years after the initial examination. Patient groups were compared by using chi2 and one-sided binomial tests. RESULTS: Sixteen (4.0%) of the 403 patients had pulmonary emboli. The highest prevalences were in patients with gynecologic malignancies (two of 13, 15%) and in those with melanoma (four of 41, 10%). Four (25%) of the 16 patients with emboli were identified at initial clinical CT image interpretation, and all had multiple emboli involving at least the lobar arteries. Missed emboli typically were solitary and involved smaller arteries; no other confounding factors were identified. Six (60%) of 10 patients with emboli who underwent any lower extremity imaging had deep vein thrombosis. With the exception of one patient, who was transferred back to the referring physician and lost to follow up, all patients with reported pulmonary emboli were treated. Two patients had subsequent embolic events: one death despite treatment and one recurrent embolus in a nontreated patient. CONCLUSION: Incidental pulmonary emboli were seen in 16 (4%) oncology patients but were initially reported in only four of them. The small size of involved arteries contributes to the failed detection at initial CT image interpretation, and patients with emboli in these small vessels may have deep vein thrombosis or recurrent emboli. PMID- 16684923 TI - Epac-mediated Ca(2+) mobilization and exocytosis in inner medullary collecting duct. AB - PKA has traditionally been thought as the binding protein of cAMP for mediating arginine vasopressin (AVP)-regulated osmotic water permeability in kidney collecting duct. It is now known that cAMP also exerts its effects via Epac (exchange protein directly activated by cAMP) and that intracellular Ca(2+) mobilization is necessary for AVP-induced apical exocytosis in inner medullary collecting duct (IMCD). The role of Epac as an effector of cAMP action in addition to PKA was investigated using confocal fluorescence microscopy in perfused IMCD. PKA inhibitors (1 microM H-89 or 10 microM KT-5720) at concentrations known to inhibit aquaporin-2 (AQP2) phosphorylation did not prevent AVP-induced Ca(2+) mobilization and oscillations. Epac-selective cAMP agonist (8-pCPT-2'-O-Me-cAMP) mimicked AVP in triggering Ca(2+) mobilization and oscillations, which was blocked by ryanodine but not by Rp-cAMP (a competitive antagonist of cAMP binding to PKA). 8-pCPT-2'-O-Me-cAMP also triggered apical exocytosis in the presence of a PKA inhibitor. Immunolocalization of AQP2 in perfused IMCD demonstrated that 8-pCPT-2'-O-Me-cAMP induces apical targeting of AQP2 and that AQP2 is abundant in junctional regions of basolateral membrane. Immunofluorescence study also confirmed the presence of Epac (isoform I) in IMCD. These results indicate that activation of Epac by an exogenous cAMP analog triggers intracellular Ca(2+) mobilization and apical exocytotic insertion of AQP2 in IMCD. PMID- 16684922 TI - Cl- channels of the distal nephron. AB - Cl- currents were observed under whole cell clamp conditions in cells of the rat cortical collecting duct (CCD), connecting tubule (CNT), and thick ascending limb of Henle's loop (TALH). These currents were much larger in intercalated cells compared with principal cells of the CCD and were also larger in the TALH and in the CNT compared with the CCD. The conductance had no strong voltage dependence, and steady-state currents were similar in inward and outward directions with similar Cl- concentrations on both sides of the membrane. Current transients were observed, particularly at low Cl- concentrations, which could be explained by solute depletion and concentration in fluid layers next to the membrane. The currents had a remarkable selectivity among anions. Among halides, Br- and F- conductances were only 15% of that of Cl-, and I- conductance was immeasurably small. SCN- and OCN- conductances were approximately 50%, and aspartate, glutamate, and methanesulfonate conductance was approximately 5% that of Cl-. No conductance could be measured for any other anion tested, including NO3-, HCO3-, formate, acetate, or isethionate; NO3- and I- appeared to block the channels weakly. Conductances were diminished by lowering the extracellular pH to 6.4. The properties of the conductance fit best with those of the cloned renal anion channel ClC-K2 and likely reflect the basolateral Cl- conductances of the cells of these nephron segments. PMID- 16684924 TI - MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells. AB - Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) --> MAPK kinase (MKK) --> MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3 transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 -->--> p38 kinase -->--> TonE. PMID- 16684925 TI - Glucose-evoked alterations in connexin43-mediated cell-to-cell communication in human collecting duct: a possible role in diabetic nephropathy. AB - Aberrant sodium absorption has been linked to the development of hypertension in both renal disease and diabetes. Efficient absorption depends on coordination of cellular activity across the entire epithelium via cell-to-cell coupling. In the current study we have utilized a model human collecting duct cell line (HCD) to assess the role of connexin43 (Cx43)-mediated gap junctions in the transfer of intracellular Ca(2+) transients within coupled cell clusters. HCD cells express Cx43 mRNA and protein, as well as that for the mechanosensitive transient receptor potential receptor (TRPV4). Mechanical stimulation of individual cells within a cluster evoked a transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) that propagated between cells via a heptanol-sensitive mechanism. The rise in [Ca(2+)](i) was dependent on both store release and Ca(2+)-influx pathways. Lucifer yellow dye transfer and Cx43 knockdown experiments confirmed direct cell-to-cell communication. Application of the Ca(2+) ionophore ionomycin, or an increase in glucose (5 to 25 mM), produced a time-dependent (48 h) increase in Cx43 protein expression. The transmission rate of touch-evoked Ca(2+) transients between coupled cells was accelerated after exposure to high glucose, providing a functional correlate to increased Cx43 expression. These data suggest a pivotal role for Cx43-mediated gap junctions in the synchronization of activity between HCD cells in response to stimuli that mimic osmotic and physical changes. Cx43 expression and cell-to-cell communication increased in response to high glucose and may protect the collecting duct from renal damage associated with more established diabetic nephropathy. PMID- 16684926 TI - Podocytes are sensitive to fluid shear stress in vitro. AB - Podocytes are exposed to mechanical forces arising from glomerular capillary pressure and filtration. It has been shown that stretch affects podocyte biology in vitro and plays a significant role in the development of glomerulosclerosis in vivo. However, whether podocytes are sensitive to fluid shear stress is completely unknown. In the present study, we therefore exposed cells of a recently generated conditionally immortalized mouse podocyte cell line to defined fluid shear stress in a flow chamber, mimicking flow of the glomerular ultrafiltrate over the surface of podocytes in Bowman's space. Shear stress above 0.25 dyne/cm(2) resulted in dramatic loss of podocytes but not of proximal tubular epithelial cells (LLC-PK(1) cells) after 20 h. At 0.015-0.25 dyne/cm(2), lamellipodia formation in podocytes was enhanced and the actin nucleation protein cortactin was redistributed to the cell margins. Shear stress further diminished stress fibers and the presence of vinculin in focal adhesions. Linear zonula occludens-1 distribution at cell-cell contacts remained unaffected at low shear stress. At 0.25 dyne/cm(2), the monolayer was broken up and remaining cell-cell contacts were reinforced by F-actin and alpha-actinin. Because the cytoskeletal changes induced by shear stress suggested the involvement of tyrosine kinases (TKs), we tested several TK inhibitors that were all without effect on podocyte number under static conditions. At 0.25 dyne/cm(2), however, the TK inhibitors genistein and AG 82 were associated with marked podocyte loss. Our data demonstrate that podocytes are highly sensitive to fluid shear stress. Shear stress induces a reorganization of the actin cytoskeleton and activates specific tyrosine kinases that are required to withstand fluid shear stress. PMID- 16684927 TI - In vivo determination of diffusive transport parameters in a superfused tissue. AB - To address the hypothesis that functional changes in tissue transport can be related to structural alterations, we combined mathematical modeling with in vivo experimentation. The model concept includes interstitial diffusion and removal by a distributed microvasculature. Transport of solute and water across the peritoneum is measured via a plastic chamber affixed to the abdominal wall of anesthetized Sprague-Dawley rats. Solutions containing [(14)C]mannitol, with or without vasoactive compounds [control (C; n = 10), C + nitroprusside (NP; n = 10), C + norepinephrine (NE; n = 10)], were infused into the chamber, and the volume and tracer concentrations were determined over 60 min to calculate the mass transfer coefficient (MTC) and the water flux. At 60 min, FITC-dextran (500 kDa) was given to mark the perfused vasculature. After euthanasia, the tissue under the chamber was frozen, dried, sliced with a cryomicrotome, and examined with fluorescent microscopy and quantitative autoradiography. The microvessel density (x10(3)/cm(2): NE, 50 +/- 10; C, 180 +/- 7.0; NP, 225 +/- 15) resulted in marked differences (P < 0.05) in water flux (mul.min(-1).cm(-2): NE, 0.1 +/- 0.1; C, 1.6 +/- 0.4; NP, 1.0 +/- 0.2) and in mannitol MTC (x10(3) cm/min: NE, 0.9 +/- 0.3; C, 3.8 +/- 0.3; NP, 3.6 +/- 0.6). Concentration profiles and calculated capillary permeability and tissue diffusivity were significantly different among the groups. These results demonstrate a direct correlation of mass transfer, diffusion, capillary permeability, and water flux with peritoneal vascular density and validate a method by which mechanistic changes in transport may be measured. PMID- 16684928 TI - Pituitary tumour pathogenesis. AB - Pituitary adenomas are the most common tumours in the central nervous system, make up approximately 10% of all primary intracerebral tumours [1] and are found incidentally in 3-27% of autopsies [2]. The predisposition to tumour formation of the highly specialized cellular phenotypes that characterize the anterior pituitary is unexplained, but it is tempting to speculate that the same hormones, growth factors and cytokines derived from intra- and extra-pituitary sites that maintain tight hypothalamic pituitary control may also contribute to pituicyte transformation. The interplay between genetic and humoural factors to promote cellular transformation is exemplified in pituitary tumorigenesis and is discussed in this review. PMID- 16684929 TI - Teaching resources. Cell culture as a model system for teaching: using PC12 cells. AB - This Teaching Resource provides an introduction to the use of cultured cells as an experimental approach in undergraduate laboratory research and the study of neuronal differentiation in PC12 cells. In addition, a thought experiment with answers is provided that can be used to assess student understanding of (i) the scientific method, (ii) signaling processes involved in cellular differentiation, and (iii) the use of pharmacological agents to manipulate a cell culture system. PMID- 16684930 TI - Teaching resources. The Sherlock Holmes lab: investigations in neurophysiology. AB - This Teaching Resource describes a research project that can be used in an advanced undergraduate course in neurobiology that covers basic electrophysiology and synaptic transmission. A thought experiment is provided that can be used to assess student understanding of (i) the scientific method, (ii) the process whereby nerve stimulation leads to muscle contraction, and (iii) the use of pharmacological agents to analyze a physiological system. PMID- 16684931 TI - Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor. AB - In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer N(G)-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells. PMID- 16684932 TI - LucTrap vectors are tools to generate luciferase fusions for the quantification of transcript and protein abundance in vivo. AB - Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra- and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level. The activity of luciferase (LUC) reporters can be detected in nondestructive qualitative and quantitative assays in vivo. Therefore, LUC reporters are particularly well suited for the detection of changes at the transcript and protein level. To the best of our knowledge, the number of plant transformation vectors for LUC fusions is very limited. In this article, we describe the LucTrap plant transformation vectors that allow generation of targeted and random transcriptional and translational fusions with the modified firefly LUC reporter LUC+. We demonstrate that LucTrap-based fusions can be used to monitor rapid changes in gene expression and protein abundance in vivo. PMID- 16684933 TI - Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species. AB - The domestication and improvement of fruit-bearing crops resulted in a large diversity of fruit form. To facilitate consistent terminology pertaining to shape, a controlled vocabulary focusing specifically on fruit shape traits was developed. Mathematical equations were established for the attributes so that objective, quantitative measurements of fruit shape could be conducted. The controlled vocabulary and equations were integrated into a newly developed software application, Tomato Analyzer, which conducts semiautomatic phenotypic measurements. To demonstrate the utility of Tomato Analyzer in the detection of shape variation, fruit from two F2 populations of tomato (Solanum spp.) were analyzed. Principal components analysis was used to identify the traits that best described shape variation within as well as between the two populations. The three principal components were analyzed as traits, and several significant quantitative trait loci (QTL) were identified in both populations. The usefulness and flexibility of the software was further demonstrated by analyzing the distal fruit end angle of fruit at various user-defined settings. Results of the QTL analyses indicated that significance levels of detected QTL were greatly improved by selecting the setting that maximized phenotypic variation in a given population. Tomato Analyzer was also applied to conduct phenotypic analyses of fruit from several other species, demonstrating that many of the algorithms developed for tomato could be readily applied to other plants. The controlled vocabulary, algorithms, and software application presented herein will provide plant scientists with novel tools to consistently, accurately, and efficiently describe two-dimensional fruit shapes. PMID- 16684934 TI - Sequencing multiple and diverse rice varieties. Connecting whole-genome variation with phenotypes. PMID- 16684936 TI - An overview of psychiatric issues in liver disease for the consultation-liaison psychiatrist. AB - Liver disease is a common cause of morbidity and mortality in the United States and elsewhere. Arising from infectious, hereditary, or toxin-induced sources, the detection of liver disease often requires a high index of suspicion. Clinical presentations are highly variable and are often accompanied by neuropsychiatric symptoms. This fact, along with an increased incidence of liver disease among patients with primary psychiatric disorders and the presence of varied drug use, complicates the tasks of providing care to patients with liver disease. To assist the consultation-liaison psychiatrist, the authors present the first of a two part series focused on psychiatric issues in liver disease. PMID- 16684937 TI - Acute stress disorder among parents of infants in the neonatal intensive care nursery. AB - The authors examined the prevalence of acute stress disorder (ASD) in parents of infants hospitalized in the neonatal intensive care unit (NICU). Forty parents were assessed after the birth of their infants. Parents completed self-report measures of ASD, parental stress, family environment, and coping style: 28% of parents developed symptoms of ASD. ASD was associated with female gender, alteration in parental role, family cohesiveness, and emotional restraint. Family environment and parental coping style are significantly associated with the development of trauma symptoms. Results from this study suggest potential interventions to help minimize psychological distress in parents. PMID- 16684938 TI - Psychiatric evaluations of heart transplant candidates: predicting post transplant hospitalizations, rejection episodes, and survival. AB - The authors assessed the validity of psychiatric evaluations for orthotopic heart transplant candidates with respect to predicting adverse post-transplant outcomes. A group of 108 transplant recipients were followed for an average of 970 days, and pre-transplant evaluations were retrospectively coded for psychiatric risk factors. Previous suicide attempts, poor adherence to medical recommendations, previous drug or alcohol rehabilitation, and depression significantly predicted attenuated survival times. Also, past suicide attempt was associated with a greater risk for post-transplant infection. Assessment and early treatment for these risk factors may reduce post-transplant morbidity and mortality. PMID- 16684939 TI - Incidence of posttraumatic stress disorder after prolonged surgical intensive care unit treatment. AB - The purpose of the study was to investigate the prevalence of posttraumatic stress disorder (PTSD) after prolonged intensive care unit (ICU) treatment and to study the differences between trauma patients and patients who were admitted with other diagnoses. Survivors of surgical intensive care of at least 30 days' duration underwent follow-up examination at about 35 months after discharge from the ICU. Thirty-seven patients were investigated. Seven patients met full DSM-IV criteria for diagnosis of PTSD. All of them had sustained severe multiple injuries. Patients with trauma were at significantly higher risk for developing PTSD than were non-trauma patients. After prolonged ICU treatment, PTSD seems more likely to occur when the reason for admission to the ICU was severe physical injury. The prevalence of PTSD does not seem to be related to injury severity or duration of ICU treatment. PMID- 16684940 TI - Symptoms of posttraumatic stress disorder in patients who have had a myocardial infarction. AB - Symptoms of posttraumatic stress disorder (PTSD) and risk factors for recurrent ischemia were evaluated in 65 survivors of a myocardial infarction (MI) at baseline and 6 months afterward. PTSD patients had more uncontrolled cardiovascular risk factors at baseline. Patients with PTSD (N=14) were offered trauma-focused cognitive-behavior treatment (CBT) plus a nonspecific intervention to improve adherence to medical recommendations. Adherence to aspirin improved in recipients of the nonspecific intervention (N=8); PTSD symptoms and cardiovascular risk improved in patients who received CBT (N=6). PTSD may be a treatable risk factor for poor post-MI outcome. Further research is needed to evaluate treatment options. PMID- 16684941 TI - Prospective study on influence of psychosocial factors on glycemic control in Japanese patients with type 2 diabetes. AB - The authors prospectively assessed the causal relationship between psychosocial factors and glycemic control in 256 Japanese outpatients with Type 2 diabetes. Using structural-equation modeling, they first developed a causal model in which glycosylated hemoglobin (HbA(1c)) at 6 months after assessment was influenced by the baseline psychosocial factors. Then, the reliability of the causal model was investigated with measurement of HbA(1c) at 12 months after baseline. Self efficacy directly reinforced adherence, and adherence had a direct association with future HbA(1c). Other psychosocial factors, including social support, diabetes-related distress, daily burden, and emotion-focused coping prospectively influenced HbA(1c) indirectly through self-efficacy. PMID- 16684942 TI - Cancer survival probability as a function of ego defense (adaptive) mechanisms versus depressive symptoms. AB - Psychological treatment studies, uncontrolled for ego defense (adaptive) styles, report conflicting survival results. The authors hypothesized that "immature" adaptive styles and frequent depression symptoms would independently predict lower survival rates. This study followed 86 consecutive, mostly late-stage, cancer outpatients for up to 5 years; their survival data were analyzed in relation to the Beck Depression Inventory and the Defense Style Questionnaire scores at study entry. Cumulative survival probability curves contrasted the extreme cases: the most (N=15) to the least (N=21) depressed, and the "immature" (N=14) to the "mature" (N=16) adaptors. Depression did not separate the groups until 30 months after diagnosis. Ego defense style separated them at 8 months; by 18 months, the "immature" survival probability had dropped to 50%, versus 87% for the "mature." At 36 months, survival probabilities were 19% and 57%, respectively. These data direct clinical attention toward ego defense mechanisms as indicators of distress and lowered survival in cancer patients. They further suggest that the maturity of adaptive mechanisms must be controlled for in behavioral-treatment trials of cancer patients. PMID- 16684943 TI - Depressive symptoms and physical/mental functioning with interferon/ribavirin treatment of posttransplant recurrent hepatitis C. PMID- 16684944 TI - Lamotrigine-associated rash and blood dyscrasias in a methadone-treatment patient with hepatitis C. PMID- 16684945 TI - A cognitive-behavioral treatment package for depression in Parkinson's disease. PMID- 16684946 TI - Torsades de pointes associated with ziprasidone. PMID- 16684947 TI - Self-mutilation of fingers after cervical spinal cord injury. PMID- 16684948 TI - Achalasia may mimic anorexia nervosa, compulsive eating disorder, and obesity problems. PMID- 16684949 TI - Postconcussional symptoms not a syndrome. PMID- 16684951 TI - Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase. AB - Although oxidative stress is known to contribute to endothelial dysfunction associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH(2)) and tetrahydrobiopterin (BH(4)) in lung tissues of Shunt and Control lambs. Our data indicated that although BH(4) levels were unchanged, BH(2) levels were significantly increased. Finally, we demonstrated that the addition of BH(2) produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH(2). PMID- 16684952 TI - HGF synthesis in human lung fibroblasts is regulated by oncostatin M. AB - Oncostatin M (OSM) is a IL-6 family cytokine locally produced in acute lung injury. Its profibrotic properties suggest a role in lung wound repair. Hepatocyte growth factor (HGF), produced by fibroblasts, is involved in pulmonary epithelial repair. We investigated the role of OSM in HGF synthesis by human lung fibroblasts. We showed that OSM upregulated HGF mRNA in MRC5 cells and in human lung fibroblasts, whereas IL-6 and leukemia inhibitory factor did not. OSM induced HGF secretion to a similar extent as IL-1beta in both a time- and dose dependent manner. HGF was released in its cleaved mature form, and its secretion was completely inhibited in the presence of cycloheximide, indicating a de novo protein synthesis. OSM in combination with prostaglandin E(2), a powerful HGF inductor, led to an additive effect. OSM and indomethacin in combination further increased HGF secretion. This could be explained, at least in part, by a moderate upregulation of specific OSM receptor beta mRNA expression through cyclooxygenase inhibition. These results demonstrate that OSM-induced HGF synthesis did not involve a PGE(2) pathway. OSM-induced HGF secretion was inhibited by PD-98059 (a specific pharmacological inhibitor of ERK1/2), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor) by 70, 82, and 100%, respectively, whereas basal HGF secretion was only inhibited by SP-600125 by 30%. Our results demonstrate a specific upregulation of HGF synthesis by OSM, most likely through a MAPK pathway, and support the suggestion that OSM may participate in lung repair through HGF production. PMID- 16684953 TI - IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM. AB - Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-alpha were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-kappaB, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL 17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-alpha-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 +/- 0.01964, t(1/2) = 12.0 h), compared with vehicle (-0.01030 +/- 0.007963, t(1/2) = 67.3 h) [results are expressed as decay constant (means +/- SE) and half-life (t(1/2) in h): P < 0.05]. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells. PMID- 16684954 TI - Modulation by bradykinin of angiotensin type 1 receptor-evoked RhoA activation of connective tissue growth factor expression in human lung fibroblasts. AB - The mechanisms regulating the opposing physiological actions of bradykinin (BK) and angiotensin II (AngII) are not well understood. Here we investigate signaling interactions between these two effectors. Connective tissue growth factor (CTGF) expression in IMR-90, human lung fibroblasts, is used as the endpoint target. In these cells the BK B2 receptor (BKB2R) is expressed constitutively, while no binding of AngII is detected. An inducible expression system is used to insert AngII receptor 1 (AT1R) and to obtain a signal level in response to AngII at the magnitude of BK. AngII and BK activate G protein-coupled targets, arachidonate release from cellular phospholipid stores, and intracellular phosphatidylinositol turnover equally. Both activate ERK, JNK, and p38 equally. However, AngII activates, whereas BK inactivates, RhoA. AngII induces a rapid (1 h) CTGF mRNA expression. RhoA siRNA and RhoA activation inhibitor, Y-27632, markedly reduce the AngII effect. Simultaneous treatment with BK and AngII attenuates the AT1R action. Additionally, BK in the absence of AngII lowers CTGF mRNA expression below basal levels over a span of 4 h. An AT1R/BKB2R chimera lacking heterotrimeric G protein coupling continues to activate MAP kinases to the same extent as wild-type (WT) AT1R and BKB2R. However, the increase of CTGF mRNA expression by this mutant is low, almost identical with that obtained by the simultaneous treatment of the WT AT1R-expressing cells with BK and AngII. In this context the chimeric receptor displays the characteristics of both receptors. These data demonstrate that, in human lung fibroblasts, BK modulates the action of AngII through the small G protein RhoA, but in a Galphai/Galphaq-independent manner. PMID- 16684955 TI - Tolerogenic dendritic cells: cytokine modulation comes of age. AB - Dendritic cells (DCs) include a heterogeneous family of professional APCs involved in initiation of immunity and in immunologic tolerance. Specifically, peripheral tolerance can be achieved and maintained by promoting regulatory T cell (Treg) responses and/or T-cell anergy or deletion. Until recently, immature developmental stages of DC differentiation were believed to induce T-cell anergy or Treg cells, whereas DCs transformed into mature DCs by activation stimuli were thought to represent immunogenic DCs capable of inciting primary T-cell responses. This paradigm has been challenged by the demonstration of Treg-cell expansion by antigen-bearing, fully mature DCs. Similarly, semimature DCs with a distinctive interleukin 10 (IL-10)+ IL-12- cytokine production profile might be endowed with tolerogenic functions, supporting the concept that DC maturation per se should no longer be considered as a distinguishing feature of immunogenic as opposed to tolerogenic DCs (TDCs). Cytokine-modulated TDCs reflect an incomplete or altered status of monocyte differentiation and promote in vitro induction of Treg cells and/or in vivo protection from autoimmune diseases. Several growth factors, including IL-10, transforming growth factor beta (TGF-beta), granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), and vasoactive intestinal peptide (VIP), modulate DC maturation and favor the differentiation of TDCs. From a therapeutic standpoint, cytokine-modulated TDCs might be beneficial for prevention and/or treatment of posttransplantation graft-versus-host disease (GVHD) and autoimmunity. PMID- 16684956 TI - The thrombospondin-1 N700S polymorphism is associated with early myocardial infarction without altering von Willebrand factor multimer size. AB - The N700S polymorphism of thrombospondin-1 (TSP-1) has been identified as a potential genetic risk factor for myocardial infarction (MI). In a large case control study of 1425 individuals who survived a myocardial infarction prior to age 45, the N700S polymorphism was a significant risk factor for myocardial infarction in both homozygous (odds ratio [OR] 1.9, 95% confidence interval [CI] 1.1-3.3, P = .01) and heterozygous carriers of the S700 allele (OR 1.4, 95% CI 1.1-3.3, P = .01). TSP-1 has been shown to reduce von Willebrand factor (VWF) multimer size, and the domain responsible for VWF-reducing activity has been localized to the calcium-binding C-terminal sequence. As the N700S polymorphism was previously shown to alter the function of this domain, we investigated whether the altered VWF-reducing activity of TSP-1 underlies the observed prothrombotic phenotype. The TSP1 N700S polymorphism did not influence VWF multimer size in patients homozygous for either allele nor was there a significant reduction of VWF multimer size following incubation with recombinant N700S fragments or platelet-derived TSP-1. PMID- 16684957 TI - Loss of inhibitory semaphorin 3A (SEMA3A) autocrine loops in bone marrow endothelial cells of patients with multiple myeloma. AB - Vascular endothelial growth factor165 (VEGF165) and semaphorin3A (SEMA3A) elicit pro- and antiangiogenic signals respectively in endothelial cells (ECs) by binding to their receptors VEGFR-2, neuropilin-1 (NRP1), and plexin-A1. Here we show that the VEGF165-driven angiogenic potential of multiple myeloma (MM) ECs is significantly higher than that of monoclonal gammopathy of undetermined significance (MGUS) ECs (MGECs) and human umbilical vein (HUV) ECs. This is probably due to a constitutive imbalance of endogenous VEGF165/SEMA3A ratio, which leans on VEGF165 in MMECs but on SEMA3A in MGECs and HUVECs. Exogenous VEGF165 induces SEMA3A expression in MGECs and HUVECs, but not in MMECs. Moreover, by counteracting VEGF165 activity as efficiently as an anti-VEGFR-2 antibody, exogenous SEMA3A restrains the over-angiogenic potential of MMECs. Our data indicate that loss of endothelial SEMA3A in favor of VEGF165 could be responsible for the angiogenic switch from MGUS to MM. PMID- 16684958 TI - Histone deacetylase inhibitors prevent exocytosis of interleukin-1beta-containing secretory lysosomes: role of microtubules. AB - A number of agents reducing interleukin-1beta (IL-1beta) activity are being developed as novel immunomodulatory and anti-inflammatory therapies. However, the elucidation of their molecular mechanism of action is required in the context of medical management of inflammatory diseases. Inhibitors of histone deacetylases (HDACs) are promising anticancer agents with pleiotropic activities. Of these, suberoylanilide hydroxamic acid has been reported to inhibit the production of several proinflammatory cytokines. In the present study, we investigated the effects of 2 HDAC inhibitors on IL-1beta secretion: suberoylanilide hydroxamic acid and a newly developed hydroxamic acid-derived compound ITF2357. These HDAC inhibitors do not affect the synthesis or intracellular localization of IL-1beta but both strongly reduce the levels of extracellular IL-1beta by preventing the exocytosis of IL-1beta-containing secretory lysosomes. At nanomolar concentrations, ITF2357 reduces the secretion of IL-1beta following ATP activation of the P2X7 receptor. Whereas the inhibition of HDACs results in hyperacetylation of tubulin, acetylation of HSP90 was unaffected. The reduction in IL-1beta secretion appears to be due to disruption of microtubules impairing lysosome exocytosis. Together, these observations indicate that a functional microtubule network is required for IL-1beta secretion and suggest that disruption of tubulin is the mechanism by which inhibitors of HDACs reduce the secretion of IL-1beta. PMID- 16684959 TI - Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure of immunosuppressive therapy. AB - In 87 patients with aplastic anemia who failed to respond to immunosuppressive treatment, we determined the minimal dose of total body irradiation (TBI) required when added to antithymocyte globulin (ATG, 30 mg/kg x 3) plus cyclophosphamide (CY, 50 mg/kg x 4) to achieve engraftment of unrelated donor marrow. TBI was started at 3 x 200 cGy, to be escalated or deescalated in steps of 200 cGy depending on graft failure or toxicity. Patients were aged 1.3 to 53.5 years (median, 18.6 years). The interval from diagnosis to transplantation was 3 to 328 months (median, 14.6 months). Donors were HLA-A, -B, -C, -DR, and -DQ identical for 62 patients, and nonidentical for 1 to 3 HLA loci at the antigen or allele level for 25. The dose-limiting toxicity was diffuse pulmonary injury. The optimum TBI dose was 1 x 200 cGy. Nine patients did not tolerate ATG and were prepared with CY + TBI. Graft failure occurred in 5% of patients. With a median follow-up of 7 years, 38 (61%) of 62 HLA-identical, and 10 (40%) of 25 HLA nonidentical transplant recipients are surviving. The highest survival rate with HLA-identical transplants was observed at 200 cGy TBI. Thus, low-dose TBI + CY + ATG conditioning resulted in excellent outcome of unrelated transplants in patients with aplastic anemia who had received multiple transfusions. PMID- 16684960 TI - Multiple myeloma cell survival relies on high activity of protein kinase CK2. AB - Casein kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase that regulates relevant biologic processes, many of which are dysregulated in malignant plasma cells. Here we investigated its role in multiple myeloma (MM). Analysis of MM cell lines and highly purified malignant plasma cells in patients with MM revealed higher protein and CK2 activity levels than in controls (normal in vitro-generated polyclonal plasma cells and B lymphocytes). The inhibition of CK2 with specific synthetic compounds or by means of RNA interference caused a cytotoxic effect on MM plasma cells that could not be overcome by IL-6 or IGF-I and that was associated with the activation of extrinsic and intrinsic caspase cascades. CK2 blockage lowered the sensitivity threshold of MM plasma cells to the cytotoxic effect of melphalan. CK2 inhibition also resulted in impaired IL-6 dependent STAT3 activation and in decreased basal and TNF-alpha-dependent I kappaB alpha degradation and NF-kappaB-driven transcription. Our data show that CK2 was involved in the pathophysiology of MM, suggesting that it might play a crucial role in controlling survival and sensitivity to chemotherapeutics of malignant plasma cells. PMID- 16684961 TI - Phosphatidylserine exposure in B lymphocytes: a role for lipid packing. AB - Plasma membrane lipids are usually distributed asymmetrically, with phosphatidylserine (PS) confined to the inner leaflet. PS exposure at the outer leaflet occurs early in apoptosis, but it is also constitutive on some nonapoptotic cell populations where it plays a role in cell signaling. How PS is transported ("flopped") to the cell surface is unknown. Contrary to previous reports that normal murine B lymphocytes lack lipid asymmetry, we show that PS is normally restricted to the inner leaflet of these cells. PS exposure on normal B cells did, however, occur spontaneously ex vivo. Consistent with the hypothesis that loss of PS asymmetry is regulated by CD45, PS is constitutively exposed on viable, CD45-deficient B cells. We show that calcium-stimulated PS exposure in B cells is strain variable, ABCA1 independent, and both preceded by and dependent on a decrease in lipid packing. This decrease in lipid packing is concomitant with cell shrinkage and consequent membrane distortion, both of which are potently inhibited by blockers of volume-regulatory K+ and Cl- ion channels. Thus, changes in plasma membrane organization precede PS translocation. The data suggest a model in which PS redistribution may occur by a translocase-independent mechanism at energetically favorable sites of membrane perturbation where lipid packing is decreased. PMID- 16684963 TI - Constitutive activation of STAT5 and Bcl-xL overexpression can induce endogenous erythroid colony formation in human primary cells. AB - The biologic hallmark of polycythemia vera (PV) is the formation of endogenous erythroid colonies (EECs) with an erythropoietin-independent differentiation. Recently, it has been shown that an activating mutation of JAK2 (V617F) was at the origin of PV. In this work, we studied whether the STAT5/Bcl-xL pathway could be responsible for EEC formation. A constitutively active form of STAT5 was transduced into human erythroid progenitors and induced an erythropoietin independent terminal differentiation and EEC formation. Furthermore, Bcl-xL overexpression in erythroid progenitors was also able to induce erythroid colonies despite the absence of erythropoietin. Conversely, siRNA-mediated STAT5 and Bcl-xL knock-down in human erythroid progenitors inhibited colony-forming unit-erythroid (CFU-E) formation in the presence of Epo. Altogether, these results demonstrate that a sustained level of the sole Bcl-xL is capable of giving rise to Epo-independent erythroid colony formation and suggest that, in PV patients, JAK2(V617F) may induce EEC via the STAT5/Bcl-xL pathway. PMID- 16684962 TI - Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1 mutants in TNFR1-associated periodic fever syndrome (TRAPS). AB - Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autosomal dominant systemic autoinflammatory disease associated with heterozygous mutations in TNF receptor 1 (TNFR1). Here we examined the structural and functional alterations caused by 9 distinct TRAPS-associated TNFR1 mutations in transfected cells and a mouse "knock-in" model of TRAPS. We found that these TNFR1 mutants did not generate soluble versions of the receptor, either through membrane cleavage or in exosomes. Mutant receptors did not bind TNF and failed to function as dominant-negative inhibitors of TNFR1-induced apoptosis. Instead, TRAPS mutant TNFR1 formed abnormal disulfide-linked oligomers that failed to interact with wild-type TNFR1 molecules through the preligand assembly domain (PLAD) that normally governs receptor self-association. TRAPS mutant TNFR1 molecules were retained intracellularly and colocalized with endoplasmic reticulum (ER) markers. The capacity of mutant receptors to spontaneously induce both apoptosis and nuclear factor kappaB (NF-kappaB) activity was reduced. In contrast, the R92Q variant of TNFR1 behaved like the wild-type receptor in all of these assays. The inflammatory phenotype of TRAPS may be due to consequences of mutant TNFR1 protein misfolding and ER retention. PMID- 16684964 TI - Identification of Y589 and Y599 in the juxtamembrane domain of Flt3 as ligand induced autophosphorylation sites involved in binding of Src family kinases and the protein tyrosine phosphatase SHP2. AB - Early signal relay steps upon ligand binding to the receptor tyrosine kinase Flt3 (ie, sites of Flt3 autophosphorylation and subsequent docking partners) are mainly unresolved. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing, we identified the tyrosine residues 572, 589, 591, and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3 ligand (FL)-mediated responses in wild-type-Flt3-(WT-Flt3-), Y589F-Flt3-, and Y599F-Flt3 expressing 32D cells. Compared with WT-Flt3-32D cells upon ligand stimulation, 32D-Y589F-Flt3 showed enhanced Erk activation and proliferation/survival, whereas 32D-Y599F-Flt3 cells hereby displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for signal relay molecules including Src family kinases and SHP2. Consistently, 32D-Y589F-Flt3 and 32D-Y599F Flt3 showed decreased FL-triggered activation of Src family kinases. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. Y599 was additionally found to interact with the protein tyrosine phosphatase SHP2 in a phosphorylation dependent manner. As Y599F-Flt3-32D was unable to associate with and to phosphorylate SHP2 and since silencing of SHP2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3 phenotype, we hypothesize that recruitment of SHP2 to pY599 contributes to FL-mediated Erk activation and proliferation. PMID- 16684965 TI - New models of frontal-subcortical skeletomotor circuit pathology in tardive dyskinesia. AB - Tardive dyskinesia (TD) is a hyperkinetic movement disorder that can occur as a side effect of treatment with antipsychotic medications. Because antipsychotics block the D2 family of dopamine receptors in the striatum, it has long been suspected this blockade contributes to the development of TD. Specifically, increased sensitivity of the dopamine receptors following chronic blockade has been thought to result in abnormal functioning of the frontal-subcortical (FSC) skeletomotor circuit and the symptoms of TD. However, this hypothesis remains unproven. In recent years, substantial research has focused on the basal ganglia and FSC circuits. This research has resulted in the development of the focused selection model of skeletomotor circuit function. This hypothesis provides a compelling model of neurocircuit abnormalities in TD. A greater understanding of the neuropathology of TD may lead to the development of better treatment and prevention strategies for this disorder. Furthermore, this information may contribute to a more complete understanding of normal skeletomotor circuit function and the role of circuit pathology in numerous neuropsychiatric conditions. PMID- 16684966 TI - Voltage-gated potassium channels: regulation by accessory subunits. AB - Voltage-gated potassium channels regulate cell membrane potential and excitability in neurons and other cell types. A precise control of neuronal action potential patterns underlies the basic functioning of the central and peripheral nervous system. This control relies on the adaptability of potassium channel activities. The functional diversity of potassium currents, however, far exceeds the considerable molecular diversity of this class of genes. Potassium current diversity contributes to the specificity of neuronal firing patterns and may be achieved by regulated transcription, RNA splicing, and posttranslational modifications. Another mechanism for regulation of potassium channel activity is through association with interacting proteins and accessory subunits. Here the authors highlight recent work that addresses this growing area of exploration and discuss areas of future investigation. PMID- 16684967 TI - Cortical networks related to human use of tools. AB - Greater manual dexterity and greater conceptual knowledge of tool use represent two main features that distinguish humans from other primates. Studies of human brain lesions suggest that the left hemisphere (at least in right-handed people) includes a system for processing manual skills that is specialized for tool use that interacts with another system involved more with conceptualizing, planning, and accessing knowledge associated with tool use. Growing evidence from recent neuroimaging studies supports this organization, and studies have begun to highlight specific brain regions and pathways that may be necessary for tool use. This review compares and summarizes results from 64 paradigms published over the past decade that have examined cortical regions associated with tool use skills and tool knowledge. A meta-analysis revealed cortical networks in both hemispheres, though with a clear left hemisphere bias, which may be organized to optimally represent action knowledge. Portions of this network appear to represent part of a system that is tightly linked with language systems, which is discussed together with the effects that handedness may have on the cortical organization for tool use. PMID- 16684968 TI - Glutamate receptors and endoplasmic reticulum quality control: looking beneath the surface. AB - Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system. The cellular regulation of glutamate receptor (GluR) ion channel function and expression is important for maintaining or adjusting target cell excitability to meet ever-changing demands, for example, in relation to developmental or use-dependent synaptic plasticity. Dysregulation of GluR function or expression may be a contributing factor in certain forms of epilepsy, stroke/ischemia, head trauma, cognitive impairments, and neurodegenerative disease. Recent years have seen substantial progress in understanding how GluRs operate in terms of their structural and functional properties, their synaptic targeting and membrane anchoring by PDZ-domain proteins, and their activity dependent cycling at the plasma membrane. Yet precious little is known about the earliest events in GluR biogenesis or the mechanisms in place to ensure the GluRs that reach the cell surface are processed, folded, and oligomerized in an appropriate manner. Indeed, only a minor fraction of the GluR content of cells is expressed at any given time on the cell surface, whereas most of the remaining receptors exist in the endoplasmic reticulum (ER). The functional competence and significance of the ER fraction of receptors are presently unknown, but they are generally thought to represent immature, unassembled, or improperly assembled subunits. Some are ultimately destined for insertion in the plasma membrane. Others may be targeted for proteosomal degradation. Still others might provide a latent pool of fully functional receptors that can be recruited to enhance cell excitability in response to specific signals or under pathological conditions. This review will explore the structural and functional elements that regulate GluR assembly and export from the ER. PMID- 16684969 TI - Phospholipase A2-generated lipid mediators in the brain: the good, the bad, and the ugly. AB - Phospholipase A2 (PLA2) generates arachidonic acid, docosahexaenoic acid, and lysophospholipids from neural membrane phospholipids. These metabolites have a variety of physiological effects by themselves and also are substrates for the synthesis of more potent lipid mediators such as eicosanoids, platelet activating factor, and 4-hydroxynonenal (4-HNE). At low concentrations, these mediators act as second messengers. They affect and modulate several cell functions, including signal transduction, gene expression, and cell proliferation, but at high concentrations, these lipid mediators cause neurotoxicity. Among the metabolites generated by PLA2, 4-HNE is the most cytotoxic metabolite and is associated with the apoptotic type of neural cell death. Levels of 4-HNE are markedly increased in neurological disorders such as Alzheimer disease, Parkinson disease, ischemia, spinal cord trauma, and head injury. The purpose of this review is to summarize and integrate the vast literature on metabolites generated by PLA2 for a wider audience. The authors hope that this discussion will jump-start more studies not only on the involvement of PLA2 in neurological disorders but also on the importance of PLA2-generated lipid mediators in physiological and pathological processes. PMID- 16684970 TI - Understanding consolidation through the architecture of memories. AB - Following its encoding, a memory undergoes consolidation. It may be possible to deepen our understanding of the mechanisms supporting consolidation by considering the complex architecture of a memory. Any behavior can be split into multiple components. For example, when learning a new skill we simultaneously learn the movement and the goal of that movement. Each of these components has a distinct representation within a memory. The "off-line" processing of each component may follow different rules, providing an explanation for the variety of performance changes supported by consolidation. By viewing a memory as a representation with multiple components, it is possible to bridge the gap between the behavioral changes, which define consolidation, and the biological mechanisms that support those changes. This is partly because different memory components can be mapped onto different neural circuits. With an increased understanding of consolidation, it may become possible to modulate these off-line processes to improve psychiatric and neurological rehabilitation. PMID- 16684971 TI - The cover. Old Woman Reading. PMID- 16684972 TI - A piece of my mind. The otoscope. PMID- 16684973 TI - Experts ponder treating prehypertension. PMID- 16684974 TI - Parkinson disease guidelines aid diagnosis, management. PMID- 16684975 TI - Breast cancer prevention strategies explored. PMID- 16684976 TI - Caffeine and incident hypertension in women. PMID- 16684977 TI - Caffeine and incident hypertension in women. PMID- 16684978 TI - Caffeine and incident hypertension in women. PMID- 16684979 TI - Caffeine and incident hypertension in women. PMID- 16684980 TI - Hospitalization trends for pneumonia among older persons. PMID- 16684981 TI - Hospitalization trends for pneumonia among older persons. PMID- 16684982 TI - Arginine therapy for acute myocardial infarction. PMID- 16684983 TI - Deceased donor characteristics and kidney transplantation. PMID- 16684984 TI - Certificate of need regulations and use of coronary revascularization after acute myocardial infarction. AB - CONTEXT: Certificate of need regulations were enacted to control health care costs by limiting unnecessary expansion of services. While many states have repealed certificate of need regulations in recent years, few analyses have examined relationships between certificate of need regulations and outcomes of care. OBJECTIVE: To compare rates of coronary revascularization and mortality after acute myocardial infarction in states with and without certificate of need regulations. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study of 1,139,792 Medicare beneficiaries aged 68 years or older with AMI who were admitted to 4587 US hospitals during 2000-2003. MAIN OUTCOME MEASURES: Thirty-day risk-adjusted rates of coronary revascularization with either coronary artery bypass graft surgery or percutaneous coronary intervention and 30-day all-cause mortality. RESULTS: The 624,421 patients in states with certificate of need regulations were less likely to be admitted to hospitals with coronary revascularization services (321,573 [51.5%] vs 323,695 [62.8%]; P<.001) or to undergo revascularization at the admitting hospital (163,120 [26.1%] vs 163,877 [31.8%]; P<.001) than patients in states without certificates of need but were more likely to undergo revascularization at a transfer hospital (73,379 [11.7%] vs 45,907 [8.9%]; P<.001). Adjusting for demographic and clinical risk factors, patients in states with highly and moderately stringent certificate of need regulations, respectively, were less likely to undergo revascularization within the first 2 days (adjusted hazard ratios, 0.68; 95% confidence interval [CI], 0.54-0.87; P = .002 and 0.80; 95% CI, 0.71-0.90; P<.001) relative to patients in states without certificates of need, although no differences in the likelihood of revascularization were observed during days 3 through 30. Unadjusted 30-day mortality was similar in states with and without certificates of need (109,304 [17.5%] vs 90,104 [17.5%]; P = .76), as was adjusted mortality (odds ratio, 1.00; 95% CI, 0.97-1.03; P = .90). CONCLUSIONS: Patients with acute myocardial infarction were less likely to be admitted to hospitals offering coronary revascularization and to undergo early revascularization in states with certificate of need regulations. However, differences in the availability and use of revascularization therapies were not associated with mortality. PMID- 16684985 TI - Effectiveness of collaborative care for older adults with Alzheimer disease in primary care: a randomized controlled trial. AB - CONTEXT: Most older adults with dementia will be cared for by primary care physicians, but the primary care practice environment presents important challenges to providing quality care. OBJECTIVE: To test the effectiveness of a collaborative care model to improve the quality of care for patients with Alzheimer disease. DESIGN, SETTING, AND PATIENTS: Controlled clinical trial of 153 older adults with Alzheimer disease and their caregivers who were randomized by physician to receive collaborative care management (n = 84) or augmented usual care (n = 69) at primary care practices within 2 US university-affiliated health care systems from January 2002 through August 2004. Eligible patients (identified via screening or medical record) met diagnostic criteria for Alzheimer disease and had a self-identified caregiver. INTERVENTION: Intervention patients received 1 year of care management by an interdisciplinary team led by an advanced practice nurse working with the patient's family caregiver and integrated within primary care. The team used standard protocols to initiate treatment and identify, monitor, and treat behavioral and psychological symptoms of dementia, stressing nonpharmacological management. MAIN OUTCOME MEASURES: Neuropsychiatric Inventory (NPI) administered at baseline and at 6, 12, and 18 months. Secondary outcomes included the Cornell Scale for Depression in Dementia (CSDD), cognition, activities of daily living, resource use, and caregiver's depression severity. RESULTS: Initiated by caregivers' reports, 89% of intervention patients triggered at least 1 protocol for behavioral and psychological symptoms of dementia with a mean of 4 per patient from a total of 8 possible protocols. Intervention patients were more likely to receive cholinesterase inhibitors (79.8% vs 55.1%; P = .002) and antidepressants (45.2% vs 27.5%; P = .03). Intervention patients had significantly fewer behavioral and psychological symptoms of dementia as measured by the total NPI score at 12 months (mean difference, -5.6; P = .01) and at 18 months (mean difference, -5.4; P = .01). Intervention caregivers also reported significant improvements in distress as measured by the caregiver NPI at 12 months; at 18 months, caregivers showed improvement in depression as measured by the Patient Health Questionnaire-9. No group differences were found on the CSDD, cognition, activities of daily living, or on rates of hospitalization, nursing home placement, or death. CONCLUSIONS: Collaborative care for the treatment of Alzheimer disease resulted in significant improvement in the quality of care and in behavioral and psychological symptoms of dementia among primary care patients and their caregivers. These improvements were achieved without significantly increasing the use of antipsychotics or sedative-hypnotics. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00246896. PMID- 16684986 TI - Prevalence of visual impairment in the United States. AB - CONTEXT: The prevalence of visual impairment in the US public has not been surveyed nationally in several decades. OBJECTIVE: To estimate the number of US individuals aged 12 years or older who have impaired distance vision due to uncorrected refractive error. DESIGN, SETTING, AND PARTICIPANTS: The National Health and Nutrition Examination Survey (NHANES), using a multistage probability sampling design, included a vision evaluation in a mobile examination center. Visual acuity data were obtained from 13,265 of 14,203 participants (93.4%) who visited the mobile examination center in 1999-2002. Visual impairment was defined as presenting distance visual acuity of 20/50 or worse in the better-seeing eye. Visual impairment due to uncorrected refractive error was defined as (presenting) visual impairment that improved, aided by automated refraction results, to 20/40 or better in the better-seeing eye. MAIN OUTCOME MEASURES: Presenting distance visual acuity (measured with usual corrective lenses, if any) and distance visual acuity after automated refraction. RESULTS: Overall, 1190 study participants had visual impairment (weighted prevalence, 6.4%; 95% confidence interval [CI], 6.0% 6.8%), and of these, 83.3% could achieve good visual acuity with correction (95% CI, 80.9%-85.8%). Extrapolating these findings to the general US population, approximately 14 million individuals aged 12 years or older have visual impairment (defined as distance visual acuity of 20/50 or worse), and of these, more than 11 million individuals could have their vision improved to 20/40 or better with refractive correction. CONCLUSIONS: Visual impairment due to uncorrected refractive error is a common condition in the United States. Providing appropriate refractive correction to those individuals whose vision can be improved is an important public health endeavor with implications for safety and quality of life. PMID- 16684987 TI - Increasing incidence of thyroid cancer in the United States, 1973-2002. AB - CONTEXT: Increasing cancer incidence is typically interpreted as an increase in the true occurrence of disease but may also reflect changing pathological criteria or increased diagnostic scrutiny. Changes in the diagnostic approach to thyroid nodules may have resulted in an increase in the apparent incidence of thyroid cancer. OBJECTIVE: To examine trends in thyroid cancer incidence, histology, size distribution, and mortality in the United States. METHODS: Retrospective cohort evaluation of patients with thyroid cancer, 1973-2002, using the Surveillance, Epidemiology, and End Results (SEER) program and data on thyroid cancer mortality from the National Vital Statistics System. MAIN OUTCOME MEASURES: Thyroid cancer incidence, histology, size distribution, and mortality. RESULTS: The incidence of thyroid cancer increased from 3.6 per 100,000 in 1973 to 8.7 per 100,000 in 2002-a 2.4-fold increase (95% confidence interval [CI], 2.2 2.6; P<.001 for trend). There was no significant change in the incidence of the less common histological types: follicular, medullary, and anaplastic (P>.20 for trend). Virtually the entire increase is attributable to an increase in incidence of papillary thyroid cancer, which increased from 2.7 to 7.7 per 100,000-a 2.9 fold increase (95% CI, 2.6-3.2; P<.001 for trend). Between 1988 (the first year SEER collected data on tumor size) and 2002, 49% (95% CI, 47%-51%) of the increase consisted of cancers measuring 1 cm or smaller; 87% (95% CI, 85%-89%) consisted of cancers measuring 2 cm or smaller. Mortality from thyroid cancer was stable between 1973 and 2002 (approximately 0.5 deaths per 100,000). CONCLUSIONS: The increasing incidence of thyroid cancer in the United States is predominantly due to the increased detection of small papillary cancers. These trends, combined with the known existence of a substantial reservoir of subclinical cancer and stable overall mortality, suggest that increasing incidence reflects increased detection of subclinical disease, not an increase in the true occurrence of thyroid cancer. PMID- 16684988 TI - Integrating palliative care for liver transplant candidates: "too well for transplant, too sick for life". AB - Chronic liver disease results in more than 1 million physician visits and more than 300,000 hospitalizations per year in the United States. More than 27,000 patients annually progress to end-stage liver disease (ESLD), liver failure, or death. Patients with ESLD experience such complications as encephalopathy, malnutrition, muscle wasting, ascites, esophagogastric variceal hemorrhage, spontaneous bacterial peritonitis, fatigue, and depression. Despite significant improvements in palliation, patients' quality of life diminishes and their disease will often inexorably progress. Liver transplantation, a valid treatment option, increases life and reduces many symptoms. With the current shortage of organs, up to 10% to 15% of these patients die without receiving an organ. Many patients also are not candidates for transplantation due to comorbid illness. In addition, some patients receive a transplant but succumb to complications of the transplant itself. Such patients and families face the conundrum of a potentially treatable yet often fatal illness. Through the case of a 55-year-old woman with a life-long history of hepatitis B virus infection who is awaiting transplant, we discuss the transplant eligibility process and the struggle with maintaining hope for a cure in the face a life-threatening illness. In all of these circumstances, the health care team must combine elements of palliative care with life sustaining therapy to maximize the patient's quality and quantity of life. PMID- 16684989 TI - Evaluating and improving the quality of care for acute myocardial infarction: can regionalization help? PMID- 16684990 TI - Managing small thyroid cancers. PMID- 16684992 TI - JAMA patient page. Liver transplantation. PMID- 16684993 TI - Structure and function of negative feedback loops at the interface of genetic and metabolic networks. AB - The molecular network in an organism consists of transcription/translation regulation, protein-protein interactions/modifications and a metabolic network, together forming a system that allows the cell to respond sensibly to the multiple signal molecules that exist in its environment. A key part of this overall system of molecular regulation is therefore the interface between the genetic and the metabolic network. A motif that occurs very often at this interface is a negative feedback loop used to regulate the level of the signal molecules. In this work we use mathematical models to investigate the steady state and dynamical behaviour of different negative feedback loops. We show, in particular, that feedback loops where the signal molecule does not cause the dissociation of the transcription factor from the DNA respond faster than loops where the molecule acts by sequestering transcription factors off the DNA. We use three examples, the bet, mer and lac systems in Escherichia coli, to illustrate the behaviour of such feedback loops. PMID- 16684994 TI - Probing the DNA sequence specificity of Escherichia coli RECA protein. AB - Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT rich sequences, as found in the recombination hot spot Chi (5'-GCTGGTGG-3'). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a lambda repressor fragment in the presence of RecA, ADP-AlF4 and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed. PMID- 16684995 TI - Complete DNA sequences of the mitochondrial genomes of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis: insight into the evolution of linear DNA genomes from mitochondrial telomere mutants. AB - We determined complete mitochondrial DNA sequences of the two yeast species, Candida orthopsilosis and Candida metapsilosis, and compared them with the linear mitochondrial genome of their close relative, C.parapsilosis. Mitochondria of all the three species harbor compact genomes encoding the same set of genes arranged in the identical order. Differences in the length of these genomes result mainly from the presence/absence of introns. Multiple alterations were identified also in the sequences of the ribosomal and transfer RNAs, and proteins. However, the most striking feature of C.orthopsilosis and C.metapsilosis is the existence of strains differing in the molecular form of the mitochondrial genome (circular mapping versus linear). Their analysis opens a unique window for understanding the role of mitochondrial telomeres in the stability and evolution of molecular architecture of the genome. Our results indicate that the circular-mapping mitochondrial genome derived from the linear form by intramolecular end-to-end fusions. Moreover, we suggest that the linear mitochondrial genome evolved from a circular-mapping form present in a common ancestor of the three species and, at the same time, the emergence of mitochondrial telomeres enabled the formation of linear monomeric DNA forms. In addition, comparison of isogenic C.metapsilosis strains differing in the form of the organellar genome suggests a possibility that, under some circumstances, the linearity and/or the presence of telomeres provide a competitive advantage over a circular-mapping mitochondrial genome. PMID- 16684996 TI - Transbronchial biopsy and usual interstitial pneumonia: a new paradigm? PMID- 16684997 TI - Pediatricians are not just small internists. PMID- 16684998 TI - "Make the cough go away". PMID- 16684999 TI - Anticoagulation control with vitamin K antagonists: how well are we doing? PMID- 16685000 TI - Reducing oral steroids with inhaled steroids: is that all that can be achieved? PMID- 16685001 TI - Transbronchial biopsy in usual interstitial pneumonia. AB - BACKGROUND: Usual interstitial pneumonia (UIP) is a slowly progressive, usually fatal form of idiopathic interstitial pneumonia for which there is no effective treatment. Transbronchial biopsy (TBB) has been utilized only to exclude other diseases such as sarcoidosis, lymphangitic carcinoma, and infection, for example, but TBB is generally considered to have little role in confirming UIP. OBJECTIVE: To determine whether diagnostic changes of UIP can be appreciated on TBB specimens. DESIGN: Retrospective analysis of TBB specimens from patients with proven UIP. SETTING: Two study sites in the United States. PARTICIPANTS: Twenty one patients with UIP confirmed by surgical lung biopsy and/or lung explant, and 1 patient with UIP confirmed by clinical and radiographic findings along with follow-up information. MEASUREMENTS AND RESULTS: Adequate tissue for diagnosis was available in 18 cases; in 7 cases (32% overall), there were varying combinations of interstitial fibrosis in a patchwork pattern along with fibroblast foci and/or honeycomb change. These features were considered diagnostic of UIP. Interstitial fibrosis along with fibroblast foci or honeycomb change were seen in two other cases, but the fibrosis lacked a patchwork pattern, and these features were considered consistent with UIP. Nonspecific interstitial fibrosis alone was found in nine cases. CONCLUSIONS: In summary, characteristic histologic features of UIP can be identified on TBB specimens more often than previously appreciated. TBB may be more useful in confirming UIP than previously recognized. PMID- 16685002 TI - Evaluation and outcome of young children with chronic cough. AB - OBJECTIVE: To evaluate the use of an adult-based algorithmic approach to chronic cough in a cohort of children with a history of > 3 weeks of cough and to describe the etiology of chronic cough in this cohort. METHODS: A prospective cohort study of children referred to a tertiary hospital with a history of > 3 weeks of cough between June 2002 and June 2004. All included children followed a pathway of investigation (including flexible bronchoscopy and evaluation of airway cytology via BAL) until diagnosis was made and/or their cough resolved. RESULTS: In our cohort of 108 young children (median age 2.6 years), the majority had wet cough (n = 96; 89%), and BAL fluid samples obtained during bronchoscopy led to a diagnosis in 45.4% (n = 49). The most common final diagnosis was protracted bacterial bronchitis (n = 43; 39.8%). These patients had neutrophil levels on BAL samples that were significantly higher than those in other diagnostic groups (p < 0.0001). Asthma, gastroesophageal reflux disease (GERD), and upper airway cough syndrome (UACS), which are common causes of chronic cough in adults, were found in < 10% of the cohort (n = 10). CONCLUSIONS: The adult based anatomic pathway, which involves the investigation and treatment of patients with asthma, GERD, and UACS first is largely unsuitable for use in the management of chronic cough in young children as the common etiologies of chronic cough in children are different from those in adults. PMID- 16685003 TI - Causes and clinical features of subacute cough. AB - STUDY OBJECTIVES: Cough lasting > 3 weeks has been defined as chronic cough. However, it might be more persuasive to divide cough into subacute, lasting 3 to 8 weeks, and chronic, lasting > 8 weeks. We evaluated the causes and clinical courses of subacute cough, and the value of the bronchoprovocation test and induced sputum examination (IS). METHODS: Nonsmoking patients with cough of 3 to 8 weeks duration were enrolled into the study. Patients with dyspnea, basal FEV1 of < 70% predicted, abnormal findings on a plain chest radiograph or physical examination were excluded. We prescribed an antihistamine-decongestant for patients who were suspected to have postinfectious cough or postnasal drip. If patients had positive results on a bronchoprovocation test or IS, therapy with inhaled corticosteroids was substituted according to an algorithmic approach. RESULTS: One hundred eighty-four patients (77 men and 107 women) were evaluated; the mean age of the study group was 47.5 years. Eighty-nine of 184 patients had postinfectious cough. Cough resolved without treatment in 62 patients. Twenty nine of 43 patients with positive bronchoprovocation test results had cough variant asthma. CONCLUSIONS: Postinfectious cough was the most common cause of subacute cough. The spontaneous resolution of cough was frequent in patients with subacute cough. Unless asthma was strongly suspected, the performance of the bronchoprovocation test could be delayed until after empirical treatment had been administered. PMID- 16685004 TI - Pulmonary function is negatively correlated with sputum inflammatory markers and cough clearability in subjects with cystic fibrosis but not those with chronic bronchitis. AB - BACKGROUND: Polymorphonuclear neutrophil (PMN)-dominated inflammation is prominent in the airways of subjects with cystic fibrosis (CF) and chronic bronchitis (CB). Interleukin (IL)-8, myeloperoxidase (MPO), and DNA are markers of neutrophilic inflammation. We hypothesized that sputum MPO, DNA, and IL-8 concentrations would negatively correlate with pulmonary function and sputum transportability. METHODS: We measured pulmonary function and analyzed sputum IL 8, MPO, and DNA concentrations, as well as the transport properties of sputum samples obtained from 16 subjects with CF and 15 subjects with CB. We also evaluated changes in these measurements in paired sputum samples from these subjects obtained 2 to 12 months apart. RESULTS: IL-8 and MPO concentrations in the sputum of CF subjects was inversely correlated with FEV(1) percent predicted (IL-8: r = -0.40; p = 0.003; MPO: r = -0.38; p = 0.003) and FVC percent predicted (IL-8: r = -0.4; p = 0.02; MPO: r = -0.4; p = 0.02). IL-8 and DNA concentrations were inversely correlated with sputum cough transportability (CTR) [IL-8: r = 0.4; p = 0.02; DNA: r = -0.36; p = 0.048]. Changes in DNA concentration in sputum samples from CF subjects over time were inversely correlated with changes in FEV(1) percent predicted (r = -0.58; p = 0.02), FVC percent predicted (r = -0.74; p = 0.002), and CTR (r = -0.59; p = 0.02). There was no correlation among pulmonary function, sputum properties, and inflammatory markers in the sputum from subjects with CB. CONCLUSIONS: The sputum concentrations of IL-8, MPO, and DNA appear to be closely associated with pulmonary function in subjects with CF but not in subjects with CB. PMID- 16685005 TI - Effect of study setting on anticoagulation control: a systematic review and metaregression. AB - BACKGROUND: For patients receiving therapy with oral anticoagulants (OACs), the proportion of time spent in the therapeutic range (ie, anticoagulation control) is strongly associated with bleeding and thromboembolic risk. The effect of study level factors, especially study setting, on anticoagulation control is unknown. OBJECTIVES: Describe anticoagulation control achieved in the published literature. We also used metaregressive techniques to determine which study-level factors significantly influenced anticoagulation control. STUDIES: All published randomized or cohort studies that measured international normalized ratios (INRs) serially in anticoagulated patients and reported the proportion of time between INRs ranging from 1.8 to 2.0 and 3.0 to 3.5. RESULTS: We identified 67 studies with 123 patient groups having 50,208 patients followed for a total of 57,154.7 patient-years. A total of 68.3% of groups were from anticoagulation clinics, 7.3% were from clinical trials, and 24.4% were from community practices. Overall, patients were therapeutic 63.6% of time (95% confidence interval [CI], 61.6 to 65.6). In the metaregression model, study setting had the greatest effect on anticoagulation control with studies in community practices having significantly lower control than either anticoagulation clinics or clinical trials (-12.2%; 95% CI, -19.5 to -4.8; p < 0.0001). Self-management was associated with a significant improvement of time spent in the therapeutic range (+7.0%; 95% CI, 0.7 to 13.3; p = 0.03). CONCLUSIONS: Patients who have received anticoagulation therapy spend a significant proportion of their time with an INR out of the therapeutic range. Patients from community practices showed significantly worse anticoagulation control than those from anticoagulation clinics or clinical trials. This should be considered when interpreting the results of, and generalizing from, studies involving OACs. PMID- 16685006 TI - Argatroban therapy in heparin-induced thrombocytopenia with hepatic dysfunction. AB - STUDY OBJECTIVES: We evaluated the dosing requirements in argatroban-treated patients with heparin-induced thrombocytopenia (HIT) and hepatic dysfunction, and compared efficacy and safety outcomes with historical control patients. DESIGN: Retrospective analysis. SETTING: Inpatient setting. PATIENTS: Patients with hepatic dysfunction, defined as total bilirubin > 25.5 micromol/L (1.5 mg/dL), aspartate aminotransferase >100 IU/L, and/or alanine aminotransferase >100 IU/L, were identified from previous multicenter, historical-controlled studies of argatroban therapy in HIT. INTERVENTIONS: Argatroban, adjusted to maintain activated partial thromboplastin times (aPTTs) 1.5 to 3 times baseline in the experimental group, vs no direct thrombin inhibition in the historical control patients. MEASUREMENTS AND RESULTS: The analysis population included 82 argatroban patients and 34 historical control patients with hepatic impairment, of whom approximately 50% in each group had renal dysfunction (defined as a serum creatinine level > 1.3 mg/dL). The argatroban dosage was 1.6 +/- 1.1 microg/kg/min (mean +/- SD) over a mean 5-day course of therapy. Significantly lower doses were used in patients with elevated vs normal total bilirubin levels (0.8 +/- 0.6 microg/kg/min vs 1.7 +/- 0.8 microg/kg/min, p = 0.0063) and in patients with hepatic/renal dysfunction vs hepatic dysfunction alone (1.2 +/- 1.1 microg/kg/min vs 2.0 +/- 1.1 microg/kg/min, p < 0.001). The aPTT 24 h after argatroban initiation was 69 +/- 22 s, with 80% of patients having a therapeutic level of anticoagulation. Thirty-four argatroban-treated patients (41.5%) and 17 control patients (50.0%) experienced the 37-day composite end point of death, amputation, or new thrombosis (p = 0.32). Argatroban significantly reduced new thrombosis (8.5% vs 26.5%, p = 0.012). Major bleeding was similar between treatment groups (4.9% vs 2.9%, p = 0.684). CONCLUSIONS: Hepatic dysfunction affects argatroban dosing, with reduced doses required particularly in patients with serum total bilirubin levels > 25.5 micromol/L (1.5 mg/dL) or combined hepatic/renal dysfunction. Individual mean aPTT-adjusted doses typically remain > or = 0.5 microg/kg/min, supporting the recommendation of 0.5 microg/kg/min as a conservative initial dose for most patients with hepatic impairment. Argatroban, with proper initial dosing and monitoring, can provide safe and effective antithrombotic therapy in patients with HIT and hepatic impairment. PMID- 16685007 TI - Ciclesonide reduces the need for oral steroid use in adult patients with severe, persistent asthma. AB - STUDY OBJECTIVES: Oral corticosteroids (OCS) may be associated with systemic adverse events (AEs), which can be reduced by replacing OCS with inhaled corticosteroids (ICS). The potential of ciclesonide, a novel ICS, to reduce OCS use in patients with severe, persistent asthma was evaluated in this study. DESIGN: A phase III, 12-week, international, multicenter, double-blind, placebo controlled, parallel-group study. PATIENTS: Adult and adolescent patients (> or = 12 years old; n = 141) with severe, persistent, oral steroid (prednisone) dependent asthma. INTERVENTIONS: Patients were randomized to receive ciclesonide (640 mug/d or 1,280 microg/d [ex-actuator]) bid or placebo for 12 weeks. Weekly evaluations determined eligibility for prednisone dose reduction based on predetermined criteria. MEASUREMENTS AND RESULTS: The prednisone dose was significantly reduced by 47% and 63% in the groups receiving ciclesonide, 640 microg/d, and ciclesonide, 1,280 microg/d, respectively, vs an increase of 4% in the placebo group (both p < or = 0.0003) at week 12. By week 12, prednisone was discontinued by approximately 30% of patients in the ciclesonide-treated groups, vs 11% of patients in the placebo group (both p < or = 0.04). FEV1 improved significantly at week 12 in the ciclesonide treatment groups vs placebo (p < 0.03). The occurrence of local and systemic AEs was comparable between all treatment groups. CONCLUSION: Study results suggest that ciclesonide significantly reduces the need for OCS in patients with severe, persistent asthma, while maintaining asthma control. PMID- 16685008 TI - Efficacy of a heat exchanger mask in cold exercise-induced asthma. AB - STUDY OBJECTIVES: To determine the efficacy of a novel mask device in limiting cold air exercise-induced decline in lung function in subjects with a history of exercise-induced asthma (EIA). SETTING: In spite of appropriate medical therapy, many asthma patients are limited in cold weather activities. DESIGN: In study 1, 13 asthmatic subjects performed two randomized, single-blind treadmill exercise tests while breathing cold air (- 25 to - 15 degrees C) through a placebo or active heat exchanger mask. In study 2, five subjects with EIA performed three treadmill exercise tests while breathing cold air: one test using the heat exchanger mask, one test without the mask but with albuterol pretreatment, and one test with neither the mask nor albuterol pretreatment (unprotected exercise). For all studies, spirometry was performed before and at 5, 15, and 30 min after exercise challenge. PATIENTS: For both studies, a total of 15 subjects with a history of asthma symptoms during cold air exercise were recruited. RESULTS: In study 1, the mean decrease (+/- SE) in FEV1 was 19 +/- 4.9% with placebo, and 4.3 +/- 1.6% with the active device (p = 0.0002). The mean decrease in maximum mid expiratory flow (FEF(25-75)) was 31 +/- 5.7% with placebo and 4.7 +/- 1.7% with the active device (p = 0.0002). In study 2, the mean decrease in FEV1 was 6.3 +/- 3.9%, 11 +/- 3.7%, and 28 +/- 10% for the heat exchanger mask, albuterol pretreatment, and unprotected exercises, respectively (p = 0.4375 for mask vs albuterol, p = 0.0625 for mask vs unprotected exercise). The mean decrease in FEF(25-75) was 10 +/- 4.8%, 23 +/- 6.0%, and 36 +/- 11%, respectively (p = 0.0625 for mask vs albuterol, p = 0.0625 for mask vs unprotected exercise). CONCLUSIONS: This heat exchanger mask blocks cold exercise-induced decline in lung function at least as effectively as albuterol pretreatment. PMID- 16685009 TI - Airway inflammation and cellular stress in noneosinophilic atopic asthma. AB - STUDY OBJECTIVES: It has been suggested that patients with noneosinophilic asthma (NEA) show increased numbers of sputum neutrophils and a lack of response to therapy with corticosteroids, which are features that are commonly related to COPD. The aim of our study was to test the hypothesis that airway inflammation in NEA patients is different from that seen in patients with eosinophilic asthma (EA) and is similar to COPD. DESIGN: Sputum cellular stress markers and neutrophilic and eosinophilic fluid-phase mediators were analyzed in asthma and COPD patients. NEA patients were identified based on a sputum eosinophil count of < or = 2.2% of the total nonsquamous cell count, and were compared to EA and COPD patients. SETTING: University Hospital of Heraklion, Department of Thoracic Medicine. PATIENTS: A total of 37 atopic asthmatic patients and 25 patients with COPD. MEASUREMENTS: Sputum cell counts, cellular expression of heme oxygenase-1, inducible nitric oxide synthase, and nitrotyrosine, and sputum levels of eosinophilic cationic protein (ECP), myeloperoxidase (MPO), interleukin-8, and granulocyte macrophage colony-stimulating factor. RESULTS: A total of 17 asthmatic patients (46%) belonged to the NEA group and 20 patients (54%) to the EA group. Patients with NEA showed no difference in neutrophil counts, fluid phase mediators, or cellular stress markers compared to patients with EA. Compared to COPD patients, NEA patients showed the following significant differences: lower total cell counts (p < 0.03); lower neutrophil counts (p < 0.01); lower nitrotyrosine positive cell counts (p < 0.003); lower ECP levels (p < 0.005); lower MPO levels (p < 0.000); higher lymphocyte counts (p < 0.01); and higher macrophage counts (p < 0.03). CONCLUSIONS: Despite low eosinophil counts, airway inflammation in NEA patients may share common features with that in EA patients but is distinct from COPD. Larger studies are needed to investigate further the clinical and inflammatory characteristics of NEA before we are able to categorize asthma patients into those with or without eosinophilic inflammation. PMID- 16685010 TI - Acute relief of exercise-induced bronchoconstriction by inhaled formoterol in children with persistent asthma. AB - STUDY OBJECTIVE: To compare the acute bronchodilatory effect of the long-acting beta2-agonist formoterol against the short-acting beta2-agonist (SABA) terbutaline during exercise-induced bronchoconstriction (EIB) in children with asthma. DESIGN: A randomized, double-blind, placebo-controlled, crossover study of the immediate effect of formoterol, 9 microg, vs terbutaline, 0.5 mg, and placebo administered as dry powder at different study days. Exercise challenge test was used as a model of acute bronchoconstriction. PATIENTS: Twenty-four 7- to 15-year-old children with persistent asthma. INTERVENTIONS: The children performed standardized treadmill exercise tests, breathing dry air, with a submaximal workload. Study medication was administered 5 min after exercise if FEV1 dropped > or = 15% within 5 min after exercise. FEV1 and forced expiratory flows were measured repeatedly until 60 min after dose. RESULTS: Formoterol and terbutaline offered a significant acute bronchodilatory effect from 3 min after dose compared with placebo (p < 0.001). There was no difference between formoterol and terbutaline in FEV1 5 min after dose (p = 0.15), with a mean increase from each predrug baseline of 62% of the maximum increase for both. Median times to recovery within 5% of baseline FEV1 were 5.0 min and 7.4 min for formoterol and terbutaline, respectively (p = 0.33). CONCLUSION: Single-dose formoterol, 9 microg, via dry powder inhaler provided an acute bronchodilatory effect similar to terbutaline during EIB in schoolchildren with persistent asthma. Formoterol is at least as effective as SABA and may be considered an alternative in the treatment of acute bronchoconstriction in school children. PMID- 16685011 TI - Clinical characteristics and treatment patterns among patients with ventilator associated pneumonia. AB - STUDY OBJECTIVES: To evaluate clinical characteristics and treatment patterns among patients with ventilator-associated pneumonia (VAP), including the implementation of and outcomes associated with deescalation therapy. DESIGN: Prospective, observational, cohort study. SETTING: Twenty ICUs throughout the United States. PATIENTS: A total of 398 ICU patients meeting predefined criteria for suspected VAP. INTERVENTIONS: Prospective, handheld, computer-based data collection regarding routine VAP management according to local institutional practices, including clinical and microbiological characteristics, treatment patterns, and outcomes. MEASUREMENTS AND RESULTS: The most frequent ICU admission diagnoses in patients with VAP were postoperative care (15.6%), neurologic conditions (13.3%), sepsis (13.1%), and cardiac complications (10.8%). The mean (+/- SD) duration of mechanical ventilation prior to VAP diagnosis was 7.3 +/- 6.9 days. Major pathogens were identified in 197 patients (49.5%) through either tracheal aspirate or BAL fluid and included primarily methicillin-resistant Staphylococcus aureus (14.8%), Pseudomonas aeruginosa (14.3%), and other Staphylococcus species (8.8%). More than 100 different antibiotic regimens were prescribed as initial VAP treatment, the majority of which included cefepime (30.4%) or a ureidopenicillin/monobactam combination (27.9%). The mean duration of therapy was 11.8 +/- 5.9 days. In the majority of cases (61.6%), therapy was neither escalated nor deescalated. Escalation of therapy occurred in 15.3% of cases, and deescalation occurred in 22.1%. The overall mortality rate was 25.1%, with a mean time to death of 16.2 days (range, 0 to 49 days). The mortality rate was significantly lower among patients in whom therapy was deescalated (17.0%), compared with those experiencing therapy escalation (42.6%) and those in whom therapy was neither escalated nor deescalated (23.7%; chi2= 13.25; p = 0.001). CONCLUSIONS: Treatment patterns for VAP vary widely from institution to institution, and the overall mortality rate remains unacceptably high. The deescalation of therapy in VAP patients appears to be associated with a reduction in mortality, which is an association that warrants further clinical study. PMID- 16685012 TI - Impact of alcohol abuse in the etiology and severity of community-acquired pneumonia. AB - BACKGROUND AND STUDY OBJECTIVES: Alcohol consumption is known to affect both systemic and pulmonary immunity, predisposing the patient to pulmonary infections. The aim of this study was to compare the etiology of disease, the antibiotic resistance of Streptococcus pneumoniae, the severity of disease, and the outcome of patients with alcohol abuse to those of nonalcoholic (NA) patients who have been hospitalized for community-acquired pneumonia (CAP). METHODS: From 1997 to 2001, clinical, microbiological, radiographic, and laboratory data, and follow-up variables of all consecutive patients who had been hospitalized with CAP were recorded. Patients were classified as alcoholic (A) [n = 128] or ex alcoholic (EA) patients (n = 54) and were compared to NA patients (n = 1,165). RESULTS: S pneumoniae was found significantly more frequently in all patients with alcohol misuse. As regards the rates of antibiotic resistance, invasive pneumococcal disease, and other microorganisms, no differences were found. The severity criteria for CAP according to the American Thoracic Society were more frequent in A patients, but mortality did not differ significantly. Multivariate analysis showed an independent association between pneumococcal CAP and alcoholism (A patients: odds ratio [OR], 1.6; p = 0.033; EA patients: OR, 2.1; p = 0.016). CONCLUSIONS: We found an independent association between pneumococcal infection and alcoholism. Current alcohol abuse was associated with severe CAP. No significant differences were found in mortality, antibiotic resistance of S pneumoniae, and other etiologies. PMID- 16685013 TI - Utilization of noninvasive ventilation in acute care hospitals: a regional survey. AB - PURPOSES: Little information is available on the utilization of noninvasive positive-pressure ventilation (NPPV) in the United States. Accordingly, we performed a survey on the use of NPPV at acute care hospitals in a region of the United States to determine variations in utilization and between hospitals, the reasons for lower rates of utilization, and the techniques used for application. METHODS: Using survey methodology, we developed a questionnaire consisting of 19 questions and distributed it by mail to directors of respiratory care at all 82 acute care hospitals in Massachusetts and Rhode Island. Nonresponders were contacted by phone to complete the survey. Responses were analyzed using standard statistics, including t tests and Mann-Whitney U tests where appropriate. RESULTS: We obtained responses from 71 of the 82 hospitals (88%). The overall utilization rate for NPPV was 20% of ventilator starts, but we found enormous variation in the estimated utilization rates among different hospitals, from none to > 50%. The top two reasons given for lower utilization rates were a lack of physician knowledge and inadequate equipment. In the 19 hospitals that provided detailed information, COPD and congestive heart failure constituted 82% of the diagnoses of patients receiving NPPV, but NPPV was still used in only 33% of patients with these diagnoses receiving any form of mechanical ventilation. CONCLUSIONS: The utilization rates for NPPV vary enormously among different acute care hospitals within the same region. The perceived reasons for lower utilization rates include lack of physician knowledge, insufficient respiratory therapist training, and inadequate equipment. Educational programs directed at individual institutions may be useful to enhance utilization rates. PMID- 16685014 TI - A new tool to assess sarcoidosis severity. AB - STUDY OBJECTIVES: Sarcoidosis is a granulomatous disorder primarily affecting the lung, but with frequent extrapulmonary organ involvement. There are no comprehensive scoring systems for sarcoidosis disease severity. Our goal was to develop and validate an objective and comprehensive sarcoidosis disease severity scoring system. DESIGN: Three sarcoidosis experts reviewed clinical data on 104 patients with biopsy-confirmed sarcoidosis. Each expert independently scored disease severity using a visual analog scale. Interrater agreement was assessed. Univariate analysis was performed, and those variables with p values < or = 0.25 were used in backward regression multivariable analysis. A model was obtained including variables with a p value of < or = 0.15 to predict severity scores. This model was subsequently validated using an independent panel of three additional international experts. SETTING: Granuloma clinic at National Jewish Medical and Research Center. PATIENTS: A total of 104 patients with biopsy confirmed sarcoidosis. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Pairwise assessment of interrater agreement yielded high degrees of correlation with Spearman correlation coefficients of 0.86 to 0.89 and an intraclass correlation coefficient of 0.87. Univariate analysis showed that smoking status, immunosuppressive therapy, percent predicted for diffusing capacity of the lung for carbon monoxide (Dlco), FEV1, FVC, and total lung capacity, FEV1/FVC ratio, disease duration, sites of organ involvement, and African-American race were associated with mean severity score. The multivariable model included cardiac and neurologic involvement, current therapy with noncorticosteroid immunosuppressive agents, Dlco percent predicted, FEV1/FVC ratio, African-American race, FVC percent predicted, and skin involvement. This model was validated using additional reviewer scores yielding Spearman correlation coefficients of 0.66 to 0.76 and an intraclass correlation coefficient of 0.74. CONCLUSIONS: We derived an objective disease severity scoring system that incorporates data on demographics, pulmonary function, and organ involvement to produce a whole-body sarcoidosis assessment. This preliminary tool has potential applicability in the assessment of disease severity in sarcoidosis research. PMID- 16685015 TI - Incidence of pulmonary hypertension and its clinical relevance in patients with sarcoidosis. AB - OBJECTIVE: To investigate the frequency of pulmonary hypertension (PH) and clinical parameters associated with PH in sarcoidosis patients. METHODS: A prospective, observational study was performed on 246 consecutive Japanese sarcoidosis patients followed up at the outpatient sarcoidosis clinic in the Central Clinic of Kyoto. The patients were evaluated for PH by Doppler echocardiography. Among these patients, 192 underwent pulmonary function tests. In addition, high-resolution CT of the lung was evaluated for the presence of lymph node enlargement, lung opacity, and thickening of bronchovascular bundles in 122 patients. PH was defined as estimated systolic pulmonary artery pressure (sPAP) > or = 40 mm Hg. The frequency of PH was evaluated, and clinical parameters were compared between patients with PH and those without PH. RESULTS: Among 212 patients who were successfully evaluated for sPAP, 12 patients (5.7%) had PH. Patients with PH had the following clinical characteristics: advanced chest radiographic stage, decreased oxygen saturation, predominantly male gender, and decreased percentage of predicted vital capacity, percentage of predicted FVC, percentage of predicted FEV1, percentage of predicted functional residual capacity, and percentage of predicted total lung capacity (%TLC). Multivariate logistic regression analysis showed that decreased %TLC was independently associated with PH. There was a weak negative correlation between sPAP and %TLC (p < 0.05). CONCLUSIONS: The frequency of PH in Japanese sarcoidosis patients was 5.7% evaluated with Doppler echocardiography. Decreased lung volume increases the risk of PH developing in patients with sarcoidosis. PMID- 16685016 TI - The prevalence of pulmonary parenchymal tuberculosis in patients with tuberculous pleuritis. AB - STUDY OBJECTIVE: To examine the prevalence and characteristics of parenchymal tuberculous pleuritis in adult patients. DESIGN: Prospective cohort study. SETTING: Three hospitals affiliated with Seoul National University in South Korea. PATIENTS: All patients > 15 years old with a diagnosis of tuberculous pleuritis were enrolled prospectively between January 1, 2004, and October 31, 2004. INTERVENTIONS: Diagnostic thoracocentesis and CT of the chest were done for each patient. Acid-fast bacilli (AFB) smears and cultures for Mycobacterium tuberculosis were requested if patients produced any sputum. A board-certified radiologist reviewed the chest radiographs for the presence and characteristics of any lesions. MEASUREMENTS AND RESULTS: One hundred six patients with tuberculous pleuritis were enrolled (median age, 53 years; range 16 to 89 years). Among them, 33 patients (31%) had sputum or bronchial washing findings positive for AFB smears or for M tuberculosis by culture. Lung parenchymal lesions were observed in 91 of the patients (86%) using chest CT; 39 patients (37%) with parenchymal lesions had radiographic characteristics of active pulmonary tuberculosis. In total, 62 patients (59%) had bacteriologically or radiographically active pulmonary tuberculosis. In addition, 78 patients (74%) had features of reactivated pulmonary tuberculosis. CONCLUSIONS: Lung parenchymal lesions were more common in this series of patients with tuberculous pleuritis than has been reported in previous studies. The patients mostly had radiographic features of reactivated, rather than primary, tuberculosis. PMID- 16685017 TI - Angiopoietin-2 levels are elevated in exudative pleural effusions. AB - OBJECTIVE: To examine the pleural fluid (PF) and serum levels of angiopoietin (Ang)-1, Ang-2, and vascular endothelial growth factor (VEGF) in patients with pleural effusions (PEs). METHODS: One hundred fifteen patients, 16 with transudative PEs due to heart failure and 99 with exudative PEs (malignant, 40; para-pneumonic, 24; tuberculous, 13; miscellaneous etiologies, 22) were included in the study. PF and serum levels of the growth factors were measured using enzyme-linked immunosorbent assay. RESULTS: PF Ang-2 and VEGF levels but not Ang 1 levels were higher (p < 0.001) in exudates than in transudates. PF Ang-2 levels were higher in tuberculous PEs than in PEs of any other etiology and were lower in heart failure PEs than in PEs of any other etiology. The highest PF VEGF levels were observed in patients with malignant and parapneumonic PEs. The lowest PF VEGF levels were observed in patients with transudates. In PEs, Ang-2 levels correlate with VEGF levels (p < 0.001), RBC count (p = 0.002), nucleated cell count (p < 0.001), total protein levels (p < 0.001), and lactate dehydrogenase levels (p < 0.001). PF Ang-1 levels were lower than serum Ang-1 levels both in patients with exudates (p < 0.001) and in those with transudates (p = 0.001). PF Ang-2 levels were higher than serum Ang-2 levels both in patients with exudates (p < 0.001) and in those with transudates (p = 0.045). PF VEGF levels were higher than serum VEGF levels in patients with malignant PEs (p < 0.001) and parapneumonic PEs (p = 0.003), but lower than serum VEGF levels in heart failure PEs (p < 0.001). In patients with tuberculous PEs and exudative PEs of miscellaneous etiology, PF and serum VEGF levels did not differ significantly. CONCLUSION: Ang-2 levels but not Ang-1 levels are elevated in exudative PEs, and they correlate with levels of VEGF and markers of pleural inflammation. It is thus possible that Ang-2 along with VEGF participate in pleural inflammation and the pathogenesis of exudative PEs. PMID- 16685018 TI - Patient perspectives on management of pneumothorax in lymphangioleiomyomatosis. AB - STUDY OBJECTIVES: The American College of Chest Physicians Delphi Consensus Statement on management of spontaneous pneumothorax recommended pleurodesis after the first secondary spontaneous pneumothorax to prevent recurrence, and evaluation of patients' perspectives regarding pneumothorax treatment was identified as a future research priority. Patients with lymphangioleiomyomatosis (LAM) are an ideal population for performing these studies, since pneumothorax occurs and recurs more commonly in LAM than in any other chronic pulmonary disorder. STUDY DESIGN AND PARTICIPANTS: A 23-item questionnaire evaluating opinions of pneumothorax treatment was distributed to 615 patients in the LAM Foundation patient database, with a response rate of 52%. RESULTS: Of respondents, 69% (216 of 314 patients) reported a history of radiographically documented pneumothorax, and 181 patients (84%) reported at least one pleurodesis procedure. Neither a history of pneumothorax nor surgical management of pneumothorax affected reported oxygen use or perception of overall lung function, yet 41% thought that their pneumothorax had contributed to a decline in lung function. Few patients (12%) worried frequently about a pneumothorax developing, but one third made lifestyle modifications due to fear of pneumothorax. Extensive pain associated with chest tube placement and inadequate pain management throughout treatment for pneumothorax were frequent concerns. Only 25% of respondents thought that pleurodesis was appropriate for a first pneumothorax, while 60% favored pleurodesis for a second pneumothorax. Despite the apparent reluctance to undergo pleurodesis, most patients agreed that pleurodesis helps prevent pneumothorax recurrence. One third of patients believed that their physicians did not consider their preferences regarding pneumothorax management. CONCLUSIONS: LAM patients and physicians may have different views about the significance of pneumothorax, in that most patients appear to favor a conservative initial approach to pneumothorax management. In conjunction with appropriate pain management, a better understanding of patients' perspectives will facilitate cooperative decision making and may ultimately improve clinical outcomes in LAM related to pneumothorax. PMID- 16685019 TI - Management of pneumothorax in lymphangioleiomyomatosis: effects on recurrence and lung transplantation complications. AB - STUDY OBJECTIVES: Pneumothorax is a common complication of lymphangioleiomyomatosis (LAM), and the optimal approach to its treatment and prevention is unknown. Chemical or surgical pleurodesis are often required to prevent recurrence. However, their efficacy in LAM is unclear, and whether they contribute to perioperative complications during lung transplantation is uncertain. SETTING: The LAM Foundation database of registered patients. DESIGN: A questionnaire was sent to all registered patients who had at least one pneumothorax to determine rates and patterns of recurrence and efficacy of interventions. A second questionnaire was sent to registered LAM patients who received a lung transplant. PATIENTS OR PARTICIPANTS: Of 395 registered patients, 260 patients (66%) reported at least one pneumothorax during their lifetime, 193 of whom (74%) completed the questionnaire. Of the 85 lung transplant patients who were sent a separate questionnaire, 80 patients (94%) responded. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Of the 193 respondents to the pneumothorax questionnaire, data on 676 episodes of pneumothorax were collected. Eighty-two percent (158 of 193 patients) had their first pneumothorax prior to a diagnosis of LAM. One hundred forty patients (73%) had at least one additional pneumothorax, either an ipsilateral recurrence (99 of 140 patients, 71%) or a contralateral pneumothorax (104 of 140 patients, 74%). Recurrence rates were 66% after conservative therapy, 27% after chemical pleurodesis, and 32% after surgery. In patients who had undergone lung transplantation, prior chemical or surgical pleurodesis was performed in 45 of 80 patients (56%). Fourteen of 80 patients (18%) reported pleural-related postoperative bleeding, 13 of whom (93%) had prior pleurodesis. CONCLUSIONS: Chemical pleurodesis or surgery are equally effective and better than conservative therapy in preventing recurrence of pneumothorax in LAM. Due to the high recurrence rate, either procedure should be considered for the initial pneumothorax in these patients. However, both contribute to increased perioperative bleeding following lung transplantation, with no effect on length of hospital stay. PMID- 16685020 TI - Pulmonary embolism as a cause of death in patients with heart disease: an autopsy study. AB - STUDY OBJECTIVE: To establish the prevalence of pulmonary embolism (PE) in autopsy material at a tertiary cardiac referral center and its importance as a cause of death in patients with heart disease (HD). DESIGN: Case series. SETTING: National Heart Institute, Mexico City. PATIENTS: One thousand thirty-two patients who died at our institution from 1985 to 1994 in whom an autopsy study was performed. MEASUREMENTS AND RESULTS: Of the 1,032 autopsies reviewed, 231 cases (24.4%) of PE were found; 100 of these patients had a diagnosis of massive PE. Massive PE (obstruction of either of the main pulmonary arteries or more than two lobar arteries) was found to be the third cause of death in this HD population. By age-group distribution, the global prevalence of massive events was higher in patients < 10 years old. Clinical suspicion (premortem) was raised in only 18% of the cases. CONCLUSIONS: PE was a frequent cause of morbidity and mortality in patients with HD who underwent autopsies. The incidence of massive PE was high in children. PMID- 16685021 TI - Exhaled metallic elements and serum pneumoproteins in asymptomatic smokers and patients with COPD or asthma. AB - STUDY OBJECTIVES: The aim of this study was to characterize the elemental composition of exhaled breath condensate (EBC) in order to identify new biomarkers of exposure and susceptibility in COPD patients. Serum pneumoproteins were used as lung-specific biomarkers of effect. DESIGN: EBC was obtained from 50 healthy subjects, 30 healthy smokers, 30 asthmatics, and 50 patients with stable COPD, and was collected by cooling exhaled air. Trace elements and toxic metals in the samples were measured by means of inductively coupled plasma-mass spectrometry and electrothermal atomic absorption spectroscopy. The serum pneumoproteins were immunoassayed. RESULTS: The EBC of COPD subjects had higher levels of such toxic elements as lead, cadmium, and aluminum, and lower levels of iron and copper, than that of the nonsmoking control subjects. There were no between-group differences in surfactant protein (SP)-A and SP-B levels. Clara cell protein and SP-D levels were negatively and positively influenced, respectively, by tobacco smoke. CONCLUSIONS: Our results show that toxic metals and transition elements are detectable in the EBC of studied subjects. We propose new biomarkers of exposure as a means of assessing the target tissue dose of carcinogenic and pneumotoxic substances from tobacco smoke or polluted workplaces, and the use of the transition elements involved in redox systems of oxidative stress as disease biomarkers associated with effect or susceptibility. Together with biomarkers of effect, such as serum pneumoproteins, the elemental composition of EBC may be clinically useful in distinguishing similar diseases. PMID- 16685022 TI - High-level expression of matrix-associated transforming growth factor-beta1 in benign airway stenosis. AB - STUDY OBJECTIVES: Acquired tracheal and subglottic stenosis frequently leads to severe airway narrowing, which requires repeated interventions, such as dilatation, laser resection, stent implantation, or surgery. To get a more detailed insight into the pathogenesis of this condition, we investigated the expression of profibrotic cytokines and the proliferation of the airway wall in benign human airway stenoses. METHODS: Specimens from patients with subglottic and tracheal stenosis and stent-related stenoses were obtained (n = 20) for reverse transcription (RT) polymerase chain reaction (PCR) analysis and immunohistochemistry testing. RESULTS: Transforming growth factor (TGF)-beta1 messenger RNA expression was significantly increased in biopsy specimens from stent-related stenoses compared to nonstenotic control sections. In contrast, TGF beta3 and interleukin-1beta showed no such differences in messenger RNA expression. Immunohistochemistry revealed a strong matrix-associated, subepithelial expression of TGF-beta1 in tracheal stenosis. Proliferating Ki-67 positive cells were mainly localized in the basal epithelial layer. Only 2 of 16 patients with tracheal stenoses and 3 of 4 patients with stent-related stenoses showed a weak expression of Ki-67-positive cells in the subepithelium. Furthermore, TGF-beta1 dose-dependently enhanced the proliferation of human lung fibroblasts in vitro, even in the presence of mitomycin-C. CONCLUSION: While a weak subepithelial proliferation occurs in stent-related stenoses, the dominant factor in late stages of untreated tracheal stenoses seems to be the high-level expression of TGF-beta1 and the deposition of extracellular matrix. PMID- 16685023 TI - Prevalence of COPD in women compared to men around the time of diagnosis of primary lung cancer. AB - PURPOSES: COPD is a well-known independent risk factor that is associated with primary lung cancer. There is, however, a striking paucity of women in studies demonstrating this association. The purpose of this study was to compare the prevalence of COPD as determined by pulmonary function tests (PFTs) between women and men at around the time of lung cancer diagnosis. METHODS: We retrospectively reviewed patients with newly diagnosed primary lung cancer who had undergone PFTs prior to their treatment. The diagnosis of airflow obstruction was made according to American Thoracic Society guidelines. Comparisons of the prevalence of COPD between men and women were performed using univariate and multivariate logistic regression analysis. RESULTS: Of the 294 patients in the study, 151 patients (51.4%) were men and 143 patient (48.6%) were women. Of the men, 110 patients (72.8%) had COPD compared with 75 patients (52.5%) among the women. This represented a significantly lower prevalence of COPD in women than in men (odds ratio [OR], 0.41; 95% confidence interval [CI], 0.25 to 0.67; p = 0.0003). When adjusted for age and smoking status, a sustained lower prevalence of COPD was noted in women compared to men (OR, 0.44; 95% CI, 0.26 to 0.74; p = 0.002). In a subset of 256 smokers, there remained a lower prevalence of COPD in women compared to men (OR, 0.45; 95% CI, 0.27 to 0.77; p = 0.003). Adjusted analysis to control for age and number of pack-years of smoking in this subset again showed a sustained reduction in the OR for women presenting with COPD (OR, 0.48; 95% CI, 0.28 to 0.83; p = 0.009). CONCLUSIONS: When COPD was examined as an end point among patients who had newly diagnosed lung cancer, a significantly higher proportion of women had normal PFT results. Gender-based differences on PFT results should be considered during the screening of lung cancer, because the stratification of high-risk patients based on the presence of COPD may miss a significant proportion of women with lung cancer. PMID- 16685025 TI - Supramaximal inflation improves lung compliance in subjects with amyotrophic lateral sclerosis. AB - RATIONALE: Lung compliance has been found to be low in patients with chronic diaphragmatic weakness or paralysis but has not been well-studied in patients with amyotrophic lateral sclerosis (ALS). Noninvasive positive-pressure ventilation (NPPV) prolongs survival in ALS patients but may also have additional beneficial effects. OBJECTIVES: This study evaluated static expiratory lung compliance (CL) in subjects with ALS and determined the effect of lung inflation with supramaximal inflation on CL. DESIGN: This was a prospective trial comparing CL before and after supramaximal lung inflation via mouthpiece-delivered positive pressure. SETTING: A single university medical center with an multidisciplinary ALS center. PARTICIPANTS: Fourteen subjects with ALS were compared to 4 healthy volunteers. INTERVENTIONS: Subjects underwent a battery of pulmonary function tests including for CL. Then they used positive pressure administered via a mouthpiece set to 10 cm H2O above their maximal static recoil pressure for 5 min. The CL measurement was then repeated. RESULTS: The mean (+/- SD) baseline CL was reduced (164.1 +/- 82.1 mL/cm H2O) in subjects with ALS and was significantly lower than that in healthy volunteers (237.5 mL/cm H2O; p = 0.04). CL increased significantly in subjects with evidence of diaphragm weakness (change in CL, 11.3 +/- 16.7 mL/cm H2O; p = 0.03). Healthy volunteers did not have an increase in CL. CONCLUSIONS: Patients with ALS and diaphragmatic weakness have reduced CL, and brief supramaximal inflation increases CL. These findings suggest that atelectasis or increased alveolar surface forces are present in ALS patients and that these patients will have increased work of breathing. Some of the beneficial effects demonstrated with NPPV therapy may be through its effects on CL and the work of breathing. PMID- 16685024 TI - Serum N-terminal brain natriuretic peptide as a prognostic parameter in patients with pulmonary hypertension. AB - STUDY OBJECTIVES: Baseline prognostic assessment in patients with pulmonary hypertension (PH) may help in the selection of treatment. High plasma levels of natriuretic peptide type B have been reported in patients with right ventricular (RV) dysfunction and suggest poor prognosis in patients with idiopathic pulmonary arterial hypertension (IPAH). We prospectively assessed the correlation of N terminal brain natriuretic peptide (NT-proBNP) with echocardiographic and hemodynamic indexes of RV function as well as with baseline functional status and long-term survival of PH patients. PATIENTS AND DESIGN: Fifty-five consecutive patients with a mean (+/- SD) age of 41 +/- 15 years and severe PH (including 36 patients with IPAH) were followed up for up to 36 months. Serum samples for NT proBNP were secured, and 6-min walk test (6 MWT), RV catheterization, and echocardiography were all performed on the same day, before the introduction of targeted treatment. RESULTS: The median baseline serum NT-proBNP concentration was 1,674 pg/mL (range, 51 to 10,951 pg/mL). NT-proBNP concentration correlated with 6MWT distance (r = 0.6; p < 0.001), cardiac index, pulmonary vascular resistance, and right atrial pressure (RAP), but not with pulmonary arterial pressure. NT-proBNP levels were also related to the ratio of the diastolic area of the RV and the LV, and to pericardial effusion during echocardiography. Receiver operating characteristic analysis identified > or = 1,400 pg/mL as the best NT-proBNP threshold predicting fatal outcome for the entire study group as well as for IPAH patients (sensitivity, 88% and 100%, respectively; specificity, 53% and 56%, respectively). In multivariate analysis, NT-proBNP, troponin T, and RAP were identified as independent factors for poor prognosis for the entire study group, while only NT-proBNP and RAP were identified as markers for poor prognosis in the IPAH subgroup. CONCLUSIONS: NT-proBNP level is related to the right heart morphology and dysfunction in PH patients. A serum NT-proBNP level of > or = 1,400 pg/mL was found to be useful in identifying patients with poor long term prognosis both in the whole studied group and in the IPAH subgroup. PMID- 16685026 TI - Closing capacity and gas exchange in chronic heart failure. AB - BACKGROUND: Although it is commonly assumed that pulmonary congestion and edema in patients with chronic heart failure (CHF) promotes peripheral airway closure, closing capacity (CC) has not been measured in CHF patients. PURPOSES: To measure CC and the presence or absence of airway closure and expiratory flow limitation (FL) during resting breathing in CHF patients. METHODS: In 20 CHF patients and 20 control subjects, we assessed CC, FL, spirometry, blood gas levels, control of breathing, breathing pattern, and dyspnea. RESULTS: The patients exhibited a mild restrictive pattern, but the CC was not significantly different from that in control subjects. Nevertheless, airway closure during tidal breathing (ie, CC greater than functional residual capacity [FRC]) was present in most patients but was absent in all control subjects. As a result of the maldistribution of ventilation and the concurrent impairment of gas exchange, the mean (+/- SD) alveolar-arterial oxygen pressure difference increased significantly in CHF patients (4.3 +/- 1.2 vs 2.7 +/- 0.5 kPa, respectively; p < 0.001) and correlated with systolic pulmonary artery pressure (r = 0.49; p < 0.03). Tidal FL is absent in CHF patients. Mouth occlusion pressure 100 ms after onset of inspiratory effort (P0.1) as a percentage of maximal inspiratory pressure (Pimax) together with ventilation were increased in CHF patients (p < 0.01 and p < 0.005, respectively). The increase in ventilation was due entirely to increased respiratory frequency (fR) with a concurrent decrease in Paco2. Chronic dyspnea (scored with the Medical Research Council [MRC] scale) correlated (r2= 0.61; p < 0.001) with fR and P0.1/Pimax. CONCLUSIONS: In CHF patients at rest, CC is not increased, but, as a result of decreased FRC, airway closure during tidal breathing is present, promoting the maldistribution of ventilation, ventilation perfusion mismatch, and impaired gas exchange. The ventilation is increased as result of increased fR, and Pimax is decreased with a concurrent increase in P0.1, implying that there is a proportionately greater inspiratory effort per breath (P0.1/Pimax). These, together with the increased fR, are the only significant contributors to increases in the MRC dyspnea score. PMID- 16685027 TI - Hemodynamic changes induced by recreational scuba diving. AB - OBJECTIVE: Cardiac changes induced by scuba diving were investigated using Doppler echocardiography. MATERIAL AND METHODS: Ten healthy scuba divers dove to a mean depth of 34.3 +/- 2.7 m of sea water (113 +/- 9 feet) and a mean duration of 25.3 +/- 3.5 min. RESULTS: One hour after the dive, microbubbles could be detected in the right-heart chambers of all subjects. Left atrial and left ventricular (LV) diameters significantly decreased after the dive. Cardiac output, assessed by aortic blood flow, remained unchanged. Heart rate increased and stroke volume (SV) decreased after the dive. LV filling was assessed on transmitral profile. An increase of the contribution of the atrial contraction to LV filling was observed. Right cavity diameters were unchanged, but an increase of the right ventricular/right atrial gradient pressure was found. CONCLUSION: The diving profile studied promotes a rather important bubble grade in all volunteers. A significantly reduced cardiac diameters and SV was found by our hemodynamic study 1 h after diving. Two factors can explain these results: low volemia secondary to immersion, and venous gas embolism induced by nitrogen desaturation. Consequently, restoration of the water balance of the body should be considered in the recovery process after diving. PMID- 16685028 TI - Clinical applications of induced sputum. AB - The development of standardized methods for sputum induction has improved the quality and reproducibility of sputum samples. This technique has been used to optimize samples in the investigation of pulmonary tuberculosis and lung cancer, but its clinical application as a noninvasive measure of airway inflammation has highlighted the enormous potential of this technique. Sputum induction has allowed researchers to characterize the inflammatory profiles of a variety of airway diseases including asthma, COPD, and chronic cough. To date, the identification of sputum eosinophilia has the greatest clinical value as this predicts a favorable response to corticosteroids and can therefore guide treatment. In asthma and COPD management, protocols aimed at normalizing the sputum eosinophil count have markedly reduced exacerbations without an overall increase in therapy. Currently, no other noninvasive measure of airway inflammation has demonstrated a benefit in reducing exacerbations. The value of sputum induction and analysis is not restricted to the recognition of sputum eosinophilia but also may be used to direct novel antineutrophilic therapies. Thus, it is time for sputum induction to move from the research laboratory to the clinic. PMID- 16685029 TI - Sepsis-associated myocardial dysfunction: diagnostic and prognostic impact of cardiac troponins and natriuretic peptides. AB - Myocardial dysfunction, which is characterized by transient biventricular impairment of intrinsic myocardial contractility, is a common complication in patients with sepsis. Left ventricular systolic dysfunction is reflected by a reduced left ventricular stroke work index or, less accurately, by an impaired left ventricular ejection fraction (LVEF). Early recognition of myocardial dysfunction is crucial for the administration of the most appropriate therapy. Cardiac troponins and natriuretic peptides are biomarkers that were previously introduced for diagnosis and risk stratification in patients with acute coronary syndrome and congestive heart failure, respectively. However, their prognostic and diagnostic impact in critically ill patients warrants definition. The elevation of cardiac troponin levels in patients with sepsis, severe sepsis, or septic shock has been shown to indicate left ventricular dysfunction and a poor prognosis. Troponin release in this population occurs in the absence of flow limiting coronary artery disease, suggesting the presence of mechanisms other than thrombotic coronary artery occlusion, probably a transient loss in membrane integrity with subsequent troponin leakage or microvascular thrombotic injury. In contrast to the rather uniform results of studies dealing with cardiac troponins, the impact of raised B-type natriuretic peptide (BNP) levels in patients with sepsis is less clear. The relationship between BNP and both LVEF and left-sided filling pressures is weak, and data on the prognostic impact of high BNP levels in patients with sepsis are conflicting. Mechanisms other than left ventricular wall stress may contribute to BNP release, including right ventricular overload, catecholamine therapy, renal failure, diseases of the CNS, and cytokine up regulation. Whereas cardiac troponins may be integrated into the monitoring of myocardial dysfunction in patients with severe sepsis or septic shock to identify those patients requiring early and aggressive supportive therapy, the routine use of BNP and other natriuretic peptides in this setting is discouraged at the moment. PMID- 16685030 TI - Psychosocial issues in the assessment and management of patients undergoing lung transplantation. AB - This review examines psychosocial issues among lung transplant patients from the time of assessment through the posttransplant period. Although psychological factors are recognized as being important in the transplant evaluation, no standard approach to psychological assessment currently exists. Lung transplant candidates often experience high levels of psychological distress while awaiting transplant, and both pretransplant and posttransplant psychological functioning have been found to predict posttransplant quality of life, adherence to treatment, and, in some cases, medical outcomes. Given the limited long-term survival following transplantation, improving psychosocial functioning is essential for enhancing outcomes among lung transplant recipients. This review summarizes the extant literature on the psychosocial factors in lung transplantation and highlights several innovative efforts to improve psychological outcomes in this challenging patient population. PMID- 16685031 TI - Some concrete ideas about manuscript abstracts. PMID- 16685032 TI - A 77-year-old farmer with respiratory failure and thrombocytopenia. PMID- 16685033 TI - Postthrombotic syndrome complicating a case of May-Thurner syndrome despite endovascular therapy: case report and review. AB - External compression of the left iliac vein is a common finding in the general population. It may predispose patients to the development of deep vein thrombosis (DVT) of the left leg and may also lead to a more complicated course than in other types of DVT. This entity has been well-described by other authors. External compression of the left iliac vein should be suspected in cases of complicated DVT or in cases of DVT with no predisposing factors. We describe a case of May-Thurner syndrome that involved a complicated treatment course, and a review of current options for diagnosis and therapy. PMID- 16685034 TI - Pharmaceutical industry sponsorship of journal supplements. PMID- 16685035 TI - Aerosol delivery devices. PMID- 16685036 TI - Mometasone and beclomethasone comparison article observations. PMID- 16685037 TI - Noninvasive ventilation and dyspnea in palliative medicine. PMID- 16685038 TI - Alternatives to low-fat diets. PMID- 16685039 TI - Zinc and pneumonia. PMID- 16685040 TI - Folate and human reproduction. AB - The influence of folate nutritional status on various pregnancy outcomes has long been recognized. Studies conducted in the 1950s and 1960s led to the recognition of prenatal folic acid supplementation as a means to prevent pregnancy-induced megaloblastic anemia. In the 1990s, the utility of periconceptional folic acid supplementation and folic acid food fortification emerged when they were proven to prevent the occurrence of neural tube defects. These distinctively different uses of folic acid may well be ranked among the most significant public health measures for the prevention of pregnancy-related disorders. Folate is now viewed not only as a nutrient needed to prevent megaloblastic anemia in pregnancy but also as a vitamin essential for reproductive health. This review focuses on the relation between various outcomes of human reproduction (ie, pregnancy, lactation, and male reproduction) and folate nutrition and metabolism, homocysteine metabolism, and polymorphisms of genes that encode folate-related enzymes or proteins, and we identify issues for future research. PMID- 16685041 TI - Ultradian ghrelin pulsatility is disrupted in morbidly obese subjects after weight loss induced by malabsorptive bariatric surgery. AB - BACKGROUND: Suppression of ghrelin production after Roux-en-Y gastric bypass that suggested its contribution to appetite reduction has been reported. OBJECTIVE: Because biliopancreatic diversion (BPD) does not affect appetite, we compared ghrelin production and 24-h pulsatility between healthy control subjects and obese subjects before and after BPD. DESIGN: A computerized algorithm identified peak heights, clearance rate, and peak frequency of ghrelin over 24 h. Twenty four-hour energy expenditure was measured in the calorimetric chamber, and energy intakes were computed. Insulin sensitivity was measured with a euglycemic hyperinsulinemic clamp. RESULTS: Mean (+/-SD) 24-h plasma ghrelin concentrations were significantly (P < 0.0001) higher in control than in obese subjects (338.17 +/- 22.09 and 164.47 +/- 29.19 microg/L, respectively), but they increased to 204.64 +/- 28.51 microg/L in the obese subjects after BPD (P < 0.01). The pulsatility index was 0.098 +/- 0.016 and 0.041 +/- 0.014 microg . L(-1) . min( 1) in control and obese subjects, respectively (P < 0.01), and decreased to 0.025 +/- 0.007 microg . l(-1) . min(-1) after BPD (P < 0.05). Energy intakes before and after BFP did not differ significantly. Although metabolizable energy after BPD was 40% of the energy intake, that (per kg fat-free mass) after BPD did not different significantly from that before BPD. CONCLUSIONS: Weight loss induced by malabsorptive bariatric surgery is associated with greater ghrelin concentrations, which, however, remain consistently lower than those in control subjects, whereas ghrelin pulsatility is subverted. Higher ghrelin concentrations may contribute to the high calorie intakes observed in post-BPD subjects. The lack of normal pulsatility may explain the new impulse of these subjects to eat very frequently. PMID- 16685042 TI - Separate effects of reduced carbohydrate intake and weight loss on atherogenic dyslipidemia. AB - BACKGROUND: Low-carbohydrate diets have been used to manage obesity and its metabolic consequences. OBJECTIVE: The objective was to study the effects of moderate carbohydrate restriction on atherogenic dyslipidemia before and after weight loss and in conjunction with a low or high dietary saturated fat intake. DESIGN: After 1 wk of consuming a basal diet, 178 men with a mean body mass index (in kg/m(2)) of 29.2 +/- 2.0 were randomly assigned to consume diets with carbohydrate contents of 54% (basal diet), 39%, or 26% of energy and with a low saturated fat content (7-9% of energy); a fourth group consumed a diet with 26% of energy as carbohydrate and 15% as saturated fat. After 3 wk, the mean weight loss (5.12 +/- 1.83 kg) was induced in all diet groups by a reduction of approximately 1000 kcal/d for 5 wk followed by 4 wk of weight stabilization. RESULTS: The 26%-carbohydrate, low-saturated-fat diet reduced triacylglycerol, apolipoprotein B, small LDL mass, and total:HDL cholesterol and increased LDL peak diameter. These changes were significantly different from those with the 54% carbohydrate diet. After subsequent weight loss, the changes in all these variables were significantly greater and the reduction in LDL cholesterol was significantly greater with the 54%-carbohydrate diet than with the 26% carbohydrate diet. With the 26%-carbohydrate diet, lipoprotein changes with the higher saturated fat intakes were not significantly different from those with the lower saturated fat intakes, except for LDL cholesterol, which decreased less with the higher saturated fat intake because of an increase in mass of large LDL. CONCLUSIONS: Moderate carbohydrate restriction and weight loss provide equivalent but nonadditive approaches to improving atherogenic dyslipidemia. Moreover, beneficial lipid changes resulting from a reduced carbohydrate intake were not significant after weight loss. PMID- 16685043 TI - Withdrawal of red meat from the usual diet reduces albuminuria and improves serum fatty acid profile in type 2 diabetes patients with macroalbuminuria. AB - BACKGROUND: Replacement of red meat in the diet with chicken has reduced the urinary albumin excretion rate (UAER) and serum cholesterol in microalbuminuric type 2 diabetes patients. The effects of withdrawing red meat are unknown in the more advanced stages of diabetic nephropathy. OBJECTIVE: Our objective was to assess the effects of replacing red meat in the usual diet (UD) with chicken (CD) and of consuming a lactovegetarian low-protein diet (LPD) on renal function, fatty acid, and lipid profile in macroalbuminuric type 2 diabetes patients. DESIGN: A crossover controlled trial was conducted in 17 type 2 diabetes patients with macroalbuminuria (24-h UAER > or = 200 microg/min). Each patient followed the UD, CD, and LPD in a random order for 4 wk. After each diet, glomerular filtration rate, UAER, serum fatty acid, lipid profile, glycemic control, anthropometric indexes, and blood pressure were measured. RESULTS: UAER [median CD: 269.4 (range: 111-1128) microg/min; LPD: 229.3 (76.6-999.3) microg/min; UD: 312.8 (223.7-1223.7) microg/min; P < 0.01] and mean (+/-SD) non-HDL cholesterol (CD: 3.92 +/- 0.99 mmol/L; LPD: 3.92 +/- 0.93 mmol/L; UD: 4.23 +/- 1.06 mmol/L; P = 0.042) were lower after CD and LPD than after UD. Compared with the UD, an increase in serum total polyunsaturated fatty acids was also observed (CD: 39.8 +/- 2.6%; LPD: 39.7 +/- 4.4%; UD: 37.3 +/- 3.1%; P = 0.029). CONCLUSION: In macroalbuminuric patients with type 2 diabetes, withdrawing red meat from the diet reduces the UAER. PMID- 16685044 TI - Consumption of coffee is associated with reduced risk of death attributed to inflammatory and cardiovascular diseases in the Iowa Women's Health Study. AB - BACKGROUND: Coffee is the major source of dietary antioxidants. The association between coffee consumption and risk of death from diseases associated with inflammatory or oxidative stress has not been studied. OBJECTIVE: We studied the relation of coffee drinking with total mortality and mortality attributed to cardiovascular disease, cancer, and other diseases with a major inflammatory component. DESIGN: A total of 41,836 postmenopausal women aged 55-69 y at baseline were followed for 15 y. After exclusions for cardiovascular disease, cancer, diabetes, colitis, and liver cirrhosis at baseline, 27,312 participants remained, resulting in 410,235 person-years of follow-up and 4265 deaths. The major outcome measure was disease-specific mortality. RESULTS: In the fully adjusted model, similar to the relation of coffee intake to total mortality, the hazard ratio of death attributed to cardiovascular disease was 0.76 (95% CI: 0.64, 0.91) for consumption of 1-3 cups/d, 0.81 (95% CI: 0.66, 0.99) for 4-5 cups/d, and 0.87 (95% CI: 0.69, 1.09) for > or =6 cups/d. The hazard ratio for death from other inflammatory diseases was 0.72 (95% CI: 0.55, 0.93) for consumption of 1-3 cups/d, 0.67 (95% CI: 0.50, 0.90) for 4-5 cups/d, and 0.68 (95% CI: 0.49, 0.94) for > or =6 cups/d. CONCLUSIONS: Consumption of coffee, a major source of dietary antioxidants, may inhibit inflammation and thereby reduce the risk of cardiovascular and other inflammatory diseases in postmenopausal women. PMID- 16685045 TI - Evaluation of Lunar Prodigy dual-energy X-ray absorptiometry for assessing body composition in healthy persons and patients by comparison with the criterion 4 component model. AB - BACKGROUND: Dual-energy X-ray absorptiometry (DXA) is widely used to assess body composition in research and clinical practice. Several studies have evaluated its accuracy in healthy persons; however, little attention has been directed to the same issue in patients. OBJECTIVE: The objective was to compare the accuracy of the Lunar Prodigy DXA for body-composition analysis with that of the reference 4 component (4C) model in healthy subjects and in patients with 1 of 3 disease states. DESIGN: A total of 215 subjects aged 5.0-21.3 y (n = 122 healthy nonobese subjects, n = 55 obese patients, n = 26 cystic fibrosis patients, and n = 12 patients with glycogen storage disease). Fat mass (FM), fat-free mass (FFM), and weight were measured by DXA and the 4C model. RESULTS: The accuracy of DXA measured body-composition outcomes differed significantly between groups. Factors independently predicting bias in weight, FM, FFM, and percentage body fat in multivariate models included age, sex, size, and disease state. Biases in FFM were not mirrored by equivalent opposite biases in FM because of confounding biases in weight. CONCLUSIONS: The bias of DXA varies according to the sex, size, fatness, and disease state of the subjects, which indicates that DXA is unreliable for patient case-control studies and for longitudinal studies of persons who undergo significant changes in nutritional status between measurements. A single correction factor cannot adjust for inconsistent biases. PMID- 16685046 TI - Ketogenic low-carbohydrate diets have no metabolic advantage over nonketogenic low-carbohydrate diets. AB - BACKGROUND: Low-carbohydrate diets may promote greater weight loss than does the conventional low-fat, high-carbohydrate diet. OBJECTIVE: We compared weight loss and biomarker change in adults adhering to a ketogenic low-carbohydrate (KLC) diet or a nonketogenic low-carbohydrate (NLC) diet. DESIGN: Twenty adults [body mass index (in kg/m(2)): 34.4 +/- 1.0] were randomly assigned to the KLC (60% of energy as fat, beginning with approximately 5% of energy as carbohydrate) or NLC (30% of energy as fat; approximately 40% of energy as carbohydrate) diet. During the 6-wk trial, participants were sedentary, and 24-h intakes were strictly controlled. RESULTS: Mean (+/-SE) weight losses (6.3 +/- 0.6 and 7.2 +/- 0.8 kg in KLC and NLC dieters, respectively; P = 0.324) and fat losses (3.4 and 5.5 kg in KLC and NLC dieters, respectively; P = 0.111) did not differ significantly by group after 6 wk. Blood beta-hydroxybutyrate in the KLC dieters was 3.6 times that in the NLC dieters at week 2 (P = 0.018), and LDL cholesterol was directly correlated with blood beta-hydroxybutyrate (r = 0.297, P = 0.025). Overall, insulin sensitivity and resting energy expenditure increased and serum gamma glutamyltransferase concentrations decreased in both diet groups during the 6-wk trial (P < 0.05). However, inflammatory risk (arachidonic acid:eicosapentaenoic acid ratios in plasma phospholipids) and perceptions of vigor were more adversely affected by the KLC than by the NLC diet. CONCLUSIONS: KLC and NLC diets were equally effective in reducing body weight and insulin resistance, but the KLC diet was associated with several adverse metabolic and emotional effects. The use of ketogenic diets for weight loss is not warranted. PMID- 16685048 TI - Negative energy balance in male and female rangers: effects of 7 d of sustained exercise and food deprivation. AB - BACKGROUND: A challenging 7-d ranger field exercise (FEX) by cadets in the Norwegian Military Academy provided a venue in which to study the effects of negative energy balance. OBJECTIVE: We quantified total energy expenditure (TEE), food intake, and changes in body composition in male and female cadets. DESIGN: TEE (measured by doubly labeled water), food intake, activity patterns (measured by accelerometry), and body composition (measured by dual-energy X-ray absorptiometry) were measured in 16 cadets (10 men and 6 women aged 21-27 y). RESULTS: The physically active (approximately 23 h/d) and semistarved (0.2-2.2 MJ/d) cadets lost weight (x +/- SD: men, -7.7 +/- 1.1 kg; women, -5.9 +/- 1.1 kg; P < 0.05). Absolute TEE differed by sex (men, 26.6 +/- 2.0 MJ/d; women, 21.9 +/- 2.0 MJ/d; P < 0.05) but body weight-specific TEE did not (men, 343 +/- 26 kJ . kg(-1) . d(-1); women, 354 +/- 18 kJ . kg(-1) . d(-1); NS). Fat-free mass (FFM) loss differed significantly by sex (men, -4.0 +/- 1.2 kg; women, -2.5 +/- 1.1 kg; P < 0.05), but percentage FFM loss did not (men, -6.3 +/- 1.9%; women, -5.6 +/- 2.4%). In contrast, absolute FM loss did not differ significantly by sex (men, 3.45 +/- 0.72 kg; women, -3.42 +/- 0.22 kg), but fat oxidation (men, 5.2 +/- 1.0 mg . min(-1) . kg FFM(-1); women, 7.3 +/- 0.5 mg . min(-1) . kg FFM(-1)) and the relative contribution of FM to TEE (men, 74 +/- 14%; women, 89 +/- 6%) were significantly greater in women than in men (P < 0.05). CONCLUSION: Female cadets maintained a significantly more fat-predominant fuel metabolism than did male cadets in response to sustained exercise and semistarvation. PMID- 16685047 TI - Small organs with a high metabolic rate explain lower resting energy expenditure in African American than in white adults. AB - BACKGROUND: African Americans have a lower resting energy expenditure (REE) relative to fat-free mass (FFM) than do whites. Whether the composition of FFM at the organ-tissue level differs between African Americans and whites and, if so, whether that difference could account for differences by race in REE are unknown. OBJECTIVE: The objectives were to quantify FFM in vivo in women and men at the organ-tissue level and to ascertain whether the mass of specific high-metabolic rate organs and tissues differs between African Americans and whites and, if so, whether that difference can account for differences in REE. DESIGN: The study was a cross-sectional evaluation of 64 women (n = 34 African Americans, 30 whites) and 35 men (n = 8 African Americans, 27 whites). Magnetic resonance imaging measures of liver, kidney, heart, spleen, brain, skeletal muscle, and adipose tissue and dual-energy X-ray absorptiometry measures of fat and FFM were acquired. REE was measured by using indirect calorimetry. RESULTS: The mass of selected high-metabolic-rate organs (sum of liver, heart, spleen, kidneys, and brain) after adjustment for fat, FFM, sex, and age was significantly (P < 0.001) smaller in African Americans than in whites (3.1 and 3.4 kg, respectively; x +/- SEE difference: 0.30 +/- 0.06 kg). In a multiple regression analysis with fat, FFM, sex, age, and race as predictors of REE, the addition of the total mass rendered race nonsignificant. CONCLUSIONS: Racial differences in REE were reduced by >50% and were no longer significant when the mass of specific high-metabolic rate organs was considered. Differences in FFM composition may be responsible for the reported REE differences. PMID- 16685049 TI - Positive association between resting energy expenditure and weight gain in a lean adult population. AB - BACKGROUND: Weight gain in adulthood is common, from modest gains in developing countries to substantial increases in Western societies. Evidence of the importance of energy expenditure in adult weight change has been limited to studies conducted in Pima Indians, in whom resting energy expenditure (REE) was found to be inversely associated with weight gain. OBJECTIVE: The aim was to determine whether REE was predictive of weight change in lean Nigerian adults. DESIGN: Weight was measured in 744 adults on 2-4 occasions over 5.5 y. REE was measured in the second follow-up examination. Sex-specific, mixed-effects models with REE, fat-free mass, and age as fixed effects were used to test the association between REE and weight change. RESULTS: Adults aged >19 y (n = 352 men and 392 women) were included in these analyses. At baseline, the mean (+/-SD) age was 45.9 +/- 16.1 y for the whole population; the mean weight was 61.4 +/- 10.7 and 58.1 +/- 12.1 kg and body mass index (in kg/m(2)) was 21.4 +/- 3.2 and 23.1 +/- 4.0 for men and women, respectively. Over a mean 5.5 y of follow-up, the age-adjusted weight gain was 0.42 kg/y for the men and 0.59 kg/y for the women. In mixed-effects models, REE was positively associated with weight gain in both men and women (P < 0.001). No significant association was observed in participants who lost weight. CONCLUSIONS: In contrast with observations in overweight Pima Indians, REE adjusted for body size and composition was positively associated with weight gain in lean Nigerian adults. This suggests either that the potential for differential regulation of body weight in lean compared with overweight populations exists or that the increased REE in this population was the result, rather than cause, of weight gain. PMID- 16685050 TI - Diets rich in Maillard reaction products affect protein digestibility in adolescent males aged 11-14 y. AB - BACKGROUND: Adolescents are nutritionally at risk because of their physiologic needs and dietary habits. Maillard reaction products (MRPs) are widely consumed by this population, mainly as a result of their high intake of fast food and snacks. OBJECTIVE: We compared the effects of diets with different MRP contents on dietary protein utilization in adolescent males aged 11-14 y. The brown diet (BD) was rich and the white diet (WD) was poor in MRP content (hydroxymethylfurfural: 3.87 and 0.94 mg/kg; fluorescence intensity: 21.04% and 7.31%, respectively). DESIGN: In a 2-period crossover trial, 18 healthy adolescent males were randomly assigned to 2 groups. The first group consumed the WD for 2 wk, observed a 40-d washout period, and then consumed the BD for 2 wk. The second group received the diets in the opposite order. Subjects collected urine and feces on the last 3 d of each dietary period. Fasting blood samples were collected after both periods. RESULTS: Compared with consumption of the WD, consumption of the BD resulted in 47% higher fecal nitrogen fecal excretion (P = 0.002), 12% lower apparent nitrogen absorption (P = 0.000), and a 6% lower nitrogen digestibility (P = 0.000). The apparent nitrogen retention and the utilization of ingested nitrogen did not differ significantly between the diets, although values after the BD tended to decrease. Serum biochemical variables related to nitrogen metabolism did not differ significantly. CONCLUSIONS: The consumption of a diet rich in MRPs negatively affects protein digestibility. The possible effects of an excessive intake of MRPs during adolescence warrant attention, and long-term effects should be considered. PMID- 16685051 TI - Efficacy of zinc in the treatment of severe pneumonia in hospitalized children <2 y old. AB - BACKGROUND: Severe pneumonia remains a leading cause of morbidity and mortality in undernourished young children in developing countries. OBJECTIVE: This study evaluated the effect of adjuvant zinc therapy on recovery from severe pneumonia by hospitalized children in southern India who were receiving standard antibiotic therapy. DESIGN: This randomized, double-blind, placebo-controlled clinical trial was conducted at the Christian Medical College Hospital, an 1800-bed teaching hospital in Tamilnadu, India. Enrollment and follow-up occurred between September 2003 and August 2004. Children aged 2-23 mo (n = 299) who were hospitalized with severe pneumonia were randomly assigned to receive 10-mg tablets of zinc sulfate or placebo twice a day during hospitalization, along with standard therapy for severe pneumonia. All clinical signs and symptoms of pneumonia were assessed and recorded at 8-h intervals. RESULTS: There were no clinical or statistically significant differences in the duration of tachypnea, hypoxia, chest indrawing, inability to feed, lethargy, severe illness, or hospitalization. Zinc supplementation was associated with a significantly longer duration of pneumonia in the hot season (P = 0.015). CONCLUSIONS: Zinc supplementation had no overall effect on the duration of hospitalization or of clinical signs associated with severe infection in young children hospitalized for severe pneumonia in southern India. This finding differs from the results of 2 previously reported trials wherein zinc supplementation was associated with a shorter period of recovery from severe pneumonia. Given the conflicting results, further research in representative settings is required to help clarify the role of zinc in the treatment of severe pneumonia. PMID- 16685052 TI - A furanocoumarin-free grapefruit juice establishes furanocoumarins as the mediators of the grapefruit juice-felodipine interaction. AB - BACKGROUND: Grapefruit juice (GFJ) enhances the systemic exposure of numerous CYP3A4 drug substrates, including felodipine, by inhibiting intestinal (but not hepatic) first-pass metabolism. Furanocoumarins have been identified as major CYP3A4 inhibitors contained in the juice, but their contribution to the GFJ effect in vivo remains unclear. OBJECTIVE: To ascertain whether furanocoumarins mediate the GFJ-felodipine interaction, a furanocoumarin-free GFJ was created and tested against orange juice and the original GFJ with respect to the oral pharmacokinetics of felodipine. DESIGN: With the use of food-grade solvents and absorption resins, furanocoumarins were removed (approximately 99%) from whole GFJ, whereas other major ingredients (flavonoids) were retained. In an open, 3 way, randomized crossover design, 18 healthy volunteers ingested felodipine (10 mg) with 1 of the 3 juices (240 mL). Blood was collected over 24 h. At least 1 wk elapsed between juice treatments. RESULTS: The median and range of the area under the curve and the maximum concentration of felodipine were significantly (P < 0.001) greater with consumption of GFJ [110 (range: 58-270) nmol . h/L and 21 (7.6-50) nmol/L, respectively] than with that of orange juice [54 (29-150) nmol . h/L and 7.6 (3.4-13.9) nmol/L, respectively] or furanocoumarin-free GFJ [48 (23 120) nmol . h/L and 8.3 (3.0-16.6) nmol/L, respectively]. GFJ, orange juice, and furanocoumarin-free GFJ did not differ significantly (P > 0.09) in median time to reach maximum plasma concentration [2.5 (1.5-6), 2.8 (1.5-4), and 2.5 (2-6) h, respectively] or terminal half-life [6.6 (4.2-13.6), 7.8 (4.4-13.2), and 6.8 (2.6 14.4) h, respectively]. CONCLUSION: Furanocoumarins are the active ingredients in GFJ responsible for enhancing the systemic exposure of felodipine and probably other CYP3A4 substrates that undergo extensive intestinal first-pass metabolism. PMID- 16685053 TI - Adiponectin is present in human milk and is associated with maternal factors. AB - BACKGROUND: Previous studies have shown that human milk has a role in the gastrointestinal, neural, and immune development of neonates. If present in milk, adiponectin would be a promising candidate for influencing infant development, given its metabolic functions. OBJECTIVES: Our objectives were to determine whether adiponectin is present in human milk and to characterize maternal factors associated with potential variation in milk adiponectin concentrations. DESIGN: We quantified adiponectin concentrations in human milk samples from donors to the Cincinnati Children's Research Human Milk Bank and randomly selected participants in a cohort study in Mexico City funded by the National Institutes of Health. Using cross-sectional and longitudinal data, we examined milk adiponectin concentrations in relation to lactation duration, maternal body mass index (BMI; in kg/m(2)), and ethnicity. RESULTS: Adiponectin was detected in human skim milk (range: 4.2-87.9 ng/mL). In cross-sectional and longitudinal analyses, duration of lactation was negatively associated with milk adiponectin concentrations (beta = -0.059 +/- 0.024 and -0.059 +/- 0.007, respectively; P < 0.02 for both). Maternal postpregnancy BMI was positively associated with milk adiponectin concentrations (beta = 0.08 +/- 0.02, P < 0.0001; longitudinal analysis). Mexican mothers had lower median milk adiponectin concentrations at 1 mo than did the non Hispanic white subjects from Cincinnati (11.5 and 19.8 ng/mL; P = 0.003). CONCLUSIONS: Adiponectin is present in human milk and its concentrations are associated with duration of lactation, maternal adiposity, and ethnicity. Given the importance of adiponectin in inflammation, insulin sensitivity, and fatty acid metabolism, future studies should examine milk adiponectin's role in infant metabolic development. PMID- 16685054 TI - Effect of iron supplementation during pregnancy on the intelligence quotient and behavior of children at 4 y of age: long-term follow-up of a randomized controlled trial. AB - BACKGROUND: Iron supplements are often prescribed during pregnancy despite the lack of intervention trials that have assessed the effects of supplementation in pregnancy on childhood development. OBJECTIVE: The objective was to determine whether iron supplementation during pregnancy influences childhood intelligence quotient (IQ) in an industrialized country. DESIGN: Pregnant women (n = 430) were randomly allocated to receive iron (20 mg/d) or placebo from 20 wk gestation until delivery, and the women and their children were followed up over the long term (4 y). Seventy percent of these families participated in the follow-up. The proportion of women with iron deficiency anemia at the end of pregnancy was 1% (2 of 146) in the iron group and 11% (15 of 141) in the placebo group. The primary outcome was the IQ of the children at 4 y of age, as assessed by the Stanford Binet Intelligence Scale. Secondary outcomes included child behavior and the general health of the mothers. RESULTS: The mean IQ was not significantly different (P = 0.980) between the children of the iron-supplemented mothers (109 +/- 11; n = 153) and the children of the mothers in the placebo group (109 +/- 11; n = 149). However, the percentage of children with an abnormal behavior score was higher in the iron group (24 of 151, or 16%) than in the placebo group (12 of 149, or 8%); the relative risk was 1.97 (95% CI: 1.03, 3.80; P = 0.037). There was no significant difference in the health of the mothers between groups, as assessed by the SF-36 Health Survey. CONCLUSIONS: Prenatal iron supplementation that reduces the incidence of iron deficiency anemia from 11% to 1% has no effect on the IQ of the offspring at 4 y of age. PMID- 16685055 TI - Soy isoflavones modulate immune function in healthy postmenopausal women. AB - BACKGROUND: The immune system may be compromised after menopause because of the effects of aging and diminishing concentrations of estrogen, an immune-modulating hormone. Isoflavones, plant-derived compounds with estrogenic and antioxidant properties, may offer immunologic benefits to women during this stage of life. OBJECTIVE: The objective of this study was to evaluate the effects of soy isoflavones, both in soymilk and in supplement form, on markers of immunity and oxidative stress in postmenopausal women. DESIGN: Postmenopausal women aged 50-65 y (n = 52) enrolled in this 16-wk double-blind, placebo-controlled trial were randomly assigned to 1 of 3 experimental groups: 1) control, 706 mL cow milk/d plus a placebo supplement; 2) soymilk, 71.6 mg isoflavones derived from 706 mL soymilk/d plus a placebo supplement; and 3) supplement, 70 mg isoflavones in a supplement plus 706 mL cow milk/d. Plasma and 24-h urine samples were obtained at baseline and at 16 wk. Immune variables included lymphocyte subsets, cytokine production, and markers of inflammation and oxidative damage. RESULTS: Isoflavone intervention in postmenopausal women resulted in higher (P < 0.05) B cell populations and lower (P < 0.05) plasma concentrations of 8-hydroxy-2-deoxy guanosine, an oxidative marker of DNA damage. Isoflavone treatment did not significantly influence concentrations of interferon gamma, interleukin 2, tumor necrosis factor alpha, or C-reactive protein in plasma or of 8-isoprostane in urine. CONCLUSIONS: Soymilk and supplemental isoflavones modulate B cell populations and appear to be protective against DNA damage in postmenopausal women. PMID- 16685056 TI - Association between fruit and vegetable consumption and oral cancer: a meta analysis of observational studies. AB - BACKGROUND: Oral cancer ranks as the seventh most common form of cancer worldwide. Recent reports have examined the effect of fruit and vegetable intake on the risk of oral cancer, but results are controversial. OBJECTIVE: A meta analysis was performed to arrive at quantitative conclusions about the contribution of fruit and vegetable intakes to the occurrence of oral cancer. DESIGN: A comprehensive, systematic bibliographic search of medical literature published up to September 2005 was conducted to identify relevant studies. Separate meta-analyses were conducted for fruit and vegetable consumption. The effect of portion or daily intake of fruit or vegetables on the risk of oral cancer was calculated. A multivariate meta-regression analysis was performed to explore heterogeneity. This multivariate meta-regression analysis examined the effect of quality score, the type of cancers included, citrus fruit and green vegetable consumption, and the time interval for dietary recall of the studies on the role of fruit or vegetable consumption in the risk of oral cancer. The presence of publication bias was assessed with a funnel plot for asymmetry. RESULTS: Sixteen studies (15 case-control studies and 1 cohort study) met the inclusion criteria and were included in the meta-analysis. The combined adjusted odds ratio (OR) estimates showed that each portion of fruit consumed per day significantly reduced the risk of oral cancer by 49% (OR: 0.51; 95% CI: 0.40, 0.65). For vegetable consumption, the meta-analysis showed a significant reduction in the overall risk of oral cancer of 50% (OR: 0.50; 95% CI: 0.38, 0.65). The multivariate meta-regression showed that the lower risk of oral cancer associated with fruit consumption was significantly influenced by the type of fruit consumed and by the time interval of dietary recall. CONCLUSION: The consumption of fruit and vegetables is associated with a reduced risk of oral cancer. PMID- 16685057 TI - Vitamin D status in adolescents and young adults with HIV infection. AB - BACKGROUND: Vitamin D status affects immune function and thus may affect the progress of HIV infection. OBJECTIVES: Our goals were to assess vitamin D intake and status in subjects with HIV infection and in matched control subjects and to determine whether HIV infection was associated with vitamin D insufficiency. DESIGN: Plasma 25-hydroxyvitamin D [25(OH)D] concentrations and vitamin D intake were measured in a cross-sectional study of members of the Reaching for Excellence in Adolescent Health (REACH) cohort. RESULTS: The subjects were aged 14-23 y; 74% were female, and 72% were black. Mean (+/-SE) vitamin D intake from food was 30% greater (P = 0.023) in HIV-positive subjects (295 +/- 18 IU/d; n = 237) than in HIV-negative subjects (227 +/- 26 IU/d; n = 121). The prevalence of vitamin D supplement use was 29% (104 of 358 subjects) and did not differ significantly by HIV status (P = 0.87). Mean plasma 25(OH)D did not differ significantly (P = 0.62) between the HIV-positive (20.3 +/- 1.1 nmol/L; n = 238) and HIV-negative (19.3 +/- 1.7 nmol/L; n = 121) subjects, nor was HIV status a significant predictor of plasma 25(OH)D when multiple regression analysis was used to adjust for other variables. The prevalence of vitamin D insufficiency [plasma 25(OH)D < or = 37.5 nmol/L] in the subjects was 87% (312 of 359 subjects). CONCLUSIONS: HIV infection did not influence vitamin D status. The prevalence of vitamin D insufficiency in both HIV-positive and HIV-negative REACH subjects was high, perhaps because these disadvantaged, largely urban youth have limited sun exposure. PMID- 16685058 TI - Frailty syndrome and skeletal muscle: results from the Invecchiare in Chianti study. AB - BACKGROUND: Frailty is a common condition in elders and identifies a state of vulnerability for adverse health outcomes. OBJECTIVE: Our objective was to provide a biological face validity to the well-established definition of frailty proposed by Fried et al. DESIGN: Data are from the baseline evaluation of 923 participants aged > or =65 y enrolled in the Invecchiare in Chianti study. Frailty was defined by the presence of > or =3 of the following criteria: weight loss, exhaustion, low walking speed, low hand grip strength, and physical inactivity. Muscle density and the ratios of muscle area and fat area to total calf area were measured by using a peripheral quantitative computerized tomography (pQCT) scan. Analyses of covariance and logistic regressions were performed to evaluate the relations between frailty and pQCT measures. RESULTS: The mean age (+/-SD) of the study sample was 74.8 +/- 6.8 y, and 81 participants (8.8%) had > or =3 frailty criteria. Participants with no frailty criteria had significantly higher muscle density (71.1 mg/cm(3), SE = 0.2) and muscle area (71.2%, SE = 0.4) than did frail participants (69.8 mg/cm(3), SE = 0.4; and 68.7%, SE = 1.1, respectively). Fat area was significantly higher in frail participants (22.0%, SE = 0.9) than in participants with no frailty criteria (20.3%, SE = 0.4). Physical inactivity and low walking speed were the frailty criteria that showed the strongest associations with pQCT measures. CONCLUSION: Frail subjects, identified by an easy and inexpensive frailty score, have lower muscle density and muscle mass and higher fat mass than do nonfrail persons. PMID- 16685059 TI - Unbalanced serum leptin and ghrelin dynamics prolong postprandial satiety and inhibit hunger in healthy elderly: another reason for the "anorexia of aging". AB - BACKGROUND: In healthy elderly, a reduction from the appetite and food intake of younger years has been defined as the "anorexia of aging," which may cause malnutrition. Leptin and ghrelin may alter the control of hunger and satiety and thus lead to anorexia. OBJECTIVE: The aim of this study was to investigate how aging affects serum leptin and ghrelin concentrations in response to a meal and the relation of those hormones to hunger and satiety sensations. DESIGN: We studied 8 community-dwelling elderly (x +/- SD age: 78 +/- 1 y) subjects and 8 younger (29.5 +/- 1 y) control subjects. Under fasting conditions and for 4 h after an 800-kcal mixed meal, satiety and hunger were evaluated at intervals, by using a visual analogic scale. Blood samples for leptin, ghrelin, and insulin measurements were collected at the following times: 30 min before and immediately and 30, 60, 120, and 240 min after the meal. RESULTS: Postprandial satiety lasted significantly longer in the elderly than in the control subjects, and hunger was suppressed in the elderly throughout the observation. Fasting leptin was higher in the elderly than in the young (x +/- SE: 4.3 +/- 1.9 and 1.3 +/- 0.4 ng/mL, respectively; P < 0.05), and postprandial fluctuation was not significant. Fasting insulin also was significantly higher in the elderly than in the young (6.8 +/- 1.3 and 3.5 +/- 0.6 mU/L, respectively; P < 0.05), and the postprandial insulin rise was greater in the elderly. Fasting and postprandial ghrelin values did not differ significantly between the 2 groups. Insulin was inversely correlated with hunger and directly correlated with satiety scores. CONCLUSIONS: In healthy elderly, anorexigenic signals prevail over orexigenic signals, and they contribute to prolonged satiety and inhibition of hunger. This condition may lead to a calorie deficit and finally to malnutrition in the elderly. PMID- 16685060 TI - A population-based study of the association between betel-quid chewing and the metabolic syndrome in men. AB - BACKGROUND: Betel-quid chewing, an established risk factor for oropharyngeal malignancy, is associated with hyperglycemia and obesity. Associations with other characteristics of the metabolic syndrome have not been reported. OBJECTIVE: This study examined associations between betel-quid chewing and the metabolic syndrome, allowing for recognized risk factors and exploring dose-response effects in a population-based study. DESIGN: Age-specific prevalence rates of the metabolic syndrome were examined in betel-quid chewing and nonchewing men (n = 19,839) recruited into the Keelung Community-based Integrated Screening program in 2001-2003. The independent effect of betel-quid chewing on metabolic syndrome risk was examined by using multiple logistic regression with control for well recognized risk factors (eg, education, physical activity, and dietary factors) and dose-response effects were examined by using trend tests. RESULTS: The age adjusted prevalence of the metabolic syndrome was highest in current chewers (25.13%), next highest in ex-chewers (22.04%), and lowest in nonchewers (15.73%) (P < 0.0001). Odds ratios (95% CIs) for the metabolic syndrome were 1.38 (1.19, 1.60) and 1.78 (1.53, 2.08) in ex-chewers and current chewers, respectively, adjusted for other significant correlates such as a family history of hypertension and diabetes mellitus. Meaningful odds ratios for the metabolic syndrome components ranged from 1.24 for hyperglycemia (95% CI: 1.09, 1.64) to 1.90 (95% CI: 1.66, 2.19) for hypertriacylglycerolemia. Increasing odds ratios for the metabolic syndrome with higher consumption of betel quid (whether by rate of use, duration of use, or cumulative exposure) suggest dose-response effects. CONCLUSIONS: After adjustment for well-established risk factors, our study showed independent predictive dose-response effects of betel-quid chewing for the metabolic syndrome and its components in a population-based study of men with a 15% prevalence of betel-nut chewing. PMID- 16685061 TI - Dietary glycemic index and load in relation to metabolic risk factors in Japanese female farmers with traditional dietary habits. AB - BACKGROUND: Little is known about the relation of dietary glycemic index (GI) and glycemic load (GL) to metabolic risk factors, particularly in non-Western populations. OBJECTIVE: We examined the cross-sectional associations between dietary GI and GL and several metabolic risk factors in healthy Japanese women with traditional dietary habits. DESIGN: The subjects were 1354 Japanese female farmers aged 20-78 y from 5 regions of Japan. Dietary GI and GL were assessed with a self-administered diet-history questionnaire. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Fasting blood samples were collected for biochemical measurements. RESULTS: The mean dietary GI was 67, and the mean dietary GL (/1000 kcal) was 88 (GI for glucose = 100). White rice (GI = 77) was the major contributor to dietary GI and GL (58.5%). After adjustment for potential dietary and nondietary confounding factors, dietary GI was positively correlated with BMI (n = 1354; P for trend = 0.017), fasting triacylglycerol (n = 1349; P for trend = 0.001), fasting glucose (n = 764; P for trend = 0.022), and glycated hemoglobin (n = 845; P for trend = 0.038). Dietary GL was independently negatively correlated with HDL cholesterol (n = 1354; P for trend = 0.004) and positively correlated with fasting triacylglycerol (P for trend = 0.047) and fasting glucose (P for trend = 0.012). CONCLUSIONS: Both dietary GI and GL are independently correlated with several metabolic risk factors in subjects whose dietary GI and GL were primarily determined on the basis of the GI of white rice. PMID- 16685062 TI - Dietary patterns and survival in older Dutch women. AB - BACKGROUND: The need to gain insight into prevailing eating patterns and their health effects is evident. OBJECTIVE: This study aimed to identify dietary patterns and their relation to total mortality in older Dutch women. DESIGN: A principal component analysis of 22 food groups was used to identify dietary patterns in 5427 women aged 60-69 y who were included in the Dutch European Prospective Investigation into Cancer and Nutrition-Elderly cohort (follow-up: approximately 8.2 y). Mortality ratios for 3 major principal components were assessed by using Cox proportional hazard analysis. RESULTS: The most relevant principal components were a Mediterranean-like dietary pattern (high intakes of vegetable oils, pasta and rice, sauces, fish, and wine), a Traditional Dutch dinner dietary pattern (high intakes of meat, potatoes, vegetables, and alcoholic beverages), and a Healthy Traditional Dutch dietary pattern (healthy variant of the Traditional Dutch dinner dietary pattern; high intakes of vegetables, fruit, nonalcoholic drinks, dairy products, and potatoes). Differences in mean intakes between the highest and lowest tertiles of the 3 patterns were greatest for fruit, dairy products, potatoes, and alcoholic beverages. Consumption of Mediterranean foods, such as fish and oils, was relatively low overall. Two hundred seventy-seven deaths occurred in 44,667 person-years. Independent of age, education, and other lifestyle factors, only the Healthy Traditional dietary pattern score was associated with a lower mortality rate. Women in the highest tertile of this pattern experienced a 30% reduction in mortality risk. CONCLUSION: A Healthy Traditional Dutch diet, rather than a Mediterranean diet, appears beneficial for longevity and feasible for health promotion in older Dutch women. This diet is comparable with other reported healthy or prudent diets that have been shown to be protective against morbidity or mortality. PMID- 16685063 TI - Dietary carbohydrate intake and glycemic index in relation to cortical and nuclear lens opacities in the Age-Related Eye Disease Study. AB - BACKGROUND: Little is known about the association between dietary carbohydrates and cataract in nondiabetic persons. OBJECTIVE: The aim was to test whether recent dietary carbohydrate intakes or glycemic index (GI; a measure of carbohydrate intake quality) was associated with the presence of cortical or nuclear opacities. DESIGN: A modified Block food-frequency questionnaire was used to obtain dietary information from 3377 participants (aged 60-80 y; 56% were women) in the Age-Related Eye Disease Study (AREDS). Lens status was evaluated by using the AREDS System for Classifying Cataracts. Associations were examined for eyes with only a single, or pure, type of lens opacity by using the generalized estimating approach to logistic regression to account for the lack of independence between the eyes of a person. RESULTS: For participants in the highest quartile, dietary GI was associated with a higher prevalence of all pure nuclear opacities [grade >2; odds ratio (OR): 1.29; 95% CI: 1.04, 1.59; P for trend = 0.02] and moderate nuclear opacities (grade > or =4; OR: 1.43; 95% CI: 0.96, 2.14; P for trend = 0.052). The OR in a comparison of the highest with the lowest quartile of intake was 1.27 (95% CI: 0.99, 1.63; P for trend = 0.09) for cortical opacities of any severity (>0% of area opaque), and the OR increased somewhat for moderate cortical opacities (>5% of area opaque; OR: 1.71; 95% CI: 1.00, 2.95; P for trend = 0.056). CONCLUSIONS: Results from the cross-sectional analysis of AREDS baseline data suggest that dietary glycemic quality and dietary carbohydrate quantity may be associated with prevalent nuclear and cortical opacities, respectively. PMID- 16685064 TI - Dietary patterns associated with bone mineral density in premenopausal Japanese farmwomen. AB - BACKGROUND: Because several nutrients are known to affect bone mineral density (BMD), the analysis of dietary patterns or combinations of foods may provide insights into the influence of diet on bone health. OBJECTIVE: We evaluated associations between dietary patterns and BMD in Japanese farmwomen. DESIGN: The study included 291 premenopausal farmwomen (aged 40-55 y) who participated in the Japanese Multi-centered Environmental Toxicant Study (JMETS; n = 1407). Forearm BMD was measured by using dual-energy X-ray absorptiometry. Diet was assessed by using a validated self-administered diet history questionnaire comprising 147 food items, from which 30 food groups were created and entered into a factor analysis. RESULTS: Four dietary patterns were identified. The "Healthy" pattern, characterized by high intakes of green and dark yellow vegetables, mushrooms, fish and shellfish, fruit, and processed fish, was positively correlated with BMD after adjustment for several confounding factors (P = 0.048). In contrast, the "Western" pattern, characterized by high intakes of fats and oils, meat, and processed meat, tended to be inversely associated with BMD; however, the association was not significant (P = 0.08). CONCLUSION: A dietary pattern with high intakes of fish, fruit, and vegetables and low intakes of meat and processed meat may have a beneficial effect on BMD in premenopausal women. PMID- 16685065 TI - Polyunsaturated fatty acid intake is adversely related to liver function in HIV infected subjects: the THUSA study. AB - BACKGROUND: Dietary fat intake in the South African population is increasing. This population also has a high prevalence of HIV infection. However, information about metabolic effects of dietary fatty acids on HIV-infected subjects is lacking. OBJECTIVE: Our objective was to investigate the relation between dietary fatty acid intake and liver function in HIV-infected compared with HIV-uninfected subjects. DESIGN: This cross-sectional epidemiologic survey included a representative sample of 1854 apparently healthy black volunteers aged > or =15 y, who were recruited from 37 randomly selected sites throughout the North West province of South Africa. Data from 216 asymptomatic HIV-infected and 1604 HIV uninfected subjects were used. RESULTS: Intakes of polyunsaturated fatty acids (PUFAs), linoleic acid (n-6), and the ratio of PUFAs to saturated fatty acids (SFAs) were positively associated with all the liver enzymes measured in HIV infected subjects (R = 0.16-0.65). Most of these R values differed significantly from the R values for HIV-uninfected subjects. No associations were seen between liver enzymes and intakes of SFAs and monounsaturated fatty acids. Vitamin E intake was positively associated with serum gamma-glutamyl transpeptidase (R = 0.23), alanine aminotransferase (R = 0.37), and aspartate aminotransferase (R = 0.58) in HIV-infected subjects; these correlations differed significantly from those of the HIV-uninfected subjects because PUFA sources are the main carriers of vitamin E. CONCLUSIONS: The results suggest that n-6 PUFA intakes may be related to liver damage in these HIV-infected asymptomatic subjects. The reasons or mechanisms responsible are not clear, and further research is necessary to determine the optimal safe amounts for intake of n-6 PUFAs by HIV-infected subjects, especially in countries with traditionally high intakes of n-6 PUFA rich vegetable oils. PMID- 16685067 TI - Dietary Reference Intakes for food labeling. AB - The annual American Society for Nutritional Sciences/American Society for Clinical Nutrition Public Information Committee symposium for 2005 titled "Dietary Reference Intakes (DRIs) for Food Labeling" served as a platform to address the use of DRIs in food labeling, a change that could affect the processing of foods and supplements and the nutrient intakes of consumers. Speakers from science, industry, trade organizations, and the government came together to provide critical thinking about the most appropriate DRI values to use in food labeling. The proceedings of the symposium cover consumer perspectives on food labels, the regulatory process for revising labeling relative to the DRIs, the food industry's perspective on the use of the new DRIs in food labeling, and differing opinions on whether to use Recommended Dietary Allowances or Estimated Average Requirements to set Daily Values. PMID- 16685066 TI - Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China. AB - BACKGROUND: In vitro and in vivo studies suggest that selected strains of probiotic bacteria can form tight complexes with aflatoxin B(1) and other carcinogens. OBJECTIVE: The aim of the present study was to determine whether administration of probiotic bacteria could block the intestinal absorption of aflatoxin B(1) and thereby lead to reduced urinary excretion of aflatoxin B(1) N(7)-guanine (AFB-N(7)-guanine), a marker for a biologically effective dose of aflatoxin exposure. Elevated urinary excretion of this aflatoxin-DNA adduct is associated with an increased risk of liver cancer. DESIGN: Ninety healthy young men from Guangzhou, China, were randomly assigned to 2 groups; one group received a mixture of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii subsp. shermanii strains 2 times/d for 5 wk, and the other group received a placebo preparation. The subjects provided 4 urine samples: at baseline, at 3 and 5 wk after starting the supplementation, and at the end of the 5-wk postintervention period. RESULTS: The percentage of samples with negative AFB N(7)-guanine values tended to be higher in the probiotic group than in the placebo group during the 5-wk intervention period (odds ratio: 2.63, P = 0.052), and a statistically significant decrease in the concentration of urinary AFB-N(7) guanine was observed in the probiotic group. The reduction was 36% at week 3 and 55% at week 5. The geometric means for the probiotic and placebo groups were 0.24 and 0.49 ng AFB-N(7)-guanine/mL, respectively, during the intervention period (P = 0.005). CONCLUSION: A probiotic supplement reduces the biologically effective dose of aflatoxin exposure and may thereby offer an effective dietary approach to decrease the risk of liver cancer. PMID- 16685068 TI - Use of population-weighted Estimated Average Requirements as a basis for Daily Values on food labels. AB - In both Canada and the United States, nutrition labeling is now mandatory for most packaged foods. The labeling is intended to help consumers select foods that can contribute to a healthful diet, but current label values are based on outdated notions of nutrient requirements. The Committee on Use of Dietary Reference Intakes in Nutrition Labeling has recommended that the reference values used for nutrition labeling be based on a population-weighted Estimated Average Requirement (EAR) for any nutrient for which requirements have been estimated. This value approximates the median of the distribution of nutrient requirements for individuals who are members of the target population for food labels. It provides the most scientifically valid, single point of comparison for an appraisal of the probable contribution of a specific food to the overall nutrient needs of individuals in the target population. In contrast, a reference value based on a population coverage approach would understate the nutrient contribution of the food item relative to the requirements of the vast majority of individuals in the target population and thus offer misinformation rather than positive guidance to the consumer. PMID- 16685069 TI - Recommended Dietary Allowances should be used to set Daily Values for nutrition labeling. AB - Guiding principles were recently suggested for revising the Daily Values (DVs) used for nutrition labels on foods and dietary supplements. These principles incorporate the new Dietary Reference Intakes, which are nutrient standards issued between 1997 and 2005 by the Institute of Medicine. Most of the principles are likely to lead to a more accurate basis for the DVs. However, the recommendation to use the Estimated Average Requirement (EAR) rather than the Recommended Dietary Allowance (RDA) should be reconsidered. Traditional public health messages to American and Canadian consumers have focused on nutrient intake levels with a high probability of being adequate. The RDA, with a 98% probability of adequacy, is designed to be the target nutrient intake for individuals; in contrast, the EAR has only a 50% probability of adequacy. Three considerations should lead to a preference for using the RDA rather than the EAR for the DVs: 1) consumers are likely to expect that a product (or a diet) with 100% of the DV has a high probability of nutrient adequacy; 2) use of the RDA for the DV will be consistent with other types of dietary guidance, such as the Dietary Guidelines for Americans 2005 and US food guides; and 3) use of the RDA as a standard for nutrient intake, rather than the EAR, has a potential benefit (a higher prevalence of adequate intakes) that exceeds potential risk (a higher prevalence of excessive intakes). PMID- 16685070 TI - The regulatory process to revise nutrient labeling relative to the Dietary Reference Intakes. AB - The Nutrition Labeling and Education Act of 1990--an amendment to the Federal Food, Drug, and Cosmetic Act--paved the way for significant changes in the labeling of foods, nutrient content, and health claims. This article gives an overview of the regulatory process used by the US Food and Drug Administration to revise the food label relative to the Dietary Reference Intakes and in ways that reflect new scientific knowledge and public health issues. PMID- 16685071 TI - The new Dietary Reference Intakes in food labeling: the food industry's perspective. AB - The food industry appreciates the complexity of applying the new Dietary Reference Intakes (DRIs) in labeling. The industry is prepared to update food labels to reflect new nutrient recommendations and views upcoming changes as an opportunity to harmonize nutrition information across the Dietary Guidelines for Americans 2005, MyPyramid.gov, and the food label. Members of the Grocery Manufacturers Association are unanimous in their belief that the food label be as useful to consumers as possible. This article raises discussion points, issues, and implications associated with implementation of the new DRIs on the food label. PMID- 16685072 TI - Consumer perspectives on food labels. AB - The symposium "Dietary Reference Intakes (DRIs) for Food Labeling" explored the scientific debate about the most appropriate DRI values to use in establishing Daily Values on food labels. An important goal of nutrition and ingredient information is to help consumers make healthful dietary choices. This summary provides highlights from recent quantitative and qualitative research on consumers' use and understanding of food label information. PMID- 16685073 TI - A tale of 2 studies: diet and breast cancer. PMID- 16685074 TI - Mindfulness meditation for oncology patients: a discussion and critical review. AB - The purpose of this article is to (1) provide a comprehensive over view and discussion of mindfulness meditation and its clinical applicability in oncology and (2) report and critically evaluate the existing and emerging research on mindfulness meditation as an intervention for cancer patients. Using relevant keywords, a comprehensive search of MEDLINE, PsycInfo, and Ovid was completed along with a review of published abstracts from the annual conferences sponsored by the Center for Mindfulness in Medicine, Health Care, and Society and the American Psychosocial Oncology Society. Each article and abstract was critiqued and systematically assessed for purpose statement or research questions, STUDY DESIGN: The search produced 9 research articles published in the past 5 years and 5 conference abstracts published in 2004. Most studies were conducted with breast and prostate cancer patients, and the mindfulness intervention was done in a clinic-based group setting. Consistent benefits--improved psychological functioning, reduction of stress symptoms, enhanced coping and well-being in cancer outpatients--were found. More research in this area is warranted: using randomized, controlled designs, rigorous methods, and different cancer diagnoses and treatment settings; expanding outcomes to include quality of life, physiological, health care use, and health-related outcomes; exploring mediating factors; and discerning dose effects and optimal frequency and length of home practice. Mindfulness meditation has clinically relevant implications to alleviate psychological and physical suffering of persons living with cancer. Use of this behavioral intervention for oncology patients is an area of burgeoning interest to clinicians and researchers. PMID- 16685075 TI - Exercise use as complementary therapy among breast and prostate cancer survivors receiving active treatment: examination of exercise intention. AB - BACKGROUND: Exercise is associated with an improved quality of life among cancer survivors. Previous research has highlighted the utility of the theory of planned behavior in understanding cancer survivors' intention to exercise. The purpose of this study was to extend the research on the theory of planned behavior in relation to exercise intention in breast cancer survivors and to provide preliminary evidence supporting its application with prostate cancer survivors during treatment. METHODS: Participants consisted of 126 breast and 82 prostate cancer survivors receiving active treatment. Participants completed self administered, mail-in questionnaires that assessed demographic and medical variables, past exercise, and the theory of planned behavior. RESULTS: For breast cancer survivors, the results revealed that attitudes, subjective norm, and perceived behavioral control explained 66% of the variance in exercise intention with the instrumental component of attitude, subjective norm, and perceived behavioral control making significant unique contributions to intention. For prostate cancer survivors, attitudes, subjective norm, and perceived behavioral control explained 57% of the variance in exercise intention, with subjective norm and perceived behavioral control making significant unique contributions to intention. CONCLUSIONS: The results provide further support that the theory of planned behavior is a useful framework for understanding determinants of exercise intention in breast cancer survivors undergoing active treatment and preliminary support for prostate cancer survivors undergoing active treatment. When designing exercise interventions, differences in breast and prostate cancer survivors' exercise intention merit consideration. PMID- 16685076 TI - Cancer as part of the journey: the role of spirituality in the decision to decline conventional prostate cancer treatment and to use complementary and alternative medicine. AB - BACKGROUND: The role of spirituality in patients' use of complementary and alternative medicine (CAM) approaches to cancer management has hardly been explored. OBJECTIVE: To explore the role of spirituality in cancer management by men with prostate cancer who have declined conventional treatment and are using CAM. METHODS: This qualitative analysis is part of a longitudinal study to assess decision making by men with prostate cancer who decline conventional treatment and use CAM. In-depth interviews were conducted at study entry (n = 29). Themes were presented to participants in focus groups to further explore and validate the interview results. For a subset of participants (n = 10), spirituality emerged as an important theme; therefore, we conducted a secondary analysis of the interview data of these men to explore the role of spirituality in cancer management and decision making. RESULTS: Spirituality appeared to influence all aspects of the cancer experience. Most participants intensified their use of spiritual practice after a diagnosis of prostate cancer. These practices included spiritual ceremonies, indigenous healing, prayer, meditation, and use of spiritual imagery. Themes related to the role of spirituality in cancer management include beliefs about Western medicine, the role of spiritual beliefs in treatment decision making, the use of spiritual imager y and metaphor in healing, and the impact of cancer on spirituality. The discussion of these themes draws on quotes and case examples, illustrating how spirituality influenced study participants' response to diagnosis, treatment decision making, and cancer care. Two case examples provide a more in-depth understanding of how some participants incorporated spiritual imagery and metaphor into treatment decision making and cancer care. Ways in which cancer influenced spirituality are also discussed. Having prostate cancer appeared to influence their spirituality by strengthening their links with a spiritual community, increasing feelings of gratitude toward life, and improving personal relationships. RELEVANCE: These findings indicate that spiritual beliefs and practices may play an important role in the formation of treatment choices for some patients. Health care providers need to be aware of and address patient concerns about how conventional treatment may conflict with their spiritual beliefs and practices. Further research and medical education is needed on spirituality and prostate cancer. PMID- 16685077 TI - Toward a core nutraceutical program for cancer management. AB - As previously suggested, it may be feasible to impede tumorevoked angiogenesis with a nutraceutical program composed of glycine, fish oil, epigallocatechin-3 gallate, selenium, and silymarin, complemented by a low-fat vegan diet, exercise training, and, if feasible, a salicylate and the drug tetrathiomolybdate. It is now proposed that the scope of this program be expanded to address additional common needs of cancer patients: blocking the process of metastasis; boosting the cytotoxic capacity of innate immune defenses (natural killer [NK] cells); preventing cachexia, thromboembolism, and tumor-induced osteolysis; and maintaining optimal micronutrient status. Modified citrus pectin, a galectin-3 antagonist, has impressive antimetastatic potential. Mushroombeta-glucans and probiotic lactobacilli can amplify NK activity via stimulatory effects on macrophages. Selenium, beta-carotene, and glutamine can also increase the number and/or cytotoxic activity of NK cells. Cachectic loss of muscle mass can be opposed by fish oil, glutamine, and beta-hydroxy-beta-methylbutyrate. Fish oil, policosanol, and vitamin D may have potential for control of osteolysis. High dose aspirin or salicylates, by preventing NF-B activation, can be expected to aid prevention of metastasis and cachexia while down-regulating osteolysis, but their impacts on innate immune defenses will not be entirely favorable. A nutritional insurance formula crafted for the special needs of cancer patients can be included in this regimen. To minimize patient inconvenience, this complex core nutraceutical program could be configured as an oil product, a powder, and a capsule product, with the nutritional insurance formula provided in tablets. It would be of interest to test this program in nude mouse xenograft models. PMID- 16685078 TI - Thematic review series: patient-oriented research. Free fatty acid metabolism in human obesity. AB - Adipose tissue lipolysis provides circulating FFAs to meet the body's lipid fuel demands. FFA release is well regulated in normal-weight individuals; however, in upper-body obesity, excess lipolysis is commonly seen. This abnormality is considered a cause for at least some of the metabolic defects (dyslipidemia, insulin resistance) associated with upper-body obesity. "Normal" lipolysis is sex specific and largely determined by the individual's resting metabolic rate. Women have greater FFA release rates than men without higher FFA concentrations or greater fatty acid oxidation, indicating that they have greater nonoxidative FFA disposal, although the processes and tissues involved in this phenomenon are unknown. Therefore, women have the advantage of having greater FFA availability without exposing their tissues to higher and potentially harmful FFA concentrations. Upper-body fat is more lipolytically active than lower-body fat in both women and men. FFA released by the visceral fat depot contributes only a small percentage of systemic FFA delivery. Upper-body subcutaneous fat is the dominant contributor to circulating FFAs and the source of the excess FFA release in upper-body obesity. We believe that abnormalities in subcutaneous lipolysis could be more important than those in visceral lipolysis as a cause of peripheral insulin resistance. Understanding the regulation of FFA availability will help to discover new approaches to treat FFA-induced abnormalities in obesity. PMID- 16685079 TI - Thematic review series: patient-oriented research. What have we learned about HDL metabolism from kinetics studies in humans? AB - Plasma measurements of lipids, lipoproteins, and apolipoproteins provide information on the static levels of these fractions without providing key information on the dynamic fluxes of lipoproteins in the circulation. Kinetics studies, in contrast, provide additional information on the production and clearance rates of lipoproteins and the flow of lipids and apolipoproteins through lipoprotein fractions. This information is crucial in accurately delineating the metabolism of HDL in plasma, because plasma concentrations of HDL are the net result of the de novo production and catabolism of HDL as well as the recycling of HDL particles and the contribution to HDL from components of other lipoproteins. Studies aimed at measuring the metabolism of HDL particles have shown that HDL metabolism in vivo is complex and consists of multiple components. Kinetics studies provide a window into the metabolism of HDL, allowing us to better understand the mechanisms of HDL decrease in human conditions and the functionality of HDL particles. Here, we review the progress in our understanding of HDL metabolism derived from in vivo kinetics studies, focusing primarily on studies in humans but also reviewing key studies in animal models. PMID- 16685080 TI - Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in a synergistic manner in neonatal rats. AB - Vitamin A (VA) is stored in tissues predominantly as retinyl esters (REs), which provide substrate for the production of bioactive retinoids. Retinoic acid (RA), a principal metabolite, has been shown to induce postnatal lung development. To better understand lung RE storage, we compared VA (given as retinyl palmitate), RA, and a nutrient-metabolite combination, VARA, given orally on postnatal days 5 7, for their ability to increase lung RE in neonatal rats. VARA increased lung RE significantly [ approximately 14, 2.4, 2.1, and <1 nmol/g for VARA, VA, RA, and control (C), respectively; P < 0.001]; the increase by VARA was more than additive compared with the effects of VA and RA alone. Lung histology and morphometry were unchanged. In a 6 h metabolic study, providing [(3)H]retinol with VARA, compared with VA or C, increased the uptake of newly absorbed (3)H by 3-fold, indicating that VARA stimulated the uptake of [(3)H]retinol and its retention as [(3)H]RE in neonatal lungs. After cessation of VARA, lung RE remained increased for 9 d afterward, through the period of alveolar development. In conclusion, VARA, a 10:1 nutrient-metabolite combination, increased lung RE significantly compared with VA alone and could be a promising therapeutic option for enhancing the delivery of VA to the lungs. PMID- 16685081 TI - QTL mapping for genetic determinants of lipoprotein cholesterol levels in combined crosses of inbred mouse strains. AB - To identify additional loci that influence lipoprotein cholesterol levels, we performed quantitative trait locus (QTL) mapping in offspring of PERA/EiJxI/LnJ and PERA/EiJxDBA/2J intercrosses and in a combined data set from both crosses after 8 weeks of consumption of a high fat-diet. Most QTLs identified were concordant with homologous chromosomal regions that were associated with lipoprotein levels in human studies. We detected significant new loci for HDL cholesterol levels on chromosome (Chr) 5 (Hdlq34) and for non-HDL cholesterol levels on Chrs 15 (Nhdlq9) and 16 (Nhdlq10). In addition, the analysis of combined data sets identified a QTL for HDL cholesterol on Chr 17 that was shared between both crosses; lower HDL cholesterol levels were conferred by strain PERA. This QTL colocalized with a shared QTL for cholesterol gallstone formation detected in the same crosses. Haplotype analysis narrowed this QTL, and sequencing of the candidate genes Abcg5 and Abcg8 confirmed shared alleles in strains I/LnJ and DBA/2J that differed from the alleles in strain PERA/EiJ. In conclusion, our analysis furthers the knowledge of genetic determinants of lipoprotein cholesterol levels in inbred mice and substantiates the hypothesis that polymorphisms of Abcg5/Abcg8 contribute to individual variation in both plasma HDL cholesterol levels and susceptibility to cholesterol gallstone formation. PMID- 16685082 TI - Metabolism of apoB lipoproteins of intestinal and hepatic origin during constant feeding of small amounts of fat. AB - We aimed to identify mechanisms by which apolipoprotein B-48 (apoB-48) could have an atherogenic role by simultaneously studying the metabolism of postprandial apoB-48 and apoB-100 lipoproteins. The kinetics of apoB-48 and apoB-100, each in four density subfractions of VLDL and intermediate density lipoprotein (IDL), were studied by stable isotope labeling in a constantly fed state with half hourly administration of almond oil in five postmenopausal women. A non-steady state, multicompartmental model was used. Despite a much lower production rate, VLDL and IDL apoB-48 shared a similar secretion pattern with apoB-100: both were directly secreted into all fractions with similar percentage mass distributions. Fractional catabolic rates (FCRs) of apoB-48 and apoB-100 were similar in VLDL and IDL. We identified a fast turnover compartment of light VLDL that had a residence time of <30 min for apoB-48 and apoB-100. Finally, a high secretion rate of apoB-48 was associated with a slow FCR of VLDL and IDL apoB-100. In conclusion, the intestine secretes a spectrum of apoB lipoproteins, similar to what the liver secretes, albeit with a much lower secretion rate. Once in plasma, intestinal and hepatic triglyceride-rich lipoproteins have similar rates of clearance and participate interactively in similar metabolic pathways, with high apoB-48 production inhibiting the clearance of apoB-100. PMID- 16685083 TI - Risk factors for injury in indoor compared with outdoor adolescent soccer. AB - BACKGROUND: There are limited data on the epidemiology of indoor soccer injuries. PURPOSE: Injury rates and risk factors for injury in adolescent indoor and outdoor soccer in the same cohort of players will be identified and compared. STUDY DESIGN: Cohort study (prevalence); Level of evidence, 2. METHODS: The study population was a random sample of 21 adolescent (ages 13-17 years) outdoor soccer teams (N = 317). The subcohort included players continuing to play in the indoor soccer season (n = 142). The injury definition included any injury occurring in soccer that resulted in medical attention, the inability to complete a session, and/or missing a subsequent session. RESULTS: The overall injury rate found in indoor soccer over 20 weeks was 4.45 injuries per 1000 player hours (95% confidence interval, 3.1-6.19). The overall injury rate found in the 13-week outdoor soccer season among a similar cohort was 5.59 injuries per 1000 player hours (95% confidence interval, 4.42-6.97). The relative risk of injury suggests that there was no significant difference between injury rates by age group or gender in indoor soccer compared with outdoor soccer. The risk of injury in the most elite division of play was greater in outdoor compared with indoor soccer (relative risk, 3.22; 95% confidence interval, 1.8-6.12). The most commonly injured body part in both indoor and outdoor soccer was the ankle, followed by the groin in indoor and the knee in outdoor soccer. CONCLUSION: There were no significant differences in overall injury rates found by gender or age group for indoor compared with outdoor soccer. Future research should focus on injury prevention strategies to reduce lower extremity injury in indoor and outdoor adolescent soccer. PMID- 16685084 TI - Central cord syndrome in patients with Klippel-Feil syndrome resulting from winter sports: report of 3 cases. PMID- 16685085 TI - Lateral epicondylitis: in vivo assessment of arthroscopic debridement and correlation with patient outcomes. AB - BACKGROUND: Surgical management of lateral epicondylitis has traditionally consisted of an open incision with debridement of the affected extensor tendon tissue. More recently, arthroscopy has been reported as a surgical option for this condition. PURPOSE: To evaluate the effectiveness of arthroscopic debridement in excising the characteristic tendinopathy of chronic lateral epicondylitis and determine if residual tendinopathy correlated with poorer patient outcomes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Data were prospectively collected on all patients who underwent arthroscopic debridement of chronic lateral epicondylitis during a 2-year period. The arthroscopic debridement was assessed in regard to its effectiveness in excising the characteristic tendinopathy through a traditional open procedure by gross and histologic analysis. Outcomes data were collected on all patients preoperatively and at a minimum of 1 year postoperatively. Patient outcomes were then correlated with the residual histologic tendinopathy after arthroscopic debridement. RESULTS: Eighteen patients were enrolled in the investigation. Gross evidence of residual tendinopathy was identified in 6 patients, with all 6 cases occurring during the first year of the study. Of the 18 patients, 10 had residual histologic evidence of tendinopathy after arthroscopic debridement. Poorer outcomes were identified in those patients who had residual histologic tendinopathy on their rating of worst level of pain (P = .03). CONCLUSION: Residual microscopic tendinopathy is often present after arthroscopic debridement. Clinically, residual microscopic tendinopathy correlated with poorer surgical outcomes in regard to patient's rating of their worst level of pain. PMID- 16685086 TI - Revision anterior cruciate ligament reconstruction with hamstring tendon autograft: 5- to 9-year follow-up. AB - BACKGROUND: The results of revision anterior cruciate ligament reconstruction are limited in the current literature, and no studies have previously documented the outcome of revision anterior cruciate ligament reconstruction using solely hamstring tendon grafts. HYPOTHESIS: Revision anterior cruciate ligament reconstruction with 4-strand hamstring tendon graft affords acceptable results and is comparable to reported outcomes with the bone-patellar tendon-bone graft. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Fifty-seven consecutive revision anterior cruciate ligament reconstructions with the hamstring tendon graft and interference screw fixation were assessed a mean time of 89 months (range, 60-109 months) after surgery. Assessment included the International Knee Documentation Committee knee ligament evaluation, instrumented laxity testing, and radiologic examination. RESULTS: Of the 50 knees reviewed, 5 (10%) had objective failure of the revision anterior cruciate ligament reconstruction. Of the 45 patients with functional grafts, knee function was normal or nearly normal in 33 patients (73%). An overall grade of normal or nearly normal was found in 56% of patients. The mean side-to-side difference on manual maximum testing was 2.5 mm (range, -1 to 4 mm). Degenerative changes on radiographs were identified in 23% of patients at the time of surgery, increasing to 56% of patients at review. The status of the articular cartilage at the time of revision surgery was the most significant contributor to successful outcome. CONCLUSION: Revision anterior cruciate ligament reconstruction with hamstring tendon graft and interference screw fixation affords acceptable results at a minimum of 5 years' follow-up. Good objective results can be obtained, but subjectively, the results appear inferior to those of primary anterior cruciate ligament reconstruction in the literature, which may be related to the high incidence of articular surface damage in this patient population. We recommend that, when available, hamstring tendon autografts should be considered for revision anterior cruciate ligament reconstruction. PMID- 16685087 TI - Biodegradable and metallic interference screws in anterior cruciate ligament reconstruction surgery using hamstring tendon grafts: prospective randomized study of radiographic results and clinical outcome. AB - BACKGROUND: The use of biodegradable screws in anterior cruciate ligament reconstruction has grown in popularity. PURPOSE: To compare the clinical and radiographic results in arthroscopically assisted anterior cruciate ligament reconstruction using 4-strand hamstring tendon grafts and either metallic (metal group) or biodegradable (PLLA group) interference screw fixation. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: A randomized series of 77 patients, all with a unilateral anterior cruciate ligament rupture, was included in the study. The preoperative assessments in both groups were similar in gender, Tegner activity level, Lysholm score, KT-1000 arthrometer measurements, and single-legged hop test results. In both groups, interference screw fixation of the graft was used at both ends, and 68 of 77 (88%) patients returned for a radiographic examination at 6 and 24 months, respectively. RESULTS: At follow-up, no significant differences were found between the 2 groups in KT-1000 arthrometer laxity measurements, Tegner activity level, or Lysholm score. The PLLA group had a significantly better outcome in the single-legged hop test and the final International Knee Documentation Committee classification (P = .007 and P = .03, respectively). At 6 and 24 months after the index operation, the PLLA group displayed significantly larger drill holes on the radiographs than did the metal group on both the tibial (8.1 vs 6.6 mm at 6 months [P = .0007]; 6.0 vs 3.2 mm at 24 months [P < .0001]) and femoral sides (7.8 vs 5.6 mm at 6 months [P < .0001]; 6.3 vs 1.9 mm at 24 months [P < .0001]). CONCLUSION: There were significantly larger radiographically visible drill holes on both the tibial and femoral sides in the PLLA group compared with the metal group at 6 and 24 months. Clinical examination at 2 years revealed no major differences between the groups. The larger drill holes in the PLLA group did not correlate with inferior clinical results. PMID- 16685088 TI - Biomechanical and anatomical effects of an external rotational torque applied to the knee: a cadaveric study. AB - BACKGROUND: External rotational torque is one of the mechanisms that may occur during a pivoting or twisting injury to the knee. HYPOTHESIS: Simulated external rotational injury by applying external rotational torque will increase knee laxity and cause soft tissue damage to the knee. STUDY DESIGN: Controlled laboratory study. METHODS: Six cadaveric knees and a custom testing system were used to produce external rotational torque of 30 degrees , 45 degrees , and 60 degrees with the knee at 30 degrees of flexion. Anterior-posterior, valgus-varus, and rotational knee laxity were quantified. After sequential rotational torque to 60 degrees , the specimens were dissected to identify injured structures. RESULTS: External rotational torque of 45 degrees and 60 degrees significantly increased knee joint laxity in all directions (P < .05). Dissection showed that all posterior cruciate ligaments were intact; all medial collateral and anterior cruciate ligaments revealed either partial or complete tears. The lateral collateral ligaments were torn in all specimens. The popliteus tendon was attenuated in 1 specimen and was completely torn in 1 specimen. The popliteofibular ligament was torn in 3 specimens. CONCLUSION: External rotational torque to 60 degrees increased knee laxity, and dissection revealed a consistent pattern of injury to the medial and lateral collateral and anterior cruciate ligaments and posterolateral corner. CLINICAL RELEVANCE: Because isolated ligament injuries are rare, recognizing these combined ligamentous injuries early is essential for repair in the acute stage. PMID- 16685089 TI - Meniscal tear characteristics in young athletes with a stable knee: arthroscopic evaluation. AB - BACKGROUND: There has been great interest in the literature regarding meniscal tears in unstable knees, but there is not as much information available on stable knees. PURPOSE: To report the characteristics of isolated meniscal tears (type and location) in athletes with intact cruciate ligaments. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Arthroscopic surgery was performed on 314 (83.1%) knees in the acute phase ( < 6 weeks) of injury and on 64 (16.9%) knees more than 6 weeks after injury for a total of 364 athletes (378 knees). Cooper's classification was used to classify the meniscal tears according to the type and location. RESULTS: Overall, 262 of 378 tears (69.3%) were located in the medial meniscus and 116 (30.7%) in the lateral meniscus. Vertical tears (77.5%) were significantly more frequent than were horizontal tears (22.5%; chi(2) test, P < .001). A total of 23.2% of tears involved the peripheral zones (zone 0 or 1), and tears that extended into the posterior horn accounted for 75.7%. Regarding the tear shape between male and female athletes, on both sides there were no statistically significant differences in the percentage of horizontal, bucket handle, longitudinal, or radial tears. CONCLUSION: The characteristics of isolated meniscal tears differ with regard to the sport, sex, and tear location and type from those seen in unstable knees. This knowledge is useful in knee injury management. PMID- 16685090 TI - The incidence and cross-sectional area of the meniscofemoral ligament. AB - BACKGROUND: The incidence, morphologic characteristics, and cross-sectional area of meniscofemoral ligaments of the knee are still not clarified. HYPOTHESIS: The incidence of meniscofemoral ligaments is very high, and they contribute significantly to the cross-sectional area of the posterior cruciate ligament complex. STUDY DESIGN: Descriptive laboratory study. METHODS: Clinical study: During arthroscopic anterior cruciate ligament reconstruction, the presence of a meniscofemoral ligament was confirmed in 38 patients (16 men, 22 women; mean age, 23.6 years) by observation and probing. Laboratory study: Posterior cruciate ligaments with the lateral meniscus attached were obtained as specimens from 30 patients (3 men, 27 women; mean age, 71.9 years) during total knee arthroplasty. The posterior cruciate ligament and meniscofemoral ligaments were observed, and the cross-sectional area was measured using an area micrometer. RESULTS: Clinical study: The anterior meniscofemoral ligament was found in 36.8% of patients, the posterior meniscofemoral ligament was found in 71.1%, and both ligaments were found in 26.7%. The overall incidence of at least 1 meniscofemoral ligament was 84.2%. Laboratory study: The anterior meniscofemoral ligament was present in 5 (16.7%) cases, and the posterior meniscofemoral ligament was present in all cases (100.0%). The cross-sectional area of the posterior cruciate ligament proper was 50.1 +/- 16.9 mm(2), that of the anterior meniscofemoral ligament was 2.3 +/- 1.2 mm(2), and that of the posterior meniscofemoral ligament was 7.5 +/- 2.5 mm(2). The mean ratio of the cross-sectional area of meniscofemoral ligaments to the posterior cruciate ligament proper was 17.2% (4.0%-38.9%). The origin of the posterior meniscofemoral ligament from the posterior horn of the lateral meniscus could be classified into 5 types. CONCLUSION: The meniscofemoral ligaments contributed significantly to the cross-sectional area of the posterior cruciate ligament complex. PMID- 16685091 TI - Thermal capsulorrhaphy for the treatment of shoulder instability. AB - Thermal capsulorrhaphy has been used to treat many different types of shoulder instability, including multidirectional instability, unidirectional instability, and microinstability in overhead-throwing athletes. A device that delivers laser energy or radiofrequency energy to the capsule tissue causes the collagen to denature and the capsule to shrink. The optimal temperature to achieve the most shrinkage without causing necrosis of the tissue is between 65 degrees and 75 degrees centigrade. This treatment causes a significant decrease in mechanical stiffness for the first 2 weeks, and then, after the tissue undergoes active cellular repair from the surrounding uninjured tissue, the mechanical properties return to near normal by 12 weeks. If the thermal energy is applied in a grid pattern, then the tissue heals with more stiffness by 6 weeks. Clinical studies on thermal capsulorrhaphy for the treatment of multidirectional instability have shown a high rate of recurrent instability (12%-64%). The clinical studies on unidirectional instability showed much better recurrence rates (4%-25%), but because most of the patients also underwent concomitant Bankart repairs and superior labral anterior posterior lesion repairs, the efficacy of the thermal treatment cannot be ascertained. A randomized controlled trial would be needed to assess whether instability with Bankart lesions requires augmentation with thermal capsulorrhaphy. For the patients with microinstability who are overhead throwing athletes, thermal capsulorrhaphy has shown varying results from a 97% rate of return to sports to a 62% rate of return to sports. Complications of this technique include temporary nerve injuries that usually involve the sensory branch of the axillary nerve and thermal necrosis of the capsule, which is rare. PMID- 16685092 TI - An analysis of the causes of failure in 57 consecutive posterolateral operative procedures. AB - PURPOSE: To investigate 57 failed posterolateral procedures in 30 consecutive knees to determine factors that may have contributed to the failure. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Thirty patient records were reviewed by an independent surgeon. The index posterolateral operations were done for 13 acute and 17 chronic knee injuries. The anterior cruciate ligament was ruptured in 17 knees, the posterior cruciate ligament was torn in 5 knees, and both cruciates were ruptured in 8 knees. In 25 knees, 46 revision posterolateral procedures had been performed, of which 27 had also failed (in 21 knees). Five knees did not undergo revision of the posterolateral structures. RESULTS: In 22 knees, multiple factors were identified that most likely contributed to the failure of the posterolateral procedures. The most common factors were nonanatomical graft reconstruction (23 knees), untreated varus malalignment (10 knees), and failure to successfully reconstruct all ruptured knee ligaments, including cruciates (27 knees). Thirty-nine anterior cruciate ligament procedures were done in 24 knees, including 24 primary and 15 revision operations. Seventeen posterior cruciate ligament procedures were done in 13 knees, including 13 primary and 4 revision operations. At the time of writing, 16 of 24 knees had a functional anterior cruciate ligament graft and 5 of 13 had a functional posterior cruciate ligament graft. CONCLUSION: The results suggest greater emphasis during the index operation for anatomical graft reconstruction of one or more of the posterolateral structures as necessary, restoration of all ruptured cruciate ligaments, and correction of varus malalignment. PMID- 16685093 TI - Mechanical and biochemical effect of monopolar radiofrequency energy on human articular cartilage: an in vitro study. AB - BACKGROUND: There are growing concerns about thermal chondroplasty using radiofrequency energy to treat partial-thickness cartilage defects. However, most studies emphasize effects on chondrocyte viability, and other factors such as mechanical properties are less studied. HYPOTHESIS: Radiofrequency energy may cause significant effects on articular cartilage other than chondrocyte viability. STUDY DESIGN: Controlled laboratory study. METHODS: Human osteoarthritic cartilage samples were obtained from total knee arthroplasty, and monopolar radiofrequency energy was applied using commercially available equipment. Material properties (compressive stiffness, surface roughness, and thickness) just before and after thermal treatment were determined using ultrasound. A series of biochemical analyses were also performed after explant culture of the samples. RESULTS: The cartilage surface became smoother by radiofrequency energy, whereas cartilage stiffness or thickness was not altered significantly. Collagen fibrils, especially in the superficial layers, were converted to denatured form, whereas proteoglycan contents released in the media as well as retained in the tissue remained unchanged. The concentrations of matrix metalloproteinases (MMP-1 and MMP-2) were reduced remarkably. CONCLUSION: Radiofrequency energy is able to create a smooth cartilage surface and reduce catabolic enzymes at the cost of collagen denaturation and chondrocyte death in the superficial layers. The stiffness of the cartilage is not changed at time zero. CLINICAL RELEVANCE: Further animal as well as clinical studies will be necessary to fully evaluate the long-term effects of radiofrequency energy. PMID- 16685094 TI - The effect of cyclic loading on rotated bone-tendon-bone anterior cruciate ligament graft constructs. AB - BACKGROUND: Single-incision anterior cruciate ligament reconstruction with a bone patellar tendon-bone construct is commonly performed with 180 degrees rotation of the graft. It has been hypothesized that further rotation of the graft to 540 degrees can effectively shorten the graft to address graft length-tunnel mismatch. Initial biomechanical failure characteristics of rotated constructs have been reported, but cyclic loading of tendons has not been performed. HYPOTHESIS: Graft rotation affects the biomechanical properties of the construct. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-five bone-patellar tendon-bone composite porcine right knee specimens were randomized into 3 groups and were externally rotated to 0 degrees , 180 degrees , or 540 degrees . Each group was then cyclically loaded in an artificial synovial fluid medium between 50 and 250 N for 5,000 cycles, loaded between 50 and 500 N for an additional 5,000 cycles, and finally subjected to load-to-failure testing. RESULTS: Graft rotation shortened constructs by 1.7 +/- 0.8 mm at 180 degrees of rotation and 7.6 +/- 2.0 mm at 540 degrees of rotation (P < .01). There was a statistically significant increase in strain during cyclic loading at 540 degrees . No significant differences in maximum load, yield stress, yield strain, or modulus of elasticity were detected in single-cycle load-to-failure testing after cyclic loading. CONCLUSION: Rotation of bone-patellar tendon-bone constructs to 540 degrees predictably shortens the effective graft length at the expense of increased strain with cyclic loading at stresses equivalent to walking and running. CLINICAL RELEVANCE: Although rotation to 540 degrees potentially addresses graft length-tunnel mismatch, further clinical evaluation is required to evaluate the impact of increased strain on knee laxity and to determine the effects of physiologic loading of rotated bone-patellar tendon-bone constructs in vivo. PMID- 16685095 TI - Path of glenohumeral articulation throughout the rotational range of motion in a thrower's shoulder model. AB - BACKGROUND: Overhead-throwing athletes have increased external and diminished internal glenohumeral rotation that may alter glenohumeral kinematics. PURPOSE: To quantify the kinematic changes present in a cadaveric model of a thrower's shoulder. STUDY DESIGN: Controlled laboratory study. METHODS: In 8 fresh-frozen cadaveric shoulders, the rotator cuff and overlying muscles were removed, and the glenohumeral capsule, coracoacromial ligament, and coracohumeral ligament were left intact. The scapula was fixed, and the humerus was placed in 90 degrees of shoulder abduction in a 6 degrees of freedom testing device. A compressive force of 44 N was applied. A thrower's shoulder model was created, and sequential conditions were examined: intact, after anterior stretching, and after the addition of posterior-inferior capsular plication. Kinematic measurements were obtained through a complete range of glenohumeral rotation. RESULTS: Glenohumeral external rotation increased 16%, from 149 degrees to 173 degrees (P < .001), after stretching in external rotation and remained increased by 11% to 166 degrees (P < .001) after posterior-inferior capsular plication. With the addition of the posterior-inferior capsular plication, internal rotation averaged 7 degrees , which was not significantly different from the intact state (11 degrees, P = .55) or the stretched state (16 degrees, P = .07). The total glenohumeral rotation after stretching followed by posterior-inferior capsular plication did not differ significantly from intact state (P = .25). At maximum external rotation, the humeral head apex was shifted posteriorly in the stretched (P = .003) and plicated (P < .001) states compared with the intact state. The humeral head apex was posteriorly displaced at 135 degrees and 150 degrees of external rotation compared with the intact condition (P = .039 and .049, respectively). In maximum internal rotation, anterior stretching had no significant effect on the humeral head apex position. However, after posterior inferior capsular plication, the humeral head apex was significantly shifted inferiorly (P = .005) and anteriorly (P = .03) in maximum internal rotation compared with the intact state. CONCLUSION: Significant changes in glenohumeral motion occur in this model during the simulated late-cocking and follow-through phases of throwing. In this model, posterior capsular tightness alters the humeral head position most profoundly during the deceleration and follow-through phases of throwing. PMID- 16685096 TI - The use of suture anchors in repair of the ruptured patellar tendon: a biomechanical study. AB - BACKGROUND: Rupture of the patellar tendon is a disabling injury that usually requires surgical treatment. The standard method of repair involves placing suture loops through transpatellar tunnels. The use of suture anchors in patellar tendon repair has not been previously described. HYPOTHESIS: No difference exists in the amount of gap formation during cyclic loading or in ultimate load-to failure strength between repairs performed with anchors and those performed with 2 types of transpatellar sutures. STUDY DESIGN: Controlled laboratory study. METHODS: Six matched pairs of cadaveric knees were tested in a custom biomechanical apparatus based on an established model. Repairs were performed using either suture anchors with No. 2 FiberWire or transpatellar suture tunnels using 2 different types of suture-No. 5 Ethibond and No. 2 FiberWire. Gap formation across the repair site during 250 cycles of extension as well as ramp up load to failure were measured for each repair. RESULTS: The mean total gap formation across the repair site at 250 cycles was 4.1 +/- 1.9 mm for the suture anchor group, 6.7 +/- 1.8 mm for the FiberWire tunnel group, and 8.5 +/- 2.7 mm for the Ethibond tunnel group. Mean load to failure was 779 +/- 183 N, 730 +/- 83 N, and 763 +/- 231 N, respectively. CONCLUSION: Significantly less gap formation throughout 250 cycles (P = .009) and no difference in load to failure occurred with patellar tendon repairs performed with suture anchors as compared with repairs performed with transpatellar tunnels. CLINICAL RELEVANCE: The newly described method, using suture anchors for repair of patellar tendon ruptures, may be clinically equal or superior to the established method of using transpatellar tunnels. PMID- 16685097 TI - Technical errors during medial patellofemoral ligament reconstruction could overload medial patellofemoral cartilage: a computational analysis. AB - BACKGROUND: The influence of reconstruction of the medial patellofemoral ligament on the patellofemoral force and pressure distributions has not yet been investigated. HYPOTHESIS: Technical errors can cause tension to develop within a reconstructed medial patellofemoral ligament, which will adversely alter the normal patellofemoral force distribution by increasing the load applied to the medial cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: Four computational knee models were used to simulate knee function from 30 degrees to 90 degrees of flexion with (1) an intact medial patellofemoral ligament, (2) an anatomically correct reconstruction using a double hamstring tendon autograft, (3) a 5-mm proximally malpositioned femoral attachment site, (4) a graft that is 3 mm shorter than the intact medial patellofemoral ligament, and (5) combined proximal malpositioning and a short graft. RESULTS: The results were similar for the intact and anatomically reconstructed medial patellofemoral ligament. Proximal malpositioning of the femoral attachment and using a short graft increased the graft tension during flexion, which decreased the lateral force and the lateral tilt moment acting on the patella. When a short graft was combined with proximal malpositioning, the compressive force applied to the medial cartilage at least doubled at low flexion angles, which increased the peak medial pressure by more than 50% at low flexion angles. CONCLUSION: When the medial patellofemoral ligament is reconstructed, small errors in graft length and position can dramatically increase the force and pressure applied to medial patellofemoral cartilage. CLINICAL RELEVANCE: Overloading the medial cartilage after medial patellofemoral ligament reconstruction could lead to degradation, pain, and arthrosis. PMID- 16685098 TI - New WHO influenza vaccine recommendations for 2005-2006 season, northern hemisphere. PMID- 16685099 TI - Influenza A outbreak in a community hospital in south east Wales, February 2005. PMID- 16685100 TI - Rates of new HIV diagnoses in Switzerland remain high but no longer rising. PMID- 16685101 TI - BSE agent in goat tissue: first known naturally occurring case confirmed. PMID- 16685102 TI - First case of LGV confirmed in Barcelona. PMID- 16685103 TI - Strep-EURO: progress in analysis and research into severe streptococcal disease in Europe, 2003-2004. PMID- 16685104 TI - Sexually transmitted infections on the rise in Northern Ireland. AB - he profile of STIs has been raised in Northern Ireland in recent months due to heightened media and political interest. PMID- 16685106 TI - How we predict the etiology of acute pancreatitis. AB - Although acute pancreatitis (AP) may be the consequence of numerous etiologic factors, more than 80% of the episodes are of biliary or alcoholic origin. Although the clinical picture is similar irrespective of the etiology, patients with severe episodes of acute pancreatitis require specific therapeutic maneuvers when biliary-induced while, in the case of alcoholic origin, they only need general support. As a consequence, the early estimation of the etiology is of particular interest, and older age and female sex are frequent characteristics of AP of biliary origin. Together with these factors, abnormal liver function tests have been classically used to identify biliary etiology. Their intrinsic value has grown in the era of new imaging techniques. From another perspective, the plasmatic level of carbohydrate-deficient transferrin seems to be the most accurate technique in differentiating cases of alcohol-induced acute pancreatitis from other etiologies. PMID- 16685107 TI - Primary pancreatic lymphomas. AB - Primary pancreatic lymphomas are extremely rare. Clinically, primary pancreatic lymphomas usually present with symptoms of carcinoma of the pancreatic head. Patients with primary pancreatic lymphomas are between 35 and 75 years of age and with a strong male predominance. Common clinical manifestations include abdominal pain, jaundice, acute pancreatitis, small bowel obstruction, and diarrhea. An accurate cytopathologic diagnosis by fine-needle aspiration (FNA) is imperative because the primary treatment is non-surgical. Cytomorphologic features include hypercellularity with discohesive cells with round nuclei, often prominent nucleoli, mitoses, and karyorrhexis. Flow cytometry analysis demonstrates a monoclonal pattern of immunoglobulin light chain expression. FNA coupled with flow cytometry analysis appears to be highly accurate in the diagnosis of primary pancreatic lymphomas. Fluorescence in-situ hybridisation technique has been established its role in the diagnosis of lymphoid malignancies, including primary pancreatic lymphomas. LDH and beta-2 microglobulin are important diagnostic and prognostic tumor markers. The differential diagnoses of primary pancreatic lymphomas include secondary lymphoma, pancreatic endocrine neoplasm, and florid chronic pancreatitis. The role of surgery is limited to the rare occasions when initial FNA and flow cytometry analysis are non-diagnostic. Treatment usually consists of a combination of chemotherapy and radiation therapy, or stem cell transplantation. Primary pancreatic lymphomas has a much better prognosis than adenocarcinoma of the pancreas. PMID- 16685108 TI - Characterization of immunoreactive trypsinogen activation peptide in urine in acute pancreatitis. AB - CONTEXT: All work on human trypsinogen activation peptide (TAP) in acute pancreatitis has been carried out with the same assay. Despite the extensive use of this original TAP assay, there is no characterization of the TAP-like immunoreactivity measured. OBJECTIVE: The aims of this study were to develop an additional TAP assay and to attempt to characterize the TAP-like immunoreactivity found in the urine of patients with acute pancreatitis. METHODS: Antibodies against the human TAP were prepared using the whole octapeptide APFD4K, conjugated at its N-terminal end. Characterization of the immunoreactivity measured with these assays was performed using gel filtration of human pancreatic juice before and after activation of trypsinogen with enterokinase. RESULTS: After activation of the pancreatic juice, there was a large initial increase in immunoreactive TAP and a decrease 6-24 hours later. Using our antiserum, we found low levels of immunoreactive TAP in urine from patients with acute pancreatitis, although many of these samples contained high levels of immunoreactive TAP when tested with the commercially available TAP kit (Biotrin). The pentapeptide D4K, used as a standard in the Biotrin kit, showed much lower immunoreactivity than the synthetic octapeptide APFD4K in our assay. The octapeptide, however, reacted similarly to D4K in the Biotrin kit assay. CONCLUSION: n Our antibody prepared against the synthetic octapeptide APFD4K is directed against the N-terminal part of the octapeptide and does not recognize the pentapeptide D4K. Immunoreactive TAP in urine in acute pancreatitis is mainly composed of the C-terminal pentapeptide, D4K. PMID- 16685109 TI - HER2/neu expression and gene alterations in pancreatic ductal adenocarcinoma: a comparative immunohistochemistry and chromogenic in situ hybridization study based on tissue microarrays and computerized image analysis. AB - CONTEXT: HER2/neu overexpression is observed in many cancers including pancreatic ductal adenocarcinoma. Although immunohistochemistry remains the basic method for evaluating HER2/neu protein expression, significant information regarding gene status cannot be assessed. DESIGN: Using tissue microarray technology, fifty histologically confirmed pancreatic ductal adenocarcinomas were cored twice and re-embedded in one paraffin block. Immunohistochemistry (clone TAB 250) and chromogenic (HER2/neu amplification Spot Light kit) in situ hybridization protocols were performed. The immunostained slides were evaluated by conventional eye microscopy and digital image analysis. The chi square test and the kappa statistic were applied by running the SPSS package. MAIN OUTCOME MEASURES: The levels of staining intensity were estimated by the performance of a semi automated image analysis system. RESULTS: HER2/neu gene amplification was detected in 8/50 cases (16%). Chromosome 17 aneuploidy was detected in 19 cases (38%). Significant improvement in interobserver agreement (kappa=0.76 vs. 0.94) was achieved correlating the immunohistochemical results obtained by conventional eye and digital microscopy, especially in the cases of overexpression (2+, 3+). Finally, 29 (58%), 11 (22%), 6 (12%) and 4 (8%) cases were characterized as 0, 1+, 2+ and 3+, respectively. HER2/neu protein expression was significantly associated with grade (P=0.019), but not with stage (P=0.466). in addition, chromosome 17 and gene status were not correlated with stage and grade. CONCLUSION: Our results indicate that a subset of pancreatic ductal adenocarcinomas is characterized by HER2/neu gene amplification. In contrast to breast cancer, protein overexpression does not predict this specific gene deregulation mechanism. This event may reflect the different biological role of the molecule in those two solid tumours, affecting the response to novel targeted agents, such as monoclonal anti-HER2/neu antibodies. Furthermore, evaluation of HER2/neu protein expression based on digital image analysis and not only on conventional eye microscopy improves the accuracy and reliability of immunohistochemical estimation, although that does not demonstrate clinical significance and prognostic value in pancreatic ductal adenocarcinoma. PMID- 16685110 TI - Functional variation in VEGF is not associated with type 2 diabetes in a United Kingdom Caucasian population. AB - CONTEXT: Vascular endothelial growth factor (VEGF) is important for pancreatic beta cell development and function. Common variation in the VEGF gene is associated with altered serum concentrations of VEGF and with several diseases, but its role in type 2 diabetes is not known. The single nucleotide polymorphisms C-2578A (rs699947), G-1154A (rs1570360) and G-634C (rs2010963) in the 5'-region of VEGF are associated with altered serum concentrations of the protein. OBJECTIVE: We performed a large case-control and family-based study to test the hypothesis that these variants are associated with type 2 diabetes in a UK Caucasian population. PARTICIPANTS: We genotyped 1,969 cases, 1,625 controls and 530 families for the three single nucleotide polymorphisms. MAIN OUTCOME MEASURES: Allele and genotype frequencies were compared between cases and controls. Family-based analysis was used to test for over- or under-transmission of alleles to affected offspring. RESULTS: Despite good power (80%) to detect odds ratios of approximately 1.2, there were no significant associations between single alleles or genotypes and type 2 diabetes. Odds ratios (and 95% confidence intervals) comparing case and control allele frequencies for single nucleotide polymorphisms C-2578A, G-1154A and G-634C were 0.97 (0.88-1.07), 0.99 (0.90-1.09) and 0.97 (0.88-1.08), respectively. CONCLUSION: This is the first large-scale study to examine the association between common functional variation in VEGF and type 2 diabetes risk. We have found no evidence that these three single nucleotide polymorphisms, shown previously to alter VEGF concentrations, are risk factors for type 2 diabetes in a large UK Caucasian case-control and family-based study. PMID- 16685111 TI - Elevated lipase and diabetic ketoacidosis associated with aripiprazole. AB - CONTEXT: Atypical antipsychotic agents are associated with diabetes mellitus and pancreatitis. Aripiprazole, a new antipsychotic, has never been implicated to cause either diabetes mellitus or pancreatitis. We present a patient who developed diabetes mellitus after being started on aripiprazole. CASE REPORT: A 33 year-old male with schizophrenia presented with fatigue, dyspepsia and epigastric pain. Patient was found to have hyperglycemia, diabetic ketoacidosis, and hyperlipasemia. Imaging studies of the pancreas were normal. Patient was started on aripiprazole treatment 18 months prior to this episode and had experienced progressive weight gain since then. Work up for other causes of pancreatitis was negative. CONCLUSIONS: Diabetes mellitus in this patient was probably a complication of aripiprazole due to progressive weight gain. In the absence of radiologic evidence of pancreatitis, hyperlipasemia was probably secondary to diabetic ketoacidosis. Possible causes of hyperlipasemia and its significance in diabetic ketoacidosis are discussed. PMID- 16685112 TI - Gemcitabine-induced rectus abdominus radiation recall. AB - CONTEXT: Radiation recall has been described in the context of gemcitabine chemotherapy. However, this phenomenon has been largely limited to skin. CASE REPORT: We hereby report a case of radiation recall dermatitis and myositis occurring on gemcitabine monotherapy, five months after completing chemoradiation for locally advanced pancreatic cancer. Radiation recall resolved spontaneously with withdrawal of gemcitabine. CONCLUSIONS: This is the second case report that describes gemcitabine-induced radiation recall in rectus abdominus muscles after gemcitabine-based radiation therapy. Given the wide use of gemcitabine following chemoradiation for pancreatic cancer, providers should be aware of this potential complication. PMID- 16685113 TI - A case of probable ibuprofen-induced acute pancreatitis. AB - CONTEXT: The incidence of drug-induced pancreatitis is rare. There have been no prior definite cases reported of ibuprofen-induced pancreatitis. CASE REPORT: We present a case of a young man with acute pancreatitis probably secondary to an ibuprofen overdose. Immediately preceding the onset of the attack he took a 51 mg/kg dose of ibuprofen. He had other causes of acute pancreatitis excluded by clinical history, serum toxicology and abdominal imaging. DISCUSSION: In the absence of re-challenge we believe it is probable that ibuprofen has a causative link with acute pancreatitis. PMID- 16685114 TI - Ansa pancreatica type of ductal anatomy in a patient with idiopathic acute pancreatitis. AB - CONTEXT: Ansa pancreatica is a type of pancreatic ductal variation. The exact clinical significance of this ductal variation is not clear. CASE REPORT: We report the case of a 21-year-old male with acute idiopathic severe pancreatitis and extensive parenchymal necrosis who later developed a large pancreatic abscess. Subsequently, transpapillary drainage of the pancreatic abscess was attempted and on endoscopic retrograde pancreatography, disruption in the mid body of the pancreas and the ansa pancreatica type of ductal anatomy was noted. A 7 Fr nasopancreatic catheter was placed across the disruption. However, due to the development of a new abscess, surgical drainage was performed. The patient has since been asymptomatic over a one-year follow up period. CONCLUSION: A pancreatic ductal variation such as ansa pancreatica may be a finding in severe acute pancreatitis; it is not clear if the presence of these two conditions is co incidental or if ansa pancreatica causes acute pancreatitis. Further studies are needed to clarify these points. PMID- 16685115 TI - Indomethacin-induced pancreatitis. A second case report. PMID- 16685117 TI - Cells lines vs. animals studies for developing new therapeutic strategies in human pancreatic cancer? PMID- 16685118 TI - Theoretical and numerical analysis of different adequacy indices for hemodialysis and peritoneal dialysis. AB - BACKGROUND: Apart from KT/V, equivalent urea clearance (EKR) and fractional solute removal (FSR) can also be used to assess the dialysis adequacy. Our objective was to analyze the relationships between these indices for different dialysis modalities and schedules, using urea kinetic modeling. METHODS: EKR and FSR were calculated for HD (three or six times per week), automatic nightly PD (ANPD) and CAPD using the following reference values of urea concentration and mass in the body: peak, peak average, time average and treatment time average. RESULTS: The standard KT/V approach is related to the treatment time average, whereas the standard EKR is related to the time average reference values. In spite of KT/V = 3.5 (K meaning dialyzer clearance or peritoneal diffusive mass transport coefficient), EKR and FSR were lower for ANPD and CAPD than for HD. The ratio of EKR to FSR was essentially the same for the different treatment modalities (range 3.48-4.07 ml/min). This could be explained by the theoretical analysis which predicts the value of EKR/FSR = V/Tc, independent of the treatment modality and schedules (V is a solute distribution volume, Tc is the time of the full dialysis cycle). CONCLUSION: Whereas the index KT/V in its standard form cannot be used to compare different dialysis regimens, EKR and FSR provide very similar evaluation of different dialysis modalities and schedules, and may be considered as equivalent measures for comparative studies of dialysis adequacy. PMID- 16685119 TI - Cerebral white matter hyperintensities on MRI: Current concepts and therapeutic implications. AB - BACKGROUND: White matter hyperintensities (WMH) are commonly observed MRI abnormalities in the elderly, which generally reflect covert vascular brain injury. WMH cumulatively produce substantial neurologic, psychiatric, and medical morbidity. This review provides an overview of current knowledge on vascular WMH, and describes some pharmacological agents that may have a role in mitigating this condition. SUMMARY OF REVIEW: This review has two main focus areas. The first is a discussion of currently available knowledge regarding the public health burden, pathogenesis, and various risk factors associated with the presence of vascular white matter lesions noted on brain MRI. The second section of the article details the mechanistic and clinical basis for promising pharmacological treatment modalities that could potentially prevent progression of ischemic cerebral white matter brain injury. Many of these therapies are already of proven efficacy in preventing recurrent stroke. CONCLUSIONS: Individuals with vascular white matter lesions on MRI may represent a potential target population likely to benefit from secondary stroke prevention therapies. PMID- 16685120 TI - Migraine and patent foramen ovale: A residual coincidence or a pathophysiological intrigue? AB - Migraine is one of the most common neurological disorders and one of the most frequent primary headaches. It imposes a significant burden on the affected individuals, society and health care system. As the etiology and pathophysiology of migraine are not well understood, treatment is largely symptomatic. Patent foramen ovale is a remnant of a fetal circulation and is highly prevalent in the general population. Its presence was linked to several disorders including migraine. The aim of this review was to search in the available data the answer to the question whether the link between migraine and patent foramen ovale is coincidental or whether they represent a pathophysiological entity. PMID- 16685121 TI - Blood lipids in brain infarction subtypes. AB - BACKGROUND: Little is known about the relationship between hypercholesterolaemia and specific aetiological subtypes of brain infarction (BI). METHODS: In a cross sectional study of 492 pairs of patients with a BI proven by MRI and matched hospital controls, we determined the blood levels of triglycerides, total, HDL and LDL cholesterol, and apolipoprotein A(1) and B, in the same centralized laboratory. We performed aetiological BI subtype classification. RESULTS: Except for triglycerides, the risk of BI increased continuously with the lipid levels, without any heterogeneity between the main BI subtypes or the group on lipid lowering therapy. The adjusted odds ratio per standard deviation in LDL cholesterol (0.93 mmol/l) was 1.74 (95% confidence interval, 1.02-2.98) in atherothrombotic strokes (n = 109) and 2.71 (95% confidence interval, 1.60-4.55) in lacunar strokes (n = 105). Eighty percent of patients were above the ATP-III guideline threshold LDL cholesterol of 2.59 mmol/l (100 mg/dl), with a major contribution of both atherothrombotic and lacunar stroke subtypes to this group. CONCLUSIONS: These findings suggest that blood lipids, particularly total and LDL cholesterol levels, are associated with all subtypes of BI, and that LDL above 2.59 mmol/l is highly prevalent. PMID- 16685122 TI - Progression of unilateral moyamoya disease: A clinical series. AB - BACKGROUND: The natural history of unilateral moyamoya disease (MMD) in adult patients is not clearly described in the literature. We present a series of 18 patients with unilateral MMD and analyze the risk factors for progression to bilateral disease. METHODS: A retrospective review of 157 MMD patients treated at Stanford University Medical Center from 1991 to 2005 identified 28 patients with unilateral MMD (defined as none, equivocal or mild involvement on the contralateral side). RESULTS: Eighteen patients (5 males and 13 females) were identified with unilateral MMD and angiographic follow-up of > or =5 months. Mean radiologic follow-up (+/- standard error of the mean) was 19.3 +/- 3.4 months and mean clinical follow-up was 24.5 +/- 3.7 months. Five patients had childhood onset MMD and 13 patients had adult onset disease. Angiographic progression from unilateral to bilateral disease was seen in 7 patients (38.9%) at a mean follow up of 12.7 +/- 2.4 months. Four of the 7 patients had significant clinical and radiologic progression requiring surgical intervention. Five of 7 patients that progressed had adult onset MMD. The presence of equivocal or mild stenotic changes of the contralateral anterior cerebral artery (ACA), middle cerebral artery (MCA) or internal carotid artery (ICA) was an important predictor of progression (p < 0.01); 6 of 8 patients (75%) with equivocal or mild contralateral disease progressed, whereas only 1 of 10 patients (10.0%) with no initial contralateral disease progressed to bilateral MMD. One patient had mild or equivocal MCA, ICA and ACA stenosis at the time of initial diagnosis and this patient progressed. CONCLUSIONS: Contralateral progression in the adult form occurs more commonly than previously reported. The presence of minor changes in the contralateral ACA, intracranial ICA and MCA is an important predictor of increased risk of progression. Patients with a completely normal angiogram on the contralateral side have a very low risk of progression. PMID- 16685123 TI - A randomised, controlled pilot study to investigate the potential benefit of intervention with insulin in hyperglycaemic acute ischaemic stroke patients. AB - BACKGROUND: Hyperglycaemia on presentation with acute ischaemic stroke (AIS) is associated with poor outcome, but intervention is unproven. We investigated the safety and tolerability of one method of glycaemic control. METHODS: Patients within 24 h of AIS and plasma glucose 8-20 mmol/l were randomised to receive either rigorous glycaemic control (RC) or standard management (SM) for 48 h. RC comprised i.v. insulin at a variable rate adjusted for target glucose concentration of 5-8 mmol/l, and intravenous crystalloid. The SM group received intravenous crystalloid alone in an open-label design. RESULTS: Thirteen patients were randomised to RC and 12 to SM (age 75 +/- 6.2 years; 40% male; 20% lacunar stroke; time to treatment 8 +/- 6.1 h; plasma glucose 10.6 +/- 0.9 mmol/l; known diabetes 52%; NIHSS 8, range 2-28). The glucose concentration-time curve was reduced in the RC group (AUC 324 +/- 15 versus 385 +/- 28 h.mmol/l, p = 0.04). By 48 h, plasma glucose in both groups was 6.8 +/- 1.1 and 7.5 +/- 1.3 mmol/l respectively, but mean hourly insulin requirements in the RC group had dropped from 3.25 +/- 0.32 units to 1.25 +/- 0.5 units (p < 0.01). One transient episode of hypoglycaemic symptoms occurred in the RC group. CONCLUSION: Glycaemic control with sliding scale insulin for 48 h is feasible and well-tolerated after AIS. Treatment after 48 h may be unnecessary. PMID- 16685128 TI - Metabolic treatment with L-carnitine in acute anterior ST segment elevation myocardial infarction. A randomized controlled trial. AB - BACKGROUND: Administration of L-carnitine in patients with anterior acute myocardial infarction (AMI) prevents left ventricular remodeling. Current study was aimed to assess the effect of L-carnitine administration on mortality and heart failure in patients with anterior AMI. METHODS: CEDIM 2 trial was a randomized, double-blind, multicenter, placebo-controlled trial planned to enroll 4,000 patients with acute anterior AMI. The trial was interrupted after the enrolment of 2,330 patients because of the lower than expected enrolment rate. The primary end point was a composite of death and heart failure at 6 months; 5 day mortality was the secondary end point. RESULTS: During the 6-month follow-up, the primary end-point was not significantly different between the L-carnitine and placebo group (9.2 vs. 10.5%, p = 0.27). A reduction in mortality was seen in the L-carnitine arm on day 5 (secondary end-point) from randomization (HR = 0.61, 95% CI 0.37-0.98, p = 0.041). CONCLUSIONS: In CEDIM 2 trial L-carnitine therapy led to a reduction in early mortality (secondary end-point) without affecting the risk of death and heart failure at 6 months in patients with anterior AMI, leading to a non-significant finding with respect to the primary end-point. PMID- 16685129 TI - Evaluation of a 12-lead digital Holter system for 24-hour QT interval assessment. AB - BACKGROUND: Drug induced QT prolongation may precipitate life threatening cardiac arrhythmias. Evaluation of the QT prolonging effect of new pharmaceutical agents in a 'thorough QT/QTc study' is being mandated by FDA. The purpose of this study was to evaluate an automated 12-lead digital Holter system for a thorough QT/QTc study. METHODS: Five healthy volunteers underwent 24-hour digital Holter monitoring. Each recording underwent a fully automated QT analysis (AQA) followed by an onscreen complete manual over read (MOR). Each recording was analyzed twice at least 2 weeks apart. The effect of data sampling (5-min segment/hour), the system sensitivity to detect 5-ms increase in QT, and the ability to assess circadian variation were evaluated. RESULTS: The AQA resulted in identical QT for the first and second analyses, but with obvious errors in QT measurements. Compared to the complete onscreen MOR, the mean QT was longer with AQA (416 +/- 41 vs. 387 +/- 30 ms, p < 0.001), correlation; r = 0.3. The reproducibility of AQA with complete MOR was very good (QT: 387 +/- 30 vs. 387 +/- 30 ms, coefficient of variation: 0.2%, r = 0.986. The 5-min mean QT intervals correlated well with the hourly mean QT intervals (r = 0.994, p < 0.001, coefficient of variation = 1 ms) and both showed a similar circadian variation. The system was sensitive to detect a 5-ms change in QT intervals (5 +/- 2 ms, coefficient of variation = 0.6%, r = 0.998, p < 0.001). CONCLUSIONS: The AQA is not an acceptable method, while the automatic analysis with complete MOR is a highly sensitive and reproducible method. Data sampling by analyzing 5-min segments per hour is sensitive and reproducible. PMID- 16685130 TI - The use of low-dose insulin in cardiogenic shock due to combined overdose of verapamil, enalapril and metoprolol. AB - We describe a case of severe heart failure due to the combined effect of verapamil and enalapril overdose in a patient treated regularly with metoprolol. The patient was dependent for 2 days on glucagon and dopamine infusion but remained oliguric, with deteriorating renal function. Marked improvement in all hemodynamic parameters was noted a short time after initiation of treatment with low-dose insulin infusion (1-2 units/h), which allowed the prompt withdrawal of glucagon and dopamine. We discuss the efficacy of glucose-insulin treatment in toxic cardiac depression and suggest that a low dose may be beneficial in similar cases. PMID- 16685131 TI - Enhanced external counterpulsation reduces lung/heart ratio at stress in patients with coronary artery disease. AB - Enhanced external counterpulsation (EECP) is a recently approved treatment modality for patients with angina and heart failure. However, the efficacy of EECP on left ventricular (LV) function has not been well established. The study was aimed to determine whether EECP leads to an improvement in objective parameters of LV function. Patients with coronary artery disease (n = 10) who showed evidence of stress-induced myocardial ischemia despite conventional medical or surgical therapies were enrolled and received EECP therapy for a total of 35 h. The therapeutic effects of EECP were examined by thallium-201 single photon emission computed tomography (201Tl-SPECT). Compared with baseline, the lung/heart ratio at stress decreased significantly from 0.40 +/- 0.08 to 0.35 +/- 0.08 (p = 0.001) at 1 month and 0.33 +/- 0.10 (p = 0.03) at 6 months following EECP treatment. LV ejection fraction marginally improved from 56.7 +/- 7.7% to 57.6 +/- 5.9% (p = 0.382) at 1 month and to 60.1 +/- 8.6% (p = 0.062) at 6 months after EECP therapy, although not statistically significant. We concluded that EECP improved LV function, shown as the reduction of lung/heart ratio at stress, in patients with coronary artery disease, up to 6 months after EECP treatment. PMID- 16685132 TI - Subclinical hypothyroidism in HIV-infected patients is not an autoimmune disease. AB - AIMS AND METHODS: A study of 350 HIV+ patients in our region showed that 16% suffered from hypothyroidism. Twenty-two HIV+ hypothyroid patients (10 with subclinical hypothyroidism, 12 with low FT4 levels (LT4) (confirmed by a dialysis equilibrium assay) and 22 HIV+ euthyroid controls receiving highly active anti retroviral therapy were included in an additional study. RESULTS: No goiter or anti-thyroid antibodies were detected. Use of stavudine was more frequent in the LT4 subgroup (p < 0.01) and subclinical hypothyroidism group (p = 0.04). Use of didanosine (OR, 12.5, p < 0.01) and ritonavir (OR, 33.0, p < 0.01) was more frequent in the LT4 subgroup, with a greater didanosine cumulative dose (616.7 mg [180.0, 1,260.0] vs. 263.7 [63.0, 948.0], p = 0.01). Reverse T3, binding protein levels, the TSH response to thyrotropin-releasing hormone, urinary iodine, plasma selenium and thiocyanate levels did not differ. IFNgamma levels were lower in the subclinical hypothyroidism group (pg/ml) (9.1 [0.0, 22.7] vs. 19.5 [0.0, 40.9], p = 0.03). CONCLUSION: None of the investigated mechanisms are able to explain the occurrence of hypothyroidism in HIV patients receiving highly active anti retroviral therapy except the anti-retroviral treatment. In light of the absence of autoimmunity, the normal adenohypophysis and thyroid responses to thyrotropin releasing hormone, central hypothyroidism is suspected and could explain LT4 and high TSH levels. Underlying mechanisms need further exploration. PMID- 16685133 TI - Inflammatory markers in adults with Prader-Willi syndrome before and during 12 months growth hormone treatment. AB - BACKGROUND: In Prader-Willi syndrome (PWS) obesity and partial growth hormone (GH) deficiency are frequently observed. The risks of cardiovascular diseases and early death are increased. We examined inflammatory markers in adult PWS, before and during 12 months of GH treatment. METHOD: Twelve PWS adults, median age 23.5 years (17-37) and median BMI 33.8 kg/m2 (21.2-50.4), participated. Serum interleukin-6, tumour necrosis factor alpha, high sensitive protein C-reactive protein (HCRP), cholesterol, triglycerides, leptin, adiponectin, glucose, insulin, insulin-like growth factor I and body composition were measured at baseline and after 6 and 12 months of GH treatment. RESULTS: Median and range at baseline for interleukin-6 was 9.87 ng/l (1.76-10.72), for tumour necrosis factor alpha 2.39 ng/l (1.00-3.26) and for HCRP 7.64 mg/l (0.41-41.1) (normal values < 5 ng/l, < 8 ng/l and<5 mg/l, respectively). At baseline correlations between inflammatory markers and age, anthropometry, body composition and the metabolic parameters were non-significant; only positive associations were found between tumour necrosis factor alpha and body weight (r = 0.617, p = 0.033) and between HCRP and BMI (r = 0.594, p = 0.041). GH treatment non-significantly decreased the levels of the inflammatory markers. CONCLUSION: In this pilot study, levels of interleukin-6 and HCRP were increased, and GH intervention did not significantly reduce the levels. Chronic inflammation might contribute to the increased cardiovascular morbidity and mortality in PWS. PMID- 16685134 TI - Prematurity--another example of perinatal metabolic programming? AB - Low birth weight is associated with both later adult diseases such as type 2 diabetes mellitus and a number of metabolic abnormalities, the foremost of which is insulin resistance. Indeed the link between an adverse perinatal environment, manifested by low birth weight, and adult life pathology may be an early, permanent reduction in insulin sensitivity. A reduction in insulin sensitivity has been demonstrated in small for gestational age (SGA), term subjects from childhood through to adulthood. Less is known about children born premature into an adverse neonatal environment. We present data demonstrating that premature infants also have metabolic abnormalities similar to those observed in term, SGA children and that these occur irrespective of whether they are SGA or appropriate for gestational age (AGA). PMID- 16685135 TI - Histamine type 1 receptor deficiency reduces airway inflammation in a murine asthma model. AB - BACKGROUND: Histamine plays an important role in immediate and late immune responses. The histamine type 1 (H1) receptor is expressed on several immune cell populations, but its role in a murine model of asthma remains unclear. The present study evaluated the role of histamine H1 receptors in airway allergic inflammation by comparing the development of bronchial asthma in histamine H1 receptor gene knockout (H1RKO) and wild-type mice. METHODS: H1RKO and wild-type mice were sensitized by intraperitoneal injection of ovalbumin (OVA) or saline, and then challenged with aerosolized OVA or saline. Ventilatory timing in response to inhaled methacholine was measured, and samples of blood, bronchoalveolar lavage, and lung tissues were taken 24 h after the last OVA challenge. RESULTS: OVA-treatedwild-type mice showed significantly increased airway eosinophilic infiltration, and airway response to methacholine compared to OVA-treated H1RKO mice. The serum level of immunoglobulin E and levels of interleukin (IL)-4, IL-5, IL-13, and TGF-beta1 in bronchoalveolar lavage fluid were lower in OVA-treated H1RKO mice than in OVA-treated wild-type mice, but there was no significant difference in interferon-gamma expression. Overall, deletion of histamine H1 receptors reduced allergic responses in a murine model of bronchial asthma. CONCLUSION: Histamine plays an important role via H1 receptors in the development of T helper type 2 responses to enhance airway inflammation. PMID- 16685136 TI - Expression of Fas ligand and CTLA4 in adenoids has a predictive value for allergic rhinitis development in children. AB - BACKGROUND: The balance of CD28/CTLA4-derived signals and Fas-dependent apoptosis activity is determined by the peripheral defense mechanisms and might play a role in the pathogenesis of allergy. OBJECTIVES: The aim of the study was to investigate the expression of costimulatory and pro- and antiapoptotic molecules in adenoid T cells of children suffering from allergic rhinitis and to find out which of these molecules have a predictive value in the development of allergic rhinitis. METHODS: The adenoids of 60 children, removed because of nasal obstruction, chronic rhinitis and recurrent respiratory infection, were evaluated. Patients were divided into two groups: group 1, suffering from chronic allergic rhinitis, and group 2, suffering from chronic rhinitis, where no specific IgE was detected, including children with a positive family history of allergy (group 2a) and children with neither a personal nor a family history of allergy (group 2b). For immunohistochemical stainings anti-CD3, anti-CD19, anti CD4, anti-CD8, anti-CD25, anti-CD28, anti-CTLA4 (CD152), anti-bcl-2, anti-Fas, and anti-FasL antibodies were used. The number of cells expressing these molecules was identified in adenoid interfollicular spaces. The results were then analyzed in allergic and nonallergic children. During a 24-month follow-up children were re-examined for allergy and results were compared to previous immunohistochemical evaluations. RESULTS: The expression of CD4, CD25, CD28, FasL, and CTLA4 was significantly increased in group 1 compared to group 2 (p < 0.05). However, the discriminant analysis confirmed that only CTLA4 and FasL expression fully discriminated allergic subjects from the others. During a 24 month period of observation 8 children from group 2a were also diagnosed with allergic rhinitis. All of them, especially those sensitized to mites, had an increased number of FasL+ and CTLA4+ in previously removed adenoids. CONCLUSION: An increased number of cells with intracellular expression of FasL and CTLA4, in interfollicular spaces of adenoids, seems to be a predictive factor of the development of allergic rhinitis. PMID- 16685137 TI - The skin allergenic properties of chemicals may depend on contaminants--evidence from studies on coumarin. AB - BACKGROUND/AIMS: Positive patch tests are considered representative of a contact allergy to the tested chemical. However, contaminants and derivatives rather than the suspected chemical itself could be responsible for the allergic skin reactions. Here, we tested the importance of contaminants in the sensitizing and allergenic properties of coumarin in mice and humans. Coumarin, an ingredient in cosmetics and fragrances, was chosen as the reference chemical since conflicting results have been obtained regarding its ability to induce contact allergy. In some chemical preparations, this could be explained by the presence of coumarin derivatives endowed with allergenic properties. METHODS: In mice, three different coumarin preparations were tested in the local lymph node assay. In humans, we assessed the irritant and allergenic properties of highly pure coumarin in nonallergic and fragrance-allergic patients. RESULTS: Pure coumarin did not exhibit irritant or sensitizing properties in the local lymph node assay. In contrast, two other commercially available coumarins and three contaminants that were detected in these coumarin preparations were identified as weak and moderate sensitizers, respectively. In humans, pure coumarin was extremely well tolerated since only 1 out of 512 patients exhibited a positive patch test to the chemical. CONCLUSIONS: These results indicate that coumarin cannot be considered as a common contact allergen and further emphasize that purity of chemicals is mandatory for the assessment of their allergenicity. PMID- 16685138 TI - Effect of isolation measures on the incidence and prevalence of hepatitis C virus infection in hemodialysis. AB - BACKGROUND: Nosocomial transmission of hepatitis C virus (HCV) in hemodialysis (HD) units is well established. In units with a high prevalence of HCV infection, the implementation of universal precautionary measures may not suffice in order to decrease the incidence and prevalence of HCV. In this setting strict isolation practices can be useful in order to achieve this goal. METHODS: The incidence and prevalence of HCV infection amongst all HD and peritoneal dialysis (PD) patients from the province of Albacete, Spain, have been studied from 1992 to 2003.Through the 1993-1995 period chronic HD patients were treated either in a room exclusively for HCV- patients or in a room shared by HCV+ and HCV- patients. Complete separation of HCV+ and HCV- patients was implemented in 1995. Acute patients have been separated since 1992. The implementation of universal precautions was applied throughout the period. RESULTS: There has not been a single seroconversion in the rooms where only HCV- patients were dialyzed during the 11 years of follow-up. There were two seroconversions in the rooms shared for 3 years by both HCV+ and HCV- patients. In 1995 the prevalence of HCV+ cases in HD and PD was 21.6 and 23.2%, respectively. Since then it has decreased steadily and in parallel for both therapies, and the current prevalence is 6.8% in HD and 5.7% in PD. CONCLUSIONS: In HD units with a high prevalence of HCV+ patients, strict isolation in combination with implementation of universal prevention measures can eliminate nosocomial transmission and obtain a long-term reduction in prevalence. PMID- 16685139 TI - Outcome and treatment of bucillamine-induced nephropathy. AB - BACKGROUND: Bucillamine (BCL), a treatment for rheumatoid arthritis, occasionally causes proteinuria. Renal specimens are reported to show segmental granular deposition of immunoglobulin G, associated with membranous nephropathy. Long-term course and optimal treatment have remained unknown, and were investigated here. METHODS: We examined clinical records of 400 patients treated with BCL for rheumatoid arthritis, at our hospital from 1998 to 2003, finding 17 with proteinuria and biopsy-proven BCL-induced nephropathy. RESULTS: In all 17 patients, proteinuria resolved without loss of renal function between 3 and 85 months after discontinuing BCL (14.1 +/- 3.4). The only factor influencing time to remission was pathologic stage of membranous nephropathy (stage I vs. stage II or III: 11.5 +/- 4.8 vs. 21.6 +/- 3.3 months; p = 0.02). Maximal proteinuria, total amount of BCL, BCL exposure time, and use of prednisolone or other immunosuppressant agents did not significantly influence time until remission. CONCLUSION: The most important therapeutic step in treating BCL-induced nephropathy is to discontinue BCL. Prednisolone or other immunosuppressant agents might not be effective. PMID- 16685140 TI - Angiotensin-converting enzyme inhibitors as a risk factor for contrast-induced nephropathy. AB - BACKGROUND/AIMS: The aim of the present study was to assess the influence of chronic angiotensin-converting enzyme (ACE) inhibitor administration on the development of contrast-induced nephropathy (CIN) in patients undergoing coronary angiography. METHODS: A total of 230 patients with renal insufficiency and age > or =65 years were divided into two groups according to prior use of ACE inhibitors (ACE inhibitor group, n = 109; control group, n = 121). CIN was defined as an increase of > or =25% in creatinine over the baseline value within 48 h of angiography. RESULTS: CIN occurred in 17 patients (15.6%) in the ACE inhibitor group and 7 patients (5.8%) in the control group (p = 0.015). Serum creatinine level increased from 1.34 +/- 0.20 to 1.53 +/- 0.27 mg/dl in the ACE inhibitor group and from 1.33 +/- 0.18 to 1.45 +/- 0.19 mg/dl in the control group (p < 0.001). Chronic ACE inhibitor administration was a risk indicator of CIN [odds ratio 3.37; 95% confidence interval 1.14-9.94; p = 0.028]. Multi-vessel coronary involvement (p = 0.001), hypoalbuminemia (p = 0.005), diabetes mellitus (p = 0.006), GFR < or =40 ml/min (p = 0.010), and congestive heart failure (p = 0.024) were other risk indicators of CIN. CONCLUSION: Chronic ACE inhibitor administration is a risk for developing CIN in elderly patients with renal insufficiency. PMID- 16685141 TI - Immediate effect of Shakuyaku-kanzo-to on muscle cramp in hemodialysis patients. AB - We administered 2.5 g of Shakuyaku-kanzo-to granule to 61 patients who had muscle cramp during hemodialysis (HD) sessions and examined its immediate effects. We selected 10 patients who wanted to take the drug at home, out of cases, for whom the drug was effective on the study described above and had them take the drug in the same way at the beginning of muscle cramp at home examined the effects. In the study during HD sessions, muscle cramp and its associated pain disappeared in 5.3 +/- 3.9 min on average in 54 out of 61 cases. In the study of patients who took the drug at home, muscle cramp disappeared within 10 min in all cases. Shakuyaku-kanzo-to is thought to be very useful for muscle cramp during HD sessions of hemodialized patients because it has immediate effects by its oral administration on the occasion of cramp. With regard to the muscle cramp, which appears at home after HD sessions, the patients can cope with it by taking the drug by themselves. This is an epoch-making therapy, for it was impossible to cope with muscle cramp except in hospitals because the therapy of muscle cramp was limited to intravenous infusion of hypertonic solutions of dextrose, mannitol, and saline during HD sessions. PMID- 16685142 TI - Coronary artery calcification, systemic inflammation markers and mineral metabolism in a peritoneal dialysis population. AB - AIMS: To assess the prevalence of coronary artery calcification (CAC) in peritoneal dialysis (PD) patients and to determine whether comorbidities such as inflammation, dyslipidemia and mineral metabolism disorders correlate with its development. METHODS: Forty-nine PD patients (45% male; median age, 52 years) were submitted to multislice computed tomography. Inflammatory markers, anti oxidized LDL antibody, calcium-phosphate balance and lipid profiles were assessed. RESULTS: Twenty-nine patients (59.2%) presented CAC (median calcium score, 234.7 Agatston units). Patients with CAC were older than those without, more frequently presented a history of coronary artery disease or hypertension and had lower HDL cholesterol levels, as well as presenting higher levels of osteoprotegerin and LDL oxidation. The logistic regression revealed that the independent determinants of CAC were age (odds ratio = 1.12; p = 0.006) and number of prescribed anti-hypertensive drugs (odds ratio = 2.38; p = 0.048). When the population was stratified by calcium score quartile, soluble Fas levels were significantly higher in patients with severe calcification. In patients younger than 45, CAC correlated positively with phosphorus levels (r = 0.52; p = 0.04). CONCLUSION: In PD patients, CAC is highly prevalent. Our results indicate that conditions such as inflammation and mineral disturbances are associated with its development. PMID- 16685143 TI - Assessment of health-related quality of life in chronic dialysis patients with the COOP/WONCA charts. AB - BACKGROUND: The aim of the present work was to determine the health-related quality of life (HRQOL) of our patients on hemodialysis using the COOP/WONCA charts. A further aim was to explore its psychometric characteristics. METHODS: The present study included all patients attending the hemodialysis unit of our hospital. The comparative study of the results was conducted with other population groups in which this questionnaire has been applied previously (primary-care patients, their accompanying persons, pregnant women, Alzheimer patient carers and drug addicts). The dimensional structure of the questionnaire was explored by factorial analysis of principal components, and internal consistency was evaluated with the Cronbach alpha factor. Concurrent validity was determined by comparative analysis of the results of the COOP/WONCA charts with those of another questionnaire, the 36-item short form of the Quality of Life Score (SF-36). To do so, 60 randomly chosen patients completed both questionnaires. RESULTS: 98.9% of patients completed the COOP/WONCA charts in their entirety. The mean scores of all the charts, with the exception of overall health, are in the range of what could be called 'favorable area of quality of life' (below 3). With respect to the rest of the groups, the dialysis patients had the best scores on the 'feelings' and 'social activities' charts and none of the worst ones. The joint factorial analysis revealed that the questionnaire has a 3-factor structure: factor 1 or daily functioning, factor 2 or quality of life and health, factor 3 or the psychosocial factor. The Cronbach alpha value for the 9 charts was 0.766. All the correlations between equivalent dimensions of questionnaires COOP/WONCA and SF-36 were statistically significant (p < 0.01). The convergent validity mean was 0.57 and the discriminant validity mean was 0.22, appreciably lower than convergent validity. CONCLUSIONS: This study shows that the process of psychological adaptation to problems deriving from dialysis is satisfactory. Moreover, the COOP/WONCA charts are a useful instrument for the determination of the HRQOL in hemodialysis patients without losing psychometric quality. PMID- 16685144 TI - Increased soluble E-cadherin in melanoma patients. AB - Cadherin switching is thought to contribute to melanoma progression. E-cadherin expression is downregulated, facilitating the release of contacts with keratinocytes, while N-cadherin expression is increased, potentially contributing to more migration. Proteolytic cleavage of the cadherin extracellular domain, a process called ectodomain shedding, is one way to decrease cadherin cell surface expression. In addition, the released ectodomain could actively contribute to a more invasive phenotype. To examine if melanoma progression correlates with increased cadherin ectodomain shedding, we tested the presence of N- and E cadherin extracellular domains in different melanoma cell lines and the presence of E-cadherin in sera of patients. Shedding occurs and is regulated in several melanoma cell lines expressing these cadherins. No correlation could be found between cadherin shedding and invasive capacity of the cell lines. However, we did find a significant increase in serum E-cadherin levels of melanoma patients with advanced disease correlating with increased S100 tumor marker values, suggesting that increased cadherin shedding may contribute to melanoma progression. PMID- 16685145 TI - Tight junction proteins in the skin. AB - It has long been accepted that tight junctions (TJ) are crucial for the formation and maintenance of the paracellular barrier and for cell polarity in simple epithelia and endothelia. Moreover, it is long known that they play a role in barrier function of amphibian skin. However, only in recent years were TJ and TJ proteins identified in the epidermis of men and mice. Their involvement in the barrier function of mammalian skin has been shown. This review summarizes our current knowledge about TJ and TJ proteins in mammalian skin. PMID- 16685146 TI - In vivo drug screening in human skin using femtosecond laser multiphoton tomography. AB - The novel femtosecond laser multiphoton imaging system DermaInspect forin vivotomography of human skin was used to study the diffusion and intradermal accumulation of topically applied cosmetic and pharmaceutical components. Near infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores and fluorescent components of a variety of ointments via a two-photon excitation process. In addition, collagen was imaged by second harmonic generation. A high submicron spatial resolution and 50 ps temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics together with a time-correlated single-photon counting module with a fast microchannel plate detector. Individual intratissue cells, intracellular mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements were clearly visualized due to NAD(P)H, melanin, elastin, and collagen imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs were detected by two-photon-excited fluorescence. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The novel noninvasive 4-D multiphoton tomography tool provides high-resolution optical biopsies with subcellular resolution, and offers for the first time the possibility to study in situ the diffusion through the skin barrier, long-term pharmacokinetics, and cellular response to cosmetic and pharmaceutical products. PMID- 16685147 TI - Differential protein expression in the epidermis of wild-type and COX-2 transgenic mice. AB - Cyclooxygenases (COX) 1 and 2 are the key enzymes of prostaglandin biosynthesis. Like in many tissues, in adult skin COX-1 is a constitutive 'housekeeping' enzyme, while COX-2 is induced transiently in stress situations such as tissue damage and regeneration. In human skin carcinomas and corresponding early-stage cancer lesions, permanent COX-2 expression and activation is a consistent feature. Knockout and various transgenic approaches and pharmacologic studies show strong evidence for a cause-and-effect relationship between the aberrant COX 2 activation and tumor formation. In skin epidermis, keratin 5 promoter-driven overexpression of COX-2 caused hyperplasia and dysplasia, and sensitized skin for carcinogenesis. Therefore, this model offers the unique possibility of identifying COX-2-dependent and prostaglandin-mediated molecular pathways leading to the formation and malignant progression of early-stage cancer lesions. PMID- 16685148 TI - Skin ultrastructure in senile lentigo. AB - Senile lentigo is a common component of photoaged skin. It is characterized by hyperpigmented macules which affect chronically irradiated skin mostly after the age of 50. This study was undertaken to assess the morphology of senile lentigo on the dorsum of the hands. A systematic comparison between lesional and perilesional skin using histology and transmission electron microscopy was done to determine whether melanocytes or keratinocytes are affected in the evolution of lesions and which tissue structure is modified. The histology study showed that lesional skin is characterized by a hyperpigmented basal layer and an elongation of the rete ridges, which seem to drive deeply into the dermis. The epidermis contained clusters of keratinocytes, which retained and accumulated the melanin pigment. Electron microscopy studies showed important modifications in the lesional skin ultrastructure in comparison with perilesional skin. In melanocytes from perilesional and lesional skin, we observed normal size melanosomes at all stages of maturation in the cytoplasm and in migration within dendrites. No pigment accumulation was observed. However, the morphology of melanocytes in lesional skin revealed an activated status with numerous mitochondria and a well-developed endoplasmic reticulum, which could reflect intense protein synthesis. In basal keratinocytes from lesional skin, we observed numerous melanosome complexes called polymelanosomes, which formed massive caps on the nuclei. Observations in colored semi-thin sections also revealed perturbed structures in the basal layer region, which could explain the skin perturbation. Indeed, we observed keratinocytes that presented important microinvaginations and pendulum melanocytes, which sank into the dermis, beneath the basal layer of keratinocytes. These cell modifications seemed to be due to a perturbation of the dermal-epidermal junction, which appeared disorganized and disrupted and could directly disturb the basal support of the cells. PMID- 16685149 TI - Semiquantitative determination of the penetration of a fluorescent hydrogel formulation into the hair follicle with and without follicular closure by microparticles by means of differential stripping. AB - Recently, hair follicles were shown to be of great importance concerning the penetration and reservoir behavior of topically applied substances. Especially microparticles are known to be efficient drug carriers into the hair follicles. It has been shown in various investigations that, depending on their size, microparticles can be utilized to enhance the penetration of substances into the hair follicles. In the present study, it was investigated whether they could also be used to block the hair follicles to stop penetration. For this purpose, the follicular penetration of a sodium fluorescein-containing hydrogel and follicular reservoir depletion were investigated by means of differential stripping in vivo. In a second study design, the skin areas were pretreated with microparticles 5 microm in diameter. Subsequently, the follicular penetration of sodium fluorescein-containing hydrogel was likewise investigated by differential stripping. The results showed that the hair follicle infundibula contained a significant fluorescent signal when sodium fluorescein in hydrogel was applied. In contrast, in the case of pretreatment with microparticles, only a very low fluorescence signal was measurable in the hair follicle infundibula. The microparticles clearly blocked the follicular orifices so that a penetration of sodium fluorescein was almost completely inhibited. This was confirmed by histological investigations. Thus, depending on their size, microparticles act as efficient drug carriers or can be utilized as follicle blockers to stop the penetration of topically applied substances. This could be very useful in skin protection to prevent the storage of harmful topically applied substances in the hair follicles, known to represent long-term reservoirs. PMID- 16685150 TI - Overcoming the stratum corneum: the modulation of skin penetration. A review. AB - It is preferred that topically administered drugs act either dermally or transdermally. For that reason they have to penetrate into the deeper skin layers or permeate the skin. The outermost layer of the human skin, the stratum corneum, is responsible for its barrier function. Most topically administered drugs do not have the ability to penetrate the stratum corneum. In these cases modulations of the skin penetration profiles of these drugs and skin barrier manipulations are necessary. A skin penetration enhancement can be achieved either chemically, physically or by use of appropriate formulations. Numerous chemical compounds have been evaluated for penetration-enhancing activity, and different modes of action have been identified for skin penetration enhancement. In addition to chemical methods, skin penetration of drugs can be improved by physical options such as iontophoresis and phonophoresis, as well as by combinations of both chemical and physical methods or by combinations of several physical methods. There are cases where skin penetration of the drug used in the formulation is not the aim of the topical administration. Penetration reducers can be used to prevent chemicals entering the systemic circulation. This article concentrates on the progress made mainly over the last decade by use of chemical penetration enhancers. The different action modes of these substances are explained, including the basic principles of the physical skin penetration enhancement techniques and examples for their application. PMID- 16685151 TI - [Trend to adjuvant systemic treatment regimens for patients with early breast cancer--meeting highlights of St. Gallen Conference 2005]. AB - The 9th International Expert Consensus Meeting on the Primary Therapy of Early Breast Cancer 2005, with 4166 participants from 78 countries, was held in January 2005 in St. Gallen, Switzerland. Its consensus recommendations were summarized in the Annals of Oncology (16:1569-1583, 2005), published on Sept. 7 that year. The Meeting made a fundamental change in the algorithm for selection of adjuvant systemic therapy for early breast cancer. Rather than the earlier approach commencing with risk assessment, the Panel affirmed that the first consideration was endocrine responsiveness. Three categories were acknowledged:endocrine responsive, endocrine non-responsive and tumors of uncertain endocrine responsiveness. The three categories were further divided according to menopausal status. Only then did the Panel divide patients into low-, intermediate-and high risk categories. It agreed that axillary lymph node involvement did not automatically define high risk. Intermediate risk included both node-negative disease (if some features of the primary tumor indicated elevated risk) and patients with one to three involved lymph nodes without additional high-risk features such as HER 2/neu gene over expression. The Panel recommended that patients be offered chemotherapy for endocrine non-responsive disease; endocrine therapy as the primary therapy for endocrine responsive disease, adding chemotherapy for some intermediate-and all high-risk groups in this category; and both chemotherapy and endocrine therapy for all patients in the uncertain endocrine response category except those in the low-risk group. PMID- 16685152 TI - [The present state and future of home care for gastric cancer patients]. AB - Recently, cancer treatment has been shift from inpatient chemotherapy to outpatient chemotherapy, because of various medical circumstances. In chemotherapy of gastric cancer, outpatient chemotherapy was not spread in the last decade, because the chemotherapy protocol of gastric cancer was not fit for outpatient chemotherapy. But the development of new drugs as TS-1 make outpatient chemotherapy more frequent. So home care of patients has been important for management of gastric cancer. Various symptoms due to obstruction at primary lesion or other lesion prevent patients from living at home in gastric cancer. But recently, technical development and spread of home parenteral nutrition make a possible home care of patients with gastric cancer. It is necessary to make a system that supports patient life at home. PMID- 16685153 TI - [Lung carcinoma]. AB - Chemotherapy for patients with advanced lung carcinoma at an early period of diseases contributes to prolonged survival. However, since survivals are limited to around 1 year, it is critical for patients to stay at home and continue their social activities under chemotherapy. As active agents such as paclitaxel, docetaxel, gemcitabine, vinorelbine and irinotecan were introduced into clinical practice, and new techniques for preventing side effects such as emesis and neutropenia were developed, chemotherapy for outpatients become feasible also in Japan. In addition, the outpatient chemotherapy, preventing oncologic emergency and early starting of palliative care are also very important for patients'quality of life (QOL) at home. This review summarizes the present status of taking care for outpatients with lung cancer. PMID- 16685154 TI - [Present conditions and future of ambulatory treatment and home care for breast cancer]. AB - Breast cancer is a disorder which occurs in the mammary gland, an organ on the body surface. Therefore, we traced the clinical course peculiar to breast cancer that is considerably different from lung and digestive cancer of a visceral origin. In addition, the standard treatment established worldwide for mammary cancer on the basis of cumulative of European and American evidence is in the surgery, internal medicine and radiologic fields. It is important that the is treated in an outpatient department setting and supported by home care. The construction of a team approach in medical care is an urgent issue for successful home care. PMID- 16685155 TI - [Home care of hematological malignancies]. AB - Hematological malignancies such as leukemia or lymphoma are mainly treated by hospitalization or in outpatient clinics. Therefore, home care and home nursing are not so intensively done in the treatment of these malignancies. However, G CSF administration against neutropenia after chemotherapy and administration of narcotics or opioids against severe pain have been performed sometimes during home care, and have been contributing to better QOL of the patients. PMID- 16685156 TI - [Present and future home therapy in advanced stage of pancreatic cancer]. AB - We describe the present and future directions of home therapy in pancreatic cancer patients according to the chemotherapy and best supportive care. Chemotherapy in an outpatient clinic is widely supported by the use of gemcitabine, and useful because of mild adverse effect, good clinical benefit response and survival benefit. Since there is no secondary chemotherapy agent after gemcitabine treatment failure, best supportive care is essential. Control of blood glucose level, ascites and pain are also important. Now pain control and home therapy are easy to perform because of the development of various useful opioid agents. PMID- 16685157 TI - [Outpatient based colorectal cancer treatment--the current status, challenges and future outlook]. AB - In recent years, chemotherapy for colorectal cancer has advanced rapidly. It has taken on the primary role in the treatment of advanced/metastatic colorectal cancer. However, surgery and radiation therapy continue to be integral measures in managing the various symptoms which occur during chemotherapy. The objective in treating non-resectable recurrent colorectal cancer is to maintain excellent QOL, while attempting to prolong the patient's life. Currently,the standard treatment in our nation is FOLFIRI/FOLFOX; however, the Western nations are starting to implement molecular targeted agents, although extremely costly. High expectations are held for research efforts oriented towards the effectiveness of oral fluoropyrimidines, anti-cancer drug sensitivity and the prognosis for side effects. It is requisite to maintain balance between QOL and life prolongation. In order to achieve this, it is imperative to listen closely to what the patient is feeling, and to continue providing support to alleviate the patient's uncertainties and troubles. Knowledge of palliative medicine and active listening methodology become necessary for the medical professional. EBM should not be the only focus; the thinking of each individual should be respected, and outpatient based chemotherapy should be attempted, allowing the individual to maintain everyday living and social activities. One looks forward to the fruition of customized medicine which corresponds to the "nature of cancer "with the "patient's individuality." PMID- 16685158 TI - [Essentials for transition of palliative care patients to palliative home care and for management of their cancer pain]. AB - Multi-disciplinary team work among visiting doctors, nurses, care managers and pharmacists located close to the patient's home is essential for smooth transition of a palliative care patient from hospital care to palliative home care and should be set up prior to the patient's discharge from the hospital. Palliative home care physicians should have knowledge of the fundamental support by the government to spare excessive cost to the patients. As for cancer pain management, opioid-centered analgesic therapies have lead to better quality home care for patients. In Japan, although oxycodone SRs and fentanyl patches are available besides morphine, there is no rescue opioid other than morphine. On the other hand, some cancer pain refractory to opioids such as neuropathic cancer pain should be carefully treated by adjuvant analgesics in conjunction with non pharmacological treatments. PMID- 16685159 TI - [Adjuvant chemotherapy with TS-1 for head and neck cancer--side effect of two week application followed by one-week rest regimen]. AB - Multimodality therapy incorporated with radiotherapy, surgery and chemotherapy are used in the treatment of head and neck cancer in order to improve the local control and survival rate. TS-1, a newly developed oral antitumor agent which could achieve the same therapeutic concentration as that of 5-FU under continuous and intravenous treatment, has been used as adjuvant therapy for carcinomas in recent years. We presented our experience applying a new regimen of TS-1 and its side effects. TS-1 has been applied for head and neck carcinomas since 2001. The oral application of TS-1 has been used in 32 cases of head and neck cancer in our department since 2003, and the agent has been applied in 22 of 32 cases as adjuvant therapy. The primary sites of malignancy included hypopharyngx (7 cases), larynx (6 cases), maxillary sinus (2 cases), oropharynx (2 cases), oral cavity (4 cases), submandibular gland (1 case) and one case in which the primary site was unknown. A regimen of four-week application followed by two-week rest had been used in 6 cases in the first part of this trial. However, a high frequency of blood toxicity was found from the third week, requiring alteration of the protocol. Thus, a new regimen of two-week application followed by one week rest was thereafter used in the other 16 cases. Blood toxicity was found in 66.7% of those cases receiving a four-week application followed by two-week rest regimen. In the 16 cases receiving the two-week application followed by one-week rest regimen, only one case showed grade 2 leucopenia while continuous application for more than eight weeks was possible in 9 cases. Mild macrocytic anemia was found in some of these cases, however none of which required any necessary interruption of the treatment. Side effects other than blood toxicity, such as edema or pigmentation of lower limbs, erythema of skin and diarrhea, were found in the other cases, requiring suspension of the treatment. But the subsequent application was possible after a break or decreasing the dosage. We concluded that the new regimen of two-week application followed by one-week rest is less likely to be interrupted by the side effects and is safer to be used outpatiently, compared with the four-week application followed by two-week rest. PMID- 16685160 TI - [Weekly administration regimen of paclitaxel (PTX) in patient with inoperable or recurrent gastric cancer]. AB - Paclitaxel is one of the new drugs against advanced/recurrent gastric cancer. We report its efficacy and toxicity with weekly administration for advanced/recurrent gastric cancer. We administered 26 patients (postoperative/non operation=9/17) PTX 80 mg/m(2)by 1-hour intravenous infusion once a week for 3 weeks followed by one week rest. Median PTX administrations were 2.0 cycles (range:1-22). Characteristics of the patients were median age of 62 (range: 37 78) and PS 0/1/2:2/17/7, male/female:18/8. Over grade 3 toxicities did not occur. The overall response rate was 14.3%, and the non-PD rate was 66.8%. Median time to treatment failure was 61 days and median survival time was 221 days. These results suggest that weekly PTX has modest activity with a favorable toxicity profile in patients with advanced/recurrent gastric cancer, and so this regimen may thus might be recommended in an outpatient treatment setting. PMID- 16685161 TI - [Thymidylate synthase and dihydropyrimidine dehydrogenase expression and histological effects of preoperative UFT in gastric cancer patients]. AB - During DNA synthesis in tumors, fluoropyrimidine anticancer agents target thymidylate synthase (TS) that catalyze the synthesis of dTMP from dUMP and are metabolized by dihydropyrimidine dehydrogenase (DPD). We administered UFT to patients with gastric cancer preoperatively to prevent cancers from advancing while they await surgery or down staging. PATIENTS AND METHODS: We administered UFT to 24 gastric cancer patients at 360 mg/m(2)/day for longer than 3 weeks as a preoperative chemotherapy. TS and DPD expression in the tumor were measured by immunohistochemistry staining before and after (during surgery) chemotherapy and compared with the results of histological assessment. RESULTS: TS and DPD expression decreased significantly after UFT administration (p<0.05). Histological assessment showed Grade 1 b or 2 in 11 of 24 patients (46%). Eight of 15 patients with high DPD (53.3%) exhibited Grade 1 b or 2. CONCLUSIONS: Histological assessment revealed the efficacy of UFT, through a DPD-inhibitory fluoropyrimidine (DIF) effect, in patients with high DPD. This suggests that preoperative administration of UFT can be a useful clinical measure. PMID- 16685162 TI - [Comparison of ramosetron and azasetron for prevention of acute and delayed cisplatin-induced emesis in lung cancer patients]. AB - We performed a retrospective study that compared the efficacy and safety of ramosetron with azasetron in a case of acute and delayed emesis induced by cisplatin (CDDP)-included chemotherapy in patients with lung cancer. The study subjects were 100 lung cancer patients treated with combination therapy of cisplatin, ifosfamide, irinotecan (CIC therapy). The ramosetron group and azasetron group received, respectively, ramosetron 0.3 mg or azasetron 10 mg intravenous injection, 30 minutes prior to CDDP. All patients received 32 mg of dexamethasone intravenously. Protection from emesis showed no significant difference between two treatment groups. However, the grade of nausea was significantly lower in the ramosetron group than in the azasetron group. Furthermore, chlorpromazine hydrochloride for use as a rescue medication was required by significantly fewer in the ramosetron group than in the azasetron group. Adverse effects were observed in 27 cases in the ramosetron group and 24 cases in the azasetron group. However, because the symptoms were all mild, we did not consider there was any safety problem. In conclusion, it was suggested that ramosetron is a clinically useful treatment for acute and delayed emesis induced by cisplatin-induced chemotherapy in patients with lung cancer. PMID- 16685163 TI - [Practical procedure of totally implanted venous access system for outpatient cancer therapy]. AB - Some outpatient cancer chemotherapy has been replacing inpatient chemotherapy. A totally implanted venous access system is now available for patients who undergo cancer chemotherapy on an outpatient basis. All eleven outpatients who underwent cancer chemotherapy with this system underwent placement of the port and administration of chemotherapy. Complications were observed in two of these eleven patients (18%). Because a subcutaneous abscess occurred in one patient between the placement of the port and her first chemotherapy session, we had to remove the port system once. Another patient developed fungemia, so we discontinued the system after 95 outpatient chemotherapy sessions. When the infection healed, we set up the system again, and no trouble was observed later in these two cases. According to the patients' survey, all patients were satisfied with outpatient chemotherapy using this system. Therefore, we concluded that this system contributes to enhanced quality of life among outpatients who undergo cancer chemotherapy. PMID- 16685164 TI - [A case of unresectable non-small cell lung cancer with pleural dissemination successfully treated with DIF]. AB - A 68-year-old woman was referred to our hospital with an abnormal shadow on her chest CT, which showed a mass lesion 3.5 cm in diameter at segment 7 in the right lung. At thoracotomy, a curative operation could not be performed because of pleural dissemination, so a partial resection was done for the histopathological examination. It showed papillary adenocarcinoma and clinical Stage IIIB. Her family hoped not to inform her that a curative operation was impossible. We explained the necessity of adjuvant chemotherapy to her, and she consented to UFT. However, the level of CEA gradually elevated, and pleural effusion on the right side appeared 2 years after operation. We converted UFT into TS-1. The level of CEA gradually reduced, and pleural effusion disappeared. We conclude that oral administration of UFT or TS-1 is useful as palliative chemotherapy for advanced non-small cell lung cancer without serious adverse events and worsening of quality of life. PMID- 16685165 TI - [A case of a second cancer of metachronous multiple primary non-small cell lung cancer successfully treated with TS-1 and CDDP chemotherapy]. AB - The patient was a 66-year-old man who had undergone right upper lobectomy and ND 2a systematic lymph node dissection for lung cancer (M/D adenocarcinoma, p-stage IB) in March of 1999 . On November 2003, postoperative routine chest computed tomography(CT) demonstrated a mass in left S6, and pathological diagnosis revealed P/D squamous cell carcinoma (cT1N2M0, stage IIIA) by CT-guided needle biopsy and mediastinoscopy. At first, we tried two courses of a combination chemotherapy consisting of carboplatin (CBDCA) and paclitaxel every 3 weeks. After 2 courses, the regimen was stopped because of grade 3 arthritis. Then, two courses of CBDCA and gemcitabine were performed. The evaluation of the response was SD by the guidelines of Response Evaluation Criteria in Solid Tumor Groups. Next, gefitinib was orally administered for 6 months but the tumor and mediastinal lymph nodes were growing. In January 2005, oral administration of TS 1 (60 mg/1, 2 courses, 75 mg/3-6 courses) was begun twice a day for 21 consecutive days while cisplatin (60 mg/m(2)) was administered intravenously on day 8. The response was PR (the tumor decreased by 46%), no serious adverse effect was observed, and the patient maintained good quality of life throughout the chemotherapy. This case suggests that TS-1+CDDP chemotherapy may be an effective treatment in patients with advanced lung cancer even after many protocols of chemotherapy. PMID- 16685166 TI - [A case of recurrent esophageal cancer with lung metastases successfully treated with docetaxel-combination chemotherapy]. AB - We report a case of a 67-year-old woman diagnosed with advanced esophageal cancer. She was treated with chemo-radiotherapy (5-FU/CDDP therapy and irradiation) initially, and primary lesion was well controlled. Two and a half years after first treatment, a chest CT showed multiple lung metastasis, which were confirmed by thoraco-laparoscopy. We chose docetaxel/CDDP combination chemotherapy, because of severe side effects due to the first treatment. After 3 courses, lung metastatic lesions were reduced. The following courses combining docetaxel-nedaplatin were done as ambulatory treatment. These regimens could be one choice for recurrent esophageal cancer, especially FP therapy-resistant or intolerant cases. PMID- 16685167 TI - [A case of gastric cancer presenting with obstructive jaundice and responding to biweekly CPT-11 and CDDP combination administration]. AB - A 74-year-old man was suffering from Borrmann type 2 advanced gastric cancer with abdominal lymph node metastases and multiple lung metastases. He started to undergo outpatient treatment with oral administration of TS-1. But pyloric stenosis was found after 6 courses of TS-1 chemotherapy, so he underwent palliative distal gastrectomy. TS-1 chemotherapy was continued afterwards, however obstructive jaundice was found. So combination chemotherapy of CPT-11 60 mg/m(2)and CDDP 30 mg/m(2)biweekly was selected as a second-line therapy after PTCD. As no side effects were found, he could be treated on an outpatient basis by CPT-11 60 mg/body and CDDP 30 mg/body biweekly. Four months has passed since the palliative operation, and the PTCD tube was successfully removed. The abdominal lymph nodes had decreased in size and the patient has maintained good QOL. Thus, combination CPT-11 and CDDP therapy could well be a new candidate for a second-line chemotherapy in outpatients. PMID- 16685168 TI - [A case of gastric cancer with DIC caused by multiple bone metastases treated with TS-1]. AB - A 71-year-old woman was admitted for anorexia. Endoscopic findings revealed type 3 gastric cancer. Histological examination of the endoscopic biopsy revealed poorly-differentiated adenocarcinoma. We performed total gastrectomy. However, 18 days after surgery, DIC due to multiple bone metastases occurred. The patient was treated with TS-1 chemotherapy in addition to anti-DIC therapy. TS-1(100 mg/day) was administered on days 1 to 5, 8 to 12, and 15 to 19 . The DIC was resolved. She was discharged after 2 courses of this regimen. This chemotherapy can be applied for the management of DIC caused by multiple bone metastases. PMID- 16685169 TI - [A resected case of stage IV gastric cancer successfully treated with TS-1/CDDP as neoadjuvant chemotherapy]. AB - A resected case of gastric cancer is described. The patient was a 60-year-old woman who presented a type 3 gastric tumor complicated by invasion of the head of the pancreas and liver. Radical resection was not indicated, and we administered the following combination chemotherapy with TS-1 and CDDP. 120 mg/day of TS-1 was orally administered for 3 weeks followed by 2 drug-free weeks as 1 course and 9 5 mg (60 mg/m(2)) of CDDP was administered intravenously on day 8. After two courses, total gastrectomy and D2 lymph node dissection were performed. Radical surgery for cure was conducted. Microscopically, the histological effect was judged to be grade 1a. One year after the operation, the patient is still alive without recurrence and metastasis. TS-1/CDDP therapy is useful for advanced gastric cancer. PMID- 16685171 TI - [A case of hepatic metastases of sigmoid colon cancer which completely responded to systemic l-leucovorin/5-FU therapy and oral LV/UFT combination therapy]. AB - A 73-year-old man was admitted for sigmoid colon cancer with multiple hepatic metastases. The patient underwent a sigmoidectomy only, because of bronchial asthma. Then, l-leucovorin (375 mg/body) and 5-FU (750 mg/body) were injected every week. After 2 cycles (1 cycle: 6 weeks of therapy followed by a 2-week treatment break), CT scanning showed almost a complete response, without side effects such as nausea, vomiting, diarrhea and bone marrow suppression. Then, oral administration of LV and UFT was started (LV 75 mg/body/day, UFT 450 mg/body/day. After 2 weeks of therapy, there was a 1-week treatment break), and 2 years after operation this therapy was stopped because the complete response on CT scanning continued. PMID- 16685170 TI - [Efficacy of the combined use of taurine for hyperbilirubinemia caused by UFT therapy after surgery for colonic cancer]. AB - For postoperative adjuvant chemotherapy, UFT was administered to 69 cases of stage II and III colonic cancer following surgery with a radical curability of A. Among these patients, 8 developed hyperbilirubinemia. UFT administration was discontinued for those who developed overt jaundice or dermatological symptoms, experienced a relapse of an earlier asthmatic respiratory difficulty, or for those who were found with multiple hepatic metastases. For the 4 who had developed subclinical jaundice with a total bilirubin level of 1.6 to 2.2 mg/dl, UFT was combined with taurine. The combination successfully eliminated hyperbilirubinemia. All 4 are currently alive with no recurrence at this writing. Taurine ameliorates one's capacity to excrete bile, blood flow, and augments the actions of hepatocytes. It is effective in treating the hyperbilirubinemia that develops during UFT therapy. PMID- 16685172 TI - [A case of liposarcoma of the sigmoid colon mesenterium successfully treated with chemotherapy]. AB - We report a case of postoperative recurrence of liposarcoma of the mesenterium successfully treated by chemotherapy using ifosfamide (IFM) and cisplatin (CDDP). A 58-year-old man presented with a strange feeling in the lower abdomen. Enhanced computed tomography showed enhanced non-epithelial tumor in the pelvic space. The tumor moved on palpation, so we diagnosed liposarcoma from the mesenterium, and resected the tumor on 22 August, 2002. The pleomorphic liposarcoma was 11 x 14 x 7 cm, growing from the mesenterium of the sigmoid colon, and weighed 640 g. We performed re-operation due to pelvic recurrence on 24 March, 2003. On 18 September of the same year, when we performed re-operation again for pelvic recurrence, there were so many recurrences on the mesenterium of small intestine that resection was impossible. We started chemotherapy 3 days later using 3.0 g/body IFM from 1 October 2003, together with 1,800 mg/body mesna for prevention of hemorrhage cystitis. We continued chemotherapy using 3.0 g/body/day IFM together with 1, 800 mg/body/day mesna on an outpatient basis, upon his weekly visit to the hospital. Patient remission was shown by abdominal enhanced computed tomography on 10 December. Some grade 2 alopecia and grade 2 leukopenia occurred, so we changed to chemotherapy once every two weeks. Then, it continued to decrease, without any side effects. The response appeared to be complete on computed tomography in August 2004, and we have been continuing the same chemotherapy without recurrence. PMID- 16685173 TI - [Development of acute type, CD 8 positive adult T-cell leukemia in a carrier of hepatitis B virus--possible therapeutic effect of lamivudine combined with chemotherapy]. AB - Cases of adult T-cell leukemia (ATL) with aberrant phenotypes have a very poor prognosis. We report the development of acute type, CD 8 positive ATL in a carrier of hepatitis B virus (HBV). The patient was treated with a combination of lamivudine and chemotherapy and consequently had longer-term survival than those reported previously. A 64-year-old(corrected 65-year-old) man was referred to our hospital in January 2002 because of ascites and abdominal tumor. He was positive for anti-HTLV-1 antibody and HBV surface antigen. Generalized computed tomography demonstrated bilateral pleural effusion, abdominal mass, and massive ascites. Cytological examination of ascitis revealed numerous atypical lymphoid cells,which were positive for CD 2, CD 5, CD 8, and CD 25. Monoclonal integration of HTLV-1 provirus was detected by Southern blot analysis on DNA extracted from lymphoid cells. A diagnosis of acute type, CD 8 positive ATL was made. Lamivudine was administered for prevention of chemotherapy induced HBV reactivation. Subsequently, he was treated with 6 cycles of CHOP and went into remission. He maintained clinical remission during a follow-up of 13 months and then relapsed. Further salvage therapies were provided with a transient effect. He died of sepsis in February 2004. The overall survival time of this patient was 25 months. It is possible that lamivudine combined with chemotherapy may have had a therapeutic effect on ATL in this case. PMID- 16685174 TI - [Case series of localized nasal NK/T-cell lymphoma treated with preceding intensified local radiation therapy before systemic chemotherapy]. AB - Nasal NK/T-cell lymphoma is EB virus-associated aggressive lymphoma, which is more prevalent in Asia. Previously, this lymphoma which was recognized as lethal midline granuloma, commonly presents with midline facial destructive lesions. In early stage I/II disease, radiation therapy exerts a powerful treatment outcome, however, toxic adverse events are indispensable and the tolerability of radiation therapy with chemotherapy has not been fully studied. It is imperative to offer an appropriate treatment for cure of this disease. We report consecutive 4 cases of nasal NK/T-cell lymphoma, which was treated with 56 Gy intensified local radiation therapy followed by systemic chemotherapy. Two cases complicated with grade 3 stomatitis during the treatment course and 3 cases were hospitalized due to the decrease of oral intake. The scheduled radiation chemotherapy was completed and resulted in complete response of disease in all cases. High intensified radiation therapy followed by chemotherapy may be effective for localized nasal NK/T-cell lymphoma. PMID- 16685175 TI - [Evidence-based management of nausea and vomiting]. AB - One of the most common chemotherapy-related adverse reactions has been nausea and vomiting. With the recent advances in supportive care, however, it has become possible to prevent most of the chemotherapy-induced nausea and vomiting. This article summarizes the ASCO guideline published in 1999 and the 2004 NCCN guideline together with the recent advances in this field. PMID- 16685176 TI - A two-year follow-up on risk status identified by the checklist for autism in toddlers. AB - We examined the characteristics of children at 4 to 5 years of age who were correctly and incorrectly classified as "at risk" for an autism spectrum diagnosis using the Checklist for Autism in Toddlers (CHAT) at age 2 to 3 years. Information is provided on the stability of risk/disorder status over a 2-year period of early development. Participants were 19 children with autism and 11 children with other developmental disabilities who had all been administered the CHAT between 2 and 3 years of age (Time 1) and received diagnostic and developmental re-evaluations between 4 and 6 years of age (Time 2). The risk status of children was discussed based on the original CHAT authors' criteria for risk of autism and the Denver modification for risk. High levels of stability in risk/diagnostic status from Time 1 assessments to Time 2 assessments were noted. Specifically, the original CHAT criteria for medium to high risk of autism applied at Time 1 predicted Time 2 diagnostic classification for 83% of the sample, and the Denver modification of the CHAT risk criteria predicted Time 2 diagnostic classification for 93% of the sample. Implications of the findings are discussed as they relate to early screening and identification of autism spectrum disorders. PMID- 16685177 TI - Early screening for autism spectrum disorders: update on the modified checklist for autism in toddlers and other measures. AB - Early intervention for autism spectrum disorders necessitates early detection. This need has led to widespread agreement across disciplines that screening is critical in very young children. Two screening issues are highlighted in this review. Level of screening refers to the type of sample: Level I is defined as an unselected sample, and Level II consists of selected children already identified as being at risk for a developmental disorder. Breadth or scope of screening refers to the range of difficulties the screening tool attempts to identify: broad screening instruments identify multiple range of developmental difficulties, whereas disorder-specific tools focus on a single disorder or class of disorders. Broad developmental instruments reviewed include the Parents' Evaluation of Developmental Status and the Ages and Stages Questionnaires; autism specific tools reviewed include the Checklist for Autism in Toddlers, the Modified Checklist for Autism in Toddlers (M-CHAT), the Pervasive Developmental Disorders Screening Test, Second Edition, and the Screening Tool for Autism in Two-year-olds. The development of the M-CHAT, a Level I and Level II screening instrument, is described, and current research and clinical use of the M-CHAT are reviewed, including description of the structured follow-up interview which reduces the false-positive rate of the parent-report M-CHAT. PMID- 16685178 TI - Early medical history of children with autism spectrum disorders. AB - Previous studies have suggested that children with autism spectrum disorders (ASD) may have different medical histories than nonspectrum children in several areas: their reactions to vaccinations, number of ear infections, chronic gastrointestinal problems, and use of antibiotics. Furthermore, some studies have found associations between regressive autism and gastrointestinal (GI) symptoms. The present study analyzes the medical records from birth to the age of 2 years of 99 children (24 typically developing; 75 with ASD, of whom 29 had parent reported regression). Data were coded in the following areas: frequency and purpose of pediatrician visits, frequency and type of illnesses and medications, type and chronicity of GI complaints, date of vaccinations, growth data, and whether the pediatrician noted behaviors indicative of an ASD before the age of 2 years. Children with ASD were found to have significantly more ear infections than the typically developing children as well as to use significantly more antibiotics. Typically developing children had significantly more illness-related fevers. There was a nonsignificant trend toward the ASD group having more chronic gastrointestinal problems. There were no significant differences between the groups for the age of vaccination or for number of pediatrician visits. Finally, pediatricians noted symptoms of onset of possible autism, including language delay, for 44 of the 75 children with ASD and 2 of the 24 typical children. Results are discussed in terms of needs for future research. PMID- 16685179 TI - Frequency of gastrointestinal symptoms in children with autistic spectrum disorders and association with family history of autoimmune disease. AB - This is a cross-sectional study that compares lifetime prevalence of gastrointestinal (GI) symptoms in children with autistic spectrum disorders (ASDs) and children with typical development and with other developmental disabilities (DDs) and examines the association of GI symptoms with a family history of autoimmune disease. A structured interview was performed in 50 children with ASD and 2 control groups matched for age, sex, and ethnicity-50 with typical development and 50 with other DDs. Seventy-four percent were boys with a mean age of 7.6 years (SD, +/-3.6). A history of GI symptoms was elicited in 70% of children with ASD compared with 28% of children with typical development (p <.001) and 42% of children with DD (p =.03). Abnormal stool pattern was more common in children with ASD (18%) than controls (typical development: 4%, p =.039; DD: 2%, p =.021). Food selectivity was also higher in children with ASD (60%) compared with those with typical development (22%, p =.001) and DD (36%, p =.023). Family history of autoimmune disease was reported in 38% of the ASD group and 34% of controls and was not associated with a differential rate of GI symptoms. In the multivariate analysis, autism (adjusted odds ratio (OR), 3.8; 95% confidence interval (CI), 1.7-11.2) and food selectivity (adjusted OR, 4.1; 95% CI, 1.8-9.1) were associated with GI symptoms. Children with ASD have a higher rate of GI symptoms than children with either typical development or other DDs. In this study, there was no association between a family history of autoimmune disease and GI symptoms in children with ASD. PMID- 16685180 TI - Autism spectrum disorders and attention-deficit/hyperactivity disorder in boys with the fragile X premutation. AB - Fragile X syndrome (FXS) is caused by a full mutation expansion (>200 CGG repeats) in the FMR1 gene that results in a deficiency of the fragile X mental retardation protein. Although most individuals with the premutation (55-200 CGG repeats) are considered unaffected by FXS, recent case studies have documented children with the premutation who have cognitive deficits, behavioral problems, and/or autism spectrum disorders. The objective of this study was to compare the prevalence of autism spectrum disorders (ASD) and attention-deficit hyperactivity disorder (ADHD) symptoms in boys with the premutation who presented as probands, in brothers with the premutation who did not present as probands, and in normal brothers of premutation and/or full mutation carriers. Participants included 43 male children: 14 probands who presented to clinic, 13 nonprobands who were identified through cascade testing (routine genetic testing of family members after identification of a proband) and confirmed to have the premutation, and a control group of 16 male siblings of individuals with the fragile X premutation or full mutation who were negative for the FMR1 mutation. Participants came from 1 of 2 collaborative sites: University of California, Davis and La Trobe University in Australia. Parents completed the Conners' Global Index-Parent Version for assessing symptoms of ADHD and the Social Communication Questionnaire (SCQ) for identifying symptoms of ASD. Children who were in the ASD range on the SCQ (n = 13) underwent further evaluation with either the Autism Diagnostic Observation Schedule-Generic (n = 10) or the Autism Diagnostic Interview-Revised (n = 3). A final diagnosis of ASD included clinical assessment utilizing DSM-IV TR criteria in addition to the standardized assessments. There was a higher rate of ASD in boys with the premutation presenting as probands (p < 0.001) or nonprobands (p < .04) compared with sibling controls without the premutation. In addition, probands had a significant increase in ADHD symptoms compared with controls (p < .0001). Of the probands, 93% had symptoms of ADHD and 79% had ASD. In the nonproband premutation group, 38% had symptoms of ADHD and 8% had ASD. Thirteen percent of sibling controls had symptoms of ADHD and none had ASD. IQ scores were similar in all 3 groups (p = .13), but the use of psychotropic medications was significantly higher in probands with the premutation compared with that in controls (p < .0001). Developmental problems have been observed in premutation carriers, particularly those who present clinically with behavioral difficulties. Although this study is based on a small sample size, it suggests that premutation carriers, even those who do not present clinically, may be at increased risk for an ASD and/or symptoms of ADHD. If the premutation is identified through cascade testing, then further assessment should be carried out for symptoms of ADHD, social deficits, or learning disabilities. PMID- 16685181 TI - Early intensive behavioral treatment: replication of the UCLA model in a community setting. AB - Although previous studies have shown favorable results with early intensive behavioral treatment (EIBT) for children with autism, it remains important to replicate these findings, particularly in community settings. The authors conducted a 3-year prospective outcome study that compared 2 groups: (1) 21 children who received 35 to 40 hours per week of EIBT from a community agency that replicated Lovaas' model of EIBT and (2) 21 age- and IQ-matched children in special education classes at local public schools. A quasi-experimental design was used, with assignment to groups based on parental preference. Assessments were conducted by independent examiners for IQ (Bayley Scales of Infant Development or Wechsler Preschool and Primary Scales of Intelligence), language (Reynell Developmental Language Scales), nonverbal skill (Merrill-Palmer Scale of Mental Tests), and adaptive behavior (Vineland Adaptive Behavior Scales). Analyses of covariance, with baseline scores as covariates and Year 1-3 assessments as repeated measures, revealed that, with treatment, the EIBT group obtained significantly higher IQ (F = 5.21, p = .03) and adaptive behavior scores (F = 7.84, p = .01) than did the comparison group. No difference between groups was found in either language comprehension (F = 3.82, p = .06) or nonverbal skill. Six of the 21 EIBT children were fully included into regular education without assistance at Year 3, and 11 others were included with support; in contrast, only 1 comparison child was placed primarily in regular education. Although the study was limited by the nonrandom assignment to groups, it does provide evidence that EIBT can be successfully implemented in a community setting. PMID- 16685182 TI - Parental perceptions and use of complementary and alternative medicine practices for children with autistic spectrum disorders in private practice. AB - The prevalence of autistic spectrum disorder (ASD) in the United States is approximately 1 in 150 children. Many health care providers are unaware of parental beliefs and treatments, both medical and complementary, that parents use for their child with ASD. Understanding these beliefs and practices concerning diagnosis, cause, and utilization of medical and complementary care may help physicians provide better comprehensive care. Parents of children with ASD from 2 private practices-one in New York and one in New Jersey-were mailed a 6-page, self-administered survey. In addition to demographics and ASD type, the survey asked parents who diagnosed their child and if there was a perceived delay in that diagnosis; whether they believed there was any causal reason for their child's autism; what chronic symptoms, if any, their child experiences; and, if they had used any complementary and/or alternative therapies and at whose recommendation. Respondents included 77 of the 150 parents (51%) contacted. Most children were diagnosed by a neurologist and/or developmental pediatrician (54% and 47%, respectively). Average perceived delay in diagnosis was 18 months. Parents most frequently cited immunizations (54%), genetic predisposition (53%), and environmental exposure (38%) as a cause of their child's autism. Approximately half of children were reported as having at least one gastrointestinal, neurological, and/or allergic symptom; more than a third had immunological symptoms. Almost all parents (95%) indicated some use of complementary and alternative medicine (CAM) therapies, with most of the self reported referrals generated from a physician or nurse (44%). Systemic complaints, parental beliefs, and use of CAM practices warrant open discussion by all health care professionals who provide care to this population. PMID- 16685183 TI - Elimination diets in autism spectrum disorders: any wheat amidst the chaff? AB - The use of complementary or alternative treatment approaches in children with autism spectrum disorders (ASDs) is increasing, and the most popular of such approaches are diets that eliminate either gluten or casein, or both. The popularity of these diets indicates a need for more rigorous research into their efficacy. Owing to significant methodological flaws, the currently available data are inadequate to guide treatment recommendations. The purpose of this review is to examine the available trials of gluten/casein diets in children with ASDs regarding the strength of their findings and also concerning points that may be useful in the design of future studies. Seven trials of these diets in ASD are critically reviewed; 6 of these were uncontrolled trials and 1 used a single blind design. All reported efficacy in reducing some autism symptoms, and 2 groups of investigators also reported improvement in nonverbal cognition. Design flaws in all of the studies weaken the confidence that can be placed in their findings. Careful double-blind, placebo-controlled studies are needed to evaluate whether actual benefit undergirds the diets' popularity and to provide better guidance to clinicians and caregivers. The literature currently available suggests that diets eliminating both gluten and casein (rather than either alone) should be studied first and that outcome measures should include assessments of nonverbal cognition. PMID- 16685186 TI - Autism: its recognition, early diagnosis, and service implications. PMID- 16685187 TI - Autism and family home movies: a comprehensive review. AB - In this article, we focus on the early development of autism studied through family home movies. We review all investigations published in English that met specific methodological standards, including the use of comparison samples, coding blind to group membership, and adequate levels of interrater reliability. After discussing in detail the pros and cons of the home-movie methodology, we review the results of all empirical studies conducted to date. We then present a summary of the features found consistently across studies that differentiate autism from typical development and mental retardation in the first 2 years of life. How family home movies can contribute to our understanding of the regression phenomenon is also addressed. Finally, the results are interpreted from both a theoretical and clinical point of view. PMID- 16685188 TI - Early language and communication development of infants later diagnosed with autism spectrum disorder. AB - It is well recognized that delayed "first words" is among the most common presenting symptoms of autistic spectrum disorders (ASD). However, data on earlier language and communication development in children with ASD are limited to retrospective reports from parents and from home videos. In this study, we prospectively collected parent report data on early communication and language development in 97 infant siblings of children with ASD and 49 low-risk controls. Parents completed the MacArthur Communicative Development Inventory--Infant Form at 12 and 18 months. Analysis compared 3 groups defined on the basis of diagnostic assessment at 24 months: (1) siblings with ASD (n = 15), (2) siblings not meeting diagnostic criteria for ASD (n = 82), and (3) low-risk controls, none of whom had ASD (n = 49). Children with ASD showed delays in early language and communication compared with non-ASD siblings and controls. At 12 months, the ASD group was reported to understand significantly fewer phrases and to produce fewer gestures. At 18 months, they showed delays in their understanding of phrases, comprehension and production of single words, and use of gestures. Siblings not diagnosed with ASD also used fewer play-related gestures at 18 months than low risk controls, even when children with identified language delays were excluded. Overall, this prospective study confirms that delays in communication and language development are apparent early in life in children with ASD, and emphasizes that developmental surveillance should include monitoring for delays in gesture, which may be among the earliest signs of ASD. PMID- 16685189 TI - Examination of the time between first evaluation and first autism spectrum diagnosis in a population-based sample. AB - Early identification of young children with an autism spectrum disorder (ASD) can lead to earlier entry into intervention programs that support improved developmental outcomes. The purpose of the present study was to examine identification and diagnostic patterns of children with ASD who live in a large metropolitan area. One hundred fifteen 8-year-old children diagnosed with ASD were identified from a population-based surveillance system at the Centers for Disease Control and Prevention. Primary variables of interest included earliest age of evaluation and earliest age of diagnosis identified from surveillance records, type of initial ASD diagnosis, evaluation sources that documented first ASD diagnosis, characteristics of professionals assigning first ASD diagnosis, and diagnostic tools used to aid the diagnostic process. We found that children with ASD identified by the surveillance system were initially evaluated at a mean of 48 months but were not diagnosed with ASD until a mean age of 61 months. There were no differences in timing of diagnosis based on sex or racial/ethnic classification, although degree of impairment associated with ASD predicted mean age at first evaluation and mean age at first ASD diagnosis. Most children were identified at non-school sources, such as hospitals and clinics; 24% of the sample did not receive a documented ASD diagnosis until entering school. Most practitioners (70%) did not use a diagnostic instrument when assigning the first ASD diagnosis. Implications for early identification of ASD are discussed. PMID- 16685190 TI - Autism spectrum disorder screening and management practices among general pediatric providers. AB - It is unclear to what extent general developmental/behavioral assessments are performed, if screening for autism spectrum disorders (ASDs) is being conducted, and what the barriers to providing such assessments are in routine pediatric practice. Therefore, this study examines (1) the factors influencing the use of general developmental and autism-specific screening tools in primary care pediatric practice, (2) the barriers to providing these assessments, and (3) pediatricians' beliefs regarding ASD prevalence. A cross-sectional survey was mailed in June 2004 to a 60% (n = 1119) random sample of Maryland and Delaware licensed pediatricians. In August 2004, a second mailing was sent to non respondents. A total of 471 (42%) of the surveys were returned, and of those, 255 (54%) who practiced in general primary care were eligible. The sample was 47% male, 69% had more than 14 years' experience, 71% were in private practices, and 56% had fewer than 10 ASD patients. Most (82%) routinely screened for general developmental delays, but only 8% screened for ASD. The main reasons reported for not screening for ASD were lack of familiarity with tools (62%), referred to a specialist (47%), or not enough time (32%). Most specialist referrals (77%) were to a developmental pediatrician. Most pediatricians (71%) believed that ASD prevalence has increased, and nearly all attributed this to changes in diagnostic criteria and treatment. Service system limitations must be overcome to increase awareness and familiarity with screening tools, provide sufficient time and resources, improve screening, and enhance provider education. PMID- 16685191 TI - Screening for autism spectrum disorders with the social communication questionnaire. AB - The Social Communication Questionnaire (SCQ) is a parent report screening measure for autism spectrum disorders (ASDs) based on the Autism Diagnostic Interview Revised (ADI-R). To examine its validity in a young sample, the SCQ was given to parents of 151 children at a mean age of 5 years, before assessment in tertiary autism or preschool clinics. Overall sensitivity was .71, the same for both clinics, but specificity was better for the preschool clinic (.62) than for the autism clinic (.53) reflecting fewer false-positives in the former. The "hit rate" was 65% with 28% of the children with autism missed by the SCQ at a cutoff score of 15 (false-negatives) and 38% of the nonautistic misidentified as having an ASD (false-positives). Item validity analysis, contrary to what was previously published, indicated that only 15 or 46% of the items distinguished between children with and without ASD in this much younger sample. False-negatives were somewhat higher functioning. The SCQ would seem to be a useful tool for identifying young children in need of further assessment and assisting in routing them to the appropriate clinic, especially if used in conjunction with a screening by a community professional. There remain questions about the "best" cutoff score to use and whether a shorter version, based on the items that distinguished autistic from nonautistic, would be more reliable and valid with younger children. Furthermore, it may be that an adjusted score is required when parents omit items or with nonverbal children who cannot be scored on some of the items. PMID- 16685192 TI - Role of the renin-angiotensin-aldosterone system in vascular remodeling and inflammation: a clinical review. AB - The concept of hypertension as primarily a consequence of altered hemodynamics has changed. Many factors are now implicated in the development of hypertensive vascular disease, and the renin-angiotensin-aldosterone system (RAAS) appears to be one of the most significant. Angiotensin II, the principal effector peptide of the RAAS, has far-reaching effects on vascular structure, growth and fibrosis, and is a key regulator of vascular remodeling and inflammation. Reactive oxygen species and a network of signaling pathways mediate angiotensin II and cellular mechanisms that promote remodeling and inflammation. The involvement of aldosterone in vessel-wall and myocardial remodeling has also come under intensive research scrutiny. Treatments that block the pathologic effects of the RAAS at several points have been shown to limit target-organ damage in hypertension and to decrease cardiovascular morbidity and mortality. Understanding the molecular and cellular mechanisms that participate in the early development of hypertensive vascular disease may lead to more targeted treatment and improved outcomes. PMID- 16685193 TI - Can the dextroenantiomer of the aromatase inhibitor fadrozole be useful for clinical investigation of aldosterone-synthase inhibition? AB - The beneficial effects of spironolactone, eplerenone, amiloride and potassium in preventing cardiovascular damage in various experimental models of salt-induced hypertension can be dissociated from blood pressure effects, and have drawn attention to the direct genomic and non-genomic actions of aldosterone at the level of the vessels, the heart and the kidneys. Exposure to endogenous aldosterone could be decreased by direct and specific aldosterone-synthase inhibition. FAD 286A, the dextroenantiomer of the aromatase inhibitor fadrozole, might be a first candidate to investigate in humans, the physiological impact and therapeutic properties of aldosterone-synthase inhibition, especially in various forms of primary aldosteronism. PMID- 16685194 TI - Spurious systolic hypertension in youth: what does it really mean in clinical practice? PMID- 16685196 TI - How to assess arterial compliance in humans. PMID- 16685195 TI - The beta2-adrenoceptor gene and hypertension: is it the promoter or the coding region or neither? PMID- 16685197 TI - Interpreting antioxidant responses to angiotensin AT1 receptor antagonists: pharmacology or chemistry? PMID- 16685198 TI - Intracrine renin and angiotensin II: a novel role in cardiovascular and renal cellular regulation. PMID- 16685199 TI - Differential persistence with initial antihypertensive therapies: a clue for understanding the needs of hypertensive patients. PMID- 16685200 TI - Hypertension in autosomal dominant polycystic kidney disease: is renin acquitted as a culprit? PMID- 16685201 TI - Spurious systolic hypertension in young adults; prevalence of high brachial systolic blood pressure and low central pressure and its determinants. AB - OBJECTIVE: To investigate the prevalence and determinants of spurious systolic hypertension (SSH) in a population-based sample of young adults and estimate their 20-year risk of coronary heart disease. POPULATION AND METHODS: Seven hundred and fifty young adults (352 men and 398 women), aged 26-31 years, from the Atherosclerosis Risk in Young Adults study were studied. Blood pressure levels were measured twice and central (aortic) pressures were derived by applanation tonometry on the radial artery using a generalized transfer function. SSH was defined as brachial systolic blood pressure (SBP) > or = 140 mmHg, brachial diastolic blood pressure (DBP) < 90 mmHg, and central SBP < 124 mmHg for men and < 120 mmHg for women. The Framingham risk score was calculated. Analysis of variance models were used to compare SSH individuals with normotensive and hypertensive males for cardiovascular risk factors. RESULTS: SSH was diagnosed in 57 men (16.1%; 95% confidence interval, 12.3-20.0) versus only three women (8%; 95% confidence interval, 0-1.6). The female population was excluded from further analysis. Compared with normotensive males, SSH individuals were heavier (88.7 versus 81.8 kg, P < 0.05) had a higher body mass index (25.8 versus 24.2 kg/m, P < 0.01) and significantly higher brachial and central SBP, DBP, pulse pressure, and mean arterial pressure. They had significantly higher pulse pressure amplification. Twenty-year Framingham risk scores based on DBP did not differ significantly between SSH subjects and normotensive individuals (2.72 versus 2.10%, respectively). CONCLUSION: SSH is predominantly found among young adult men. Apart from weight and body mass index, no other cardiovascular risk factors differed significantly between subjects with SSH and normotension or hypertension. When calculating the 20-year risk of coronary heart disease based on brachial DBP, SSH individuals were at intermediate risk between normotensive and hypertensive participants, but differences were not statistically significant. PMID- 16685202 TI - Predicting outcomes on head-up tilt based on orthostatic hypotension patterns. AB - OBJECTIVE: To assess the frequency of different orthostatic hypotension (OH) patterns in patients having supine hypertension with OH ('SHOH') versus patients with OH and normal supine blood pressure ('OH alone'); and to relate OH patterns with outcomes on head-up tilt. METHODS: Consecutive patients with nonspecific dizziness were studied with a 10-min supine, 30-min head-up tilt test. Supine hypertension was diagnosed when supine systolic blood pressure (SBP) was at least 140 mmHg and/or supine diastolic blood pressure was at least 90 mmHg. OH was defined as SBP reduction of at least 20 mmHg within 3 min of tilt. OH patterns were identified corresponding to SBP time-curves during the initial 5 min of tilt: progressive, sustained and transient patterns. RESULTS: Among 400 patients tested, 31 had 'SHOH' and 39 had 'OH alone'. Frequencies of OH patterns were similar in both groups. The progressive OH pattern predicted symptomatic hypotension, leading to early tilt termination in all 'SHOH' and 88% of 'OH alone' patients. In comparison, tilt was early terminated in 33-48% of patients with sustained OH, transient OH and without OH. Early tilt termination was unrelated to age, gender, magnitude of supine SBP, pulse pressure and nadir SBP within 5 min tilt. CONCLUSIONS: Five minutes of postural challenge permitted assessing OH patterns. Outcome on protracted tilt was related to OH patterns, the worse outcome being likened to progressive OH, both in patients with 'SHOH' and in patients with 'OH alone'. Future studies will show whether OH patterns may serve as guidance for blood pressure therapy in selected patients. PMID- 16685203 TI - Association of the beta2-adrenergic receptor gene with essential hypertension in the non-Han Chinese Yi minority human population. AB - OBJECTIVE: The human beta2-adrenergic receptor (ADRB2) gene is a candidate for contributing to the pathophysiology of essential hypertension. The aims of the present study were to investigate the associations of differing single nucleotide polymorphisms (SNPs) and haplotypes of the ADRB2 gene promoter and coding regions with essential hypertension in genetically homogeneous Hani and Yi minority groups that are non-Han Chinese. METHODS: Four SNPs in the regulatory and seven SNPs in the coding region were genotyped in 271 essential hypertension individuals and 267 controls, and eight haplotypes in the regulatory and five haplotypes in the coding region were determined and tested for association using the likelihood test statistic. RESULTS: There were significant associations of essential hypertension with separate SNPs located in both the regulatory and coding regions in the Yi minority group. In contrast, no associations of essential hypertension were detected with any of single SNPs in the Hani minority group. There is a significant difference in haplotype frequency distributions between the hypertensive participants and the controls in two groups (P < 10). CONCLUSION: The results indicate that variants at the ADRB2 locus may play a role in the pathophysiology of hypertension specifically in the Yi minority group. PMID- 16685204 TI - Association of alpha1A adrenergic receptor gene variants on chromosome 8p21 with human stage 2 hypertension. AB - OBJECTIVE AND DESIGN: We previously reported a significant linkage between human chromosome 8p22 with essential hypertension and systolic blood pressure levels. On the basis of this, we used an efficient age, sex and area-matched case-control scheme to test the association of the polymorphisms in the human alpha1A adrenergic receptor (ADRA1A) gene, located on chromosome 8p21-p11.2, with essential hypertension in a northern Han Chinese population. METHODS: Seven polymorphisms were identified by direct sequencing of genomic DNA derived from 48 randomly recruited hypertensive and 48 healthy subjects. They were also examined for association with essential hypertension in 480 stage 2 hypertensive individuals and their individually matched controls. RESULTS: We observed significantly higher frequencies of the 347Arg allele and 2547G alleles in the cases compared with their controls (P = 0.04 and 0.007, respectively). McNemar's test revealed that carriers of 2547G alleles were at a greater risk of essential hypertension with an odds ratio of 3.00 [95% confidence interval (CI) 1.23-8.35]. We then performed a conditional logistic regression to adjust the effects of conventional risk factors, revealing an odds ratio of 2.84 for carriers of the 2547G allele (95% CI 1.15-6.99). With the haplotypic probabilities estimated using PHASE software, we performed haplotype trend regression analysis, showing a significant association between haplotype 7 and essential hypertension (P = 0.02), after adjustment for conventional risk factors. CONCLUSIONS: Our findings suggest that the genetic variations in the ADRA1A gene are significantly associated with essential hypertension, and may play an important role in the development of essential hypertension in this Chinese population. PMID- 16685205 TI - Functional variants of the angiotensinogen gene determine antihypertensive responses to angiotensin-converting enzyme inhibitors in subjects of African origin. AB - OBJECTIVE: To determine whether the response to angiotensin-converting enzyme inhibitor (ACEI) monotherapy in subjects of African origin is determined by genetic variants within the angiotensinogen (AGT) gene. METHODS: A total of 194 hypertensive patients of African ancestry were recruited from district clinics in Johannesburg, South Africa. Eighty patients received open-label ACEI (enalapril or lisinopril) monotherapy, and 114 open-label calcium antagonist (nifedipine) as a drug class comparator. Twenty-four hour ambulatory blood pressure (ABP) monitoring was performed at baseline (off medication) and after 2 months of therapy. DNA was analysed for functional variants (-217G-->A and -20A-->C) of the AGT gene. The impact of genotype on ABP responses to ACEI monotherapy or calcium antagonists; and on plasma aldosterone and renin levels after ACEI monotherapy was assessed. RESULTS: Adjusting for baseline ABP and type of ACEI in the ACEI treated group, the -217G-->A variant predicted ABP responses to ACEI (n = 77; P < 0.01), but not to nifedipine (n = 108). ACEI in patients with the AA genotype of the -217G-->A variant failed to elicit an antihypertensive response [change in ABP, mmHg: systolic blood pressure (SBP) +0.84 +/- 2.89, P = 0.78; diastolic blood pressure (DBP) -0.47 +/- 1.74, P = 0.79]. In contrast, those patients with at least one copy of the -217G allele developed a 7.23 +/- 1.55 and 5.38 +/- 1.12 mmHg decrease (P < 0.0001) in SBP and DBP, respectively, after ACEI administration. Similarly, the -20A-->C variant predicted ABP responses to ACEI monotherapy (P < 0.01) but not to nifedipine. Moreover, patients who were AA genotype for both variants failed to develop an antihypertensive response to ACEI (change in ABP, mmHg: SBP +1.06 +/- 3.05, P = 0.73; DBP -0.39 +/- 1.83, P = 0.83); whereas patients with at least one copy of both the -217G and the -20C allele developed substantial decreases in ABP (change in ABP, mmHg: SBP -14.08 +/ 3.72, P < 0.0001; DBP -9.62 +/- 2.74, P < 0.0001). Patients with at least one copy of the -217G allele demonstrated a significant reduction in the aldosterone to-renin ratio (-0.098 +/- 0.035, P < 0.01), whereas in those patients who were 217AA genotype the ratio was unchanged (-0.03 +/- 0.16, P = 0.85). CONCLUSION: Functional variants of the AGT gene contribute to the variability of antihypertensive responses to ACEI monotherapy in individuals of African ancestry, with genotype determining whether or not responses occur. PMID- 16685206 TI - Chlorogenic acid attenuates hypertension and improves endothelial function in spontaneously hypertensive rats. AB - BACKGROUND AND OBJECTIVES: Epidemiologic studies indicate that ingestion of vegetables and fruit inhibits the development of cardiovascular disease. Chlorogenic acids are abundant phenolic compounds contained in vegetables and fruits, but the impact of dietary chlorogenic acids on vascular function in hypertension is not known. We therefore examined the effects of 5-caffeoylquinic acid (CQA), a representative chlorogenic acid, on blood pressure and vascular function in age-matched normotensive Wistar-Kyoto rats and spontaneously hypertensive rats. METHODS AND RESULTS: A single ingestion of CQA (30-600 mg/kg) reduced blood pressure in spontaneously hypertensive rats, an effect that was blocked by administration of a nitric oxide synthase inhibitor, N(G)-nitro-L arginine methyl ester. When spontaneously hypertensive rats were fed diets containing 0.5% CQA for 8 weeks (approximately 300 mg/kg per day), the development of hypertension was inhibited compared with the control diet group. CQA ingestion increased urinary excretion of nitric oxide metabolites and decreased urinary excretion of hydrogen peroxide; decreased NADPH-dependent superoxide anion production in the aorta, suggesting that dietary CQA inhibited vascular NADPH oxidase activity; significantly improved acetylcholine-induced endothelium-dependent vasodilation in the aorta; and markedly reduced the degree of immunohistochemical staining for nitrotyrosine and media hypertrophy in aorta sections. In contrast, CQA had no effects in Wistar-Kyoto rats. CONCLUSIONS: Dietary CQA reduces oxidative stress and improves nitric oxide bioavailability by inhibiting excessive production of reactive oxygen species in the vasculature, and leads to the attenuation of endothelial dysfunction, vascular hypertrophy, and hypertension in spontaneously hypertensive rats. PMID- 16685207 TI - A comparison of three different methods to determine arterial compliance in the elderly: the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study. AB - BACKGROUND: Several different techniques to evaluate arterial compliance have been described but have not been simultaneously tested in a large-scale, population-based setting. This study aimed to evaluate the feasibility and relation to cardiac risk of three of these techniques in the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study. METHODS AND RESULTS: In the population-based PIVUS study (1016 participants aged 70), assessment of arterial distensibility by ultrasound in the carotid artery, by pulse wave analysis (augmentation index) and the stroke volume to pulse pressure ratio by echocardiography were successfully employed in 86, 92, 91 and 77% of the sample, respectively. All three indices of arterial compliance were inter-related (r = 0.19-0.34, P < 0.0001 for all). Although all three indices were significantly related to the Framingham risk score (r = 0.12-0.32, P = 0.0005-0.0001), only carotid artery distensibility and the stroke volume to pulse pressure ratio were independently associated with the Framingham score in multiple regression analysis (P < 0.0001 for both). CONCLUSIONS: All three indices to evaluate arterial compliance were feasible to obtain in a general elderly population and were inter-related. Although all of the techniques were correlated to Framingham risk score, only carotid artery distensibility and the stroke volume to pulse pressure ratio were independently related to coronary risk, suggesting complementary use of these two indices of arterial compliance in the future. PMID- 16685208 TI - Interleukin 6 is associated with subclinical atherosclerosis: a link with soluble intercellular adhesion molecule 1. AB - BACKGROUND: Among the markers of inflammation, a cytokine, interleukin (IL)-6, promotes the expression of intercellular adhesion molecule 1 (ICAM-1), C-reactive protein (CRP) synthesis, and leads to a series of procoagulant actions with potential major implications on the progression of atherosclerosis. AIM OF THE STUDY: To analyse in a population-based study, the relationship between IL-6 and atherosclerotic lesions and the role of serum ICAM-1 and CRP on this relationship. POPULATION: Among 1015 individuals randomly recruited between 1995 and 1997 in Haute-Garonne, a French region with a low cardiovascular risk, 953 subjects with complete data for all measurements were analysed. Common carotid intima-media thickness (IMT) and the presence of plaques in the carotid and femoral arteries were assessed by ultrasonography. RESULTS: Quartiles of IL-6, serum ICAM-1 and CRP were positively associated with plaques and IMT. After adjustment for traditional risk factors, IL-6 (P < 0.001) and serum ICAM-1 (P < 0.002) remained positively associated with plaques but not CRP (P = 0.20). Neither IL-6, nor serum ICAM-1, nor CRP were independently associated with IMT. When serum ICAM-1 was entered into the model in addition to traditional risk factors and IL-6, the percentage of variance in the number of plaques explained by the model did not increase significantly. CONCLUSION: IL-6 levels are associated with subclinical atherosclerotic lesions independently of traditional risk factors; the influence of IL-6 on ICAM-1 secretion may play a role in this association. These results argue the interest of IL-6 in the stratification of cardiovascular risk. PMID- 16685209 TI - Synergistic effect of mechanical stretch and angiotensin II on superoxide production via NADPH oxidase in vascular smooth muscle cells. AB - OBJECTIVE: Mechanical forces and angiotensin II influence the structure and function of vascular cells, and play an important role in reactive oxygen species production. In this study, we examined the effects of mechanical stretch and angiotensin II on the expression of p22-phox and Nox-1, essential membrane components of NADPH oxidase, and superoxide production in rat vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Neither a stretch force nor angiotensin II alone altered p22-phox and Nox-1 expression in VSMCs. Combined stimulation markedly increased p22-phox and Nox-1 mRNA, however, which was associated with increased NADPH oxidase activity, superoxide production and total 8-iso-prostaglandin F2alpha concentration. The increases in p22-phox mRNA levels induced by a stretch force in combination with angiotensin II were prevented by treatment with an angiotensin type I (AT1) receptor antagonist, RNH-6270 (100 nmol/l). Protein expression of the AT1 receptor was upregulated by a stretch force. CONCLUSIONS: These data indicate that mechanical stretch and angiotensin II synergistically increase NADPH oxidase expression in VSMCs, and suggest that part of this mechanism is mediated through an upregulation of the AT1 receptor induced by mechanical stretch. The combined effects of mechanical strain and angiotensin II might promote vascular damage through the production of superoxide in a hypertensive state. PMID- 16685210 TI - Contribution of reactive oxygen species to the pathogenesis of left ventricular failure in Dahl salt-sensitive hypertensive rats: effects of angiotensin II blockade. AB - OBJECTIVE: We investigated the contribution of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) generation to the pathogenesis of diastolic heart failure (DHF) in Dahl salt sensitive (DS) hypertensive rats, with the aim of testing our hypothesis that the cardioprotective effects of angiotensin II (Ang II) blockade are provided by the suppression of this pathway. METHODS: DS rats were maintained on high (H: 8.0% NaCl) or low (L: 0.3% NaCl) salt diets from age 7 to 17 weeks. DS/H rats were also treated with candesartan cilexetil (10 mg/kg per day, orally) or a superoxide dismutase mimetic, tempol (3 mmol/l in drinking water) from age 7 to 17 weeks. RESULTS: DS/H rats represented hypertension, left ventricular (LV) relaxation abnormality and myocardial stiffening with preserved systolic heart function. As compared with DS/L rats, DS/H rats showed higher levels of transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), p22phox and gp91phox mRNA expression, NADPH oxidase activity and thiobarbituric acid-reactive substance (TBARS) contents in LV tissues. Gene expression of uncoupling protein-2 (UCP-2), an inner mitochondrial membrane proton transporter, was also 2.8 +/- 0.5-fold higher. In DS/H rats, treatment with candesartan did not alter blood pressure, but resulted in a marked improvement of the hemodynamic deterioration; these therapeutic effects were accompanied by decreases in myocardial NADPH oxidase activity, TBARS contents and the expression of TGF-beta, CTGF, p22phox, gp91phox and UCP-2. Similar therapeutic effects were provided by treatment with tempol in DS/H rats. CONCLUSIONS: Our data suggest that NADPH oxidase-mediated ROS production contributes to the pathogenesis of DHF in DS hypertensive rats, and that the cardioprotective effects of AngII blockade are, at least partially, mediated through the suppression of this pathway. PMID- 16685211 TI - Transient receptor potential channels in essential hypertension. AB - OBJECTIVE: The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated. METHODS: We studied TRPCs in 51 patients with essential hypertension and 51 age-matched and sex-matched normotensive control subjects. Calcium and gadolinium influx into human monocytes was determined using the fluorescent dye technique. TRPC expression was measured using reverse transcriptase-polymerase chain reaction and in-cell western assay. Gene silencing by small interfering RNA for specific TRPC knockdown was also performed. RESULTS: We observed an increased gadolinium/calcium-influx ratio through TRPC in essential hypertensive patients compared with normotensive control subjects [cation influx ratio (mean +/- SEM), 125 +/- 14 versus 80 +/- 7%; each n = 51; P < 0.01], due to an increase of gadolinium influx in hypertensive patients compared with normotensive control subjects (48 +/- 4 versus 36 +/- 3%; each n = 51; P < 0.05). We observed a significant increase of TRPC3 and TRPC5 protein expression in essential hypertensive patients compared with normotensive control subjects (normalized TRPC3 expression, 3.21 +/- 0.59 versus 1.36 +/- 0.07; each n = 20; P < 0.01; normalized TRPC5 expression, 2.10 +/- 0.28 versus 1.40 +/- 0.52; each n = 12; P < 0.05). We used small interfering RNA for knockdown of TRPC5. The thereby reduced channel expression caused a significant attenuation of calcium and gadolinium influx. CONCLUSION: This study points to an important role of TRPCs in essential hypertension. PMID- 16685212 TI - Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II. AB - CONTEXT: RGS2 (regulators of G-protein signaling) is a negative regulator of Galphaq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. OBJECTIVE: To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. METHODS: PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3' untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). RESULTS: RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. CONCLUSION: Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans. PMID- 16685214 TI - Differential regional haemodynamic changes during mineralocorticoid hypertension. AB - BACKGROUND: Primary aldosteronism is a cause of hypertension in up to 10% of hypertensive patients, but the mechanisms by which excess aldosterone raises arterial pressure remain unclear. OBJECTIVE: To investigate the systemic and regional haemodynamic changes during the development and maintenance of aldosterone-induced hypertension and the effect of sympathetic and vasopressin blockade. METHODS: Responses to intravenous infusion of aldosterone (10 microg/h) for 4 weeks were determined in five conscious sheep. The effects of sympathetic blockade with propranolol and phentolamine or vasopressin V1-receptor blockade with SR59049 were investigated in six further sheep infused with aldosterone. RESULTS: Aldosterone progressively increased the mean arterial pressure by 20 mmHg over 4 weeks (P < 0.001). The changes in cardiac output were variable between animals, resulting in no overall significant change. Total peripheral conductance was significantly decreased due to selective reductions in mesenteric conductance (from 6.17 +/- 0.27 to 4.46 +/- 0.15 ml/min per mmHg, P < 0.001) and iliac conductance (from 1.54 +/- 0.21 to 1.27 +/- 0.15 ml/min per mmHg, P < 0.001). In contrast, renal and coronary conductance were unchanged and renal blood flow increased from 290 +/- 17 to 350 +/- 28 ml/min (P < 0.01) and coronary blood flow from 34.7 +/- 3.0 to 44.6 +/- 2.5 ml/min (P < 0.05). These aldosterone induced changes were not inhibited by sympathetic or vasopressin V1-receptor blockade. CONCLUSION: Excess aldosterone caused a slow progressive increase in arterial pressure, which in the long term depended on reduced total peripheral conductance. This resulted from vasoconstriction in the gut and skeletal muscle, but not the kidney. These effects were not mediated by the sympathetic nervous system or vasopressin. PMID- 16685213 TI - Blood pressure variability is more important than blood pressure level in determination of end-organ damage in rats. AB - OBJECTIVE: This study was designed to determine how important a novel risk factor of elevated blood pressure variability (BPV) is in the determination of end-organ damage by comparison with the classic risk factor of a high blood pressure (BP) level. METHODS AND RESULTS: The effects of haemodynamics on cardiovascular morphology were evaluated by univariate and multivariate regression analysis in two different rat models with an enlarged distribution of haemodynamics. In male sham-operated and sinoaortic-denervated Wistar-Kyoto rats and spontaneously hypertensive rats (n = 34), BPV was more important than BP in cardiac and renal damage and aortic hypertrophy. BPV and BP had independent effects, explaining 59.4% of the variation in damage to these organs. In male (n = 44) and female (n = 46) F1 hybrids of Sprague-Dawley rats and spontaneously hypertensive rats, the greater importance of BPV than BP was further demonstrated in left ventricular hypertrophy, glomerular damage and aortic hypertrophy. The phenomenon was more evident in females than males for cardiovascular hypertrophy. BPV and BP or BPV alone had independent effects, explaining 46.9% (male) or 37.5% (female) of the variation in damage to these organs. CONCLUSION: BPV is a more critical determinant than BP level for cardiac damage, renal lesions and aortic hypertrophy in rats, strongly suggesting the significance of BPV control for the protection of these organs. PMID- 16685215 TI - Investigation of aldosterone-synthase inhibition in rats. AB - BACKGROUND: In-vivo investigation of aldosterone-synthase inhibitors requires experimental models to characterize the biological effects of these compounds. METHODS: Seven successive experiments were performed in groups of 2-month-old male spontaneously hypertensive rats. Urinary free aldosterone was the main end point measured during two contrasted diets: low sodium-high potassium (LS), inducing high urinary aldosterone (839 pmol/24 h, 95% confidence interval 654 1077), and high sodium-normal potassium (HS), inducing low urinary aldosterone (38.1 pmol/24 h; 95% confidence interval, 32.4-44.9). RESULTS: FAD 286 A (10 and 30 mg/kg) decreased urinary free aldosterone by 53 and 87% on the LS diet, and 50 and 75% on the HS. Plasma renin concentration increased three-fold after a 4-week treatment of 30 mg/kg FAD 286 A on the LS diet and did not change on the HS. The combination of FAD 286 A (30 mg/kg) and spironolactone (30 mg/kg) on the LS diet induced a biological picture of severe hypoaldosteronism and was not tolerated, whereas the HS diet prevented these abnormalities. The combination of FAD 286 A (30 mg/kg) and furosemide (30 mg/kg) on the HS diet corrected the diuretic induced hypokalemia (4.1 +/- 0.2 versus 3.7 +/- 2.2 mEq/l, P < 0.033). CONCLUSION: This experimental model will be useful to screen future aldosterone synthase inhibitors and study their biological effects in various experimental conditions. PMID- 16685216 TI - Waist circumference and the metabolic syndrome predict the development of elevated albuminuria in non-diabetic subjects: the DESIR Study. AB - OBJECTIVE: Metabolic determinants of microalbuminuria remain poorly understood in non-diabetic individuals and particularly in women. We investigated in both sexes whether an elevated waist circumference (WC) or the presence of the metabolic syndrome (MetS) predict the development of elevated albuminuria at 6 years. DESIGN AND PATIENTS: We studied 2738 subjects from the DESIR cohort without microalbuminuria or diabetes at baseline and who were followed up for 6 years. RESULTS: At 6 years, 254 individuals [9.3%; 95% confidence interval (CI) 8.2 10.4%] had developed elevated albuminuria (> or = 20 mg/l), which was significantly and positively associated with WC and blood pressure, but not with fasting glucose, lipids or body mass index in either sex. In both sexes, subjects with a high WC or with MetS at baseline were more likely to develop elevated albuminuria at 6 years compared with those with a normal WC or absence of MetS. In multivariate logistic analysis, WC as a continuous variable or a WC of 94 cm or greater for men and a WC greater than 88 cm for women were predictive of the development of elevated albuminuria, after adjusting for age, hypertension, the use of angiotensin-converting enzyme inhibitors, fibrinogen and glycaemia. MetS was a risk factor for elevated albuminuria in men (odds ratio 1.87; 95% CI 1.25 2.81), with differences according to the MetS definition. CONCLUSION: Abdominal adiposity is related to the development of elevated albuminuria in both sexes, suggesting that the measurement of WC may improve the identification of non diabetic individuals at risk of developing microalbuminuria and emphasizing the interest of screening for albuminuria among those with MetS. PMID- 16685217 TI - Hemodynamic and metabolic responses to interstitial angiotensin II in normal weight and obese men. AB - OBJECTIVE: The expression of the AT1 receptor in adipose tissue is not decreased or even increased in obese subjects despite systemic activation of the renin angiotensin system. Therefore, we hypothesized that peripheral tissues of obese subjects are hypersensitive to angiotensin (Ang) II. METHODS: We characterized the effect of locally applied Ang II in skeletal muscle and subcutaneous adipose tissue of healthy non-obese (n = 12) and obese (n = 11) men using the microdialysis technique. Tissues were perfused with Ringer's solution + ethanol and incremental doses of Ang II (0.01, 0.1 and 1 micromol/l). Dialysate ethanol, glycerol, glucose, lactate, and pyruvate concentrations were measured to assess changes in blood flow (ethanol dilution technique), lipolysis and glycolysis, respectively. RESULTS: In adipose tissue, basal ethanol ratio was significantly higher and dialysate metabolite concentrations were significantly lower in obese versus non-obese men. In muscle, basal dialysate glycerol was significantly higher in obese versus non-obese men. Ang II elicited small increases in ethanol ratio and decreases in dialysate glucose in adipose tissue and skeletal muscle in both non-obese and obese men. Dialysate lactate increased significantly in both tissues of obese, but not non-obese men. Dialysate glycerol increased in adipose tissue of non-obese (+ 40%) but not of obese and remained almost unchanged in muscle of both groups. CONCLUSIONS: Interstitially applied Ang II elicits subtle changes in tissue perfusion and metabolism. However, we did not find a major increase in interstitial Ang II responsiveness in obese men. PMID- 16685218 TI - Left atrial volume in end-stage renal disease: a prospective cohort study. AB - BACKGROUND: End-stage renal disease (ESRD) is a high-risk condition and left ventricular hypertrophy (LVH) is the strongest risk factor in this population. OBJECTIVE AND METHODS: Since the prognostic value of left atrial (LA) size in ESRD is still unknown, we performed a prospective cohort study aimed at testing the prognostic value of LA volume in a cohort of 249 ESRD patients. RESULTS: Both un-indexed and indexed LA volume (LAV) was significantly higher in dialysis patients than in healthy subjects (P < 0.001). On multivariate analysis only left ventricular mass index (LVMI), LV ejection fraction (LVEF), ratio of early (E) to late atrial (A) mitral Doppler peak flow velocity (E/A ratio) and antihypertensive treatment maintained an independent association with LAV. During the follow-up 113 patients died. LAV added significant prognostic power to a multivariate Cox model of all-cause death and the model based on height provided the best data fit. Notably, this index maintained an independent predictive value for death (P = 0.03) also when LVMI and LVEF were jointly forced into the Cox's model. Neither crude nor body surface area (BSA)-adjusted LAV had an independent association with death when tested in the Cox model including LVMI and LVEF. CONCLUSIONS: In patients with ESRD, LAV indexed for height displays prognostic value beyond and above that provided by LV mass and function. PMID- 16685219 TI - Renin increments the inward calcium current in the failing heart. AB - BACKGROUND: Evidence is available that activation of the renin-angiotensin system is involved in cardiac remodeling. It is unknown whether renin can change the inward calcium current (ICa) in the failing heart. This problem was investigated in the present study. METHODS: Cardiomyocytes were isolated from the ventricle of 4-month-old cardiomyopathic hamsters and measurements of the L-type ICa were performed using the patch-clamp technique in a whole-cell configuration. RESULTS: Extracellular renin (128 pmol Ang I/ml per min) plus angiotensinogen (110 pmol angiotensin I generated by renin to exhaustion) incremented the peak ICa density significantly, an effect suppressed by enalapril maleate (10 mol/l) or by losartan (10 mol/l) added to the bath, indicating that the effect of renin plus angiotensinogen was related to the formation of angiotensin I and its conversion to angiotensin II at the surface cell membrane. Renin internalization seems to increment the ICa because intracellular dialysis of renin (128 pmol Ang I/ml per min) plus angiotensinogen (110 pmol angiotensin I generated by renin to exhaustion) also increased the peak ICa density significantly, an effect suppressed by intracellular losartan (10 mol/l) but not by extracellular losartan (10 mol/l). CONCLUSIONS: Extracellular renin plus angiotensinogen increases the ICa in isolated myocytes from the failing heart of cardiomyopathic hamsters through the formation of angiotensin II and the activation of angiotensin type 1 receptors at the surface cell membrane. A similar increment of ICa was found with intracellular administration of renin plus angiotensinogen. This finding might indicate that renin internalization is involved in control of inward calcium current in the failing heart. PMID- 16685220 TI - Improvement in hypertension management in England: results from the Health Survey for England 2003. AB - OBJECTIVE: To evaluate the mean levels of blood pressure and hypertension (> or = 140 mmHg systolic or > or = 90 mmHg diastolic pressure or on treatment for hypertension) in the adult English population, and to evaluate any changes in the efficacy of hypertension management between 1994 and 2003. DESIGN/METHODS: Cross sectional surveys. England, 2003. A nationally representative sample of 8834 non institutionalized adults (aged > or = 16 years). Rates of awareness, treatment and control of hypertension. RESULTS: Since 1994, mean systolic blood pressure has fallen by 1.6 and 4.3 mmHg in male and female adults, respectively. The rates of awareness and treatment have increased, and control rates (< 140 mmHg systolic and < 90 mmHg diastolic) among hypertensive men and women have approximately doubled to 21.5 and 22.8%, respectively. Of those on treatment for hypertension, the majority (56%) are on two or more agents compared with 40% in 1994 and 1998. CONCLUSION: Hypertension management has improved greatly since 1994, with more awareness, treatment and control. Nevertheless, in 2003 the majority of hypertensive adults in England had blood pressure levels above the currently recommended targets. PMID- 16685221 TI - Lower target blood pressures are safe and effective for the prevention of recurrent stroke: the PROGRESS trial. AB - OBJECTIVE: To explore the likely optimum blood pressure (BP) level for patients with a history of cerebrovascular disease. METHODS: The Perindopril Protection Against Recurrent Stroke Study (PROGRESS) was a randomized, placebo-controlled trial that established the beneficial effects of BP lowering in 6105 patients with cerebrovascular disease. The present study comprises two series of post hoc analyses. The first was designed to investigate the effects of randomized treatment on recurrent stroke by baseline BP levels, and the second was a corresponding observational analysis investigating the association between achieved follow-up BP levels and recurrent stroke risk. RESULTS: Analyses of the randomized treatment comparisons showed that BP lowering with combination therapy produced similar risk reductions in each of four subgroups defined by baseline BP of less than 120, 120-139, 140-159, and 160 mmHg or greater (P homogeneity = 0.5). The effects of single-drug therapy were also comparable across these subgroups (P homogeneity = 0.2), but consistently greater benefits were observed with combination compared to single-drug therapy. The analyses of achieved follow up BP showed that the lowest risk of recurrence was among the one-quarter of participants with the lowest follow-up BP levels (median 112/72 mmHg), and that risks rose progressively with higher follow-up BP levels. Minor side-effects were progressively more common at lower BP levels (P homogeneity = 0.04), but there was no excess of serious complications (all P homogeneity > 0.2). CONCLUSION: These analyses provide no evidence of a J-curve relationship between BP level and stroke risk among patients with cerebrovascular disease, and identify no patient group among whom more intensive BP lowering would not be expected to produce greater risk reductions. PMID- 16685223 TI - A1079T transversion in the gene for the alpha1 isophorm of the Na+/K+ ATPase in the Dahl S rat. PMID- 16685222 TI - Discontinuation of antihypertensive drugs among newly diagnosed hypertensive patients in UK general practice. AB - OBJECTIVES: To evaluate antihypertensive drug discontinuation among newly diagnosed hypertensive patients. METHODS: This was a population-based cohort study using the UK General Practice Research Database (GPRD). Patients newly diagnosed with hypertension between 1991 and 2001 and subsequently treated with antihypertensive drugs were included. Overall antihypertensive drug discontinuation was evaluated from a patient's first-ever antihypertensive prescription. Class-specific discontinuations were evaluated from a patient's first-ever prescriptions of angiotensin-converting enzyme (ACE) inhibitors (ACE I), alpha antagonists, angiotensin-2 antagonists (AIIA), beta blockers, calcium channel blockers (CCB), miscellaneous, potassium-sparing diuretics, and thiazides. Discontinuation occurred when no antihypertensive prescription was issued within 90 days following the most recent prescription expiration. RESULTS: The study population comprised 109 454 patients, with 223 228 antihypertensive drug-class episodes contributing to the class-specific analysis. Overall antihypertensive drug discontinuation was 20.3% [95% confidence interval (CI): 20.0, 20.5%] at 6 months and 28.5% (95% CI: 28.2, 28.7%) at 1 year, with a median time to discontinuation of 3.07 years. The median time to antihypertensive class discontinuation was longest for AIIAs (2.90 years) followed by ACE-I (2.24), CCB (1.86), beta blockers (1.50), thiazides (1.50), alpha antagonists (1.35), potassium-sparing diuretics (0.40), and miscellaneous (0.39). One-year discontinuation ranged from 29.4% (95% CI: 28.0, 30.7) for AIIAs to 64.1% (95% CI: 62.1, 66.3) for potassium-sparing diuretics. Forty-four percent who discontinue their first-ever antihypertensive drug class failed to switch to a different drug class within 90 days of discontinuation. CONCLUSION: It is important that general practitioners (GPs) monitor patients closely in the first year following antihypertensive drug initiation, due to the high early risk of discontinuation, and the low percentage of patients who switch to a different antihypertensive drug class after a drug-class discontinuation. AIIA, followed by ACE-I and CCB, had the lowest risk of discontinuation among antihypertensive drug classes. PMID- 16685225 TI - FEVER study: a trial further supporting the concept of a blood pressure independent stroke protective effect by dihydropyridines. PMID- 16685230 TI - Strategies of medical intervention in the management of acute spinal cord injury. AB - STUDY DESIGN: : Literature review. OBJECTIVE: : The purpose of this paper is to review clinical treatment strategies and future developments in the treatment of acute spinal cord injury. SUMMARY OF BACKGROUND DATA: : The treatment of acute spinal cord injury continues to be supportive. The search for specialized pharmacologic agents to prevent secondary injury and promote repair or regeneration remains heated. METHODS: : Medline search from 1996 to present limited to clinical research and basic science review articles in the English Language. RESULTS: : Steroids continue to be administered in the clinical setting of acute spinal cord injury primarily out of peer pressure and fear of litigation. Basic science experiments suggest that modulation of post-traumatic inflammation may provide the best opportunity to arrest the secondary injury cascade. Protein kinase and metalloproteinase inhibition are promising treatment strategies. Regeneration techniques are concentrating on cell transplantation and manipulating glial receptors and protein production. Clinical investigations are limited to Phase III trials on a very select few of these drugs. CONCLUSIONS: : While many advances in the basic science of spinal cord injury provide optimism for future treatments, clinical science lags. At present, there are no pharmacologic strategies of proven benefit. Although steroids continue to be given to patients with spinal cord injury in many institutions, evidence of deleterious effects continues to accumulate. Current standard of care management includes support of arterial oxygenation and spinal cord perfusion pressure. PMID- 16685231 TI - Changing face of spine trauma care in North America. AB - STUDY DESIGN: An evidence-based review and summary of literature from multiple disciplines involved in spine trauma. OBJECTIVES: To outline epidemiologic, clinical, and research issues influencing spine trauma in a longitudinal perspective. In addition, to provide guidance to clinicians and researchers to ensure that philosophies pertaining to the betterment of spine trauma care are understood and supported. SUMMARY OF BACKGROUND DATA: Epidemiologic data have provided insight into future demands the elderly patient with spine injury will place on the health care system. Regional trauma programs have emerged with further specialization resulting in regionalized spine trauma care. Evidence based guidelines have streamlined imaging, and biomaterial advancements have facilitated the stabilization of the spinal column and decompression of the spinal cord. Promising experimental therapies promoting axonal regeneration and neuroprotective agents are beginning clinical trials, generating cautious optimism that effective therapies for spinal cord injuries will emerge. The unsustainable economics of increasing technology and patient expectations will make economic evaluation critical. METHODS: Evidence-based review of current literature and expert opinion. CONCLUSIONS: Multicenter spine trauma registries with patient-reported outcomes will allow many questions around spine trauma to be answered using the highest levels of evidence. This process in synergy with technical and biologic developments should ensure progress toward optimal care of the spine trauma patient. Future challenges will be to treat the breadth and magnitude of the discoveries within the fiscal restraints of the health care system and ensure its affordability for society. PMID- 16685232 TI - Nonoperative management and treatment of spinal injuries. AB - STUDY DESIGN: Review of literature. OBJECTIVE: To delineate and discuss nonoperative treatment and treatment of spinal injuries. SUMMARY OF BACKGROUND DATA: Nonoperative methods have been a mainstay of care for spinal injuries since ancient Egypt. The vast majority of all spinal injuries should be treated in the nonoperative fashion. The indications and methods continue to evolve. METHODS: A PubMed search of the literature returned more than 1000 articles related to spine trauma. A total of 270 were references to nonoperative treatment, and 100 were thought to be relevant and included in this review. RESULTS: All spine injuries are treated in a nonoperative manner, at least initially. The vast majority of injuries are successfully and appropriately treated in a definitive manner with nonsurgical methods. Over the past 10-15 years, the advent of better rigid cervical fixation has decreased the use of halo vests as definitive treatment of many cervical injuries. In contrast, during the same time, more thoracolumbar injuries are being treated in a nonsurgical fashion because the outcomes have been shown to be similar or superior. CONCLUSIONS: As with all of medicine, the treatment of spine trauma will continue to evolve with time. It is paramount that the physician selects the treatment that will provide the best short-term recovery with the least impact on long-term function. PMID- 16685233 TI - The timing of surgical intervention in the treatment of spinal cord injury: a systematic review of recent clinical evidence. AB - STUDY DESIGN: Evidence-based literature review. OBJECTIVE: To provide updated evidence-based recommendations regarding spinal cord decompression in patients with acute spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: It is controversial whether early decompression following SCI conveys a benefit in neurologic outcome. METHODS: MEDLINE search of experimental and clinical studies showing the effect of decompression on neurologic outcome following SCI. We focused on articles published within the last 10 years, with a particular emphasis on research conducted within the past 5 years. RESULTS: A total of 66 articles were retrieved. Animal studies consistently show that neurologic recovery is enhanced by early decompression. There was 1 randomized controlled trial that showed no benefit to early (<72 hours) decompression. Several recent prospective series suggest that early decompression (<72 hours) can be performed safely and may improve neurologic outcomes. A recent systematic review showed that early decompression (<24 hours) resulted in statistically better outcomes compared to both delayed decompression and conservative treatment. CONCLUSIONS: There are currently no standards regarding the role and timing of decompression in acute SCI. We recommend urgent decompression of bilateral locked facets in a patient with incomplete tetraplegia or in a patient with SCI with neurologic deterioration. Urgent decompression in acute cervical SCI remains a reasonable practice option and can be performed safely. There is emerging evidence that surgery within 24 hours may reduce length of intensive care unit stay and reduce post-injury medical complications. PMID- 16685235 TI - Classification of lower cervical spine injuries. AB - STUDY DESIGN: Blinded assessment by multiple observers of consecutive case series. OBJECTIVES: Measure the reliability of a new system of determining stability in subaxial cervical spine injuries. SUMMARY OF BACKGROUND DATA: Classification is fundamental to allow communication, determine prognosis, and direct treatment. Current systems have many limitations, including difficultly of use, lack of proven reliability and validity, and no assessment of stability. A new system to assess instability is proposed. METHODS: A literature review of the most commonly described classification systems is reported. The Cervical Spine Injury Severity Score was tested for reliability by 10 examiners who graded 35 consecutive cases of cervical trauma. Plain radiographs and CT were saved as read using Efilm Lite in random order. Each was scored and intraobserver and interobserver agreement was measured using intraclass correlation coefficients (ICC). RESULTS: Intraobserver agreement was excellent with ICC ranging from 0.97 to 0.99. Interobserver agreement was also excellent with mean 0.80 ranging from 0.75 to 0.98. CONCLUSION: A new cervical spine classification system of injury is paramount to treatment and outcomes. A new system may increase reliability and therefore allow more accurate determination of stability and dictate treatment. PMID- 16685236 TI - The evolution of spinal instrumentation for the management of occipital cervical and cervicothoracic junctional injuries. AB - STUDY DESIGN/SETTING: Independent computer-based literature review of articles pertaining to instrumentation and fusion of junctional injuries of the cervical spine. OBJECTIVE: To review and discuss the evolution of instrumentation techniques and systems used in the treatment of cervical spine junctional injuries. SUMMARY OF BACKGROUND DATA: Instrumentation of junctional injuries of the cervical spine has been limited historically by failure to achieve rigid internal fixation in multiple planes. The evolution of these techniques has required increased insight into the morphology and unique biomechanics of the structures to be instrumented. METHODS: Computer-based literature search of Ovid and PubMed databases. RESULTS: Extensive literature search yielded insights into the evolution of systems initially based on onlay bone graft combined with wiring techniques. Such techniques have come to include systems incorporating rigid, longitudinal struts that accommodate multiplanar screws placed in the lateral masses, pedicles, transarticular regions, and occipital bone. CONCLUSIONS: Despite a rapid evolution of techniques and instrumentation technologies, it remains incumbent on the physician to provide the patient with a surgical procedure that balances the likelihood of a favorable outcome with the risk inherent in the implementation of the procedure. PMID- 16685237 TI - The contemporary treatment of odontoid injuries. AB - STUDY DESIGN: Review article. OBJECTIVE: To outline current concepts regarding the assessment and treatment of odontoid fractures. SUMMARY OF BACKGROUND DATA: Odontoid fractures account for 9% to 15% of adult, cervical spine fractures. These injuries usually result from hyperflexion or hyperextension of the cervical spine during low-energy impacts in the elderly or high-energy impacts in the young and middle aged. Neurologic injury associated with these fractures is rare. METHODS: A review of pertinent literature was conducted. The information gleaned from this review was summarized. RESULTS: Odontoid fractures should be evaluated with appropriate imaging to assess the fracture itself as well as exclude other contiguous or noncontiguous fractures. The Anderson and D'Alonzo classification system is most commonly used. True type I and III odontoid fractures are generally thought to be relatively stable and are often treated nonoperatively with immobilization. Type II fractures at the base of the odontoid are less stable, and there are differing opinions regarding the precise definition and optimal treatment of these injuries. Nonoperative treatment options for odontoid fractures include external immobilization with a collar or halo. Operative treatment options for odontoid fractures include one of several posterior C1-C2 fusion constructs or anterior odontoid fixation if the fracture pattern is amenable. CONCLUSIONS: Despite the frequency of odontoid fractures, there is still much debate regarding the optimal treatment of these fractures, especially the type II fractures. This fact may be because of the absence of an ideal solution for this clinical problem. Certainly, prospective controlled clinical studies are needed. PMID- 16685239 TI - Reliability of a novel classification system for thoracolumbar injuries: the Thoracolumbar Injury Severity Score. AB - STUDY DESIGN: Prospective study of 5 spine surgeons rating 71 clinical cases of thoracolumbar spinal injuries using the Thoracolumbar Injury Severity Score (TLISS) and then re-rating the cases in a different order 1 month later. OBJECTIVE: To determine the reliability of the TLISS system. SUMMARY OF BACKGROUND DATA: The TLISS is a recently introduced classification system for thoracolumbar spinal column injures designed to simplify injury classification and facilitate treatment decision making. Before being widely adopted, the reliability of the TLISS must be studied. METHODS: A total of 71 cases of thoracolumbar spinal trauma were distributed on CD-ROM to 5 attending spine surgeons, including clinical/radiographic data, details of the TLISS, and a scoring sheet in which cases would be scored using the system. The surgeons were later assigned the task with the cases reordered. Intraobserver and interobserver reliability was calculated for TLISS components, total score, and surgeon's treatment decision using the Cohen unweighted kappa coefficients and Spearman rank-order correlation. RESULTS: Interrater reliability assessed by generalized kappa coefficients was 0.33 +/- 0.03 for injury mechanism, 0.91 +/- 0.02 for neurologic status, 0.35 +/- 0.03 for posterior ligamentous complex status, 0.29 +/- 0.02 for TLISS total, and 0.52 +/- 0.03 for treatment recommendation. Respective results using the Spearman correlation were 0.35 +/- 0.04, 0.94 +/- 0.01, 0.48 +/- 0.04, 0.65 +/- 0.03, and 0.51 +/- 0.04. Surgeons agreed with the TLISS recommendation 96.4% of the time. Intrarater kappa coefficients were 0.57 +/- 0.04 for injury mechanism, 0.93 +/- 0.02 for neurologic status, 0.48 +/- 0.04 for posterior ligamentous complex status, 0.46 +/- 0.03 for TLISS total, and 0.62 +/- 0.04 for treatment recommendation. Respective results using the Spearman correlation were 0.70 +/- 0.04, 0.95 +/- 0.02, 0.59 +/- 0.05, 0.77 +/- 0.04, and 0.59 +/- 0.05. CONCLUSIONS: The TLISS has good reliability and compares favorably to other contemporary thoracolumbar fracture classification systems. PMID- 16685240 TI - The biomechanics of long versus short fixation for thoracolumbar spine fractures. AB - STUDY DESIGN: Literature review. OBJECTIVE: Provide an overview of biomechanical strengths and weaknesses of long and short fixation constructs applied in thoracolumbar fractures, along with a discussion of specific indications for selecting an instrumentation construct for a given fracture. SUMMARY OF BACKGROUND DATA: Previous clinical and biomechanical studies have shown that segmental spinal instrumentation provides superior torsional, axial, and sagittal stability relative to nonsegmental systems. Multilevel fixation (long constructs) has proven reliable and effective in treating thoracic injuries, with or without anterior reconstruction. Short-segment pedicle instrumentation (short constructs) have proven effective in stabilizing thoracolumbar and lumbar fractures while limiting the disruption of lower lumbar motion segments. Loss of anterior column integrity leads to fixation failure when short constructs are not supplemented with further fixation or an anterior reconstruction. METHODS: Review of the applicable clinical and biomechanical literature. CONCLUSIONS: Long constructs serve well in thoracic and thoracolumbar fractures, while short-segment fixation offers advantages in selected thoracolumbar and lumbar fractures. Anterior column integrity determines the risk of sagittal collapse and kyphosis at the thoracolumbar junction. Recognition of fundamental biomechanical principles is necessary to make either construct work reliably. PMID- 16685241 TI - Complications associated with surgical stabilization of high-grade sacral fracture dislocations with spino-pelvic instability. AB - STUDY DESIGN: Retrospective evaluation of 19 consecutive patients with sacral fracture dislocations and cauda equina syndrome. OBJECTIVE: To review the safety and patient impact of early surgical decompression, and rigid segmental stabilization in patients with high-grade sacral fracture dislocations. SUMMARY OF BACKGROUND DATA: The ideal treatment for patients presenting with fracture dislocations of the sacrum resulting from high-energy mechanisms remains unknown. Previous studies consisted of multicenter case reviews that showed satisfactory outcomes with either nonoperative or a variety of surgical methods. However, over the last 20 years, no consistent treatment algorithm for these severe injuries has emerged. The advent of rigid, low-profile segmental fixation of the lumbar spine to the pelvic ring has offered a solution to many of the surgical challenges. This study evaluates the rate of complications of this method. It is intended to serve as a foundation for further evaluation and development of this treatment strategy, and as a basis for future comparison studies. METHODS: Patients were treated with a formally established algorithm, including resuscitation, and clinical assessment with detailed neurologic assessment and radiographic workup with pelvic computerized tomography and reformatted views. Electrophysiologic testing was conducted to confirm the presence of sacral plexus injuries in patients who were unable to be examined. Patients received neural element decompression and open reduction with segmental internal fixation through a midline posterior approach by connecting lower lumbar pedicle screws to long iliac screws when the patient's general medical condition allowed for surgical intervention. A formal sacroiliac arthrodesis was not performed. For the purposes of this study, patients were assessed specifically for the following adverse events: (1) infection, (2) wound healing, (3) neurologic deterioration following surgical treatment, (4) postoperative loss of sacral fracture reduction, (5) instrumentation failure, (6) axial lumbopelvic pain requiring further treatment, and (7) unplanned secondary surgery. RESULTS: There were 19 patients with an average age of 32 years treated according to this algorithm. Fracture reduction was successfully maintained in all patients. During the index surgical intervention, 14/19 patients (74%) had had either a traumatic dural tear or nerve root avulsion. Major complications involved fracture of the connecting rods in 6/19 patients (31%) and wound healing disturbances in 5/19 (26%). There were no lasting complications such as chronic osteomyelitis noted. In patients followed over a 1-year period, the visual analog score, referable to the sacral injury, averaged 5.5 on a scale of 0-10. CONCLUSIONS: Rigid segmental lumbopelvic stabilization allowed for reliable fracture reduction of the lumbosacral spine and posterior pelvic ring, permitting early mobilization without external immobilizaton and neurologic improvement in a large number of patients. Complications were primarily related to infection, wound healing, and asymptomatic rod breakage, and were without long-term sequelae. PMID- 16685242 TI - Cement augmentation techniques in traumatic thoracolumbar spine fractures. AB - STUDY DESIGN: Review of human cadaveric and in vivo animal studies and clinical trial. OBJECTIVE: To develop less invasive surgical techniques for reconstruction of the anterior column in thoracolumbar fractures. SUMMARY OF BACKGROUND DATA: Persistent central endplate depression can cause anterior column insufficiency after posterior surgery for traumatic thoracolumbar fractures. Reduction of the central endplate followed by intravertebral cement augmentation could restore weight-bearing capacity. MATERIALS AND METHODS: In human cadaveric burst fracture models, balloon-assisted endplate reduction (BAER) and vertebroplasty techniques have been investigated in terms of their safety and biomechanical properties. The histologic properties of different cement polymers were studied in an animal vertebral body and endplate defect model. In addition, the clinical outcome of percutaneous cement augmentation in the setting of a burst fracture examining the BAER technique and vertebroplasty with adjunctive posterior pedicle screw fixation is reviewed. RESULTS: These techniques have proven to be safe and effective, although cement leakage outside the confines of the vertebral body may occur. Calcium phosphate cements are preferable over methylmethacrylate because of their in vivo histologic properties. Using the BAER technique and posterior pedicular fixation, anterior vertebral height restoration is possible. Following balloon removal, some loss of fracture height restoration is observed. Further loss of vertebral height reduction was not observed following cement curing clinically. CONCLUSIONS: These studies show that less invasive anterior vertebral reconstruction using percutaneous cement augmentation techniques is feasible following traumatic vertebral fractures. PMID- 16685243 TI - The initial assessment and management of the multiple-trauma patient with an associated spine injury. AB - STUDY DESIGN: Review and summary of the relevant literature from multiple disciplines. OBJECTIVE: Provide the readership with evidence-based guidelines on the initial assessment and treatment of the multiple-trauma patient with an associated spinal column injury. SUMMARY OF BACKGROUND DATA: Early operative stabilization of the isolated spinal column injury has decreased hospital and intensive care unit length of stay. Early intervention has not provided consistently improved neurologic outcomes. The timing of spinal column stabilization in the multiple-trauma patient continues to be a source of discussion. METHODS: Review of published English literature from 1990 to present using key words: spinal trauma, multiple-trauma with spinal injury; timing of spinal injury treatment; spinal fracture management; and Advanced Trauma Life Support. CONCLUSIONS: The treatment of the poly-trauma patient with an associated spinal column injury requires strict adherence to Advanced Trauma Life Support principles. Once life and limb-threatening injuries have been identified and addressed, spinal column assessment and neurologic protection must be maintained at the highest priority. Early spinal stabilization can be performed safely in the multiple-trauma patient in medical centers, in which medical and ancillary staff is available on a 24-hour basis and is familiar with these procedures. PMID- 16685244 TI - Use of minimally invasive surgical techniques in the management of thoracolumbar trauma: current concepts. AB - STUDY DESIGN: Literature review and expert opinion. OBJECTIVE: To provide an overview of the current concepts of minimally invasive surgical (MIS) techniques for the management of thoracolumbar (TL) spinal trauma. SUMMARY OF BACKGROUND DATA: Current surgical treatment of thoracolumbar trauma typically involves open placement of spinal instrumentation with fusion. Conventional open spinal exposures can be associated with significant muscle morbidity that can lead to subsequent paraspinal muscular atrophy, scarring, decreased extensor strength and endurance, as well as pain. This approach-related morbidity is the main impetus for application MIS techniques to spinal procedures including trauma. METHODS: A review of the relevant English literature was performed. RESULTS: The current rationale, clinical applications, outcomes, and limitation of MIS management of TL injuries are summarized. CONCLUSION: The application of MIS techniques to spinal trauma is theoretically sound. However, the indications and technology are currently in evolution. Although very limited information is available, the results of current MIS techniques for the management of TL trauma are encouraging. PMID- 16685245 TI - Raloxifene (Evista) for breast cancer prevention in postmenopausal women. AB - Preliminary results from a new study, unpublished but reported in a press release from the National Cancer Institute and widely disseminated in the public press, suggest that raloxifene (Evista) might be a better choice than tamoxifen (Nolvadex, and others) for prevention of breast cancer in high-risk postmenopausal women. PMID- 16685246 TI - Acupuncture. Does it work? For which indications? PMID- 16685247 TI - Invader UGT1A1 molecular assay for irinotecan toxicity. A genetic test for an increased risk of toxicity from the cancer chemotherapy drug irinotecan (Camptosar). PMID- 16685248 TI - Estimating ambient concentration and cancer risk for 1,3-butadiene in Japan. AB - A detailed assessment of 1,3-butadiene exposure was performed for the purpose of risk assessment in Japan. The concentration of 1,3-butadiene and the 1,3 butadiene-exposure-related lifetime excess cancer risk in the general environment and in the vicinity of industrial point sources were estimated using two different types of diffusion models: the National Institute of Advanced Industrial Science and Technology-atmospheric dispersion model for exposure and risk assessment (AIST-ADMER) model ver. 1.0 for the estimation of regional scale concentrations and the Ministry of Economy, Trade and Industry - low rise industrial source dispersion (METI-LIS) model ver. 2.01 for the estimation of local concentrations near industrial sources. The calculated results indicate that the annual mean concentrations of 1,3-butadiene in residential areas are generally less than 0.5 microg/m(3), but in a few area near industrial point sources they exceed 1.7 microg/m(3), corresponding to a lifetime excess cancer risk of 10(-5). Using data on exposure concentrations and cancer unit risk, the lifetime excess cancer risk for persons exposed to 1,3-butadiene in Japan was evaluated. The results indicate that an extremely small number of people have a risk of developing 1,3-butadiene-exposure-related cancer that is greater than 10( 5), while that of most of the population in Japan is between 10(-5) and 10(-6). The total 1,3-butadiene-exposure-related cancer risk in Japan was calculated as 2.0 cases/year. A large proportion of the cancer risk was associated with general environmental areas. However, the individual risks of the population living in the vicinity of industrial point sources were significantly higher than those of the population living in the general environment. PMID- 16685249 TI - Estimation of daily bisphenol a intake of Japanese individuals with emphasis on uncertainty and variability. AB - The purpose of this study was to comprehensively assess the exposure of Japanese individuals to bisphenol A (BPA) with emphasis on uncertainty and variability in available information. The uncertainty and variability in parameters were numerically analyzed using Monte Carlo simulation. The uncertainty in the functional relationship between sources and exposure was treated by comparing two approaches: one was to aggregate ingestion and inhalation through all possible exposure pathways and the other was to estimate the intake from urinary excretion by backward calculation. For individuals aged 6 months or above, food was the most significant source of intake. The alteration of the method used in inactivating the inside surface of drink cans slightly contributed to the decrease in daily intake. By the backward calculation approach based on urinary excretion, 95% confidence intervals for the daily intake for high-exposure populations were estimated to be 0.037-0.064 microg/kg/day for males and 0.043 0.075 microg/kg/day for females. Even conservatively estimated daily intakes were lower than the EU's temporary tolerable daily intake (TDI) of 10 microg/kg/day as well as the U.S. Environmental Protection Agency (US EPA)'s reference dose (RfD) of 50 microg/kg/day. Thus, it is unlikely that humans, including infants and young children, are at unacceptable risk from possible BPA exposure. PMID- 16685250 TI - Assessment of exposure to and risk posed by toluene in Japanese residents: combining exposure from indoor and outdoor sources. AB - Toluene is a chemical substance whose release to the atmosphere is the largest among the pollutant release and transfer registers (PRTR) chemicals and is regarded as one of the major indoor air pollutants. We have developed a method of estimating the distribution of personal exposures of Japanese residents, combining exposure from indoor and outdoor sources. Personal exposure was divided into contributions of indoor and outdoor sources. Ambient toluene concentration was estimated for a 5 x 5 km square grid spatial resolution using the National Institute of Advanced Industrial Science and Technology-atmospheric dispersion model for exposure and risk assessment (AIST-ADMER) using PRTR emission data. The contribution of indoor sources was assumed to be the difference between indoor and outdoor concentrations, which was regarded to have a lognormal distribution. The health risk from toluene exposure in Japanese residents is expressed as the number of people whose personal exposure exceeded the reference values, which were set on the basis of a review of epidemiological studies. Finally, a risk reduction strategy is suggested according to the proposed principle of risk management. PMID- 16685251 TI - Estimating health risk from exposure to 1,4-dioxane in Japan. AB - Exposure to 1,4-dioxane from the atmosphere around high-emission plants and from consumer products used in daily life that contain the substance may have adverse health effects; however, its emission into the atmosphere is not regulated. In this study, the health risk posed by 1,4-dioxane is assessed to investigate whether measures should be undertaken to reduce exposure to 1,4-dioxane. The notion of the margin of exposure (MOE), given by the ratio of no observed adverse effect level (NOAEL) to actual or projected exposure level, is used to assess risk. In exposure assessment, two types of exposure channel are considered: (a) the use of consumer products that contain 1,4-dioxane and (b) the inhalation of air around high-emission plants. To estimate exposure via channel (a), we measured the concentration of 1,4-dioxane in consumer products and estimated the interindividual variability of exposure by Monte Carlo simulation that reflects the measured data. To estimate exposure via channel (b), we employed a local level atmospheric dispersion model to estimate the concentration of 1,4-dioxane immediately around high-emission plants. For hazard assessment, we derived the inhalatory and oral NOAELs for liver adenomas and carcinomas and the uncertainty factor. The results suggest that measures are not needed to reduce exposure to 1,4-dioxane from consumer products. As for inhalation exposure around high emission plants, some residents may be exposed to health risks if certain conservative analytical conditions are assumed. Even in this case, we conclude that it is not necessary for Plant A to stop the use of 1,4-dioxane immediately and that medium- to long-term emission reduction measures should be sufficient. PMID- 16685252 TI - Estimation of aggregate population cancer risk from dichloromethane for Japanese using atmospheric dispersion model. AB - The aggregate population cancer risk from dichloromethane exposure for each prefecture and for all of Japan was estimated using an atmospheric dispersion model and by considering the population within each calculation mesh (about 5 x 5 km). Indoor dichloromethane exposure was also taken into consideration. The number of lifetime dichloromethane-exposure-induced cancer cases for all of Japan was estimated to be only 1.3 (of 125 million people) using a most recently reported unit risk value. It was also found that the average ratio of the contribution to the aggregate population cancer risk attributable to outdoor emission sources (industrial factories) to the total emission sources was no more than 40% for all of Japan. From these results, it is believed that further reductions in dichloromethane emissions from industrial factories on a prefectural or a nationwide scale would not be effective in reducing cancer risk. It was also revealed that the average ambient concentration of dichloromethane measured at monitoring stations for hazardous air pollutants in each prefecture is a good measure of the average ambient dichloromethane concentration to which people in that prefecture are exposed. Therefore, it was suggested that the aggregate population cancer risk from dichloromethane exposure can be effectively estimated for entire Japan by simply using the average ambient concentration measured at monitoring stations in all of Japan taking into consideration indoor dichloromethane exposure. PMID- 16685253 TI - Measurement of methylphenidate-induced change in extrastriatal dopamine concentration using [11C]FLB 457 PET. AB - [(11)C]FLB 457 is a very high-affinity radiotracer that allows the measurement of dopamine D(2/3) receptor availability in regions of the brain where densities are very low, such as the cerebral cortex. It is not known if [(11)C]FLB 457 binding is sensitive to the concentration of endogenous dopamine in humans in a manner analogous to [(11)C]raclopride and [(123)I]IBZM in the striatum. To test this possibility, extrastriatal [(11)C]FLB 457 binding was measured at baseline and after the oral administration of 40 to 60 mg of the psychostimulant methylphenidate (MP) in 12 healthy volunteers using positron emission tomography (PET) in a balanced-order, double-blind design. The dynamic PET data were quantified using a two-tissue compartment model with a metabolite-corrected arterial plasma input function. Two volunteers were excluded because of excessive head movement. In the remainder, MP caused significant reductions in the volume of distribution (VD) in temporal and frontal cortical regions and thalamus, suggesting that [(11)C]FLB 457 binding is sensitive to endogenous dopamine concentration. Moreover, the change in [(11)C]FLB 457 binding after MP correlated with the dose of MP (in mg/kg body weight) in all regions assessed. We conclude that MP in doses within the therapeutic range for the treatment of attention deficit hyperactivity disorder causes increases in dopamine concentrations in extrastriatal regions and that [(11)C]FLB 457 PET may be a useful tool for the assessment of change in dopamine concentration in these areas in humans. PMID- 16685254 TI - Multiphoton imaging of ultrasound/Optison mediated cerebrovascular effects in vivo. AB - Ultrasound (US) enhanced with microbubble contrast agents may transiently disrupt the blood-brain barrier (BBB) with minimal damage, providing a technique for noninvasive, localized drug-delivery deep within the brain. The mechanism and temporal profile of disruption are not understood, owing to the limitations of imaging modalities used previously. In this study, we monitored US-induced BBB disruption with multiphoton microscopy, providing high-resolution temporal and spatial information about the permeabilization mechanism and immediate effects of US exposure. Anesthetized C57 mice were prepared with a craniotomy and injected intravenously with fluorescent dyes to permit visualization of the vasculature and BBB integrity. The animals were imaged through a cranial window while exposed to low-intensity US (f=1.029 MHz, power=0.2 W) with a coincident intravenous injection of Optison (a microbubble contrast agent). We observed arteriolar vasoconstriction on US exposure that disrupted blood flow and lasted up to 5 mins; BBB disruption occurred via two characteristically distinct processes perivascular fluorescence gradually increased (over minutes) along the length of the affected vessel without apparent rupture of the vessel wall or rapidly (seconds) increased in select, focal regions. These data corroborated previous studies suggesting increased endothelial transcytosis and breached tight junctions and demonstrated vasoconstriction, which might alter BBB permeability by modifying cerebral blood flow. PMID- 16685255 TI - Transgenic overexpression of adenosine kinase aggravates cell death in ischemia. AB - Adenosine is an endogenous neuromodulator with anticonvulsive and neuroprotective activity. Adenosine levels are normally kept in the range of 20 to 200 nmol/L by low basal expression of its main metabolic enzyme, adenosine kinase (ADK). Dysfunction of the adenosinergic system has been demonstrated to contribute to epileptogenesis. To investigate whether upregulation of ADK may render the brain more susceptible to ischemic cell death, mutant mice overexpressing an Adk transgene in brain were subjected to middle cerebral artery occlusion (MCAO). One day after either 15 or 60 mins of MCAO, wild-type (WT) animals had infarct areas encompassing about 5% and 50% of their ischemic hemisphere, respectively. In marked contrast, the volume of the infarcts increased three-fold in Adk transgenic mutants after 15 mins of MCAO, and after 60 mins of MCAO all mutants died within 24 h. Pretreatment of the mutants with the ADK inhibitor 5 iodotubercidin led to lesions similar to those in WT mice. Thus, low levels of ADK are essential to maintain adenosine-mediated neuroprotection. We conclude that pathologic overexpression of ADK as in epilepsy may also render the brain more susceptible to injury from ischemia. Consequently, ADK emerges as a rational therapeutic target to enhance neuroprotection. PMID- 16685257 TI - Infarct prediction and treatment assessment with MRI-based algorithms in experimental stroke models. AB - There is increasing interest in using algorithms combining multiple magnetic resonance imaging (MRI) modalities to predict tissue infarction in acute human stroke. We developed and tested a voxel-based generalized linear model (GLM) algorithm to predict tissue infarction in an animal stroke model in order to directly compare predicted outcome with the tissue's histologic outcome, and to evaluate the potential for assessing therapeutic efficacy using these multiparametric algorithms. With acute MRI acquired after unilateral embolic stroke in rats (n=8), a GLM was developed and used to predict infarction on a voxel-wise basis for saline (n=6) and recombinant tissue plasminogen activator (rt-PA) treatment (n=7) arms of a trial of delayed thrombolytic therapy in rats. Pretreatment predicted outcome compared with post-treatment histology was highly accurate in saline-treated rats (0.92+/-0.05). Accuracy was significantly reduced (P=0.04) in rt-PA-treated animals (0.86+/-0.08), although no significant difference was detected when comparing histologic lesion volumes. Animals that reperfused had significantly lower (P<0.01) GLM-predicted infarction risk (0.73+/ 0.03) than nonreperfused animals (0.81+/-0.05), possibly reflecting less severe initial ischemic injury and therefore tissue likely more amenable to therapy. Our results show that acute MRI-based algorithms can predict tissue infarction with high accuracy in animals not receiving thrombolytic therapy. Furthermore, alterations in disease progression due to treatment were more sensitively monitored with our voxel-based analysis techniques than with volumetric approaches. Our study shows that predictive algorithms are promising metrics for diagnosis, prognosis and therapeutic evaluation after acute stroke that can translate readily from preclinical to clinical settings. PMID- 16685258 TI - Measurement of serotonin 5-HT1A receptor binding using positron emission tomography and [carbonyl-(11)C]WAY-100635-considerations on the validity of cerebellum as a reference region. AB - [Carbonyl-(11)C]WAY-100635 has been used extensively in positron emission tomography (PET) imaging of serotonin 1A receptors (5-HT1A) in vivo in the human brain. Specific binding to receptors is usually estimated using compartmental modeling with arterial plasma input function. The use of reference tissue input (cerebellum) enables quantification without the need of arterial blood sampling, but the accuracy of this method is highly dependent on the validity of the reference region in terms of both specific and nonspecific binding. In this paper, we report exceptionally high uptake of [carbonyl-(11)C]WAY-100635 in the gray matter of cerebellum in one healthy male subject, which was reproducible in repeated PET scanning and most likely represents specific binding to 5-HT1A receptors in cerebellar gray matter. Serotonin 1A receptors are transiently expressed in the human cerebellum during early childhood and usually level off until adolescence but may persist in some individuals. As a methodological implication, the results of this study with regard to test-retest characteristics of [carbonyl-(11)C]WAY-100635 measurements in healthy volunteers using both arterial plasma and reference tissue input functions support the use of cerebellar white matter as reference region, to avoid the potential bias originating from binding of [carbonyl-(11)C]WAY-100635 to 5-HT1A receptors in cerebellar gray matter. PMID- 16685256 TI - HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across blood-brain barrier: implication for viral neuropathogenesis. AB - Human immunodeficiency virus-1 (HIV-1) encephalitis is characterized by brain infiltration of virus-infected monocytes and macrophages. Cellular products and viral proteins secreted by infected cells likely play an important role in blood brain barrier (BBB) impairment and the development of HIV-1-associated dementia (HAD). We previously demonstrated that HIV-1 envelope glycoprotein gp120 induces toxicity and alters expression of tight junction proteins in human brain microvascular endothelial cells (HBMECs). Here, we delineate the mechanisms of gp120-induced BBB dysfunction. Human brain microvascular endothelial cells expressed HIV-1 co-receptors (CCR5 and CXCR4). Exposure of HBMECs to gp120 derived from macrophage (CCR5) or lymphocyte (CXCR4)-tropic viruses decreased BBB tightness, increased permeability, and enhanced monocyte migration across in vitro BBB models. Blood-brain barrier integrity was restored after gp120 removal. CCR5 antibodies and inhibitors of myosin light chain kinase or protein kinase C (PKC) blocked gp120-enhanced monocyte migration and permeability of BBB in vitro. Exposure of HBMECs to gp120 induced release of intracellular calcium ([Ca(2+)](i)) that was prevented by CCR5 antibody and partially blocked by CXCR4 antagonist. Human immunodeficiency virus-1 gp120 activated three PKC isoforms in HBMECs [PKC-alpha/betaII, PKC(pan)-betaII and PKC-zeta/lambda]. Furthermore, specific PKC inhibitors (acting at the ATP-binding and calcium release site) blocked gp120-induced PKC activation and prevented increase in BBB permeability, supporting the biologic significance of these results. Thus, gp120 can cause dysfunction of BBB via PKC pathways and receptor mediated [Ca(2+)](i) release leading to cytoskeletal alterations and increased monocyte migration. PMID- 16685259 TI - Quantitative analysis for estimating binding potential of the peripheral benzodiazepine receptor with [(11)C]DAA1106. AB - [(11)C]DAA1106 is a potent and selective ligand for the peripheral benzodiazepine receptor (PBR) with high affinity. It has been reported that the density of PBR is related to brain damage, so a reliable tracer method for the evaluation of PBR would be of use. We evaluated a quantification method of [(11)C]DAA1106 binding in simulated data and human brain data. In the simulation study, the reliability of parameters estimated from the nonlinear least-squares (NLS) method, graphical analysis (GA), and multilinear analysis (MA) was evaluated. In GA, variation of the estimated distribution volume (DV) was small. However, DV was underestimated as noise increased. In MA, bias was smaller, and variation of the estimated DV was larger than in GA. In NLS, although variation was larger than in GA, it was small enough in regions of interest analysis, and not only DV but also binding potential (BP), determined from the k(3)/k(4) without any constraint, could be estimated. The variation of BP estimated with NLS became larger as k(3) or k(4) became smaller. In human studies with normal volunteers, regions of interest were drawn on several brain regions, BP was calculated by NLS, and DV was also estimated by NLS, GA, and MA. As a result, DVs estimated with each method were well correlated. However, there was no correlation between BP with NLS and DV with NLS, GA, and MA, because of the variation of K(1)/k(2) between individuals. In conclusion, BP is estimated most reliably by NLS with the two-tissue compartment model. PMID- 16685260 TI - Validation of a tracer kinetic model for the quantification of 5-HT(2A) receptors in human brain with [(11)C]MDL 100,907. AB - The positron emission tomography (PET) ligand [(11)C]MDL 100,907 has previously been introduced to image the serotonin 2A (5-HT(2A)) receptor in human brain. The aim of this work was to contribute to the verification of the tracer kinetic modelling in human studies. Five healthy volunteers were scanned twice after intravenous bolus injection of approximately 370 MBq [(11)C]MDL 100,907 using dynamic PET. One scan was performed under baseline condition, the other scan commenced 90 mins after a single oral dose of 30 mg of the antidepressant mirtazapine, which binds to the 5-HT(2A) receptor. There did not appear to be radiolabelled metabolites of [(11)C]MDL 100,907 in human plasma, which are likely to cross the blood-brain barrier. Total volumes of distribution VD in 11 different brain regions were estimated using a reversible, two tissue, four rate constants compartment model with a variable fractional blood volume term and the metabolite-corrected plasma input function. There were no significant changes of the VD in the cerebellum between the baseline and the blocked scans confirming the cerebellum as a region devoid of displaceable binding. Regional estimates of binding potential were then obtained indirectly using the cerebellar VD and occupancies calculated. The mean occupancy with this clinically effective dose of mirtazapine was 60% without significant regional differences. This study confirmed the use of an arterial input kinetic model for the quantification of 5 HT(2A) receptor binding with [(11)C]MDL 100,907 and the use of the cerebellum as a reference region for the free and nonspecific binding. PMID- 16685261 TI - Personal and microenvironmental concentrations of particles and microbial aerosol in relation to health symptoms among teachers. AB - A total of 81 randomly selected elementary school teachers participated in two sampling campaigns conducted 2 weeks apart during the winter. A 24-h sample collection was performed using personal and microenvironmental sampling from homes, and an 8-h sample collection was performed from workplaces of the studied subjects. Filters were analyzed for particle mass, absorption coefficient of the filter, and for both total and viable microorganisms. Comprehensive questionnaire responses were collected from the teachers concerning weekly occurred symptoms during the previous 12-month period, and they filled in symptom diaries immediately after each sampling campaign concerning symptoms during the previous 24-h and 7-day periods. The effect of different recall periods on agreement between questionnaire responses was assessed. Factor analysis was used in order to identify factors explaining the pattern of correlations within the personal, home, and work measurements. Moreover, associations between personal, home, and work measurements of pollutants and symptoms were analyzed using general estimation equations. The recall period of 7 days seemed to provide the most reliable data for the health effect assessment. Information from the factor analysis may allow reduction of variables related to the exposure assessment, and better interpretation of results. Both personal exposure and concentrations of pollutants at home were more frequently associated with health symptoms than concentrations at work. In multipollutant analyses, absorbance coefficient was positively associated with eye symptoms, and total bacteria with both cough and blocked nose. PMID- 16685262 TI - Individual solar-UV doses of pupils and undergraduates in China. AB - The solar-UV exposures of school pupils and medical undergraduates were studied over four seasons in Shenyang (latitude 41 degrees 11'-43 degrees 2', longitude 122 degrees 25'-123 degrees 48'), Liaoning, PR China. Ultraviolet exposure was measured using personal ultraviolet radiation (UVR) sensor on sunny days along with simultaneous monitoring and recording of direct-ambient UVR dose (perpendicular UV dose) for comparison. We observed that the UV doses of pupils on weekdays were higher than those on weekends and that the UV doses of the pupils were significantly higher than those of the undergraduates during both weekdays and weekends. The percent of the mean individual daily UV dose to the environmental counterpart are 2.88% (Autumn), 7.76% (Winter), 3.77% (Spring) and 2.06% (Summer) for the pupils, and corresponding percent for the undergraduates are 0.88%, 0.32%, 0.67% and 0.81%. The means of minimum erythemal dose values with pupils for weekday (5 days), weekend (2 days) and week (7 days) through the whole year are 0.302 (0.275 ~ 0.328), 0.215 (0.170 ~ 0.261) and 0.278 (0.255 ~ 0.301), respectively. The corresponding means with undergraduates are 0.097 (0.081~ 0.113), 0.091 (0.070 ~ 0.112) and 0.095 (0.082 ~ 0.108), respectively. Although the season is a major factor influencing the personal direct-ambient percent, it is emphasized that the impact of the lifestyle cannot be ignored. PMID- 16685263 TI - Long-term complete responses after 131I-tositumomab therapy for relapsed or refractory indolent non-Hodgkin's lymphoma. AB - We present the long-term results of 18 chemotherapy relapsed indolent (N = 12) or transformed (N = 6) NHL patients of a phase II anti-CD20 (131)I-tositumomab (Bexxar) therapy study. The biphasic therapy included two injections of 450 mg unlabelled antibody combined with (131)I-tositumomab once as dosimetric and once as therapeutic activity delivering 75 or 65 cGy whole-body radiation dose to patients with normal or reduced platelet counts, respectively. Two patients were not treated due to disease progression during dosimetry. The overall response rate was 81% in the 16 patients treated, including 50% CR/CRu and 31% PR. Median progression free survival of the 16 patients was 22.5 months. Median overall survival has not been reached after a median observation of 48 months. Median PFS of complete responders (CR/CRu) has not been reached and will be greater than 51 months. Short-term side effects were mainly haematological and transient. Among the relevant long-term side effects, one patient previously treated with CHOP chemotherapy died from secondary myelodysplasia. Four patients developed HAMA. In conclusion, (131)I-tositumomab RIT demonstrated durable responses especially in those patients who achieved a complete response. Six of eight CR/CRu are ongoing after 46-70 months. PMID- 16685264 TI - Modified nucleosides: an accurate tumour marker for clinical diagnosis of cancer, early detection and therapy control. AB - Modified nucleosides, regarded as indicators for the whole-body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies. To test their usefulness as tumour markers and to compare them with the conventional tumour markers, fractionated urine samples were analysed using chromatography. The excretion patterns of nucleosides of 68 cancer patients with malignant and benign tumours and 41 healthy controls have been studied. Significant elevations in the total sum and the concentrations of at least three (or four) of indicator nucleosides cytidine, pseudouridine, 2-pyridone-5-carboxamide-N1-ribofuranoside, N2,N2-dimethylguanine, 1-methylguanosine, 2-methylguanosine and 1-methyladenosine indicate a tumour with a sensitivity of 54% (77%) and a specificity of 86% (98%). Using an artificial neural network analysis, a sensitivity of 97% and a specificity of 85% were achieved in differentiating between tumour and control volunteers. The comparison with carcinoembryonic antigen, cancer antigen 15-3 und tissue polypeptide antigen indicates that urinary nucleosides may be useful tumour markers. This study suggests that the simultaneous determination of modified nucleosides and creatinine in urine samples of patients with cancer leads to an advantage to current methods and is a useful method to detect cancer early and to control the success of therapy. PMID- 16685265 TI - Pulmonary toxicity in patients treated with gemcitabine and a combination of gemcitabine and a taxane: investigation of a signal using postmarketing data. PMID- 16685266 TI - Single-nucleotide polymorphisms in the RB1 gene and association with breast cancer in the British population. AB - A substantial proportion of the familial risk of breast cancer may be attributable to genetic variants each contributing a small effect. pRb controls the cell cycle and polymorphisms within it are candidates for such low penetrance susceptibility alleles, since the gene has been implicated in several human tumours, particularly breast cancer. The purpose of this study was to determine whether common variants in the RB1 gene are associated with breast cancer risk. We assessed 15 tagging single-nucleotide polymorphisms (SNPs) using a case control study design (n< or = 4474 cases and n < or = 4560 controls). A difference in genotype frequencies was found between cases and controls for rs2854344 in intron 17 (P-trend = 0.007) and rs198580 in intron 19 (P-trend = 0.018). Carrying the minor allele of these SNPs appears to confer a protective effect on breast cancer risk (odd ratio (OR) = 0.86 (0.76-0.96) for rs2854344 and OR = 0.80 (0.66-0.96) for rs198580). However, after adjusting for multiple testing these associations were borderline with an adjusted P-trend = 0.068 for the most significant SNP (rs2854344). The RB1 gene is not known to contain any coding SNPs with allele frequencies > or = 5% but several intronic variants are in perfect linkage disequilibrium with the associated SNPs. Replication studies are needed to confirm the associations with breast cancer. PMID- 16685267 TI - The clinical benefit of pegylated liposomal doxorubicin in patients with metastatic breast cancer previously treated with conventional anthracyclines: a multicentre phase II trial. AB - This study evaluates the clinical benefit of pegylated liposomal doxorubicin (PLD) in patients with metastatic breast cancer (MBC), previously treated with conventional anthracyclines. Seventy-nine women with MBC previously treated with anthracyclines received PLD 50 mg m(-2) every 4 weeks. All patients were previously treated with chemotherapy and 30% of patients had > or =3 prior chemotherapies for metastatic disease. Patients were considered anthracycline resistant when they had disease progression on anthracycline therapy for MBC or within 6 months of adjuvant therapy. The overall clinical benefit rate (objective response+stable disease > or =24 weeks) was 24% (16.1% in patients with documented anthracycline resistance vs 29% in patients classified as having non anthracycline-resistant disease). There was no difference with respect to the clinical benefit between patients who received PLD >12 months and those who received PLD < or =12 months since last anthracycline treatment for metastatic disease (clinical benefit 25 vs 24.1%, respectively). Median time to progression and overall survival were 3.6 and 12.3 months, respectively. The median duration of response was 12 months, and the median time to progression in patients with stable disease (any) was 9.5 months. Fourteen patients (17.7%) had a prolonged clinical benefit lasting > or =12 months. In conclusion, PLD was associated with an evident clinical benefit in anthracycline-pretreated patients with MBC. PMID- 16685268 TI - CD13/Aminopeptidase N overexpression by basic fibroblast growth factor mediates enhanced invasiveness of 1F6 human melanoma cells. AB - CD13/Aminopeptidase N (CD13) is known to play an important role in tumour cell invasion. We examined whether basic fibroblast growth factor (bFGF) is involved in the regulation of CD13 expression in human melanoma cells. 1F6 human melanoma cells were stably transfected with constructs encoding either the 18 kDa (18 kD) or all (ALL) bFGF isoform proteins. We observed highly increased CD13 mRNA and protein expression in the 1F6 clones regardless of the overexpression of either the 18 kD or all isoform proteins. Neutral aminopeptidase activity was increased five-fold and could be inhibited by bestatin and the CD13-neutralising antibody WM15. The enhanced invasion through Matrigel, but not migration in a wound assay, was efficiently abrogated by both bestatin and WM15. Upregulation of CD13 expression was the result of increased epithelial and myeloid promoter activity up to 4.5-fold in 1F6-18 kD and 1F6-ALL clones. Interestingly, in a panel of human melanoma cell lines, a significant correlation (r(2)=0.883, P<0.05) between bFGF and CD13 mRNA and protein expression was detected. High bFGF and CD13 expression were clearly related with an aggressive phenotype. Taken together, our data indicate that high bFGF expression upregulates CD13 expression in human melanoma cells by activating both the myeloid and the epithelial CD13 promoter. In addition, we show that high bFGF and CD13 expression results in enhanced invasive capacity and metastatic behaviour of human melanoma cells. PMID- 16685269 TI - The relation between survival and expression of HER1 and HER2 depends on the expression of HER3 and HER4: a study in bladder cancer patients. AB - Increased expression of the epidermal growth factor (EGF) receptors, HER1 and HER2 are related to poor prognosis in most cancers studied. Recently, a high expression of the two remaining receptors of the EGF system, HER3 and HER4 has been related to a favourable prognosis. However, prognostic significance of HER1 and HER2 receptors in bladder cancer is controversial and the effect of the expression of different combinations of these receptors on patient survival is not well understood. Therefore, we examined the mRNA expression of all four EGF receptors with real-time polymerase chain reaction in biopsies from 88 patients with bladder cancer, where the survival was followed for a median of 38.5 months (range 1-117 months). Expression of HER1 and HER2 alone showed no correlation with survival. However, a high expression of HER1 together with high expression of HER3 and HER4 correlated to a better prognosis compared to the high expression of HER1 together with low expression of HER3 and HER4 (P=0.0006). Also, a significantly longer survival was observed in patients expressing high HER2 when coexpressed with high HER3 and HER4, as compared to the survival in patients with tumours expressing high HER2 but low HER3 and HER4 (P=0.0005). Our results suggest that the final outcome of patients with high HER1- and HER2-expressing tumours depends on the expression of HER3 and HER4. PMID- 16685270 TI - Expression of DNA damage response proteins and complete remission after radiotherapy of stage IB-IIA of cervical cancer. AB - The primary aim of this study was to investigate if the expression of the DNA damage identifying protein DNA-PKcs known to be involved in DNA repair after treatment with ionising radiation can be used as a predictive marker for radiotherapy (RT) response in cervical cancer. Formalin-fixed primary tumour biopsies from 109 patients with cervical cancer, FIGO-stage IB-IIA, treated with preoperative brachytherapy followed by radical surgery were analysed by immunohistochemistry. In addition, correlation studies between early pathological tumour response to radiation and expression of Ku86, Ku70, Mdm-2, p53 and p21 in primary tumours were also performed. We found that tumour-transformed tissue shows positive immunostaining of DNA-PKcs, Ku86 and Ku70, while non-neoplastic squamous epithelium and tumour-free cervix glands show negative immunoreactivity. Expression of DNA-PKcs positively correlated with both Ku86 and Ku70, and a statistically significant correlation between the Ku subunits was also found. After RT, 85 patients demonstrated pathologic complete remission (pCR), whereas 24 patients had residual tumour in the surgical specimen (non-pCR). The main finding of our study is that there was no correlation between the outcome of RT and the expression of DNA-PK subunits. Positive p53 tumours were significantly more common among non-pCR cases than in patients with pCR (P=0.031). Expression of p21 and Mdm-2 did not correlate with the outcome of RT. PMID- 16685271 TI - An elevated C-reactive protein concentration, prior to surgery, predicts poor cancer-specific survival in patients undergoing resection for gastro-oesophageal cancer. AB - There is increasing evidence that the presence of an ongoing systemic inflammatory response is associated with poor outcome in patients undergoing resection for a variety of tumours. The aim of the present study was to examine the relationship between clinico-pathological status, preoperative C-reactive protein concentration and cancer-specific survival in patients undergoing resection for gastro-oesophageal cancer. One hundred and twenty patients attending the upper gastrointestinal surgical unit in the Royal Infirmary, Glasgow, who were selected for potentially curative surgery, were included in the study. Laboratory measurements of haemoglobin, white cell, lymphocyte and platelet counts, albumin and C-reactive protein were carried out at the time of diagnosis. All patients underwent en-bloc resection with lymphadenectomy and survived at least 30 days following surgery. On multivariate analysis, only the positive to total lymph node ratio (hazard ratio (HR) 2.02, 95% confidence interval (CI) 1.44-2.84, P<0.001) and preoperative C-reactive protein concentration (HR 3.53, 95% CI 1.88-6.64, P<0.001) were independent predictors of cancer-specific survival. The patient group with no evidence of a preoperative systemic inflammatory response (C-reactive protein < or =10 mg l(-1)) had a median survival of 79 months compared with 19 months in the elevated systemic inflammatory response group (P<0.001). The results of the present study indicate that in patients selected to undergo potentially curative resection for gastro oesophageal cancer, the presence of an elevated preoperative C-reactive protein concentration is an independent predictor of poor cancer-specific survival. PMID- 16685272 TI - Dermcidin expression in hepatic cells improves survival without N-glycosylation, but requires asparagine residues. AB - Proteolysis-inducing factor, a cachexia-inducing tumour product, is an N glycosylated peptide with homology to the unglycosylated neuronal survival peptide Y-P30 and a predicted product of the dermcidin gene, a pro-survival oncogene in breast cancer. We aimed to investigate whether dermcidin is pro survival in liver cells, in which proteolysis-inducing factor induces catabolism, and to determine the role of potentially glycosylated asparagine residues in this function. Reverse cloning of proteolysis-inducing factor demonstrated approximately 100% homology with the dermcidin cDNA. This cDNA was cloned into pcDNA3.1+ and both asparagine residues removed using site-directed mutagenesis. In vitro translation demonstrated signal peptide production, but no difference in molecular weight between the products of native and mutant vectors. Immunocytochemistry of HuH7 cells transiently transfected with V5-His-tagged dermcidin confirmed targeting to the secretory pathway. Stable transfection conferred protection against oxidative stress. This was abrogated by mutation of both asparagines in combination, but not by mutation of either asparagine alone. These findings suggest that dermcidin may function as an oncogene in hepatic as well as breast cells. Glycosylation does not appear to be required, but the importance of asparagine residues suggests a role for the proteolysis-inducing factor core peptide domain. PMID- 16685273 TI - Prostaglandin E(2) stimulates progression-related gene expression in early colorectal adenoma cells. AB - Upregulation of cyclooxygenase-2 (COX-2) and prostaglandin-dependent vascularisation in small adenomatous polyps is an essential part of colon carcinogenesis. To study the underlying cellular mechanisms, LT97 and Caco2 human colorectal tumour cells not expressing endogenous COX-2 were exposed to 1 microM prostaglandin E(2) (PGE(2)) in their medium. At 30 min after addition, expression of c-fos was stimulated 5-fold and 1.3-fold, respectively, depending on the activation of both extracellular signal-regulated kinase and p38. The amount of c jun in nuclear extracts was increased 20% in LT97 cells. Expression of COX-2 was upregulated 1.7-fold in LT97 cells and 1.5-fold in Caco2 2 h after prostaglandin (PG) addition by a p38-mediated pathway. The known PGE(2) target gene vascular endothelial growth factor (VEGF) was not modulated. Effects of sustained PGE(2) production were studied in VACO235 cells that have high endogenous COX-2 and in LT97 cells infected with an adenovirus expressing COX-2. Prostaglandin E(2) secretion into the medium was 1-2 nM and 250 pM, respectively. Expression of both VEGF and c-fos was high in VACO235 cells. In LT97 cells, COX-2 upregulated c-fos expression and c-jun content in nuclear extracts 1.7- and 1.2-fold, respectively, in a PG-dependent way. This shows that exogenous PGE(2) as well as COX-2 overexpression affect signalling and gene expression in a way that enhances tumour progression. PMID- 16685274 TI - Role of lymphangiogenesis in epithelial ovarian cancer. AB - We investigated the significance of lymphatic count, vascular count and angiogenic growth factors using immunohistochemistry in 108 tumour specimens of epithelial ovarian cancer with antibodies to lymphatic vessel endothelial hyaluronan receptor (LYVE-1), platelet endothelial cell adhesion molecule CD31, vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP) in epithelial ovarian cancer to understand the pathogenesis of metastasis in ovarian cancer. The effect of prognostic variables on progression-free and overall survival was assessed. On multivariate analysis, bulky residual disease after surgery was the best prognostic indicator (P<0.001) for progression-free and overall survival (P<0.001). Lymphatic count was statistically significant as a prognostic factor for progression-free (P=0.05) and overall survival (P=0.04). However, lymphatic count did not impact on survival curves. No correlation was found between lymphatic count and age, histological subtype, FIGO stage or residual disease. Vascular count, VEGF or TP expressions were not significant in either analysis. Lymphatic spread may be significant in aiding metastases in ovarian cancer but requires other biological factors to act in conjunction, as it does not have clearcut prognostic significance. Dissemination of ovarian cancer does not occur primarily through vascular or lymphatic routes but may occur through direct intraperitoneal spread of disease. PMID- 16685275 TI - Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases. AB - Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process. PMID- 16685277 TI - Clinical outcome and risk factors for recurrence in borderline ovarian tumours. AB - We investigated the long-term prognosis of borderline ovarian tumours and determined risk factors for recurrence. One hundred and twenty-one borderline ovarian tumours treated between 1994 and 2003 at the participating institutions in the Tohoku Gynecologic Cancer Unit were retrospectively investigated for clinical stage, histopathological subtype, surgical technique, postoperative chemotherapy, the presence or absence of recurrence, and prognosis. The median follow-up period was 57 months (1-126 months). One hundred and nine cases (90.6%) were at clinical stage I. The histopathological subtypes consisted of 91 cases of mucinous tumour (75.2%), 27 cases of serous tumour (22.3%), and three cases of endometrioid tumour. Conservative surgery was used in 53 cases (43.8%), radical surgery in 68 cases (56.2%), a staging laparotomy in 43 cases (35.5%), and postoperative adjuvant therapy in 30 cases (24.8%). Recurrence was found in eight cases, but no tumour-related deaths were reported. Although no significant difference in disease-free survival rate was seen between different clinical stages, the difference in disease-free survival rate between serous and non serous (mucinous and endometrioid) types was significant (P<0.05). The 10-year disease-free survival rate was 89.1% for the radical surgery group and 57.4% for the conservative surgery group -- this difference was significant (P<0.05). In the conservative surgery group, cystectomy and serous tumour were independent risk factors for recurrence. Although recurrence was observed, the long-term prognosis of borderline ovarian tumour was favourable, without tumour-related deaths. Considering the favourable prognosis, conservative surgery can be chosen as far as the patient has a non-serous tumour and receive adnexectomy. However, in cases of serous type and/or receiving cystectomy special care should be given as relative risk rates of recurrence elevate by 2-4-folds. PMID- 16685276 TI - Phase II study of gefitinib in combination with docetaxel as first-line therapy in metastatic breast cancer. AB - We have evaluated the activity and safety of gefitinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in combination with docetaxel as first-line treatment of women with metastatic breast cancer (MBC). In total, 41 patients with MBC were enrolled in a first-line combination therapy study with oral gefitinib (250 mg day(-1)) and intravenous docetaxel (75 mg m(-2), the first 14 patients; or 100 mg m(-2), the following 27 patients, on day 1 of a 3-week cycle). Out of 41 patients, 38 received at least one cycle of therapy. There were no differences in activity or tolerability between the two docetaxel doses. G3/4 toxicities were neutropenia (49%), diarrhoea (10%), acne like rash (5%), and anaemia (2%). Complete plus partial responses (CR+PR) were observed in 22 out of 41 patients with a 54% response rate (95% confidence interval (CI) 45-75%). The 22 patients that achieved a response following six cycles of docetaxel plus gefitinib continued gefitinib monotherapy (median duration, 24 weeks; range, 2-108+ weeks). Two patients with PR following combination therapy achieved a CR during gefitinib monotherapy. Complete plus partial responses correlated with oestrogen receptor (ER) status, since they occurred in 19 out of 27 (70%) patients with ER-positive tumours as compared to three out of 14 (21%) patients with ER-negative tumours (P=0.01). PMID- 16685278 TI - Docetaxel induces apoptosis in hormone refractory prostate carcinomas during multiple treatment cycles. AB - Caspase-cleaved proteins are released from disintegrated apoptotic cells and can be detected in the circulation. We here addressed whether caspase-cleaved cytokeratin 18 (CK18-Asp396) can be used as a serum biomarker for assessment of the clinical efficiency of chemotherapy in hormone-refractory prostate cancer (HRPC). A total of 82 patients with HRPC were evaluated during 751 treatment cycles, either with estramustine (EMP)/vinorelbine or with EMP/docetaxel. The levels of CK18-Asp396 and of total CK18 were measured in patient serum before and during therapy by ELISA. Docetaxel induced significant increases in serum CK18 Asp396 (P<0.0001) and total CK18 (P<0.0002), suggesting induction of apoptosis. Similarly, vinorelbine induced increases in both CK18-Asp396 and CK18 (P<0.001 and 0.011). In contrast, EMP induced increases in total serum CK18 (P<0.0001), but not in CK18-Asp396 (P=0.13). The amplitudes of docetaxel-induced increases were associated with baseline prostate-specific antigen (PSA) and CK18 serum levels in these patients, consistent with tumoral origin of caspase-cleaved fragments. Docetaxel induced significant increases in CK18-Asp396 during second-, third- and fourth-line therapy and induced increased levels of CK18-Asp396 during treatment cycles 1-8. In contrast, vinorelbine induced significant increases only during cycles 1-3. In a subgroup of 32 patients that received EMP/vinorelbine in second line followed by EMP/docetaxel in third line, docetaxel induced stronger increases than vinorelbine (P=0.008). These results show that the CK18-Asp396 serum marker can be used to assess tumour apoptosis in vivo and suggest that the clinical efficiency of docetaxel in HRPC is due to induction of apoptosis during multiple treatment cycles. PMID- 16685279 TI - Molecular phylogeny of wild hops, Humulus lupulus L. AB - We have analysed wild hops collected widely from the Northern Hemisphere, assessing the genetic diversity and the geographical distribution of haplotypes, to investigate the evolution and phylogeny of hops, Humulus lupulus. The haplotypes were characterized by the nuclear ribosomal DNA spacer region (length and DNA sequence) and chloroplast DNA noncoding regions (DNA sequences). The results indicated that primary divergence into European (including Caucasus and Altai hops), and Asian-North American types, was 1.05+/-0.28 to 1.27+/-0.30 million years ago. Although an Eastern boundary for European nuclear haplotype distribution was unclear due to the ambiguous origin of Northern Chinese samples, the European hop group showed a wide geographical distribution across Eurasia from the Altai region to Portugal. The low genetic variation in this group suggested rapid and recent expansion. The North American hop group showed high diversity, and is considered to include hops that have migrated from Asia. Japanese and Chinese hops were identified as genetically distinct. This study has shown that wild hops in each growing region are genetically differentiated with considerable genetic diversity. It gives insights into the evolution and domestication of hops that are discussed. PMID- 16685280 TI - Comparative population genetic study of two oligophagous insects associated with the same hosts. AB - A parallel study of the genetic structure of two oligophagous species associated with the same hosts was conducted to determine the main factors shaping the distribution of genetic diversity. The bark beetle Tomicus piniperda and the pine processionary moth (PPM) Thaumetopoea pityocampa are both associated with the genus Pinus and belong to different guilds (xylophagous vs defoliating species). The PPM is an ectophagous species that feeds on the needles of living trees, whereas T. piniperda is endophagous and bores galleries in the inner bark of weakened trees. Both species were sampled in the main regions of France, and their genetic structure was assessed after genotyping with five microsatellite markers. Populations of the PPM were significantly structured. A pattern of isolation by distance was found when distances were calculated as bypassing the Massif Central, whereas no such pattern could be found with raw geographic distances. On the contrary, most populations of T. piniperda were not differentiated. No effect of host species could be detected in either of the two species. We conclude that the two taxa have contrasting effective dispersal rates per generation, and we hypothesize that this reflects the different selection pressures acting on individual fitness via different strategies of host use. PMID- 16685281 TI - Speciation: Marathon mollusc migrations. PMID- 16685283 TI - Power of QTL mapping experiments in commercial Atlantic salmon populations, exploiting linkage and linkage disequilibrium and effect of limited recombination in males. AB - Whereas detection and positioning of genes that affect quantitative traits (quantitative trait loci (QTL)) using linkage mapping uses only information from recombinants in the genotyped generations, linkage disequilibrium (LD) mapping uses historical recombinants. Thus, whereas linkage mapping requires large family sizes to detect and accurately position QTL, LD mapping is more dependent on the number of families sampled from the population. In commercial Atlantic salmon breeding programmes, only a small number of individuals per family are routinely phenotyped for traits such as disease resistance and meat colour. In this paper, we assess the power and accuracy of combined linkage disequilibrium linkage analysis (LDLA) to detect QTL in the commercial population using simulation. When 15 half-sib sire families (each sire mated to 30 dams, each dam with 10 progeny) were sampled from the population for genotyping, we were able to detect a QTL explaining 10% of the phenotypic variance in 85% of replicates and position this QTL within 3 cM of the true position in 70% of replicates. When recombination was absent in males, a feature of the salmon genome, power to detect QTL increased; however, the accuracy of positioning the QTL was decreased. By increasing the number of sire families sampled from the population to be genotyped to 30, we were able to increase both the proportion of QTL detected and correctly positioned (even with no recombination in males). QTL with much smaller effect could also be detected. The results suggest that even with the existing recording structure in commercial salmon breeding programmes, there is considerable power to detect and accurately position QTL using LDLA. PMID- 16685282 TI - Finding of male-killing Spiroplasma infecting Drosophila melanogaster in Africa implies transatlantic migration of this endosymbiont. AB - We report the identification of male-killing Spiroplasma in a wild-caught female Drosophila melanogaster from Uganda, the first such infection to be found in this species outside of South America. Among 38 female flies collected from Namulonge, Uganda in April, 2005, one produced a total of 41 female offspring but no males. PCR testing of subsequent generations revealed that females retaining Spiroplasma infection continued to produce a large excess of female progeny, while females that had lost Spiroplasma produced offspring with normal sex ratios. Consistent with earlier work, we find that male-killing and transmission efficiency appear to increase with female age, and we note that males born in sex ratio broods display much lower survivorship than their female siblings. DNA sequence comparisons at three loci suggest that this Spiroplasma strain is closely related to the male-killing strain previously found to infect D. melanogaster in Brazil, although part of one locus appears to show a recombinant history. Implications for the origin and history of male-killing Spiroplasma in D. melanogaster are discussed. PMID- 16685284 TI - Nutrient determinants of postprandial triglyceride response in a population-based sample of type II diabetic patients. AB - BACKGROUND: Nutrient determinants of postprandial triglyceride (TG) are matter of debate, especially for type II diabetes. OBJECTIVE: This study was performed to evaluate the impact of dietary habits on postprandial TG response in a population based sample of type II diabetic patients. DESIGN: One-hundred and forty type II diabetic patients (63 men/77 women, age 45-70 years) referring to the same health district, not on hypolipidemic drugs and without any other chronic disease, performed four TG profiles (at fasting, before, 2 and 3 h after lunch) with a specific device (Accutrend GCT, Roche Diagnostics Mannheim, Germany) validated previously. Dietary habits were recorded by a dietitian utilizing a previously validated semiquantitative questionnaire. RESULTS: Triglyceride values (mmol/l, mean +/- s.d.) were 2.22 +/- 0.93 at fasting, decreased before lunch (2.03 +/- 0.81), reached peak values 3 h after lunch (2.73 +/- 1.11). Postprandial TG increments (3 h after lunch minus pre-lunch concentration) significantly correlated with the intake (g/day) of animal protein (r = 0.20, P < 0.02), total fat (r = 0.21, P < 0.01), animal fat (r = 0.19, P < 0.03) and vegetable fat (r = 0.19, P < 0.03), also after adjusting for fasting TG and high-density lipoprotein cholesterol levels. Expressing nutrient intake as percentage of total calorie intake, total and animal fat remained significantly and directly related to postprandial TG increment (r = 0.21, P < 0.01 for total fat; r = 0.19, P < 0.03 for animal fat) whereas the percentage of carbohydrates was inversely related (r = -0.23, P < 0.007). CONCLUSIONS: Fat intake seems the major nutritional determinant of postprandial TG response in type II diabetic patients. PMID- 16685285 TI - Response to: "Rescuing the NIH before it is too late" from the Deputy Director for Extramural Research. AB - For a number of reasons, the NIH and the biomedical research community are facing a period of fiscal constraint after pronounced growth. In these difficult times, it is important that we all speak from the facts and work together to do a better job of explaining the importance of the nation's investment in biomedical research. PMID- 16685286 TI - A MULTICHANNEL FIBER-OPTIC MAPPING SYSTEM FOR INTRAMURAL RECORDING OF CARDIAC ACTION POTENTIALS. AB - Measuring cardiac action potentials at many sites within the ventricular wall is important for understanding cardiac arrhythmias; however, recording in the depth of the heart wall presents many difficulties. We have developed a multichannel optical mapping system for recording cardiac action potentials transmurally. Each channel uses a single small-diameter optical fiber to transmit and collect light from the cardiac tissue. Excitation light is supplied by low-power green lasers. Wavelength separation is performed with a dichroic mirror, and fluorescence is detected with a photodiode. We have recorded action potentials with an unfiltered signal-to-noise ratio (SNR) as high as 60:1 and a temporally filtered SNR as high as 200:1. The collection of fluorescence is optimized so that low excitation light intensity can be used, which increases the available recording time. Channels are modular and compact, and the system can be easily expanded to include additional channels, ratiometry or dual-dye mapping. In addition, the system is highly flexible and can be used for virtually any experiment from single cell recording to surface and transmural mapping of the whole heart. PMID- 16685288 TI - The Everyday Violence of Hepatitis C Among Young Women Who Inject Drugs in San Francisco. AB - A theoretical understanding of the gendered contours of structural, everyday and symbolic violence suggests that young addicted women are particularly vulnerable to the infectious diseases caused by injection drug use-especially hepatitis C. Participant-observation fieldwork among heroin and speed addicts in San Francisco's Haight Ashbury neighborhood reveals that extreme levels of violence against women are normalized in the common sense of street-youth drug culture. Physical, sexual and emotional violence, as well as the pragmatics of income generation, including drug and resource sharing in the moral economy of street addicts, oblige most young homeless women to enter into relationships with older men. These relationships are usually abusive and economically parasitical to the women. Sexual objectification and a patriarchal romantic discourse of love and moral worth leads to the misrecognition of gender power inequities by both the men and women who are embroiled in them, as well as by many of the public services and research projects designed to help or control substance abusers. Despite deep epistemological, theoretical and logistical gulfs between quantitative and qualitative methods, applied public health research and the interventions they inform can benefit from the insights provided by a theoretical and cross-methodological focus on how social power contexts shape the spread of infectious disease and promote disproportional levels of social suffering in vulnerable populations. PMID- 16685289 TI - Efficacy and safety of a novel stabilized stannous fluoride and sodium hexametaphosphate dentifrice for dentinal hypersensitivity. AB - PURPOSE: Dentinal hypersensitivity is a common complaint among dental patients. Recently, a novel 0.454% stabilized stannous fluoride dentifrice containing sodium hexametaphosphate (SHMP) was introduced that offers a desensitizing benefit. This trial was conducted to assess the desensitizing efficacy of this new dentifrice relative to a sodium fluoride control dentifrice. METHODS AND MATERIAL: This was a double-blind, parallel-group, randomized clinical trial conducted according to the American Dental Association (ADA) Guidelines for the Acceptance of Products for the Treatment of Dentinal Hypersensitivity. Ninety subjects who met the entrance criteria were stratified based on age, gender, and baseline sensitivity scores and randomly assigned to either the stabilized stannous fluoride + SHMP dentifrice (Crest Pro-Health) or the sodium fluoride control dentifrice. Subjects were instructed to brush twice daily for eight weeks. Efficacy assessments were made, including tactile (Yeaple probe) and thermal (Schiff Air Index) sensitivity, and an oral soft tissue examination was conducted at baseline, week four, and week eight. RESULTS: The mean sensitivity score based on the Schiff Air Index for the stannous fluoride + SHMP group was statistically significantly lower than that of the control group, at both weeks four and eight (P < .0001). At week eight, the stannous fluoride + SHMP dentifrice group had an adjusted mean 44% lower than that of the control group. The mean tactile sensitivity score for the stannous fluoride + SHMP group was statistically significantly higher, indicating a reduction in sensitivity, than that of the control group, at both weeks four and eight (P < .0001). At week eight, the stannous fluoride + SHMP dentifrice group had a mean desensitizing improvement of 71% greater than the control. CONCLUSION: The stabilized stannous fluoride + SHMP dentifrice provided statistically significant reductions in dentinal hypersensitivity at four and eight weeks compared to the sodium fluoride control dentifrice. PMID- 16685290 TI - Evaluation of shear bond strength and SEM observation of all-in-one self-etching primer used for bonding of fissure sealants. AB - OBJECTIVES: To evaluate and compare enamel shear bond strength of an all-in-one self-etching primer (Prompt L-Pop) to regular acid etch material when bonded to two-fissure sealant systems (Concise and Dyract Seal). METHOD AND MATERIALS: Forty newly extracted non-carious first permanent molars were embedded in a Teflon mold. The teeth were divided into four groups and each consisted of ten specimens. The bonding surfaces were treated with either Prompt L-Pop as recommended by the manufacturer or etched with phosphoric acid. After 24 hours of water storage, the specimens were evaluated for shear bond strength using an Instron testing machine. Scanning electron microscope (SEM) examinations were carried out to evaluate the failure sites of the sealants. RESULTS: The mean shear bond strengths using Prompt L-Pop were Concise: 23.46 MPa and Dyract Seal: 20.34 MPa. These values were higher than and statistically different from those of Dyract Seal (9.99 MPa) and Concise (8.85 MPa) when phosphoric acid was used. The failure was predominantly of the adhesive type. The SEM examination showed the failure of debonding was predominantly related to the type of etching systems used rather than the type of fissure sealants. CONCLUSION: The use of the all-in one self-etching adhesive Prompt L-Pop improves the mean enamel shear bond strength of fissure sealants. PMID- 16685291 TI - Clinical evaluation of posterior composite restorations in endodontically treated teeth. AB - OBJECTIVE: The purpose of this study was to evaluate the two year clinical performance of posterior composite restorations in endodontically treated premolars and molars using a hybrid composite (Filtek Z-250, 3M ESPE) and a total etch bonding system (Single Bond, 3M ESPE). METHOD AND MATERIALS: Thirty-nine class II restorations in endodontically treated premolars (n=11) and molars (n=28) of 27 patients (14 female, 13 male, mean age 36.51) in 16 maxillar and 23 mandibular teeth were placed by one operator. Restorations were evaluated by two experienced investigators at baseline, 12 months, and 24 months according to the modified United States Public Health Service (USPHS) criteria that included retention, color match, marginal discoloration, secondary caries, anatomic form, marginal adaptation, and surface texture. All restorations were able to be evaluated at baseline, 12 months, and 24 months. RESULTS: Paired samples t-test showed only marginal discoloration showed a statistically significant difference (p<0.05) at the end of 24 months, and no other significant differences were observed for the other variables examined over the duration of the study. None of the restored teeth showed periapical pathology at the end of 24 months. CONCLUSION: At two years, limited deterioration in marginal discoloration was detected. The clinical performance of posterior composite restorations in endodontically treated teeth using Filtek Z250 was found clinically acceptable after two years. PMID- 16685292 TI - Bond strength of total-etch and self-etch dentin adhesive systems on peripheral and central dentinal tissue: a microtensile bond strength test. AB - OBJECTIVE: The purpose of this study was to evaluate the microtensile bond strength (mu TBS) of four total-etch adhesives [Excite (EB), Prime&Bond NT (PBNT), Single Bond (SB), and One Coat Bond (OCB)] by comparing them to five self etching adhesives (Clearfil SE Bond (CSEB), Xeno III (X III), Prompt L-Pop (PLP), AQ Bond (AQB), and Tyrian/One Step plus (TOSP)] at different dentinal areas. In addition the interface between the adhesive resins and dentin was examined using scanning electron microscopy (SEM). METHODS AND MATERIALS: Superficial occlusal dentin of extracted human molars was exposed and finished with wet 800-grit silicon carbide paper. A block of composite resin was then bonded to the molar samples with the above adhesives according to the manufacturers' instructions. After 24 hours in water at 37 degrees C, the specimens were sectioned into 1 mm thick slices and divided into two regional subgroups according to their relationship to pulp tissue using visual criteria: pulp center and pulp periphery. The slices were then trimmed for the microtensile bond test and subjected to a tensile force and crosshead speed of 1 mm/min. Two-way analysis of variance was performed for statistical analyses. In addition the bond strengths for nine adhesive systems for each dentinal area were compared using the Post-Hoc test. The resin-dentin interfaces for each adhesive system were observed under a SEM. RESULTS: Mean mu TBS ranged from (25.2 MPa) for TOSP to (48.9 MPa) for PBNT. The bond strengths of total-etch adhesive systems were not significantly different, and were higher than self-etch adhesive systems, except for CSEB (p<0.05). No significant regional difference was observed for all of the nine adhesive systems (p>0.05). SEM observation showed there is not a standard hybridization for adhesive systems. CONCLUSION: Different dentinal areas may not exhibit as great an influence on bond strengths using new adhesive systems. PMID- 16685293 TI - Cost analysis of hand hygiene using antimicrobial soap and water versus an alcohol-based hand rub. AB - Proper hand hygiene is acknowledged as the most critical element of an adequate infection control program in the oral healthcare setting. However, adherence to proper hand hygiene protocols is often lacking. Poor compliance with hand hygiene protocols has been attributed to such factors as lack of time, hand irritation, hand dryness, forgetfulness, skepticism over importance, understaffing, perceived low risk of cross-infection, inconvenience, and the belief gloves alone offer protection. In the medical environment the use of alcohol-based hand rubs now represent the preferred method of performing hand hygiene when delivering non surgical care. In this study we compared the costs associated with traditional hand washing against an alcohol-based hand rub protocol in the dental setting. The results indicate an alcohol-based hand rub protocol is less costly and less time consuming when compared to traditional handwashing in the dental setting, creating a new paradigm for hand hygiene in the dental office. PMID- 16685294 TI - Prevalence of dental caries, severity, and pattern in age 6 to 7-year-old children in a selected community in Saudi Arabia. AB - AIMS: The aim of this study was to determine the prevalence, severity, and pattern of dental caries in 6 to 7-year-old children in military primary schools in Jeddah city, Saudi Arabia. METHODS AND MATERIALS: A random sample of 300 children (6 to 7-year-olds) was drawn from six schools of military dependents. Clinical examinations were carried out under standardized conditions by two trained and calibrated examiners (MAM) and (YR). Caries were diagnosed using the British Association for the Study of Community Dentistry (BASCD) criteria. RESULTS: Caries were diagnosed in 288 (96%) of the children, and only 4% were clinically caries free. Mean decayed, missing, and filled teeth (dmft) for the 300 children was 8.06 (+ 4.04) per child and mean decayed, missing, and filled surfaces (dmfs) was 23.18 (+ 15.64). The teeth most affected by caries were mandibular primary second molars (83.35%), and the least affected were mandibular primary central incisors (1.15%). CONCLUSIONS: The level of caries was higher than seen in children of equivalent age in other studies. The level of caries is expected to increase in permanent dentition. The above findings stress the need for an effective program of oral prevention in these children, such as a school dental health education program for children and their parents in order to improve their oral health status. PMID- 16685295 TI - Blood pressure assessment practices of dental hygienists. AB - An estimated 50 million Americans have high blood pressure (HBP), with 30% of them unaware of their condition. Both the American Dental Association (ADA) and the American Dental Hygienists' Association (ADHA) have advocated including recording blood pressure during the dental appointment. Recording blood pressure is also a standard procedure in patient care. This study surveyed 236 dental hygienists attending a continuing education program to document their blood pressure assessment practices. The majority (55%) of participants indicated they rarely or never record blood pressure. The primary reason cited by 51% of the participants was a lack of time in the appointment. Based on these findings, a recommendation was made for dental offices to modify their patient check-in procedures to include recording blood pressure. PMID- 16685296 TI - Comparative clinical evaluation of acellular dermal matrix allograft and connective tissue graft for the treatment of gingival recession. AB - AIMS: "Gingival recession is a condition reported to occur due to abnormal periodontal anatomy, poor hygiene, excessive occlusal forces, toothbrush abrasion, and even iatrogenic or factitious causes. Though various surgical techniques are available to treat this problem, the most common is the palatal soft tissue autograft. Recently, an acellular dermal matrix allograft (ADMA) has been available as a substitute for the palatal tissue harvest. The aim of this study is to compare the ADMA with the conventional subepithelial connective tissue graft (SCTG) in the treatment of gingival recession." METHODS AND MATERIALS: Fourteen patients with 20 gingival recessions of Miller's grade I and II were selected and randomized in two groups of control (SCTG ) and test (ADMA). In each group ten recession defects were treated. The following parameters were measured at baseline and then at six months post surgery: recession height (RH), recession width (RW), probing depth (PD), attached gingiva (AG), keratinized gingiva (KG), and clinical attachment level (CAL). All parameters were analyzed using the two-sample t-test. Data analysis was performed using SPSS (version 11) software. RESULTS: The following mean changes (mm) occurred in SCTG and ADMA, respectively: 2.60+/-0.97 and 2.90+/-0.81 decrease in RH; 1.70+/-1.01 and 1.65+/ 0.67 decrease in RW; 2.50+/-0.97 and 2.95+/-0.69 increase in KG; 2.25+/-0.92 and 2.65+/-0.85 increase in AG; 2.60+/-1.08 and 2.75+/-0.92 decrease in CAL; and finally 0.05+/-0.50 and 0.10+/-0.46 decrease in PD for the SCTG and ADMA groups, respectively. The percentage of root coverage for the two groups was 70.12%+/ 22.81% and 72.08%+/-14.12%, respectively. The changes from baseline to the six month visit were significant for both groups in terms of all parameters but PD. However, the differences in mean changes were not significant between the two groups in any of the parameters. CONCLUSION: These findings imply the ADMA and SCTG techniques could produce the same results when used for the successful treatment of gingival recessions. In addition the ADMA could be used as an adequate alternative treatment modality for conventional techniques. PMID- 16685297 TI - The relationship of the inferior dental canal to the roots of impacted mandibular third molars in a Jordanian population. AB - OBJECTIVE: The aim of this study was to evaluate the topographic relationship between the inferior dental canals (IDCs) and the roots of impacted mandibular third molars. METHODS: Preoperative orthopantomograms (OPGs) were examined and the proximity of the IDC to the roots of impacted mandibular third molars was categorized into the following: groups: superimposition, adjacent, perforation, grooving, notching, or none. The categories notching, grooving, and perforation were regrouped together and called the true relationship between the IDC and the root apices. The type of impaction, age, and sex of the patient were also noted. RESULTS: The positional category of 96.1% of the radiographs with bilateral impactions was identical on both sides of the mandible. Out of 2526 impacted mandibular third molars examined, 1146 (45.3%) belonged to the superimposition category, 663 (26.2%) were adjacent, 312 (12.3%) showed grooving, 78 (3.08%) showed notching, and 9 (0.35%) were actually perforating the IDC. The results showed 15.7% of the total cases were in true relationship with the IDC. There was a significant association (p = 0.000) between patient's age and true relationship. CONCLUSIONS: Identical positional relationship of the bilateral impacted third molars to the IDC was noted in 96.1% of the radiographs. The position of the IDC in relation to the roots of impacted third molars varied according to the patient's age. PMID- 16685298 TI - Comparison of two chlorhexidine rinse protocols on the incidence of alveolar osteitis following the surgical removal of impacted third molars. AB - PRINCIPLES: Alveolar osteitis (dry socket) is the most common complication following the extraction of permanent teeth. This study was undertaken to compare the effect of two chlorhexidine rinse protocols on the incidence of alveolar osteitis in patients undergoing surgical removal of impacted mandibular third molar teeth. METHODS: A prospective randomized clinical trial was conducted among 99 subjects. Patients were randomly assigned into two groups. Subjects were instructed to rinse twice daily with 15 ml of chlorhexidine rinse 30 seconds for one week before and one week after surgery (group I) or one week after surgery (group II). Postoperatively, all patients were instructed to return in one week or sooner if bothersome pain increased or persisted. Data were collected regarding abnormal healing, presence of necrotic tissue, exposed bone, and absence of clot. RESULTS: The results indicated group I and group II were not statistically significant different in the reduction of alveolar osteitis. CONCLUSIONS: To reduce alveolar osteitis after impacted third molar surgery, it was observed use of postoperative chlorhexidine rinse was adequate. The postoperative use of chlorhexidine is more feasible than both preoperative and postoperative use. PMID- 16685299 TI - Orthodontic treatment need in an accredited graduate orthodontic center in north america: a pilot study. AB - AIM: To assess the objective orthodontic treatment needs of patients treated in an accredited Orthodontic Clinic in North America using a retrospective cross sectional study and the Dental Aesthetic Index (DAI). METHODS AND MATERIALS: One hundred pre- and post-treatment study models were randomly selected from the model storage facility of the clinic. Only the pre-treatment study models were assessed using the DAI. The pre-treatment and post-treatment ages for each case were noted as recorded on the corresponding study models. The range for the pre treatment age was 10 to 52 years, and from 12 years, 4 months to 54 years for the post-treatment age range. The duration of treatment for each case was calculated by subtracting the pre-treatment age from the post-treatment age (range of treatment duration, 9 to 125 months). Descriptive statistics as well as chi square statistics were employed to analyze the data. RESULTS: Fifteen cases had normal or minor malocclusions with no treatment or slight need for treatment. Definite malocclusions with treatment highly desirable accounted for 22 cases and severe malocclusion with treatment highly desirable was found in 16 cases. Forty seven cases had handicapping malocclusion with treatment considered mandatory. While a statistically significant association (p<0.05) was found between duration of treatment and severity levels of malocclusion (DAI scores), the association between pre-treatment age and DAI scores was not significant (p > 0.05). CONCLUSIONS: Eighty-five percent of the study sample needed orthodontic treatment with different malocclusion severity levels, while 47% of the cases qualified for publicly subsidized treatment due to handicapping malocclusions. The association between duration of treatment and DAI score groups (malocclusion severity levels) was found statistically significant. It could be helpful for more clinic-based (demand populations) studies on treatment needs be undertaken across the globe using the DAI, at least for the purposes of comparison. PMID- 16685300 TI - Fixed space maintainers combined with open-face stainless steel crowns. AB - OBJECTIVE: This study investigates the clinical performance of fixed space maintainers placed on seriously damaged abutment teeth. METHODS: Crowns were placed on damaged abutment primary teeth. Fixed space maintainers were prepared by using rectangular wire between the window in the facial surface of the crowns and other abutment teeth and were subsequently bonded with a flowable resin composite. This procedure was introduced clinically, and the cases were observed over a period of twelve months. RESULTS: Twenty-seven fixed space maintainers (25 on lower jaw, two on upper jaw) were included in this study. No clinical failure was recorded in any of the cases in the observation time, and the rate of clinical performance was 100%. CONCLUSION: The study shows the effectiveness of fixed space maintainers combined with stainless steel crowns ("open-face fixed space maintainers") which were placed on primary molar teeth used as abutments in cases with extensive caries and loss of occlusogingival dimension. PMID- 16685301 TI - The composition of unstimulated whole saliva of healthy dental students. AB - The purpose of this study was to determine the unstimulated whole saliva biochemical parameters in healthy dental students of Tehran University of Medical Sciences (TUMS) living in the students' dormitory with a mean age of 22 years. Five ml whole saliva samples were obtained by expectoration. The saliva composition was measured by a spectrophotometer and affiliated kits. The data was analyzed through the student's unpaired t-test using the SPSS program. In the male students (n=50) the mean concentrations of glucose (mmol/l), inorganic phosphate (mmol/l), total protein (mg/ml), magnesium (mmol/l), chloride (mmol/l), and calcium (mmol/l) were 0.75+/-0.44, 1.52+/-0.63, 6.69+/-2.89, 1.27+/-0.45, 27.60+/-11.06, and 2.17+/-0.76, respectively. In the female students (n=50) they were 0.73+/-0.47, 1.58+/-0.63, 7.26+/-3.78, 1.37+/-0.44, 30.42+/-12.74, and 1.87+/-0.78, respectively. There were no significant differences between the whole saliva values in male and female students. PMID- 16685302 TI - Torus palatinus and torus mandibularis in edentulous patients. AB - AIM: To determine the prevalence of tori in Jordanian edentulous patients, the sex variation in their distribution, and their clinical characteristics. METHODS: Three hundred and thirty eight patients were examined in the Prosthodontic Clinic in the Department of Restorative Dentistry at Jordan University of Science and Technology. The location, extent, and clinical presentation of tori were recorded related to the age and sex of patients. RESULTS: The overall prevalence of tori was 13.9%. The prevalence of torus palatinus was 29.8% (14/47), while that of torus mandibularis was significantly higher 42.6% (20/47). Both types of tori were associated with each other in 27.7% of cases (13/47). CONCLUSIONS: There was no significant difference in the prevalence of tori between males and females. There seems to be a strong association between mandibular and palatal tori. PMID- 16685303 TI - Smoking habits of adolescents and the role of dentists. AB - Tobacco smoking has a devastating effect on the health and well-being of the public and remains the nation's leading avoidable cause of premature mortality and disability. On an average, each smoker who dies would have lived another 15 years if he/she was a non-smoker. Though adult smoking has decreased markedly, there is an alarming rise in the use of tobacco among adolescents. One unique aspect of dentistry is some of the adverse health effects of tobacco uses are clinically apparent in the oral cavity at a relatively early stage of use. More than one-half of the adult population and nearly three-quarters of the student population see a dentist each year, and yet 80% of dentists do not routinely ask about tobacco use and advise tobacco users to quit. Nicotine is a highly addictive substance, and the urge to continue smoking is powerful. As doctors our motto in advising patients should be, "If you smoke, quit. If you don't, then don't try it." The second part of the message is especially important for adolescents, since most adult smokers started their habit as adolescents. The authors have highlighted the harmful effects of smoking and the role dentists have to impart as responsible citizens in educating their patients, especially in the younger generation to help them lead a healthier and more meaningful tomorrow. PMID- 16685304 TI - Retrobulbar hemorrhage: a case report. AB - Retrobulbar hemorrhage is a rare complication that may occur after mid-face injuries or following soft and hard tissue surgery around the eyes. The cardinal signs and symptoms of retrobulbar hemorrhage are pain, diplopia, ophthalmoplegia, a progression of increasing proptosis, and decreasing visual acuity leading to blindness. The diagnosis can be confirmed with computed tomography (CT) of the orbit or with ocular ultrasound. These diagnostic images are also important to define the size of the hematoma. This report describes a traumatic retrobulbar hemorrhage. In this case there were no signs of acute visual loss, and conservative treatment was possible without surgical intervention. PMID- 16685305 TI - Dentoalveolar and mandibular body fractures caused by a horse kick: report of a case. AB - A rare case of multiple mandibular fractures and severe facial laceration is presented. The cause was a horse hoof kick to an unmounted equestrian. The pertinent literature is reviewed and the maxillofacial injuries associated with animal interaction are discussed. PMID- 16685306 TI - The reinforced removable retainer. AB - The aim of this paper is to present a new type of orthodontic removable retainer, which is of great help to the orthodontist as well as to the patient. The procedures of fabrication are described. The Reinforced Removable Retainer (Triple "R" Retainer) is well tolerated, adaptable, and easy to fit and remove. Its main advantage is that it is not easy to break, less bulky, and very retentive. PMID- 16685307 TI - A lighting approach for clinical photographs of the face. AB - This article describes an alternative approach of lighting setup for taking clinical photographs of the face. The lighting techniques presented in this article will help the clinician obtain good-quality clinical photographs. PMID- 16685308 TI - AAOMP case challenge: "erythematous burning lips". PMID- 16685309 TI - The pacemaker and implantable cardioverter defibrillator recall issue - a Canadian perspective. AB - In 2005, an unprecedented number of recalls were issued on pacemakers and implantable cardioverter defibrillators. While recalls in the cardiac rhythm device industry are not new, the sheer magnitude of potentially affected patients in 2005 led to a great deal of concern, frustration, and even anger. Physicians have, in many instances, been uncertain when (or if) to recommend device replacement in an environment where the magnitude of the risk and the potential consequences of device failure have not been well defined in a timely way. Doctors and patients are now calling for reform of postmarket analysis and reporting mechanisms. The present article provides a uniquely Canadian perspective on this international issue. Potential solutions include the development of a set of realistic and common expectations, a restoration of confidence in postmarket analysis and reporting mechanisms, increased data transparency, and an increased role for patient and physician groups. PMID- 16685310 TI - Is early- and late-onset depression after acute myocardial infarction associated with long-term survival in older adults? A population-based study. AB - BACKGROUND: Early-onset depression after acute myocardial infarction (AMI) affects short-term survival in clinical samples of patients. There is no information on the impact of early-onset depression or late-onset depression on long-term survival. OBJECTIVE: To investigate the impact of early- and late-onset depression on survival using administrative data. METHODS: A historical inception cohort design was used, commencing in 1994 with up to eight years of follow-up. A province-wide administrative data set from British Columbia was used to select the cohort and construct the variables. Data regarding hospitalizations, physician visits and prescription drugs were available. All individuals 66 years of age and older who had an AMI in 1994 or 1995 were selected (n=4874). Individuals were categorized as depressed, possibly depressed or not depressed based on physician or hospital visits indicating depression as a diagnosis and/or prescriptions for antidepressants. Early-onset depression was assessed during the first six months post-AMI, and late-onset depression was assessed between six months and five years post-AMI. All-cause mortality up to eight years post-AMI was the outcome. RESULTS: Both early- and late-onset depression were associated with long-term mortality. The hazard ratio was 1.34 (95% CI 1.04 to 1.73) for early-onset depression and 1.79 (95% CI 1.38 to 2.35) for late-onset depression. CONCLUSIONS: Both early- and late-onset depression post-AMI were significantly associated with mortality up to eight years post-AMI. Depression is a strong independent predictor of post-AMI mortality in older adults. PMID- 16685311 TI - An alternate technique to pacing in complex congenital heart disease: assessment of the left thoracotomy approach. AB - BACKGROUND: Pacing in children with congenital heart disease often requires alternate approaches to standard transvenous pacing. The surgical approach used to implant the pacemaker leads has been shown to impact lead survival. There is a paucity of pediatric literature describing the experience using a left thoracotomy approach. OBJECTIVES: To report on short- and mid-term experiences with pacemaker implant via the left thoracotomy approach in children with complex congenital heart disease. METHODS AND RESULTS: Data were abstracted retrospectively from patients' hospital charts. To date, the left thoracotomy technique has been used in 11 patients with complex heart disease, with a median of three prior cardiac operations. The median patient age was five years (range of two months to 23 years of age). The pacing indications were acquired postoperative atrioventricular block (n=5), sinus node dysfunction (n=5) and long QT syndrome (n=1). There were no intraoperative complications or long-term complications from this approach. The pacing thresholds at implant and follow-up were acceptable in all patients. One patient died in follow-up for reasons unrelated to the pacemaker or arrhythmia. CONCLUSIONS: The placement of epicardial pacemaker leads via the left thoracotomy approach is a safe and effective alternative to transvenous pacing in pediatric patients with complex congenital heart disease. PMID- 16685312 TI - Stroke prevention in patients with atrial fibrillation: the diagnosis and management of hypertension by specialists. AB - BACKGROUND: Hypertension is common in patients with atrial fibrillation (AF) and is an important cause of stroke. OBJECTIVES: To determine how effectively hypertension is managed among specialist-treated outpatients with AF. METHODS: Investigators reviewed the charts of patients with a diagnosis of AF cared for by medical specialists to determine the change in blood pressure, patterns of antihypertensive drug use and the role of the specialist in the management of hypertension. RESULTS: Of 209 patients with AF, 118 had a history of hypertension or an office blood pressure greater than 140/90 mmHg. Blood pressure was measured at 73% of all visits. Hypertension was identified as an important problem in 57% of patients and antihypertensive therapy was either initiated or suggested in 77%. One year after the initial specialist visit, systolic blood pressure was significantly lower (140+/-20 mmHg at one year versus 148+/-23 mmHg initially; P=0.015); however, there was no change in diastolic blood pressure (80+/-12 mmHg at one year versus 81+/-16 mmHg initially; P=0.602) and only 50% of patients had a blood pressure less than 140/90 mmHg. In contrast, the percentage of patients receiving warfarin increased from 46% to 78% (P=0.0001). CONCLUSIONS: In patients treated by specialists for AF, systolic blood pressure is significantly reduced during follow-up; however, 50% of patients continue to have suboptimal blood pressure control. In many patients, hypertension is not identified as an important comorbid illness and antihypertensive therapy is neither recommended nor initiated by the specialist. Greater specialist involvement in the identification and treatment of hypertension in patients with AF could lead to an important, additional reduction in stroke. PMID- 16685313 TI - Exercise capacity and impact of exercise training in patients after a Fontan procedure: a review. AB - After a successful Fontan procedure, children and adolescents should improve their exercise capacity. However, several studies have shown that these children have a reduced maximal oxygen consumption compared with healthy children. The lower exercise performance in these patients was mainly explained by a reduced cardiorespiratory functional capacity. However, it has recently been reported that the lower exercise performance may also be related to altered skeletal muscle function. Moreover, exercise training had a beneficial impact on several parameters related to exercise tolerance in these patients. The main studies supporting these observations are reviewed, with a focus on the physiological adaptation and limitation of the exercise performance as well as the benefits of exercise training in patients after a Fontan procedure. PMID- 16685315 TI - In search of 'truth' and standards for reporting cardiac procedure waiting times. PMID- 16685314 TI - True versus reported waiting times for valvular aortic stenosis surgery. AB - OBJECTIVES: To produce a universally accepted waiting time definition for cardiovascular surgery, present the rationale for this definition, and compare data on current waiting times in British Columbia based on this definition versus the current definition in patients waiting for aortic stenosis surgery. STUDY DESIGN: The present study is a retrospective data analysis. SETTING: The fixed dollar, single-payer health care delivery system in British Columbia. PATIENTS: All residents of British Columbia who were at least 22 years of age and who were placed on a waiting list for aortic valve surgery with the diagnosis of aortic stenosis between January 1, 1991, and December 31, 2000, were eligible for the present study. INTERVENTIONS: Dates of physician visits, procedures and surgery were obtained from the British Columbia Cardiac Registries and Medical Services Plan databases. True waiting times from physician visits to procedures and surgery were calculated. RESULTS: Of the 2516 patients booked for aortic valve surgery with a primary diagnosis of aortic stenosis, 2237 subjects (88.9%) were eligible for analysis after exclusions. The eligible patients ranged in age from 22 to 95 years, and 36.8% were female. The true median waiting time was 243 days (148 days [25th percentile], 397 days [75th percentile]), which was 3.2 times the interval currently reported as the waiting time (75 days [42, 127]). Thirty-nine patients died while waiting for surgery. Patients used more resources while waiting for surgery than after surgery. CONCLUSIONS: True waiting times for surgery for aortic stenosis in British Columbia are significantly longer than reported waiting times. The authors propose that the wait list time for cardiovascular surgery be redefined as "the time interval between the patient's first contact with a medical care provider with symptoms or signs which ultimately lead to cardiovascular surgery and the date of that surgery". PMID- 16685316 TI - Successful explantation of a left ventricular assist device following acute fulminant myocarditis. AB - A left ventricular (LV) assist device was implanted in a 53-year-old woman in cardiogenic shock secondary to fulminant myocarditis. LV function recovered to normal after one week of support from an LV assist device. The device was explanted and the patient is showing a good outcome with a normalized LV function. PMID- 16685317 TI - Papillary fibroelastoma of the pulmonary valve. AB - There is a lack of information regarding the diagnosis and management of papillary fibroelastoma of the pulmonary valve due to the rarity of the tumour at this location. A case of pulmonary valve papillary fibroelastoma in a 60-year-old woman is reported and the approach for diagnosis and management is described. PMID- 16685320 TI - Combination vs. disproportionation in dialkyl biradicals. Selectivity reversal in a crystalline solid. AB - While 1,6-biradicals produced by photodecarbonylation of dimethyl 11 oxodibenzo[c,h]bicyclo[4.4.1]undeca-3,8-diene-1,6-dicarboxylate (1) react exclusively by disproportionation in benzene solution, reactions in crystals lead to radical-radical combination reactions in almost quantitative yield. PMID- 16685318 TI - Catheter thrombosis during primary percutaneous coronary intervention for acute ST elevation myocardial infarction despite subcutaneous low-molecular-weight heparin, acetylsalicylic acid, clopidogrel and abciximab pretreatment. AB - BACKGROUND: Subcutaneous enoxaparin is increasingly employed as the antithrombin of choice in non-ST elevation myocardial infarction and in conjunction with various fibrinolytic regimens in acute ST elevation myocardial infarction (STEMI). Few data exist describing the use of subcutaneous or intravenous enoxaparin as an anticoagulant in the highly thrombotic setting of primary percutaneous coronary intervention (PCI) for STEMI. METHODS: The Which Early ST Elevation Therapy (WEST) study compared fibrinolysis (with and without early cardiac catheterization) with primary PCI in a setting that expedited both strategies on first medical contact. Patients assigned primary PCI are administered acetylsalicylic acid 325 mg, clopidogrel 300 mg and subcutaneous enoxaparin 1 mg/kg before transport to a PCI centre. Of 36 initial patients treated with primary PCI, three patients had procedures that were complicated by extensive thrombosis within coronary catheters and on PCI equipment. RESULTS: Index cases were men aged 43 to 68 years who presented with confirmed STEMI and angiographically proven acute total or subtotal occlusion of a major epicardial coronary segment. During PCI, performed 76 min to 102 min following enoxaparin administration, a clot developed within the guide catheter or on the coronary guidewires and balloon catheter shafts, thus necessitating the replacement of all PCI equipment. In one case, there was evidence of continued intracoronary clot propagation and embolization. CONCLUSION: A single, conventional, weight-adjusted dose of subcutaneous enoxaparin before expedited primary PCI for STEMI may not provide a reliable antithrombotic effect. Supplementary intravenous enoxaparin is now strongly recommended within the WEST study, and a substudy evaluating pre- and postprocedural antifactor Xa activity has been initiated. PMID- 16685321 TI - Photodynamic therapy with 5-aminolevulinic acid induces distinct microcirculatory effects following systemic or topical application. AB - In photodynamic therapy (PDT) the photosensitiser 5-aminolaevulinic acid (ALA) can be used by systemic or topical application. Previous experiments showed that the photodynamic effects might not be mediated solely by porphyrins localized in the parenchyma, but also by porphyrins in the microvasculature. Therefore, the microcirculatory effects of PDT following systemic versus topical application of ALA have been investigated. Amelanotic melanomas were implanted in the dorsal skin fold chamber of Syrian Golden hamsters. ALA was injected i.v. for systemic PDT before irradiation, whereas ALA was applied to the chambers for topical PDT before irradiation with an incoherent lamp. FITC-labelled erythrocytes were injected to determine red blood cell velocity (RBCV) and functional vessel density (FVD). Twenty-four hours after PDT tissue was taken for histology and immunohistochemistry to reveal the degree of apoptosis and to show the accumulation of leukocytes. FVD or RBCV was not altered significantly by systemic or topical low-dose PDT (10 J cm(-2)), whereas a significant reduction of RBCV and FVD was detected after high-dose PDT (100 J cm(-2)) following systemic or topical application of ALA. Systemic PDT with 100 J cm(-2) stopped the flow only in the tumor center, whereas topical PDT with 100 J cm(-2) lead to a breakdown of RBCV in all chamber areas. Two hours and 24 h after systemic high-dose PDT, perfused microvessels and capillaries could be detected in normal tissue and tumor periphery, in contrast to topical high-dose PDT leading to a shut down of FVD 24 h after irradiation in all areas of the chamber tissue. Histological staining revealed a more pronounced intracellular oedema and swelling of cells after topical high-dose PDT than systemic high-dose PDT. These results indicate that topical high-dose PDT with ALA has a more pronounced effect on microcirculation as compared to systemic high-dose PDT in this model. PMID- 16685322 TI - Photophysical behaviour of 1-(4-N,N-dimethylaminophenylethynyl)pyrene (DMAPEPy) in homogeneous media. AB - The photophysical behaviour of a new pyrene derivative, 1-(4-N,N dimethylaminophenylethynyl)pyrene (DMAPEPy), in various solvents has been studied. Due to the presence of an ethynyl link with a cylindrical pi cloud between the donor (N,N-dimethyl group) and the acceptor (pyrene), the molecule shows efficient intramolecular charge transfer, with a high extinction coefficient in all the solvents. There is significant solvatochromism in the fluorescence with a large increase in the Stokes' shift of around 125 nm between n-hexane and acetonitrile. The solvent-dependent spectral data show a good correlation with the Kamlet-Taft solvent polarity parameter (pi*). The plots of Stokes' shifts with E(T)(30) are linear for non-protic solvents and for protic solvents but with different slopes. The fluorescence quantum yields are high for non-polar solvents and decrease as the solvent polarity increases. Unlike the parent molecule pyrene, DMAPEPy shows a short lifetime, which is fairly insensitive to oxygen-induced quenching and is dependent on solvent polarity. The molecule shows high steady-state fluorescence anisotropy, which is very sensitive to the viscosity change of the medium. PMID- 16685323 TI - UV-B light induces an adaptive response to UV-C exposure via photoreactivation activity in Euglena gracilis. AB - Phytoplankton such as Euglena are constantly exposed to solar light which is used for photosynthesis. Although the solar ultraviolet (UV) induces DNA damage such as cyclobutane-pyrimidine dimers (CPDs), many kinds of living organisms can repair CPDs by photoreactivation (PR) utilizing the near-UV/blue light component in sunlight. Euglena cells are known to possess such PR activity. In the present paper, the formation of CPDs induced by UV-C exposure and the photoreactivation PR repair of these CPDs by UV-A are demonstrated. To clarify the adaptive responses prior UV-B irradiation on PR activity, cells were cultured in the dark or under UV-B light. When the cells were cultured in the dark for 3 d prior to UV C exposure, PR activity decreased. When the cells were cultured under UV-B light, however, PR activity increased. These results suggest that exposing the cells to UV-B prior to exposure to UV-C induced an adaptive response towards DNA damage caused by UV-C exposure, and this UV-C induced damage was repaired through PR activity. PMID- 16685324 TI - Reaction pathways involved in the production of hydroxyl radicals in thylakoid membrane: EPR spin-trapping study. AB - It has been suggested that both free metals and reduced ferredoxin (Fd) participate in the light-induced production of hydroxyl radicals (OH*) in thylakoid membranes of chloroplasts. The most direct evidence for the involvement of Fd in OH* formation under physiological conditions was reported by Jakob and Heber (Plant Cell Physiol., 1996, 37, 629-635), who used the oxidation of dimethylsulfoxide to methane sulfinic acid as an indicator of OH* production. We confirmed their conclusions using a more sensitive and reliable EPR spin-trapping method and extended their work by additional findings. Free metal-dependent and ferredoxin-dependent OH* production was studied simultaneously and strong metal chelator Desferal was used to distinguish between these reaction pathways. The participation of protein-bound iron within photosystem I was confirmed by partial suppression of OH* generation in broken chloroplasts by methyl viologen. The enhancement in the production of OH* in thylakoid membranes by externally added ferredoxin can be considered as a straightforward evidence of the involvement of ferredoxin in OH* formation. PMID- 16685325 TI - Photocatalytic degradation of pesticide methomyl: determination of the reaction pathway and identification of intermediate products. AB - The degradation of pesticide methomyl in aqueous solution by UV-irradiation in the presence of TiO2 "Degussa P-25" has been studied. It was found that mineralisation to carbon dioxide, water, sulfate and ammonia took place during the process. The rate of photodecomposition of methomyl was measured using high performance liquid chromatography (HPLC), while its mineralization was followed using ion chromatography (IC), and total organic carbon (TOC) analysis. The identification of reaction intermediate products was carried out using coupled techniques HPLC-MS (electrospray ionization in positive mode) and a degradation pathway was proposed. Under our conditions, complete disappearance of 1.23 x 10( 4) mol l(-1) of pure pesticide occurred within 45 min of illumination and 80% TOC removal occurred in less than 4 h. Three main intermediates were identified resulting from (i) the rupture of the ester bond (or the N-O bond), (ii) the hydroxylation of methyl group borne by the nitrogen atom and (iii) the product resulting from the decarboxylation of the oxidized hydroxylated methyl group (photo-Kolbe reaction). In order to be sure that the photocatalytic results were consistent, hydrolysis and photolysis tests were performed. Photocatalysis proved to be an excellent new advanced oxidation technology (AOT) to eliminate methomyl present in water. PMID- 16685326 TI - Ultraviolet-induced autofluorescence characterization of normal and tumoral esophageal epithelium cells with quantitation of NAD(P)H. AB - Cellular autofluorescence was characterized in normal human esophageal cells and in malignant esophageal epithelial cells. The study was performed under excitation at 351 nm where the cell fluorescence is mainly due to the reduced pyridine nucleotides (NAD(P)H) with a very small contribution from the oxidized flavins (FMN, FAD) or lipopigments. The autofluorescence emission of squamous cell carcinoma, adenocarcinoma on Barrett's mucosa and normal cells was characterized by microspectrofluorimetry on monolayers and by spectrofluorimetry on cell suspensions. The relative contribution of each fluorophore to the fluorescence emission of the different cell types was evaluated by a curve fitting analysis. A statistically highly significant difference was observed between the average intensity of the raw spectra of the different cell types. Tumoral cells had a fluorescence intensity approximately twice as high as that of normal cells. The results of the NAD(P)H quantitation analyzed by microspectrofluorimetry on single living cells and spectrofluorimetry on cell suspensions were consistent with those obtained by biochemical cycling assays, showing that the amount of intracellular NAD(P)H is higher in tumoral cells than in normal cells. Bound NAD(P)H concentration was found to be quite stable whatever the cell type while the amount of free NAD(P)H showed a very important increase in tumoral cells. PMID- 16685327 TI - Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences. AB - We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design. PMID- 16685328 TI - Ultraviolet radiation-induced non-melanoma skin cancer in the Crl:SKH1:hr-BR hairless mouse: augmentation of tumor multiplicity by chlorophyllin and protection by indole-3-carbinol. AB - Over 1 million new cases of ultraviolet radiation-induced non-melanoma skin cancers (NMSC) per year now occur in the USA and the incidence of these diseases continues to increase. New preventative strategies are required. The hypothesis tested was that dietary administration of the putative cancer chemopreventatives sodium-copper-chlorophyllin (Chlor) or indole-3-carbinol (I3C) would inhibit UV induced skin carcinogenesis in the Crl:SKH1:hr-BR hairless mouse. Groups of 20 mice were pre-fed isocaloric/isonutritive 20% corn-oil AIN-76a based diets that contained either Chlor (1.52 g%), I3C (5.08 g%) or no chemopreventative (control) for 2 weeks followed by exposure of their dorsal skin to a 10 week incremental, sub-erythemal, carcinogenic simulated solar UV exposure regime. Feeding was continued for the duration of the experiment. Matched non-UV exposed dietary groups were also included in the experimental design. The diets had no significant (p > 0.05) effect on body weight, feed consumption, cutaneous methanol-extractable UV photoprotective substances or on cutaneous UV-reflective characteristics. By day 180, UV-irradiated mice fed the Chlor had a significantly (p < 0.05) higher tumor multiplicity (33.6 +/- 4.72; mean +/- SEM) than UV irradiated control animals (22.8 +/- 4.25). UV-irradiated mice fed I3C had a significantly (p < 0.001) lower tumor multiplicity (13.0 +/- 2.42) than that of both the UV-irradiated control and UV-irradiated Chlor-fed mice. The Chlor or I3C diets did not significantly (p > 0.05) affect UV-induced systemic suppression of contact hypersensitivity responses. These results demonstrate augmentation of the UV-induced cutaneous carcinogenic process by dietary chlorophyllin and protection from this carcinogenic process by indole-3-carbinol via mechanisms that do not involve changes in skin optical properties, modulation of photoimmunosuppression or caloric/nutrient effects. PMID- 16685329 TI - A model considering light reabsorption processes to correct in vivo chlorophyll fluorescence spectra in apples. AB - Chlorophyll-a contained in the peel of Granny Smith apples emits fluorescence upon excitation with blue light. The observed emission, collected by an external detector and corrected by its spectral response, is still distorted by light reabsorption processes taking place in the fruit skin and differs appreciably from the true spectral distribution of fluorescence emerging from chlorophyll molecules in the biological tissue. Reabsorption processes particularly affect the ratio of fluorescence intensities at 680 nm and at 730 nm. A model to obtain the correct spectral distribution of the emission, from the experimental fluorescence recorded at a fluorometer detector and corrected for the detector spectral sensitivity, is developed in the present work. Measurements of the whole fruit reflectance, the peel transmittance and the flesh reflectance allow the calculation of the reabsorption-corrected spectra. The model is validated by comparing the corrected emission spectra with that obtained for a thin layer of apple-peel-chloroplasts, where no reabsorption takes place. It is recommended to correct distortions in emission spectra of intact fruits due to light reabsorption effects whenever a correlation between the physiological state of the fruit and its fluorescence spectra is investigated. PMID- 16685330 TI - Strategy in a fishbowl: an invitation to determine the shape of IMIA in 2015. AB - OBJECTIVES: The International Medical Informatics Association (IMIA) today is an inclusive organization that represents the medical and health informatics world through its multiple member countries as well as affiliate, corporate and academic institutions, plus working groups and regions. The IMIA leadership deemed this is an excellent time to create a strategic alignment of IMIA's goals and in turn to create a framework of the IMIA agenda for the future. METHODS: The process began in early 2004, with a survey distributed to all IMIA members seeking members' views. The initial views were presented to the IMIA Board and General Assembly at Medinfo 2004 in San Francisco, USA. A Strategic Planning Task Force was established to take forward the development of a Strategic Plan. Through a combination of e-mail exchanges, face-to-face planning-discussion sessions in Geneva, Switzerland, and Washington DC, USA, and use of mediated conference calls, the IMIA Strategic Plan was evolved. RESULTS: The framework model (also known as the IMIA rainbow umbrella) seeks to represent, in visual and descriptive terms, the numerous possibilities for connections and integration. Knowledge is the central core of IMIA. All of our strategies, interactions and efforts, emanate from this knowledge core. Using a concentric circle model, the next circle (from the central core) represents science. This is followed by the application layer circle, then the impact layer/circle and finally the outermost circle represents the people layer. Another dimensionality of the IMIA Strategic Plan is the need to represent various key sectors. There are six sectors superimposed on the five concentric circle layers of IMIA's integration and connection to others. These sectors represent: health (our vision), research and science, behavioral responsibility, education, relationships and reach. CONCLUSION: We are still at a relatively early stage of planning. The Strategic Framework and Plan will be discussed by the IMIA Board and the IMIA General Assembly meeting (November 2006). PMID- 16685331 TI - A global socio-economic-medico-legal model for the sustainability of longitudinal electronic health records. Part 1. AB - OBJECTIVES: This paper pursues the challenge of sustaining lifetime electronic health records (EHRs) based on a comprehensive socio-economic-medico-legal model. The notion of a lifetime EHR extends the emerging concept of a longitudinal and cross-institutional EHR and is invaluable information for increasing patient safety and quality of care. METHODS: The challenge is how to compile and sustain a coherent EHR across the lifetime of an individual. Several existing and hypothetical models are described, analyzed and compared in an attempt to suggest a preferred approach. RESULTS: The vision is that lifetime EHRs should be sustained by new players in the healthcare arena, who will function as independent health record banks (IHRBs). Multiple competing IHRBs would be established and regulated following preemptive legislation. They should be neither owned by healthcare providers nor by health insurer/payers or government agencies. The new legislation should also stipulate that the records located in these banks be considered the medico-legal copies of an individual's records, and that healthcare providers no longer serve as the legal record keepers. CONCLUSIONS: The proposed model is not centered on any of the current players in the field; instead, it is focussed on the objective service of sustaining individual EHRs, much like financial banks maintain and manage financial assets. This revolutionary structure provides two main benefits: 1) Healthcare organizations will be able to cut the costs of long-term record keeping, and 2) healthcare providers will be able to provide better care based on the availability of a lifelong EHR of their new patients. PMID- 16685332 TI - Assessing the difficulty and time cost of de-identification in clinical narratives. AB - OBJECTIVE: To characterize the difficulty confronting investigators in removing protected health information (PHI) from cross-discipline, free-text clinical notes, an important challenge to clinical informatics research as recalibrated by the introduction of the US Health Insurance Portability and Accountability Act (HIPAA) and similar regulations. METHODS: Randomized selection of clinical narratives from complete admissions written by diverse providers, reviewed using a two-tiered rater system and simple automated regular expression tools. For manual review, two independent reviewers used simple search and replace algorithms and visual scanning to find PHI as defined by HIPAA, followed by an independent second review to detect any missed PHI. Simple automated review was also performed for the "easy" PHI that are number- or date-based. RESULTS: From 262 notes, 2074 PHI, or 7.9 +/- 6.1 per note, were found. The average recall (or sensitivity) was 95.9% while precision was 99.6% for single reviewers. Agreement between individual reviewers was strong (ICC = 0.99), although some asymmetry in errors was seen between reviewers (p = 0.001). The automated technique had better recall (98.5%) but worse precision (88.4%) for its subset of identifiers. Manually de-identifying a note took 87.3 +/- 61 seconds on average. CONCLUSIONS: Manual de-identification of free-text notes is tedious and time-consuming, but even simple PHI is difficult to automatically identify with the exactitude required under HIPAA. PMID- 16685333 TI - A framework for characterizing terminological systems. AB - OBJECTIVES: The notion of a terminological system (TS) is complex due to the broad range of systems, applications, and clinical domains. A uniform approach to describe the characteristics of TSs is lacking. This impedes furthering understanding, applicability, mutual comparison and development of TSs. For these reasons we propose a terminological systems characterization framework. METHODS: Relevant issues pertaining to TSs and terminology servers have been extracted from literature describing requirements and functionality of TSs. From these issues, features have been distilled and further refined. A categorization has been developed to provide a convenient arrangement of these features. RESULTS: The framework distinguishes between application-dependent and application independent features of TSs. Definitions are provided for measures of content coverage, which was identified as the only application-dependent feature. Application-independent features are categorized along two axes: their respective type of TS and the particular element within that system, i.e. the formalism, the content, or the functionality. For each feature we provide an explicit question, the answer to which yields a feature value. The framework has been applied to SNOMED CT and the CLUE browser. CONCLUSIONS: We present and apply a framework to support a feature-based characterization of terminological systems. Standardized methods for content coverage studies reduce the effort of assessing the applicability of a TS for a specific clinical setting. A two-axial categorization provides a convenient arrangement of the large number of application-independent features. Application of the framework increases comparability of terminological systems. This framework may also help TS developers determine how their system can be improved. PMID- 16685334 TI - Quality assurance of medical ontologies. AB - OBJECTIVE: To review the literature concerning the quality assurance of medical ontologies. METHODS: scholar.google.com was searched using the search strings (+ontology +"quality assurance") and (+ontology +"evaluation/evaluating"). Relevant publications were selected by manual review. Other work already familiar to the author, or suggested by other researchers contacted by the author, were included. The papers were analysed for common themes. RESULTS: Four broad properties of an ontology were identified that may be quality-assured: philosophical validity, compliance with meta-ontological commitments, 'content correctness', and fitness for purpose. Each published methodology addressed only a subset of these properties. 'Content' may be divided into domain knowledge content, and metadata describing either the provenance of domain knowledge content, or relationships between it and lexical information (e.g. for display and retrieval). 'Correctness' (whether of domain knowledge content or metadata) may also be further subdivided into truth, completeness, parsimony and internal consistency. CONCLUSIONS: Understanding of how to assure the quality of ontologies, or evaluate their fitness for specific purposes, is improving but remains poor. A combination of methodologies is required, but tools to support a comprehensive quality assurance programme remain lacking. Perfect quality of an ontology is not provable and may not be desirable: an ontology compliant with all current philosophical theories, following necessary ontological commitments, and with entirely 'correct' content, may be too complex to be directly usable or useful. The extent to which an ontology's fitness for purpose is predicted or influenced by its other properties remains to be determined. Field studies of ontologies in use, including interrater effects, are required. PMID- 16685335 TI - Assessing the state of population health by age-adjusted life expectancies. AB - OBJECTIVES: A gain in life expectancy of a population is commonly interpreted as an effect of improved health care. After the reunion of Germany in 1990 life expectancy at birth grew extremely in the new federal states. Within one decade after reunion the new federal states had a gain in life expectancy of about five years while the gain in the old federal states was only about two years. METHODS: It has been widely argued that this phenomenon is caused by an obviously improved public health service and environmental protection or even by an increased social status in the new federal states. On the other hand, the median population age grew rapidly in the new federal states, caused by a dramatic reduction of the birth rate as well as a high emigration rate of young people. Using real time series for three selected federal states and for the total Federal Republic of Germany, it is derived that most of the gain in life expectancy is explained by population ageing. RESULTS: An elementary probabilistic procedure is proposed allowing for estimating the amount in life expectancy not attributable to population ageing. CONCLUSIONS: The age-adjusted life expectancy can be regarded as an unbiased measure of a populations' state of health that stays comparable both over time and across countries. PMID- 16685336 TI - Symposium on "biomedical informatics and biomedical statistics education". PMID- 16685337 TI - Where to in the next ten years of health informatics education? AB - OBJECTIVES: To explore whether education in health/medical informatics should continue to evolve along the lines pursued since the early seventies, or whether a change is advisable. METHODS: Roots and key resulting characteristics for European and US American approaches HI education are identified. In Europe holistic approaches based on a synthesis of medicine and informatics (= computer science) with programs ranging from vocational training through university programs to doctoral and postdoctoral programs were characteristic. The US American approaches emphasized the higher levels of education and a diverse selection of specialized subjects. Changes in health and health informatics are summarized. RESULTS: Two types of changes are identified: high-tech applications arising at the interface of imaging, robotics, and the -omics (genomics, proteomics, metabolomics), and invasive applications centering on consumer health informatics and a move from curative to prospective health care. CONCLUSIONS: It is proposed that curative medicine is adequately served by current educational approaches, but that the move towards prospective health care requires a move towards education and change management for health professionals and health informatics professionals. PMID- 16685338 TI - Changing academic roles--new approaches to teaching and distance learning in Australia. AB - OBJECTIVES: A primary objective of this study was to identify a valid method for academic workload allocation. This required the identification of significant variables that may be used to indicate and measure academic workload. A supporting objective was to illustrate how the adoption of a flexible learning mode and supporting technologies across one university with multiple campuses and an international student cohort has impacted upon academic roles and teaching delivery methods. METHODS: An extensive literature review focusing primarily on the teaching aspects of academic roles was undertaken. These roles were defined as teaching, including curriculum development, undertaking research, provide professional and community services and undertake some administrative work. This review was followed by the documentation of a case study. RESULTS: Significant changes to the roles and responsibilities of academics working in higher education are now discernable. The adoption of Web-based applications and other communication technologies have made it possible to not only extend traditional distance education offerings but also to teach large multicultural classes across multiple campuses simultaneously. This in turn necessitates a review of teaching strategies and of organization-wide student and staff support infrastructures to ensure that the teaching quality is maintained or improved whilst meeting individual student learning needs. CONCLUSIONS: Changes to academics' roles are occurring due to the globalisation of higher education as well as the adoption of new educational delivery methods and the use of new technologies. The resulting complexity of academic workload measurement and the need to equitably allocate this workload to individual academics has become more challenging than ever. More traditional universities can learn from such experiences to better prepare for these inevitable changes. PMID- 16685339 TI - Medical informatics education needs information system practicums in health care settings--experiences and lessons learned from 32 practicums at four universities in two countries. AB - OBJECTIVES: To report about the themes and about experiences with practicums in the management of information systems in health care settings (health information management) for medical informatics students. METHODS: We first summarize the topics of the health information management practicums/projects that the authors organized between 1990 and 2003 for the medical informatics programs at Heidelberg/Heilbronn, Germany, UMIT, Austria, as well as for the informatics program at the University of Leipzig, Germany. Experiences and lessons learned, obtained from the faculty that organized the practicums in the past 14 years, are reported. RESULTS: Thirty (of 32) health information management practicums focused on the analysis of health information systems. These took place inside university medical centers. Although the practicums were time-intensive and required intensively tutoring students with regard to health information management and project management, feedback from the students and graduates was mainly positive. DISCUSSION: It is clearly recommended that students specializing in medical informatics need to be confronted with real-world problems of health information systems during their studies. PMID- 16685340 TI - Professional competence and computer literacy in e-age, focus on healthcare. AB - OBJECTIVES: The healthcare sector is facing an enormous acceleration due to the emergence of new knowledge, drugs, devices and diseases. Professional competence, continuing education, service excellence and patient benefits can be facilitated by the developments in information and computer technology--computer literacy is becoming imperative for all who are involved in healthcare delivery. The paper attempts to identify solutions that can aid the process of ICT uptake for full benefit of patients and healthcare professionals. METHODS: With the support of published literature, the article considers the importance of ICT skills in general and in healthcare and presents some advantages of generic vendor independent methods of ICT certification. RESULTS: Discussed are the preliminary results of the United Kingdom's National Health Service information technology reform which addresses the need for intensified use of ICT and applies the ECDL concept. CONCLUSIONS: It is useful to complement the introduction of computer literacy as a qualification concept by a standardized accreditation of ICT skills. Solid level of computer literacy creates a reliable and efficient background for everyday activities of healthcare professionals, enables the application of further domain-specific training modules and prepares suitable environments for the introduction and acceptance of new technologies such as electronic health records and electronic transfer of prescriptions by positively transforming the attitudes of users towards them. PMID- 16685341 TI - EGOOZ: specifying the components of electronic patient record-related education. AB - OBJECTIVE: To determine whether educators consider electronic patient record (EPR)-related education necessary and if so, what subjects have to be taught more extensively in the future. METHODS: A list of possibly relevant subjects was determined from the literature. A questionnaire was designed which contained those subjects and the respondents were asked to indicate, for each subject, its competency level and required competency level in current teaching. Since the response rate was low a second questionnaire was developed to have the results of the analysis of the first questionnaire validated by a larger group of educators. RESULTS: In total 45 learning goals were identified from the literature. The questionnaire was sent to representatives of several disciplines: basic medical education, medical specializations, pharmacy, dentistry and nursing. The analysis of the first questionnaire resulted in nine subjects that needed more attention in the future. Because of the low response the needs could not be specified for the individual disciplines. This insight was obtained from a second questionnaire. The response to this questionnaire was high. From the analysis of the second questionnaire differences between views of educators involved in the training of GPs and educators involved in the training of other specializations were observed. CONCLUSION: Educators find EPR-related education important. There are different opinions about the phase in which EPR-related education should be given. PMID- 16685342 TI - The new features of the ExaMe evaluation system and reliability of its fixed tests. AB - OBJECTIVES: The ExaMe system for the evaluation of targeted knowledge has been in development since 1998. The new features of the ExaMe system are introduced in this paper. Especially, the new three-layer architecture is described. Besides the system itself, the properties of fixed tests in the ExaMe system are studied. In special detail, the reliability of the fixed tests is discussed. The theory background is explained and some limitations of the reliability are pointed out. METHODS FOR ESTIMATION OF THE RELIABILITY: Three characteristics used for estimation of reliability of educational tests are discussed: Cronbach's alpha, standardized item alpha and split half coefficient. The relation between these characteristics and reliability and between characteristics themselves is investigated. In more detail, the properties of Cronbach's alpha, the characteristics mostly used for the estimation of reliability, are discussed. A confidence interval is introduced for the characteristics. RESULTS AND CONCLUSIONS: Since 2000, the serviceability of the ExaMe evaluation system as the supporting evaluation tool has been repeatedly shown at the courses of Ph.D. studies in biomedical informatics at Charles University in Prague. The ExaMe system also opens new possibilities for self-evaluation and distance learning, especially when connected with electronic books on the Internet. The estimation of reliability of tests contains some limitations. Keeping them in mind, we can still get some information about the quality of certain educational tests. Therefore, the estimation of reliability of the fixed tests is implemented in the ExaMe system. PMID- 16685343 TI - Using computer and internet-based resources to teach health care planning and administration in an undergraduate medical program. AB - OBJECTIVE: We describe the teaching methods, involving computer and Internet based resources, used in the "Administration and Planning in Health Care" course of the undergraduate medical program at the Federal University of Rio Grande do Sul. METHOD: Description on how the curriculum guidelines for the undergraduate medical education in Brazil have been implemented at this university. The guidelines specify that graduates should be skilled and knowledgeable in health care administration and management, understand the market dynamics of health care services, and be prepared to contribute to the development of health policy. RESULTS: A required 60-hour course provides students with an opportunity to learn about the structure, planning and administration of the Brazilian and of other health care systems, and their roles, as health care professionals, within those systems. The course is also intended to allow students to develop the minimal skill set required for manipulation of health care data available from national and international databases, and to use the Internet as a source of information in health care. The curriculum includes: Module 1 - basic computer skills, an introduction to networks as an infrastructure for management, the use of spreadsheets and databases for data processing and system modelling, retrieval of Internet-based health data and on-line bibliographic searches; Module 2 - health system financing and service quality management, using a university hospital as a case study; and Module 3 - a comparison of the Brazilian public health care system (SUS) with other national health systems resulting in a term paper formatted for journal submission and presented at a simulated conference at the end of the course. CONCLUSION: Progressive shift in emphasis from theory to practice in this course has resulted in better development of the skill set required for the students. PMID- 16685344 TI - Developing an NLP and IR-based algorithm for analyzing gene-disease relationships. AB - OBJECTIVES: High-throughput techniques such as cDNA microarray, oligonucleotide arrays, and serial analysis of gene expression (SAGE) have been developed and used to automatically screen huge amounts of gene expression data. However, researchers usually spend lots of time and money on discovering gene-disease relationships by utilizing these techniques. We prototypically implemented an algorithm that can provide some kind of predicted results for biological researchers before they proceed with experiments, and it is very helpful for them to discover gene-disease relationships more efficiently. METHODS: Due to the fast development of computer technology, many information retrieval techniques have been applied to analyze huge digital biomedical databases available worldwide. Therefore we highly expect that we can apply information retrieval (IR) technique to extract useful information for the relationship of specific diseases and genes from MEDLINE articles. Furthermore, we also applied natural language processing (NLP) methods to do the semantic analysis for the relevant articles to discover the relationships between genes and diseases. RESULTS: We have extracted gene symbols from our literature collection according to disease MeSH classifications. We have also built an IR-based retrieval system, "Biomedical Literature Retrieval System (BLRS)" and applied the N-gram model to extract the relationship features which can reveal the relationship between genes and diseases. Finally, a relationship network of a specific disease has been built to represent the gene disease relationships. CONCLUSIONS: A relationship feature is a functional word that can reveal the relationship between one single gene and a disease. By incorporating many modern IR techniques, we found that BLRS is a very powerful information discovery tool for literature searching. A relationship network which contains the information on gene symbol, relationship feature, and disease MeSH term can provide an integrated view to discover gene-disease relationships. PMID- 16685345 TI - [Treatment of cleft lip and palate: past, present and future]. AB - Comparing the survey results of 224 Centers of Cleft Lip and Palate in the United States with the current status in China, and reviewing the experience and lessons systemically, we could get a conclusion that the repair of cleft lip and palate depends on the experience and excellent skills of the surgeons, and its important role in combined and sequential treatment (team approach) of cleft lip and palate should not be ignored. The team approach program requires multidisciplinary cooperation and good compliance of the patients. In order to improve the treatment outcomes of cleft lip and palate, it is necessary for us to study the advanced program and methods and combined the situation of our country. PMID- 16685346 TI - [Root canal therapy of resinified teeth with surgical operating microscope and ultrasonic instruments]. AB - PURPOSE: To evaluate the role of surgical operating microscope and ultrasonic instruments in endodontic treatment of patients who underwent previous resinifying therapy. METHODS: 63 premolars and molars that had been treated with resinifying therapy before were selected for root canal therapy. The coronal portion of resinified root canals were negotiated with ultrasonic instruments under surgical operating microscope, and the apical portion was managed with small size hand files and 15% EDTA. The root canals of all cases were shaped by Nickel-Titanium rotary instruments Hero 642, and obturated with lateral condensation technique. The negotiation of root canal system, instrument fracture, alterations of canal morphology, and operation time were recorded. The efficiency of preparation and obturation was analyzed by radiographs before and after treatment. RESULTS: The root canals of 54 teeth were negotiated, enlarged and obturated, with a success rate of 85.7%. No complications were found, such as vertical fracture, ledge, perforation and instrument separation. Good result of treatment was achieved in the 54 cases. CONCLUSIONS: The use of surgical operating microscope and ultrasonic instruments is proved to be effective in negotiation of coronal portion of the resinified root canals. Nevertheless, the use of ultrasonic instruments in apical or curved portion of root canals is not encouraged. PMID- 16685347 TI - [Fabrication and application of orbital template with part-opening channels for dental implantation]. AB - PURPOSE: To observe the effect of orbital dental template with part-opening channels used in dental implant surgery. METHODS: Working casts were made for 13 cases with missing teeth after impressions were taken. The dentitions of the cases were then restored with wax teeth. Part-opening channels were made through the wax teeth. After that, impressions and working casts were made again to fabricate the template with part-opening channels by thermo-forming technique. During the implant surgery, the drilling of the bur and inserting of the implants were guided by the part-opening channels on the templates. RESULTS: The working cast and X-ray films of the 12 cases showed that all implants were placed in desirable location and accurate direction with satisfactory prostheses. CONCLUSION: The dental template with part-opening channels could help the operator to place the implants in desirable location and direction successfully. Its fabrication was simple, but it could offer us great convenience. PMID- 16685348 TI - [Study of the dento-facial features in children of Han nationality with normal occlusion in Shandong district]. AB - PURPOSE: To establish the standard values of roentgengraphic cephalometry in children with normal occlusion in Shandong district and to make the facial model. METHODS: 156 children were selected to take lateral cephalograph, and were divided into two groups: mixed dentition and early permanent dentition. Cephalograms were analyzed for all subjects with WinCeph7.0. Data were obtained for student's t test between the two groups and between males and females. RESULTS: The norms with derivation for all measurement of X ray cephalometrical analysis on hard tissues were established. Meanwhile, the difference between males and females was compared and the facial model of children with normal occlusion was made. CONCLUSION: Gender difference was shown in linear measurements, with greater growth in males in general. The growing tendency of children from mixed dentition to early permanent dentition was that the maxilla of the male grows faster while the mandible of the female grows faster. PMID- 16685349 TI - [Orthodontic traction of impacted maxillary canine: a comparison of different ages]. AB - PURPOSE: To evaluate the success rate and the length of orthodontic treatment of impacted maxillary canines in adult and adolescent patients. METHODS: 17 adults who had been treated for a total of 21 impacted maxillary canines, was compared with a younger control group. The control group were chosen for a similar degree of impaction by carefully matching the positions of the impacted canines in 3 dimension. SPSS11.0 software package was used for Chi-square and paired t test. RESULTS: The success rate among the adults was 85.71% compared with 100% among the control group. There was significant difference between the two groups (P<0.01). The adults showed significant increase in the duration of treating the impacted canines (P<0.001). CONCLUSION: The success rate for orthodontic resolution of an impacted canine in an adult is significantly lower than in a younger patient and the duration of treatment in adults is expected to take considerably longer. PMID- 16685350 TI - [Expression and significance of PTEN protein in tongue squamous cell carcinoma]. AB - PURPOSE: To study the expressions and clinical significance of PTEN protein in tongue squamous cell carcinoma (TSCC). METHODS: The expressions of PTEN at protein level was investigated in 74 cases of TSCC and 15 cases of normal peripheral tissues around cancer (as control) by strep avidin-biotin complex (SABC) immunohistochemical technique. Mann-Whitney U-test, Pearson's Chi-square test and Fisher exact test were used to analyze the data. RESULTS: The rates of the positive expression in normal tissue and TSCC of PTEN were 100% and 66.2% respectively (P<0.05). Pathological grade and T stage were not significantly related with the level of PTEN expression (P>0.05). There was significant correlation between the level of PTEN expression and cervical lymph node status (P<0.05). CONCLUSION: Expressions of PTEN is correlated with cervical lymphnode metastasis in tongue squamous cell carcinoma. PMID- 16685351 TI - [Intralesional injection of Pingyangmycin for treatment of venous-venular malformation in the maxillofacial regions of children: report of 306 cases]. AB - PURPOSE: To evaluate the effect of intralesional injection of Pingyangmycin (PYM) for treatment of venous-venular malformation in the maxillofacial regions of children. METHODS: The medical records of 306 patients with venous-venular malformation in the maxillofacial regions between January 1997 and June 2005 were reviewed. One hundred and twenty-three of the patients were men, and one hundred and eighty-three were woman, the sex ratio was 1:1.49. The patients aged from one month to 12 years, 172 were less than 6 months (56.21%). Pingyangmycin was injected into the center or in the vicinity of the venous-venular malformations, and repeated every 7 to 10 days. This cycle were repeated 3 to 5 times when necessary. RESULTS: Three hundred and six patients were followed up for 6 to 102 months. Complete response was obtained in 156 cases (50.98%), partial response in 116 cases (37.91%), and improvement in 34 cases (11.11%). The overall response rate was 100%, and the curative rate was 88.89%. CONCLUSION: This method is safe, simple and effective for venous-venular malformation in the maxillofacial regions of children. PMID- 16685352 TI - [A study on the accuracy of electronic root canal length measurement and its influential factors]. AB - PURPOSE: To study the accuracy of JUSTY-II electronic apex locator and the influential factors on the accuracy of root canal working length measurement. METHODS: 148 teeth, including 71 teeth with pulpitis, 46 with necrotic pulp and 31 with apical periodontitis, were divided into two groups in random. The accuracy of electronic root canal working length measurement group was compared with that of the manual measurement group by Chi(2) test. The influence of clinical types, root canal in dry or moisture conditions and root canal preparation on the accuracy of root canal working length measurement was analyzed. RESULTS: The accuracy of electronic apex locator was 87.84%, while it was 43.24% in manual measurement, the difference was significant (P<0.01). The accuracy in the group of apical periodontitis was significantly lower than that of the other groups (P<0.05). Root canal in dry or moisture conditions had no effect on the accuracy of root canal length measurement, but root canal preparation could decrease the accuracy of measurement (P<0.05). CONCLUSION: Electronic apex locator is an accurate and convenient device in measurement of root canal working length. Apical periodontitis and root canal preparation can decrease the accuracy of measurement. PMID- 16685353 TI - [Ultrasonic atomization of Shaduolika and dexamethasone for pain relief in patients with foliate papillitis]. AB - PURPOSE: To evaluate the effect of ultrasonic atomization of Shaduolika (a traditional Chinese herb), dexamethasone for pain relief in patients with foliate papillitis. METHODS: 84 patients with foliate papillitis were divided into two groups randomly with the single-blind method. Patients in the experimental group were treated with ultrasonic atomization of Shaduolika and dexamethasone, while patients in the control group were treated by taking Niuhuangjiedu tablets (mixed traditional Chinese medicine). All the patients were observed for five days. Pain relief degree by VAS and the response to the interventions between the experimental and the control group were recorded and compared. The data was analyzed with Ridit test, Chi-square test and Student's t test using SAS6.12 software package, respectively. RESULTS: Pain scores between the experimental and the control group prior to treatment had no significant difference (U=0.1859<1.96, P>0.05), while significant difference existed in pain relief after treatment (U=5.773, P<0.01). The effect of ultrasonic atomization of Shaduolika and dexamethasone for pain relief was significantly better than the control group (U=5.233,P<0.01). CONCLUSIONS: For patients with foliate papillitis, ultrasonic atomization of Shaduolika and dexamethasone is a safe and effective method. PMID- 16685354 TI - [Perioperative nursing care of patients with zygomatic complex fracture]. AB - PURPOSE: To summarize the key points of nursing around operation and cautions that are needed to take in zygomatic complex fracture(ZCF). METHODS: 99 surgically treated ZCF cases were given preoperative nursing, psychological nursing, disease observation, preoperative preparation, postoperative nursing of general anesthesia, incision, nutrition, open-mouth training and observation of complications. RESULTS: Fracture reduction and fixation in 99 cases were satisfactory. The mouth opening raised from 2.2 cm to 3.9 cm. The wound healed primarily in 99% cases. No patient developed psychological disturbance. The degree of satisfactory about nursing exceeded 95%. CONCLUSION: The key points of perioperative nursing are psychological nursing, including elimination of fear and pessimism. When the patients have craniocerebral traumas, we should observe the changes of vital signs. When the patients have open fractures, we should take active hemostatic, antishock as well as, anti-infection measures with good postoperative care of incision, nutrition and observation of complications. PMID- 16685355 TI - [The influence of bonding strength of denture soft reline resin by different surface treatments]. AB - PURPOSE: This study evaluated the influence of bonding strength of heat polymerized denture soft reline resin by 4 different surface treatments bonded to denture base resin. METHODS: Denture base resin (8mm in diameter and 15 mm in length) were fabricated from heat-polymerized acrylic resin (Densply). And heat polymerized denture soft reline resin disks (Physio liner) were also made (4 mm thick and 10 mm diameter). Two denture base resins were bonded to the denture reline resin disk at the centure on the two sides of the disk. All the specimen were divided into 4 groups (n=10) with 3 different surface treatments: (1) no treatment(control), (2) sandblasted, (3) surface liner application, (4) surface liner application+sandblasted. Bond strength (MPa) was measured by tension bond testing at a 12.7 mm/min speed until the soft reline resin debonded from the base resin. Data were statistically analyzed by SPSS 10.0 software package for ANOVA. RESULTS: The mean bond strengths of group 1, 2, 3, 4 were 3.518, 2.834, 4.077, 3.852MPa respectively. The bonding strength of all the three groups showed significant difference (P<0.0014). The group with surface liner application showed significantly highest bonding strength, while the group with sandblasted showed the lowest bonding strength. CONCLUSIONS: The use of surface liner can significantly improve the bonding strength of heat-polymerized denture soft reline resin to the denture base resin, while the bonding strength decreased after using sandblasting. PMID- 16685356 TI - [The establishment of B(a)P and TPA transformed cell line and its biological characteristics]. AB - PURPOSES: To transform HPV E6/E7 immortalized human oral epithelial cell line HIOEC cells by benzo(a)pyrene B(a)P and tetradecanoyl phorbol acetate (TPA) in vitro, and establish a carcinogenesis model of oral squamous cell carcinoma. METHODS: HIOEC cells were treated with 0.1 microg/ml -1.2 microg/ml B(a)P for 6 months. Some of these cells were treated with 0.1 microg/ml TPA 24 hours at 4th passage and 10th passage, respectively. The cells were cloned at 18th passage, and then were cultured with DMEM medium contain 10% FBS at 21st passage. The cells were cultured in vitro for 1 year and developed into a malignant cell line HIOEC-B(a)P-TPA. The morphological changes of the cells were observed with differential interference contrast microscope and HE staining. The expression of cytokeratin and vimentin was identified with immunohistochemical staining. The soft agar colonies forming ability and tumorigenesity of the cells were identified to confirm the malignant characteristics of HIOEC-B(a)P-TPA cells. RESULTS: (1) After HIOEC cells were treated with B(a)P plus TPA for 6 months, HIOEC-B(a)P-TPA cells grew well in DMEM medium containing with physical concentration of calcium and 10% FBS. (2) During HIOEC cells were treated with chemical carcinogens, the morphology of the cells was changed. HIOEC-B(a)P-TPA cells showed as fibroblast-like cells with many atypical mitosis. (3) The expression of cytokeratin decreased in the cells while that of vimentin increased in the cells. (4) HIOEC-B(a)P-TPA cells had strong soft agar colony formation ability and the colony formation ratio was 24.5%. (5) HIOEC-B(a)P-TPA cells have no tumorigenisity till now. CONCLUSIONS: We established a biological factors and chemical carcinogens induced malignant cell line-HIOEC-B(a)P-TPA after a long period. It will provide a good multiple factors, multistage carcinogenesis model of OSCC for further research. PMID- 16685357 TI - [The temporal and spatial expression of CBFalpha1 in rabbit condylar cartilage during forward mandibular positioning]. AB - PURPOSE: To identify the relationship between the expression of Core binding factor alpha1 (Cbfalpha1) in condylar cartilage and condylar growth modifications in growing rabbits following forward positioning mandibles with functional appliance. METHODS: Sixty rabbits with age of 8 weeks were randomly divided into the experimental group (n=36) and control group (n=24). Rabbits in the experimental groups were induced to forward their mandibles by a functional appliance. The animals in the two groups were killed after 3 days and 1, 2, 4, 8 and 12 weeks, respectively. The temporal and spatial expression of Cbfalpha1 in condylar cartilage was examined by immunohistochemical staining and measured with Image Computer Analysis Software. Date was statistically analyzed by Student's t test and one-way ANOVA. RESULTS: The expression of Cbfalpha1 was mainly found in the chondrocytes from the transitive zone and hypertrophic zone, and was also observed in the chondrocytes and osteoblasts of the mineralized zone. Compared with those of the age-matched controls, the positive signals for Cbfalpha1 of each zone in the experimental animals were significantly stronger (P<0.05), and the highest expression of transitive zone, hypertrophic zone and mineralized zone was detected after 4 weeks of treatment (71.00+/-1.52, 50.00+/-0.75, 82.00+/ 0.39). CONCLUSION: The pattern of Cbfalpha1 expression is concerned with the adaptive remodeling of the condylar cartilage after functional appliance. PMID- 16685358 TI - [Effects of ADAM28 AS-ODN on proliferation and AKPase of HDFCs]. AB - PURPOSE: To study the effects of ADAM28 antisense oligodeoxynucleotide (AS-ODN) on proliferation of human dental follicle cells (HDFCs) and activity of alkaline phosphatase (AKPase). METHODS: Cell culture, gene transfection, MTT chromatometry and enzyme kinetics methods were used to detect the possible mechanisms of ADAM28 AS-ODN on proliferation, differentiation of HDFCs and effect of the activity of AKPase. Statistical significance was assessed by multiple comparison (q test, SNK) in one-way analysis of variance (ANOVA). RESULTS: ADAM28 AS-ODN was transfected into HDFCs successfully. MTT and AKPase activity assays indicated that cell proliferation and AKPase activity of ADAM28 AS-ODN group were significantly lower than those of S-ODN and vacuity control group (P<0.01). MTT assay revealed P value between AS-ODN and S-ODN, blank control group was 0.0002, 0.0001 respectively, P<0.01; AKPase assay showed P value between AS-ODN and S ODN, vacuity control group was 0.0007, 0.0003 respectively, P<0.01. ADAM28 AS-ODN could inhibit the proliferation and AKPase activity of HDFCs significantly. CONCLUSIONS: ADAM28 may promote proliferation and differentiation of HDFCs by participating in Notch signal pathway. PMID- 16685359 TI - [Study on the osteogenesis ability of co-culturing bone marrow stromal cells (BMSCs) and small intestinal submucosa]. AB - PURPOSE: Using co-culturing of bone marrow stromal cells (BMSCs) and small intestinal submucosa (SIS), to evaluate the cellular compatibility and the osteogenesis ability as well as the feasibility of SIS to serve as a scaffold in tissue engineering. METHODS: Bone marrow was aspirated from the healthy adult goat and culture-expanded in vitro by the method of total marrow. The third passage cells were transplanted into SIS after being induced to differentiate into osteoblasts for 4 days and then seeded into SIS for 3 to 10 days. BMSCs alone were cultured at the same condition to act as controls. The cellular morphology and function (attachment, proliferation and differentiation) and the ectopia osteogenesis in vivo were assessed separately by means of phase contrast microscope, SEM and MTT, ALP activity, histology detection. The difference between the two groups were analyzed by Student's t test statistically. RESULTS: The BMSCs could adhere to SIS and proliferate and grow on the surface of SIS normally. The cellular activity and function were not affected by SIS, no statistical difference was found between the two groups (P>0.05). The BMSCs/SIS compounds had good abilities of ectopia osteogenesis in vivo, and with time going on new bone formation increased. There was no bone formation in the control group. The endochondral bone formatin was the main pattern of osteogenesis. CONCLUSIONS: As a natural, absorbable, acellular scaffold, SIS is biocompatible and can be used as a tissue engineering scaffold. And for a membrane feature, there is the possibility to construct tissue engineered bone membrane with SIS. PMID- 16685360 TI - [Culture and characteristics of porcine dental papilla cells (pDPCs) in vitro]. AB - PURPOSE: To culture porcine dental papilla cells (pDPCs) and to study the cytobiological characteristics of the cells in vitro. METHODS: Dental papillae were collected from postnatal (1-3 days) pigs, then the pDPCs were isolated and cultured through the method of enzyme-digestion. Vimentin and cytokeratin (CK) were used to demonstrate the cells' mesenchymal derivation. Collagen I collagen III and dentin sialoprotein (DSP) were detected in pDPCs by immunohistochemistry. RESULTS: The pDPCs were well cultured in the medium of DMEM/F12 containing 10% FBS in vitro. Vimentin was positively expressed in pDPCs but CK was not. Collagen I, collagen III, and DSP were all positive in the plasma of the pDPCs. CONCLUSIONS: The pDPCs were successfully isolated and cultured in vitro. The pDPCs come from mesenchymal tissue and have the capability of synthesizing extracellular matrix (collagen I, collagen III, and DSP) of dentin-pulp complex. This study indicated that the pDPCs may have the potential to be used as seed cell in regeneration of dentin-pulp complex. PMID- 16685361 TI - [Primary culture and identification of mouse odontoblast-like cells]. AB - PURPOSE: To culture primary mouse odontoblast and to provide a base for study on inducing ES cells to odontoblast. METHODS: Lower incisor germs were removed from 1-week-old mouse. The dental papillae were isolated in microscope, and the dental papillae cells were dispersed using 0.25% collagenase and 0.25% trypsin. The cell clones, which had similar morphology to odontoblasts, were selected for further culturing. The primary cultured cells were identified by light and electron microscopes and mRNA expression of mouse dentin sialophoprotein. RESULTS: The cultured cells had the same morphology and ultrastructure. They were rich in Golgi's complex, ribosome and rough endoplasmic reticulum. These cells expressed DSPP at mRNA levels. CONCLUSION: The cultured cells were mouse odontoblast-like cells. The method could be used for the study of odontal cells in vitro. PMID- 16685362 TI - [Generation of transgenic mouse of dentin sialoprotein and transgene expression analysis]. AB - PURPOSE: To generate the transgenic mouse model of DSP and perform transgene expression analysis by RT-PCR. METHODS: Plasmid pcDNA3.1-CX was constructed by substituting promoter cbeta-actin for CMV promoter of pcDNA3.1, and the ultimate transgenic vector, pcDNA3.1-CX-dsp, was constructed by cloning DSP coding sequence into pcDNA3.1-CX. The pcDNA3.1-CX-dsp plasmid was linearized and microinjected into the male pronucleus of the zygotes. The tail DNA of pups was tested by PCR and Southern blot. A member of F1 generation of one positive mouse was used to perform transgene expression analysis by RT-PCR. RESULTS: 717 embryos were implanted to 29 recipient pseudopregnant mice, 4 of the 67 pups carrying the transgene. Expression of DSP was detected in a member of F1 generation of one positive mouse by RT-PCR. CONCLUSION: Founders of the DSP transgenic mouse were obtained successfully, and the expression of DSP was primarily confirmed. PMID- 16685363 TI - [Experimental study of target-specific adriamycin nanoparticles in treating tongue squamous cell carcinoma]. AB - PURPOSE: This study was designed to explore the method for increasing the target specific action of anticancer agent in treating tongue squamous cell carcinoma. METHODS: 30 purebred New Zealand rabbits burdened with tongue squamous cell carcinoma were randomly divided into 2 groups. Group A was treated with adriamycin and group B treated with adriamycin nanoparticles, with 15 animals in each group. The adriamycin and poly (buty1-2-cyanoacrylate)-adriamycin nanoparticles (mean diameter 93.1 nm+/-11.7 nm) with 1 mg/ml in concentration were administered respectively to subjects through super-selective lingual arterial tuber. The dose was 2 mg/kg for each group. 1, 5 and 15 hours after administration, 5 subjects were sacrificed and the tongue, carcinoma in tongue tissue, heart, liver, spleen, kidney and plasma were examined through high performance liquid chromatography (HPLC) to identify the drug concentration. The data then underwent Student's t test. RESULTS: The drug concentration in the tongue and carcinoma in group B were significantly higher than those in group A (P<0.01). The drug concentration in heart, liver, spleen, kidney and plasma of group B were significantly lower than those of group A (P<0.01). CONCLUSION: The super-selective lingual arterial administration of adriamycin nanoparticles can reinforce the target-specific action of anticancer agent, increase the drug concentration within the lesion and decrease it in non-specific organs. PMID- 16685364 TI - [The effect of overdose fluoride on the expression of TGF-beta1 in rat's dental pulps]. AB - PURPOSE: To observe the effect of overdose fluoride on the expression of TGF beta1 in rat's dental pulps. METHODS: 20 wister rats were divided into two groups. In the control group, equal dose distilled water were given to the rats. In the experimental group, 20 mg.kg(-1).d(-1) NaF were given. After 8 weeks of treatment, immunohistochemical staining was adopted for detection of the expression of TGF-beta1 in dental pulps of the rats. SPSS10.0 software package was used for Student's t test. RESULTS: Image analysis results showed that the expression of TGF-beta1 in the dental pulp and inner dentin were inhibited in the experimental group as compared with the control group (P<0.01). CONCLUSION: The overdose fluoride will inhibit the secretion of TGF-beta1, which leads to abnormal development of the teeth. PMID- 16685365 TI - [Effect of overdose fluoride on expression of bone sialoprotein in developing dental tissues of rats]. AB - PURPOSE: To investigate the changes of bone sialoprotein (BSP) in developing dental tissues of rats exposed to fluoride. METHODS: Twenty rats were randomly divided into two groups, one was with distilled water (control group), the other was with distilled water treated by fluoride (experimental group). When the fluorosis model was established, the changes of the expression of BSP were investigated and compared between the two groups. HE staining was used to observe the morphology of the cell, and immunohistochemisty assay was used to determine the expression of BSP in rat incisor. Student's t test was used for statistical analysis. RESULTS: The ameloblasts had normal morphology and arranged orderly. Immunoreactivitis of BSP was present in matured ameloblasts, dentinoblasts, cementoblasts, and the matrix in the control group. But in the experimental group the ameloblasts arranged in multiple layers, the enamel matrix was confused and the expression of BSP was significantly lower than that of the control group. Statistical analysis showed significant differences between the two groups (P<0.01). CONCLUSION: Fluoride can inhibit the expression of BSP in developing dental tissues of rats, and then inhibit differentiation of the tooth epithelial cells and secretion of matrix. This is a probable intracellular mechanism of dental fluorosis. PMID- 16685366 TI - [Study on abutment movement of distal removable dentures retained by attachments]. AB - PURPOSE: To study the influence on the abutment movement with three kinds of precision attachments designed for distal-extension removable partial dentures (RPDs). METHODS: Snap attachment, Ceka attachment, Mini-SG attachment were used to retain RPDs. Then food chip was masticated between the upper and lower dentitions on the attachments side. Placement of abutment was measured by LB-72 high-sensitive-laser-traneferring-detector and the data were statistically analyzed for random block analysis of variance. RESULTS: The displacement, inclination, and torsion of the abutment movements in the mesial-distal, bucco lingual and occluso-gingival directions all had statistical significance (P<0.05). The largest mesial-distal inclination occurred with the Mini-SG attachment. The Ceka attachment produced more abutment displacement in mesial distal and apical directions. Occlusal displacement and anti-clockwise rotation were the least with the Snap attachment. CONCLUSION: The most equitable distribution of stresses on the supporting structures was achieved by Snap attachment with its resilent assemble, Ceka attachment transfers most of the loading forces to the abutments, Mini-SG attachment can not reduce the excessive torque applied to the abutments effectively. PMID- 16685367 TI - [Three dimensional finite element stress analysis in the vertical fractured first premolar after bonding and replantation]. AB - PURPOSE: To evaluate the change of stress distribution after bonding and replantation of vertical fractured first maxillary premolar. METHODS: The stress distribution in two types of loading and different treatments on the vertical fractured first maxillary premolar were analyzed by three dimensional finite element. The gap was respectively filled with Clearfil SE Bond and Super Bond C & B; and vertical and lateral stress force was calculated. RESULTS: The result of the FEM study of adhesive fracture tooth indicated that the stress distribution was similar to the normal tooth, Clearfil SE Bond was better than Super Bond C & B. CONCLUSION: The treatment of bonding and replantation for vertical fractured tooth is effective. PMID- 16685368 TI - [Study on the stress distribution of different superstructures for tooth-implant supported denture]. AB - PURPOSE: To compare the stress distributions of fixed bridge, cement-retained telescopic prostheses and extracoronal precision attachment (CEKA attachment) when connect the implants to the natural teeth, in order to find a more suitable design for it. METHODS: 3-D finite element models of tooth-implant partial fixed dentures were constructed by using the ANSYS software. The partial fixed denture was supported by the second premolar and an implant (substitute the second molar). A total load of 200N was distributed to the three units of the denture in vertical and lateral (45 degree) directions. RESULTS: Both of the cement-retained telescopic and the CEKA attachment made the stress of the osseous tissues surrounding the implant and the tooth declined obviously under the loads of the two directions. The stress of the cement-retained telescopic was lower under vertical load,the stress of Ceka attachment was lower under lateral load while it caused the stress of the natural tooth increasing slightly. The stress under the lateral load was higher than that under the vertical load. CONCLUSION: Both of the cement-retained telescopic and the CEKA attachment can improve the stress distribution of tooth-implant support denture. The CEKA attachment is better under lateral loads. PMID- 16685369 TI - [An in vitro experiment on the antimicrobial effects of ethanol extract from salvia miltiorrhiza bunge on several oral pathogenic microbes]. AB - PURPOSE: To study the in vitro antimicrobial activity of ethanol extract of salvia miltiorrhiza bunge on several oral pathogenic microbes. METHODS: Antimicrobial activities of ethanol extract of salvia miltiorrhiza bunge for porphyromonas gingivalis ATCC 33277, A. actinomycetes comitans ATCC 24523, Streptococcus mutans, and Lactobacillus were determined using the cup-plate method. The minimum antibacterial concentrations of ethanol extract of salvia miltiorrhiza bunge were measured. SPSS10.0 software package was used for Student's t test. RESULTS: Ethanol extract of salvia miltiorrhiza bunge had antimicrobial activities on Porphyromonas gingivalis, A. actinomycetemcomitans, Streptococcus mutans, Lactobacillus, and the minimum inhibitory concentrations were 15.62 mg/ml, 15.62 mg/ml, 62.50mg/ml and 15.62 mg/ml; The pH of the solution was influential to its antimicrobial activity. CONCLUSION: Ethanol extract of salvia miltiorrhiza bunge has an antimicrobial activity on oral pathogenic microbes. PMID- 16685370 TI - [Design of geometric models of the removable partial denture framework]. AB - PURPOSE: The aim of this study was to introduce a method on computer aided design (CAD) of removable partial denture(RPD) framework based on domestic CAD/CAM software system, which contributes to the further research to develop the domestic software system applied in restorative dentistry. METHODS: Point cloud data of a partially edentulous cast, a mandibular Kennedy Class II modification 2 arch, was captured by an optical scanning system with projective grating and high resolution digital camera. Using domestic CAD/CAM software system, the above point cloud data was reduced, digital surveying line and inserting path were determined. In terms of the principle of clinical design, the tissue surfaces and polishing surfaces of every component of RPD were built such as clasp, rest, lingual bar, and mesh construction, et al. As well as the characteristic structures of the framework, including the half-pear shaped cross section of lingual bar, the internal and external finish lines, the tissue stop at the tissue surface of mesh construction and so on, were fabricated on the base of reduced data model. 3-D surface model of the RPD framework was created. RESULTS: Geometric model of the RPD framework was fabricated successfully, which took on good fitting, high visibility and editing conveniently. And the data converted to STL file format that could be read by any other CAD/CAM software system and was in preparation for subsequent computer aided manufacture (CAM) of RPD framework. CONCLUSIONS: It is suggested that this method based on domestic software in CAD of RPD framework be feasible. PMID- 16685371 TI - [Antifungal susceptibility testing of commensal and pathogenic clinical isolates of oral Candida]. AB - PURPOSE: To evaluate and compare the sensitivity and resistance of commensal and pathogenic oral Candida to antifungal agents. METHODS: YBC Test Kit was used to identify species of commensal and pathogenic oral Candida; The in vitro susceptibilities of oral Candida to 5-flucytosine (5-FC), itraconazole (ITR) and fluconazole (FLU) were determined by NCCLS M27-A2 method. RESULTS: All strains were sensitive to 5-flucytosine, drug resistant strains to fluconazole and itraconazole were found both in commensal and pathogenic oral Candida spp; The proportion of no-C. albicans and antifungal drug resistant strains isolated from head and neck cancer patients treated with radiotherapy or chemotherapy were higher than oral commensal Candida and oral Candidiasis. CONCLUSIONS: The no-C. albicans and drug resistant strains to fluconazole and itraconazole can be found both in commensal and pathogenic oral Candida spp now. PMID- 16685372 TI - [Personal suggestions to write and publish SCI cited papers]. AB - The Science Citation Index (SCI) was introduced primarily as a method of information retrieval and has also been used as an objective measure of the quality of an academic journal. How to write and publish a SCI cited paper has been posed to the daily schedule of scientific researchers. This paper gives some suggestions according to the author's experience in writing and publishing SCI cited papers. In preparing a manuscript of high quality in English, one should possess an excellent English ability, carefully read the "Instructions for authors" of the journal which you will submit to, carefully and seriously prepare the manuscript in English and revise it according to the peer reviewed comments. PMID- 16685373 TI - siRNA mediated knockdown of fibroblast growth factor receptors 1 or 3 inhibits FGF-induced anchorage-independent clonogenicity but does not affect MAPK activation. AB - Supplementation with exogenous growth factors such as fibroblast growth factors (FGFs) is essential for anchorage-independent growth of the SW-13 human adrenal adenocarcinoma cell line. We have found that SW-13 cells express mRNAs for FGFRs 1, 3, and 4, but not FGFR2. To assess the roles of individual FGFRs, in anchorage independent growth, we determined the effects of down-regulation of each FGFR on FGF2- and FGF4-mediated soft agar colony formation in these cells. Using RNAi strategies we found that knockdown of either FGFR1 or FGFR3 leads to inhibition of FGF2- or FGF4-induced soft agar clonogenicity without affecting that induced by heregulin beta1. However, this inhibition is independent of ERK1/2 activation as levels of FGF-induced phospho-ERK 1/2 remain unchanged upon knockdown of either FGFR1 or FGFR3. Conversely, RNAi-mediated knockdown of FGFR4 appeared to have no significant effect on either FGF2- or FGF4-induced anchorage-independent colony formation, or ERK1/2 phosphorylation. These results suggest that constitutive levels of both FGFR1 and FGFR3, but not FGFR4 are essential for FGF stimulated anchorage-independent growth of SW-13 cells. PMID- 16685374 TI - Thymidylate synthase expression in oral squamous cell carcinoma predicts response to S-1. AB - The purpose of this research was to evaluate the predictive value of expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), or orotate phosphoribosyltransferase (OPRT) genes for response to S-1. Twenty-five patients with oral squamous cell carcinoma (OSCC) received S-1 80 mg/m2/day. Pretreatment tumor biopsies were analyzed for TS, DPD, TP or OPRT mRNA expression by real-time reverse transcription-PCR. TS protein expression was evaluated by immunohistochemistry using a polyclonal TS antibody. Twenty-five patients were evaluable for response and gene expression. Six of the 25 (24%) achieved complete remission and 4 of the 25 (16%) had a partial response. Median TS/beta-actin was 2.51 (range 0.98-7.07). Median TS/beta-actin was 1.26 in responding patients and 3.43 in non-responders (P=0.0001). Ten of 11 patients with TS/beta-actin <1.80 and 0 of 15 with higher values responded (P<0.0001). Overall survival was 29.7 months in patients with TS/beta-actin <1.80 and 41.7 months in patients with higher values (P=0.0013). No correlations were seen between expression of DPD, TP or OPRT mRNA and response or survival. Weak TS staining was seen in 6 of 25 tumors evaluable for immunohistochemistry, including 5 responders. All 4 of the patients with both weak staining and TS/beta-actin <1.80 responded. High TS mRNA expression predicts non-response to S-1. On the other hand, high levels of DPD or TP mRNA and low levels of OPRT mRNA are not associated with S-1 resistance. TS mRNA expression is considered to be a useful prognostic factor in OSCC patients with S-1 single-agent therapy. PMID- 16685375 TI - BARD1 content correlates with increased DNA fragmentation associated with muscle wasting in tumour-bearing rats. AB - Apoptotic events have been clearly associated with muscle wasting in different types of experimental cancer cachexia. In these conditions, cell death is triggered by cytokines or tumour-produced factors. BARD1 is a nuclear protein that is also involved in apoptosis both in vitro and in vivo. The results presented here demonstrate that BARD1 content in skeletal muscle correlates with increased DNA fragmentation during experimental cancer cachexia. It is suggested that BARD1 acts as a modulator of muscle apoptosis or, alternatively, that BARD1 participates in the protein degradation by functioning as ubiquitin ligase. PMID- 16685376 TI - Chinese medicine Angelica sinensis suppresses radiation-induced expression of TNF alpha and TGF-beta1 in mice. AB - Radiotherapy of thoracic cancer often causes pulmonary inflammation leading to pneumonitis and fibrosis. We favor the hypothesis that cytokine-mediated multicellular interactions may result in the overexpression of proinflammatory cytokines such as TNF-alpha and TGF-beta1, which promotes progressive radiation induced lung injury. The root of Angelica sinensis, known as 'Danggui' in Chinese medicine, is widely used to treat radiation-induced pneumonitis in humans and shows clinical efficacy and low/no toxicity with an unclear mechanism. Using quantitative RT-PCR and immunohistochemistry (IHC) methods, we investigated radiation-induced lung injury in a mouse model. C57BL/6 mice were assigned to 4 groups: no treatment (NT), Angelica Sinensis treatment only (AS), X-ray irradiation only (XRT, single fraction of 12 Gy irradiation to the thoraces) and AS treatment plus XRT (AS/XRT). Mice in NT and AS groups exhibited low TNF-alpha and TGF-beta1 mRNA levels and few positive cell counts for TNF-alpha (8-17 cells per field, x400 magnification) and TGF-beta1 (9-31 cells per field), respectively. In XRT mice, there were increased inflammatory cells positive for TNF-alpha and TGF-beta1 in lung tissue compared with NT mice (P<0.01). However, when XRT mice received AS treatment (AS/XRT), the number of inflammatory cells in lung tissue positive for both TNF-alpha and TGF-beta1 was decreased compared with XRT-only mice (P<0.01) accompanied by moderately decreased mRNA levels of TNF alpha and TGF-beta1. We conclude that radiation induces expression of TNF-alpha and TGF-beta1 in the inflammatory cells of irradiated lung tissue during the pneumonic phase. The predominant localization of TNF-alpha and TGF-beta1 in inflammatory cell infiltrates suggests these cytokines' involvement in the process of radiation-induced pneumonitis. Moreover, effective down-regulation of TNF-alpha and TGF-beta1 in irradiated lung tissue by Angelica Sinensis is, at least in part, indicative of its clinical efficacy in treating radiation-induced pneumonitis. PMID- 16685377 TI - Robo1/Robo4: differential expression of angiogenic markers in colorectal cancer. AB - The family of roundabout (Robo) proteins is related to the transmembrane receptors and plays a major role in the process of axonal guidance in neurogenesis. It has recently been shown that Robo proteins are also associated with tumor angiogenesis with Slit2 acting as the corresponding ligand. The aim of this study was to validate the differential expression by means of microarray analysis and real-time PCR and to analyze the in situ expression of Robo1 and Robo4 in colorectal cancer. Quantitative analyses of Robo1, Robo4 and Slit2 mRNA expression measured by large scale gene expression studies (Affymetrix U133A) showed a significant up-regulation of Robo1 in tumor vs. normal tissue, whereas Robo4 and Slit2 showed no significant deregulation. For subsequent real-time PCR experiments, paired colorectal tissue samples from cancerous and corresponding non-cancerous tissues were obtained from 50 colorectal cancer patients who underwent surgical resection. Robo1 mRNA overexpression in cancerous tissue compared with normal counterparts was observed in 80% of the patients with a 4 fold expression in 45% and a 12-fold expression in 15%. For Robo4, an up regulation was detected in >70% (36/50). For Slit2, no differential expression was observed. The overexpression of Robo1 and Robo4 in tumor vs. normal tissue was verified using real-time PCR. The histological analysis revealed an expression of Robo1 mainly in tumor cells, whereas Robo4 is located primarily in endothelial cells of tumor vessels. Therefore, the Robo proteins provide potential target structures for the anti-tumorigenic and anti-angiogenic therapy of colorectal carcinoma. PMID- 16685378 TI - Activation of STAT3 is a marker of poor prognosis in human colorectal cancer. AB - It is known that the signal transducer and activator of transcription 3 (STAT3) is a key signaling molecule implicated in the regulation of growth and malignant transformation. Constitutive activation of STAT3 has been observed in a number of tumour-derived cell lines, as well as in a wide variety of human malignancies. The present study was conducted to examine p-STAT3 (activated form of STAT3) expression and its association with clinicopathological factors and prognosis in human colorectal adenocarcinomas. Expression of p-STAT3 was immunohistochemically examined in 108 cases of colorectal adenocarcinoma tissue obtained at surgery. and was found in 57.4% of tumours (62 of 108). p-STAT3 immunoreactivity significantly correlated with the depth grading of tumour invasion (p<0.001), lymphatic invasion (p<0.05), Dukes' classification (p<0.05), stage (p<0.001) and prognosis after operation (p<0.001). Expression of p-STAT3 was a marker of poor prognosis in overall survival (p<0.01). Expression of p-STAT3 was detected by Western blot analysis in three colon carcinoma tissue samples obtained at surgery. To our knowledge, this is the first study on the poor prognosis of p STAT3 in human colorectal adenocarcinomas. These findings suggest that expression of p-STAT3 is an important factor related to tumour invasion and poor prognosis of human colorectal adenocarcinoma. PMID- 16685379 TI - Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer. AB - Understanding the molecular action of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, might allow us to perform more effective therapies for hormone-independent advanced prostate cancer. A DNA microarray study was undertaken to comprehensively analyze the alteration of levels of 1,081 genes after gefitinib treatment in androgen-independent PC3 and DU145 cells and androgen-dependent LNCaP cells. The proliferation of PC3, DU145 and LNCaP cells was significantly inhibited by 50.2%, 83.8% and 55.2%, respectively, 6 days after 10 microM gefitinib administration. Of the above 1,081 genes, we identified 23, 13 and 33 genes with significantly different expression in PC3, DU145 and LNCaP cells, respectively, 24 h after 10 microM-gefitinib exposure. Among the identified genes, only Quiescin Q6, a negative cell cycle regulator, was increased after gefitinib treatment in all three cell lines regardless of gefitinib sensitivity. Except for Quiescin Q6, there were no overlapping genes between PC3 and DU145 cells. However, levels of several oncogenes or proliferation-related genes were changed after gefitinib treatment in the 2 androgen-independent cell lines. We also identified 7 unique genes [glycyl-tRNA synthetase, interferon, alpha-inducible protein, stratifin, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, dual specificity phosphatase 9, guanine nucleotide binding protein (G protein) beta polypeptide 2, neural retina leucine zipper] whose levels were altered exclusively after gefitinib administration in gefitinib-resistant PC3 and LNCaP cells, but not in DU145 cells, suggesting that these 7 genes could be targets for overcoming gefitinib resistance. Collectively, our molecular profiling data will serve as a framework for understanding the molecular action of gefitinib for prostate cancer. PMID- 16685380 TI - Siegesbeckia glabrescens induces apoptosis with different pathways in human MCF-7 and MDA-MB-231 breast carcinoma cells. AB - Breast cancer is one of the most common malignancies diagnosed in women and it is increasing in incidence. Siegesbeckia glabrescens (SG) has been used in traditional oriental medicine to treat cardiovascular diseases such as hypertension and angina pectoris. This study examined whether or not SG could induce apoptosis in human breast carcinoma cells. The treatment of estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cells with a variety of SG concentrations (0-1.0 mg/ml) resulted in a dose-dependent sequence of events that were marked by apoptosis. Furthermore, this apoptosis was accompanied by the cleavage of procaspase-9 and -3, and poly(ADP-ribose) polymerase (PARP) in the MCF-7 cells, and procaspase-8 and -3 and PARP in the MDA-MB-231 cells. Although, the SG-induced apoptosis was associated with a decrease in the level of Bcl-2 mRNA expression and an increase in the level of Bax mRNA expression in MCF 7 cells, there was no detectable change in the MDA-MB-231 cells. This suggests that SG might exert anti-proliferative action in human breast carcinoma cells via two different apoptotic pathways, namely an intrinsic signal in MCF-7 cells and an extrinsic signal in MDA-MB-231 cells. Therefore, regardless of the ER status, SG might be a promising pro-apoptotic agent for treating breast cancer. PMID- 16685381 TI - Duodenal juice stimulates oesophageal stem cells to induce Barrett's oesophagus and oesophageal adenocarcinoma in rats. AB - the present study was performed to examine the sequential process of the development of Barrett's oesophagus (BE) and oesophageal adenocarcinoma (ADC) induced by duodeno-oesophageal reflux (DER) in rats. Total gastrectomy was performed in male Wistar rats weighing approximately 250 g followed by reconstruction with oesophago-jejunostomy, which causes unavoidable DER without exposure to exogenous carcinogens. Animals were selected at random and sacrificed every 10 weeks after surgery until 50 weeks. Severe squamous oesophagitis with erosion, regenerative thickening (RT), and basal cell hyperplasia (BCH) were observed on the 10th week after surgery. On the 20th week, glandular structures that stained positively with Galactose oxidase-Schiff (foveolar metaplasia) were observed in the basal layer of the oesophageal squamous epithelium. On the 30th week, the glands developed and formed cysts that stained positively with concanavalin A (pyloric glandular metaplasia) and/or high-iron diamine and Alcian blue (intestinal metaplasia). From the 40th week after surgery, ADC cells surrounded by columnar-lined epithelium were found. Persistent stimulation with DER can alter the stem cells in the squamous epithelial basal layer leading to the formation of columnar-lined cells and subsequent ADC. Foveolar metaplasia was observed as part of the sequence of events leading to the development of columnar lined epithelium (CLE), followed by the appearance of pyloric glandular metaplasia and intestinal metaplasia, completing the histogenesis of BE. PMID- 16685383 TI - Chronic colitis promotes tumor development. AB - Patients with chronic ulcerative colitis have a significantly increased risk of colorectal cancer development. This study was aimed at clarifying whether colitis promotes tumor development or not. A dose of 200 mg/kg body weight 1,2 dimethylhydrazine was given to male Wistar rats. Four weeks later, 5% acetic acid (colitis group) or 0.9% saline (control group) was administered intrarectally once a week for 12 weeks and the rats were sacrificed after 27 weeks of dimethylhydrazine injection. Macroscopic lesions (ML) were more frequently detected in the colitis group than in the control group without statistical significance. However, the number of ML per rat with ML was largest in the colitis group (4.50 vs. 1.33; p=0.039). Eleven of 13 tumors were sessile in the colitis group, while three of five were pedunculated in the control group (p=0.044). All ML of 3 mm or more in diameter in the control group were intramucosal well-differentiated tumors. In the colitis group, 4 of 13 tumors were poorly or moderately differentiated or mucinous carcinomas, and 11 of 13 invaded the submucosal layer or deeper (p=0.003). The number of aberrant crypt foci per rat was smaller in the colitis group than in the control group. The number of crypt orifices was larger in the colitis group than in the control group (23.6 vs. 8.8: p<0.001). Significantly higher proliferative activity of normal-appearing mucosa was noted in the colitis group in all three parts of the colon. Colitis is suggested to promote colonic tumor development. PMID- 16685382 TI - Potential role of HER-2; in primary breast tumor with bone metastasis. AB - HER-2 is an important prognostic factor in breast cancer, and its overexpression is observed in 20-60% of cases with micrometastases in the bone marrow. The aim of this study was to explore the potential functional role of HER-2 in primary breast tumors with bone metastases. Forty-eight primary breast tumors with simultaneous or non-simultaneous bone metastases were studied. The expression of hormone receptors, and metastasis and growth factor-related proteins were assessed by immunohistochemical staining. The correlation in statistical significance was assessed by the Chi-square test. Of the 48 breast tumors, 11 (22.9%) were HER-2-positive and 37 were HER-2-negative. There was no significant difference in HER-2 status and clinicopathologic factors between the two groups. Of the 11 HER-2-positive tumors, there were 2 and 3 cases that showed positive nuclear expression for estrogen and progesterone receptors, respectively. No extranuclear expression of HRs was detected in these tumors. For metastasis related proteins such as c-Met, VEGF, and MTA-1, which are activated by HER-2, only some insignificant focal expression of these proteins was observed. An increased level of pAkt was observed in 9 (81.8%) of 11 tumors, and an increased expression of CXCR4 was observed in 6 (54.5%) of 11 tumors. The frequency of increased levels of pAkt and CXCR4 was not significant between HER-2-positive and -negative tumors. The increased levels of pAkt and CXCR4 are induced by factors that are both dependent and independent of HER-2, and the activation of HER 2/CXCR4/ Akt signaling pathway in primary breast tumors may contribute to the formation of bone metastases in breast cancer. PMID- 16685384 TI - Relation of DNA ploidy to genetic aberrations detected by chromosomal CGH and FISH in gastric adenocarcinomas. AB - We analyzed DNA copy number aberrations (DCNAs) by chromosomal comparative genomic hybridization (CGH) in 93 consecutive sporadic gastric adenocarcinomas. In addition, numerical aberrations in chromosomes 7, 11, 17, and 18 were evaluated by fluorescence in situ hybridization (FISH). Gastric cancers were divided on the basis of nuclear DNA content measured by laser scanning cytometry (LSC) into two groups, 36 DNA diploid (1.0 or= 1.2) cancers. The most frequent gain and loss of DNA copy number were found at 8q21-23 and 19p13.3, respectively, in both diploid and aneuploid cancers. Diploid cancers were further divided on the basis of genetic aberrations into major type and subtype cancers. The diploid cancer group included nine subtype cancers that showed large numbers of DCNAs; the mean number of DCNAs detected by CGH was 26.7 per tumor. This value was much larger in these diploid subtype cancers than diploid major type cancers (mean, 5.2 per tumor, p<0.0001). These nine cancers were also characterized by large intercellular variations in chromosome copy numbers that were not detected in the 27 major diploid type cancers. The aneuploid cancer group included only three subtype tumors that showed only a small number of DCNAs (mean, 3 per tumor) and minimal intercellular variations in chromosomal copy number. These data indicate that gastric adenocarcinomas can be divided into three types; aneuploid, major diploid type and diploid subtype cancers. Large-scale studies are necessary to clarify the differences in biological characteristics and underlying genetic mechanisms between these types. PMID- 16685385 TI - Expression of thymidine phosphorylase and VEGF in esophageal squamous cell carcinoma. AB - The purpose of this study was to determine the role of TP and VEGF in angiogenesis and its clinical significance in prognosis of patients with esophageal carcinoma. Expressions of TP and VEGF, microvascular density and cell proliferation activity were evaluated by using 40 immunohistochemically stained resected esophageal carcinoma tissues, and the survival rate of the patients was analyzed. Significant positive correlation and regression were found between the VEGF expression level of tumor and microvascular density (r=0.73, p<0.0001). Not statistically strong but significant positive correlation and regression were found between the TP expression level of tumor and microvascular density (r=0.32, p=0.046). No significant relationships were found between TP and VEGF expressions. Pathological T-factor and pathological N-factor were significant prognostic factors. Tumor length, site of lesion, gender, age, and Ki67 labeling index were not significant prognostic factors. The VEGF expression level was one of the unfavorable prognostic factors (risk ratio =1.035, 95% CI=1.007-1.065, p=0.01). The patients with high TP expression showed a tendency for unfavorable prognosis, but it was not statistically significant (RR=1.017, 95% CI=0.996 1.042, p=0.1). The prognosis of patients in the TP/VEGF[+/+] group was significantly poorer than that of the patients in the TP/VEGF[-/-] group and TP/VEGF[+/- or -/+] group (RR=0.488 for TP/VEGF[-/-] group, =0.717 for TP/VEGF[+/ or -/+] group, p=0.005). In conclusion, VEGF and TP expression seems to have a relationship with tumor angiogenesis, and co-expression of TP and VEGF seemed to be one of the unfavorable prognostic factors. PMID- 16685386 TI - A useful protocol for analyses of mutations of the epidermal growth factor receptor gene. AB - Recent studies have reported that mutations of the epidermal growth factor receptor (EGFR) gene are associated with the responsiveness of tyrosine kinase inhibitors (TKIs), which are molecular targets for non-small cell lung cancer (NSCLC). To provide genetic analyses for NSCLC patients, a simple and reliable method using paraffin-embedded materials is needed. The DEXPAT DNA extraction kit was used for DNA extraction from paraffin-embedded materials. DNA was amplified using the nested PCR technique, then analyzed by direct sequencing for EGFR mutations (exons 18 to 21). The phenol/chloroform extraction for DNA was also performed for comparison. When the DEXPAT kit was used, distinct bands were observed in all products after nested PCR assays of paraffin-embedded materials. Distinct sequencing signals were obtained. Results from the sequencing analysis of paraffin-embedded materials and frozen materials were completely concordant. The current study suggests that DNA extraction with the DEXPAT kit followed by nested PCR is a simple and reliable technique for analyzing the EGFR gene status with paraffin-embedded samples. PMID- 16685387 TI - GEP associates with wild-type p53 in hepatocellular carcinoma. AB - Granulin-epithelin precursor (GEP) is a novel growth factor whose up-regulation we previously reported in 72% of hepatocellular carcinoma (HCC). GEP expression has been reported to be associated with p53 protein accumulation in a breast cancer study, though the p53 mutation status was not revealed. We aim to investigate whether p53 protein and mutation status correlates with GEP expression in HCC. The statistical comparison of p53 and GEP data revealed an overall positive association between the two protein expression patterns (P<0.001). Upon detailed analysis, the association of p53 and GEP protein expression was found to be highly significant only in HCCs with wild-type p53 (P=0.001); there was no association in HCCs with p53 mutation (P=0.669). The GEP levels in the HepG2 hepatoma cell line with a wild-type p53 background were modulated by transfection experiments. Overexpression of the GEP protein resulted in an increased p53 protein level and suppression of the GEP protein resulted in a decreased p53 protein level in HepG2 cells. In summary, we demonstrated that p53 wild-type protein nuclei accumulation is associated with GEP protein expression in human HCC specimens, and GEP modulates p53 wild-type protein levels in vitro. PMID- 16685388 TI - Th1/Th2 cytokine imbalance in meningioma, anaplastic astrocytoma and glioblastoma multiforme patients. AB - The balance between Th1 and Th2 cytokines is thought to be an important factor in terms of tumour prognosis. Serum samples from 61 newly diagnosed patients with brain tumours and 50 age- and sex-matched non-tumour controls were analysed by ELISA for circulating levels of interleukin-12 (IL-12p70 and p40) and interleukin 10 (IL-10); pivotal Th1 and Th2 cytokines, respectively. Patients were divided into various groups depending on their histological diagnosis: meningioma (n=11), anaplastic astrocytoma (n=4) and glioblastoma multiforme (GBM; n=46). Significant reduction in serum IL-12 was seen in all groups as compared with the controls: meningioma, p=0.03; anaplastic astrocytoma, p<0.001; and GBM, p<0.001. Conversely, serum IL-10 was significantly increased in anaplastic astrocytoma, p=0.02, and GBM, p=0.03. The changes in the serum cytokines were not caused by the effects of steroids, as sequential analysis of patients pre- and post-steroid treatment commencement showed no difference. This study shows that patients with advanced primary intracranial malignancies have decreased circulating IL-12 and increased circulating IL-10, demonstrating that brain tumours have a major systemic effect on the immune system. PMID- 16685389 TI - Antitumor effect of combination of S-1 and docetaxel on the human breast cancer xenograft transplanted into SCID mice. AB - In vivo experiments were performed on breast cancer xenografts to examine whether the combination therapy with S-1, an oral dihydrouracil dehydrogenase (DPD) inhibitory fluoropyrimidine, plus docetaxel functions as an additive/synergistic modulator in tumor growth. The human breast cancer xenograft, MDA-MB-435SHM, was inoculated into SCID female mice. The tumor growth and thymidylate synthase (TS)/DPD activity of tumors treated with the agents were investigated. The T/C value (relative mean tumor weight of the treated group/relative tumor weight of the control group) of the group treated with docetaxel, S-1 and combination therapy were 45.3, 63.1 and 29.8%, respectively; suggesting the positive antitumor effects of the combination therapy in particular. In addition, significant down-regulation of DPD activity was also observed in the tumors treated with S-1, docetaxel and their combination. Down-regulation of the DPD activity of the tumors is also considered to be correlated with the antitumor effect of the treated groups, suggesting its influence on the synergistic effect of the combination therapy. PMID- 16685390 TI - G-CSF and/or M-CSF accelerate differentiation of bone marrow cells into endothelial progenitor cells in vitro. AB - It has been reported that granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can mobilize endothelial progenitor cells (EPCs) in bone marrow cells (BMCs) into peripheral blood (PB) in vivo. Previously, we also reported that macrophage-colony stimulating factor (M-CSF) can mobilize EPCs into PB, which results in the rapid recovery of blood flow in induced-ischemia limbs by augmenting the number of intramuscular capillaries in vivo. In the present study, we demonstrate that M CSF and/or G-CSF can increase EPCs from lineage (CD3, B220, Gr-1, Mac-1, CD11c, Ter119, NK1.1 or CD31)-negative BMCs in vitro. Lineage-negative BMCs were cultured with or without M-CSF and/or G-CSF. Three days after culture with M-CSF and/or G-CSF, the number of Flk-1+/CD45-, Sca-1+/CD45-, CD31+/CD45- or CD146+/CD45- cells increased in comparison with no cytokines. When the cultured BMCs with or without G-CSF and/or M-CSF were intravenously injected into ischemia induced hindlimbs of mice, the number of intramuscular capillaries in the ischemia-induced legs increased; BMCs cultured with G-CSF and/or M-CSF were more effective than those of cytokine non-treated BMCs. These results suggest that M CSF and/or G-CSF can induce the differentiation of BMCs into EPCs, even in vitro. PMID- 16685391 TI - Expression of the TRAG-3 gene in human esophageal cancer: the frequent synchronous expression of MAGE-3 gene. AB - We previously reported some cancer testis antigens, especially for MAGE genes, to be expressed in a relatively high population of gastro-intestinal and breast cancers. TRAG-3 (Taxol resistant associated gene-3) may be another cancer testis antigen; however, its expression has still not been fully studied. The TRAG-3 expression was evaluated in a total of 57 cancer cell lines and 322 cancer samples of gastrointestinal and breast cancers by RT-PCR. TRAG-3 was expressed in 23/57 (40%) of the cell lines: the highest expression was found in gastric cancer (6/9: 67%), followed by esophageal (13/28: 46%), colon (3/11: 27%) and liver (1/4: 25%) cancers. In clinical samples, the expression was the highest in esophageal cancer (32/58: 55%), followed by liver (13/50: 26%), bile duct (5/27: 19%), gastric (5/50: 10%), breast (5/50: 10%) and colon (2/87: 2.3%) cancers. The TRAG-3 expression significantly correlated with the expression of MAGE-3 in esophageal cancer (p<0.05). As the TRAG-3 gene is located on Xq28, which is the same locus as the MAGE gene family, we found a frequent synchronous expression pattern with TRAG-3 and MAGE-3 in esophageal cancer. PMID- 16685392 TI - In vitro sensitivity to platinum-derived drugs is associated with expression of thymidylate synthase and dihydropyrimidine dehydrogenase in human lung cancer. AB - Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are critical enzymes in nucleic acid metabolism. Proliferating cell nuclear antigen (PCNA) is a specific protein that is correlated with proliferative activity of cells. The TS gene has a variable number of tandem repeats (VNTR) in its 5'-untranslated region and a single nucleotide polymorphism (SNP) in the VNTR area. We examined the association of in vitro sensitivity to anticancer drugs with TS polymorphism, TS, DPD, and PCNA mRNA expression using human lung cancer tissues. Seventy-eight surgically resected lung cancer tissues were tested for in vitro sensitivity to 5 fluorouracil, cisplatin (CDDP), carboplatin (CBDCA), irinotecan, docetaxel, and gemcitabine by histoculture and MTT assay. The TS polymorphisms were analyzed by PCR and PCR-RFLP. TS, DPD, and PCNA mRNA expression levels were quantified by real-time RT-PCR and normalized relative to beta-actin mRNA expression. The inhibition rates (IRs) of CDDP and CBDCA were significantly correlated with TS/PCNA, the ratio of TS/actin and PCNA/actin, and DPD/PCNA, the ratio of DPD/actin and PCNA/actin. This correlation was further explored by subgroup analyses according to TS VNTR or TS functional type, in which 2R/3G, 3C/3G, or 3G/3G were classified into H-type group and 2R/2R, 2R/3C, or 3C/3C into L-type group. The associations of TS/PCNA and DPD/PCNA with the IRs of CDDP, CBDCA remained significant in the 3R/3R group and H-type group. These results suggest that in vitro sensitivity to platinum-derived drugs, CDDP and CBDCA, is associated with PCNA-normalized mRNA expression of TS and DPD in human lung cancer tissues, as affected by the TS polymorphism. The clinical significance of these pharmacogenomic markers for chemotherapy regimens with platinum-derived drugs should be investigated further for personalized treatment of lung cancer. PMID- 16685393 TI - Interferon-alpha resistance can be reversed by inhibition of IFN-alpha-induced COX-2 expression potentially via STAT1 activation in A549 cells. AB - The current study demonstrates that COX-2 expression is positively regulated by IFN-alpha, which is mediated by activation of STAT1 in A549 cells. The IFN-alpha induced COX-2 expression and STAT1 activation were markedly inhibited by the addition of curcumin to the IFN-alpha-pretreated cells. While IFN-alpha or COX-2 inhibitors alone did not result in growth inhibition of A549 cells, the combination of IFN-alpha and celecoxib or curcumin resulted in a significant growth inhibition of A549 cells, which was associated with down-regulation of CDK2, 4, and 6 and up-regulation of p27. We demonstrate that the expression of COX-2 was induced by IFN-alpha possibly via STAT1 activation in the A549 human non-small cell lung cancer cell line, which may partly account for its IFN-alpha resistance. The addition of curcumin or celecoxib to the IFN-alpha-pretreated A549 cells altered the IFN-alpha sensitivity of cell growth inhibition. PMID- 16685394 TI - Expression of ACP6 is an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma. AB - ACP6 (acid phosphatase 6, lysophosphatidic) is a lysophosphatidic acid (LPA) specific phosphatase that hydrolyzes LPA to monoacylglycerol and is involved in lipid metabolism in the mitochondria. Its role in oncogenesis and cancer progression has not been studied. In this study, we examined the expression of ACP6 mRNA and evaluated its clinical significance in esophageal squamous cell carcinoma (ESCC). Expression of ACP6 mRNA was quantified by real-time reverse transcription polymerase chain reaction using the LightCycler in 70 esophageal ESCC specimens and their paired normal esophageal mucosa. The data were analyzed with reference to clinicopathological factors. ACP6 mRNA expression in esophageal cancer tissue was significantly lower than that in corresponding normal esophageal mucosa (P=0.0301). Among the esophageal cancer tissues, ACP6 mRNA expression significantly correlated with local tumor invasion (T factor, P=0.0461) and lymph node metastasis (P=0.0128). Furthermore, low ACP6 mRNA expression was associated with a significantly shorter survival time compared with high expression (log-rank test, P=0.0358). In multivariate analysis, ACP6 mRNA expression emerged as a significant independent factor (P=0.0148). Impaired ACP6 expression may lead to more aggressive invasion of ESCC, and ACP6 mRNA expression level could be an independent prognostic factor for patients with ESCC. PMID- 16685395 TI - Curcumin inhibits hypoxia-induced angiogenesis via down-regulation of HIF-1. AB - Hypoxia-inducible factor-1 (HIF-1) has a central role in cellular responses to hypoxia, including the transcriptional activation of a number of genes involved in angiogenesis in tumors. We found that curcumin, a natural, biologically active compound isolated from the commonly used spice turmeric, significantly decreases hypoxia-induced HIF-1alpha protein levels in HepG2 hepatocellular carcinoma cells. Moreover, curcumin suppressed the transcriptional activity of HIF-1 under hypoxia, leading to a decrease in the expression of vascular endothelial growth factor (VEGF), a major HIF-1 target angiogenic factor. Curcumin also blocked hypoxia-stimulated angiogenesis in vitro and down-regulated HIF-1alpha and VEGF expression in vascular endothelial cells. These findings suggest that curcumin may play pivotal roles in tumor suppression via the inhibition of HIF-1alpha mediated angiogenesis. PMID- 16685396 TI - G3139 induces cell death by caspase-dependent and -independent apoptosis on human melanoma cell lines. AB - G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors. PMID- 16685397 TI - Increased expression of pituitary tumor-transforming gene (PTTG)-1 is correlated with poor prognosis in glioma patients. AB - Pituitary tumor-transforming gene (PTTG), which is homologous to a mammalian securin, plays a pivotal role in cell transformation and is overexpressed in numerous cancer cell lines and tissues. PTTG functions in the control of mitosis, cell transformation, DNA repair and gene regulation. In the present study, we investigated whether the expression of PTTG1 is correlated with tumorigenicity and prognosis in glioma patients. Expression of PTTG1 was confirmed in three glioma cell lines at the mRNA and protein levels using RT-PCR analysis and Western blotting, respectively. Furthermore, PTTG1 protein was detected in 44 glioma tissue samples using immunohistochemical techniques, markedly increased in high-grade gliomas compared to low-grade gliomas and associated with an unfavorable patient outcome. Moreover, siRNA against the PTTG1 gene inhibited cell proliferation and invasion in glioma cell lines. These data suggest that increased expression of PTTG1 contributes to the tumorigenicity of glioma cells and may be useful as a prognostic marker for glioma patients. PMID- 16685398 TI - Structure-activity studies with cytotoxic anthrapyrazoles. AB - Anthrapyrazoles have been investigated as cancer chemotherapeutic agents. The mechanism of action of these compounds is thought to involve inhibition of DNA topoisomerase II. A structure-activity study was carried out to determine the in vitro cytotoxic activity of nine novel anthrapyrazoles against human breast carcinoma, head and neck squamous cell carcinoma and leukemia cells, and against Chinese hamster ovary cells. The activity of these anthrapyrazole analogues was compared with that of two clinically tested anthrapyrazoles, losoxantrone and piroxantrone. Inhibition of topoisomerase II as a mechanism of action for the analogues was also investigated. The cytotoxic activity of the analogues was determined in vitro by MTT cell growth inhibition assay and inhibition of catalytic topoisomerase II activity by each compound was measured using a fluorometric DNA decatenation assay. All of the anthrapyrazole analogues inhibited the growth of the four cell lines with IC50 values that ranged from 0.1 to 45.2 microM. Losoxantrone was the most potent of the anthrapyrazole analogues studied. A tertiary amine in the basic side chain at N-2 increased the cytotoxic activity compared with a secondary amine in this side chain for many of the analogues, but not if there was a basic side chain at the C-5 position. A chlorine substituent on the basic side chain at N-2 did not have a consistent effect on activity. Moving the position of a chlorine substituent from C-5 to C-7 or introducing a basic side chain at C-5 did not have a consistent effect on cytotoxic activity. Anthrapyrazole analogues showed a broad range of activity for inhibiting topoisomerase II decatenation activity. Losoxantrone and piroxantrone were the most potent inhibitors of topoisomerase II activity. There was no significant correlation between the cytotoxic activity of the anthrapyrazole analogues and their ability to inhibit decatenation by topoisomerase II. PMID- 16685399 TI - Zanthoxyli Fructus induces growth arrest and apoptosis of LNCaP human prostate cancer cells in vitro and in vivo in association with blockade of the AKT and AR signal pathways. AB - Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types. PMID- 16685400 TI - p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions. AB - Esophageal squamous cell carcinoma (ESCC) is a common cancer with a very poor prognosis. New methods are needed to screen high-risk populations and identify curable tumors and precursor lesions early. Molecular markers may be useful in such screening efforts. This study was designed to determine the prevalence of p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in patients with ESCC to evaluate the variation of gene methylation across a spectrum of preneoplastic lesions, and assess the feasibility of using gene methylation in a primary screening test utilizing frozen esophageal cells collected by balloon cytology samplers. Samples were obtained from high-risk subjects from north central China. These samples included 11 foci of histologically normal mucosa, 8 foci of low grade squamous dysplasia, 7 foci of high-grade squamous dysplasia, and 13 foci of ESCC from 6 fully embedded resection specimens; endoscopic biopsies from 6 individuals with no histological evidence of disease; and frozen esophageal balloon samples from 12 asymptomatic subjects. Promoter CpG site-specific hypermethylation status was determined for each gene using real-time methylation specific PCR (qMS-PCR) based on Taqman chemistry. Of the 6 ESCC patients, 5 showed methylation of at least one gene. For most genes, methylation occurred with increasing frequency during neoplastic progression, with the largest increase found between low- and high-grade dysplasia. There was considerable variation in methylation patterns among different foci of the same histological grade, even within individual patients, but 16/20 (80%) of high-grade dysplastic and cancer foci had >or= 2 methylated genes, while 17/19 (89%) of normal and low grade dysplastic foci had <2 methylated genes. These genes were rarely methylated in histologically normal mucosa from patients with or without ESCC. Gene methylation was common and easily detectable in the frozen esophageal cells collected by balloon cytology samplers. Our data suggest that methylation of p16, MGMT, RARbeta2, CLDN3, CRBP, and MT1G is common in the esophageal mucosa of patients with ESCC in this high-risk population, and tends to increase in prevalence in foci with increasing histological severity of disease. Methylation data from panels of genes may be able to identify patients with high-grade lesions. Balloon cytology may be able to screen the length of the esophagus effectively for a subset of cells with abnormal methylation, and may be useful in a primary screening test for ESCC and its precursor lesions. PMID- 16685401 TI - Functional analyses of C13orf19/P38IP in prostate cell lines. AB - Human C13orf19 was previously identified to be downregulated in prostate cancer (PCa) but its function is unknown to date. In the present study, C13orf19 mRNA expression was inhibited by siRNA transfection. Furthermore, a possible regulation by androgens and the previously postulated interaction with p38 MAP kinase (p38MAPK) was investigated. The siRNA-mediated downregulation of the C13orf19 mRNA expression in the prostate cell lines PC-3 and BPH-1 was examined by quantitative PCR. Cellular viability, apoptosis, cell cycle distribution and clonogenic survival were investigated. In addition, the effects of C13orf19 downregulation in combination with chemotherapy on overall cell survival were studied. The inhibition of C13orf19 mRNA expression to 12% (after 12 h) and 55% (after 96 h) in PC-3 cells attested to a strong and persistent molecular effect provoked by the siRNA-D5 construct. However, no obvious effects on doubling time and cellular morphology were observed. Cell cycle distribution, clonogenic survival, apoptosis and cell viability showed no alterations, even after combining siRNA transfection with chemotherapy. Therefore, it can be concluded that the reduced expression of C13orf19 in PCa is not involved in the malignant transformation of the cells. A possible androgen dependence of C13orf19 mRNA expression was investigated by treating LNCaP cells with the androgen R1881 and in combination with the antiandrogen, bicalutamide. C13orf19 is expressed independently of the androgen. To analyze the putative interaction between C13orf19 and p38MAPK, PC-3 and BPH-1 cells were treated with the p38MAPK inhibitor, SB203580, and C13orf19 mRNA expression was examined. Additionally, the expression and phosphorylation status of p38MAPK after the inhibition of C13orf19 was investigated by Western blotting. No interaction between C13orf19 and p38MAPK was identified. Therefore, the gene should forthwith be named C13orf19 or Fam48A and not P38IP. PMID- 16685402 TI - PD98059-inhibited invasion of Dunning rat prostate cancer cells involves suppression of motility but not MMP-2 or uPA secretion. AB - Up-regulation of extracellular-regulated kinases 1/2 (ERK1/2) has been implicated in tumor progression and metastasis in many types of cancer. We have previously shown that ERK1/2 is necessary for invasiveness of Dunning rat prostatic adenocarcinoma cell lines in which levels of activated ERK1/2 correlate with the metastatic potential. Here, we further examined the biological effects of elevated ERK1/2 in the highly metastatic Dunning cell line, MLL, in which the abilities to invade and metastasize are enhanced relative to its progenitor strain. Inhibition of ERK1/2 activation by the MEK1 inhibitor, PD98059, dose dependently reduced MLL cell invasiveness and motility with similar IC50 values. On the other hand, the abilities of MLL cells to adhere to the extracellular matrix, phosphorylate myosin regulatory light chain and secrete matrix-degrading enzymes, matrix metalloproteinase (MMP)-2 and urokinase plasminogen activator (uPA) were marginally, if at all, affected by PD98059 treatment. These data indicated that the inhibitory effect of PD98059 on the invasiveness of MLL cells was primarily due to the suppression of cell motility, and the up-regulation of ERK1/2 is, at least in part, responsible for the enhanced cellular motility and invasiveness of the MLL cells. PMID- 16685403 TI - Current bioinformatics tools in genomic biomedical research (Review). AB - On the advent of a completely assembled human genome, modern biology and molecular medicine stepped into an era of increasingly rich sequence database information and high-throughput genomic analysis. However, as sequence entries in the major genomic databases currently rise exponentially, the gap between available, deposited sequence data and analysis by means of conventional molecular biology is rapidly widening, making new approaches of high-throughput genomic analysis necessary. At present, the only effective way to keep abreast of the dramatic increase in sequence and related information is to apply biocomputational approaches. Thus, over recent years, the field of bioinformatics has rapidly developed into an essential aid for genomic data analysis and powerful bioinformatics tools have been developed, many of them publicly available through the World Wide Web. In this review, we summarize and describe the basic bioinformatics tools for genomic research such as: genomic databases, genome browsers, tools for sequence alignment, single nucleotide polymorphism (SNP) databases, tools for ab initio gene prediction, expression databases, and algorithms for promoter prediction. PMID- 16685404 TI - Adiponectin receptor expression in the human adrenal cortex and aldosterone producing adenomas. AB - Adiponectin is an adipocyte-derived circulating peptide that plays an important role in adipose tissue metabolism, insulin sensitivity and cardiovascular disease. The adrenal gland, by secreting glucorticoid and mineralocorticoid hormones, intervenes in cardiovascular and glucose metabolism regulation and is surrounded by adipose tissue. Hence, we investigated the hypothesis that adiponectin receptor types 1 and 2 (adipo-R1 and adipo-R2) are expressed in the human adrenal gland and in adrenocortical zona glomerulosa cell-derived aldosterone-producing adenoma (APA) tissue. We used real-time reverse transcription-polymerase chain reaction to demonstrate the mRNA of adipo-R1 and adipo-R2 in 10 histologically normal human adrenal cortexes that were obtained from patients with renal cancer undergoing nephrectomy with ipsilateral adrenalectomy and in 10 APAs. Melting curve analysis and sequencing were used to confirm the specificity of the amplicons obtained. Results consistently showed the expression of specific mRNAs of adiponectin receptors in all histologically normal human adrenal cortexes and APAs. This novel finding suggests that adiponectin could play a regulatory role in adrenocortical function and growth in humans. PMID- 16685405 TI - An image analysis of the spatial distribution of perivascular mast cells in human melanoma. AB - A mutual spatial and functional relationship occurs between mast cells (MCs) and endothelial cells and the density of MCs is highly correlated with the extent of tumor angiogenesis. The aim of this study was to investigate the pattern of MCs around the blood vessels in melanoma samples by means of an approach derived from spatial statistics, based on the analysis of the distribution of the distances of MCs from vessels to objectively establish if the two structures (MCs and vessels) are distributed independently over the studied area or if they displayed any kind of spatial association. Results showed that a higher number of vessels and MCs can be observed in melanoma as compared with samples from common acquired nevi (control group). The percent of area covered by vessel profiles was significantly higher in the melanoma group than the control group and the MC density was also significantly different; the melanoma group showing a number of MCs per unit area twice as high as the number measured in the control group. Furthermore, in the melanoma group, MCs were closer to each other and to the vessels. In fact, both the mean distance from vessels and the mean distance from the nearest cell profile were significantly lower than in the control group. This close association between MCs and the endothelium does not necessarily imply a participation of MCs in angiogenic processes, but might rather indicate that MCs are involved in the maintenance reaction necessary for the long lasting functional integrity of the endothelium. PMID- 16685406 TI - Expression of thioredoxin and thioredoxin-binding protein-2 in the liver of patients with chronic hepatitis C as a predictor of response to interferon therapy. AB - Oxidative stress contributes to the pathogenesis of various hepatic injuries. Thioredoxin (TRX) is an indicator of oxidative stress, reported to be increased in the serum of patients with chronic hepatitis C with the progression of fibrosis. The aim of this study was to evaluate the clinical significance of the expression of TRX and thioredoxin-binding protein-2 (TBP-2), which is a negative regulator of TRX function, in the liver of patients with chronic hepatitis C and the relationship of this to the efficacy of interferon (IFN) treatment. A retrospective study was performed using the liver biopsy specimens obtained before IFN treatment from 69 patients with chronic serotype 1 hepatitis C virus (HCV) infection. TRX and TBP-2 mRNA levels in the liver biopsy specimens were amplified by real-time RT-PCR. The serum TRX protein level was estimated with a sandwich enzyme-linked immunosorbent assay kit, and the expression of TRX protein in the liver was examined immunohistochemically in 19 patients. There was no association between the serum TRX level and the TRX level in the liver. There was a significant correlation between the expression level of TRX protein in the liver and the TRX mRNA level in the liver. TRX and TBP-2 levels in the liver tended to decrease slightly with increased fibrosis stage, although not significantly. The TRX level in the liver tended to increase with hepatitis activity index, although not significantly. TBP-2 mRNA levels in the liver were significantly higher in responders than non-responders to the IFN therapy (p<0.05). Among patients who had a high viral load of >850 KIU/ml, the TRX level in the livers of non-responders was significantly lower than that in the livers of responders (p<0.05). TRX and TBP-2 mRNA levels in the liver before IFN therapy may predict the outcome of IFN therapy in patients with chronic serotype 1 HCV infection. PMID- 16685408 TI - Polysiphonia japonica extract suppresses the Wnt/beta-catenin pathway in colon cancer cells by activation of NF-kappaB. AB - Abnormal activation of the Wnt/beta-catenin pathway and subsequent up-regulation of beta-catenin response transcription (CRT) are associated with the development of colon cancer. Thus, the Wnt/beta-catenin pathway is an attractive target for chemoprevention and treatment of this cancer. We used a cell-based screen to identify a methanol extract of Polysiphonia japonica (EPJ) that suppresses the Wnt/beta-catenin pathway without altering the level of beta-catenin protein and reduces the expression of cyclin D1, which is a known beta-catenin/T cell factor (TCF)-dependent gene. EPJ inhibited the growth of various colon cancer cells. In addition, EPJ induced the nuclear translocation of nuclear factor-kappaB (NF kappaB) in SW480 colon cancer cells. Our findings suggest that EPJ attenuates Wnt/beta-catenin signaling via activation of NF-kappaB and can potentially be used as a chemopreventive agent against colon cancer. PMID- 16685407 TI - Rosuvastatin, a new HMG-CoA reductase inhibitor, reduces the colonic inflammatory response in dextran sulfate sodium-induced colitis in mice. AB - The aim of the present study was to elucidate the beneficial effects of rosuvastatin, a new HMG-CoA reductase inhibitor, on colonic mucosal damage and on the inflammatory response in a dextran sulfate sodium (DSS) colitis model. Acute colitis was induced using 8% DSS in female BALB/c mice. Colonic mucosal inflammation was evaluated clinically, biochemically, and histologically. Mucosal protein contents and mRNA levels of tumor necrosis factor (TNF)-alpha were determined by immunoassay and real time-PCR. The mRNA levels of endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. Disease activity scores in DSS-induced colitis model mice, as determined by weight loss, stool consistency, and blood in stool, were significantly lower in the rosuvastatin treated mice than in control mice. Shortening of the colon was significantly reversed by rosuvastatin. Increases in tissue-associated myeloperoxidase activity and thiobarbituric acid-reactive substances after DSS administration were both significantly inhibited by treatment with rosuvastatin. Rosuvastatin also inhibited increases in intestinal TNF-alpha protein and mRNA expression after DSS administration, respectively. The mucosal mRNA levels of eNOS were decreased after DSS administration, but preserved in mice treated with rosuvastatin. These results suggest that rosuvastatin prevents the development of DSS-induced colitis in mice via the inhibition of mucosal inflammatory responses associated with the preservation of eNOS transcription. PMID- 16685409 TI - Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP. AB - Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation. PMID- 16685410 TI - Growth promoting in vitro effect of synthetic cyclic RGD-peptides on human osteoblast-like cells attached to cancellous bone. AB - In tissue engineering, the application of biofunctional compounds on biomaterials such as integrin binding RGD-peptides has gained growing interest. Anchorage dependent cells like osteoblasts bind to these peptides thus ameliorating the integration of a synthetic implant. In case sterilized bone grafts are used as substitutes for reconstruction of bone defects, the ingrowth of the implanted bone is often disturbed because of severe pretreatment such as irradiation or autoclaving, impairing the biological and mechanical properties of the bone. We report for the first time on the in vitro coating of the surface of freshly resected, cleaned bone discs with synthetic, cyclic RGD-peptides. For this approach, two different RGD-peptides were used, one containing two phosphonate anchors, the other peptide four of these binding moieties to allow efficient association of these reactive RGD-peptides to the inorganic bone matrix. Human osteoblast-like cells were cultured on RGD-coated bone discs and the adherence and growth of the cells were analyzed. Coating of bone discs with RGD-peptides did not improve the adhesion rate of osteoblast-like cells to the discs but significantly (up to 40%) accelerated growth of these cells within 8 days after attachment. This effect points to pretreatment of bone implants, especially at the critical interface area between the implanted bone and the non-resected residual bone structure, before re-implantation in order to stimulate and enhance osteointegration of a bone implant. PMID- 16685411 TI - A novel MLH1 mutation harbored as a germ line aberration by a young woman of an HNPCC-like family and exhibited by a CML patient when occurring prior to the initiation of the blast phase concomitant with a c-MYC amplification. AB - Germ line mutations in the MLH1 and MSH2 genes account for the majority of hereditary nonpolyposis colorectal cancer (HNPCC) families. Here, we describe a family that does not meet the international criteria for HNPCC, of which a young woman harbors a missense mutation (D132H). This novel germ line mutation has not previously been reported. Of the mismatch repair (MMR) genes, MLH1 has been shown to play an important role in hematologic malignancies. The novel mutation was also revealed to be a somatic aberration occurring prior to the initiation of the blast phase in a chronic myelogenous leukemia (CML) patient. Among the possible MLH1 partners involved in signaling MMR or apoptosis is the proto-oncogene c-MYC, which is closely related to cellular proliferation. We further revealed a concomitant c-MYC dramatic amplification in the CML-MLH1-mutation carrier patient, also occurring at the pre-blast phase. Our data contribute further to characterizing the mutational spectrum of the MLH1 gene. Furthermore, given the role of c-MYC and its interaction with MLH1, taken together with the mutational status of both genes revealed at the pre-blast phase in the CML patient, a plausible increased genetic instability might be expected to take place, possibly contributing to blast triggering. Our results may provide additional insight into the complex interplay between the MMR system and other cellular pathways. PMID- 16685412 TI - The differential expression of SPARC in esophageal squamous cell carcinoma. AB - Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that modulates cell adhesion and growth. The aim of this study was to assess the prevalence of SPARC mRNA and protein expression in esophageal squamous cell carcinoma (ESCC) and to investigate the role of SPARC in proliferation and metastasis of ESCC. The differential expression of SPARC between esophageal squamous cell carcinoma and its corresponding normal esophageal mucosa was analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT PCR), immunohistochemistry, as well as Western blotting. The expression of SPARC in ESCC was significantly higher than that in the corresponding normal esophageal mucosa as shown by semi-quantitative RT-PCR, immunohistochemistry and Western blotting. Meanwhile, SPARC protein was also significantly up-regulated in ESCC compared with the corresponding normal esophageal mucosa. SPARC protein was located in the cytoplasm and nuclei of ESCC cells. The results of Western blotting showed that out of 18 paired samples, SPARC was up-regulated in 13 cases, no significant differences were found for the other 5 cases. These findings revealed that SPARC was up-regulated in ESCC and was closely associated with ESCC metastasis. This study provides new insights into nuclei location of SPARC. PMID- 16685413 TI - Altered calcium handling is an early sign of streptozotocin-induced diabetic cardiomyopathy. AB - The main objective of the present study was to determine alterations of calcium handling in the diabetic rat heart during the transition from adaptive to maladaptive phase of cardiomyopathy. By inhibiting the nuclear enzyme poly(ADP ribose) polymerase (PARP), we also investigated the possible role of this enzyme in the sequence of pathological events. Six weeks after induction of type I diabetes by injection of streptozotocin in rats, the hearts were perfused according to Langendorff. Intracellular-free calcium (Ca(2+)(i)) levels were measured by surface fluorometry using Indo-1 AM. Cyclic changes in Ca(2+)(i) concentrations and hemodynamic parameters were measured simultaneously. The hearts were challenged by infusion of isoproterenol. Six weeks of diabetes resulted in reduced inotropy and lusitropy. The diabetic hearts (DM) expressed a significantly elevated end-diastolic Ca(2+)(i) level (control, 111-/+20 vs DM, 221-/+35 nM). The maximal transport capacity of SERCA2a and conductance of RyR2 were reduced. These changes were not accompanied by major alterations in the tissue content of SERCA2a, RyR2, phospholamban and Na(+)/Ca(2+) exchanger. In response to beta-adrenergic activation, SERCA2a transport capacity and RyR2 conductance were stunted in the DM hearts. Inhibition of PARP induced minor changes in the mechanical function and calcium handling of the DM hearts. In conclusion, the observed changes in contractility and in Ca(2+)(i) handling are most likely attributable to functional disturbances of SERCA2a and RyR2 in this transitional phase of diabetes. At this stage of diabetes, PARP does not appear to play a significant pathogenetic role in the alterations in contractile function and calcium handling. PMID- 16685414 TI - Evaluating strategies to enhance the anti-tumor immune response to a carbohydrate mimetic peptide vaccine. AB - Carbohydrate mimetic peptides of tumor associated carbohydrate antigens (TACA) are T-cell-dependent antigens and, therefore, immunization with these surrogates is predicted to overcome the low immunogenicity of carbohydrate antigens. Consistent with this hypothesis, we show that among the potential immune cells involved, peptide immunization led to an increase in T-cell populations. While peptide mimetics may also function as TLR binding ligands, we did not observe evidence of involvement of NK cells. Examining tumor challenged animals, we observed that peptide immunization and not tumor cells rendered IL-12 responsiveness to T-cells, as T-cells from peptide-immunized mice produced IFN gamma upon stimulation with IL-12. Cyclophosphamide administration enhanced the anti-tumor efficacy of the vaccine, which was achieved by enhancing T-cell responses with no effect on NK cell population. Prophylactic immunization of mice with a DNA construct encoding carbohydrate mimetic peptides indicated a specific role for the mimotope vaccine in anti-tumor immune responses. These data suggest a role for both CD4(+) and CD8(+) T-cells induced by mimotopes of TACA in protective immunity against tumor cells. PMID- 16685415 TI - 57Arg in the bHLH transcription factor DEC2 is essential for the suppression of CLOCK/BMAL2-mediated transactivation. AB - The basic helix-loop-helix (bHLH) transcription factors, DEC2 and DEC1, play critical roles in the circadian rhythm of the suprachiasmatic nucleus (SCN). It is known that mammalian circadian rhythms are regulated by molecular clockwork systems based on a negative-feedback loop, and CLOCK/BMAL1 and CLOCK/BMAL2 enhance DEC2 transcription via CACGTG E-boxes. To understand the role of arginine 57 ((57)Arg) within the basic region of DEC2, we examined the effect of substituting this residue into DEC2 on CLOCK/BMAL2-mediated transactivation. A luciferase assay showed that substituting (57)Arg for Ala or Lys in DEC2 diminished the suppressive activity of wild-type DEC2 on CLOCK/ BMAL2-mediated transactivation, while substituting (48)Pro for Ala in DEC2 did not alter it, and the same was true for wild-type DEC2. We also showed that proteins which were wild-type and substitution mutants of DEC2 were expressed at nearly equivalent levels by Western blotting. These findings demonstrate that (57)Arg in the basic region of DEC2 is essential for its activity in suppressing CLOCK/BMAL2-mediated transactivation. PMID- 16685416 TI - hCAR-EGFP fusion receptor in human follicular lymphoma B cells - a model for adenoviral gene therapy for B cell malignancies. AB - Adenovirus-mediated gene therapy for hematopoietic malignancies, especially those derived from B cells, is difficult due to systemic nature of these diseases. More importantly, most tumor cells derived from B cell lineage express a very low level of the adenovirus receptor hCAR; thus, warranting the design of adenoviral vectors with high affinity to abundant B cell surface molecules. To mimic this approach and to test the validity of adenoviral vectors in gene therapy of disseminated malignancies, we created an hCAR-expressing follicular lymphoma B cell line. The cell line was generated with the aid of a lentivirus vector carrying a novel fusion gene with EGFP replacing the cytoplasmic domain of hCAR. After verifying that this cell line was expressing the hybrid receptor in a correct manner and enrichment of the bright EGFP positive population, the cells were transduced with adenoviruses expressing the red fluorescent protein DsRed2. It was shown that regular transduction with a low viral dose (1 pfu/cell) increased the gene transfer rate by a factor of 5. Furthermore, experiments with adenovirus vector carrying the HSV-TK-GFP transgene demonstrated that the modified follicular lymphoma B cells became sensitive to ganciclovir while the parental cells remained virtually resistant to this form of gene therapy. In summary, we show here with this simple model system that adenoviral gene therapy of B cell malignancies is possible provided that correct receptors for adenovirus attachment are present on the surface of the target cells. Thus, our results warrant further modifications of adenovirus capsid to obtain vectors with specific affinity to B cell epitopes. PMID- 16685417 TI - Hepatocyte 'priming' and increase in transforming growth factor-beta1 mRNA expression are delayed in hypothyroid versus euthyroid rats during liver regeneration. AB - Hypothyroidism decreases liver weight and delays the compensatory liver growth after partial hepatectomy (PH) as compared with the euthyroid condition. The aim of this study was to investigate, in hypothyroid rats, the mRNA expression of genes modulating these effects, focusing on c-fos and c-myc, hallmarks of hepatocyte 'priming', and on transforming growth factor-beta1 (TGF-beta1) and its receptor, the transforming growth factor-beta1 receptor-type II (TbetaR-II), negative regulators of liver growth. Euthyroid and hypothyroid male Wistar rats underwent 70% PH and total RNA was isolated from frozen liver samples removed at basal state and during regeneration, 0-144 h after surgery. In this study, we show for the first time that, in the basal liver state, hypothyroidism increased TGF-beta1 and TbetaR-II mRNA levels by 45% and 30%, respectively, as compared with the euthyroid condition and, after PH, resulted in a approximately 12-h delay in the activation of c-fos and c-myc mRNA expression. Moreover, the increase in TGF-beta1 mRNA levels, detected 24-48 h after PH in euthyroid rats, was delayed by 72 h in hypothyroid rats, occurring when a concomitant reduction in TbetaR-II was measured. These results suggest that, in hypothyroid rats, at the basal liver level, the increase in mRNA expression of genes that negatively regulate liver growth might be involved in the decrease in liver weight and that, after PH, the delay of hepatocyte 'priming' and coordinated changes in mRNA expression of negative regulators of liver regeneration might be involved in delaying the regenerative process. PMID- 16685418 TI - Resveratrol inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in beta-amyloid-treated C6 glioma cells. AB - Resveratrol has been reported to exert a variety of important pharmacological effects including anti-inflammatory, cardioprotective and cancer chemopreventive properties; however, its mechanisms of action are not completely under-stood. beta-amyloid protein is considered to be responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer disease. In the present study, we investigated the protective effect of resveratrol on beta amyloid-induced cytoxicity in cultured rat astroglioma C6 cells. Preincubation of C6 cells with resveratrol concentration-dependently protected the cells from the growth inhibition induced by beta-amyloid treatment. beta-amyloid treatment led to increased nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) expression; however, cells pretreated with resveratrol showed a dose dependent inhibition of NO production and iNOS expression following beta-amyloid treatment. Resveratrol also attenuated beta-amyloid-induced prostaglandin E2 (PGE2) release, which was associated with the inhibition of cyclooxygenase (COX) 2 expression. Furthermore, beta-amyloid treatment induced nuclear translocation of NF-kappaB, which was suppressed by resveratrol pretreatment. Collectively, the present results indicate that modulation of nuclear factor-kappaB (NF-kappaB) activity is involved in the neuroprotective action of resveratrol against beta amyloid-induced toxicity. PMID- 16685419 TI - Creatine supplementation increases soleus muscle creatine content and lowers the insulinogenic index in an animal model of inherited type 2 diabetes. AB - Creatine supplementation may exert beneficial effects on muscle performance and facilitate peripheral glucose disposal in both rats and human subjects. The present study was undertaken to explore the effects of creatine supplementation on the ATP, creatine, phosphocreatine and glycogen content of white and red gastrocnemius and soleus muscles and on blood D-glucose and plasma insulin concentrations before and during an intravenous glucose tolerance test in Goto Kakizaki rats, a current animal model of inherited type 2 diabetes mellitus. Creatine supplementation increased muscle creatine content, especially in the soleus muscle of young rats (+35.5-/+15.8%; d.f.=10; p<0.05), and lowered the insulinogenic index, i.e. the paired ratio between plasma insulin and blood D glucose concentrations. The latter change was mainly attributable to a lowering of plasma insulin concentration. It is proposed, therefore, that creatine supplementation may improve the sensitivity to insulin in extrapancreatic sites in the present animal model of type 2 diabetes. PMID- 16685420 TI - Molecular basis for the involvement of thymidine phosphorylase in cancer invasion. AB - Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), has been implicated in bladder cancer angiogenesis and invasion. However, the molecular basis of its role in invasion remains unclear. We investigated the expression of TP and 10 invasion-related genes in bladder cancers from 72 randomly selected patients by real-time two-step RT-PCR assay. We found that the expression levels of TP, MMP-9, uPA, and MMP-2 were significantly higher in invasive tumors than those in superficial tumors. Also, the expression level of TP significantly correlated with that of uPA, MMP-1, MMP-9, PAI-1 and VEGF. KK47/TP cells, bladder cancer cells that overexpress TP, had a higher expression of MMP-7 and MMP-9 than KK/CV cells that express lower level of TP in hypoxic condition. PC/TP cells, prostate cancer cells that overexpress TP, also had a higher expression of MMP-1 and MMP-7 than PC/CV cells that express no detectable TP. Taken together these data indicate that TP enhances the invasion of tumor cells through the induction of invasion-related genes. PMID- 16685421 TI - Human membrane protein Tim-3 facilitates hepatitis A virus entry into target cells. AB - In this study, a cellular surface membrane protein of immunoglobulin (Ig) superfamily (IgSF) was identified from a human dendritic cell (DC) cDNA library by large-scale random sequencing, which is identical to previously reported Tim-3 (T-cell Ig- and mucin-domain-containing molecule 3). Recent data have suggested the association of the 281-residue mouse Tim-3 molecule with Th1-related T cell responses and disease in mice. Human Tim-3 is a 301-residue type I membrane protein whose extracellular region contains a Cys-rich Ig-like domain and a mucin domain, the characteristics of Tim proteins. It shows significant homology to human hepatitis A virus (HAV) cellular receptor-1 (HuHAVcr-1)/Tim-1. Human Tim-3 mRNA was highly expressed in monocytes or monocyte-derived cells, and the expression level decreased when DC underwent maturation and activation. There is no previous report on the biological functions of human Tim-3, especially the involvement in virus infection. We demonstrated that HeLa cells, which are refractory to HAV infection, acquired a limited susceptibility to HAV infection after stably overexpressing human Tim-3 as confirmed by Western blot analysis using anti-Tim-3 antibody, but Tim-3-Fc fusion protein had no direct HAV-binding activity. The results indicated that human Tim-3 can promote HAV entry into target cells but itself may not function as a cellular receptor of HAV. PMID- 16685422 TI - In vitro culture on Matrigel favors the long-term maintenance of rat zona glomerulosa-cell differentiated phenotype. AB - Zona glomerulosa (ZG) cells cultured on plastic within few days dedifferentiate losing their capacity to secrete aldosterone (ALDO) in appreciable amounts. Evidence indicates that extracellular matrix modulates the secretory behavior of adrenocortical cells cultured in vitro. Hence, we compared the morphology and function of rat ZG cells grown on plastic and Matrigel basement membrane matrix (hereinafter Matrigel) for up to 12 days. At day 3, no significant differences were observed between cells cultured on plastic and Matrigel. Starting from day 6, ZG cells cultured on plastic lost their ultrastructural differentiated features (mitochondria with tubular cristae, smooth endoplasmic reticulum cisternae and lipid droplets), exhibiting a fibroblast-like appearance. The mRNA expression of the main steroidogenic enzymes, as evaluated by real-time polymerase chain reaction, the baseline secretion of ALDO and other post pregnenolone hormones, as evaluated by high pressure liquid chromatography, and the secretory response to ACTH, angiotensin-II and K(+), as evaluated by radioimmunoassay, displayed a time-dependent decrease. Matrigel was found to maintain unchanged both the ultrastructure and the expresion of steroidogenic enzymes of ZG cells until day 12 of culture. Baseline and agonist-stimulated steroid-hormone secretion decreased with the duration of culture on Matrigel, but was always higher than that of ZG cells grown on plastic. Hence, our study clearly indicates that the culture on Matrigel favors the maintenance of rat ZG cell differentiated phenotype, allowing the conclusion that this technique is suitable for long-term in vitro investigations. PMID- 16685423 TI - Urotensin-II and UII-receptor expression and function in the rat adrenal cortex. AB - Urotensin-II (UII) is a potent hypertensive peptide, which has been recognized as an endogenous ligand of the G protein-coupled receptor (GPR)-14, now named UT-R. Real-time PCR demonstrated the expression of UII and UT-R mRNAs in both dispersed and in vitro cultured rat adrenocortical cells. UII concentration-dependently decreased basal, but not ACTH-stimulated, corticosterone secretion from cultured adrenocortical cells, and the effect was abolished by the UT-R antagonist Palosuran. UII did not affect the proliferation rate of cultured cells. Taken together, these findings suggest that UII may be included in the group of peptides (adrenomedullin, atrial natriuretic peptide, neurotensin and beacon), that, acting in an autocrine-paracrine manner, are involved in the inhibitory tuning of adrenocortical secretion. PMID- 16685424 TI - Characterization of a novel human protein phosphatase 2C family member, PP2Ckappa. AB - A novel member of human protein phosphatase 2C gene named PP2Ckappa was isolated from a human fetal brain cDNA library. The 2.0 kb cDNA encodes a 372 amino acid polypeptide with an intact protein phosphatase 2C (PP2C) catalytic domain. Reverse transcription-PCR (RT-PCR) revealed that the PP2Ckappa was widely expressed in normal human tissues. Transient transfection suggested that PP2Ckappa was localized in the nucleus in AD293 cells. Recombinant Trx-His PP2Ckappa showed phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing phospho-threonine residues. Furthermore, the overexpression of PP2Ckappa distinctly activated the heat shock transcription factor pathway in eukaryotic cells. PMID- 16685425 TI - The expression profile of PKC isoforms during MC3T3-E1 differentiation. AB - Protein kinase C (PKC) is a family of kinases whose isoforms show subtle differences in physiological and biochemical responses, with their expression being cell- specific. We hypothesize that there may be a specific profile of expression of PKC isoforms in differentiating osteoblastic cells (OBC) with individual isoforms having specific functions. Herein, the MC3T3-E1 cell line was used as a differentiating model, which was induced from the pre-osteoblast stage to mature osteoblast and characterized with several phenotypic markers, including alkaline phosphatase activity, osteocalcin and bone sialoprotein. The expression of PKC isoforms was monitored using Western blot analysis. Upon induction of osteogenesis, the intracellular localization of PKC eta and theta was determined using immunofluorescence. Lastly, the effect of P38 MAP kinase inhibition was determined using SB203580. Results show 1) PKC alpha, delta, lambda were all highly expressed in MC3T3-E1 osteoblastic cells, 2) the expression of PKC theta was significantly down-regulated upon induction of osteoblastic differentiation; 3) PKC eta was non-detectable at certain cell culture days; however, was up regulated as the cells transit from each differentiation phase. The increased expression of PKC eta correlated with increases in OC, BSP levels and alkaline phosphatase activity. Immunofluorescence procedure confirmed the Western blot results with an increase in PKC eta and a decrease in PKC theta upon osteogenic stimulation. The inhibition of p38 resulted in a marked down-regulation of PKC eta. The data demonstrate that there is a specific profile of expression of PKC isoforms in differentiating osteoblasts; the different expression pattern of individual isoforms may be either a consequence of the differentiation itself or plays a role in the regulatory mechanism of osteoblastic differentiation. This study has provided primary information on the temporal pattern of expression of PKC isoforms in the differentiating osteoblast and further insight into their possible role in osteoblastic cell maturation. PMID- 16685426 TI - Effect of GLP-1 on D-glucose transport, lipolysis and lipogenesis in adipocytes of obese subjects. AB - GLP-1 has anorectic properties and regulates fuel homeostasis through both its insulinotropic and insulinotrophic actions and effects in extrapancreatic tissue. This study is aimed at characterizing the response to GLP-1 of adipocytes from obese patients, in terms of D-glucose transport and lipid metabolism, in comparison with data from normal subjects. Adipocytes were obtained by enzymatic digestion from the abdominal fat tissue of 25 morbidly obese patients and 8 normal subjects undergoing bariatric or inguinal hernia surgery, respectively. Basal GLUT4 expression, D-glucose transport, glycerol release and lipogenesis were measured in cells treated, when required, with 10(-12)-10(-9) M GLP-1, insulin, glucagon and the GLP-1 structurally related peptides, exendin-4 and exendin-9. In obese patients, versus normal subjects, a trend towards lower values was found in GLUT4 protein or mRNA, although the differences were not statistically significant; insulin-stimulated glucose uptake was higher and cells did not respond to GLP-1, while both exendins (10(-10) and 10(-9) M) exerted an inhibitory action; basal lipolysis was higher and so was the effect of GLP-1 and glucagon, whereas insulin abolished the lipolytic action of all peptides; both basal lipogenesis and the response to insulin were higher while GLP-1 and exendin 4 were ineffective. These results document the analogies and dissimilarities between the response to GLP-1, exendin-4 and exendin-9, as well as to insulin and glucagon, relative to glucose transport and lipid metabolism of fat tissue from obese patients versus normal subjects, the reduced lipogenic effect and enhanced lipolytic action of GLP-1 being, perhaps, adequate for its therapeutic use in obesity. PMID- 16685427 TI - Desflurane differentially affects the release of proinflammatory cytokines in plasma and bronchoalveolar fluid of endotoxemic rats. AB - Previous studies have indicated that volatile anaesthetics can attenuate the inflammatory response to lipopolysaccharide (LPS) and other proinflammatory stimuli in vitro and in vivo. Thus far, no studies are available on the influences of desflurane on the cytokine-release. We therefore aimed to investigate the effects of desflurane on the systemic and pulmonary release of proinflammatory cytokines in endotoxemic rats. Eighteen anaesthetized and ventilated Sprague-Dawley rats were randomly assigned to the following groups: LPS-only: Six animals received LPS (5 mg/kg, i.v.) with no further intervention. LPS-Desflurane: Six animals received continuous inhalation of 1MAC Desflurane before and during endotoxemia with LPS (5 mg/kg, i.v.). Sham: Six animals served as control without inhalation of desflurane and endotoxemia. After 4 h, levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in plasma and bronchoalveolar fluid were analyzed. Nitrite production as a readout for nitric oxide (NO) release from alveolar macrophages was measured by Griess assay. IkappaB-alpha degradation and iNOS-protein in macrophage homogenates were determined by Western Blotting. Inhalation of desflurane during endotoxemia showed a significant decrease in release of the proinflammatory cytokines TNF-alpha (-61%, P< or =0.05) and IL-1beta (-47%, P< or =0.05) in plasma as compared to LPS-only group, whereas the release of IL-6 was not significantly affected by desflurane. Within the lung, the NO-release was notably increased in supernatants of cultured alveolar macrophages from desflurane-group compared to both LPS-only and Sham group. IkappaB-alpha degradation in alveolar macrophages was impaired in the Desflurane-group as compared to the LPS-only group. Our data implicate that inhalation of 1MAC Desflurane during experimental endotoxemia differentially affects the inflammatory response in rats. PMID- 16685428 TI - Comparative integromics on Angiopoietin family members. AB - Angiopoietin-1 (ANGPT1), Angiopoietin-4 (ANGPT4), VEGF, FGF2, FGF4, HGF, Ephrin, IL8 and CXCL12 (SFD1) are pro-angiogenic factors (angiogenic activators), while Angiopoietin-2 (ANGPT2), Angiostatin, Endostatin, Tumstatin, Canstatin, THBS1, THBS2, TNFSF15 (VEGI) and Vasohibin (VASH1) are anti-angiogenic factors (angiogenic inhibitors). ANGPT1 and ANGPT2 are ligands for TIE family receptor tyrosine kinases, TIE1 and TIE2 (TEK). Angiopoietin family consists of ANGPT1, ANGPT2, ANGPT4, ANGPTL1 (ANGPT3), ANGPTL2, ANGPTL3 (ANGPT5), ANGPTL4, ANGPTL5, ANGPTL6 and ANGPTL7. TCF/LEF binding sites within the promoter region of human Angiopoietin family members were searched for by using bioinformatics and human intelligence (Humint). Because four TCF/LEF-binding sites were identified within the human ANGPTL7 promoter, comparative genomics analyses on ANGPTL7 orthologs were further performed. ANGPTL7 gene at human chromosome 1p36.22 was located within intron 28 of FRAP1 gene encoding mTOR protein. Chimpanzee ANGPTL7 gene, consisting of five exons, was located within NW_101546.1 genome sequence. Chimpanzee ANGPTL7 showed 99.4% and 86.1% total-amino-acid identity with human ANGPTL7 and mouse Angptl7, respectively. Human ANGPTL7 mRNA was expressed in neural tissues, keratoconus cornea, trabecular meshwork, melanotic melanoma and uterus endometrial cancer, while mouse Angptl7 mRNA was expressed in four-cell embryo, synovial fibroblasts, thymus, uterus and testis. Four TCF/LEF-binding sites within human ANGPTL7 promoter were conserved in chimpanzee ANGPTL7 promoter; however, only an unrelated TCF/LEF-binding site occurred in mouse and rat Angptl7 promoters. Human ANGPTL7, characterized as potent target gene of WNT/ beta-catenin signaling pathway, is a pharmacogenomics target in the fields of oncology and regenerative medicine. PMID- 16685430 TI - Cytoprotective effects of Lycium barbarum against reducing stress on endoplasmic reticulum. AB - Chinese medicinal herbs have been consumed for thousands of years for the purpose of healthy aging. Lycium barbarum is valued in Chinese culture for its benefits to anti-aging, vision, kidney and liver. Recent studies showed that extracts from L. barbarum possess biological activities including anti-aging, anti-tumor, immune-stimulatory and cytoprotection. Most of these studies emphasized that the protective function of L. barbarum is due to its anti-oxidative effects. We have previously demonstrated that extract from L. barbarum can protect neurons against beta-amyloid (Abeta) peptide-induced apoptosis. Since Abeta toxicity may be mediated via oxidative stress, it is still unclear whether the extract from L. barbarum is a simple anti-oxidant exhibiting cytoprotective effects. We hypothesized that extract from L. barbarum is not simply an anti-oxidant in order to function as a neuroprotective agent. The aim of this study is to investigate whether the extract from L. barbarum (LBG) protect neurons via mechanisms independent of anti-oxidative effects. Using a reducing agent, dithiothreitol (DTT), we found that LBG exhibits cytoprotective effects against reducing stress by lowering the DTT-induced LDH release and caspase-3 activity. DTT can trigger endoplasmic reticulum (ER) stress leading to PKR-like ER kinase (PERK) activation. We also showed that LBG attenuates DTT-induced PERK phosphorylation. The extract from L. barbarum is not simply an anti-oxidant; it can also exhibit cytoprotective effects against reducing stress by DTT. PMID- 16685429 TI - Evidence of circulating donor genetic material in bone allotransplantation. AB - Musculoskeletal allotransplantion is the most common form of human tissue transplantation. Unlike solid organ transplants, bone allotransplants undergo rigorous processing and are considered non-viable tissue. In this study, we propose that donor genetic material may exist in circulation after bone allotransplantation. Fifty-one female patients who received bone allotransplants from male donors were assessed. Blood plasma samples were analyzed using real time quantitative polymerase chain reaction (PCR) with dual labeled fluorogenic probes for the presence of the SRY gene on the Y chromosome. Of the total 51 patients, the SRY sequence was detected in 6 patients. Five were positive at day 1 postoperatively and negative thereafter, with the remaining patient positive at 3 months post-transplantation. Our results document, for the first time, the presence of donor DNA in the circulation of recipients after bone allotransplantation. Our findings suggest a potential new investigative tool to assess the postoperative status of bone allotransplants. PMID- 16685431 TI - Canonical WNT signaling pathway and human AREG. AB - AREG (Amphiregulin), BTC (beta-cellulin), EGF, EPGN (Epigen), EREG (Epiregulin), HBEGF, NRG1, NRG2, NRG3, NRG4 and TGFA (TGFalpha) constitute EGF family ligands for ERBB family receptors. Cetuximab (Erbitux), Pertuzumab (Omnitarg) and Trastuzumab (Herceptin) are anti-cancer drugs targeted to EGF family ligands, while Gefitinib (Iressa), Erlotinib (Tarceva) and Lapatinib (GW572016) are anti cancer drugs targeted to ERBB family receptors. AREG and TGFA are biomarkers for Gefitinib non-responders. The TCF/LEF binding sites within the promoter region of human EGF family members were searched for by using bioinformatics and human intelligence (Humint). Because three TCF/LEF-binding sites were identified within the 5'-promoter region of human AREG gene, comparative genomics analyses on AREG orthologs were further performed. The EPGN-EREG-AREG-BTC cluster at human chromosome 4q13.3 was linked to the PPBP-CXCL segmental duplicons. AREG was the paralog of HBEGF at human chromosome 5q31.2. Chimpanzee AREG gene, consisting of six exons, was located within NW_105918.1 genome sequence. Chimpanzee AREG was a type I transmembrane protein showing 98.0% and 71.4% total amino-acid identity with human AREG and mouse Areg, respectively. Three TCF/LEF-binding sites within human AREG promoter were conserved in chimpanzee AREG promoter, but not in rodent Areg promoters. Primate AREG promoters were significantly divergent from rodent Areg promoters. AREG mRNA was expressed in a variety of human tumors, such as colorectal cancer, liver cancer, gastric cancer, breast cancer, prostate cancer, esophageal cancer and myeloma. Because human AREG was characterized as potent target gene of WNT/beta-catenin signaling pathway, WNT signaling activation could lead to Gefitinib resistance through AREG upregulation. AREG is a target of systems medicine in the field of oncology. PMID- 16685432 TI - Biology of insulin-like growth factor binding protein-4 and its role in cancer (review). AB - Insulin-like growth factor binding protein-4 (IGFBP-4) is an important member of the insulin-like growth factor (IGF) system. The IGFBP-4 has three domains of which the N-terminal sequence is important for the binding of IGF. It acts as a transport protein for IGF-I and IGF-II and modulates their biological effects. There is increasing evidence that IGFBP-4 inhibits IGF-induced cellular growth both in vitro and in vivo. IGFBP-4 can also mediate its actions through a mechanism independent of IGFs. IGFBP-4 level and expression in various tissues are influenced by IGFBP protease, nutrition, several growth factors and hormones. Overexpression of IGFBP-4 in transgenic animal models causes reduced growth of organs containing smooth muscle. Most cancers express IGFBP-4 at levels which correlate with their state of differentiation. However, the effects of IGFBP-4 on tumor growth are uncertain. In vitro studies have shown that overexpression of IGFBP-4 inhibit the growth of some colon cancer cells. Overexpression of IGFBP-4 in vivo has been reported to decrease the growth of prostate cancer. The effect of altered expression of IGFBP-4 in vivo in colon and other cancers needs to be explored as locally available IGFs appear to stimulate mitogenesis. PMID- 16685433 TI - Identification of genes with altered expression in medullary breast cancer vs. ductal breast cancer and normal breast epithelia. AB - Medullary breast cancer (MCB) is a morphologically and biologically distinct subtype that, despite cytologically highly malignant characteristics, has a favorable prognosis compared to the more common infiltrating ductal breast carcinoma. MCB metastasizes less frequently, which has been attributed to both immunological and endogenous cellular factors, although little is known about the distinct biology of MCB that may contribute to the improved outcome of MCB patients. To identify candidate genes, we performed gene array expression analysis of cell lines of MCB, ductal breast cancer and normal breast epithelia, and the differential expression of a panel of candidate genes was further validated by quantitative PCR and immunohistochemical analysis of cell lines and tumor biopsies. A limited number of genes, including several members of the GAGE and insulin growth factor binding protein (IGFBP) gene families, Vav1, monoglyceride lipase and NADP+-dependent malic enzyme, exhibited altered expression in MCB vs. ductal breast cancer, and the differences for some of these genes were confirmed on an extended panel of cell lines by quantitative PCR. Immunohistochemical analysis further established that the expression of monoglyceride lipase was restricted to ductal breast cancer and present in 77% of these tumors, while Vav1 was restricted to MCB and present in 60% of tumors. In this study, we have identified genes that are differentially expressed in MCB vs. ductal breast cancer and further analysis of the gene products should illuminate the biological differences between MCB and ductal breast cancer. PMID- 16685434 TI - Dendritic cell-based therapy for mantle cell lymphoma. AB - Mantle cell lymphoma (MCL) is a B cell malignancy that is resistant to conventional therapies. High-dose therapy (HDT) followed by stem cell transplantation is effective in inducing remission. However, residual lymphoma cells are eventually responsible for the subsequent relapse. Effective therapeutic strategies to eliminate the residual lymphoma is required. In this study, we have examined the in vitro and in vivo anti-lymphoma effects of MCL specific cytotoxic T lymphocytes (CTLs) that were generated using dendritic cells (DCs) fused with MCL cells for immunostimulation. Dendritic cells were generated in vitro using dendritic cell-specific medium, cytomorphology, immunophenotypes and functional capabilities of the generated DCs were studied. Such DCs were then used for the preparation of DC-MCL hybrids and the DC-MCL hybrids were used to generate CTLs against MCL cells and tested for their MCL-specific cytotoxicity in vitro and in vivo. The CTLs demonstrated MCL-specific cytotoxicity in vitro against GRANT-519, a human MCL cell line. These CTLs did not show significant effect against an irrelevant target. To test the in vivo therapeutic effect of DC MCL hybrid-stimulated CTLs, a preclinical model consisting of NOD-SCID mice bearing Granta 519 was developed. The NOD-SCID mice bearing Granta-519 MCL tumors were treated with DC-MCL hybrids and the same donor T lymphocytes. There was an increase in survival (60% in mice treated with DC-MCL hybrid approach compared to 20% in the untreated group). Histological analysis of liver from control and treated mice displayed a decrease in the number of the tumor nodules in the treatment group. These results indicate the potential of DC-based therapy for the treatment of MCL. PMID- 16685435 TI - Expression profiling of esophageal squamous cell carcinoma patients treated with definitive chemoradiotherapy: clinical implications. AB - In esophageal squamous cell carcinoma (ESCC), chemoradiotherapy (CRT) has a curative potential even in cases of locally advanced carcinoma. However, only about half of the patients benefit from CRT, and an accurate prediction of sensitivity to CRT is eagerly awaited. Using microarrays, we analyzed gene expression patterns of pretreatment biopsy specimens from 33 patients with CRT alone including long-term survivors, more than 3 years (14 cases) and short-term survivors, less than 1 year (11 cases). The expression patterns of about 12,600 genes were used to identify genes correlated with survival terms. Fifty-seven genes correlating with short-term survival and 120 genes with long-term survival were identified. The genes involved in the immune response were characteristically upregulated in the long-term survivors, and an immunohistochemical staining confirmed an increased CD8-positive T cell number in the long-term survivors over that in the short-term survivors. In the short-term survivors, on the other hand, increased expression of the genes involved in drug resistance was observed. Our gene list should contribute to the elucidation of the mechanisms of CRT response and contains useful markers for predicting the prognosis of individual ESCC patients treated with CRT alone. PMID- 16685437 TI - Deletion of the KIT gene is associated with liver metastasis and poor prognosis in patients with gastrointestinal stromal tumor in the stomach. AB - The goal of this study was to investigate the association of mutations in the KIT gene and the platelet-derived growth factor receptor alpha (PDGFRA) gene with clinicopathological features of patients with gastrointestinal stromal tumor (GIST) localized in the stomach. We evaluated 56 gastric GISTs for KIT and PDGFRA mutations. DNA was extracted from paraffin-embedded tumor specimens, and exons 9, 11, 13 and 17 of the KIT gene and exons 12 and 18 of the PDGFRA gene were amplified by polymerase chain reaction and sequenced. The genetic features were then compared with the clinicopathological features. Immunohistochemistry was performed for KIT, CD34, Ki-67 (as a marker of cell proliferation) and CD31 (as a marker of microvessel density), and apoptosis was assessed by in situ DNA nick end labeling. Thirty-four (61%) of the 56 GISTs had a mutation in exon 11 of KIT, and 2 (4%) had a mutation in exon 13 of KIT. Deletions in exon 11 of KIT were the most common mutation encountered in the present study. No mutations were found in exon 9 or 17 of KIT. Six of the 20 GISTs lacking KIT mutations had a mutation in exon 18 of PDGFRA, and 1 had a mutation in exon 12 of PDGFRA. The KIT mutation positive GISTs showed more frequent liver metastases and higher mortality than KIT mutation-negative GISTs. Our data indicate that KIT mutations, especially deletions in exon 11, are markers of poor prognosis for gastric GISTs. PMID- 16685436 TI - RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth. AB - In recent years, RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Several groups are actively exploring the use of small interfering RNA (siRNA) for therapeutic applications to treat cancer. Our previous studies have demonstrated the inhibition of various proteases, including serine proteases, cysteine proteases and matrix metalloproteases, via RNA interference (RNAi) in gliomas. Similar to gliomas, malignant meningiomas also exhibit elevated protease levels in comparison to normal brain and benign meningiomas. Here, we used siRNA to simultaneously target urokinase plasminogen activator (uPA) and its receptor, uPAR. A human CMV promoter-driven mammalian expression vector (pU2) was used to produce hairpin double-stranded RNA (hp RNA) to target uPA and uPAR. As determined by Western blotting and fibrin zymography, pU2 effectively inhibited uPAR protein levels and uPA enzymatic activity in meningioma cells (IOMM-Lee). In vitro studies (Matrigel invasion and spheroid migration) revealed reduced meningioma cell invasion and migration. Intratumoral injections of the plasmid vector expressing siRNA for uPA and uPAR resulted in regression of pre-established, subcutaneous tumors in mice. In addition, in vivo studies of mice injected with pU2-transfected meningioma cells revealed inhibition of intracranial tumor formation. These findings suggest that siRNA can be used as a potent and specific therapeutic tool for the treatment of malignant meningiomas in humans. PMID- 16685438 TI - Poor outcome of patients with pulmonary adenocarcinoma showing decreased E cadherin combined with increased S100A4 expression. AB - A loss or reduced expression of E-cadherin, the main cell-to-cell adhesion molecule, correlates with distant metastasis in various cancers. Recent studies have reported a close correlation between the expression of E-cadherin and that of S100A4, calcium-binding protein. In this study, we investigated the expression of E-cadherin and S100A4 status in relation to the clinicopathological parameters of pulmonary adenocarcinoma. We finely and quantitatively examined the expression of E-cadherin and S100A4 using real-time polymerase chain reaction (PCR) in a total of 92 pulmonary adenocarcinomas obtained by surgical resection. All of the pulmonary adenocarcinomas showed significant expression of E-cadherin and S100A4. Real-time PCR showed lower E-cadherin expression in 21 adenocarcinomas, while 71 adenocarcinomas expressed a higher expression of E-cadherin. Of 21 adenocarcinomas with lower-expressing E-cadherin, 12 showed a higher expression of S100A4. These 12 cases significantly showed a poorer prognosis than others (p=0.047, Kaplan-Meier, log-rank test) and significantly showed more frequent venous involvement than others (p=0.042, chi2 test). These results suggested that reduced E-cadherin expression combined with higher S100A4 expression is related to a poor prognosis through hematogenous metastasis in pulmonary adenocarcinoma. PMID- 16685439 TI - Genome-wide gene expression profile analysis of esophageal squamous cell carcinomas. AB - To identify the molecules involved in esophageal carcinogenesis and those applicable as novel tumor markers and for the development of new molecular therapies, we performed gene expression profile analysis of 19 esophageal squamous cell carcinoma (ESCC) cells purified by laser microbeam microdissection (LMM). Using a cDNA microarray representing 32,256 genes, we identified 147 genes that were commonly up-regulated and 376 transcripts that were down-regulated in ESCC cells compared with non-cancerous esophageal epithelial cells. A comparison of clinicopathological data with the expression profiles of the 19 ESCCs identified 20 genes whose expression levels could most significantly separate cases with lymph node metastasis from those without. In addition, immunohistochemical analysis of candidate tumor markers on tissue microarrays demonstrated transactivation of a secretory protein, transforming growth factor alpha (TGFA) in the great majority of 228 ESCC cases and an association of their expression with the poor prognosis of patients. Our data provide valuable information for establishing novel diagnostic markers for early diagnosis and choice of therapy, and identifying therapeutic target molecules for the development of novel anti-cancer drugs and immunotherapy in esophageal cancer treatment. PMID- 16685440 TI - Synergistic effect and condition of pegylated interferon alpha with paclitaxel on glioblastoma. AB - Glioblastomas are highly vascularized tumors and anti-angiogenic strategy is one of the most promising therapeutic approaches to treat brain tumors. Interferon alpha (IFN-alpha) as a single agent or combined with standard chemo-therapy has been shown to inhibit various tumors, but the effect of combination anti angiogenic therapy on brain tumors has not been well studied. We determined the optimal dose and schedule of pegylated IFN-alpha (PEG-IFN-alpha) against U-87MG human glioblastoma cells growing orthotopically in nude mice, since several clinical trials reported that PEG-IFN-alpha administered at higher or lower doses was less effective. The group treated two times per week with injections of 10 KU of PEG-IFN-alpha for 4 weeks showed significant decreases in cell proliferation and angiogenesis. Moreover, the optimal dose and schedule of PEG-IFN-alpha determined in this study and combined with paclitaxel treatment potently inhibited tumor growth in vivo. The mechanisms of the significant therapeutic effects were most likely caused by directly inhibiting cell proliferation and angiogenesis, and rendering apoptosis increased. Specifically PEG-IFN alpha/paclitaxel combination induced apoptosis of tumor-associated endothelial cells more than that of tumor cells. These results suggest that optimal biological dosage and scheduling of PEG-IFN-alpha and paclitaxel combination is a potent strategy for glioblastoma patients as a new synergistic anti-endothelial treatment. PMID- 16685441 TI - Effects of ghrelin on anorexia in tumor-bearing mice with eicosanoid-related cachexia. AB - Ghrelin is a novel brain-gut peptide that stimulates food intake and may secondarily increase body weight via a growth hormone secretagogue receptor (GHS R). Tumor-bearing mice (MCG101), characterized by anorexia, fat loss and muscle wasting due to increased concentration of PGE2 and proinflammatory cytokines (IL 1beta, IL-6, TNF-alpha), were provided ghrelin i.p. at a low (20 microg/day) and high dose (40 microg/day) to examine the ability of ghrelin to counteract tumor induced anorexia. Immunohistochemical staining and Western blot analyses were used to identify GHS-R expression in the brain as well as its relationship to NPY expression in hypothalamic neurons. GHS-R mRNA in hypothalamus and ghrelin mRNA in gastric fundus were quantified by RT-PCR. Body composition was determined by carcass extractions. GHS-R expression in hypothalamus and plasma ghrelin levels were significantly increased in freely-fed tumor-bearing mice, while gastric fundus expression of ghrelin was unaltered compared to non-tumor-bearing mice (controls). Ghrelin treatment increased food intake, body weight and whole body fat at both low and high doses of ghrelin in normal controls, while tumor-bearing mice showed improved intake and body composition at the high dose of ghrelin only. Exogenous ghrelin normalized the GHS-R expression in hypothalamus from tumor-bearing mice without alterations in the gastric fundus expression of ghrelin. Tumor growth was not altered by exogenous ghrelin. Our results indicate that MCG 101-bearing mice became ghrelin resistant despite upregulation of hypothalamic GHS-R expression, which confirms similar indirect observations in cancer patients. Thus, other factors downstream of the ghrelin-GHS-R system appear to be more important than ghrelin to explain cancer-induced anorexia. PMID- 16685442 TI - Combination treatment with arsenic trioxide and sulindac augments their apoptotic potential in lung cancer cells through activation of caspase cascade and mitochondrial dysfunction. AB - Non-steroidal anti-inflammatory drugs (NSAIDs) are known to enhance the responsiveness of tumor cells toward chemotherapeutic drugs and radiation. However, the precise mechanism of synergistic enhancement in tumoricidal activity is not clearly known. Herein, we demonstrate that the combination treatment of arsenic trioxide (As2O3) and sulindac resulted in a synergistic augmentation of cytotoxicity toward NCI-H157 lung cancer cells, which was revealed as apoptosis accompanied by chromatin fragmentation and an increase in sub-G0/G1 fraction. In addition, combination treatment with As2O3 and sulindac increased the catalytic activity of caspase-3, -8, and -9 along with induction of Fas/FasL expression and cytosolic release of cytochrome c. Pharmacologic scavenging study of reactive oxygen species (ROS) revealed that synergistic augmentation of cytotoxicity was achieved by generation of ROS, which might modulate the expression of Bcl-2 family proteins, the activity of caspase-3, and mitochondrial membrane potential transition. PMID- 16685443 TI - LAGlambda-1: a clinically relevant drug resistant human multiple myeloma tumor murine model that enables rapid evaluation of treatments for multiple myeloma. AB - We set out to generate new human myeloma tumors that grow in immunodeficient mice and can be used for pathophysiological studies and rapid evaluation of new therapies. Fresh whole core bone marrow (BM) biopsies taken from 33 myeloma patients were engrafted into the hind limb muscle of severe combined immunodeficient (SCID) mice. Human Ig was detected in 28/33 mice and three grew palpable tumors displaying many features of human myeloma including morphology, immunophenotype and BM plasmacytosis. Following intramuscular passage, we generated large numbers of mice with predictable increases in tumor growth and human paraprotein levels. We further characterized the model generated from an IgGlambda-producing tumor known as LAGlambda-1 and determined the effects of the proteasome inhibitor bortezomib, the alkylating agent melphalan, and the DNA damaging agent liposomal doxorubicin, on the growth of this tumor. LAGlambda-1 bearing mice receiving higher doses of bortezomib showed reduced tumor growth whereas a lower dose had no effect. In contrast, melphalan did not significantly alter tumor growth, except minimally at high doses, reflecting the resistance of this patient's tumor to this drug. We also used our intramuscular (i.m.) LAGlambda-1 model to optimize the dosing schedule of liposomal doxorubicin. Low doses administered once daily three days per week decreased tumor growth and human paraprotein levels whereas much higher doses given once weekly had no anti myeloma effects. Furthermore, LAGlambda-1 cells produce local tumors when injected subcutaneously and lytic lesions when injected intravenously allowing for multiple methods of evaluating the anti-myeloma effects of a variety of agents. Our new clinically relevant SCID models of human myeloma should greatly facilitate drug development and enable novel therapies to quickly move from the laboratory to the clinic. PMID- 16685444 TI - Specific immune recognition of pancreatic carcinoma by patient-derived CD4 and CD8 T cells and its improvement by interferon-gamma. AB - Pancreatic carcinoma is a very aggressive disease and little is known about its immunobiology. We here describe the presence in pancreatic cancer patients of spontaneously induced functional CD4 and CD8 memory/effector T cells reactive to autologous tumor cells or to the pancreatic cancer associated antigen, MUC-1. Such specific cells were present in the bone marrow or peripheral blood of most of the 23 tested patients. Low dose stimulation of primary cultures of pancreatic cancer cells with 500 IU/ml IFN-gamma for 72 h enhanced HLA-I expression and induced the de novo expression of HLA-II molecules. This led to a much better immune recognition by autologous HLA-I restricted and purified CD8 T cells and allowed tumor cell recognition by HLA-II restricted purified CD4 T-helper cells. Thus, interferon-gamma appears to be a useful adjuvant cytokine to enhance the immunogenicity of a patients' tumor cells and their recognition by tumor reactive immune cells. PMID- 16685446 TI - Transcriptome characterization of human mammary cell lines expressing different levels of ERBB2 by serial analysis of gene expression. AB - Over-expression of ERBB2, a member of the family of transmembrane receptor tyrosine kinases, occurs in 15-30% of primary breast tumors and is associated with poor prognosis and chemoresistance to a variety of anticancer drugs. In this study, aiming to identify differentially-expressed genes involved in erbB2 mediated transformation of the breast, we generated SAGE libraries from two human mammary cell lines, derived from normal luminal cells, expressing different levels of erbB2. The parental cell line HB4a expresses basal levels and the C5.2 expresses high levels of erbB2. A total of 161,632 tags was generated by sequencing, 81,684 from HB4a cells (30,854 unique tags) and 79,948 from C5.2 cells (30,568 unique tags). The comparison between the HB4a and C5.2 libraries revealed 334 distinct transcripts more expressed in HB4a cells and 328 distinct transcripts more expressed in C5.2 cells. The expression pattern of some of these transcripts was further validated by RT-PCR. The C5.2 cell line, which over express ERBB2, showed in comparison to HB4a cells a higher percentage of genes involved in transport, RNA processing, apoptosis and protein folding. A higher percentage of the genes more expressed in HB4a cells compared to C5.2 were found to be involved in signal transduction and cytoskeleton organization. The use of SAGE analysis allowed us to identify a significant number of genes implicated in different cellular pathways up- or down-regulated in the presence of ERBB2 over expression, including genes not previously implicated in breast cancer that could be considered as potential candidate markers for prognosis and therapy. PMID- 16685445 TI - Estrogen enhances the efficacy of an oncolytic HSV-1 mutant in the treatment of estrogen receptor-positive breast cancer. AB - Oncolytic herpes simplex virus-1 (HSV-1) mutants selectively replicate in and lyse tumor cells. Viral replication is dependent on the cellular proliferative mechanism. Estrogen increases cellular proliferation and decreases apoptosis in estrogen receptor-positive (ER+) human breast cancer cells. We hypothesize that the cellular changes produced by estrogen may enhance oncolytic viral replication and improve the treatment of ER+ breast cancer cells. Estrogen increased proliferation and replication of the HSV-1 mutant, NV1066, in ER+ breast cancer cells. Additionally, cells grown with estrogen had lower rates of apoptosis and higher bcl-2 levels at baseline and after infection. Estrogen enhanced the oncolytic effect of NV1066, with cell kills of 95% and 97% at MOIs of 0.1 and 0.5, compared to 53 and 87% respectively without estrogen (p<0.001). Therapy of ER+ human breast cancer cells with a replication-competent HSV-1 mutant is improved in the presence of estrogen, in contrast to more standard therapies, such as chemotherapy and radiation, which demonstrate decreased efficacy in similar conditions. These data provide the mechanistic basis for the use of oncolytic HSV-1 in patients with hormone receptor-positive breast cancer, particularly if the disease progresses with conventional therapies. PMID- 16685447 TI - Urokinase-type plasminogen activator induces proliferation in breast cancer cells. AB - Urokinase-type plasminogen activator (uPA) is implicated in various pathophysiological processes, including extracellular matrix turnover, cell migration and invasion. Our study aimed to determine the role of uPA in both proliferation and mitogen-activated protein kinase (MAPK) pathway. Hence, we analyzed the effects induced by exogeneous addition of domain-specific uPA antibodies and uPA-interacting molecules on proliferation of uPA-suppressed MDA MB-231 breast cancer cells. uPA expression was reduced to 53% by stable transfection with an antisense/vector construct and to 65% by siRNA transfection. Immunocytochemical Ki67 staining and flow cytometry (S-phase) analysis indicated a strong decrease of cellular proliferation activity (35% and 38%, respectively). Exogenous addition of high molecular weight-uPA (HMW-uPA) or incubation with the amino terminal fragment (ATF), which lacks the enzymatic activity of uPA, lead to increased cell proliferation. A strong increase of proliferation was absent when the monoclonal anti-uPAR antibody IIIF10 (blocking uPA binding site), soluble uPAR (scavenger effect) and phosphatidyl-inositol-specific phospholipase C (PI PLC, degrading uPAR) was added prior to the addition of HMW-uPA. In conclusion, HMW-uPA and ATF induce proliferation of breast cancer cells by binding to uPAR. Thereby, integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the MAPK pathway. PMID- 16685448 TI - Differential targeting of protein kinase B in cell death induced by sulindac and its metabolite sulindac sulfide. AB - Non-steroidal anti-inflammatory drugs such as sulindac inhibit human colorectal carcinogenesis through a mechanism involving the direct inhibition of cyclooxygenase (Cox)-2. However, a wealth of recent evidence indicates that these agents might elicit their effects through mechanisms independently of Cox-2. In this study, we investigated the effects of sulindac and its metabolite, sulindac sulfide on modulation of the critical survival kinase, protein kinase B (PKB). Here, we demonstrate for the first time that treatment with either sulindac or sulindac sulfide results in a decrease in PKB activity, and we provide compelling evidence that this occurs through two distinct mechanisms. Additionally, we report that overexpression of, and conditional activation of PKB attenuates the apoptotic effects of sulindac, but not for sulindac sulfide - the metabolic metabolite of sulindac. We also demonstrate that treatment with sulindac sulfide, but not sulindac, results in a very early robust activation of both caspase-8 and -9. Furthermore, we show that the apoptotic effects of sulindac sulfide can be reverted by both the caspase-8 and -9 inhibitors. Evidence is provided to indicate that PKB is targeted by robust caspase activation due to sulindac sulfide. Hence, further investigation into the mechanisms regulating conversion of sulindac to sulindac sulfide (or direct use of the latter compound), may enhance our ability to target cancers with enhanced signaling through the growth factor-->phosphatidylinositol 3-kinase pathway. PMID- 16685449 TI - Mature dendritic cells generated from patient-derived peripheral blood monocytes in one-step culture using streptococcal preparation OK-432 exert an enhanced antigen-presenting capacity. AB - Dendritic cells (DCs) have been shown to be potent in inducing cytotoxic T cell (CTL) response leading to the efficient anti-tumor effect in active immunotherapy. Myeloid DCs are conventionally generated from human peripheral blood monocytes in the presence of interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Streptococcal preparation OK-432, which is known to be a multiple cytokine inducer, has been extensively studied as to its maturation effects on immature DCs using an in vitro culture system. The purpose of this study was to examine whether it could be possible to generate mature DCs directly from peripheral monocytes using OK-432. We specifically focused on the possibility that recombinant cytokines, which are considered to be essential for in vitro DC generation, could be substituted by OK-432. Human peripheral monocytes, which were obtained from patients with advanced cancer, were cultured with IL-4 and OK-432 for 7 days. Cultured cells were compared with DCs generated in the presence of IL-4 and GM-CSF with or without OK-432 with regard to the surface phenotype as well as the antigen-presenting capacity. As a result, the culture of monocytes in the presence of IL-4 followed by the addition of OK-432 on day 4 (IL-4/OK-DC) induced cells with a fully mature DC phenotype. Functional assays also demonstrated that IL-4/OK-DCs had a strong antigen-presenting capacity determined by their enhanced antigen-specific CTL response and exerted a Th1-type T cell response which is critical for the induction of anti-tumor response. In conclusion, human peripheral blood monocytes cultured in the presence of IL-4 and OK-432 without exogenous GM-CSF demonstrated a fully mature DC phenotype and strong antigen-presenting capacity. This one-step culture protocol allows us to generate fully mature DCs directly from monocytes in 7 days and thus, this protocol can be applicable for DC-based anti-tumor immunotherapy. PMID- 16685450 TI - Induction of DNA double-strand breaks in A549 and normal human pulmonary epithelial cells by cigarette smoke is mediated by free radicals. AB - DNA double-strand breaks (DSBs) are potentially mutagenic/carcinogenic lesions. Induction of DSBs triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX (gammaH2AX) can be detected immunocytochemically, and the intensity of gammaH2AX immunofluorescence (IF), reflecting the number of gammaH2AX-IF foci per nucleus, reveals the frequency of DSBs. Using multiparameter cytometric analysis of gammaH2AX-IF, we previously observed that DSBs are induced in normal human bronchial epithelial (NHBE) and A549 pulmonary adenocarcinoma cells following exposure to cigarette smoke (CS) or smoke condensate. In the present study, we show that N-acetyl L-cysteine (NAC) and glutathione, both effective scavengers of free radicals, prevented induction of DSBs by CS in these cells. In contrast, the glutathione synthesis inhibitor, DL Buthionine-[S,R]-sulfoximine (BSO), enhanced the induction of DSBs by CS. The observed reduction of DSBs by NAC correlated with protection of the reproductive capability (clonogenicity) of A549 cells treated with CS. The data implicate formation of free radicals by CS as factors generating DSBs and affecting cell survival. Interestingly, at the conditions of exposure to CS when clonogenicity was only moderately affected, S-phase cells showed significantly higher sensitivity in terms of induction of DSBs compared with G1 or G2M cells. In light of the evidence that CS increases oxidative stress and induces cell proliferation in the lungs of smokers, the high propensity of S-phase cells to develop DSBs upon exposure to CS has to be considered as a potentially pathogenic event in smoke-induced tumor development. This is the first report to reveal cell cycle phase specificity in both the induction of DSBs by CS and their prevention by free radical scavengers. The detection of gammaH2AX to assess the induction of CS induced DSBs and their relationship to cell cycle phase provides a convenient tool to explore approaches to protect cells from this type of genotoxic damage. PMID- 16685451 TI - Effective therapy of experimental human malignant melanomas with a targeted cytotoxic somatostatin analogue without induction of multi-drug resistance proteins. AB - Malignant melanomas are characterized by a high intrinsic resistance to chemotherapy. Multiple drug resistance (MDR) can be mediated by transport proteins such as MDR-1, multidrug resistance-associated protein (MRP) or lung resistance protein (LRP). The cytotoxic analogue of somatostatin AN-238 consisting of 2-pyrrolinodoxorubicin (AN-201) linked to a somatostatin analogue RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. We evaluated the expression of somatostatin receptors on human malignant melanoma tumor lines MRI-H255 and MRI-H187 and examined the effects of the targeted analogue AN-238 and its cytotoxic radical AN-201 on growth of these tumors in nude mice. We also studied the effects of AN-238 and AN-201 on the expression of MDR-1, MRP-1 and LRP by real-time PCR. AN-238 inhibited the growth of MRI-H255 and MRI-H187 tumors while AN-201 was ineffective. Blockade of somatostatin receptors by somatostatin analogue RC-121 abolished the effects of AN-238. Targeted therapy with AN-238 did not produce an induction of mRNA of MDR 1, MRP-1 or LRP. Our findings show that targeted chemotherapy with cytotoxic somatostatin analogue AN-238 inhibits the growth of malignant melanomas. AN-238 could provide a novel treatment approach for advanced malignant melanomas. PMID- 16685452 TI - Polyamine depletion and cell cycle manipulation in combination with HSV thymidine kinase/ganciclovir cancer gene therapy. AB - We have shown earlier that polyamine biosynthesis inhibition is accompanied by cell cycle alterations that can be utilized to enhance the efficacy of herpes simplex virus thymidine kinase - ganciclovir (HSV-TK/GCV) cancer gene therapy. In the present study, we asked 1) can the activated polyamine catabolism instead of biosynthesis inhibition be utilized to enhance the efficacy of HSV-TK/GCV gene therapy, and 2) can other known cell cycle inhibitors be used to make tumor cells more sensitive to this form of gene therapy? We show, using rat (9L) and human (U251-MG) glioma cell populations with 15% of HSV-TK-positive cells that DENSPM induced activation of polyamine catabolism caused a profound polyamine deprivation in U251-MG cells, but there were no associated cell cycle effects in these cells. Consequently, we did not see any enhancement of the HSV-TK/GCV system. Aphidicolin, hydroxyurea, mimosine and resveratrol, but not lovastatin induced an apparent cell cycle arrest, followed by an intense but transient increase of the S phase cells after removal of the drug. This effect was shown to potentiate the HSV-TK/GCV cytotoxicity to some extent, especially in 9L cells and when the GCV treatment was started 0-24 h before the drug treatment. However, the enhancement was weaker than observed earlier with DFMO-induced cell cycle arrest and a considerable degree of the effect appeared to result from the growth inhibitory actions of the drugs. In summary, we demonstrate that polyamine deprivation via DENSPM action is not associated with cell cycle effects and is not sufficient to cause enhancement of the HSV-TK/GCV system. Also, drugs with a rapid effect to the cell cycle are weak boosters of the HSVTK/GCV gene therapy, thus being less useful than DFMO for enhancement of this gene therapy form in animal studies and clinical trials. PMID- 16685453 TI - Prevention of early-onset familial/hereditary colon cancer: new models and mechanistic biomarkers (review). AB - Human colon cancer is a multi-factorial, multi-step disease wherein genetic and dietary factors represent important regulators of initiation, promotion and progression. While the etiology of sporadic colon cancer remains largely unidentified, familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) represent predisposing genetic syndromes for early-onset familial/hereditary colon cancer. These syndromes are characterized by germ-line mutations in the adenomatous polyposis coli (APC) and/or DNA mismatch repair genes, respectively. Currently available preclinical animal models for human FAP and HNPCC syndromes, expressing clinically relevant germ-line mutations, exhibit adenomas in the small intestine rather than in the colorectum. These models are, therefore, subject to extrapolation for direct clinical translatability of the data for colon carcinogenesis and chemoprevention. Experimental models expressing clinically relevant genetic defects (APC and/or DNA mismatch repair gene mutations) in an appropriate target site (colon) may represent novel approaches that reduce extrapolation of the data for their clinical relevance. This report provides an overview on carcinogenesis and chemoprevention in preclinical models of FAP and HNPCC syndromes, and summarizes recent data on i) development of new cell culture models for FAP and HNPCC syndromes; and ii) validation of developed models for rapid, mechanism-based screening of new pharmacological or naturally occurring chemopreventive agents. PMID- 16685454 TI - Effective silencing of EGFR with RNAi demonstrates non-EGFR dependent proliferation of glioma cells. AB - The epidermal growth factor receptor (EGFR, ErbB1) is frequently dysregulated in a variety of solid human tumors, including malignant glioma. EGFR expression has been associated with disease progression, resistance to standard therapies and poor survival. The application of small interfering RNAs (siRNAs) has become an effective and highly specific tool to modulate gene expression, and a wide range of oncogenes have been silenced successfully. Here we show the siRNA-mediated down-regulation of EGFR in two established glioma cell lines with different EGFR expression levels (U373 MG, LN18). The expression of EGFR mRNA and protein was down-regulated by 70-90%. However, siRNA treatment had no inhibitory effect on cell proliferation, migration and activation status of EGFR-coupled signaling cascades. In accordance with these results, gene expression analysis with microarrays revealed only small, albeit specific changes in expression patterns. In conclusion, these data indicate that the specific down-regulation of EGFR might not be sufficient for a single agent therapeutic approach in malignant glioma. PMID- 16685455 TI - Hyperthermia enhances cytotoxicity of amine oxidase and spermine on drug resistant LoVo colon adenocarcinoma cells. AB - Hyperthermia is currently receiving widespread attention when associated with other therapeutic modalities, such as irradiation or chemotherapy, in the treatment of cancer. The occurrence of resistance to cytotoxic pharmacological agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. We investigated a new strategy to overcome multidrug resistance (MDR) cancer cells, using bovine serum amine oxidase (BSAO), which forms toxic products from spermine (H2O2 and aldehydes). The cytotoxicity of the products was evaluated in drug-sensitive (LoVo WT) and multidrug-resistant (LoVo DX) colon adenocarcinoma cells at 37 and 42 degrees C, using a clonogenic cell survival assay. Cytotoxicity was considerably enhanced at 42 degrees C. Both toxic species contributed to the thermal enhancement of cytotoxicity induced by BSAO and spermine. Cytotoxicity was eliminated in the presence of catalase and aldehyde dehydrogenase (ALDH). An interesting finding was that BSAO and spermine at <1 microM, which were non toxic at 37 degrees C, became cytotoxic at 42 degrees C and resemble thermosensitizers. Cell survival results and electron microscopy investigations suggest that, at 42 degrees C, LoVo DX cells are not resistant to the cytotoxic enzymatic oxidation products of spermine, as was already demonstrated in these cells at 37 degrees C. Moreover, microscopy modifications caused by both toxic products were more pronounced in LoVo DX than in LoVo WT cells, where morphological cytoplasmatic alterations were shown. Our findings suggest that hyperthermia combined with the enzymatic toxic oxidation products of spermine might be a promising anticancer strategy, mainly against MDR tumor cells. PMID- 16685456 TI - Telomerase activity and hTERT mRNA expression in glial tumors. AB - Human telomerase is a structurally complex ribonucleoprotein that is responsible for the maintenance of telomeric DNA at the ends of the chromosomes. The enzyme is proposed as having an important role in cell immortalization and oncogenesis. A limited number of studies have been performed on the telomerase system in brain tumors, and these studies are somewhat conflicting. The relative ineffectiveness of current therapies for malignant gliomas led to the need for novel targets for more promising approaches. In order to clarify the prognostic significance of telomerase expression in gliomas and to speculate on therapeutic implications, we examined telomerase activity by the telomeric repeat amplification protocol (TRAP) assay in 42 gliomas, (32 multiform glioblastomas, 4 anaplastic astrocytomas, 4 differentiated astrocytomas, 1 oligoastrocytoma and 1 oligosarcoma). Telomerase messenger expression (hTERT mRNA) was evaluated by reverse transcription-PCR analysis in the same group of tumors. High telomerase activity was detected in 21/42 gliomas (50%). The levels of telomerase in terms of its messenger level expression overlapped the activity; in fact, a significant association between telomerase activity and hTERT mRNA expression was found (chi2 test; p<0.0001). At univariate analysis, advanced age as well as high telomerase activity and hTERT mRNA levels were seen to be significant predictors of worse prognosis regarding both overall survival (p=0.007, p=0.007, p=0.04, respectively) and disease-free interval (p=0.008, p=0.008, p=0.04, respectively). All these variables maintained a significant independent prognostic role in multivariate analysis. Telomerase may represent an indicator of progression and poor prognosis in this type of cancer, with interesting therapeutic implications. PMID- 16685457 TI - Synthesis and in vitro investigation of cationic 5,15-diphenyl porphyrin monoclonal antibody conjugates as targeted photodynamic sensitisers. AB - The synthesis of three cationic 5,15-diphenyl porphyrins, bearing an isothiocyanate group for conjugation to proteins is described. The potential of these compounds as targeted photosensitisers for use in photodynamic therapy (PDT) is demonstrated by their efficient conjugation to monoclonal antibodies and the ability of the bioconjugates to bind and photodynamically inactivate cancer cells that express the corresponding antigen. PMID- 16685458 TI - Primary bone lymphoma: a retrospective analysis. AB - The aim of this study was to retrospectively define those patients with unequivocal primary bone lymphoma presenting to the Sheffield Lymphoma Group and document patient and tumour characteristics and management strategies, and correlate these with survival. Thirty-seven patients were documented from a total of 3148 cases of non-Hodgkin's lymphoma seen over 34 years. There were 17 males and 20 females, with a mean age of 55.4 years (range, 27-78). Pain was the most commonly presented symptom (67.5%), and the pelvis was the most frequently presented site (21.3%). Grade 2 and diffuse large B cell lymphoma comprised the majority of histologies (78.7% and 70.3%, respectively). Treatment was most often with radiotherapy alone (41.8%) or combined with CHOP-like chemotherapy (37.9%). The overall response rate was 56.7%, and 5- and 10-year survival rates were 64.5% and 49.6%, respectively. Univariate analysis showed an age of <60 years and complete response to be favourable prognostic factors. There was a trend toward better survival with combined modality therapy involving CHOP-like chemotherapy. Bone lymphoma has a better survival than other extranodal lymphomas. Younger age and complete response are favourable predictive factors. Combined modality treatment is likely to be the treatment of choice but this remains to be confirmed in large prospective multicentre studies. PMID- 16685459 TI - Progression of squamous cell carcinoma of the head and neck is associated with down-regulation of the opioid growth factor receptor. AB - Opioid growth factor (OGF) is a native opioid peptide ([Met5]-enkephalin) that interacts with the OGF receptor (OGFr). OGF serves as a tonically active negative growth factor in neoplasia, and the OGF-OGFr axis contributes to the maintenance of an equilibrium in cell proliferation by targeting the cyclin-dependent inhibitory kinase pathway. To inquire whether the expression of OGFr is related to tumor progression, cell lines of human squamous cell carcinoma of the head and neck (SCCHN) were transplanted into nude mice, and small, medium, and large tumors were assessed for OGFr by receptor binding assays and quantitative immunohistochemistry, and for gene expression of OGFr mRNA. Large tumors had a reduction of 3- to 7-fold in OGFr binding sites relative to small tumors, and medium size tumors showed a progressive diminishment in OGF receptors that ranged between that of small and large neoplasias. Tumors with xenografts of three different cell lines of SCCHN, representing poorly- and well-differentiated cancers, exhibited similar results. Quantitative densitometric immunohistochemistry revealed data comparable to receptor binding assays. Receptor affinity and the gene expression of OGFr mRNA were unchanged in tumors of different sizes. These data demonstrate that OGFr is reduced in SCCHN with tumor progression and that translation/posttranslation of OGFr protein, but not transcriptional levels of the OGFr gene, is (are) involved. The attenuated levels of OGFr binding capacity may serve as a marker of SCCHN. These subnormal levels of OGFr may diminish the efficacy of the OGF-OGFr axis in maintaining cell proliferative activity, and contribute to more active cell replication. Gene therapy to reinstate more OGFr, and thus enhance OGFr function, could serve as a useful treatment for inhibiting tumor progression. PMID- 16685460 TI - Comparative integromics on VEGF family members. AB - VEGF, Hedgehog, FGF, Notch, and WNT signaling pathways network together for vascular remodeling during embryogenesis, tissue regeneration, and carcinogenesis. VEGFA (VEGF), VEGFB, VEGFC, VEGFD (FIGF) and PGF (PlGF) are VEGF family ligands for receptor tyrosine kinases, including VEGFR1 (FLT1), VEGFR2 (KDR) and VEGFR3 (FLT4). Bevacizumab (Avastin), Sunitinib (Sutent) and Sorafenib (Nexavar) are anti-cancer drugs targeted to VEGF signaling pathway. TCF/LEF binding sites within the promoter region of human VEGF family members were searched for by using bioinformatics and human intelligence (Humint). Because four TCF/LEF-binding sites were identified within the 5'-promoter region of human VEGFD gene within AC095351.5 genome sequence, comparative genomics analyses on VEGFD orthologs were further performed. ASB9-ASB11-VEGFD locus at human chromosome Xp22.2 and ASB5-VEGFC locus at human chromosome 4q34 were paralogous regions within the human genome. Human VEGFD mRNA was expressed in lung, small intestine, uterus, breast, neural tissues, and neuroblastoma. Mouse Vegfd mRNA was expressed in kidney, pregnant oviduct, and neural tissues. Chimpanzee VEGFD promoter, cow Vegfd promoter, mouse Vegfd promoter and rat Vegfd promoter were identified within NW_121675.1, AC161065.2, AL732475.6 and AC130036.3 genome sequences, respectively. Three out of four TCF/LEF-binding sites within human VEGFD promoter were conserved in chimpanzee VEGFD promoter, and one in cow Vegfd promoter. TCF/LEF-binding site, not conserved in human VEGFD promoter, occurred in cow, mouse and rat Vegfd promoters. At least five out of six bHLH-binding sites within human VEGFD proximal promoter region were conserved in chimpanzee VEGFD proximal promoter region, while only one in cow Vegfd proximal promoter region. Together these facts indicate that relatively significant promoter evolution occurred among mammalian VEGFD orthologs. Human VEGFD was characterized as a potent target gene of WNT/beta-catenin signaling pathway. VEGFD, implicated in angiogenesis and lymphatic metastasis, is a pharmacogenomics target in the field of oncology. PMID- 16685461 TI - Effect of thioridazine on gap junction intercellular communication in connexin 43 expressing cells. AB - Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 micromol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a "hyperphosphorylated" connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications. PMID- 16685462 TI - Antitumour activity of crocetin in accordance to tumor incidence, antioxidant status, drug metabolizing enzymes and histopathological studies. AB - Lung cancer is the leading cause of cancer related mortality worldwide. Crocetin, saffron plant derivative known to play a role in cancer chemoprevention. In the present study the effects of crocetin was tested against lung cancer-bearing mice in both pre-initiation and post-initiation periods. Healthy male Swiss albino mice (6-8 weeks old) were used throughout the study. Experiment was designed with the treatment regimen of crocetin [20 mg/kg body weight dissolved in dimethyl sulphoxide (DMSO)] for 4 weeks before (pre-initiation) and from 12th week after Benzo(a) pyrene B(a)p (50 mg/kg body weight) induced lung carcinoma(post initiation). The level of lipid peroxidation (LPO) and marker enzymes markedly increased in carcinogen administered animals, which was brought back to near normal by crocetin treatment. The activities of the enzymic antioxidants and glutathione metabolizing enzymes were decreased in B(a)p induced animals and increased upon drug treatment. Crocetin profoundly reverted back the pathological changes observed in cancerous animals. From the results crocetin proves to scavenge free radical and plays an important role in cellular function. Tumor incidence and histopathological studies proves crocetin is a potent antitumour agent. PMID- 16685463 TI - Low ethanol intake prevents salt-induced hypertension in WKY rats. AB - Low alcohol intake in humans lowers the risk of coronary heart disease and may lower blood pressure. In hypertension, insulin resistance with altered glucose metabolism leads to increased formation of aldehydes. We have shown that chronic low alcohol intake decreased systolic blood pressure (SBP) and tissue aldehyde conjugates in spontaneously hypertensive rats and demonstrated a strong link between elevated tissue aldehyde conjugates and hypertension in salt-induced hypertensive Wistar-Kyoto (WKY) rats. This study investigated the antihypertensive effect of chronic low alcohol consumption in high salt-treated WKY rats and its effect on tissue aldehyde conjugates, platelet cytosolic free calcium ([Ca2+]i, and renal vascular changes. Animals, aged 7 weeks, were divided into three groups of six animals each. The control group was given normal salt diet (0.7% NaCl) and regular drinking water; the high salt group was given a high salt diet (8% NaCl) and regular drinking water; the high salt + ethanol group was given a high salt diet and 0.25% ethanol in drinking water. After 10 weeks, SBP, platelet [Ca2+]i, and tissue aldehyde conjugates were significantly higher in rats in the high salt group as compared with controls. Animals on high salt diets also showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidney. Ethanol supplementation prevented the increase in SBP and platelet [Ca2+]i and aldehyde conjugates in liver and aorta. Kidney aldehyde conjugates and renal vascular changes were attenuated. These results suggest that chronic low ethanol intake prevents salt-induced hypertension and attenuates renal vascular changes in WKY rats by preventing an increase in tissue aldehyde conjugates and cytosolic [Ca2+]i. PMID- 16685464 TI - Age at diagnosis and loss of heterozygosity on chromosome 1p and 19q in oligodendroglial tumors. AB - The age distribution and incidence of loss of heterozygosity (LOH) of 1p and 19q was analyzed in 85 oligodendroglial tumors WHO II and III. The peak of tumor manifestation was in the age group of 35 to 55 years. There was no association between age at diagnosis and LOH incidence. We conclude that the prognostic effect of age on survival is not mediated by LOH 1p/19q. PMID- 16685465 TI - Levetiracetam therapy in patients with brain tumour and epilepsy. AB - Epilepsy is a common clinical problem in patients with brain tumours, strongly affecting patients' quality of life. Tumour-related seizures are often difficult to control, and the clinical picture is complicated by frequent interactions between antiepileptic drugs (AEDs) and antineoplastic agents. We studied the safety and efficacy of levetiracetam (LEV), a new AED with a different pharmacological profile from traditional anticonvulsants, in 19 patients (6 females; age range 28-70 years, mean 48 years) with supratentorial gliomas and epilepsy. Seizure types were simple partial in four patients, complex partial in 4, complex partial with secondary generalization in 7, and generalized tonic clonic in 4. LEV was added to the existing AED treatment on account of persisting seizures, and titrated at dosages of 1,000-3,000 mg/day. Patients were seen at the Outpatient's Centre every 1-3 months, and followed-up for 7-50 months (mean 25 months, median 20 months). At the end of the observation period, nine patients were seizure free (seizure free period ranging from 7 to 33 months, mean 16, median 12) and five patients reported an improvement in seizure-frequency from daily to weekly (n=1) or from weekly to monthly (n=3). Seizure frequency was unmodified in four patients and increased (from monthly to weekly) in one. No LEV related adverse effects were observed. LEV plasma concentrations monitored in 12 subjects ranged from 11.9 to 82.1 microg/ml. Our preliminary open data indicate that add-on treatment with LEV in patients with brain tumours is safe and appears to be effective in reducing seizure frequency. Controlled studies on larger populations are warranted to confirm these open observations. PMID- 16685466 TI - Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH dependent dicarbonyl reductase expressed in vascular endothelial tissue. AB - Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components. PMID- 16685467 TI - Contribution of the C-terminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS-1. AB - Chaperonin is a double ring-shaped oligomeric protein complex, which captures a protein in the folding intermediate state and assists its folding in an ATP dependent manner. The chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, is a group II chaperonin and is composed of two distinct subunits, alpha and beta. Although these subunits are highly homologous in sequence, the homo-oligomer of the beta-subunit is more thermostable than that of the alpha-subunit. To identify the region responsible for this difference in thermostability, we constructed domain-exchange mutants. The mutants containing the equatorial domain of the beta-subunit were more resistant to thermal dissociation than the mutants with that of the alpha-subunit. Thermostability of a beta-subunit mutant whose C-terminal 22 residues were replaced with those of the alpha-subunit decreased to the comparable level of that of the alpha-subunit homo-oligomer. These results indicate that the difference in thermostability between alpha- and beta-subunits mainly originates in the C-terminal residues in the equatorial domain, only where they exhibit substantial sequence difference. PMID- 16685468 TI - Update on clinical trials in Dysphagia. AB - Randomized clinical trials (RCTs) are often known as the gold standard in treatment efficacy studies. This article defines the characteristics of RCTs and the factors that investigators must consider in designing clinical trials in dysphagia. Design issues unique to behavioral treatments often used in dysphagia are discussed. Ongoing RCTs in dysphagia are described including studies of (1) the effectiveness of the Shaker exercise versus standardized treatment in patients with severe dysphagia resulting from stroke or treatment for head and neck cancer who have been nonoral for at least three months; (2) the comparative effects of nectar- and honey-thickened liquids versus chin tuck posture and in patients with dementia or Parkinson's disease with or without dementia who aspirate on thin liquids; and (3) the comparative effects of muscle exercise versus sensory postural therapy for dysphagia resulting from treatment for head and neck cancer. Issues in generalizing from the results of clinical trials are also described. PMID- 16685469 TI - Measures of tongue function related to normal swallowing. AB - The availability of objective measures of tongue function presents a possible supplement to the clinical dysphagia evaluation. The purpose of this study was to improve our understanding of normal tongue physiology during swallowing and maximum isometric tasks, establish a preliminary database of tongue function variables, and determine if differences existed among the variables as a function of age, gender, or varied bolus consistency. Ninety subjects, divided into age and gender groups, participated in tasks that determined maximum isometric tongue pressure, mean tongue pressure during swallowing, and percentage of maximum isometric pressure used during swallowing. Descriptive statistics, correlations, and analyses of variance were computed to analyze the data. Results indicated that males had significantly higher maximum isometric pressures than females, and the youngest group had significantly higher maximum pressures than the oldest group. Mean swallowing pressures and percentage of maximum isometric pressures used during swallowing differed as a function of bolus type but did not differ as a function of age or gender. In addition, maximum isometric pressures were correlated with mean swallowing pressures, and mean swallowing pressures and percentage of maximum isometric pressures used during swallowing were correlated between consistencies. PMID- 16685473 TI - Suicide in Austrian children and young adolescents aged 14 and younger. AB - INTRODUCTION: Research on suicide in childhood and early adolescence is sparse. We investigated suicide cases of children and young adolescents in terms of prevalence, gender differences, suicide methods and monthly distribution during a period of 32 years. METHODS: Registered suicides aged 14 or younger occurring between 1970 and 2001 (n = 275) in Austria were studied. RESULTS: The mean suicide rate for 10 to 14-year-olds was 1.4 per 100,000 with a male-female ratio of 3.1:1. The total child and young adolescent suicide rate and boys' suicide rates decreased over the study period. However, there was no significant fluctuation in girls' suicide rates. Hanging was the predominant suicide method in both genders. Use of this method decreased steadily over the study period, whereas the percentage of suicides by other methods, i.e., jumping and firearms suicides increased. Greater number of suicides in children and young adolescents was observed during the months of April/May and October/November. CONCLUSION: Suicide rates of children and young adolescents in Austria are on the decrease, in accordance with a reported decrease in the general suicide rate in Austria. PMID- 16685474 TI - Ethnic differences in parental detection of externalizing disorders. AB - BACKGROUND: Previous research has reported lower treatment rates for externalizing disorders among non-Western children as compared to Western children. Ethnic differences in parental detection may be an explanation for this discrepancy. AIMS: In a cross-sectional study among the four largest ethnic groups in the Netherlands, namely Dutch, Moroccan, Turkish and Surinamese, we examined the influence of ethnicity on parental detection of behavioural disorders. METHOD: A total of 270 children (aged 6-10 years) and their parents were interviewed regarding psychiatric disorders and socio-demographic data. Sensitivity and specificity were calculated by using standard definitions, with adjustment for parental educational level. RESULTS: Sensitivity to detect any externalizing disorder and ADHD in particular was significantly lower among Moroccan and Surinamese parents when compared to Dutch parents. Sensitivity to detect ADHD tended to be lower among Turkish parents. Specificity to detect any externalizing disorder was higher among Moroccan and Turkish parents. Specificity to detect ADHD was higher among Moroccan parents and tended to be higher among Turkish parents. CONCLUSIONS: The detection rate of externalizing disorders is markedly lower among non-Dutch parents than among Dutch parents. This finding emphasizes the importance of taking parents' cultural context into account when appraising their report on possible externalizing disorders in their children. PMID- 16685475 TI - Reactive attachment disorder--a theoretical model beyond attachment. AB - Despite its importance in public health, reactive attachment disorder (RAD) is an under-researched and little used clinical category. Abnormalities of social relatedness have long been documented in children who have been abused, neglected or institutionalised, but there have been more recent efforts to define these behaviours within the psychiatric nosology. There has been an implicit assumption that the central deficit in RAD is in the attachment system, but this has caused controversy and may have blocked research. We propose that RAD is better construed within the framework of intersubjectivity, which has a central role in the development of core brain and social functions and may also have had an important role in the evolution of a key human characteristic-complex social functioning. This broader framework may potentially explain apparently diverse symptoms such as indiscriminate friendliness and negative or unpredictable reunion responses. Finally, we suggest that a change of name may be useful in progressing the field, but accept that this may be difficult until there is better agreement in the clinical and scientific communities about the core features and aetiology of this disorder. PMID- 16685478 TI - Eating disorders and body image in Spanish and Mexican female adolescents. AB - OBJECTIVE: To compare the prevalence of eating disorders (ED), socio-cultural risk factors, and body image characteristics in two populations of female adolescents, one Mexican and one Spanish, from similar socio-economic backgrounds. METHOD: A total of 467 Spanish and 329 Mexican girls aged from 11-12 to 17-18 years were assessed using the Eating Attitude Test-26 (EAT-26), the CETCA (Eating Disorders Assessment Questionnaire), the CIMEC (Questionnaire on Influences on Body Shape Model), and a survey of socio-demographic and risk factors produced for this study. RESULTS: One out of four subjects showed a significant risk of an eating disorder and 6-7% probably already had one. No significant differences were found between the two samples. Significant differences were found in risk behaviors: more Spanish girls reported body dissatisfaction and binging; more Mexican girls had a history of psychiatric and psychological treatment, pressure from parents and friends to lose weight, dieting, physical activity and vomiting to lose weight, and a history of greater weight loss. In both the groups around 50% of subjects wanted to increase the size of their breasts. However, significantly more Mexican girls desired thinner arms and narrower shoulders and back, and more Spanish girls wanted thinner hips, buttocks, and legs, parts of the body that many Mexicans wanted to increase. CONCLUSIONS: Risk behaviors and the ideal body models of these Spanish and Mexican adolescent girls varied significantly, indicating major socio-cultural differences. However, the prevalence of ED was similar. Further research should aim to clarify whether the similarities found between this Spanish sample and a Mexican sample taken from an upper-middle class urban environment, a minority that is unrepresentative of the general population, are also observed in samples from other sectors of Mexican society. PMID- 16685476 TI - Social phobia, anxiety, oppositional behavior, social skills, and self-concept in children with specific selective mutism, generalized selective mutism, and community controls. AB - We compared social phobia, anxiety, oppositional behavior, social skills, and self-concept in three groups: (1) 28 children with specific mutism (who did not speak to teachers but were more likely to speak to parents and peers at home and school); (2) 30 children with generalized mutism (whose speaking was restricted primarily to their homes); and (3) 52 community controls. Children with generalized mutism evidenced higher anxiety at school, and more separation anxiety, OCD, and depressive symptoms at home. Parents and teachers reported that the social phobia and anxiety scores of children in both the specific and generalized mutism subgroups were higher than controls. Children in both the specific and generalized mutism groups evidenced greater deficits in verbal and nonverbal social skills at home and school than controls. Teachers and parents did not report differences in nonverbal measures of social cooperation and conflict resolution and we found no evidence that selective mutism was linked to an increase in externalizing problems such as oppositional behavior or ADHD. Although children with specific mutism speak in a wider range of situations and appear less anxious to their teachers than children with generalized mutism, significant socially phobic behavior and social skills deficits are present in both groups. PMID- 16685479 TI - Do community treatment orders for mental illness reduce readmission to hospital? An epidemiological study. AB - BACKGROUND: It has been suggested that community treatment orders (CTOs) will prevent readmission to hospital, but controlled studies have been inconclusive. We aimed to test the hypothesis that hospital discharges made subject to CTOs are associated with a reduced risk of readmission. The use of such a measure is likely to change after its introduction as clinicians acquire familiarity with it, and we also tested the hypothesis that the characteristics of patients subject to CTOs changed over time in the first decade of their use in Victoria, Australia. METHOD: A database from Victoria, Australia (total population 4.8 million) was used. Cox proportional hazard models compared the hazard ratios of readmission to hospital before the end of the study period (1992-2000) for 16,216 discharges subject to a CTO and 112,211 not subject to a CTO. RESULTS: Community treatment orders used on discharge from a first admission to hospital were associated with a higher risk of readmission, but CTOs following subsequent admissions were associated with lower readmission risk. The risk also declined over the study period. CONCLUSIONS: The effect of using a CTO depends on the patient's history. At a population level their introduction may not reduce readmission to hospital. Their impact may change over time. DECLARATION OF INTEREST: None. PMID- 16685481 TI - [Computer-based health care research in the competence network inflammatory bowel disease. Handling of bowel diseases in general practice]. AB - BACKGROUND: Consultations due to most different bowel diseases and abdominal symptoms are common in primary care. However, little is known about the concrete situation in the general practitioner's office so far. In this study, practitioners involved in the actual treatment of patients with bowel diseases document their medical strategies by the use of an internet-based data entry system. Hence, with the support of an internet-based data collection tool, health care research has started to break new ground. METHODS: Following the programming of a digital data collection system and the installation of a nationwide research network of general practitioners interested in this matter, participating physicians communicated anonymised data on medical care of patients with various abdominal problems and intestinal/bowel diseases to the principal study investigators for 12 months, thereby using a computer-based data entry system (via internet). RESULTS: Within the 12 months of observation, 94 general practitioners documented 1,584 patient contacts by using the new data collection system. Accordingly, the most frequent reasons for consultation were abdominal pain (39%) and a change in stool habits (28%), each over a period of > 21 days. Women reported abdominal pain more often than men. Furthermore, the willingness to cooperate with specialists (hospitalization, referrals) was high among participating physicians, in reference to abdominal problems, thereby not demonstrating gender specificity. In the majority of cases, computer-assisted data entry took place outside regular consultation hours, and physicians frequently documented more than one patient within one session. CONCLUSION: High numbers of hospitalizations and specialist referrals, as shown in this study, points toward a high intensity of medical attendance necessary for patients with unspecific symptoms of the bowel or related abdominal problems. Computer-assisted data entry systems, however, represent a challenging yet still realizable way of data collection. This procedure should be brought forward for future projects in health care research. PMID- 16685482 TI - [Striking a new path in medical education. CAMPUS, an interactive, case-based training system]. AB - BACKGROUND AND PURPOSE: Computer-assisted teaching and learning tools offer new opportunities for improving education and training of medical professionals. CAMPUS represents a software for computer-based, problem-oriented learning. It is a case-based training system which provides the patient's history within a highly realistic, multimedia format. Thus, the interactive design is expected to challenge, test and improve the medical knowledge and the diagnostic skills of the students. The objective of the present study was to introduce CAMPUS as a computer-based learning tool and to present preliminary results with regard to acceptance and user-friendliness. METHODS: CAMPUS was evaluated by 52 students regarding quality and experienced learning success. A tutorial was conducted within separate, small-numbered groups of students, each working on one learning case. The virtual case started with a summary of the patient's leading symptoms. The students independently took the patient history and carried out the physical examination. Then, they were asked to suggest differential diagnoses, refer the patient to appropriate diagnostic examinations and were encouraged to choose adequate therapeutic strategies. Subsequently, the quality of CAMPUS and the subjective learning success were evaluated with a standardized questionnaire. RESULTS: Nearly all students described the user interface as visually attractive (51/52) and clearly structured (52/52). In particular, the students found the use of videos to be advantageous. A marked learning success was described by most students (46/52) and all students considered learning with CAMPUS to be effective. CONCLUSION: CAMPUS offers an innovative training program to improve medical education and to enhance conventional teaching methods efficiently. PMID- 16685483 TI - [Psychiatric disorders following myocardial infarction]. AB - BACKGROUND: The number of patients who survive acute myocardial infarction has increased during recent decades. In addition, demographic development results in a rising incidence of cardiovascular diseases. Based on these facts, also the significance of psychiatric disorders is growing that may occur after myocardial infarction, such as depression, posttraumatic stress and anxiety disorders. CLINICAL SIGNIFICANCE: Physicians are faced with the challenge to identify these clinical entities, that show a syndromal overlap with somatic complaints after myocardial infarction. After differentiation prompt start of adequate psychiatric psychotherapeutic interventions is of relevance, not only regarding the patient's quality of life, but also in terms of cardiovascular prognosis. Indeed, depressive and anxiety disorders are known to be associated with a poor compliance as for rehabilitation and secondary prevention of cardiovascular disorders. Moreover, some studies suggest depression to be an independent risk factor of coronary heart disease. CONCLUSION: Consequently, early recognition and treatment, most often primarily in the hands of internists and cardiologists, are of enormous importance for the course and prognosis of the psychiatric disorder but also of cardiovascular disease. PMID- 16685484 TI - [Virus persistence in hepatitis C: lifelong infection despite therapy?]. AB - BACKGROUND: Chronic hepatitis C infection still represents a clinical and scientific challenge. Exciting progress has been achieved by the use of combined therapy regimens with pegylated interferon and ribavirin resulting in sustained virological response rates of 60-80%, depending on the genotype. VIRUS PERSISTENCE DESPITE SUCCESSFUL THERAPY: Despite favorable longterm data with regard to viremia, liver histology and serum liver enzymes in treated patients who comply with the criteria of sustained virological response, a complete elimination of the hepatitis C virus (HCV) is rarely observed. Besides liver tissue, peripheral blood mononuclear cells (PBMCs) could be proven as locations of HCV persistence. It is assumed that there are further extrahepatic compartments in the host organism in which virus particles capable of replication remain, in spite of a seemingly successful therapy. OCCULT HEPATITIS C: The problem of the existence of small amounts of potentially replicative viruses becomes apparent even in occult hepatitis C, a constellation in which anti-HCV antibodies are missing, but HCV RNA in liver tissue and mostly also in PBMCs exist. CONCLUSION: The precise significance of the HCV persistence in the host organism is still inconclusive; according to first research results, however, it can lead to a deterioration of the liver histology. At present, it is also unclear if patients with occult hepatitis C as well as with evidence of HCV RNA in the liver and/or extrahepatic compartments after seemingly successful antiviral treatment are to be regarded as infectious. PMID- 16685485 TI - [Costs of the treatment of hyperglycemia in patients with diabetes mellitus. The impact of age, type of therapy and complications: results of the German CoDiM study]. AB - BACKGROUND AND PURPOSE: Diabetes mellitus is a chronic progressive disease with multiple complications and due to its high prevalence represents a true economic challenge to the health care system. However, the specific cost structure is not known. It was the aim of this study to analyze the costs of antihyperglycemic treatment in relation to age, treatment regimen and presence of complications. MATERIAL AND METHODS: This study is part of the CoDiM study. The analysis was based on the data of a random sample (n = 306,736) of all members of the "Allgemeine Ortskrankenkasse Hessen" (AOK Hessen, Local Statutory Health Insurance of Hessen) and data provided by the "Kassenarztliche Vereinigung Hessen" (KV Hessen, Association of Statutory Health Insurance Physicians in Hessen). Patients with diabetes were identified by the ICD-10 classification and prescriptions of blood glucose-lowering drugs. Costs of antihyperglycemic therapy were calculated per patient and year computing procedures and antidiabetic drugs in outpatient care as well as hospital costs. RESULTS: The mean costs of treatment of hyperglycemia were 542 Euros per patient in 2001 and represented 10% of total costs. The major parts in this segment were expenses for hypoglycemic drugs (46%) and for blood glucose self-monitoring (21%). The costs were highest in the groups treated by either insulin alone or a combination of insulin and oral hypoglycemic agents (1,366 Euros and 1,479 Euros, respectively) as compared to patients on oral hypoglycemic agents alone or on diet (296 Euros and 59 Euros, respectively). Stripes for blood glucose self-monitoring were almost exclusively prescribed to insulintreated patients and caused average costs in this group of 446 Euros. By contrast, costs for self-monitoring were almost negligible in the two other groups. A subdivision of costs according to complications revealed that patients with microangiopathy (retinopathy, nephropathy) and foot complications caused twice as much expenses as patients with macroangiopathy (vascular diseases) or without complications (779 Euros vs. 370 Euros and 401 Euros, respectively). CONCLUSION: The expenses for the antihyperglycemic treatment of patients with diabetes represent only a minority of total per capita costs and these were slightly more than half due to insulin treatment and blood glucose self-monitoring. PMID- 16685486 TI - [Magnetic resonance tomography in patients with cardiomyopathies]. PMID- 16685487 TI - [Gastrointestinal hemorrhage]. PMID- 16685488 TI - [Mesenchymal stem cells from bone marrow. Phenotype, aspects of biology, and clinical perspectives]. AB - BACKGROUND: Since their initial description by Friedenstein et al. 30 years ago, mesenchymal stem cells have been characterized as a cell population of the bone marrow, which is easily accessible and readily manageable for in vitro manipulation. RESULTS: Cell biology studies have delineated the phenotype of mesenchymal stem cells, optimized the conditions for in vitro expansion, and documented their differentiation capacity along mesodermal lineages. First clinical applications as an adjunct to stem cell transplantation regimens have shown some potential in osteogenic regeneration as well as immunosuppressive effects after allogeneic stem cell transplantation. Animal studies have further shown, that mesenchymal stem cells may serve as vehicles for the transfer of genetic material. Preclinical studies remain, however, to answer numerous questions with regard to populational heterogeneity and the physiological roles of this cell compartment. CONCLUSION: Further preclinical studies as well as clinical cell therapy studies are essential to fully exploit the potential of mesenchymal stem cells in regenerative medicine, cellular immunotherapy, and gene therapy. PMID- 16685489 TI - [Clinical research within the German competence net "acute and chronic leukemias"]. AB - BACKGROUND: Leukemias are a challenge and a cost factor to society because of their frequency in all age groups. They also serve as a model for a variety of diseases and possess exemplary relevance for basic research and patient care. Leukemia research and therapy have achieved high standards and even a leading position in Germany with regard to clinical trials, standardization of diagnostics and molecular studies of prognostic factors, signal transduction and gene expression. Progress is hampered, however, by fragmentation of leukemia trial groups, diagnostic approaches and treatment research activities. GOALS: A network was therefore created to integrate the leading leukemia trial groups on chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphatic leukemia (ALL), myelodysplastic syndromes (MDS) and chronic myeloproliferative diseases (CMPD) and their interdisciplinary partners (diagnostics, treatment research, biometry) in cooperation with basic research and pharmaceutical industry to foster advancements in leukemiarelated research and health care through clinical trials, promotion of translational research, introduction of standards for diagnostics and therapy, and development of evidence-based guidelines. ACHIEVEMENTS: Achievements include establishment of central information, communication and management structures, creation of an AML intergroup comprising five study groups, formation of an MDS study group and establishment of platforms for diagnostics, genomics and proteomics, and medical informatics. Exchange of scientific progress is mediated by intra- and internet, biannual newsletters, regular project group meetings, and annual network symposia. PERSPECTIVES: On the basis of experience with the Competence Network "Acute and chronic leukemias" with its management, communication and information structures, the "European LeukemiaNet" (ELN) has been established within the 6th Framework Program of the European Union. The ELN integrates 78 leading leukemia trial groups (AML, ALL, CML, CLL, MDS, and CMPD), their 83 interdisciplinary partner groups (diagnostics, treatment research, registry, guidelines), industry and SMEs (small- and medium-sized enterprises) across Europe to form a cooperative network for advancements in leukemia-related research and health care. PMID- 16685490 TI - [Chloroquine-induced cutaneous hyperpigmentation]. PMID- 16685491 TI - [Limits in feasibility of restoring health: against the totalitarian tendency in modern health care]. PMID- 16685492 TI - [Visualized medicine. Macroscopy, molecular anatomy and medical art]. AB - Quite frequently clinicians avail themselves of works of well known artists in order to illustrate certain diseases. In contrast, pathologists find it harder to use art to demonstrate morphology. My approach to compensate for this is twofold: on the one hand, with 8 mostly three dimensional wooden objects with either mirror or aluminium elements, on the other with 449 collages consisting of pictures exclusively with a medical background (histology, radiology, nuclear medicine) or showing a combination of histological or cytological photos with pictures of flowers, trees or landscapes. My experience is that this technique arouses interest in complex medical issues even with medically unqualified individuals. PMID- 16685494 TI - Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery. AB - Clostridial acetone-butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis. PMID- 16685495 TI - Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405. AB - We have investigated hydrogen (H2) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H2-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H2, carbon dioxide (CO2), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H2/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H2, CO2, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H2-production continued even in stationary phase and H2/CO2 ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H2 production rate of 14.6 mmol g dry cell( 1) h(-1) was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product. PMID- 16685496 TI - [Fracture diagnosis in osteoporosis]. AB - As life expectancy rises the prevalence of osteoporosis also increases, which represents a growing burden for the populace and the healthcare system. Vertebral fractures are the most frequent type of all osteoporotic fractures. Since they can be diagnosed by radiology, these examinations are particularly important for devising suitable treatment strategies. The goal of activities undertaken by osteoporosis organizations is to put across to radiologists the importance of accurate evaluation of these fractures. Identification of osteoporotic vertebral fractures is however problematic since the transition from healthy vertebral bodies to those deformed by osteoporosis constitutes a continuum and it is often difficult in deformed vertebral bodies to distinguish between an osteoporotic fracture and a non-osteoporotic deformity. This overview attempts to heighten awareness of the significant role the radiologist plays in the diagnostic work-up of osteoporosis and to provide diagnostic aids for assessing osteoporotic vertebral fractures including differential diagnoses. PMID- 16685502 TI - Effects of statins on the adipocyte maturation and expression of glucose transporter 4 (SLC2A4): implications in glycaemic control. AB - AIMS/HYPOTHESIS: Hyperlipidaemia often occurs in patients with type 2 diabetes mellitus. Though HMG-CoA reductase inhibitors (statins) are widely used for controlling hypercholesterolemia, atorvastatin has also been reported to have an adverse effect on glucose metabolism. Based on these findings, the aim of this study was to investigate the effects of statins on adipocytes, which play pivotal roles in glucose metabolism. METHODS: In 3T3-L1 cells, effects of statins on adipocyte maturation were determined morphologically. Protein and mRNA levels of SLC2A4 and adipocyte marker proteins were determined by immunoblotting and RT PCR, respectively. Type 2 diabetic NSY mice were treated with atorvastatin for 15 weeks, followed by glucose and insulin tolerance tests and examination of SLC2A4 expression in white adipose tissue (WAT). Seventy-eight Japanese subjects with type 2 diabetes and hypercholesterolaemia were treated with atorvastatin (10 mg/day), and its effects on lipid and glycaemic profiles were measured 12 weeks after treatment initiation. RESULTS: Treatment with atorvastatin inhibited adipocyte maturation, SLC2A4 and C/EBPalpha expressions and insulin action in 3T3 L1 cells. Atorvastatin also attenuated SLC2A4 and C/EBPalpha expressions in differentiated 3T3-L1 adipocytes. These effects were reversed by L. mevalonate or geranylgeranyl pyrophosphate. In NSY mice, atorvastatin accelerated glucose intolerance as a result of insulin resistance and decreased SLC2A4 expression in WAT. In addition to improving hyperlipidaemia, atorvastatin treatment significantly increased HbA(1c) but not fasting glucose levels in diabetic patients, and this effect was greater in the non-obese subgroup. CONCLUSIONS/INTERPRETATION: These results demonstrate that atorvastatin attenuates adipocyte maturation and SLC2A4 expression by inhibiting isoprenoid biosynthesis, and impairs glucose tolerance. These actions of atorvastatin could potentially affect the control of type 2 diabetes. PMID- 16685503 TI - Increased serum resistin in elite endurance athletes with high insulin sensitivity. AB - AIMS/HYPOTHESIS: Resistin is an adipokine associated with obesity and type 2 diabetes in animal models, but in humans its role remains uncertain. This study was undertaken to test whether serum resistin is related to insulin resistance and markers of low-grade inflammation in elite athletes taken as a model of extreme insulin sensitivity. SUBJECTS MATERIALS AND METHODS: In 23 elite athletes (sprinters, middle-distance and marathon runners) and in 72 sedentary men including lean and obese individuals with NGT, and obese individuals with IGT or new-onset type 2 diabetes, we assessed insulin sensitivity using a whole-body insulin-sensitivity index (WBISI) derived from a 3-h OGTT; energy homeostasis was also assessed by means of indirect calorimetry, along with circulating adipokines and low-grade pro-inflammatory cyto-chemokines. RESULTS: Professional athletes had increased WBISIs (p<0.001) and lipid oxidation (p<0.03); they also showed higher serum resistin concentrations (p<0.001), although the pro-inflammatory chemokines were not increased in comparison with the other study groups. Resistin was independently associated only with fasting plasma NEFA. Increased resistin was detected in the middle-distance and marathon runners, but not in the sprinters when compared with the lean, young, sedentary individuals. CONCLUSIONS/INTERPRETATION: Serum resistin concentration is increased in elite athletes, providing evidence against the notion that resistin levels reflect insulin resistance in humans, as seen in animal studies. Increased resistin was observed in aerobic-endurance, but not sustained-power athletes and this feature appeared to be independently associated with parameters of fatty acid metabolism. PMID- 16685504 TI - Scientific papers presented at the European Congress of Radiology: a two-year comparison. AB - The purpose of this report was to determine the rate at which abstracts orally presented at the European Congress of Radiology (ECR) 2001 were published in 2001 2005 Medline-indexed journals and to compare publication rates and factors with presentations at the ECR in two different periods (2001 and 2000). Absolute and relative publication rates (APR, RPR) and different publication-related factors were analysed. From 991 abstracts originating from 52 countries, 449 articles (APR 45%) were subsequently published in 125 journals, most frequently in European Radiology (n=79, 18%). Country of origin statistically (p<0.0001) influences subsequent publication of the abstract, with Germany having the highest number of presentations (n=300) and derived articles (n=175, RPR 58%) whereas Sweden had the highest RPR (82%). Interventional and physics studies had the highest RPR (59% and 58%, respectively). The ECR meeting has a very high and stable APR (ECR 2001: 45% vs ECR 2000: 47%), and the journal European Radiology had the larger number of related publications (18% RPR following ECR 2001 compared with 14% from ECR 2000). Germany had the highest number of presentations and publications for both meetings. The highest RPR for ECR 2001 was found in interventional and physics studies whereas chest and cardiac studies had the highest RPR for ECR 2000. PMID- 16685505 TI - Quantifying peri-meniscal synovitis and its relationship to meniscal pathology in osteoarthritis of the knee. AB - The objectives of this study were to validate a semiquantitative scoring system for estimating perimeniscal synovitis in osteoarthritic (OA) knees and to examine the relationship between the extent of synovitis and the degree of meniscal pathology using gadolinium-enhanced magnetic resonance imaging (MRI). Forty-three subjects with clinically diagnosed OA knee were assessed for peri-meniscal synovitis using gadolinium-enhanced MRI. Quantitative measurements of synovitis were made by summing areas in consecutive slices within generated regions of interest, and the synovitis was also scored semi-quantitatively using a 0-3 scale. Meniscal pathology (extrusion, degeneration and tearing) was also scored semiquantitatively. Establishment of a correlative relationship was undertaken using Spearman's rho (rho). A total of 86 sites were assessed. The semi quantitative synovitis score correlated well with the quantitative synovitis score (rho>0.9). A moderate association between medial meniscal extrusion and synovitis was demonstrated (rho=0.762, P<0.000), although this association was not as strong in the lateral compartment (rho=0.524, P<0..000). The results suggest the semiquantitative scoring system is valid for assessing perimeniscal synovitis. The relationship between meniscal pathology and adjacent synovitis requires further study. PMID- 16685506 TI - Postinterventional percutaneous closure of femoral artery access sites using the Clo-Sur PAD device: initial findings. AB - The purpose of this study was to evaluate a percutaneous hemostatic device in patients to achieve immediate hemostasis at the vascular access site as well as early ambulation after vascular interventional procedures. In a randomized trial, a hemostatic device (Clo-Sur PAD, Medtronic AVE, Inc., Santa Rosa, CA, USA; n=60) was compared with manual compression (n=60) in patients after endoluminal intervention through an inguinal access (sheath sizes: 5-7 French). Device safety was evaluated by assessing complications within 24 h and 14 days. System efficacy was measured by the percentage of achieved immediate hemostasis and early ambulation. Device application was possible in 57 cases (95.0%), with 93.3% (56/60) of the patients rising 2 h after application. Hemostasis time was 10.15+/ 1.96 min (control group: 16.20+/-1.79 min), with a pressure bandage time of 3.47+/-5.53 h (control group: 13.8+/-4.32 h). Ambulation was possible after 2.13+/-0.50 h (control group: 8.57+/-3.47 h). Complications encountered were access-site bleeding with hematoma (device: n=3; control: n=9). All complications were managed conventionally without blood transfusion or surgical intervention. The system is an easy to use device permitting early ambulation without additional pressure bandaging in the majority of patients. Preliminary data show that hemostasis does not depend on the level of anticoagulation. PMID- 16685508 TI - Dynamical structure of center-of-pressure trajectories in patients recovering from stroke. AB - In a recent study, De Haart et al. (Arch Phys Med Rehabil 85:886-895, 2004) investigated the recovery of balance in stroke patients using traditional analyses of center-of-pressure (COP) trajectories to assess the effects of health status, rehabilitation, and task conditions like standing with eyes open or closed and standing while performing a cognitive dual task. To unravel the underlying control processes, we reanalyzed these data in terms of stochastic dynamics using more advanced analyses. Dimensionality, local stability, regularity, and scaling behavior of COP trajectories were determined and compared with shuffled and phase-randomized surrogate data. The presence of long-range correlations discarded the possibility that the COP trajectories were purely random. Compared to the healthy controls, the COP trajectories of the stroke patients were characterized by increased dimensionality and instability, but greater regularity in the frontal plane. These findings were taken to imply that the stroke patients actively (i.e., cognitively) coped with the stroke-induced impairment of posture, as reflected in the increased regularity and decreased local stability, by recruiting additional control processes (i.e., more degrees of freedom) and/or by tightening the present control structure while releasing non-essential degrees of freedom from postural control. In the course of rehabilitation, dimensionality stayed fairly constant, whereas local stability increased and regularity decreased. The progressively less regular COP trajectories were interpreted to indicate a reduction of cognitive involvement in postural control as recovery from stroke progressed. Consistent with this interpretation, the dual task condition resulted in less regular COP trajectories of greater dimensionality, reflecting a task-related decrease of active, cognitive contributions to postural control. In comparison with conventional posturography, our results show a clear surplus value of dynamical measures in studying postural control. PMID- 16685509 TI - The growth-mortality tradeoff: evidence from anuran larvae and consequences for species distributions. AB - A tradeoff affecting the ability to grow under high versus low resource levels has been commonly hypothesized to influence species distributions across resource gradients in a wide variety of taxa. This influence is dependent on individual growth being proportional to traits that affect demographic processes such as mortality. However, data on how individual growth scales with demographic performance are rare. We conducted a mesocosm experiment, and re-analyzed data from a similarly designed field experiment, to examine the relationship between growth and mortality in two tadpole species that segregate across a resource gradient. Overall, environmental conditions leading to faster growth also lead to lower mortality rates. However, species differed in this relationship. Leopard frogs achieved faster growth than wood frogs, but their absolute mortality was greater and increased steeply as growth decreased. Conversely, absolute mortality of wood frogs was lower and less strongly dependent on growth. These interspecific differences suggest a second tradeoff, that between maximizing growth rates or minimizing mortality, with potentially important demographic consequences. Leopard frogs grow faster than wood frogs in productive ponds, but are excluded from unproductive ponds dominated by wood frogs due to accelerating mortality rates with declining realized growth. A review of the literature suggests that in diverse taxa, including plants, microcrustaceans and drosophilids, patterns in mortality are consistent with this tradeoff indicating that the mechanism we demonstrate could be a link between individual performance and demographic rates influencing species distributions in other systems. PMID- 16685510 TI - Invasion of an exotic forb impacts reproductive success and site fidelity of a migratory songbird. AB - Although exotic plant invasions threaten natural systems worldwide, we know little about the specific ecological impacts of invaders, including the magnitude of effects and underlying mechanisms. Exotic plants are likely to impact higher trophic levels when they overrun native plant communities, affecting habitat quality for breeding songbirds by altering food availability and/or nest predation levels. We studied chipping sparrows (Spizella passerina) breeding in savannas that were either dominated by native vegetation or invaded by spotted knapweed (Centaurea maculosa), an exotic forb that substantially reduces diversity and abundance of native herbaceous plant species. Chipping sparrows primarily nest in trees but forage on the ground, consuming seeds and arthropods. We found that predation rates did not differ between nests at knapweed and native sites. However, initiation of first nests was delayed at knapweed versus native sites, an effect frequently associated with low food availability. Our seasonal fecundity model indicated that breeding delays could translate to diminished fecundity, including dramatic declines in the incidence of double brooding. Site fidelity of breeding adults was also substantially reduced in knapweed compared to native habitats, as measured by return rates and shifts in territory locations between years. Declines in reproductive success and site fidelity were greater for yearling versus older birds, and knapweed invasion appeared to exacerbate differences between age classes. In addition, grasshoppers, which represent an important prey resource, were substantially reduced in knapweed versus native habitats. Our results strongly suggest that knapweed invasion can impact chipping sparrow populations by reducing food availability. Food chain effects may be an important mechanism by which strong plant invaders impact songbirds and other consumers. PMID- 16685511 TI - Persistence of host and parasite populations subject to experimental size selective removal. AB - Predators have the potential to limit the spread of pathogens not only by selecting infected prey but also by shaping prey demographics. We tested this idea with an epidemiological experiment in which we simulated variable levels of size-selective predation on zooplankton hosts and monitored the persistence of host and parasite populations. In the absence of simulated predation, the virulent protozoan Caullerya mesnili frequently drove its host Daphnia galeata to extinction. Uninfected control populations showed lower extinction rates and higher average densities than infected populations in the absence of simulated predation (all of the latter went extinct or remained infected). With a weak removal rate of the largest hosts, the proportion of populations in which the parasite drove the host to extinction decreased, while the number of populations in which the host persisted and the parasite went extinct increased. Host parasite coexistence was also observed in some cases. With intermediate levels of removal, most of the parasite populations went extinct, while the host populations persisted. With an even higher removal rate, Daphnia were driven to extinction as well. Thus, variation in one factor, size-selective mortality, resulted in four different patterns of population dynamics. Our results highlight the potential role of predation in shaping the epidemiology and community structure of host-parasite systems. PMID- 16685512 TI - Production of two novel monoclonal antibodies that distinguish mouse lymphatic and blood vascular endothelial cells. AB - We produced two novel rat monoclonal antibodies (LA102 and LA5) to identify mouse lymphatic vessels and blood vessels, respectively. We characterized the two antibodies as to the morphological and functional specificities of endothelial cells of both types of vessels. The antibodies were produced by a rapid differential immunization of DA rats with collagenase- and neuraminidase-treated mouse lymphangioma tissues. LA102 specifically reacted with mouse lymphatic vessels except the thoracic duct and the marginal sinus of lymph nodes, but not with any blood vessels. In contrast, LA5 reacted with most mouse blood vessels with a few exceptions, but not with lymphatics. LA102 recognized a protein of 25 27 kDa, whereas LA5 recognized a molecule of 12-13 kDa. Neither antibody recognized any currently identified lymphatic or vascular endothelial cell antigens. Immunoelectron microscopy revealed that the antigens recognized by LA102 and LA5 were localized on both luminal and abluminal endothelial cell membranes of each vessel type. Interestingly, LA102 immunoreactivity was strongly expressed on pinocytic or transport vesicle membrane in the cytoplasm of lymphatic endothelium. Besides endothelial cells, both antibodies also recognized some types of lymphoid cells. Since, the LA102 antigen molecule is expressed on some lymphoid cells, it may play important roles in the migration of lymphoid cells and in some transport mechanisms through lymphatic endothelial cells. PMID- 16685513 TI - Stratification of medulloblastoma on the basis of histopathological grading. AB - Medulloblastoma (WHO grade IV) is an embryonal tumour of the cerebellum and the most common malignant central nervous system tumour in children. Despite significant advances in treatment, 5-year survival rates are still less than 70%, suggesting the presence of subgroups with different response to radio/chemotherapy. In the present study, we re-evaluated a series of 347 medulloblastomas from the SIOP II clinical trial of the International Society of Paediatric Oncology to identify features predictive of clinical outcome. Relapse free survival for medulloblastomas with severe anaplasia [5-year rate: S(60)=49.5%], was significantly shorter than for tumours with moderate or mild anaplasia S(60)=65.4%; P=0.001). The difference between both groups was even larger when the presence or absence of extensive apoptosis was included (46.5 vs. 66.7%; P=0.0216). Other histological features including nodularity, necrosis, vascular proliferation and the presence of beta-catenin mutations (7% of cases) were not predictive for relapse free survival. These findings indicate that degree of anaplasia is the most significant histologic feature predictive of the survival of medulloblastoma patients. PMID- 16685517 TI - Affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for SALDI MS analysis of peptides and proteins. AB - Silane-immobilized magnetic iron oxide particles were used as the assisting material in surface-assisted laser desorption/ionization (SALDI) mass spectrometric analysis. This approach can be used to analyze small proteins and peptides. The upper detectable mass range is approximately 16 kDa. The detection limit for peptides is about 20 fmol. Silanized iron oxide particles with negatively charged functionalities can also be used as the affinity probes to selectively trap oppositely charged species from sample solutions by adjusting the pH of the solution. A tryptic digest product of cytochrome C at a concentration as low as 10 nM can be enriched by the particles and directly analyzed by iron oxide SALDI MS without the need for elution steps. Affinity based mass spectrometry using the bifunctional silanized magnetic iron oxide particles as the SALDI matrix and concentrating probe is demonstrated in this study. PMID- 16685518 TI - Preparation and characterization of methacrylate-based semi-micro monoliths for high-throughput bioanalysis. AB - Hexyl methacrylate (HMA)-based monolithic semi-micro columns were prepared by in situ polymerization within the confines of 1.02-mm-i.d. silicosteel tubing for reversed-phase and/or precipitation-redissolution liquid chromatography. Practically useful monolithic columns with adequate separation efficiency, high permeability, and good mechanical strength were successfully obtained using a polymerization mixture comprising 24% hexyl methacrylate (HMA), 6% ethylene dimethacrylate (EDMA), 44.5% 1-propanol, and 25.5% 1,4-butanediol. The column performance was evaluated through the separations of a series of alkylbenzenes. At a normal flow rate of 50 microL min(-1), the produced HMA-based monolithic columns typically exhibited 3,000 theoretical plates for a 20-cm-long column, and the pressure drop was generally less than 1 MPa per 20 cm. The monolithic columns were resistant to at least 15 MPa, and could be properly operated at 15-20 times higher flow rate than normal, reducing the separation time to 1/15-1/20. The HMA based monolithic columns were applied to rapid and efficient separations of proteins such as ribonuclease A, cytochrome c, transferrin, and ovalbumin in the precipitation-redissolution mode. Using a CH(3)CN gradient elution at a flow rate of 1,000 microL min(-1), four proteins were baseline separated within 20 s. PMID- 16685519 TI - Comparison of three strategies to evaluate uncertainty from in-house validation data. A case study: mercury determination in sediments. AB - In the present paper, three approaches are compared for the evaluation of the combined uncertainty in the determination of mercury in aquatic sediments by an aqua regia extraction procedure. For this, the data obtained in validation studies from five certified reference materials (CRMs), covering a range of concentrations from 0.8 to 130 mg kg(-1) of mercury and analysed by three atomic spectroscopic techniques (cold vapour generation atomic fluorescence spectrometry, CV-AFS, cold vapour generation atomic absorption spectroscopy, and inductively coupled plasma mass spectroscopy), were considered. The combined uncertainty was firstly assessed by considering separately the data obtained for each CRM analysed (approach A). Moreover, this assessment was also performed with two other calculation approaches (B and C) based on the pooled data obtained from the validation step. The comparison of the results obtained for the different techniques showed a clear bias effect when using CV-AFS with nitric acid as a diluent. In relation to the strategies tested for the combined uncertainty assessment, approach C proved to be the easiest and friendliest method for uncertainty assessment. PMID- 16685520 TI - Four-way calibration applied to the simultaneous determination of folic acid and methotrexate in urine samples. AB - First-, second- and third-order calibration methods were investigated for the simultaneous determination of folic acid and methotrexate. The interest in the determination of these compounds is related to the fact that methotrexate inhibits the body's absorption of folic acid and prolonged treatment with methotrexate may lead to folic acid deficiency, and to the use of folic acid to cope with toxic side effects of methotrexate. Both analytes were converted into highly fluorescent compounds by oxidation with potassium permanganate, and the kinetics of the reaction was continuously monitored by recording the kinetics curves of fluorescence emission, the evolution with time of the emission spectra and the excitation-emission matrices (EEMs) of the samples at different reaction times. Direct determination of mixtures of both drugs in urine was accomplished on the basis of the evolution of the kinetics of EEMs by fluorescence measurements and four-way parallel-factor analysis (PARAFAC) or multiway partial least squares (N-PLS) chemometric calibration. The core consistency diagnostic (CORCONDIA) was employed to determine the correct number of factors in PARAFAC and the procedure converged to a choice of three factors, attributed to folic acid, methotrexate and to the sum of fluorescent species present in the urine. PMID- 16685521 TI - Self-management in lower urinary tract symptoms: the next major therapeutic revolution. AB - The standard treatments for men with lower urinary tract symptoms (LUTS) range from watchful waiting to medical and finally surgical intervention. However, the role of self-management interventions such as education and reassurance, lifestyle modification and behavioural changes has not been formally investigated, although they are widely advocated and utilised for LUTS. Self management interventions are well established in other chronic diseases such as diabetes, arthritis and asthma. These interventions, if successfully organised within a structured program for LUTS, could improve patient outcomes as well as reduce the economic burden of LUTS treatment, by replacing or augmenting other treatments. Recent studies showing that long-term urodynamic and symptomatic deterioration of LUTS is minimal suggest that this is a safe and valid treatment option. This is supported by a recent pilot study of a LUTS self-management program which showed significant improvements in I-PSS and frequency-volume parameters. The results of a recently completed randomised controlled trial are awaited. PMID- 16685524 TI - Combined effects of water stress and high temperature on photosynthesis, nitrogen metabolism and lipid peroxidation of a perennial grass Leymus chinensis. AB - Drought and high-temperature stresses have been extensively studied; however, little is known about their combined impact on plants. In the present study, we determined the photosynthetic gas exchange, chlorophyll fluorescence, nitrogen level, and lipid peroxidation of the leaves of a perennial grass (Leymus chinensis (Trin.) Tzvel.) subjected to three constant temperatures (23, 29 and 32 degrees C), and five soil-moisture levels (75-80%, 60-65%, 50-55%, 35-40% and 25 30% of field capacity, respectively). High temperature significantly decreased plant biomass, leaf green area, leaf water potential, photosynthetic rate (A), maximal efficiency of PSII photochemistry (F (v)/F (m)), actual PSII efficiency (Phi(PSII)), the activities of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2), but markedly increased the ratio of leaf area to leaf weight (SLA), endopeptidase (EP; EC 3.4.24.11) activity, and malondialdehyde (MDA) content, especially under severe water stress conditions. The A and F (v)/F (m) were significantly and positively correlated with leaf-soluble protein content, and the activities of NR and GS. However, both photosynthesis parameters were significantly and negatively correlated with EP activity and MDA content (P < 0.05). It is suggested that high temperature, combined with severe soil drought, might reduce the function of PSII, weaken nitrogen anabolism, strengthen protein catabolism, and provoke lipid peroxidation. The results also indicate that severe water stress might exacerbate the adverse effects of high temperature, and their combination might reduce the plant productivity and distribution range of L. chinensis in the future. PMID- 16685527 TI - Polycystic ovary syndrome: etiology and pathogenesis. AB - OBJECTIVE: To provide a review of the pathogenesis of polycystic ovary syndrome. DESIGN: Literature survey. RESULT(S): Three major pathophysiologic hypotheses have been proposed to explain the clinical findings of polycystic ovary syndrome (PCOS) related to three major laboratory findings: the LH hypothesis, the insulin hypothesis and the ovarian hypothesis. Although the presence of many small follicles with a high androgen to estrogen ratio was first thought to represent a high rate of follicular atresia in polycystic ovaries, recent studies have demonstrated that the granulosa cells are viable and able to respond to FSH stimulation with normal increases in estradiol production. Thus, a new hypothesis has arisen that FSH activity is somehow blocked at the ovarian level. CONCLUSION(S): PCOS is a syndrome involving defects in primary cellular control mechanisms that result in the expression of chronic anovulation and hyperandrogenism. In this syndrome, the relation between the various parameters is of particular interest. These relations constitute the cornerstone of the pathogenesis of PCOS. The fact that the pathogenesis of PCOS has not yet been clarified, despite the plethora of relative information, may be the result of a general way of thinking in the interpretation of several scientific data, and especially those that refer to biochemical phenomena. The use of the various models of the theory of chaos, that permits a concrete approach for the interpretation of data, may constitute an optional procedure for the future understanding of the association of different parameters and their disturbances in the pathogenesis of the polycystic ovary syndrome. PMID- 16685525 TI - Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes. AB - Two photorespiratory mutants from Lotus japonicus, namely Ljgln2-1 and Ljgln2-2, deficient in plastidic glutamine synthetase (GLN2), were analysed at the molecular level. Both mutants showed normal levels of Gln2 mRNA, indicating that they were affected post-transcriptionally. Complete sequencing of full-length Gln2 cDNAs revealed the presence of a single point mutation on each mutant, leading to G85R and L278H amino acid replacements, respectively. Different types of experimental approaches, including heterologous expression and complementation tests in Escherichia coli, showed that both GLN2 mutant proteins completely lacked of biosynthetic and transferase enzyme activities. Moreover, it was also shown that while GLN2-1 mutant protein was assembled into a less stable inactive octamer, GLN2-2 mutant protein was unable to acquire a proper quaternary structure and was rapidly degraded. Therefore, the mutations analysed are the first of their type affecting the stability and/or the quaternary structure of the GLN2 enzyme. The kinetic parameters of purified recombinant GLN2 were determined. The enzyme showed positive cooperativity towards ammonium and Mg(2+). Thiol compounds stimulated by twofold the biosynthetic activity but not the transferase activity of recombinant GLN2 and were able to alter the kinetics towards glutamate of the enzyme. Moreover, the biosynthetic activity of recombinant GLN2 was stimulated by more than tenfold by the presence of free Mg(2+). PMID- 16685528 TI - Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori. AB - piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1 98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species. PMID- 16685529 TI - Thalidomide and celecoxib as potential modulators of irinotecan's activity in cancer patients. AB - PURPOSE: Nuclear factor-kappaB (NF-kappaB) activation induces resistance to irinotecan. Preclinically, thalidomide and COX-2 inhibitors reduce NF-kappaB activation. We tested the feasibility of combining irinotecan with thalidomide and thalidomide/celecoxib in patients with refractory malignancies. PATIENTS/METHODS: The study was conducted in two parts. First, the optimal dose of thalidomide (400 or 200 mg daily) in combination with irinotecan 125 mg/m(2) days 1 and 8 every 3 weeks was determined. In the second part, celecoxib 400 mg twice-daily was added to irinotecan/thalidomide. Pharmacokinetics of irinotecan and thalidomide alone or concurrently were evaluated. Tumor necrosis factor alpha, beta-fibroblast growth factor, and NF-kappaB activation were measured in blood mononuclear cells (PBMC). No CYP450 enzyme inducers/inhibitors were allowed. RESULTS: Thirty-six patients were enrolled: Eleven received thalidomide 400 mg, 13 thalidomide 200 mg and 12 thalidomide 400 mg and celecoxib, with irinotecan. For the two-drug combination, there was a higher rate of moderate/severe diarrhea/myelosuppression with thalidomide 200 mg. Thus thalidomide 400 mg was combined with celecoxib. The triple combination resulted in similar toxicity as the doublet with the lower thalidomide dose. Concurrent administration of irinotecan/thalidomide did not influence pharmacokinetics. Anti tumor responses occurred in two patients and prolonged stabilization in eight others. NF-kappaB activation increased over time. Patients experiencing tumor response or prolonged stabilization had lower NF-kappaB activation, albeit not statistically significant (P = 0.124). CONCLUSIONS: The combination of thalidomide/irinotecan is safe and devoid of PK interactions. Thalidomide 400 mg appeared more suitable for combination, whereas the addition of celecoxib did not improve tolerability. Tumor-specific studies in patients with lesser prior treatment will be necessary to establish the therapeutic impact of the combinations. PMID- 16685530 TI - Development of adrenal chromaffin cells in Sf1 heterozygous mice. AB - Adrenal medullary chromaffin cells are derivatives of the neural crest and are widely believed to share a common sympathoadrenal (SA) progenitor with sympathetic neurons. For decades, the adrenal cortical environment was assumed to be essential for channelling SA progenitors towards an endocrine chromaffin cell fate. Our recent analysis of steroidogenic factor 1(Sf1) -/- mice, which lack an adrenal cortex, has challenged this view: in Sf1 -/- mice chromaffin cells migrate to the correct "adrenal" location and undergo largely normal differentiation. In contrast to Sf1 homozygous mutants, heterozygous animals have an adrenal cortex, which, however, is smaller than in wildtype littermates. We show here that the Sf1 +/- adrenal cortical anlagen attract normal numbers of chromaffin progenitor cells into their vicinity by embryonic day 13.5 (E13.5). Two days later, however, only a few scattered cells with highly immature features have immigrated into the adrenal cortex, whereas the remainder form a coherent cell assembly ectopically located at the medial surface of the gland. These cells appear more mature than the scattered intracortical chromaffin progenitors and express the adrenaline synthesizing enzyme PNMT with a delay of 1 day in comparison with wildtype littermates. Nevertheless, chromaffin progenitor cells undergo a numerical reduction of approximately 30% by E17.5. Together, our data suggest that normal adrenocortical development is critical for the correct immigration of chromaffin progenitors into the cortical anlagen, for the timing of PNMT expression and for the regulation of chromaffin cell numbers. PMID- 16685531 TI - Morphology and metamorphosis of the peptidergic Va neurons and the median nerve system of the fruit fly, Drosophila melanogaster. AB - Metamorphosis is a fundamental developmental process and has been intensively studied for various neuron types of Drosophila melanogaster. However, detailed accounts of the fate of identified peptidergic neurons are rare. We have performed a detailed study of the larval morphology and pupal remodelling of identified peptidergic neurons, the CAPA-expressing Va neurons of D. melanogaster. In the larva, Va neurons innervate abdominal median and transverse nerves that are typically associated with perisympathetic organs (PSOs), major neurohaemal release sites in insects. Since median and transverse nerves are lacking in the adult, Va neurites have to undergo substantial remodelling during metamorphosis. We have examined the hitherto uncharacterised gross morphology of the thoracic PSOs and the abdominal median and transverse nerves by scanning electron microscopy and found that the complete reduction of these structures during metamorphosis starts around pupal stage P7 and is completed at P9. Concomitantly, neurite pruning of the Va neurons begins at P6 and is preceded by the high expression of the ecdysone receptor (EcR) subtype B1 in late L3 larvae and the first pupal stages. New neuritic outgrowth mainly occurs from P7-P9 and coincides with the expression of EcR-A, indicating that the remodelling of the Va neurons is under ecdysteroid control. Immunogold-labelling has located the CAPA peptides to large translucent vesicles, which are released from the transverse nerves, as suggested by fusion profiles. Hence, the transverse nerves may serve a neurohaemal function in D. melanogaster. PMID- 16685532 TI - Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture. AB - A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions. PMID- 16685534 TI - Superselective embolization of a post-biopsy renal pseudoaneurysm in a 13-month old infant. AB - Renal artery pseudoaneurysms are infrequent and are most often secondary to surgical or percutaneous renal biopsies. Their rupture can cause rapid clinical deterioration by massive haemorrhage. Diagnosis and treatment must, therefore, be rapid. Currently, surgical treatment remains the gold standard in most institutions while paediatric cases of endovascular embolization have seldom been described. We report a 13-month-old infant with a post-biopsy renal pseudoaneurysm successfully treated by selective arterial embolization. PMID- 16685540 TI - [Choroidal effusion syndrome after embolization of an indirect cavernous sinus fistula via the superior ophthalmic vein]. AB - Embolization of a cavernous sinus fistula (SCF) via the superior ophthalmic vein (SOV) was reported to be an almost uncomplicated procedure, even after ligature of the vein at the end of the procedure. We report about a complication of this therapy. A 58-year-old female had a successful embolization of a right indirect cavernous sinus fistula via the SOV. At the end of the operation the SOV was ligated because of the danger of bleeding. Directly after surgery she experienced general worsening of the right eye with signs of venous congestion and marked effusion syndrome. The patient underwent total heparinization to achieve an opening of venous collaterals. Under local therapy with atropine 1% eye drops a decrease of the intraocular pressure was observed. The effusion syndrome was completely resolved within 1 month. If embolization of a cavernous sinus fistula is performed via the SOV, the ligature of the vein at the end of the procedure leads to thrombosis, which can reduce the venous stream from the eye and orbit. A secondary effusion syndrome with ocular hypertension because of a ciliolenticular block situation is possible and requires appropriate therapy. It is not possible to assess the capacity and time of opening of the venous collateral system before surgery. Therefore a transient outflow disturbance should be considered. PMID- 16685541 TI - Immunohistochemical evaluation of a novel clone of monoclonal anti-MYCN antibody B8.4B in neuroblastic tumours: a correlation with MYCN gene status. PMID- 16685542 TI - Topical furosemide versus oral steroid in preoperative management of nasal polyposis. AB - The efficacy of topical nasal furosemide treatment has been shown in the protection of nasal polyp recurrence. The aim of the study was to compare the effect of oral steroid, as standard preoperative treatment, and inhaled furosemide, as alternative treatment, for 7 days preoperatively in terms of subjective improvement of nasal symptoms, polyp size reduction, inflammation in the polyp tissue, and intraoperative blood loss. A group of 40 patients with nasal polyposis entered the study and they were randomly allocated to 7-day preoperative treatment with either oral methylprednisolon (1 mg/kg/day) or topical furosemide by inhalation (6.6 mmol/l solution). Subjective scores of rhinosinusitis symptoms, polyp scores at endoscopy, and biopsy of the most superficial polyp were taken at inclusion. All procedures were repeated on day 7. Intraoperative blood loss was estimated (scores 0-10) by the surgeon at the operation. Eosinophils, mastocytes, and oedema were quantified by histomorphometry. Subjective symptoms and endoscopy scores did not differ significantly between the groups after the treatment although improvement of olfaction was insignificantly better in the steroid group. Steroid treatment significantly reduced eosinophil count, with no effect on mastocytes and oedema. Furosemide treatment did not affect inflammatory cells count significantly, but it has significantly reduced oedema in previously unoperated patients. No difference in intraoperative bleeding was observed between the groups. PMID- 16685543 TI - A prospective randomised controlled trial comparing Merocel and Rapid Rhino nasal tampons in the treatment of epistaxis. AB - A prospective study was performed to compare the efficacy and patient tolerance of Merocel and Rapid Rhino nasal tampons in the treatment of epistaxis. A total of 42 patients were studied. There was no significant difference between the two types of pack in efficacy or patient discomfort with pack in situ. Rapid Rhino produced significantly lower scores for subjective patient discomfort during insertion and removal of pack. PMID- 16685545 TI - Proposal of "evolution theory in cerebrospinal fluid dynamics" and minor pathway hydrocephalus in developing immature brain. AB - BACKGROUND: The specificity of cerebrospinal fluid (CSF) dynamics in the immature brain still remains unknown. In our data previously published, the transependymal intraparenchymal CSF pathway (the minor pathway) plays a significant role in various degrees in the alternative CSF passage. Now, there is a growing consensus in the age differences in the outcome of neuroendoscopic ventriculostomy in treatment of non-communicating types of hydrocephalus. The authors discuss the clinical significance of the specific CSF dynamics and propose the new aspect of classification of hydrocephalus with a theory in the development of CSF from the pathophysiological point of view. PATIENTS AND METHODS: Between January 2001 and March 2004, 122 hydrocephalic children were registered at the Jikei University Hospital Women's & Children's Medical Center (JWCMC), Tokyo. Our retrospective study for the efficacy of neuroendoscopic ventriculostomy confirmed the significantly high failure rate of neuroendoscopic ventriculostomy in treating hydrocephalus in neonates and infants with non-communicating hydrocephalus as the initial impression. The prospective CSF dynamic studies using cine-mode MRI and CT ventriculo-cisternography were then routinely started. Altogether, 9 out of 29 (31%) endoscopically treated cases needed shunt placement 3-30 weeks (mean 7.9 weeks) after the endoscopic procedure(s) (publication in preparation). Five out of 11 (45%) neonates/infants under 3 months, 3/5 (60%) infants at 7-12 months, 10/10 (100%) toddlers at 1-4 years and 3/3 (100%) schoolchildren at 5-17 years were cured, as in the condition of "post-endoscopic ventriculostomy arrested hydrocephalus". The pattern of ventriculo-cisternography in neonatal/infantile cases revealed intraparenchymal predominant pattern (minor pathway) of the CSF dynamics rather than passage in the major pathway. DISCUSSION: The various basic investigations in rodents, cats and monkeys have suggested that CSF is absorbed not via Pacchionian bodies as the last end of the major pathway, which do not exist in these animals and are recognized after infantile period in human, but through the choroids plexus and the periventricular fenestrated venous capillaries into the deep venous channel. The high incidence of "failure to arrest hydrocephalus" by neuroendoscopic ventriculostomy in fetal, neonatal and infantile periods was considered to depend on the specific CSF dynamics, in which the major CSF pathway has not developed and the minor pathway has a significant role. PROPOSAL OF THEORY: We herein propose a new aspect of classification for hydrocephalus with special reference to the CSF circulation in the minor CSF pathway, i.e. "minor pathway hydrocephalus", differentiating the conventional classification by Dandy (communicating and non-communicating) or Russell (non obstructive and obstructive) as "major pathway hydrocephalus". We also herein propose a hypothesis that the CSF dynamics develop in the theory of evolution from the immature brain, as in the animals with the minor CSF pathway predominance, towards matured adult human brain together with completion of the major CSF pathway: the "evolution theory in CSF dynamics". PMID- 16685546 TI - Pituitary-adrenal responses to arm versus leg exercise in untrained man. AB - The purpose of this study was to examine pituitary-adrenal (PA) hormone responses [beta-endorphin (beta-END), adrenocorticotropic hormone (ACTH) and cortisol] to arm exercise (AE) and leg exercise (LE) at 60 and 80% of the muscle-group specific VO2 peak. Eight healthy untrained men (AE VO2 peak=32.4+/-3.0 ml kg(-1) min(-1), LE VO2 peak=46.9+/-5.3 ml kg(-1) min(-1)) performed two sub-maximal AE and LE tests in random order. Plasma beta-END, ACTH and cortisol were not different (P>0.05) between AE and LE at either exercise intensity; the 60% testing elicited no changes from pre-exercise (PRE) values. For 80% testing, plasma beta-END, ACTH and cortisol were consistently, but not significantly, greater during LE than AE. In general, plasma beta-END and ACTH were higher (P<0.05) during 80% exercise, than PRE, for both AE and LE. Plasma cortisol was elevated (P<0.05) above PRE during 80% LE, and following 80% for both AE and LE. Plasma ACTH was higher (P<0.05) during 80% LE and AE versus 60% LE and AE, respectively. Plasma beta-END and cortisol were significantly higher during and immediately after 80% LE than 60% LE. Thus, plasma beta-END, ACTH and cortisol responses were similar for AE and LE at the two relative exercise intensities, with the intensity threshold occurring somewhere between 60 and 80% of VO2 peak. It appears that the smaller muscle mass associated with AE was sufficient to stimulate these PA axis hormones in a manner similar to LE, despite the higher metabolic stress (i.e., plasma La-) associated with LE. PMID- 16685547 TI - The effect of 2 weeks vitamin C supplementation on immunoendocrine responses to 2.5 h cycling exercise in man. AB - An increased systemic concentration of stress hormones (of the hypothalamic pituitary adrenal axis) and some cytokines may contribute to the depression of immune cell function typically observed after prolonged exercise. The aim of the present study was to determine the effect of 2 weeks of supplementation with vitamin C (VC) on cortisol, adrenocorticotrophic hormone, interleukin-6, oxidative stress and neutrophil responses to a single bout of endurance exercise. Nine healthy endurance-trained males exercised for 2.5 h at 60% VO2max after 2 weeks of placebo (PLA) or VC (1,000 mg day(-1)) supplementation. All participants completed both trials utilising a randomised crossover design with a minimum 14 day washout period between trials. There was a significant trial x time interaction effect for plasma cortisol concentration (P = 0.039) which tended to be lower in the VC trial but post hoc analysis found no specific between trial differences. There was a significantly lower post-exercise neutrophilia (P < 0.014) in the VC trial, compared with the PLA trial. There was no trial x time interaction for measures of neutrophil function (bacteria-stimulated elastase release, fMLP or PMA-stimulated oxidative burst). However, there was a trend for higher fMLP-stimulated neutrophil oxidative burst in the VC compared with PLA trial (trial x time interaction, P = 0.075). These results suggest that supplementation with VC for a period of up to 2 weeks provides little to no protection against the depression of neutrophil function which typically occurs after endurance exercise. PMID- 16685548 TI - The effect of varying the time of concentric and eccentric muscle actions during resistance training on skeletal muscle adaptations in women. AB - This study investigated the effect of manipulating the time to complete both the concentric (CON) and eccentric (ECC) muscle actions during resistance training on strength, skeletal muscle properties and cortisol in women. Twenty-eight women (mean +/- SE age = 24.3 +/- 1.1 year) with strength training experience completed three training sessions per week for 9 weeks. Two sets of four lower body exercises (leg press, parallel squat, knee extension and knee flexion) were completed using 6-8 RM intensity. The long CON (LC) group performed the CON action for 6 s and the ECC action for 2 s, while the long ECC (LE) group completed the CON and ECC phases for 2 and 6 s, respectively. Both groups experienced significant increases in leg press CON only, ECC only and combined ECC and CON maximal strength (1 RM). Immunohistochemical analyses demonstrated that both types I and IIA vastus lateralis fibre areas significantly increased following LC training while only type I fibre area increased following LE training. There was a decrease in MHCIId(x) with a concomitant increase in MHCIIa (P < 0.05) in both groups. Twenty-four hour urinary cortisol significantly increased after LC training only. It was concluded that LC resistance training was more effective than LE for increasing both types I and IIA fibre area and cortisol when time under tension and intensity of muscle actions were matched between the two modes of resistance training in young healthy women. PMID- 16685549 TI - Hyperhydration prior to a simulated second day of the 3-day moderate intensity equestrian competition does not cause arterial hypoxemia in Thoroughbred horses. AB - Dehydration and the associated impairment of cardiovascular and thermoregulatory function comprise major veterinary problems in horses performing prolonged exercise, particularly under hot and humid conditions. For these reasons, there is considerable interest in using pre-exercise hyperhydration to help maintain blood volume in the face of the excessive fluid loss associated with sweat production during prolonged exertion. However, recently it was reported that pre exercise hyperhydration causes arterial hypoxemia in horses performing moderate intensity exercise simulating the second day of an equestrian 3-day event competition (E3DEC) which may adversely affect performance (Sosa Leon et al. in Equine Vet J Suppl 34:425-429, 2002). These findings are contrary to data from horses performing short-term maximal exertion, wherein hyperhydration did not affect arterial O2 tension/saturation. Thus, our objective in the present study was to examine the impact of pre-exercise hyperhydration on arterial oxygenation of Thoroughbred horses performing an exercise test simulating the second day of an E3DEC. Control and hyperhydration studies were carried out on seven healthy Thoroughbred horses in random order, 7 days apart. In the control study, horses received no medications. In the hyperhydration experiments, nasogastric administration of NaCl (0.425 g/kg) 5 h pre-exercise induced a plasma volume expansion of 10.9% at the initiation of exercise. This methodology for inducing hypervolemia was different from that of Sosa Leon et al. (2002). Blood-gas tensions/pH as well as plasma protein, hemoglobin and blood lactate concentrations were measured pre-exercise and during the exercise test. Our data revealed that pre-exercise hyperhydration neither adversely affected arterial O2 tension nor hemoglobin-O2 saturation at any time during the exercise test simulating the second day of an E3DEC. Further, it was observed that arterial blood CO2 tension, pH, and blood lactate concentrations also were not affected by pre-exercise hyperhydration. However, hemodilution in hyperhydrated horses caused an attenuation of the expansion in the arterial to mixed-venous blood O2 content gradient during phases B and D of the exercise protocol, which was likely offset by an increase in cardiac output. It is concluded that pre-exercise hyperhydration of horses induced in the manner described above is not detrimental to arterial oxygenation of horses performing an exercise test simulating the second day of an E3DEC. PMID- 16685550 TI - Optimal power-to-mass ratios when predicting flat and hill-climbing time-trial cycling. AB - The purpose of this article was to establish whether previously reported oxygen to-mass ratios, used to predict flat and hill-climbing cycling performance, extend to similar power-to-mass ratios incorporating other, often quick and convenient measures of power output recorded in the laboratory [maximum aerobic power (W(MAP)), power output at ventilatory threshold (W(VT)) and average power output (W(AVG)) maintained during a 1 h performance test]. A proportional allometric model was used to predict the optimal power-to-mass ratios associated with cycling speeds during flat and hill-climbing cycling. The optimal models predicting flat time-trial cycling speeds were found to be (W(MAP)m( 0.48))(0.54), (W(VT)m(-0.48))(0.46) and (W(AVG)m(-0.34))(0.58) that explained 69.3, 59.1 and 96.3% of the variance in cycling speeds, respectively. Cross validation results suggest that, in conjunction with body mass, W(MAP) can provide an accurate and independent prediction of time-trial cycling, explaining 94.6% of the variance in cycling speeds with the standard deviation about the regression line, s=0.686 km h(-1). Based on these models, there is evidence to support that previously reported VO2-to-mass ratios associated with flat cycling speed extend to other laboratory-recorded measures of power output (i.e. Wm( 0.32)). However, the power-function exponents (0.54, 0.46 and 0.58) would appear to conflict with the assumption that the cyclists' speeds should be proportional to the cube root (0.33) of power demand/expended, a finding that could be explained by other confounding variables such as bicycle geometry, tractional resistance and/or the presence of a tailwind. The models predicting 6 and 12% hill-climbing cycling speeds were found to be proportional to (W(MAP)m( 0.91))(0.66), revealing a mass exponent, 0.91, that also supports previous research. PMID- 16685551 TI - Human muscle fatigue and elastic compressive stockings. AB - The present study was performed to test if elastic compressive stockings (ECSs) increase muscle fatigability during sustained muscle contraction or if it improves the recovery after fatigue. Surface electromyograms (EMGs) were recorded on 4 leg and thigh muscles, and static ankle dorsal flexion force levels were measured in the right limb of 15 healthy subjects. The subjects maintained a 50% maximum ankle dorsal flexion force (MVF) for as long as possible without and, after a 30 min rest, with a European class I ECS. Finally, after another 30 mn rest, the pressure exerted by the ECS on the skin was measured at standard points on the limb, using a Salzmann apparatus. During the first 10 min of both rest periods, the subjects performed brief static maximum ankle dorsal flexions every 30 s. ECS exerted a 14.3 mm Hg mean pressure at tibial level C. Linear relationships, whose slopes were not influenced by ECS, existed between the maintenance time and both the mean power frequency and the logarithm of the total power of the tibialis anterior and gastrocnemius lateralis EMGs. The endurance times, the force recovery times after fatigue and the linear relationships between the logarithm of the time elapsing after exhaustion and the MVF reached during the recovery period were also independent of ECS. The results show that class I ECSs are not responsible for greater muscle fatigability; but they do not improve force recovery during rest following static fatiguing voluntary contractions. PMID- 16685552 TI - Indices of electromyographic activity and the "slow" component of oxygen uptake kinetics during high-intensity knee-extension exercise in humans. AB - The control of pulmonary oxygen uptake (VO2) kinetics above the lactate threshold (LT) is complex and controversial. Above LT, VO2 for square-wave exercise is greater than predicted from the sub-LT VO2-WR relationship, reflecting the contribution of an additional "slow" component (VO2(sc)). Investigators have argued for a contribution to this slow component from the recruitment of fast twitch muscle fibres, which are less aerobically efficient than slow-twitch fibres. Six healthy subjects performed a rapid-incremental bilateral knee extension exercise test to the limit of tolerance for the estimation of VO2(peak), ventilatory threshold (VT), and the difference between VO2(peak) and VO2 at VT (Delta). Subjects then completed three repetitions of square-wave exercise at 30% of VT for 10 min (moderate intensity), and at VT + 25%Delta (heavy intensity) for 20 min. Pulmonary gas exchange was measured breath-by breath. Surface EMG was recorded from m. rectus femoris; integrated EMG (IEMG) and mean power frequency (MPF) were derived for successive contractions. In comparison to moderate-intensity exercise, the phase 2 VO2 kinetics in heavy exercise were marginally slower than for moderate-intensity exercise (time constant (+/- SD) 25 +/- 9 and 22 +/- 10 s, respectively; NS), with a discernible VO2(sc) (VO2 difference between minutes 6 and 3 of exercise: 74 +/- 21 and 0 +/- 20 ml min(-1), respectively). However, there was no significant change in IEMG or MPF, either in the moderate domain or in the heavy domain over the period when the slow component was manifest. These observations argue against an appreciable preferential recruitment of fast-twitch units with high force-generating characteristics and fast sarcolemmal conduction velocities in concert with the development of the VO2 slow component during heavy-intensity knee-extensor exercise. The underlying mechanism(s) remains to be resolved. PMID- 16685553 TI - Changes in muscle and joint elasticity following long-term strength training in old age. AB - The current investigation was designed (1) to examine the effect of a 48-week strength training on musculotendinous (MT) and musculoarticular (MA) stiffness characteristics in older men and women; and (2) to evaluate the influence of gender on stiffness behaviour in response to such training. The training was performed twice per week and mainly consisted of three series of 10 repetitions of calf-rise at 75% of the 3-repetition maximum. Two methods were used to perform stiffness measurements during plantar flexion: (1) the use of quick-release movements, allowing the calculation of MT stiffness; (2) the application of sinusoidal perturbations to the joint, allowing the calculation of MA stiffness. In each case, stiffness was linearly related to torque, leading to the calculation of a normalized stiffness index (SI) as the slope of this stiffness torque relationship: SI(MT) and SI(MA), respectively. Results showed a similar decrease in SI(MT) among older men (-27%, P < 0.05) and women (-29%, P < 0.05) following training. A decrease in SI(MA) was only observed among women (-11%, P < 0.05). The results suggest that (1) MT stiffness decreases following training in older individuals, counterbalancing the effect of ageing; and (2) older men and women respond differently to the same resistance-training stimulus in terms of MA stiffness. Gender-related differences in MA stiffness response may originate from passive MA elastic structures. PMID- 16685555 TI - [The cost of sepsis]. AB - In recent years great efforts in clinical sepsis research have led to a better understanding of the underlying pathophysiology and new therapeutic approaches including drugs and supportive care. Despite this success, severe sepsis remains a serious health care problem. Each year approximately 75,000 patients in Germany and approximately 750,000 patients in the USA suffer from severe sepsis. The length of stay and the cost of laborious therapies lead to high intensive care unit (ICU) costs. Sepsis causes a significant national socioeconomic burden if indirect costs due to productivity loss are included and in Germany severe sepsis has been estimated to generate costs between 3.6 and 7.7 billion Euro annually. Thus, this complex and life-threatening disease has been identified as a high cost driver not only for the ICU, but also from the perspectives of hospitals and society. To improve the outcome of severe sepsis, innovative drugs and treatment strategies are urgently needed. Some drugs and strategies already offer promising results and will probably play a major role in the future. Even though their cost effectiveness is likely, intensive care medicine has to carry a substantial economic burden. This article summarizes studies focusing on the evaluation of direct or indirect costs of sepsis and the cost-effectiveness of new therapies. PMID- 16685556 TI - [Modified Hardinge approach with limited incision]. AB - BACKGROUND: The technique of a minimally invasive anterolateral, intracapsular, modified Hardinge approach with transosseous refixation developed by the senior author is described in detail. MATERIAL AND METHODS: Clinical and radiographic data after cemented total hip arthroplasty reveal adequate outcome without increased complication rates despite limited incision technique (average 10 cm). This technique can be safely applied by surgeons in training and performed in acceptable operating times. RESULTS: A comparison of the described technique to a standard incision cannot be made from our data, but current literature suggests the main benefit to be cosmetic. A technically well performed operation with adequate long-term outcome remains far more important than a short incision. PMID- 16685560 TI - Are autopsies of help to the parents of SIDS victims? A follow-up on SIDS families. AB - Little is known about what bereaved parents feel about the autopsy performed on their child. A multi-centre case control study of sudden infant death syndrome (SIDS) victims was carried out in Germany between 1998 and 2001, in which all infants had been autopsied. We performed a follow-up study 4-7 years after the parents had lost their child. A total of 141 parents filled in the questionnaire, which were sent to them by the study centre. Of these, 71% had had another child after the SIDS/sudden unexpected death in infancy. The majority (83%) of the participating parents found the autopsy helped them to cope better with the death. A large proportion (46%) did not want any professional help after the death, and 55% did not wish to have any contact with a self-help group. We conclude that the autopsy is helpful to the majority of bereaved parents. Professional help and self-help groups should be offered to the parents even if the majority in our study did not want to use either. PMID- 16685561 TI - Variability in the population pharmacokinetics of pyrazinamide in South African tuberculosis patients. AB - OBJECTIVE: This study was designed to characterize the population pharmacokinetics of pyrazinamide in South African pulmonary tuberculosis patients, with special reference to interindividual and interoccasional variability (IIV and IOV, respectively). METHODS: Concentration-time measurements obtained from 227 patients receiving oral doses of pyrazinamide were pooled to create a dataset containing 3,092 data points spanning multiple dosing occasions. The software program NONMEM was used to analyze the data. RESULTS: A one compartment model with first-order absorption, including a zero-order component describing release from formulation, and first-order elimination best described the data. The absorption rate constant was estimated to be bimodally distributed between two distinct subgroups, fast and slow, in approximately even proportion. Absorption rate was threefold greater in fast absorbers (3.56 h(-1)) in comparison to slow absorbers (1.25 h(-1)). Typical values of oral clearance and apparent volume of distribution were estimated as 3.42 L h(-1) and 29.2 l, respectively. IOV was supported in oral clearance (0.0238, variance) and absorption rate (0.623, variance). The duration of zero-order absorption was estimated as 0.290 h, and was quite variable between patients (0.957, variance). CONCLUSION: The absorption of pyrazinamide in the studied population was highly variable and two separate subpopulations were identified. IOV accounted for a proportion of the variability in clearance and the absorption rate constant. PMID- 16685562 TI - The use of angiotensin-converting enzyme inhibitors and other drugs with cardiovascular effects by non-demented and demented elderly with a clinical diagnosis of heart failure. A population-based study of the very old. AB - OBJECTIVES: The aim of this study was to investigate drug treatment patterns for heart failure (HF) in the very elderly and, in particular, to determine if angiotensin-converting enzyme inhibitors (ACEIs) were under-used by demented persons. METHODS: The 265 participants investigated in this study were all 75 years and older, with HF and using cardiovascular drugs, and were part of the Nordanstig cohort (919 persons) of the population-based Kungsholmen project. Data on demographics, medical conditions, including dementia and HF from the baseline investigation 1995-1998, and drug use data from the baseline and follow-up (1999 2001) investigations were used. RESULTS: ACEIs were used by 25.7% of the participants. After adjustment for sociodemographic and medical background factors, there was no significant difference in ACEI use by dementia status, but use was lower with increasing age: the odds ratio (OR) was 0.11 and the 95% confidence interval (95%CI) was 0.01-0.95 between participants 90 years and older and those 75-79 years old (p=0.045). Use was also lower in those persons living in an institution compared to community-living elderly (OR: 0.28; 95% CI: 0.09 0.91; p=0.034). Only 15.8% of the participants used beta-blockers. Of the 12.8% using calcium channel blockers, 82% used preparations with negative inotropic effects. Non-steroid antiinflammatory drugs (NSAIDS), contraindicated in HF, were used by 10.6%. CONCLUSIONS: No significant difference in ACEI utilization related to dementia diagnosis was shown, but the study did reveal a significantly lower use in the oldest age group and in elderly persons living in institutions. The low utilization rates of ACEIs and beta-blockers, the high proportion of calcium channel blockers with negative inotropic effects, and the fairly frequent use of NSAIDs in the study cohort suggest that the quality in drug treatment of very old people with HF can be improved. PMID- 16685563 TI - Nonmuscle myosin, force maintenance, and the tonic contractile phenotype in smooth muscle. AB - Recent studies have demonstrated that nonmuscle (NM) myosin II forms filaments and can generate and maintain force in smooth muscle tissue [Lofgren et al. (2003) J Gen Physiol 121:301-310; Morano et al. (2000) Nat Cell Biol 2:371-375]. To further investigate the mechanical contribution of NM myosin to force maintenance during smooth muscle contraction, we utilized a selective inhibitor of the NM myosin ATPase, blebbistatin [Straight et al. (2003) Science 299:1743 1747]. Force and myosin light chain (MLC(20)) phosphorylation were measured during KCl stimulation of small strips of intact mouse bladder and aorta at 22 degrees C. The bladder strips contracted with a typical phasic force response, characterized by a large, rapid, transient increase in force followed by a decline to a lower, steady-state level. The addition of blebbistatin did not alter the peak force, but decreased force maintenance. KCl depolarization of aortic strips resulted in a tonic contraction; force increased to a sustained steady state. Similar to the bladder tissue, blebbistatin substantially decreased the steady-state force in the aorta. Blebbistatin did not influence the MLC(20) phosphorylation transient in either tissue type. Additionally, blebbistatin did not change the maximum shortening velocity (V (max)) during KCl depolarization of the aorta. Our results also suggest that NMIIA and NMIIB isoforms are differentially expressed. The expression of NMIIA is more prominent in the bladder, while NMIIB expression is predominant in the aorta. These results suggest that NM myosin contributes to the mechanism of force maintenance in smooth muscle, and could suggest that the expression of NMIIB is a factor for determining the tonic contractile phenotype. PMID- 16685564 TI - Renal Ca2+ handling in sgk1 knockout mice. AB - Coexpression studies in Xenopus oocytes revealed the ability of the serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate the renal epithelial Ca(2+) channel TRPV5. SGK1 increases the abundance of the channel protein in the plasma membrane, an effect requiring the participation of the Na(+)/H(+) exchanger regulating factor 2 (NHERF2). The present study was performed to explore the role of SGK1 in the regulation of renal Ca(2+) handling in vivo. To this end, TRPV5, calbindin D-28K abundance, and renal Ca(2+) excretion were analyzed in gene targeted mice lacking functional SGK1 (sgk1( -/- )) and their age- and sex matched littermates (sgk1( +/+ )). Immunohistochemistry revealed lower abundance of TRPV5 and calbindin D-28K protein in sgk1( -/- ) mice than in sgk1( +/+ ) mice, both fed with control diet. Feeding the mice a Ca(2+)-deficient diet marked ly increased TRPV5 protein abundance in both genotypes. Renal Ca(2+) excretion under control diet was significantly lower in sgk1 ( -/- ) than in sgk1( +/+ ) mice. The Ca(2+)-deficient diet decreased renal excretion of Ca(2+) to the same levels in both phenotypes. Furosemide increased fractional Ca(2+) excretion and dissipated the difference between phenotypes. We conclude that lack of SGK1 may lead to decrease in TRPV5 abundance in connecting tubules but does not abrogate TRPV5 regulation. The decrease in abundance of TRPV5 in connecting tubules of sgk1( -/- ) mice is presumably compensated for by enhanced Ca(2+) reabsorption in upstream nephron segments such as the loop of Henle, which may indirectly result from impaired SGK1-dependent Na(+) reabsorption in the aldosterone-sensitive distal part of the nephron, salt loss, and enhanced Na(+) (and Ca(2+)) reabsorption in those upstream nephron segments. PMID- 16685566 TI - [Oral EBV-associated diffuse large B-cell lymphomas in HIV-negative immunocompromised patients]. AB - BACKGROUND: Diffuse large B-cell lymphomas (DLBCL) are non-Hodgkin's lymphomas (NHL) and with a prevalence of 30-40% they comprise the most frequent NHL in adults. Although their etiology is still unknown, a virus induction, especially by the Epstein-Barr-virus (EBV), is the subject of discussion. Patients with congenital or acquired immunodeficiency are primarily afflicted. PATIENT 1: A 39 year-old female patient developed an EBV-associated DLBCL of the plasmablastic subtype in the maxillary alveolar ridge in the region of teeth 11 and 21 after 24 years of immunosuppressive therapy with azathioprine due to myasthenia gravis. Clinically the lesion presented as a localized acute necrotizing periodontitis that was resistant to symptomatic therapy. After polychemotherapy the disease is in complete remission until today. PATIENT 2: A 56-year-old male patient developed an EBV-associated DLBCL of immunoblastic variant of the right maxillary edentulous alveolar ridge in the posterior region 7 weeks after heart transplantation and immunosuppressive therapy with azathioprine and cyclosporine A. Clinically, a soft, nonpainful, swelling measuring 1.5x0.5x0.5 cm with a central ulceration was evident. The tumor was excised followed by local radiation therapy. No recurrence was noted during a 15-year-follow-up. DISCUSSION: The presented clinical cases demonstrate the increased risk of occurrence of oral malignant B-cell lymphomas as adverse effects of immunosuppressive therapy. The demonstration of EBV in the tumor cells in both cases underlines the involvement of this virus in the pathogenesis of oral DLBCL arising in the setting of an immunodeficiency as has been shown previously in patients with HIV. CONCLUSIONS: Due to the varying and often nonspecific clinical appearance of oral DLBCL, an early biopsy and work-up by an institute specialized in hematopathology is essential for diagnosis, because these tumors may disseminate in early stages. PMID- 16685565 TI - 8-Hydroxy-2'-deoxyguanosine as a marker of oxidative DNA damage related to occupational and environmental exposures. AB - Oxidative DNA damage is considered to play an important role in pathophysiological processes, ageing and cancer. So far major interest has been on measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG), the preferred methods relying on HPLC or GC-mass spectrometry. The high biological relevance of 8-OHdG is due to its ability to induce G-->T transversions, which are among the most frequent somatic mutations found in human cancers. Effects of workplace exposures on the level of white blood cell 8-OHdG or urinary 8-OHdG have been reported with controversial results. Exposures examined include asbestos, azo-dyes, benzene, fine particulate matter (PM(2.5)), glassworks, polycyclic aromatic hydrocarbons (PAHs), rubber manufacturing, silica, metals, styrene, toluene and xylenes. The available data indicate that there is still a lack of well established dose response relations between occupational or environmental exposures and the induction of 8-OHdG. Smoking has been most consistently identified as a confounder for 8-OHdG, but various occupational studies did not reveal higher levels of 8-OHdG in smokers. Despite the conflicting results, the reported studies show promise for 8-OHdG as a biomarker of oxidative stress associated with chemical exposure. However, there are critical aspects related to the analytical challenge, artifactual production of 8-OHdG, inter- and intra individual variation, confounding factors and inter-laboratory differences, implying that further work is needed to reach a consensus on the background level of 8-OHdG. PMID- 16685567 TI - [Craniofacial augmentation with porous polyethylene implants (Medpor: first clinical results]. AB - SUBJECT: Porous polyethylene (Medpor) is an alloplastic material commonly used in craniofacial reconstruction. We report about our first clinical experiences with Medpor for facial augmentation procedures. PATIENTS AND METHODS: We treated 27 patients between 2001 and 2005 (11 female, 16 male) with 48 Medpor-implants. The indications for application of porous polyethylene implants in our clinic were congenital malformations (15), post-traumatic defects (10), and reconstructions after tumor resection (2). The implants were used for nasal/paranasal augmentations (16), for zygomatico-orbital augmentations (18), and for augmentations of the chin and malar region (11). The procedures were performed in a standardized manner. We used prefabricated, self-contoured implants and fixed them subperiosteally with titanium osteosynthesis screws. All operations were performed under general anesthesia. We evaluated the aesthetic results and the ingrowth behavior clinically and histologically. RESULTS: We achieved good aesthetic results and the patients showed no signs of discomfort or rejection. Four patients required a second intervention. These revision surgeries included two cases of local infections and two for aesthetic contouring. The necessary reduction of the implants allowed the harvesting of tissue and implant samples for microscopy. CONCLUSION: Porous polyethylene implants showed a good fibrovascular integration without encapsulation under the light microscope. Giant cells were detected on the surface of the implants. Besides this there was evidence for resorption of the implant material. Fixation with titanium screws is very effective. No implant dislocation or implant fracture occurred. The implants showed high volume stability and were easily handled and contoured. It is not possible to visualize Medpor implants with current imaging techniques, because polyethylene shows no contrast. PMID- 16685568 TI - [Evaluation of individual ceramic implants made of Bioverit with CAD/CAM technology to reconstruct multidimensional craniofacial defects of the human skull]. AB - BACKGROUND: Restrictions in the bone structure of the craniomandibular region caused by malformation, traumata or malignant tumours are currently of interest in reconstructive oral and maxillofacial surgery. Methods of autologous bone transplantation are well established for reconstruction of those defects. The reconstruction and remodeling of contour-shaping defects is more difficult due to atrophy and resorption of free-transplantable tissues. Artificially induced harmful effects have been reported on harvesting in the donor area. Further available methods of alloplastic reconstruction are computer-assisted design and manufacturing systems (CAD/CAM). The advantages of individual design and fabrication are obvious in the manufacturing of defect-specific implants. MATERIAL AND METHODS: In the present study the application of individual CAD based reconstructed bioceramic implants made of Bioverit II was evaluated in the region of the facial skull. Clinical results, patient acceptance and the analysis of the postoperative observation period of 30 months are reviewed. RESULTS: Altogether 25 individual Bioverit ceramics were implanted in the facial region. All patients were satisfied with the aesthetic results of the implantations after primary surgery. Three patients developed a need for further correction during the observation period; one implant fracture was observed. CONCLUSION: Finally, it can be stated that the preoperative expenditures in time, experts, technology and fabrication of individual CAD/CAM planned and manufactured implants are justified by the following advantages: fixed volume, reduced operating time, lack of donor morbidity, easy subsequent treatment of the material and the aesthetic results achieved. PMID- 16685573 TI - Field surveys of ozone symptoms on spontaneous vegetation. Limitations and potentialities of the European programme. AB - Within the European intensive forest monitoring programme, the native vegetation on permanent Level II plots has been monitored for visible ozone injuries. The main purpose of the programme is to assess the potential risks for the forest vegetation and the natural ecosystems at the intensive monitoring plots. During the first years of the programme the surveys were qualitative, reporting only the number and the name of the symptomatic species in selected Light Exposed Sampling Site. In 2003 a new plot design was tested, based on the distribution of a number of miniplots along the edge of the forest, so as to obtain quantitative findings about the occurrence and distribution of the symptoms. The problems that still persist are related to: (i) the forest edge assessed for ozone symptoms may have a different floristic composition from the Level II plot itself; (ii) the anthropic pressure and the disturbances affecting the forest edge alters the floristic composition; (iii) the variability of the plant composition in the forest edge, which makes comparability difficult between different sites; and (iv) the evaluation of symptoms in several species that have not yet been experimentally tested. Further difficulties are due to the fact that symptoms observed in the field are often aspecific and cannot, therefore, be attributed solely to the phytotoxic action of ozone. To improve the effectiveness of the European programme, it is necessary: (i) to individualise and select common sensitive plant species for homogeneous ecological regions; (ii) to enhance experimental activities to test the sensitivity of a large number of plant species. PMID- 16685574 TI - Wade Hampton Frost, pioneer epidemiologist. PMID- 16685576 TI - Derivative ratio as a measure of effect: sex over age of occurrence of myocardial infarction in Brazil. AB - The concept of relative risk (RR) as an incidence rate ratio is extended to a ratio of derivatives of cumulative occurrences of events with respect to any continuous variable whose domain is partitioned in sub-domains of study and referent groups to be contrasted. RR, thus expressed as a function, allows the identification of key values of the independent variable through which effect, as a relation between sub-domains, is modulated. The effect of sex over age of occurrence of myocardial infarction in a sample of Brazilian patients is analysed. Male sex, as contrasted to female, is acknowledged as a risk factor for the disease but it is known that this risk wanes with aging. Derivatives of cumulative occurrences with respect to age are calculated for men and women. Given the normal distribution of these occurrences, derivatives are found to be very similar to the corresponding normal density functions, with difference not greater than 0.005. This provides an easy way of identifying the derivatives whose ratio is likewise easily calculated. Results suggest that, in the Brazilian sample of patients studied, the greatest men to women RR is 1.9 at the age of 23.5 years and that at the age of 61.2 years risks become evens. PMID- 16685575 TI - Generalizability of relative risk estimates from a well-defined population to a general population. AB - We investigated the degree of generalizability of relative risk (RR) estimates for a sample from a well-defined population to a general population using actual data. A total of 45,452 men aged 40-69 years who completed a self-administered questionnaire were followed from 1990-1994 to the end of 1999-2000 in the Japan Public Health Center-based Prospective Study. We considered those who responded to the self-administered questionnaire (45,452 men) as representing the general population (population), and those who underwent a health check-up as representing a sample from a well- defined population (sample) (12,162 men). Exposure distributions, mortality rates, and the confounder-adjusted RRs of all cause mortality according to cigarette smoking or body mass index (BMI) were compared between the sample and the population using empirical sampling distributions from the population. The sample had significantly different prevalences of exposures and lower mortality rates than the population. Adjusted RRs were significantly higher in current smokers (RR = 1.83) and in subjects who smoked > or =40 cigarettes per day (RR = 2.67) in the sample than the respective values in the corresponding categories (RR = 1.48 and 1.62, respectively) of the population. The adjusted RR of those with the lowest BMI in the sample (RR = 1.30) were also significantly lower than those of the corresponding category in the population (RR = 2.06). Our results suggest that even for RRs, the extrapolation of estimates for a sample from a well-defined population to a general population may not be possible. PMID- 16685577 TI - Interviewer variability - quality aspects in a case-control study. AB - Quality assurance and quality control are important for the reliability of case control studies. Here we describe the procedures used in a previously published study, with emphasis on interviewer variability. To evaluate risk factors for acute pancreatitis, information including previous diagnoses and medication was collected from medical records and by telephone interviews from 462 cases and 1781 controls. Quality assurance procedures included education and training of interviewers and data validity checks. Quality control included a classification test, annual test interviews, expert case validation, and database validation. We found pronounced variations between interviewers. The maximal number of interviews per day varied from 3 to 9. The adjusted average (95% CI) number of diagnoses captured per interview of cases was 4.1 (3.8-4.3) and of controls 3.5 (3.4-3.7) (excluding one deviating interviewer). For drugs, the average (95% CI) number per interview was 3.9 (3.7-4.1) for cases and 3.3 (3.2-3.4) for controls (excluding one deviating interviewer). One of the fourteen interviewers deviated significantly from the others, and more so for controls than for cases. This interviewer's data ;were excluded. Nonetheless, data concerning controls more frequently needed correction and supplementation than for cases. Erroneous coding of diagnoses and medication was also more frequent among controls. Thus, a system for quality control of coding practices is crucial. Variability in interviewers' ability to ascertain information is a possible source of bias in interview-based case-control studies when "blinding" cannot be achieved. PMID- 16685578 TI - Prevalence of peripheral arterial disease - results of the Heinz Nixdorf recall study. AB - BACKGROUND: This report presents population-based estimates of the prevalence of peripheral arterial disease (PAD), chronic critical limb ischemia (CLI), and Moenckeberg's medial calcinosis (MC) in Germany. PATIENTS AND METHODS: From the year 2000 to 2003, a total of 4,814 subjects aged 45-75 years were included in the study. In 30 of the subjects (0.6%), determination of the ankle brachial index (ABI) was not possible, leaving 4,735 subjects (99.4%) in the data set. PAD was considered present in all subjects with an ABI < 0.9 in one leg, and/or a history of prior treatment for PAD. CLI was considered present if the highest ankle artery pressure measured < 70 mmHg. Prevalence of MC was calculated for ABI cut-off values of 1.3 and 1.5. FINDINGS: The overall prevalence of PAD according to the ABI criteria was 6.4% among men and 5.1% among women. After accounting for history of PAD, the prevalence increased to 8.2% among men and 5.5% among women. Taking the ABI criteria and medical history into account, males had a higher prevalence of PAD, with large increases in males aged 65-69 and 70-75 years. Chronic CLI was rare in the investigated population, and was found in only five older subjects (0.1%). With the criterion of ABI > 1.3, about 13.3% of males and 6.9% of females had MC. In contrast to PAD, the prevalence of MC did not increase with age. With the criterion of ABI > 1.5, MC was present in only 1.1% and 0.5% of men and women, respectively, but only 30 (0.6%) subjects had incompressible ankle arteries with a cuff pressure > 260 mmHg. CONCLUSION: Prevalences of PAD based only on ABI generally underestimate the true prevalence of PAD in population-based studies. CLI predominantly affects older subjects. In addition, cut-off values for MC must be newly determined. PMID- 16685579 TI - Associations between RAS Gene polymorphisms, environmental factors and hypertension in Mongolian people. AB - OBJECTIVE: To explore the association between RAS system genes (AGT, ACE and AT(1)R) polymorphisms, environmental factors and hypertension in Mongolian people. METHODS: On the basis of cross-sectional study, a case-control study with 299 hypertensives and 281 nomotensives was conducted, and the conditions of environmental factors were acquired by questionnaire. Serum lipid and insulin were detected by using biochemical experiments. Six single nucleotide polymorphisms of RAS system were genotyped by polymerase chain reaction/restriction fragment length polymorphism and polymerase chain reaction/single strand conformation polymorphism. RESULTS: Overweight or obesity, high serum TG and insulin resistance were risk factors of hypertension by single factor analysis. All the RAS genotype distributions were compatible with Hardy Weinberg expectations. There were no significant differences to be found between cases and controls for genotype frequencies or allele frequencies of the six polymorphisms of RAS system, except in men group, OR value of men carried ACE ID+DD genotype vs. men carried II genotype was 2.20 (95%CI 1.21-4.02), and OR of people who carried both ACE ID (or DD) and AGT M235T MT (or MM) vs. people with both ACE ID (or DD) and AGT M235T TT was 1.59 (95%CI 1.06-2.38). CONCLUSIONS: Overweight or obesity, dyslipidemia and insulin resistance were risk factors of hypertension in Mongolian people. ACE gene ID+DD genotype was the risk factor of hypertension in men group. People who carried both ACE ID (or DD) and AGT M235T MT (or MM) had more risk to have hypertension. PMID- 16685580 TI - Intrafamilial transmission of hepatitis C virus infection. AB - BACKGROUND: The role of intrafamilial HCV transmission is still controversial. METHODS: An overall sample of 2856 consecutive HCV-infected patients was studied. All index cases were interviewed to identify potential risk factors for transmission and underwent the following tests: HBsAg, anti-HBc, HIV, and, qualitative HCV-RNA and genotyping. RESULTS: Coinfection with HBsAg was recorded in 4.7%, and with HIV in 2.6% of the HCV-infected index cases. Anti-HCV was detected in 2.1% of the members of their original families, and in 13.8% of 2662 sexual partners. The overall rate of infection for offspring was 2.3%, but the risk was significantly higher when the index case was female. The risk for sexual partners was significantly higher when the risk factor for the index case was intravenous drug (IVD) use rather than blood transfusion. Logistic regression analysis showed that female gender and drug addiction in sexual partners of index case were independent factors significantly associated with HCV transmission to sexual partners. CONCLUSIONS: Among all family members of index cases, sexual partners of IVD users were at greatest risk of HCV infection. Sexual transmission may not be the main route of transmission though, since IVD use in the sexual partners themselves was independently associated with HCV infection. PMID- 16685581 TI - Trends in hepatitis B and hepatitis C virus among blood donors over 16 years in Turkey. AB - BACKGROUND: We document in the present study the trends over the sixteen years in HBV and HCV seroprevalence among blood donors in Turkish populations. METHODS: In this study, serologic test results of whole blood (n = 6.240.130) donors at 22 Red Crescent Centers between 1989 and 2004 were evaluated retrospectively. RESULTS AND CONCLUSION: The overall prevalence was 4.19% for HBsAg and 0.38% for HCV antibody during the study period. The annual prevalence of HBsAg gradually increased from 4.92% in 1989 to 5.23% in 1991 (p=0.001, t=21.00, CI(95), 17237 22490) and gradually decreased from that to 2.10% in 2004 (p=0.001, t=17.27, CI(95), 12869-21342). The seroprevalence of HCV antibody gradually increased from 18 per 10.000 in 1996, to 56 per 10.000 in 1998 (p=0.073, t=3.81, CI(95), 459.62 5721.23), while that decreased to 34 per 10.000 in 2004 (p=0.021, t=7.49, CI(95), 743.98-3980.11). The seroprevalence of hepatitis B and C has decreased markedly between 1989 and 2004 in Turkey. This could be related to the significant increase in the number of volunteer blood donors that increased from 135,779 to 197,815. PMID- 16685582 TI - Doping use among tertiary education students in six developed countries. AB - Data on doping among young non-professional athletes are scarce. In order to estimate the prevalence and predictors of doping use, a standardized, anonymous questionnaire was self-administered by 2650 tertiary education students from five European Union countries (Finland, France, Germany, Greece, Italy) and Israel. The reported usage rate of a doping agent (at least once) was 2.6%, with no significant variation in the frequency of doping reporting among the participating countries. Doping was, however, less common among students of biomedical schools (OR: 0.49, 95% CI: 0.27-0.89) and was higher among males (OR: 2.16, 95% CI: 1.25-3.74). Students, who use to drink coffee or recall frequent occasions of involvement in drunkenness episodes, were more likely (twice and three times, respectively) to report doping, and students using nutritional supplements or having participated in a major athletic event were more likely (four times and twice, respectively) to report doping in comparison with students who do not. Of note is the high odds ratio for reporting individual doping when having a friend who uses doping (OR: 8.61, 95% CI: 4.49-16.53). Given the large size of the physically active young individuals in the population and the small number of professional athletes, doping in the general population may be, in absolute terms, as sizeable problem as it is among the professional athletes. There was evidence that high-risk behaviour and supplement use increased the risk of doping. PMID- 16685583 TI - Underreporting of meningococcal disease incidence in the Netherlands: results from a capture-recapture analysis based on three registration sources with correction for false positive diagnoses. AB - In order to come to a reliable evaluation of the effectiveness of the chosen vaccination policy regarding meningococcal disease, the completeness of registrations on meningococcal disease in the Netherlands was estimated with the capture-recapture method. Data over 1993-1998 were collected from (A) mandatory notifications (n = 2926); (B) hospital registration (n = 3968); (C) laboratory surveillance (n = 3484). As the standard capture-recapture method does not take into account false positive diagnoses, we developed a model to adjust for the lack of specificity of our sources. We estimated that 1363 cases were not registered in any of the three sources in the period of study. The completeness of the three sources was therefore estimated at 49% for source A, 67% for source B and 58% for source C. After adjustment for false positive diagnoses, the completeness of source A, B, and C was estimated as 52%, 70% and 62%, respectively. The capture-recapture methods offer an attractive approach to estimate the completeness of surveillance sources and hence contribute to a more accurate estimate of the disease burden under study. However, the method does not account for higher-order interactions or presence of false positive diagnoses. Being aware of these limitations, the capture-recapture method still elucidates the (in)completeness of sources and gives a rough estimate of this (in)completeness. This makes a more accurate monitoring of disease incidence possible and hence attributes to a more reliable foundation for the design and evaluation of health interventions such as vaccination programs. PMID- 16685584 TI - Import of West Nile virus infection in the Czech Republic. AB - We report West Nile virus infection of the central nervous system in a 69-year old man, residing in North Moravia (Czech Republic), who visited the USA from 6 July to 31 August 2002. He developed fever with fatigue at the end of his US stay, and was hospitalized in Ostrava after his return on 3 September with fever (up to 39.5 degrees Celsius), fatigue, anorexia, moderate laryngotracheitis, dizziness, insomnia, blurred speech, and a marked bradypsychism. EEG demonstrated a slow bifrontal theta-delta activity, and CT of the brain a slight hydrocephalus. A significant increase of antibodies neutralizing West Nile virus was detected between the first (1:16) and second (1:256) blood serum sample. The patient recovered gradually and was released from hospital on 16 September. This is the first recorded human case of West Nile fever (WNF) imported to the Czech Republic. Nine similar cases of WNF import from the USA have already been reported in other European countries - France, Denmark, the Netherlands, and Germany. PMID- 16685585 TI - Clinical-environmental surveillance of legionellosis: an experience in Southern Italy. AB - In Italy, although the number of cases of legionellosis notified to the health authorities has significantly increased in recent years, the incidence is still believed to be underestimated. To verify the true frequency and identify the sources of infection, an active clinical-environmental surveillance program was instituted in three hospital facilities in Southern Italy. Between January 2001 and March 2005, a total of 1000 patients admitted to the three hospitals with a diagnosis of pneumonia were enrolled. The urinary antigen and anti-Legionella antibody titre were assayed in each subject, and direct searches for the microorganism were made in biological specimens. Legionellosis was found to be present in 5.9% of the patients. For each of the cases of legionellosis, microbiological surveys were made of the water supply in the public and/or private facilities involved. Overall, 197 water samples of hospital origin and 218 of community origin were analysed: Legionella spp was isolated in 44.2 and 36.7% of the cases, respectively. Comparison of our data with those of the routine surveillance system for the same area (only 7 cases during the period 1997-2000), showed that the frequency of legionellosis is grossly underestimated in Southern Italy. It is therefore necessary to set up more rigorous controls in both hospital and community facilities, so that timely preventive measures can be taken to avoid any further spread of the disease. PMID- 16685587 TI - Infertility, ovulation induction treatments and the incidence of breast cancer--a historical prospective cohort of Israeli women. AB - CONTEXT: Ovulation induction drugs may be associated with increased breast cancer risk. Results so far have been inconclusive. OBJECTIVE: To evaluate the association between infertility, exposure to ovulation induction drugs and the incidence of breast cancer. DESIGN: Historical prospective cohort and nested case control study. SETTING: Institutional practice PATIENTS: About 5,788 women attending five infertility centers in Israel between 1964 and 1984. INTENTION: Abstracting of medical records and telephone interviews. MAIN OUTCOME MEASURE: Breast cancer incidence was determined through linkage with the National Cancer Registry database. Standardized incidence ratios (SIRs) and 95% confidence intervals were computed by comparing the observed to the expected cancer rates in the general population. In addition, a nested case-control study within the cohort was performed with interviews of breast cancer cases and two matched controls. RESULTS: The study cohort included 120,895 women years of follow-up. Compared to 115.2 expected breast cancer cases, 131 cases were observed (SIR = 1.1; 95% CI 0.9-1.4). Risk for breast cancer was significantly higher for women treated with clomiphene citrate (SIR = 1.4; 95% CI 1.0-1.8). Similar results were noted when comparisons were carried out between treated and untreated women, and when multivariate models were applied. In the nested case-control study, higher cycle index (OR = 2.2; 95% CI 1.0-4.8) and treatment with clomiphene citrate (OR=2.7; 95% CI 1.3-5.7) were associated with higher risk for breast cancer. CONCLUSION: Infertility and usage of infertility drugs in general are not associated with increased risk for breast cancer. However, for infertile women treated with clomiphene citrate, breast cancer risk is elevated. PMID- 16685588 TI - Effects of estrogen receptor expression and histopathology on annual hazard rates of death from breast cancer. AB - BACKGROUND: Breast cancer incidence rates vary according to estrogen receptor expression (ER) and histopathology. We hypothesized that annual mortality rates from breast cancer after initial diagnosis (hazard rates) might also vary by ER and histopathology. METHODS: We accessioned the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER, 1992-2002) program to estimate hazard rates according to ER (positive and negative) and histopathology (duct, tubular, lobular, medullary, inflammatory, papillary, and mucinous types). We used spline functions to model hazard rates free of strongly parametric assumptions for ER negative and positive cases overall and by histopathology. RESULTS: Hazard rates for ER negative and ER positive cases were distinct and non proportional. At 17 months, ER negative hazard rates peaked at 7.5% per year (95% CI, 7.3-7.8% per year) then declined, whereas ER positive hazard rates lacked a sharp early peak and were comparatively constant at 1.5-2% per year. Falling ER negative and constant ER positive hazard rates crossed at 7 years; after which, prognosis was better for ER negative cases. Among ER positive and negative cases, there were proportional and non-proportional hazards according to histopathologic type, but the two basic ER-associated patterns were maintained. CONCLUSIONS: Hazard rates differed quantitatively and qualitatively according to ER and histopathology. These large-scale population-based results seem consistent with genomic studies, demonstrating two main classes of breast cancers with distinct prognoses according to ER expression. PMID- 16685589 TI - Health-related quality of life in breast cancer: A cross-cultural survey of German, Japanese, and South Korean patients. AB - PURPOSE: The aim of this study was to evaluate health-related quality of life (HRQOL) in breast cancer patients across countries, and to cross-culturally examine the impact of psychosocial factors on HRQOL. PATIENTS AND METHODS: A total of 413 women with breast cancer from Germany (n = 195), Japan (n = 112), and Korea (n = 106) completed a survey assessing HRQOL and HRQOL-related factors. HRQOL was measured using the Short-Form Health Survey (SF-8). Measures of psychological distress (Hospital Anxiety and Depression Scale and Impact of Event Scale-Revised), coping (Dealing with Illness Inventory-German Revised), and social support (Illness-Specific Social Support Scale-German Revised) were included. RESULTS: The effect of the factor country on physical QOL was seen to be significant, but small (P = 0.049, ES = 0.018). The scales of General Health (P = 0.023), Vitality (P = 0.004), and Role Emotional (P = 0.003) differed across countries, with the South Korean patients having lower scores compared to the German and Japanese patients. The nature of the impact of psychosocial factors on HRQOL did not differ greatly across countries except with regard to avoidance, however, the degree to which these factors influence HRQOL did differ greatly. Overall, depression, depressive coping, and problematic support showed a strong detrimental effect on the HRQOL of breast cancer patients. CONCLUSIONS: Results from this study suggest that strategies which target an improvement of HRQOL in cancer patients should also consider the patients' cultural and healthcare system contexts. Interventions are needed to improve detrimental psychosocial factors. PMID- 16685590 TI - XPD common variants and their association with melanoma and breast cancer risk. AB - There are suggestions in the literature that common variants in the XPD gene may be associated with an altered risk of melanoma and breast cancer. To establish if the XPD common variants Asp312Asn and Lys751Gln are associated with an increased melanoma or breast cancer risk we performed an association study based on genotyping 426 unselected patients with malignant melanoma (MM) and 1830 consecutive breast cancer cases and compared the results to 1262 geographically matched newborns, 621 adults from the region of Szczecin (unselected for age and cancer family history), 421 healthy adults age- and sex-matched with the melanoma cases and 511 healthy controls matched with the breast cancer patients from the region of Szczecin. Additionally we examined the prevalence of three additional XPD variants, Gly156Gly, Leu485Pro and Arg112His amongst the 421 unselected melanoma patients. All of the variants when evaluated singularly were found not to be associated either with melanoma or breast cancer risk in younger or older patients. A modest association was observed with breast cancer risk when the Lys751Gln_CC/Asp312Asn_AA genotype (OR=1.5, p<0.05) segregated together. Individuals harboring the Lys751Gln_CC/Gly156Gly_CC genotype were significantly over-represented among late-onset melanoma cases (OR=1.7, p<0.05). The results of analyses of linkage disequilibrium and haplotype frequency support the thesis that a combination of at least two SNPs (Lys751Gln_CC/Gly156Gly_CC or Lys751Gln_CC/Asp312Asn_AA) inherited as a haplotype was associated with disease. These two pairs of SNPs could therefore be regarded as a single hereditary unit that would have a very small probability of being disrupted by recombination. Additional studies are required to determine whether these particular changes can be associated with an increased risk of other malignancies at different sites of origin. PMID- 16685591 TI - Presence of epithelial cells in nipple aspirate fluid is associated with subsequent breast cancer: a 25-year prospective study. AB - Fluid and epithelial cells obtained from the breasts of non-pregnant, non lactating women by nipple aspiration, can be used for early diagnosis of breast neoplasms. However, since nipple aspirate fluid (NAF) with cells is obtainable from less than half of women sampled, the question arises: Is this method capable of targeting the women most likely to develop breast cancer? We approached this question with a 25-year prospective study to determine if subjects yielding NAF with or without epithelial cells were more likely to develop breast cancer during the follow-up period than subjects from whom no NAF or epithelial cells were obtained. Logistic regression analysis was used to determine relative risk (RR) with 95% confidence intervals (CI). The follow-up cohort of 972 was representative of the eligible cohort of 1605 for factors related to breast cancer risk and nipple aspiration outcome, and representative of the general population for breast cancer risk. After a mean follow-up period of 25 years, women with epithelial cells in NAF were significantly more likely to develop breast cancer (RR=1.92; CI=1.22-3.01; p1500 g had stage III ROP in Chang Gung Memorial Hospital at Kaohsiung (Taiwan, Republic of China). All six infants were male and had received oxygen supplementation during hospitalization. Only one infant had received previous surgery and frequent packed red blood cell transfusions. Three infants received laser photocoagulation or cryotherapy, and all infants have good outcomes regardless of whether they received treatment. This report provides convincing evidence for expanding the screening criteria in our nursery and emphasizes the importance of assessing the risk of ROP for larger preterm infants. PMID- 16685609 TI - A noise-sensor light alarm reduces noise in the newborn intensive care unit. AB - This one-group pre-post test design was to evaluate sound distribution and sudden peak noise frequencies (SPNs) and the associated events after using a noise sensor light alarm in a tertiary neonatal intensive care unit (NICU). The alarm is activated as the sound level reaches>or=65 A-weighted decibel (dBA). The environmental sound level was monitored continuously for a period of 1 week before and 1 month after using the alarm. The mean sound level in the incubator of patients receiving ventilator support before and after using the device were 58.0+/-0.6 and 56.4+/-0.7 dBA (t=8.619; p<0.001), whereas those at the radiant heated bed were 58.0+/-2.4 and 58.1+/-2.0 dBA (t=0.715; p=0.476). The percentage of observation time of sound levels<58 dBA increased by 28% in the incubator and 4% at the radiant heated bed (p<0.001). Episodes of SPN decreased from 630 to 185 times/d in the incubator and from 2069 to 748 times/d at the radiant heated bed after using the device. The noise-sensor light alarm effectively reduces sound level and episodes of SPN in the NICU. This may alleviate stress of noise for newborns with critical illness. PMID- 16685610 TI - Minimally invasive repair of pectus excavatum - shifting the paradigm? AB - PURPOSE: The "minimally invasive repair of pectus excavatum" (MIRPE) has increasingly become the standard operation for the repair of pectus excavatum. The aim of the present study was to report on our first-line postoperative results, including a survey of patients and to elucidate the acceptance of MIRPE. PATIENTS AND METHODS: All MIRPE patients, who were operated on between 2000 and 2004 in our departments, were included in a retrospective study. In 2002, a retrospective questioning of 57 patients was initiated by sending identical questionnaires separately to both the patients and their parents asking for individual reasons for choosing and/or agreeing to MIRPE. RESULTS: Complications were seen in 25 out of 84 MIRPE patients (mean 14 years, range from 5 to 20 years), but most of them were minor. Removal of the bar was necessary in 6 cases, due to bar displacement, secondary haematothorax, pericardial effusion, and local infection, respectively. No major complications occurred in the age group between 9 and 14 years. More than 90 % of the patients and their parents were satisfied or highly satisfied with the MIRPE procedure. CONCLUSION: General and specific complications are similar to other series and they decrease with the surgeon's experience. The most severe problem, occurring in older patients, is how to avoid dislocation of the pectus bar. In the light of the complication rate, the acceptance of MIRPE is still high, especially in adolescents and young adults who had refused operation with the open techniques. In this way, a shifting of a paradigm is taking place. PMID- 16685611 TI - Respiratory pressure monitoring as an indirect method of intra-abdominal pressure measurement in gastroschisis closure. AB - AIM OF STUDY: Abdominal compartment syndrome (ACS) is a rare but potentially fatal complication of gastroschisis closure. The liberal use of a staged reduction technique has become a well-established method of avoiding this problem. Unfortunately the use of silos is associated with a high rate of sepsis, prolonged ileus, and ventilation. A method of predicting an impending ACS would help surgeons to decide more objectively which patients would benefit from a staged reduction. A new simple method is presented here which predicts intra abdominal pressure based on airway pressure readings. METHOD: Over a four-year period, 34 neonates with gastroschisis underwent measurement of Pplateau respiratory pressures and simultaneous intra-vesical pressures. RESULT: The Pplateau pressures were approximately 10 cmH2O higher than any concurrent intra vesical pressure readings. ACS occurred, in one patient, when pressure measurements were above 15 cmH2O (intra-vesical) or 25 cmH2O (Pplateau). CONCLUSION: By measuring Pplateau pressures, it is possible to predict the intra abdominal pressure and hence avoid the development of an abdominal compartment syndrome on closing the abdominal wall in gastroschisis. PMID- 16685612 TI - Gastroschisis and omphalocele: retrospective study of initial postoperative management in the ICU. AB - AIM OF THE STUDY: The purpose of this study is to describe the management of infants with gastroschisis (G) and omphalocele (O) during the first 7 days after surgery. METHODS: A retrospective review of all cases of O or G managed at the ICU of the Robert Debre Teaching Hospital between January 1993 and July 2000 was carried out. PATIENTS: 29 infants with G, 15 with O (12 unruptured O [UO] and 3 ruptured O [RO]). RESULTS: Ventilatory support consisted of conventional mechanical ventilation (46 %) and/or in high-frequency oscillatory ventilation (61 %). After day 4, ventilatory requirements evaluated by mean airway pressure (MAP) differed significantly between G (n = 10/29) and O (n = 7/15; group vs. day of life, p = 0.04). The average of MAP measured on days 5, 6, and 7 was significantly higher in O than in G (14.7 +/- 3.0 versus 10.9 +/- 2.8, p < 0.01, respectively). Volume expansion was required at least once in 90 % of patients. Mean fluid requirements were significantly lower in UO than in G and in RO (41 +/ 31 ml/kg, 91 +/- 73 ml/kg, and 137 +/- 25 ml/kg, respectively; p = 0.02 for each comparison). Patients with G were significantly more likely to receive norepinephrine (59 % vs. 20 %, p = 0.027) than patients with O. Twenty-six infants with G (90 %) and 11 with O (73 %) were discharged alive from ICU. CONCLUSIONS: Haemodynamic instability can be expected in patients with G or RO, and ventilatory requirements were higher in infants with O than in infants with G during the first week after surgery. PMID- 16685613 TI - Crohn's disease in children. Preliminary experience with a laparoscopic approach. AB - BACKGROUND: Laparoscopic surgery in patients with Crohn's disease (CD) has been demonstrated to have advantages over a conventional approach in children. The aim of this study was to review the children treated for CD with a laparoscopic approach, to report our indications, the surgical procedure, the complications, and to compare the children with pancolitis or ileocaecal (segmental) Crohn's disease. PATIENTS AND METHOD: We reviewed the files of 11 children treated for CD in a single institution between 1999 and 2004 for a retrospective study of clinical and surgical data. Mann-Whitney U-test was used for statistical analysis of nonparametric data. RESULTS: Eleven children were operated. The average age when initial clinical symptoms became apparent was 12.1 years (range 6.6 - 15), and surgery was performed after an average of 3.4 years of disease (range 1 - 7.6). The surgical indications were stenosis in 6 cases, failure to thrive in 1 case (segmental CD, SCD group) and pancolitis refractory to medical treatment in 4 cases (pancolitis group, PCD group). Mean operative time was 207 minutes (range 140 - 270) for the SCD group and 285 minutes (range 260 - 300) for the PCD group (p < 0.05). Three cases needed a conversion to open surgery (2 in PCD group, one in SCD group), mainly in relation to anastomosis performed with an EEA stapler. The average length of surgical unit stay was 6.5 days (range 4 - 8) for the PCD group and 6.4 days (range 4 - 8) for the SCD group; average follow-up was 16 months (range 3 - 38). Two patients had a relapse of CD (stenosis of the anastomosis in one, skin fistula in the other). CONCLUSION: A laparoscopic approach for ileocolic resection in Crohn's disease is a feasible procedure, even in cases of pancolitis. We recommend an extra-corporeal anastomosis because, in relation to the inflammatory bowel, the mechanical anastomosis is not a safe procedure in cases of pancolitis. PMID- 16685614 TI - Peritonitis in childhood: clinical relevance of cytokines in the peritoneal exudate. AB - BACKGROUND: The aim of the study was to assess pro-inflammatory cytokine (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6]) and anti-inflammatory cytokine (interleukin 10 [IL-10]) responses in children with peritonitis secondary to hollow viscus perforation, and to evaluate the influence of peritoneal microbial status on cytokine expression and morbidity. METHODS: The peritoneal fluid of 13 children with perforated appendicitis was examined with qualitative analysis of bacteria, and measurement of cytokine levels, which were compared to cytokine plasma levels, over a five-day period following operation. RESULTS: All fluid specimens showed permanently elevated levels of TNF-alpha and IL-10. IL-6 tended to decrease to normal levels by the 5th postoperative day. Peritoneal exudate levels of TNF-alpha and IL-10 were more than 100 - 1000 times greater than those in plasma. The most common bacterial species isolated in the peritoneal fluid was Escherichia coli. Despite persisting high cytokine concentrations and bacterial load of the peritoneal cavity for 5 days postoperatively, the children recovered uneventfully and the systemic signs of infection disappeared rapidly. CONCLUSION: Neither the bacterial nor the pro inflammatory cytokine load of the peritoneal cavity proved to be associated with the clinical course. We hypothesize that in peritonitis in childhood a significant and clinically relevant cytokine-mediated inflammatory response is compartmentalized in the peritoneal cavity. Therefore adjuvant surgical measures in addition to appendectomy and intraoperative debridement seem not to be necessary, at least for peritonitis due to perforated appendicitis in children. PMID- 16685615 TI - Postoperative obstructive symptoms are common after Rehbein's procedure for Hirschsprung's disease. AB - Rehbein's procedure for Hirschsprung's disease has been questioned because several centimetres of aganglionic bowel are left behind, considered to result in obstructive symptoms. However, the reported outcome is similar to that after operations with other techniques. The purpose of this study was to review our experience of Rehbein's procedure during an 8-year period, focusing particularly on obstructive symptoms. We retrospectively studied 44 patients operated with Rehbein's procedure for biopsy-verified Hirschsprung's disease from October 1993 to October 2001. Three patients with total colonic aganglionosis and two patients who underwent total colectomy as a result of severe enterocolitis finally had a distal ileo-rectal anastomosis. Four (9 %) patients had a sphincteromyectomy before the Rehbein procedure. The patients' bowel function was evaluated by a questionnaire and an interview carried out 27 to 123 (mean 82) months after the operation. Two patients could not be traced and one patient, with total colonic aganglionosis, had an ileostomy at follow-up. Nineteen (46 %) of the 41 patients were treated with oral laxatives or enemas (12 patients), required repeated Botox injections (4 patients), or had undergone sphincteromyectomy (9 patients) due to obstructive symptoms. Eleven (27 %) of the 41 evaluated patients had soiling at least once a week at follow-up. Eleven (29 %) of 38 patients with a colorectal anastomosis had good bowel function without additional therapy. In conclusion, Rehbein's procedure for the treatment of Hirschsprung's disease is often complicated by obstructive problems. PMID- 16685616 TI - Technical problems and complications of a transanal pull-through for Hirschsprung's disease. AB - BACKGROUND: The purpose of this study was to evaluate the technical difficulties and complications of a transanal pull-through for Hirschsprung's disease. MATERIAL AND METHODS: This report was based on a multicentric retrospective study of 65 cases. Pull-through procedures were transanal Swenson or Soave procedures in 26 and 39 cases, respectively. RESULTS: Evaluation of the aganglionic level, peri-rectal dissection, and anastomosis were the three steps in the procedure where surgeons encountered difficulties. Such difficulties led to serious complications in 3 cases. A patient with a colon biopsy before the pull-through procedure had a postoperative pneumoperitoneum requiring a second laparoscopy for suture and washing. Another patient had peritonitis due to anastomotic leakage. Finally, a difficult rectal dissection in a neonate led to a urethral injury requiring secondary urethral repair. Only 41 of the 65 patients had no abdominal scars (63 %). CONCLUSION: We considered the transanal pull-through for Hirschsprung's disease to be a reliable technique. Nevertheless, it requires an urethral stent, precise dissection, careful anastomosis and selected indications in order to avoid major complications. PMID- 16685617 TI - An external device for faecal incontinence. AB - AIM: The aim of the study was to describe a simple external device that enables the incontinent patient to control the time, frequency and place of defecation. PRINCIPLE: The device is based on the principle of a "ball & socket" valve. The "ball" is an inflatable silastic balloon while the "socket" is the anorectal junction. The device can be used with minor modifications in patients with terminal colostomy to make them continent and avoid the need for colostomy bags. PATIENTS AND METHODS: The inflatable plug has been used successfully in eighteen incontinent children for a period ranging from six months to 8 years. The child decides the amount of air inside the inflatable plug that is comfortable and yet adequate to prevent soiling. This usually ranged between 10 to 25 cm of air. RESULTS AND COMPLICATIONS: To date, the device has been manufactured manually. All the children tolerated the plug without discomfort. Deflation of the balloon occurred after 3 - 5 days of use due to defective manufacturing. None of the patients developed ischaemia of the bowel or skin excoriations. CONCLUSION: This conservative, simple, inflatable plug enhances both qualitative and quantitative faecal continence in children with faecal soiling. An improvement in quality of life was also perceived by the patients and their parents. Better manufacturing may improve the results. PMID- 16685618 TI - Effective esophageal balloon dilation for esophageal stenosis in recessive dystrophic epidermolysis bullosa. AB - Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder of squamous epithelium that results in dystrophic scarring of the skin after minor trauma. RDEB is classified into two subtypes: Hallopeau-Siemens (HS) and non Hallopeau-Siemens (nHS). Although severe scarring of the skin is the most common and obvious complication of the disease, esophageal scarring with formation of long strictures may also develop. Treatment options for esophageal stenosis in patients with RDEB include steroids, hyperalimentation, esophageal dilation and replacement. This report describes a child who was dilated immediately after diagnosis of severe esophageal stenosis subsequent to nHS-RDEB and managed successfully. Endoscopic esophageal balloon dilation under fluoroscopic control was very useful for detecting the region of stenosis and bougienage. The literature on such injuries is reviewed here, and the problems associated with the treatment of children with esophageal stenosis associated with RDEB are discussed. PMID- 16685619 TI - Cystic lymphangioma of Retzius space manifested as acute abdomen. AB - The authors describe a rare pediatric case of cystic lymphangioma arising from the Retzius space. A 9-year-old boy underwent an appendectomy in a nearby hospital after a sudden onset of severe hypogastralgia. When laparotomy revealed a retroperitoneal mass, he was referred to our hospital. After diagnosis of a multicystic mass in the Retzius space, extirpation of the cystic lesion was performed. Histological evaluation of the resected specimens revealed cystic lymphangioma. The patient has been free of symptoms for 6 years since the operation. PMID- 16685620 TI - Morphology of the enteric nervous system in the hindgut of an infant with cloacal exstrophy. AB - The authors describe the morphology of the enteric nervous system in the hindgut of an infant with cloacal exstrophy. Cloacal exstrophy was diagnosed at 32 weeks' gestation using prenatal ultrasonography. The baby was delivered at 34 weeks' gestation and underwent a separation of the cecum from bladder halves, reapproximation of hemibladders, closure of the omphalocele and pubic symphysis, and a distal colostomy. Intestinal wall specimens were obtained at colostomy from the distal end of the rudimentary hindgut. Serial frozen sections were prepared for histochemical acetylcholinesterase staining. Histological investigations demonstrated a strikingly crowded, immature enteric ganglia and prominent bundles of wandering cholinergic nerves. These findings suggest the unique pathology of the enteric nervous system development in cloacal exstrophy, in which the rudimentary hindgut behaves as a blind alley of the migratory pathway for neural crest-derived cells during embryogenesis. Histological examinations of the hindgut enteric nervous system in cloacal exstrophy may be beneficial for evaluating the postnatal development of the distal colon which might be utilized for a pull-through procedure. PMID- 16685621 TI - Completely isolated, noncontiguous duplication cyst. AB - Enteric duplication cysts are hollow, epithelium-lined, cystic, or tubular structures that are intimately attached to a portion of the gastrointestinal tract. Completely isolated duplication cysts are an extremely rare variety of gastrointestinal duplications with their own exclusive blood supply. Only two such previous cases have been reported in the literature so far. We report a third case of completely isolated, giant duplication cyst with a vascular pedicle in a four-day-old neonate with a review of the literature. PMID- 16685622 TI - Congenital patent urachus associated with incomplete urethral duplication: a rare association. AB - Congenital patent urachus (CPU) is an uncommon anomaly. Blind incomplete urethral duplication is encountered more frequently than other types of urethral duplication (UD) in clinical practice. We describe a child with CPU in association with distal blind incomplete UD. A possible pathogenesis of this extremely rare coexistence is discussed. PMID- 16685623 TI - Late obstruction after subureteral collagen injection. AB - We present a case with distal ureteral obstruction after a single subureteral collagen injection. Postnatally, the girl presented with bilateral vesicoureteral reflux (VUR). At one year of age, a bilateral subureteral collagen injection was carried out. Postoperatively, the girl was followed up regularly and showed no pathological findings. But three years later, hydronephrosis with a distal ureteral obstruction on the right side was found. An ureteral reimplantation was performed on that side and the histological finding was a ureteral stenosis, consisting of collagen with a knotty sclerosis and a histiocytic and granulomatous reaction. This is the first case with a late obstruction at the ureteral orifice three years after a single collagen injection. We found an inflammatory reaction against the collagen, which led to a sclerosis of the implant. Further surveillance of patients after subureteral injections should focus on this kind of complication. PMID- 16685624 TI - Rokitansky-Kustner-Hauser syndrome - a case report. AB - Rokitansky-Kuster-Hauser syndrome, also called utero-vaginal aplasia, was first described at the beginning of the 19th century by Mayer (1829). It affects 1.2 % of girls and consists of a complete absence of the vagina with severe developmental anomalies of the uterus. Most often it consists of rudimentary cornua uteri, a normal female karyotype 46 XX and secondary female sexual characteristics. PMID- 16685625 TI - Ursodeoxycholic acid as a supplementary treatment in corrected biliary atresia. PMID- 16685628 TI - [Predictive value of an ischemia test in men and women with stable angina pectoris in clinical practice. Results of the heart catheter registry of the Working Society of Senior Hospital Cardiologists]. AB - BACKGROUND AND OBJECTIVE: Symptoms of coronary artery disease (CAD) and the accuracy of non-invasive tests differ between men and women. This study sought to evaluate the difference between the predictive value of a stress test in clinical practice for the diagnosis of significant coronary heart disease (CHD: stenosis > 50%) between women and men with stable angina. PATIENTS AND METHODS: 143,848 consecutive patients undergoing diagnostic coronary angiography at 99 hospitals during 2002 were included in the prospective cardiac catheter registry of the Working Party of Senior Hospital Cardiologists (Arbeitsgemeinschaft Leitende Kardiologische Krankenhausarzte [ALKK]). All patients with stress test and stable angina CCS class I-III (n=27387; 20.4%) were included, 10,911 (39.8%) of them female. 70.6% of women and 73.2% of men had a positive stress test. RESULTS: In 46.1% of women and 71.5% of men with positive test and stable angina had relevant CHD (p<0,001). Diabetes increased the prevalence of CHD in patients with a positive test both in women (65.5%) and men (80.5%), with CCS class III angina to 63.3% and 85.8%, respectively. CONCLUSIONS: In clinical practice a positive stress test in women with stable angina is associated significantly less often with clinically relevant CHD than in men. The low positive predictive value of 46.1% underlines the need for additional clinical features like diabetes or cardiac symptoms (CCS class) before invasive diagnosis is performed. PMID- 16685629 TI - [Pulmonary nocardiasis with abscesses spreading to cerebrum, cerebellum and orbits]. AB - HISTORY AND ADMISSION FINDINGS: A 71-year-old woman presented with suspected tuberculosis. She reported having productive coughs, unwanted weight loss and subfebrile temperature in the preceding 3 months. She was known to have chronic obstructive pulmonary disease treated with corticoids given systemically and by inhalation. She was a heavy smoker. INVESTIGATIONS: Computed tomography revealed a left apical lung abscess. In the further course of the disease magnetic resonance imaging of the head demonstrated multiple abscesses in both cerebral hemispheres and an abscess, 3.4 cm in diameter, in the right side of the cerebellum, as well as a intra-orbital tumor on the right. Needle aspirate of the eyeball grew Nocardia farcinica. TREATMENT AND COURSE: Over 3 weeks antimicrobial treatment was given with imipenem and amikacin, followed by oral cotrimoxazole for 12 months. The abscesses completely regressed and after 12 months no recurrence was demonstrated either radiologically or clinically. CONCLUSION: Although nocardiasis is rare in Germany it must be included in the differential diagnosis of pneumonia with abscesses. This is especially so if acid-fast bacilli are found. As the resistance pattern of N. farcinica to antibiotics varies, early treatment is essential with antibiotics to which it is sensitive. PMID- 16685631 TI - [Breast cancer]. PMID- 16685632 TI - [Dual antithrombotic therapy after implantation of coronary stents]. AB - Dual antiplatelet-aggregation treatment with aspirin and clopidogrel after coronary stent implantation is nowadays standard peri-interventional practice, although its use is not yet licensed for this indication in many European countries. Clopidogrel administration is initiated before PCI with a loading dose of 300 mg when given at least 6 hours before PCI, otherwise 600 mg. The required duration of combined (aspirin + clopidogrel) antiplatelet-aggregation treatment after coronary stent implantation depends on the type of stent and the pre existing disease. After bare-metal stent implantation dual antiplatelet medication is needed for at least 3-4 weeks, after drug-eluting stent implantation 6 months, after coronary brachytherapy 12 months, and 9 months after an acute coronary syndrome. These time intervals should also be respected before any elective surgical intervention. Early operations, because postponement is impossible, should be performed under antiplatelet-aggregation treatment after assessment of bleeding risk in the individual case. Premature termination of this treatment carries an increased risk of serious cardiovascular events, especially stent thrombosis and myocardial infarction. Prolonged antiplatelet-aggregation treatment is of benefit especially in patients with a high risk of serious cardiovascular events. Patients with an indication for long-term anticoagulation may require, during the period of highest risk of stent thrombosis after stent implantation, administration of combined aspirin, clopidogrel and anticoagulants with an INR target value in the lower therapeutic range. The increased risk of bleeding must be weighed up against the potential benefit. PMID- 16685633 TI - Bayesian analysis of correlated evolution of discrete characters by reversible jump Markov chain Monte Carlo. AB - We describe a Bayesian method for investigating correlated evolution of discrete binary traits on phylogenetic trees. The method fits a continuous-time Markov model to a pair of traits, seeking the best fitting models that describe their joint evolution on a phylogeny. We employ the methodology of reversible-jump (RJ) Markov chain Monte Carlo to search among the large number of possible models, some of which conform to independent evolution of the two traits, others to correlated evolution. The RJ Markov chain visits these models in proportion to their posterior probabilities, thereby directly estimating the support for the hypothesis of correlated evolution. In addition, the RJ Markov chain simultaneously estimates the posterior distributions of the rate parameters of the model of trait evolution. These posterior distributions can be used to test among alternative evolutionary scenarios to explain the observed data. All results are integrated over a sample of phylogenetic trees to account for phylogenetic uncertainty. We implement the method in a program called RJ Discrete and illustrate it by analyzing the question of whether mating system and advertisement of estrus by females have coevolved in the Old World monkeys and great apes. PMID- 16685634 TI - Intrinsic parent-offspring correlation in inbreeding level in a song sparrow (Melospiza melodia) population open to immigration. AB - The extent to which offspring resemble their parents in genotype and phenotype underpins patterns of genetic and phenotypic variation, selection, and evolution in natural populations. Genetic and phenotypic resemblance can clearly result from additive genetic variance and can be shaped by nongenetic parental and common environmental influences. In contrast, there is no straightforward expectation that inbreeding coefficient (f), a nonadditive component of genetic "quality," should be correlated across parents and offspring in sexually reproducing species or consequently cause resemblance across generations. Here, we report a significant parent f-offspring f correlation within a free-living pedigreed population of song sparrows (Melospiza melodia) on Mandarte Island, Canada. Across 15 years, relatively inbred parents had relatively inbred offspring on average. We show that rather than requiring nonrandom pairing with respect to f and kinship, parent f-offspring f correlations arise as an intrinsic consequence of random pairing within Mandarte's open population, where immigrants interbreed with Mandarte-hatched natives. However, on Mandarte, parent f offspring f correlations may have been exacerbated because relatively inbred individuals paired with more closely related mates than expected by chance. Such intrinsic parent f-offspring f correlations have major implications for the understanding of resemblance, selection, and evolution in natural populations. PMID- 16685635 TI - Asymptotic size determines species abundance in the marine size spectrum. AB - The majority of higher organisms in the marine environment display indeterminate growth; that is, they continue to grow throughout their life, limited by an asymptotic size. We derive the abundance of species as a function of their asymptotic size. The derivation is based on size-spectrum theory, where population structure is derived from physiology and simple arguments regarding the predator-prey interaction. Using a hypothesis of constant satiation, which states that the average degree of satiation is independent of the size of an organism, the number of individuals with a given size is found to be proportional to the weight raised to the power -2.05, independent of the predator/prey size ratio. This is the first time the spectrum exponent has been derived solely on the basis of processes at the individual level. The theory furthermore predicts that the parameters in the von Bertalanffy growth function are related as K ? L 1infinity PMID- 16685636 TI - Scale, environment, and trophic status: the context dependency of community saturation in rocky intertidal communities. AB - Our understanding of the relative influence of different ecological drivers on the number of species in a place remains limited. Assessing the relative influence of local ecological interactions versus regional species pools on local species richness should help bridge this conceptual gap. Plots of local species richness versus regional species pools have been used to address this question, yet after an active quarter-century of research on the relative influence of local interactions versus regional species pools, consensus remains elusive. We propose a conceptual framework that incorporates spatial scale and ecological interaction strength to reconcile current disparities. We then test this framework using a survey of marine rocky intertidal algal and invertebrate communities from the northeast Pacific. We reach two main conclusions. First, these data show that the power of regional species pools to predict local richness disintegrates at small spatial scales coincident with the scale of biological interactions, when studying ecologically interactive groups of species, and in generally more abiotically stressful habitats (e.g., the high intertidal). Second, conclusions of past studies asserting that the regional species pool is the primary driver of local species richness may be artifacts of large spatial scales or ecologically noninteractive groups of species. PMID- 16685637 TI - Estimating density dependence from time series of population age structure. AB - Population fluctuations are caused by demographic and environmental stochasticity, time lags due to life history, and density dependence. We model a general life history allowing density dependence within and among age or stage classes in a population undergoing small or moderate fluctuations around a stable equilibrium. We develop a method for estimating the overall strength of density dependence measured by the rate of return toward equilibrium, and we also consider a simplified population description and forecasting using the density dependent reproductive value. This generality comes at the cost of requiring a time series of the population age or stage structure instead of a univariate time series of adult or total population size. The method is illustrated by analyzing the dynamics of a fully censused population of red deer (Cervus elaphus) based on annual fluctuations of age structure through 21 years. PMID- 16685638 TI - Effects of productivity and disturbance on species richness: a neutral model. AB - Productivity and disturbance are major determinants of species diversity, and results from theoretical models predict that species richness should peak at intermediate levels of both factors. Such "unimodal" responses have been documented in many field and laboratory studies and have usually been attributed to differences among species in competitive ability and/or trade-offs between competitive ability and tolerance to disturbance. Here we show that most documented patterns of disturbance-richness and productivity-richness relationships, as well as the observed interactions between the two factors, can be explained by a simple neutral model where all species are ecologically identical and lack trade-offs in species characteristics. This finding suggests that current neutral theories can be extended to explain patterns of species responses to productivity and disturbance. PMID- 16685639 TI - Combining population-dynamic and ecophysiological models to predict climate induced insect range shifts. AB - Hundreds of species are shifting their ranges in response to recent climate warming. To predict how continued climate warming will affect the potential, or "bioclimatic range," of a skipper butterfly, we present a population-dynamic model of range shift in which population growth is a function of temperature. We estimate the parameters of this model using previously published data for Atalopedes campestris. Summer and winter temperatures affect population growth rate independently in this species and therefore interact as potential range limiting factors. Our model predicts a two-phase response to climate change; one range-limiting factor gradually becomes dominant, even if warming occurs steadily along a thermally linear landscape. Whether the range shift accelerates or decelerates and whether the number of generations per year at the range edge increases or decreases depend on whether summer or winter warms faster. To estimate the uncertainty in our predictions of range shift, we use a parametric bootstrap of biological parameter values. Our results show that even modest amounts of data yield predictions with reasonably small confidence intervals, indicating that ecophysiological models can be useful in predicting range changes. Nevertheless, the confidence intervals are sensitive to regional differences in the underlying thermal landscape and the warming scenario. PMID- 16685640 TI - Local and global costs of adaptive plasticity to density in Arabidopsis thaliana. AB - Although phenotypic plasticity is demonstrably adaptive in a range of settings, organisms are not perfectly plastic. Costs of plasticity comprise one factor predicted to counter the evolution of this adaptive strategy, yet evidence of costs is rare. Here, we test the fitness effects of plastic life-history and morphological responses to density and costs of this plasticity in recombinant inbred lines of Arabidopsis thaliana. Several costs of plasticity and homeostasis were detected. Of particular relevance, there was a significant cost of plasticity to active stem-elongation responses, an adaptive trait in many species. There was also a cost of plasticity to apical branch production at both high and low density, which resulted from the greater suppression of basal branching in genotypes with plastic apical branch production relative to genotypes with fixed apical branch production. The presence of a cost in multiple environments (i.e., a global cost) is predicted to counter the evolution of plasticity. Experimental segregating progenies such as the one used here are expected to have higher genetic costs of plasticity than arrays of genotypes sampled from natural populations because selection should remove genotypes with costs resulting from linkage disequilibrium or epistasis. The use of experimental progeny arrays therefore increases the ability to evaluate genetic costs. PMID- 16685641 TI - From hawks and doves to self-consistent games of territorial behavior. AB - Explaining the "prior-residence effect" (automatic owner status of individuals who arrived first in an area) was one of the very first applications of game theory in animal behavior. These models, however, predict paradoxical solutions where intruders always win, with no satisfactory explanation for the absence of such cases in nature. We propose a solution based on new developments in evolutionary game theory. A self-consistent model with feedbacks between individual behavior and population dynamics produces qualitatively different frequency-dependent selection on intruders (floaters) than on territory owners. Starting with an ancestral population with no respect for ownership, the most likely evolutionary end point is complete or partial respect. Conventional rules of conflict resolution thus can rely on "uncorrelated asymmetries" without differences in resource-holding power or territory value, although they will be strengthened by such differences. We also review the empirical literature on animal contests, testing whether asymmetries in resource-holding power are required to explain the observations. Despite much empirical effort, results remain inconclusive, because experiments are often unable to distinguish between the motivation of individuals to fight and the behavioral outcome of a contest. To help arrive at conclusive answers, we suggest a standardized empirical approach to quantify prior-residence effects. PMID- 16685642 TI - Multilocus association mapping using variable-length Markov chains. AB - I propose a new method for association-based gene mapping that makes powerful use of multilocus data, is computationally efficient, and is straightforward to apply over large genomic regions. The approach is based on the fitting of variable length Markov chain models, which automatically adapt to the degree of linkage disequilibrium (LD) between markers to create a parsimonious model for the LD structure. Edges of the fitted graph are tested for association with trait status. This approach can be thought of as haplotype testing with sophisticated windowing that accounts for extent of LD to reduce degrees of freedom and number of tests while maximizing information. I present analyses of two published data sets that show that this approach can have better power than single-marker tests or sliding-window haplotypic tests. PMID- 16685643 TI - Variance calculations for identity-by-descent estimation. AB - Nonparametric linkage strategies often involve estimation of identity by descent (IBD) with the use of affected sibling pairs. Methods for IBD estimation are well established and have been successful for mapping complex traits. However, the majority of linkage approaches involving IBD have focused on statistical testing, rather than on the effect estimates themselves. Through a bootstrap procedure developed for linkage-scan data sets, we provide standard errors for the estimated mean IBD that are broadly applicable. Applications that benefit from the availability of standard errors include effect-size estimates and confidence intervals; meta-analyses, including tests for heterogeneity; and discordant sibling-pair evaluation. We demonstrate the use of estimated mean IBD and its standard errors in the National Institute of Mental Health Human Genetics Initiative linkage samples for bipolar disorder and Alzheimer disease. Mean IBD and its standard errors are valuable tools for the further assessment and evaluation of linkage-scan samples involving complex disease. PMID- 16685644 TI - Online system for faster multipoint linkage analysis via parallel execution on thousands of personal computers. AB - Computation of LOD scores is a valuable tool for mapping disease-susceptibility genes in the study of Mendelian and complex diseases. However, computation of exact multipoint likelihoods of large inbred pedigrees with extensive missing data is often beyond the capabilities of a single computer. We present a distributed system called "SUPERLINK-ONLINE," for the computation of multipoint LOD scores of large inbred pedigrees. It achieves high performance via the efficient parallelization of the algorithms in SUPERLINK, a state-of-the-art serial program for these tasks, and through the use of the idle cycles of thousands of personal computers. The main algorithmic challenge has been to efficiently split a large task for distributed execution in a highly dynamic, nondedicated running environment. Notably, the system is available online, which allows computationally intensive analyses to be performed with no need for either the installation of software or the maintenance of a complicated distributed environment. As the system was being developed, it was extensively tested by collaborating medical centers worldwide on a variety of real data sets, some of which are presented in this article. PMID- 16685645 TI - Promoter mutations that increase amyloid precursor-protein expression are associated with Alzheimer disease. AB - Genetic variations in promoter sequences that alter gene expression play a prominent role in increasing susceptibility to complex diseases. Also, expression levels of APP are essentially regulated by its core promoter and 5' upstream regulatory region and correlate with amyloid beta levels in Alzheimer disease (AD) brains. Here, we systematically sequenced the proximal promoter (-766/+204) and two functional distal regions (-2634/-2159 and -2096/-1563) of APP in two independent AD series with onset ages < or =70 years (Belgian sample, n=180; Dutch sample, n=111) and identified eight novel sequence variants. Three mutations (-118C-->A, -369C-->G, and -534G-->A) identified only in patients with AD showed, in vitro, a nearly twofold neuron-specific increase in APP transcriptional activity, similar to what is expected from triplication of APP in Down syndrome. These mutations either abolished (AP-2 and HES-1) or created (Oct1) transcription-factor binding sites involved in the development and differentiation of neuronal systems. Also, two of these clustered in the 200-bp region (-540/-340) of the APP promoter that showed the highest degree of species conservation. The present study provides evidence that APP-promoter mutations that significantly increase APP expression levels are associated with AD. PMID- 16685646 TI - A mutation of beta -actin that alters depolymerization dynamics is associated with autosomal dominant developmental malformations, deafness, and dystonia. AB - Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for beta -actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia. PMID- 16685647 TI - Total-genome analysis of BRCA1/2-related invasive carcinomas of the breast identifies tumor stroma as potential landscaper for neoplastic initiation. AB - We have shown that the tumor microenvironment of sporadic breast cancer is diverse in genetic alterations and contributes to the cancer phenotype. The dynamic morphology of the mammary gland might be of special interest in hereditary breast/ovarian cancer syndrome (HBOC). We hypothesized that hotspots of loss of heterozygosity or allelic imbalance (LOH/AI) within the tumor stroma of BRCA1/2-related breast cancers provide an impaired mammary stroma that could facilitate later malignant transformation of the breast epithelium. We conducted a total genome LOH/AI scan of DNA derived from the epithelium and stroma of 51 BRCA1/2-related breast cancers, using 372 microsatellite markers. We compared these data with those from a set of 134 sporadic breast cancers. HBOC-related breast cancers accumulated significantly more genetic alterations than did sporadic breast cancers. BRCA1/2-related breast cancer stroma showed LOH/AI at 59.7% of all loci analyzed, similar to the average frequency of LOH/AI observed in the epithelium (66.2%). This is remarkably different from sporadic breast cancers, for which the average epithelial LOH/AI frequency (36.7%) far exceeds the average stromal LOH/AI frequency (28.4%) (P=.03). We identified 11 hotspot loci of LOH/AI in the BRCA1/2 stroma, encompassing genes such as POLD1, which functions in DNA replication, and SDHB. In a subset of samples, enriched for BRCA1 cases, we found 45.0% overall LOH/AI in the stroma, which was significantly higher than the 41.8% LOH/AI observed in corresponding epithelium (P=.04). Together, our data indicate that, in HBOC-related breast cancers, the accumulation of genomic instability in the cancer stroma coincides with that in the neoplastic epithelium, and we postulate that such a genetically unstable stroma might facilitate a microenvironment that functions as a landscaper that promotes genomic instability in the epithelium and, subsequently, neoplastic transformation. PMID- 16685649 TI - Cathepsin D deficiency is associated with a human neurodegenerative disorder. AB - Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D. PMID- 16685648 TI - Diplotype trend regression analysis of the ADH gene cluster and the ALDH2 gene: multiple significant associations with alcohol dependence. AB - The set of alcohol-metabolizing enzymes has considerable genetic and functional complexity. The relationships between some alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes and alcohol dependence (AD) have long been studied in many populations, but not comprehensively. In the present study, we genotyped 16 markers within the ADH gene cluster (including the ADH1A, ADH1B, ADH1C, ADH5, ADH6, and ADH7 genes), 4 markers within the ALDH2 gene, and 38 unlinked ancestry-informative markers in a case-control sample of 801 individuals. Associations between markers and disease were analyzed by a Hardy Weinberg equilibrium (HWE) test, a conventional case-control comparison, a structured association analysis, and a novel diplotype trend regression (DTR) analysis. Finally, the disease alleles were fine mapped by a Hardy-Weinberg disequilibrium (HWD) measure (J). All markers were found to be in HWE in controls, but some markers showed HWD in cases. Genotypes of many markers were associated with AD. DTR analysis showed that ADH5 genotypes and diplotypes of ADH1A, ADH1B, ADH7, and ALDH2 were associated with AD in European Americans and/or African Americans. The risk-influencing alleles were fine mapped from among the markers studied and were found to coincide with some well-known functional variants. We demonstrated that DTR was more powerful than many other conventional association methods. We also found that several ADH genes and the ALDH2 gene were susceptibility loci for AD, and the associations were best explained by several independent risk genes. PMID- 16685650 TI - The origin of EFNB1 mutations in craniofrontonasal syndrome: frequent somatic mosaicism and explanation of the paucity of carrier males. AB - Craniofrontonasal syndrome (CFNS) is an X-linked disorder that exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis, and additional minor malformations, but males are usually mildly affected with hypertelorism only. Despite this, males appear underrepresented in CFNS pedigrees, with carrier males encountered infrequently compared with affected females. To investigate these unusual genetic features of CFNS, we exploited the recent discovery of causative mutations in the EFNB1 gene, which encodes ephrin-B1, to survey the molecular alterations in 59 families (39 newly investigated and 20 published elsewhere). We identified the first complete deletions of EFNB1, catalogued 27 novel intragenic mutations, and used Pyrosequencing and analysis of nearby polymorphic alleles to quantify mosaic cases and to determine the parental origin of verified germline mutations. Somatic mosaicism was demonstrated in 6 of 53 informative families, and, of 17 germline mutations in individuals for whom the parental origin of mutation could be demonstrated, 15 arose from the father. We conclude that the major factor accounting for the relative scarcity of carrier males is the bias toward mutations in the paternal germline (which present as affected female offspring) combined with reduced reproductive fitness in affected females. Postzygotic mutations also contribute to the female preponderance, whereas true nonpenetrance in males who are hemizygous for an EFNB1 mutation appears unusual. These results highlight the importance of considering possible origins of mutation in the counseling of families with CFNS and provide a generally applicable approach to the combined analysis of mosaic and germline mutations. PMID- 16685651 TI - Reconstruction of a functional human gene network, with an application for prioritizing positional candidate genes. AB - Most common genetic disorders have a complex inheritance and may result from variants in many genes, each contributing only weak effects to the disease. Pinpointing these disease genes within the myriad of susceptibility loci identified in linkage studies is difficult because these loci may contain hundreds of genes. However, in any disorder, most of the disease genes will be involved in only a few different molecular pathways. If we know something about the relationships between the genes, we can assess whether some genes (which may reside in different loci) functionally interact with each other, indicating a joint basis for the disease etiology. There are various repositories of information on pathway relationships. To consolidate this information, we developed a functional human gene network that integrates information on genes and the functional relationships between genes, based on data from the Kyoto Encyclopedia of Genes and Genomes, the Biomolecular Interaction Network Database, Reactome, the Human Protein Reference Database, the Gene Ontology database, predicted protein-protein interactions, human yeast two-hybrid interactions, and microarray co-expressions. We applied this network to interrelate positional candidate genes from different disease loci and then tested 96 heritable disorders for which the Online Mendelian Inheritance in Man database reported at least three disease genes. Artificial susceptibility loci, each containing 100 genes, were constructed around each disease gene, and we used the network to rank these genes on the basis of their functional interactions. By following up the top five genes per artificial locus, we were able to detect at least one known disease gene in 54% of the loci studied, representing a 2.8-fold increase over random selection. This suggests that our method can significantly reduce the cost and effort of pinpointing true disease genes in analyses of disorders for which numerous loci have been reported but for which most of the genes are unknown. PMID- 16685652 TI - Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia. AB - DNA polymerase gamma (pol gamma ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol gamma (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G-->A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol gamma , that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)-deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID- 16685653 TI - Ordered genotypes: an extended ITO method and a general formula for genetic covariance. AB - Traditionally, the stochastic ITO transition matrices provide a simple general method for obtaining the joint genotype distribution and genotypic correlations between any specified pair of noninbred relatives. The ITO method has been widely used in modern genetic analysis; however, since it was originally derived for unordered genotypes, it is not very useful in some new applications -- for example, when one is modeling genomic imprinting and must keep track of the parental origin of alleles. To address these new, emerging problems, here we extend the ITO method to handle ordered genotypes. Our extended method is applied to calculate the covariance in unilineal and bilineal relatives under genomic imprinting, and some generalized linear functions of the transition matrices are given. Since the ITO method is limited to biallelic loci and to unilineal and bilineal relatives, we derive a general formula for calculating the genetic covariance using ordered genotypes for any type of relative pair. PMID- 16685654 TI - Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy. AB - In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7). PMID- 16685655 TI - Haplotype homozygosity and derived alleles in the human genome. AB - Haplotype-based techniques are being used to estimate the relative age of alleles -particularly in screening loci for signals of recent positive selection--but does this approach capture even coarse age differences? Using simulations and empirical data from the International HapMap Project, we show that a simple pairwise metric of haplotype homozygosity gives significantly higher mean values for human single-nucleotide-polymorphism alleles that appear to be derived than for those that appear to be ancestral, as determined by comparison with the chimpanzee genome. Our results support the use of haplotype-based techniques, such as extended haplotypic homozygosity, to assess the age of alleles. PMID- 16685656 TI - Elevated expression and genetic association links the SOCS3 gene to atopic dermatitis. AB - In a systematic analysis of global gene-expression patterns, we found that SOCS3 messenger RNA was significantly more highly expressed in skin from patients with atopic dermatitis than in skin from healthy controls, and immunohistochemical analysis confirmed a similar elevation of SOCS3 protein. Furthermore, we found a genetic association between atopic dermatitis and a haplotype in the SOCS3 gene in two independent groups of patients (P<.02 and P<.03). These results strongly suggest that SOCS3, located in a chromosomal region previously linked to the disease (17q25), is a susceptibility gene for atopic dermatitis. PMID- 16685657 TI - Contiguous gene deletion within chromosome arm 10q is associated with juvenile polyposis of infancy, reflecting cooperation between the BMPR1A and PTEN tumor suppressor genes. AB - We describe four unrelated children who were referred to two tertiary referral medical genetics units between 1991 and 2005 and who are affected with juvenile polyposis of infancy. We show that these children are heterozygous for a germline deletion encompassing two contiguous genes, PTEN and BMPR1A. We hypothesize that juvenile polyposis of infancy is caused by the deletion of these two genes and that the severity of the disease reflects cooperation between these two tumor suppressor genes. PMID- 16685658 TI - Fibulin-4: a novel gene for an autosomal recessive cutis laxa syndrome. AB - Cutis laxa is a condition characterized by redundant, pendulous, and inelastic skin. We identified a patient with recessive inheritance of a missense mutation (169G-->A; E57K) in the Fibulin-4 gene. She had multiple bone fractures at birth and was diagnosed with cutis laxa, vascular tortuosity, ascending aortic aneurysm, developmental emphysema, inguinal and diaphragmatic hernia, joint laxity, and pectus excavatum by age 2 years. Her skin showed markedly underdeveloped elastic fibers, and the extracellular matrix laid down by her skin fibroblasts contained dramatically reduced amounts of fibulin-4. We conclude that fibulin-4 is necessary for elastic fiber formation and connective tissue development. PMID- 16685659 TI - Considerations for genomewide association studies in Parkinson disease. PMID- 16685660 TI - Conflicting results regarding the semaphorin gene (SEMA5A) and the risk for Parkinson disease. PMID- 16685662 TI - No evidence for association with Parkinson disease for 13 single-nucleotide polymorphisms identified by whole-genome association screening. PMID- 16685661 TI - Genomewide association, Parkinson disease, and PARK10. PMID- 16685663 TI - A case-control association study of the 12 single-nucleotide polymorphisms implicated in Parkinson disease by a recent genome scan. PMID- 16685665 TI - A note on permutation tests in multistage association scans. PMID- 16685669 TI - Sildenafil in dialysis patients. AB - Some of the highest rates of erectile dysfunction are seen in patients with renal failure, especially those on dialysis. The treatment of erectile dysfunction has been revolutionized in the last decade by the introduction of sildenafil. The literature on sildenafil in dialysis patients is here reviewed. Sildenafil seems to be both safe and effective in the treatment of sexual dysfunction in the dialysis population. PMID- 16685670 TI - Informed consent for therapy and research in continuous renal replacement therapy: an international survey. AB - OBJECTIVE: To study the approach of health care workers (HCW) to informed consent for therapy and research in the field of continuous renal replacement therapy (CRRT). DESIGN: Administration of questionnaire. SETTING: Two International Courses on Critical Care Nephrology (CCN) held in Vicenza and Melbourne. PARTICIPANTS: Eight hundred and twenty one course participants. RESULTS: We obtained 349 analysable questionnaires (42.5% of participants). Only 22.5% of responders always obtain informed consent for CRRT; 70.3% just inform patients/relatives without seeking consent, 7.1% never obtain informed consent. In ICU patients, informed consent is considered 'good, correct and feasible' for therapy and for research by only 13% and 27% of responders, respectively. Consent for clinical research obtained from the next of kin or legal guardian is considered good, correct and feasible' by 56.3% of respondents, while 39.1% believe that next of kin or legal guardians can not really make informed decisions. Finally, nearly half of responders think that present rules hamper research in ICU. For many questions, significant variability of responses was found according to profession, specialty and origin of responders. CONCLUSIONS: In the field of CRRT, stated practice, beliefs and currently accepted ethical standards vary greatly according to profession, specialty and origin. A significant disagreement between what is widely promoted to be the 'correct' approach and what is currently done is evident. PMID- 16685671 TI - Design of a subcutaneous vascular access device using Ni-Ti SMA clamps for hemodialysis. AB - A subcutaneous vascular access device which utilizes an Ni-Ti shape memory alloy (Ni-Ti SMA) spring, has been developed as a new method for subcutaneous vascular access in the treatment of hemodialysis patients. Until now, the connection between the SVAD and needle cannot be externally monitored, and the clamp must be opened and closed with a percutaneous needle by a nephrologist. The SMA-SVAD accomplishes the opening and closing of its clamp using two Ni-Ti SMA springs, and a transcutaneous energy transmission system (TET) transmits energy to the Ni Ti SMA springs without the need for percutaneous wires. Two SMA springs open and close the clamp of the SMA-SVAD, without affecting any of the other parts of the system. Wasted thermal energy is reduced to minimal values via electrical regional heating methods. The state of the SMA-SVAD can be monitored according to the amount of power consumed by the external energy transmitter. In in-vitro experiments, when the clamp was opened and the pressure difference in the hemodialysis machine was set to 50 mmHg, water flow through SMA-SVAD reached 500 ml/min. The maximal surface temperatures of the SVAD and catheter were successfully maintained at proper levels (approximately 38-39 degrees C), approximately 2-3 degrees higher than the temperature of the surrounding tissues. The time elapsed from the initiation of energy transmission until the opening of the SMA clamp was 5 seconds. PMID- 16685672 TI - Residual blood loss in single use dialyzers: effect of different membranes and flux. AB - AIM/BACKGROUND: The aim of this study was to evaluate the residual blood loss in new type single use dialyzers under the usually prevailing conditions during hemodialysis and to investigate whether or not this loss is dependent on dialyzer membrane composition or flux characteristics. MATERIALS AND METHODS: In 158 hemodialysis (HD) patients, 158 single used dialyzers were studied in corresponding HD sessions. 52/158 dialyzers were made from modified cellulose (acetate, CA or triacetate, CTA) membrane and 106 from synthetic ones (58 with ethyl-vinyl-alcohol (EVAL), 48 with polyacrilonitrile (AN69)). Of those dialyzers 85/158 (58 EVAL+27CA) were low flux (LF) while the other 73 were high flux (HF). Patients underwent 4 hour HD sessions and at the end of the session blood was drawn for the measurement of hematocrit (Ht) and hemoglobin (Hb). Additionally, after the end of dialysis the used dialyzers were rinsed with 1000 mL of 0.05% NH(3) solution in distilled water. The wash was collected and subsequently Hb was measured using the benzidine method. From the volume of the solution and its concentration of Hb, total Hb of the solution was measured and blood loss in terms of red blood cell (RBC) volume was estimated by the use of the formula: RBC (mL) = Total Hb (g) in the solution x patient's Ht (ml/dL) / Patient's Hb (g/dL). For results to be comparable between dialyzers, RBC volume/m(2) of dialyzer membrane was expressed. In 5/158 patients blood loss was also estimated in 6 consecutive HD sessions using the same type of dialyzer. RESULTS: For the sum of the dialyzers, blood loss / dialyzer in terms of RBC volume, expressed as median (range), was 0.978 mL (0.01-23.9). There was statistically significant (p<0.001 or p<0.05) higher blood loss with the use of AN69 dialyzer than with the other three. RBC HF >RBC LF (p<0.001) constrained the first group of patients to use a 6% higher dosage of ferrum and 3.5% higher dosage of erythropoietin than the other group to achieve the optimal hemoglobulin values. No difference existed regarding RBC between CA, CTA and EVAL membranes. RBC measured in the small group of 5/158 patients for 6 consecutive HD sessions with the same dialyzer showed a wide range of RBC loss indicating an effect of the human factor. CONCLUSIONS: Blood loss during HD sessions due to residual blood cell volume inside dialyzers is usually slight using new type single use dialyzers but, sometimes, it can be significant and may contribute to the development or deterioration of preexisting iron deficiency anemia. The results of this study indicated that this loss can be attributed to the membrane composition of the dialyzer or to the human factor and has nothing to do with the ultrafiltration coefficient of the dialyzer. PMID- 16685673 TI - Effects of high-volume continuous hemofiltration on experimental pancreatitis associated lung injury in pigs. AB - OBJECTIVE: To investigate the effect of high-volume continuous hemofiltration on experimental pancreatitis associated lung injury (PALI) in pigs. METHODS: Animals had severe acute pancreatitis (SAP) induced by intraductal injection of sodium taurocholate and trypsin and were randomly assigned to three groups: 1) Controls, 2) Low-volume (LV) (20 ml/kg/h) continuous venovenous hemofiltration (CVVH) and 3) High-volume (HV) (100 ml/kg/h) CVVH at the onset of the induction of SAP. Systemic and pulmonary hemodynamic index were monitored intermittently. At the same time, arterial oxygen tension (PaO 2 ), cytokines and activated NF- e B levels of peripheral blood mononuclear cell were measured. After the animals died, the degree of microscopic lung injury was judged and scored. RESULTS: The median survival times of control, low-volume and high-volume groups were respectively 41 h, 50 h and 65 h. Temperatures in high-volume CVVH group were more steady than in control and LV CVVH groups (p<0.01). MPAP was significantly decreased by 4-5 mmHg in the HV CVVH group after 12 h of treatment. PaO(2) was significantly higher in HV group than in LV group at 6 h , 12 h, 24 h and 48 h (p<0.01). HV CVVH resulted in significant reductions not only in interstitial edema and atelectasis but also hemorrhages, hyaline membranes, microthrombi and total lung injury histology score. Plasma cytokines in the high-volume group were significantly lower than in the LV and control groups. In the HV group, the expression of NF- e B activation at 6 h, 12 h and 24 h was lower than in the control and LV groups respectively. CONCLUSION: CVVH can reduce pulmonary edema and the severity of PALI in pigs with high-volume CVVH being significantly better than low-volume CVVH. The beneficial effects of CVVH on arterial oxygenation and pulmonary function may be connected with improvements in systemic hemodynamics, reduction in plasma cytokine concentration and decreased activity of NF- e B in peripheral blood mononuclear cells. PMID- 16685674 TI - Observation and quantification of cavitation on a mechanical heart valve with an electro-hydraulic total artificial heart. AB - In previous studies, we investigated the cavitation phenomenon in a mechanical heart valve using an electro-hydraulic total artificial heart. With this system, a 50% glycerin solution kept at 37 degrees C was used as the working fluid. We reported that most of the cavitation bubbles were observed near the valve stop and were caused by the squeeze flow. However, in these studies, the effect of the partial pressure of CO(2) on the mechanical heart valve cavitation was neglected. In this study, in order to investigate the effect of the partial pressure of CO(2) on mechanical heart valve cavitation using an electro-hydraulic total artificial heart, we controlled the partial pressure of the CO(2) in vitro. A 25 mm Medtronic Hall valve was installed in the mitral position of an electro hydraulic total artificial heart. In order to quantify the mechanical heart valve cavitation, we used a high-speed camera. Even though cavitation intensity slightly increased with increases in the PCO(2) at heart rates of 60, 70 and 100 bpm, throughout the experiment, there was no significant difference between the PCO(2) and cavitation intensity. PMID- 16685675 TI - In vitro set-up of modified Blalock Taussig shunt: vascular resistance-flow relationship. AB - BACKGROUND: A modified Blalock-Taussig (mBT) shunt is an anastomosis created between the systemic and pulmonary arterial tree in order to improve pulmonary blood flow in neonates and children with congenital heart disease. The aim of this study was to assess vascular resistance-flow relationship in an in vitro set up of a modified Blalock Taussig shunt. METHODS: A shunt set-up was constructed with the vessels of a sheep. A modified BT shunt was anastomosed between an innominate (brachiocephalic) and a right pulmonary artery. A Medos pump (ventricular assist device) was used to create pulsatile flow. Three different mean pulmonary artery flow rates (Q PA ) were applied. Once mean pulmonary and mean aortic flows (Q AO ) were fixed, shunt flow rates for twelve different pulmonary vascular resistances (R p ) were investigated. RESULTS: For all three pulmonary flow rates, the shunt flow decreased with increasing pulmonary resistance. In addition, systemic flow decreased compared to pulmonary flow. When pulmonary flow rate was set at 800 ml/min and aorta flow rate at 900 ml/min, the distribution of flow between pulmonary and systemic organs flow rates ranged between 69% - 70% and 30% - 31% respectively. Similarly, when both pulmonary and aorta flow rates were set at 900 ml/min, pulmonary and systemic organ flows ranged between 73% - 77% and 23% - 27% respectively. For pulmonary and aorta flow rates of 1000 ml/min and 900 ml/min, respectively, the distribution of flow between pulmonary and systemic organ flow rates varied between 79% - 83% and 17% 21% respectively. CONCLUSION: Knowledge of the relationship between vascular resistances and flow in this surgically created in vitro mBT shunt set-up may be helpful in the clinical management of the patients whose survival is crucially dependent on the blood flow distribution between the pulmonary and systemic circulation. PMID- 16685676 TI - Formation of a sheet-shaped organoid using rat primary hepatocytes for long-term maintenance of liver-specific functions. AB - In recent years, use of hepatocyte aggregates has led to development of a hybrid artificial liver support system (HALSS) that has high performance. However, in general, their thickness is 100 microm or more, and generation of a dead cell layer due to oxygen exhaustion inside the aggregates has been a universal problem. The present study proposes a novel organoid culture method with better performance than previous organoid culture methods by forming a sheet-shaped organoid (organoid-sheet) with a thickness of approximately 100 microm. The cell number of the organoid-sheet was maintained at approximately 75% of the initial number at 4 days of culture. On the other hand, that of a cylindrical organoid (cylindroid), which formed inside of a plasma separation hollow fiber with 285 microm inner diameter in our previous study, decreased to approximately 50% within 2 days. The ammonia removal rate of the cells in the organoid-sheet was higher than that of the cells in the cylindroid on the first day, but it decreased during the culture time. At day 15, the rate was reduced by almost 50% with respect to the value on the first day. The cells in the cylindroid displayed a lower ammonia removal rate. A significant difference was not observed between the albumin synthesis rates of the two cultures on the first day. However, over a period of time the cells in the organoid-sheet showed a higher albumin synthesis rate than cells in the cylindroid. As this novel organoid maintains these functions for at least 1 month, it is expected to be applied for the development of a HALSS with higher performance. PMID- 16685677 TI - Ischemic type biliary lesions in histidine-tryptophan-ketoglutarate (HTK) preserved liver grafts. AB - Ischemic type biliary lesions lead to considerable morbidity following orthotopic liver transplantation. The exact pathogenesis is unknown. One major hypothesis is that insufficient perfusion of the arterial vessels of the biliary tree, especially under perfusion with the high viscous University of Wisconsin solution, might be responsible for ischemic type biliary lesions. Due to low viscosity, HTK solution is reported to have a lower incidence of biliary complications. However, there is no data concerning ischemic type biliary lesions in HTK preserved livers. In this paper we report our results after orthotopic liver transplantation with special regard to ischemic type biliary lesions in liver grafts preserved with HTK solution. Between 09/1997 and 01/2005 300 liver transplantations were performed in our center. Thirty-two (10.7%) liver grafts were preserved with HTK solution, 268 (89.3%) were preserved with UW solution. Six and 43 grafts showed ischemic type biliary lesions after orthotopic liver transplantation in HTK- (18.8%) and UW- (16.0%) groups, respectively (p=0.696). There was no statistical significant difference between the two groups. Donor related factors, recipient age, indication for transplantation, transplantation technique, immunosuppression and ischemia time were comparable in both groups. Ischemic type biliary lesions occurred with the same frequency in HTK preserved livers compared to UW preserved organs. We suggest that low viscosity of the preservation fluid by itself does not guarantee reliable perfusion of the small arteries of a liver graft and a pressure perfusion might be beneficial even in HTK solution. PMID- 16685678 TI - Splenic rupture following colonoscopy in a hemodialysis patient. PMID- 16685679 TI - Reply to: Comments on paper: 'Hybrid test bench for evaluation of any device related to mechanical cardiac assistance'. PMID- 16685680 TI - Julio Cortazar quotes on normal and abnormal movements: magical realism or reality? AB - Together with Mario Vargas Llosa and Gabriel Garcia Marquez, Julio Cortazar was one of the most representative authors of the Latin American magical realism genre. Within his extensive body of work, many descriptions of characters suffering physical disabilities, as well as situations suggesting such medical conditions, can be extracted. In this review, two short stories by Cortazar are presented. In the first one, the main character could easily be a man suffering from corticobasal degeneration; in the second, an old woman with symptoms suggestive of progressive supranuclear palsy is clearly depicted. Despite the fact that one of the main ingredients in Cortazar's magical realism is fiction, cases described here fit real medical conditions quite well, making it hard to believe that they represent purely fantastic descriptions rather than the product of Cortazar's inquisitive observation and the description of real patients. PMID- 16685681 TI - Effect of celery (Apium graveolens) extracts on some biochemical parameters of oxidative stress in mice treated with carbon tetrachloride. AB - Extracts of celery leaves and roots in ether, chloroform, ethyl acetate, n butanol and water were evaporated to dryness and dissolved in 50% ethanol to make 10% (w[sol ]v) solutions. The potential protective action of the extracts was assessed by the corresponding in vitro and in vivo tests. In the in vitro experiments crude methanol extracts were tested as potential scavengers of free OH* and DPPH* radicals, as well as inhibitors of liposomal peroxidation (LPx). Analogous experiments were also carried out with the extracts of celery root, for comparison. The results obtained show that both the extracts of root and leaves are good scavengers of OH* and DPPH* radicals and reduce LPx intensity in liposomes, which points to their protective (antioxidant) activity. In vivo experiments were concerned with antioxidant systems (activities of GSHPx, GSHR, Px, CAT, XOD, GSH content and intensity of LPx) in liver homogenate and blood of mice after their treatment with extracts of celery leaves, or in combination with CCl4. On the basis of the results obtained it can be concluded that the examined extracts showed a certain protective effect. Of all the extracts the n-butanol extract showed the highest protective effect. Combined treatments with CCl4 and extracts showed both positive and negative synergism - inducing or suppressing the impact of CCl4 alone. The differences observed in the action of particular extracts are probably due to the different contents of flavonoids and some other antioxidant compounds. PMID- 16685682 TI - Isolation of flavonoids, a biscoumarin and an amide from the flower buds of Daphne genkwa and the evaluation of their anti-complement activity. AB - As part of an ongoing study aimed at identifying the anti-complement active compound from the flower buds of Daphne genkwa, ten flavonoids, one biscoumarin and one amide were isolated. Their structures were identified from spectroscopic and physicochemical data as genkwanin 5-O-beta-d-primveroside (1), apigenin 7-O beta-d-glucuronide (2), genkwanin 5-O-beta-d-glucopyranoside (3), apigenin 5-O beta-d-glucopyranoside (4), genkwanin (5), apigenin (6), luteolin 7-methyl ether (7), luteolin (8), daphnoretin (9), velutin (10), 7-methoxyacacetin (11) and aurantiamide acetate (12). Among them, compounds 9, 10 and 12 were isolated from this plant for the first time. The anti-complement activity of these compounds was tested against the classical pathway of the complement system. Among the compounds, daphnoretin (9) exhibited significant anti-complement activity with an IC(50) value of 11.4 microm, whereas the other compounds were not active in the assay. PMID- 16685683 TI - The pull test: a history. AB - The pull test (PT) is used as a measure of postural instability in Parkinson's disease (PD) and other movement disorders. In 1987, it was incorporated into the Unified Parkinson's Disease Rating Scale (UPDRS), a scale used to measure the severity and treatment response in PD both in research studies and in clinical practice. However, the origins of the observation of postural instability in movement disorders and the attempt to quantify it are much older. Here, we trace the history of postural instability first described as a feature of PD by Romberg in 1853. Attempts to evaluate postural instability began with the first measurement by Charcot in the 1880s by pulling the clothes of patients and progressed to the push on the sternum by Hoehn and Yahr in the 1960s. Eventually, this evolved into the formal PT proposed by Fahn in the 1980s. Despite the widespread use of the PT as part of the UPDRS, variability exists in its execution. Recommendations have been made for training of examiners in clinical trials to improve its accuracy in assessing postural instability. We agree with improving PT technique for clinical trials and advocate for its routine use in clinical practice when diagnosing and treating movement disorders. Further, we propose the name "Fahn pull test" for the maneuver based on his significant contribution to its development. PMID- 16685684 TI - Postural tremor suppression is dependent on thalamic stimulation frequency. AB - Deep brain stimulation (DBS) of the ventral intermediate nucleus (VIM) reduces tremor in people with essential tremor (ET), yet the dependence of tremor suppression on stimulation frequency remains unclear. To address this issue, we tested tremor suppression for three 15-second measurements during a variety of stimulation frequencies in 11 ET patients treated with VIM DBS. Stimulation frequencies at or above 100 Hz produced maximal benefit; higher frequencies provided no additional benefit. If this short-term measure predicts long-term response in routine activities at home, then this stimulation frequency setting will prolong battery half-life compared to higher frequency settings. These findings suggest that ET patients treated with VIM DBS may receive adequate benefit from stimulation frequencies about 100 Hz and this setting compared to commonly used higher settings will prolong battery life of surgically implanted pulse generators. PMID- 16685685 TI - Evaluation of clinical diagnostic criteria for psychogenic movement disorders. AB - Previous studies showed that there are certain features suggestive of a psychogenic disorder, and Fahn and Williams proposed criteria for psychogenic movement disorders. Data on the sensitivity and specificity of these criteria are lacking. We expanded on the Fahn and Williams criteria to create a new set of diagnostic criteria. We retrospectively reviewed 79 patients in a movement disorders specialty clinic. We applied the proposed diagnostic criteria to both cases and controls and analyzed sensitivity and specificity. The diagnostic criteria correctly identified "clinically probable" (or better) psychogenic movement disorders with a sensitivity of 83% and specificity of 100%. For "clinically possible" or greater, sensitivity was 97% and specificity was 96%. In addition, a significantly higher proportion of patients with psychogenic disease were female and reported either a family history of neurological disease or other exposure to neurological disease (P = 0.001 and 0.01, respectively). The diagnostic criteria are simple to apply and have high sensitivity and specificity for psychogenic movement disorders. Also, we report a correlation between previous exposure to a disease model and psychogenic disease. PMID- 16685686 TI - Restless legs syndrome: epidemiological and clinicogenetic study in a South Tyrolean population isolate. AB - Genetic contributions to restless legs syndrome (RLS) have been consistently recognized from population and family studies. To determine the clinical and genetic features of RLS in a population isolate and explore linkage to three previously described susceptibility loci on chromosomes 12q, 14q, and 9p, respectively, an isolated population in the South Tyrolean Alps was identified and 530 adults participated in the study. Using a two-step strategy, 47 patients with idiopathic RLS were ascertained. The prevalence in the population was 8.9%. Twenty-eight patients (59.6%) had at least one affected first-degree relative and were classified as hereditary cases. In a single extended pedigree, linkage to known RLS loci was investigated specifying autosomal dominant and recessive models; parametric and nonparametric multipoint linkage scores were computed. None of the calculated linkage scores was suggestive of linkage between RLS and any of the three investigated loci. This study was conducted in a population isolate providing for a homogeneous genetic and environmental background. The absence of a suggestive linkage signal at the three known RLS susceptibility loci is indicative of further locus heterogeneity of this frequent disorder and encourages further studies to unveil the genetic causes of RLS. PMID- 16685687 TI - Minor depression and brain perfusion images in Parkinson's disease. AB - Depression is common in individuals with Parkinson's disease. However, the pathophysiology of depression in Parkinson's disease remains obscure. Here we compared brain perfusion images of Parkinson's disease patients with and without depression to investigate correlations between depression and brain perfusion images in Parkinson's disease. We divided 40 consecutive patients with Parkinson's disease into two groups: patients with minor depression (n = 22) and patients without depression (n = 18). We then compared brain perfusion images between the two groups. As a result, hypoperfusion of the left superior and inferior frontal gyrus was demonstrated in depressed patients. These results were partially in agreement with previous studies on de novo and parkinsonian major depression. We could not conclude on whether pathophysiological mechanisms differed between de novo depression and depression with Parkinson's disease, and between major and minor depressions. PMID- 16685688 TI - Determination of chitin and protein contents during the isolation of chitin from shrimp waste. AB - Accurate determination of chitin and protein contents in crustacean biomass and the intermediate products during the industrial isolation of chitin cannot be made directly from the total nitrogen content, unless the appropriate corrections are applied. This method, however, is affected by the presence of other nitrogen containing chemical species that are formed endogenously or by the action of microorganisms during the handling of the sample. Therefore, an alternative rapid method to estimate the contents of these components can be very useful both in research and in various fields of application. An original method has been developed to address this problem. The method consists of the development of a set of equations based on the stoichiometric contents of nitrogen of chitin and protein whereby the amounts of each component can be estimated from the value of the total nitrogen content, provided the rest of the proximate composition of the sample is accurately known. In order to validate the procedure, a set of model mixtures of pure chitin and protein concentrate in the solid state, both extracted from shrimp head waste, are used. Excellent agreement between the predicted and real values of chitin and protein are obtained (R2=0.98, slope=0.90). When the proposed method is tested in the analysis of real samples obtained from five different processing protocols of pretreatment of raw shrimp head, it is found that in general the values of protein and chitin contents throughout the various stages of the process vary as expected. [GRAPH: SEE TEXT] Variation of the measured total nitrogen versus calculated stoichiometric total nitrogen of the chitin-protein mixtures. PMID- 16685689 TI - Biodegradable heparin-loaded microspheres: carrier molecular composition and microsphere structure. AB - Microspheres of amphiphilic triblock polymers PLLA-PEG-PLLA were investigated as carriers for heparin delivery. Two series of PLLA-PEG-PLLA triblock were synthesized and prepared into microspheres with heparin loaded. The microspheres were hollow and the surface morphology varied from smooth to porous. The pore size increased with increasing PEG content. The microsphere size distribution showed that higher PEG content increased the average microsphere size. The release rate of heparin was closely related to the surface morphology of the microspheres. DSC spectra showed that both cold crystalline temperature (Tc) and crystalline melting temperature (Tm) of heparin-loaded microspheres were related to the copolymer composition and the Tc was lower than those of corresponding pure microspheres. [IMAGES: SEE TEXT] PMID- 16685690 TI - Effect of dopamine agonists on fatigue and somnolence in Parkinson's disease. AB - Dopamine agonists are becoming increasingly important in the treatment of early and advanced symptoms of Parkinson disease (PD). Although dopamine agonists are known to increase somnolence, their effect on fatigue and the relationship between fatigue and somnolence have not been investigated thoroughly. The objective of this study is to quantitatively measure fatigue in patients with PD treated with dopamine agonists and to correlate fatigue with somnolence. Fifteen patients with PD (mean age, 60.6 +/- 9.7 years; mean disease duration, 4.1 +/- 1.9 years) underwent a continuous (30-second) motor task using four muscle groups before and after 3 months of treatment with dopamine agonists. A fatigue index, defined as the decay of maximal force during continuous exercise, was calculated. Findings were compared with 15 healthy, age-matched control subjects. Patients also completed the Multidimensional Fatigue Inventory (MFI), the Epworth Sleepiness Scale (ESS), and the Hamilton Depression Scale at the same time points. Mean fatigue index (FI) before treatment was significantly higher in PD patients than controls (31.37% +/- 3.81% vs. 23.39% +/- 3.03%, P < 0.001). There was no significant between-group difference after 3 months of treatment. There was no difference in FI of the more affected side before and after treatment (33.33% +/- 6.18% vs. 34.08% +/- 5.43%, P > 0.1). No significant change in MFI scores were noted after treatment, although scores on the ESS increased significantly (6.6 +/- 2.63 vs. 11.7 +/- 5.16; P < 0.05). Fatigue is prevalent in patients with PD but is not influenced by dopamine agonists. Somnolence cannot be attributed to the increase in fatigability and apparently involves a different mechanism. PMID- 16685691 TI - Eyelid movements during blinking in patients with Parkinson's disease. AB - We examined eyelid movements during spontaneous, voluntary, and trigeminal reflex blinks in 16 patients with mild to moderate Parkinson's disease (PD) off medication and 14 controls. Voluntary and reflex blink amplitudes tended to be smaller than normal for PD patients, whereas eyelid kinematics (amplitude-maximum velocity relationship) for all three blink types were normal. Spontaneous blink rate was less than normal for 10 patients and abnormally high for 6 patients. A significant positive correlation between spontaneous blink amplitude and blink rate was found. These observations suggest that PD modifies the gain of a premotor blink circuit shared by spontaneous, voluntary, and reflex blinks. PMID- 16685692 TI - Valvular heart disease in Parkinson's disease treated with ergot derivative dopamine agonists. AB - Valvular heart disease, associated with ergot derivative dopamine agonists, has been reported in patients with Parkinson's disease (PD). There are few comparative studies on the frequency, severity, and dose dependency of valvular disease associated with ergot derivatives. We analyzed these factors in 58 PD patients who were taking ergot derivatives (22 were taking bromocriptine and 36 were taking pergolide) and compared them with 20 age-matched controls based on the two-dimensional echocardiographic findings. Aortic, mitral, and tricuspid valvular thicknesses, as well as tenting areas and tenting distance of the mitral valve were measured. We also assessed the correlation between the cumulative or daily doses of ergot derivatives and each valvular parameter. There was no significant increase in the frequency of valvulopathy in the PD patients taking bromocriptine or pergolide. We identified a positive correlation between the daily dose of pergolide and the tenting area of the mitral valve (r = 0.385; P = 0.020). However, other valvular parameters were not found to correlate with the age, disease duration, treatment duration, or dosage of medication. Our study failed to uncover definitive harmful effects of ergot derivative dopamine agonists on cardiac valves; this is considering the relatively lower daily doses than other reported cases. To establish a dosage dependency with valvulopathy, a more detailed study with higher doses of ergot derivatives for longer duration of treatment may be needed. PMID- 16685693 TI - Bayesian meta-analysis and meta-regression for gene-disease associations and deviations from Hardy-Weinberg equilibrium. AB - Violation of Hardy-Weinberg equilibrium (HWE) can raise doubts about the validity of the conclusions from genetic association studies. However, for most currently performed gene-disease association studies, the available tests have low power to detect deviations from HWE. We consider this issue from a meta-analysis perspective, and suggest an approach to estimate the deviation and investigate its relationship with the observed genetic effects. Different degrees of deviation from HWE have previously been proposed as a potential source of heterogeneity across studies. We present a hierarchical meta-regression model that can be applied to test this assumption, using the concept of the fixation coefficient. We re-analyse seven meta-analyses to illustrate these methods. The uncertainty in the genetic effect estimate tended to increase once the fixation coefficient was taken into account. Dependence of the genetic effect size on the deviation from HWE was found in one meta-analysis, while in the other six examples, deviations from HWE did not clearly explain between-study heterogeneity in the genetic effects. The proposed hierarchical models allow the synthesis of data across gene-disease association studies with appropriate consideration of HWE issues. PMID- 16685694 TI - Electromyography patterns of propriospinal myoclonus can be mimicked voluntarily. AB - In order to investigate the clinical impact of polymyographic evaluation on the diagnosis of propriospinal myoclonus (PSM), we performed electromyography recordings of various truncal muscles in eight healthy volunteers while they mimicked PSM symptoms. Before the experiment, each volunteer learned how to mimic PSM by watching a videotape that showed typical PSM characteristics, i.e., brief symmetric flexion of the trunk. The recorded polymyographic patterns of all volunteers were quite compatible with those found in the previous reports. The present study demonstrates that previously known polymyographic patterns of PSM can be voluntarily mimicked. Additional studies, such as jerk-locked cortical potential, are required to confirm the diagnosis of true PSM. PMID- 16685695 TI - How long does it take for epilepsy to become intractable? A prospective investigation. AB - OBJECTIVE: To determine prospectively when in the course of epilepsy intractability becomes apparent. METHODS: Data are from a prospective cohort of 613 children followed for a median of 9.7 years. Epilepsy syndromes were grouped: focal, idiopathic, catastrophic, and other. Intractability was defined in two ways: (1) 2 drugs failed, 1 seizure/month, on average, for 18 months (stringent), and (2) failure of 2 drugs. Delayed intractability was defined as 3 or more years after epilepsy diagnosis. RESULTS: Eighty-three children (13.8%) met the stringent and 142 (23.2%) met the two-drug definition. Intractability depended on syndrome (p < 0.0001): 26 (31.3%) children meeting stringent and 39 (27.5%) meeting the 2-drug definition had delayed intractability. Intractability was delayed more often in focal than catastrophic epilepsy (stringent: 46.2 vs 14.3%, p = 0.003; two-drug: 40.3 vs 2.2%, p